World Journal of Gastroenterology

Transcription

1 ISSN (print) ISSN (online) World Journal of Gastroenterology World J Gastroenterol 2014 April 7; 20(13):

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 13 April 7, 2014 TOPIC HIGHLIGHT 3391 Challenges of recurrent hepatitis C in the liver transplant patient Dhanasekaran R, Firpi RJ 3401 Computational biology approach to uncover hepatitis C virus helicase operation Flechsig H 3410 Chronic hepatitis C virus infection and atherosclerosis: Clinical impact and mechanisms Adinolfi LE, Zampino R, Restivo L, Lonardo A, Guerrera B, Marrone A, Nascimbeni F, Florio A, Loria P 3418 Adaptive immune response during hepatitis C virus infection Larrubia JR, Moreno-Cubero E, Lokhande MU, García-Garzón S, Lázaro A, Miquel J, Perna C, Sanz-de-Villalobos E 3431 Tools for the diagnosis of hepatitis C virus infection and hepatic fibrosis staging Saludes V, González V, Planas R, Matas L, Ausina V, Martró E 3443 Immunologic, metabolic and genetic factors in hepatitis C virus infection Fierro NA, Gonzalez-Aldaco K, Torres-Valadez R, Martinez-Lopez E, Roman S, Panduro A 3457 How hepatitis C virus invades hepatocytes: The mystery of viral entry Zhu YZ, Qian XJ, Zhao P, Qi ZT 3468 Restoring the gut microbiome for the treatment of inflammatory bowel diseases Allegretti JR, Hamilton MJ 3475 Therapeutic drug monitoring in patients with inflammatory bowel disease Yarur AJ, Abreu MT, Deshpande AR, Kerman DH, Sussman DA 3485 Advanced therapeutic endoscopist and inflammatory bowel disease: Dawn of a new role Modha K, Navaneethan U April 7, 2014 Volume 20 Issue 13

12 Contents World Journal of Gastroenterology Volume 20 Number 13 April 7, From conception to delivery: Managing the pregnant inflammatory bowel disease patient Huang VW, Habal FM 3507 Glucose intolerance and diabetes mellitus in ulcerative colitis: Pathogenetic and therapeutic implications Maconi G, Furfaro F, Sciurti R, Bezzio C, Ardizzone S, de Franchis R 3516 Hepatitis B and C virus reactivation in immunosuppressed patients with inflammatory bowel disease Sansone S, Guarino M, Castiglione F, Rispo A, Auriemma F, Loperto I, Rea M, Caporaso N, Morisco F 3525 Clinical management of inflammatory bowel disease in the organ recipient Indriolo A, Ravelli P 3534 Pharmacogenetics of azathioprine in inflammatory bowel disease: A role for glutathione-s-transferase? Stocco G, Pelin M, Franca R, De Iudicibus S, Cuzzoni E, Favretto D, Martelossi S, Ventura A, Decorti G 3542 Anemia in inflammatory bowel disease: A neglected issue with relevant effects Guagnozzi D, Lucendo AJ 3552 Psychiatric comorbidity in the treatment of patients with inflammatory bowel disease Filipovic BR, Filipovic BF 3564 Colon-specific prodrugs of 4-aminosalicylic acid for inflammatory bowel disease Dhaneshwar SS REVIEW 3572 Remote ischemic preconditioning as treatment for non-ischemic gastrointestinal disorders: Beyond ischemia-reperfusion injury Camara-Lemarroy CR 3582 What physicians should know about the management of chronic hepatitis B in children: East side story Choe HJ, Choe BH II April 7, 2014 Volume 20 Issue 13

13 Contents World Journal of Gastroenterology Volume 20 Number 13 April 7, 2014 MINIREVIEWS 3590 Contrast-enhanced ultrasound in the diagnosis of nodules in liver cirrhosis Kim TK, Jang HJ ORIGINAL ARTICLE 3597 Clinicopathological study of primary biliary cirrhosis with interface hepatitis compared to autoimmune hepatitis Kobayashi M, Kakuda Y, Harada K, Sato Y, Sasaki M, Ikeda H, Terada M, Mukai M, Kaneko S, Nakanuma Y RESEARCH REPORT 3609 Association of rs , rs and rs polymorphisms with anti-tnf medication response in Greek patients with Crohn's disease Thomas D, Gazouli M, Karantanos T, Rigoglou S, Karamanolis G, Bramis K, Zografos G, Theodoropoulos GE 3615 Rabeprazole, clarithromycin, and amoxicillin Helicobacter pylori eradication therapy: Report of an efficacy study Onyekwere CA, Odiagah JN, Igetei R, Duro Emanuel AO, Ekere F, Smith S 3620 Influence of the safety and diagnostic accuracy of preoperative endoscopic ultrasound-guided fine-needle aspiration for resectable pancreatic cancer on clinical performance Kudo T, Kawakami H, Kuwatani M, Eto K, Kawahata S, Abe Y, Onodera M, Ehira N, Yamato H, Haba S, Kawakubo K, Sakamoto N 3628 Tumors of the angle of Treitz: A single-center experience Xie YB, Liu H, Cui L, Xing GS, Yang L, Sun YM, Bai XF, Zhao DB, Wang CF, Tian YT 3635 Seroepidemiology of Helicobacter pylori infection in elderly people in the Beijing region, China Zhang M, Zhou YZ, Li XY, Tang Z, Zhu HM, Yang Y, Chhetri JK 3640 Prognostic value of number of examined lymph nodes in patients with nodenegative gastric cancer Jiao XG, Deng JY, Zhang RP, Wu LL, Wang L, Liu HG, Hao XS, Liang H 3649 Prevalence and characteristics of dyspepsia among college students in Zhejiang Province Li M, Lu B, Chu L, Zhou H, Chen MY 3655 Lack of association between apolipoprotein C3 gene polymorphisms and risk III April 7, 2014 Volume 20 Issue 13

14 Contents World Journal of Gastroenterology Volume 20 Number 13 April 7, 2014 of nonalcoholic fatty liver disease in a Chinese Han population Niu TH, Jiang M, Xin YN, Jiang XJ, Lin ZH, Xuan SY E VIDENCE-BASED MEDICINE 3663 Psychosocial issues in evidence-based guidelines on inflammatory bowel diseases: A review Häuser W, Moser G, Klose P, Mikocka-Walus A RETROSPECTIVE STUDY 3672 Transcription factor ERG is a specific and sensitive diagnostic marker for hepatic angiosarcoma Wang ZB, Yuan J, Chen W, Wei LX META-ANALYSIS 3680 Evidence based medicine and surgical approaches for colon cancer: Evidences, benefits and limitations of the laparoscopic vs open resection Lorenzon L, La Torre M, Ziparo V, Montebelli F, Mercantini P, Balducci G, Ferri M CASE REPORT 3693 Colonic and anal metastases from pancreato-biliary malignancies Ejtehadi F, Chatzizacharias NA, Brais RJ, Hall NR, Godfrey EM, Huguet E, Praseedom RK, Jah A 3698 Colonoscopy-induced ischemic colitis in patients without risk factors Lee SO, Kim SH, Jung SH, Park CW, Lee MJ, Lee JA, Koo HC, Kim A, Han HY, Kang DW 3703 Successful treatment of liver abscess secondary to foreign body penetration of the alimentary tract: A case report and literature review Chong LW, Sun CK, Wu CC, Sun CK 3712 Hemobilia and other complications caused by percutaneous ultrasoundguided liver biopsy Zhou HB 3716 Ulcerative colitis flair induced by mesalamine suppositories hypersensitivity Ding H, Liu XC, Mei Q, Xu JM, Hu XY, Hu J IV April 7, 2014 Volume 20 Issue 13

15 Contents World Journal of Gastroenterology Volume 20 Number 13 April 7, 2014 APPENDIX I-VI Instructions to authors ABOUT COVER Editorial Board Member of World Journal of Gastroenterology, YSalvatore Gruttadauria, MD, Associate Professor, Abdominal Transplant Surgery, Istituto Mediteraneo Trapianti e Terapie ad Alta Specializzazione (ISMETT), University of Pittsburgh Medical Center, Palermo 91124, 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: Cai-Hong Wang Proofing Editor-in-Chief: Lian-Sheng Ma Responsible Science Editor: Yuan Qi Proofing Editorial Office Director: Jin-Lei Wang 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: bpgoffice@wjgnet.com PUBLISHER Baishideng Publishing Group Co., Limited Flat C, 23/F., Lucky Plaza, Lockhart Road, Wan Chai, Hong Kong, China Fax: Telephone: bpgoffice@wjgnet.com PUBLICATION DATE April 7, 2014 COPYRIGHT 2014 Baishideng Publishing Group Co., Limited. Articles published by this Open-Access 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. SPECIAL STATEMENT All articles published in this journal represent the viewpoints of the authors except where indicated otherwise. INSTRUCTIONS TO AUTHORS Full instructions are available online at wjgnet.com/ /g_info_ htm ONLINE SUBMISSION V April 7, 2014 Volume 20 Issue 13

16 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (2): Hepatitis C virus TOPIC HIGHLIGHT Challenges of recurrent hepatitis C in the liver transplant patient Renumathy Dhanasekaran, Roberto J Firpi Renumathy Dhanasekaran, Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN 55901, United States Roberto J Firpi, Division of Gastroenterology, Hepatology and Nutrition, University of Florida, Gainesville, FL 32610, United States Author contributions: Dhanasekaran R wrote the manuscript; Firpi RJ conceptualized the paper, edited the manuscript and supervisor. Correspondence to: Roberto J Firpi, MD, MS, Associate Professor, Division of Gastroenterology, Hepatology and Nutrition, University of Florida, 1600 SW Archer Rd, MSB M440, Gainesville, FL 32610, United States. roberto.firpi@medicine.ufl.edu Telephone: Fax: Received: September 29, 2013 Revised: November 22, 2013 Accepted: March 7, 2014 Published online: April 7, 2014 Abstract Cirrhosis secondary to hepatitis C virus (HCV) is a very common indication for liver transplant. Unfortunately recurrence of HCV is almost universal in patients who are viremic at the time of transplant. The progression of fibrosis has been shown to be more rapid in the post-transplant patients than in the transplant naïve, hence treatment of recurrent HCV needs to be considered for all patients with documented recurrent HCV. Management of recurrent HCV is a challenging situation both for patients and physicians due to multiple reasons as discussed in this review. The standard HCV treatment with pegylated interferon and Ribavarin can be considered in these patients but it leads to a lower rate of sustained virologic clearance than in the nontransplanted population. Some of the main challenges associated with treating recurrent HCV in post-transplant patients include the presence of cytopenias; need to monitor drug-drug interactions and the increased incidence of renal compromise. In spite of these obstacles all patients with recurrent HCV should be considered for treatment since it is associated with improvement in survival and a delay in fibrosis progression. With the arrival of direct acting antiviral drugs there is renewed hope for better outcomes in the treatment of post-transplant HCV recurrence. This review evaluates current literature on this topic and identifies challenges associated with the management of post-transplant HCV recurrence Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Cirrhosis; Hepatitis C; Recurrence; Transplant; Interferon Core tip: Management of recurrent hepatitis C in posttransplant patient is challenging but can be rewarding as treatment has been shown to improve survival and slow fibrosis progression. This review summarizes major challenges in this population and discusses the natural history of post-transplant Hepatitis C virus (HCV), risk factors for HCV recurrence, management of immunosuppression and current treatment options. The preliminary data on use of newer direct acting antiviral drugs in the post-transplant setting has also been included. Dhanasekaran R, Firpi RJ. Challenges of recurrent hepatitis C in the liver transplant patient. World J Gastroenterol 2014; 20(13): Available from: URL: com/ /full/v20/i13/3391.htm DOI: org/ /wjg.v20.i INTRODUCTION Cirrhosis related to hepatitis C virus (HCV) infection is a significant cause of morbidity and mortality worldwide. The World Health Organization estimates that around 3391 April 7, 2014 Volume 20 Issue 13

17 Dhanasekaran R et al. Challenges of post-transplant hepatitis C recurrence 150 million people worldwide are chronically infected with HCV, and that more than people die every year from HCV related liver diseases [1]. Liver transplantation (LT) is the best curative option for patients with HCV related cirrhosis and this is the leading indication for LT in the United States [2]. Unfortunately post-transplant HCV recurrence is almost universal, and it presents a challenging situation to both patients and transplant hepatologists alike. Patients who are wrestling posttransplant surgical complications, medication related side effects and rejection episodes have to also worry about ongoing allograft injury secondary to HCV recurrence. In addition, physicians face the challenge of identifying candidates eligible for antiviral therapy, ascertaining the ideal time to initiate therapy and find means to combat side effects of therapy, in this cohort of very sick patients. NATURAL HISTORY OF RECURRENCE Recurrence of HCV viremia occurs very early after transplant, with initial studies showing presence of HCV particles in serum of 96% of the patients who underwent LT for HCV [3]. Allograft infection is believed to occur during reperfusion of liver immediately after transplantation and in fact the viral particles have been found to replicate within few hours of transplantation [4]. HCV core and NS3 viral peptide sequences have been found to be identical before and after LT in most patients, suggesting that the sequence is largely preserved [5]. Also of concern is the fact that HCV infection is found to be more rapidly progressive in the post LT setting with two major forms being reported [6]. Severe cholestatic hepatitis is the more aggressive form and this presentation is unique to patients on immunosuppression who are exposed to very high viral load. The second form is the more common, chronic hepatitis with associated rapid progression of fibrosis. As mentioned above, the recurrence of HCV viremia post LT is universal but the severity of recurrence and rate of fibrosis progression are variable and are influenced by multiple donor and recipient factors. And unfortunately liver tests are not sensitive or specific in identifying graft dysfunction or degree of fibrosis in post LT patients, which is the reason several centers propose performing annual protocol biopsies in patients transplanted for HCV. Early diagnosis of allograft injury can lead to earlier initiation of therapy, with the caveat that interpretation of liver biopsies is not straightforward in this population due to frequent overlapping pathologies like reperfusion injury or rejection episodes. These protocol biopsies have also been useful in understanding the natural history of recurrent HCV. Several studies have demonstrated accelerated rate of fibrosis progression leading to increased graft loss with reported rates of cirrhosis ranging from 20% to 54% at 5 years [7-10]. We routinely perform protocol biopsies in our center and early experience demonstrated accelerated rate of fibrosis in post LT patients at a rate of 0.8 per year and we found that the one year protocol biopsy was useful to predict rate of progression [11]. In a recent large multicenter study involving 1264 patients, the cumulative risk of cirrhosis at 3 years was 38% for women and 33% for men [12]. This rapid progression in fibrosis translates to overall worse clinical outcomes in HCV patients. In a retrospective analysis of UNOS database HCV recurrence was found to significantly and independently impair patient and allograft survival after liver transplantation [13]. In this large study involving more than patients, HCV was associated a 20% increased risk for mortality and 30% increased risk for graft failure. RISK FACTORS FOR RECURRENCE Donor factors Several studies have found that HCV recurrence is more rapid and more severe in patients who received organs from older donors [14-16]. The reason for this is not entirely well understood, but it is probably multifactorial and is likely related to age related changes in liver including decreased hepatocyte volume, pseudocapillarization of sinusoids and reduced microcirculation [17,18]. Older livers also have changes in hepatic stellate cell activation and decreased liver regenerative capacity, which in the background of ongoing recurrence of hepatitis could contribute to the rapid progression of fibrosis in post-transplant patients [19]. Though donor age significantly affects outcome, it essentially is a non-modifiable risk factor. In this era of organ shortage, avoiding older age donors for hepatitis C patients is not a practical solution since more than a third of adult deceased donors are older than fifty years [2]. A few early studies had suggested than recipients of living donor liver transplantation (LDLT) were at higher risk for recurrence than deceased donor transplants (DDLT) [20]. Garcia-Retortillo et al [20] reported that LDLT patients had a 2.8-fold higher risk of developing severe recurrence when compared to DDLT. But other well designed larger studies have refuted these results [21-23]. And one of the studies with long term follow up actually showed better outcomes in LDLT with improved survival and lower fibrosis scores [24]. In a recent meta-analysis of fourteen studies, with a total of 2024 participants, no significant differences were found between LDLT and DDLT in terms of long-term patient survival, graft survival, or HCV recurrence. In summary, LDLT can be safely offered in experienced centers to HCV patients, without impacting significantly outcomes. Other donor related factors that have been studied include HLA matching, degree of steatosis of donor liver, occurrence of reperfusion injury and cold ischemic time. But none of these factors have been established as playing a causative role in fibrosis progression. Donor livers with > 40%-50% steatosis are usually not accepted by transplant centers since these livers have been associated with higher rates of primary non function and graft failure [25,26]. Also, presence of significant donor steatosis 3392 April 7, 2014 Volume 20 Issue 13

18 Dhanasekaran R et al. Challenges of post-transplant hepatitis C recurrence has also been associated with more rapid progression of HCV recurrence; raising the question of whether such livers should even be used in this subset. But data on milder degree of steatosis is not clear or uniform. A few studies have proposed that it is safe to use donor livers with mild steatosis, as they do not have any impact on graft survival [27,28]. But Briceño et al [29] reported that three year post LT graft survival was 95%, if steatosis was < 30% and 69% if donor steatosis was > 30% (P = ). This study goes so far as to suggest that LT with > 30% steatotic donor livers should be precluded for HCV recipients. This appears to be safe approach, but we know that 20%-25% of the general population has hepatic steatosis secondary to nonalcoholic fatty liver disease. And the people in the donor pool, which includes people involved in motor vehicle accidents, usually have a history of alcohol use related steatosis too. From a practical standpoint it might not be entirely possible to direct all livers with greater than 30% steatosis away from hepatitis C patients, but recognition of risk for rapid progression of HCV recurrence can lead to closer follow up and earlier treatment initiation. Viral factors Viral replication can be detected very early in the postoperative period [30] but viral kinetics are variable. Serum HCV RNA levels typically increase rapidly from the second week post LT and peak by the fourth postoperative month [31]. Outside of the transplant setting, the level of viremia has not been shown to correlate with disease severity in patients with HCV [32,33]. However, it appears to play a significant role in progression of post LT recurrence. A few early studies did not show correlation between level of viremia and risk for progression and even suggested that a carrier state might exist with high levels of viremia and absent inflammation [8,34]. Several other well conducted studies have shown that high level of post LT viremia was independently associated with more rapid progression of hepatitis [7,35-37]. Shackel et al [36] reported that a one year peak viral load > 10 7 IU/mL was associated with a hazard ratio of 8.68 for worse patient survival. An international consensus panel has indeed accepted both pre and early post LT viral load as established risk factors for severe recurrence [38]. A correlation between early levels of viremia and subsequent allograft injury suggests that initiation of antiviral therapy early in the post LT course might be desirable. Another viral factor that has been explored is genotype, with early studies suggesting than genotype1b was associated with higher risk for recurrent hepatitis [26,39]. But later studies did not find any influence of genotype on outcomes [39,40]. Polymorphism in IL28B gene, which encodes interferon-lambda-3 (IFN-λ-3), has been established as a predictor of response to IFN based therapy in the nontransplant setting [41]. Charlton et al [42] examined the impact of IL28B polymorphisms in the post LT setting and evaluated the role of both donor and recipient IL28B status. Both recipient and donor liver IL28B genotype were strongly and independently associated with IFN-based treatment response in patients after LT. And interestingly recipient IL28B TT genotype was associated with more severe histological recurrence of HCV. In contrast, Lange et al [43] showed that donor IL28B had significant impact on the natural course and treatment outcome of HCV liver graft reinfection. Our center published data found that the rate of sustained viral response (SVR) to HCV therapy was 100% if both recipient and donor were CC genotype, while the SVR was only 25% if neither donor nor recipient had a CC genotype. Recipients and donors with CC genotype also had less fibrosis than recipients with genotypes CT and TT [44]. Overall, IL28B genotype appears to affect both the treatment response and also the natural history of recurrence. Consequently, once the relationships are more clearly established, there might be a role for preferentially offering donor livers with CC genotype to HCV patients. IMMUNOSUPPRESSION Corticosteroids Corticosteroids are used routinely as part of immunosuppressive regimen in LT patients both for induction and for management of episodes of acute cellular rejection (ACR). Initial studies in the non-transplant setting showed that use of steroids lead to a dose dependent increase in HCV viral load [45]. Gane et al [7] studied viral replication in post LT patients receiving steroids and showed that a more dramatic increase in viral load occurred in this population and methylprednisolone treatment for ACR was found to lead to a fold increase in serum HCV RNA. The mechanism for this is not clearly understood, but in vitro studies using a replicon model showed that treatment of hepatocytes with clinically relevant concentrations of steroids actually resulted in a slight decrease in HCV replication [46]. Therefore, the rapid replication noted in vivo is probably more related to the suppressed host immune response than to the virus. Steroids have been shown to mediate suppression of virus-specific plasmacytoid dendritic cells and T-cell responses potentially leading to unchecked viral replication [47,48]. Keeping in line with these findings, Berenguer et al [49] demonstrated that avoiding rapid steroid tapering and using a steroid sparing double induction immunosuppression regimen led to less severe disease. And other studies have similarly suggested that slow tapering of steroids and use of lower doses lead to better outcomes in HCV patients [50]. The results from clinical observational studies mentioned above appeared to support the idea of avoiding steroid boluses and avoid high dose steroids for induction, but the results from randomized controlled trials have been mixed. A recently published randomized controlled trial divided 75 HCV positive recipients to receive tacrolimus (TAC) plus a corticosteroid or TAC plus mycophenolate mofetil. They found that the steroid-avoidance regimen had no apparent impact on LDLT outcomes like survival 3393 April 7, 2014 Volume 20 Issue 13

19 Dhanasekaran R et al. Challenges of post-transplant hepatitis C recurrence or fibrosis progression [51]. But steroid boluses were used for episodes of acute cellular rejection in both arms of the study. In another two year prospective randomized study, steroid free immunosuppression was compared to steroid use and they found that steroid avoidance with basiliximab, calcineurin inhibitor, and mycophenolate sodium was safe and as effective as steroid containing immunosuppression in adult OLT [52]. Overall there appeared to be no benefit to the steroid free protocol in terms of ACR, fibrosis progression, patient survival, or graft survival rates [53]. In summary, steroids should be used cautiously in post LT HCV patients and, when needed, lower doses should be used with a slow taper. But there is not enough evidence to switch all HCV patients to a completely steroid free immunosuppression regimen. Calcineurin inhibitors TAC and Cyclosporine A (CysA) are the most widely used maintenance immunosuppression in post LT patients. In vitro studies have shown that CysA has a suppressive effect on the HCV replication and protein expression in cultured human hepatocyte cells and this effect appears to be independent of its immunosuppressive function [54,55]. We found similar effects in-vivo in two clinical trials at our center where the use of CysA was found to be associated with a modest HCV RNA drop and appeared to enhance the antiviral response to interferon based antiviral therapy when compared to TAC [56,57]. Similarly a meta-analysis of 17 studies concluded that CysA was associated with a marginally higher relative risk (RR) for achieving SVR with antiviral therapy when compared to TAC [58]. Though the data on antiviral effects of CysA are convincing, the same is not true regarding clinical outcome parameters like survival or rejection rates. A meta-analysis of 16 randomized trials including patients transplanted for all etiologies concluded that TAC was superior to CysA in improving survival and preventing acute rejection [59]. There is a lack of large randomized controlled trials addressing this question specifically in HCV patients. A retrospective study concluded that in patients undergoing LT for HCV-related liver disease, post-transplantation outcome was not related to the Calcineurin Inhibitors used, with no differences in bridging fibrosis, cirrhosis, cholestatic hepatitis, allograft loss or mortality between TAC and CysA [60]. In another retrospective analysis of data from UNOS database suggested that using CysA was associated with increased risk of patient death and graft failure [61]. Adding to the complexity of the situation, recent data has shown that the use of TAC is associated with ACR in patients with certain IL- 28B polymorphism and CysA might be beneficial in this specific subset [62]. Data on the role of other maintenance immunosuppressants like Azathioprine and Rapamycin in HCV recurrence or fibrosis progression is not very clear. Samonakis et al [63] have suggested that there may be a beneficial effect of maintenance azathioprine given for 6 mo or longer based on observational studies. Mycophenolic acid (MPA) has also been shown to be an inhibitor of HCV replication [64,65]. MPA was shown to have a distinct anti-hcv mechanism of action, independent of cell proliferation and guanosine depletion. Further clinical studies are needed exploring its use in HCV patients. Antilymphocyte agents which are used to treat rejection are associated with worse recurrence and hence have to be employed with caution [63]. TREATMENT Preemptive therapy Treating HCV patients on liver transplant waiting list can be considered as a prophylactic approach since absence of viremia at the time of transplant can prevent recurrence. Unfortunately, tolerance for treatment is very low in these patients with decompensated liver disease [66]. When newer drugs for HCV are available in the future there is hope that higher proportion of patients on the LT waiting list will be virus negative. Another treatment strategy is termed pre-emptive therapy, where patients are initiated on antiviral therapy early in the post LT period before the recurrence of viremia. The rationale behind this strategy is to prevent liver inflammation or fibrosis by early suppression of the virus. Although the reasoning behind this hypothesis is sound, data from clinical observational studies and trials do not support this strategy. High rates of discontinuation due to side effects and poor SVR rates plague this approach. In one of the earlier studies addressing this question Mazzaferro et al [67] initiated antiviral therapy at a median duration of 18 d post LT and demonstrated SVR rate of 33% with milder graft injury both in patients who remained HCV negative and in patients who turned HCV positive despite therapy and similar results were demonstrated in patients who received a living donor liver transplant [68]. These results have not been consistently reproduced. In a small randomized controlled trial by Singh et al [69] pre-emptive treatment delayed the occurrence of HCV hepatitis, but did not decrease the incidence or the severity of HCV hepatitis and this delay has been demonstrated in studies with long term follow-up too [70]. Unfortunately many studies have shown that both eligibility for therapy and tolerability for these medications are low in this post LT cohort. In Shergill et al [70] randomized trial only 41% of the post LT patients were eligible to receive pre-emptive antiviral therapy and dose reductions and discontinuations were required in 85% and 37% of patients, respectively. Other studies have shown similar side effect profile and low SVR rates [71]. In the immediate post LT period, most patients are battling renal dysfunction, cytopenias, immunosuppression related side effects and episodes of acute rejection which is probably why the physiologic reserve to endure side effects from PEG IFN or Ribavarin is low. So until safer and more effective antiviral drugs are available the pre-emptive strategy cannot be recommended for routine practice April 7, 2014 Volume 20 Issue 13

20 Dhanasekaran R et al. Challenges of post-transplant hepatitis C recurrence Table 1 Clinical outcome of treatment for post-transplant hepatitis C recurrence Ref. I Study design SVR Fibrosis progression Discontinuance Roche et al [85] (2008) 113 Retrospective study Carrión et al [81] (2007) 81 Randomized controlled trial Neff et al [86] (2004) 57 Retrospective study Oton et al [87] (2006) 55 Prospective cohort study Samuel et al [83] (2003) 52 Randomized controlled trial Fernández et al [82] (2006) Mukherjee et al [84] (2006) 47 Prospective cohort study 39 Prospective cohort study 38% Fibrosis stage remained stable (78.5%) in patients with SVR and increased (44%) in non-responders 48% in early fibrosis and 18% in advanced fibrosis 24.5% virus negative at 48 wk Fibrosis progression -26% in treated group vs 70% in untreated group Responders to therapy trended toward improvement in level of fibrosis 24% overall did not complete therapy. 38% required the premature discontinuation of either IFN, RBV, or both agents Treatment interruption in 22% and 56% of patients with mild and advanced fibrosis respectively Overall discontinuation rate 31.5%. Dose adjustments in 74% 43.60% No improvement in fibrosis progression 29% discontinued due to intolerance 21% No impact on fibrosis progression Treatment interruption 43% 23% Significant histological improvement in 23% 33.30% Improved or stable fibrosis scores were also demonstrated in 66.7% of non-responder Treatment interruption in 21% 43.6% discontinuation rates SVR: Sustained virologic response; IFN: Interferon; RBV: Ribavarin. Treatment of established recurrence Most centers initiate treatment of HCV after histological recurrence has been documented on liver biopsy. The AALSD guidelines on HCV therapy also recommend treatment initiation in appropriate candidates after demonstration of recurrent histologic disease, but the stage of fibrosis at which therapy should optimally be started is not clear ant it does vary between centers. Results from initial studies evaluating efficacy of IFN monotherapy were disappointing [72]. But subsequent studies with dual therapy of IFN and Ribavarin, more specifically with pegylated IFN (PEG IFN) have been encouraging [73]. In a systematic review by Berenguer et al [74] of 19 studies including 611 patients who received PEG-IFN alfa with ribavirin for established histologic recurrence of HCV, the mean SVR rate was found to be 30.2% despite high rate of treatment discontinuation and dose reductions due to adverse events. Therapy has also been found to be cost effective in a study by Logge et al [75] where they used a Markov model for disease progression. Several studies have looked at prognostic factors for achievement of SVR and have demonstrated an association with lower pretreatment HCV RNA, HCV genotype 2 or 3, adherence to therapy, and achievement of an early virological response [76,77]. Clinically, the most important question is whether treatment is associated with improvement in clinical outcomes like survival or fibrosis progression. Selzner et al [78] retrospectively analyzed long term outcome of 446 patients treated with dual therapy found that treatment was independently associated with significantly prolonged survival. Similar encouraging survival outcome has also been demonstrated in few other studies [79,80]. Another important clinical outcome is rate of fibrosis progression and data on the effect of treatment on fibrosis progression has not been uniform (Table 1) [81-87]. In one of the early observational studies analyzing histological response from our center, we demonstrated that fibrosis progression was slower in patients who underwent HCV therapy in a cohort of 34 patients [88]. Selzner et al [78] also reported improved histologic outcome with PEG IFN based treatment for HCV recurrence but paired biopsies were available only in 52% of this study group. Results from a few other smaller studies also suggest that treatment leads to improvement in fibrosis but there has been variability in how fibrosis data is reported [89,90]. This probably is the reason that a meta-analysis of 14 studies which analyzed histologic response by Berenguer et al [74] found a significant heterogeneity among studies and only a minority of patients underwent liver biopsy both prior to and after antiviral therapy. Despite these limitations it did appear that achieving SVR led to improvement in the stage of fibrosis and the necroinflammatory grade, while nonresponders generally experienced progressive fibrosis. The face of HCV therapy is rapidly changing with the arrival of newer, more potent antiviral drugs. Direct acting antiviral drugs like Boceprevir and Telaprevir were approved in 2011 for treatment of HCV and they have shown significant increase in SVR when used in combination with PEG IFN and Ribavarin. In phase Ⅲ trials in the non-transplant setting 63%-75% of treatment naïve patients receiving triple therapy achieved SVR [91,92]. But triple therapy does come with its own set of challenges which are more conspicuous in the transplant setting including drug interactions, need for frequent dosing, increased incidence of cytopenias, high costs with proven efficacy only in HCV genotype 1 patients. Telaprevir is an inhibitor of the enzyme cytochrome P450 3A which is involved in the metabolism of both CysA and TAC. Garg et al [93] conducted a phase Ⅰ study to assess the interaction between Telaprevir and CysA or TAC. And they showed that Telaprevir increased CysA exposure by approximately 4.6-fold and increased TAC exposure by approximately 70-fold. In a pilot study to assess the safety and efficacy of triple therapy in LT patients, data were gathered for 12 wk in nine HCV patients who were treated with a combi April 7, 2014 Volume 20 Issue 13

21 Dhanasekaran R et al. Challenges of post-transplant hepatitis C recurrence nation of Telaprevir, PEG IFN, and ribavirin along with TAC, CysA or Sirolimus [94]. It was found that drug-drug interactions between TVR and immunosuppressants could be managed with close monitoring of trough levels and adequate dosage adjustments. A recent cohort study of patients treated with Boceprevir (n = 18) or Telaprevir (n = 19) based triple therapy demonstrated that SVR rates were comparable to dual therapy but around a third of patients developed severe anemia requiring transfusion [95]. Results from ongoing trials involving triple therapy in this special population are expected in the near future. The potential for newer drugs which can potentially be used in regimens without IFN and Ribavarin will change the treatment scenario drastically. A case report of a patient with severe recurrent HCV who was treated with Sofosbuvir, an HCV polymerase inhibitor, and Daclatasvir, an HCV NS5A replication complex inhibitor for 24 wk demonstrated undetectable HCV RNA within 4 wk of initiating treatment, and the patient achieved SVR at 9 mo [96]. If these promising results hold good in clinical trials, several of the challenges associated with posttransplant hepatitis C recurrence could be tackled more effectively. CONCLUSION Recurrence of HCV presents a clinically challenging situation in post-transplant patients. The recurrence of viremia is universal and the progression of fibrosis is rapid. Incidence of cirrhosis and graft failure is significantly higher in untreated patients translating to poor overall clinical outcome. Multiple donor, viral and immunosuppression related factors have been found to influence rate of fibrosis progression and clinical outcome. Treatment of recurrence with PEG IFN and Ribavarin is efficacious in this population albeit with lower SVR rates and worse side effect profile. Therapy is associated with multiple challenges including severe cytopenias, drug interactions and renal failure. But given the improvement in survival and fibrosis progression associated with treatment, physicians should try to treat all eligible patients with recurrence, despite obstacles. Direct acting antiviral drugs have ushered a new era in the management of HCV patients. Emerging data on direct acting antivirals in the post-transplant setting shows promise in terms of safety and efficacy with appropriate monitoring and dose adjustment of immunosuppressants. Though multiple studies have investigated the role of antiviral therapy, well designed randomized controlled trials further exploring various aspects of HCV therapy in post LT population are urgently needed. REFERENCES 1 World Health Organization. Available from: URL: Af. 2 Organ Procurement and Transplantation Network. Data on September 16th, Available from: URL: transplant.hrsa.gov/data/ 3 Wright TL, Donegan E, Hsu HH, Ferrell L, Lake JR, Kim M, Combs C, Fennessy S, Roberts JP, Ascher NL. Recurrent and acquired hepatitis C viral infection in liver transplant recipients. Gastroenterology 1992; 103: [PMID: ] 4 Garcia-Retortillo M, Forns X, Feliu A, Moitinho E, Costa J, Navasa M, Rimola A, Rodes J. Hepatitis C virus kinetics during and immediately after liver transplantation. Hepatology 2002; 35: [PMID: DOI: / jhep ] 5 Rosen HR, Marousek G, Chou S. A longitudinal analysis of T-cell epitope-coding regions of hepatitis C virus after liver transplantation. Transplantation 2002; 74: [PMID: DOI: / ] 6 McCaughan GW, Zekry A. Pathogenesis of hepatitis C virus recurrence in the liver allograft. Liver Transpl 2002; 8: S7-S13 [PMID: ] 7 Gane EJ, Naoumov NV, Qian KP, Mondelli MU, Maertens G, Portmann BC, Lau JY, Williams R. A longitudinal analysis of hepatitis C virus replication following liver transplantation. Gastroenterology 1996; 110: [PMID: DOI: /gast.1996.v110.pm ] 8 Neumann UP, Berg T, Bahra M, Seehofer D, Langrehr JM, Neuhaus R, Radke C, Neuhaus P. Fibrosis progression after liver transplantation in patients with recurrent hepatitis C. J Hepatol 2004; 41: [PMID: DOI: / j.jhep ] 9 Yilmaz N, Shiffman ML, Stravitz RT, Sterling RK, Luketic VA, Sanyal AJ, Mills AS, Contos MJ, Coterell A, Maluf D, Posner MP, Fisher RA. A prospective evaluation of fibrosis progression in patients with recurrent hepatitis C virus following liver transplantation. Liver Transpl 2007; 13: [PMID: DOI: /lt.21117] 10 Walter T, Dumortier J, Guillaud O, Hervieu V, Scoazec JY, Boillot O. Factors influencing the progression of fibrosis in patients with recurrent hepatitis C after liver transplantation under antiviral therapy: a retrospective analysis of 939 liver biopsies in a single center. Liver Transpl 2007; 13: [PMID: DOI: /lt.21000] 11 Firpi RJ, Abdelmalek MF, Soldevila-Pico C, Cabrera R, Shuster JJ, Theriaque D, Reed AI, Hemming AW, Liu C, Crawford JM, Nelson DR. One-year protocol liver biopsy can stratify fibrosis progression in liver transplant recipients with recurrent hepatitis C infection. Liver Transpl 2004; 10: [PMID: DOI: /lt.20238] 12 Lai JC, Verna EC, Brown RS, O Leary JG, Trotter JF, Forman LM, Duman JD, Foster RG, Stravitz RT, Terrault NA. Hepatitis C virus-infected women have a higher risk of advanced fibrosis and graft loss after liver transplantation than men. Hepatology 2011; 54: [PMID: DOI: / hep.24390] 13 Forman LM, Lewis JD, Berlin JA, Feldman HI, Lucey MR. The association between hepatitis C infection and survival after orthotopic liver transplantation. Gastroenterology 2002; 122: [PMID: DOI: /gast ] 14 Cameron AM, Ghobrial RM, Hiatt JR, Carmody IC, Gordon SA, Farmer DG, Yersiz H, Zimmerman MA, Durazo F, Han SH, Saab S, Gornbein J, Busuttil RW. Effect of nonviral factors on hepatitis C recurrence after liver transplantation. Ann Surg 2006; 244: [PMID: ] 15 Berenguer M, Prieto M, San Juan F, Rayón JM, Martinez F, Carrasco D, Moya A, Orbis F, Mir J, Berenguer J. Contribution of donor age to the recent decrease in patient survival among HCV-infected liver transplant recipients. Hepatology 2002; 36: [PMID: DOI: / jhep ] 16 Féray C, Caccamo L, Alexander GJ, Ducot B, Gugenheim J, Casanovas T, Loinaz C, Gigou M, Burra P, Barkholt L, Esteban R, Bizollon T, Lerut J, Minello-Franza A, Bernard PH, Nachbaur K, Botta-Fridlund D, Bismuth H, Schalm SW, Samuel D. European collaborative study on factors influ April 7, 2014 Volume 20 Issue 13

22 Dhanasekaran R et al. Challenges of post-transplant hepatitis C recurrence encing outcome after liver transplantation for hepatitis C. European Concerted Action on Viral Hepatitis (EUROHEP) Group. Gastroenterology 1999; 117: [PMID: DOI: /S (99) ] 17 Sersté T, Bourgeois N. Ageing and the liver. Acta Gastroenterol Belg 2006; 69: [PMID: ] 18 Le Couteur DG, Warren A, Cogger VC, Smedsrød B, Sørensen KK, De Cabo R, Fraser R, McCuskey RS. Old age and the hepatic sinusoid. Anat Rec (Hoboken) 2008; 291: [PMID: DOI: /ar.20661] 19 Schmucker DL. Age-related changes in liver structure and function: Implications for disease? Exp Gerontol 2005; 40: [PMID: DOI: /j.exger ] 20 Garcia-Retortillo M, Forns X, Llovet JM, Navasa M, Feliu A, Massaguer A, Bruguera M, Fuster J, Garcia-Valdecasas JC, Rimola A. Hepatitis C recurrence is more severe after living donor compared to cadaveric liver transplantation. Hepatology 2004; 40: [PMID: DOI: / hep.20357] 21 Shiffman ML, Stravitz RT, Contos MJ, Mills AS, Sterling RK, Luketic VA, Sanyal AJ, Cotterell A, Maluf D, Posner MP, Fisher RA. Histologic recurrence of chronic hepatitis C virus in patients after living donor and deceased donor liver transplantation. Liver Transpl 2004; 10: [PMID: DOI: /lt.20232] 22 Guo L, Orrego M, Rodriguez-Luna H, Balan V, Byrne T, Chopra K, Douglas DD, Harrison E, Moss A, Reddy KS, Williams JW, Rakela J, Mulligan D, Vargas HE. Living donor liver transplantation for hepatitis C-related cirrhosis: no difference in histological recurrence when compared to deceased donor liver transplantation recipients. Liver Transpl 2006; 12: [DOI: /lt.20660] 23 Russo MW, Galanko J, Beavers K, Fried MW, Shrestha R. Patient and graft survival in hepatitis C recipients after adult living donor liver transplantation in the United States. 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Liver Int 2007; 27: [PMID: DOI: / j x] 28 Burra P, Loreno M, Russo FP, Germani G, Galligioni A, Senzolo M, Cillo U, Zanus G, Fagiuoli S, Rugge M. Donor livers with steatosis are safe to use in hepatitis C virus-positive recipients. Liver Transpl 2009; 15: [PMID: DOI: /lt.21761] 29 Briceño J, Ciria R, Pleguezuelo M, de la Mata M, Muntané J, Naranjo A, Sánchez-Hidalgo J, Marchal T, Rufián S, López- Cillero P. Impact of donor graft steatosis on overall outcome and viral recurrence after liver transplantation for hepatitis C virus cirrhosis. Liver Transpl 2009; 15: [PMID: DOI: /lt.21566] 30 Charlton M. Liver biopsy, viral kinetics, and the impact of viremia on severity of hepatitis C virus recurrence. Liver Transpl 2003; 9: S58-S62 [PMID: DOI: / jlts ] 31 Guerrero RB, Batts KP, Burgart LJ, Barrett SL, Germer JJ, Poterucha JJ, Wiesner RH, Charlton MR, Persing DH. Early detection of hepatitis C allograft reinfection after orthotopic liver transplantation: a molecular and histologic study. Mod Pathol 2000; 13: [PMID: DOI: /modpathol ] 32 Hollingsworth RC, Sillekens P, van Deursen P, Neal KR, Irving WL. Serum HCV RNA levels assessed by quantitative NASBA: stability of viral load over time, and lack of correlation with liver disease. The Trent HCV Study Group. J Hepatol 1996; 25: [PMID: DOI: / S (96) ] 33 Anand BS, Velez M. Assessment of correlation between serum titers of hepatitis C virus and severity of liver disease. World J Gastroenterol 2004; 10: [PMID: ] 34 Chazouilleres O, Kim M, Combs C, Ferrell L, Bacchetti P, Roberts J, Ascher NL, Neuwald P, Wilber J, Urdea M. Quantitation of hepatitis C virus RNA in liver transplant recipients. Gastroenterology 1994; 106: [PMID: ] 35 Sreekumar R, Gonzalez-Koch A, Maor-Kendler Y, Batts K, Moreno-Luna L, Poterucha J, Burgart L, Wiesner R, Kremers W, Rosen C, Charlton MR. Early identification of recipients with progressive histologic recurrence of hepatitis C after liver transplantation. 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Hepatology 1998; 28: [PMID: DOI: / hep ] 40 Zhou S, Terrault NA, Ferrell L, Hahn JA, Lau JY, Simmonds P, Roberts JP, Lake JR, Ascher NL, Wright TL. Severity of liver disease in liver transplantation recipients with hepatitis C virus infection: relationship to genotype and level of viremia. Hepatology 1996; 24: [PMID: DOI: /hep ] 41 Ge D, Fellay J, Thompson AJ, Simon JS, Shianna KV, Urban TJ, Heinzen EL, Qiu P, Bertelsen AH, Muir AJ, Sulkowski M, McHutchison JG, Goldstein DB. Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature 2009; 461: [PMID: DOI: /nature08309] 42 Charlton MR, Thompson A, Veldt BJ, Watt K, Tillmann H, Poterucha JJ, Heimbach JK, Goldstein D, McHutchison J. Interleukin-28B polymorphisms are associated with histological recurrence and treatment response following liver transplantation in patients with hepatitis C virus infection. Hepatology 2011; 53: [PMID: DOI: / hep.24074] 43 Lange CM, Moradpour D, Doehring A, Lehr HA, Müllhaupt B, Bibert S, Bochud PY, Antonino AT, Pascual M, Farnik H, Shi Y, Bechstein WO, Moench C, Hansmann ML, Sarrazin C, Lötsch J, Zeuzem S, Hofmann WP. Impact of donor and recipient IL28B rs genotypes on hepatitis C virus liver 3397 April 7, 2014 Volume 20 Issue 13

