World Journal of Gastroenterology

Transcription

1 ISSN (print) ISSN (online) World Journal of Gastroenterology World J Gastroenterol 2014 July 14; 20(26): Published by Baishideng Publishing Group Inc

2 Editorial Board The World Journal of Gastroenterology Editorial Board consists of 1353 members, representing a team of worldwide experts in gastroenterology and hepatology. They are from 68 countries, including Albania (1), Algeria (1), Argentina (7), Australia (31), Austria (9), Belgium (10), Brazil (20), Brunei Darussalam (1), Bulgaria (2), Cambodia (1), Canada (25), Chile (4), China (161), Croatia (1), Cuba (1), Czech (6), Denmark (2), Egypt (9), Estonia (2), Finland (6), France (17), Germany (56), Greece (31), Guatemala (1), Hungary (14), Iceland (1), India (33), Indonesia (2), Iran (10), Ireland (9), Israel (18), Italy (195), Japan (151), Jordan (1), Kuwait (1), Lebanon (7), Lithuania (1), Malaysia (1), Mexico (10), Morocco (1), Netherlands (5), New Zealand (4), Nigeria (3), Norway (6), Pakistan (6), Poland (12), Portugal (8), Puerto Rico (1), Qatar (1), Romania (10), Russia (3), Saudi Arabia (2), Singapore (7), Slovenia (2), South Korea (64), Spain (51), Sri Lanka (1), Sudan (1), Sweden (12), Switzerland (5), Thailand (7), Trinidad and Tobago (1), Tunisia (2), Turkey (56), United Kingdom (47), United States (173), Venezuela (1), and Vietnam (1). EDITORS-IN-CHIEF Stephen C Strom, Stockholm Saleh A Naser, Orlando Andrzej S Tarnawski, Long Beach Damian Garcia-Olmo, Madrid GUEST EDITORIAL BOARD MEMBERS Jia-Ming Chang, Taipei Jane CJ Chao, Taipei Kuen-Feng Chen, Taipei Tai-An Chiang, Tainan Yi-You Chiou, Taipei Seng-Kee Chuah, Kaohsiung Wan-Long Chuang, Kaohsiung How-Ran Guo, Tainan Ming-Chih Hou, Taipei Po-Shiuan Hsieh, Taipei Ching-Chuan Hsieh, Chiayi county Jun-Te Hsu, Taoyuan Chung-Ping Hsu, Taichung Chien-Ching Hung, Taipei Chao-Hung Hung, Kaohsiung Chen-Guo Ker, Kaohsiung Yung-Chih Lai, Taipei Teng-Yu Lee, Taichung City Wei-Jei Lee, Taoyuan Jin-Ching Lee, Kaohsiung Jen-Kou Lin, Taipei Ya-Wen Lin, Taipei Hui-kang Liu, Taipei Min-Hsiung Pan, Taipei Bor-Shyang Sheu, Tainan Hon-Yi Shi, Kaohsiung Fung-Chang Sung, Taichung Dar-In Tai, Taipei Jung-Fa Tsai, Kaohsiung Yao-Chou Tsai, New Taipei City Chih-Chi Wang, Kaohsiung Liang-Shun Wang, New Taipei City Hsiu-Po Wang, Taipei Jaw-Yuan Wang, Kaohsiung Yuan-Huang Wang, Taipei Yuan-Chuen Wang, Taichung Deng-Chyang Wu, Kaohsiung Shun-Fa Yang, Taichung Hsu-Heng Yen, Changhua MEMBERS OF THE EDITORIAL BOARD Albania Saadi Berkane, Algiers Algeria Samir Rouabhia, Batna Argentina N Tolosa de Talamoni, Córdoba Eduardo de Santibanes, Buenos Aires Bernardo Frider, Capital Federal Guillermo Mazzolini, Pilar Carlos Jose Pirola, Buenos Aires Bernabé Matías Quesada, Buenos Aires María Fernanda Troncoso, Buenos Aires Australia Golo Ahlenstiel, Westmead Minoti V Apte, Sydney Jacqueline S Barrett, Melbourne Michael Beard, Adelaide Filip Braet, Sydney Guy D Eslick, Sydney Christine Feinle-Bisset, Adelaide Mark D Gorrell, Sydney Michael Horowitz, Adelaide Gordon Stanley Howarth, Roseworthy Seungha Kang, Brisbane Alfred King Lam, Gold Coast Ian C Lawrance, PerthFremantle Barbara Anne Leggett, Brisbane Daniel A Lemberg, Sydney Rupert W Leong, Sydney Finlay A Macrae, Victoria Vance Matthews, Melbourne David L Morris, Sydney Reme Mountifield, Bedford Park Hans J Netter, Melbourne Nam Q Nguyen, Adelaide Liang Qiao, Westmead Rajvinder Singh, Adelaide Ross Cyril Smith, StLeonards Kevin J Spring, Sydney Debbie Trinder, Fremantle Daniel R van Langenberg, Box Hill David Ian Watson, Adelaide Desmond Yip, Garran Li Zhang, Sydney I March 26, 2014

3 Austria Felix Aigner, Innsbruck Gabriela A Berlakovich, Vienna Herwig R Cerwenka, Graz Peter Ferenci, Wien Alfred Gangl, Vienna Kurt Lenz, Linz Markus Peck-Radosavljevic, Vienna Markus Raderer, Vienna Stefan Riss, Vienna Belgium Michael George Adler, Brussels Benedicte Y De Winter, Antwerp Mark De Ridder, Jette Olivier Detry, Liege Denis Dufrane Dufrane, Brussels Nikos Kotzampassakis, Liège Geert KMM Robaeys, Genk Xavier Sagaert, Leuven Peter Starkel, Brussels Eddie Wisse, Keerbergen Brazil SMP Balzan, Santa Cruz do Sul JLF Caboclo, Sao jose do rio preto Fábio Guilherme Campos, Sao Paulo Claudia RL Cardoso, Rio de Janeiro Roberto J Carvalho-Filho, Sao Paulo Carla Daltro, Salvador José Sebastiao dos Santos, Ribeirao Preto Eduardo LR Mello, Rio de Janeiro Sthela Maria Murad-Regadas, Fortaleza Claudia PMS Oliveira, Sao Paulo Júlio C Pereira-Lima, Porto Alegre Marcos V Perini, Sao Paulo Vietla Satyanarayana Rao, Fortaleza Raquel Rocha, Salvador AC Simoes e Silva, Belo Horizonte Mauricio F Silva, Porto Alefre Aytan Miranda Sipahi, Sao Paulo Rosa Leonôra Salerno Soares, Niterói Cristiane Valle Tovo, Porto Alegre Eduardo Garcia Vilela, Belo Horizonte Brunei Darussalam Vui Heng Chong, Bandar Seri Begawan Bulgaria Tanya Kirilova Kadiyska, Sofia Mihaela Petrova, Sofia Cambodia Francois Rouet, Phnom Penh Canada Brian Bressler, Vancouver Frank J Burczynski, Winnipeg Wangxue Chen, Ottawa Francesco Crea, Vancouver Mirko Diksic, Montreal Jane A Foster, Hamilton Hugh J Freeman, Vancouver Shahrokh M Ghobadloo, Ottawa Yuewen Gong, Winnipeg Philip H Gordon, Quebec Rakesh Kumar, Edmonton Wolfgang A Kunze, Hamilton Patrick Labonte, Laval Zhikang Peng, Winnipeg Jayadev Raju, Ottawa Maitreyi Raman, Calgary Giada Sebastiani, Montreal Maida J Sewitch, Montreal Eldon A Shaffer, Alberta Christopher W Teshima, Edmonton Jean Sévigny, Québec Pingchang Yang, Hamilton Pingchang Yang, Hamilton Eric M Yoshida, Vancouver Bin Zheng, Edmonton Chile Marcelo A Beltran, La Serena Flavio Nervi, Santiago Adolfo Parra-Blanco, Santiago Alejandro Soza, Santiago China Zhao-Xiang Bian, Hong Kong San-Jun Cai, Shanghai Guang-Wen Cao, Shanghai Long Chen, Nanjing Ru-Fu Chen, Guangzhou George G Chen, Hong Kong Li-Bo Chen, Wuhan Jia-Xu Chen, Beijing Hong-Song Chen, Beijing Lin Chen, Beijing Yang-Chao Chen, Hong Kong Zhen Chen, Shanghai Ying-Sheng Cheng, Shanghai Kent-Man Chu, Hong Kong Zhi-Jun Dai, Xi an Jing-Yu Deng, Tianjin Yi-Qi Du, Shanghai Zhi Du, Tianjin Hani El-Nezami, Hong Kong Bao-Ying Fei, Hangzhou Chang-Ming Gao, Nanjing Jian-Ping Gong, Chongqing Zuo-Jiong Gong, Wuhan Jing-Shan Gong, Shenzhen Guo-Li Gu, Beijing Yong-Song Guan, Chengdu Mao-Lin Guo, Luoyang Jun-Ming Guo, Ningbo Yan-Mei Guo, Shanghai Xiao-Zhong Guo, Shenyang Guo-Hong Han, Xi an Ming-Liang He, Hong Kong Peng Hou, Xi an Zhao-Hui Huang, Wuxi Feng Ji, Hangzhou Simon Law, Hong Kong Yu-Yuan Li, Guangzhou Meng-Sen Li, Haikou Shu-De Li, Shanghai Zong-Fang Li, Xi an Qing-Quan Li, Shanghai Kang Li, Lasa Han Liang, Tianjin Xing e Liu, Hangzhou Zheng-Wen Liu, Xi an Xiao-Fang Liu, Yantai Bin Liu, Tianjin Quan-Da Liu, Beijing Hai-Feng Liu, Beijing Fei Liu, Shanghai Ai-Guo Lu, Shanghai He-Sheng Luo, Wuhan Xiao-Peng Ma, Shanghai Yong Meng, Shantou Ke-Jun Nan, Xi an Siew Chien Ng, Hong Kong Simon SM Ng, Hong Kong Zhao-Shan Niu, Qingdao Bo-Rong Pan, Xi an Di Qu, Shanghai Rui-Hua Shi, Nanjing Bao-Min Shi, Shanghai Xiao-Dong Sun, Hangzhou Si-Yu Sun, Shenyang Guang-Hong Tan, Haikou Wen-Fu Tang, Chengdu Anthony YB Teoh, Hong Kong Wei-Dong Tong, Chongqing Eric Tse, Hong Kong Hong Tu, Shanghai Rong Tu, Haikou Jian-She Wang, Shanghai Kai Wang, Jinan Xiao-Ping Wang, Xianyang Dao-Rong Wang, Yangzhou De-Sheng Wang, Xi an Chun-You Wang, Wuhan Ge Wang, Chongqing Xi-Shan Wang, Harbin Wei-hong Wang, Beijing Zhen-Ning Wang, Shenyang Wai Man Raymond Wong, Hong Kong Chun-Ming Wong, Hong Kong Jian Wu, Shanghai Sheng-Li Wu, Xi an Wu-Jun Wu, Xi an Bing Xia, Wuhan Qing Xia, Chengdu Yan Xin, Shenyang Dong-Ping Xu, Beijing Jian-Min Xu, Shanghai Wei Xu, Changchun Ming Yan, Jinan Xin-Min Yan, Kunming Yi-Qun Yan, Shanghai Feng Yang, Shanghai Yong-Ping Yang, Beijing He-Rui Yao, Guangzhou Thomas Yau, Hong Kong Winnie Yeo, Hong Kong Jing You, Kunming Jian-Qing Yu, Wuhan Ying-Yan Yu, Shanghai Wei-Zheng Zeng, Chengdu Zong-Ming Zhang, Beijing II March 26, 2014

4 Dian-Liang Zhang, Qingdao Ya-Ping Zhang, Shijiazhuang You-Cheng Zhang, Lanzhou Jian-Zhong Zhang, Beijing Ji-Yuan Zhang, Beijing Hai-Tao Zhao, Beijing Jian Zhao, Shanghai Jian-Hong Zhong, Nanning Ying-Qiang Zhong, Guangzhou Ping-Hong Zhou, Shanghai Yan-Ming Zhou, Xiamen Tong Zhou, Nanchong Li-Ming Zhou, Chengdu Guo-Xiong Zhou, Nantong Feng-Shang Zhu, Shanghai Jiang-Fan Zhu, Shanghai Zhao-Hui Zhu, Beijing Croatia Tajana Filipec Kanizaj, Zagreb Cuba Damian Casadesus, Havana Czech Jan Bures, Hradec Kralove Marcela Kopacova, Hradec Kralove Otto Kucera, Hradec Kralove Marek Minarik, Prague Pavel Soucek, Prague Miroslav Zavoral, Prague Denmark Vibeke Andersen, Odense E Michael Danielsen, Copenhagen Egypt Mohamed MM Abdel-Latif, Assiut Hussein Atta, Cairo Ashraf Elbahrawy, Cairo Mortada Hassan El-Shabrawi, Cairo Mona El Said El-Raziky, Cairo Elrashdy M Redwan, New Borg Alrab Zeinab Nabil Ahmed Said, Cairo Ragaa HM Salama, Assiut Maha Maher Shehata, Mansoura Estonia Margus Lember, Tartu Tamara Vorobjova, Tartu Finland Marko Kalliomäki, Turku Thomas Kietzmann, Oulu Kaija-Leena Kolho, Helsinki Eija Korkeila, Turku Heikki Makisalo, Helsinki Tanja Pessi, Tampere France Armando Abergel Clermont, Ferrand Elie K Chouillard, Polssy Pierre Cordelier, Toulouse Pascal P Crenn, Garches Catherine Daniel, Lille Fanny Daniel, Paris Cedric Dray, Toulouse Benoit Foligne, Lille Jean-Noel Freund, Strasbourg Nathalie Janel, Paris Majid Khatib, Bordeaux Jacques Marescaux, Strasbourg Jean-Claude Marie, Paris Hang Nguyen, Clermont-Ferrand Hugo Perazzo, Paris Alain L Servin, Chatenay-Malabry Chang Xian Zhang, Lyon Germany Stavros A Antoniou, Monchengladbach Erwin Biecker, Siegburg Hubert E Blum, Freiburg Thomas Bock, Berlin Katja Breitkopf-Heinlein, Mannheim Elke Cario, Essen Güralp Onur Ceyhan, Munich Angel Cid-Arregui, Heidelberg Michael Clemens Roggendorf, München Christoph F Dietrich, Bad Mergentheim Valentin Fuhrmann, Hamburg Nikolaus Gassler, Aachen Andreas Geier, Wuerzburg Markus Gerhard, Munich Anton Gillessen, Muenster Thorsten Oliver Goetze, Offenbach Daniel Nils Gotthardt, Heidelberg Robert Grützmann, Dresden Thilo Hackert, Heidelberg Joerg Haier, Muenster Claus Hellerbrand, Regensburg Harald Peter Hoensch, Darmstadt Jens Hoeppner, Freiburg Richard Hummel, Muenster Jakob Robert Izbicki, Hamburg Gernot Maximilian Kaiser, Essen Matthias Kapischke, Hamburg Michael Keese, Frankfurt Andrej Khandoga, Munich Jorg Kleeff, Munich Alfred Koenigsrainer, Tuebingen Peter Christopher Konturek, Saalfeld Michael Linnebacher, Rostock Stefan Maier, Kaufbeuren Oliver Mann, Hamburg Marc E Martignoni, Munic Thomas Minor, Bonn Oliver Moeschler, Osnabrueck Jonas Mudter, Eutin Sebastian Mueller, Heidelberg Matthias Ocker, Berlin Andreas Ommer, Essen Albrecht Piiper, Frankfurt Esther Raskopf, Bonn Christoph Reichel, Bad Brückenau Elke Roeb, Giessen Udo Rolle, Frankfurt Karl-Herbert Schafer, Zweibrücken Andreas G Schreyer, Regensburg Manuel A Silva, Penzberg Georgios C Sotiropoulos, Essen Ulrike S Stein, Berlin Dirk Uhlmann, Leipzig Michael Weiss, Halle Hong-Lei Weng, Mannheim Karsten Wursthorn, Hamburg Greece Alexandra Alexopoulou, Athens Nikolaos Antonakopoulos, Athens Stelios F Assimakopoulos, Patras Grigoris Chatzimavroudis, Thessaloniki Evangelos Cholongitas, Thessaloniki Gregory Christodoulidis, Larisa George N Dalekos, Larissa Maria Gazouli, Athens Urania Georgopoulou, Athens Eleni Gigi, Thessaloniki Stavros Gourgiotis, Athens Leontios J Hadjileontiadis, Thessaloniki Thomas Hyphantis, Ioannina Ioannis Kanellos, Thessaloniki Stylianos Karatapanis, Rhodes Michael Koutsilieris, Athens Spiros D Ladas, Athens Theodoros K Liakakos, Athens Emanuel K Manesis, Athens Spilios Manolakopoulos, Athens Gerassimos John Mantzaris, Athens Athanasios D Marinis, Piraeus Nikolaos Ioannis Nikiteas, Athens Konstantinos X Papamichael, Athens George Sgourakis, Athens Konstantinos C Thomopoulos, Patras Konstantinos Triantafyllou, Athens Christos Triantos, Patras Georgios Zacharakis, Athens Petros Zezos, Alexandroupolis Demosthenes E Ziogas, Ioannina Guatemala Carlos Maria Parellada, Guatemala Hungary Mihaly Boros, Szeged Tamás Decsi, Pécs Gyula Farkas, Szeged Andrea Furka, Debrecen Y vette Mandi, Szeged Peter L Lakatos, Budapest Pal Miheller, Budapest Tamás Molnar, Szeged Attila Olah, Gyor Maria Papp, Debrecen Zoltan Rakonczay, Szeged III March 26, 2014

5 Ferenc Sipos, Budapest Miklós Tanyi, Debrecen Tibor Wittmann, Szeged Iceland Tryggvi Bjorn Stefánsson, Reykjavík India Brij B Agarwal, New Delhi Deepak N Amarapurkar, Mumbai Shams ul Bari, Srinagar Sriparna Basu, Varanasi Runu Chakravarty, Kolkata Devendra C Desai, Mumbai Nutan D Desai, Mumbai Suneela Sunil Dhaneshwar, Pune Radha K Dhiman, Chandigarh Pankaj Garg, Mohali Uday C Ghoshal, Lucknow Kalpesh Jani, Vadodara Premashis Kar, New Delhi Jyotdeep Kaur, Chandigarh Rakesh Kochhar, Chandigarh Pradyumna K Mishra, Mumbai Asish K Mukhopadhyay, Kolkata Imtiyaz Murtaza, Srinagar P Nagarajan, New Delhi Samiran Nundy, Delhi Gopal Pande, Hyderabad Benjamin Perakath, Vellore Arun Prasad, New Delhi D Nageshwar Reddy, Hyderabad Lekha Saha, Chandigarh Sundeep Singh Saluja, New Delhi Mahesh Prakash Sharma, New Delhi Sadiq Saleem Sikora, Bangalore Sarman Singh, New Delhi Rajeev Sinha, Jhansi Rupjyoti Talukdar, Hyderabad Rakesh Kumar Tandon, New Delhi Narayanan Thirumoorthy, Coimbatore Indonesia David Handojo Muljono, Jakarta Andi Utama, Jakarta Iran Arezoo Aghakhani, Tehran Seyed Mohsen Dehghani, Shiraz Ahad Eshraghian, Shiraz Hossein Khedmat, Tehran Sadegh Massarrat, Tehran Marjan Mohammadi, Tehran Roja Rahimi, Tehran Farzaneh Sabahi, Tehran Majid Sadeghizadeh, Tehran Farideh Siavoshi, Tehran Ireland Gary Alan Bass, Dublin David J Brayden, Dublin Ronan A Cahill, Dublin Glen A Doherty, Dublin Liam J Fanning, Cork Barry Philip McMahon, Dublin RossMcManus, Dublin Dervla O Malley, Cork Sinead M Smith, Dublin Israel Dan Carter, Ramat Gan Jorge-Shmuel Delgado, Metar Eli Magen, Ashdod Nitsan Maharshak, Tel Aviv Shaul Mordechai, Beer Sheva Menachem Moshkowitz, Tel Aviv William Bahij Nseir, Nazareth Shimon Reif, Jerusalem Ram Reifen, Rehovot Ariella Bar-Gil Shitrit, Jerusalem Noam Shussman, Jerusalem Igor Sukhotnik, Haifa Nir Wasserberg, Petach Tiqwa Jacob Yahav, Rehovot Doron Levi Zamir, Gedera Shira Zelber-Sagi, Haifa Romy Zemel, Petach-Tikva Italy Ludovico Abenavoli, Catanzaro Luigi Elio Adinolfi, Naples Carlo Virginio Agostoni, Milan Anna Alisi, Rome Piero Luigi Almasio, Palermo Donato Francesco Altomare, Bari Amedeo Amedei, Florence Pietro Andreone, Bologna Imerio Angriman, Padova Vito Annese, Florence Paolo Aurello, Rome Salavtore Auricchio, Naples Gian Luca Baiocchi, Brescia Gianpaolo Balzano, Milan Antonio Basoli, Rome Gabrio Bassotti, San Sisto Mauro Bernardi, Bologna Alberto Biondi, Rome Ennio Biscaldi, Genova Massimo Bolognesi, Padua Luigi Bonavina, Milano Aldo Bove, Chieti Raffaele Bruno, Pavia Luigi Brusciano, Napoli Giuseppe Cabibbo, Palermo Carlo Calabrese, Bologna Daniele Calistri, Meldola Vincenza Calvaruso, Palermo Lorenzo Camellini, Reggio Emilia Marco Candela, Bologna Raffaele Capasso, Naples Lucia Carulli, Modena Renato David Caviglia, Rome Luigina Cellini, Chieti Giuseppe Chiarioni, Verona Claudio Chiesa, Rome Michele Cicala, Roma Rachele Ciccocioppo, Pavia Sandro Contini, Parma Gaetano Corso, Foggia Renato Costi, Parma Alessandro Cucchetti, Bologna Rosario Cuomo, Napoli Giuseppe Currò, Messina Paola De Nardi, Milano Giovanni D De Palma, Naples Raffaele De Palma, Napoli Giuseppina De Petro, Brescia Valli De Re, Aviano Paolo De Simone, Pisa Giuliana Decorti, Trieste Emanuele Miraglia del Giudice, Napoli Isidoro Di Carlo, Catania Matteo Nicola Dario Di Minno, Naples Massimo Donadelli, Verona Mirko D Onofrio, Verona Maria Pina Dore, Sassari Luca Elli, Milano Massimiliano Fabozzi, Aosta Massimo Falconi, Ancona Ezio Falletto, Turin Silvia Fargion, Milan Matteo Fassan, Verona Gianfranco Delle Fave, Roma Alessandro Federico, Naples Francesco Feo, Sassari Davide Festi, Bologna Natale Figura, Siena Vincenzo Formica, Rome Mirella Fraquelli, Milan Marzio Frazzoni, Modena Walter Fries, Messina Gennaro Galizia, Naples Andrea Galli, Florence Matteo Garcovich, Rome Eugenio Gaudio, Rome Paola Ghiorzo, Genoa Edoardo G Giannini, Genova Luca Gianotti, Monza Maria Cecilia Giron, Padova Alberto Grassi, Rimini Gabriele Grassi, Trieste Francesco Greco, Bergamo Luigi Greco, Naples Antonio Grieco, Rome Fabio Grizzi, Rozzano Laurino Grossi, Pescara Salvatore Gruttadauria, Palermo Simone Guglielmetti, Milan Tiberiu Hershcovici, Jerusalem Calogero Iacono, Verona Enzo Ierardi, Bari Amedeo Indriolo, Bergamo Raffaele Iorio, Naples Paola Iovino, Salerno Angelo A Izzo, Naples Loreta Kondili, Rome Filippo La Torre, Rome Giuseppe La Torre, Rome Giovanni Latella, L Aquila Salvatore Leonardi, Catania Massimo Libra, Catania Anna Licata, Palermo C armela Loguercio, Naples Amedeo Lonardo, Modena Carmelo Luigiano, Catania Francesco Luzza, Catanzaro Giovanni Maconi, Milano Antonio Macrì, Messina Mariano Malaguarnera, Catania IV March 26, 2014

6 Francesco Manguso, Napoli Tommaso Maria Manzia, Rome Daniele Marrelli, Siena Gabriele Masselli, Rome Sara Massironi, Milan Giuseppe Mazzarella, Avellino Michele Milella, Rome Giovanni Milito, Rome Antonella d Arminio Monforte, Milan Fabrizio Montecucco, Genoa Giovanni Monteleone, Rome Mario Morino, Torino Vincenzo La Mura, Milan Gerardo Nardone, Naples Riccardo Nascimbeni, Brescia Gabriella Nesi, Florence Giuseppe Nigri, Rome Erica Novo, Turin Veronica Ojetti, Rome Michele Orditura, Naples Fabio Pace, Seriate Lucia Pacifico, Rome Omero Alessandro Paoluzi, Rome Valerio Pazienza, San Giovanni Rotondo Rinaldo Pellicano, Turin Adriano M Pellicelli, Rome Nadia Peparini, Ciampino Mario Pescatori, Rome Antonio Picardi, Rome Alberto Pilotto, Padova Alberto Piperno, Monza Anna Chiara Piscaglia, Rome Maurizio Pompili, Rome Francesca Romana Ponziani, Rome Cosimo Prantera, Rome Girolamo Ranieri, Bari Carlo Ratto, Tome Barbara Renga, Perugia Alessandro Repici, Rozzano Maria Elena Riccioni, Rome Lucia Ricci-Vitiani, Rome Luciana Rigoli, Messina Mario Rizzetto, Torino Ballarin Roberto, Modena Roberto G Romanelli, Florence Claudio Romano, Messina Luca Roncucci, Modena Cesare Ruffolo, Treviso L ucia Sacchetti, Napoli Rodolfo Sacco, Pisa Lapo Sali, Florence Romina Salpini, Rome Giulio Aniello, Santoro Treviso Armando Santoro, Rozzano Edoardo Savarino, Padua Marco Senzolo, Padua Annalucia Serafino, Rome Giuseppe S Sica, Rome Pierpaolo Sileri, Rome Cosimo Sperti, Padua Vincenzo Stanghellini, Bologna Cristina Stasi, Florence Gabriele Stocco, Trieste Roberto Tarquini, Florence Mario Testini, Bari Guido Torzilli, Milan Guido Alberto Massimo, Tiberio Brescia Giuseppe Toffoli, Aviano Alberto Tommasini, Trieste Francesco Tonelli, Florence Cesare Tosetti Porretta, Terme Lucio Trevisani, Cona Guglielmo M Trovato, Catania Mariapia Vairetti, Pavia Luca Vittorio Valenti, Milano Mariateresa T Ventura, Bari Giuseppe Verlato, Verona Alessandro Vitale, Padova Marco Vivarelli, Ancona Giovanni Li Volti, Catania Giuseppe Zanotti, Padua Vincenzo Zara, Lecce Gianguglielmo Zehender, Milan Anna Linda Zignego, Florence Rocco Antonio Zoccali, Messina Angelo Zullo, Rome Japan Yasushi Adachi, Sapporo Takafumi Ando, Nagoya Masahiro Arai, Tokyo Makoto Arai, Chiba Takaaki Arigami, Kagoshima Itaru Endo,Yokohama Munechika Enjoji, Fukuoka Shunji Fujimori, Tokyo Yasuhiro Fujino, Akashi Toshiyoshi Fujiwara, Okayama Yosuke Fukunaga, Tokyo Toshio Fukusato, Tokyo Takahisa Furuta, Hamamatsu Osamu Handa, Kyoto Naoki Hashimoto, Osaka Yoichi Hiasa, Toon Masatsugu Hiraki, Saga Satoshi Hirano, Sapporo Keiji Hirata, Fukuoka Toru Hiyama, Higashihiroshima Akira Hokama, Nishihara Shu Hoteya, Tokyo Masao Ichinose, Wakayama Tatsuya Ide, Kurume Masahiro Iizuka, Akita Toshiro Iizuka, Tokyo Kenichi Ikejima, Tokyo Tetsuya Ikemoto, Tokushima Hiroyuki Imaeda, Saitama Atsushi Imagawa, Kan-onji Hiroo Imazu, Tokyo Akio Inui, Kagoshima Shuji Isaji, Tsu Toru Ishikawa, Niigata Toshiyuki Ishiwata, Tokyo Soichi Itaba, Kitakyushu Yoshiaki Iwasaki, Okayama Tatehiro Kagawa, Isehara Satoru Kakizaki, Maebashi Naomi Kakushima, Shizuoka Terumi Kamisawa, Tokyo Akihide Kamiya, Isehara Osamu Kanauchi, Tokyo Tatsuo Kanda, Chiba Shin Kariya, Okayama Shigeyuki Kawa, Matsumoto Takumi Kawaguchi, Kurume Takashi Kawai, Tokyo Soo Ryang Kim, Kobe Shinsuke Kiriyama, Gunma Tsuneo Kitamura, Urayasu Masayuki Kitano, Osakasayama Hirotoshi Kobayashi, Tokyo Hironori Koga, Kurume Takashi Kojima, Sapporo Satoshi Kokura, Kyoto Shuhei Komatsu, Kyoto Tadashi Kondo, Tokyo Yasuteru Kondo, Sendai Yasuhiro Kuramitsu, Yamaguchi Yukinori Kurokawa, Osaka Shin Maeda, Yokohama Koutarou Maeda, Toyoake Hitoshi Maruyama, Chiba Atsushi Masamune, Sendai Hiroyuki Matsubayashi, Suntogun Akihisa Matsuda, Inzai Hirofumi Matsui, Tsukuba Akira Matsumori, Kyoto Yoichi Matsuo, Nagoya Y Matsuzaki, Ami Toshihiro Mitaka, Sapporo Kouichi Miura, Akita Shinichi Miyagawa, Matumoto Eiji Miyoshi, Suita Toru Mizuguchi, Sapporo Nobumasa Mizuno, Nagoya Zenichi Morise, Nagoya Tomohiko Moriyama, Fukuoka Kunihiko Murase, Tusima Michihiro Mutoh, Tsukiji Akihito Nagahara, Tokyo Hikaru Nagahara, Tokyo Hidenari Nagai, Tokyo Koichi Nagata, Shimotsuke-shi Masaki Nagaya, Kawasaki Hisato Nakajima, Nishi-Shinbashi Toshifusa Nakajima, Tokyo Hiroshi Nakano, Kawasaki Hiroshi Nakase, Kyoto Toshiyuki Nakayama, Nagasaki Takahiro Nakazawa, Nagoya Shoji Natsugoe, Kagoshima City Tsutomu Nishida, Suita Shuji Nomoto, Naogya Sachiyo Nomura, Tokyo Takeshi Ogura, Takatsukishi Nobuhiro Ohkohchi, Tsukuba Toshifumi Ohkusa, Kashiwa Hirohide Ohnishi, Akita Teruo Okano, Tokyo Satoshi Osawa, Hamamatsu Motoyuki Otsuka, Tokyo Michitaka Ozaki, Sapporo Satoru Saito, Yokohama Chouhei Sakakura, Kyoto Naoaki Sakata, Sendai Ken Sato, Maebashi Toshiro Sato, Tokyo Tomoyuki Shibata, Toyoake H Shimada, Tokyo Tomohiko Shimatani, Kure Yukihiro Shimizu, Nanto Tadashi Shimoyama, Hirosaki Masayuki Sho, Nara Ikuo Shoji, Kobe Atsushi Sofuni, Tokyo Takeshi Suda, Niigata M Sugimoto, Hamamatsu Ken Sugimoto, Hamamatsu Haruhiko Sugimura, Hamamatsu Shoichiro Sumi, Kyoto Hidekazu Suzuki, Tokyo Masahiro Tajika, Nagoya Hitoshi Takagi, Takasaki Toru Takahashi, Niigata V March 26, 2014

7 Yoshihisa Takahashi, Tokyo Shinsuke Takeno, Fukuoka Akihiro Tamori, Osaka Kyosuke Tanaka, Tsu Shinji Tanaka, Hiroshima Atsushi Tanaka, Tokyo Yasuhito Tanaka, Nagoya Shinji Tanaka, Tokyo Minoru Tomizawa, Yotsukaido City Kyoko Tsukiyama-Kohara, Kagoshima Takuya Watanabe, Niigata Kazuhiro Watanabe, Sendai Satoshi Yamagiwa, Niigata Takayuki Yamamoto, Yokkaichi Hiroshi Yamamoto, Otsu Kosho Yamanouchi, Nagasaki Ichiro Yasuda, Gifu Yutaka Yata, Maebashi-city Shin-ichi Yokota, Sapporo Norimasa Yoshida, Kyoto Hiroshi Yoshida, Tama-City Hitoshi Yoshiji, Kashihara Kazuhiko Yoshimatsu, Tokyo Kentaro Yoshioka, Toyoake Nobuhiro Zaima, Nara Jordan Khaled Ali Jadallah, Irbid Kuwait Islam Khan, Kuwait Lebanon Bassam N Abboud, Beirut Kassem A Barada, Beirut Marwan Ghosn, Beirut Iyad A Issa, Beirut Fadi H Mourad, Beirut AIa Sharara, Beirut Rita Slim, Beirut Lithuania Antanas Mickevicius, Kaunas Malaysia Huck Joo Tan, Petaling Jaya Mexico Richard A Awad, Mexico City Carlos R Camara-Lemarroy, Monterrey Norberto C Chavez-Tapia, Mexico City Wolfgang Gaertner, Mexico City Diego Garcia-Compean, Monterrey Arturo Panduro, Guadalajara OT Teramoto-Matsubara, Mexico City Felix Tellez-Avila, Mexico City Omar Vergara-Fernandez, Mexico City Saúl Villa-Trevino, Cuidad de México Morocco Samir Ahboucha, Khouribga Netherlands Robert J de Knegt, Rotterdam Tom Johannes Gerardus Gevers, Nijmegen Menno Hoekstra, Leiden BW Marcel Spanier, Arnhem Karel van Erpecum, Utrecht New Zealand Leo K Cheng, Auckland Andrew Stewart Day, Christchurch Jonathan Barnes Koea, Auckland Max Petrov, Auckland Nigeria Olufunmilayo Adenike Lesi, Lagos Jesse Abiodun Otegbayo, Ibadan Stella Ifeanyi Smith, Lagos Norway Trond Berg, Oslo Trond Arnulf Buanes, Krokkleiva Thomas de Lange, Rud Magdy El-Salhy, Stord Rasmus Goll, Tromso Dag Arne Lihaug Hoff, Aalesund Pakistan Zaigham Abbas, Karachi Usman A Ashfaq, Faisalabad Muhammad Adnan Bawany, Hyderabad Muhammad Idrees, Lahore Saeed Sadiq Hamid, Karachi Yasir Waheed, Islamabad Poland Thomas Brzozowski, Cracow Magdalena Chmiela, Lodz Krzysztof Jonderko, Sosnowiec Anna Kasicka-Jonderko, Sosnowiec Michal Kukla, Katowice Tomasz Hubert Mach, Krakow Agata Mulak, Wroclaw Danuta Owczarek, Kraków Piotr Socha, Warsaw Piotr Stalke, Gdansk Julian Teodor Swierczynski, Gdansk Anna M Zawilak-Pawlik, Wroclaw Portugal Marie Isabelle Cremers, Setubal Ceu Figueiredo, Porto Ana Isabel Lopes, LIsbon M Paula Macedo, Lisboa Ricardo Marcos, Porto Rui T Marinho, Lisboa Guida Portela-Gomes, Estoril Filipa F Vale, Lisbon Puerto Rico Caroline B Appleyard, Ponce Qatar Abdulbari Bener, Doha Romania Mihai Ciocirlan, Bucharest Dan LucianDumitrascu, Cluj-Napoca Carmen Fierbinteanu-Braticevici, Bucharest Romeo G Mihaila, Sibiu Lucian Negreanu, Bucharest Adrian Saftoiu, Craiova Andrada Seicean, Cluj-Napoca Ioan Sporea, Timisoara Letiţia Adela Maria Streba, Craiova Anca Trifan, Iasi Russia Victor Pasechnikov, Stavropol Vasiliy Ivanovich Reshetnyak, Moscow Vitaly Skoropad, Obninsk Saudi Arabia Abdul-Wahed N Meshikhes, Dammam M Ezzedien Rabie, Khamis Mushait Singapore Brian KP Goh, Singapore Richie Soong, Singapore Ker-Kan Tan, Singapore Kok-Yang Tan, Singapore Yee-Joo Tan, Singapore Mark Wong, Singapore Hong Ping Xia, Singapore Slovenia Matjaz Homan, Ljubljana Martina Perse, Ljubljana South Korea Sang Hoon Ahn, Seoul Soon Koo Baik, Wonju Soo-Cheon Chae, Iksan Byung-Ho Choe, Daegu VI March 26, 2014

8 Suck Chei Choi, Iksan Hoon Jai Chun, Seoul Yeun-Jun Chung, Seoul Young-Hwa Chung, Seoul Ki-Baik Hahm, Seongnam Sang Young Han, Busan Seok Joo Han, Seoul Seung-Heon Hong, Iksan Jin-Hyeok Hwang, Seoungnam Jeong Won Jang, Seoul Jin-Young Jang, Seoul Dae-Won Jun, Seoul Young Do Jung, Kwangju Gyeong Hoon Kang, Seoul Sung-Bum Kang, Seoul Koo Jeong Kang, Daegu Ki Mun Kang, Jinju Chang Moo Kang, Seodaemun-gu Gwang Ha Kim, Busan Sang Soo Kim, Goyang-si Jin Cheon Kim, Seoul Tae Il Kim, Seoul Jin Hong Kim, Suwon Kyung Mo Kim, Seoul Kyongmin Kim, Suwon Hyung-Ho Kim, Seongnam Seoung Hoon Kim, Goyang Sang Il Kim, Seoul Hyun-Soo Kim, Wonju Jung Mogg Kim, Seoul Dong Yi Kim, Gwangju Kyun-Hwan Kim, Seoul Jong-Han Kim, Ansan Ja-Lok Ku, Seoul Kyu Taek Lee, Seoul Hae-Wan Lee, Chuncheon Inchul Lee, Seoul Jung Eun Lee, Seoul Sang Chul Lee, Daejeon Song Woo Lee, Ansan-si Hyuk-Joon Lee, Seoul Seong-Wook Lee, Yongin Kil Yeon Lee, Seoul Jong-Inn Lee, Seoul Kyung A Lee, Seoul Jong-Baeck Lim, Seoul Eun-Yi Moon, Seoul SH Noh, Seoul Seung Woon Paik, Seoul Won Sang Park, Seoul Sung-Joo Park, Iksan Kyung Sik Park, Daegu Se Hoon Park, Seoul Yoonkyung Park, Gwangju Seung-Wan Ryu, Daegu Dong Wan Seo, Seoul Il Han Song, Cheonan Myeong Jun Song, Daejeon Yun Kyoung Yim, Daejeon Dae-Yeul Yu Daejeon Spain Mariam Aguas, Valencia Raul J Andrade, Málaga Antonio Arroyo, Elche Josep M Bordas, Barcelona Lisardo Boscá, Madrid Ricardo Robles Campos, Murcia Jordi Camps, Reus Carlos Cervera Barcelona Alfonso Clemente, Granada Pilar Codoner-Franch, Valencia Fernando J Corrales, Pamplona Fermin Sánchez de Medina, Granada Alberto Herreros de Tejada, Majadahonda Enrique de-madaria, Alicante JE Dominguez-Munoz, Santiago de Compostela Vicente Felipo, Valencia CM Fernandez-Rodriguez, Madrid Carmen Frontela-Saseta, Murcia Julio Galvez, Granada Maria Teresa García, Vigo MI Garcia-Fernandez, Málaga Emilio Gonzalez-Reimers, La Laguna Marcel Jimenez, Bellaterra Angel Lanas, Zaragoza Juan Ramón Larrubia, Guadalajara Antonio Lopez-Sanroman, Madrid Vicente Lorenzo-Zuniga, Badalona Alfredo J Lucendo, Tomelloso Vicenta Soledad Martinez-Zorzano, Vigo José Manuel Martin-Villa, Madrid Julio Mayol, Madrid Manuel Morales-Ruiz, Barcelona Alfredo Moreno-Egea, Murcia Albert Pares, Barcelona Maria Pellise, Barcelona José Perea, Madrid Miguel Angel Plaza, Zaragoza María J Pozo, Cáceres Enrique Quintero, La Laguna Jose M Ramia, Madrid Francisco Rodriguez-Frias, Barcelona Silvia Ruiz-Gaspa, Barcelona Xavier Serra-Aracil, Barcelona Vincent Soriano, Madrid Javier Suarez, Pamplona Carlos Taxonera, Madrid M Isabel Torres, Jaén Manuel Vazquez-Carrera, Barcelona Benito Velayos, Valladolid Silvia Vidal, Barcelona Sri Lanka Arjuna Priyadarsin De Silva, Colombo Sudan Ishag Adam, Khartoum Sweden Roland G Andersson, Lund Bergthor Björnsson, Linkoping Johan Christopher Bohr, Örebro Mauro D Amato, Stockholm Thomas Franzen, Norrkoping Evangelos Kalaitzakis, Lund Riadh Sadik, Gothenburg Per Anders Sandstrom, Linkoping Ervin Toth, Malmö Konstantinos Tsimogiannis, Vasteras Apostolos V Tsolakis, Uppsala Switzerland Gieri Cathomas, Liestal Jean Louis Frossard, Geneve Christian Toso, Geneva Stephan Robert Vavricka, Zurich Dominique Velin, Lausanne Thailand Thawatchai Akaraviputh, Bangkok P Yoysungnoen Chintana, Pathumthani Veerapol Kukongviriyapan, Muang Vijittra Leardkamolkarn, Bangkok Varut Lohsiriwat, Bangkok Somchai Pinlaor, Khaon Kaen D Wattanasirichaigoon, Bangkok Trinidad and Tobago B Shivananda Nayak, Mount Hope Tunisia Ibtissem Ghedira, Sousse Lilia Zouiten-Mekki, Tunis Turkey Sami Akbulut, Diyarbakir Inci Alican, Istanbul Mustafa Altindis, Sakarya Mutay Aslan, Antalya Oktar Asoglu, Istanbul Yasemin Hatice Balaban, Istanbul Metin Basaranoglu, Ankara Yusuf Bayraktar, Ankara Süleyman Bayram, Adiyaman Ahmet Bilici, Istanbul Ahmet Sedat Boyacioglu, Ankara Züleyha Akkan Cetinkaya, Kocaeli Cavit Col, Bolu Yasar Colak, Istanbul Cagatay Erden Daphan, Kirikkale Mehmet Demir, Hatay Ahmet Merih Dobrucali, Istanbul Gülsüm Ozlem Elpek, Antalya Ayse Basak Engin, Ankara Eren Ersoy, Ankara Osman Ersoy, Ankara Yusuf Ziya Erzin, Istanbul Mukaddes Esrefoglu, Istanbul Levent Filik, Ankara Ozgur Harmanci, Ankara Koray Hekimoglu, Ankara Abdurrahman Kadayifci, Gaziantep Cem Kalayci, Istanbul Selin Kapan, Istanbul Huseyin Kayadibi, Adana Sabahattin Kaymakoglu, Istanbul Metin Kement, Istanbul Mevlut Kurt, Bolu Resat Ozaras, Istanbul VII March 26, 2014

9 Elvan Ozbek, Adapazari Cengiz Ozcan, Mersin Hasan Ozen, Ankara Halil Ozguc, Bursa Mehmet Ozturk, Izmir Orhan V Ozkan, Sakarya Semra Paydas, Adana Ozlem Durmaz Suoglu, Istanbul Ilker Tasci, Ankara Müge Tecder-ünal, Ankara Mesut Tez, Ankara Serdar Topaloglu, Trabzon Murat Toruner, Ankara Gokhan Tumgor, Adana Oguz Uskudar, Adana Mehmet Yalniz, Elazig Mehmet Yaman, Elazig Veli Yazisiz, Antalya Yusuf Yilmaz, Istanbul Ozlem Yilmaz, Izmir Oya Yucel, Istanbul Ilhami Yuksel, Ankara United Kingdom Nadeem Ahmad Afzal, Southampton Navneet K Ahluwalia, Stockport Yeng S Ang, Lancashire Ramesh P Arasaradnam, Coventry Ian Leonard Phillip Beales, Norwich John Beynon, Swansea Barbara Braden, Oxford Simon Bramhall, Birmingham Geoffrey Burnstock, London Ian Chau, Sutton Thean Soon Chew, London Helen G Coleman, Belfast Anil Dhawan, London Sunil Dolwani, Cardiff Piers Gatenby, London Anil T George, London Pasquale Giordano, London Paul Henderson, Edinburgh Georgina Louise Hold, Aberdeen Stefan Hubscher, Birmingham Robin D Hughes, London Nusrat Husain, Manchester Matt W Johnson, Luton Konrad Koss, Macclesfield Anastasios Koulaouzidis, Edinburgh Simon Lal, Salford John S Leeds, Aberdeen Hongxiang Liu, Cambridge Michael Joseph McGarvey, London Michael Anthony Mendall, London Alexander H Mirnezami, Southampton J Bernadette Moore, Guildford Claudio Nicoletti, Norwich Savvas Papagrigoriadis, London David Mark Pritchard, Liverpool James A Ross, Edinburgh Kamran Rostami, Worcester Xiong Z Ruan, London Dina Tiniakos, Newcastle upon Tyne Frank I Tovey, London Dhiraj Tripathi, Birmingham Vamsi R Velchuru, Great Yarmouth Nicholas T Ventham, Edinburgh Diego Vergani, London Jack Westwood Winter, Glasgow Terence Wong, London Ling Yang, Oxford United States Daniel E Abbott, Cincinnati Ghassan K Abou-Alfa, New York Julian Abrams, New York David William Adelson, Los Angeles Jonathan Steven Alexander, Shreveport Tauseef Ali, Oklahoma City Mohamed R Ali, Sacramento Rajagopal N Aravalli, Minneapolis Hassan Ashktorab, Washington Shashi Bala, Worcester Charles F Barish, Raleigh P Patrick Basu, New York Robert L Bell, Berkeley Heights David Bentrem, Chicago Henry J Binder, New Haven Joshua Bleier, Philadelphia Wojciech Blonski, Johnson City Kenneth Boorom, Corvallis Brian Boulay, Chicago Carla W Brady, Durham Kyle E Brown, Iowa City Adeel AButt, Pittsburgh Weibiao Cao, Providence Andrea Castillo, Cheney Fernando J Castro, Weston Adam S Cheifetz, Boston Adam S Cheifetz, Boston Xiaoxin Luke Chen, Durham Ramsey Cheung, Palo Alto Parimal Chowdhury, Little Rock Edward John Ciaccio, New York Dahn L Clemens, Omaha Yingzi Cong, Galveston Laura Iris Cosen-Binker, Boston Joseph John Cullen, Lowa Mark J Czaja, Bronx Mariana D Dabeva, Bronx Christopher James Damman, Seattle Isabelle G De Plaen, Chicago Abhishek Deshpande, Cleveland Punita Dhawan, Nashville Hui Dong, La Jolla Wael El-Rifai, Nashville Sukru H Emre, New Haven Paul Feuerstadt, Hamden Josef E Fischer, Boston Laurie N Fishman, Boston Joseph Che Forbi, Atlanta Temitope Foster, Atlanta AmyEFoxx-Orenstein, Scottsdale Daniel E Freedberg, New York Shai Friedland, Palo Alto Virgilio George, Indianapolis Ajay Goel, Dallas Oliver Grundmann, Gainesville Stefano Guandalini, Chicago Chakshu Gupta, St. Joseph Grigoriy E Gurvits, New York Xiaonan Han, Cincinnati Mohamed Hassan, Jackson Martin Hauer-Jensen, Little Rock Koichi Hayano, Boston Yingli Hee, Atlanta Samuel B Ho, San Diego Jason Ken Hou, Houston Lifang Hou, Chicago K-Qin Hu, Orange Jamal A Ibdah, Columbia Robert Thomas Jensen, Bethesda Huanguang Charlie Jia, Gainesville Rome Jutabha, Los Angeles Andreas M Kaiser, Los Angeles Avinash Kambadakone, Boston David Edward Kaplan, Philadelphia Randeep Kashyap, Rochester Rashmi Kaul, Tulsa Ali Keshavarzian, Chicago Amir Maqbul Khan, Marshall Nabeel Hasan Khan, New Orleans Sahil Khanna, Rochester Kusum K Kharbanda, Omaha Hyun Sik Kim, Pittsburgh Joseph Kim, Duarte Jae S Kim, Gainesville Miran Kim, Providence Timothy R Koch, Washington Burton I Korelitz, New York Betsy Kren, Minneapolis Shiu-Ming Kuo, Buffalo Michelle Lai, Boston Andreas Larentzakis, Boston Edward Wolfgang Lee, Los Angeles Daniel A Leffler, Boston Michael Leitman, New York Suthat Liangpunsakul, Indianapolis Joseph K Lim, New Haven Elaine Y Lin, Bronx Henry C Lin, Albuquerque Rohit Loomba, La Jolla James David Luketich, Pittsburgh Mohammad F Madhoun, Oklahoma City Thomas C Mahl, Buffalo Ashish Malhotra, Bettendorf Pranoti Mandrekar, Worcester John Marks, Wynnewood Wendy M Mars, Pittsburgh Julien Vahe Matricon, San Antonio Craig J McClain, Louisville George K Michalopoulos, Pittsburgh Tamir Miloh, Phoenix Ayse Leyla Mindikoglu, Baltimore Huanbiao Mo, Denton Klaus Monkemuller, Birmingham John Morton, Stanford Adnan Muhammad, Tampa Michael J Nowicki, Jackson Patrick I Okolo, Baltimore Giusepp Orlando, Winston Salem Natalia A Osna, Omaha Virendra N Pandey, Newark Mansour A Parsi, Cleveland Michael F Picco, Jacksonville Daniel S Pratt, Boston Xiaofa Qin, Newark Janardan K Reddy, Chicago Victor E Reyes, Galveston Jon Marc Rhoads, Houston Giulia Roda, New York Jean-Francois Armand Rossignol, Tampa Paul A Rufo, Boston Madhusudana Girija Sanal, New York Miguel Saps, Chicago Sushil Sarna, Galveston Ann O Scheimann, Baltimore Bernd Schnabl, La Jolla VIII March 26, 2014

10 Matthew J Schuchert, Pittsburgh Ekihiro Seki, La Jolla Chanjuan Shi, Nashville David Quan Shih, Los Angeles William B Silverman, Iowa City Shashideep Singhal, New York Bronislaw L Slomiany, Newark Steven F Solga, Bethlehem Byoung-Joon Song, Bethesda Dario Sorrentino, Roanoke Scott R Steele, Fort Lewis Branko Stefanovic, Tallahassee Arun Swaminath, New York Kazuaki Takabe, Richmond Naoki Tanaka, Bethesda Hans Ludger Tillmann, Durham George Triadafilopoulos, Stanford John Richardson Thompson, Nashville Andrew Ukleja, Weston Miranda AL van Tilburg, Chapel Hill Gilberto Vaughan, Atlanta Vijayakumar Velu, Atlanta Gebhard Wagener, New York Kasper Saonun Wang, Los Angeles Xiangbing Wang, New Brunswick Daoyan Wei, Houston Theodore H Welling, Ann Arbor C Mel Wilcox, Birmingham Jacqueline Lee Wolf, Boston Laura Ann Woollett, Cincinnati Harry Hua-Xiang Xia, East Hanover Wen Xie, Pittsburgh Guang Yu Yang, Chicago Michele T Yip-Schneider, Indianapolis Kezhong Zhang, Detroit Huiping Zhou, Richmond Xiao-Jian Zhou, Cambridge Richard Zubarik, Burlington Venezuela Miguel Angel Chiurillo, Barquisimeto Vietnam Van Bang Nguyen, Hanoi IX March 26, 2014

11 S Contents Weekly Volume 20 Number 26 July 14, 2014 TOPIC HIGHLIGHT 8325 Proteomic and genomic studies of non-alcoholic fatty liver disease - clues in the pathogenesis Lim JW, Dillon J, Miller M 8341 Modern approach to the clinical management of non-alcoholic fatty liver disease Del Ben M, Polimeni L, Baratta F, Pastori D, Loffredo L, Angelico F 8351 Nonalcoholic fatty liver disease and polycystic ovary syndrome Vassilatou E 8364 Experimental models of non-alcoholic fatty liver disease in rats Kucera O, Cervinkova Z 8377 Non-alcoholic fatty liver disease and diabetes: From physiopathological interplay to diagnosis and treatment Leite NC, Villela-Nogueira CA, Cardoso CRL, Salles GF 8393 Clinical differences between alcoholic liver disease and nonalcoholic fatty liver disease Toshikuni N, Tsutsumi M, Arisawa T 8407 Nonalcoholic fatty liver disease and cardiovascular disease Liu H, Lu HY 8416 Role of small bowel capsule endoscopy in the diagnosis and management of iron deficiency anemia in elderly: A comprehensive review of the current literature Muhammad A, Vidyarthi G, Brady P 8424 Endoscopic ultrasound-guided treatments: Are we getting evidence based - a systematic review Fabbri C, Luigiano C, Lisotti A, Cennamo V, Virgilio C, Caletti G, Fusaroli P 8449 Colonic polyps: Is it useful to characterize them with advanced endoscopy? Lopez-Ceron M, Sanabria E, Pellise M July 14, 2014 Volume 20 Issue 26

12 Contents World Journal of Gastroenterology Volume 20 Number 26 July 14, c-met signaling in the development of tumorigenesis and chemoresistance: Potential applications in pancreatic cancer Delitto D, Vertes-George E, Hughes SJ, Behrns KE, Trevino JG 8471 Pancreatic cancer organotypics: High throughput, preclinical models for pharmacological agent evaluation Coleman SJ, Watt J, Arumugam P, Solaini L, Carapuca E, Ghallab M, Grose RP, Kocher HM 8482 hent1 expression is predictive of gemcitabine outcome in pancreatic cancer: A systematic review Nordh S, Ansari D, Andersson R REVIEW 8491 Liver zonation: Novel aspects of its regulation and its impact on homeostasis Gebhardt R, Matz-Soja M 8505 Gastroenteric tube feeding: Techniques, problems and solutions Blumenstein I, Shastri YM, Stein J ORIGINAL ARTICLE 8525 High fat diet feeding results in gender specific steatohepatitis and inflammasome activation Ganz M, Csak T, Szabo G 8535 Novel diagnostics for aggravating pancreatic fistulas at the acute phase after pancreatectomy Kanda M, Fujii T, Takami H, Suenaga M, Inokawa Y, Yamada S, Kobayashi D, Tanaka C, Sugimoto H, Koike M, Nomoto S, Fujiwara M, Kodera Y 8545 Clobenpropit enhances anti-tumor effect of gemcitabine in pancreatic cancer Paik WH, Ryu JK, Jeong KS, Park JM, Song BJ, Lee SH, Kim YT, Yoon YB 8558 Changes in circulating Foxp3 + regulatory T cells and interleukin-17-producing T helper cells during HBV-related acute-on-chronic liver failure Liang XS, Li CZ, Zhou Y, Yin W, Liu YY, Fan WH 8572 Zinc protoporphyrin IX enhances chemotherapeutic response of hepatoma cells to cisplatin Liu YS, Li HS, Qi DF, Zhang J, Jiang XC, Shi K, Zhang XJ, Zhang XH RESEARCH REPORT 8583 Prognostic significance of preoperative fibrinogen in patients with colon cancer Sun ZQ, Han XN, Wang HJ, Tang Y, Zhao ZL, Qu YL, Xu RW, Liu YY, Yu XB II July 14, 2014 Volume 20 Issue 26

13 Contents World Journal of Gastroenterology Volume 20 Number 26 July 14, 2014 CASE CONTROL STUDY 8592 Risk of gastric cancer is associated with PRKAA1 gene polymorphisms in Koreans Kim YD, Yim DH, Eom SY, Moon SI, Yun HY, Song YJ, Youn SJ, Hyun T, Park JS, Kim BS, Lee JY, Won HK, Kim H RETROSPECTIVE STUDY 8599 Endoscopic ultrasound-guided fine-needle aspiration for suspected malignancies adjacent to the gastrointestinal tract Gambitta P, Armellino A, Forti E, Vertemati M, Colombo PE, Aseni P 8606 Birthplace is not a determinant of colorectal adenomas Tran F, Koo JH 8612 Follow-up of patients with pseudotumoral chronic pancreatitis: Outcome and surveillance Téllez-Ávila FI, Villalobos-Garita Á, Giovannini M, Chan C, Hernández-Calleros J, Uscanga L, Ramírez-Luna MÁ 8617 Need for pancreatic stenting after sphincterotomy in patients with difficult cannulation Nakahara K, Okuse C, Suetani K, Michikawa Y, Kobayashi S, Otsubo T, Itoh F 8624 Selection of appropriate endoscopic therapies for duodenal tumors: An openlabel study, single-center experience Matsumoto S, Yoshida Y 8631 Impact of tumor location on clinical outcomes of gastric endoscopic submucosal dissection Yoon JY, Shim CN, Chung SH, Park W, Chung H, Lee H, Shin SK, Lee SK, Lee YC, Park JC 8638 Outcomes of simple saline-coupled bipolar electrocautery for hepatic resection Guo JY, Li DW, Liao R, Huang P, Kong XB, Wang JM, Wang HL, Luo SQ, Yan X, Du CY CLINICAL TRIALS STUDY 8646 Lipid levels in serum and cancerous tissues of colorectal cancer patients Zhang X, Zhao XW, Liu DB, Han CZ, Du LL, Jing JX, Wang Y 8653 Plasma free amino acid profiling of esophageal cancer using highperformance liquid chromatography spectroscopy Ma H, Hasim A, Mamtimin B, Kong B, Zhang HP, Sheyhidin I III July 14, 2014 Volume 20 Issue 26

14 Contents World Journal of Gastroenterology Volume 20 Number 26 July 14, Outcomes of autologous bone marrow mononuclear cell transplantation in decompensated liver cirrhosis Bai YQ, Yang YX, Yang YG, Ding SZ, Jin FL, Cao MB, Zhang YR, Zhang BY OBSERVATIONAL STUDY 8667 Pro-atherosclerotic markers and cardiovascular risk factors one year after liver transplantation Alvares-da-Silva MR, Oliveira CPMS, Stefano JT, Barbeiro HV, Barbeiro D, Soriano FG, Farias AQ, Carrilho FJ, Carneiro D Albuquerque LA 8674 Combination of symptoms, syndrome and disease: Treatment of refractory diabetic gastroparesis Li JL, Li M, Pang B, Zhou Q, Tian JX, Liu HX, Zhao XY, Tong XL 8681 Expression of P450 and nuclear receptors in normal and end-stage Chinese livers Chen H, Shen ZY, Xu W, Fan TY, Li J, Lu YF, Cheng ML, Liu J PROSPECTIVE STUDY 8691 Drain amylase value as an early predictor of pancreatic fistula after cephalic duodenopancreatectomy Dugalic VD, Knezevic DM, Obradovic VN, Gojnic-Dugalic MG, Matic SV, Pavlovic-Markovic AR, Dugalic PD, Knezevic SM 8700 APE1 polymorphisms are associated with colorectal cancer susceptibility in Chinese Hans Zhang SH, Wang LA, Li Z, Peng Y, Cun YP, Dai N, Cheng Y, Xiao H, Xiong YL, Wang D RANDOMIZED CLINICAL TRIAL 8709 I.31, a new combination of probiotics, improves irritable bowel syndromerelated quality of life Lorenzo-Zúñiga V, Llop E, Suárez C, Álvarez B, Abreu L, Espadaler J, Serra J CASE REPORT 8717 Acute abdomen: Rare and unusual presentation of right colic xanthogranulomatosis Addario Chieco P, Antolino L, Giaccaglia V, Centanini F, Cunsolo GV, Sparagna A, Uccini S, Ziparo V 8722 HBsAg clearance by Peg-interferon addition to a long-term nucleos(t)ide analogue therapy Barone M, Iannone A, Di Leo A 8726 Perihepatic adhesions: An unusual complication of hemolysis, elevated liver enzymes and low platelet syndrome Koeneman MM, Koek GH, Bemelmans M, Peeters LL IV July 14, 2014 Volume 20 Issue 26

15 Contents World Journal of Gastroenterology Volume 20 Number 26 July 14, Two-stage treatment with hepatectomy and carbon-ion radiotherapy for multiple hepatic epithelioid hemangioendotheliomas Komatsu S, Iwasaki T, Demizu Y, Terashima K, Fujii O, Takebe A, Toyokawa A, Teramura K, Fukumoto T, Ku Y, Fuwa N 8736 Tubulopapillary adenoma of the gallbladder accompanied by bile duct tumor thrombus Yamamoto K, Yamamoto F, Maeda A, Igimi H, Yamamoto M, Yamaguchi R, Yamashita Y 8740 First case of IgG4-related sclerosing cholangitis associated with autoimmune hemolytic anemia Masutani H, Okuwaki K, Kida M, Yamauchi H, Imaizumi H, Miyazawa S, Iwai T, Takezawa M, Koizumi W 8745 Laparoscopic segmental colectomy for colonic lymphangiomas: A definitive, minimally invasive surgical option Zhuo CH, Shi DB, Ying MG, Cheng YF, Wang YW, Zhang WM, Cai SJ, Li XX V July 14, 2014 Volume 20 Issue 26

16 Contents World Journal of Gastroenterology Volume 20 Number 26 July 14, 2014 APPENDIX I-VI Instructions to authors ABOUT COVER Editorial Board Member of World Journal of Gastroenterology, Anna Chiara Piscaglia, MD, PhD, Research Scientist, State Hospital - Republic of San Marino, Catholic University, Gemelli Hospital, Rome, Italy AIMS AND SCOPE World Journal of Gastroenterology (World J Gastroenterol, WJG, print ISSN , online ISSN , DOI: ) is a peer-reviewed open access journal. WJG was established on October 1, It is published weekly on the 7 th, 14 th, 21 st, and 28 th each month. The WJG Editorial Board consists of 1353 experts in gastroenterology and hepatology from 68 countries. The primary task of WJG is to rapidly publish high-quality original articles, reviews, and commentaries in the fields of gastroenterology, hepatology, gastrointestinal endoscopy, gastrointestinal surgery, hepatobiliary surgery, gastrointestinal oncology, gastrointestinal radiation oncology, gastrointestinal imaging, gastrointestinal interventional therapy, gastrointestinal infectious diseases, gastrointestinal pharmacology, gastrointestinal pathophysiology, gastrointestinal pathology, evidence-based medicine in gastroenterology, pancreatology, gastrointestinal laboratory medicine, gastrointestinal molecular biology, gastrointestinal immunology, gastrointestinal microbiology, gastrointestinal genetics, gastrointestinal translational medicine, gastrointestinal diagnostics, and gastrointestinal therapeutics. WJG is dedicated to become an influential and prestigious journal in gastroenterology and hepatology, to promote the development of above disciplines, and to improve the diagnostic and therapeutic skill and expertise of clinicians. INDEXING/ABSTRACTING World Journal of Gastroenterology is now indexed in Current Contents /Clinical Medicine, Science Citation Index Expanded (also known as SciSearch ), Journal Citation Reports, Index Medicus, MEDLINE, PubMed, PubMed Central, Digital Object Identifier, and Directory of Open Access Journals. ISI, Journal Citation Reports, Gastroenterology and Hepatology, 2012 Impact Factor: (34/74); Total Cites: (6/74); Current Articles: 944 (1/74); and Eigenfactor Score: (6/74). FLYLEAF I-IX Editorial Board EDITORS FOR THIS ISSUE Responsible Assistant Editor: Xiang Li Responsible Electronic Editor: Dan-Ni Zhang Proofing Editor-in-Chief: Lian-Sheng Ma Responsible Science Editor: Su-Xin Gou Proofing Editorial Office Director: Xiu-Xia Song NAME OF JOURNAL World Journal of Gastroenterology ISSN ISSN (print) ISSN (online) LAUNCH DATE October 1, 1995 FREQUENCY Weekly EDITORS-IN-CHIEF Damian Garcia-Olmo, MD, PhD, Doctor, Professor, Surgeon, Department of Surgery, Universidad Autonoma de Madrid; Department of General Surgery, Fundacion Jimenez Diaz University Hospital, Madrid 28040, Spain Saleh A Naser, PhD, Professor, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL 32816, United States Stephen C Strom, PhD, Professor, Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Stockholm , Sweden Andrzej S Tarnawski, MD, PhD, DSc (Med), Professor of Medicine, Chief Gastroenterology, VA Long Beach Health Care System, University of California, Irvine, CA, 5901 E. Seventh Str., Long Beach, CA 90822, United States EDITORIAL OFFICE Jin-Lei Wang, Director Xiu-Xia Song, Vice Director World Journal of Gastroenterology Room 903, Building D, Ocean International Center, No. 62 Dongsihuan Zhonglu, Chaoyang District, Beijing , China Telephone: Fax: editorialoffice@wjgnet.com Help Desk: PUBLISHER Baishideng Publishing Group Inc 8226 Regency Drive, Pleasanton, CA 94588, USA Telephone: Fax: bpgoffice@wjgnet.com Help Desk: PUBLICATION DATE July 14, 2014 COPYRIGHT 2014 Baishideng Publishing Group Inc. Articles published by this Open-Access journal are distributed under the terms of the Creative Commons Attribution Noncommercial License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited, the use is non commercial and is otherwise in compliance with the license. SPECIAL STATEMENT All articles published in journals owned by the Baishideng Publishing Group (BPG) represent the views and opinions of their authors, and not the views, opinions or policies of the BPG, except where otherwise explicitly indicated. INSTRUCTIONS TO AUTHORS Full instructions are available online at wjgnet.com/ /g_info_ htm ONLINE SUBMISSION VI July 14, 2014 Volume 20 Issue 26

17 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. WJG 20 th Anniversary Special Issues (12): Nonalcoholic fatty liver disease TOPIC HIGHLIGHT Proteomic and genomic studies of non-alcoholic fatty liver disease - clues in the pathogenesis Jun Wei Lim, John Dillon, Michael Miller Jun Wei Lim, John Dillon, Michael Miller, Department of Gastroenterology, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, United Kingdom Author contributions: Lim JW, Dillon J and Miller M contributed equally to this paper. Correspondence to: Michael Miller, MBChB, MRCP, PhD, Specialist Registrar, Department of Gastroenterology, University of Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, United Kingdom. m.miller@dundee.ac.uk Telephone: Fax: Received: October 24, 2013 Revised: January 14, 2014 Accepted: April 1, 2014 Published online: July 14, 2014 Abstract Non-alcoholic fatty liver disease (NAFLD) is a widely prevalent hepatic disorder that covers wide spectrum of liver pathology. NAFLD is strongly associated with liver inflammation, metabolic hyperlipidaemia and insulin resistance. Frequently, NAFLD has been considered as the hepatic manifestation of metabolic syndrome. The pathophysiology of NAFLD has not been fully elucidated. Some patients can remain in the stage of simple steatosis, which generally is a benign condition; whereas others can develop liver inflammation and progress into non-alcoholic steatohepatitis, fibrosis, cirrhosis and hepatocellular carcinoma. The mechanism behind the progression is still not fully understood. Much ongoing proteomic researches have focused on discovering the unbiased circulating biochemical markers to allow early detection and treatment of NAFLD. Comprehensive genomic studies have also begun to provide new insights into the gene polymorphism to understand patientdisease variations. Therefore, NAFLD is considered a complex and mutifactorial disease phenotype resulting from environmental exposures acting on a susceptible polygenic background. This paper reviewed the current status of proteomic and genomic studies that have contributed to the understanding of NAFLD pathogenesis. For proteomics section, this review highlighted functional proteins that involved in: (1) transportation; (2) metabolic pathway; (3) acute phase reaction; (4) antiinflammatory; (5) extracellular matrix; and (6) immune system. In the genomic studies, this review will discuss genes which involved in: (1) lipolysis; (2) adipokines; and (3) cytokines production Baishideng Publishing Group Inc. All rights reserved. Key words: Non-alcoholic fatty liver disease; Proteomics; Genomics; Metabolic syndrome; Pathophysiology Core tip: Non-alcoholic fatty liver disease (NAFLD) is a widely prevalent hepatic disorder in Western populations. NAFLD can occur as a spectrum diseases, from simple steatosis, to non-alcoholic steatohepatitis characterised by hepatocellular injury and inflammation, to cirrhosis and hepatocellular carcinoma. This paper reviewed the current status of proteomic and genomic studies that have contributed to the understanding of NAFLD pathogenesis. This review highlighted several functional proteins and genetic polymoprhisms; particular those involved in insulin resistance, triglycerides metabolism and hepatic inflammation. It is hoped that this review will offer further insights into the pathophysiology of NAFLD. Lim JW, Dillon J, Miller M. Proteomic and genomic studies of non-alcoholic fatty liver disease - clues in the pathogenesis. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: INTRODUCTION Non-alcoholic fatty liver disease (NAFLD) is a widely prevalent hepatic disorder, affecting up to 30% of the 8325 July 14, 2014 Volume 20 Issue 26

18 Lim JW et al. Review of NAFLD pathogenesis general population of Western countries [1]. The term NAFLD comprises an entire pathological spectrum of diseases, ranging from simple steatosis (SS), non-alcoholic steatohepatitis (NASH), progressive inflammation, fibrosis, cirrhosis to hepatocellular carcinoma (HCC) [2]. Furthermore, NAFLD has been shown to be closely associated with obesity, insulin resistance, dyslipidemia, type Ⅱ diabetes mellitus (T2DM) and cardiovascular disease [2-4]. Therefore, NAFLD has been considered as the hepatic manifestation of metabolic syndrome (MetS). In the majority of patients, SS is a relatively benign course. However, SS may evolve into NASH, which is a more aggressive liver disease that tends to be progressive and may lead to cirrhosis [5]. The mechanism behind the progression of SS to NASH is still not fully understood. Currently, liver biopsy remains the gold standard in diagnosis of NAFLD. The histological hallmark of NAFLD is lipid accumulation in the hepatocytes in absence of pathologies such as viral hepatitis or alcohol abuse [4]. However, liver biopsy is an invasive procedure and the liver is not necessarily uniformly affected in NAFLD [6,7]. Therefore, non-invasive biomarkers for evaluation of liver disease and fibrosis are urgently needed. Proteomics is a large-scale analysis of protein changes of different patient populations. The identification of specific proteins, either as novel markers or as over/under expressed markers, may have a huge impact by increasing the availability of biomarkers for early diagnosis and therapy [8]. On the other hand, genome-wide association (GWA) studies aim to identify the genetic influences on common diseases with common genetic variation of observable traits. The technologies of high-throughput genotyping are able to assay the common single nucleotide polymorphism (SNPs) and relate them to clinical conditions and measurable traits [9]. The development of NAFLD is a complex multifactorial process involving the perturbation of multiple gene and protein regulations. Initially, Day and James proposed a two-hit hypothesis to explain the concept of steatohepatitis: first hit is the production of steatosis and second hit is the source of oxidative stress capable of initiating significant lipid peroxidation [10]. However, the traditional two-hit hypothesis has been modified to multiple parallel hits hypothesis. The significant overlaps among insulin resistance, hepatic de novo lipogenesis and subsequent hepatocyte injury have been proposed to contribute to the progression from SS to NASH [11]. Furthermore, numerous candidate gene studies on the effects of genes on the presence of NAFLD and its progression have further supported the multiple parallel hits hypothesis [12]. This review aims to summarise the current proteomic and genetic studies to provide better understanding about the pathogenesis of NAFLD. HEPATIC FIBROGENESIS DEVELOPMENT Accumulating laboratory evidence suggests that insulin resistance and excess of free fatty acids can trigger the oxidative stress in hepatocytes and lead to chronic inflammation and fibrogenesis [13,14]. Hepatic fibrogenesis is the final consequences of chronic liver disease and constitutes a model of wound-healing process. Regardless of the cause of hepatic damage, hepatic stellate cells (HSCs) are activated within the extracellular matrix (ECM) into myofibroblasts (activated HSCs). These activated HSCs result in the deposition of fibrotic matrix. Hepatic macrophages have been long recognised to be the source of matrix metalloproteinases (MMP) which can degrade the scar tissue in liver [15]. The development of fibrogenesis is a dynamic process due to an imbalance in ECM deposition and degradation. Hepatic fibrosis is associated with increased ECM, which include collagen, laminin, hyaluron, elastin and fibronectin. As fibrogenesis progresses, the quantity and composition of ECM changes [16]. Therefore, the protein expression may vary at different stages of fibrogenesis due to endogenous adaptive responses. Targeting the optimum levels of different protein expression may shed a light in both disease prediction and disease mechanism. PROTEOMIC STUDIES The advancement of proteomics analysis has provided us with powerful tools for studying NAFLD diagnosis and discovering biomarkers. The first study of serum protein profiles in NAFLD was published by Younossi et al [17]. The author performed a surface-enhanced laser desorption ionization time of flight mass spectrometry (SELDI- TOF MS) on 98 obese patients; where 91 patients were diagnosed with NAFLD (12 steatosis alone, 52 steatosis with nonspecific inflammation, 27 NASH) and 7 patients without NAFLD as obese control. The proteomic analysis revealed 12 significant protein peaks. Due to the inherent limitation of low mass accuracy in SELDI-TOF MS, only fibrinogen γ was identified and suggested that it may be related to fibrosis. Later, Bell et al [18] utilised an ion-intensity based, label-free quantitative proteomics approach (LFQP) and discovered 55 proteins that changed significantly between NAFLD and NASH with advanced fibrosis. Bell et al [18] further revealed 15 proteins that changed significantly between early NASH and NASH with advanced fibrosis. A 6 proteins diagnostic method (fibrinogen β chain, retinol binding protein 4, serum amyloid P component, lumican, transgelin 2 and CD5 antigen-like) and a 3 proteins diagnostic method (component C7, insulin-like growth factor acid labile subunit and transgelin 2) were developed to diagnose the different stages of NAFLD (AUROC ranging from 0.83 to 0.91). Additionally, alanine aminotransferase (ALT) has been found to be significantly inferior in diagnosis the stages of NAFLD (AUROC = 0.53) [18]. Several studies have subsequently demonstrated that ALT is a poor NAFLD workup marker and there is no optimal ALT levels to predict advanced fibrosis [19]. Despite the lack of single protein in differentiating SS and NASH, Bell et al [18] have provide an insight into the pathogenesis of NAFLD and 8326 July 14, 2014 Volume 20 Issue 26

19 Lim JW et al. Review of NAFLD pathogenesis NASH. Interestingly, most of the proteins identified in the study were involved in immune system regulation, inflammation, hepatic ECM structure and protein carriers in blood. Protein carriers Apolipoproteins, are important structural and functional proteins that transport lipids in blood circulation. As NAFLD is considered the hepatic manifestation of MetS, it is not surprising that apolipoprotein serum levels are altered in patients with chronic liver disease. Previous publications have revealed that the level of ApoA1 decreased with progression of hepatic fibrosis [20-22]. A recent proteomic study conducted by Choe et al [21] using rate nephelometry and particle-enhanced immunonephelometry in non-diabetic patients has concluded that ApoB/ApoA1 ratio was associated with the prevalence of NAFLD and was independent of obesity and other metabolic components. Therefore, ApoB/ApoA1 ratio can be used as a predictable marker for cardiovascular risk in NAFLD patients. CD5 antigen-like (CD5L) protein, also known as human Sp alpha, is a soluble protein that is expressed by macrophages present in lymphoid tissues and may have an important role in the regulation of the innate and adaptive immune systems [23]. A serum proteomic study using 2-dimensional differential in gel electrophoresis (DIGE) was performed on immune depleted sera from 3 groups of patients (pre-cirrhotic NAFLD, cirrhotic NAFLD and cirrhotic NAFLD with co-existing HCC) [22]. This study has identified a pattern of serum apolipoproteins (ApoA1, Pro-ApoA1 and ApoA4) and CD5L to distinguish between pre-cirrhotic NAFLD and cirrhotic NAFLD [22]. However, CD5L does not increase incrementally with the degree of fibrosis and still falls short as cirrhosis marker with AUC of [22]. Moreover, CD5L is a poor biomarker for HCC. Hence, there could be another trigger factors from the progression of NAFLD, NASH to HCC. Metabolic pathways Rodríguez-Suárez et al [24] analysed the protein expression of liver samples with DIGE in combination with MALDI TOF/TOF. Ten of the proteins were further validated by Western blotting to confirm the observed changes of protein expression [24]. This study has successfully demonstrated that both serum concentration of carbamoyl phosphate synthase 1 (CPS1) and 78 kda glucose-regulated protein (GRP78) decreased gradually from cholecystectomy controls with normal liver function and histology subjects to steatosis and NASH patients [24]. Further validation of CPS1 and GRP78 in higher number of patients is still warranted. However, this study has provided a useful insight for NAFLD progression. GRP78, also know as immunoglobulin binding protein (BiP), is a central regulator of endoplasmic reticulum (ER) function, which is crucial for cells survival. GRP78 has been recognised for its roles in protein folding, misfolded protein targeting for degradation and ER Ca 2+ homeostasis [25,26]. Laboratory researches further demonstrated that GRP78 can inhibit both insulin-dependent and ER-stress-dependent SREBP-1c proteolytic cleavage that plays an important role in de novo lipogenesis [27]. In addition, compelling laboratory evidences have indicated that mitochondrial abnormalities may be involved in the pathogenesis of NAFLD [28-30]. CPS1 is a ligase enzyme located in the mitochondria that involved in the production of urea. The down-regulation of CPS1 may lead to an increase of urea, which subsequently becomes uric acid [31]. This mechanism could potentially explain the observed significant elevated uric acid levels in NAFLD patient [32]. Uric acid has been shown to exert pro-inflammatory and pro-oxidant effect both in adipose tissue and vascular smooth muscle cells [33]. A recent largest observational population based cohort of non-diabetic American adult has suggested that uric acid might play a role in NAFLD [33]. Sirota et al [33] revealed that increased serum uric acid levels are associated with greater severity of NAFLD by ultrasonography. Several prospective and retrospective studies have also provided evidences supporting the association of high serum uric acid, the risk of T2DM, cardiovascular diseases and hepatitis [34-36]. These have further supported that NAFLD is the hepatic manifestation of MetS. It has been hypothesised that uric acid may contribute to insulin resistance by inducing local adipose tissue inflammation [37]. This may reduced the production of adiponectin, a protein that is involved in glucose haemostasis [37,38]. Furthermore, uric acid may stimulate hepatic lipogenesis, mediated by uric-acid dependent intracelullar and mitochondrial oxidative stress [33,37]. Whether such downstream effects of CPS1 and uric acid play a pathogenic role in NAFLD still requires further laboratory and clinical investigation. In general, mitochondrial oxidative damage and impaired mitochondrial β-oxidation could contribute to hepatic steatosis. Subsequently, the progressive loss of mitochondrial function in conjunction with ER stress could play a key role in the transition of insulin resistance and NAFLD progression. However, little is known about the spectrum of changes of mitochondrial and ER function and their potential role in the natural history of NAFLD. Acute phase proteins An observational study in Japanese population study, high sensitivity C-reactive protein (hs-crp) serum levels has been reported to be significantly higher in biopsy-proven NASH compared with SS [39]. Furthermore, hs-crp levels were significantly elevated in advanced NASH compared with those with mild NASH [39]. However, the role hs- CRP in differentiating severity of NASH remains controversial. Zimmermann et al [40] demonstrated a strong positive association between BMI and hs-crp levels, which were measured with nephelometry, throughout the broad range of obesity. However, there were no significant associations between inflammation or fibrosis, respectively 8327 July 14, 2014 Volume 20 Issue 26

20 Lim JW et al. Review of NAFLD pathogenesis Extracellular matrix While the presence of inflammation is due to lipid accumulation, an imbalance of ECM production and degradation will therefore lead to fibrogenensis. The association of serum concentration of ECM components, especially increased serum hyaluronic acid, type Ⅳ collagen 7S and laminin levels measured with routine laboratory parameters and the degree of fibrosis in NAFLD have been studied extensively [56-59]. Hyaluronic acid is a component of ECM, which synthesised by mesenchymal cells. The increased production and delayed clearance of hyaluronic acid have been demonstrated to be associated with the severity of hepatic inflammation [56]. Type Ⅳ collagen is the main collagen component of basement membrane whereas laminin is the main non-collagenous glycoproand hs-crp observed [40]. Serum haemoglobin levels have been observed to be significantly elevated in extremely obese NAFLD patient and shown to be significantly associated with prevalence of NAFLD [41-43]. Yu et al [42] performed a serum protein fingerprint analysis, utilising the SELDI-TOF-MS technique combined with bioinformatic approaches and discovered that haemoglobin subunit alpha was significantly up-regulated in NAFLD group. It has been proposed that the relationship between serum haemoglobin and NAFLD may be partially modulated by haptoglobin (Hpt), an α2-glycoprotein acute phase protein produced in response to an inflammatory insult [44]. Serum haemoglobin levels may be a marker of hepatic steatosis but not of severity of NAFLD in obese patients. Acute phase proteins are the key to the hepatic innate defences against insults and reduce hepatic tissue damage. Therefore, acute phase proteins may have an immediate role in hepatocytes injuries and could be used as NAFLD predictive markers in obese patients. However, the concentration of acute phase proteins does not reflect the severity of disease, suggesting different protein mechanism for NAFLD progression. A recent study by Kamada et al [44] has shed a light into the role of acute phase protein and progression of NAFLD. Kamada et al [44] utilised lectin antibody enzymelinked immunosorbent assay (ELISA) and demonstrated that serum fucosylated haptoglobin (Fuc-Hpt) underwent a stepwise increased with increasing hepatocyte ballooning scores in biopsy-proven NAFLD patients (AUROC ranging from to 0.759). Fucosylated alpha-fetoproteins have been previously shown to be more selectively secreted into bile [45]. Kamada et al [44] hypothesised that an increase in ballooning hepatocytes could lead to the disruption of the fucosylation-based machinery and subsequently an increased secretion of Fuc-Hpt into the serum. Pentraxin 3 (PTX-3), another acute-phase protein that resembles CRP in function and a possible modulator of complement system has been used as the marker for disease severity in cardiovascular, inflammatory and infectious diseases [46]. Yoneda et al [47] measured plasma PTX-3 levels with ELISA in biopsy-proven NAFLD, based on the method of Brunt. This study revealed a profound elevation of plasma PTX-3 levels in NASH and further suggested that PTX-3 could be a promising clinical marker for fibrosis severity in NASH [47]. CRP and serum amyloid protein belong to the short pentraxins group, which have reported to be produced by the liver as a systemic response to local inflammation. On the other hand, PTX-3 is the prototypic long pentraxin, which is produced in damaged tissue and in response to proinflammatory stimuli such as interleukin 1β (IL- 1β) and tumour necrosis factor-α (TNF-α). It has been anticipated that PTX-3 expression pattern may be more tissue specific than CRP [47,48]. However, conflicting studies have been published regarding the association of PTX-3 levels, hs-crp levels and the severity of MetS [48]. Further validation of PTX-3 is warranted to assess the usefulness of PTX-3 in understanding NAFLD. Anti-inflammatory and anti-oxidants A large cross-sectional study has demonstrated the inverse relationship between total serum bilirubin levels with vanadate oxidation method and the prevalence of NAFLD independent of known metabolic risk factors [49]. Similar inverse relationship of NAFLD was discovered with conjugated and unconjugated bilirubin respectively [50,51]. In addition, serum bilirubin has been long recognised to have an inverse relationship with the development of coronary artery disease [52]. Bilirubin is the metabolic end product of heme catabolism as haem oxygenase-1 (HO-1), a stress-responsive protein that degrades the pro-oxidant heme to biliverdin. Subsequently, biliverdin reductase reduces biliverdin to bilirubin [53]. The intrinsic amplification properties of biliverdin reductase redox cycle could readily augment the antioxidant effects of bilirubin by fold [54]. The interesting challenge here is to link the clinical studies, which focus on serum levels of bilirubin with molecular mechanisms to understand the pathogenesis of NAFLD. Recent cardiovascular research that focused on HO-1 activation in rat model of chronic heart failure has demonstrated significant improved survival due to the reduction in leukocyte activation and cardiomyocytes apoptosis. Furthermore, chronic HO-1 activation has shown to suppress the elevated levels of myeloperoxidase (MPO) activity, IL-1β production and TNF-α production [53]. Li et al [55] characterised the expression of HO-1 in biopsy specimens of normal and active cirrhosis liver specimen with immunohistochemistry; and cultured human hepatic myofibroblasts with Northern and Western blot analysis. Li et al [55] discovered that HO-1 can inhibit the proliferation of hepatic myofibroblasts. Therefore, it is anticipated that bilirubin can inhibit the pathogenesis of NAFLD and NASH through the potent antioxidant, anti-inflammatory and anti-fibrogenic effects. Further research is needed to elucidate the mechanisms underlying this association and to establish the role of serum bilirubin as a potential protective marker for NAFLD risk July 14, 2014 Volume 20 Issue 26

21 Lim JW et al. Review of NAFLD pathogenesis tein of the ECM that deposited in the space of Disse during sinusoidal capillarisation [58,60]. It is believed that hepatocytes ballooning and releases of type Ⅳ collagen and laminin are the main causes of increased their serum levels. However, the mechanism of this interaction is still unknown. It is important to note that hyaluronic acid, type Ⅳ collagen 7S and laminin have been shown to increased in cases of fibrosis resulting from alcoholic, primary biliary cirrhosis and chronic viral hepatitis [56,61,62]. Therefore, hyaluronic acid, type Ⅳ collagen 7S and laminin may be useful in detecting the presence of severe fibrosis in chronic liver disease, but not specific to NAFLD. Lumican, a highly biologically active 40-kDa keratin sulfate proteoglycan member of the leucine-rich repeat protein superfamily, is known to be progressively overexpressed with the increasing severity of NAFLD and NASH [63,64]. Similar to the liver, the maintenance of myocardial ECM and regulation of fibrotic myocardial remodelling are essential for normal cardiac physiological function [65]. A recent cardiac failure research discovered that lumican production is induced by a combination of inflammation and mechanical stimuli from cardiac fibroblasts [66]. Further laboratory research also revealed that in the absence of lumican, hepatocytes are protected against fibrosis despite ongoing injury [64]. Therefore, lumican might have an important role in tissue remodelling and contribute to fibrogenesis. ECM degradation occurs predominantly as a consequence of the action of MMPs [67]. MMPs has been known to be an important regulatory enzyme in inflammatory process to modulate leukocyte influx, either through regulation of barrier function, cytokine or chemokine activity, or gradient formation [68]. An upregulation of tissue inhibitors of metalloproteinases (TIMPs) in liver fibrosis may impair MMPs activity and lead to altered ECM production [69]. Interestingly, a growing body of evidences has demonstrated MMPs role in liver fibrogenesis [67,69]. Serum MMP-1 level assayed by ELISA has been found to be negatively correlates with the severity of fibrosis whereas MMP-1 mrna quantified with spectrophotometer and single reverse transcription polymerase chain reaction (PCR) has been found to be elevated in liver tissue as fibrosis progressed [70,71]. The decreased synthesis of MMP-1 is believed to due to HSC apoptosis or necrosis. However, the link between increased hepatic MMP-1 expression and decreased serum MMP-1 remained unanswered. Notable increase in serum MMP-9 level with ELISA was found in NASH and hepatitis C infected patients [72]. Furthermore, immunohistology of the liver specimen and revealed a striking difference of the immunolabelling patterns between NASH and hepatitis C infected liver [72]. Hepatitis C infected liver demonstrated an essentially localised labelling in the cytoplasm of hepatocytes, biliary canaliculi and bile ducts whereas NASH liver demonstrated a discrete labeling of neutrophil subpopulation [72]. The difference of MMP-9 patterns suggested a different pathophysiological involvement of this protease in the fibrogenesis underlying different etiology. On the other hand, Wanninger et al [67] utilised sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) and Western blotting analysis in primary human hepatocytes, steatosis human hepatocytes and murine NASH hepatocytes to study the activity of MMP-9. Wanninger et al [67] concluded that MMP-9 activity is associated with hepatic inflammation and fibrosis but not with hepatic steatosis and body weight. This raises the question of peripheral adipocytes involvement with other inflammatory mediators that could have contributed to the progression of NAFLD and NASH. Furthermore, Wanninger et al [67] revealed a negative correlation of MMP-9 activity with serum adiponectin in murine NASH models. This finding highlighted the role of inflammation in the progression of NAFLD. The anti-inflammatory activity of adiponectin will be further discussed in the genomic section. A study utilising reverse transcription and quantitative real-time reverse transcription PCR in animal models has demonstrated an accumulation of macrophages and increased gene expression of MMP-12, MMP-13 and TIMP-1 in predominantly adipose tissue compared with liver tissue [73]. The author proposed that attenuated hepatic MMP expression in both livers and adipose tissue might be responsible for the shifting balance of fibrogenesis [73]. However, the key source of these enzymes and tight relationship between adipose tissue dysfunction and developments of hepatic fibrosis remain unclear. Despite the growing understanding of MMPs and TIMPs, the conclusive role of each MMPs and TIMPs subtypes in appropriate human model is still limited. However, this review highlighted the increased recognition of liver fibrosis as dynamic pathological process that involves both progression and regression of ECM. Immune cells and cytokines A paper by Zhan and An has comprehensively reviewed the role of liver innate immune cells such as T helper cells (Th cells), activated kupffer cells (KC) and natural killer T (NKT) cells in the development of NAFLD [74]. Hereby, we further reviewed related cytokine papers that being published later. It has been long proposed that it was Th1 cytokine excessive production in NAFLD results in hepatic insulin resistance and NASH [74]. However, a recent laboratory research on C57BL/6 and Balb/c mice with mrna extraction and real-time PCR demonstrated that imbalance between Th1 and Th2 did not account for the increased NASH severity and suggested that macrophage responses are the important contributors to NAFLD progression [75]. In addition, cytokines are not only being produced by local sources but also by peripheral blood cells. The presence of an altered phenotype and functionality of circulating immune cells has been speculated to be a distinctive characteristic in NASH patients [76,77]. Innate immune cells have the ability to secrete cyto July 14, 2014 Volume 20 Issue 26

22 Lim JW et al. Review of NAFLD pathogenesis kines and to promote influx of inflammatory cells. To date, emerging strong evidences suggested that cytokines such as TNF-α and IL-1 are sensitising factors acting upon leukocyte infiltration of the liver [76,78]. These cytokines and chemokines effects contributed to intracellular oxidative stress and mitochondrial dysfunction, which amplified hepatocytes damage. The cytokines mechanism appears to be like a vicious cycle. The further disrupted cellular mitochondrial respiratory chain can thereby cause the formation reactive oxidative species of nearby hepatocytes [76]. As a consequence, HSCs is activated and lead to hepatic fibrosis. The development and progression of NASH act in a cooperative manner with pro-inflammatory cytokines. IL-6 is a pro-inflammatory cytokine that has been studied extensively for its wide range of biological function. IL-6 has been proposed to have direct and indirect deleterious role such as induction of inflammation, hepatoprotector, regulators of acute phase response and insulin signalling [79]. Wieckowska et al [80] demonstrated a markedly increased hepatic IL-6 expression assayed with immunohistochemistry in patients with NASH as compared to SS or normal liver. The hepatic IL-6 expression in this study cohort has showed to be correlates with the severity of inflammation and fibrosis [80]. At the same time, a positive correlation was observed between the plasma IL-6 levels measured with ELISA and hepatic IL-6 expression [80]. However, discrepant results have been reported regarding IL-6 in NAFLD studies. Haukeland et al [81] determined IL-6 in 47 biopsy-proven NAFLD and 30 controls with enzyme immunoassays and revealed no significant changes in NASH group compared to SS. Interestingly, Haukeland et al [81] further evaluated serum level of CC-chemokine ligand 2 /monocyte chemoattractant protein-1 (CCL2/MCP1) and demonstrated an increasing levels from healthy controls to SS and reached the highest levels in NASH. It has been proposed that an increased circulating CCL2/MCP-1 levels may be related to the development of NASH [79,81]. CCL2, an acute and chronic phase chemokine has been known to be secreted by HSCs. CCL2 is responsible for monocyte and macrophage infiltration of liver and maintaining hepatic inflammation and fibrogenesis [82]. On the other hand, MCP-1 is known to be derived from visceral adipose tissue and reached liver via portal circulations [83]. Many more studies are still warranted to understand the pivotal roles of CCL2 and MCP-1 in the development of inflammatory responses in the liver. The lipid accumulation from visceral tissue and hepatocellular could significantly contribute to the expression of CCL2 and MCP-1. Therefore, the concentrations of both chemokines are crucial for the recruitment of immune cells to sites of inflammation. It is believed that adipose tissue-derived cytokine could also affects liver innate immune cells [74]. With LFQP approach, Bell et al [18] demonstrated that retinol binding protein 4 (RBP4), an adipokine that associated with insulin resistance and T2DM was significantly decreased in expression with increasing NAFLD severity. Utilising ELISA, Alkhouri et al [84] also demonstrated an inverse relationship between RBP4 and liver fibrosis. However, there were also contrary reports that demonstrated significantly elevated serum RBP4 in NAFLD patients [85,86]. The lack of consistency prompted us to reevaluate RBP4 as a potential novel marker to assess the progression of fibrogenesis in NAFLD. On the other hand, the link between RBP4 and T2DM are more obvious. Graham et al [87] revealed an increased serum RBP4 with ELISA in patients with T2DM and a decreased expression of glucose transporter 4 (GLUT4). The mechanism behind this remains unclear, but raises the possibility of adipocytes involvement in glucose sensing. A nicely summarised review article by Christou et al [88] indicated that RBP4 might have the ability to induce insulin resistance in an autocrine or paracrine mechanism in the adipose tissue. It is noteworthy that circulating RBP4 can be influenced by non-metabolic condition such as renal failure and intervention such as anti-diabetic drugs and exercise [88,89]. Therefore, the association of RBP4 level and MetS could be affected and should be interpret with caution. The pathogenic role of RBP4 has contributed to genetic studies of RBP4 locus in human chromosome 10q, which have been linked to hyperinsulinemia or early onset T2DM [89]. The polymorphism of RBP4 gene polymorphism has been linked with the development of T2DM and obesity in different populations [89,90]. However, the associations among RBP4 polymorphism, serum RBP4 levels and measures of MetS have not been universally described [90]. GENOMIC STUDIES The first GWA study was performed by Romeo et al [91] using a phenotype based on magnetic resonance spectroscopy. The authors identified that an allele in human patatin-like phospholipase domain containing 3 gene (PNPLA3) (rs738409; I148M) was strongly associated with increased hepatic lipids levels and hepatic inflammation. This study importantly raised that genetic factors could account towards the susceptibility of NAFLD [91]. With regards to the pathophysiology of NAFLD, variations in genes involved in hepatic lipid metabolism, ECM balance, cytokines and insulin resistance will be discussed in this review. Genes that affect lipolysis Polymorphisms of PNPLA3 gene have been found to be strongly associated with the pathogenic status of NAFLD in different populations [91-93]. PNPLA3 gene, encoding adiponutrin is known to have lipase activity in vitro and has been found to be involved in glucose and lipid metabolism [91]. Despite several publications on the association between PNPLA3 polymorphisms and NAFLD, the physiological functions of the PNPLA3 still remain unclear. A well-conducted meta-analysis revealed that GG genotype of PNPLA3 (rs738409; I148M) exerts a stronger effect on hepatic lipids accumulations and patients 8330 July 14, 2014 Volume 20 Issue 26

23 Lim JW et al. Review of NAFLD pathogenesis with GG phenotype are more susceptible to aggressive disease associated with higher fibrosis scores than CC genotype [94]. Furthermore, Sookoian et al [94] found no correlation between increased liver disease severity and heterozygous or homozygous mutants of G-allele. The link between NAFLD stage and PNPLA3 is well established, but the association of insulin resistance and obesity with PNPLA3 are less clear [95,96]. A recent review of PNPLA3 polymorphism suggested that PNPLA3 is more likely to influence lipid content and liver disease severity but not insulin resistance [95]. Lallukka et al [97] demonstrated that NAFLD patients with polymorphism of PNPLA3 is not characterised by adipose tissue inflammation, insulin resistance and features of the metabolic syndrome such as hyperinsulinaemia, hypertriacylglycerolaemia and a low HDL-cholesterol concentration. Despite the less clear relationship in insulin resistance, the severity of NAFLD has been proven to be related to the PNPLA3 in T2DM populations. Therefore, screening analysis of the PNPLA3 polymorphism could still be useful to select T2DM patients who need specific interventions as prevention for NAFLD [98]. A recent transplant research revealed that PNPLA3 of non-cc genotype is associated with post-transplant obesity but not independently with diabetes. Furthermore, the donor genotypes were not associated with obesity or diabetes [99]. This research highlighted the potential of peripheral mechanism for insulin resistance and could reflect a multi-factorial gene involvement in NAFLD. One point to note is that the effect on allograft steatosis was not studied. Ample evidences exist in the literatures that triglycerides (TG) accumulation in liver is a result of obesity and diabetes [100]. Surprisingly, an accumulation of TG has been suggested to be a protective mechanism to prevent progressive liver damage in NAFLD [101]. Adipose tissue triglyceride lipase (ATGL) has been found to be a ratelimiting enzyme for degradation of TG and has been implicated in the pathogenesis and progression of NAFLD. A deficiency of ATGL was proposed to lead to TG accumulation in many organs, including the liver [102,103]. In murine model, ATGL knock out (ATGL-KO) mice demonstrated increased hepatic lipids accumulation during induced-er stress but suppressed gene expression of inflammatory markers. Further evaluation suggested that an enrichment of oleic acid in the TG pool plays the main role for ATGL in protection from potentially harmful consequences of ER stress [103]. Furthermore, ATGL- KO animal model demonstrated a high insulin sensitivity despite liver TG storage [102]. Therefore, the progressions of NAFLD in obese and diabetes patients have a distinguishable phenotypic imprint on the hepatocyte lipidome. Further research in ATGL polymorphism and lipidome could potential elucidates the lipolytic catabolism pathway in NAFLD. Apolipoprotein C3 (ApoC3) is the most important components of very low density lipoprotein (VLDL). ApoC3, also a strong inhibitor lipoprotein lipase (LPL) has been found to enhance fatty acid uptake from plasma TG in adipose tissue in mice [104]. Therefore, a deficiency of ApoC3 could leads to higher susceptibility to dietinduced obesity followed by more severe development of insulin resistance [104]. Approximately between 50% and 75% of patients with T2DM have fatty liver diagnosed by ultrasound [4]. Insulin resistance has been anticipated to play a major role in the pathogenesis of T2DM and may enhance hepatic fat accumulations by increasing free fatty transportation due to the effect of hyperinsulinemia to stimulate anabolic process [4,105]. Petersen et al [105] demonstrated a 3- to 4-fold increased in prevalence of insulin resistance in Asian-Indian men compared with Caucasian men. Later, Petersen et al [106] further demonstrated that the polymorphisms of ApoC3 (C-482T and T-455C) are associated with NAFLD and insulin resistance in Indian men. However, there is a contrary report suggested that the genetic variants in ApoC3 does not contribute to NAFLD in Finnish populations [107]. The reason regarding the divergent results remains unknown but importantly highlighted the ethnic differences in disease susceptibility. Given the essential contribution of lipogenesis pathway, another common gene variant, glucokinase regulatory protein (GCKR) gene has been studied extensively [ ]. GCKR regulates glucokinase, a phosphorylating enzyme that responsible for hepatic glucose metabolism and activates hepatic lipogenesis [108]. Polymorphism of GCKR was found to be significantly associated with increased risk of T2DM, particularly in Asian population [109,110]. Furthermore, polymorphism of GCKR (rs and rs ) has been shown to be associated with NAFLD in Chinese people and obese youth respectively [108,111]. In addition, Tan et al [112] further suggested that the interactions of GCKR and PNPLA3 genes can increase susceptibility to NAFLD. The protein peroxisome proliferator-activated receptor gamma coactivator 1-alpha PGC-1α), encoded by the PPARGC1A gene, has also been recognised to be the transcriptional co-activator of peroxisome proliferator activated receptor gamma (PPAR-γ) gene [113]. The PGC- 1α and PPAR-γ pathway play a central role in energy metabolism, mitochondrial biogenesis and function, oxidative phosphorylation, gluconeogenesis and lipogenesis and induction of apoptosis [113,114]. PPARGC1A knock out mice have been reported to develop hepatic steatosis, as a result of the combination of reduced mitochondrial respiratory capacity and increased expression of lipogenic genes [115]. However, the role of PCG-1α in MetS is less clear than PPAR-γ. Controversial reports exist in the literatures, regarding PPARGC1A gene in patient s susceptibility for NAFLD. Hui et al [116] demonstrated that only polymorphism of PPAR-γ (C161T) are associated with NAFLD susceptibility possibly through the adiponectin pathway, not PPARGC1A gene. Subsequent reports demonstrated that PPARGC1A (rs ) risk A allele is associated with an increased risk of NAFLD and is independent of the 8331 July 14, 2014 Volume 20 Issue 26

24 Lim JW et al. Review of NAFLD pathogenesis effect of the PNPLA3 (rs738409) polymorphism in Taiwanese obese children population [117]. The role of PCG- 1α remains unclear but has been hypothesised to be associated with exercise [118]. On the other hand, the genetic variation in PPAR-γ (Pro12Ala and C1431T) has been studied extensively in different ethic cohorts [ ]. Polymorphism of PPAR-γ has been concluded to be strongly associated with NAFLD whereas polymorphism of G allele in PPAR-γ gene (rs ) is only associated with lobular inflammation [119]. Genes that affect adipokines Adipocytes play multiple endocrine roles in the body. As body mass index (BMI) increases, the metabolic role of adipocytes changes [122]. An increased understanding of the role of the adipocyte and its associated adipokines could give us some clues in understanding MetS and NAFLD. Adiponectin is the most abundant and adipose-specific adipokine. Contrary to other adipokines, adiponectin correlates negatively with BMI, insulin resistance, plasma TG, LDL-cholesterol, hepatic fat content and progression to NASH in NAFLD patients [38,123]. Adiponectin has been reported to be higher in women and in Caucasian compared with Asian or African individuals [123,124]. Adiponectin is known to have insulin-sensitising, antifibrogenic and anti-inflammatory properties. Studies have found that Adiponectin is involved in the activation of AMPK and downregulates the expression of sterol regulatory element-binding protein 1c (SREBP1c), a transcription factor that regulates cholesterol and lipid synthesis. As a result, this lead to decreased gluconeogenesis, decreased free fatty acid influx into the liver, increased free fatty acid oxidation and decreased de novo lipogenesis [38,125]. Two common SNPs in Adiponectin gene (45GT and 276GT) have been found to be significantly associated with the presence of NAFLD in non-obese, non-diabetic and normolipidemic patients and were subsequently correlated to the severity of NASH. Furthermore, Adiponectin gene (45GT and 276GT) has been proven to be the acute modulation of postprandial adiponectin levels and lipoprotein responses after fat ingestion [126]. However, controversy exists in the literatures. Some authors have reported a lack of association between Adiponectin gene and NAFLD [38,127]. Furthermore, several studies demonstrated a weak association between adiponectin gene polymorphism and cardiovascular disease [128,129]. The association between adiponectin polymorphisms and T2DM has been inconsistent and recent meta-analysis concluded that adiponectin gene polymorphism does not influence the development of T2DM [130,131]. Interestingly, two recent meta-analyses have revealed the potential protective factor of Adiponectin gene of different allele for cancer risk [132,133]. One point to note was that these meta-analyses were not specific to liver aetiology. However, recent evidences demonstrated a correlation between NAFLD and colorectal cancer [134]. The mechanism behind this remains unknown but raises the possibility of Adiponectin s anti-inflammatory effect that suppress TNF-α and other proinflammatory cytokines [38]. Further research is warranted to elucidate the link between cytokines and progression of NAFLD. Leptin (LEP), an adipocyte-derived hormone exerts its action through leptin receptor (LEPR), a single-transmembrane-domain receptor of the cytokine-receptor family has been shown to be responsible for satiety and energy homeostasis [135,136]. LEP and LEPR mutations have been shown to be strongly associated with obesity and endocrine dysfunction [135,137]. In study of NAFLD, Leptin-deficient ob/ob mouse has demonstrated a dramatic up-regulations of hapetic de novo lipogenesis markers and a significant impairment in hepatic mitochondrial function [138]. Recently, an association between variants of LEPR and PNPLA3 has been reported. The interactions between LEPR and PNPLA3 genes showed an increased risk of NAFLD compared to either gene alone [139]. Genes that affect cytokines production Little is known about the evolvement of NAFLD, a relative benign condition to NASH, an irreversible inflamed stage of liver disease. Inflammatory cytokines has been proposed to play a central role in the progression of NAFLD [140]. Cytokines are central mediators of hepatic inflammation, cells apoptosis and cells regeneration. TNF-α and IL-6 have been found to be the initial cytokines chemokines that being produced after hepatic insults [140,141]. TNF-α has been known to interact between fat accumulation and hepatic inflammation [141]. Coulon et al [141] suggested that TNF-α was significantly higher in NASH patients compared to SS patients. This highlighted the potential role of TNF-α in NASH. Furthermore, a recent meta-analysis has demonstrated that polymophisms of TNF-α (-238) is particularly associated with the susceptibility to NAFLD [142]. TNF-α has also been previously known to be an important cytokine that regulates insulin resistance by affecting insulin receptor substrate-1 (IRS-1) and insulin receptor kinase (IRK) in the insulin signal transduction pathway [142]. Nevertheless, the involvement of TNF-α in insulin resistance is debatable. Polymorphisms in gene such as IRS-1 (972 Arg) and ectoenzyme nucleotide pyrophosphate phosphodiesterase 1 (ENPP1) that directly affect insulin receptor activity have demonstrated that insulin resistance does contribute to the progression of NAFLD [143]. However, a recent study by Aparicio-Vergara et al [144] demonstrated a disassociation of TNF-α induced hepatic inflammation and insulin resistance. IL-6 is another cytokine that involved in the regulation of several cellular processes and has been demonstrated to be associated with NASH in several ethic cohorts [141,145]. However, the polymorphism of G/C allele is questionable. Carulli et al [146]. demonstrated that polymorphism of IL-6-174C is more prevalent in NAFLD and associated with insulin resistance while other studies demonstrated that polymorphism of C allele was unlikely 8332 July 14, 2014 Volume 20 Issue 26

25 Lim JW et al. Review of NAFLD pathogenesis Table 1 Proteomic studies of non-alcoholic fatty liver disease Protein categories Protein markers Ref. Year Findings Implications Protein carrier Apolipoproteins Choe et al [21] 2013 Independent association between ApoB/ ApoA1 ratio and NAFLD Metabolic pathways Acute phase protein Antiinflammatory and anti-oxidant CD5L Gray et al [22] 2009 Identified a pattern of serum apolipoproteins and CD5L to distinguish between pre-cirrhotic NAFLD and cirrhotic NAFLD 2010 Identified CD5L as poor biomarkers for HCC 2010 Decreased in NASH compared to control CPS1 Rodríguez-Suárez et al [24] Decreased in NASH compared to control patients GRP78 Rodríguez-Suárez et al [24] patients Uric Acid Sirota et al [33] 2013 Association of increased serum uric acid level and severity of NAFLD hs-crp Yoneda et al [39] 2007 Increased hs-crp serum level in advanced NASH Haemoglobin Yu et al [42] 2012 Association of elevated serum haemoglobin levels and prevalence of NAFLD Fuc-Hpt Kamada et al [44] 2013 Stepwise increased with increasing hepatocyte ballooning scores in biopsy-proven NAFLD patients PTX-3 Yoneda et al [47] 2008 A profound elevation of plasma PTX-3 level in NASH patients Bilirubin Kwak et al [49] 2012 An inverse relationship between total serum bilirubin level and the prevalence of NAFLD NAFLD is the hepatic manifestation of MetS ApoB/ApoA1 ratio as a predictable marker of cardiovascular risk in NAFLD Possibility of different trigger factors for the progression of NAFLD, NASH to HCC Down-regulation of CPS1 may lead to an increase of uric acid [31] Increased de novo lipogenesis might play a role in NAFLD pathogenesis [27] Mitochondrial oxidative damage could contribute to hepatic steatosis Acute phase proteins may have an immediate role in hepatocytes injuries predictive markers Serum levels of acute phase proteins do not necessary reflect the severity of disease NAFLD progression has a more complex underlying protein mechanism Bilirubin has antioxidant, antiinflammatory and anti-fibrogenic effects Extracellular matrix Immune cells and cytokines Hyaluronic acid Kaneda et al [56] 2006 Association of increased serum concentration An imbalance of ECM production and Type Ⅳ collagen Yoneda et al [59] 2007 of ECM components and degree of fibrosis in degradation could lead to fibrogenensis, 7S NAFLD but not specific to NAFLD Laminin Gabrielli et al [60] 1996 Lumican Krishnan et al [64] 2012 Over-expressed of Lumican with increasing severity of NAFLD and NASH MMP-9 D'Amico et al [72] 2010 An increase in serum MMP-9 levels in NASH and hepatitis C infected patients Demonstrated a difference of immunolabelling patterns between NASH and hepatitis C infected liver Wanninger et al [67] 2011 Association of MMP-9 activity and hepatic inflammation A negative correlation of MMP-9 activity and serum adiponectin CCL2/MCP1 Haukeland et al [81] 2006 Demonstrated an increasing level of CCL2/ MCP1, from healthy controls to NASH RBP4 Bell et al [18] 2010 A decreased expression of RBP4 with increasing NAFLD severity Alkhouri et al [84] 2009 An inverse relationship between RBP4 levels and liver fibrosis Graham et al [87] 2006 Association of increased serum RBP4 with T2DM and a decreased expression of glucose transporter 4 Christou et al [88] 2012 RBP4 has the ability to induce insulin resistance in the adipose tissue Circulating RBP4 can be influenced by nonmetabolic conditions and interventions Unregulated tissue remodelling and fibrogenesis could contribute to the progression of NAFLD Possibility of a different pathophysiological involvement of protease in the fibrogenesis of different liver etiology Possibility of peripheral adipocytes involvement in NAFLD progression The lipid accumulation from visceral tissue and hepatocellular could be responsible for the recruitment of immune cells RBP4 could contribute to the fibrogenesis pathway in NAFLD RBP4 is associated with insulin resistance and T2DM The association of RBP4 level and MetS is still not conclusive and should be interpret with caution NAFLD: Non-alcoholic fatty liver disease; MetS: Metabolic syndrome; NASH: Non-alcoholic steatohepatitis; HCC: Hepatocellular carcinoma; CPS1: Carbamoyl phosphate synthase 1; hs-crp: High sensitivity C-reactive protein; PTX-3: Pentraxin 3; ECM: Extracellular matrix; MMP: Matrix metalloproteinases; CCL2/MCP1: CC-chemokine ligand 2/monocyte chemo-attractant protein-1; T2DM: Type Ⅱ diabetes mellitus July 14, 2014 Volume 20 Issue 26

26 Lim JW et al. Review of NAFLD pathogenesis Table 2 Genomic studies of non-alcoholic fatty liver disease Gene categories Genes Ref. Year Findings Implications Gene that affect lipolysis Genes that affect adipokines Genes that affect cytokines PNPLA3 Romeo et al [91] 2008 PNPLA3 polymorphism is strongly associated with the pathogenic status of NAFLD in different populations Sookoian et al [94] 2011 GG genotype of PNPLA3 (rs738409; I148M) exerts a stronger effect on hepatic lipids accumulations Dubuquoy et al [95] 2013 PNPLA3 polymorphism is more likely to influence lipid content and liver disease severity but not insulin resistance ATGL Chitraju et al [102] 2013 Demonstrated that ATGL-KO animal models have high insulin sensitivity ApoC3 Petersen et al [106] 2010 ApoC3 polymorphism is associated with NAFLD and insulin resistance in Indian populations Hyysalo et al [107] 2012 ApoC3 polymorphism does not associated with NAFLD in Finnish populations GCKR Santora et al [108] 2012 GCKR polymorphism is associated with NAFLD Tan et al [112] 2013 Interactions of GCKR and PNPLA3 genes can increase susceptibility to NAFLD PPARGC1A and PPAR-γ Hui et al [116] 2008 PPARGC1A gene polymorphism is not associated with NAFLD PPAR-α is associated with NAFLD susceptibility Gawrieh et al [119] 2012 PPAR-γ polymorphism is associated with NAFLD Adiponectin Musso et al [126] 2008 Adiponectin gene polymorphism is associated with the presence of NAFLD Han et al [130] 2011 Adiponectin gene polymorphism does Li [20] 2012 not influence the development of T2DM Muhidin et al [134] 2012 Demonstrated a correlation between NAFLD and colorectal cancer LEP and LEPR Zain et al [139] 2013 Demonstrated an association between variants of LEPR and PNPLA3 TNF-α Wang et al [142] 2012 Polymophisms of TNF-α are associated with the susceptibility to NAFLD IL-6 Carulli et al [146] 2009 Polymorphisms of IL-6 are more prevalent in NAFLD and associated with insulin resistance Giannitrapani et al [145] 2013 Polymorphisms of IL-6 are unlikely to associate with insulin resistance A well established link between PNPLA3 and NAFLD progression Identified the patients group who is more susceptible to aggressive diseases A less clear association of insulin resistance with PNPLA3 polymorphism An accumulation of TG could be a protective mechanism in NAFLD Highlighted the ethnic differences in ApoC3 polymorphism and disease susceptibility Lipogenesis pathway plays a role in NAFLD pathogenesis The PGC-1α and PPAR-γ pathway play a role in NAFLD progression The role of PCG-1α (encoded by the PPARGC1A gene) in MetS is less clear than PPAR-γ De novo lipogenesis plays a role in NAFLD Possibility of different mechanisms for insulin sensitising in NAFLD Raises the possibility and association of antiinflammatory effect in NAFLD progression and oncological disease Patients with mutations of both LEPR and PNPLA3 are highly susceptible to NAFLD Insulin resistance and hepatic inflammation are related Insulin signal transduction pathway does contribute to the progression of NAFLD NAFLD: Non-alcoholic fatty liver disease; MetS: Metabolic syndrome; NASH: Non-alcoholic steatohepatitis; HCC: Hepatocellular carcinoma; CPS1: Carbamoyl phosphate synthase 1; hs-crp: High sensitivity C-reactive protein; PTX-3: Pentraxin 3; ECM: Extracellular matrix; MMP: Matrix metalloproteinases; CCL2/MCP1: CC-chemokine ligand 2/monocyte chemo-attractant protein-1; T2DM: Type Ⅱ diabetes mellitus; PPAR-γ: Peroxisome proliferator activated receptor gamma. to associate with insulin resistance [145]. Hence, insulin resistance and augmentation of hepatic inflammation could be modulated by other factors than TNF-α and IL-6. CURRENT KNOWLEDGE There is no doubt that there is a complex interplay between the body environment, predispose genetics and external environment (diet) for the development and progression of NAFLD. From the literatures, we know that NAFLD is characterised by insulin resistance, metabolic hyperlipidaemia and hepatic inflammation (Tables 1 and 2). Insulin resistance, accumulation of lipids (both visceral and hepatocellular) and persistent hepatic inflammation can result in a complex biochemical interaction. This could alter the cytokine profile and cell biology. A resultant disequilibrium in lipid homeostasis leads to hepatocellular lipid accumulation and an increase in oxidative stress due to mitochondrial dysfunction. Many studies have shed light on the role inflammation in progression of NAFLD to NASH. The activation of cell death pathways such as apoptosis and necrosis has redundant roles in further triggering liver damage and fibrosis. One of the crucial aspects from this review is that promising anti-inflammatory and innate immune mediators such as bilirubin, TNF-α and IL-6 are also commonly associated with cardiovascular disease, obesity and diabetes mellitus. The background of MetS further con July 14, 2014 Volume 20 Issue 26

27 Lim JW et al. Review of NAFLD pathogenesis founds these shared observations. An increased understanding of the roles of the liver in metabolic diseases or vice versa could lead to development of improved targeted strategies for disease prevention and treatment. This review also highlighted a considerable disease variation exists in the prevalence and severity of NAFLD. Therefore, suggesting that the risk of morbidity and mortality that might be influenced by a combination of genetic and environmental factors, which has strongly proven by the PNPLA3 polymorphisms. The improve understanding and further identification of genetic polymorphisms could lead to two potential applications for clinical practice. Firstly, genetic biomarkers could allow early disease diagnosis of higher risk individuals or family. Secondly, genetic biomarkers could potentially lead to the development of novel preventive and targeting measures of NAFLD. CONCLUSION Advances in proteomic technologies have contributed to the discovery of clinically important protein biomarkers, which are the key molecules that reflect biological reactions. Extensive literature searches revealed several potential serum protein markers that could differentiate NAFLD and NASH. Further validations of these proteins markers in larger cohorts are required to reflect the degree of hepatic fibrosis. In addition, genomic studies have further revealed the importance of genetic polymorphisms in various steps of the pathogenesis of NAFLD. Clearly, unfavourable genetic polymorphism coupled with unfavourable biological environment can increase a patient s susceptibility to the development of NAFLD and its progression to NASH. In general, further breakthrough and investigation of changes in protein expression levels are still warranted to understand the pathophysiology of NAFLD. REFERENCES 1 Vernon G, Baranova A, Younossi ZM. 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World J Gastroenterol 2013; 19: [PMID: DOI: / wjg.v19.i6.802] 126 Musso G, Gambino R, De Michieli F, Durazzo M, Pagano G, Cassader M. Adiponectin gene polymorphisms modulate acute adiponectin response to dietary fat: Possible pathogenetic role in NASH. Hepatology 2008; 47: [PMID: DOI: /hep.22142] 127 Wong VW, Wong GL, Tsang SW, Hui AY, Chan AW, Choi PC, So WY, Tse AM, Chan FK, Sung JJ, Chan HL. Genetic polymorphisms of adiponectin and tumor necrosis factor-alpha and nonalcoholic fatty liver disease in Chinese people. J Gastroenterol Hepatol 2008; 23: [PMID: DOI: /j x] 128 Zhang H, Mo X, Hao Y, Gu D. Association between polymorphisms in the adiponectin gene and cardiovascular disease: a meta-analysis. BMC Med Genet 2012; 13: 40 [PMID: DOI: / ] 129 Xi B, He D, Wang Q, Xue J, Liu M, Li J. Common polymorphisms (rs and rs ) in the ADIPOQ gene are not associated with hypertension susceptibility among the Chinese. 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32 Lim JW et al. Review of NAFLD pathogenesis +276G>T, C>G, G>A) of adiponectin gene and type 2 diabetes mellitus: a systematic review and metaanalysis. Diabetologia 2011; 54: [PMID: DOI: /s ] 131 Li Y, Yang Y, Shi L, Li X, Zhang Y, Yao Y. The association studies of ADIPOQ with type 2 diabetes mellitus in Chinese populations. Diabetes Metab Res Rev 2012; 28: [PMID: DOI: /dmrr.2309] 132 Zhou W, Liu Y, Zhong DW. Adiponectin (ADIPOQ) rs G/T polymorphism is associated with risk of cancer: evidence from a meta-analysis. Tumour Biol 2013; 34: [PMID: DOI: /s ] 133 Xu Y, He B, Pan Y, Gu L, Nie Z, Chen L, Li R, Gao T, Wang S. The roles of ADIPOQ genetic variations in cancer risk: evidence from published studies. Mol Biol Rep 2013; 40: [PMID: DOI: /s ] 134 Muhidin SO, Magan AA, Osman KA, Syed S, Ahmed MH. The relationship between nonalcoholic fatty liver disease and colorectal cancer: the future challenges and outcomes of the metabolic syndrome. J Obes 2012; 2012: [PMID: DOI: /2012/637538] 135 Lu H, Sun J, Sun L, Shu X, Xu Y, Xie D. Polymorphism of human leptin receptor gene is associated with type 2 diabetic patients complicated with non-alcoholic fatty liver disease in China. J Gastroenterol Hepatol 2009; 24: [PMID: DOI: /j x] 136 Swellam M, Hamdy N. Association of nonalcoholic fatty liver disease with a single nucleotide polymorphism on the gene encoding leptin receptor. IUBMB Life 2012; 64: [PMID: DOI: /iub.597] 137 Dubern B, Clement K. Leptin and leptin receptor-related monogenic obesity. Biochimie 2012; 94: [PMID: DOI: /j.biochi ] 138 Perfield JW, Ortinau LC, Pickering RT, Ruebel ML, Meers GM, Rector RS. Altered Hepatic Lipid Metabolism Contributes to Nonalcoholic Fatty Liver Disease in Leptin-Deficient Ob/Ob Mice. J Obes 2013; 2013: 8 [DOI: /2013/296537] 139 Zain SM, Mohamed Z, Mahadeva S, Cheah PL, Rampal S, Chin KF, Mahfudz AS, Basu RC, Tan HL, Mohamed R. Impact of leptin receptor gene variants on risk of non-alcoholic fatty liver disease and its interaction with adiponutrin gene. J Gastroenterol Hepatol 2013; 28: [PMID: DOI: /jgh.12104] 140 Tilg H. The role of cytokines in non-alcoholic fatty liver disease. Dig Dis 2010; 28: [PMID: DOI: / ] 141 Coulon S, Francque S, Colle I, Verrijken A, Blomme B, Heindryckx F, De Munter S, Prawitt J, Caron S, Staels B, Van Vlierberghe H, Van Gaal L, Geerts A. Evaluation of inflammatory and angiogenic factors in patients with nonalcoholic fatty liver disease. Cytokine 2012; 59: [PMID: DOI: /j.cyto ] 142 Wang JK, Feng ZW, Li YC, Li QY, Tao XY. Association of tumor necrosis factor-α gene promoter polymorphism at sites -308 and -238 with non-alcoholic fatty liver disease: a meta-analysis. J Gastroenterol Hepatol 2012; 27: [PMID: DOI: /j x] 143 Dongiovanni P, Valenti L, Rametta R, Daly AK, Nobili V, Mozzi E, Leathart JB, Pietrobattista A, Burt AD, Maggioni M, Fracanzani AL, Lattuada E, Zappa MA, Roviaro G, Marchesini G, Day CP, Fargion S. Genetic variants regulating insulin receptor signalling are associated with the severity of liver damage in patients with non-alcoholic fatty liver disease. Gut 2010; 59: [PMID: DOI: /gut ] 144 Aparicio-Vergara M, Hommelberg PP, Schreurs M, Gruben N, Stienstra R, Shiri-Sverdlov R, Kloosterhuis NJ, de Bruin A, van de Sluis B, Koonen DP, Hofker MH. Tumor necrosis factor receptor 1 gain-of-function mutation aggravates nonalcoholic fatty liver disease but does not cause insulin resistance in a murine model. Hepatology 2013; 57: [PMID: DOI: /hep.26046] 145 Giannitrapani L, Soresi M, Balasus D, Licata A, Montalto G. Genetic association of interleukin-6 polymorphism (-174 G/ C) with chronic liver diseases and hepatocellular carcinoma. World J Gastroenterol 2013; 19: [PMID: DOI: /wjg.v19.i ] 146 Carulli L, Canedi I, Rondinella S, Lombardini S, Ganazzi D, Fargion S, De Palma M, Lonardo A, Ricchi M, Bertolotti M, Carulli N, Loria P. Genetic polymorphisms in non-alcoholic fatty liver disease: interleukin-6-174g/c polymorphism is associated with non-alcoholic steatohepatitis. Dig Liver Dis 2009; 41: [PMID: DOI: / j.dld ] P- Reviewers: De Petro G, Gerova VA S- Editor: Ma YJ L- Editor: A E- Editor: Ma S 8340 July 14, 2014 Volume 20 Issue 26

33 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. TOPIC HIGHLIGHT WJG 20 th Anniversary Special Issues (12): Nonalcoholic fatty liver disease Modern approach to the clinical management of non-alcoholic fatty liver disease Maria Del Ben, Licia Polimeni, Francesco Baratta, Daniele Pastori, Lorenzo Loffredo, Francesco Angelico Maria Del Ben, Licia Polimeni, Francesco Baratta, Daniele Pastori, Lorenzo Loffredo, Department of Internal Medicine and Medical Specialities, Rome, Italy Francesco Angelico, Department of Public Health and Infectious Diseases, Sapienza University, Rome, Italy Author contributions: Del Ben M and Polimeni L contributed equally to this work; Del Ben M, Polimeni L and Baratta F analyzed the literature data; Pastori D and Loffredo L reviewed and edited the manuscript; Polimeni L and Angelico F designed the study and wrote the manuscript; all authors read and approved the manuscript. Correspondence to: Francesco Angelico, MD, Professor of Internal Medicine, Department of Public Health and Infectious Diseases, Sapienza University, 155 viale del Policlinico, Rome, Italy. francesco.angelico@uniroma1.it Telephone: Fax: Received: October 2, 2013 Revised: November 5, 2013 Accepted: March 7, 2014 Published online: July 14, 2014 Abstract Non-alcoholic fatty liver disease (NAFLD) is the most common and emerging form of chronic liver disease worldwide. It includes a wide spectrum of liver diseases ranging from simple fatty liver to steatohepatitis, which may progress to cirrhosis, liver cancer, and liver mortality. Common metabolic diseases, which are well established cardiovascular risk factors, have been associated to NAFLD and cardiovascular disease is the single most important cause of morbidity and mortality in this patient population. The pathogenesis of NAFLD appears multifactorial and many mechanisms have been proposed as possible causes of fatty liver infiltration. Management of fatty liver has become a major challenge to healthcare systems as the consequence of the increasing rates of obesity worldwide. First-line management focuses on lifestyle modifications. Moderate weight reduction either by dietary restriction or by increased habitual physical activity is safe and highly recommended. Several therapeutic interventions have been proposed. These include insulin sensitizer agents, lipid lowering drugs, antioxidants such as vitamin E and supplementation of vitamin D3. However, therapeutic strategies have been largely empirical so far, and experimental trials have mostly been carried out in uncontrolled settings with small sample sizes. Metabolic conditions such as diabetes mellitus, obesity, hypertension and hyperlipidemia, should be strongly considered and a multidisciplinary approach should be personalized for individual patients. Treatment of co-morbidities should be regarded as of paramount importance in the management of these patients. The purpose of this review is to examine different approaches for the clinical management of non-alcoholic fatty liver disease Baishideng Publishing Group Inc. All rights reserved. Key words: Non-alcoholic fatty liver disease; Nonalcoholic steatohepatitis; Insulin resistance; Oxidative stress; Cardiovascular risk; Statins; Vitamin D; Vitamin E; Pioglitazone Core tip: Management of fatty liver has become a major challenge to healthcare systems as the consequence of the increasing rates of obesity worldwide. Several therapeutic interventions have been proposed, which include moderate weight loss, insulin sensitizer agents, lipid lowering drugs, antioxidants such as vitamin E and treatment of vitamin D3 deficiency. However, therapeutic approaches have been largely empirical and experimental trials have mostly been carried out in uncontrolled settings with small sample sizes so far. Treatment of coexisting metabolic conditions should be regarded as of paramount importance in the management of these patients. Del Ben M, Polimeni L, Baratta F, Pastori D, Loffredo L, Angelico F. Modern approach to the clinical management of non-alcoholic fatty liver disease. World J Gastroenterol 2014; 20(26): Available from: URL: com/ /full/v20/i26/8341.htm DOI: July 14, 2014 Volume 20 Issue 26

34 Del Ben M et al. Management of non-alcoholic fatty liver disease org/ /wjg.v20.i INTRODUCTION Non-alcoholic fatty liver disease (NAFLD) represents the most common and emerging form of chronic liver disease worldwide. It is increasingly detected by routine abdominal ultrasound examination. Indeed, NAFLD reached epidemic proportions and the general prevalence of this condition is reported ranging between 20%-30% and 70%-90% respectively in patients with severe obesity or with type 2 diabetes mellitus [1-4]. NAFLD includes a wide spectrum of liver diseases ranging from simple fatty liver to non-alcoholic steatohepatitis (NASH), which may progress to fibrosis and even cirrhosis and hepatocellular carcinoma. Simple steatosis generally represents a benign condition following a non-progressive clinical course. By contrast, a subset of patients with NASH, in particular those with a more severe fibrosis, are at higher risk for progressing to liver disease complications [5]. In fact, about 25% of patients affected by NASH can develop cirrhosis with the subsequent risk of hepatocellular carcinoma (HCC) [6]. Moreover, the rising number of HCCs developing into non cirrhotic NASH liver is becoming a cause for concern [7,8]. The pathogenesis of non-alcoholic and virus-negative liver steatosis and NASH appears multifactorial and many mechanisms have been proposed as possible causes of fatty liver infiltration [9]. The association of steatosis with a number of different clinical conditions has been suggested. Common metabolic diseases, such as hyperlipidaemia, type 2 diabetes and central obesity, are well established cardiovascular risk factors and have been associated to both benign liver steatosis and progressive NASH [10]. Moreover, it has recently been proposed that the metabolic syndrome may play a causal role in the pathogenesis of NASH [11-14]. Management of NAFLD and NASH has become a major challenge to healthcare systems as the consequence of the increasing rates of obesity worldwide. Since NAFLD is usually associated to a number of insulin resistance related conditions, treatment of comorbidity should be regarded as of paramount importance in the management of these patients. Several therapeutic interventions have been proposed, which include weight reduction, insulin sensitizer agents, lipid lowering drugs, antioxidants such as vitamin E and treatment of vitamin D3 deficiency (Table 1). However, therapeutic strategies have been largely empirical so far and experimental trials have mostly been carried out in uncontrolled settings with small sample sizes. NAFLD AND OBESITY: A ROLE FOR LOW-CALORIE DIET, EXERCISE AND BARIATRIC SURGERY Diet is an important contributor to the pathogenesis of Table 1 Major proposed interventions for nonalcoholic fatty liver disease Lifestyle intervention Calorie restriction Mediterranean diet Exercise Medication Weight loss drugs Orlistat Sibutramine Antidiabetics Metformin Pioglitazone DPP IV inhibitors GLP-1 receptors agonists Antioxidants Vitamin E Statins Pravastatin Pitavastatin Atorvastatin Rosuvastatin PUFA Vitamin D UDCA Probiotics Berberine Silymarin Silybin Bariatric surgery NAFLD and a healthy low-fat diet may have benefits independent of weight loss. Some studies demonstrated favourable effects of the Mediterranean diet on NAFLD. Recently, Ryan et al [15] demonstrated in an insulin-resistant population with NAFLD a reduction of liver steatosis and an improvement of insulin sensitivity after 6 wk of the Mediterranean diet, compared to current dietary advice. Moreover, Kontogianni et al [16] reported that higher adherence to the Mediterranean diet was not associated with a less likelihood of having NAFLD, but it was associated with a lower degree of insulin resistance and less severe liver disease among patients with NAFLD. Finally, in a post hoc analysis of a controlled trial conducted in Israel, a modified Mediterranean diet was associated with the greatest reduction in alanine aminotransferase levels (ALT) in obese type 2 diabetes patients [17]. There is evidence that saturated fat and fructose are more likely to stimulate hepatic lipid accumulation and progression in NASH, whereas unsaturated fat, choline, antioxidants and high-protein diets rich in isoflavones seem to have a more preventive effect [18-22]. Among polyunsaturated fatty acids, in particular, the n-3 fatty acids seem to reduce the accumulation of triglycerides and ameliorate hepatic steatosis [23]. Since the majority of patients with NAFLD are overweight or obese, weight reduction either by dietary restriction or by increased habitual physical activity or by both, is highly recommended. Weight loss is safe in patients with nonalcoholic fatty liver disease, and it improves liver histology [24]. Moreover, diet is also effective 8342 July 14, 2014 Volume 20 Issue 26

35 Del Ben M et al. Management of non-alcoholic fatty liver disease for the treatment of metabolic syndrome and is able to control its clinical and metabolic features; in fact, central obesity, insulin resistance, blood pressure and atherogenic dyslipidemia are improved after a modest weight loss of 5%-10% [25]. In addition, lifestyle modifications in the form of moderate calorie restriction and weight loss have also been consistently associated with reduced oxidative stress [25]. The only two registered drugs for pharmacological weight reduction, Orlistat and Sibutramine, gave some positive results on serum liver enzymes, but not on liver histology in patients with NAFLD [26]. Bariatric surgery is considered a quite safe [27] and effective [28] option for obese patients in reducing weight. Despite its known efficacy on decreasing obesity related disease [29], at present there is a lack of randomized controlled trials evaluating the effects of bariatric surgery in patients with NAFLD and NASH. The available studies in fact are retrospective or prospective cohort studies. Based on these data, a recent Cochrane review concluded that because of a high risk of bias, a reliable summary of their data is not possible [30]. Thus, well-designed randomised trials to assess benefits and harms of bariatric surgery as a therapeutic approach for patients with NASH are required. Short-term aerobic exercise can improve fatty liver composition in patients with NAFLD and recent studies reported reduction in hepatic lipid content also after exercise intervention programs which did not induce weight loss [31-33]. However, the independent role of regular physical activity for NAFLD has been assessed only in few studies and the optimal physical activity regimen still remains to be determined. INSULIN RESISTANCE AND THE METABOLIC SYNDROME: A POSSIBLE ROLE FOR INSULIN SENSITIZER AGENTS AND INCRETINS Insulin resistance is a frequent metabolic abnormality affecting approximately 20% of the non-diabetic population and occurring in association with many cardiovascular and metabolic abnormalities. It has been suggested that the presence of insulin resistance is an essential requirement for the accumulation of hepatocellular fat. In fact, according to current opinion, the pathogenesis of NAFLD is based on a two hits hypothesis [5,34]. The first hit involves hepatic triglyceride accumulation (i.e., simple steatosis) and is mainly due to insulin resistance thus increasing vulnerability to further damage. In fact, insulin resistance is responsible for causing abnormalities of lipid storage and lipolysis in insulin sensitive tissues, which may induce an increased fatty acid flux from adipose tissue to the liver and cause steatosis. Insulin resistance may also cause lipid peroxidation which in turn may activate inflammatory cytokines and promote the progression of innocent steatosis to nonalcoholic steato-hepatitis and liver fibrosis. In a previous study by our group, when non-diabetic subjects were simultaneously categorized by insulin resistance and insulin secretion status, those with a predominant insulin resistance had a higher prevalence of severe liver steatosis at ultrasound, thus confirming the pathophysiological role of hyperinsulinemia in the events leading to the development of fatty liver [11]. The same subjects had significantly higher body mass index and waist circumference and similar blood pressure and serum lipids as compared to those with a prevalent insulin secretory defect. Moreover, insulin resistance was associated with severe steatosis, independently from potentially confounding factors such as age, BMI, high fasting glucose and high serum triglycerides [9]. Insulin resistance has been demonstrated to unify NAFLD to the metabolic syndrome. In fact, it has been suggested that fatty liver can be considered as the hepatic consequence of the insulin resistance metabolic syndrome, emerging as a distinct high cardiovascular risk entity which has been recently indicated as the secondary target of therapy for the reduction of coronary risk after serum low-density-lipoprotein cholesterol [35]. Metabolic syndrome is characterized by the clustering of glucose intolerance and/or diabetes, hyperinsulinemia, increased levels of triglycerides and decreased HDL cholesterol, hypertension and central and overall obesity [36]. In a previous study, we demonstrated a strong positive association between the prevalence of some common metabolic diseases and the severity of liver steatosis. Obesity, impaired glucose tolerance and type 2 diabetes mellitus were 5.3-fold, 3.6-fold and 4.0-fold, more common among subjects with more severe steatosis, as compared to controls without steatosis. In the same group the prevalence of hypertriglyceridemia was seven times more frequent than in the control group and about three times higher than in the group with mild steatosis. Among subjects with severe steatosis obesity, impaired glucose metabolism and hyperlipidemia were present in over 50% of patients. These findings were paralleled by the presence of a statistically significant increase in mean BMI, plasma glucose and triglyceride levels from the group of subjects without steatosis to the three groups with increasing severity of fatty liver [10]. Based on the above considerations, the use of drugs improving insulin resistance has been proposed for patients with fatty liver. Metformin was the first insulin sensitizer used; however, only marginal benefits on transaminases and no improvement in steatosis or inflammation have been reported so far, and metformin is not recommended for patients with NAFLD [37]. Recently, it has been suggested that metformin may prevent liver tumorigenesis via suppression of liver fat accumulation in the early stage, before the onset of NAFLD. However, the suggested potential role of metformin as antitumor agent should be further confirmed by large-scale clinical trials [38,39]. Concerning thiazolidinediones, treatment with pioglitazone was associated with improvements of liver 8343 July 14, 2014 Volume 20 Issue 26

36 Del Ben M et al. Management of non-alcoholic fatty liver disease steatosis and inflammation in recent controlled clinical trials [40-42]. Therefore, pioglitazone may be recommended for the treatment of adults with biopsy proven NASH [43]. Recently, some studies investigated the possible effects of analogues of glucagon-like peptide-1 (GLP-1), an incretin that increases insulin sensitivity and aids glucose metabolism, on NAFLD. In vivo and in vitro studies demonstrated that GLP-1 analogues reduce fatty acid accumulation and endoplasmic reticulum stress-related apoptosis in human hepatocytes treated with fatty acids as well as in an animal model of NAFLD [35]. This fact could improve hepatic steatosis by modulation of lipid metabolism and hepatic insulin signaling in NAFLD animal models [45,46]. These effects could be explained by possible direct stimulation of hepatic GLP-1 receptor. Cuthbertson et al [46] recently conducted a prospective study in 25 obese, T2DM patients with hepatic steatosis, treated with metformin and sulphonylureas/dpp-iv inhibitors. After a 6 mo treatment with GLP-1 receptor agonists (exenatide or liraglutide), reduction of mean HbA1c, body weight and intrahepatic lipid was demonstrated [46]. The relative reduction in intrahepatic lipid correlated with that in HbA1c but was not significantly correlated with that in total body weight. Adequately powered, randomised, placebo-controlled intervention studies using GLP-1 analogues in patients with NAFLD, with or without T2DM, are needed to demonstrate the potential role of incretins in ameliorating NAFLD and to investigate the inter-relationship between the biochemical, metabolic and histological responses. OXIDATIVE STRESS AND REDUCED ANTIOXIDANT STATUS: A POSSIBLE ROLE FOR VITAMIN E Several lines of evidence suggest that chronic oxidative stress is one of the key mechanisms responsible for liver damage and disease progression in NAFLD. In particular, according to the two-hit theory, oxidative stress is a major player triggering the progression of steatosis to NASH as the result of an imbalance between prooxidant and anti-oxidant chemicals that lead to liver cell damage [5,34]. Indeed, the increased production of reactive oxygen species (ROS) is known to cause lipid peroxidation, followed by inflammation, and activation of stellate cells leading to fibrogenesis. Consistent with the above theory, subjects with NASH had significantly increased levels of lipid peroxidation products [47-52]. Moreover, in a small study performed in 21 subjects with NASH and 19 controls, subjects with NASH had significantly higher levels of oxidized LDL and of thiobarbituric acid-reacting substances (TBARS) suggesting an increased cardiovascular risk [51]. Similar results were reported in India, where TBARS levels were significantly elevated and GSH/GSSG ratio was significantly decreased in NAFLD subjects without and with type 2 diabetes [49]. Increased systemic levels of malondialdehyde were ob- served in 58 male patients with histologically proven NAFLD compared to healthy age matched males [48]. In a further study, NAFLD children with immune responses against MDA derived antigens showed more severe lobular inflammation and had a 13-fold higher prevalence of overt NASH suggesting the presence of oxidative stress in a high proportion of NAFLD children [50]. In two more studies the percentage of hepatocytes, positive for 8-OHdG expression, and serum 8-OHdG levels were significantly higher in patients with NASH than simple fatty liver, while the oxidative stress marker GGT was increased in both conditions [51,53]. Finally, in a recent cross-sectional study, oxidative stress, detected as the ratio of plasma total antioxidant status to total oxidant status, was associated with insulin resistance in obese adolescents with NAFLD [52]. In a previous study [54], we demonstrated an increased systemic oxidative stress in subjects with fatty liver, as compared to those without. This increase was associated to severity of liver steatosis evaluated either by liver ultrasonography, or by fatty liver index or by serum cytokeratin-18 levels, a marker of apoptosis reflecting liver disease severity. In fact, we found increased systemic oxidative stress assessed by increased levels of urinary 8-iso-PGF2α, currently regarded as the best measure of oxidative stress in vivo and of serum snox2-dp, a marker of NOX2 activation by blood cells, which plays an important role in ROS production. The same findings were also observed after the exclusion of obese subjects, or subjects with diabetes or with metabolic syndrome and in those not taking statin medication. In our study, a correlation was also found between HOMA-IR, urinary 8-iso PGF2α and snox2- dp, confirming the interdependency of insulin resistance and oxidative stress in the pathogenesis of NAFLD. Moreover, both oxidative stress markers were also highly correlated with serum cytokeratin-18, thus suggesting a possible role in the progression from simple fatty liver to NASH. Therefore, although the mechanisms underlying disease progression remain poorly understood, a therapeutic strategy targeting oxidative stress reduction has been proposed and supplementation with vitamin E has been suggested by recent guidelines for the treatment of NASH in non-diabetic subjects [37]. In pioglitazone vs vitamin E vs placebo for the Treatment of nondiabetic patients with nonalcoholic steatohepatitis (PIVENS) trial, vitamin E therapy demonstrated a significant improvement in steatosis, inflammation, ballooning, and resolution of steatohepatitis in adult patients with aggressive NASH, who did not have diabetes or cirrhosis [42]. Conversely, in the TONIC trial, where 173 children were randomized to receive vitamin E (400 IU twice daily), metformin (500 mg twice daily), or placebo for 96 wk, neither agent was superior to placebo in attaining the primary outcome, a reduction in ALT level 50% or less of the baseline [55]. Finally, it should be taken into consideration that vitamin E supplementation has 8344 July 14, 2014 Volume 20 Issue 26

37 Del Ben M et al. Management of non-alcoholic fatty liver disease not been associated to benefits in cardiovascular death or cerebrovascular events [56]. Moreover, the results of two recent reviews showed no evidence of benefits by vitamin E supplementation for the prevention of cardiovascular events and there was no evidence for a possible association of vitamin E with higher all-cause mortality [57,58]. NAFLD AND CARDIOVASCULAR RISK: A POSSIBLE ROLE FOR STATINS NAFLD has been traditionally interpreted as a condition, which may progress into liver related complications such as cirrhosis, liver cancer, and liver mortality. In particular, liver fibrosis predicts disease progression and the risk for hepatocellular carcinoma. However, most people with NAFLD in the absence of significant hepatic fibrosis do not develop serious liver problems. Conversely, people with NAFLD have an increased chance of developing cardiovascular diseases, such as myocardial infarction and stroke, which represent the major causes of death in this setting [25]. Indeed, cardiovascular disease is the single most important cause of morbidity and mortality in this patient population [59]. Therefore, it appears that the increased mortality of patients is primarily a result of cardiovascular diseases and, to a lesser extent, of liver related diseases. Indeed, many epidemiological, clinical, and pathophysiological observations support a strong association between NAFLD and increased cardiovascular risk, which in some studies was found to be independent of traditional risk factors and aspects of the metabolic syndrome. Moreover, patients with NASH seem to be at higher risk for atherosclerosis than patients with simple steatosis as a consequence of chronic inflammation and oxidative stress. Finally, an increased risk of developing type 2 diabetes mellitus has also been demonstrated among NAFLD patients [60]. Therefore, based on the above considerations, we may perhaps consider patients with NAFLD at increased global cardiometabolic risk, although the precise mechanisms by which NAFLD contributes to cardiovascular disease (CVD) and diabetes are still the subject of ongoing research. In a recent review, we discussed the epidemiological, clinical, and physiopathological evidence of an association between NAFLD and cardiovascular diseases [61]. Recent genetic studies [62,63], such as those showing a strong correlation between presence of the I148M polymorphism in the PNPLA3 gene and elevated serum levels of ALT and AST and liver inflammation, will probably be helpful in improving the assessment of cardiovascular risk in NAFLD patients with and without metabolic syndrome. NASH is also linked to accelerated atherogenesis through the presence of abnormal production of triglyceride- and cholesterol-rich remnant particles, leading to accelerated atherosclerosis, together with other features of the metabolic syndrome. Dyslipidemia is characterized by increased serum triglycerides, low high-density lipoprotein (HDL) cholesterol and increased small, dense, low-density lipoprotein (LDL) particles, i.e., the so-called atherogenic lipid triad [64,65]. The presence of small dense LDL particles is associated with increased cardiovascular risk. Therefore, aggressive treatment of dyslipidemia and the other features of the metabolic syndrome should be a primary target in patients with NAFLD. Statins are first line drugs for the treatment of LDL cholesterol. According to ATPIII Guidelines, the current recommendation that liver biochemistries be checked before and periodically after starting statin therapy is not evidence-based and is controversial [36]. On the other end, measuring aminotransferases at baseline cannot adequately identify those who have underlying liver disease and there is no sound rationale why statins should not be used in patients with chronic liver disease who otherwise need statin therapy. In fact, in the GRACE Study, atorvastatin treatment was safe in patients with mild to moderately abnormal liver tests that were potentially attributable to NAFLD [66]. Moreover, in small uncontrolled studies, pravastatin, atorvastatin, rosuvastatin and pitavastatin were safe and effective in NASH patients with dyslipidemia [67,68]. In all these studies a decrease of serum aminotransferase was also observed and liver histology improved in some patients. Therefore, the AASLD Guidelines recommend that statins can be used to treat dyslipidemia in patients with NAFLD and NASH (Strenght 1; Quality B) [37]. VITAMIN D DEFICIENCY: A POSSIBLE ROLE FOR VITAMIN D3 Vitamin D deficiency is a highly prevalent condition worldwide. Low serum 25(OH)D3 concentrations have been reported among adults and children affected by NASH [69,70]. Furthermore, we have recently demonstrated the existence of a strong and independent association between NAFLD and low 25(OH)D3 levels in a large adult population with normal serum liver enzymes [71]. Vitamin D is present in the diet and dietary supplements, but its primary source is the photo-mediated conversion of 7-dehydrocholesterol in the skin [72]. To become biologically active, vitamin D requires 25-hydroxylation in the liver and subsequent 1-hydroxylation in the kidney [73]. The 25-hydroxylation occurs exclusively in the hepatocyte and is mediated by CYP27A1 and CYP2R1, two liverexpressed cytochromes characterized by different intracellular locations, specificity and affinity for vitamin D3. The biological effects of vitamin D3 are mediated by the vitamin D receptor (VDR) [74]. In a recent study [75], we suggest that the vitamin D-vitamin D receptor system may play an important role in the response of the liver to chronic damage induced by different pathogenic stimuli. In fact, low hepatic VDR expression which was closely associated with more severe liver histology in this study, could represent the primary event leading to progression of hepatitis July 14, 2014 Volume 20 Issue 26

38 Del Ben M et al. Management of non-alcoholic fatty liver disease Based on the above considerations, treatment of vitamin D3 deficiency to prevent or treat NAFLD to NASH progression has been proposed. However, large placebocontrolled randomized trials have not been performed so far. Thus, it is premature to recommend vitamin D3 for the specific treatment of NAFLD/NASH. OTHER TREATMENTS Many other agents have been investigated as potential therapeutic options in NAFLD and NASH in small, proof-of-concept studies. Omega-3 fatty acids are currently approved to treat severe hypertriglyceridemia and have also proven benefitial for cardiovascular disease. Recently, they have been suggested as a treatment for NAFLD and some studies support the use of omega-3 fatty acids in this setting [24]. Their potential mechanisms of action may be mediated by regulation of hepatic gene expression, improvement of insulin sensitivity, reduction of inflammation and oxidative stress. In a cross-sectional study by Zelber-Sagi et al [76] dietary habits of NAFLD patients have been investigated, finding that the NAFLD group consumed less fish rich in omega-3 and almost double the quantity of soft drinks and also consumed more meat compared with the general population. Moreover, these dietary differences were associated with an increased risk of NAFLD independent of traditional risk factors. Evidence emerging from animal studies show a reduction in hepatic steatosis, inflammation and oxidative stress and an improvement in insulin sensitivity. Moreover, some preliminary clinical studies in humans reported a reduction in hepatic steatosis on imaging and an improvement of insulin sensitivity and serum liver function tests. In any case, these studies have small sample size and methodological flaws. Results from ongoing large multicenter studies with histological outcomes are needed [77]. Thus, AASLD Guidelines currently concluded that it is premature to recommend omega-3 fatty acids for the specific treatment of NAFLD or NASH, but they may be considered as first line agents to treat hypertriglyceridemia in patients with NAFLD (Strength-1; Quality-B) [37]. Furthermore, ursodeoxycholic acid (UDCA) has been proposed as a treatment for NAFLD and NASH and several studies have been conducted concerning this treatment [78,79]. All but one study are with small sample size and/or surrogate end-points. The only available large RCT with histological endpoints showed lack of any histological benefit of UDCA in patients with NASH [79]. During the last years, there has been renewed interest in the potential efficacy of silymarin and silybin, two natural products derived from the milk thistle plant Silybum marianum, traditionally used in classical Greece to treat hepatic and biliary diseases. A higher bioavailability of silybin has been shown and experimental studies have demonstrated its antifibrotic, antioxidant and metabolic effects [80,81]. Treatment with silymarin was associated with improvement in liver enzymes in a small placebo-controlled study in patients with NAFLD [82]. Silybin, combined with phosphatidylcholine and vitamin E, induced improvements of insulin resistance and liver histology in a large multicenter RCT [83]. A growing body of evidence supports a relation between overgrowth of gut microbiota with NAFLD and NASH. In fact, it has been suggested that liver injury could be partly caused by exposure to bacterial and fermentation agents produced by microflora which are metabolized in the liver. Therefore, the use of probiotics to better balance the gut flora has been proposed. So far, various experimental studies and clinical trials revealed promising effects [84]. However, larger trials with longerterm follow-up are needed. Recently, treatment with berberine has been suggested for patients with NAFLD, although its clinical application should be better defined. In fact, few small experimental and clinical studies have demonstrated favorable effects on insulin resistance, hepatic lipoprotein secretion and gut microenvironment modifications [85]. Finally, mitochondrial dysfunction may induce overproduction of free radicals that, in turn, trigger lipid peroxidation and cell death. As a result, impaired mitochondrial function is thought to contribute to NAFLD and insulin resistance [86,87]. However, so far, improvement of mitochondrial dysfunction has only been associated to increased physical exercise and few experimental studies have assessed the potential role of antioxidant molecules targeted to mitochondria [88,89]. CONCLUSION NAFLD is a multifactorial disease and the exact mechanism is unknown. Therefore, it is important to carefully evaluate for competing etiologies and clinically important co-morbidities. Metabolic conditions, such as diabetes mellitus, obesity, hypertension, and hyperlipidemia, should be treated aggressively and a multidisciplinary approach should be personalized for individual patients. The main targets of therapy are reported in Table 2. Since NAFLD is largely a manifestation of obesity and metabolic syndrome, first- line management of NAFLD focuses on lifestyle modifications. Based on the available evidence, Mediterranean diet seems to be the best dietary regimen for patients with NAFLD. Weight loss generally reduces hepatic steatosis, and is achieved either by low-calorie diet alone or in conjunction with increased physical activity. Many patients with NAFLD have severe obesity. The role of bariatric surgery for these patients is still under evaluation. As insulin resistance is nearly universal in patients with NASH, therapeutic measures to reduce insulin resistance have been suggested. Metformin was the first insulin sensitizer used; however, only marginal benefits on transaminases and no significant effect on liver steatosis or inflammation have been reported so far. Treatment 8346 July 14, 2014 Volume 20 Issue 26

39 Del Ben M et al. Management of non-alcoholic fatty liver disease Table 2 Main targets of intervention in nonalcoholic fatty liver disease Overweight and obesity Sedentary lifestyles Insulin resistance Oxidative stress Atherogenic dyslipidemia Vitamin D deficiency Hepatocyte protection Cardiovascular risk Treatment of comorbidities Mitochondrial dysfunction with pioglitazone is associated with improvements of liver histology and therefore may be recommended for the treatment of patients with biopsy-proven NASH. Proposed pharmacologic therapies for NASH include antioxidants such as vitamin E (a-tocopherol) administered at daily dose of 800 IU/d, which could improve liver histology in non-diabetic adults with biopsy-proven NASH. The cardiovascular morbidity and mortality is perhaps one of the most important and new aspects of NAFLD and several recent studies have shown that statins can be safely used to treat dyslipidemia in patients with NAFLD, but are not recommended to specifically treat NASH. UDCA is not recommended and it is premature to recommend omega-3 fatty acids and vitamin D supplementation. Patients should be advised about the limited effectiveness of most drug therapies except possibly insulin sensitizers for some patients with diabetes and vitamin E for those patients without diabetes. Finally, it should be considered that there are no medications approved by the United States Food and Drug Administration. Our clinical approach to the treatment of NAFLD is based on few relatively small studies demonstrating benefits from some of the above treatments. REFERENCES 1 Browning JD, Szczepaniak LS, Dobbins R, Nuremberg P, Horton JD, Cohen JC, Grundy SM, Hobbs HH. 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42 Del Ben M et al. Management of non-alcoholic fatty liver disease 75 Barchetta I, Carotti S, Labbadia G, Gentilucci UV, Muda AO, Angelico F, Silecchia G, Leonetti F, Fraioli A, Picardi A, Morini S, Cavallo MG. Liver vitamin D receptor, CYP2R1, and CYP27A1 expression: relationship with liver histology and vitamin D3 levels in patients with nonalcoholic steatohepatitis or hepatitis C virus. Hepatology 2012; 56: [PMID: DOI: /hep.25930] 76 Zelber-Sagi S, Nitzan-Kaluski D, Goldsmith R, Webb M, Blendis L, Halpern Z, Oren R. Long term nutritional intake and the risk for non-alcoholic fatty liver disease (NAFLD): a population based study. J Hepatol 2007; 47: [PMID: DOI: /j.jhep ] 77 Janczyk W, Socha P, Lebensztejn D, Wierzbicka A, Mazur A, Neuhoff-Murawska J, Matusik P. Omega-3 fatty acids for treatment of non-alcoholic fatty liver disease: design and rationale of randomized controlled trial. BMC Pediatr 2013; 13: 85 [PMID: DOI: / ] 78 Wu SD, Li L, Wang JY. Ursodeoxycholic acid for nonalcoholic steatohepatitis. Eur J Gastroenterol Hepatol 2012; 24: [PMID: DOI: /MEG.0b013e ec0] 79 Ratziu V, de Ledinghen V, Oberti F, Mathurin P, Wartelle- Bladou C, Renou C, Sogni P, Maynard M, Larrey D, Serfaty L, Bonnefont-Rousselot D, Bastard JP, Rivière M, Spénard J. A randomized controlled trial of high-dose ursodesoxycholic acid for nonalcoholic steatohepatitis. J Hepatol 2011; 54: [PMID: DOI: /j.jhep ] 80 Cacciapuoti F, Scognamiglio A, Palumbo R, Forte R, Cacciapuoti F. Silymarin in non alcoholic fatty liver disease. World J Hepatol 2013; 5: [PMID: DOI: /wjh.v5.i3.109] 81 Loguercio C, Festi D. Silybin and the liver: from basic research to clinical practice. World J Gastroenterol 2011; 17: [PMID: DOI: /wjg.v17.i ] 82 Hashemi SJ, Hajiani E, Sardabi EH. A Placebo-Controlled Trial of Silymarin in Patients with Nonalcoholic Fatty Liver Disease. Hepat Mon 2009; 9: Loguercio C, Andreone P, Brisc C, Brisc MC, Bugianesi E, Chiaramonte M, Cursaro C, Danila M, de Sio I, Floreani A, Freni MA, Grieco A, Groppo M, Lazzari R, Lobello S, Lorefice E, Margotti M, Miele L, Milani S, Okolicsanyi L, Palasciano G, Portincasa P, Saltarelli P, Smedile A, Somalvico F, Spadaro A, Sporea I, Sorrentino P, Vecchione R, Tuccillo C, Del Vecchio Blanco C, Federico A. Silybin combined with phosphatidylcholine and vitamin E in patients with nonalcoholic fatty liver disease: a randomized controlled trial. Free Radic Biol Med 2012; 52: [PMID: DOI: /j.freeradbiomed ] 84 Kelishadi R, Farajian S, Mirlohi M. Probiotics as a novel treatment for non-alcoholic Fatty liver disease; a systematic review on the current evidences. Hepat Mon 2013; 13: e7233 [PMID: DOI: /hepatmon.7233] 85 Liu Y, Zhang L, Song H, Ji G. Update on berberine in nonalcoholic Fatty liver disease. Evid Based Complement Alternat Med 2013; 2013: [PMID: DOI: /2013/308134] 86 García-Ruiz C, Baulies A, Mari M, García-Rovés PM, Fernandez-Checa JC. Mitochondrial dysfunction in non-alcoholic fatty liver disease and insulin resistance: cause or consequence? Free Radic Res 2013; 47: [PMID: DOI: / ] 87 Serviddio G, Bellanti F, Vendemiale G, Altomare E. Mitochondrial dysfunction in nonalcoholic steatohepatitis. Expert Rev Gastroenterol Hepatol 2011; 5: [PMID: DOI: /egh.11.11] 88 Ascensão A, Martins MJ, Santos-Alves E, Gonçalves IO, Portincasa P, Oliveira PJ, Magalhães J. Modulation of hepatic redox status and mitochondrial metabolism by exercise: therapeutic strategy for liver diseases. Mitochondrion 2013; 13: [PMID: DOI: /j.mito ] 89 Mukhopadhyay P, Horváth B, Zsengellėr Z, Bátkai S, Cao Z, Kechrid M, Holovac E, Erdėlyi K, Tanchian G, Liaudet L, Stillman IE, Joseph J, Kalyanaraman B, Pacher P. Mitochondrial reactive oxygen species generation triggers inflammatory response and tissue injury associated with hepatic ischemia-reperfusion: therapeutic potential of mitochondrially targeted antioxidants. Free Radic Biol Med 2012; 53: [PMID: DOI: /j.freeradbiomed ] P- Reviewers: Abdel-Raheem IT, Grattagliano I, Nadir A, Skrypnyk IN, Schuchmann M, Trovato GM S- Editor: Qi Y L- Editor: A E- Editor: Ma S 8350 July 14, 2014 Volume 20 Issue 26

43 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. WJG 20 th Anniversary Special Issues (12): Nonalcoholic fatty liver disease Nonalcoholic fatty liver disease and polycystic ovary syndrome TOPIC HIGHLIGHT Evangeline Vassilatou Evangeline Vassilatou, Endocrine Unit, 2 nd Department of Internal Medicine - Propaedeutic, Attikon University Hospital, Athens, Greece Author contributions: Vassilatou E solely contributed to this paper. Correspondence to: Evangeline Vassilatou, MD, PhD, Endocrine Unit, 2 nd Department of Internal Medicine - Propaedeutic, Attikon University Hospital, 1 Rimini st., Athens, Greece. niadas@hol.gr Telephone: Fax: Received: October 28, 2013 Revised: February 17, 2014 Accepted: April 15, 2014 Published online: July 14, 2014 Abstract Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease in the Western world comprising a spectrum of liver damage from fatty liver infiltration to end-stage liver disease, in patients without significant alcohol consumption. Increased prevalence of NAFLD has been reported in patients with polycystic ovary syndrome (PCOS), one of the most common endocrinopathies in premenopausal women, which has been redefined as a reproductive and metabolic disorder after the recognition of the important role of insulin resistance in the pathophysiology of the syndrome. Obesity, in particular central adiposity and insulin resistance are considered as the main factors related to NAFLD in PCOS. Moreover, existing data support that androgen excess, which is the main feature of PCOS and is interrelated to insulin resistance, may be an additional contributing factor to the development of NAFLD. Although the natural history of NAFLD remains unclear and hepatic steatosis seems to be a relatively benign condition in most patients, limited data imply that advanced stage of liver disease is possibly more frequent in obese PCOS patients with NAFLD. PCOS patients, particularly obese patients with features of the metabolic syndrome, should be submitted to screening for NAFLD comprising assessment of serum aminotransferase levels and of hepatic steatosis by abdominal ultrasound. Lifestyle modifications including diet, weight loss and exercise are the most appropriate initial therapeutic interventions for PCOS patients with NAFLD. When pharmacologic therapy is considered, metformin may be used, although currently there is no medical therapy of proven benefit for NAFLD. Long-term follow up studies are needed to clarify clinical implications and guide appropriate diagnostic evaluation, follow-up protocol and optimal treatment for PCOS patients with NAFLD Baishideng Publishing Group Inc. All rights reserved. Key words: Nonalcoholic fatty liver disease; Polycystic ovary syndrome; Insulin resistance; Obesity; Hyperandrogenism; Premenopausal women Core tip: Nonalcoholic fatty liver disease (NAFLD) is frequent in patients with polycystic ovary syndrome (PCOS). Obesity and insulin resistance are considered as the main factors related to NAFLD in PCOS. Androgen excess may be an additional contributing factor to the development of NAFLD. Limited data imply that advanced stage of liver disease is possibly more frequent in obese PCOS patients with NAFLD. PCOS patients, particularly obese patients with the metabolic syndrome, should be screened for NAFLD. Long-term follow up studies are needed to clarify clinical implications, appropriate diagnostic evaluation and optimal treatment for PCOS patients with NAFLD. Vassilatou E. Nonalcoholic fatty liver disease and polycystic ovary syndrome. World J Gastroenterol 2014; 20(26): Available from: URL: v20/i26/8351.htm DOI: i July 14, 2014 Volume 20 Issue 26

44 Vassilatou E. NAFLD and PCOS INTRODUCTION Nonalcoholic fatty liver disease (NAFLD) is an increasingly recognised chronic disorder characterised by fat accumulation in the liver, histologically identical to alcoholic liver disease, in patients with no or minimal alcohol consumption. For the diagnosis, exclusion of nutritional disorders, drugs and diseases known to cause secondary fatty liver disease is a prerequisite [1]. There is a wide spectrum of liver damage ranging from simple steatosis to cirrhosis [2]. The clinical relevance of NAFLD is related to its high prevalence (20%-30%) [1,3] in the general population and its possible evolution to end-stage liver disease and rarely to hepatocellular carcinoma [4]. Given that NAFLD prevalence is markedly increased in obesity, in type 2 diabetes mellitus and in dyslipidemia [1] the role of insulin resistance in the pathogenesis of this entity has been studied and a strong association has been shown between the two entities [5,6]. Existing data support that NAFLD is the hepatic component of the metabolic syndrome [7]. Polycystic ovary syndrome (PCOS) is one of the most common endocrinopathies in premenopausal women, affecting 5%-18% of this population depending on the used diagnostic criteria [8,9] and is characterised by hyperandrogenism and ovulatory dysfunction. Other diseases causing the same symptoms have to be excluded before considering the diagnosis of PCOS. Insulin resistance has been shown to be an essential feature of the syndrome in the 1980s [10,11] long after the syndrome s first description, affecting both obese and lean patients [11]. Increased prevalence of impaired glucose tolerance and diabetes mellitus [12], abdominal adiposity [13] and dyslipidemia [14] has been shown in PCOS patients implicating that these patients are at increased risk for the metabolic syndrome [15]. During the last years there is increasing evidence of an association of nonalcoholic fatty liver disease and polycystic ovary syndrome. The pathophysiologic link and the clinical significance of this association remain to be determined, in order to clarify issues concerning evaluation and management of these patients. In this review a detailed Pubmed search of all available data concerning the association of nonalcoholic fatty liver disease and polycystic ovary syndrome was performed in order to summarize current knowledge on this topic. NONALCOHOLIC FATTY LIVER DISEASE AND INSULIN RESISTANCE Liver damage in the absence of significant alcohol consumption (> 20 g/d) has been reported since the 1950s [16], however it was mostly recognised after the description of histopathological findings characteristic of alcohol liver disease in patients with none or minimal alcohol consumption by Ludwig et al [17] in 1980, who introduced the term non-alcoholic steatohepatitis (NASH). NASH, which develops in a subset of patients with NAFLD, is an advanced stage of the disease characterised by severe steatohepatitis with lobular necroinflammation and variable degrees of fibrosis and can further progress to advanced fibrosis and cirrhosis in some cases [1,2]. Once cirrhosis is present, hepatocellular carcinoma may also develop [4]. NAFLD is considered as the most common cause of cryptogenic cirrhosis [18]. Simple steatosis is the early stage of NAFLD which does not progress to severe disease in the majority of patients and is considered as a relatively benign condition [1,2]. However, it is still not possible to predict who is at risk for advanced NAFLD. Although the prevalence of NAFLD remains poorly defined due to variation of the characteristics of the studied populations, of the evaluating methods and of the diagnostic criteria that are used (e.g., liver function tests, liver imaging studies, liver biopsy) it is estimated to affect 20%-30% of adults in the general population in developed countries [19-23]. Evaluation of fatty liver content by proton magnetic resonance spectroscopy ( 1 H-MRS), which can accurately measure hepatic triglyceride content, led to the estimation of an even higher percentage, 33.6%, in a large urban population in the United States [24]. Data showing an increased prevalence of NAFLD in obesity [22,24] reaching 90% in morbidly obese [25] and in diabetic patients up to 70% [26,27] drew attention to the possible role of insulin resistance and hyperinsulinemia in the pathophysiology of the disease. Insulin resistance assessed using the homeostasis model assessment (HOMA-IR) was demonstrated in patients with NAFLD independently of obesity and diabetes [5,28]. These findings were confirmed by studies in nonobese, nondiabetic patients with NAFLD, using the euglycemic insulin clamp technique, the gold standard method for the assessment of insulin sensitivity, which determined the sites of insulin resistance: hepatic and peripheral insulin resistance (skeletal muscle and adipose tissue) [6,7,29,30]. These data supported that insulin resistance is a primary defect in NAFLD, superimposed by obesityand diabetes-associated insulin resistance, in obese and/ or diabetic patients. Thus given that insulin resistance is the key component of the metabolic syndrome, it was suggested that NAFLD is the hepatic manifestation of the metabolic syndrome [7]. Insulin resistance in adipose tissue results in accelerated lipolysis, causing an increased flow of free fatty acids to the liver, thus favouring hepatic fat accumulation. The distribution of adipose tissue is also important since it has been shown that visceral adipose tissue is more insulin resistant than subcutaneous adipose tissue. Consequently visceral adipose tissue by increased portal free fatty acid flow to the liver seems to be an important regulator of fatty liver [31]. Insulin resistance and compensatory hyperinsulinemia are also related to increased de novo lipogenesis (synthesis of free fatty acids in the liver) which is another contributor to hepatic fat accumulation. Insulin has been shown to stimulate the expression of lipogenic enzymes through sterol regulatory element binding protein 1 (SREBP-1), even in the setting of insulin resistance [32]. Moreover 8352 July 14, 2014 Volume 20 Issue 26

45 Vassilatou E. NAFLD and PCOS hyperinsulinemia contributes to a decrease in lipid oxidation as increased insulin levels have been shown to inactivate the forkhead box transcription factor Foxa2, which regulates expression of genes encoding enzymes of fatty acid oxidation [33]. In normal conditions this transcription factor is active in the fasting state, when insulin levels are low and inactive in the fed state when insulin levels are increased [33]. The enlargement of adipose tissue that characterises obesity and in particular enlargement of visceral adipose tissue with associated chronic lowgrade inflammation and monocyte infiltration, leads to secretion of proinflammatory cytokines and modified release of adipose tissue hormones that modulate insulin sensitivity. Elevated levels of proinflammatory cytokines interleukin-6 and tumor necrosis factor-α, leptin resistance and decreased levels of adiponectin are considered as mediators of insulin resistance [34]. However it has been suggested as an alternative hypothesis that liver fat accumulation may develop independently of peripheral insulin resistance. Hepatic steatosis resulting in hepatic insulin resistance without changes in body weight and evidence of peripheral insulin resistance has been described in animal studies [35,36]. Data from human studies suggest that liver fat accumulation may contribute to insulin resistance and the metabolic syndrome [35,36]. The cause/effect relationship between hepatic steatosis and insulin resistance still remains unclear [37]. The association of NAFLD with insulin resistance and features of the metabolic syndrome led to the investigation of its putative role in the development and progression of cardiovascular disease. There is increasing evidence showing that NAFLD is a risk factor for cardiovascular disease. In addition existing data report that cardiovascular disease is the most common cause of death in NAFLD patients before advanced liver disease develops, as only patients with NASH have an increased liver-related mortality rate. However, additional research is needed to demonstrate that NAFLD should be considered as marker of cardiovascular disease [38]. POLYCYSTIC OVARY SYNDROME AND INSULIN RESISTANCE PCOS was first described as a reproductive disorder comprising menstrual irregularity, infertility, hirsutism and enlarged polycystic ovaries, by Stein and Leventhal [39], in In the 1960s the term polycystic ovary syndrome was introduced and it is still considered as the most appropriate. Hyperandrogenism clinically expressed as hirsutism, acne and/or androgenic alopecia and anovulation clinically expressed as oligomenorrhea or amenorrhea and infertility are the main characteristics of the syndrome. The syndrome was classified as normogonadotropic, normoestrogenic anovulation (type 2) by World Health Organisation. Hyperandrogenism and anovulation were accepted as the required criteria for the diagnosis, after exclusion of other disorders with these manifestations, according to the National Institutes of Health Consensus Development in 1990 (NIH criteria). In 2003, in a conference in Rotterdam a third diagnostic criterion was added - the polycystic morphology of the ovaries on ultrasound, and the presence of at least two out of three criteria is required for the diagnosis (Rotterdam criteria) [40]. This definition raised controversy, because women with anovulation and polycystic ovarian morphology but without hyperandrogenism were diagnosed with the syndrome. In 2006, a panel of the Androgen Excess Society recommended that hyperandrogenism should be one of the at least two out of three criteria (hyperandrogenism, anovulation and polycystic ovarian morphology) required for the diagnosis, considering that hyperandrogenism is an essential component of PCOS [41]. The presence of hyperandrogenism in women with syndromes of extreme insulin resistance [42] was the initiative for the investigation of the possible role of insulin resistance and compensatory hyperinsulinemia in the pathophysiology of PCOS. In 1980 Burghen et al [10] reported the presence of hyperinsulinemia in obese PCOS patients. Following studies confirmed this finding and in addition showed that it was independent of obesity [43-45]. Studies using the euglycemic insulin clamp technique demonstrated peripheral insulin resistance, independently of obesity [11,46], although obesity and central adiposity have an important independent adverse effect on insulin sensitivity [47]. Further studies showed that patients with PCOS have an increased prevalence of impaired glucose tolerance and diabetes mellitus [12,48], abdominal adiposity [13] and dyslipidemia [14] and an evident impact of racial and ethnic differences [49]. It was also shown that metabolic syndrome is frequent in these patients, especially in obese patients. Thus it was established that PCOS is a metabolic disorder as well. Metabolic dysfunction in PCOS patients leads to increased risk for cardiovascular disease with aging, particularly after menopause. Classic components of an adverse cardiovascular risk profile (central adiposity, impaired glucose tolerance and diabetes mellitus, dyslipidemia and hypertension) are frequently present in PCOS patients of all ages, occurring independently of obesity [50]. However, all these components are worsened when obesity is present [49,50]. In addition, increased levels of several biochemical inflammatory and thrombotic markers of cardiovascular risk are more prevalent in PCOS patients. A very recent small study compared biochemical inflammatory and thrombotic markers of cardiovascular risk between PCOS patients with and without NAFLD, without demonstrating differences between the two groups [51]. In that study, NAFLD was assessed by ultrasonography and in some patients was confirmed by biopsy [51]. In a recent meta-analysis, significantly elevated serum levels of C-reactive protein, homocysteine, plasminogen activator inhibitor-1 (PAI-1) antigen and PAI-1 activity, advanced glycation end products, lipoprotein A, asymmetric dimethylarginine and vascular endothelial growth factor were demonstrated in PCOS patients compared to controls [52]. Moreover, PCOS patients have earlier subclinical 8353 July 14, 2014 Volume 20 Issue 26

46 Vassilatou E. NAFLD and PCOS cardiovascular disease, assessed by various methods such as coronary artery calcification, carotid intima-media thickness, pulse wave velocity, flow-mediated dilation of the branchial artery compared to controls [53-55]. However, despite the demonstrated increased cardiovascular risk markers and early subclinical atherosclerosis in PCOS patients, data from observational population studies showing an increase in cardiovascular events are controversial [56]. A 31 year follow-up study of 786 PCOS patients diagnosed by ovarian wedge resection histology, showed no increased risk of death from coronary heart disease [57]. This finding was confirmed in a subsequent retrospective study of 319 women with PCOS, although increased nonfatal cerebrovascular events were reported [58]. Similarly, there was no evidence for increased cardiovascular events in postmenopausal PCOS patients vs postmenopausal controls in the first long-term prospective followup study [59]. Conversely, the Nurses Health Study with a participation of female nurses showed that women with a history of menstrual irregularity (as a surrogate marker for PCOS) had an increased risk of both non-fatal and fatal coronary heart disease compared to women reporting normal menses [60]. Two more studies showed that postmenopausal women with existent hyperandrogenemia and premenopausal menstrual irregularity (considered features of PCOS) had a larger number of cardiovascular events than postmenopausal controls [61,62]. Thus, large prospective long-term follow-up studies, adequately powered [56], are needed. Nevertheless, the androgen excess and polycystic ovary syndrome (AE-PCOS) Society recommends CVD risk assessment in PCOS patients at any age and appropriate interventions when needed [63]. Insulin resistance in PCOS is due to a post-receptor defect in insulin signal transduction which is present mainly in skeletal muscle and adipose tissue and concerns insulin action on glucose and lipid metabolism [49]. In the setting of this selective insulin resistance, other tissues which express insulin receptors like the ovaries are insulin sensitive and are exposed to increased circulating insulin levels (hyperinsulinemia). It has been shown that in normal women insulin acts as a co-gonadotropin to increase LH-induced androgen synthesis in theca cells and FSHinduced estrogen production in granulosa cells. This insulin action on steroidogenesis is preserved in PCOS and is enhanced due to hyperinsulinemia. Moreover, there is evidence that theca cells from polycystic ovaries are more responsive to androgen synthesis stimulation by insulin [64]. In addition, hyperinsulinemia decreases hepatic production of sex-hormone binding globulin (SHBG) which is the main binding protein for testosterone and prolongs its metabolic clearance, resulting in increased testosterone bioavailability. Thus insulin resistance, a prevalent finding in PCOS patients, is an important contributing factor to the ovarian androgen excess that characterizes PCOS. Androgen excess in turn may contribute to insulin resistance by modulating insulin action in muscle and adipose tissue, by increasing visceral adiposity and by reducing the secretion of adiponectin, the main insulin-sensitizing hormone of adipose tissue [49]. DIAGNOSIS AND PREVALENCE OF NAFLD IN PATIENTS WITH PCOS Most patients with NAFLD are asymptomatic at diagnosis and symptoms, when present, are not specific, such as fatigue, malaise and right upper quadrant discomfort. Hepatomegaly may be the only physical finding, while signs of chronic liver disease are rare [1,2]. Thus, diagnosis is based on laboratory evaluation of liver function and/or liver imaging studies after the exclusion of excess alcohol consumption and secondary causes of fatty liver disease (drugs, toxins, viral infections, bariatric surgery, nutritional and metabolic factors, autoimmune liver disease, genetic causes etc.) [1-3]. Liver biopsy is the gold standard for diagnosis and staging of the disease [1,2], however, being an invasive method, is not routinely performed. NAFLD was first diagnosed in a PCOS patient in 2005, as reported by Brown et al [65]. A 24-year-old woman with PCOS, obese, nondiabetic, with no history of alcohol consumption and no known cause of liver disease, underwent a liver biopsy because of elevated aminotransferase levels, which showed severe steatohepatitis [nonalcoholic steatohepatitis (NASH)]. It was suggested that NALFD might occur in some patients with PCOS given that insulin resistance is a common feature in both NAFLD and PCOS and both disorders are linked with metabolic syndrome. Thus a concern was raised for the frequency of this hepatic disease in these patients and the importance of screening them with liver function tests. Laboratory evaluation of NAFLD in patients with PCOS Elevated serum aminotransferase levels are the most common and often the only laboratory abnormality in patients with NAFLD, while elevated serum alkaline phosphatase and γ-glutalyltranferase are detected less frequently [1]. However, elevations of aminotransferase levels can be used only as a crude estimate of the presence of NAFLD given that the majority of patients with fatty liver do not have laboratory abnormalities [1,66]. In the Third National Health and Nutrition Examination Survey, 2.8% of participants had an abnormal aminotransferase level with no identifiable cause of liver disease, attributable to NAFLD [3]. The first study examining the presence of elevated aminotransferase levels in PCOS patients as a surrogate marker of NAFLD, reported elevated alanine (ALT) and aspartate (AST) aminotransferase levels in 30% and 12%, respectively, of 70 patients, evaluated in an infertility clinic [67]. The used cut-off levels for elevated ALT and AST levels were > 35 U/L and > 40 U/L, respectively. In this retrospective study, patients were of several ethnicities (Hispanic 63%, White 17%, Black 10%, Asian 10%), most of them (74%) obese and there were no controls [67]. Another retrospective study with no controls too, demonstrated elevated ALT and/or AST levels in 15% of 8354 July 14, 2014 Volume 20 Issue 26

47 Vassilatou E. NAFLD and PCOS Table 1 Studies investigating the presence of nonalcoholic fatty liver disease in polycystic ovary syndrome patients by biochemical and/or ultrasound evaluation Ref. Patients (n ) Obese patients PCOS diagnostic criteria NAFLD laboratory diagnosis NAFLD ECHO diagnosis Schwimmer et al [67] 70 74% NIH 30% 1 - Setji et al [68] 200 ND NIH 15% 100% 2 Cerda et al [72] % Rotterdam 39% 41.5% Gambarin-Gelwan et al [83] 88 42% NIH 15% 3 55% Preiss et al [94] % Rotterdam 36% - Economou et al [74] 83 45% 4 NIH 12% - Barfield et al [69] % Rotterdam 15.4% - Markou et al [75] 17 0 Rotterdam 0% 0% Vassilatou et al [73] % AES 22.8% 36.8% Tan et al [79] % AES 28.7% - Chen et al [76] 279 ND Rotterdam 20.9% 61.4% 5 Lerchbaum et al [77] % NIH 19.2% - Gangale et al [71] 140 ND Rotterdam 57.8% 57.8% Ma et al [84] 117 ND Rotterdam ND 39.3% Zueff et al [85] % Rotterdam % 1 Elevated alanine aminotransferase (ALT) levels; 2 ECHO evaluation in a subgroup: 16/29 patients with elevated aminotransferase levels; 3 Laboratory evaluation in a subgroup: all patients with ECHO findings of hepatic steatosis; 4 Obese and overweight patients were reported in the same subgroup; 5 ECHO evaluation in patients with elevated ALT levels. ND: Not defined; NIH: National Institutes of Health; AES: Androgen Excess Society. 200 PCOS patients, evaluated in an academic endocrine clinic [68]. This cohort was also of mixed race (Caucasian 68%, Black 20%) and the used cut-off levels for elevated ALT and AST levels were > 60 U/L. Interestingly, 6 of these patients, years-old, underwent liver biopsy because of persistently elevated aminotransferase levels which documented the presence of NASH with fibrosis [68], an unexpected finding for this age group. Another retrospective study performed in two centers, evaluated 39 adolescent, obese PCOS patients and reported elevated ALT and/or AST levels in 15.4% of patients. Patients were of several ethnicities (Hispanic 61.5%, Caucasian 10.3%, Black 12.8%, Asian 15.4%) and no controls were included [69]. In an intervention study examining the effects of metformin on NAFLD in overweight/obese PCOS patients, elevated ALT levels using a cut-off > 19 U/L [70] were shown in 57.8% of 140 patients, at baseline assessment [71]. A prospective, case-control study from Chile showed a statistically significant difference in elevated ALT levels between 41 PCOS patients compared to 31 age- and body mass index (BMI)- matched healthy women (39% vs 3.1%, respectively), using a cut-off > 25 U/L, according to normal values for healthy Chilean women [72]. More than half of the patients were obese. In agreement, a prospective, case-control study from Greece showed a significant difference of ALT and/or AST levels between 57 PCOS patients compared to 60 age- and BMI-matched healthy women (22.8% vs 3.3%, respectively), using a cut-off > 40 U/L [73]. This study comprised lean (40.3%), overweight (22.9%) and obese (36.8%) patients and only one lean patient had abnormal tests. In another case-control study from Greece although no significant difference in elevated ALT levels was reported between 83 PCOS patients compared to 64 age- and BMI-matched healthy women (12% vs 4.6%, respectively), using a different cut-off (> 25 U/L), when data were analysed according to BMI subgroups, the difference in abnormal tests remained non-significant for the lean subgroup of patients and controls (4.5% vs 5.9%, respectively), but a significant difference was demonstrated for the overweight/obese subgroup (20.5% vs 3%, respectively) [74]. In accordance, a case-control study with 17 PCOS patients, all lean, years-old, and 17 age- and BMI-matched healthy women showed normal aminotransferase levels in all patients [75]. Two subsequent larger case-control studies also showed a significant difference in elevated ALT levels between patients and controls, one study from Taiwan with 279 PCOS patients and 279 age-matched healthy women [76] and one study from Austria with 611 PCOS patients and 139 BMI-matched control women [77]. Thus existing evidence clearly shows a significantly higher prevalence of elevated aminotransferase levels in PCOS patients and the reported variation (15% to 57.8%) is due to differences in laboratory diagnostic criteria (different cut-off values) and different characteristics of the studied cohorts (ethnicity, age, BMI and PCOS diagnostic criteria) (Table 1). The use of other laboratory markers for identifying subjects at risk for NAFLD has been also examined in PCOS patients. A case-control study showed that the caspase 3-cleaved fragment of cytokeratin 18 (CK18), an established serum marker for NASH reflecting an increased hepatic apoptosis [78], was significantly elevated, after correction for BMI, in 186 PCOS patients compared to 73 age-matched controls [79]. Moreover, 27.4% of patients had CK18 levels 395 U/L, indicating NASH, compared to 1.4% of controls [79]. Another case-control study examined the presence of hepatic steatosis by calculating the fatty liver index (FLI), an algorithm based on BMI, waist circumference, triglycerides and gamma-glutamyl transferase, considered a simple and accurate predictor of hepatic steatosis in the general population [80] and two fibrosis indices: AST-to-platelet ratio index and FIB July 14, 2014 Volume 20 Issue 26

48 Vassilatou E. NAFLD and PCOS index. Significantly higher FLI levels were detected in 611 PCOS patients than in 139 BMI-matched control women [77]. However, significantly increased prevalence of elevated FLI levels (> 60) were found only in obese patients compared to obese controls, whereas a similar prevalence of elevated FLI levels was noted in overweight patients and controls and no elevated FLI levels were shown in lean patients and controls. No elevated fibrosis indices were found in either patients or controls [77]. Imaging evaluation of NAFLD in patients with PCOS Imaging modalities are widely used for the detection of NAFLD including ultrasonography, computerised tomography (CT), magnetic resonance imaging (MRI) and 1 H MRS. Ultrasonography, CT and MRI are qualitative or semiquantitative methods, whereas 1 H MRS is a quantitative method as it can accurately measure hepatic triglyceride content (HTGC) [24]. However none of these imaging modalities can assess inflammation and hepatic fibrosis [66]. Thus, as aforementioned, liver biopsy is the gold standard for diagnosing and staging NAFLD and for monitoring the efficacy of therapeutic interventions. However, biopsy is an invasive method, associated with potential morbidity and mortality and is prone to sampling errors [66]. Because of these limitations regarding liver biopsy, several non-invasive methods have been proposed for assessing hepatic inflammation and fibrosis, but none has been proven adequate to substitute for liver biopsy. However, non-invasive methods may be useful for selecting patients for liver biopsy. Among such methods, semiquantitative ultrasonographic scores assessing the extent of hepatic steatosis [81], including the recently described ultrasonographic fatty liver indicator (US-FLI) [82], which have been shown to correlate with histological evaluation of NAFLD may help to identify patients with increased risk for steatohepatitis needing liver biopsy. Abdominal ultrasonography has been used extensively as a screening method for detecting fatty liver infiltration, since it has been shown to have an acceptable level of sensitivity for detecting fatty liver (sensitivity 80% in the presence of > 30% of fatty infiltration), a short examination time and a low cost [66]. Availability of 1 H MRS in clinical practice is limited due to high cost and long scan time, while the use of CT is limited due to the exposure of the patients to radiation. Two studies investigated the presence of hepatic steatosis in PCOS patients by abdominal ultrasonography, without including controls. The first study, retrospective, demonstrated hepatic steatosis in 55% of 88 PCOS patients and interestingly, more than one third of hepatic steatosis patients were lean [83]. It has to be noted that patients were of several ethnicities and one third of them were on oral contraceptives when evaluated. The second study reported hepatic steatosis in 39.3% of 117 PCOS Chinese patients [84]. A prospective case-control study showed a statistically significant difference of hepatic steatosis by abdominal ultrasonography between 41 PCOS patients compared to 31 age- and BMI- matched healthy women (41.5% vs 19.4%, respectively) [72]. Another prospective, case-control study, using the same imaging modality, reported significant difference of hepatic steatosis between 57 PCOS patients compared to 60 age- and BMI-matched healthy women (36.8% vs 20.0%, respectively) [73]. Most patients with hepatic steatosis were obese and only two were lean. In agreement, a small prospective case-control study with only lean PCOS patients and age- and BMI- matched healthy women showed the absence of hepatic steatosis by abdominal ultrasonography in all study participants and by CT evaluation in PCOS patients [75]. In a case control study with only obese PCOS patients and agematched controls, evaluated by ultrasonography, hepatic steatosis was detected in 73.3% of patients [85]. In contrast with the above data, a very recent study case-control study showed no differences in the frequency and severity of hepatic steatosis among 55 PCOS patients, 25 control women and 26 men, evaluated by ultrasonography [86]. CT evaluation of fatty liver was used in a very recent study with 30 overweight and obese adolescent girls with PCOS detecting fatty liver, as determined by a ratio of liver to spleen hounsfield attenuation units < 1, in 2 patients (6.7%) [87]. An intervention study examining the effects of omega-3 fatty acid supplementation on liver fat content in PCOS patients, assessed hepatic steatosis by 1 H MRS. Hepatic steatosis, defined as liver fat percentage greater than 5%, was detected in 12 out of 25 PCOS patients at baseline evaluation [88]. A case-control study showed a statistically significant difference of hepatic steatosis defined as liver fat percentage greater than 5.5% [24] assessed by 1 H MRS between 29 PCOS patients compared to 22 ageand BMI-matched healthy women (6.1% vs 1.9%, respectively) [89]. All these data provide evidence that NAFLD is more prevalent in PCOS patients including adolescent patients [69,87] (Table 1). In 2007, Carmina [90] proposed the need for liver evaluation in PCOS patients and in a prescient remark suggested that conversely women of reproductive age with NAFLD should be investigated for the presence of PCOS. Two years later, a small prospective study reported that 10 out of 14 premenopausal women with NAFLD (7 with biopsy proven NAFLD) were diagnosed with PCOS by the Rotterdam criteria and importantly all biopsied patients with PCOS (5 patients) had NASH, providing evidence of an increased prevalence of PCOS in premenopausal NAFLD patients [91]. Mechanisms linking NAFLD to PCOS Given that NAFLD is rather uncommon in premenopausal women [92] the observed increased prevalence of NAFLD in PCOS patients raises the question of the factor or factors that render women with PCOS more prone to the development of NAFLD July 14, 2014 Volume 20 Issue 26

49 Vassilatou E. NAFLD and PCOS Clinical data Clinical studies have shown a significant association of elevated ALT levels in PCOS patients, with age [76], obesity [67,74,76,77,93], waist circumference [67,77], serum triglycerides [67,76,77,93], HDL-cholesterol [68,76,77,93], LDLcholesterol [76,77], sex-hormone binding globulin (SHBG) levels [74] and degree of insulin resistance assessed by indices like quantitative insulin sensitivity index (QUICKI) and HOMA-IR [67,72,76,77] and by the euglycemic insulin clamp technique [93]. Increased values of FLI, an algorithm indicating hepatic steatosis, were associated with age, obesity, waist circumference, HDL- and LDL-cholesterol and degree of insulin resistance assessed by HOMA- IR [77]. Increased levels of the apoptotic serum marker CK18 were associated with obesity, HDL- and LDLcholesterol [79]. A significant association of hepatic steatosis in PCOS patients, detected by imaging modalities, was also shown with age [73,85,87], obesity [72,73,83-85,87-89], waist circumference [72,73,84,85], serum triglycerides [71,84,85,88], HDLcholesterol [73,83,84], LDL-cholesterol [87], SHBG levels [71,73,88] and degree of insulin resistance assessed by QUICKI and HOMA-IR [71-73,83-85,88,89] and by the euglycemic insulin clamp technique [87]. All these data support that obesity, in particular central adiposity and insulin resistance are the main factors related to NAFLD in PCOS. This is also supported by data showing that lifestyle modifications including diet, weight loss and exercise, either alone or in combination with metformin have beneficial effects in PCOS patients with NAFLD [65,68,71,94]. Isolated features of the metabolic syndrome are also related to the presence of NAFLD in PCOS, while a significant percentage of NAFLD patients with PCOS [73,77] including adolescent patients [69,87], reaching 100% in some cohorts [71], meet the criteria for the diagnosis of metabolic syndrome. However, a question that should be addressed is whether NAFLD is associated with PCOS as a consequence of the shared risk factors, or whether PCOS contributes to NAFLD independently of these factors. Two studies showed that PCOS diagnosis is significantly associated with NAFLD, after adjustment for age, obesity and waist circumference in the first [73] and after adjustment for age, obesity and dyslipidemia in the second study [76]. Since PCOS is a predominantly hyperandrogenic syndrome, investigation of the role of androgens as a putative contributing factor to the development of NAFLD in these patients may clarify this issue. Some of the studies that used laboratory evaluation for the diagnosis of NAFLD in PCOS patients have shown a significant association of NAFLD with androgens. One study showed a significant association of elevated ALT levels in 70 PCOS patients with the presence of hirsutism, the main clinical expression of hyperandrogenism, however serum testosterone levels were not measured [67]. A case-control study reported a positive correlation of ALT levels with total testosterone levels and free-androgen index (FAI) values only in overweight/obese PCOS patients and controls [74]. Similarly, a case-control study with the largest, up to now, number of patients demonstrated that PCOS patients with elevated FLI levels (> 60) (detected only in obese patients) indicating the presence of NAFLD, had higher free testosterone levels than PCOS patients with FLI levels < 60 [77]. In addition another large case-control study showed that FAI values and total testosterone levels were positively associated with elevated ALT levels with a cut-off > 33 U/L, and this association remained after adjustment for the possible confounding effects of obesity, dyslipidemia and insulin resistance [76]. Studies that used imaging modalities for the diagnosis of NAFLD in PCOS patients have also demonstrated a significant association of NAFLD with androgens. An intervention study examining the effects of metformin on NAFLD in overweight/obese PCOS patients showed that PCOS patients with NAFLD detected by abdominal ultrasonography had significantly higher FAI values compared to PCOS patients without NAFLD, at baseline evaluation [71]. A prospective, case-control study showed that NAFLD in PCOS patients and controls, detected by abdominal ultrasonography, was significantly related to increased FAI values [73]. Similarly another case-control study with only obese PCOS patients and age-matched controls showed that NAFLD detected by abdominal ultrasonography was positively correlated with FAI values and negatively correlated with SHBG levels [85]. Moreover, a case-control study demonstrated that hyperandrogenic (defined with a FAI 7) PCOS patients had significantly higher liver fat measured by 1 H MRS compared to normoandrogenic PCOS patients, which remained higher after adjustment for total adipose tissue and visceral adipose tissue volumes and insulin resistance [89]. Similarly, a very recent study which evaluated liver fat by CT in overweight/ obese adolescent girls with PCOS demonstrated that age and total testosterone levels were independent contributors to fatty liver [87]. In contrast, a small, retrospective study did not find any difference in FAI values and DHEA-S levels between PCOS patients with and without NAFLD [95]. Similarly an intervention study examining the effects of omega-3 fatty acid supplementation on liver fat content in PCOS patients, did not report differences in FAI values and total testosterone levels between PCOS patients with and without NAFLD, evaluated by 1 H MRS, at baseline evaluation [88]. A very recent study showed that levels of the serum apoptotic marker M30 [caspase 3-cleaved fragment of cytokeratin 18 (CK18)] were significantly elevated in 12 PCOS patients with biopsy-proven non-nash (only steatosis) NAFLD, compared to 12 BMI-matched patients without PCOS and with biopsy-proven non-nash NAFLD [96]. This finding, indicating a more intense proapoptotic environment in PCOS patients with NAFLD, seems to be an early feature of NAFLD since the presence of NASH was excluded by biopsy in these patients and may be attributed to hyperandrogenism. Another finding of the study was the altered expression of two genes in the adipose tissue of NAFLD patients with PCOS compared to NAFLD patients without PCOS: a 8357 July 14, 2014 Volume 20 Issue 26

50 Vassilatou E. NAFLD and PCOS Obesity Insulin resistance/ hyperinsulinemia Central adiposity PCOS NAFLD Hyperandrogenism Figure 1 Mechanisms linking nonalcoholic fatty liver disease to polycystic ovary syndrome. Obesity and insulin resistance, features highly prevalent in polycystic ovary syndrome (PCOS) patients and hyperandrogenism, a predominant characteristic of PCOS, as contributing factors to the development of nonalcoholic fatty liver disease (NAFLD). Hyperandrogenism may exert direct effects on the liver and indirect effects by modulating insulin sensitivity and favoring visceral adiposity. Insulin resistance/hyperinsulinemia contributes to hyperandrogenism by affecting the production, the clearance and bioavailability of ovarian androgens. decrease in LDL receptor mrna expression, which may be also attributed to hyperandrogenism, and an increase in protein ninein (NIN) mrna expression [96]. All things considered, there is increasing evidence that hyperandrogenism is related to NAFLD in PCOS. Androgen excess may contribute to NAFLD in PCOS patients by direct effects on the liver, by indirect effects through modulations of insulin sensitivity and secretion, as aforementioned or by increasing visceral adiposity and by combination of these actions (Figure 1). Insulin resistance in turn is an important contributing factor to the ovarian androgen excess by affecting the production, the clearance and bioavailability of ovarian androgens, as already mentioned (Figure 1). Further studies are needed to assess the role of this vicious cycle of hyperandrogenism and insulin resistance in the development of NAFLD. However it can be suggested that hyperandrogenism should be considered as an additional link in synergy with obesity and insulin resistance for the development of NAFLD in PCOS. Putative genetic links Genetic susceptibility to PCOS has been shown by family studies and twin records estimating a heritability of approximately 70% [97]. In addition increased testosterone secretion from theca cells and defects in insulin action in skin fibroblasts from PCOS patients, persist in cultured cells over many passages suggesting they are genetically determined [49,97]. Most of the existing genetic studies on PCOS used a candidate gene approach based on hypotheses concerning the pathogenesis of the syndrome, which is complex and incompletely understood. Thus, genes related to components of PCOS pathogenesis such as genes influencing obesity and insulin resistance, β-cell dysfunction, steroid production and metabolism, androgen receptor and X-inactivation and ovarian folliculogenesis have been studied as candidate genes [97]. Several PCOS genetic susceptibility loci have been identified so far and some may be also implicated in the pathogenesis of NAFLD [98]. However, many of candidate gene studies have been limited by small sample size and small number of investigated gene variants [49,97]. It has to be mentioned that few findings have been replicated in separate cohorts. Additional limitations of these studies are the use of different diagnostic criteria for PCOS and the fact that only premenopausal women can be phenotyped for PCOS [97]. Genome-wide association studies (GWAS) have been used since 2005 to localize susceptibility genes for complex disorders in a hypothesis-free concept. The understanding of the pathogenesis of a disorder is not a prerequisite for GWAS and study results may reveal unknown or unexpected pathogenetic pathways. The first GWAS in PCOS was conducted in Chinese PCOS patients diagnosed by the Rotterdam criteria, and identified susceptibility loci for PCOS on chromosome 2p16.3, 2p21 and 9q33.3 [99]. Subsequent studies in European PCOS cohorts have replicated some of the Chinese PCOS GWAS signals [100,101]. Interestingly, the finding that the same susceptibility loci contribute to disease risk in Chinese and European PCOS cohorts suggests that PCOS is an ancient trait present in ancestral populations [49]. The role of genetic factors in the development and progression of NAFLD has been suggested by family and twin studies and inter-ethnic variation studies [102]. Most of the studies investigating the genetic basis of NAFLD used the hypothesis driven candidate gene approach. Thus, numerous genes involved in lipid metabolism, insulin signaling, inflammation, fibrotic mediators and oxidative stress have been studied and multiple genetic susceptibility loci have been identified [102]. However, many of these studies are underpowered resulting in inconsistencies and their findings were not replicated in larger cohorts [102]. The first GWAS in NAFLD identified 8358 July 14, 2014 Volume 20 Issue 26

51 Vassilatou E. NAFLD and PCOS a missense mutation (I148M) in patatin-like phospholipase domain-containing (PNPLA) 3 gene (adiponutrin gene) which was strongly associated with increased hepatic fat, independently of visceral adiposity and insulin resistance [103]. Subsequent studies conducted in different ethnicities confirmed that the I148M variant of PNPLA3 gene is not only a major determinant of liver fat content, but a predisposing factor to the full spectrum of liver damage in NAFLD - steatohepatitis and progressive fibrosis - as well [104]. Moreover, it was shown that this variant is a major determinant of liver disease progression in other liver diseases such as alcoholic liver disease and chronic hepatitis C [104]. Future genetic studies are expected to elucidate the genetics of NAFLD and PCOS and the possible genetic links between the two entities. Implications and management Although it is debated whether simple hepatic steatosis truly represents a disease, it is known that a yet-to-be determined percentage of patients will develop NASH, which may in turn putatively progress to end-stage liver disease. The natural history of NAFLD remains poorly understood, mainly due to the lack of prospective longterm follow-up studies. Focusing on NAFLD patients with PCOS there are scarce data of patients with biopsy proven NASH [51,65,68,91,105]. However the fact that 6 obese, young PCOS patients in a cohort of 200 PCOS patients, had biopsy documented NASH with fibrosis [68] raises concern for a possibly more frequent advanced stage of liver disease in PCOS patients. Benign hepatocellular tumors in obese patients with nonalcoholic steatohepatitis have been reported. Recently, a 50-year-old, obese PCOS patient with metabolic syndrome and focal liver lesions on imaging evaluation was diagnosed with inflammatory hepatocellular adenomatosis and biopsy proven NASH [106]. Early detection of NAFLD in PCOS patients is important because appropriate intervention at the stage of simple steatosis or steatohepatitis may decrease or even eliminate the possibility of disease progression. In this context, PCOS patients should have a liver evaluation comprising assessment of aminotransferase levels and of hepatic steatosis by ultrasound, especially those with metabolic syndrome. Lifestyle modifications including diet, weight loss and exercise, either alone or in combination with pharmacologic therapy have been shown to have beneficial effects in patients with simple steatosis or NASH. However there are no long-term data confirming a better prognosis [1]. Therapeutic interventions primarily target the risk factors for NAFLD - obesity and adverse metabolic profile (insulin resistance, dyslipidemia, hyperglycemia). Thus diet, weight loss and exercise are the cornerstone of treatment and may be combined with insulin-sensitizers (metformin or pioglitazone), hypolipidemic drugs and hepatoprotective agents like antioxidants and anti-inflammatory agents. Few interventional data exist for NAFLD in PCOS patients. There is one case with response to diet, moderate weight loss and exercise with an improvement in her histology findings at a post-treatment liver biopsy [65]. Four out of the 6 aforementioned PCOS patients with biopsy documented NASH, demonstrated normalization of aminotransferase levels in response to diet and exercise alone or in combination with metformin [68]. In addition, 9 PCOS patients with elevated aminotransferases from the same cohort responded to interventions with normalization of aminotransferases - 6 patients underwent diet, weight loss and exercise, 2 were treated with metformin and 1 underwent bariatric surgery [68]. It has to be mentioned that 7 PCOS patients with elevated aminotransferases demonstrated a spontaneous normalization at repeated evaluation [68]. A randomized, crossover study examined the effects of omega-3 fatty acid supplementation on liver fat content in obese PCOS patients, assessed by 1 H MRS and showed a reduction in fat content mainly in the NAFLD subgroup of patients, after a 8 wk treatment [88]. This beneficial effect was attributed to omega-3 fatty acids modulation of intrahepatic lipid metabolism, probably through activation of peroxisome proliferatoractivated receptor-α [88]. The effect of metformin on NAFLD in obese PCOS patients has been assessed in two prospective studies, after 8 and 12 mo of treatment respectively [71,94]. In both studies a significant reduction of liver enzyme levels was shown in patients who completed the treatment. In addition a reduction of insulin resistance assessed by HOMA-IR and in FAI values together with an increase of SHBG and HDL levels were observed in the second study [71]. Metformin, a drug that improves the sensitivity of peripheral tissues to insulin, resulting in a decrease of circulating insulin levels, has been used in PCOS since Many studies have shown that metformin not only has a beneficial effect on glucose metabolism but also has an effect on lipid metabolism and on androgen excess by increasing SHBG levels and decreasing androgen levels [107]. These actions result in amelioration of the adverse metabolic profile and in restoration of ovulatory menstrual cycles in treated PCOS patients. Similar actions have been reported in PCOS patients with thiazolidinediones, however pioglitazone is not recommended for use in nondiabetic PCOS because of concerns about cardiovascular safety [107]. Thus metformin is recommended in combination with lifestyle modifications in overweight and obese women with PCOS who have impaired glucose tolerance and other features of metabolic syndrome [107]. In that context, metformin might be the drug of choice for treating PCOS patients with NAFLD when pharmacologic therapy is considered. However randomised control studies are needed, as there is still no proven effective medication for NAFLD. CONCLUSION NAFLD is more prevalent in PCOS, which has been 8359 July 14, 2014 Volume 20 Issue 26

52 Vassilatou E. NAFLD and PCOS redefined as a reproductive and metabolic disorder after the recognition of the important role of insulin resistance in the pathophysiology of the syndrome. Obesity and insulin resistance are the main factors related to NAFLD in PCOS. Androgen excess which is the main feature of PCOS and is interrelated to insulin resistance may be an additional contributing factor to the development of NAFLD in PCOS. Limited data imply that advanced stage of liver disease is possibly more frequent in obese PCOS patients with NAFLD. PCOS patients, in particular obese patients with features of the metabolic syndrome, should be submitted to liver evaluation comprising assessment of aminotransferase levels and abdominal ultrasound. 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54 Vassilatou E. NAFLD and PCOS 58 Wild S, Pierpoint T, McKeigue P, Jacobs H. Cardiovascular disease in women with polycystic ovary syndrome at longterm follow-up: a retrospective cohort study. Clin Endocrinol (Oxf) 2000; 52: [PMID: ] 59 Schmidt J, Landin-Wilhelmsen K, Brännström M, Dahlgren E. Cardiovascular disease and risk factors in PCOS women of postmenopausal age: a 21-year controlled follow-up study. J Clin Endocrinol Metab 2011; 96: [PMID: DOI: /jc ] 60 Solomon CG, Hu FB, Dunaif A, Rich-Edwards JE, Stampfer MJ, Willett WC, Speizer FE, Manson JE. Menstrual cycle irregularity and risk for future cardiovascular disease. J Clin Endocrinol Metab 2002; 87: [PMID: ] 61 Shaw LJ, Bairey Merz CN, Azziz R, Stanczyk FZ, Sopko G, Braunstein GD, Kelsey SF, Kip KE, Cooper-Dehoff RM, Johnson BD, Vaccarino V, Reis SE, Bittner V, Hodgson TK, Rogers W, Pepine CJ. Postmenopausal women with a history of irregular menses and elevated androgen measurements at high risk for worsening cardiovascular event-free survival: results from the National Institutes of Health--National Heart, Lung, and Blood Institute sponsored Women s Ischemia Syndrome Evaluation. J Clin Endocrinol Metab 2008; 93: [PMID: DOI: /jc ] 62 Krentz AJ, von Mühlen D, Barrett-Connor E. Searching for polycystic ovary syndrome in postmenopausal women: evidence of a dose-effect association with prevalent cardiovascular disease. Menopause 2007; 14: [PMID: ] 63 Wild RA, Carmina E, Diamanti-Kandarakis E, Dokras A, Escobar-Morreale HF, Futterweit W, Lobo R, Norman RJ, Talbott E, Dumesic DA. Assessment of cardiovascular risk and prevention of cardiovascular disease in women with the polycystic ovary syndrome: a consensus statement by the Androgen Excess and Polycystic Ovary Syndrome (AE- PCOS) Society. 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Clin Endocrinol (Oxf) 2011; 75: [PMID: DOI: /j x] 72 Cerda C, Pérez-Ayuso RM, Riquelme A, Soza A, Villaseca P, Sir-Petermann T, Espinoza M, Pizarro M, Solis N, Miquel JF, Arrese M. Nonalcoholic fatty liver disease in women with polycystic ovary syndrome. J Hepatol 2007; 47: [PMID: DOI: /j.jhep ] 73 Vassilatou E, Lafoyianni S, Vryonidou A, Ioannidis D, Kosma L, Katsoulis K, Papavassiliou E, Tzavara I. Increased androgen bioavailability is associated with non-alcoholic fatty liver disease in women with polycystic ovary syndrome. Hum Reprod 2010; 25: [PMID: DOI: /humrep/dep380] 74 Economou F, Xyrafis X, Livadas S, Androulakis II, Argyrakopoulou G, Christakou CD, Kandaraki E, Palioura E, Diamanti-Kandarakis E. In overweight/obese but not in normal-weight women, polycystic ovary syndrome is associated with elevated liver enzymes compared to controls. Hormones (Athens) 2009; 8: [PMID: ] 75 Markou A, Androulakis II, Mourmouris C, Tsikkini A, Samara C, Sougioultzis S, Piaditis G, Kaltsas G. Hepatic steatosis in young lean insulin resistant women with polycystic ovary syndrome. Fertil Steril 2010; 93: [PMID: DOI: /j.fertnstert ] 76 Chen MJ, Chiu HM, Chen CL, Yang WS, Yang YS, Ho HN. Hyperandrogenemia is independently associated with elevated alanine aminotransferase activity in young women with polycystic ovary syndrome. J Clin Endocrinol Metab 2010; 95: [PMID: DOI: /jc ] 77 Lerchbaum E, Gruber HJ, Schwetz V, Giuliani A, Möller R, Pieber TR, Obermayer-Pietsch B. Fatty liver index in polycystic ovary syndrome. Eur J Endocrinol 2011; 165: [PMID: DOI: /EJE ] 78 Feldstein AE, Wieckowska A, Lopez AR, Liu YC, Zein NN, McCullough AJ. Cytokeratin-18 fragment levels as noninvasive biomarkers for nonalcoholic steatohepatitis: a multicenter validation study. Hepatology 2009; 50: [PMID: DOI: /hep.23050] 79 Tan S, Bechmann LP, Benson S, Dietz T, Eichner S, Hahn S, Janssen OE, Lahner H, Gerken G, Mann K, Canbay A. Apoptotic markers indicate nonalcoholic steatohepatitis in polycystic ovary syndrome. J Clin Endocrinol Metab 2010; 95: [PMID: DOI: /jc ] 80 Bedogni G, Bellentani S, Miglioli L, Masutti F, Passalacqua M, Castiglione A, Tiribelli C. The Fatty Liver Index: a simple and accurate predictor of hepatic steatosis in the general population. BMC Gastroenterol 2006; 6: 33 [PMID: DOI: / X-6-33] 81 Hamaguchi M, Kojima T, Itoh Y, Harano Y, Fujii K, Nakajima T, Kato T, Takeda N, Okuda J, Ida K, Kawahito Y, Yoshikawa T, Okanoue T. The severity of ultrasonographic findings in nonalcoholic fatty liver disease reflects the metabolic syndrome and visceral fat accumulation. Am J Gastroenterol 2007; 102: [PMID: ] 82 Ballestri S, Lonardo A, Romagnoli D, Carulli L, Losi L, Day CP, Loria P. Ultrasonographic fatty liver indicator, a novel score which rules out NASH and is correlated with metabolic parameters in NAFLD. Liver Int 2012; 32: [PMID: DOI: /j x] 83 Gambarin-Gelwan M, Kinkhabwala SV, Schiano TD, Bodian C, Yeh HC, Futterweit W. Prevalence of nonalcoholic fatty liver disease in women with polycystic ovary syndrome. Clin Gastroenterol Hepatol 2007; 5: [PMID: DOI: /j.cgh ] 84 Ma RC, Liu KH, Lam PM, Cheung LP, Tam WH, Ko GT, Chan MH, Ho CS, Lam CW, Chu WC, Tong PC, So WY, Chan JC, Chow CC. Sonographic measurement of mesenteric fat predicts presence of fatty liver among subjects with polycystic ovary syndrome. J Clin Endocrinol Metab 2011; 96: 8362 July 14, 2014 Volume 20 Issue 26

55 Vassilatou E. NAFLD and PCOS [PMID: DOI: /jc ] 85 Zueff LF, Martins WP, Vieira CS, Ferriani RA. Ultrasonographic and laboratory markers of metabolic and cardiovascular disease risk in obese women with polycystic ovary syndrome. Ultrasound Obstet Gynecol 2012; 39: [PMID: DOI: /uog.10084] 86 Borruel S, Fernández-Durán E, Alpañés M, Martí D, Alvarez-Blasco F, Luque-Ramírez M, Escobar-Morreale HF. Global adiposity and thickness of intraperitoneal and mesenteric adipose tissue depots are increased in women with polycystic ovary syndrome (PCOS). J Clin Endocrinol Metab 2013; 98: [PMID: DOI: / jc ] 87 Michaliszyn SF, Lee S, Tfayli H, Arslanian S. Polycystic ovary syndrome and nonalcoholic fatty liver in obese adolescents: association with metabolic risk profile. Fertil Steril 2013; 100: [PMID: DOI: /j.fertnst ert ] 88 Cussons AJ, Watts GF, Mori TA, Stuckey BG. Omega-3 fatty acid supplementation decreases liver fat content in polycystic ovary syndrome: a randomized controlled trial employing proton magnetic resonance spectroscopy. J Clin Endocrinol Metab 2009; 94: [PMID: DOI: /jc ] 89 Jones H, Sprung VS, Pugh CJ, Daousi C, Irwin A, Aziz N, Adams VL, Thomas EL, Bell JD, Kemp GJ, Cuthbertson DJ. Polycystic ovary syndrome with hyperandrogenism is characterized by an increased risk of hepatic steatosis compared to nonhyperandrogenic PCOS phenotypes and healthy controls, independent of obesity and insulin resistance. J Clin Endocrinol Metab 2012; 97: [PMID: DOI: /jc ] 90 Carmina E. Need for liver evaluation in polycystic ovary syndrome. J Hepatol 2007; 47: [PMID: DOI: /j.jhep ] 91 Brzozowska MM, Ostapowicz G, Weltman MD. An association between non-alcoholic fatty liver disease and polycystic ovarian syndrome. J Gastroenterol Hepatol 2009; 24: [PMID: DOI: /j x] 92 Hamaguchi M, Kojima T, Ohbora A, Takeda N, Fukui M, Kato T. Aging is a risk factor of nonalcoholic fatty liver disease in premenopausal women. World J Gastroenterol 2012; 18: [PMID: DOI: /wjg.v18.i3.237] 93 Targher G, Solagna E, Tosi F, Castello R, Spiazzi G, Zoppini G, Muggeo M, Day CP, Moghetti P. Abnormal serum alanine aminotransferase levels are associated with impaired insulin sensitivity in young women with polycystic ovary syndrome. J Endocrinol Invest 2009; 32: [PMID: DOI: /6375] 94 Preiss D, Sattar N, Harborne L, Norman J, Fleming R. The effects of 8 months of metformin on circulating GGT and ALT levels in obese women with polycystic ovarian syndrome. Int J Clin Pract 2008; 62: [PMID: DOI: /j x] 95 Kauffman RP, Baker TE, Baker V, Kauffman MM, Castracane VD. Endocrine factors associated with non-alcoholic fatty liver disease in women with polycystic ovary syndrome: do androgens play a role? Gynecol Endocrinol 2010; 26: [PMID: DOI: / ] 96 Baranova A, Tran TP, Afendy A, Wang L, Shamsaddini A, Mehta R, Chandhoke V, Birerdinc A, Younossi ZM. Molecular signature of adipose tissue in patients with both non-alcoholic fatty liver disease (NAFLD) and polycystic ovarian syndrome (PCOS). J Transl Med 2013; 11: 133 [PMID: DOI: / ] 97 Barber TM, Franks S. Genetics of polycystic ovary syndrome. Front Horm Res 2013; 40: [PMID: DOI: / ] 98 Baranova A, Tran TP, Birerdinc A, Younossi ZM. Systematic review: association of polycystic ovary syndrome with metabolic syndrome and non-alcoholic fatty liver disease. Aliment Pharmacol Ther 2011; 33: [PMID: DOI: /j x] 99 Chen ZJ, Zhao H, He L, Shi Y, Qin Y, Shi Y, Li Z, You L, Zhao J, Liu J, Liang X, Zhao X, Zhao J, Sun Y, Zhang B, Jiang H, Zhao D, Bian Y, Gao X, Geng L, Li Y, Zhu D, Sun X, Xu JE, Hao C, Ren CE, Zhang Y, Chen S, Zhang W, Yang A, Yan J, Li Y, Ma J, Zhao Y. Genome-wide association study identifies susceptibility loci for polycystic ovary syndrome on chromosome 2p16.3, 2p21 and 9q33.3. Nat Genet 2011; 43: [PMID: DOI: /ng.732] 100 Goodarzi MO, Jones MR, Li X, Chua AK, Garcia OA, Chen YD, Krauss RM, Rotter JI, Ankener W, Legro RS, Azziz R, Strauss JF, Dunaif A, Urbanek M. Replication of association of DENND1A and THADA variants with polycystic ovary syndrome in European cohorts. J Med Genet 2012; 49: [PMID: DOI: /jmedgenet ] 101 Welt CK, Styrkarsdottir U, Ehrmann DA, Thorleifsson G, Arason G, Gudmundsson JA, Ober C, Rosenfield RL, Saxena R, Thorsteinsdottir U, Crowley WF, Stefansson K. Variants in DENND1A are associated with polycystic ovary syndrome in women of European ancestry. J Clin Endocrinol Metab 2012; 97: E1342-E1347 [PMID: DOI: / jc ] 102 Day CP. Genetic and environmental susceptibility to nonalcoholic fatty liver disease. Dig Dis 2010; 28: [PMID: DOI: / ] 103 Romeo S, Kozlitina J, Xing C, Pertsemlidis A, Cox D, Pennacchio LA, Boerwinkle E, Cohen JC, Hobbs HH. Genetic variation in PNPLA3 confers susceptibility to nonalcoholic fatty liver disease. Nat Genet 2008; 40: [PMID: DOI: /ng.257] 104 Dongiovanni P, Donati B, Fares R, Lombardi R, Mancina RM, Romeo S, Valenti L. PNPLA3 I148M polymorphism and progressive liver disease. World J Gastroenterol 2013; 19: [PMID: ] 105 Hossain N, Stepanova M, Afendy A, Nader F, Younossi Y, Rafiq N, Goodman Z, Younossi ZM. Non-alcoholic steatohepatitis (NASH) in patients with polycystic ovarian syndrome (PCOS). Scand J Gastroenterol 2011; 46: [PMID: DOI: / ] 106 Nascimbeni F, Ballestri S, Di Tommaso L, Piccoli M, Lonardo A. Inflammatory hepatocellular adenomatosis, metabolic syndrome, polycystic ovary syndrome and non-alcoholic steatohepatitis: chance tetrad or association by necessity? Dig Liver Dis 2014; 46: [PMID: ] 107 Dunaif A. Drug insight: insulin-sensitizing drugs in the treatment of polycystic ovary syndrome--a reappraisal. Nat Clin Pract Endocrinol Metab 2008; 4: [PMID: DOI: /ncpendmet0787] P- Reviewers: Ballestri S, Hashemi M S- Editor: Zhai HH L- Editor: A E- Editor: Ma S 8363 July 14, 2014 Volume 20 Issue 26

56 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. WJG 20 th Anniversary Special Issues (12): Nonalcoholic fatty liver disease TOPIC HIGHLIGHT Experimental models of non-alcoholic fatty liver disease in rats Otto Kucera, Zuzana Cervinkova Otto Kucera, Zuzana Cervinkova, Department of Physiology, Charles University in Prague, Faculty of Medicine in Hradec Kralove, Hradec Kralove, Czech Republic Author contributions: Kucera O and Cervinkova Z solely contributed to this paper. Supported by Programme PRVOUK P37/02 Correspondence to: Otto Kucera, MD, PhD, Department of Physiology, Charles University in Prague, Faculty of Medicine in Hradec Kralove, Simkova 870, Hradec Kralove, Czech Republic. kucerao@lfhk.cuni.cz Telephone: Fax: Received: October 29, 2013 Revised: January 15, 2014 Accepted: February 17, 2014 Published online: July 14, 2014 Abstract Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the Western world, and it persists at a high prevalence. NAFLD is characterised by the accumulation of triglycerides in the liver and includes a spectrum of histopathological findings, ranging from simple fatty liver through non-alcoholic steatohepatitis (NASH) to fibrosis and ultimately cirrhosis, which may progress to hepatocellular carcinoma. The pathogenesis of NAFLD is closely related to the metabolic syndrome and insulin resistance. Understanding the pathophysiology and treatment of NAFLD in humans has currently been limited by the lack of satisfactory animal models. The ideal animal model for NAFLD should reflect all aspects of the intricate etiopathogenesis of human NAFLD and the typical histological findings of its different stages. Within the past several years, great emphasis has been placed on the development of an appropriate model for human NASH. This paper reviews the widely used experimental models of NAFLD in rats. We discuss nutritional, genetic and combined models of NAFLD and their pros and cons. The choice of a suitable animal model for this disease while respecting its limitations may help to improve the understanding of its complex pathogenesis and to discover appropriate therapeutic strategies. Considering the legislative, ethical, economical and health factors of NAFLD, animal models are essential tools for the research of this disease Baishideng Publishing Group Inc. All rights reserved. Key words: Animal model; High-fat diet; Methionineand choline-deficient diet; Non-alcoholic fatty liver disease; Non-alcoholic steatohepatitis; Otsuka-Long- Evans-Tokushima fatty rats; Zucker rats Core tip: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the Western world, but its pathogenesis is still not fully understood. This paper reviews the commonly used experimental models of NAFLD in rats. We discuss nutritional, genetic and combined models of NAFLD, and we highlight their pros and cons with special emphasis on nutritional models. To date, there is not an ideal model that reflects all aspects of the complex etiopathogenesis of human NAFLD and the typical histological features of its different stages. Kucera O, Cervinkova Z. Experimental models of non-alcoholic fatty liver disease in rats. World J Gastroenterol 2014; 20(26): Available from: URL: com/ /full/v20/i26/8364.htm DOI: org/ /wjg.v20.i INTRODUCTION Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the Western world, where its prevalence is estimated to be approximately 20%-30% of the adult population [1]. A steep increase in 8364 July 14, 2014 Volume 20 Issue 26

57 Kucera O et al. Experimental models of NAFLD in rats the prevalence of NAFLD correlates to increased obesity in the world. NAFLD is characterised by the accumulation of triglycerides in the liver and includes a spectrum of histopathological findings, including simple fatty liver, non-alcoholic steatohepatitis (NASH), fibrosis and ultimately cirrhosis, which may progress to hepatocellular carcinoma. The pathogenesis of NAFLD is closely related to metabolic syndrome and insulin resistance. NAFLD is thought to be the hepatic manifestation of metabolic syndrome [2]. Despite intensive research over the past decades, the pathogenesis of NAFLD is not completely understood, and treatment of the disease has not been fully defined. PATHOGENESIS NAFLD develops over years as a result of the accumulation of different risk factors involving varying degrees of genetic susceptibility. Nutritional habits, amount and composition of diet, physical inactivity, chronic stress and lifestyle are the major non-genetic factors determining the manifestation and severity of the disease. Similar to the pathogenesis of metabolic syndrome, of which NAFLD is a constituent [2], there is presumed to be a polygenic genetic background to this disease [3]. The pathogenesis of NAFLD in humans is very complex, and there are many players in the field, each of which may interact with one another. Moreover, some risk factors may be more or less pronounced and may arise or disappear over time. Currently, it is not fully understood why some patients with similar risk factors do not develop fatty accumulation, while others develop steatosis and steatohepatitis. The pathogenesis of NAFLD appears to be unique in each patient, but the risk factors are likely to be the same. The natural history of NAFLD has been not fully clarified and appears to be slow in most patients. The progress from simple steatosis to advanced stages of the disease often takes years. It is not clear whether all patients with simple steatosis are at risk of steatohepatitis or advanced fibrosis. In contrast to patients with simple steatosis, those with NASH have a higher risk of death from cardiovascular and liver-related diseases [4]. NAFLD with elevated liver enzymes is associated with a clinically significant risk of developing end-stage liver disease, and most NAFLD patients will develop diabetes or impaired glucose tolerance in the long term [4]. In the United States, an estimated one-third of the population has NAFLD, and approximately 2%-5% have NASH [1]. The accumulation of lipids in the liver results from an imbalance among hepatic lipid intake, synthesis, degradation and secretion [5]. Fatty acids for the synthesis of triglycerides are acquired from different sources, including de novo formation in the liver, from portal blood as free fatty acids (FFAs) and from circulating lipoproteins, mainly from chylomicrons. Insulin resistance plays a key role in the hepatic accumulation of fats [5]. Serum FFAs are elevated by accelerated lipolysis in the peripheral adipose tissue and visceral fat in the state of insulin resistance. Hyperglycaemia caused by peripheral insulin resistance also promotes the synthesis of fatty acids in the liver. The elevation of chylomicrons is typical after intake of food containing high amounts of fats. Peripheral insulin resistance is often accompanied by increased insulin levels, and hyperinsulinaemia is known to inhibit the formation and secretion of very low-density lipoproteins (VLDL) from the liver in NASH patients [6]. Another effect of hyperinsulinaemia and hyperglycaemia is the inhibition β-oxidation of fatty acids and the promotion of lipogenesis in the liver [7]. Insulin is the primary stimulator of hepatic lipogenesis through activation of the sterol regulatory element-binding protein-1c (SREBP-1c) transcription factor [8]. Insulin resistance is found in the majority of patients with primary NAFLD and is more severe in NASH [9]. Some patients with simple steatosis progress to NASH. According to the two-hit theory [10], liver steatosis sensitises hepatocytes to the second hits, leading to hepatocyte damage, inflammation and fibrosis. These second insults may be increased oxidative stress (induction of microsomal cytochrome P450) and lipid peroxidation, mitochondrial dysfunction, cytokine/adipokine imbalance [tumour necrosis factor α (TNF-α), interleukin 6 (IL-6), adiponectin, leptin, resistin, etc.], lipotoxicity of FFAs, hepatic accumulation of cholesterol, lipopolysaccharide derived from GUT and the activation of innate immunity [10,11]. Exceeding defence and self-reparative mechanisms of hepatocytes lead to cell death. The importance of particular factors and their interactions are still not fully understood. The main difference between simple steatosis and NASH is in the degrees of hepatocyte injury and apoptosis [12]. Increased apoptosis is thought by some authors to be the third hit leading to advanced fibrosis and cirrhosis [13]. Overall chronic liver injury with a subsequent chronic regenerative response in the terrain of NASH leads to the activation of hepatic stellate cells and a fibrogenic response. With regards to fibrogenesis in NAFLD, special emphasis should be placed on chronic inflammation, oxidative stress/lipid peroxidation, leptin, adiponectin, platelet-derived growth factor, transforming growth factor β1 (TGF-β1), renin-angiotensin system and hepatocyte death [11]. The possible progression of human NAFLD is briefly depicted in Figure 1. Recently, a more realistic concept of the progression to NASH was introduced, which takes into account cumulative small pathogenic hits [14]. HISTOLOGY NAFLD is histologically defined by the presence of liver steatosis exceeding 5% of hepatocytes, regardless of whether it is macrovesicular, mixed or microvesicular [15]. In adults, NAFLD is characterised predominantly by macrovesicular steatosis that is usually present in a zone 3 or panacinar distribution [16]. NASH is defined by the presence of hepatocellular ballooning (most frequently in zone 3), lobular or portal inflammation (lymphocytes, monocytes, eosinophils, and macrophages) and eventually fibrosis in the terrain of steatotic liver [17]. The presence 8365 July 14, 2014 Volume 20 Issue 26

58 Kucera O et al. Experimental models of NAFLD in rats Intact liver Genetic susceptibility, dietary factors, lifestyle, etc. Simple steatosis Oxidative stress, lipid peroxidation, mitochondrial dysfunction, cytokines, adipokines, etc. NASH Chronic hepatocyte injury, chronic inflammation, liver regeneration, TGF-β1, activation of stellate cells, etc. Advanced fibrosis Hepatocellular carcinoma Cirrhosis Figure 1 Schematic diagram of the possible progression of non-alcoholic fatty liver disease. TGF-β1: Transforming growth factor β1; NASH: Non-alcoholic steatohepatitis. of a characteristic ballooning injury is the key feature to the diagnosis of NASH. Hepatocyte apoptosis is often observed in NASH, and the severity of steatohepatitis correlates with the degree of apoptosis [18]. Examination of histological samples from liver biopsies remains the gold standard for accurately assessing the degree of steatosis, necroinflammatory changes and fibrosis, and these may distinguish NASH from simple steatosis [19]. ANIMAL MODELS OF NAFLD Understanding the pathophysiology and treatment of NAFLD in human beings has been limited by the lack of satisfactory animal models. The ideal animal model for NAFLD should reflect all aspects of the intricate etiopathogenesis of human NAFLD and the typical histological findings of its different stages. Until now, there was no such animal model. Thus, researchers have attempted to introduce a suitable model of NAFLD, imitating at least the most important pathogenic and histological features of NAFLD. Animal models should be reproducible, reliable, simple, affordable and technically available with minimal disadvantages. Mice and rats have mostly been used as experimental animals of NAFLD [20]. Most models have been genetic models, high-fat diets or choline-methionine-deficient diets. The development of hepatic steatosis is common, and some also develop histological features of steatohepatitis; only a minority progress to fibrosis [20]. In the past several years, great emphases have been placed on the development of an appropriate model for human NASH. Most models successfully simulate the histological patterns of the disease, but they often lack the important metabolic or genetic context of human NASH (i.e., obesity, insulin resistance, hyperglycaemia, hyperinsulinaemia, dyslipidaemia, cytokine/adipokine imbalance) [14]. It should also be mentioned that, according to the natural history and pathogenesis of human NAFLD, there cannot be an ideal model. The progress from simple steatosis to NASH likely lasts years, and only a minority of patients with simple fatty liver progress to steatohepatitis. Thus, the models of steatohepatitis do not reflect this aspect even if the shorter lifespan of rats is taken into account. From this point of view, models of chronic overnutrition with a spontaneous progression of steatosis to steatohepatitis may be more valid than other models [20]. Moreover, these models exhibit a more complex interplay between metabolic abnormalities and liver injury [20]. In contrast to humans, data obtained from animal experiments are quite homogenous due to different sampling, even in wild type strains. Animal studies are generally performed on well-defined populations (species, strain, sex, weight or age, laboratory conditions, type of diet, etc.). In contrast, there is considerable heterogeneity in the human population, even if the study is defined by sex, place, age, ethnicity, and weight. There remains high interindividual variability concerning genetic background, comorbidities, physical activity, medication, diet composition, life style, etc. Thus, the interpretability of the data to humans is limited. Moreover, rodents have unique characteristics that affect the development of NAFLD and metabolic syndrome, which further hinder simple interpretations of the data. Crucial findings in animal models must be verified by clinical research when possible. This review attempts to describe and briefly summarise the existing experimental models of NAFLD in rats. The paper is mainly focused on commonly used rat models of NAFLD in vivo. We discuss nutritional, genetic 8366 July 14, 2014 Volume 20 Issue 26

59 Kucera O et al. Experimental models of NAFLD in rats Table 1 Characteristics of the most frequently used models of non-alcoholic fatty liver disease in rats Model Obesity Insulin resistance Plasma cytokines/ adipokines Dyslipidaemia Elevation of transaminases Liver histology Steatosis Inflammation Hepatocyte ballooning Fibrosis Nutritional models High-fat diets 1 Yes [27,28,34,35] /no [29,36] / Yes [27-29,34,35] not describe [37,38] / not described [36-38] TNF-α [27,34] adiponectin [28,34,35] Yes [27,28] /no [36] / not described [29,34,35,37,38] Yes [27,28,34,38] / no [23,29,35,36] Yes [23,27-29,34-38] / / Yes [27-29,34,37,38] Yes [34,37] Yes [28,34] no [26] no [23,35,36] not described [23,27-29,35,36,38] / /no [23,35,36] / not described [27,37,38] Atherogenic diet [41-43] (10% w/w Yes Yes TNF-α Yes Yes Yes Yes Yes Yes lard, 2% w/w cholesterol) High-fructose/sucrose diet [45-47] No Yes IL-1β, IL-2, IL-6 Yes No Yes Yes [46] /No [45] Not described Yes [46] /not described [45] High-fructose, high-fat diet [48] Yes Yes leptin Yes Yes Yes Yes Not described Yes Cafeteria diet [49] Yes Yes Not described Not described Not described Yes Yes Not described Not described Methionine- and choline-deficient Weight loss! No IL-1β, IL-6, TNF-α triglycerides, Yes Yes Yes Not described Yes diet [52,54] cholesterol Choline-deficient diet [52-53] Yes Yes Not described Yes No or mild Yes Yes [52] Not described Mild after 90 d [53] Genetic models Zucker fatty rats [72-74,77,78] Yes Yes adiponectin, leptin, TNF-α, Yes Yes Yes No Not described No Zucker fatty rats + high-fat diet [78] Yes Yes TNF-α Not described Yes Yes Yes Yes Yes Otsuka Long-Evans Tokushima Yes Yes leptin, IL-6 Yes Yes (AST) Yes Yes Yes No fatty rats [98-100] adiponectin 1 Characteristics of high-fat models depend mainly on the rat strain, amount and composition of fat, duration of feeding, experimental design, etc. AST: Aspartate aminotransferase; IL: Interleukin; TNF: Tumour necrosis factor. and combined models of the disease. The basic characteristics of the most widely employed models described in the text are summarised in Table 1. We do not review pharmacologic or toxic models of liver steatosis (dexamethasone, tamoxifen, orotic acid-diet, etc.), which are distinct from the natural etiopathogenesis of primary human NAFLD. Suitable animal models may help us to understand the complex pathogenesis of NAFLD and to discover new non-pharmacological or drug interventions for the treatment of disease. It can also further reveal risk factors of the progression of disease and uncover therapeutic strategies against the progression of NAFLD. NUTRITIONAL MODELS Nutritional models of NAFLD can be classified according to the mechanism by which steatosis and other changes are induced [21]. The first group includes models with increased lipid import or synthesis in the liver (high-fat diets, high-fructose/sucrose diets, combined diets). The second group is defined by reduced lipid export or catabolism (methionine- and choline-deficient diet; choline-deficient, L-amino acid-defined diet). Because few NAFLD patients exhibit evident genetic defects, the use of dietary models of NAFLD is more relevant to human disease than genetic models [20]. Currently, the most common rat model of NAFLD uses high-fat diets. Additionally, methionine- and choline-deficient diets and high-fructose diets are widely employed. High-fat diets First, we should define what a high-fat diet (HFD) means for rats. Standard rat diets contain lower amounts of fat than the recommended human diet. In contrast to approximately 30% of total energy intake from fats in humans, the standard rat diets contain less than 10% of kcal fat, whereas high-fat diets and very high-fat diets contain 30%-50% and more than 50% of kcal fat, respectively [22]. Through literature review, we found that the use of high-fat diets is very popular, but there are many variations of high-fat diets with different compositions and experimental designs; thus, the results obtained from these studies are inconsistent. In some experiments, HFD induced hepatic steatosis of 8367 July 14, 2014 Volume 20 Issue 26

60 Kucera O et al. Experimental models of NAFLD in rats varying degrees and patterns [23-25], while in others, HFD did not lead to the development of fat accumulation in the liver [26]. Moreover, HFD led to histopathological hepatic changes similar to human NASH in several other experiments [27-29]. Studies also differ in regards to the development of insulin resistance, obesity and dyslipidaemia, and other known pathogenic factors of human NAFLD. These studies vary in the time of feeding; rat strain; sex and age of rats; the amount of energy derived from fats; the origin of fats; the ratio of saturated (SFA), monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acids; and the proportion of ω-3 and ω-6 PUFA. Regarding the composition of fats, fatty acids considerably influence the expression of many genes, particularly in the liver [30]. Diets also vary in the composition of nonfat nutrients (the percentage of proteins and saccharides; the ratio of mono-, oligo- and polysaccharides). Most diets are fed ad libitum, but in some papers, controlled feeding via intragastric cannula was used [27]. All laboratory rodent species are susceptible to the induction of metabolic alterations upon HFD feeding [31]. HFDs are not only used for the induction of fatty liver changes [32] but are also employed as models of metabolic syndrome, dyslipidaemia, obesity and insulin resistance [22]. Because insulin resistance is one of the most important pathogenic factors of NAFLD [2], feeding rats with a HFD is legitimate and more closely resembles the pathogenesis of human NAFLD than genetic models and nutrient-deficient diets. There are interstrain differences in the susceptibility of rats to HFD. Examination of 3 strains of male rats fed a HFD (71% of kcal fat) for 3 wk showed that all three strains developed steatosis affecting > 66% of liver cells, but the histological patterns were different [24]. Microvesicular, mixed, and macrovesicular steatosis were found in Lewis, Wistar, and Sprague-Dawley rats, respectively. Moreover, the highest degree of fibrosis, hepatocyte damage, and reduced blood flow velocity in central veins were observed in the Sprague-Dawley strain [24]. Another study compared Lewis and Sprague-Dawley rats of both genders fed a HFD for 3 wk. The livers of all experimental groups on a HFD demonstrated evidence of steatosis, but similarly to the previous article, Lewis rats developed microvesicular steatosis, whereas Sprague-Dawley rats presented macrovesicular steatosis accompanied by pronounced fibrosis [25]. This experiment also confirmed the higher susceptibility of male rats (of both strains) to the development of steatosis [25], which corresponds to observations in humans, where NAFLD is preferentially found in men or postmenopausal women [33]. Our previous observations showed that HFD (71% of kcal fat) feeding does not differ in the degree and pattern of steatosis and liver triglyceride content in male Sprague-Dawley and Wistar rats, respectively [23]. It was also confirmed that the time of feeding and the amount of dietary fats corresponded to the degree of liver pathology and other markers of liver tissue injury [28,34]. In 2006, Buettner et al [35] compared the metabolic and molecular effects of different HFDs. They fed male Wistar rats with a HFD containing 42% of energy in fats. They used four different sources of fats-lard (comparable quantities of SFA and MUFA), olive oil (mainly MUFA), coconut fat (mainly SFA) and fish oil (mainly ω-3 PUFA). Histopathological samples of the livers of rats fed by diets containing lard, olive oil and coconut fat exhibited mainly microvesicular steatosis without inflammation or fibrosis. These diets also up-regulated key genes of lipid synthesis. In contrast, the major enzymes of fatty acid oxidation were down-regulated in these groups. SREBP- 1c was up-regulated in all HFDs, but peroxisome proliferator activated receptor-α (PPAR-α), the major regulator of fatty acid oxidation, was up-regulated only in the liver of rats fed a diet containing ω-3 PUFA. The diets based on lard and olive oil induced more pronounced obesity and insulin resistance and the highest accumulation of fats in the liver. In another study, a beneficial effect of ω-3 PUFA was shown [34]. Giving ω-3 PUFA reduced the histological severity of HFD-induced NAFLD in Sprague- Dawley rats. Hunting for a new high-fat animal model of NASH In 2004, Lieber et al [29] introduced a new high-fat model of NASH in rats. Feeding Sprague-Dawley rats a liquid HFD (71% of kcal fat) for 3 wk incurred liver steatosis, inflammation and mitochondrial abnormalities without a significant elevation of serum transaminases. The histological observations were accompanied by increased hepatic TNF-α and TNF-α mrna, collagen type 1 and procollagen mrna, induction of cytochrome P450 2E1 and elevated markers of lipid peroxidation. Although there was no difference in final bodyweight, plasma insulin concentrations were increased. In this work, a control group received a diet containing 35% of kcal fat without significant liver changes [29], but a diet containing 35% of kcal fat is thought to be a high-fat diet for rats [22]. Our study showed that Wistar rats fed a diet with 35% of kcal fat exhibited considerable microvesicular steatosis [23]. Significant steatosis was not found in Sprague-Dawley rats, although the liver triglyceride content was significantly increased (3-fold) [23]. Using similar or equivalent diets in Sprague-Dawley rats, we [23] and others [36] failed to reproduce the histological picture of NASH, even when the time of feeding was extended to 5 or 6 wk. In another study, feeding male Sprague-Dawley rats a high-fat emulsion by gavage for 6 wk successfully induced obesity, insulin resistance, hyperlipidaemia, hepatic steatosis, pericentral necrosis, inflammation and mitochondrial lesions [27]. These findings were accompanied by lipid peroxidation, elevated expression of cytochrome P450 2E1, reduced PPAR-α in the liver, and high aminotransferase activity and serum TNF-α levels [27]. This model reproduces several characteristics of human NASH but also introduces several limitations, such as forced overfeeding, high content of polyunsaturated fatty acids, technical requirements and skills. Another attempt to establish a rat model of NASH used total enteral nutrition to overfeed male Sprague- Dawley rats a HFD (5%-70% of kcal corn oil) for d [28]. The hepatic triglyceride content was dependent on 8368 July 14, 2014 Volume 20 Issue 26

61 Kucera O et al. Experimental models of NAFLD in rats the percentage of corn oil in the diet. Histopathological findings of NASH (steatosis, macrophage infiltration, apoptosis, and focal necrosis) were present in the 70% corn oil group. In this group, markers of lipid peroxidation, TNF-α expression, expression of cytochrome P450 2E1 in the liver and serum alanine aminotransferase (ALT) were elevated. Although the intragastric infusion of HFD results in pathology that is clinically similar to NASH, the method of feeding does not reflect human NAFLD. During the last few years, there has been an upswing of HFD models of NASH [28,34,37,38]. These models successfully induce histopathological patterns of human NASH and the main metabolic perturbations of metabolic syndrome. The histological picture shows liver steatosis, lobular inflammatory infiltrates, hepatocyte necrosis and apoptosis, hepatocyte ballooning, and zone 3 fibrosis. Other characteristic features of NAFLD/metabolic syndrome are also observed (visceral obesity, insulin resistance, dyslipidaemia, elevated hepatic cytochrome P450 2E1 and TNF-α expression, oxidative stress and lipid peroxidation, increased serum ALT, and decreased adiponectin and PPAR-α expression). The majority of nutritional HFD models of NASH use fat with high amounts of corn oil, which mainly contains PUFA, of which > 98% are ω-6 PUFA. In contrast, saturated or monounsaturated fats are primarily found in the human diet. Thus, the use of such diets does not correlate with the dietary aetiology of NAFLD in humans. Moreover, ω-6 PUFAs exert proinflammatory characteristics in the body and enhance lipid peroxidation [39]. When male Wistar rats were fed ad libitum with a HFD containing mainly SFA (67% of energy from fats, coconut fat or butter) for 14 wk, neither liver steatosis nor increased liver triglyceride content were induced [26]. In conclusion, there is a high variability in the different diets and experimental protocols. Choosing suitable diet compositions, durations of feeding, strains and sexes of rats are crucial events in the experimental design. The Sprague-Dawley strain and male gender appear more susceptible to the development of NAFLD by HFD feeding. The specific mechanisms of interstrain differences have not been fully clarified. Using a HFD with high amounts of ω-6 PUFA can induce histopathological changes similar to NASH with some features of metabolic syndrome, but liver inflammation may be at least partially caused by the proinflammatory effect of ω-6 PUFA. Another limiting factor may be the lower reproducibility of nutritional models of NASH based on HFD in different laboratories. High-cholesterol and high-fat, high-cholesterol diets (atherogenic diets) Diets with supplemented cholesterol (up to 3% w/w) were originally used for the study of atherosclerosis pathogenesis. It was observed that diets containing cholesterol, particularly with the combination of higher fat and cholic acid in the chow, induced fatty liver changes. Dietary cholesterol is an important risk factor for the progression of simple steatosis to NASH in a mouse model [40]. Thus, the employment of high-fat, high-cholesterol diets has become popular for the induction of NAFLD in rats. Atherogenic diets may be easily prepared by the addition of extra fat and cholesterol (i.e., 10% w/w lard and 2% w/w cholesterol) to standard pellet chow, and they induce liver steatosis, necrosis, hepatocyte ballooning, steatohepatitis and fibrosis [41-43]. Furthermore, obesity, dyslipidaemia, insulin resistance and elevated serum aminotransferases accompany these liver changes. High-fructose/sucrose diet A shift in the eating habits of Western countries in recent decades has been characterised by an increase in monoand disaccharides, primarily sucrose (50% fructose) and fructose (i.e., candy, soft drinks). Fructose and other saccharides in excess are known to promote de novo lipogenesis. In humans, higher amounts of fructose in the diet are associated with metabolic syndrome, obesity and NAFLD [44]. Fructose promotes protein fructosylation and the formation of reactive oxygen species in the liver [44]. For rat models of metabolic syndrome or NAFLD, fructose or sucrose can be added into the diet or drinking water. A fructose-enriched diet induces steatosis and, in some experiments, inflammation and periportal fibrosis [45-47]. The histopathological findings were accompanied by insulin resistance, dyslipidaemia, elevated proinflammatory cytokines and markers of hepatic lipid peroxidation. Combined diets Various fast food diets containing higher amounts of fat and fructose are used to induce NAFLD in rats. Moreover, these diets can be supplemented with cholesterol or trans-fatty acids. Feeding male Wistar rats a highfructose, high-fat diet together with fructose in the drinking water for 16 wk resulted in marked obesity, impaired glucose tolerance, dyslipidaemia, hyperinsulinaemia, increased plasma leptin and liver steatosis, inflammation, and fibrosis [48]. The cardiovascular system (increased systolic blood pressure, endothelial dysfunction, inflammation, fibrosis and hypertrophy of the left ventricle), kidneys (inflammation and fibrosis) and pancreas were also affected. The cafeteria diet is a special combined diet that more accurately reflects the variety of highly palatable, energy dense, unhealthy foods in Western countries. Feeding a cafeteria diet is a better model for human obesity and metabolic syndrome compared to a simple HFD [49]. Male Wistar rats fed standard chow with concurrently offered cafeteria food (cookies, cereals, cheese, processed meats, crackers, etc.) ad libitum for 15 wk developed hyperphagia, resulting in severe obesity and prediabetes (glucose and insulin intolerance). This diet induced panlobular microvesicular steatosis, steatohepatitis and chronic inflammation in white and brown adipose tissues. Liver histopathological and metabolic alterations were more pronounced in the cafeteria diet compared to the lardbased HFD [49]. Voluntary feeding a cafeteria diet closely reflects the etiopathogenesis of human NAFLD July 14, 2014 Volume 20 Issue 26

62 Kucera O et al. Experimental models of NAFLD in rats According to the point of view of changing eating behaviour in Western populations, combined diets (highfructose, high-fat diet or cafeteria diet) are the most relevant model for metabolic syndrome and NAFLD in rats. Methionine- and choline-deficient diet One of the most commonly used dietary models of NAFLD and NASH is the methionine- and cholinedeficient diet (MCDD). Choline is an essential substance that is involved in many metabolic reactions in rats (e.g., methylation or transport of lipids). It is used for the formation of many biologically important compounds, such as phosphatidylcholine, sphingomyelin and acetylcholine. In humans, choline deficiency can be found in malnutrition, alcohol abuse or during pregnancy and lactation, and it may lead to the development of hepatic steatosis [50]. When choline is lacking in the diet, methionine can be used for the synthesis of choline. The absence of both choline and methionine substantially disturbs the formation of phosphatidylcholine. Phosphatidylcholine is essential for the normal formation of VLDL and its secretion from the liver [51]. Feeding rats a MCDD results in a rapid accumulation of triglycerides in the liver with the subsequent development of steatohepatitis and fibrogenesis [52]. In contrast to MCDD, a choline-deficient diet with sufficient supplementation of methionine does not result in the blockage of VLDL secretion, thereby leading to steatosis with a lower degree of inflammation in the rat liver [52,53]. In 2006, Veteläinen et al [52] described in a detail course of histological changes in an MCDD model in Wistar rats. At 1 wk, feeding MCDD induced mainly microvesicular steatosis with a macrovesicular component, predominantly in the acinar zone 3. At 3 wk, the pattern of steatosis changed to macrovesicular and scattered foci of inflammatory cells. At 5 wk, diffuse and extensive macrovesicular steatosis with foci of mononuclear inflammatory cells together with the occasional spotty necrosis occurred. At 7 wk, extensive macrovesicular steatosis, accumulation of cellular debris and numerous clusters of inflammatory cells developed. At this time, increased perivenular fibrosis with occasional centrilobular fibrosis was reported. Biochemically confirmed hepatic steatosis was accompanied by the accumulation of cholesterol in the liver [52]. Feeding rats with MCDD results in malnutrition; thus, rats lose weight, and the weight of the liver proportionally decreases [52]. Liver injury induced by MCDD is accompanied by elevated serum transaminases and proinflammatory cytokines, such as IL-1β, IL-6 and TNF-α [54]. Due to hepatic blockage of VLDL, serum levels of triglycerides and cholesterol are reduced [52]. In contrast to the pathogenesis of human NAFLD, MCDD does not induce insulin resistance in rats [52,55]. Feeding MCDD increases lipid peroxidation and lowers the hepatic content of reduced glutathione (GSH) [52,56]. The lack of methionine may be partially responsible for the decreased glutathione synthesis. An increase in oxidative stress and lipid peroxidation precedes the onset of steatohepatitis in MCDD-fed rats, and lipid peroxidation corresponds to hepatocellular damage and increased hepatic TNF-α [52]. By contrast, liver regeneration following a 2/3 partial hepatectomy in Wistar rats is not significantly impaired by MCDD feeding [57]. Liver mitochondria from MCDD rats show higher oxidative activity but a lower efficiency of oxidative phosphorylation; thus, mitochondrial ATP synthesis efficiency was decreased [58]. Liver mitochondria isolated from MCDD-fed rats exhibit higher hydrogen peroxide production, decreased respiratory control index of complex Ⅰ, up-regulation of uncoupling protein-2, increased permeability of the inner mitochondrial membrane for H + and, as a consequence, a reduction in mitochondrial membrane potential [56,59,60]. In a study by Kirsch et al [61], species (rat and mouse), strain (Wistar, Long-Evans and Sprague-Dawley rats, and C57/BL6 mice) and sex differences were investigated in an MCDD model of NASH. The degrees of steatosis, hepatic lipid content and serum ALT activity were significantly higher in male rats than in female rats. Wistar rats had higher liver lipid levels, serum ALT levels, and liver mass/body mass ratios than Long-Evans and Sprague- Dawley rats. The mechanisms underlying these strain differences are uncertain. Feeding rats with MCDD for 4 wk induced only minor necrosis and inflammation, and fibrosis was absent [61]. In contrast to mice, rats fed MCDD develop steatohepatitis more slowly [21,61]. The choline-deficient, L-amino acid-defined (CDAA) diet has similar characteristics as the MCDD. The CDAA diet contains a low amount of methionine; thus the mechanism by which this diet induces typical hepatic changes is similar to MCDD. Feeding a CDAA diet induces liver steatosis, which is followed by more severe steatosis with inflammation, increased oxidative stress and fibrosis, cirrhosis, preneoplastic lesions and hepatocellular carcinoma [62-64]. Similar to MCDD, rats fed a CDAA diet do not develop typical characteristics of metabolic syndrome. This model may be employed for the study of hepatocarcinogenesis from steatohepatitis. Although the MCDD model is one of the best defined rat models of NASH and is widely used to study NASH, it does not reflect several causative features of human NASH [65]. In contrast to humans, rats fed MCDD lose weight, have low serum triglyceride and cholesterol levels and have normal insulin sensitivity. This model induces changes in the liver by nutritional deficiency and does not reflect the metabolic profile and aetiology of NAFLD in humans. MCDD imitates several histopathological features of human NAFLD, but the distribution of steatosis may vary. Depending on the time of feeding, MCDD induces hepatic steatosis, steatohepatitis or fibrosing steatohepatitis and thus simulates the histological picture of NAFLD. This model is more suitable for studying the consequences of fat accumulation, inflammation, oxidative stress and fibrogenesis in the liver than the complex pathogenesis of human NAFLD. In contrast to most other NAFLD models, MCDD induces 8370 July 14, 2014 Volume 20 Issue 26

63 Kucera O et al. Experimental models of NAFLD in rats reproducible histological changes with significant inflammation and fibrogenesis. In conclusion, the MCDD model mimics only histopathological features but lacks the main aetiopathogenic factors of human disease. Choline-deficient diet Choline-deficient diet (CDD) induces hepatic steatosis, and the degree of steatosis is dependent upon the time of feeding [52]. In contrast to MCDD, CDD-fed rats do not develop severe fibrosis, and the inflammation is of a lower extent [52]. The accumulation of fats in the liver is accompanied by non-significant changes or mild elevations in serum transaminases [52,53]. Feeding CDD for 90 d leads to the development of moderate fibrotic alterations in the liver of male Wistar rats [53]. Liver steatosis in this model is characterised by the development of obesity, dyslipidaemia (increase in plasma concentrations of triglycerides and cholesterol) and insulin resistance after 7 wk [52]. Hepatic GSH and lipid peroxidation were reported to be unaltered [52]. In contrast, Vendemiale et al [66] and Hensley et al [67] showed a greater mitochondrial content of oxidised lipids and proteins, lower concentration of glutathione and increased mitochondrial ROS formation. The activity of mitochondrial complex Ⅰ, mitochondrial ATP synthase activity and hepatic ATP levels were significantly reduced in the fatty livers of CDD-fed rats [66,67]. In summary, the CDD and MCDD are two distinct models in rats, which differ not only in their histological findings but also in the metabolic and other characteristics of rats (insulin resistance, inflammatory response, oxidative stress) [52]. CDD induces fatty liver accompanied by metabolic alterations typical of metabolic syndrome. GENETIC MODELS Genetic models can be further subdivided into naturally occurring genetic models and models of induced genetic mutations. There is a variety of genetic models of NAFLD in mice [68]. In view of the polygenic genetic background of this disease [3], monogenic mouse models of NAFLD do not fully replicate the etiopathogenesis of human NAFLD, but they may provide valuable results concerning particular events in the pathogenesis of NAFLD. Moreover, genetically modified mice are less available and more expensive. In contrast to mice, there are very few genetically modified rats. The Zucker fatty rats One of the most commonly used models of NAFLD in rats is a genetic model of the obese Zucker rats (the Zucker fatty rats, fa/fa rats, ZFR). ZFR have a natural mutation in the leptin receptor (fa allele), which decreases the affinity of the receptor for leptin and alters signal transduction [69]. ZFR are homozygotes for the fa allele, while heterozygotes for the fa allele (lean Zucker rats) serve as controls. Leptin is a peptide hormone secreted from adipose tissue, which participates in the regulation of feeding behaviour and energy expenditure [70]. This hormone is also involved in the modulation of cell death and fibrogenesis [71]. Thus, the mutation in the leptin receptor leads to leptin resistance. ZFR are widely used as an animal model of genetic obesity and metabolic syndrome [22]. ZFR develop severe obesity and are hyperleptinaemic, hyperphagic, inactive, obese and insulin-resistant (hyperinsulinaemia, mild hyperglycaemia, hyperlipidaemia) [72,73]. ZFR rats are also used as a model of insulin resistance. Hyperlipidaemia in ZFR is characterised by increased VLDL and high-density lipoproteins (HDL) without significant changes in low-density lipoprotein (LDL) cholesterol but with reduced expression of the hepatic LDL receptor [74,75]. It was also observed that the systolic arterial blood pressure increased (at 28 wk) [76], and plasma adiponectin levels were higher [73] in ZFR than in lean controls. Macrovesicular or microvesicular steatosis is present in ZFR without signs of progression to steatohepatitis [77]. The accumulation of fat in the liver of Zucker obese rats is localised to the periportal area [78]. The liver of obese Zucker rats have lower amounts of GSH and vitamin E and decreased activity of catalase [79]. The regenerative capacity of the liver of fa/fa rats is significantly decreased after 2/3 partial hepatectomy [80]. As a consequence of leptin resistance, increased expressions of SREBP-1c and the carbohydrate response element binding protein (ChREBP) were reported [81,82]. The elevations of SREBP- 1c mrna was accompanied by increased expression of lipogenic enzymes and the accumulation of triglycerides in the liver [81]. As in ob/ob mice, ZFR do not spontaneously develop steatohepatitis, and they require a second hit for the progression of steatosis to steatohepatitis. A second hit could be exposure to lipopolysaccharide [77], a combination of diet rich in disaccharide and low-dose lipopolysaccharide [83], a high-fat diet [78] or the combination of MCDD with a high fat diet [84]. Because the dietary models (except MCDD) more closely resemble the pathogenesis of human NAFLD compared to the genetic models, the use of dietary interventions for genetic models has become more popular. After feeding fa/fa rats a high-saturated fat diet (60% of energy of diet derived from lard) for 8 wk, more severe micro- and macrovesicular steatosis and steatohepatitis developed [78]. Liver injury was accompanied by fasting hyperglycaemia and increased serum activity of ALT, TNF-α and TGF-β, increased collagen deposition, and greater activation of hepatic stellate cells. Markers of oxidative stress, such as lipid peroxidation, protein carbonyls, glutathione, and antioxidant enzymes, were significantly aggravated in ZFR fed a high-fat diet. A special genetic model of obesity with type 2 diabetes, the Zucker diabetic fatty (ZDF) rats, was derived from ZFR by the selective breeding of hyperglycaemic ZFR. ZDF male rats typically develop hyperinsulinaemia, peripheral insulin resistance, hyperglycaemia and noninsulin dependent diabetes by 12 wk of age [85,86]. ZDF females are more resistant to the spontaneous development of diabetes, and the induction of type Ⅱ diabetes in female ZDF rats is successful only when they are fed a 8371 July 14, 2014 Volume 20 Issue 26

64 Kucera O et al. Experimental models of NAFLD in rats high-fat diet [87]. ZFR partially simulates human metabolic syndrome (obesity, insulin resistance, dyslipidaemia, hyperinsulinaemia, steatosis), but they have several disadvantages. Because leptin or leptin receptor mutations are rare in humans [88], ZFR do not reflect the multifactorial etiopathogenesis of human NAFLD. Moreover, ZFR do not spontaneously develop steatohepatitis and are resistant to liver fibrosis. ZFR require further interventions for the progression from simple steatosis to steatohepatitis. There are several other rat models utilising altered leptin pathways in which hepatic steatosis develops. These are spontaneously hypertensive obese rats (Koletsky s rats), stroke-prone spontaneously hypertensive rats (SHRSP5/Dmcr) or corpulent JCR:LA-cp rats, which have a recessive mutation in the leptin receptor gene that blocks expression of the protein [89]. These rats exhibit several similar phenotypic characteristics to ZFR. They are hyperphagic, obese, hyperlipidaemic and insulin resistant [90,91]. Although they develop simple fatty liver [91,92], these models have not been widely employed for the study of NAFLD. The exposure of stroke-prone spontaneously hypertensive rats to a high-fat, high-cholesterol diet results in steatosis, necrosis with lymphocyte infiltrations, hepatocyte ballooning with Mallory-Denk bodies and fibrosis [93]. Transgenic spontaneously hypertensive rats overexpressing SREBP-1a Recently, a new transgenic model of SREBP-1a overexpression was introduced in the spontaneously hypertensive rat [94]. SREBP-1 gene polymorphisms are associated with obesity, severe insulin resistance and type 2 diabetes in humans [95]. Transgenic spontaneously hypertensive rats (SHR) overexpressing SREBP-1a rats are non-obese, but they spontaneously develop hypertension, fatty liver, and several important characteristics of metabolic syndrome (hyperinsulinaemia, hyperglycaemia, hypertriglyceridaemia, and hypercholesterolaemia) [94]. Biochemical examination confirmed the accumulation of triglycerides in the liver and found elevated hepatic levels of cholesterol [94]. Transgenic SHR overexpressing SREBP-1a rats fed with a high-fructose diet exhibit significantly increased oxidative stress in the liver (enhanced lipid peroxidation, decreased activities of antioxidative enzymes and lower glutathione), which was more pronounced in older rats [96]. Similarly, the progression of histopathological findings was age-dependent. Centrilobular steatosis without inflammation was observed at 2 mo of age, whereas prominent fatty changes accompanied by focal liver cell necrosis and inflammatory infiltrate similar to human NASH were found after 16 mo [96]. Transgenic SHR overexpressing SREBP-1a rats may provide a tool for studying the pathogenic mechanisms and treatment of metabolic syndrome associated with NAFLD [94]. Otsuka Long-Evans Tokushima fatty rats Otsuka Long-Evans Tokushima fatty rats (OLETF) were obtained in 1984 by the selective breeding of an outbred colony of Long-Evans rats exhibiting a diabetic phenotype (polyuria, polydipsia, obesity). OLETF rats have a deletion in the promoter region of the gene encoding the cholecystokinin-1 (CCK-1) receptor and in the first and second exons of this gene, resulting in low or absent expression of CCK-1 receptor mrna [97]. A control strain, the LETO rats, which originated from the same colony of rats, expresses the CCK-1 receptor and does not exhibit the phenotype of OLETF rats. CCK-1 participates in satiety control in the hypothalamus; thus, OLETF rats exhibit hyperphagia, and they are mildly obese, hyperlipidaemic, hyperglycaemic (after the age of 18 wk), hyperinsulinaemic, insulin resistant and have diabetes mellitus [55,98-100]. Serum concentrations of leptin and IL-6 are elevated, while adiponectin is lower in these rats [98]. OLETF rats are used as a model for metabolic syndrome with diabetes [22]. OLETF rats spontaneously develop liver steatosis when fed a standard diet ad libitum [100]. At wk of age, micro- and macrovesicular steatosis (with a maximum at approximately 30 wk) and hepatocyte ballooning without collagen deposition or fibrosis were observed, but the steatosis disappeared after 42 wk [100]. mrna expressions of lipogenic genes, such as SREBP- 1c and ChREBP, were significantly elevated in the liver of OLETF rats prior to the onset of hepatic steatosis (10 wk) and then gradually decreased to control values (50 wk) [100]. Contrarily, PPAR-α is down-regulated in younger OLETF rats (12 and 28 wk) [101]. For the induction of steatohepatitis in OLETF rats, MCDD or a combined diet with choline and methionine deficiency and high-fat content can be used [55,102]. In the OLETF rats, MCDD induces hepatic lobular inflammation and perivenular and pericellular fibrosis in zone 3 with increased expression of TGF-β1 and activation of stellate cells [102]. The advantage of a combination of MCDD and HFD in OLETF rats is the development of steatohepatitis with enhanced insulin resistance, more pronounced inflammation and fibrosis (pre-cirrhosis), and increased mrna expression of lipogenic genes [55]. In contrast to the finding of Ota et al [55], Song et al [100] described hepatocyte ballooning in OLETF (and LETO) rats only when a combined diet (MCDD + HFD) was used. Similar to the Zucker fatty rats, OLETF rats do not reflect the etiopathogenesis of human NAFLD (mutation in CCK-1 receptor). OLETF rats are recommended at the age of wk as animal models of hepatic steatosis, but they are not a suitable model for NASH without an additional dietary hit. Prague hereditary hypercholesterolaemic rats Prague hereditary hypercholesterolaemic rats were crossbred from Wistar rats as a model for polygenic hypercholesterolaemia induced by dietary cholesterol (2% w/w cholesterol diet). Hypercholesterolaemia is caused by the elevation of cholesterol in VLDL, intermediatedensity lipoproteins, and LDL but not in HDL [103]. When fed standard chow, serum cholesterol is only mildly elevated [103]. In contrast to 5% dietary fat alone (standard 8372 July 14, 2014 Volume 20 Issue 26

65 Kucera O et al. Experimental models of NAFLD in rats chow), a high cholesterol diet in these rats induces the development of fatty liver and the accumulation of cholesterol [103]. Thus, the presence of cholesterol in the diet is necessary for the development of fatty liver. This combined model of fatty liver is also not widely used. CONCLUSION Although there is not an ideal model for human nonalcoholic fatty liver disease that reflects all the clinical, histological, aetiological and pathogenic aspects of human disease, choosing an appropriate model for studying particular events of NAFLD while respecting its limitations has contributed greatly to the understanding of this disease, its progression and treatment. Considering the legislative, ethical, economical and health factors of NAFLD, animal models are and will continue to be an essential tool for further research of this disease. REFERENCES 1 Vernon G, Baranova A, Younossi ZM. 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68 Kucera O et al. Experimental models of NAFLD in rats A, Pandya PK, Considine RV. Does leptin play a role in the pathogenesis of human nonalcoholic steatohepatitis? Am J Gastroenterol 2003; 98: [PMID: DOI: /j x] 89 Takaya K, Ogawa Y, Hiraoka J, Hosoda K, Yamori Y, Nakao K, Koletsky RJ. Nonsense mutation of leptin receptor in the obese spontaneously hypertensive Koletsky rat. Nat Genet 1996; 14: [PMID: DOI: /ng ] 90 Koletsky S. Pathologic findings and laboratory data in a new strain of obese hypertensive rats. Am J Pathol 1975; 80: [PMID: ] 91 Elam MB, Wilcox HG, Cagen LM, Deng X, Raghow R, Kumar P, Heimberg M, Russell JC. Increased hepatic VLDL secretion, lipogenesis, and SREBP-1 expression in the corpulent JCR: LA-cp rat. J Lipid Res 2001; 42: [PMID: ] 92 Wexler BC. Hyperlipidemia, hyperglycemia and hypertension in repeatedly bred parents of the obese spontaneously hypertensive rat (obese/shr) unaccompanied by arteriosclerosis. Atherosclerosis 1984; 51: [PMID: ] 93 Kitamori K, Naito H, Tamada H, Kobayashi M, Miyazawa D, Yasui Y, Sonoda K, Tsuchikura S, Yasui N, Ikeda K, Moriya T, Yamori Y, Nakajima T. Development of novel rat model for high-fat and high-cholesterol diet-induced steatohepatitis and severe fibrosis progression in SHRSP5/Dmcr. Environ Health Prev Med 2012; 17: [PMID: DOI: /s ] 94 Qi NR, Wang J, Zidek V, Landa V, Mlejnek P, Kazdová L, Pravenec M, Kurtz TW. A new transgenic rat model of hepatic steatosis and the metabolic syndrome. Hypertension 2005; 45: [PMID: DOI: /01. HYP b] 95 Eberlé D, Clément K, Meyre D, Sahbatou M, Vaxillaire M, Le Gall A, Ferré P, Basdevant A, Froguel P, Foufelle F. SREBF-1 gene polymorphisms are associated with obesity and type 2 diabetes in French obese and diabetic cohorts. Diabetes 2004; 53: [PMID: ] 96 Malínská H, Oliyarnyk O, Hubová M, Zídek V, Landa V, Simáková M, Mlejnek P, Kazdová L, Kurtz TW, Pravenec M. Increased liver oxidative stress and altered PUFA metabolism precede development of non-alcoholic steatohepatitis in SREBP-1a transgenic spontaneously hypertensive rats with genetic predisposition to hepatic steatosis. Mol Cell Biochem 2010; 335: [PMID: DOI: / s ] 97 Takiguchi S, Takata Y, Funakoshi A, Miyasaka K, Kataoka K, Fujimura Y, Goto T, Kono A. Disrupted cholecystokinin type-a receptor (CCKAR) gene in OLETF rats. Gene 1997; 197: [PMID: ] 98 Jung TS, Kim SK, Shin HJ, Jeon BT, Hahm JR, Roh GS. α-lipoic acid prevents non-alcoholic fatty liver disease in OLETF rats. Liver Int 2012; 32: [PMID: DOI: /j x] 99 Kawano K, Hirashima T, Mori S, Saitoh Y, Kurosumi M, Natori T. Spontaneous long-term hyperglycemic rat with diabetic complications. Otsuka Long-Evans Tokushima Fatty (OLETF) strain. Diabetes 1992; 41: [PMID: ] 100 Song YS, Fang CH, So BI, Park JY, Lee Y, Shin JH, Jun DW, Kim H, Kim KS. Time course of the development of nonalcoholic Fatty liver disease in the Otsuka long-evans Tokushima Fatty rat. Gastroenterol Res Pract 2013; 2013: [PMID: DOI: /2013/342648] 101 Yeon JE, Choi KM, Baik SH, Kim KO, Lim HJ, Park KH, Kim JY, Park JJ, Kim JS, Bak YT, Byun KS, Lee CH. Reduced expression of peroxisome proliferator-activated receptor-alpha may have an important role in the development of non-alcoholic fatty liver disease. J Gastroenterol Hepatol 2004; 19: [PMID: DOI: / j x] 102 Uno M, Kurita S, Misu H, Ando H, Ota T, Matsuzawa-Nagata N, Kita Y, Nabemoto S, Akahori H, Zen Y, Nakanuma Y, Kaneko S, Takamura T. Tranilast, an antifibrogenic agent, ameliorates a dietary rat model of nonalcoholic steatohepatitis. Hepatology 2008; 48: [PMID: DOI: /hep.22338] 103 Kovár J, Tonar Z, Heczková M, Poledne R. Prague hereditary hypercholesterolemic (PHHC) rat - a model of polygenic hypercholesterolemia. Physiol Res 2009; 58 Suppl 2: S95-S99 [PMID: ] P- Reviewers: Gwak GY, Mueller-Wieland D S- Editor: Qi Y L- Editor: A E- Editor: Ma S 8376 July 14, 2014 Volume 20 Issue 26

69 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. WJG 20 th Anniversary Special Issues (12): Nonalcoholic fatty liver disease TOPIC HIGHLIGHT Non-alcoholic fatty liver disease and diabetes: From physiopathological interplay to diagnosis and treatment Nathalie C Leite, Cristiane A Villela-Nogueira, Claudia R L Cardoso, Gil F Salles Nathalie C Leite, Cristiane A Villela-Nogueira, Claudia R L Cardoso, Gil F Salles, Internal Medicine Department, University Hospital Clementino Fraga Filho, School of Medicine, Universidade Federal do Rio de Janeiro, Rio de Janeiro , Brazil Author contributions: Leite NC and Villela-Nogueira CA contributed equally to this work, both reviewed the subject and drafted the manuscript; Cardoso CRL and Salles GF reviewed the manuscript and contributed with intellectual content. Supported by Conselho Brasileiro de Desenvolvimento Científico e Tecnológico (CNPq-Brasil) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ-Brasil) Correspondence to: Gil F Salles, MD, PhD, Professor, Internal Medicine Department, University Hospital Clementino Fraga Filho, School of Medicine, Universidade Federal do Rio de Janeiro, Rua Croton 72, Rio de Janeiro , Brazil. gilsalles@hucff.ufrj.br Telephone: Fax: Received: October 26, 2013 Revised: December 1, 2013 Accepted: January 19, 2014 Published online: July 14, 2014 Abstract Non-alcoholic fatty liver disease (NAFLD) is highly prevalent in patients with diabetes mellitus and increasing evidence suggests that patients with type 2 diabetes are at a particularly high risk for developing the progressive forms of NAFLD, non-alcoholic steatohepatitis and associated advanced liver fibrosis. Moreover, diabetes is an independent risk factor for NAFLD progression, and for hepatocellular carcinoma development and liver-related mortality in prospective studies. Notwithstanding, patients with NAFLD have an elevated prevalence of prediabetes. Recent studies have shown that NAFLD presence predicts the development of type 2 diabetes. Diabetes and NAFLD have mutual pathogenetic mechanisms and it is possible that genetic and environmental factors interact with metabolic derangements to accelerate NAFLD progression in diabetic patients. The diagnosis of the more advanced stages of NAFLD in diabetic patients shares the same challenges as in non-diabetic patients and it includes imaging and serological methods, although histopathological evaluation is still considered the gold standard diagnostic method. An effective established treatment is not yet available for patients with steatohepatitis and fibrosis and randomized clinical trials including only diabetic patients are lacking. We sought to outline the published data including epidemiology, pathogenesis, diagnosis and treatment of NAFLD in diabetic patients, in order to better understand the interplay between these two prevalent diseases and identify the gaps that still need to be fulfilled in the management of NAFLD in patients with diabetes mellitus Baishideng Publishing Group Inc. All rights reserved. Key words: Non-alcoholic fatty liver disease; Diabetes mellitus; Pathogenesis; Diagnosis; Treatment Core tip: This review addresses the important interplay between non-alcoholic fatty liver disease and diabetes mellitus, with particular emphasis on physiopathological mechanisms, diagnosis and treatment. Leite NC, Villela-Nogueira CA, Cardoso CRL, Salles GF. Nonalcoholic fatty liver disease and diabetes: From physiopathological interplay to diagnosis and treatment. World J Gastroenterol 2014; 20(26): Available from: URL: wjgnet.com/ /full/v20/i26/8377.htm DOI: org/ /wjg.v20.i INTRODUCTION Diabetes mellitus (DM) is a public-health concern: 347 million people worldwide have diabetes and the WHO projects that diabetes will be the 7 th leading cause of death in It is a group of metabolic diseases charac July 14, 2014 Volume 20 Issue 26

70 Leite NC et al. NAFLD and diabetes: A review terized by hyperglycemia resulting from defects in insulin secretion, insulin action or both. There are two most important etiopathogenetic categories: type 1 DM (T1DM), which accounts for 5%-10% of the cases of DM and results from autoimmune destruction of the beta-cells of the pancreas and absolute deficiency of insulin; and type 2 DM (T2DM), which encompasses patients who have insulin resistance with relative insulin deficiency and accounts for 90%-95% of diabetic patients. Both T1DM and T2DM patients are at high risk of developing chronic macrovascular and microvascular degenerative complications. Nevertheless, beyond these classic complications, DM is also associated with liver-related mortality and increasing risk of hepatocellular carcinoma [1,2]. Non-alcoholic fatty liver disease (NAFLD) prevalence is increased in patients with DM [3,4]. Increasing evidence suggests that patients with T2DM are at a particularly high risk for non-alcoholic steatohepatitis (NASH), with varying degrees of liver fibrosis [5,6]. Likewise, preexisting diabetes is an independent risk factor for NAFLD progression and for liver-related mortality and hepatocellular carcinoma in prospective studies [1,7-9]. On the other hand, patients with NAFLD have an elevated prevalence of prediabetes [10] and recent data has shown that NAFLD presence predicts the development of T2DM [10,11]. There are pathogenetic mechanisms linking NAFLD and DM. Besides insulin resistance and hyperinsulinemia, disordered lipid metabolism, increased oxidative stress and inflammation contribute to both entities. It is possible that genetic and environmental factors interact with metabolic derangements to accelerate NAFLD progression in diabetic patients. Ultrasound is the method of choice for screening patients for NAFLD. In patients with diabetes and histologically-proven NASH, abnormal liver enzymes may be seen in less than 20% of patients [5,6,12,13]. Liver biopsy is still the gold standard method to diagnose and stage NAFLD. However, it is a costly and invasive procedure with some limitations, such as sampling error and intra-and inter-observer variability among pathologists [6]. It was previously suggested that liver biopsy should be considered in all patients with DM and hepatic steatosis on ultrasound [6]. Newer radiological techniques, biomarkers and clinical algorithms are being currently studied and may provide, in the near future, valuable noninvasive alternatives to histological diagnosis. Treatment is based mainly on lifestyle changes and antioxidants and numerous drugs have been studied searching for histological improvement with variable outcomes. The following review aims to discuss all these aspects from physiopathological mechanisms to diagnosis and treatment of NAFLD in DM. DEFINITION OF NAFLD In 1980, Ludwig et al [14] described a small series of patients with liver histology characterized by fat accumulation, hepatic necroinflammation and, in most cases, fibrosis in the absence of a history of excessive alcohol consumption. Since this original description, the histologic criterion for diagnosing NAFLD has evolved, and several grading systems have been proposed to assess histologic severity. In 1999, Matteoni et al [15] proposed pathological NAFLD subtypes based on long-term outcome studies, and Brunt et al [16] proposed a specific classification for NASH based on criteria used in other forms of chronic liver disease, using grades of necroinflammatory lesions and stages of fibrosis. Subsequently, the US Pathology Committee of the NASH Clinical Research Network [17] proposed and validated a histological scoring system of specific lesions that addressed the full spectrum of NAFLD. In addition, a separate scoring system known as the NAFLD activity score (NAS) has been developed for use in clinical trials. It measures steatosis semi-quantitatively, as well as lobular inflammation and hepatocellular ballooning, to enable systematic documentation of changes in activity of NASH [17]. However, a numerical value alone is not accepted for definitive diagnosis of steatohepatitis. In general, accumulation of greater than 5% of fat, particularly in the form of triglycerides, is considered to be the minimal requirement for histologic diagnosis of steatosis. NASH, in turn, encompasses steatosis plus lobular inflammation and ballooning degeneration, with or without a varying degrees of fibrosis, which is originally centered on the hepatic venulae. CLINICAL PRESENTATION NAFLD is generally asymptomatic at presentation and is frequently found among individuals with conditions such as obesity, T2DM, metabolic syndrome and its individual components [18]. The most common signs and symptoms are fatigue, right upper quadrant pain and hepatomegaly, as well as acanthosis nigricans, which are more frequently observed in the pediatric population. In fact, most patients with NAFLD are diagnosed by incidental elevated liver enzymes or imaging studies suggesting hepatic steatosis [18]. By definition, NAFLD is established in patients who consume little or no alcohol presenting a histological picture that resembles alcohol-induced liver disease. Most studies defined a threshold for excessive alcohol consumption as more than 20 g/d in women and more than 30 g/d in men [15,19]. In contrast, intake levels of 20 g/d (140 g weekly) for men, and 10 g/d (70 g weekly) for women have been endorsed as the acceptable thresholds to define non-alcoholic in the guidelines proposed by the Asia-Pacific Working Party for NAFLD (APWP- NAFLD) and by the National Institutes of Health Clinical Research Network [20,21]. Additionally, definitive diagnosis of NAFLD requires exclusion of other secondary causes of hepatic steatosis, such as medications like prednisolone, tamoxifen, amiodarone and methotrexate among others, exposure to toxins as vinyl chloride, total parenteral nutrition, cachexia, intestinal bypass surgery, viruses infections like genotype 3 hepatitis C virus and human immunodeficiency virus July 14, 2014 Volume 20 Issue 26

71 Leite NC et al. NAFLD and diabetes: A review Table 1 Studies with histopathological evaluation of diabetic patients with non-alcoholic fatty liver disease Ref. Sample size Metabolic syndrome and its components Diabetes duration (yr) Diabetes-related complications Elevated enzyme levels AST/ALT/GGT Risk factors for NASH Fibrosis Amarapurka % et al [12] Gupte et al [13] 32 ALT and/or AST 31% No risk factors 22% Kemmer et al [38] 22 (females) No risk factors Leite et al [6] 92 Hypertension 88% 7.8 Microvascular 46% 14%/16%/13% Hypertriglyceridemia 34%-60% High ALT Dyslipidaemia 86% Macrovaascular 26% Low HDL-chol Prashanth et al [5] 83 Metabolic syndrome 77% 8.2 ALT 7% MS components 37% High ALT High AP ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; GGT: Gammaglutamyltransferase; AP: Alkaline phosphatase; NASH: Non-alcoholic steatohepatitis; HDL-chol: HDL-cholesterol. Epidemiology NAFLD is the commonest cause of liver disease in western countries, present in over 30% of the general population [22,23]. The prevalence in Asian populations ranges from 6% up to 25% [24]. The prevalence of NAFLD among children is 3%-10%, rising up to 40%-70% among obese children [25]. The prevalence of NASH is difficult to assess because it requires a histological diagnosis, which is impractical in all patients with NAFLD. In a recent study in a cohort of 400 military personnel and their relatives, ultrasound prevalence of NAFLD was 46%, and NASH was found in 30% of these individuals, resulting in a NASH prevalence of 12% for the entire cohort [26]. NASH cirrhosis is now the third most common indication for liver transplantation in the United States, behind hepatitis C and alcoholic liver disease, and is the only liver-related transplant indication that continues to increase [27]. Liver disease is the third leading cause of death in NAFLD patients [8,28], with hepatocellular carcinoma being the most frequent cause of liver-related death [29]. T2DM is not only a risk factor for NAFLD but it is also related to a higher prevalence of NASH and fibrosis [1,5,6]. Data regarding T1DM and NAFLD are scarce. Although T1DM is characterized by insulin deficiency, obesity and metabolic syndrome may eventually occur in these patients and lead to NAFLD [30]. Targher et al [31] found a NAFLD prevalence of 44% in patients with T1DM by ultrasound imaging, without histological confirmation. Nevertheless, the prevalence of NAFLD in patients with T2DM has been reported to be as high as 74% [3,4,26,32,33]. Most of the studies evaluating NAFLD in T2DM have relied exclusively on ultrasonography or on elevated liver enzymes for diagnosis. Studies describing the histopathological spectrum of NAFLD in type 2 diabetes mellitus are still scarce [6]. NAFLD and Diabetes Mellitus: the natural history is a two-way traffic NAFLD represents a wide spectrum of conditions ranging from fatty liver, which follows in general a benign and stable clinical course, to NASH that may progress to cirrhosis. Less than 1% to 4% of patients with simple steatosis progress to advanced fibrosis. By contrast, NASH can lead to cirrhosis in 15% to 25% of individuals, with further liver-related complications and death [8,11,34]. Among those with NASH-related cirrhosis, about 25% will develop major complications of portal hypertension within 3 years [35]. The role of diabetes in NASH and fibrosis was initially evaluated in patients undergoing bariatric surgery. The prevalence of T2DM ranged from 14% to 28% in these studies and its presence was a predictor of NASH and fibrosis in morbidly obese patients [36,37]. Younossi et al [1], in a retrospective cohort study with 132 patients with histological diagnosis of NAFLD, reported that patients with DM had greater rates of cirrhosis and mortality than those with NAFLD without DM. In a recent prospective study of 328 asymptomatic patients, 16.5% had an established diagnosis of T2DM. The prevalence of NAFLD and NASH in the entire cohort was 46% and 12%, respectively. However, in diabetic patients, NAFLD was observed in 74% and NASH in 22% [26]. Few studies evaluated the prevalence of NAFLD and the correlated factors with histopathological stages of NAFLD in patients with T2DM. Table 1 outlines a summary of these studies. The sample sizes varied from 32 to 92 patients, and NASH was present in 63%-87% of the patients, while the prevalence of any fibrosis ranged from 22% to 60% in these studies [5,6,13]. In the largest single-centre study, we found high prevalence of the more severe stages of NAFLD: 78% for NASH and 34%-60% for moderate-to-severe fibrosis [6]. No diabetesrelated variable, such as glycemic control, diabetes duration or the presence of micro- and macro-vascular complications, was associated with the more severe stages of NAFLD [6]. Alanine aminotransferase (ALT), high triglycerides and low HDL-cholesterol were independently associated with NASH. On the other hand, male gender, older age and elevated values of gammaglutamyl transferase (GGT) were associated with moderate-to July 14, 2014 Volume 20 Issue 26

72 Leite NC et al. NAFLD and diabetes: A review severe fibrosis [6]. Prashanth et al [5] reported prevalence of NASH and of any stage of fibrosis of 62.6% and 37.3%, respectively. Serum ALT and alkaline phosphatase levels, although within normal limits, were significantly higher in patients with NASH [5]. Of note, NASH prevalence increased proportionally to the number of metabolic syndrome components presented. Also, no diabetes-related variable was predictive of the severity of NAFLD. In a study conducted in Mexico, 60 patients with DM were evaluated and 22 of them (37%) had elevated liver enzymes and/or steatosis on radiological examination [38]. These patients underwent liver biopsy and the prevalence of NASH was 64%. There was no association of liver enzymes, lipid profile, glycated hemoglobin or body mass index with the presence of NASH. In another small group of 32 T2DM patients submitted to liver biopsies, 49% had NAFLD on histopathology, of which 87.5% had NASH. There was no significant correlation between liver enzymes and NASH or fibrosis [13]. Subsequently, these researchers further reported that in 36 patients with NASH, 30.5% had any stage of fibrosis. Patients with any stage of fibrosis had higher levels of aminotransferases and a higher aspartate aminotransferase (AST) to ALT ratio [12]. Although in most of the studies diabetic patients with NASH had higher serum ALT than those without NASH, and ALT levels were independently associated with the presence of NASH on liver biopsy, abnormal ALT levels were uncommon and did not have enough predictive value to be indicated as a screening test for NASH detection [6]. Moreover, in patients with T2DM, serum liver enzymes could be less representative of the severity of intrahepatic fat accumulation. In a case-control study, patients with T2DM showed approximately 80% more intra-hepatic fat content by magnetic resonance spectroscopy than age, sex, and body weight-matched non-diabetic controls [39]. Liver fat content was underestimated by serum ALT compared with equally obese nondiabetic subjects. A recent cross-sectional study demonstrated that in addition to diabetes, a family history of diabetes also increased the risk of NASH and fibrosis in non-diabetic individuals [40]. The association between family history of diabetes with NASH and fibrosis remained significant even after adjusting for prediabetes status, suggesting that a family history of diabetes may provide additional risk stratification in non-diabetic patients with NAFLD. In spite of apparent absence of association between any diabetes-related characteristic and the presence of more severe stages of NAFLD in previous studies, evidence is mounting that NAFLD may be associated with the occurrence of microvascular and macrovascular degenerative complications in diabetic patients [3,31,41,42]. In patients with T1DM and T2DM, NAFLD was associated with higher rates of microalbuminuria, reduced glomerular filtration rate and retinopathy [43,44]. Prospective studies have demonstrated a higher incidence of chronic kidney disease in patients with T2DM and NAFLD, independent of several established risk factors [42,43]. Both subclinical atherosclerosis markers, like increased carotid intimamedia thickness and aortic stiffness, as well as clinical cardiovascular diseases, were also more frequent in patients with T1DM and T2DM with the diagnosis of NAFLD than in their counterparts without NAFLD [3,31,45]. It was demonstrated that the association between macrovascular disease and NAFLD was independent of classic cardiovascular risk factors and components of metabolic syndrome [3,31]. Another issue to be considered is whether diabetes also accelerates the progression of NAFLD. The presence of DM was independently associated with advanced liver fibrosis in cross-sectional studies [46,47]. However, findings from prospective studies with serial liver biopsies are still scarce and controversial. In a cohort study with 129 patients with biopsy-proven NAFLD reevaluated after a mean follow-up of 13.7 years, the progression of liver fibrosis occurred in 41% [8]. More pronounced insulin resistance during follow-up was associated with liver fibrosis progression. Unfortunately, fasting plasma glucose was not measured at baseline, and thus the prevalence of diabetes at onset of the study could not be reported [8]. In another cohort of 103 NAFLD patients who underwent a second liver biopsy at an average interval of 3.2 years, fibrosis staging progressed in 37%. Preexisting diabetes and early stages of fibrosis at first biopsy were predictors of fibrosis progression [7]. Otherwise, in a systematic review including ten longitudinal studies comprising 221 patients who had NASH on their initial biopsy, received no intervention of proven benefit regarding histology and underwent a second liver biopsy at least one year apart, only age and any degree of inflammation in the initial biopsy were the risk factors related to progression to advanced fibrosis. Other traditional parameters such as obesity, diabetes and hypertension were not statistically significant predictors [48]. Overall, inclusion of heterogeneous studies, disagreement on criteria for NASH diagnosis, liver biopsy sampling error and variability among pathologists are remarkable limitations of long-term histological studies. It is not clearly defined yet if NAFLD worsens glycemic control in patients with T2DM. NAFLD patients have shown both an impaired ability of insulin to suppress endogenous glucose production related to hepatic insulin resistance, and a reduction in glucose disposal, a measure of whole-body insulin sensitivity [49]. Indeed, intrahepatic triglyceride content may influence insulin requirements in diabetic patients via an effect on the sensitivity of endogenous glucose production to insulin [50]. On the other hand, NAFLD is known to be associated with insulin resistance, hyperinsulinemia and prediabetes, which includes both impaired fasting glucose (IFG) and impaired glucose tolerance (IGT). In an Australian study of 70 patients with NAFLD determined by ultrasound, 24% had IGT and 10% had diabetes on standard oral glucose tolerance test (OGTT). Over half of the NAFLD patients with a normal fasting glucose had 8380 July 14, 2014 Volume 20 Issue 26

73 Leite NC et al. NAFLD and diabetes: A review Lipolysis Peripheral glucose uptake Insulin resistance Delivery and synthesis of FFA Type 2 DM Hyperglycemia Hyperinsulinemia Gluconeogenesis Glycogenolysis De novo lipogenesis Key lipogenic Transcription factors NAFLD Figure 1 Pivotal role of insulin resistance in non-alcoholic fatty liver disease pathophysiology. : Decrease; : Increase. FFA: Free fatty acids; DM: Diabetes mellitus; NAFLD: Non-alcoholic fatty liver disease. abnormal glucose tolerance, as detected by OGTT. In addition, irrespective of the status of glucose tolerance, 2-h hyperinsulinemia during the OGTT occurred in all subjects with NAFLD, and fasting insulin resistance was found in 73%; fasting insulin resistance was assessed by the homeostasis model assessment method (HOMA-IR) and was defined by a HOMA-IR equal or greater than 2 [51]. In the Framingham Heart study with 2589 individuals, fatty liver was associated with T2DM, IFG, hypertension, metabolic syndrome, HDL-cholesterol, triglycerides, and adiponectin levels, even after multivariate adjustment for other fat depots, such as visceral adipose tissue, waist circumference, and body mass index [52]. Moreover, several prospective observational studies have shown an increased incidence of T2DM in patients with NAFLD diagnosed by ultrasonography or by liver biopsy. However, most of them were not adjusted for main covariates, such as family history of T2DM, physical activity and fasting glucose and insulin levels [8,53]. In a recent prospective community-based study in an urban adult population from Sri Lanka, individuals with ultrasonographic NAFLD showed an increased risk of developing T2DM. After three years follow-up, T2DM incidence rates were 64.2 and 34 per 1000 person/years for those with and without NAFLD, respectively, and NAFLD was an independent predictor of T2DM development [54]. In a longitudinal cohort study of 7849 Korean individuals followed-up for 5 years, those who had both elevated ALT and ultrasonographic liver steatosis had an increased risk of future diabetes development [55]. Another Korean study demonstrated that NAFLD had an independent and additive effect on the development of T2DM in patients with IFG at baseline [56]. Similarly, Musso et al [11] reported an increased risk for incident diabetes in patients with evidence of ultrasonographic and histological NAFLD in a recent meta-analysis of three large community-based cohorts with a range of followup from 4 to 10 years. Overall, it appears that DM and NAFLD are two conditions with intense interplay roles, one adversely affecting the natural history of the other, and vice-versa. PATHOGENETIC MECHANISMS The pathophysiological hallmark of NAFLD is the underlying insulin resistance (Figure 1). Insulin resistance at the level of the adipocyte seems to be the primary defect in NAFLD, leading to increased lipolysis. Any grade of insulin secretion deficit associated with DM further increases lipase activity in adipose tissue. This leads first to elevated circulating and portal free fatty acids (FFAs) and subsequently to their increased skeletal muscle and hepatic delivery and uptake, which decreases insulin action in these tissues [57,58]. Insulin resistance in these tissues leads to increased gluconeogenesis and glycogenolysis in liver as well as reduction in peripheral glucose disposal result July 14, 2014 Volume 20 Issue 26

74 Leite NC et al. NAFLD and diabetes: A review ing in hyperglycemia. The pancreatic beta islet cells adapt to hyperglycemia by increasing insulin secretion, leading to hyperinsulinemia. Hyperinsulinemia and hyperglycemia also upregulate several key lipogenic transcription factors, including sterol regulatory element-binding protein 1c (SREBP1c) and carbohydrate response element binding protein (ChREBP), promoting hepatic lipid synthesis or de novo lipogenesis [59]. In patients with NAFLD, studies have shown that the vast majority of hepatic fat originates from FFAs (59%), but 26% comes from de novo lipogenesis and 15% originates from the diet [60]. Hepatic steatosis results when the balance between delivery and synthesis of FFAs exceeds the liver capacity to oxidize or export them. Lipids are exported in the form of very-low-density lipoprotein (VLDL), and the synthesis of apolipoprotein B-100, which is the apolipoprotein contained in VLDL particles, is reduced in patients with NASH [61]. Accumulation of lipids can exert toxic effects on the liver by inefficient oxidation or activation of inflammatory pathways. Although hepatic triglycerides (triacylglycerol) are thought to be protective for NAFLD progression, certain lipid metabolites such as diacylglycerol and ceramides may themselves cause cell injury and death and contribute to NASH development [62]. FFAs may be oxidized within mitochondria, peroxisomes and microsomal system. Increased FFA oxidation causes an oxidative stress that further uncouples mitochondrial oxidation/phosphorylation and generates more reactive oxygen species (ROS). Indeed, abnormal mitochondrial morphology and function is frequently present in the hepatocytes of patients with NASH. These abnormalities might render hepatocytes even more susceptible to oxidative damage [63]. Oxidative stress generated from ROS promotes lipid peroxidation and augments inflammation by up-regulating key factors and pathways of NFκB and toll-like receptor (TLR) signaling [64]. Systemic subclinical inflammation also appears to be involved in the pathogenesis of NAFLD and NASH. Patients with NAFLD have higher circulating markers of inflammation than healthy controls. It has been shown that diabetic patients with NAFLD have higher circulating markers of inflammation than diabetic patients without NAFLD [65]. It has been also described that, as obesity increases, there is an increase in macrophage infiltration in adipose tissue. These activated macrophages secrete inflammatory cytokines such as TNF-alpha and IL-6 that may exacerbate insulin resistance by decreasing insulin signaling. In turn, serum levels of adiponectin, an anti-inflammatory insulin sensitizing and potentially hepatoprotective adipokine, are reduced in patients with NAFLD and are lower in NASH than in simple steatosis [66,67]. Furthermore, adiponectin levels fall in prediabetes and in T2DM and there could be a link between adipocyte dysfunction and NASH in these patients [68]. It is now recognized that NAFLD and particularly NASH progression results from a complex interplay between insulin resistance with hyperinsulinemia, increased oxidative stress, hepatic and systemic inflammation. Hyperinsulinemia combined with ongoing liver inflammation and hepatocyte apoptosis may induce profibrotic factors. Thus, profibrotic factors can contribute to fibrosis progression by activating hepatocyte stellate cells. NAFLD has recently been linked to alterations of gut microbiota and its metabolic effects. Increased absorption of lipopolysaccharides (LPS) resulting from a leaky small intestinal mucosa may cause activation of the innate immune system, by direct stimulation of TLR-signaling, leading to inflammation and insulin resistance [69]. Of note, patients with T2DM had mean plasma levels of LPS higher than controls due to a higher small intestinal bacterial overgrowth and increased leakiness of the intestinal mucosa [70]. Genetic factors may also play a role in the development of NAFLD. Based on the complex mechanisms involved in the pathogenesis of NAFLD, there is a low likelihood of finding a single candidate gene responsible for NAFLD or a clear genetic link between T2DM and NAFLD. Through several genome-wide association studies, the missense rs C/G single-nucleotide polymorphism implying an amino acid change from isoleucine (I) to methionine (M) at the position 148 (I148M) of the protein encoding by the patatin-like phospholipase domaincontaining 3 gene (PNPLA3) was strongly associated with increased hepatic fat content and NAFLD histological severity [71,72]. Genetic variation at PNPLA3 seems to confer a markedly increased risk of severe histological features of NAFLD, but there is no association of this genetic polymorphism with body mass index, triglyceride, HDLand LDL-cholesterol levels, or diabetes [73]. Other polymorphisms in the microsomal triglyceride transfer protein or in the gene of superoxide dismutase 2 could play a role in the interaction between NAFLD and diabetes [74,75]. The main physiopathological mechanisms involved in NAFLD progression from simple steatosis to NASH and fibrosis are summarized in Figure 2. Diagnosis When NAFLD is suspected, the first step to define its diagnosis is to exclude other known etiologies of chronic liver diseases like drug-related steatosis [76,77], viruses and alcohol. As previously described, a careful history of alcohol ingestion must be taken. Of note, diabetic patients with excessive alcohol intake may have both alcoholicand NAFLD [78]. Liver enzymes may be elevated, but normal aminotransferases do not exclude the diagnosis of NAFLD, even in diabetic individuals [4,79]. Aminotransferase levels have been reevaluated and new thresholds have been suggested for normal levels when considering patients with NAFLD. These levels are 19 U/L for men and 30 U/L for women and this has improved the sensitivity for diagnosing NAFLD [80], although the diagnosis of NASH still cannot be performed based solely on aminotransferases. Although unspecific, serum ferritin levels may be high and it is important to discard hemochromatosis 8382 July 14, 2014 Volume 20 Issue 26

75 Leite NC et al. NAFLD and diabetes: A review Steatosis progression to NASH Lipid metabolites (diacylglycerol and ceramides): lipotoxic effects FFA oxidation: Reactive oxygen species Adiponectin serum levels Hepatic and systemic inflammation Altered gut microbiota and its metabolic effects Profibrotic factors activating hepatocyte stellate cells Figure 2 Main physiopathological mechanisms influencing non-alcoholic fatty liver disease. Progression to non-alcoholic steatohepatitis and fibrosis. : Decrease; : Increase. NASH: Non-alcoholic steatohepatitis; FFA: Free fatty acids. in patients with a high transferrin saturation index [81]. Low-titer autoantibodies, such as anti-nuclear and antismooth muscle, can be found as an epiphenomenon in NAFLD [82], although a liver biopsy may be indicated to exclude autoimmune liver disease. The diagnostic approach to patients with NAFLD is based mainly on imaging, serological and histopathological methods. Apart from DM, other clinical conditions are associated with NAFLD, such as essential hypertension, obesity, hypertriglyceridemia, polycystic ovary disease and metabolic syndrome [83-85]. Thus, NAFLD should also be investigated in these clinical settings, and routinely in DM. The spectrum of NAFLD is similar in diabetic and non-diabetic individuals, and it develops from simple steatosis to advanced fibrosis, cirrhosis and hepatocellular carcinoma [86]. The only reliable method that identifies these different stages is liver biopsy. However, owing to its potential complications and limitations like cost, sampling error and procedure risks, many non-invasive methods have been proposed to diagnose NAFLD and to predict those patients with a higher risk of having NASH [87,88]. Imaging methods have a variable accuracy to identify liver steatosis [89]. Liver ultrasonography (US) is a safe, inexpensive and readily available method. It is the most used technique to diagnose NAFLD with a sensitivity of 60%-94% and a specificity of 66%-95% for detecting steatosis [90,91]. Its main limitation is that it is operatordependent and cannot detect mild steatosis (5%-30%) [90-92]. Additional Doppler fluxometry helps identify indirect signs of advanced liver disease. Recently, Ballestri et al [93] developed the ultrasonographic fatty liver indicator (US- FLI), a new score ranging from 2 to 8 points, which is capable of ruling out NASH based on US parameters like the intensity of liver and kidney contrast, posterior attenuation of US beam, vessel blurring, difficult visualization of gallbladder wall, difficult visualization of the diaphragm and areas of focal sparing. An US-FLI < 4 has a negative predictive value for NASH of 94% and can be easily assessed [93]. Computed tomography (CT) allows quantitative and qualitative evaluation of liver steatosis with a higher accuracy. Based on the difference of the hepatic-splenic attenuation, unenhanced CT can detect liver steatosis grades as low as 5% [94]. Magnetic resonance (MR) imaging with appropriate sequences also provides high sensitivity and specificity. MR spectroscopy is one of the most accurate methods for the evaluation of liver steatosis, has a strong correlation with histology and can detect very low levels of steatosis [95]. Recently, MR elastography has showed a high predictive value for excluding advanced fibrosis and a good accuracy for detecting NASH with an area under ROC curve of MR elastography discriminated NASH from steatosis with a sensitivity of 94% and specificity of 73% with a cutoff of 2.74 kilopascals units (kpa) [96]. However, MR is too expensive to be used routinely, but might be useful in patients under study protocols and in those with a strong suspicious of NAFLD with normal liver echogenicity on ultrasound [97]. A novel method to diagnose and quantify steatosis is the controlled attenuated parameter (CAP) [98]. CAP is software that can be used simultaneously with liver transient elastography available by Fibroscan [99,100]. It is a simple and easily performed method that can detect liver steatosis at levels as low as 5%. Sasso et al [99] defined the best cutoff value of 292 for severe steatosis (> 66%) detection, with a negative predictive value of 100%. Its main limitation is the difficulty of obtaining reliable measurements in obese patients. When liver steatosis is estimated using CAP, liver stiffness is also evaluated by Fibroscan elastography. In this method, vibrations of mild amplitude and low frequency are transmitted by the 8383 July 14, 2014 Volume 20 Issue 26

76 Leite NC et al. NAFLD and diabetes: A review transducer, inducing an elastic shear wave that propagates through the liver. The velocity of wave propagation relates directly to liver stiffness or fibrosis: the stiffer the tissue, the faster the shear wave propagates. The velocity of the shear wave propagation is measured in kilopascals (kpa). Higher tissue stiffness corresponds to increasing severity of fibrosis. Wong et al [101] defined a cutoff of 10.3 kpa in NAFLD patients to predict advanced fibrosis with a sensitivity of 92% and specificity of 88%. The negative predictive value of this cutoff for advanced fibrosis was 99%. The possibility of evaluating fibrosis and steatosis simultaneously makes the Fibroscan a valuable tool in the study of NAFLD. Many serological methods have been evaluated in the diagnosis of NAFLD regarding their accuracy for detecting NASH [102]. In this setting, the AST/ALT ratio [103], FIB-4 index [87], the BARD score [47], NAFLD Fibrosis Score [46], and the enhanced liver fibrosis (ELF) test shall be addressed. The AST/ALT ratio has been used to identify patients with advanced fibrosis and a value > 1 may predict advanced fibrosis in patients with NAFLD [103]. The FIB-4 index is easily calculated using the following formula: [age (years) AST (U/L)]/[platelet (10 9 /L) square root of ALT (U/L)] being useful for the diagnosis of liver fibrosis, but not capable of diagnosing NASH. It was described that a FIB-4 index 2.67 had an 80% positive predictive value and a FIB-4 index 1.30 had a 90% negative predictive value for fibrosis in patients with NAFLD, although other non-invasive tests like the ELF and Fibrotest were more accurate than FIB-4 index to predict advanced fibrosis [87,104]. The NAFLD fibrosis score and the BARD score include DM as a variable in their formula. The BARD score is composed of three variables: an AST/ALT ratio 0.8 sums 2 points; a BMI 28 sums 1 point; presence of diabetes sums 1 point. The possible score ranges from 0 to 4 points. According to the results by Harrison et al [47], score values of 0 or 1 would have a high negative predictive value (NPV) for severe fibrosis. The NAFLD score comprises demographic and easily obtained laboratory variables as age, BMI, hyperglycemia, platelet count, albumin and AST/ALT ratio. The formulae { [0.037 age (years)] + (0.094 BMI) + [1.13 IFG/diabetes (yes = 1, no = 0)] + (0.99 AST/ALT) - [0.013 platelet (10 9 /L)] - [0.66 albumin (g/dl)]} can be assessed online at com. According to several studies it has a high accuracy to predict advanced fibrosis. Musso et al [11] showed in a meta-analysis that the NAFLD score had a sensitivity and specificity higher than 90% to predict advanced fibrosis and suggested that the combination of two non-invasive methods like the NAFLD score and Fibroscan elastography could be a useful tool in this setting. The ELF panel, obtained through the assessment of three matrix turnover proteins (hyaluronic acid, TIMP-1 and PIIINP) displayed a high accuracy in predicting fibrosis, although its use may be limited by cost and local availability [105]. Cytokeratin-18 (CK-18) is a serological marker of apoptosis that can be used alone or in combination and was highly accurate for NASH detection [106]. Its main limitation for clinical use is that a well-established cutoff is not yet defined. So far, studies evaluating the different serum biomarkers are comprised of general patients with NAFLD and there is no specific test for patients with DM. Recently, we studied several serum biomarkers in 78 biopsyproven NAFLD diabetic patients and showed an association between low levels of adiponectin and TGF-β1 with severe NAFLD stages [67]. Maybe the combination of two methods like serum biomarkers and imaging methods might be the best tool for predicting NASH and advanced fibrosis. The histological diagnosis of NAFLD is defined as the presence of lipid deposit in more than 5% of the hepatocytes independent of the localization into the hepatic lobule. However, the most important issue is the definition of NASH, owing to its prognostic value. Brunt et al [16] in 1999 classified NAFLD into three different stages: mild, moderate and severe. Likewise, Matteoni s classification of NAFLD was based on the severity of hepatic lesion as follows: type 1, isolated steatosis; type 2, steatosis and lobular inflammation; type 3, steatosis and ballonization of hepatocytes; and type 4, which added the presence of hyaline bodies and fibrosis to the previous stages. Stages 3 and 4 were considered as NASH [15]. Kleiner et al [17] in 2005 proposed an update on Brunt s classification and defined a score named NAS, based on the sum of three criteria: steatosis (graded 0 to 3), lobular inflammation (graded 0 to 3) and ballonization (graded 0 to 2). A NAS 5 points implies an advanced inflammatory activity. However, this score should not be applied to diagnose NASH because many patients have NASH with a NAS < 4 points. The NAS is a useful tool to evaluate treatment response and should be used in this situation. Thus, the hallmarks to the diagnosis of NASH are the histological findings observed in liver biopsy and not its intensity. Currently, NASH is defined by the combination of steatosis and necroinflammatory lesions, like ballonization, with or without fibrosis [107]. Treatment There are very few randomized, blinded and controlled clinical trials of drugs with sufficient duration and adequate histological outcomes in patients with NAFLD and DM. Hence, data on treatment of NAFLD in diabetic patients are scarce, and treatment of NAFLD in diabetic patients is conducted based on evidences from mixed populations of diabetic and non-diabetic individuals. Weight loss following caloric restriction and physical exercise improves insulin sensitivity and cardiometabolic risk factors. However, both implementation and maintenance of these lifestyle interventions pose challenges for most of the individuals [108,109]. A 5% weight loss through lifestyle modification improved liver biochemistry and reduced hepatic steatosis [110], however at least a 10% weight reduction was required for a significant improvement in inflammation, ballooning, and NAS [110]. Notably, seden July 14, 2014 Volume 20 Issue 26

77 Leite NC et al. NAFLD and diabetes: A review Table 2 Current data on non-pharmacological treatments of non-alcoholic fatty liver disease Ref. Sample size Type 2 diabetes Type of intervention Study design/ duration Liver enzymes Imaging Histology Kistler et al [111] 813 adults 25% Inactive or moderate or vigorous exercise Hallsworth 19 adults Resistance et al [112] exercise Bacchi et al [113] 31 adults 100% Aerobic (AER) or resistance (RES) training Mathurin 381 adults 25% Bariatric et al [114] surgery Mummadi et al [115] 766 paired liver biopsies Bariatric surgery Retrospective analysis of biopsyproven NAFLD Randomly assigned to either exercise or standard care. 8 wk Randomized controlled study. 4 mo Prospective study. Follow-up of 5 yr Systematic review and meta-analysis (15 studies) Vigorous recommendations was associated with GGT levels compared with being inactive No significant changes in ALT levels Significant in ALT and GGT levels 1 and 5 yr after bariatric surgery Resistance exercise: 13% relative in liver lipid by 1 H-MRS Vigorous exercise was associated with a adjusted odds of NASH Hepatic fat content was in both by in-opposedphase MR imaging Significant in NASH Fibrosis, 96% with F1 or resolution 81.3% in NASH and 65.5% in fibrosis : Decrease; : Increase. ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; GGT: Gammaglutamyl transferase; AP: Alkaline phosphatase; NAFLD: Non-alcoholic fatty liver disease; NASH: Non-alcoholic steatohepatitis; 1 H-MRS: Proton magnetic resonance spectroscopy; F1: Stage 1 of fibrosis. tary patients with NAFLD and DM should undergo a cardiovascular risk assessment before initiating a fitness program, especially before a high intensive training. In a retrospective study with 813 individuals with biopsyproven NAFLD from the Nonalcoholic Steatohepatitis Clinical Research Network, neither moderate intensity exercise nor total exercise per week were associated with NASH or stage of fibrosis. In this study, meeting vigorous recommendations was associated with decreased adjusted odds of having NASH. This study suggested that maybe the intensity of the exercise could be more important than its duration [111]. Notwithstanding, Hallsworth et al [112] showed that independent of weight loss, moderate anaerobic exercise seemed to improve insulin sensitivity and hepatic steatosis. In a recent randomized controlled trial, Bacchi et al [113] compared the effects of aerobic (AER) or resistance (RES) training on hepatic fat content in 31 type 2 diabetic subjects with NAFLD. Hepatic fat content was markedly reduced in both AER and RES training groups. In addition, hepatic steatosis, defined as hepatic fat content > 5.56% by an in-opposedphase magnetic resonance imaging technique, was not detected in about one-quarter of the patients in each intervention group. Because NAFLD is present in the majority of patients who undergo bariatric surgery, there has been growing interest in evaluating the role of foregut surgery in NAFLD treatment. In a prospective study with 381 severely obese adults followed-up for 5 years after surgery, significant improvements in steatosis, ballooning, NAS and resolution of NASH were observed, changes already present at the first year [114]. After 5 years, levels of fibrosis increased, but 95.7% of patients maintained a grade 1 fibrosis. As none of the patients had advanced fibrosis at entry, the effect of bariatric surgery on liver fibrosis could not be evaluated [114]. In a meta-analysis that evaluated the influence of bariatric surgery on liver histology in adults with NAFLD, Mummadi et al [115] found that steatosis, NASH, and fibrosis improved or completely resolved in a significant proportion of patients. At this moment, there is still no clear evidence indicating foregut bariatric surgery as an established option to specifically treat NASH, but it may provide benefit in NAFLD treatment in otherwise eligible obese individuals [115]. Table 2 summarizes the principal studies concerning non-pharmacological interventions in patients with NAFLD. High doses of omega-3 polyunsaturated fatty acids (PUFAs) are effective in treating hypertriglyceridemia, which is often a feature of NAFLD and T2DM. The efficacy of omega-3 PUFAs supplementation in NAFLD has recently been examined in a systematic review of nine eligible studies, involving 355 patients with NAFLD [116]. This systematic review with different doses of omega-3 PUFAs demonstrated significant reductions in hepatic fat content. However, at this point, the optimal dose and duration of this therapy is not yet established. A large randomized placebo-controlled trial of two doses of eicosapentanoic acid is under way in the United States. Many drugs have been evaluated in NAFLD management. The main studies on pharmacological treatments of NAFLD are resumed on Table 3. Statin therapy is recommended in patients with overt cardiovascular disease and in almost all patients with T2DM. Additionally, these drugs can be used in dyslipidemic subjects with increased baseline liver enzymes and may even produce some histological benefit in NASH [117,118]. Ursodesoxycholic acid (UDCA) is a secondary bile acid with lipid lowering, antiapoptotic and anti-inflammatory properties. There has been initial interest in the use of UDCA to treat NAFLD, although double-blind, randomized, placebo-controlled 8385 July 14, 2014 Volume 20 Issue 26

78 Leite NC et al. NAFLD and diabetes: A review Table 3 Current data on pharmacological treatments of non-alcoholic fatty liver disease Ref. Sample size Type 2 diabetes Type of intervention/ drug Study design/ duration Liver enzymes Imaging Histology Parker et al [116] 355 Omega-3 PUFA: g/d Lindor et al [119] 166 UDCA: mg/kg per day Leuschner et al [120] 185 UDCA: mg/kg per day Systematic review and meta-analysis (9 studies) median duration of treatment: 6 mo Randomized placebo-controlled study (24 mo) Randomized placebo-controlled study (18 mo) Uygun et al [121] 36 0% Metformin: 1.7 g/d Randomized placebo-controlled study (6 mo) Haukeland et al [122] 48 27% Metformin: g/d Randomized placebo-controlled study (6 mo) Shields et al [123] 19 0% Metformin: 500 mg-1 g/d Randomized placebo-controlled trial (12 mo) Lutchman et al [126] 18 0% Pioglitazone: 30 mg/d Prospective open study (12 mo) Belfort et al [127] 55 48% Pioglitazone: 45 mg/d Randomized placebo-controlled study (6 mo) Aithal et al [128] 74 0% Pioglitazone: 30 mg/d Randomized placebo-controlled trial (12 mo) Sanyal et al [129] 247 0% Vitamin E: 800 UI/d Pioglitazone: 30 mg/d Randomized placebo-controlled trial (24 mo) Significant efficacy of PUFA on ALT and AST levels No significant changes in ALT, AST and GGT levels with UDCA No significant changes in ALT and AST, GGT levels with UDCA Significant in ALT and AST levels with metformin No significant changes in ALT, AST levels with metformin No significant changes in ALT and AST levels with metformin ALT levels normalized in 72% Significant efficacy of pioglitazone on ALT and AST levels Significant in ALT and GGT levels Significant in ALT, AST and GGT levels with both treatments Significant efficacy of PUFA on liver fat (US, 1 H-MRS) Significant efficacy of metformin on liver fat (US) No significant on liver fat (CT) with metformin Hepatic fat content was by MR imaging Significant efficacy of pioglitazone on liver fat ( 1 H-MRS) No significant changes in NASH or fibrosis with UDCA No significant changes in NASH or fibrosis with UDCA No significant in inflammatory activity or fibrosis with metformin No significant changes in NASH with metformin No significant changes in NASH or fibrosis with metformin significant in necroinflammation and fibrosis with pioglitazone Significant in necroinflammation but not in fibrosis with pioglitazone Significant in inflammatory activity and fibrosis with pioglitazone Significant of NASH with vitamin E. No changes in fibrosis with either treatment : Decrease; : Increase. ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; GGT: Gammaglutamyl transferase; AP: Alkaline phosphatase; NAFLD: Non-alcoholic fatty liver disease; NASH: Non-alcoholic steatohepatitis; PUFA: Polyunsaturated fatty acids; UDCA: Ursodeoxycholic acid; US: Ultrasonography; MR: Magnetic resonance; 1 H-MRS: Proton magnetic resonance spectroscopy; CT: Computed tomography. trials with doses ranging from 13 to 28 mg/kg per day and pre- and post-treatment liver biopsies have yielded disappointing results [119,120]. Given the importance of insulin resistance in the pathogenesis of NAFLD, insulin-sensitizing agents have been investigated in the treatment of this condition in patients with and without diabetes. Metformin reduces endogenous glucose production and improves whole-body insulin sensitivity. It is the first-line choice in oral therapy for patients with T2DM. Metformin has beneficial effects on serum aminotransferases and insulin resistance. However, in patients with NAFLD without T2DM, a number of small randomized placebo-controlled clinical trials with different doses ( mg/d) and short durations (6-12 mo) have failed to demonstrate an improvement in liver steatosis, inflammation or fibrosis [ ]. In spite of these poor results, there is evidence from case-control and population-based studies that the use of metformin was associated with risk reduction for the development of hepatocellular carcinoma in diabetic patients [124,125]. Rosiglitazone and pioglitazone are peroxisome proliferator-activated receptor γ (PPAR γ) agonists that redistribute fat from the muscle and liver to peripheral adipose tissue and, thereby, improve insulin resistance. Concerns have been raised regarding an association between increased cardiovascular risk with rosiglitazone and its use has been restricted. Three studies of pioglitazone with doses ranging from 30 to 45 mg found a significant improvement in liver histology when compared with placebo in patients with NASH [ ], but improvement of fibrosis was demonstrated in only one study [128]. Moreover, among these studies only one examined a cohort of patients with T2DM or impaired glucose tolerance with NASH; in this study, pioglitazone significantly improved steatosis, hepatocellular ballooning, inflammation and 8386 July 14, 2014 Volume 20 Issue 26

79 Leite NC et al. NAFLD and diabetes: A review necroinflammation, compared with placebo. Improvement in NAS was seen in 73% of patients treated with pioglitazone compared to 24% of placebo-treated patients, and there was a trend toward improvement in fibrosis in patients receiving pioglitazone [127]. The PIVENS study [129] is a recent clinical trial that randomized 247 non-diabetic patients with biopsy-proven NASH to pioglitazone 30 mg/d, vitamin E 800 IU/d, or placebo for 24 mo. The primary outcome was histological improvement in the features of NASH. Pioglitazone, as compared to placebo, was not associated with a significantly higher rate of improvement in the composite NAS score. However, both vitamin E and pioglitazone treatment improved the scores of steatosis, inflammation, ballooning, and serum aminotransferase levels [129]. It seems that liver histology benefits obtained with pioglitazone therapy may disappear with its discontinuation. Nonetheless, there is a debate surrounding the long-term risk-benefit ratio of pioglitazone therapy. The most frequent side-effects of pioglitazone are weight gain of 2-5 kg and bone loss with fractures [130]. Pioglitazone treatment can also precipitate congestive heart failure in patients with preexisting cardiac failure [131]. In addition, increased bladder cancer risk has been recently associated with pioglitazone use in diabetic patients [132]. Glucagon-like peptide-1 (GLP-1) agonists and dipeptidyl peptidase-Ⅳ (DPP-Ⅳ) inhibitors are new pharmacological agents with multiple anti-hyperglycemic actions. The biological activities of GLP-1 agonists include glucose-dependent insulin secretion, suppression of postprandial glucagon to reduce hepatic glucose release and slowing of gastric emptying. There is also evidence that GLP-1 agonists have beneficial effects on the liver, including suppression of hepatic lipogenesis and stimulation of lipid oxidation [133,134]. A recent meta-analysis of two GLP-1 agonists, liraglutide and exenatide, in populations with and without diabetes, including data on liver enzyme tests from 12 of the 25 trials included, found that ALT concentrations decreased after treatment with the liraglutide but not with exenatide [135]. Clinical trials with these agents in patients with NAFLD with or without T2DM are ongoing and their results are awaited in the next years. Increased oxidative stress occurs in NAFLD and T2DM. Among antioxidant compounds, vitamin E has the most significant evidence supporting its use. In the PIVENS study [129], vitamin E supplementation, 800 UI/d, resulted in significant improvement in pathological features of NASH. The improvement in NAS was observed in 42% of patients receiving vitamin E compared with 19% of patients receiving placebo. Nevertheless, caution must be applied regarding the long-term safety of vitamin E, especially in doses greater than 400 IU/d, which may be associated with increased risk of all-cause mortality [136]. Currently, there is no evidence regarding vitamin E effectiveness and safety in diabetic patients with NASH or in patients with NASH-related cirrhosis. GFT505, a dual peroxisome proliferator-activated receptor (PPAR)-α/δ agonist, improved peripheral and hepatic insulin sensitivity in a randomized crossover study to subsequent 8-week treatment periods with GFT505 (80 mg/d) or placebo. GFT505 also reduced liver enzyme concentrations and could be a promising drug candidate for the treatment of T2DM and NAFLD. There was no indication of PPARγ activation and no safety concern with GFT505 [137]. Obeticholic acid (OCA), a farnesoid X agonist receptor, is a semi-synthetic human bile acid that regulates glucose and lipid metabolism. Data from a small pilot study demonstrate that OCA improves insulin sensitivity compared with placebo. Also, of importance, OCA appears to improve liver injury in patients with T2DM and NAFLD [138]. Larger studies with longer duration of therapy and follow-up are needed to evaluate long-term efficacy of these emerging therapies. CONCLUSION Patients with DM and NAFLD are prone to the severest stages of liver diseases and to cardiovascular and liverrelated outcomes. The major challenge is to identify these patients by accurate non-invasive methods. Many algorithms and new imaging methods are available but they still need to be validated in this specific population. The ideal treatment would be effective for both NASH and diabetes, but it is not yet available. Given the importance of cardiovascular and liver outcomes in diabetic patients, effective interventions are urgently required in order to prevent progression to these life-threatening and prevalent complications. 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84 Leite NC et al. NAFLD and diabetes: A review land K. Metformin in patients with non-alcoholic fatty liver disease: a randomized, controlled trial. Scand J Gastroenterol 2009; 44: [PMID: DOI: / ] 123 Shields WW, Thompson KE, Grice GA, Harrison SA, Coyle WJ. The Effect of Metformin and Standard Therapy versus Standard Therapy alone in Nondiabetic Patients with Insulin Resistance and Nonalcoholic Steatohepatitis (NASH): A Pilot Trial. Therap Adv Gastroenterol 2009; 2: [PMID: DOI: / X ] 124 Hassan MM, Curley SA, Li D, Kaseb A, Davila M, Abdalla EK, Javle M, Moghazy DM, Lozano RD, Abbruzzese JL, Vauthey JN. Association of diabetes duration and diabetes treatment with the risk of hepatocellular carcinoma. Cancer 2010; 116: [PMID: DOI: / cncr.24982] 125 Lai SW, Chen PC, Liao KF, Muo CH, Lin CC, Sung FC. Risk of hepatocellular carcinoma in diabetic patients and risk reduction associated with anti-diabetic therapy: a populationbased cohort study. Am J Gastroenterol 2012; 107: [PMID: DOI: /ajg ] 126 Lutchman G, Modi A, Kleiner DE, Promrat K, Heller T, Ghany M, Borg B, Loomba R, Liang TJ, Premkumar A, Hoofnagle JH. The effects of discontinuing pioglitazone in patients with nonalcoholic steatohepatitis. Hepatology 2007; 46: [PMID: DOI: /hep.21661] 127 Belfort R, Harrison SA, Brown K, Darland C, Finch J, Hardies J, Balas B, Gastaldelli A, Tio F, Pulcini J, Berria R, Ma JZ, Dwivedi S, Havranek R, Fincke C, DeFronzo R, Bannayan GA, Schenker S, Cusi K. A placebo-controlled trial of pioglitazone in subjects with nonalcoholic steatohepatitis. N Engl J Med 2006; 355: [PMID: DOI: /NEJMoa060326] 128 Aithal GP, Thomas JA, Kaye PV, Lawson A, Ryder SD, Spendlove I, Austin AS, Freeman JG, Morgan L, Webber J. Randomized, placebo-controlled trial of pioglitazone in nondiabetic subjects with nonalcoholic steatohepatitis. Gastroenterology 2008; 135: [PMID: DOI: /j.gastro ] 129 Sanyal AJ, Chalasani N, Kowdley KV, McCullough A, Diehl AM, Bass NM, Neuschwander-Tetri BA, Lavine JE, Tonascia J, Unalp A, Van Natta M, Clark J, Brunt EM, Kleiner DE, Hoofnagle JH, Robuck PR. Pioglitazone, vitamin E, or placebo for nonalcoholic steatohepatitis. N Engl J Med 2010; 362: [PMID: DOI: /NEJMoa ] 130 Kelly IE, Han TS, Walsh K, Lean ME. Effects of a thiazolidinedione compound on body fat and fat distribution of patients with type 2 diabetes. Diabetes Care 1999; 22: [PMID: ] 131 Hernandez AV, Usmani A, Rajamanickam A, Moheet A. Thiazolidinediones and risk of heart failure in patients with or at high risk of type 2 diabetes mellitus: a meta-analysis and meta-regression analysis of placebo-controlled randomized clinical trials. Am J Cardiovasc Drugs 2011; 11: [PMID: DOI: / ] 132 Lewis JD, Ferrara A, Peng T, Hedderson M, Bilker WB, Quesenberry CP, Vaughn DJ, Nessel L, Selby J, Strom BL. Risk of bladder cancer among diabetic patients treated with pioglitazone: interim report of a longitudinal cohort study. Diabetes Care 2011; 34: [PMID: DOI: /dc ] 133 Ben-Shlomo S, Zvibel I, Shnell M, Shlomai A, Chepurko E, Halpern Z, Barzilai N, Oren R, Fishman S. Glucagonlike peptide-1 reduces hepatic lipogenesis via activation of AMP-activated protein kinase. J Hepatol 2011; 54: [PMID: DOI: /j.jhep ] 134 Svegliati-Baroni G, Saccomanno S, Rychlicki C, Agostinelli L, De Minicis S, Candelaresi C, Faraci G, Pacetti D, Vivarelli M, Nicolini D, Garelli P, Casini A, Manco M, Mingrone G, Risaliti A, Frega GN, Benedetti A, Gastaldelli A. Glucagonlike peptide-1 receptor activation stimulates hepatic lipid oxidation and restores hepatic signalling alteration induced by a high-fat diet in nonalcoholic steatohepatitis. Liver Int 2011; 31: [PMID: DOI: / j x] 135 Vilsbøll T, Christensen M, Junker AE, Knop FK, Gluud LL. Effects of glucagon-like peptide-1 receptor agonists on weight loss: systematic review and meta-analyses of randomised controlled trials. BMJ 2012; 344: d7771 [PMID: DOI: /bmj.d7771] 136 Miller ER, Pastor-Barriuso R, Dalal D, Riemersma RA, Appel LJ, Guallar E. Meta-analysis: high-dosage vitamin E supplementation may increase all-cause mortality. Ann Intern Med 2005; 142: [PMID: DOI: / ] 137 Cariou B, Hanf R, Lambert-Porcheron S, Zaïr Y, Sauvinet V, Noël B, Flet L, Vidal H, Staels B, Laville M. Dual peroxisome proliferator-activated receptor α/δ agonist GFT505 improves hepatic and peripheral insulin sensitivity in abdominally obese subjects. Diabetes Care 2013; 36: [PMID: DOI: /dc ] 138 Mudaliar S, Henry RR, Sanyal AJ, Morrow L, Marschall HU, Kipnes M, Adorini L, Sciacca CI, Clopton P, Castelloe E, Dillon P, Pruzanski M, Shapiro D. Efficacy and safety of the farnesoid X receptor agonist obeticholic acid in patients with type 2 diabetes and nonalcoholic fatty liver disease. Gastroenterology 2013; 145: e1 [PMID: DOI: /j.gastro ] P- Reviewers: Buchler C, Takahashi Y S- Editor: Ma YJ L- Editor: O Neill M E- Editor: Ma S 8392 July 14, 2014 Volume 20 Issue 26

85 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. WJG 20 th Anniversary Special Issues (12): Nonalcoholic fatty liver disease TOPIC HIGHLIGHT Clinical differences between alcoholic liver disease and nonalcoholic fatty liver disease Nobuyuki Toshikuni, Mikihiro Tsutsumi, Tomiyasu Arisawa Nobuyuki Toshikuni, Tomiyasu Arisawa, Department of Gastroenterology, Kanazawa Medical University, Ishikawa , Japan Mikihiro Tsutsumi, Department of Hepatology, Kanazawa Medical University, Ishikawa , Japan Author contributions: Toshikuni N wrote the manuscript; Arisawa T and Tsutsumi M supervised the work. Correspondence to: Nobuyuki Toshikuni, MD, Department of Gastroenterology, Kanazawa Medical University, 1-1 Daigaku, Uchinada-machi, Ishikawa , Japan. n.toshikuni@gmail.com Telephone: Fax: Received: October 28, 2013 Revised: January 29, 2014 Accepted: March 19, 2014 Published online: July 14, 2014 Abstract Alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD) are serious health problems worldwide. These two diseases have similar pathological spectra, ranging from simple hepatic steatosis to steatohepatitis, liver cirrhosis, and hepatocellular carcinoma. Although most subjects with excessive alcohol or food intake experience simple hepatic steatosis, a small percentage of individuals will develop progressive liver disease. Notably, both ALD and NAFLD are frequently accompanied by extrahepatic complications, including cardiovascular disease and malignancy. The survival of patients with ALD and NAFLD depends on various disease-associated conditions. This review delineates the clinical characteristics and outcomes of patients with ALD and NAFLD by comparing their epidemiology, the factors associated with disease susceptibility and progression, and the predictors and characteristics of outcomes. A comprehensive understanding of the characteristics and outcomes of ALD and NAFLD is imperative in the management of these chronic liver diseases Baishideng Publishing Group Inc. All rights reserved. Key words: Alcoholic liver disease; Nonalcoholic fatty liver disease; Clinical characteristics; Outcomes; Chronic liver disease Core tip: Although alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD) have similar pathological spectra, from simple hepatic steatosis to steatohepatitis and liver cirrhosis, the epidemiological and clinical characteristics of these two diseases differ. Comparative analyses of the factors associated with disease susceptibility and progression and the predictors and characteristics of outcomes would be helpful in the management of these diseases. Notably, both ALD and NAFLD are frequently accompanied by extrahepatic complications, including cardiovascular disease and malignancy, which can influence patient survival. Toshikuni N, Tsutsumi M, Arisawa T. Clinical differences between alcoholic liver disease and nonalcoholic fatty liver disease. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: INTRODUCTION Excessive alcohol intake is associated with various diseases, including alcoholic liver disease (ALD), cardiovascular disease, and cancer [1]. The prevalence of alcohol use disorders increased dramatically in the sixteenth century when alcohol distillation became more widespread, and alcohol-associated diseases have continued to afflict humanity [2]. A recent review reported that alcohol substantially contributes to the global burden of disease and is responsible for 4.6% of disability-adjusted life-years and 3.8% of all deaths [1]. Moreover, alcohol has a negative effect on socioeconomic activities; the cost of excessive social drinking is 1% or more of the gross domestic 8393 July 14, 2014 Volume 20 Issue 26

86 Toshikuni N et al. Clinical differences between ALD and NAFLD product in high-income countries [3]. Excessive food intake can lead to overweight and obesity. An estimated 1.46 billion adults worldwide had a body mass index (BMI) of 25 kg/m 2 or higher in 2008, which reflects modern overnutrition [4]. Metabolic syndrome often develops in obese individuals, and nonalcoholic fatty liver disease (NAFLD) has been recognized as the liver manifestation of metabolic syndrome [5]. A recent study from the United States found that the annual healthcare costs were 1.6-fold higher for subjects with metabolic syndrome than for those without metabolic syndrome [6]. ALD and NAFLD are both serious health and socioeconomic problems worldwide. Although these diseases have similar pathological spectra, ranging from simple hepatic steatosis to steatohepatitis and liver cirrhosis [7], ALD and NAFLD differ from each other in many characteristics, ranging from differences in clinical features to patient outcomes. A comparison of these diseases may result in a better understanding and management of both ALD and NAFLD. Therefore, in this review, we comprehensively characterized ALD by comparing its clinical features and outcomes with those of NAFLD. EPIDEMIOLOGY Table 1 displays the epidemiology of ALD and NAFLD. Comprehensive data regarding the prevalence of ALD are scarce. Geographic differences in alcohol intake have been reported. For example, Eastern Europe has the highest annual per capita intake (15.7 L per person), while North Africa and the Middle East have the lowest (1.0 L per person) [8]. Although patterns of alcohol intake may affect the prevalence of ALD, studies from Europe, the United States, and Asia, in which hepatic steatosis was assessed using ultrasonography or magnetic resonance imaging, reported that the prevalence of NAFLD ranged from 12.9% to 46.0% [9-12]. A study from Japan demonstrated that the prevalence of NAFLD had increased over time from 12.6% in 1989 to 28.4% in 2000 [13]. Moreover, a recent study from China, where the per capita annual alcohol intake is 4-6 L per person [8], found that the prevalence rates of ALD and NAFLD were 4.5% and 15.0%, respectively, and both rates are expected to increase in the future [14]. Age A study from the United States revealed that the peak prevalence of hospitalization for alcohol-related conditions was between 45 and 69 years of age [15]. In contrast, the prevalence of NAFLD in a large Japanese study peaked at years of age in males and at age years in females [12] ; however, a large study in China found that the prevalence of NAFLD in both males and females peaked at years of age [16]. In addition, NAFLD is commonly observed in children; a recent autopsy study from the United States revealed that approximately 10% of children had NAFLD [17]. Gender ALD is predominantly observed in males. For example, a cross-sectional study revealed that the male to female ratio of patients with alcoholic liver cirrhosis was 9:1 [18]. A nationwide study in the United States that assessed patients discharged from the hospital following a diagnosis of ALD found that 4.5 per persons had acute alcoholic hepatitis, with a male to female ratio of 1.83:1, and that 13.7 per persons had chronic alcoholic hepatitis with cirrhosis, with a male to female ratio of 2.64:1 [19]. Studies have also revealed a male predominance in the prevalence of NAFLD. For example, a study from the United States found that 58.9% of patients with NAFLD were male [11], and a large study from Japan reported that the prevalence rates of NAFLD in males and females were 41.0% and 17.7%, respectively. The prevalence rates of NAFLD were higher at all ages in males than in females, but the rate gradually increased in females with age, from 3.3% in the second decade of life to 31.3% beyond the sixth decade [12]. Similar trends were observed in the Chinese population [20]. Ethnicity Studies have reported ethnic differences in the prevalence rates of ALD and NAFLD. For example, the prevalence rate of alcoholic liver cirrhosis was higher among South Asian males (32.8%) than among Afro-Caribbean males (1.1%) in the United Kingdom [21]. A recent large study of patients with NAFLD indicated that the prevalence of this disease was higher in Hispanics (24.2%) than in non-hispanic Whites (17.8%) and non-hispanic Blacks (13.5%) [22]. DIAGNOSTIC PROCEDURES Differentiation As mentioned above, ALD and NAFLD have similar pathological spectra, from simple steatosis to liver cirrhosis, which makes confident differentiation of the two diseases difficult. However, the following histological findings are helpful in the differential diagnosis [7]. The fatty degeneration of liver cells occurs to a greater degree in NAFLD than in ALD. In contrast, inflammatory cell infiltration is more pronounced in ALD than in NAFLD. Furthermore, venous or perivenular fibrosis, phlebosclerosis, and (less commonly) lymphocytic phlebitis are more common in ALD than in NAFLD. Clinically, the differentiation between ALD and NAFLD is usually performed by taking a history of a patient s alcohol intake combined with laboratory and imaging examinations; however, the reliability of these methods may not be high [23]. Therefore, discriminant indices consisting of clinical parameters have been developed [24-26]. For example, the ALD/NAFLD index (ANI) is calculated from a patient s gender, BMI, aspartate aminotransferase (AST)/alanine aminotransferase (ALT) ratio, and mean corpuscular volume (MCV) [25]. This index has exhibited a high diagnostic accuracy, with an area un July 14, 2014 Volume 20 Issue 26

87 Toshikuni N et al. Clinical differences between ALD and NAFLD Table 1 Epidemiology of alcoholic liver disease and nonalcoholic fatty liver disease ALD NAFLD Ref. Prevalence rate (overall) Unclear 12.9%-46.0% [8-12] (rates may reflect geographical differences in the amount of alcohol intake) Prevalence rate (China) 4.50% 15.0% [14] Trend (Japan) Increasing (12.6% in 1989; 28.4% in 2000) [8] Peak age yr (United States) yr in males; yr in females (Japan) [12,15] yr (China) [16] Gender Male dominant Male dominant [11,12,18-20] Ethnicity South American males > Afro-Caribbean males Hispanics > Whites > Blacks [21,22] ALD: Alcoholic liver disease; NAFLD: Nonalcoholic fatty liver disease. der the receiver operating characteristic curve (AUROC) of (cut-off value, 0; sensitivity, 93.5%; specificity, 92.0%) in the derivation set and AUROCs of 0.974, 0.989, and in the three validation sets. A recent validation study confirmed the high accuracy of the index [27]. However, the ANI may be less reliable in patients with endstage liver disease because these patients frequently have an elevated MCV and an increased AST/ALT ratio [25]. Because the ANI was formulated based on data sets mainly obtained from Caucasians, this index may not be applicable to other ethnic groups, suggesting the need for indices specifically for these groups. Assessment of disease stage Following a differential diagnosis, the assessment of disease severity and stage is vital for optimal patient management. Although a liver biopsy is the gold standard, this procedure is invasive and costly and is accompanied by patient discomfort, which affects its feasibility. Noninvasive methods for assessing the stage of fibrosis have been recently developed, including fibrosis indices and transient elastography [28]. Many fibrosis indices have been devised based on clinical parameters, and the usefulness of these indices has been tested in patients with chronic liver diseases. The AST/platelet ratio index (APRI) [29], the FIB-4 index [30], and the FibroTest [31] have been validated for use in assessing the fibrosis stage in patients with both ALD and NAFLD. The AUROC of the FibroTest for the diagnosis of liver cirrhosis in patients with ALD was 0.95 (cut-off value, 0.7; sensitivity, 91%; specificity, 87%) [32], while a more recent study found that the AU- ROCs of the APRI, the FIB-4 index, and the FibroTest for liver cirrhosis in patients with ALD were 0.67, 0.80, and 0.94 (cut-off value, 0.7; sensitivity, 86.6%; specificity, 86.0%), respectively [33]. In patients with NAFLD, the AU- ROCs of the APRI and the FIB-4 index for the diagnosis of advanced fibrosis were 0.73 and 0.80 (cut-off value, 2.67; sensitivity, 33%; specificity, 98%), respectively [34], and in another study, the AUROCs for the APRI and the FIB-4 index were 0.82 (cut-off value, 1; sensitivity, 67%; specificity, 81%) and 0.87 (cut-off value, 3.25; sensitivity, 48%; specificity, 95%), respectively [35]. Another study in patients with NAFLD indicated that the AUROCs of the FibroTest for the diagnosis of advanced fibrosis in two independent cohorts were 0.92 (cut-off value, 0.7; sensitivity, 25%; specificity, 97%) and 0.81(cut-off value, 0.7; sensitivity, 25%; specificity, 99%), respectively [36]. Transient elastography has also been shown to be useful in assessing the disease stage. For example, the accuracy of this method in assessing the fibrosis stage was validated for ALD, with AUROCs of 0.94 (cut-off value, kpa; sensitivity, 87%; specificity, 89%) for advanced fibrosis and 0.87 (cut-off value, kpa; sensitivity, 84%; specificity, 83%) for liver cirrhosis [37]. The AUROCs of transient elastography, the APRI, and the FIB-4 index for the diagnosis of advanced fibrosis in patients with NAFLD were 0.93 (cut-off value, 9.6 kpa; sensitivity, 63.6%; specificity, 83.7%), 0.74 (cut-off value, 0.5; sensitivity, 65.1%; specificity, 72.3%), and 0.80 (cut-off value, 2.67; sensitivity, 20.6%; specificity, 95.5%), respectively [38]. Generally, transient elastography cannot differentiate simple hepatic steatosis from mild hepatic fibrosis but can assess the degree of hepatic fibrosis. Recently, a novel measurement method for assessing the hepatic steatosis grade was developed, which has been designated as the controlled attenuation parameter (CAP) [39]. This method is based on transient elastography and permits a simultaneous evaluation of the fibrosis stage and the steatosis grade. Studies have demonstrated the usefulness of CAP in quantitatively assessing the steatosis grade with a high accuracy [40-42]. A large cohort study found that the AUROCs of the CAP for the diagnosis of steatosis > 10%, steatosis > 33%, and steatosis > 66% were 0.79, 0.84, and 0.84, respectively, in 440 patients who underwent a liver biopsy [42]. This study also found that elevated CAP values were significantly associated with excessive alcohol intake, indicating a possible clinical application of CAP in the diagnosis and management of ALD. Further validation studies are required to establish the usefulness of these non-invasive methods in patients with ALD and NAFLD. Scoring systems for ALD and NAFLD Scoring systems specific for ALD or NAFLD have been developed to assess disease severity and stage 8395 July 14, 2014 Volume 20 Issue 26

88 Toshikuni N et al. Clinical differences between ALD and NAFLD Table 2 Rates of alcoholic liver disease and nonalcoholic fatty liver disease progression and the factors associated with susceptibility and progression Rate of progression from simple hepatic steatosis to liver cirrhosis Rate of progression from steatohepatitis to liver cirrhosis Environmental factors associated with disease susceptibility Environmental factors associated with disease progression ALD NAFLD Ref. 1.0%-3.1% per year 0%-2.5% per year [51-55] 3.2%-12.2% per year 1.3%-3.5% per year [51-54] Increased alcohol intake Increased calorie intake [18,50,57] Daily heavy drinking, not episodic or binge drinking [18,60] Fructose [62] Increased amount of alcohol intake Higher intake of soft drinks [58,61] and meats Spirits rather than beer or wine Fructose [59,62] Host factors associated with disease susceptibility Age Older age [65,66] Gender Female [57] Body mass index Obesity Obesity [22,58,67] Metabolic syndrome Presence Presence [22,71,72] Type 2 diabetes Presence Presence [71,72] Ethnicity Hispanic, Black Hispanic [11,73,74] Genetic variant PNPLA3 rs G PNPLA3 rs G [80-84] Host factors associated with disease progression Age Older age Older age [58,67,68] Gender Female No difference [52,55,58,67] Body mass index Obesity [58,67] Metabolic syndrome Type 2 diabetes Ethnicity Genetic variant PNPLA3 rs G PNPLA3 rs G [80-84] ALD: Alcoholic liver disease; NAFLD: Nonalcoholic fatty liver disease. and/or to predict patient outcomes. Scoring systems for ALD [43,44] include Maddrey s discriminant function (mdf), the glasgow alcoholic hepatitis score (GAHS), the Age-Bilirubin-international normalized ratio (INR)- Creatinine (ABIC) score, and the Lille score. Studies have demonstrated that these systems are useful in predicting the short-term survival of patients with alcoholic hepatitis [44,45]. The Brunt score [46] and the NAFLD activity score (NAS) [47] are histological scoring systems for NAFLD. Studies have suggested that the NAS has an excellent ability to differentiate simple hepatic steatosis from nonalcoholic steatohepatitis (NASH) [48]. Other scoring systems for the assessment of NAFLD stage, such as the NAFLD fibrosis score and the BARD score, have shown promising results [49]. FACTORS ASSOCIATED WITH DISEASE SUSCEPTIBILITY AND PROGRESSION Simple hepatic steatosis occurs in most subjects who ingest excessive amounts of alcohol [2] or food [50]. However, only a small percentage of these individuals will develop advanced liver fibrosis or liver cirrhosis. Repeat liver biopsies at 4-year intervals in patients with ALD indicated that liver cirrhosis developed in 11% of the patients with simple hepatic steatosis and 39% of those with alcoholic hepatitis [51]. A recent cohort study of patients with ALD estimated that the 5-year risk of cirrhosis was 6.9% for patients with simple hepatic steatosis and 16.0% for patients with steatohepatitis [52]. Studies examining the histological course of NAFLD over a mean follow-up period of years found that liver cirrhosis developed in 0%-8% of the patients without hepatic fibrosis and 11.3%-17.6% of those with hepatic fibrosis [53,54]. A longterm (approximately 20 years) follow-up study on simple hepatic steatosis observed that 22% of patients with ALD and 1.2% of those with NAFLD developed liver cirrhosis [55]. A recent autopsy study found that the ratios of simple hepatic steatosis to steatohepatitis or liver cirrhosis were 2.33:1 in ALD patients and 3.60:1 in NAFLD patients [56]. The differences in disease progression may be caused by environment-host interactions. Table 2 contains the environmental and host factors that have been suggested by clinical and/or basic research. Recent advances in genetics have provided further insight into the host factors that can affect disease progression. Environmental factors The risks for the development and progression of ALD are increased as the intake of alcohol increases [18,57,58]. The type of beverage may also modify ALD progression. In a pooled cross-sectional time-series analysis, the consumption of spirits, rather than beer or wine, was associated with mortality from cirrhosis in primarily beer-drinking countries [59]. Drinking patterns are also factors associated with ALD. Studies have demonstrated that daily or 8396 July 14, 2014 Volume 20 Issue 26

89 Toshikuni N et al. Clinical differences between ALD and NAFLD near-daily heavy drinking, not episodic or binge drinking, is closely associated with ALD development [18,60]. Moreover, it was demonstrated that alcohol intake outside of mealtimes and the intake of multiple, different beverages increase the risk of developing ALD [18]. Similarly, a high total energy intake is positively associated with the development of NAFLD [50], and specific dietary components affect the pathogenesis of this disease. A cross-sectional study found that a greater intake of soft drinks and meats was associated with an increased risk of NAFLD [61]. Fructose may contribute to disease progression [62] and disease development [63], whereas the ingestion of n-3 polyunsaturated fatty acids may decrease intrahepatic fat deposition [64]. Host factors Age: The ability to metabolize alcohol decreases with age because aging causes reductions in liver size and blood flow to the liver and decreases the activity of enzymes related to alcohol metabolism, such as alcohol dehydrogenase, acetaldehyde dehydrogenase, and cytochrome P-4502E1 [65,66]. Thus, the livers of older subjects become more vulnerable to alcohol toxicity. Indeed, studies in patients with ALD revealed positive correlations between age and advanced fibrosis or liver cirrhosis [58,67]. Similarly, a systematic review of NASH patients revealed that aging was significantly associated with increased fibrosis [68]. The aging of the human population worldwide will likely have an impact on the progression of ALD and NAFLD. Gender: Females have been found to be more susceptible to alcohol-induced liver damage than males. For example, a large prospective study found that the amount of alcohol intake at which the relative risk of ALD is greater than 1 was lower in females (7-13 beverages per week) than in males (14-27 beverages per week) [57], and multivariate analyses indicated that female gender was significantly associated with increased fibrosis in patients with ALD [58,67]. Studies of patients with simple alcoholic steatosis found that females were at a higher risk for cirrhosis than males [52,55]. In contrast, although a gender difference was observed in the prevalence of NAFLD, no significant gender difference was observed in the risk of increased fibrosis among patients with NASH [68]. Obesity, metabolic syndrome, and type 2 diabetes: Obesity predisposes the individual to the development of both ALD [58] and NAFLD [22]. Although a higher BMI was significantly associated with increased fibrosis in ALD patients [58,67], obesity was not significantly associated with increased fibrosis in patients with NAFLD [68]. Insulin resistance (IR) is a key factor in the development of metabolic syndrome [69] and is largely responsible for the development of type 2 diabetes. Recent studies have demonstrated the close relationship between ALD and IR [70] and have suggested that metabolic syndrome and type 2 diabetes are associated with the development of ALD [71]. In contrast, IR or metabolic syndrome and type 2 diabetes were found to be highly associated with NAFLD development [22,71,72] but not NAFLD progression [68]. Ethnicity: Ethnicity may influence the pathogenesis of ALD and NAFLD. For example, a large cross-sectional study revealed that, among current drinkers, the activities of the liver enzymes AST and γ-glutamyl transpeptidase (GGT) were likely to be 2-fold higher in black non-hispanic and Mexican Americans than in white non-hispanic Americans [73]. Another study suggested that African- Americans were more susceptible to alcohol-induced hepatotoxicity than whites [74]. Although the homeostasis model assessment of insulin resistance (HOMA-IR) was not a significant risk factor for NASH among Latinos (OR = 0.93; 95%CI: ), the HOMA-IR was significant among non-latino whites (OR = 1.06; 95%CI: ), which suggests that ethnicity may modulate the effects of IR on the risk of NASH [75]. Genetic factors: Considerable effort has been expended to identify genetic factors that contribute to the pathogenesis of ALD and NAFLD [76,77]. Close links have been found between single nucleotide polymorphisms (SNPs) in DNA and the susceptibility to and severity of many diseases. The patatin-like phospholipase domain-containing 3 gene, PNPLA3, encodes adiponutrin [78], a protein thought to play an important role in lipid metabolism, such as the hydrolysis of triacylglycerols [79]. A recent genome-wide association study (GWAS) of NAFLD suggested that a PNPLA3 SNP, I148M (rs C/G), is associated with increased hepatic fat content and hepatic inflammation [80]. Moreover, a recent meta-analysis [81] confirmed that this SNP strongly correlates with both hepatic fat content (patients with the GG genotype had 73% more hepatic fat than those with the CC genotype) and disease progression (the pooled ORs of the GG vs the CC genotype were 3.25 for having higher necroinflammatory scores and 3.26 for developing fibrosis). Notably, these findings were consistent across ethnic groups. Similar results were obtained in patients with ALD [82-84]. For example, a multivariate analysis revealed that the PNPLA3 SNP was more frequently found in ALD patients than in controls (OR = 1.54) and that this SNP was the strongest independent predictor of the progression of alcoholic liver cirrhosis (OR = 2.08) [84]. These findings regarding the PNPLA3 SNP may explain, at least in part, the ethnic differences in the susceptibility to and prevalence rates of ALD and NAFLD. For example, Hispanics have been reported to be more susceptible to ALD [73] and have the highest prevalence rate of NAFLD [11] ; this is consistent with the frequency of the PNPLA3 SNP, which is highest among Hispanics [80]. Recent studies have found other PNPLA3 SNPs associated with NAFLD [85,86]. A recent GWAS of Japanese NAFLD patients identified rs and rs381062, SNPs that the investigators suggested are associated with the NAFLD grade and/or stage [86]. Additionally, this GWAS also found SAMM50 and PARVB SNPs, both 8397 July 14, 2014 Volume 20 Issue 26

90 Toshikuni N et al. Clinical differences between ALD and NAFLD Table 3 Outcome predictors in patients with alcoholic liver disease and nonalcoholic fatty liver disease Outcomes ALD NAFLD Ref. Hepatocarcinogenesis Underlying liver disease Compensated cirrhosis Not identified [90] Decompensated cirrhosis Older age [91] Compensated/decompensated cirrhosis PNPLA3 rs G Older age, any alcohol intake [92-94] Not identified Older age, elevated GGT, high CP score [96] Unknown stage Older age, type 2 diabetes, elevated AST, low PLT [95] Hepatic decompensation Persistent alcohol intake [90] Mortality (overall) Underlying liver disease Simple hepatic steatosis Low ALB, severe steatosis Low ALB [55] Simple hepatic steatosis/steatohepatitis Older age, type 2 diabetes [101] Advanced fibrosis/cirrhosis Persistent alcohol intake, older age, smoking, AST/ALT > 1, older age [99,100] low ALB Compensated cirrhosis Persistent alcohol intake [90] Decompensated cirrhosis Older age, alcohol abuse, elevated ALP [97] Older age, persistent alcohol intake, low [98] ALB, high MELD score Older age, poor liver function [91] Unknown stage Metabolic syndrome, older age, smoking, [102] Black Type 2 diabetes, insulin resistance Type 2 diabetes, insulin resistance [103] ALD: Alcoholic liver disease; NAFLD: Nonalcoholic fatty liver disease; ALB: Albumin; ALP: Alkaline phosphatase; MELD: Model for end-stage liver disease; GGT: γ-glutamyl transpeptidase; CP: Child-Pugh; AST: Aspartate aminotransferase; PLT: Platelet; ALT: Alanine aminotransferase. of which were considered to be involved in the second hit of NAFLD, leading to a shift from simple steatosis to NASH. In contrast, a GWAS of Caucasian NAFLD patients identified FDFT1 and COL13A1 SNPs [87]. Another GWAS of Caucasian NAFLD patients identified NCAN and PPP1R3B SNPs, as well as a PNPLA3 SNP (rs738409), and demonstrated that these SNPs correlate with hepatic steatosis. Furthermore, it was found that SNPs in the NCAN, GCKR, LYPLAL1, and PNPLA3 genes correlated with histological lobular inflammation/ fibrosis [88]. In addition, accumulated data have suggested that some SNPs, other than the PNPLA3 SNP, may be associated with the pathogenesis of ALD. A meta-analysis revealed a close relationship between a TNFA SNP (rs361525) and ALD [89]. Moreover, several studies have found a higher frequency of the CD C/T SNP in patients with alcoholic cirrhosis than in those without this disease [76]. Despite extensive genome-wide searching, the PNPLA3 rs G genotype is one of the few confirmed genetic factors contributing to a patient s susceptibility to both ALD and NAFLD and to the progression of both diseases. Because this SNP may be useful in screening and managing high-risk patients and may constitute a therapeutic target, further studies are warranted. PREDICTORS OF OUTCOMES Predictors of hepatocarcinogenesis, hepatic decompensation, and mortality have been identified in patients with ALD and NAFLD (Table 3). Hepatocarcinogenesis Although a recent study of patients with compensated alcoholic cirrhosis failed to identify any risk factors significantly associated with hepatocarcinogenesis [90], a study of patients with decompensated alcoholic cirrhosis found that older age was significantly associated with hepatocarcinogenesis [91]. Moreover, the PNPLA3 SNP was reported to be closely associated with the susceptibility to hepatocellular carcinoma (HCC) in patients with ALD [92,93]. Older age and alcohol intake were found to be independent risk factors for hepatocarcinogenesis in patients with NASH-related cirrhosis [94]. A multivariate analysis of a large number of Japanese patients with NAFLD found that older age, type 2 diabetes, an elevated serum AST concentration, and a low platelet count were significantly associated with hepatocarcinogenesis [95]. A recent study compared hepatocarcinogenesis in patients with alcoholic liver cirrhosis and NASH-related cirrhosis. A multivariate analysis revealed that older age, an elevated serum GGT concentration, and a higher Child-Pugh score were risk factors for the development of HCC in patients with NASH-related cirrhosis, whereas no factor was found to be significantly associated with hepatocarcinogenesis in patients with alcoholic cirrhosis [96]. Hepatic decompensation Persistent alcohol intake has been reported to predict hepatic decompensation in patients with alcoholic cirrhosis [90]. To our knowledge, however, no studies to date 8398 July 14, 2014 Volume 20 Issue 26

91 Toshikuni N et al. Clinical differences between ALD and NAFLD have assessed the risk factors for hepatic decompensation in patients with compensated NASH-related cirrhosis. Mortality A long-term cohort study analyzed the predictors of mortality in patients with ALD and simple hepatic steatosis and reported that a low serum albumin concentration and severe steatosis were significantly associated with increased mortality [55]. Moreover, persistent alcohol intake was found to be the only factor predictive of mortality in patients with compensated alcoholic cirrhosis [90]. A long-term follow-up study, in which most of the patients suffered from decompensated alcoholic cirrhosis, found that older age, alcohol abuse, and elevated alkaline phosphatase levels were risk factors for mortality [97]. In another long-term follow-up study involving patients with decompensated alcoholic cirrhosis, older age, persistent alcohol intake, a low serum albumin concentration, and a higher baseline model for end-stage liver disease (MELD) score were predictive of mortality [98]. Similarly, older age and poorer liver function were significantly associated with increased mortality in patients with decompensated alcoholic cirrhosis [91]. More recently, an analysis of patients with advanced, non-decompensated ALD revealed that smoking, a low serum albumin concentration, persistent alcohol intake, and older age were associated with increased mortality [99]. In patients with NAFLD and simple hepatic steatosis, only a low serum albumin concentration was found to be significantly associated with patient mortality [55]. A longterm cohort study involving patients with NAFLD found that an AST/ALT ratio > 1 and older age were associated with overall mortality, whereas higher serum bilirubin concentrations and stage 4 fibrosis were associated with liver-related mortality [100]. Older age and accompanying type 2 diabetes were significantly associated with increased mortality in patients with biopsy-proven NAFLD, whereas NASH, older age and type 2 diabetes were risk factors for liver-related mortality [101]. A recent, large population-based study revealed that metabolic syndrome was independently associated with overall mortality, liverspecific mortality, and cardiovascular mortality and that older age, smoking, and black race were risk factors for overall mortality [102]. A large cohort study found that type 2 diabetes and/ or IR were independent predictors of overall mortality in patients with both ALD and NAFLD. Furthermore, type 2 diabetes, IR, obesity, and metabolic syndrome were independent predictors of liver-related mortality in both patient subsets [103]. COMORBIDITIES ALD and NAFLD are frequently accompanied by extrahepatic complications. Therefore, we compared the comorbidities of patients with these two types of liver disease. Metabolic syndrome and its related diseases Excessive alcohol intake is highly associated with metabolic syndrome-related diseases, including hypertension, type 2 diabetes, and dyslipidemia [ ], whereas NAFLD and metabolic syndrome are closely related [108]. Few studies to date have compared the associations of ALD and NAFLD with metabolic syndrome and/or related diseases. A recent study, however, found that an alcoholic fatty liver was more strongly associated with hypertension than a nonalcoholic fatty liver, whereas the latter was more strongly associated with dyslipidemia than the former [109]. Further studies are required to confirm these findings. Cerebrovascular and cardiovascular diseases Although little is known regarding the relationships between ALD and cerebrovascular and cardiovascular diseases, excessive alcohol intake has been reported to enhance the risks of these diseases, whereas light-tomoderate alcohol intake lowers these risks [ ]. In comparison, a systematic review confirmed that NAFLD is an independent risk factor for cardiovascular disease [113], but it remains unclear whether NAFLD is a risk factor for cerebrovascular disease. Extrahepatic malignancy Although alcohol per se is not a carcinogen, excessive alcohol intake has been associated with HCC and other cancers, including oral cavity, pharyngeal, laryngeal, esophageal, colorectal, and female breast cancer [96,114]. A recent systematic review suggested a close relationship between NAFLD and colorectal cancer [113]. CHARACTERISTICS OF OUTCOMES ALD and NAFLD are heterogeneous disorders that encompass a wide range of pathologies, from simple hepatic steatosis to liver cirrhosis and HCC. Furthermore, both diseases are frequently accompanied by extrahepatic complications. Patient survival can depend on various disease conditions. Although few studies to date have directly compared the outcomes of patients with ALD and NAFLD, we have delineated the characteristics of the reported outcomes (Table 4). Hepatocarcinogenesis The cumulative incidence rates of HCC were 6.8% at 10 years in ALD patients with compensated cirrhosis [90] and 7.1% at 5 years in patients with decompensated cirrhosis [91]. In contrast, a systematic review found that the cumulative incidence rate of HCC at up to 20 years in patients with NAFLD or NASH, of whom few or none had cirrhosis, was 0%-3% [115]. In patients with NASH and cirrhosis, the cumulative rates of HCC ranged from 2.4% over 7 years to 12.8% over 3 years [115]. A large Japanese study of patients with NAFLD found that the annual rate of new HCC cases was 0.043% [95]. More recently, the 5-year cumulative rates of HCC were found to be com July 14, 2014 Volume 20 Issue 26

92 Toshikuni N et al. Clinical differences between ALD and NAFLD Table 4 Outcome characteristics of patients with alcoholic liver disease and nonalcoholic fatty liver disease Outcomes ALD NAFLD Ref. Hepatocarcinogenesis Incidence rate Simple hepatic steatosis/steatohepatitis 0%-0.2% per year [115] Steatohepatitis/cirrhosis 0.3%-4.3% per year [115] Compensated cirrhosis 0.7% per year [90] Decompensated cirrhosis 1.4% per year [91] Compensated/decompensated cirrhosis 2.5% per year 2.1% per year [96] Unknown stage 0.043% per year [95] Prevalence rate of non-cirrhotic liver as 5.3%-12.0% 25.0%-58.3% [ ] underlying liver disease Comparison of survival Similar to non-ald, NBNC HCC Better than ALD- or HCV-related HCC [119,120] Hepatic decompensation Incidence rate Compensated cirrhosis 4.4% per year 4.5% per year [90,121] Mortality (overall) Incidence rate Simple hepatic steatosis 3.3% per year [52] Steatohepatitis 5.0% per year [52] Steatohepatitis/cirrhosis 1.8% per year [100] Compensated cirrhosis 3.2% per year [90] Decompensated cirrhosis 5.7%-6.0% per year [91,97] Compensated/decompensated cirrhosis 5.0% per year [124] Compared with liver diseases due to other causes Similar to HCV-related compensated liver disease [100] Similar to HCV-related compensated cirrhosis Similar to HCV-related decompensated cirrhosis Better than HCV-related compensated cirrhosis Similar to HCV-related decompensated cirrhosis [90,122] [91,122] Similar to HCV-related cirrhosis [124] Improved survival Abstinence [123] Causes of death Simple hepatic steatosis [55] Liver-related causes 17% 2% Arteriosclerosis 20% 38% Extrahepatic malignancy 14% 17% Infection 3% 8% Cirrhosis [90,97,98,122,124] HCC 10%-13% 6.9%-47.4% Liver failure 25%-60% 17.2%-31.6% Cardiovascular disease 1% 27.6% Cerebrovascular disease 1%-4% Infection 8.9%-25% 41.4% Extrahepatic malignancy 8%-25% ALD: Alcoholic liver disease; NAFLD: Nonalcoholic fatty liver disease; HCC: Hepatocellular carcinoma; NBNC: Non-B, non-c; HCV: Hepatitis C virus. parable in patients with alcoholic (12.5%) and NASHrelated (10.5%) cirrhosis [96]. Collectively, the incidence of HCC seems to be somewhat higher in patients with ALD than in patients with NAFLD. Most (88.0%-94.7%) ALD-related HCCs arise from cirrhosis, whereas 41.7%-75.0% of NAFLD-related HCCs arise from a non-cirrhotic liver [ ]. Moreover, there was no difference in age between the patients with ALD-related HCC and those with NAFLD-related HCC, although the percentage of females was lower in the former than in the latter [116,119]. To our knowledge, no studies to date have compared the outcomes of patients with ALD- and NAFLD-related HCC. A study of patients who had non-hepatitis B/nonhepatitis C HCC found that the survival rates were similar between the patients with ALD-related HCC and those with non-ald-related HCC, whereas the tumor recurrence rate was higher in the ALD group [120]. A comparison of the outcomes of curative treatment for HCC in patients with NASH and those with ALD or hepatitis C-related liver disease revealed that the cause of liver disease did not influence the recurrence-free survival according to a multivariate analysis, whereas NASH was significantly associated with an improved overall survival [119]. Hepatic decompensation A study of the natural history of alcoholic cirrhosis found that the cumulative 10 year hepatic decompensation rate was 37.4% [90]. During the follow-up, only approximately one-fourth of these patients abstained from 8400 July 14, 2014 Volume 20 Issue 26

93 Toshikuni N et al. Clinical differences between ALD and NAFLD alcohol. In contrast, 45% of patients with compensated NASH-related cirrhosis developed hepatic decompensation during a follow-up of 10 years [121]. Ascites, a manifestation of hepatic decompensation, is frequently observed in patients with alcoholic cirrhosis [90,98,122], but the incidence of ascites is relatively low in patients with NASHrelated cirrhosis [121]. Mortality and causes of death The cumulative 5-year survival rates of patients with ALD were reported to be 83.3% in patients with simple hepatic steatosis and 74.9% in those with alcoholic steatohepatitis [52]. Furthermore, the cumulative 5-year survival rates in patients with compensated and decompensated alcoholic cirrhosis were reported to be 83.9% [90] and 71.3% [91], respectively. Abstinence from alcohol has the potential to increase the survival of patients with alcoholic cirrhosis [123]. In a study of the survival of patients with decompensated alcoholic and hepatitis C-related cirrhosis, a multivariate analysis revealed that the cause of liver disease did not affect survival [91]. A comparison of patients with NASH-related cirrhosis (mean Child-Pugh score 6.1) and hepatitis C-related cirrhosis (mean Child-Pugh score 6.1) revealed that the cumulative 5-year survival rates were 75.2% and 73.8%, respectively [124]. A similar study examining the natural history of advanced fibrosis or compensated cirrhosis due to either NASH or hepatitis C found that the 10-year cumulative survival rates were 81.6% and 82.0%, respectively [100]. A recent study of patients with NASH-related or alcoholic cirrhosis who underwent liver transplantation found no significant differences in the post-transplant survival and cardiovascular mortality rates. However, cardiovascular causes of post-transplant death were more frequent in the NASH group, while malignancies were more frequent in the alcoholic group [125]. In addition, studies have reported that the overall survival after liver transplantation was excellent in patients with HCC and ALD or NAFLD [126,127]. The causes of death have been found to differ in patients with ALD and NAFLD. A long-term followup of patients with simple hepatic steatosis revealed that many of the patients with ALD and NAFLD died of extrahepatic, rather than hepatic, causes. For example, the main causes of death in ALD patients were arteriosclerosis (20%), liver cirrhosis (17%), unknown causes (16%), and extrahepatic cancers (14%), whereas the main causes of death in NAFLD patients were arteriosclerosis (38%), unknown causes (19%), extrahepatic cancers (17%), and infections (8%) [55]. Similarly, studies of patients with alcoholic cirrhosis indicated that a significant percentage of individuals died of extrahepatic causes, with the leading causes being liver failure (25.0%-36.0%), bacterial infection (11.5%-25.0%), extrahepatic cancers (8.0%-25.0%), and HCC (12.5%-13.0%) [90,98]. A population-based study found that ALD contributed to liverrelated but not cardiovascular mortality [128]. Moreover, recent studies of patients with histologically proven NAFLD, of whom 41.6%-59.2% had NASH, found that the leading causes of death were cardiovascular disease (27.8%-28.2%), liver-related causes (15.4%-26.1%), and extrahepatic malignancy (15.7%-17.9%) [101,129]. Similar patterns were observed in other studies of patients with NAFLD, with the leading causes of death being extrahepatic malignancy (28.0%-33.3%), liver-related complications (12.8%-19.0%), and cardiovascular disease (19.0%-25.0%) [ ]. In one study of patients with NASH-related cirrhosis, the major causes of death were infection (41.4%), cardiovascular disease (27.6%), and liver-related complications (24.1%) [121], whereas the causes were HCC (47.4%) and liver failure (31.6%) in a second study [124]. In summary, the mortality rates of patients with ALD and NAFLD were found to be similar, although the causes of death differed somewhat. When compared with patients with hepatitis C, larger percentages of patients with ALD and NAFLD died of extrahepatic causes; many ALD patients died of infection and extrahepatic cancers, and many NAFLD patients died of cardiovascular disease. CONCLUSION In this review, we attempted to delineate the characteristics and outcomes of patients with ALD and NAFLD, particularly from a clinical aspect. Both liver diseases are generally related to unhealthy lifestyle habits, including excessive alcohol and food intake, and both are likely to be serious health problems in the future. 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97 Toshikuni N et al. Clinical differences between ALD and NAFLD effects on metabolic traits. PLoS Genet 2011; 7: e [PMID: DOI: /journal.pgen ] 89 Marcos M, Gómez-Munuera M, Pastor I, González-Sarmiento R, Laso FJ. Tumor necrosis factor polymorphisms and alcoholic liver disease: a HuGE review and meta-analysis. Am J Epidemiol 2009; 170: [PMID: DOI: /aje/kwp236] 90 Toshikuni N, Izumi A, Nishino K, Inada N, Sakanoue R, Yamato R, Suehiro M, Kawanaka M, Yamada G. Comparison of outcomes between patients with alcoholic cirrhosis and those with hepatitis C virus-related cirrhosis. J Gastroenterol Hepatol 2009; 24: [PMID: DOI: /j x] 91 Solà R, Alvarez MA, Ballesté B, Montoliu S, Rivera M, Miquel M, Cirera I, Morillas RM, Coll S, Planas R. Probability of liver cancer and survival in HCV-related or alcoholic-decompensated cirrhosis. A study of 377 patients. 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98 Toshikuni N et al. Clinical differences between ALD and NAFLD 28: 164S-168S [PMID: ] 117 Kawada N, Imanaka K, Kawaguchi T, Tamai C, Ishihara R, Matsunaga T, Gotoh K, Yamada T, Tomita Y. Hepatocellular carcinoma arising from non-cirrhotic nonalcoholic steatohepatitis. J Gastroenterol 2009; 44: [PMID: DOI: /s ] 118 Ertle J, Dechêne A, Sowa JP, Penndorf V, Herzer K, Kaiser G, Schlaak JF, Gerken G, Syn WK, Canbay A. Non-alcoholic fatty liver disease progresses to hepatocellular carcinoma in the absence of apparent cirrhosis. Int J Cancer 2011; 128: [PMID: DOI: /ijc.25797] 119 Reddy SK, Steel JL, Chen HW, DeMateo DJ, Cardinal J, Behari J, Humar A, Marsh JW, Geller DA, Tsung A. Outcomes of curative treatment for hepatocellular cancer in nonalcoholic steatohepatitis versus hepatitis C and alcoholic liver disease. Hepatology 2012; 55: [PMID: DOI: /hep.25536] 120 Kim SK, Marusawa H, Eso Y, Nishikawa H, Ueda Y, Kita R, Kimura T, Chiba T, Osaki Y, Kudo M. Clinical characteristics of non-b non-c hepatocellular carcinoma: a singlecenter retrospective study. Digestion 2011; 84 Suppl 1: [PMID: DOI: / ] 121 Sanyal AJ, Banas C, Sargeant C, Luketic VA, Sterling RK, Stravitz RT, Shiffman ML, Heuman D, Coterrell A, Fisher RA, Contos MJ, Mills AS. Similarities and differences in outcomes of cirrhosis due to nonalcoholic steatohepatitis and hepatitis C. Hepatology 2006; 43: [PMID: DOI: /hep.21103] 122 Wiegand J, Kühne M, Pradat P, Mössner J, Trepo C, Tillmann HL. Different patterns of decompensation in patients with alcoholic vs. non-alcoholic liver cirrhosis. Aliment Pharmacol Ther 2012; 35: [PMID: DOI: /j x] 123 Xie YD, Feng B, Gao Y, Wei L. Effect of abstinence from alcohol on survival of patients with alcoholic cirrhosis: A systematic review and meta-analysis. Hepatol Res 2013 Apr 11; Epub ahead of print [PMID: DOI: /hepr.12131] 124 Yatsuji S, Hashimoto E, Tobari M, Taniai M, Tokushige K, Shiratori K. Clinical features and outcomes of cirrhosis due to non-alcoholic steatohepatitis compared with cirrhosis caused by chronic hepatitis C. J Gastroenterol Hepatol 2009; 24: [PMID: DOI: /j x] 125 Bhagat V, Mindikoglu AL, Nudo CG, Schiff ER, Tzakis A, Regev A. Outcomes of liver transplantation in patients with cirrhosis due to nonalcoholic steatohepatitis versus patients with cirrhosis due to alcoholic liver disease. Liver Transpl 2009; 15: [PMID: DOI: /lt.21927] 126 Sotiropoulos GC, Beckebaum S, Lang H, Frilling A, Molmenti EP, Cicinnati VR, Saner FH, Erim Y, Baba HA, Malagó M, Broelsch CE. Single-center experience on liver transplantation for hepatocellular carcinoma arising in alcoholic cirrhosis: results and ethical issues. Eur Surg Res 2008; 40: 7-13 [PMID: DOI: / ] 127 Afzali A, Berry K, Ioannou GN. Excellent posttransplant survival for patients with nonalcoholic steatohepatitis in the United States. Liver Transpl 2012; 18: [PMID: DOI: /lt.22435] 128 Trimble G, Zheng L, Mishra A, Kalwaney S, Mir HM, Younossi ZM. Mortality associated with alcohol-related liver disease. Aliment Pharmacol Ther 2013; 38: [PMID: DOI: /apt.12432] 129 Rafiq N, Bai C, Fang Y, Srishord M, McCullough A, Gramlich T, Younossi ZM. Long-term follow-up of patients with nonalcoholic fatty liver. Clin Gastroenterol Hepatol 2009; 7: [PMID: DOI: /j.cgh ] 130 Adams LA, Lymp JF, St Sauver J, Sanderson SO, Lindor KD, Feldstein A, Angulo P. The natural history of nonalcoholic fatty liver disease: a population-based cohort study. Gastroenterology 2005; 129: [PMID: ] 131 Ong JP, Pitts A, Younossi ZM. Increased overall mortality and liver-related mortality in non-alcoholic fatty liver disease. J Hepatol 2008; 49: [PMID: DOI: /j.jhep ] 132 Adams LA, Harmsen S, St Sauver JL, Charatcharoenwitthaya P, Enders FB, Therneau T, Angulo P. Nonalcoholic fatty liver disease increases risk of death among patients with diabetes: a community-based cohort study. Am J Gastroenterol 2010; 105: [PMID: DOI: / ajg ] 133 Treeprasertsuk S, Björnsson E, Enders F, Suwanwalaikorn S, Lindor KD. NAFLD fibrosis score: a prognostic predictor for mortality and liver complications among NAFLD patients. World J Gastroenterol 2013; 19: [PMID: DOI: /wjg.v19.i8.1219] P- Reviewers: Fan JG, Malnick S, Po-Shiuan H, Qin P S- Editor: Qi Y L- Editor: A E- Editor: Ma S 8406 July 14, 2014 Volume 20 Issue 26

99 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. WJG 20 th Anniversary Special Issues (12): Nonalcoholic fatty liver disease TOPIC HIGHLIGHT Nonalcoholic fatty liver disease and cardiovascular disease Hong Liu, Hong-yun Lu Hong Liu, Hong-yun Lu, Department of Endocrinology and Metabolism, the Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai , Guangdong Province, China Author contributions: Liu H and Lu HY equally contributed to this paper. Correspondence to: Dr. Hong-yun Lu, Department of Endocrinology and Metabolism, the Fifth Affiliated Hospital of Sun Yat-sen University, Zhuhai , Guangdong Province, China. luhongyun2013@163.com Telephone: Fax: Received: October 26, 2013 Revised: January 4, 2014 Accepted: April 8, 2014 Published online: July 14, 2014 Abstract Nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD) are two diseases that are common in the general population. To date, many studies have been conducted and demonstrate a direct link between NAFLD and CVD, but the exact mechanisms for this complex relationship are not well established. A systematic search of the PubMed database revealed that several common mechanisms are involved in many of the local and systemic manifestations of NAFLD and lead to an increased cardiovascular risk. The possible mechanisms linking NAFLD and CVD include inflammation, oxidative stress, insulin resistance, ectopic adipose tissue distribution, dyslipidemia, endothelial dysfunction, and adiponectin, among others. The clinical implication is that patients with NAFLD are at an increased risk of CVD and should undergo periodic cardiovascular risk assessment Baishideng Publishing Group Inc. All rights reserved. Key words: Nonalcoholic fatty liver disease; Cardiovascular disease; Metabolic syndrome; Risk assessment Core tip: The link between nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD) should be carefully evaluated in future research, which represents an intriguing field of investigation. A better understanding of the role of inflammation, oxidative stress, and insulin resistance, among other mechanisms, may be necessary to clarify the pathogenesis of NAFLD and CVD, and thereby contribute to the development of new therapies. Liu H, Lu HY. Nonalcoholic fatty liver disease and cardiovascular disease. World J Gastroenterol 2014; 20(26): Available from: URL: v20/i26/8407.htm DOI: i INTRODUCTION Nonalcoholic fatty liver disease (NAFLD) is a common liver disease seen in clinical practice. It comprises a wide disease spectrum ranging from simple steatosis to nonalcoholic steatohepatitis (NASH), which can progress to end stage liver disease, cirrhosis and hepatocellular carcinoma [1]. Initially, NAFLD was considered a benign liver disease, but it is now regarded as the liver manifestation of metabolic syndrome (MetS), a highly atherogenic condition. Most patients are overweight or obese with insulin resistance (IR), hypertension, and dyslipidemia. When compared with control subjects who do not have NAFLD, patients with NAFLD have a higher prevalence of atherosclerosis, which is independent of obesity and other established risk factors [2]. The role of NAFLD as a potential independent cardiovascular disease (CVD) risk factor has recently gained considerable prominence. The purpose of this review is to discuss the pathogenesis of NAFLD, the relationship between NAFLD and CVD, the mechanisms that link both conditions, and the clinical implications that may influence NAFLD and risk of CVD July 14, 2014 Volume 20 Issue 26

100 Liu H et al. NAFLD and CVD Table 1 Published studies on the association between nonalcoholic fatty liver disease and cardiovascular disease Ref. Population sample size Diagnosis methods Outcomes Main results Targher et al [9] 702 patients with T2 DM Liver ultrasound AF NAFLD is strongly associated with an increased prevalence of persistent and permanent AF in patients with T2 DM Lu et al [8] 7042 participants Liver ultrasound and CT Defilippis et al [10] 3362 subjects aged CT yr Feitosa et al [11] 2756 subjects CT and elevated ALT c-imt and CAD Atherogenic dyslipidemia CHD NAFLD was significantly associated with cardiovascular outcomes independent of conventional risk factors CT-diagnosed NAFLD was associated with atherogenic dyslipidemia even after adjustment for several metabolic risk factors FL and ALT (> 40 U/L) were each individually associated with prevalent CHD. However, when accounting for traditional metabolic risk factors in a multivariate model FL and no predictive value for CHD Akin et al [12] 157 obese patients Liver ultrasound c-imt Obese patients with NAFLD had markedly increased c-imt than those without NAFLD Catena et al [14] Colak et al [15] 68 patients with essential hypertension 51 patients in study group and 21 in control group Liver ultrasound AASI In hypertensive patients, AASI and symmetric AASI were higher than in normotensive subjects (P < 0.001), but both indices of vascular stiffness were comparable in patients with and without NAFLD Liver biopsy c-imt C-IMT was significantly higher in patients with NAFLD group(p < 0.001) T2 DM: Type 2 diabetes mellitus; AF: Atrial fibrillation; c-imt: Carotid intima media thickness; FT: Fatty liver; CHD: Coronary heart disease; CAD: Coronary atherogenic dyslipidemia; AASI: Ambulatory arterial stiffness index. NAFLD, METS AND DIABETES In general, NAFLD is diagnosed based on the following criteria: liver biopsy showing steatosis in at least 5% of hepatocytes or imaging study confirmation; exclusion of liver disease of other etiology, including alcohol-induced liver disease (history of excessive alcohol consumption greater than 20 g/d), drug-induced liver disease, autoimmune or viral hepatitis, as well as cholestatic or metabolic/genetic liver disease [3]. A joint interim statement of the International Diabetes Federation defines MetS with three of the five criteria, including elevated waist circumference, elevated triglycerides, reduced high-density lipoprotein (HDL), elevated blood pressure, and elevated fasting-glucose levels [4]. Approximately 90% of patients with NAFLD have at least one of the features of MetS, and approximately 33% meet the complete diagnosis, placing NAFLD as the hepatic representation of MetS [5]. The prevalence of metabolic abnormalities such as diabetes and hypertension were increased up to 15-fold in patients with NASH compared to steatosis independent of age or body mass index (BMI). The relationship between diabetes and NAFLD remains poorly understood; thus, further prospective study of the association and outcomes should be performed. NAFLD predicts the development of type 2 diabetes mellitus (T2 DM) and vice versa, and each condition may serve as a progression factor for the other. In a recent study of T2 DM patients with an average BMI of 36 kg/m 2, over 60% of patients who underwent weight reduction surgery (n = 64 of 92) had moderate to severe NAFLD on liver biopsy [6]. A study by Leite et al [7] in patients with T2 DM found that three of the 92 patients had histological evidence of cirrhosis secondary to NAFLD without clinical evidence of liver disease. To approach the relationship between NAFLD and diabetes from another perspective, multiple studies supported by a recent meta-analysis have shown that NAFLD was associated with IR and diabetes and that NAFLD presence predicted the development of diabetes [8]. In a Korean study of individuals without diabetes at baseline, where fasting insulin levels were also measured and divided into quartiles, the 5-year (crude) OR for developing T2 DM in the presence of ultrasound evidence of fatty liver at baseline was 5.05 (95%CI: ) for the lowest insulin quartile and was 6.34 (95%CI: ) for the highest insulin quartile [9]. After multivariate adjustment, including for baseline glucose level, the OR for the highest insulin quartile remained significant at 2.42 (95%CI: ) [10]. NAFLD AND CARDIOVASCULAR DISEASE PREVALENCE The clinical manifestations of NAFLD, such as steatosis and inflammation, are additional risk factors of CVD, although the precise mechanisms by which NAFLD contributes to CVD are still the subject of ongoing research. The age of onset of CVD events in NAFLD patients ranged from 45 to 65 years [11]. All had significantly higher estimated CVD risk at 10 years (17% vs 10%) by the Framingham risk score (FRS) than NAFLD patients without new CVD events [12]. The mortality rate among patients with NAFLD followed for 8 years was higher than that in the general population. In another study consisting of biopsy-diagnosed NAFLD patients who were followed for 18 years, CVD was among the common causes of death after all of the cancers combined [13]. Clinical studies Table 1 summarized several latest clinical studies on the relationship between NAFLD and CVD. Targher et al [9] 8408 July 14, 2014 Volume 20 Issue 26

101 Liu H et al. NAFLD and CVD showed a significant increase of atrial fibrillation (AF) in the presence of NAFLD (retrospective, n = 702, Caucasians). Lu et al [8] suggested that NAFLD was a strong independent predictor of CVD and may play a central role in the cardiovascular risk of MetS. DeFilippis et al [10] showed that CT-diagnosed NAFLD was associated with the atherogenic dyslipidemia phenotype in a dose dependent fashion. These relationships persisted after adjustment for several metabolic risk factors and HOMA- IR, suggesting a possible independent pathophysiologic role between NAFLD and dyslipidemia (prospective, n = 3362, Caucasian, Chinese, African American, Hispanic). Feitosa et al [11] showed that ALT ( 40 U/L) was a predictor of prevalent coronary heart disease (CHD) in men but not in women, while CT measured liver fat (FL) was not significant in either sex (prospective, n = 2756, European-American). Akın et al [12] showed that obese children and adolescents with NAFLD are at an risk of early atherosclerotic changes. As liver function tests are not sufficient to identify patients with fatty liver, ultrasonographic evaluation of NAFLD might be considered in all obese children and adolescents (cross-sectional, n = 157, Turkey). Catena et al [14] showed that in essential hypertensive patients without additional cardiovascular risk factors, NAFLD is associated with IR but not with increased arterial stiffness (observational, cross-sectional, n = 68, Italy). Colak et al [15] showed that NAFLD is associated with earlier endothelial dysfunction in patients with atherosclerosis compared to control subjects (observational case-control, n = 51, Turkey). Based on the evidence enumerated above, NAFLD patients have higher incidence of AF, atherosclerotic changes, dyslipidemia and coronary heart disease. Therefore, our primary goal was to systematically evaluate the possible mechanisms linking NAFLD and CVD, their implication in clinical practice and various treatment modalities. Classical and new emerging risk factors The new risk factors for CVD include markers such as inflammation (e.g., C-reactive protein, lipoprotein A), homocystine, and markers of fibrinolytic and homeostatic function [e.g., fibrinogen, tissue plasminogen activator, and plasminogen activator inhibitor-1 (PAI-1)]. These markers are also associated with NAFLD [13]. A case-control study of 35 patients diagnosed with NAFLD by liver biopsy and 45 healthy controls showed that the plasma homocysteine level was higher in NAFLD patients compared to the control group (p = ) [16]. The classic common risk factors for NAFLD and CVD are age and gender, physical inactivity, T2DM, hyperlipidemia, obesity, and hypertension [16]. POSSIBLE MECHANISMS LINKING NAFLD AND CVD The association of NAFLD with MetS and diabetes maybe partially explain the increased risk of CVD with NAFLD. Additionally, several studies showed that NAFLD in itself may contribute to the increased risk of CVD [17-19]. However, the exact mechanisms for this complex relationship are not clear. It is likely that several highly interrelated factors contribute to the enhanced risk of diabetes and metabolic syndrome in persons with NAFLD. The following factors are possible explanations of underlying mechanisms of the association between CVD and NAFLD. Oxidative stress and inflammation It has recently been shown that NAFLD is an independent risk factor for death from CVD. Although the precise mechanism linking NAFLD and CVD is unclear, a systematic review and meta-analysis have shown that a marker of NAFLD (and oxidative stress) may be the key link between NAFLD and CVD [20]. These data suggest that some component of oxidative stress, perhaps induced by the disease process in NAFLD, may be involved in the pathogenesis of CVD. Oxidative stress plays an important role in the progression from simple steatosis to steatohepatitis [15]. The association between oxidative stress and NAFLD in humans has been demonstrated by the immunohistochemical detection of lipid peroxidation products and 8-hydroxy-deoxyguanosine in the plasma and liver biopsies from patients with NAFLD [21]. Inflammation is crucial in the pathogenesis of NAFLD, and adipose tissue is now considered a metabolically active endocrine organ that produces pro-inflammatory cytokines, including TNF-α, IL-6, C-reactive protein (CRP) and IL-8. There is also evidence to support the activation of other inflammatory pathways, oxidative stress, as well as de novo pathways by TNF-α. A cross-sectional survey of 360 people indicated that an increase in CRP (OR = 1.37; 95%CI: ) per 1 SD (1.48 mg/l) was an independent risk factor for NAFLD [21]. IR Increased IR is an undisputed major contributor to NAFLD, MetS, and atherosclerosis. In fact, liver fat content appears to be the best independent predictor of IR in skeletal muscle, adipose tissue, and the liver [12]. Similarly, adverse CV outcome is likely to be associated with liver fat/inflammation in a monotonic relationship, progressively increasing with more advanced stages of NAFLD. While IR promotes fatty acid accumulation in the liver, the latter causes hepatic IR characterized by a lack of suppression of endogenous liver glucose production [22]. Therefore, NAFLD might act as a stimulus for further increased whole-body IR and dyslipidemia (with a characteristic overproduction of triglyceride- and cholesterol-rich remnant particles), leading to accelerated atherosclerosis [1]. Visceral fat Individuals with obesity have large amounts of visceral adipose tissue (VAT). VAT is a metabolically active endocrine organ that can secrete pro-inflammatory cytokines, adipokines and hormones that mediate inflammation 8409 July 14, 2014 Volume 20 Issue 26

102 Liu H et al. NAFLD and CVD Pro-coagulation and hypofibrinolysis The prothrombotic state in the atherosclerosis process encompasses platelet hyper-aggregability, hypercoagulability and hyper-fibrinolysis [14,58,59]. Markers of fibrinolytic and hemostatic function (e.g., fibrinogen, tissue plasminogen activator, and PAI-1-antigens) are strongly associated with NAFLD. PAI-1 is expressed in visceral adipose tisand IR, which, in turn, affect CV risk factors [23]. VAT is defined as intra-abdominal fat bounded by parietal peritoneum or transversalis fascia. Recently, increased VAT assessed by CT showed a significant association with CVD, which was defined by the presence of plaque calcification [24,25]. However, the mechanisms linking visceral fat or obesity to CV disease are strongly related to IR, which itself is robustly associated with CV risk and atherosclerosis, already reviewed in detail [26]. It is therefore unclear whether VAT actually confers direct CV risk through secreted factors, or indirectly via IR-related processes, or both [16]. Further studies to identify the association between NAFLD and CVD through VAT should be considered. Ectopic adipose tissue distribution In keeping with the adverse cardiovascular effects of ectopic hepatic fat, recent studies have also described the effects of ectopic fat accumulation in the heart, which correlates with visceral fat, IR and MetS parameters [27]. Additionally, ectopic hepatic fat has been shown to be independently associated with NAFLD [28]. In a recent metaanalysis of 16 studies (n = 2872), epicardial fat thickness or volume was significantly associated with the presence of coronary artery disease [2,29]. Adiponectin Mature adipocytes act as an active endocrine and paracrine organ, secreting an increasing number of growth factors that participate in diverse metabolic processes, particularly IR [4,30,31]. Patients with NAFLD exhibit reduced levels of adiponectin, which are inversely correlated with the severity of NAFLD histology [32]. The reduced production of adiponectin associated with obesity may contribute to the progression of NAFLD [33]. Dyslipidaemia Dyslipidaemia causes upregulation of the transcription factor sterol regulatory element binding protein-1c (SREBP-1c), and both insulin and SREBP-1c synergistically stimulate genes involved in de-novo lipogenesis. SREBP-1c also causes inhibition of FFA oxidation, leading to increased hepatic lipid content [34-36]. To compensate for the increased hepatic triglycerides, the liver forms an atherogenic lipid profile, consisting of high TG levels, low high-density lipoprotein (HDL) cholesterol, increased small, dense low-density lipoprotein (LDL) particles, increased very low-density lipoprotein (VLDL) cholesterol levels and elevated apolipoprotein B100 concentration; all of which are strongly associated with adverse cardiovascular outcomes [37-39]. Postprandial hyperlipidemia NAFLD may be linked to accelerated atherogenesis through the presence of abnormal lipoprotein metabolism, especially during the post-prandial phase [11]. Postprandial hyperlipidemia is a risk factor for both NAFLD and CVD [40-42]. The atherosclerotic risk of postprandial hyperlipidemia is derived from an increase of remnant lipoproteins (RLPs) [15]. In patients with IR, an increase of postprandial RLP values usually occurs and becomes a risk factor for coronary heart disease [43-46]. Swarbrick et al [47] showed that consumption of fructose-sweetened, but not glucose-sweetened, beverages for 10 wk increases de novo lipid synthesis as well as the 24-h postprandial TG, which includes increased levels of apob, LDL, oxidized LDL, RLP triglyceride, and the apob/apoa1 ratio (all biomarkers were increased for CVD). Therefore, postprandial hyperlipidaemia may explain, at least in part, why some lean, overweight, and obese individuals with NAFLD may encounter CVD despite normal fasting lipid profiles or taking lipid-lowering medication [48-51]. Endothelial dysfunction Endothelial dysfunction is now recognized as the earliest detectable component in the development of atherosclerosis. In both diabetic and non-diabetic cohorts, studies have shown an independent association between impaired endothelium-dependent flow-mediated dilation (FMD) and NAFLD. In addition, lower FMD was observed in NASH compared with simple steatosis, again confirming the graded association of CV risk with severity of NAFLD [52-54]. Chronic kidney disease NAFLD may also be associated with a detrimental effect on other organs that may have a direct or indirect influence on CVD or organs that may accelerate the presentation of CVD [55]. For example, NAFLD has been shown to be associated with the development of chronic kidney disease (CKD) in Korean individuals. Therefore, NAFLD may indirectly modulate the risk of CVD through CKD [56]. Obstructive sleep apnea Obstructive sleep apnea (OSA) is characterized by loud and frequent snoring, periods of apnea during sleep, and excessive day somnolence [9,12,57]. Interestingly, OSA is also regarded as one of the factors that accelerate the progression of NAFLD to NASH. Importantly, a considerable number of studies have shown an increase in the incidence of CVD in people with OSA. Animal studies have shown that OSA can lead to an increase in IR and alterations in lipid metabolism in the presence of NAFLD [14]. The conclusion of that study was that OSA is associated with an increased risk of CVD, which has also been demonstrated in epidemiological, clinical and physiological studies [22] July 14, 2014 Volume 20 Issue 26

103 Liu H et al. NAFLD and CVD Antiobesity drugs Low physical activity, smoking, etc. Visceral obesity Metabolic abnormalities (metabolic syndrome, type 2 diabetes, etc.) Cardiovascular disease (macrovascular and microvascular disease) Cardiometabolic status Insulin sensitizers NAFLD Morbidity Mortality Liver status Antioxidative drugs Anti-inflammatory drugs Anti-fibrosis drugs NASH Cirrhosis Hepatic carcinoma Figure 1 Evaluated treatments for nonalcoholic fatty liver disease. NAFLD: Nonalcoholic fatty liver disease; NASH: Nonalcoholic steatohepatitis. sue. Plasma PAI-1 levels are more closely related to fat accumulation and PAI-1 expression in the liver than in adipose tissue, suggesting that, among insulin-resistant individuals, fatty liver is an important site of PAI-1 production. Fibrinogen, von Willebrand factor (vwf) and PAI-1 are also considered markers of the acute-phase reaction of inflammation and thrombosis, and have been closely linked to CVD [7,59-62]. CLINICAL IMPLICATIONS It is evident that patients with NASH are more prone to developing CVD (increased mortality by 86%) than patients with simple steatosis (increased mortality by 55%). We suggest adding a new modality of approaching patients with NAFLD [23]. The primary objective of any NAFLD therapy is to improve steatohepatitis and fibrosis, with the ultimate goal of preventing CVD and liverrelated death. Once the diagnosis of NAFLD is made, the first step will be a lifestyle intervention using a combination of diet, active walking, and behavior modification, with a goal of a 10% weight reduction [10]. Recently, Koskinen et al [63] showed that modest wine drinking (20-30 g/daily) offers protection against suspected NAFLD. Lifestyle modification remains the cornerstone of management. Weight loss and increased physical activity are effective mediators of NAFLD, and their role in CVD risk reduction is well established. However, before initiating any significant increase in exercise level, patients at risk should be evaluated for underlying CVD. The second step is to assess the risk of hepatic fibrosis. The noninvasive methods for fibrosis evaluation include plasma cytokeratine 18 fragments and Angulo score [9]. The invasive method (liver biopsy) remains the only reliable means to determine prognosis based on the severity of fibrosis. The third step will include the assessment of cardiovascular risk stratification. We suggest the use of measurements of the carotid arteries (IMT) in non-diabetic NAFLD patients and/or the Framingham score with an effort test, as well as biomarkers of inflammation (Creactive protein, fibrinogen), oxidative stress, (MDA, Paraoxonase), IR (HOMA), lipotoxicity (TG, HDL, LDL, TC), OGTT, and microalbumin/creatinine ratio [13]. The final step is to initiate appropriate therapy according to the comorbidities and the clinical status of each patient [21] (Figure 1). Insulin sensitizers Metformin was associated with improvement in hepatocellular ballooning, but not fibrosis, steatosis, inflammation or NAFLD activity score (NAS). TZDs should be reserved for second-line treatment in the majority of patients. One exception may be patients with T2DM and NAFLD in whom TZDM therapy may rectify both conditions. TZDs improved insulin sensitivity and steatohepatitis in a large, multicenter randomized controlled trial Pioglitazone or Vitamin E for NASH Study (PIV- ENS). However, we should notice its drawback of lower extremity edema and weight gain (average 2 to 5 kg). Incretin-based therapies A direct relationship between the gastrointestinal and 8411 July 14, 2014 Volume 20 Issue 26

104 Liu H et al. NAFLD and CVD endocrine systems has recently been appreciated with the discovery of neuroendocrine hormones known as incretins. The two primary incretins are GLP-1 and glucosedependent insulinotropic polypeptide (GIP). There is accumulating and convincing pre-clinical evidence that GLP-1 and its analogues have the ability to decrease hepatic steatosis in animal models, suggesting that there is potential for incretin-based medications to reverse or delay progression through the stages of NAFLD to cirrhosis [64]. There is an urgent need for prospective clinical trials designed to scrutinize the potential of these agents for treatment of NAFLD beyond improvements in metabolic parameters, such as weight loss. Cytoprotective and antioxidant agents Two randomized trials [65,66] showed that high-dose bile acids are unlikely to provide significant benefit, although their use was routinely advocated. Two recently published, large, randomized controlled trials, PIVENS and TONIC [67,68], assessed the effect of vitamin E on adult and pediatric NAFLD populations, respectively. Vitamin E treatment resulted in improvements in hepatocellular ballooning and NAS in both trials. These results for vitamin E are quite promising and suggest that patients with biopsy-proven steatohepatitis associated with hepatocellular ballooning (NAS 4) may benefit from its use. Antitumour necrosis factor-alpha agents This type of therapy has been studied in a number of small NAFLD trials, two of which have assessed histological response and demonstrated improvement in steatosis, inflammation and ballooning [69,70]. Lipid-lowering agents One recently published large study [71] provided compelling evidence that lipid-lowering agents, such as statins, are safe and efficacious in patients with NAFLD/NASH and that the agents can induce a reduction in the extent of the hepatic steatosis. Phlebotomy One prospective phase Ⅱ study [72,73] of phlebotomy with paired liver biopsies that evaluated phlebotomy therapy in NAFLD patients suggested that iron reduction may improve liver histology. Surgical intervention and anti-obesity drugs Bariatric surgery is an increasingly popular therapeutic option among morbidly obese patients. One recently published study [71] reported that surgery should complement treatment of obesity-related comorbidity, but not replace established therapy. Theoretically, improving NAFLD via weight loss is an ideal approach in obese or overweight people because other complications are simultaneously ameliorated with weight loss. Of several commonly used antiobesity medications, orlistat and sibutramine are available for longterm prescription. Harrison [74] reported that subjects who lost 5% of their body weight over 9 mo experienced improvements in IR and steatosis, while subjects who lost 9% of their body weight also experienced improvements in hepatic histology [75,76]. Both orlistat and sibutramine have been shown to have beneficial effects on body weight, lipid profiles, glucose metabolism, and inflammatory markers in several trials [77]. However, insufficient safety data are available regarding the long-term outcomes of antiobesity therapy. Indeed, sibutramine was reported to increase blood pressure and heart rate, which may limit its use in clinical practice [76]. CONCLUSION NAFLD is regarded as the hepatic component of metabolic syndrome and is associated with a high risk of developing CVD. 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105 Liu H et al. NAFLD and CVD ciation between Nonalcoholic Fatty Liver Disease and Cardiovascular Disease: A Systematic Review and Meta-Analysis. Int J Endocrinol 2013; 2013: [PMID: DOI: /2013/124958] 9 Targher G, Mantovani A, Pichiri I, Rigolon R, Dauriz M, Zoppini G, Morani G, Vassanelli C, Bonora E. Non-alcoholic fatty liver disease is associated with an increased prevalence of atrial fibrillation in hospitalized patients with type 2 diabetes. Clin Sci (Lond) 2013; 125: [PMID: DOI: /CS ] 10 DeFilippis AP, Blaha MJ, Martin SS, Reed RM, Jones SR, Nasir K, Blumenthal RS, Budoff MJ. Nonalcoholic fatty liver disease and serum lipoproteins: the Multi-Ethnic Study of Atherosclerosis. Atherosclerosis 2013; 227: [PMID: DOI: 10.] 11 Feitosa MF, Reiner AP, Wojczynski MK, Graff M, North KE, Carr JJ, Borecki IB. Sex-influenced association of nonalcoholic fatty liver disease with coronary heart disease. Atherosclerosis 2013; 227: [PMID: DOI: / j.atherosclerosis ] 12 Akın L, Kurtoglu S, Yikilmaz A, Kendirci M, Elmalı F, Mazicioglu M. Fatty liver is a good indicator of subclinical atherosclerosis risk in obese children and adolescents regardless of liver enzyme elevation. Acta Paediatr 2013; 102: e107-e113 [PMID: DOI: /apa.12099] 13 Huang Y, Bi Y, Xu M, Ma Z, Xu Y, Wang T, Li M, Liu Y, Lu J, Chen Y, Huang F, Xu B, Zhang J, Wang W, Li X, Ning G. Nonalcoholic fatty liver disease is associated with atherosclerosis in middle-aged and elderly Chinese. Arterioscler Thromb Vasc Biol 2012; 32: [PMID: DOI: / ATVBAHA ] 14 Catena C, Bernardi S, Sabato N, Grillo A, Ermani M, Sechi LA, Fabris B, Carretta R, Fallo F. Ambulatory arterial stiffness indices and non-alcoholic fatty liver disease in essential hypertension. Nutr Metab Cardiovasc Dis 2013; 23: [PMID: DOI: / ] 15 Colak Y, Senates E, Yesil A, Yilmaz Y, Ozturk O, Doganay L, Coskunpinar E, Kahraman OT, Mesci B, Ulasoglu C, Tuncer I. Assessment of endothelial function in patients with nonalcoholic fatty liver disease. Endocrine 2013; 43: [PMID: ] 16 de Carvalho SC, Muniz MT, Siqueira MD, Siqueira ER, Gomes AV, Silva KA, Bezerra LC, D Almeida V, de Oliveira CP, Pereira LM. Plasmatic higher levels of homocysteine in non-alcoholic fatty liver disease (NAFLD). Nutr J 2013; 12: 37 [PMID: ] 17 Thakur ML, Sharma S, Kumar A, Bhatt SP, Luthra K, Guleria R, Pandey RM, Vikram NK. Nonalcoholic fatty liver disease is associated with subclinical atherosclerosis independent of obesity and metabolic syndrome in Asian Indians. Atherosclerosis 2012; 223: [PMID: DOI: /j.atherosclerosis ] 18 Arslan U, Kocaoğlu I, Balcı M, Duyuler S, Korkmaz A. The association between impaired collateral circulation and nonalcoholic fatty liver in patients with severe coronary artery disease. J Cardiol 2012; 60: [PMID: DOI: /j.jjcc ] 19 Beaton MD. Current treatment options for nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. Can J Gastroenterol 2012; 26: [PMID: ] 20 Cakır E, Ozbek M, Colak N, Cakal E, Delıbaşi T. Is NAFLD an independent risk factor for increased IMT in T2DM? Minerva Endocrinol 2012; 37: [PMID: ] 21 Bhatia LS, Curzen NP, Byrne CD. Nonalcoholic fatty liver disease and vascular risk. Curr Opin Cardiol 2012; 27: [PMID: DOI: /HCO.0b013e c] 22 Severova MM, Saginova EA, Galliamov MG, Ermakov NV, Rodina AV, Fomin VV, Mukhin NA. [Clinicopathogenetic characteristics of cardiorenal syndrome in non-alcoholic fatty liver disease]. Ter Arkh 2012; 84: [PMID: ] 23 Heuer M, Kaiser GM, Kahraman A, Banysch M, Saner FH, Mathé Z, Gerken G, Paul A, Canbay A, Treckmann JW. Liver transplantation in nonalcoholic steatohepatitis is associated with high mortality and post-transplant complications: a single-center experience. Digestion 2012; 86: [PMID: DOI: / ] 24 Wang CC, Tseng TC, Hsieh TC, Hsu CS, Wang PC, Lin HH, Kao JH. Severity of fatty liver on ultrasound correlates with metabolic and cardiovascular risk. 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Liver Int 2012; 32: [PMID: DOI: /j x] 30 Kim D, Choi SY, Park EH, Lee W, Kang JH, Kim W, Kim YJ, Yoon JH, Jeong SH, Lee DH, Lee HS, Larson J, Therneau TM, Kim WR. Nonalcoholic fatty liver disease is associated with coronary artery calcification. Hepatology 2012; 56: [PMID: DOI: /hep.25593] 31 Stepanova M, Younossi ZM. Independent association between nonalcoholic fatty liver disease and cardiovascular disease in the US population. Clin Gastroenterol Hepatol 2012; 10: [PMID: DOI: /j.cgh ] 32 Li G, Hu H, Shi W, Li Y, Liu L, Chen Y, Hu X, Wang J, Gao J, Yin D. Elevated hematocrit in nonalcoholic fatty liver disease: a potential cause for the increased risk of cardiovascular disease? Clin Hemorheol Microcirc 2012; 51: [PMID: DOI: /CH ] 33 Bonapace S, Perseghin G, Molon G, Canali G, Bertolini L, Zoppini G, Barbieri E, Targher G. Nonalcoholic fatty liver disease is associated with left ventricular diastolic dysfunction in patients with type 2 diabetes. Diabetes Care 2012; 35: [PMID: DOI: /dc ] 34 Ahmed MH, Abu EO, Byrne CD. Non-Alcoholic Fatty Liver Disease (NAFLD): new challenge for general practitioners and important burden for health authorities? Prim Care Diabetes 2010; 4: [PMID: DOI: / j.pcd ] 35 Hacihamdioğlu B, Okutan V, Yozgat Y, Yildirim D, Kocaoğlu M, Lenk MK, Ozcan O. Abdominal obesity is an independent risk factor for increased carotid intima- media thickness in obese children. Turk J Pediatr 2011; 53: [PMID: ] 36 Dogru T, Genc H, Tapan S, Ercin CN, Ors F, Aslan F, Kara M, Sertoglu E, Bagci S, Kurt I, Sonmez A. Elevated asymmetric dimethylarginine in plasma: an early marker for endothelial dysfunction in non-alcoholic fatty liver disease? Diabetes Res Clin Pract 2012; 96: [PMID: DOI: / j.diabres ] 37 Alkhouri N, Kistangari G, Campbell C, Lopez R, Zein NN, Feldstein AE. Mean platelet volume as a marker of increased cardiovascular risk in patients with nonalcoholic steatohepatitis. Hepatology 2012; 55: 331 [PMID: DOI: / hep.24721] 38 Motta AB. Report of the international symposium: polycystic ovary syndrome: first Latin-American consensus. Int J 8413 July 14, 2014 Volume 20 Issue 26

106 Liu H et al. NAFLD and CVD Clin Pract 2010; 64: [PMID: DOI: / j x] 39 Gawrieh S, Baye TM, Carless M, Wallace J, Komorowski R, Kleiner DE, Andris D, Makladi B, Cole R, Charlton M, Curran J, Dyer TD, Charlesworth J, Wilke R, Blangero J, Kissebah AH, Olivier M. Hepatic gene networks in morbidly obese patients with nonalcoholic fatty liver disease. Obes Surg 2010; 20: [PMID: DOI: / s ] 40 Hessheimer AJ, Forner A, Varela M, Bruix J. Metabolic risk factors are a major comorbidity in patients with cirrhosis independent of the presence of hepatocellular carcinoma. Eur J Gastroenterol Hepatol 2010; 22: [PMID: DOI: /MEG.0b013e32833aa19b] 41 Lee YJ, Shim JY, Moon BS, Shin YH, Jung DH, Lee JH, Lee HR. The relationship between arterial stiffness and nonalcoholic fatty liver disease. Dig Dis Sci 2012; 57: [PMID: DOI: /s ] 42 Ahmed MH, Barakat S, Almobarak AO. Nonalcoholic fatty liver disease and cardiovascular disease: has the time come for cardiologists to be hepatologists? J Obes 2012; 2012: [PMID: DOI: /2012/483135] 43 Hurjui DM, Niţă O, Graur LI, Mihalache L, Popescu DS, Huţanaşu IC, Ungureanu D, Graur M. Non-alcoholic fatty liver disease is associated with cardiovascular risk factors of metabolic syndrome. Rev Med Chir Soc Med Nat Iasi 2012; 116: [PMID: ] 44 Cameron I, Alam MA, Wang J, Brown L. Endurance exercise in a rat model of metabolic syndrome. Can J Physiol Pharmacol 2012; 90: [PMID: DOI: /y ] 45 Santos RD, Agewall S. Non-alcoholic fatty liver disease and cardiovascular disease. Atherosclerosis 2012; 224: [PMID: DOI: /j.atherosclerosis ] 46 Hurjui DM, Niţă O, Graur LI, Mihalache L, Popescu DS, Graur M. The central role of the non alcoholic fatty liver disease in metabolic syndrome. Rev Med Chir Soc Med Nat Iasi 2012; 116: [PMID: ] 47 Swarbrick MM, Stanhope KL, Elliott SS, Graham JL, Krauss RM, Christiansen MP, Griffen SC, Keim NL, Havel PJ. Consumption of fructose-sweetened beverages for 10 weeks increases postprandial triacylglycerol and apolipoprotein-b concentrations in overweight and obese women. Br J Nutr 2008; 100: [PMID: DOI: / Sooo ] 48 Senateş E, Colak Y, Yeşil A, Coşkunpinar E, Sahin O, Kahraman OT, Erkalma Şenateş B, Tuncer I. Circulating resistin is elevated in patients with non-alcoholic fatty liver disease and is associated with steatosis, portal inflammation, insulin resistance and nonalcoholic steatohepatitis scores. Minerva Med 2012; 103: [PMID: ] 49 Vlachopoulos C, Manesis E, Baou K, Papatheodoridis G, Koskinas J, Tiniakos D, Aznaouridis K, Archimandritis A, Stefanadis C. Increased arterial stiffness and impaired endothelial function in nonalcoholic Fatty liver disease: a pilot study. Am J Hypertens 2010; 23: [PMID: DOI: /ajh ] 50 Brandt ML, Harmon CM, Helmrath MA, Inge TH, McKay SV, Michalsky MP. Morbid obesity in pediatric diabetes mellitus: surgical options and outcomes. Nat Rev Endocrinol 2010; 6: [PMID: DOI: /nrendo ] 51 Fan JG, Zhou Q, Wo QH. [Effect of body weight mass and its change on the incidence of nonalcoholic fatty liver disease]. Zhonghua Gan Zang Bing Zazhi 2010; 18: [PMID: DOI: /cma.j.issn ] 52 Rivera R, Vanaclocha F. [Nonalcoholic fatty liver disease and psoriasis]. Actas Dermosifiliogr 2010; 101: [PMID: ] 53 Ashburn DD, Reed MJ. Gastrointestinal system and obesity. Crit Care Clin 2010; 26: [PMID: DOI: /j.ccc ] 54 Roĭtberg GE, Sharkhun OO, Ushakova TI. [Non-alcoholic fatty liver disease as an atherosclerosis risk factor]. Eksp Klin Gastroenterol 2010; (7): [PMID: ] 55 Kwon YM, Oh SW, Hwang SS, Lee C, Kwon H, Chung GE. Association of nonalcoholic fatty liver disease with components of metabolic syndrome according to body mass index in Korean adults. Am J Gastroenterol 2012; 107: [PMID: DOI: /ajg ] 56 Băloşeanu CL, Streba CT, Vere CC, Comănescu V, Rogoveanu I. Association between liver histology, carotid ultrasonography and retinal vascular changes in patients with nonalcoholic fatty liver disease (NAFLD). Rom J Morphol Embryol 2012; 53: [PMID: ] 57 Kubes P, Mehal WZ. Sterile inflammation in the liver. Gastroenterology 2012; 143: [PMID: DOI: /j.gastro ] 58 Li X, Xia M, Ma H, Hofman A, Hu Y, Yan H, He W, Lin H, Jeekel J, Zhao N, Gao J, Gao X. Liver fat content is associated with increased carotid atherosclerosis in a Chinese middleaged and elderly population: the Shanghai Changfeng study. Atherosclerosis 2012; 224: [PMID: DOI: /j.atherosclerosis ] 59 Yang YY, Huang YT, Lee TY, Chan CC, Yeh YC, Lee KC, Lin HC. Rho-kinase-dependent pathway mediates the hepatoprotective effects of sorafenib against ischemia/reperfusion liver injury in rats with nonalcoholic steatohepatitis. Liver Transpl 2012; 18: [PMID: DOI: / lt.23520] 60 Saleh DA, Ismail MA, Ibrahim AM. Non alcoholic fatty liver disease, insulin resistance, dyslipidemia and atherogenic ratios in epileptic children and adolescents on long term antiepileptic drug therapy. Pak J Biol Sci 2012; 15: [PMID: ] 61 Ampuero J, Romero-Gómez M. [Influence of non-alcoholic fatty liver disease on cardiovascular disease]. Gastroenterol Hepatol 2012; 35: [PMID: DOI: / j ] 62 Mantovani A, Zoppini G, Targher G, Golia G, Bonora E. Non-alcoholic fatty liver disease is independently associated with left ventricular hypertrophy in hypertensive Type 2 diabetic individuals. J Endocrinol Invest 2012; 35: [PMID: ] 63 Koskinen J, Magnussen CG, Kähönen M, Loo BM, Marniemi J, Jula A, Saarikoski LA, Huupponen R, Viikari JS, Raitakari OT, Juonala M. Association of liver enzymes with metabolic syndrome and carotid atherosclerosis in young adults. The Cardiovascular Risk in Young Finns Study. Ann Med 2012; 44: [PMID: DOI: / ] 64 Samson SL, Bajaj M. Potential of incretin-based therapies for non-alcoholic fatty liver disease. J Diabetes Complications 2013; 27: [PMID: ] 65 Leuschner UF, Lindenthal B, Herrmann G, Arnold JC, Rössle M, Cordes HJ, Zeuzem S, Hein J, Berg T. High-dose ursodeoxycholic acid therapy for nonalcoholic steatohepatitis: a double-blind, randomized, placebo-controlled trial. Hepatology 2010; 52: [PMID: DOI: / hep.23727] 66 Ratziu V, de Ledinghen V, Oberti F, Mathurin P, Wartelle- Bladou C, Renou C, Sogni P, Maynard M, Larrey D, Serfaty L, Bonnefont-Rousselot D, Bastard JP, Rivière M, Spénard J. A randomized controlled trial of high-dose ursodesoxycholic acid for nonalcoholic steatohepatitis. J Hepatol 2011; 54: [PMID: DOI: /j.jhep ] 67 Sanyal AJ, Chalasani N, Kowdley KV, McCullough A, Diehl AM, Bass NM, Neuschwander-Tetri BA, Lavine JE, Tonascia J, Unalp A, Van Natta M, Clark J, Brunt EM, Kleiner DE, Hoofnagle JH, Robuck PR. Pioglitazone, vitamin E, or place July 14, 2014 Volume 20 Issue 26