23 Dhanasekaran R et al. Challenges of post-transplant hepatitis C recurrence graft reinfection. J Hepatol 2011; 55: [PMID: DOI: /j.jhep ] 44 Firpi RJ, Dong H, Clark VC, Soldevila-Pico C, Morelli G, Cabrera R, Norkina O, Shuster JJ, Nelson DR, Liu C. CC genotype donors for the interleukin-28b single nucleotide polymorphism are associated with better outcomes in hepatitis C after liver transplant. Liver Int 2013; 33: [PMID: DOI: /liv.12013] 45 Fong TL, Valinluck B, Govindarajan S, Charboneau F, Adkins RH, Redeker AG. Short-term prednisone therapy affects aminotransferase activity and hepatitis C virus RNA levels in chronic hepatitis C. Gastroenterology 1994; 107: [PMID: ] 46 Henry SD, Metselaar HJ, Van Dijck J, Tilanus HW, Van Der Laan LJ. Impact of steroids on hepatitis C virus replication in vivo and in vitro. Ann N Y Acad Sci 2007; 1110: [PMID: DOI: /annals ] 47 Rosen HR, Hinrichs DJ, Gretch DR, Koziel MJ, Chou S, Houghton M, Rabkin J, Corless CL, Bouwer HG. Association of multispecific CD4(+) response to hepatitis C and severity of recurrence after liver transplantation. Gastroenterology 1999; 117: [PMID: DOI: / S (99) ] 48 Boor PP, Metselaar HJ, Mancham S, Tilanus HW, Kusters JG, Kwekkeboom J. Prednisolone suppresses the function and promotes apoptosis of plasmacytoid dendritic cells. Am J Transplant 2006; 6: [PMID: DOI: / j x] 49 Berenguer M, Aguilera V, Prieto M, San Juan F, Rayón JM, Benlloch S, Berenguer J. Significant improvement in the outcome of HCV-infected transplant recipients by avoiding rapid steroid tapering and potent induction immunosuppression. J Hepatol 2006; 44: [PMID: DOI: /j.jhep ] 50 Brillanti S, Vivarelli M, De Ruvo N, Aden AA, Camaggi V, D Errico A, Furlini G, Bellusci R, Roda E, Cavallari A. Slowly tapering off steroids protects the graft against hepatitis C recurrence after liver transplantation. Liver Transpl 2002; 8: [PMID: DOI: /jlts ] 51 Takada Y, Kaido T, Asonuma K, Sakurai H, Kubo S, Kiuchi T, Inomata Y, Isaji S, Tsumura H, Teramukai S, Matsubara Y, Sakabayashi S, Uemoto S. Randomized, multicenter trial comparing tacrolimus plus mycophenolate mofetil to tacrolimus plus steroids in hepatitis C virus-positive recipients of living donor liver transplantation. Liver Transpl 2013; 19: [PMID: DOI: /lt.23679] 52 Ramirez CB, Doria C, Frank AM, Armenti ST, Marino IR. Completely steroid-free immunosuppression in liver transplantation: a randomized study. Clin Transplant 2013; 27: [PMID: DOI: /ctr.12119] 53 Klintmalm GB, Davis GL, Teperman L, Netto GJ, Washburn K, Rudich SM, Pomfret EA, Vargas HE, Brown R, Eckhoff D, Pruett TL, Roberts J, Mulligan DC, Charlton MR, Heffron TG, Ham JM, Douglas DD, Sher L, Baliga PK, Kinkhabwala M, Koneru B, Abecassis M, Millis M, Jennings LW, Fasola CG. A randomized, multicenter study comparing steroidfree immunosuppression and standard immunosuppression for liver transplant recipients with chronic hepatitis C. Liver Transpl 2011; 17: [PMID: DOI: / lt.22417] 54 Watashi K, Hijikata M, Hosaka M, Yamaji M, Shimotohno K. Cyclosporin A suppresses replication of hepatitis C virus genome in cultured hepatocytes. Hepatology 2003; 38: [PMID: DOI: /jhep ] 55 Miroux C, Morales O, Ghazal K, Othman SB, de Launoit Y, Pancré V, Conti F, Delhem N. In vitro effects of cyclosporine A and tacrolimus on regulatory T-cell proliferation and function. Transplantation 2012; 94: [PMID: DOI: /TP.0b013e d8f] 56 Firpi RJ, Soldevila-Pico C, Morelli GG, Cabrera R, Levy C, Clark VC, Suman A, Michaels A, Chen C, Nelson DR. The use of cyclosporine for recurrent hepatitis C after liver transplant: a randomized pilot study. Dig Dis Sci 2010; 55: [PMID: DOI: /s ] 57 Firpi RJ, Zhu H, Morelli G, Abdelmalek MF, Soldevila- Pico C, Machicao VI, Cabrera R, Reed AI, Liu C, Nelson DR. Cyclosporine suppresses hepatitis C virus in vitro and increases the chance of a sustained virological response after liver transplantation. Liver Transpl 2006; 12: [PMID: DOI: /lt.20532] 58 Rabie R, Mumtaz K, Renner EL. Efficacy of antiviral therapy for hepatitis C after liver transplantation with cyclosporine and tacrolimus: a systematic review and meta-analysis. Liver Transpl 2013; 19: [PMID: DOI: / lt.23516] 59 Haddad EM, McAlister VC, Renouf E, Malthaner R, Kjaer MS, Gluud LL. Cyclosporin versus tacrolimus for liver transplanted patients. Cochrane Database Syst Rev 2006; (4): CD [PMID: DOI: / CD pub2] 60 Berenguer M, Aguilera V, San Juan F, Benlloch S, Rubin A, López-Andujar R, Moya A, Pareja E, Montalva E, Yago M, de Juan M, Mir J, Prieto M. Effect of calcineurin inhibitors in the outcome of liver transplantation in hepatitis C viruspositive recipients. Transplantation 2010; 90: [PMID: DOI: /TP.0b013e3181fa93fa] 61 Irish WD, Arcona S, Bowers D, Trotter JF. Cyclosporine versus tacrolimus treated liver transplant recipients with chronic hepatitis C: outcomes analysis of the UNOS/OPTN database. Am J Transplant 2011; 11: [PMID: DOI: /j x] 62 Bitetto D, Fabris C, Falleti E, Fornasiere E, Avellini C, Cmet S, Cussigh A, Fontanini E, Pirisi M, Corradini SG, Merli M, Molinaro A, Toniutto P. Recipient interleukin-28b Rs C/T polymorphism and acute cellular rejection after liver transplantation: role of the calcineurin inhibitor used. Transplantation 2012; 93: [PMID: DOI: / TP.0b013e31824df7f3] 63 Samonakis DN, Germani G, Burroughs AK. Immunosuppression and HCV recurrence after liver transplantation. J Hepatol 2012; 56: [PMID: DOI: / j.jhep ] 64 Henry SD, Metselaar HJ, Lonsdale RC, Kok A, Haagmans BL, Tilanus HW, van der Laan LJ. Mycophenolic acid inhibits hepatitis C virus replication and acts in synergy with cyclosporin A and interferon-alpha. Gastroenterology 2006; 131: [PMID: DOI: / j.gastro ] 65 Chen H, Ye L, Su JM, Li Y, Zeng JR, Huo WZ. [Inhibition of hepatitis C virus replication by mycophenolic acid in hepatocytes]. Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi 2013; 27: [PMID: ] 66 Kuo A, Tan V, Lan B, Khalili M, Feng S, Roberts JP, Terrault NA. Long-term histological effects of preemptive antiviral therapy in liver transplant recipients with hepatitis C virus infection. Liver Transpl 2008; 14: [PMID: DOI: /lt.21548] 67 Mazzaferro V, Tagger A, Schiavo M, Regalia E, Pulvirenti A, Ribero ML, Coppa J, Romito R, Burgoa L, Zucchini N, Urbanek T, Bonino F. Prevention of recurrent hepatitis C after liver transplantation with early interferon and ribavirin treatment. Transplant Proc 2001; 33: [PMID: ] 68 Sugawara Y, Makuuchi M, Matsui Y, Kishi Y, Akamatsu N, Kaneko J, Kokudo N. 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24 Dhanasekaran R et al. Challenges of post-transplant hepatitis C recurrence a prospective, randomized, controlled trial. Transplantation 1998; 65: [PMID: ] 70 Shergill AK, Khalili M, Straley S, Bollinger K, Roberts JP, Ascher NA, Terrault NA. Applicability, tolerability and efficacy of preemptive antiviral therapy in hepatitis C-infected patients undergoing liver transplantation. Am J Transplant 2005; 5: [PMID: DOI: / j x] 71 Bzowej N, Nelson DR, Terrault NA, Everson GT, Teng LL, Prabhakar A, Charlton MR. PHOENIX: A randomized controlled trial of peginterferon alfa-2a plus ribavirin as a prophylactic treatment after liver transplantation for hepatitis C virus. Liver Transpl 2011; 17: [PMID: DOI: /lt.22271] 72 Cotler SJ, Ganger DR, Kaur S, Rosenblate H, Jakate S, Sullivan DG, Ng KW, Gretch DR, Jensen DM. Daily interferon therapy for hepatitis C virus infection in liver transplant recipients. Transplantation 2001; 71: [PMID: DOI: / ] 73 Gane EJ, Lo SK, Riordan SM, Portmann BC, Lau JY, Naoumov NV, Williams R. A randomized study comparing ribavirin and interferon alfa monotherapy for hepatitis C recurrence after liver transplantation. Hepatology 1998; 27: [PMID: DOI: /hep ] 74 Berenguer M. Systematic review of the treatment of established recurrent hepatitis C with pegylated interferon in combination with ribavirin. J Hepatol 2008; 49: [PMID: DOI: /j.jhep ] 75 Logge C, Vettorazzi E, Fischer L, Nashan B, Sterneck M. Cost-effectiveness analysis of antiviral treatment in liver transplant recipients with HCV infection. Transpl Int 2013; 26: [PMID: DOI: /tri.12085] 76 Sharma P, Marrero JA, Fontana RJ, Greenson JK, Conjeevaram H, Su GL, Askari F, Sullivan P, Lok AS. Sustained virologic response to therapy of recurrent hepatitis C after liver transplantation is related to early virologic response and dose adherence. Liver Transpl 2007; 13: [PMID: ] 77 Bizollon T, Pradat P, Mabrut JY, Chevallier M, Adham M, Radenne S, Souquet JC, Ducerf C, Baulieux J, Zoulim F, Trepo C. Benefit of sustained virological response to combination therapy on graft survival of liver transplanted patients with recurrent chronic hepatitis C. Am J Transplant 2005; 5: [PMID: DOI: /j x] 78 Selzner N, Renner EL, Selzner M, Adeyi O, Kashfi A, Therapondos G, Girgrah N, Herath C, Levy GA, Lilly L. Antiviral treatment of recurrent hepatitis C after liver transplantation: predictors of response and long-term outcome. Transplantation 2009; 88: [PMID: DOI: / TP.0b013e3181bd783c] 79 Picciotto FP, Tritto G, Lanza AG, Addario L, De Luca M, Di Costanzo GG, Lampasi F, Tartaglione MT, Marsilia GM, Calise F, Cuomo O, Ascione A. Sustained virological response to antiviral therapy reduces mortality in HCV reinfection after liver transplantation. J Hepatol 2007; 46: [PMID: DOI: /j.jhep ] 80 García-Reyne A, Lumbreras C, Fernández I, Colina F, Abradelo M, Magan P, San-Juan R, Manrique A, López- Medrano F, Fuertes A, Lizasoain M, Moreno E, Aguado JM. Influence of antiviral therapy in the long-term outcome of recurrent hepatitis C virus infection following liver transplantation. Transpl Infect Dis 2013; 15: [PMID: DOI: /tid.12097] 81 Carrión JA, Navasa M, García-Retortillo M, García-Pagan JC, Crespo G, Bruguera M, Bosch J, Forns X. Efficacy of antiviral therapy on hepatitis C recurrence after liver transplantation: a randomized controlled study. Gastroenterology 2007; 132: [PMID: ] 82 Fernández I, Meneu JC, Colina F, García I, Muñoz R, Castellano G, Fuertes A, Abradelo M, Lumbreras C, Moreno E, Solís-Herruzo JA. Clinical and histological efficacy of pegylated interferon and ribavirin therapy of recurrent hepatitis C after liver transplantation. Liver Transpl 2006; 12: [PMID: DOI: /lt.20883] 83 Samuel D, Bizollon T, Feray C, Roche B, Ahmed SN, Lemonnier C, Cohard M, Reynes M, Chevallier M, Ducerf C, Baulieux J, Geffner M, Albrecht JK, Bismuth H, Trepo C. Interferon-alpha 2b plus ribavirin in patients with chronic hepatitis C after liver transplantation: a randomized study. Gastroenterology 2003; 124: [PMID: DOI: /gast ] 84 Mukherjee S, Lyden E. Impact of pegylated interferon alpha-2b and ribavirin on hepatic fibrosis in liver transplant patients with recurrent hepatitis C: an open-label series. Liver Int 2006; 26: [PMID: DOI: /j x] 85 Roche B, Sebagh M, Canfora ML, Antonini T, Roque-Afonso AM, Delvart V, Saliba F, Duclos-Vallee JC, Castaing D, Samuel D. Hepatitis C virus therapy in liver transplant recipients: response predictors, effect on fibrosis progression, and importance of the initial stage of fibrosis. Liver Transpl 2008; 14: [PMID: DOI: /lt.21635] 86 Neff GW, Montalbano M, O Brien CB, Nishida S, Safdar K, Bejarano PA, Khaled AS, Ruiz P, Slapak-Green G, Lee M, Nery J, De Medina M, Tzakis A, Schiff ER. Treatment of established recurrent hepatitis C in liver-transplant recipients with pegylated interferon-alfa-2b and ribavirin therapy. Transplantation 2004; 78: [PMID: DOI: /01.TP C] 87 Oton E, Barcena R, Moreno-Planas JM, Cuervas-Mons V, Moreno-Zamora A, Barrios C, Garcia-Garzon S, Moreno A, Boullosa-Graña E, Rubio-Gonzalez EE, Garcia-Gonzalez M, Blesa C, Mateos ML. Hepatitis C recurrence after liver transplantation: Viral and histologic response to full-dose PEGinterferon and ribavirin. Am J Transplant 2006; 6: [PMID: DOI: /j x] 88 Firpi RJ, Abdelmalek MF, Soldevila-Pico C, Reed A, Hemming A, Howard R, Van Der Werf W, Lauwers G, Liu C, Crawford JM, Davis GL, Nelson DR. Combination of interferon alfa-2b and ribavirin in liver transplant recipients with histological recurrent hepatitis C. Liver Transpl 2002; 8: [PMID: ] 89 Abdelmalek MF, Firpi RJ, Soldevila-Pico C, Reed AI, Hemming AW, Liu C, Crawford JM, Davis GL, Nelson DR. Sustained viral response to interferon and ribavirin in liver transplant recipients with recurrent hepatitis C. Liver Transpl 2004; 10: [PMID: DOI: /lt.20074] 90 Kornberg A, Küpper B, Tannapfel A, Thrum K, Bärthel E, Habrecht O, Settmacher U. Sustained clearance of serum hepatitis C virus-rna independently predicts long-term survival in liver transplant patients with recurrent hepatitis C. Transplantation 2008; 86: [PMID: DOI: /TP.0b013e31817c0e20] 91 Poordad F, McCone J, Bacon BR, Bruno S, Manns MP, Sulkowski MS, Jacobson IM, Reddy KR, Goodman ZD, Boparai N, DiNubile MJ, Sniukiene V, Brass CA, Albrecht JK, Bronowicki JP. Boceprevir for untreated chronic HCV genotype 1 infection. N Engl J Med 2011; 364: [PMID: DOI: /NEJMoa ] 92 Sherman KE, Flamm SL, Afdhal NH, Nelson DR, Sulkowski MS, Everson GT, Fried MW, Adler M, Reesink HW, Martin M, Sankoh AJ, Adda N, Kauffman RS, George S, Wright CI, Poordad F. Response-guided telaprevir combination treatment for hepatitis C virus infection. N Engl J Med 2011; 365: [PMID: DOI: /NEJMoa ] 93 Garg V, van Heeswijk R, Lee JE, Alves K, Nadkarni P, Luo X. Effect of telaprevir on the pharmacokinetics of cyclosporine and tacrolimus. Hepatology 2011; 54: [PMID: DOI: /hep.24443] 94 Werner CR, Egetemeyr DP, Lauer UM, Nadalin S, König April 7, 2014 Volume 20 Issue 13

25 Dhanasekaran R et al. Challenges of post-transplant hepatitis C recurrence srainer A, Malek NP, Berg CP. Telaprevir-based triple therapy in liver transplant patients with hepatitis C virus: a 12-week pilot study providing safety and efficacy data. Liver Transpl 2012; 18: [PMID: DOI: / lt.23542] 95 Coilly A, Roche B, Dumortier J, Leroy V, Botta-Fridlund D, Radenne S, Pageaux GP, Si-Ahmed SN, Guillaud O, Antonini TM, Haïm-Boukobza S, Roque-Afonso AM, Samuel D, Duclos-Vallée JC. Safety and efficacy of protease inhibitors to treat hepatitis C after liver transplantation: a multicenter experience. J Hepatol 2014; 60: [PMID: ] 96 Fontana RJ, Hughes EA, Bifano M, Appelman H, Dimitrova D, Hindes R, Symonds WT. Sofosbuvir and daclatasvir combination therapy in a liver transplant recipient with severe recurrent cholestatic hepatitis C. Am J Transplant 2013; 13: [PMID: ] P- Reviewers: Liu B, Zhu F S- Editor: Qi Y L- Editor: A E- Editor: Zhang DN 3400 April 7, 2014 Volume 20 Issue 13

26 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (2): Hepatitis C virus TOPIC HIGHLIGHT Computational biology approach to uncover hepatitis C virus helicase operation Holger Flechsig Holger Flechsig, Department of Physical Chemistry, Fritz Haber Institute of the Max Planck Society, Berlin, Germany Author contributions: Flechsig H wrote the paper. Correspondence to: Dr. Holger Flechsig, Department of Physical Chemistry, Fritz Haber Institute of the Max Planck Society, Faradayweg 4-6, Berlin, Germany. flechsig@fhi-berlin.mpg.de Telephone: Fax: Received: September 27, 2013 Revised: January 6, 2014 Accepted: March 6, 2014 Published online: April 7, 2014 Abstract Hepatitis C virus (HCV) helicase is a molecular motor that splits nucleic acid duplex structures during viral replication, therefore representing a promising target for antiviral treatment. Hence, a detailed understanding of the mechanism by which it operates would facilitate the development of efficient drug-assisted therapies aiming to inhibit helicase activity. Despite extensive investigations performed in the past, a thorough understanding of the activity of this important protein was lacking since the underlying internal conformational motions could not be resolved. Here we review investigations that have been previously performed by us for HCV helicase. Using methods of structure-based computational modelling it became possible to follow entire operation cycles of this motor protein in structurally resolved simulations and uncover the mechanism by which it moves along the nucleic acid and accomplishes strand separation. We also discuss observations from that study in the light of recent experimental studies that confirm our findings Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Hepatitis C virus; Viral replication; Helicase protein; Adenosine-triphosphate-induced operation; Conformational motions; Nucleic acid unzipping; Computational biology; Coarse-grained modelling; Elasticnetwork model Core tip: Proteins that are involved in the replication cycle of hepatitis C virus are relevant targets for antiviral therapies. Their dynamical properties, however, cannot be sufficiently studied in experiments yet. Alternatively, owing to the available power of modern computers, the mechanism by which they operate can be investigated in computer experiments using appropriate models. In this regard, considerable progress has now been achieved for the viral helicase protein and the obtained results, in combination with experimental data, have significantly improved our understanding of its activity. Flechsig H. Computational biology approach to uncover hepatitis C virus helicase operation. World J Gastroenterol 2014; 20(13): Available from: URL: com/ /full/v20/i13/3401.htm DOI: org/ /wjg.v20.i INTRODUCTION Hepatitis C is a liver disease caused by infection with the hepatitis C virus (HCV). According to the world health organization, about 150 million people are chronically infected by HCV with 3-4 million additional infections every year. About 75%-85% of newly infected individuals develop chronic disease with long-term complications including liver cirrhosis and liver cancer. Since to date no vaccine is available and more than patients die yearly in consequence of hepatitis C disease, the virus poses a major health concern. HCV belongs to the family of Flaviviridae and has a positive sense single-stranded RNA genome that consists of a single open reading frame, which is translated 3401 April 7, 2014 Volume 20 Issue 13

27 Flechsig H. Operation cycles of HCV helicase into a single protein of approximately amino acids. This polyprotein is further cleaved by viral and cellular proteases into three structural and seven non-structural proteins, which organize replication and packaging of the viral genome. A key role in the multiplication process of the virus is occupied by non-structural proteins NS5A and NS3, which function as molecular motors directly interacting with RNA. NS5A is a polymerase that synthesizes new viral RNA while NS3 contains a large portion that operates as a helicase, capable of translocating along single nucleic acids strands and separating their complementary strands or remove proteins bound to them [1]. Due to their eminent role in the replication machinery of HCV, these proteins have attracted much attention as possible targets for anti-hcv drugs and thus towards the development of effective treatments [2-4]. HCV helicase Helicases are motor proteins that are powered by adenosine-triphosphate (ATP) molecules and ubiquitous in virtually all processes that involve manipulation of DNA and RNA [5]. They are capable of displacing proteins bound to the nucleic acids, removing secondary structures and, most prominently, separating their duplex structures into single-stranded components [6,7]. Hence, they play a central role in replication, recombination and repair of nucleic acid substrates. Likewise generally in molecular motors, the operation of helicase proteins relies on cyclic internal conformational motions driven by binding of an ATP molecule, its hydrolysis into adenosine-diphosphate (ADP) and a phosphate Pi, and the dissociation of the chemical products [8,9]. These internal structural changes are used to generate forces on the nucleic acid strand and allow the helicase to translocate along it and eventually implement its particular function. The NS3 helicase was the first HCV protein that could be successfully crystallized and to date represents the probably best-characterized helicase molecule. The first structures of this protein were reported in 1997 by Yao et al [10] and, bound to a short fragment of DNA oligonucleotides, in 1998 by Kim et al [11]. Figure 1 shows the molecular architecture of HCV helicase. The protein consists of three domains that are arranged in a Y-shaped form. Domains Ⅰ and Ⅱ are structurally related and referred to as the helicase motor domains. At the open interface between these domains, ATP molecules can arrive and bind to a pocket that is located close to the surface of domain Ⅰ. The motor domains are separated from the third helicase domain by a large deep cleft in which a single nucleic acid strand can be bound and hold. In the past the HCV helicase protein has been the subject of numerous investigations, aiming to explore its functional properties and understand the mechanism by which it operates. Despite its clinical relevance it has thus become also a prototype helicase representing a broad class of structurally and functionally related proteins [12]. Already the first crystallographic studies of HCV helicase have indicated the mobility of motor domain Ⅱ, which is flexibly linked to motor domain Ⅰ, and discussed its possible functional role. Based on the early studies timeresolved single molecule experiments have been designed and important aspects of HCV helicase operation were investigated in optical tweezer setups and using fluorescence analysis [13,14]. These experiments have elucidated the ATP-dependent unwinding activity of the helicase on DNA and RNA duplex structures and greatly contributed to the understanding of helicase function. Although models for the operation of HCV helicase have been established and theoretical investigations were performed additionally [6,11,15], the internal conformational motions related to interactions with ATP molecules in this protein could not be sufficiently resolved and thus, the particular mechanism by which HCV helicase moves along nucleic acid strands and performs strand separation remained elusive hitherto. In this situation, modelling studies that are based on present structures of HCV helicase and employ methods from computational biology may play an important role. In this article we review investigations that have been previously performed by us for HCV helicase. We shall focus here on the conceptual aspects of our analysis and present our findings in a rather general framework, while a more profound description including details can be gathered from our original publication [16]. Moreover, we will discuss our findings in the light of novel crystal structures of HCV helicase complexed with ATP-mimicking nucleotides and DNA and RNA. Computational biology methods The function of any protein relies on its intrinsic structural flexibility and is caused by internal conformational motions. Since these motions can hardly be resolved to a sufficient extend by experimental methods and, furthermore, available structural data provides only static snapshots, capturing a protein at a few steps along its functional cycle, our knowledge about the operation principles of proteins is often limited. On the other side, the tremendous increase of computational power that has become available over the last decades have enabled the investigation of protein dynamics in computer simulations and opened promising new possibilities in protein research employing numerical modelling. Widespread approaches that have become a standard tool of computer-assisted molecular biology are molecular dynamics simulations [17-19]. These descriptions take into account all atoms of a protein and allow to model complicated interactions between them by using multiparameter force-fields. Molecular dynamics simulations allow to investigate dynamical processes in proteins at high resolution. Their application, however, is limited by the size of the protein and the timescales of internal motions. While usually these methods are capable of resolving rapid processes with motions on timescales up to nanoseconds, they fail to cover the biologically relevant slow conformational motions associated with the ATPrelated turnover cycles in protein machines and molecular 3402 April 7, 2014 Volume 20 Issue 13

28 Flechsig H. Operation cycles of HCV helicase A Ⅰ ATP pocket Ⅱ B DNA cleft 5' end Ⅲ Figure 1 Molecular architecture of hepatitis C virus helicase. A: Structure of hepatitis C virus (HCV) helicase in ribbon representation (Protein Data Bank code 1A1V). Motor domains Ⅰ and Ⅱ are coloured golden and blue, respectively. Domain Ⅲ is shown in grey. A sulfate ion, shown as beads, is located at the surface of domain Ⅰ in the proximity of the adenosine-triphosphate (ATP) binding pocket. The short DNA fragment (shown in bead representation) occupies a large cleft separating the motor domains from the third domain; B: Network representation of HCV helicase. The chain of beads representing a single DNA strand is shown in green. motors. To follow large-amplitude motions with typical timescales of tens of milliseconds in molecular dynamics simulations would require a massive computational expense, which is beyond what can be reached nowadays, even with modern computer architectures. To overcome these difficulties, coarse-grained models of protein dynamics have been developed and became increasingly popular in the past [20]. Being based on rigorous approximations, they allow a straightforward numerical implementation and can drastically reduce the computational burden. In such models, the structure of a protein is simplified and the complicated intra-molecular interactions are replaced by effective potentials. A coarsegrained approach commonly used to investigate dynamics of protein machines is the elastic-network model [21,22]. Within this description, entire amino acid residues of a protein structure are typically replaced by single identical beads and interactions between them are mediated by empiric harmonic potentials that depend only on the distance between any two beads. Within the framework of this model, a protein is therefore viewed as an elastic object, i.e., a network of beads connected by deformable elastic springs. Despite the coarse-grained nature of elastic-network descriptions and the gross simplification made, these models have been proved powerful in describing ATP-induced slow motions in molecular machines and advanced the understanding of important aspects of their collective dynamics [23-28]. In the first part of this review we will explain how an elastic-network description of HCV helicase that incorporated interactions with ATP ligands was established and individual operation cycles were followed in computer experiments. We will then show how the dynamical modelling was extended to include also interactions with nucleic acid strands, which allowed us to study how internal conformational motions are coupled to progressive helicase locomotion and follow the mechanism of strand separation. ATP-INDUCED OPERATION CYCLES OF HCV HELICASE We have used the nucleotide-free structure determined by Yao et al [10] [Protein Data Bank (PDB) code 1HEI] to construct the elastic network of HCV helicase. The network (Figure 1B) consisted of 443 identical beads, which represented entire amino acid residues, and elastic springs connecting any two beads if their spatial distance was below a prescribed interaction radius of 8 Angstroms. A spring connecting two beads was assigned the natural length that corresponded exactly to the distance between the two beads as extracted from the PDB file. Therefore, by construction, the initial network represented a steady conformation of the protein. When, however, interactions between the network and ligands were taken into account, network dynamics could be induced and related conformational motions were analyzed. The internal motions of proteins in solution are dominated by friction and internal forces are negligible already on timescales slower than tens of picoseconds. The slow ATPrelated conformational motions in protein machines and molecular motors are certainly overdamped. Therefore, within the elastic-network model, slow conformational motions of a protein are described as relaxation motions of its corresponding elastic network. If, for simplicity, thermal fluctuations and hydrodynamical interactions are neglected, the dynamics of the network can be described by equations of motion that correspond to Newton mechanics [16]. This set of equations was numerically integrated in the computer simulations to obtain the position of all network beads at every instant of time. In this way structural changes of the protein network could be conveniently traced. To follow ATP-induced dynamics in HCV helicase, we have established a ligand-elastic-network model that incorporated binding of ATP, its hydrolysis, and the dissociation of the product in an approximate fashion. ATP 3403 April 7, 2014 Volume 20 Issue 13

29 Flechsig H. Operation cycles of HCV helicase Substrate Product Figure 2 Cyclic ligand-induced conformational motions in hepatitis C virus helicase. Top row: binding of the substrate ligand led to switching from open to closed conformation. Release of the product ligand induced motions back to the open form; Bottom row: motions of the motor domains relative to the nucleic acid binding cleft that are critical for grip control are indicated by arrows. Open Closed DNA cleft Side view itself is a relatively complex molecule and its interactions with the protein rely on complicated processes and involve chemical details that could not be accounted for in our description. To remain consistent with the approximate nature of the model, we have described an ATP molecule by a single ligand bead (the substrate ligand) and modelled its binding process to the ATP-pocket by allowing it to physically interact with network beads present there. Specifically, upon binding, the substrate ligand could create spring connections to four selected beads inside the binding pocket. These particular interaction beads corresponded to amino acid residues that belong to sequence motifs which have been shown to be conserved among all helicases and are crucially involved in interactions with ATP. The additional ligand springs were initially stretched so that initiated by the binding process, attractive forces between the ligand and the four pocket beads were generated and network deformations became induced. First localized in the binding pocket, they gradually spread and resulted in conformational motions of the entire protein network until eventually a steady network state that corresponded to the network-ligand complex was reached. In this conformation, to roughly emulate the hydrolysis of ATP, we assumed that the substrate ligand became converted into the product ligand, which represented the chemical products ADP and Pi being ready to dissociate from the protein. Therefore, we assumed that the product ligand, in contrast to the substrate ligand, could no longer interact with the ligand pocket and thus removed the four ligand springs. Since then the ligandfree network found itself in a conformation that was different from the native one, with the network springs be- ing deformed, conformational motions bringing it back to its initial state occurred. The reset ligand-free network could then in principle bind another substrate ligand and the next ATP-induced operation cycle would be initiated. The cyclic ligand-induced conformational changes in HCV helicase as obtained from our simulations are depicted in Figure 2. We observed that subsequent to binding of the substrate ligand pronounced relative motions of the two motor domains, bringing the HCV helicase from an open-shape conformation in which these domains were well separated into a closed-shape form where they were found in tight contact, were induced. In this conformation of the network-ligand complex the substrate ligand was converted into the product ligand, which was then removed from the network. Under subsequent conformational motions, bringing the helicase network back to its initial configuration, the motor domains moved away from each other and restored the openshape form. In addition to these large-scale domain rearrangements, we recognized another component of the same dynamics that was more subtle and consisted in motions of the domains with respect to the cleft that holds the nucleic acid strand. Such changes become apparent in the side view perspective of the protein (Figure 2): Under binding of the ligand, motor domain Ⅰ moved upwards and therefore broadened the cleft at one side, whereas motor domain Ⅱ moved downwards narrowing the cleft at the opposite side. Along the back-motion of the network, after removal of the product ligand, the situation was reversed. Domain Ⅰ moved back downwards, whereas domain Ⅱ was lifted away from the nucleic acid 3404 April 7, 2014 Volume 20 Issue 13

30 Flechsig H. Operation cycles of HCV helicase cleft. Our findings suggest that by performing these motions relative to the cleft that holds the nucleic acid strand, each of the motor domains is capable of controlling its grip on the strand. Consequently, by weakening or tightening the grip depending on the presence of the substrate ligand, the large-amplitude opening and closing motions of the motor domains can be translated into steady translocation along the single nucleic acid strand by means of a ratchet mechanism. To demonstrate the mode by which HCV helicase is able to move along nucleic acid strands, we have performed simulations in which an approximate description of a DNA strand was included and interactions between the protein and DNA were accounted for in a simplified way. RATCHETING INCHWORM TRANSLOCATION We have extended our dynamical description to incorporate interactions between the HCV helicase and a single DNA strand. While apparently detailed models of DNA and its dynamical properties are available, it was more important for our purposes to employ a proper mechanical description of this molecule. To remain consistent with the coarse-grained modelling implemented so far, a single DNA strand was described as a chain of identical beads which were consecutively linked by elastic springs (see Figure 1B). The chain, which was representing the sugarphosphate backbone of DNA, was placed roughly in the center of the nucleic acid binding cleft and was allowed to undergo deformations such as stretching and bending when interacting with the helicase. Regarding the interactions between HCV helicase and the nucleic acid it is known that, instead of being distributed over the entire cleft, major contacts to the sugarphosphate backbone of DNA are established to specific conserved amino acid residues of the protein [11]. These are Thr269 from motor domain Ⅰ and Thr411 from motor domain Ⅱ. In our description we have restricted helicase-dna interactions to those two key residues and modelled them only roughly via additional elastic links that were established when a motor domain moved towards the DNA strand and removed when a motor domain was lifted from the strand. As described in the previous section, upon binding of the substrate ligand to the helicase network, motor domain Ⅱ moved down and therefore tightened its grip on the DNA strand (hand-on state), whereas at the same time motor domain Ⅰ moved away from the strand and weakened its grip (hand-off state). Therefore, when the substrate ligand was bound, we have created a stiff bond between the Thr411-bead from motor domain Ⅱ and the nearest bead in the DNA chain, while a bond connecting the Thr269-bead from motor domain Ⅰ to the chain was absent. Then, along the formation of the network-ligand complex, the largeamplitude slow conformational changes occurred under which motor domain Ⅰ moved towards domain Ⅱ until they were in tight contact. At this stage, when the ligand after its conversion from substrate into product was removed, the grip of the motor domains on the DNA was reversed. Consequently, the bond between motor domain Ⅱ and the DNA was removed (hand-off) and, at the same time, a bond between the Thr269-bead from motor domain Ⅰ and the nearest bead in the DNA was established (hand-on). Then, during the ligand-free part of the operation cycle, the motor domains slowly moved away from each other, this time with motor domain Ⅰ holding the DNA chain. After one ligand-induced operation cycle the helicase is found again in the initial open-shape conformation, with the motor domains being separated, but became effectively transported along the DNA - as we found - by one single base in the chain. The translocation mechanism is illustrated in Figure 3, where snapshots from the simulation are depicted. With the described model extensions we could successfully uncover, in a structurally resolved way, the mechanism by which HCV helicase is able to move along a single DNA strand. The style of locomotion along DNA used by HCV helicase is reminiscent of the mechanism by which worms deform themselves to crawl along trees. The ATP-dependent opening and closing motions of the two motor domains are converted into directed base-by-base translocation along the DNA strand by an integrated ratcheting mechanism of these domains, i.e. the alternating gripping (hand-on and hand-off style) on the DNA. Because of that analogy, the described mechanism of propagation along DNA employed by HCV helicase is referred to as the ratcheting inchworm translocation. STRAND-SEPARATION BY HCV HELICASE In the performed computer experiments we finally aimed to demonstrate how steady translocation of HCV helicase along the single nucleic acid strand is coupled to the separation of the second complementary strand. Therefore, we have set up a model of duplex DNA that consisted of two opposite single chains that were bridged by additional links. These connections were intended to effectively mimic the attractive interactions between complementary bases of the opposing single chains that stabilize the duplex form of DNA. On the other side, however, these bridge-links could also be broken when the two strands become separated and interactions between them gradually vanished. The duplex DNA was positioned in such a way that the upper strand was again centered in its putative binding cleft and the complementary strand was located below the motor domains of the protein (Figure 4). While the two motor domains of HCV helicase serve as essential modules, whose opening and closing motions make inchworm translocation possible, the role of the third protein domain is less clear. As revealed from our previous simulations it is rigidly connected to motor domain Ⅰ and, therefore, has to follow its motion. Hence, 3405 April 7, 2014 Volume 20 Issue 13

31 Flechsig H. Operation cycles of HCV helicase A B Figure 3 Ratcheting inchworm translocation. Four consequent snapshots (A-D) within a single ligandinduced operation cycle are shown. Red bonds indicate connections between a motor domain and the DNA chain. After one cycle, the helicase is found in a similar open-shape conformation, but is propagated by one base along the DNA strand (compare A and D). D C A B Figure 4 Unzipping of duplex DNA by hepatitis C virus helicase. Shown are three consequent snapshots (A-C) from a simulation that demonstrates ratcheting inchworm translocation and mechanical strand separation. C during progressive translocation of the motor domains the third domain will always be dragged by domain Ⅰ. This fact may suggest a significant role of domain Ⅲ during the process of duplex separation. Although little is known about interactions between HCV helicase and the duplex DNA, we can, based on the previously proposed mechanism [11], assume the following mechanical scenario of action. Under steady translocation of the two motor domains along the upper DNA strand, the third helicase domain encounters the free end of the complementary strand at the DNA fork and interactions between them set in. Particularly, repulsive forces shall appear that tend to push apart the lower DNA strand and prevent it from penetrating into the protein structure, where it would cause partial unfolding. In the simulation we have therefore assumed that the lower DNA strand was always 3406 April 7, 2014 Volume 20 Issue 13