107 Liu H et al. NAFLD and CVD bo for nonalcoholic steatohepatitis. N Engl J Med 2010; 362: [PMID: DOI: /NEJMoa ] 68 Lavine JE, Schwimmer JB, Van Natta ML, Molleston JP, Murray KF, Rosenthal P, Abrams SH, Scheimann AO, Sanyal AJ, Chalasani N, Tonascia J, Ünalp A, Clark JM, Brunt EM, Kleiner DE, Hoofnagle JH, Robuck PR. Effect of vitamin E or metformin for treatment of nonalcoholic fatty liver disease in children and adolescents: the TONIC randomized controlled trial. JAMA 2011; 305: [PMID: DOI: /jama ] 69 Adams LA, Zein CO, Angulo P, Lindor KD. A pilot trial of pentoxifylline in nonalcoholic steatohepatitis. Am J Gastroenterol 2004; 99: [PMID: ] 70 Satapathy SK, Sakhuja P, Malhotra V, Sharma BC, Sarin SK. Beneficial effects of pentoxifylline on hepatic steatosis, fibrosis and necroinflammation in patients with non-alcoholic steatohepatitis. J Gastroenterol Hepatol 2007; 22: [PMID: ] 71 Nseir W, Mograbi J, Ghali M. Lipid-lowering agents in nonalcoholic fatty liver disease and steatohepatitis: human studies. Dig Dis Sci 2012; 57: [PMID: DOI: /s ] 72 Dixon JB. Surgical management of obesity in patients with morbid obesity and nonalcoholic fatty liver disease. Clin Liver Dis 2014; 18: [PMID: ] 73 Beaton MD, Chakrabarti S, Levstik M, Speechley M, Marotta P, Adams P. Phase II clinical trial of phlebotomy for nonalcoholic fatty liver disease. Aliment Pharmacol Ther 2013; 37: [PMID: DOI: /apt.12255] 74 Harrison AG. Fragmentation reactions of methionine-containing protonated octapeptides and fragment ions therefrom: an energy-resolved study. J Am Soc Mass Spectrom 2013; 24: [PMID: DOI: /s x] 75 Harrison SA, Fecht W, Brunt EM, Neuschwander-Tetri BA. Orlistat for overweight subjects with nonalcoholic steatohepatitis: A randomized, prospective trial. Hepatology 2009; 49: [PMID: DOI: /hep.22575] 76 Tziomalos K, Krassas GE, Tzotzas T. The use of sibutramine in the management of obesity and related disorders: an update. Vasc Health Risk Manag 2009; 5: [PMID: ] 77 Filippatos TD, Derdemezis CS, Gazi IF, Nakou ES, Mikhailidis DP, Elisaf MS. Orlistat-associated adverse effects and drug interactions: a critical review. Drug Saf 2008; 31: [PMID: ] P- Reviewers: Kim SH, Xu Y S- Editor: Ma YJ L- Editor: Wang TQ E- Editor: Ma S 8415 July 14, 2014 Volume 20 Issue 26

108 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. WJG 20 th Anniversary Special Issues (13): Gastrointestinal endoscopy TOPIC HIGHLIGHT Role of small bowel capsule endoscopy in the diagnosis and management of iron deficiency anemia in elderly: A comprehensive review of the current literature Adnan Muhammad, Gitanjali Vidyarthi, Patrick Brady Adnan Muhammad, Patrick Brady, Division of Digestive Diseases and Nutrition, University of South Florida, Morsani College of Medicine, Tampa, FL 33620, United States Gitanjali Vidyarthi, Division of Gastroenterology, James A Haley VA Hospital, Tampa, FL 33620, United States Author contributions: Muhammad A, Vidyarthi G and Brady P contributed equally to the manuscript. Correspondence to: Adnan Muhammad, MD, Division of Digestive Diseases and Nutrition, University of South Florida, Morsani College of Medicine, Bruce B. Downs Blvd, Tampa, FL 33620, United States. adnan_muhd@hotmail.com Telephone: Fax: Received: November 6, 2013 Revised: January 30, 2014 Accepted: March 12, 2014 Published online: July 14, 2014 Abstract Iron deficiency anemia (IDA) is common and often under recognized problem in the elderly. It may be the result of multiple factors including a bleeding lesion in the gastrointestinal tract. Twenty percent of elderly patients with IDA have a negative upper and lower endoscopy and two-thirds of these have a lesion in the small bowel (SB). Capsule endoscopy (CE) provides direct visualization of entire SB mucosa, which was not possible before. It is superior to push enteroscopy, enteroclysis and barium radiography for diagnosing clinically significant SB pathology resulting in IDA. Angioectasia is one of the commonest lesions seen on the CE in elderly with IDA. The diagnostic yield of CE for IDA progressively increases with advancing age, and is highest among patients over 85 years of age. Balloon assisted enteroscopy is used to treat the lesions seen on CE. CE has some limitations mainly lack of therapeutic capability, inability to provide precise location of the lesion and false positive results. Overall CE is a very safe and effective procedure for the evaluation of IDA in elderly Baishideng Publishing Group Inc. All rights reserved. Key words: Small bowel; Capsule endoscopy; Iron deficiency anemia; Elderly; Diagnostic yield Core tip: Iron deficiency anemia (IDA) is a common problem especially in the elderly. Small bowel (SB) lesions may be the source of IDA. Capsule endoscopy (CE) provides direct visualization of entire SB mucosa. Angioectasia is one of the commonest lesions seen on the CE in elderly with IDA. The diagnostic yield of CE for IDA increases with advancing age. Balloon assisted enteroscopy is used to treat the lesions seen on CE causing IDA. Muhammad A, Vidyarthi G, Brady P. Role of small bowel capsule endoscopy in the diagnosis and management of iron deficiency anemia in elderly: A comprehensive review of the current literature. World J Gastroenterol 2014; 20(26): Available from: URL: v20/i26/8416.htm DOI: i INTRODUCTION Iron-deficiency anemia (IDA) is the most common cause of anemia worldwide, causing significant disease-related morbidity, and has a negative impact on patient s well-being and overall outcome. Anemia is defined by the World Health Organization (WHO) as a hemoglobin concentration of less than 13 mg/dl in men and less than 12 mg/dl in women [1]. The gold standard for the diagnosis of iron deficiency is the absence of iron staining (Prussian blue stain) on bone marrow biopsy. In clinical prac July 14, 2014 Volume 20 Issue 26

109 Muhammad A et al. Capsule endoscopy for anemia in elderly tice, this invasive test is replaced by evaluation with more readily available laboratory parameters. The classic hallmarks of IDA are low serum ferritin (< 20 ng/l), low serum iron (< 33 g/dl), high serum total iron-binding capacity (> 400 g/dl) and low mean corpuscular volume (< MCV 80 fl). Trial of empiric iron OGIB and IDA (negative EGD/colonoscopy) SB Evaluation Repeat EGD/ colonoscopy IDA IN ELDERLY In the year 2010, 40.3 million people (13.0% of the total population in United States) were 65 years of age and older (elderly), and 5.5 million were above 85 years of age. The elderly population is expected to increase to more than 20% of the total population by 2030, with individuals 85 years and older representing the fastest growing segment of this group. The Third National Health and Nutrition Examination Survey (NHANES Ⅲ, 1988 to 1994) indicated that the prevalence of IDA was nearly 10.6% in elderly. This means almost 4 million elderly Americans have IDA [2]. IDA is common and often under recognized problem especially in elderly with increased morbidity and mortality. Anemia, however, is not simply a consequence of aging, but also a marker of underlying disease, requiring investigation for an etiology. Anemia is recognized to be associated with increased frailty, poor exercise, performance, diminished cognitive function, dementia, decreased mobility, increased risk for falls, lower bone and muscle density, depression and delirium [3,4]. Anemia is a marker for increased disease-related morbidity, including hospitalization and mortality in elderly [5]. IDA in elderly may be the result of multiple factors. Major considerations include iron deficiency secondary to a bleeding lesion in the gastrointestinal (GI) tract and this is one of the major indications for referral to gastroenterologists (13% of referrals). In clinical practice esophagogastroduodenoscopy (EGD) and colonoscopy are performed in the initial evaluation of IDA to exclude a source of chronic blood loss from the GI tract. Despite undergoing standard endoscopic evaluation, up to 30% of patients with IDA have no definitive diagnosis [6]. Twenty percent of elderly patients have a negative upper and lower endoscopy and two-thirds of them have a lesion in the small bowel (SB) [7]. It is important to investigate the SB in all patients with unexplained IDA, after negative standard dual endoscopic evaluations (ASGE Practice Guideline 2010) [8] (Figure 1). SB CAPSULE ENDOSCOPE The original capsule endoscope (PillCam SB) was developed by Given Imaging in 2001 [9]. It is a disposable capsule, 11 by 26 mm in size and weighs 3.7 g. There is a set of short focal length lenses located in front of the camera to collect an image in a 140 degree field of view and magnify it by a factor of 8. This optical system provides a resolution of 0.1 mm. The capsule communicates with the external world through a radio transmitter. Each CT enterography/ct angiography second two pictures are transmitted at 432 MHz to an array of sensors taped to the patient s abdomen. These sensors in turn relay information to a data recording device worn on a belt. The capsule is powered by two silver oxide batteries with a battery life of 8 h. The new capsule (PillCam SB 3) captures up to 6 frames per second with a 30% improvement in picture resolution. ROLE OF CE FOR IDA IN ELDERLY Since the initial presentation at DDW of 2000, the fantastic voyage of capsule endoscopy (CE) has made significant strides [10]. One of the most significant impacts of the CE has been in the elderly, where it provides a less invasive and virtually complete exam. It has expanded our area of visual survey to include direct visualization of entire SB mucosa, which was not possible before. CE had emerged as a light in the darkness for the identification and localization of SB mucosal diseases [9,11]. It has developed an important role in the investigation of patients with IDA when EGD and colonoscopy are negative. CE COMPARED TO OTHER DIAGNOSTIC MODALITIES SB capsule endoscopy Deep enteroscopy Figure 1 Diagnostic approach to evaluate obscure gastrointestinal bleeding/iron deficiency anemia (ASGE Practice Guideline 2010). OGIB: Obscure gastrointestinal bleeding; IDA: Iron deficiency anemia; SB: Small bowel; EGD: Esophagogastroduodenoscopy. There are a number of other diagnostic modalities, old and new to evaluate the SB, but they all have limitations. CE has made fluoroscopic imaging of SB almost obsolete [12]. Traditional SB X-ray series have the lowest yield and fail to detect many mucosal lesions. CE is superior to SB enteroclysis for detecting lesions in patients with unexplained IDA and should be the next diagnostic test of choice after unremarkable standard endoscopic evaluation [13]. Although push enteroscopy (PE) offers direct visual inspection of the SB mucosa beyond the reach of the standard upper endoscopes, it reaches only cm 8417 July 14, 2014 Volume 20 Issue 26

110 Muhammad A et al. Capsule endoscopy for anemia in elderly A B C D E Figure 2 Angioectasia with active bleeding (A), ulcer caused by nonsteroidal anti-inflammatory drugs (B), tumor with active bleeding (C), celiac disease with scalloping and mosaic patternand (D), and active bleeding (E) seen in the small bowel. beyond the ligament of Treitz and its sensitivity in identifying the source of bleeding is limited [14]. CE is superior to PE and SB barium radiography for diagnosing clinically significant SB pathology in patients with IDA [15]. CE PROCEDURE CE is performed as per the standard protocols endorsed by American Society for Gastrointestinal Endoscopy (ASGE) [16]. An informed consent is obtained prior to the procedure. Patients usually present in an out-patient setting, after fasting for 8 h. A bowel preparation is optional. They swallow the capsule with few sips of water in a sitting position. A clear liquid breakfast after 2 h and a light meal after 4 h are permitted. After 8 h, patient returns to the endoscopy unit, the data recorder is removed and images are downloaded to the computer. The recordings are then viewed by an experienced reader. FINDINGS OF CE IN ELDERLY The common SB findings seen on CE when performed for IDA in elderly are [17] : (1) Angioectasia or arteriovenous malformation (Figure 2A); (2) Ulceration [related to nonsteroidal anti-inflammatory drugs (NSAIDs) or Crohn s disease] (Figure 2B); (3) Tumor or mass lesion (Figure 2C); (4) Celiac disease (CD) (mosaic pattern and scalloping) (Figure 2D); and (5) Active bleeding (Figure 2E). Angioectasia is one of the commonest found lesion seen on the CE in elderly with IDA. It accounts for 40% of cases of bleeding of obscure origin [18,19] It is seen more commonly in patients with hereditary hemorrhagic telangiectasia, renal disease and in elderly patients with multiple comorbidities [20]. The incidence of angioectasia is about 23% [21]. It varies from a flat lesion greater in diameter than one villus to a pulsatile red protrusion with surrounding venous dilation. They can be treated with argon plasma coagulation or multipolar electrocautery at the time of enteroscopy. Another common finding, which causes IDA in elderly is the inflammation induced by NSAIDS. The mechanism of action of NSAIDS is COX-1 inhibition and prostaglandin depletion. The Spectrum of NSAID induced SB injury became clear only after the advent of CE, as before 2000 this knowledge was available only from case reports and autopsy series. NSAID induces ulcers may be single or multiple. NSAID induced strictures can be a cause of capsule retention if the stricture was not suspected before administering the capsule. The differential of NSAID induced ulceration includes Crohn s disease, CD (ulcerative jejuno-ileitis), infection (Cytomegalovirus, Tuberculosis, Yersinia), radiation, ischemia, vasculitis and chemotherapy (mucositis) [22] July 14, 2014 Volume 20 Issue 26

111 Muhammad A et al. Capsule endoscopy for anemia in elderly Table 1 Studies on the utility of capsule endoscopy for the evaluation of iron deficiency anemia along with their findings n (%) Ref. Total patients Patients with positive findings (DY) Angioectasia Inflammatory lesions (erosions/ulcers) Mass lesions Active bleeding Other findings (celiac disease, Crohn s disease) Holleran et al [46] (53) 17 (49) 12 (34) 4 (11) 2 (6) Sidhu et al [26] (42) 141 (58) 16 (11) Tong et al [47] (26) 6 (24) 16 (64) 2 (8) 1 (4) Koulaouzidis et al [48] (31) 49 (72) 19 (28) Yamada et al [49] (63) 6 (20) 7 (37) 2 (12) 4 (21) Efthymiou et al [50] (38) Milano et al [51] (78) 13 (37) 9 (26) 6 (17) 7 (20) Goenka et al [52] (37) Katsinelos et al [53] (34) 6 (46) 4 (31) 1 (8) 2 (15) Riccione et al [54] (66) 51 (56) 18 (20) 9 (10) 13 (14) Van Turenhourt et al [55] (44) Laine et al [56] (33) 4 (31) 9 (69) 0 0 Sheibani et al [57] (61) 21 (60) 4 (12) 5 (14) 5 (14) Kim et al [58] (48) 8 (66) 2 (17) 0 2 (17) Sidhu et al [25] (48) 84 (56) 25 (16) 10 (6) 33 (22) Muhammad et al [27] (55) 35 (28) 64 (50) 0 15 (12) 13 (10) Chami et al [59] 12 4 (33) Carey et al [60] (46) 35 (56) 16 (26) 4 (6) 7 (12) Apostolopoulos et al [61] (57) 12 (41) 13 (45) 4 (14) 0 Estevez et al [62] (63) Van Tuyl et al [63] (33) Qvigstaad et al [64] (28) Kalantzis et al [65] (42) De Leusse et al [66] 20 6 (30) Ben Soussan et al [67] 18 7 (38) Enns et al [68] 14 7 (50) 2 (29) 3 (42) 2 (29) 0 Fireman et al [69] (52) 18 (49) 11 (30) 0 8 (21) Pennazio et al [17] (44) 4 (21) 9 (47) 0 6 (32) CD is another condition where the diagnosis is frequently missed. Because of the increased prevalence of undiagnosed CD (iceberg effect), any test that has a promise of increased detection is of great interest. IDA may be the only manifestation of CD in elderly. Another utility of CE in elderly with CD lies in patient population with refractory CD to evaluate for malignancy such as lymphoma, carcinoma and GISTs. SB tumors are a relatively uncommon diagnosis and account for 3% of all GI malignancies. After gastrointestinal stromal tumor (GIST), adenocarcinoma of the SB is the most common malignancy found in the elderly. In a study of 5129 patients, 129 (2.4%) were found to have SB malignancy [23]. Current data suggests that CE can shorten the diagnostic work up for SB tumor and can influence further management and outcome. Given the second peak of Crohn s disease in the elderly, CE can play an important role in the diagnosis of this condition. CE can detect minor (distorted villi, erosion or scars) or major changes (ulcers or strictures) related to Crohn s disease [24]. DIAGNOSTIC YIELD OF CE FOR IDA IN ELDERLY The DY of CE in the evaluation of unexplained IDA progressively increases with advancing age, and is highest among patients over 85 years of age. This may be explained by the fact that ASA, NSAIDs, and warfarin usage is more common in elderly as compared with younger patients. Elderly patients with high diagnostic yield (DY) for IDA are likely to benefit more from CE than younger patients. IDA in the elderly with or without obscure GI bleeding (OGIB) is the major indication for CE after a negative EGD and colonoscopy. There have been a numbers of papers written on the DY of CE when performed for IDA (Table 1). However, there are only few studies which specifically reported on the DY in elderly, and have shown that it is significantly higher as compared to younger age group. Sidhu et al [25] performed a retrospective review of 779 consecutive patients that underwent CE over a 7-year period ( ) for OGIB and recurrent IDA. The DY of CE in elderly was 53%. The most common diagnosis in the elderly was angioectasia (34%). When compared to younger patients, the DY was significantly higher in elderly for IDA (51% vs 37%, P = 0.003, OR = 1.8, 95%CI: ). Management was also altered in a significant greater proportion in the elderly (P = 0.002, OR = 1.8, 95%CI: ). The authors finally concluded that CE has a positive impact on the management of IDA in elderly and there should be no barrier to performing CE in this age group. The authors of this study published another paper recently where they reported the DY of CE for IDA is significantly higher in patients who were aged 70 years or older (P < 0.001, OR = 1.9, 95%CI: ) [26]. Similarly, we have also published on the DY of CE for IDA which was found to be 69% among patients of > 85 years of age and 56% for the age group of years [27]. In another study, we have reported on the utility 8419 July 14, 2014 Volume 20 Issue 26

112 Muhammad A et al. Capsule endoscopy for anemia in elderly A B Figure 3 Capsule endoscopy (A) and single balloon enteroscopy (B) showing jejunal carcinoma with subsequent marking of the lesion. of CE in the diagnosis of CD in elderly for the first time, when they presented for IDA. We found that out of 279 elderly patients with IDA, 7 (2.5%) had mucosal abnormalities suggestive of CD (atrophy, scalloping, mosaic pattern, layering, and nonspecific ulcerating jejuno-ileitis). Subsequent evaluation with serum antibody testing +/- multiple distal duodenal biopsies confirmed the diagnosis in all patients [28]. Recently Koulaouzidis et al [29] published a systematic review on the DY of CE for IDA in all age groups. The pooled DY of CE in studies focused solely on patients with IDA was 66.6% (95%CI: 61.0%-72.3%). CE VS DEEP ENTEROSCOPY FOR IDA IN ELDERLY CE has a number of advantages compared with deep enteroscopy especially in elderly patients. These include the potential ability to visualize the entire SB with a non-invasive procedure carrying a minimal risk of complications, a high sensitivity for lesions and high patient acceptance. These advantages make CE the preferred initial procedure for evaluation of IDA. However, it also has a number of disadvantages including the lack of biopsy and therapeutic capability, the potential for retention above a stricture, and the potential for missed lesions. The overall miss rate for CE is estimated to be 10%-30% in patients with OGIB, and solitary lesions are more likely to be missed [30]. Deep enteroscopy is needed in patients with continued IDA despite a negative CE, and for biopsy and therapy of lesions demonstrated by CE. The currently available types of enteroscopy include PE, intra-operative enteroscopy (IOE), balloon assisted enteroscopy (BAE), and spiral enteroscopy (SE). PE has limited applicability because only the duodenum and proximal jejunum can be visualized. IOE has the capability of visualizing the entire SB but carries a significant operative morbidity, and a mortality of up to 4% [31]. SE is a promising new technique which allows deep enteroscopy in a shorter period of time than BAE. This shortens anesthesia time which may be a significant advantage in the elderly. However, it is not widely available, and the system is not currently being sold while modifications to enhance its utility are being developed. This leaves BAE as the most viable option for deep enteroscopy. The greatest advantage of BAE is the ability to biopsy and tattoo the lesion seen on the CE (Figure 3). Other advantages include the therapeutic benefit which includes hemostasis, polypectomy, balloon dilation of strictures and foreign body removal. The disadvantages are that it is invasive with higher risks for complications, time consuming, requires sedation and the entire SB is not visualized in one procedure. When used by both oral and anal route in a given patient, it has the potential to visualize the entire SB. In two prospective randomized studies of double balloon enteroscopy (DBE) vs single balloon enteroscopy, total enteroscopy was achieved more frequently with DBE. However, there was no difference in DY or therapeutic outcome between the two techniques [32-34]. BAE is frequently a long procedure lasting 60 min or more, and requires deep sedation. Elderly patients may be at increased risk for complications due to prolonged endoscopic procedures, and prolonged sedation due to co-morbidities such as cardiac and pulmonary disease. In addition they are at greater risk of aspiration, have an increased response to sedatives, and have a blunted response to hypoxia and hypercarbia [35]. The complications of BAE include perforation, pancreatitis, and GI bleeding. These occur in 1%-2% of patients which is a higher complication rate compared with standard endoscopic procedures (EGD and colonoscopy) [36,37]. There are only few studies reported on the safety and efficacy of BAE in elderly patients. In a single center, retrospective study on the efficacy and safety of DBE, a total of 60 patients older than 75 years with 110 younger patients were evaluated [38]. Elderly patients were more likely to have angioectasia (39% vs 23%, P = 0.01), and to require endoscopic therapy (48.6% vs 29.2%, P = 0.01). The overall complication rate was 0.9% with no serious complications. There was no difference in the success and complication rates in both age groups in this study. In another retrospective study, 137 patients (80 years or older) underwent DBE after CE. The correlation between the findings on CE and DBE occurred in 78.9% of patients. False negative findings on CE were present 8420 July 14, 2014 Volume 20 Issue 26

113 Muhammad A et al. Capsule endoscopy for anemia in elderly in 6 patients with false positive findings in 18 patients. The DY for DBE in patients with OGIB was 90.2%. No complications related to DBE were encountered in these octogenarians in the 48 h following the procedure. The authors finally concluded that DBE was safe in octogenarians, and that elderly patients with positive findings on CE should be considered for DBE [39]. The majority of studies have reported a good correlation between the findings at capsule endoscopy and BAE and a comparable yield of positive findings [40]. In general, they can be considered complementary studies. BAE may be contraindicated in some elderly patients with severe cardiopulmonary co-morbidities. For these reasons, CE should be the first investigation in elderly patients with IDA and other suspected disorders requiring endoscopic SB visualization. BAE is needed for therapy, biopsy and marking of lesions requiring surgery. A cost-effectiveness analysis for diagnosis of IDA in elderly favors CE over BAE and other radiologic studies. A cost-effectiveness analysis for management of bleeding favors BAE over all other therapeutic modalities. CE is patient friendly, guides BAE approach in positive studies, and predicts a low re-bleeding rate in negative studies. BAE is invasive and requires significant resources and time. It may be occasionally needed to extract a retained capsule endoscope at the SB stricture. Finally, BAE should be considered in patients with a negative CE who continue to have unexplained IDA and OGIB. LIMITATIONS OF CE CE enables visualization of the entire SB but lacks the potential for therapeutic intervention. Other limitations include inability to provide precise location of a lesion, false-positive results, potential for erratic passage resulting in missed lesions and limited battery life causing incomplete studies. COMPLICATION OF CE CE is a very safe procedure with few reported adverse events. There have been concerns about the theoretical interaction of CE with cardiac defibrillators, although no adverse events have been reported in the literature [41,42]. Inability to swallow the capsule, battery failure before capsule reaches the cecum, and capsule retention are some of the important problems associated with CE in elderly as well as in younger patients [43]. In patients who are unable to swallow the capsule, it can be successfully placed in the duodenum by using a capsule delivery device. In patients with suspected SB stricture, a new capsule (AGILE Patency system by Given Imaging) is available, which can be given before SB capsule to evaluate for SB patency [44]. The true remaining contraindications to CE are SB obstruction/pseudo-obstruction and pregnancy [45]. CONCLUSION The use of GI endoscopy in geriatric patients is increasing as a larger proportion of the population is reaching an advanced age. IDA is a major problem in elderly and requires further evaluation and management. SB CE plays an important role in the evaluation of IDA in all age groups, especially in the elderly with a very high DY. SB angioectasia, ulceration, tumor, CD and active bleeding are the common findings seen on CE in elderly with IDA. BAE is used to treat the lesions seen on CE. CE has some limitations mainly lack of therapeutic capability, inability to provide precise location of the lesion and false positive results. Overall CE is a very safe and effective procedure for the evaluation of IDA in elderly. REFERENCES 1 Herbert V. Megaloblastic anemia as a problem in world health. Am J Clin Nutr 1968; 21: [PMID: ] 2 Looker AC, Dallman PR, Carroll MD, Gunter EW, Johnson CL. Prevalence of iron deficiency in the United States. 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Does capsule endoscopy improve outcomes in obscure gastrointestinal bleeding? Randomized trial versus dedicated small bowel radiography. Gastroenterology 2010; 138: e1; quiz e11-2 [PMID: DOI: /j.gastro ] 57 Sheibani S, Levesque BG, Friedland S, Roost J, Gerson LB. Long-term impact of capsule endoscopy in patients referred for iron-deficiency anemia. Dig Dis Sci 2010; 55: [PMID: DOI: /s ] 58 Kim S, Kedia PS, Jaffe DL, Ahmad NA. Impact of capsule endoscopy findings on patient outcomes. Dig Dis Sci 2009; 54: [PMID: DOI: /s y] 59 Chami G, Raza M, Bernstein CN. Usefulness and impact on management of positive and negative capsule endoscopy. Can J Gastroenterol 2007; 21: [PMID: ] 60 Carey EJ, Leighton JA, Heigh RI, Shiff AD, Sharma VK, Post JK, Fleischer DE. A single-center experience of 260 consecutive patients undergoing capsule endoscopy for obscure gastrointestinal bleeding. Am J Gastroenterol 2007; 102: [PMID: DOI: /j x] 61 Apostolopoulos P, Liatsos C, Gralnek IM, Giannakoulopoulou E, Alexandrakis G, Kalantzis C, Gabriel P, Kalantzis N. The role of wireless capsule endoscopy in investigating unexplained iron deficiency anemia after negative endoscopic evaluation of the upper and lower gastrointestinal tract. Endoscopy 2006; 38: [PMID: DOI: / s ] 62 Estévez E, González-Conde B, Vázquez-Iglesias JL, de Los Angeles Vázquez-Millán M, Pértega S, Alonso PA, Clofent J, Santos E, Ulla JL, Sánchez E. Diagnostic yield and clinical outcomes after capsule endoscopy in 100 consecutive patients with obscure gastrointestinal bleeding. Eur J Gastroenterol Hepatol 2006; 18: [PMID: ] 63 Van Tuyl SA, Van Noorden JT, Kuipers EJ, Stolk MF. Results of videocapsule endoscopy in 250 patients with suspected small bowel pathology. Dig Dis Sci 2006; 51: [PMID: DOI: /s ] 64 Qvigstad G, Hatlen-Rebhan P, Brenna E, Waldum HL. Capsule endoscopy in clinical routine in patients with suspected disease of the small intestine: a 2-year prospective study. Scand J Gastroenterol 2006; 41: [PMID: DOI: / ] 65 Kalantzis N, Papanikolaou IS, Giannakoulopoulou E, Alogari A, Kalantzis C, Papacharalampous X, Gabriel P, Alexandrakis G, Apostolopoulos P. Capsule endoscopy; the cumulative experience from its use in 193 patients with suspected small bowel disease. Hepatogastroenterology 2005; 52: [PMID: ] 66 De Leusse A, Landi B, Edery J, Burtin P, Lecomte T, Seksik P, Bloch F, Jian R, Cellier C. Video capsule endoscopy for investigation of obscure gastrointestinal bleeding: feasibility, results, and interobserver agreement. Endoscopy 2005; 37: [PMID: DOI: /s ] 67 Ben Soussan E, Antonietti M, Hervé S, Savoye G, Ramirez S, Lecleire S, Ducrotté P, Lerebours E. Diagnostic yield and therapeutic implications of capsule endoscopy in obscure gastrointestinal bleeding. Gastroenterol Clin Biol 2004; 28: [PMID: ] 68 Enns R, Go K, Chang H, Pluta K. Capsule endoscopy: a single-centre experience with the first 226 capsules. Can J Gastroenterol 2004; 18: [PMID: ] 69 Fireman Z, Eliakim R, Adler S, Scapa E. Capsule endoscopy in real life: a four-centre experience of 160 consecutive patients in Israel. Eur J Gastroenterol Hepatol 2004; 16: [PMID: ] P- Reviewers: Ersoy O, Iakovidis DK S- Editor: Zhai HH L- Editor: A E- Editor: Zhang DN 8423 July 14, 2014 Volume 20 Issue 26

116 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. WJG 20 th Anniversary Special Issues (13): Gastrointestinal endoscopy TOPIC HIGHLIGHT Endoscopic ultrasound-guided treatments: Are we getting evidence based - a systematic review Carlo Fabbri, Carmelo Luigiano, Andrea Lisotti, Vincenzo Cennamo, Clara Virgilio, Giancarlo Caletti, Pietro Fusaroli Carlo Fabbri, Vincenzo Cennamo, Unit of Gastroenterology and Digestive Endoscopy, AUSL Bologna Bellaria-Maggiore Hospital, Bologna, Italy Carmelo Luigiano, Clara Virgilio, Unit of Gastroenterology and Digestive Endoscopy, ARNAS Garibaldi, Catania, Italy Andrea Lisotti, Giancarlo Caletti, Pietro Fusaroli, Department of Medical and Surgical Sciences - DIMEC, University of Bologna, Hospital S Maria della Scaletta, Imola, Italy Author contributions: Fabbri C and Fusaroli P designed research, edited and finalized the text; Lisotti A and Luigiano C performed literature search, analyzed the data and wrote the text; Virgilio C, Cennamo V and Caletti G reviewed the paper for important intellectual content. Correspondence to: Pietro Fusaroli, MD, Department of Medical and Surgical Sciences - DIMEC, University of Bologna, Hospital S Maria della Scaletta, Via Montericco 4, Imola, Italy. pietro.fusaroli@unibo.it Telephone: Fax: Received: November 1, 2013 Revised: January 30, 2014 Accepted: March 12, 2014 Published online: July 14, 2014 Abstract The continued need to develop less invasive alternatives to surgical and radiologic interventions has driven the development of endoscopic ultrasound (EUS)-guided treatments. These include EUS-guided drainage of pancreatic fluid collections, EUS-guided necrosectomy, EUS-guided cholangiography and biliary drainage, EUSguided pancreatography and pancreatic duct drainage, EUS-guided gallbladder drainage, EUS-guided drainage of abdominal and pelvic fluid collections, EUS-guided celiac plexus block and celiac plexus neurolysis, EUSguided pancreatic cyst ablation, EUS-guided vascular interventions, EUS-guided delivery of antitumoral agents and EUS-guided fiducial placement and brachytherapy. However these procedures are technically challenging and require expertise in both EUS and interventional endoscopy, such as endoscopic retrograde cholangiopancreatography and gastrointestinal stenting. We undertook a systematic review to record the entire body of literature accumulated over the past 2 decades on EUS-guided interventions with the objective of performing a critical appraisal of published articles, based on the classification of studies according to levels of evidence, in order to assess the scientific progress made in this field Baishideng Publishing Group Inc. All rights reserved. Key words: Endoscopic ultrasound; Pseudocyst drainage; Necrosectomy; Celiac plexus neurolysis; Levels of evidence; Fine needle injection Core tip: Endoscopic ultrasound (EUS)-guided interventions have become increasingly popular. The advantages of EUS guidance over percutaneous and surgical routes are well established for pseudocyst drainage and celiac plexus neurolysis as they have been assessed in high level of evidence literature. However, for other very fashionable procedures such as bile duct and pancreatic duct drainage, the role of EUS guidance has only been reported as preliminary studies in limited number of patients. The level of evidence of each EUSguided intervention is accurately reported in this review in order to provide the readers with the current status of knowledge and allow insights into potential future direction of research. Fabbri C, Luigiano C, Lisotti A, Cennamo V, Virgilio C, Caletti G, Fusaroli P. Endoscopic ultrasound-guided treatments: Are we getting evidence based - a systematic review. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: org/ /wjg.v20.i July 14, 2014 Volume 20 Issue 26

117 Fabbri C et al. Levels of evidence in EUS-guided treatments INTRODUCTION Endoscopic ultrasound (EUS) has evolved from a purely diagnostic imaging modality to an interventional procedure that provides a minimally invasive alternative to interventional radiologic and surgical techniques. Several innovative techniques now constitute the portfolio of interventional EUS, such as EUS-guided drainage (GD) of pancreatic fluid collections (PFCs), EUSguided necrosectomy, EUS-guided cholangiography and biliary drainage (BD), EUS-guided pancreatography and pancreatic duct drainage (PDD), EUS-guided gallbladder drainage, EUS-GD of abdominal and pelvic fluid collections, EUS-guided celiac plexus block (CPB) and celiac plexus neurolysis (CPN), EUS-guided pancreatic cyst ablation, EUS-guided delivery of antitumoral agents and EUS-guided fiducial placement, brachytherapy and EUSguided vascular interventions. However, EUS-guided treatments are technically challenging and require expertise in both standard diagnostic EUS and endoscopic interventional procedures, such as endoscopic retrograde cholangiopancreatography (ERCP) and gastrointestinal stenting. For such a reason, it is important that we carefully monitor the results of our EUS-guided treatments in order to either implement them in clinical practice or abandon/thoroughly revise them. Evidence based medicine is known as a strategic tool to do so. Following our previous systematic analysis of the levels of evidence (LE) of the EUS literature [1-4], we reviewed the entire body of literature accumulated over the past 2 decades on EUS-guided treatments. Our main aim was to critically appraise the published articles, based on the classification of studies according to LE, in order to assess the scientific progress made in this field. All articles relevant to EUS-guided interventional procedures were extracted up to September Moreover, the references of reviewed articles were scrutinized to obtain any other reference that eluded the primary search. This review is based on the results of searches carried out in PubMed and Google Scholar.Original research articles [randomized controlled trials (RCT), prospective studies (PS) and retrospective studies (RS)], meta-analyses, systematic reviews and surveys pertinent to EUSguided interventional procedures were included. Studies enrolling up to 10 patients were categorized as case series. We also included letters and case reports describing recent, innovative or original EUS-guided treatments. Commentaries, non-english language articles, congress proceedings and abstracts, and articles in which EUS did not represent the principal matter were not included. In regard to data collection, priority was assigned to the study subject, design and methods, the type and year of publication and the number of patients enrolled. The content of each study was further analyzed to identify relevant clinical issues. In particular, when the same group of patients from the same institution was included in two consecutive papers (e.g., preliminary study and final results study), we included only the data from the most recent one to avoid duplicated results. Levels of evidence were stratified according to the North of England evidence-based guidelines [5,6]. LE Ⅰ a: Evidence obtained from meta-analysis of RCTs; LE Ⅰ b: Evidence obtained from at least one RCT; LE Ⅱa: Evidence obtained from at least one well designed controlled study without randomization; LE Ⅱb: Evidence obtained from at least one other type of well-designed quasi-experimental study; LE Ⅲ: Evidence obtained from well-designed non-experimental descriptive studies such as comparative studies, correlation studies, and case studies; LE Ⅳ: Evidence obtained from expert committee reports or opinions, or clinical experiences of respected authorities. A total of 381 pertinent articles were finally included for the purpose of this systematic review. Published research focused primarily on EUS-guided cholangiography and biliary drainage (85 studies), followed by EUS-GD of pancreatic fluid collections (84 studies), EUS-guided CPN or CPB (52 studies), EUS-guided tumor ablation (34 studies), EUS-guided ethanol ablation (28 studies), EUS-guided fiducial placement (26 studies), EUS-guided vascular interventions (23 studies), EUS-guided necrosectomy (20 studies), EUS-guided pancreatography and pancreatic duct drainage (15 studies), EUS-guided gallbladder drainage (7 studies) and EUS-GD of abdominal (non-peripancreatic) and pelvic collections (7 studies). A detailed classification of the studies according to the subclasses and the corresponding LE is presented in Table 1. As expected, we identified a predominance of LE Ⅲ and Ⅳ articles in all types of EUS-guided treatments, reflecting the relative novelty of these techniques. Nevertheless, a fair number of high LE articles (LE Ⅰa and Ⅰb) were identified for EUS-GD of pancreatic fluid collections and EUS-guided CPN, forming a solid base of evidence for these established indications. On the other hand, novel therapeutic applications, such as EUS-guided cholangiography and biliary drainage and EUS-guided tumor ablation, still lack relevant clinical data and should still be considered strictly investigational. A focused description of all forms of EUS-guided treatment is given below, in a schematic format. EUS-GUIDED DRAINAGE OF PFCs EUS-GD is regarded as an established technique for the treatment of PFCs. Up to now, the reported evidence pertains about 2115 patients enrolled in safety and efficacy studies overall [7-64]. Mean technical and clinical success rates reported in series with more than 10 patients were 97% and 90%, respectively and mean overall recurrence rate was 8% [8-64] (Table 2). The mean overall complication rate was 17% including bleeding (69 cases), superinfection (52 cases), stents migration that required endoscopic reintervention (51 cases), perforation treated with surgery 8425 July 14, 2014 Volume 20 Issue 26

118 Fabbri C et al. Levels of evidence in EUS-guided treatments Table 1 Level of evidence per subject Level of evidence Ⅰa Ⅰb Ⅱa Ⅱb Ⅲ Ⅳ Total EUS-GD of pancreatic fluid collections EUS-guided necrosectomy EUS-guided cholangiography and biliary drainage EUS-guided pancreatography and pancreatic duct drainage EUS-guided gallbladder drainage EUS-GD of abdominal (non-peripancreatic) and pelvic collections EUS-guided Celiac Plexus Neurolysis or Block EUS-guided ethanol ablation EUS-guided tumor ablation EUS-guided fiducial placement EUS-guided vascular intervention Total EUS-GD: Endoscopic ultrasound-guided drainage. (27 cases) and pneumoperitoneum treated conservatively (18 cases). However, only 5 cases of death were deemed to be procedure related [8-64]. EUS vs surgical drainage A recent RCT [62] comparing EUS and surgery for pancreatic pseudocyst drainage, showed no pseudocyst recurrence during the follow-up in the former group and no evidence that surgical cystogastrostomy was superior to EUS. Moreover, EUS treatment was associated with shorter hospital stay, better physical and mental health of patients, and lower costs. EUS-GD of PFCs is not inferior to surgical drainage in terms of safety and efficacy (LE Ⅰb). EUS vs blind endoscopic drainage Meta-analysis of EUS-GD of PFCs showed superior technical and treatment success rates and more favorable safety profiles than traditional non-eus guided drainage [65] (LE Ⅰa). Varadarajulu et al [26] published the first RCT, randomizing 30 patients to undergo either EUS-GD or endoscopic conventional transmural drainage (ECTD). All patients assigned to EUS underwent successful drainage (100%), while the procedure was technically successful in only 5/15 patients (33%) assigned to ECTD. All 10 patients who failed drainage by ECTD underwent successful drainage of the PFC on a crossover to EUS. Major procedure-related bleeding was encountered in 2 patients in whom ECTD was performed (LE Ⅰb). Park et al [30] enrolled 60 patients in a RCT with the same design as above. Technical success of the drainage was significantly higher in the EUS group (94%) than in the ECTD group (72%) (P = 0.039) in intention-to-treat analysis. In 8 cases where ECTD had failed because of non-bulging PFCs, crossover to EUS-GD was always successful. Complications occurred in 7% of the EUS group vs 10% of the ECTD group (P = NS). During follow-up, PFC resolution was achieved in 97% in the EUS group and in 91% in the ECTD group (P = NS) (LE Ⅰb). EUS-GD of PFCs has superior technical and clinical outcomes compared to blind endoscopic drainage (LE Ⅰa). Forward view vs linear scanning EUS EUS-GD of PFCs is commonly performed with linear scanning echoendoscopes, whose tangential approach to PFCs may be challenging for operators. Theoretically, technical difficulties might be overcome using a forwardviewing echoendoscope which allows a straight approach to PFCs. However, a recent RCT [45] comparing the performance of linear vs forward-viewing echoendoscopes in draining PFCs failed to demonstrate any significant difference in technical success, mean procedure time, safety or efficacy between the two types of echoendoscopes. The use of forward-viewing echoendoscope for EUSguided drainage of PFCs does not confer any significant advantage in terms of safety and efficacy compared to the use of linear scanning echoendoscope (LE Ⅰb). Timing of stent removal In order to evaluate the incidence of PFCs recurrence after successful EUS-GD, 28 patients were randomized either to stent removal (n = 13) or to stent left in place (n = 15) and were followed up for a median period of 14 months. PFCs recurrence was observed in 5 patients in the stent retrieval group, as opposed to none in the other group (P = 0.013) [20]. After successful EUS-GD of PFCs, stent retrieval is associated with higher recurrence rate than leaving stent in place (LE Ⅰb). Nasocystic drainage to maintain patency: Siddiqui et al [60] evaluated in a RS EUS-guided nasocystic drainage alongside transmural stents in PFCs with viscous solid debris. Association with the nasocystic drainage resulted in lower stent occlusion rate and better short-term clinical outcomes compared to those patients who underwent standard EUS-GD. The placement of a nasocystic drainage may increase the clinical success rate, especially in PFCs containing abundant debris (LE Ⅲ). Multiple transluminal gateway technique: Varadarajulu et al [42], showed that drainage of necrotic PFCs with multiple instead of a single transmural access, placing multiple stents and a nasocystic drainage in each tract, led to better long-term clinical outcomes. Multiple instead of 8426 July 14, 2014 Volume 20 Issue 26

119 Fabbri C et al. Levels of evidence in EUS-guided treatments Table 2 Endoscopic ultrasound-guided drainage of pancreatic fluid collections Ref. Design Cases Technical success Clinical success Recurrence Complications 1 Binmoeller et al [8] RS 27 93% 78% 22% 52% Pfaffenbach et al [9] PS 11 91% 82% 18% None Giovannini et al [10] PS % 89% 9% 3% Norton et al [11] RS 14 93% 93% 23% 14% Vosoghi et al [12] RS % 93% 7% 7% Enya et al [13] PS % 85% 0% None Hookey et al [14] RS 32 96% 93% 12% 11% Krüger et al [15] PS 35 94% 88% 12% 33% Azar et al [16] RS 23 91% 82% 18% 4% Antillon et al [17] PS 33 94% 87% 4% 15% Kahaleh et al [18] PS % 93% NR 19% Ahlawat et al [19] PS % 82% 18% 18% Arvanitakis et al [20] RCT % 94% 11% 22% Lopes et al [21] RS 51 94% 84% 17% 25% Varadarajulu et al [22] PS % 95% 0% None Lopes et al [23] PS % 94% 19% 26% Ardengh et al [24] PS 77 94% 91% 11% 6% Varadarajulu et al [25] RS % 95% NR None Varadarajulu et al [26] RCT % 96% NR 4% Varadarajulu et al [27] PS 60 95% 93% 4% 2% Barthet et al [28] PS % 89% NR 25% Talreja et al [29] PS % 95% 0% 44% Park et al [30] RCT 39 95% 95% 6% 7% Yasuda et al [31] RS 26 92% 87% 17% None Itoi et al [32] PS % 100% 0% None Varadarajulu et al [33] PS % 90% 0% None Ang et al [34] PS % 100% 0% 10% Ahn et al [35] RS 47 98% 100% 11% 11% Jazrawi et al [36] RS % 100% 10% None Sadik et al [37] PS % 88% 4% 15% Will et al [38] PS % 96% 15% 29% Seicean et al [39] PS 24 83% 79% 0% 17% Heinzow et al [40] RS 42 88% 78% 21% 21% Varadarajulu et al [41] PS % 99% NR 5% Varadarajulu et al [42] RS % 69% 0% 8% Varadarajulu et al [43] RS % 100% 5% None Zheng et al [44] PS 14 90% 90% 0% 19% Voermans et al [45] RCT % 82% 9% 11% Mangiavillano et al [46] PS 21 86% 81% 14% 5% Seewald et al [47] RS 80 97% 83% 13% 26% Itoi et al [48] RS % 100% 0% 6% Puri et al [49] PS % 97% 2% 7% Fabbri et al [50] PS % 95% 5% 15% Rasmussen et al [51] RS 22 86% 86% 18% 18% Khashab et al [52] RS % 100% 0% None Penn et al [53] PS % 85% 18% 15% Weilert et al [54] PS % 78% NR 33% Rana et al [55] RS % 100% 0% 5% Binmoeller et al [56] RS % 79% NR 21% Nan et al [57] RS % 100% NR 5% Kato et al [58] RS 67 88% 83% 15% 1% Künzli et al [59] RS % 84% 18% 20% Siddiqui et al [60] RS 88 99% 79% 3% 30% Rische et al [61] RS % 94% 6% 33% Varadarajulu et al [62] RCT % 95% 0% None Total 55 studies % (83%-100%) 90% (69%-100%) 8% (0%-23%) 17% (0%-52%) 1 Complications include: early and late, procedural and stent related; 2 Only patients with walled-off pancreatic necrosis. RCT: Randomized controlled trial; PS: Prospective study; RS: Retrospective study; NR: Not reported. single transmural points of access allow better drainage of the necrotic contents and improve treatment success (LE Ⅲ). Use of covered self-expandable metal stents: Covered self-expandable metal stents have been recently tested for drainage of PFCs and walled-off pancreatic necrosis with the intent of creating a larger fistula compared to plastic stents. Increased success rate and reduced time to resolution were shown in case series and pilot studies [48,50,53,54] 8427 July 14, 2014 Volume 20 Issue 26

120 Fabbri C et al. Levels of evidence in EUS-guided treatments Table 3 Endoscopic ultrasound-guided necrosectomy Ref. Design Cases Technical success Clinical success Recurrence Complications 1 Seewald et al [70] RS % 85% 15% 30% Charnley et al [71] RS % 92% 0% None Voermans et al [72] RS % 93% 7% 40% Hocke et al [73] RS 30 97% 83% 3% 23% Schrover et al [74] RS % 75% 12% 25% Mathew et al [75] RS 6 100% 100% 0% None Escourrou et al [76] RS % 100% 0% 46% Jürgensen et al [77] RS % 97% 0% 17% Bakker et al [78] RCT % 100% 20% 40% Will et al [79] RS % 100% 11% 17% Rische et al [61] RS % 86% 14% 36% Yamamoto et al [80] RS 4 100% 50% NR 25% Hritz et al [81] RS 4 100% 100% 0% None Yasuda et al [82] RS % 75% 7% 33% Ang et al [83] RS 8 100% 87% 13% None Sarkaria et al [84] RS % 88% 0% 6% Total 16 studies % (97%-100%) 88% (50%-100%) 7% (0%-20%) 28% (0%-46%) 1 Complications include: early and late, procedural and stent related. RCT: Randomized controlled trial; PS: Prospective study; RS: Retrospective study; NR: Not reported. (LE Ⅱb). However stents designed for other indications were used. Recently, new devices have been introduced for the purpose of PFCs drainage, provided with larger diameter and antimigration features such as the NAGI stent (Taewoong-Medical Co, Seoul, South Korea) or the AXIOS stent (Xlumena Inc., Mountain View, California, United States) [66,67]. A case series [68] described the use of the AXIOS stent in 9 patients who underwent EUS-guided drainage of PFCs. The technical success rate was 89% (8/9) due to one failure of the delivery system and all patients had successful outcome achieving complete PFC resolution. One patient developed a tension pneumothorax immediately after transesophageal drainage. No migrations were reported, and all stents were removed easily. Only one patient presented a recurrence 4 wk after stent removal. Use of covered self-expandable metal stents seems to improve the clinical outcome in these patients; however, larger studies comparing metal and plastic stents are warranted (LE Ⅱb). EUS-GUIDED NECROSECTOMY Debridement of pancreatic necrosis has traditionally been managed surgically. In recent years, EUS-guided endoscopic necrosectomy has become an alternative. This technique involves a transmural (transgastric or transduodenal) EUS-guided access to the necrotic area, followed by large caliber (e.g., 18 mm) balloon dilation of the tract between the collection and the gastrointestinal wall, allowing for passage of a gastroscope into the collection to visualize the necrotic material. A variety of tools, such as baskets, snares, and nets have been used to remove the necrotic tissue. EUS-guided necrosectomy has been reported in 283 published cases so far. In the published studies a median of 4 (1-35) sessions was required to achieve resolution of the necrotic collection [69]. Mean technical and clinical success rates reported were 100% and 88%, respectively; mean overall complication rate was 28% and mean overall recurrence rate was 7% [61,70-84] (Table 3). A recent RCT [78] by the Dutch Pancreatitis Study Group showed a lower rate of proinflammatory response, organ failure and major complications in patients undergoing EUS-guided necrosectomy as compared to surgical necrosectomy (LE Ⅰb). EUS-GUIDED CHOLANGIOGRAPHY AND BILIARY DRAINAGE When biliary ductal access via endoscopic retrograde cholangiopancreatography (ERCP) fails, rescue measures include precut papillotomy, percutaneous transhepatic biliary drainage (PTBD), surgical bypass and EUS-guided BD. Three different EUS-guided BD approaches have been described: direct transluminal stenting via transgastric or transduodenal route, rendezvous technique passing a guidewire through an intrahepatic or extrahepatic access to the papilla, and antegrade stent placement. EUS-guided BD has currently been performed in 1127 published cases, with mean technical and clinical success rates of 91% and 88%, respectively. However, mean overall complication rate was 26% with mortality of 0.4% (4/1127 patients) [85-113] (Table 4). EUS-guided BD vs percutaneous BD In a recent RCT 25 patients with unresectable malignant biliary obstruction and a previous failed ERCP attempt were assigned either to EUS-guided or to percutaneous transhepatic BD. The authors reported 100% technical and clinical success in both study groups, with no difference in incidence of adverse events [99] (LE Ⅰb). Combining EUS and ERCP in the same procedure was a cost saving strategy compared to referring the patient for 8428 July 14, 2014 Volume 20 Issue 26

121 Fabbri C et al. Levels of evidence in EUS-guided treatments Table 4 Endoscopic ultrasound-guided cholangiography and biliary drainage Ref. Design Cases Technical success Clinical success Complications 1 Bories et al [86] RS 11 91% 80% 72% Maranki et al [87] RS 49 84% 80% 18% Brauer et al [88] PS 12 92% 72% 16% Horaguchi et al [89] PS % 94% 37% Kim et al [90] RS 15 80% 80% None Fabbri et al [91] PS 16 75% 75% 8% Park et al [92] RS 57 96% 89% 47% Hara et al [93] PS 18 94% 94% 77% Komaki et al [94] RS % 100% 46% Ramírez-Luna et al [95] PS 11 91% 82% 18% Shah et al [96] RS 68 85% 85% 9% Iwashita et al [97] RS 40 73% 73% 12% 2 Dhir et al [98] RS 58 98% 98% 3% Artifon et al [99] RCT % 100% 15% Song et al [100] PS 15 87% 87% 47% Kim et al [101] PS 13 92% 84% 38% Vila et al [102] RS % 70% 23% Horaguchi et al [103] RS % 100% 10% Hara et al [104] PS 18 94% 89% 27% Park et al [105] PS 45 91% 87% 11% Kawakubo et al [106] RS % 100% 14% Dhir et al [107] RS 35 97% 97% 23% Khashab et al [108] RS 35 94% 91% 14% Gornals et al [109] RS 15 87% 73% 40% Gupta et al [110] RS % 87% 35% Dhir et al [111] RS 68 97% 97% 21% 3 Kawakubo et al [112] RS 64 95% 95% 42% Total 27 studies % (70%-100%) 87% (70%-100%) 29% (3%-77%) 1 Complications include: early and late, procedural- and stent-related; 2 2.5% mortality (1 patient ); 3 4% mortality (3 patients). RCT: Randomized controlled trial; PS: Prospective study; RS: Retrospective study; NR: Not reported. percutaneous transhepatic BD [109] (LE Ⅲ). EUS-guided BD appears to be a valid alternative to percutaneous BD, showing similar efficacy and safety (LE Ⅰb). However, data are still very preliminary and large RCT are needed to demonstrate whether EUS can represent a valid alternative to percutaneous route in this setting. EUS-guided rendezvous BD vs precut papillotomy The outcome of 58 patients undergoing EUS-guided rendezvous drainage because of bile duct obstruction, after failed selective biliary cannulation, was compared to an historical cohort of 144 patients treated with precut papillotomy. Treatment success was significantly higher for the EUS-guided rendezvous patients than for those who underwent precut papillotomy, while there was no difference in complications rate [98]. EUS-guided rendezvous drainage seems to be superior to precut papillotomy in patients with bile duct obstruction after failed ERCP (LE Ⅲ). EUS-guided rendezvous BD vs EUS-guided transluminal BD A recent RS (33 patients) compared the outcome of two different techniques in patients who underwent a standardized approach to EUS-guided BD, with an initial attempt at using the rendezvous technique (n = 13) followed by the transluminal approach (n = 20) in case of rendezvous failure. The Authors reported that both techniques achieved the same effectiveness and safety [108]. Transluminal EUS-guided BD may represent a safe and effective alternative in case of failure of rendezvous technique (LE Ⅲ). EUS-guided transhepatic BD vs EUS-guided extrahepatic BD EUS-guided BD can be performed either via intrahepatic (through the stomach) or via extrahepatic (through the duodenum) route. In a recent RS, despite similar technical and clinical success rate, extrahepatic access was associated with significantly shorter procedure and hospitalization time and with less complications [107] (LE Ⅲ). Another multicenter RS enrolling 68 patients who underwent transluminal EUS-guided BD for malignant obstructive jaundice showed similar technical and clinical success both in patients who underwent transhepatic and extrahepatic drainage. However, transhepatic access was burdened with a significantly higher complication rate compared to the extrahepatic route (30.5% vs 9.3%, P = 0.03); multivariate analysis identified the transhepatic route as the only factor independently related to the risk of procedure-related adverse event [111] (LE Ⅲ). EUSguided BD shows similar technical and clinical success rate with both transhepatic and extrahepatic access. However, extrahepatic access seems to be safer than transhepatic access (LE Ⅲ) July 14, 2014 Volume 20 Issue 26

122 Fabbri C et al. Levels of evidence in EUS-guided treatments Table 5 Endoscopic ultrasound-guided pancreatography and pancreatic duct drainage Ref. Design Cases Technical success Clinical success Complications 1 Will et al [114] RS % (SPDD: 67%) 50% 43% Tessier et al [115] RS 36 92% (SPDD: 92%) 69% 55% Kahaleh et al [116] RS % (SPDD: 77%) 77% 15% Barkay et al [117] RS 21 86% (SPDD: 48%) 86% 10% Ergun et al [118] RS % (SPDD: 90%) 72% 20% Shah et al [96] RS % (SPDD: 86%) 100% 16% Vila et al [102] RS 19 58% (SPDD: NR) NR 26% Kurihara et al [119] RS % (SPDD: 93%) 93% 7% Fujii et al [120] RS 45 98% (SPDD: 73%) 53% 24% Total 9 studies % (58%-100%) 74.5% (53%-100%) 20%(7%-55%) 1 Complications include: early and late, procedural and stent related. SPDD: Successful pancreatic duct drainage; RS: Retrospective study; NR: Not reported. EUS-GUIDED PANCREATOGRAPHY AND PANCREATIC DUCT DRAINAGE EUS-guided PDD has been reported in 248 published cases so far. They are usually indicated after failed ERCP in patients with benign conditions such as ductal stones, strictures or post-surgical stenosis [85,96,102, ] (Table 5). Outcomes of EUS-guided PDD EUS-guided PDD is a challenging procedure and it is technically more demanding than EUS-guided BD. As a result, technical and clinical outcomes of EUS-guided PDD were less favorable than for EUS-guided BD with an overall technical success rate of 78% [96,102, ] (LE Ⅲ). Technical failures were mainly due to difficult orientation of the echoendoscope along the axis of the pancreatic duct, inability to dilate the transmural tract because of dense fibrosis, and impossible endotherapy because of too acute angle of access to the pancreatic duct [96,102, ]. As a note of interest, successful ERCP was reported in some cases after EUS-guided pancreatography by needle injection of contrast medium with or without methyleneblue [96,117] (LE Ⅲ). EUS-guided PDD is a challenging procedure, showing suboptimal clinical success and relevant complication rate (LE Ⅲ). Technical issues and complications EUS-guided rendezvous technique was usually attempted first, followed by the transenteric EUS-guided PDD in case of rendezvous failure [119] (LE Ⅲ). EUS-guided transenteric stenting required more dilation of the needle tract than rendezvous technique, leading to serious adverse events such as pancreatitis (4%), pancreatic juice leakage (3%), bleeding (3%), and perforation (3%) [119] (LE Ⅲ). The most common site for pancreatic duct access was through the gastric body, in view of the straight and stable echoendoscope position and the ease of access to the pancreatic duct [96,102, ] (LE Ⅲ). Plastic stents were used for EUS-guided PDD unlike metal stents. In fact, covered metal stents can block side branches leading to obstructive pancreatitis and uncovered metal stents can cause pancreatic juice leakage between the stomach and pancreas [96,102, ] (LE Ⅲ). EUS-guided PDD via transenteric route shows higher complication rate than via rendezvous route (LE Ⅲ). EUS-GUIDED GALLBLADDER DRAINAGE Patients with acute cholecystitis unresponsive to medical therapy, require decompression of the gallbladder if they are unsuitable for emergency surgery. Available treatments are percutaneous transhepatic gallbladder drainage and EUS-guided gallbladder drainage. The latter has been performed in 97 published cases with mean technical and clinical success rates are 98% and 98%, respectively; overall mean complication rate was 16% [48, ] (Table 6). EUS-guided vs percutaneous gallbladder drainage Recently a non-inferiority RCT [131] was conducted to evaluate the technical feasibility, efficacy and safety of EUS-guided vs percutaneous drainage in this setting. The authors enrolled 59 patients and reported similar technical success rate (97% vs 97%), clinical success (100% vs 96%) and rate of adverse events (7% vs 3%) in the two study groups (LE Ⅰb). Transgastric vs transduodenal approach Both transgastric and transduodenal approaches have been performed to achieve EUS-guided gallbladder drainage. In a pilot study, plastic stent migration was observed in a patient 3 wk after trans-gastric drainage. The authors suggested that transduodenal approach toward the gallbladder neck could avoid plastic stent migration [127] (LE Ⅱ b). On these basis, specific lumen-apposing metal stents with large distal and proximal flares have been developed [48,130,133]. EUS-guided gallbladder drainage shows similar feasibility, efficacy and safety profiles to percutaneous drainage (LE Ⅰb). EUS-GUIDED DRAINAGE OF ABDOMINAL NON-PERIPANCREATIC AND PELVIC COLLECTIONS EUS-GD represents a valid treatment of fluid collections located in anatomic regions adjacent to the gastrointes July 14, 2014 Volume 20 Issue 26

123 Fabbri C et al. Levels of evidence in EUS-guided treatments Table 6 Endoscopic ultrasound-guided drainage of gallbladder Ref. Design Cases Technical success Clinical success Complications 1 Baron et al [121] CR 1 100% 100% None Kwan et al [122] RS 3 100% 100% 33% Lee et al [123] PS 9 100% 100% 11% Takasawa et al [124] CR 1 100% 100% None Kamata et al [125] CR 1 100% 100% None Kamata et al [126] CR 1 100% 100% None Song et al [127] PS 8 100% 100% 37% Súbtil et al [128] RS 4 100% 100% 25% Itoi et al [129] CR 2 100% 100% None Jang et al [130] PS % 100% 13% Jang et al [131] RCT 30 97% 97% 7% Itoi et al [48] RS 5 100% 100% None Itoi et al [132] CR 1 100% 100% None de la Serna-Higuera et al [133] RS 13 85% 85% 15% Widmer et al [134] RS 3 100% 100% None Total 15 studies % (85%-100%) 100% (85%-100%) 0% (0%-37%) 1 Complications include: early and late, procedural and stent related. RCT: Randomized controlled trial; PS: Prospective study; RS: Retrospective study; CR: Case report. Table 7 Endoscopic ultrasound-guided drainage of non-peripancreatic and pelvic collections Ref. Design Cases Technical success Clinical success Complications 1 Attwell et al [135] CR 1 100% 100% None Giovannini et al [136] PS % 75% 25% Seewald et al [137] CR 2 100% 100% None Seewald et al [138] CR 1 100% 100% None Kahaleh et al [139] CR 2 100% 100% None Lee et al [140] CR 1 100% 100% None Jah et al [141] CR 1 100% 100% None Shami et al [142] RS 5 100% 100% None Ang et al [143] CR 1 100% 100% None Piraka et al [144] PS 7 100% 100% 28% Noh et al [145] PS 3 100% 100% None Puri et al [146] RS % 93% None Itoi et al [147] CR 1 100% 100% None Decker et al [148] CR 1 100% 100% None Gupta et al [149] RS 20 90% 90% 35% Ulla-Rocha et al [150] RS 6 100% 100% None Varadarajulu et al [151] CR 1 100% 100% None Knuth et al [152] CR 1 100% 100% None Ramesh et al [153] RS % 87% None Luigiano et al [154] CR 2 100% 100% None Total 20 studies % (90%-100%) 100% (75%-100%) 0% (0%-35%) 1 Complications include: early and late, procedural and stent related. PS: Prospective study; RS: Retrospective study; CR: Case report. tinal tract (i.e., subphrenic space, perihepatic, left lobe of the liver, proximal small bowel, left colon, perirectal space, etc.). EUS-GD of abdominal (non-peripancreatic) and pelvic collections has been performed in 120 published cases so far, with mean technical and clinical success rates of 99% and 92%, respectively [ ] (LE Ⅱ b). Overall complication rate was 13% (Table 7). Pelvic collections may present a clinical challenge because of their location, usually surrounded by major organs and anatomic structures (urinary bladder, rectum, prostate, vagina or uterus). All published data available reported the use of a drainage catheter or plastic stents [136,146,153] (LE Ⅲ). Fully covered metal stents have recently been adopted for the drainage of pelvic abscesses [154] in order to minimize the risk of peritoneal leaks, to provide a larger diameter fistula and to avoid early stent occlusion; all these characteristics were shown to increase the clinical success rate and the time to collection resolution (LE Ⅲ). EUS-guided drainage represents a preferential treatment of deep-seated abdominal fluid collections (LE Ⅱb). EUS-GUIDED CELIAC PLEXUS NEUROLYSIS AND BLOCK CPN and CPB provide pain relief and reduces narcotic use in patients with intra-abdominal malignancies and chronic pancreatitis [155]. The injection of a neurolytic drug into the celiac plexus disrupts the signal transmission to 8431 July 14, 2014 Volume 20 Issue 26

124 Fabbri C et al. Levels of evidence in EUS-guided treatments Table 8 Endoscopic ultrasound-guided plexus neurolysis/celiac plexus block n (%) Ref. Design Indications Techniques Technical success Clinical success (pain relief) Complications Wiersema et al [167] RS PC (n = 25) CPN 100% 79%-88% 4 transient diarrhea Metastases (n = 5) Gress et al [163] RCT CP (n = 10) EUS-guided 100% 50% None CP (n = 8) CT-guided 25% Gunaratnam et al [168] PS PC (n = 58) CPN 100% 78% 5 transient abdominal pain Gress et al [169] PS CP (n = 90) CPB 100% 55% 3 diarrhea Tran et al [170] RS PC (n = 10) CPN 100% 70% NR Ramirez-Luna et al [171] RS PC (n = 11) CPN 100% 72.20% None Levy et al [172] RS PC (n = 18) CGN (n = 17) NR 16/17 (94) 12 hypotension CGB (n = 1) 0/1 (0) 6 diarrhea CP (n = 18) CGN (n = 5) NR 4/5 (80) CGB (n = 13) 5/13 (38) O'Toole et al [173] RS PC (n = 2) CPB (n = 189) NR NR 2 post-procedural pain CP (n = 187) 1 retroperitoneal abscess PC (n = 21) CPN (n = 31) NR NR 1 hypotension CP (n = 10) Santosh et al [164] RCT CP (n = 27) EUS-CPB 100% 70% 2 diarrhea CP (n = 29) Percutaneous-CPB - 30% Leblanc et al [165] RCT CP (n = 23) CPB (central) 100% 15/23 (65) None CP (n = 27) CPB (bilateral) 16/27 (59) Sahai et al [174] RS PC (n = 34)/ Central CPN 100% 45.90% 1 adrenal artery bleeding CP (n = 37) PC (n = 45)/ Bilateral CPN 70.40% CP (n = 44) Sakamoto et al [175] PS PC (n = 67) 34CPN 100% 72%-79% None 33 BPN 96.90% 19%-78% Wyse et al [158] RCT PC (n = 96) 48 CPN 100% 60.70% None 48 control - - LeBlanc et al [160] RCT PC (n = 29) CPB (central) 100% 20/29 (69) None PC (n = 21) CPB (bilateral) 17/21 (81) Téllez-Ávila et al [161] RS PC (n = 53) Central (n = 21) NR 10/21 (48) None Bilateral (n = 32) 18/32 (56) Iwata et al [176] RS PC (n = 47) CPN 100% 68.10% NR Ascunce et al [177] RS PC (n = 64) CPN 100% 50% 1 hypotension Stevens et al [166] RCT CP (n = 40) Triamcinolone + bupivacaine (n = 21) 100% 68.4%-85.7% 1 severe hypertension Bupivacaine (n = 19) 4 pain exacerbation 1 gastric hematoma Wiechowska- Kozlowska et al [178] RS PC (n = 29) CPN 100% 86% 3 diarrhea 1 hypotonia 2 post-procedural pain Wang et al [179] PS PC (n = 23) Celiac ganglion 100% 82.60% None irradiation Leblanc et al [180] PS PC (n = 20) 10 ml (n = 10) 100% 80% 3 nausea and vomiting 20 ml (n = 10) 100% 2 diarrhea 1 lightheadness Seicean et al [181] PS PC (n = 32) CPN 100% 75% NR Doi et al [162] RCT PC (n = 68) CPN (n = 34) 100% 45.50% 1 GI bleeding CGN (n = 34) 88.20% 73.50% 3 hypotension 5 diarrhea 17 pain exacerbation Total 23 studies % (88.2%-100%) 71.9% (45.5%-90%) - RCT: Randomized controlled trial; PS: Prospective study; RS: Retrospective study; PC: Pancreatic cancer; CP: Chronic pancreatitis; CPN: Celiac plexus neurolysis; CPB: Celiac plexus block; CT: Computed tomography; CGB: Celiac ganglia block; CGN: Celiac ganglia neurolysis; NR: Not reported. spinal cord and central nervous system. Due to the anatomical location of the celiac plexus around the origin of the celiac trunk and the superior-mesenteric artery, EUS- CPN provides direct, real-time visualization leading to a safer approach than trans-abdominal or posterior access (Table 8). EUS-CPN in patients with pancreatic cancer EUS-CPN vs analgesics: EUS-CPN (8 studies, 283 patients) was demonstrated safe and effective in alleviating refractory pain due to pancreatic cancer: pooled proportion 80.1% (74.5%-85.2%) [156] (LE Ⅰa). Alcoholbased EUS-CPN was found safe and effective in this 8432 July 14, 2014 Volume 20 Issue 26

125 Fabbri C et al. Levels of evidence in EUS-guided treatments setting: the pooled proportion of patients (5 studies, 119 patients) that experienced pain relief was 72.5% [157] (LE Ⅰ a). In a recent RCT, 96 patients with advanced pancreatic cancer were randomly assigned to early EUS-guided CPN or to conventional pain management; the authors observed greater pain relief in the early EUS-CPN group at three months than in conventional management group [-67% (-87 to -25), P = 0.01] [158] (LE Ⅰb). Finally, compared to opioids, EUS-CPN (6 studies, 358 patients) was demonstrated to reduce pain at four and eight wk [visual analog score (-0.70 to -0.13) and (-0.89 to -0.01)] and significantly reduced opioid consumptions in the EUS-CPN group (P < ) [159]. EUS-CPN is superior to analgesic therapy in reducing pain (LE Ⅰa). Single central injection vs bilateral injections: Leblanc et al [160] randomized 50 patients with pancreatic cancer to receive one or two injections of alcohol for CPN without observing any difference in onset or duration of pain relief in the two groups [161]. There is no difference between central vs bilateral injections in EUS-CPN (LE Ⅰ b). EUS-CPN vs EUS-direct celiac ganglia neurolysis: Thirty-four patients were assigned to undergoing either EUS-celiac ganglia neurolysis (CGN) or classical EUS- CPN. The authors observed higher treatment response rate (73.5% vs 45.5%, P = 0.026) and complete response rate (50.0% vs 18.2%, P = 0.010) in the EUS-CGN group compared to the EUS-CPN group [162]. EUS-CGN is superior to conventional EUS-CPN in inducing pain relief (LE Ⅰb). EUS-CPN and EUS-CPB in patients with chronic pancreatitis EUS-CPN vs analgesics: In patients with pain due to chronic pancreatitis (9 studies, 376 patients) alcoholbased EUS-CPN provided pain relief in 59.4% (95%CI: ) [157]. EUS-CPN is effective in pain control due to chronic pancreatitis; however, in this setting, due to the relative lower efficacy than in oncologic disease, the development of techniques or new injected drugs seem to be needed (LE Ⅰa). EUS-CPB vs analgesic: Meta-analysis for efficacy of steroid-based EUS-guided celiac plexus block (EUS-CPB) in patients with refractory pain due to chronic pancreatitis (6 studies, 221 patients) showed an effective alleviation of abdominal pain only in 51.46% of them [158]. EUS-CPB is moderately effective in pain control due to chronic pancreatitis. In this setting, the development of new techniques and/or injected drugs is needed (LE Ⅰa). EUS-guided vs percutaneous-cpb: An RCT comparing the safety and efficacy of EUS-guided vs CT-guided celiac plexus block in patients with chronic pancreatitis showed that EUS-CPB was significantly more effective in short-term (50% vs 25% at 4 wk) and long-term (30% vs 12% at the end of follow-up) pain control [163] (LE Ⅰ b). Another RCT comparing EUS-guided (29 patients) vs percutaneous fluoroscopy-guided (27 patients) CPB with bupivacaine (10 ml) and triamcinolone (3 ml) in patients with chronic pancreatitis demonstrated an improvement in pain scores (visual analog score) in 70% of cases in the EUS group vs 30% of cases in the percutaneous group (P = 0.044) [164] (LE Ⅰb). EUS-CPB provides better pain control than percutaneous-cpb (LE Ⅰb). Single central injection vs bilateral injections: LeBlanc et al [165] randomized 50 patients with chronic pancreatitis to receive one or two injections of bupivacaine and triamcinolone without observing any difference in duration of pain relief or onset of pain in the two groups. There is no difference between central vs bilateral injections in EUS-CPB (LE Ⅰb). Bupivacaine and triamcinolone vs bupivacaine alone: In order to evaluate the effect of the addition of triamcinolone to bupivacaine in EUS-CPB, 40 patients were randomized to receive either bupivacaine alone or bupivacaine and triamcinolone. There was no significant difference in pain control between the two groups (14.3% vs 15.8% for controls), therefore the trial was stopped for futility [166]. There is no advantage of adding triamcinolone to bupivacaine for EUS-CPB (LE Ⅰb). Complications of EUS-CPN and EUS-CPB Most frequent (up to 30% of patients) adverse events related to EUS-CPN/CPB are represented by diarrhea, abdominal pain and hypotension; however, they are usually mild (grade Ⅰ-Ⅱ) and self-limiting [ ] (Table 8). Nevertheless, we found reports of serious adverse events related to EUS-CPN/CPB including bleeding, abscess, abdominal ischemia, permanent paralysis and also death (LE Ⅲ) (Table 9). In our opinion, the risk of serious morbidity and mortality should be weighed against expected benefits particularly in patients with a long life expectancy (i.e., patients with chronic pancreatitis). EUS-GUIDED ETHANOL INJECTION Pancreatic cystic lesions The initial steps for performing EUS-guided ethanol cyst ablation are similar to those for pancreatic EUS- FNA including antibiotic prophylaxis and puncturing the cysts with a 22-gauge needle. After partial or total evacuation of cystic fluid for diagnostic purposes, a volume of ethanol equal to that aspirated should be injected and maintained for 3-5 min. After aspiration of the total amount of ethanol injected, a chemotherapeutic agent (i.e., paclitaxel) may be injected and left inside the cystic cavity [ ] (Table 10). Ethanol vs saline: Ethanol injection with EUS led to a greater reduction in cyst size compared to simple saline injection (43% vs 11%); moreover, ethanol injection re July 14, 2014 Volume 20 Issue 26

126 Fabbri C et al. Levels of evidence in EUS-guided treatments Table 9 Serious adverse events of endoscopic ultrasound-guided celiac plexus neurolysis/celiac plexus block Ref. Journal Year Complication Indication Technique Gress et al [247] Gastrointest Endosc retroperitoneal bleeding CP EUS-CPN 1 retroperitoneal abscess CP EUS-CPB Mahajan et al [248] Gastrointest Endosc empyema CP EUS-CPB Muscatiello et al [249] Endoscopy retroperitoneal abscess PC EUS-CPN Sahai et al [174] Am J Gastroenterol retroperitoneal bleeding CP EUS-CPB O Toole et al [173] Endoscopy retroperitoneal abscess CP EUS-CPB Ahmed et al [250] Endoscopy ischemia CP EUS-CPN Shin SK et al [251] Korean J Pain ejaculatory failure CP EUS-CPB Lalueza et al [252] Endoscopy brain abscess CP EUS-CPN Gimeno-Garcia et al [253] Endoscopy ischemia/death CP EUS-CPN Fujii et al [254] Endoscopy spinal cord infarction/paralysis PC EUS-CPN-G Mittal et al [255] Neurology spinal cord infarction/paralysis PC EUS-CPN-G Loeve et al [256] Gastrointest Endosc gastric necrosis/death PC EUS-CPN Jang et al [257] Clin Endosc hepatic-bowel infarction/death PC EUS-CPN Doi et al [162] Endoscopy GI bleeding (puncture site) PC EUS-CGN CP: Chronic pancreatitis; PC: Pancreatic cancer; CPN: Celiac plexus neurolysis; CPB: Celiac plexus block. Table 10 Endoscopic ultrasound-guided ethanol injection of abdominal solid and cystic tumors Ref. Design Indications Lesion size (mm) Techniques Clinical success Complications Gan et al [187] PS Pancreatic cystic lesions (n = 25) 6-30 Ethanol 35% None Oh et al [185] PS Pancreatic cystic lesions (n = 14) Ethanol and paclitaxel 79% 1 acute pancreatitis 6 hyperamylasemia 1 abdominal pain Oh et al [182] PS Septated pancreas cysts (n = 10) Ethanol and paclitaxel 60% 1 acute pancreatitis DeWitt et al [183] RCT Pancreatic cystic lesions (n = 42) Ethanol vs saline 33% 1 acute pancreatitis 5 abdominal pain 1 cystic bleeding DeWitt et al [184] PS Pancreatic cystic lesions (n = 12) Ethanol 75% at follow-up - Oh et al [186] PS Pancreatic cystic lesions (n = 52) Ethanol and paclitaxel 62% 1 acute pancreatitis 1 abdominal pain 1 fever 1 splenic vein thrombosis DiMaio et al [189] RS Pancreatic cystic lesions (n = 13) 20.1 ± 7.1 Ethanol (single/multi) 38% 1 abdominal pain Oh et al [190] RS Pancreatic cystic lesions (n = 1) 5.2 Ethanol 99% 28 ml + paclitaxel Jurgensen et al [192] RS Pancreatic NET (n = 1) 13 Ethanol 95% 8 ml Muscatiello et al [193] RS Pancreatic NET (n = 1) 11 and 7 Ethanol 40% 2 ml Deprez et al [194] RS Pancreatic NET (n = 1) 13 Ethanol 98% 3.5 ml Vleggaar et al [195] RS Pancreatic NET (n = 1) 10 Ethanol 96% 0.3 ml Levy et al [191] RS Pancreatic NET (n = 5) 8-21 Ethanol 95-99% ml Barclay et al [196] RS Solid Hepatic Metastasis (n = 1) 33 Ethanol 98% 6 ml Failure, underwent surgery Complete remission No recurrence at 18 mo Complete remission Asymptomatic at 6 mo 60% symptoms resolution Good condition at 5.5 yr Portal vein thrombosis Pain + lipase increase Small pancreatic necrosis Hematoma and duodenal ulcer None None Liver hematoma Gunter et al [197] RS GI stromal tumor (n = 1) 40 Ethanol 95% Complete remission Abdominal pain 1.5 ml Mucosal ulceration Hu et al [198] RS Liver metastasis (n = 1) 35 Ethanol 100% 10 ml Artifon et al [199] RS Left adrenal metastasis (n = 1) 50 Ethanol 98% 15 ml Local control and decrease in size Palliation of related pain DeWitt et al [200] RS Metastatic lymph node (n = 1) Ethanol Locally successful None ml Total (cystic lesion) 8 studies 169 patients % (33%-79%) - Fever None RCT: Randomized controlled trial; PS: Prospective study; RS: Retrospective study; NR: Not reported; NET: Neuroendocrine tumor. sulted in complete cyst ablation in 33% of cases (12 out of 36) [183] (LE Ⅰb). Follow-up by CT scan at 2 years of patients who had obtained complete cyst ablation after treatment showed persistent resolution of pancreatic cystic lesions in 75% of cases [184] (LE Ⅱb). Ethanol injection and lavage induces a significantly greater reduction in cyst 8434 July 14, 2014 Volume 20 Issue 26

127 Fabbri C et al. Levels of evidence in EUS-guided treatments Table 11 Endoscopic ultrasound-guided tumor ablation Ref. Design Indications Techniques Type Tumor response Complications Chang et al [202] PS Pancreatic cancer (n = 8) Injection Cytoimplant 2 partial; None 1 minor Hecht et al [203] PS Pancreatic cancer (n = 21) Injection ONYX iv gemcitabine Chang et al [211] RS Pancreatic cancer (n = 1) Injection TNFerade + chemoradiotx Hecht et al [205] PS Pancreatic cancer (n = 50) Injection (27 EUS-guided) TNFerade + chemoradiotx Irisawa et al [204] PS Pancreatic cancer (n = 7) Injection Immature dendritic cells Hanna et al [207] PS Pancreatic cancer (n = 9) Injection (6 EUS-guided) BC chemoradiotx 2 partial; 2 sepsis 2 minor; 2 duodenal perforations 6 stable; 11 progression Surgical resection None 1 complete; 6 GI bleeding 3 partial; 6 deep vein thrombosis 4 minor; 2 pulmonary embolism 12 stable 2 pancreatitis 6 cholangitis 2 mixed; None 2 stable; 3 progressive 2 surgically resectable; None 3 partial Chang et al [206] PS Esophageal cancer (n = 24) Injection TNFerade 6 complete; 5 thromboembolic events 2 stable (highest dose) Arcidiacono [208] PS Pancreatic cancer (n = 22) Cryothermal Ablation EUS-CTP 6 partial response (only 6 patients analyzed) 3 hyperamylasemia Maier et al [212] PS Head/neck cancer (n = 21) Brachytx Ir-192 needles 4 full; None 15 partial; 3 none Lah et al [213] RS Metastatic celiac lymph Brachytx I-125 seeds Response None nodes (n = 1) Martinez- RS Metastatic mediastinal Brachytx I-125 seeds Response None Monge et al [214] lymph node (n = 1) Sun et al [209] PS Pancreatic cancer (n = 15) Brachytx I-125 seeds 4 partial; 1 site infection 3 minor; 3 hematologic side effects 5 stable; 3 progressive Jin et al [210] PS Pancreatic cancer (n = 22 ) Brachytx I-125 seeds 4 partial; 1 seed migration 10 stable RCT: Randomized controlled trial; PS: Prospective study; RS: Retrospective study; NR: Not reported; CTP: Cryothermal probe; GI: Gastrointestinal. size and allows a significantly higher rate of cyst ablation than saline alone (LE Ⅰb). Ethanol plus paclitaxel: In their experience on 52 patients with uniloculated or oligoloculated pancreatic cyst treated with ethanol lavage followed by paclitaxel injection, Oh et al [186] observed complete resolution in 62% of patients after 1-year follow-up. The authors identified small cyst size as a positive predictive factor of treatment response. Addition of paclitaxel to ethanol injection is safe and effective and leads to a greater treatment rate of pancreatic cystic lesions compared to ethanol alone (LE Ⅱb). Solid lesions EUS-guided injection of ethanol has been applied to a variety of solid tumors including pancreatic endocrine tumors, hepatic metastases, and submucosal tumors [ ]. In a single-center RS, Levy et al [191] reported safety and efficacy of EUS-guided ethanol injection in five patients with pancreatic insulinoma. The authors obtained symptoms resolution in 60% of patients with no complications [191] (LE Ⅲ). Ethanol injection is feasible and safe in solid pancreatic insulinomas (LE Ⅲ). EUS-GUIDED TUMOR ABLATION EUS-guided fine needle injection EUS-fine needle injection (FNI) is a simple technique to deliver chemotherapeutic agents into tumoral tissue for the treatment of locally advanced pancreatic or esophageal cancer. The technical outcome of all the studies about EUS-FNI reached 100%, paralleling the ability of performing EUS-FNA for cytological diagnosis. However, the clinical outcome varied greatly according to the different chemical or biological agents being tested [201] (Table 11). Allogeneic mixed lymphocyte culture: The first study assessing EUS-FNI for pancreatic cancer tested the safety and efficacy of allogeneic mixed lymphocyte culture in locally advanced pancreatic adenocarcinoma in 8 patients. The procedure (single session of EUS-guided injection) was safe and two partial responses and one minor response were reported (median survival 13.2 mo) [202] (LE Ⅱb). Adenovirus ONYX-015: ONYX-015, a modified adenovirus (deletion in the E1B gene) which replicate in tumor 8435 July 14, 2014 Volume 20 Issue 26

128 Fabbri C et al. Levels of evidence in EUS-guided treatments cells leading to cell death, was used for EUS-FNI in pancreatic cancer in combination with systemic gemcitabine. The authors enrolled 21 patients in this phase Ⅰ study and reported two patients with partial regression and two with minor response. However, 4 serious adverse events were observed (two sepsis and two duodenal perforations) [203] (LE Ⅱb). Immature dendritic cells: Irisawa et al [204] reported a pilot study (phase Ⅰ) with injection of immature dendritic cells (DCs). DCs were used for EUS-FNI in view of their potent induction of primary T-cell response against tumor antigens. Among 7 patients with locally advanced pancreatic adenocarcinoma, one complete and three partial responses were reported. No adverse events were described [204] (LE Ⅱb). TNFerade: EUS-FNI of TNFerade, a replication-deficient adenovirus vector carrying the tumor necrosis factor-α gene, was tested in a multicenter study on 50 patients with locally advanced pancreatic cancer in combination with systemic fluorouracil. The authors observed 1 complete response, 3 partial responses, and 12 patients with stable disease after treatment. Interestingly, seven patients became suitable for surgery after EUS-FNI and 6 of them underwent R0 resection. According to the authors, an RCT is warranted to further assess these encouraging results [205] (LE Ⅱb). The efficacy of EUS-FNI of TNFerade was also assessed in 24 patients with locally advanced but still resectable esophageal cancer (20% stage Ⅱ, 80% stage Ⅲ). EUS-FNI of TNFerade was combined with cisplatin, 5-fluorouracil and radiation therapy. Six complete responses and 2 stable diseases were observed. The median survival was 47.8 mo and 5-year survival and disease-free survival rates were 41% and 38%, respectively. Additionally, EUS-FNI proved to be safe [206] (LE Ⅱb). BC-819: The safety, tolerability and preliminary efficacy of EUS-FNI of BC-819, a DNA plasmid developed to target the expression of diphtheria-toxin gene under the control of H19 regulatory sequences, was recently tested in 6 patients with pancreatic cancer in combination with chemoradiotherapy. Three patients showed partial response and other two patients who were downstaged were able to undergo surgical resection [207]. Intratumoral EUS-FNI in patients with advanced pancreatic and esophageal cancer is technically easy, safe and can induce tumor downstaging in some cases (LE Ⅱb). EUS-guided cryothermal ablation The safety and efficacy of cryothermal ablation was assessed using a newly developed cryotherm probe (CTP) in 22 patients with locally advanced pancreatic cancer. CTP is a large bore flexible bipolar device that combines radiofrequency with cryogenic cooling in the same session. EUS-guided CTP ablation was feasible in 16 patients. CT scan was performed in all cases after treatment; in 6/16 patients a reduction in tumor size was clearly seen. The procedure was well tolerated in all cases [208] (LE Ⅱb). EUS-guided brachytherapy The feasibility, safety and efficacy of EUS-guided implantation of radioactive seeds in patients with locally advanced pancreatic cancer were assessed in a few studies [ ]. Partial tumor response ranged from 13.6% to 27% while a stable disease was observed in 45.5%-53% of cases in two pilot studies [209,210]. In both series, up to 30% of patients reported transient pain reduction within the first period after treatment. Adverse event rate range was 0%-20% (pancreatitis and pseudocyst formation) in association to systemic, non-eus-related, adverse events (LE Ⅱb). EUS-guided CTP ablation and brachytherapy are feasible in a subset of patients with locally advanced pancreatic cancer. However, their safety and clinical outcome have to be further investigated (LE Ⅱb). EUS-guided fiducial placement Imaging-guided radiation therapy is based upon a realtime tracking system to target the tumor to be irradiated. In order to minimize irradiation of adjacent normal tissue in pancreatic malignancies, the placement of radiopaque fiducials inside or near the tumor allows a radiographic marking enabling precise tumor targeting. Firstly, fiducials were placed in patients with advanced pancreatic cancer were placed with surgical or radiological techniques. In the last decade, the less invasive EUS-guided fiducial placement was shown to be safe and precise [ ] (Table 12). Safety and effectiveness: Two PSs enrolling a total of 101 patients with locally advanced or recurrent pancreatic cancer reported high technical and clinical success rates (88%-90%). Overall complication rate was low with only few minor adverse events (one patient experienced minor bleeding from the site of EUS needle entrance and one experienced mild pancreatitis). Migration of the gold fiducials was reported in 7% of cases [216,217] (LE Ⅱb). Traditional vs coiled fiducials: Khashab et al [218] compared the technical success, safety, visibility and migration of two different types of fiducials (traditional vs coiled). In their RS, no differences were observed in visibility, degree of fiducial migration, number of fiducial placement, technical difficulty or complication rate (LE Ⅲ). Ideal fiducial geometry: A recent study compared the achievement of the iideal fiducial geometry (IGF) (defined as the placement of 3 fiducials with at least 2 cm of distance, at least 15 degrees angle, and non-planar placement) in 39 patients who underwent EUS-guided fiducial placement vs 38 who underwent surgical fiducial placement. In this RS, the authors identified a significantly higher rate of IGF reached with surgical vs EUS placement (47% vs 18%, P = ). However, it was ob July 14, 2014 Volume 20 Issue 26

129 Fabbri C et al. Levels of evidence in EUS-guided treatments Table 12 Endoscopic ultrasound-guided fiducial placement n (%) Ref. Design Indications Techniques Technical success Needle Complications Pishvaian et al [215] PS Abdominal/mediastinal Fiducial placement 11/13 (84.6) 19 Gauge 1 infection cancer (n = 13) Varadarajulu et al [222] RS Pancreatic cancer (n = 9) Fiducial placement 9/9 (100) NR None DiMaio et al [223] RS Abdominal/mediastinal Fiducial placement 29/30 (97) 22 Gauge None cancer (n = 30) Sanders et al [217] PS Pancreatic cancer (n = 51) Fiducial placement 46/51 (90) 19 Gauge 1 mild pancreatitis Park et al [216] PS Pancreatic cancer (n = 57) Fiducial placement 50/57 (88) 19 Gauge None Ammar et al [224] RS Abdominal cancer/lymph nodes (n = 13) Single fiducial marker 9/9 trans-gastric 22 Gauge None 4/4 trans-duodenal Varadarajulu et al [225] PS Pancreatic cancer (n = 2) Fiducial placement 2/2 (100) 19 Gauge flexible None Khashab et al [218] RS Pancreatic cancer (n = 39) Fiducial placement 39/39 (100) 19 and 22 Gauge None (traditional vs coiled) Law et al [226] RS Small pancreatic NET (n = 2) Fiducial placement 2/2 (100) 22 Gauge None Majumder et al [219] RS Pancreatic cancer (n = 39) Fiducial placement 35/39 (89.7) 19 Gauge 1 mild pancreatitis 4 abdominal pain Yang et al [220] RS Prostate cancer (n = 16) Fiducial placement 16/16 (100) 19 Gauge None Yang et al [221] RS Prostate cancer recurrence Fiducial placement 6/6 (100) 19 Gauge None (n = 6) Trevino et al [227] RS Rectal cancer (n = 1) Fiducial placement 3/3 (100) 19 Gauge None (forward-view EUS) Total 13 studies % (84.6%-100%) - 0% RCT: Randomized controlled trial; PS: Prospective study; RS: Retrospective study; NR: Not reported; NET: Neuroendocrine tumor: served that despite the lower IGF rate in the EUS group, fiducial tracking for irradiation therapy was successful in a similar percentage of patients from the two groups (> 80%) [219] (LE Ⅲ). EUS-guided fiducial placement is safe and leads to technical and clinical success in about 90% of patients (LE Ⅱb). Non-pancreatic cancer: Two recent retrospective case series reported the feasibility and safety of fiducial placement in 16 patients with prostate cancer and in 6 with prostate cancer recurrence. The authors reported extremely high success rates (16/16 and 6/6 respectively) with no incidence of adverse events [220,221] (LE Ⅲ). EUSguided fiducial placement was feasible and safe in patients with prostate cancer or prostate cancer recurrence (LE Ⅲ). EUS-GUIDED VASCULAR INTERVENTIONS EUS combined with color/power Doppler allows precise identification of vascular anatomy, potential high risk vessels with/without portal hypertension, and occult sources of bleeding such as Dieulafoy s lesions and pseudoaneurysms. Moreover, EUS provides direct access to vascular structures next to gastrointestinal wall, allowing precise vascular interventions [ ] (Table 13). EUS-guided treatment of non-variceal bleeding The efficacy of EUS-guided treatments of non-variceal upper gastrointestinal bleeding was reported only in form of small case series and case reports. Fockens et al [229] first reported about the usefulness of EUS in the diagnosis of small abnormal vessels in 8 patients with Dieulafoy s lesions. In 50% of cases it was possible to perform EUSguided injection of sclerosing agent into the aberrant vessels [229] (LE Ⅲ). EUS-guided treatment of portal hypertension Endoscopic vs EUS-guided sclerotherapy of esophageal collateral veins: An RCT compared the safety and efficacy of EUS-guided and endoscopic sclerotherapy (ethanolamine oleate injection) in 50 patients affected by liver cirrhosis. The authors did not observe any difference in variceal eradication, number of sessions needed to achieve the eradication, variceal recurrence and adverse event rates [230] (LE Ⅰb). EUS-guided sclerotherapy does not confer any significant advantage in terms of safety and efficacy compared to classical endoscopic sclerotherapy (LE Ⅰb). Gastric variceal bleeding: In a RS, EUS-assisted cyanoacrylate (CYA) injection until obliteration of all gastric varices collateral was compared to an historical group of cirrhotic patients who underwent standard endoscopic injection, only in case of recurrent bleeding. While early re-bleeding rate was similar in the two groups (7.4% vs 12.8%, respectively, P = NS), late recurrent bleeding was significantly reduced in patients who underwent CYA injection under EUS control to check for complete obliteration (18.5% vs 44.7%, P = , OR = 0.28) [231] (LE Ⅱb). EUS guidance allows an higher rate of gastric variceal obliteration and reduces recurrent bleeding (LE Ⅱb). Coil embolization vs CYA injection for gastric varices: A multicenter RS compared feasibility, safety and applicability of coil embolization vs sclerotherapy (CYA 8437 July 14, 2014 Volume 20 Issue 26

130 Fabbri C et al. Levels of evidence in EUS-guided treatments Table 13 Endoscopic ultrasound-guided vascular interventions n (%) Ref. Design Indications Techniques Technical success Rebleeding Complications Fockens et al [229] RS Dieulafoy s lesion (n = 4) Polidocanol injection 4/4 (100) 2/4 (50) None Levy et al [234] RS Dieulafoy s lesion (n = 1) Alcohol 99% injection 1/1 (100) No None Gonzalez et al [235] RS Dieulafoy s lesion (n = 2) Polidocanol 2/2 (100) No None or CYA injection Levy et al [234] RS Various (n = 4) Alcohol 99% or CYA 4/4 (100) No None injection Gonzalez et al [235] RS Pseudo-aneurysm (n = 3) CYA injection 3/3 (100) No None Gonzalez et al [235] RS Gastric varices (n = 2) CYA injection 2/2 (100) No None Lee et al [231] RS Gastric varices (n = 101) EUS-assisted CYA injection - Early 4/54 (7.4) None Late 10/54 (18) Lahoti et al [236] RS Esophageal varices (n = 5) Sclerotherapy 5/5 (100) No 1 esophageal stricture Romero-Castro et al [237] RS Gastric varices (n = 5) CYA injection 5/5 (100) No None De Paulo et al [230] RCT Esophageal varices (n = 50) Levy et al [238] RS Choledochojejunal anastomotic varices (n = 1) Endo vs EUS-guided CYA injection 24/25 (96) 2/24 recurrence of varices (8.3) None Coil embolization 1/1 (100) No None Romero-Castro et al [239] RS Gastric varices (n = 4) Coil embolization 3/4 (75) No None Binmoeller et al [233] RS Gastric varices (n = 30) CYA injection + coil 30/30 (100) 4/24 (16.6) None embolization Romero-Castro et al [232] RS Gastric varices (n = 30) CYA injection (n = 19) vs coils (n = 11) Weilert et al [240] RS Rectal varices (n = 1) CYA injection plus coils Gonzalez et al [241] RS Splenic artery aneurism (n = 1) Roberts et al [242] RS Visceral pseudoaneurysm (n = 1) 97.4 % vs 90.9% NR 9 CYA embolization; 1 chest pain; 1 fever; 1 variceal bleeding 100% No None CYA injection 1/1 (100) No None HistoAcryl injection 1/1 (100) No None Roach et al [243] RS SMA aneurysm (n = 1) Thrombin injection 1/1 (100) No None Chaves et al [244] RS SMA aneurysm (n = 1) Thrombin injection 1/1 (100) No None Robinson et al [245] RS Splenic artery aneurysm (n = 1) Thrombin injection 1/1 (100) No None Lameris et al [246] RS Visceral pseudoaneurysm (n = 1) Thrombin + collagen injection 1/1 (100) No None RCT: Randomized controlled trial; PS: Prospective study; RS: Retrospective study; NR: Not reported; CYA: Cyanoacrylate; SMA: Superior mesenteric artery. injection) under EUS guidance. Thirty patients (11 coil group vs 19 CYA group) underwent EUS-guided treatment for gastric varices. The rate of variceal obliteration was similar in the two groups (90.9% vs 94.7%, respectively) without differences in number of EUS sessions. Eleven patients (11/19) in the sclerotherapy group experienced adverse events; in 9 of them an asymptomatic pulmonary glue embolism was found on CT scan, while 1 patient experienced fever and another experienced chest pain; on the other hand, only one patient treated with coil embolization experienced an adverse event (esophageal variceal bleeding). The comparison among the two treatment groups demonstrated a significantly lower incidence of any grade adverse events in the embolization group (58% vs 9%, P < 0.01); only 3 patients, two in the CYA and one in the coil group, experienced symptomatic adverse events [232] (LE Ⅱb). Combined coil embolization and CYA injection for gastric varices: The authors reported about 30 patients who underwent EUS-guided trans-esophageal combined embolization and sclerotherapy of gastric varices using in the majority of cases a forward-view echoendoscope. Successful treatment was achieved in all cases (30 out of 30, 100%) after a mean of 1.3 EUS sessions, including 2 cases with active bleeding. Rebleeding occurred in 16% of cases and no procedure-related adverse events were reported [233] (LE Ⅲ). EUS-guided coil embolization and CYA injection are both effective for gastric varices treatment in patients with cirrhosis (LE Ⅱb). While both sclerotherapy and embolization monotherapy present a high complication rate, combined coil embolization and CYA injection seems to be safe and effective in patients with gastric varices (LE Ⅲ). CONCLUSION Several EUS-guided treatments are now available in endosonographer s armamentarium. The usefulness of EUS-GD of PFCs and of EUS-CPN has been well established in studies with high LE. Other techniques including EUS-guided biliary drainage have been tested only in studies with medium-low LE and thus should still be performed either in referral centers by experienced endosonographers or in investigational/research settings. Well-designed RCTs are warranted to further elucidate 8438 July 14, 2014 Volume 20 Issue 26

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134 Fabbri C et al. Levels of evidence in EUS-guided treatments M, Rolshud D, Widmer J, Kahaleh M. Pancreatic necrosectomy using covered esophageal stents: a novel approach. J Clin Gastroenterol 2014; 48: [PMID: DOI: /MCG.0b013e ] 85 Binmoeller KF, Nguyen-Tang T. Endoscopic ultrasoundguided anterograde cholangiopancreatography. J Hepatobiliary Pancreat Sci 2011; 18: [PMID: DOI: /s ] 86 Bories E, Pesenti C, Caillol F, Lopes C, Giovannini M. Transgastric endoscopic ultrasonography-guided biliary drainage: results of a pilot study. Endoscopy 2007; 39: [PMID: DOI: /s ] 87 Maranki J, Hernandez AJ, Arslan B, Jaffan AA, Angle JF, Shami VM, Kahaleh M. Interventional endoscopic ultrasound-guided cholangiography: long-term experience of an emerging alternative to percutaneous transhepatic cholangiography. Endoscopy 2009; 41: [PMID: DOI: /s ] 88 Brauer BC, Chen YK, Fukami N, Shah RJ. Single-operator EUS-guided cholangiopancreatography for difficult pancreaticobiliary access (with video). 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136 Fabbri C et al. Levels of evidence in EUS-guided treatments approach. Dig Endosc 2013; Epub ahead of print [PMID: DOI: /den.12163] 135 Attwell AR, McIntyre RC, Antillon MR, Chen YK. EUSguided drainage of a diverticular abscess as an adjunct to surgical therapy. Gastrointest Endosc 2003; 58: [PMID: DOI: /S (03) ] 136 Giovannini M, Bories E, Moutardier V, Pesenti C, Guillemin A, Lelong B, Delpéro JR. Drainage of deep pelvic abscesses using therapeutic echo endoscopy. Endoscopy 2003; 35: [PMID: DOI: /s ] 137 Seewald S, Brand B, Omar S, Yasuda I, Seitz U, Mendoza G, Holzmann T, Groth S, Thonke F, Soehendra N. EUS-guided drainage of subphrenic abscess. Gastrointest Endosc 2004; 59: [PMID: DOI: / S (03) ] 138 Seewald S, Imazu H, Omar S, Groth S, Seitz U, Brand B, Zhong Y, Sikka S, Thonke F, Soehendra N. EUSguided drainage of hepatic abscess. Gastrointest Endosc 2005; 61: [PMID: DOI: / S (04) ] 139 Kahaleh M, Wang P, Shami VM, Tokar J, Yeaton P. 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138 Fabbri C et al. Levels of evidence in EUS-guided treatments ultrasound. Endoscopy 2008; 40 Suppl 2: E83 [PMID: DOI: /s ] 194 Deprez PH, Claessens A, Borbath I, Gigot JF, Maiter D. Successful endoscopic ultrasound-guided ethanol ablation of a sporadic insulinoma. Acta Gastroenterol Belg 2008; 71: [PMID: ] 195 Vleggaar FP, Bij de Vaate EA, Valk GD, Leguit RJ, Siersema PD. Endoscopic ultrasound-guided ethanol ablation of a symptomatic sporadic insulinoma. Endoscopy 2011; 43 Suppl 2 UCTN: E328-E329 [PMID: DOI: / s ] 196 Barclay RL, Perez-Miranda M, Giovannini M. EUS-guided treatment of a solid hepatic metastasis. Gastrointest Endosc 2002; 55: [PMID: ] 197 Günter E, Lingenfelser T, Eitelbach F, Müller H, Ell C. EUSguided ethanol injection for treatment of a GI stromal tumor. Gastrointest Endosc 2003; 57: [PMID: ] 198 Hu YH, Tuo XP, Jin ZD, Liu Y, Guo Y, Luo L. Endoscopic ultrasound (EUS)-guided ethanol injection in hepatic metastatic carcinoma: a case report. 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139 Fabbri C et al. Levels of evidence in EUS-guided treatments j.gie ] 222 Varadarajulu S, Trevino JM, Shen S, Jacob R. The use of endoscopic ultrasound-guided gold markers in imageguided radiation therapy of pancreatic cancers: a case series. Endoscopy 2010; 42: [PMID: DOI: / s ] 223 DiMaio CJ, Nagula S, Goodman KA, Ho AY, Markowitz AJ, Schattner MA, Gerdes H. EUS-guided fiducial placement for image-guided radiation therapy in GI malignancies by using a 22-gauge needle (with videos). Gastrointest Endosc 2010; 71: [PMID: DOI: /j.gie ] 224 Ammar T, Coté GA, Creach KM, Kohlmeier C, Parikh PJ, Azar RR. Fiducial placement for stereotactic radiation by using EUS: feasibility when using a marker compatible with a standard 22-gauge needle. Gastrointest Endosc 2010; 71: [PMID: DOI: /j.gie ] 225 Varadarajulu S, Bang JY, Hebert-Magee S. Assessment of the technical performance of the flexible 19-gauge EUS-FNA needle. 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Treatment of esophageal varices: a randomized controlled trial comparing endoscopic sclerotherapy and EUS-guided sclerotherapy of esophageal collateral veins. Gastrointest Endosc 2006; 63: ; quiz 463 [PMID: ] 231 Lee YT, Chan FK, Ng EK, Leung VK, Law KB, Yung MY, Chung SC, Sung JJ. EUS-guided injection of cyanoacrylate for bleeding gastric varices. Gastrointest Endosc 2000; 52: [PMID: ] 232 Romero-Castro R, Ellrichmann M, Ortiz-Moyano C, Subtil- Inigo JC, Junquera-Florez F, Gornals JB, Repiso-Ortega A, Vila-Costas J, Marcos-Sanchez F, Muñoz-Navas M, Romero- Gomez M, Brullet-Benedi E, Romero-Vazquez J, Caunedo- Alvarez A, Pellicer-Bautista F, Herrerias-Gutierrez JM, Fritscher-Ravens A. EUS-guided coil versus cyanoacrylate therapy for the treatment of gastric varices: a multicenter study (with videos). Gastrointest Endosc 2013; 78: [PMID: DOI: /j.gie ] 233 Binmoeller KF, Weilert F, Shah JN, Kim J. EUS-guided transesophageal treatment of gastric fundal varices with combined coiling and cyanoacrylate glue injection (with videos). Gastrointest Endosc 2011; 74: [PMID: DOI: /j.gie ] 234 Levy MJ, Wong Kee Song LM, Farnell MB, Misra S, Sarr MG, Gostout CJ. Endoscopic ultrasound (EUS)-guided angiotherapy of refractory gastrointestinal bleeding. Am J Gastroenterol 2008; 103: [PMID: ] 235 Gonzalez JM, Giacino C, Pioche M, Vanbiervliet G, Brardjanian S, Ah-Soune P, Vitton V, Grimaud JC, Barthet M. Endoscopic ultrasound-guided vascular therapy: is it safe and effective? Endoscopy 2012; 44: [PMID: DOI: /s ] 236 Lahoti S, Catalano MF, Alcocer E, Hogan WJ, Geenen JE. Obliteration of esophageal varices using EUS-guided sclerotherapy with color Doppler. Gastrointest Endosc 2000; 51: [PMID: ] 237 Romero-Castro R, Pellicer-Bautista FJ, Jimenez-Saenz M, Marcos-Sanchez F, Caunedo-Alvarez A, Ortiz-Moyano C, Gomez-Parra M, Herrerias-Gutierrez JM. 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Endoscopy 2009; 41: [PMID: DOI: /s ] 242 Roberts KJ, Jones RG, Forde C, Marudanayagam R. Endoscopic ultrasound-guided treatment of visceral artery pseudoaneurysm. HPB (Oxford) 2012; 14: [PMID: DOI: /j x] 243 Roach H, Roberts SA, Salter R, Williams IM, Wood AM. Endoscopic ultrasound-guided thrombin injection for the treatment of pancreatic pseudoaneurysm. Endoscopy 2005; 37: [PMID: ] 244 Chaves DM, Costa FF, Matuguma S, Lera Dos Santos ME, de Moura EG, Maluf Filho F, Sakai P. Splenic artery pseudoaneurysm treated with thrombin injection guided by endoscopic ultrasound. Endoscopy 2012; 44 Suppl 2 UCTN: E [PMID: DOI: /s ] 245 Robinson M, Richards D, Carr N. Treatment of a splenic artery pseudoaneurysm by endoscopic ultrasound-guided thrombin injection. Cardiovasc Intervent Radiol 2007; 30: [PMID: ] 246 Lameris R, du Plessis J, Nieuwoudt M, Scheepers A, van der Merwe SW. A visceral pseudoaneurysm: management by EUS-guided thrombin injection. Gastrointest Endosc 2011; 73: [PMID: DOI: /j.gie ] 247 Gress F, Ciaccia D, Kiel S. Endoscopic ultrasound (EUS) guided celiac plexus block (CB) for management of pain due to chronic pancreatitis (CP): a large single centre experience. Gastrointest Endosc 1997; 45: AB Mahajan RJ, Nowel W, Theerathron P, Lipscomb A, Hart R, Adams L. Empyema after endoscopic ultrasound guided celiac plexus block (EUS-CPB) in chronic pancreatitis (CP): experience at an academic center [Abstract]. Gastrointest Endosc 2002; 55: AB Muscatiello N, Panella C, Pietrini L, Tonti P, Ierardi E. Complication of endoscopic ultrasound-guided celiac plexus neurolysis. Endoscopy 2006; 38: 858 [PMID: ] 250 Ahmed HM, Friedman SE, Henriques HF, Berk BS. Endorgan ischemia as an unforeseen complication of endoscopic-ultrasound-guided celiac plexus neurolysis. Endoscopy 2009; 41 Suppl 2: E218-E219 [PMID: DOI: / s ] 251 Shin SK, Kweon TD, Ha SH, Yoon KB. Ejaculatory failure af July 14, 2014 Volume 20 Issue 26

140 Fabbri C et al. Levels of evidence in EUS-guided treatments ter unilateral neurolytic celiac plexus block. Korean J Pain 2010; 23: [PMID: DOI: /kjp ] 252 Lalueza A, López-Medrano F, del Palacio A, Alhambra A, Alvarez E, Ramos A, Pérez A, Lizasoain M, Meije Y, García- Reyne A, Aguado JM. Cladosporium macrocarpum brain abscess after endoscopic ultrasound-guided celiac plexus block. Endoscopy 2011; 43 Suppl 2 UCTN: E9-10 [PMID: DOI: /s ] 253 Gimeno-García AZ, Elwassief A, Paquin SC, Sahai AV. Fatal complication after endoscopic ultrasound-guided celiac plexus neurolysis. Endoscopy 2012; 44 Suppl 2 UCTN: E267 [PMID: DOI: /s ] 254 Fujii L, Clain JE, Morris JM, Levy MJ. Anterior spinal cord infarction with permanent paralysis following endoscopic ultrasound celiac plexus neurolysis. Endoscopy 2012; 44 Suppl 2 UCTN: E265-E266 [PMID: DOI: / s ] 255 Mittal MK, Rabinstein AA, Wijdicks EF. Pearls & amp; oy-sters: Acute spinal cord infarction following endoscopic ultrasound-guided celiac plexus neurolysis. Neurology 2012; 78: e57-e59 [PMID: DOI: / WNL.0b013e318248df51] 256 Loeve US, Mortensen MB. Lethal necrosis and perforation of the stomach and the aorta after multiple EUS-guided celiac plexus neurolysis procedures in a patient with chronic pancreatitis. Gastrointest Endosc 2013; 77: [PMID: DOI: /j.gie ] 257 Jang HY, Cha SW, Lee BH, Jung HE, Choo JW, Cho YJ, Ju HY, Cho YD. Hepatic and splenic infarction and bowel ischemia following endoscopic ultrasound-guided celiac plexus neurolysis. Clin Endosc 2013; 46: [PMID: DOI: /ce ] P- Reviewers: Chatterjee S, Gornals JB, Tellez-Avila F S- Editor: Zhai HH L- Editor: A E- Editor: Zhang DN 8448 July 14, 2014 Volume 20 Issue 26

141 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. WJG 20 th Anniversary Special Issues (13): Gastrointestinal endoscopy Colonic polyps: Is it useful to characterize them with advanced endoscopy? TOPIC HIGHLIGHT Maria Lopez-Ceron, Erwin Sanabria, Maria Pellise Maria Lopez-Ceron, Erwin Sanabria, Maria Pellise, Endoscopy Unit, Department of Gastroenterology, Institut de Malalties Digestives i Metabòliques, Hospital Clinic, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Institut d Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain Author contributions: Lopez-Ceron M and Sanabria E performed the literature review and wrote the manuscript; Pellise M contributed to topic selection, critical revision and approval of the final manuscript. Supported by The CIBERehd (Centro de Investigación Biomédica en Red, enfermedades hepaticas y digestivas) to Sanabria E Correspondence to: Maria Pellise, MD, PhD, Endoscopy Unit, Department of Gastroenterology, Institut de Malalties Digestives i Metabòliques, Hospital Clinic, Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Institut d Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Villarroel 170, Catalonia, Barcelona, Spain. mpellise@clinic.ub.es Telephone: Fax: Received: November 5, 2013 Revised: February 14, 2014 Accepted: March 12, 2014 Published online: July 14, 2014 Abstract There have been major developments in endoscopic imaging techniques in recent years. Endoscopes with high definition and magnification can provide high quality images that allow for the histological estimation of lesions in vivo and in situ when combined with ancillary enhancement techniques such as chromoendoscopy (CE) and virtual CE (narrow band imaging fujinon intelligent chromoendoscopy, or i-scan). Despite the enormous potential for these advanced techniques, their value and feasibility in the clinic are still doubted, particularly in cases of colonic polyps that are slated for removal, where in vivo characterization may be deemed unnecessary. However, there are several advantages offered by such advanced endoscopic imaging. CE with or without magnification demonstrates highly accurate histology and invasion depth prediction, and virtual CE is a feasible and less cumbersome alternative to CE in terms of histological estimation, though not sufficiently accurate for depth invasion prediction. Furthermore, the supplementary information provided by advanced imaging systems can assist the endoscopist in the selection of a strategic approach, such as in deciding whether a colonic lesion should be resected, left in situ, or requires more intensive surgical treatment. Lastly, advanced high-resolution imaging techniques may be more cost effective, such that histopathology of lowrisk lesions following resection can be eliminated. The results of these evaluations and comparisons with traditional CE are presented and discussed. Taken together, the benefits provided by these advanced capabilities justify their development, and advocates their use for the treatment and management of colonic polyps Baishideng Publishing Group Inc. All rights reserved. Key words: Colon polyps; Chromoendoscopy; Narrow band imaging; Fujinon intelligent chromoendoscopy; i-scan; Colonoscopy; Optical biopsy Core tip: Endoscopic characterization of colonic polyps by virtual histology is a currently accessible tool for identification of lesion type, thus enabling endoscopists to determine optimal treatment strategies. Chromoendoscopy (CE) has shown high accuracy for differentiating polyp histologies (neoplastic vs non-neoplastic) and for estimating the depth of invasion. Furthermore, digital systems such as narrow band imaging are a viable alternative to CE regarding lesion characterization, ease of use, reversibility, and cleanliness. Lopez-Ceron M, Sanabria E, Pellise M. Colonic polyps: Is it useful to characterize them with advanced endoscopy? World J Gastroenterol 2014; 20(26): Available from: URL: DOI: July 14, 2014 Volume 20 Issue 26

142 Lopez-Ceron M et al. Endoscopic characterization of colonic polyps COLORECTAL CANCER Colorectal cancer (CRC) is the second most common cancer in women and the third most common in men from developed countries [1], which contrasts with the preventable nature of gastrointestinal cancers. While colorectal adenomas have long been considered as CRC precursors, recent evidence shows that that 20%-30% of all CRCs emerge from a distinct group of lesions, generically named serrated polyps, consisting of traditional serrated adenomas, sessile serrated adenomas (SSA) and hyperplastic polyps (HP) [2]. While HPs are generally understood to show little or no potential for degeneration, the other serrated polyps are capable of malignant transformation, similar to classic adenomas [3]. Currently, endoscopy is the most viable method for CRC prevention, which not only allows for diagnosis, but also for the removal of premalignant lesions. Furthermore, in addition to its preventative effects, endoscopic polypectomy has also been shown to reduce CRC mortality [4]. VIRTUAL HISTOLOGY Substantial progress in the advancement of endoscopic imaging has been achieved in recent years. The acquisition of higher quality images allows for visualization of subtle details in the gastrointestinal mucosa. Prediction of lesion type prior to histological processing is made possible through examination of mucosal surface architecture and microvasculature, a method referred to as virtual histology or optical biopsy. The prediction of polyp histology and degree of invasion depth that can be ascertained with virtual histology has important therapeutic implications. Before resecting a lesion, the endoscopist must determine if the lesion contains an invasive cancer, if it can be left in situ, and if required, what the optimal resection method is. Enhanced endoscopic imaging can assist the endoscopist in these determinations. Whereas resection of small HPs ( 5 mm) of the rectum and sigmoid colon, juvenile polyps and inflammatory polyps is not necessary considering their non-existent potential for degeneration, endoscopic resection is generally suitable for the remaining lesion types, excluding those containing cancer foci in deeper layers that require surgical management. Tumors with superficial submucosal invasion (< 1000 µm, Vienna classification sm-1) [5] have a negligible rate of lymph node metastasis. Hence, endoscopic resection of these lesions may be the definitive treatment provided they are well- or moderately-differentiated, do not show vascular or lymphatic invasion, and the resection margin is > 1 mm [6]. Importantly, these types of lesions can be accurately identified as a result of advancements in endoscopic imaging and ancillary techniques. High-definition endoscopes display images of a superior quality, and the use of magnification devices allows for a highly detailed inspection of the mucosal surface. Whereas amplified digital images lack sharpness, these optical systems can achieve a 150 magnification without loss of resolution [7]. In addition to these advances, there are auxiliary enhancement techniques that provide additional detail. The use of optical systems is generally implied in studies using magnification, yet endoscopes providing optical magnification are not available in the United States or in Europe (with some exceptions in the United Kingdom and a few other European centers). Thus, studies using this technology are mainly from Asian countries. CHROMOENDOSCOPY Chromoendoscopy (CE) is the topical instillation of dye through an endoscope channel to highlight small details of the mucosa. The two most commonly used dye types are vital or absorptive dyes that are taken up by intestinal cells, such as methylene blue and crystal violet, and contrast dyes such as indigo carmine, which are deposited in the mucosal grooves and enhance subtle mucosal unevenness. Before the dyes are administered, a solution containing mucolytic and de-foaming agents (for example, 300 mg of N-acetylcysteine and 100 mg of simethicone per liter of water) is pumped through the tissue to remove mucus and fecal remains. Dyes can be applied with a catheter for the staining of large mucosal areas (Figure 1), or directly through the endoscope channel with the aid of a syringe (Figure 2) (e.g., 5 cc of dye followed by 15 cc of air in a 20 cc syringe) for occasional application. Absorptive contrasts should be left to stand after instillation for one minute before rinsing. Generally, methylene blue is used at a concentration of 0.1%, while indigo carmine is used at concentrations of 0.1%-0.5% for large and small areas, respectively. Due to its potential toxicity [7], 0.05% crystal violet should be applied in small quantities with a special non-traumatic catheter combined with magnification devices. CE has been reliably used to distinguish neoplastic from non-neoplastic lesions. Additionally, it can predict the degree of invasion of colorectal tumors with high accuracy. Pit patterns of colonic lesions have been classified by Kudo et al [8] into seven categories (Table 1). To simply this complex classification for clinical use, pit patterns are grouped into three basic types: non-neoplastic lesions, Kudo Ⅰ or Ⅱ that do not require treatment (Figure 3A); Kudo non-invasive neoplasia, Kudo Ⅲs, ⅢL, Ⅳ and selected cases of Ⅴi, corresponding to adenomas and cancers with superficial submucosal invasion that are endoscopically treatable (Figure 3B); and invasive, Kudo Ⅴ n and some Ⅴi, requiring surgical treatment due to deep submucosal invasion [9]. Invasive patterns are typically not evenly distributed throughout the lesion, and usually found in lesions located in confined areas with steep edges, depressions or nodules larger than 1 cm, thereby necessitating the use of magnification for identification. Using magnification endoscopy for pit pattern assessment, Japanese studies have demonstrated that neoplastic lesions can be distinguished from non-neoplastic lesions with an accuracy of up to 99.1% [10], and the degree of invasion (superficial vs deep) can be estimated in up to 8450 July 14, 2014 Volume 20 Issue 26

143 Lopez-Ceron M et al. Endoscopic characterization of colonic polyps A B C Figure 1 Indigo carmine staining for chromoendoscopy. A: Colorectal mucosa after cleansing with mucolytic and de-foaming agents; B: Instillation of dye using a spray catheter; C: Indigo carmine staining result. with CE and magnification. A new category with wider and rounder crypts was thus described (type Ⅱ-O) (Figure 4), which results in a more accurate (81%) diagnosis of SSA [18]. Figure 2 Local application of chromoendoscopy with a single-use syringe through the endoscope channel. 98.8% of cases [11]. Furthermore, the application of these pattern assessments shows inter- and intra-observer agreement of 72%-86% [12]. CE using high-resolution endoscopes without magnification also allows for differentiation of neoplastic from non-neoplastic lesions, though with slightly reduced accuracy (87%-92%) [13-16]. The recent rise in cases of serrated polyps (SP) highlights limitations in the simple categorization of neoplastic and non-neoplastic lesions for identification of malignant types. Although SPs are generally non-neoplastic and show no dysplasia, their resection is indicated considering their potential for malignancy [17], except in instances of diminutive ( 5 mm) HPs in the rectum or sigmoid colon. These HPs are not considered malignant and so can be left in situ. Consequently, the method for classification should be amended to accurately distinguish between adenoma, SSA and HP lesion types. Specifically, endoscopic differentiation between SSA and HP is indeed valuable as not all serrated lesions have the same malignant potential, with some indications that HPs can have a more benign behavior than SSAs and adenomas, though both HPs and diminutive SSAs can be located in the sigmoid colon and rectum. Although medical literature regarding endoscopic characterization of SPs is heterogeneous and often confusing, SSAs and HPs are typically described as pale and flat lesions, exhibiting poorly defined edges and often a layer of mucus or stool attached to their surface. The pit pattern in SSA was evaluated in a Japanese study DIGITAL CE For its proven clinical applications, CE represents the benchmark for enhanced imaging techniques that have been subsequently developed. However, as the technique is labor- and time-intensive, CE has not been universally implemented, and thus has precipitated the development of several digital CE devices. Digital CE techniques provide similar information to standard CE, with the advantage of reversibility (turned on and off by pressing a button on the head of the endoscope), speed, and hygiene. All of them provide a high-resolution image. The first system marketed was narrow band imaging (NBI) by Olympus Medical Systems (Tokyo, Japan). Latter systems were designed and commercialized to provide a similar view via digital image post-processing, such as fujinon intelligent chromo endoscopy (FICE) from Fujinon (Saitama, Japan) and i-scan from Pentax (Tokyo, Japan). NBI is an endoscopic enhancement tool based on spectrum filtration to only allow the passage of green and blue light, thereby providing contrasted images that highlight subtle mucosal changes, revealing the inner structure and vascular pattern of the tissue. Thus, NBI provides information not only on the surface architecture as traditional CE, but also on vascularization, both of which are altered in neoplastic lesions of the gastrointestinal tract. Comparisons of Kudo s pit patterns determined by NBI and CE show considerable but not total agreement [19-21]. NBI allows for the evaluation of vascularization intensities with relative ease, which are often not consistently reported, providing diagnostic accuracy equal or superior to the pit pattern [22,23]. Neoplastic lesions exhibit a dark color due to high vascularity, in contrast with lightercolored non-neoplastic tissues. The combination of pit pattern and vascularization intensity is the most common diagnostic criterion, reaching a high diagnostic accuracy with (90%-91%) [22,23] and without (80%-95%) [24-28] magnification (Figure 5). Several classification strategies have been documented 8451 July 14, 2014 Volume 20 Issue 26

144 Lopez-Ceron M et al. Endoscopic characterization of colonic polyps Table 1 Pit pattern classification Kudo et al [8] class Description Most likely histology Neoplastic/non-neoplastic Treatment Ⅰ Round crypts Normal mucosa Non-neoplastic None Ⅱ Regular wider or stellar crypts Hyperplastic polyp (also sessile serrated adenomas) Non-neoplastic None if rectosigmoid and 5 mm/ endoscopic resection ⅢL Elongated or roundish crypts Adenoma Neoplastic Endoscopic resection Ⅲs Tubular or roundish pits smaller Intramucosal/superficial invasive Neoplastic Endoscopic resection than the normal crypts cancer Ⅳ Branch-like or gyrus-like crypts Adenoma Neoplastic Endoscopic resection Ⅴi Irregular crypts Superficial invasive/deep invasive cancer Neoplastic Surgery/endoscopic resection in selected cases Ⅴn Non-structural crypts Deep submucosal invasive cancer Neoplastic Surgery A B Figure 4 Sessile serrated adenoma type Ⅱ-O. Figure 3 High-resolution endoscopic images of indigo carmine staining for Kudo s pit pattern classification. A: Type Ⅱ diminutive rectal hyperplastic polyp; B: Type ⅢL tubular adenoma with low-grade dysplasia. in the literature for the assessment of NBI technology. One report evaluating the use of NBI without magnification classified results using a confidence measure, with an observation of at least one histological characteristic designated as a high confidence diagnosis prediction, and a low confidence prediction indicating there were questionable traits or features belonging to both neoplastic and non-neoplastic categories [28]. The determination of diagnostic predictions with high confidence allows therapeutic decisions to be made at the time of endoscopy. A classification based on capillary pattern (CP) has been introduced by Sano et al [29] for use with magnification endoscopes (Table 2, Figure 6). This classification method has demonstrated reliable discrimination of category CP Ⅰ (non-neoplastic lesions), with correct classification of 95.3% of lesions [30]. Application of Sano s classification without the use of magnification yields reduced but satisfactory accuracy (91%) [31]. Similar accuracies have also been reported by studies directly comparing the performance of NBI against CE to distinguish non-neoplastic from neoplastic lesions [19,21,32,33]. Recently, a study using high-resolution white-light images and NBI described several characteristic features of SSA, including a cloud-like surface, indistinct borders, irregular shape, and dark spots within the crypts. The presence of all these features was sufficient to distinguish SSA from HP with an accuracy of 93% [34]. Moreover, the ability to estimate the degree of invasion of colorectal neoplasms with NBI has also been assessed, largely in Asian studies using magnification with a focus on microvasculature characteristics. Application of Sano s classification allowed for the discrimination of cancerous lesions with superficial submucosal invasion (sm1) from those with deeper invasion with an accuracy of 87.7% (sensitivity 84.8% and specificity 88.7%) [35], though accuracy obtained by CE with magnification is superior (98.8%) [11]. Hence, CE with magnification is preferred in cases with suspected invasive lesions. Since the use of magnification is not widespread, a joint classification with Asian and Western endoscopists has been recently designed. NICE classification (NBI International Colorectal Endoscopic Classification) [36] is based on polyp color as well as surface and vessel patterns (Table 3, Figure 7). Although these features are best 8452 July 14, 2014 Volume 20 Issue 26

145 Lopez-Ceron M et al. Endoscopic characterization of colonic polyps A B Figure 5 High-resolution endoscopic image of narrow band imaging. A: Hyperplastic polyp with weak vascular pattern intensity and type Ⅱ Kudo s pit pattern classification; B: Tubular adenoma with foci of high grade dysplasia exhibiting strong vascular pattern intensity and type ⅢL Kudo s pit pattern classification. Table 2 Capillary pattern classification determined from narrow band imaging with magnification Sano et al [29] classification CPⅠ CPⅡ CPⅢA CPⅢB Capillary pattern Absent Present Present Meshed capillary vessels with blind endings, branching and curtailed irregularity Capillary characteristics - Vessels surrounding glands Lack of uniformity, high density of vessels Most likely histology Normal or hyperplastic polyp Adenoma or intramucosal carcinoma Treatment None Polypectomy or endoscopic mucosal resection Superficial submucosal invasive carcinoma (sm1) Polypectomy or endoscopic mucosal resection/surgery Nearly avascular or loose microvessels Deep submucosal invasive carcinoma Surgery visualized with magnification, NICE categorization is still feasible without it, as preliminary studies differentiated Type 1 from Type 2 in polyps < 1 cm with a sensitivity of 98% and negative predictive value of 95% [37]. The characterization of lesions with NBI has been applied in clinical practice using a Resect and Discard concept [38]. Histological analysis and endoscopic resection can be avoided under certain conditions with this strategy, such as in cases with HPs smaller than 5 mm in the rectum and sigmoid. Furthermore, lesions with low carcinogenic potential (adenomas less than 10 mm) can be safely removed and discarded also without a need for histopathological analysis. In both cases, prediction of histology needs to be done with a high degree of confidence, and lesions greater than 10 mm should routinely be sent for histology. This strategy also allows for the establishment of a proper surveillance interval immediately following the colonoscopy, without a need to wait for the pathology report. Initial evaluations of this strategy demonstrated predictions with a high degree of confidence in 89% of polyps and allocation of proper surveillance intervals in 98% of cases [38]. It has been estimated that application of this strategy in the United States would save $33 million dollars annually [39]. The American Association of Gastrointestinal Endoscopy (ASGE) has subsequently stated that polyps 5 mm can safely be resected and discarded only after an optical diagnosis with a high degree of confidence has been performed. To determine if these smaller lesions in the sigmoid colon or rectum can be safely left in situ, optical diagnostic methods should provide a negative predictive value (NPV) for the diagnosis of adenoma of at least 90%. Moreover, in combination with routine histology of polyps > 5 mm, proper surveillance intervals should be achieved in at least 90% of cases [40]. NBI achieves these requirements when performed by proficient endoscopists, with one recent study demonstrating proper surveillance intervals in polyps 5 mm in 92%-99% of cases (according to American or European guidelines, respectively), and a 92% NPV for the diagnosis of rectosigmoid adenomas [41]. However, this threshold was not reached when performed by non-academic centers or non-tertiary hospitals [42-44]. Therefore, NBI interpretation should be applied with caution before making these results generalizable in clinical practice. Furthermore, the medicolegal consequences of the Resect and Discard strategy must be taken into account. While the resection and pathologic assessment of low risk lesions carries a huge time and cost burden, advanced histological features (i.e., villous component, high grade dysplasia, or cancer), though rare, can occur in lesions < 1 cm [45]. The guidelines of endoscopy societies should therefore provide legal standards and include recommendations regarding advanced imaging interpretation to support endoscopists. In this regard, it has been proposed that high quality pictures of discarded and un-resected lesions should be included in the endoscopic report [46]. Alternative digital CE techniques, such FICE and 8453 July 14, 2014 Volume 20 Issue 26

146 Lopez-Ceron M et al. Endoscopic characterization of colonic polyps Sano s Capillary pattern classification [29] Ⅰ Ⅱ ⅢA ⅢB Figure 6 Sano s capillary vessel classification. Adapted from Uraoka et al [29]. Table 3 Narrow band imaging-international colorectal endoscopic classification as determined by narrow band imaging with/without magnification Type 1 Type 2 Type 3 Color Same or lighter than background Brown Brown or dark brown Occasional white patchy areas Vessels None or isolated lacy vessels Brown vessels surrounding white structures Areas of disrupted or missing vessels Surface pattern Most likely histology Dark or white spots of uniform size or homogeneous absence of pattern Hyperplastic polyp Oval, tubular or branched white structures surrounded by brown vessels Adenoma or intramucosal or superficial submucosal invasive carcinoma (sm1) Amorphous or absent surface pattern Deep submucosal invasive carcinoma Treatment None Polypectomy or endoscopic mucosal resection Surgery A B C Figure 7 Narrow band imaging-international colorectal endoscopic classification. A: Hyperplastic polyp, narrow band imaging-international colorectal endoscopic (NICE) Type 1; B: Tubular adenoma with low-grade dysplasia, NICE Type 2; C: Deep invasive cancer, NICE Type 3. i-scan, have not been as widely implemented as NBI, thus less evidence is available for evaluation and comparison. As FICE and i-scan enhance vascularization (with additional selectable modes in which the surface structure is also enhanced), studies to date have been based on inspection of the vascular pattern and/or pit pattern. FICE [47-53] and i-scan [54-59] systems have been evaluated for the discrimination of neoplastic and non-neoplastic lesions, demonstrating similar results to NBI, although poorer than CE in some cases. FICE combined with magnification has also been tested for prediction of submucosal invasion depth, with accuracies similar to NBI, but significantly lower than CE [51]. CONCLUSION The exponential increase in lesions detected in the bowel after the implementation of colorectal cancer screening programs along with the improvements in image quality have made the endoscopic characterization of polyps especially relevant. A rigorous estimation of the histologic lesion type facilitates the determination of appropriate endoscopic treatment, if needed, or to indicate surgery. The development of ancillary techniques to white-light endoscopy like CE has enabled accurate discrimination between neoplastic and non-neoplastic lesions along with an estimation of the degree of invasion when combined 8454 July 14, 2014 Volume 20 Issue 26

147 Lopez-Ceron M et al. Endoscopic characterization of colonic polyps with magnification systems. Nevertheless, this technique has not been fully implemented in Western countries since it is cumbersome and time-consuming. Digital CE systems, such as NBI, have attempted to overcome these drawbacks with their ease of use, reversibility and cleanliness. Their capability to accurately distinguish non-neoplastic from neoplastic lesions has been demonstrated, though their use in estimating the degree of invasion still needs further evaluation. NBI, along with the Resect and Discard strategy, has proven to be accurate in academic centers and tertiary hospitals, with the potential for significant financial savings. The available evidence suggests that conventional CE is the most accurate technique for the characterization of colonic polyps, though NBI is a viable alternative for experienced endoscopists. 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148 Lopez-Ceron M et al. Endoscopic characterization of colonic polyps intensity. Endoscopy 2008; 40: [PMID: DOI: /s ] 23 Tischendorf JJ, Wasmuth HE, Koch A, Hecker H, Trautwein C, Winograd R. Value of magnifying chromoendoscopy and narrow band imaging (NBI) in classifying colorectal polyps: a prospective controlled study. Endoscopy 2007; 39: [PMID: DOI: /s ] 24 Rogart JN, Jain D, Siddiqui UD, Oren T, Lim J, Jamidar P, Aslanian H. Narrow-band imaging without high magnification to differentiate polyps during real-time colonoscopy: improvement with experience. Gastrointest Endosc 2008; 68: [PMID: DOI: /j.gie ] 25 Sikka S, Ringold DA, Jonnalagadda S, Banerjee B. Comparison of white light and narrow band high definition images in predicting colon polyp histology, using standard colonoscopes without optical magnification. Endoscopy 2008; 40: [PMID: DOI: /s ] 26 Rastogi A, Keighley J, Singh V, Callahan P, Bansal A, Wani S, Sharma P. 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149 Lopez-Ceron M et al. Endoscopic characterization of colonic polyps 48 Pohl J, Lotterer E, Balzer C, Sackmann M, Schmidt KD, Gossner L, Schaab C, Frieling T, Medve M, Mayer G, Nguyen-Tat M, Ell C. Computed virtual chromoendoscopy versus standard colonoscopy with targeted indigocarmine chromoscopy: a randomised multicentre trial. Gut 2009; 58: [PMID: DOI: /gut ] 49 Togashi K, Osawa H, Koinuma K, Hayashi Y, Miyata T, Sunada K, Nokubi M, Horie H, Yamamoto H. A comparison of conventional endoscopy, chromoendoscopy, and the optimalband imaging system for the differentiation of neoplastic and non-neoplastic colonic polyps. Gastrointest Endosc 2009; 69: [PMID: DOI: /j.gie ] 50 dos Santos CE, Lima JC, Lopes CV, Malaman D, Salomão AD, Garcia AC, Teixeira CR. Computerized virtual chromoendoscopy versus indigo carmine chromoendoscopy combined with magnification for diagnosis of small colorectal lesions: a randomized and prospective study. Eur J Gastroenterol Hepatol 2010; 22: [PMID: DOI: /MEG.0b013e32833a5d63] 51 Yoshida N, Naito Y, Kugai M, Inoue K, Uchiyama K, Takagi T, Ishikawa T, Handa O, Konishi H, Wakabayashi N, Kokura S, Yagi N, Morimoto Y, Yanagisawa A, Yoshikawa T. Efficacy of magnifying endoscopy with flexible spectral imaging color enhancement in the diagnosis of colorectal tumors. J Gastroenterol 2011; 46: [PMID: DOI: / s ] 52 Longcroft-Wheaton GR, Higgins B, Bhandari P. Flexible spectral imaging color enhancement and indigo carmine in neoplasia diagnosis during colonoscopy: a large prospective UK series. Eur J Gastroenterol Hepatol 2011; 23: [PMID: DOI: /MEG.0b013e328349e276] 53 Yoshida N, Naito Y, Inada Y, Kugai M, Inoue K, Uchiyama K, Handa O, Takagi T, Konishi H, Yagi N, Morimoto Y, Wakabayashi N, Yanagisawa A, Yoshikawa T. The detection of surface patterns by flexible spectral imaging color enhancement without magnification for diagnosis of colorectal polyps. Int J Colorectal Dis 2012; 27: [PMID: DOI: /s ] 54 Hoffman A, Sar F, Goetz M, Tresch A, Mudter J, Biesterfeld S, Galle PR, Neurath MF, Kiesslich R. High definition colonoscopy combined with i-scan is superior in the detection of colorectal neoplasias compared with standard video colonoscopy: a prospective randomized controlled trial. Endoscopy 2010; 42: [PMID: DOI: / s ] 55 Lee CK, Lee SH, Hwangbo Y. Narrow-band imaging versus I-Scan for the real-time histological prediction of diminutive colonic polyps: a prospective comparative study by using the simple unified endoscopic classification. Gastrointest Endosc 2011; 74: [PMID: DOI: / j.gie ] 56 Hoffman A, Kagel C, Goetz M, Tresch A, Mudter J, Biesterfeld S, Galle PR, Neurath MF, Kiesslich R. Recognition and characterization of small colonic neoplasia with high-definition colonoscopy using i-scan is as precise as chromoendoscopy. Dig Liver Dis 2010; 42: [PMID: DOI: /j.dld ] 57 Chan JL, Lin L, Feiler M, Wolf AI, Cardona DM, Gellad ZF. Comparative effectiveness of i-scan and high-definition white light characterizing small colonic polyps. World J Gastroenterol 2012; 18: [PMID: DOI: / wjg.v18.i ] 58 Neumann H, Vieth M, Fry LC, Günther C, Atreya R, Neurath MF, Mönkemüller K. Learning curve of virtual chromoendoscopy for the prediction of hyperplastic and adenomatous colorectal lesions: a prospective 2-center study. Gastrointest Endosc 2013; 78: [PMID: DOI: / j.gie ] 59 Pigò F, Bertani H, Manno M, Mirante V, Caruso A, Barbera C, Manta R, Bassotti G, Olivetti G, Conigliaro RL. i-scan highdefinition white light endoscopy and colorectal polyps: prediction of histology, interobserver and intraobserver agreement. Int J Colorectal Dis 2013; 28: [PMID: DOI: /s ] P- Reviewers: Bujanda L, Kato J, Rosty C, Sabbagh LC S- Editor: Zhai HH L- Editor: A E- Editor: Zhang DN 8457 July 14, 2014 Volume 20 Issue 26

150 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. WJG 20 th Anniversary Special Issues (14): Pancreatic cancer TOPIC HIGHLIGHT c-met signaling in the development of tumorigenesis and chemoresistance: Potential applications in pancreatic cancer Daniel Delitto, Eva Vertes-George, Steven J Hughes, Kevin E Behrns, Jose G Trevino Daniel Delitto, Steven J Hughes, Kevin E Behrns, Jose G Trevino, Department of Surgery, University of Florida-Gainesville, Gainesville, FL 32610, United States Eva Vertes-George, Department of Pathology, University of Florida-Gainesville, Gainesville, FL 32610, United States Author contributions: All the authors contributed to this manuscript. Correspondence to: Jose G Trevino, MD, Department of Surgery, University of Florida-Gainesville, 1600 SW Archer Rd, Rm 6175, PO Box , Gainesville, FL 32610, United States. jose.trevino@surgery.ufl.edu Telephone: Fax: Received: September 23, 2013 Revised: December 18, 2013 Accepted: April 1, 2014 Published online: July 14, 2014 Abstract Pancreatic ductal adenocarcinoma is the 4 th leading cause of cancer deaths in the United States. The majority of patients are candidates only for palliative chemotherapy, which has proven largely ineffective in halting tumor progression. One proposed mechanism of chemoresistance involves signaling via the mesenchymalepithelial transition factor protein (MET), a previously established pathway critical to cell proliferation and migration. Here, we review the literature to characterize the role of MET in the development of tumorigenesis, metastasis and chemoresistance, highlighting the potential of MET as a therapeutic target in pancreatic cancer. In this review, we characterize the role of c-met in the development of tumorigenesis, metastasis and chemoresistance, highlighting the potential of c-met as a therapeutic target in pancreatic cancer Baishideng Publishing Group Inc. All rights reserved. Key words: Pancreatic adenocarcinoma; c-met; Chemoresistance; Receptor tyrosine kinase Core tip: As one of the leading causes of cancer-related deaths, pancreatic cancer remains elusive to our current therapeutic options. These modest advances in current therapies for pancreatic cancer have led to the recognition and development of targeted therapies toward tyrosine kinase receptors such as the c-met receptor. In this review, we characterize the role of c-met in the development of tumorigenesis, metastasis and chemoresistance, highlighting the potential of c-met as a therapeutic target in pancreatic cancer. Delitto D, Vertes-George E, Hughes SJ, Behrns KE, Trevino JG. c-met signaling in the development of tumorigenesis and chemoresistance: Potential applications in pancreatic cancer. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: INTRODUCTION Pancreatic cancer is the 4 th leading cause of cancer deaths in the United States [1]. Currently, surgical resection is the only treatment option with the potential of cure. However, only 17% of patients are surgical candidates upon diagnosis and surgical resection in combination with chemotherapy and radiation therapy results in a 5-year survival of approximately 23% in specialized centers focused on pancreatic cancer [2]. While chemotherapy has the potential to delay tumor progression, innate or acquired chemoresistance and subsequent tumor resurgence is the norm [3,4]. Biologically diverse mechanisms have been identified to be involved in the chemoresistant phenotype, ranging from genetic and epigenetic changes to microenvironmental adaptation [4,5]. The goal of this review is to focus on the signaling of the mesenchymal July 14, 2014 Volume 20 Issue 26

151 Delitto D et al. c-met as a therapeutic target in pancreatic cancer epithelial transition factor protein (MET) in pancreatic cancer. The mesenchymal-epithelial transition factor gene (cmet) encodes for a membrane-bound receptor tyrosine kinase (RTK) expressed predominantly by epithelial cells. MET is activated and signals downstream pathways following induction of phosphorylation in response to binding of its ligand, hepatocyte growth factor (HGF), also referred to as scatter factor. These ligands are secreted by cells of mesenchymal origin. The resulting HGF/MET pleiotropic signaling cascade activates mediators of cell proliferation and motility and has been heavily implicated in tumorigenesis via identification of amplification, activating mutation, and/or overexpression of MET in most solid organ neoplasms. Here, we review the literature to characterize the role of MET in the development of tumorigenesis, invasion, metastasis and chemoresistance, highlighting the potential of MET as a therapeutic target in pancreatic cancer. PHYSIOLOGIC HGF-MET SIGNALING MET activation propagates a complex system of intracellular signaling cascades that act to affect cell proliferation and migration. HGF is secreted by mesenchymal cells in close proximity to MET-expressing epithelial cells during embryogenesis or in response to tissue injury, thus functioning as a paracrine signaling mechanism that promotes cell proliferation and migration. MET is translated as a 180 kda protein that is subsequently cleaved to form a heterodimer consisting of a short alpha (approximately 40 kda) and long beta (approximately 140 kda) chain of residues. The mature protein is then transported to and inserted in the plasma membrane. Upon HGF ligand binding to MET, autophosphorylation at multiple tyrosine residues within the cytoplasmic domain occurs, catalyzed by intrinsic ATPase activity. This results in changes in the tertiary structure of MET facilitating the formation of a signaling complex including GAB1 and GRB2 proteins that subsequently activates multiple downstream pathways (Figure 1). Known effector molecules of this signaling cascade include Src, mitogenactivated kinase, extracellular signal-regulated kinase 1 and 2, phosphoinositide 3-kinase (PI3K), protein kinase B (Akt), signal transducer and activator of transcription (STAT), nuclear-factor-κb, and mammalian target of rapamycin [6-9]. MET-mediated induction of these pathways acts to positively influence cell proliferation, migration, and survival (Figure 2). Via these down-stream effectors, HGF-MET signaling plays a crucial role in important physiologic processes including embryonic development, organ regeneration and wound healing. MET is essential for embryonic development and hgf- or c-met-null embryos die in utero [10]. In early embryonic development, HGF and its receptor MET are coexpressed by progenitor cells, suggesting autocrine signaling is an early homeostatic mechanism for stem cell survival [11]. HGF-MET signaling is necessary to ensure the growth and survival of placental trophoblast cells as well as embryonic hepatocytes. MET signaling is also necessary for the proper migration of muscle progenitor cells, development of the embryonic nervous system, and epithelial branching morphogenesis [12,13]. Later in development, paracrine HGF-MET signaling is critical for properly orchestrating organogenesis. Assays evaluating the ability of epithelial cells to form tubules in vitro, a process which recapitulates organ development, demonstrate that HGF signaling induces cells to undergo an epithelialto-mesenchymal (EMT) transition. This transition allows host cells to relocate during embryonic development. Ultimately, these cells reclaim their epithelial identity, but the EMT marks a critical event in organogenesis. [11] Inflammation and wound healing following injury are also highly dependent on HGF-MET signaling. HGF increases dramatically following renal or hepatic damage, inducing a diverse array of anti-apoptotic responses [9,14,15]. In cases of chronic or repetitive injury, HGF acts to oppose fibrosis by inducing apoptosis of myofibroblasts and by antagonizing transforming growth factor-β (TGF-β) [9,13,16]. Peptic ulcer disease represents a specific example of MET s protective effect. The loss of HGF signaling in a murine model led to decreased gastric mucosal cell proliferation and delayed healing from mucosal injury [17]. In fact, HGF-MET signaling has been implicated as essential to the protection, regeneration, and antifibrotic activity of cutaneous, pulmonary, hepatic, and gastrointestinal tissues in response to injury [13]. With respect to pancreatic endocrine physiology, the beta cell, responsible for insulin secretion, is dependent on HGF-MET signaling to hypertrophy and proliferate in response to persistent hyperglycemia [18]. In effect, MET is essential for the hyperinsulinemia seen in Type Ⅱ diabetes. c-met knockdown mice exhibit increased beta cell apoptosis during development and are more susceptible to streptozotocin-induced diabetes [19]. Additionally, c-met knockdown mice displayed reduced beta cell expansion during pregnancy leading to an increase in gestational diabetes [20]. Multiple investigations have confirmed that these knockdown mice have decreased glucose tolerance and reduced insulin secretion after stimulation [21,22]. In fact, stimulation of the HGF/MET pathway has been suggested to encourage beta cell proliferation after islet cell transplantation. Thus, MET plays a critical role in pancreatic neuroendocrine cell proliferation and development. Relatively little data is available concerning MET signaling and normal pancreatic exocrine development. A recent investigation by Anderson et al [23] examined the phenotype of a point mutation in c-met that impaired localization and activation of MET. Zebrafish with this mutation exhibited mislocalization of pancreatic ductal cells compared with wild-type animals. Interestingly, ductal proliferation was unaffected. Further, inhibition of MET proteindownstream signaling with PI3K and STAT inhibitors produced a similar phenotype, suggesting an essential role for MET in migration and localization of 8459 July 14, 2014 Volume 20 Issue 26

152 Delitto D et al. c-met as a therapeutic target in pancreatic cancer MET receptor (inactive) MET activation, dimerization and autophosphorylation HGF Extracellular Intracellular P P P P P P Figure 1 The mesenchymal-epithelial transition factor receptor functions as a transmembrane tyrosine kinase receptor. Ligand binding from hepatocyte growth factor (HGF)/scatter factor induces receptor dimerization and autophosphorylation of intracellular tyrosine residues, which serves as a catalytic site for the SH2 domains of numerous cytosolic signaling proteins. MET: Mesenchymal-epithelial transition factor. HGF-MET signaling HGF HGF P P P P P P MET RTK PLC Grb2 PI3K Jak IP3 DAG Sos Src Ras Ca 2+ Raf AKT MEK STAT PKC ERK 1 and 2 FAK mtor Cell survival, motility and proliferation Figure 2 Hepatocyte growth factor activation of the mesenchymal-epithelial transition tyrosine kinase receptor induces a pleiotropic response involving a host of intracellular signaling to induce cell survival, migration and proliferation. HGF: Hepatocyte growth factor; MET: Mesenchymal-epithelial transition factor; RTK: Receptor tyrosine kinase; JAK: Janus kinase; STAT: Signal transducer and activator of transcription; PLC: Phospholipase C; IP3: Inositol triphosphate; DAG: Diacylglycerol; Ca 2+ : Calcium; PKC: Protein kinase C; Grb2: Growth factor receptor-bound protein 2; Sos: Son of sevenless homolog; Ras: Harvey rat sarcoma viral oncogene homolog; Raf: Rapidly accelerating fibrosarcoma; MEK: Mitogen activated protein kinase kinase; ERK: Extracellular-signal-regulated kinase; FAK: Focal adhesion kinase; PI3K: Phosphoinositide 3-kinase; AKT: Protein kinase B; mtor: Mammalian target of rapamycin July 14, 2014 Volume 20 Issue 26

153 Delitto D et al. c-met as a therapeutic target in pancreatic cancer express HGF or MET become highly tumorigenic when implanted in vivo [34,35]. Therefore, while MET activity may not be the inciting mechanism in the formation of many cancers, overexpression in pre-clinical models appears to confer a more aggressive phenotype. In fact, MET expression has been correlated with more aggressive disease and worse clinical outcomes in many cancers. In NSCLC, MET overexpression correlates with an unfavorable prognosis and has been implicated as a primary mechanism of resistance to epidermal growth factor receptor (EGFR) inhibitor therapy [36,37]. In hepatocellular carcinoma the expression level of MET is directly correlated to metastatic behavior and inversely correlated to the level of tumor differentiation and patient survival [38-41]. In a prospective cohort analysis of 554 patients with renal cell carcinoma, a particular single nucleotide polymorphism (SNP) in c-met was associated with a decline in median recurrence-free survival from 50 to 19 mo [42]. While the functional outcome of this SNP remains to be elucidated, an activating point mutation is highly suspected. Likewise, MET overexpression is a HER2/neu-independent prognostic marker for nodepositive breast cancer, signifying increased tumor aggressiveness [43]. MET expression significantly correlated with the depth of invasion and regional lymph node metastasis in colorectal cancer [44]. Thus, the list of solid organ neoplasms for which upregulation of HGF-MET signaling portends a more aggressive phenotype is extensive [45,46]. Taken together, this data demonstrates that dysregulation of the HGF-MET pathway contributes to tumor progression. This data also has implications regarding the status of the HGF-MET pathway on the effectiveness of certain biologic therapies, a concept we will expand upon later. Concerning pancreatic adenocarcinoma, evidence is accumulating that correlates dysregulated MET activity with an aggressive phenotype. In a recent investigation thirty-six pancreatic tumor samples were analyzed and MET expression levels were directly proportional to tumor grade [47]. Similar histopathologic analyses showed an approximate five to seven-fold increase in MET protein expression levels in pancreatic cancer compared to normal pancreas samples [48,49]. Histopathologic evaluation of our own resected patient population support these findings (Figure 3). A larger collection of pancreatic tumor specimens subsequently confirmed increased MET protein expression compared with normal controls and MET protein overexpression significantly correlated with increased TNM stage [50]. In fact, secreted HGF protein from surrounding stromal tissue has been correlated with MET overexpression in patients with pancreatic cancer and associated with worsened overall survival [51]. Given the known pathophysiologic actions of MET in cancer and a well-demonstrated overexpression pattern in pancreatic adenocarcinoma, inhibition would seem a logical therapeutic avenue. Unfortunately, targeting MET alone as a therapeutic strategy appears to be overly optimistic. Despite conembryonic pancreatic ductal cells. In summary, physiologic HGF-MET signaling is essential for appropriate embryonic development and organ repair. The function of the HGF/MET pathway observed in multiple organ systems appears to drive cell proliferation and mobility. Unfortunately, dysregulation of this pathway clearly could result in tumor initiation and/or progression. Amplification, mutation or overexpression of c-met become deleterious, contributing to malignant transformation and metastasis. Activating and sustaining HGF-MET signaling in this pathologic context drives tumor progression and is responsible, at least in part, to the development of chemoresistance. PATHOLOGIC HGF-MET SIGNALING IN CANCER Excessive MET activity is a feature of many cancers, although inciting mechanisms appear to be tumor-specific [24]. c-met received early attention as a proto-oncogene when activating mutant alleles were implicated in cases of hereditary papillary renal cell carcinoma [25]. The resulting MET receptor was constitutively activated, undergoing spontaneous ligand-independent phosphorylation [11]. In an analysis of seven families with hereditary papillary renal carcinoma, four displayed activating c-met mutations, all of which were located in the tyrosine kinase domain of the MET protein [25]. Sporadic c-met mutations have also been described in gastric carcinomas, glioblastomas, and squamous cell carcinomas of the head and neck [11,12,26]. Furthermore, aberrant positive feedback systems involving autocrine and paracrine signaling in the HGF-MET axis contribute to tumorigenic phenotypes in melanomas, osteosarcomas, breast cancer and gliomas [26]. One retrospective histopathologic analysis observed MET overexpression in 87% of renal cell carcinoma specimens [27]. Additionally, a strong correlation between MET expression and the esophageal metaplasia-dysplasia-adenocarcinoma continuum has been shown in surgical specimens from patients with esophageal adenocarcinoma [28]. In fact, c-met amplification occurs in approximately 9% of esophageal cancers [29]. These investigations provide compelling evidence that c-met is a potent oncogene. The association between MET activity and neoplastic progression has been investigated in animal models. Hypoxia-induced tumor cell invasion is dependent upon upregulated MET signaling, suggesting another mechanism driving growth and metastasis [30,31]. Overexpression of wild-type MET in hepatocytes led to spontaneous hepatocellular carcinoma development that regressed upon MET inactivation [30,32]. Thus, overexpression of nonmutated MET is sufficient to induce tumor development. Moreover, inhibition of MET caused established tumors to regress, suggesting that MET signaling is necessary for tumor growth and maintenance. Subsequent animal models have proposed that the frequency of many carcinomas and lymphomas is greatly increased by MET overexpression [33]. Non-neoplastic cell lines forced to constitutively 8461 July 14, 2014 Volume 20 Issue 26

154 Delitto D et al. c-met as a therapeutic target in pancreatic cancer HE c-met Pancreatic cancer Normal pancreas 200 mm 200 mm 200 mm 200 mm Figure 3 Immunoperoxidase staining. Immunoperoxidase staining of formalin fixed, paraffin embedded human pancreatic specimens demonstrate over expression of c-met receptor in pancreatic cancer patients when compared to adjacent normal pancreatic tissue controls (right panel). HE staining demonstrate histological confirmation of diseased (pancreatic cancer) or normal tissue (left panel). vincing evidence of primarily MET-induced tumors, a growing body of evidence supports secondary MET involvement in a synergistic crosstalk with other RTKs such as EGFR, vascular endothelial growth factor receptor and insulin-like growth factor-1 receptor (IGF-1R) to promote malignant cell migration, invasion, and chemoresistance [52-55]. In hepatocellular carcinoma cells, EGFR co-immunoprecipitates with MET and activated EGFR leads to ligand-independent activation of the MET pathway [36]. MET and IGF-1R synergistically promote migration and invasion in pancreatic adenocarcinoma. Downregulation of MET via adenoviral infection with a MET ribozyme abrogated the effects of IGF-1, suggesting codependence of IGF-1R and MET in directing tumor invasion and migration [56]. These complex, multifactorial interactions among RTKs play a key role in growth and maintenance of a variety of tumor types and are under intense scrutiny for potential therapeutic value or mechanisms of therapeutic resistance. These discoveries will be essential to the evolving reality of personalized cancer treatment strategies. MET AND TUMOR METASTASIS The microenvironment of a tumor may be as instrumental to the progression of disease as the tumor itself. In fact, stromal support in the form of angiogenesis, mitogenic signaling and cytoskeletal attachments are necessary for tumors to grow and metastasize in vivo. As previously mentioned, HGF secretion by stromal cells mediates MET activity in a paracrine manner. Additionally, HGF- MET signaling encourages angiogenesis by inducing VEGF expression by cancer cells [57,58]. However, neovascularization alone is not sufficient for metastasis to occur. Recall that in embryonic development and tissue repair, MET plays an essential, physiologic role in cellular migration and subsequent organogenesis. Unfortunately, overexpression of MET and its subsequent downstream pathways, including PI3K and Src, similarly enable growth and invasion of malignant cell populations. An initial step in tumor migration involves clearing a path through the extracellular matrix (ECM). This is accomplished primarily by the actions of secreted matrix metalloproteinases (MMPs), which digest surrounding ECM. Not surprisingly, MMPs have been shown to be upregulated by MET signaling [24]. Cells must also respond to chemotactic factors in the ECM for effective migration. As previously mentioned, an EMT endows epithelial cells with certain properties of mesenchymal cells that enable migration. Furthermore, it has recently been proposed that the EMT may be coupled with a transition to a more stem-cell-like state, suggesting further importance of the EMT to metastasis and tumor progression [59]. In embryogenesis, MET controls the EMT necessary to enable myogenic progenitor cell migration [9]. Additionally, EMT is further driven by Wnt signaling, a pathway that is also stimulated by MET via glycogen synthase kinase 3-β [60]. The mechanism by 8462 July 14, 2014 Volume 20 Issue 26

155 Delitto D et al. c-met as a therapeutic target in pancreatic cancer Table 1 Cancer stem cell markers are listed with previously described functions CSC marker Proposed function CD44 ECM binding, organization of actin cytoskeleton, modulation of mitogenic signaling [112] CD24 P-Selectin binding, cell migration [113] ESA Mediation of epithelial intercellular adhesion [114] CD133 Activation of Wnt signaling and angiogenesis [115,116] CXCR4 Receptor of SDF-1, hematopoietic stem cell homing, invasion [117] MET Receptor of HGF, promotes cell growth, proliferation, migration [11] u-pa ECM degradation, cell migration [118] Note the pattern of migratory functions associated with cancer stem cell (CSC) markers. ECM: Extracellular matrix; ESA: Epithelial specific antigen; CXCR4: Chemokine receptor type 4; SDF-1: Stromal cell-derived factor 1; MET: Mesenchymal-epithelial transition factor; HGF: Hepatocyte growth factor; u-pa: Urokinase-type plasminogen activator. which MET governs the EMT directly in tumor metastases remains to be elucidated. Finally, malignant cells must take up residence in a distant organ as a metastatic focus. Remarkably, HGF- MET signaling plays a role both in cellular dissociation within the primary tumor and cellular re-association within the metastatic niche [24]. HGF triggers destabilization of adherens junctions within the primary tumor through FAK-mediated integrin signaling [61]. As tumor cells invade and metastasize, failure of proper interaction with foreign microenvironments leads to programmed cell death. HGF-MET signaling upregulates cytoskeleton adhesion receptors and enables tumor cells to effectively engage their new surroundings and elude apoptosis, thereby facilitating metastatic development [24]. Thus, in addition to fostering primary tumor growth, MET appears to act at multiple regulatory points in the development of metastatic disease. MET AND CANCER STEM CELLS A growing body of evidence suggests that a hierarchy exists in cancer cell populations, a notion initially discovered in hematopoietic malignancies. Cancer stem cells (CSCs) actually comprise a small minority of tumor cells but appear to be the only group capable of unlimited self-renewal and formation of xenografts. Interestingly, these cells appear to have a limited potential for further differentiation [62,63]. CSC populations have subsequently been identified in a variety of solid organ neoplasms including brain, breast, melanoma, pancreas, prostate and colon. While CSC identification is specific to each tumor type, common themes include cell surface markers such as CD24, CD44, CD133, epithelial surface antigen (ESA), chemokine receptor type 4, and urokinase plasminogen activator (Table 1) [64-72]. Importantly, in pancreatic cancer stem cell (PCSC) populations, MET overexpression conferred an equally tumorigenic phenotype to CD44 + /CD24 + /ESA + cells [73]. Restated, MET overexpression alone may sustain a pancreatic cancer stem cell phenotype. Conversely, MET overexpression may prompt cancer cells to dedifferentiate into CSCs. MET activation in prostate cancer cells induces a stem-like phenotype and endows cells with more invasive potential [74]. In head and neck squamous cell carcinoma, cells overexpressing MET can recapitulate the heterogeneity of parental tumors in vivo and exhibit increased self-renewal, invasion, and metastasis [75]. In glioblastomas, overexpression of MET leads to a stem-like phenotype resistant to terminal differentiation signals [76]. Regardless of the origin of CSCs, MET overexpression is associated with a stem-cell-like phenotype in a wide range of cancers. MET AND CHEMORESISTANCE Chemoresistance is an important factor contributing to the high mortality rate of most cancers and is germane to treatment failure in pancreatic cancer. With few exceptions, tumor metastasis precludes surgical therapy and leaves chemotherapy as the only therapeutic option. In borderline cases, neoadjuvant chemotherapy protocols may offer opportunities for attempts at a surgical resection. After surgery, adjuvant chemotherapy protocols are beneficial in avoiding recurrence, especially in more aggressive tumor types. Unfortunately, the development of chemoresistance is a real oncologic dilemma. In the face of chemoresistant tumor populations, no effective treatments exist. Therefore, understanding the molecular regulators of chemoresistance has major implications in therapeutic intervention. Several lines of evidence converge to suggest that MET overexpression may confer a chemoresistant phenotype. We have outlined the close relationship between MET and CSCs. In fact, CSCs have been shown to be largely responsible for chemoresistant phenotypes in glioblastomas, hematopoietic, pancreatic and colorectal cancers [77-83]. Mechanisms range from reducing drug delivery to repairing cytotoxic injury and ultimately result in tumor cell repopulation [77-83]. Furthermore, a higher proportion of cells bearing CSC markers has been associated with poor outcomes in glioblastomas, breast and pancreatic cancer [84-86]. Thus, investigative directions have become particularly focused on identifying factors that drive and sustain CSCs. Given the significance of HGF- MET signaling in PCSC populations, the role of MET in this process would seem to be particularly relevant in pancreatic cancer. The activation of the HGF-MET axis has been directly implicated in acquiring and maintaining chemoresistance in several tumor cell populations (Table 2). HGF stimulation protects NSCLC cells from cisplatin toxicity, in part mediated by downregulation of apoptosis-inducing factor [87]. c-met amplification is associated with NSCLC resistance to the EGFR inhibitor Gefitinib via modulation of the PI3K pathway [88]. Multiple investigations have revealed that MET inhibition sensitizes ovarian carcinoma to carboplatin plus paclixatel, whereas MET over July 14, 2014 Volume 20 Issue 26

156 Delitto D et al. c-met as a therapeutic target in pancreatic cancer Table 2 Mechanisms of hepatocyte growth factor-mesenchymal-epithelial transition factor induced chemoresistance in different cancer types Cancer type Chemotherapy Mechanism of HGF-MET signaling in chemoresistance Multiple myeloma Bortezomib MET overexpression: Apoptotic resistance via PI3K-Akt activation [92] Glioblastoma Radiation, cisplatin, camptothecin, Addition of HGF: Anti-apoptotic effects via PI3K-Akt dependent pathways [91] adriamycin, and taxol groups Rhabdomyosarcoma Vincristine/etoposide, radiation Addition of HGF: Enhanced migration, MMP secretion, PI3K-Akt activation [119] Non-small cell lung carcinoma Cisplatin Addition of HGF: Downregulation of apoptosis-inducing factor (AIF) [87] Non-small cell lung carcinoma Erlotinib c-met amplification: Activation of EGFR, preservation of PI3K-Akt activation [88] Gastric adenocarcinoma Adriamycin Addition of HGF: Anti-apoptotic effects via PI3K-Akt upregulation [93] Pancreatic adenocarcinoma Gemcitabine MET overexpression: Anti-apoptotic effects via PI3K-Akt activation, induction of EMT-like changes [94,95] Ovarian adenocarcinoma Carboplatin/paclitaxel MET overexpression: Apoptotic resistance via PI3K-Akt activation [89,90] MET: Mesenchymal-epithelial transition factor; PI3K: Phosphoinositide 3-kinase; Akt: Protein kinase B; HGF: Hepatocyte growth factor; MMP: Matrix metalloproteinase; EGFR: Epidermal growth factor receptor; EMT: Epithelial-mesenchymal transition. expression imparts chemoresistance [89,90]. Furthermore, stimulation of the HGF-MET pathway confers protection against chemotherapeutic agents by upregulation of PI3K/Akt signaling in multiple myeloma, glioblastoma and gastric adenocarcinoma [91-93]. Our group has found that pharmacologic MET inhibition using a small molecule inhibitor sensitizes esophageal adenocarcinoma cells to pyrimidine analog chemotherapy (unpublished data). Additionally, preclinical studies have demonstrated that overexpression of MET has also been associated with EMT-like changes in acquired-gemcitabine-resistant pancreatic cancer cells [94]. These findings are not surprising as pancreatic cancer is known for rapid acquisition of chemoresistant behavior and also MET overexpression. Additionally, MET inhibition in pancreatic adenocarcinoma leads to gemcitabine sensitization [95]. Although consisting largely of in vitro data, these investigations demonstrate a strong correlation between MET overexpression and chemoresistance in a variety of malignancies. The mechanism by which MET overexpression confers chemoresistance in pancreatic cancer likely involves the mesenchymal support network. Tumors most heavily invested with stroma are often those most refractory to chemotherapy [4]. Stroma is the predominant source of HGF, suggesting MET activation is, at least in part, a result of paracrine signaling. In breast cancer, HGF-MET signaling augments tumor cell adhesion to ECM components by upregulating integrin synthesis and inducing conformational changes that activate integrins [24,96]. This integrin-mediated adhesion is actually a mechanism by which tumor cells can oppose the cytotoxic effect of chemotherapy [97]. Indeed, studies have shown that integrin expression, specifically a β, is upregulated in cases of relapsed leukemia. This finding suggests that increased integrin expression may contribute to generating minimal residual disease, defined as tumor cell persistence following therapy [4]. Further investigation is necessary to characterize the mechanism by which MET-driven integrin upregulation imparts chemoresistance and whether this principle is applicable to other tumor types. However, disruption of the HGF-MET axis may result in biochemical dissociation from the protective mesenchymal environment, thereby imparting sensitivity to cytotoxic therapies. Data specific to the pancreatic cancer microenvironment regarding MET signaling is forthcoming. Animal models that utilize VEGF inhibitors to impart ischemia actually result in increased tumor growth and invasion but inhibition of MET abrogates this proliferative response to hypoxia [98]. As previously mentioned, PCSCs can be defined by comparatively high MET expression. Pharmacologic inhibition of MET in PCSC populations blocked self-renewal capacity, reduced the overall PCSC population and significantly slowed tumor growth in vivo [99]. Treatment with MetMAb, a monovalent antibody against MET, has shown decreased pancreatic tumor growth in orthotopic models in vivo [100]. Further, recent preclinical data suggest cabozantinib, a novel small molecule MET inhibitor, overcomes gemcitabine resistance. These studies will likely lead to phase 3 clinical trials using this inhibitor in pancreatic cancer patients [101]. Finally, the interplay between RTKs and the potential for redundancy deserves emphasis when discussing therapeutic intervention. MET and other RTKs are involved in a complex signaling network that may exist as a redundant system with controlled feedback. For example, MET induction has been associated with anti-egfr therapy and resultant MET overexpression confers resistance to EGFR inhibitors in lung and colorectal cancer [88, ]. Thus, MET inhibition may potentiate therapeutic effects aimed against other RTKs, and vice versa. In fact, effective sirna inhibition of c-met transcripts in NSCLC confers sensitization to gefitinib, an inhibitor of EGFR [88]. Further, concomitant administration of EGFR and MET inhibitors eliminated NSCLC cells more effectively than either drug alone [55,105]. Similarly, MET inhibition led to increased sensitivity of her2-positive breast cancer cells to trastuzumab [106]. Not surprisingly, combination RTK inhibition is quickly becoming the standard in targeted oncologic chemotherapies involving MET inhibition. CONCLUSION In summary, c-met encodes a versatile RTK crucial to physiologic cell proliferation, organogenesis and wound healing. Its mechanism of action involves multiple antiapoptotic, pro-mitogenic, and pro-motility downstream 8464 July 14, 2014 Volume 20 Issue 26

157 Delitto D et al. c-met as a therapeutic target in pancreatic cancer Table 3 Mesenchymal-epithelial transition factor inhibitors are shown with specific targets and evidence of anti-tumor effect Drug Target(s) Impact Cabozantinib MET Induced apoptosis in gemcitabine-resistant pancreatic cancer cell lines, currently in phase Ⅰ clinical trials [101] Crizotinib ALK, MET Inhibited growth of gemcitabine resistant pancreatic cancer cell lines [95], FDA approved for ALK-expressing NSCLC and myofibroblastic sarcomas Foretinib MET, VEGFR Inhibited tumor growth in lung metastasis animal model but failed to show benefit in multiple phase Ⅱ clinical trials [110,120,121] Tivantinib MET Inhibited growth in multiple cancer cell lines via MET targeting as well as inhibition of microtubule formation [122] E7050 MET, VEGFR Inhibited growth in xenograft models of lung, gastric and pancreatic cancer [123] PF MET Inhibited growth and metastasis of pancreatic neuroendocrine tumors [124] SU11274 MET Inhibited growth and proliferation in colon cancer cell lines [125] T MET, VEGFR Inhibited tumor growth in a variety of murine xenograft models [126] MET: Mesenchymal-epithelial transition factor; ALK: Anaplastic lymphoma kinase; NSCLC: Non-small cell lung carcinoma; VEGFR: Vascular endothelial growth factor receptor. effectors. Unfortunately, dysregulated HGF-MET signaling is implicated in multiple oncologic mechanisms, including tumor growth, invasion and chemoresistance. Not surprisingly, clinical studies have consistently revealed MET overexpression as a negative prognostic indicator in a wide variety of malignancies. HGF-MET signaling mediates mesenchymal-cellmediated mitogenic support to developing tumor cell populations. MET activity enhances ECM degradation and integrin-mediated adhesion. In addition to promoting mobility and invasion, this appears to confer a protective microenvironment conducive to the development of chemoresistant clones. MET signaling is a marker of cancer stem cell populations, a recently characterized subgroup of cancer cells resistant to cytotoxic therapies. A better understanding of tumor growth signaling pathways and chemoresistant mechanisms carries the potential of immense therapeutic value, especially in aggressive tumors such as pancreatic adenocarcinoma. 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162 Delitto D et al. c-met as a therapeutic target in pancreatic cancer DOI: / CD ] 125 Gao SH, Liu C, Wei J, Feng Y. Effect of c-met inhibitor SU11274 on human colon cancer cell growth. Chin Med J (Engl) 2013; 126: [PMID: ] 126 Awazu Y, Nakamura K, Mizutani A, Kakoi Y, Iwata H, Yamasaki S, Miyamoto N, Imamura S, Miki H, Hori A. A novel inhibitor of c-met and VEGF receptor tyrosine kinases with a broad spectrum of in vivo antitumor activities. Mol Cancer Ther 2013; 12: [PMID: DOI: / MCT ] P- Reviewers: Bramhall S, Barreto S, Chowdhury P, Symeonidis NG S- Editor: Gou SX L- Editor: A E- Editor: Zhang DN 8470 July 14, 2014 Volume 20 Issue 26

163 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. WJG 20 th Anniversary Special Issues (14): Pancreatic cancer TOPIC HIGHLIGHT Pancreatic cancer organotypics: High throughput, preclinical models for pharmacological agent evaluation Stacey J Coleman, Jennifer Watt, Prabhu Arumugam, Leonardo Solaini, Elisabeta Carapuca, Mohammed Ghallab, Richard P Grose, Hemant M Kocher Stacey J Coleman, Jennifer Watt, Prabhu Arumugam, Leonardo Solaini, Elisabeta Carapuca, Mohammed Ghallab, Richard P Grose, Hemant M Kocher, Centre for Tumour Biology, Barts Cancer Institute - a CR-UK Centre of Excellence, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, United Kingdom Jennifer Watt, Prabhu Arumugam, Leonardo Solaini, Hemant M Kocher, Barts and the London HPB Centre, The Royal London Hospital, Barts Health NHS Trust, London EC1M 6BQ, United Kingdom Author contributions: Coleman SJ designed and wrote the paper; Watt J, Arumugam P, Solaini L, Carapuca E, Ghallab M and Grose RP contributed equally to the writing of the paper; Kocher hm made final amendments of the paper and approval of the version to be published. Correspondence to: Hemant M Kocher, MS, MD, FRCS, Centre for Tumour Biology, Barts Cancer Institute - a CR-UK Centre of Excellence, Queen Mary University of London, John Vane Science Centre, Charterhouse Square, London EC1M 6BQ, United Kingdom. h.kocher@qmul.ac.uk Telephone: Fax: Received: October 18, 2013 Revised: January 15, 2014 Accepted: April 1, 2014 Published online: July 14, 2014 Abstract Pancreatic cancer carries a terrible prognosis, as the fourth most common cause of cancer death in the Western world. There is clearly a need for new therapies to treat this disease. One of the reasons no effective treatment has been developed in the past decade may in part, be explained by the diverse influences exerted by the tumour microenvironment. The tumour stroma cross-talk in pancreatic cancer can influence chemotherapy delivery and response rate. Thus, appropriate preclinical in vitro models which can bridge simple 2D in vitro cell based assays and complex in vivo models are required to understand the biology of pancreatic cancer. Here we discuss the evolution of 3D organotypic models, which recapitulare the morphological and functional features of pancreatic ductal adenocarcinoma (PDAC). Organotypic cultures are a valid high throughput preclinical in vitro model that maybe a useful tool to help establish new therapies for PDAC. A huge advantage of the organotypic model system is that any component of the model can be easily modulated in a short timeframe. This allows new therapies that can target the cancer, the stromal compartment or both to be tested in a model that mirrors the in vivo situation. A major challenge for the future is to expand the cellular composition of the organotypic model to further develop a system that mimics the PDAC environment more precisely. We discuss how this challenge is being met to increase our understanding of this terrible disease and develop novel therapies that can improve the prognosis for patients Baishideng Publishing Group Inc. All rights reserved. Key words: 3D organotypic model; Pancreatic cancer; Pancreatic stellate cell; Stroma; Preclinical models Core tip: Pancreatic cancer carries a terrible prognosis, as the fourth most common cause of cancer death in the Western world. One of the reasons no effective treatment has been developed in the past decade may in part, be explained by the influences exerted by the tumour microenvironment. The tumour stroma crosstalk in pancreatic cancer can influence chemotherapy delivery and response rate. Organotypic models of pancreatic cancer allow new therapies that can target the cancer, the stromal compartment or both to be tested in a model that mirrors the in vivo situation and can help improve patient prognosis. Coleman SJ, Watt J, Arumugam P, Solaini L, Carapuca E, Ghallab M, Grose RP, Kocher HM. Pancreatic cancer organotypics: high throughput, preclinical models for pharmacological agent evaluation. World J Gastroenterol 2014; 20(26): July 14, 2014 Volume 20 Issue 26

164 Coleman SJ et al. Pancreatic cancer organotypics as preclinical models Available from: URL: v20/i26/8471.htm DOI: i Pancreatic cancer Pancreatic cancer has one of the highest mortality rates among malignancies, and is the fourth most common cause of cancer death in the Western world [1,2]. With an overall 5-year survival rate of 6% and median survival of less than six months, pancreatic ductal adenocarcinoma (PDAC) carries one of the bleakest prognoses in all of medicine. Surgery offers the only hope of a possible cure for patients; however even of those 10% of patients eligible for curative resection, only 21% will survive to five years [3]. This is due to the fact that, at diagnosis, distant metastases are common [4]. Clearly there is an urgent need for therapies for PDAC. One of the possible reasons that targeted therapies fail to improve the prognosis of patients with PDAC may, in part, be explained by the diverse influences exerted by the tumour microenvironment. Delineating the signalling networks within the tumour microenvironment, may help to explain the huge discrepancy between relative success and effectiveness of therapies in preclinical assay (predominately 2D cell based assays and xenograft mouse models) and their abject failure in human PDAC. Many epithelial malignancies, including breast, prostate, skin and pancreatic cancers, often exhibit a significant stromal reaction around the tumour cells [5-9]. Once thought to be a bystander, it is becoming increasingly evident that the stroma not only functions as a mechanical barrier but also constitutes a dynamic compartment that is critically involved in the process of tumour formation, progression, invasion, and metastasis [10,11]. In particular, PDAC shows the most prominent stromal reaction or desmoplasia (defined as proliferation of fibrotic tissue with an altered ECM which contributes to tumour growth and metastasis) (Figure 1) [12]. This surrounding tumour environment is an highly heterogeneous and complex mixture of cells from different lineages; fibroblasts, pancreatic stellate cells, smooth muscle cells, immune, inflammatory, neural, adipose and endothelial cells [13-16]. The high proportion of stromal cells in pancreatic cancer (up to 80% of the tumour volume [17] ) is associated with overexpression of a number of paracrine and autocrine signalling factors, such as vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), transforming growth factor β (TGFβ), insulin-like growth factor Ⅰ (IGF-Ⅰ), fibroblast growth factor (FGF) and their respective receptors as well as secretion of Matrix metalloproteinases (MMPS) and proteases which serve to fuel pancreatic cancer proliferation, metastasis and invasion [18-22]. In turn, pancreatic cancer cells secrete growth factors such as FGFs, TGFβ, IGF and platelet derived growth factor (PDGF) [23]. This interaction between cancer cells and stroma leads to altered transcription in stromal components, such as fibroblasts and inflammatory cells, promoting cancer cell motility and resistance to hypoxia. The net result is an unique tumour micro-environment, where tumour cells become inaccessible to chemotherapy and metastasise readily, leading to poor chemotherapy response rate [17]. These studies have highlighted the importance of stroma-cancer cross-talk. Thus, just studying pancreatic cancer cells without any stromal representation does not reflect accurately the in vivo situation. Cells grown on 2D tissue culture plates or in Transwell inserts differ in their morphology, differentiation and cell-cell and cellmatrix interactions compared to cells in vivo [24,25]. There is a need for physiologically relevant in vitro model systems that allow us to investigate and interrogate cancer and stromal cell behaviour and their interactions. Thus, 3D organotypic models are an invaluable research tool [26]. MODELLING PDAC In vitro (2D) studies of tumour stroma interactions in PDAC Improved understanding of the mechanisms that mediate epithelial-stromal interactions in PDAC is now possible due to the isolation, and in vitro culture, of pancreatic stellate cells (PSC), the key cells driving the desmoplastic reaction [21]. In the healthy pancreas, PSCs make up 4%-7% of all pancreatic cell types and exist in a quiescent state [27]. Quiescent PSCs are characterised by lipid droplets rich in vitamin A, resembling hepatic stellate cells (HSCs) first described by in the 19 th century [28]. They express desmin and glial fibrillary acid protein (GFAP) marker which serve to distinguish them from pancreatic fibroblasts [29]. In acute and chronic inflammatory conditions, PSCs are activated. This is characterised a loss of fat droplets, expression of α-smooth muscle actin (αsma), and an increased synthesis and secretion of several ECM proteins such as fibronectin, laminin and collagen type Ⅰ and Ⅲ [27,30,31]. The isolation and immortalisation of PSCs from human and rat pancreas has provided an additional tool for studying PSC activation and can overcome the limitations of culturing primary stellate cells. While immortalised stellate cells have provided a valuable tool in the study of PSC function, it is important to validate findings using primary PSCs [14]. PSCs have been immortalised using either SV40 large T antigen or human telomerase in human PSCs as we have previously successfully done in our laboratory [24,32-38]. Immortalised PSCs display an activated phenotype in 2D culture. Importantly PSC cell line is comparable to activated PSCs, which include expression of αsma and ECM proteins. Importantly, expression profiling of primary and PSC cell lines have shown only a few differences, with differential differences expression of ECM proteins, cytokines and integrins [37]. In addition, both immortalised and primary PSCs respond to TGF-β or PDGF in a similar manner [33]. Thus primary and immortalised PSCs have facilitated for the dissection of 8472 July 14, 2014 Volume 20 Issue 26

165 Coleman SJ et al. Pancreatic cancer organotypics as preclinical models A B Figure 1 Human pancreatic ductal adenocarcinoma has a dense desmoplastic stromal component. A: HE of human pancreatic cancer shows an area of invasive tumour; B: Stromal and epithelial components of the tumour are highlighted from figure A (scale bar 100 µm). Cancer Stroma important cross-talk between PSCs and pancreatic cancer cells and are an important source to explore the tumour promoting aspects of tumour myofibroblasts in PDAC [16]. The bidirectional interaction between PSCs and pancreatic cancer cells has been studied using co-culture or well-established 2D in vitro assays such as wound assays or Transwell inserts to study migration [39]. Co-culturing of PSCs and pancreatic cancer cells showed that PSCs can increase the proliferation and migration of pancreatic cancer cells, while inhibiting apoptosis by the release of several cytyokines and growth factors. Similarly, culturing PSCs in the conditioned medium of pancreatic cancer cells increases the proliferation, matrix synthesis and motility of PSCs, most likely via FGF-2, PDGF and TGF-β [22,40]. The desmoplasia in PDAC is believed to have a detrimental effect on the successful response to chemotherapy and radiotherapy [40,41]. In vitro experiments have shown that PSCs can increase the stem cell characteristic of pancreatic cancer cells, a possible mechanism of resistance to therapy [42]. Furthermore, in areas of the tumour that are hypoxic as a result of hypovascularity and profuse stroma provides a micro-environment in which pancreatic cancer cells thrive [43]. In vitro studies have shown that, co-culturing PSCs and pancreatic cancer cells under hypoxic conditions, PSCs are able to influence PCC invasion more strongly than in normoxic conditions [44]. Thus, pharmacological targeting of PSCs is an attractive option in treating PDAC. role of the stroma in PDAC-in vivo studies Animal models, such as xenografts, orthotopic grafts or genetically engineered mice (GEM), have validated many in vitro findings. Early subcutaneous mouse models, in which PSCs and pancreatic cancer cells were injected into the flanks of immunocompromised mice, demonstratied that, in the presence of PSCs, pancreatic cancer cell proliferation increased and tumours formed more rapidly than when pancreatic cancer cells were injected alone [22]. Apte and colleagues showed that injection of pancreatic cancer cells (MiaPaCa-2 and AsPC-1 cell lines), together with primary human PSC into the mouse pancreas was able to stimulate fibrosis, tumour growth and metastasis [40]. More recently, sex mismatch studies (injection of male PSCs and female pancreatic cancer cells into the pancreas of female mice), have shown that Y chromosome positive PSCs are able to migrate through blood vessels, together with cancer cells, localising to distant sites, such as the liver and diaphragm, where they are able to facilitate seeding, survival and growth of pancreatic cancer cells [45]. The development of genetically engineered mouse (GEM) models of PDAC has provided the most physiologically relevant model that closely mimics the situation in human cancer. Most of the GEM models of PDAC are based on the conditional, pancreas-specific, expression of the Kras oncogene (KRAS G12D ), present in 90% of human PDAC cases [46], this is facilitated by expressing Cre recombinase under the control of the embryonic pancreas lineage determining transcription factor Pdx-1 or Ptf1/p48 ( KC mice). KC mice develop pancreatic tumours ranging from precursor pancreatic intra-ductal neoplasms (PanINs) to fully invasive and metastatic disease [47,48], albeit with a long latency period of up to a year. These KC mice have been crossed with mice harbouring several additional mutations, to investigate their contribution to the rapid progression to PDAC. GEM models of PDAC have been developed with activating mutations in TGFβ receptor and/or inactivation of tumoral suppressors such as p53 ( KPC mice), INK4A/ARF and Smad4, which are the most common PDAC drivers [49]. There are several excellent reviews on the various GEM models that have been developed for studying the development of PDAC [50-52]. The generation of complex allele combinations together with the latency period involved in the development of tumour makes these models inherently expensive. Further criticism against GEM models of PDAC has focused on the multi-focality of their PDAC, involvement of whole pancreas with tumours, histological variants commonly observed, presence of tumours in other organs as well as genetic homogeneity; features missing in the human PDAC [53]. Thus, 3D organotypic models may be an attractive option as a preclinical tool, bridging the gap between traditional 2D cell culture assays and the complex GEM models. Organotypic models used in other cancers The idea of recapitulating the physiomimetic 3D envi July 14, 2014 Volume 20 Issue 26

166 Coleman SJ et al. Pancreatic cancer organotypics as preclinical models A C E Day 1 Day 1 B D Day 0 Day 1 F Cancer cell Stellate cell Extra-cellular matrix Cell culture media membrane (e.g., Matrigel) undergoing glandular differentiation forming with apico-basal polarity and a central hollow lumen [66], have led to similar experiments for the liver, salivary gland, bone, lung, skin, intestine, kidney and thyroid glands [64,67-71]. The choice of cell source and ECM is critical in developing a representative model. For example, human luminal epithelial cells, grown in laminin rich basement membrane analogue (Matrigel) form acini [72] ; however when grown in collagen I, these same cells show an altered integrin profile and abnormal polarity [73]. These 3D models have increased our understanding of how cells perceive biochemical and physical cues from the surrounding microenvironment [74]. For example, β1 integrin is expressed in normal breast epithelial cells but is lost when cells transform into a malignant phenotype. Re-expression of β1 integrin in 3D matrices induces the reversion of the tumor phenotype by allowing the malignant cells to differentiate into glands [75]. The incorporation of tissue specific stromal cells is critical for approximation to the in vivo condition. Thus, the isolation and availability of human PSCs hase been critical to the development of PDAC organotypic cultures [26]. Figure 2 Submerged organotypic culture models used to investigate pancreatic ductal adenocarcinoma. A: Cancer cells were embedded in the extracellular matrix mixture before it was allowed to polymerise. These cells were then fed with culture media placed on top of the gel; B: Representative configuration of cells within the gel after 7 d of culture. The cancer cells forming duct-like structures within the gel. This model mimics the behaviour of invading cancer cells; C, D: show the same model with both pancreatic stellate cells and cancer cells embedded within the extracellular matrix gel. Using this model the interaction between stellate cells and invading cancer cells can be examined; E: Stellate cells embedded in the gel prior to polymerisation, with cancer cells seeded on top of the gel 24 h later. In this model cancer cell invasion can be analysed in the presence of pancreatic stellate cells (F). Representatice HE of these organotypic models are reviewed in Froeling et al [26]. ronment started in the 1960 s growing 3D tissue explants in tissue culture media (organ cultures). Organ cultures of neural tissue explants are perhaps the best established model [54]. These models are still being used to study the basis of neurological diseases and injuries [55]. Organ cultures are also used in the study of cardiovascular function [56], angiogenesis [57], thymus [58], skin [59], bone [60], and urogenital tissues [61]. In cancer research, there has been an abundance of evidence suggesting that 3D models are superior to the conventional 2D culture in plastic flasks. However, current preclinical research still relies heavily on the latter [62]. From the simplest form: the monotypic cell model, comprising just one epithelial cell type, 3D co cultures have progressively evolved to contain multiple cell types, thus enabling study of their respective contributions [63]. An early example was the the skin equivalent, achieved by culturing keratinocytes either on de-epidermalised dermis or on collagen gels embedded with dermal fibroblasts [64,65]. The success of pioneering studies with breast epithelial cells cultured in, or on, a reconstituted basement Pancreatic cancer organotypics Pancreatic cancer cell lines and normal pancreatic ductal epithelial cells (HPDE) previously have been cultured on type Ⅰ glycosaminoglycan scaffolds and in collagen type Ⅰ or Matrigel. Given only epithelial cells were in these models, the effect of the stroma on tumour cell behaviour was absent [76-78]. However, these studies were able to show that pancreatic cancer cells embedded into Matrigel formed spheroids with a distinct morphology and loss of apico-basal polarity as compared to culturing in 2D [76]. The introduction of stromal cells in PDAC 3D organotypic cultures was first demonstrated by our laboratory [24]. Depending on the hypothesis being explored, the flexible 3D models of PDAC can be set up distinctly. Pancreatic cancer cells can be embedded into the ECM gel consisting of collagen and Matrigel to simulate cells that have already invaded into the stroma However, in order to understand the influence of PSCs on the behaviour of invaded pancreatic cancer cells these cells can be embedded in an ECM gel together with cancer cells (Figure 2). Submerged ECM gels (when pancreatic cancer cells are grown on top of the gel and PSCs are embedded) are designed to model the early events in tumour progression. When pancreatic cancer cells are cultured on top of this model, they form luminal structures that resemble ducts (Figure 3). Using this model, we have shown that PSCs induce Ezrin translocation from the apical to the basal compartment of the cells is an early event in pancreatic cancer cell invasion [24,79]. This phenomenon has been validated across a range of human gastro-intestinal tumours [80,81]. Finally, in order to study the invasion of pancreatic cancer cells in the 3D model, the submerged 8474 July 14, 2014 Volume 20 Issue 26

167 Coleman SJ et al. Pancreatic cancer organotypics as preclinical models A Day -1 Day 0 Day 1 B Cancer cell Stellate cell Extra-cellular matrix Cell culture media Metal grid DAPI Cytokeratin a-sma C D 100 µm 100 µm Figure 3 Raised organotypic model of pancreatic ductal adenocarcinoma with embedded pancreatic stellate cells. A: Extracellular matrix gels containing stellate cells were polymerised in 24-well plates before cancer cells were seeded on top and allowed to attach. These gels were then raised onto metal grids and fed from below creating a chemotactic gradient. Cells were cultured for up to 14 d; B: Illustration of cancer cells in a raised model containing stellate cells showing proliferation and invasion into the gel; C: HE section of a raised gel containing stellate cells with cancer cells seeded on top; D: Immunofluorscence in the same gel showing strong cytokeratin expression in the cancer cells and α-smooth muscle actin (α-sma) expression in the embedded stellate cells. culture system can be raised upon a grid ( air-liquid model) and fed from underneath, creating a gradient that stimulates pancreatic cancer cells to invade while at the same time recapitulates cancer-stellate cell interaction in vivo (Figure 4). Using the air liquid 3D model we have shown that the presence of PSCs leads to a significant increase, and altered sub-cellular distribution, of β-catenin in pancreatic cancer cells. Treating these 3D co cultures with All Trans Retinoic Acid (ATRA, which renders PSC quiescent) dampens Wnt-β catenin signalling resulting in reduced pancreatic cancer invasion [16]. Importantly, these results were confirmed in vivo, whereby treating KPC mice with ATRA led to disruption lead to disruption of the activated stroma and increase in apoptosis of tumour cells. These sets of observations validate the use of the organotypic model as a tool to assess new therapies in PDAC. 3D organotypic models provide a perfect intermediate between 2D cultures and GEM. Use of distinct cell types in these co-culture allows assessment of changes in signalling cascades and molecular targets resulting from cancer-stroma cross-talk in the absence of noise from other stromal elements present in vivo. Thus the relative contribution of each cell type in the complex microinvrionment can be assessed. Using this approach Kadaba et al [14] isolated cancer cells from organotypic models of various organ including pancreas, skin and oesophagus after the cancer cells were exposed in 3D to their respective stromal cells (Figure 5). They demonstrated that cancer cell stromal interactions significantly alter proliferation, cell cycle, cell movement, cell signalling and inflammatory response in addition to changing stiffness in the ECM gel. Importantly, changes in stiffness of ECM gels was particularly prominent as the proportion of PSC in the ECM gel increased, a finding highly pertinent to drug delivery and perfusion in PDAC [41]. This study also highlighted the possible need for multidrug targeting or use of pleiotropic agents in PDAC therapy. Despite the importance of multiple pathways in PDAC, the proto-oncogene Src has been heralded as a potential single molecular therapeutic target [82]. The conundrum of promise of Src inhibitors in combination with chemotherapy in vitro and the in vivo reduction of metastasis in KPC mice by 50%, was explored in organotypic cultures [82]. Using fluorescence lifetime imaging microscopy (FILM) to measure fluorescence resonance 8475 July 14, 2014 Volume 20 Issue 26

168 Coleman SJ et al. Pancreatic cancer organotypics as preclinical models A Day -1 Day 0 Day 1 B Cancer cell Stellate cell Extra-cellular matrix Cell culture media Metal grid DAPI Cytokeratin a-sma C D 100 µm 100 µm Figure 4 Raised organotypic model of pancreatic ductal adenocarcinoma with cancer cells and pancreatic stellate cell interaction. A: Extracellular matrix gels containing were polymerised in 24-well plates before cancer stellate cells were seeded on top and allowed to interact and attach. These gels are then raised onto metal grids and fed from below creating a chemotactic gradient. Organotypic models were cultured for up to 14 d; B: Illustration of cancer cells and stellate cells in a raised model showing increased proliferation and invasion of both stellate and cancer cells into the gel; C: HE section of a raised gel with cancer cells and pancreatic stellate cells seeded on top; D: Immunofluorescence in the same gel showing strong cytokeratin expression in the cancer cells and α-smooth muscle actin (α-sma) expression in the stellate cells which form a layer below the cancer cells. energy transfer (FRET) an ECFP-YFP Src reporter, in PDAC cells in organotypic cultures Anderson and colleagues investigated the influence of tumour microenvironment on Dasatinib delivery in PDAC [83]. In organotypic PDAC models with cancer cells expressing the Src biosensor cultured on top of an ECM gel with embedded primary human fibroblasts, they were able to show quantitatively that the microenvironment contribution to poor drug delivery to tumour cells is dependent on distance of cells from the invasive edge. This was validated in subcutaneous in vivo models due to the limitations of microscopy techniques precluding orthotopic or GEM models. This study demonstrated the adaptability of the organotypic model as powerful tool to address hypotheses at the molecular level in a complex microenvironment. Future applications and challenges 3D organotypic models that mimic the morphological and functional features of their in vivo parental tissues have potential for bridging the gap between cell-based discovery research and animal models [84,85]. A huge advantage of the organotypic system is that any component of the model can readily be modulated in a short time-frame. For example, the matrix composition can be altered to reflect the in vivo situation. The increase in ECM stiffness exerts elevated force on transformed cells increasing cellular response and resulting in increased tumour growth, survival and motility [14,86]. The relative paucity of primary stellate cells to conduct all the experiments in sufficient replicates lead us to generate a mini organotypic culture system (Figure 6) which give comparable results to the conventional air liquid co culture model [14]. Additional cell types can be titrated in such as stellate cells [14] or endothelial cells (Di Maggio, unpublished observations). For example, to assess the role of stroma on angiogenesis, in oesophageal cancer endothelial cells on a 2D monolayer have been cultured with fibroblast and cancer cells embedded in a collagen gel layered on top [87]. Elsewhere investigation of the role of macrophages in malignant growth of human squamous cell carcinoma has been investigated in organotypic cultures [88]. Immune response and inflammation play an important role in the desmoplastic reaction and inflammation is thought to activate pancreatic stellate cells [13,89]. Therapeutic agents such as chemotherapy (Gemen July 14, 2014 Volume 20 Issue 26

169 Coleman SJ et al. Pancreatic cancer organotypics as preclinical models A Cancer cell Stellate cell Extra-cellular matrix B Figure 5 Use of organotypic model to isolate cell types grown together by laser microdisection. By using the raised organotypic model with pancreatic stellate cells embedded within the gel (Figure 4B) the sellate and cancer cells are kept separate. A, B: Laser microdissection of the cancer cells can then be performed to allow analysis of cancer cells grown in the presence of pancreatic stellate cells. Scale bar 100 µm. A Cancer cell Stellate cell Extra-cellular matrix Cell culture media Metal grid Day -1 Day 0 Day 1 DAPI Cytokeratin Vimentin B C 100 µm 100 µm Figure 6 Mini-organotypic model of pancreatic ductal adenocarcinoma. Extra cellular matrix gel is polymerised within the insert of a standard migration assay plate. A: Cancer and pancreatic stellate cells are seeded on top of the gel and allowed to attach. The media is then removed and then replaced only in the bottom of the well to again create a chemotactic gradient cells are cultured for 7-10 d; B: HE image demonstrating a similar pattern of cell proliferation and invasion is seen, as in the raised model; C: Immunofluorscence in the same gel showing strong cytokeratin expression in the cancer cells and vimentin expression in the stellate cells which form a layer below the cancer cells and invade into the gel ahead of cancer cells. zitdis and Carapuca and Ghallab, unpublished observations), small molecules [90] or RNAi (Arumugam and Watt, unpublished observations) can be tested in these organotypic cultures. The best dosage and regimen can then be taken in small animals thus reducing animal usage [16,83]. Examples from other related fields include testing Metkinase inhibitor or COX-2 inhibitor in skin cancer models [91], tyrosine kinase inhibitors for breast cancers [92] and Eps8 and HAX1 or β6 integrin [93] RNAi in cancer cells prior to their incorporation into organotypic cultures to assess the effects on cell invasion. Finally, many PDAC patients present very late with their disease when metastasis have already occurred. Thus, treating PDAC cells immediately after seeding in a 3D environment does not reflect the true clinical setting as tumours are well established at the time of patient treatment. We currently are investigating the effect of treating organotypic models once they are established and invasion of PDAC and/or stromal cells has begun. It is likely this would give a better understanding of the treatment regimen that is required when novel therapies emerge into a preclinical setting. CONCLUSION Organotypic culture models are valuable tools for studying the mechanisms of pancreatic cancer, providing an easily manipulated system in which specific questions can be addressed, thus facilitating the translation of basic science to the clinic. Allowing manipulation of cell types, matrix composition, and exogenous therapies, these physiologically relevant model systems are reproducible, experimentally flexible and offer targeted highthroughput platforms. Although the organotypic model provides a physiologically relevant means to study the tumour stroma interactions and the use of new therapies to target the cross talk, it remains a simplified representation of the complex in vivo situation and it still remains critical to test new therapies in orthotopic or transgenic models of the disease. However, the use of the organotypic model as a preclinical tool is becoming increasingly important and our group, as well as others, are modulating the 3D cultures to recapture other important aspects of the tumour microenvironment that can influence cancer cell behaviour. Thus, 3D organotypic models have potential for bridging the gap between cell based discovery and complex animal models. By providing an environment in which cell behaviour and novel treatment options can be investigated in an easily reproducible and controlled manner, these models more precisely mimic pancreatic cancer, thus providing a major contribution to preclinical drug and therapeutic discovery July 14, 2014 Volume 20 Issue 26

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173 Coleman SJ et al. Pancreatic cancer organotypics as preclinical models 90 Coleman SJ, Chioni AM, Ghallab M, Anderson RK, Lemoine NR, Kocher HM, Grose RP. Nuclear translocation of FGFR1 and FGF2 in pancreatic stellate cells facilitates pancreatic cancer cell invasion. EMBO Mol Med 2014; 6: [PMID: ] 91 Nystrom ML, McCulloch D, Weinreb PH, Violette SM, Speight PM, Marshall JF, Hart IR, Thomas GJ. Cyclooxygenase-2 inhibition suppresses alphavbeta6 integrin-dependent oral squamous carcinoma invasion. Cancer Res 2006; 66: [PMID: DOI: / CAN ] 92 Chioni AM, Grose R. FGFR1 cleavage and nuclear translocation regulates breast cancer cell behavior. J Cell Biol 2012; 197: [PMID: DOI: /jcb ] 93 Ramsay AG, Keppler MD, Jazayeri M, Thomas GJ, Parsons M, Violette S, Weinreb P, Hart IR, Marshall JF. HS1- associated protein X-1 regulates carcinoma cell migration and invasion via clathrin-mediated endocytosis of integrin alphavbeta6. Cancer Res 2007; 67: [PMID: DOI: / CAN ] P- Reviewers: Apte MV, Liu QD S- Editor: Ma YJ L- Editor: A E- Editor: Zhang DN 8481 July 14, 2014 Volume 20 Issue 26

174 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. WJG 20 th Anniversary Special Issues (14): Pancreatic cancer TOPIC HIGHLIGHT hent1 expression is predictive of gemcitabine outcome in pancreatic cancer: A systematic review Stina Nordh, Daniel Ansari, Roland Andersson Stina Nordh, Daniel Ansari, Roland Andersson, Department of Surgery, Clinical Sciences Lund, Lund University and Skåne University Hospital, SE Lund, Sweden Author contributions: Nordh S performed the literature search; Nordh S and Ansari D were involved in data analysis and manuscript writing; Andersson R designed the study and revised the manuscript; all authors read and approved the final manuscript. Correspondence to: Roland Andersson, MD, PhD, Department of Surgery, Clinical Sciences Lund, Lund University and Skåne University Hospital, Paradisgatan 2, SE Lund, Sweden. roland.andersson@med.lu.se Telephone: Received: October 8, 2013 Revised: March 3, 2014 Accepted: March 12, 2014 Published online: July 14, 2014 High human equilibrative nucleoside transporter 1 (hent1)-expression has shown a survival benefit in pancreatic cancer patients treated with gemcitabine in several studies. The aim of this systematic review was to summarize the results and try to assess the predictive value of hent1 for determining gemcitabine outcome in pancreatic cancer. Relevant articles were obtained from PubMed, Embase and Cochrane databases. Studies evaluating hent1-expression in pancreatic tumor cells from patients treated with gemcitabine were selected. Outcome measures were overall survival, disease-free survival (DFS), toxicity and response rate. The database searches identified 10 studies that met the eligibility criteria, and a total of 855 patients were included. Nine of 10 studies showed a statistically significant longer overall survival in univariate analyses in patients with high hent1-expression compared to those with low expression. In the 7 studies that reported DFS as an outcome measure, 6 had statistically longer DFS in the high hent1 groups. Both toxicity and response rate were reported in only 2 articles and it was therefore hard to draw any major conclusions. This review provides evidence that hent1 is a predictive marker for pancreatic cancer patients treated with gemcitabine. Some limitations of the review have to be taken into consideration, the majority of the included studies had a retrospective design, and there was no standardized scoring protocol for hent1-expression Baishideng Publishing Group Inc. All rights reserved. Key words: Pancreatic cancer; Gemcitabine; hent1; Predictive; Survival Core tip: Human equilibrative nucleoside transporter 1 is a predictive marker for pancreatic cancer patients treated with gemcitabine. Abstract Nordh S, Ansari D, Andersson R. hent1 expression is predictive of gemcitabine outcome in pancreatic cancer: A systematic review. World J Gastroenterol 2014; 20(26): Available from: URL: i26/8482.htm DOI: INTRODUCTION Gemcitabine is the standard chemotherapy treatment for pancreatic cancer [1-3], but its efficacy is limited; only 15% of patients with advanced pancreatic cancer [4] and up to 30% in general [5] can be expected to respond to treatment. Gemcitabine is hydrophilic and therefore passive diffusion through hydrophobic cellular membranes is slow [1]. Permeation through the membranes requires specialized membrane transporters [1,3], and human equilibrative nucleoside transporter 1 (hent1) is the most important for gemcitabine [6,7]. Because gemcitabine is a prodrug, it has to be phosphorylated after intracellular uptake [1] in order to have a cytotoxic effect [6]. This ratelimiting step is carried out by the enzyme deoxycytidine kinase (dck) [8]. Recent research has revealed that differences in the 8482 July 14, 2014 Volume 20 Issue 26

175 Nordh S et al. hent1 expression in pancreatic cancer expression of genes, including hent1 [9,10] and enzymes involved with gemcitabine metabolism, such as dck, may be predictors of the efficacy of gemcitabine treatment for pancreatic cancer [11]. Several studies have indicated that high expression of hent1 is associated with longer overall survival (OS) and longer disease-free survival (DFS) [9,10,12]. The aim of this review was to evaluate and summarize the potential predictive value of hent1 expression in pancreatic tumor cells in patients treated with gemcitabine. STUDY SELECTION To identify all relevant English-language articles published from 1966 to March 2013, a computerized search of PubMed, Embase and Cochrane databases was performed. The following search terms were used: (hent1 OR nucleoside transporter), (gemcitabine OR gemzar), (pancreatic OR pancreas), (cancer OR adenocarcinoma OR neoplasm). The Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) [13] was used as a guideline for the processing and reporting of the results. The initial search yielded 230 publications (54 in PubMed, 1 in the Cochrane database, 175 in Embase). To find studies that might have been missing in the database search, a manual search was made by reading through reference lists of relevant articles and systematic reviews. The results of the search and the selection of studies are shown in Figure 1. The quality of the included articles was assessed using the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) [14]. ELIGIBILITY CRITERIA For inclusion in this systematic review, the following criteria had to be met: retrospective or prospective studies of patients with pancreatic cancer, all stages, treated with gemcitabine with or without additional radiation; the expression of hent1 had to be reported and related to patient outcome; and the articles had to be available in full text and published in English. Exclusion criteria were conference abstracts, overlapping patient cohorts and studies in which the relevant outcomes of interest were not addressed. DATA EXTRACTION A data extraction form was completed before the extraction process began. The form was reviewed by a second author (RA) to ensure that all relevant information was being extracted. The data extraction was done by a single reviewer (SN) and the following data were extracted from each study: publication details [author(s), date of publication, location, study center], study design, population details (age, sex, pt-stage, pn-stage), patient number, type of intervention (dose, schedule, duration), method to determine hent1-expression, hent1-scoring, primary and secondary outcome measurements, and results correlated with hent1-expression. OUTCOMES OF INTEREST AND DEFINITION The primary outcome measures were OS and DFS correlated with hent1 expression. Secondary outcome measures were toxicity according to the Common Toxicity Criteria ( and response rate according to RECIST [15] criteria. LITERATURE SEARCH The search identified 230 references in the 3 databases. Of these, 120 were excluded after identification of duplicates and exclusion based on irrelevant titles. Abstracts from 110 articles were screened and 75 were excluded. The main reasons for exclusion were: nonclinical trials (such as review articles), hent1-expression was not being assessed, and irrelevant outcome measurements. The remaining 35 studies were retrieved for further assessment. Of these, 25 references were excluded for the following reasons: they were conference abstracts and not full-text articles; hent1-subgroups were measured [16,17] ; hent1 was evaluated as a prognostic factor rather than a predictive factor of gemcitabine treatment [18] ; there were overlapping patient populations [19] ; and too small sample size/case reports [20]. An additional 5 article abstracts were screened for eligibility after identification in a manual search of reference lists of relevant articles. All of these were excluded based on irrelevance. In total, 10 studies fulfilled our inclusion criteria and were included in this systemic review. CHARACTERISTICS OF SELECTED STUDIES The included articles were published between 2004 and They originated from Belgium (2 studies) [1,3], Canada (one study) [9], United States (one study) [12] and Japan (5 studies) [2,8,21-23]. The 5 studies from Japan originated from 5 different universities, Kyushu, Osaka, Yokohama, Mie and Hiroshima. One author had published 2 articles [1,3], for one of these the patient population was recruited from 2 centers, while for the other the patient population was recruited from 5 centers. The potential bias of overlapping patient populations was therefore small, but must nevertheless be taken into consideration in the analysis of the results. Nine of 10 studies were retrospective [1-3,8-10,21-23] and one was a post hoc analysis of a randomized controlled study [12]. The 10 studies involved a total of 855 patients and the sample size varied from 21 to 234 (Tables 1 and 2). Four studies [1,8,10,12] used parallel groups, while the remainder were single-arm studies. The treatment protocols, which differed between the studies, included ad July 14, 2014 Volume 20 Issue 26

176 Nordh S et al. hent1 expression in pancreatic cancer Included Eligibility Screening Identification Records identified through database searches n = 230 Abstracts screened for relevance n = 110 Full-text articles assessed for eligibility n = 35 Studies included in the systematic review n = 10 Duplicates and irrelevant titles excluded n = 120 Abstracts excluded (review, hent1 not assessed, irrelevant outcome measurements) n = 75 Articles identified by manual search of reference lists n = 5 Full-text articles excluded as irrelevant (conference abstracts, hent1-genotypes measured, other chemotherapeutic agents than gemcitabine. Overlapping populations) n = 30 Figure 1 Flowchart showing article selection process. Table 1 Characteristics of the identified studies Ref. Year of publication Country Inclusion period No. of patients Study design Follow-up median (95%CI), mo Spratlin et al [9] 2004 Canada RS NR Giovannetti et al [10] 2006 Italy RS 11.2 ( ) Farrell et al [12] 2009 United States Post hoc 2 NR Maréchal et al [3] 2009 Belgium RS 21.9 ( ) Fujita et al [8] 2010 Japan RS 15.7 ( ) Maréchal et al [1] 2012 Belgium RS 55.7 ( ) Kawada et al [2] 2012 Japan RS 31 Morinaga et al [21] 2012 Japan RS NR Murata et al [22] 2012 Japan RS 15 ( ) Nakagawa et al [23] 2012 Japan RS 39.7 (2 122) Total: n = 81 with complete hent1; 2 Post hoc analysis of randomized controlled trial. NR: Not reported; RS: Retrospective. juvant gemcitabine monotherapy, palliative gemcitabine treatment, neoadjuvant gemcitabine chemotherapy, adjuvant gemcitabine chemotherapy + radiation, neoadjuvant gemcitabine + radiation (and adjuvant 5-fluorouracil), resection only and neoadjuvant gemcitabine-based chemoradiation + adjuvant gemcitabine (Table 2). All protocols were based on gemcitabine treatment and resection of the tumor. hent1 EXPRESSION To quantify hent1-expression, 8 studies used immunohistochemistry (IHC) and 2 used reverse transcription polymerase chain reaction. Grading of the expression differed between the studies, as is described in more detail in Table 3. The majority of the studies dichotomized the expression in high/positive vs low/negative hent1 expression. There were no standardized scoring procedures available. OVERALL SURVIVAL Nine of the 10 included studies had OS as an outcome measurement of interest (Table 4). Kawada et al [2] was the sole study that only reported disease-specific survival (DSS) as the primary outcome. The definition of DSS is the length of time from either the date of diagnosis or start of treatment for a specific disease (e.g., pancreatic cancer), and that the patients with the disease still are alive. The difference from OS is that OS measures death 8484 July 14, 2014 Volume 20 Issue 26

177 Nordh S et al. hent1 expression in pancreatic cancer Table 2 Characteristics of the identified studies Ref. Age median, yr (range) Sex m/tot n (%) hent1 method Chemotherapy Radiation dose Gy/(Gy/frac) Outcome measurement Quality REMARK Spratlin et al [9] 58 (51-64) 1 11 (52) IHC Pall Gem No OS 10 Giovannetti et al [10] 65 (22-83) 53 (50) RT-PCR Pall Gem 45 OS, DFS, TTP, RR 12 Adj Gem Farrell et al [12] 53/63/ (49) IHC Adj Gem 50.4 OS, DFS, Tox 17 Maréchal et al [3] 56 (34-83) 23 (51) IHC Adj Gem OS, DFS, Tox 13 Fujita et al [8] 65 (36-86) 42 (60) RT-PCR Adj Gem or No OS, DFS 12 Resection only Maréchal et al [1] NR 129 (53) IHC Adj Gem 50.4 OS 15 Kawada et al [2] - (41-81) 33 (52) IHC Neo Gem 50/2 DSS 9 Adj 5-FU Morinaga et al [21] 64 (45-74) 17 (63) IHC Adj Gem No OS, DFS 12 Murata et al [22] 68 (44-87) 38 (69) IHC Neo Gem 45/2 OS, DFS, RR 13 Adj Gem Nakagawa et al [23] 67 (41-83) 52 (48) IHC Adj Gem + S1 No OS, DFS %CI; 2 Medians in the different hent1 expression groups. IHC: Immunohistochemistry; RT-PCR: Reverse transcription polymerase chain reaction; Adj: Adjuvant; Neo: Neoadjuvant; Radio: Radiotherapy; Gem: Gemcitabine; Pall: Palliative; OS: Overall survival; DFS: Disease-free survival; Tox: Toxicity; TTP: Time to progression; DSS: Disease-specific survival; RR: Response rate. from any cause, not just death from a particular disease. Survival times for the individual studies were calculated based on: diagnosis, in one study [10] ; the start of gemcitabine treatment in 2 studies [9,22] ; resection, in 5 studies [1,3,8,21,23] ; and randomization in one study [12]. In univariate analyses, all 9 studies that had OS as an outcome measurement of interest showed a survival benefit with gemcitabine treatment and high/positive expression of hent1 compared with patients with low/ negative hent1 expression. Multivariate analyses were conducted in 8 of the 9 studies. Seven of these identified high/positive hent1 as an indicator of longer OS in patients with pancreatic cancer who received gemcitabine treatment. One study [8] indicated a trend towards better OS in the multivariate analysis, but this was not statistically significant (P = 0.2). DISEASE-FREE SURVIVAL In 7 studies, DFS was reported as an outcome measurement (Table 4). DFS was calculated from the same starting points as reported above for OS. Six of these studies showed a statistically significant longer DFS in univariate analyses. One study [8] was not statistically significant with regard to hent1. Multivariate analyses were conducted in 5 studies but of these only 3 [3,12,23] reported statistically significant results with longer DFS in regard to hent1 in patients with pancreatic cancer treated with gemcitabine. Morinaga et al [21] and Murata et al [22] also performed multivariate analyses, but the results did not prove to be statistically significant with reported P-values ranging from to TOXICITY Only 2 studies addressed the issue of toxicity [3,12], but the numbers published were inadequate for further analy- sis. Farrell et al [12] tried to find a relationship between hent1-levels and the incidence of grade Ⅲ or higher toxicities; however no relationship was found using a logistic regression model. The analysis data were not shown in the article. Maréchal et al [3] reported that grade Ⅲ/Ⅳ hematological toxicities were noted in 10/45 patients and grade Ⅲ/Ⅳ nonhematological in 3/45 patients. They did not relate this finding to hent1 expression and no further data or data analysis was shown in the article with regard to toxicity. RESPONSE RATE Of the 10 included studies only 2 [10,22] reported the outcome measurement response rate (RR). Giovannetti et al [10] evaluated RR in 34/36 patients in a group of patients receiving palliative treatment with gemcitabine. Two of the patients were not evaluable because of early death and refusal. The results showed that 5 patients had a partial response, 13 had stable disease and 16 had progressive disease. No further evaluation or data analysis was made with respect to RR. Murata et al [22] evaluated RR in respect to hent1 expression; radiographic RR was judged according to RECIST (Response Evaluation Criteria in Solid Tumors). Radiographic RR was not significantly correlated with hent1 expression (P = 0.665). DISCUSSION Based on the data collected from the selected studies, there is evidence that hent1 expression is a predictive marker for pancreatic cancer patients treated with gemcitabine. Patients with high expression of hent1 had significantly longer OS in all included studies that evaluated this outcome measurement. These results are in accordance with other studies of gemcitabine outcome correlated with hent1 expression in other types of tumors, 8485 July 14, 2014 Volume 20 Issue 26

178 Nordh S et al. hent1 expression in pancreatic cancer Table 3 hent1 expression levels, cut-offs and grouping Ref. Method Grading Reference cells Groups (n ) Spratlin et al [9] IHC 0-2 based on relative intensities of staining. Langerhans cells, Dichotomized: 0 = absence of staining lymphocytes. Low = 0 (12) 1 = intermediate staining High = 1 and 2 (9) 2 = most intense staining Giovannetti et al [10] RT-PCR Gene-expression ratio with GAPDH, expressed as tertiles Gene expression tertiles: GAPDH/target gene ratio Low < 1.06 (27) Intermediate (28) High 1.38 (26) Dichotomized: By medians Low < 1.23 (44) High 1.23 (37) Farrell et al [12] IHC Based on relative intensities. Lymphocytes Dichotomized: High = strong reactivity in > 50% of neoplastic cells. No (18) 1 No = no staining in > 50% vs Low = all cases between High and No. Low/high (73) 1 Maréchal et al [3] IHC 0-3 based on staining intensities Langerhans cells Dichotomized: 0 = no staining Lymphocytes Low < 80 (26) 1 = weakly positive (final score) 2 = moderately positive 3 = strongly positive High = 80 (19) Final score calculated: multiplying intensity score and the percentage of the specimen. Weighted score Fujita et al [8] RT-PCR Level of mrna calculated from standard curve constructed with total RNA from Capan-1, a human pancreatic cancer cell line mrna split into high/low groups using recursive descent partitioning. Cut-off 0.5 Low (26) 1 High (14) Maréchal et al [1] IHC 0-2 based on staining intensities Lymphocytes Dichotomized: Quantified as Farrell Low/moderate (136) 1 High (86) 1 Kawada et al [2] IHC 0-2 based on staining intensities. Langerhans cells Negative = 0-1 (41) 1 = same intensity as control. Positive = 2 (22) Morinaga et al [21] IHC Staining intensity and percentage of positive tumor cells Low = hent1 score 0-3 (11) scored and given a hent1-score by calculating the two Staining 0-3 where High = hent1 score 4-6 (16) 0 = no 1= weakly pos 2 = moderately pos 3 = strongly pos Percentage: 0 = no positive 1 50% positive cells 2 = 50%-80% positive cells 3 = 80% Murata et al [22] IHC Staining intensity + extent of positive staining Langerhans cells Dichotomized: Intensity: Negative = low and intermediate (16) 0 = no staining 1 = weakly positive Positive = high (39) 2 = moderately positive 3 = strongly positive Extent staining: High = score 3 > 50% cells Low = score 0 or 1 > 50% Intermediate = all others Nakagawa et al [23] IHC Staining intensities: Langerhans cells Low = grade 0 or 1 in > 50% (31) 0 = not stained High = grade 2 or 3 in > 50% of cells (78) 1 = faintly stained 2 = weakly stained 3 = as strongly as islet cells 1 In gem arm. GAPDH: Glyceraldehyde 3-phosphate dehydrogenase. including biliary tract cancer [24], cholangiocarcinoma [25], bladder cancer [26] and non-small cell lung cancer [27]. Since there is no standardized protocol for the grading of hent1 expression, the methods used differed between the included studies (Table 3). The majority used immunohistochemistry to evaluate hent1 expression 8486 July 14, 2014 Volume 20 Issue 26

179 Nordh S et al. hent1 expression in pancreatic cancer Table 4 Results Ref. Median survival all patients (95%CI) Univariate analysis median (95%CI) or HR (95%CI) P -value OS DFS Main conclusions Multivariate analysis HR (95%CI) Univariate analysis median (95%CI) or HR (95%CI) P -value Multivariate analysis HR (95%CI) Spratlin et al [9] ( ) (mo): NR Pat with detectable hent1 had High = 13 ( ) ( ) Low = 4 ( ) P = 0.01 Giovannetti et al [10] 13.3 ( ) (mo): Low = 8.48 ( ) Low = 5.34 ( ) Palliative (mo): Low = 5.85 ( ) Inter = ( ) Inter = 1.07 ( ) Inter = ( ) High = ( ) High = 1 High = ( ) P P < P = groups: 2 groups: HR = 4.21 Adjuvant (mo): Low = ( ) P Low = 9.26 ( ) High = ( ) Inter = ( ) P High = ( ) P 0.01 Farrell et al [12] NR (HR): Low/high = 0.40 (HR): Low/high = Low/High = 0.51 ( ) ( ) Low/High = 0.57 ( ) 0.39 ( ) No = 1 No = 1 No = 1 No = 1 P = 0.02 P = 0.03 P = 0.05 P = sig longer OS compared with pat with low hent1 hent1 expression was significantly correlated with outcome - pat with high hent1 had longer OS hent1 expression was ass with longer OS, DFS in pat receiving gem. hent1 is a relevant predictive marker for gem outcome Maréchal et al [3] 21.9 ( ) (HR): High = 1 (HR): High = 1 Pat with high hent1 had sig High = 1 Low = 3.42 ( ) High = 1 Low = 3.17 ( ) longer OS and DFS compared to low hent1 Low = 3.88 ( ) P = Low = 3.55 ( ) P = P = P = 0.02 Fujita et al [8] NR (mo): (RR): (mo): NR Low hent1 ass with shorter OS High = 45 Low = High = 25 in gem-group Low = 16.5 ( ) Low = 8 P = P = 0.2 (not sig) P = 0.11 (not sig) Maréchal et al [1] 32.0 ( ) (HR): n = NR NR High hent1 predicts longer High = 0.43 ( ) High = 0.34 ( ) (GEM-group) Low/Mod = 1 Low/Mod = 1 P < P < Kawada et al [2] NR Positive vs negative Positive/ negative P = P = OS in pat treated with adj gem. Absence of gem - hent1 lacks prognostic value NR NR DSS tended to be better in the hent1-neg group but not statistically sig Morinaga et al [21] NR (mo): Low = 1 (mo): Low = 1 High hent1 sig ass with longer Low = 11.8 ( ) High = Low = 7.3 ( ) High = OS in pat receiving adj gem after High = 22.2 ( ) ( ) High = 9.3 ( ) ( ) resection P = P = P = P = (HR): (HR): Low = 1 Low = 1 High = ( ) High = ( ) P = P = Murata et al [22] 24.3 (HR): Positive = 1 (HR): Positive = 1 Sig longer OS, RFS in pat with Positive = 1 Negative = 3.15 Positive = 1 Negative = 1.76 pos hent1 Negative = 3.04 ( ) ( ) Negative = 2.34 ( ) ( ) P = P = P = P = Nakagawa et al [23] OS: 34.9 (5y-SR %): High = 1 (5y-SR %): High = 1 hen1 expression is predictive DFS: 17.8 High = 38 Low = 3.16 High = 30 Low = 2.70 Low = 13 ( ) Low = 17 ( ) P = P = P = P = of the efficacy of adj gem-based chemotherapy after resection 1 From diagnosis/from treatment; 2 n = 222 in multivariate analysis. Ass: Associated; pat: Patient; sig: Significant; adj: pos: Positive; op: Operation; HR: Hazard ratio; SR: Survival rate; RFS: Recurrence-free survival July 14, 2014 Volume 20 Issue 26

180 Nordh S et al. hent1 expression in pancreatic cancer in the tumor cells. This is the main method for assessing biomarkers in histopathology. Most studies in this review using this method of evaluation had 2 independent assessors (blinded to each other and to patient outcomes) to make the grading for higher quality and better precision. The different ways of grading protein expression is an issue that needs to be considered when assessing the results of independent studies as well as the results of this review. There is a need for standardized protocols to achieve better homogeneity across studies when it comes to grading the protein expression of hent1 in pancreatic tumor cells. Gemcitabine is the standard treatment for patients with pancreatic cancer. This is based on several studies where gemcitabine has shown a survival benefit compared with other treatment regimes [1,28-30]. In this review, the treatment differed amongst the included studies, but they all used gemcitabine as the basis for chemotherapy. Most studies were performed in resectable patients, but the predictive value of hent1 was also confirmed in unresectable patients [9,10]. Kawada et al [2] used neoadjuvant chemoradiation and their results showed, in contrast with all the others, a trend towards better DSS in patients with low expression of hent1, although the results were not statistically significant. They did use a slightly different outcome measurement that may have influenced the results. Even though the result was not statistically significant it raises some questions that are important in the discussion about the different treatment regimens across the studies. In the case of neoadjuvant treatment or neoadjuvant chemoradiation, tumor cells with high expression of hent1 may be destroyed before the tumor samples are collected and will therefore give misleading information. This creates interesting issues as to when and how the tumor cells should be analyzed. Fine needle aspiration is discussed as an option for retrieving tumor cells for evaluation of hent1. This method can be used before resection and may therefore be an effective tool to identify which patients may benefit from neoadjuvant treatment with gemcitabine. Future studies are needed in this area. One study [12] was a post hoc analysis of a randomized controlled trial, which is of course rated higher methodologically than are retrospective cohort studies. The common opinion is that a systematic review exhibits the greatest strength if the majority of the included studies are randomized controlled trials or at least prospective trials. However, no such trials have been made within this area, but the need for a review was still considered to be necessary. The retrospective design of the included studies implies that we need to consider reporting and selection bias when analyzing the results. REMARK [14], which is a relatively new assessment tool, was used for quality evaluation in this review. The maximum score in REMARK is 20, and the average score in the included articles was 12.6 within the range of Since this is a relatively new tool, there is not much information as to what quality is considered high, and what is low. In this review, the included articles were of relatively similar quality (Table 2) according to REMARK. The results of this review are important to the consideration of future treatment options for patients with pancreatic cancer. According to this review, hent1 has been proven to be a predictive marker for gemcitabine outcome, thus there are a few considerations to be made. First, if we can alter the expression of hent1 in the tumor cells to create a higher expression, more patients would benefit from gemcitabine treatment and survive longer. Pretreatment with thymidylate synthase inhibitors has proven to increase the expression of hent1 in tumor cells in vitro [6]. This might be a way to alter the expression in vivo as well, but further studies are needed. The second option is to find another way for gemcitabine to enter the tumor cells and exert its toxic effect. Research in this area is currently under way, and progress should enable more personalized treatment options for pancreatic cancer patients. Another aspect for the future is the cost of overtreatment with gemcitabine in patients who do not benefit from it. According to one study conducted on a Swedish pancreatic cancer cohort, EUR 8.6 million would be saved each year in Sweden if hent1 testing were used to select patients for gemcitabine therapy [31]. CONCLUSION This review provides evidence that hent1 is a predictive marker for pancreatic cancer patients treated with gemcitabine. 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Ann Surg 2012; 256: [PMID: DOI: / SLA.0b013e a42] 26 Matsumura N, Nakamura Y, Kohjimoto Y, Inagaki T, Nanpo Y, Yasuoka H, Ohashi Y, Hara I. The prognostic significance of human equilibrative nucleoside transporter 1 expression in patients with metastatic bladder cancer treated with gemcitabine-cisplatin-based combination chemotherapy. BJU Int 2011; 108: E110-E116 [PMID: DOI: /j X x] 27 Oguri T, Achiwa H, Muramatsu H, Ozasa H, Sato S, Shi July 14, 2014 Volume 20 Issue 26

182 Nordh S et al. hent1 expression in pancreatic cancer mizu S, Yamazaki H, Eimoto T, Ueda R. The absence of human equilibrative nucleoside transporter 1 expression predicts nonresponse to gemcitabine-containing chemotherapy in non-small cell lung cancer. Cancer Lett 2007; 256: [PMID: DOI: /j.canlet ] 28 Burris HA, Moore MJ, Andersen J, Green MR, Rothenberg ML, Modiano MR, Cripps MC, Portenoy RK, Storniolo AM, Tarassoff P, Nelson R, Dorr FA, Stephens CD, Von Hoff DD. Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial. J Clin Oncol 1997; 15: [PMID: ] 29 Regine WF, Winter KA, Abrams RA, Safran H, Hoffman JP, Konski A, Benson AB, Macdonald JS, Kudrimoti MR, Fromm ML, Haddock MG, Schaefer P, Willett CG, Rich TA. Fluorouracil vs gemcitabine chemotherapy before and after fluorouracil-based chemoradiation following resection of pancreatic adenocarcinoma: a randomized controlled trial. JAMA 2008; 299: [PMID: DOI: / jama ] 30 Oettle H, Post S, Neuhaus P, Gellert K, Langrehr J, Ridwelski K, Schramm H, Fahlke J, Zuelke C, Burkart C, Gutberlet K, Kettner E, Schmalenberg H, Weigang-Koehler K, Bechstein WO, Niedergethmann M, Schmidt-Wolf I, Roll L, Doerken B, Riess H. Adjuvant chemotherapy with gemcitabine vs observation in patients undergoing curative-intent resection of pancreatic cancer: a randomized controlled trial. JAMA 2007; 297: [PMID: DOI: / jama ] 31 Ansari D, Tingstedt B, Andersson R. Pancreatic cancer - cost for overtreatment with gemcitabine. Acta Oncol 2013; 52: [PMID: DOI: / X ] P- Reviewers: Macedo FI, Rossi RE, Yun S, Zhang Q S- Editor: Wen LL L- Editor: Cant MR E- Editor: Zhang DN 8490 July 14, 2014 Volume 20 Issue 26

183 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. REVIEW Liver zonation: Novel aspects of its regulation and its impact on homeostasis Rolf Gebhardt, Madlen Matz-Soja Rolf Gebhardt, Madlen Matz-Soja, Institute of Biochemistry, Faculty of Medicine, University of Leipzig, Leipzig, Germany Author contributions: Gebhardt R and Matz-Soja M conceived, designed, wrote and approved the paper. Supported by Bundesministerium für Forschung und Technologie (BMBF) in the framework of the Systems Biology initiative Virtual Liver Network, No Correspondence to: Rolf Gebhardt, PhD, Institute of Biochemistry, Faculty of Medicine, University of Leipzig, Johannissallee 30, Leipzig, Germany. rgebhardt@medizin.uni-leipzig.de Telephone: Fax: Received: October 28, 2013 Revised: February 20, 2014 Accepted: April 5, 2014 Published online: July 14, 2014 Abstract Liver zonation, the spatial separation of the immense spectrum of different metabolic pathways along the liver sinusoids, is fundamental for proper functioning of this organ. Recent progress in elucidating localization and interactions of different metabolic pathways by using omics techniques and novel approaches to couple them with refined spatial resolution and in characterizing novel master regulators of zonation by using transgenic mice has created the basis for a deeper understanding of core mechanisms of zonation and their impact on liver physiology, pathology and metabolic diseases. This review summarizes the fascinating technical achievements for investigating liver zonation and the elucidation of an emerging network of master regulators of zonation that keep the plethora of interrelated and sometimes opposing functions of the liver in balance with nutritional supply and specific requirements of the entire body. In addition, a brief overview is given on newly described zonated functions and novel details on how diverse the segmentation of metabolic zonation may be. From these facts and developments a few fundamental principles are inferred which seem to rule zonation of liver parenchyma. In addition, we identify important questions that still need to be answered as well as interesting fields of research such as the connection of zonation with circadian rhythm and gender dimorphism which need to be pushed further, in order to improve our understanding of metabolic zonation. Finally, an outlook is given on how disturbance of liver zonation and its regulation may impact on liver pathology and the development of metabolic diseases Baishideng Publishing Group Inc. All rights reserved. Key words: Homeostasis; Liver; Metabolic zonation; Metabolism; Morphogen signalling; Optimization; Pathology; Regulatory network; Sinusoid; Systems biology Core tip: Liver metabolism comprises an immense spectrum of interrelated anabolic and catabolic functions which are performed simultaneously without wasting energy. To cope with this challenge, liver parenchyma shows a considerable heterogeneity and functional plasticity known as metabolic zonation. This review summarizes fascinating technical achievements in this field, covers recent progress in characterizing and visualizing zonation, and outlines an emerging network of master regulators that ensures proper maintenance of homeostasis and energetic optimization of liver function. Finally, an outlook is given on how disturbance of liver zonation and its regulation may impact on liver pathology and the development of metabolic diseases. Gebhardt R, Matz-Soja M. Liver zonation: Novel aspects of its regulation and its impact on homeostasis. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: org/ /wjg.v20.i INTRODUCTION The liver shows a remarkable uniform anatomical struc July 14, 2014 Volume 20 Issue 26

184 Gebhardt R et al. Liver zonation: Regulation and impact ture composed of the regular arrangement of mainly hexagonal lobules in a honey comb-like pattern. At the periphery of these lobules blood from the portal venules and the hepatic arterioles enters the sinusoids, the smallest capillaries of the liver which form an anastomosing network that runs concentrically to the central vein where the blood drains into the hepatic venules [1-3]. The hepatocytes lined up in a sponge-like arrangement between the sinusoids along the porto-central axis show a remarkable heterogeneity with respect to the biochemical and physiological functions they perform. This dynamic structural and functional heterogeneity, known as metabolic zonation [4], seems to be the precondition for simultaneously performing the plethora of functions necessary to maintain metabolic homeostasis of the organisms under the various physiological conditions. Progress in metabolic zonation and its regulation was comprehensively reviewed from time to time [5-7], and culminated in the discovery of Wnt/β-catenin signalling as a master regulator of zonation [8,9]. The present review tries to give an update on the impressive development of research on zonation during the last decade. It will be conjectured that perturbation of the sophisticated regulation of zonation may presumably have an enormous impact on the development of various metabolic diseases. Therefore, a major aim of our review is to identify important open questions that need to be answered, in order to gain deeper insight into the mechanisms of liver zonation, its regulation and possible malfunction in disease. NEW METHODS A survey of traditionally used methods for studying liver zonation has recently be provided [10]. Therefore, we will focus here mainly on novel developments facilitating progress in elucidating hepatocyte heterogeneity and its consequences on a very broad scale, particularly at the omics level. Collection of intact tissue from periportal and pericentral zones by laser-capture microdissection (LCM) [11] certainly marks an improvement compared to digitonin-collagenase perfusion (DCP) [12], because it avoids tissue damage and extracted RNA can be directly used for microarray studies. In contrast to DCP which provides data for the most important cell-type in the liver, the hepatocyte, LCM cannot, however, discriminate between different contributions of hepatocytes and nonparenchymal cells [11]. Microscopic inspection of immunolabelled or otherwise stained sections is still one of the best and most reliable methods to reveal liver cell heterogeneity, because the antigen content of each cell, parenchymal or non-parenchymal, can separately be investigated and discerned, and gradients in expression across the periportalpericentral axis can easily be visualised. In the case of proteins, detection of individual proteins is possible by specific antibodies and has been used efficiently since the early times of investigation in this field. Nowadays, whole slide scans can be prepared that favour inspection of the heterogeneity from the global level (liver lobe) down to the sub-microscopic level. Concerning human liver, the collection of various immunostainings in the Human Protein Atlas ( can actually be used for an initial orientation on whether a certain protein is heterogeneously expressed in liver or not. Although this possibility is very helpful, the reliability of these findings is often limited by the small number of liver donors, the small size of the sections, and the questionable quality of the antibodies despite the honourable effort invested into their evaluation. With respect to other parameters, such as lipids or metabolites no comparable methods were available in the past. This has changed, because novel techniques based on mass spectrometry have been developed. For instance, in situ lipidomics analysis by cluster time-of-flightsecondary ion mass spectrometry (TOF-SIMS) imaging was used to map liver biopsies of patients with in nonalcoholic fatty liver disease (NAFLD) [13]. This technique allowed to map lipids on liver sections at the micrometer scale and to simultaneously characterize their molecular distribution. Briefly, accumulations of triacylglycerols, diacylglycerols, monoacylglycerols, fatty acids (with the apparition of myristic acid) and a selective macrovacuolar localization of cholesterol were observed in steatotic areas of fatty livers compared to control livers [13]. Remarkably, the pronounced zonation of lipid droplet accumulation NAFLD was underscored by the detection of very fine differences in lipid localizations depending on alkyl acid chain lengths and other molecular features. The drawback of this fascinating method is that TOF- SIMS is a hard ionization technology hampering the detection and discrimination of intact phospholipids. In a more recent study, this drawback was circumvented by using matrix assisted laser desorption ionization-imaging mass spectrometry (MALDI-IMS), a more soft technology [14]. These workers investigated hepatic phospholipid abundance by quantitative lipidomic profiling and phospholipid localization on sections of patients with simple steatosis and non-alcoholic steatohepatitis (NASH). In essence, this pioneering study shows several new aspects relevant for normal and pathologic phosopholipid metabolism: (1) phospholipid metabolism is strongly zonated in normal liver of obese patients; (2) the marked zonation of several molecular species of phosphatidyl choline present in controls and patients with simple steatosis is lost in NASH specimens; and (3) phospholipid zonation is associated with the presence of an intrahepatic proinflammatory phenotype [14]. Although these studies strongly suggest that loss, shift or gain of zonation of phospholipid metabolism is characterisitic of the development of progressive phenotypes in NAFLD, it remains unclear whether they may be causative or merely consequences of this process. Another, even broader methodological approach is in situ sequencing of RNA in tissue sections [15] which allows the association of sequencing results to histologi July 14, 2014 Volume 20 Issue 26

185 Gebhardt R et al. Liver zonation: Regulation and impact cal structures. Unfortunately, this technique has not yet been used for studying liver zonation. Nonetheless, these developments may provide us with a first glimpse on the potential of future research for studying liver zonation. REGULATION OF ZONATION Obviously, all types of signals such as gradients of oxygen, nutrients, metabolites, hormones and cytokines do have an influence on zonation in so far as they modulate the activity of various enzymes involved in the metabolic pathways. This modulation can, but not necessarily has to, result in the shift of zonation, since the protein content or the enzyme activity within the cells can vary independently of the number of cells involved. Indeed, in contrast to many dynamic metabolic pathways several apparently stable functions have been identified over the years the localization of which is almost invariable under a large variety of physiological and even pathological conditions [6]. One of these, hepatic glutamine synthesis via glutamine synthetase shows a very peculiar and stable pericentral localization in less than 3 rows of hepatocytes surrounding the central veins [16]. It was this fascinating all or none expression which lead to the discovery of morphogens as master regulators of metabolic zonation (for review [9] ). Morphogen signalling Morphogens are signalling molecules which play a decisive role in embryogenesis, morphogenesis, and organogenesis [17,18]. Characteristically, their pathway activity is high on average during these processes, but may fluctuate considerably in different phases of development. Usually, in the mature organism, their activity is strongly down-regulated. Reactivation, if any, occurs during tissue regeneration following damage or loss of tissue [19] and, in particular, during development of various types of cancer [20,21]. Under all conditions, one outstanding feature of morphogen signalling is gradient formation over short distances coupled with discrete interpretation of this gradient to regulate a finite number of events along the gradient [22-24]. Therefore, morphogen signalling pathways are involved in all kinds of tissue patterning. In the last decade, accumulating evidence was collected demonstrating that morphogen signalling pathways are deeply involved in regulating metabolism in the mature liver. The first example was Wnt/β-catenin signalling [25,26] an inhibitory component of which, the adenomatous polyposis coli (APC) gene product, later became apparent as zonation keeper [8,27,28]. Further examples for regulation of liver metabolism by morphogens comprise signalling through various members of the fibroblast growth factor (FGF) family [29,30], the transforming growth factor (TGF)-beta family including the bone morphogenetic proteins (BMPs) [31,32], the notch receptor [33-35] and hepatocyte growth factor (HGF) [36,37]. Although it is very likely that many, if not all, morphogens which regulate different aspects of liver metabolism are likewise involved in shaping liver zonation, we will concentrate herein only on Wnt/β-catenin, HGF and Hedgehog signalling for which most evidence is available at present. Wnt signalling The dominant role of Wnt/β-catenin signalling in governing metabolic zonation in the liver is now well established and has been reviewed in many excellent reviews [9,28,38,39]. Its activity is highest around the central vein of the liver lobules. Therefore, it is particularly involved in the regulation of pathways limited to or predominating in the pericentral zone such as glutamine synthesis, drug metabolism, bile acid and heme synthesis [8,40,41]. However, the expression of certain periportal functions (e.g., gluconeogenesis, and ureogenesis) is also influenced by the Wnt/β-catenin signalling pathway [8]. Usually, these portal functions are down-regulated, if β-catenin signalling is induced [39,42]. Whether these diverse actions have anything to do with the puzzling and apparently mutually exclusive expression of E-cadherin and N-cadherin in the periportal and pericentral zone, respectively [43], remains an open question that needs to be addressed in the future. Because of the broad influence of the activity of this pathway on liver zonation, it seems appropriate to consider Wnt/β-catenin signalling as a master regulator of zonation. Nonetheless, there may be some exceptions from the rule. In several liver functions such as lipid and ethanol metabolism β-catenin signalling may be essential, but most probably is not sufficient for proper regulation. Thus, Wnt signalling does not seem to affect lipid metabolism per se, but rather in response to additional challenges such as ethanol or methionine and choline-deficient diet [44,45]. HGF and RAS HGF was identified in 1991 as a morphogen able to induce 3-dimensional (3-D) morphogenesis [46], while other functions, e.g., as a growth factor, migration stimulus, and cell survival, were already known before [47-50]. With respect to liver, HGF is primarily seen as a mitogen for hepatocytes, transiently activated in the early phase of liver regeneration after partial hepatectomy [51-53]. Its proliferation stimulating activity seems to be higher in the periportal zone than in the perivenous zone [54]. HGF is usually synthesized in mesenchymal cells, while its receptor, Met, is expressed in the epithelia in close vicinity [48]. The contribution of HGF to the regulation of zonation was suggested on the basis of cell culture experiments using HGF as an inducer of rat sarcoma (RAS) signalling [55]. The idea that opposing signalling pathways triggered by Ha-ras- and β-catenin-dependent factors might determine zonation of (periportal) gene expression in murine liver was developed by the Schwarz group [55,56] based on comparisons of expression patterns of periportal and pericentral hepatocytes with those of liver tumors carrying activating mutations in either the Ha-ras or the ctnnb1 gene. Definitive prove of this hy July 14, 2014 Volume 20 Issue 26

186 Gebhardt R et al. Liver zonation: Regulation and impact A B 200 μm 200 μm Figure 1 Immunohistochemical staining of Indian hedgehog in normal liver and after up-regulation of β-catenin signalling. Ihh protein was detected in liver sections of wild type (WT) mice (A) and mice with down-regulated expression of APC protein after three month of age (B). Ihh protein in WT mice shows a clear gradient which is highest in hepatocytes surrounding the central veins. After up-regulation of β-catenin signalling the zone of Ihh-positive hepatocytes is considerably extended as are other known pericentral markers (e.g., glutamine synthetase; c.f. [8,42] ). Sections were stained with Rabbit anti-mouse Ihh polyclonal antibody (Abcam) followed by the EnVision + Dual Link System-horse radish peroxidase (DAKO) and counterstained with hemalum (Mayer solution). pothesis as well as of the contribution of HGF in this mechanism is still lacking. Perhaps, RAS signalling is kind of a nodal point integrating various signals that lead to activation or inhibition of ras signalling. Furthermore, the downstream effectors of RAS are also still unclear, although members of the mitogen-activated protein kinase (MAPK) cascade are potent candidates [57]. Concerning the influence on zonation, interactions between RAS signalling and WNT/β-catenin signalling as well as Hedgehog (Hh) signalling are of special interest. Such interactions have been recently reviewed mainly with respect to proliferation, differentiation and tumorigenesis [57-59]. It remains to be demonstrated whether they are relevant, at least partially, also for regulating zonation in adult liver especially as these interactions are highly context-dependent. Furthermore, the relative strength of each pathway activity compared to the others may be a major point distinguishing these interactions in cancer development from those in liver zonation (see discussion in [10] ). Hedgehog signalling Components of Hh signalling have been found to be expressed at very low levels in hepatocytes [60], whereas much higher levels could be measured in hepatic stellate cells [61,62] and in cholangiocytes [63]. These findings have lead to the widespread opinion that Hedgehog signalling in hepatocytes is not functionally active under normal conditions, a view that has paralyzed research in this field for almost a decade [10]. Only in certain states of liver damage, for instance, in heavily steatotic (ballooned) hepatocytes [64,65] or in response to lipotoxic challenge [66] it was realized that Hh signalling was upregulated resulting in enhanced synthesis of sonic hedgehog (Shh). Another state in which Hh signalling is induced in apparently normal hepatocytes is partial hepatectomy (PH) [67]. After 70% PH, the mrna level of Indian hedgehog (Ihh) increases up to 48 h and precedes that of Shh which mainly increases between 48 and 72 h. In all cases, increased Hh signalling seems to aid in regeneration and repair acting not only on the hepatocytes but also on other cell types including progenitor cell populations [68,69]. Despite the apparently low activity in hepatocytes we found many hints from literature, mainly derived from other organs that, by analogy, pointed to an involvement of Hh signalling in the regulation of metabolism in healthy mature hepatocytes. We have recently compiled these arguments in a hypothesis strongly suggesting that hepatocellular Hh signalling may contribute to liver zonation in spite of its low activity [10]. Indeed, using transgenic mice with conditional deletion of smoothened (Smo) we were able to demonstrate that even the very low activity of this pathway has a profound influence on mature liver function in particular on the expression of Igf1 in hepatocytes and, consequently, on the level of insulin-like growth factor Ⅰ (IGF-Ⅰ) protein in the serum [70]. Actually, these changes are due to the downregulation of the transcription factor GLI3 in response to the deletion of Smo in hepatocytes. Interestingly, the levels of all three GLI transcription factors are so low that their changes can be detected only by sensitive quantitative RT-PCR, but not by ordinary microarray gene expression analyses. Another fact is also very interesting: As we have hypothesized [10], Ihh is the main ligand involved and its expression is downregulated in the Smo-knockout mice [70]. On the other hand, immunohistochemistry showed that Ihh is expressed exclusively in the most distal pericentral area [70] (Figure 1). This is in line with the known fact that Ihh is a target of Wnt/β-catenin signalling [71]. The dependence of Ihh production on the activity of β-catenin signalling is further demonstrated by the extension of its pericentral distribution in transgenic mice with enhanced β-catenin signalling (Figure 1). Taken together, these findings indicate that expression of Ihh by (pericentral) hepatocytes is under the control of both, Hh and Wnt signalling. Though this fits nicely to another feature predicted by our hypothesis, namely, the tight crosstalk between Wnt/ β-catenin and Hh signalling pathways [10], the specific contribution of Hh signalling to the regulation of zonation remains still to be established July 14, 2014 Volume 20 Issue 26

187 Gebhardt R et al. Liver zonation: Regulation and impact Gradient formation Morphogens usually act through gradients over short distances (less than cells) that can be visualized by immunofluorescence in developing tissues [22,23]. In adult liver similar attempts failed, because they are too low and often only the highest activity is detectable [9,10,72]. Thus, the direction of the morphogen gradients (Wnt, central to portal; HGF, portal to central; Hedgehog, still unknown) can only be deduced from downstream effects and/or target activities. Likewise, the origin of these gradients is still unknown as is the possible (local) source of Wnt, HGF, and Hh signals. In the case of Hh signalling, the pericentral localization of Ihh in healthy mature hepatocytes has been reported [70], but it is unclear whether this is cause or consequence of pathway activity. Concerning Wnt factors, many different molecular species were found to be expressed in all liver cell types [73], but their function in determining zonation remains elusive. There are some hints that the crucial Wnt signal is derived from endothelial cells of the central veins in line with very early assumptions of the source of an inducing factor for distribution of GS [9,74], but direct experimental evidence is still lacking. This uncertainty about the morphogen gradients is a considerable drawback, because it not only hampers our deeper understanding of how zonation is controlled, but also our possibilities to explore the dominant role of the morphogens for therapeutic intervention. On the other hand, it may well be that we are asking the wrong question, if we ask for the crucial source of the morphogens. Perhaps, there are various sources, but the intensive molecular crosstalk between different morphogen pathways through direct and indirect feedback loops is able to produce stable gradients in pathway activity along the sinusoids. The fact that manipulating just one specific inhibitory component of Wnt signalling, the tumor suppressor gene product APC, is sufficient to eradicate the entire gradient [8], can be interpreted in favour of this assumption. If so, we need to learn more about the crosstalk of morphogen signalling pathways, in particular in the adult stage, in order to understand liver zonation. One recent observation from our laboratory may be key concerning the crosstalk between the Wnt- and the Hedgehog signalling pathways. As mentioned above, we observed that Ihh expression in hepatocytes shows a strong pericentral dominance in normal mice (c.f. Figure 1) that becomes less pronounced upon knockout of Smo [70]. In transgenic mice with a moderate increase in the Wnt signalling expression of Ihh was strongly enhanced and extended towards the portal tract (Figure 1) as does the expression pattern of other pericentral markers in these mice, e.g., glutamine synthetase (GS). Even under these conditions many periportal hepatocytes remain free of any staining indicating that Ihh is not expressed in these cells. Taken together these results demonstrate that the expression of Ihh in normal hepatocytes is positively coupled with both, Hh and Wnt signalling. It should be emphasized that under these conditions, at least in mice younger than 4 mo, hepatocellular production of Ihh is physiological [70] and does not indicate damaged or death hepatocytes as has been inferred from certain disease models [63,64]. CURRENT PICTURE OF METABOLIC ZONATION The plethora of metabolic functions of the liver and their complex underlying network of metabolic reactions make it impossible to provide a complete picture of the zonation of all individual pathways and molecular steps that have been investigated so far. Instead, we are aiming at providing a rough overview on novel progress and findings that were recently be achieved. Some information mainly based on a recent proteome study [42] is assembled in Figure 2 in a schematic way. Other important aspects are briefly summarized in the text. Central metabolism The picture of zonation of carbohydrate metabolism has not seen much improvement/refinement over the last years, since most phenomena were already comprehensively studied and described in the past [75]. What has changed most, are some regulatory aspects and certain connections between carbohydrate, energy and lipid metabolism. Though the importance of hormonal signals (e.g., insulin and glucagon) or metabolic factors (e.g., oxygen tension) emphasized earlier [7] is beyond doubt, it became apparent that Wnt/β-catenin signalling unexpectedly plays a dominant role also in controlling zonation of many aspects of carbohydrate metabolism [8,42,76]. On the basis of enhanced lactate dehydrogenase protein content it was suggested that up-regulation of Wnt/ β-catenin signalling may result in a Warburg-like rewiring of glycolysis [76]. However, this interpretation may be too simple, since a more careful proteomic study clearly showed that except for lactate dehydrogenase all other glycolytic enzymes were strongly down-regulated [42]. Since up-regulation of Wnt/β-catenin in hepatocytes simultaneously results in the up-regulation of GS expression [8], these findings strongly support the conclusion that GS-positive cells (including GS-positive HCC [77] generally run with a lower glycolysis rate than the rest of the hepatocytes, even the GS-negative pericentral ones (Figure 2). Concerning amino acid metabolism, the picture of zonation that could be drawn in former reviews was still relatively coarse [6,78,79] but meanwhile could be considerably refined, at least for the mouse. On the basis of microarray gene expression studies on periportal and pericentral hepatocyte preparations, especially three genes encoding enzymes of histidine catabolism and of an enzyme of histamine metabolism were found to be preferentially expressed in periportal hepatocytes [80]. Likewise, several genes encoding enzymes of glycine and serine metabolism were also periportally expressed where they might support gluconeogenesis as well as urea synthesis from serine. Most uncertainties with respect to a possible zona July 14, 2014 Volume 20 Issue 26

188 Gebhardt R et al. Liver zonation: Regulation and impact PP PC Wnt β-catenin/tcf Oxidative phosphorylation Complex Ⅰ Complex Ⅲ Complex Ⅴ Glycogenesis Gluconeogenesis Glycolysis TCA cycle Ammonia production Glutaminase Serine dehydratase Urea cycle Glutamine synthesis Lipogenesis Figure 2 Scheme illustrating aspects of zonation of central metabolism in normal mice. Selected functions of central metabolism are depicted as conical boxes showing the direction of their porto-central gradients with specific emphasis of the strength of this gradient in the glutamine synthetase-positive zone (right of the vertical line). Dashed boxes indicate gradients that have been determined using a single method only (proteome analysis), instead of at least two independent methods (other boxes). Since the data is valid for normal mice, the presumably corresponding gradient of Wnt/β-catenin signalling is depicted at the top of the scheme, above the row of the hepatocytes. PP: Periportal; PC: Pericentral. tion were formerly reported for certain aspects of lipid metabolism, because of incomplete or controversial findings [6,79]. The reasons for the high incidence of conflicting results in lipid metabolism appear enigmatic, but may be due to relatively shallow gradients of pathway activites, on the one hand, and a greater variability in different physiological states, on the other. The last two decades have seen a considerable improvement of the situation with many novel results which have solved some of the ambiguities. However, we are still far from a satisfactory understanding and several new questions have emerged. In the framework of this review, it is beyond scope to provide a comprehensive overview on all the novel results concerning zonation of lipid metabolism. Recently, an excellent review on this subject was published by Hijmans et al [81], and additional aspects will be reviewed specifically elsewhere (Schleicher et al in preparation). Also in the case of lipid metabolism, the 8496 July 14, 2014 Volume 20 Issue 26

189 Gebhardt R et al. Liver zonation: Regulation and impact gene expression study [80] of provided novel information by showing the periportal expression of Apolipoprotein CII, of phosphatide phosphatase type 2c and of ATP citrate lyase in the mouse. Preferential localization of apolipoprotein (Apo)E was also found in rats [82], but genderassociated modifications/variations were observed (see below). Energy metabolism The heterogeneity of mitochondria of periportal and pericentral hepatocytes was already noted very early based on morphologic features of these organelles [83]. Apart from the description of the heterogeneity of some mitochondrial enzymes such as succinate dehydrogenase [84] very few functional characteristics are known (for work before 1992 see [6] ). Importantly, a significantly higher cytosolic ATP/ADP ratio in the periportal compared to the pericentral hepatocytes was shown by Quistorff et al [85]. However, the situation seems rather variable and mitochondrial function, in particular mitochondrial redox state, responds dynamically to alterations of hormones and oxygen supply [86]. Recently, the proteome study performed on mice with conditional knockout of APC revealed interesting differences between hepatocytes with enhanced β-catenin signalling and different wild type mice [42]. Many enzymes of citric acid cycle were upregulated up to 3.8-fold with the exception of succinyl-coa ligase which was down-regulated 1.5-fold. Proteins of complexes Ⅰ to Ⅲ were mostly downregulated between 1.7-fold and 3.6-fold. In contrast, some proteins of complex Ⅴ were upregulated [42]. These findings seem to hold particularly for cells with (enhanced) expression of GS and suggest a very specific effect of Wnt/β-catenin signalling on the zonation of oxidative phosphorylation (OXPHOS) and energy production (Figure 2). Interestingly, deletion of β-catenin was found to induce inverse changes determined by using functional parameters [87]. Thus, they nicely fit to the proteome data and are in line with the periportalization of the hepatocytes indicated also by other functions in β-catenin-ko mice [40]. Drug metabolism Zonation of drug metabolism is of considerable importance for liver function, particularly under conditions of liver diseases. Therefore, comprehensive reviews have been published in the past for fetal [88] and adult liver [80,89] as well as with special emphasis on enterohepatic circulation [90]. Here, we would like to focus on recent progress in understanding of the regulation of a large part of enzymes of phase Ⅰ and phase Ⅱ biotransformation. The dependence of the pericentral expression of several cytochrome P450 isozymes on the activity of the Wnt/ β-catenin signalling was already noticed in 2006 by Hailfinger et al [56] and Sekine et al [40]. Obviously, the influence of β-catenin on cytochrome P450 expression is very complex including interactions with RAS-signalling [91] and signalling through the constitutive androstane receptor (CAR) [92]. Work on these subjects has excellently been reviewed by Braeuning et al [91,93]. Similarly, the zonal expression of certain phase Ⅱ enzymes such as glutathione-s-transferases is affected by Wnt/β-catenin signalling [94]. It will be a considerable challenge for future work to elucidate the influence of other morphogen pathways on the heterogeneous expression of drug metabolizing enzymes and drug transporters in the liver. Protein synthesis and autophagy Recently, a novel Forkhead box O (FOXO)-dependent mechanism for inducing autophagy via up-regulation of glutamine synthetase was discovered [95]. Since a FOXOand β-catenin-dependent regulation is found in liver only in a narrow zone of the lobules around the central veins [28], this mechanism is confined to about 7% of the hepatocytes indicating an extreme form of zonation. Though it is not known at present how autophagy is regulated in the remaining part of liver parenchyma, we have recently forwarded a hypothesis about a possible alternative mechanism based on characteristics derived from studies of the regulation of autophagy in whole liver [96]. The intriguing idea of this mechanism is that the extent of autophagy in liver might be determined by the extracellular concentration of glutamine (in the periportal and the proximal pericentral zone), while it is determined by the intracellular concentration of glutamine in the distal pericentral zone. Moreover, we have hypothesized that the assumed mechanism for the regulation of autophagy in the periportal zone may be controlled by Hh signalling which has just been found to play a role in regulating zonation [10]. Indeed, there is strong evidence from other organs for a close crosstalk of the mammalian target of rapamycin (mtor) and hedgehog pathways [97,98] that may be relevant also for the liver. Given the novel influence of liver hedgehog activity on the IGF-I regulatory network [70] and the known interrelationship between IGF signalling, FOXO activity and longevity [99,100], the question arises whether the extent of liver autophagy in total is harmonized through such a circuit with body size and the life-span of the organism. Besides these hints on zonation of autophagy, there is preliminary information from new proteome data in the Smo-KO mice [70] suggesting a prominent zonation of total protein biosynthesis (Gebhardt R, Matz-Soja M, and Shevchenco, unpublished observation). These findings generalize the long known fact that the synthesis of the main plasma protein, albumin, is zonated with a periportal predominance [6]. This dual scenario, zonation of both, autophagy and protein biosynthesis, leads to a number of important questions: (1) what happens in the liver after a long-lasting fasting period? How is the extraorbitant loss of liver protein reported under such conditions by the Lamers group [101] distributed among the different liver zones? (2) is the dominance of urea cycle, tricarboxylic-acid cycle, oxidative phosphorylation and, later on, of gluconeogenesis after fasting not only a results of the switch from 8497 July 14, 2014 Volume 20 Issue 26

190 Gebhardt R et al. Liver zonation: Regulation and impact insulin to glucagon signalling, but also the result of loss of bulk protein in pericentral hepatocytes? or (3) which other (pericentral) liver functions (e.g., heme or bile acid synthesis) may be affected by such changes and what is their impact on the entire organism? So far, it is obvious that our knowledge base for answering these questions with the necessary precision is not sufficient yet. Antioxidative mechanisms Mitochondria as well as peroxisomes are major sources of reactive oxygen species (ROS) [102] far exceeding that produced by the cytochrome P450 system of biotransformation. Protection of these organelles from damaging effects of ROS, especially of superoxide, is provided by superoxide dismutase 2 which generates H2O2, a potent oxidant. The removal of H2O2 is performed by the antioxidant enzymes catalase, GSH peroxidase 1 and peroxiredoxin-Ⅲ [103]. Zonation of ROS production and removal is determined by the zonal differences in mitochondrial structure and function, the pericentral predominance of peroxisomes, and zonal differences in GSH production and content (for review [6] ). The role of MnSOD in zonal gene expression is exemplified in the case of GS expression in MnSOD-KO mice, where the ordinary pericentral localization of this enzyme is lost and replaced by a scattered expression within the parenchyma [104]. Importantly in this context, the β-catenin pathway has been suggested to act as a switch between various transcription programs sensitive to hypoxia and oxidative stress [105]. Since most of these effects are mediated via hypoxia-inducing factor (HIF) and FOXO transcription factors, these interactions could affect the influence of the oxygen gradient and other stress factors across the porto-central axis on various zonated metabolic pathways described to be oxygen-dependent [73]. The fact that H2O2 is considered a key regulatory component in this concept appears in a new light given the possible zonation of the antioxidative mechanisms via peroxiredoxin which were not yet known at that time. The zonation of peroxiredoxins, thioredoxins, and glutaredoxins has not yet been studied, but it can be assumed that they predominate in the pericentral zone due to their association with both, mitochondria and peroxisomes. These mechanisms may be of critical importance in NAFLD, particularly in the development of NASH [106,107] and, because of their presumable zonation, may influence the precipitation of the first signs of this disease in the pericentral zone. Specific functions An entire synthetic pathway the zonation of which was described only recently is represented by heme biosynthesis [55,108]. An interesting characteristic of this pathway is that although it is present in all hepatocytes with a definitive pericentral dominance, the distribution of the individual enzyme proteins is not directly comparable. While it can be very broad for one enzyme (e.g., δ-aminolevulinate synthase), it may be much more restricted to the pericentral zone for another one (e.g., coproporphyrinogen oxidase). These variations suggest that this pathway is not simply a linear sequence of reactions taking place in each cell (although with different speed), but rather may frequently be diverted for exchanging certain metabolites with other organs in the body. This may result in various feedback mechanisms ensuring the proper adaptation of heme synthesis to whole body requirements. This exchange may be controlled by Wnt/ β-catenin signalling and ras signalling, since both pathways reciprocally influence heme biosynthesis [108]. In a recent study focussing on sexual dimorphism in the heterogeneity of gene expression in mouse liver several new zonated functions for either males or females were described by using LCM technology [11]. Among certain canonical pathways known to be predominantly localized to the pericentral zone, retinoic acid biosynthesis was newly described [11]. Most probably, however, this function which is related to vitamin A storage and metabolism must be assigned to the heterogeneity of hepatic stellate cells [109] rather than to that of hepatocytes. FUNCTIONAL SIGNIFICANCE OF ZONATION About 50 years ago when more and more examples of heterogeneous distributions of enzymes in liver parenchyma were discovered this phenomenon was still considered a curiosity by most biochemists and physiologists. Soon, however, it became apparent that such examples were not exemptions but the rule and are characteristic for many metabolic pathways. This was the time when the term metabolic zonation was coined by Jungermann and Sasse [4]. In this concept mainly based on findings concerning carbohydrate metabolism the spatial separation of anabolic and catabolic reactions was central, and the avoidance of futile cycles of ATP hydrolysis between key gluconeogenic and glycolytic reactions provided an easy explanation for this concept/separation. Another reason was that the simultaneous performance of opposite metabolic pathways in an organ might aid in quickly adapting to different metabolic needs by simply switching from one function to the opposite one without time- and energy-consuming biosynthesis of respective enzymes (for review see [6] ). Although metabolic zonation was realized to play a certain role in maintenance of body homeostasis, this connection received less attention. In the light of the recent discoveries mentioned above, two functional characteristics seem most important for understanding the power of metabolic zonation: (1) maintenance of metabolic homeostasis; and (2) optimization of liver function. Appropriate maintenance of metabolic homeostasis in the body probably implicates that every adaptation to exogenous nutritional challenge or any specific endogenous demand is reflected by a corresponding alteration of liver zonation, irrespective of whether it is almost negligible or very intensive. Of course, profound changes are immediately obvious and 8498 July 14, 2014 Volume 20 Issue 26

191 Gebhardt R et al. Liver zonation: Regulation and impact may be brought about by hormones and other signals required for regulating daily metabolism. The corresponding metabolic pathways appear dynamic. In contrast, the minor ones are probably due to slight changes in the signalling activity of the morphogen signalling network. The corresponding pathways appear static and need much longer time until visible changes can be detected. However, their consequences in the long run may be likewise dramatic, since under these conditions body homeostasis may gradually get lost. It is tempting to speculate that certain changes in liver zonation exerted by altered activities of one or more morphogen pathway(s) play a causative role in metabolic diseases. Examples for such a connection may be found in the association of certain polymorphisms in Wnt signalling with type 2 diabetes [ ]. The fundamental role of the optimization principle became obvious during a study on model-based optimization [113] within the framework of the Virtual Liver project ( The aims of this study were (1) to extend an existing 2-compartment model of ammonia metabolism (2-CM; representing the periportal and pericentral zones) to a 16-compartent model (16-CM; where each compartment represents an individual hepatocyte in a mouse liver lobule); and (2) to determine optimal enzyme activity distributions alongside the 16 compartments using a non-linear programming algorithm. The optimization problem is formulated based on biologically motivated enzyme constraints (limited capacity for protein synthesis) and objective functions that represent different physiological strategies of the liver. Using this approach, several characteristics of zonation of ammonia metabolism could be predicted that were in surprising accord with experimental findings [113]. These include a small periportal compartment for glutamine breakdown, a larger periportal down-stream compartment dedicated to ureogenesis without glutaminase support, and a very small pericentral compartment dedicated exclusively to glutamine synthesis. Evidence for the existence of a periportal down-stream region with a glutaminase-independent ureogenesis was provided by Comar et al [114] using a sophisticated liver perfusion system with cannulation of both, the portal vein and the hepatic artery. By generalizing these findings, zonation appears as a perfect means for optimizing the metabolic capacity of the liver. The regulatory systems determining zonated expression can be viewed as mere parts of sophisticated mechanisms transforming complex metabolic cues into the proper zonation. Future research using systems biology approaches should aim at identifying further examples of the optimization principle. EMERGING SUBJECTS AND MISSING RESEARCH AREAS As pointed out above, the fundamental role of zonation in the control of body homeostasis at the metabolic level which is emerging from novel regulatory mechanisms dominated by morphogen pathways is still far from being understood. However, it may bear a considerable potential for explaining metabolic adaptation to different nutritional states and requirements as well as the development of metabolic diseases. Adaptive processes may profit from the fact that metabolic zonation is associated with different gradients of transcription factors along the porto-central axis. Since combinatorial interactions among transcription factors are critical for directing tissue-, cell-type- and cell position-specific gene expression [115,116], independent regulation of the transcription factors by several signalling pathways creates the possibility to shift the expression of individual enzymes or transporters as well as that of whole pathways or of subgroups of them. How such combinatorial interactions may contribute to liver zonation was illustrated in a recent review for β-catenin and several other TFs coregulating pericentral GS expression [28]. Further interactions with β-catenin were described for the androgen receptor [117], many members of the steroid receptors [118], and for several other nuclear receptors such as hepatocyte nuclear factor 4 (HNF4) [119]. Even though HNF4 has been shown to be almost homogeneously expressed throughout the liver lobules [120], the large diversity of HNF4 variants at the protein level (Gaunitz, Gebhardt, unpublished observation) calls for careful interpretation of these results. Interestingly, conditional knockout of HNF4 in liver did not affect pericentrally expressed genes, but resulted in the additional expression of some of these genes in the periportal zone, albeit at a lower level [121]. Such shifts could result in the dynamic overlap of opposing metabolic pathways, e.g., of gluconeogenesis and glycolysis, resulting in more or less regulatory influence of metabolic intermediates and other metabolic (feedback) regulators on the activity of these pathways. But the contrary could also take place, as suggested by the almost static expression of GS: despite varying or shifting profiles of individual transcription factors, the invariability of one of them, here of β-catenin, may guarantee the exclusive expression of GS in some few hepatocytes around the central vein [9,28]. Under certain, possibly rare conditions, however, a third scenario may occur. The combinatorial game of transcription factors in zonation could lead to aberrant events of gene expression resulting in either gain of functions that are not characteristic of mature liver (e.g., selective gain of embryonic features) or loss of definitively liver-specific functions (e.g., partial dedifferentiation). All these events could contribute to the development of metabolic diseases. The reflections outlined above illustrate how important it will be to profoundly understand the regulatory network underlying metabolic zonation. In this context it needs to be pointed out that research on zonation with respect to two important features of metabolism, namely circadian rhythm and gender dimorphism is largely missing. It is known for a long time that liver metabolism underlies profound rhythmic changes, daily and seasonally [ ]. Because of lack of research in this field, these 8499 July 14, 2014 Volume 20 Issue 26

192 Gebhardt R et al. Liver zonation: Regulation and impact changes are not yet adequately mirrored at the level of metabolic zonation. Only occasionally respective data was reported. Long ago, for instance, daily alterations in glycogen metabolism were investigated [126] and a remarkable rhythmicity in time and zonal localization was found. Recently, we have observed that liver glutaminase protein shows a strong circadian rhythm being highest in the morning and lowest at night (Gebhardt, Kalkhoff, von Bergen, unpublished observation). This finding points to corresponding changes in the periportal expression zone of this enzyme and challenges the currently prevailing view that urea cycle activity is continuously fuelled (and mainly driven) by the prior breakdown of glutamine entering the liver via portal vein and hepatic artery [127]. Perhaps, there are periods where such fuelling is neither necessary, nor helpful for ensuring a balanced nitrogen metabolism. Likewise, gender dimorphism of liver metabolism is well studied globally [124, ], but not sufficiently comprehensive on the level of lobular heterogeneity. Some examples should be mentioned. It has long been discovered that the very small pericentral zone where glutamine synthetase is expressed is larger in males than in females of many mouse strains [132]. However, whether and how this difference impacts on amino acid and ammonia metabolism is completely unknown. Furthermore, the difference in the size of the expression zone of this enzyme seems to hint - according to the current molecular explanation for the limited range of the expression of this gene - at a higher pericentral activity of Wnt/ β-catenin signalling in males than in females. Although it was frequently emphasized that this may be explained by the molecular interaction between the androgen receptor and β-catenin, final prove of this obvious guess is still missing. Similar phenomena with sex differences were occasionally reported such as zonation of ApoE synthesis [82] or gender-specific interplay through Wnt/β-catenin signalling and constitutive androgen receptor in drug metabolism [92], but remained isolated. In case of Apo E synthesis, a clear periportal to pericentral gradient of mrna levels was observed in male rats, whereas a bowllike distribution with lowest expression in the midzonal area was found in female rats by in situ hybridization [82]. A considerable step forward was achieved by Saito et al [11], who used LCM combined with micro-arrays in order to compare gender effects on zonal gene expression. Interestingly, they found that sexual dimorphism seems more pronounced in zone 1 than in zone 3. CONCLUSION Applications of omics technologies, novel approaches for refined spatial localization, and the use of transgenic mice with liver-specific knockout of different morphogens have strongly improved our understanding of liver zonation. In particular, the emerging network of several morphogens that work as master regulators of zonation has provided new insight into the basic mechanisms that govern zonation. Instead of Wnt/β-catenin signalling acting alone, it is now certain that this pathway acts in close cooperation with Hedgehog and RAS signalling. Of course, many important questions remain unanswered so far and, because of the complexity of the crosstalk, a clear picture will require analysis by a thorough systems biology approach based on space- and time-dependent data. These should incorporate data on alterations of zonation during the daily rhythm of metabolism and should also include gender dimorphism. However, even with the first rough idea of this emerging regulatory network, it can be concluded that disturbance of liver zonation will considerably impact on body homeostasis, particularly in diseased states. 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196 Gebhardt R et al. Liver zonation: Regulation and impact Ilmenau: ISLE-Verl, 2010: Comar JF, Suzuki-Kemmelmeier F, Constantin J, Bracht A. Hepatic zonation of carbon and nitrogen fluxes derived from glutamine and ammonia transformations. J Biomed Sci 2010; 17: 1 [PMID: DOI: / ] 115 Fedorova E, Zink D. Nuclear architecture and gene regulation. Biochim Biophys Acta 2008; 1783: [PMID: DOI: /j.bbamcr ] 116 Ravasi T, Suzuki H, Cannistraci CV, Katayama S, Bajic VB, Tan K, Akalin A, Schmeier S, Kanamori-Katayama M, Bertin N, Carninci P, Daub CO, Forrest AR, Gough J, Grimmond S, Han JH, Hashimoto T, Hide W, Hofmann O, Kamburov A, Kaur M, Kawaji H, Kubosaki A, Lassmann T, van Nimwegen E, MacPherson CR, Ogawa C, Radovanovic A, Schwartz A, Teasdale RD, Tegnér J, Lenhard B, Teichmann SA, Arakawa T, Ninomiya N, Murakami K, Tagami M, Fukuda S, Imamura K, Kai C, Ishihara R, Kitazume Y, Kawai J, Hume DA, Ideker T, Hayashizaki Y. An atlas of combinatorial transcriptional regulation in mouse and man. Cell 2010; 140: [PMID: DOI: /j.cell ] 117 Truica CI, Byers S, Gelmann EP. Beta-catenin affects androgen receptor transcriptional activity and ligand specificity. Cancer Res 2000; 60: [PMID: ] 118 Mulholland DJ, Dedhar S, Coetzee GA, Nelson CC. Interaction of nuclear receptors with the Wnt/beta-catenin/Tcf signaling axis: Wnt you like to know? Endocr Rev 2005; 26: [PMID: DOI: /er ] 119 Colletti M, Cicchini C, Conigliaro A, Santangelo L, Alonzi T, Pasquini E, Tripodi M, Amicone L. Convergence of Wnt signaling on the HNF4alpha-driven transcription in controlling liver zonation. Gastroenterology 2009; 137: [PMID: DOI: /j.gastro ] 120 Lindros KO, Oinonen T, Issakainen J, Nagy P, Thorgeirsson SS. Zonal distribution of transcripts of four hepatic transcription factors in the mature rat liver. Cell Biol Toxicol 1997; 13: [PMID: ] 121 Stanulović VS, Kyrmizi I, Kruithof-de Julio M, Hoogenkamp M, Vermeulen JL, Ruijter JM, Talianidis I, Hakvoort TB, Lamers WH. Hepatic HNF4alpha deficiency induces periportal expression of glutamine synthetase and other pericentral enzymes. Hepatology 2007; 45: [PMID: DOI: /hep.21456] 122 Kornmann B, Schaad O, Bujard H, Takahashi JS, Schibler U. System-driven and oscillator-dependent circadian transcription in mice with a conditionally active liver clock. PLoS Biol 2007; 5: e34 [PMID: DOI: /journal. pbio ] 123 Ando H, Takamura T, Matsuzawa-Nagata N, Shima KR, Nakamura S, Kumazaki M, Kurita S, Misu H, Togawa N, Fukushima T, Fujimura A, Kaneko S. The hepatic circadian clock is preserved in a lipid-induced mouse model of non-alcoholic steatohepatitis. Biochem Biophys Res Commun 2009; 380: [PMID: DOI: /j.bbrc ] 124 Hirao J, Nishimura M, Arakawa S, Niino N, Mori K, Furukawa T, Sanbuissho A, Manabe S, Nishihara M, Mori Y. Sex and circadian modulatory effects on rat liver as assessed by transcriptome analyses. J Toxicol Sci 2011; 36: 9-22 [PMID: ] 125 Schmutz I, Albrecht U, Ripperger JA. The role of clock genes and rhythmicity in the liver. Mol Cell Endocrinol 2012; 349: [PMID: DOI: /j.mce ] 126 Sasse D. Dynamics of liver glycogen: the topochemistry of glycogen synthesis, glycogen content and glycogenolysis under the experimental conditions of glycogen accumulation and depletion. Histochemistry 1975; 45: [PMID: ] 127 Häussinger D. Regulation of hepatic ammonia metabolism: the intercellular glutamine cycle. Adv Enzyme Regul 1986; 25: [PMID: ] 128 Bur IM, Cohen-Solal AM, Carmignac D, Abecassis PY, Chauvet N, Martin AO, van der Horst GT, Robinson IC, Maurel P, Mollard P, Bonnefont X. The circadian clock components CRY1 and CRY2 are necessary to sustain sex dimorphism in mouse liver metabolism. J Biol Chem 2009; 284: [PMID: DOI: /jbc.M ] 129 Waxman DJ, Holloway MG. Sex differences in the expression of hepatic drug metabolizing enzymes. Mol Pharmacol 2009; 76: [PMID: DOI: / mol ] 130 Lorbek G, Perše M, Horvat S, Björkhem I, Rozman D. Sex differences in the hepatic cholesterol sensing mechanisms in mice. Molecules 2013; 18: [PMID: DOI: /molecules ] 131 Lu YF, Jin T, Xu Y, Zhang D, Wu Q, Zhang YK, Liu J. Sex differences in the circadian variation of cytochrome p450 genes and corresponding nuclear receptors in mouse liver. Chronobiol Int 2013; 30: [PMID: DOI: / ] 132 Sirma H, Williams GM, Gebhardt R. Strain- and sex-specific variations in hepatic glutamine synthetase activity and distribution in rats and mice. Liver 1996; 16: [PMID: ] P- Reviewers: Kim JS, Takahashi T, Takahashi JG, Zhang XL S- Editor: Qi Y L- Editor: A E- Editor: Liu XM 8504 July 14, 2014 Volume 20 Issue 26

197 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. Gastroenteric tube feeding: Techniques, problems and solutions REVIEW Irina Blumenstein, Yogesh M Shastri, Jürgen Stein Irina Blumenstein, Department of Gastroenterology and Clinical Nutrition, Johann Wolfgang Goethe University Clinic, Frankfurt, Germany Yogesh M Shastri, Jürgen Stein, Gastroenterology and Clinical Nutrition, Hospital Sachsenhausen, Frankfurt, Germany Jürgen Stein, Crohn Colitis Clinical Research Center Rhein- Main, Frankfurt, Germany Author contributions: All authors made substantial contributions to conception and design of the article and to acquisition, analysis and interpretation of data; Stein J drafted the manuscript; All authors reviewed the manuscript for important intellectual content and approved the final version for publication. Correspondence to: Jürgen Stein, MD, PhD, Crohn Colitis Clinical Research Center Rhein-Main, Schifferstr. 59, Frankfurt, Germany. j.stein@em.uni-frankfurt.de Telephone: Fax: Received: November 10, 2013 Revised: February 23, 2014 Accepted: April 15, 2014 Published online: July 14, 2014 Abstract Gastroenteric tube feeding plays a major role in the management of patients with poor voluntary intake, chronic neurological or mechanical dysphagia or gut dysfunction, and patients who are critically ill. However, despite the benefits and widespread use of enteral tube feeding, some patients experience complications. This review aims to discuss and compare current knowledge regarding the clinical application of enteral tube feeding, together with associated complications and special aspects. We conducted an extensive literature search on PubMed, Embase and Medline using index terms relating to enteral access, enteral feeding/ nutrition, tube feeding, percutaneous endoscopic gastrostomy/jejunostomy, endoscopic nasoenteric tube, nasogastric tube, and refeeding syndrome. The literature showed common routes of enteral access to include nasoenteral tube, gastrostomy and jejunostomy, while complications fall into four major categories: mechanical, e.g., tube blockage or removal; gastrointestinal, e.g., diarrhea; infectious e.g., aspiration pneumonia, tube site infection; and metabolic, e.g., refeeding syndrome, hyperglycemia. Although the type and frequency of complications arising from tube feeding vary considerably according to the chosen access route, gastrointestinal complications are without doubt the most common. Complications associated with enteral tube feeding can be reduced by careful observance of guidelines, including those related to food composition, administration rate, portion size, food temperature and patient supervision Baishideng Publishing Group Inc. All rights reserved. Key words: Enteral tube feeding; Percutaneous endoscopic gastrostomy; Refeeding syndrome; Enteral nutrition; Buried bumper syndrome; Nasoenteral tubes; Colocutaneous fistulae Core tip: Keeping up with new developments in the fast-moving field of enteral nutrition is a challenge for any gastroenterologist. While enteral tube feeding plays a major role in the care of critically ill patients and those with poor voluntary intake, chronic neurological or mechanical dysphagia or gut dysfunction, mechanical, gastrointestinal, infectious and metabolic complications can lead to serious conditions or death. We have undertaken a comprehensive review of current literature assessing the safety and effectiveness of various endoscopic, sonographic, radiologic, electromagnetic and fluoroscopic application techniques. In addition, we address prophylactic measures to prevent complications, problem solutions and special aspects. Blumenstein I, Shastri YM, Stein J. Gastroenteric tube feeding: Techniques, problems and solutions. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: org/ /wjg.v20.i July 14, 2014 Volume 20 Issue 26

198 Blumenstein I et al. Gastroenteric tube feeding INTRODUCTION Enteral nutritional support plays a very significant part in the management of patients with poor voluntary oral intake [1,2], chronic neurological or mechanical dysphagia [3-5], or intestinal failure [6,7], and in the critically ill [8,9]. Enteral feeding is not only more physiological than parenteral nutrition (PN), but has also been shown to improve patient outcomes, decrease costs [10] and reduce septic complications [11,12] in comparison to PN. In a meta-analysis of 82 randomized controlled trials (RCTs), PN was associated with a significantly increased risk of infectious complications, especially in patients receiving therapy for malignancy [13]. In patients who cannot be fed using routine bedside methods, endoscopically or radiologicallyaided methods must be employed to achieve enteral access. Complicated access situations require the additional expertise of a gastroenterologist, who is an integral part of the nutrition support team [14]. While the range and ease of enteral access procedures has been greatly enhanced in recent years by the introduction of new techniques and improved equipment and accessories, many studies have nonetheless demonstrated a high prevalence of tube- and/or feeding-related complications in patients receiving long-term enteral nutrition [15-19]. The purpose of this review is to provide an overview of current knowledge and practice in the rapidly changing and developing field of endoscopic enteral tube feeding (ETF), covering routes of access as well as problems associated with enteral feeding and their solutions. In principle, tube systems for gastric or jejunal nutrition can be placed by nasal insertion (nasoenteral tubes; NETs), guided percutaneous application, or surgical techniques. Nasal tubes are mainly used for short-term enteral feeding (4-6 wk) and in situations where other methods of enteral feeding are contraindicated. In the long term, however, NETs are often poorly tolerated by the conscious patient, since they not only elicit a foreign body sensation in the pharynx, but may also cause reflux esophagitis and pressure ulcers, and have a tendency to dislocate. NETs can also be a source of psychological stress to the patient, the presence of the tube being a visible sign of his/her illness. Enteral feeding via transnasal tubes is often poorly tolerated by geriatric patients with an acute confusional state, and the need for repeated insertion of tubes following voluntary removal by the patient is demanding and time-consuming for nursing staff. Nasal tubes are not suitable for patients who are to undergo orofacial therapy for potentially reversible dysphagia (in most cases due to cerebrovascular accident). Furthermore, the presence of a nasal tube significantly interferes with swallowing training [20]. Techniques for achieving NET placement include unguided bedside insertion or placement under fluoroscopic, endoscopic, electromagnetic or direct surgical guidance. Depending on experience, the success rate of endoscopic transnasal and transoral NET feeding tube placement has been described to range from 86% to 97% [21]. Blind insertion, the most common technique for nasoenteral intubation, results in malposition in 0.5%-16% of cases, with tracheal, pulmonary, or pleural malposition in 0.3%-15%. This may result in pulmonary or pleural formula infusion, pneumothorax or pulmonary abscess [22]. A study by de Aguilar-Nascimento and Kudsk [23] demonstrated that of 932 blind postpyloric tube placement attempts, 433 (46%) failed and 20 (1.6%) were airway misplacements. Air instillation and auscultation are inaccurate methods for validating the position, and misplacement is often not suspected unless a radiograph is obtained [24,25]. After gastric positioning, spontaneous or prokinetically-assisted transpyloric tube migration occurs only in 5%-15% of patients, compared to 14%-60% using guided assistance. Duodenal intubation can successfully be achieved in 70%-93% of patients using right lateral positioning, gastric insufflation, tube tip angulation, and clockwise rotation during insertion. However, such tube placements require experience and an average of min to perform [26]. In a recent study of 616 patients in an intensive care unit (ICU), Rivera et al [27] demonstrated that the use of an electromagnetic tube placement device (ETPD) to monitor tip position of the feeding tube resulted in no adverse events, reduced costs, and earlier initiation of EN. Failure of bedside NET placement is an indication for the use of fluoroscopy or endoscopy. Enteral intubation under fluoroscopic guidance is successful in 90% of cases, achieving jejunal positioning in 53%, but requires on average 22 min of fluoroscopy room time [28]. While endoscopically-guided (ENET) nasoenteral access techniques have been in use since 1984 [29], ENET placement can be very tedious and unrewarding for the endoscopist when compared to other procedures such as polypectomies, PEG or endoscopic retrograde cholangiopancreatography (ERCP). Most endoscopy training programs do not adequately teach techniques for ENET placement. However, a lack of perseverance on the part of the endoscopist may result in unnecessary administration of total parenteral nutrition (TPN) in patients who are in fact suited for enteral feeding. The positioning of feeding tubes distal of the ligament of Treitz may cause some uncertainty even amongst experienced endoscopists [30]. A new, recently-described nasoendoscopical placement method shows promise as a more successful technique for tube placement beyond the duodenal flexure [31-33]. One in ten patients experiences procedure-related complications following NET [34], either at the time of insertion or subsequently. Reported complications of nasal tube feeding include nasopharyngeal lesions, sinusitis, aspiration, diarrhea, intestinal ischemia and metabolic derangements. Aspiration is reported in up to 89% of patients, with no clear advantage of nasoenteric over gastroenteric feeding. Distal duodenal or jejunal feeding may prevent regurgitation of feeding formula [24]. About 2%-5% of patients may present with epistaxis following ENET placement [35,36], a complication shown to be equally common in patients undergoing transnasal vs 8506 July 14, 2014 Volume 20 Issue 26

199 Blumenstein I et al. Gastroenteric tube feeding Table 1 Tube-related complications of enteral tube feeding [203] Mechanical complications Infectious complications Metabolic complications transoral endoscopy for ENET placement [37]. Tube-related complications (Table 1) depend not only on the route of enteral access, but also on the material and diameter of the feeding tube. A critical review of pulmonary complications associated with the blind placement of narrow-bore nasoenteric tubes (NETs) was recently performed by Sparks and colleagues [38]. Of the 9931 NET placements reviewed, a total of 187 were improper tube placements in the tracheobronchial tree, translating to a 1.9% mean overall malposition rate. These 187 misplacements included 35 (18.7%) reported pneumothoraces, at least 5 of which resulted in patient death. NET malpositioning was reported in 13%-32% of subsequent repositioning attempts [38]. Occlusion of the NET is an underestimated and underreported complication of ETF. It has been reported to occur in 9%-35% of patients [36,39,40], but actual incidence is much higher. The most important underlying cause is ignorance of tube feeding care among nursing staff. The following technical factors predispose to tube occlusion: inadequate irrigation with water, especially after feed or medicine administration; instillation of medications, particularly crushed tablets; narrow lumen; long tubes (for further details, see occlusion of the PEG tube, below). TECHNICAL ASPECTS OF PERCUTANEOUS FEEDING TUBE PLACEMENT Tube obstruction Primary malposition Perforation of the intestinal tract Secondary displacement of the feeding tube Knotting of the tube Accidental tube removal Breakage and leakage of the tube Leakage and bleeding from insertion site Erosion, ulceration and necrosis of skin and mucosa Intestinal obstruction (ileus) Hemorrhage Inadvertent Ⅳ infusion of enteral diet Infection at the tube insertion site Aspiration pneumonia Nasopharyngeal and ear infections Peritonitis Infective diarrhea Electrolyte disturbances Hyper- and hypoglycemia Vitamin and trace element deficiency Tube feeding syndrome ( Refeeding syndrome ) Percutaneous endoscopic gastrostomy Percutaneous endoscopic gastrostomy (PEG) is indicated for patients requiring long-term nutritional support (> 30 d) who have a functional gastrointestinal (GI) tract but insufficient oral intake of nutrients. The most common indications include inadequate swallowing as a result of a neurological event, oropharyngeal or esophageal cancer, and severe facial trauma [41,42]. Based on data from 1327 patients, Kurien et al [43] demonstrated in a recently-published trial that patients who underwent gastrostomy had significantly lower mortality compared to those who deferred the procedure. PEG techniques In principle, there are three techniques for PEG tube placement; the peroral pull technique, the peroral push technique and the direct percutaneous procedure. The most widely-used technique for PEG placement is the pull method introduced by Gauderer et al [44] in 1980, which has replaced surgical gastrostomy as the mediumand long-term solution to enteral nutrition delivery [45], being safer and more cost-effective, with lower procedure-related mortality (0.5%-2%) and lower complication rates [46-48]. Furthermore, tube displacement occurs less frequently than with nasogastric tubes (NGT). The Sack-Vine push variant (placing a catheter over a Seldinger wire) yields comparable results [49]. Alternative procedures such as sonographically-controlled PEG are not yet sufficiently technically developed to be adopted on a wide scale [50]. The introducer PEG, using a balloon catheter placed transabdominally into the stomach, was described by Russell et al [51]. The main problem initially associated with this technique was deflection of the stomach wall due to the puncture, combined with the risk of catheter misplacement. However, its safety has since been improved by the use of an intragastrically-positioned T-fastener under fluoroscopic or endoscopic control to fix the stomach to the abdominal wall [52,53]. A new, safe introducer method (Freka Pexact ) has recently become available for patients in whom the standard pull PEG cannot be used or would involve an increased risk during passage of the internal bumper. Its main advantage is the combination of a double gastropexy with a peel-away sheath introducer to effect secure fixation of the stomach wall, analogous to surgical gastropexy [54,55]. The success rate of PEG tube placement is as high as 99.5% (range 76%-100%). Reasons for failure include inadequate transillumination, complete oropharyngeal or esophageal obstruction, and gastric resections [21]. The average life span of PEG tubes has been described to be one to two years, with tube degradation being the most common reason for tube replacement [21]. Jejunal tubes through the PEG and direct percutaneous endoscopic jejunostomy Long-term jejunal feeding can be achieved endoscopically with jejunal tubes through the PEG (JET-PEG) and direct percutaneous endoscopic jejunostomy (DPEJ). Jejunal feeding is appropriate in patients with recurrent vomiting and/or tube feeding-related aspiration, severe gastroesophageal reflux, gastroparesis, gastric outlet ob July 14, 2014 Volume 20 Issue 26

200 Blumenstein I et al. Gastroenteric tube feeding struction, or total or partial gastrectomy [56]. Though not definitively proven to reduce tube feeding-related aspiration (see below)], the combination of gastric decompression via PEG and simultaneous jejunal nutrition shows clinical benefit in many patients. JET-PEG placement can be carried out by pushing a jejunal feeding tube through the previously-placed PEG using a beneath the scope (BTS) or over the wire tube technique. Initial positioning of the tube beyond the ligament of Treitz is essential to reduce the retrograde migration rate. Although feeding tube placement beyond the ligament of Treitz may be considered a technical success, its functional success is largely disappointing because of frequent retrograde tube migration into the stomach and tube dysfunction caused by kinking or obstruction (as the diameter of the jejunal tube is restricted to 12 F). Furthermore, JET-PEG has not been demonstrated to effectively decrease enterorespiratory aspiration compared with PEG tube feeding alone [57,58]. In combined gastric decompression/feeding jejunostomy tubes, the small diameter of the tube often provides inadequate gastric venting and may cause jejunal lumen clogging. JET-PEG tubes have a high success rate of up to 93%. The mean functional duration of the tubes was found to be 55 d in adults and 39 d in children [21]. Retrograde dislodgment of the jejunal extension tube, tube obstruction and mechanical failure have been described as the most common device-related complications [21]. Endoscopically-placed clips may secure the tube and prevent retrograde migration [59], but this does not overcome the problem of occlusion common to smallcaliber tubes. DPEJ, a modification of the pull PEG technique, appears to be the ideal procedure for long-term jejunal feeding [60]. A colonoscope or an enteroscope is introduced orally into the small bowel, and transillumination is observed on the anterior abdominal wall as an indicator of the scope s position in the jejunum. A trocar is passed through the anterior abdominal wall directly into the jejunum. As a sounding device for needle puncture, a 21-gauge 1.5 inch needle used for infiltration of local anesthetic may be used. The puncture should be performed with a purposeful, swift stabbing motion of the wrist. Grasping the tip of the sounding needle with a forceps helps to stabilize the jejunal segment and allows proper orientation for insertion of the larger trocar/needle assembly alongside the indwelling sounding needle. A 120-inch insertion wire is advanced through the cannula and grasped by the awaiting forceps or snare, and the procedure is completed as described for a pull-type PEG placement. Though similar to PEG placement, DPEJ is a considerably more difficult technique [60,61]. There are three retrospective studies on DPEJ outcome involving a total of 230 patients from a cancer center, a surgical unit, and a gastroenterological referral center. Successful placement was achieved in 72%-88% [62]. Skin-level gastrostomy Skin-level gastrostomy was introduced to reduce skin irritation, minimize granulation tissue and improve the patients quality of life. It provides easy and convenient access for enteral nutrition and is well established in pediatric patients [63]. The most popular system is the button skin-level nonrefluxing device, first described by Foutch et al [64]. Currently, three button types with two different retaining elements (retention dome and balloon type) are available [65]. Indications for this device are usually peristomal problems and/or the patient s wish to be independent from the PEG tube. Contraindications are active peristomal infection, a stoma existing less than four weeks after primary PEG insertion, a fistulous stoma channel, and a stomal tract longer than 4.5 cm. Initial application of the button should be carried out under endoscopic control or under fluoroscopic guidance via a guide wire to prevent misplacement and to allow removal of the previously-placed gastrostomy catheter [56]. Despite the potential advantages of a skin-level device, the largest prospective multicenter evaluation of a single-step button (86 patients) reported serious placement problems and a high complication rate [66]. Initiation of feeding There are various techniques for administering feeds in ETF patients, the most common of which are summarized below (Table 2). The recommendation to delay feeding initiation until h after PEG or transabdominal gastrostomy placement was based on a presumption that the GI system would return to normal function during this period of time, allowing a better seal of the enteral opening [46,67]. More recently, however, several prospective randomized studies have clearly demonstrated that, at least in the case of PEG placement, much earlier initiation of feeding after as little as 1-3 h is equally safe [68-71]. This was confirmed in a meta-analysis by Bechtold et al [72]. COMPLICATIONS ASSOCIATED WITH PEG PLACEMENT: INCIDENCE AND MANAGEMENT About 13%-40% of patients with PEG placement experience minor complications such as maceration due to leakage of gastric contents around the tube, and peristomal pain [46,47,73,74]. Serious complications requiring further intervention have been reported in 0.4%-4.4% of procedures and include peristomal leakage with peritonitis, necrotizing fasciitis of the anterior abdominal wall, gastric bleeding, injury to internal organs, tumor seeding at the PEG site, and death. The 30-d incidence of mortality after PEG has been reported to be in the range of 6.7%-26% [46,47,73]. This high value is due rather to the underlying comorbid factors of these patients than to the 8508 July 14, 2014 Volume 20 Issue 26

201 Blumenstein I et al. Gastroenteric tube feeding Table 2 Techniques for delivery of feeds in enteral tube feeding Method of feeding Indication Comments Bolus intermittent (by syringe or bulb) Cyclic intermittent (by gravity or pump) Ambulatory patients Partially recumbent ml over 5-10 min multiple times, high risk of aspiration and diarrhea, cheap and convenient for NGT Higher infusion rate for a shorter period (8-16 h); while changing from tube feeds to oral Intermittent drip Home enteral feeding L over 8-12 h overnight, no daytime feeds (by gravity or pump) Constant infusion (by gravity or pump) Bedridden patients ICU patients Initiate with ml/h, altered periodically depending on gastric residual volume, increased chances of aspiration and metabolic abnormalities; incline head end of bed to 45 to reduce aspiration and regurgitation NGT: Nasogastric tube; ICU: Intensive care unit. procedure itself [21,75,76]. Peristomal wound infections Incidence and causes: Peristomal wound infections are the most common complication associated with the PEG procedure, with an incidence ranging from 4%-30% [77]. About three quarters of these are minor and resolve when treated with antibiotics. While regular skin and stomal care are crucial for the prevention of local infection, bandaging techniques also play an important role: In a comparative study, Chung et al [78] showed that excessive traction on the gastrostomy tube significantly increases the rate of local infection. Factors predisposing to infection can be (1) technique-related, such as a narrow incision or lack of antibiotic prophylaxis; (2) host factors, e.g., malnutrition, obesity, diabetes, malignancy, drug therapy (immunosuppressive medication, chronic corticosteroid therapy); and (3) nursing care-related, such as improper wound dressing or excessive traction between the internal bumper and the stomach wall. Prevention and treatment: Two recently-published metaanalyses involving 1100 patients from 10 randomized controlled trials assessing the efficacy of antibiotic prophylaxis in PEG insertion have demonstrated antibiotic prophylaxis to effect a relative risk reduction of 64% and an absolute risk reduction of 15%. Either a first-generation cephalosporin or a penicillin-based prophylaxis should be selected [79,80]. During the last decade, methicillin-resistant Staphylococcus aureus (MRSA) has emerged as an important cause of PEG-related wound infection. Horiuchi et al [81] suggested that, in addition to standard prophylactic antibiotics, MRSA decolonization using oral and nasallydelivered preparations might reduce the risk of MRSArelated peristomal infections in these patients. A recently-published randomized controlled study demonstrated that the use of glycerin hydrogel (GHG) dressings significantly decreases the number of peristomal infections, and that the frequency of dressing changes can be safely extended to 7 d during the first week, making it a less labor-intensive and more cost-effective option for post- PEG wound management. In the case of transabdominal gastrostomy, where direct transabdominal access of the gastrostomy tube avoids its exposure to oropharyngeal flora, another recent randomized controlled trial demonstrated no statistically significant difference in the rate of peristomal infection with or without preprocedural administration of antibiotics [55]. Clogged feeding tubes Incidence and causes: The incidence of clogged feeding tubes in PEG is reported to be as high as 23%-35%. Clogging is especially common when thick enteral feeds, bulking agents and medications are delivered through relatively small PEG tubes (i.e., 9 F). Tube occlusion is classified as either obstruction of the internal lumen or mechanical tube failure [39,82]. Prevention and treatment: Since ph values below 4 have been described to promote protein coagulation, repeated gastric residual aspiration should be avoided or minimized [83]. Tubes should also be flushed with ml water before and after delivering medications or bulking agents (i.e., psyllium, resins). If possible, all medications should be completely dissolved in water prior to flushing or applied as liquid formulations [84]. Saline should be avoided, since it can crystallize within the tube and promote gradual clogging [85]. Pancreatic enzymes mixed with bicarbonate have been reported to prevent tube clogging effectively [39,82]. Furthermore, they were found superior to carbonated beverages in dissolving clogs [86]. In a recent systematic review, water flushes have been shown to be the most effective method of preventing enteral feeding tube clogging [87]. Finally, clogged tubes can be cleared mechanically using various endoscopic catheters, braided quid wires, or special declogging plastic brush devices [25,88]. Peristomal leakage Incidence and causes: Although its reported incidence appears low (1%-2%), peristomal leakage is in fact a much more common complication [85,88,89]. Several factors contributing to the risk of peristomal leakage have been identified, including excessive cleansing with hydroperoxide, infections, gastric hypersecretion and excessive side torsion along the PEG tube, as well as patient July 14, 2014 Volume 20 Issue 26

202 Blumenstein I et al. Gastroenteric tube feeding specific factors that inhibit wound healing (malnutrition, immunodeficiency, diabetes). Furthermore, the risk of peristomal leakage is increased if the tube is not stabilized by means of an external polster [25,85]. Prevention and treatment: Prevention of peristomal leakage must focus on the reduction of risk factors (e.g., antisecretory therapy with PPIs), while barrier creams containing zinc and skin protectants are also recommended [85]. If these fail, the PEG tube can be removed. After waiting 4-6 d to allow the tract to partially close, a new PEG can then be placed through the same tract [85,90]. However, this procedure should only be attempted if sufficient time has passed to ensure scarring of the stomach to the abdominal wall [85,90]. If this is not the case, the PEG tube must be removed and a new PEG tube placed at a different site. On no account should the original PEG tube be replaced by a larger diameter tube, as this may cause enlargement of the tract, resulting in exacerbation of the leakage [85,90]. Conversion of the PEG to a PEG/double-lumen system has been reported to be a successful alternative [25,88], while conversion to a gastrojejunal (GJ) tube can also be considered for feeding distal to the stomach. Bleeding Incidence and causes: Acute bleeding is not uncommon after PEG tube placement, with a reported incidence of up to 2.5% [46,91,92]. The most common causes of acute bleeding immediately following PEG tube placement are local vessel injury at skin level, and mucosal tear in the upper GI tract. Risk factors include previous anatomic alteration, anticoagulation, and antiplatelet therapy [41]. In a recent single-center study of 990 patients, Richter et al [93] recently demonstrated the use of serotonin reuptake inhibitors (SRIs) during the 24 h before PEG placement to be a risk factor for increased bleeding. In this cohort, however, no association with aspirin or clopidogrel intake (at any dose) either before or after the procedure was found. The influence of SRI intake on bleeding risk is of particular significance in geriatric patients, since these drugs are administered to a large proportion of this patient population. Delayed bleeding can be caused by esophagitis, gastritis, gastric erosion, gastric or duodenal ulceration, and the buried bumper syndrome [94]. One study has demonstrated that esophagitis was the cause of delayed GI bleeding in 39% of PEG patients undergoing endoscopy [95]. Prevention and treatment: Current American Society for Gastrointestinal Endoscopy (ASGE) guidelines recommend the continuation of aspirin and non-steroidal anti-inflammatory drugs (NSAIDs), especially in high-risk patients, and the discontinuation of Clopidogrel therapy in low-risk patients, and where appropriate also in individuals with a higher risk of bleeding [92]. Discontinuation of warfarin therapy should also be considered, with the application of unfractionated heparin as bridging therapy [92]. Furthermore, in the light of their aforementioned study, Richter et al [93] recommend the interruption of SRI intake 24 h before the procedure. Colonic fistulae Incidence, types and causes: Colonic misplacement of the PEG tube may lead to serious complications, in particular the development of gastrocolic, colocutaneous or gastrocolocutaneous fistulae [96,97]. While these fistulae are rare and cited mostly only as isolated case-reports, Friedmann et al [97] describe 28 cases of colocutaneous fistula resulting from colonic malpositioning of PEG, including 12 cases of gastrocolocutaneous fistula and one of jejunocolocutaneous fistula. In contrast to the gastrocolic fistula, a fistulous passage connecting the stomach with the colon, the gastrocolocutaneous fistula is defined as an epithelial connection between the mucosa of the stomach, the colon, and the skin. Its probable etiology is the penetration of a bowel loop (mostly transverse colon) interposed between the stomach and the abdominal wall, either by inadvertent puncture during tube placement or, more commonly, due to gradual erosion of the tube into the adjacent bowel [98]. Factors predisposing to its occurrence in acute settings are insufficient gastric insufflation, past history of laparotomy causing adhesions and consecutive trapping of bowel loops, and improper transillumination. Up to 45% of colocutaneous fistulae are seen in patients with prior abdominal surgery [99]. These patients should undergo contrast study to rule out an overlap of stomach and colon. Prevention and treatment: To prevent colonic misplacement, the transillumination of the gastroscope through the abdominal wall, and the endoscopically visible imprint of a finger (or needle) on the skin is still a conditio sine qua non before introduction of the needle into the stomach [100]. In the absence of appropriate transillumination, the use of ultrasound and/or computed tomography is recommended to exclude abnormal abdominal anatomy [101], with slow advancement of a small-gauge anesthetic needle (e.g., the needle used to infiltrate local anesthetic into the PEG site) through the abdominal wall into the stomach, aspirating on the attached syringe. A safe tract is established by endoscopic visualization of the needle in the gastric lumen and simultaneous return of air into the syringe. Return of fluid or gas into the syringe before the needle is endoscopically visualized within the gastric lumen suggests entry into bowel interposed between the abdominal wall and the stomach [85,97,102]. The most common clinical symptoms associated with fistulae are watery diarrhea containing feed, or the presence of stool around the PEG tube. Rarely, fistulae present acutely with peritonitis, infection, fasciitis, or failure of the formula infusion [97]. Fistulae are diagnosed using contrast agent given via the PEG tube. Several approaches have been suggested for fistula management, ranging from conservative re July 14, 2014 Volume 20 Issue 26

203 Blumenstein I et al. Gastroenteric tube feeding moval of the PEG tubes without laparotomy, hereby allowing the fistula tract to close spontaneously within a few days, to invasive exploration of the colon. Colonic clipping has been reported by Kim et al [103]. While challenging the necessity for such a sophisticated endoscopic intervention in this case (after only 10 d of conservative treatment), Gyökeres et al [104] suggest that the method may be considered an option for the closure of persistent gastrocolic fistulae associated with PEG placement. Buried bumper syndrome Incidence and causes: Buried bumper syndrome (BBS) is a rare, mainly long-term complication of PEG, in which the internal bolster migrates from the gastric lumen and lodges in the gastric wall (incomplete BBS) or anywhere along the GI tract outside the gastric lumen (complete BBS) [ ]. It has a reported prevalence of 1. 5%-8.8% [74,88, ]. Though normally a late complication (usually presenting not until at least four months post- PEG), BBS has also been reported to occur as early as 21 d after PEG placement [89,94,112]. Common symptoms include immobilization of the PEG tube, feeding difficulties or the need for more pressure when giving feeds, peritubular leakage, complete occlusion of the tube, and the occurrence of abdominal pain. The main causative factor of BBS is excessive traction between internal and external bumper. Other possible contributing factors include malnutrition, poor wound healing, weight gain due to PEG feeding, and a stiff internal bumper (polyurethane). Diagnosis is mainly clinical but requires endoscopy for confirmation, and may reveal anything from simple ulceration and mucosal overgrowth around the internal bumper to complete outward erosion of the tube with non-visualization of the internal bumper. Prevention and treatment: To prevent BBS, it is advisable to allow an additional 1.5 cm of space between the external bumper of the PEG tube and the skin in order to minimize the risk of pressure-induced necrosis. Mucosal overgrowth of the inner bumper can be prevented by mobilizing and loosening the PEG from the outside at least every other day. The incidence of BBS is lower in patients with PEG tubes made from Foleys urinary catheter-type silicone tube and in patients with balloonassisted PEG-introducer devices than in routine PEG patients and those with balloon-tipped replacement tubes (probably because fluid inside the balloon can regulate the pressure more effectively [113] ). Even if it is asymptomatic, buried bumper must be removed once diagnosed, as continued bumper migration may lead to bleeding, perforation, peritonitis and death. Various techniques can be employed to remove the buried device and either reaccess the luminal tract with a new tube or secure an entirely new access site: The needle-knife technique can be used in cases of partial or superficial burial [110,114]. Alternatively, the buried tube can be pulled out and simultaneously replaced with a new pull-type feeding tube following insertion of a guide wire through the old tube [109]. Müller-Gerbes et al [115] describe a minimally-invasive technique (push method) where the inner bumper is cut by means of a papillotome brought into the stomach from the outside through the shortened PEG while under constant endoscopic control. Last but not least, it has been reported that the buried tube may be safely removed simply by external traction [111,113,116]. In cases of deep impaction or migration into the abdominal wall, surgical intervention in the form of laparotomy or a laparoscopic approach is required [111,117]. Pneumoperitoneum Pneumoperitoneum (PNP) following PEG or JET-PEG placement is a known finding occurring after 8%-18% [ ] of procedures. PNP usually has a benign and self-resolving course which does not warrant any further intervention. However, the recent reported higher incidence of complications requiring intervention in ICU patients with post- PEG PNP suggests the need for more intensive investigation of patients with this finding [119]. Liver injury Hepatic injury during PEG placement (e.g., transhepatic PEG placement) occurs infrequently, but is a potentially life-threatening and probably underdiagnosed complication [ ]. Unexplained pain after PEG placement in the absence of wound infection should always raise the suspicion of liver injury. If hepatomegaly is suspected or improper transillumination is present at the puncture site, transabdominal ultrasound must be performed. In addition, the tube insertion site should generally be chosen left of the upper abdominal midline and combined with the safe tract technique (see above). Abdominal wall metastasis at the PEG site Abdominal wall metastasis as a late complication at the PEG site has been reported with an incidence of < 1% [126,127]. Although it is a rare complication, the malignant seeding of tumor cells is associated with an extremely poor prognosis [126]. Risk factors for abdominal wall metastasis include primary pharyngoesophageal cancer, squamous cell histology, less differentiated and large-sized cancers, and an advanced cancer stage [128]. Since direct mechanical tumor implantation is the most likely mechanism, the pull-string or direct-introducer technique for PEG placement is preferable [55]. If these techniques are not available, an overtube should be used [129] or the PEG placement should be performed after surgical removal of the primary cancer [ ]. GASTROINTESTINAL COMPLICATIONS The most common complications observed with ETF involve GI function [31, ]. These complications and their possible causes and solutions are listed in Table July 14, 2014 Volume 20 Issue 26

204 Blumenstein I et al. Gastroenteric tube feeding Table 3 Gastrointestinal complications of enteral nutrition; causes, prevention and treatment Complication Cause Prevention/treatment Diarrhea Too rapid increase in amount of feed per day Observe adaptation phase Too rapid infusion rate Reduce/control infusion rate Feed temperature too cold Increase to room temperature Hyperosmolar feedings (> 300 mosm) Use isotonic feeding solution, initially dilute hyperosmolar feeding solutions Lactose intolerance Use low-lactose or lactose-free diet Fat malabsorption Use low-fat or MCT-containing diet Hypoalbuminemia Use chemically defined diet and/or feed Antibiotic therapy or medications Review medications Chemotherapy/radiotherapy Prescribe antidiarrheal medications Nausea/vomiting Too rapid infusion rate Reduce/control infusion rate Bacterial contamination of formula feed/delivery equipment contamination Handle administration systems hygienically, change delivery equipment every 24 h, keep opened bottles of formula no more than 24 h in refrigerator Cramps/bloating Too rapid infusion rate Reduce/control infusion rate Lactose intolerance Use low-lactose or lactose-free diet Fat malabsorption Use low-fat or MCT-containing diet Regurgitation/aspiration Gastric retention Reduce/control infusion rate, use duodenal tubes, incline patient during food administration Constipation Inadequate fluid intake Increase fluid intake, check fluid balance Fiber intake too low Use fiber-containing formulas Fecal impaction Enemas Electrolyte and hormonal derangement Osmotic laxatives (lactulose ml), peristaltic agents (e.g., prostigmine mg iv) MCT: Medium-chain triglyceride. Nausea occurs in 10%-20% of patients, while abdominal bloating and cramps from delayed gastric emptying are also common. Additional complications include aspiration and nonocclusive bowel necrosis, which are associated with high mortality [130,133]. In a multicenter observational study of 400 patients, Montejo et al [130] found that 251 patients (63%) experienced one or more GI complications during their feeding course. In a subsequent study, the same group evaluated the incidence of GI complications in gastric- and jejunally-fed patients, and found it to be 57% and 24%, respectively [130,134]. Diarrhea Incidence and causes: Diarrhea is the most commonly reported GI side effect in patients receiving ETF. Depending on definition [135], diarrhea occurs in up to 30% of patients in medical and surgical wards and more than 80% of patients in the intensive care unit (ICU) [ ]. The pathogenesis of diarrhea in enterally-fed patients is multi-factorial. Of those factors unrelated to the enteral formula or administration method, the use of antibiotics and/or specific medications is the most common reason for the development of diarrhea [136,139,140]. Diarrhea may be caused either by the medication itself (e.g., oral magnesium or phosphate supplements, antacids, prokinetic agents), or by the formulation in which it is delivered. Medications containing sorbitol can cause diarrhea due to osmotic effects, while antibiotics alter the intestinal flora, favoring the growth of Clostridium difficile (C. difficile), E. coli and Klebsiellae. Thus, courses of antibiotic treatment should be kept as short as possible and the use of prophylactic antibiotics limited. Pseudomembranous colitis is a complication observed with increasing frequency, especially during antibiotic exposure (for reviews see [136,141] ). Patients receiving ETF are nine times more likely to develop C. difficile-associated diarrhea than matched non tube-fed patients [142]. Antibiotics can also reduce colonic bacterial production of short chain fatty acids from insoluble carbohydrates and fiber [143]. Hypoalbuminemia (serum albumin level < 2.5 g/dl) has long been implicated as a cause of diarrhea as a result of intestinal edema. However, studies aiming to confirm this have yielded conflicting results [144]. Prevention and treatment: The early identification of diarrhea risk factors and the development of a diarrhea risk management algorithm are recommended. Over the last two decades, several RCTs investigating the use of fibers in the treatment and prevention of ETF-associated diarrhea have been carried out [136]. Results from a metaanalysis suggest fiber to be highly effective in reducing the incidence of diarrhea in patients at increased risk (e.g., postsurgical, critically ill patients) [145]. However, mixed results have been reported regarding the use of different types of fiber (insoluble or soluble) to prevent ETF-induced diarrhea. Schultz et al [146] demonstrated the relative effectiveness of pectin combined with an insoluble fiber formula compared to an insoluble fiber formula alone. The usefulness of soluble fibers for the treatment of diarrhea during enteral nutrition has been demonstrated only in two small studies [147,148]. Partially hydrolyzed guar gum (PHGG) has been shown to reduce the incidence of ETF-induced diarrhea compared with standard, fiber-free formula in both general ward and ICU settings [147,149]. In contrast, supplementation with inulins or fructooligosaccharide (FOS) has been shown to increase 8512 July 14, 2014 Volume 20 Issue 26

205 Blumenstein I et al. Gastroenteric tube feeding Table 4 Randomized controlled trials measuring the impact of probiotics on enteral nutrition-related diarrhea Ref. Study population Treatment groups Sample size (placebo) Heimburger et al [204] Adults starting EN Lactobacillus acidophilus and L. bulgaricus Alberda et al [205] Frohmader et al [206] Ferrie et al [207] Barraud et al [208] Bleichner et al [209] Adults starting EN on ICU Adults starting EN on ICU Adults with diarrhea during EN on ICU Adults starting EN on ICU Adults starting EN on ICU Daily dose Probiotics 41 (23) 3000 CFU/d 31% developed diarrhea Outcome Controls 11% developed diarrhea VSL#3 - live cells 10/9 (9) mg/d 14%/12% 1 of days 23% of days with diarrhea with diarrhea VSL#3 45 (25) mg/d 0.5 liquid stools/d 1.1 liquid stools/d L. rhamnosus GG 36 (18) ( cells/d) and inulin (560 mg/d) 3.8 d duration of diarrhea Ergyphilus 167 (80) ( CFU/d 55% developed diarrhea Saccharomyces boulardii Schlotterer et al [210] Burnt adults Saccharomyces boulardii Tempe et al [211] Adults in ICU Saccharomyces boulardii 128 (64) CFU/d 7.7% of days with diarrhea 18 (9) CFU/d 1.5% of days with diarrhea 40 (20) CFU/d 8.7% of days with diarrhea 2.6 d duration of diarrhea 53% developed diarrhea 9.1% of days with diarrhea 14% of days with diarrhea 16.9% of days with diarrhea 1 Viable probiotics/probiotic sonicates. VSL#3 consists of Lactobacillus casei, Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus delbrueckii, Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium infantis, and Streptococcus salivarius. Ergyphilus consists of mainly Lactobacillus rhamnosus GG, Lactobacillus casei, Lactobacillus acidophilus, and Bifidobacterium bifidum. flatulence and bowel movement frequency in enterally fed patients [150,151]. The fiber consensus panel of the European Society for Parenteral and Enteral Nutrition (ESPEN) recommends supplementing ETF with PHGG to prevent ENinduced diarrhea in both ICU and postsurgical patients (grade A recommendation) [152]. Guidelines of the American Society for Parenteral and Enteral Nutrition (AS- PEN) recommend the use of PHGG in ICU patients (grade D recommendation). However, insoluble fibers should be avoided in all ICU patients (grade C recommendation), as their use may increase the risk of bowel obstruction in the critically ill [9]. A recent systematic review suggests that the use of different fiber mixtures may be the most promising strategy for the prevention of ETF-induced diarrhea [145]. Despite the potential for manipulation of the intestinal microflora in ETF, few studies have investigated the effects of prebiotics or fermentable carbohydrates on the incidence of ETF-associated diarrhea [136,153,154]. To date, eight RCTs have assessed the impact of probiotics in the prevention of ETF-associated diarrhea (Table 4). Of these, five were shown to be beneficial (for review, see [136,155] ). The safety of probiotics in critically ill and/ or immunocompromised patients has, however, been called into question following an RCT which reported increased mortality in a group of patients with severe acute pancreatitis who took probiotics [156]. Whelan and Myers performed a systematic review focused on adverse events related to probiotics in patients receiving enteral nutrition [157]. Only 3 of 53 trials showed increased complications, which were largely non-infectious in nature and occurred in specific patient groups (e.g., transplant and pancreatitis). The total of 50 trials (> 4000 patients) showed either no effect or a positive effect on outcomes related to safety (e.g., mortality and infections). Thus, in spite of contrasting evidence, it may be concluded that the use of probiotics in enteral formula is a useful tool in preventing ETF-associated diarrhea, but should not be used in transplant patients or the critically ill. A systematic approach to the management of diarrhea in ETF patients is depicted in Figure 1. Constipation Constipation is less common than diarrhea during ETF, and more prevalent in patients requiring long-term ETF. The primary goal of constipation management in these patients is prevention. Despite the theoretical rationale for fiber supplementation of enteral formulae, there are still no convincing data demonstrating the benefits of fiber supplementation in terms of improved bowel function or prevention of constipation. A meta-analysis from seven RCTs in the acute setting (two ICUs, three surgical and two medical wards) showed that fiber supplementation effected at least a downwards trend in the percentage of patients reporting constipation. Furthermore, a significant reduction in laxative use in patients receiving fiber formula was observed in four studies [145]. In spite of the lack of sufficient data, fiber formulae should therefore be used at least in patients requiring long-term ETF. pulmonary complications Pneumonia is a potentially life-threatening complication which is usually a consequence of pulmonary aspiration of oral secretions or, less commonly, of gastric and small-bowel contents. It may occur with no obvious evidence of vomiting. Pulmonary aspiration is more common when patients are fed with NGT in a supine 8513 July 14, 2014 Volume 20 Issue 26

206 Blumenstein I et al. Gastroenteric tube feeding 1 Provide adequate fluids to maintain hydration and electrolyte balance (1) Provide soluble fiber 2 Reduce fluid and electrolyte losses (2) Change to continuous duodenal infusion (3) Reduce rate of infusion 3 Determine etiology Enteric pathogen or inflammation or disease process? Yes No Treat accordingly Pharmacological Enteric pathogen Disease/inflammation If possible, change offending medication C. difficile Salmonella Shigella Campylobacter Yersinia E. coli Malabsorption syndromes Diabetes Pancreatic insufficiency Bile salt malabsorption Fecal impaction Antibiotics Sorbitol-containing medications H2 receptor blockers Lactulose/laxatives Magnesium-containing antacids Potassium and phosphorus supplements Antineoplastics Quinidine Diarrhea continues Antimotility medication Loperamide HCl or diphenoxylate HCI, atropine sulfate codeine, paregoric, deodorized tincture of opium Treatment worked Treatment failed Gradually increase the tube feeding rate to goal as diarrhea resolves Change to peptide-based or elemental enteral formula Treatment worked Treatment failed Increase rate as tolerated to goal Discontinue tube feeding and provide PN until diarrhea resolves deliver 0.2 L normal saline via feeding tube Figure 1 Management of diarrhea in enteral tube-fed patients. Adapted from reference [212]. position [25, ] and is caused by a combination of gravitational back-flow, impairment of the lower esophageal sphincter (LES), lack of swallow-induced LES relaxation, infrequent esophageal body contractions, and the presence of the tube across the gastric cardia [161]. It is very common in patients with impaired consciousness or poor gag reflexes, occurring in up to 30% of those with tracheotomies [162] and 12.5% of neurology patients [163] July 14, 2014 Volume 20 Issue 26

207 Blumenstein I et al. Gastroenteric tube feeding Table 5 Patients at high risk of refeeding syndrome Patients with anorexia Patients with chronic alcoholism Oncology patients Postoperative patients Elderly patients (comorbidities, decreased physiological reserves) Patients with uncontrolled diabetes mellitus (electrolyte depletion, diuresis) Patients with chronic malnutrition: Marasmus Prolonged fasting or low energy diet Morbid obesity with profound weight loss High stress unfed for > 7 d Malabsorptive syndromes (inflammatory bowel disease, cystic fibrosis, short bowel syndrome) Adapted from reference [188]. However, while feeding via PEG may reduce the risk of aspiration, it will not eliminate it altogether [164], especially in patients in whom GI motility disturbances inhibit this route of feeding [158,165]. Since the incidence of reflux depends on the position of the tip of the feeding tube (6% with the tube tip in the duodenum, 4% near the ligament of Treitz, and 0.4% distal to the ligament of Treitz), several published guidelines favor small bowel over gastric feeding in patients who are at risk for aspiration. This applies in particular to critically ill patients, in whom gastroduodenal atony, caused by increased intracranial pressure, hyperglycemia and stress, can lead to delayed or impaired gastric emptying and decreased transpyloric transport of nutrients (for extended review see Waseem et al [161,166] ). However, the results of more than 10 controlled clinical trials [134, ] are contrary and inconclusive, particularly with regard to nasoenteral tube feeding [164,168]. Meta-analyses of these studies have also presented conflicting views [164,176,177]. Since it has been shown that even in patients with normal intestinal motility both naso- and gastrojejunal tubes, once correctly positioned, will be moved in a proximal direction by the phase Ⅱ migrating motor complex, placement of enteral nutrition tubes 40 cm distal to the ligament of Treitz is considered the optimal method [165,168]. METABOLIC COMPLICATIONS Artificial feeding may cause a variety of metabolic problems including deficiency or excess of fluids, electrolytes, vitamins and trace elements. Overhydration occurs frequently, particularly when ETF patients are also receiving supplementary intravenous nutrition or fluids. Furthermore, underlying metabolic diseases including diabetes mellitus and renal or hepatic insufficiency must be taken into account while administering ETF. Refeeding syndrome Refeeding syndrome (RFS) was first described in malnourished Far East prisoners of war after the Second World War who developed cardiac and neurological symptoms soon after starting eating [178,179], and remains an often forgotten condition. RFS is characterized by electrolyte depletion, fluid shifts and glucose derangements that occur upon oral, enteral or parenteral reinstitution of nutrition in malnourished patients [180]. Incidence: The true incidence of RFS is still not known. The majority of reported cases are prospective and retrospective cohort studies or case series. Patients with anorexia represent the prototypical population of RFS [181], and a recently-published multicenter study from France reported an incidence of 10% in these patients [182]. Other common conditions associated with RFS include hyperemesis, alcoholism, cancer, and malabsorptive syndromes such as short bowel syndrome, inflammatory bowel disease, cystic fibrosis, and various forms of bariatric surgery [181]. Pathophysiology: The pathophysiology of RFS remains poorly understood. It occurs because the body adapts to undernutrition by down-regulating membrane pumping in order to conserve energy. This, in turn, causes leakage of intracellular potassium, magnesium, calcium and phosphate, with subsequent whole-body depletion. Simultaneously, sodium and water also leak into the cells. Sudden refeeding reverses these processes and, along with insulin, drives electrolytes into the cells, potentially leading to a precipitous fall in circulating levels of the aforementioned electrolytes. This may be accompanied by an acute increase in circulating and extracellular fluid (exogenous administration or endogenous movement of sodium and water out of the cells). To further aggravate the situation, undernourished kidneys have a limited capability to handle salt and water load. Specific micronutrient deficiencies can compound these problems. Hypophosphatemia is the hallmark of RFS, and is responsible for significant morbidity and even mortality [180]. It can manifest as clinical features of RFS, e.g., rhabdomyolysis, leukocyte dysfunction, respiratory failure, cardiac failure, hypotension, arrhythmia, seizure, coma and sudden death [183]. In adult patients, refeeding hypophosphatemia is more common in enteral than parenteral feeding. This may be due to the incretin effect from absorption of glucose [184]. Cardiac arrest has been reported as a complication of RFS in patients presenting with less than 70% of prior body weight. Prolonged starvation results in a reduction of total cardiac volume, end diastolic volume, and left ventricular mass. During RFS, ventricular volume returns to normal while left ventricular mass remains reduced, leading to fluid retention and congestive cardiac failure. In addition, hypophosphatemia may lead to decreased sarcomere contractility and cause myocardial damage [185,186]. Prevention and treatment: Awareness of RFS and identification of patients at risk are the first steps in pre July 14, 2014 Volume 20 Issue 26

208 Blumenstein I et al. Gastroenteric tube feeding Table 6 Therapy and prevention of refeeding syndrome Careful evaluation of cardiovascular system, check for any electrolyte abnormalities before initiating refeeding In severe cases, an initial starting volume of 50%-75% of daily requirements should be used < 7 yr old: kcal/kg bw/d 7-10 yr: 75 kcal/kg bw/d yr: 60 kcal/kg bw/d yr: 50 kcal/kg bw/d > 18 yr: 25 kcal/kg bw/d (or an average 1000 kcal/d initially) If the initial food challenge is tolerated, caloric intake may be increased over the next 3-5 d. Each requirement should be tailored to the individual s needs, and the above values may need to be adjusted by as much as 30%. Frequent administration of small feeds is recommended. Feeds should provide a minimum of 1 kcal/ml to minimize volume overload Protein Initial regimen for malnourished patients: g/kg bw/d The feed should be rich in essential amino acids, and should gradually be increased, as an intake of g/kg bw/d is needed for anabolism to occur Vitamins/trace elements Thiamine, folic acid, riboflavin, ascorbic acid and pyridoxine should be supplemented, as well as the fat-soluble vitamins A, D, E, and K 300 mg thiamine should be given Ⅳ at least 30 min. before refeeding is initiated, and should be continued with 100 mg iv for at least 7 d. Later on, oral thiamine can be supplemented as 100 mg tablets Iron should be supplemented iv according to the Ganzoni formula {iron deficit (mg) = bw (kg) [(target Hb - actual Hb (g/l )] depot iron (500 mg)} Minerals Sodium should be restricted (about 1 mmol/kg bw/ or 1.5 g/d), but liberal amounts of phosphorus, potassium and magnesium should be given to patients with normal renal function Magnesium (normal range: mmol/l ) Mild to moderate hypomagnesemia ( mmol/l ) Initially 0.5 mmol/kg bw/d over 24 h iv, then 0.25 mmol/kg bw/d for 5 d iv Maintenance requirement 0.2 mmol/kg bw per day iv or 0.4 mmol/kg bw per day orally Phosphate (normal range: mmol/l) Mild hypophosphatemia ( mmol/l) mmol/kg bw per day orally Moderate hypophosphatemia ( mmol) mmol/kg bw per day orally Severe hypophosphatemia (< 0.3 mmol/l ) iv supplementation with either potassium phosphate or sodium phosphate (e.g., 0.8 mmol/kg bw monobasic potassium phosphate in half-normal saline by continuous infusion over 8-12 h) Plasma phosphate, calcium, magnesium and potassium should be monitored, and the infusion should be stopped once plasma phosphate concentration exceeds 0.30 mmol/l Adapted from references [185,191]. venting refeeding problems [187]. Any patient with no or negligible food intake for more than five days is at risk of developing refeeding problems. High-risk patients include the chronically undernourished and those who have diminished physiological reserves and/or are critically ill [188]. Table 5 summarizes criteria from the National Institute for Health and Clinical Excellence (NICE) for the identification of patients at high risk of RFS. Recommendations for therapy and prevention are shown in Table 6. Despite the lack of high level (level A/B) recommendations, overall consensus favors the gradual introduction and advancement of feeding over several days while closely monitoring electrolytes and - to a lesser extent - vitamins and trace elements. Serum phosphate, magnesium, calcium, potassium, urea, and creatinine concentrations should be measured before feeding and repeated daily during the first week after feeding is started. Caloric intake should generally start with approximately 1000 kcal or kcal/kg (25%-50% of estimated requirements) daily, particularly during the first week of refeeding, and be increased by approximately 20% daily until the determined goal is reached. The average weekly weight gain, particularly in extremely undernourished patients (e.g., anorexia nervosa) should not exceed 0.5 kg/wk [189,190]. If hypophosphatemia occurs (< 0.50 mmol/l), it should be corrected with 50 mmol intravenous phosphate over 24 h [185,186,191,192]. In the case of mild to moderate hypomagnesemia ( mmol/l), 1 g magnesium should be given every 6 h intravenously. Should severe, clinically symptomatic hypomagnesemia occur, 8-12 g magnesium must be given daily in divided doses and serum magnesium monitored every 8-12 h [181,193]. Few data are available regarding optimal vitamin and trace element supplementation. Some authors recommend 300 mg of thiamine (parenteral or enteral) before starting refeeding and 100 mg daily thereafter. In the presence of Wernicke s encephalopathy, even higher doses of thiamine ( mg) may be warranted [193]. In order to avoid fluid overload, fluid repletion should be carefully controlled. Some authors have recommended initial fluid and sodium restriction to prevent congestive heart failure [189] July 14, 2014 Volume 20 Issue 26

209 Blumenstein I et al. Gastroenteric tube feeding ROLE OF NUTRITION SUPPORT TEAMS Although still limited in number, studies of team vs nonteam enteral management provide clear evidence for the positive effects of an organized multidisciplinary approach using protocols and recommendations based on published guidelines [194]. A study in a community hospital showed that management of ETF patients by a nutrition support team (NST) was associated with reductions in mortality rate, length of hospital stay and readmission rate [195]. In a prospective study comparing team vs non-team management of ETF patients at a Veterans Administration Medical Center, Powers and colleagues demonstrated that the former reduced complications and improved patients nutrition status [196]. This finding was confirmed at a university teaching hospital [197]. In addition, cost-utility analyses of patients receiving ETF either as hospitalized patients or as outpatients have demonstrated a significant lowering of healthcare costs through the reduction of metabolic and mechanical complications [ ]. Organization and tasks of nutrition support teams A well-organized NST should include a physician, a nurse, a nutritionist and a pharmacist [202]. The goal of the NST is to provide high quality nutritional care. This is accomplished through (1) identification of patients who are at risk nutritionally; (2) performance of a comprehensive nutritional assessment to serve as a guide to nutritional therapy; and (3) provision of safe and effective nutritional support. To accomplish these goals, the NST should offer services including inpatient consultations, quality assurance protocols, research programs, home nutrition services, and last but not least, staff educational programs. Proper training of staff on the correct use of nutritional support is crucial, especially with regard to the reduction of complications. CONCLUSION Endoscopic methods in particular have facilitated a variety of enteral feeding access options. These should be tried in all patients who are unable to ingest adequate amounts of food but have adequate absorptive capacity of the intestine. Well-trained endoscopists who are extremely proficient in these techniques are indispensable for the successful provision of hospital nutrition support and care. Optimal techniques for tube placement, together with the prompt recognition and immediate management of complications, can significantly reduce overall morbidity and mortality due to ETF-associated complications. To further promote positive patient outcomes and reduce the incidence of complications, ETF should always be managed by multidisciplinary nutrition support teams. 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216 Blumenstein I et al. Gastroenteric tube feeding 208 Barraud D, Blard C, Hein F, Marçon O, Cravoisy A, Nace L, Alla F, Bollaert PE, Gibot S. Probiotics in the critically ill patient: a double blind, randomized, placebo-controlled trial. Intensive Care Med 2010; 36: [PMID: DOI: /s ] 209 Bleichner G, Bléhaut H, Mentec H, Moyse D. Saccharomyces boulardii prevents diarrhea in critically ill tube-fed patients. A multicenter, randomized, double-blind placebocontrolled trial. Intensive Care Med 1997; 23: [PMID: DOI: /s ] 210 Schlotterer M, Bernasconi P, Lebreton F. Value of Saccharomyces boulardii in the digestive acceptability of continuousflow enteral nutrition in burnt patients. Nutr Clin Metabol 1987; 1: [DOI: /S (87)80016-X] 211 Tempé JD, Steidel AL, Blehaut H, Hasselmann M, Lutun P, Maurier F. [Prevention of diarrhea administering Saccharomyces boulardii during continuous enteral feeding]. Sem Hop 1983; 59: [PMID: ] 212 Fuhrman MP. Diarrhea and Tube Feeding. Nutr Clin Pract 1999; 14: [DOI: / ] P- Reviewers: Campo SMA, Crary MA S- Editor: Zhai HH L- Editor: A E- Editor: Liu XM 8524 July 14, 2014 Volume 20 Issue 26

217 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. ORIGINAL ARTICLE High fat diet feeding results in gender specific steatohepatitis and inflammasome activation Michal Ganz, Timea Csak, Gyongyi Szabo Michal Ganz, Timea Csak, Gyongyi Szabo, Department of Medicine, University of Massachusetts Medical School, Worcester, MA 01605, United States Author contributions: All the authors contributed to studying concept and design, acquisition, analysis and interpretation of data; Ganz M and Szabo G contributed to drafting of the manuscript; Csak T and Szabo G contributed to critical revision of the manuscript for important intellectual content; Szabo G contributed to obtained funding, study supervision. Supported by National Institutes of Health Grant, No. DK Correspondence to: Gyongyi Szabo, MD, PhD, Department of Medicine, University of Massachusetts Medical School, 364 Plantation St, LRB-208, Worcester, MA 01605, United States. gyongyi.szabo@umassmed.edu Telephone: Fax: Received: December 18, 2012 Revised: April 11, 2013 Accepted: June 1, 2013 Published online: July 14, 2014 Abstract AIM: To develop an animal model that encompasses the different facets of non-alcoholic steatohepatitis (NASH), which has been a challenge. METHODS: In this study, we used a high fat diet (HFD) feeding supplemented with fructose and sucrose in the water mimicking the high-fructose corn syrup that is abundant in the diet in the United States. We used C57Bl/6 wild-type mice for short and long-term feedings of 6 and 16 wk respectively, and evaluated the extent of liver damage, steatosis, and inflammasome activation. Our methods included histopathological analysis to assess liver damage and steatosis, which involved H and E and oil-red-o staining; biochemical studies to look at ALT and triglyceride levels; RNA analysis using quantitative polymerase chain reaction; and cytokine analysis, which included the enzyme-linked immunosorbent assay method to look at interleukin (IL)-1β and tumor necrosis factor-α (TNFα) levels. Furthermore, at each length of feeding we also looked at insulin resistance and glucose tolerance using insulin tolerance tests (ITT) and glucose tolerance tests. RESULTS: There was no insulin resistance, steatosis, or inflammasome activation at 6 wk. In contrast, at 16 wk we found significant insulin resistance demonstrated by impaired glucose and ITT in male, but not female mice. In males, elevated alanine aminotransferase and triglyceride levels, indicated liver damage and steatosis, respectively. Increased liver TNFα and monocyte chemoattractant protein-1 mrna and protein, correlated with steatohepatitis. The inflammasome components, adaptor molecule, Aim2, and NOD-like receptor 4, increased at the mrna level, and functional inflammasome activation was indicated by increased caspase-1 activity and IL-1β protein levels in male mice fed a long-term HFD. Male mice on HFD had increased α-smooth muscle actin and pro-collagen-1 mrna indicating evolving fibrosis. In contrast, female mice displayed only elevated triglyceride levels, steatosis, and no fibrosis. CONCLUSION: Our data indicate gender differences in NASH. Male mice fed a long-term HFD display steatohepatitis and inflammasome activation, whereas female mice have steatosis without inflammation Baishideng Publishing Group Inc. All rights reserved. Key words: Gender differences; Non-alcoholic steatohepatitis; Inflammasome; High fat diet Core tip: Our work shows that there are gender differences in the development of non-alcoholic steatohepatitis. We used a high fat diet feeding supplemented with fructose and sucrose in mice, to mimic the highfructose corn syrup that is abundant in the western diet. There is preferential steatohepatitis and inflammasome activation in male mice, whereas female mice display steatosis without inflammation July 14, 2014 Volume 20 Issue 26

218 Ganz M et al. Gender differences in NASH Ganz M, Csak T, Szabo G. High fat diet feeding results in gender specific steatohepatitis and inflammasome activation. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: INTRODUCTION Non-alcoholic fatty liver disease (NAFLD) affects approximately one third of the Western population and 75% of obese individuals [1,2]. It has become the most common cause of elevated liver enzymes in the Western world and will continue to be on the rise with the obesity epidemic [3-6]. The spectrum of NAFLD ranges from simple steatosis, which consists of intracellular lipid accumulation resulting in a fatty liver, to non-alcoholic steatohepatitis (NASH), which is characterized by the presence of inflammatory cells and necroinflammation, and fibrosis that develops in 20% of patients with NASH. These patients can progress to cirrhosis and hepatocellular carcinoma [1,7-9]. Factors determining the progression of NAFLD and strategies for effective treatment remain elusive. NASH is two to three times more common in men than women until after the age of 60, after which the prevalence is higher in women [10]. In animal models, there has been a lack of consensus as to the role of gender on the development of NAFLD [11-13]. The progression of NAFLD is centered around inflammation, and inflammasomes are major contributors to this process. Inflammasomes are intracellular multiprotein complexes that sense endogenous and exogenous danger signals through NOD-like receptors (NLRs) [14-16]. Components of intracellular inflammasome complexes are the sensors (Aim2, NLRP3 or NLRC4), adaptor molecule (ASC), and pro-caspase-1 [14,17]. Inflammasome activation results in cleavage of pro-caspase-1 to activated caspase-1, which promotes cleavage and activation of pro-inflammatory cytokines [pro-interleukin-1β (pro-il- 1β) and pro-il-18] resulting in a sustained inflammatory response [18,19]. It has been shown that the methioninecholine deficient (MCD) diet, induces significant upregulation of the NACHT, LRR, and PYD domainscontaining protein 3 (NALP3) inflammasome, and that a 9-mo HFD feeding can also cause NALP3 activation [20]. In this study, we performed a HFD feeding for a short-term (6-wk) and long-term (16-wk) duration that included both male and female mice. Our data demonstrates that steatohepatitis and inflammasome upregulation and activation are observed in male mice, but not female mice, after a 16-wk HFD feeding. MATERIALS AND METHODS Animal studies This study was approved by the Institutional Animal Care and Use Committee of the University of Massachusetts Medical School. Principles of laboratory animal care were followed. Male and Female C57Bl/6 wild-type mice that were 8-10 wk old (n = 10 per group) (Jackson Laboratory) were fed either a control chow diet or a high-fat, high-carbohydrate diet [Surwit diet (58% kcal % fat); Research Diets, New Brunswick, NJ, United States] with drinking water supplemented with a high-fructose corn syrup equivalent consisting of a total of 42 g/l of carbohydrates at a ratio of 55% fructose (Acros Organics, Morris Plains, NJ, United States) and 45% sucrose (Sigma-Aldrich, St. Louis, MO, United States) by weight, based on the study by Kohli et al [21]. The mice were given ad libitum access to the food for 6 or 16 wk. Biochemical analysis and cytokine measurements Serum alanine aminotransferase levels were measured using a kinetic method (D-TEK, Bensalem, PA, United States), and liver triglyceride levels were determined using an L-type triglyceride H kit (Wako Chemicals United States, Inc., Richmond, VA, United States). Mouse IL- 1β enzyme-linked immunosorbent assay (ELISA) kit was purchased from R and D Systems (Minneapolis, MN). Mouse TNF-α kit was purchased from BD Bioscience (San Jose, CA, United States), monocyte chemoattractant protein 1 (MCP-1) ELISA kit was purchased from Biolegend (San Diego, CA, United States). Histopathological analysis Sections of formalin-fixed livers were stained with hematoxylin-eosin. Frozen samples were also stained with Oil-Red-O that were made at the optimal cutting temperature. Slides were analyzed using microscopy using Image J and Microsuite (Olympus Soft Imaging Solutions GmbH, Munster, Germany). Glucose tolerance tests Glucose levels were measured after 14 wk feeding following an overnight fast. Mice were injected IP with 40% glucose (Sigma, St. Louis, MO, United States) at 2 mg/kg body weight and glucose levels were obtained at 0, 30, 75, 120 and 180 min. Blood samples were obtained by tail nick and glucose levels were measured using a GlucCell glucose monitor and strips. A subset of mice had blood collected by facial vein puncture at time point 0 and 30 min for serum insulin measurement using a mouse enzyme-linked immunosorbent assay kit (ALPCO, Salem, NH, United States). Insulin tolerance tests After 15 wk of feeding and after an overnight fast, mice were injected IP with 0.75 U/kg body weight of recombinant human insulin (Novolin, Novo Nordisk, Bagsvaerd, Denmark). Glucose levels were measured in the same way as in the glucose tolerance tests (GTT) at time points 0, 30, 75 and 120 min following insulin injection. RNA analysis RNA was purified using a RNeasy kit (Qiagen Sciences, Germantown, MD, United States) with the on-column 8526 July 14, 2014 Volume 20 Issue 26

219 Ganz M et al. Gender differences in NASH A Weight-short-term feeding B Weight-long-term feeding Weight (g) b Male-chow Female-chow Male-HFD Female-HFD Weight (g) b b b b b b b Male-chow Female-chow Male-high fat Female-high fat t (wk) t (wk) Figure 1 Effect of high fat diet feeding on body weight. Male and female C57Bl/6 mice were fed with either a high fat diet (HFD) supplemented with sucrose and fructose in the drinking water, or with a control chow diet for 6 or 16 wk. Body weights were recorded during both the short-term (6 wk, A), and long-term (16 wk, B) feedings. b P < 0.01 vs chow fed controls, n = 10/group. DNA digestion (Qiagen Sciences, Germantown, MD, United States). Complementary DNA was transcribed using a reverse-transcription system (Promega Corp., Madison, WI, United States). Real-time quantitative polymerase chain reaction (qpcr) was performed with an icycler (Bio-Rad Laboratories, Inc., Hercules, CA, United States) using SYBR Green as the detector of doublestarted DNA, as previously described [22]. Caspase activity assay Caspase-1 activity was determined using freshly prepared whole liver lysates with a colorimetric assay. The analysis was based on the cleavage of the WEHD-pNA (Trp- Glu-His-Asp-p-nitroanilide) substrate (R and D Systems, Minneapolis, MN, United States). Statistical analysis Statistical significance was determined using the nonparametric Kruskal-Wallis test and the Mann-Whitney test when appropriate. Data are presented as means and standard errors and are considered statistically significant for P RESULTS Long-term HFD results in increased body weight and insulin resistance Male mice fed a short-term 6-wk HFD gained significantly more weight than chow-fed controls. Female mice did not gain significant weight on the HFD compared to chow fed mice at 6 wk (Figure 1A). In contrast, at 16 wk, both male and female mice gained significantly more weight on a HFD than chow fed mice (Figure 1B). GTT were performed on a subset of mice one week before being euthanized. There was no difference in glucose levels following glucose injection at any time point in male or female mice after a short-term HFD (data not shown). In mice fed a long-term HFD, males showed significantly higher glucose levels 180 min after glucose injection than those on a chow diet (Figure 2A). Female mice on a long-term HFD diet showed no significant difference in glucose levels at any time point following glucose injection compared to controls (Figure 2B). Insulin tolerance test was also performed one week prior to euthanasia. In the short-term feedings, male mice had significantly increased glucose levels compared to chow fed mice at the 120-min time point (Figure 2C), whereas female mice had no difference in glucose levels between the groups at any time point (Figure 2D). In the group that received a long-term HFD, males had significantly higher glucose levels at all time points following insulin injection compared to chow fed males (Figure 2E), whereas females had no difference in glucose levels compared to chow fed controls (Figure 2F). Serum insulin levels before and 30 min after glucose injection showed no significant difference in either gender after a 6-wk HFD feeding compared to chow fed controls (Figure 3A and B). In contrast, insulin levels were elevated in long-term HFD male mice 30 min after glucose injection compared to chow fed controls (Figure 3C). No change in insulin levels were detected in female mice fed the long-term HFD (Figure 3D). Long-term HFD feeding results in steatohepatitis in male mice, but only steatosis in female mice To assess liver steatosis and inflammation, liver sections were evaluated histologically using hematoxylin and eosin and Oil-Red-O staining. Neither gender showed changes in histology after a short-term (6-wk) HFD feeding compared to chow diet (data not shown). However, both male and female mice undergoing a long-term HFD feeding had significantly more micro and macrovesicular steatosis than chow fed controls (Figure 4A and B). Steatosis was further evaluated by triglyceride levels in the liver. Both male and female mice had significantly elevated liver triglyceride levels compared to chow fed mice at 16 wk (Figure 4C). However, male mice on a long-term HFD had higher triglyceride levels than female mice on the same diet (Figure 4C). Liver damage was assessed by alanine aminotrans July 14, 2014 Volume 20 Issue 26

220 Ganz M et al. Gender differences in NASH A GTT-males, 16 wk Chow High fat B GTT-females, 16 wk Chow High fat Glucose (mg/dl) a Glucose (mg/dl) Minutes after glucose stimulation Minutes after glucose stimulation C 200 ITT-males, 6 wk Chow High fat b D 200 ITT-females, 6 wk Chow High fat Glucose (mg/dl) Glucose (mg/dl) Minutes after insulin stimulation Minutes after insulin stimulation E Glucose (mg/dl) b ITT-males, 16 wk b b Chow High fat F Glucose (mg/dl) ITT-females, 16 wk Chow High fat Minutes after insulin stimulation Minutes after insulin stimulation Figure 2 High fat diet results in insulin resistance in male mice after 16 wk high fat diet feeding. Male and female mice were fed with either high fat diet (HFD) or chow diet for 6 or 16 wk. Glucose tolerance test (GTT) was performed: glucose levels were measured 0, 30, 75, 120 and 180 min after ip glucose injection in male (A) and female mice (B) fed with HFD for 16 wk. Insulin tolerance test (ITT) was performed: glucose levels were measured 0, 30, 75 and 120 min after ip insulin injection in male and female mice fed with HFD for 6 wk (C and D, respectively) or 16 wk (E and F, respectively). a P < 0.05, b P < 0.01 vs chow fed controls, n = 5/group, P = 0.03 vs chow fed controls in GTT-males, 16 wk, n = 5/group. ferase (ALT) levels. No change in ALT levels in either gender was noted after a short-term HFD feeding (data not shown). Following a 16-wk HFD, male mice showed significantly higher levels of ALT than chow fed controls, whereas no significant change in ALT was noted in the long-term female HFD group (Figure 4D). Next, we evaluated the presence of inflammation in the liver. There was no change in protein levels of tumor necrosis factor-α (TNFα) in the livers of either male or female mice in the short-term HFD group (Figure 5A). In the livers of long-term HFD males, TNFα mrna levels were increased approximately 4-fold, whereas no significant change was noted in the females (Figure 5B). A similar trend was seen in TNFα protein levels in the liver, with a 2-fold increase in the long-term HFD males, and no increase in the long-term HFD females (Figure 5C). MCP-1 is a chemokine that contributes to steatosis and inflammatory cell infiltration. In a pattern similar to TNFα, no change in levels of MCP-1 protein was seen in mice fed a short-term HFD compared to chow-fed 8528 July 14, 2014 Volume 20 Issue 26

221 Ganz M et al. Gender differences in NASH A Plasma insulin (ng/ml) Insulin-males, 6 wk Before After B Plasma insulin (ng/ml) Insulin-females, 6 wk Before After 0.0 Chow High fat 0.0 Chow High fat Minutes after glucose stimulation Minutes after glucose stimulation C Plasma insulin (ng/ml) Insulin-males, 16 wk a Before After D Plasma insulin (ng/ml) Insulin-females, 16 wk Before After 0.0 Chow High fat 0.0 Chow High fat Minutes after glucose stimulation Minutes after glucose stimulation Figure 3 Elevated insulin levels in male mice after long-term high fat diet feeding. Insulin levels were measured using enzyme-linked immunosorbent assay prior to and 30 min after glucose stimulation in males (A) and females (B), following a short-term high fat diet (HFD) feeding, and males (C) and females (D), after a long-term HFD feeding. a P < 0.05 vs chow fed controls, n = 5/group. controls (Figure 5D). MCP-1 mrna levels in the liver were increased in the long-term HFD males compared to chow fed mice, whereas no change was noted in the longterm HFD females (Figure 5E). Long-term HFD male mice showed a 2-fold increase in MCP-1 protein in the liver compared to chow-fed mice, whereas no change in MCP-1 protein was seen in female mice (Figure 5F). Inflammasome components are upregulated and activated in male mice following a long-term HFD, but not females We evaluated inflammasome activation only in the longterm 16-wk HFD feeding based on previous studies that a longer feeding is needed for inflammasome activation [20]. We found an increase in mrna in components of the inflammasome only in male mice fed a long-term HFD, including ASC, Aim2, and NLRC4 (Figure 6A and B). Inflammasome activation was indicated by increased caspase-1 activity (Figure 6C) and increased total IL-1β protein levels (Figure 6D) in the liver of male mice fed a long-term HFD. Fibrosis markers are elevated in males who received long-term HFD Because liver inflammation can trigger fibrosis, fibrotic markers were evaluated in mice fed a long-term HFD. Elevation in mrna levels of both α-smooth muscle actin (αsma) (Figure 7A) and pro-collagen-1 (Figure 7B) are seen in male mice who received long-term HFD feeding, whereas no significant change was seen in females compared to chow fed controls. Because of the early stage of fibrosis, we found no increase in αsma protein levels (data not shown). DISCUSSION NASH has become a global epidemic and continued research into the pathogenesis resulting in inflammation and steatosis requires a robust animal model, which has remained a challenge [23]. Here we show that mice fed a HFD supplemented with fructose and sucrose in the drinking water for 16 wk, results in steatohepatitis and moderate activation of the inflammasome pathway in male, but not female, mice. Our study evaluated gender differences on the degree of inflammation, steatosis, and fibrosis between male and female mice fed a high fat diet enriched in fructose and sucrose for 6 or 16 wk. We found evidence of steatohepatitis in male mice fed a long-term HFD as demonstrated by an increase in ALT, triglycerides, TNFα, and MCP-1, whereas in females only an increase in triglycerides was seen, consistent with the presence of steatosis, but no 8529 July 14, 2014 Volume 20 Issue 26

222 Ganz M et al. Gender differences in NASH A Males, 16 wk Females, 16 wk Males, 16 wk B Chow Chow HFD HFD C Mg triglyceride/100 mg protein P < 0.01 Triglyceride, 16 wk P = 0.01 P < 0.01 Chow High fat D ALT (IU/mL) P = 0.01 ALT, 16 wk Chow High fat 0 Male Female 0 Male Female Figure 4 Long-term high fat diet feeding results in steatohepatitis in male mice, and steatosis in female mice. Male and female mice were fed with either high fat diet (HFD) or control chow diet for 6 or 16 wk. A, B: Liver tissue was subjected to HE of male (A) and Oil-Red-O staining (B) ( 100). Dark arrow: Macrovesicular steatosis, white arrow: Microvesicular steatosis. One representative slide from n = 5/group is shown; C, D: Liver triglyceride levels (C) were evaluated in mice fed with HFD for 16 wk. Serum alanine aminotransferase (ALT) levels were measured after long-term HFD feeding (D). steatohepatitis. Several studies have investigated gender on the role of NAFLD progression with no consistent outcome. Kamada et al [13] show that estrogen plays a role in protection of liver injury following high fat diet, whereas another group showed that there was no protection in MCD diet induced steatohepatitis in female mice that received an ovariectomy or antiestrogen treatment [11]. Yet another study showed that female mice fed a chow diet supplemented with 30% fructose in the drinking water, displayed more pronounced inflammation than male mice [12]. In terms of other liver diseases in humans, men are about three to five times more likely than women to develop hepatocellular carcinoma (HCC) [24]. The underlying mechanism leading to a decreased incidence in women is thought to be due to estrogen suppression of IL-6 in Kupffer cells [25] and due to increased androgen receptor signaling by testosterone in men, resulting in increased hepatocyte proliferation [26]. Furthermore, studies have shown that the male gender is an independent risk factor in the progression of cirrhosis [27,28], and that young women have the lowest rate of developing cirrhosis from chronic hepatitis C [29]. Further studies need to be done looking into the differences of liver injury in males and females. Our study in mice indicates gender differences that were dependent on the length of HFD feeding and/ or the estrogen status of female mice. Previous work has shown that various high fat diet feedings induce the inflammasome and that knock-outs of parts of the inflammasome pathway can result in protection from HFD induced obesity [20,30]. Both upregulation of inflammasome expression and activation occurred in male mice after 16 wk. We show that there 8530 July 14, 2014 Volume 20 Issue 26

223 Ganz M et al. Gender differences in NASH A 50 TNFa protein, 6 wk D 300 MCP-1 protein, 6 wk TNFa (pg/mg tissue) Chow High fat MCP-1 (pg/mg tissue) Chow High fat 0 Males Females 0 Males Females B TNFa mrna (fold change) b TNFa mrna, 16 wk Chow High fat E MCP-1 mrna (fold change) MCP-1 mrna, 16 wk b Chow High fat 0 Males Females 0 Males Females C TNFa (pg/mg tissue) b TNFa protein, 16 wk Chow High fat F MCP-1 (pg/mg tissue) b MCP-1 protein, 16 wk Chow High fat 0 Males Females 0 Males Females Figure 5 Long-term high fat diet results in hepatic inflammation in male mice. A-C: Liver tumor necrosis factor-a (TNFa) protein levels (A) were evaluated in the short-term feeding levels of TNFα in the liver were analyzed at the mrna (B) and protein (C) level after a long-term feeding; D-F: Monocyte chemoattractant protein 1 (MCP-1) protein levels (D) were evaluated in the short-term feeding. Levels of MCP-1 in the liver were analyzed at the mrna (E) and protein (F) level after a longterm feeding. b P < 0.01 vs chow fed controls, n = 10/group. is an increase in mrna in parts of the inflammasome pathway, including ASC, Aim2, and NLRC4, as well as an increase in caspase-1 activity and IL-1β protein in the liver, after a 16-wk HFD feeding in male mice, whereas mice fed a HFD for 9 mo, show an increase in mrna in all parts of the NALP3 inflammasome (NALP3, ASC, Caspase-1, Pannexin, and IL-1β) [20]. This finding suggests that longer access to a HFD is required to obtain complete inflammasome activation and thus more inflammation and liver damage. Another possibility is that changing the composition of the diet could result in more extensive liver damage and fibrosis, such as the combination of dietary fat and dietary cholesterol [31]. It is conceivable that changes in fatty acid composition due to the diet affects inflammation, for example saturated fatty acids result in inflammasome activation [20]. The limitation of existing animal models of NASH that are based on genetic modifications and no single model captures the different aspects of human fatty liver disease [23,32,33]. MCD extensively used in our previous studies results in severe steatohepatitis in a short period of time [20,34], however in contrast to human fatty liver disease, mice lose weight and lack insulin resistance [23]. To use a model that results in steatohepatitis, in addition to weight gain and insulin resistance, we chose HFD model consisting mainly of medium chain hydrogenated saturated trans fatty acids supplemented with fructose and sucrose in the drinking water, similar to the high-fructose corn syrup that is so widespread in the United States [21]. Of note, there has been a wide variety in the extent of 8531 July 14, 2014 Volume 20 Issue 26

224 Ganz M et al. Gender differences in NASH A 5 4 Liver mrna-males, 16 wk b a Chow High fat b B Liver mrna-females, 16 wk Chow High fat Fold change 3 2 Fold change NLRP3 pro-caspase 1 ASC Aim2 NLRC4 0.0 pro-il-1b pro-caspase 1 ASC C 3 Caspase-1 activity-males, 16 wk b D IL-1b protein-males, 16 wk b Fold change 2 1 IL-1b (pg/ml) Chow High fat 0 Chow High fat Figure 6 Long-term high fat diet induced inflammasome upregulation and activation in male mice. Hepatic mrna expression of the inflammasome components NOD-like receptors3 (NLRP3), caspase-1, adaptor molecule, Aim2, and NLRC4 were measured using real-time quantitative polymerase chain reaction in male (A) and female (B) mice after a long-term high fat diet. The functional activity of the inflammasome was evaluated by measurements of caspase-1 activity (C) and total nterleukin (IL)-1β protein (D) levels, in the liver. a P < 0.05, b P < 0.01 vs chow fed controls, n = 10/group. A Fold change asma mrna, 16 wk b Chow High fat B Fold change Pro-collagen-1 mrna, 16 wk a Chow High fat 0 Males Females 0 Males Females Figure 7 Long-term male mice fed a high fat diet display increase in fibrotic markers. Hepatic mrna expression of α-smooth muscle actin (αsma) (A) and procollagen-1 (B), were measured using real-time quantitative polymerase chain reaction in male mice following a long-term high fat diet feeding. a P < 0.05, b P < 0.01 vs chow fed controls, n = 10/group. inflammation and steatosis seen in high fat diet feedings. Some groups have shown fibrosis after only 6 wk of feeding [35], whereas others have needed 24 wk or longer to see fibrosis [36,37]. This variation could be due to variances in the composition of the diet and in differences in gut microbiota in the mice in each individual study. In summary, our data shows that gender differences exist in the development of steatohepatitis and inflammasome activation following a long-term high fat diet feeding. Male mice display steatohepatitis in addition to inflammasome upregulation and activation, whereas female mice develop steatosis and do not show activation of the inflammasome pathway. ACKNOWLEDGMENTS The authors would like to thank Jan Petrasek for his help with editing the manuscript, and Dora Lippai for assis July 14, 2014 Volume 20 Issue 26

225 Ganz M et al. Gender differences in NASH tance with the glucose and insulin tolerance tests. COMMENTS Background Non-alcoholic fatty liver disease is one of the most common causes of liver disease in the Western world and continues to be on the rise. Developing an animal model that closely resembles human non-alcoholic fatty liver disease has remained a challenge. Furthermore, it is known that males are more likely than females to develop cirrhosis. Research frontiers Developing an animal model that resembles non-alcoholic fatty liver disease in humans is an important area. Further knowledge into non-alcoholic fatty liver disease can help further knowledge in the field. Innovations and breakthroughs Previous studies have shown that feeding mice a high fat diet with added sucrose and fructose results in steatohepatitis and fibrosis. A number of studies have looked at gender differences in non-alcoholic fatty liver disease with no consistent outcome. In the present study, the authors looked at the effect of both a short-term and long-term high fat diet feeding supplemented with sucrose and fructose in both male and female wild-type mice. This paper showed that male mice fed a long-term high fat diet display steatohepatitis and inflammasome activation, whereas female mice have steatosis without inflammation. Applications Further studies looking into why there are gender differences in developing nonalcoholic fatty liver disease are warranted. These studies could lead to potential treatments for non-alcoholic fatty liver disease. Peer review The manuscript presents some animal data on the gender differences regarding the consequences of high fat feeding. The manuscript is in a very good condition. Although the animal model for gender difference may be questioned and translation of the data to the clinical area is difficult, the data may be attractive for some of the readers. REFERENCES 1 Sheth SG, Gordon FD, Chopra S. 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Critical role of toll-like receptors and the common TLR adaptor, MyD88, in induction of granulomas and liver injury. J Hepatol 2006; 45: [PMID: DOI: /j.jhep ] 23 Schattenberg JM, Galle PR. Animal models of non-alcoholic steatohepatitis: of mice and man. Dig Dis 2010; 28: [PMID: DOI: / ] 24 Bosch FX, Ribes J, Díaz M, Cléries R. Primary liver cancer: worldwide incidence and trends. Gastroenterology 2004; 127: S5-S16 [PMID: ] 25 Naugler WE, Sakurai T, Kim S, Maeda S, Kim K, Elsharkawy AM, Karin M. Gender disparity in liver cancer due to sex differences in MyD88-dependent IL-6 production. Science 2007; 317: [PMID: DOI: /science ] 26 Yu MW, Chen CJ. Elevated serum testosterone levels and risk of hepatocellular carcinoma. Cancer Res 1993; 53: July 14, 2014 Volume 20 Issue 26

226 Ganz M et al. Gender differences in NASH [PMID: ] 27 Poynard T, Ratziu V, Charlotte F, Goodman Z, McHutchison J, Albrecht J. Rates and risk factors of liver fibrosis progression in patients with chronic hepatitis c. J Hepatol 2001; 34: [PMID: ] 28 El-Serag HB. Hepatocellular carcinoma. N Engl J Med 2011; 365: [PMID: DOI: /NEJMra ] 29 Kim WR, Poterucha JJ, Benson JT, Therneau TM. The impact of competing risks on the observed rate of chronic hepatitis C progression. Gastroenterology 2004; 127: [PMID: ] 30 Stienstra R, van Diepen JA, Tack CJ, Zaki MH, van de Veerdonk FL, Perera D, Neale GA, Hooiveld GJ, Hijmans A, Vroegrijk I, van den Berg S, Romijn J, Rensen PC, Joosten LA, Netea MG, Kanneganti TD. Inflammasome is a central player in the induction of obesity and insulin resistance. Proc Natl Acad Sci USA 2011; 108: [PMID: DOI: /pnas ] 31 Savard C, Tartaglione EV, Kuver R, Haigh WG, Farrell GC, Subramanian S, Chait A, Yeh MM, Quinn LS, Ioannou GN. Synergistic interaction of dietary cholesterol and dietary fat in inducing experimental steatohepatitis. Hepatology 2013; 57: [PMID: DOI: /hep.25789] 32 Takahashi Y, Soejima Y, Fukusato T. Animal models of nonalcoholic fatty liver disease/nonalcoholic steatohepatitis. World J Gastroenterol 2012; 18: [PMID: DOI: /wjg.v18.i ] 33 Almonacid-Urrego CC, Sánchez-Campos S, Tuñón MJ, González-Gallego J. Non-alcoholic steatohepatitis: what can we learn from animal models? Curr Med Chem 2012; 19: [PMID: ] 34 Csak T, Dolganiuc A, Kodys K, Nath B, Petrasek J, Bala S, Lippai D, Szabo G. Mitochondrial antiviral signaling protein defect links impaired antiviral response and liver injury in steatohepatitis in mice. Hepatology 2011; 53: [PMID: DOI: /hep.24301] 35 Gäbele E, Dostert K, Dorn C, Patsenker E, Stickel F, Hellerbrand C. A new model of interactive effects of alcohol and high-fat diet on hepatic fibrosis. Alcohol Clin Exp Res 2011; 35: [PMID: DOI: / j x] 36 Xu ZJ, Fan JG, Ding XD, Qiao L, Wang GL. Characterization of high-fat, diet-induced, non-alcoholic steatohepatitis with fibrosis in rats. Dig Dis Sci 2010; 55: [PMID: DOI: /s ] 37 Ito M, Suzuki J, Tsujioka S, Sasaki M, Gomori A, Shirakura T, Hirose H, Ito M, Ishihara A, Iwaasa H, Kanatani A. Longitudinal analysis of murine steatohepatitis model induced by chronic exposure to high-fat diet. Hepatol Res 2007; 37: [PMID: DOI: /j X x] P- Reviewer: Tasci I S- Editor: Zhai HH L- Editor: A E- Editor: Wang CH 8534 July 14, 2014 Volume 20 Issue 26

227 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. ORIGINAL ARTICLE Novel diagnostics for aggravating pancreatic fistulas at the acute phase after pancreatectomy Mitsuro Kanda, Tsutomu Fujii, Hideki Takami, Masaya Suenaga, Yoshikuni Inokawa, Suguru Yamada, Daisuke Kobayashi, Chie Tanaka, Hiroyuki Sugimoto, Masahiko Koike, Shuji Nomoto, Michitaka Fujiwara, Yasuhiro Kodera Mitsuro Kanda, Tsutomu Fujii, Hideki Takami, Masaya Suenaga, Yoshikuni Inokawa, Suguru Yamada, Daisuke Kobayashi, Chie Tanaka, Hiroyuki Sugimoto, Masahiko Koike, Shuji Nomoto, Michitaka Fujiwara, Yasuhiro Kodera, Department of Gastroenterological Surgery (Surgery Ⅱ), Nagoya University Graduate School of Medicine, Nagoya , Japan Author contributions: Kanda M, Fujii T, Takami H and Suenaga M analyzed the data and wrote the manuscript; Kanda M and Fujii T designed the study; Inokawa Y, Yamada S, Kobayashi D, Tanaka C, Sugimoto H, Koike M, Nomoto S, Fujiwara M and Kodera Y revised the manuscript for important intellectual content. Correspondence to: Tsutomu Fujii, MD, PhD, FACS, Department of Gastroenterological Surgery (Surgery Ⅱ), Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showaku, Nagoya , Japan. fjt@med.nagoya-u.ac.jp Telephone: Fax: Received: December 24, 2013 Revised: January 28, 2014 Accepted: March 6, 2014 Published online: July 14, 2014 Abstract AIM: To identify sensitive predictors of clinically relevant postoperative pancreatic fistula (POPF) at the acute phase after pancreatectomy. METHODS: This study included 153 patients diagnosed as having POPFs at postoperative day (POD) 3 after either open pancreatoduodenectomy or distal pancreatectomy between January 2008 and March The POPFs were categorized into three grades based on the International Study Group on Pancreatic Fistula Definition, and POPFs of grades B or C were considered to be clinically relevant. The predictive performance for the clinically relevant POPF formation of values at PODs 1, 3 and 5 as well as time-dependent changes in levels of inflammatory markers, including white blood cell count, neutrophil count, total lymphocyte count, C-reactive protein (CRP), procalcitonin level, plateletto-lymphocyte ratio and neutrophil-to-lymphocyte ratio, and amylase content in the drain fluid were compared using the receiver operating characteristic (ROC) curve and multivariable analyses. A scoring system for the prediction of clinically relevant POPFs was created using five risk factors identified in this study, and its diagnostic performance was also evaluated. RESULTS: Over time, 77 (50%) of 153 enrolled patients followed a protracted course and were categorized as having clinically relevant POPFs. ROC curve analyses revealed that changes in CRP levels from POD 1 to POD 3 had the greatest area under the curve value (0.767) and that an elevated CRP level of 28.4 mg/l yielded the most optimal predictive value for clinically relevant POPFs. Multivariable analyses for the risk factors of clinically relevant POPFs identified invasive carcinomas of the pancreas and elevation of the CRP level ( 28.4 mg/l, from POD 1 to POD 3) as independent diagnostic factors for clinically relevant POPFs (OR 2.94, 95%CI: , P = and OR 4.82, 95%CI: , P = 0.022, respectively). A gradual increase in the prevalence of clinically relevant POPFs in proportion to the risk classification score was confirmed. A highly elevated CRP level and a risk score 8 were significantly associated with a prolonged duration of drain placement and postoperative hospitalization. CONCLUSION: A steep rise in the serum CRP level from POD 1 to POD 3 was a highly predictive factor for subsequent clinically relevant POPFs Baishideng Publishing Group Inc. All rights reserved. Key words: Pancreatectomy; Pancreatic fistula; Prediction; Inflammatory marker; C-reactive protein Core tip: The diagnostic performance of inflammatory markers and amylase content in drain fluid were com July 14, 2014 Volume 20 Issue 26

228 Kanda M et al. Predictors of aggravating pancreatic fistula pared to identify sensitive predictors of clinically relevant postoperative pancreatic fistulas (POPFs) at the acute phase after pancreatectomy. Receiver operating characteristic curve analyses revealed that the changes in C-reactive protein (CRP) levels from postoperative day (POD) 1 to POD 3 had the greatest area under the curve value for clinically relevant POPFs. Multivariable analyses identified increased elevation of the CRP level as an independent diagnostic factor for clinically relevant POPFs. A steep rise in the CRP level was a highly diagnostic factor for clinically relevant POPFs and may be helpful in selecting the appropriate management for POPFs. Kanda M, Fujii T, Takami H, Suenaga M, Inokawa Y, Yamada S, Kobayashi D, Tanaka C, Sugimoto H, Koike M, Nomoto S, Fujiwara M, Kodera Y. Novel diagnostics for aggravating pancreatic fistulas at the acute phase after pancreatectomy. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: INTRODUCTION In general, pancreatectomy is recognized to be a highly invasive surgery. Despite recent advances in surgical methodologies, devices and perioperative management, pancreatectomy is associated with a mortality of approximately 5% and a morbidity of 35%-60% [1,2]. The incidence of postoperative pancreatic fistulas (POPFs), a surgical complication of particular concern, has been reported to be 20%-40% after pancreatoduodenectomy and 30%-55% after distal pancreatectomy, thereby having a decisive influence on the clinical course after pancreatectomy [3-6]. Diverse anastomotic procedures, treatment of the pancreatic stump, and the administration of somatostatin analogs have been evaluated by randomized clinical trials with the aim of reducing the prevalence of POPFs [7-9], but they have not been associated with appreciable improvement. The aggravation of POPFs can lead to prolonged hospitalization, burgeoning costs of medical treatment, and development into fatal conditions, such as sepsis through abdominal abscesses and intra-abdominal hemorrhages from ruptured aneurysms [4,5]. In addition, clinically relevant POPFs have been reported to be risk factors for peritoneal recurrences [10]. Thus, management of POPFs is a high-priority issue in pancreatic surgery. Sensitive diagnostic factors of clinically relevant POPF are needed to identify high-risk patients and strengthen measures aimed at preventing the aggravation of POPFs. The clinical stratification of POPFs was established by the International Study Group on Pancreatic Fistula Definition (ISGPF) in 2005 [11]. The presence or absence of POPFs can be determined at postoperative day (POD) 3 by the amylase content in the drained fluid. The most important issue is whether the POPF subsides promptly or deteriorates into severe leakage, thereby influencing the clinical course. Predicting clinically relevant POPFs is often difficult because the amylase content in the drained fluid does not always represent the actual conditions of leakage [12,13]. Numerous pre- or intraoperative risk factors for postoperative POPFs have been reported: a soft pancreas, a small main pancreatic duct, a fatty pancreas, malnutrition, and obesity [14-18]. Nevertheless, physicians frequently vacillate on whether or not a POPF becomes prolonged after pancreatectomy. The prediction of the course of POPFs during the early postoperative phase (i.e., POD 3) would enable the appropriate selection from two contrary treatment strategies: the early removal of drainage tubes or retention of the tubes accompanied by scrutiny of the abdomen by imaging studies or the administration of broad-spectrum antibiotics and the use of somatostatin analogs. The early removal of drainage tubes in patients with clinically irrelevant POPFs and the early recovery of patients from clinically relevant POPFs through optimal management would both lead to shortened hospitalizations. In terms of monitoring inflammation, measurement of the white blood cell (WBC) count, neutrophil count, total lymphocyte count (TLC), C-reactive protein (CRP) levels, and procalcitonin levels, as well as being alert to the presence of the systemic inflammatory response syndrome (SIRS), have been in widespread use [19-21]. Once a POPF is aggravated, pancreatic fluid leaks into the abdominal cavity and causes local inflammation, secondary infection and, eventually, expansion to a systemic inflammatory response. Thus, the hypothesis that inflammatory markers represent the momentum of subsequent POPFs was raised because POPFs induce local and systemic inflammatory responses. No reports have evaluated the associations between the time-dependent changes of postoperative inflammatory markers and the subsequent course of POPF focusing on patients diagnosed as having POPFs at POD 3. To test the hypothesis and to identify sensitive diagnostic factors of clinically relevant POPFs, the inflammatory markers in patients who underwent pancreatectomy were investigated at the acute phase of the postoperative course over time, and their diagnostic values for POPF aggravation were evaluated. MATERIALS AND METHODS Ethics This study conforms to the ethical guidelines of the World Medical Association Declaration of Helsinki- Ethical Principles for Medical Research Involving Human Subjects. Written informed consent for the surgery and the usage of clinical data, as required by the Review Board of Nagoya University (Nagoya, Japan), was obtained from all patients. Patient characteristics and surgical procedure Between January 2008 and March 2013, 337 major pan July 14, 2014 Volume 20 Issue 26

229 Kanda M et al. Predictors of aggravating pancreatic fistula createctomies (i.e., pancreatoduodenectomy, distal pancreatectomy and total pancreatectomy) were performed at the Department of Gastroenterological Surgery of Nagoya University. Of these, 23 patients who underwent total pancreatectomy were excluded. Patients without POPFs were excluded because the principal purpose of the current study was to identify the diagnostic factors of aggravation among patients with POPFs. Additionally, patients with postoperative infectious complications, such as wound infection and pneumonia, which might influence the inflammatory status, were excluded. Ultimately, 153 patients who were diagnosed as having POPFs at 3 d after pancreatoduodenectomy or distal pancreatectomy were enrolled. The preoperative backgrounds of the patients, intraoperative conditions, and postoperative course (POPF severity, duration of placement of drainage tube, and postoperative inflammatory markers) were investigated to identify sensitive diagnostic factors of clinically relevant POPFs. For pancreatoduodenectomies, pancreatic reconstruction was performed by a direct anastomosis between the pancreatic duct and mucosal layer of the jejunal loop. Except for patients with a remarkably dilated main pancreatic duct, a stent tube was inserted into the pancreatic duct. Adhesion of the jejunal wall and the pancreatic stump by one-layer sutures was added [22,23]. Silastic flexible drains (Blake drain, Ethicon, Somerville, NJ, United States) were placed at the anterior and posterior surfaces of the pancreatojejunostomy and choledochojejunostomy. For distal pancreatectomies, closure of the main pancreatic duct was performed using a continuous suture. Silastic flexible drains were placed at the anterior and posterior surface of the pancreatic stump. Definition of pancreatic fistula Patients were categorized as having developed a grade A, B or C fistula based on the definitions of the ISGPF [11]. Briefly, POPFs were categorized into three grades: biochemical leak without peripancreatic fluid collection, defined as output containing pancreatic amylase on or after the third postoperative day from an operatively positioned drain with a pancreatic amylase level more than three times the upper serum reference value (grade A); leaks with changes in management and a delay of discharge from the hospital (grade B); and leaks that required percutaneous drainage of a fluid collection or reexploration for critically ill patients and were associated with an extended stay in the hospital (grade C). POPFs of grades B or C were considered to be clinically relevant POPFs in this study. Perioperative management Cephalosporin was administered immediately before surgery and every 3 h during surgery. In all patients, the administration of antibiotics and H2 blockers was continued routinely thorough POD 3. Oral intake was started routinely 5 d after surgery unless postoperative complications, such as delayed gastric emptying, occurred. Bloodsugar level was routinely monitored, and appropriate levels were maintained (70 mg/dl blood-sugar level < 200 mg/dl). The amylase content in the discharge from the closed-suction drains was examined at PODs 1, 3 and 5. Continuous low pressure suction was applied to the abdominal drains to minimalize the fluid cavity through POD 4. Abdominal drains were removed at POD 4 in patients without POPFs or replaced as needed until a cure of the POPFs was confirmed in patients with POPFs. Abdominal ultrasonography was performed as needed postoperatively. Percutaneous drainage or the replacement of tubes was employed when signs of inadequate drainage were found. Evaluation of inflammatory markers Blood examinations were conducted routinely before surgery and at PODs 1, 3 and 5. The inflammatory markers included the WBC, neutrophil count, TLC, platelet count, and CRP, procalcitonin and serum amylase levels. The procalcitonin level was tested routinely only at POD 3. In addition to the markers mentioned above, the plateletto-lymphocyte ratio and neutrophil-to-lymphocyte ratio were calculated. Time-dependent changes of these factors were also investigated. As indications of systemic inflammatory status, the duration of a high fever ( 38 ), SIRS, and the use of catecholamines were evaluated. The amylase content and bacterial culture in the drain fluids were also examined at PODs 1, 3 and 5 or until drain removal. To identify the appropriate cutoff value of each marker and time-dependent changes, analyses of receiver operating characteristic (ROC) curves were conducted. The diagnostic performance of inflammatory markers for clinically relevant POPFs was compared using optimal cutoff values determined by analyses of the ROC curves. Selected markers with known risk factors for POPFs were included in the multivariable analyses. Moreover, the influence of the selected diagnostic factors on the duration of drain placement and hospitalization was evaluated. Risk classification scale for clinically relevant POPFs A scoring scale for prediction of clinically relevant POPFs was introduced using the risk factors identified in the present study, and the diagnostic performance was evaluated. Parameters that demonstrated significant associations with clinically relevant POPFs in univariate analyses were included in this scoring scale. Then, individual scores were allocated to each parameter in accordance with their odds ratios. Further evaluation of potent factors The sensitivity, specificity, positive predictive value, negative predictive value, likelihood ratio of a positive result and likelihood ratio of a negative result for clinically relevant POPFs were further evaluated among the potent factors identified in this study to compare diagnostic performance. The CRP level on POD 3, increased eleva July 14, 2014 Volume 20 Issue 26

230 Kanda M et al. Predictors of aggravating pancreatic fistula Table 1 Demographics and clinical characteristics of 153 patients n (%) Characteristic tion of the CRP level from PODs 1 to 3, drain amylase content on POD 3 and the risk score were included in this analysis. Statistical analysis The qualitative χ 2 test and quantitative Mann-Whitney s U test were used to compare the two groups. ROC curve analyses were conducted to evaluate the discrimination of clinically relevant POPFs by the markers of interest. The goodness of fit for clinically relevant POPFs was assessed by comparing the area under the curve (AUC) value, and optimal cutoff values were also determined using the Youden Index. The risk factors for clinically relevant POPFs were evaluated using binomial logistic analyses, and the variables with P < 0.1 were entered into the final model. The statistical analyses were performed using JMP ver10 (SAS Institute, NC, United States). A P < 0.05 was considered to be significant. RESULTS Value Age (yr), mean (range) 65.2 (28-89) Male/female 101/52 Preoperative symptom(s) 37 (24) Diabetes mellitus (preoperative ) 39 (25) History of pancreatitis 20 (13) Body mass index, mean ± SD 22.5 ± 3.5 Procedure Pancreatoduodenectomy 103 Distal pancreatectomy 50 Resection of the portal vein 21 (14) Pathological diagnosis Invasive carcinoma of the pancreas 68 Intraductal papillary mucinous neoplasm 35 Carcinoma of the ampulla 13 Cholangiocarcinoma 10 Pancreatic neuroendocrine tumor 8 Other 19 Grade of postoperative pancreatic fistula 1 A 76 B 70 C 7 1 Defined according to the International Study Group on Pancreatic Fistula Definition. Patient characteristics The patients characteristics are summarized in Table 1. The ages of the 153 subjects ranged from 28 years to 89 years (65.2 ± 10.3 years; mean ± SD), and the male-tofemale ratio was 101:52. Surgery consisted of pancreatoduodenectomy in 103 patients and distal pancreatectomy in 50 patients. In terms of the pathological diagnoses, 68 (44%) subjects had invasive carcinoma of the pancreas. Thirty-nine (25%) patients had diabetes mellitus preoperatively, and 20 (13%) patients had a history of pancreatitis. None of the enrolled participants had active inflam- matory disease (e.g., cholangitis or pancreatitis) before surgery, suggesting that the postoperative inflammatory status was assessed accurately. All patients were afebrile, and the CRP levels were < 20 mg/l preoperatively. The postoperative mortality rate was 0%. Eventually, 77 (50%) of the 153 enrolled patients followed a protracted course and were categorized as having clinically relevant POPFs. There was no significant difference in the incidence rate of clinically relevant POPFs between the patients who underwent pancreatoduodenectomy and the patients who underwent distal pancreatectomy (54% vs 42%, respectively, P = 0.151). ROC curve analyses of postoperative inflammatory markers The predictive values of postoperative inflammatory markers at each time point and time-dependent changes were evaluated by ROC curve analyses. The AUC values, P values for differences between grade-a and grade-b/ C POPFs, and the most optimal cutoff point to predict clinically relevant POPFs are listed in Table 2. The change in the CRP levels from POD 1 to POD 3 had the highest AUC value (0.767), and a further elevation of the CRP level of 28.4 mg/l yielded the most optimal diagnostic value for clinically relevant POPFs. In general, changes from POD 3 to POD 5 were less diagnostic than those from POD 1 to POD 3. Among the markers determined at POD 3, the platelet count and CRP level showed relatively high AUC values (> 0.6). The amylase content in the drain fluid at POD 3 was significantly associated with clinically relevant POPFs (P = 0.003; AUC = 0.640; optimal cutoff value 1918 IU/L). Representative ROC curves are shown in Figure 1. Comparison of diagnostic performance for clinically relevant POPFs Next, the diagnostic values for clinically relevant POPFs were compared among the well-known risk factors (e.g., the size and texture of the pancreatic duct), postoperative high fever, SIRS, the use of catecholamines, and the inflammatory markers selected by ROC curve analyses and amylase content in the drain fluid at POD 3. Univariate analyses showed that invasive carcinoma of the pancreas, platelet count (< /mm 3, POD 3), CRP level ( 158 mg/l, POD 3), CRP elevation ( 28.4 mg/l, from POD 1 to POD 3) and amylase content in the drain fluid ( 1918 IU/L, POD 3) were significant adverse factors for POPFs (Table 3). Multivariable analyses identified invasive carcinoma of the pancreas and elevation of the CRP level ( 28.4 mg/l, from POD 1 to POD 3) as independent diagnostic factors for clinically relevant POPFs (OR 2.94, 95%CI: , P = and OR 4.82, 95% CI: , P = 0.022, respectively; Table 3). In patients with invasive carcinoma of the pancreas, the ratio of clinically relevant POPF to mild POPF was higher but the total prevalence of POPFs was lower compared with the whole population (Figure 2) July 14, 2014 Volume 20 Issue 26

231 Kanda M et al. Predictors of aggravating pancreatic fistula Table 2 Inflammatory markers for clinically relevant postoperative pancreatic fistulas Marker POD 1 POD 3 POD 5 Change from POD 1 to 3 Change from POD 3 to 5 AUC P value AUC P value Cutoff AUC P value AUC P value Cutoff AUC P value White blood cell count (/mm 3 ) Neutrophil count (/mm 3 ) Total lymphocyte count (/mm 3 ) Platelet count (/mm 3 ) Serum amylase (IU/mL) C-reactive protein (mg/l) < < < Procalcitonin (ng/ml) Neutrophil-to-lymphocyte ratio Platelet to lymphocyte ratio P values were of the comparison between grade A and grade B/C as calculated by the Mann Whitney U test. Cutoff values were determined by receiver operating characteristic curve analyses. POD: Postoperative day; AUC: Area under the curve. CRP POD 1 POD 3 Change PODs 1 to Sensitivity Sensitivity Sensitivity Cut off 28.4 mg/l; Sensitivity = 0.86 Specificity = AUC = AUC = AUC = specificity specificity specificity Platelet count POD 3 Drain amylase POD Sensitivity Sensitivity AUC = AUC = specificity specificity Figure 1 Receiver operating characteristic curves. Receiver operating characteristic curve for the C-reactive protein (CRP) level, platelet count, and amylase content in drain fluid as diagnostic factors for clinically relevant postoperative pancreatic fistulas. The change in the CRP levels from postoperative day (POD) 1 to POD 3 demonstrated the highest area under the curve (AUC) value. Association of elevation in the CRP level with postoperative course and other markers Patients with a further elevation in the CRP levels 28.4 mg/l from POD 1 to POD 3 (high elevation of the CRP level) had a significantly longer duration of drain placement than patients without elevation in the CRP levels (24 and 12 d, respectively; median, P < 0.001; Figure 3A). Consequently, the duration of postoperative hospitalization was significantly greater in patients with high CRP elevations than in patients without high CRP elevations (38.5 and 25.5 d, respectively; median, P < 0.001; Figure 3B). High CRP elevation was not significantly associated with the WBC count, neutrophil count, procalcitonin level or bacterial culture in the drain fluid at POD July 14, 2014 Volume 20 Issue 26

232 Kanda M et al. Predictors of aggravating pancreatic fistula Table 3 Diagnostic value of clinical factors for clinically relevant postoperative pancreatic fistula Variable Univariate Multivariable Odds ratio 95%CI P value Odds ratio 95%CI P value Age ( 65 yr) Gender (male) Diabetes mellitus History of pancreatitis Body mass index ( 22) Operative procedure (DP) Invasive carcinoma of the pancreas Operative time ( 360 min) Blood loss ( 600 ml) Intraoperative transfusion Pancreas texture (soft) Main pancreatic duct (< 4 mm) Persistence of high fever ( 38, 3 d) Persistence of SIRS ( 3 d) Usage of catecholamines Total lymphocyte count (< 600/mm 3, POD 3) Platelet count (< /mm 3, POD 3) CRP ( 158 mg/l, POD 3) < Neutrophil-to-lymphocyte ratio ( 9.6, POD 3) CRP elevation ( 28.4 mg/l, POD 1 to 3) < Drain amylase ( 1918 IU/mL, POD 3) Drain fluid bacterial culture (positive, POD 3) Analyzed by binomial logistic analyses. DP: Distal pancreatectomy; SIRS: Systemic inflammatory response syndrome; CRP: C-reactive protein; POD: Postoperative day. Overall (n = 314) Grade B or C Invasive carcinoma of the pancreas (n = 193) Grade B or C Figure 2 Distribution of postoperative pancreatic fistulas grade in the entire study population as well as patients with invasive carcinoma of the pancreas. POPF: Postoperative pancreatic fistulas. Grade A Without POPF Grade A Without POPF POPF/total = 49% Grade B or C/POPF = 50% POPF/total = 35% Grade B or C/POPF = 65% Risk scoring scale for clinically relevant POPFs A scoring scale for the prediction of clinically relevant POPFs was developed to enhance the clinical utility of the diagnostic factors detected in this study. Five diagnostic factors (invasive carcinoma of the pancreas, platelet count < /mm 3 at POD 3, CRP 158 mg/l at POD 3, CRP elevation 28.4 mg/l from POD 1 to POD 3, and amylase content in the drain fluid 1918 IU/L at POD 3) were included in the scoring scale because these factors demonstrated a significant association with clinically relevant POPFs. The prevalence of clinically relevant POPFs increased gradually in proportion with the score, from 0% to 100%. The score had a remarkably high goodness of fit for clinically relevant POPFs (AUC = 0.848). A score of 8 yielded the most optimal diagnostic value for clinically relevant POPFs, thereby supporting the clinical utility of the scoring scale (Figure 4). In addition, patients with a risk score 8 had a significantly longer duration of drain placement and postoperative hospitalization than patients with a score < 8 (25.5 ± 18.2 and 12 ± 10.6, 39.5 ± 29.0 and 26.0 ± 10.1 d, respectively; mean ± SD, P < for both). Further evaluation of important factors Table 4 lists the sensitivity, specificity, positive predictive value, negative predictive value, likelihood ratio of a positive result and likelihood ratio of a negative result of four important factors: the CRP level on POD 3, the increased elevation of the CRP level from POD 1 to 3, the drain amylase content on POD 3 and the risk score. It is worth noting that an increased elevation of the CRP level demonstrated a high negative predictive value (0.857) and 8540 July 14, 2014 Volume 20 Issue 26

233 Kanda M et al. Predictors of aggravating pancreatic fistula A t (d) Duration of drain placement Median 12 d P < Median 24 d B t (d) Length of postoperative hospital stay Median 25.5 d P < Median 38.5 d Figure 3 Comparison of duration of drain placement and postoperative hospitalization between two groups categorized by elevation in the C-reactive protein level from postoperative days 1 to 3. A: The duration of drain placement was significantly longer in patients with high elevation of the C-reactive protein (CRP) level than in those without high elevation of the CRP level (P < 0.001); B: The duration of postoperative hospitalization was significantly longer in patients with high elevation of the CRP level than in patients without high elevation of the CRP level (P < 0.001). POD: Postoperative day < 28.4 mg/l 28.4 mg/l 0 < 28.4 mg/l 28.4 mg/l CRP elevation (PODs 1 to 3) CRP elevation (PODs 1 to 3) A 5 CRP elevation 28.4 mg/l, PODs 1 to 3 3 Diagnosis Invasive carcinoma of the pancreas 2 CRP 158 mg/l, POD 3 2 Drain amylase 1918 IU/mL, POD 3 1 Platelet count < /mm 3, POD 3 B Sensitivity Cutoff score 8 Sensitivity = 0.80 Specificity = % 0.2 AUC = % 60% specificity 40% 20% 0% Risk score POPF grade A POPF grade B or C Figure 4 Risk classification scoring scale for clinically relevant postoperative pancreatic fistulas calculated using five parameters. A: Parameters with allocated scores and a bar graph of the prevalence of clinically relevant postoperative pancreatic fistulas (POPFs). Prolonged POPFs increased gradually in proportion to the score from 0% to 100%; B: Receiver operating characteristic curve for the risk score as a diagnostic factor of clinically relevant POPFs. A remarkably high goodness of fit was demonstrated. CRP: C-reactive protein; POD: Postoperative day. a low likelihood ratio of a negative result (0.212), indicating that it may be useful especially for the prediction of the absence of a clinically relevant POPF. The risk score scale was found to have an additional impact on detecting the presence of clinically relevant POPF. DISCUSSION Aggravated POPFs can involve critical complications, such as sepsis and intra-abdominal hemorrhage from ruptured aneurysms, resulting in prolonged hospitalization, increased medical expense, and in the most extreme cases, mortality [4,5]. In addition, clinically relevant POPFs can lead to an increased risk of recurrences through peritoneal dissemination [10]. Clinically relevant POPFs are the most serious problem in the field of pancreatic surgery. The realistic goal is to minimize the extent of POPFs and to limit it to grade A, which has less impact on the postoperative clinical course. To achieve this goal, the early identification of high-risk patients with clinically relevant POPFs is necessary for their subsequent management, including the thorough control of infection, effective drainage, and nutritional care with the aim to localize inflammation and achieve early recovery. When should surgeons manage POPFs, and what are the definitive criteria in clinical practice? An important crossroads may occur at POD 3, when POPFs are usually diagnosed. Management (including drainage and antibiotic administration) after the diagnosis of a POPF at POD 3 depends on whether the POPF is likely to be aggravated July 14, 2014 Volume 20 Issue 26

234 Kanda M et al. Predictors of aggravating pancreatic fistula Table 4 Comparison of diagnostic performance of potent factors for clinically relevant postoperative pancreatic fistula Odds ratio 95%CI P value Sensitivity Specificity Positive predictive value Negative predictive value Likelihood ratio of positive result Likelihood ratio of negative result CRP 158 mg/l < CRP elevation 28.4 mg/l < Drain amylase 1918 IU/mL Risk score < Values on postoperative day (POD) 3; 2 Change from POD 1 to POD 3. P values were analyzed by binomial logistic analyses. CRP: C-reactive protein. Drainage tubes are removed at the surgeon s request. However, it would be too late to prevent the expansion of a POPF if a course of treatment is determined at POD 5 or later. In the present study, patient inclusion was limited to those diagnosed as having a POPF, and all of the evaluated factors should have been available at POD 3. POPFs are associated with inflammation that is a combination of pancreatic fluid with secondary inflammation by bacterial infection, so inflammatory markers are expected to reflect the momentum of POPFs [6]. Thus, POD 3 was the ideal time point for the early prediction of clinically relevant POPFs, and the inflammatory markers at POD 3 were evaluated by focusing on universally prevalent markers. Elevation of the CRP level from POD 1 to POD 3 was found to be an independent risk factor for clinically relevant POPFs with the largest odds ratio, suggesting that it was the most sensitive diagnostic factor of a subsequent clinically relevant POPF. Surprisingly, elevation of the CRP level from POD 1 to POD 3 demonstrated a greater diagnostic performance than well-known major risk factors, such as the size and texture of the pancreatic duct and the amylase content of drain fluid. Within 6 h after stimulation, the CRP level exceeded normal values, and it peaked after 48 h [24]. CRP has a nearly constant half-life in serum of 19 h [25]. Therefore, the CRP concentration in serum is determined by its rate of synthesis and reflects the intensity of the stimulus for acute inflammatory responses. The high diagnostic value of elevation of the CRP level from POD 1 to POD 3 could also be explained. First, an inevitable elevation of the CRP level due to surgical stress and local inflammation in resected tissues was confirmed at POD 1 in all patients. The halflife of CRP is 19 h, so the CRP level starts to show a gradual decline at PODs 2-3 in patients without additional inflammatory events, whereas it continues to increase in patients with expanding POPFs. Second, the CRP level can comprehensively reflect the chemical inflammation of the peritoneum due to leakage of the pancreatic fluid as well as coexisting inflammation due to bacterial infection. The well-known inflammatory markers of WBC and neutrophil counts showed less diagnostic value compared with the CRP level, most likely because of their wide normal range, thereby debilitating the decrease by severe infection and influences by factors other than inflammation (e.g., medications) [26-28]. Procalcitonin is specific only to bacterial infection and does not reflect a comprehensive inflammatory status (including chemical inflammation caused by pancreatic fluids) [29]. In addition, elevation in the CRP level from POD 1 to POD 3 is an ideal marker because it is readily measurable in hospitals at low cost and has high diagnostic value for POPFs. Drain amylase content is superior for determining the presence of POPFs because it directly reflects the leakage of pancreatic fluids. However, the concentration can be strongly influenced by the amount of exudative ascites fluid and the efficacy of drainage, indicating that it does not always increase parallel to the exacerbation of POPFs. Pancreatic texture, which is a well-known risk factor of POPF, is problematic in that it is determined by surgeons subjectively. On the other hand, the CRP level has the advantages that it can be measured objectively and that it enables the evaluation of the degree of comprehensive inflammation easily. As shown in Figure 2, subjects with invasive carcinoma of the pancreas were less likely to have POPFs, but the POPFs tended to be protracted when they did occur. It has been postulated that the surrounding tissues and the bloodstream around the anastomotic site are diminished by the dissection of lymph nodes, leading to poorer wound healing [30]. Additionally, the size and texture of the pancreatic duct (which have been recognized as major risk factors for POPFs) were not significantly associated with aggravated POPFs in the present study. Patient assessment revealed that the size and texture of the pancreatic duct increased the formation of POPFs but did not always lead directly to persistent POPFs. We proposed a novel risk scoring scale for clinically relevant POPFs. The score demonstrated good compatibility with the prevalence of clinically relevant POPFs. The score included five parameters that are available at POD 3 (including changes in the CRP level) that achieved a better goodness of fit for the aggravation of POPFs than changes in the CRP level. The scoring scale can be easily calculated at POD 3, indicating that it would be a convenient and useful method for patient selection. After the diagnosis of POPFs at POD 3, changes in the CRP level and the risk score scale were helpful in determining the subsequent management. In patients with a continuous elevation of the CRP level or a high risk score, frequent physical assessment, a computed tomography, and the culture of drainage fluids may be considered to allow early management of aggravated POPFs. Additionally, the strict control of infection, thorough management 8542 July 14, 2014 Volume 20 Issue 26

235 Kanda M et al. Predictors of aggravating pancreatic fistula of drains, and administration of somatostatin analogs might be recommended, depending on a patient s condition. Conversely, patients without elevations of the CRP level or a high risk score are not at risk of clinically relevant POPFs, so an early removal of the drains and early discharge from the hospital should be considered. The benefits of early drain removal have been reported by Bassi et al [31] and Kawai et al [32], but subsequent abdominal abscesses and in-hospital death have also been observed. Changes in the CRP levels and the risk score scale could help in the selection of patients eligible for the early removal of drains. Welsch et al [33] reported that a high CRP level (140 mg/l) at POD 4 is a diagnostic factor for overall infectious complications after pancreatectomy. Our results would apply better to clinical practice due to their focus on clinically relevant POPFs at an earlier time point. The present study had limitations, including a low number of enrolled patients and the retrospective determination of cut-off values in an explorative setting. The inflammatory markers and the amylase content in drain fluid were not examined every day. The measurement of the levels of proinflammatory cytokines such as interleukin-6 would have made this study more complete. Because the results of the current study were exploratory, this analysis may lead to prospective studies evaluating the hypothesis that an elevation of the CRP level from PODs 1 to 3 might have a diagnostic value for the identification of patients with clinically relevant POPFs. Welldesigned prospective clinical studies based on changes in the CRP level or the use of a high-performance risk scoring scale at the acute phase after pancreatectomy could help to establish the therapeutic strategies for POPFs. In conclusion, a steep rise in the serum CRP level from 1 POD to 3 PODs was shown to be a highly diagnostic factor for clinically relevant POPFs and could be helpful in selecting the most appropriate management for POPFs. The scoring scale consisting of five diagnostic factors identified in the current study may provide additional assistance for patient classification. COMMENTS Background Aggravation of postoperative pancreatic fistulas (POPFs) leads to prolonged hospitalization and burgeoning medical costs and can develop into fatal conditions. Research frontiers The identification of sensitive diagnostic factors of clinically relevant POPFs are needed to identify high-risk patients and strengthen measures aimed at preventing the aggravation of POPFs. Innovations and breakthroughs To date, many pre- and intraoperative risk factors of POPF formation have been reported. However, the selection methods for high-risk patients with aggravated POPFs at the acute phase after pancreatectomy have not been fully discussed. In this study, the elevation of the C-reactive protein (CRP) level was found to be a strong diagnostic factor of a clinically relevant POPF. Applications The changes in CRP levels and the risk score can provide a good indication for the management of a POPF, including the early removal of the drainage tubes. The inclusion of proinflammatory cytokines (e.g., interleukin-6) may achieve a further refinement of the risk score. Terminology POPF is the most common complication in pancreatic surgeries and can lead to fatal condition. How to control POPFs is one of the most important issues in the field of pancreatic surgery. Peer review This retrospective study identifies an increase in CRP as a good marker for predicting aggravation of a POPF. The conclusion of this study can assist surgeons, especially in the appropriate management of POPFs. 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237 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. ORIGINAL BRIEF ARTICLE Clobenpropit enhances anti-tumor effect of gemcitabine in pancreatic cancer Woo Hyun Paik, Ji Kon Ryu, Kyoung-Sin Jeong, Jin Myung Park, Byeong Jun Song, Sang Hyub Lee, Yong-Tae Kim, Yong Bum Yoon Woo Hyun Paik, Department of Internal Medicine, Inje University Ilsan Paik Hospital, Koyang , South Korea Ji Kon Ryu, Kyoung-Sin Jeong, Jin Myung Park, Byeong Jun Song, Sang Hyub Lee, Yong-Tae Kim, Yong Bum Yoon, Division of Gastroenterology, Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, Seoul , South Korea Author contributions: Paik WH performed the experiments and wrote the manuscript; Jeong KS performed the majority of the experiments; Ryu JK designed the study and revised the manuscript; Park JM and Song BJ performed the experiments; Lee SH, Kim YT and Yoon YB critically revised the manuscript. Supported by Korean Society of Internal Medicine Research Fund (2012) and the Seoul National University College of Medicine Research Fund (2011) Correspondence to: Ji Kon Ryu, MD, PhD, Associate Professor, Division of Gastroenterology, Department of Internal Medicine and Liver Research Institute, Seoul National University College of Medicine, 101 Daehak-ro, Jongno-gu, Seoul , South Korea. jkryu@snu.ac.kr Telephone: Fax: Received: December 1, 2013 Revised: February 27, 2014 Accepted: April 5, 2014 Published online: July 14, 2014 Abstract AIM: To evaluate the anti-tumor effect of clobenpropit, which is a specific H3 antagonist and H4 agonist, in combination with gemcitabine in a pancreatic cancer cell line. METHODS: Three kinds of human pancreatic cancer cell lines (Panc-1, MiaPaCa-2, and AsPC-1) were used in this study. Expression of H3 and H4 receptors in pancreatic cancer cells was identified with Western blotting. Effects of clobenpropit on cell proliferation, migration and apoptosis were evaluated. Alteration of epithelial and mesenchymal markers after administration of clobenpropit was analyzed. An in vivo study with a Panc-1 xenograft mouse model was also performed. RESULTS: H4 receptors were present as 2 subunits in human pancreatic cancer cells, while there was no expression of H3 receptor. Clobenpropit inhibited cell migration and increased apoptosis of pancreatic cancer cells in combination with gemcitabine. Clobenpropit up-regulated E-cadherin, but down-regulated vimentin and matrix metalloproteinase 9 in real-time polymerase chain reaction. Also, clobenpropit inhibited tumor growth (gemcitabine 294 ± 46 mg vs combination 154 ± 54 mg, P = 0.02) and enhanced apoptosis in combination with gemcitabine (control 2.5%, gemcitabine 25.8%, clobenpropit 9.7% and combination 40.9%, P = 0.001) by up-regulation of E-cadherin and downregulation of Zeb1 in Panc-1 xenograft mouse. CONCLUSION: Clobenpropit enhanced the anti-tumor effect of gemcitabine in pancreatic cancer cells through inhibition of the epithelial-mesenchymal transition process Baishideng Publishing Group Inc. All rights reserved. Key words: Clobenpropit; Epithelial-mesenchymal transition; Histamine; Histamine receptors; Pancreatic neoplasm Core tip: Histamine is associated with carcinogenesis through activation of its 4 membrane-specific receptors. Clobenpropit, which is an agonist of H4 receptor, inhibited cell migration and increased apoptosis of pancreatic cancer cells in combination with gemcitabine. Clobenpropit up-regulated an epithelial marker, but down-regulated mesenchymal markers in real-time polymerase chain reaction. In addition, clobenpropit inhibited tumor growth and enhanced apoptosis in combination with gemcitabine by up-regulation of E-cadherin and down-regulation of Zeb1 in Panc-1 xenograft mouse. In conclusion, clobenpropit enhanced anti-tumor effects of gemcitabine in pancreatic cancer cells through inhibition of epithelial-mesenchymal transition process July 14, 2014 Volume 20 Issue 26

238 Paik WH et al. Anti-tumor effect of clobenpropit in pancreatic cancer Paik WH, Ryu JK, Jeong KS, Park JM, Song BJ, Lee SH, Kim YT, Yoon YB. Clobenpropit enhances anti-tumor effect of gemcitabine in pancreatic cancer. World J Gastroenterol 2014; 20(26): Available from: URL: com/ /full/v20/i26/8545.htm DOI: org/ /wjg.v20.i INTRODUCTION Pancreatic cancer is a very aggressive human cancer and has dismal prognosis with only 6% of patients surviving 5 years after diagnosis [1]. In spite of the progress of treatments, attempts at survival prolongation, especially in the advanced stage, have failed and resulted in no significant improvement [2]. Surgical resection is the only potentially curative treatment in pancreatic cancer, but only 15% of patients are candidates for resection [3,4]. Some chemotherapeutic agents are used in pancreatic cancer, and gemcitabine became the standard chemotherapeutic agent in pancreatic cancer after a randomized trial in 1997 [5]. Gemcitabine is a nucleoside pyrimidine analogue which exerts its cytotoxic actions primarily by the incorporation of gemcitabine triphosphate into DNA, leading to masked chain termination [6]. However, pancreatic cancer is highly resistant to chemotherapy including gemcitabine [7], and the disappointing circumstance of pancreatic cancer is mainly due to late diagnosis at which chemoresistance in patients is a critical issue [8]. Resistance to gemcitabine has been increasing and the effectiveness of gemcitabine has been reduced to less than 20% [9]. Hence, new therapeutic targets and chemotherapeutic agents of pancreatic cancer are desperately required. A high concentration of histamine has been shown in melanoma [10], small cell lung carcinoma [11], breast carcinoma [12] and colorectal carcinoma [13]. Histamine participates in tumor proliferation or apoptosis through activation of its four membrane-specific receptors, H1, H2, H3 and H4 [14]. As in other human cancers, expression of histidine decarboxylase and histamine contents have been reported in pancreatic cancer [15,16]. Histamine inhibits cell proliferation through the H1 and H2 receptor, which is associated with a partial differentiation in pancreatic cancer [17]. Through the H2 receptor, histamine induces G0/G1 phase arrest and modulation of mitogen activated protein kinase and Bcl-2 family proteins [18-20]. Furthermore, a previous study suggests that H3 and H4 receptors are involved in pancreatic cancer cell growth, with proliferation increased through H3 receptor and diminished via H4 receptor [21]. However, the mechanism of the anti-cancer effect through the histamine receptor still remains unclear. Clobenpropit, which is a specific H3 antagonist and H4 agonist, inhibits the spread of mammary adenocarcinoma by decreasing invasion potential [22]. A recent study suggested that the modulation of H4 receptor by clobenpropit disrupts epithelial-mesenchymal transition (EMT) processes, extracellular matrix (ECM) breakdown, and invasion potential and decreases tumor growth in cholangiocarcinoma [23]. Similarly, EMT plays a crucial role in tumor progression and metastasis of pancreatic cancer [24]. Furthermore, EMT regulators including Zeb1 are known to induce chemoresistance of human pancreatic cancer cells [25]. Thus, therapeutic agents targeting the EMT process could restore the chemoresistance of pancreatic cancer. Therefore, we aimed to investigate the anticancer efficacy of clobenpropit with gemcitabine combination in human pancreatic cancer cells. Additionally, we evaluated the alteration of EMT markers after administration of clobenpropit with in vitro and in vivo studies. MATERIALS AND METHODS Pancreatic cancer cell lines Human pancreatic cancer cell lines, Panc-1, MiaPaCa-2 and AsPC-1 were obtained from Korea Cell Line Bank and maintained in Dulbecco modified Eagle medium containing 10% fetal bovine serum. They were incubated at 37 and 5% CO2. Western blotting After washing with PBS, three kinds of pancreatic cancer cells (Panc-1, MiaPaCa-2 and AsPC-1) at 70-80% confluence were processed and lysed in NP-40 buffer (20 mmol Tris-HCl, ph 7.4, 100 mmol NaCl, 1% NP-40, 0.5% sodium deoxycholate, 5 mmol MgCl2, 0.1 mmol phenylmethylsulfonyl fluoride, and 10 mg/ml protease inhibitor mixture). Then, proteins were quantified with a BCA protein assay kit (Pierce, Rockford, IL). Proteins were separated by 10% to 15% SDS-polyacrylamide denaturing gels, transblotted onto nitrocellulose membranes and probed with rabbit antihuman H3 and H4 receptor antibodies (Millipore, Billerica, MA). Immunoreactivity was developed using a peroxidase conjugate antiserum (Sigma-Aldrich, St Louis, MO) and detected by enhanced chemiluminescence reagents (Amersham Biosciences, Baied Urfe, Quebec, Canada). Western blotting of Panc-1, MiaPaCa-2 and AsPC-1 treated with 50 or 100 μmol/l of clobenpropit (Sigma-Aldrich) alone or in combination with 5 μmol/l of gemcitabine (Yuhan, Seoul, South Korea) was also performed. Wound healing assay Three kinds of pancreatic cancer cells ( ) were seeded in 6-well plates and cultured until reaching 70%-80% confluence as a monolayer. A straight scratch was made on cell plates, and then cell plates were gently rinsed to remove the detached cells. After adding 0, 10 and 50 μmol/l of clobenpropit to each plate, cells were grown for an additional 48 h. After washing the cells with PBS twice, photos were taken on a confocal microscope (Leica, Wetzlar, Germany). Wound healing ranges were measured by Aperio ImageScope V (Aperio Technologies, Vista, CA). Additional wound healing assays after administration of gemcitabine (5 μmol/l) and/or clobenpropit (50 μmol/l) in Panc-1 and gemcitabine (15 μmol/l) and/or clobenpropit (50 μmol/l) 8546 July 14, 2014 Volume 20 Issue 26