32 Flechsig H. Operation cycles of HCV helicase subject to an overall repulsive force that was generated by beads from the third helicase domain and which became strong at short distances. With the implemented setup we have traced three ligand-induced operation cycles of HCV helicase within a single simulation and observed how progressive baseby-base inchworm translocation along the upper DNA strand lead to separation of the lower strand. In Figure 4 snapshots taken at different time moments during the simulation are provided. As the two motor domains moved along the upper DNA strand, the third helicase domain was dragged into the free space at the duplex fork. As a result of the repulsive interactions that tried to prevent contact of this domain with the lower strand, forces pushing away that strand were generated. As these forces built up, stress was accumulated in the bridge-links that held together the DNA duplex structure. Eventually these connections became broken one after another and the duplex DNA was successively unzipped. Our simulation show that the third helicase domain acted like a wedge that, in the course of translocation along the upper strand, was pressed between the two DNA strands and mechanically separated them. We found that HCV helicase had broken three base-pairs after three translocation steps, one base-pair at a time. CONCLUSION Molecular machines and motors are nanoscale engines that operate by performing cyclic changes of their conformation, powered by the chemical energy that is derived from the hydrolysis of ATP molecules. To observe these conformational motions directly in singlemolecule experiments in sufficient detail is however not yet possible due to the limited resolution of nowadays techniques. Therefore, important aspects of the activity of motor proteins remained elusive so far. On the other side, the growing capacities of modern computers have facilitated the investigation of protein dynamics in computer simulations. Here, we present a review of computer-assisted investigations that have been previously performed for the helicase protein of HCV by employing coarse-grained molecular dynamics methods. HCV helicase is a molecular motor that translocates over nucleic acid strands and splits their duplex structure. Since it plays a crucial role in the replication cycle of the virus, HCV helicase presents a major target for drugs and understanding of the functional properties underlying its operation is a key step towards the development of efficient anti-hcv therapies. Despite a lot of past studies, the mechanism by which HCV helicase moves over nucleic acid and drives strand separation remained speculative. By establishing a structure-based mechanical model of this protein that included interactions with ATP molecules and DNA in an approximate manner, we were able to follow entire operation cycles of HCV helicase in computer experiments. We have found that the ATP-induced dynamics in this protein consisted in well-organized large-amplitude conformational changes and involved concerted relative motions of the two motor domains. These motions are functional and, as identified from our simulations, can lead to progressive base-by-base inchworm translocation along a single DNA strand my means of a ratchet mechanism. Along this process, the third helicase domain acts as a wedge that is pressed between the two DNA strands and actively separates the duplex structure by breaking base-pairs one at a time. The ratcheting inchworm translocation had been previously proposed based on experimental data. Furthermore these results suggested that DNA unwinding occurs in bursts, whereas our findings reveal that basepairs are broken step by step. After our work was published Charles M. Rice reported in 2010, in his Inaugural Article in PNAS, novel crystal structures for a set of HCV helicase complexes with ATP-mimics and bound single-stranded DNA [29]. They provide, for the first time, conformational snapshots along the functional cycle of HCV helicase and allow to discuss the molecular basis of its activity. The obtained data shows that upon binding of the ATP- mimicking nucleotide the two motor domains come into tight contact, and, while interactions between domain Ⅱ and the DNA are maintained, they appear to be decreased between the protein domain Ⅰ and the 3 end of the DNA. In a transition conformation corresponding to a post-hydrolysis state of the helicase, however, distances to the DNA are changed and new connections between domain Ⅰ and the DNA strand appear, whereas domain Ⅱ may be able to slide along the strand when returning to the initial open-shape conformation of the helicase. Taken together, these novel crystal structures provide insights into ATP-induced conformational changes in HCV helicase and offer a molecular-level explanation of the ratcheting single-base translocation mechanism based on the observed loss and recovery of interactions between the protein and the single DNA strand. Furthermore, in 2011 crystal structures capturing HCV helicase with its natural substrate RNA became available in its nucleotide-free conformation and bound to a non-hydrolyzable analog [30]. They also evidence largescale domain motions and the switching from open to closed conformation in response to binding of the ATPmimicking ligand and point towards a mechanism of unidirectional stepping along RNA by one base-pair per hydrolysis cycle, caused by alternating affinities of the helicase to bind the RNA. The novel structural data has unravelled important aspects of the molecular mechanisms that underlie the activity of HCV helicase and thereby largely contributed to the understanding of how this important molecular motor performs its operation. In Figure 5, observations from our simulation are compared with the experimentally determined structures. The structural data is in well agreement with the predictions of conformational motions as revealed from our computer experiments, as 3407 April 7, 2014 Volume 20 Issue 13

33 Flechsig H. Operation cycles of HCV helicase A Ligand-free B Ligand-complex Figure 5 Comparison with novel hepatitis C virus crystal structures. Structural changes between ligand-free conformation and ligand-bound complex as predicted by our simulations (A and B) are compared with structures determined in recent experiments (C and D, Protein Data Bank codes 3KQH and 3KQU; ADP.BeF3 was used as ATP analog). ATP: Adenosinetriphosphate; ADP: Adenosine-diphosphate. C Ligand-free D ADP.BeF3 they confirm both the essential relative motions of the motor domains and the ratchet mechanism employed by the protein to translate them into locomotion along the nucleic acid strand with a step-size of one single base. In addition to the static structures, results from high-resolution optical tweezers experiments, aimed to follow the unwinding of double-stranded RNA by HCV helicase, have been reported in 2011 by the Bustamante lab [31]. They observe an opening of the duplex structure in single base-pair steps, which is consistent with what we have found in our computer experiments. It should be mentioned that recently coarse-grained modelling of HCV helicase has been combined with atomistic simulations [32]. The obtained results are in support of the inchworm model of helicase locomotion and propose key amino acid residues crucial for the translocation machinery. In summary, using coarse-grained structure-based modelling that included interactions with ATP molecules and DNA in an approximate fashion, we were able to follow entire operation cycles of an important molecular motor in computer experiments for the first time. Our findings are today confirmed by experimental data that has been acquired recently. Generally, our study demonstrates the feasibility of approximate mechanical models in computer experiments of molecular machines and motors. Despite their coarse-grained nature, such dynamical descriptions are capable of explaining functional aspects of these nanoscale engines and, therefore, help to understand the general principles by which they operate. Although approaches from computational biology have been proven remarkably successful in assisting in protein research, their explanatory power is also limited and apparently they cannot replace the experiments. Recently, large progress has been achieved in single-molecule techniques to investigate protein dynamics. In experiments that are based on the high-speed atomic force microscopy it became possible to observe protein machines as they perform their operation [33,34]. These approaches offer new possibilities towards an understanding of the important machines that control life at the nanoscale. Researchers may also use such experimental methods to investigate helicase motors in the future. REFERENCES 1 Frick DN, Lam AM. Understanding helicases as a means of virus control. Curr Pharm Des 2006; 12: [PMID: DOI: / ] 2 De Francesco R, Migliaccio G. Challenges and successes in developing new therapies for hepatitis C. Nature 2005; 436: [PMID: DOI: /nature04080] 3 Kwong AD, Rao BG, Jeang KT. Viral and cellular RNA helicases as antiviral targets. Nat Rev Drug Discov 2005; 4: [PMID: DOI: /nrd1853] 4 Moradpour D, Penin F, Rice CM. Replication of hepatitis C virus. Nat Rev Microbiol 2007; 5: [PMID: DOI: /nrmicro1645] 5 Caruthers JM, McKay DB. Helicase structure and mechanism. Curr Opin Struct Biol 2002; 12: [PMID: DOI: /S X(02) ] 6 Lohman TM, Bjornson KP. Mechanisms of helicase-catalyzed DNA unwinding. Annu Rev Biochem 1996; 65: [PMID: DOI: /annurev.bi ] 7 Jankowsky E, Fairman ME. RNA helicases--one fold for many functions. Curr Opin Struct Biol 2007; 17: April 7, 2014 Volume 20 Issue 13

34 Flechsig H. Operation cycles of HCV helicase [PMID: DOI: /j.sbi ] 8 Spudich JA. How molecular motors work. Nature 1994; 372: [PMID: DOI: /372515a0] 9 Enemark EJ, Joshua-Tor L. On helicases and other motor proteins. Curr Opin Struct Biol 2008; 18: [PMID: DOI: /j.sbi ] 10 Yao N, Hesson T, Cable M, Hong Z, Kwong AD, Le HV, Weber PC. Structure of the hepatitis C virus RNA helicase domain. Nat Struct Biol 1997; 4: [PMID: DOI: /nsb ] 11 Kim JL, Morgenstern KA, Griffith JP, Dwyer MD, Thomson JA, Murcko MA, Lin C, Caron PR. Hepatitis C virus NS3 RNA helicase domain with a bound oligonucleotide: the crystal structure provides insights into the mode of unwinding. Structure 1998; 6: [PMID: DOI: / S (98) ] 12 Frick DN. The hepatitis C virus NS3 protein: a model RNA helicase and potential drug target. Curr Issues Mol Biol 2007; 9: 1-20 [PMID: ] 13 Dumont S, Cheng W, Serebrov V, Beran RK, Tinoco I, Pyle AM, Bustamante C. RNA translocation and unwinding mechanism of HCV NS3 helicase and its coordination by ATP. Nature 2006; 439: [PMID: DOI: /nature04331] 14 Myong S, Bruno MM, Pyle AM, Ha T. Spring-loaded mechanism of DNA unwinding by hepatitis C virus NS3 helicase. Science 2007; 317: [PMID: DOI: / science ] 15 Zheng W, Liao JC, Brooks BR, Doniach S. Toward the mechanism of dynamical couplings and translocation in hepatitis C virus NS3 helicase using elastic network model. Proteins 2007; 67: [PMID: DOI: /prot.21326] 16 Flechsig H, Mikhailov AS. Tracing entire operation cycles of molecular motor hepatitis C virus helicase in structurally resolved dynamical simulations. Proc Natl Acad Sci USA 2010; 107: [PMID: DOI: / pnas ] 17 McCammon JA, Gelin BR, Karplus M. Dynamics of folded proteins. Nature 1977; 267: [PMID: DOI: /267585a0] 18 Tuckerman ME, Martyna GJ. Understanding modern molecular dynamics: techniques and applications. J Phys Chem B 2000; 104: [DOI: /jp992433y] 19 Karplus M, McCammon JA. Molecular dynamics simulations of biomolecules. Nat Struct Biol 2002; 9: [PMID: DOI: /nsb ] 20 Tozzini V. Coarse-grained models for proteins. Curr Opin Struct Biol 2005; 15: [PMID: DOI: / j.sbi ] 21 Tirion MM. Large Amplitude Elastic Motions in Proteins from a Single-Parameter, Atomic Analysis. Phys Rev Lett 1996; 77: [PMID: DOI: /Phys- RevLett ] 22 Bahar I, Atilgan AR, Erman B. Direct evaluation of thermal fluctuations in proteins using a single-parameter harmonic potential. Fold Des 1997; 2: [PMID: DOI: /S (97) ] 23 Hinsen K. Analysis of domain motions by approximate normal mode calculations. Proteins 1998; 33: [PMID: DOI: /(SICI) ( )33:3<417:: AID-PROT10>3.0.CO;2-8] 24 Bahar I, Atilgan AR, Demirel MC, Erman B. Vibrational dynamics of folded proteins: significance of slow and fast motions in relation to function and stability. Phys Rev Lett 1998; 80: [DOI: /PhysRevLett ] 25 Tama F, Sanejouand YH. Conformational change of proteins arising from normal mode calculations. Protein Eng 2001; 14: 1-6 [PMID: DOI: /protein/14.1.1] 26 Zheng W, Doniach S. A comparative study of motor-protein motions by using a simple elastic-network model. Proc Natl Acad Sci USA 2003; 100: [PMID: DOI: /pnas ] 27 Yang L, Song G, Jernigan RL. How well can we understand large-scale protein motions using normal modes of elastic network models? Biophys J 2007; 93: [PMID: DOI: /biophysj ] 28 Flechsig H, Popp D, Mikhailov AS. In silico investigation of conformational motions in superfamily 2 helicase proteins. PLoS One 2011; 6: e21809 [PMID: DOI: / journal.pone ] 29 Gu M, Rice CM. Three conformational snapshots of the hepatitis C virus NS3 helicase reveal a ratchet translocation mechanism. Proc Natl Acad Sci USA 2010; 107: [PMID: DOI: /pnas ] 30 Appleby TC, Anderson R, Fedorova O, Pyle AM, Wang R, Liu X, Brendza KM, Somoza JR. Visualizing ATP-dependent RNA translocation by the NS3 helicase from HCV. J Mol Biol 2011; 405: [PMID: DOI: / j.jmb ] 31 Cheng W, Arunajadai SG, Moffitt JR, Tinoco I, Bustamante C. Single-base pair unwinding and asynchronous RNA release by the hepatitis C virus NS3 helicase. Science 2011; 333: [PMID: DOI: /science ] 32 Zheng W, Tekpinar M. Structure-based simulations of the translocation mechanism of the hepatitis C virus NS3 helicase along single-stranded nucleic acid. Biophys J 2012; 103: [PMID: DOI: /j.bpj ] 33 Ando T. Molecular machines directly observed by highspeed atomic force microscopy. FEBS Lett 2013; 587: [PMID: DOI: /j.febslet ] 34 Ando T, Uchihashi T, Kodera N. High-speed AFM and applications to biomolecular systems. Annu Rev Biophys 2013; 42: [PMID: DOI: /annurevbiophys ] P- Reviewers: Bock T, Diamantis I S- Editor: Gou SX L- Editor: A E- Editor: Ma S 3409 April 7, 2014 Volume 20 Issue 13

35 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (2): Hepatitis C virus TOPIC HIGHLIGHT Chronic hepatitis C virus infection and atherosclerosis: Clinical impact and mechanisms Luigi E Adinolfi, Rosa Zampino, Luciano Restivo, Amedeo Lonardo, Barbara Guerrera, Aldo Marrone, Fabio Nascimbeni, Anna Florio, Paola Loria Luigi E Adinolfi, Rosa Zampino, Luciano Restivo, Barbara Guerrera, Aldo Marrone, Department of Medical, Surgical, Neurological, Metabolic, and Geriatric Sciences, Second University of Naples, Naples, Italy Amedeo Lonardo, Fabio Nascimbeni, Paola Loria, Department of Internal Medicine, Endocrinology, Metabolism and Geriatrics, University of Modena and Reggio Emilia, Modena, Italy Anna Florio, Vascular Surgery, Second University of Naples, Naples, Italy Author contributions: Adinolfi LE conceived and drafted the article, and approved the final version; Zampino R contributed to drafting the article and approved the final version; Restivo L, Guerrera B, Marrone A, Nascimbeni F and Florio A reviewed the literature and approved the final version of this article; Lonardo A and Loria P critically reviewed the manuscript and approved the final version of this article. Supported by A grant by Regione Campania, Italy Correspondence to: Luigi E Adinolfi, MD, Professor, Director of Internal Medicine, Department of Medical, Surgical, Neurological, Metabolic, and Geriatric Sciences, Second University of Naples, Marcianise (CE), Naples, Italy. luigielio.adinolfi@unina2.it Telephone: Fax: Received: September 27, 2013 Revised: October 30, 2013 Accepted: January 6, 2014 Published online: April 7, 2014 Abstract Hepatitis C virus (HCV) infection represents a major health issue worldwide due to its burden of chronic liver disease and extrahepatic manifestations including cardiovascular diseases, which are associated with excess mortality. Analysis of published studies supports the view that HCV infection should be considered a risk factor for the development of carotid atherosclerosis, heart failure and stroke. In contrast, findings from studies addressing coronary artery disease and HCV have yielded conflicting results. Therefore, meta- analytic reviews and prospective studies are warranted. The pathogenic mechanisms connecting HCV infection, chronic liver disease, and atherogenesis are not completely understood. However, it has been hypothesized that HCV may promote atherogenesis and its complications through several direct and indirect biological mechanisms involving HCV colonization and replication within arterial walls, liver steatosis and fibrosis, enhanced and imbalanced secretion of inflammatory cytokines, oxidative stress, endotoxemia, mixed cryoglobulinemia, perturbed cellular and humoral immunity, hyperhomocysteinemia, hypo-adiponectinaemia, insulin resistance, type 2 diabetes and other components of the metabolic syndrome. Understanding these complex mechanisms is of fundamental importance for the development of novel therapeutic approaches to prevent and to treat vascular complications in patients with chronic HCV infection. Currently, it seems that HCV clearance by interferon and ribavirin treatment significantly reduces non-liver-related mortality; moreover, interferon-based treatment appears to decrease the risk of ischemic stroke Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Hepatitis C virus; Atherosclerosis; Coronary artery disease; Stroke; Inflammation Core tip: Hepatitis C virus (HCV) infection represents a risk factor for carotid atherosclerosis, heart failure and ischemic stroke. However, findings from studies addressing coronary artery disease and HCV have yielded conflicting results. Moreover, an excess of cardiovascular mortality among anti-hcv positive subjects has been reported. HCV promotes atherogenesis through direct and indirect mechanisms. Inflammation, cytokines activation, cellular and humoral immunity, metabolic derangement, oxidative stress, liver steatosis and 3410 April 7, 2014 Volume 20 Issue 13

36 Adinolfi LE et al. HCV and cardiovascular risk fibrosis have been postulated as potential atherogenic mechanisms. Knowledge of such complex mechanisms may be important for understanding disease progression and promoting novel therapeutic approaches. At present, interferon-based treatment of chronic hepatitis C seems to reduce the risk of stroke as well as nonliver-related mortality. Adinolfi LE, Zampino R, Restivo L, Lonardo A, Guerrera B, Marrone A, Nascimbeni F, Florio A, Loria P. Chronic hepatitis C virus infection and atherosclerosis: Clinical impact and mechanisms. World J Gastroenterol 2014; 20(13): Available from: URL: i13/3410.htm DOI: INTRODUCTION Hepatitis C virus (HCV) infection is endemic worldwide, with an estimated global prevalence of 3%, resulting in approximately 170 million infected people [1]. HCV infection is the major cause of chronic liver diseases leading to a wide range of hepatic diseases, including cirrhosis and hepatocellular carcinoma. In addition, there is a growing body of data concerning the role of HCV in extrahepatic manifestations [2], including metabolic derangements [3], and, more recently, accelerated atherosclerosis eventually triggering cardiovascular events [4]. Atherosclerosis, either subclinical or manifest, is a chronic inflammatory disease. The chief clinical manifestations are coronary artery disease (CAD), stroke and ischemic limbs. In addition to the traditional factors of atherosclerosis other novel factors have been hypothesized. The possible role of an infectious agent in the development of experimental atherosclerosis in rodents was first reported more than 120 years ago [5], and this concept has gained new life in recent years [6]. In the last few years, a strict association between atherosclerosis and nonalcoholic fatty liver disease (NAFLD) has been reported, challenging the old paradigm that chronic liver disease protects from atherosclerosis [7,8]. Similar to NAFLD, HCV infection is increasingly identified as a potential atherogenic condition. In fact, chronic HCV infection causes hepatic and systemic inflammation [4] via increased levels of pro-atherogenic chemokines and cytokines [4] and hepatic steatosis, a distinguishing feature of this infection [9]. In addition, it has been demonstrated that HCV colonizes and replicates within carotid plaques [10,11] likely causing vascular inflammation. Earlier studies conducted in the general population showed that HCV markers were independently associated with atherosclerosis [12,13]. Subsequent research, however, yielded conflicting results, some studies confirming [14,15] and others denying such an association [16,17]. However, recent data have shown excess cardiovascular mortality during the course of chronic HCV infection [18,19]. Given the high prevalence of HCV infection on a worldwide basis and the primacy of cardiovascular diseases among the causes of mortality, the role of HCV as a cardiovascular risk factor needs to be analysed in-depth. Accordingly, in this review we evaluated the literature regarding the association between chronic HCV infection and atherosclerosis, the clinical impact on the development of cardiovascular diseases and the possible pathogenic mechanisms underlying increased cardiovascular risk in those with HCV infection. HCV AND ATHEROSCLEROSIS Table 1 reports the findings of the studies evaluating the association between HCV and carotid atherosclerosis. Ishizaka et al [12,13], assessing a large number of subjects in two studies conducted in the general population, were first in reporting an association between HCV infection and atherosclerosis. The first study was published in 2002 and showed that HCV infection was associated with an increased risk of carotid atherosclerosis [12]. Similar results were obtained in a second study published in 2003 [13]. Irrespective of known atherogenic risk factors, Ishizaka et al [12,13] found that HCV was an independent predictor of carotid atherosclerosis. Another study, which enrolled a large cohort of Japanese subjects from the general population, showed that HCV-infected subjects had increased arterial stiffness compared to HCV-negative controls [20]. Fukui et al [21] confirmed these results in a series of patients with type 2 diabetes in which anti-hcv positivity was demonstrated to be an independent risk factor of both increased carotid intima-media-thickness (IMT) and plaque. Following these pioneer Japanese reports, two Italian studies further confirmed the independent association between HCV infection and atherosclerosis. Boddi et al [10] showed that the prevalence of an IMT greater than 1 mm was significantly higher in anti-hcv positive subjects than in controls. At multivariate analysis, anti- HCV positivity was an independent predictor of IMT. Similarly, Targher et al [15] showed that HCV positivity was an independent predictor of increased carotid IMT. A large study in Egyptian individuals, including HCV infected patients, HCV subjects with viral clearance, and subjects never infected (controls), showed that the prevalence of carotid atherosclerosis did not vary when patients with active infection were compared to those with past infection. However, HCV infected patients showed a higher risk of atherosclerosis following adjustment for known cardiovascular risk factors [22]. Petta et al [23] evaluating carotid atherosclerosis in a cohort of biopsy-proven chronic hepatitis C genotype 1 patients, reported that HCV patients had a significantly higher prevalence of atherosclerosis than matched control subjects (41.9% vs 22.9%, respectively). In addition, the severity of hepatic fibrosis was independently associated with a higher risk of carotid plaques. Recently, our group assessed carotid atherosclerosis in a large cohort of consecutive liver biopsy-proven chronic hepatitis C patients with and without steatosis [24]. HCV patients showed a significantly higher prevalence of atherosclerosis than that observed in the HCV April 7, 2014 Volume 20 Issue 13

37 Adinolfi LE et al. HCV and cardiovascular risk Table 1 Main characteristics of the studies which evaluated the association between hepatitis C virus and atherosclerosis Ref. Type of study Country HCV+ HCV- (n ) (n ) Studies showing association Ishizaka et al [12], 2002 Population Japan Ishizaka et al [13], 2003 Population Japan Tomiyawa et al [20], 2003 Cohort Japan Fukui et al [21], 2003 Cohort Japan Boddi et al [10], 2007 Cohort Italy Targher et al [15], 2007 Cohort Italy Butt et al [35], 2009 Observational cohort United States Mostafa et al [22], 2010 Cross-sectional Egypt Petta et al [23], 2012 Cohort Italy Adinolfi et al [9], 2013 Cohort Italy Studies showing no association Bilora et al [28], 2008 Cohort Italy Caliskan et al [29], 2009 Cohort Turkey Tien et al [30], 2009 Cohort United States Masiá et al [31], 2011 Cohort Spain HCV: Hepatitis C virus. negative control group (53.7% vs 34.3%, respectively, P < ). HCV patients without steatosis showed a higher prevalence of atherosclerosis than that observed in the HCV-negative control group without steatosis (26.0% vs 14.8%, P < 0.015). HCV patients with steatosis had a prevalence of atherosclerosis significantly higher than NAFLD patients (77.7% vs 57.8%, P < ). These results provided definite evidence that HCV was strictly associated with the development of atherosclerosis and that patients with HCV-related steatosis, irrespective of HCV genotype, age, gender and degree of histological liver damage, exhibited the highest prevalence of atherosclerosis. Moreover, at multivariate analysis, HCV-related steatosis was an independent risk factor for carotid atherosclerosis (OR = 32.35; 95%CI: , P < ). HCV-related steatosis predicted atherosclerosis with an AUC of 0.78 (95%CI: , P < ; with a positive specificity of 81.7% and sensitivity of 74.2%). These data suggest that steatosis is both a good marker for identifying atherosclerosis-prone individuals and an early mediator of atherosclerosis. Evidence for the latter role is based on steatosis being associated with traditional proatherogenic factors such as metabolic syndrome, insulin resistance, hyper-homocysteinaemia, liver inflammation and fibrosis scores, hypo-adiponectinaemia and hypertumour necrosis factor-alpha (TNF-α) [25] and oxidative stress [26]. In brief, our study [24] suggests that HCV-related steatosis modulates atherogenic factors such as inflammation and the dysmetabolic milieu, therefore, favouring development of atherosclerosis. Furthermore, our study [24] showed that amongst the younger HCV population (e.g., < 50 years old) about 34% showed atherosclerosis and a significant proportion (24.1%) had plaques, whereas in the control group the prevalence of atherosclerosis was significantly lower (16.0%) and the presence of plaques (3.9%) was a relatively uncommon finding. Such data support the view that chronic HCV infection predisposes individuals to the premature development of atherosclerosis and advanced carotid changes despite the more favourable cardiovascular risk profile featuring lower lipid levels and lower prevalence of metabolic syndrome [24]. An increased risk of carotid atherosclerosis was also demonstrated in HIV-infected patients. In this setting, Sosner et al [27] reported an increased prevalence of carotid plaque in HIV/HCV co-infected patients, strengthening the view that HCV infection is an independent risk factor of atherosclerosis. In contrast to the above studies which highlighted a strict association between chronic HCV infection and carotid atherosclerosis, other studies did not confirm such an association. For example, Bilora et al [28] and Caliskan et al [29], in the setting of hemodialysis patients, did not find any association between HCV infection and atherosclerosis. Similarly, in a cohort of HCV-infected women, with and without HIV co-infection, Tien et al [30], after adjustment for confounding factors, were unable to demonstrate an association between HCV infection and atherosclerosis. Likewise, Masiá et al [31] did not observe any association between HCV infection and atherosclerosis in a cohort of HIV co-infected patients. It is important to underline that those studies showing a positive association, generally included a very large population of subjects, whereas the studies showing no association were generally conducted in smaller cohorts of patients at high risk for atherosclerosis (e.g., either hemodialysed or HCV/HIV co-infected) (Table 1). Considering that atherosclerosis is a multi-factorial disorder, studies with low power may have generated false negative results of doubtful clinical significance. In addition, inconsistency among studies may be the result of different features in the populations under examination, differences in the assessment of HCV infection and stage of liver disease, technique used to evaluate atherosclerosis and, of primary importance, comparator control groups and adjustment for confounding factors. Overall, we deem that an analysis of available studies supports the view that HCV infection should be considered a risk factor for the development of atherosclerosis. Meta-analytic reviews and adequate prospective studies are eagerly awaited to gauge the impact of HCV on the development of atherosclerosis more precisely. IMPACT OF HCV-ASSOCIATED ATHEROSCLEROSIS ON CARDIAC AND CEREBRAL VASCULAR DISEASES A consistent body of evidence indicates that chronic HCV infection should be considered a risk factor for subclinical atherosclerosis. Accordingly, the question is: is there evidence that HCV is a risk factor for the development of cardiac and cerebral vascular diseases? Clearly 3412 April 7, 2014 Volume 20 Issue 13

38 Adinolfi LE et al. HCV and cardiovascular risk Table 2 Main characteristics of the studies which evaluated the association between hepatitis C virus and cardiac diseases Ref. Type of study Country HCV+ (n ) HCV- (n ) Comment Studies showing association Vassalle et al [14], 2004 Cohort Italy CAD (491), HCV 6.3% vs control (195), 2% Alyan et al [37], 2008 Cohort Turkey Butt et al [35], 2009 Cohort United States CAD patients Yelken et al [38], 2009 Cohort Turkey Renal transplanted Maruyama et al [39], 2013 Cohort Japan 217 Myocardial perfusion defect Younossi et al [40], 2013 Population United States Congestive heart failure Adinolfi et al [9], 2013 Retrospective Italy Hospitalized Studies showing no association Völzke et al [16], 2004 Cross-sectional Germany Arcari et al [17], 2006 Case-control United States Myocardial infarction Forde et al [36], 2012 Population cohort United Kingdom Myocardial infarction Younossi et al [40], 2013 Population United States Myocardial infarction CAD: Coronary artery disease; HCV: Hepatitis C virus. Vassalle et al [14] showed that HCV infection was an independent predictor of angiographically-documented CAD (adjusted OR = 4.2; 95%CI: ). Similar results were reported in another study conducted in patients with CAD [37] and in a study on hemodialysed patients [38]. Two recent studies have further reinforced the possible role of HCV in the development of cardiac diseases. Maruyama et al [39] showed myocardial perfusion defects in 87% of patients with chronic hepatitis C; of interest, such defects improved after viral eradication with interferon treatment. Younossi et al [40], evaluated United States subjects from the National Health and Nutrition Examination Survey (NHANES), and showed that chronic HCV infection was independently associated with the presence of metabolic conditions, such as insulin resistance, type 2 diabetes mellitus and hypertension, and with congestive heart failure. Recently, we conducted a retrospective study including 78 HCV-positive patients compared to 742 HCVnegative subjects [41]. We observed higher ischemic heart events in the HCV-positive patients than in the HCVnegative patients (22% vs 13%, respectively, P = 0.031). In contrast to the above studies, several other studies did not show an association between HCV infection and cardiac diseases, particularly myocardial infarction. In this context, in a large study including subjects from the NHANES database, Younossi et al [40] showed that chronic HCV infection was independently associated with congestive heart failure, but not ischaemic heart disease. Similarly, Arcari et al [17] and Maruyama et al [39] did not find any association between HCV infection and myocardial infarction. Völzke et al [16], in their study conducted in the Pomeranian general population, Germany, enrolled 233 HCV-Ab positive cases and 4033 control individuals, and did not show an association between HCV seropositivity and cardiovascular diseases. In conclusion, the findings from a consistent number of studies which showed a strict association between HCV infection and cardiac diseases are in conflict with negative studies, which mainly addressed myocardial inthe answer is not straightforward and involves different levels of assessment. First, it is important to evaluate a hard outcome, such as mortality related to cardiovascular diseases in those with HCV infection, and next, studies evaluating such an association need to be weighted. Evidence for a significant excess of cardiovascular mortality among anti-hcv positive subjects is provided by a large study which assessed HCV-associated all-cause mortality in the general United States population [32]. Moreover, a study conducted in the general population showed that HCV infection was an independent predictor of cerebrovascular deaths and that such mortality was correlated with serum HCV RNA levels [33]. Of importance, HCV clearance by interferon and ribavirin treatment significantly reduces non-liver-related mortality in these patients [34]. The above data suggest a possible direct impact of HCV infection on cardiovascular diseases. HCV and cardiac diseases In the last few years many studies have evaluated the association between HCV infection and atherosclerosis. Table 2 reports the main findings of these published papers. Epidemiological and cohort studies have reported conflicting results. Butt et al [35] conducted the largest epidemiological study (82083 HCV-infected and HCVuninfected subjects) in United States veterans over a 5-year period. The data showed that HCV infected subjects had a significantly higher prevalence of cardiac diseases (myocardial infarction, congestive heart failure, coronary artery bypass grafting or coronary angioplasty) despite being younger and having a more favourable cardiometabolic risk profile. In contrast, Forde et al [36] in a retrospective analysis which included HCV-infected and -uninfected subjects in a United Kingdom general practice, failed to show any difference in the incidence of myocardial infarction during a median observational period of 3.2 years. However, as acknowledged by these authors [36], the nature of the study, the outcome evaluated and the relative short follow-up period may have affected the results. In a case-control study, which included 686 patients, 3413 April 7, 2014 Volume 20 Issue 13

39 Adinolfi LE et al. HCV and cardiovascular risk Table 3 Main characteristics of the studies which evaluated the association between hepatitis C virus and stroke Ref. Type of study Country HCV+ (n ) HCV- (n ) Comment Studies showing association Forssen et al [42], 2009 Population retrospective United States Gutierrez et al [43], 2012 Population retrospective United States - - NHANES ( ) Liao et al [44], 2012 Population cohort Taiwan Hsu et al [45], 2013 Cohort retrospective Taiwan Adinolfi et al [9], 2013 Cohort retrospective Italy Hospitalized Studies showing no association Younossi et al [40], 2013 Population retrospective United States NHANES ( ) NHANES: National Health and Nutrition Examination Survey; HCV: Hepatitis C virus. farction as the outcome. Accordingly, at present, due to the different methodology and parameters evaluated, it is difficult to draw definitive conclusions. Despite such limitations, it seems reasonable to conclude that chronic HCV infection appears to be linked with excess cardiovascular risk, except for myocardial infarction. Again, meta-analytic reviews and prospective studies are warranted. HCV and stroke In the last 5 years, six papers, reported in Table 3, were published concerning the association between HCV and stroke. In contrast to those previously reported studies on cardiovascular diseases, 5 out of these 6 reports showed an association between HCV infection and ischemic stroke. In a large retrospective population-based study, Forssen et al [42] showed an association between HCV infection and stroke (OR = 1.76; 95%CI: ). This report, however, was published in abstract form alone. Similarly, another retrospective report published by Gutierrez et al [43] in abstract form, showed a close association between HCV infection and stroke (OR = 9.61; 95%CI: ) in subjects enrolled from the NHANES cohort during In contrast, in another retrospective study from the NHANES database, between , Younossi et al [40] were not able to demonstrate an association between HCV infection and stroke. However, many confounding factors, such as gender, race, and hypertension, were significantly different between the HCV population and the control group. Therefore, heterogeneity may have generated unreliable results. In a large prospective study conducted in Taiwan, utilizing the Taiwan National Health Insurance Research Database, Liao et al [44] demonstrated a strict association between HCV infection and stroke (HR = 1.22; 95%CI: ). Similarly, Hsu et al [45] showed that HCV infection was a risk factor for stroke (HR = 1.23; 95%CI: ) in a large retrospective study including Taiwan subjects from the Longitudinal Health Insurance database Of importance, these authors demonstrated that an interferon-based therapy reduced the risk of stroke in HCV patients [45]. We recently conducted a study with the aims of evaluating the prevalence and role of HCV infection in patients with stroke [41]. The study enrolled 820 consecutive individuals, 123 cases with stroke and 697 age- and gender-matched controls. We found that the prevalence of HCV was higher in cases with stroke than in the control group (26.8% vs 6.6%, P = ). Moreover, HCV patients with stroke were younger and had lower risk factors and higher inflammation indices than HCV-negative stroke patients. Multivariate analysis showed HCV infection to be an independent risk factor for stroke (OR = 2.04; 95%CI: , P = ). Taken collectively, these data [41,42,44,45] suggest that HCV infection increases the risk of stroke. Moreover, in HCV patients, stroke occurs at younger age, irrespective of sex and in subjects with lower risk of stroke/atherosclerosis such as lower cholesterol/triglyceride levels, and lower prevalence of hypertension [41]. Finally, HCV viral load appears to be associated with an increased risk of mortality in patients with stroke [33]. A meta-analysis of the studies which evaluated whether HCV infection was a risk factor for stroke has been conducted [46]. The results suggested that HCV infection significantly increased the risk of stroke (OR = 1.97; 95%CI: ). However, due to the relatively low number of studies performed, the authors suggest caution in the interpretation of the data and advocate further studies to confirm the results [46]. HCV AND ATHEROSCLEROSIS: PATHOGENIC MECHANISMS The precise pathogenic mechanisms connecting HCV infection, chronic liver disease, and atherogenesis are not completely understood. Figure 1 depicts the potential pathogenic factors involved. It is logical to hypothesize that HCV may promote atherogenesis through several direct and indirect biological mechanisms. Atherosclerotic plaques are widely recognized to develop and to destabilize as a result of persistent inflammatory changes. Chronic HCV infection is associated with both hepatic and systemic inflammation, which is triggered by HCV either directly or indirectly. Such inflammatory pathways involve cytokines release and increased oxidative stress. HCV RNA sequences have been isolated within carotid plaques, supporting the hypothesis that HCV plays a direct pro-atherogenic role by inducing arterial inflammation, likely via the pro-inflammatory cytokine interleukin 3414 April 7, 2014 Volume 20 Issue 13

40 Adinolfi LE et al. HCV and cardiovascular risk HCV Steatosis IR diabetes Pro-inflammatory cytokines Cryoglobulin Increased: Homocysteine Oxidative stress IR Lipid peroxidation Decreased: Adiponectine Antioxidants Oxidative stress Living and replicating within plaque Chronic local and systemic inflammation Cardiac disease Cerebrovascular disease Atherosclerosis Ischemic stroke Figure 1 Pathogenic mechanisms associated with the development of atherosclerosis in chronic hepatitis C infection. HCV: Hepatitis C virus; IR: Insulin resistance. 1β [47]. In addition, HCV structural and non-structural proteins play a major role in initiating and maintaining chronic inflammation. HCV promotes an imbalanced T helper (Th)1/Th2 cytokines ratio perturbing the equilibrium between cellular immunity, promoted and maintained by interleukin (IL)-2, TNF-α and interferon-γ, and humoral immunity, sustained by IL-4, IL-5, IL-6 and IL-10 [48]. Compared to HCV negative individuals, patients with chronic HCV infection have been shown to exhibit higher TNF-α levels which are associated with an increased risk of heart failure and death [49]. Moreover, an intermediate cardiovascular risk, higher levels of proinflammatory cytokines (IL-6 and TNF-α), and a higher ratio of pro-inflammatory/anti-inflammatory cytokines (TNF-α/IL-10 and IL-6/IL-10) have been reported in non-obese, non-diabetic, HCV-infected patients than that observed in the control group [50]. HCV-driven secretion of inflammatory cytokines may contribute to the development of cardiovascular disease through several effector mechanisms, including enhanced synthesis of matrix metalloproteinase-9, intracellular adhesion molecules, expression of anti-endothelium antibodies, and generation of oxidative stress and insulin resistance. The higher prevalence of atherosclerosis observed in chronic HCV infection is correlated with liver steatosis [24], a key feature of HCV infection which is associated with several pro-atherogenic factors including inflammatory cytokines, hyper-homocysteinaemia, hypo-adiponectinaemia, insulin resistance, and components of the metabolic syndrome [24]. Atherosclerosis in HCV patients is also associated with liver fibrosis [23] which is an expected finding given the tight association between steatosis and fibrosis in those with chronic hepatitis C [9]. Histological hepatic changes play a pivotal role in regulating local and systemic inflammation through proteins secreted by the diseased liver [4]. HCV is considered a metabolic virus and is associated with metabolic disorders, in particular insulin resistance and type 2, which are diabetes pro-atherogenic conditions. HCV infection is associated with increased levels of endotoxaemia, which promotes a strong inflammatory reaction via TNF-α and toll-like receptors, which are both able to induce a marked inflammatory reaction. Of interest, HCV is also the agent causing mixed cryoglobulinaemia a paradigmatic vasculitic condition [4]. Based on the above considerations it is clear that chronic HCV infection represents a condition directly and indirectly promoting a milieu of pro-atherogenic factors, mainly pro-inflammatory molecules, which trigger atherogenesis and favour its complications eventually leading to an increased prevalence of cardiovascular diseases. A more detailed understanding of the complex mechanisms underlying HCV-related inflammation and atherogenesis is of key importance for the development of novel therapeutic approaches targeting the different 3415 April 7, 2014 Volume 20 Issue 13

41 Adinolfi LE et al. HCV and cardiovascular risk steps of the inflammatory response to prevent and treat atherosclerosis in patients with chronic HCV infection. CONCLUSION The literature seems to support the view that chronic HCV infection is a risk factor for atherosclerosis, the development of cardiovascular diseases and significant cardiovascular mortality. However, further well-conducted studies are necessary to better assess the impact of HCV on the different cardiovascular conditions, in particular on myocardial infarction. The pathogenic mechanisms should be further elucidated due to their potential impact on the development of novel therapeutic approaches to prevent and to treat cardiovascular complications in patients with chronic HCV infection. Based on the evidence discussed in the present article, we feel it reasonable to recommend to screen noninvasively for atherosclerosis all patients with chronic HCV infection. Knowledge of the whole pathologic burden will be of help in making a reasoned decision for the management of chronic HCV infection. In this respect, it is important to underline that existing data seem to indicate that interferon-based treatment reduces the risk of stroke and of cardiovascular-related mortality. Future studies should clarify the full impact and the right timing of antiviral treatment in preventing, improving or reversing HCV-related atherosclerosis. REFERENCES 1 Lauer GM, Walker BD. Hepatitis C virus infection. N Engl J Med 2001; 345: [PMID: DOI: / NEJM ] 2 Zignego AL, Craxì A. Extrahepatic manifestations of hepatitis C virus infection. Clin Liver Dis 2008; 12: , ix [PMID: DOI: /j.cld ] 3 Adinolfi LE, Restivo L, Zampino R, Lonardo A, Loria P. Metabolic alterations and chronic hepatitis C: treatment strategies. Expert Opin Pharmacother 2011; 12: [PMID: DOI: / ] 4 Zampino R, Marrone A, Restivo L, Guerrera B, Sellitto A, Rinaldi L, Romano C, Adinolfi LE. 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Gut 2010; 59: [PMID: DOI: /gut ] 23 Petta S, Torres D, Fazio G, Cammà C, Cabibi D, Di Marco V, Licata A, Marchesini G, Mazzola A, Parrinello G, Novo S, Licata G, Craxì A. Carotid atherosclerosis and chronic hepatitis C: a prospective study of risk associations. Hepatology 2012; 55: [PMID: DOI: / hep.25508] 24 Adinolfi LE, Restivo L, Zampino R, Guerrera B, Lonardo A, Ruggiero L, Riello F, Loria P, Florio A. Chronic HCV infection is a risk of atherosclerosis. Role of HCV and HCVrelated steatosis. Atherosclerosis 2012; 221: [PMID: DOI: /j.atherosclerosis ] 25 Durante-Mangoni E, Zampino R, Marrone A, Tripodi MF, 3416 April 7, 2014 Volume 20 Issue 13