239 Paik WH et al. Anti-tumor effect of clobenpropit in pancreatic cancer Table 1 Sequences of primers used for real-time polymerase chain reaction Gene H4 receptor GAPDH E-cadherin Vimentin Fibronectin MMP-9 CK-19 Sequences Forward: 5 -GTGGTTAGCATAGGTTATAC-3 Reverse: 5 -ATGCCACTGCACTCCTGC-3 Forward: 5 -ACGGATTTGGTCGTATTGGG-3 Reverse: 5 -TGATTTTGGAGGGATCTCGC-3 Forward: 5 -GCCTCCTGAAAAGAGAGTGGAAG-3 Reverse: 5 -TGGCAGTGTCTCTCCAAATCCG-3 Forward: 5 -AGGCAAAGCAGGAGTCCACTGA-3 Reverse: 5 -ATCTGGCGTTCCAGGGACTCAT-3 Forward: 5 -ACAACACCGAGGTGACTGAGAC-3 Reverse: 5 -GGACACAACGATGCTTCCTGAG-3 Forward: 5 -TTCTGCCCCAGCGAGAGA-3 Reverse: 5 -GTGCAGGCGGAGTAGGATTG-3 Forward: 5 -GAAGAACCATGAGGAGGAAATCA-3 Reverse: 5 -ACCTCATATTGGCTTCGCATGT-3 were performed to evaluate the change of cell migration after gemcitabine and clobenpropit combination treatment. Real-time polymerase chain reaction Gene expression was evaluated in mrna from all pancreatic cancer cell lines. Total RNA was extracted using Trizol Reagent (Invitrogen, Carlsbad, CA). RNA samples were diluted to a final concentration of 0.5 mg/ml in RNase-free water and stored at -80 until use. Synthesis of the cdna was performed with 1 mg of total RNA with M-MLV reverse transcription reagents (Invitrogen), and real-time polymerase chain reaction (PCR) reaction was carried out on the ABI PRISM 7000 Sequence Detection System (Applied Biosystems, Foster City, CA) in 20 μl TaqMan Gene Expression Master Mix (Applied Biosystems) using 200 ng cdna. Human primers sets were ordered and used according to their protocols. The specific primers were described in Table 1 [26-29]. The human β-actin gene was used as an endogenous reference to control for the independent expression of sample-to-sample variability. The relative expression of target genes was normalized by dividing the target Ct value by the endogenous Ct values. All equipment was purchased from Applied Biosystems and used according to manufacturer s protocols. MTS proliferation assay Cells were plated into 96-well plates at a density of cells/well and stimulated with clobenpropit (1 to 100 μmol/l) for up to 48 h to determine optimal dose and stimulation time. 3-(4,5-Demethylthiazole-2- yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2htetrazolium (MTS; Promega, Madison, WI) was added to the cells, and the numbers of live cells were counted after allowing development for 1 h. The plates were read on a Wallac-1420 plate reader (Perkin-Elmer, Boston, MA) at an absorbance of 490 nm. Data are expressed as fold change of treated cells as compared with basal treated controls. Apoptosis determination Pancreatic cancer cells were cultured and divided into 4 groups according to the treatment: (1) control; (2) gemcitabine (5 μmol/l) alone; (3) clobenpropit (50 μmol/l) alone; and (4) gemcitabine (5 μmol/l) and clobenpropit (50 μmol/l) combination. After trypsinization, cells were incubated with annexin V-fluorescein isothiocyanate and propidium iodide (BD Biosciences, Franklin Lakes, NJ) for 15 min at room temperature in the dark. The degree of apoptosis was analyzed by fluorescence activated cell sorting. The proportion of stained cells in each quadrant was quantified with CellQuest software (BD Biosciences). Animal experiments Five-week-old male BALB/c nude mice were purchased from Orient (Gyeonggi-do, Korea). Mice were housed under specific pathogen-free conditions, and a γ-rayirradiated laboratory rodent diet (Purina Korea, Gyeonggi-do, South Korea) and autoclaved water were provided ad libitum. All the protocols for the animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at Seoul National University Hospital (IACUC No ). All animal procedures were consistent with the Guide for the Care and Use of Laboratory Animals issued by the Institute of Laboratory Animal Resources Commission on Life Science, United States National Research Council. To generate tumors, Panc-1 was subcutaneously inoculated with cells suspended in 0.15 ml of Matrigel. All mice were divided into 4 groups randomly, and each group consisted of 5 mice: (1) control (vehicle alone); (2) gemcitabine (twice-a-week intraperitoneal injection at 125 mg/kg for 40 d); (3) clobenpropit (every other day intraperitoneal injection at 20 μmol/l per kilogram for 40 d); and (4) gemcitabine (twice-a-week intraperitoneal injection at 125 mg/kg for 40 d) and clobenpropit (every other day intraperitoneal injection at 20 μmol/l per kilogram for 40 d) [23,30]. The body weight of each mouse was measured weekly with an electronic scale. Tumor size was measured every week with electronic calipers and the volume was calculated by the following formula: tumor volume = (length width 2 ) π/6 [31]. One week later, after finishing the treatment schedule, mice were anesthetized with isoflurane and tissue, organs and tumors were harvested for analysis. The expression of EMT markers was investigated by real-time PCR in whole tumor mrna. Tumor samples were fixed in 10% buffered formalin, embedded in lowtemperature fusion paraffin, and sectioned for terminal deoxynucleotidyl transferase (TdT)-mediated dutp nick end labeling (TUNEL) staining and immunohistochemical staining. Western blots were conducted for the assessment of E-cadherin, vimentin, MMP-9 and Zeb1 expressions. Antibodies of E-cadherin, vimentin, MMP-9 and Zeb1 were purchased from Santa Cruz. Statistical analysis All experimental results represent at least 3 indepen July 14, 2014 Volume 20 Issue 26

240 Paik WH et al. Anti-tumor effect of clobenpropit in pancreatic cancer A Panc MiaPaCa AsPc H4 receptor 1: Control; 70 kda 2: Clobenpropit 43 kda 50 μmol/l; 3: Clobenpropit β-actin 100 μmol/l B Panc-1 MiaPaCa-2 AsPc H4 receptor 1: Control; 2: Gemcitabine 70 kda 5 μmol/l; 3: Clobenpropit 50 μmol/l; 4: Gemcitabine β-actin 5 μmol/l + Clobenpropit 50 μmol/l Figure 1 Western blotting of histamine receptors in pancreatic cancer cells. Cellular extracts of Panc-1, MiaPaCa-2 and AsPC-1 were separated by SDS-PAGE, transblotted onto nitrocellulose membranes and detected using anti-h4 receptor antibodies. Pancreatic cancer cell lines were treated with different concentrations of clobenpropit and/or gemcitabine, and Western blotting was performed. The specific antibody to H4 receptor showed immunoreactivity mainly as a band at and Da. Expression of H4 receptor was still present after administration of clobenpropit alone (A) or combination with gemcitabine (B). Xenograft mouse model In the Panc-1 xenograft mouse model, the body weight of mice was checked every week (Figure 7A). There was no significant difference in mean body weight between the groups. There was no mortality of mice until the end of treatment. The combination treatment of gemcitabine with clobenpropit showed significant tumor growth inhibition compared with other treatment groups (control 501 ± 92 mg, gemcitabine 294 ± 46 mg, clobenpropit 444 ± 167 mg, and combination 154 ± 54 mg, Figure 7B and C). E-cadherin was up-regulated after clobenpropit administration in real-time PCR (Figure 8A). Immunohistochemical staining also showed up-regulation of E-cadherin in clobenpropit alone and combination group (Figure 8C). To quantify apoptosis of tumors, TUNEL staining was performed. The percentage of TUNELdent experiments using cells from a minimum of three separate isolations. Results for continuous variables are expressed as mean ± SE of mean and compared with the Kruskal-Wallis ANOVA followed by Dunn s multiple comparison test or repeated measures ANOVA. P < 0.05 was considered statistically significant. Analysis was performed with GraphPad Prism version 5.04 (GraphPad Software Inc., La Jolla, CA). RESULTS H4 receptor was present in pancreatic cancer cells The specific antibody to H4 receptor showed immunoreactivity mainly as a band at and Da in Panc-1, MiaPaCa-2 and AsPC-1 (Figure 1A). However, there was no H3 receptor expression in all cell lines (data not shown). When clobenpropit (50 μmol/l) and/or gemcitabine (5 μmol/l) were added, H4 receptor expression was also seen but was not significantly changed in all cell lines (Figure 1B). Clobenpropit inhibited cell migration by inhibition of EMT process The inhibition of cell migration was assessed by a wound healing assay. Clobenpropit inhibited the migration of pancreatic cancer cells in a wound healing assay (Figure 2). Wound healing ranges were decreased when clobenpropit concentration was higher. In addition, the migration rate was also inhibited after treatment with gemcitabine and clobenpropit combination compared with control and gemcitabine or clobenpropit alone (Figure 3). To investigate the mechanism of clobenpropit on cell migration, real-time PCR for epithelial and mesenchymal markers was performed. E-cadherin was about 4-fold increased after treatment of clobenpropit in Panc-1. MMP-9 was reduced in MiaPaCa-2, and vimentin and MMP-9 were reduced to about half after treatment of clobenpropit in AsPC-1 (Figure 4). Therefore, clobenpropit down-regulated epithelial markers, while up-regulating mesenchymal markers; this means that clobenpropit might disrupt EMT process of pancreatic cancer cells. Clobenpropit enhanced gemcitabine-induced apoptosis Clobenpropit alone did not affect the proliferation of pancreatic cancer cells (Figure 5). However, the exposure to gemcitabine (5 μmol/l) and/or clobenpropit (50 μmol/l) induced apoptosis of pancreatic cancer cells. Gemcitabine and clobenpropit combination therapy significantly increased apoptosis of Panc-1, MiaPCa-2 and AsPC-1 compared with control (Figure 6). However, gemcitabine alone did not increase apoptosis of pancreatic cancer cells significantly compared with control July 14, 2014 Volume 20 Issue 26

241 Paik WH et al. Anti-tumor effect of clobenpropit in pancreatic cancer A Panc-1 Control Clobenpropit 10 μmol/l Clobenpropit 50 μmol/l 80 a Wound healing range (%) a Clobenpropit (μmol/l) B MiaPaCa-2 Control Clobenpropit 10 μmol/l Clobenpropit 50 μmol/l Wound healing range (%) NS a Clobenpropit (μmol/l) 8549 July 14, 2014 Volume 20 Issue 26

242 Paik WH et al. Anti-tumor effect of clobenpropit in pancreatic cancer C AsPC-1 Control Clobenpropit 10 μmol/l Clobenpropit 50 μmol/l 80 NS b Wound healing range (%) Clobenpropit (μmol/l) Figure 2 Effect of clobenpropit on cell migration. Clobenpropit inhibits the migration of Panc-1 (A), MiaPaCa-2 (B) and AsPC-1 (C) in wound healing assay. Wound healing range was inversely correlated with clobenpropit concentration in MiaPaCa-2 and AsPC-1. a P < 0.05, b P < 0.01 vs control. A 1.2 B Fold change Fold change Basal Clobenpropit (μmol/l) Basal Clobenpropit (μmol/l) Figure 3 Effect of clobenpropit on pancreatic cancer cell proliferation was evaluated by MTS assay. A: Panc-1, B: AsPC-1. Clobenpropit showed no cytotoxicity in pancreatic cancer cells. stained cells was increased more in the gemcitabine and clobenpropit combination group compared with other groups (control 2.5%, gemcitabine 25.8%, clobenpropit 9.7% and combination 40.9%, Figure 8B). The expression of E-cadherin was also increased in clobenpropit alone and combination group by Western blotting, whereas Zeb1 expression, a repressor of E-cadherin, was decreased in combination group (Figure 8D). DISCUSSION The role of histamine and its receptors in carcinogenesis is complex and somehow confusing. We evaluated the effect of a specific histamine receptor and its agonist against human pancreatic cancer cells in this study. The significant findings in the present study are that clobenpropit emphasized gemcitabine-induced apoptosis of hu July 14, 2014 Volume 20 Issue 26

243 Paik WH et al. Anti-tumor effect of clobenpropit in pancreatic cancer A Panc-1 80 Wound healing range (%) Control G 0 Control G C G + C C G + C B MiaPaCa-2 80 Wound healing range (%) Control G 0 Control G C G + C C G + C Figure 4 Migration of pancreatic cancer cells was inhibited after gemcitabine and clobenpropit combination in wound healing assay. The concentrations of gemcitabine in Panc-1 (A) and MiaPaCa-2 (B) were 5 and 15 μmol/l, respectively. The concentration of clobenpropit was 50 μmol/l. G: Gemcitabine; C: Clobenpro- man pancreatic cancer cells in vitro in cell culture as well as in vivo in tumor xenograft mice. There was no adverse health effect due to clobenpropit in mice as monitored by body weight. The enhanced cytotoxicity of gemcitabine and clobenpropit combination might result from disruption of EMT through H4 receptor. Three kinds of pancreatic cancer cells (Panc-1, Mia- PaCa-2 and AsPC-1) were used in this study, and all the cells had H4 receptors. The H4 specific antibody showed immunoreactivity mainly as a band at and Da, which is consistent with a previous report [32]. Increasing evidence indicates that cancer cells are subjected to the EMT, a process by which epithelial cells undergo remarkable morphologic changes characterized by a transition from an epithelial to a mesenchymal phenotype leading to increased motility and invasion [33]. EMT is a developmental process which plays an important role in tumor progression and metastasis in diverse solid tumors, including pancreatic cancer [25]. E-cadherin, a calcium-dependent transmembrane glycoprotein, is one of the most important molecules in cell-cell adhesion in epithelial tissue [34], which localized on the surface of epithelial cells in regions of adherent junctions [35]. It plays a major role in malignant cell transformation, and especially in tumor development and progression. Loss of E-cadherin is associated with invasion and metastasis of 8551 July 14, 2014 Volume 20 Issue 26

244 Paik WH et al. Anti-tumor effect of clobenpropit in pancreatic cancer A 5 4 Panc-1 Control 0.1 mmol/l 1 mmol/l 10 mmol/l A NS Panc-1 a Fold change 3 2 Apoptotic cell (%) B H4 E-cadherin Vimentin MiaPaCa-2 Fibronectin CK-19 MMP-9 B 0 60 Control G C G + C MiaPaCa-2 a NS Fold change Apoptotic cell (%) H4 E-cadherin Vimentin Fibronectin CK-19 MMP-9 C 0 50 Control G C G + C AsPC-1 a C Fold change AsPC-1 Apoptotic cell (%) NS H4 E-cadherin Vimentin Fibronectin CK-19 MMP-9 Figure 5 Changes of epithelial and mesenchymal markers of pancreatic cancer cells by clobenpropit. Real-time polymerase chain reaction shows that E-cadherin was up-regulated (A), whereas MMP-9 and vimentin were down-regulated (B, C) in pancreatic cancer cells after treatment with different concentrations of clobenpropit. tumors [36]. Furthermore, the loss of E-cadherin expression has been associated with a poor clinical outcome in several cancers [25,37,38]. The H4 receptor agonist increased the expression of E-cadherin in this study, both in vitro in cell culture and in vivo in xenograft mouse. Clobenpropit 0 Control G C G + C Figure 6 Enhanced apoptosis of pancreatic cancer cells by gemcitabine and clobenpropit combination treatment. Clobenpropit enhanced apoptotic cell death in combination of gemcitabine in human pancreatic cancer cells. The percentage of apoptotic cells was determined by fluorescein isothiocyanate-labeled annexin V assay followed by flow cytometry. Statistically significant differences ( a P < 0.05 vs control group) of the combination treatment of gemcitabine (5 μmol/l) and clobenpropit (50 μmol/l) compared with control in Panc-1 (A), MiaPaCa-2 (B) and AsPC-1 (C). G: Gemcitabine; C: Clobenpropit. could play an important role with interfering cell migration and increasing chemosensitivity of gemcitabine in pancreatic cancer cells through inhibition of EMT process and up-regulation of E-cadherin. Transcriptional repressors of E-cadherin such as Zeb1, Zeb2, Twist, Snail and Slug are associated with 8552 July 14, 2014 Volume 20 Issue 26

245 Paik WH et al. Anti-tumor effect of clobenpropit in pancreatic cancer A 25 C Body weight (g) Control G C G + C t /d B Tumor volume (mm 2 ) Control G C G + C b b a Control G C G + C t /d Figure 7 Inhibition of tumor growth in Panc-1 xenograft mouse by gemcitabine and clobenpropit combination treatment. Body weight (A) and tumor volume (B) curves for Panc-1 xenograft mouse model with administration of vehicle (control), gemcitabine, clobenpropit or their combination. There was no significant difference of body weight between the groups. Tumor volume of gemcitabine and clobenpropit combination therapy group was significant lower than control and other treatment groups. Tumor bearing mice and excised tumor of each treatment group (C). G: Gemcitabine; C: Clobenpropit. a P < 0.05, b P < 0.01 vs control. EMT [39-42]. Diverse signal pathways such as Wnt cascade, TGF-β and PI3K/Akt pathway are connected with these transcriptional repressors of E-cadherin [43,44]. Zeb1 expression was decreased in the clobenpropit treated mice group compared with the control and gemcitabine alone groups. Zeb1 could act as the main transcriptional repressor of E-cadherin in this study although the relationship between Zeb1 and H4 receptor remains unsolved. Vimentin is a mesenchymal marker which is up-regulated with EMT [45]. Down-regulation of vimentin after clobenpropit administration suggests that H4 receptor agonist disrupts the EMT process. For the invasion and metastasis of tumors, breakdown of the ECM should be present [46]. Clobenpropit may protect the ECM from breakdown by down-regulation of MMP-9, preventing invasion or metastasis of pancreatic cancer. The change of epithelial markers or mesenchymal markers after administration with clobenpropit was different according to the cells. E-cadherin was increased in Panc-1 only, while vimentin was decreased in AsPC-1 only. MMP-9 was decreased in MiaPaCa-2 and AsPC-1. This can be explained by the different expressions of EMT markers according to the cancer cells [25]. However, it was coherent that the change of EMT markers indicated the disruption of the EMT process by clobenpropit in this study. The chemosensitivity of pancreatic cancer cells varies, and Panc-1, MiaPaCa-2 and AsPC-1 are known to be resistant to gemcitabine [25]. In order to clarify the effect of clobenpropit, we used the most chemoresistant cells (Panc-1) in the xenograft model. It is known that pancreatic cancer cells undergoing EMT with increased expression of Snail and Twist become invasive and develop chemoresistance [47]. In addition, EMT reversion by silencing Zeb1 increases cellular sensitivity to gemcitabine [25]. The apoptosis of pancreatic cancer cells was significantly increased after gemcitabine and clobenpropit combination treatment in present study. Moreover, the tumor volume of the xenograft mouse was significantly decreased in the combination group compared with control and clobenpropit or gemcitabine alone groups, and TUNEL stain also showed increased apoptosis in the combination group. These results support the idea that therapeutic targeting to reverse EMT could increase chemosensitivity in pancreatic cancer. However, further studies are needed to clarify the molecular alterations which reverse EMT through H4 receptor. In conclusion, clobenpropit enhanced gemcitabineinduced apoptosis in human pancreatic cancer cells by inhibition of EMT process. The novel role of the H4 re July 14, 2014 Volume 20 Issue 26

246 Paik WH et al. Anti-tumor effect of clobenpropit in pancreatic cancer A 3 H4 5 a E-cadherin a 4 2 a Fold change 1 Fold change Control G C G + C 0 Control G C G + C 3 Vimentin 3 Fibronectin 2 2 Fold change 1 Fold change 1 0 Control G C G + C 0 Control G C G + C 2 CK-19 2 MMP-9 Fold change 1 Fold change 1 0 Control G C G + C 0 Control G C G + C B 50 b a 40 % TUNEL-positive cells a 0 Control G C G + C 8554 July 14, 2014 Volume 20 Issue 26

247 Paik WH et al. Anti-tumor effect of clobenpropit in pancreatic cancer C Control G C G + C Vimentin E-cadherin TUNEL HE 100 mm 100 mm 100 mm 100 mm 50 mm 50 mm 50 mm 50 mm 50 mm 50 mm 50 mm 50 mm D Control G C G + C E-cadherin Vimentin MMP-9 Zeb1 β-actin Figure 8 Effect of gemcitabine and clobenpropit combination treatment in Panc-1 xenograft mouse. A: Real-time polymerase chain reaction shows increased E-cadherin expression after clobenpropit treatment compared with gemcitabine alone; B: Immunohistochemical staining shows up-regulation of E-cadherin in gemcitabine and clobenpropit combination group. Apoptotic index calculated with TUNEL staining shows increased apoptosis in gemcitabine and clobenpropit combination group; C: Pathological evaluation of tumor tissue determined by hematoxylin and eosin staining, TUNEL staining and immunohistochemistry of E-cadherin and vimentin; D: E-cadherin was also increased in clobenpropit alone and combination group by Western blotting, whereas Zeb1, the repressor of E-cadherin, was decreased in combination group. G: Gemcitabine; C: Clobenpropit. a P < 0.05, b P < 0.01 vs control. ceptor in carcinogenesis of pancreatic cancer represents a new therapeutic molecular target and clobenpropit could be a promising drug. Further studies are required to reveal the mechanism of EMT inhibition via H4 receptor. ACKNOWLEDGMENTS Grateful thanks to Hye Jo Ryu for assistance with experiments. COMMENTS Background Histamine is associated with carcinogenesis through activation of its 4 membrane-specific receptors. Pancreatic cancer, which is a very aggressive human cancer, is highly resistant to conventional chemotherapy. Therefore, new treatment options including targeting histamine receptors in pancreatic cancer are required. Research frontiers Recently, the modulation of the histamine receptor by clobenpropit disrupts 8555 July 14, 2014 Volume 20 Issue 26

248 Paik WH et al. Anti-tumor effect of clobenpropit in pancreatic cancer the epithelial-mesenchymal transition (EMT) process, and results in inhibition of tumor growth in cholangiocarcinoma. Similarly, EMT plays a critical role in tumor progression and metastasis of pancreatic cancer. The research hotspot is to evaluate the anti-tumor effect and mechanism of clobenpropit in pancreatic cancer. Innovations and breakthroughs Clobenpropit emphasized gemcitabine-induced apoptosis of pancreatic cancer. The enhanced cytotoxicity of gemcitabine and clobenpropit may result from disruption of EMT through H4 receptor. Applications The study results suggest that the combination of clobenpropit and gemcitabine may be a promising treatment in pancreatic cancer. Peer review The present study reported that clobenpropit enhanced the anti-tumor effect of gemcitabine in pancreatic cancer cells through inhibition of the EMT process. 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249 Paik WH et al. Anti-tumor effect of clobenpropit in pancreatic cancer Dermatol 2010; 163: [PMID: DOI: / j x] 27 Nakamura K, Ninomiya I, Oyama K, Inokuchi M, Kinami S, Fushida S, Fujimura T, Kayahara M, Ohta T. Evaluation of immune response according to the metastatic status in the regional lymph nodes in patients with gastric carcinoma. Oncol Rep 2010; 24: [PMID: ] 28 Chang ZG, Wei JM, Qin CF, Hao K, Tian XD, Xie K, Xie XH, Yang YM. Suppression of the epidermal growth factor receptor inhibits epithelial-mesenchymal transition in human pancreatic cancer PANC-1 cells. Dig Dis Sci 2012; 57: [PMID: DOI: /s ] 29 Kapral M, Wawszczyk J, Jurzak M, Hollek A, Węglarz L. The effect of inositol hexaphosphate on the expression of selected metalloproteinases and their tissue inhibitors in IL- 1β-stimulated colon cancer cells. Int J Colorectal Dis 2012; 27: [PMID: DOI: /s ] 30 Lee SH, Ryu JK, Lee KY, Woo SM, Park JK, Yoo JW, Kim YT, Yoon YB. 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Cancer Res 2006; 66: [PMID: DOI: / can ] 39 Yang J, Mani SA, Donaher JL, Ramaswamy S, Itzykson RA, Come C, Savagner P, Gitelman I, Richardson A, Weinberg RA. Twist, a master regulator of morphogenesis, plays an essential role in tumor metastasis. Cell 2004; 117: [PMID: DOI: /j.cell ] 40 Comijn J, Berx G, Vermassen P, Verschueren K, van Grunsven L, Bruyneel E, Mareel M, Huylebroeck D, van Roy F. The twohanded E box binding zinc finger protein SIP1 downregulates E-cadherin and induces invasion. Mol Cell 2001; 7: [PMID: ] 41 Hajra KM, Chen DY, Fearon ER. The SLUG zinc-finger protein represses E-cadherin in breast cancer. Cancer Res 2002; 62: [PMID: ] 42 Eger A, Aigner K, Sonderegger S, Dampier B, Oehler S, Schreiber M, Berx G, Cano A, Beug H, Foisner R. DeltaEF1 is a transcriptional repressor of E-cadherin and regulates epithelial plasticity in breast cancer cells. Oncogene 2005; 24: [PMID: DOI: /sj.onc ] 43 Iwatsuki M, Mimori K, Yokobori T, Ishi H, Beppu T, Nakamori S, Baba H, Mori M. 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Ann Surg Oncol 2007; 14: [PMID: DOI: /s ] P- Reviewers: Freund JN, Takao S S- Editor: Qi Y L- Editor: O Neill M E- Editor: Zhang DN 8557 July 14, 2014 Volume 20 Issue 26

250 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. ORIGINAL BRIEF ARTICLE Changes in circulating Foxp3 + regulatory T cells and interleukin-17-producing T helper cells during HBV-related acute-on-chronic liver failure Xue-Song Liang, Cheng-Zhong Li, Yin Zhou, Wei Yin, Ya-Yun Liu, Wen-Han Fan Xue-Song Liang, Cheng-Zhong Li, Yin Zhou, Wei Yin, Ya- Yun Liu, Wen-Han Fan, Department of Infectious Diseases, Changhai Hospital, Second Military Medical University, Shanghai , China Yin Zhou, Department of Biochemistry and Molecular Biology, College of Basic Medical Sciences, Second Military Medical University, Shanghai , China Author contributions: Liang XS conceived the study, participated in the design of the study, performed the statistical analysis and drafted the manuscript; Li CZ participated in the design of the study and enrolled the subjects; Zhou Y performed most of the experiments; Yin W collected all of the human material; Liu YY and Fan WH were responsible for patient follow up; all authors read and approved the final manuscript. Supported by Grants from Shanghai Natural Science Fund, No. 09ZR ; National Natural Science Foundation of China No ; and Shanghai Health Bureau Fund, No Correspondence to: Xue-Song Liang, Associate professor, Department of Infectious Diseases, Changhai Hospital, Second Military Medical University, No. 168 Changhai Road, Yangpu District, Shanghai , China. liangxuesong2000@163.com Telephone: Fax: Received: October 15, 2013 Revised: December 31, 2013 Accepted: April 8, 2014 Published online: July 14, 2014 Abstract AIM: To longitudinally investigate cytokine gene expression and protein levels in Th17 and Treg cells, to observe T-cell phenotypes during hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACHBLF) and to analyze changes in Th17 and Treg phenotypes during disease progression. METHODS: We measured the expression of seven Th17/Treg differentiation-related genes and serum concentrations of the corresponding cytokines in 18 ACHBLF, 18 chronic hepatitis B (CHB) disease controls and 10 healthy controls (HCs) by real-time quantitative PCR and enzyme linked immunosorbent assay. Peripheral Th17 and Treg cell frequencies were analyzed by flow cytometry. RESULTS: From the onset of ACHBLF, patients presented with a conductive Th17 differentiation cytokine environment accompanied by high Th17 frequency and high serum IL-17 levels, which were sustained throughout the disease course. The Treg-related cytokine IL-2 and Foxp3 were also up-regulated from disease onset, and Foxp3 gene expression showed a gradually increasing trend during ACHBLF. The circular phenotype of Treg and Th17 cells showed changes from the onset of ACHGLF. At disease onset, Th17 frequency increased significantly compared with both CHB and HCs, but Treg cell frequency decreased significantly compared with CHB. During the ACHBLF event, Th17 frequency remained higher compared with HCs, but decreased sharply from the peak point to the recovery point; Treg cell frequency increased gradually during the ACHBLF event. Treg and Th17 cell counts correlated with ACH- BLF development; in all patients, serum IL-17 levels significantly correlated with patient serum ALT levels. In survivors, Th17 frequency at the onset point and the Treg to Th17 ratio at the peak point correlated with the patient s model for end stage liver disease (MELD) plus sodium (MELD-Na) score. The Treg to Th17 ratio and the Th17 frequency at onset were significant predictors of patient survival. Low Treg/Th17 cell ratios at the onset predicted poor survival. Survivors exhibited an initial decrease in the circulating Treg/Th17 ratio from the onset to the peak time, and subsequently displayed a continuous increase. CONCLUSION: Treg and Th17 cells showed changes in genes, protein levels and T cell phenotypes during ACHBLF events. An increased Treg/Th17 ratio was associated with the survival of ACHBLF patients Baishideng Publishing Group Inc. All rights reserved July 14, 2014 Volume 20 Issue 26

251 Liang XS et al. Immune slopes in ACHBLF Key words: Hepatitis B virus; Treg; Th17; Immune; Hepatitis B virus-related acute-on-chronic liver failure Core tip: In this study, we longitudinally investigated Foxp3 + Treg cells and IL-17 + Th cells by measuring gene levels, protein levels and T-cell phenotypes during HBVrelated acute-on-chronic liver failure (ACHBLF) progression. From the onset of ACHBLF, there were changes in Foxp3 + Treg and Th17 cell frequencies. An increased Treg/Th17 ratio was associated with the survival of ACHBLF patients. Liang XS, Li CZ, Zhou Y, Yin W, Liu YY, Fan WH. Changes in circulating Foxp3 + regulatory T cells and interleukin-17- producing T helper cells during HBV-related acute-on-chronic liver failure. World J Gastroenterol 2014; 20(26): Available from: URL: v20/i26/8558.htm DOI: i INTRODUCTION Hepatitis B virus (HBV) is a major human pathogen, infecting 350 million people worldwide. Adults infected with HBV can either exhibit acute self-limited HBV infection or progress to chronic infection, while viral infection in utero or early in life generally results in chronic hepatitis B (CHB) [1]. Some CHB patients may rapidly progress towards liver failure, a condition referred to as acute-onchronic liver failure (ACLF) [2]. HBV-related acute-onchronic liver failure (ACHBLF) is one of the most severe consequences of HBV infection. ACHBLF refers to liver failure occurring in patients with CHB, CHB-related liver cirrhosis, or chronic asymptomatic HBV carriers [3]. In China, as a result of the high prevalence of HBV infection, ACHBLF cases account for more than 80% of ACLF cases [4,5]. The precise mechanisms underlying the deterioration of liver function occurring in ACLF remain unclear; however, the impairment of cellular immunity is believed to be a contributing factor [6-9]. Recently, a novel and unique pro-inflammatory T cell subset, interleukin-17 (IL-17)-producing CD4 + T helper cells (Th17), was identified. Several key cytokines, including IL-1β, IL-6, tumor necrosis factor alpha (TNF-α) and IL-23, create a cytokine milieu that regulates the differentiation and expansion of human Th17 cells [10-13]. Th17 and its related cytokines may play an important role in the pathogenesis of HBV infection [6-9,14-16]. Conversely, another CD4 + T cell subset, Foxp3 + regulatory T cells (Treg), which are characterized by their constitutive expression of CD25 and Foxp3 and by immunological suppression [17-21], can restrain the immune response, thus limiting liver damage [9,22-24]. In chronic HBV infection, an imbalance between Tregs and effector T cells has recently been described by several groups. These studies suggest that either the number or functional imbalance of Tregs in the blood or liver may be a reason for persistent HBV infection and disease progression [9,24-26]. Although increased Th17 and Treg frequencies in patients with CHB have been reported, little is known about the relationship between circulating Th17 and Tregs and their role in ACHBLF development. Recently, we studied the changes in Treg and Th17 cell balance in the development of acute and chronic hepatitis B virus infection and found that in ACHBLF patients, peripheral blood Th17 cell frequency increased significantly compared with that seen in healthy controls (HCs), but the frequency of Treg cells did not increase synchronously, creating an imbalance between Treg and Th17 cells in ACHBLF [9]. Zhai et al [27] also found that at the onset of ACHBLF, the frequency of Th17 cells increased in ACHBLF patients and that the ratio of Th17 to Treg cells correlated with patient survival. However, Th17 and Treg cell dynamics in ACHBLF patients have not been previously reported. Our study longitudinally analyzed the frequency of Treg and Th17 cells and related cytokine protein and gene expression in a cohort of 18 patients with ACH- BLF. Our data document changes in Treg and Th17 cell populations during ACHBLF. MATERIALS AND METHODS Subjects A total of 18 patients with ACHBLF have been described in our recent study [9]. In brief, inclusion criteria included (1) HBV-related liver cirrhosis or chronic HBV infection based on a histopathologic diagnosis or compatible laboratory data and sonographic findings; (2) recent development of jaundice, ascites, hemodynamic instability and/or encephalopathy grade Ⅲ-Ⅳ, compatible with the definition of hepatic decompensation and necessitating further treatment in the ICU; (3) no evidence of hepatocellular carcinoma or other metastatic liver tumors that could affect liver function; and (4) no immunosuppressive medication within the last 3 mo prior to study entry [9,28]. All of the patients were longitudinally followed. Table 1 summarizes the clinical data acquired from the investigations of these patients at the onset point (1-2 wk after clinical onset), peak point (the time of peak total bilirubin level, 2-3 wk after clinical onset), and recovery point (total bilirubin levels decreased by more than 30%, typically 7-8 wk after clinical onset) [29]. The disease control group was composed of 18 age-, gender- and Child- Pugh stage-matched patients with CHB. The diagnostic criteria for CHB were as follows: positive for hepatitis B surface antigen (HBsAg) and anti-hepatitis B core (anti- HBc) for more than 6 mo; negative for antibodies to hepatitis C virus, hepatitis D virus, hepatitis G virus, and HIV-1 and -2; no other causes of chronic liver damage; and persistently elevated serum ALT levels and positive serum HBV DNA for at least 6 mo. Ten healthy adults (age range: years, 7 females and 3 males) were included as HCs. All patients and controls were Chinese. The study was approved by the local medical ethics committee of Shanghai Changhai Hospital, and informed 8559 July 14, 2014 Volume 20 Issue 26

252 Liang XS et al. Immune slopes in ACHBLF Table 1 Clinical characteristics of patients with hepatitis B virus-related acute-on-chronic liver failure No. Age (yr) Sex Onset Peak Recovery Week after clinical onset ALT (U/L) TBIL (μmol/l) HBV DNA (Log 10 copies/ml) PTA MELD- Na Week after clinical onset ALT (U/L) TBIL (μmol/l) HBV DNA (Log 10 copies/ml ) PTA MELD-Na Week after clinical onset ALT (U/L) TBIL (μmol/l) HBV DNA (Log 10 copies/ ml) PTA MELD- Na 1 58 M % % % M % % % M % % Died 4 50 F % Died 5 59 M % % % M % % % M % % % M % % % M % % % M % % % M % % % F % ND 27.24% % M % ND 4.70% Died M % % % F % % % M % % % M % % Died M % % % consent was acquired from each individual. Peripheral blood mononuclear cell isolation, total RNA extraction and complementary DNA synthesis Peripheral blood mononuclear cells (PBMCs) were isolated from the heparinized peripheral blood of the studied subjects by standard Ficoll-Paque (GE Healthcare, Uppsala, Sweden) density centrifugation. Total RNA was extracted from PBMCs using the RNeasy kit (Qiagen, Valencia, CA, and United States). All samples were treated with DNaseI to eliminate potential genomic DNA contamination. The quality and quantity of the RNA were determined using an ultraviolet spectrophotometer (NANODROP1000, Thermo). Target RNA was reverse-transcribed using the Omniscript RT Kit (Qiagen, Valencia, CA, United States). All samples were treated according to identical protocols and in parallel. RNA and cdna were stored at -80 until further processing. Quantification of gene expression The panels of genes of interest (GOIs) are listed in Table 2. The GAPDH gene was selected as an endogenous reference. Candidate primer sets were first designed by Primer Express Software (Applied Biosystems, Foster City, CA, United States) and then validated manually to satisfy the following criteria: (1) amplicon length between 50 and 150 base pairs; (2) amplification efficiencies, as determined by a template linear dilution method as described previously [30], were approximately equal for the GOIs and 18S rrna; and (3) primers were verified to generate a single product specific to target genes by both blast algorithm ( and melting curve analysis. The expression of GOIs was determined by RQ-PCR using an ABI PRISM 7000 Sequence Detection System (Applied Biosystems). PCR was performed using SYBR Green PCR Master Mix (Applied Biosystems) in a final volume of 50 µl with the following thermal conditions: 50 for 2 min, 95 for 10 min followed by 40 cycles of 95 for 10 s and 60 for 30 s. Samples were assayed in triplicate. The expression ratio of each GOI between different groups was analyzed using the delta comparative cycle threshold method [31] July 14, 2014 Volume 20 Issue 26

253 Liang XS et al. Immune slopes in ACHBLF Table 2 T helper type 17 and T regulatory differentiation-related cytokines Genes GenBank accession number Primers (5' to 3') Reported function IL-1β NM_ Forward: GCTGATGGCCCTAAACAGATGAA Induce human Th17 polarization [17] Reverse: TGAAGCCCTTGCTGTAGTGGTG IL-6 NM_ Forward: AAGCCAGAGCTGTGCAGATGAGTA Induce human Th17 polarization [17] Reverse: TGTCCTGCAGCCACTGGTTC IL-23/p19 NM_ Forward: GCAGCCTGAGGGTCACCACT Unique subunit of IL-23 Reverse: GGCGGCTACAGCCACAAA IL-17A NM_ Forward: TGTCCACCATGTGGCCTAAGAG Main effective cytokine of Th17 cells [7] Reverse: GTCCGAAATGAGGCTGTCTTTGA TGF-β 1 NM_ Forward: AGCGACTCGCCAGAGTGGTTA Induce mouse Th17 and Treg polarization, suppress Reverse: GCAGTGTGTTATCCCTGCTGTCA human Th17 polarization [9,10,17] IL-2 NM_ Forward: CAACTCCTGTCTTGCATTGCACTAA Induce human and mouse Treg polarization [10] Reverse: AATGTGAGCATCCTGGTGAGTTTG FoxP3 NM_ Forward: GTTCACACGCATGTTTGCCTTC Master regulatory transcription factors of Treg Reverse: CACAAAGCACTTGTGCAGACTCAG lineage [21] Flow cytometric analysis For Th17 cell examination, the PBMCs were isolated from peripheral blood. PBMCs ( ) were stimulated further for 5 h with 50 ng/ml phorbol myristate acetate, 1 mmol/l ionomycin (both from Sigma, St Louis, MO, United States) and 10 mg/ml brefeldin A (TocrisCookson, Bristol, United Kingdom) in complete RPMI-1640 (Invitrogen, Carlsbad, CA, United States) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, Grand Island, NY, United States). Upon harvest, cells were first surface-stained with fluorescein isothiocyanate (FITC)-conjugated anti-human CD4 antibodies for 20 min, fixed and permeabilized with Perm/Fix solution, and then stained intracellularly with phycoerythrin (PE)- conjugated anti-human IL-17A. For Treg cell examination, peripheral blood (100 μl) was first surface-stained with FITC-conjugated antihuman CD4 antibodies and allophycocyanin (APC)- conjugated anti-human CD25 antibodies for 30 min, then lysed with FACSTM lysing solution (BD PharMingen) and treated with ebioscience fix/perm mixture (ebiosciences) according to the manufacturer s instructions. Finally, the cells were incubated with PE-conjugated antihuman Foxp3 antibodies overnight. Isotope controls were used to ensure antibody specificity. Flow cytometry was performed using a FACSCalibur (Becton Dickinson, San Jose, CA). FACS data were analyzed using CellQuest software (Becton Dickinson Rutherford, NJ) [28]. All antibodies were purchased from BD Biosciences (San Jose, CA, United States). Enzyme linked immunosorbent assay Serum concentrations of IL-1β, IL-6, IL-23, IL-17A, TGF-β1, and IL-2 were measured by commercially available enzyme linked immunosorbent assay kits (R and D Systems, Minneapolis, MN, United States) according to the protocols provided by the manufacturer. All samples were assessed in triplicate. Virological and biochemical assessments The levels of HBsAg, HBeAg, anti-hbs, anti-hbc, anti- HBe, anti-hcv, anti-hdv, anti-hgv, anti-hiv-1, and anti-hiv-2 were measured using commercially available kits (Abbot Laboratories, North Chicago, IL) in our clinical lab. Serum HBV-DNA levels were measured by fluorescent quantitative PCR with commercially available kits (PE/B/MJ/L, Shenzhen, China) according to the manufacturer s instructions. The threshold of the HBV DNA detection limit was 500 copies/ml. Statistical analysis All data were analyzed using IBM SPSS statistic version 19 (IBM, Com). The Kruskal-Wallis test was used to evaluate the differences among more than 2 groups. The Mann-Whitney U test was used to evaluate the difference between two groups. Spearman s correlation test was used to assess the correlation of immune factors and clinical characters. The area under the receiver operating characteristic (ROC) curve was used to compute predictive values of different factors on survival rates. For all tests, two-sided P < 0.05 was considered significant. RESULTS Clinical characteristics and treatment of patients with ACBHLF The clinical and biochemical details of the studied patients are listed in Table 1. We measured the expression of 7 genes and the serum concentrations of six corresponding cytokines, which have been reported previously to contribute to the differentiation of Th17 and Treg cells [32,33]. The name, GenBank accession number, primer sequences and reported function of each gene are presented in Table 2. Patients with ACHBLF received conservative management, including nutritional support, hepatoprotective drugs, antiviral therapy (lamivudine 100 mg/d) and prevention and control of complications, but did not receive any immune-modulating therapy. Patients were divided subsequently into non-survivor (NS) patients (who died or received a liver graft) and survivors (who survived or whose total bilirubin level decreased by more than 30% by the end of study) according to an earlier report [29]. Four patients died during the study, and one patient received a liver graft July 14, 2014 Volume 20 Issue 26

254 Liang XS et al. Immune slopes in ACHBLF A 20 B 15 Gene expression ratio of IL Gene expression ratio of IL CHB ACHBLF HCs 0 CHB ACHBLF HCs C 15 D 20 Gene expression ratio of IL-1b 10 5 b Gene expression ratio of IL-17A CHB ACHBLF HCs 0 CHB ACHBLF HCs E 3 F 8 Gene expression ratio of TGF-b 2 1 Gene expression ratio of IL b b 0 CHB ACHBLF HCs 0 CHB ACHBLF HCs G 20 Gene expression ratio of FoxP CHB ACHBLF HCs Figure 1 Expression of interleukin-17-producing T helper cell and Foxp3 + regulatory T cell-related genes in patients with hepatitis B virus-related acute-onchronic liver failure at the onset point. Compared with healthy controls (HCs), all interleukin-17-producing T helper cell (Th17)-related cytokines were up-regulated, but only IL-1β was up-regulated significantly. Compared with general chronic hepatitis B (CHB), there were no significant differences in Th17-related cytokine gene expression at the onset of hepatitis B virus-related acute-on-chronic liver failure (ACHBLF). For Foxp3 + regulatory T cell (Treg)-related gene expression, IL-2 expression was significantly up-regulated compared with both CHB patients and HCs. Data are presented as whisker-box plots, and the difference between ACHBLF and the HC or CHB groups was analyzed by a Mann-Whitney test. Boxes represent the interquartile range, with the median represented by the line inside the box. Upper whisker, the highest value less than or equal to the 75 percentile plus 1.5 times interquartile range; lower whisker, the lowest value greater than or equal to the 25 percentile minus 1.5 times interquartile range. Outliers were excluded. b P < 0.01 vs ACHBLF group July 14, 2014 Volume 20 Issue 26

255 Liang XS et al. Immune slopes in ACHBLF A 20 a a B 20 Gene expression ratio of IL Gene expression ratio of IL Onset Peak Recovery HC ACHBLF Onset Peak Recovery HC ACHBLF C Gene expression ratio of IL-b a a D Gene expression ratio of IL-17A a b E Gene expression ratio of TGF-b Onset Peak Recovery HC ACHBLF a b F Gene expression ratio of IL Onset Peak Recovery HC ACHBLF d b d b 0.0 Onset Peak Recovery HC ACHBLF 0 Onset Peak Recovery HC ACHBLF G 25 c b Gene expression ratio of FoxP Onset Peak Recovery HC ACHBLF Figure 2 Dynamic cytokine milieu for interleukin-17-producing T helper cells and Foxp3 + regulatory T cells in patients with hepatitis B virus-related acuteon-chronic liver failure. The gene expression of pro-th17 cytokines showed higher levels throughout the hepatitis B virus-related acute-on-chronic liver failure (ACH- BLF) event compared with HCs, especially for IL-6 and IL-1β (A and C). FoxP3 gene expression was up-regulated gradually during ACHBLF events (G). Data are presented as whisker-box plots. Boxes represent the interquartile range, with the median represented by the line inside the box. Upper whisker, the highest value less than or equal to the 75 percentile plus 1.5 times interquartile range; lower whisker, the lowest value greater than or equal to the 25 percentile minus 1.5 times interquartile range. Outliers were excluded. Differences between different time points were analyzed by the Kruskal-Wallis test and with HCs by the Mann-Whitney test. a P < 0.05, b P < 0.01 vs HC; c P < 0.05, d P < 0.01 vs recovery group July 14, 2014 Volume 20 Issue 26

256 Liang XS et al. Immune slopes in ACHBLF A pg/ml 40 b b b B pg/ml 30 a Serum level of IL Serum Level of IL-1β Onset Peak Recovery HC ACHBLF 0 Onset Peak Recovery HC ACHBLF C Serum level of IL-23 pg/ml D Serum level of IL-17 pg/ml b b b 0 Onset Peak Recovery HC ACHBLF 0 Onset Peak Recovery HC ACHBLF E Serum Level of IL-2 pg/ml d d b b F Serum level of TGF-β 10 4 pg/ml b b 0 Onset Peak Recovery HC ACHBLF 0 Onset Peak Recovery HC ACHBLF Figure 3 Serum levels of interleukin-17-producing T helper cell and Foxp3 + regulatory T cell-related cytokines in patients with hepatitis B virus-related acute-on-chronic liver failure. A: Interleukin (IL)-6; B: IL-1β; C: IL-23; D: IL-17; E: IL-2; F: transforming growth factor (TGF)-β1 were tested. Data are presented as the mean ± standard deviation. Differences between different time points were analyzed by the Kruskal-Wallis test and with HCs by the Mann-Whitney test. a P < 0.05; b P < 0.01 vs HC; d P < 0.01 vs recovery group. Both Th17 and Treg cell-related cytokines were upregulated at the onset of ACHBLF Compared with HCs, the gene expression of the pro- Th17 cytokines IL-1β, IL-6 and IL-23/p29 was up-regulated, especially IL-1β, at the onset of ACHBLF (Figure 1A-C). We also observed higher expression of IL-17A (Figure 1D). However, there were no significant differences between the ACHBLF and CHB patients. Although the expression of TGF-β1 in the patients with ACHBLF did not differ from that of the HCs and CHB patients (Figure 1E), IL-2 gene expression was upregulated significantly compared with the HCs and CHB patients (Figure 1F). We also observed higher levels of Foxp3 gene expression (Figure 1G). Dynamic Treg and Th17 cell differentiation environment at both gene and protein levels during ACHBLF During ACHBLF, the differentiation environment was conducive to Th17 cell differentiation (Figure 2A-C and Figure 3A-C), which was demonstrated by significantly 8564 July 14, 2014 Volume 20 Issue 26

257 Liang XS et al. Immune slopes in ACHBLF A HC CHB ACHBLF B 15 IL-17A CD IL-17A + CD4 + T cells on total CD4 + T cells (%) 10 5 a a FoxP3 0 CHB ACHBLF HCs CD25 + CD4 C 15 D 15 FoxP3 + CD25 + CD4 + T cells on total CD4 + T cells (%) 10 5 b Ratio of FoxP3 + Treg cells toth17 cells 10 5 b b 0 CHB ACHBLF HCs 0 CHB ACHBLF HCs Figure 4 population of interleukin-17-producing T helper cells as a percentage of total CD4 + cells in the peripheral blood mononuclear cells was evaluated by flow cytometry. A: Representative dot plots of intracellular IL-17 and Foxp3 staining in patients with HBV-related acute-on-chronic liver failure (ACHBLF); B: Percentage of IL-17A + CD4 + T cells in total CD4 + T cells; C: Percentage of FoxP3 + CD25 + CD4 + T cells in total CD4 + T cells; D: Ratio of Treg cells to Th17 cells. CHB: Chronic hepatitis B. Differences between ACHBLF and HCs or CHB group were analyzed by the Mann-Whitney test. a P < 0.05, b P < 0.01 vs ACHBLF group. elevated IL-6 and IL-1β levels both at the gene and protein expression level (Figures 2A, C and 3A, B). However, Treg cell-related cytokine expression levels gradually decreased during ACHBLF (Figures 2E, F and 3E, F). Corresponding to this Th17 differentiation environment, patients possessed higher IL-17A levels at both the gene and protein levels during the ACHBLF event (Figure 2D and Figure 3D). Interestingly, Foxp3 gene expression was up-regulated gradually during the ACHBLF event. Foxp3 gene levels at the recovery point were significantly higher than HCs and at onset time point (Figure 2G). Treg and Th17 cell numbers during ACHBLF Based on our knowledge of the cytokine milieu in patients with ACHBLF at the onset point, we further evaluated the Treg and Th17 cell subsets in PBMCs, defined as the percentage of Th17 cells and Treg cells in the total CD4 + T cell population using flow cytometry (Figure 4A). The Th17 cell population was increased in patients with ACHBLF (2.83% ± 2.61%) compared with patients with CHB (1.48% ± 0.71%, p = 0.04,) and HCs (1.37% ± 0.51%, p = 0.01; Figure 4B), whereas Treg decreased in ACHBLF patients (4.08% ± 2.28%) compared with CHB patients (6.71% ± 1.76%, p = 0.001) (Figure 4C). Given the nonsynchronous changes of Treg and Th17 cells at the onset of ACHBLF, to better understand the relationship between these two types of immune cells, we used the Treg/Th17 ratio. The Treg/Th17 ratio of ACHBLF at the onset point was decreased significantly (2.83% ± 2.60%) compared with HCs (4.09% ± 1.39%, p = 0.003) and the CHB patients (4.67% ± 2.30%, p = 0.007) (Figure 4D). These data indicate that a significant imbalance in the numbers of circulating CD4 + T cells occurs in patients with ACHBLF. increased ratio of Treg to Th17 cells in patients with ACHBLF To characterize the changes in the circulating CD4 + T subset, all 18 patients with ACHBLF were longitudinally 8565 July 14, 2014 Volume 20 Issue 26

258 Liang XS et al. Immune slopes in ACHBLF A IL-17A Onset Peak Recovery CD4 B IL-17A + CD4 + T cells on total CD4 + T cells (%) b Onset Peak Recovery HCs ACHBLF FoxP3 CD25 + CD4 C 15.0 D 15.0 FoxP3 + CD25 + CD4 + T cells on total CD4 + T cells (%) Onset Peak Recovery HCs ACHBLF Ratio of Treg cells to Th17 cell b Onset Peak Recovery HCs ACHBLF b Figure 5 ratio of Foxp3 + regulatory T cells to interleukin-17-producing T helper cells was dynamically increased during hepatitis B virus-related acute-onchronic liver failure. A: Representative dot plots of intracellular IL-17 and Foxp3 staining in different stages of a hepatitis B virus-related acute-on-chronic liver failure (ACHBLF) individual. Values in the upper right quadrant indicate the circulating Th17 cells percentage and the FoxP3 + percentage of total CD4 T cells; B: The dynamics of Th17 cell frequency at the onset, peak and recovery phases of ACHBLF; C: The dynamics of Treg cells at the onset, peak and recovery phases of ACHBLF; D: The dynamics of the ratio of Treg to Th17 cell frequency at the onset, peak and recovery phases of acute-on-chronic HBV-related liver failure. The data were analyzed by one-way ANVOA Kruskal-Wallis test, p < 0.05 indicates significance. b P < 0.01 vs HCs. followed. Intracellular IL-17 and FoxP3 staining in different stages of ACHBLF were tested using flow cytometry (Figure 5A). As shown in Figure 5B, during ACHBLF events, patients had a sustained higher frequency of Th17 cells in the peripheral blood compared with HCs. The frequency of Th17 cells at the peak point was increased slightly compared with at the onset point, but there was no significant difference between the two clinical phases. However, at the recovery time point, the frequency of Th17 cells (1.69% ± 0.97%) was decreased sharply from the peak time point (2.84% ± 2.92%). The frequency of Treg cells was not changed significantly during the entire clinical phase (Figure 5C). We further analyzed the changes in the ratio of Tregs to Th17 cells during ACHBLF events. The ratio of Treg to Th17 cells was increased gradually from the onset point to the recovery point, and at the recovery point, the ratio of Treg to Th17 (3.07% ± 1.80%) was similar to that of the HCs (4.09% ± 1.39%, p = 0.17) (Figure 5D). Relationship between disease development and the Th17-Treg dynamic in patients with ACHBLF To identify the relationship between host immune changes and disease development, we used total bilirubin (TBIL), alanine aminotransferase (ALT) and a model for end stage liver disease (MELD) plus sodium (MELD-Na) score to evaluate disease development [34,35]. We performed correlation analyses between these main biochemical measures and the MELD-Na score and Th17 frequency, Treg frequency and the ratio of Treg to Th17 cells at the onset, peak, and recovery phases of the disease among our 18 ACHBLF patients. The data revealed that all of these host immune characteristics positively or negatively correlated with disease development (table 3). However, in all patients, only serum IL-17 levels at peak point markedly correlated with the ALT level (r = -0.56, p = 0.02). We 8566 July 14, 2014 Volume 20 Issue 26

259 Liang XS et al. Immune slopes in ACHBLF Table 3 Correlations between clinical characteristics and host immune data during the onset, peak, and recovery phases of hepatitis B virus-related acute-on-chronic liver failure Onset Peak Recovery Th17 Treg Treg/Th17 IL-17 Th17 Treg Treg/Th17 IL-17 Th17 Treg Treg/Th17 IL-17 r P -value r P -value r P -value r P -value r P -value r P -value r P -value r P -value r P -value r P -value r P -value r P -value Total patients ALT TBIL MELD-Na Survivors ALT TBIL MELD-Na ALT: Alanine aminotransferase; TBIL: Total bilirubin; PTA: Prothrombin activity; MELD-Na: Model for end-stage liver diseases and sodium; Th17: Interleukin-17-producing T helper cells; Treg: Foxp3 + regulatory T cell; Treg/ Th17: Ratio of Treg to Th17 cells. Data were analyzed by Spearman correlation test. further analyzed patients in the survival group and found that the ratio of Treg to Th17 at the peak point positively correlated with ALT levels (r = 0.58, p = 0.04). Furthermore, we also found that in the survival group, changes in the Treg to Th17 ratio from the onset to the peak point markedly correlated with TBIL levels (r = -0.57, p = 0.04). Predictive value of Treg, Th17 and the Treg to Th17 ratio on 28-d survival in ACHBLF Because Treg, Th17, and IL-17 levels and the ratio of Treg to Th17 were shown to be associated with ACHBLF development, we further evaluated the early predictive value of these factors on ACHBLF outcome. We evaluated the value of these factors at onset and at peak point in predicting ACHBLF survival by the end of a 28-d follow-up period. ROC curve analysis showed that Th17 frequency and the ratio of Treg to Th17 at the onset point were significant in predicting 28-d ACHBLF survival. The Th17 frequency was a negative predictor and the Treg to Th17 ratio was a positive predictor [the area under the ROC was 0.1 (95%CI: , p = 0.01) for Th17 frequency at onset and (95%CI: , p = 0.02) for the ratio of Treg to Th17 at onset]. There was no significant predictive value of Treg frequency during the event, IL-17 levels during the event, and changes in the ratio of Treg to Th17 from the onset to peak point (Table 4). To further investigate the predictive value of the Treg to Th17 frequency ratio during ACHBLF events on the survival of ACHBLF patients, we divided the ACHBLF patients into survivors and non-survivors (NS) [29]. As shown in Figure 6A, we found that the ratio of Treg to Th17 frequency at the onset point in NS patients (0.91% ± 0.24%) was significantly lower than that in survivors (2.64% ± 2.19%, p = 0.02). Furthermore, we also found that survivors exhibited an initial decrease in the circulating ratio of Treg to Th17 frequency from the onset point to the peak point. Subsequently, survivors displayed a continuous increase of the Treg to Th17 ratio, accompanied by a total bilirubin level decrease of more than 30% (Figure 6B). However, dead/transplanted patients lacked these sequential responses compared with survivors. The Treg to Th17 ratio remained persistently low in these patients (Figure 6C). These data suggest that the restoration of the balance of Treg and Th17 cells may represent a prognostic marker for a favorable clinical result in ACHBLF patients. DISCUSSION In the present study, we demonstrated that a Treg and Th17 immune imbalance exists in ACHBLF patients by measuring gene, protein and T cell phenotypes. We also longitudinally identified the pattern of dynamic change in Treg and Th17 cells during ACHBLF. We found that compared with CHB, ACHBLF patients possessed a favorable Th17 differentiation environment, accompanied by a sustained higher Th17 frequency and IL-17 levels. The results suggest that Th17 and related pro-inflammatory cytokine up-regulation played a vital role in disease progression from CHB to ACHBLF and also played a critical role in ACHBLF development. Zhang et al [7] previously found that circulating Th17 cells increased with disease progression from CHB to ACHBLF. Another CD4 + T cell subset, Tregs, plays a critical role in HBV persistence during CHB events July 14, 2014 Volume 20 Issue 26

260 Liang XS et al. Immune slopes in ACHBLF Table 4 Predictive value of Foxp3 + regulatory T cell and interleukin-17-producing T helper cells in hepatitis B virus-related acuteon-chronic liver failure patient survival Variables Area Standard error Asymptotic sign 95%CI Low boundary Upper boundary Treg frequency at onset Th17 frequency at onset Ratio of Treg to Th17 at onset Serum IL-17 level at onset Treg frequency at peak Th17 frequency at peak Ratio of Treg to Th17 at peak Ratio of Treg to Th17 change from onset to peak Serum IL-17 level at peak We found that when disease progressed from CHB to ACHBLF, although the Treg differentiation cytokine IL-2 was up-regulated, circulating Treg frequency began to decrease and showed lower levels during ACHBLF events compared with CHB events. These data suggest that Treg down-regulation may promote liver inflammation and accelerate disease progression from CHB to ACHBLF and ACHBLF development. The development of ACHBLF involves dysregulation of both the innate and adaptive immune systems. Many different cell types, including T lymphocytes, monocytes and dendritic cells, are primed in ACHBLF. These cells are believed to play a pivotal role in the pathogenesis of ACHBLF [4,6,8,12,26]. Treg and Th17 cells are both involved in the pathogenesis of HBV infection. Tregs and Th17 cells are closely associated with each other; the two types of T cells not only share the same origin but are also mutually antagonistic in function. Thus, the balance between the two types of T cells could impact inflammation control and autoimmune inflammation [36,37]. Many studies have found that an imbalance between Th17 and Treg cells is closely related to the development of a number of diseases, including HBV infection [9,27,36-39]. Recently, two different groups have demonstrated imbalances between Treg and Th17 cells in ACHBLF patients and that this imbalance plays an important role in the progression of ACHBLF [27,40]. Furthermore, several studies have demonstrated that antiviral therapy induces a viral load reduction that partly restores antiviral immunity in patients with CHB [41-44]. We also previously reported that for acute and chronic HBV infected patients, the balance between Treg and Th17 cell frequency was different. Treg and Th17 immune imbalance only exists in CHB and ACHBLF [9]. In this study, we further demonstrated that immune imbalance of Treg and Th17 cells exists in ACHBLF measured by both gene and protein levels from the clinical onset. Furthermore, the imbalance of Treg to Th17 changed along the progression of the disease and correlated with the outcome of the disease. Furthermore, to avoid a quick viral load reduction that induces immune restoration in the early part of the ACHBLF event, in this study, all patients accepted lamivudine therapy to improve the long-term prognosis of ACHBLF. Although it is now recognized that an imbalance between Th17 and Tregs exists in ACHBLF patients, our knowledge about the dynamic interplay between Tregs and Th-17 cells during ACHBLF events is limited. In this study, we longitudinally followed ACHBLF patients and demonstrated that the immune imbalance between Treg and Th17 dynamically changed along ACHBLF progression, but mechanistic data were absent in our study. At onset, the ratio of Treg to Th17 cells, with a significant Th17 increase, was decreased significantly compared with CHB patients and HCs. Then, the Treg to Th17 ratio dynamically increased from the onset to the recovery point, and finally, the ratio of Treg to Th17 was increased to similar levels as HCs at the recovery point. These data suggest that the Treg and Th17 balance modulates liver damage and the outcome of ACHBLF. Based on the role of Th17 and Treg in ACHBLF and the relationship between the two types of T cells, we studied the relationship between the two types of T cells and ACHBLF progression. We used the MELD-Na score to evaluate ACHBLF disease development, ALT and TBIL to evaluate liver inflammation, and the ratio of Treg to Th17 to represent the interplay of the two types of T cells. We found that in all patients, the frequency of the two types of T cells and their ratio during ACHBLF events correlated with the ALT, TBIL and MELD-Na score positively or negatively, but only serum IL-17 levels at peak point significantly correlated with ALT levels (table 4). However, in survivors, we found that Th17 frequency at onset and recovery showed a relationship with MELD-Na (Table 4), and the ratio of Treg to Th17 at the peak point significantly correlated with ALT levels. These data suggested that MELD-Na is an improved model for the prediction of mortality in patients with end-stage liver disease awaiting liver transplantation but cannot exactly represent the stage of ACHBLF, especially in patients whose acute liver damage developed quickly [45]. The mortality of ACHBLF is high and there are few early predictors for survival in ACHBLF. Zhai et al [27] found that the ratio of Th17 to Treg cells was inversely associated with survival in HBV-related ACLF patients. In this study, we not only found that a lower ratio of circulating Treg to Th17 frequency at the onset predicts a poor survival rate but also found that survivors exhibited an initial decrease in the circulating ratio of Treg to Th July 14, 2014 Volume 20 Issue 26

261 Liang XS et al. Immune slopes in ACHBLF A Ratio of Treg cells to Th17 cells P = 0.02 In summary, our findings suggest that an immune imbalance between Treg and Th17 cells exists in ACHBLF patients, and the immune imbalance dynamically changes with disease progression. The ratio of Treg to Th17 cell frequency is dynamically associated with survival in ACH- BLF patients. A low Treg to Th17 cell ratio at onset predicts poor survival. Survivors exhibit an initial decrease in the circulating ratio of Treg to Th17 cells from onset to peak time and subsequently display a continuous increase in the ratio of Treg to Th17 cells. B Ratio of Treg cells to Th17 cell C Ratio of Treg cells to Th17 cells Survival P = Non-survival Onset Peak Recovery Onset Peak Recovery Figure 6 Predictive value of the Foxp3 + regulatory T cells to interleukin- 17-producing T helper cells cell ratio during disease development on the survival of hepatitis B virus-related acute-on-chronic liver failure patients. A: A lower ratio of Treg cells to Th17 frequency in non-survival ACHBLF patients at clinical onset; B: The distribution of the ratio of Treg cells to Th17 cells in surviving ACHBLF patients; C: The distribution of the ratio of Treg cells to Th17 cells in non-surviving ACHBLF patients. Each symbol represents one individual, and each line represents the changes in an individual patient s ratio of Treg to Th17 frequency. The solid line depicts the mean ratio of all patients. cells from onset to peak time and subsequently displayed a continuous increase in the ratio of Treg to Th17 cells, accompanied by a total bilirubin level decrease of more than 30%. According to these data, we speculate that the interplay between Tregs and Th-17 is important for maintaining the balance between a limited immune response and pathological damage. ACKNOWLEDGMENTS We would like to thank all of the patients enrolled in this study for their kind understanding and support. COMMENTS Background FoxP3 + Treg cells and IL-17 + Th cells are both involved in the progression of hepatitis B virus (HBV) infection. During chronic HBV infection there is an imbalance between the two related types of immune T cells. Little is known about the change in the pattern of the two types of T cells during HBV-related acuteon-chronic liver failure (ACHBLF) progression. Research frontiers Studies have previously demonstrated that there is an imbalance between FoxP3 + Treg and IL-17 + Th cells during ACHBLF. The ratio of Th17 to Treg cells is inversely associated with survival in HBV-related ACLF patients. Innovations and breakthroughs In this study, the authors longitudinally investigated FoxP3 + Treg cells and IL Th cells using gene expression and protein levels and also T-cell phenotypes during ACHBLF. At the onset of ACHBLF, patients presented with an environment conducive for Th17 differentiation, but Treg cell-related cytokines showed a gradually increasing trend over time. FoxP3 + Treg and IL-17 + T cells showed changes in gene, protein level and T cell phenotypes during ACHBLF events. An increased Treg/Th17 ratio was associated with survival in ACHBLF patients. Applications ACHBLF is one of the most severe consequences of HBV infection. The mortality of ACHBLF is high, and there are few early predictors for survival in ACHBLF. The ratio of Treg to Th17 cell frequency is dynamically associated with survival in ACHBLF patients. A low Treg to Th17 cell ratio at disease onset predicts poor survival, and survivors exhibit an initial decrease in the circulating ratio of Treg to Th17 cells from onset to peak time; therefore, monitoring the changes in the Treg to Th17 cell ratio could predict survival in ACHBLF. Terminology ACHBLF refers to acute liver failure occurring in patients with chronic HBV infection (CHB), CHB-related liver cirrhosis, or chronic asymptomatic HBV carriers. ACHBLF manifests as jaundice and coagulopathy, complicated within 4 wk by ascites and/or encephalopathy. Peer review The manuscript aimed to longitudinally investigate the expression levels of Treg cells in patients with ACHBLF and to analyze changes in Th17 and Treg cells during the follow-up period. Overall, the data are interesting. Although the numbers of patients are small, they are well characterized. However, an absence of mechanistic data was observed in this manuscript. REFERENCES 1 Sen S, Williams R, Jalan R. The pathophysiological basis of acute-on-chronic liver failure. Liver 2002; 22 Suppl 2: 5-13 [PMID: DOI: /j x] 2 Rehermann B, Nascimbeni M. Immunology of hepatitis B virus and hepatitis C virus infection. Nat Rev Immunol 2005; 5: [PMID: DOI: /nri1573] 8569 July 14, 2014 Volume 20 Issue 26

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263 Liang XS et al. Immune slopes in ACHBLF Delta C(T)) Method. Methods 2001; 25: [PMID: DOI: /meth ] 32 Lee YK, Mukasa R, Hatton RD, Weaver CT. Developmental plasticity of Th17 and Treg cells. Curr Opin Immunol 2009; 21: [PMID: DOI: /j.coi ] 33 Hemdan NY, Birkenmeier G, Wichmann G. Key molecules in the differentiation and commitment program of T helper 17 (Th17) cells up-to-date. Immunol Lett 2012; 148: [PMID: DOI: /j.imlet ] 34 Biggins SW, Kim WR, Terrault NA, Saab S, Balan V, Schiano T, Benson J, Therneau T, Kremers W, Wiesner R, Kamath P, Klintmalm G. Evidence-based incorporation of serum sodium concentration into MELD. Gastroenterology 2006; 130: [PMID: DOI: / j.gastro ] 35 Yuen MF, Sablon E, Hui CK, Li TM, Yuan HJ, Wong DK, Doutreloigne J, Bogaerts V, Wong BC, Fan ST, Lai CL. Prognostic factors in severe exacerbation of chronic hepatitis B. Clin Infect Dis 2003; 36: [PMID: DOI: /374226] 36 Li J, Wang L, Wang S, Zhu H, Ye P, Xie A, Shen B, Liu C, Guo C, Fu Q, Zhang K, Xia J. The Treg/Th17 imbalance in patients with idiopathic dilated cardiomyopathy. Scand J Immunol 2010; 71: [PMID: DOI: / j x] 37 Li N, Bian H, Zhang J, Li X, Ji X, Zhang Y. The Th17/Treg imbalance exists in patients with heart failure with normal ejection fraction and heart failure with reduced ejection fraction. Clin Chim Acta 2010; 411: [PMID: DOI: /j.cca ] 38 Li J, Qiu SJ, She WM, Wang FP, Gao H, Li L, Tu CT, Wang JY, Shen XZ, Jiang W. Significance of the balance between regulatory T (Treg) and T helper 17 (Th17) cells during hepatitis B virus related liver fibrosis. PLoS One 2012; 7: e39307 [PMID: DOI: /journal.pone ] 39 Xu D, Fu J, Jin L, Zhang H, Zhou C, Zou Z, Zhao JM, Zhang B, Shi M, Ding X, Tang Z, Fu YX, Wang FS. Circulating and liver resident CD4+CD25+ regulatory T cells actively influence the antiviral immune response and disease progression in patients with hepatitis B. J Immunol 2006; 177: [PMID: ] 40 Niu Y, Liu H, Yin D, Yi R, Chen T, Xue H, Zhang S, Lin S, Zhao Y. The balance between intrahepatic IL-17(+) T cells and Foxp3(+) regulatory T cells plays an important role in HBV-related end-stage liver disease. BMC Immunol 2011; 12: 47 [PMID: DOI: / ] 41 You J, Sriplung H, Geater A, Chongsuvivatwong V, Zhuang L, Li YL, Lei H, Liu J, Chen HY, Tang BZ, Huang JH. Impact of viral replication inhibition by entecavir on peripheral T lymphocyte subpopulations in chronic hepatitis B patients. BMC Infect Dis 2008; 8: 123 [PMID: DOI: / ] 42 Boni C, Penna A, Bertoletti A, Lamonaca V, Rapti I, Missale G, Pilli M, Urbani S, Cavalli A, Cerioni S, Panebianco R, Jenkins J, Ferrari C. Transient restoration of anti-viral T cell responses induced by lamivudine therapy in chronic hepatitis B. J Hepatol 2003; 39: [PMID: DOI: / s (03) ] 43 Akbar SM, Horiike N, Chen S, Michitaka K, Abe M, Hiasa Y, Matsuura B, Onji M. Mechanism of restoration of immune responses of patients with chronic hepatitis B during lamivudine therapy: increased antigen processing and presentation by dendritic cells. J Viral Hepat 2011; 18: [PMID: DOI: /j x] 44 TrehanPati N, Kotillil S, Hissar SS, Shrivastava S, Khanam A, Sukriti S, Mishra SK, Sarin SK. Circulating Tregs correlate with viral load reduction in chronic HBV-treated patients with tenofovir disoproxil fumarate. J Clin Immunol 2011; 31: [PMID: DOI: /s ] 45 Sun QF, Ding JG, Xu DZ, Chen YP, Hong L, Ye ZY, Zheng MH, Fu RQ, Wu JG, Du QW, Chen W, Wang XF, Sheng JF. Prediction of the prognosis of patients with acute-on-chronic hepatitis B liver failure using the model for end-stage liver disease scoring system and a novel logistic regression model. J Viral Hepat 2009; 16: [PMID: DOI: / j x] P- Reviewers: Chen DY, Yan YQ S- Editor: Ma YJ L- Editor: Logan S E- Editor: Zhang DN 8571 July 14, 2014 Volume 20 Issue 26

264 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. ORIGINAL BRIEF ARTICLE Zinc protoporphyrin IX enhances chemotherapeutic response of hepatoma cells to cisplatin Yang-Sui Liu, Huan-Song Li, Dun-Feng Qi, Jun Zhang, Xin-Chun Jiang, Kui Shi, Xiao-Jun Zhang, Xin-Hui Zhang Yang-Sui Liu, Huan-Song Li, Dun-Feng Qi, Jun Zhang, Xin- Chun Jiang, Kui Shi, Xiao-Jun Zhang, Xin-Hui Zhang, The Central Hospital of Xuzhou City, Xuzhou Hospital Affiliated to Medical College of Southeast University, Xuzhou , Jiangsu Province, China Author contributions: Liu YS and Zhang XH performed the majority of experiments; Li HS, Zhang J and Zhang XJ provided vital reagents and analytical tools and were also involved in revising the manuscript; Qi DF, Jiang XC and Shi K analyzed the data; Liu YS, Zhang XJ and Zhang XH designed the study and wrote the manuscript. Supported by Xuzhou City Health Bureau Project, China, No. XZZD1128; and Xuzhou City Municipal Science and Technology Bureau Project, China, No. XWJ Correspondence to: Xin-Hui Zhang, MD, The Central Hospital of Xuzhou City, Xuzhou Hospital Affiliated to Medical College of Southeast University, Jiefang Rd, Xuzhou , Jiangsu Province, China. 88liuyin888@163.com Telephone: Fax: Received: November 13, 2013 Revised: January 14, 2014 Accepted: April 2, 2014 Published online: July 14, 2014 METHODS: Cytotoxicity was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was determined by a flow cytometric assay. Western blotting was used to measure protein expression. Heme oxygenase (HO)-1 activity was measured by determining the level of bilirubin generated in isolated microsomes. Reactive oxygen species (ROS) production was monitored by flow cytometry. Caspase-3 activity was measured with a colorimetric assay kit. Mice were inoculated with tumor cells subcutaneously into the right flanks. All mice were sacrificed 6 wk after the first treatment and tumors were weighed and measured. RESULTS: Overexpression of HO-1 in HepG2 cell line was associated with increased chemoresistance to cisdiaminedichloroplatinum (cisplatin; CDDP) compared to other cell lines in vitro. Inhibition of HO-1 expression or activity by zinc protoporphyrin IX (ZnPP IX) markedly augmented CDDP-mediated cytotoxicity towards all liver cancer cell lines in vitro and in vivo. In contrast, induction of HO-1 with hemin increased resistance of tumor cells to CDDP-mediated cytotoxicity in vitro and in vivo. Furthermore, cells treated with ZnPP IX plus CDDP exhibited marked production of intracellular ROS and caspase-3 activity, which paralleled the incidence of cell apoptosis, whereas hemin decreased cellular ROS and caspase-3 activity induced by CDDP. CONCLUSION: ZnPP IX increases cellular sensitivity and susceptibility of liver cancer cell lines to CDDP and this may represent a mechanism of increasing ROS Baishideng Publishing Group Inc. All rights reserved. Abstract AIM: To investigate the effect of zinc protoporphyrin IX on the response of hepatoma cells to cisplatin and the possible mechanism involved. Key words: Zinc protoporphyrin IX; Heme oxygenase-1; Liver cancer cell lines; Cisplatin; Chemotherapy Core tip: Overexpression of heme oxygenase (HO)-1 in HepG2 cell line was associated with increased chemoresistance to cis-diaminedichloroplatinum (cisplatin; CDDP) compared to other cell lines. Inhibition of HO-1 expression by zinc protoporphyrin IX (ZnPP IX) markedly augmented CDDP-mediated cytotoxicity towards other hepatoma cells. Induction of HO-1 with hemin increased resistance of tumor cells to CDDP-mediated cytotoxicity. Furthermore, cells treated with ZnPP IX plus CDDP exhibited marked production of intracellular reactive oxygen species (ROS) and caspase-3 activity, whereas hemin decreased cellular ROS and caspase-3 activity induced by CDDP. Therefore, administration 8572 July 14, 2014 Volume 20 Issue 26

265 Liu YS et al. Zinc protoporphyrin IX and hepatoma of HO-1 inhibitors may evolve into a new liver cancer treatment strategy. Liu YS, Li HS, Qi DF, Zhang J, Jiang XC, Shi K, Zhang XJ, Zhang XH. Zinc protoporphyrin IX enhances chemotherapeutic response of hepatoma cells to cisplatin. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: org/ /wjg.v20.i INTRODUCTION Hepatocellular carcinoma (HCC) is the fourth most common cancer and the third leading cause of cancerrelated death worldwide [1,2]. Surgical resection is the best treatment to reduce the growth of HCC and improve life expectancy of HCC patients. However, more than 80% of HCC patients are diagnosed with an advancedstage or unresectable disease. In patients who undergo resection, the prognosis is poor with a recurrence rate as high as 50% at two years [3,4]. Current chemotherapy is also not effective and the development of chemoresistance is the major limitation. Therefore, a new understanding of the molecular mechanisms of drug resistance is urgently needed for treatment of this deadly disease. Heme oxygenase (HO) is a microsomal initial and rate-limiting enzyme that catalyzes the degradation of heme to produce equimolar quantities of biliverdin, CO and free iron, which play crucial roles in the defense against oxidative and cellular stress [5]. To date, three distinct mammalian HO isoforms have been identified: HO-1, HO-2 and HO-3. HO-2 and HO-3 are constitutively expressed, whereas HO-1 is known to be highly induced by a vast array of stress-inducing stimuli such as H2O2, UV irradiation, hypoxia, and extracellular acidosis [6-9]. Induction of the HO-1 protein represents a cytoprotective defense mechanism in the adaptive response to cellular stress [10]. Moreover, new observations indicate that HO-1 and its products also exert anti-inflammatory effects and influence the growth and proliferation of tumor cells. Elevated expression and increased activity of HO-1 have been found in various malignant tumors such as human renal cell carcinoma, pancreatic cancer, hepatoma, prostate cancer, and Kaposi sarcoma [11-15]. Furthermore, overexpression of HO-1 in tumor cells can be further elevated by chemotherapy, radiotherapy or photodynamic therapy [16]. Its overexpression in human cancers may give cancer cells a growth advantage and enhance resistance to chemotherapy and other stressors [16-18]. Inhibition of HO-1 expression or activity suppresses cellular proliferation and increases responsiveness of tumor cells to some anticancer treatments in vitro and in vivo [18-20]. In contrast, induction of HO-1 expression or activity decreases cell sensitivity to antitumor drugs. Accumulating evidence suggests that HO-1 can be a therapeutic target for antitumor treatment. In this study, we investigated whether constitutively overexpressed HO-1 in liver cancer cells was associated with resistance to apoptosis induction by cis-diaminedichloroplatinum (cisplatin; CDDP), and then explored the role of HO-1 in protecting tumor cells against chemotherapeutic agents in vitro and in vivo. MATERIALS AND METHODS Reagents CDDP, zinc protoporphyrin IX (ZnPP IX), hemin, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and 2,7-dichlorodihydrofluo-recin diacetate (DCFH-DA) were from Sigma (St. Louis, MO, United States). Dulbecco s Modified Eagle s Medium, fetal bovine serum (FBS), and other cell culture reagents were obtained from Gibco BRL Life Technologies (Grand Island, NY, United States). Rabbit anti-human HO-1 monoclonal antibodies were from Cell Signaling Technology (Danvers, MA, United States). Goat anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) polyclonal antibody and horseradish peroxidase-conjugated goat anti-mouse antibody were from Santa Cruz Biotechnology (Santa Cruz, CA, United States). Cell lines and cell culture The human liver cancer cell lines HepG2, SMMC7721, and 97H were purchased from the Cell Bank, Chinese Academy of Sciences (Shanghai, China). The three cell lines were cultured in RPMI 1640 containing 100 U/ ml penicillin, 0.1 mg/ml streptomycin and 10% heatinactivated FBS at 37 in an atmosphere of 5% CO2 and 95% air. To induce or inhibit the activity of HO-1, hemin or ZnPP IX was added 1 h prior to the addition of CDDP. Cell viability analysis In vitro, the cytostatic and/or cytotoxic effects of treatments were determined using the MTT assay. Tumor cells were seeded in a 96-well plate at a concentration of cells per well and cultured overnight for cell attachment. The following day, cells were treated with the investigational agents for 48 h. After an appropriate time, 20 μl MTT [5 mg/ml in phosphate-buffered saline (PBS)] was added to each well for 4 h. The supernatant was discarded and 150 μl DMSO was added to each well. The plates were shaken until the crystals had dissolved. Absorbance was measured at a wavelength of 570 nm using an enzyme-linked immunosorbent assay reader with background subtraction at 690 nm. Cell viability was expressed as percentage of untreated controls. Experiments were completely randomized in design and repeated six times. In vitro apoptosis assay Induction of apoptosis in vitro was determined by a flow cytometric assay with an annexin V and propidium iodide apoptosis kit according to the manufacturer s instructions 8573 July 14, 2014 Volume 20 Issue 26

266 Liu YS et al. Zinc protoporphyrin IX and hepatoma (Invitrogen, United States). Cells were plated in six-well plates at cells/well and treated for 48 h. After treatment, the cells were harvested from the plate using trypsin and washed twice with PBS, and then incubated with annexin V-fluorescein isothiocyanate and propidium iodide for 15 min. The number of apoptotic cells was analyzed by flow cytometry using a FACScan Analyzer. Experiments were completely randomized in design and repeated six times. Protein preparation and Western blotting Western blotting was used to measure protein expression as follows. Cells were harvested after treatment, and washed twice with PBS. The suspension was resuspended in a buffer containing 1% Triton X-100 with PBS and Halt Protease Inhibitor Cocktail for 30 min on ice and then centrifuged at g for 20 min. Protein concentration was measured with the bicinchoninic acid protein assay reagent according to the manufacturer s instructions (Thermo Scientific, United States). Equivalent amounts of total proteins (80 mg) from each sample were separated by 10% gradient SDS-PAGE and electrophoretically transferred to polyvinylidene difluoride membranes. After blocking with 10% milk, the membranes were incubated with the primary antibody for 3 h at room temperature. The dilutions of the primary antibodies were as follows: 1:1000 for anti-hho-1 antibody and 1:2000 for anti-gapdh antibody. The membranes were washed four times with 0.1% Tween 20 in Tris-buffered saline and then incubated with a secondary antibody for 1 h. The membranes were washed extensively again and the protein bands were visualized with the ECL-Plus chemiluminescence system according to the manufacturer s instructions (Applygen Technologies, Beijing, China). The relative optical density of each Western blotting band was measured using the Quantity One Quantification Software according to the manufacturer s guidelines (Bio-Rad Laboratories). HO-1 activity HO-1 activity was measured by determining the level of bilirubin generated in isolated microsomes. After treatment, cells were collected and homogenized in a homogenization buffer [20 mmol/l potassium phosphate buffer (ph 7.4), 250 mmol/l sucrose, 2 mmol/l EDTA, 2 mmol/l phenylmethyl sulfonyl fluoride (PMSF) and 10 μg/ml leupeptin]. Homogenates were centrifuged at g for 30 min at 4. The resulting supernatants were centrifuged at g for 1 h at 4. The pellet was suspended in phosphate buffer (ph 7.0) and designated the microsome fraction. An aliquot of the microsomal fraction was then added to a reaction mixture containing cytosol of the cells (2 mg cytosolic protein), hemin (20 μmol/l), glucose-6-phosphate (2 mmol/l), glucose-6-phosphate-dehydrogenase (0.2 units), and NADPH (0.8 mmol/l). The reaction mixture was incubated for 60 min at 37 in the dark and terminated by the addition of 1 ml chloroform. The bilirubin concentration was calculated by measuring the difference in absorbance between 465 and 530 nm using a Shimadzu UV-160A spectrophotometer with a molar extinction coefficient of 40/mmol/L/cm. Experiments were completely randomized in design and repeated six times. Measurement of oxidative stress ROS production in each sample was monitored by flow cytometry using the DCFH-DA fluorescent probe. DCFH-DA is a stable compound that rapidly diffuses into cells and is activated by intracellular esterases to DCFH, which is converted by H2O2 and peroxidases to the DFC fluorescent derivate. Thus, the fluorescence intensity is proportional to the amount of peroxide produced by cells. After treatment, the cells were incubated with 10 μmol/l DCFH-DA for 30 min at 37 in the dark. Cells were then washed twice with PBS and resuspended again. The intracellular ROS was quantitated as a function of fluorescence intensity measured by flow cytometry. Measurement of caspase-3 activity Caspase-3 activity was measured with a Colorimetric Assay Kit according to the manufacturer s instructions (Roche). After treatment cells were collected and lysed with chilled lysis buffer [50 mmol/l HEPES (ph 7.5), 150 mmol/l NaCl, 20 mmol/l EDTA, 0.2% Triton X-100, 1 mmol/l PMSF, 10 μg/ml aprotinin, and 5 mmol/l dithiothreitol] for 10 min on ice. The supernatant containing 100 μg protein was incubated with 0.2 mmol/l AcDEVD-pNA, a specific substrate for caspase-3. Caspase-3 activity was measured at 405 nm with background subtraction at 570 nm and expressed in AcpNA cleavage or released absorbance. Liver cancer xenografts in mice Female athymic nude mice (6 wk old) were purchased from the Central Animal Laboratory, Nanjing University, Nanjing, China. Mice were maintained according to Nanjing University of Traditional Chinese Medicine institutional policies. Mice were inoculated subcutaneously into the right flanks with tumor cells, resuspended in 0.2 ml PBS. When tumors reached mm 3 in volume, mice were randomly divided into 6 groups of 8 mice each: control (drug vehicle), hemin (10 mg/kg), ZnPP IX (5 mg/kg), CDDP (5 mg/kg), combination of hemin (10 mg/kg) plus CDDP (5 mg/kg), and combination of ZnPP IX (5 mg/kg) plus CDDP (5 mg/kg). All the drugs were injected weekly into their peritoneal cavities four times. The control group was injected with PBS. All mice were sacrificed 6 wk after the first treatment. Tumors were weighed as previously described [21]. Statistical analysis All data are expressed as mean ± standard deviation (SE). Statistical analysis was performed using one-way analysis of variance and independent-sample t test for each paired experiment with Windows version In all assays, P < 0.05 was considered statistically significant July 14, 2014 Volume 20 Issue 26

267 Liu YS et al. Zinc protoporphyrin IX and hepatoma A HO-1 HepG2 97H SMMC7721 HepG2 CDDP (μg/ml) Control GAPDH HO-1 GAPDH B Cellgrowth inhibition (% of control) H HepG2 SMMC H HO-1 GAPDH CDDP (μg/ml) Control CDDP (μg/ml) (mg/ml) SMMC7721 HO-1 Control Figure 1 Liver cancer cell lines showed divergent heme oxygenase-1 expression levels, which were associated with variable susceptibility to chemotherapy. In Western blotting, high levels of heme oxygenase (HO)-1 protein were detected in HepG2 cell line, whereas other cell lines revealed lower expression (A) HepG2 > 97H > SMMC7721. Cell viability was assessed 48 h after application of cis-diaminedichloroplatinum (cisplatin; CDDP) via MTT assay. All cell lines showed dose-dependent growth inhibition upon treatment with CDDP. The HepG2 cell line, with the highest HO-1 expression, was significantly more chemoresistant to CDDP than the other cell lines with lower HO-1 expression (B). GAPDH Figure 2 Heme oxygenase-1 could be induced by cis-diaminedichloroplatinum in liver cancer cell lines. Western blotting showed that heme oxygenase (HO)-1 protein expression was significantly increased after treatment with cisdiaminedichloroplatinum (cisplatin; CDDP) for 24 h and achieved a plateau at a concentration of 10 μg/ml in all liver cancer cells. RESULTS HO-1 expression in liver cancer cells is associated with susceptibility to chemotherapy Liver cancer cell lines showed different expression levels of HO-1 (Figure 1A). Native HO-1 expression was higher in HepG2 than in other cells, without either hemin or antitumor drug treatment. Furthermore, we compared CDDP-induced growth inhibition in three kinds of human liver cancer cells. HepG2 cells with high native HO-1 expression showed significantly greater in vitro chemoresistance to CDDP than the other cell lines with low native HO-1 expression (Figure 1B). HO-1 could be induced by CDDP in liver cancer cell lines The level of HO-1 was significantly increased in all liver cancer cell lines when cells were treated with CDDP (Figure 2). The highest level of HO-1 expression in all cell lines was achieved with CDDP at concentrations of 5-10 μg/ml. The expression of HO-1 achieved a plateau when CDDP was used at a concentration of 20 μg/ml in all liver cancer cells. Targeted inhibition of HO-1 activity by ZnPP IX in liver cancer cell lines As shown in Figure 3, HO-1 activity was significantly elevated in all liver cancer cell lines after treatment with CDDP compared with untreated cells in vitro (P < 0.05). ZnPP IX decreased HO-1 activity as well as CDDPinduced HO-1 activity (P < 0.05). In contrast, administration of CDDP in combination with hemin significantly increased HO-1 activity in all liver cancer cell lines compared to CDDP treatment alone (P < 0.05). Inhibition of HO-1 activity increases susceptibility to chemotherapy in vitro To demonstrate further the cytoprotective properties of HO-1, the HO-1 inductor hemin and ZnPP IX were added to tumor cells 1 h prior to the application of CDDP. The cytotoxic effect induced by CDDP was largely increased by addition of ZnPP IX, which suggests that inhibition of HO-1 activity by ZnPP IX boosted the anticancer effects of CDDP (Figure 4). In addition, hemin treatment significantly decreased apoptosis induced by CDDP. These findings suggest that augmentation of HO-1 activity induced by hemin is associated with reduced CDDP-induced apoptosis in all liver cancer cell lines. However, it seemed that apoptosis induced by CDDP plus ZnPP IX in the HepG2 cell line was higher than that in the other cell lines. Cytotoxicity of ZnPP IX is related to increased oxidative stress To investigate whether apoptosis induced by the addition 8575 July 14, 2014 Volume 20 Issue 26

268 Liu YS et al. Zinc protoporphyrin IX and hepatoma HepG2 was performed using the oxidant-sensitive fluorescence probe DCDHF. As shown in Figure 5, ZnPP IX (10 μmol/l) increased the fluorescence intensity and CDDP (10 μg/ml)-induced ROS in liver cancer cell lines (P < 0.05). In contrast, hemin (10 μmol/l) decreased CDDPinduced ROS in all liver cancer cell lines (P < 0.05). These findings suggest that the ZnPP IX-induced increase in apoptosis after exposure to CDDP is related to increased intracellular ROS Control ZnPPIX Hemin CDDP 97H ZnPPIX + CDDP Hemin + CDDP Caspase-3 activity in treated and untreated liver cancer cells Caspase-3 activity of the cells paralleled cellular apoptotic vulnerability. Caspase-3 activity in cells was not significantly affected by hemin at the concentration used (10 μmol/l) compared with untreated cells (Figure 6). However, caspase-3 activity was significantly increased in all liver cancer cells after treatment with ZnPP IX (10 μmol/l) or CDDP (10 μg/ml) (P < 0.05). Furthermore, after treatment with CDDP, caspase-3 activity was re-elevated by the addition of ZnPP IX (P < 0.05). Caspase-3 activity induced by CDDP was markedly reduced in the presence of hemin (10 μmol/l) (P < 0.05) Control ZnPPIX Hemin CDDP SMMC7721 ZnPPIX + CDDP Hemin + CDDP Inhibition of HO-1 expression increases susceptibility to chemotherapy in vivo Mice treated with ZnPP IX (5 mg/kg) or CDDP (5 mg/kg) showed significantly reduced tumor growth in comparison with untreated control mice (Figure 7). Inhibition of HO-1 activity by ZnPP IX (5 mg/kg) boosted the anticancer effects of CDDP (5 mg/kg), resulting in a significant reduction of tumor growth in CDDP-treated mice, compared to tumors treated with CDDP alone. In contrast, administration of hemin (10 mg/kg) in combination with CDDP tended to increase tumor weight compared to tumors treated with CDDP alone Control ZnPPIX Hemin CDDP ZnPPIX + CDDP Hemin + CDDP Figure 3 Targeted inhibition of heme oxygenase-1 activity by zinc protoporphyrin IX in liver cancer cell lines. Heme oxygenase (HO)-1 activity was measured by determining the level of bilirubin generated in isolated microsomes in cells treated with 10 μmol/l zinc protoporphyrin (ZnPP) IX, 10 μmol/l hemin, and/or 10 μg/ml cis-diaminedichloroplatinum (cisplatin; CDDP) for 24 h. ZnPP IX significantly inhibited HO-1 activity and decreased cis-diaminedichloroplatinum (cisplatin; CDDP)-induced HO-1 activity in all liver cancer cell lines compared with controls or cells treated with cisplatin alone (P < 0.05). In contrast, hemin increased CDDP-induced HO-1 activity in all liver cancer cell lines (P < 0.05). of ZnPP IX was related to the increase in intracellular production of ROS in liver cancer cells, flow cytometry DISCUSSION Accumulating evidence indicates that overexpression of HO-1 increases the proliferation of cancer cells or renders them resistant to apoptosis induced by chemicals, radiotherapy, photodynamic therapy, and other stressors [12,13,17]. Downregulation of HO-1 expression or activity can suppress cell proliferation or increase cellular sensitivity to some therapeutic regimens. For example, inhibition of HO-1 activity could suppress the proliferation of lung cancer, pancreatic cancer and leukemia cells, and sensitize cells to antitumor drugs in vitro [22-25]. Moreover, inhibition of HO-1 activity could also lead to increased pancreatic cancer cell susceptibility to chemotherapy in vivo [26]. However, little is known about the chemoresistant and poor prognostic function of HO-1 in liver tumors. In this study, we found that constitutive overexpression of HO-1 was specific to HepG2 cells, and this overexpression of HO-1 was associated with higher resistance to CDDP in vitro (Figure 1). We also found that CDDP increased HO-1 activity in liver cancer cells, and 8576 July 14, 2014 Volume 20 Issue 26

269 Liu YS et al. Zinc protoporphyrin IX and hepatoma A 100 HepG H Survival (% of controls) Survival (% of controls) (CDDP mg/ml) (CDDP mg/ml) Survival (% of controls) SMMC7721 CDDP ZnPP IX (10 mmol/l) + CDDP Hemin (10 mmol/l) + CDDP (CDDP mg/ml) B HepG2 Control ZnPP IX (10 mmol/l) Hemin (10 mmol/l) AV AV AV CDDP (10 mg/ml) ZnPP IX (10 mmol/l) + CDDP (10 mg/ml) Hemin (10 mmol/l) + CDDP (10 mg/ml) AV AV AV 8577 July 14, 2014 Volume 20 Issue 26

270 Liu YS et al. Zinc protoporphyrin IX and hepatoma 97H Control ZnPP IX (10 mmol/l) Hemin (10 mmol/l) AV AV AV 10 4 CDDP (10 mg/ml) 10 4 ZnPP IX (10 mmol/l) + CDDP (10 mg/ml) 10 4 Hemin (10 mmol/l) + CDDP (10 mg/ml) AV AV AV SMMC7721 Control ZnPP IX (10 mmol/l) Hemin (10 mmol/l) AV AV AV 10 4 CDDP (10 mg/ml) 10 4 ZnPP IX (10 mmol/l) + CDDP (10 mg/ml) 10 4 Hemin (10 mmol/l) + CDDP (10 mg/ml) AV AV AV Figure 4 Inhibition of heme oxygenase-1 activity led to increased susceptibility to chemotherapy in vitro. Cell viability was assessed via MTT assay. zinc protoporphyrin (ZnPP) IX (10 μmol/l) significantly increased apoptosis in cis-diaminedichloroplatinum (cisplatin; CDDP)-treated cells, compared with those treated with CDDP alone (A) (P < 0.05). In contrast, hemin decreased apoptosis induced by CDDP in all liver cancer cell lines. FACS analysis with propidium iodide staining also showed that downregulation of heme oxygenase (HO)-1 by ZnPP IX increased apoptosis after exposure to CDDP in comparison with the control group (P < 0.05), whereas increased expression of HO-1 by hemin resulted in decreased sensitivity to CDDP (B) July 14, 2014 Volume 20 Issue 26

271 Liu YS et al. Zinc protoporphyrin IX and hepatoma 80 HepG2 400 HepG2 Mean fluorescence intensity Caspase-3 activity (nmol/mg protein) Control ZnPPIX Hemin CDDP ZnPPIX + CDDP Hemin + CDDP 0 Control ZnPPIX Hemin CDDP ZnPPIX + CDDP Hemin + CDDP 80 97H H Mean fluorescence intensity Caspase-3 activity (nmol/mg protein) Control ZnPPIX Hemin CDDP ZnPPIX + CDDP Hemin + CDDP 0 Control ZnPPIX Hemin CDDP ZnPPIX + CDDP Hemin + CDDP 80 SMMC SMMC7721 Mean fluorescence intensity Caspase-3 activity (nmol/mg protein) Control ZnPPIX Hemin CDDP ZnPPIX + CDDP Hemin + CDDP 0 Control ZnPPIX Hemin CDDP ZnPPIX + CDDP Hemin + CDDP Figure 5 Cytotoxicity of zinc protoporphyrin IX is related to increased oxidative stress. Induction of intracellular ROS was evaluated by flow cytometry by measuring CM-H2DCFDA fluorescence. Each cell line was treated with 10 μmol/l zinc protoporphyrin (ZnPP) IX, 10 μmol/l hemin, and/or 10 μg/ml cis-diaminedichloroplatinum (cisplatin; CDDP) for 24 h. Mean fluorescence intensity was quantified for these treatments. ZnPP IX increased the fluorescence intensity of the cells and drug-induced ROS in liver cancer cell lines compared with controls or cells treated with CDDP alone. In contrast, hemin decreased CDDP-induced ROS in all liver cancer cell lines. inhibition of HO-1 activity by ZnPP IX boosted the anticancer effects of CDDP. These results suggest that the cellular sensitivity of liver cancer cells to CDDP is Figure 6 Caspase-3 activity in treated and untreated liver cancer cells. Inhibition of heme oxygenase (HO)-1 activity increased caspase-3 induced by cis-diaminedichloroplatinum (cisplatin; CDDP) in all liver cancer cell lines. Caspase-3 activity was significantly increased in all cells after treatment with cisplatin (10 μg/ml) and re-elevated by addition of zinc protoporphyrin (ZnPP) IX (10 μmol/l). The caspase-3 activity induced by CDDP was markedly reduced in the presence of hemin (10 μmol/l) in all liver cancer cell lines. augmented by downregulation of HO-1 activity by ZnPP IX. In support of this conclusion, induction of HO-1 activity by the addition of hemin resulted in a decrease in CDDP-induced apoptosis in all liver cancer cells (Figure 1). These findings suggest that HO-1, as a stress response 8579 July 14, 2014 Volume 20 Issue 26

272 Liu YS et al. Zinc protoporphyrin IX and hepatoma Tumor weight (mg) Tumor weight (mg) Control Control ZnPPIX ZnPPIX Hemin HepG2 SMMC7721 Hemin CDDP CDDP ZnPPIX + CDDP ZnPPIX + CDDP Hemin + CDDP Hemin + CDDP Figure 7 Inhibition of heme oxygenase-1 expression increases susceptibility to chemotherapy in vivo. Each nude mouse was treated with 5 mg/kg zinc protoporphyrin (ZnPP) IX, 10 mg/kg hemin, and/or 5 mg/kg cis-diaminedichloroplatinum (cisplatin; CDDP) for 6 wk and tumor weight was assessed after the last treatment. ZnPP IX treatment significantly reduced tumor growth in CDDP-treated mice, compared with those treated with CDDP alone. CDDP in combination with hemin in mice significantly increased tumor weight compared with CDDP treatment alone. gene, plays an important role in the determination of the sensitivity of liver cancer cells to CDDP. Recent studies in other models support this view: induction of HO-1 expression made colon cancer cells and leukemia resistant to antitumor drugs such as merocyanine and pyrrolidine dithiocarbamate [27,28]. Furthermore, inhibition of HO-1 activity by polyethylene glycol-znpp IX augmented the cytotoxic effect of several chemotherapeutic agents in an experimental murine sarcoma model [17]. However, opposite effects were observed in breast cancer and B lymphoblasts, in which HO-1 did not protect the cells from chemotherapy-induced apoptosis [29]. This might be because HO-1 has different biological actions in different cancer cells. The signaling pathways mediating these effects on cancer are poorly understood, although there is accumulating evidence showing that HO-1 is an antiapoptotic agent in several types of cells. Some previous studies have demonstrated that apoptosis of human oral and gastric cancer cells could be regulated by Nrf2 and p21 [30,31]. Overexpression of HO-1 resulted in a significant increase in Nrf2 and p21 levels and decreased cellular sensitivity to chemotherapy. In contrast, inhibition of HO-1 expression or activity leads to the downregulation of Nrf2 and p21 and decreases cellular sensitivity to chemotherapy. However, we have also shown that the mechanism of resistance to apoptosis by upregulation of HO-1 may be related to increased ROS level and caspase-3 activity. The destructive mechanisms of chemotherapy on cancer cells are mainly based on the generation of oxidative stress and/or induction of apoptosis [32]. The accumulation of ROS results in the subsequent loss of mitochondrial membrane potential and activation of the caspase-9/3 pathway. The present study showed that the activity of caspase-3 and ROS level induced by CDDP were significantly inhibited by elevated hemin (Figures 5 and 6). In contrast, ROS level and caspase-3 activity were significantly re-elevated by the addition of ZnPP IX in liver cancer cells after treatment with CDDP. Therefore, our study suggests that the resistance to apoptosis by HO-1 may be through a pathway with elevated ROS level and caspase-3 activity in human liver cancer cells. In addition, the products of HO-1 enzymatic activity directly influence several other cell signaling pathways, such as the Ras-Raf-ERK pathway, which have crucial roles in human renal cancer cells [33]. However, the mechanisms involved in increased chemosensitivity need additional investigation. A more important finding, consistent with in vitro results, is that inhibition of HO-1 activity by ZnPP IX boosts anti-cancer effects of CDDP, resulting in a significant reduction of tumor growth in mice, compared to those treated with CDDP alone (Figure 7). In contrast, administration of hemin in combination with CDDP tends to increase tumor weight. These findings suggest that inhibition of HO-1 expression leads to increased sensitivity of liver cancer cells to CDDP in vivo. In conclusion, the findings in this study suggest that high HO-1 levels in HepG2 cells are, at least partially, responsible for resistance to chemotherapy and environmental stress. Inhibition of HO-1 activity by ZnPP IX results in sensitizing tumor cells to chemotherapy such as CDDP by increasing the cellular ROS level and caspase-3 activity in vitro and in vivo. The combined treatment with HO-1 inhibitors may intensify the antitumor activity of chemotherapeutics and may open up new perspectives in the treatment of liver cancer. COMMENTS Background Inhibition of heme oxygenase (HO)-1 expression or activity was shown to suppress cellular proliferation and increase responsiveness of tumor cells to some anti-cancer treatments. In contrast, induction of HO-1 expression or activity decreases cell sensitivity to anti-tumor drugs. Accumulating evidence suggests that HO-1 could be a therapeutic target for anti-tumor treatment. Research frontiers The cytoprotective enzyme HO-1 is significantly overexpressed in many tumors and seems to play an important role in cellular resistance to chemotherapy and 8580 July 14, 2014 Volume 20 Issue 26

273 Liu YS et al. Zinc protoporphyrin IX and hepatoma radiotherapy. In this study, the authors examined whether constitutively overexpressed HO-1 in liver cancer cells was associated with resistance to apoptosis induction by cis-diaminedichloroplatinum (cisplatin; CDDP), and explored the role of HO-1 in protecting tumor cells against chemotherapeutic agents in vitro and in vivo. Innovations and breakthroughs The results suggested that overexpression of HO-1 in HepG2 cell lines was associated with increased chemoresistance to CDDP compared with other cell lines in vitro. The inhibition of HO-1 by zinc protoporphyrin IX (ZnPP IX) was associated with increased cellular sensitivity and susceptibility of liver cancer cell lines to CDDP in vivo or in vitro. Reactive oxygen species (ROS) and caspase-3 seem to be involved in HO-1-mediated resistance to anti-cancer treatment. Therefore, administration of HO-1 inhibitors may evolve into a new liver cancer treatment strategy. Applications The findings in this study suggest that high HO-1 levels in HepG2 cells may, at least partially, be responsible for their resistance to chemotherapy and environmental stress. The combined treatment with HO-1 inhibitors may intensify the anti-tumor activity of chemotherapeutic agents and open up new perspectives in the treatment of liver cancer. Terminology High HO-1 levels in HepG2 cells may be responsible for their resistance to chemotherapy and environmental stress. Inhibition of HO-1 activity by ZnPP IX sensitizes tumor cells to chemotherapy (e.g., with CDDP) by increasing the cellular ROS level and caspase-3 activity in vitro and in vivo. Peer review This manuscript reports the results of a study on the effect of ZnPP IX, an HO-1 inhibitor, on the cellular sensitivity and susceptibility of liver cancer cell lines to CDDP in vitro and in vivo. The results are interesting and may open up new perspectives in the treatment of liver cancer. 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274 Liu YS et al. Zinc protoporphyrin IX and hepatoma 22 Kim HR, Kim S, Kim EJ, Park JH, Yang SH, Jeong ET, Park C, Youn MJ, So HS, Park R. Suppression of Nrf2-driven heme oxygenase-1 enhances the chemosensitivity of lung cancer A549 cells toward cisplatin. Lung Cancer 2008; 60: [PMID: ] 23 Do MT, Kim HG, Khanal T, Choi JH, Kim DH, Jeong TC, Jeong HG. Metformin inhibits heme oxygenase-1 expression in cancer cells through inactivation of Raf-ERK-Nrf2 signaling and AMPK-independent pathways. Toxicol Appl Pharmacol 2013; 271: [PMID: DOI: /j.taap] 24 Mayerhofer M, Gleixner KV, Mayerhofer J, Hoermann G, Jaeger E, Aichberger KJ, Ott RG, Greish K, Nakamura H, Derdak S, Samorapoompichit P, Pickl WF, Sexl V, Esterbauer H, Schwarzinger I, Sillaber C, Maeda H, Valent P. Targeting of heat shock protein 32 (Hsp32)/heme oxygenase-1 (HO-1) in leukemic cells in chronic myeloid leukemia: a novel approach to overcome resistance against imatinib. Blood 2008; 111: [PMID: ] 25 Tibullo D, Barbagallo I, Giallongo C, La Cava P, Parrinello N, Vanella L, Stagno F, Palumbo GA, Li Volti G, Di Raimondo F. Nuclear translocation of heme oxygenase-1 confers resistance to imatinib in chronic myeloid leukemia cells. Curr Pharm Des 2013; 19: [PMID: ] 26 Nuhn P, Künzli BM, Hennig R, Mitkus T, Ramanauskas T, Nobiling R, Meuer SC, Friess H, Berberat PO. Heme oxygenase-1 and its metabolites affect pancreatic tumor growth in vivo. Mol Cancer 2009; 8: 37 [PMID: DOI: / ] 27 Lin F, Girotti AW. Hyperresistance of leukemia cells to photodynamic inactivation after long-term exposure to hemin. Cancer Res 1996; 56: [PMID: ] 28 Hellmuth M, Wetzler C, Nold M, Chang JH, Frank S, Pfeilschifter J, Mühl H. Expression of interleukin-8, heme oxygenase-1 and vascular endothelial growth factor in DLD-1 colon carcinoma cells exposed to pyrrolidine dithiocarbamate. Carcinogenesis 2002; 23: [PMID: ] 29 Andreadi CK, Howells LM, Atherfold PA, Manson MM. Involvement of Nrf2, p38, B-Raf, and nuclear factor-kappab, but not phosphatidylinositol 3-kinase, in induction of hemeoxygenase-1 by dietary polyphenols. Mol Pharmacol 2006; 69: [PMID: ] 30 Lee YM, Auh QS, Lee DW, Kim JY, Jung HJ, Lee SH, Kim EC. Involvement of Nrf2-mediated upregulation of heme oxygenase-1 in mollugin-induced growth inhibition and apoptosis in human oral cancer cells. Biomed Res Int 2013; 2013: [PMID: DOI: /2013/210604] 31 Liu ZM, Chen GG, Ng EK, Leung WK, Sung JJ, Chung SC. Upregulation of heme oxygenase-1 and p21 confers resistance to apoptosis in human gastric cancer cells. Oncogene 2004; 23: [PMID: ] 32 Simizu S, Takada M, Umezawa K, Imoto M. Requirement of caspase-3(-like) protease-mediated hydrogen peroxide production for apoptosis induced by various anticancer drugs. J Biol Chem 1998; 273: [PMID: ] 33 Banerjee P, Basu A, Datta D, Gasser M, Waaga-Gasser AM, Pal S. The heme oxygenase-1 protein is overexpressed in human renal cancer cells following activation of the Ras-Raf- ERK pathway and mediates anti-apoptotic signal. J Biol Chem 2011; 286: [PMID: DOI: /jbc. M ] P- Reviewers: Lee HC, Mehdi I, Yamasaki T, Zhang YJ S- Editor: Gou SX L- Editor: Wang TQ E- Editor: Zhang DN 8582 July 14, 2014 Volume 20 Issue 26

275 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. RESEARCH REPORT Prognostic significance of preoperative fibrinogen in patients with colon cancer Zhen-Qiang Sun, Xiao-Na Han, Hai-Jiang Wang, Yong Tang, Ze-Liang Zhao, Yan-Li Qu, Rui-Wei Xu, Yan-Yan Liu, Xian-Bo Yu Zhen-Qiang Sun, Hai-Jiang Wang, Ze-Liang Zhao, Xian-Bo Yu, Department of Gastrointestinal Surgery, Affiliated Tumor Hospital, Xinjiang Medical University, Urumqi , Xinjiang Uygur Autonomous Region, China Zhen-Qiang Sun, Research Laboratory of Disease Genomics, Cancer Research Institute, Central South University, Changsha , Hunan Province, China Xiao-Na Han, Yong Tang, Yan-Li Qu, Department of Digestive Oncology, Affiliated Tumor Hospital, Xinjiang Medical University, Urumqi , Xinjiang Uygur Autonomous Region, China Rui-Wei Xu, Infection and Statistical Office, Affiliated Tumor Hospital, Xinjiang Medical University, Urumqi , Xinjiang Uygur Autonomous Region, China Yan-Yan Liu, Clinical Pharmacy, Tengzhou Central People s Hospital, Tengzhou , Shandong Province, China Author contributions: Sun ZQ, Han XN and Wang HJ conceived and designed the study; Sun ZQ wrote the first draft of the manuscript; Zhao ZL and Yu XB assisted with measurements; Tang Y and Xu RW helped analyze the data; Qu YL and Liu YY collected clinicopathological materials and postoperative samples; all authors read and approved the final manuscript. Supported by The Science and Technology Innovation Fund of Xinjiang Medical University, No. XJC Correspondence to: Hai-Jiang Wang, MD, Department of Gastrointestinal Surgery, Affiliated Tumor Hospital, Xinjiang Medical University, 789 Suzhou East Street, Urumqi , Xinjiang Uygur Autonomous Region, China. wanghaijiang0129@163.com Telephone: Fax: Received: November 5, 2013 Revised: February 22, 2014 Accepted: March 12, 2014 Published online: July 14, 2014 Abstract AIM: To investigate the prognostic significance of preoperative fibrinogen levels in colon cancer patients. METHODS: A total of 255 colon cancer patients treated at the Affiliated Tumor Hospital of Xinjiang Medical University from June 1 st 2005 to June 1 st 2008 were enrolled in the study. All patients received radical surgery as their primary treatment method. Preoperative fibrinogen was detected by the Clauss method, and all patients were followed up after surgery. Preoperative fibrinogen measurements were correlated with a number of clinicopathological parameters using the Student t test and analysis of variance. Survival analyses were performed by the Kaplan-Meier method and Cox regression modeling to measure 5-year disease-free survival (DFS) and overall survival (OS). RESULTS: The mean preoperative fibrinogen concentration of all colon cancer patients was 3.17 ± 0.88 g/l. Statistically significant differences were found between preoperative fibrinogen levels and the clinicopathological parameters of age, smoking status, tumor size, tumor location, tumor-node-metastasis (TNM) stage, modified Glasgow prognostic scores (mgps), white blood cell (WBC) count, neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), and carcinoembryonic antigen (CEA) levels. Univariate survival analysis showed that TNM stage, tumor cell differentiation grade, vascular invasion, mgps score, preoperative fibrinogen, WBC, NLR, PLR and CEA all correlated with both OS and DFS. Alpha-fetoprotein (AFP) and body mass index correlated only with OS. Kaplan-Meier analysis revealed that both OS and DFS of the total cohort, as well as of the stage Ⅱ and Ⅲ patients, were higher in the hypofibrinogen group compared to the hyperfibrinogen group (all P < 0.05). In contrast, there was no significant difference between OS and DFS in stage Ⅰ patients with low or high fibrinogen levels. Cox regression analysis indicated preoperative fibrinogen levels, TNM stage, mgps score, CEA, and AFP levels correlated with both OS and DFS. CONCLUSION: Preoperative fibrinogen levels can serve as an independent prognostic marker to evaluate patient response to colon cancer treatment Baishideng Publishing Group Inc. All rights reserved July 14, 2014 Volume 20 Issue 26

276 Sun ZQ et al. Preoperative fibrinogen and colon cancer Key words: Fibrinogen; Colon cancer; Clinicopathological parameters; Relationship; Prognosis Core tip: Inflammatory cytokines have been implicated in the development of several malignancies, including colon cancer. Specifically, fibrinogen has been correlated with some clinicopathological factors of cancer. However, the prognostic value of fibrinogen measurements remains unclear, particularly in colon cancer patients. In this study, preoperative fibrinogen levels were correlated with a poor prognosis in a cohort of colon cancer patients. These data suggest that preoperative fibrinogen levels can serve as an independent prognostic factor for colon cancer patients following radical surgery. Sun ZQ, Han XN, Wang HJ, Tang Y, Zhao ZL, Qu YL, Xu RW, Liu YY, Yu XB. Prognostic significance of preoperative fibrinogen in patients with colon cancer. World J Gastroenterol 2014; 20(26): Available from: URL: com/ /full/v20/i26/8583.htm DOI: org/ /wjg.v20.i INTRODUCTION Recently, the incidence of gastrointestinal cancer has increased as standards of living have improved. Colon cancer is one of the most common malignancies of the digestive track in humans. Early detection is strongly correlated with an improved prognosis, thus it is important to develop simple, non-invasive methods to accurately detect colon cancer and assess patient prognosis. Recent evidence indicates that fibrinogen, a major protein in the blood clotting process, is linked to a number of malignancies [1-4]. Elevated fibrinogen levels have been correlated with tumor progression, invasiveness, and metastasis [5-7]. However, the role of fibrinogen in colon cancer remains unclear. To assess the prognostic significance of fibrinogen for colon cancer patients, preoperative fibrinogen levels, clinicopathological parameters, and follow-up data were analyzed from a cohort of 255 colon cancer patients undergoing radical surgery. The results indicate that preoperative fibrinogen levels correlate with a number of cancer clinicopathological parameters, and are negatively correlated with 5-year survival rates. Therefore, fibrinogen measurements could provide a useful prognostic tool for colon cancer patients. MATERIALS AND METHODS Patients and clinicopathological parameters Clinicopathological parameters and follow-up data were assessed from 255 colon cancer patients (135 male, 120 female) who received radical surgery in the Affiliated Tumor Hospital of Xinjiang Medical University between June 1 st 2005 and June 1 st Mean patient age was ± years. Prior to participation, a diagnosis of colon cancer was confirmed by histopathology for all patients. Tumor-node-metastasis (TNM) stage was determined according to the American Joint Committee on Cancer/International Union Against Cancer TNM staging system of colorectal cancer (2010, 7 th edition). The American Society of Anesthesiologists score of the patients was 1 in 198 cases and 2 in 57 cases. No patient received preoperative chemotherapy or immunotherapy. The presence of complex metastases, such as uncertain lumps, micrometastases (particularly in the liver), and abdominal/pelvic lymph node metastases, were diagnosed using enhanced computed tomography, magnetic resonance imaging, positron emission tomography-computed tomography, and puncture biopsies. The following criteria were used to exclude patients: confirmed metastasis (39 cases), use of procoagulant or anticoagulant drugs within 8 wk of the enrollment (11 cases), hepatitis infection (23 cases), kidney disease (9 cases), myocardial infarction (14 cases), hypertension (47 cases), or other infectious or hematologic diseases which could influence fibrinogen levels (22 cases). Postoperative adjuvant chemotherapy was administered to stage Ⅱ patients with high-risk factors (including poor differentiation, large lumps, T4 stage, less than 1 cm of tumor resection margin, fewer than 12 lymph nodes for postoperative biopsy) and stage Ⅲ patients. Blood samples were obtained by phlebotomy; 5-7 d prior to surgery, 2 ml of blood was collected from each patient. Preoperative fibrinogen levels were measured by the Clauss standard method with bovine thrombin (100 NIH U/mL). Follow-up assessments After surgery, patients underwent follow-up assessments once every 3 mo for the first 2 years, and then once every 6 mo until 5 years after surgery. Follow-ups were either by outpatient or inpatient review, or by telephone. Thirty patients did not participate in follow-up analyses because they did not maintain any communication with physicians after surgery. In addition, 3 patients developed dysthymia and were unable to cooperate with the study procedures, one patient committed suicide due to the burden of the cancer, and 2 patients did non participate in follow-up assessments for unknown reasons. Therefore, the total follow-up rate in the study was 88.2%. Ethics The study design and procedures described below were approved by the Ethics Committee of the Affiliated Tumor Hospital of Xinjiang Medical University (No. W ). All patients provided informed written consent prior to their participation. Statistical analysis All data is presented as mean ± SD. Univariate analyses of preoperative fibrinogen and each clinicopathological parameter were performed using the Student t-test and 8584 July 14, 2014 Volume 20 Issue 26

277 Sun ZQ et al. Preoperative fibrinogen and colon cancer Table 1 Correlations between preoperative fibrinogen and clinicopathological parameters Parameters n Fibrinogen (g/l, mean ± SD) P value Age (yr) < ± ± 0.73 Nationality Han ± Uygur ± 0.92 Others ± 0.94 BMI (kg/m 2 ) < ± ± ± ± 0.79 Smoking Never ± Light ± 0.74 Heavy ± 0.99 Grade (tumor differentiation) Low ± High ± 0.92 TNM Ⅰ ± Ⅱ ± 0.95 Ⅲ ± 0.78 Tumor size (cm) < ± ± ± 0.98 Type Mess type ± Ulceration ± 0.89 Infiltrative ± 0.13 Tumor location Left ± Right ± 0.97 Transverse ± 0.55 Perineural invasion Yes ± No ± 0.88 Vascular invasion Yes ± No ± 0.87 CEA (ng/ml) < ± ± 0.84 AFP (ng/ml) < ± ± 0.82 mgps ± ± ± 1.00 WBC count < /L ± /L ± 0.90 NLR < ± ± 1.02 PLR < ± ± 0.96 > ± 0.83 AFP: Alpha-fetoprotein; BMI: Body mass index; CEA: Carcinoembryonic antigen; mgps: Modified Glasgow prognostic score; NLR: Neutrophillymphocyte ratio; PLR: Platelet-lymphocyte ratio; TNM: Tumor-nodemetastasis; WBC: White blood cell. analysis of variance. Survival rates were determined using the Kaplan-Meier method and the log-rank test to perform comparisons between the hypofibrinogen (< 2.61 g/l) and hyperfibrinogen ( 2.61 g/l) groups. Multivariate survival analysis was performed using the Cox regression model to determine relative risk (RR) and 95% confidence interval (CI). All statistical analyses were carried out using SPSS for Windows, version 18 (SPSS Inc., Chicago, IL, United States). Statistical significance was defined by P RESULTS Correlations between preoperative fibrinogen levels and clinicopathological parameters of colon cancer patients The mean level of preoperative fibrinogen levels of all 255 colon cancer patients was 3.17 ± 0.88 g/l. Statistical analyses were performed to assess any relationships between fibrinogen levels and clinicopathological factors (Table 1). Preoperative fibrinogen levels were associated with age, smoking status, tumor size, tumor location, TNM stage, modified Glasgow prognostic (mgps) score, white blood cell (WBC) count, neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), and carcinoembryonic antigen (CEA) levels (P < 0.05 for all parameters). Meanwhile, preoperative fibrinogen was not related to the patient s nationality, body mass index (BMI), tumor grade, gross tumor type, tumor invasion into the nervous or lymphatic system, or serum alpha-fetoprotein (AFP) levels. Prognostic analysis of clinicopathological parameters in colon cancer patients To determine the prognostic significance, preoperative fibrinogen and other clinicopathological factors were correlated with the 5-year disease-free survival (DFS) and overall survival (OS) rates of the patient cohort (Table 2). Results show that TNM stage, tumor grade, vascular invasion status, mgps score, preoperative fibrinogen, WBC count, NLR, PLR, and CEA levels were all significantly correlated with both OS and DFS rates (all P < 0.05). In addition, AFP and BMI were significantly correlated with OS (P < 0.05). In contrast, no significant relationship was observed between prognosis and age, nationality, smoking status, gross tumor type, tumor location, or perineural invasion status. Survival analysis of patients with different preoperative fibrinogen levels Given the correlation observed between preoperative fibrinogen and DFS and OS rates, the effect of fibrinogen levels on patient survival was examined. Patients were divided into hyperfibrinogen (> 2.61 g/l) or hypofibrinogen (< 2.61 g/l) groups. Kaplan-Meier survival analyses were performed to determine 5-year OS and DFS rates for each group. Among the entire cohort, both OS and DFS rates were higher in the hypofibrinogen group compared with the hyperfibrinogen group (Figure 1A; P < 8585 July 14, 2014 Volume 20 Issue 26

278 Sun ZQ et al. Preoperative fibrinogen and colon cancer Table 2 Univariate survival analyses of clinicopathological parameters Parameters n 5-yr DFS χ 2 P value 5-yr OS χ 2 P value Age (yr) < % % % 61.80% Nationality Han % % Uygur % 48.80% Others % 76.20% BMI (kg/m 2 ) < % % % 55.60% % 74.00% % 59.30% Smoking Never % % Light % 60.20% Heavy % 61.40% Grade Low % % High % 49.30% TNM Ⅰ % % Ⅱ % 70.60% Ⅲ % 43.90% Type Mess type % % Ulceration % 59.30% Infiltrative % 0 Tumor location Left % % Right % 54.30% Transverse % 81.80% Perineural invasion Yes % % No % 63.90% Vascular invasion Yes % % No % 65.10% CEA (ng/ml) < % % % 49.10% AFP (ng/ml) < % % % 43.20% mgps % % % 49.80% % 4.80% WBC count < /L % % /L % 59.30% NLR < % % % 43.80% PLR < % % % 57.20% > % 33.30% Fibrinogen (g/l) < % % % 53.60% DFS: Disease-free survival; OS: Overall survival; BMI: Body mass index; TNM: Tumor-node-metastasis; CEA: Carcinoembryonic antigen; AFP: Alphafetoprotein; mgps: Modified Glasgow prognostic scores; WBC: White blood cell; NLR: Neutrophil-lymphocyte ratio; PLR: Platelet-lymphocyte ratio. 0.05). Furthermore, hyperfibrinogen was associated with significantly reduced OS and DFS rates in stage Ⅱ and Ⅲ colon cancer patients (Figure 1C, D; P < 0.05), but not in patients with stage I disease (Figure 1B). However, 8586 July 14, 2014 Volume 20 Issue 26

279 Sun ZQ et al. Preoperative fibrinogen and colon cancer A OS% DFS% Fibrinogen < 2.61 g/l Fibrinogen 2.61 g/l Censcored Censcored Fibrinogen < 2.61 g/l Fibrinogen 2.61 g/l Censcored Censcored t /mo t /mo B OS% 0.4 DFS% Fibrinogen < 2.61 g/l Fibrinogen 2.61 g/l Censcored Censcored Fibrinogen < 2.61 g/l Fibrinogen 2.61 g/l Censcored Censcored t /mo t /mo C OS% DFS% Fibrinogen < 2.61 g/l Fibrinogen 2.61 g/l Censcored Censcored Fibrinogen < 2.61 g/l Fibrinogen 2.61 g/l Censcored Censcored t /mo t /mo D OS% 0.4 DFS% Fibrinogen < 2.61 g/l Fibrinogen 2.61 g/l Censcored Censcored Fibrinogen < 2.61 g/l Fibrinogen 2.61 g/l Censcored Censcored t /mo t /mo Figure 1 Kaplan-Meier curves of hyperfibrinogen and hypofibrinogen colon cancer groups. A: Entire patient cohort; B: Stage Ⅰ; C: Stage Ⅱ; D: Stage Ⅲ. 1 and 2 stand for the 5-yr overall survival (OS) and 5-yr disease-free survival (DFS), respectively. Both OS and DFS of the entire cohort, stage Ⅱ, and stage Ⅲ patients were significantly higher in the hypofibrinogen (< 2.61 g/l) group than in the hyperfibrinogen ( 2.61 g/l) group (P < 0.05) July 14, 2014 Volume 20 Issue 26

280 Sun ZQ et al. Preoperative fibrinogen and colon cancer Table 3 Cox proportional hazards model analyses for overall survival and disease-free survival Parameters 5-yr OS 5-yr DFS P value RR 95%CI P value RR 95%CI PLR NLR WBC count AFP CEA Vascular invasion Fibrinogen BMI TNM Grade mgps RR: Relative risk; CI: Confidence interval; PLR: Platelet-lymphocyte ratio; NLR: Neutrophil-lymphocyte ratio; WBC: White blood cell; AFP: Alphafetoprotein; CEA: Carcinoembryonic antigen; BMI: Body mass index; TNM: Tumor-node-metastasis; mgps: Modified Glasgow prognostic scores. Table 4 Studies on fibrinogen in different malignancies Ref. Country n Pre-therapeutic plasma fibrinogen (g/l) Cancer type Prognosis DFS/OS Takeuchi et al [1] (2007) Japan ± 1.02 Esophageal cancer No, neither Polterauer et al [2] (2009) Austria ± 1.30 Cervical cancer Yes, both Polterauer et al [3] (2009) Austria ± 1.50 Ovarian cancer Yes, both Ghezzi et al [4] (2010) Italy 336 hyper: 40.8% Endometrial cancer Yes, both Zhao et al [5] (2012) China ± 1.46 Non-small cell lung cancer DFS, no; OS, yes Son et al [6] (2013) South Korea ± 0.88 Non-metastatic colon cancer Yes, both Zhu et al [7] (2013) China ( ) Non-small cell lung cancer Yes, both DFS: Disease-free survival; OS: Overall survival. the lack of a significant association with stage Ⅰ disease may be related to the relatively small sample size for this patient group. Survival analysis of different clinicopathological parameters Cox regression analyses showed that preoperative fibrinogen, TNM stage, mgps score, CEA and AFP levels were significantly associated with both 5-year OS and 5-year DFS (P < 0.05). For DFS, the strongest predictive factor was preoperative fibrinogen levels (RR: 2.940; 95%CI: , P < 0.05), followed by TNM stage, mgps, AFP and CEA levels. Similarly, fibrinogen was the strongest predictor for OS (RR: 3.324; 95%CI: , P < 0.05), followed by mgps, TNM stage, AFP and CEA (Table 3). These data indicate that preoperative fibrinogen plays an important role in the prognosis of colon cancer patients who receive radical surgery. DISCUSSION Recent evidence has indicated that there are links between inflammation and cancer development [8]. In addition to the genetic basis of cancer, the host inflammatory response can play an important role in tumor progression [9,10]. In particular, inflammatory cytokines are associated with a number of cancer types and contribute to tumorigenesis by promoting tumor cell proliferation, invasion, or metastasis, which can have dramatic effects on patient prognosis. Several inflammatory markers that are readily detectable in plasma are currently being investigated as prognostic factors for cancer patients, as well as for their role in tumor biology. These markers include monocyte chemo-attractant proteins [11,12], WBC count, NLR [13,14], PLR [11,15], and fibrinogen [16], among others. However, there is currently no consensus concerning their efficacy to assess patient prognosis [1-3]. Fibrinogen is synthesized in the liver as a 350 kda glycoprotein. Fibrinogen plays an important role in blood coagulation, thrombosis, wound healing, and platelet aggregation. Moreover, recent studies suggest fibrinogen is associated with cancer development (Table 4). As a component of the extracellular matrix, fibrinogen can affect cell behavior through interactions with other matrix proteins, or the cell itself. Fibrinogen has been associated with increased tumor growth and metastatic potential, although the precise mechanism remains unclear. One potential mechanism involves fibrinogen s influence on tumor cell proliferation, migration, and signaling through interactions with multiple integrin and non-integrin receptors. Another possible mechanism is the promotion of tumor angiogenesis, as fibrinogen has been shown to cooperate with growth factors, including vascular endothelial and fibroblast growth factors, to stimulate angiogenesis [17-19]. Furthermore, the fibrinolytic system derived from fibrinogen also plays a facilitating role in both an July 14, 2014 Volume 20 Issue 26

281 Sun ZQ et al. Preoperative fibrinogen and colon cancer giogenesis and the proliferation process of tumor cells [20]. Several lines of evidence indicate that fibrinogen can promote tumor cell metastasis. The fibrinolytic system stimulates adhesion between tumor cells and platelets or endothelial cells, thereby promoting the dissemination of tumor cells into the bloodstream [21]. Studies using fibrinogen-deficient mice have indicated that fibrinogen is an important factor in the metastasis of circulating tumor cells [22]. In addition, fibrinogen can suppress natural killer cell-mediated tumor cell clearance, leading to an increase in the number of metastatic cells [23]. Taken together, these data suggest that fibrinogen is involved in tumor cell invasion and metastasis, further supporting its potential as a prognostic factor for cancer patients. In clinical studies, pre-treatment fibrinogen levels have been associated with clinicopathological factors in a number of cancers. Fibrinogen has been correlated with the depth of tumor invasion, tumor size, and WBC and platelet counts [1]. Elevated fibrinogen levels are associated with lymph node metastasis and distant metastasis. Fibrinogen has also been correlated with clinicopathological factors such as age and BMI [3]. In our study, we analyzed additional clinicopathological parameters in colon cancer patients, and found that preoperative fibrinogen levels correlated with several cancer-relevant factors, including age, smoking status, tumor size, tumor location, TNM stage, mgps score, WBC count, NLR, and PLR. The results of this study also support previous reports demonstrating that smoking, which confers a high risk of tumor development, is correlated with serum fibrinogen levels [24]. However, the mechanisms linking smoking and fibrinogen in cancer patients remain unclear. Tumor progression is the result of complex interactions between tumor cells and the host environment [25] and the host inflammatory response. Tumors can occur at sites of inflammation, such as during hepatitis virus or H. pylori infection, and can also trigger the release of pro-inflammatory cytokines to promote an inflammatory microenvironment that favors tumorigenesis [26,27]. The elevation of fibrinogen levels in colon cancer patients may be the result of tissue damage and subsequent inflammatory responses [28]. For example, tumors can induce a hypercoagulable and hypoxic state through a variety of mechanisms, including the stimulation of an inflammatory response [18,21,29]. Thus, it is likely that elevated fibrinogen reflects the host-tumor response [15]. However, tumor cells can produce endogenous fibrinogen, suggesting that other sources may also contribute [18]. Consistent with this evidence, results from this study indicate that serum fibrinogen is closely related with other inflammatory markers, such as the mgps score, WBC count, NLR and PLR, suggesting that fibrinogen reflects the inflammatory status of colon cancer tumors. A systemic inflammatory response prior to surgery may reflect the presence of an additional disease or a change in the patient s physiology [30]. Several studies have demonstrated that fibrinogen levels can serve as an independent prognostic factor for cancer patients. For example, fibrinogen levels were closely associated with both 5-year DFS and OS in a cohort of 313 cervical cancer patients [2]. A similar analysis of a cohort of 422 ovarian cancer patients led to the same conclusion [3]. In colon cancer patients, systemic inflammatory markers can predict patient survival following radical resection [15] and provide independent prognostic risk factors for tumor specificity and OS for these patients [31]. A Cox regression statistical analysis in the present study showed that preoperative fibrinogen level was closely associated with prognosis of patients with colon cancer, which is in contrast to the results of Takeuchi et al [1], who found that serum fibrinogen was not associated with prognosis in 105 esophageal cancer patients. Furthermore, survival analyses indicated that both the 5-year OS and DFS of the entire cohort, as well as of stage Ⅱ and stage Ⅲ patients, were significantly higher in the hypofibrinogen group compared to the hyperfibrinogen group. These results are consistent with previous studies investigating inflammation and colon cancer, and suggest that fibrinogen can serve as a useful prognostic marker for colon cancer patients. In conclusion, this study demonstrates that fibrinogen is closely associated with several clinicopathological features and 5-year survival rates of colon cancer patients. Furthermore, elevated preoperative fibrinogen levels were found to predict poor survival in colon cancer patients. These data suggest that preoperative fibrinogen is a promising prognostic marker for colon cancer patients receiving radical surgery. Moreover, fibrinogen-based therapies may provide a novel therapeutic approach for treating colon cancer. COMMENTS Background Inflammation, including inflammatory cytokines, plays an important role in the incidence, invasion, and metastasis of several tumors. However, as an inflammation precursor, the role of fibrinogen in tumors, particularly colon cancer, remains unclear. Research frontiers Increasing evidence indicates that the host inflammatory response plays an important role in tumor development and progression. Inflammatory mediators and cytokines secreted by inflammatory cells are elevated in tumors and promote proliferation, invasion or metastasis, which consequently have an impact on patient prognosis. These inflammatory markers, including monocyte chemoattractant proteins, white blood cell counts, neutrophil-lymphocyte ratio, plateletlymphocyte ratio, and fibrinogen, are easily detected in plasma and therefore could represent useful prognostic markers. However, the prognostic value of inflammatory markers, particularly fibrinogen, remains unclear. Innovations and breakthroughs While there have been a number of studies on the relationship of fibrinogen and cancer, few have analyzed this relationship in colon cancer patients. In this study, the prognostic significance of preoperative fibrinogen in colon cancer patients was investigated. The results illustrate that preoperative fibrinogen is negatively correlated with prognosis in colon cancer patients who received radical surgery, suggesting that preoperative fibrinogen is a promising prognostic marker for colon cancer patients. Applications The study indicates that preoperative fibrinogen is an independent prognostic factor for colon cancer patients that undergo radical surgery. Consequently, preoperative fibrinogen may serve as a potential prognostic marker for colon 8589 July 14, 2014 Volume 20 Issue 26

282 Sun ZQ et al. Preoperative fibrinogen and colon cancer cancer patients. Moreover, anti-fibrinogen therapies may provide a novel strategy for treating colon cancer. Terminology Fibrinogen is a plasma glycoprotein that is involved in blood clot formation. Levels of this protein have been associated with tumor progression and metastasis in many cancer types. Peer review This study comprehensively analyzes the prognostic significance of preoperative fibrinogen levels in patients with colon cancer. The results suggest that preoperative fibrinogen is a promising independent prognostic factor for patients with colon cancer, and may provide a new target for colon cancer therapy. REFERENCES 1 Takeuchi H, Ikeuchi S, Kitagawa Y, Shimada A, Oishi T, Isobe Y, Kubochi K, Kitajima M, Matsumoto S. Pretreatment plasma fibrinogen level correlates with tumor progression and metastasis in patients with squamous cell carcinoma of the esophagus. J Gastroenterol Hepatol 2007; 22: [PMID: DOI: /j x] 2 Polterauer S, Seebacher V, Hefler-Frischmuth K, Grimm C, Heinze G, Tempfer C, Reinthaller A, Hefler L. Fibrinogen plasma levels are an independent prognostic parameter in patients with cervical cancer. Am J Obstet Gynecol 2009; 200: 647.e1-647.e7 [PMID: DOI: / j.ajog ] 3 Polterauer S, Grimm C, Seebacher V, Concin N, Marth C, Tomovski C, Husslein H, Leipold H, Hefler-Frischmuth K, Tempfer C, Reinthaller A, Hefler L. Plasma fibrinogen levels and prognosis in patients with ovarian cancer: a multicenter study. Oncologist 2009; 14: [PMID: DOI: /theoncologist] 4 Ghezzi F, Cromi A, Siesto G, Giudici S, Serati M, Formenti G, Franchi M. Prognostic significance of preoperative plasma fibrinogen in endometrial cancer. Gynecol Oncol 2010; 119: [PMID: ] 5 Zhao J, Zhao M, Jin B, Yu P, Hu X, Teng Y, Zhang J, Luo Y, Zhang L, Zheng S, Zhou Q, Li H, Liu Y, Qu X. 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Fibrinogen synthesized by cancer cells augments the proliferative effect of fibroblast growth factor-2 (FGF-2). J Thromb Haemost 2008; 6: [PMID: DOI: /j x] 19 Fitzgerald DJ. Fibrinogen receptor and platelet signalling. Blood Coagul Fibrinolysis 1999; 10 Suppl 1: S77-S79 [PMID: ] 20 Gerner C, Steinkellner W, Holzmann K, Gsur A, Grimm R, Ensinger C, Obrist P, Sauermann G. Elevated plasma levels of crosslinked fibrinogen gamma-chain dimer indicate cancer-related fibrin deposition and fibrinolysis. Thromb Haemost 2001; 85: [PMID: ] 21 Palumbo JS, Kombrinck KW, Drew AF, Grimes TS, Kiser JH, Degen JL, Bugge TH. Fibrinogen is an important determinant of the metastatic potential of circulating tumor cells. Blood 2000; 96: [PMID: ] 22 Palumbo JS, Talmage KE, Massari JV, La Jeunesse CM, Flick MJ, Kombrinck KW, Jirousková M, Degen JL. Platelets and fibrin(ogen) increase metastatic potential by impeding natural killer cell-mediated elimination of tumor cells. Blood 2005; 105: [PMID: DOI: / blood ] 23 Palumbo JS, Talmage KE, Massari JV, La Jeunesse CM, Flick MJ, Kombrinck KW, Hu Z, Barney KA, Degen JL. Tumor cell-associated tissue factor and circulating hemostatic factors cooperate to increase metastatic potential through natural killer cell-dependent and-independent mechanisms. Blood 2007; 110: [PMID: DOI: / blood ] 24 Everett CJ, Wells BJ, Frithsen IL, Koopman RJ. Smoking, fibrinogen and cancer mortality. J Natl Med Assoc 2007; 99: [PMID: ] 25 Balkwill F, Mantovani A. Inflammation and cancer: back to Virchow? Lancet 2001; 357: [PMID: DOI: /S (00) ] 26 Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inflammation. Nature 2008; 454: [PMID: DOI: /nature07205] 8590 July 14, 2014 Volume 20 Issue 26

283 Sun ZQ et al. Preoperative fibrinogen and colon cancer 27 Grivennikov SI, Greten FR, Karin M. Immunity, inflammation, and cancer. Cell 2010; 140: [PMID: DOI: /j.cell ] 28 Beer JH, Haeberli A, Vogt A, Woodtli K, Henkel E, Furrer T, Fey MF. Coagulation markers predict survival in cancer patients. Thromb Haemost 2002; 88: [PMID: ] 29 Lawrence SO, Simpson-Haidaris PJ. Regulated de novo biosynthesis of fibrinogen in extrahepatic epithelial cells in response to inflammation. Thromb Haemost 2004; 92: [PMID: DOI: /TH ] 30 Roxburgh CS, Salmond JM, Horgan PG, Oien KA, McMillan DC. Comparison of the prognostic value of inflammationbased pathologic and biochemical criteria in patients undergoing potentially curative resection for colorectal cancer. Ann Surg 2009; 249: [PMID: DOI: / SLA.0b013e3181a3e738] 31 Richards CH, Leitch EF, Horgan PG, Anderson JH, McKee RF, McMillan DC. The relationship between patient physiology, the systemic inflammatory response and survival in patients undergoing curative resection of colorectal cancer. Br J Cancer 2010; 103: [PMID: DOI: / sj.bjc ] P- Reviewers: Chen KF, García MT, Murtaza I, Wasserberg N S- Editor: Zhai HH L- Editor: Cant MR E- Editor: Wang CH 8591 July 14, 2014 Volume 20 Issue 26

284 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. CASE CONTROL STUDY Risk of gastric cancer is associated with PRKAA1 gene polymorphisms in Koreans Yong-Dae Kim, Dong-Hyuk Yim, Sang-Yong Eom, Sun In Moon, Hyo-Yung Yun, Young-Jin Song, Sei-Jin Youn, Taisun Hyun, Joo-Seung Park, Byung Sik Kim, Jong-Young Lee, Hee Kwan Won, Heon Kim Yong-Dae Kim, Dong-Hyuk Yim, Sang-Yong Eom, Sun In Moon, Heon Kim, Department of Preventive Medicine, College of Medicine, Chungbuk National University, Cheongju , South Korea Hyo-Yung Yun, Young-Jin Song, Department of Surgery, College of Medicine, Chungbuk National University, Cheongju , South Korea Sei-Jin Youn, Department of Internal Medicine, College of Medicine, Chungbuk National University, Cheongju , South Korea Taisun Hyun, Department of Food and Nutrition, Chungbuk National University, Cheongju , South Korea Joo-Seung Park, Department of Surgery, College of Medicine, Eulji University, Daejon , South Korea Byung Sik Kim, Department of Surgery, Asan Medical Center, College of Medicine, Ulsan University, Seoul , South Korea Jong-Young Lee, Center for Genome Science, National Institute of Health, Cheongju , South Korea Hee Kwan Won, Department of Internal Medicine, Division of Endocrinology and Metabolism, School of Medicine, Konyang University, Daejeon , South Korea Author contributions: Kim YD, Lee JY, and Kim H designed the study protocol; Yim DH, Eom SY, and Moon SI performed the statistical analysis and data interpretation; Song YJ, Yun HY, Park JS, Youn SJ, Kim BS, Won HK and Hyun T contributed equally to this study through the selection of subjects, sampling, and clinical data acquisition; Kim YD and Kim H drafted the manuscript. Supported by A grant from the National R & D Program for Cancer Control, Ministry of Health and Welfare, South Korea, No Correspondence to: Heon Kim, Professor, Department of Preventive Medicine and Medical Research Institute, College of Medicine, Chungbuk National University, 52 Naesudong-ro, Hungdok-gu, Cheongju , South Korea. kimheon@cbu.ac.kr Telephone: Fax: Received: December 24, 2013 Revised: January 29, 2014 Accepted: April 2, 2014 Published online: July 14, 2014 Abstract AIM: To evaluate the association between genetic polymorphisms of the gene encoding AMP-activated protein kinase (PRKAA1 ) and the risk of gastric cancer. METHODS: The study subjects consisted of 477 ageand sex-matched case-control pairs. Genotyping was performed for 5 tag single nucleotide polymorphisms (SNPs): rs , rs154268, rs , rs , and rs Associations between gastric cancer and putative risk factors (including the SNPs) were analyzed with multivariate conditional logistic regression models, after adjusting for potential confounding factors. Multiple testing corrections were implemented following methodology for controlling the false discovery rate. Gene-based association tests were performed by using the versatile gene-based association study (VEGAS) method. RESULTS: In the dominant model, SNPs rs [odds ratio (OR) = 1.51, 95%CI: )], rs (OR = 1.65, 95%CI: ), rs (OR = 1.48, 95%CI: ), and rs (OR = 1.53, 95%CI: ) were significantly associated with an increased risk of gastric cancer. In the recessive model, SNPs rs (OR = 1.66, 95%CI: ), rs (OR = 0.63, 95%CI: ), and rs (OR = 1.47, 95%CI: ) were significant risk or protective factors for gastric cancer. In the codominant model, the ORs of each of the 5 SNPs were statistically significant. All SNPs in the model showed a dose-response relationship between the minor allele frequency and the risk of gastric cancer. Most notably, subjects with a homozygous minor allele in SNP rs showed 2.15 times the risk of gastric cancer as subjects without a minor allele. The PRKAA1 gene showed a significant gene-based association with gastric cancer in the VEGAS test. CONCLUSION: All 5 tested tag SNPs of the PRKAA1 gene (rs , rs154268, rs , rs , and rs ) were significantly associated with gastric cancer July 14, 2014 Volume 20 Issue 26

285 Kim YD et al. PRKAA1 polymorphisms and gastric cancer 2014 Baishideng Publishing Group Inc. All rights reserved. Key words: AMP-activated protein kinase; Gastric cancer; PRKAA1 ; Single nucleotide polymorphism; Casecontrol study Core tip: There were a few studies to evaluate association between PRKAA1 gene and gastric cancer. However, in previous study, only one single nucleotide polymorphism (SNP) (rs ) of PRKAA1 gene was focused. The purpose of this study was to evaluate the association between 5 SNPs of the gene encoding AMP-activated protein kinase (PRKAA1 ) and the risk of gastric cancer. All SNPs in the model showed a dose-response relationship between the minor allele frequency and the risk of gastric cancer. The PRKAA1 gene showed a significant gene-based association with gastric cancer in the versatile gene-based association study test. Kim YD, Yim DH, Eom SY, Moon SI, Yun HY, Song YJ, Youn SJ, Hyun T, Park JS, Kim BS, Lee JY, Won HK, Kim H. Risk of gastric cancer is associated with PRKAA1 gene polymorphisms in Koreans. World J Gastroenterol 2014; 20(26): Available from: URL: v20/i26/8592.htm DOI: i INTRODUCTION Gastric cancer is the second most common cause of cancerrelated mortality worldwide [1]. Since the 1980s, South Korea, Japan, China, and other Asian countries have had a particularly high incidence of this disease, despite general trends of decreasing incidence and mortality [2,3]. A model of gastric carcinogenesis in humans has been derived based on evidence from various epidemiological and pathological studies. According to this model, gastric cancer arises in a sequence of stages: chronic gastritis, atrophic gastritis, intestinal metaplasia, and dysplasia [4]. Helicobacter pylori, high salt intake, alcohol intake, smoking, diet, and genetic factors have been reported to be involved in gastric carcinogenesis [5-9]. AMP-activated protein kinase (AMPK) is an energy sensing/signaling intracellular protein, and a conserved serine/threonine kinase that regulates energy homeostasis and metabolic stress [10]. AMPK is activated by phosphorylation when the AMP/ATP ratio is high [11]. Activated AMPK switches on ATP-generating (catabolic) pathways and switches off ATP-consuming (anabolic) pathways [12,13]. AMPK activation is known to inhibit the accumulation of lipid in the body, decrease the biosynthesis of fatty acids and cholesterol, and increase the oxidation of fatty acids [12]. Considerable evidence indicates that AMPK activation suppresses cell proliferation in both tumor and nonmalignant cells. These results of AMPK activation are mediated through various mechanisms, including G1 phase arrest in the cell cycle [14], and the inhibition of fatty acid and protein synthesis [13,15]. Regulation of the cell cycle by AMPK is mediated through activation of the p53-p21 axis pathway, activation of tumor suppressor LKB1, inhibition of the mammalian target of rapamycin pathway, and other similar mechanisms [16]. Based on this evidence, research on AMPK function has focused on its important role in development, and on its potential use as a therapeutic target for some cancers [16,17]. It is possible that AMPK plays an important role in gastric carcinogenesis and, therefore, polymorphic alleles of the encoding gene could modify individual susceptibility to gastric cancer. Recently, Song et al [18] reported that the rs single nucleotide polymorphism (SNP) of the protein kinase, AMP-activated alpha 1 catalytic subunit (PRKAA1) gene was associated with an increased risk of gastric cancer in the Korean population. However, because their study only examined the rs SNP of the PRKAA1 gene, it remains important to elucidate the associations between other SNPs of PRKAA1 and gastric cancer. Accordingly, the aim of the present study was to evaluate the associations between 5 polymorphic alleles of PRKAA1, the gene that encodes AMPK, and gastric carcinogenesis in Koreans. MATERIALS AND METHODS Ethics This work has been carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association. This study was approved by the institutional review boards of Chungbuk National University Hospital, South Korea (IRB No ). All subjects had provided written informed consent. Study subjects The subjects included in this study consisted of 477 newly diagnosed gastric cancer patients and an equal number of age- (within 3 years) and sex-matched controls. The diagnoses of patients with gastric cancer were confirmed at Chungbuk National University Hospital and Eulji University Hospital, which are located in a geographically central region of the South Korea. Control subjects did not have a previous diagnosis of any type of cancer, and were selected from individuals who had received routine medical examinations at these hospitals. The case and control groups each included 301 men and 176 women. The mean ± SD age was 58.7 ± 9.9 years in the case group and 57.8 ± 10.2 years in the control group. Trained interviewers interviewed all subjects by using a structured questionnaire, which included questions on demographic factors, smoking habits, alcohol consumption, and dietary habits. Peripheral blood and urine samples were collected from all the subjects. Selection of PRKAA1 SNPs We selected SNPs of PRKAA1 from several prominent 8593 July 14, 2014 Volume 20 Issue 26

286 Kim YD et al. PRKAA1 polymorphisms and gastric cancer Table 1 Frequencies of PRKAA1 polymorphisms in cases and controls SNP Position Genotype Case Control case/control Freq 1 HWE 2 Freq 1 HWE 2 rs CC CA AA N / /125 9/8 476/475 rs AA AG GG N / / /94 475/474 rs TT TC CC N / / /96 475/473 rs TT TC CC N / /149 18/15 477/475 rs TT TC CC N / /199 21/40 474/472 1 Freq: Allele frequency; 2 P value deviation from Hardy-Weinberg Equilibrium (HWE). SNP: Single nucleotide polymorphism. online databases (GeneCards, HUGE navigator, NCBI; because this gene may be related to diet risk factors for gastric carcinogenesis. To select tagging SNP, we identified functional elements from the Functional Elements SNPs Database, used the tagger pairwise method from the International HapMap Project, and finally selected SNPs with a minor allele frequency 0.05 in JPT (Japanese in Tokyo, Japan) and CHB (Han Chinese in Beijing, China) samples. SNPs that significantly deviated from the Hardy-Weinberg equilibrium were discarded. Genomic DNA was extracted from whole blood by using a QuickGene-810 nucleic acid isolation system (Fujifilm, Tokyo, Japan) and QuickGene DNA Whole Blood Kit S (Kurabo, Osaka, Japan), in accordance with the manufacturer s instructions. DNA was stored at 4 until use. SNP genotyping was performed by using a GoldenGate Genotyping Assay with VeraCode technology (Illumina, San Diego, CA, United States). A custom GoldenGate assay was designed for the analysis of the selected SNPs in the PRKAA1 gene. Those SNPs were then assessed for suitability for the GoldenGate genotyping platform, and the analysis was carried out on the validated SNPs. The average call rate was 99.40%. Genotyping was carried out by Macrogen (Seoul, South Korea). Statistical analysis Testing for deviation from the Hardy-Weinberg equilibrium was performed for each SNP in both cases and controls by using Pearson s χ 2 test. D values were measured by using Lewontin s method for all combinations of biallelic loci, and linkage disequilibrium blocks were structured by using Haploview version 4.2 (Daly Lab at the Broad Institute Cambridge, MA, United States). Haplotype blocks were constructed and statistically compared between cases and controls by using SNP Analyzer version 2.0 (ISTEC, Goyang, South Korea). Haplotype blocks which frequency over 5% were selected for analysis. Student s t-test was used to compare the values of continuous variables in the patient and control groups. Associations between gastric cancer and putative risk factors (including the SNPs) were estimated by using odds ratios (ORs) and their corresponding 95%CI, as derived from multivariate conditional logistic regression models, after adjusting for potential confounding factors such as age, sex, smoking history, alcohol intake amount, total calorie intake, and education level. Homozygous reference genotypes, heterozygous alleles, and homozygous risk alleles in each SNP were coded as 0, 1, and 2 in the codominant model; 0, 1, and 1 in the dominant model; and 0, 0, and 1 in the recessive model, respectively. Benjaminin and Hochberg s methods for control of the false discovery rate (FDR) were used for multiple testing corrections [19]. Two-sided adjusted P values < 0.05 were considered to be statistically significant. FDR Q values were calculated separately for the SNPs and haplotypes based on those numbers. Gene-based association tests were performed by using the versatile gene-based association study (VEGAS) method [20]. The study power calculations were performed by using the case - control for discrete traits mode in the Genetic Power Calculator created by Shaun Purcell ( The following parameters were applied: a risk allele frequency of 0.4, an alpha error of 0.01, and a disease prevalence of 0.1%. The power of a codominant model was when the heterozygous odds ratio was set to 1.5. The power of a dominant model was when the odds ratio for a genotype with 1 or 2 risk allele(s) was taken to be 2. The power of a recessive model was when a value of 2 was input for the odds ratio for a genotype with 2 risk allele(s). SAS version 9.2 (SAS Institute, Cary, NC) was used for all statistical analyses. RESULTS We explored the associations between 5 DNA polymorphisms in the PRKAA1 gene, which encodes AMPK, and gastric cancer risk. A total of 954 subjects were included in the analysis, comprising 477 gastric cancer cases and equal number of matched controls. There were no significant differences between the two groups in terms of age, gender, smoking status, or alcohol intake July 14, 2014 Volume 20 Issue 26

287 Kim YD et al. PRKAA1 polymorphisms and gastric cancer A rs rs rs rs rs B Haplotypes ht1 +rs rs rs rs rs C G C T T Freq. Cases Controls Block 1 (30 kb) ht2 C A T T T ht3 C A T T C ht4 A G C C T Figure 1 Haplotype linkage disequilibrium blocks and haplotype frequencies for PRKAA1. A: Linkage disequilibrium blocks and correlation coefficients among PRKAA1 polymorphisms. Black squares indicate statistically significant allelic association between the pair of single nucleotide polymorphisms (SNPs), as measured by using the D statistic; darker gray indicate higher values of D, up to a maximum of 1; B: Haplotype frequencies of PRKAA1 polymorphisms in the case and control groups. Freq: Allele frequency. Table 2 Association between PRKAA1 polymorphisms and gastric cancer SNP Dominant model Recessive model OR (95%CI) P value 1 Q 2 OR (95%CI) P value 1 Q 2 rs ( ) ( ) rs ( ) ( ) rs ( ) ( ) rs ( ) ( ) rs ( ) ( ) VEGAS statistics (P) (0.0054) (0.0001) Reference alleles of each single nucleotide polymorphism (SNP) in logistic analysis are as follows; CC for rs , AA for rs , and TT for rs , rs and rs P values for logistic analysis of two alternative models (dominant and recessive) adjusted with calorie intake, smoking history, alcohol intake and educational level; 2 False discovery rate Q value. Table 3 Codominant model odds ratios of the PRKAA1 polymorphisms for gastric cancer SNPs Adjusted OR (95%CI) P value 1 Q 2 rs CC CA 1.46 ( ) AA 1.75 ( ) rs AA AG 1.30 ( ) GG 2.15 ( ) rs TT TC 1.29 ( ) CC 2.05 ( ) rs TT TC 1.63 ( ) CC 1.77 ( ) rs TT TC 0.72 ( ) CC 0.54 ( ) VEGAS statistics (P) (0.0004) 1 P values for logistic analysis of codominant model adjusted with age, sex, calorie intake, smoking history, alcohol intake and educational level; 2 False discovery rate Q value. SNP: Single nucleotide polymorphism; VEGAS: Versatile gene-based association study. Table 1 presents the frequencies of the 5 selected SNPs in the study subjects. The genotype distributions at all 5 SNPs were in Hardy-Weinberg equilibrium, with non-significant χ 2 values. Haplotype linkage disequilibrium block and haplotype frequencies for PRKAA1 are presented in Figure 1. Common haplotypes (frequency > 5%) of the block were found in 93.6% of cases, and 95.7% of controls. The frequencies and distribution of genotypes are presented in Tables 2 and 3, which also report the odds ratios for the associations between each polymorphism and gastric cancer. We performed the PRKAA1 gene association analysis with dominant, recessive, and codominant models by using conditional logistic regression. In the dominant model, 4 of 5 SNPs we observed were significantly associated with an increased risk of gastric cancer. SNPs rs ( C allele, OR = 1.51, 95%CI: , P = 0.018, FDR Q = 0.023), rs ( C allele, OR = 1.65, 95%CI: , P = 0.001, FDR Q = 0.006), rs ( A allele, OR = 1.48, 95%CI: , P = 0.012, FDR Q = 0.023), and rs ( G allele, OR = 1.53, 95%CI: , P = 0.014, FDR Q = 0.023) were significantly associated with an increased risk of gastric cancer. In the recessive model, SNP rs (OR = 1.66, 95%CI: , P = 8595 July 14, 2014 Volume 20 Issue 26

288 Kim YD et al. PRKAA1 polymorphisms and gastric cancer Table 4 Association between PRKAA1 haplotypes and gastric cancer Haplotypes Codominant Dominant Recessive OR (95%CI) P value 1 Q 2 OR (95%CI) P value 1 Q 2 OR (95%CI) P value 1 Q 2 PRKAA1 CGCTT 1.57 ( ) ( ) ( ) haplotype CATTT 0.79 ( ) ( ) ( ) block 1 CATTC 0.44 ( ) ( ) ( ) CGCCT 1.44 ( ) ( ) ( ) P values for logistic analysis of three alternative models (codominant, dominant and recessive), the P values for haplotype association were calculated by the single nucleotide polymorphism Analyzer 2.0 software; 2 False discovery rate Q value , FDR Q = 0.005), rs (OR = 0.63, 95%CI: , P = 0.003, FDR Q = 0.005) and rs (OR = 1.47, 95%CI: , P = 0.002, FDR Q = 0.005) were significant risk factors for gastric cancer (Table 2). In the codominant model, the odds ratios were statistically significant for all 5 SNPs. All SNPs in the model showed dose-response relationships between minor allele frequency and the risk of gastric cancer. Most notably, subjects with a homozygous minor allele in SNP rs showed 2.15 times of risk for gastric cancer, compared with subjects who did not have a minor allele (Table 3). To evaluate the association between gastric cancer and all SNPs within the PRKAA1 gene (rather than each SNP individually) we performed a gene-based analysis following the VEGAS method, the results of which indicated that SNPs in PRKAA1 had a statistically significant association with gastric cancer in all 3 models (P = , , and for the dominant, recessive, and codominant models, respectively). The haplotype block was also evaluated for an association with the risk of gastric cancer (Table 4), but none of the results were significant in each of the 3 models. DISCUSSION In this study, we hypothesized that genetic polymorphisms in PRKAA1 might contribute to gastric cancer development by affecting the regulation of energy metabolism. Activated AMPK inactivates a number of metabolic enzymes involved in ATP-consuming cellular events (such as fatty acid, cholesterol, and protein synthesis) and also activates ATP-generating processes (such as the uptake and oxidation of glucose and fatty acids) [12,13]. Besides energy metabolism, AMPK also functions as a suppressor of cell proliferation [14]. Consequently, some research on AMPK has focused on its potential role as a therapeutic target for cancer. A recent genome-wide association study identified a new SNP (rs ) in the PRKAA1 gene that is significantly associated with increased susceptibility to gastric cancer [21,22]. Additionally, Song et al [18] reported that the rs SNP was associated with an increased risk of gastric cancer in the Korean population. In their replication study, rs TC and CC genotypes were associated with a significantly increased risk of gastric cancer (OR = 1.29 for TC vs TT, OR = 1.68 for CC vs TT). In agreement with these findings, our result also showed that the rs SNP is associated with gastric cancer (OR = 1.29 for TC vs TT, OR = 2.05 for CC vs TT). Together, these results suggest that rs SNP might play an important role in the development of gastric cancer. However, since rs is not the only SNPs found in this gene, it remained important to examine associations between other SNPs of PRKAA1 and gastric cancer development. In the present study, we evaluated the associations of 5 SNPs of PRKAA1 gene with gastric cancer. Interestingly, we found that all 5 of the tested SNPs of PRKAA1 we tested were associated with a significantly increased risk of gastric cancer. Most notably, subjects with a homozygous minor allele in SNP rs were at 2.15 times the risk of gastric cancer, compared with subjects who did not have a minor allele. After controlling the FDR, the associations of these SNPs remained statistically significant in the codominant model. In a gene-based association test, the PRKAA1 gene was found to be significantly associated with gastric cancer. These results suggest that genetic polymorphism of PRKAA1 might play an important role in gastric carcinogenesis. Although the biological mechanism underlying the association between PRKAA1 and gastric cancer has not been clarified, these significant associations could potentially be explained by the ability of activated AMPK phosphorylates p53 to induce G1/S arrest. Further, the AMPK-p53 connection may represent a cell cycle checkpoint [23]. Therefore, individuals with mutant PRKAA1 alleles, which encode inactive AMPK, may be vulnerable to gastric cancer. Anti-inflammatory action by AMPK could provide another explanation of the association between SNPs of PRKAA1 and gastric cancer. A recent study has reported that activated AMPK can counter-regulate macrophage inflammatory function [24] and activate some anti-inflammatory agents [25]. Loss of anti-inflammatory action by AMPK in the body of individuals with mutant PRKAA1 alleles results in more severe injury of the epithelium [26]. Bone marrow-derived cells are recruited at these sites of epithelial damage, and these cells can be a potential source of malignancy [27]. To our knowledge, there is no study addressed association between PRKAA1 gene and gastric cancer except in Chinese and Korean population. Since SNPs frequency are different according to the population, it is need to 8596 July 14, 2014 Volume 20 Issue 26

289 Kim YD et al. PRKAA1 polymorphisms and gastric cancer further study in different races other than Asian. The present study has several limitations. First, a relatively small number of patients and controls were enrolled in this study. Second, we could not obtain detailed data on the histological tumor types for the cases of gastric cancer. Finally, because the data of environmental factor for gastric cancer such as H. pylori infection and diet was not available in this study, we could not evaluate the gene-environmental interaction. It is needed further study about it. In summary, the PRKAA1 gene and 5 of its SNPs (rs , rs154268, rs , rs , and rs ) were associated with an increased risk of gastric cancer in Koreans. COMMENTS Background Gastric cancer is the second most common cause of cancer-related mortality worldwide. AMP-activated protein kinase (AMPK) is an energy sensing/signaling intracellular protein, and a conserved serine/threonine kinase that regulates energy homeostasis and metabolic stress. It is known that AMPK activation suppresses cell proliferation in both tumor and non-malignant cells. Therefore, it is possible that AMPK plays an important role in gastric carcinogenesis and polymorphic alleles of the encoding gene could modify individual susceptibility to gastric cancer. The aim of the present study was to evaluate the associations between 5 polymorphic alleles of PRKAA1, the gene that encodes AMPK, and gastric carcinogenesis in Koreans. Research frontiers In the present study, the authors evaluated the associations of 5 single nucleotide polymorphisms (SNPs) of PRKAA1 gene with gastric cancer. To our knowledge, this is the first replication study to indicate an association between rs154268, rs , rs , and rs and gastric cancer development. Interestingly, all 5 SNPs of PRKAA1 tested SNP were associated with a significantly increased risk of gastric cancer. Most notably, subjects with a homozygous minor allele in SNP rs were at 2.15 times the risk of gastric cancer, compared with subjects who did not have a minor allele. Innovations and breakthroughs There were a few studies to evaluate association between PRKAA1 gene and gastric cancer. However, in previous study, only one SNP (rs ) of PRKAA1 gene was focused. In this study, authors evaluated 5 SNPs of PRKAA1 gene including rs in associated with gastric cancer risk. Applications The result of this study showed that PRKAA1 gene and its 5 SNPs (rs , rs154268, rs , rs , and rs ) were associated with an increased risk of gastric cancer in Koreans. Further studies are needed to determine the mechanism which by PRKAA1 and other environmental factors interact and influence the development of gastric cancer. Peer review This is a nice paper with a good summary of the issue and a well described methodology for studying 5 polymorphic alleles of PRKAA1, the gene that encodes AMPK, and gastric cancer in Koreans. In this study, the authors test the association between 5 SNPs of PRKAA1 gene and gastric cancer in a Korean population. The PRKAA1 gene has been implicated in carcinogenesis at several levels and the authors provide a reasonable rationale for this selection. The SNPs show nominally significant association to gastric cancer. REFERENCES 1 Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden: Globocan Int J Cancer 2001; 94: [PMID: DOI: /ijc.1440] 2 Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, CA Cancer J Clin 2005; 55: [PMID: DOI: /canjclin ] 3 Soerjomataram I, Lortet-Tieulent J, Parkin DM, Ferlay J, Mathers C, Forman D, Bray F. Global burden of cancer in 2008: a systematic analysis of disability-adjusted life-years in 12 world regions. 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290 Kim YD et al. PRKAA1 polymorphisms and gastric cancer j.bbrc ] 18 Song HR, Kim HN, Kweon SS, Choi JS, Shim HJ, Cho SH, Chung IJ, Park YK, Kim SH, Choi YD, Joo KW, Shin MH. Genetic variations in the PRKAA1 and ZBTB20 genes and gastric cancer susceptibility in a Korean population. Mol Carcinog 2013; 52 Suppl 1: E155-E160 [PMID: ] 19 Benjamini Y, Hochberg Y. Controlling the false discovery rate: a practical and powerful pproach to multiple testing. J R Stat Soc Ser B Methodol 1995; 57: Liu JZ, McRae AF, Nyholt DR, Medland SE, Wray NR, Brown KM, Hayward NK, Montgomery GW, Visscher PM, Martin NG, Macgregor S. A versatile gene-based test for genome-wide association studies. Am J Hum Genet 2010; 87: [PMID: DOI: /j.ajhg ] 21 Abnet CC, Freedman ND, Hu N, Wang Z, Yu K, Shu XO, Yuan JM, Zheng W, Dawsey SM, Dong LM, Lee MP, Ding T, Qiao YL, Gao YT, Koh WP, Xiang YB, Tang ZZ, Fan JH, Wang C, Wheeler W, Gail MH, Yeager M, Yuenger J, Hutchinson A, Jacobs KB, Giffen CA, Burdett L, Fraumeni JF, Tucker MA, Chow WH, Goldstein AM, Chanock SJ, Taylor PR. A shared susceptibility locus in PLCE1 at 10q23 for gastric adenocarcinoma and esophageal squamous cell carcinoma. Nat Genet 2010; 42: [PMID: DOI: /ng.649] 22 Wang LD, Zhou FY, Li XM, Sun LD, Song X, Jin Y, Li JM, Kong GQ, Qi H, Cui J, Zhang LQ, Yang JZ, Li JL, Li XC, Ren JL, Liu ZC, Gao WJ, Yuan L, Wei W, Zhang YR, Wang WP, Sheyhidin I, Li F, Chen BP, Ren SW, Liu B, Li D, Ku JW, Fan ZM, Zhou SL, Guo ZG, Zhao XK, Liu N, Ai YH, Shen FF, Cui WY, Song S, Guo T, Huang J, Yuan C, Huang J, Wu Y, Yue WB, Feng CW, Li HL, Wang Y, Tian JY, Lu Y, Yuan Y, Zhu WL, Liu M, Fu WJ, Yang X, Wang HJ, Han SL, Chen J, Han M, Wang HY, Zhang P, Li XM, Dong JC, Xing GL, Wang R, Guo M, Chang ZW, Liu HL, Guo L, Yuan ZQ, Liu H, Lu Q, Yang LQ, Zhu FG, Yang XF, Feng XS, Wang Z, Li Y, Gao SG, Qige Q, Bai LT, Yang WJ, Lei GY, Shen ZY, Chen LQ, Li EM, Xu LY, Wu ZY, Cao WK, Wang JP, Bao ZQ, Chen JL, Ding GC, Zhuang X, Zhou YF, Zheng HF, Zhang Z, Zuo XB, Dong ZM, Fan DM, He X, Wang J, Zhou Q, Zhang QX, Jiao XY, Lian SY, Ji AF, Lu XM, Wang JS, Chang FB, Lu CD, Chen ZG, Miao JJ, Fan ZL, Lin RB, Liu TJ, Wei JC, Kong QP, Lan Y, Fan YJ, Gao FS, Wang TY, Xie D, Chen SQ, Yang WC, Hong JY, Wang L, Qiu SL, Cai ZM, Zhang XJ. Genome-wide association study of esophageal squamous cell carcinoma in Chinese subjects identifies susceptibility loci at PLCE1 and C20orf54. Nat Genet 2010; 42: [PMID: ] 23 Jones RG, Plas DR, Kubek S, Buzzai M, Mu J, Xu Y, Birnbaum MJ, Thompson CB. AMP-activated protein kinase induces a p53-dependent metabolic checkpoint. Mol Cell 2005; 18: [PMID: DOI: / j.molcel ] 24 Sag D, Carling D, Stout RD, Suttles J. Adenosine 5 -monophosphate-activated protein kinase promotes macrophage polarization to an anti-inflammatory functional phenotype. J Immunol 2008; 181: [PMID: ] 25 O'Neill LA, Hardie DG. Metabolism of inflammation limited by AMPK and pseudo-starvation. Nature 2013; 493: [PMID: DOI: /nature11862] 26 MacDonald TT, Horton MA, Choy MY, Richman PI. Increased expression of laminin/collagen receptor (VLA-1) on epithelium of inflamed human intestine. J Clin Pathol 1990; 43: [PMID: DOI: /jcp ] 27 Houghton J, Stoicov C, Nomura S, Rogers AB, Carlson J, Li H, Cai X, Fox JG, Goldenring JR, Wang TC. Gastric cancer originating from bone marrow-derived cells. Science 2004; 306: [PMID: ] P- Reviewers: Bader EL Din NGBG, Garcia-Elorriaga G, Park WS S- Editor: Gou SX L- Editor: A E- Editor: Liu XM 8598 July 14, 2014 Volume 20 Issue 26

291 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. RETROSPECTIVE BRIEF ARTICLE STUDY Endoscopic ultrasound-guided fine-needle aspiration for suspected malignancies adjacent to the gastrointestinal tract Pietro Gambitta, Antonio Armellino, Edoardo Forti, Maurizio Vertemati, Paola Enrica Colombo, Paolo Aseni Pietro Gambitta, Antonio Armellino, Edoardo Forti, Unità di Chirurgia Endoscopica ed Ecoendoscopia Ospedale Niguarda Ca Granda, Milano, Italy Maurizio Vertemati, Dipartimento di Scienze Biomediche e Cliniche L. Sacco, Università degli Studi di Milano, Milano, Italy Paola Enrica Colombo, Servizio di Fisica Sanitaria, Ospedale Niguarda Ca Granda, Milano, Italy Paolo Aseni, HPB and Transplantation Surgery, Ospedale Niguarda Ca Granda, Milano, Italy Author contributions: Gambitta P and Aseni P contributed equally to this work by designing the study, performing the research, and writing the paper; Gambitta P, Armellino A, Forti E and Aseni P performed the research; Forti E, Vertemati M, and Colombo PE analyzed data; Vertemati M and Aseni P reviewed and edited the manuscript. Correspondence to: Paolo Aseni, MD, HPB and Transplantation Surgery, Ospedale Niguarda Ca Granda, Piazza Ospedale Maggiore 3, Milano, Italy. paoloaseni@gmail.com Telephone: Fax: Received: January 9, 2014 Revised: March 7, 2014 Accepted: April 21, 2014 Published online: July 14, 2014 Abstract AIM: To investigate the impact of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) in association with a multidisciplinary team evaluation for the detection of gastrointestinal malignancies. METHODS: A cohort of 1019 patients with suspected malignant lesions adjacent to the gastrointestinal tract received EUS-FNA after a standardized multidisciplinary team evaluation (MTE) and were divided into 4 groups according to their specific malignant risk score (MRS). Patients with a MRS of 0 (without detectable risk of malignancy) received only EUS without FNA. For patients with a MRS score ranging from 1 (low risk) - through 2 (intermediate risk) - to 3 (high risk), EUS-FNA cytology of the lesion was planned for a different time and was prioritized for those patients at higher risk for cancer. The accuracy, efficiency and quality assessment for the early detection of patients with potentially curable malignant lesions were evaluated for the whole cohort and in the different classes of MRSs. The time to definitive cytological diagnosis (TDCD), accuracy, sensitivity, specificity, positive and negative predictive values, and the rate of inconclusive tests were calculated for all patients and for each MRS group. RESULTS: A total of 1019 patients with suspected malignant lesions were evaluated by EUS-FNA. In 515 patients of 616 with true malignant lesions the tumor was diagnosed by EUS-FNA; 421 patients with resectable lesions received early surgical treatment, and 94 patients received chemo-radiotherapy. The overall diagnostic accuracy for the 1019 lesions in which a final diagnosis was obtained by EUS-FNA was When patients were stratified by MTE into 4 classes of MRSs, a higher rate of patients in the group with higher cancer risk (MRS-3) received early treatment and EUS- FNA showed the highest level of accuracy (1.0). TDCD was also shorter in the MRS-3 group. The number of patients who received surgical treatment or chemo-radiotherapy was significantly higher in the MRS-3 patient group (36.3% in MRS-3, 10.7% in MRS-2, and 3.5% in MRS-1). CONCLUSION: EUS-FNA can effectively detect a curable malignant lesions at an earlier time and at a higher rate in patients with a higher cancer risk that were evaluated using MTE Baishideng Publishing Group Inc. All rights reserved. Key words: Gastrointestinal neoplasm; Endoscopic ultrasonography; Clinical scoring system; Fine needle aspiration; Clinical decision support system 8599 July 14, 2014 Volume 20 Issue 26

292 Gambitta P et al. EUS-FNA in gastrointestinal malignancies Core tip: Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) has become paramount in establishing a diagnosis for all suspected malignant lesions of the gastrointestinal tract. Due to its increasing demand, the diagnostic yield of EUS-FNA and the length of time to determine a definitive cytological diagnosis may not be satisfactory in clinical practice. We found that EUS- FNA, when combined with the clinical evaluation of malignancy risk, was associated with a reliable level of accuracy. When prioritized for those patients with the highest clinical suspicion of cancer risk, EUS-FNA provides a shorter time to diagnosis for those patients with a higher cancer risk who can benefit from early therapy. Gambitta P, Armellino A, Forti E, Vertemati M, Colombo PE, Aseni P. Endoscopic ultrasound-guided fine-needle aspiration for suspected malignancies adjacent to the gastrointestinal tract. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: INTRODUCTION Endoscopic ultrasound (EUS) provides excellent visualization of almost all of the submucosal layers of the gastrointestinal tract as well as the organs and structures adjacent to the gastrointestinal tract, including mediastinal lymph nodes. However, the ability of EUS to differentiate between inflammatory masses and cancer is limited [1-3]. With the advent of curvilinear echoendoscopes, transesophageal, transgastric, trans-duodenal or trans-rectal endoscopic ultrasound guided fine needle aspiration (EUS-FNA) biopsies have become common practice. The role of EUS-FNA has also become paramount in establishing a correct tissue diagnosis in patients with abnormal lymph nodes during the diagnosis of unexplained mediastinal lymphadenopathy and during the staging of non-small-cell lung cancer (NSCLC) when histo-pathological findings may guide the correct therapeutic management [4-6]. Due to emerging evidence that supports its utility [7,8], the indications and requests for EUS-FNA for all suspected malignant lesions are increasing despite its limited availability. Given the increasingly widespread clinical use of EUS-FNA, it seems justified to implement strategies to prioritize the procedure for those patients with resectable disease who can benefit from early surgical treatment and for those patients with unresectable malignancies who can take advantage of specific chemotherapy or radiation therapy. The aim of this study was to assess the potential benefit of EUS-FNA when scheduled on a priority basis in patients stratified for cancer risk by a multidisciplinary team evaluation (MTE) according to a specific malignancy risk score (MRS). We evaluated a large cohort for possible benefit of EUS-FNA when patients were stratified into 4 different classes of MRS by analyzing and comparing the time to obtain a definitive cytological diagnosis (TDCD), the rate of patients who underwent surgery, and the rate of patients with unresectable lesions who could receive specific cancer treatment in different groups. Detailed analyses of accuracy and quality assessments of EUS-FNA as well as their relevance in the process of clinical decision making, were also evaluated and discussed in the whole cohort and in the different groups. MATERIALS AND METHODS Study design, and identification of the cohort From November 1998 to May 2011, all patients with suspected thoracic or abdominal malignant lesions of the gastrointestinal submucosal layer as well as lesions of the organs and structures lying in close vicinity to the gastrointestinal tract including lymph nodes, were recruited in Ospedale Niguarda Ca Granda, Milano for this study. Data were collected in a prospectively dedicated computerized database and then retrospectively analyzed 30 mo after the end of the study (November 2013, end of the study: May 2011). All relevant clinical data with cytological findings, TDCD, and final clinical diagnoses for all patients with inconclusive tests were recorded, as well as all complications associated with the procedure. Inclusion criteria All patients with a suspected malignant lesion who could benefit from possible treatment with surgery, chemotherapy or radiotherapy were included. All patients with a known primary tumor requiring surgical evaluation for resectability by tissue diagnosis of a suspected metastatic lesion were also included in the study provided that EUS- FNA was evaluated as technically feasible by the attending gastroenterologist (Gambitta P). The Institutional Review Board and the Ethical Committee approved the study. All patients provided written informed consent to undergo the procedure. Criteria for MRS assessment The clinical records of each patient were reviewed by one gastroenterologist and collegially discussed during the MTE; the team was composed of surgeons, oncologists and radiologists. Three specialists (one radiologist, one oncologist and one surgeon) expressed their clinical judgements by subjective evaluation and by objective clinical criteria following the National Comprehensive Cancer Network (NCCN) guidelines for diagnosis [9]. They assigned a score of 0 if the lesion was judged as most likely benign, or a score of 1 if the lesion was judged as most likely malignant. The final clinical score was the sum of the scores of each specialist. The clinical judgment of each single specialist was blind to the other two judgements. All patients were then divided into 4 groups with scores ranging from MRS-0 to MRS-3. Patients with MRS-0 without detectable oncological risk were submit July 14, 2014 Volume 20 Issue 26

293 Gambitta P et al. EUS-FNA in gastrointestinal malignancies ted to EUS without FNA. These patients were followedup for at least 30 mo or until death. Further investigations were planned whenever clinically required. In all patients with MRS ranging from 1 to 3 (1 = low risk, 2 = intermediate risk, 3 = high risk of malignancy), EUS was first planned, followed by FNA. Prioritization of the EUS-FNA and TDCD The procedure was performed within 3 d of the MTE for patients with a MRS of 3, within 10 d for those with a MRS of 2 and within 15 d for those with a MRS of 1. TDCD was evaluated as the number of days required from the last clinical evaluation to obtain the final report of cytological evaluation. The time for the cytological processing technique did not receive any different prioritization for the whole cohort of patients and it was always performed in the shortest time possible. EUS-FNA procedure and technique Patients were placed in the left lateral decubitus position and were sedated with iv meperidine plus midazolam, according to the judgment of the endoscopist. Standard EUS was performed using either an FG-36UX or EG-383 OUT linear-array echoendoscope (Pentax) for the evaluation and staging of the lesions. Once a lesion was identified, EUS-FNA was then performed. Solid masses in the head and uncinate process of the pancreas were biopsied by a transduodenal approach with a 22 Gauge or 19 Gauge ultrasound needle (Wilson-Cook Medical Inc., United States). Following infiltration of the target lesion, the mandarin was pulled back and an excess of 50 passes into the lesion were performed before the needle was finally retracted. Sample collection, processing, and diagnostic yield The mandarin was reintroduced to push any collected tissue fragments that possessed a wormlike appearance into a cytorich red medium. This technique allowed for the optimal use of collected material avoiding the routine handling of this material as a smear, leading to a reduction in processing time [10]. No cytopathologist was present in the examination room, and the determination of adequacy was based on macroscopic inspection of the aspirate by the operator. A cloudy sediment suggesting cellularity or core tissue was used for determination of adequacy. The biopsy procedure was repeated until sufficient material was aspirated. Criteria for cytological and final diagnoses The cytological diagnoses were then categorized into three groups: (1) positive for malignancy; (2) benign; and (3) inconclusive. The cytological material obtained by EUS guided aspiration was recorded as positive (diagnostic for cancer) when malignant cells were present in the aspirate, or as benign if only benign cells from the target organ were present. However, if no cells were present or no cells from the target organ were observed, or if destroyed cytological material was found, the cytological diagnosis was recorded as inconclusive. Lymph node aspirates without lymphatic cells and without cancer cells were also recorded as cases with an inconclusive cytological diagnosis. All cytology reports that were benign, malignant or inconclusive were confirmed or invalidated by one or more of the following: (1) definitive histology by resection specimen after surgery; (2) clinical evaluation after a clinical course of at least 30 mo as a benign lesion without evidence of malignant disease, or as a malignant lesion when confirmed clinically by evaluation of the typical clinical course characteristic of malignant diseases; and (3) analysis of specimen obtained during autopsy. Evaluation of a standard accuracy rate True positive (TP), true negative (TN), false positive (FP) and false negative (FN) results, accuracy (AC), sensitivity (ST), specificity (SP), positive and negative predictive value (PPV, NPV), number of patients with positive and negative final diagnosis (D+, D-), likelihood ratios for a positive or negative test (+LR and -LR), and the rate of inconclusive tests (IT) were calculated. The results were calculated by standard statistical computing as follows: AC as (TP+TN)/N, ST as TP/N(D+), SP as TP/N(D-), PPV as TP/N(T+), NPV as TN/N(T-), +LR as (ST/1- SP) and -LR as SP/1-ST. To ensure that all relevant information was present we utilized the standards for reporting of diagnostic accuracy statement and checklist to improve the quality of diagnostic accuracy [11] ; Inconclusive results were grouped in accordance with recent recommendations [12]. Statistical analysis To analyze significant differences among the three groups (i.e., MRS-1, MRS-2 and MRS-3) such as number of patients who underwent surgery an the mean values of TDCD a one-way ANOVA with Scheffè s post-hoc test was employed. A two-tailed P < 0.05 was considered statistically significant. SPSS 20.0 software was used for all statistical analyses and calculations. RESULTS Anatomical location of the lesions and surgical procedures adopted A total of 1136 patients were evaluated. In 12 patients the procedure was abandoned, 19 patients were lost to follow up, 86 with MRS of 0 received EUS without FNA, and 1019 completed the EUS-FNA procedure. Of the 1019 lesions evaluated by EUS-FNA, 932 were extraluminal and 87 were submucosal. In 616 patients a malignant lesion was diagnosed and the lesions in 515 patients were detected by EUS-FNA. In 570 patients a specific treatment was adopted (455 surgical procedure and 115 chemo-radiotherapy procedures). The different anatomical locations for all the lesions, the number of patients who required surgical treatment and the different surgical procedures employed are summarized in Table July 14, 2014 Volume 20 Issue 26

294 Gambitta P et al. EUS-FNA in gastrointestinal malignancies Table 1 Different anatomical localizations for the 1019 lesions in which endoscopic ultrasound-guided fine-needle aspiration was employed with the 431 surgical procedures adopted Macroscopic (> 1 cm) (n = 932) Surgical procedures (n = 393) 512 pancreatic masses 201 pancreatic resections 135 mediastinal lymphadenopathy 44 pneumonectomy, 12 lobectomy, 14 wedge resection 107 enlarged abdominal lymph nodes 11 Debulking, 19 nephrectomy, 6 splenectomy 23 lung cancer adjacent to the esophagus 11 pneumonectomy, 2 wedge resection 21 perirectal masses 6 low anterior resection, 5 Miles procedures 18 cancer of the extrahepatic bile duct 4 bile duct resections, 2 hepatic resections 18 perigastric masses 7 gastrectomy, 2 wedge resections 15 mediastinal masses 9 pneumonectomy 12 perirectal node 7 transanal rectal resection 13 pleural thickening and pleural effusion 7 pleurectomy, decortication 10 masses of the retroperitoneum/ 3 retroperitoneal debulking 10 peritoneal thickening and ascites 4 peritonectomy 10 left lobe of the liver 3 liver resection 9 left adrenal gland 5 adrenalectomy 8 prostate nodules with rectal involvement 4 prostatectomy and rectal resection 6 ovarian cyst 5 salpingo-oophorectomy 3 thyroid 3 thyroidectomy 1 spleen 1 splenectomy 1 cyst of CBD 1 biliary duct resection Microscopic (< 1 cm submucosal) (n = 87) Surgical procedures (n = 23) 49 gastric 12 gastrectomy, 1 sleeve gastrectomy 14 esophageal 2 esophagectomy 13 rectal 4 low rectal ant resection 8 duodenal 2 DCP 3 colorectal anastomoses 2 redo surgery for anastomotic recurrence CBD: Common bile duct; DCP: Duodenocephalopancreatectomy. Patients with of MRS 0 A total of 86 patients were evaluated as MRS-0; these patients were submitted to EUS without FNA. All of these patients who exhibited no clinical evidence of malignancy as evaluated by MTE, were followed up for a period of at least 30 mo or until death. In 83 patients, the clinical evaluation of the presumed benign lesion was confirmed by further clinical investigations during the follow up period. In three patients (3.48%), a malignancy was detected at 6, 7 and 10 mo after the first clinical evaluation. In two patients, non-functioning neuroendocrine tumors of the pancreatic head and tail, considered small benign pancreatic cysts by EUS, were subsequently detected after surgical resection; in one other patient, with a pancreatic lesion evaluated as pancreatitis by EUS, an adenocarcinoma of the pancreatic head was subsequently diagnosed based on the surgical specimen. Patients with MRS of 1-3 In 1019 patients the EUS-FNA was completed. Among 616 patients with a proven final diagnosis of cancer, EUS-FNA resulted in the detection of 511 (83%) patients with a TP diagnosis. In 431 patients the lesion was considered resectable and surgery was performed (Table 1). Ninety-four patients with unresectable lesions were submitted to different specific chemotherapies and radiation therapies as suggested by ongoing protocols. In the group of MRS of 3 we identified the highest number of patients (34.6%) who could benefit from different surgical procedures and chemo-radiotherapy. Complications related to the procedure Major complications occurred in two patients (0.2%). One esophageal perforation unrelated to FNA occurred during pharyngo-esophageal intubation in a patient who recovered uneventfully after 14 d in the intensive care unit (Clavien Ⅳa stage). In the other patient one sigmoid perforation that required surgery occurred (Clavien Ⅲa stage). Minor complications (Clavien Ⅱ) related to FNA were observed in five other patients. Accuracy and efficiency and quality assessment Table 2 shows all of the data including the number of patients with a positive and negative final diagnosis, the number of TP, TN, FP, FN diagnoses, and the number of inconclusive diagnoses. In 515 of the 616 patients with true malignant lesions (83%) the EUS-FNA was able to demonstrate the presence of malignant cells, in 359 of the 399 patients (89%) with true benign lesions, benign cells were observed. In 129 patients (12.6%), the specimen diagnosis was considered inconclusive. There were no false positive diagnoses in relation to a malignant cell diagnosis and 16 false negative diagnoses (7 of them in the pancreatic mass group). The overall diagnostic accuracy for the 1019 lesions in which a final diagnosis was obtained was 0.95 with an average of 2.2 passes per lesion (range 1 to 5). In Table 3 all values for AC, ST, SP, PPV, NPV, +LR, -LR, IT are reported, as well as the rate of patients who could receive treatment and the TDCD for each MRS group and for the whole group. TDCD and the number of patients who could receive surgical 8602 July 14, 2014 Volume 20 Issue 26

295 Gambitta P et al. EUS-FNA in gastrointestinal malignancies Table 2 Cytological findings and final diagnoses of cancer n (%) Table 3 Accuracy and efficiency and quality assessment n (%) Cytology Positive Negative Inconclusive (treated/untreated) Patients (n ) MRS1 (n = 312) MRS2 (n = 276) MRS3 (n = 431) Overall (n = 1019) Final diagnosis D (FN) 85 (55/30) 616 (TP) D- 0 (FP) (0/44) 403 (TN) 515 (50.5) 375 (36.8) 129 (55/74) (12.7) 1019 D+ patients with positive final cytological diagnosis, D- patients with negative final cytological diagnosis, true positive (TP), true negative (TN), false positive (FP) and false negative (FN). Inconclusive tests are splitted into 2 groups: those that were treated (with surgery, or with chemo-radiotherapy) or those left untreated. treatment or chemo-radiotherapy were significantly different in MRS-3 patients. DISCUSSION AC: (TP+TN)/N ST: TP/N (D+) SP: TP/N (D-) PPV: TP/N (T+) NPV: TN/N (T-) LR (ST/1-SP) LR (SP/1-ST) TP 36 (3.5) 109 (10.6) 370 (36) 616 (60.4) TN 231 (22.6) 101 (9.9) 26 (2.5) 403 (39.5) IT 39 (12.5) 55 (19.9) 35 (8.1) 129 (12.6) TDCD mean 25.4 (8-32) c 16.2 (11-24) a 12.4 (10-21) a 16.3 (8-32) days (range) Patients TP 36 (3.5) c 109 (10.7) a 370 (36.3) a 515 (50.5) treated with surgery or CRT Patients with inconclusive tests treated (surgery- CRT) 16 (9-7) 21 (12-9) 18 (13-5) 55 (34-21) Calculation of accuracy (AC), sensitivity (ST), specificity (SP), and positive (PPV) and negative (NPV) predictive values for malignant risk score (MRS)-1, MRS-2 and MRS-3, likelihood ratios for positive and negative test (+LR, -LR) for different groups of patients. D+: Number of patients with positive final diagnosis; D-: Number of patients with negative final diagnosis; IT: Inconclusive test; TDCD: Time to definitive cytological diagnosis; CRT: Chemo-Radio-Therapy. a P < 0.05 vs MRS1; c P < 0.05 vs MRS3 according to the Scheffé post-hoc test. In our study, the AC of the EUS-FNA was high (0.95) for the whole cohort of patients irrespectively of MTE, and ST (0.93), SP (1.0), PPV (1.0) and NPV (0.90) were all in line with results reported by other studies [13-15]. In patients with a high clinical suspicion of malignant disease, as in the MRS-3 group, the AC was higher, reaching 1 with a ST of 0.99, SP of 1, PPV 1 and NPV of In the same group of patients with a MRS of 3, we did not find FP or FN diagnoses. The mean time to final cytological diagnosis as well as the rate of inconclusive tests were lower than that found in the other groups. Interestingly, no FP diagnoses were observed in all groups of patients. These findings represent the main advantages of EUS-FNA for the entire population of patients. We documented 3 of 86 (3.48%) FN diagnoses in the group with a MRS of 0: These patients received EUS without FNA and two of them presented with a cystic lesion of the pancreas. It is widely accepted that the evaluation of cystic lesions of the pancreas poses a diagnostic challenge for the radiologist, the endoscopist and the pathologist [16,17]. At the moment all patients with pancreatic cystic lesions with a diameter larger than 3 cm are considered to be potentially suspect for malignant or premalignant lesions and are subjected to EUS-FNA with determination of carbohydrate antigen (CA 19-9, and CA 72-4), amylase, cyst fluid viscosity, and various stains in the fluid. Seven of the 16 FN diagnoses were related to solid pancreatic lesions. This finding suggests the possibility that neoplastic cells can be difficult to obtain from pancreatic tumors when the perilesional pancreatic tissue has a consistent amount of inflammatory cells [18]. Although our finding of 12.6% inconclusive diagnoses is in accordance with other studies [19-21], these data can be explained by some technical aspects of the procedure itself which can present some difficulties during the puncture of the target lesion depending on several factors including the anatomical level of the lesion, the nature of the lesion itself, the small size of the lesion, and finally the absence of an onsite cytotechnologist, who verify in real time the presence of an adequate amount of tissue sampling. A consistent part of our study was performed for the evaluation of patients with focal pancreatic lesions that are often smaller than 2 cm in diameter. The accuracy of EUS-FNA in focal pancreatic lesions is usually less impressive than for mediastinal lesions [22] and the sensitivity and diagnostic accuracy of EUS-FNA for solid pancreatic lesions is reported to be strongly related to tumor size [23]. The low rate of inconclusive diagnoses by EUS-FNA in the group with a MRS of 3 (8.1% vs 19.9% in MRS-2 and 12.5% for MRS-1) can be explained, at least in part, by more adequate lesional and perilesional tissue in the group with the highest MRS, and above all in the group with pancreatic lesions. In these patients the cytological material obtained by the FNA can allow a more consistent and adequate sample for correct cytological evaluation [24,25]. The choice of the trans-esophageal procedure that we adopted for patients with mediastinal lymphadenopathy, in accord with other centers, most likely balanced the overall number of inconclusive tests. In our experience, this approach seems easier than a the trans-bronchial approach, facilitating detection and tissue sampling of mediastinal masses [26,27] and it most likely enables better diagnostic accuracy. Although limitation of our study was the lack of an 8603 July 14, 2014 Volume 20 Issue 26

296 Gambitta P et al. EUS-FNA in gastrointestinal malignancies accurate and reproducible method for cancer risk evaluation, our MTE was always homogeneously and uniformly carried out during the 12 years of the study, which was strictly in accord with the NCCN guidelines. This simple clinical practice allowed us to stratify patients with MRSs. In the group of MRS of 3 we found the highest number of TP tests, indicating patients who could benefit from early surgical treatment and showed the lowest number of TN tests. In the same high risk group with a MRS of 3, EUS-FNA reached the highest level of AC, ST, SP, PPV, NPV, without FP or FN diagnoses. We conclude that a simple but standardized clinical evaluation by a multidisciplinary team can improve the diagnostic yield of EUS-FNA optimizing the clinical workup for all patients with curable malignant lesions. Our study offers some important indications on how to optimize diagnostic procedures in patients with suspected malignant lesions and can be considered a first step methodology for further evaluations of diagnostic efficiency in the setting of clinical and surgical oncology. ACKNOWLEDGMENTS The authors thank Ryan Wade and Vittoria Aseni for their assistance in preparing and revising the language of the manuscript. COMMENTS Background Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) has become paramount in establishing the diagnosis of all suspected malignant lesions of the gastrointestinal tract. The time to a definitive cytological diagnosis when using EUS-FNA may not be satisfactory in clinical practice due to its increasing demand during the clinical decision-making process for both benign and malignant lesions. Inadequate selection of patients may hamper the process of clinical decision making. Possible strategies to improve diagnostic efficiency in the clinical and surgical oncology settings are currently under investigation. Research frontiers The current study demonstrates that the practice of stratifying patients into different groups of cancer risk may improve the impact and efficiency of EUS-FNA in the early detection of those patients with malignant lesions; these patients may benefit from early surgery or chemo-radiotherapy, allowing for a very high level of accuracy in all patient groups. Innovations and breakthroughs Authors found that EUS-FNA was associated with a reliable level of accuracy in all patients when its use was prioritized for those patients with the highest cancer risk. EUS-FNA combined with a multidisciplinary clinical evaluation of the risk of malignancy enables a shorter time to diagnosis for those particular patients who can benefit from early therapy. Applications The present study offers some important indications about how to optimize diagnostic procedures in patients with suspected malignant lesions, and this report can be considered a first step methodology for further evaluations of diagnostic efficiency in the clinical and surgical oncology settings. Peer review This retrospective analysis was conducted in a cohort of 1019 patients with suspected malignant lesions adjacent to the gastrointestinal tract who underwent EUS-FNA during a period of 12 years. They found that EUS-FNA, when associated with a specific multidisciplinary team evaluation, enables a useful stratification of the patients on the basis of their specific cancer risk, allowing for better efficiency and a shorter time to diagnosis in those patients who can benefit from early surgery or chemo-radiotherapy treatment. REFERENCES 1 Rösch T, Schusdziarra V, Born P, Bautz W, Baumgartner M, Ulm K, Lorenz R, Allescher HD, Gerhardt P, Siewert JR, Classen M. 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BMJ 2013; 346: f2778 [PMID: DOI: / bmj.f2778] 13 Rocca R, De Angelis C, Daperno M, Carucci P, Ravarino N, Bruno M, Crocellà L, Lavagna A, Fracchia M, Pacchioni D, Masoero G, Rigazio C, Ercole E, Sostegni R, Motta M, Bussolati G, Torchio B, Rizzetto M, Pera A. Endoscopic ultrasound-fine needle aspiration (EUS-FNA) for pancreatic lesions: effectiveness in clinical practice. Dig Liver Dis 2007; 39: [PMID: DOI: /j.dld ] 8604 July 14, 2014 Volume 20 Issue 26

297 Gambitta P et al. EUS-FNA in gastrointestinal malignancies 14 Eloubeidi MA, Tamhane A. Prospective assessment of diagnostic utility and complications of endoscopic ultrasoundguided fine needle aspiration. Results from a newly developed academic endoscopic ultrasound program. Dig Dis 2008; 26: [PMID: DOI: / ] 15 Coté GA, Hovis CE, Kohlmeier C, Ammar T, Al-Lehibi A, Azar RR, Edmundowicz SA, Mullady DK, Krigman H, Ylagan L, Hull M, Early DS. Training in EUS-Guided Fine Needle Aspiration: Safety and Diagnostic Yield of Attending Supervised, Trainee-Directed FNA from the Onset of Training. Diagn Ther Endosc 2011; 2011: [PMID: DOI: /2011/378540] 16 Ahmad NA, Kochman ML, Lewis JD, Ginsberg GG. Can EUS alone differentiate between malignant and benign cystic lesions of the pancreas? Am J Gastroenterol 2001; 96: [PMID: DOI: /j x] 17 Scheiman JM. Management of cystic lesions of the pancreas. J Gastrointest Surg 2008; 12: [PMID: ] 18 de la Fuente SG, Ceppa EP, Reddy SK, Clary BM, Tyler DS, Pappas TN. Incidence of benign disease in patients that underwent resection for presumed pancreatic cancer diagnosed by endoscopic ultrasonography (EUS) and fine-needle aspiration (FNA). J Gastrointest Surg 2010; 14: [PMID: DOI: /s ] 19 Iglesias-Garcia J, Dominguez-Munoz JE, Abdulkader I, Larino-Noia J, Eugenyeva E, Lozano-Leon A, Forteza-Vila J. Influence of on-site cytopathology evaluation on the diagnostic accuracy of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) of solid pancreatic masses. Am J Gastroenterol 2011; 106: [PMID: DOI: /ajg ] 20 Turner BG, Cizginer S, Agarwal D, Yang J, Pitman MB, Brugge WR. Diagnosis of pancreatic neoplasia with EUS and FNA: a report of accuracy. Gastrointest Endosc 2010; 71: [PMID: DOI: /j.gie ] 21 Cleveland P, Gill KR, Coe SG, Woodward TA, Raimondo M, Jamil L, Gross SA, Heckman MG, Crook JE, Wallace MB. An evaluation of risk factors for inadequate cytology in EUS-guided FNA of pancreatic tumors and lymph nodes. Gastrointest Endosc 2010; 71: [PMID: DOI: /j.gie ] 22 Fritscher-Ravens A, Topalidis T, Bobrowski C, Krause C, Thonke E, Jäckle S, Soehendra N. Endoscopic ultrasoundguided fine-needle aspiration in focal pancreatic lesions: a prospective intraindividual comparison of two needle assemblies. Endoscopy 2001; 33: [PMID: DOI: /s ] 23 Siddiqui AA, Brown LJ, Hong SK, Draganova-Tacheva RA, Korenblit J, Loren DE, Kowalski TE, Solomides C. Relationship of pancreatic mass size and diagnostic yield of endoscopic ultrasound-guided fine needle aspiration. Dig Dis Sci 2011; 56: [PMID: DOI: /s z] 24 Devereaux BM, Leblanc JK, Yousif E, Kesler K, Brooks J, Mathur P, Sandler A, Chappo J, Lehman GA, Sherman S, Gress F, Ciaccia D. Clinical utility of EUS-guided fine-needle aspiration of mediastinal masses in the absence of known pulmonary malignancy. Gastrointest Endosc 2002; 56: [PMID: DOI: /mge ] 25 Harewood GC, Wiersema MJ. Diagnosis of pancreatic cancer- EUS/FNA to the rescue? Am J Gastroenterol 2001; 96: [PMID: DOI: /j x] 26 Arima M, Tada M. Endoscopic ultrasound-guided fine needle aspiration biopsy in esophageal and mediastinal diseases: clinical indications and results. Dig Endosc 2003; 15: [DOI: /j x] 27 Hernandez LV, Mishra G, George S, Bhutani MS. A descriptive analysis of EUS-FNA for mediastinal lymphadenopathy: an emphasis on clinical impact and false negative results. Am J Gastroenterol 2004; 99: [PMID: DOI: /j x] P- Reviewer: Tulassay ZJ S- Editor: Gou SX L- Editor: A E- Editor: Zhang DN 8605 July 14, 2014 Volume 20 Issue 26

298 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. RETROSPECTIVE BRIEF ARTICLE STUDY Birthplace is not a determinant of colorectal adenomas Fiona Tran, Jenn Hian Koo Fiona Tran, Jenn Hian Koo, Department of Gastroenterology and Hepatology, Liverpool Hospital, NSW 1871, Australia Fiona Tran, Jenn Hian Koo, Department of Gastroenterology, The University of New South Wales, NSW 2052, Australia Author contributions: Tran F was involved in study concept and design, acquisition of data, analysis and interpretation of data and drafting and critical revision of the manuscript; Koo JH was involved in study concept and design, analysis and interpretation of data, critical revision of the manuscript and final approval of article. Correspondence to: Fiona Tran, MBBS, Department of Gastroenterology and Hepatology, Liverpool Hospital, Sydney South West Local Health District, Locked Mailbag 7103, Liverpool, NSW 1871, Australia. f.tran88@gmail.com Telephone: Fax: Received: November 25, 2013 Revised: February 10, 2014 Accepted: April 21, 2014 Published online: July 14, 2014 Abstract AIM: To examine the impact of the patient s birthplace on the prevalence of colonic polyps and histopathological subtypes. METHODS: This is a retrospective audit of the colonoscopy practice of one Gastroenterologist in a tertiaryreferral hospital from 2008 to Data collected include demography, birthplace, language spoken, details of the colonoscopy including indications, completion rates, complications, results including prevalence and histopathology of polyps. Statistical methods used were binary logistic regression, χ 2 and Mann-Whitney U. RESULTS: A total of 623 patients (48% male, 67% aged over 50 years) were recruited and categorised according to birthplace: Australia/New Zealand 42%, European 20%, Asian 15%, Middle Eastern/African 11%, South American 9% and Pacific Islander 3%. The median age of the cohort was 56.3 years (range: years), median body mass index 27.3 kg/m 2 (range: kg/m 2 ), 25% were smokers, 25% had hypercholesterolemia, 20% had diabetes mellitus 16% were on aspirin and 7% were on non-steroidal antiinflammatory drugs. A total of 651 colonoscopies were performed for standard indications. The prevalence of polyps varied according to patient s birthplace: Europe 45.1%, Australia and New Zealand 39.5%, Pacific Islands 33.3%, Asia 30.3%, Middle East and Africa 26.9% and South America 24.5% (P = 0.027, df = 6). However, multivariate analysis revealed that birthplace was not an independent predictor of developing polyps, including adenomas and advanced adenomas after correcting for age and male sex. CONCLUSION: Birthplace is not a predictor for developing colorectal neoplasia, including adenomas and advanced adenomas; hence, should not influence the recommendations for colorectal cancer screening Baishideng Publishing Group Inc. All rights reserved. Key words: Ethnicity; Polyps; Adenomas; Advanced adenomas; Colorectal cancer Core tip: The detection and removal of colorectal adenomas is a vital component of colorectal cancer prevention. The provision of colorectal cancer screening by medical practitioners is influenced by patient s ethnicity. However, birthplace is not a predictor for developing colorectal neoplasia, including polyps, adenomas and advanced adenomas; hence, should not influence the recommendations for colorectal cancer screening. Tran F, Koo JH. Birthplace is not a determinant of colorectal adenomas. World J Gastroenterol 2014; 20(26): Available from: URL: v20/i26/8606.htm DOI: i July 14, 2014 Volume 20 Issue 26

299 Tran F et al. Birthplace is not a determinant of colorectal adenomas INTRODUCTION Colorectal cancer (CRC) is the second leading cause of cancer death in Australia with the age-standardised incidence rate of 46 and 32.1 per for men and women respectively [1]. The incidence rate is lower than parts of Asia, Africa and the Middle East. Nonetheless, it has been recognised that the risk of CRC of immigrants will gradually increase over time, attributed to adoption of the western lifestyle including diet and exercise [2,3]. Despite this, the provision of CRC screening by primary care providers has been shown to be influenced by the patient s ethnicity and birthplace [4] and that doctor-patient ethnic concordance was associated with decrease provision of preventive medicine [5]. The detection and removal of colorectal adenomas decreases the incidence and mortality of CRC [6,7]. The exact incidence and prevalence of polyps in different countries is not well described; however it is expected to mirror the incidence of CRC. Recent studies from Asia have revealed comparable prevalence of advanced neoplasm compared with Western nations, especially in Japanese and Korean populations [8]. However, there is conflicting evidence regarding the prevalence of colorectal adenomas between ethnic groups living in the same country. In an Australian cohort, the prevalence of advanced adenomas was significantly higher in Caucasians compared with Chinese; however, this study did not adjust for confounders including smoking, diabetes and body mass index in multivariate analysis [9]. A study examining Asian Americans (n = 2723) found no difference in advanced adenomas between Asian and non-asian participants [10]. Similarly, there is emerging evidence that there is no difference between adenoma detection rates between ethnic groups previously thought as low risk for developing CRC, such as Hispanics compared with those of high risk [11,12]. Nonetheless, there is no study to date that examines the effect of patient s birthplace on detection of colorectal neoplasia. The aim of this study was to examine the effect of birthplace on the detection of colorectal adenomas and advanced adenomas. MATERIALS AND METHODS We conducted a retrospective review of the colonoscopy practice of a single gastroenterologist (Koo JH) in a tertiary-referral hospital from 2008 to The hospital serves an ethnically diverse population, with greater than 40% of residents born overseas [13]. Patients were eligible if they were aged over 16 years. Demographic and clinical details were obtained from medical record review. Characteristics obtained include age, sex, anthropometric data, birthplace, language spoken at home, employment status, co-morbidities including diabetes and hypercholesterolemia, medications including aspirin and non-steroidal anti-inflammatory drugs (NSAIDs) and smoking. Colonoscopy details were recorded including primary indication, number, location and size of colonic polyps, histopathological type of resected polyps. The endoscopic size of the polyp was based on comparison with the known diameter of open biopsy forceps. The colonoscopies were performed by Koo JH and advanced gastroenterology trainees under his supervision. All resected and retrieved colonic polyps were formalin-fixed and paraffin-embedded. These specimens were stained with haematoxylin and eosin and examined by a gastrointestinal pathology service. Advanced adenomas were defined as size 10 mm, had villous histology or any evidence of high grade dysplasia [14]. The study was approved by the Ethics Committee of the Sydney South West Local Health District. Statistical analysis Statistical analysis was performed using SPSS, version 22.0 (SPSS Inc, Chicago, IL, United States). Descriptive statistics are reported as mean ± SD or median (range) unless otherwise stated. The prevalence of polyps, adenomas and advanced adenomas were compared between ethnic groups using independent t-tests, Kruskal-Wallis one-way analysis of variance or Mann-Whitney U tests for continuous non-parametric variables and χ 2 tests for categorical data. Binary logistic regression analysis was used to examine factors associated with polyp, adenomas and advanced adenoma detection. In each model, polyp, adenoma and advanced adenoma detection were the dependent variables respectively, with age, sex, ethnicity, body mass index, co-existing diabetes mellitus and hypercholesterolemia, use of aspirin and NSAIDs, and smoking included as independent variables. The adenoma detection rate was compared between birthplaces using chi-squared tests. Adenoma detection rate (ADR) was defined as the number of colonoscopies where one or more adenomas were detected divided by the total number of colonoscopies [15]. RESULTS Patient characteristics A total of 623 patients (48% male, 67% aged over 50 years) were recruited and categorised according to birthplace: Australia/New Zealand 42%, European 20%, Asian 15%, Middle Eastern/African 11%, South American 9% and Pacific Islander 3%. Among patients where data was available (n = 446), 66.8% reported English as their language spoken at home and 49% were employed. The median age of the cohort was 56.3 years (range: years), median body mass index (BMI) 27.3 kg/m 2 (range: kg/m 2 ), 25% were smokers, 25% had hypercholesterolemia, 20% had diabetes mellitus 16% were on aspirin and 7% were on NSAIDs (Table 1). In the study period, a total of 651 colonoscopies were performed for standard indications: 29% rectal bleeding, 20% altered bowel habits, 16% anaemia, 16% abdominal pain, 9% colorectal neoplasia surveillance, 8% positive faecal occult blood test (FOBT), 6% asymptomatic colorectal cancer screening, 4% abnormal imaging 8607 July 14, 2014 Volume 20 Issue 26

300 Tran F et al. Birthplace is not a determinant of colorectal adenomas Table 1 Clinical characteristics according to birthplace Characteristics Australia/New Zealand Asia Europe Middle East/Africa South America Pacific Islands P -value Age > 50 yr 56% 70% 85% 65% 74% 67% < 0.01 Aspirin 17% 12% 17% 12% 9% 14% 0.16 NSAID 8% 5% 11% 10% 13% 0% 0.15 Smoking 2% 12% 21% 19% 26% 18% 0.09 Hypercholesterolemia 18% 25% 35% 25% 30% 20% 0.03 Diabetes 18% 19% 23% 21% 15% 60% 0.05 NSAID: Non-steroidal anti-inflammatory drugs. Table 2 Adenoma detection rate according to birthplace Birthplace Adenoma detection rate 1 P -value 3 Advanced adenoma detection rate 2 P -value 3 Australia and New Zealand 47.50% 18.80% Asia 29.50% % 0.03 Europe 50.40% % 0.25 Middle East and Africa 38.00% % 0.07 South America 40.70% < % 0.04 Pacific Islands 31.80% % Adenoma detection rate is defined as number of colonoscopies where one or more adenomas were detected divided by the total number of colonoscopies; 2 Advanced adenoma detection rate is defined as number of colonoscopies where one or more advanced adenomas were detected divided by the total number of colonoscopies; 3 χ 2 test between Australia and New Zealand-born and those born elsewhere. findings, 4% progress colonoscopy for inflammatory bowel disease and 2% weight loss. Of the 14% who were asymptomatic, there was no statistically significant difference in birthplace. The caecal intubation rate was 93%. A total of 21 colorectal cancers and 635 polyps (281 adenomas and 91 advanced adenomas, 166 hyperplastic polyps, 24 normal histology, 3 inflammatory polyps, 1 hamartomatous, 69 polyps were not retrieved) were detected in 227 patients. The overall ADR was 20.4%. Of all the colorectal cancers, 18 were adenocarcinomas (14 moderately differentiated, 4 poorly differentiated), 1 mucinous colorectal cancer, 1 lymphoma, 1 gastrointestinal stromal tumour. Prevalence of colonic polyps and colorectal cancers The prevalence of polyps varied according to patient s birthplace: Europe 45.1%, Australia/New Zealand 39.5%, Pacific Islands 33.3%, Asia 30.3%, Middle East/ Africa 26.9% and South America 24.5% (P = 0.027, df = 6). The number of adenomas according to patient s birthplace: Australia/New Zealand 46.2% (n = 129), Europe 25.4% (n = 71), Middle East/Africa 13.3% (n = 37), Asia 10.0% (n = 28), South America 3.9% (n = 11), Pacific Islands 1.2% (n = 3). The prevalence of adenomas and advanced adenomas was not statistically different according to birthplace (P = and P = respectively). There was no statistically significant difference in detected adenomas and advanced adenomas between patients born in Australia/New Zealand compared with those born elsewhere (P = 0.21, P = 0.65 respectively). However, in patients aged > 50 years, greater proportion of those born in Australia/NZ had advanced adenomas compared with those born elsewhere (25/133, 19% vs 27/257, 11%, P = 0.022). There was a greater proportion of European-born patients with polyps detected compared with Australia/ New Zealand (125/195, 64% vs 102/258, 40%, P < 0.001) There was a trend towards a significant difference in adenomas detected in this group compared with Asianborn (P = 0.07) and European-born patients (P = 0.06). However, there was no statistically significant difference in polyps, adenomas or advanced adenomas detected between Australian/New Zealand-born and those born in South America, the Middle East/Africa and the Pacific Islands. In those aged > 50 years, there was a difference between advanced adenomas detected in those born in Australia/New Zealand (25/137, 18%) compared to those born in Asia (8/90, 9%) (P = 0.05). In patients with adenomas detected, the median age of Asian-born patients was lower than Australia/New- Zealand-born (Median age 63 years vs 58 years, P = 0.016, df = 1). Similarly, in all patients with advanced adenomas detected, there was a statistically significant difference in median age of Asian-born patients compared to Australia/New-Zealand-born (P = 0.015). The ADR and advanced adenoma detection rate varied according the birthplace (Table 2). There was a significant difference between ADR of Australia/New Zealand-born patients compared with those born in Asia (P = 0.002), South America (P < 0.001) and Pacific Islands (P = 0.03). Similarly, there was a significant difference between advanced ADR of Australia/New Zealandborn patients compared with Asia (P = 0.03), and South America (P = 0.04). There was no statistically significant difference in the prevalence of colorectal cancers by patients birthplace (P = 0.49) July 14, 2014 Volume 20 Issue 26

301 Tran F et al. Birthplace is not a determinant of colorectal adenomas Table 3 Predictors of polyp detection, adenoma detection, advanced adenoma detection Univariate analysis OR 95%CI P -value Multivariate analysis OR 95%CI P -value Polyp detection Male sex < Male sex Older age < Older age < Aspirin use Positive FOBT Hypercholesterolemia Diabetes Anaemia Positive FOBT < BMI Smoking Birthplace Adenoma detection Male sex Male sex Older age Older age NSAID use Positive FOBT Positive FOBT Birthplace Advanced adenoma detection Male sex Male sex Older age < Rectal bleeding Rectal bleeding Positive FOBT < Positive FOBT < Birthplace FOBT: Faecal occult blood test; BMI: Body mass index. Predictors of colorectal neoplasia Binary logistic regression was performed to identify predictors of colonic polyps, adenomas and advanced adenomas to determine whether birthplace was a risk factor. The predictors of polyp detection in multivariate analysis included male sex, older age and positive FOBT (Table 3). In univariate analysis, the predictors for adenoma detection were male sex, older age. In multivariate analysis, the predictors for adenoma detection were male sex, positive FOBT (Table 3). The predictors for advanced adenoma in univariate analysis include male sex, older age, smoking, rectal bleeding, positive FOBT. In multivariate analysis, the predictors for advanced adenomas included male sex, rectal bleeding and positive FOBT (Table 3). Other risk factors and confounders including, body mass index, diabetes mellitus, hypercholesterolemia, smoking, aspirin and NSAID use were not statistically significant in both univariate and multivariate analysis for adenoma and advanced adenomas. DISCUSSION In an ethnically diverse population in Australia, this study revealed that birthplace was not a significant predictor for the detection of colonic polyps, adenomas or advanced adenomas. It further reinforced previously demonstrated risk factors for developing colorectal neoplasia, such as older age and male sex. In addition, Asian-born patients had adenomas and advanced adenomas detected at younger ages compared with Australia/New Zealandborn patients. The prevalence of colorectal neoplasia differed ac- cording to patient s birthplace, with higher prevalence among Western-born and Australian/New Zealand-born patients. However, after correcting for risk factors and confounders on multivariate analysis, birthplace was no longer a predictor of colorectal neoplasia. This important finding supports recent studies conducted in a multiethnic Asian population which demonstrated that race was not a predictor for colorectal adenomas in multivariate analysis [16] and a retrospective study in the United States which also revealed no significant differences in the adenoma detection rate in Hispanic patients compared with Whites [11]. In contrast, a recent multi-ethnic Australian population study reported polyp detection varied with race with significantly higher prevalence of advanced adenomas in Caucasians compared with Chinese, although the study did not correct for risk factors and confounders [9]. Furthermore, it did not clarify the category of Chinese - Chinese patients may have originated from different Asian countries, and therefore had differing intrinsic risks for developing colorectal neoplasia. Our study importantly reinforces the observation that birthplace is not a predictor of colorectal neoplasia and that recommendations and provision of CRC screening tests should not be based on patients birthplace. Previous studies have reported medical practitioners recommendation of CRC screening tests varied according to their patients birthplace, resulting in lower recommendations to ethnic minority patients who were considered at lower risk of developing colorectal neoplasia compared with Western-born patients [4,17]. As medical practitioner recommendation is the single most important predictor of CRC screening participation [18], appropriate and timely recommendations for screening irrespective of patients 8609 July 14, 2014 Volume 20 Issue 26

302 Tran F et al. Birthplace is not a determinant of colorectal adenomas birthplace is crucial to improve outcomes. This study demonstrated that Asian-born patients had adenomas and advanced adenomas detected at a younger age compared with Australian/New Zealand-born patients. This has been previously reported in a study comparing concurrent cohorts of Chinese and Western patients, where Chinese patients had a slightly lower ageadjusted prevalence of adenomas [19]. Notwithstanding the incomplete family history data, this has significant implications for colorectal cancer screening, in particular the age of initiation of screening. The younger ages of diagnosis of CRC in African Americans had subsequently resulted in recommendations for earlier age of initiation of screening in the United States [20]. Additional prospective studies are therefore recommended to determine the appropriate age to start screening in Asian patients, given the complexities that already exist with current CRC screening guidelines. This study adjusted for important predictors of developing colorectal neoplasia such as demographic factors including age, sex and clinical factors such as body mass index, diabetes mellitus, hypercholesterolemia, smoking and aspirin use. Factors such as quality of bowel preparation were also considered as this has been demonstrated to affect adenoma detection [21]. Correcting for these variables further strengthens the validity of the study, and reinforces the well-established independent predictors for developing adenomas and advanced adenomas including male sex, older age and positive FOBT. The predictors for polyp detection also include smoking and higher BMI. This is supported by a study, which demonstrated a higher risk of advanced adenomas in female smokers without a family history of colorectal cancer [22]. Similarly, a recent meta-analysis demonstrated a 5-unit increase in BMI was associated with a relative risk increase of developing colorectal adenomas by 1.19 (95%CI: ) [23]. It has been well documented that there are differences in the provision and uptake of colorectal cancer screening based on patients ethnicity and birthplace. Patient s ethnicity and birthplace have been associated with participation in colorectal cancer screening. In screening studies in the United Kingdom, return of FOBT, colonoscopy attendance following a positive FOBT and attendance rate for flexible sigmoidoscopy screening were significantly lower in Asians compared with Whites, after controlling for demographic factors [24,25]. The Australian National bowel cancer screening pilot study also revealed lower FOBT return rates among subjects who spoke a language other than English [26]. In addition, foreign-born Asians and Hispanics had lower CRC screening participation compared with United States-born Asians and Hispanics living in the United States [27]. The finding from our study that birthplace does not influence polyp detection, especially among subjects previously considered low risks such as Asians, reinforces the need to promote CRC screening participation irrespective of subjects birthplace and ethnicity. A study examining a multi-ethnic community revealed that fewer immigrants had their colorectal cancer diagnosed through screening practices compared with Australian-born patients [28]. Furthermore, patient s ethnicity has been demonstrated to influence the physician s likelihood of recommending colorectal cancer screening [4]. However, the findings of this study demonstrate that birthplace is not a predictor developing colorectal neoplasia and Asian-born patients have colorectal neoplasia detected at a lower age compared to Australia/New Zealand-born patients. There are several limitations to this study. It is a retrospective, single-centre study, which limits the external validity of the conclusion. Additionally, there is limited data on the duration of residency of immigrants in Australia, which could influence their colorectal polyp and cancer risk [2]. This can be addressed in future, multi-centre, prospective studies, including this demographic data. There is also heterogeneity of colorectal cancer risk within the ethnic groups. For example, in patients who are classified as Asian, the CRC risk varies between those who are South East Asian, such as Vietnamese patients, who are known to be low risk, compared with North East Asian, such as Koreans and Japanese patients whose risks are comparable with Western patients. In this study, there was no significant difference in adenoma and advanced adenomas detected between South East Asian and North East Asian patients. Furthermore, the proportion of patients classified as North East Asian was small (n = 7/89, 8%) and hence, the Asian subgroup was predominantly a lower risk group. Nonetheless, studies have demonstrated a similar risk in colorectal cancer between Asian migrants and those born in Western countries [29]. Birthplace is not a predictor for developing colorectal neoplasia, including polyps, adenomas and advanced adenomas; hence, should not influence the recommendations for colorectal cancer screening. COMMENTS Background The detection and removal of colorectal adenomas is a vital component of colorectal cancer prevention. A lower incidence of colorectal polyps has been previously reported in non-western populations. Research frontiers Colorectal cancer is a leading cause of morbidity and mortality. Currently, there exist multiple complexities in guidelines for colorectal cancer screening. Innovations and breakthroughs The result of this study suggests that birthplace is not a predictor for developing colorectal neoplasia, including polyps, adenomas and advanced adenomas. Applications The results of this study suggest that patient s ethnicity should not influence the recommendations for colorectal cancer screening. Terminology Advanced adenomas were defined as size 10 mm, had villous histology or any evidence of high grade dysplasia. Adenoma detection rate was defined as the number of colonoscopies where one or more adenomas were detected divided by the total number of colonoscopies. Peer review This is an important topic to study as, if differences in adenoma detection rate are identified among different ethnic populations, it could lead to changes in recommendations for colorectal cancer screening. Although similar data has been explored, more data is still needed on the topic July 14, 2014 Volume 20 Issue 26

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Breast and bowel cancer screening uptake patterns over 15 years for UK south Asian ethnic minority populations, corrected for differences in socio-demographic characteristics. BMC Public Health 2008; 8: 346 [PMID: ] 25 Robb KA, Power E, Atkin W, Wardle J. Ethnic differences in participation in flexible sigmoidoscopy screening in the UK. J Med Screen 2008; 15: [PMID: ] 26 Ward PR, Javanparast S, Matt MA, Martini A, Tsourtos G, Cole S, Gill T, Aylward P, Baratiny G, Jiwa M, Misan G, Wilson C, Young G. Equity of colorectal cancer screening: cross-sectional analysis of National Bowel Cancer Screening Program data for South Australia. Aust N Z J Public Health 2011; 35: [PMID: ] 27 Goel MS, Wee CC, McCarthy EP, Davis RB, Ngo-Metzger Q, Phillips RS. Racial and ethnic disparities in cancer screening: the importance of foreign birth as a barrier to care. J Gen Intern Med 2003; 18: [PMID: ] 28 Koo JH, Kin S, Wong C, Jalaludin B, Kneebone A, Connor SJ, Leong RW. Clinical and pathologic outcomes of colorectal cancer in a multi-ethnic population. Clin Gastroenterol Hepatol 2008; 6: [PMID: ] 29 McCracken M, Olsen M, Chen MS, Jemal A, Thun M, Cokkinides V, Deapen D, Ward E. Cancer incidence, mortality, and associated risk factors among Asian Americans of Chinese, Filipino, Vietnamese, Korean, and Japanese ethnicities. CA Cancer J Clin 2007; 57: [PMID: ] P- Reviewers: Cantisano BGJ, Kim JK S- Editor: Gou SX L- Editor: A E- Editor: Zhang DN 8611 July 14, 2014 Volume 20 Issue 26

304 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. RETROSPECTIVE STUDY Follow-up of patients with pseudotumoral chronic pancreatitis: Outcome and surveillance Félix Ignacio Téllez-Ávila, Álvaro Villalobos-Garita, Marc Giovannini, Carlos Chan, Jorge Hernández-Calleros, Luis Uscanga, Miguel Ángel Ramírez-Luna Félix Ignacio Téllez-Ávila, Miguel Ángel Ramírez-Luna, Endoscopy Department, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, CP 14000, México Álvaro Villalobos-Garita, Gastroenterology Department, Hospital Calderón Guardia, CCSS, San José, CP 10105, Costa Rica Marc Giovannini, Endoscopic Unit, Paoli-Calmettes Institute, 232 Bd St-Marguerite, Marseille cedex 9, France Carlos Chan, Surgery Department, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, CP 14000, México Jorge Hernández-Calleros, Luis Uscanga, Gastroenterology Department, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, CP 14000, México Author contributions: Téllez-Ávila FI design the report; Téllez- Ávila FI, Villalobos-Garita A, Chan C, Hernández-Calleros J, Uscanga L, and Ramírez-Luna MÁ were attending doctors for patients; Téllez-Ávila FI and Ramírez-Luna MÁ performed endoscopies; Téllez-Ávila FI, Villalobos-Garita A, Hernández- Calleros J, Chan C, and Giovannini M organized the report; and Téllez-Ávila FI, Villalobos-Garita A, and Giovannini M wrote the paper. Correspondence to: Félix Ignacio Téllez-Ávila, MD, MSc, PhD, Endoscopy Department, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Vasco de Quiroga 15. Col. Sección XVI. Del. Tlalpan, Mexico City, CP 14000, Mexico. felixtelleza@gmail.com Telephone: Fax: Received: November 26, 2013 Revised: February 14, 2014 Accepted: April 5, 2014 Published online: July 14, 2014 Abstract AIM: To follow up patients with pseudotumoral chronic pancreatitis (PCP) to assess their outcome and identify an optimal surveillance interval. METHODS: Data obtained prospectively were analyzed in a retrospective manner. Patients with clinical evidence of chronic pancreatitis (abdominal pain in the epigastrium, steatorrhea, and diabetes mellitus), endoscopic ultrasound (EUS) criteria > 4, and EUS-fine needle aspiration (FNA) were included. A pseudotumor was defined as a non-neoplastic space-occupying lesion, a cause of chronic pancreatitis that may mimic changes typical of pancreatic cancer on CT or endoscopic ultrasound but without histological evidence. A real tumor was defined as a neoplastic space-occupying lesion because of pancreatic cancer confirmed by histology. RESULTS: Thirty-five patients with chronic pancreatitis were included, 26 (74.2%) of whom were men. Nine (25.7%) patients were diagnosed with pseudotumoral chronic pancreatitis and two (2/35; 5.7%) patients with pseudotumoral chronic pancreatitis were diagnosed with pancreatic cancer on follow-up. The time between the diagnosis of pseudotumoral chronic pancreatitis and pancreatic adenocarcinoma was 35 and 30 d in the two patients. Definitive diagnosis of pancreatic adenocarcinoma was made by surgery. In the remaining six patients with pseudotumoral chronic pancreatitis, the median of follow-up was 11 mo (range 1-22 mo) and they showed no evidence of malignancy on surveillance. In the follow-up of patients without pseudotumoral chronic pancreatitis but with chronic pancreatitis, none were diagnosed with pancreatic cancer. According to our data, older patients with chronic pancreatitis are at risk of pseudotumoral chronic pancreatitis. CONCLUSION: According to characteristics of patient, detection of PCP should lead a surveillance program for pancreatic cancer with EUS-FNA in < 1 mo or directly to surgical resection Baishideng Publishing Group Inc. All rights reserved. Key words: Chronic pancreatitis; Pseudotumoral chronic pancreatitis; Surveillance; Endoscopic ultrasound 8612 July 14, 2014 Volume 20 Issue 26

305 Téllez-Ávila FI et al. Solid mass lesions in chronic pancreatitis Core tip: Actually, there are no clear recommendations for follow-up of patients with chronic pancreatitis and solid pancreatic mass lesions. We followed-up patients with chronic pancreatitis and solid pancreatic mass lesions and we assessed the final outcome and identified an optimal surveillance interval. We found that almost one-third of patients with chronic pancreatitis had pseudotumoral chronic pancreatitis, and 22.2% had unresectable pancreatic adenocarcinoma less than 2 mo after the initial diagnosis. Endoscopic ultrasound fine needle aspiration can miss malignancy in nearly 25% of patients with pseudotumoral chronic pancreatitis. Téllez-Ávila FI, Villalobos-Garita Á, Giovannini M, Chan C, Hernández-Calleros J, Uscanga L, Ramírez-Luna MÁ. Followup of patients with pseudotumoral chronic pancreatitis: Outcome and surveillance. World J Gastroenterol 2014; 20(26): Available from: URL: v20/i26/8612.htm DOI: i INTRODUCTION Pancreatic cancer (PC) at diagnosis is unresectable in 70% of cases [1]. The risk of developing PC is increased in patients with chronic pancreatitis. Surveillance in patients with chronic pancreatitis may represent an opportunity for early detection of PC [2]. The increase in risk for PC in patients with chronic pancreatitis ranges from times in 10-year follow-up [1,3]. It is difficult to differentiate by images between pancreatic carcinoma and pseudotumor in the context of chronic pancreatitis [4,5]. In case of endoscopic ultrasound (EUS) some criteria have been proposed, but even using the fine needle aspiration (FNA) biopsy, the results have not been satisfactory [6,7]. Actually, there are no clear recommendations for follow-up of patients with chronic pancreatitis and solid pancreatic mass lesions [8]. The aim of this study was to follow up patients with chronic pancreatitis and solid pancreatic mass lesions to assess the final outcome and identify an optimal surveillance interval. MATERIALS AND METHODS Data obtained prospectively were analyzed in a retrospective manner. Electronic and paper records of consecutive patients evaluated from March 2005 to December 2012 were evaluated. Patients with clinical evidence of chronic pancreatitis, EUS criteria > 4, and EUS FNA were included [9]. According to the local Ethics Committee, all patients signed an informed consent document. Before the procedure, all patients had laboratory tests including prothrombin time and full blood count. The patients were placed in a left decubitus position and sedated using a combination of midazolam, propofol, and fentanyl by an anesthetist. Patients were continually monitored with an automated noninvasive blood pressure device, electrocardiogram, and pulse oximetry throughout the procedure. EUS was performed with a linear array echoendoscope, GFUCT-140 (Olympus America Inc; Center Valley, PA), by two echoendoscopists. All patients were hospitalized, and after the procedure they were observed with an automatic monitor for at least 4 h for surveillance of possible complications. EUS FNA (standard needle) At first, the transducer was brought into a stable position in front of the targeted lesion. The metal spiral was then introduced into the biopsy channel, observing carefully that the needle piston was securely locked and the needle was completely retracted. The spiral was inserted entirely and the handle with the Luer-lock firmly screwed onto the biopsy channel. To ensure that the sheath was protecting the entire length of the working channel, we used the optic of the endoscope. With the stylet retracted but still inside the needle, the biopsy needle was moved forward into the lesion under full real-time ultrasound control. After penetration into the middle of a lesion, the stylet was completely removed. Upon reaching the optimal needle position in the middle of the lesion, a 10 ml syringe with a locking device was firmly screwed on the needle, and the syringe piston was pulled to create a low pressure. The syringe piston was locked in this position for permanent suction. The needle was moved to and fro 5-10 times inside the lesion under complete ultrasonic control. With the needle tip still in the lesion, suction was released and the needle was safely retracted inside the needle sheath and locked in a secure position. All patients had a CT with a 64-slice multidetector CT (Somatom, Sensation 64; Siemens München Germany) and images were obtained with a section thickness of 3 mm with a reconstruction interval of mm. All cases were analyzed on a workstation with the capability to produce coronal reformatted images. Patients received intravenous (Ⅳ) contrast; 120 ml of Conray (Mallinckrodt Baker Inc., St Louis Missouri, United States) was given 45 s prior to the CT examination. Forty milliliters of ioditrast M60 (Justesa Imagen Mexicana) was diluted in 1000 ml of water and given to all patients orally 1 h prior to CT. All patients received Ⅳ and oral contrast. All CT images were analyzed by at least two certified radiologists and discussed with the endoscopic team before the procedure (EUS-FNA). All CT and endoscopic studies were performed in the same center. A pseudotumor (Figure 1) was defined as a non-neoplastic space-occupying lesion, a cause of chronic pancreatitis that may mimic changes typical of pancreatic cancer on CT or endoscopic ultrasound but without histological evidence. It should be recognized, however, that even this definition of pseudotumor is highly subjective since it relies on the quality of the preoperative diagnostic evaluation as well as the skills of the interpreters of the tests performed [10]. A real tumor was defined as a neoplastic space-occupying lesion because of pancreatic cancer confirmed by histology. Clinical characteristics considered associated with chronic pancreatitis were: abdominal pain 8613 July 14, 2014 Volume 20 Issue 26

306 Téllez-Ávila FI et al. Solid mass lesions in chronic pancreatitis A B Figure 1 Pseudotumoral chronic pancreatitis in included patient. A: Computed tomography image; B: Endoscopic ultrasound image. Table 1 Clinical and demographic characteristics of included patients classified by the presence/absence of pseudotumor n (%) Chronic pancreatitis (n = 26) Pseudotumoral chronic pancreatitis (n = 9) P value Female 5 (19.2) 4 (44.4) NS Age, yr 1 30 (18-74) 53 (18-75) Number of EUS criteria 1 4 (4-8) 4 (4-6) NS Follow-up 1 24 (1-67) 5 (1-35) NS Aetiology, alcohol 21 (81) 6 (67) NS DM 20 (77) 7 (78) NS 1 Expressed in median (range). EUS: Endoscopic ultrasound; DM: Diabetes mellitus; NS: Not significant. Table 2 Imaging characteristics of included patients with pseudotumor and chronic pancreatitis Patient Age, yr Gender Number of diagnostic criteria for chronic pancreatitis by EUS Evidence of pseudotumor on CT Interval ( time between ) or follow-up 1 64 F 4 No 1 mo 2 48 F 4 No 13 mo 3 53 M 4 No 21 mo 4 44 F 4 Yes 5 mo 5 75 F 4 Yes 22 mo 6 69 M 4 Yes 13 mo 7 18 M 6 Yes 30 d 8 56 M 5 Yes 30 d 9 52 M 4 Yes 30 d Time between: Represents the time between diagnosis of pseudotumor and pancreatic cancer; Follow-up: Time of follow-up after diagnosis of pseudotumor without diagnosis of cancer. F: Female; M: Male; CT: Computed tomography; EUS: Endoscopic ultrasound. in the epigastrium, often with radiation to the back; steatorrhea; and diabetes mellitus [11]. Statistical analysis Medians, ranges, and proportions were used to summarize the demographics and clinical variables. Using the χ 2 test or Mann-Whitney U test, according variables, differences between groups were tested. A two-tailed P value < 0.05 was considered significant. All analyses were performed by SPSS V.20 for Mac. RESULTS A total of 200 pancreatic EUS were performed because of clinical suspicion of chronic pancreatitis (abdominal pain in the epigastrium with radiation to the back, or exocrine pancreatic insufficiency with chronic diarrhea and/or steatorrhea). Thirty-five patients with diagnosis of chronic pancreatitis were included. Twenty-six (74.2%) patients were men and 9 (25.8%) patients were women. The median age was 38 years (range years). All patients had clinical and EUS criteria. Twenty-two (62.8%) patients had 4 EUS criteria, 6 (17%) patients had five criteria, and 7 (20%) patients had 6 criteria. Nine (25.7%) patients were diagnosed with pseudotumoral chronic pancreatitis. Clinical and demographic characteristics of included patients classified by the presence/absence of pseudotumoral chronic pancreatitis are shown in Table 1. In Tables 2 and 3, clinical data, demographics, and imaging characteristics of included patients with pseudotumor and chronic pancreatitis are shown. Two of nine (22.2%) patients with pseudotumoral chronic pancreatitis were diagnosed with pancreatic cancer on follow-up, although basal EUS FNA did not reveal malignant cells. One (11.1%) patient was diagnosed on follow-up with myofibroblastic tumor of the pancreas. The time between the diagnosis of pseudotumoral chronic pancreatitis and pancreatic adenocarcinoma was 35 and 30 d. The diagnosis of myofibroblastic tumor was 30 d after the pseudotumoral chronic pancreatitis diagnosis. The two patients with pancreatic adenocarcinoma had an unresectable pancreatic adenocarcinoma at the moment of final diagnosis. Definitive diagnosis of pancreatic adenocarcinoma was made by surgery. In the remaining six patients with pseudotumoral chronic pancreatitis, the median of follow-up was 11 mo (range 1-22 mo) and they showed no evidence of malignancy on surveillance. In the follow-up of patients with chronic pancreatitis but without pseudotumoral chronic pancreatitis, none were diagnosed with pancreatic cancer. The median follow-up was 22 mo (range 1-67 mo) (Figure 2) July 14, 2014 Volume 20 Issue 26

307 Téllez-Ávila FI et al. Solid mass lesions in chronic pancreatitis Table 3 Imaging characteristics of the pseudotumors and final diagnosis Pseudotumor/ patient Maximum diameter (mm) Localization Vascular involvement Presence of lymphadenopathy EUS FNA/adequate sample Surgery Final diagnosis 1 35 Body Yes No Normal/yes No CP 2 30 Head No Yes CP/yes No CP 3 20 Head No No Normal/yes No CP 4 35 Head Yes Yes Inflammation/yes Yes CP 5 35 Neck No No CP/yes No CP 6 28 Body No No CP/yes No CP 7 30 Neck No Yes Non-neoplastic cells/yes Yes Myofibroblastic tumor 8 40 Head Yes Yes Normal/yes Yes Pancreatic cancer 9 40 Head Yes Yes CP/yes Yes Pancreatic cancer CP: Chronic pancreatitis; EUS: Endoscopic ultrasound; FNA: Fine needle aspiration. 35 patients with CP Table 4 Frequency of pseudotumoral chronic pancreatitis in patients with chronic pancreatitis according to studies reported in the literature PCP 9 patients CP 26 patients Ref. Year Number of patients (n ) Frequency of pseudotumor Frequency of pancreatic cancer Barthet et al [12] % 3.5% Burski et al [8] % 13% Current study % 22% 2 patients with pancreatic cancer 7 patients without pancreatic cancer No patients with cancer 1 The time between diagnosis of pseudotumor and pancreatic cancer was not reported, only time of survival after diagnosis of pancreatic cancer; 2 The time between diagnosis of pseudotumor and pancreatic cancer re- Figure 2 Pseudotumoral chronic pancreatitis and development of pancreatic cancer on follow-up. PCP: Pseudotumoral chronic pancreatitis; CP: Chronic pancreatitis. DISCUSSION Almost one-third of patients with chronic pancreatitis had pseudotumoral chronic pancreatitis, and two of them (2/9; 22.2%) had unresectable pancreatic adenocarcinoma less than 2 mo after the initial diagnosis. The frequency of pseudotumoral chronic pancreatitis is not well known and little data exist. In one study with 85 patients with chronic pancreatitis, 6% (n = 5) of these patients had pseudotumoral chronic pancreatitis and 3.5% (n = 3) of them were diagnosed with pancreatic cancer [12]. In a more recent study, Burski et al [8] found that 29% (125/436) of patients with chronic pancreatitis had pseudotumoral chronic pancreatitis and 13% (16/125) of them were diagnosed with pancreatic adenocarcinoma on follow-up. In Table 4, published data about patients with chronic pancreatitis and pseudotumoral chronic pancreatitis are shown. Regarding follow-up of patients with pseudotumoral chronic pancreatitis, there are not clear recommendations regarding the ideal imaging study and time for subsequent imaging relative to the initial diagnosis. Therefore, the surveillance for pancreatic cancer in patients with pseudotumoral chronic pancreatitis is not well established and it has a negative impact in this population [1,13-16]. In this study, in patients with pseudotumoral chronic pancreatitis for whom pancreatic cancer was diagnosed on follow-up, pancreatic cancer was confirmed ported was 4.2 mo. at an advanced stage in a period of less than 2 mo after the initial detection. This data suggest a misdiagnosis rather than new onset of the neoplasm during followup. Because of that, surveillance programs for pancreatic cancer with intervals greater than 6 mo seem to be insufficient in patients with pseudotumoral chronic pancreatitis. In the study by Burski et al [8], it was concluded that an interval of 3-6 mo for surveillance for pancreatic cancer in patients with pseudotumoral chronic pancreatitis was not optimal due to rapid disease progression. Several studies have attempted to establish EUS imaging criteria (without tissue sampling) for the discrimination of benign inflammatory pseudotumors and tumors. Despite the high resolution of EUS, it does not provide reliable differentiation of benign and malignant lesions of the pancreas [17]. New technologies, such as EUS elastography and contrast-enhanced EUS (CE-EUS) could be important tools for differential diagnosis. In a multicenter study, 30 cases with benign nodule of chronic pancreatitis were studied with EUS elastography [18]. All nodules of chronic pancreatitis presented benign aspects (mixed green and low intensity of blue) and elastography showed malignant aspects (intense blue coloration) for all pancreatic adenocarcinomas, endocrine tumors, pancreatic metastases, and pancreatic sarcomas. In the study of Hocke et al [19], adenocarcinoma developed on chronic pancreatitis was non-enhanced after contrast injection. Conversely, pseudotumoral chronic pancreatitis was hypervascularized (91%) after SonoVue injection. According to our data, older patients with chronic pancreatitis 8615 July 14, 2014 Volume 20 Issue 26

308 Téllez-Ávila FI et al. Solid mass lesions in chronic pancreatitis are at risk of pseudotumoral chronic pancreatitis, and could be candidates for closer follow-up (Table 1). The limitations of our work are the small sample size and retrospective analysis. The nature of disease makes it difficult for a single center to have a bigger sample size. Multicenter studies must be considered for future designs. Our data are useful for future systematic reviews and meta-analyses. In conclusion, we suggest that according to specific characteristics of patient, detection of pseudotumoral chronic pancreatitis should lead a close surveillance program for pancreatic cancer with EUS in less than 1 mo or directly to surgical resection. EUS FNA can miss malignancy in nearly 25% of patients with pseudotumoral chronic pancreatitis. COMMENTS Background Pancreatic cancer at diagnosis is unresectable in 70% of cases. It is difficult to differentiate by images between pancreatic carcinoma and pseudotumor in the context of chronic pancreatitis. The authors followed-up patients with chronic pancreatitis and solid pancreatic mass lesions and we assessed the final outcome and identified an optimal surveillance interval. Innovations and breakthroughs According to characteristics of patient, detection of pseudotumoral chronic pancreatitis should lead a surveillance program for pancreatic cancer with endoscopic ultrasound fine needle aspiration in < 1 mo or directly to surgical resection. Peer review This is an interesting retrospective analysis of patients with pseudotumoral lesions in the context of chronic pancreatitis. The results are alarming. REFERENCES 1 Brand RE, Lerch MM, Rubinstein WS, Neoptolemos JP, Whitcomb DC, Hruban RH, Brentnall TA, Lynch HT, Canto MI. Advances in counselling and surveillance of patients at risk for pancreatic cancer. Gut 2007; 56: [PMID: DOI: /gut ] 2 Queneau PE, Adessi GL, Thibault P, Cléau D, Heyd B, Mantion G, Carayon P. Early detection of pancreatic cancer in patients with chronic pancreatitis: diagnostic utility of a K-ras point mutation in the pancreatic juice. Am J Gastroenterol 2001; 96: [PMID: DOI: / j x] 3 Lowenfels AB, Maisonneuve P, Cavallini G, Ammann RW, Lankisch PG, Andersen JR, Dimagno EP, Andrén-Sandberg A, Domellöf L. Pancreatitis and the risk of pancreatic cancer. International Pancreatitis Study Group. N Engl J Med 1993; 328: [PMID: DOI: / NEJM ] 4 Farrell JJ. Diagnosing pancreatic malignancy in the setting of chronic pancreatitis: is there room for improvement? Gastrointest Endosc 2005; 62: [PMID: DOI: /j.gie ] 5 Pery C, Meurette G, Ansquer C, Frampas E, Regenet N. Role and limitations of 18F-FDG positron emission tomography (PET) in the management of patients with pancreatic lesions. Gastroenterol Clin Biol 2010; 34: [PMID: DOI: /j.gcb ] 6 Brand B, Pfaff T, Binmoeller KF, Sriram PV, Fritscher-Ravens A, Knöfel WT, Jäckle S, Soehendra N. Endoscopic ultrasound for differential diagnosis of focal pancreatic lesions, confirmed by surgery. Scand J Gastroenterol 2000; 35: [PMID: DOI: / ] 7 Brimienė V, Brimas G, Strupas K. Differential diagnosis between chronic pancreatitis and pancreatic cancer: a prospective study of 156 patients. Medicina (Kaunas) 2011; 47: [PMID: ] 8 Burski C, Varadarajulu S, Trevino J. Diagnosing cancer in chronic pancreatitis: The struggle persists. Gastrointest Endosc 2012; 75: AB193 [DOI: /j.gie ] 9 Varadarajulu S, Eltoum I, Tamhane A, Eloubeidi MA. Histopathologic correlates of noncalcific chronic pancreatitis by EUS: a prospective tissue characterization study. Gastrointest Endosc 2007; 66: [PMID: DOI: / j.gie ] 10 Adsay NV, Basturk O, Klimstra DS, Klöppel G. Pancreatic pseudotumors: non-neoplastic solid lesions of the pancreas that clinically mimic pancreas cancer. Semin Diagn Pathol 2004; 21: [PMID: DOI: /j.semdp ] 11 Feldman M, Friedman L, Brand L. Sleissenger and Fordtran s Gastrointestinal and Liver disease. 9th ed. Philadelphia: Saunders Elsevier, 2010: Barthet M, Portal I, Boujaoude J, Bernard JP, Sahel J. Endoscopic ultrasonographic diagnosis of pancreatic cancer complicating chronic pancreatitis. Endoscopy 1996; 28: [PMID: DOI: /s ] 13 Balthazar EJ. Pancreatitis associated with pancreatic carcinoma. Preoperative diagnosis: role of CT imaging in detection and evaluation. Pancreatology 2005; 5: [PMID: DOI: / ] 14 Vitone LJ, Greenhalf W, McFaul CD, Ghaneh P, Neoptolemos JP. The inherited genetics of pancreatic cancer and prospects for secondary screening. Best Pract Res Clin Gastroenterol 2006; 20: [PMID: DOI: /j.bpg ] 15 Howes N, Neoptolemos JP. Risk of pancreatic ductal adenocarcinoma in chronic pancreatitis. Gut 2002; 51: [PMID: DOI: /gut ] 16 Gemmel C, Eickhoff A, Helmstädter L, Riemann JF. Pancreatic cancer screening: state of the art. Expert Rev Gastroenterol Hepatol 2009; 3: [PMID: DOI: / ] 17 Harewood GC, Wiersema MJ. Endosonography-guided fine needle aspiration biopsy in the evaluation of pancreatic masses. Am J Gastroenterol 2002; 97: [PMID: DOI: /j x] 18 Giovannini M, Thomas B, Erwan B, Christian P, Fabrice C, Benjamin E, Geneviève M, Paolo A, Pierre D, Robert Y, Walter S, Hanz S, Carl S, Christoph D, Pierre E, Jean-Luc VL, Jacques D, Peter V, Andrian S. Endoscopic ultrasound elastography for evaluation of lymph nodes and pancreatic masses: a multicenter study. World J Gastroenterol 2009; 15: [PMID: DOI: /wjg ] 19 Hocke M, Schulze E, Gottschalk P, Topalidis T, Dietrich CF. Contrast-enhanced endoscopic ultrasound in discrimination between focal pancreatitis and pancreatic cancer. World J Gastroenterol 2006; 12: [PMID: ] P- Reviewers: Chao CT, El-Sayed M, Keck T, Luo HS, Zhou GX S- Editor: Wen LL L- Editor: A E- Editor: Liu XM 8616 July 14, 2014 Volume 20 Issue 26

309 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. RETROSPECTIVE BRIEF ARTICLE STUDY Need for pancreatic stenting after sphincterotomy in patients with difficult cannulation Kazunari Nakahara, Chiaki Okuse, Keigo Suetani, Yosuke Michikawa, Shinjiro Kobayashi, Takehito Otsubo, Fumio Itoh Kazunari Nakahara, Chiaki Okuse, Keigo Suetani, Yosuke Michikawa, Fumio Itoh, Department of Gastroenterology and Hepatology, St. Marianna University School of Medicine, Kawasaki , Japan Shinjiro Kobayashi, Takehito Otsubo, Department of Gastroenterological and General Surgery, St. Marianna University School of Medicine, Kawasaki , Japan Author contributions: Nakahara K and Okuse C contributed equally to this work; Nakahara K and Okuse C designed the report; Nakahara K, Suetani K, Michikawa Y, and Kobayashi S were attending doctors for the patients; Otsubo T and Itoh F organized the report; and Nakahara K wrote the paper. Correspondence to: Kazunari Nakahara, PhD, Department of Gastroenterology and Hepatology, St. Marianna University School of Medicine, , Sugao, Miyamae-ku, Kawasaki , Japan. nakahara@marianna-u.ac.jp Telephone: Fax: Received: January 11, 2014 Revised: March 10, 2014 Accepted: April 21, 2014 Published online: July 14, 2014 Abstract AIM: To investigate the need for pancreatic stenting after endoscopic sphincterotomy (EST) in patients with difficult biliary cannulation. METHODS: Between April 2008 and August 2013, 2136 patients underwent endoscopic retrograde cholangiopancreatography (ERCP)-related procedures. Among them, 55 patients with difficult biliary cannulation who underwent EST after bile duct cannulation using the pancreatic duct guidewire placement method (P-GW) were divided into two groups: a stent group (n = 24; pancreatic stent placed) and a no-stent group (n = 31; no pancreatic stenting). We retrospectively compared the two groups to examine the need for pancreatic stenting to prevent post-ercp pancreatitis (PEP) in patients undergoing EST after biliary cannulation by P-GW. RESULTS: No differences in patient characteristics or endoscopic procedures were observed between the two groups. The incidence of PEP was 4.2% (1/24) and 29.0% (9/31) in the Stent and no-stent groups, respectively, with the no-stent group having a significantly higher incidence (P = 0.031). The PEP severity was mild for all the patients in the stent group. In contrast, 8 had mild PEP and 1 had moderate PEP in the no-stent group. The mean serum amylase levels (means ± SD) 3 h after ERCP (183.1 ± vs ± IU/L, P = 0.006) and on the day after ERCP (209.5 ± vs ± IU/L, P = 0.002) were significantly higher in the no-stent group. A multivariate analysis identified the absence of pancreatic stenting (P = 0.045; odds ratio, 9.7; 95%CI: ) as a significant risk factor for PEP. CONCLUSION: In patients with difficult cannulation in whom the bile duct is cannulated using P-GW, a pancreatic stent should be placed even if EST has been performed Baishideng Publishing Group Inc. All rights reserved. Key words: Pancreatic stenting; Pancreatic guidewire placement; Post-endoscopic retrograde cholangiopancreatography pancreatitis; Endoscopic sphincterotomy; Endoscopic retrograde cholangiopancreatography Core tip: We retrospectively examined the need for pancreatic stenting after endoscopic sphincterotomy (EST) in patients with difficult biliary cannulation in whom the bile duct was cannulated using the pancreatic duct guidewire placement method (P-GW). The incidences of post-endoscopic retrograde cholangiopancreatography pancreatitis (PEP) were 4.2% and 29.0% in the Stent and no-stent groups, respectively, with the no-stent group having a significantly higher incidence (P = 0.031). A multivariate analysis identified the absence of pancreatic stenting as a significant risk factor 8617 July 14, 2014 Volume 20 Issue 26

310 Nakahara K et al. Pancreatic stenting in patients with EST for PEP. Therefore, in patients with difficult cannulation in whom the bile duct is cannulated using P-GW, a pancreatic stent should be placed even if EST has been performed. Nakahara K, Okuse C, Suetani K, Michikawa Y, Kobayashi S, Otsubo T, Itoh F. Need for pancreatic stenting after sphincterotomy in patients with difficult cannulation. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: org/ /wjg.v20.i INTRODUCTION While various causes of post-endoscopic retrograde cholangiopancreatography pancreatitis (PEP) have been noted, the most common cause is obstruction to the outflow of pancreatic juice due to edema of the papilla of Vater [1,2]. In recent years, many randomized controlled trials have shown the usefulness of pancreatic stenting for preventing the development of PEP [1-5]. Endoscopic sphincterotomy (EST) is also considered an effective procedure for preventing PEP because it enables the outflow of pancreatic juice [6-8]. Thus, it is still unclear whether there is an additional need for pancreatic stenting to prevent PEP in patients who undergo EST. Because the pancreatic duct orifice remains patent after EST, the placement of a pancreatic spontaneous dislodgement stent without flaps for the prevention of PEP often results in the premature dislodgement of the stent. Thus, in this study, we examined the additional need for pancreatic stenting to prevent PEP in patients undergoing EST. Patients with difficult biliary cannulation who underwent EST after selective biliary cannulation by the pancreatic duct guidewire placement method (P-GW) [9-11] were divided into two groups according to whether pancreatic stenting had occurred, and we compared the treatment outcomes and incidence of complications between the two groups. A multivariate analysis was performed to identify risk factors for the development of PEP in patients undergoing EST after biliary cannulation by P-GW. MATERIALS AND METHODS Patients Endoscopic retrograde cholangiopancreatography (ERCP)-related procedures were performed in 2136 cases at the Division of Gastroenterology and Hepatology of the St. Marianna University School of Medicine Hospital between April 2008 and August In our department, biliary cannulation is first attempted using the conventional contrast-assisted cannulation (CC) method. However, in cases where bile duct cannulation is difficult to perform using CC but in which a guidewire can be placed in the pancreatic duct, P-GW is employed as the procedure of first choice to achieve biliary cannulation. A second cannula was passed into the same working channel of the scope alongside the guidewire using the two-devices-in-one-channel method [12], and biliary cannulation was attempted. Of the 2136 patients who underwent ERCP-related procedures during the study period, 1454 had a native papilla. After 316 patients who did not undergo biliary cannulation or who had pancreatic diseases were excluded, 1138 patients remained. Biliary cannulation was achieved using CC in 977 of these patients. Then, among the 161 patients who experienced difficult biliary cannulation using CC, P-GW was attempted in 144, and successful biliary cannulation was achieved in 122 patients. Among these 122 patients with successful biliary cannulation using P-GW, EST was performed in 55, endoscopic papillary balloon dilation (EPBD) was performed in 18, and no papillary procedure was performed in 49 patients. EST was performed using an electrosurgical generator in the 120-Watt Endocut mode (ICC 200: ERBE Corp., Tuebingen, Germany). Following EST, a pancreatic duct stent was placed in 24 patients (stent group), whereas no pancreatic stenting was performed in the remaining 31 cases (no-stent group) (Figure 1). The pancreatic duct stents used were all 5-Fr, 3-cm-long spontaneous dislodgement stents with a single duodenal pigtail (Pit-stent, Gadelius Medical, Tokyo, Japan). Decisions regarding whether pancreatic stenting was necessary were left to the discretion of the endoscopist performing each procedure. All the procedures were supervised by a single expert, who performs approximately ERCPs per year. Because our hospital is an educational institution, trainees performed approximately half of the procedures. However, when deep cannulation of the bile duct was not achieved within 15 min, the expert took over the procedure. In all cases, 600 mg of gabexate mesilate was administered on the day of the procedure to prevent PEP. The patients serum amylase levels (normal range: IU/L) were measured prior to, 3 h after, and 1 d after the ERCP procedure. Measurements The stent group (n = 24), in which pancreatic stenting was performed after EST, and the no-stent group (n = 31), in which no pancreatic duct stent was placed after EST, were retrospectively compared in terms of patient characteristics (age, sex, primary disease, history of pancreatitis, and peripapillary diverticulum), endoscopic procedures (diameter of the pancreatic duct guidewire, range of EST incision, biliary maneuver, and procedure time), incidence of PEP, complication rate, and serum amylase levels. Moreover, univariate and multivariate logistic regression analyses were performed to identify the risk factors for PEP in patients undergoing EST after biliary cannulation by P-GW July 14, 2014 Volume 20 Issue 26

311 Nakahara K et al. Pancreatic stenting in patients with EST Total number of ERCPs (n = 2136) Patients with a native papilla of Vater (n = 1454) Patients with pancreatic disease (n = 316) Conventional contrast-assisted cannulation attempted (n = 1138) Success (n = 977) Failure (n = 161) P-GW attempted (n = 144) Success (n = 122) Failure (n = 22) EPBD (n = 18) EST (n = 55) Non EST/EPBD (n = 49) PD stenting (n = 18) No-PD stenting (n = 0) PD stenting (n = 24) No-PD stenting (n = 31) PD stenting (n = 48) No-PD stenting (n = 1) Figure 1 Flow chart showing the clinical courses of the patients. EST: Endoscopic sphincterotomy; P-GW: Pancreatic duct guidewire placement; ERCP: Endoscopic retrograde cholangiopancreatography; PD: Pancreatic duct; EPBD: Endoscopic papillary balloom dilation. Table 1 Comparison of patient characteristics and diagnoses between the stent and no-stent groups Stent group (n = 24) No-stent group (n = 31) P -value Age (mean ± SD) 70.8 ± ± Sex (male/female) 11/13 16/ History of pancreatitis 0 0 Periampullary diverticulum Choledocholithiasis Cholangiocarcinoma Acute cholecystitis Lymph node metastasis Gallbladder carcinoma Sphincter of Oddi dysfunction Liver metastasis The ranges of the EST incisions were defined as follows: an incision up to the hooding fold was considered a small incision, an incision up to the upper border of the oral protrusion was a large incision, and an incision with a range between those of small and large incisions was an intermediate incision. The diagnosis of pancreatitis and the determination of its severity were based on consensus guidelines proposed by Cotton et al [13]. Moreover, complications, such as bleeding, perforation, and cholangitis, were also diagnosed according to the consensus guidelines proposed by Cotton et al [13]. Statistical analysis The statistical analysis was performed using Fisher s exact test or Welch s t-test, as appropriate. To identify risk factors for post-ercp pancreatitis, variables found to be possibly significant (P < 0.2) by univariate analysis were entered into a multiple logistic regression model. P values < 0.05 were regarded as denoting significance. The statistical analysis was performed using the Prism 5 program (GraphPad Software, Inc., CA, United States) and SPSS (version 19; SPSS, Chicago, IL, United States). RESULTS Patient characteristics and endoscopic procedures No significant differences in age, sex, distribution of primary diseases, history of pancreatitis, or presence/ absence of peripapillary diverticulum were observed between the Stent and no-stent groups (Table 1). Moreover, the analysis of endoscopic procedure-related variables revealed no significant differences in the diameter of the pancreatic duct guidewire, range of EST incision, biliary maneuver, or procedure time between the Stent and no-stent groups (Table 2). Incidence of PEP The incidence of PEP was 4.2% (1/24) in the stent group and 29.0% (9/31) in the no-stent group, with the no-stent group having a significantly higher incidence (P = 0.031) (Table 3). The PEP severity was mild for all the patients in the stent group. In contrast, among the 9 PEP patients in the no-stent group, 8 had mild PEP and 1 had moderate PEP. Intermediate EST incisions were performed in all the patients in the stent group with PEP, whereas among the 9 patients in the no-stent group with PEP, 3 received small incisions, 5 received intermediate incisions, and 1 received a large incision. Conserva July 14, 2014 Volume 20 Issue 26

312 Nakahara K et al. Pancreatic stenting in patients with EST Table 2 Comparison of endoscopic procedures between the stent and no-stent groups 1500 No-stent group Stent group P = Stent group (n = 24) No-stent group (n = 31) P -value Pancreatic guidewire 12/12 16/ diameter (0.025 inch or inch) Incision range of EST Small Medium Large Endoscopic biliary stenting Bile duct stone removal Intraductal ultrasonography Endoscopic nasobiliary drainage Biopsy of the bile duct Cytology of the bile juice Endoscopic naso-gallbladder drainage Peroral cholangioscopy Procedure time (min, mean ± SD) 59.3 ± ± EST: Endoscopic sphincterotomy. Table 3 Comparison of complications between the stent and no-stent groups n (%) Stent group (n = 24) No-stent group (n = 31) P -value Overall complications 3 (12.5) 11 (35.5) Pancreatitis 1 (4.2) 9 (29.0) Bleeding 2 (8.3) 2 (6.5) Perforation 0 (0) 0 (0) Cholangitis 0 (0) 0 (0) AMY (IU/l) P = P = Before 3 h after 1 d after Figure 2 Comparison of serum amylase levels between the Stent and nostent groups. ference in the serum amylase levels before ERCP was observed between the Stent and no-stent groups (P = 0.238), the levels 3 h after and one day after ERCP were significantly higher in the no-stent group (P = and P = 0.002, respectively) (Figure 2). Risk factors for PEP The univariate analysis identified the absence of pancreatic duct stenting (P = 0.031; OR, 9.4; 95%CI: ) and the incidence of endoscopic nasobiliary drainage (P = 0.047; OR, 5.3; 95%CI: ) as significant risk factors for PEP (Table 4). The multivariate analysis identified the absence of pancreatic stenting (P = 0.045; OR, 9.7; 95%CI: ) as the only significant risk factor (Table 5). The precut [14] and P-GW techniques [9-11,15,16] have been employed for patients with difficult biliary cannulation. The precut technique is difficult to perform and is reported to be associated with a high incidence of complications, such as bleeding, perforation, and pancreatitis [17]. Therefore, we perform P-GW as a first choice for patients in whom a guidewire can be placed in the pancreatic duct. Previous studies of the efficacy of P-GW have reported varied results, with the success rate of biliary cannulation ranging from 43.8% to 92.6% [9-11,15,16]. However, the studies reporting low success rates (in the 40% range) for biliary cannulation also included patients for whom the placement of a guidewire in the pancreatic duct was unsuccessful; such patients are not considered suitable candidates for P-GW [15,16]. When such cases are excluded, the success rate of biliary cannulation using P-GW is high, ranging from 72.6% to 92.6% [10,11]. Another advantage of P-GW, if the procedure can be completed with the guidewire placed in the pancreatic duct, is the ease of placing a pancreatic stent at the end of the procedure. Difficult biliary cannulation is considered a procedure-related risk factor for PEP [18]. It is crititive therapy without additional endoscopic procedures resulted in improvement in all the patients. Overall incidence of complications With regard to complications other than PEP, bleeding was observed in 2 patients each in the stent group (2/24; 8.3%) and the no-stent group (2/31; 6.5%), with no significant difference in incidence between the two groups (P = 1.000). No other complications, such as perforation or cholangitis, were observed in either group. The overall complication rate, including PEP, was 12.5% (3/24) in the stent group and 35.5% (11/31) in the no-stent group. Although the difference was not significant, the rate tended to be higher in the no-stent group (P = 0.067) (Table 3). Serum amylase levels The serum amylase levels (means ± SD) in the stent group were 118 ± IU/L before ERCP, ± IU/L 3 h after ERCP, and ± IU/L one day after ERCP; the corresponding values in the nostent group were 85.5 ± 56.0, ± and ± IU/L, respectively. Although no significant dif- DISCUSSION 8620 July 14, 2014 Volume 20 Issue 26

313 Nakahara K et al. Pancreatic stenting in patients with EST Table 4 Risk factors for post-endoscopic retrograde cholangiopancreatography pancreatitis in patients undergoing endoscopic sphincterotomy after biliary cannulation by pancreatic duct guidewire placement Univariate analysis Pancreatitis (+) (n = 10) Pancreatitis (-) (n = 45) P -value OR (95%CI) Age (< 60 yr) ( ) Female gender ( ) Periampullary diverticulum ( ) Pancreatic guidewire (0.035 inch) ( ) EST incision range (small) ( ) No pancreatic duct stenting (1.1-81) Endoscopic biliary stenting ( ) Bile duct stone removal ( ) Intraductal ultrasonography ( ) Endoscopic nasobiliary drainage (1.1-26) Biopsy of the bile duct ( ) Cytology of the bile juice ( ) Endoscopic naso-gallbladder drainage ( ) Peroral cholangioscopy ( ) Procedure time (> 60 min) ( ) EST: Endoscopic sphincterotomy. Table 5 Risk factors for post-endoscopic retrograde cholangiopancreatography pancreatitis in patients undergoing endoscopic sphincterotomy after biliary cannulation by pancreatic duct guidewire placement Multivariate analysis P -value OR (95%CI) Incision range of EST ( small) ( ) No pancreatic duct stenting (1.1-90) Endoscopic nasobiliary drainage ( ) EST: Endoscopic sphincterotomy. cal to conduct additional research into the prevention of PEP in patients with difficult biliary cannulation who require P-GW. In recent years, a number of randomized controlled trials have demonstrated the usefulness of pancreatic stenting for the prevention of PEP [1-5]. Ito et al [4] reported the usefulness of pancreatic stenting for preventing PEP in patients with difficult biliary cannulation for whom bile duct cannulation was achieved using P-GW. According to their results, the success rate of pancreatic stenting after biliary cannulation with P-GW was 92.9% (26/28 patients), and the 22.9% incidence of PEP in the group without pancreatic stent placement was significantly higher than the 2.9% incidence in the group that underwent pancreatic stent placement (relative risk, 0.13; 95%CI: ). Thus, in addition to improving the success rate of biliary cannulation in patients with difficult biliary cannulation, the use of P-GW results in a higher success rate of pancreatic stenting and facilitates the prevention of PEP. EST is also considered effective for the prevention of PEP because EST reduces the obstruction of the pancreatic juice outflow [6-8]. However, it remained unclear whether additional pancreatic stenting might also be necessary for preventing PEP in patients undergoing EST. Because the pancreatic duct remains patent after EST, the placement of a pancreatic spontaneous dislodgement stent without flaps to prevent PEP often results in the premature dislodgement of the stent. There have also been concerns that the stents may not be sufficiently effective. However, in our study, the incidence of PEP in the no-stent group was 29.0%, which was significantly higher than the 4.2% incidence in the stent group. The serum amylase levels after ERCP were also significantly higher in the no-stent group compared with the stent group. The multivariate analysis identified the absence of pancreatic duct stenting as the only significant risk factor for PEP. Thus, these findings support that in patients with difficult biliary cannulation for whom P-GW is used to achieve bile duct cannulation, a pancreatic duct stent should be placed to prevent PEP even if EST has been performed. The need for pancreatic stenting after EST is presumed to be due to the insufficient opening of the pancreatic duct orifice by EST and the potential obstruction of the free outflow of pancreatic juice by the thermocoagulation degeneration of the pancreatic duct orifice. Furthermore, because pancreatography was performed in all of the patients in this study, the increased internal pressure of the pancreatic duct compared with the opening of the pancreatic duct orifice might have influenced the results. Additionally, this study included patients with difficult cannulation in whom edema of the papilla of Vater may have spread extensively. Theoretically, creating a large EST incision by applying electric discharges for a brief period of time will open the pancreatic duct without causing electrosurgical current-induced edema, thereby preventing PEP. However, according to the results of this study, PEP occurred in only one patient in the no-stent group, who received a large incision. Moreover, neither the univariate nor the multivariate analysis identified a small EST incision as a risk factor for PEP. Thus, the association between the 8621 July 14, 2014 Volume 20 Issue 26

314 Nakahara K et al. Pancreatic stenting in patients with EST range of the incision and the incidence of PEP remains unclear. However, because PEP occurred in 3 of the 4 patients in the no-stent group who received a small EST incision, a small incision cannot be excluded as a risk factor for PEP. Further studies with larger sample sizes would be needed to clarify this issue. In conclusion, in patients with difficult biliary cannulation who undergo successful biliary cannulation using P-GW, it appears that a pancreatic duct stent should be placed to prevent PEP even if EST is performed. However, because of the limitations of our study, including the small sample size and the retrospective design, further prospective studies using larger sample sizes will be needed to confirm our findings. COMMENTS Background The pancreatic duct guidewire placement method is reported to be effective in patients with difficult biliary cannulation. However, difficult biliary cannulation is associated with a high incidence of post-endoscopic retrograde cholangiopancreatography pancreatitis (PEP). Research frontiers The efficacy of pancreatic stenting to prevent PEP in patients with difficult cannulation has been reported. However, endoscopic sphincterotomy (EST) is also considered effective for preventing PEP because it facilitates the outflow of pancreatic juice. Innovations and breakthroughs The authors examined the incidence of PEP in patients with difficult cannulation for whom the bile duct was cannulated using P-GW with or without pancreatic stenting. The incidence of PEP was 4.2% and 29.0% in the stent and no-stent groups, respectively, with the incidence in the no-stent group being significantly higher. A multivariate analysis identified the absence of pancreatic stenting as a significant risk factor for PEP. Applications In patients with difficult cannulation for whom the bile duct is cannulated using P-GW, a pancreatic stent should be placed even if EST has been performed. Peer review The authors report the incidence of post-endoscopic retrograde cholangiopancreatography (ERCP)-pancreatitis in patients with difficult ERCP undergoing the pancreatic duct guidewire placement method of biliary cannulation with or without a PD stent. This is an interesting study. REFERENCES 1 Tarnasky PR, Palesch YY, Cunningham JT, Mauldin PD, Cotton PB, Hawes RH. Pancreatic stenting prevents pancreatitis after biliary sphincterotomy in patients with sphincter of Oddi dysfunction. Gastroenterology 1998; 115: [PMID: DOI: /S (98) ] 2 Fazel A, Quadri A, Catalano MF, Meyerson SM, Geenen JE. Does a pancreatic duct stent prevent post-ercp pancreatitis? A prospective randomized study. Gastrointest Endosc 2003; 57: [PMID: DOI: /mge ] 3 Sofuni A, Maguchi H, Itoi T, Katanuma A, Hisai H, Niido T, Toyota M, Fujii T, Harada Y, Takada T. Prophylaxis of postendoscopic retrograde cholangiopancreatography pancreatitis by an endoscopic pancreatic spontaneous dislodgement stent. Clin Gastroenterol Hepatol 2007; 5: [PMID: DOI: /j.cgh ] 4 Ito K, Fujita N, Noda Y, Kobayashi G, Obana T, Horaguchi J, Takasawa O, Koshita S, Kanno Y, Ogawa T. Can pancreatic duct stenting prevent post-ercp pancreatitis in patients who undergo pancreatic duct guidewire placement for achieving selective biliary cannulation? A prospective randomized controlled trial. J Gastroenterol 2010; 45: [PMID: DOI: /s ] 5 Kawaguchi Y, Ogawa M, Omata F, Ito H, Shimosegawa T, Mine T. Randomized controlled trial of pancreatic stenting to prevent pancreatitis after endoscopic retrograde cholangiopancreatography. World J Gastroenterol 2012; 18: [PMID: DOI: /wjg.v18.i ] 6 Simmons DT, Petersen BT, Gostout CJ, Levy MJ, Topazian MD, Baron TH. Risk of pancreatitis following endoscopically placed large-bore plastic biliary stents with and without biliary sphincterotomy for management of postoperative bile leaks. Surg Endosc 2008; 22: [PMID: DOI: /s ] 7 Nakai Y, Isayama H, Togawa O, Kogure H, Tsujino T, Yagioka H, Yashima Y, Sasaki T, Ito Y, Matsubara S, Hirano K, Sasahira N, Toda N, Tada M, Kawabe T, Omata M, Koike K. New method of covered wallstents for distal malignant biliary obstruction to reduce early stent-related complications based on characteristics. Dig Endosc 2011; 23: [PMID: DOI: /j x] 8 Jeong YW, Shin KD, Kim SH, Kim IH, Kim SW, Lee KA, Jeon BJ, Lee SO. [The safety assessment of percutaneous transhepatic transpapillary stent insertion in malignant obstructive jaundice: regarding the risk of pancreatitis and the effect of preliminary endoscopic sphincterotomy]. Korean J Gastroenterol 2009; 54: [PMID: DOI: /kjg ] 9 Dumonceau JM, Devière J, Cremer M. A new method of achieving deep cannulation of the common bile duct during endoscopic retrograde cholangiopancreatography. Endoscopy 1998; 30: S80 [PMID: DOI: /s ] 10 Maeda S, Hayashi H, Hosokawa O, Dohden K, Hattori M, Morita M, Kidani E, Ibe N, Tatsumi S. Prospective randomized pilot trial of selective biliary cannulation using pancreatic guide-wire placement. Endoscopy 2003; 35: [PMID: DOI: /s ] 11 Ito K, Fujita N, Noda Y, Kobayashi G, Obana T, Horaguchi J, Takasawa O, Koshita S, Kanno Y. Pancreatic guidewire placement for achieving selective biliary cannulation during endoscopic retrograde cholangio-pancreatography. World J Gastroenterol 2008; 14: ; discussion 5599 [PMID: DOI: /wjg ] 12 Fujita N, Noda Y, Kobayashi G, Kimura K, Yago A. ERCP for intradiverticular papilla: two-devices-in-one-channel method. Endoscopic Retrograde Cholangiopancreatography. Gastrointest Endosc 1998; 48: [PMID: DOI: /S (98) ] 13 Cotton PB, Lehman G, Vennes J, Geenen JE, Russell RC, Meyers WC, Liguory C, Nickl N. Endoscopic sphincterotomy complications and their management: an attempt at consensus. Gastrointest Endosc 1991; 37: [PMID: DOI: /S (91) ] 14 Huibregtse K, Katon RM, Tytgat GN. Precut papillotomy via fine-needle knife papillotome: a safe and effective technique. Gastrointest Endosc 1986; 32: [PMID: DOI: /S (86) ] 15 Xinopoulos D, Bassioukas SP, Kypreos D, Korkolis D, Scorilas A, Mavridis K, Dimitroulopoulos D, Paraskevas E. Pancreatic duct guidewire placement for biliary cannulation in a single-session therapeutic ERCP. World J Gastroenterol 2011; 17: [PMID: DOI: /wjg.v17. i ] 16 Herreros de Tejada A, Calleja JL, Díaz G, Pertejo V, Espinel J, Cacho G, Jiménez J, Millán I, García F, Abreu L. Doubleguidewire technique for difficult bile duct cannulation: a multicenter randomized, controlled trial. 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315 Nakahara K et al. Pancreatic stenting in patients with EST ME, Dorsher PJ, Moore JP, Fennerty MB, Ryan ME, Shaw MJ, Lande JD, Pheley AM. Complications of endoscopic biliary sphincterotomy. N Engl J Med 1996; 335: [PMID: DOI: /NEJM ] 18 Freeman ML, DiSario JA, Nelson DB, Fennerty MB, Lee JG, Bjorkman DJ, Overby CS, Aas J, Ryan ME, Bochna GS, Shaw MJ, Snady HW, Erickson RV, Moore JP, Roel JP. Risk factors for post-ercp pancreatitis: a prospective, multicenter study. Gastrointest Endosc 2001; 54: [PMID: DOI: /mge ] P- Reviewers: Shehata MMM, Sofi A S- Editor: Gou SX L- Editor: A E- Editor: Zhang DN 8623 July 14, 2014 Volume 20 Issue 26

316 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. RETROSPECTIVE STUDY Selection of appropriate endoscopic therapies for duodenal tumors: An open-label study, single-center experience Satohiro Matsumoto, Yukio Yoshida Satohiro Matsumoto, Yukio Yoshida, Department of Gastroenterology, Saitama Medical Center, Jichi Medical University, Saitama , Japan Author contributions: Matsumoto S conceived and designed the experiments, performed the experiments and analyzed the data; Matsumoto S contributed reagents/materials/analysis tools, wrote the paper; Matsumoto S and Yoshida Y approved the final manuscript. Correspondence to: Satohiro Matsumoto, MD, PhD, Department of Gastroenterology, Saitama Medical Center, Jichi Medical University, Amanuma, Omiya, Saitama , Japan. s.w.himananon@ac.auone-net.jp Telephone: Fax: Received: January 23, 2014 Revised: March 11, 2014 Accepted: April 15, 2014 Published online: July 14, 2014 ± 6.7 mm (4-25 mm); en bloc resection rate, 86.7% vs 83.9%; complete resection rate, 86.7% vs 74.2%; procedure time, 86.5 ± 63.1 min ( min) vs 13.2 ± 17.0 min (2-89 min) (P < ); intraprocedural perforation, 3 cases vs none (P = ); delayed perforation, none in either group; postprocedural bleeding, 1 case vs none; mean postoperative length of hospitalization, 8.2 ± 2.9 d (5-16 d) vs 6.1 ± 2.0 d (2-12 d) (P = ); recurrence, none vs 1 case (occurring at 7 mo postoperatively). CONCLUSION: ESD was associated with a longer procedure time and a higher incidence of intraprocedural perforation; EMR was associated with a lower rate of complete resection Baishideng Publishing Group Inc. All rights reserved. Abstract AIM: To determine an appropriate compartmentalization of endoscopic submucosal dissection (ESD) or endoscopic mucosal resection (EMR) for duodenal tumors. METHODS: Forty-six duodenal lesions (excluding papillary lesions) from 44 patients with duodenal tumors treated endoscopically between 2005 and 2013 were divided into the ESD and EMR groups for retrospective comparison and analysis. RESULTS: The mean age was 65 ± 9 years (35-79 years). There were 24 lesions from men and 22 from women. The lesions consisted of 6 early cancers, 31 adenomas and 9 neuroendocrine tumors. Lesion location was the duodenal bulb in 15 cases and the descending part of the duodenum in 31 cases. The most common macroscopic morphology was elevated type in 21 cases (45.6%). Mean tumor diameter was 11.9 ± 9.7 mm (3-60 mm). Treatment procedure was ESD (15 cases) vs EMR (31 cases). The examined parameters in the ESD vs EMR groups were as follows: mean tumor diameter, 12.9 ± 14.3 mm (3-60 mm) vs 11.4 Key words: Duodenal tumor; Endoscopic submucosal dissection; Endoscopic mucosal resection; Cancer; Adenoma; Neuroendocrine tumor Core tip: Endoscopic treatment of duodenal lesions is associated with a high incidence of complications. In particular, duodenal endoscopic submucosal dissection (ESD) is technically difficult. Therefore, the indications for duodenal ESD are not yet to be established. This study aimed to determine an appropriate compartmentalization of duodenal ESD or endoscopic mucosal resection (EMR). ESD was associated with a longer procedure time and a higher incidence of intraprocedural perforation; EMR was associated with a lower rate of complete resection. For early duodenal cancer and neuroendocrine tumors, which require en bloc resection, ESD is preferable if en bloc resection by EMR is difficult, while EMR is sufficient for endoscopic treatment of adenomas. Matsumoto S, Yoshida Y. Selection of appropriate endoscopic therapies for duodenal tumors: An open-label study, single-center experience. World J Gastroenterol 2014; 20(26): July 14, 2014 Volume 20 Issue 26

317 Matsumoto S et al. Duodenal endoscopic therapies: ESD or EMR? Available from: URL: v20/i26/8624.htm DOI: i INTRODUCTION Endoscopic submucosal dissection (ESD) is widely recognized as a useful treatment procedure for early gastric cancer [1,2]. In recent years, the indications for ESD have been expanded to include esophageal and colorectal cancer [3-5]. There have also been some reports of the application of ESD for duodenal tumors [6-9]. However, the technical difficulty of this procedure for lesions located in the duodenum is extremely high, because of the poor operability of endoscopes in this location, the thin duodenal wall, high degree of fibrillization of the submucosal layer. The risk of accidental complications such as delayed bleeding and perforation due to exposure to bile and duodenal juice is also high. Therefore, the indications for ESD remain controversial [1]. There have been no reports of comparison between ESD and endoscopic mucosal resection (EMR) for the treatment of duodenal tumors. In this study, we retrospectively compared and analyzed the data of patients with duodenal tumors, excluding papillary lesions, treated by ESD or EMR. MATERIALS AND METHODS Study population This study included 46 duodenal lesions (excluding papillary lesions) from 44 patients who underwent endoscopic treatment for duodenal tumors at the Saitama Medical Center between 2005 and Clinical background, including tumor size, location, macroscopic type and histology, was analyzed in all the cases. The duodenal lesions were then divided into two groups, the ESD and EMR groups, for retrospective comparison and analysis of the treatment outcomes, including the procedure time, en bloc resection rate, complete resection rate, complication rate and postoperative length of hospitalization, as well as the clinical background. Patient selection criteria We discussed the choice of therapy between ESD and EMR at the preoperative conference. We usually choose EMR for duodenal adenomas. On the other hand, we choose ESD for (1) well-differentiated intramucosal carcinoma; (2) neuroendocrine tumors (NET) measuring less than 1 cm diameter and invading deeper than submucosal layer; and (3) adenomas suspected of being cancerous in endoscopic findings, which are unlikely to be amenable to en bloc resection by EMR. We made our definitive decision in consideration of the background factors of the patients in addition to the above. Histopathological evaluation All patients were examined by gastrointestinal endoscopy and biopsy prior to the endoscopic treatment. The diagnoses of all the duodenal lesions were confirmed by histopathologic examination. Histologically, complete resection was defined as the absence of tumor cells at the resection margin of the specimen plus endoscopic en-bloc resection. EMR procedure We usually used a single-channel upper GI endoscope with a water-jet system, the GFI-Q260J (Olympus, Japan). For the treatment of lesions located near the duodenal papilla, the duodenal endoscope TJF-260V (Olympus) was used. Physiological saline was used for local injection into the submucosal layer, and a Snare Master (Olympus) and a captivator (Boston Scientific, Japan) were used for the resection of the lesions (Figure 1). ESD procedure The ESD procedures were carried out by operators who had at least 5 years of experience in performing ESD. The GIF-Q260J (Olympus) endoscope was used. Hyaluronic acid solution is essential as the submucosal injection solution; a 1% sodium hyaluronate solution (Suvenyl; Chugai Pharmaceutical, Japan) (used until 2007) or 0.4% sodium hyaluronate solution for submucosal injection (Mucoup; Johnson and Johnson, Japan) (used from 2008) was mixed in 10% glycerin containing 5% fructose and 0.9% NaCl (Glyceol; Chugai Pharmaceutical). A mucosal incision around the lesion and submucosal dissection for complete removal of the lesion were performed using the Flex knife (Olympus) or Dual knife (Olympus). The Hook knife (Olympus) was also used, particularly in cases where dissection of the submucosa was difficult. A high-frequency generator (VIO 300D; ERBE, Germany) was used during incision of the mucosa. We have used carbon dioxide insufflation during the ESD since 2009 (Figure 2). Statistical analysis Data are expressed as mean ± SD or percentage. Statistical analysis was performed using the Student s t-test and Fisher s exact test. The macroscopic type, histology, and complications were compared by the χ 2 test. All the data analyses were performed using the StatView software (version 5.0, SAS Institute Inc., United States). Differences at P values of less than 0.05 were regarded as significant. RESULTS The mean age of the patients was 65 ± 9 years (range: years). There were 24 lesions from men and 22 from women. The 46 duodenal tumors included 6 early cancers (13.0%), 31 adenomas (67.4%) and 9 NETs 8625 July 14, 2014 Volume 20 Issue 26

318 Matsumoto S et al. Duodenal endoscopic therapies: ESD or EMR? A B C D Figure 1 Endoscopic mucosal resection of a duodenal cancer. A: A protruded-type tumor 2.5 cm 2.5 cm in size was identified; B: We performed a submucosal injection; C: The tumor was grasped with a snare; D: Complete mucosal resection was finished. A B C D Figure 2 Endoscopic submucosal dissection of a duodenal adenoma. A: A depressed type tumor 1.5 cm 1.5 cm in size was identified; B: We performed a submucosal injection. The lifting of the lesion after submucosal injection was poor; C: Then, submucosal dissection was done; D: Complete submucosal dissection was finished July 14, 2014 Volume 20 Issue 26

319 Matsumoto S et al. Duodenal endoscopic therapies: ESD or EMR? Table 1 Clinical characteristics of 15 endoscopic submucosal dissection and 31 endoscopic mucosal resection performed in 44 patients n (%) Total ESD (n = 15) EMR (n = 31) P value Age, yr, mean ± SD (range) 65 ± 9 (35-79) 68 ± 5 (57-79) 64 ± 10 (35-77) Male/female 24/22 6/9 18/ Location in duodenum Bulb 15 (32.6) 8 (53.3) 7 (22.6) Second portion 31 (67.4) 7 (46.7) 24 (77.4) Size, mm, mean ± SD (range) 11.9 ± 9.7 (3-60) 12.9 ± 14.3 (3-60) 11.4 ± 6.7 (4-25) mm 8 (17.4) 2 (13.3) 6 (19.4) < 20 mm 38 (82.6) 13 (86.7) 25 (80.6) Macroscopic type Protruded 9 (19.6) 0 9 (29.0) Elevated 21 (45.6) 4 (26.7) 17 (54.8) Depressed 7 (15.2) 4 (26.7) 3 (9.7) SMT 9 (19.6) 7 (46.6) 2 (6.5) Histology Adenocarcinoma 6 (13.0) 2 (13.3) 4 (12.9) Adenoma 31 (67.4) 6 (40.0) 25 (80.6) NET 9 (19.6) 7 (46.7) 2 (6.5) ESD: Endoscopic submucosal dissection; EMR: Endoscopic mucosal resection; SMT: Submucosal tumor; NET: Neuroendocrine tumor. (19.6%). The lesions were located in the duodenal bulb in 15 cases (32.6%) and the descending part of the duodenum in 31 cases (67.4%). The most common macroscopic morphology was the elevated type in 21 cases (45.6%), followed by the protruded-type and submucosal tumor (SMT) in 9 cases each (19.6%). The mean tumor diameter was 11.9 ± 9.7 mm (3-60 mm). As for the treatment procedure, ESD was performed in 15 cases, and EMR in 31 cases. The mean tumor diameter was 12.9 ± 14.3 mm (3-60 mm) in the ESD group and 11.4 ± 6.7 mm (4-25 mm) in the EMR group. There were many cases of depressed-type and SMT lesions in the ESD group, while all the protruded-type lesions were included in the EMR group. According to the tumor type, NET accounted for a half of the cases in the ESD group, while the most common tumor type in the EMR group was adenoma, accounting for 80.6% of the tumors in this group. The proportion of cases of adenocarcinoma was similar between the two groups, although the preoperative pathological diagnosis was adenoma in 3 of 4 cases of adenocarcinoma in the EMR group (Table 1). The en bloc resection rate was 86.7% in the ESD group and 83.9% in the EMR group, and the complete resection rate was 86.7% in the ESD group and 74.2% in the EMR group. En bloc resection of the lesions was not achieved in 2 cases of the ESD group and 5 cases of the EMR group. To describe in further detail, the 2 cases of the ESD group in which en bloc resection was not achieved included one elevated-type adenoma measuring 60 mm in diameter and one NET measuring 8 mm in diameter showing invasion beyond the submucosal layer. Both the lesions were located in the duodenal bulb. For the latter case, treatment was discontinued because of the difficulty in detachment of the tumor. In the EMR group, there were a total of 7 cases in which en bloc resection was not achieved, consisting of 3 protruded-type lesions, 3 elevated-type lesions, and 1 depressed-type lesion. The cases in which complete resection was not achieved included the 2 above-described cases of the ESD group in which en bloc resection was not achieved, and 8 cases of the EMR group. Of these 8 cases from the EMR group, the lesion was located in the duodenal bulb in 4 cases and in the descending part of the duodenum in 4 cases; the macroscopic type was the protruded type in 5 cases, the elevated type in 2 cases, and the depressed type in 1 case. The procedure time was 86.5 ± 63.1 min (range: min) in the ESD group and 13.2 ± 17.0 min (range: 2-89 min) in the EMR group (P < ). As for complications, intraprocedural perforation occurred in 3 cases of the ESD group, but in none of the cases of the EMR group (P = ). The 3 cases of intraprocedural perforation included 2 cases of NET located in the duodenal bulb, and 1 case of early cancer of the depressed type located in the descending part of the duodenum. Delayed perforation was not observed in any of the cases in either group. Emergency surgery was necessitated in 2 cases of the ESD group, but not in any of the cases of the EMR group. The observed postprocedural bleeding was acute bleeding that occurred within 24 h of the procedure in 1 case of the ESD group. On the other hand, no postprocedural bleeding was observed in any of the cases of the EMR group. In the ESD group, closure of the wound with clips after the procedure was not performed in any of the 9 cases of lesions located in the duodenal bulb, but the wound was closed with clips in 4 of the 5 cases of lesions located in the descending part of the duodenum. In the remaining case where closure by clipping was not performed, postprocedural bleeding occurred on the day after the procedure. In the EMR group, the wound was closed with clips in 6 of the 7 cases of lesions located in the duodenal bulb and 18 of the 24 cases of lesions located in the descending part of the duodenum. The mean postoperative length of hospitalization was 8.2 ± 2.9 d (range: 5-16 d) in the ESD group and 6.1 ± 2.0 d (range: 2-12 d) in the EMR group (P = ) (Table 2) July 14, 2014 Volume 20 Issue 26

320 Matsumoto S et al. Duodenal endoscopic therapies: ESD or EMR? Table 2 Clinical outcomes of 15 endoscopic submucosal dissection and 31 endoscopic mucosal resection performed in 44 patients Total ESD (n = 15) EMR (n = 31) P value En bloc resection rate 84.8% 86.7% 83.9% > Complete resection rate 78.3% 86.7% 74.2% Procedure time, min, mean ± SD (range) 37.1 ± 51.4 (2-217) 86.5 ± 63.1 (15-217) 13.2 ± 17.0 (2-89) < Complication 6 (13.0%) 5 (35.7%) 1 (3.4%) Perioperative perforation Perioperative bleeding Postoperative bleeding Emergency surgery Postoperative hospital stay, day, mean ± SD (range) 6.8 ± 2.5 (2-16) 8.2 ± 2.9 (5-16) 6.1 ± 2.0 (2-12) As of December 2013, follow-up by endoscopy had been completed in 26 cases (56.5%). The mean followup period was 9.7 ± 12.6 mo (range: mo). Recurrence was observed at 7 mo after the procedure in only 1 case of the EMR group. DISCUSSION Endoscopic treatment of duodenal lesions is associated with a high incidence of complications, such as bleeding and perforation, because of poor operability using a scope and the thin duodenal wall. In particular, duodenal ESD is technically difficult, requires a longer procedure time, and is associated with a high risk of bleeding and perforation [6,9]. Therefore, the operator should be sufficiently skilled, with experience of safe and steady implementation of ESD at least for lesions of the stomach, esophagus, and large bowel. Indications for ESD in patients with duodenal tumors should be determined taking into consideration the histopathology, macroscopic morphology, and size of the lesions. Duodenal adenomas are well known to have the potential for malignant transformation [10,11]. In particular, adenomas measuring 2 cm or greater in diameter or those that histopathologically show highgrade dysplasia are more likely to become malignant [12-14] ; therefore, resection of such lesions is recommended. In a study that investigated 128 lesions of early duodenal cancer treated by surgery or endoscopic polypectomy, no lymph node metastasis was detected in any of the cases of intramucosal carcinoma [15]. Based on these findings, endoscopic treatment can be considered for differentiated non-invasive carcinomas not showing invasion of the submucosal layer. Although EMR has been reported to be safe and useful for the treatment of duodenal tumors [16-22], lesions greater than 2 cm in diameter are likely to require piecemeal resection [16,22]. We have previously reported the use of ESD for carcinoids, which are currently called NETs. EMR is adequate for lesions measuring less than 1 cm in diameter that are superficial, reaching only up to the submucosal layer, especially those with a polypoid morphology. However, ESD may be useful in cases where en bloc resection by EMR is difficult. Surgical treatment should be considered for tumors whose lower edges are found to be widely adjoining the muscular layer, because treatment of such tumors by ESD is associated with a high risk of perforation, and because accurate pathologic diagnosis of the deep margin may be difficult [7]. Although the present study did not reveal any significant differences between the EMR and ESD groups, both the en bloc and complete resection rates were lower in the EMR group. During EMR of lesions located in the duodenal bulb, the pyloric ring may become an obstacle to snaring. In the descending part of the duodenum, the distance between the mucosal folds is short, and there are relatively numerous lesions extending over the folds. Based on these observations, it may be difficult to ensure snaring of lesions located in the descending part of the duodenum by EMR. The rates of persistence and recurrence are higher in cases of piecemeal resection than in the case of en bloc resection [22]. Moreover, because en bloc resection allows accurate pathological evaluation of the deep and lateral resection margins [23], ESD may be preferable for endoscopic treatment of cancer and NET, which require en bloc resection, if en bloc resection by EMR is difficult. On the other hand, EMR may be sufficient for endoscopic treatment of adenomas, because piecemeal resection seems to be acceptable for these lesions. However, because 3 of the 4 cancers in the EMR group in this study had been diagnosed as adenomas preoperatively, caution is necessary, especially in cases of preoperative diagnosis of adenoma. Among the complications associated with endoscopic treatment for duodenal lesions, bleeding is the most frequent and usually occurs within 24 h after the procedure. The reported incidence of acute bleeding after EMR for adenomas is 4%-33% [16-22]. As for perforation, caution is required not only against intraprocedural perforation, but also against delayed perforation due to exposure to bile and pancreatic juice [6]. The wound should be closed by clipping after resection in order to prevent complications such as postprocedural bleeding and delayed perforation [9]. In this study, the frequency of perforation was significantly higher and the postoperative length of hospitalization was significantly longer in the ESD group than in the EMR group. Although delayed perforation was not observed in any of the cases, either in the ESD or in the EMR group, postprocedural bleeding was observed in 1 case of the ESD group, where closure of the wound with clips was not performed. In an effort to ensure safer treatment, we have been performing ESD 8628 July 14, 2014 Volume 20 Issue 26

321 Matsumoto S et al. Duodenal endoscopic therapies: ESD or EMR? for duodenal tumors under general anesthesia in the presence of a surgeon in an operating room since The limitations of this study were that it was a singleinstitution study and the sample size was small. Because of the retrospective design, ESD was often selected as the treatment procedure for lesions for which en bloc resection by EMR was expected to be difficult, such as depressed-type lesions and NET; inevitably therefore, there would have been a selection bias. In conclusion, we consider that duodenal ESD may be indicated for well-differentiated intramucosal carcinoma and NETs measuring less than 1 cm in diameter and not invading deeper than the submucosal layer lesions, which are unlikely to be amenable to en bloc resection by EMR. However, because duodenal ESD is associated with a relatively high incidence of complications, its use should be considered carefully. Accumulation of further clinical data is required for a clearer elucidation of the short-term and long-term prognoses. COMMENTS Background In recent years, the indications for endoscopic submucosal dissection (ESD) have been expanded to include esophageal and colorectal cancer. There have also been some reports of the application of ESD for duodenal tumors. However, there have been no reports of comparison between ESD and endoscopic mucosal resection (EMR) for the treatment of duodenal tumors. Research frontiers The technical difficulty of ESD for lesions located in the duodenum is extremely high, because of the poor operability of endoscopes in this location, the thin duodenal wall, high degree of fibrillization of the submucosal layer. The risk of accidental complications such as delayed bleeding and perforation due to exposure to bile and duodenal juice is also high. Therefore, the indications for ESD remain controversial. Innovations and breakthroughs ESD was associated with a longer procedure time and a higher incidence of intraprocedural perforation. EMR was associated with a lower rate of complete resection. For early duodenal cancer and neuroendocrine tumors (NET), which require en bloc resection, ESD is preferable if en bloc resection by EMR is difficult, while EMR is sufficient for endoscopic treatment of adenomas. Applications Duodenal ESD may be indicated for well-differentiated intramucosal carcinoma and NETs measuring less than 1 cm in diameter and not invading deeper than the submucosal layer lesions, which are unlikely to be amenable to en bloc resection by EMR. Terminology Gastrointestinal tract neuroendocrine cells sometimes go through certain changes that cause them to grow too much and form tumors. These tumors are known as NET; in the past, they were called carcinoids. But in 2000, the World Health Organization reclassified carcinoids as neuroendocrine tumors and neuroendocrine cancers. Peer review In this study authors aimed to determine an appropriate compartmentalization of ESD or EMR for duodenal tumors. Although the study group is relatively small, it is observed that ESD was associated with a longer procedure time and a higher incidence of perforation. On the other hand EMR was associated with a lower rate of complete resection. It is concluded that while ESD is preferable for early duodenal cancer, EMR is sufficient for endoscopic treatment of adenomas. This is basically well written paper of an interesting topic. REFERENCES 1 Kim KO, Kim SJ, Kim TH, Park JJ. Do you have what it takes for challenging endoscopic submucosal dissection cases? World J Gastroenterol 2011; 17: [PMID: DOI: /wjg.v17.i ] 2 Gotoda T, Yamamoto H, Soetikno RM. Endoscopic submucosal dissection of early gastric cancer. J Gastroenterol 2006; 41: [PMID: ] 3 Fujishiro M, Yahagi N, Kakushima N, Kodashima S, Muraki Y, Ono S, Yamamichi N, Tateishi A, Shimizu Y, Oka M, Ogura K, Kawabe T, Ichinose M, Omata M. Endoscopic submucosal dissection of esophageal squamous cell neoplasms. Clin Gastroenterol Hepatol 2006; 4: [PMID: ] 4 Kakushima N, Yahagi N, Fujishiro M, Kodashima S, Nakamura M, Omata M. Efficacy and safety of endoscopic submucosal dissection for tumors of the esophagogastric junction. Endoscopy 2006; 38: [PMID: ] 5 Yamamoto H, Yahagi N, Oyama T. Mucosectomy in the colon with endoscopic submucosal dissection. Endoscopy 2005; 37: [PMID: ] 6 Honda T, Yamamoto H, Osawa H, Yoshizawa M, Nakano H, Sunada K, Hanatsuka K, Sugano K. Endoscopic submucosal dissection for superficial duodenal neoplasms. Dig Endosc 2009; 21: [PMID: DOI: / j x] 7 Matsumoto S, Miyatani H, Yoshida Y, Nokubi M. Duodenal carcinoid tumors: 5 cases treated by endoscopic submucosal dissection. Gastrointest Endosc 2011; 74: [PMID: DOI: /j.gie ] 8 Suzuki S, Ishii N, Uemura M, Deshpande GA, Matsuda M, Iizuka Y, Fukuda K, Suzuki K, Fujita Y. Endoscopic submucosal dissection (ESD) for gastrointestinal carcinoid tumors. Surg Endosc 2012; 26: [PMID: DOI: /s y] 9 Matsumoto S, Miyatani H, Yoshida Y. Endoscopic submucosal dissection for duodenal tumors: a single-center experience. Endoscopy 2013; 45: [PMID: DOI: /s ] 10 Galandiuk S, Hermann RE, Jagelman DG, Fazio VW, Sivak MV. Villous tumors of the duodenum. Ann Surg 1988; 207: [PMID: ] 11 Miller JH, Gisvold JJ, Weiland LH, McIlrath DC. Upper gastrointestinal tract: villous tumors. AJR Am J Roentgenol 1980; 134: [PMID: ] 12 Rosen M, Zuccaro G, Brody F. Laparoscopic resection of a periampullary villous adenoma. Surg Endosc 2003; 17: [PMID: ] 13 Lépilliez V, Napoléon B, Ponchon T, Saurin JC. [Duodenal adenomas: diagnostic and treatment]. Gastroenterol Clin Biol 2009; 33: [PMID: DOI: / j.gcb ] 14 Okada K, Fujisaki J, Kasuga A, Omae M, Kubota M, Hirasawa T, Ishiyama A, Inamori M, Chino A, Yamamoto Y, Tsuchida T, Nakajima A, Hoshino E, Igarashi M. Sporadic nonampullary duodenal adenoma in the natural history of duodenal cancer: a study of follow-up surveillance. Am J Gastroenterol 2011; 106: [PMID: DOI: /ajg ] 15 Nagatani K, Takekoshi T, Baba Y, Kaku Y, Koizumi K, Fujii A. Indications for endoscopic treatment of early duodenal cancer: based on cases reported in the literature (in Japanese with English abstract). Endosc Dig 1993; 7: Kim HK, Chung WC, Lee BI, Cho YS. Efficacy and longterm outcome of endoscopic treatment of sporadic nonampullary duodenal adenoma. Gut Liver 2010; 4: [PMID: DOI: /gnl ] 17 Apel D, Jakobs R, Spiethoff A, Riemann JF. Follow-up after endoscopic snare resection of duodenal adenomas. Endoscopy 2005; 37: [PMID: ] 18 Hirasawa R, Iishi H, Tatsuta M, Ishiguro S. 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322 Matsumoto S et al. Duodenal endoscopic therapies: ESD or EMR? ] 19 Ahmad NA, Kochman ML, Long WB, Furth EE, Ginsberg GG. Efficacy, safety, and clinical outcomes of endoscopic mucosal resection: a study of 101 cases. Gastrointest Endosc 2002; 55: [PMID: ] 20 Oka S, Tanaka S, Nagata S, Hiyama T, Ito M, Kitadai Y, Yoshihara M, Haruma K, Chayama K. Clinicopathologic features and endoscopic resection of early primary nonampullary duodenal carcinoma. J Clin Gastroenterol 2003; 37: [PMID: ] 21 Lépilliez V, Chemaly M, Ponchon T, Napoleon B, Saurin JC. Endoscopic resection of sporadic duodenal adenomas: an efficient technique with a substantial risk of delayed bleeding. Endoscopy 2008; 40: [PMID: DOI: /s ] 22 Alexander S, Bourke MJ, Williams SJ, Bailey A, Co J. EMR of large, sessile, sporadic nonampullary duodenal adenomas: technical aspects and long-term outcome (with videos). Gastrointest Endosc 2009; 69: [PMID: DOI: /j.gie ] 23 Sohn JW, Jeon SW, Cho CM, Jung MK, Kim SK, Lee DS, Son HS, Chung IK. Endoscopic resection of duodenal neoplasms: a single-center study. Surg Endosc 2010; 24: [PMID: ] P- Reviewers: Arroyo A, Elpek GO, Hara K, Rolle U, Tang WF S- Editor: Qi Y L- Editor: A E- Editor: Liu XM 8630 July 14, 2014 Volume 20 Issue 26

323 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. RETROSPECTIVE STUDY Impact of tumor location on clinical outcomes of gastric endoscopic submucosal dissection Ji Young Yoon, Choong Nam Shim, Sook Hee Chung, Wan Park, Hyunsoo Chung, Hyuk Lee, Sung Kwan Shin, Sang Kil Lee, Yong Chan Lee, Jun Chul Park Ji Young Yoon, Choong Nam Shim, Sook Hee Chung, Wan Park, Hyunsoo Chung, Hyuk Lee, Sung Kwan Shin, Sang Kil Lee, Yong Chan Lee, Jun Chul Park, Department of Internal Medicine, Institute of Gastroenterology, Yonsei University College of Medicine, Seoul , South Korea Author contributions: Yoon JY and Shim CN contributed equally to this work; all the authors participated in this study. Supported by A Faculty Research Grant of Yonsei University College of Medicine for 2010, No Correspondence to: Jun Chul Park, MD, Assistant Professor, Department of Internal Medicine, Institute of Gastroenterology, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul , South Korea. junchul75@yuhs.ac Telephone: Fax: Received: November 5, 2013 Revised: February 13, 2014 Accepted: March 12, 2014 Published online: July 14, 2014 Abstract AIM: To determine whether there is a correlation between the location of the lesion and endoscopic submucosal dissection (ESD) outcome. METHODS: From January 2008 to December 2010, ESD of 1443 gastric tumors was performed. En bloc resection rate, complete resection rate, procedure time and complication rate were analyzed according to the tumor location. RESULTS: The rates of en bloc resection and complete resection were 91% (1318/1443) and 89% (1287/1443), respectively. The post-esd bleeding rate was 4.3%, and perforation rate was 2.7%. Tumors located in the upper third of the stomach were associated with a longer procedure time and significantly higher rates of incomplete resection, piecemeal resection, and perforation than tumors below the upper third of the stomach. Posterior wall lesions had significantly longer procedure times and higher rates of incomplete resection and piecemeal resection than lesions in other locations. In multivariate analysis, posterior wall lesions and upper third lesions were significantly associated with incomplete resection and perforation, respectively. In post-esd bleeding analysis, location was not a significant related factor. CONCLUSION: More advanced endoscopic techniques are required during ESD for lesions located in the upper third or posterior wall of the stomach to decrease complications and improve therapeutic outcomes Baishideng Publishing Group Inc. All rights reserved. Key words: Endoscopic submucosal dissection; Gastric neoplasm; Location; Complication; Outcomes Core tip: Location of the tumor is one of the most important clinical factors for complete resection and complications of endoscopic submucosal dissection (ESD) for early gastric cancer. Nonetheless, few studies have evaluated clinicopathologic outcomes of ESD according to the subdivision of tumor location. Based on our data, posterior wall lesions and upper third lesions were significantly associated with incomplete resection and perforation, respectively. Therefore, endoscopists should recognize the need for more advanced endoscopic techniques when performing ESD for lesions located in the upper third or posterior wall of the stomach to decrease the rate of serious complications and improve clinical outcomes. Yoon JY, Shim CN, Chung SH, Park W, Chung H, Lee H, Shin SK, Lee SK, Lee YC, Park JC. Impact of tumor location on clinical outcomes of gastric endoscopic submucosal dissection. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: July 14, 2014 Volume 20 Issue 26

324 Yoon JY et al. Impact of tumor location on ESD INTRODUCTION Advances in diagnostic technology and the increasing prevalence of screening programs have increased the rate of early gastric cancer (EGC) detection. EGCs that are confined to the mucosa and lack lymph node metastasis can be cured by endoscopic resection, such as endoscopic mucosal resection (EMR) or endoscopic submucosal dissection (ESD) [1-4]. Compared with EMR, ESD may achieve complete resection not only of larger lesions but also of ulcerative lesions. In addition, ESD allows for a precise histological assessment of resected specimens and may reduce the risk of residual disease and local recurrence [5]. The factors affecting successful ESD include several characteristics of lesions such as location, presence of ulceration and histology [6,7]. In a recent multicenter study, scarred lesions, undifferentiated lesions and lesions located in the upper third required more advanced ESD techniques, because the complete resection rate is lower with these lesions than with other lesions [7]. In addition, the location of the tumor is one of the most important clinical factors for whether or not complete resection is possible or whether or not complication occurs. However, few studies have evaluated clinicopathologic outcomes of ESD according to the subdivision of tumor location in the longitudinal portions of the stomach and cross-sectional circumference divided into four equal parts. Therefore, we conducted this study to evaluate the level of difficulty of the procedure and clinicopathologic outcomes according to the tumor location. MATERIALS AND METHODS Patients We analyzed 1319 patients with 1443 lesions who underwent ESD for gastric tumors at Yonsei University Health Care Center between January 2008 and December Endoscopy with standard upper gastrointestinal endoscopes (GIF Q260 and H260, Olympus, Japan), chromoendoscopy with indigo carmine, and biopsies of the lesions with standard biopsy forceps (FB-21K-1; Olympus, Japan) were initially performed to determine the feasibility of ESD. Endoscopic ultrasonography (EUS) with radial scanning echoendoscopes (EG-3679URK, Pentax, Japan and GF-UE260, Olympus, Japan) was performed in case of carcinoma to evaluate the depth of invasion. Patients with lesions confirmed to be gastric cancer underwent abdominal computed tomography (CT) scans to determine if lymph node or distant metastasis was present. For this study, the endoscopic findings of EGC were classified as elevated (types Ⅰ or Ⅱa), flat (type Ⅱb), depressed (types Ⅱc, Ⅱc+Ⅲ, or Ⅱa+Ⅱc) or mixed (types Ⅱa+Ⅱb, Ⅱb+Ⅱc, Ⅱa + Ⅱc, Ⅲ+Ⅱa or Ⅲ+Ⅱb). Gastric tumor lesions were classified according to their location in the upper, middle, and lower thirds of the stomach, and also to location in the cross-sectional circumference divided into four equal parts (anterior wall, posterior wall, lesser curvature or greater curvature). Eligibility criteria for ESD were as follows: (1) differentiated adenocarcinoma (well- to moderately-differentiated tubular adenocarcinoma) or dysplasia confirmed histologically by forceps biopsy; (2) depth of invasion limited to the mucosa or submucosa ( 500 μm penetration into the submucosa) as determined by EUS; (3) lesions without ulceration, regardless of size, or 30 mm or less in size with ulceration; or (4) undifferentiated adenocarcinoma or intramucosal cancer without ulcer findings 20 mm in size [8]. Endoscopic resection All procedures were performed by an attending gastroenterologist, and five attending physicians were involved in the procedures. All ESDs were performed under conscious sedation using intravenous propofol or midazolam. Vital signs were continuously monitored during the procedure. After identifying the target lesion, marking dots were made circumferentially about 5 mm lateral to the margin of the lesion using a needle knife (KD-10Q, Olympus, Japan) or argon plasma coagulation (ERBE Elektromedizin, Germany). Epinephrine (1:10000 dilution) was then injected submucosally around the lesion, and an initial short incision was made in the mucosa with a needle knife to allow submucosal insertion of the tip of an insulation-tipped (IT) knife (KD-611L, Olympus, Japan). Circumferential mucosal cutting was performed outside the marking dots, and an additional submucosal injection was carried out. Finally, direct dissection of the submucosal layer was performed, and endoscopic hemostasis with specialized hemostatic forceps (FD-410LR, Olympus, Japan) was performed when needed. Histologic evaluation of resection efficacy All resected specimens were systematically sectioned at 2 mm intervals centered on the part of the lesion closet to the margin and the site of the deepest invasion. Histological assessment was based on the Vienna classification [9]. Final pathologic diagnoses were classified as low grade dysplasia (LGD), high grade dysplasia (HGD), differentiated EGC, and undifferentiated EGC. Outcome measures Patient data, including patient age, gender, previous medication history, the size, number and location of lesions, procedure start and end times, endoscopic findings, pathology, and complications, were collected. Complete resection of en bloc resected tumors was defined as the lateral and vertical margins being free of tumor cells on histologic examination. Complete resection of tumors resected in a piecemeal fashion was defined as complete removal of the entire lesion, including sufficient tumor-free margins after perfect reconstruction of all pieces. Procedure time was defined as the time from marking to complete removal, including the time required 8632 July 14, 2014 Volume 20 Issue 26

325 Yoon JY et al. Impact of tumor location on ESD Table 1 Baseline characteristics of gastric tumors n (%) Characteristic Value Gender (female:male) 1:2.18 (454:989) Age, yr, mean ± SD 63.0 ± 9.4 Tumor size, mm < (71.7) (28.3) Macroscopic appearance Elevated 310 (21.5) Flat 128 (8.9) Depressed 90 (6.2) Mixed 915 (63.4) Ulcer 71 (4.9) Invasion depth Mucosa 612 (42.4) Submucosa 106 (7.3) LocationⅠ Lower third 1233 (85.4) Middle third 112 (7.8) Upper third 98 (6.8) Location Ⅱ Anterior wall 294 (20.4) Lesser curvature 481 (33.3) Posterior wall 405 (28.1) Greater curvature 263 (18.2) Histology Low grade dysplasia 534 (37.0) High grade dysplasia 176 (12.2) Differentiated EGC 655 (45.4) Undifferentiated EGC 78 (5.4) Procedure time, min 61.8 (± 47.0) Curability Complete resection 1287 (89.0) Incomplete resection 156 (11.0) Resectability En bloc 1318 (91.3) Piecemeal 125 (8.7) Perforation 39 (2.7) Post-ESD bleeding 63 (4.3) EGC: Early gastric cancer; ESD: Endoscopic submucosal dissection. for hemostasis. Complication data included whether a complication occurred and details regarding bleeding, perforation and other factors related to the type of complication. Clinicopathologic evaluation To identify factors affecting the success of ESD, we analyzed lesion characteristics, procedure, and the procedure result. Analyzed lesion characteristics included the presence of ulceration, macroscopic morphology, size and location of the tumor. Procedure results were analyzed for curability. Resection was deemed complete when removal was achieved with tumor-free lateral and vertical margins and there was no lymphovascular involvement or lymph node metastasis. Incomplete resection was defined as any resection that did not meet the curative criteria described above. Follow-up Endoscopic surveillance by esophagogastroduodenoscopy (EGD) was performed 3, 6, 12, and 24 mo after ESD for EGC to exclude local recurrence, as well as synchronous, and metachronous lesions. After 24 mo, EGD was carried out annually. Moreover, abdominal CT scans were performed every 6 mo for the first year and annually thereafter, to detect lymph node or distant metastasis. In cases with adenomas, endoscopic surveillance by EGD was scheduled for 3, 12, and 24 mo after ESD. Statistical analysis The data were analyzed using Pearson s χ 2 test, unpaired t-test, Fisher s exact test, and the Mann-Whitney U test. P values < 0.05 were considered significant. To identify related risk factors for complications and complete resection, predictors with P values < 0.05 in the univariate analysis were included in a backward, stepwise multiple logistic regression model. All data analyses were conducted using a statistical software package (SPSS version 18.0, Chicago, IL, United States). RESULTS Gastric tumor characteristics During the study period, ESD was performed in 1319 patients with 1443 gastric tumors. Baseline clinicopathologic characteristics of the gastric tumors and the clinical outcomes of ESD are shown in Table 1. Mean age was 63.0 ± 9.4 years. The lesions consisted of 733 (50.8%) EGCs and 710 (49.2%) dysplastic lesions. Submucosal invasion occurred in 7.3% of cases. Mixed-type endoscopic morphology was the most common (63.4%). With respect to size and location, tumors less than 20 mm in size (71.7%), those located in the lower third (85.4%) and those located in the lesser curvature (33.3%) were most common. The mean tumor size was ± 8.81 mm. The mean procedure time was 61.8 ± 47.0 min. The complete resection rate was 89% (1287/1443), and the en bloc resection rate was 91.3% (Table 1). The post-esd bleeding rate was 4.3%, and the perforation rate was 2.7%. Most cases of bleeding (60/63) were treated by endoscopic hemostasis such as hemoclipping, argon plasma coagulation or epinephrine injection. Two cases were treated by angiographic embolization. Only one case required surgery for bleeding control. Around half of all perforation cases (20/39) were minute or micro-perforations, while the remaining ones were overt perforations. Only two such cases required surgery. All other cases were treated by conservative care. There was no mortality in the present study. Endoscopic outcomes according to the location We compared the clinical outcomes of ESD in relation to detailed tumor location. Upon division into upper third and other lesions, the upper third lesion group had significantly higher percentages of incomplete resections (19.4% vs 10.2%, P = 0.005) and piecemeal resections (15.3% vs 8.2%, P = 0.015) compared with other tumor locations. Additionally, upper third lesions required a longer procedure time (90.51 min vs min, P < 0.001) and were associated with a higher perforation rate (9.2% 8633 July 14, 2014 Volume 20 Issue 26

326 Yoon JY et al. Impact of tumor location on ESD Table 2 Comparison of the upper third and non-upper third groups n (%) Upper third Non-upper third P value Tumor size, mm < (71.4) 964 (71.7) (28.6) 381 (28.3) Ulcer 2 (2.0) 69 (5.1) Pathology Dysplasia 45 (45.9) 665 (49.4) Carcinoma 53 (54.1) 680 (50.6) Curability Incomplete resection 19 (19.4) 137 (10.2) Complete resection 79 (80.6) 1208 (89.8) Resectability En bloc 83 (84.7) 1235 (91.8) Piecemeal 15 (15.3) 110 (8.2) Procedure time, min, mean ± SD ± ± < Perforation 9 (9.2) 30 (2.2) < Post-ESD bleeding 4 (4.1) 57 (4.2) ESD: Endoscopic submucosal dissection. Table 3 Comparison of the posterior wall and non-posterior wall groups n (%) Posterior wall Non-posterior wall P value Tumor size, mm < (68.6) 756 (72.8) (31.4) 282 (27.2) Ulcer 21 (5.2) 50 (4.8) Pathology Dysplasia 216 (53.3) 494 (47.6) Carcinoma 189 (46.7) 544 (52.4) Procedure time, min, mean ± SD ± ± < Curability Incomplete resection 60 (14.8) 96 (9.2) Complete resection 345 (85.2) 942 (90.8) Resectability En bloc 358 (88.4) 960 (92.5) Piecemeal 47 (11.6) 78 (7.5) Perforation 14 (3.5) 25 (2.4) 0.27 Post-ESD bleeding 17 (4.2) 44 (4.2) ESD: Endoscopic submucosal dissection. vs 2.2%) (Table 2). There was no significant difference in the frequency of post-esd bleeding. After dividing location according to posterior wall and other lesions, the posterior wall lesion group had a significantly longer procedure time (69.41 min vs min, P < 0.001) and higher rates of incomplete resections (14.8% vs 9.2%, P = 0.002) and piecemeal resections (11.6% vs 7.5%, P = 0.013) than lesions in other locations (Table 3). There was no significant difference in the frequency of post-esd bleeding or perforation between the two groups. Table 4 Multivariate analysis for incomplete resection Factors related to incomplete resection and complications We analyzed the factors associated with complete resection complications of ESD such as perforation or bleeding. In univariate analyses, lesion size larger than 20 mm, upper third location, posterior wall location, carcinoma and procedure time longer than 60 min were significantly related to incomplete resection. In multivariate analysis, lesion size larger than 20 mm, posterior wall location, carcinoma and procedure time longer than 60 min were significantly related to incomplete resection (Table 4). In addition, univariate predictors of perforation were lesion size larger than 20 mm, upper third location, procedure time longer than 60 min and piecemeal resection. In multivariate analysis, upper third location, procedure time longer than 60 min and piecemeal resection were statistically significantly related to perforation (Table 5). Moreover, univariate predictors of post-esd bleeding were lesion size larger than 20 mm, procedure time longer than 60 min and piecemeal resection. In multivariate analysis, procedure time longer than 60 min and piecemeal resection were statistically significantly related to post-esd bleeding (Table 6). DISCUSSION Univariate analysis Tumor size, mm < 20 1 (reference) ( ) Ulcer - 1 (reference) ( ) SM invasion - 1 (reference) ( ) Location Non-upper third 1 (reference) Upper third ( ) Location Non-posterior wall 1 (reference) Posterior wall ( ) Pathology Dysplasia 1 (reference) Carcinoma ( ) Procedure time, min < 60 1 (reference) ( ) SM: Submucosa. Multivariate analysis OR (95%CI) P value OR (95%CI) P value < ( ) ( ) ( ) < ( ) < ( ) < < ESD has been widely accepted as an effective and safe treatment for gastric tumors [5,10]. To summarize previous studies, it is believed that the location of a lesion affects both the completeness of resection and whether complications are likely to occur [6,7,11-14]. These previous studies analyzed tumor location which is anatomically divided 8634 July 14, 2014 Volume 20 Issue 26

327 Yoon JY et al. Impact of tumor location on ESD Table 5 Multivariate analysis for perforation Univariate analysis Tumor size, mm < 20 1 (reference) ( ) Ulcer - 1 (reference) ( ) SM invasion - 1 (reference) ( ) Location Non-upper third 1 (reference) Upper third ( ) Location Non-posterior wall 1 (reference) Posterior wall ( ) Pathology Dysplasia 1 (reference) Carcinoma ( ) Procedure time, min < 60 1 (reference) ( ) Curability Complete resection 1 (reference) Incomplete resection ( ) Resectability En bloc 1 (reference) Piecemeal ( ) SM: Submucosa. Multivariate analysis OR (95%CI) P value OR (95%CI) P value ( ) < ( ) < ( ) < ( ) < < Table 6 Multivariate analysis for post-endoscopic submucosal dissection bleeding Univariate analysis Tumor size, mm < 20 1 (reference) ( ) Ulcer - 1 (reference) ( ) SM invasion - 1 (reference) ( ) Location Non-upper third 1 (reference) Upper third ( ) Location Non-posterior wall 1 (reference) Posterior wall ( ) Pathology Dysplasia 1 (reference) Carcinoma ( ) Procedure time, min < 60 1 (reference) ( ) Curability Complete resection 1 (reference) Incomplete resection ( ) Resectability En bloc 1 (reference) Piecemeal ( ) SM: Submucosa. Multivariate analysis OR (95%CI) P value OR (95%CI) P value ( ) < ( ) < ( ) into three portions (upper, middle, and lower thirds) as a factor related to the clinical outcomes of ESD. Many studies reported that upper third location was associated with incomplete resection, longer procedure time and a higher rate of perforation [7,11,12]. However, even among tumors located in the same thirds of the stomach, the difficulty of the procedure and the clinical outcomes may be different according to the cross-sectional circumference, which is divided into four equal parts. In our study, after dividing the upper third lesions from other lesions, it was found that the upper third lesion group had significantly higher percentages of incomplete resections (19.4% vs 10.2%) and piecemeal resections (15.3% vs 8.2%), longer procedure time (91 min vs 60 min) and a higher perforation rate (9.2% vs 2.2%). Furthermore, we analyzed clinical outcomes between posterior wall lesions and non-posterior lesions. The posterior wall lesion group had significantly longer procedure times and more frequent piecemeal and incomplete resections, which are likely explained in part by the difference in technical difficulty and poor visual field. In previous studies, procedure time during ESD became longer as tumor locations became higher [7,15]. In addition, longer procedure time was needed for tumors located in the posterior wall [15]. These findings were consistent with our study results. As longer procedure times have been shown to be associated with increased risks of complications [16,17], we attributed the relationship between tumor location and complications to the longer procedure time of ESD for gastric tumors in the upperthird or the posterior wall of the stomach. As mentioned above, prolonged procedure time in cases involving the upper-third or the posterior wall location was caused primarily by technical difficulties and a poor visual field. During ESD for gastric tumors in the upper-third of the stomach, endoscopists cannot let the knife encroach on the submucosal layer beneath the tumor, and cannot control the direction and depth well adhering to the dissection plan [7]. Along these lines, the worse outcomes after ESD for those lesions in this study were also consistent with earlier studies that demonstrated lower rates of en bloc and curative resections in lesions of the upper portion of the stomach [6,7,12] July 14, 2014 Volume 20 Issue 26

328 Yoon JY et al. Impact of tumor location on ESD It is very important that we determine the factors related to complete resection, because clinically complete resection is the ultimate goal of ESD, and it is closely related to tumor recurrence rate after ESD [18,19]. We analyzed incomplete resection rates according to the tumor location, and independent factors related to incomplete resection. In multivariate analysis, lesions larger than 20 mm, posterior wall location, carcinoma and procedure time longer than 60 min were statistically significantly associated with incomplete resection. Interestingly, the significant factor of location for incomplete resection was being in the posterior wall in our study. The posterior wall location makes it technically difficult to use a knife during ESD in comparison with the anterior wall or lesser curvature locations. Furthermore, in this study, almost all cases of endoscopic resection were performed using a single channel endoscope and because this endoscope s accessory channel opening is oriented at the 7 o clock position, which is the opposite direction needed for posterior wall dissections, and makes ESD of posterior wall lesions difficult. During the ESD procedure, serious complications such as bleeding or perforation may occur. Risk factors for perforation were identified in a recent study by Yoo et al [20], who proposed that risk of perforation is associated with age, depth of invasion and length of the procedure. In our study, the perforation rate was significantly associated with upper third location. This may be due to the fact that lesions in the lower third are easily approached and manipulated by endoscopy, and thus it is technically easier to perform ESD and there is a lower chance of applying sufficient tension on the gastric wall to cause perforation. Another possible reason is that the lower or the mid-portion of the gastric wall is thicker than the upper portion of the stomach. Previous reports showed that bleeding occurred more frequently in the corpus than in the antrum [11,21]. Okada et al [22] demonstrated that having a tumor located in the mid-third of the stomach was an independent risk factor for post-esd bleeding. However, the relationship between tumor location and post-esd bleeding remains controversial to date. In our post-esd bleeding analysis, location was not a significant factor for post-esd bleeding. Besides tumor location, various factors such as the patient s underlying medical and drug history, the endoscopist s experience and preventive coagulation of visible vessels in the resection area after ESD may affect post-esd bleeding. A recent study demonstrated that post-esd bleeding depends on how meticulously coagulation of visible vessels is performed after dissection [23]. Regrettably, we could not analyze the effect of underlying disease or anti-platelet agents on post-esd bleeding because we had relatively few patients with chronic disease such as chronic renal failure or cirrhosis, and all the patients in our study who underwent ESD discontinued anti-platelet agents prior to the procedure. Limitations of this study include the fact that it was a retrospective single-center study with a limited followup duration. Therefore, there may be a bias according to the endoscopist who performed the ESD. In addition, we did not include long-term follow-up data concerning recurrence, disease-free survival, and overall survival, which are important for evaluating the effect of risk factors on outcomes. Nevertheless, this study focused on outcomes after ESD for gastric tumors with reference to therapeutic efficacy and complications according to lesion location. Although we did not address long-term outcomes, this study revealed that the two main components for the feasibility of ESD, acceptable complete resection and complication rates, change according to the location of gastric tumors. In addition, the clinical implications of tumor location were based on largevolume data. Accordingly, we suggest that attention to gastric tumor location, particularly in the upper third or posterior wall, during ESD is needed to avoid incomplete resection and complications. We should also explain the possibility of further surgical treatment after ESD to patients with such lesions. Nonetheless, further investigations with long-term follow-up data concerning the clinical significance of tumor location are needed to support our recommendations. In conclusion, ESD for gastric tumor is an effective and safe therapy. However, endoscopists should recognize the need for more advanced endoscopic techniques when performing ESD for lesions located in the upper third or posterior wall of the stomach in order to decrease the rate of serious complications and improve clinical outcomes. COMMENTS Background Advances in diagnostic technology and the increasing prevalence of screening programs have increased the rate of early gastric cancer (EGC) detection. EGCs that are confined to the mucosa and lack lymph node metastasis can be cured by endoscopic resection, such as endoscopic mucosal resection and endoscopic submucosal dissection (ESD). Research frontiers Previous studies analyzed tumor location which is anatomically divided into three portions (upper, mid, and lower thirds) as a factor related to the clinical outcomes of ESD. Innovations and breakthroughs In this study, the perforation rate was significantly associated with upper third location. This may be due to the fact that lesions at lower third are easily approached and manipulated by endoscopy, and thus it is technically easier to perform ESD and there is a lower chance of applying sufficient tension on the gastric wall to cause perforation. Applications ESD for gastric tumor is an effective and safe therapy. However, endoscopists should recognize the need for more advanced endoscopic techniques when performing ESD for lesions located in the upper third or posterior wall of the stomach in order to decrease the rate of serious complications and improve clinical outcomes. Peer review This is an interesting study regarding on the technical aspect of gastric ESD, focused on the tumor location. The number of the subjects is large and the analysis is simple with clear results. REFERENCES 1 Cho WY, Cho JY, Chung IK, Kim JI, Jang JS, Kim JH. Endoscopic submucosal dissection for early gastric cancer: 8636 July 14, 2014 Volume 20 Issue 26

329 Yoon JY et al. Impact of tumor location on ESD quo vadis? World J Gastroenterol 2011; 17: [PMID: DOI: /wjg.v17.i ] 2 Chun HJ, Keum B, Kim JH, Seol SY. Current status of endoscopic submucosal dissection for the management of early gastric cancer: a Korean perspective. World J Gastroenterol 2011; 17: [PMID: DOI: /wjg.v17. i ] 3 Lee JH, Hong SJ, Jang JY, Kim SE, Seol SY. Outcome after endoscopic submucosal dissection for early gastric cancer in Korea. World J Gastroenterol 2011; 17: [PMID: DOI: /wjg.v17.i ] 4 Lee JH, Kim JJ. Endoscopic mucosal resection of early gastric cancer: Experiences in Korea. World J Gastroenterol 2007; 13: [PMID: ] 5 Oka S, Tanaka S, Kaneko I, Mouri R, Hirata M, Kawamura T, Yoshihara M, Chayama K. Advantage of endoscopic submucosal dissection compared with EMR for early gastric cancer. Gastrointest Endosc 2006; 64: [PMID: DOI: /j.gie ] 6 Imagawa A, Okada H, Kawahara Y, Takenaka R, Kato J, Kawamoto H, Fujiki S, Takata R, Yoshino T, Shiratori Y. 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Gastric Cancer 2000; 3: [PMID: ] 9 Schlemper RJ, Riddell RH, Kato Y, Borchard F, Cooper HS, Dawsey SM, Dixon MF, Fenoglio-Preiser CM, Fléjou JF, Geboes K, Hattori T, Hirota T, Itabashi M, Iwafuchi M, Iwashita A, Kim YI, Kirchner T, Klimpfinger M, Koike M, Lauwers GY, Lewin KJ, Oberhuber G, Offner F, Price AB, Rubio CA, Shimizu M, Shimoda T, Sipponen P, Solcia E, Stolte M, Watanabe H, Yamabe H. The Vienna classification of gastrointestinal epithelial neoplasia. Gut 2000; 47: [PMID: ] 10 Goto O, Fujishiro M, Kodashima S, Ono S, Omata M. Outcomes of endoscopic submucosal dissection for early gastric cancer with special reference to validation for curability criteria. Endoscopy 2009; 41: [PMID: DOI: /s ] 11 Jeon SW, Jung MK, Cho CM, Tak WY, Kweon YO, Kim SK, Choi YH. Predictors of immediate bleeding during endoscopic submucosal dissection in gastric lesions. Surg Endosc 2009; 23: [PMID: DOI: / s ] 12 Ohnita K, Isomoto H, Yamaguchi N, Fukuda E, Nakamura T, Nishiyama H, Mizuta Y, Akiyama M, Nakao K, Kohno S, Shikuwa S. Factors related to the curability of early gastric cancer with endoscopic submucosal dissection. Surg Endosc 2009; 23: [PMID: DOI: / s ] 13 Goto A, Nishikawa J, Okamoto T, Hamabe K, Nishimura J, Nakamura M, Kiyotoki S, Saito M, Miura O, Sakaida I. Outcomes of endoscopic submucosal dissection for early gastric cancer and factors associated with incomplete resection. Hepatogastroenterology 2013; 60: [PMID: DOI: /hge12533] 14 Mannen K, Tsunada S, Hara M, Yamaguchi K, Sakata Y, Fujise T, Noda T, Shimoda R, Sakata H, Ogata S, Iwakiri R, Fujimoto K. Risk factors for complications of endoscopic submucosal dissection in gastric tumors: analysis of 478 lesions. J Gastroenterol 2010; 45: [PMID: DOI: /s ] 15 Ahn JY, Choi KD, Choi JY, Kim MY, Lee JH, Choi KS, Kim do H, Song HJ, Lee GH, Jung HY, Kim JH. Procedure time of endoscopic submucosal dissection according to the size and location of early gastric cancers: analysis of 916 dissections performed by 4 experts. Gastrointest Endosc 2011; 73: [PMID: DOI: /j.gie ] 16 Lee IL, Wu CS, Tung SY, Lin PY, Shen CH, Wei KL, Chang TS. Endoscopic submucosal dissection for early gastric cancers: experience from a new endoscopic center in Taiwan. J Clin Gastroenterol 2008; 42: [PMID: DOI: /01.mcg ff] 17 Yamamoto S, Uedo N, Ishihara R, Kajimoto N, Ogiyama H, Fukushima Y, Yamamoto S, Takeuchi Y, Higashino K, Iishi H, Tatsuta M. Endoscopic submucosal dissection for early gastric cancer performed by supervised residents: assessment of feasibility and learning curve. Endoscopy 2009; 41: [PMID: DOI: /s ] 18 Isomoto H, Shikuwa S, Yamaguchi N, Fukuda E, Ikeda K, Nishiyama H, Ohnita K, Mizuta Y, Shiozawa J, Kohno S. Endoscopic submucosal dissection for early gastric cancer: a large-scale feasibility study. Gut 2009; 58: [PMID: DOI: /gut ] 19 Jang JS, Choi SR, Qureshi W, Kim MC, Kim SJ, Jeung JS, Han SY, Noh MH, Lee JH, Lee SW, Baek YH, Kim SH, Choi PJ. Long-term outcomes of endoscopic submucosal dissection in gastric neoplastic lesions at a single institution in South Korea. Scand J Gastroenterol 2009; 44: [PMID: DOI: / ] 20 Yoo JH, Shin SJ, Lee KM, Choi JM, Wi JO, Kim DH, Lim SG, Hwang JC, Cheong JY, Yoo BM, Lee KJ, Kim JH, Cho SW. Risk factors for perforations associated with endoscopic submucosal dissection in gastric lesions: emphasis on perforation type. Surg Endosc 2012; 26: [PMID: DOI: /s x] 21 Shiba M, Higuchi K, Kadouchi K, Montani A, Yamamori K, Okazaki H, Taguchi M, Wada T, Itani A, Watanabe T, Tominaga K, Fujiwara Y, Hayashi T, Tsumura K, Arakawa T. Risk factors for bleeding after endoscopic mucosal resection. World J Gastroenterol 2005; 11: [PMID: ] 22 Okada K, Yamamoto Y, Kasuga A, Omae M, Kubota M, Hirasawa T, Ishiyama A, Chino A, Tsuchida T, Fujisaki J, Nakajima A, Hoshino E, Igarashi M. Risk factors for delayed bleeding after endoscopic submucosal dissection for gastric neoplasm. Surg Endosc 2011; 25: [PMID: DOI: /s ] 23 Takizawa K, Oda I, Gotoda T, Yokoi C, Matsuda T, Saito Y, Saito D, Ono H. Routine coagulation of visible vessels may prevent delayed bleeding after endoscopic submucosal dissection--an analysis of risk factors. Endoscopy 2008; 40: [PMID: DOI: /s ] P- Reviewers: Kita H, Puzziello A S- Editor: Zhai HH L- Editor: Wang TQ E- Editor: Liu XM 8637 July 14, 2014 Volume 20 Issue 26

330 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. RETROSPECTIVE STUDY Outcomes of simple saline-coupled bipolar electrocautery for hepatic resection Jian-Yang Guo, De-Wei Li, Rui Liao, Ping Huang, Xian-Bing Kong, Ji-Ming Wang, Hong-Lin Wang, Shi-Qiao Luo, Xiong Yan, Cheng-You Du Jian-Yang Guo, De-Wei Li, Rui Liao, Ping Huang, Xian-Bing Kong, Ji-Ming Wang, Hong-Lin Wang, Shi-Qiao Luo, Xiong Yan, Cheng-You Du, Department of Hepatobiliary Surgery, The First Affiliated Hospital, Chongqing Medical University, Chongqing , China Author contributions: Guo JY and Du CY designed the research; Guo JY, Liao R, Luo SQ and Yan X performed the research; Wang JM, Wang HL and Du CY contributed new reagents and analytic tools; Li DW, Liao R, Huang P and Kong XB analyzed the data; Guo JY, Li DW and Du CY wrote the paper. Correspondence to: Cheng-You Du, MD, PhD, Department of Hepatobiliary Surgery, The First Affiliated Hospital, Chongqing Medical University, Yixueyuan Rd 1, Chongqing , China. duchengyou@126.com Telephone: Fax: Received: January 9, 2014 Revised: February 14, 2014 Accepted: April 2, 2014 Published online: July 14, 2014 Abstract AIM: To evaluate the application of bipolar coagulation (BIP) in hepatectomy by comparing the efficacy of BIP alone, cavitron ultrasonic surgical aspirator (CUSA) + BIP and conventional clamp crushing (CLAMP). METHODS: Based on our database of patient records, a total of 380 consecutive patients who underwent hepatectomy at our hospital were retrospectively studied for the efficacy of BIP alone, CUSA + BIP and CLAMP. Of all the patients, 75 received saline-coupled BIP (Group A), 53 received CUSA + BIP (Group B), and 252 received CLAMP (Group C). The pre-, mid-, and postoperative clinical manifestations were compared, and the effects of those maneuvers were evaluated. RESULTS: There was no obvious difference among the preoperative indexes between the different groups. The operative time was longer in Groups A and B than in Group C (P < for both). The amount of bleeding and the rate of transfusion during the operation were significantly higher in Group C than in Groups A and B (P < for all). The incidence of postoperative complications in Group C (46.43%) was higher than that in Groups A (30.67%, P = 0.015) and B (28.30%, P = 0.016). The patients liver function recovery and postoperative hospital stay were not significantly different. BIP could decrease intraoperative hemorrhage and postoperative complications compared to CLAMP. CONCLUSION: Simple saline-coupled BIP should be considered a safe and reliable technique for liver resection to decrease intraoperative hemorrhage and postoperative complications Baishideng Publishing Group Inc. All rights reserved. Key words: Hepatectomy; Surgical procedures; Blood loss; Complications; Hospital stay; Comparative study Core tip: The aim of this clinical study is to recommend a simplified and feasible surgical technique for liver resection. In this study, we found that simple salinecoupled bipolar electrocautery (BIP) could reduce blood loss, blood transfusion and complications compared with clamp crushing. Therefore, saline-coupled BIP can accomplish hepatectomy excellently and would be a safe and reliable technique that is easily applied in liver resection. Guo JY, Li DW, Liao R, Huang P, Kong XB, Wang JM, Wang HL, Luo SQ, Yan X, Du CY. Outcomes of simple salinecoupled bipolar electrocautery for hepatic resection. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: July 14, 2014 Volume 20 Issue 26

331 Guo JY et al. Application of bipolar electrocautery INTRODUCTION Hepatectomy and its related surgical complications remain major concerns for surgeons operating on the liver. Controlling hemorrhage, shortening the time of inflow occlusion, and performing anatomical limited resections are important strategies for safe and careful dissection of the liver parenchyma. For patients with hepatocellular carcinoma (HCC), massive hemorrhage and blood transfusions are the powerful determinants of early liver failure and immune repression, which cause adverse effects, often leading to early tumor recurrence, and seriously affecting long-term survival after resection [1,2]. Advancements in the theory and practice of liver surgery have led to the invention of different approaches, such as the cavitron ultrasonic surgical aspirator (CUSA), water jet scalpel, monopolar floating ball, ligasure, microwave and other procedures to transect the liver parenchyma favorably. CUSA combined with bipolar coagulation (BIP) and conventional clamp crushing (CLAMP) are favored by most surgeons at many medical centers [3-6]. Notably, it is critical to expose vasculature and decrease blood loss during hepatic resection. As a device for hemostasis, BIP has aroused increased interest because of its excellent hemostatic effect and low thermal damage to surrounding tissues. Because of these potential benefits, increasing numbers of surgeons are also applying BIP in hepatectomy as a preferred method. Although various BIP devices and techniques have been developed in some centers, the efficacy of BIP alone is actually unclear, hindering its wide adoption. This retrospective cohort analysis investigated the efficacy of BIP alone, CUSA + BIP and CLAMP. MATERIALS AND METHODS Patient characteristics Between April 2011 and May 2013, data from 380 consecutive patients who underwent liver resection at our hospital were collected, based on our database of patient records. Through CT, MRI or pathologic examinations of these patients, the following was detected: 245 malignant tumors (including 179 HCC), 47 benign tumors (such as hemangioma, hamartoma, and focal nodular hyperplasia), 70 cases of hepatolithiasis and 18 cases of other diseases (such as liver abscess, polycystic liver, and hepatic hydatid). Detailed characteristics of those patients are shown in Table 1. This study was approved by the ethics board of The First Affiliated Hospital of Chongqing Medical University, and written informed consent was obtained from all patients. Study design Patients were divided into 3 groups: Group A, patients treated by BIP, with BIP administered by Shuyou surgical instrument Inc, Zhejiang, China, and Force EZ Electrosurgical Generator; Covidien Inc, Boulder, Colo, United States; bipolar, 70W, Figure 1; Group B, patients treated by CUSA, with CUSA administered by Cavitron Ultrasonic Surgical Aspirator System 200; Valleylab Inc, Boulder, Colo, United States; amplitude, 70W; and Group C, patients receiving combined treatment of BIP and CLAMP. All patients received general anesthesia. For fair comparison and avoidance of the potential for patient selection bias with these various techniques by different surgeons, three experienced hepatic surgeons in our unit performed all operations. We chose one technique over the other due to equipment availability or change over time. CUSA and CLAMP were performed between April 2011 and April 2012, and BIP was performed from April 2012 to May There is no significant difference in the characteristics of the three groups of patients (P > 0.05), including the fibrotic or cirrhotic liver and tumor condition. Except for BIP, no other hemostatic device (i.e., argon beam coagulator) was used on any of these patients undergoing hepatic resection. The operation time (min), intraoperative bleeding (ml), the rate of blood transfusion and the amount of packed red blood cells transfusion (the amount of bleeding more than 25% of the blood volume or hemoglobin lower than 70 g/l was the indication of transfusion) were analyzed. Surgical procedure Hepatectomy was performed following standard procedures. The hemostatic forcep tip was moved vertically to the resectional line when transecting the parenchyma by CLAMP. The clamp held approximately 1 cm of the liver parenchyma each time. Hepatic parenchyma cells were disrupted and emulsified by vibration, and the bile ducts and vessels were preserved using CUSA; hemostasis was performed by BIP. The procedure of hepatic resection by saline-coupled BIP alone had water dripping from two sides of the bipolar electrocoagulator, and the power was set at 70 watt. BIP was combined with scissors and sucker for disruption, washing, hemostasis and suction. However, vessels of more than 3 mm in diameter were exposed to ligate, and vessels less than 3 mm in diameter were coagulated (Figure 2). Our results demonstrated that most of the hepatectomies did not require blocking the portal vein blood supply. Postoperative management Postoperatively, all patients were admitted to the ICU ward for rehabilitation. Postoperative outcomes included recovery of liver function, length of postoperative hospitalization, and the rate of complications. To monitor liver function, albumin (ALB, g/l), total bilirubin (TB, μmol/l), alanine transaminase (ALT, μmol/l), and aspertate aminotransferase (AST, μmol/l) levels were measured on days 1 and 3 after resection. Complications for monitoring included: abdominal cavity infection, bile leakage, acute hepatic failure, respiratory failure, severe hemorrhage, reoperation within 30 d of surgery, hyperbilirubinemia, sepsis, intestinal obstruction, pulmonary embolism, hepatorenal syndrome, death within 30 d of surgery, pleural effusion, incision infection, incision dehiscence, pulmonary infection, delayed recovery of liver 8639 July 14, 2014 Volume 20 Issue 26

332 Guo JY et al. Application of bipolar electrocautery Table 1 Characteristics of patients undergoing liver resection n (%) Baseline characteristic BIP (n = 75) CUSA + BIP (n = 53) CLAMP (n = 252) P value Age 1 (yr) 54.1 (17-81) 50.7 (16-85) 53.7 (19-81) Sex Male 47 (62.67) 33 (62.26) 155 (61.51) Cause of surgery HCC 38 (50.67) 20 (37.74) 121 (48.02) Other malignancy 14 (18.67) 9 (16.98) 43 (17.06) Benign tumour 8 (10.66) 10 (18.87) 29 (11.51) Intrahepatic stone 12 (16.00) 11 (20.75) 47 (18.65) Others 3 (4.00) 3 (5.66) 12 (4.76) Child-Pugh A 56 (74.67) 41 (77.36) 210 (83.33) B 19 (25.33) 12 (22.64) 42 (16.67) ICG-R15 < (89.33) 46 (86.79) 219 (86.90) (10.67) 7 (13.21) 33 (13.10) Background liver Normal 31 (41.33) 31 (58.49) 130 (51.58) Chronic hepatitis 24 (32.00) 12 (22.64) 61 (24.21) Cirrhosis 20 (26.67) 10 (18.87) 61 (24.21) Level of surgeon Chief doctor 31 (20.67) 29 (27.36) 127 (25.20) Deputy director doctor 56 (37.33) 40 (37.74) 226 (44.84) Attending doctor 63 (42.00) 37 (34.90) 151 (29.96) Type of resection Extended 13 (17.33) 8 (15.09) 35 (13.89) Hemihepatectomy Hemihepatectomy 33 (44.00) 20 (37.74) 108 (42.85) Lobectomy 29 (38.67) 21 (39.62) 92 (36.51) Segmentectomy 0 4 (7.55) 13 (5.16) Wedge resection (1.59) Tumor Yes 60 (80.00) 39 (73.58) 193 (76.59) Tumor condition Size, < 30 mm 14 (23.33) 7 (17.95) 56 (29.02) mm 46 (76.67) 32 (82.05) 137 (70.98) Single 36 (60.00) 31 (79.49) 115 (59.59) Multiple 24 (40.00) 8 (20.51) 78 (40.41) Superficial 36 (60.00) 28 (71.79) 141 (73.06) Deep 24 (40.00) 11 (28.21) 52 (26.94) 1 P analysis of variance (F). HCC: Hepatocellular carcinoma; BIP: Bipolar coagulation; CUSA: Cavitron ultrasonic surgical aspirator; CLAMP: Conventional clamp crushing; ICG-R15: Indocyanine green retention rate at 15 min. A B Figure 1 Operative devices used for single saline-coupled bipolar electrocautery. A: Force EZ electrosurgical generator; B: Bipolar electrocautery (bilateral dribbling water) for hepatectomy. function, seroperitoneum, pulmonary atelectasis and deep vein thrombosis. Statistical analysis For statistical analysis, measurement values are expressed as mean with standard deviation or median with interquartile range. The differences in the measurement values were detected by ANOVA (F) or Kruskal-Wallis (H) rank test. The differences in enumeration data were compared by χ 2 or Kruskal-Wallis (H) rank test. The difference 8640 July 14, 2014 Volume 20 Issue 26

333 Guo JY et al. Application of bipolar electrocautery A B C D Figure 2 A representative case with left liver cancer resected by single saline-coupled bipolar electrocautery. A: Computerized tomography imaging on admission; B-D: The surgical process of hepatectomy by single bipolar electrocautery (BIP). The arrow indicates a liver vessel exposed by BIP. Table 2 Surgical outcomes n (%) Outcome BIP (n = 75) CUSA+BIP (n = 53) CLAMP (n = 252) P value Blood loss ml 73 (97.33) 48 (90.57) 192 (76.19) < > 1000 ml 2 (2.67) 5 (9.43) 60 (23.81) Intraoperative transfusion 1 12 (16.00) 7 (13.21) 117 (46.43) < Blood loss (ml) median ( ) 328 ( ) 583 ( ) < Transfusion of RBC (ml) median 37 (0-800) 56 (0-1200) 69 (0-2800) < Operative time (min) median 315 ( ) 335 (80-730) 265 (60-590) < P (χ 2 ); 2 Variables are expressed as median (minimum-maximum). P: Multiple samples rank sum test (H). BIP: Bipolar coagulation; CUSA: Cavitron ultrasonic surgical aspirator; CLAMP: Conventional clamp crushing. between both groups was determined via Bonferroni correction. P < 0.05 was considered statistically significant. All data were analyzed using SPSS Version RESULTS Patient characteristics A total of 380 consecutive patients underwent liver resection. Preoperative data for patients in each group are presented in Table 1. The following was not significantly different between each group (P > 0.05 for all): age, gender, the cause of operation, liver function, indocyanine clearance (ICG-R15), liver disease, tumor condition (tumors located 2 cm or less from the liver surface were defined as superficial; those more than 2 cm from the surface were defined as deep), and extent of liver resection (resection range: right trilobectomy, extended left hepatactomy, right hepatactomy; right anterior lobe, right posterior lobe, left liver, central portion resection, S5+7, S5+6; Couinaud lobectomy; local hepatectomy; enucleation). Blood loss Intraoperative data are shown in Table 2. Median blood loss during surgery was 315 ml in Group A, 328 ml in Group B and 583 ml in Group C (P < 0.001). The percentage of patients whose hemorrhage volume was more than 1000 ml was 2.67% in Group A, 9.43% in Group B and 23.81% in Group C (P < 0.001). The blood transfusion rate was 16.00% for Group A, 13.21% for Group B and 46.43% for Group C (P < 0.001). The median amount of transfusion with packed red cells was 37 ml in Group A, 56 ml in Group B and 69 ml in Group C (P < 0.001). There was no significant difference between 8641 July 14, 2014 Volume 20 Issue 26

334 Guo JY et al. Application of bipolar electrocautery Table 3 The differences in indexes between the groups n (%) BIP (A), CUSA + BIP (B), CLAMP (C) Group A and B (P > 0.05, Table 3). (A vs B) (B vs C) (A vs C) P value P value P value Blood loss, > 1000 ml < < Intraoperative transfusion < < Blood loss (ml), median < < Transfusion of RBC (ml), median < < Operative time (min), median < < Complications Yes Abdominal infection Bile leak Ascites BIP: Bipolar coagulation; CUSA: Cavitron ultrasonic surgical aspirator; CLAMP: Conventional clamp crushing; RBC: Red blood cell. Postoperative complications Postoperative complications of each group were compared and are presented in Table 4. The incidence of total complications in Group C was higher than that in Groups A and B (46.43% vs 30.67% vs 28.30%, P = 0.007). There were significant differences in single complication rates including abdominal infection (group A 2.67% vs Group B 1.89% vs Group C 8.73%, P = 0.035), bile leakage (Group A 1.33% vs Group B 1.89% vs Group C 7.14%, P = 0.039) and seroperitoneum (Group A 1.33% vs Group B 3.77% vs Group C 11.51%, P = 0.002). The results demonstrated that significant differences existed between Groups A and C for seroperitoneum (1.33% vs 11.51%, P = 0.014). Deaths within 30 d of surgery were not statistically significant among the groups. In Group A, one patient died from pulmonary embolism. No patient died in Group B. In Group C, one patient died from severe hemorrhage and multiple organ dysfunction (1.33% vs 0% vs 0.79%, P = 0.710). The incidence of other complications was not significantly different among the three groups (P > 0.05 for all). Postoperative recovery conditions are shown in Table 5. No differences were noted in ALB, TB, ALT or AST levels on postoperative day 3 and preoperation (all P > 0.05). Elevated ALB levels were found in Group B when compared to Groups A and C (preoperative: 40.9 ± 5.3 vs 37.5 ± 6.1 vs 39.4 ± 5.8, P = 0.003; day 1: 29.4 ± 4.3 g/l vs 26.9 ± 5.6 g/l vs 26.2 ± 4.9 g/l, P < 0.001; day 3: 29.9 ± 4.8 g/l vs 27.2 ± 4.3 g/l vs 27.5 ± 4.3 g/l, P < 0.001). ALT levels were lower in Group C than in Groups A and B on postoperative day 1 (200 μmol/l vs 314 μmol/l vs 279 μmol/l, P < 0.001). There was no significant difference among the groups for other liver function recovery tests. Table 4 Postoperative complications n (%) Complication BIP CUSA + BIP CLAMP P value Operation time and hospitalization These results demonstrated that the operation time in Group C was shorter than that in the other two groups (median time: 315 min in Group A, 335 min in Group B, 265 min in Group C, P < 0.001). There was no difference between Groups A and B (P = 0.409). There was no significant difference for hospital stays after surgery (Group A 16.6 ± 7.4 d vs Group B 15.3 ± 9.2 d, vs Group C 16.5 ± 9.6 d; P = 0.666). DISCUSSION (n = 75) (n = 53) (n = 252) Yes 23 (30.67) 15 (28.30) 117 (46.43) Abdominal infection 2 (2.67) 1 (1.89) 22 (8.73) Bile leak > 2 wk 1 (1.33) 1 (1.89) 18 (7.14) Acute liver failure 1 (1.33) 0 2 (0.79) Return to operating room 0 1 (1.89) 3 (1.19) Respiratory failure 0 1 (1.89) 2 (0.79) Bleeding > 500 ml within 1 (1.33) 1 (1.89) 7 (2.78) h Hyperbilirubinemia 3 (4.00) 5 (9.43) 8 (3.17) Sepsis (0.79) Ileus (1.19) Pulmonary embolism 1 (1.33) 0 1 (0.40) Hepatorenal syndrome (0.40) d postoperative death 1 (1.33) 0 2 (0.79) Deep venous thrombosis (0.40) Pleural effusion 4 (5.33) 3 (5.66) 24 (9.52) Wound infection 16 (21.33) 12 (22.64) 50 (19.84) Wound dehiscence (0.79) Pneumonia 2 (2.67) 2 (3.77) 16 (6.35) Hepatic insufficiency 7 (9.33) 2 (3.77) 20 (7.94) Ascites 1 (1.33) 2 (3.77) 29 (11.51) Atelectasis 1 (1.33) 1 (1.89) 3 (1.19) BIP: Bipolar coagulation; CUSA: Cavitron ultrasonic surgical aspirator; CLAMP: Conventional clamp crushing. Controlling bleeding and choosing the appropriate technique to transect the liver are the key points in hepatectomy [7-9]. For CLAMP, the whole surgical procedure is simple and fast during parenchyma transaction. However, the operation is relatively rough and can easily lead to severe liver damage. For CUSA + BIP, the surgeon can gradually grind and suction out parenchyma with clear anatomical structure under direct vision. Obviously, blood loss and postoperative complications were decreased using this method. Although Takayama et al [10] reported hepatectomy with CLAMP, which was associated with a significantly higher grade and appearance of the landmark hepatic vein on the cut surface than CUSA + BIP. Of note, the results of that study were based on the surgeon s familiarity with CLAMP. Not every surgeon has such skilled operation levels. Therefore, some research centers could not fully agree with them [11,12]. Moreover, other methods to transect the liver parenchyma, such as water jet scalpel, habib procedures, monopolar floating ball and ligasure are not commonly used worldwide [13-16]. To our best knowledge, this is the first report about comparative analysis of the application of saline-coupled BIP alone in hepatectomy for the excellent hemostasis and satisfactory surgical results. The majority of bleeding for liver resection comes from the process of transecting the liver parenchyma July 14, 2014 Volume 20 Issue 26

335 Guo JY et al. Application of bipolar electrocautery Table 5 Laboratory values and hospital stay n (%) Recovery evaluation BIP CUSA+BIP CLAMP 1 P value (n = 75) (n = 53) (n = 251) TB (μmol/l) Pre ( ) 15.5 ( ) 13.5 ( ) Day ( ) 23.4 ( ) 25.9 ( ) Day ( ) 18.3 ( ) 21.9 ( ) ΔTB ( ) 2.9 ( ) 6 ( ) ALT (μmol/l) Pre- 32 (11-337) 48 (10-579) 36 (7-984) Day ( ) 279 ( ) 200 ( ) < Day ( ) 135 ( ) 130 ( ) ΔALT 2 85 ( ) 84 ( ) 84 ( ) AST (μmol/l) Pre- 35 (14-261) 40 (6-531) 33 (8-698) Day ( ) 294 ( ) 247 ( ) Day 3 88 ( ) 59 ( ) 72 (12-940) ΔAST 2 38 ( ) 24 ( ) 31 ( ) ALB (g/l) 3 Pre ± ± ± mean ± SD Day ± ± ± 4.9 < Day ± ± ± 4.3 < ΔALB ± ± ± Hospital stay (d) mean ± SD 16.6 ± ± ± One case in group C who died from massive bleeding and multiple organ failure was excluded; 2 Δtotal bilirubin (TB, μmol/l) = TB (day 3 - pre-), Δalanine transaminase (ALT, μmol/l) = ALT (day 3 - pre-), Δaspertate aminotransferase (AST, μmol/l) = AST (day 3 - pre-), Δalbumin (ALB, g/l) = ALB (pre- - day 3); 3 Variables are expressed as mean ± SD, PANOVA (F). Pre-: Pre-operation; BIP: Bipolar coagulation; CUSA: Cavitron ultrasonic surgical aspirator; CLAMP: Conventional clamp crushing. The present study demonstrated that liver resection was relatively rough by CLAMP, especially for deep tissue. Because of long and large hemostatic forceps and its small operative field, the maneuver results in unnecessary vascular damage. For the rate of blood transfusion, BIP and CUSA + BIP were lower than CLAMP. A meta-analysis performed by Liu et al [17] revealed that perioperative blood transfusion was associated with adverse clinical outcomes for HCC. Moreover, blood loss of more than 2000 ml was also a poor prognostic factor for HCC [18]. Our results support that BIP and CUSA + BIP could improve quality of life and survival of HCC patients. Further comparison between BIP and CUSA + BIP showed that BIP could get the same effect for blood loss, blood transfusion, operational security and prognosis after simplified surgical procedures. Our results demonstrate that the incidence of postoperative complications, such as abdominal infection, bile leakage and seroperitoneum, were higher in the CLAMP group compared to the BIP and CUSA + BIP groups. The reason for that phenomenon was less blood transfusion and section integrity [19-21]. Delva et al [22] supported that the recovery of liver function was related to the Pringle maneuver length, cirrhosis, the quantity and quality of the remnant liver parenchyma and blood transfusion. Massive parenchyma was grasped and stitched with the clamp. Stable fibrillar structure features were formed, vessels and biliary ducts were closed ideally and less thermal damage was made by BIP. Postoperative complications were associated with operational technique, blood loss, blood transfusion and individual differences for surgery [23-26]. Therefore, BIP could be safer and decrease the risk of complications for hepatectomy. Our results showed that, for an experienced hepatobiliary surgeon, the intrahepatic bile duct could be clearly freed by a single BIP without CUSA. Because the resectional extent of the ultrasonic section device was approximately 4 mm, it was easy to expose the tumor using CUSA when the surgeon could not ensure the distance between the section and tumor. There was a potential danger of spreading hepatitis when CUSA disrupted hepatic cells [27]. Compared to CLAMP, the cutting speed by BIP was lower, and the operative time was prolonged approximately 20%-30%. It is actually not surprising that the use of these instruments resulted in increased operative time because there is more attention paid to dissecting out blood vessels and coagulating or ligating them before dividing them. However, the operative time of BIP could be decreased if performed by highly skilled surgeons. BIP could not only separate the liver parenchyma but also reduce hemorrhage. Additionally, the operative time in the BIP group was an independent prognostic factor for postoperative complications. In conclusion, simple saline-coupled BIP could reduce blood loss, blood transfusion and complications compared to CLAMP. Therefore, saline-coupled BIP would be a safe and reliable technique easily applied in liver resection. ACKNOWLEDGMENTS The authors gratefully acknowledge the assistance of Bin Peng from Department of Epidemiology, Public Health College of Chongqing Medical University, who helped us with the statistics. The authors also thank Professor C Michael Dabney (National University, San Diego, United States) for the critical reading of the manuscript July 14, 2014 Volume 20 Issue 26

336 Guo JY et al. Application of bipolar electrocautery COMMENTS Background Liver resections are high risk procedures, the key points in which are controlling bleeding and choosing appropriate technique. Advancements in the theory and practice of liver surgery have led to the invention of multiple approaches to transect liver parenchyma. For these reasons, choosing a safe and reliable method of hepatectomy is important in decreasing intraoperative hemorrhaging and postoperative complications. Research frontiers Cavitron ultrasonic surgical aspirator (CUSA) combined with bipolar coagulation (BIP) and conventional clamp crushing (CLAMP) are favored by most surgeons at many medical centers. As a device for hemostasis, more and more surgeons are applying BIP in hepatectomy as a preferred method. However, compared to CUSA and CLAMP, the efficacy of BIP alone is actually unclear. Innovations and breakthroughs The present study is the first report about comparative analysis of the application of saline-coupled BIP alone in hepatectomy for excellent hemostasis and satisfactory surgical results. Applications The study results suggest that saline-coupled BIP could reduce blood loss, blood transfusion and complications compared to CLAMP. Saline-coupled BIP can accomplish hepatectomy excellently and would be a safe and reliable technique easily applied in liver resection. Terminology CUSA is a powerful ultrasonic aspirator that allows the surgeon to accomplish tissue resection with accurate control while fragmentation, suction, and irrigation occur simultaneously; BIP is a technique for surgical dissection and hemostasis that consists of two tips with current pass confined in a small amount of tissue. Peer review This is an interesting manuscript describing outcomes of three different methods of parenchymal transection in hepatic surgery. 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337 Guo JY et al. Application of bipolar electrocautery sponse in patients undergoing liver resection. Br J Surg 2013; 100: [PMID: DOI: /bjs.9035] 22 Delva E, Camus Y, Nordlinger B, Hannoun L, Parc R, Deriaz H, Lienhart A, Huguet C. Vascular occlusions for liver resections. Operative management and tolerance to hepatic ischemia: 142 cases. Ann Surg 1989; 209: [PMID: ] 23 Neal CP, Mann CD, Pointen E, McGregor A, Garcea G, Metcalfe MS, Berry DP, Dennison AR. Influence of hepatic parenchymal histology on outcome following right hepatic trisectionectomy. J Gastrointest Surg 2012; 16: [PMID: DOI: /s ] 24 Chok KS, Ng KK, Poon RT, Lo CM, Fan ST. Impact of postoperative complications on long-term outcome of curative resection for hepatocellular carcinoma. Br J Surg 2009; 96: [PMID: DOI: /bjs.6358] 25 Cheung TT, Poon RT, Yuen WK, Chok KS, Jenkins CR, Chan SC, Fan ST, Lo CM. Long-term survival analysis of pure laparoscopic versus open hepatectomy for hepatocellular carcinoma in patients with cirrhosis: a single-center experience. Ann Surg 2013; 257: [PMID: DOI: /SLA.0b013e31827b947a] 26 Farid SG, Aldouri A, Morris-Stiff G, Khan AZ, Toogood GJ, Lodge JP, Prasad KR. Correlation between postoperative infective complications and long-term outcomes after hepatic resection for colorectal liver metastasis. Ann Surg 2010; 251: [PMID: DOI: /SLA.0b013e3] 27 Matsumata T, Kanematsu T, Okadome K, Sugimachi K. Possible transmission of serum hepatitis in liver surgery with the ultrasonic dissector. Surgery 1991; 109: [PMID: ] P- Reviewer: Kapoor S S- Editor: Gou SX L- Editor: Wang TQ E- Editor: Wang CH 8645 July 14, 2014 Volume 20 Issue 26

338 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. CLINICAL BRIEF TRIALS ARTICLE STUDY Lipid levels in serum and cancerous tissues of colorectal cancer patients Xin Zhang, Xian-Wen Zhao, Dong-Bo Liu, Cun-Zhi Han, Li-Li Du, Jie-Xiang Jing, Yan Wang Xin Zhang, Dong-Bo Liu, Department of Anus and Intestine Surgery, Provincial Cancer Hospital of Shanxi Province, Taiyuan 30013, Shanxi Province, China Xian-Wen Zhao, Cun-Zhi Han, Li-Li Du, Jie-Xiang Jing, Yan Wang, Laboratory of Cancer Etiology, Provincial Cancer Institute of Shanxi Province, Taiyuan 30013, Shanxi Province, China Author contributions: Zhang X and Du LL designed the study in addition to providing financial support for this work; Liu DB collected materials from the patients and obtained patient information; Han CZ, Du LL and Wang Y performed the majority of experiments; Jing JX and Zhao XW provided key reagents and analytical tools and were also involved in editing the manuscript; Du LL and Han CZ wrote the manuscript. Correspondence to: Li-Li Du, Associate Senior Technologist, Laboratory of Cancer Etiology, Provincial Cancer Institute of Shanxi Province, Zhigong New Street 3, Taiyuan 30013, Shanxi Province, China. byshcz@vip.163.com Telephone: Fax: Received: January 9, 2014 Revised: March 28, 2014 Accepted: April 21, 2014 Published online: July 14, 2014 Abstract AIM: To investigate the correlations between lipid metabolism disorder and the occurrence and development of colorectal cancer by monitoring the alterations in lipid levels in cancerous tissue and serum in patients with colorectal cancer. METHODS: The levels of total and free cholesterol (TCH and FCH), triglycerides (TG), low density lipoprotein-cholesterol (LDL-C), high density lipoprotein- cholesterol (HDL-C), apolipoprotein A1 (ApoA-1) and ApoB in serum of 206 patients with colorectal cancer, 70 patients with benign colorectal disease and 300 healthy participants, and in the cancerous tissue and paracancerous tissue of 152 patients with colorectal cancer were measured with an Olympus 600 auto-biochemical analyzer. The obtained data were statistically analyzed. RESULTS: Serum FCH level was significantly higher (1.9 ± 0.4 mmol/l vs 1.3 ± 0.3 mmol/l, 1.9 ± 0.4 mmol/l vs 1.2 ± 0.4 mmol/l, P < 0.05), whereas serum levels of TCH, LDL-C, ApoA-I and ApoB were significantly lower in patients with colorectal cancer than in patients with benign colorectal disease and healthy controls. The levels of FCH and TG in cancerous tissue were significantly lower (14.5 ± 9.6 μmol/g vs 19.3 ± 13.9 μmol/g, P < 0.05; 16.3 ± 19.8 μmol/g vs 44.1 ± 38.1 μmol/g, P < 0.05), whereas HDL-C level was significantly higher (7.9 ± 4.5 μmol/g vs 5.7 ± 3.9 μmol/g, P < 0.01) in cancerous tissue than in paracancerous tissue. The levels of TCH and TG in serum and the levels of TCH and HDL-C in cancerous tissue in patients with colorectal cancer were significantly correlated with TNM stage. The levels of TCH and LDL-C in serum were significantly lower, whereas HDL-C level in cancerous tissue was significantly higher in patients with lymph node metastasis than in patients without lymph node metastasis. The levels of TCH, FCH, TG, HDL-C and LDL-C in cancerous tissue were not significantly different from those in paracancerous tissue. The serum levels of FCH and TG were significantly higher, whereas serum HDL-C levels were significantly lower in patients with rectum cancer than in patients with colon cancer. CONCLUSION: The disordered and abnormally altered levels of lipids in cancerous tissue and serum of patients with colorectal cancer may be correlated with the occurrence and development of colorectal cancer Baishideng Publishing Group Inc. All rights reserved. Key words: Colorectal cancer; Correlation; Lipid level; Occurrence; Progression Core tip: The disordered and abnormally altered levels of lipids in cancerous tissue and serum of patients with colorectal cancer may be correlated with the occurrence and development of colorectal cancer July 14, 2014 Volume 20 Issue 26

339 Zhang X et al. Lipid levels in serum and cancerous tissues Zhang X, Zhao XW, Liu DB, Han CZ, Du LL, Jing JX, Wang Y. Lipid levels in serum and cancerous tissues of colorectal cancer patients. World J Gastroenterol 2014; 20(26): Available from: URL: v20/i26/8646.htm DOI: i INTRODUCTION In recent years, a large number of studies have demonstrated that abnormal levels of lipids are closely correlated with the initiation and progression of breast cancer [1,2]. In particular, there have been a number of reports in the literature regarding the relationship between abnormal lipid levels and colorectal cancer [3-6]. Dessi et al [7,8] observed changes in cholesterol levels in both an in vitro experimental study on cancer cells and an experimental study on the proliferation of normal cells. However, there have been inconsistencies as to whether this relationship is due to the altered lipid metabolism taking place after tumor formation or due to altered lipid metabolism favorable for tumor formation. In order to clarify the association between abnormal lipid metabolism and colorectal cancer, this study was designed to elucidate the associations between abnormal alterations in in vivo lipid metabolism and the occurrence and development of colorectal cancer by examining alterations in the levels of a number of relevant lipids in cancerous tissue, paracancerous tissue and serum in patients with colorectal cancer and compare them with those from patients with benign colorectal disease and healthy participants. MATERIALS AND METHODS Clinical data Patient group: A total of 260 patients with colorectal cancer including 166 patients with rectum carcinoma and 94 patients with colon cancer were recruited for this study. These patients were inpatients with colorectal cancer who were admitted to the Department of Anus and Intestine Surgery, Provincial Cancer Hospital of Shanxi Province from March 2008 to May Of these patients, 152 were male and 108 were female with a median age of 54 years, ranging from 35 to 75 years. Seventy patients with benign colorectal disease were also enrolled, including 20 with colonic polyps, 14 with adenoma of the colon, 21 with chronic colitis and 15 with ulcerative colitis. Of these patients, 45 were male and 25 were female with a median age of 53 years, ranging from 36 to 70 years. Disease in all the recruited patients was pathologically confirmed by both biopsy and colonoscopy, and by post-operative histopathologic examination. Control group: A total of 300 healthy participants were recruited as controls, including 80 who accompanied the inpatients and 220 who were considered healthy following physical examination. Of these participants, 170 were male and 130 were female with a median age of 52 years, ranging from 35 to 70 years. A history of colorectal cancer in these participants was eliminated. The protocols for this study were approved by The Ethic Committee of the Provincial Cancer Hospital of Shanxi Province. Informed consent was obtained from each of the recruited patients and the healthy participants. Collection of serum samples, cancerous tissue and paracancerous tissue Approximately 3 ml of venous blood was drawn from each of the patients with colorectal cancer and from the patients with benign colorectal disease who had not received any pre-operative therapies as well as the healthy participants who had fasted in the morning. The blood samples were centrifuged at 3000 rpm for 10 min. The separated serum samples were saved for subsequent measurement of blood lipid levels. Approximately 0.5 g of cancerous tissue and normal tissue at a distance of about 4 cm from the cancerous tissue (paracancerous tissue) were collected from 152 patients with colorectal cancer. These tissue samples were first rinsed with physiological saline solution and then frozen at -80 for the subsequent biochemical and histological examinations described below. Experimental methods Treatment of patient biopsies: The frozen cancerous tissue and paracancerous tissue were removed from the freezer and thawed naturally. Approximately 0.2 g of the thawed samples was taken from each sample and ground in a grinder with a small amount of arenaceous quartz into a homogeneous paste and 2 ml of ethanolpetroleum ether (1:1) mixture was added. This homogenate mixture was transferred to a 10-mL centrifuge tube, mixed thoroughly, extracted by shaking in an oscillator for 2 min and then centrifuged at 3000 rpm for 10 min. Approximately 0.5 ml of the supernatant was taken from each sample to measure the lipid levels in these tissues. Measurement of lipid levels: Total cholesterol (TCH), free cholesterol (FCH) and triglyceride (TG) were measured using enzymatic methods. High density lipoproteincholesterol (HDL-C) and low density lipoprotein-cholesterol (LDL-C) were measured using the three generation of homogeneous method. Apolipoprotein A1 (ApoA-1) and apolipoprotein B (ApoB) were measured using immunoturbidimetry. Instruments and reagents: An Olympus 600 autobiochemical analyzer was obtained from the Olympus Corporation (Tokyo, Japan). All the reagents used in this study were provided by DiaSys Diagnostic Systems (Shanghai) Co., Ltd (Shanghai, China) July 14, 2014 Volume 20 Issue 26

340 Zhang X et al. Lipid levels in serum and cancerous tissues Table 1 Comparison of serum lipid levels in patients with colorectal cancer, patients with benign colorectal disease and healthy participants (mmol/l) Items Statistical analysis SPSS13.0 software was used to perform t-test, variance and correlation analyses. The difference(s) between groups with P < 0.05 were regarded as statistically significant. RESULTS Patients with colorectal cancer (n = 260) Patients with benign colorectal disease (n = 70) Healthy participants (n = 300) P value TCH 4.8 ± 0.9 1,2 5.9 ± ± 0.9 < 0.05 < 0.01 FCH 1.9 ± 0.4 1,2 1.3 ± ± 0.4 < 0.01 < 0.01 TG 1.5 ± ± ± 0.7 > 0.05 HDL-C 1.4 ± ± ± 0.3 >0.05 LDL-C 3.1 ± 0.8 1,2 3.9 ± ± 0.8 < 0.01 < 0.01 ApoA-I (g/l) 1.4 ± 0.2 1,2 2.1 ± ± 1.5 < 0.01 < ApoB (g/l) 0.9 ± 0.3 1,2 1.8 ± ± 0.3 < 0.01 < Comparison between patients with colorectal cancer and patients with benign colorectal disease, P < 0.05 and P < 0.01, respectively; 2 Comparison between patients with colorectal cancer and healthy participants, P < 0.01 and P < 0.01, respectively. ApoA-1: Apolipoprotein A1; ApoB: Apolipoprotein B; FCH: Free cholesterol; HDL-C: High density lipoprotein-cholesterol: LDL-C: Low density lipoprotein-cholesterol; TCH: Total cholesterol; TG: Triglyceride. Comparison of the serum levels of lipids in patients with colorectal cancer, patients with benign colorectal disease and healthy participants The serum levels of TCH, LDL-C, ApoA-I and Apo-B in patients with colorectal cancer were significantly lower than those in patients with benign colorectal disease and healthy participants (P < 0.05), whereas the serum level of FCH was significantly lower in patients with colorectal cancer than in patients with benign colorectal disease and healthy participants (P < 0.01). However, there were no statistically significant differences in the serum levels of TG and HDL-C among the three groups (P < 0.05). There were no statistically significant differences in the serum levels of all lipids examined (TCH, FCH, TG, HDL-C, LDL-C, ApoA-I and Apo-B) between the patients with benign colorectal disease and the healthy participants (P < 0.05). These data are presented in Table 1. Table 2 Lipid levels in cancerous tissue and paracancerous tissue in patients with colorectal cancer (μmol/g) Group Cancerous tissue (n = 152) Paracancerous tissue (n = 152) P value TCH 17.8 ± ± 18.9 < 0.05 FCH 14.5 ± ± 13.9 < 0.01 TG 16.3 ± ± 38.1 < HDL-C 7.9 ± ± 3.9 < 0.01 LDL-C 4.4 ± ± 3.3 > 0.05 FCH: Free cholesterol; HDL-C: High density lipoprotein-cholesterol: LDL-C: Low density lipoprotein-cholesterol; TCH: Total cholesterol; TG: Triglyceride. Comparison of the levels of lipids in cancerous tissue and paracancerous tissue of patients with colorectal cancer The levels of TCH, FCH and TG in cancerous tissue were lower, whereas HDL-C level was significantly higher in cancerous tissues than those in paracancerous tissue of patients with colorectal cancer (P < 0.05 and P < 0.01, respectively). However, there was no statistically significant difference in LDL-C between the two types of tissues (P > 0.05). These data are presented in Table 2. Correlations between lipid levels in serum and cancerous tissue in patients with colorectal cancer and TNM stages The analysis of variance regarding lipid levels in serum and cancerous tissue of patients with colorectal cancer and TNM stages revealed that there were significant correlations between the levels of TCH, TG and HDL-C in cancerous tissue and serum of patients with colorectal cancer and clinical TNM stages of colorectal cancer. With pathogenic progression, the levels of TCH and TG in cancerous tissue and in serum of patients at TNM stages Ⅲ and Ⅳ were significantly reduced, whereas HDL-C level in cancerous tissue was significantly increased as compared to those in patients at TNM stages Ⅰ and Ⅱ (P < 0.05 and P < 0.01), respectively. These data are presented in Table 3. Comparison of the lipid levels in cancerous tissue and serum of patients with and without lymph node metastasis The levels of TCH and HDL-C in the cancerous tissue of patients with lymph node metastasis were significantly higher than those of patients without lymph node metastasis (P < 0.05), whereas TG levels in the cancerous tissue of patients with lymph node metastasis were significantly lower than those of patients without lymph node metastasis (P < 0.01). These data are presented in Table 4. Comparison of the levels of various lipids in cancerous tissue, paracancerous tissue and serum samples of patients with rectal cancer and those of patients with colon cancer There were no significant differences in the levels of FCH, TG, TCH, HDL-C and LDL-C in the cancerous tissue and paracancerous tissue between patients with rectal cancer and patients with colon cancer (P > 0.05). However, the levels of FCH and TG in the serum of patients with rectal cancer were 1.5 ± 0.47 mmol/l and 1.4 ± 0.59 mmol/l, respectively, which were significantly higher than those in the serum of patients with colon 8648 July 14, 2014 Volume 20 Issue 26

341 Zhang X et al. Lipid levels in serum and cancerous tissues Table 3 Correlations between serum lipid levels and cancerous tissue of patients with colorectal cancer with the tumor node metastasis stages Groups Case (n ) TCH FCH TG HDL-C LDL-C Tissue (μmol/g) Ⅰ ± ± ± ± ± 2.3 Ⅱ ± ± ± ± ± 2.9 Ⅲ ± ± ± ± ± 3.4 Ⅳ ± 5.3 a 13.1 ± ± 9.4 a 9.3 ± 3.2 a 4.2 ± 2.7 Serum (mmol/l) Ⅰ ± ± ± ± ± 1.3 Ⅱ ± ± ± ± ± 0.9 Ⅲ ± ± ± ± ± 0.9 Ⅳ ± 0.9 d 1.3 ± ± 0.5 d 1.1 ± ± 0.8 a P < 0.05 vs the levels of total cholesterol (TCH) and high density lipoprotein-cholesterol (HDL-C) in cancerous tissues with tumor node metastasis (TNM) stages; d P < 0.01 vs the levels of TCH and triglyceride (TG) in serum with TNM stages. LDL-C: Low density lipoprotein-cholesterol. Table 4 Lipid levels in serum and cancerous tissues of patients with and without lymph node metastasis Group Case (n ) TCH FCH TG HDL-C LDL-C Tissue (μmol/g) With lymph node metastasis ± ± ± ± ± 3.4 Without lymph node metastasis ± ± ± ± ± 2.9 P < > 0.05 < < > 0.05 Serum (mmol/l) With lymph node metastasis ± ± ± ± ± 0.8 Without lymph node metastasis ± ± ± ± ± 0.7 P < < > 0.05 < > Indicates the comparison between lipid levels in cancerous tissue of patients with and without lymph node metastasis; 2 Indicates the comparison between lipid levels in serum of patients with and without lymph node metastasis. FCH: Free cholesterol; HDL-C: High density lipoprotein-cholesterol; LDL-C: Low density lipoprotein-cholesterol; TCH: Total cholesterol; TG: Triglyceride. total of Korean adults and standardized their medical examinations once every two years. They found that a total of males and females were diagnosed with primary cancers. Among them, the occurrence of rectum cancer and prostate cancer in males and breast cancer in females was positively correlated with TCH levels with a HR value of 1.12, 95%CI of and P value of 0.05 for male rectum cancer; HR value of 1.12, 95%CI: and P value < 0.01 for prostate cancer, and a HR value of 1.17, 95%CI: and P = 0.03 for female breast cancer, respectively. Yamada et al [9] also found that after adjustment for such factors as age, sex, body mass index, cigarette smoking and alcohol intake, the TCH level in serum was positively correlated with the occurrence of colorectal cancer in situ. A study by Chung et al [6] indicated a negative correlation of serum TCH level with the risk of colorectal cancer occurrence with an OR value of 0.3 and 95%CI: The reason for this correlation may be due to nutritional alterations and abnormal metabolism in patients with colorectal cancer. Tomiki et al [10] conducted a casecontrol study and their results indicated that TCH levels in patients with colorectal cancer, gastric cancer, and esophageal cancer were significantly lower than those in healthy control participants. The study by Bird et al [11] indicated that after adjustment for relevant influencing factors such as obesity, physical activity, refined carbohycancer, which were 1.4 ± 0.38 and 1.3 ± 0.36 mmol/l, respectively (P < 0.01). The serum HDL-C level in patients with rectum cancer was 1.21 ± 0.37 mmol/l, which was significantly lower than 1.43 ± 0.75 mmol/l in patients with colon cancer (P < 0.01). DISCUSSION Cholesterol is present in every tissue/organ and is the most abundant steroid compound in the human body. As an important component of cell membranes, cholesterol plays important physiological roles. When cholesterol is deficient in humans, cellular rigidity is increased and the cells are easily fractured. TCH is the sum of a variety of lipid molecules and lipoproteins that contain cholesterol, including FCH, HDL-C and LDL-C. Cholesterol is present in the human body in many forms. For instance, when present as HDL-C it is transported from the extrahepatic tissues via HLD to the liver where it is further metabolized and finally secreted out of the body. When it is present as LDL-C, it is transported via LDL-C to various tissues/organs. Apolipoproteins Apo-A1 and Apo-B are correlated with the metabolism and function of HDL and LDL. Both HDL and LDL are involved in cholesterol transport, and thus have certain relationships with the occurrence and progression of cancers. Kitahara et al [5] conducted a 14-year follow-up in a 8649 July 14, 2014 Volume 20 Issue 26

342 Zhang X et al. Lipid levels in serum and cancerous tissues drates and alcohol intake, the serum TCH level was not inversely related to the risk of colorectal polyps. Kostić et al [12] performed a study on the correlations between the levels of total lipids and TCH and benign and malignant tumors of the colon and rectum. Their statistical analysis of the obtained data revealed that hypocholesterolemia was associated with the incidence of adenocarcinoma. Gaard et al [13] conducted a 7-13 year prospective study in participants and found 186 cases with rectum cancer and 106 cases with colon cancer. Their statistical analyses revealed no association between the levels of blood lipids and lipoproteins and the risk of rectum cancer and colon cancer. While there was a statistically significant difference in this risk among females, this was regarded as incidental. The results obtained in the present study indicated that the serum FCH level in patients with colorectal cancer was significantly increased, whereas TCH levels were significantly decreased as compared to those in patients with benign colorectal disease and healthy participants. Additionally, in this study, we also found that the FCH level in the cancerous tissue of patients with colon cancer was significantly lower than that in their paracancerous tissue. These results are completely inconsistent with those described above. This disparity requires further investigation. TG is one of the most abundant lipids in the human body. Most tissues utilize the energy derived from the products of TG hydrolysis. Under normal conditions, the storage, transport and exchange of lipids in the human body are maintained in a state of dynamic balance. When cancer occurs, the physiological balance of lipid levels is destroyed, leading to lipid metabolism disorders. Yamada et al [9] found that TG level was positively correlated with the occurrence of colorectal carcinoma in situ. The results of a population study conducted by Sun et al [14] indicated that higher serum TG level was generally related to a higher risk of tubulovillous/villous adenoma in the rectosigmoid colon. A study by Chung et al [6] indicated that serum TG level was negatively and strongly correlated with the risk of colorectal cancer with an OR value of 0.2 and 95%CI: , whereas the study by Bird et al [11] indicated that after adjustment for factors such as obesity, physical activity, refined carbohydrates and alcohol intake, high serum TG level was correlated with the risk of adenomas in the left colon and rectum. A case-control study conducted by Tomiki et al [10] indicated no significant difference in TG between cancer-bearing cases and controls. The results of the present study revealed no statistically significant difference in serum TG level between patients with colorectal cancer, patients with benign colorectal disease and the healthy control group. These results are consistent with those reported by Tomiki et al [10]. The results of the present study also indicated that TG levels in cancerous tissue of patients with rectum cancer and patients with colon cancer were significantly lower than those in their corresponding paracancerous tissue. However, the mechanisms underlying these differences are not clear at present and require further investigation. Apolipoproteins ApoA-I and ApoB are the major components of serum lipoproteins and are correspondingly related to the metabolism and function of HDL and LDL. They are also related, to an extent, to the occurrence and development of tumors. Kostić et al [12] investigated the relations between the levels of total lipids, TCH, LDL, HDL, phospholipids, gastrin and insulin and both benign and malignant tumors of the colon and rectum. After conducting statistical analyses of the obtained results, they found that only hyperlipidemic LDL-C was related to the risk of colorectal cancer. A case-control study by Tomiki et al [10] indicated that the levels of TC and LDL-C in the serum of patients with colorectal cancer were significantly lower than those in the controls, whereas no significant differences in serum levels of HDL-C and TG were seen between patients with gastrointestinal cancer and controls. van Duijnhoven et al [15] reported that the concentrations of HDL-C and ApoA-1 were negatively correlated with the risk of colorectal cancer with a RR value of 0.78 and 95%CI: for HDL-C, and a RR value of 0.82 and 95%CI: for ApoA-1, respectively. Their results indicate that high concentrations of serum HDL-C are related to the reduced risk of rectum cancer. However, the mechanisms underlying these correlations require further investigation. The results of the present study show that serum Apo-A1 concentration in patients with colorectal cancer was significantly lower than those in patients with benign colorectal disease and in healthy controls. These results are consistent with those reported by van Duijnhoven et al [15]. There were no statistically significant differences in serum HDL-C levels between these groups. In this study, we also found that lipid alterations in patients with colorectal cancer were closely related to several clinical characteristics. With the progression of TNM stage, the serum levels of TCH and TG and the levels of TCH in cancerous tissues were decreased, whereas HDL-C levels were increased. The serum TCH and HDL-C levels in patients with lymph node metastasis were decreased, whereas HDL-C level was decreased and HDL-C in cancerous tissue was increased. There were no significant differences in the levels of lipids in both serum and cancerous tissue between the histological types. Notarnicola et al [16] suggested that the levels of TCH, LDL-C and the ratio of LDL-C/HDL-C were significant higher in patients with distant metastasis and in patients without distant metastasis, and the increased levels of these lipids may promote distant metastasis in patients with colorectal cancer. These results showed that lipid alterations in patients with colorectal cancer may be related to the degree of malignancy of tumors, i.e., the higher the degree of malignancy and the later the TNM stage, there is a higher demand for cholesterol which is taken up by cancer cells. A case-control study conducted by Tomiki et al [10] indicated that in patients with earlier TNM stage, the TCH level was significantly lower. However, the low level of TCH was not correlated with clinical stage. There are a large number of reports in the literature 8650 July 14, 2014 Volume 20 Issue 26

343 Zhang X et al. Lipid levels in serum and cancerous tissues indicating that abnormally altered lipid levels are related to colorectal adenoma. For instance, Shinomiya et al [17] conducted a comparative investigation on serum lipid levels and ApoE genotype in 205 Japanese patients with colorectal adenoma and 220 cases that were confirmed to be normal by colonoscopy, to examine the relation between both serum lipids and ApoE genotype and colorectal adenomas. They reported that after adjustment for body mass index, cigarette smoking, alcohol intake and other related factors, the odds ratios of proximal and distal adenomas were associated with the presence of allele E4. Serum levels of TC and LDL-C were not related to both proximal and distal adenomas. However, serum TG was positively related to distal adenomas. These data suggest that altered lipid metabolism may be differentially associated with tumorigenesis of the proximal and distal colorectum. The study by Zhongyin et al [18] indicated that the risk of colorectal adenomas for populations carrying ApoE E3/E4 genotype was lower than that for populations carrying other genotypes, and altered lipid metabolism may reduce the risk of colorectal cancer. Tabuchi et al [19] conducted a multiple logistic regression analysis of the correlation between the incidence of colorectal adenoma or carcinoma and fasting serum levels of TC and TG in Japanese patients and revealed that TG was an independent correlation factor in male (P < 0.01), but not in female patients. The TG level in patients with invasive carcinoma was not significantly higher than that in patients with adenoma, suggesting that hypertriglyceridemia may be an independent risk factor for colonic adenoma in men. In conducting an analysis of plasma lipid metabolism in patients with colorectal adenoma, Li et al [20] found that dyslipidemia may affect the incidence of colorectal adenoma, particularly at hypertriglyceridemia and low HDL-C levels, which may be related to the occurrence of colorectal adenoma. Yang et al [21] performed a large scale cross-sectional study on Korean participants and identified 5958 participants with colorectal adenomas including 5504 with non-advanced adenomas and 454 with advanced adenomas. They found that higher serum TG level was significantly associated with an increased prevalence of both non-advanced and advanced colorectal adenomas, while higher levels of ApoA-1 and HDL-C were significantly associated with an increased prevalence of non-advanced adenomas. The results of the present study showed no statistically significant differences in serum lipid levels and tissue lipid levels between patients with benign colorectal disease and healthy controls. Under normal conditions, the storage, transport and exchange of lipids within the human body are maintained in a normal state of dynamic balance. The data obtained from the present study indicated that when colorectal cancer occurred, the serum levels of TCH, LDL-C, Apo- AI, and ApoB were reduced, whereas FCH level was increased. The levels of TCH, FCH and TG in cancerous tissue were reduced and HDL-C level was increased, indicating that when colorectal cancer developed, the physiological balance of lipids is destroyed, leading to lipid metabolism disorder. The mechanisms underlying lipid metabolism disorder are not completely understood. In vitro studies have confirmed that in cancer cells, in order to meet the increasing demand for cell proliferation, cholesterol anabolism in these cells is significantly enhanced, which is characterized by increased absorption, increased synthesis and increased activity of a key rate-limiting enzyme of cholesterol synthesis, 3-hydroxy-3-methylglutaryl-CoA reductase [5]. This may be the major reason for the decreased serum cholesterol level in patients with colorectal cancer. The decreased FCH level in cancerous tissue is presumed to be related to the reason why the synthesis and transport of cholesterol cannot meet the demand for the rapid proliferation of cancer cells. Dessi et al [22] reported that the cholesterol level in cancerous tissue was increased due to the deposition of cholesterol in this tissue. However, this appears to contradict the growth characteristics of cancer cells and requires further investigation. HDL-C level was decreased in serum, but was increased in cancerous tissue and the LDL-C level was decreased in serum, indicating that both HDL and LDL are involved in cholesterol transport during proliferation of cancerous tissue. It is likely that the rate of cholesterol transport via HDL from extra-hepatic tissues is reduced, whereas the rate of cholesterol via LDL to other tissues is accelerated. Apolipoproteins ApoA-I and ApoB serve as structural proteins for HDL and LDL, respectively. Their alteration may be related to the parallel alterations in lipoproteins. Whether the decreased TG level in cancerous tissue shares a similar mechanism remains to be investigated. ACKNOWLEDGMENTS We thank Drs. Xiu-Ying Liu and Min-Sheng Lei of the Department of Clinical Laboratory, Cancer Hospital of Shanxi Province for their help with this research. COMMENTS Background Abnormal lipid metabolism has been found to be associated with several types of cancer including colorectal cancer. However, whether this association is due to the altered lipid metabolism taking place after tumor formation or due to altered lipid metabolism favorable for tumor formation is not clear and requires clarification. Research frontiers One of the hotspots in current cancer research is the determination of the correlations between metabolic disorders, including lipid metabolism disorders, and the occurrence and development of colorectal cancer. Innovations and breakthroughs This study attempted to elucidate the associations between abnormal alterations in in vivo lipid metabolism and the occurrence and development of colorectal cancer. Applications The results of this study indicate that when colorectal cancer develops in humans, the physiological balance of lipids is destroyed, leading to lipid metabolism disorders. Thus, the application of pharmacological approaches targeting abnormal lipid metabolism in cancer cells to maintain the physiological balance of lipids may be a potentially promising treatment option in patients with colorectal cancer July 14, 2014 Volume 20 Issue 26

344 Zhang X et al. Lipid levels in serum and cancerous tissues Peer review In this paper, authors compared the serum lipids levels of the colorectal cancer and benign illnesses and the health participants, also compared the lipids levels of the cancerous and paracancerous tissue. Moreover they also assessed relation between the lipids levels and the tumor node metastasis stages and lymph node metastasis in patients with colorectal cancer. Therefore authors provided a lot of useful information for readers in this paper. REFERENCES 1 Han CZ, Du LL, Liu XY, Jing JX, Zhao XW, Tian BG. Study on the correlation between serum leptin and lipids level and breast cancer. Yingyang Xuebao 2005; 5: Chang SJ, Hou MF, Tsai SM, Wu SH, Hou LA, Ma H, Shann TY, Wu SH, Tsai LY. The association between lipid profiles and breast cancer among Taiwanese women. Clin Chem Lab Med 2007; 45: [PMID: DOI: / CCLM ] 3 Rose G, Blackburn H, Keys A, Taylor HL, Kannel WB, Paul O, Reid DD, Stamler J. 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The association of serum lipids with colorectal adenomas. Am J Gastroenterol 2013; 108: [PMID: DOI: /ajg ] 22 Dessì S, Batetta B, Pulisci D, Spano O, Anchisi C, Tessitore L, Costelli P, Baccino FM, Aroasio E, Pani P. Cholesterol content in tumor tissues is inversely associated with highdensity lipoprotein cholesterol in serum in patients with gastrointestinal cancer. Cancer 1994; 73: [PMID: ] P- Reviewer: Gao CM S- Editor: Gou SX L- Editor: Webster JR E- Editor: Zhang DN 8652 July 14, 2014 Volume 20 Issue 26

345 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. CLINICAL BRIEF TRIALS ARTICLE STUDY Plasma free amino acid profiling of esophageal cancer using high-performance liquid chromatography spectroscopy Hong Ma, Ayshamgul Hasim, Batur Mamtimin, Bin Kong, Hai-Ping Zhang, Ilyar Sheyhidin Hong Ma, Hai-Ping Zhang, Ilyar Sheyhidin, Department of Thoracic Surgery, The First Affiliated Hospital, Medical University of Xinjiang, Urumqi , Xinjiang Uyghur Autonomous Region, China Hong Ma, Ayshamgul Hasim, Department of Pathology, College of Basic Medicine, Medical University of Xinjiang, Urumqi , Xinjiang Uyghur Autonomous Region, China Batur Mamtimin, Centers of Pharmaceutical Analysis, College of Pharmacy, Medical University of Xinjiang, Urumqi , Xinjiang Uyghur Autonomous Region, China Bin Kong, Pharmacy Department of Production and Construction Corps General Hospital of Xinjiang, Urumqi , Xinjiang Uyghur Autonomous Region, China Author contributions: Ma H and Hasim A contributed equally to this work; Ma H and Sheyhidin I designed the research; Kong B performed the experiments; Mamtimin B analyzed the data; Zhang HP collected plasma samples; and Ma H wrote the manuscript. Supported by National Natural Science Foundation of China, Grant No ; and Scientific Research Foundation of Xinjiang Medical University, Grant No. XJC Correspondence to: Ilyar Sheyhidin, Professor, Department of Thoracic Surgery, The First Affiliated Hospital, Medical University of Xinjiang, 137 Liyushan Road, Urumqi , Xinjiang Uyghur Autonomous Region, China. ilyarsha@hotmail.com Telephone: Fax: Received: January 27, 2014 Revised: March 17, 2014 Accepted: April 27, 2014 Published online: July 14, 2014 Abstract AIM: To perform plasma free amino acid (PFAA) profiling of esophageal squamous cell carcinoma (ESCC) patients at different pathological stages and healthy subjects. METHODS: Plasma samples from ESCC patients (n = 51) and healthy control adults (n = 60) were analyzed by high-performance liquid chromatography (HPLC). The ESCC patients included moderate/poorly-differentiation (n = 24), lymph node metastasis (n = 17) and clinical stage > Ib2 (n = 36). Partial least squares discriminant analysis was performed to demonstrate that the PFAA metabolic patterns enabled discrimination between ESCC patients and controls, and the Student t test was applied to assess significant differences in PFAA concentrations between the two groups. RESULTS: There were significant differences in the PFAA profiles between controls and ESCC patients. Compared with healthy controls, the levels of Asp, Glu, Gly, His, Thr, Tau, Ala, Met, Ile, Leu, and Phe were decreased in ESCC patients, but Cys was increased. There exists a strong correlation between PFAA profiles and clinicopathological characteristics in ESCC patients. The levels of many PFAAs (i.e., Glu, Asp, Ser, Gly, Tau, Ala, Tyr, Val, Ile, and Leu) were related to pathological grading, lymph node metastasis, and ESCC clinical stage. Very good discrimination between ESCC patients and control subjects was achieved by multivariate modeling of plasma profiles. CONCLUSION: HPLC-based plasma profiling analysis was shown to be an effective approach to differentiate between ESCC patients and controls. PFAA profiles may have potential value for screening or diagnosing ESCC Baishideng Publishing Group Inc. All rights reserved. Key words: Metabolomics; High-performance liquid chromatography; Esophageal squamous cell cancer; Plasma; Amino acids Core tip: Recently, metabolomics-based techniques have been developed to identify cancer-related metabolic signatures for early cancer detection. However, studies on esophageal squamous cell carcinoma (ESCC) remain limited. This study used high-performance liquid chromatography to quantitatively study plasma free amino acid (PFAA) changes in ESCC patient, and analyze the correlation between PFAA profiles and clinicopathological characteristics of ESCC. The results 8653 July 14, 2014 Volume 20 Issue 26

346 Ma H et al. Amino acid profiling of esophageal cancer showed that most of the amino acids were differentially expressed in ESCC patients and control subjects, and were related to the ESCC clinicopathological characteristics. The study suggests that PFAA profiling is of potential value for screening or diagnosing ESCC. Ma H, Hasim A, Mamtimin B, Kong B, Zhang HP, Sheyhidin I. Plasma free amino acid profiling of esophageal cancer using high-performance liquid chromatography spectroscopy. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: INTRODUCTION Esophageal cancer (EC) is the 8 th most common cancer worldwide, and ranked 6 th as the leading cause of cancerrelated deaths [1]. Approximately individuals worldwide die from EC every year, with cases in China [2]. Esophageal squamous cell carcinoma (ESCC) is the most common histologic EC type in China. Xinjiang is one of the high-risk areas in China, with an adjusted mortality rate of Kazakh EC as high as 155.9/ in Toli county of northern Xinjiang [3], which is higher than different ethnicities of the same area and the average rate in other nations. EC has become the leading cause of cancer deaths in the Kazakh ethnic group. Because of the lack of specific signs or symptoms at an early stage, the majority of patients present at an advanced stage. Despite recent advances in multimodal treatment, overall 5-year survival rates for advanced EC are only 5%-15% due to lymph node metastasis and local recurrence [4,5]. Currently, conventional endoscopic detection combining biopsy is the main method for EC diagnosis; however, it is associated with significant limitations because dysplastic and early carcinomatous lesions are not macroscopically visible. Therefore, a screening method that facilitates early detection of EC is of crucial clinical importance. Amino acids are biologically important organic compounds, which play central roles both as building blocks of proteins and as intermediates in metabolism. The free amino acids are distributed throughout the body to participate in metabolism, and are termed the amino acid pool. Malignancy is characterized by the fast speed of cell multiplication and enhanced metabolism, showing that malignant cells require a large number of amino acids from the amino acid pool to synthesize protein and nucleic acid [6]. Consequently, changes in the plasma free amino acid (PFAA) profile might reflect the cancer-induced protein metabolism in tumors, skeletal muscle, and liver in cancer patients. However, previous studies reported that the PFAA profile is not the same in different cancers [7]. Miyagi et al [8] investigated the differences in PFAA profiles from five types of cancer patients, including lung cancer, gastric cancer, colorectal cancer, breast cancer, and prostate cancer. The results showed that some of the amino acids (such as tyrosine, glycine, leucine, phenylalanine) had a close link with the specific cancers, indicating that PFAA profiles correlate with the organ-site origin among the five different cancers. Regarding ESCC, few studies have focused on the quantitation of PFAA profiles. Recently, metabolomics has developed rapidly as a branch of systems biology. Metabolomics studies the global assessment and validation of endogenous smallmolecule biochemicals (metabolites) within a biological system, including cells, tissues, or organisms [9,10]. These metabolites may directly or indirectly interact with molecular targets and thereby influence the risk and complications associated with various diseases, including cancer [11]. Metabolomics-based methods have been widely used in disease diagnosis, biomarker screening, gene modifications, drug efficacy and toxicity [12-14]. The main analytical techniques for metabolomic studies are based on nuclear magnetic resonance (NMR), gas chromatography/liquid chromatography-mass spectrometry and highperformance liquid chromatography (HPLC). HPLC as an outstanding metabolomic tool together with multivariate statistics is widely used in metabolite identification and quantification. Compared with other analytical techniques, HPLC has already shown excellent discrimination and high sensitivity, with minimal effort and at a reasonable cost [15]. Previously, we described the application of 1 HNMRbased metabolomics in differentiating the metabolic profiles of plasma in ESCC patients and healthy controls. We discovered that a series of metabolites in glucose metabolism, fatty acid metabolism and the tricarboxylic acid cycle showed an altered expression level in ESCC patient s plasma [16]. In this study, we used HPLC to quantitatively detect PFAA profiles of ESCC patients and healthy people, and systematically analyzed the discriminating metabolites in the different pathological stages of ESCC. Our study may be helpful for improving non-invasive ESCC screening or diagnosis and for providing novel insights about EC metabolism. MATERIALS AND METHODS Patients and samples This study was approved by the Ethics Committee of the Medical University of Xinjiang. Written informed consent was obtained from all participants before study participation. Plasma samples were collected preoperatively from Kazakh patients from Xinjiang with ESCC (n = 51) at the Department of Thoracic Surgery of the First Affiliated Hospital in Medical University of Xinjiang, between June 2010 and March Patients were between 37 and 80 years old (mean age: 57 years), and had not received chemoradiation therapy. Tumors were pathologically confirmed and classified according to World Health Organization classification standards. The patients included 27 cases of well-differentiated and 24 cases of moderately/ poorly-differentiated ESCC. Of these patients, 17 cases 8654 July 14, 2014 Volume 20 Issue 26

347 Ma H et al. Amino acid profiling of esophageal cancer Val His Thr Ala Pro Leu Lys Phe EU Asp Glu Asn Ser Gly Tau Arg Cys Tyr Ile met t /min Figure 1 Chromatogram of 19 amino acids. Retention times of 6.1, 7.7, 10.1, 12.4, 12.8, 13.8, 14.1, 17.0, 17.3, 17.8, 18.2, 23.9, 24.1, 25.0, 25.8, 27.9, 28.3, 28.5 and 29.4 min correspond to aspartate (Asp), glutamate (Glu), asparagine (Asn), serine (Ser), glycine (Gly), histidine (His), arginine (Arg), threonine (Thr), taurine (Tau), alanine (Ala), proline (Pro), cysteine (Cys), tyrosine (Tyr), valine (Val), methionine (Met), lysine (Lys), isoleucine (Ile), leucine (Leu), and phenylalanine (Phe), respectively. were positive for lymph node metastasis, 15 cases had stage Ib2 disease and 36 cases had stage > Ib2 disease. Controls (n = 60) were healthy age- and sex-matched male and female volunteers. Exclusion criteria included prior or concurrent neoplasm, or cardiovascular, hepatic, renal, or inflammatory disease. Blood samples were collected from patients and controls between 7-8 am, were centrifuged at 4000 rpm for 10 min, and plasma samples were isolated and stored at -80 until use. HPLC measurement of PFAAs Plasma samples were thawed in a 4 water bath, and vortexed with 400 μl of methyl cyanide for 30 s to precipitate proteins. This solution was again centrifuged at rpm for 15 min, and the separated supernatant was transferred to 1.5 ml microtubes and vortexed for 30 s. Ten microliter aliquots of supernatant were sampled by amino acid-calibrated HPLC (Alliance e2695 HPLC unit; Waters Corp., Milford, MA, United States). Ten microliters of plasma supernatant were mixed in 60 μl of boric acid solution (ph 8.8) and vortexed for 30 s, followed by warming in a 55 water bath for 15 min. Five microliters of each sample were injected into the HPLC system for analysis. Each standard was individually run on the gradient noted in the LC parameters program (reaction temperature 35, gradient elution analysis). The extracted phenols were detected by an octadecylsilyl column with a gradient elution and programmable fluorescence wavelength detector, column size 250 mm 4.6 mm, flow rate 1.0 ml per detection, excitation wavelength 235 nm, emission wavelength 395 nm, injection volume 5 μl). Two consecutive runs with the same retention times were taken, and the means were plotted for calibration graphs. From the chromatograms thus obtained, the areas of the peaks were recorded with the standards and used for calibration. The following 19 amino acids were analyzed: aspartate (Asp), glutamate (Glu), asparagine (Asn), serine (Ser), glycine (Gly), histidine (His), arginine (Arg), threonine (Thr), taurine (Tau), alanine (Ala), proline (Pro), cysteine (Cys), tyrosine (Tyr), valine (Val), methionine (Met), lysine (Lys), isoleucine (Ile), leucine (Leu), and phenylalanine (Phe). The absolute concentration of each amino acid was expressed in μmol/l. Statistical analysis The metabolic data were analyzed statistically using SPSS v.17.0 software (SPSS Inc., Chicago, IL, United States), and the mean amino acid concentrations standard deviations (SDs) were calculated to determine PFAA profiles for both ESCC patients and controls. The Student t-test was used to assess significant differences in the PFAA concentrations between cancer patients and controls. P-values < 0.05 were considered statistically significant. After the chromatographic peak area was normalized, partial least squares-discriminant analysis (PLS-DA) was performed to construct plasma amino acid metabolic profiles of ESCC patients and controls. RESULTS Chromatographic separation The representative chromatograms of human plasma amino acids are shown in Figure 1. The method demonstrated good chromatographic separation of all the amino acids within a short analysis time. Retention times for Asp, Glu, Asn, Ser, Gly, His, Tau, Arg, Thr, Ala, Pro, Cys, Tyr, Val, Met, Ile, Leu, Lys, and Phe were approximately 6.1, 7.7, 10.1, 12.4, 12.8,13.8, 14.1, 17.0, 17.3, 17.8, 18.2, 23.9, 24.1, 25.0, 25.8, 27.9, 28.3, 28.5 and 29.4 min, respectively. There was no interference from other amino acids or other endogenous plasma compounds. Analysis of human plasma samples Univariate analysis was used to compare the PFAA profiles of controls vs ESCC patients. The levels of each amino acid are shown in Table 1. There were significant differences in the PFAA profiles between the controls and the ESCC patients. Compared with healthy controls, the plasma concentrations of all PFAAs were decreased in ESCC patients, except for Cys. The plasma concentrations of Asp, Glu, Gly, His, Thr, Tau, Ala, Met, Ile, Leu, 8655 July 14, 2014 Volume 20 Issue 26