42 Adinolfi LE et al. HCV and cardiovascular risk Rinaldi L, Restivo L, Cioffi M, Ruggiero G, Adinolfi LE. Hepatic steatosis and insulin resistance are associated with serum imbalance of adiponectin/tumour necrosis factoralpha in chronic hepatitis C patients. Aliment Pharmacol Ther 2006; 24: [PMID: DOI: / j x] 26 Vidali M, Tripodi MF, Ivaldi A, Zampino R, Occhino G, Restivo L, Sutti S, Marrone A, Ruggiero G, Albano E, Adinolfi LE. Interplay between oxidative stress and hepatic steatosis in the progression of chronic hepatitis C. J Hepatol 2008; 48: [PMID: DOI: /j.jhep ] 27 Sosner P, Wangermez M, Chagneau-Derrode C, Le Moal G, Silvain C. Atherosclerosis risk in HIV-infected patients: the influence of hepatitis C virus co-infection. Atherosclerosis 2012; 222: [PMID: DOI: /j.atheroscl erosis ] 28 Bilora F, Campagnolo E, Rinaldi R, Rossato A, Arzenton M, Petrobelli F. Carotid and femoral atherosclerosis in chronic hepatitis C: a 5-year follow-up. Angiology 2008; 59: [PMID: DOI: / ] 29 Caliskan Y, Oflaz H, Pusuroglu H, Boz H, Yazici H, Tamer S, Karsidag K, Yildiz A. Hepatitis C virus infection in hemodialysis patients is not associated with insulin resistance, inflammation and atherosclerosis. Clin Nephrol 2009; 71: [PMID: ] 30 Tien PC, Schneider MF, Cole SR, Cohen MH, Glesby MJ, Lazar J, Young M, Mack W, Hodis HN, Kaplan RC. Association of hepatitis C virus and HIV infection with subclinical atherosclerosis in the women s interagency HIV study. AIDS 2009; 23: [PMID: DOI: / QAD.0b013e32832d7aa8] 31 Masiá M, Padilla S, Robledano C, Ramos JM, Gutiérrez F. Evaluation of endothelial function and subclinical atherosclerosis in association with hepatitis C virus in HIV-infected patients: a cross-sectional study. BMC Infect Dis 2011; 11: 265 [PMID: DOI: / ] 32 Lee MH, Yang HI, Lu SN, Jen CL, You SL, Wang LY, Wang CH, Chen WJ, Chen CJ. Chronic hepatitis C virus infection increases mortality from hepatic and extrahepatic diseases: a community-based long-term prospective study. J Infect Dis 2012; 206: [PMID: DOI: /infdis/ jis385] 33 Lee MH, Yang HI, Wang CH, Jen CL, Yeh SH, Liu CJ, You SL, Chen WJ, Chen CJ. Hepatitis C virus infection and increased risk of cerebrovascular disease. Stroke 2010; 41: [PMID: DOI: /STROKEAHA ] 34 Berenguer J, Rodríguez E, Miralles P, Von Wichmann MA, López-Aldeguer J, Mallolas J, Galindo MJ, Van Den Eynde E, Téllez MJ, Quereda C, Jou A, Sanz J, Barros C, Santos I, Pulido F, Guardiola JM, Ortega E, Rubio R, Jusdado JJ, Montes ML, Gaspar G, Esteban H, Bellón JM, González-García J. Sustained virological response to interferon plus ribavirin reduces non-liver-related mortality in patients coinfected with HIV and Hepatitis C virus. Clin Infect Dis 2012; 55: [PMID: DOI: /cid/cis500] 35 Butt AA, Xiaoqiang W, Budoff M, Leaf D, Kuller LH, Justice AC. Hepatitis C virus infection and the risk of coronary disease. Clin Infect Dis 2009; 49: [PMID: DOI: /599371] 36 Forde KA, Haynes K, Troxel AB, Trooskin S, Osterman MT, Kimmel SE, Lewis JD, Lo Re V. Risk of myocardial infarction associated with chronic hepatitis C virus infection: a population-based cohort study. J Viral Hepat 2012; 19: [PMID: DOI: /j x] 37 Alyan O, Kacmaz F, Ozdemir O, Deveci B, Astan R, Celebi AS, Ilkay E. Hepatitis C infection is associated with increased coronary artery atherosclerosis defined by modified Reardon severity score system. Circ J 2008; 72: [PMID: ] 38 Yelken B, Gorgulu N, Caliskan Y, Elitok A, Cimen AO, Yazici H, Oflaz H, Turkmen A, Sever MS. Association between chronic hepatitis C infection and coronary flow reserve in dialysis patients with failed renal allografts. Transplant Proc 2009; 41: [PMID: DOI: /j.transproceed ] 39 Maruyama S, Koda M, Oyake N, Sato H, Fujii Y, Horie Y, Murawaki Y. Myocardial injury in patients with chronic hepatitis C infection. J Hepatol 2013; 58: [PMID: DOI: /j.jhep ] 40 Younossi ZM, Stepanova M, Nader F, Younossi Z, Elsheikh E. Associations of chronic hepatitis C with metabolic and cardiac outcomes. Aliment Pharmacol Ther 2013; 37: [PMID: DOI: /apt.12234] 41 Adinolfi LE, Restivo L, Guerrera B, Sellitto A, Ciervo A, Iuliano N, Rinaldi L, Santoro A, Li Vigni G, Marrone A. Chronic HCV infection is a risk factor of ischemic stroke. Atherosclerosis 2013; 231: [PMID: DOI: /j.atherosclerosis ] 42 Forssen U, McAfee A, Enger C, Bennett D, Shantakumar S. Risk of thromboembolic events (TEs) among patients infected with hepatitis C. Hepatology 2009; 50: 672A 43 Gutierrez J, Elkind MSV. Chronic inflammatory diseases and stroke: Evidence for heterogeneous mechanisms. Ann Neurol 2012; 72: S6-S7 44 Liao CC, Su TC, Sung FC, Chou WH, Chen TL. Does hepatitis C virus infection increase risk for stroke? A populationbased cohort study. PLoS One 2012; 7: e31527 [PMID: DOI: /journal.pone ] 45 Hsu CS, Kao JH, Chao YC, Lin HH, Fan YC, Huang CJ, Tsai PS. Interferon-based therapy reduces risk of stroke in chronic hepatitis C patients: a population-based cohort study in Taiwan. Aliment Pharmacol Ther 2013; 38: [PMID: DOI: /apt.12391] 46 He Huang R, Zhao Z. Hepatitis C virus infection and risk of stroke: a systematic review and meta-analysis. PLoS One 2013; 8: e81305 [PMID: DOI: /journal. pone ] 47 Costantini S, Capone F, Guerriero E, Maio P, Colonna G, Castello G. Serum cytokine levels as putative prognostic markers in the progression of chronic HCV hepatitis to cirrhosis. Eur Cytokine Netw 2010; 21: [PMID: DOI: /ecn ] 48 Abbas Z, Moatter T. Interleukin (IL) 1beta and IL-10 gene polymorphism in chronic hepatitis C patients with normal or elevated alanine aminotransferase levels. J Pak Med Assoc 2003; 53: [PMID: ] 49 Tsui JI, Whooley MA, Monto A, Seal K, Tien PC, Shlipak M. Association of hepatitis C virus seropositivity with inflammatory markers and heart failure in persons with coronary heart disease: data from the Heart and Soul study. J Card Fail 2009; 15: [PMID: DOI: / j.cardfail ] 50 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 ] P- Reviewers: Dang SS, Fusco DN S- Editor: Gou SX L- Editor: Webster JR E- Editor: Ma S 3417 April 7, 2014 Volume 20 Issue 13

43 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (2): Hepatitis C virus Adaptive immune response during hepatitis C virus infection TOPIC HIGHLIGHT Juan Ramón Larrubia, Elia Moreno-Cubero, Megha Uttam Lokhande, Silvia García-Garzón, Alicia Lázaro, Joaquín Miquel, Cristian Perna, Eduardo Sanz-de-Villalobos Juan Ramón Larrubia, Elia Moreno-Cubero, Megha Uttam Lokhande, Silvia García-Garzón, Alicia Lázaro, Joaquín Miquel, Cristian Perna, Eduardo Sanz-de-Villalobos, Translational Hepatology Unit, Guadalajara University Hospital, University of Alcalá, E Guadalajara, Spain Juan Ramón Larrubia, Department of Medicine and Medical Specialities, School of Medicine, University of Alcalá, E Alcalá de Henares, Spain Author contributions: Larrubia JR contributed towards the conception and design of the review; Larrubia JR, Moreno-Cubero E and Lokhande MU co-wrote and Larrubia JR revised the manuscript; García-Garzón S, Lázaro A, Miquel J, Perna C and Sanzde-Villalobos E contributed equally to the supportive work and supervision. Supported by Grants from Instituto de Salud Carlos Ⅲ, Spain and European Regional Development Fund (ERDF), a way of making Europe, E.U., No. PI12/00130; and Fundación de Investigación Médica Mutua Madrileña, Spain, No. 8922/2011; and Lokhande MU was funded by a research grant from Asociación de Hepatología Translacional No. AHT-2010/01, Spain Correspondence to: Juan Ramón Larrubia, MD, MSc, PhD, Translational Hepatology Unit, Guadalajara University Hospital, University of Alcalá, Donante de Sangre st, E Guadalajara, Spain. juan.larrubia@uah.es Telephone: Fax: Received: August 21, 2013 Revised: September 28, 2013 Accepted: November 28, 2013 Published online: April 7, 2014 Abstract Hepatitis C virus (HCV) infection affects about 170 million people worldwide and it is a major cause of liver cirrhosis and hepatocellular carcinoma. HCV is a hepatotropic non-cytopathic virus able to persist in a great percentage of infected hosts due to its ability to escape from the immune control. Liver damage and disease progression during HCV infection are driven by both viral and host factors. Specifically, adaptive immune response carries out an essential task in controlling non-cytopathic viruses because of its ability to recognize infected cells and to destroy them by cytopathic mechanisms and to eliminate the virus by non-cytolytic machinery. HCV is able to impair this response by several means such as developing escape mutations in neutralizing antibodies and in T cell receptor viral epitope recognition sites and inducing HCV-specific cytotoxic T cell anergy and deletion. To impair HCV-specific T cell reactivity, HCV affects effector T cell regulation by modulating T helper and Treg response and by impairing the balance between positive and negative co-stimulatory molecules and between pro- and antiapoptotic proteins. In this review, the role of adaptive immune response in controlling HCV infection and the HCV mechanisms to evade this response are reviewed Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Hepatitis C; Adaptive immune response; Hepatitis C virus-specific cytotoxic T cells; Hepatitis C virus-specific T helper cells; T regs; Hepatitis C virus escape mutations; Anergy; Apoptosis; Chemotaxis Core tip: In the last few years, the knowledge about the role of adaptive immune response in hepatitis C pathogenesis has increased exponentially. This review summarizes our current understanding of the role of antigen-specific responses in hepatitis C virus (HCV) control and liver damage and discusses recent findings that identify costimulatory molecules modulation, apoptosis induction and chemokine regulation as major HCV mechanisms to evade immune control. Larrubia JR, Moreno-Cubero E, Lokhande MU, García-Garzón S, Lázaro A, Miquel J, Perna C, Sanz-de-Villalobos E. Adaptive immune response during hepatitis C virus infection. World J Gastroenterol 2014; 20(13): Available from: URL: 3418 April 7, 2014 Volume 20 Issue 13

44 Larrubia JR et al. HCV-specific immune response DOI: INTRODUCTION Hepatitis C virus (HCV) is a hepatotropic non-cytopathic virus which is able to evade immune system efficiently as mechanism to persist in infected hosts. To fight against a viral infection the host displays two kinds of immune responses, the innate and the adaptive immune response. The innate response is the first immunological barrier and it is essential in controlling cytopathic viruses but not enough in non-cytopathic infections. This primary response limits viral spreading but also acts as adaptive response activator through antigen presentation to viral specific cells. Adaptive response is the second line in the immunological defense and it plays a major role in noncytopathic viral infections due to the ability of this kind of infections to remain occult to the innate system. The current knowledge about the role of the adaptive response role in viral control and pathogenesis during HCV infection will be reviewed in the following paragraphs. GENERAL FEATURES OF ADAPTIVE IMMUNE RESPONSE Non-cytopathic viruses have developed evolutionary mechanisms to remain hidden to the immune system, which is an advantage for their persistence. They are usually not highly infectious but produce long-lasting diseases that allow them to spread the infection over time. The host/non-cytopathic-virus relationship is a dynamic process in which the virus tries to decrease its visibility, whereas the host attempts to prevent and eradicate infection with minimal collateral damage to itself [1]. To control non-cytopathic viral infections, the activation of the adaptive immune system, especially the cellular immune response, is necessary (Figure 1). Naïve specific CD4 + and CD8 + T cells are primed by dendritic cells in the lymph nodes. Once these cells become activated, they change their phenotype into effector cells and migrate to the infected tissue attracted by the chemokines produced by the parenchymal cells. Primed specific CD4 + cells are essential to allow the adequate activation of specific cytotoxic T cells by secretion of T helper (Th)-1 cytokines [2]. Subsequently, these specific cytotoxic T lymphocytes (CTL) play a major role in resolution of spontaneous infection because they are able to recognize the infected cells and destroy them by cytolytic mechanisms. On the other hand, they also produce type-1 cytokines that eliminate the virus without tissue damage. Both CD4 + and CD8 + cell activation depends on the engagement between T cell receptor and the Major Histocompatibility Complex (MHC)/epitope complex as well as the interaction between co-stimulatory molecules with their ligands and the adequate cytokine milieu [3]. When these cells have finished their effector task, they express negative co-stimulatory molecules and pro-apoptotic factors to switch-off their activity, and a subsequent constriction in the specific T cell population is produced. After this event, a memory T cell population is maintained for years to come to respond faster to a new infection and in certain cases to keep under control occult viral infection [4]. ADAPTIVE IMMUNE RESPONSE IN HCV INFECTION The definitive barrier to control HCV infection is the adaptive immunity. This response has two arms to fight against pathogens; humoral and cellular immune response. Humoral immune response that means neutralizing and non-neutralizing antibodies (Non-nAbs), can endorse antiviral activity [5]. Cellular immune response shows antiviral immunity by means of virus specific CTLs and CD4 T helper cells, which play key effector and regulatory roles respectively. These T cells take part in viral clearing and pathogenesis of HCV by direct killing of infected cells or producing soluble factors that are able to clear the virus in a non-cytolytic manner, it also can lead to HCV pathogenic events, favoring direct liver damage and attracting non-specific inflammatory cells to perpetuate the liver inflammation [5]. Humoral immune response nabs generally play a critical role for controlling initial viremia and protecting from re-infection in viral infections. However, the role of the humoral immune response in the clearance of HCV infection is a controversial issue and there are still gaps in the knowledge of the interplay between HCV and nabs. Antibodies generated during acute infection may be targeted against epitopes within structural and non-structural viral proteins; however, the majority of nab have been mapped to the envelope glycoproteins E1 and E2 [6]. HCV can be controlled in patients with hypogammaglobulinemia [7], suggesting this fact as a minor role for nab in HCV control. In this situation, HCV-specific T cells may compensate for lack of neutralizing antibodies to obtain HCV clearance. Few studies supports the idea that HCV clearance occurs mostly in the absence of nabs and these Abs alone are inadequate to eradicate HCV [8-12]. therefore, these data also confer a minor role to nab in HCV control, although other studies propose, at least in some cases, a more important role for nab in HCV clearing. In fact, there are also proofs supporting a maior role of nab to neutralize virus infectivity during acute infection. The earliest studies in chimpanzees showed that hyperimmune serum agaist hypervariable region-1 induced protection against homologous HCV infection [13]. Evidence that nab could protect from natural infection in humans arose form a cohort of women accidentally exposed to the same HCV-strain. In this cohort rapid induction of nab during the early phase of infection contributed to HCV control [14]. Therefore, rapid 3419 April 7, 2014 Volume 20 Issue 13

45 Larrubia JR et al. HCV-specific immune response Th2 MHC class Ⅱ CD4 + T cell HCV peptides CD8 + T cell Y B cell Antigen-presenting cell MHC class Ⅰ HCV HCV antigens MHC class Ⅰ Y B cell HCV-antibodies Y Y Peripheral blood Y Figure 1 Hepatitis C virus-specific immune response activation. Graph showing the priming of naïve T cells by professional antigen-presenting cells in the lymph nodes after antigen up-take in the liver. After specific-t cell activation, these cells become effector T helper (Th) and cytotoxic T cells (CTL) and they migrate into the liver. Th2 response regulates B cells while Th1 response controls CTLs effector function. Specifc-CTLs are able to destroy hepatitis C virus (HCV) by cytolytic and non-cytolytic mechanisms. Th1 TNF-α interferon-γ CD8 + T cell HCV peptides Infected hepatocyte Lymph node Perforin granzyme-b Liver induction of nab early during infection is associated with spontaneous recovery and these antibodies appear to be more cross-neutralizing [15]. Nevertheless, these nab are not detected in most of acute infections, while delayed appearance of high titer cross reactive nabs in chronically infected patients suggests that selective mechanisms may operate to prevent the appearance of these Abs during acute infection [11]. Perhaps, the long-term persistence of these nabs in chronically infected patients may regulate viral replication. In any case, delayed and inefficient neutralizing antibody response during chronic infection induces HCV escape mutations at Abs recognition sites [16], causing the selection of viral mutants. Moreover, it has been proposed that HCV stimulates B cells in a B cell receptor-independent manner during chronic infection [17] and may favor the development of lymphoproliferative and autoimmune diseases [5]. Immune complexes induced during HCV infection are believed to play a pathogenetic role in the development of manifestations such as cryoglobulinemia, glomerulonephritis, porphyria cutanea tarda, and necrotizing cutaneous vasculitis [18-20]. Cellular immune response A strong, multispecific and long-lasting T-cell immune response plays an important role for control of viral infection [8,21]. This efficient response has been observed both in patients as well as experimental models controlling HCV infection [10,12]. Efficiency of antiviral CTL response depends on where these cells are primed. Efficient antiviral CTL response is observed when it is primed in lymphoid organs, whereas within the liver, priming tends to induce T cell inactivation, tolerance or apoptosis [5]. Effector T cells fail to control persistant HCV due to multiple causes, such as: HCV escape mutation at T cell receptor recognition site, immunosuppressive effects exertion, Tregs induction, or T effector exhaustion or deletion [22-24]. Adaptive cellular response during acute HCV infection: Vigorous CD4 + and CD8 + T cell responses targeting multiple HCV regions with intrahepatic production of interferon (IFN)-γ emerge in acute hepatitis C infection [10,24-26]. The appearance of HCV-specific T cells can be detectable in the peripheral blood or in the liver compartment several weeks after infection in humans or in experimental chimpanzee models [8,27], in association to primary peak of transaminases. Moreover, HCV titer is reduced when these cells start producing IFN-γ and correlates with transaminases level decrease [27] (Figure 2). The protective function of CD4 + T cells appears to be due to the production of antiviral cytokines, but also due to their helping nature to antiviral B and CD8 + T cells. The HCV clearance has been observed and correlated with vigorous proliferation of specific CD4 + T cells [28,29] with concurrent interleukin (IL)-2 and IFN-γ production [30,31]. The early, sustained development of CD4 + T cell response needs to be successful for viral clearance [31], whereas HCV-specific CD4 + T cell responses are not observed in chronic HCV infection. Moreover, the recurrent viremia has been correlated with loss of previous strong CD4 + T cell responses after several months of viral clearance [32,33]. CD4 help during acute HCV infection is essential to develop a spontaneous recovery [34], since CTL priming in presence of CD4 help is a critical factor in developing a protective response [31]. On the other hand, the magnitude of CD8 + T cells response in acute HCV infection does not correlate with the clinical or viral outcome [31,35,36]. Expression of a dysfunctional phenotype with weak proliferation, IFN-γ production, cytotoxicity and increased levels of well known exhausted phenotype- programmed death-1 receptor (PD-1) are found in HCV infection, irrespective of infection progression [37-41]. Antigen-dependent reactivity of HCV-specific CD8 + T cells has been proved during the early phase of acute infection associated with sub April 7, 2014 Volume 20 Issue 13

46 Larrubia JR et al. HCV-specific immune response HCV titer ALT level HCV-specific CTL detection γ-interferon secretion HCV infection Figure 2 Cytolytic and non-cytolytic mechanisms to eliminate hepatitis C virus by specific cytotoxic T cells. Scheme showing the hepatitis C virus (HCV) viral load and alanine-amminotransferase (ALT) dynamics after acute HCV infection in relation to appearance of HCV-specific cytotoxic T cells and gamma-interferon secretion. Once HCV-specific cytotoxic T cells are detectable an ALT peak is observed, while when gamma interferon is secreted HCV titers decreased and ALT value becomes normal. CTL: Cytotoxic T lymphocyte. t /wk Escape mutations Tregs (+) HCV (-) Th response (+) (-) Positive costimulatory molecules Negative costimulatory molecules PD-1 CTLA-4 BTLA Tim-3 (+) (-) Bim Mcl-1 CD28 CD27 ICOS 4-1BB GITR Chronic infection Anergy Apoptosis Reactivity Infection resolution HCV-specific CTLs Figure 3 Scheme showing the hepatitis C virus strategies to escape from hepatitis C virus-specific cytotoxic T cells control. Hepatitis C virus (HCV) modulates the balance between positive and negative co-stimulatory molecules, between pro- and anti-apoptotic molecules, and between Th and Treg cells and develops escape mutations at TCR recognition site. PD-1: Programmed cell death protein 1; CTLA-4: Cytotoxic T-lymphocyte antigen 4; BTLA: B- and T-lymphocyte attenuator; Tim-3: T-cell immunoglobulin domain and mucin domain 3; ICOS: Inducible T-cell Costimulator; GITR: Glucocorticoid induced tumor necrosis factor receptor family related gene; Bim: Bcl-2-interacting mediator; Mcl-1: Myeloid leukemia cell differentiation protein; (-) inhibition; (+) induction. sequent rapid decay of CD8 + T cell responses in treatment responders, when these patients were submitted to antiviral therapy [42]. However, the development of selfsustaining memory T cells (CD127 + HCV-specific CD8 + T and CD4 + T cells) are associated with HCV infection control [10,24,31]. In fact, years following sustained virologic response after anti-hcv treatment; it is possible to find HCV traces in association with HCV-specific T cell reactivity. These data suggest that HCV-specific memory T cells are essential to control HCV reservoirs completely in treated patients after the initial treatment induced viral control [43]. Adaptive cellular response during chronic HCV infection: Patients controlling HCV infection exhibit broader CTL responses with higher functional avidity and wider cross-recognition ability than patients with persistent HCV infection [44]. The main reasons for HCV persistence are the appearance of rapid HCV escape mutations [45,46], T cell exhaustion [47], and deletion [48], immune regulatory cytokines secretion [49] and immune modulatory T reg cell induction [50] (Figures 3 and 4). HCV polymerase has high replication rate and lack of proof-reading capacity, which permits a rapid viral escape from emerging humoral and cellular immune responses, leading to persistent infection [45,51]. Mutation development in MHC class I restricted epitopes targeted by CD8 + T cells are associated with persistence [52,53], which proved indirectly that MHC-restricted CD8 + T cells exert selection pressure. Selection of viral escape mutations could occur early in acute infection and remained fixed thereafter, indicating that viral escape may indeed be a premature causative mechanism of CD8 + T cell failure and viral persistence [54,55]. Furthermore, the MHC alleles can influence infection outcome; protective T cell responses target epitopes that can not allow escape mutations due to high 3421 April 7, 2014 Volume 20 Issue 13

47 Larrubia JR et al. HCV-specific immune response CD8 positive cells directly exvivo PBMC after specific stimulation Blocking apoptosis Viral control Pent + cells MFI = 70 MFI = 171 MFI = 163 MFI = No expansion Counts MFI = N cells Viral persistence Pent + cells MFI = 142 MFI = 12 MFI = 110 MFI = 10 MFI = 68 N cells PD-1-FITC CD127-FITC γ IFN-FITC CD8-Cy5 Bim-Alexa488 Mcl1-Alexa488 Figure 4 FACS dot-plots and histograms of hepatitis C virus-specific CD8 + cells from hepatitis C virus patients with different viral control. Hepatitis C virus (HCV)-specific CD8 + cells were stained with Abs anti γ-ifn, anti-pd-1, anti- CD127, anti-mcl-1, anti-bim, anti-cd8 plus pentameric HLA-A2/NS31406 peptide complexes. PD1, γ-ifn and CD127 was analyzed directly ex vivo. Antigen specific proliferation was assessed after blocking or not apoptosis. After expansion Bim and Mcl-1 expression was analyzed. HCV-specific CTLs controlling HCV infection expressed a CD127 high, PD-1 low, Mcl-1 high and Bim low phenotype while CTLs not controlling HCV displayed the opposite phenotype. MFI: mean fluorescence intensity, Pent: Pe-HLA-A2/NS31406 pentameric complexes. PBMC: Peripheral blood mononuclear cells. PD-1: Programmed cell death protein 1; IFN: Interferon; Bim: Bcl-2-interacting mediator; Mcl-1: Myeloid leukemia cell differentiation protein April 7, 2014 Volume 20 Issue 13

48 Larrubia JR et al. HCV-specific immune response costs for viral replicative fitness [56-59]. In fact, there is an association between expression of some MHC class- Ⅰ molecules, such as HLA-B27, and protection following HCV infection. This is explained because mutations in immunodominant epitopes recognized by these MHC class-Ⅰ molecules lead to substantial viral loss of fitness or because a cluster of mutations is needed to impair T cell recognition. The secretion of certain immune-regulatory cytokines is also related with HCV persistence. IL-10 cytokine is found to increase in chronic HCV infection [60]. In chronic HCV patients, the suppression of IFN-γ production and proliferation of virus-specific CD4 + and CD8 + T cells have been observed in livers with IL-10-producing HCVspecific CD8 + T cells [61]. IL-10 produced by monocytes or natural killer (NK) cells also downregulates effector T cell responses. For instance, monocytes secrete IL-10 in response to HCV core-mediated Toll like receptor (TLR) 2 stimulation, in vitro [62]. IL-10 producing HCV-specific CD8 + T cells inhibit IFN-α production [63], but also promote apoptosis of plasmocytoid dendritic cells [62]. In addition, intrahepatic HCV-specific IL-10 producing CD8 + T cells prevent liver damage during chronic infection [64]. Moreover, tumor growth factor (TGF)-β is also involved in antiviral immune suppression and chronic HCV infection evolution [49]. Finally, HCV infection can also shift the ratio between T cell-sustaing cytokines such as IL-2 [65]. To sum up these data, regulatory cytokines such as IL-10 or TGF-β develop a dual task. First of all, they impair T cell responses to allow viral persistence but also decrease liver damage to extend host survival. Regulatory T cells (Tregs) are important to control the balance between host damage and viral control produced by specific immune response. In cases of excessive immune response that could be harmful for the host, these cells can induce immune-tolerance to the viral epitopes. The T cell subset with suppressive function, CD4 + CD25 + FoxP3 + regulatory T (Treg) cells, engages in the control of auto-immunity and immune responses through various mechanisms including the inhibition of antigen presenting cell maturation and T-cell activation [66]. HCV infection increases the frequency of Treg cells and the extent of suppression irrespective of the outcome of the infection [67]. However, higher Tregs frequency has been observed in chronic HCV infected patients than in resolved cases [50,68-70]. Interestingly, depletion of CD25 + cells enhance in vitro responsiveness of the remaining HCV-specific effector cells [50,68,69], which suggests a fundamental role of Tregs in the establishment of chronic HCV infection. Moreover, Treg cells are induced and proliferated in chronic HCV infection and appeared to alter liver inflammation [70]. Conversely, Programmed Death Ligand-1 mediated inhibition limits the expansion of Tregs by controlling STAT-5 phosphorylation (pstat-5) [71], which can diminish suppressive function of Tregs, leading to viral load control and ultimately ensuring long-lasting host survival. On the other hand, HCV is able to induce the upregulation of different negative co-stimulatory molecules in order to provoke an anergic status on HCV-specific T cells. These effector cells are featured by their inability to kill infected hepatocytes, and by the impairment of proliferation and production of type-1 cytokines after antigen recognition. These features are more intense in the intrahepatic comparment due to the higher antigenemia and the tolerogenic hepatic enviorment [72]. Overall, T cell exhaustion follows a predictable pattern. T cells that undergo exhaustion first lose their capacity to produce IL-2, a cytokine that supports proliferation. IL-2 is predominantly produced by CD4 + T cells, whereas CD8 + T cells produce little IL-2 themselves and depend on CD4 + T cell help. This is followed by sequential loss of cytotoxicity and TNF-α and IFN-γ production [73]. PD-1 is one of that molecules involved in the generation of a state of exhaustion on HCV-specific CD8 + T cells during chronic HCV infection [74]. Importance of PD-1 expression on HCV-specific T cell effector dysfunction has been described [75-77]. In addition, blocking of PD-1 signaling results in the functional restoration of blood-derived HCV-specific CD8 + T cell responses in chronic infection [47,77]. However, to restore function of more exhausted HCV-specific T cells isolated from liver biopsies of infected patients, there is need of Cytotoxic T-Lymphocyte Antigen 4 (CTLA4) blockade in addition to PD-1 blockade [78]. On top of this, the co-expression of other inhibitory receptors, besides PD-1 and CTLA-4, such as 2B4, CD160, KLRG1 [79] and, T cell immunoglobulin and mucin domain containing molecule 3 (Tim-3) [80] occurred in about half of HCV-specific CD8 + T cell responses and correlate with low or intermediate level of CD127 expression, impaired proliferative capacity, an intermediate T cell differentiation stage [81] (Figures 3 and 4). These data indicate that HCV infection modulates different negative co-stimulatory molecules to favor the development of HCV-specific CD8 + T cell exhaustion. On the other hand, HCV infection is also able to regulate pro-apoptotic pathways to induce HCV-specific T cell deletion, in order to escape from immune response [82] (Figures 3 and 4). HCV-specific CTLs from chronic patients targeting the virus express an exhausted phenotype associated to the up-regulation of the pro-apoptotic molecule Bcl-2-interacting mediator (Bim) and downregulation of induced myeloid leukemia cell differentiation protein (Mcl-1). Bim activity is blocked by the antiapoptotic molecule Mcl-1. The reactivity of these cells is impaired due to the imbalance between Mcl-1 and Bim during chronic infection but can be restored by blocking apoptosis [48,75] (Figures 3 and 4). In Table 1 the phenotype and reactivity of peripheral HCV-specific CTLs according to viral control after modulating apoptosis and different co-stimulatory molecules is summarized. Persistent HCV infection also affects CD4 + T cell responses but can be only partially restored by PD-1/ PD-L1 blockade or by antigen removal [83,84]. In chronic HCV infection, NK cells develop a regulatory function against CD4 + T cells that may be involved in the antigenspecific defective T helper immunity [85,86] April 7, 2014 Volume 20 Issue 13

49 Larrubia JR et al. HCV-specific immune response Table 1 Summary of the phenotypic and functional features of hepatitis C virus-specific CD8 + cells according to hepatitis C virus control directly ex vivo and after different in vitro treatments PD-1 ex vivo CD127 ex vivo Mcl-1 ex vivo /after TCR triggering Bim ex vivo /after TCR triggering Expansion 1 Expansion (αpd-l1) 2 Expansion (zvadfmk) 3 Expansion (αpd-l1 and αctla-4) 4 Expansion (αpd-l1 and α41bb) 5 Persistent infection PBMC (+) (-) Low/low Low/high Impaired Restored Restored Restored Restored IHMC (++) (-) Impaired Restored Impaired Resolved infection PBMC (-) (++) High/low Low/low Positive Positive Positive (-) Low expression; (+) Intermediate expression; (++) High expression. Expansion: proliferation after specific in vitro challenge 1 without any other treatment; 2 in presence of anti-pd-l1 mab; 3 blocking apoptosis after treatment with z-vad-fmk, in presence of anti-pd-l1 mab and anti-ctla4 mab 4 and adding anti-pd-l1 mab plus stimulating anti-41bb mab 5. PBMC: Peripheral blood mononuclear cells; IHMC: Intrahepatic mononuclear cells [47,48,75,76,78,105]. Bim: Bcl- 2-interacting mediator; CTLA4: Cytotoxic T-Lymphocyte Antigen 4. ACCEPTED MODEL OF HCV PATHOGENESIS As previously commented, specific CTLs play a central role in HCV immunopathogenesis. These cells are not only able to kill some infected hepatocytes inducing a minor liver damage, but also they secrete type-i cytokines responsible for non-cytopathic virus clearing. To attract these cells into the liver, the infected hepatocytes secrete different chemokines. The migration of lymphocytes to the liver is a complex process including adhesion, rolling, and transendothelial migration. Chemokines and their receptors play an essential role in this multistep pathway [87]. During primo infection, when the adaptive immune system is not able to control infection, the infected hepatocytes continue secreting chemokines to try to attract more defensive cells. In viral chronic hepatitis, the expression of different chemokines in the liver has been described. CXCL-10 is increased in the liver and peripheral blood during chronic viral hepatitis [88,89]. This molecule is produced by hepatocytes and sinusoidal endothelial cells. Moreover, CXCL9 and CXCL11 are also increased in serum and liver of subjects with chronic viral hepatitis [90]. CXCL9 is detected primarily on sinusoidal endothelial cells, while CXCL-11 is produced mainly by hepatocytes [91]. CCL5 intrahepatic expression is also elevated in viral chronic hepatitis and is produced by hepatocytes, sinusoidal endothelial cells and biliary epithelium. Finally, several studies have reported an increased level of CCL3 and CCL4 either in the liver or in serum. These molecules are detected on endothelial cells, on some hepatocytes and biliary epithelial cells [92]. The expression of all these chemokines in the liver can be induced directly by viral proteins. Previous reports have shown a high hepatocyte synthesis of CXCL10, CXCL9 and CCL5, induced by some HCV proteins such as NS5A and core [93], although a recent in vitro study suggests that HCV proteins could also decrease CCL5 and CXCL10 genes expression [94]. All these chemokines recruit T cells with a Th1/Tc1 phenotype, expressing specific chemokine receptors such as CCR5 and CXCR3. The non-elr-cxc chemokine attracts CXCR3 expressing T cells while CC chemokine attract CCR5 expressing T cells to the liver. Consequently, in viral chronic hepatitis, an intrahepatic enrichment of CCR5 and CXCR3 expressing T cells, located in hepatic lobule and portal tracts has been shown, while these populations are very infrequent in uninfected subjects [95] (Figure 5). Persistent HCV infection is characterized by a nonspecific inflammatory infiltrate in the liver, mainly of CD8 + cells [96], responsible for liver damage. These cells are attracted by the interaction between the intrahepatic secreted chemokines and the chemokine receptors expressed on T cells. Actually, previous reports have shown a correlation between liver inflammation and liver infiltrating CXCR3/CCR5 expressing T cells. The frequency of these cells was positively correlated with portal and lobular inflammation. These data suggest that CCR5 and CXCR3 could play an important role in chronic liver damage by means of inflammatory T cells recruitment into the liver. Obviously, several previous studies have also shown a correlation between liver inflammation and chemokine levels. Intrahepatic CXCL10 mrna levels are associated with intralobular inflammation [97] and also CXCL9 and CXCL11 correlate with the grade of liver inflammation [98]. Furthermore, CC chemokines are also correlated with the intrahepatic inflammatory activity [99]. Clearly, intrahepatic CCL5 level correlates with the inflammatory activity but not with liver fibrosis. Bearing in mind all the previous data, it is possible to speculate that chemokines are secreted in the infected liver to attract an adaptive immune response able to clear the virus. Unfortunately, when the specific response fails, these chemokines also attract non-specific inflammatory cells, which are not able to remove the virus but produce by-standard liver inflammation. Therefore, as chemokines are nonspecific chemoattractants, intrahepatic inflammatory infiltrate during chronic infection is mainly non-virus-specific and consequently unable to eliminate the infection, but able to produce cytokines capable of initiating and perpetuating hepatic damage and fibrogenesis [95, 100, 101] (Figure 6). STRATEGIES TO RESTORE ADAPTIVE IMMUNE RESPONSE IN HCV INFECTION Taking into account that specific T cell responses are essential to control HCV during natural immune response, 3424 April 7, 2014 Volume 20 Issue 13

50 Larrubia JR et al. HCV-specific immune response CXCR3-FITC CXCR3 FITC CXCR3 FITC % 35% CD8-PE 90% 10% CD8-PE CCR5PECy5 CCR5PECy5 CCR5PECy % 40% CD8-PE 85% 15% CD8-PE LA: 1 PPA: 1 LA: 3 PPA: 3 CD8-PE Figure 5 Chemokine receptor expression of liver infiltrating CD8 T cells according to liver damage. Immunohistochemical CD8 staining of liver samples from patients with different degree of liver inflammation. CCR5 and CXCR3 expression on CD8 T cells from those samples was studied by FACS analysis after staining with the appropriate mabs. A positive correlation between CCR5 and CXCR3 expression on intrahepatic total CD8 + T cells and liver inflammation was observed. LA: Lobular activity; PPA: Peri-portal activity according to Metavir Index. Efficient specific immune response Inefficient specific immune response Viral control Viral persistence Memory T cells patrolling the liver Massive infiltration by non-specific mononuclear cells recruited by intrahepatic chemokines Viral control without liver damage Viral persistence and liver damage Specific CTL Non specific CTL HCV Figure 6 Scheme showing the role of T cell intrahepatic recruitment according to the degree of liver damage and viral control. In resolved hepatitis C virus (HCV) infection an adequate effector T cell response is attracted to the liver to clear the virus. After that, a memory T cell population is continuously patrolling the liver to keep under control viral traces. Nevertheless, in persistent infection, after HCV-specific T cell failure to control infection, a non-specific inflammatory infiltrate is sequestered into the liver, responsible of the persistent liver damage. CTL: Cytotoxic T lymphocytes April 7, 2014 Volume 20 Issue 13