348 Ma H et al. Amino acid profiling of esophageal cancer Table 1 Concentration of plasma free amino acids in controls and esophageal squamous cell carcinoma patients Amino acid (μmol/l) Control ESCC P value Aspartate 0.04 ± ± 0.07 < Glutamate 0.37 ± ± 0.06 < Asparagine 0.06 ± ± Serine 0.27 ± ± Glycine 0.14 ± ± 0.03 < Histidine 0.28 ± ± 0.11 < Arginine 0.09 ± ± Threonine 0.21 ± ± 0.08 < Taurine 0.26 ± ± 0.12 < Alanine 0.98 ± ± 0.18 < Proline 0.08 ± ± Cystine 0.01 ± ± Tyrosine 0.07 ± ± Valine 0.24 ± ± Methionine 0.12 ± ± 0.01 < Lysine 0.09 ± ± Isoleucine 0.29 ± ± 0.08 < Leucine 0.21 ± ± 0.08 < Phenylalanine 0.12 ± ± 0.03 < and Phe were significantly decreased (P < 0.05) in ESCC patients compared with controls. Association of PFAA profiles with ESCC clinicopathological characteristics We further evaluated the association of PFAA profiles with tumor invasiveness, metastasis, and differentiation. All ESCC cases were classified by different pathologic criteria, including lymph node metastasis, differentiation grade, and clinical stage. Statistical analysis showed that the concentrations of most amino acids were significantly associated with lymph node metastasis, differentiation grade, and clinical stage (P < 0.05 in all cases). When comparing positive lymph node metastases vs negative node metastases, except for Arg, Thr, and Cys, the concentrations of the remaining tested amino acids were decreased (Table 2). The PFAA levels were significantly lower in patients with late-stage disease (> Ib2) vs earlystage disease ( Ib2), except for Asp, Arg, Pro, Cys, and Lys (Table 2). The PFAAs were also lower in all cancer patients with middle/poorly differentiated cancers vs well differentiated cancers, except for His, Pro, Met, and Phe (Table 2). Pattern recognition SIMCA-P 11 (Umetrics Inc., Umea, Sweden) software is used for PLS-DA of the data from HPLC. The scores plot shows each set of two groups scattering into different regions, representing a good separation of the cancer from non-cancer cases with corresponding plasma amino acid pattern, and suggested that the ESCC have a specific profile distinguishable from healthy controls (Figure 2). DISCUSSION EC is one of the most frequent malignancies in China, and is a serious threat to human health. Effectively im- proving EC prognoses is directly dependent upon early diagnosis and treatment. Tumor biological markers such as carcinoembryonic antigen and epithelial membrane antigen are not useful for early EC diagnosis, because of low sensitivity and specificity. HPLC is one of the most powerful chromatographic techniques, and can often easily achieve separations and analyses that would be difficult or impossible using other forms of chromatography. In the present study, we performed PFAA profiling using HPLC spectroscopy, and identified useful metabolic markers for ESCC screening or diagnosis. The results showed that most of the amino acids were differentially expressed in ESCC patients and control subjects, and were related to the ESCC clinicopathological characteristics. The PLS-DA model identified patients with ESCC and healthy controls, suggesting that PFAA profiling is useful for screening or diagnosis of ESCC. In previous studies, the changes in PFAA profiles in cancer patients were sometimes inconsistent [17]. Some discrepancies exist between our current study and other prior reports. For example, Shingyoji et al [18] reported that HPLC-determined plasma Gly levels are increased in lung cancer patients. However, plasma Gly was decreased in ESCC patients in our study. This difference may be due not only to sample size but also to cancers from different organs having different PFAA profiles. There are also many similarities between our results and previous studies. Our study showed a significant reduction in gluconeogenic amino acids (such as Ala, His, and Gly) in ESCC patients, which is similar to that seen in esophageal adenocarcinoma plasma metabolomics as well as in other cancers [14,19]. This reveals a typical signature in cancer patients, and it has previously been proven that tumors rely on glycolysis as a main source of energy, even in the presence of oxygen [20]. Recent research has shown that several non-essential amino acids (i.e., Glu, Ser, and Gly) play critical roles in cancer metabolism [21]. In the present study, the levels of Gly, Ala, Glu, and Asp were significantly decreased in ESCC patients vs healthy controls. Gly is the best discriminating amino acid because it is an important intermediate in folate metabolism, which is especially altered in colon cancer [22]. Due to its recently demonstrated bowel-protective effects and its easy administration, Gly is a highly interesting candidate for therapeutic approaches [23]. It has been reported that Gly consumption and expression of the mitochondrial Gly biosynthetic pathway proteins are strongly correlated with rapid cancer cell proliferation [24]. In our study, plasma Gly concentrations were decreased in ESCC patients, and were associated with poorly-differentiation, non-lymph node metastasis, and late stage disease (> Ib2). These results further confirm that disturbances in Gly metabolism can promote tumor growth and proliferation. Ala was described as a precursor of gluconeogenesis because it is the key protein-derived glucose precursor used by the liver [25]. Tessem et al [26] used 1 H high-resolution magic angle spinning spectroscopy approach to investigate the potential role of Ala as metabolic biomarker 8656 July 14, 2014 Volume 20 Issue 26

349 Ma H et al. Amino acid profiling of esophageal cancer Table 2 Association of plasma free amino acid profiles with clinicopathological characteristics Pathological differentiation Lymph node metastasis Clinical stage Amino acid Mid/poor Well P value Positive Negative P value Ib2 > Ib2 P value Aspartate 0.06 ± ± ± ± ± ± Glutamate 0.03 ± ± 0.05 < ± ± 0.06 < ± ± 0.06 < Asparagine 0.07 ± ± 0.16 < ± ± ± ± 0.02 < Serine 0.11 ± ± 0.11 < ± ± 0.14 < ± ± Glycine 0.06 ± ± 0.02 < ± ± 0.10 < ± ± Histidine 0.06 ± ± ± ± 0.03 < ± ± 0.03 < Arginine 0.05 ± ± 0.09 < ± ± ± ± Threonine 0.10 ± ± ± ± ± ± 0.07 < Taurine 0.12 ± ± 0.20 < ± ± 0.13 < ± ± Alanine 0.04 ± ± ± ± 0.18 < ± ± Proline 0.02 ± ± ± ± ± ± Cystine 0.04 ± ± 0.03 < ± ± ± ± Tyrosine 0.12 ± ± 0.38 < ± ± 0.03 < ± ± 0.02 < Valine 0.02 ± ± 0.01 < ± ± ± ± Methionine 0.07 ± ± ± ± 0.01 < ± ± 0.01 < Lysine 0.07 ± ± 0.06 < ± ± ± ± Isoleucine 0.06 ± ± 0.07 < ± ± 0.07 < ± ± 0.05 < Leucine 0.05 ± ± 0.02 < ± ± 0.08 < ± ± 0.05 < Phenylalanine 0.00 ± ± ± ± 0.03 < ± ± 0.02 < t [2] t [1] Figure 2 Profiling of high-performance liquid chromatography spectra from patients with ESCC and healthy donors by partial least squares-discriminant analysis. Partial least squares-discriminant analysis (PLS-DA) model with plasma of ESCC subjects (filled square) and healthy subjects (filled triangle). Model parameters r 2 x = 0.53, r 2 y = 0.58, and Q 2 = in prostate biopsy tissues, and found that Ala levels were significantly higher in prostate cancer vs benign prostate tissues. In our ESCC patients, especially those with latestage disease, Ala levels were decreased significantly compared with healthy controls. This observation may reflect that malignant tumors are associated with increased glycolytic flux. This is consistent with the need for increased protein synthesis in tumors, which leads to increased Ala consumption by the tumor tissues, causing low plasma Ala levels in ESCC. Additionally, the transamination to Ala from pyruvate is catalyzed by the enzyme alanine transaminase (ALT); therefore, further studies are needed to establish the activity of ALT and find the underlying biochemical pathways for Ala use in ESCC. Previous work suggests that Glu oxidation may be a major source of respiratory energy for tumor cells [27]. Our present results showed that Glu levels was decreased in plasma of ESCC patients, which is in agreement with other studies that showed Glu transamination primarily to pyruvate to form Ala [28]. This increased Ala concentration may enhance gluconeogenesis by proliferating cancer cells. The branched-chain amino acids, Leu, Ile, and Val, are essential amino acids that serve both as important energy substrates and as precursors for the synthesis of other amino acids and proteins [29]. In the present study, the Leu and Ile levels in the plasma of patients with ESCC were significantly lower than those in normal controls. The cause may be that a large number of amino acids were consumed as a substrate for synthesis of proteins and nucleic acids and other substances. As the source of one carbon unit, histidine supplies the formanimo to tetrahydrofolate, which participates in the synthesis of purines and pyrimidines. Because tumor cells have an active nucleic acid metabolism, histidine is excessively absorbed into tumor tissue, and consumption is markedly increased, leading to lower levels in the body July 14, 2014 Volume 20 Issue 26

350 Ma H et al. Amino acid profiling of esophageal cancer Furthermore, we noticed that the levels of a large group of PFAAs (Glu, Asp, Ser, Gly, Tau, Ala, Tyr, Val, Ile, and Leu) were decreased in poorly-differentiated, lymph node metastasizing, late stage (> Ib2) ESCC vs well-differentiated, non-lymph node metastasizing, and early stage ( Ib2) disease. The results showed that PFAA profiles were strongly correlated with ESCC tumor progression, and may probably reflect an increased demand for amino acids to supply the growing tumor. Thus, PFAA profiles may be valuable molecular markers for tumor progression, recurrence, and prognosis. In conclusion, PFAA profiles in ESCC patients are significantly different from those in healthy persons, using HPLC spectroscopy. Additionally, there exists a strong correlation between PFAA profiles and clinicopathological characteristics in ESCC patients. We believe that the non-invasive HPLC-based metabolic method has a potential role in improving early diagnosis and screening of high-escc risk populations, and in helping judge disease prognosis. In prospective studies, we will further verify our results using a larger sample size, and aim to identify the underlying mechanisms involved in the coupling of amino acid metabolism to altered reprogramming of gene expression in ESCC. COMMENTS Background Recently, metabolomics-based techniques have been developed to identify cancer-related metabolic signatures for early cancer detection. Among metabolites, profiling of plasma free amino acids (PFAAs) is a promising approach because PFAAs link all organ systems and have important roles in metabolism. However, several investigators have reported that PFAA profiles are not the same in different cancers, including esophageal squamous cell carcinoma (ESCC). Therefore, this study aimed to identify the PFAA profile of human plasma as a means to identify an ESCC-related metabolic signature, which may be helpful for improving non-invasive ESCC screening or diagnosis and for providing novel insights about ESCC metabolism. Research frontiers PFAA profiles have been shown to be influenced by metabolic variations in different solid tumors. Regarding ESCC, few studies have focused on the quantitation of PFAA profiles. In this study, authors used high-performance liquid chromatography to determine the PFAA profiles of patients with ESCC and compared the alterations between ESCC patients and controls using univariate analyses. Innovations and breakthroughs This is the first study to quantitatively detect PFAA profiles of ESCC patients and healthy people, and systematically analyze the discriminating metabolites in the different pathological stages of ESCC. The results showed that most of the amino acids were differentially expressed in ESCC patients and control subjects, and were related to the ESCC clinicopathological characteristics. The partial least squares-discriminant analysis model identified patients with ESCC and healthy controls, suggesting that PFAA profiling is useful for screening or diagnosis of ESCC. Applications PFAA profiles may be helpful for improving non-invasive ESCC screening or diagnosis and for providing novel insights about ESCC metabolism. Terminology Metabolomics is a quantitative measurement of the dynamic multiparametric metabolic response of living systems to pathophysiological stimuli or genetic modification. Amino acids and their metabolites, which play important roles as both basic substrates and regulators in many metabolic pathways. Amino acid metabolism in cancer patients differs from that in healthy people. Some studies indicated that free amino acid profiling is of potential value for screening or diagnosing cancer. Peer review The authors performed PFAA profile of ESCC and its association with clinicopathological characteristics. It revealed that most of the amino acids were differentially expressed in ESCC patients and controls, and were related to the ESCC clinicopathological characteristics. The results derived from this study are well-presented and has potential clinical value for the screening and diagnosis of esophageal cancer. REFERENCES 1 Kamangar F, Dores GM, Anderson WF. Patterns of cancer incidence, mortality, and prevalence across five continents: defining priorities to reduce cancer disparities in different geographic regions of the world. J Clin Oncol 2006; 24: [PMID: ] 2 Ruol A, Castoro C, Portale G, Cavallin F, Sileni VC, Cagol M, Alfieri R, Corti L, Boso C, Zaninotto G, Peracchia A, Ancona E. 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Metabolic profiling and the metabolome-wide association study: significance level for biomarker identification. J Proteome Res 2010; 9: [PMID: DOI: /pr ] 12 Hasim A, Ali M, Mamtimin B, Ma JQ, Li QZ, Abudula A. Metabonomic signature analysis of cervical carcinoma and precancerous lesions in women by (1)H NMR spectroscopy. Exp Ther Med 2012; 3: [PMID: ] 13 Spratlin JL, Serkova NJ, Eckhardt SG. Clinical applications of metabolomics in oncology: a review. Clin Cancer Res 2009; 15: [PMID: DOI: / CCR ] 14 Mamas M, Dunn WB, Neyses L, Goodacre R. The role of me July 14, 2014 Volume 20 Issue 26

351 Ma H et al. Amino acid profiling of esophageal cancer tabolites and metabolomics in clinically applicable biomarkers of disease. Arch Toxicol 2011; 85: 5-17 [PMID: DOI: /s ] 15 Shimbo K, Kubo S, Harada Y, Oonuki T, Yokokura T, Yoshida H, Amao M, Nakamura M, Kageyama N, Yamazaki J, Ozawa S, Hirayama K, Ando T, Miura J, Miyano H. Automated precolumn derivatization system for analyzing physiological amino acids by liquid chromatography/mass spectrometry. Biomed Chromatogr 2010; 24: [PMID: DOI: / bmc.1346] 16 Hasim A, Ma H, Mamtimin B, Abudula A, Niyaz M, Zhang LW, Anwer J, Sheyhidin I. Revealing the metabonomic variation of EC using ¹H-NMR spectroscopy and its association with the clinicopathological characteristics. Mol Biol Rep 2012; 39: [PMID: DOI: /s z] 17 Kubota A, Meguid MM, Hitch DC. Amino acid profiles correlate diagnostically with organ site in three kinds of malignant tumors. Cancer 1992; 69: [PMID: ] 18 Shingyoji M, Iizasa T, Higashiyama M, Imamura F, Saruki N, Imaizumi A, Yamamoto H, Daimon T, Tochikubo O, Mitsushima T, Yamakado M, Kimura H. The significance and robustness of a plasma free amino acid (PFAA) profile-based multiplex function for detecting lung cancer. BMC Cancer 2013; 13: 77 [PMID: DOI: / ] 19 Wang L, Chen J, Chen L, Deng P, Bu Q, Xiang P, Li M, Lu W, Xu Y, Lin H, Wu T, Wang H, Hu J, Shao X, Cen X, Zhao YL. 1H-NMR based metabonomic profiling of human esophageal cancer tissue. Mol Cancer 2013; 12: 25 [PMID: DOI: / ] 20 Garber K. Energy boost: the Warburg effect returns in a new theory of cancer. J Natl Cancer Inst 2004; 96: [PMID: ] 21 Phang JM, Liu W, Hancock C. Bridging epigenetics and metabolism: role of non-essential amino acids. Epigenetics 2013; 8: [PMID: DOI: /epi.24042] 22 Ong ES, Zou L, Li S, Cheah PY, Eu KW, Ong CN. Metabolic profiling in colorectal cancer reveals signature metabolic shifts during tumorigenesis. Mol Cell Proteomics 2010; Epub ahead of print [PMID: ] 23 de Aguiar Picanço E, Lopes-Paulo F, Marques RG, Diestel CF, Caetano CE, de Souza MV, Moscoso GM, Pazos HM. L-arginine and glycine supplementation in the repair of the irradiated colonic wall of rats. Int J Colorectal Dis 2011; 26: [PMID: DOI: /s ] 24 Jain M, Nilsson R, Sharma S, Madhusudhan N, Kitami T, Souza AL, Kafri R, Kirschner MW, Clish CB, Mootha VK. Metabolite profiling identifies a key role for glycine in rapid cancer cell proliferation. Science 2012; 336: [PMID: DOI: /science ] 25 Stumvoll M, Meyer C, Perriello G, Kreider M, Welle S, Gerich J. Human kidney and liver gluconeogenesis: evidence for organ substrate selectivity. Am J Physiol 1998; 274: E817-E826 [PMID: ] 26 Tessem MB, Swanson MG, Keshari KR, Albers MJ, Joun D, Tabatabai ZL, Simko JP, Shinohara K, Nelson SJ, Vigneron DB, Gribbestad IS, Kurhanewicz J. Evaluation of lactate and alanine as metabolic biomarkers of prostate cancer using 1H HR- MAS spectroscopy of biopsy tissues. Magn Reson Med 2008; 60: [PMID: DOI: /mrm.21694] 27 Moreadith RW, Lehninger AL. The pathways of glutamate and glutamine oxidation by tumor cell mitochondria. Role of mitochondrial NAD(P)+-dependent malic enzyme. J Biol Chem 1984; 259: [PMID: ] 28 Leichtle AB, Nuoffer JM, Ceglarek U, Kase J, Conrad T, Witzigmann H, Thiery J, Fiedler GM. Serum amino acid profiles and their alterations in colorectal cancer. Metabolomics 2012; 8: [PMID: ] 29 Tajiri K, Shimizu Y. Branched-chain amino acids in liver diseases. World J Gastroenterol 2013; 19: [PMID: DOI: /wjg.v19.i ] P- Reviewer: Chen GS S- Editor: Gou SX L- Editor: Cant MR E- Editor: Zhang DN 8659 July 14, 2014 Volume 20 Issue 26

352 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. CLINICAL TRIALS STUDY Outcomes of autologous bone marrow mononuclear cell transplantation in decompensated liver cirrhosis Yang-Qiu Bai, Yu-Xiu Yang, Ya-Ge Yang, Song-Ze Ding, Fang-Li Jin, Ming-Bo Cao, Yan-Rui Zhang, Bing-Yong Zhang Yang-Qiu Bai, Yu-Xiu Yang, Song-Ze Ding, Ming-Bo Cao, Yan-Rui Zhang, Bing-Yong Zhang, Department of Gastroenterology and Hepatology, Zhengzhou University People s Hospital, Zhengzhou , Henan Province, China Ya-Ge Yang, Department of Gastroenterology and Hepatology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou , Henan Province, China Fang-Li Jin, The Seventh People s Hospital of Zhengzhou City, Zhengzhou , Henan Province, China Author contributions: Yang YX and Zhang BY designed the experiment; Bai YQ, Yang YG and Jin FL performed the experiments; Bai YQ and Yang YG wrote the manuscript; Cao MB and Zhang YR coordinated the study; Ding SZ revised the manuscript. Supported by Grants for Key Bio-Medical Research Projects in Henan Province, China, No , No Correspondence to: Bing-Yong Zhang, MD, PhD, Department of Gastroenterology and Hepatology, Zhengzhou University People s Hospital, No. 7 Wei Wu Road, Zhengzhou , Henan Province, China. zhbingyong@sina.com.cn Telephone: Fax: Received: January 27, 2014 Revised: April 10, 2014 Accepted: May 12, 2014 Published online: July 14, 2014 Abstract AIM: To determine the long-term efficacy of autologous bone marrow mononuclear cells (BM-MNCs) transplantation in terms of improving liver function and reducing complications in patients with decompensated cirrhosis. METHODS: A total of 47 inpatients with decompensated liver cirrhosis were enrolled in this trial, including 32 patients undergoing a single BM-MNCs transplantation plus routine medical treatment, and 15 patients receiving medical treatment only as controls. Fortythree of 47 patients were infected with hepatitis B virus. Bone marrow of ml was obtained from each patient and the BM-MNCs suspension was transfused into the liver via the hepatic artery. The efficacy of BM-MNCs transplantation was monitored during a 24-mo follow-up period. RESULTS: Liver function parameters in the two groups were observed at 1 mo after BM-MNCs transfusion. Prealbumin level was ± 25.3 mg/l vs ± 28.7 mg/l (P = 0.047); albumin level was 33.5 ± 3.6 g/l vs 30.3 ± 2.2 g/l (P = 0.002); total bilirubin 36.9 ± 9.7 mmol/l vs 45.6 ± 19.9 mmol/l (P = 0.048); prothrombin time 14.4 ± 2.3 s vs 15.9 ± 2.8 s (P = 0.046); prothrombin activity 84.3% ± 14.3% vs 74.4% ± 17.8% (P = 0.046); fibrinogen 2.28 ± 0.53 g/l vs 1.89 ± 0.44 g/l (P = 0.017); and platelet count 74.5 ± /L vs 63.3 ± /L (P = 0.027) in the treatment group and control group, respectively. Differences were statistically significant. The efficacy of BM-MNCs transplantation lasted 3-12 mo as compared with the control group. Serious complications such as hepatic encephalopathy and spontaneous bacterial peritonitis were also significantly reduced in BM-MNCs transfused patients compared with the controls. However, these improvements disappeared 24 mo after transplantation. CONCLUSION: BM-MNCs transplantation is safe and effective in patients with decompensated cirrhosis. It also decreases the incidence of serious complications Baishideng Publishing Group Inc. All rights reserved. Key words: Autologous; Bone marrow mononuclear cells; Transplantation; Liver cirrhosis; Hepatitis B virus Core tip: We aimed to study the efficacy of autologous bone marrow mononuclear cells (BM-MNCs) transplantation in terms of improving liver function and reducing complications in patients with decompensated cirrhosis. Liver function parameters were improved one month after BM-MNCs transfusion in cirrhosis patients, 8660 July 14, 2014 Volume 20 Issue 26

353 Bai YQ et al. Autologous bone marrow mononuclear cell transplantation firstly in prealbumin level, followed by albumin level, total bilirubin, prothrombin time, prothrombin activity, fibrinogen and platelet count. The efficacy of BM-MNCs transplantation lasted 3-12 mo as compared with the control group. However, these improvements disappeared 24 mo after transplantation. Bai YQ, Yang YX, Yang YG, Ding SZ, Jin FL, Cao MB, Zhang YR, Zhang BY. Outcomes of autologous bone marrow mononuclear cell transplantation in decompensated liver cirrhosis. World J Gastroenterol 2014; 20(26): Available from: URL: v20/i26/8660.htm DOI: i INTRODUCTION Hepatitis B virus (HBV) infection is a major health care challenge in Asian countries including China. In some patients, HBV infection can lead to stepwise complications at a later stage of the disease, including liver cirrhosis, liver failure and hepatocellular carcinoma [1]. Decompensated liver cirrhosis is a critical condition due to its high morbidity and mortality [2]. Patients presenting with one or more of the symptoms such as jaundice, ascites, portal hypertension, gastrointestinal bleeding, and encephalopathy are considered to have decompensation [3]. Despite improvements in the management of liver cirrhosis, the overall outcome of the disease remains poor. Orthotopic liver transplantation is by far the best treatment option for patients with decompensated cirrhosis; however, limitations such as a shortage of organ donors, surgical complications, post-transplantation rejection, and high cost of the procedure have made this treatment not easily available [4]. Therefore, the development of regeneration therapy for liver cirrhosis is urgently required. Recent studies have indicated that stem cell therapy may be a potential alternative to liver transplantation. Autologous bone marrow mononuclear cells (BM- MNCs) transplantation has been reported to be safe and effective in several studies [5-10]. In the short-term, it improves liver function and Child-Pugh scores after 4-24 wk of therapy in both HBV infected and alcoholinduced cirrhosis. In liver biopsies of transplanted patients, increased alpha-fetoprotein and proliferating cell nuclear antigen expression are detected, suggesting that these patients have undergone partial liver regeneration processes [5-10]. A prospective cohort study by Peng et al [9] in HBV infected patients indicated that the short-term efficacy of BM-MNCs transfusion was favorable, but long-term outcomes were not markedly improved. At 192 wk of follow-up, there were no marked differences in the incidence of hepatocellular carcinoma (HCC) or mortality between the transfusion and control groups [9]. This method appears preferable in patients with both HBV infected and alcohol-induced liver cirrhosis. It may also have the potential to reduce the incidence of HCC and mortality [9]. The above results clearly indicate the advantages and disadvantages of this therapy; however, due to the geographic variation in the disease and the variations in treatment, it is not clear how long the effects may last following a single infusion plus a routine medical treatment regimen. In addition, it is unknown if BM-MNCs transplantation may reduce serious complications in cirrhotic patients and improve quality of life. We aimed to determine the short- and long-term efficacy and safety of BM-MNCs transplantation in patients with decompensated cirrhosis receiving a normal treatment regimen with a focus on the incidence of complications during the study. The results showed that transplantation of autologous BM-MNCs is safe and effective in patients with decompensated cirrhosis. The beneficial effect seemed to last for at least 12 mo. This approach improves liver function, blood coagulation, and short-term quality of life, and reduces the incidence of serious complications. Therefore, repeated transplantation of autologous BM-MNCs may represent a routine therapeutic approach to improve the general condition of patients with decompensated cirrhosis. MATERIALS AND METHODS Patients In this trial, 47 patients with decompensated liver cirrhosis were recruited. The patients were admitted to the Department of Gastroenterology at Henan Provincial People s Hospital from March 2009 to March Written informed consent was obtained from 32 patients after explaining the benefits and risks of the study and its complications. Fifteen patients declined the infusion and received only routine medical treatment. These patients served as controls. In the treatment group, 32 patients were treated with BM-MNCs plus a conventional internal medicine regimen including drugs for anti-hbv virus, liver cell protection, transaminase and jaundice reducing drugs. In the control group, patients only received the above conventional medical treatment without BM-MNCs infusion. The treatment group included 30 patients with HBV infection and cirrhosis and two patients with alcoholic liver cirrhosis; in the control group, there were 13 patients with HBV infection and cirrhosis and two patients with alcoholic liver cirrhosis. The baseline clinical parameters of these patients are presented in Table 1. The diagnosis of decompensated cirrhosis was made based on their Child-Pugh classification of grade B and C. Inclusion criteria included ultrasonographic evidence of liver cirrhosis with ascites, portal hypertension, low serum albumin (ALB), high total bilirubin (TBIL), prolonged prothrombin time (PT), normal alpha fetoprotein level, and no hepatocellular carcinoma on hepatic artery angiographic imaging. The exclusion criteria were as follows: combined heart and lung function abnormality, blood system diseases, acquired immunodeficiency 8661 July 14, 2014 Volume 20 Issue 26

354 Bai YQ et al. Autologous bone marrow mononuclear cell transplantation Table 1 Comparison of clinical and laboratory parameters between the two groups before transplantation Parameter Control BM-MNCs P value Patient (n) ND Age (yr) 47.4 ± ± Gender Male 9 20 Female HBsAg Positive Negative Liver function Alanine aminotransferase 51.7 ± ± (IU/L) Aspartate aminotransferase 63.7 ± ± (IU/L) Serum albumin (g/l) 28.1 ± ± Prealbumin (mg/l) 85.1 ± ± Total bilirubin (mmol/l) 53.5 ± ± Coagulation function Prothrombin time (s) 16.3 ± ± Prothrombin activity 71.8% ± 19.4% 67.8% ± 15.0% Fibrinogen (g/l) 1.83 ± ± Routine blood indices White blood cell counts ( 2.83 ± ± /L) Hemoglobin (g/l) ± ± Platelet counts ( 10 9 /L) 69.0 ± ± Data are expressed as the mean ± SD. 1 Pearson χ 2 test; 2 t test; 3 Fisher s exact test. BM-MNCs: Bone marrow mononuclear cells; HBsAg: Hepatitis B surface antigen; ND: No data. Table 2 Comparison of the occurrence of complications between the two groups Patient group n Esophageal Hepatic Spontaneous Hepatocellular variceal hemorrhage encephalopathy bacterial peritonitis carcinoma Control BM MNCs P value Fisher s exact test. BM-MNCs: Bone marrow mononuclear cells. disease, malignant tumor, acute or chronic thrombosis of the hepatic vein or portal vein, a history of severe infection, refractory ascites, and moderate to severe hepatic encephalopathy or variceal bleeding during the last two months before enrollment. This study was approved by the Ethics Committee of Henan Provincial People s Hospital, Zhengzhou, China. Preparation of BM-MNCs from patients BM-MNCs were prepared following the procedure previously reported by Peng et al [9]. Briefly, marrow aspiration was performed in the bilateral posterior superior iliac spine under local anesthesia. Then ml of human bone marrow was obtained with 1000 U/mL of Liquaemin as anti-coagulant. The bone marrow mononuclear cells were separated and purified by Ficoll-Paque density centrifugation [11]. The cells were placed into two 50 ml sterile centrifugal tubes, cells in the middle layer were collected, and an equal amount of physiological saline was added and mixed. The mixed cell suspension was gently added to the upper part of the Ficoll-Paque solution, followed by centrifugation at 2000 rpm for 20 min. Interphase-containing cells were obtained and washed three times with 10 ml of normal saline; cells were adjusted with physiological saline to a density of /L. Cell viability was determined by trypan blue staining. Autologous BM-MNCs transplantation On the day of transfusion, a femoral artery puncture was made and a catheter was inserted into the hepatic artery. Intrahepatic blood vessels were observed following the application of ioversol angiography. The prepared cell suspension was administered into the hepatic artery at a speed of 10 ml/h using a micro-pump. Patient follow-up Patients were followed for up to 24 mo after BM-MNCs transfusion. During this period, data on the patient s general condition and complications such as gastrointestinal bleeding, hepatic encephalopathy, spontaneous bacterial peritonitis, hepatocellular carcinoma, and changes in clinical symptoms and signs such as abdominal distension, physical strength, appetite, ascites, pleural effusion, lower extremity edema in the two groups were collected and recorded. One week and 1, 3, 6, 12, and 24 mo after transplantation, the following blood biochemistry tests were performed: liver function tests: alanine aminotransferase (ALT), aspartate aminotransferase (AST), ALB, prealbumin (PA), TBIL; coagulation function tests: PT, prothrombin activity (PTA), fibrinogen (FIB); routine blood indices: white blood cell counts (WBC), hemoglobin, and platelet counts. Statistical analysis All data were analyzed by SPSS 17.0 software (SPSS Inc., Chicago, IL, United States) and a value of P < 0.05 was considered statistically significant. Data from clinical and biochemical analyses are expressed as mean ± SD and compared using the χ 2 and t tests. Fisher s exact test was used when appropriate. RESULTS Patient characteristics and complications When we compared patient age, gender, liver function, blood coagulation function and routine blood indices, there were no significant differences between the two groups (P > 0.05, Table 1). The incidence of complications (Table 2) was monitored throughout the 24-mo follow-up period, and no significant differences were observed in the occurrence of esophageal variceal hemorrhage between the two groups. However, we noted a significant reduction in the occurrence of hepatic en July 14, 2014 Volume 20 Issue 26

355 Bai YQ et al. Autologous bone marrow mononuclear cell transplantation Table 3 Levels of albumin, prealbumin and total bilirubin in the two groups at different time points after transplantation Liver function Group 1 wk 1 mo 3 mo 6 mo 12 mo 24 mo Serum albumin (g/l) 1 Control 29.2 ± ± ± ± ± ± 3.4 BM-MNCs 28.4 ± ± ± ± ± ± 3.8 P value Prealbumin (mg/l) 1 Control 92.5 ± ± ± ± ± ± 17.7 BM-MNCs 91.1 ± ± ± ± ± ± 22.7 P value Total bilirubin (mmol/l) 1 Control 51.3 ± ± ± ± ± ± 14.2 BM-MNCs 50.9 ± ± ± ± ± ± 10.7 P value Data are expressed as the mean ± SD. 1 t test. BM-MNCs: Bone marrow mononuclear cells. Table 4 Levels of prothrombin time, prothrombin activity and fibrinogen in the two groups at different time points after transplantation Function Group 1 wk 1 mo 3 mo 6 mo 12 mo 24 mo Prothrombin time (s) 1 Control 16.2 ± ± ± ± ± ± 2.3 BM-MNCs 16.8 ± ± ± ± ± ± 2.1 P value Prothrombin activity 1 Control 72.5% ± 17.8% 72.0% ± 17.7% 74.4% ± 17.8% 75.9% ± 18.2% 77.4%± 18.2% 75.6% ± 14.6% BM-MNCs 68.7% ± 16.0% 77.3% ± 15.7% 84.3% ± 14.3% 87.2% ± 13.3% 86.9% ± 13.5% 84.0% ± 13.6% P value Fibrinogen (g/l) 1 Control 1.90 ± ± ± ± ± ± 0.28 BM-MNCs 1.86 ± ± ± ± ± ± 0.52 P value Data are expressed as the mean ± SD. 1 t test. BM-MNCs: Bone marrow mononuclear cells. cephalopathy and spontaneous bacterial peritonitis in the treatment group compared with the control group. One patient in each group developed HCC during the 24-mo follow-up period (P = 0.541). Liver function tests One month after transplantation, ALB and PA levels in patients in the treatment group gradually increased. ALB levels in patients in the treatment group were significantly improved compared with those in the control group from 3 to 12 mo after transplantation (Table 3). The improvement in PA levels in the treatment group was significant at 1-12 mo after transplantation compared with the control group (Table 3). Autologous BM-MNCs transplantation also significantly decreased TBIL levels after 1 mo, and particularly at 3-12 mo after transplantation (Table 3). However, these differences were not observed at 24 mo after transplantation (Table 3). A comparison of liver function between baseline and 24 mo after transplantation indicated that there were no marked differences in ALT and AST levels between the two groups (data not shown). Coagulation function test Blood coagulation in patients in the treatment group began to improve one month after transplantation. PT levels in the treatment group gradually decreased and PTA levels gradually increased (Table 4). Significant differences in PT and PTA levels were observed in the treatment group at 3-6 mo after transplantation (Table 4). No significant differences were observed in PT and PTA levels at mo after transplantation (Table 4). FIB levels in the treatment group gradually increased one month after transplantation, and particularly at 3-12 mo (Table 4). These differences were no longer observed 24 mo after transplantation (Table 4). Routine blood tests The peripheral WBC level in the treatment group was higher than that in the control group 6 mo after treatment; however, no significant differences in peripheral WBC levels were found between the treatment group and the control group at other time points (Table 5). Hemoglobin levels were significantly higher in the treatment group than in the control group at 3-6 mo after transplantation (Table 5). Platelet counts were significantly higher in the treatment group than in the control group at 3-12 mo after transplantation (Table 5). DISCUSSION Decompensated liver cirrhosis is an end-stage liver disease due to various causes of liver fibrosis, and a lack of safe and effective clinical treatments. Chronic infection with HBV and HCV, and alcohol consumption are major global causes of cirrhosis. Alcohol and HCV infection are common causes of cirrhosis in European, North American and other developed countries, whereas HBV infection is the major cause in many Asian and African countries, including China [12,13]. The majority of patients 8663 July 14, 2014 Volume 20 Issue 26

356 Bai YQ et al. Autologous bone marrow mononuclear cell transplantation Table 5 Levels of white blood cell counts, hemoglobin, and platelet counts in the two groups at different time points after transplantation Routine blood indices Group 1 wk 1 mo 3 mo 6 mo 12 mo 24 mo White blood cell counts ( 10 9 /L) 1 Control 2.82 ± ± ± ± ± ± 0.92 BM-MNCs 2.69 ± ± ± ± ± ± 0.72 P value Hemoglobin (g/l) 1 Control ± ± ± ± ± ± 14.3 BM-MNCs ± ± ± ± ± ± 16.8 P value Platelet counts ( 10 9 /L) 1 Control 68.9 ± ± ± ± ± ± 15.6 BM-MNCs 69.3 ± ± ± ± ± ± 15.2 P value Data are expressed as the mean ± SD. 1 t test. BM-MNCs: Bone marrow mononuclear cells. in the present study had HBV infection and cirrhosis. BM-MNCs is a general term for single nucleus cells in the bone marrow, including mesenchymal stem cells (MSCs), hematopoietic stem cells, endothelial progenitor cells and stromal cells. BM-MNCs is a mixed cell population containing a variety of cellular components that can generate various cell types found in other tissues [14-16]. MSCs have the ability of self-renewal and pluripotency, they secrete a number of cytokines and growth factors to regulate cellular functions, including promoting liver regeneration, inhibiting inflammation and activation of liver astrocytes, blocking the production of and facilitating the degradation of excessive extracellular matrix to repair injured liver tissues [17,18]. Clinical trials have also shown the potential of MSCs to reduce liver fibrosis [19]. Transplantation of MSCs [19] and MNCs [6,8] has been explored in clinical trials to treat chronic liver disease with various efficacies. In the current study, we used a mixture of BM-MNCs to treat HBV infected cirrhosis, and the efficacy likely reflected the overall effects of these individual stem cells. Recent reports have demonstrated the therapeutic effect of stem cell transplantation in liver cirrhosis. Kharaziha et al [8] reported that eight patients with endstage liver disease did not experience discomfort and their liver function improved significantly after administration of MSCs. Kim et al [20] and Lyra et al [21,22] showed significant improvements in albumin and quality of life in patients with liver cirrhosis caused by hepatitis B. The results indicated that the mean serum bilirubin and international normalized ratio levels decreased, and the levels of serum albumin increased. Another study by Peng et al [9] confirmed the above results in a short-term study; however, a longer-term investigation suggested that these beneficial effects did not last more than two years, despite the fact that there was no increase in the incidence of HCC. In our study, we observed the efficacy and safety of BM-MNCs transplantation in patients with decompensated cirrhosis and determined how long the efficacy lasted in order to find whether repeated transplantation of autologous BM-MNCs at regular time intervals might be an approach for improving the conditions of decompensated cirrhotic patients. The overall outcomes appear similar to those reported previously. In addition, it should be noted that this procedure significantly reduced serious complications such as hepatic encephalopathy and spontaneous bacterial peritonitis in the transfused patients. As the effect did not last more than two years, we consider that an annual treatment regimen may significantly improve the patient s general condition and quality of life. The mechanism by which BM-MNCs contribute to hepatocyte regeneration or liver repair is still under investigation, trans-differentiation of MSCs into hepatocytes represents genomic plasticity in response to the microenvironment [23]. In a CCl4 mouse model, persistent injury was found to induce efficient trans-differentiation of bone marrow cells (BMCs) into functional hepatocytes [24]. Green fluorescent protein (GFP)-transfected BMCs efficiently migrated into the peri-portal area of liver lobules after one day, and repopulated 25% of the recipient liver by four weeks in mice with liver cirrhosis induced by CCl4. In contrast, no GFP-positive BMCs were detected in control mice with undamaged livers following transplantation. Transfused cells were first seen to differentiate into hepatoblasts and later became albumin-producing hepatocytes. The improved liver function following BMCs transplantation suggests that recipient conditions and microenvironments are key factors for successful cell therapy using BMCs [24]. At present, it is difficult to estimate which factors may affect the engulfment of BM-MNCs into the liver. Cells administered via peripheral veins achieved similar results to cells administered via the hepatic artery. In addition, the synergistic effect of fibroblast growth factor or granulocyte-colony stimulating factor can help mobilize BMCs, and will probably increase hepatic engulfment and improve efficacy. However, further research is required to optimize the protocol and explore the mechanisms. One drawback in our experimental design is that there was no additional time point between 12 and 24 mo during the follow-up period; this may have resulted in missed patient information to determine the exact time point at which efficacy decreased after transplantation. However, it can be speculated that the improvement in patients liver function and blood coagulation 8664 July 14, 2014 Volume 20 Issue 26

357 Bai YQ et al. Autologous bone marrow mononuclear cell transplantation was maintained for at least 12 mo or more. With regard to cirrhosis complications, no significant difference was observed in the occurrence of esophageal variceal hemorrhage and hepatocellular carcinoma between the two groups. Liver function and coagulation function in patients in the treatment group began to improve one month after transplantation. ALB, PA, PTA and FIB gradually increased; TBIL and PT gradually decreased. Significant alterations in these markers were observed between the treatment group and the control group at 3-12 mo after transplantation. In addition, it should be noted that after transfusion, white blood cell count, hemoglobin and platelet count in patients in the treatment group increased gradually, which are signs of improved hypersplenism. During the trial, we found a reduction in spleen volume on ultrasonography in some patients in the treatment group (data not shown). Whether reduced portal hypertension occurs is currently under investigation. Although clinical trials have shown some improvement in liver function, it must be remembered that the natural history of cirrhosis tends to be variable. The question of how to further optimize cell transplant type, the infusion method, the number of transplant operations and their impact on clinical efficacy remains to be answered. Future studies will be required to optimize BM-MNCs transplantation for the treatment of cirrhosis. In conclusion, infusion of autologous BM-MNCs is safe and effective in patients with decompensated cirrhosis for at least 12 mo. This approach significantly improved liver function, blood coagulation and short-term quality of life, and reduced the incidence of serious complications, therefore can be considered a suitable regenerative therapy in patients with decompensated cirrhosis. ACKNOWLEDGMENTS The authors are grateful to the staff at the Center of Translation Medicine, Henan Provincial People s Hospital for their assistance with this project. COMMENTS Background Decompensated liver cirrhosis presents with several symptoms such as jaundice, ascites, portal hypertension, and encephalopathy. Decompensated liver cirrhosis is a critical medical condition due to its high morbidity and mortality. The bone marrow mononuclear cells (BM-MNCs) population is a mixed cell population that has self-renewal properties and the ability to produce multiple differentiation progenitors. Thus, in this study authors evaluated the efficacy of autologous BM-MNCs transplantation in patients with decompensated cirrhosis. Research frontiers Orthotopic liver transplantation is the final treatment for decompensated cirrhosis, however, donor shortage has resulted in this treatment being unavailable. Stem cell therapy may be a potential alternative to liver transplantation. A research hotspot is the development of safe and effective regeneration therapy to improve liver function and reduce the complications of decompensated cirrhosis. Innovations and breakthroughs The authors determined the long-term efficacy and safety of BM-MNCs transplantation in patients with decompensated cirrhosis. The efficacy was monitored during a follow-up period of 24 mo. The results of this study showed that this approach is a suitable therapy for patients with decompensated cirrhosis. Applications This study suggested that the transplantation of autologous BM-MNCs is safe and effective, and repeated transplantation of autologous BM-MNCs may represent a routine therapeutic approach to improve decompensated cirrhosis. Terminology BM-MNCs is the general term for single nucleus cells in the bone marrow, including mesenchymal stem cells, hematopoietic stem cells, endothelial progenitor cells and stromal cells. The BM-MNCs population is a mixed cell population containing a variety of cellular components which have self-renewal properties and the ability to produce multiple differentiation progenitors. Peer review The article presents a significant and valuable clinical research describing the short- and long-term efficacy of BM-MNCs transplantation in improving liver function and reducing the complications in decompensated cirrhosis patients. REFERENCES 1 Lian JS, Zeng LY, Chen JY, Jia HY, Zhang YM, Xiang DR, Yu L, Hu JH, Lu YF, Zheng L, Li LJ, Yang YD. De novo combined lamivudine and adefovir dipivoxil therapy vs entecavir monotherapy for hepatitis B virus-related decompensated cirrhosis. World J Gastroenterol 2013; 19: [PMID: DOI: /wjg.v19.i ] 2 Tsochatzis EA, Bosch J, Burroughs AK. New therapeutic paradigm for patients with cirrhosis. Hepatology 2012; 56: [PMID: DOI: /hep.25915] 3 Mukerji AN, Patel V, Jain A. Improving survival in decompensated cirrhosis. Int J Hepatol 2012; 2012: [PMID: DOI: /2012/318627] 4 Takami T, Terai S, Sakaida I. Advanced therapies using autologous bone marrow cells for chronic liver disease. Discov Med 2012; 14: 7-12 [PMID: ] 5 Takami T, Terai S, Sakaida I. Stem cell therapy in chronic liver disease. Curr Opin Gastroenterol 2012; 28: [PMID: DOI: /MOG.0b013e d6a] 6 Terai S, Ishikawa T, Omori K, Aoyama K, Marumoto Y, Urata Y, Yokoyama Y, Uchida K, Yamasaki T, Fujii Y, Okita K, Sakaida I. Improved liver function in patients with liver cirrhosis after autologous bone marrow cell infusion therapy. Stem Cells 2006; 24: [PMID: DOI: /stemcells ] 7 Park CH, Bae SH, Kim HY, Kim JK, Jung ES, Chun HJ, Song MJ, Lee SE, Cho SG, Lee JW, Choi JY, Yoon SK, Han NI, Lee YS. A pilot study of autologous CD34-depleted bone marrow mononuclear cell transplantation via the hepatic artery in five patients with liver failure. Cytotherapy 2013; 15: [PMID: DOI: /j.jcyt ] 8 Kharaziha P, Hellström PM, Noorinayer B, Farzaneh F, Aghajani K, Jafari F, Telkabadi M, Atashi A, Honardoost M, Zali MR, Soleimani M. Improvement of liver function in liver cirrhosis patients after autologous mesenchymal stem cell injection: a phase I-II clinical trial. Eur J Gastroenterol Hepatol 2009; 21: [PMID: DOI: / MEG.0b013e32832a1f6c] 9 Peng L, Xie DY, Lin BL, Liu J, Zhu HP, Xie C, Zheng YB, Gao ZL. Autologous bone marrow mesenchymal stem cell transplantation in liver failure patients caused by hepatitis B: short-term and long-term outcomes. Hepatology 2011; 54: [PMID: DOI: /hep.24434] 10 Levicar N, Pai M, Habib NA, Tait P, Jiao LR, Marley SB, Davis J, Dazzi F, Smadja C, Jensen SL, Nicholls JP, Apperley JF, Gordon MY. Long-term clinical results of autologous infusion of mobilized adult bone marrow derived CD34+ cells in patients with chronic liver disease. Cell Prolif 2008; 41 Suppl 1: [PMID: DOI: / j x] 11 Yeo C, Saunders N, Locca D, Flett A, Preston M, Brookman P, Davy B, Mathur A, Agrawal S. Ficoll-Paque versus 8665 July 14, 2014 Volume 20 Issue 26

358 Bai YQ et al. Autologous bone marrow mononuclear cell transplantation Lymphoprep: a comparative study of two density gradient media for therapeutic bone marrow mononuclear cell preparations. Regen Med 2009; 4: [PMID: DOI: /rme.09.44] 12 Perz JF, Armstrong GL, Farrington LA, Hutin YJ, Bell BP. The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. J Hepatol 2006; 45: [PMID: DOI: / j.jhep ] 13 Seeff LB, Hoofnagle JH. Epidemiology of hepatocellular carcinoma in areas of low hepatitis B and hepatitis C endemicity. Oncogene 2006; 25: [PMID: DOI: /sj.onc ] 14 Khurana S, Mukhopadhyay A. In vitro transdifferentiation of adult hematopoietic stem cells: an alternative source of engraftable hepatocytes. J Hepatol 2008; 49: [PMID: DOI: /j.jhep ] 15 Yan Y, Xu W, Qian H, Si Y, Zhu W, Cao H, Zhou H, Mao F. Mesenchymal stem cells from human umbilical cords ameliorate mouse hepatic injury in vivo. Liver Int 2009; 29: [PMID: DOI: /j x] 16 Banas A, Teratani T, Yamamoto Y, Tokuhara M, Takeshita F, Osaki M, Kato T, Okochi H, Ochiya T. Rapid hepatic fate specification of adipose-derived stem cells and their therapeutic potential for liver failure. J Gastroenterol Hepatol 2009; 24: [PMID: DOI: /j x] 17 Parekkadan B, van Poll D, Megeed Z, Kobayashi N, Tilles AW, Berthiaume F, Yarmush ML. Immunomodulation of activated hepatic stellate cells by mesenchymal stem cells. Biochem Biophys Res Commun 2007; 363: [PMID: DOI: /j.bbrc ] 18 Zhou P, Hohm S, Olusanya Y, Hess DA, Nolta J. Human progenitor cells with high aldehyde dehydrogenase activity efficiently engraft into damaged liver in a novel model. Hepatology 2009; 49: [PMID: DOI: / hep.22862] 19 Houlihan DD, Newsome PN. Critical review of clinical trials of bone marrow stem cells in liver disease. Gastroenterology 2008; 135: [PMID: DOI: / j.gastro ] 20 Kim JK, Park YN, Kim JS, Park MS, Paik YH, Seok JY, Chung YE, Kim HO, Kim KS, Ahn SH, Kim do Y, Kim MJ, Lee KS, Chon CY, Kim SJ, Terai S, Sakaida I, Han KH. Autologous bone marrow infusion activates the progenitor cell compartment in patients with advanced liver cirrhosis. Cell Transplant 2010; 19: [PMID: DOI: / X506863] 21 Lyra AC, Soares MB, da Silva LF, Fortes MF, Silva AG, Mota AC, Oliveira SA, Braga EL, de Carvalho WA, Genser B, dos Santos RR, Lyra LG. Feasibility and safety of autologous bone marrow mononuclear cell transplantation in patients with advanced chronic liver disease. World J Gastroenterol 2007; 13: [PMID: ] 22 Lyra AC, Soares MB, da Silva LF, Braga EL, Oliveira SA, Fortes MF, Silva AG, Brustolim D, Genser B, Dos Santos RR, Lyra LG. Infusion of autologous bone marrow mononuclear cells through hepatic artery results in a short-term improvement of liver function in patients with chronic liver disease: a pilot randomized controlled study. Eur J Gastroenterol Hepatol 2010; 22: [PMID: DOI: / MEG.0b013e32832eb69a] 23 Cho KA, Lim GW, Joo SY, Woo SY, Seoh JY, Cho SJ, Han HS, Ryu KH. Transplantation of bone marrow cells reduces CCl4 -induced liver fibrosis in mice. Liver Int 2011; 31: [PMID: DOI: /j x] 24 Terai S, Sakaida I, Yamamoto N, Omori K, Watanabe T, Ohata S, Katada T, Miyamoto K, Shinoda K, Nishina H, Okita K. An in vivo model for monitoring trans-differentiation of bone marrow cells into functional hepatocytes. J Biochem 2003; 134: [PMID: ] P- Reviewers: Dang SS, Morales-Gonzalez JA, Vento S S- Editor: Gou SX L- Editor: O Neill M E- Editor: Liu XM 8666 July 14, 2014 Volume 20 Issue 26

359 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. OBSERVATIONAL BRIEF ARTICLE STUDY Pro-atherosclerotic markers and cardiovascular risk factors one year after liver transplantation Mario Reis Alvares-da-Silva, Claudia Pinto Marques Souza de Oliveira, José Tadeu Stefano, Hermes V Barbeiro, Denise Barbeiro, Francisco G Soriano, Alberto Queiroz Farias, Flair José Carrilho, Luiz Augusto Carneiro D Albuquerque Mario Reis Alvares-da-Silva, Luiz Augusto Carneiro D Albuquerque, Division of Liver and Gastrointestinal Transplant (LIM-37), Department of Gastroenterology, University of São Paulo School of Medicine, São Paulo, Brazil Mario Reis Alvares-da-Silva, Division of Gastroenterology, Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcellos, Porto Alegre, Brazil Claudia Pinto Marques Souza de Oliveira, José Tadeu Stefano, Alberto Queiroz Farias, Flair José Carrilho, Division of Clinical (LIM-07), Department of Gastroenterology, University of São Paulo School of Medicine, São Paulo, Brazil Hermes V Barbeiro, Denise Barbeiro, Francisco G Soriano, Division of Emergency Medicine (LIM-51), University of São Paulo School of Medicine, São Paulo, Brazil Author contributions: Alvares-da-Silva MR and Oliveira CPMS designed the study, collected the data and performed and co-wrote the manuscript; Stefano JT provided and coordinated the collection of all the human materials; Barbeiro HV, Barbeiro D and Soriano FG performed most of the laboratory analyses; Farias AQ coordinated the collection of all the human materials; Carrilho FJ and Carneiro D Albuquerque LA co-designed the study, and provided financial support for this work; all co-authors reviewed and approved the final manuscript. Supported by Department of Gastroenterology LIM-37/LIM07, School of Medicine, University of São Paulo, Brazil Correspondence to: Mario Reis Alvares-da-Silva, MD, PhD, Division of Gastroenterology, Hospital de Clínicas de Porto Alegre, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcellos, 2350, sala 2033, Porto Alegre, Brazil. marioreis@live.com Telephone: Fax: Received: January 30, 2014 Revised: March 18, 2014 Accepted: April 15, 2014 Published online: July 14, 2014 Abstract AIM: To investigate pro-atherosclerotic markers (endothelial dysfunction and inflammation) in patients one year after liver transplantation. METHODS: Forty-four consecutive liver transplant (LT) outpatients who were admitted between August 2009 and July 2010, were followed-up by for 1 year, exhibited no evidences of infection or rejection, all of them underwent tacrolimus-based immunosuppressive regimens were consecutively enrolled. Inflammatory cytokines (TNFα, IFNγ, IL-8, and IL-10), endothelial biomarkers (svcam-1, sicam-1, MPO, adiponectin, PAI-1, SAP, SAA, E-selectin, and MMP-9), high sensitive C-reactive protein, and Framingham risk score (FRS) were assessed. The anthropometric data, aminotransferases, metabolic syndrome features, glucose and lipid profiles, and insulin resistance data were also collected. The LT recipients were compared to 22 biopsy-proven non-alcoholic steatohepatitis (NASH) patients and 20 healthy controls (non-obese, non-diabetics, and nondyslipidemic). RESULTS: The LT recipients had significantly younger ages and lower body mass indices, aminotransferases, fasting glucose and insulin levels, glucose homeostasis model and metabolic syndrome features than the NASH patients. Classic cardiovascular risk markers, such as Hs-CRP and FRS [2.0 ( )], were lower in the LT patients compared to those observed in the NASH patients (P = 0.009). In contrast, the LT recipients and NASH patients had similar inflammatory and endothelial serum markers compared to the controls (pg/ml): lower IL-10 levels (32.3 and 32.3 vs 62.5, respectively, P = 0.019) and higher IFNγ (626.1 and vs 67.9, respectively, P < 0.001), E-selectin (48.5 and vs 35.7, respectively, P < 0.001), svcam-1 ( and vs , respectively, P < 0.001), and sicam-1 (230.3 and vs 152.9, respectively, P = 0.015) levels July 14, 2014 Volume 20 Issue 26

360 Alvares-da-Silva MR et al. Pro-atherosclerotic markers after liver transplantation CONCLUSION: Non-obese LT recipients have similar pro-atherosclerotic serum profiles after a short 1-year follow-up period compared to NASH patients, suggesting a high risk of atherosclerosis in this population Baishideng Publishing Group Inc. All rights reserved. Key words: Cell adhesion molecules; Endothelial biomarkers; Cardiovascular disease; Nonalcoholic steatohepatitis; Metabolic syndrome Core tip: Liver transplant (LT) patients a have high risk of long-term development of cardiovascular disease (CVD), which is currently recognized as an important cause of death 5 to 10 years after transplant in this population. Atherosclerosis is a hallmark of CVD, with both disorders involving a prolonged asymptomatic phase and often leading to morbidity and mortality upon initial clinical presentation. Regardless, endothelial dysfunction is the first step in developing early atherosclerosis. In the present study, we evaluated inflammatory and endothelial markers one year after transplantation in asymptomatic LT recipients in comparison to high-cvd-risk biopsy-proven nonalcoholic steatohepatitis (NASH) patients and healthy controls. We found that LT recipients had pro-inflammatory profiles and endothelial dysfunction similar to those of NASH patients, both of which were higher than those in the compared controls. These findings suggest that LT recipients, even in a short 1-year follow-up period, display a high atherosclerotic risk and should be carefully monitored to effectively prevent CVD. Alvares-da-Silva MR, Oliveira CPMS, Stefano JT, Barbeiro HV, Barbeiro D, Soriano FG, Farias AQ, Carrilho FJ, Carneiro D Albuquerque LA. Pro-atherosclerotic markers and cardiovascular risk factors one year after liver transplantation. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: INTRODUCTION Liver transplantation is the standard treatment for acute and chronic end-stage liver disease. Advances in medical therapy and surgical techniques have increased the life span of liver transplant (LT) recipients. As a result, medical complications that accompany long-term survival, including atherosclerotic cardiovascular disease (CVD), metabolic bone disease, and de novo malignancy, have accounted for an increasing proportion of late morbimortality in these patients. CVD, which is responsible for 19% to 42% of all non-liver related mortality, is a major cause of morbidity and mortality after LT [1-6]. Atherosclerosis is the hallmark of CVD and remains an important health issue in the modern world despite research aimed at understanding its underlying pathogenesis. This condition involves a prolonged asymptomatic phase; symptoms only develop when blood flow is insufficient to ensure tissue vitality. The first clinical presentation often leads to morbidity and mortality [7]. Arterial plaque with no symptoms is called subclinical atherosclerosis, and chronic inflammation is a risk factor for plaque rupture. High sensitivity C-reactive protein (Hs-CRP) is an inflammatory marker that predicts CVD in healthy individuals [8,9]. Endothelial dysfunction is the first step in developing early atherosclerosis. Several studies confirm that elevated plasma levels of endothelial markers, such as von Willebrand factor, and soluble vascular cell adhesion molecule-1 (svcam-1) may serve as molecular markers for atherosclerosis and are independent risk factors for the development of coronary heart disease [9]. Risk estimation for atherosclerotic and cardiovascular events that is based only on the presence of classical risk factors is often insufficient. Therefore, efforts have been made to identify blood markers that indicate the presence of preclinical disease. This study was designed to investigate pro-atherosclerotic markers (endothelial dysfunction and inflammation) in patients one year after LT. MATERIALS AND METHODS Population Between August 2009 and July 2010, 44 consecutive adult (older than 18 years old) outpatients who underwent orthotopic LT at the LT Unit of the University of São Paulo School of Medicine, Brazil, were followed for 1 year. The results were compared to 20 age-matched (10-year age classes) controls [body mass index (BMI) < 30 kg/m 2, non-diabetics, and non-dyslipidemic]. Additionally, because NASH is an important risk factor for CVD, 22 patients with biopsy-proven NASH were also compared with the LT recipients and controls. One experienced pathologist graded the liver biopsies from the NASH patients, according to the NAFLD activity score (NAS) [10]. The LT recipients had no evidence of infection or rejection and were evaluated during regular outpatient clinic visits. The transplant data were reviewed from the patient s charts. At the 1-year follow-up, features of MS, glucose and lipid profiles, HOMA-IR, inflammatory cytokines, and endothelial biomarkers were determined. MS was defined using the American Diabetes Association criteria [11]. Framingham risk scoring system and physical activity Framingham risk scoring system (FRS) was calculated by assigning gender-specific points for age, smoking, diabetes, blood pressure, low-density lipoprotein cholesterol (LDL-cholesterol), and high-density lipoprotein cholesterol (HDL-cholesterol). The gender-specific FRS equations were then used to calculate the risk of developing cardiovascular events over the next 10 years [11]. The patients were graded as low risk (< 10%), intermediate risk (10%-20%), and high risk (> 20%), according to the National Cholesterol Education Program Adult Treat July 14, 2014 Volume 20 Issue 26

361 Alvares-da-Silva MR et al. Pro-atherosclerotic markers after liver transplantation ment Panel Ⅲ guidelines [12]. The International Physical Activity Questionnaire assessed physical activity. Sedentary lifestyle was described as less than 10 min/wk of continuous exercises [13]. Laboratory evaluation The laboratory evaluation in all patients included a blood cell count and the measurement of aspartate aminotransferase (AST), alanine aminotransferase (ALT), total cholesterol and fractions, triglycerides, and fasting glucose and insulin levels. These parameters were measured using the standard techniques of clinical chemistry laboratories (Modular P800, Hitachi, Roche Applied Science, Indianapolis, IN, United States). Insulin resistance was measured using the glucose homeostasis model (HOMA-IR): the product of fasting plasma glucose level (mg/dl) and insulin concentration (miu/l), divided by 405. Serum cytokine measurements For the cytokine and chemokine measurements, the serum was stored at -80 until use. The serum cytokine levels (TNF-α, IL-8, IFNγ, and IL-10) were then measured using a sensitive sandwich enzyme-linked immunosorbent assay (ELISA) kit (RD System Inc., Minneapolis, MN, United States). All measurements were performed in duplicate, and the average values were used in the statistical analyses. Inflammation and endothelial dysfunction markers To detect changes in inflammation and endothelial dysfunction markers, we analyzed the levels of high-sensitive C-reactive protein (Hs-CRP), soluble intercellular adhesion molecule-1 (sicam-1), soluble vascular cell adhesion molecule-1 (svcam-1), E-selectin, adiponectin, plasminogen activator inhibitor 1 (PAI-1), serum amyloid P (SAP), serum amyloid A (SAA); matrix metallopeptidase 9 (MMP-9), and myeloperoxidase (MPO). Each measurement was performed in pg/ml in a multiplex assay on the Luminex platform, as described by the manufacturer (Milliplex CVD Panel 1, Millipore, Copenhagen, Denmark). The readers of the index tests and reference standard were blinded to the other test results. Ethical concerns The study was performed in accordance with the ethical standards of the Helsinki Declaration. An institutional ethics review board approved the protocol, and written informed consent was obtained from each patient. The Department of Gastroenterology (LIM-37/LIM07), School of Medicine, University of São Paulo, Brazil, supported this work. The authors have no conflicts of interest to disclose. Table 1 Demographic and clinical data from liver transplant recipients included n (%) Variable Results (n = 44) Recipient age (yr) ± 14.3 Donor age (yr) ± 17.6 Gender Male 29 (65.9) Female 15 (34.1) Etiology of liver disease 1 HCV-related 16 (36.4) Non-HCV-related 28 (63.6) Patients with NAFLD 4 (9.1) Patients with hepatocellular carcinoma 15 (34.1) Pre-transplant MELD ± 9.4 Donor liver weight (g) ± Recipient liver weight (g) ± Cold ischemia time (min) ± Warm ischemia time (min) ± 8.9 Intraoperative blood requirements 1 Packed red cells (Units) 1.09 ± 1.6 Plasma (Units) 0.71 ± 1.8 Platelets (Units) 1.03 ± 3.53 Intraoperative albumin infusion (10 g bottles) ± 4.8 Tacrolimus-based immunosuppression 44 (100) Steroids withdrawal (d) ± Pre-transplant diabetes mellitus 16 (36.4) Diabetes mellitus 1 yr after transplant 17 (38.6) Pre-transplant BMI ± 5.3 BMI 1 yr after transplant ± 4.3 Abdominal circumference 1 yr after transplant (cm) ± 8.7 Tobacco consumption 8 (18.2) Arterial hypertension 16 (36.4) Sedentarism 28 (66.3) Metabolic syndrome 10 (22.7) Framingham risk score (10-yr) ( ) 1 Mean and standard deviation; 2 Median and 25 th -75 th percentiles. BMI: Body mass index. Statistical analysis Sample estimation was not performed, as there was no similar study on which to base the calculation. The data are expressed as mean ± standard deviation (SD) for variables with normal distribution and compared using oneway analysis of variance (ANOVA). The median and 25 th and 75 th percentiles were used for variables with skewed distribution, which were compared using the Mann-Whitney U-test. Multiple comparisons were conducted, and a significance level of 5% was established. RESULTS Table 1 describes the demographic and clinical data from the LT recipients and donors. Most of the patients were males, with non-hepatitis C virus (HCV)-related liver disease. Only 4 (9.1%) patients were transplanted for NAFLD cirrhosis. In addition, most of the included patients had no hepatocellular carcinoma diagnosed during the pre-transplant period or at the explant analysis. The mean age of the donors was young (41.9 years), and the intraoperative data (ischemia times, intraoperative blood requirements, and intraoperative albumin infusion) demonstrated that undergoing the procedure was uneventful. All included LT recipients underwent tacrolimusbased immunosuppressive treatment, and steroids were withdrawn in d. During the 1-year followup, obesity was not a prevalent comorbidity, with a mean BMI of kg/m 2. The mean abdominal circumfer July 14, 2014 Volume 20 Issue 26

362 Alvares-da-Silva MR et al. Pro-atherosclerotic markers after liver transplantation Table 2 Demographic and clinical characteristics of the studied population Variable (mean ± SEM) otherwise indicated NASH (n = 22) LT (n = 44) Controls (n = 20) P 1 Age 58.5 ± ± ± 9.31 < Sex %male/female 36.3/ / /46.7 BMI 31.7 ± ± ± 2.7 < Fasting glucose ± ± 46.8 NA < Fasting insulin 17.6 ± ± 7.7 NA 0.03 HOMA-IR ± ± 2.4 NA < AST 45.5 ± ± 18.1 NA ALT 58.8 ± ± 15.3 NA < Total cholesterol ± ± 26.8 NA NS HDL cholesterol 50.1 ± ± 15.9 NA NS 1 LT vs NASH. SEM: Standard error of the mean; NS: Non-significant; NA: Not applied; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; HDL: High-density lipoprotein. Table 3 Inflammatory cytokines in liver transplant recipients, non-alcoholic steatohepatitis and controls Variable (median - 25 th -75 th percentile) NASH (n = 22) LT (n = 44) 1 Controls (n = 20) P TNFa (pg/ml) 13.4 ( ) 12.1 ( ) 10.1 ( ) IFNg (pg/ml) ( ) ( ) 67.9 ( ) < IL-8 (pg/ml) 57.8 ( ) 36.5 ( ) 40.7 ( ) < IL-10 (pg/ml) 32.3 ( ) 32.3 ( ) 62.5 ( ) Comparisons: IFNγ (NASH = LT-P = 0.3; NASH and LT > controls - P < 0.001); IL-8 (NASH > LT - P < 0.001; NASH > controls - P = 0.04; LT = controls - P = 0.11); IL-10 (NASH = LT - P = 0.84; NASH < controls - P = 0.02; LT < controls - P = 0.04). CVD is a major cause of morbimortality after LT, and identifying those candidates who are at the greatest risk of postoperative complications is a cornerstone strategy for optimizing outcomes [14,15]. The present study demonstrated that at 1 year post-transplant, LT recipients have similar pro-atherosclerotic profiles, as measured by endothelial biomarkers and inflammatory cytokines, as patients with NASH, even when conventional cardiovasence 1 year post-transplant was cm. MS features were present in only 22.7% of the LT recipients. While 38.7% of the patients had DM, and high blood pressure was identified in 36.4%. Only 18.2% of the patients smoked tobacco. Conversely, sedentarism was present in 66.3% of the studied sample. The mean FRS was low (2.0), consistent with the low MS prevalence. Table 2 shows the laboratory results 1 year post-lt. The hepatic profiles were approximately normal and significantly lower than those of the NASH patients. The fasting glucose, insulin, and HOMA-IR levels were also lower than those in the NASH patients. Although NASH patients had higher total cholesterol levels, their HDL and LDL-cholesterol and triglyceride levels were not significantly different from those of the LT patients, likely because the NASH patients were taking medications. MS was observed more frequently in the NASH patients (100%) than in the LT recipients. Regarding inflammation, the IFNγ level was comparable in the NASH patients and LT recipients (P = 0.3); the levels in both groups were higher than that in the controls (P < 0.001). The anti-inflammatory IL-10 was similar in the LT and NASH patients (P = 0.84), and significantly lower in both groups compared to the controls (P 0.05), as shown in Table 3. There were no betweengroup differences in the TNF-α level, and the IL-8 levels were similar between the LT recipients and controls (P > 0.05). The CVD risk (i.e., Hs-CRP) was similar in the LT recipients and controls (P = 0.41) but significantly lower in the LT patients compared to the NASH (P = 0.007) patients (Table 4). Regarding endothelial biomarkers, the liver recipients and NASH patients were comparable considering sv- CAM-1 and sicam-1 (P = 0.5), and these levels in both groups were higher than in the controls; svcam-1 was significantly higher in the LT recipients than in the controls (P < 0.001), and sicam-1 exhibited an insignificant tendency to be higher between these groups (P = 0.05). The E-selectin level was higher in the NASH patients than in the transplanted patients, but it was also higher in the transplanted patients compared to the controls (P = 0.04). MPO and PAI-1 were significantly lower in the LT recipients than in the other groups, while the SAP and SAA levels were significantly lower only when comparing the LT recipients with the NASH patients. The serum adiponectin levels were higher in the transplanted patients than in NASH patients (P = 0.007). Only one patient (a patient with post-transplant myocardial infarction) developed cardiovascular events during the follow-up period. No patients presented with recurrent hepatocellular carcinoma within the 1-year followup. DISCUSSION 8670 July 14, 2014 Volume 20 Issue 26

363 Alvares-da-Silva MR et al. Pro-atherosclerotic markers after liver transplantation Table 4 Comparison among liver transplant recipients, non-alcoholic steatohepatitis and controls regarding endothelial biomarkers Variable (pg/ml) NASH (n = 22) LTR (n = 44) Controls (n = 10) P 3 svcam ± ± ± < sicam ± ± ± MPO ± ± ± < Adiponectin ± ± ± PAI ± ± ± 58.4 < SAP ± ± ± < SAA ( ) ( ) ( ) < E-selectin ( ) 48.5 ( ) 35.7 ( ) < MMP ( ) 50.5 ( ) ( ) HsCRP ( ) 0.53 ( ) 0.29 ( ) Mean ± SD; 2 Median (25 th -75 th percentiles); 3 Comparisons: svcam-1 (LTR = NASH - P = 0.5; LTR > controls - P < 0.001); s-icam-1 (LTR = NASH - P = 0.48; LTR > controls - P = 0.05; MPO (LTR < NASH - P = 0.02; LTR < controls - P < 0.001); Adiponectin (LTR > NASH - P = 0.007; LTR = controls - P = 0.27); PAI-1 (LTR < NASH and controls - P < 0.001); SAP (LTR < NASH - P < 0.001; LTR = controls - P = 0.22); SAA (LTR < NASH - P = 0.006; LTR = controls - P = 0.91); E-selectin (LTR < NASH - P = 0.001; LTR > controls - P = 0.04); MMP-9 (LTR < NASH and controls - P < 0.001); CRP (LTR < NASH - P = 0.007; LTR = controls - P = 0.41). svcam-1: Soluble vascular cell adhesion molecule-1; sicam-1: Soluble intracellular cell adhesion molecule-1; PAI-1: Plasminogen activator inhibitor 1; SAP: Serum amyloid P; SAA: Serum amyloid A; MMP-9: Matrix metallopeptidase 9; HsCRP: High-sensitivity C-reactive protein; LTR: Liver transplant recipients; NASH: Non-alcoholic steatohepatitis. cular risk factors, such as obesity or elevated Hs-CRP or/ and high FRS, are not observed. In liver disease, NASH patients represent the major leading intersection between metabolic syndrome and CVD; therefore, they represent a good comparison group when considering CVD risk in a given population. Post-transplant MS is an important risk factor for CVD, and it should be monitored [16,17]. In our study, patients presented relatively low BMIs pre-transplant, and diabetes was uncommon. Moreover, after LT, the prevalence of diabetes, hypertension, and MS remained low. This finding contrasts with several other studies that demonstrated relatively higher post-lt MS prevalence [6,16]. Additionally, BMI did not increase at the end of the 1-year follow-up. Correspondingly, in the present study, the LT recipients displayed a normal lipid profile, and FRS was not elevated; the mean FRS was 2%, which indicates lowrisk. Hs-CRP is a well-established predictive marker of risk of coronary events. CRP can induce endothelial lectin-like oxidized low-density lipoprotein (ox-ldl) receptor-1, which is the primary endothelial receptor for ox- LDL and may lead to the activation of pro-inflammatory genes, including IL-8, sicam-1, and svcam-1 [17]. Here, Hs-CRP was higher in the LT recipients than in the controls, but the difference was not significant. Indeed, this result is consistent with the aforementioned LT recipient characteristics and highlights the strength of our main results. Moreover, differences regarding age or BMI most likely have not influenced the results, as the LT recipients and controls had quite similar characteristics. Increased levels of selectins and adhesion molecules are considered to be important indicators of atherosclerosis [18]. One of the key initial events in the development of atherosclerosis is the adhesion of monocytes to the endothelial cells, with subsequent transmigration into the vascular intima. Soluble leukocyte and vascular cell adhesion molecules (CAM), such as selectins, integrins, svcam-1, and sicam-1, play critical roles in the adhesion of monocytes to endothelial cells [17]. In the present study, the svcam-1 and sicam-1 levels were higher in the LT recipients than in the controls. Remarkably, the CAM levels were comparable between the LT recipients and NASH patients. In addition, the E-selectin levels in the LT recipients were significantly higher than in the controls. These results could indicate the initiation of an atherosclerotic disease process. In contrast, the LT recipients displayed lower MPO, MMP-9, and PAI-1 levels than the controls and NASH patients. It is unknown why these markers were low in our study; although tacrolimus itself can negatively impact MMP-9 [19], and PAI-1 can be low in association with thrombocytopenia, as approximately 90% of blood PAI-1 is found in the platelet compartment [20]. Regarding MPO, which promotes atherosclerosis via oxidative stress [21], levels under < 115 ng/ml were recently correlated with a longer event-free period in a high-risk population suffering from peripheral arterial disease [22]. In our study, only the LT recipients had mean MPO levels 115. Inflammation plays a leading role in atherosclerosis. Most of the studies on cytokines in LT patients indicate immediate complications, such as ischemia-reperfusion injury or rejection. In the current study, the TNFα levels were similar among the LT recipients, NASH patients, and controls, but a previous study from our group demonstrated that TNFα does not increase in NAFLD [23]. IFNγ levels were lower in the controls than in the LT recipients, suggesting that LT recipients have more inflammation than normal. This result is confirmed by antiinflammatory IL-10, a pivotal anti-inflammatory cytokine that showed higher levels in the controls compared to the LT patients. Down-regulation of IL-10 has also been recently demonstrated in NASH patients [24], and low circulating levels have been demonstrated in obese patients. The role of IL-8 is not well documented, even in NAFLD. It has been suggested that cirrhosis itself (with hepatic shunts and liver dysfunction) can partially explain 8671 July 14, 2014 Volume 20 Issue 26

364 Alvares-da-Silva MR et al. Pro-atherosclerotic markers after liver transplantation the higher systemic levels of pro-inflammatory cytokines [25]. We hypothesized that hepatic clearance is not the cause of the cytokine profile in our LT population, as we only included outpatients who had no significant liver function damage. HCV was also recently associated with the pro-inflammatory profile [26] ; thus, this variable must be considered when analyzing our results, as 36.4% of the patients were HCV-positive. Adiponectin, an anti-inflammatory adipokine that acts as an anti-obesity hormone, is usually down-regulated in NAFLD [27]. Few studies have investigated adiponectin in LT patients. In the present study, the LT recipients had higher adiponectin levels than the NASH patients, and there were no differences between the patients and controls. Immunosuppressive therapy should be considered, as steroids and calcineurin inhibitors are related to a high risk of metabolic syndrome [28]. Calcineurin inhibitors are also linked to renal injury and are prone to increase oxidative stress and lipid peroxidation. Thus, immunosuppressive agents might be somehow associated with our main results. Most LT studies focused on CVD after a long-term follow-up. Longer follow-ups are associated with an increased likelihood that a patient will suffer from MS, and it is presumably difficult to alter this path. Studies predicting cardiac complications based on short follow-ups are scarce and do not focus on atherosclerotic disease [29-32]. The present study was not designed to assess long-term prognosis but rather to evaluate the risk of LT recipients within 1-year post-transplant. This study has several strengths that should be emphasized. The sample selection was adequate, as only outpatients without clinically evident inflammatory complications, such as rejection or infection, were included. Inclusion at the end of the first year post-lt enabled the authors to evaluate cardiac risk factors late enough to avoid specific LT complication biases and early enough to allow to prevent disease progression. The LT recipients were not obese; the MS prevalence was low, and the LT recipients were compared to both controls and NASH patients. Several limitations should also be noted. The sample size was small, and the study was conducted at a single-center. Liver biopsies were not performed following a protocol schedule; baseline endothelial function and inflammatory profile information was not available for the LT recipients; and no cardiovascular imaging studies were performed. Finally, although unlikely, it is impossible for us to determine whether the inflammatory cytokines and endothelial marker profiles were related to some inherent transplant issues, such as a continuous rejection stimulus. In conclusion, we confirmed our hypothesis that LT recipients, even after a short follow-up period of 1-year post-transplant, are a population with a high atherosclerotic risk, as demonstrated by their inflammatory profiles and endothelial biomarkers. These results suggest that LT recipients should be carefully followed to prevent future CVD. COMMENTS Background Advances in medical therapy and surgical techniques made common the longterm survival of liver transplant recipients. Indeed, liver transplant recipients have a high risk of long-term development of cardiovascular disease. Research frontiers Most post-transplantation studies are focused on noticeable cardiovascular after a long-term follow-up. Studies predicting cardiac complications based on short follow-ups are scarce and do not focus on atherosclerotic disease. The present study was not designed to assess long-term prognosis but rather to evaluate the risk of liver transplant recipients within 1-year post-transplant. Innovations and breakthroughs This study demonstrated a high pro-inflammatory profile and endothelial dysfunction in low-risk liver transplant recipients one year post-transplantation. Applications Liver transplant recipients should be evaluated for cardiovascular early after transplantation to allow for effective preventative strategies. Terminology Endothelial dysfunction is the first step and inflammation plays an important role in developing early atherosclerosis. One of the key initial events is the adhesion of monocytes to the endothelial cells, with subsequent transmigration into the vascular intima. Soluble leukocyte and vascular cell adhesion molecules (CAM), such as selectins, integrins, svcam-1, and sicam-1, play critical roles in the adhesion of monocytes to endothelial cells. Peer review The manuscript provides information about pro-atherosclerotic markers and cardiovascular risk factors one year after liver transplantation. This subject would certainly contribute to improve the knowledge about liver transplantation. REFERENCES 1 Desai S, Hong JC, Saab S. Cardiovascular risk factors following orthotopic liver transplantation: predisposing factors, incidence and management. Liver Int 2010; 30: [PMID: DOI: /j x] 2 Stravitz RT, Carl DE, Biskobing DM. Medical management of the liver transplant recipient. Clin Liver Dis 2011; 15: [PMID: DOI: /j.cld ] 3 Hanouneh IA, Feldstein AE, McCullough AJ, Miller C, Aucejo F, Yerian L, Lopez R, Zein NN. The significance of metabolic syndrome in the setting of recurrent hepatitis C after liver transplantation. Liver Transpl 2008; 14: [PMID: DOI: /lt.21524] 4 Albeldawi M, Aggarwal A, Madhwal S, Cywinski J, Lopez R, Eghtesad B, Zein NN. Cumulative risk of cardiovascular events after orthotopic liver transplantation. 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365 Alvares-da-Silva MR et al. Pro-atherosclerotic markers after liver transplantation dation of a histological scoring system for nonalcoholic fatty liver disease. Hepatology 2005; 41: [PMID: ] 11 Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults. Executive Summary of The Third Report of The National Cholesterol Education Program (NCEP) Expert Panel on Detection, Evaluation, And Treatment of High Blood Cholesterol In Adults (Adult Treatment Panel III). JAMA 2001; 285: [PMID: ] 12 Grundy SM, Hansen B, Smith SC, Cleeman JI, Kahn RA. Clinical management of metabolic syndrome: report of the American Heart Association/National Heart, Lung, and Blood Institute/American Diabetes Association conference on scientific issues related to management. Arterioscler Thromb Vasc Biol 2004; 24: e19-e24 [PMID: ] 13 Matsudo SM, Matsudo VR, Araujo TL, Andrade DR, Andrade EL, de Oliveira LC, Braggion GF. 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The release of cytokines, adhesion molecules, and extracellular matrix parameters during and after reperfusion in human liver transplantation. Transplantation 1996; 62: [PMID: ] 22 Elias-Miro M, Massip-Salcedo M, Jimenez-Castro M, Peralta C. Does adiponectin benefit steatotic liver transplantation? Liver Transpl 2011; 17: [PMID: DOI: /lt.22358] 23 Rabelo F, Oliveira CP, Faintuch J, Mazo DF, Lima VM, Stefano JT, Barbeiro HV, Soriano FG, Alves VA, Carrilho FJ. Proand anti-inflammatory cytokines in steatosis and steatohepatitis. Obes Surg 2010; 20: [PMID: ] 24 Brogren H, Wallmark K, Deinum J, Karlsson L, Jern S. Platelets retain high levels of active plasminogen activator inhibitor 1. PLoS One 2011; 6: e26762 [PMID: DOI: /journal.pone ] 25 Wiest R, Weigert J, Wanninger J, Neumeier M, Bauer S, Schmidhofer S, Farkas S, Scherer MN, Schäffler A, Schölmerich J, Buechler C. Impaired hepatic removal of interleukin-6 in patients with liver cirrhosis. Cytokine 2011; 53: [PMID: DOI: /j.cyto ] 26 Oliveira CP, Kappel CR, Siqueira ER, Lima VM, Stefano JT, Michalczuk MT, Marini SS, Barbeiro HV, Soriano FG, Carrilho FJ, Pereira LM, Alvares-da-Silva MR. Effects of hepatitis C virus on cardiovascular risk in infected patients: a comparative study. Int J Cardiol 2013; 164: [PMID: DOI: /j.ijcard ] 27 Buechler C, Wanninger J, Neumeier M. Adiponectin, a key adipokine in obesity related liver diseases. World J Gastroenterol 2011; 17: [PMID: DOI: /wjg. v17.i ] 28 Oliveira CP, Stefano JT, Alvares-da-Silva MR. Cardiovascular risk, atherosclerosis and metabolic syndrome after liver transplantation: a mini review. Expert Rev Gastroenterol Hepatol 2013; 7: [PMID: DOI: /egh.13.19] 29 Estep JM, Baranova A, Hossain N, Elariny H, Ankrah K, Afendy A, Chandhoke V, Younossi ZM. Expression of cytokine signaling genes in morbidly obese patients with nonalcoholic steatohepatitis and hepatic fibrosis. Obes Surg 2009; 19: [PMID: DOI: /s x] 30 Laish I, Braun M, Mor E, Sulkes J, Harif Y, Ben Ari Z. Metabolic syndrome in liver transplant recipients: prevalence, risk factors, and association with cardiovascular events. Liver Transpl 2011; 17: [PMID: DOI: /lt.22198] 31 Dec GW, Kondo N, Farrell ML, Dienstag J, Cosimi AB, Semigran MJ. Cardiovascular complications following liver transplantation. Clin Transplant 1995; 9: [PMID: ] 32 Fouad TR, Abdel-Razek WM, Burak KW, Bain VG, Lee SS. Prediction of cardiac complications after liver transplantation. Transplantation 2009; 87: [PMID: DOI: /TP.0b013e318198d734] P- Reviewers: Fourtounas C, Silva R, Zou HQ S- Editor: Qi Y L- Editor: A E- Editor: Zhang DN 8673 July 14, 2014 Volume 20 Issue 26

366 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. Combination of symptoms, syndrome and disease: Treatment of refractory diabetic gastroparesis OBSERVATIONAL BRIEF ARTICLE STUDY Jun-Ling Li, Min Li, Bing Pang, Qiang Zhou, Jia-Xing Tian, Hong-Xing Liu, Xi-Yan Zhao, Xiao-Lin Tong Jun-Ling Li, Bing Pang, Qiang Zhou, Xi-Yan Zhao, Xiao-Lin Tong, Department of Endocrinology, Guang anmen Hospital, China Academy of Chinese Medical Sciences, Beijing , China Jun-Ling Li, Jia-Xing Tian, Hong-Xing Liu, Beijing University of Traditional Chinese Medicine, Beijing , China Min Li, Molecular Biology Laboratory, Guang anmen Hospital, China Academy of Chinese Medical Sciences, Beijing , China Author contributions: Tong XL, Li JL, Li M and Zhou Q designed the research; Li JL, Pang B, Tian JX, Liu HX, Zhao XY performed the research; Li JL and Pang B analyzed the data and wrote the paper. Supported by The National Key Basic Research And Development Plan, No. 2010CB530601; National Natural Science Foundation of China, No Correspondence to: Xiao-Lin Tong, Professor of Medicine, Department of Endocrinology, Guang anmen Hospital, China Academy of Chinese Medical Sciences, 5 Beixiange Street, Xuanwu District, Beijing , China. xiaolintong66@sina.com Telephone: Fax: Received: December 17, 2013 Revised: February 24, 2014 Accepted: April 15, 2014 Published online: July 14, 2014 Abstract AIM: To assess effect of combination of symptoms, syndrome and disease on treatment of diabetic gastroparesis with severe nausea and vomiting. METHODS: Professor Tong Xiaolin s clinical electronic medical records of patients who were treated between January 1, 2006 and October 1, 2012 were used as a database. Patients who met the inclusion criteria were enrolled. General information (name, sex and age), symptoms and blood glucose levels were obtained from the clinic electronic medical record, which was supplemented by a telephone interview. The patient-rated Gastroparesis Cardinal Symptom Index (GCSI) was used to evaluate the severity of the symptoms of gastroparesis. The effects of the treatment were assessed by the change in the severity of the symptoms of gastroparesis and the change in blood glucose between the baseline levels and the post-treatment levels at 1, 2, 4, 8 and 12 wk. RESULTS: Forty-five patients had a mean GCSI nausea and vomiting severity score of 4.21 ± 0.67 and a total GCSI score of 2.77 ± 0.63 before treatment. There was a significant improvement in the nausea and vomiting score at every return visit compared with the baseline score (1 wk: 3.02 ± 1.04 vs 4.18 ± 0.71, P < 0.001; 2 wk: 2.32 ± 1.25 vs 4.16 ± 0.73, P < 0.001; 4 wk: 2.12 ± 1.26 vs 4.12 ± 0.73, P < 0.001; 8 wk: 1.79 ± 1.09 vs 4.24 ± 0.77, P < 0.001; 12 wk: 0.69 ± 0.92 vs 4.25 ± 0.70, P < 0.001). Twenty-five of the 45 patients had complete resolution of vomiting during the observation period (mean time to resolution was 37.9 ± 27.3 d). The postprandial fullness and early satiety subscale, bloating subscale and total GCSI scores were also improved. Finally, the blood glucose levels improved after treatment, although the change was not significant. CONCLUSION: Use of the combination of symptoms, syndrome and disease to treat diabetic gastroparesis with refractory nausea and vomiting may be a new treatment option Baishideng Publishing Group Inc. All rights reserved. Key words: Diabetic gastroparesis; Refractory nausea and vomiting; Traditional Chinese medicine; Treatment; Gastroparesis Cardinal Symptom Index Core tip: Limited therapeutic options exist for the treatment of diabetic gastroparesis (DGP) with refractory nausea and vomiting. Traditional Chinese medicine (TCM) has supplied important complementary and alternative treatments. Professor Tong Xiaolin is an expert in the use of TCM for severe gastroparesis in China. The combination of symptoms, syndrome and disease is a concept that he incorporates into his clini July 14, 2014 Volume 20 Issue 26

367 Li JL et al. Treatment of refractory diabetic gastroparesis cal practice. This article introduces how he uses this concept to treat DGP and assesses its effect by analysis of his clinical electronic medical records. It may provide a new treatment option for refractory disease associated with DGP. Li JL, Li M, Pang B, Zhou Q, Tian JX, Liu HX, Zhao XY, Tong XL. Combination of symptoms, syndrome and disease: Treatment of refractory diabetic gastroparesis. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: org/ /wjg.v20.i INTRODUCTION Gastroparesis is a chronic motility disorder of the stomach that is characterized by delayed gastric emptying in the absence of mechanical obstruction. The cardinal symptoms include postprandial fullness (early satiety), nausea, vomiting and bloating [1]. Gastroparesis is commonly found in patients with diabetes mellitus [2] and can lead to weight loss, poor nutritional status and poor glycemic control. Common treatments for gastroparesis include erythromycin, metoclopramide, domperidone and cisapride [3]. Some patients experience severe symptoms and therefore do not respond to traditional treatment modalities. These patients are unable to maintain sufficient oral nutrition and have frequent visits to the emergency room or are hospitalized [4]. Nausea and vomiting, the most bothersome symptoms reported by patients [5], contribute to electrolyte imbalance, dehydration and increased healthcare utilization [6]. Diabetic patients with gastroparesis can experience severe nausea and vomiting with impaired glycemic control and nutritional status [7-9]. The current treatment options for severe symptoms are surgery and gastric electrical stimulation (GES). These options both require hospitalization and are associated with a high cost and high risk of infection. Thus, new therapeutic options to alleviate severe nausea and vomiting are needed. Traditional Chinese medicine (TCM) has been widely used in the treatment of diabetes mellitus in China and often provides a good curative effect. Professor Xiao-Lin Tong is one of the academic leaders in the field of TCM and is an expert in the use of TCM to treat severe gastroparesis in present-day China. He has worked in this field for > 30 years and has formed his own TCM theoretical system for diabetes and its complications. A combination of symptoms, syndrome and disease is his primary concept for the treatment of diabetic gastroparesis (DGP) accompanied by severe nausea and vomiting, and is associated with good curative effects. The aim of this study was to evaluate his TCM method for the treatment of severe DGP and to introduce a new treatment option for clinicians by analyzing his clinical electronic medical records. MATERIALS AND METHODS Patients Professor Xiao-Lin Tong s clinic electronic medical records were used as the database for this study. Patients who met the inclusion criteria and who were seen in the clinic between January 1, 2006 and October 1, 2012 were enrolled in this nonblinded study. The inclusion criteria were as follows: (1) documented diagnosis of DGP for > 1 year; (2) being refractory to conventional medical therapy such as antiemetics and prokinetics; and (3) a Gastroparesis Cardinal Symptom Index (GCSI) nausea/ vomiting subscale severity score 3.5 [1]. Patients without follow-up, including those who did not return to professor Xiao-Lin Tong s clinic after the first visit and could not be contacted by telephone, were excluded. Patients with another planned intervention (such as placement of a gastric electrical stimulator) or a new medication for the treatment of severe gastroparesis were excluded. Finally, those patients with primary eating or swallowing disorders, including rumination syndrome, psychogenic vomiting and cyclic vomiting syndrome, or an active malignancy were also excluded. Study protocol General information (name, sex and age) and blood glucose levels were reviewed retrospectively from the clinical electronic medical records. The baseline and post-treatment symptoms were obtained from the clinical records and were supplemented by information obtained from a telephone interview. Patient evaluations were performed at 1-, 2-, 4-, 8- or 12-wk return visits. The symptoms of gastroparesis were evaluated using the GCSI, which uses a six-point scale ranging from none (0) to very severe (5) [1]. The severity of each of nine symptoms was evaluated individually and grouped according to the three subscales (nausea/vomiting, postprandial fullness/early satiety and bloating) and by the total GCSI score. The severity of the symptoms of gastroparesis was the main standard used in evaluating the TCM therapy. TCM as a therapy for severe DGP Professor Tong Xiaolin writes a prescription according to the idea of a combination of symptoms, syndrome and disease. First, he chooses some herbs according to the patients symptoms. Patients with severe DGP often experience vomiting and abdominal bloating as their main symptoms. Xiao-Banxia-Tang (consisting of Pinelliaternata and ginger) and Suye-Huanglian-Tang (consisting of perilla leaves and Rhizoma Coptidis) were used as traditional antiemetic prescriptions. Zhizhu wan (consisting of Fructus Aurantii Immaturus and Bighead Atractylodes rhizome) was used as a traditional medication to relieve distention and other symptoms of abdominal bloating. Second, he selects some herbs according to the syndrome. In TCM theory, syndrome is the fundamental cause of the symptoms and can be established according to the types of symptoms. Patients with excessive heat 8675 July 14, 2014 Volume 20 Issue 26

368 Li JL et al. Treatment of refractory diabetic gastroparesis Table 1 Change in nausea/vomiting subscale Score followup (wk) of the spleen and stomach (symptoms of a bitter taste in the mouth and dry stool) were treated with Dahuang- Huanglian-Xiexin-Tang (consisting of Rheumofficinale and the rhizome of Chinese goldthread). Patients with heat in the upper and cold in the lower (symptoms of fulminant vomiting and cold pain in the stomach) were treated with Xiexin-Tang (consisting of Pinellia ternata, ginger, Rhizoma Coptidis, Radix Scutellariae, Rhizoma Zingiberis, ginseng and liquorice). Patients who experienced deficiency-cold of the spleen and kidney (symptoms of spitting or drooling, diarrhea, cold limbs and a deep thready pulse) were given Fuzi-Lizhong-Tang (consisting of ginseng, Bighead Atractylodes rhizome, Rhizoma Zingiberis and monkshood). Finally, as the primary index to monitor the diabetic disease process, the patient s blood glucose level was also monitored. Rhizoma Coptidis, Rhizoma Anemarrhenae and Radix Trichosanthis were subsequently prescribed to control high blood glucose levels. Patients with severe vomiting or abdominal distension were re-evaluated in 7 d. Patients were advised to sip their medication (which was provided as a decoction) as tolerated if they experienced vomiting. After the severe symptoms were relieved, the return period was changed to 2, 4, 8 or 12 wk, according to the severity of their symptoms and their response to treatment. Patients took their TCMs twice daily and maintained regular clinic visits until the symptoms resolved. Statistical analysis Patient identification, data registration and data entry were performed by two clinicians. A third clinician checked the database for entry errors. All of the data were analyzed using SPSS 17.0 software (SPSS Inc., Chicago, IL, United States). The severity of the symptoms and blood glucose levels before treatment and at 1, 2, 4, 8 and 12 wk after treatment were compared using a paired t test. All data were presented as the mean ± SD, with P < 0.05 considered statistically significant. RESULTS No. Before treatment After treatment Before P value minus after ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± Study population Forty-five eligible patients were treated from January 1, 2006 to October 1, The treatment group comprised 32 women (71.1%) and 13 men (28.9%), with a mean age of 43.7 ± 15.3 years (range: years). Of these, 24 patients (54.3%) were diagnosed with type 1 Table 2 Change in postprandial fullness/early satiety and bloating subscale scores Followup No. Symptom Before (wk) treatment After treatment Before P value minus after 1 33 Fullness/ 2.53 ± ± ± early satiety Bloating 1.23 ± ± ± Fullness/ 2.48 ± ± ± early satiety Bloating 1.30 ± ± ± Fullness/ 2.64 ± ± ± early satiety Bloating 1.44 ± ± ± Fullness/ 2.82 ± ± ± early satiety Bloating 1.57 ± ± ± Fullness/ 2.81 ± ± ± early satiety Bloating 1.25 ± ± ± diabetes mellitus, and 21 (45.7%) with type 2 diabetes mellitus. The mean duration of diabetes for the 45 patients was 11 years (range: 1-36 years), and the mean duration of gastroparesis was 30.6 ± 43.3 mo (range: mo). Ten patients experienced chronic gastritis, eight had reflux esophagitis, and two presented with a history of incomplete intestinal obstruction. In addition, one patient had undergone a cholecystectomy, and one was diagnosed with gallbladder polyps. The 45 patients had a nausea/vomiting subscale severity score of 4.21 ± 0.67, a postprandial fullness/early satiety subscale severity score of 2.7 ± 0.97, a bloating subscale severity score of 1.38 ± 0.82, and a total GCSI score of 2.77 ± Change in symptom severity The severity of the symptoms was evaluated before and after 1, 2, 4, 8 and 12 wk of treatment (Tables 1-3). This was a retrospective study based on Professor Tong s clinical practice, therefore, patients may have had different follow-up periods according to the severity of their symptoms. Additionally, once the severe symptoms were relieved, a patient s follow-up period may have been changed to 2, 4, 8 or 12 wk or even longer to limit the number of follow-up visits. Thus, many of the patients, due to great improvement in their symptoms after one or two follow-up appointments, did not return to the clinic within the 12-wk time frame. Consequently, most of their treatment records did not appear until after the 12-wk observation period. As a result, most of the patients in this study did not have treatment records representative of the five follow-up time points as the number of clinic visits decreased over time. Out of 45 patients, seven had follow-up consultations at each time point, and five had follow-up appointments at 1, 2, 4 and 8 wk. Four patients had follow-up appointments at 1, 2 and 4 wk, five had follow-up consultations at 1 and 2 wk, and five had follow-up appointments at 1 wk. Change in the nausea/vomiting subscale score: Table 1 shows the changes in the nausea/vomiting subscale 8676 July 14, 2014 Volume 20 Issue 26

369 Li JL et al. Treatment of refractory diabetic gastroparesis Table 3 Change in total Gastroparesis Cardinal Symptom Index score Follow-up (wk) No. Before treatment After treatment Before minus after P value ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± These percentages reflect the lack of association between the two types of diabetes and the susceptibility to DGP. There were more female than male (33 women vs 13 men) patients with refractory nausea and vomiting, which suggests that women are more prone to severe DGP. This finding is consistent with previous reports that proposed that severe DGP may be related to a high estrogen level in women [11-13]. The pathogenesis of DGP is not clear, and there is no standard evaluation for DGP. Most studies evaluate gastric emptying time because gastroparesis is defined as delayed gastric emptying. Radionuclide scanning at 15-min intervals for 4 h after the intake of labeled food is considered the gold standard for measuring gastric emptying [9]. This test is relatively expensive, is associated with radiation exposure, and does not have a standardized application at different medical centers [14]. In addition, completing a gastric emptying study in DGP patients with severe nausea and vomiting is difficult. Many reports show a poor correlation between improvement of the symptoms of gastroparesis and tests for gastric emptying [15,16]. Severity of symptom and quality of life outcomes are needed for a better evaluation of the effectiveness of treatment [17-20]. Patient-reported symptom severity is important in the evaluation of this disease and is the only measure that directly reflects each patient s experience with symptom severity, function and well-being. In clinical practice, clinicians rely on the patients reports of their symptoms to manage DGP and to monitor the effectiveness of treatment. The GCSI is a widely used symptom standard in studies of gastroparesis [21-23]. This scale was developed based on reviews of the medical literature, clinician interviews and patient focus groups and has been validated in patients with gastroparesis [1,24]. The total GCSI score has been used as a standard to diagnose gastroparesis [25]. We used the GCSI as the principal tool to evaluate patients with DGP and refractory nausea and vomiting. Glycemic control is an important part of the management of DGP [26], therefore, we also monitored the blood glucose levels during TCM treatment. TCM greatly reduced the nausea/vomiting subscale score, postprandial fullness/ early satiety subscale score, bloating subscale score and total GCSI score. There was a tendency toward improvement in blood glucose levels after treatment. The current treatment options for severe DGP are limited. The most widely used treatment reported is gastric electrical stimulation (GES), and although GES treatscore. The nausea/vomiting subscale scores after 1, 2, 4, 8 and 12 wk of treatment were significantly improved compared with the baseline score (P < 0.05).The patient symptom severity score also improved over time. Out of 45 patients, 25 had complete resolution of vomiting by 12 wk based on their medical records. The mean time to resolution was 37.9 ± 27.3 d (range: 7-90 d). Change in the postprandial fullness/early satiety and bloating subscale scores: Similar improvements were found in the postprandial fullness/early satiety and bloating subscale scores (Table 2). The patient-reported symptom severity score also improved over time. Change in overall symptoms: Table 3 shows that the total GCSI score significantly improved at all follow-up time points. The curative effect also increased as time progressed. The general feeling of well-being of the patients was then evaluated. The evaluation of overall wellbeing included sleep status, physical capacity and psychological status. Out of 45 patients, 43 reported that they feel better after treatment. The mean time from the onset of treatment to this report was 19.6 ± 11.7 d (range: 2-56 d). Change in blood glucose levels Patients who were evaluated for DGP suffered from severe nausea or vomiting. The patients symptoms were the primary focus, thus, their blood glucose levels were not always assessed. The fasting blood glucose levels were used to evaluate blood glucose levels (Table 4). As shown in Table 4, the blood glucose levels tended to improve with treatment. DISCUSSION DGP with refractory nausea and vomiting is a difficult clinical problem to resolve. Current treatment options for severe gastroparesis are limited because the pathogenesis of DGP is not completely understood [10]. TCM has a long history of use as a complementary and alternative medicine. Many patients with incurable diseases such as severe DGP, especially in China, are advised to see a traditional Chinese doctor. However, there have been few reports that have evaluated the clinical efficacy of TCM treatments. This study was based on the nearly 6-year TCM clinical practice of Professor Tong. Twentyfive patients (54.3%) were diagnosed with type 1 diabetes mellitus, and 21 (45.7%) with type 2 diabetes mellitus. Table 4 Change in fasting blood glucose level Follow-up (wk) No. Before treatment After treatment Before minus after P value ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± July 14, 2014 Volume 20 Issue 26

370 Li JL et al. Treatment of refractory diabetic gastroparesis ment is effective [27,28], it is expensive [29] and poses risks of device infection, accidental deactivation by a magnetic field, and gastric perforation by the electrical leads [30]. The endoscopic pyloric injection of botulinum toxin has also been used to treat severe gastroparesis [31,32], but in patients who have vomiting as a major symptom, there was no predictable response to this treatment [31]. In contrast, treatment with TCM is inexpensive and noninvasive. TCM takes patient symptoms into account before the treatments are selected. Vomiting is often the most troublesome symptom in these patients, therefore, Xiao- Banxia-Tang combined with Suye-Huanglian-Tang was used to relieve the symptoms of vomiting. Xiao-Banxia- Tang effectively alleviates vomiting by inhibiting NK1 receptor activity, antagonizing motilin activity and releasing intestinal serotonin [33-35]. Suye-Huanglian-Tang has also been used to treat intractable vomiting [36-38]. The use of these two prescriptions may explain the rapid improvement in vomiting that we observed. Abdominal distention is a prominent symptom of DGP, and some reports [39,40] have shown that abdominal bloating and fullness alone may be associated with DGP. Zhizhu wan is used to improve abdominal distention by enhancing gastrointestinal motility [41,42]. Other Chinese medicines were added according to the syndrome presentation. This combined treatment may explain why the symptoms improved with prolonged treatment and why the curative effects increased over time. Modern medical findings were also applied to this TCM therapy. Chinese medicines that are known to lower blood glucose levels were used, including Chinese goldthread rhizomes [43,44] and Rhizoma Anemarrhenae [45-47] Blood glucose levels were improved in the patients who were evaluated. There is considerable documentation of TCM as a complementary and alternative medicine source. Although identifying the exact pharmacological composition of these medicines is difficult due to their complex components, their clinical activities cannot be ignored. The aim of this study was to evaluate a TCM treatment of DGP in patients with refractory nausea and vomiting and to provide a new treatment option for clinicians. This is a large study of patients with severe DGP, but it has several limitations, including its retrospective nature and use of clinical electronic medical records supplemented with detailed telephone interviews to identify the patients symptoms. Patients in this study did not undergo gastric emptying scintigraphy during treatment to determine whether there was a correlation between improved gastric emptying and relief of their symptoms. This study used the patients symptoms to determine the clinical follow-up period, thus, the timing of the return visits of the patients was not consistent, and there was not an equal number of patients at every follow-up time point. Despite the above limitations, we believe that this study offers a useful treatment option for DGP with refractory nausea and vomiting. Prospective studies are needed to better evaluate this form of TCM. COMMENTS Background Diabetic patients with severe nausea and vomiting have impaired glycemic control and nutritional status. However, limited therapeutic options exist for the treatment of diabetic gastroparesis (DGP) with refractory nausea and vomiting. Finding new treatment options for this refractory disease is a complex clinical problem that needs to be solved. Research frontiers The most common treatment options for severe gastroparesis are surgery and gastric electrical stimulation, but these treatments require hospitalization and are associated with a high cost and high risk of infection. Innovations and breakthroughs Limited therapeutic options exist for the treatment of DGP with refractory nausea and vomiting. Based on nearly 7 years of clinical medical records, this study analyzed the effects of Professor Tong s treatment on this refractory disease and found significant improvement in nausea and vomiting as well as in other symptoms of gastroparesis. This study provides a new treatment option for clinicians. Applications The study results suggest that the use of the combination of symptoms, syndrome and disease model within the Traditional Chinese medicine (TCM) framework could be a treatment option for DGP with refractory nausea and vomiting. Terminology Syndrome is a specific diagnostic concept in TCM; it is a pathological summary of the location, cause, nature and condition of a disease at a certain stage and also describes a conclusion about the pathological nature of the disease. Peer review This manuscript provides a detailed data analysis to assess the effects of Professor Tong Xiaolin s methods in the treatment of DGP with severe nausea and vomiting. The use of combination of symptoms, syndrome and disease to treat DGP with refractory nausea and vomiting may be a new treatment option. REFERENCES 1 Revicki DA, Rentz AM, Dubois D, Kahrilas P, Stanghellini V, Talley NJ, Tack J. Development and validation of a patient-assessed gastroparesis symptom severity measure: the Gastroparesis Cardinal Symptom Index. Aliment Pharmacol Ther 2003; 18: [PMID: DOI: / j x] 2 Horowitz M, O Donovan D, Jones KL, Feinle C, Rayner CK, Samsom M. Gastric emptying in diabetes: clinical significance and treatment. 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372 Li JL et al. Treatment of refractory diabetic gastroparesis Horowitz M. Predictors of delayed gastric emptying in diabetes. Diabetes Care 2001; 24: [PMID: DOI: /diacare ] 40 Tack J, Lee KJ. Pathophysiology and treatment of functional dyspepsia. J Clin Gastroenterol 2005; 39: S211-S216 [PMID: DOI: /01.mcg d1] 41 Liu Y, Zhao XR, Wang R, Qiu GQ, Zhang M. The impact of Zhizhu Pill on the serum gastrointestinal hormones of type 2 diabetes functional constipation. Zhongguo Zhongyao Zazhi 2008; 33: Ma XH, Shang YZ. Experimental study on the effect of Zhizhu pill and Zhizhu Decoction on gastrointestinal movement. Lishizhen Guoyi Guoyao 2005; 7: Li ZQ, Zuo DY, Qie XD, Qi H, Zhao MQ, Wu YL. Berberine acutely inhibits the digestion of maltose in the intestine. J Ethnopharmacol 2012; 142: [PMID: DOI: /j.jep ] 44 Li J, Meng X, Fan X, Lai X, Zhang Y, Zeng Y. [Pharmacodyamic material basis of rhizoma coptidis on insulin resistance]. Zhongguo Zhong Yao Zazhi 2010; 35: [PMID: ] 45 Miura T, Ichiki H, Iwamoto N, Kato M, Kubo M, Sasaki H, Okada M, Ishida T, Seino Y, Tanigawa K. Antidiabetic activity of the rhizoma of Anemarrhena asphodeloides and active components, mangiferin and its glucoside. Biol Pharm Bull 2001; 24: [PMID: DOI: / bpb ] 46 Miura T, Ichiki H, Hashimoto I, Iwamoto N, Kato M, Kubo M, Ishihara E, Komatsu Y, Okada M, Ishida T, Tanigawa K. Antidiabetic activity of a xanthone compound, mangiferin. Phytomedicine 2001; 8: [PMID: DOI: / ] 47 Ichiki H, Miura T, Kubo M, Ishihara E, Komatsu Y, Tanigawa K, Okada M. New antidiabetic compounds, mangiferin and its glucoside. Biol Pharm Bull 1998; 21: [PMID: DOI: /bpb ] P- Reviewer: Wang XP S- Editor: Zhai HH L- Editor: Kerr C E- Editor: Zhang DN 8680 July 14, 2014 Volume 20 Issue 26

373 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. OBSERVATIONAL BRIEF ARTICLE STUDY Expression of P450 and nuclear receptors in normal and end-stage Chinese livers Hong Chen, Zhong-Yang Shen, Wang Xu, Tie-Yan Fan, Jun Li, Yuan-Fu Lu, Ming-Liang Cheng, Jie Liu Hong Chen, Zhong-Yang Shen, Wang Xu, Tie-Yan Fan, Jun Li, The Institute of Organ Transplantation, The General Hospital of Chinese People s Armed Police Forces, Beijing , China Yuan-Fu Lu, Jie Liu, Department of Pharmacology, Kay Lab for Basic Pharmacology of Ministry of Education, Zunyi Medical College, Zunyi , Guizhou Province, China Ming-Liang Cheng, Department of Infectious Diseases, Guiyang Medical College Hospital, Guiyang , Guizhou Province, China Author contributions: Chen H, Shen ZY, Cheng ML and Liu J designed the experiments; Chen H, Xu W, Fan TY and Li J collected samples and performed the experiments; Lu YF performed RT-PCR analysis; Cheng ML contributed reagents/analytic tools; Chen H and Liu J analyzed the data and wrote the manuscript. Supported by Grants from the Chinese 863 project, No. 2012AA022409; and Guizhou Science and Technology Foundation, No Correspondence to: Dr. Zhong-Yang Shen, The Institute of Organ Transplantation, The General Hospital of Chinese People s Armed Police Forces, No. 69 Yong-Ding Road, Beijing , China. zhongyangshen@gmail.com Telephone: Fax: Received: January 9, 2014 Revised: February 19, 2014 Accepted: April 8, 2014 Published online: July 14, 2014 in HCC, 80% in alcoholic cirrhosis, and 65% in severe cirrhosis. For CYP2 family, the expression of CAR was decreased 50% in HCC, but increased 50% in peri-hcc tissues. Similar decreases (about 50%) of CYP2B6, CY- P2C9, CYP2C19, CYP2D6 and CYP2E1 were observed in HCC, as compared to peri-hcc tissues and normal livers. CYP2C19 were decreased in all end-stage liver diseases and CYP2E1 also decreased in alcoholic cirrhosis and severe cirrhosis. For CYP3 family, the expression of PXR was decreased 60% in HCC, together with decreases in CYP3A4, CYP3A5, and CYP3A7. In contrast, the expression of CYP3A7 was slightly increased in HBV cirrhosis. The expression of CYP4A11 was decreased 85% in HCC, 7% in alcoholic cirrhosis and severe liver cirrhosis, along with decreases in PPARα. The 93 endstage livers had much higher inter-individual variations in gene expression than 35 normal livers. CONCLUSION: the expression of CYP enzyme genes and corresponding nuclear receptors was generally decreased in end-stage liver diseases, and significant differences in gene expression were evident between peri-hcc and HCC Baishideng Publishing Group Inc. All rights reserved. Abstract AIM: To investigate the expression of P450 enzyme genes by using end-stage liver disease samples and trimmed normal Chinese donor livers. METHODS: The end-stage liver disease samples [n = 93, including hepatocellular carcinoma (HCC), peri-hcc tissue, hepatitis B virus cirrhosis, alcoholic cirrhosis, and severe cirrhosis] and trimmed normal Chinese donor livers (n = 35) from The Institute of Organ Transplantation in Beijing, China. Total RNA was extracted, purified, and subjected to real-time RT-PCR analysis. RESULTS: For cytochrome P450 enzymes 1 (CYP1) family, the expression of CYP1A2 was decreased 90% Key words: Cytochrome P450; Nuclear receptors; mrna expression; End-stage livers; Chinese donor livers; Hepatocellular carcinoma Core tip: This study examined the expression of P450 enzyme (CYP) genes in end-stage liver diseases, including hepatocellular carcinoma (HCC), peri-hcc tissue, hepatitis B virus cirrhosis, alcoholic cirrhosis, and severe cirrhosis, and compared to normal donor livers by real-time RT-PCR analysis. The 93 end-stage livers had much higher inter-individual variations in gene expression than 35 normal livers. In general, the end-stage livers had decreased expression of CYP1A2, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, CYP3A5, CYP3A7 and CYP4A11, as well as their corresponding nuclear receptor AhR, CAR, PXR, and PPARα. HCC had 8681 July 14, 2014 Volume 20 Issue 26

374 Chen H et al. P450 expression in end-stage liver diseases dramatic decreases in CYP gene expression and nuclear receptors compared with peri-hcc tissues. Table 1 Sample demographics (n = 128) Liver disease Gender Age (yr) Chen H, Shen ZY, Xu W, Fan TY, Li J, Lu YF, Cheng ML, Liu J. Expression of P450 and nuclear receptors in normal and end-stage Chinese livers. World J Gastroenterol 2014; 20(26): Available from: URL: v20/i26/8681.htm DOI: i INTRODUCTION Cytochrome P450 enzymes are important in the metabolism of endogenous substances and xenobiotics [1]. Functional human P450 isozyme variations are implicated with drug toxicities, such as S-acenocoumarol, phenytoin and warfarin (CYP2C9), metoclopramide, codeine, mirtazapin and tramadol (CYP2D6), acetaminophen and alcohol (CYP2E1), methadone and efavirenz (CYP2B6), clopidogrel and desipramine (CYP2C19), and tacrolimus (CYP3A5) [2-4]. The expression of CYP enzymes is influenced by endogenous factors, such as genetic polymorphisms [5,6], sex [7], age [8], and hormone levels [7]. The expression of CYP enzymes is also influenced by exogenous factors such as drugs and environmental chemicals [9] as well as the physiopathological conditions [10]. Hepatic cytochrome P450 (CYP) expression is subjected to changes during various disease conditions, such as steatosis [11], chronic hepatitis C [12], alcoholic liver diseases [13], nonalcoholic fatty liver disease [14], liver fibrosis [15], cirrhosis [16], hepatoblastoma [17], and hepatocellular carcinoma [18,19]. Hepatic CYP expression is also under control of nuclear receptors in that AhR regulates CYP1 gene family, CAR regulates CYP2 gene family, PXR regulates CYP3 family, and PPARα regulates CYP4 family [20-22]. The end-stage livers removed for liver transplantation are precious resources to investigate gene expression changes, including P450 enzyme genes [10,23-25]. However, little is known about end-stage liver P450 profiling from the Chinese population. This study was initiated to fill the gap. A total of 93 end-stage livers and 35 trimmed normal donor livers were obtained from the Oriental Liver Transplant Center in Beijing, China, and total RNA was isolated and purified to profile the expression of CYP1-4 family genes and corresponding nuclear receptors. The results demonstrated the generalized reduction of CYP gene expression in end-stage liver diseases. MATERIALS AND METHODS Liver samples and dimorphism The end-stage human liver samples (n = 93) were collected from Oriental Liver Translation Center (Beijing, China), and the sample demographics are shown in Table 1. Samples included hepatocellular carcinoma (HCC, n = 24), and the corresponding HCC surrounding tissues (peri-hcc, n = 24), hepatitis B virus (HBV) cirrhosis (n Normal donors End-stage livers = 27), severe cirrhosis (n = 13), and alcoholic cirrhosis (n = 5). The diagnosis of end-stage liver diseases was made by Pathology Department of the Institute of Organ Transplantation. This study received Institutional Review Board exemption status by the Oriental Liver Transplant Center and the Guiyang Medical College Human Subject Committee because the specimens were obtained without identification to the authors of the study. Total RNA isolation All 128 human liver samples were used for real-time RT- PCR analysis. Total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA) and purified with RNeasy columns (Qiagen, Valencia, CA). The quality and integrity of purified RNA was determined by spectrophotometry with 260/280 ratio > 1.8 and 260/230 ratio > 1.5, as well as by agarose gel electrophoresis showing clear 18S and 28S bands without degradation. Real-time RT-PCR analysis Total RNA was reverse-transcribed into cdna with Multiscript reverse transcriptase using High Capacity RT kits from Applied Biosystems (Foster City, CA), and amplified with Power SYBR Green PCR Master Mix in a 7900HT PCR System (Applied Biosystems, Foster City, CA). Oligonucleotide primers were designed with Primer3 software (version 4), and are listed in Table 2. Relative expression of genes was calculated by the 2 -ΔΔCt method and normalizing to the house-keeping gene GAPDH. Statistical analysis Data was calculated as mean ± SE. The SPSS17 software was used for statistical analysis. For comparisons among three or more groups, data were analyzed using a one-way analysis of variance (ANOVA), followed by Duncan s multiple range test. For the comparisons between HCC and peri-hcc, Student t-test was used. The significance level was set at p < 0.05 in all cases. RESULTS Trimmed normal livers Men Women HCC Peri-HCC HBV cirrhosis Severe cirrhosis Alcoholic cirrhosis HCC: hepatocellular carcinoma; HBV: hepatitis B virus. Expression of AhR and CYP1 family genes The expression of AhR and AhR-regulated CYP1A2 is shown in Figure 1 and Table 3. The average expression 8682 July 14, 2014 Volume 20 Issue 26

375 Chen H et al. P450 expression in end-stage liver diseases Table 2 Primer sequence for real-time RT-PCR analysis Gene GenBank Forward Reverse number AhR NM_ CTGCCTTTCCCACAAGATGT AGTTATCCTGGCCTCCGTTT CAR NM_ CTTCTCTCCTGACCGACCTG AGGCCTAGCAACTTCGCATA CYP1A2 NM_ GGACAGCACTTCCCTGAGAG GAGGCAGTCTCCACGAACTC CYP2B6 NM_ TCCAGTCTCAGCTCCCAAGT CTGGCCAACATGTCCCTACT CYP2C9 NM_ CCACATGCCCTACACAGATG TGCCCTTGGGAATGAGATAG CYP2C19 NM_ CAACAACCCTCGGGACTTTA GTCTCTGTCCCAGCTCCAAG CYP2D6 NM_ CAGAGATGGAGAAGGCCAAG AGAACAGGTCAGCCACCACT CYP2E1 NM_ CCCAAAGGATATCGACCTCA AGGGTGTCCTCCACACACTC CYP3A4 NM_ ACCGTGACCCAAAGTACTGG GTTTCTGGGTCCACTTCCAA CYP3A5 NM_ GGAGATGTTCCCCATCATTG GCCCCAAAGATGTCTTTCAA CYP3A7 NM_ GAAACACAGATCCCCCTGAA AGGGAAATCAGGCTCCACTT CYP4A11 NM_ CACCACAACCCAAAAGTGTG ATTGTTTCCCGATGCAGTTC FXR NM_ ATTTTGACGGAAATGGCAAC AGACCCTTTCAGCAAAGCAA GADPH NM_ ACAGTCAGCCGCATCTTCTT ACGACCAAATCCGTTGACTC PPARα Y07619 ACGATTCGACTCAAGCTGGT GTTGTGTGACATCCCGACAG PXR NM_ GGAATGTTGGCTGAATGCTT CTGCATGCTGCTTCACATTT A AhR a B 300 CYP1A2 AhR mrna (% of GAPDH) a CYP1A2 mrna (% of GAPDH) a a a 0 Normal liver Peri-HCC HCC HBV cirrhosis Alcoholic cirrhosis Severe cirrhosis 0 Normal liver Peri-HCC HCC HBV cirrhosis Alcoholic cirrhosis Severe cirrhosis C 600 CYP1A2 Peri-HCC HCC CYP1A2 mrna (% of GAPDH) Patient number Figure 1 expression of cytochrome P450 enzymes 1 family genes. A: AhR; B: CYP1A2; C: CYP1A2 in 24 paired HCC and peri-hcc tissue from individual patients. The end-stage livers (HCC and corresponding peri-hcc tissue, n = 24, HBV cirrhosis, n = 27, alcoholic cirrhosis, n = 5 and severe cirrhosis, n = 13) and normal donor trimmed livers (n = 35) were collected at the Oriental Liver Transplant Center, Beijing, China. The data are mean ± SE, Significantly different from normal livers, a P < 0.05 vs normal liver; HCC different from peri-hcc tissues. AhR: aryl hydrocarbon receptor; CYP: Cytochrome P450 enzymes; HCC: hepatocellular carcinoma; HBV: hepatitis B virus. of AhR in 35 normal livers (14.7% ± 0.7% of GADPH) was not significantly different from 93 diseased livers (17.4% ± 1.5% of GADPH), but diseased livers had huge inter-individual variations compared with normal livers (47-fold vs 5-fold) (Table 3). peri-hcc had 50% higher expression of AhR compared with normal livers, 8683 July 14, 2014 Volume 20 Issue 26

376 Chen H et al. P450 expression in end-stage liver diseases Table 3 mrna expression of Cytochrome P450 enzymes and corresponding nuclear receptors Normal liver (n = 35) End-stage livers (n = 93) Gene Expression% -fold mean ± SE Expression% -fold mean ± SE AhR ± ± 1.52 CYP1A ± ± 23.9 a CAR ± ± 0.86 CYP2B ± ± 2.77 a CYP2C ± ± 34.8 a CYP2C ± ± 23.9 a CYP2D ± ± 15.6 CYP2E ± ± 75.4 a PXR ± ± 1.81 CYP3A ± ± 48.6 a CYP3A ± ± 19.1 a CYP3A ± ± 47.7 PPARα ± ± 2.43 CYP4A ± ± 11.1 a Relative transcript levels (% of GAPDH), -fold indicates the inter-individual variations within normal or end-stage livers. Significantly different from normal livers, a p < but this was not statistically significant. Compared with peri-hcc, AhR mrna levels in HCC were significantly lower (Figure 1). There were no major changes in AhR expression in HBV cirrhosis, alcoholic cirrhosis, and severe cirrhosis. The average expression of CYP1A2 in normal livers (234% ± 94% of GADPH) was significantly higher than diseased livers (136% ± 24% of GADPH), and diseased livers had 8.5-fold higher inter-individual variations than normal livers (224-fold vs 26-fold) (Table 3). CYP1A2 in HCC was decreased by 90%, significantly lower than normal livers and peri-hcc (Figure 1). CYP1A2 expression was also decreased in alcoholic cirrhosis by 79% and severe cirrhosis by 66%, as compared to normal livers, but was unchanged in HBV cirrhosis and peri-hcc tissues. The paired individual values from peri-hcc and HCC from 24 patients are shown in the bottom panel of Figure 1. CYP1A2 expression was low in HCC, except for patient No. 14, as compared to peri-hcc. Some patients showed huge differences (i.e., patient No. 22), while others were moderate (i.e., patient No. 4). Expression of CAR and CYP2 family genes The expression of CAR and CAR-regulated CYPs are shown in Figure 2 and Table 3. The average expression of CAR in 35 normal livers (7.87% ± 0.5% of GADPH) was not significantly different from 93 diseased livers (7.73% ± 0.8% of GADPH), but diseased livers had huge inter-individual variations (1390-fold vs 26-fold) (Table 3). The basal expression of CAR was about 8% of GADPH. CAR mrna levels in HCC was decreased to 3.7% of GADPH, 50% lower than normal livers and 70% lower than peri-hcc (Figure 2). There were no major changes in CAR expression in HBV cirrhosis and severe cirrhosis. Alcoholic cirrhosis tended to be 45% higher than normal livers, but was not significant. The average expression of CYP2B6 in normal livers (17.1% ± 2.7% of GADPH) was lower than 93 diseased livers (27.2% ± 2.8% of GADPH), and diseased livers had higher inter-individual variations (158-fold vs 78-fold) (Table 3). CYP2B6 mrna levels in peri-hcc were increased 120%, and were decreased 50% in HCC as compared to normal livers (Figure 2). Expression of CYP2B6 was also increased in HBV cirrhosis and alcoholic cirrhosis, but was not altered in severe cirrhosis. The average expression of CYP2D6 in normal livers (149% ± 12%) was not different from diseased livers (156% ± 16% of GADPH), and diseased livers had greater inter-individual variations (220-fold vs 30-fold) (Table 3). Similarly, CYP2D6 mrna levels in peri-hcc were increased 53%, while in HCC CYP2D6 mrna was decreased 40% as compared to normal livers (Figure 2). There were no major changes in CYP2D6 expression in HBV cirrhosis, alcoholic cirrhosis and severe cirrhosis. The average expression of CYP2E1 in normal livers (1710% ± 115%) was significantly higher than diseased livers (990% ± 75% of GADPH), and diseased livers had huge inter-individual variations than normal livers (250-fold vs 17-fold) (Table 3). CYP2E1 mrna levels in HCC were decreased by 75%, significantly lower than normal livers and peri-hcc. CYP2E1 expression was also decreased in alcoholic cirrhosis and in severe cirrhosis. Both CYP2C9 and CYP2C19 are highly expressed in liver and are shown in Figure 3. The average expression of CYP2C9 in normal livers (786% ± 58% of GAPDH) was higher than in diseased livers (418% ± 35% of GADPH), and diseased livers had greater inter-individual variations (390-fold vs 110-fold) (Table 3). HCC had dramatic decreases in mrna levels of CYP2C9 (13% of normal livers), which is also significantly lower than peri- HCC. The expression of CYP2C9 tended to be lower in HBV cirrhosis, alcoholic cirrhosis and severe cirrhosis, but was not significant. The average expression of CY- P2C19 in normal livers (594% ± 40% of GAPDH) was higher than diseased livers (256% ± 39% of GADPH), 8684 July 14, 2014 Volume 20 Issue 26

377 Chen H et al. P450 expression in end-stage liver diseases 16 CAR 300 CYP2D6 Relative mrna levels (% of GAPDH) a Relative mrna levels (% of GAPDH) a a 0 Normal liver Peri-HCC HCC HBV cirrhosis Alcoholic cirrhosis Severe cirrhosis 0 Normal liver Peri-HCC HCC HBV cirrhosis Alcoholic cirrhosis Severe cirrhosis Relative mrna levels (% of GAPDH) CYP2B6 Normal liver a Peri-HCC a HCC a HBV cirrhosis a Alcoholic cirrhosis Severe cirrhosis Relative mrna levels 10 2 (% of GAPDH) CYP2E1 Normal liver Peri-HCC a HCC HBV cirrhosis a Alcoholic cirrhosis a Severe cirrhosis Figure 2 expression of cytochrome P450 enzymes 2 family genes. Right, CAR and CYP2B6; Left, CYP2D6 and CYP2E1. The 93 end-stage livers and normal donor trimmed livers (n = 35) were compared. The data are mean ± SE, Significantly different from normal livers, a P < 0.05 vs normal liver; HCC different from peri- HCC tissues. CAR: constitutive androstane receptor; CYP: Cytochrome P450 enzymes; HCC: hepatocellular carcinoma. and diseased livers had huge inter-individual variations compared with normal livers (788-fold vs 25-fold) (Table 3). CYP2C19 expression was decreased in all end-stage livers, with dramatic decreases in HCC by over 90% (Figure 3). peri-hcc had decreased CYP2C19, but the levels were significantly higher than HCC. The individual pair values of CYP2C19 in peri-hcc and HCC are shown in the bottom panel of Figure 3. In all 24 patients, CYP2C9 mrna levels were consistently lower in HCC, some showed huge differences (i.e., patient No. 13), while others moderate (i.e., patient No. 4) or mild (i.e., patient No. 7). Expression of PXR and CYP3 family genes The expression of PXR and PCR-regulated CYPs are shown in Figure 4 and Table 3. The average expression of PXR in normal livers (41.4% ± 5.0% of GAPDH) was not significantly different from diseased livers (33.0% ± 1.8% of GADPH), and diseased livers had higher inter-individual variations (29-fold vs 13-fold) (Table 3). PXR mrna levels in HCC were decreased by 60%, significantly lower than peri-hcc and normal livers (Figure 4). There were no major changes in PXR expression in HBV cirrhosis, alcoholic cirrhosis, and severe cirrhosis. The average expression of CYP3A4 in normal livers (798% ± 49% of GAPDH) was significantly higher than diseased livers (498% ± 49% of GADPH), and diseased livers had much higher inter-individual variations than normal livers (240-fold vs 8-fold) (Table 3). The mrna expression of CYP3A4 in HCC was decreased 85%, significantly lower than normal livers and peri-hcc. The mrna levels of CYP3A4 were also lower in severe cirrhosis. The average expression of CYP3A5 in normal livers (278% ± 27% of GAPDH) was significantly different from diseased livers (203% ± 19% of GADPH), and diseased livers had higher inter-individual variations (99-fold vs 30-fold) (Table 3). The expression of CYP3A5 was decreased 75% in HCC, significantly lower than normal livers and peri-hcc (Figure 4). The average expression of CYP3A7 in normal livers (450% ± 37% of GAPDH) was not significantly different from diseased livers (479% ± 48% of GADPH), and diseased livers had huge inter-individual variations compared with normal 8685 July 14, 2014 Volume 20 Issue 26

378 Chen H et al. P450 expression in end-stage liver diseases A 1000 CYP2C9 B CYP2C19 CYP2C9 mrna (% of GAPDH) a CYP2C19 mrna (% of GAPDH) a a a a a a 0 Normal liver Peri-HCC HCC HBV cirrhosis Alcoholic cirrhosis Severe cirrhosis 0 Normal liver Peri-HCC HCC HBV cirrhosis Alcoholic cirrhosis Severe cirrhosis C CYP2C19 mrna (% of GAPDH) CYP2C19 Peri-HCC HCC Patient number Figure 3 expression of cytochrome P450 enzymes 2C family genes. A: CYP2C9; B: CYP2C19; C: CYP1A2 in 24 paired HCC and peri-hcc tissue from individual patients. The 93 end-stage livers and 35 normal donor trimmed livers were compared. The data are mean ± SE, Significantly different from normal livers, a P < 0.05 vs normal liver; HCC different from peri-hcc tissues. CYP: Cytochrome P450 enzymes; HCC: hepatocellular carcinoma. livers (1415-fold vs 12-fold) (Table 3). The expression of CYP3A7 was decreased 80% in HCC, significantly lower than normal livers and peri-hcc (Figure 4). The mrnas of CYP3A7 in HBV cirrhosis were 70% significantly higher than normal livers and 50% higher than in alcoholic cirrhosis (not significant). The mrnas of CYP3A7 in peri-hcc and severe cirrhosis were not different from normal livers. Expression of PPARα and CYP4 family genes The average expression of PPARα in normal livers (29.4% ± 1.9% of GAPDH) was not different from diseased livers (27.6% ± 2.4% of GADPH), and diseased livers had much higher inter-individual variations than normal livers (65-fold vs 4-fold) (Table 3). PPARα mrnas were 65% higher in peri-hcc, but were 35% lower in HCC, as compared to normal livers. The mrnas of PPARα were also lower in alcoholic cirrhosis and severe cirrhosis (Figure 5). The average expression of CYP4A11 in normal livers (198% ± 17% of GAPDH) was significantly higher than in diseased livers (89.2% ± 11% of GADPH), but diseased livers had much higher inter-individual variations than normal livers (524-fold vs 32-fold) (Table 3). The expression of CYP4A11 was decreased nearly 90% in HCC, followed by severe cirrhosis (78%), and alcoholic cirrhosis (72%), but was not significantly altered in peri-hcc and HBV cirrhosis. The individual paired mrnas of CYP4A11 in peri-hcc and HCC are shown in the bottom panel of Figure 5C. CYP4A11 mrnas were decreased in 21 of 24 patients, except for patient No. 10, No. 16 and No. 24, as compared to peri-hcc. Some patients showed huge differences (patient No. 15, No. 17, and No. 21), while others had mild differences (patient No. 2 and No. 5). DISCUSSION The present study examined the expression of major drug metabolizing P450 genes and their corresponding nuclear receptors in 93 end-stage Chinese livers and compared with 35 normal Chinese donor liver trimmings from the Oriental Liver Transplant Center (Beijing, China), including HCC and peri-hcc tissues, HBV cirrhosis, sever cirrhosis and alcoholic cirrhosis. The results clearly demonstrate that the CYP1-4 family genes were generally down-regulated in end-stage liver diseases, with much greater inter-individual variations in gene expression than normal livers. Significant differences in gene expression 8686 July 14, 2014 Volume 20 Issue 26

379 Chen H et al. P450 expression in end-stage liver diseases 60 PXR 400 CYP3A5 Relative mrna levels (% of GAPDH) a Relative mrna levels (% of GAPDH) a 0 Normal liver Peri-HCC HCC HBV cirrhosis Alcoholic cirrhosis Severe cirrhosis 0 Normal liver Peri-HCC HCC HBV cirrhosis Alcoholic cirrhosis Severe cirrhosis Relative mrna levels (% of GAPDH) CYP3A4 Normal liver Peri-HCC a HCC HBV cirrhosis Alcoholic cirrhosis a Severe cirrhosis Relative mrna levels 10 2 (% of GAPDH) CYP3A7 Normal liver Peri-HCC a HCC a HBV cirrhosis a a Alcoholic cirrhosis Severe cirrhosis Figure 4 expression of Cytochrome P450 enzymes 3 family genes. Right, PXR and CYP3A4, left, CYP3A5 and CYP3A7. The data are mean ± SE, Significantly different from normal livers, a P < 0.05 vs normal liver; HCC different from peri-hcc tissues. PXR: pregnane X receptor; CYP: Cytochrome P450 enzymes; HCC: hepatocellular carcinoma. were also evident between peri-hcc and HCC. CYP1 family genes are regulated by AhR. CYP1A2 is an abundant CYP1A isoform in the liver. In chronic hepatitis C in association with fibrosis, both AhR and CYP1A2 were decreased with the development of fibrosis [12,14] ; their down-regulation appeared to be associated with oxidative stress in the liver diseases [19]. CYP1A2 was reported to be decreased in end-stage liver diseases [16,23,25], and the same phenomenon occurs for Chinese end-stage livers in the present study. CYP1A1 and CYP1B1 are also AhR target genes; although CYP1A1 was down-regulated in end-stage liver diseases [10,15], CYP1B1 was increased together with ABCB4 [15], indicating they are differentially regulated. The down-regulation of CYP1A2 in end-stage liver diseases is consistent with liver dysfunction with the progress of the diseases. CYP2 family had large members of enzyme genes and 5 of them (CYP2B6, CYP2C9, CYP2C19, CYP2E1, and CYP2D6) were investigated in the present study. CYP2E1 is a major P450 isoform for ethanol and is responsible for the metabolism of a variety of xenobiotics [2], CYP2C19 is important in the metabolism of clopidogrel and lacosamide [26,27], while CYP2B6 is an overlooked P450 isozyme, which is now recognized to be important for xenobiotic metabolism [3]. In general, CYP2E enzyme genes were decreased depending on the end-stage liver diseases, with most decreases seen in HCC. CAR is a nuclear receptor regulating CYP2 family gene expression [19]. In end-stage livers, the expression of CAR was generally decreased [10,12], consistent with present observations. Accordingly, CYP2 family enzyme genes were decreased with the liver diseases progressed to the end-stage. In nonalcoholic fatty liver disease [28] and early stages of HCC [18], increased expression of CYP2E1 was reported, but at the end-stage of liver diseases, CYP2E1 was decreased [12-13,25]. In the present study, CYP2E1 decreased not only in HCC, but also in alcoholic cirrhosis and severe cirrhosis (Figure 4). The function of cytochrome P450 2C19 (CYP2C19) is decreased in patients with end-stage liver disease that require liver transplantation [25,29]. In the present study, CYP2C19 was decreased in all end-stage liver diseases, including decreases in peri- HCC. Among CYP2 family genes, CYP2C19 was affected the most, and decreased up to 90% in HCC. All CYP July 14, 2014 Volume 20 Issue 26

380 Chen H et al. P450 expression in end-stage liver diseases A 1000 PPARa a B CYP4A11 PPARa mrna (% of GAPDH) a a,c a a CYP4A11 mrna (% of GAPDH) a a a 0 Normal liver Peri-HCC HCC HBV cirrhosis Alcoholic cirrhosis Severe cirrhosis 0 Normal liver Peri-HCC HCC HBV cirrhosis Alcoholic cirrhosis Severe cirrhosis C 600 CYP4A11 Peri-HCC HCC CYP1A2 mrna (% of GAPDH) Patient number Figure 5 expression of Cytochrome P450 enzymes 4 family genes. A: PPARa; B: CYP4A11; C: CYP4A11 in 24 paired HCC and peri-hcc tissue from individual patients. Data are mean ± SE, Significantly different from normal livers, a P < 0.05 vs normal liver; HCC different from peri-hcc tissues. PPARα: peroxisome proliferator-activated receptor; CYP: Cytochrome P450 enzymes; HCC: hepatocellular carcinoma. family genes are dramatically down-regulated in HCC as compared to peri-hcc and normal tissues, and decreases in CYP2C (2C9 and 2C19) were also evident in end-stage liver diseases. CYP3 family metabolizes the majority of drugs [1]. CYP3 family genes are mainly regulated by PXR and three major CYP3A family members (CYP3A4, CYP3A5 and CYP3A7) were studied in the present study. In endstage liver diseases, the expression of these genes was decreased only in HCC. The nuclear receptor PXR is expressed in liver and intestine and is activated by a wide variety of endobiotics and xenobiotics, including the majority of drugs. PXR serves as a master transcriptional regulator of CYP3A isozymes [22]. PXR is decreased in end-stage liver diseases [10,12]. CYP3A4 is the most important drug metabolizing enzyme in adult humans because of its prominent expression in liver and its broad substrate specificity [30]. Expression of CYP3A4 decreased in the end-stage liver diseases [13,25]. CYP3A5 is important for metabolizing the immunosuppressive drugs used for solid organ transplantation, such as tacrolimus, sirolimus, and ciclosporin [31], and in the present study, it was decreased in end-stage HCC, but it tended to be slightly higher in HBV cirrhosis. Little is known about the expression of CYP3A7 in end-stage liver diseases, and this research would add to our understanding of this CYP3A isoforms in human liver diseases. In general, CYP3A family gene expressions were down-regulated in HCC, but were variably affected in other end-stage liver diseases. CYP4 family genes are regulated by PPARα. Both PPARα and CYP4A11 were investigated in the present study. PPAR is important in lipid metabolism and PPARregulated fatty acid oxidation gene CYP4A11 plays important roles in lipid metabolism [32]. In the present study, the expression of PPARα was slightly decreased in HCC only, but the expression of CYP4A11 was decreased over 85% in HCC, and 70% in severe cirrhosis and alcoholic cirrhosis. The down-regulation of PPARα and its target genes would affect the function of liver in lipid metabolism. The end-stage liver diseases are characterized by a decrease in functional hepatocytes, a decrease in P450 enzyme activities, and reduced blood flow to the liver, which could affect drug metabolism and systemic clearance [33,34]. The decreased CYP1-4 family gene expressions could have a significant impact on drug metabolism and xenobiotic elimination. For example, ropivacaine is metabolized by CYP1A2 and CYP3A4, and its clearance 8688 July 14, 2014 Volume 20 Issue 26

381 Chen H et al. P450 expression in end-stage liver diseases is decreased in chronic end-stage liver diseases [24] ; Fluvoxamine-induced inhibition of theophylline clearance decreased from 62% in healthy subjects to 52% and 12% in patients with mild cirrhosis and those with severe cirrhosis, respectively, due to CYP1A2 dysfunction [35]. Thus, during end-stage liver diseases, liver dysfunction would dramatically reduce drug efficacy and may increase drug toxicity. It should also be noted that end-stage livers had huge individual variations in CYP gene expression, as compared to normal livers. Such a huge individual variation in gene expression among end-stage livers was reported before [24]. CYP polymorphisms and mutations [29,31,32] could also contribute to this huge inter-individual variation. Epigenetic mechanisms such as aberrant microrna expression would also affect CYP expression [36]. Compared to effects of steatosis on CYP expression [11,37], cirrhosis is the end-stage liver disease and the effects are much more pronounced [16,38]. In summary, this study demonstrates that end-stage livers had compromised liver P450 enzyme gene expression, together with their corresponding nuclear receptors. All the tested P450s were decreased in HCC, as compared to peri-hcc and normal livers. Other end-stage liver diseases, namely severe cirrhosis, alcoholic cirrhosis and HBV cirrhosis also had decreased hepatic P450 enzyme gene expressions, albeit to varying degrees. The decreased P450 enzymes would result in liver dysfunction in reducing drug efficacy and increasing drug toxicities. COMMENTS Background The end-stage livers removed for liver transplantation are precious resources to investigate gene expression changes, including P450 enzyme genes. However, little is known about end-stage liver P450 expression from the Chinese population. Research frontiers Hepatic cytochrome P450 (CYP) expression is subjected to changes in liver diseases, and to profile their expression is of basic and clinical significance. Innovations and breakthroughs The end-stage livers had compromised liver P450 enzyme gene expression, together with varying expression of their corresponding nuclear receptors. All the tested P450s were decreased in hepatocellular carcinoma, as compared to peri-hcc and normal livers. Applications The decreased P450 enzymes would result in liver dysfunction, in reducing drug efficacy and increasing drug toxicities. Terminology Real-time RT-PCR is a sensitive technology to study the expression of CYP genes in the end-stage liver diseases. Peer review This study is among the first to study CYP and nuclear receptors in the Chinese population. REFERENCES 1 Parkinson A, Ogilvie BW. Biotransformation of xenobiotics. In: Klaassen CD. Casarett and Doull s Toxicology: The Basic Science of Poisons. 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Association of liver stiffness with hepatic expression of pharmacokinetically important genes in alcoholic liver disease. Alcohol Clin Exp Res 2013; 37 Suppl 1: E17-E22 [PMID: DOI: /j x] 14 Fisher CD, Lickteig AJ, Augustine LM, Ranger-Moore J, Jackson JP, Ferguson SS, Cherrington NJ. Hepatic cytochrome P450 enzyme alterations in humans with progressive stages of nonalcoholic fatty liver disease. Drug Metab Dispos 2009; 37: [PMID: DOI: /dmd ] 15 Haas S, Merkelbach-Bruse S, Justenhoven C, Brauch H, Fischer HP. Expression of xenobiotic and steroid hormone metabolizing enzymes in hepatocellular tumors of the noncirrhotic liver. Pathol Res Pract 2009; 205: [PMID: DOI: /j.prp ] 16 Elbekai RH, Korashy HM, El-Kadi AO. The effect of liver cirrhosis on the regulation and expression of drug metabolizing enzymes. Curr Drug Metab 2004; 5: [PMID: ] 17 Schmidt A, Braeuning A, Ruck P, Seitz G, Armeanu-Ebinger S, Fuchs J, Warmann SW, Schwarz M. Differential expression of glutamine synthetase and cytochrome P450 isoforms in human hepatoblastoma. Toxicology 2011; 281: 7-14 [PMID: DOI: /j.tox ] 18 Wang YX, Meng XW, Wang YD, Liu Y. MRNA levels of two different enzymes in hepatocellular carcinoma. Hepatogastroenterology 2010; 57: [PMID: ] 19 Tanaka S, Mogushi K, Yasen M, Ban D, Noguchi N, Irie T, Kudo A, Nakamura N, Tanaka H, Yamamoto M, Kokudo N, Takayama T, Kawasaki S, Sakamoto M, Arii S. Oxidative stress pathways in noncancerous human liver tissue to predict hepatocellular carcinoma recurrence: a prospective, multicenter study. Hepatology 2011; 54: [PMID: DOI: /hep.24536] 20 Waxman DJ. P450 gene induction by structurally diverse xenochemicals: central role of nuclear receptors CAR, PXR, 8689 July 14, 2014 Volume 20 Issue 26

382 Chen H et al. P450 expression in end-stage liver diseases and PPAR. Arch Biochem Biophys 1999; 369: [PMID: ] 21 Aleksunes LM, Klaassen CD. Coordinated regulation of hepatic phase I and II drug-metabolizing genes and transporters using AhR-, CAR-, PXR-, PPARα-, and Nrf2-null mice. Drug Metab Dispos 2012; 40: [PMID: DOI: /dmd ] 22 Koutsounas I, Theocharis S, Patsouris E, Giaginis C. Pregnane X receptor (PXR) at the crossroads of human metabolism and disease. Curr Drug Metab 2013; 14: [PMID: ] 23 George J, Murray M, Byth K, Farrell GC. Differential alterations of cytochrome P450 proteins in livers from patients with severe chronic liver disease. Hepatology 1995; 21: [PMID: ] 24 Jokinen MJ, Neuvonen PJ, Lindgren L, Höckerstedt K, Sjövall J, Breuer O, Askemark Y, Ahonen J, Olkkola KT. Pharmacokinetics of ropivacaine in patients with chronic endstage liver disease. Anesthesiology 2007; 106: [PMID: ] 25 De Bock L, Boussery K, Van Winckel M, De Paepe P, Rogiers X, Stephenne X, Sokal E, Van Bocxlaer J. In vitro cytochrome p450 activity decreases in children with high pediatric endstage liver disease scores. Drug Metab Dispos 2013; 41: [PMID: DOI: /dmd ] 26 Bentué-Ferrer D, Tribut O, Verdier MC. [Therapeutic drug monitoring of lacosamide]. Therapie 2012; 67: [PMID: ] 27 Lee JB, Lee KA, Lee KY. Cytochrome P450 2C19 polymorphism is associated with reduced clopidogrel response in cerebrovascular disease. Yonsei Med J 2011; 52: [PMID: DOI: /ymj ] 28 Aubert J, Begriche K, Knockaert L, Robin MA, Fromenty B. Increased expression of cytochrome P450 2E1 in nonalcoholic fatty liver disease: mechanisms and pathophysiological role. Clin Res Hepatol Gastroenterol 2011; 35: [PMID: DOI: /j.clinre ] 29 Chiu KW, Nakano T, Hu TH, Tseng HP, Cheng YF, Jawan B, Eng HL, Goto S, Chen CL. Influence of CYP2C19 genotypes on graft pathological findings and postoperative liver function in recipients after living-donor liver transplantation. Ann Transplant 2010; 15: [PMID: DOI: / j x] 30 Klein K, Zanger UM. Pharmacogenomics of Cytochrome P450 3A4: Recent Progress Toward the Missing Heritability Problem. Front Genet 2013; 4: 12 [PMID: DOI: /fgene ] 31 MacPhee IA. Pharmacogenetic biomarkers: cytochrome P450 3A5. Clin Chim Acta 2012; 413: [PMID: DOI: /j.cca ] 32 Goldwasser J, Cohen PY, Yang E, Balaguer P, Yarmush ML, Nahmias Y. Transcriptional regulation of human and rat hepatic lipid metabolism by the grapefruit flavonoid naringenin: role of PPARalpha, PPARgamma and LXRalpha. PLoS One 2010; 5: e12399 [PMID: ] 33 Parkinson A, Mudra DR, Johnson C, Dwyer A, Carroll KM. The effects of gender, age, ethnicity, and liver cirrhosis on cytochrome P450 enzyme activity in human liver microsomes and inducibility in cultured human hepatocytes. Toxicol Appl Pharmacol 2004; 199: [PMID: DOI: / journal.pone ] 34 Johnson TN, Boussery K, Rowland-Yeo K, Tucker GT, Rostami-Hodjegan A. A semi-mechanistic model to predict the effects of liver cirrhosis on drug clearance. Clin Pharmacokinet 2010; 49: [PMID: DOI: / ] 35 Orlando R, Padrini R, Perazzi M, De Martin S, Piccoli P, Palatini P. Liver dysfunction markedly decreases the inhibition of cytochrome P450 1A2-mediated theophylline metabolism by fluvoxamine. Clin Pharmacol Ther 2006; 79: [PMID: ] 36 Rieger JK, Klein K, Winter S, Zanger UM. Expression variability of absorption, distribution, metabolism, excretionrelated micrornas in human liver: influence of nongenetic factors and association with gene expression. Drug Metab Dispos 2013; 41: [PMID: DOI: / dmd ] 37 Buechler C, Weiss TS. Does hepatic steatosis affect drug metabolizing enzymes in the liver? Curr Drug Metab 2011; 12: [PMID: ] 38 Albarmawi A, Czock D, Gauss A, Ehehalt R, Lorenzo Bermejo J, Burhenne J, Ganten TM, Sauer P, Haefeli WE. CYP3A activity in severe liver cirrhosis correlates with Child-Pugh and model for end-stage liver disease (MELD) scores. Br J Clin Pharmacol 2014; 77: [PMID: DOI: / bcp.12182] P- Reviewers: Buchler C, Dietrich CG, Laguna JC S- Editor: Ma YJ L- Editor: O Neill M E- Editor: Zhang DN 8690 July 14, 2014 Volume 20 Issue 26

383 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. PROSPECTIVE STUDY Drain amylase value as an early predictor of pancreatic fistula after cephalic duodenopancreatectomy Vladimir D Dugalic, Djordje M Knezevic, Vladan N Obradovic, Miroslava G Gojnic-Dugalic, Slavko V Matic, Aleksandra R Pavlovic-Markovic, Predrag D Dugalic, Srbislav M Knezevic Vladimir D Dugalic, Djordje M Knezevic, Slavko V Matic, Srbislav M Knezevic, Department of Hepatobiliary and Pancreatic Surgery, First Surgical University Hospital, Clinical Center of Serbia, School of Medicine, University of Belgrade, Belgrade, Serbia Vladan N Obradovic, St. Joseph s Hospital Health Center, Syracuse, NY 13413, United States Miroslava G Gojnic-Dugalic, Clinic for Gynecology and Obstetrics, Clinical Center of Serbia, School of Medicine, University of Belgrade, Belgrade, Serbia Aleksandra R Pavlovic-Markovic, Clinic for Gastroenterology and Hepatology, Clinical Center of Serbia, School of Medicine, University of Belgrade, Belgrade, Serbia Predrag D Dugalic, Clinic for Gastroenterology and Hepatology, University Hospital Zemun, Belgrade, Serbia Author contributions: Dugalic VD designed the study, performed surgery, collected and analyzed data and wrote the paper; Knezevic DM, Matic SV and Knezevic SM performed surgery, analyzed and collected data; Pavlovic-Markovic AR and Dugalic PD performed diagnostic procedures; Obradovic VN analyzed data and contributed to manuscript preparation; Gojnic-Dugalic MG analyzed data. Correspondence to: Vladimir D Dugalić, MD, Department of Hepatobiliary and Pancreatic Surgery, First Surgical University Hospital, Clinical Center of Serbia, 6 Koste Todorovića St., Belgrade, Serbia. vanjadug@yahoo.com Telephone: Received: January 17, 2014 Revised: March 7, 2014 Accepted: April 15, 2014 Published online: July 14, 2014 Abstract AIM: To determine predictors of clinically relevant pancreatic fistulas (CRPF) by measuring drain fluid amylase (DFA) in the early postoperative period. METHODS: This prospective clinical study included 382 patients with periampullary tumors that were surgically resected at our department between March 2005 and October A cephalic duodenopancreatectomy (DP) was performed on all patients. Two closed suction drains were placed at the end of the surgery. The highest postoperative DFA value was recorded and analyzed during the first three postoperative days and on subsequent days if the drains were kept longer. Pancreatic fistula (PF) was classified according to the International Study Group of Pancreatic Fistula (ISGPF) criteria. Postoperative complications were defined according to the Dindo-Clavien classification. All data were statistically analyzed. The optimal thresholds of DFA levels on the first, second and third postoperative days were estimated by constructing receiver operating curves, generated by calculating the sensitivities and specificities of the DFA levels. The DFA level limits were used to differentiate between the group without PF and the groups with biochemical pancreatic fistula (BPF) and CRPF. RESULTS: Pylorus-preserving duodenopancreatectomy was performed on 289 (75.6%) patients, while the remaining patients underwent a classic Whipple procedure (CW). The total incidence of PF was 37.7% (grade A 22.8%, grade B 11.0% and grade C 3.9%). Soft pancreatic texture (SPT) was present in 58.3% of patients who developed PF. Mortality was 4.2%. The median DFA value on the first postoperative day (DFA1) in patients who developed PF was 4520 U/L (range U/L) for grade A fistula (BPF) with a SPT and a diameter of the main pancreatic duct (MPD) of 3 mm. For grade B/C (CRPF), the median DFA1 value was 8501 U/L (range U/L) with a SPT and MPD of 3 mm. These values were significantly higher when compared to the patients who did not have PF (122; range U/L). The upper limit of DFA values for the first 3 postoperative days in the examined stages of PF were: DFA U/L for the BPF and CRPF; DFA3 350 U/L for BPF and DFA3 800 U/L for CRPF. The determined values were highly significant and demonstrated a reliable diagnostic test for both BPF and CRPF. CONCLUSION: DFA U/L is an important pre July 14, 2014 Volume 20 Issue 26

384 Dugalic VD et al. Significance of postpancreatectomic drain amylase values dictive factor for PF of any degree. The trend of DFA3 (decrease of < 50%) compared to DFA1 is a significant factor in the differentiation of CRPF from transient BPF Baishideng Publishing Group Inc. All rights reserved. Key words: Cephalic duodenopancreatectomy; Periampullary tumors; Pancreatic fistula; Drain fluid amylase level; Prediction Core tip: The aim of the study is to determine the possibility of early prediction of the occurrence of clinically relevant pancreatic fistula (CRPF) during the postoperative period after cephalic duodenopancreatectomy for periampullary carcinoma by measuring drain fluid amylase (DFA) values during the first 3 postoperative days. Three-hundred and eighty-two surgically treated patients with resectable periampullary tumors were prospectively analyzed between 2005 and The total incidence of pancreatic fistula was 37.7%. The median DFA values were significantly higher in patients who developed CRPF. We concluded DFA could represent a reliable predictive parameter for CRPF development. Dugalic VD, Knezevic DM, Obradovic VN, Gojnic-Dugalic MG, Matic SV, Pavlovic-Markovic AR, Dugalic PD, Knezevic SM. Drain amylase value as an early predictor of pancreatic fistula after cephalic duodenopancreatectomy. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: org/ /wjg.v20.i INTRODUCTION Pancreatic fistula (PF), especially clinically relevant pancreatic fistula (CRPF), represents one of the most common and serious complications in pancreatic surgery. Early prediction of the PF occurrence is of the utmost importance [1]. If we can reliably exclude the development of CRPF during the early postoperative period (first three days), patients could be treated with a fast track protocol, which includes early mobilization, early removal of drains and oral feeding, adequate pain control, a shorter hospital stay and reduced treatment costs [2]. However, if there is a risk of CRPF development, which is accompanied by a higher percentage of life-threatening complications, treatment of patients is more extensive and involves delayed oral feeding and drain removal, possible introduction of somatostatin analogues, antibiotics, and/or interventional radiological procedures. The incidence of PF in the international literature is variable, from 2%-51% depending on applied criteria [1,3,4]. In 2005, the International Study Group on Pancreatic Fistula (ISGPF) precisely defined pancreatic fistula as the appearance of any measurable volume of drain fluid on or after the 3 rd postoperative day with an amylase content 3 times greater than the upper normal serum value [1]. They also proposed a classification scheme upon which the severity of PF was graded precisely according to the clinical procedures and outcomes (grades A, B, and C). Because the biochemical pancreatic fistula (BPF) does not significantly affect the postoperative course, it is very important to differentiate between those patients who will and will not develop PF and also to distinguish the fistula stages (A vs B/C). Although it allows for comparison of results between centers, the ISGPF definition of PF is determined largely on the basis of outcomes, making it impossible to determine guidelines for prompt treatment of postoperative PF. One cannot predict the clinical course of patients with verified PF on the third postoperative day based solely on the ISGPF classification [5,6]. There are several risk factors for the occurrence of PF: disease-related (pancreatic texture, pancreatic duct diameter, pathologic diagnosis), patient-related (demographics, co-morbidities, jaundice, neoadjuvant therapy) and intraoperative (surgical technique, type of anastomosis, type of reconstruction, intraoperative blood loss and duration of surgery) [7]. It is crucial to recognize risk factors that can lead to development of CRPF and thus determine further postoperative treatment. The most important factors for pancreatic anastomotic dehiscence are soft pancreatic texture (without marked fibrosis, prone to suture tear) and main pancreatic duct diameter of less than 3 mm. There are few published studies that analyzed the correlation of drain fluid amylase (DFA) level with the risk of developing PF [8-15]. Even fewer studies analyzed the risk factors for early prediction of postoperative CRPF and its distinction from BPF [13,14,16-20]. The aim of this study was to determine if DFA value can be used as a predictive criterion for the development of CRPF and to distinguish CRPF from BPF in the early postoperative course. MATERIALS AND METHODS A prospective clinical study was conducted at our department from March 2005 to October 2012, during which 382 patients with a surgically resectable periampullary tumor were followed. A cephalic duodenopancreatectomy (DP) was performed in all patients. Several patients had been previously treated elsewhere and were referred to our institution for definitive treatment. All surgical procedures were performed by 5 senior surgeons. The surgeon was responsible for choosing the type of surgery [pylorus-preserving DP (PPDP) or classic Whipple procedure (CW)] and the type of anastomosis [pancreaticojejunal (PJ) with duct to mucosa or by invagination, or pancreaticogastric anastomosis (PG) with or without stent placement], because none of these surgical techniques have a statistically proven advantage over the others [21]. We began performing PG anastomosis at our department two years ago, primarily in the patients with a narrow main pancreatic duct (MPD) and soft pancreatic texture (SPT). In all patients, two closed suction drains were placed at the end of the surgery, one close to the pancreatic 8692 July 14, 2014 Volume 20 Issue 26

385 Dugalic VD et al. Significance of postpancreatectomic drain amylase values Table 1 Clinical, intraoperative and postoperative characteristics in relation to the occurrence and stage of pancreatic fistula-univariate analysis n (%) Parameter Pancreatic fistula P value No1 BPF2 CRPF3 P 12 P 13 P 23 Gender (M/F) 137/101 50/37 44/13 1 Age 61.1 ± ± ± PF 238 (62.3) 87 (22.8) 57 (14.9) Pancreas texture-soft 9 (10.7) 32 (38.0) 43 (51.2) MPD diameter 3 mm 45 (23.4) 74 (38.5) 73 (38.1) Soft/MPD 3 mm 7/9 (77.8) 31/32 (96.9) 40/43 (93.0) Surgical procedure PPDP 133 (46.0) 74 (25.6) 82 (28.4) Whipple 31 (33.6) 35 (38.0) 26 (28.4) Pathology Pancreatic ductal adeno Ca 87 (60.0) 33 (22.8) 25 (17.2) Papila Vateri Ca 28 (26.9) 35 (33.6) 41 (39.5) Common bile duct/duodenal Ca 16 (30.8) 21 (40.4) 15 (28.8) Other Blood transfusion (pt s) 147/238(61.8) 46/87 (52.9) 33/57 (57.9) Blood transfusion (ml) ± ± ± Hospital stay 14.1 ± ± ± Mortality - 2/87 (2.3) 14/57 (24.6) Significant P < 0.05, n/n, mean ± SD. MPD: Main pancreatic duct; PPDP: Pylorus-preserving duodenopancreatectomy. anastomosis and the other near the bilio-enteric anastomosis. Drains were removed if there was no significant drainage (blood, bile, intestinal contents, pus or unusual color raising suspicion of PF), if the DFA values were low, and at the discretion of the surgeon. Somatostatin analogues were predominantly used in patients with high risk anastomosis (SPT, narrow MPD). All patients received intravenous antibiotics at the induction of anesthesia and for three days after surgery. Low molecular weight heparin was administered subcutaneously for the duration of hospitalization. In all patients, we recorded the overall amount of drain secretion and DFA values taken from the drain with higher amylase values during the first three postoperative days or more if the drains were kept longer. The upper limit value for serum amylase in our laboratory is 100 U/L. PF was classified according to ISGPF criteria [1]. Postoperative complications were defined according to the Dindo-Clavien classification [22]. Patients who received a total pancreatectomy because of positive surgical margins, distal pancreatectomy or enucleation of the tumor were not included in the study. The overall analysis of this study included the following parameters: (1) Preoperative: patient characteristics (age and gender); (2) Intraoperative: type of resection (PPDP or CW), type of reconstruction (PJ or PG), duration of surgery, red blood cell transfusion and texture of the pancreas (hard or soft; estimated during surgery in correlation with postoperative histological finding); and (3) Postoperative: Incidence of postoperative complications and reoperation, histopathological analysis of the resected specimen, type and the degree of tumor differentiation, resectional margin, TNM stage, mortality and hospital stay. Statistical analysis Continuous variables were expressed as the mean ± SD or as median (range). Patient characteristics, perioperative and postoperative factors were compared between groups using χ 2 statistics and Student-t test. Median DFA levels were compared by means of the non-parametric Mann-Whitney U test. The optimal thresholds of DFA levels on the first, second and third postoperative days were estimated by constructing receiver operating curves (ROC), generated by calculating the sensitivities and specificities of the DFA level. These thresholds were used for differentiation between the group without PF and the groups with BPF and CRPF. P values less than 0.05 were considered statistically significant. The statistical analysis was performed with the SPSS 16.0 software. RESULTS PPDP was performed on 289 of the 382 patients (75.6%), and CW was performed on the remaining 93 patients (PJ 190, PG 16). The median age of the patients was 60 years (range years), with a male preponderance (60.5% vs 39.5%). The male prevalence was significantly higher in cases of CRPF (Table 1). The majority of patients had a pre-surgical diagnosis of periampullary carcinoma (80.6%). The additional indications for surgery were IPMN, neuroendocrine tumors, chronic pancreatitis, metastatic renal cell carcinoma and melanoma. Median operative time was 394 min (range min). The percentage of postoperative complications according to the Dindo-Clavien classification is shown in table 2. Overall mortality rate was 4.2% (16 of 382 patients). Causes of death were PF with sepsis (8), bleeding (3), liver failure (2), cardiac (2) and pulmonary embolism 8693 July 14, 2014 Volume 20 Issue 26

386 Dugalic VD et al. Significance of postpancreatectomic drain amylase values Table 2 Postoperative complications by Dindo-Clavien classification n (%) Fistula NO A B/C Dindo-clavien (84.4) 56 (64.4) 0 (0.0) 1 15 (6.3) 12 (13.8) 1 (1.8) 2 8 (3.4) 9 (10.3) 30 (52.6) 3 10 (4.2) 4 (4.6) 8 (14.0) 4 4 (1.7) 4 (4.6) 4 (7.0) (2.3) 14 (24.6) Total 238 (100.0) 87 (100.0) 57 (100.0) Table 3 Drain fluid amylase values on postoperative days 1-3 in relation to the occurrence of pancreatic fistula, pancreatic texture and diameter of main pancreatic duct for patients with pancreatic fistula grade A Table 4 Drain fluid amylase values on postoperative days 1-3 in relation to the occurrence of pancreatic fistula, pancreatic texture and diameter of main pancreatic duct for patients with pancreatic fistula grade B/C Grade B/C PF Postoperative days No clinical fistula (n = 238) Hard pancreatic texture (n = 18) ( ) 8250 ( ) Soft pancreatic texture duct diameter 3 mm (n = 40) P = P = P = (6-1374) 2990 ( ) Sig ( ) P = ( ) P = P = P = P = (3-600) 2298 ( ) 6010 ( ) P = Significant P < 0.05, median (range). PF: Pancreatic fistula. Grade A PF Postoperative day No clinical fistula (n = 238) Hard pancreatic texture (n = 28) Soft pancreatic texture diameter 3 mm (n = 31) (1). Thirty-three patients returned to the OR (8.6%) for bleeding and sepsis caused by PF, and 8 of these patients died. Positive posterior and/or medial (SMA) surgical margins were present in 37.3% of cases, and 61.3% of patients had positive lymph nodes. In 226 patients (59.1%) intraoperative blood transfusion (hemoglobin < 80 g/l) was required. Intraoperative blood transfusion did not significantly influence the occurrence of any degree of PF (Table 1). There was no statistically significant difference in the amount of postoperative drainage fluid in relation to the occurrence of PF. PF of any degree occurred in 37.7% of all patients, with no statistically significant difference in occurrence of BPF (22.8%) compared to CRPF (14.9%). Forty-three (51.2%) out of 84 patients with SPT developed CRPF. CRPF was associated more often with adenocarcinoma of the papilla of Vater (39.5%). Patients with SPT had a significantly higher incidence of MPD diameter of 3 mm in all PF groups (78%-97%) (Table 1). The median concentration of DFA exhibited much lower values in the first three postoperative days in patients without postoperative PF (Table 3). Therefore, the data from this group are shown as a control in tables 3 and 4. Median amylase value (MAV) in the drains on the 1 st postoperative day was significantly higher in patients with BPF and the presence of SPT combined with the MPD diameter of 3 mm, compared to the patients Sig ( ) ( ) P = ( ) P = P = P = (6-1374) ( ) P = ( ) P = P = P = (3-600) 1130 ( ) 1050 ( ) P = Significant P < 0.05, median (range). PF: Pancreatic fistula. with hard pancreatic texture (HPT) and any diameter of MPD. MAV on the 2 nd and 3 rd postoperative day was not significantly different between the same groups. In the patients with BPF and SPT, MAV did not differ significantly on the 1 st and 2 nd postoperative day, while it was significantly lower on the 3 rd postoperative day. The observed trend is similar to the trend seen in the group without postoperative PF (Table 3). MAV from the drain fluid was also analyzed in the group of patients with postoperative CRPF (Table 4). We observed that the MAV value was similar regardless of the pancreatic texture on the 1 st postoperative day. On the 2 nd and 3 rd postoperative day, MAV was higher in the group with SPT and a MPD diameter of 3 mm compared to the HPT group, but the difference was not statistically significant (p = and p = 0.208, respectively). As opposed to SPT, the incidence of CRPF was significantly lower in cases with HPT (18 out of 40). CRPF with SPT and a MPD diameter of 3 mm had higher but not statistically significant (p = 0.877) MAV on the 2 nd postoperative day compared to the 1 st postoperative day. There was no significant decrease in MAV on the 3 rd postoperative day. We concluded that the persistently higher concentration of DFA without significant decrease until the 3 rd postoperative day was the main prognostic value of measuring DFA in patients with CRPF with SPT. By ROC analysis, we determined the DFA threshold level at each postoperative day for BPF and CRPF with SPT and a MPD diameter of 3 mm. As we have previously shown, the presence of SPT, especially in CRPF, was the greatest predictor of the occurrence of PF based on the DFA level on the first three postoperative days. Therefore, all further analysis to determine value thresholds for DFA in the development of these fistulas compared with BPF referred to these conditions. Highly significant values of AUC showed that the given DFA limits for each postoperative day are reliable as diagnostic tests for both BPF (AUC = , p < ) 8694 July 14, 2014 Volume 20 Issue 26

387 Dugalic VD et al. Significance of postpancreatectomic drain amylase values Sensitivity Grade A PF 0.4 Pancreatic texture-soft duct diameter 3 mm 0.2 AUC 1 = AUC 2 = AUC 3 = Sensitivity Grade B/C PF 0.4 Pancreatic texture-soft duct diameter 3 mm 0.2 AUC 1 = AUC 2 = AUC 3 = Days DFA Reference line 1-specificity 1-specificity Figure 1 Receiver operating characteristics curve identified a threshold value of drain fluid amylase on postoperative days 1, 2 and 3 to predict biochemical pancreatic fistula vs clinically relevant pancreatic fistulas with soft pancreatic texture. PF: Pancreatic fistula; DFA: drain fluid amylase. Table 5 Prognostic characteristics of threshold (cut-off) drain fluid amylase levels by postoperative days in the occurrence of biochemical pancreatic fistula and clinically relevant pancreatic fistulas in relation to the texture of the pancreas Grade PF Postoperative days Cut off Sensitivity Specificity FN FP U/L A Pancreatic texture-soft, duct diameter 3 mm 1 > % 87.5% 6.9% 12.5% 2 > % 94.3% 10.3% 5.7% 3 > % 97.8% 1.3% 2.2% B/C Pancreatic texture-soft duct, diameter 3 mm 1 > % 87.5% 7.7% 12.5% 2 > % 97.7% 10.3% 2.3% 3 > % 98.9% 9.8% 1.1% PF: Pancreatic fistula; FN: False negative; FP: False positive. and CRPF (AUC = , p < ) (Figure 1). By selecting thresholds with higher sensitivity (89.7%-98.7%), the false negative rate was reduced to 1%-10% with a specificity of 87.5%-98.9%, and the false positive rate was 1%-12%. CRPF developed with DFA1 values below 1200 U/L in only 3 patients. In patients with SPT, the DFA threshold of 1200 U/L on the 1 st postoperative day did not differ between the compared fistula grades (A vs B/C). Lower threshold values of DFA on the 2 nd postoperative day in patients with BPF corresponded with the previously analyzed MAV by postoperative days in the case of this fistula type. MAV on the 3 rd postoperative day is a diagnostic criterion for predicting the occurrence of CRPF compared to BPF (Table 5). During the development of CRPF, the ratio of DFA concentrations shows only slight day-to-day changes or remains quasi constant (persistent). Thus, in CRPF the marginal threshold values at the 1 st and 2 nd postoperative days did not differ and were relatively lower on 3 rd postoperative day. However, in BPF the threshold DFA values were several times lower on the 3 rd postoperative day than on the first day (> 50%). Values of the defined thresholds overlap, as the threshold of > 800 for BPF is contained within the threshold of > 1200 for CRPF. Therefore, in practical implementation of diagnostic tests that are based on certain threshold values, apart from using threshold values on the first postoperative day, the DFA trend should be followed on a daily basis until the 3 rd postoperative day. DISCUSSION Pancreatic fluid leakage into the peritoneal cavity in the early postoperative period after DP occurs due to several reasons. It originates either from the pancreatic parenchyma or the MPD (anastomotic leakage). Parenchymal leakage is either from the cut surface of the pancreas (tear through the stitches) or trans-parenchymal, when the pancreas acts as a sweating gland that releases an exudate with a high concentration of amylase, similar to pancreatic ascites, seen in patients with acute pancreatitis [10]. The concentration of drain amylase on the first postoperative day could also be affected by the amount of intraoperative leakage of pancreatic fluid into the abdomen. It is often difficult to differentiate between parenchymal and anastomotic leakage, especially in the early postoperative period. Parenchymal leakage is identified by persistently high levels of drain fluid amylase without extravasation of contrast on fistulography and usually stops spontaneously, while the anastomotic leakage shows contrast extravasation at the level of PJ and has no tendency to resolve spontaneously [23-25]. Although consensus has not been reached, measurement of DFA in the early postoperative period after 8695 July 14, 2014 Volume 20 Issue 26

388 Dugalic VD et al. Significance of postpancreatectomic drain amylase values Pancreatic fistula grade A B/C U/L Postoperative days Drain fluid amylase Figure 2 Incidence of biochemical pancreatic fistula (A) or clinically relevant pancreatic fistulas (B/C) in a soft pancreas depending on the drain fluid amylase values on the first three postoperative days. DP can be very useful in predicting the development of PF [8,9,10,13,14,16,20,26]. In their study, Molinari et al [8] defined a DFA threshold value of 5000 U/L as the only significant predictive factor for PF of any grade, with a sensitivity of 92.6% and a specificity of 83.6%. They assumed that high concentrations of DFA1 were caused either by intraoperative leakage of pancreatic fluid or by early and imperceptible spills at the level of anastomosis, which usually preceded the appearance of CRPF. During a successful postoperative recovery, pancreatic function is reduced until the fifth postoperative day and then slowly begins to recover. Complications developed only in those patients in whom DFA1 was 5000 U/L and DFA5 was 200 U/L, and these complications were explained as a small area of ischemic necrosis at the anastomotic site on the 5 th postoperative day. The study was performed on patients after DP and distal pancreatectomy, which represented heterogeneous groups with regard to the different behavior of PF in the presence of anastomosis with the bowel. They did not explicitly 8696 July 14, 2014 Volume 20 Issue 26

389 Dugalic VD et al. Significance of postpancreatectomic drain amylase values Table 6 Development of clinically relevant pancreatic fistulas depending on a threshold DFA1 (our results in comparison with different series, molinari, sutcliffe) DFA U/L Fistula B/C No fistula Sensitivity Specificity Fn Fp Molinari > % 99.1% 31.6% 0.9% Sutcliffe > % 67.2% 5.3% 32.8% DFA: Drain fluid amylase. discuss grades of PF and therefore did not separately observe BPF and CRPF. Sutcliffe et al [9] defined much lower threshold values of DFA1 ( 350 U/L) as a predictor of PF occurrence. Seventy patients with DP treated with a standardized protocol (homogeneous group) were analyzed. However, they did not analyze BPF and CRPF separately and only nine patients had PF. The data they obtained supported the hypothesis that the PF was due to technical error during PJA, which appears immediately and can be detected on the 1 st postoperative day. As we found in our results, this study indicated that the concept of late fistula is questionable, given that none of the patients who had normal DFA1 values and in whom the drains were removed on the 5 th postoperative day developed PF [27]. Our study indicated that DFA1 had significant value in predicting PF, especially with SPT. The established threshold value of 1200U/L based on ROC analysis confidently predicts the development of any type of PF (sensitivity 92.3%-93.1%, specificity 87.5%). Significantly higher median DFA1 concentrations were measured in patients with PF compared to those without PF. We noticed that in the group of patients with BPF, DFA3 had the same trend of significant decline as the group of patients without PF. These results differ significantly from the group of patients with CRPF, in which the decline of DFA3 is only moderate (< 50%) (Figure 2). Our results were analyzed using the threshold DFA1 values defined in the works of Molinari and Sutcliffe (Table 6) [8,9]. We noticed a low sensitivity in the case of a DFA1 threshold value of 5000 U/L (68.4% and 31.6% false negatives) and a low specificity for a threshold value of 350 U/L (67.2% and 32.8% false positives). Isolated measurement of DFA in the early postoperative period is not sufficient, but it is very useful for predicting the occurrence of PF and for the differentiation between BPF and CRPF. Few options have been available for early prediction of CRPF thus far. Several authors tried to answer this problem [12,13,17,18,20,26,28,29] using parameters other than DFA values, such as inflammatory (WBC, temperature, albumin level), pre and intraoperative (pancreatic texture, MPD diameter, intraoperative blood transfusion), high risk pathology, etc. [30-32]. However, clinical implementation of these parameters is questionable. Male sex is more often associated with CRPF. Surprisingly, the amount of intraoperative blood transfusion was not significantly different in patients with and without PF. We do not have an explanation for this, and the explanation for the relationship between intraoperative blood transfusion and the occurrence of CRPF is not entirely convincing. The assumption is that blood loss, especially when rapid, leads to ischemia and impaired healing of the PJ anastomosis. Aggressive intraoperative volume replacement can cause tissue edema in the area of anastomosis, which can lead to the occlusion of the MPD or disruption of the stitches [29]. However, the amount of administered blood depends on the preoperative hemoglobin levels, intraoperative blood loss, the operative technique, and post-operative blood losses. We found a higher percentage of BPF compared to CRPF in our study. Given that BPF does not affect the postoperative course, we may question the ISGPF definition of PF. The high percentage of BPF could be due to strict usage of the ISGPF definition of PF, and therefore a significant number of patients who have had marginal DFA levels on the 3 rd postoperative day were classified as BPF (DFA U/L). With this in mind, it may be more appropriate to introduce a 5 th day of DFA values for the definition of PF [33,34]. Apart from DFA3, the patient s clinical aspect on the 5 th postoperative day is of great importance in our opinion. Recovery of pancreatic function, an increase of DFA in CRPF, infection in CRPF (which can be confirmed in drainage fluid only on day 5) and changes in the appearance of drainage fluid in CRPF (sinister fluid) all occur on the 5 th postoperative day [35,36]. It is at this time that we may distinguish CRPF from BPF. However, the problem still remains that early differentiation is needed to allow us to determine a strict protocol in the postoperative management of these patients [3,37,38]. In conclusion, our suggestion is that regardless of the consistency of the pancreatic texture (hard or soft), patients with DFA1 values below 1200 U/L, with a significant drop in DFA3 (> 50%) and with the absence of inflammatory reaction (WBC, fever) could be treated with early drain removal on the 3 rd postoperative day to avoid the development of late pancreatic fistula July 14, 2014 Volume 20 Issue 26

390 Dugalic VD et al. Significance of postpancreatectomic drain amylase values COMMENTS Background Pancreatic fistula is one of the most common and serious complications after cephalic duodenopancreatectomy. Research frontiers Early prediction of clinically relevant pancreatic fistula (CRPF) is of the utmost importance because its appearance may be accompanied by a higher percentage of life-threatening complications, delayed oral feeding and drain removal, possible introduction of somatostatin analogues, antibiotics and/or interventional radiological procedures. Innovations and breakthroughs The authors studied 382 patients who underwent cephalic duodenopancreatectomy for periampullary tumors. The overall amount of drain secretion and drain fluid amylase (DFA) values were measured from the drain with the higher amylase level during the first three postoperative days or more if the drains were kept longer. The total incidence of pancreatic fistula (PF) was 37.7% (grade A 22.8%, grade B 11.0% and grade C 3.9%). Soft pancreatic texture was present in the majority of patients who developed PF. DFA values on the first postoperative day (DFA1) were significantly higher in the patients who developed PF, and a DFA higher than 1200 U/L was a predictive factor of PF of any degree. The trend of a DFA decline to less than 50% of DFA1 on the third postoperative day was a significant factor in the differentiation of CRPF from transient BPF. Applications Reliably excluding the possibility of CRPF development during the early postoperative period after cephalic duodenopancreatectomy enables patient treatment with the fast track protocol, which includes early mobilization, early removal of drains and oral feeding, adequate pain control, a shorter hospital stay and reduced treatment costs. Peer review The authors evaluated the significance of drain fluid amylase to determine the occurrence of CRPF during the early postoperative period after cephalic duodenopancreatectomy for periampullary carcinoma. They analyzed 387 patients and demonstrated that the total incidence of pancreatic fistula was 37.7%. An accurate statistical analysis demonstrated that drain fluid amylase of 1200 U/L is an important predictive factor of pancreatic fistula of any degree. REFERENCES 1 Bassi C, Dervenis C, Butturini G, Fingerhut A, Yeo C, Izbicki J, Neoptolemos J, Sarr M, Traverso W, Buchler M. Postoperative pancreatic fistula: an international study group (ISGPF) definition. Surgery 2005; 138: 8-13 [PMID: DOI: /j.surg ] 2 Kehlet H, Wilmore DW. Multimodal strategies to improve surgical outcome. Am J Surg 2002; 183: [PMID: DOI: /S (02) ] 3 Crippa S, Salvia R, Falconi M, Butturini G, Landoni L, Bassi C. Anastomotic leakage in pancreatic surgery. 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Hepatogastroenterology 2003; 50: [PMID: ] 12 Shinchi H, Wada K, Traverso LW. The usefulness of drain data to identify a clinically relevant pancreatic anastomotic leak after pancreaticoduodenectomy? J Gastrointest Surg 2006; 10: [PMID: DOI: /j.gassur ] 13 Okano K, Kakinoki K, Suto H, Oshima M, Kashiwagi H, Yamamoto N, Akamoto S, Fujiwara M, Takama T, Usuki H, Hagiike M, Suzuki Y. Persisting ratio of total amylase output in drain fluid can predict postoperative clinical pancreatic fistula. J Hepatobiliary Pancreat Sci 2011; Epub ahead of print [PMID: ] 14 Moskovic DJ, Hodges SE, Wu MF, Brunicardi FC, Hilsenbeck SG, Fisher WE. Drain data to predict clinically relevant pancreatic fistula. HPB (Oxford) 2010; 12: [PMID: DOI: /j ] 15 Pratt WB, Callery MP, Vollmer CM. Risk prediction for development of pancreatic fistula using the ISGPF classification scheme. 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How do we predict the clinically relevant pancreatic fistula after pancreaticoduodenectomy?--an analysis in 244 consecutive patients. World J Surg 2009; 33: [PMID: DOI: /s ] 19 Frymerman AS, Schuld J, Ziehen P, Kollmar O, Justinger C, Merai M, Richter S, Schilling MK, Moussavian MR. Impact of postoperative pancreatic fistula on surgical outcome--the need for a classification-driven risk management. J Gastrointest Surg 2010; 14: [PMID: DOI: / s ] 20 Noji T, Nakamura T, Ambo Y, Suzuki O, Nakamura F, Kishida A, Hirano S, Kondo S, Kashimura N. Clinically relevant pancreas-related infectious complication after pancreat July 14, 2014 Volume 20 Issue 26

391 Dugalic VD et al. Significance of postpancreatectomic drain amylase values icoenteral anastomosis could be predicted by the parameters obtained on postoperative day 3. Pancreas 2012; 41: [PMID: DOI: /MPA.0b013e31823e7705] 21 Hayashibe A, Kameyama M. Duct-to-mucosa pancreaticojejunostomies with a hard pancreas and dilated pancreatic duct and duct-to-mucosa pancreaticojejunostomies with a soft pancreas and non-dilated duct. HPB (Oxford) 2008; 10: [PMID: DOI: / ] 22 Dindo D, Demartines N, Clavien PA. Classification of surgical complications: a new proposal with evaluation in a cohort of 6336 patients and results of a survey. Ann Surg 2004; 240: [PMID: DOI: /01.sla ae] 23 Murakami Y, Uemura K, Hayasidani Y, Sudo T, Hashimoto Y, Nakagawa N, Ohge H, Sueda T. A soft pancreatic remnant is associated with increased drain fluid pancreatic amylase and serum CRP levels following pancreatoduodenectomy. J Gastrointest Surg 2008; 12: [PMID: ] 24 Nguyen JH. Distinguishing between parenchymal and anastomotic leakage at duct-to-mucosa pancreatic reconstruction in pancreaticoduodenectomy. World J Gastroenterol 2008; 14: [PMID: DOI: /wjg ] 25 Mahvi D. Defining, controlling, and treating a pancreatic fistula. J Gastrointest Surg 2009; 13: [PMID: DOI: /s x] 26 Kosaka H, Kuroda N, Suzumura K, Asano Y, Okada T, Fujimoto J. Multivariate logistic regression analysis for prediction of clinically relevant pancreatic fistula in the early phase after pancreaticoduodenectomy. J Hepatobiliary Pancreat Sci 2014; 21: [PMID: DOI: / jhbp.11] 27 Pratt WB, Callery MP, Vollmer CM. The latent presentation of pancreatic fistulas. Br J Surg 2009; 96: [PMID: DOI: /bjs.6614] 28 Kurahara H, Shinchi H, Maemura K, Mataki Y, Iino S, Sakoda M, Ueno S, Takao S, Natsugoe S. Indicators of complications and drain removal after pancreatoduodenectomy. J Surg Res 2011; 170: e211-e216 [PMID: DOI: / j.jss ] 29 Callery MP, Pratt WB, Kent TS, Chaikof EL, Vollmer CM. A prospectively validated clinical risk score accurately predicts pancreatic fistula after pancreatoduodenectomy. J Am Coll Surg 2013; 216: 1-14 [PMID: DOI: / j.jamcollsurg ] 30 Yamamoto Y, Sakamoto Y, Nara S, Esaki M, Shimada K, Kosuge T. A preoperative predictive scoring system for postoperative pancreatic fistula after pancreaticoduodenectomy. World J Surg 2011; 35: [PMID: DOI: /s x] 31 Daskalaki D, Butturini G, Molinari E, Crippa S, Pederzoli P, Bassi C. A grading system can predict clinical and economic outcomes of pancreatic fistula after pancreaticoduodenectomy: results in 755 consecutive patients. Langenbecks Arch Surg 2011; 396: [PMID: DOI: /s x] 32 Schmidt CM, Choi J, Powell ES, Yiannoutsos CT, Zyromski NJ, Nakeeb A, Pitt HA, Wiebke EA, Madura JA, Lillemoe KD. Pancreatic fistula following pancreaticoduodenectomy: clinical predictors and patient outcomes. HPB Surg 2009; 2009: [PMID: DOI: /2009/404520] 33 Strasberg SM, Linehan DC, Clavien PA, Barkun JS. Proposal for definition and severity grading of pancreatic anastomosis failure and pancreatic occlusion failure. Surgery 2007; 141: [PMID: DOI: /j.surg ] 34 Reid-Lombardo KM, Farnell MB, Crippa S, Barnett M, Maupin G, Bassi C, Traverso LW. Pancreatic anastomotic leakage after pancreaticoduodenectomy in 1,507 patients: a report from the Pancreatic Anastomotic Leak Study Group. J Gastrointest Surg 2007; 11: ; discussion 1459 [PMID: DOI: /s ] 35 Bassi C, Molinari E, Malleo G, Crippa S, Butturini G, Salvia R, Talamini G, Pederzoli P. Early versus late drain removal after standard pancreatic resections: results of a prospective randomized trial. Ann Surg 2010; 252: [PMID: DOI: /SLA.0b013e3181e61e88] 36 Kawai M, Tani M, Terasawa H, Ina S, Hirono S, Nishioka R, Miyazawa M, Uchiyama K, Yamaue H. Early removal of prophylactic drains reduces the risk of intra-abdominal infections in patients with pancreatic head resection: prospective study for 104 consecutive patients. Ann Surg 2006; 244: 1-7 [PMID: DOI: /01.sla a6] 37 Bassi C, Malleo G, Pederzoli P. In reply to Pancreatic surgery 101. Drain, no drain, early drain removal or late drain removal. What are the data? Where do we go from here. Ann Surg 2011; 253: [PMID: DOI: / / SLA.0b013e d] 38 Yeo CJ. Pancreatic surgery 101: drain, no drain, early drain removal, or late drain removal. What are the data? Where do we go from here? Ann Surg 2010; 252: [PMID: DOI: /SLA.0b013e3181e89e1e] P- Reviewers: Galli A, Nakazawa T S- Editor: Ma YJ L- Editor: A E- Editor: Liu XM 8699 July 14, 2014 Volume 20 Issue 26

392 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. PROSPECTIVE BRIEF ARTICLE STUDY APE1 polymorphisms are associated with colorectal cancer susceptibility in Chinese Hans Shi-Heng Zhang, Lin-Ang Wang, Zheng Li, Yu Peng, Yan-Ping Cun, Nan Dai, Yi Cheng, He Xiao, Yan-Li Xiong, Dong Wang Shi-Heng Zhang, Lin-Ang Wang, Zheng Li, Yu Peng, Yan- Ping Cun, Nan Dai, Yi Cheng, He Xiao, Yan-Li Xiong, Dong Wang, Cancer Center, Daping Hospital and Research Institute of Surgery, Third Military Medical University, Chongqing , China Lin-Ang Wang, Chongqing Medical University, Chongqing , China Author contributions: Zhang SH and Wang LA contributed equally to this work; Zhang SH, Wang LA, Li Z, Peng Y, and Wang D designed the research; Zhang SH, Wang LA, Peng Y, Cheng Y, and Xiong YL performed the research; Zhang SH, Li Z, and Xiao H analyzed the data; Zhang SH and Wang LA wrote the paper; Cun YP, Dai N, and Wang D contributed to editing and revision of the manuscript. Supported by Grants from the National Natural Science Foundation of China, No and No Correspondence to: Dong Wang, MD, PhD, Cancer Center, Daping Hospital and Research Institution of Surgery, Third Military Medical University, No. 10 Changjiang Zhi Rd, Yuzhong District, Chongqing , China. dongwang64@hotmail.com Telephone: Fax: Received: November 30, 2013 Revised: March 27, 2014 Accepted: April 21, 2014 Published online: July 14, 2014 Abstract AIM: To study the association between four base excision repair gene polymorphisms and colorectal cancer risk in a Chinese population. METHODS: Two hundred forty-seven colorectal cancer (CRC) patients and three hundred cancer-free controls were enrolled in this study. Four polymorphisms (OGG1 Ser326Cys, APE1 Asp148Glu, -141T/G in the promoter region, and XRCC1 Arg399Gln) in components of the base excision repair pathway were determined in patient blood samples using polymerase chain reaction with confronting two-pair primers. The baseline information included age, gender, family history of cancer, and three behavioral factors [smoking status, alcohol consumption, and body mass index (BMI)]. χ 2 tests were used to assess the Hardy-Weinberg equilibrium, the distributions of baseline characteristics, and the four gene polymorphisms between the cases and controls. Multivariate logistic regression analyses were conducted to analyze the correlations between the four polymorphisms and CRC risk, adjusted by the baseline characteristics. Likelihood ratio tests were performed to analyze the gene-behavior interactions of smoking status, alcohol consumption, and BMI on polymorphisms and CRC susceptibility. RESULTS: The APE1 148 Glu/Glu genotype was significantly associated with an increased risk of colorectal cancer (OR = 2.411, 95%CI: , P < relative to Asp/Asp genotype). There were no associations between OGG1, XRCC1, or APE1 promoter polymorphisms and CRC risk. A multivariate analysis including three behavioral factors showed that the APE1 148 Glu/Glu genotype was associated with an increased risk for CRC among both smokers and non-smokers, non-drinkers and individuals with a BMI 25 kg/m 2 (ORs = 2.356, 3.299, 2.654, and 2.581, respectively). The XRCC1 399 Arg/Gln genotype was associated with a decreased risk of CRC among smokers and drinkers (OR = 0.289, 95%CI: , P < 0.001, and OR = 0.327, 95%CI: , P < 0.05, respectively). The APE1 promoter polymorphism -141 T/G genotype was associated with a reduced risk of colorectal cancer among subjects with a BMI < 25 kg/m 2 (OR = 0.214, 95%CI: , P < 0.05 relative to T/T genotype). There were significant gene-behavior interactions between smoking status and XRCC1 Arg399Gln, as well as BMI and APE1-141T/G polymorphism (all P < 0.05). CONCLUSION: APE1 Asp148Glu is associated with increased CRC risk and smoking alters the association between XRCC1 Arg399Gln and CRC risk in the Chinese 8700 July 14, 2014 Volume 20 Issue 26

393 Zhang SH et al. APE1 polymorphisms influence cancer risk Han population Baishideng Publishing Group Inc. All rights reserved. Key words: Apurinic endonuclease 1; Base excision repair; Single nucleotide polymorphisms; Colorectal cancer; X-ray repair cross-complementing groups Core tip: There are discrepancies in reports concerning the association between polymorphisms in genes involved in the base excision DNA repair pathway (OGG1, APE1, and XRCC1) and colorectal cancer susceptibility. This study examines four of these polymorphisms in a Chinese Han population from the southwest region of China. Results show that the APE1 148 Glu/Glu genotype is associated with an increased risk of CRC. Moreover, analyses examining gene-behavior interactions revealed that smoking may influence the relationship between the XRCC1 Arg399Gln polymorphism and CRC risk. Zhang SH, Wang LA, Li Z, Peng Y, Cun YP, Dai N, Cheng Y, Xiao H, Xiong YL, Wang D. APE1 polymorphisms are associated with colorectal cancer susceptibility in Chinese Hans. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: INTRODUCTION Colorectal cancer (CRC) is the third most commonly diagnosed cancer in males and the second most common cancer in females. Incidence rates of CRC are highest in Australia, New Zealand, Europe, and North America and lowest in Africa and South-Central Asia. However, CRC incidence rates are rapidly increasing in several areas historically at low risk, including Spain and a number of countries within Eastern Europe and Eastern Asia [1]. Approximately deaths from CRC are estimated worldwide, accounting for 8% of all cancer deaths, making it the fourth most deadly cancer [2]. Many cancers, including CRC, can be initiated by DNA damage induced by chemical agents, smoking, alcohol consumption, and fat metabolism [3]. To avoid accumulation of such damage, cells have developed precise and effective DNA repair systems. Base excision repair (BER) is the major DNA repair pathway for most oxidative DNA lesions [4] that recognizes and repairs base modifications and single strand breaks (SSB) [5]. BER is initiated by a mono- or bifunctional DNA glycosylase and involves: (1) base lesion recognition, excision, and cleavage of an abasic site; (2) end-processing of SSB termini to generate 3 hydroxyl (OH)/5 phosphate (P) group ends; (3) gap-filling after lesion excision; and (4) nick sealing by DNA ligases [6]. Three key human proteins involved in the BER and SSB pathways [7] are X-ray repair cross-complementing protein 1 (XRCC1), which acts as a scaffold to recruit BER proteins, 8-oxoguanine DNAglycosylase 1 (OGG1), which participates in generating SSBs with 3 P αβ unsaturated aldehyde and 5 P termini [6], and apurinic endonuclease 1 (APE1), the key enzyme responsible for the incision of the apurinic/apyrimidinic sites and the generation of 3 -OH termini [8]. Polymorphisms in genes coding for these three key proteins could affect the accumulation of DNA lesions in colorectal mucosa, thus influencing CRC risk [9]. Among all the polymorphisms in these proteins, OGG1 Ser- 326Cys, APE1 Asp148Glu, and XRCC1 Arg399Gln are the most common and well-studied [4]. Although the associations of OGG1 Ser326Cys and XRCC1 Arg399Gln gene polymorphisms and CRC risk have been inconsistent [3,10-12], the APE1 Asp148Glu polymorphism is associated with an increased risk of CRC in Turkish and Polish populations [7,13]. Studies focused on the promoter polymorphism in APE1 (-141T/G) are rare, with only two articles reporting that the APE1-141G/G genotype was a protective factor in lung cancer risk [14,15], and one study showing that the variant allele G is associated with a significantly decreased risk for glioblastoma [16]. A genomewide association study investigating gene polymorphisms and CRC risk conducted in 2011 identified XRCC1 Arg399Gln among the 6216 single nucleotide polymorphisms within 100 kb of the 157 DNA repair loci, but revealed no relationship between this polymorphism and CRC risk [17]. Another meta-analysis studied 455 polymorphisms in 110 different genes and showed a protective effect of homozygous variants of XRCC1 [18]. As the associations between these four key gene polymorphisms and CRC are still uncertain in the Chinese population, the aim of this study was to define the relationships between CRC risk and the OGG1 Ser326Cys, APE1 Asp148Glu and the promoter variation -141T/G, and XRCC1 Arg- 399Gln polymorphisms in the Chinese Han population. MATERIALS AND METHODS Study population The study population included 247 cancer patients (60 with colon cancer and 187 with rectal cancer) and 300 cancer-free controls admitted to the Daping Hospital from January 2006 to December All subjects were Han Chinese from the southwest area of China between 20 and 90 years of age with complete demographic and behavioral information. Patients and controls were matched for age and gender. Pathological confirmation for all cancer patients was performed, and control subjects had no tumor history before or during the study. The study protocol was approved by the ethics committee of Daping Hospital and informed consent was obtained from each participant. A uniform questionnaire was used for all subjects regarding sociodemographic characteristics, smoking status, alcohol consumption, body mass index (BMI), and other potential confounding factors. Subjects were considered smokers if they were a current or past smoker before the 8701 July 14, 2014 Volume 20 Issue 26

394 Zhang SH et al. APE1 polymorphisms influence cancer risk Table 1 Primer sequences Target gene Sequence of primers Allele and PCR product size (bp) OGG1 F1: 5 -CAGCCCAGACCCAGTGGACTC-3 C allele (252 bp) Ser326Cys R1: 5 -TGGCTCCTGAGCATGGCGGG-3 F2: 5 -CAGTGCCGACCTGCGCCAATG-3 G allele (194 bp) R2: 5 -GGTAGTCACAGGGAGGCCCC-3 XRCC1 F1: 5 -TCCCTGCGCCGCTGCAGTTTCT-3 G allele (447 bp) Arg399Gln R1: 5 -TGGCGTGTGAGGCCTTACCTCC-3 F2: 5 -TCGGCGGCTGCCCTCCCA-3 A allele (222 bp) R2: 5 -AGCCCTCTGTGACCTCCCAGGC-3 APE1 F1: 5 -CCTACGGCATAGGTGAGACC-3 G allele (167 bp) Asp148Glu R1: 5 -TCCTGATCATGCTCCTCC-3 F2: 5 -TCTGTTTCATTTCTATAGGCGAT-3 T allele (236 bp) R2: 5 -GTCAATTTCTTCATGTGCCA-3 APE1 promoter F1: 5 -CTAACTGCCAGGGACGCCGA-3 T allele (136 bp) -141T/G R1: 5 -ACACTGACTTAAGATTCTAACTA-3 F2: 5 -ACTGTTTTTTTCCCTCTTGCACAG-3 G allele (335 bp) R2: 5 -TGAGCAAAAGAGCAACCCCG-3 PCR: Polymerase chain reaction; OGG1: 8-oxoguanine DNA-glycosylase 1; APE1: Apurinic endonuclease 1; XRCC1: X-ray repair cross-complementing protein 1. date of enrollment, whereas non-smokers were defined as those who reported that they had never smoked before or during the study duration. For alcohol consumption, drinkers were described as having alcohol every day, whereas non-drinkers did not consume alcohol daily. Height and current body weight were recorded to analyze the BMI (kg/m 2 ), with a cut-off threshold of 25 kg/m 2. DNA isolation and genotyping Genomic DNA was extracted from peripheral whole blood obtained from all participants by using an EZNASE Blood DNA kit (Omega Bio-tek, Norcross, GA, United States) and stored at -80. The extracted samples were used for characterization of the following polymorphic DNA repair genes: OGG1 (rs ; Ser- 326Cys, C/G in Exon 7), APE1 (rs ; Asp148Glu, T/G in Exon 5 and rs ; -141T/G in the promoter region), and XRCC1 (rs25487; Arg399Gln, G/A in Exon 10). Polymerase chain reaction with confronting two-pair primers (PCR-CTPP) was used for genotyping, which is an inexpensive and time-saving method applicable for most single nucleotide polymorphisms [19]. For detecting a single nucleotide polymorphism (base X or Y), one primer for the X allele is set to include X (antisense of X) at the 3 end, with the counterpart sense primer upstream. For the Y allele, a sense primer including Y at the 3 end is set, with the antisense primer downstream. One common band and one specific band for each allele are amplified, allowing for direct genotyping by electrophoresis [20]. Primer pairs were designed using GenBank reference sequences to provide unique product lengths for each allele (Table 1). PCR amplification was performed in glass capillaries in 25 μl reaction mixtures containing 2 μl genomic DNA, 12.5 μl Go Taq MIX (2x), 1 μl primer for each of the four primers, and 6.5 μl dh2o. Reaction conditions included initial denaturation at 95 for 10 min, then 30 cycles of denaturation at 95 for 1 min, annealing for 1 min at 60 (APE1 Asp148Glu), 58 (APE1-141T/G), 66 (XRCC1 Arg399Gln), or 64 (OGG1 Ser326Cys), and elongation at 72 for 1 min. PCR products were analyzed by agarose gel electrophoresis. Statistical analysis Statistical analyses were performed with SPSS version 19.0 (IBM, Armonk, NY, United States). A χ 2 test was conducted for assessing the differences in demographic variables, family history of cancer, behavioral risk factors, and distribution of the four gene polymorphisms between case and control subjects, as well as the Hardy- Weinberg equilibrium. Unconditional logistic regression was undertaken to estimate odds ratios (OR) and 95%CI after adjustment for age, gender, family history of cancer, smoking status, alcohol consumption, and BMI. As the distribution of the XRCC1 Gln/Gln genotype was relatively rare in both groups, the homozygous Arg/Arg and Gln/Gln genotype were combined as the adverse genotype in the subgroup analysis, but not the interaction test. Statistical significance for the interaction was tested by the likelihood ratio test, which compared logistic models with and without interaction terms. Linkage disequilibrium was assessed using Haploview (v.4.2, 2008, Daly Lab at the Broad Institute, Cambridge, MA, United States). RESULTS Geographic characteristics of subjects The clinical and demographic characteristics of research subjects are summarized in Table 2. There were no statistically significant differences between cases and controls in terms of age, gender, family history of cancer, or the three behavior factors (smoking, alcohol consumption, and BMI) July 14, 2014 Volume 20 Issue 26

395 Zhang SH et al. APE1 polymorphisms influence cancer risk Table 2 Demographic characteristics of colorectal cancer cases and control participants n (%) Variables Cases (n = 247) Controls (n = 300) P value Age 1 Mean age ± SE ± ± < 60 yr 120 (48.58) 153 (51.00) 60 yr 127 (51.42) 147 (49.00) Gender Male 145 (58.70) 175 (58.33) Female 102 (41.30) 125 (41.67) Family history of cancer No 214 (86.64) 264 (88.00) Yes 33 (13.36) 36 (12.00) Smoking Nonsmokers 159 (64.37) 190 (63.33) Smokers 88 (35.63) 110 (36.67) Alcohol consumption Nondrinkers 176 (71.25) 210 (70.00) Drinkers 71 (28.75) 90 (30.00) Body mass index < (3.64) 12 (4.00) (14.98) 46 (15.33) (81.38) 242 (80.67) 1 Age is a continuous variable in the regression model analysis. Single gene distribution and CRC risk Genotype distributions of the four polymorphisms in control subjects were consistent with the Hardy-Weinberg equilibrium. The distributions of OGG1 Ser326Cys, APE1 Asp148Glu, -141T/G in the promoter region, and XRCC1 Arg399Gln genotypes in CRC patients and controls are shown in Table 3. The APE1 148 Glu/Glu genotype was higher in CRC patients than in controls (28.34% vs 13.67%, P < 0.001). Compared with the APE1 148 Asp/Asp genotype, the Glu/Glu genotype showed a statistically significant increased risk of CRC (OR = 2.411, 95%CI: , P < 0.001). Frequency of the APE1 Glu allele was significantly higher in CRC patients than in controls (46.56% vs 36.50%, OR = 1.516, 95%CI: , P < 0.001). There were no statistically significant differences between cases and controls in APE1-141T/G, XRCC1 Arg399Gln, or OGG1 Ser- 326Cys genotypes, or allelic frequencies. As the APE1 Asp148Glu and -141T/G polymorphisms are located on the same chromosome, the linkage disequilibrium test was conducted to determine if there was a chain expression between the two loci. The result showed weak linkage disequilibrium between APE1 Asp148Glu and -141T/G polymorphisms (D = 0.28, r 2 = 0.065, data not shown), indicating no need for haplotype analysis. Gene polymorphisms and smoking status Table 4 summarizes the combined effects of smoking and the four gene polymorphisms, adjusted for age, gender, alcohol consumption, BMI, and family history of cancer. An increased OR of CRC associated with the APE1 148 Glu/Glu genotype was found in smokers and non-smokers (smoker: OR = 3.299, 95%CI: ; non-smoker: OR = 2.336, 95%CI: , all P < 0.05). Compared with the XRCC1 399 homozygous combined genotype (Arg/Arg and Gln/Gln), the OR for the Arg/Gln genotype was significantly decreased in smokers (OR = 0.289, 95%CI: , P < 0.001), but not in non-smokers (OR = 1.120, 95%CI: ), with an interaction observed between smoking status and XRCC1 Arg399Gln polymorphism (P < 0.05). Compared with the homozygous wild type, the adjusted ORs for APE1-141 T/G and OGG1 Ser326Cys polymorphisms showed no statistically significant differences between smokers and non-smokers. There were no significant gene-behavior interactions observed between smoking status and OGG1 Ser326Cys, APE1 Asp148Glu, or -141T/G. Gene polymorphisms and alcohol consumption Table 5 shows the genotypes of subjects stratified by alcohol consumption, adjusted for age, gender, smoking status, BMI, and family history of cancer. Although there was an increased risk for CRC in those who were classified as non-drinkers with the APE1 Glu/Glu genotype (OR = 2.654, 95%CI: , P < 0.05) but not in drinkers (OR = 2.095, 95%CI: ), there was no significant interaction between alcohol use and APE1. Compared with the combined XRCC1 Arg/Arg and Gln/Gln genotypes, the Arg/Gln genotype showed a decreased risk of CRC in drinkers (OR = 0.327, 95%CI: , P < 0.05). There was no significant risk for CRC observed among non-drinkers, and there was no gene-behavior interaction between alcohol use and XRCC1. There were no statistically significant differences in APE1-141T/G or G/G genotypes compared with the T/T genotype, and no differences between the OGG1 326 Ser/Cys and Cys/Cys genotypes compared with the Ser/Ser genotype. No significant gene-behavior interactions between alcohol consumption and APE1-141T/G and OGG1 Ser326Cys genotypes were observed. Gene polymorphisms and BMI The distributions of the four gene polymorphisms stratified by BMI are presented in Table 6. Subjects with a BMI 25 kg/m 2 and the APE1 Glu/Glu genotype showed a higher risk of CRC (OR = 2.581, 95%CI: , P < 0.05). In contrast, no clear interaction with regard to risk was found between the BMI and APE1 Asp148Glu polymorphism. The APE1-141T/G genotype indicated a lower risk of CRC in subjects with BMI < 25 kg/m 2 (OR = 0.214, 95%CI: , P < 0.05). There was a significant interaction between BMI and APE1-141T/G (P < 0.05), but not with OGG1 Ser- 326Cys or XRCC1 Arg399Gln gene polymorphisms. DISCUSSION This study is the first report showing the association between the Glu allele of the APE1 Asp148Glu gene variant and an increased risk of CRC in a Chinese Han population, consistent with the results of Jelonek et al [13] 8703 July 14, 2014 Volume 20 Issue 26

396 Zhang SH et al. APE1 polymorphisms influence cancer risk Table 3 Gene distributions n (%) Genes Cases (n = 247) Controls (n = 300) Crude OR (95%CI) 1 P value 1 Adjusted OR (95%CI) 2 P value 2 OGG1 Genotype Ser/Ser 44 (17.81) 48 (16.00) Ser326Cys Ser/Cys 111 (44.94) 139 (46.33) ( ) ( ) Cys/Cys 92 (37.25) 113 (37.67) ( ) ( ) Allele Ser 199 (40.28) 235 (39.17) Cys 295 (59.72) 365 (60.83) ( ) XRCC1 Genotype Arg/Arg 131 (53.04) 142 (47.33) Gln399Arg Arg/Gln 91 (36.84) 132 (44.00) ( ) ( ) Gln/Gln 25 (10.12) 26 (8.67) ( ) ( ) Allele Arg 353 (71.46) 416 (69.33) Gln 141 (28.54) 184 (30.67) ( ) APE1 Genotype Asp/Asp 87 (35.22) 122 (40.66) Asp148Glu Asp/Glu 90 (36.44) 137 (45.67) ( ) ( ) Glu/Glu 70 (28.34) 41 (13.67) ( ) < ( ) < Allele Asp 264 (53.44) 381 (63.50) Glu 230 (46.56) 219 (36.50) ( ) APE1 promoter Genotype TT 93 (37.65) 93 (31.00) T/G TG 102 (41.30) 140 (46.67) ( ) ( ) GG 52 (21.05) 67 (22.33) ( ) ( ) Allele T 288 (58.30) 326 (54.33) G 206 (41.70) 274 (45.67) ( ) Obtained by χ 2 test, and the significance level was adjusted to ; 2 Adjusted with age, gender, smoking status, alcohol consumption, body mass index, and family history of cancer. OGG1: 8-oxoguanine DNA-glycosylase 1; APE1: Apurinic endonuclease 1; XRCC1: X-ray repair cross-complementing protein 1. Table 4 Distribution of genotypes and odds ratios for colorectal cancer stratified by smoking status Non-smokers Smokers P interaction Case/control OR (95%CI) 1 P value Case/control OR (95%CI) 1 P value OGG1 Ser326Cys Ser/Ser 30/ / Ser/Cys 75/ ( ) / ( ) Cys/Cys 54/ ( ) / ( ) XRCC1 Arg399Gln Arg/Arg + Gln/Gln 93/ / Arg/Gln 66/ ( ) / ( ) < APE1 Asp148Glu Asp/Asp 58/ / Asp/Glu 60/ ( ) / ( ) Glu/Glu 41/ ( ) / ( ) APE1-141T/G TT 59/ / TG 66/ ( ) / ( ) GG 34/ ( ) / ( ) Adjusted for age, gender, alcohol consumption, body mass index, and family history of cancer. OGG1: 8-oxoguanine DNA-glycosylase 1; APE1: Apurinic endonuclease 1; XRCC1: X-ray repair cross-complementing protein 1. in a Polish population and Canbay et al [7] in a Turkish population. Kasahara et al [21] also reported that the Glu allele was associated with a 2-3 fold increased risk for CRC in a Japanese population. In the present study, no genebehavior interactions were observed between the three behavioral factors and the APE1 Asp148Glu polymorphism, but the risk among smokers was slightly higher. Additionally, individuals with the APE1 Glu/Glu genotype that were non-drinkers or had a BMI 25 kg/m 2 showed an increased risk of CRC. The APE1 gene consists of five exons and four introns within a 2.21 kb span on chromosome 14 at q11.2-q12 and encodes a 317 amino acid protein [14]. APE1 has 3 -phosphodiesterase activity and efficiently removes 3 phosphoglycolate groups [22]. It initiates repair of abasic sites in DNA by hydrolyzing the phosphodiester 5 backbone [23]. Additionally, APE1/Ref-1 also functions as a redox agent, maintaining transcription factors such as AP-1, nuclear factor-κb, Myb, hypoxia-inducible factor-1α, HLF, PAX, and p53 that are involved in cancer promotion and progression in an active reduced state [14,23-25]. However, biochemical characterization of this variant shows normal endonuclease and abasic-dna binding activity [4], though severe functional damage in the APE1 protein may cause early death or abortion, making it difficult to assess function in adults. A report in 2002 indicated that a prolonged cell-cycle delay observed in breast cancer samples was associated with the number 8704 July 14, 2014 Volume 20 Issue 26

397 Zhang SH et al. APE1 polymorphisms influence cancer risk Table 5 Distribution of genotypes and odds ratios for colorectal cancer stratified by alcohol consumption Non-drinkers Drinkers P interaction Case/control OR (95%CI) 1 P value Case/control OR (95%CI) 1 P value OGG1 Ser326Cys Ser/Ser 33/ / Ser/Cys 87/ ( ) / ( ) Cys/Cys 56/ ( ) / ( ) XRCC1 Arg399Gln Arg/Arg + Gln/Gln 106/ / Arg/Gln 70/ ( ) / ( ) APE1 Asp148Glu Asp/Asp 62/ / Asp/Glu 64/ ( ) / ( ) Glu/Glu 50/ ( ) / ( ) APE1-141T/G TT 68/ / TG 72/ ( ) / ( ) GG 36/ ( ) / ( ) Adjusted for age, gender, smoking status, body mass index, and family history of cancer. OGG1: 8-oxoguanine DNA-glycosylase 1; APE1: Apurinic endonuclease 1; XRCC1: X-ray repair cross-complementing protein 1. Table 6 Distribution of genotypes and odds ratios for colorectal cancer stratified by body mass index < 25 kg/m 2 25 kg/m 2 P interaction Case/control OR (95%CI) 1 P value Case/control OR (95%CI) 1 P value OGG1 Ser326Cys Ser/Ser 9/ / Ser/Cys 17/ ( ) / ( ) Cys/Cys 20/ ( ) / ( ) XRCC1 Arg399Gln Arg/Arg + Gln/Gln 27/ / Arg/Gln 19/ ( ) / ( ) APE1 Asp148Glu Asp/Asp 17/ / Asp/Glu 13/ ( ) / ( ) Glu/Glu 16/ ( ) / ( ) APE1-141T/G TT 17/ / TG 15/ ( ) / ( ) GG 14/ ( ) / ( ) Adjusted for age, gender, smoking status, body mass index, and family history of cancer. OGG1: 8-oxoguanine DNA-glycosylase 1; APE1: Apurinic endonuclease 1; XRCC1: X-ray repair cross-complementing protein 1. of APE1 Asp148Glu and XRCC1 Arg399Gln variant alleles [26]. Furthermore, increased APE1 transcription was observed in CRC patients with the OGG1 326 Ser allele [27]. These findings suggest that the APE1 Asp148Glu polymorphism may act as a co-factor with other genes in tumorigenesis, though further research is needed to reveal the exact mechanism. Promoter polymorphisms can affect transcription factor recognition, and thus impact gene expression. Although the APE1-141T/G promoter polymorphism has not been thoroughly examined in CRC, Jing et al [28] reported that the G allele of this variant is associated with a decreased risk of prostate cancer. Lu et al [15] also found that the APE1-141T/G polymorphism was associated with a decreased risk of lung cancer. Their study showed that the -141 G allele was associated with reduced promoter activity and lower APE1 mrna levels in human peripheral blood mononuclear cells and normal lung tissues. However, Zhou et al [16] found no significant association between APE1 promoter -141T/G polymorphism and glioma risk, though the stratified histologic analysis revealed a decreased glioblastoma risk with the G allele variant. Results from the present study also indicate no association between the APE1-141T/G variants and CRC susceptibility. However, subgroup analysis stratified by BMI revealed a protective effect of the T/G genotype on development of CRC among subjects with a BMI < 25 kg/m 2. As this analysis was limited by a small sample size, further in-depth studies are needed to confirm this effect. The XRCC1 gene is located at 19q13.2 and consists of 17 exons encoding a 633 amino acid non-enzymatic nuclear scaffold protein that interacts with enzymatic factors such as polyadenosine diphosphate-ribose poly July 14, 2014 Volume 20 Issue 26

398 Zhang SH et al. APE1 polymorphisms influence cancer risk merase, DNA ligase Ⅲ, and DNA polymerase β to facilitate protein-protein responses [4] and efficient repair of DNA SSBs [29]. Although the XRCC1 Arg399Gln polymorphism has been shown to rescue sensitivity to the alkylating agent methyl methanesulfonate and the DNA repair defect in the XRCC1-deficient Chinese hamster ovary cell line EM9 [4], there are inconsistent reports concerning XRCC1 variants and CRC. Some reports have revealed an increased risk for CRC with the Gln/Gln genotype [11,13,30,31], whereas results from studies in Turkey and the Czech Republic report no relationship between the gene variant and CRC risk [7,32,33]. A meta-analysis published last year showed that XRCC1 Arg399Gln polymorphism is significantly associated with increased CRC risk [34]. The results of this study show that the XRCC1 Arg- 399Gln polymorphism is not associated with CRC risk, though a protective effect against CRC was observed in smokers with the Asp/Gln genotype. The observed gene-behavior interaction suggests that tobacco use may modify the association of the XRCC1 Arg399Gln variant and CRC risk. It has been shown that the XRCC1 Gln399Gln genotype is linked with an increased risk of tobacco-related cancers among light smokers, but a decreased risk among heavy smokers [35], which is consistent with these findings. The same protective effect was also seen among drinkers with the Arg/Gln genotype, though no significant interaction between alcohol consumption and XRCC1 polymorphism was observed. This protective effect did not correspondingly increase with the number of Gln alleles. It is possible that polymorphisms of other repair genes, such as ERCC1 or XRCC2, which are in linkage disequilibrium with XRCC1 Arg399Gln, may potentially modify the effect of this polymorphism on the risk of CRC. However, the Gln/Gln genotype was rare in the study cohort; large-scale investigations are needed to determine the exact interaction. The OGG1 gene is located at 3p26.2 and encodes the major repair enzyme for the excision of 8-oxoguanine, a mutagenic base byproduct resulting from exposure to reactive oxygen. A well-characterized OGG1 polymorphism replacing C with G at nucleotide 1245 results in a substitution of serine for cysteine [36]. The research of Yamane et al [37] reveals this cysteine reduces the ability of OGG1 to prevent mutagenesis by 8-hydroxyguanine in human cells in vivo. However, the clinical investigations between the OGG1 Ser326Cys polymorphism and the risk of CRC are inconsistent. Several studies suggest that the Cys/Cys genotype may increase the CRC risk in different ethnic populations [7,32,38,39], yet results from a study by Hansen et al [40] suggest that carriers of the Cys allele have a lower risk of CRC. Furthermore, a meta-analysis conducted by Zhang et al [3] indicated no association between the OGG1 Ser326Cys gene variant and risk of CRC, which is consistent with the results of the present study. In conclusion, the data presented here indicate that the homozygous APE1 148 Glu/Glu variant genotype is significantly associated with an elevated risk of CRC, especially for smokers and overweight individuals. These results suggest that the APE1 gene polymorphism and BER pathway contribute to the possible harmful effects of smoking and obesity. Tobacco use may modify the association between CRC risk and the XRCC1 Arg399Gln polymorphism. However, these findings are limited by uncontrolled biases that were present in the selection of participants and the low penetrance of these variants in CRC susceptibility. Further large-population based studies are therefore needed to confirm the results of this study. COMMENTS Background Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide, and a leading cause of cancer-related death. DNA damage plays an important role in the development and occurrence of CRC. Precise and effective DNA repair systems, such as the base excision repair pathway, have evolved to maintain genomic stability and minimize accumulation of damage. Research frontiers Gene polymorphisms in DNA repair pathways could affect DNA repair capacity, and thus influence the risk of CRC. Several polymorphisms in genes encoding components of these repair pathways have been identified, including 8-oxoguanine DNA-glycosylase 1 (OGG1), apurinic endonuclease 1 (APE1), and X-ray repair cross-complementing protein 1 (XRCC1). Among these, OGG1 Ser326Cys, APE1 Asp148Glu, and XRCC1 Arg399Gln are the most studied polymorphisms. An additional polymorphism in the promoter region of APE1 has been identified, though less studied in cancer research. Innovations and breakthroughs This research is the first to study base excision repair gene polymorphisms in a Chinese Han population from the southwest part of China. Results from this study demonstrate an interaction between smoking and the XRCC1 Arg399Gln variant. Applications These results provide population characteristic data and corresponding gene polymorphisms associated with a risk for CRC, which will help to identify a potential high-risk population. Terminology Single nucleotide polymorphisms are DNA sequence variations occurring when a single nucleotide in the genome differs between members of a biological species or paired chromosomes. Gene-environment interaction is the association between genes and environmental factors. Some environmental factors could influence the association between genes and some certain diseases, and cause an increase or decrease in risk in a particular group of people. Peer review The authors analyzed an important aspect of risk factors for CRC. The number of patients included in this study is adequate and it is very interesting because it has been conducted in an Asian population that has different genetic characteristics with respect to a Western population. This study is of clinical relevance due to the potential in identifying a high-risk CRC population. REFERENCES 1 Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. 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Environ Mol Mutagen 2002; 39: [PMID: ] 27 Obtulowicz T, Swoboda M, Speina E, Gackowski D, Rozalski R, Siomek A, Janik J, Janowska B, Ciesla JM, Jawien A, Banaszkiewicz Z, Guz J, Dziaman T, Szpila A, Olinski R, Tudek B. Oxidative stress and 8-oxoguanine repair are enhanced in colon adenoma and carcinoma patients. Mutagenesis 2010; 25: [PMID: DOI: /mutage/geq028] 28 Jing B, Wang J, Chang WL, Li B, Chen J, Niu YJ. Association of the polymorphism of APE1 gene with the risk of prostate cancer in Chinese Han population. Clin Lab 2013; 59: [PMID: ] 29 Shim HJ, Yun JY, Hwang JE, Bae WK, Cho SH, Lee JH, Kim HN, Shin MH, Kweon SS, Lee JH, Kim HJ, Chung IJ. BRCA1 and XRCC1 polymorphisms associated with survival in advanced gastric cancer treated with taxane and cisplatin. Cancer Sci 2010; 101: [PMID: DOI: / j x] 30 Zhao Y, Deng X, Wang Z, Wang Q, Liu Y. Genetic polymorphisms of DNA repair genes XRCC1 and XRCC3 and risk of colorectal cancer in Chinese population. Asian Pac J Cancer Prev 2012; 13: [PMID: DOI: / apjcp ] 31 Nissar S, Lone TA, Banday MZ, Rasool R, Chowdri NA, Parray FQ, Abdullah S, Sameer AS. Arg399Gln polymorphism 8707 July 14, 2014 Volume 20 Issue 26

400 Zhang SH et al. APE1 polymorphisms influence cancer risk of XRCC1 gene and risk of colorectal cancer in Kashmir: A case control study. Oncol Lett 2013; 5: [PMID: DOI: /ol ] 32 Pardini B, Naccarati A, Novotny J, Smerhovsky Z, Vodickova L, Polakova V, Hanova M, Slyskova J, Tulupova E, Kumar R, Bortlik M, Barale R, Hemminki K, Vodicka P. DNA repair genetic polymorphisms and risk of colorectal cancer in the Czech Republic. Mutat Res 2008; 638: [PMID: DOI: /j.mrfmmm ] 33 Engin AB, Karahalil B, Karakaya AE, Engin A. Association between XRCC1 ARG399GLN and P53 ARG72PRO polymorphisms and the risk of gastric and colorectal cancer in Turkish population. Arh Hig Rada Toksikol 2011; 62: [PMID: DOI: / ] 34 Zeng FR, Ling Y, Yang J, Tian XC, Yang X, Luo RC. X-ray repair cross-complementing group 1 Arg399Gln gene polymorphism and susceptibility to colorectal cancer: a metaanalysis. Tumour Biol 2013; 34: [PMID: DOI: /s ] 35 Tudek B. Base excision repair modulation as a risk factor for human cancers. Mol Aspects Med 2007; 28: [PMID: DOI: /j.mam ] 36 Karahalil B, Bohr VA, Wilson DM. Impact of DNA polymorphisms in key DNA base excision repair proteins on cancer risk. Hum Exp Toxicol 2012; 31: [PMID: DOI: / ] 37 Yamane A, Kohno T, Ito K, Sunaga N, Aoki K, Yoshimura K, Murakami H, Nojima Y, Yokota J. Differential ability of polymorphic OGG1 proteins to suppress mutagenesis induced by 8-hydroxyguanine in human cell in vivo. Carcinogenesis 2004; 25: [PMID: DOI: /carcin/ bgh166] 38 Curtin K, Samowitz WS, Wolff RK, Ulrich CM, Caan BJ, Potter JD, Slattery ML. Assessing tumor mutations to gain insight into base excision repair sequence polymorphisms and smoking in colon cancer. Cancer Epidemiol Biomarkers Prev 2009; 18: [PMID: DOI: / EPI ] 39 Moreno V, Gemignani F, Landi S, Gioia-Patricola L, Chabrier A, Blanco I, González S, Guino E, Capellà G, Canzian F. Polymorphisms in genes of nucleotide and base excision repair: risk and prognosis of colorectal cancer. Clin Cancer Res 2006; 12: [PMID: DOI: / CCR ] 40 Hansen R, Saebø M, Skjelbred CF, Nexø BA, Hagen PC, Bock G, Bowitz Lothe IM, Johnson E, Aase S, Hansteen IL, Vogel U, Kure EH. GPX Pro198Leu and OGG1 Ser326Cys polymorphisms and risk of development of colorectal adenomas and colorectal cancer. Cancer Lett 2005; 229: [PMID: DOI: /j.canlet ] P- Reviewers: Berretta M, Giannopoulos GA S- Editor: Gou SX L- Editor: Rutherford A E- Editor: Zhang DN 8708 July 14, 2014 Volume 20 Issue 26

401 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. RANDOMIZED CLINICAL TRIAL I.31, a new combination of probiotics, improves irritable bowel syndrome-related quality of life Vicente Lorenzo-Zúñiga, Elba Llop, Cristina Suárez, Beatriz Álvarez, Luis Abreu, Jordi Espadaler, Jordi Serra Vicente Lorenzo-Zúñiga, Jordi Serra, Gastroenterology, Motility and Functional Gut Disorders Unit, Hospital Universitari Germans Trias i Pujol/CIBERehd, Badalona, Spain Elba Llop, Cristina Suárez, Beatriz Álvarez, Luis Abreu, Gastroenterology, Hospital Puerta de Hierro, Madrid, Spain Jordi Espadaler, AB-Biotics, Badalona, Spain Author contributions: All authors contributed equally to this paper. Supported by Spanish Ministry of Health, Instituto de Salud Carlos Ⅲ (in part), No. PI10/02135 Correspondence to: Jordi Serra, MD, PhD, Gastroenterology, Motility and Functional Gut Disorders Unit, Hospital Universitari Germans Trias i Pujol/CIBERehd, Carretera del Canyet s/n, Badalona, Spain. jserrap.germanstrias@gencat.cat Telephone: Fax: Received: November 6, 2013 Revised: February 5, 2014 Accepted: March 19, 2014 Published online: July 14, 2014 by 18 ± 3 and 22 ± 4 points in the high and the low dose groups, respectively (P = and P = vs placebo), but only 9 ± 3 in the placebo group. Gutspecific anxiety, as measured with VSI, also showed a significantly greater improvement after 6 wk of treatment in patients treated with probiotics (by 10 ± 2 and 14 ± 2 points, high and low dose respectively, P < 0.05 for both vs 7 ± 1 score increment in placebo). Symptom relief showed no significant changes between groups. No adverse drug reactions were reported following the consumption of probiotic or placebo capsules. CONCLUSION: A new combination of three different probiotic bacteria was superior to placebo in improving IBS-related quality of life in patients with IBS and diarrhea Baishideng Publishing Group Inc. All rights reserved. Abstract AIM: To determine the dose-related effects of a novel probiotic combination, I.31, on irritable bowel syndrome (IBS)-related quality of life (IBS-QoL). METHODS: A multicenter, randomized, double-blind, placebo-controlled intervention clinical trial with three parallel arms was designed. A total of 84 patients (53 female, 31 male; age range years) with IBS and diarrhea according to Rome-Ⅲ criteria were randomly allocated to receive one capsule a day for 6 wk containing: (1) I.31 high dose (n = 28); (2) I.31 low dose (n = 27); and (3) placebo (n = 29). At baseline, and 3 and 6 wk of treatment, patients filled the IBSQoL, Visceral Sensitivity Index (VSI), and global symptom relief questionnaires. RESULTS: During treatment, IBS-QoL increased in all groups, but this increment was significantly larger in patients treated with I.31 than in those receiving placebo (P = 0.008). After 6 wk of treatment, IBS-QoL increased Key words: Irritable bowel syndrome; Probiotic combination; Irritable bowel syndrome; Quality-of-life Core tip: Irritable bowel syndrome (IBS) is a benign functional gut disorder, and its severity is closely related to the impact of the disorder on quality of life. Probiotic bacteria have been shown to have a modest beneficial effect on abdominal symptoms in patients with IBS, but the effect of probiotics on IBS-related quality of life (IBS-QoL) is unclear. The present study was designed to specifically address the effect of a probiotic combination (I.31) on IBS-QoL, and demonstrates that I.31 is superior to placebo in improving IBS-QoL. These data suggest that I.31 may be beneficial for the global management of this complex disorder. Lorenzo-Zúñiga V, Llop E, Suárez C, Álvarez B, Abreu L, Espadaler J, Serra J. I.31, a new combination of probiotics, improves irritable bowel syndrome-related quality of life. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: 8709 July 14, 2014 Volume 20 Issue 26

402 Lorenzo-Zúñiga V et al. I.31 improves quality of life in IBS Irritable bowel syndrome (IBS) is a chronic functional gut disorder that affects about 8%-10% of the population in Western countries, mainly young and middleaged women [1]. Although IBS, as with other functional gut disorders, is a benign disorder with a good long-term prognosis, it has an important impact on a patient s quality of life [2,3]. IBS also produces a significant economic burden due to both direct health care-related costs and indirect costs due to impaired work productivity [4]. In fact, IBS has been proposed as the second leading cause of absenteeism after the common cold [5]. The severity of IBS ranges from mild, sporadic symptoms, to severe, invalidating symptoms. It has been shown that severity is closely related to the impact of the disease on a patient s quality of life [6]. IBS is a complex functional gut disorder of unknown origin. Several factors, including gastrointestinal hypersensitivity, motility, low-grade inflammation, and psychosocial factors seem to interplay to produce abdominal symptoms. In the last few years, increasing evidence for the role of gut bacteria in the control of gut function has been recognized [3], and recent studies using novel techniques for the quantification of gut microflora have demonstrated differences in the flora of patients with IBS compared to healthy subjects [7]. In parallel, several publications during the last decade have shown that changes in gut microflora, by supplementation of probiotic bacteria, may have beneficial effects in IBS symptoms [8,9]. However, despite deterioration in quality of life being one of the main health-related problems for IBS patients, the vast majority of published controlled trials assess the effects of probiotics on abdominal symptoms [8,9], whereas the effect of probiotics on IBS-related quality of life remains unclear [10]. We designed a pilot clinical trial with the main objective being to determine the dose-related effects of a novel probiotic combination on IBS-related quality of life. Because the effects of probiotics depend on the specific bacteria combinations used, we administered a mixture of equal parts of three probiotic bacteria: two Lactobacillus plantarum (CECT7484 and CECT7485) and one Pediococcus acidilactici (CECT7483). This formula was chosen among more than 100 strains of lactic acid bacteria due to their ability to survive gut passage and adhere to intestinal mucus in vitro. Moreover, when combined, the three strains produced significant amounts of butyric, propionic, and acetic acid in a ratio similar to that found in the healthy gut [11], and reduced inflammation and diarrhea in two different animal models of gut inflammation (J. Espadaler, personal communication). IBS-related quality of life was assessed using a specific questionnaire (IBS-QoL) previously translated and validated into Spanish [12]. As secondary objectives, we evaluated the effect of probiotic intake on gut related anxiety and global symptom relief by means of specific questionnaires [13,14]. INTRODUCTION MATERIALS AND METHODS Subjects A total of 84 patients (53 female, 31 male) aged between 20 and 70 years were enrolled in the study from January 2010 to December All patients met Rome- Ⅲ criteria for IBS with diarrhea. Inflammatory bowel disease and celiac disease were excluded with clinical and analytical data, including blood chemistry, CRP, and tissue anti-transglutaminase antibodies. Subjects suffering from chronic or acute illness that could interfere with the study, that were taking medications that could interfere in the study (including anti-inflammatory drugs, PPIs, antidepressants, anti-diarrheal, prokinetics, and antispasmodic agents), and patients who consumed antibiotics or probiotics in the four weeks prior to entering into the study were excluded. Pregnant or lactating women were also excluded. If the subjects fulfilled all the inclusion and exclusion criteria no run-in period was considered, and patients entered the randomization period immediately. All subjects gave written informed consent to participate. The study was performed in accordance with the Declaration of Helsinki, adhered to the CONSORT 2010 statement ( and the protocol was approved by the Ethics Committees of Hospital Puerta de Hierro (Madrid, Spain), and of Hospital Germans Trias i Pujol (Badalona, Spain). Treatment We used a combination of three strains of lactic acid bacteria: two Lactobacillus plantarum (CECT7484 and CECT7485) and one Pediococcus acidilactici (CECT7483). Two different doses of this combination were administered in separate groups of subjects: a high dose combination (effective dose cfus/capsule throughout the study) and a low dose combination (effective dose cfus/capsule throughout the study). Concentration of viable cells was measured from probiotic preparation at the beginning and end of the study. The proportion of the three strains was the same in both doses (1:1:1). Placebo capsules were indistinguishable in form, color, and taste to the capsules containing bacteria. Capsules were stored for stability analyses at 25 and 65% relative humidity in stability chambers following ICH guidelines. Study design The study was designed as a multicenter, randomized, double-blind, placebo-controlled intervention clinical trial with three parallel arms. Randomization lists were computer generated, and identical capsules containing the allocated treatment and blisters were produced by ABbiotics, so that both patients and physicians were blinded to the actual treatment given to each patient. Each patient was randomly allocated to one of the following treatments for 6 wk (42 d): (1) I.31 high dose capsule once 8710 July 14, 2014 Volume 20 Issue 26

403 Lorenzo-Zúñiga V et al. I.31 improves quality of life in IBS Table 1 Baseline characteristics of the subjects recruited High dose (n = 28) Low dose (n = 27) Placebo (n = 29) P value Age (yr) 47.5 ± ± ± 13.1 NS Male/female 9/19 7/20 15/14 NS BMI 24.7 ± ± ± 5.2 NS IBSQoL 54.2 ± ± ± 18.5 NS VSI 43.0 ± ± ± 15.3 NS Glucose 95.1 ± ± ± 14.5 NS (mg/dl) Cholesterol ± ± ± 30.5 NS (mg/dl) LDL (mg/dl) ± ± ± 50.2 NS HDL (mg/dl) 56.6 ± ± ± 45.5 NS Creatinine 0.79 ± ± ± 0.18 NS (mg/dl) GGT (IU/L) 18.1 ± ± ± 15.6 NS GOT (IU/L) 19.6 ± ± ± 4.1 NS GPT (IU/L) 21.4 ± ± ± 10.6 NS BMI: Body mass index; IBSQoL: Irritable bowel syndrome-related quality of life; VSI: Visceral Sensitivity Index; LDL: Low density lipoprotein cholesterol; HDL: High density lipoprotein; GGT: Gamma glutamyl aminotransferase; GOT: G glutamic-oxaloacetic transaminase; GPT: Glutamic-pyruvic transaminase. daily; (2) I.31 low dose capsule once daily; and (3) a placebo capsule once daily. Efficacy and safety assessment The primary efficacy endpoint was the improvement in health-related quality of life (HRQoL) at the end of the 6-wk study period, assessed with a specific questionnaire for IBS: the validated Spanish version [12] of the IBS- QoL [15]. Scores of IBS-QoL were standardized to a scale. Improvement was calculated as the difference between the midpoint (day 21) or endpoint (day 42) scores and the baseline score for each group. All subjects with information in 1 or more of the 9 individual domains of the IBS-QoL questionnaire were included in the ITT analysis. The validated Visceral Sensitivity Index (VSI) scale [13] was used to assess anxiety specifically related to gastrointestinal sensations and symptoms. VSI improvement was calculated as the difference between the baseline score and the midpoint (day 21) or endpoint (day 42) scores for each group. Symptom relief was evaluated with a 5-point scale as proposed by Müller-Lissner et al [14] : 1, symptom worsening; 2, no relief; 3, somewhat relieved; 4, considerably relieved; and, 5, completely relieved. Patients filled IBS- QoL and VSI questionnaires at baseline (day 1) and during follow-up visits on days 21 and 42. Symptom relief was calculated in each individual as the weekly average of the scores recorded during the last four weeks of treatment for each group. All subjects with information in 1 or more weeks over the last 4 were included in the analysis. Patients were defined as responders when answered considerably relieved or completely relieved at least 50% of the time during the last four weeks, as recommended by EMA guideline CPMP/EWP/785/97 [14]. The empty blisters delivered by patients were counted to confirm treatment compliance. No analysis of fecal samples was performed. Adverse events were monitored following the directives of the Spanish Pharmacovigilance System for standard clinical trials with drugs. Statistical analysis Results were expressed as mean ± SE. Statistical analysis was performed on the ITT population. For betweengroup comparisons of quantitative variables, an ANOVA test was used if application conditions were satisfied according to Levene s test for homogeneity of variances and the Shapiro-Wilk test for normality; alternatively a non-parametric ANOVA (Kruskal-Wallis test) was used. Reported P values have been corrected using the Bonferroni-Holm method for multiple comparisons in ANOVA and Kruskall-Wallis post-hoc tests. Correlation between qualitative variables was tested using t test or Mann-Whitney U test depending on data normality, and correlation between quantitative variables was likewise tested using Pearson s or Spearman s rank test. According to the increment in IBS-QoL, patients were divided as good responders (IBS-QoL score increment 15 points), poor responders (IBS-QoL score increment between 10 and 15 points), and non-responders (IBS-QoL score increment < 10 points), and contingency tables were constructed. Differences between groups were tested using the χ 2 test. The study was powered to detect an increment of 10 points over placebo in the 0 to 100 IBSQoL scale at the end of the study period, with α = 0.05 and β = 0.80, a drop-out rate of 20% and SD = 10, resulting in a target n of 33 subjects per arm, after adjusting for comparisons between the three arms. RESULTS At baseline, there were no differences between the patients allocated to the different treatment groups in none of the measured parameters (Table 1). The number of subjects lost to follow-up or with insufficient data in the questionnaires was low for all parameters in all groups, and valid data could be obtained from the majority of patients in all treatment groups at the end of the study (Figure 1). IBS-related quality of life All groups of patients showed an improvement in IBS- QoL after 3 wk of treatment, and statistically significant differences between the treatment groups were observed after three and six weeks of supplementation (P = and P = 0.008, respectively). After three weeks, mean score increments were 18 ± 2 for the group allocated to high dose probiotics (P = vs placebo), 17 ± 3 for the low dose group (P = vs placebo), and 12 ± 2 for the placebo group. Differences among groups became even more significant after six weeks of supplementation, and both the high and the low dose groups (18 ± 3 and 22 ± 8711 July 14, 2014 Volume 20 Issue 26

404 Lorenzo-Zúñiga V et al. I.31 improves quality of life in IBS Enrollment Assessed for eligibility (n = 84) Excluded (n = 0) Not meeting inclusion criteria (n = 0) Decline to participate (n = 0) Other reasons (n = 0) Randomized (n = 84) Allocation Allocated to high dose (n = 28) Received intervention (n = 28) Did not received intervention (n = 0) Allocated to low dose (n = 27) Received intervention (n = 27) Did not received intervention (n = 0) Allocated to placebo (n = 29) Received intervention (n = 29) Did not received intervention (n = 0) Follow-up Loss to follow-up (n = 3) Discontinued intervention (n = 3) Loss to follow-up (n = 3) Discontinued intervention (n = 3) Loss to follow-up (n = 5) Discontinued intervention (n = 5) Relief VSI IBSQoL Intent to treat (ITT) analysis (n = 24) Evaluable per protocol (n = 21) Insufficient data (n = 7) Intent to treat (ITT) analysis (n = 23) Evaluable per protocol (n = 20) Insufficient data (n = 8) Intent to treat (ITT) analysis (n = 24) Evaluable per protocol (n = 22) Insufficient data (n = 6) Intent to treat (ITT) analysis (n=23) = 23) Evaluable per protocol (n=21) = 21) Insufficient data (n = 6) Intent to treat (ITT) analysis (n=24) = 24) Evaluable per protocol (n=19) = 19) Insufficient data (n = 8) Intent to treat (ITT) analysis (n = 25) Evaluable per protocol (n = 24) Insufficient data (n = 3) Intent to treat (ITT) analysis (n=24) = 24) Evaluable per protocol (n=20) = 20) Insufficient data (n = 9) Intent to treat (ITT) analysis (n=24) = 24) Evaluable per protocol (n=22) = 22) Insufficient data (n = 7) Intent to treat (ITT) analysis (n=24) = 24) Evaluable per protocol (n=23) (n = 23) Insufficient data (n = 6) Figure 1 Patient flow through the study according to CONSORT guidelines [37]. Note the similar number of loss to follow-up in all treatment arms. IBSQoL: Irritable bowel syndrome-related quality of life; VSI: Visceral Sensitivity Index. 4, respectively), achieved a significant greater increment in IBS-QoL compared to 9 ± 3 in the placebo group (P = and P = 0.023, for the high and low dose vs placebo, respectively; Figure 2A) without statistical differences between the high and the low probiotic doses (P = 0.392). IBS-QoL data did not follow a normal distribution, so we used a non-parametric ANOVA (Kruskall-Wallis test). A linear mixed model with repeated measures, adjusted for age, BMI, and sex, obtained a P = Per domain analysis showed a greater improvement in almost all the domains in the patients treated with the probiotic combination (both high and low doses) than in those treated with a placebo (Figure 2B), and this difference reached statistical significance in the Mental Health domain (P = 0.030). In a post hoc analysis, when the individual response to treatment was analyzed, the number of patients with a good response to the treatment (defined as score improvement 15 points), was significantly larger in those treated with probiotics (both with the high and low dose) than in those treated with placebo (P = 0.009; Figure 3). Slightly changing this cutoff (e.g., 14 points or 16 points) yields similar results (data not shown). Likewise, the number of subjects showing some improvement (defined as score improvement >10 points) was also signifi- cantly larger in those treated with probiotics than in those treated with a placebo (P = 0.038; Figure 3). Despite a fivefold difference in the concentration of probiotic between the high and low doses, no differences in the effect on IBS-QoL could be observed between doses at the end of the study (Figure 2A). When all patients treated with probiotics (high and low dose) are pooled together after 6 wk of treatment, the number of patients needed to treat (NNT) to achieve a good improvement ( 15 points increment; i.e., good responders) in health-related quality of life is 2.6 patients. VSI Gut-related anxiety, as measured with the VSI scale, also showed a significantly greater improvement in patients treated with the probiotic combination for both the high (10 ± 2 score increment; P = vs placebo) and the low dose groups (14 ± 2 score increment; P = vs placebo) compared to those treated with placebo (7 ± 1 score increment). However, this effect needed a longer time than that observed with IBS-related quality of life, and became evident only after 6 treatment weeks, whereas at three weeks there were no differences between the treatment groups (VSI score increments after three weeks were 6 ± 2, 7 ± 2, and 6 ± 1 for the high dose, low dose, 8712 July 14, 2014 Volume 20 Issue 26

405 Lorenzo-Zúñiga V et al. I.31 improves quality of life in IBS A IBS-QoL score difference P = P = High Low Placebo B IBS-QoL score difference P = Emotional health Mental health Sleep Energy Physical functioning Diet Social role Physical role Sex 0 High Low Placebo High Low Placebo High Low Placebo High Low Placebo High Low Placebo High Low Placebo High Low Placebo High Low Placebo High Low Placebo Figure 2 Irritable bowel syndrome related quality of life score improvement compared to baseline after 42 d of treatment. A: Global scores improved significantly more in both treatment groups than placebo (Kruskall-Wallis test); B: Among the different domains, the mental status showed a significant improvement when compared to placebo. High dose n = 24 Low dose n = 23 Placebo n = 24 5 (21%) 5 (22%) 4 (17%) 13 (54%) 13 (56%) 13 (54%) 6 (25%) 5 (22%) 7 (29%) Good response (ΔIBS-QoL 15) Poor response (ΔIBS-QoL 10-15) Non-response (ΔIBS-QoL < 10) P = for the difference among groups Figure 3 Irritable bowel syndrome-related quality of life score response to probiotic and placebo therapy. Good response was defined as score improvement 15 points; poor response as score improvement points; and non-response as score improvement < 10 points. The number of responders (score increment > 10) was significantly larger in both groups of patients treated with probiotics than in those treated with placebo (χ 2 test). IBSQoL: Irritable bowel syndrome-related quality of life. and placebo groups, respectively). Relief of symptoms When considering data from the last four weeks of treatment, the number of responders ( considerably relieved or completely relieved at least 50% of the time) was somewhat, but not significantly, greater in both treatment groups (42% in the high dose group, 32% in the low dose 8713 July 14, 2014 Volume 20 Issue 26

406 Lorenzo-Zúñiga V et al. I.31 improves quality of life in IBS group) than in the placebo group (25%; P = 0.467). Safety No rescue medication was reported to be used by any subject during their participation in the study. No adverse drug reactions were reported following the consumption of probiotic or placebo capsules. Additionally, the dropout rate did not differ between study groups (3 patients in the high and low dose groups, and 5 patients in the placebo group). A small increment of liver enzyme levels (less than 3 times over normal ranges) was observed in 4 patients: two in the high dose group, one in the low dose, and one in the placebo group. Of note, one patient in the high probiotic dose group and the one in the low probiotic dose group already had liver enzyme levels above the normal range at baseline. DISCUSSION The most relevant finding of the present study is that a new combination of 3 different probiotic bacteria (I.31 probiotics) taken daily for 6 wk had a positive impact on IBS-related quality of life, with the effect not being related to the dose of probiotics. The higher probiotic dose appeared to achieve a slightly faster effect on IBS-QoL, which was significantly larger than in the placebo group after 3 wk of treatment. However, at the end of the study no differences could be observed between doses, neither in quality of life nor in the other parameters measured. These results are a bit surprising, given that the higher dose contained 5 times more viable probiotic cells than the lower dose, and suggests that a plateau effect could have been achieved at the lower dose. IBS is a complex, heterogeneous condition of unknown origin, with a variety of different factors involved in symptom generation. These include: increased visceral sensitivity [16], altered motility and gas transport [17], lowgrade inflammation [18], psychological disturbances [19], and early life experiences [20]. The final symptoms present in each individual patient and the severity of the disease are the result of the interplay between all these factors [21]. IBS has an important impact in the quality of life of the patients [2,3], and the degree of alteration of quality of life is closely related to the severity of IBS in each individual patient [6]. Hence, in the absence of a curative strategy, improvement of quality of life should be an important objective of IBS treatment. IBS-QoL was evaluated using a specific questionnaire [15] that was previously translated and validated to the Spanish language [12]. This questionnaire has been previously used in large clinical trials to assess the effect of drugs in IBS-QoL [22]. We decided that a cut-off of 15 points in IBS-QoL score improvement should define good responders, and a cut-off of 10 points should distinguish responders from non-responders. These cut-off points, which are arbitrary, are in the same line as used in other studies assessing the clinical impact of treatments on QoL [23]. Using this methodology, we found that 55% of patients treated with probiotics (high as well as low dose) were good responders, whilst only 17% of placebo-treated patients did, and more than 75 % of the patients were responders. Hence, the benefit of probiotic treatment on IBS-QoL was not only statistically significant, but also clinically relevant. When the effect over the specific domains was analyzed, we found an improvement of quality of life in all the domains, but this difference was only statistically significant for the mental status domain. Improvement of quality of life was associated to a significant improvement in gut related anxiety, as measured by a specifically developed questionnaire: VSI [13]. This finding is also relevant, because mental disorders, like anxiety and depression, are often present in IBS and may have an impact on the severity of the disease and quality of life [6,24,25]. VSI has been shown to be a strong predictor of current IBS symptom severity [13,24]. Improvement in VSI took longer than IBS-QoL improvement, and became evident only after 6 wk of treatment, suggesting that other factors influenced IBS-QoL. Abdominal symptom relief during probiotic treatment was somewhat greater, but not statistically significant, in patients treated with probiotics. These differences were in line with previous studies showing a modest effect of probiotics on individual symptoms [9,10]. The lack of effect of probiotics on symptom relief may be due to the small number of subjects included in the study. The sample size in this pilot study was specifically powered to detect differences in IBS-QoL. In fact, based on data from previous clinical studies with probiotics [9], over 100 patients per arm should have been included in order to detect a significant difference in global symptom relief, with α = 0.05 and β = 0.80 after adjusting for comparisons between three arms and accounting for drop-outs. However, considering this limitation of the present study, our data suggest that the effect of probiotics on IBS seems not to be limited to the area of GI-symptoms, but is also evident for other aspects outside the abdomen, like mental health status, gut related anxiety, and IBS-related quality of life. During the last few years, the role of intestinal microbiota in the modulation of gut function has received increasing attention. Studies in mice showed that intestinal microbiota modulates immune and smooth muscle function, epithelial cell permeability, enteric neurotransmission, and visceral sensitivity [26]. Most of these factors are altered to some degree in patients with IBS [4,27-29]. Modulation of intestinal microflora by probiotics can decrease visceral sensitivity in mice [30,31] and the inflammatory responses in humans, an effect that correlated with symptom improvement in IBS patients [32]. However, the effects of intestinal microbiota go beyond the limits of the GI-tract, and several studies suggest that they are also involved in modulation of body weight, cutaneous perception, and behavior [33-35]. Moreover, a recent study from McMaster shows that intestinal microbiota can influence the central nervous system and behavior in adult mice in the absence of discernible changes in local or circulating 8714 July 14, 2014 Volume 20 Issue 26

407 Lorenzo-Zúñiga V et al. I.31 improves quality of life in IBS cytokines or specific gut neurotransmitter levels, suggesting the existence of a direct gut microbiota-brain axis [36]. Hence, it seems possible that a direct effect of probiotics on the central nervous system could also have contributed to the effects of probiotics in the present study. Our results do not provide evidence for a doserelated effect of the tested probiotics. The explanation for such an outcome is unclear, but may be due to the intrinsic nature of probiotics, which may not follow the typical pharmacological rules or to a saturation of the effect. The effects of probiotics are not universal for all bacteria, not even for strains of the same species, as each specific bacterial strain may have particular effects on gut function, which is probably also true for other functions outside the GI-tract. Likewise, there may be synergistic or antagonistic effects when a bacterial combination is administered [8]. In the present study, we used a mixture of three probiotic bacteria, two strains of Lactobacillus plantarum (CECT7484 and CECT7485) and one Pediococcus acidilactici (CECT7483), which was previously found to reduce inflammation and diarrhea in two different animal models of gut inflammation. Using this formula, we found a rapid and clinically relevant effect of the probiotic combination on IBS-related quality of life, which was associated to an improvement of gut related anxiety, but not to similar relief in abdominal symptoms. Hence, although our study was not designed to determine mechanistic factors involved in the effects induced by probiotics, our results suggest that the mechanisms involved in improvement of IBS-related quality of life may include both local and central effects. If these results were reproduced in larger studies, they open the possibility of developing treatment strategies using probiotics that are not only addressed against the abdominal symptoms of patients with functional gut disorders, but can also influence other important aspects of the disorder and other conditions often associated with IBS like behavior, anxiety, or depression. In conclusion, we found that a new combination of three different probiotic bacteria was superior to placebo in improving IBS-related quality of life in patients with IBS and diarrhea. After 6 wk of treatment, the difference was evident in both high and low doses of bacteria, and the increment in quality of life was mainly due to an increment in the mental status domain and an associated to an improvement in gut related anxiety. Hence, this probiotic combination can be useful for the treatment of patients with IBS that impacts their quality of life. ACKNOWLEDGMENTS The authors thank the Translation Service of the Autonoma University of Barcelona for English editing of the manuscript. COMMENTS Background To determine the dose-related effects of the novel probiotic combination I.31 on irritable bowel syndrome (IBS)-related quality of life (IBS-QoL). Research frontiers Changes in gut microflora, by supplementation of probiotic bacteria, may have beneficial effects in IBS symptoms. Innovations and breakthroughs I.31 probiotic formula had effects on IBS-quality of life at 3 and 6 wk, as well as on Visceral Sensitivity Index (VSI) at 6 wk, but had only a modest effect on abdominal symptoms. Applications This probiotic combination can be useful for the treatment of patients with IBS that impacts their quality of life. Terminology IBS-QoL is a standardized score to a scale. The VSI scale is used to assess anxiety specifically related to gastrointestinal sensations and symptoms. Peer review This is a study on the effects of IBS symptoms of a probiotic formula consisting of three different probiotic strains. Data are interesting, but the presentation of the data needs to be more focused. REFERENCES 1 Lovell RM, Ford AC. Global prevalence of and risk factors for irritable bowel syndrome: a meta-analysis. Clin Gastroenterol Hepatol 2012; 10: e4 [PMID: DOI: /j.cgh ] 2 Gralnek IM, Hays RD, Kilbourne A, Naliboff B, Mayer EA. The impact of irritable bowel syndrome on health-related quality of life. Gastroenterology 2000; 119: [PMID: ] 3 Simrén M, Svedlund J, Posserud I, Björnsson ES, Abrahamsson H. Health-related quality of life in patients attending a gastroenterology outpatient clinic: functional disorders versus organic diseases. Clin Gastroenterol Hepatol 2006; 4: [PMID: ] 4 Paré P, Gray J, Lam S, Balshaw R, Khorasheh S, Barbeau M, Kelly S, McBurney CR. 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408 Lorenzo-Zúñiga V et al. I.31 improves quality of life in IBS [PMID: ] 11 D Argenio G, Mazzacca G. Short-chain fatty acid in the human colon. Relation to inflammatory bowel diseases and colon cancer. Adv Exp Med Biol 1999; 472: [PMID: ] 12 Badia X, Herdman M, Mearin F, Pérez I. Adaptación al español del cuestionario IBSQoL para la medición de la calidad de vida en pacientes con síndrome de intestino irritable. Rev Esp Enferm Dig 2000; 92: Labus JS, Bolus R, Chang L, Wiklund I, Naesdal J, Mayer EA, Naliboff BD. The Visceral Sensitivity Index: development and validation of a gastrointestinal symptom-specific anxiety scale. Aliment Pharmacol Ther 2004; 20: [PMID: ] 14 Müller-Lissner S, Koch G, Talley NJ, Drossman D, Rueegg P, Dunger-Baldauf C, Lefkowitz M. Subject s Global Assessment of Relief: an appropriate method to assess the impact of treatment on irritable bowel syndrome-related symptoms in clinical trials. 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Early life risk factors that contribute to irritable bowel syndrome in adults: a systematic review. Am J Gastroenterol 2008; 103: ; quiz 775 [PMID: DOI: / j x] 21 Drossman DA, Chang L, Bellamy N, Gallo-Torres HE, Lembo A, Mearin F, Norton NJ, Whorwell P. Severity in irritable bowel syndrome: a Rome Foundation Working Team report. Am J Gastroenterol 2011; 106: ; quiz 1760 [PMID: DOI: /ajg ] 22 Watson ME, Lacey L, Kong S, Northcutt AR, McSorley D, Hahn B, Mangel AW. Alosetron improves quality of life in women with diarrhea-predominant irritable bowel syndrome. Am J Gastroenterol 2001; 96: [PMID: ] 23 Carson RT, Tourkodimitris S, Lewis BE, Johnston JM. Small bowel II: PWE-127 Two randomised, double-blind, placebocontrolled phase 3 trials of linaclotide in adults with irritable bowel syndrome: Effects on quality of life. Gut 2011; 61 (Suppl 2): A348-A Jerndal P, Ringström G, Agerforz P, Karpefors M, Akkermans LM, Bayati A, Simrén M. Gastrointestinal-specific anxiety: an important factor for severity of GI symptoms and quality of life in IBS. Neurogastroenterol Motil 2010; 22: 646-e179 [PMID: DOI: /j x] 25 Sugaya N, Nomura S, Shimada H. Relationship between cognitive factors and anxiety in individuals with irritable bowel syndrome. Int J Behav Med 2012; 19: [PMID: DOI: /s ] 26 Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, Gordon JI. Molecular analysis of commensal host-microbial relationships in the intestine. Science 2001; 291: [PMID: ] 27 Eugenio MD, Jun SE, Cain KC, Jarrett ME, Heitkemper MM. Comprehensive self-management reduces the negative impact of irritable bowel syndrome symptoms on sexual functioning. Dig Dis Sci 2012; 57: [PMID: DOI: /s ] 28 Farndale R, Roberts L. Long-term impact of irritable bowel syndrome: a qualitative study. Prim Health Care Res Dev 2011; 12: [PMID: DOI: /S ] 29 Drossman D, Morris CB, Hu Y, Toner BB, Diamant N, Whitehead WE, Dalton CB, Leserman J, Patrick DL, Bangdiwala SI. Characterization of health related quality of life (HRQOL) for patients with functional bowel disorder (FBD) and its response to treatment. Am J Gastroenterol 2007; 102: [PMID: ] 30 Bravo JA, Forsythe P, Chew MV, Escaravage E, Savignac HM, Dinan TG, Bienenstock J, Cryan JF. Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proc Natl Acad Sci USA 2011; 108: [PMID: DOI: /pnas ] 31 Verdú EF, Bercik P, Verma-Gandhu M, Huang XX, Blennerhassett P, Jackson W, Mao Y, Wang L, Rochat F, Collins SM. Specific probiotic therapy attenuates antibiotic induced visceral hypersensitivity in mice. Gut 2006; 55: [PMID: ] 32 O Mahony L, McCarthy J, Kelly P, Hurley G, Luo F, Chen K, O Sullivan GC, Kiely B, Collins JK, Shanahan F, Quigley EM. Lactobacillus and bifidobacterium in irritable bowel syndrome: symptom responses and relationship to cytokine profiles. Gastroenterology 2005; 128: [PMID: ] 33 Bäckhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. Host-bacterial mutualism in the human intestine. Science 2005; 307: [PMID: ] 34 Amaral FA, Sachs D, Costa VV, Fagundes CT, Cisalpino D, Cunha TM, Ferreira SH, Cunha FQ, Silva TA, Nicoli JR, Vieira LQ, Souza DG, Teixeira MM. Commensal microbiota is fundamental for the development of inflammatory pain. Proc Natl Acad Sci USA 2008; 105: [PMID: DOI: /pnas ] 35 Lyte M, Varcoe JJ, Bailey MT. Anxiogenic effect of subclinical bacterial infection in mice in the absence of overt immune activation. Physiol Behav 1998; 65: [PMID: ] 36 Bercik P, Denou E, Collins J, Jackson W, Lu J, Jury J, Deng Y, Blennerhassett P, Macri J, McCoy KD, Verdu EF, Collins SM. The intestinal microbiota affect central levels of brainderived neurotropic factor and behavior in mice. Gastroenterology 2011; 141: , 609.e1-3 [PMID: DOI: /j.gastro ] 37 Schulz KF, Altman DG, Moher D. CONSORT 2010 Statement: updated guidelines for reporting parallel group randomised trials. Trials 2010; 11: 32 [PMID: DOI: / ] P- Reviewers: Carter D, Iovino P, Ohman L, Rahimi R S- Editor: Zhai HH L- Editor: Rutherford A E- Editor: Wang CH 8716 July 14, 2014 Volume 20 Issue 26

409 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. CASE REPORT Acute abdomen: Rare and unusual presentation of right colic xanthogranulomatosis Paola Addario Chieco, Laura Antolino, Valentina Giaccaglia, Francesca Centanini, Gaetano Vincenzo Cunsolo, Alessandra Sparagna, Stefania Uccini, Vincenzo Ziparo Paola Addario Chieco, Laura Antolino, Valentina Giaccaglia, Francesca Centanini, Gaetano Vincenzo Cunsolo, Alessandra Sparagna, Vincenzo Ziparo, Department of General Surgery, General Surgery 1 Unit, S. Andrea University Hospital, Rome, Italy Paola Addario Chieco, Laura Antolino, Valentina Giaccaglia, Francesca Centanini, Gaetano Vincenzo Cunsolo, Alessandra Sparagna, Vincenzo Ziparo, Stefania Uccini, Department of Pathology, S. Andrea University Hospital, Rome, Italy Author contributions: Antolino L, Centanini F, Cunsolo GV and Sparagna A contributed to acquisition of the data; Antolino L and Giaccaglia V contributed to analysis and interpretation of the data; Antolino L, Giaccaglia V, Cunsolo GV, Sparagna A and Uccini S contributed to drafting of the manuscript; Addario Chieco P, Giaccaglia V and Ziparo V contributed to critical revision of the paper. Correspondence to: Paola Addario Chieco, MD, Department of General Surgery, General Surgery 1 Unit, S. Andrea University Hospital, Via di Grottarossa 1085, Rome, Italy. paddariochieco@ospedalesantandrea.it Telephone: Fax: Received: February 1, 2013 Revised: April 23, 2013 Accepted: May 18, 2013 Published online: July 14, 2014 Abstract Xanthogranulomatous inflammation (XGI) is a disease of unknown origin, most frequently described in the kidney and gallbladder; its localization in the colorectal tract is extremely rare. The extension of the typical inflammatory process to the surrounding tissues may lead to misdiagnosis as cancer. We report the case of a 56-year-old woman presenting to the Emergency Department with pain, increased levels of α1 and α2 proteins and C-reactive protein (17.5 mg/dl; normal value 0-0.5), and a palpable mass, localized in the right lower quadrant of the abdomen. A computed tomography scan showed a large right cecal mass with necrotic areas, local inflammation of retroperitoneal fat, and enlargement of local lymph nodes. Because of the high suspicion of colic abscess as well as malignancy and worsening of the clinical condition, the patient underwent right colectomy after 4 d of antibiotic treatment. Pathology revealed xanthogranulomatous inflammation involving the ileocecal valve. We review the reports of large bowel tract XGI in the international literature Baishideng Publishing Group Inc. All rights reserved. Key words: Xanthogranulomatosis; Large bowel xanthogranulomatosis; Abdominal mass; Abdominal abscess Core tip: Xanthogranulomatous inflammation (XGI) is a disease of unknown origin, most frequently described in the kidney and gallbladder, and extremely rare in the colon. The extension of inflammation to the surrounding tissues may lead to misdiagnosis as cancer. We report the case of a 56-year-old woman presenting to the Emergency Department with pain, increased levels of acute-phase proteins, and a palpable mass in the right lower abdominal quadrant. Computed tomography showed a large cecal mass with necrotic areas and enlarged lymph nodes. Due to suspicion of malignancy and worsening of the clinical conditions, she underwent right colectomy. Pathology revealed XGI of the ileocecal valve. Addario Chieco P, Antolino L, Giaccaglia V, Centanini F, Cunsolo GV, Sparagna A, Uccini S, Ziparo V. Acute abdomen: Rare and unusual presentation of right colic xanthogranulomatosis. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: INTRODUCTION Xanthogranulomatous inflammation (XGI) is a rare 8717 July 14, 2014 Volume 20 Issue 26

410 Addario Chieco P et al. Unusual presentation of right colic xanthogranulomatosis A B Figure 1 Contrast enhanced computed tomography showing a large mass in the right abdominal quadrant (A), and inflammatory involvement of retroperitoneal fat and local lymph nodes (B). pathologic entity with characteristic macroscopic and microscopic features. Typical histologic findings are: aggregation of lipid-laden foamy macrophages accompanied by giant cells, few plasma cells and lymphocytes leading to parenchymal destruction [1]. XGI is an uncommon entity that can involve any organ, and the most common reported sites are the kidney and gallbladder. Other described rare localizations are: epididymis, ovary, fallopian tube, endometrium, thyroid, adrenal gland, appendix, lymph nodes, lung, bone, soft tissue, and skin [2]. The large bowel has rarely been involved in the xantogranulomatous process, with only a few reports in the international literature [2-6]. It is often difficult to differentiate the lesion from an infiltrative cancer because XGI frequently presents as a mass-like lesion with an extension of fibrosis and inflammation to the surrounding tissues mimicking an advanced neoplasia. We report the case of a middleaged woman suffering from a right colic XGI mimicking a malignant tumor or an abscess mass. CASE REPORT A 56-year-old female presented to our Emergency Department with severe abdominal pain in the right lower quadrant which developed gradually over 3 mo, weight loss and intermittent fever. She denied other symptoms such as vomiting, hematemesis and melena. Her past medical history was positive for diabetes, hyperthyroidism treated with total thyroidectomy in 2008 and replacement therapy with levothyroxine, and class I obesity (body mass index: 30.7 kg/m 2 ). She denied smoking, alcohol use, or any illicit drug use. Two months previously she had 3 consecutive fecal occult blood tests which were all negative. Physical examination showed a sore and tender abdomen, particularly painful in the right lower quadrant where a rigid, suspicious mass was palpable along with a positive Blumberg maneuver. Bowel sounds in all 4 quadrants and rectal examination were normal. Blood tests revealed microcytic, hypochromic anemia [hemoglobin (Hb) 9.5 g/dl; normal value (NV) 12-16], normal leukocyte count, and elevated C-reactive protein (17.5 mg/dl; NV 0-0.5). Aspartate aminotransferase and alanine aminotransferase values were slightly increased (57 U/L; NV and 145 U/L; NV 11-66, respectively). Gamma-glutamyltransferase, triglyceride and cholesterol levels were within the normal range. Oncomarkers (carcinoembryonic antigen and carbohydrate antigen) values were within the normal range. An abdominal computed tomography (CT) scan showed a solid irregular mass, measuring 96 mm 91 mm 66 mm, with large necrotic areas and several enlarged lymph nodes around it (Figure 1A and B). The lesion also seemed to be adherent to the VI liver segment. Clinical and radiological findings raised the suspicion of right colon cancer involving the surrounding structures with a clinical preoperative staging of: ct4 cn+ cm0. After 24 h, because of worsening of clinical conditions, the patient underwent median laparotomy. Intraoperatively, a large mass, fixed to nearby structures, and involving the ileocecal valve and cecum was found. A frozen section of the specimen was performed and revealed chronic inflammation. A right colectomy was performed. Postoperative recovery was uneventful. Pathological findings Histological examination revealed the intestinal wall distorted by a diffuse infiltration of lipid-laden macrophages (macrophage cytoplasm containing large amounts of lipid) and giant cells, mainly filling the sub-serosal surface, associated with suppurative peritonitis. The intestinal wall of the ileocecal valve was closely adhered to the appendix, causing luminal stenosis. In the appendix, the mucosa was histologically not involved in the disease, but the outer part of the appendix wall was closely adhered to the ileocecal junction and was equally heavily infiltrated by a large amount of lipid-laden macrophages (Figure 2). DISCUSSION XGI is a rare disease first reported in the genitourinary tract [7,8]. Any organ can be involved, but it is most common in the kidney and in the gallbladder. In particular, XGI of the gallbladder is found in 1%-4% of resected specimens [9]. In some cases, when the inflammatory process involves adjacent organs, it may clinically resemble 8718 July 14, 2014 Volume 20 Issue 26

411 Addario Chieco P et al. Unusual presentation of right colic xanthogranulomatosis A B C Figure 2 Pathological findings. Hematoxylin and eosin staining of the intestinal wall of the ileocecal valve and appendix showing heavy infiltration of a large amount of lipid-laden macrophages in which scattered multinucleated giant cells are shown (A, 160). In the field, numerous ectatic small blood vessels consistent with the edematous features of the wall are visible (B, 250; C, 400). invasive gallbladder carcinoma leading to extensive surgical resection. As reported by Rastogi et al [9] in 2010, most patients with xanthogranulomatous cholecystitis are female, presenting with vomiting, right upper quadrant pain, and a previous or concomitant diagnosis of gallstones. Xanthogranulomatous pyelonephritis constitutes less than 1% of chronic pyelonephritis, is frequently associated with urinary tract obstruction, nephrolitiasis and concomitant infection, and is preponderant in females and younger age groups [10]. Due to its vague and nonspecific clinical presentation, equivocal laboratory and radiological investigations may mimic renal tuberculosis or renal carcinoma [10]. XGI is a disease with specific macroscopic and microscopic features. Typical findings on macroscopic examination include a bright yellow mass with abscessual cavities and micro-abscesses; on microscopic examination, it includes large numbers of lipid-laden macrophages, combined with a minor component of chronic and acute inflammatory cells [4]. Other lesions containing foam cells, such as xanthoma, malacoplakia and pseudoxanthogranulomatous inflammation should be considered in the differential diagnosis. Xanthomatous lesions are not accompanied by fibrosis or giant cells. Malacoplakia is an unusual inflammatory condition characterized by inflammatory xanthomatous proliferation with the presence of Michaelis-Gutmann bodies. Pseudoxanthogranulomatous inflammation is characterized by pigmented foam cell collections with brown lipofuscin or hemosiderin pigment in their cytoplasm that are positive for Fontana-Masson or Prussian blue stains [4]. In the present case, no pigmentcontaining cells were found and the Fontana-Masson stain was negative, therefore pseudoxanthogranulomatous inflammation was exluded in the differential diagnosis. The exact pathogenesis of XGI is not well known. There have been many hypotheses, from inflammation and chronic suppuration, to hemorrhages and digestion of blood cells from macrophages, to defective lipid transportation, defective chemotaxis of macrophages and lymphatic obstruction [1-6]. Most recent studies are suggest that XGI probably represents a chronic suppurative process leading to tissue destruction and localized proliferation of macrophages containing large amounts of lipid, which are the characteristic histological features of the disease [2,6]. In particular, for the colon, it is possible that the inflammatory processes following diverticulitis, perforation, or appendicitis may cause a chronic suppurative process, with tissue destruction, that serves as an environment in which the host and microorganisms interact, leading to aggregation of lipid-accumulating macrophages [11]. Specifically, our patient was suffering from hyperthyroidism and diabetes, so had disrupted lipid and carbohydrate metabolism. Those conditions could play a role in development of XGI. When XGI presents as a mass with an extension of fibrosis and inflammation to the surrounding tissues, it is not possible to differentiate it from an infiltrative cancer [2]. For this reason, symptoms, signs and imaging studies showing an irregular contrast-enhanced lesion extending to surrounding tissues, can easily mislead the surgeon. To the best of our knowledge, there are only 5 reported cases regarding colonic XGI in the international literature (Table 1), 3 women and 2 men, and the age of the patients ranged from 30 to 72 years, all of them undergoing radical surgical intervention with no described relapse of the disease [2,6]. The first paper was published in 1986 by Davis et al [3], reporting the case of a 30-year-old woman with a double cervix and uterus didelphys presenting with rectal pain and palpable rectal mass. The final diagnosis, after a posterior surgical approach because of the suspicion of a tumor involving the sacrum, was an anorectal xanthogranulomatous abscess in a Müllerian duct remnant. Then, in 1996, Lo et al [4] presented the case of a 72-year-old male with abdominal distension and a palpable mass in the left 8719 July 14, 2014 Volume 20 Issue 26

412 Addario Chieco P et al. Unusual presentation of right colic xanthogranulomatosis Table 1 Reported cases of large bowel xanthogranulomatous inflammation Ref Age Involved organs Clinical presentation CT findings preoperative diagnosis Surgical operation/ pathology (yr)/sex Anadol et al [2] 57/F Cecum and appendix RLQ abdominal pain, rectorrhagia, anemia Davis et al [3] 30/F Anorectal area Rectal and sacral pain, nausea, weight loss, palpable tender mass on rectal examination Lo et al [4] 72/M Sigmoid colon Intestinal obstruction, palpable tender mass in the LIF, tenesmus, nausea and weight loss Oh et al [5] 38/F Sigmoid colon, appendix, salpinx Dhawan et al [6] 60/M Ascending colon Present case 56/F Cecum, jejunal loop CT: Solid mass in the RLQ with poorly defined margins and mesenteric fatty tissue infiltration Cecum cancer CT: two masses involving the intramural portion of the distal rectal wall Tumor involving the sacrum Barium enema: stricture at the sigmoid colon causing obstruction Sigmoid cancer or diverticulitis with stricture Abdominal pain, diarrhoea, CT: Colonic loop around the rectosigmoid junction with mesenteric fever, mild RLQ tenderness without any palpable mass fat infiltration Sigmoid cancer Abdominal pain, constipation, vomiting Abdominal pain, palpable mass in the RIF, fever and weight loss CT: irregular circumferential thickening involving 6 cm of the ascending colon. Multiple small pericolic lymph nodes Right colon cancer Appendectomy Frozen section Right colectomy XGI of the cecum Posterior approach, removal of the tumors, reapproximation of the rectal wall XGI of a Müllerian duct remnant involving the anorectum Excision of the mass including sigmoid colon, proximal jejunum and part of the peritoneum XGI of the sigmoid colon Resection of the sigmoid colon, appendectomy, right salpingectomy XGI of the sigmoid colon Right colectomy XGI of the ascending colon with mucosal involvement CT: expansive mass originating Right colectomy from the cecum with necrotic areas that seems to involve VI liver segment. Many lymph nodes enlarged, especially next to the mass. Right colon cancer XGI of the ileocecal valve CT: Computer tomography; M: Male; F: Female; NA: Not available; XGI: Xanthogranulomatous inflammation; RLQ: Right lower quadrant; LIF: Left iliac fossa; RIF: Right iliac fossa. iliac fossa. With suspicion of an occluding carcinoma of the sigmoid colon or diverticulitis with stricture, the patient underwent resection of the sigmoid, part of the jejunum and the peritoneum. The final pathology showed a XGI of the sigmoid colon. Later, in 2005, Oh et al [5] published the case of a 38-year-old female with diarrhea, fever and left lower quadrant abdominal pain. She underwent low anterior resection with a final diagnosis of XGI of the sigmoid colon. Anadol et al [2] reported in 2009 a case very similar to ours. The patient was a 57-year-old women presenting with right lower quadrant abdominal pain, rectorrhagia, anemia, and a solid mass of the right colon on a CT scan. She also underwent right colectomy with a pathological report showing a xanthogranulomatous colitis of the cecum. One of the differences with our case is that our patient presented to the Emergency Department with local peritonitis in the right lower quadrant and was admitted as having an acute abdomen; because of the suspicion of an abscess with diverticula and/or a neoplasm we did not perform colonoscopy. The worsening of the clinical conditions led to the surgical intervention. Finally, very recently, Dhawan et al [6] reported a 60-year-old male with abdominal pain, vomiting and constipation, who underwent right colectomy. The pathology showed right colic XGI with mucosal involvement; this is in contrast to our case, and all other reports about colic XGI, where intes- tinal wall involvement was mainly sub-serosal. All these 5 cases show that the symptoms, signs and radiological examination are very similar to those of colorectal cancer. In our experience, we suggest considering XGI also in the event of acute abdomen, or inflammatory abscess. The consensus is that it might be useful to confirm the diagnosis intraoperatively, as a frozen section did not help us in our surgical strategy, as was also reported by Anadol et al [2]. XGI is a rare entity, with kidney and gallbladder as the most common reported sites. The large intestine is an unusual localization of XGI, with only 5 cases being reported in the literature. It is very difficult to differentiate from advanced cancer and/or a local abscessed intestinal mass because inflammation and fibrosis of the lesion extend to the surrounding tissues. We think that the suspicion of malignancy is likely to remain unclear until the final pathological examination is performed. Colonic XGI is a benign disease, often requiring extended surgical resection, and has an excellent postoperative outcome. REFERENCES 1 Cozzutto C, Carbone A. The xanthogranulomatous process. Xanthogranulomatous inflammation. Pathol Res Pract 1988; 183: [PMID: DOI: / S (88) ] 8720 July 14, 2014 Volume 20 Issue 26

413 Addario Chieco P et al. Unusual presentation of right colic xanthogranulomatosis 2 Anadol AZ, Gonul II, Tezel E. Xanthogranulomatous inflammation of the colon: a rare cause of cecal mass with bleeding. South Med J 2009; 102: [PMID: ] 3 Davis M, Whitley ME, Haque AK, Fenoglio-Preiser C, Waterman R. Xanthogranulomatous abscess of a mullerian duct remnant. A rare lesion of the rectum and anus. Dis Colon Rectum 1986; 29: [PMID: DOI: / BF ] 4 Lo CY, Lorentz TG, Poon CS. Xanthogranulomatous inflammation of the sigmoid colon: a case report. Aust N Z J Surg 1996; 66: [PMID: DOI: / j tb00839.x] 5 Oh YH, Seong SS, Jang KS, Chung YW, Paik CH, Park YW, Han DS. Xanthogranulomatous inflammation presenting as a submucosal mass of the sigmoid colon. Pathol Int 2005; 55: [PMID: DOI: / j x] 6 Dhawan S, Jain D, Kalhan SK. Xanthogranulomatous inflammation of ascending colon with mucosal involvement: report of a first case. J Crohns Colitis 2011; 5: [PMID: DOI: /j.crohns ] 7 Peterson RO. Urologic pathology. 3rd ed. Philadelphia: JB Lippincott Co, 2006: Selzer DW, Dahlin DC, Deweerd JH. Tumefactive xanthogranulomatous pyelonephritis. Surgery 1957; 42: [PMID: ] 9 Rastogi A, Singh DK, Sakhuja P, Gondal R. Florid xanthogranulomatous cholecystitis masquerading as invasive gallbladder cancer leading to extensive surgical resection. Indian J Pathol Microbiol 2010; 53: [PMID: DOI: / ] 10 Goyal S, Gupta M, Goyal R. Xanthogranulomatous pyelonephritis: A rare entity. N Am J Med Sci 2011; 3: [PMID: DOI: /najms ] 11 Antonakopoulos GN, Chapple CR, Newman J, Crocker J, Tudway DC, O Brien JM, Considine J. Xanthogranulomatous pyelonephritis. A reappraisal and immunohistochemical study. Arch Pathol Lab Med 1988; 112: [PMID: ] P- Reviewer: Lipar M S- Editor: Zhai HH L- Editor: Cant MR E- Editor: Wang CH 8721 July 14, 2014 Volume 20 Issue 26

414 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. BRIEF CASE ARTICLE REPORT HBsAg clearance by Peg-interferon addition to a long-term nucleos(t)ide analogue therapy Michele Barone, Andrea Iannone, Alfredo Di Leo Michele Barone, Gastroenterology Unit, Department of Medical and Surgical Science, University of Foggia, Foggia, Italy Andrea Iannone, Alfredo Di Leo, Gastroenterology Unit, Department of Emergency and Organ Transplantation (D.E.T.O.), University of Bari, Bari, Italy Author contributions: Barone M and Iannone A contributed equally to this work and wrote the paper; Di Leo A approved the final version. Correspondence to: Michele Barone, MD, PhD, Professor of Medicine, Chief of Gastroenterology Unit, Department of Medical and Surgical Science, University of Foggia, Viale Pinto 1, Foggia, Italy. michele.barone@unifg.it Telephone: Fax: Received: January 20, 2014 Revised: March 10, 2014 Accepted: April 5, 2014 Published online: July 14, 2014 Abstract The ideal endpoint of hepatitis B virus (HBV) antiviral therapy is HBsAg loss, a difficult goal to obtain, especially in HBeAg negative patients. Herein, we report the results obtained by the addition of peg-interferon α-2a to a long-lasting nucleos(t)ide analogue therapy in a HBeAg negative, genotype D patient with steadily HBV-DNA negative/hbsag positive values. In 2002, our Caucasian 44-year-old male patient received lamivudine and, 4 years later, added adefovir because of a virological breakthrough. In 2011, considering his young age, liver stiffness (4.3 kpa) and HBsAg levels (3533 IU/mL), we added Peg-interferon α-2a for six months (3 in combination with nucleos(t)ide analogues followed by 3 mo of Peg-interferon α-2a monotherapy). A decrease of HBsAg levels was observed after 1 mo (1.21 log) of Peg-interferon and 3 mo (1.88 log) after the discontinuation of all drugs. Later, a complete clearance of HBsAg was obtained with steadily undetectable HBV- DNA serum levels (< 9 IU/mL). HBsAg clearance by the addition of a short course of Peg-interferon α-2a represents an important result with clinical and pharmacoeconomic implications, considering that nucleos(t)ide analogues therapy in HBeAg negative chronic hepatitis B patients is considered a long-lasting/life-long treatment Baishideng Publishing Group Inc. All rights reserved. Key words: Addition; HBeAg negative; HBsAg clearance; Nucleos(t)ide analogues; Peg-interferon Core tip: The ideal endpoint of antiviral therapy is HBsAg loss, a difficult goal to obtain, especially in HBeAg negative patients. A Caucasian 44-year-old male patient, HBeAg negative, genotype D, received lamivudine and, 4 years later, added adefovir because of a virological breakthrough. Five years later, considering his age, liver stiffness (4.3 kpa) and HBsAg levels (3533 IU/mL), we added Peg-interferon α-2a for six months (3 in combination with nucleos(t)ide analogues followed by 3 of Peg-interferon monotherapy), obtaining a complete HBsAg clearance. This result has important clinical and pharmaco-economic implications, since nucleos(t)ide analogues therapy in HBeAg negative patients is considered a long-lasting/life-long treatment. Barone M, Iannone A, Di Leo A. HBsAg clearance by Peginterferon addition to a long-term nucleos(t)ide analogue therapy. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: INTRODUCTION Hepatitis B virus infection is a worldwide health problem. World Health Organization estimates that about 2 billion people have been infected by the virus (1/3 of the entire world population) and about 350 million (6%) are carriers of a chronic infection [1]. Italy ranks among the countries with low endemicity [2] July 14, 2014 Volume 20 Issue 26

415 Barone M et al. HBsAg clearance by Peg-IFN plus analogues Currently, the treatment of chronic hepatitis B (CHB) is based on either a 12 mo-period of peg-interferon α-2a (PEG-IFN α-2a) or a long-lasting nucleos(t)ide analogues (NAs) administration [1-3]. The ideal endpoint of antiviral therapy, even if uncommon, is the loss of HBsAg ± seroconversion to anti-hbs [1-3]. HBe/anti-HBe seroconversion in HBeAg positive patients and disappearance of hepatitis B virus (HBV)-DNA from serum, however, represent significant end-points since they are correlated with a reduced risk of liver disease progression [4]. In HBeAg negative patients, HBsAg loss is exceptionally observed during the first 4-5 years of NAs treatment [1]. In fact, in patients undergoing lamivudine monotherapy HBsAg clearance rates are 1.9% and 11.7% at 5 and 7 years, respectively [5,6]. Also patients treated with adefovir show low percentages of HBsAg loss, ranging from 0% to 5% at 1 and 5 years, respectively [1,7]. Moreover, the combination treatment with lamivudine + adefovir does not increase their efficacy compared to the monotherapies (2.4% at 4 years) [8]. On the other hand, after 12 mo of PEG-IFN α-2a therapy, HBsAg clearance in HBeAg negative patients is 9% and 12% at 3 and 5 years of follow-up, respectively [1,9,10]. In these subjects, a rapid decrease in serum HBsAg is an important predictor of response to PEG- IFN α-2a treatment [11]. The combination therapy with PEG-IFN α-2a + NAs does not seem to increase the HBsAg loss rate as compared to the PEG-IFN α-2a monotherapy. In fact, 3 years after the administration of PEG-IFN α-2a (180 µg/wk) + lamivudine for 12 mo a 8% HBsAg loss was observed [9]. However, a combination therapy with PEG-IFN α-2a (180 µg/wk) + adefovir for 48 wk determined a 17% HBsAg loss after 2 years of follow-up [12]. Finally, only a few cases in the literature describe the effect of PEG-IFN α-2a as add-on therapy in patients undergoing long-lasting NAs treatment with steadily HBV- DNA negative/hbsag positive values. Mangano et al [13] report a HBsAg/anti-HBs seroconversion obtained by the addition of PEG-IFN α-2a for 12 mo in a young CHB patient undergoing lamivudine therapy. However, they did not assess HBsAg titre before the add-on therapy, neither report HBV genotype, two well-known predictors of response to PEG-IFN α-2a. Kittner et al [14] after a 12-mo add-on therapy with PEG-IFN α-2a in 12 CHB patients undergoing NAs treatment report 2 cases of HBsAg/anti-HBs seroconversion. However, the first patient was a HBV genotype A (known to respond better to PEG-IFN α-2a [15] ), while the second had a very low HBsAg titre (16 IU/mL) before the PEG-IFN α-2a administration. CASE REPORT Case history A 44-year-old Caucasian male with CHB came to our attention in November His mother and one brother were affected by HBV-related cirrhosis and CHB, respectively, suggesting a mother-to-child transmission of the Liver stiffness (kpa) Figure 1 Liver stiffness assessments by transient elastography. Progressive reduction of liver stiffness values, assessed every year from 2002 to virus. The medical history of our patient was characterized by first determination of elevated alanine aminotransferase (ALT) levels in 1996 and diagnosis of HBV infection in June In November 2002, he was admitted to our Gastroenterology Unit and blood examinations were performed that demonstrated ALT 1.2 the upper limit of normal (ULN), negative HBeAg, positive anti-hbe, positive HBV-DNA ( copies/ml, determined by the Versant HBV-DNA 3.0 assay), HBV genotype D and negative anti-hcv and anti-hdv IgG. Thus, he underwent liver biopsy, that showed a grade 2 stage 2 CHB (by METAVIR score system), and transient elastography, that resulted 8.5 kpa (IQR 0.7 kpa, SR 100%). According to these results and the guidelines in force at that time and because of the patient refusal to undergo interferon therapy, we started an antiviral treatment with lamivudine in December 2002 obtaining ALT normalization and a HBV DNA value < cp/ml. In January 2006, a virological breakthrough to lamivudine was observed, with increased ALT levels ( 2.3 ULN) and HBV-DNA serum levels of cp/ml (by the COBAS Amplicor HBV Monitor assay). Thus, adefovir (10 mg/day), the only NA rescue therapy available for lamivudine resistance at that time, was added, reaching undetectable HBV- DNA (< copies/ml, by the COBAS Amplicor HBV Monitor assay) and normal ALT levels after 6 mo of combined therapy. Liver stiffness, assessed every year by transient elastography (Figure 1), progressively decreased, reaching a value of 4.3 kpa (IQR 0.6 kpa, SR 100%) in February HBV testing From 2002 to 2005 HBV-DNA was tested by the Versant HBV-DNA 3.0 assay (Bayer Corporation, Tarrytown, NY) with a lower limit of quantification (LLQ) of copies/ml, while from 2006 to 2010 it was determined by the COBAS Amplicor HBV Monitor assay (Roche Diagnostics, Indiannapolis, IN) with a LLQ of copies/ml. Finally, from 2011, HBV-DNA serum July 14, 2014 Volume 20 Issue 26

416 Barone M et al. HBsAg clearance by Peg-IFN plus analogues HBsAg (IU/mL) Feb 2011 Oct 2011 levels were determined by the Roche TaqMan PCR Real Time assay (Roche Diagnostics, Indiannapolis, IN) with a LLQ of 9 IU/mL. HBsAg has been tested since 2010 by the Roche Elecsys HBsAg II quantitative assay (Roche Diagnostics, Indiannapolis, IN). Serological HBV markers (HBsAg, anti-hbsag, HBeAg, anti-hbe and anti-hbc IgG and IgM) and anti-hdv IgG were all detected using standard laboratory techniques. Anti-HCV was determined by commercial EIA (HCV 3.0 Ortho Clinical Diagnostics, Amersham, Bucks, United Kingdom). DISCUSSION Nov 2011 Jun 2012 Jan 2013 Feb 2013 HBsAg HBV-DNA < 9 IU/mL ULN (ALT) ALT value Apr 2013 Jun 2013 Figure 2 HBsAg, hepatitis B virus-dna and alanine aminotransferase serum level assessment. A decrease of the starting HBsAg titre (3533 IU/mL) was observed 1 mo after the beginning of Peg-interferon α-2a (1.21 log; HBsAg titre 218 IU/mL) and 3 mo after the discontinuation of all drugs (1.88 log; HBsAg titre 47 IU/mL). Seven months later, the quantitative determination of HBsAg resulted negative. The patient showed steadily undetectable HBV-DNA (< 9 IU/mL) and normal ALT levels (< ULN). IU: International unit; ULN: Upper limit of normal. In February 2011, HBV-DNA was < 9 IU/mL, HBsAg titre was high (3,441 IU/mL) [16] and ALT was lower than ULN ( 0.6) (Figure 2). In October 2011, on the basis of these results and taking in consideration the young age of the patient (42 years), we decided to add PEG-IFN α-2a (180 µg/wk) for 6 mo with the intent of promoting HBsAg loss, also considering that the patient never underwent this kind of treatment. At this time HBV-DNA was < 9 IU/mL, HBsAg value 3533 IU/mL and ALT 0.4 ULN (Figure 2). PEG-IFN α-2a (180 μg/wk) was added to NAs for 3 mo, then NAs treatment was interrupted while PEG-IFN α-2a was continued for additional 3 mo. In November 2011, one month after the beginning of PEG-IFN α-2a, a decrease of HBsAg titre higher than one log was obtained (HBsAg 218 IU/mL, 1.21 log decrease). Moreover, three months after the discontinuation of the PEG-IFN α-2a therapy, a further decrease of HBsAg serum levels was observed (HBsAg 47 IU/mL, 1.88 log decrease), in presence of undetectable HBV-DNA (< 9 IU/mL) and normal ALT levels. Seven months later, the quantitative determination of HBsAg resulted negative. Later on, we performed 4 other HBsAg determinations (the last on June 2013), and all were negative, with HBV-DNA serum levels steadily undetectable (< 9 UI/ ml) and normal ALT (Figure 2). The advent of HBsAg titre determination has revived the possibility to start a course of Peg-interferon α-2a in HBV patients who never received interferon, considering that this prognostic parameter makes interferon treatment cost/effective and minimizes the possibility of adverse effects. The rates of HBsAg clearance reported in the literature refer to either NAs [1,5-8] or PEG-IFN α-2a [1,9,10] monotherapies or to the combination of these drugs [9,12]. On the other hand, only few data are available about the overlap of PEG-IFN α-2a in steadily HBV-DNA negative/hbsag positive patients, undergoing long-lasting NAs therapy, with the exception of the findings reported by Mangano et al [13] and Kittner et al [14], that present some limitations (lack of HBsAg titre and HBV genotype determination before the add-on therapy, or treatment of HBV genotype A, which is known to respond better to PEG-IFN α-2a [15], or treatment of a patient with a HBsAg titre as low as 16 IU/mL). In the present study, we propose a therapeutical approach that takes in consideration a new combination of nucleos(t)ide analogues therapy and PEG-IFN α-2a. In fact, we used for 3 mo nucleos(t)ide analogues in combination with Peg-interferon α-2a followed by 3 mo of Peg-interferon α-2a monotherapy. Moreover, even if our original intent was to promote HBsAg loss, this schedule gave us the possibility to evaluate if the treatment with Peg-interferon alone prevents sudden HBV reactivation with hepatitis flares, after interruption of nucleos(t)ide analogues therapy. Our results do not exclude the possibility that HBsAg clearance could be due to the beneficial effects of 5 years of therapy with analogues. However, a casual striking decrease of HBsAg after one month of PEG-IFN α-2a therapy seems unlikely. Finally, our findings need to be verified by a prospective trial on patients under long-lasting NAs therapy with clinical (young age, low stage of fibrosis) and virological (HBeAg negative, steadily undetectable HBV-DNA, genotype D and high HBsAg titre) characteristics similar to those observed in our patient, in order to verify the efficacy of PEG-IFN α-2a add-on treatment on HBsAg clearance. This therapeutic strategy would produce obvious clinical and pharmaco-economic advantages, especially if a HBsAg-guided PEG-IFN α-2a therapy is adopted. COMMENTS Clinical diagnosis Patient was a 44-year-old Caucasian male with chronic hepatitis B. Differential diagnosis According to the liver stiffness value assessed in February 2011 (4.3 kpa), his chronic hepatitis was characterized by mild fibrosis and not by moderate or severe fibrosis July 14, 2014 Volume 20 Issue 26

417 Barone M et al. HBsAg clearance by Peg-IFN plus analogues Laboratory diagnosis In October 2011, HBV-DNA was < 9 IU/mL (Roche TaqMan PCR Real Time assay), HBsAg value 3533 IU/mL (Roche Elecsys HBsAg II quantitative assay) and alanine aminotransferase 0.4 ULN. Imaging diagnosis Liver stiffness, determined by transient elastography in February 2011, was 4.3 kpa (IQR 0.6 kpa, SR 100%). Pathological diagnosis Liver biopsy was performed only in 2002 and showed a grade 2 stage 2 chronic hepatitis B (by METAVIR score system). Treatment Patient started lamivudine in December 2002, added adefovir in January 2006, because of a virological breakthrough to lamivudine, and, in October 2011, added Peg-interferon α-2a to nucleos(t)ide analogues for 3 mo, then nucleos(t)ide analogues were interrupted while Peg-interferon α-2a was continued for additional 3 mo. Related reports Only few data are available about the overlap of Peg-interferon α-2a to nucleos(t)ide analogues in steadily HBV-DNA negative/hbsag positive patients with the exception of the findings reported by Mangano et al and Kittner et al, that present some limitations (lack of HBsAg titre and HBV genotype determination before the add-on therapy, or treatment of HBV genotype A, which is known to respond better to Peg-interferon α-2a, or treatment of a patient with a HBsAg titre as low as 16 IU/mL). Experiences and lessons In the present case, the authors propose a new therapeutical approach to promote HBsAg clearance, based on the overlap of Peg-interferon α-2a in steadily HBV-DNA negative/hbsag positive patients undergoing long-lasting NAs therapy. Peer review This case report offers a new approach to promote HBsAg clearance in patients with CHB treated with NUCs for long-time. This interesting observation must be checked in a clinical trial. REFERENCES 1 European Association For The Study Of The Liver. EASL clinical practice guidelines: Management of chronic hepatitis B virus infection. J Hepatol 2012; 57: [PMID: DOI: /j.jhep ] 2 Stroffolini T, Gaeta GB, Mele A. AASLD Practice Guidelines on chronic hepatitis B and HBV infection in Italy. Hepatology 2007; 46: ; author reply 609 [PMID: ] 3 Moucari R, Korevaar A, Lada O, Martinot-Peignoux M, Boyer N, Mackiewicz V, Dauvergne A, Cardoso AC, Asselah T, Nicolas-Chanoine MH, Vidaud M, Valla D, Bedossa P, Marcellin P. High rates of HBsAg seroconversion in HBeAgpositive chronic hepatitis B patients responding to interferon: a long-term follow-up study. J Hepatol 2009; 50: [PMID: DOI: /j.jhep ] 4 Iloeje UH, Yang HI, Su J, Jen CL, You SL, Chen CJ. Predicting cirrhosis risk based on the level of circulating hepatitis B viral load. Gastroenterology 2006; 130: [PMID: ] 5 Idilman R, Cinar K, Seven G, Bozkus Y, Elhan A, Bozdayi M, Yurdaydin C, Bahar K. Hepatitis B surface antigen seroconversion is associated with favourable long-term clinical outcomes during lamivudine treatment in HBeAg-negative chronic hepatitis B patients. J Viral Hepat 2012; 19: [PMID: DOI: /j x] 6 Fasano M, Lampertico P, Marzano A, Di Marco V, Niro GA, Brancaccio G, Marengo A, Scotto G, Brunetto MR, Gaeta GB, Rizzetto M, Angarano G, Santantonio T. HBV DNA suppression and HBsAg clearance in HBeAg negative chronic hepatitis B patients on lamivudine therapy for over 5 years. J Hepatol 2012; 56: [PMID: DOI: / j.jhep ] 7 Hadziyannis SJ, Tassopoulos NC, Heathcote EJ, Chang TT, Kitis G, Rizzetto M, Marcellin P, Lim SG, Goodman Z, Ma J, Brosgart CL, Borroto-Esoda K, Arterburn S, Chuck SL. Longterm therapy with adefovir dipivoxil for HBeAg-negative chronic hepatitis B for up to 5 years. Gastroenterology 2006; 131: [PMID: ] 8 Ghany MG, Feld JJ, Zhao X, Heller T, Doo E, Rotman Y, Nagabhyru P, Koh C, Kleiner DE, Wright EC, Liang TJ, Hoofnagle JH. Randomised clinical trial: the benefit of combination therapy with adefovir and lamivudine for chronic hepatitis B. Aliment Pharmacol Ther 2012; Epub ahead of print [PMID: DOI: /j x] 9 Marcellin P, Bonino F, Lau GK, Farci P, Yurdaydin C, Piratvisuth T, Jin R, Gurel S, Lu ZM, Wu J, Popescu M, Hadziyannis S. Sustained response of hepatitis B e antigen-negative patients 3 years after treatment with peginterferon alpha-2a. Gastroenterology 2009; 136: e1-4 [PMID: DOI: /j.gastro ] 10 Marcellin P, Piratvisuth T, Brunetto M. Increasing rates of HBsAg clearance and seroconversion in patients with HBeAg-negative disease treated with peginterferon alfa-2a ± lamivudine: results of 5-year post-treatment follow up. J Hepatol 2009; 50: S Moucari R, Mackiewicz V, Lada O, Ripault MP, Castelnau C, Martinot-Peignoux M, Dauvergne A, Asselah T, Boyer N, Bedossa P, Valla D, Vidaud M, Nicolas-Chanoine MH, Marcellin P. Early serum HBsAg drop: a strong predictor of sustained virological response to pegylated interferon alfa-2a in HBeAg-negative patients. Hepatology 2009; 49: [PMID: DOI: /hep.22744] 12 Takkenberg RB, Jansen L, de Niet A, Zaaijer HL, Weegink CJ, Terpstra V, Dijkgraaf MG, Molenkamp R, Jansen PL, Koot M, Rijckborst V, Janssen HL, Beld MG, Reesink HW. Baseline hepatitis B surface antigen (HBsAg) as predictor of sustained HBsAg loss in chronic hepatitis B patients treated with pegylated interferon-α2a and adefovir. Antivir Ther 2013; 18: [PMID: DOI: /IMP2580] 13 Mangano C, Squadrito G, Cacciola I, Carpentieri M, Foti G, Raimondo G. Effectiveness of add-on pegylated interferon alfa-2a therapy in a lamivudine-treated patient with chronic hepatitis B. Ann Hepatol 2011; 10: [PMID: ] 14 Kittner JM, Sprinzl MF, Grambihler A, Weinmann A, Schattenberg JM, Galle PR, Schuchmann M. Adding pegylated interferon to a current nucleos(t)ide therapy leads to HBsAg seroconversion in a subgroup of patients with chronic hepatitis B. J Clin Virol 2012; 54: [PMID: DOI: /j.jcv ] 15 Buster EH, Hansen BE, Lau GK, Piratvisuth T, Zeuzem S, Steyerberg EW, Janssen HL. Factors that predict response of patients with hepatitis B e antigen-positive chronic hepatitis B to peginterferon-alfa. Gastroenterology 2009; 137: [PMID: DOI: /j.gastro ] 16 Brunetto MR, Oliveri F, Colombatto P, Moriconi F, Ciccorossi P, Coco B, Romagnoli V, Cherubini B, Moscato G, Maina AM, Cavallone D, Bonino F. Hepatitis B surface antigen serum levels help to distinguish active from inactive hepatitis B virus genotype D carriers. Gastroenterology 2010; 139: [PMID: DOI: /j.gastro ] P- Reviewers: Guo XZ, Karatapanis S S- Editor: Qi Y L- Editor: A E- Editor: Zhang DN 8725 July 14, 2014 Volume 20 Issue 26

418 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. CASE REPORT Perihepatic adhesions: An unusual complication of hemolysis, elevated liver enzymes and low platelet syndrome Margot M Koeneman, Ger H Koek, Marc Bemelmans, Louis L Peeters Margot M Koeneman, Louis L Peeters, Department of Obstetrics and Gynecology, Maastricht University Medical Center, 6202 AZ Maastricht, The Netherlands Ger H Koek, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Maastricht University Medical Center, 6202 AZ Maastricht, The Netherlands Marc Bemelmans, Department of Surgery, Maastricht University Medical Center, 6202 AZ Maastricht, The Netherlands Author contributions: Koek GH, Bemelmans M and Peeters LL were the treating physicians; Koeneman MM drafted the manuscript; Koek GH and Peeters LL reviewed the manuscript; all authors performed data collection and contributed to approve of the final version of the manuscript. Correspondence to: Margot M Koeneman, MD, Department of Obstetrics and Gynecology, Maastricht University Medical Center, Postbus 5800, 6202 AZ Maastricht, The Netherlands. m.koeneman@alumni.maastrichtuniversity.nl Telephone: Fax: Received: August 13, 2013 Revised: November 15, 2013 Accepted: January 6, 2014 Published online: July 14, 2014 Abstract We present a case of symptomatic perihepatic adhesions, which developed after a pregnancy complicated by hemolysis, elevated liver enzymes and low platelet (HELLP) syndrome, in which a subcapsular liver hematoma occurred. Our patient presented with complaints of persistent, severe right-sided upper abdominal pain. The complaints developed gradually, one year after a pregnancy that had been complicated by HELLP syndrome with a subcapsular liver hematoma. The hematoma had resolved spontaneously. An upperabdominal magnetic resonance imaging revealed a density between liver and diaphragm at the site of the former subcapsular hematoma, suspect of perihepatic adhesions. The presence of perihepatic adhesions was confirmed during a laparoscopy and treated by adhesiolysis in the same session. The adhesions may have developed in response to the degradation process of the subcapsular liver hematoma during conservative treatment. This case of perihepatic adhesions may therefor be the first presentation of a long term sequel of subcapsular liver hematoma in HELLP syndrome Baishideng Publishing Group Inc. All rights reserved. Key words: Hemolysis, elevated liver enzymes and low platelet syndrome; Liver; Haematoma; Long-term effects; Adhesions; Tissue Core tip: This case of perihepatic adhesions may be the first presentation of a long term sequel of subcapsular liver hematoma in hemolysis, elevated liver enzymes and low platelet syndrome, which occured in pregnancy. Koeneman MM, Koek GH, Bemelmans M, Peeters LL. Perihepatic adhesions: An unusual complication of hemolysis, elevated liver enzymes and low platelet syndrome. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: org/ /wjg.v20.i INTRODUCTION Subcapsular liver hematoma is a rare but severe complication of the hemolysis, elevated liver enzymes and low platelet (HELLP) syndrome in pregnancy, with a reported incidence of 0.9% in a series of 442 pregnancies complicated by HELLP syndrome [1]. To the best of our knowledge, there are no reports on long-term sequels of subcapsular liver hematoma after HELLP syndrome [2]. CASE REPORT A 29-year-old primipara presented with complaints of persistent, severe right -sided upper abdominal pain during deep inspiration, physical exercise and on local pres July 14, 2014 Volume 20 Issue 26

419 Koeneman MM et al. Perihepatic adhesions after HELLP syndrome solution) into the peritoneal cavity to prevent the formation of new adhesions. On follow-up until three years post-surgery, she was free of symptoms. Figure 1 T2 weighted coronal magnetic resonance imaging image of subcapsular liver hematoma. The arrows indicate a hyperdense area suggestive of the subcapsular liver hematoma. Diaphragm Figure 2 Laparoscopy image of perihepatic adhesions. The arrow indicates the location of the perihepatic adhesions to the diaphragm that were found during laparoscopy. sure. These complaints had developed gradually after giving birth one year earlier. This pregnancy had been complicated by HELLP syndrome with a subcapsular liver hematoma sized 20 cm 5 cm, which had resolved spontaneously. At current presentation, liver function tests were normal. She assured having never experienced signs suggestive of sexually transmitted diseases or pelvic inflammatory disease (PID). She had never undergone intraabdominal interventions. An upper-abdominal magnetic resonance imaging revealed a density between liver and diaphragm at the site of the former subcapsular hematoma, suspect of perihepatic adhesions (Figure 1). She was referred to the hepatobiliary surgeon, who performed a laparoscopy and confirmed the presence of a thick adhesion between liver segment Ⅴ/Ⅷ and the diaphragm (Figure 2). Adhesiolysis was performed in the same session by monopolar diathermia and sharp dissection, followed by the instillation of 1.5 L Adept (4% icodextrin DISCUSSION Clinical symptoms and imaging in our patient suggested the presence of perihepatic adhesions subsequently confirmed by laparoscopy. Adhesion formation is believed to be promoted by the inflammatory response to mesothelial injury, resulting from subcapsular liver parenchymal necrosis as part of the HELLP syndrome in our patient. Blood and other necrotic material are well-known triggers of inflammation [3]. Therefore, the formation of perihepatic adhesions in our patient may have been triggered by the degradation process of the resolving subcapsular liver hematoma. The aspect of the adhesion found in our patient differed clearly from that found in Fitz-Hugh- Curtis syndrome, occurring as a complication of PID. This case may well be the first report of a long-term hepatic sequel of a subcapsular liver hematoma complicating HELLP syndrome. Perihepatic adhesions must be suspected in patients with complaints of persistent rightsided upper abdominal pain after conservative treatment of a liver hematoma. Biochemical markers such as liver function tests are expected to be normal in these patients and are therefore not of diagnostic value, however imaging studies can be suggestive of the presence of perihepatic adhesions. Key in confirming the diagnosis of perihepatic adhesions seems to be laparoscopy, which also enables immediate adhesiolysis as an effective therapeutic modality. COMMENTS Case characteristics The authors present a case of symptomatic perihepatic adhesions, which developed after a pregnancy complicated by hemolysis, elevated liver enzymes and low platelet (HELLP) syndrome in which a subcapsular liver hematoma occurred. Clinical diagnosis The patient presented with complaints of persistent, severe right-sided upper abdominal pain. Differential diagnosis The complaints developed gradually, one year after a pregnancy that had been complicated by HELLP syndrome with a subcapsular liver hematoma. Imaging diagnosis An upper-abdominal magnetic resonance imaging revealed a density between liver and diaphragm at the site of the former subcapsular hematoma, suspect of perihepatic adhesions. Treatment Adhesiolysis was performed in the same session by monopolar diathermia and sharp dissection, followed by the instillation of 1.5 L Adept (4% icodextrin solution) into the peritoneal cavity to prevent the formation of new adhesions. Experiences and lessons Key in confirming the diagnosis of perihepatic adhesions seems to be laparoscopy, which also enables immediate adhesiolysis as an effective therapeutic modality. Peer review The authors describes for the first time a long-term hepatic sequel of a subcapsular liver hematoma complicating HELLP syndrome. It is suitable for publication as a case report observation July 14, 2014 Volume 20 Issue 26

420 Koeneman MM et al. Perihepatic adhesions after HELLP syndrome REFERENCES 1 Sibai BM, Ramadan MK, Usta I, Salama M, Mercer BM, Friedman SA. Maternal morbidity and mortality in 442 pregnancies with hemolysis, elevated liver enzymes, and low platelets (HELLP syndrome) Am J Obstet Gynecol 1993; 169: [PMID: DOI: / (93) I] 2 van Pampus MG, Aarnoudse JG. Long-term outcomes after preeclampsia. Clin Obstet Gynecol 2005; 48: [PMID: DOI: /01.grf d] 3 Lauder CI, Garcea G, Strickland A, Maddern GJ. Abdominal adhesion prevention: still a sticky subject? Dig Surg 2010; 27: [PMID: DOI: / ] P- Reviewers: Ni Y, Resende R S- Editor: Zhai HH L- Editor: A E- Editor: Wang CH 8728 July 14, 2014 Volume 20 Issue 26

421 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. CASE REPORT Two-stage treatment with hepatectomy and carbon-ion radiotherapy for multiple hepatic epithelioid hemangioendotheliomas Shohei Komatsu, Takeshi Iwasaki, Yusuke Demizu, Kazuki Terashima, Osamu Fujii, Atsushi Takebe, Akihiro Toyokawa, Kazuhiro Teramura, Takumi Fukumoto, Yonson Ku, Nobukazu Fuwa Shohei Komatsu, Atsushi Takebe, Takumi Fukumoto, Yonson Ku, Division of Hepato-Biliary-Pancreatic Surgery, Department of Surgery, Kobe University Graduate School of Medicine, Hyogo , Japan Takeshi Iwasaki, Department of Surgery, National Hospital Organization, Kobe Medical Center, Hyogo , Japan Yusuke Demizu, Kazuki Terashima, Osamu Fujii, Nobukazu Fuwa, Department of Radiology, Hyogo Ion Beam Medical Center, Hyogo , Japan Akihiro Toyokawa, Department of Surgery, Yodogawa Christian Hospital, Osaka , Japan Kazuhiro Teramura, Department of Pathology, Yodogawa Christian Hospital, Osaka , Japan Author contributions: Komatsu S wrote the manuscript and performed additional data analysis; Iwasaki T helped by supervising and approving the final manuscript; Demizu Y, Terashima K, Fujii O and Fuwa N performed particle radiotherapy; Takebe A, Toyokawa A were attending doctors of the patient; Teramura K made a pathological diagnosis; Fukumoto T and Ku Y developed a new treatment procedure and supervised the manuscript. Correspondence to: Shohei Komatsu, MD, PhD, Division of Hepato-Biliary-Pancreatic Surgery, Department of Surgery, Kobe University Graduate School of Medicine, Kusunoki-cho, Chuo-ku, Hyogo , Japan. komasho8@gmail.com Telephone: Fax: Received: November 19, 2013 Revised: February 10, 2014 Accepted: March 7, 2014 Published online: July 14, 2014 Abstract Hepatic epithelioid hemangioendothelioma (HEH) is a rare neoplasm of vascular origin with variable malignant potential. Because most patients with this condition have multiple bilobar lesions, liver transplantation is the standard treatment, and hepatectomy is much less frequently indicated. We describe a case of a 35-yearold woman with unresectable multiple bilobar HEH successfully treated by combination treatment with hepatectomy and carbon-ion radiotherapy. This case is very meaningful since it demonstrated the effectiveness of carbon-ion radiotherapy for HEH and the possibility of expanding the curative treatment options for multiple bilobar hepatic tumors Baishideng Publishing Group Inc. All rights reserved. Key words: Two-stage treatment; Hepatic epithelioid hemangioendothelioma; Hepatectomy; Carbon-ion radiotherapy; Particle radiotherapy; Omental flap spacer Core tip: Hepatic epithelioid hemangioendothelioma (HEH) is a rare neoplasm with variable malignant potential. Liver transplantation is the most frequently reported treatment because most patients have multifocal bilobar lesions. Here, we describe a case of a 35-yearold woman with unresectable multiple bilobar HEH that was successfully treated by combination treatment of hepatectomy and carbon-ion radiotherapy. This case demonstrated two significant points: the effectiveness of carbon-ion radiotherapy for HEH and the possibility of expanding the curative treatment options for multiple bilobar hepatic tumors. Komatsu S, Iwasaki T, Demizu Y, Terashima K, Fujii O, Takebe A, Toyokawa A, Teramura K, Fukumoto T, Ku Y, Fuwa N. Twostage treatment with hepatectomy and carbon-ion radiotherapy for multiple hepatic epithelioid hemangioendotheliomas. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: INTRODUCTION Hepatic epithelioid hemangioendothelioma (HEH) is 8729 July 14, 2014 Volume 20 Issue 26

422 Komatsu S et al. Two-stage treatment for HEH A B C D Figure 1 Abdominal computed tomography revealed a total of 7 lesions located in both lobes. A: The tumor of segment 4 (short arrow) hinders the possibility of right hemihepatectomy, because it invades the roots of the middle and left hepatic veins (arrowhead indicates the tumor of segment 3); B-D: The 3 deep-seated tumors in the right lobe (long arrows) hinder the possibility of left hemihepatectomy. a rare vascular tumor characterized by the presence of epithelioid endothelial cells [1,2], which presents varying clinical behaviors reflecting borderline malignancy to aggressive disease. Although several nonsurgical modalities such as chemotherapy, radiotherapy, interferon treatment, antiangiogenic chemotherapy, or transcatheter arterial chemoembolization (TACE) has been explored [3-5], surgical treatments by liver transplantation (LT) or hepatectomy remains the mainstay of treatment for patients with HEH [1]. Here, we report a case of unresectable multiple bilobar HEH that showed complete remission after combination treatment with hepatectomy and carbon-ion radiotherapy. To our knowledge, this is the first case report describing the application of particle radiotherapy in the treatment of this rare hepatic malignant tumor. CASE REPORT A 35-year-old female patient was admitted to our department for the surgical treatment of multiple bilobar liver tumors. Four years previously, a routine medical examination had revealed multiple liver tumors, the largest measuring 36 mm in diameter. A histological diagnosis of HEH had been made by fine needle tumor biopsy. Because she had been in the late stage of pregnancy and chest computed tomography (CT) had shown the presence of multiple small nodules in both lungs, the patient was closely followed up by CT every 4 mo. After a normal delivery, both liver and lung tumors did not show any obvious signs of growth until CT showed enlargement of the liver tumor (segment 6) from 36 to 43 mm in diameter. Laboratory data did not indicate any abnormality; the patient was negative for hepatitis B and C virus infection and the serum levels of tumor markers, including α-fetoprotein, prothrombin induced by vitamin K absence or antagonist Ⅱ, carcinoembryonic antigen, carbohydrate antigen 19-9, and carbohydrate antigen 125, were within normal ranges. The patient had no remarkable medical, surgical, or family history. Abdominal CT showed a total of seven tumors in both lobes, suggestive of an unresectable disease stage (Figure 1). LT was not indicated because of the unavailability of a living donor candidate and a cadaveric donor was unavailable as per the donor organ allocation policy of the Japan Society for Transplantation and organ shortage in Japan. Accordingly, we opted for a treatment strategy that involved right hemihepatectomy and partial resection of the left lateral segment followed by particle radiotherapy to the residual and solitary tumor in segment 4. Furthermore, the residual tumor was predicted to be close to the transection surface to which the colon would adhere and would hamper postoperative radiotherapy. Therefore, we planned to cover the cut surface using an omental flap to ensure spacing between the tumor and bowels. At laparotomy, multiple well-delineated white nodules were seen on the liver surface (Figure 2A). The tumor of segment 6 adhered firmly to Gerota s fascia (Figure 2B), indicating its invasive nature. We performed right lobectomy with excision of the infiltrated portion of Gerota s fascia as well as partial hepatectomy of segment 3 (Figure 8730 July 14, 2014 Volume 20 Issue 26

423 Komatsu S et al. Two-stage treatment for HEH A B C Tumor of Segment 4 D Round ligament MHV Cut end of RHV Greater omentum Figure 2 Intraoperative images. A: The tumors were well-circumscribed, whitish, and firm; B: The tumor of segment 6 invaded Gerota s fascia; C: After right lobectomy, the tumor of segment 4 was very close to the cut surface of the liver; D: A flap of the greater omentum (arrows) was sutured over the cut surface of the liver to cover the tumor of segment 4. MHV: Middle hepatic vein; RHV: Right hepatic vein. 2C) and then placed the omental flap on the cut surface of the liver (Figure 2D) for spacing. Moreover, to prevent postoperative irradiation to the pericardium, the round ligament was placed below the diaphragm (Figure 2D). Histopathological examination revealed atypical cells containing many vacuolated cells and dendritic cells, and the neoplastic cells were positive for vimentin, factor Ⅷ-related antigen, CD31, and CD34, leading to the establishment of a definite diagnosis of HEH (Figure 3). Abdominal CT after the operation showed that the omental flap was maintaining sufficient space between the tumor and the gastrointestinal tract (Figure 4A). The postoperative course was uneventful, and the patient was discharged on postoperative day 12. One month after the hepatectomy, carbon-ion radiotherapy of 76 GyE (gray equivalents) was administered in 20 fractions over 30 d (Figure 4B). No acute or late treatment-related toxicities (grade 2) were observed, and the liver function test results were normal. Follow-up imaging studies showed gradual regression of the tumor until 2 years after irradiation, and no enlargement or vascularity was observed thereafter, indicating complete local control (Figure 4C-E). Chest CT showed no changes in the small lung nodules. The patient remains free of recurrence almost 4 years after combination treatment. DISCUSSION Surgical intervention has been accepted as first-line treatment, and several studies have suggested that even extrahepatic disease is not a contraindication for surgical treatment [1,6,7]. However, a clear indication for either LT or hepatectomy has yet to be determined. Orthotopic LT has been most frequently indicated in the Western world with good outcomes [8] because > 80% of patients have multiple bilobar lesions at the time of diagnosis [1]. Lerut et al [6] reviewed the cases of 59 patients from the European Transplant Registry and reported 5- and 10- year survival rates of 83% and 72%, respectively, with disease-free survival rates of 82% and 64%, respectively. The United Network for Organ Sharing Registry reported a 5-year survival rate of 64% among 110 patients who underwent LT [9]. In contrast, hepatectomy has been indicated in only a small proportion of patients with limited unilobar disease [1,10]. Mehrabi et al [1] pooled 434 cases from the literature and reported that hepatectomy was performed in only 9.4% (22 patients) of these cases, whereas LT was performed in 45% (110 patients). Nevertheless, it is noteworthy that cases for which hepatectomy was performed with curative intent showed the best 5-year survival rate of 75% compared to 54.5% for cases of LT and 30% for cases of chemotherapy and/or radiation. In this regard, Grotz et al [7] from the Mayo Clinic recently reported comparable results with hepatectomy (11 patients) and LT (11 patients) and suggested that hepatectomy was appropriate for patients with resectable disease, even for those with bilobar lesions by parenchyma-saving resection (partial hepatectomy). They also analyzed the favorable predictive factors such as the largest tumor diameter of 10 cm and multifocal disease with 10 nodules. In any case, 8731 July 14, 2014 Volume 20 Issue 26

424 Komatsu S et al. Two-stage treatment for HEH A B C D E Figure 3 Pathological findings. A: The specimen was composed of many atypical vacuolatd and dendritic cells; B-E: The tumor cells were immunoreactive for vimentin (B), factor VIII-related antigen (C), CD31 (D), and CD34 (E). tissue toxicities. None of the other nonsurgical therapeutic modalities has been widely accepted since there is little evidence of their efficacy, because of the rarity of this disease. However, TACE was recently recommended by a few researchers. Wang et al [13] reported comparable results with hepatectomy (17 patients) and TACE (12 patients), with 3-year survival rates of 74.1% and 81.6%, respectively. Cardinal et al [5] reported better results with TACE in patients with metastatic diseases than with LT or hepatectomy in a retrospective study including 25 patients, even though the TACE arm included only four patients. There are even patients in the spontaneous course alive after 5 years without treatment [2,14], indicating the borderline malignant nature of the disease resembling neuroendocrine tumors where patients even with metastases maintains stable disease for years. In fact, our paour patient had unresectable disease because the presence of the tumor in segment 4 and the central location of multiple tumors in the right lobe hindered parenchymasaving resection and negatively affected the possibility of curative resection by either right or left hemihepatectomy, respectively (Figure 1). Although LT would have been indicated, it is not feasible in Japan unless a living donor is available. A combination of left hemihepatectomy and radiofrequency ablation was a possible alternative approach but was not suitable in our case. Radiofrequency ablation was relatively contraindicated because of the tumor sizes and the perivascular location, which has been reported to increase the local recurrence rate due to the heat-sink effect [11,12]. Conventional radiotherapy is not usually indicated for multiple lesions because of excessive normal liver 8732 July 14, 2014 Volume 20 Issue 26

425 Komatsu S et al. Two-stage treatment for HEH A C D E B Figure 4 Treatment course. A: Post-operative abdominal computed tomography (CT) showed the greater omental flap (arrows) around the tumor, maintaining a sufficient space between the tumor and the gastrointestinal tract; B: Treatment plan for carbon-ion radiotherapy with a total dose of 76 GyE in 20 fractions. Isodose lines demonstrate 100% of the prescribed dose at the center and decreasing by 10% of the dose from the inside to the outside. Owing to the greater omental flap, the tumor was entirely irradiated with full dose, and the gastrointestinal tract was successfully spared; C: Comparison of abdominal CT and magnetic resonance imaging scans taken over a period of 24 mo after carbon-ion radiotherapy indicated shrinkage of the tumor with decreasing tumor vascularity. The maximum tumor diameter was 35 mm before treatment, and tumor size decreased thereafter to 27 mm after 6 mo; D: Tumor size of 26 mm after 12 mo; E: Tumor size of 24 mm after 24 mo. tient has been stable for at least 4 years. However, further investigations are needed to clarify whether such nonsurgical interventions indeed influence the patients natural history. Nonetheless, since the biological and clinical behaviors of HEH are varied and unpredictable, no clinical or pathological prognostic factors have yet been identified. Thus, we opted for the more curative treatment option, and consequently chose to administer particle radiotherapy to the single residual lesion in segment 4 following right hemihepatectomy and partial hepatectomy. Particle radiotherapy using protons or carbon-ions is a new modality that elicits an inherent antitumor effect against various malignant tumors [15]. Owing to the defined range of protons or carbon-ions as exhibited by the Bragg peak, they allow reduction of the irradiated volume and the dose administered to the normal tissues [15,16]. Hepatocellular carcinoma (HCC) is one of the malignancies most suitable for treatment with particle radiotherapy. We recently reported good clinical treatment results with particle radiotherapy for HCC with a local control rate of approximately 90%, indicating that particle radiotherapy may be a useful alternative to conventional local therapies [17,18]. Further, due to our abundant experience with particle radiotherapy for HCC [17], we believe that HEH could be controlled as long as a sufficient dose is administered. Particle radiotherapy is generally not recommended for tumors that are adjacent to the gastrointestinal tract because the dose tolerated by the intestine is extremely low [19,20]. Unlike conventional photon radiotherapy, a distance of only 5-10 mm between the tumor and the gastrointestinal tract is sufficient for safe and curative irradiation with particle radiotherapy. In our case, we avoided potential bowel adherence to the target lesions using an omental flap as a spacer intraoperatively to facilitate irradiation after hepatectomy. We recently reported the effectiveness of a new two-step treatment with surgical prosthetic spacer placement and subsequent particle radiotherapy for HCC or pelvic malignancies [21,22]. These spacing techniques provide new strategies for radical treatment with radiotherapy. In summary, this case report described two new im July 14, 2014 Volume 20 Issue 26

426 Komatsu S et al. Two-stage treatment for HEH portant findings: the effectiveness of carbon-ion radiotherapy for HEH and the efficacy and safety of a new strategy for managing multiple bilobar hepatic tumors by combining hepatectomy and particle radiotherapy. These findings provide a new treatment option for HEH and multiple bilobar hepatic tumors. COMMENTS Case characteristics A 35-year-old female patient presented with no sign of subjective symptoms. Clinical diagnosis The patient was diagnosed as multiple bilobar hepatic epithelioid hemangioendotheliomas. Differential diagnosis Hepatocellular carcinoma, metastatic liver tumor. Laboratory diagnosis The patient was negative for hepatitis B and C virus infection and the serum levels of tumor markers, including α-fetoprotein, prothrombin induced by vitamin K absence or antagonist II, carcinoembryonic antigen, carbohydrate antigen 19-9, and carbohydrate antigen 125, were within normal ranges. Imaging diagnosis Abdominal CT showed a total of seven liver tumors in both lobes, and the maximal tumor size was 43 mm in diameter. Pathological diagnosis Histopathological examination revealed atypical cells containing many vacuolated cells and dendritic cells, and neoplastic cells were positive for vimentin, factor Ⅷ-related antigen, CD31, and CD34, resulting in the definite diagnosis of hepatic epithelioid hemangioendothelioma. Treatment The patient was treated with right hemihepatectomy and partial resection of the left lateral segment followed by particle radiotherapy to the residual and solitary tumor in segment 4. Related reports Hepatic epithelioid hemangioendothelioma is a rare vascular tumor that occurs exclusively in adults, but there have been few reports describing effective alternative treatment option except for liver transplantation or hepatectomy because of its rarity. Term explanation Particle radiotherapy, such as proton or carbon-ions, is a new modality that allows reduction of the irradiated volume and the dose administered to the normal tissues owing to the excellent dose distribution. Experiences and lessons This case report provides a new treatment option for hepatic epithelioid hemangioendothelioma and multiple bilobar hepatic tumors. Peer review Although this is only a single case report that lack convincing evidence of benefit, our treatment strategy may have a possibility to expand the treatment options for hepatic epithelioid hemangioendothelioma and multiple bilobar hepatic tumors. REFERENCES 1 Mehrabi A, Kashfi A, Fonouni H, Schemmer P, Schmied BM, Hallscheidt P, Schirmacher P, Weitz J, Friess H, Buchler MW, Schmidt J. Primary malignant hepatic epithelioid hemangioendothelioma: a comprehensive review of the literature with emphasis on the surgical therapy. Cancer 2006; 107: [PMID: DOI: /cncr.22225] 2 Makhlouf HR, Ishak KG, Goodman ZD. Epithelioid hemangioendothelioma of the liver: a clinicopathologic study of 137 cases. Cancer 1999; 85: [PMID: ] 3 Bocci G, Nicolaou KC, Kerbel RS. Protracted low-dose effects on human endothelial cell proliferation and survival in vitro reveal a selective antiangiogenic window for various chemotherapeutic drugs. Cancer Res 2002; 62: [PMID: ] 4 Galvão FH, Bakonyi-Neto A, Machado MA, Farias AQ, Mello ES, Diz ME, Machado MC. Interferon alpha-2b and liver resection to treat multifocal hepatic epithelioid hemangioendothelioma: a relevant approach to avoid liver transplantation. Transplant Proc 2005; 37: [PMID: DOI: /j.transproceed ] 5 Cardinal J, de Vera ME, Marsh JW, Steel JL, Geller DA, Fontes P, Nalesnik M, Gamblin TC. Treatment of hepatic epithelioid hemangioendothelioma: a single-institution experience with 25 cases. Arch Surg 2009; 144: [PMID: DOI: /archsurg ] 6 Lerut JP, Orlando G, Adam R, Schiavo M, Klempnauer J, Mirza D, Boleslawski E, Burroughs A, Sellés CF, Jaeck D, Pfitzmann R, Salizzoni M, Söderdahl G, Steininger R, Wettergren A, Mazzaferro V, Le Treut YP, Karam V. The place of liver transplantation in the treatment of hepatic epitheloid hemangioendothelioma: report of the European liver transplant registry. Ann Surg 2007; 246: ; discussion 957 [PMID: DOI: /SLA.0b013e31815c2a70] 7 Grotz TE, Nagorney D, Donohue J, Que F, Kendrick M, Farnell M, Harmsen S, Mulligan D, Nguyen J, Rosen C, Reid-Lombardo KM. Hepatic epithelioid haemangioendothelioma: is transplantation the only treatment option? HPB (Oxford) 2010; 12: [PMID: DOI: / j x] 8 Agrawal N, Parajuli S, Zhao P, Satoskar R, Laurin J, Azumi N, Matsumoto C, Shetty K. Liver transplantation in the management of hepatic epithelioid hemangioendothelioma: a single-center experience and review of the literature. Transplant Proc 2011; 43: [PMID: DOI: /j.transproceed ] 9 Rodriguez JA, Becker NS, O Mahony CA, Goss JA, Aloia TA. Long-term outcomes following liver transplantation for hepatic hemangioendothelioma: the UNOS experience from 1987 to J Gastrointest Surg 2008; 12: [PMID: DOI: /s ] 10 Mosoia L, Mabrut JY, Adham M, Boillot O, Ducerf C, Partensky C, Baulieux J. Hepatic epithelioid hemangioendothelioma: long-term results of surgical management. J Surg Oncol 2008; 98: [PMID: DOI: / jso.21132] 11 Mulier S, Ni Y, Jamart J, Ruers T, Marchal G, Michel L. Local recurrence after hepatic radiofrequency coagulation: multivariate meta-analysis and review of contributing factors. Ann Surg 2005; 242: [PMID: ] 12 Berber E, Siperstein A. Local recurrence after laparoscopic radiofrequency ablation of liver tumors: an analysis of 1032 tumors. Ann Surg Oncol 2008; 15: [PMID: DOI: /s ] 13 Wang LR, Zhou JM, Zhao YM, He HW, Chai ZT, Wang M, Ji Y, Chen Y, Liu C, Sun HC, Wu WZ, Ye QH, Zhou J, Fan J, Tang ZY, Wang L. Clinical experience with primary hepatic epithelioid hemangioendothelioma: retrospective study of 33 patients. World J Surg 2012; 36: [PMID: DOI: /s x] 14 Otrock ZK, Al-Kutoubi A, Kattar MM, Zaatari G, Soweid A. Spontaneous complete regression of hepatic epithelioid haemangioendothelioma. Lancet Oncol 2006; 7: [PMID: DOI: /S (06) ] 15 Schulz-Ertner D, Tsujii H. Particle radiation therapy using proton and heavier ion beams. J Clin Oncol 2007; 25: [PMID: DOI: /JCO ] 16 Brada M, Pijls-Johannesma M, De Ruysscher D. Proton therapy in clinical practice: current clinical evidence. J Clin Oncol 2007; 25: [PMID: DOI: / JCO ] 17 Komatsu S, Fukumoto T, Demizu Y, Miyawaki D, Terashima K, Sasaki R, Hori Y, Hishikawa Y, Ku Y, Murakami 8734 July 14, 2014 Volume 20 Issue 26

427 Komatsu S et al. Two-stage treatment for HEH M. Clinical results and risk factors of proton and carbon ion therapy for hepatocellular carcinoma. Cancer 2011; 117: [PMID: DOI: /cncr.26134] 18 Komatsu S, Murakami M, Fukumoto T, Hori Y, Hishikawa Y, Ku Y. Risk factors for survival and local recurrence after particle radiotherapy for single small hepatocellular carcinoma. Br J Surg 2011; 98: [PMID: DOI: / bjs.7397] 19 Ishikawa H, Tsuji H, Kamada T, Hirasawa N, Yanagi T, Mizoe JE, Akakura K, Suzuki H, Shimazaki J, Tsujii H. Risk factors of late rectal bleeding after carbon ion therapy for prostate cancer. Int J Radiat Oncol Biol Phys 2006; 66: [PMID: DOI: /j.ijrobp ] 20 Kawashima M, Furuse J, Nishio T, Konishi M, Ishii H, Kinoshita T, Nagase M, Nihei K, Ogino T. Phase II study of radiotherapy employing proton beam for hepatocellular carcinoma. J Clin Oncol 2005; 23: [PMID: DOI: /JCO ] 21 Fukumoto T, Komatsu S, Hori Y, Murakami M, Hishikawa Y, Ku Y. Particle beam radiotherapy with a surgical spacer placement for advanced abdominal leiomyosarcoma results in a significant clinical benefit. J Surg Oncol 2010; 101: [PMID: DOI: /jso.21417] 22 Komatsu S, Hori Y, Fukumoto T, Murakami M, Hishikawa Y, Ku Y. Surgical spacer placement and proton radiotherapy for unresectable hepatocellular carcinoma. World J Gastroenterol 2010; 16: [PMID: DOI: /wjg. v16.i ] P- Reviewers: Akyuz U, Fernandez-Pineda I, Kapischke M S- Editor: Qi Y L- Editor: A E- Editor: Wang CH 8735 July 14, 2014 Volume 20 Issue 26

428 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. CASE REPORT Tubulopapillary adenoma of the gallbladder accompanied by bile duct tumor thrombus Kentaroh Yamamoto, Fumio Yamamoto, Atsuhiro Maeda, Hirotsune Igimi, Mami Yamamoto, Ryosuke Yamaguchi, Yuichi Yamashita Kentaroh Yamamoto, Fumio Yamamoto, Hirotsune Igimi, Mami Yamamoto, Department of Surgery, Yamamoto Memorial Hospital, Saga , Japan Atsuhiro Maeda, Department of Internal Medicine, Maeda Hospital, Saga , Japan Ryosuke Yamaguchi, Yuichi Yamashita, Department of Gastroenterological Surgery, Fukuoka University School of Medicine, Fukuoka , Japan Author contributions: Yamamoto K, Yamamoto F and Yamashita Y designed the report; Yamamoto K, Yamamoto M and Igimi H performed the genetic analyses; Yamamoto K, Maeda A and Yamaguchi R collected the patient s clinical data; Yamamoto K analyzed the data and wrote the paper. Supported by Yamamoto Memorial Hospital, Imari City, Saga, Japan Correspondence to: Kentaroh Yamamoto, MD, PhD, Department of Surgery, Yamamoto Memorial Hospital, Hachiyakarami 88-4, Niri-cho, Imari, Saga , Japan. kentaroh-y@kenjin-kai.com Telephone: Fax: Received: January 7, 2014 Revised: January 31, 2014 Accepted: March 6, 2014 Published online: July 14, Baishideng Publishing Group Inc. All rights reserved. Key words: Intraductal papillary mucinous neoplasm of the bile duct; Tumor thrombi Core tip: Intraductal papillary mucinous neoplasm of the bile duct (IPNB) has relatively recently been recognized as a separate disease entity with an unclear pathogenesis. We present a case of IPNB developing from the gallbladder accompanied by a bile duct tumor thrombus in a 79-year-old female. Although this is not a malignant lesion, it has the potential to mimic a malignant lesion, and hence needs aggressive treatment. Yamamoto K, Yamamoto F, Maeda A, Igimi H, Yamamoto M, Yamaguchi R, Yamashita Y. Tubulopapillary adenoma of the gallbladder accompanied by bile duct tumor thrombus. World J Gastroenterol 2014; 20(26): Available from: URL: DOI: Abstract Intraductal papillary mucinous neoplasm of the bile duct (IPNB) is recognized as a precancerous lesion; however, both its pathogenesis and progression remain unclear. We present here a case of IPNB arising from the gallbladder accompanied by bile duct tumor thrombus in a 79-year-old female. The resected specimen revealed a tubulopapillary adenoma with no malignant cells. This case suggests that even in the absence of malignant cells, these tumors can behave as malignant tumors requiring aggressive treatment. Even if no malignant cells are present, intraepithelial neoplasms occurring in the ampullopancreatobiliary tract can behave as malignant tumors. INTRODUCTION Intraductal papillary mucinous neoplasm (IPMN) of the bile duct (IPNB) is a disease entity that was proposed in 2001 by Chen et al [1]. IPNB is regarded as a counterpart of IPMN of the pancreas, and is considered to be a precancerous lesion [2-5]. A similar spectrum of lesions also exists in the gallbladder [6]. However, these lesions have yet to be fully characterized and their pathogenesis and progression remain unclear. Most reported cases of invasive IPNB ultimately become cancerous. Generally, the tumors accompanied by a bile duct tumor thrombus are cancerous. Here we report a rare gallbladder tumor without cancerous changes accompanied by a bile duct tumor thrombus July 14, 2014 Volume 20 Issue 26

429 Yamamoto K et al. Adenoma of the gallbladder A B C D Figure 1 A 79-year-old female presented to our hospital for an incidentally-diagnosed gallbladder tumor. A: Coronal contrast-enhanced computed tomography (CT) image. The white arrow points to the gallbladder tumor with bile duct tumor thrombus; B: Coronal drip infusion cholangiographic CT image. The white arrow indicates the gallbladder tumor with bile duct tumor thrombus; C: Endoscopic retrograde cholangiopancreatography image. The white arrow indicates the defect due to the bile duct tumor thrombus; D: Magnetic resonance cholangiopancreatography image. The white arrow indicates the defect due to the bile duct tumor thrombus. Common bile duct Figure 2 The resected specimen. Dissection of the common bile duct demonstrated a mucin-producing tumor thrombus. CASE REPORT A 79-year-old female presented to our hospital for an incidentally-diagnosed gallbladder tumor. No abnormalities were seen in blood test results, including tumor markers. Laboratory data (normal range) were as follows: aspartate aminotransferase, 18 U/L (5-35 U/L); alanine aminotransferase, 14 U/L (5-30 U/L); alkaline phosphatase, 151 U/L ( U/L); gamma-glutamyl transferase, 20 U/mL (0-50 U/mL); lactate dehydrogenase, 170 U/L ( U/L); albumin, 4.0 g/dl ( g/dl); and total bilirubin, 0.64 g/dl ( g/dl). The concentrations of carcinoembryonic antigen and carbohydrate antigen 19-9 were 2.2 ng/ml (< 5.0 ng/ml) and 4.0 U/mL (< 37.0 U/mL), respectively. No serological evidence of hepatitis B or C was seen. Contrast enhanced computed tomography (CT) revealed a tumor measuring 40 mm in diameter in the cystic duct, accompanied by a common bile duct tumor thrombus (Figure 1A). Drip infusion cholangiographic CT revealed a defect in the common bile duct (Figure 1B). Magnetic resonance cholangiopancreatography and endoscopic retrograde cholangiopancreatography revealed similar findings (Figure 1C and D). Cytological examination revealed the absence of malignant cells in bile. Without the evidence of malignant cells, we diagnosed it as gallbladder cancer or bile duct cancer because of the common bile duct tumor thrombus. The patient underwent choledochectomy and cholecystectomy. Macroscopic examination of the resected specimen revealed a 40-mm tumor located in the neck of the gallbladder and a 30-mm tumor thrombus in the common bile, with rich mucilage (Figure 2). Microscopically, hematoxylin and eosin staining demonstrated the tumor to be a pyloric type tubulopapillary adenoma with moderate epithelial atypia, without evidence of stromal invasion (Figure 3). On immunological staining, the tumor cells were positive for MUC5AC, but negative for 8737 July 14, 2014 Volume 20 Issue 26

430 Yamamoto K et al. Adenoma of the gallbladder A B Figure 3 Pathological examination of the tumor. A: Hematoxylin and eosin staining demonstrated the tumor to be a tubulopapillary adenoma with moderate epithelial atypia ( 400); B: MUC5AC staining showing positive expression ( 600). MUC1 and CK20. DISCUSSION Recently, intraepithelial neoplasms occurring in the ampullopancreatobiliary tract have attracted a substantial amount of attention. These include the so-called IPNB, IPMN of the pancreas, intraductal tubulopapillary neoplasms (ITPN) of the pancreas, intra-ampullary-tubular neoplasms (IAPN) and intracystic papillary neoplasms (ICPN) of the gallbladder [1-6]. IPNB, IPMN, and ITPN are recognized by the World Health Organization [7]. IPNB, on the other hand, is a relatively recently proposed (in 2001) disease entity [1] which has been shown to be histologically similar to IPMN [2-5]. A similar spectrum of lesions also exists in IAPN and ICPN; however, these have not been fully characterized [6]. IPNBs are histologically classified as low- or intermediate-grade intraepithelial neoplasia corresponding to adenomas or borderline malignancy, high grade intraepithelial neoplasia corresponding to carcinoma in situ, or as having an associated invasive carcinoma [5,6]. The neoplasms are regarded as precancerous lesions; therefore, radical resection is recommended in operable patients [4,5,8,9]. In our case, although the epithelial atypia was moderate, it was accompanied by a bile duct tumor thrombus. The neoplasms accompanied with tumor thrombus are often ordinary invasive carcinoma [10,11]. What is curious is that no symptoms of biliary tract obstruction were observed in our patient, although we cannot rule out the possibility that such symptoms could have occurred in the immediate future. Furthermore, it is reported that pyloric type adenocarcinoma of the gallbladder has a poor prognosis [12]. So, in our case, surgical treatment is considered reasonable and proper. Even if no malignant cells are present, intraepithelial neoplasms occurring in the ampullopancreatobiliary tract can behave as malignant tumors. Hence, these patients should be treated aggressively. COMMENTS Case characteristics A 79-year-old female with an incidentally-diagnosed gallbladder tumor accompanied by bile duct tumor thrombus. Clinical diagnosis The patient was diagnosed with gallbladder carcinoma by the imaging study. Differential diagnosis Differential diagnoses were bile duct carcinoma invaded to the gallbladder, malignant lymphoma and intraductal papillary mucinous neoplasm of the bile duct. Laboratory diagnosis All of the laboratory tests were within normal limits. Imaging diagnosis Computed tomography revealed a tumor measuring 40 mm in diameter in the cystic duct, accompanied by a common bile duct tumor thrombus. Drip infusion cholangiographic-computed tomography, magnetic resonance imaging, endoscopic retrograde cholangiopancreatography revealed a defect in the common bile duct. Pathological diagnosis Cytological examination revealed the absence of malignant cells in bile. Microscopically, resected specimen revealed a pyloric type tubulopapillary adenoma with moderate epithelial atypia. Treatment The patient underwent choledochectomy and cholecystectomy. Related reports Intraductal papillary mucinous neoplasms of the bile duct are histologically classified as low- or intermediate-grade intraepithelial neoplasia corresponding to adenomas or borderline malignancy, high grade intraepithelial neoplasia corresponding to carcinoma in situ, or as having an associated invasive carcinoma. The neoplasms accompanied with tumor thrombus are often ordinary invasive carcinoma. Term explanation Intraductal papillary mucinous neoplasm of the bile duct is a recently recognized disease entity whose behavior is still unclear. Experiences and lessons Although the epithelial atypia was moderate, it was accompanied by a bile duct tumor thrombus. Even if no malignant cells are present, intraepithelial neoplasms occurring in the ampullopancreatobiliary tract can behave as malignant tumors. Hence, these patients should be treated aggressively. Peer review This article applies the validity of surgical treatment for intraepithelial neoplasms occurring in the ampullopancreatobiliary tract, even if it is an adenoma. REFERENCES 1 Chen TC, Nakanuma Y, Zen Y, Chen MF, Jan YY, Yeh TS, Chiu CT, Kuo TT, Kamiya J, Oda K, Hamaguchi M, Ohno Y, Hsieh LL, Nimura Y. Intraductal papillary neoplasia of the liver associated with hepatolithiasis. Hepatology 2001; 34: [PMID: ] 2 Bickenbach K, Galka E, Roggin KK. Molecular mechanisms of cholangiocarcinogenesis: are biliary intraepithelial neoplasia and intraductal papillary neoplasms of the bile duct precursors to cholangiocarcinoma? Surg Oncol Clin 8738 July 14, 2014 Volume 20 Issue 26

431 Yamamoto K et al. Adenoma of the gallbladder N Am 2009; 18: , vii [PMID: DOI: / j.soc ] 3 Nakanuma Y, Zen Y, Harada K, Ikeda H, Sato Y, Uehara T, Sasaki M. Tumorigenesis and phenotypic characteristics of mucin-producing bile duct tumors: an immunohistochemical approach. J Hepatobiliary Pancreat Sci 2010; 17: [PMID: DOI: /s ] 4 Rocha FG, Lee H, Katabi N, DeMatteo RP, Fong Y, D Angelica MI, Allen PJ, Klimstra DS, Jarnagin WR. Intraductal papillary neoplasm of the bile duct: a biliary equivalent to intraductal papillary mucinous neoplasm of the pancreas? Hepatology 2012; 56: [PMID: DOI: /hep.25786] 5 Kubota K, Nakanuma Y, Kondo F, Hachiya H, Miyazaki M, Nagino M, Yamamoto M, Isayama H, Tabata M, Kinoshita H, Kamisawa T, Inui K. Clinicopathological features and prognosis of mucin-producing bile duct tumor and mucinous cystic tumor of the liver: a multi-institutional study by the Japan Biliary Association. J Hepatobiliary Pancreat Sci 2014; 21: [PMID: DOI: /jhbp.23] 6 Adsay V, Jang KT, Roa JC, Dursun N, Ohike N, Bagci P, Basturk O, Bandyopadhyay S, Cheng JD, Sarmiento JM, Escalona OT, Goodman M, Kong SY, Terry P. Intracholecystic papillary-tubular neoplasms (ICPN) of the gallbladder (neoplastic polyps, adenomas, and papillary neoplasms that are 1.0 cm): clinicopathologic and immunohistochemical analysis of 123 cases. Am J Surg Pathol 2012; 36: [PMID: ] 7 Kloppel G, Hruban RH, Longnecker DS, Adler G, Kern SE, Partanen TJ. Ductal adenocarcinoma of the pancreas. In: World Health Organization classification of tumours. Pathology and genetics of tumours of the digestive system. Lyon: IARC Press, 2000: Nanashima A, Kinoshita N, Nakanuma Y, Zen Y, Sumida Y, Abo T, Hidaka S, Takeshita H, Yasutake T, Hayashi T, Nagayasu T. Clinicopathological features of intraductal papillary neoplasm of the bile duct and patient outcome after surgical resection. Hepatogastroenterology 2008; 55: [PMID: ] 9 Jung G, Park KM, Lee SS, Yu E, Hong SM, Kim J. Long-term clinical outcome of the surgically resected intraductal papillary neoplasm of the bile duct. J Hepatol 2012; 57: [PMID: DOI: /j.jhep ] 10 Midorikawa Y, Kubota K, Komatsu Y, Hasegawa K, Koike Y, Mori M, Makuuchi M. Gallbladder carcinoma with a tumor thrombus in the common bile duct: an unusual cause of obstructive jaundice. Surgery 2000; 127: [PMID: ] 11 Xin-Wei Y, Jue Y, Bao-Hua Z, Feng S. An unusual gallbladder carcinoma with tumor thrombus in the common bile duct. J Cancer Res Ther 2013; 9: [PMID: DOI: / ] 12 Albores-Saavedra J, Chablé-Montero F, Méndez-Sánchez N, Mercado MÁ, Vilatoba-Chapa M, Henson DE. Adenocarcinoma with pyloric gland phenotype of the extrahepatic bile ducts: a previously unrecognized and distinctive morphologic variant of extrahepatic bile duct carcinoma. Hum Pathol 2012; 43: [PMID: DOI: / j.humpath ] P- Reviewer: Wang DS S- Editor: Gou SX L- Editor: Wang TQ E- Editor: Wang CH 8739 July 14, 2014 Volume 20 Issue 26

432 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. BRIEF CASE ARTICLE REPORT First case of IgG4-related sclerosing cholangitis associated with autoimmune hemolytic anemia Hironori Masutani, Kosuke Okuwaki, Mitsuhiro Kida, Hiroshi Yamauchi, Hiroshi Imaizumi, Shiro Miyazawa, Tomohisa Iwai, Miyoko Takezawa, Wasaburo Koizumi Hironori Masutani, Kosuke Okuwaki, Mitsuhiro Kida, Hiroshi Yamauchi, Hiroshi Imaizumi, Shiro Miyazawa, Tomohisa Iwai, Miyoko Takezawa, Wasaburo Koizumi, Department of Gastroenterology, Kitasato University East Hospital, Kanagawa , Japan Author contributions: Masutani H and Okuwaki K contributed equally to this work; Masutani H, Okuwaki K, and Yamauchi H performed the research; Kida M, Miyazawa S, Iwai T, Takezawa M and Imaizumi H contributed to technical support; Kida M, Imaizumi H and Koizumi W contributed to critical revision of the manuscript for important intellectual content; Masutani H wrote the paper. Correspondence to: Hironori Masutani, MD, Department of Gastroenterology, Kitasato University East Hospital, Asamizodai, Minami-ku, Sagamihara, Kanagawa , Japan. masutani@kitasato-u.ac.jp Telephone: Fax: Received: January 21, 2014 Revised: March 1, 2014 Accepted: April 5, 2014 Published online: July 14, 2014 Abstract To our knowledge, patients with immunoglobulin G4- related sclerosing cholangitis (IgG4-SC) associated with autoimmune hemolytic anemia (AIHA) have not been reported previously. Many patients with IgG4-SC have autoimmune pancreatitis (AIP) and respond to steroid treatment. However, isolated cases of IgG4-SC are difficult to diagnose. We describe our experience with a patient who had IgG4-SC without AIP in whom the presence of AIHA led to diagnosis. The patient was a 73-year-old man who was being treated for dementia. Liver dysfunction was diagnosed on blood tests at another hospital. Imaging studies suggested the presence of carcinoma of the hepatic hilus and primary sclerosing cholangitis, but a rapidly progressing anemia developed simultaneously. After the diagnosis of AIHA, steroid treatment was begun, and the biliary stricture improved. IgG4-SC without AIP was thus diagnosed Baishideng Publishing Group Inc. All rights reserved. Key words: IgG4-related sclerosing cholangitis; Immunoglobulin G4-related sclerosing cholangitis; Autoimmune hemolytic anemia; Autoimmune hemolytic anemia; Autoimmune pancreatitis Core tip: Patients with immunoglobulin G4-related sclerosing cholangitis (IgG4-SC) associated with autoimmune hemolytic anemia (AIHA) have not been reported previously. Many patients with IgG4-SC have autoimmune pancreatitis (AIP) and respond to steroid treatment. However, isolated cases of IgG4-SC are difficult to diagnose. We describe our experience with a patient who had IgG4-SC without AIP in whom the presence of AIHA led to diagnosis. Masutani H, Okuwaki K, Kida M, Yamauchi H, Imaizumi H, Miyazawa S, Iwai T, Takezawa M, Koizumi W. First case of IgG4- related sclerosing cholangitis associated with autoimmune hemolytic anemia. World J Gastroenterol 2014; 20(26): Available from: URL: v20/i26/8740.htm DOI: i INTRODUCTION Immunoglobulin G4-related sclerosing cholangitis (IgG4- SC) was first reported in Japan and refers to sclerosing cholangitis of unknown cause, characterized by increased serum IgG4 levels and fibrosis associated with marked infiltration of local lesions by lymphocytes and IgG4- positive plasma cells [1]. Recently, Clinical Diagnostic Criteria of IgG4-related Sclerosing Cholangitis 2012 have been reported [2]. Many patients with IgG4-SC have autoimmune pancreatitis (AIP) and respond to steroid treatment [3]. It can be difficult to differentially diagnose IgG July 14, 2014 Volume 20 Issue 26

433 Masutani H et al. Case of IgG4-SC Associated with AIHA SC from conditions such as primary sclerosing cholangitis (PSC) and biliary cancer, particularly in patients who have IgG4-SC alone. We describe our experience with a patient who had IgG4-SC without AIP in whom the presence of autoimmune hemolytic anemia (AIHA) led to diagnosis. To our knowledge, patients with IgG4-SC and AIHA have not been reported previously, suggesting that such cases are extremely rare. CASE REPORT The patient was a 73-year-old man who was receiving treatment for dementia. Liver dysfunction was diagnosed on blood tests performed at another hospital. The family history was not relevant to the current disorder. The patient was not a smoker and did not drink alcohol and had no physical abnormalities or symptoms. The results of blood tests are shown in Table 1. The serum aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and γ-glutamyl transpeptidase levels were elevated, but there was no evidence of jaundice. The IgG (18.0 g/l) and IgG4 (2.3 g/l) levels were also high. Abdominal ultrasonography showed wall thickening in the region extending from the common bile duct to the intrahepatic bile duct (Figure 1). Contrast-enhanced, early-phase computed tomography showed wall thickening with contrast enhancement in the region from the upper biliary tract to the intrahepatic bile duct and strong contrast enhancement in the hepatic parenchyma in the portal region (Figure 2). Endoscopic retrograde cholangiopancreatography (ERCP) revealed no abnormalities of the pancreatic ducts. Imaging studies of the bile ducts showed dilation after a confluent stricture, with no strictures of the lower common bile duct. However, findings consistent with a band-like stricture and beaded appearance, frequently associated with PSC (Figure 3), were evident. Initially, hilar cholangiocarcinoma and PSC were suspected. Cytologic examination of bile specimens obtained at the time of ERCP and biliary brush cytology showed no clinically significant findings of malignancy. The patient was therefore observed while receiving ursodeoxycholic acid, and liver dysfunction transiently improved. However, obstructive cholangitis developed after about 3 mo, and a biliary stent was placed to drain the biliary tract. Subsequently, jaundice worsened with a high direct bilirubin level, and anemia progressed (Table 1). Although the patient had jaundice with a very high direct bilirubin level, he had a positive direct Coombs test with a high reticulocyte count, a low haptoglobin level, and an elevated lactate dehydrogenase level. AIHA was thus diagnosed. Prednisolone (1 mg/kg per day, 60 mg) was administered, and the anemia as well as the jaundice gradually improved (Table 1). ERCP images obtained 1 mo after starting steroid therapy are shown in Figure 4. The biliary stricture at the portal region had markedly improved. The biliary images, elevated serum IgG4 level, and response to steroid treatment met the diagnostic criteria for a probable diagnosis of IG4-SC according to the Clinical Diagnostic Criteria of IgG4-related Sclerosing Table 1 Laboratory data Cholangitis 2012, and IgG4-SC was thus diagnosed. DISCUSSION Initial At time of After steroid presentation cholangitis treatment White blood cells ( 10 9 /L) Red blood cells ( /L) Hemoglobin (g/l) Hematocrit 38% 11.5% 39.2% Platelets ( 10 9 /L) Reticulocytes Haptoglobin (mg/l) Total bilirubin (µmol/l) Direct bilirubin (µmol/l) Aspartate aminotransferase (IU/L) Alanine aminotransferase (IU/L) Alkaline phosphatase (IU/L) γ-glutamyl transpeptidase (IU/L) Lactate dehydrogenase (IU/L) C-reactive protein (nmol/l) < 10 IgG (g/l) (NR ) IgG4 (g/l) (NR ) Carbohydrate antigen 19-9 (U/mL) NR: Normal range. A B Figure 1 Abdominal ultrasonograms, showing wall thickening in the region extending from the upper bile duct (A) to the intrahepatic bile duct (B). A literature search of the PubMed/MEDLINE and 8741 July 14, 2014 Volume 20 Issue 26

434 Masutani H et al. Case of IgG4-SC Associated with AIHA Figure 2 Early-phase, abdominal computed tomographic scans, showing wall thickening with a contrast effect in the region from the upper biliary tract to the intrahepatic bile duct (arrows) and a strong contrast effect mainly in the hepatic parenchyma in the portal region (circle). A B C D Figure 3 Endoscopic retrograde cholangiopancreatography images obtained before treatment. A: There were no abnormalities of the pancreatic ducts; B: Images of the right bile duct showed stricture and simple dilation after a relatively long confluent stricture (arrows), with no strictures of the lower common bile duct (circle); C, D: A band-like stricture was found in the left bile duct (circle), and a beaded appearance was seen from the upper bile duct to the left hepatic duct (arrows). Embase databases indicated that cases of IgG4-SC associated with AIHA have not been reported previously. Many patients with IgG4-SC have AIP and respond to steroid therapy. In patients who have IgG4-SC alone, it is particularly difficult to differentiate IgG4-SC from PSC and biliary cancer, making the diagnosis challenging. We described our experience with a patient who had IgG4- SC without AIP in whom the presence of AIHA led to diagnosis. Clinically, IgG4-SC is characterized by jaundice leading to diagnosis as well as stricture of the lower bile duct on imaging studies of the biliary tract. Our patient initially presented with liver dysfunction, with no evidence of jaundice on blood tests, and had no findings suggesting AIP or stricture of the lower bile duct on imaging studies. Blood tests at initial presentation showed an elevated serum IgG4 concentration (Table 1). There was a time lag of about 1 wk until the results of specialized tests became available. IgG4-SC is characterized by elevated serum IgG4 levels, but elevated levels may also be found in PSC. Boonstra et al [4] reported that the IgG4/ IgG1 ratio is useful for differentiating IgG4-SC from PSC. However, the IgG4/IgG1 ratio was unavailable because we did not measure serum IgG1 levels. Cholangiographic images in our patient were consistent with type 4 IgG4-SC according to the classification proposed by Nakazawa et al [5,6] and are thus very rarely encountered. These atypical clinical and imaging findings made diagnosis very difficult July 14, 2014 Volume 20 Issue 26

435 Masutani H et al. Case of IgG4-SC Associated with AIHA Figure 4 Endoscopic retrograde cholangiopancreatography image obtained after steroid treatment, showing that the biliary stricture situated mainly in the portal region had markedly improved. AIHA is a disease that responds to steroid treatment, but can be associated with connective-tissue diseases such as systemic lupus erythematosus, autoimmune lymphoproliferative syndrome, common variable immune deficiency, and hematologic diseases such as chronic lymphocytic leukemia [7]. Several patients with PSC and AIHA have been reported on [8-11], but to our knowledge patients with IgG4-SC associated with AIHA have not been documented previously. In AIHA, IgG antibodies bound to erythrocyte membranes might be identified by IgG Fc receptors of phagocytes and phagocytized. IgG Fc receptors of phagocytes are specific for IgG1 and IgG3 and essentially show no activity for IgG2 or IgG4. Phagocytes have receptors for complement C3b, whereas IgG4 has been reported to lack complement activation activity [12-14]. These findings suggest that IgG4 does not contribute to the development of AIHA. Our patient probably had one of the many potential combinations of autoimmune disorders. In other words, congenital or acquired factors may have caused autoimmune disorders in our patient, resulting in the simultaneous development of IgG4-SC and AIHA. In our patient, difficulty was encountered in the diagnosis of biliary stricture seen mainly in the portal region, and treatment was required for a rapidly progressing anemia. There was no response to transfusion therapy. Because the patient had a positive direct Coombs test, and the results of blood tests supported a diagnosis of hemolysis, AIHA was diagnosed. Steroid therapy was started to treat AIHA, and the biliary lesions improved relatively promptly, indicating a diagnosis of IgG4-SC. At the time of this writing, 18 mo after the onset of the current disorder, the patient is receiving 3 mg of prednisolone on consecutive days, with no recurrence. COMMENTS Case characteristics The patient presented with no symptoms, and liver dysfunction was diagnosed on blood tests. Clinical diagnosis The presence of autoimmune hemolytic anemia (AIHA) led to the diagnosis of immunoglobulin G4-related sclerosing cholangitis (IgG4-SC) in a patient without autoimmune pancreatitis (AIP). Differential diagnosis It can be difficult to differentially diagnose IgG4-SC from conditions such as primary sclerosing cholangitis (PSC) and biliary cancer, particularly in patients who have IgG4-SC alone. Laboratory diagnosis The serum IgG, IgG4, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, and γ-glutamyl transpeptidase levels were elevated, but there was no evidence of jaundice. Imaging diagnosis Endoscopic retrograde cholangiopancreatography (ERCP) revealed no abnormalities of the pancreatic ducts, and imaging studies of the bile ducts showed dilation after a confluent stricture, with no strictures of the lower common bile duct. Pathological diagnosis Cytologic examination of bile specimens obtained at the time of ERCP and biliary brush cytology showed no clinically significant findings of malignancy. Treatment Prednisolone (1 mg/kg per day, 60 mg) was administered, and the anemia as well as the jaundice gradually improved. Related reports In patients who have IgG4-SC alone, it is particularly difficult to differentiate IgG4-SC from PSC and biliary cancer, making the diagnosis challenging. IgG4/ IgG1 ratio might be useful for distinguish PSC from IgG4-SC as reported by Boonstra et al in Hepatology. Term explanation IgG4-SC was first reported in Japan and refers to sclerosing cholangitis of unknown cause, characterized by increased serum IgG4 levels and fibrosis associated with marked infiltration of local lesions by lymphocytes and IgG4-positive plasma cells. Experiences and lessons In the patient, the presence of AIHA led to the diagnosis of IgG4-SC unassociated with AIP. Peer review To our knowledge, patients with IgG4-SC and AIHA have not been reported previously, suggesting that such cases are extremely rare. In patients who have IgG4-SC alone, it is particularly difficult to differentiate IgG4-SC from PSC and biliary cancer, making the diagnosis challenging. Boonstra et al reported that the IgG4/IgG1 ratio is useful for differentiating IgG4-SC from PSC. However, the IgG4/IgG1 ratio was unavailable because we did not measure serum IgG1 levels. The authors described their experience with a patient who had IgG4-SC without AIP in whom the presence of AIHA led to diagnosis. REFERENCES 1 Kamisawa T, Funata N, Hayashi Y, Eishi Y, Koike M, Tsuruta K, Okamoto A, Egawa N, Nakajima H. A new clinicopathological entity of IgG4-related autoimmune disease. J Gastroenterol 2003; 38: [PMID: ] 2 Ohara H, Okazaki K, Tsubouchi H, Inui K, Kawa S, Kamisawa T, Tazuma S, Uchida K, Hirano K, Yoshida H, Nishino T, Ko SB, Mizuno N, Hamano H, Kanno A, Notohara K, Hasebe O, Nakazawa T, Nakanuma Y, Takikawa H. Clinical diagnostic criteria of IgG4-related sclerosing cholangitis J Hepatobiliary Pancreat Sci 2012; 19: [PMID: DOI: /s y] 3 Ghazale A, Chari ST, Zhang L, Smyrk TC, Takahashi N, Levy MJ, Topazian MD, Clain JE, Pearson RK, Petersen BT, Vege SS, Lindor K, Farnell MB. Immunoglobulin G4-associated cholangitis: clinical profile and response to therapy. Gastroenterology 2008; 134: [PMID: DOI: /j.gastro ] 4 Boonstra K, Culver EL, de Buy Wenniger LM, van Heerde MJ, van Erpecum KJ, Poen AC, van Nieuwkerk KM, Spanier BW, Witteman BJ, Tuynman HA, van Geloven N, van Buuren H, Chapman RW, Barnes E, Beuers U, Ponsioen CY. Serum immunoglobulin G4 and immunoglobulin G1 for 8743 July 14, 2014 Volume 20 Issue 26

436 Masutani H et al. Case of IgG4-SC Associated with AIHA distinguishing immunoglobulin G4-associated cholangitis from primary sclerosing cholangitis. Hepatology 2014; 59: [PMID: DOI: /hep.26977] 5 Nakazawa T, Naitoh I, Hayashi K, Miyabe K, Simizu S, Joh T. Diagnosis of IgG4-related sclerosing cholangitis. World J Gastroenterol 2013; 19: [PMID: DOI: / wjg.v19.i ] 6 Nakazawa T, Ohara H, Sano H, Ando T, Joh T. Schematic classification of sclerosing cholangitis with autoimmune pancreatitis by cholangiography. Pancreas 2006; 32: 229 [PMID: ] 7 Lechner K, Jäger U. How I treat autoimmune hemolytic anemias in adults. Blood 2010; 116: [PMID: DOI: /blood ] 8 Holm T, Kildahl-Andersen O, Valle PC. Primary sclerosing cholangitis with autoimmune hemolytic anemia and pulmonary infiltrations. Scand J Gastroenterol 1998; 33: [PMID: ] 9 Eilam O, Goldin E, Shouval D, Gimon T, Brautbar C. Sclerosing cholangitis associated with Crohn s disease and autoimmune haemolytic anaemia. Postgrad Med J 1993; 69: [PMID: ] 10 Moeller DD. Sclerosing cholangitis associated with autoimmune hemolytic anemia and hyperthyroidism. Am J Gastroenterol 1985; 80: [PMID: ] 11 Pineau BC, Pattee LP, McGuire S, Sekar A, Scully LJ. Unusual presentation of primary sclerosing cholangitis. Can J Gastroenterol 1997; 11: [PMID: ] 12 von dem Borne AE, Beckers D, van der Meulen FW, Engelfriet CP. IgG4 autoantibodies against erythrocytes, without increased haemolysis: a case report. Br J Haematol 1977; 37: [PMID: ] 13 Engelfriet CP, Borne AE, Beckers D, Van Loghem JJ. Autoimmune haemolytic anaemia: serological and immunochemical characteristics of the autoantibodies; mechanisms of cell destruction. Ser Haematol 1974; 7: [PMID: ] 14 Komine M. [Autoimmune hemolytic anemia]. Rinsho Ketsueki 1992; 33: [PMID: ] P- Reviewers: Balzano G, Gong ZJ, Hoff DAL, Yazisiz V S- Editor: Qi Y L- Editor: A E- Editor: Zhang DN 8744 July 14, 2014 Volume 20 Issue 26

437 Submit a Manuscript: Help Desk: DOI: /wjg.v20.i World J Gastroenterol 2014 July 14; 20(26): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. CASE REPORT Laparoscopic segmental colectomy for colonic lymphangiomas: A definitive, minimally invasive surgical option Chang-Hua Zhuo, De-Bing Shi, Min-Gang Ying, Yu-Fan Cheng, Yu-Wei Wang, Wen-Ming Zhang, San-Jun Cai, Xin-Xiang Li Chang-Hua Zhuo, De-Bing Shi, Yu-Wei Wang, San-Jun Cai, Xin-Xiang Li, Department of Colorectal Surgery, Fudan University Shanghai Cancer Center, Shanghai , China Chang-Hua Zhuo, De-Bing Shi, Yu-Fan Cheng, Yu-Wei Wang, Wen-Ming Zhang, San-Jun Cai, Xin-Xiang Li, Department of Oncology, Shanghai Medical College, Fudan University, Shanghai , China Chang-Hua Zhuo, Min-Gang Ying, Department of Surgical Oncology, Fujian Provincial Cancer Hospital, Teaching Hospital of Fujian Medical University, Fuzhou , Fujian Province, China Yu-Fan Cheng, Department of Pathology, Fudan University Shanghai Cancer Center, Shanghai , China Wen-Ming Zhang, Department of Endoscopy, Fudan University Shanghai Cancer Center, Shanghai , China Author contributions: Zhuo CH and Shi DB conceived and drafted the manuscript; Li XX, Shi DB and Cai SJ performed the operation; Shi DB, Ying MG, Wang YW and Li XX assisted in reviewing the literature and drafting the manuscript; Cheng YF made the histopathological diagnosis; Zhang WM reviewed the endoscopic diagnosis; Ying MG, Cai SJ and Li XX made critical revisions to the manuscript and edited the final version of the manuscript; all authors read and approved the final manuscript. Supported by National Natural Science Foundation of China, No ; and the Basic Research Project of Shanghai Science and Technology Commission, No. 13JC Correspondence to: Xin-Xiang Li, MD, Department of Colorectal Surgery, Fudan University Shanghai Cancer Center, 270 Dong-an Road, Xu-Hui District, Shanghai , China. lxx1149@163.com Telephone: Fax: Received: December 8, 2013 Revised: January 29, 2014 Accepted: March 7, 2014 Published online: July 14, 2014 Colonic lymphangioma is an unusual benign malformation. We herein describe two cases. A 36-year-old woman was admitted with one year of intermittent abdominal pain; colonoscopy, abdominopelvic computed tomography and endoscopic ultrasonography (EUS) revealed enlarged cystic masses at the ascending colon. In another 40-year-old man, colonoscopy and EUS revealed an asymptomatic lobulated cystic mass with four small sessile polyps at the sigmoid colon. Both patients underwent laparoscopic segmental colectomy. Both masses were histologically confirmed as cystic lymphangiomas, and the patients were discharged without complications. The management of colonic lymphangioma depends on the individual situation; close surveillance or endoscopic therapy may be appropriate for asymptomatic lesions smaller than 2.5 cm in diameter. Surgical intervention can be considered for larger lesions or in patients who develop complication risks. Laparoscopic segmental colon resection may be recommended to excise relatively large submucosal lesions because it is a definitive, minimally invasive intervention with a fast postoperative recovery Baishideng Publishing Group Inc. All rights reserved. Key words: Cystic lymphangioma; Colon neoplasm; Laparoscopic surgery; Colectomy; Segmental resection Core tip: We herein present two unusual cases of colonic cystic lymphangiomas that required surgical intervention. Both patients underwent laparoscopic segmental colectomy and had uneventful postoperative recoveries with confirmed histopathological diagnoses. This minimally invasive approach is an attractive alternative for rare benign malformation cases. Abstract Zhuo CH, Shi DB, Ying MG, Cheng YF, Wang YW, Zhang WM, Cai SJ, Li XX. Laparoscopic segmental colectomy for colonic lymphangiomas: A definitive, minimally invasive surgical option. World J Gastroenterol 2014; 20(26): Available from: 8745 July 14, 2014 Volume 20 Issue 26

438 Zhuo CH et al. Laparoscopic resection for colonic cystic lymphangiomas URL: DOI: A INTRODUCTION Cystic lymphangiomas occur most often in the head, neck, trunk and extremities of children and young adults. Intraabdominal lymphangiomas, which mostly originate from the mesentery and retroperitoneum, are rare and account for fewer than 5% of all lymphangiomas [1-3]. The actual incidence of lymphangiomas in the colon or rectum is unclear. Stout [4] reported 4 lymphangiomas among 292 (1.4%) colorectal neoplasms, but as endoscopic examination has become more widespread, colonic lymphangiomas have been reported more frequently [5]. Most colonic lymphangiomas are asymptomatic and do not require treatment, but resection is necessary in some specific situations. We herein describe two cases of colonic lymphangiomas treated using laparoscopic segmental bowel resection and we review the relevant medical literature. CASE REPORT First case: laparoscopic segmental resection of the ascending colon for a cystic lesion with a fast growth speed A 36-year-old female patient visited our department with a one-year history of mild intermittent abdominal pain at the right lower quadrant. Five years prior, she had been examined at a local hospital with a complaint of chronic constipation; at the time, colonoscopy revealed a 1.5 cm 1.0 cm submucosal cystic mass at the ascending colon. The patient received conservative management coupled with endoscopic yearly follow-up, and the cystic lesion had recently become enlarged. There was no significant family history or other medical history. The patient was 170 cm in height and weighed 65 kg (BMI, 22 kg/m 2 ); the ASA grade was 1. She appeared well, and physical examination revealed no remarkable abdominal abnormalities. Laboratory tests, including those measuring tumour markers (CEA, CA125 and AFP), were all within normal limits. Radiographies of the chest and abdomen were normal. Colonoscopy revealed a round, semi-transparent submucosal lesion with a gentle slope and smooth surface at the middle ascending colon (Figure 1A). An abdominopelvic computed tomography (CT) scan showed a 4 cm 4 cm sized, low-density, non-enhancing cystic mass located submucosally at the ascending colon (Figure 2). Endoscopic ultrasonography (EUS) (EUS2000, Olympus, Japan) revealed a 3.9 cm 3.5 cm lesion visible as an echo-free cyst in the third (submucosal) layer without blood flow. The lesion had a positive cushion (pillow) sign (Figure 3A). The patient underwent laparoscopic segmental resection of the ascending colon. In brief, five ports were made, with two 12 mm and three 5 mm cannulas placed as previously described [6]. The ascending mesocolon was mobilised using a lateral- B Figure 1 Endoscopic findings for the two cases. A round, semi-transparent and wide-based submucosal lesion at the ascending colon in Case 1 (A) and a sharply marginated, lobular submucosal lesion at the sigmoid colon in Case 2 (B) with gentle slopes and smooth surfaces are noted. Left bottom of Figure 1A shows that when the patient's position was altered, the shape of the mass changed and was fluctuant on palpation. A [R] B [R] [A] [P] [A] [P] Figure 2 Abdominopelvic computed tomography scan for Case 1 revealed a cystic mass (indicated by white arrow) located at the ascending colon, which was visible in a non-contrast scan (A) and was not enhanced after the arterial phase (B). A: Anterior; R: Right; L: Left; P: Posterior. [L] [L] 8746 July 14, 2014 Volume 20 Issue 26

439 Zhuo CH et al. Laparoscopic resection for colonic cystic lymphangiomas A B Figure 3 Endoscopic ultrasonography images of the two cases obtained using a catheter EUS probe. A: Elevated lesions as echo-free, homogenous cysts in Case 1 (frequency 6 MHz); B: Multiple septal walls (indicated by white arrows) in Case 2 (frequency 12 MHz); EUS revealed that both lesions were located in the third (submucosal) layer. A B Figure 4 Histopathological examination of the two cases after hematoxylin and eosin stain (at 40 magnification for low power field images). A: Case 1 revealed a dilated submucosal cyst lined with a single layer of endothelial cells [left bottom, hematoxylin and eosin (HE) stain, 200]; B: Case 2 displayed lymphatic vessels of varied sizes, which were covered by a flattened layer of lymphatic epithelium. The smooth muscle layer of the vessels is also noted (right bottom, HE stain, 400). to-medial approach. The ileocolic pedicle was resected, and the right flexure taken down. The ascending colon was freed and the specimen was extracted through an incision extended from the supraumbilical port site. A segment of involved colon approximately 6-7 cm in length was resected and an side-to-side anastomosis was accomplished extracorporeally using a linear-cutter (Proximate, Ethicon Endo-Surgery, Cincinnati, OH, United States). The patient had an uneventful recovery and was discharged seven days post-operation. Pathological analysis identified the mass as a submucosal macrocystic lymphangioma (Figure 4A). Annual endoscopic follow-up for 3 years was recommended for the patient. Second case: laparoscopic segmental sigmoidectomy for the cystic mass with concurrent polyps A 50-year-old male patient with a history of type 2 diabetes mellitus was admitted for an incidental finding of a lobulated cystic mass at the sigmoid colon. The patient had a history of colorectal polyps and had undergone endoscopic polypectomy 2 years prior. He was asymptomatic and appeared well with a BMI of 22 kg/m 2 and ASA grade 2. Blood chemistry and normal tumour maker (CEA, AFP, CA50 and CA125) tests were normal. Abdominal CT did not show significant abnormality due to bowel flatulence. Colonoscopy revealed that in addition to four sessile polyps ranging from 0.3 to 0.5 cm in diameter, the patient had an extra 3.0 cm 3.5 cm lobular submucosal lesion located cm above the anal verge. The lesion colour did not differ from that of the surrounding normal mucosa, and there were no ulcerations or erosions on the smooth surface (Figure 1B). Endoscopic ultrasonography (EUS2000, Olympus, Japan) revealed multiple internal submucosal septa (Figure 3B). In order to remove the section affected by the cystic lesion and polyps, the patient underwent laparoscopic segmental sigmoidectomy. In brief, five ports were made with two 12 mm and three 5 mm cannulas placed as previously described [7]. The inferior mesenteric artery was identified and resected. The descending and sigmoid mesocolon was mobilised, and the specimen was extracted through an incision extending from the suprapubic port. A segment of sigmoid colon, approximately 8 cm in length, was resected extracorporeally. The distal colon was reintroduced into the abdominal cavity after the anvil of the circular stapler was introduced and fixed inside. Pneumoperitoneum was reestablished and a double-stapled anastomosis between the distal colon and the proximal 8747 July 14, 2014 Volume 20 Issue 26

440 Zhuo CH et al. Laparoscopic resection for colonic cystic lymphangiomas rectal stump was accomplished intra-abdominally. An uncomplicated postoperative recovery was achieved, and the patient was discharged at the sixth day post-operation. The pathologic features were diagnostic of macrocystic lymphangioma (Figure 4B). Annual endoscopic follow-up for at least 3 years was recommended. DISCUSSION Lymphangioma may be developmentally malformed or malpositioned lymphatic tissue, and, though its exact aetiology remains unclear, genetic abnormalities might also play a role [8]. We herein discuss the pathology, diagnostic modality and treatment options for this unusual lymphatic malformation of the colon based on the cases described above. Macroscopically, colonic lymphangiomas can be classified as one of the following three types: simple (capillary), cavernous and cystic. The cystic type is the most frequently reported [5]. Cystic lymphangiomas are yellow, greyish, or yellow-pink in colour, and they often appear as multiple cysts or spongy masses with cavities containing watery or milky fluids [9]. Cystic lymphangiomas may be classified into microcystic, macrocystic, and mixed subtypes according to the cyst volume (2 cm 3 is used as the cutoff value) [10]. Both of our cases were classified as colonic macrocystic lymphangioma. Microscopically, as shown in Figure 4, the macrocystic mass is characterised by thin-walled spaces, which are frequently surrounded by lymph and/or lymphocytes, and is lined by flattened endothelium. The mass is also situated in close proximity to collagenous elastic tissue and smooth muscle [11]. Colonic lymphangiomas usually present as submucosal polypoid lesions without any symptomatic presentation. Some patients may experience episodic constipation with vague abdominal distress, diarrhea or other nonspecific symptoms [9,12]. Haemorrhage or anaemia [13,14] and protein-losing enteropathy [5,15] are always associated with surface ulceration or erosion. Acute abdomen obstruction of the volvulus, intussusceptions and bowel may occur when a mass is sufficiently enlarged [16-18]. Abdominal pain may mimic acute appendicitis [12,19]. Most colonic lymphangiomas are preoperatively diagnosed as submucosal tumours using colonoscopy, which is the most commonly used diagnostic modality. Lymphangioma appears as a well-defined, submucosal cystic lesion, typically with a smooth, pinkish, translucent surface [20]. Endoscopic ultrasonography is useful for diagnosing colonic lymphangioma. When using EUS, the characteristic features of colonic lymphangioma are lesions localised at the submucosal layer with a homogenous echo pattern and multilocular cysts that are echofree or contain septal structures [21]. Endoscopic aspiration for cytology evaluation or biopsy often has no diagnostic value and may result in an efflux of lymphatic fluid [20] or even local infection [22]. Abdominal CT may detect nonenhancing submucosal cystic masses and can be recommended to rule out coexisting intraperitoneal co-morbid lesions [5]. Both of the cases described here underwent these routine diagnostic modalities, which revealed the characteristic features mentioned above. Management of colonic lymphangiomas is dependent on their size, growth speed, symptoms and degree of suspected malignancy [17]. Malignant transformation of colonic lymphangiomas does not occur [11]. Therefore, close endoscopic surveillance can be considered and spontaneous resolution may occur [13,23]. With respect to sclerotherapy for lymphangiomas, agents such as Bleomycin, sodium tetradecyl sulphate, or OK-432 (lyophilised incubation mixture of group A streptococcus pyogenes of human origin) are routinely used. Macrocystic lesions, but not microcystic lesions, respond well to OK-432 injections [24]. Most of the reported cases of lymphangioma have occurred in the head and neck of children [1,25-27]. To the best of our knowledge, the use of endoscopic sclerotherapy to treat colonic lesions has not previously been reported. The complications resulting from the use of this therapy, which include local infection, perforation and bleeding, should be considered [28]. Other non-surgical therapy, including steroids, fibrin glue, or Ethibloc, has not been established as superior to surgery [29,30]. Colonic lymphangioma can be complicated by concurrent colon cancer or adenoma [12]. The mass might form secondarily as a result of lymphatic obstruction and inflammatory processes due to various causes [13,30-32]. Additionally, the potential to grow and invade adjacent structures introduce risks of life-threatening complications [17], and, as a result, surgical interventions may be required. In the current cases, they were implicated to surgical intervention for the reason of relatively large and fast growing lesion or lesion with concurrent polyps. Endoscopic resection has been recommended for intraluminal tumours with a maximum diameter 2.5 cm or smaller [12,21]. Matsuda et al [12] reviewed 279 Japanese cases of colorectal lymphangiomas. Of them, 104 (37.4%) patients underwent endoscopic resection. Removing the entire lesion as completely as possible, rather than using mere puncture and drainage, was the most important factor for reducing the risk of recurrence [20,33-35]. In 1996, Kenney et al [36] reported the first case of laparoscopic excision of a cystic lymphangioma from the mesentery of the proximal jejunum. Ryu et al [37] reported two cases of huge mesenteric cystic lymphangioma (13 and 11 cm in diameter), both of which were treated laparoscopically, with partial aspiration of the cysts using a spinal needle. Hoffmann et al [38] reported a 4.5 cm cystic lesion in the cecal region. The cyst, including its base and a portion of the colon, was resected laparoscopically. However, these cases were all lymphangiomas originating from the mesentery, which is the most commonly involved intra-abdominal site [20]. In both of our cases, it was difficult to find the masses laparoscopically because they were located at the submucosal layer. Additionally, both masses were relatively large and could not be removed either laparoscopically or endoscopically while preserving the involved colon. It 8748 July 14, 2014 Volume 20 Issue 26

441 Zhuo CH et al. Laparoscopic resection for colonic cystic lymphangiomas is critical to preoperatively identify and locate masses using endoscopic staining [39]. According to our experience, neither technique makes it challenging nor time consuming to remove the specimen and facilitate anastomosis with a small incision (approximately 4 cm in length). Compared to traditional open approaches, laparoscopic surgery has various advantages, including less intraoperative blood loss, decreased postoperative pain, a shorter hospital stay, minimal scarring, and a faster return to normal activities [40]. ACKNOWLEDGMENTS The authors thank Associate Professor Andy Tsun, PhD, from the Unit of Molecular Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, for his help with editing this manuscript. COMMENTS Case characteristics A 36-year-old female (Case 1) and another 40-year-old male (Case 2) presented with mild intermittent abdominal pain and without specific symptoms, respectively; initially, cystic lesions at the colon were accidentally found using colonoscopy. Clinical diagnosis An enlarged submucosal cystic mass at the middle ascending colon for Case 1 and a lobular submucosal lesion coupled with four polyps located at the sigmoid colon for Case 2. Differential diagnosis Other submucosal tumours, such as leiomyoma, lipoma or other cystic lesions, such as enteric duplication cysts and colitis cystica profunda as well as carcinoid tumours or mucocele, should be considered as the differential diagnoses. Laboratory diagnosis In both cases, the blood chemistry and tumour marker level tests were all within normal limits. Imaging diagnosis In Case 1, colonoscopy revealed a round semi-transparent submucosal lesion (4 cm 4 cm), abdominal CT revealed a non-enhancing submucosal cystic mass and EUS revealed an echo-free cyst in the third layer without blood flow. In Case 2, colonoscopy revealed a lobular submucosal lesion (3.0 cm 3.5 cm) coupled with four polyps ( cm in diameter) at cm above the anal verge; EUS revealed multiple submucosal internal septa. Pathological diagnosis Pathological analysis revealed the colonic masses in both cases to be macrocystic lymphangiomas. Treatment Laparoscopic segmental ascending colectomy and sigmoidectomy were performed on Case 1 and Case 2, respectively. Related reports Both cases had uneventful postoperative recoveries. Term explanation A laparoscopic segmental colectomy is a minimally invasive procedure that involves removing a portion or segment of the colon. Experiences and lessons This report not only represents two unusual cases of colonic cystic lymphangiomas, it also suggests that surgical intervention is required in some situations and that the laparoscopic approach is an attractive option for this rare benign malformation. Peer review This article describes a minimally invasive surgical alternative for managing an unusual condition compared to an endoscopic approach or open surgery. This definitive surgical resection offers more success than endoscopic excision in terms of the relapse rate. REFERENCES 1 Alqahtani A, Nguyen LT, Flageole H, Shaw K, Laberge JM. 25 years experience with lymphangiomas in children. J Pediatr Surg 1999; 34: [PMID: ] 2 Bill AH, Sumner DS. A unified concept of lymphangioma and cystic hygroma. Surg Gynecol Obstet 1965; 120: [PMID: ] 3 Roisman I, Manny J, Fields S, Shiloni E. Intra-abdominal lymphangioma. Br J Surg 1989; 76: [PMID: ] 4 Stout AP. Tumors of the colon and rectum (excluding carcinoma and adenoma). 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443 Submit a Manuscript: Help Desk: World J Gastroenterol 2014 July 14; 20(26): I-VI ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Inc. All rights reserved. INSTRUCTIONS TO AUTHORS GENERAL INFORMATION World Journal of Gastroenterology (World J Gastroenterol, WJG, print ISSN , online ISSN , DOI: ) is a peer-reviewed open access (OA) journal. WJG was established on October 1, It is published weekly on the 7 th, 14 th, 21 st, and 28 th each month. The WJG Editorial Board consists of 1353 experts in gastroenterology and hepatology from 68 countries. Aims and scope The primary task of WJG is to rapidly publish high-quality original articles, reviews, and commentaries in the fields of gastroenterology, hepatology, gastrointestinal endoscopy, gastrointestinal surgery, hepatobiliary surgery, gastrointestinal oncology, gastrointestinal radiation oncology, gastrointestinal imaging, gastrointestinal interventional therapy, gastrointestinal infectious diseases, gastrointestinal pharmacology, gastrointestinal pathophysiology, gastrointestinal pathology, evidence-based medicine in gastroenterology, pancreatology, gastrointestinal laboratory medicine, gastrointestinal molecular biology, gastrointestinal immunology, gastrointestinal microbiology, gastrointestinal genetics, gastrointestinal translational medicine, gastrointestinal diagnostics, and gastrointestinal therapeutics. WJG is dedicated to become an influential and prestigious journal in gastroenterology and hepatology, to promote the development of above disciplines, and to improve the diagnostic and therapeutic skill and expertise of clinicians. WJG is published by Baishideng Publishing Group (BPG) in both electronic and online forms. All WJG articles are published in WJG website and PubMed Central. The major advantages of OA journals are faster release and delivery, no page or graph restrictions, and increased visibility, usage and impact. Full-text PDF articles and electronic/online versions are freely available to global readers. After the paper is published, the author(s) can obtain high-quality PDF files, which contain the journal cover, a list of editorial board members, table of contents, text, and back cover of the journal. BPG has a strong professional editorial team composed of editorial board members, editors-in-chief, science editors, language editors, and electronic editors. BPG currently publishes 43 OA clinical medical journals, including 42 in English, has a total of editorial board members or peer reviewers, and is a world first-class publisher. Columns The columns in the issues of WJG will include: (1) Editorial: The editorial board members are invited to make comments on an important topic in their field in terms of its current research status and future directions to lead the development of this discipline; (2) Frontier: The editorial board members are invited to select a highly cited cutting-edge original paper of his/her own to summarize major findings, the problems that have been resolved and remain to be resolved, and future research directions to help readers understand his/her important academic point of view and future research directions in the field; (3) Diagnostic Advances: The editorial board members are invited to write high-quality diagnostic advances in their field to improve the diagnostic skills of readers. The topic covers general clinical diagnosis, differential diagnosis, pathological diagnosis, laboratory diagnosis, imaging diagnosis, endoscopic diagnosis, biotechnological diagnosis, functional diagnosis, and physical diagnosis; (4) Therapeutics Advances: The editorial board members are invited to write high-quality therapeutic advances in their field to help improve the therapeutic skills of readers. The topic covers medication therapy, psychotherapy, physical therapy, replacement therapy, interventional therapy, minimally invasive therapy, endoscopic therapy, transplantation therapy, and surgical therapy; (5) Field of Vision: The editorial board members are invited to write commentaries on classic articles, hot topic articles, or latest articles to keep readers at the forefront of research and increase their levels of clinical research. Classic articles refer to papers that are included in Web of Knowledge and have received a large number of citations (ranking in the top 1%) after being published for more than years, reflecting the quality and impact of papers. Hot topic articles refer to papers that are included in Web of Knowledge and have received a large number of citations after being published for no more than 2 years, reflecting cutting-edge trends in scientific research. Latest articles refer to the latest published high-quality papers that are included in PubMed, reflecting the latest research trends. These commentary articles should focus on the status quo of research, the most important research topics, the problems that have now been resolved and remain to be resolved, and future research directions. Basic information about the article to be commented (including authors, article title, journal name, year, volume, and inclusive page numbers; (6) Minireviews: The editorial board members are invited to write short reviews on recent advances and trends in research of molecular biology, genomics, and related cutting-edge technologies to provide readers with the latest knowledge and help improve their diagnostic and therapeutic skills; (7) Review: To make a systematic review to focus on the status quo of research, the most important research topics, the problems that have now been resolved and remain to be resolved, and future research directions; (8) Topic Highlight: The editorial board members are invited to write a series of articles (7-10 articles) to comment and discuss a hot topic to help improve the diagnostic and therapeutic skills of readers; (9) Medical Ethics: The editorial board members are invited to write articles about medical ethics to increase readers knowledge of medical ethics. The topic covers international ethics guidelines, animal studies, clinical trials, organ transplantation, etc.; (10) Clinical Case Conference or Clinicopathological Conference: The editorial board members are invited to contribute high-quality clinical case conference; (11) Original Articles: To report innovative and original findings in gastroenterology and hepatology; (12) Brief Articles: To briefly report the novel I July 14, 2014 Volume 20 Issue 26

444 Instructions to authors and innovative findings in gastroenterology and hepatology; (13) Meta-Analysis: Covers the systematic review, mixed treatment comparison, meta-regression, and overview of reviews, in order to summarize a given quantitative effect, e.g., the clinical effectiveness and safety of clinical treatments by combining data from two or more randomized controlled trials, thereby providing more precise and externally valid estimates than those which would stem from each individual dataset if analyzed separately from the others; (14) Case Report: To report a rare or typical case; (15) Letters to the Editor: To discuss and make reply to the contributions published in WJG, or to introduce and comment on a controversial issue of general interest; (16) Book Reviews: To introduce and comment on quality monographs of gastroenterology and hepatology; and (17) Autobiography: The editorial board members are invited to write their autobiography to provide readers with stories of success or failure in their scientific research career. The topic covers their basic personal information and information about when they started doing research work, where and how they did research work, what they have achieved, and their lessons from success or failure. Name of journal World Journal of Gastroenterology ISSN ISSN (print) ISSN (online) Launch date October 1, 1995 Frequency Weekly Editors-in-chief Damian Garcia-Olmo, MD, PhD, Doctor, Professor, Surgeon, Department of Surgery, Universidad Autonoma de Madrid; Department of General Surgery, Fundacion Jimenez Diaz University Hospital, Madrid 28040, Spain Saleh A Naser, PhD, Professor, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL 32816, United States Stephen C Strom, PhD, Professor, Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Stockholm , Sweden Andrzej S Tarnawski, MD, PhD, DSc (Med), Professor of Medicine, Chief Gastroenterology, VA Long Beach Health Care System, University of California, Irvine, CA, 5901 E. Seventh Str., Long Beach, CA 90822, United States Editorial office Jin-Lei Wang, Director Xiu-Xia Song, Vice Director World Journal of Gastroenterology Room 903, Building D, Ocean International Center, No. 62 Dongsihuan Zhonglu, Chaoyang District, Beijing , China Telephone: Fax: editorialoffice@wjgnet.com Help Desk: Publisher Baishideng Publishing Group Inc 8226 Regency Drive, Pleasanton, CA 94588, USA Telephone: Fax: bpgoffice@wjgnet.com Help Desk: Instructions to authors Full instructions are available online at com/ /g_info_ htm Indexed and abstracted in Current Contents /Clinical Medicine, Science Citation Index Expanded (also known as SciSearch ), Journal Citation Reports, Index Medicus, MEDLINE, PubMed, PubMed Central, Digital Object Identifier, and Directory of Open Access Journals. ISI, Thomson Reuters, 2011 Impact Factor: (32/74 Gastroenterology and Hepatology). SPECIAL STATEMENT All articles published in journals owned by the BPG represent the views and opinions of their authors, and not the views, opinions or policies of the BPG, except where otherwise explicitly indicated. Biostatistical editing Statistical review is performed after peer review. We invite an expert in Biomedical Statistics to evaluate the statistical method used in the paper, including t test (group or paired comparisons), chi-squared test, ridit, probit, logit, regression (linear, curvilinear, or stepwise), correlation, analysis of variance, analysis of covariance, etc. The reviewing points include: (1) Statistical methods should be described when they are used to verify the results; (2) Whether the statistical techniques are suitable or correct; (3) Only homogeneous data can be averaged. Standard deviations are preferred to standard errors. Give the number of observations and subjects (n). Losses in observations, such as drop-outs from the study should be reported; (4) Values such as ED50, LD50, IC50 should have their 95% confidence limits calculated and compared by weighted probit analysis (Bliss and Finney); and (5) The word significantly should be replaced by its synonyms (if it indicates extent) or the P value (if it indicates statistical significance). Conflict-of-interest statement In the interests of transparency and to help reviewers assess any potential bias, WJG requires authors of all papers to declare any competing commercial, personal, political, intellectual, or religious interests in relation to the submitted work. Referees are also asked to indicate any potential conflict they might have reviewing a particular paper. Before submitting, authors are suggested to read Uniform Requirements for Manuscripts Submitted to Biomedical Journals: Ethical Considerations in the Conduct and Reporting of Research: Conflicts of Interest from International Committee of Medical Journal Editors (ICMJE), which is available at: ethical_4conflicts.html. Statement of informed consent Manuscripts should contain a statement to the effect that all human studies have been reviewed by the appropriate ethics com- II July 14, 2014 Volume 20 Issue 26

445 Instructions to authors mittee or it should be stated clearly in the text that all persons gave their informed consent prior to their inclusion in the study. Details that might disclose the identity of the subjects under study should be omitted. Authors should also draw attention to the Code of Ethics of the World Medical Association (Declaration of Helsinki, 1964, as revised in 2004). Sample wording: [Name of individual] has received fees for serving as a speaker, a consultant and an advisory board member for [names of organizations], and has received research funding from [names of organization]. [Name of individual] is an employee of [name of organization]. [Name of individual] owns stocks and shares in [name of organization]. [Name of individual] owns patent [patent identification and brief description]. Statement of human and animal rights When reporting the results from experiments, authors should follow the highest standards and the trial should conform to Good Clinical Practice (for example, US Food and Drug Administration Good Clinical Practice in FDA-Regulated Clinical Trials; UK Medicines Research Council Guidelines for Good Clinical Practice in Clinical Trials) and/or the World Medical Association Declaration of Helsinki. Generally, we suggest authors follow the lead investigator s national standard. If doubt exists whether the research was conducted in accordance with the above standards, the authors must explain the rationale for their approach and demonstrate that the institutional review body explicitly approved the doubtful aspects of the study. Before submitting, authors should make their study approved by the relevant research ethics committee or institutional review board. If human participants were involved, manuscripts must be accompanied by a statement that the experiments were undertaken with the understanding and appropriate informed consent of each. Any personal item or information will not be published without explicit consents from the involved patients. If experimental animals were used, the materials and methods (experimental procedures) section must clearly indicate that appropriate measures were taken to minimize pain or discomfort, and details of animal care should be provided. SUBMISSION OF MANUSCRIPTS Manuscripts should be typed in 1.5 line spacing and 12 pt. Book Antiqua with ample margins. Number all pages consecutively, and start each of the following sections on a new page: Title Page, Abstract, Introduction, Materials and Methods, Results, Discussion, Acknowledgements, References, Tables, Figures, and Figure Legends. Neither the editors nor the publisher are responsible for the opinions expressed by contributors. Manuscripts formally accepted for publication become the permanent property of Baishideng Publishing Group Co., Limited, and may not be reproduced by any means, in whole or in part, without the written permission of both the authors and the publisher. We reserve the right to copy-edit and put onto our website accepted manuscripts. Authors should follow the relevant guidelines for the care and use of laboratory animals of their institution or national animal welfare committee. For the sake of transparency in regard to the performance and reporting of clinical trials, we endorse the policy of the ICMJE to refuse to publish papers on clinical trial results if the trial was not recorded in a publiclyaccessible registry at its outset. The only register now available, to our knowledge, is sponsored by the United States National Library of Medicine and we encourage all potential contributors to register with it. However, in the case that other registers become available you will be duly notified. A letter of recommendation from each author s organization should be provided with the contributed article to ensure the privacy and secrecy of research is protected. Authors should retain one copy of the text, tables, photographs and illustrations because rejected manuscripts will not be returned to the author(s) and the editors will not be responsible for loss or damage to photographs and illustrations sustained during mailing. Online submissions Manuscripts should be submitted through the Online Submission System at: Authors are highly recommended to consult the ONLINE INSTRUC- TIONS TO AUTHORS ( g_info_ htm) before attempting to submit online. For assistance, authors encountering problems with the Online Submission System may send an describing the problem to bpgoffice@wjgnet.com, or by telephone: If you submit your manuscript online, do not make a postal contribution. Repeated online submission for the same manuscript is strictly prohibited. MANUSCRIPT PREPARATION All contributions should be written in English. All articles must be submitted using word-processing software. All submissions must be typed in 1.5 line spacing and 12 pt. Book Antiqua with ample margins. Style should conform to our house format. Required information for each of the manuscript sections is as follows: Title page Title: Title should be less than 12 words. Running title: A short running title of less than 6 words should be provided. Authorship: Authorship credit should be in accordance with the standard proposed by ICMJE, based on (1) substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; (2) drafting the article or revising it critically for important intellectual content; and (3) final approval of the version to be published. Authors should meet conditions 1, 2, and 3. Institution: Author names should be given first, then the complete name of institution, city, province and postcode. For example, Xu-Chen Zhang, Li-Xin Mei, Department of Pathology, Chengde Medical College, Chengde , Hebei Province, China. One author may be represented from two institutions, for example, George Sgourakis, Department of General, Visceral, and Transplantation Surgery, Essen 45122, Germany; George Sgourakis, 2nd Surgical Department, Korgialenio-Benakio Red Cross Hospital, Athens 15451, Greece. Author contributions: The format of this section should be: Author contributions: Wang CL and Liang L contributed equally to this work; Wang CL, Liang L, Fu JF, Zou CC, Hong F and Wu XM designed the research; Wang CL, Zou CC, Hong F and Wu XM performed the research; Xue JZ and Lu JR contributed new reagents/analytic tools; Wang CL, Liang L and Fu JF analyzed the data; and Wang CL, Liang L and Fu JF wrote the paper. Supportive foundations: The complete name and number of supportive foundations should be provided, e.g. Supported by National Natural Science Foundation of China, No Correspondence to: Only one corresponding address should be III July 14, 2014 Volume 20 Issue 26

446 Instructions to authors provided. Author names should be given first, then author title, affiliation, the complete name of institution, city, postcode, province, country, and . All the letters in the should be in lower case. A space interval should be inserted between country name and address. For example, Montgomery Bissell, MD, Professor of Medicine, Chief, Liver Center, Gastroenterology Division, University of California, Box 0538, San Francisco, CA 94143, United States. montgomery.bissell@ucsf.edu Telephone and fax: Telephone and fax should consist of +, country number, district number and telephone or fax number, e.g., Telephone: Fax: Peer reviewers: All articles received are subject to peer review. Normally, three experts are invited for each article. Decision on acceptance is made only when at least two experts recommend publication of an article. All peer-reviewers are acknowledged on Express Submission and Peer-review System website. Abstract There are unstructured abstracts (no less than 200 words) and structured abstracts. The specific requirements for structured abstracts are as follows: An informative, structured abstract should accompany each manuscript. Abstracts of original contributions should be structured into the following sections: AIM (no more than 20 words; Only the purpose of the study should be included. Please write the Aim in the form of To investigate/study/ ), METH- ODS (no less than 140 words for Original Articles; and no less than 80 words for Brief Articles), RESULTS (no less than 150 words for Original Articles and no less than 120 words for Brief Articles; You should present P values where appropriate and must provide relevant data to illustrate how they were obtained, e.g., 6.92 ± 3.86 vs 3.61 ± 1.67, P < 0.001), and CONCLUSION (no more than 26 words). Key words Please list 5-10 key words, selected mainly from Index Medicus, which reflect the content of the study. Core tip Please write a summary of less than 100 words to outline the most innovative and important arguments and core contents in your paper to attract readers. Text For articles of these sections, original articles and brief articles, the main text should be structured into the following sections: INTRODUCTION, MATERIALS AND METHODS, RE- SULTS and DISCUSSION, and should include appropriate Figures and Tables. Data should be presented in the main text or in Figures and Tables, but not in both. Illustrations Figures should be numbered as 1, 2, 3, etc., and mentioned clearly in the main text. Provide a brief title for each figure on a separate page. Detailed legends should not be provided under the figures. This part should be added into the text where the figures are applicable. Keeping all elements compiled is necessary in line-art image. Scale bars should be used rather than magnification factors, with the length of the bar defined in the legend rather than on the bar itself. File names should identify the figure and panel. Avoid layering type directly over shaded or textured areas. Please use uniform legends for the same subjects. For example: Figure 1 Pathological changes in atrophic gastritis after treatment. A:...; B:...; C:...; D:...; E:...; F:...; G: etc. It is our principle to publish high resolution-figures for the E-versions. Tables Three-line tables should be numbered 1, 2, 3, etc., and mentioned clearly in the main text. Provide a brief title for each table. Detailed legends should not be included under tables, but rather added into the text where applicable. The information should complement, but not duplicate the text. Use one horizontal line under the title, a second under column heads, and a third below the Table, above any footnotes. Vertical and italic lines should be omitted. Notes in tables and illustrations Data that are not statistically significant should not be noted. a P < 0.05, b P < 0.01 should be noted (P > 0.05 should not be noted). If there are other series of P values, c P < 0.05 and d P < 0.01 are used. A third series of P values can be expressed as e P < 0.05 and f P < Other notes in tables or under illustrations should be expressed as 1 F, 2 F, 3 F; or sometimes as other symbols with a superscript (Arabic numerals) in the upper left corner. In a multi-curve illustration, each curve should be labeled with,,,,,, etc., in a certain sequence. Acknowledgments Brief acknowledgments of persons who have made genuine contributions to the manuscript and who endorse the data and conclusions should be included. Authors are responsible for obtaining written permission to use any copyrighted text and/or illustrations. REFERENCES Coding system The author should number the references in Arabic numerals according to the citation order in the text. Put reference numbers in square brackets in superscript at the end of citation content or after the cited author s name. For citation content which is part of the narration, the coding number and square brackets should be typeset normally. For example, Crohn s disease (CD) is associated with increased intestinal permeability [1,2]. If references are cited directly in the text, they should be put together within the text, for example, From references [19,22-24], we know that.... When the authors write the references, please ensure that the order in text is the same as in the references section, and also ensure the spelling accuracy of the first author s name. Do not list the same citation twice. PMID and DOI Pleased provide PubMed citation numbers to the reference list, e.g., PMID and DOI, which can be found at nlm.nihgov/sites/entrez?db=pubmed and org/simpletextquery/, respectively. The numbers will be used in E-version of this journal. Style for journal references Authors: the name of the first author should be typed in boldfaced letters. The family name of all authors should be typed with the initial letter capitalized, followed by their abbreviated first and middle initials. (For example, Lian-Sheng Ma is abbreviated as Ma LS, Bo-Rong Pan as Pan BR). The title of the cited article and italicized journal title (journal title should be in its abbreviated form as shown in PubMed), publication date, volume number (in black), start page, and end page [PMID: IV July 14, 2014 Volume 20 Issue 26

447 Instructions to authors DOI: /wjg ]. Style for book references Authors: the name of the first author should be typed in boldfaced letters. The surname of all authors should be typed with the initial letter capitalized, followed by their abbreviated middle and first initials. (For example, Lian-Sheng Ma is abbreviated as Ma LS, Bo-Rong Pan as Pan BR) Book title. Publication number. Publication place: Publication press, Year: start page and end page. Format Journals English journal article (list all authors and include the PMID where applicable) 1 Jung EM, Clevert DA, Schreyer AG, Schmitt S, Rennert J, Kubale R, Feuerbach S, Jung F. Evaluation of quantitative contrast harmonic imaging to assess malignancy of liver tumors: A prospective controlled two-center study. World J Gastroenterol 2007; 13: [PMID: DOI: /wjg ] Chinese journal article (list all authors and include the PMID where applicable) 2 Lin GZ, Wang XZ, Wang P, Lin J, Yang FD. Immunologic effect of Jianpi Yishen decoction in treatment of Pixudiarrhoea. Shijie Huaren Xiaohua Zazhi 1999; 7: In press 3 Tian D, Araki H, Stahl E, Bergelson J, Kreitman M. Signature of balancing selection in Arabidopsis. Proc Natl Acad Sci USA 2006; In press Organization as author 4 Diabetes Prevention Program Research Group. Hypertension, insulin, and proinsulin in participants with impaired glucose tolerance. Hypertension 2002; 40: [PMID: PMCID: DOI: /01.HYP ] Both personal authors and an organization as author 5 Vallancien G, Emberton M, Harving N, van Moorselaar RJ; Alf-One Study Group. Sexual dysfunction in 1, 274 European men suffering from lower urinary tract symptoms. J Urol 2003; 169: [PMID: DOI: /01. ju ] No author given 6 21st century heart solution may have a sting in the tail. BMJ 2002; 325: 184 [PMID: DOI: /bmj ] Volume with supplement 7 Geraud G, Spierings EL, Keywood C. Tolerability and safety of frovatriptan with short- and long-term use for treatment of migraine and in comparison with sumatriptan. Headache 2002; 42 Suppl 2: S93-99 [PMID: DOI: /j s2.7.x] Issue with no volume 8 Banit DM, Kaufer H, Hartford JM. Intraoperative frozen section analysis in revision total joint arthroplasty. Clin Orthop Relat Res 2002; (401): [PMID: DOI: / ] No volume or issue 9 Outreach: Bringing HIV-positive individuals into care. HRSA Careaction 2002; 1-6 [PMID: ] Books Personal author(s) 10 Sherlock S, Dooley J. Diseases of the liver and billiary system. 9th ed. Oxford: Blackwell Sci Pub, 1993: Chapter in a book (list all authors) 11 Lam SK. Academic investigator s perspectives of medical treatment for peptic ulcer. In: Swabb EA, Azabo S. Ulcer disease: investigation and basis for therapy. New York: Marcel Dekker, 1991: Author(s) and editor(s) 12 Breedlove GK, Schorfheide AM. Adolescent pregnancy. 2nd ed. Wieczorek RR, editor. White Plains (NY): March of Dimes Education Services, 2001: Conference proceedings 13 Harnden P, Joffe JK, Jones WG, editors. Germ cell tumours V. Proceedings of the 5th Germ cell tumours Conference; 2001 Sep 13-15; Leeds, UK. New York: Springer, 2002: Conference paper 14 Christensen S, Oppacher F. An analysis of Koza s computational effort statistic for genetic programming. In: Foster JA, Lutton E, Miller J, Ryan C, Tettamanzi AG, editors. Genetic programming. EuroGP 2002: Proceedings of the 5th European Conference on Genetic Programming; 2002 Apr 3-5; Kinsdale, Ireland. Berlin: Springer, 2002: Electronic journal (list all authors) 15 Morse SS. Factors in the emergence of infectious diseases. Emerg Infect Dis serial online, , cited ; 1(1): 24 screens. Available from: URL: ncidod/eid/index.htm Patent (list all authors) 16 Pagedas AC, inventor; Ancel Surgical R&D Inc., assignee. Flexible endoscopic grasping and cutting device and positioning tool assembly. United States patent US Aug 1 Statistical data Write as mean ± SD or mean ± SE. Statistical expression Express t test as t (in italics), F test as F (in italics), chi square test as χ 2 (in Greek), related coefficient as r (in italics), degree of freedom as υ (in Greek), sample number as n (in italics), and probability as P (in italics). Units Use SI units. For example: body mass, m (B) = 78 kg; blood pressure, p (B) = 16.2/12.3 kpa; incubation time, t (incubation) = 96 h, blood glucose concentration, c (glucose) 6.4 ± 2.1 mmol/l; blood CEA mass concentration, p (CEA) = mg/l; CO 2 volume fraction, 50 ml/l CO 2, not 5% CO 2 ; likewise for 40 g/l formaldehyde, not 10% formalin; and mass fraction, 8 ng/g, etc. Arabic numerals such as 23, 243, 641 should be read The format for how to accurately write common units and quantums can be found at: g_info_ htm. Abbreviations Standard abbreviations should be defined in the abstract and on first mention in the text. In general, terms should not be abbreviated unless they are used repeatedly and the abbreviation is helpful to the reader. Permissible abbreviations are listed in Units, Symbols and Abbreviations: A Guide for Biological and Medical Editors and Authors (Ed. Baron DN, 1988) published by The Royal Society of Medicine, London. Certain commonly used abbreviations, such as DNA, RNA, HIV, LD50, PCR, HBV, ECG, WBC, RBC, CT, ESR, CSF, IgG, ELISA, PBS, ATP, EDTA, mab, can be used directly without further explanation. V July 14, 2014 Volume 20 Issue 26

448 Instructions to authors Italics Quantities: t time or temperature, c concentration, A area, l length, m mass, V volume. Genotypes: gyra, arg 1, c myc, c fos, etc. Restriction enzymes: EcoRI, HindI, BamHI, Kbo I, Kpn I, etc. Biology: H. pylori, E coli, etc. Examples for paper writing All types of articles writing style and requirement will be found in the link: Info.aspx?id=15. RESUBMISSION OF THE REVISED MANUSCRIPTS Authors must revise their manuscript carefully according to the revision policies of Baishideng Publishing Group Co., Limited. The revised version, along with the signed copyright transfer agreement, responses to the reviewers, and English language Grade A certificate (for non-native speakers of English), should be submitted to the online system via the link contained in the sent by the editor. If you have any questions about the revision, please send to esps@wjgnet.com. Language evaluation The language of a manuscript will be graded before it is sent for revision. (1) Grade A: priority publishing; (2) Grade B: minor language polishing; (3) Grade C: a great deal of language polishing needed; and (4) Grade D: rejected. Revised articles should reach Grade A. Copyright assignment form Please download a Copyright assignment form from Responses to reviewers Please revise your article according to the comments/suggestions provided by the reviewers. The format for responses to the reviewers comments can be found at: com/ /g_info_ htm Proof of financial support For papers supported by a foundation, authors should provide a copy of the approval document and serial number of the foundation. STATEMENT ABOUT ANONYMOUS PUBLICA- TION OF THE PEER REVIEWERS COMMENTS In order to increase the quality of peer review, push authors to carefully revise their manuscripts based on the peer reviewers' comments, and promote academic interactions among peer reviewers, authors and readers, we decide to anonymously publish the reviewers comments and author s responses at the same time the manuscript is published online. PUBLICATION FEE WJG is an international, peer-reviewed, open access, online journal. Articles published by this journal are distributed under the terms of the Creative Commons Attribution Non-commercial License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited, the use is non commercial and is otherwise in compliance with the license. Authors of accepted articles must pay a publication fee. Publication fee: 1398 USD per article. All invited articles are published free of charge. VI July 14, 2014 Volume 20 Issue 26

449 Published by Baishideng Publishing Group Inc 8226 Regency Drive, Pleasanton, CA 94588, USA Telephone: Fax: Help Desk: I S S N Baishideng Publishing Group Inc. All rights reserved.