51 Larrubia JR et al. HCV-specific immune response several studies have been performed to analyze the role of different therapeutic approaches on T cell response to know whether it is possible to reverse dysfunction of these cells. One possible mechanism to restore T cell function could be to decrease exhaustion by viral load reduction. During HCV infection, a clear HCV-specific cytotoxic T cell response restoration during anti-hcv therapy has not been shown, as could be expected after treatment induced HCV load decrease [102,103], although specific T helper response restoration in sustained viral responders has been described [104]. Nevertheless, patients presenting a better HCV-specific CD8 cell proliferative potential at baseline, are more likely to present a rapid and sustained viral response. Therefore, currently there are contradictory data about the effect of HCV titter reduction on specific T cell response during chronic hepatitis treatment. In any case, several pre-clinical studies using different strategies have been performed to try to restore HCVspecific responses in vitro. Modulation of co-stimulatory molecules (Table 1), in addition to blocking immunosuppressive cytokines could be promising strategies to restore an effective T cell response. The blockade of negative co-stimulatory molecules, such as PD-1, CTLA-4, Tim-3, has shown in vitro to increase specific-t cell reactivity. This strategy can be combined with the stimulation of positive co-stimulatory molecules such as 4-1BB [105]. Finally, after restoring a T cell response could be necessary to boost that response using a therapeutic vaccine [106]. Moreover, redirecting CD8 + T cells to recognize HCVepitopes by HCV-specific Vβ and Vα T cell receptor gene transduction has been demonstrated in vitro and it could be a future strategy to induce a new HCV-specific T cell population in chronic patients with deletion of these responses [107]. Although all these results seem to be quite promising, the blockade of negative co-stimulatory pathways, stimulation of positive ones and generation of new specific T cells could lead to the development of autoimmune or lymphoproliferative diseases, which could prevent the use of this strategy as a therapeutic tool in humans in the near future. Therefore, more research is necessary in this field before these strategies are suitable for the treatment of chronic viral infections [75,78,106]. CONCLUSION HCV is a hepatotropic non-cytopathic virus able to produce a chronic liver disease. HCV clearing resides in the efficacy of adaptive immune response and, particularly HCV specific CTL response plays a central role in viral control through cytopathic and non-cytopathic mechanisms. Nevertheless, during persistent infection, specific- CTL response is impaired due to its exhaustion and deletion, emergence of HCV escape mutations and lack of T helper cooperation. 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55 Larrubia JR et al. HCV-specific immune response 94 Sillanpää M, Kaukinen P, Melén K, Julkunen I. Hepatitis C virus proteins interfere with the activation of chemokine gene promoters and downregulate chemokine gene expression. J Gen Virol 2008; 89: [PMID: DOI: /vir ] 95 Larrubia JR, Benito-Martínez S, Calvino M, Sanz-de-Villalobos E, Parra-Cid T. Role of chemokines and their receptors in viral persistence and liver damage during chronic hepatitis C virus infection. World J Gastroenterol 2008; 14: [PMID: DOI: /wjg ] 96 Sprengers D, van der Molen RG, Kusters JG, Kwekkeboom J, van der Laan LJ, Niesters HG, Kuipers EJ, De Man RA, Schalm SW, Janssen HL. Flow cytometry of fine-needleaspiration biopsies: a new method to monitor the intrahepatic immunological environment in chronic viral hepatitis. J Viral Hepat 2005; 12: [PMID: DOI: / j x] 97 Harvey CE, Post JJ, Palladinetti P, Freeman AJ, Ffrench RA, Kumar RK, Marinos G, Lloyd AR. Expression of the chemokine IP-10 (CXCL10) by hepatocytes in chronic hepatitis C virus infection correlates with histological severity and lobular inflammation. J Leukoc Biol 2003; 74: [PMID: DOI: /jlb ] 98 Helbig KJ, Ruszkiewicz A, Semendric L, Harley HA, McColl SR, Beard MR. Expression of the CXCR3 ligand I-TAC by hepatocytes in chronic hepatitis C and its correlation with hepatic inflammation. Hepatology 2004; 39: [PMID: DOI: /hep.20167] 99 Kusano F, Tanaka Y, Marumo F, Sato C. Expression of C-C chemokines is associated with portal and periportal inflammation in the liver of patients with chronic hepatitis C. Lab Invest 2000; 80: [PMID: DOI: /labinvest ] 100 Bertoletti A, Maini MK. Protection or damage: a dual role for the virus-specific cytotoxic T lymphocyte response in hepatitis B and C infection? Curr Opin Microbiol 2000; 3: [PMID: DOI: /S (00) ] 101 Friedman SL. Liver fibrosis -- from bench to bedside. J Hepatol 2003; 38 Suppl 1: S38-S53 [PMID: DOI: / S (02) ] 102 Barnes E, Harcourt G, Brown D, Lucas M, Phillips R, Dusheiko G, Klenerman P. The dynamics of T-lymphocyte responses during combination therapy for chronic hepatitis C virus infection. Hepatology 2002; 36: [PMID: DOI: /jhep ] 103 Pilli M, Zerbini A, Penna A, Orlandini A, Lukasiewicz E, Pawlotsky JM, Zeuzem S, Schalm SW, von Wagner M, Germanidis G, Lurie Y, Esteban JI, Haagmans BL, Hezode C, Lagging M, Negro F, Homburger Y, Neumann AU, Ferrari C, Missale G. HCV-specific T-cell response in relation to viral kinetics and treatment outcome (DITTO-HCV project). Gastroenterology 2007; 133: [PMID: DOI: /j.gastro ] 104 Kamal SM, Fehr J, Roesler B, Peters T, Rasenack JW. Peginterferon alone or with ribavirin enhances HCV-specific CD4 T-helper 1 responses in patients with chronic hepatitis C. Gastroenterology 2002; 123: [PMID: DOI: /gast ] 105 Fisicaro P, Valdatta C, Massari M, Loggi E, Ravanetti L, Urbani S, Giuberti T, Cavalli A, Vandelli C, Andreone P, Missale G, Ferrari C. Combined blockade of programmed death-1 and activation of CD137 increase responses of human liver T cells against HBV, but not HCV. Gastroenterology 2012; 143: e4 [PMID: DOI: / j.gastro ] 106 Ferrari C. Therapeutic vaccination for hepatitis C: can protective T-cell responses be restored after prolonged antigen exposure? Gastroenterology 2008; 134: [PMID: DOI: /j.gastro ] 107 Zhang Y, Liu Y, Moxley KM, Golden-Mason L, Hughes MG, Liu T, Heemskerk MH, Rosen HR, Nishimura MI. Transduction of human T cells with a novel T-cell receptor confers anti-hcv reactivity. PLoS Pathog 2010; 6: e [PMID: DOI: /journal.ppat ] P- Reviewers: Ciotti M, El Din NGB, Marcelo LL, Maggi F S- Editor: Wen LL L- Editor: A E- Editor: Wu HL 3430 April 7, 2014 Volume 20 Issue 13

56 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (2): Hepatitis C virus TOPIC HIGHLIGHT Tools for the diagnosis of hepatitis C virus infection and hepatic fibrosis staging Verónica Saludes, Victoria González, Ramon Planas, Lurdes Matas, Vicente Ausina, Elisa Martró Verónica Saludes, Victoria González, Lurdes Matas, Vicente Ausina, Elisa Martró, Microbiology Service, Fundació Institut d Investigació en Ciències de la Salut Germans Trias i Pujol, Hospital Universitari Germans Trias i Pujol (Universitat Autònoma de Barcelona), Badalona, Spain Verónica Saludes, Victoria González, Lurdes Matas, Elisa Martró, CIBER Epidemiología y Salud Pública (CIBERESP), Barcelona, Spain Victoria González, Centre for Epidemiological Studies on HIV/ STI in Catalonia (CEEISCAT)-ICO, Badalona, Spain Ramon Planas, Liver Unit, Hospital Universitari Germans Trias i Pujol, Badalona, Spain Ramon Planas, CIBER Enfermedades Hepáticas y Digestivas (CIBEREHD), Barcelona, Spain Vicente Ausina, CIBER Enfermedades Respiratorias (CI- BERES), Bunyola, Spain Author contributions: Martró E conceived the topic; Saludes V, González V and Martró E reviewed the literature and wrote the manuscript; Planas R, Matas L and Ausina V provided overall intellectual input into the review s topic and edited the manuscript; all authors approved the final version to be published. Supported by A Miguel Servet contract No. MS09/00044 funded by FIS-ISCIII (Spanish Government) to Martró E; grant PI10/01734 within the Plan Nacional de I+D+I and co-financed by ISCIII-Subdirección General de Evaluación y el Fondo Europeo de Desarrollo Regional (FEDER) to González V, Saludes V, Martró E Correspondence to: Elisa Martró, PhD, Researcher in the National Health System, Microbiology Service, Fundació Institut d Investigació en Ciències de la Salut Germans Trias i Pujol, Hospital Universitari Germans Trias i Pujol (Universitat Autònoma de Barcelona), Ctra. del Canyet s/n, Badalona, Spain. emartro.igtp.germanstrias@gencat.cat Telephone: Fax: Received: September 27, 2013 Revised: December 7, 2013 Accepted: March 6, 2014 Published online: April 7, 2014 Abstract Hepatitis C virus (HCV) infection represents a major public health issue. Hepatitis C can be cured by therapy, but many infected individuals are unaware of their status. Effective HCV screening, fast diagnosis and characterization, and hepatic fibrosis staging are highly relevant for controlling transmission, treating infected patients and, consequently, avoiding end-stage liver disease. Exposure to HCV can be determined with high sensitivity and specificity with currently available third generation serology assays. Additionally, the use of point-of-care tests can increase HCV screening opportunities. However, active HCV infection must be confirmed by direct diagnosis methods. Additionally, HCV genotyping is required prior to starting any treatment. Increasingly, high-volume clinical laboratories use different types of automated platforms, which have simplified sample processing, reduced hands-on-time, minimized contamination risks and human error and ensured full traceability of results. Significant advances have also been made in the field of fibrosis stage assessment with the development of non-invasive methods, such as imaging techniques and serum-based tests. However, no single test is currently available that is able to completely replace liver biopsy. This review focuses on approved commercial tools used to diagnose HCV infection and the recommended hepatic fibrosis staging tests Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Hepatitis C virus; Diagnosis; Real-time polymerase chain reaction; Serology; Hepatitis C virus-rna quantification; Hepatitis C virus genotyping; Hepatic fibrosis staging Core tip: About 150 million people are chronically infected with hepatitis C virus (HCV) worldwide, making them at risk for cirrhosis, hepatocellular carcinoma and end-stage liver disease. Recent advances in hepatitis C therapy may bring the opportunity of eradicating this infection. However, ongoing HCV transmission, under April 7, 2014 Volume 20 Issue 13

57 Saludes V et al. HCV diagnosis and hepatic fibrosis staging diagnosis of HCV-infected persons, and difficulties in accessing treatment remain great challenges that require public health responses. In this review, we focus on diagnostic methods used to control HCV infection, including laboratory and point-of-care tests. We also discuss available non-invasive methods to assess liver fibrosis, as the severity of liver disease has important implications in the prognosis and treatment of hepatitis C. Saludes V, González V, Planas R, Matas L, Ausina V, Martró E. Tools for the diagnosis of hepatitis C virus infection and hepatic fibrosis staging. World J Gastroenterol 2014; 20(13): Available from: URL: v20/i13/3431.htm DOI: i INTRODUCTION HCV is an enveloped, single-strand RNA virus that belongs to the Flaviviridae family. Its genome of approximately 9.6 kb contains a single open reading frame that encodes for three structural (core, E1 and E2) and seven non-structural proteins (p7, NS2, NS3, NS4A, NS4B, NS5A and NS5B) and is flanked by untranslated regions (UTR) [1]. The HCV genome exhibits significant genetic variability, which has lead to its classification into seven genotypes and multiple subtypes within each genotype [2,3]. While genotypes 1, 2 and 3 are distributed worldwide, the prevalence of HCV genotypes and subtypes varies geographically according to transmission route and ethnicity [4]. Hepatitis C virus (HCV) has a seroprevalence of 2.8% (over 185 million people) worldwide, making it the major causative agent of chronic liver disease, cirrhosis and hepatocellular carcinoma [5,6]. Although acute HCV infection can be spontaneously cleared, it leads to a chronic infection in the majority of persons. Given the asymptomatic nature of a high proportion of acute and chronic HCV infections, unrecognized infection is a global public health problem that should be promptly addressed using appropriate screening strategies and diagnostic assays. Serological and molecular markers of HCV infection are key to correctly diagnose past exposure versus active infection, and acute versus chronic infection, as well as to assess treatment indication. Pegylated-interferon alpha (PegIFN-α) and ribavirin (RBV) combination therapy is the current standard of care for treating chronic hepatitis C by non-1 genotypes. A triple therapy that also contains an HCV-specific protease inhibitor has recently been approved to treat chronic infection by HCV genotype 1 in many countries around the world [7]. Over the past decade, HCV genotyping assays have been improved and ultrasensitive quantitative molecular assays have been developed. These technical improvements are mainly due to changes in the treatment algorithms and the use of response-guided therapy, which is based on how rapidly HCV responds to treatment ( on-treatment virologic response ). Acutely infected patients have higher response rates to antiviral treatment than those with an established chronic infection. Thus, effective screening and fast diagnosis of HCV are highly relevant steps in preventing disease progression and virus spread, since they allow infected persons to be identified and treated. We discuss the assays approved for in vitro diagnostics in the following sections. TOOLS FOR THE DIAGNOSIS AND MANAGEMENT OF HCV INFECTION Two major types of assays have been developed to diagnose and manage HCV infection: those that detect HCVspecific antibodies that are used to indirectly diagnose infection, and those that detect viral components (e.g., the core antigen or the viral genome) that are used to directly diagnose hepatitis C and manage HCV infected patients. Indirect diagnosis Serologic assays detect HCV-specific total antibodies (IgM and IgG) and are used to screen and diagnose HCV exposure. However, these assays do not discriminate between active and resolved infections. Screening assays: Since 1989, when HCV was discovered and its immunodominant epitopes were identified, HCV infection has been mainly diagnosed by detecting HCV antibodies from serum samples using enzyme immunoassays (EIA). Over time, serologic assays have evolved, and current third-generation assays now include multiple recombinant HCV antigens from the core, NS3, NS4 and NS5 regions. This has resulted in the reduction of the window period and in an overall improved detection of patients exposed to HCV (with excellent sensitivity and specificity) [8]. Currently, immunoassays can be fully automated using high-throughput, random access instruments that are widely used in clinical laboratories. Characteristics of the most commonly used assays are summarized in Table 1. Most of these are chemiluminescence immunoassays, which have improved the specificity and positive predictive value (PPV) of conventional EIAs [9]. Briefly, HCV antigens are immobilized on different types of solid phases (microwell, magnetic and paramagnetic particles). The presence of HCV-specific antibodies in the clinical specimen is then detected with a conjugate antibody (anti-human IgG labeled with acridinium or horseradish peroxidase) that catalyzes the oxidation of a luminol, producing light. The light signal is measured by the system and then normalized relative to the cut-off value [signal/ cut-off (S/CO)] or as relative lights units [9,10]. Anti-HCV assays have several disadvantages, including: (1) the prolonged duration of the window period between the time of infection and the detection of HCV antibodies (approximately d) [11] ; (2) the low PPV in low-risk populations (as false-positive results may result from the presence of multiple circulating immunoglobulins that can interact non-specifically with HCV 3432 April 7, 2014 Volume 20 Issue 13

58 Saludes V et al. HCV diagnosis and hepatic fibrosis staging Table 1 Main commercial immunoassays to detect anti-hepatitis C virus antibodies approved for in vitro diagnostics Analyzer and manufacturer Assay principle Solid phase HCV antigens Reaction sample volume (μl) Time of reaction (min) IVD registration Architect i2000sr, Abbott Laboratories CMIA Paramagnetic particles AxSYM, Abbott Laboratories MEIA Paramagnetic particles LiaisonXL, DiaSorin CLIA Paramagnetic particles VITROS ECi, VITROS 3600, Ortho-Clinical Diagnostics Elecsys, Roche Diagnostics ECLIA Paramagnetic particles ADVIA Centaur, Siemens CLIA Magnetic particles HCr43 (Core and NS3), c100-3 (NS4A) FDA, CE HCr43 (Core), c200 (NS3), FDA, CE c100-3 (NS4A) Core, NS3, NS CE CLIA Microwell c22-3 (Core), c200 (NS3 and NS4), NS FDA, CE Core, NS3, NS FDA, CE c22-3 (Core), NS3, c200, NS FDA, CE IVD: Certified as in vitro diagnostic test or device; CE: Conformité Européenne (European Union); FDA: Food and Drug Administration (United States of America); ECLIA: Electrochemiluminescence immunoassay; CMIA: Chemiluminescent microparticle immunoassay; CLIA: Chemiluminescence immunoassay; HCV: Hepatitis C viru; MEIA: Microparticle capture enzyme immunoassay. antigens); and (3) the possibility of false-negative results in immune-compromised persons or in those who are undergoing haemodialysis due to an inadequate antibody response. Furthermore, all available assays have a gray zone from which results are not interpretable. In cases with uninterpretable results, the sample should be centrifuged to completely remove all cells, cellular debris and fibrin, and the assay should be repeated in duplicate to verify its status. If the results of the duplicated repetition are below the assay cut-off for both replicates, the sample should be considered negative. If either duplicate retest result is above or equal to the cut-off, the sample should be tested by supplementary assays to confirm the result. Confirmatory assays: Recombinant immunoblot assays (RIBA) can be used to confirm the presence of HCVspecific antibodies for individuals who have tested positive by EIA, especially when screening populations with a low prevalence of HCV infection. This assay is highly specific, as the presence of antibodies against each of several HCV proteins is assessed as individual bands on a membrane strip. The CHIRON RIBA HCV 3.0 SIA assay, which was previously cleared by the United States Food and Drug Administration (FDA), has been recently discontinued. The INNO-LIA HCV Score (Fujirebio) assay is CE-marked and can be automated on the Auto-LIA 48 instrument. This assay includes recombinant proteins and synthetic peptides from the E2 hypervariable region, the helicase (NS3), and the NS4A, NS4B and NS5A regions. However, a main problem of RIBA is the occurrence of indeterminate results, especially in those specimens with grey-zone results in the screening assays. Currently, this assay has been substituted as a confirmatory test by widely-used molecular techniques, which can additionally distinguish between active and resolved infections. Rapid, point-of-care screening tests: Both serologic and molecular assays to detect HCV infection have to be carried out in a laboratory, which forces patients to return for their results. Improving this by obtaining the results during the patient s visit has lead to the development of simple, rapid and non-instrumented point-of-care tests (POCTs). POCTs also make it possible to test for HCV outside of clinical settings in hard-to-reach, high-risk populations, such as injecting drug users (IDU), which are unlikely to be screened following conventional testing [12]. POCT technologies have been successfully used to detect human immunodeficiency virus (HIV) infections [13] and may be useful in addressing the problem of underdiagnosis of HCV infection. Several POCTs have been developed to detect HCVspecific antibodies with a relatively high sensitivity and specificity [14]. However, results from such tests should be interpreted with caution, as population-selection biases and the use of different reference standards used to ascertain true disease status could influence test performances. The only test currently approved by the FDA is the OraQuick HCV Rapid assay (OraSure). This test detects HCV antibodies in fingerstick and venipuncture whole blood, serum, plasma, or oral fluid specimens by an indirect lateral flow immunoassay. Core, NS3 and NS4 antigens are immobilized on a nitrocellulose membrane, and the results are directly visualized using colloidal gold labeled with protein A, which generates a reddish-purple line within 20 min in the presence of HCV-specific antibodies. This test showed a sensitivity of 97.8%-99.3%, and a specificity of 99.5%-99.6%, in serum specimens from a population of IDU, depending on the reference standard used [15]. A sensitivity of 83.3% was observed from oral fluids [16]. In another study, the sensitivity varied between 98.1% (oral fluid) and 99.9% (plasma or serum), with a specificity between 99.6% (oral fluid) and 99.9% (blood, plasma or serum), in a population of individuals who were symptomatic for hepatitis or asymptomatic for hepatitis but with risk factors for HCV infection (mostly IDU and their sex partners, incarcerated individuals and 3433 April 7, 2014 Volume 20 Issue 13

59 Saludes V et al. HCV diagnosis and hepatic fibrosis staging Table 2 Hepatitis C virus-rna qualitative assays approved for in vitro diagnostics Assay and manufacturer Method Reaction sample volume (μl) Lower limit of detection (IU/mL) Instrumentation for automated processing IVD registration COBAS AMPLICOR HCV Test v2.0, Roche Molecular Systems COBAS AmpliPrep/COBAS AMPLICOR HCV Test v2.0, Roche Molecular Systems COBAS AmpliPrep/COBAS TaqMan HCV Qualitative Test v2.0, Roche Molecular Systems APTIMA HCV RNA Qualitative Assay 1, Hologic - Gen-Probe VERSANT HCV RNA Qualitative Assay, Siemens RT-PCR (plasma) 60 (serum) RT-PCR (plasma) 60 (serum) COBAS AMPLICOR Analyzer (amplification and detection) COBAS AmpliPrep (extraction), COBAS AMPLICOR Analyzer (amplification and detection) Real-time RT-PCR Fully automated: cobas p 630 Instrument (primary tube handling), COBAS AmpliPrep (extraction and MM setup), COBAS TaqMan Analyzer or the COBAS TaqMan 48 Analyzer (amplification and detection) TMA Not automated. PANTHER System s functionality currently in development CE, FDA, Japan, Canada CE, FDA, Canada CE, FDA TMA TMA modules (TCS, luminometer HC+, etc.) CE, FDA FDA 1 The performance of this assay has not been demonstrated for monitoring hepatitis C virus (HCV) infected patients. IVD: Certified as in vitro diagnostic test or device; CE: Conformité Européenne (European Union); FDA: Food and Drug Administration (United States of America); RT-PCR: Reverse transcriptionpolymerase chain reaction; MM: Master mix. individuals with other sexually-transmitted diseases, including HIV) [17]. Additional studies would be required to assess: (1) the performance of this test in populations with a lower HCV prevalence; (2) the effect of the HCV genotype on the test results; and (3) the influence of HIV coinfection on the test accuracy. Further studies are also required to determine the cost-effectiveness of POCT testing in resource-limited countries with high HCV prevalence. Recently, the Home Access Hepatitis C test system (Home Access Health Corporation) has been cleared by the FDA. The kit can be purchased online, by fax, or by mail and is based on collecting a blood sample on a blood specimen card using a fingerstick at home, shipping the sample anonymously to an accredited laboratory and calling back for test results. Pre- and post-test counseling and referrals are also provided. The use of POCTs may lead to low proportions of false-negative results (i.e., in immunosuppressed individuals) and false-positive results that may require additional confirmatory tests. Even so, by reaching more at-risk populations, POCTs could allow more cases to be identified and treated than laboratory-based tests [18]. Direct diagnosis The detection of viral components is needed to diagnose an active HCV infection. HCV Core antigen detection and quantification: The HCV Core antigen can be detected in the serum of HCV-infected patients, and its levels are significantly related to those of HCV-RNA [19-21]. HCV Core assays cost less than molecular assays and can be easily performed in an immunoassay format. Therefore, they could be used as an alternative to HCV-RNA assays for three different situations: (1) to distinguish active from resolved HCV infections [22,23] ; (2) to identify HCV infection in the antibody window period [24] ; and (3) to identify HCV infection in seronegative individuals at high risk for HCV infection, such as in hemodialysis patients [25]. Several studies have suggested that quantification of the Core antigen could be used to monitor the response to IFN-α plus RBV therapy in chronically infected patients [26-28]. Currently, Core antigen detection can be fully automated in the Architect HCV Core antigen test (Abbott Laboratories) [22]. Although this assay is not sensitive enough to replace HCR-RNA testing for treatment monitoring according to the current clinical practice guidelines, it could be used as a supplemental test in resource-constrained settings [28]. Several combination assays that detect both HCV antibodies and Core antigen have been developed. Currently, the MONOLISA HCV Ag-Ab ULTRA (Bio- Rad) is a CE-marked microtiter-based assay that, despite not being as sensitive as the HCV antigen-specific assays, improves the detection of HCV infection within the window period of antibody assays [29]. Molecular HCV assays: Molecular assays to detect the HCV genome are used for several purposes in the clinical setting. First, the presence of circulating HCV-RNA reflects viral replication, such that sensitive molecular assays (with a lower limit of detection < 50 IU/mL) are used to diagnose active HCV infection in patients with a positive antibody test. Second, molecular testing is required for an early diagnosis of acute HCV infection, as the HCV-RNA can be detected before specific antibodies become detectable (within 1-3 wk after exposure). Finally, the diagnosis of a chronic HCV infection is confirmed by the presence of both HCV antibodies (with the exception of severely immunosuppressed patients) and HCV- RNA over 6 mo [7,30-32]. Commercially available assays that have been approved for in vitro diagnostics are listed in Tables 2 and April 7, 2014 Volume 20 Issue 13

60 Saludes V et al. HCV diagnosis and hepatic fibrosis staging Table 3 Hepatitis C virus-rna quantitative assays approved for in vitro diagnostics Assay and manufacturer Method Reaction sample volume (μl) Lower limit of detection (IU/mL) Linear range of quantification (IU/mL) Instrumentation for automated processing IVD registration Abbott RealTime HCV, Abbott Molecular artus HCV RG RT-PCR Kit, Qiagen artus HCV QS-RGQ Kit, Qiagen COBAS AmpliPrep/ COBAS TaqMan HCV Test, Roche Molecular Systems COBAS AmpliPrep/ COBAS TaqMan HCV Quantitative Test v2.0, Roche Molecular Systems COBAS TaqMan HCV Test v2.0 for use with the High Pure System, Roche Molecular Systems VERSANT HCV RNA 1.0 Assay (kpcr), Siemens VERSANT HCV RNA 3.0 Assay, Siemens Real-time RT-PCR Real-time RT-PCR Real-time RT-PCR Real-time RT-PCR Real-time RT-PCR Real-time RT-PCR Real-time RT-PCR ( ) m2000sp (extraction and assay setup), m2000rt (amplification and detection) ( ) Manual extraction (QIAamp DSP Virus Kit) and assay setup, Rotor-Gene Q (amplification and detection) ( ) QIAsymphony RGQ: QIAsymphony SP (extraction), QIAsymphony AS (assay setup), Rotor-Gene Q (amplification and detection) ( ) COBAS AmpliPrep (extraction), COBAS TaqMan Analyzer or COBAS TaqMan 48 Analyzer (amplification and detection) ( ) Fully automated: cobas p 630 (primary tube handling), COBAS AmpliPrep (extraction), COBAS TaqMan Analyzer or COBAS TaqMan 48 Analyzer (amplification and detection) ( ) (CE) Manual extraction (High Pure System 25 - ( ) (FDA) Viral Nucleic Acid Kit), COBAS TaqMan Analyzer (amplification and detection) ( ) VERSANT kpcr Molecular System Sample Preparation (SP) Module and Amplification/Detection (AD) Module bdna ( ) No nucleic acid extraction needed. System 340 bdna Analyzer (S340) (US) or VERSANT 440 Molecular System (CE) CE, FDA CE CE CE, FDA, Canada, Japan CE, FDA CE, FDA CE CE, FDA 1 The intended use of this assay is limited to the measurement of hepatitis C virus (HCV) viral loads at baseline and after 12 wk of therapy. IVD: Certified as in vitro diagnostic test or device; CE: Conformité Européenne (European Union); FDA: Food and Drug Administration (United States of America); kpcr: Real-time kinetic polymerase chain reaction; bdna: Branched DNA; RT-PCR: Reverse transcription-polymerase chain reaction. The highly conserved HCV 5 UTR region is the target of choice for HCV genome detection across different genotypes. For this, several laboratory processes are necessary, including nucleic acid extraction from clinical specimens (serum or plasma), nucleic acid or signal amplification, and detection. These steps may be fully or partly automated with commercial platforms. However, these assays are time-consuming and require sophisticated technical equipment, trained personnel, dedicated laboratory areas and expensive reagents. Historically, qualitative assays were more sensitive than quantitative tests. Nowadays, most quantitative assays are highly sensitive and could replace qualitative tests. Real-time reverse-transcription PCR (RT-PCR) is the method of reference for the quantification of HCV- RNA levels in clinical practice according to European and American guidelines [7,30], given its high sensitivity and wide dynamic range of quantification. Reverse transcription of the HCV-RNA as well as PCR amplification and real-time detection is performed in a closed system, thus avoiding carryover contamination with amplified products. The COBAS TaqMan assays (Roche Molecular Systems) are the most widely used worldwide. Although the first version of the assay performed worse with HCV genotype 4 than with the other genotypes, this has been improved in version 2.0 [33,34]. Abbott Molecular [35], Siemens and Qiagen [36,37] also offer real-time RT-PCR assays. Alternatively, Siemens offers an assay based on the branched-dna (bdna) signal-amplification technology [38,39]. No nucleic acid extraction is needed, and the process can be fully automated in the VERSANT 440 Molecular System. Although both the 5 UTR and core regions are targeted, the lower limit of detection of this assay limits its applicability. Quantitative tests are also used to monitor antiviral therapy. In order to minimize side-effects, emergence of resistance and costs, HCV-RNA must be periodically quantified to strictly follow treatment stopping rules. With the advent of new treatment regimens that include a protease inhibitor and the response-guided treatment algorithms, only assays with a lower limit of quantification of 25 IU/mL and a lower limit of detection of approximately IU/mL, should be used [31]. Additionally, the presence of detectable but not quantifiable HCV-RNA below those levels is clinically relevant, as it reflects true viremia [40,41]. HCV genotyping assays: Since the HCV genotype is 3435 April 7, 2014 Volume 20 Issue 13

61 Saludes V et al. HCV diagnosis and hepatic fibrosis staging Table 4 Hepatitis C virus genotyping assays approved for in vitro diagnostics Assay and manufacturer Method Reaction sample volume (μl) Lower limit of detection (IU/mL) Target Instrumentation IVD registration Abbott HCV Genotype Ⅱ, Abbott Molecular LINEAR ARRAY Hepatitis C Virus Genotyping Test, Roche Molecular Systems VERSANT HCV Genotype 2.0 Assay Line Probe Assay (LiPA), Siemens Real-time RT- PCR RT-PCR and hybridization RT-PCR and hybridization 'UTR NS5B m2000sp (extraction and assay setup), m2000rt (amplification and detection) 'UTR AMPLICOR and COBAS AMPLICOR HCV Test v2.0 (amplification and detection). Manual hybridization 'UTR core Conventional PCR instrument, Auto-LiPA 48 or AutoBlot 3000H automated Systems (hybridization), LiPA Scan Software CD (reading) CE, FDA CE CE IVD: Certified as in vitro diagnostic test or device; CE: Conformité Européenne (European Union); FDA: Food and Drug Administration (United States of America); RT-PCR: Reverse transcription-polymerase chain reaction. predictive of the response to IFN-α-based therapy, genotyping is mandatory to tailor dose and duration of treatment [42]. Furthermore, it is necessary for deciding on triple therapy eligibility with currently approved protease inhibitors, which are effective against HCV genotype 1 [7,43,44]. The reference method for HCV genotyping is genome sequencing of the core/e1 or the NS5B regions and subsequent phylogenetic analysis [45]. However, this in-house method is restricted to reference centers. Similar to diagnostic assays, the target of choice for commercially available genotyping assays has classically been the highly conserved 5 UTR. However, discrimination among subtypes and the genotypes 1 and 6 is not always reliable using this region [2]. HCV subtyping is important for epidemiological studies, especially in the case of outbreaks. While subtyping is not considered to be clinically relevant for PegIFN-α and RBV treatment regimens, it may be clinically relevant in the era of directly acting antivirals. For instance, subtypes 1a and 1b have been described to have subtype-specific resistance profiles to linear protease inhibitors [46,47], and subtype 1g has been reported to naturally harbor the resistance mutation T54S [48]. Commercialized HCV genotyping assays approved for in vitro diagnostic use are summarized in Table 4. Assays targeting other regions in addition to the 5 UTR have been recently developed to better discriminate between subtypes 1a and 1b within HCV genotype 1. The Versant HCV genotype 2.0 assay (Siemens) targets the 5 UTR and core regions [49,50]. This colorimetric assay is based on conventional PCR amplification followed by reverse hybridization onto membrane strips containing specific probes. The obtained band pattern can be either visually interpreted or read by a scanner. On the other hand, the Abbott RealTime HCV Genotype Ⅱ (Abbott Molecular) targets the 5 UTR and NS5B regions [51-53]. This assay is based on a single-step real-time RT-PCR with labeled genotype-/subtype-specific probes that minimizes contamination with amplified products. Finally, the Linear Array Hepatitis C Virus Genotyping Test (Roche Molecular Systems) is also based on reverse hybridization but targets the 5 UTR only [54]. Unlike direct sequencing, these three assays facilitate the detection of mixed genotype infections, which can be found especially in IDUs. Additionally, assays targeting two different genomic regions could help to detect recombinant variants; the frequency of recombinants may have been underestimated by the wide use of assays targeting only the 5 UTR in the clinical practice before assays were developed that additionally target the core or NS5B regions. Commercialized genotyping assays may result in < 5% of indeterminate results [42,55,56] due to the high genetic variability of HCV. Clinicians are forced to treat patients with an indeterminate result as if they were infected with genotypes 1 or 4 (resulting in longer treatment duration and higher RBV doses than for genotypes 2 and 3) and cannot decide on triple therapy eligibility. Therefore, patients with an indeterminate genotype result should be retested either using an alternative commercial assay or the reference method. TOOLS FOR HEPATIC FIBROSIS STAGING Chronic hepatitis C is characterized by a continuous process of liver inflammation that progresses to liver fibrosis. Liver fibrosis leads to cirrhosis over the decades in 10%-40% of the cases. The most important end stages of liver cirrhosis are esophageal varices, ascites, hepatic encephalopathy, hepatocellular carcinoma and, finally, liver failure. Importantly, cirrhosis can progress to hepatocellular carcinoma with an incidence of 1%-5% per year, and death associated to complications of cirrhosis has an incidence of 4% per year [30,57]. Liver fibrosis results from the accumulation of an extracellular matrix mainly composed of collagens, proteoglycans, fibronectin and hyaluronic acid (HA). Hepatic stellate cells are the main producers of this matrix after its transdifferentiation from a quiescent to a myofibroblast phenotype in the setting of chronic liver injury. Unfortunately, a specific therapy against liver fibrogenesis does not currently exist because, among other reasons, the exact mechanisms involved in this process are not completely understood yet [58-60]. This is especially problematic for patients who do not respond to current antiviral therapies. The management of chronic hepatitis C strongly depends on hepatic fibrosis staging, since this is important 3436 April 7, 2014 Volume 20 Issue 13

62 Saludes V et al. HCV diagnosis and hepatic fibrosis staging for treatment decision making and disease prognosis. The presence of an advanced (METAVIR score F3-F4) or moderate (METAVIR score F2) stage of liver fibrosis is an indication for antiviral therapy [30,61], as these patients are at high risk for disease progression. Additionally, liver fibrosis progression is mostly reverted in successfullytreated patients [62,63]. Thus, fibrosis staging methods ideally should be able to differentiate between non-significant (METAVIR scores F0-F1) and significant (score F2) fibrosis. Furthermore, hepatic fibrosis staging helps in the early detection of cirrhosis, which allows clinicians to monitor the appearance of related decompensation events and hepatocellular carcinoma. Liver biopsy Liver biopsy remains the best accepted standard method for fibrosis staging [30]. Typically, liver fibrosis is classified semi-quantitatively in several stages by different scoring methods [64]. METAVIR is the best evaluated system in patients with chronic hepatitis C; fibrosis is scored as F0 (no fibrosis), F1 (portal fibrosis without septa), F2 (portal fibrosis with few septa), F3 (numerous septa without cirrhosis) or F4 (cirrhosis) [65]. However, liver biopsy presents several limitations: this procedure is expensive, time consuming and invasive with possible complications, and its interpretation is associated with sampling error and intraand inter-observer variability. As a result, liver biopsy often does not accurately assess the fibrosis stage and is not useful for monitoring the disease progression [64]. Due to the limitations of liver biopsy, an extensive number of alternative non-invasive tests have been developed, including both imaging and serum-based tests. However, only some of these have been validated and accepted for chronic hepatitis C management [30,66], as reviewed below. These alternative tests are useful for establishing the two ends of the fibrosis spectrum (of minimal fibrosis and cirrhosis) but are less helpful in assessing the mid-ranges of fibrosis. Transient elastography Transient elastography (FibroScan, Echosens) is so far the most common, accurate and validated alternative method [67]. It is an ultrasound-based imaging technology that measures liver stiffness, which directly correlates with the degree of fibrosis [68]. FibroScan is painless, fast (5-10 min) and easy to analyze and has low intraand inter-observer variability [69]. Consequently, it can be performed periodically, allowing monitoring of either fibrosis progression during its natural course [70] or regression while on treatment [63]. In addition, it predicts 5-year clinical outcomes in chronic hepatitis C [71]. FibroScan reports the liver stiffness measurements (LSM) as the median expressed in kilopascals (kpa), the interquartile range (IQR) and the percentage of valid measurements. According to the manufacturer s instructions, results are not reliable when the number of valid measurements is < 10, the IQR of the median LSM value is 30% and/or the percentage of successful measurements is 60%. This situation takes place in about 16% of all cases, mainly due to factors such as obesity and inadequate operator experience [72]. More recently, the introduction of the XL probe has increased the reliability of liver stiffness results as compared to the M probe when used in obese patients [73]. Several meta-analyses have evaluated the diagnostic accuracy of transient elastography in the staging of liver fibrosis. Those meta-analyses that evaluated accuracy by means of the AUROC concluded that FibroScan is able to diagnose the presence of cirrhosis with excellent accuracy (AUROC 0.90) and the presence of significant fibrosis with moderate accuracy (AUROC from 0.80 to < 0.90) [74,75]. Similar conclusions were obtained from metaanalyses that evaluated FibroScan accuracy by calculating sensitivity and specificity values: sensitivity and specificity were 70%-72% and 82%-84%, respectively, for the diagnosis of significant fibrosis, and 83%-87% and 89%-95% for cirrhosis [76-78]. Thus, transient elastography is now recommended in the clinical practice for assessing liver fibrosis in patients with chronic hepatitis C, and especially for detecting cirrhosis when other clinical manifestations are not present [30,66]. However, different stiffness cut-off values have been proposed to discriminate among stages of liver fibrosis, and a general consensus is still needed. In Spain, the use of FibroScan instead of liver biopsy is accepted in patients with chronic hepatitis C in order to identify significant fibrosis (cut-off 7.6 kpa) and cirrhosis (cut-off 14.6 kpa) [79,80]. Serum-based tests and scores Certain cytokines involved in fibrosis and factors associated with extracellular matrix deposition or degradation may be used as individual biomarkers of liver fibrosis. Additionally, numerous serum-based tests combining direct and/or indirect markers of fibrosis have been proposed as alternatives to liver biopsy. Direct biomarkers reflect the deposition of extracellular matrix in the liver by fibrogenesis/fibrolysis and include HA, pro-collagen Ⅲ amino-terminal peptide, tissue inhibitor of matrix metalloproteinase-1, alpha 2-macroglobulin, haptoglobin and matrix metalloproteinase-1. Indirect biomarkers include routine laboratory data on the levels of alanine aminotransferase, aspartate aminotransferase, gammaglutamyltransferase, apolipoprotein A1, cholesterol, bilirubin and urea as well as the platelet count and the prothrombin index. Nonetheless, only some serum-based tests have been widely validated in chronic hepatitis C and are recommended in the clinical practice for detecting significant fibrosis [30,66] (Table 5). However, these tests are not helpful for tracking fibrosis progression. ELF, Fibrospect Ⅱ, Hepascore, Fibrometer and FibroTest are patented and commercially available tests. Although abundant studies have been performed to assess the different tests, we have only included contrasted results of meta-analysis studies in this review. Meta-analyses aimed to assess the diagnostic accuracy of serum-based tests to identify significant fibrosis/cirrhosis in comparison with liver biopsy have shown moderate 3437 April 7, 2014 Volume 20 Issue 13

63 Saludes V et al. HCV diagnosis and hepatic fibrosis staging Table 5 Serum-based tests recommended in the clinical practice for the detection of significant fibrosis Serum-based tests Parameters considered Ref. Tests combining direct markers of liver fibrosis Enhanced liver HA, PⅢNP, TIMP-1, age [88] Fibrosis test (ELF ) MP3 MMP-1, PⅢNP [89] Fibrospect Ⅱ HA, TIMP-1, alpha-2-macroglobulin [90] Tests including indirect markers of liver fibrosis AST to platelet ratio AST, platelet count [91] index (APRI) AST/ALT ratio AST, ALT [92] Forns index GGT, platelet count, cholesterol, age [93] Tests including combinations of direct and indirect markers of fibrosis Hepascore Bilirubin, GGT, HA, alpha-2-macroglobulin, [94] gender, age Fibrometer HA, AST, platelet count, prothrombin [95] index, alpha-2-macroglobulin, urea, age FibroTest alpha-2-macroglobulin, haptoglobin, apolipoprotein A1, GGT, total bilirubin [96] HA: Hyaluronic acid; PⅢNP: Pro-collagen Ⅲ amino-terminal peptide; TIMP-1: Tissue inhibitor of matrix metalloproteinase-1; MMP-1: Matrix metalloproteinase-1; AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; GGT: Gamma-glutamyltransferase. accuracies in patients with chronic hepatitis C. The most widely validated tests are APRI and FibroTest. A comprehensive meta-analysis on the APRI index performed by Lin et al [81] revealed an AUROC of 0.77 for the diagnosis of significant fibrosis and of 0.83 for cirrhosis. The authors concluded that the APRI index is a good alternative for its use in clinical practice for confirming the presence of severe fibrosis/cirrhosis when other tests or symptoms are non-conclusive. The main strengths of the APRI index are its low cost and wide availability, which make this simple test an excellent first-line screening in developing countries. Poynard et al [82] analyzed the diagnostic accuracy of FibroTest for significant fibrosis and showed a mean standardized AUROC of 0.85 in patients with chronic hepatitis C. However, the disadvantage of FibroTest, similar to the other patented tests, is that it is relatively expensive and is not readily available in all centers. In addition, co-morbidities such as hemolysis, inflammation or Gilbert s syndrome can lead to potential mistakes by changing the levels of some of the serum markers included in the test. In a later meta-analysis performed by Leroy et al [83], Fibrometer had a higher AUROC (0.84) than FibroTest (0.80), APRI (0.79) or Hepascore (0.78) for significant fibrosis. In a recent meta-analysis assessing tests that combine direct and/or indirect markers, a moderate accuracy for the identification of significant fibrosis was described for ELF (median AUROC, 0.81), Fibrometer (0.82) and FibrospectII (0.86), and for the identification of cirrhosis in the case of APRI (0.84), Fibrotest (0.86), Forns Index (0.87), ELF (0.88) and Hepascore (0.89). Fibrometer showed an excellent accuracy for diagnosing cirrhosis (0.91) [84]. Thus, in patients with chronic hepatitis C, both transient elastography and serum-based markers have a similar moderate accuracy for diagnosing significant fibrosis. Nevertheless, transient elastography is the most accurate non-invasive method for cirrhosis diagnosis. To increase the accuracy of diagnosing significant fibrosis and to decrease the number of liver biopsies needed, several algorithms based on a combination of serum-based tests or a combination of transient elastography and a serum-based test have been recommended for use in clinical practice [30]. The sequential algorithm for fibrosis evaluation (SAFE) first uses the APRI index and then the FibroTest. When a final diagnosis cannot be reached, liver biopsy is needed [85]. The Castéra algorithm uses transient elastography and FibroTest and relies on liver biopsy to resolve discrepancies [86]. Both SAFE and Castéra algorithms have shown an excellent accuracy in diagnosing significant fibrosis, but a higher number of liver biopsies could be avoided using the second algorithm (of 72% with Castéra vs 48% with SAFE). CONCLUSION Despite great advances in HCV treatment, the lack of recognition of infection will hamper the control of hepatitis C. The World Health Organization announced in 2012 a framework for global action to prevent and control viral hepatitis infections [87]. Early diagnosis provides the best opportunity for effective medical support and prevention of further spread of the infection. Expanded HCV testing is required, and diagnostic tests with high sensitivity and specificity are available. Additionally, POCT tests could improve the number of diagnosed individuals and their access to care. Despite significant advances in the field of fibrosis stage assessment, a single non-invasive method that is able to completely replace the liver biopsy does not exist at the current time. Liver biopsy should be performed when non-invasive methods or algorithms cannot be used or when inconclusive results are obtained, but it is not needed when clinical manifestations of cirrhosis are present. 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67 Saludes V et al. HCV diagnosis and hepatic fibrosis staging 86 Castéra L, Vergniol J, Foucher J, Le Bail B, Chanteloup E, Haaser M, Darriet M, Couzigou P, De Lédinghen V. Prospective comparison of transient elastography, Fibrotest, APRI, and liver biopsy for the assessment of fibrosis in chronic hepatitis C. Gastroenterology 2005; 128: [PMID: DOI: /j.gastro ] 87 World Health Organization. Prevention & Control of Viral Hepatitis Infection: Framework for Global Action Available from: URL: 88 Rosenberg WM, Voelker M, Thiel R, Becka M, Burt A, Schuppan D, Hubscher S, Roskams T, Pinzani M, Arthur MJ. Serum markers detect the presence of liver fibrosis: a cohort study. Gastroenterology 2004; 127: [PMID: DOI: /j.gastro ] 89 Leroy V, Monier F, Bottari S, Trocme C, Sturm N, Hilleret MN, Morel F, Zarski JP. Circulating matrix metalloproteinases 1, 2, 9 and their inhibitors TIMP-1 and TIMP-2 as serum markers of liver fibrosis in patients with chronic hepatitis C: comparison with PIIINP and hyaluronic acid. Am J Gastroenterol 2004; 99: [PMID: DOI: / j x] 90 Patel K, Gordon SC, Jacobson I, Hézode C, Oh E, Smith KM, Pawlotsky JM, McHutchison JG. Evaluation of a panel of non-invasive serum markers to differentiate mild from moderate-to-advanced liver fibrosis in chronic hepatitis C patients. J Hepatol 2004; 41: [PMID: DOI: /j.jhep ] 91 Wai CT, Greenson JK, Fontana RJ, Kalbfleisch JD, Marrero JA, Conjeevaram HS, Lok AS. A simple noninvasive index can predict both significant fibrosis and cirrhosis in patients with chronic hepatitis C. Hepatology 2003; 38: [PMID: DOI: /jhep ] 92 Williams AL, Hoofnagle JH. Ratio of serum aspartate to alanine aminotransferase in chronic hepatitis. Relationship to cirrhosis. Gastroenterology 1988; 95: [PMID: ] 93 Forns X, Ampurdanès S, Llovet JM, Aponte J, Quintó L, Martínez-Bauer E, Bruguera M, Sánchez-Tapias JM, Rodés J. Identification of chronic hepatitis C patients without hepatic fibrosis by a simple predictive model. Hepatology 2002; 36: [PMID: DOI: /jhep ] 94 Adams LA, Bulsara M, Rossi E, DeBoer B, Speers D, George J, Kench J, Farrell G, McCaughan GW, Jeffrey GP. Hepascore: an accurate validated predictor of liver fibrosis in chronic hepatitis C infection. Clin Chem 2005; 51: [PMID: DOI: /clinchem ] 95 Calès P, Oberti F, Michalak S, Hubert-Fouchard I, Rousselet MC, Konaté A, Gallois Y, Ternisien C, Chevailler A, Lunel F. A novel panel of blood markers to assess the degree of liver fibrosis. Hepatology 2005; 42: [PMID: DOI: /hep.20935] 96 Imbert-Bismut F, Ratziu V, Pieroni L, Charlotte F, Benhamou Y, Poynard T. Biochemical markers of liver fibrosis in patients with hepatitis C virus infection: a prospective study. Lancet 2001; 357: [PMID: DOI: /S (00) ] P- Reviewers: De Re V, Takaki A S- Editor: Gou SX L- Editor: A E- Editor: Ma S 3442 April 7, 2014 Volume 20 Issue 13

68 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (2): Hepatitis C virus TOPIC HIGHLIGHT Immunologic, metabolic and genetic factors in hepatitis C virus infection Nora A Fierro, Karina Gonzalez-Aldaco, Rafael Torres-Valadez, Erika Martinez-Lopez, Sonia Roman, Arturo Panduro Nora A Fierro, Karina Gonzalez-Aldaco, Rafael Torres-Valadez, Erika Martinez-Lopez, Sonia Roman, Arturo Panduro, Department of Molecular Biology in Medicine, Civil Hospital of Guadalajara, Fray Antonio Alcalde and Health Sciences Center, University of Guadalajara, Guadalajara 44280, Jalisco, Mexico Author contributions: Fierro NA analyzed data, wrote the paper and critically revised the manuscript; Gonzalez-Aldaco K, Torres- Valadez R and Martinez-Lopez E performed the research; Roman S analyzed the data; Panduro A conceived, drafted, analyzed data and critically revised the manuscript; all authors revised and approved the final version. Supported by The National Council of Science and Technology (CONACYT-FONDO SECTORIAL, Mexico), Grant No. Salud to Roman S; CONACYT-CIENCIA BA- SICA, Mexico, Grant No , COECYTJAL-UDG, Mexico, Grants No. S , CONACYT-INFR, Mexico and Grant No to Fierro NA Correspondence to: Arturo Panduro, MD, PhD, Department of Molecular Biology in Medicine, Civil Hospital of Guadalajara, Fray Antonio Alcalde and Health Sciences Center, University of Guadalajara, 278, Col El Retiro, Guadalajara 44280, Jalisco, Mexico. apanduro@prodigy.net.mx Telephone: Fax: Received: September 27, 2013 Revised: January 16, 2014 Accepted: March 6, 2014 Published online: April 7, 2014 Abstract The mechanisms that regulate disease progression during hepatitis C virus (HCV) infection and the response to treatment are not clearly identified. Numerous studies have demonstrated that a strong host immune response against HCV favors HCV clearance. In addition, genetic factors and metabolic machinery, particularly cholesterol modulation, are involved in HCV infection. It is likely that the interplay between all of these factors contributes to the outcome of HCV infection. In recent years, the world has experienced its largest epidemic of obesity. Mexico and the United States are the leading sufferers from this epidemic at the global level. Obesity is associated with the development of numerous pathologies including hypercholesterolemia which is one of the eight most important risk factors for mortality in Mexico. This may be related to the course of HCV infection in this population. Here, we focus on the urgent need to study the progression of HCV infection in relation to ethnic characteristics. Discoveries are discussed that hold promise in identifying immune, metabolic and genetic factors that, in conjunction, could be therapeutic targets or predictors of the progression of HCV infection Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Hepatitis C virus; Immune response; Lipids; Metabolism; Genetics Core tip: Immunologic, metabolic and genetic factors are involved in the progression of hepatitis C virus (HCV) infection and the response to treatment. The significant increase in obesity worldwide, including in Mexico, imposes a new metabolic stress factor in patients with HCV infection. Given that the lipid components associated with HCV infection are finely modulated, it is possible that the progression of HCV infection may be regulated by the characteristics of the population s lipid composition. Fierro NA, Gonzalez-Aldaco K, Torres-Valadez R, Martinez- Lopez E, Roman S, Panduro A. Immunologic, metabolic and genetic factors in hepatitis C virus infection. World J Gastroenterol 2014; 20(13): Available from: URL: wjgnet.com/ /full/v20/i13/3443.htm DOI: org/ /wjg.v20.i INTRODUCTION Hepatitis C infection is caused by the hepatitis C virus 3443 April 7, 2014 Volume 20 Issue 13

69 Fierro NA et al. Immunity, metabolism, genetics and HCV (HCV), a positive-stranded ribonucleic acid (RNA) virus of the Flaviviridae family with a hepatotropic lifecycle. HCV infection is an important cause of chronic liver disease and the third-leading cause of all death from cirrhosis and hepatocellular carcinoma (HCC) worldwide. Approximately 3% of the world s population (160 million people) are currently infected with HCV, which in most cases establishes a lifelong chronic infection [1]. However, 25%-30% of infected individuals spontaneously clear the virus during acute infection. Because HCV is a noncytopathic virus, it is accepted that the interplay between the virus and the host immune response may influence the outcome of infection [2]. In addition, host genetic factors, such as polymorphisms in cytokine and chemokine receptor genes promoters [3,4], are thought to be important contributors to the modulation of HCV outcome. A key role for the low-density lipoprotein receptor (LDL-R) has been demonstrated during the first steps of HCV attachment to the cell surface [5]. LDL is responsible for transporting most of the cholesterol in plasma [6] and cholesterol levels are tightly modulated during HCV infection. Virus entry into the cell is also mediated by other lipoprotein receptors including scavenger receptor class B type Ⅰ (SRB-Ⅰ). In addition to providing a docking site for HCV particles, SRB-Ⅰ facilitates entry of the virus into the hepatocyte [6,7]. The E2 envelope protein determines viral attachment to SRB-Ⅰ. Currently, Latinos represent the fastest growing ethnic group in the United States and are the most overweight. Mexico and the United States are experiencing the largest obesity epidemic in the world. In conjunction, genetic and cultural components, lifestyle and environmental factors are all associated with the development of obesity. In addition to representing a public health problem by itself, obesity is also associated with the development of numerous pathologies, including hypercholesterolemia. A recent analysis of the burden of disease revealed that hypercholesterolemia was among the eight most important risk factors for mortality in Mexico [8]. Thus, the specific characteristics of the lipid components already described suggest a unique mechanism of regulation of metabolic machinery among the Mexican people that could impact the progression of HCV infection. Crosstalk between metabolic and immune components in conjunction with viral and genetic host factors may occur and predict HCV disease outcome. To date, the exact mechanisms responsible for HCV clearance and recovery are unknown. An understanding of these mechanisms will contribute to the development of novel, individualized, preventive strategies and an urgently needed vaccine. This review summarizes recent advances in understanding the crosstalk between the immune response, metabolism and genetics in HCV viral clearance and emphasizes characteristics of the Mexican population and their association with this process. IMMUNE RESPONSE TO HCV INFECTION Innate and adaptive immune response to HCV A robust innate immune response is activated when HCV infects the liver. This response includes the induction of several interferon-stimulated genes (ISGs) [9,10] and is mediated by the specific production of inflammatory and antiviral cytokines by macrophages, natural killer (NK) cells and neutrophils, which release perforin, granzyme B, interferon-gamma (IFN-γ) and tumor necrosis factorbeta. Immune cells also express Fas ligand which cause cell death in infected hepatocytes [11]. Animal models have enabled the precise description of the kinetics of viremia throughout infection. In particular, chimpanzee models have revealed that HCV RNA levels increase rapidly upon initial infection [12]. Thereafter, a slow decrease in viremia is observed. It is accepted that the innate immune response in hepatocytes contributes to the second phase of slowed viral replication [13]. This response includes type Ⅰ interferon responses and the antiviral activity of plasmacytoid dendritic cells and NK cells. Thus, an ineffective innate immune response can result in disease progression. The adaptive immune response to HCV is mediated by the humoral and cellular immune systems. HCV-specific T lymphocytes are detectable five to nine weeks after infection [14,15], which coincides with the onset of hepatitis. Both CD4 + and CD8 + T cells have been shown to play major roles in the outcome of HCV infection. CD8 + T cells inhibit HCV replication by cytolytic and non-cytolytic effector mechanisms [16] that are highly dependent on CD4 + T cell activation. CD4 + T cells are critical for the elimination of viruses through tightly regulated mechanisms. CD4 + T-helper cells are divided into four subsets (Th1, Th2, T regulatory, Th17) based on their expression profile of transcription factors and secreted cytokines. An effective Th1 cellular function is crucial for an adequate immune response against HCV, given the specific production of IFN-γ by this cellular subtype. Unbalanced Th1/Th2 T-cell responses in the liver are a characteristic of hepatic inflammation and subsequent liver fibrosis as a result of HCV infection [17]. Clinical data reveals increased interleukin-17 levels in HCV-infected patients who had increased alanine transaminase (ALT) values, suggesting that Th17 cells in chronic hepatitis C infection might be associated with control of liver injury. However, these observations are preliminary and not fully conclusive [18,19]. Indeed, vigorous peripheral and intrahepatic virus-specific T cell responses that target multiple epitopes have been described in patients who recover from HCV infection [13], while a weak and functionally impaired T cell response has been reported in patients who fail to clear the virus. Consistent with these findings, the role of T regulatory cells in HCV seems to range from suppressing T-cell responses directed against HCV to down-regulating the immune responses causing liver damage [20]. In addition, animal models have 3444 April 7, 2014 Volume 20 Issue 13

70 Fierro NA et al. Immunity, metabolism, genetics and HCV A: Innate immunity Liver B: Adaptive immunity FasL IFNg/TNFa NK NK IL-12 DC MHC Ⅰ TCR CD8 + T Clearance of virus Activated DC HCV-infected IFNa/b DC MHC Ⅱ CD4 + T IFNg/TNFa DC TCR LSEC Hepatocytes MHC Ⅱ CD40 CD80 IL-12 Cell-mediated immune response CD4 + T IL-2 IFNg TNFa HCV-infected Fas Perforin/granzyme B DC IFNa/b Th1 NK IFNg/TNFa Humoral immune response Clearance of virus FasL Interfering HCV-induced Abs nabs Figure 1 Mechanisms of Immune response to hepatitis C virus. Multiple innate (A) and adaptive immune (B) components are involved in hepatitis C virus (HCV) infection (see text), dendritic cells (DC), natural killer (NK) cells, CD4 +, CD8 + T cells and B-cell mediated humoral immune response are crucial for disease outcome. IFN-γ: interferon-gamma; TNF-α: tumor necrosis factor-α; IL: Interleukin; nabs: Neutralizing anti-hcv antibodies. indicated that early expression of memory precursor markers on HCV-specific T cells or the expression of ligands for these memory markers in the liver predicts the outcome of acute infection [17] (Figure 1). Neutralizing antibodies The generation of neutralizing antibodies represents a protective strategy in host immunity. Neutralizing anti- HCV antibodies (nabs) were originally described in chimpanzees. These antibodies target epitopes within the hypervariable region 1 (HVR1) of envelope glycoprotein E2 [21]. In humans, nabs were identified in a cohort study of transplant recipients in which the incidence of HCV viremia was lower in patients receiving immunotherapy (anti-hbv immunoglobulins contaminated with anti- HCV immunoglobulins) compared to patients whose therapy did not include anti-hcv antibodies [22], indicating the potential contribution of neutralizing antibodies to viral control. Functional analysis and neutralization experiments using sera from chronically HCV-infected patients have demonstrated that host neutralizing responses target viral entry at a step after initial HCV binding. Initial HCV attachment to the cell surface is likely facilitated by interactions with attachment factors that include the 3445 April 7, 2014 Volume 20 Issue 13

71 Fierro NA et al. Immunity, metabolism, genetics and HCV LDL receptor [5]. Upon initial attachment, at least six host entry factors including scavenger SRB-Ⅰ, CD81, the tight junction proteins claudin 1 and occludin [23], receptor tyrosine kinases [24] and the Niemann-Pick C1-like 1 cholesterol absorption receptor [25] are important for particle internalization. Indeed, several E2 domains have been shown to play pivotal roles in viral entry and neutralization. Two regions in the E2 viral envelope glycoprotein have increased genetic variability within quasispecies and among genotypes and have been identified as hypervariable regions. Antibodies that demonstrate broadly neutralizing activity tend to be directed against conserved and conformational epitopes within E2, which inhibit the interaction between CD81 and E2 [26-30]. Mechanisms of immune evasion by HCV The strategies of HCV persistence and evasion of the immune response involve multiple mechanisms. The high genetic variability of HCV is a major contributor to the development of chronic HCV infection. It has been shown that HCV constantly circulates in the patient and rapidly evolves into genetically distinct but closely related variants within quasispecies. The simultaneous presence of different variants has been postulated to allow for the rapid selection of mutants that are best adapted to changes in the host environment. Genetic analysis reveals a positive correlation between distinct HCV quasispecies, viral clearance and a slowly adapting viral population, whereas distinct patterns of progression to chronicity are related to selective pressure on the HVR1 region of HCV E2. Moreover, chronic progression is associated with the rapid evolution of quasispecies [31], and single point mutations that result in glycosylation site modifications during viral genetic and conformational changes have been suggested to be responsible for masking the envelope glycoprotein binding sites [32-36]. The result is viral persistence in the body despite the presence of an immune response. During the innate immune response, HCV interferes with innate signaling pathways using its structural and non-structural proteins to interact with factors that regulate ISGs, thus attenuating innate responses [37-39]. In addition, patients who fail to clear the virus show a weak and functionally impaired T cell response. This result strongly suggests that HCV also employs strategies to escape host adaptive immunity. The action of nabs may be blocked by the presence of interfering HCV-induced Abs [32,40,41]. There are two proposed mechanisms through which lipoproteins may provide HCV with protection from nabs: masking viral epitopes by associating with LDL and very-low density lipoprotein (VLDL) or accelerating viral entry by interactions with high-density lipoprotein (HDL). In both cases, HCV lipoprotein-dependent protection results in limiting the exposure of viral epitopes to nabs [42]. Recently, it has been suggested that in HCV genotype 2a, the viruses that combine with LDL and VLDL and are consequently distributed in low-density fractions are more capable of escaping neutralizing antibodies compared to viruses dis- tributed in high-density fractions [43]. In addition, HCV is able to infect B-lymphocytes and induce hypermutations in the heavy chains of immunoglobulins. These hypermutations may decrease the affinity and specificity of anti- HCV antibodies and may allow the virus to escape from the immune system [44]. HCV dissemination by cell-tocell transmission may also contribute to viral persistence by avoiding surveillance of nabs present in the serum [45] (Figure 1). HCV VIRUS AND LIPID METABOLISM HCV entry After its discovery, HCV was found to be associated with lipoproteins. Thomssen et al [46] demonstrated the existence of distinct HCV particles categorized as high and low density particles. These particles, also recognized as lipo viro particles (LVP), are rich in triglycerides (TG) and can be almost completely precipitated by anti- Apo lipoprotein B and E. LVPs contain HCV RNA and structural viral proteins, mainly E1 and E2 that attach the virus to the hepatocyte surface through specific receptors [6,47]. Attachment and internalization of HCV into the cell represent the first steps in the viral replication cycle. The LDL receptor is likely the mediator of viral entry through Apolipoprotein E (ApoE), the major structural agent of infectious LVP and the natural ligand of LDL-R [48]. ApoE exists in three common isoforms (E2, E3 and E4) that affect LDL receptor binding [49]. Allele 4 is associated with clearance of HCV and protection against severe liver damage [50]. Allele 2 is associated with clearance and binds poorly to LDL-R; it is plausible that this defective binding could result in poor uptake of HCV LVP into hepatocytes, thus altering the balance between virus replication and the immune response by decreasing viral replication and favoring clearance. In contrast, allele 3 is associated with persistence [51]. Recently, an association between levels of ApoE and interferon sensitivity was found, and ApoE was shown to induce a higher LDL concentration, potentially inhibiting the binding of HCV to LDL-R. The association between ApoE and the peg-interferon treatment response suggests that lipid modulation is a potential target for modifying interferon sensitivity, favoring the clearance of HCV and avoiding progression to chronic infection [52] (Figure 2). HCV replication, virion assembly and secretion: Role of apolipoprotein B and microsomal triglyceride transfer protein HCV RNA replication is strongly influenced by the intracellular levels and composition of fatty acids, including cholesterol. Recent studies have demonstrated that the expression of genes involved in biosynthesis, degradation and intracellular transport of lipids is altered during HCV infection [53,54]. The positive RNA strand of HCV induces remodeling of the intracellular membranes to generate compartments where RNA replication takes place April 7, 2014 Volume 20 Issue 13

72 Fierro NA et al. Immunity, metabolism, genetics and HCV nm Apo C Apo B 100 LVP Apo B 100 Apo C LVP HCV Apo E E1 E2 HCV Apo E E1 E2 GAGs LDL-R SRB-Ⅰ CD81 CLDN1 OCLN Hepatocyte cytosol Figure 2 Hepatitis C virus entry. Hepatitis C virus (HCV) binds to lipoproteins and forms the lipo viro particle (LVP). LVP is attached by the hepatic low density lipoprotein (LDL) receptors through viral surface membrane proteins E1 and E2. After the initial phase, the LVP interacts with several input factors: the scavenger receptor class B type Ⅰ (SRB-Ⅰ), the membrane receptor CD81, coreceptors claudin 1 (CLDN1) and ocludin (OCLN). LVP is internalized into the cytosol by endocytosis. The NS5B viral protein is responsible for generating the membranous web required for RNA replication through diverse mechanisms [55]. Transcription of genes related to lipid metabolism is augmented during HCV infection. Sterol regulatory element-binding protein (SREBP) expression, which controls the transcription of specific genes required for cholesterol biosynthesis, is increased by the NS2 and NS4B HCV proteins. However, the exact mechanism responsible for disruption of host lipid metabolism by HCV infection is not yet clear. Understanding the components involved in this process will allow the possible design of specific therapeutic targets. Some authors have reported that 30% of the total protein in complexes associated with HCV RNA is functionally involved in lipid metabolism [56]. Many lipids are crucial for the viral lifecycle, and inhibitors of cholesterol/fatty acid biosynthetic pathways inhibit viral replication, maturation and secretion [57]. In humans, apolipoprotein B (ApoB)-100 and ApoB-48 are obligatory proteins for the assembly of hepatic VLDL and intestinal chylomicron, respectively. VLDL assembly occurs via a two-step mechanism involving the formation of ApoBcontaining VLDL precursor particles in the lumen of the endoplasmic reticulum, a step that requires microsomal triglyceride transfer protein (MTP) [58]. Hepatic VLDL assembly and secretion are also profoundly influenced by alterations in the de novo biosynthesis of phospholipids, such as phosphatidylethanolamine and phosphatidylcholine. Increased VLDL-TG concentration is a central feature of diabetic dyslipidemia and is largely caused by increased VLDL-TG secretion [59]. According to clinical data and experimental models, the HCV core protein has been shown to inhibit the MTP protein [60]. This enzyme plays a rate-limiting role in VLDL assembly and ApoB secretion [61]. Inhibition of VLDL assembly and secretion also affects virion morphogenesis and secretion, leading to the notion that HCV may hijack the VLDL secretion pathway for virion maturation and secretion. The reliance of HCV on host lipid metabolic pathways for its replication, morphogenesis and release requires the modulation of host lipid pathways by HCV to create a lipid-rich intracellular environment favorable for replication. HCV assembly and maturation in hepatocytes depend on MTP and ApoB in a manner that parallels the formation of VLDL [56,62]. Inhibitors of MTP and reduction of the expression of ApoB lowers virion production [56,63]. Mechanism of HCV-associated steatosis Steatosis, or the accumulation of hepatocellular lipid droplets, and altered serum lipid profiles are common consequences of HCV infection. The presence of ste April 7, 2014 Volume 20 Issue 13

73 Fierro NA et al. Immunity, metabolism, genetics and HCV Inflammation (inflammatory cytokines) ROS Accumulation of lipids (steatosis) Lipid peroxidation RX- Ralpha PPARa SREB1c b-oxidation Figure 3 Hepatitis C virus effect in lipid metabolism and development of steatosis. Hepatitis C virus (HCV) alters intracellular lipid metabolism to create an environment rich in lipids to facilitate its replication. This allows the accumulation of lipids, decrement in exportation of very low density lipoprotein (VLDL) and development of steatosis, a pathological phenotype associated with HCV chronic infection. Reactive oxygen species (ROS) generated during infection cause lipid peroxidation triggering inflammation. MTP: Microsomal triacylglycerol transfer protein; RXRα: Retinoid X receptoralpha; PPARα: Peroxisome proliferators-activated receptor alpha; SREB1c: cholesterol regulatory element binding protein-1c; FAS: Fatty acid synthase. MTP FAS Decreased export of cholesterol VLDL assembly Secretion and catabolism Lipogenesis atosis in infected patients with HCV varies from 30% to 70% depending on ethnic and other factors, such as alcohol consumption, being overweight, obesity and factors that are also risk factors for non-alcoholic steatohepatitis [64]. In patients with chronic HCV, serum cholesterol is significantly reduced when compared to appropriately matched controls. This has been specifically analyzed in HCV genotype 3 infections in specific populations [65], and this metabolic effect can be reversed after successful HCV eradication. However, large-scale studies including samples from distinct populations and different genotypes are necessary to determine the roles of viral genotypes and host genetic factors in this process. HCV modulates lipid metabolism to create an environment rich in lipids favorable for viral replication. Consistent with this lipid modification, each step of the viral replication cycle appears related to lipid metabolism [66]. The interaction of HCV proteins with hepatic cellular components contributes to the interference with lipid and carbohydrate metabolism, resulting in the release of cytokines, insulin resistance, inflammation, oxidative stress and steatosis [67,68]. The mechanism of triglyceride accumulation induced by HCV infection is multifactorial. de Gottardi et al [69] reported that host lipid metabolism may be modulated by HCV at three levels: impaired lipoprotein secretion, enhanced lipogenesis, and impaired fatty acid degradation. These detrimental alterations incurred during HCV infection then manifest as the pathological basis for some HCV-associated diseases, most notably steatosis and metabolic syndromes such as insulin resistance, obesity, and HCC [70-72]. MicroRNAs (mirnas) exert regulatory control through modulation of many targets. In the liver, mirna-122 is important for regulating lipid metabolism [73,74] and aberrant expression of mir- NAs is linked to HCV infection [75]. It has recently been reported that HCV replication induces the expression of mir-27 in vitro and in vivo. This results in larger and more abundant lipid droplets and coincides with the repression of regulators of triglyceride homeostasis including peroxisome proliferator-activated receptor alpha (PPARα), identifying HCV s up-regulation of mir-27 as a novel mechanism that may contribute to the development of steatosis [76]. In conjunction, the main lipid alterations in HCV are enhanced lipogenesis, reduced secretion and β-oxidation of lipids regulated by transcription factors, hormones and key enzymes that are briefly described below (Figure 3). PPARs: PPARs are ligand-dependent transcription factors. The three subtypes of PPARs (PPARalpha, PPARdelta, and PPARgamma) are differentially expressed in tissues, and play pivotal roles in lipid, lipoprotein and glucose homeostasis [77]. PPARalpha, PPARdelta, and PPARgamma are differentially involved in HCV infection. A clear effect of PPARα in HCV RNA replication has been described and a recent report has shown that PPARα-selective antagonists inhibit HCV replication, while PPARα is not involved in this process [78]. PPARα is a transcription factor in the nuclear hormone receptor superfamily; it is involved in the differentiation of normal adipocytes, and its major function is to control fatty acid oxidation and activation. PPARα participates in enhancing the expression of adiponectin messenger RNA (mrna) and serum adiponectin levels. PPARα deficiency causes defective hepatic fatty acid oxidation [79,80], while persistent activation of PPARα is essential for the pathogenesis of hepatic steatosis and HCC induced by HCV infection [81]. Dharancy et al [82] reported that PPARα mrna expression was significantly reduced in the liver of chronic HCV patients infected with HCV genotype 3448 April 7, 2014 Volume 20 Issue 13

74 Fierro NA et al. Immunity, metabolism, genetics and HCV 3 when compared with HCV genotype 1 infections. A better understanding of the role of PPARα and its interaction with HCV proteins may help in developing novel therapies against HCV-induced steatosis and HCC. Adiponectin and leptin: Adiponectin is an insulinsensitizing protein that is abundantly expressed in white adipose tissue [83] and is a member of the adipocytokine family. Adiponectin improves hepatic insulin sensitivity, decreases lipid accumulation in macrophages and has anti-inflammatory properties [84-86]. Serum adiponectin levels in humans depend upon metabolic activity; it is present as a low molecular weight trimer and a high molecular weight (HMW) hexamer. HMW adiponectin is the biologically active form [87]. Adiponectin exerts its effect via two receptors, the adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). AdipoR1 is expressed in skeletal muscles, and AdipoR2 is expressed in the liver [88]. AdipoR1 is associated with AMP-activated protein kinase (AMPK) activation, while AdipoR2 is associated with PPARα activity. Some insights regarding the pathways of steatohepatitis have been identified by impaired lipid accumulation due to hepatic loss of adiponectin receptors, which play an important role in fatty acid accumulation by up-regulating the expression of enzymes during HCV infection, including AMPK, fatty acid synthase (FAS), acetyl-coa carboxylase, liver gluconeogenic enzyme and phosphoenolpyruvate-carboxykinase [79,80]. Adiponectin is assumed to protect hepatocytes from triglyceride accumulation by increasing the β-oxidation of free fatty acids or decreasing de novo production of free fatty acid in hepatocytes [89]. In contrast to the anti-inflammatory role of adiponectin, leptin is a pro-inflammatory adipocytokine identified as one of the best markers of total body fat; its elevated expression can result in stimulation of cellular lipolysis and fatty oxidation, promoting a negative energy balance. Both adiponectin and leptin are crucial for hepatic steatosis [90]. However, data concerning the roles of adiponectin and leptin on HCV-related steatosis remain divergent. This is mainly due to the multiple functions involving adipocytokines. By using a HCV core-transgenic mouse model, a recent report reveals that HCV coreinduced, non-obese hepatic steatosis is associated with down regulation of the leptin gene in visceral fat and hypoadiponectinemia [91]. A better understanding of this process may be valuable in the design of new therapeutic interventions, particularly in the cases of non-obese hepatic steatosis involving HCV infection. AMPK: AMPK is a heterotrimeric protein with serine/ threonine protein kinase activity that works as a sensor of cellular energy status and enables metabolic adaptation to the environment, such as in nutritional stress [92-94]. AMPK plays a key role in the regulation of both lipid and glucose metabolism. Activated AMPK inhibits energy-consuming biosynthetic pathways such as lipogenesis and activates ATP-producing catabolic pathways such as β-oxidation. A study demonstrated that phosphorylation of AMPK at threonine 172 causes dramatically reduced AMPK activity in cells infected with HCV or harboring an HCV subgenomic replicon. Thus, inhibition of AMPK is required for HCV replication and restoration of AMPK activity may represent a target for anti-hcv therapies [95,96]. The anti-hcv activity of 2-octynoic acid (2-OA), a compound used in perfumes, lipstick, and many food flavorings, has recently been revealed in vitro. This activity seems to be associated with the activation of AMPK by 2-OA, which regulates ISGs and suppresses mirna-122 expression, inhibiting HCV infection. This represents a novel mechanism to explain inhibition of infection by AMPK [97]. SREBP: SREBP plays an important role in the regulation of lipid synthesis and cholesterol metabolism. The SREBP gene encodes three isoforms (SREBP-1a, SREBP-1c and SREBP-2 [98] that regulate genes involved in cholesterol synthesis. SERBP-1a is an activator of cholesterol and fatty acid synthesis, while SERBP-1c regulates genes involved in lipid synthesis [99-102]. SREBP-1c activates the lipogenesis pathway in response to insulin. HCV infection and the HCV core protein up-regulate the expression of SREBP-1c and cause the development of fatty liver [103]. In non-alcoholic fatty liver disease, SERBP- 1c is expressed at a level up to five-fold higher than observed in controls [104]. It has been demonstrated that HCV infection enhances the proteolytic cleavage of SREBP precursors in hepatic cells. The HCV NS2 and NS4B proteins can upregulate SERBP-1c and consequently promote enhanced transcriptional activity of fatty acid synthase [105,106]. Animal models have been used to investigate the effect of the HCV core and NS proteins on SREBP gene regulation, and these models have indicated that HCV proteins that interfere with SREBP lead to steatosis [105,106]. The host subtilisin/kexin/isozyme/1 (SKI-1) or site 1 (S1P) plays a crucial role in the proteolytic activation of SREBP. The use of a SKI-1/S1P-specific proteinbased inhibitor recently demonstrated that SKI-1/S1P inhibition blocks HCV infection in hepatoma cells by a mechanism that is associated with dramatic reduction in the number of lipid droplets and adipose differentiationrelated protein/perilipin 2. The inhibition of virus assembly from infected cells identifies SKI-1/S1P as both a regulator of the HCV lifecycle and a potential hostdirected therapeutic target against HCV infection [107]. Retinoid X receptor alpha: The retinoid X receptor (RXR) family is a family of nuclear receptors that target and regulate multiple signaling pathways. RXRs control gene expression by binding cooperatively as a dimer to hormone responsive elements [108,109]. RXRs form homodimers and are involved in 9-cis retinoic acid-mediated gene activation. RXRs exist as three isoforms, namely RXRα, RXRβ and RXRγ; the RXRα isoform is abundantly expressed in the liver and regulates cell proliferation and differentiation April 7, 2014 Volume 20 Issue 13

75 Fierro NA et al. Immunity, metabolism, genetics and HCV Table 1 Genes and hepatitis C infection Lipid metabolism genes altered by HCV Gene Protein Effect SREBP PPARα MTP PEPCK Sterol regulatory element-binding protein Peroxisome proliferatorsactivated receptor alpha Microsomal triacylglycerol transfer protein Phosphoenolpyruvate carboxykinase Core protein increases the gene expression [110] HCV infection led to reduction in gene expression [126] Core protein led to reduction in MTP activity and lower gene expression [127,128] NS5A increases gene expression and development of steatosis [129] FAS Fatty acid synthase Core protein induces FAS promoter activity and severity of steatosis [115] RXRα Retinoid X receptoralpha Core protein enhances the transcriptional activity and contributes to the pathogenesis of infection [112] APOE Apolipoprotein E HCV forms lipoviroparticles and hijacks ApoE for entry into hepatocyte [48] Genes associated with spontaneous clearance of HCV Gene Protein Allele IL28B Interleukin 28B rs CC [130] IL28B Interleukin 28B rs TT [131] IL28B Interleukin 28B ss dg [132] KIR Natural killer cell KIR3DS1 [133] immunoglobulin-like receptor TNFα Tumor necrosis -863CC [134] factor-α APOB Apolipoprotein B rs TT [135] MTP: Microsomal triacylglycerol transfer protein; FAS: Fatty acid synthase; HCV: Hepatitis C virus; SREBP: Sterol regulatory element-binding protein; PPARα: Peroxisome proliferator-activated receptor alpha; RXRα: Retinoid X receptor alpha. Some reports have suggested an interaction between HCV core proteins with RXRα and shown that the HCV core protein binds to the DNA-binding domain of RXRα, leading to an increase in binding of RXRα to its DNA response element [110,111]. In addition, RXRα is activated in cells expressing the HCV core protein and in the livers of HCV core-transgenic mice that develop hepatic steatosis and HCC. The HCV core protein may compete with p50- and p65-nuclear factor kappa-lightchain-enhancer of activated B cells (NF-κB) subunits for direct interaction with RXRα. This competition may influence signaling via NF-κB, a regulator of many cellular functions and lipid synthesis, and up-regulate the enzymes acyl CoA oxidase and retinol binding protein Ⅱ, exacerbating steatosis by increasing oxidative stress and decreasing β-oxidation [111,112]. These reports indicate that modulation of RXRα controls gene expression by interacting with the core protein and contributes to pathogenesis of HCV infection [112]. FAS: FAS is a protein that is directly linked to intracellular lipid synthesis and plays a central role in triglyceride accumulation in the liver. FAS catalyzes the conversion of acetyl CoA and malonyl CoA to saturated fatty acids, which are then converted to TG after etherification [106,113]. Yang et al [114] have demonstrated that HCV infection directly induces FAS expression, while Jackel-Cram et al [115] have reported that HCV-3a is a stronger teratogenic factor than HCV-1b. These authors reported that a single amino acid substitution at position 164 (phenylalanine) of the HCV- 3a core protein can up-regulate FAS activity. Therefore, the short sequence YATG in HCV-1b and FATG in HCV-3a is responsible for FAS up-regulation at the transcriptional level [114]. However, further studies are required to understand the molecular mechanism of FAS activation. GENETICS AND HCV INFECTION Recent progress in the field of molecular genetics has revealed that the clinical outcome and response to treatment for HCV may be predetermined by genetic polymorphisms. Genes related to lipids, metabolism and the immune response have been studied (Table 1). Largescale genetic epidemiological studies are required to attain adequate statistical power. Identifying the relationship between specific genes and the progression of HCV infection is a crucial step toward understanding the pathogenesis of infection. DYSLIPIDEMIAS AND HCV INFECTION The lipid profile in chronic HCV-infected patients presents low levels of total cholesterol, HDL and LDL regarding non-infected individuals. In addition, these levels are significantly lower in patients who present with fatty liver. HCV-genotype 3-infected patients show a higher incidence of fatty liver development. The relationship of these lipid alterations with HCV infection is important for the lifecycle of the virus, and it is positively correlated with the response to antiviral treatment, especially in the setting of genotype 3 infections [57]. Studies have demonstrated the presence of hypobetalipoproteinemia in patients infected with HCV [116]. The pathogenesis of hypobetalipoproteinemia in HCV patients is unclear. Recently, hepatic steatosis has been correlated with this type of dyslipidemia. However, the influence of viral infection on these parameters has not been thoroughly investigated. It has been demonstrated that hepatic fibrosis is increased as serum levels of ApoB decrease. Thus, there is a strong negative correlation between fibrosis and ApoB levels [117]. Studies report that serum triglyceride levels are decreased in patients with HCV; however, in these studies, more than 50% of patients had liver cirrhosis and HCC [118]. Thus, the decrease in TG cannot be attributed to the presence of virus alone. In more recent studies, where HCV patients without cirrhosis or HCC were analyzed, the prevalence of TG was similar to control patients without HCV. However, TGs in the fasting state, transported 3450 April 7, 2014 Volume 20 Issue 13

76 Fierro NA et al. Immunity, metabolism, genetics and HCV only by VLDL, are found at significantly lower levels in HCV-infected patients compared to individuals without HCV infection. The levels of TGs in different stages of fibrosis in patients with HCV have been compared and no differences were found. Differences were only found in patients with cirrhosis. In contrast, the levels of TGs transported by VLDL declined with progression of liver damage until cirrhosis was reached. These data indicate that hypertriglyceridemia in HCV patients is not of viral origin. In contrast, the decreased serum levels of TG can be explained by the interference in maturation and excretion of VLDL caused by the viral lifecycle [119]. Dyslipidemias in Mexico Chronic diseases associated with diet and lifestyles, including obesity and dyslipidemias, have genetic components. Currently, Mexico and the United States are experiencing the largest obesity epidemic in the world. The significant increase in prevalence of these diseases in recent years strongly suggests the influence of environmental factors in their development. High levels of cholesterol and lipid disorders are important risk factors for developing diseases including hepatic and cardiovascular diseases. Pathologies associated with high levels of cholesterol represent one of the eight main risk factors for mortality in Mexico [8,120]. Hypercholesterolemia has been described as a public health problem in Mexico since 1988 when the Secretariat of Health conducted the first national survey related to serum cholesterol levels among the Mexican population. In 1993, a new study was performed (National Survey of Chronic Diseases) revealing that the prevalence of hypercholesterolemia had risen to 35.5%, a 10% increase compared to the previous study. This trend continued during the coming years, resulting in an increase in prevalence of hypercholesterolemia from 35.3% to 42.6% [121]. Additionally, serum TGs are higher and HDL is lower among Mexicans when compared to other populations worldwide. In 2000, data from the Secretariat of Health revealed that fasting serum samples from 2351 adults had a mean total cholesterol of mg/dl, HDL cholesterol of 38.4 mg/dl and TGs of mg/dl. Only 6.1% of the study population was diagnosed with dyslipidemia and 85.88% did not know that they were ill [121]. Detailed analysis of the data revealed that the most frequent dyslipidemia was hypobetalipoproteinemia (low HDL), followed by hypertriglyceridemia and hypercholesterolemia. The most common combinations were high levels of TGs and low HDL cholesterol, and high levels of TGs with high total cholesterol (mixed dyslipidemia). It is currently accepted that there is an important association between being overweight, obesity and various types of dyslipidemia [121]. As in the United States, the obesity epidemic in Mexico has dramatically increased in children and adults. In 2000, the Secretariat of Health in Mexico revealed an association between obesity and hypertriglyceridemia. Obese adults compared with individuals of normal weight were four times more at risk for a diagnosis of high cholesterol, low cholesterol HDL, high TGs or any combination of these conditions [121]. Currently, even children have been detected with high blood levels of cholesterol and triglycerides, most likely due to the marketing of processed foods with high sugar or fat content, changes in dietary patterns and the abuse of food rich in animal fat. It has been described that 100 mg of dietary cholesterol for each 1000 kcal results in 12 mg/dl increase in the concentration of blood cholesterol, and that children and adolescents with high levels of cholesterol are more likely to continue having high levels into adult stage [122]. Mexico has been reported to be a low endemic area for HCV [123,124]. Moreover, a low association between HCV infection and HCC has been reported in the country [125]. Given that lipid components associated with HCV infection are finely modulated in the Mexican population, it is plausible to hypothesize that the progression of infection may be regulated by the characteristics of this population s lipid composition. FINAL REMARKS Ineffective viral escape from the immune response and effective T cell activity, together with the absence of interfering HCV-induced Abs, are factors that may contribute in combination to the spontaneous clearance of HCV. In contrast, exhausted T cell responses resulting in impaired T cell activity and viral adaptation to the host immune response may result in chronicity. In addition to the recognized role of lipoproteins during the initial steps of HCV infection, lipoproteins can also provide HCV with protection from nabs, either by masking viral epitopes by associating with LDL and VLDL or by accelerating viral entry via HDL and thus limiting the exposure of viral epitopes to nabs. ApoE alleles 2 and 4 are associated with HCV clearance and protection against severe liver damage. Furthermore, association between ApoE levels and IFN sensitivity suggest that lipid modulation is a potential target for preventing HCV disease progression. When comparing serum lipid concentrations with those of other populations of the world, it is notable that Mexicans have higher concentrations of TGs and lower HDL cholesterol. Hypercholesterolemia is among the eight most important risk factors for mortality in the country. Moreover, in Mexico, as in the United States and other developed countries, obesity has significantly increased in recent years. Analysis of the 2000 Mexican National Health Survey significantly associated obesity with hypertriglyceridemia, followed by hypercholesterolemia. This reveals a unique lipid profile in the Mexican population that, in conjunction with genetics and lifestyle, might be responsible for the immune response during chronic diseases, including HCV infection. CONCLUSION Advances in understanding HCV disease outcome suggest that individualized therapies that account for fac April 7, 2014 Volume 20 Issue 13

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80 Fierro NA et al. Immunity, metabolism, genetics and HCV CM, Ho YP, Chen TH, Yeh CT. HCV core-induced nonobese hepatic steatosis is associated with hypoadiponectinemia and is ameliorated by adiponectin administration. Obesity (Silver Spring) 2012; 20: [PMID: DOI: /oby ] 92 Hardie DG. Minireview: the AMP-activated protein kinase cascade: the key sensor of cellular energy status. Endocrinology 2003; 144: [PMID: DOI: / en ] 93 Hardie DG. The AMP-activated protein kinase pathway-- new players upstream and downstream. J Cell Sci 2004; 117: [PMID: DOI: /jcs.01540] 94 Khan M, Jahan S, Khaliq S, Ijaz B, Ahmad W, Samreen B, Hassan S. Interaction of the hepatitis C virus (HCV) core with cellular genes in the development of HCV-induced steatosis. Arch Virol 2010; 155: [PMID: DOI: /s ] 95 Nakashima K, Takeuchi K, Chihara K, Hotta H, Sada K. Inhibition of hepatitis C virus replication through adenosine monophosphate-activated protein kinase-dependent and -independent pathways. 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Up-regulation of fatty acid synthase promoter by hepatitis C virus core protein: genotype-3a core has a stronger effect than genotype-1b core. J Hepatol 2007; 46: [PMID: DOI: /j.jhep ] 116 Bima AI, Hooper AJ, van Bockxmeer FM, Burnett JR. Hypobetalipoproteinaemia secondary to chronic hepatitis C virus infection in a patient with familial hypercholesterolaemia. Ann Clin Biochem 2009; 46: [PMID: DOI: /acb ] 117 Petit JM, Benichou M, Duvillard L, Jooste V, Bour JB, Minello A, Verges B, Brun JM, Gambert P, Hillon P. Hepatitis C virus-associated hypobetalipoproteinemia is correlated with plasma viral load, steatosis, and liver fibrosis. Am J Gastroenterol 2003; 98: [PMID: DOI: /j x] 118 Dai CY, Yeh ML, Huang CF, Hou CH, Hsieh MY, Huang JF, Lin IL, Lin ZY, Chen SC, Wang LY, Chuang WL, Yu ML, Tung HD. Chronic hepatitis C infection is associated with insulin resistance and lipid profiles. 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81 Fierro NA et al. Immunity, metabolism, genetics and HCV 122 Barquera S, Flores M, Olaiz-Fernández G, Monterrubio E, Villalpando S, González C, Rivera JÁ, Sepúlveda J. Dyslipidemias and obesity in Mexico. Salud Publica Mex 2007; 49 Suppl 3: S338-S347 Available from: URL: scielo.org.mx/pdf/spm/v49s3/04.pdf 123 Panduro A, Roman S, Khan A, Tanaka Y, Kurbanov F, Martinez-Lopez E, Campollo O, Hernandez-Nazara Z, Mizokami M. Molecular epidemiology of hepatitis C virus genotypes in west Mexico. Virus Res 2010; 151: [PMID: DOI: /j.virusres ] 124 Panduro A, Escobedo Meléndez G, Fierro NA, Ruiz Madrigal B, Zepeda-Carrillo EA, Román S. [Epidemiology of viral hepatitis in Mexico]. Salud Publica Mex 2011; 53 Suppl 1: S37-S45 [PMID: ] 125 Roman S, Fierro N, Moreno-Luna LE, Panduro A. Hepatitis B virus genotype H and environmental factors associated to the low prevalence of hepatocellular carcinoma in Mexico. J Cancer Ther 2013; 4: [DOI: /jct A044] 126 Cheng Y, Dharancy S, Malapel M, Desreumaux P. Hepatitis C virus infection down-regulates the expression of peroxisome proliferator-activated receptor alpha and carnitine palmitoyl acyl-coa transferase 1A. World J Gastroenterol 2005; 11: [PMID: ] 127 Yasui K, Harano Y, Mitsuyoshi H, Tsuji K, Endo M, Nakajima T, Minami M, Itoh Y, Zen Y, Nakanuma Y, Yoshikawa T, Okanoue T. Steatosis and hepatic expression of genes regulating lipid metabolism in Japanese patients infected with hepatitis C virus. J Gastroenterol 2010; 45: [PMID: DOI: /s ] 128 Perlemuter G, Sabile A, Letteron P, Vona G, Topilco A, Chrétien Y, Koike K, Pessayre D, Chapman J, Barba G, Bréchot C. Hepatitis C virus core protein inhibits microsomal triglyceride transfer protein activity and very low density lipoprotein secretion: a model of viral-related steatosis. FASEB J 2002; 16: [PMID: DOI: / fj com] 129 Qadri I, Choudhury M, Rahman SM, Knotts TA, Janssen RC, Schaack J, Iwahashi M, Puljak L, Simon FR, Kilic G, Fitz JG, Friedman JE. Increased phosphoenolpyruvate carboxykinase gene expression and steatosis during hepatitis C virus subgenome replication: role of nonstructural component 5A and CCAAT/enhancer-binding protein β. J Biol Chem 2012; 287: [PMID: DOI: / jbc.m ] 130 Thomas DL, Thio CL, Martin MP, Qi Y, Ge D, O Huigin C, Kidd J, Kidd K, Khakoo SI, Alexander G, Goedert JJ, Kirk GD, Donfield SM, Rosen HR, Tobler LH, Busch MP, McHutchison JG, Goldstein DB, Carrington M. Genetic variation in IL28B and spontaneous clearance of hepatitis C virus. Nature 2009; 461: [PMID: DOI: /nature08463] 131 Grebely J, Petoumenos K, Hellard M, Matthews GV, Suppiah V, Applegate T, Yeung B, Marks P, Rawlinson W, Lloyd AR, Booth D, Kaldor JM, George J, Dore GJ. Potential role for interleukin-28b genotype in treatment decision-making in recent hepatitis C virus infection. Hepatology 2010; 52: [PMID: DOI: /hep.23850] 132 Prokunina-Olsson L, Muchmore B, Tang W, Pfeiffer RM, Park H, Dickensheets H, Hergott D, Porter-Gill P, Mumy A, Kohaar I, Chen S, Brand N, Tarway M, Liu L, Sheikh F, Astemborski J, Bonkovsky HL, Edlin BR, Howell CD, Morgan TR, Thomas DL, Rehermann B, Donnelly RP, O Brien TR. A variant upstream of IFNL3 (IL28B) creating a new interferon gene IFNL4 is associated with impaired clearance of hepatitis C virus. Nat Genet 2013; 45: [PMID: DOI: /ng.2521] 133 Rivero-Juarez A, Gonzalez R, Camacho A, Manzanares- Martin B, Caruz A, Martinez-Peinado A, Torre-Cisneros J, Pineda JA, Peña J, Rivero A. Natural killer KIR3DS1 is closely associated with HCV viral clearance and sustained virological response in HIV/HCV patients. PLoS One 2013; 8: e61992 [PMID: DOI: /journal.pone ] 134 Lio D, Caruso C, Di Stefano R, Colonna Romano G, Ferraro D, Scola L, Crivello A, Licata A, Valenza LM, Candore G, Craxì A, Almasio PL. IL-10 and TNF-alpha polymorphisms and the recovery from HCV infection. Hum Immunol 2003; 64: [PMID: ] 135 Zhu C, Zhang R, Liu D, Mukhtar MM, Liu W, Peng G, Wang K, Hao Q, Xu Y, Liu F, Zhu Y, Wu J. Association of functional polymorphism of ApoB promoter with hepatitis C virus infection. Clin Chim Acta 2009; 401: [PMID: DOI: /j.cca ] P- Reviewers: Jin B, Sener A S- Editor: Gou SX L- Editor: A E- Editor: Ma S 3456 April 7, 2014 Volume 20 Issue 13

82 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (2): Hepatitis C virus TOPIC HIGHLIGHT How hepatitis C virus invades hepatocytes: The mystery of viral entry Yong-Zhe Zhu, Xi-Jing Qian, Ping Zhao, Zhong-Tian Qi Yong-Zhe Zhu, Xi-Jing Qian, Ping Zhao, Zhong-Tian Qi, Department of Microbiology, Shanghai Key Laboratory of Medical Biodefense, Second Military Medical University, Shanghai , China Author contributions: All authors contributed to this work. Supported by Research Grants from National S and T Major Project for Infectious Diseases Control, No. 2012ZX ; National Natural Science Foundation of China, No , No and No ; Medical Youth Science Program, No. 13QNP100; Shanghai Municipal Natural Science Foundation, No. 13ZR ; Shanghai LAD Project, No. B901 Correspondence to: Zhong-Tian Qi, PhD, Department of Microbiology, Shanghai Key Laboratory of Medical Biodefense, Second Military Medical University, 800 Xiangyin Road, Shanghai , China. qizt@smmu.edu.cn Telephone: Fax: Received: September 28, 2011 Revised: December 15, 2013 Accepted: January 3, 2014 Published online: April 7, 2014 Abstract Hepatitis C virus (HCV) infection is a global health problem, with an estimated 170 million people being chronically infected. HCV cell entry is a complex multi-step process, involving several cellular factors that trigger virus uptake into the hepatocytes. The high- density lipoprotein receptor scavenger receptor class B type I, tetraspanin CD81, tight junction protein claudin-1, and occludin are the main receptors that mediate the initial step of HCV infection. In addition, the virus uses cell receptor tyrosine kinases as entry regulators, such as epidermal growth factor receptor and ephrin receptor A2. This review summarizes the current understanding about how cell surface molecules are involved in HCV attachment, internalization, and membrane fusion, and how host cell kinases regulate virus entry. The advances of the potential antiviral agents targeting this process are introduced Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Hepatitis C virus; Virus entry; Hepatocytes; Receptor; Host kinase; Antiviral target Core tip: Cell entry is the first step in viral infection and replication, which offers an important target for antiviral therapy. Hepatitis C virus (HCV) cell entry is a complex multi-step process, involving a several cellular factors that trigger virus uptake into the hepatocytes. This review summarizes the current understanding about how cell surface molecules are involved in HCV attachment, internalization, and membrane fusion, and how host cell kinases regulate virus entry. The advances of the potential antiviral agents targeting this process are introduced. Zhu YZ, Qian XJ, Zhao P, Qi ZT. How hepatitis C virus invades hepatocytes: The mystery of viral entry. World J Gastroenterol 2014; 20(13): Available from: URL: DOI: org/ /wjg.v20.i INTRODUCTION Hepatitis C virus (HCV) infection is a global health problem. About million people are chronically infected and at risk of developing liver cirrhosis and/or liver cancer [1]. More than people die from hepatitis-crelated liver diseases every year. Current antiviral treatment of chronic HCV infection is based on pegylated interferon (PEG-IFN) and ribavirin, which is limited by side effects and suboptimal response rates [2]. Since 2011, standard of care (SOC) treatment of HCV includes the addition of direct-acting antivirals with a protease inhibitor to the PEG-IFN and ribavirin [3,4]. However, resistance and severe side effects are still issues [5]. There is currently no vaccine for hepatitis C. Thus, novel preventive and therapeutic strategies are urgently needed. Cell entry is the first step in viral infection and replica April 7, 2014 Volume 20 Issue 13

83 Zhu YZ et al. HCV cell entry tion, which offers an important target for antiviral therapy. The establishment of infectious HCV pseudoparticles (HCVpps) [6,7], cell-culture-derived HCV (HCVcc) [8-10], and progress in small animal models of HCV infection [11-14] have allowed us to identify host factors contributing to HCV entry and to develop antiviral compounds. HCV entry into hepatocytes is thought to be a highly coordinated process that involves several cell surface molecules in sequential steps [15]. More than two decades of intense research has provided a detailed understanding of these entry factors, and considerable progress has been made to decipher how they trigger and facilitate HCV cell entry. Recent data also demonstrate the important role of host cell tyrosine protein kinases in regulating HCV entry [16]. In this review, we summarize the current knowledge about how HCV uses these host factors to invade hepatocytes, and how host cell kinases regulate virus entry, and the recent progress of antiviral therapy targeting this process. HCV PARTICLES HCV is an enveloped, positive-stranded RNA virus belonging to the Hepacivirus in the Flaviviridae family. The HCV virion is comprised of a nucleocapsid surrounded by a host-derived membrane containing the E1 and E2 HCV glycoproteins [17]. Functional virion-associated E1 and E2 are thought to form a non-covalent heterodimer stabilized by disulfide bridges [18,19], which mediate the majority of cell-entry processes of the virion, including interaction with host receptors and fusion in the low ph environment of early endosomes [20-23]. The first 27 amino acids residues of the N terminus of E2, called hypervariable region 1 (HVR1), are the most divergent among HCV isolates. Recent data show that HVR1 contains three different functional microdomains that cooperate to confer HCV cell entry and immune evasion [24]. Based on similarities with envelope proteins of the Flaviviridae family, E2 was previously proposed to be the fusion protein [25-27]. However, recent data on pestiviruses and HCV are no longer in favor of E2 being a fusion protein. The structure of E2 protein from the pestivirus bovine viral diarrhea virus does not show any evidence of a fusion peptide in this protein [28,29]. More recently, the crystal structure of the E2 core bound to broadly neutralizing antibody AR3C was resolved and revealed a compact architecture composed of a central immunoglobulin-fold β sandwich flanked by two additional protein layers, which differs markedly from predictions of an extended, threedomain, class Ⅱ fusion protein fold [25]. E1 was recently demonstrated to be a modulator of HCV binding to receptors and membrane fusion [30]. The characterization of HCV particles circulating in patient sera is the heterogeneity of size and density, due to the association of virions with different serum components, such as immunoglobulins [31] and different classes of lipoproteins [32].The nature of the association between HCV and lipoproteins remains unclear. Circulating virions might directly bind to low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) in the blood, or interact with lipoprotein components during virus assembly and are secreted together with VLDL [33]. Highly infectious HCV particles are associated with VLDL, termed lipoviral particles (LVPs). LVPs are lipoprotein-like structures composed of triglyceride-rich lipoproteins containing apolipoprotein (Apo)B, ApoE and ApoC1, viral nucleocapsids, and envelope glycoproteins E1 and E2 [34-38]. Several functional roles for the formation of HCV-lipoprotein complexes have been proposed, including interacting with lipoprotein receptors for attachment and entry, concealing epitopes to facilitate immune escape, and hijacking host factors for HCV maturation and secretion [39]. PARTICIPANTS AND THE CELL-ENTRY PATHWAY The HCV entry process into human hepatocytes requires numerous host proteins, including two binding factors glycosaminoglycans (GAGs) [40,41] and the LDL receptor (LDLR) [42-45], four receptors scavenger receptor class B type Ⅰ (SR-BI) [46], tetraspanin CD81 [47], tight junction proteins, claudin-1 (CLDN1) [48] and occludin (OCLN) [49], and several entry factors including epidermal growth factor receptor (EGFR), ephrin receptor A2 (EphA2) [50], transferrin receptor 1 (TfR1) [51], and cholesterol transporter Niemann-Pick C1-like 1 (NPC1L1) [52] (Table 1). The entry pathway consists of three steps: (1) viral attachment to the hepatocyte; (2) receptor-mediated endocytosis of the viral particle; and (3) endosomal fusion (Figure 1). ATTACHMENT OF HCV PARTICLES TO THE CELL SURFACE In vivo, circulating HCV enters the liver through the sinusoidal blood. The liver sinusoidal endothelial cells (LSECs) and Kupffer cells express liver/lymph-node-specific intercellular adhesion molecule 3-grabbing integrin (L- SIGN) and dendritic-cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN), respectively. Both L-SIGN and DC-SIGN bind HCV E2 with high affinity, suggesting they may capture particles from blood and transmit virus to hepatocytes [53,54]. Successful traverse from the endothelium allows the virus to contact hepatocytes at the basal surface. GAGs and LDLR are proposed to contribute to concentrate HCV particles on target cell surface [41,44]. On hepatocytes, highly sulfated GAGs (HS GAGs) serve as the first attachment sites [55]. While direct E2 binding to HS GAGs is observed [55], E1 and ApoE are also suggested to attach to GAGs [56]. As a result of the association between HCV and lipoproteins, the LDLR has been proposed as another potential attachment factor for HCV [43]. Although HCV particles bind to the LDLR, it seems that this interaction does not lead to a productive entry process [57] April 7, 2014 Volume 20 Issue 13

84 Zhu YZ et al. HCV cell entry Table 1 Characterization and the main function of cell surface molecules required for hepatitis C virus entry ENDOCYTOSIS MEDIATED BY CO-RECEPTORS AND SPECIFIC ENTRY FACTORS Characterization Main function in HCV entry GAGs Binding factor Attachment to HCV E1 and ApoE LDL-R Binding factor Binding to virion-associated lipoprotein SR-BI Receptor Binding to virion-associated lipoprotein; lipid transfer; binding to E2 HVR1 CD81 Receptor Interaction with E2; signaling pathway activation which promotes actin remodeling CLDN1 Receptor Interaction with HCV-CD81 complex OCLN Receptor Function at the late stage of HCV entry EGFR and EphA2 Entry factor Signaling pathways activation which lead to the formation of CD81-CLDN1 complexes TfR1 Entry factor Possibly involved in virus internalization NPC1L1 Entry factor Possibly involved in endosomal fusion HCV: Hepatitis C virus; GAGs: Glycosaminoglycans; LDL: Low-density lipoprotein; CLDN1: Claudin-1; OCLN: Occludin; EGFR: Epidermal growth factor receptor; EphA2: Ephrin receptor A2; TfR1: Transferrin receptor 1; NPC1L1: Niemann-Pick C1-like 1. After initial attachment to the host cell surface, the virus interacts with co-receptors and other specific entry factors, which leads to molecular rearrangements at the plasma membrane and subsequently results in viral internalization. SR-BI is highly expressed in the liver and steroidogenic tissues [58], can bind a variety of lipoproteins including high-density lipoprotein (HDL), LDL, and oxidized LDL [59], and plays a key role in mediating selective cholesterol uptake from HDL into hepatocytes to maintain lipid homeostasis [60]. SR-BI is a glycoprotein with two N- and C-terminal cytoplasmic domains separated by a large extracellular domain that is involved in lipid metabolism. The large extracellular loop of SR-BI was initially identified to bind directly to the HVR1 of HCV E2 [61], and then interact with virus-associated lipoproteins [62]. Recent data show that HCV particles utilize SR-BI in a manifold manner [63]. First, the initial attachment of HCV particles to SR-BI is independent of E2 but, rather, is mediated by lipoprotein components, such as ApoE [63,64]. Second, the lipid transfer function of SR-BI may facilitate the viral particles access to the next entry step, which would allow exposure of CD81 binding sites on HCV E2 and transfer the virus particle to CD81 [63]. Finally, direct interactions between E2 HVR1 and SR-BI enhance infectivity of the particle at post-attachment levels [65]. CD81 is a member of the tetraspanin superfamily defined by four transmembrane domains, a conserved CCG motif, and four cysteine residues that form critical disulfide bonds in the large extracellular loop (LEL). CD81 interacts with HCV E2 glycoprotein via a series of discontinuous amino acid residues in the LEL [66]. Several studies suggest that CD81 acts as a post-binding entry molecule. HCV- CD81 interaction induces a conformational rearrangement of the E1 and E2 glycoproteins that facilitates ph-dependent fusion and endocytosis of the virus [67]. Upon CD81 engagement, HCV activates signals allowing the lateral movement of the virus particle/cd81 complex and its delivery to areas of cell-cell contact [68]. Tetraspanins associate with each other to carry out many biological functions, and several CD81 partners and CD81-associated tetraspanins are proposed to play a role in HCV entry [69-71]. CLDN1 is a member of the claudin family of tight junction proteins, consisting of a large and small extracellular loop (EL1 and EL2, respectively), and four transmembrane domains. The highly conserved EL1 is critical for HCV entry [48]. CLDN6 and CLDN9 can replace CLDN1 for HCV entry, but they are expressed only at low levels in the liver [72]. CD81 and CLDN1 act at closely related time-points during HCV entry [73], and direct CD81-CLDN1 interaction is observed [74], strongly suggesting they form a complex to facilitate virus entry. The HCV envelope glycoproteins do not directly interact with CLDN1, but CLDN1 interacts with CD81 and thereby plays an important role during post-binding steps of the HCV entry process [48,73,74]. CLDN1 is expressed both at the basolateral and apical membranes [75]. Interestingly, CLDN1- CD81 complexes are absent from apically located tight junctions, suggesting that virus engagement of basolateral pools of CD81 and CLDN1 may initiate the particle internalization process [75]. EGFR is a receptor tyrosine kinase (RTK) that regulates several key processes, including cell proliferation, survival, and differentiation during development, tissue homeostasis and tumorigenesis [76]. EphA2 mediates cell positioning, cell morphology, polarity and motility [77]. EGFR and EphA2 were recently identified to be required for HCV entry [50] and modulate interactions between CD81 and CLDN1 by EGFR-dependent signaling pathways [78]. OCLN, also belongs to the tight junction protein family, and has been identified as another co-receptor required for a late post-binding event [49]. OCLN is composed of four transmembrane regions, two extracellular loops and N- and C-terminal cytoplasmic regions [79]. The determinants on OCLN of HCV infectivity and species specificity are mapped to the second extracellular loop [80]. Although OCLN has been reported to interact with the HCV E2 glycoproteins [81], whether the interaction is mediated via a direct E2-OCLN binding or by indirect interactions with CD81/CLDN1 complexes is unclear. Internalization of HCV particles is dependent on clathrin-mediated endocytosis [22]. HCV promotes CD81 endocytosis via a clathrin- and dynamin-dependent process in association with CLDN1 [82]. Recently, TfR1 was identified as a HCV entry factor [51]. TfR1 is ubiquitously expressed in all tissues and is the main receptor for cellular iron uptake into cells via clathrin-mediated endocytosis. TfR1 plays a role in HCV infection at the level of glycoprotein-mediated entry, acts after CD81, and possibly is involved in HCV particle internalization [51] April 7, 2014 Volume 20 Issue 13

85 Zhu YZ et al. HCV cell entry 1 HCV lipoviral particle ApoC1 3 TfR1 2 CLDN1 CD81 SRB1 ApoE LDLR GAGs ApoB EGFR EphA2 PI4K PI3K + Actin Grb2/Shc1 CLDN1 Clathrin CLDN1? AKT Rho GTPase camp HRas OCLN OCLN H + H PKA Raf MEK MKNK1 Bile canaliculus NPC1L1 Early endosome ERK? Cholesterol Viral fusion Figure 1 Hepatitis C virus entry into hepatocytes. 1 Hepatitis C virus (HCV) LVP binds to glycosaminoglycans (GAGs) and low-density lipoprotein receptor (LDLR), which have high affinity with ApoE. SRBI plays an important role in both binding and post-binding steps through interaction with virion-associated lipoprotein or HCV E2. 2 Binding of SRBI to HCV particles allows exposure of CD81 binding sites on HCV E2 and transfer of the virus particles to CD81. Upon interaction with HCV E2, CD81 activates Rho GTPase family members that promote actin remodeling, thus allowing the delivery of the E2-CD81 complex to come into contact with claudin-1 (CLDN1). Two RTKs, epidermal growth factor receptor (EGFR) and EphA2 facilitate the formation of the CD81-CLDN1 complex by the EGFR-activated EGFR/Shc1/Grb2/HRas signaling pathway. The CD81-CLDN1 association is also regulated by PKA in a camp-dependent manner to retain CLDN1 on the plasma membrane. MKNK1 was recently identified as a host factor in HCV entry, which possibly acts to facilitate the downstream of EGFR. 3 The virion is primed by the low-ph fusion activity of CD81 and CLDN1 and translocates to the tight junctions in order to be endocytosed. Viral internalization is dependent on clathrin-mediated endocytosis. Junction protein occludin (OCLN) may contribute to this process. TfR1 plays a role in HCV infection at the level of glycoprotein-mediated entry, acts after CD81, and possibly is involved in HCV particle internalization. PI3K-AKT and PI4K pathways are engaged in the late step of HCV entry. However the mechanisms are yet to investigated. 4 Following internalization, HCV fusion occurs in the early endosomes. Low ph environment and virion-associated cholesterol are required for the fusion process. NPC1L1 may play a role in the process by cholesterol transport. After fusion between the viral envelope and an endosomal membrane, the viral genome is released into the cytosol and replication take place. ENDOSOMAL FUSION The fusion within the endosomal cell compartment between viral and host membranes is the final step of HCV entry to release the viral RNA for the following HCV life cycle. It is believed that this process is triggered in a receptor-independent but ph-dependent manner, and is closely linked with lipid composition [83]. The virus-receptor interactions and low ph might cause glycoprotein rearrangements to trigger fusion-related protein transition from a pre-fusion state to a post-fusion structure [84]. Of the two envelope protein, E1 acts as a protein chaperone. It has been shown that specific residues of fusion-peptide-like domain on E1 are required for mediating cell fusion and entry of HCV [27,85,86]. Still, the role of this domain in HCV entry needs further investigation. However, the role of E2 in viral fusion is controversial. Previous studies have suggested that a specific region of E2 is a key fusion determinant of HCV [26,27], while Law s team has found no presence of a fusion peptide in HCV E2 by crystal structure analysis [25]. The conformational changes of the envelope proteins finally lead to a fusion pore to release the nucleocapsid into the cytosol [87,88]. Besides the acidic endosomal ph environment, the cholesterol of the target membranes is also found to have a strong promoting effect on the membrane fusion capacity of flaviviruses [89-91]. The NPC1L1 cholesterol uptake receptor has been identified recently to be one of key entry factors for HCV [52]. It has been revealed that it might promote HCV entry by modulating cholesterol homeostasis or influencing cholesterol level of LVPs, thus affecting membrane composition required for membrane fusion [92]. REGULATION OF HCV ENTRY BY HOST KINASES It has been recently demonstrated that HCV uses host kinases during the entry process to facilitate virus-receptor interaction, and appropriate membrane traffic. There is strong evidence that CD81 and CLDN-1 work together to facilitate HCV entry. The association of CD81 and CLDN-1 appears to be regulated by mul April 7, 2014 Volume 20 Issue 13

86 Zhu YZ et al. HCV cell entry Table 2 Antiviral agents targeting viral entry Stages of HCV entry process HCV particle/ attachment Receptormediated endocytosis Endosomal fusion Target Antiviral agents Ref. HCV E1 and E2 Neutralizing antibodies [102,103] Heparin [41,55] Lectins [106,107] EGCG [109,110] Oleanane-type [111] triterpenes Virion-associated lipoprotein Anti-apoE mab [108] SRBI Anti-SR-BI mab [113] ITX 5061 [117] CD81 Anti-CD81 mab [114] CLDN1 Anti-CLDN1mAb [115] CLDN1-derived peptide [116] EGFR Erlotinib [50] NPC1L1 Ezetimibe [52] TfR1 Anti-TfR1 mab [51] Internalization Amphipathic DNA [118] polymers Arbidol [122] Tamoxifen [123] Fusion E2-derived peptide [121] CD81-derived peptide [124] Curcumin [125] Phenothiazines [126] Ferroquine [127] auy11 [128] HCV Ⅱ-1/GS [129] Regulation Ras Tipifarnib [78] PKA H89 [96] MKNK1 RO [93] PI3K Wortmanin [99] Targets within the different stages of Hepatitis C virus (HCV) entry process are depicted, followed by examples of compounds targeting the respective entry step. Stages of development and references are indicated. GAGs: Glycosaminoglycans; CLDN1: Claudin-1; OCLN: Occludin; LDLR: Lowdensity lipoprotein receptor; EGFR: Epidermal growth factor receptor. tiple signaling pathways. Using a functional RNAi kinase screen, two RTKs, EGFR and EphA2, are identified as host cofactors for HCV entry by prompting CD81- CLDN1 complex formation [50]. EGFR regulates CD81- CLDN1 co-receptor association by the activation of the EGFR/Shc1/Grb2/HRas signaling pathway [78]. Proteomic analysis has revealed that HRas associates with CD81 and CLDN1, suggesting HRas acts as a molecular switch promoting RTK-mediated HCV entry [78]. Following EGFR stimulation, the Ras/MEK/ERK pathway is activated, which could lead to the activation of MAPK interacting serine/threonine kinase 1 (MKNK1) [93]. MKNK1 was recently identified as a host factor in HCV entry, which possibly acts to facilitate the downstream effect of EGFR [93]. For a multi-receptor virus, successful entry is based on the lateral movement of the virus on the plasma membrane and interaction with sufficient receptors to initiate internalization [94]. In the case of HCV, after the interaction with HCV E2, CD81 activates Rho GTPase family members Rac, Rho and cell division cycle 42 that promote actin remodeling, thus allowing the delivery of the E2-CD81 complex to come into contact with CLDN1 [68]. If the localization of either co-receptor is disrupted, the association of CD81 with CLDN1 will be perturbed [95]. By screening a series of kinase inhibitors for their effects on HCV infection, protein kinase A (PKA) was identified as having an important role in HCV entry [96]. Initiation of HCV infection activates PKA in a camp-dependent manner to retain CLDN1 on the plasma membrane, which then promotes the entry of HCV [96]. The class Ⅰ phosphatidylinositol 3-kinase (PI3K) is activated by G-protein-coupled receptors and tyrosine kinase receptors. Upon its activation, it converts phosphatidylinositol 4,5-biphosphate to phosphatidylinositol 3,4,5-triphosphate, which binds to and recruits AKT to the membrane for its phosphorylation. Many flaviviruses regulate the PI3K-AKT pathway for their entry [97,98]. HCV rapidly and transiently activates the PI3K-AKT pathway in the early stage of infection to enhance its entry, and the activation is mediated by the interaction between HCV E2 and its co-receptors, CD81 and CLDN1 [99]. However, the exact step of the entry process that the AKT pathway regulates remains elusive. In addition, phosphatidylinositol 4-kinases type Ⅲ α (PI4KⅢα) and β (PI4KⅢβ) have been suggested to play a role in membrane remodeling and trafficking during HCV entry [100,101]. However, the underlying molecular mechanism is unclear. HCV ENTRY AS AN ANTIVIRAL TARGET HCV entry has been revealed as a highly complex process requiring the involvement of viral envelope proteins and multiple host proteins, which offer a large number of promising therapeutic targets (Table 2). Each stage of HCV entry can be designed as an antiviral target, including virus particles, virus attachment, receptor-mediated endocytosis, endosomal fusion, and the regulatory pathways. HCV entry can be inhibited with monoclonal or polyclonal neutralizing antibodies [ ]. However, crossreactive neutralizing antibodies seem to appear later during HCV infection, and several mechanisms contribute to reduce their accessibility to their cognate epitopes. These include the masking of major conserved neutralizing epitopes by HVR1, specific N-linked glycans, and the lipid moiety of the viral particle. Other potential mechanisms of evasion from the neutralizing antibody response include a modulation by HDLs and interfering antibodies, as well as the capacity of the virus to be transferred by cell-to-cell contact [105]. Several non-hcv specific molecules interfering with HCV envelope glycoproteins and abrogating viral attachment have been described. Heparin, a structural analog of GAGs, has been demonstrated to inhibit HCV entry [41,55]. Lectins, such as cyanovirin-n and griffithsin, bind to the high-mannose glycans present on the HCV particles and thereby inhibit HCV entry [106,107]. ApoE antibodies and peptides are able to inhibit HCV entry by 3461 April 7, 2014 Volume 20 Issue 13

87 Zhu YZ et al. HCV cell entry blocking its binding to target cells [108]. Some natural products have been shown to prevent virus binding, such as the green tea polyphenol epigallocatechin-3-gallate [109,110] and oleanane-type trierpene [111]. To overcome the high variability of the viral envelope proteins, small-molecule host-targeting agents (HTAs) are also being investigated [112]. Antibodies or peptides targeting CD81, SR-B1 and CLDN1 have been shown to be an efficacious way to prevent HCV infection both in vitro and in vivo in a genotype-independent manner [ ]. ITX 5061, one of the most promising HTAs, which promotes HDL levels in animals and patients by targeting SRB1 [117], is currently in phase 2 clinical trials. Clathrindependent endocytosis [ ] and endosomal fusion [121] are also potential targets for the development of anti- HCV compounds. Arbidol suppresses clathrin-mediated endocytosis by hindering HCV endosomal trafficking and impairing dynamin-2-induced membrane scission [122]. Tamoxifen is a selective estrogen receptor modulator, and affects both viral binding and post-binding events including endocytosis [123]. The stapled peptides are designed according to the linear peptide sequence of the large extracellular loop of CD81, which has been reported to play an important role in HCV E2 binding interaction, and has an inhibitory effect on HCV membrane fusion [124]. Curcumin affects membrane fluidity, therefore impeding viral binding and fusion [125]. Phenothiazines inhibit HCV infection at an early stage of the viral cycle by interfering with virion-cell fusion [126]. They insert into cholesterolrich domains of target membranes and perturb cholesterol distribution in lipid membranes, creating a barrier to virion-cell fusion. Ferroquine, an anolog of chloroquine, suppresses HCV infection at a late post-binding step by impairing the fusion process [127]. An arabino-based rigid amphipathic fusion inhibitor, auy11, inhibits HCV infection at the early fusion process by interacting with envelope lipids [128]. HCV-II/GS suppresses HCV endosomal fusion by locking the viral envelope prefusion state or formation of an incapable envelope conformation for viral fusion [129]. Given the relevance of host cell kinases for HCV entry and the number of kinase inhibitors being developed to treat a wide variety of human diseases, kinase inhibitors have been suggested as a novel class of antivirals for the prevention and treatment of HCV infection [78,93,96,99]. Erlotinib, a clinically approved inhibitor of EGFR, and the NPC1L1 inhibitor ezetimibe have been shown to impair HCV infection in vivo [50,52]. CONCLUSION The importance of HCV entry owes to its critical role in the initiation of infection, the major target of immune responses and the determination of tissue and species tropism [24]. In recent years, substantial progress has been made to decipher the mystery of HCV entry. Current data support a complex interplay between the HCVencoded glycoproteins E1 and E2 and four receptors - SR-BI, CD81, and tight junction proteins CLDN1 and OCLN in defining HCV entry. EGFR, NPC1L1 and TfR1 are important cofactors for HCV entry. HCV entry has been revealed as a highly complex process requiring orchestration of several cell surface molecules and regulation of multiple host kinases, which offer a multitude of promising targets for antivirals. The ideal therapeutic regimen is thought to be an alloral, IFN-free combination cocktail with pan-genotype coverage, minimal side effects and high virological cure rates in all patient groups [1]. Given the chronic nature and genetic diversity of HCV, targeting host entry factors with antibodies or small-molecule inhibitors could block the spread of DAA-resistant variants and disturb infection dynamics necessary to maintain chronic liver infection. The most advanced inhibitor of HCV entry is ITX 5061, a small molecule compound that was initially identified to promote HDL levels in animals and patients by targeting SRBI. ACKNOWLEDGMENTS We are grateful to all of our colleagues for their help over the years and apologize if we have missed citing some articles due to space restrictions. REFERENCES 1 Scheel TK, Rice CM. Understanding the hepatitis C virus life cycle paves the way for highly effective therapies. Nat Med 2013; 19: [PMID: DOI: /nm.3248] 2 Manns MP, Wedemeyer H, Cornberg M. Treating viral hepatitis C: efficacy, side effects, and complications. 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90 Zhu YZ et al. HCV cell entry 66 Flint M, von Hahn T, Zhang J, Farquhar M, Jones CT, Balfe P, Rice CM, McKeating JA. Diverse CD81 proteins support hepatitis C virus infection. J Virol 2006; 80: [PMID: DOI: /JVI ] 67 Sharma NR, Mateu G, Dreux M, Grakoui A, Cosset FL, Melikyan GB. Hepatitis C virus is primed by CD81 protein for low ph-dependent fusion. J Biol Chem 2011; 286: [PMID: DOI: /jbc.M ] 68 Brazzoli M, Bianchi A, Filippini S, Weiner A, Zhu Q, Pizza M, Crotta S. CD81 is a central regulator of cellular events required for hepatitis C virus infection of human hepatocytes. J Virol 2008; 82: [PMID: DOI: / JVI ] 69 Rocha-Perugini V, Montpellier C, Delgrange D, Wychowski C, Helle F, Pillez A, Drobecq H, Le Naour F, Charrin S, Levy S, Rubinstein E, Dubuisson J, Cocquerel L. The CD81 partner EWI-2wint inhibits hepatitis C virus entry. 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91 Zhu YZ et al. HCV cell entry DOI: /JVI ] 97 Lee CJ, Liao CL, Lin YL. Flavivirus activates phosphatidylinositol 3-kinase signaling to block caspase-dependent apoptotic cell death at the early stage of virus infection. J Virol 2005; 79: [PMID: DOI: /JVI ] 98 Zhu YZ, Cao MM, Wang WB, Wang W, Ren H, Zhao P, Qi ZT. Association of heat-shock protein 70 with lipid rafts is required for Japanese encephalitis virus infection in Huh7 cells. J Gen Virol 2012; 93: [PMID: DOI: /vir ] 99 Liu Z, Tian Y, Machida K, Lai MM, Luo G, Foung SK, Ou JH. Transient activation of the PI3K-AKT pathway by hepatitis C virus to enhance viral entry. J Biol Chem 2012; 287: [PMID: DOI: /jbc.M ] 100 Trotard M, Lepère-Douard C, Régeard M, Piquet-Pellorce C, Lavillette D, Cosset FL, Gripon P, Le Seyec J. Kinases required in hepatitis C virus entry and replication highlighted by small interference RNA screening. 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A human monoclonal antibody targeting scavenger receptor class B type I precludes hepatitis C virus infection and viral spread in vitro and in vivo. Hepatology 2012; 55: [PMID: DOI: / hep.24692] 114 Meuleman P, Hesselgesser J, Paulson M, Vanwolleghem T, Desombere I, Reiser H, Leroux-Roels G. Anti-CD81 antibodies can prevent a hepatitis C virus infection in vivo. Hepatology 2008; 48: [PMID: DOI: / hep.22547] 115 Fofana I, Krieger SE, Grunert F, Glauben S, Xiao F, Fafi-Kremer S, Soulier E, Royer C, Thumann C, Mee CJ, McKeating JA, Dragic T, Pessaux P, Stoll-Keller F, Schuster C, Thompson J, Baumert TF. Monoclonal anti-claudin 1 antibodies prevent hepatitis C virus infection of primary human hepatocytes. Gastroenterology 2010; 139: , 964.e1-4 [PMID: DOI: /j.gastro ] 116 Si Y, Liu S, Liu X, Jacobs JL, Cheng M, Niu Y, Jin Q, Wang T, Yang W. A human claudin-1-derived peptide inhibits hepatitis C virus entry. 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Proc Natl Acad Sci USA 2010; 107: [PMID: DOI: /pnas ] 121 Liu R, Tewari M, Kong R, Zhang R, Ingravallo P, Ralston R. A peptide derived from hepatitis C virus E2 envelope protein inhibits a post-binding step in HCV entry. Antiviral Res 2010; 86: [PMID: DOI: / j.antiviral ] 122 Blaising J, Lévy PL, Polyak SJ, Stanifer M, Boulant S, Pécheur EI. Arbidol inhibits viral entry by interfering with clathrindependent trafficking. Antiviral Res 2013; 100: [PMID: DOI: /j.antiviral ] 123 Murakami Y, Fukasawa M, Kaneko Y, Suzuki T, Wakita T, Fukazawa H. Selective estrogen receptor modulators inhibit hepatitis C virus infection at multiple steps of the virus life cycle. Microbes Infect 2013; 15: [PMID: DOI: /j.micinf ] 124 Cui HK, Qing J, Guo Y, Wang YJ, Cui LJ, He TH, Zhang L, Liu L. Stapled peptide-based membrane fusion inhibitors of hepatitis C virus. Bioorg Med Chem 2013; 21: [PMID: DOI: /j.bmc ] 125 Anggakusuma, Colpitts CC, Schang LM, Rachmawati H, 3466 April 7, 2014 Volume 20 Issue 13

92 Zhu YZ et al. HCV cell entry Frentzen A, Pfaender S, Behrendt P, Brown RJ, Bankwitz D, Steinmann J, Ott M, Meuleman P, Rice CM, Ploss A, Pietschmann T, Steinmann E. Turmeric curcumin inhibits entry of all hepatitis C virus genotypes into human liver cells. Gut 2013; Epub ahead of print [PMID: DOI: /gutjnl ] 126 Chamoun-Emanuelli AM, Pecheur EI, Simeon RL, Huang D, Cremer PS, Chen Z. Phenothiazines inhibit hepatitis C virus entry, likely by increasing the fluidity of cholesterol-rich membranes. Antimicrob Agents Chemother 2013; 57: [PMID: DOI: /AAC ] 127 Vausselin T, Calland N, Belouzard S, Descamps V, Douam F, Helle F, François C, Lavillette D, Duverlie G, Wahid A, Fénéant L, Cocquerel L, Guérardel Y, Wychowski C, Biot C, Dubuisson J. The antimalarial ferroquine is an inhibitor of hepatitis C virus. Hepatology 2013; 58: [PMID: DOI: /hep.26273] 128 Colpitts CC, Ustinov AV, Epand RF, Epand RM, Korshun VA, Schang LM. 5-(Perylen-3-yl)ethynyl-arabino-uridine (auy11), an arabino-based rigid amphipathic fusion inhibitor, targets virion envelope lipids to inhibit fusion of influenza virus, hepatitis C virus, and other enveloped viruses. J Virol 2013; 87: [PMID: DOI: / JVI ] 129 Bush CO, Pokrovskii MV, Saito R, Morganelli P, Canales E, Clarke MO, Lazerwith SE, Golde J, Reid BG, Babaoglu K, Pagratis N, Zhong W, Delaney WE, Paulson MS, Beran RK. A small-molecule inhibitor of hepatitis C virus infectivity. Antimicrob Agents Chemother 2014; 58: [PMID: DOI: /AAC ] P- Reviewers: Dubuisson J, Song LT S- Editor: Cui XM L- Editor: Kerr C E- Editor: Zhang DN 3467 April 7, 2014 Volume 20 Issue 13

93 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (3): Inflammatory bowel disease Restoring the gut microbiome for the treatment of inflammatory bowel diseases TOPIC HIGHLIGHT Jessica R Allegretti, Matthew J Hamilton Jessica R Allegretti, Matthew J Hamilton, Division of Gastroenterology, Brigham and Women s Hospital and Harvard Medical School, Boston, MA 02115, United States Author contributions: All authors contributed to this work. Correspondence to: Matthew J Hamilton, MD, Division of Gastroenterology, Brigham and Women s Hospital and Harvard Medical School, 75 Francis St, Boston, MA 02115, United States. mjhamilton@partners.org Telephone: Received: September 28, 2013 Revised: November 27, 2013 Accepted: February 26, 2014 Published online: April 7, 2014 Abstract Fecal microbiota transplantation (FMT) is considered to be a highly successful therapy for recurrent and refractory Clostridium difficile infection (CDI) based on recent clinical trials. The pathogenesis of inflammatory bowel diseases (IBD) is thought to be due in part to perturbations in the gut microflora that disrupt homeostasis. FMT restores essential components of the microflora which could reverse the inflammatory processes observed in IBD. Case reports and series for the treatment of IBD by FMT have shown promise with regards to treatment success and safety despite the limitations of the reporting. Future studies will determine the optimal delivery and preparation of stool as well as the conditions under which the recipient will derive maximal benefit. The long term consequences of FMT with regards to infection, cancer, auto-immune, and metabolic diseases are not known and will require continued regulation and study. Despite these limitations, FMT may be beneficial for the treatment of ulcerative colitis and Crohn s disease, particularly those with concurrent CDI or with pouchitis Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Crohn s; Ulcerative colitis; Microbiota; Fecal transplantation; Dysbiosis; Pouchitis; Clostridium difficile ; Probiotic Core tip: Advances into the understanding of the pathogenesis of inflammatory bowel diseases (IBD) have highlighted the importance of a dysbiosis in the intestinal microbiome. A perturbed microbiota with loss of colonization resistance is a main driver of Clostridium difficile infection and exciting new data exists that microbial restoration through the use of fecal microbiota transplantation (FMT) is highly successful. Therefore, it is logical to conclude that FMT will have therapeutic efficacy in IBD. Preliminary studies that have evaluated FMT for IBD are reviewed with an emphasis on subpopulations that may benefit the most. The limitations and unknowns for this novel therapy are also discussed. Allegretti JR, Hamilton MJ. Restoring the gut microbiome for the treatment of inflammatory bowel diseases. World J Gastroenterol 2014; 20(13): Available from: URL: DOI: org/ /wjg.v20.i INTRODUCTION Ulcerative colitis (UC) and Crohn s disease, the two main classifications of inflammatory bowel diseases (IBD) are characterized by chronic intestinal inflammation resulting in recurrent episodes of disease exacerbations with associated abdominal pain, diarrhea, weight loss and rectal bleeding. IBD remains poorly understood and medical therapies continue to be inadequate. The current mainstays of conventional therapy for these diseases include 5-aminosalicylates (5-ASAs), corticosteroids, thiopurines, and anti-tumour necrosis factor agents. However, despite continued advances in therapy, a significant number of 3468 April 7, 2014 Volume 20 Issue 13

94 Allegretti JR et al. Fecal microbiota transplantation for IBD patients remain refractory to standard therapies. Overall, 20%-30% of patients with UC will require a colectomy. At least 50% of patients with Crohn s disease will require surgical treatment in the first 10 years of disease and 70%-80% will require surgery within their lifetime [1]. The etiology of IBD is complex and several factors are believed to play a role in its development and progression. The host genotype is important and twin studies have shown concordance rates of 16% for UC and 35% for Crohn s disease [2]. However, these numbers also indicate that non-genetic factors play a substantial role in the development of IBD [3]. The most important of these is likely the intestinal microbiota (reviewed extensively in this issue of WJG). Humans have evolved with the microbes in the intestine which are known to provide critical functions to the host such as metabolism, digestion, development and maintenance of the immune system, and mucosal barrier function. The microbes exist in the various niches to carry out their function and are relatively stable over time [4]. In disease states such as IBD, the microbial balance that favored homeostasis is perturbed and studies that have analyzed the composition of the gut microbiome in IBD have found a loss in the richness and diversity of the bacterial components including under representation of the anti-inflammatory phyla Bacteroides and Firmicutes and a relative plume of pro-inflammatory Proteobacteria [5-7]. This shift in the composition of the microbiota ( dysbiosis ) may favor the appearance of distinct pathogens that perpetuate the inflammatory response. In this regard, several studies have revealed an increase in adherent/invasive E. coli in the terminal ileum of patients with Crohn s disease and Mycobacterium avium paratuberculosis has been casually linked to Crohn s pathogenesis although a direct link has not been proven [8]. Opportunistic microbes such as C. difficile may also be able to establish pathogenicity in niches that may be present in the colons of IBD patients. Whether the dysbiosis directly leads to inflammation or is a consequence of an inflammatory environment is yet to be determined. Nonetheless, antibiotics and fecal diversion have been successful in treating various forms of IBD [9], and it is possible that restoring a healthy microbiota through Fecal microbiota transplantation (FMT) may prove to be more effective [2]. FMT has been suggested as a therapy for IBD, given the observed intestinal dysbiosis [10]. FMT has also been termed fecal bacteriotherapy, human probiotic infusion, stool transplant, intestinal microbiome restoration and fecal transfer in the literature. FMT involves collecting stool from a healthy pre-screened donor and delivering a prepared slurry into the gastrointestinal tract of the individual with disease via nasogastric tube, EGD, colonoscopy, or enema [10]. Multiple studies have investigated the role of FMT for the treatment of colitis and diarrhea caused by the opportunistic pathogen C. difficile. The accumulated data suggests that FMT is a safe and highly effective therapy for C. difficile infections (CDIs) refractory to standard medical treatment with antibiotics [11-22]. In this review, we will discuss the literature on the use of FMT for the treatment of IBD with a focus on special populations of patients with IBD who are predicted to respond to this treatment. We also discuss the limitations of FMT and remaining questions for this exciting novel therapy. PUBLISHED EXPERIENCE WITH FMT AS A THERAPY FOR IBD There are currently no published clinical trials on FMTs in either UC or Crohn s disease. The literature consists of various case reports and case series, mainly in UC. The first report of FMT for UC was presented by Bennet and Brinkman in 1989 [23]. Bennet, who had UC, self treated with fecal retention enemas. Six months after his experimentation, he remained symptom-free and off of medications. This report was followed by a case series, by Borody and colleagues in the same year, of 55 patients with a mixture of gastrointestinal disorders including UC, Crohn s disease (only one patient), and irritable bowel syndrome who were treated with FMT by retention enemas [24]. They reported that 20 of the 55 patients were cured after one FMT and 9 had significant symptom reduction. This study was very limited and did not provide details as to how clinical outcomes were measured and which patient groups may have derived the greatest treatment benefit. Furthermore, details were not provided as to the frequency and duration of treatment or the length of follow up. The one patient with Crohn s disease was reportedly symptom free after four months after suffering from steroid refractory disease [24]. In a later review, Borody reported that this Crohn s patient had relapsed at 18 mo [25]. Borody followed up this report up with another case series highlighting 6 patients with UC [26]. Each of the 6 had at least five years of disease and had either failed what was described as maximal medical therapy (steroids, 5-ASAs and mercaptopurine) or quickly relapsed upon withdrawal of medications. Each patient was confirmed to have active inflammation on colonoscopy and was negative for CDI. Prior to the treatment, each patient received antibiotics for 7-10 d in order to suppress the Clostridia (vancomycin 500 milligrams (mg) twice daily, metronidazole 400 mg twice daily, and rifampicin 150 mg twice daily). Each patient also underwent a one time 3 L lavage with an oral polyethylene glycol solution. These patients provided their own donors and received daily fecal retention enemas for five days. They were encouraged to retain the enemas for as long as possible (6-8 h). Each of these patients was in complete remission four months after treatment and remained off of IBD medications. In several of the patients, follow up colonoscopy revealed no active inflammation. The follow up time was variable but remission was reportedly sustained over many years (1-13) [26]. A recent systematic review on the topic found nine articles and 26 patients (18 UC, 6 CD, 2 indeterminate) who had received FMT for management of IBD, several of which are included 3469 April 7, 2014 Volume 20 Issue 13

95 Allegretti JR et al. Fecal microbiota transplantation for IBD in the series described above [27]. Of these 26 patients, results were reported in 17. After FMT, 13/17 patients were able to cease all IBD medications within 6 wk and all had symptom reduction or resolution at 4 mo [28]. It is important to note that these cases varied significantly in the route of administration, preparation of stool, and screening protocols. Angelberger et al [29] characterized the bacteria communities present both pre and post FMT in 5 patients with moderate or severe UC. They found that none of the 5 patients achieved remission by week 12 and response was only noted in one patient. In two of the patients, further deterioration of their UC was noted at 4 wk post FMT. Upon analysis of the microbial compositions, they found that the UC patients pre FMT displayed a low phylotype richness and an overrepresentation of Enterococcaceae and Enterobacteriaceae and an underrepresentation of Lachnospiraceae, Ruminococcaceae and Bacteroidaceae when compared to healthy donors [29]. They found that post FMT the microbiota of the patients became similar to that of the donor, however the duration of that change was patient dependent. The one patient with a clinical response maintained a similar microflora to the donor extending to 12 wk post FMT. However, the 2 patients who experienced disease deterioration showed increased microbiota dissimilarity by 4 wk post FMT. This small study raises several important questions such as what IBD phenotypes may respond best to FMT and how many infusions are necessary to establish a healthy microbiota and a sustained clinical response. Future studies for the treatment of IBD should carefully consider whether disease severity at the time of FMT affects treatment outcome, and at what point in the IBD disease process FMT may be optimal. FMT FOR CDI IN PATIENTS WITH IBD The incidence of CDI continues to rise [30]. First line treatment for CDI consists of antibiotic therapy, however recurrence rates have been reported between 15%-35% [30]. FMTs are best studied in CDI infections refractory to standard treatment. Current literature consists of multiple case series, systematic reviews and a recent randomized controlled trial [31]. Impressively, cure rates have been reported between 81% and 100% [30]. CDI infections are more common in patients with IBD, with a higher prevalence among patients with UC (3.7%) and Crohn s disease (1.1%) compared with the background general population (0.45%) [32]. While IBD itself is thought to be an independent risk factor for CDI, the increased prevalence has also been linked to immunosuppressive medications, increased antibiotic use and multiple surgeries and hospitalizations [33]. CDI may have adverse effects on the underlying IBD and so effective therapy to eradicate the organism is necessary to promote disease remission [33]. A recent systematic review identified eight articles that reported on 15 patients (9 UC, 6 Crohn s disease) who underwent FMT for recurrent or refractory CDI, however outcomes were only reported in 12 of these patients [27]. All patients had resolution of C. difficile as measured by stool specific testing. Several patients were noted to have fever and abdominal pain post FMT in this cohort but no major adverse events were reported. We will await the results of future trials to verify that FMT is a safe and effective therapy for IBD patients with C. difficile. UC PATIENTS WITH ILEAL POUCHES Up to 20% of people with UC undergo an ileal pouch anal anastomosis (IPAA) [34]. Over 60% of individuals undergoing an IPAA for UC have at least one episode of pouchitis [35]. This complication is uncommon in those undergoing IPAA for non-uc related reasons, such as familial adenomatous polyposis suggesting that genetic and environmental factors such as the composition of microbiota play a role in the pathogenesis of pouchitis [36]. Its development in relationship to microbiota likely has two sides: a dysbiosis which reflects changes in bacterial composition possibly at the core of the pathogenesis of UC as well as the emergence of pathogenic bacteria such as C. difficile [37]. Pouchitis often responds to a course of antibiotics but may recur and require multiple courses of the same antibiotic or a switch to a different antibiotic [34]. Various types of probiotic preparations have been demonstrated to maintain remission in pouchitis when used daily [38,39]. Unfortunately, pouchitis becomes a chronic, refractory condition in 5%-30% of patients undergoing IPAA for UC [40] and more effective therapy is needed. Because dysbiosis likely propagates pouchitis in IBD and bacterial manipulation with antibiotics and probiotics has proven to be successful, it stands to reason that FMT will prove to be a successful treatment for many pouchitis patients. There is currently no published literature or ongoing trials exploring this possibility. LIMITATIONS AND UNANSWERED QUESTIONS FOR FMT Screening The process of screening the donor stool and what tests should be ordered prior to FMT continues to evolve. Ideally, experts from gastroenterology and infectious disease can form a consensus regarding the appropriate screening of donor stool. In our practice, we ask the donor initially about high risk sexual behaviors, whether they have been diagnosed with any gastrointestinal diseases such as IBD, colon polyps, or irritable bowel syndrome, and whether or not they have taken antibiotics within the previous 3 mo. We then screen both the donor and recipient s blood for Hepatitis A (IgG and IgM), Hepatitis B (HBsAg/Ab and HBc), Hepatitis C (Ab), HIV-1/2 (Ab and viral load), and Syphilis (TP- IgG). The donor s stool is screened for C. difficile (by culture), routine stool bacterial culture, Giardia antigen, 3470 April 7, 2014 Volume 20 Issue 13

96 Allegretti JR et al. Fecal microbiota transplantation for IBD Cryptosporidium antigen, and test for ova and parasites. More extensive screening protocols have been used in other studies that include additionally screening the donor for strongyloides, CMV, HTLV 1 and 2, EBV and Entamoeba histolytica [31]. Given regional and geographic differences, we recommend consulting with an infectious disease specialist and infection control in order to determine the appropriate screening tests for an individual practice setting. Whether or not the efficacy of FMT is improved with a related donor vs unrelated donor is not clear at this time. One recent systematic review suggested that stool from a related donor resulted in a higher resolution rate (90.5%) for CDI than an unrelated donor (84%) [30]. However, other studies where universal donor pools were used have yielded similar overall results [30]. Identification of individual bacterial components within the donor microbiota which could potentially influence efficacy are being investigated [41]. Patient preparation Most published reviews have recommended largevolume bowel lavage before the procedure, regardless of upper or lower tract administration in order to mechanically reduce Clostridial organisms that are still present [30]. This concept has never been tested formally. For recurrent C. difficile, reports generally have recommended discontinuing antibiotics 1-3 d prior to the FMT [13,30], however this has also not been compared to continuing antibiotics up until the day of the procedure. At our Institution, we have patients discontinue antibiotics the night before the FMT. Stool delivery Another issue that will need clarification through future study is the mode by which the stool is delivered to the bowel. Although the efficacy of FMT has been shown to be similar when delivered by endoscopy, nasogastric tube, enema or colonoscopy, it is possible that one method may be superior to another in IBD. The type and location of IBD may drive this decision. Upper gastrointestinal delivery of stool may be more efficacious for patients with small bowel Crohn s disease vs delivery by colonoscopy for patients with colonic disease. Lastly, tolerability and relative safety of each procedure will have to be considered when deciding between upper gastrointestinal delivery vs lower. In this regard, belching, nausea and abdominal cramps have been reported with upper gastrointestinal administration on the day of the procedure in 8/16 patients, however these symptoms resolved upon follow up [31]. Although no major adverse events have been reported with any intestinal administration of stool, the safety of the proposed procedure should be considered at the time of treatment. Although colonoscopy is generally considered to be a safe treatment in the setting of active IBD, the perforation rate may be increased and other modes of stool delivery should be considered in patients with moderate or severe inflammation or stricturing disease. Lastly, it is possible that FMT delivered by retention enema is effective in a subset of IBD patients and would obviate the need for an endoscopic procedure and hospital visit. Processing and storage Another unanswered question is whether donor stool may be frozen and then thawed prior to FMT. This has obvious practical implications but whether or not the key components of the stool will be adequately preserved is not known. The University of Minnesota reported similar C. difficile cure rates among patients who received fresh vs frozen (minus 80 degrees Celsius from 1-8 wk) specimens (n = 33) [42]. In this series, 10 patients had underlying IBD. Interestingly, only 4 patients required a second FMT for recurrent symptoms and 3 of them had underlying IBD [42]. Stool frozen at -80 therefore may be equally effective for FMT as fresh stool, however the viability of organisms after exposure to atmospheric oxygen may be an important consideration. Facultative anaerobes in stool may be inactivated by oxygen and thus transplants under anaerobic conditions may be more efficacious [4]. It is conceivable that oral preparations that mimic human stool may be manufactured in the near future. Although probiotics have yielded modest treatment effects in certain populations of IBD [43-46], it is likely that the various probiotics lacked critical organisms and possibly other factors that help successfully restore the gut microbiota back to health. Long term complications Whether or not FMT may exacerbate underlying bowel disease in some patients may be an important question. A case of a UC flare after FMT for CDI was recently reported [47]. The patient had quiescent disease for twenty years and was not on immunosuppressive medications. He developed symptoms nine days after the FMT. C. difficile testing was negative and sigmoidoscopy revealed the appearance of inflammation and ulceration that was not present on the FMT colonoscopy. There is a theoretical concern for the transmission of infections that may have escaped the screening process [48]. A recent case series reports two patients who experienced gastroenteritis only 2 d and 2 wk after FMT respectively. Both patients were found to be C. difficile negative by PCR but norovirus positive. The donor stool however was negative for norovirus and the authors concluded that there was not direct transmission from donor to patient. Whether or not FMT may influence non-gastrointestinal diseases in the long term such as metabolic disease, obesity, and cardiovascular disease is not known at this time [49-53]. This may be the reason why several regulatory agencies such as the United States Food and Drug Administration have asked for more research on FMT 3471 April 7, 2014 Volume 20 Issue 13

97 Allegretti JR et al. Fecal microbiota transplantation for IBD before this can be recommended as a first line treatment. Regulation While initial reports of FMTs for IBD are promising, several unresolved issues remain. Treatment of IBD with FMTs may be considered investigational and so many health care providers may not cover the cost of the procedure (colonoscopy and stool preparation). This is the case in the United States where the Food and Drug Administration has required that providers who would like to perform FMT must file an Investigational New Drug application. Many patients may end up having to pay the hospital charge for this treatment out of their own funds. This burden may be greater when it is possible that patients with IBD may require several treatments. Nonetheless, it is conceivable that long term costs may be reduced if FMT leads to treatment success and the patient is able to avoid expensive medical therapies, hospitalizations, or surgeries. CONCLUSION FMT is now considered to be a highly successful therapy for recurrent and refractory CDI based on recent clinical trials. The pathogenesis of IBD is thought to be due in part to perturbations in the gut microflora that disrupt homeostasis. Therefore, it is logical to extend the successes of FMT in CDI to the treatment of IBD. Case reports and series for the treatment of IBD by FMT have shown promise with regards to treatment success and safety despite the limitations of the reporting. While several questions remain unanswered such as the long term consequences of FMT on the recipient, this therapy may be beneficial for the treatment of UC and Crohn s disease, particularly those with concurrent CDI or with pouchitis. The study of the gut microbiome has opened an exciting new world in medicine raising as many questions as it seems to answer. It is nonetheless here to stay with additional data from randomized controlled trials much needed. 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Clin Gastroenterol Hepatol 2013; 11: [PMID: DOI: / j.cgh ] 48 Schwartz M, Gluck M, Koon S. Norovirus gastroenteritis after fecal microbiota transplantation for treatment of Clostridium difficile infection despite asymptomatic donors and lack of sick contacts. Am J Gastroenterol 2013; 108: 1367 [PMID: DOI: /ajg ] 49 Wang Z, Klipfell E, Bennett BJ, Koeth R, Levison BS, Dugar B, Feldstein AE, Britt EB, Fu X, Chung YM, Wu Y, Schauer P, Smith JD, Allayee H, Tang WH, DiDonato JA, Lusis AJ, Hazen SL. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature 2011; 472: [PMID: DOI: /nature09922] 50 Nicholson JK, Holmes E, Kinross J, Burcelin R, Gibson G, Jia W, Pettersson S. Host-gut microbiota metabolic interac April 7, 2014 Volume 20 Issue 13

99 Allegretti JR et al. Fecal microbiota transplantation for IBD tions. Science 2012; 336: [PMID: DOI: /science ] 51 Turnbaugh PJ, Ley RE, Mahowald MA, Magrini V, Mardis ER, Gordon JI. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 2006; 444: [PMID: DOI: /nature05414] 52 Samuel BS, Shaito A, Motoike T, Rey FE, Backhed F, Manchester JK, Hammer RE, Williams SC, Crowley J, Yanagisawa M, Gordon JI. Effects of the gut microbiota on host adiposity are modulated by the short-chain fatty-acid binding G protein-coupled receptor, Gpr41. Proc Natl Acad Sci USA 2008; 105: [PMID: DOI: / pnas ] 53 Vijay-Kumar M, Aitken JD, Carvalho FA, Cullender TC, Mwangi S, Srinivasan S, Sitaraman SV, Knight R, Ley RE, Gewirtz AT. Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5. Science 2010; 328: [PMID: DOI: /science ] P- Reviewers: Eglinton TW, Limdi JK, Misiakos EPP, Sollano JDD, Skrypnyk IN S- Editor: Wen LL L- Editor: A E- Editor: Zhang DN 3474 April 7, 2014 Volume 20 Issue 13

100 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (3): Inflammatory bowel disease TOPIC HIGHLIGHT Therapeutic drug monitoring in patients with inflammatory bowel disease Andres J Yarur, Maria T Abreu, Amar R Deshpande, David H Kerman, Daniel A Sussman Andres J Yarur, Maria T Abreu, Amar R Deshpande, David H Kerman, Daniel A Sussman, Division of Gastroenterology, University of Miami Leonard Miller School of Medicine, Miami, FL 33136, United States Author contributions: All authors substantially contributed to the writing of this article and approved the final manuscript. Correspondence to: Daniel A Sussman, MD, Division of Gastroenterology, University of Miami Leonard Miller School of Medicine, 1120 NW 14th Street, Suite 310F3 (D-49), Miami, FL 33136, United States. dsussman@med.miami.edu Telephone: Fax: Received: October 30, 2013 Revised: January 15, 2014 Accepted: February 17, 2014 Published online: April 7, 2014 Abstract Thiopurine analogs and anti-tumor necrosis factor (TNF) agents have dramatically changed the therapeutics of inflammatory bowel diseases (IBD), improving short and long-term outcomes. Unfortunately some patients do not respond to therapy and others lose response over time. The pharmacokinetic properties of these drugs are complex, with high inter-patient variability. Thiopurine analogs are metabolized through a series of pathways, which vary according to the patients pharmacogenetic profile. This profile largely determines the ratios of metabolites, which are in turn associated with likelihoods of clinical efficacy and/or toxicity. Understanding these mechanisms allows for manipulation of drug dose, aiming to reduce the development of toxicity while improving the efficacy of treatment. The efficacy of anti-tnf drugs is influenced by many pharmacodynamic variables. Several factors may alter drug clearance, including the concomitant use of immunomodulators (thiopurine analogs and methotrexate), systemic inflammation, the presence of anti-drug antibodies, and body mass. The treatment of IBD has evolved with the understanding of the pharmacologic profiles of immunomodulating and TNF-inhibiting medications, with good evidence for improvement in patient outcomes observed when measuring metabolic pathway indices. The role of routine measurement of metabolite/drug levels and antibodies warrants further prospective studies as we enter the era of personalized IBD care Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Inflammatory bowel disease; Anti-tumor necrosis factor; Infliximab; Adalimumab; Drug level; Azathioprine; Thiopurines; Antibodies; Drug monitoring; Thioguanine Core tip: The use of thiopurine analogs and anti-tumor necrosis factor (TNF) agents in patients with inflammatory bowel diseases (IBD) has improved outcomes. The pharmacokinetics of thiopurines is variable among patients, and some do not benefit from these drugs. The manipulation and monitoring of thiopurines can potentially increase response to treatment and/or reduce the development of toxicity. The efficacy of anti-tnf drugs is also variable and several factors can modify drug clearance, including the concomitant use of immunomodulators, systemic inflammation, the presence of anti-drug antibodies, and body mass. The treatment of IBD has advanced with the understanding of the pharmacologic profiles of immunomodulating and TNFinhibiting medications. Yarur AJ, Abreu MT, Deshpande AR, Kerman DH, Sussman DA. Therapeutic drug monitoring in patients with inflammatory bowel disease. World J Gastroenterol 2014; 20(13): Available from: URL: i13/3475.htm DOI: INTRODUCTION Inflammatory bowel diseases (IBD) are chronic disor April 7, 2014 Volume 20 Issue 13

101 Yarur AJ et al. Therapeutic drug monitoring in IBD ders of the intestinal tract characterized by relapsing and remitting intestinal inflammation; Crohn s disease (CD) and ulcerative colitis (UC) are the best-recognized of these entities. The etiology of IBD is not clear, but it is thought to occur when the intestinal flora induces an exaggerated immune response in the context of genetic predisposition. Most therapeutic regimens focus on immunosupression as the hallmark of treatment with medications including corticosteroids, purine anti-metabolites, and newer biologic agents that target specific molecules of the inflammatory cascade like tumor necrosis factor (TNF). Dramatic advances in the treatment of IBD have been seen with the availability of new drugs and our ability to tailor the treatment strategy for each patient. Drug monitoring is relevant not only when trying to achieve treatment efficacy, but also when trying to mitigate toxic side effects and optimize costs. In medicine, we usually measure levels in drugs with a narrow therapeutic range and that pose significant deleterious side effects with overdosing (e.g., theophylline, cyclosporine, and lithium). With some antibiotics (e.g., vancomycin), we measure levels in order to assure the patient receives the required dose to induce a therapeutic effect. Accordingly, the treatment of IBD has evolved with advances in laboratory medicine. We will review the latest evidence concerning measurement of genomic risk for metabolic side effects and levels of metabolites, drugs and antibodies used in the treatment of IBD, including thiopurines (purine anti-metabolites) and anti tumor necrosis factor (anti-tnf) agents. THIOPURINE ANALOGS In IBD patients, thiopurines have been successfully employed for decades. The thiopurine analogs with activity against IBD include mercaptopurine (MP) and its prodrug azathioprine (AZA). The general consensus is that the thiopurine medications are effective at preventing relapse of quiescent disease, but not at inducing remission [1]. They also exert a beneficial effect when combined with biologic drugs such as infliximab (an anti-tnf agent), improving rates of clinical remission and mucosal healing [2]. These medications antagonize endogenous purines, interfering with the synthesis of DNA. They were first developed to treat leukemia in pediatric patients, but because of their observed anti-proliferative effect on T-cells, they have been successfully used to treat autoimmune disease and to prevent graft rejection after solid organ transplantation. While the exact molecular mechanism by which thiopurines exert their immunosuppressive effect is not well-comprehended, several theories have been proposed. One hypothesis suggests that 6-thioguanine (6-TGN), a metabolite of AZA, accumulates in lymphocytes and blocks the expression of inflammatory-related cytokines, including TNF-related apoptosis-inducing ligand, TNF receptor S7, and alpha-4-integrins, ultimately inhibiting the inflammatory response induced by T-cells in the intestinal lamina propria in patients with IBD [3]. Drug metabolism The metabolism of AZA is quite complex, and involves many enzymatic pathways producing active, inactive, and potentially toxic metabolites (Figure 1). After getting absorbed from the gastrointestinal tract, 88% of AZA is converted to MP and methyl-nitro-thioimidazole in red blood cells (RBC) [4]. MP can then be metabolized through three of the following competing pathways: conversion to 6-thiouric acid by xanthine oxidase (XO), methylation by thiopurine methyltransferase (TPMT) into 6-methyl mercaptopurine (6-MMP) (which is associated with potential hepatotoxicity), or conversion to thioinosine monophosphate (TIMP) by hypoxanthine phosphoribosyltransferase (HGPRT) [5]. TIMP is subsequently converted into 6-TGN, which are thought to be desirable therapeutic metabolites of AZA/MP [4,6]. Though 6-TGN is a favorable metabolite, high levels of this agent can result in life-threatening myelosuppression. Measuring the thiopurine methyltransferase phenotype/ genotype Thiopurine drugs have a complex metabolism, and some metabolites may cause life-threatening toxicity, including myelosuppression and hepatotoxicity. TPMT catalyzes one of the rate-limiting pathways in AZA/MP metabolism. Because TPMT has variable activity among patients, checking TPMT activity prior to initiation of thiopurine therapy is routinely used in clinical practice and is recommended. TPMT induces the metabolism of MP to 6-MMP, competing with the HGPRT and XO enzymatic pathways (Figure 1). TPMT also metabolizes TIMP to 6-methyl-thioinosine monophosphate (6-MeTIMP); thus, low TPMT activity results in greater conversion of MP to 6-TGN, as less TIMP is converted to 6-MeTIMP. Measuring TPMT before prescribing AZA/MP can help predict those patients who will accumulate high levels of 6-TGN relative to 6-MMP, a profile which increases the risk of leukopenia early in the treatment course. The TPMT status of a patient may be identified by requesting the genotype or the phenotype from a commercial laboratory, with the phenotype testing being in general more clinically useful. TPMT genotype: Studies in different racial and ethnic backgrounds have shown that most of the population (89%) are homozygous for the wild type (WT) TPMT gene (high TPMT metabolism), 10% are heterozygous for the WT and a low metabolic polymorphism (intermediate TPMT metabolism), and 1 in 300 are homozygous for low TPMT metabolic polymorphism (low TPMT metabolism) [7]. While higher 6-TGN levels are associated with a better clinical response, they also increase risk of myelotoxicity with AZA/MP; therefore, determining TPMT phenotype/genotype is currently used to predict early leukopenia [6]. Further research in this area has resulted in the identification of fourteen single nucleotide 3476 April 7, 2014 Volume 20 Issue 13

102 Yarur AJ et al. Therapeutic drug monitoring in IBD 6-MMP Inhibits purine synthesis TPMT TPMT 6-MeTIMP IMPDH GMPS AZA MP 6-TIMP 6-TXMP 6-TGN XO ITPA 6-TGMP 6-TA TPMT 6-TITP 6-TGDP 6-MeTITP 6-TGTP Inhibition of Rac 1 T-Cell apoptosis Figure 1 Metabolic pathway of azathioprine/mercaptopurine metabolism. AZA: Azathioprine; MP: Mercaptopurine; TPMT: Thiopurine methyltransferase; XO: Xanthine oxidase; IMPDH: Inosine-5-monophosphate dehydrogenase; GMPS: Guanidine- 5-monophosphate synthetase; 6-TA: 6-Thiouric acid; 6-MMP: 6-Methylmercaptopurine; 6-TIMP, 6-Thioinosine monophosphate; 6-MeTIMP: 6-Methylthioinosine monophosphate; 6-MeTITP: 6-Methyl thioinosine triphosphate pyrophosphate; 6-TITP: 6-Thioinosine triphos- phate pyrophosphate; TXMP: Thioxanthosine monophosphate; 6-TGN: 6-Thioguanine nucleotides; 6-TGMP: 6-Thioguanine monophosphate; 6-TGDP: 6-Thioguanine diphosphate; 6-TGTP: 6-Thioguanine triphosphate. polymorphisms for the TPMT gene that cause a diminished or absent enzymatic activity. TPMT phenotype: As with genotype, enzyme activity (or phenotype) may also be measured and sub-divided into three major groups (high, intermediate, and low TPMT metabolizers). The correlation between TPMT genotype and phenotype varies between 65 and 89% [8,9]. The cause of this variance is unclear, but measuring phenotype has a better predictive value for myelosuppression when compared to genotype [8]. Select situations exist where the genotype can theoretically be more reliable than the phenotype. Because TPMT is measured in erythrocytes and uremia may affect the assay, measuring TPMT genotype and not phenotype may be reasonable when a patient has had a recent transfusion of red blood cells or has a high blood urea nitrogen, (usually in patients requiring dialysis) [10]. Also, some medications including azathioprine itself and some diuretics may increase TPMT activity, but the clinical significance of this effect is not clear [11]. Conversely, mesalamines and sulfasalazine inhibit TPMT, theoretically increasing the risk of leukopenia, though this claim is unproven [12]. Monitoring thiopurine metabolites Once the decision has been made to treat patients and at a particular dose, monitoring thiopurine metabolite levels is a clinical option. Measuring metabolites has two important applications, increasing the likelihood of treatment efficacy and reducing the risk of treatment-related toxicities. The two metabolites that are commercially available are 6-TGN and 6-MMP. 6-TGN has been the metabolite most associated with treatment efficacy; as such, its measurement has been proposed as a strategy to optimize treatment in patients with IBD receiving AZA/MP. 6-TGN is a metabolite of TIMP, which goes through a series of phosphorylation events resulting in 6-thioguanine diphosphate. A 6-TGN level > 230 pmol/ RBC has been correlated with clinical remission in both adults and children with IBD [6,13]. Another study using a different assay that included only adult patients failed to show a relation between 6-TGN levels and clinical activity [14]. The need to follow 6-TGN levels during treatment has not been well-established. In a prospective cohort study, Wright et al [15] found that patients on a stable dose of azathioprine present with variable levels of 6-TGN over time, bringing into question the value of interpreting any single 6-TGN level. The difference in outcomes among studies is unclear, but could be related to the heterogeneity in the instrument used to determine IBD activity and the use of different assays to measure the 6-TGN levels. Another added potential use for metabolite measurement is to assess adherence to medical therapy. If both 6-TGN and 6-MMP are low, it is likely the patient is not ingesting or absorbing the medication. Randomized controlled trials looking at the role of serial measurements of thiopurine metabolites and the effect of subsequent dose adjustment on outcomes are needed. AZA metabolite measurement can also be used to help prevent drug-related toxicity. 6-MMP is a metabolite produced from MP by TPMT. Higher 6-MMP levels have been found to correlate with a higher risk of hepatotox April 7, 2014 Volume 20 Issue 13

103 Yarur AJ et al. Therapeutic drug monitoring in IBD icity. Even though patients with 6-MMP levels > 5700 pmol/ RBC have a three-fold increased risk of hepatotoxicity, not all patients with a high 6-MMP level will develop elevated liver enzymes, and having a low 6-MMP level does not preclude the development of hepatotoxicity [6,16]. As with 6-MMP, some patients with very high 6-TGN levels do not develop myelotoxicity while some with low 6-TGN levels may still develop this abnormality. Thus, measuring 6-TGN and 6-MMP levels do not replace monitoring liver enzymes and blood counts. 6-TGN level measurements can also be useful to identify those patients who will not experience clinical benefit despite an optimal AZA/MP dose. Patients with normal TPMT activity and 6-TGN levels > 400 pmol/ RBC who do not achieve clinical remission will likely remain refractory to treatment even if the treatment is continued for 6 more months or the dose is increased [17]. Patients resistant to AZA/MP therapy and the use of allopurinol Patients who fail therapy with AZA/MP may not always benefit from dose escalation. The metabolic pathways in some patients favor an increased conversion of medication into 6-MMP rather than 6-TGN. Metabolite levels confirm this phenomenon when increasing thiopurine dose in patients who are clinically not responding [18]. This finding supports the metabolic heterogeneity that exists among the IBD population and how some patients preferentially favor particular AZA/MP metabolic pathways. The identification of these patients represents another potential use for metabolite measurement. Up to 24% of patients preferentially produce 6-MMP while maintaining low levels of 6-TGN despite increasing the dose [18] ; these patients usually have high TPMT activity, which is genetically determined and are usually labeled as thiopurine resistant. In this setting, one option is to switch to another therapeutic class (methotrexate or anti-tnfs), but manipulation of the known pathways provides an alternative option to optimize therapy in these so-called shunters. Inhibition of TPMT could reduce the production of 6-MMP, but drugs with this effect (e.g., mesalamines/sulfasalazine) have failed to reliably optimize the metabolic profile, likely due to their weak inhibitory effect on the enzyme. Another potential target is XO. This enzyme converts MP into 6-thiouric acid, which has no known biologic effect. When this enzyme is inhibited, much of the MP is shunted down the 6-TGN pathway. Allopurinol, a XO-inhibitor used in the treatment of gout, has been successfully used to shunt metabolism of AZA/MP down this more favorable 6-TGN pathway. The mechanism by which this occurs is not completely understood, as the sole inhibition of XO would not explain the rise in 6-TGN. Sparrow et al [19] selected a group of patients who had a preferable metabolism towards 6-MMP and initiated therapy with 100 mg of allopurinol while reducing the AZA/MP dose by 25%-50%. After 2-4 wk on this regimen, they found that the level of 6-TGN had significantly increased while the 6-MMP level had decreased. A subsequent study by the same group looking at clinical response in AZA/MP non-responders when starting allopurinol found that the allopurinol-immunomodulator regimen also improved disease activity [20]. This beneficial effect was not only observed for disease activity, but also reversed hepatotoxicity, presumably by reducing the 6-MMP levels. However, there are several issues that must be addressed when starting patients on a combination of a thiopurine and allopurinol. First, we recommend serial blood counts, checking a complete blood count (CBC) with differential weekly for the first month and monthly thereafter. Thiopurine metabolites may be checked one month after initiating therapy in order to ensure the goal of increased 6-TGN and decreased 6-MMP is achieved. If leukopenia develops, the dose of AZA/MP can be reduced. Another proposed strategy to overcome the shunting of MP towards 6-MMP is splitting the thiopurine into twice daily dosing. A retrospective study in patients with preferential 6-MMP metabolism showed that dividing the daily dose of the thiopurine (MP or AZA) modifies how the drug is metabolized, resulting in a significant reduction in 6-MMP levels and decreasing toxicity without having an adverse impact on clinical disease activity or 6-TGN levels [21]. The exact mechanism explaining the beneficial outcomes seen when implementing this strategy is unknown, and further studies are needed to confirm the results. In summary, measuring TPMT activity prior to thiopurine initiation has improved the clinical care for IBD patients, both by improving treatment efficacy and by ameliorating iatrogenic toxicities. Many clinicians, including our group, also follow thiopurine metabolite levels with the intent of positively impacting patient care. The enclosed algorithm represents a logical approach to the care of IBD patients on thiopurine therapy (Figure 2). ANTI-TUMOR NECROSIS FACTOR Anti-TNF agents have dramatically changed the management of CD and UC. Though four anti-tnf agents are currently approved to treat IBD, we will focus on infliximab (IFX) and adalimumab (ADA), as these are the two anti-tnf drugs with commercially available assays to measure drug levels and anti-drug antibodies. IFX is a chimeric IgG1 (human-constant and murine-variable regions) monoclonal antibody consisting of human constant and murine variable regions; it is indicated for induction and maintenance of clinical remission in patients with moderate-to-severely active CD and UC [22,23]. ADA is a recombinant fully human IgG1 monoclonal antibody, also approved for the induction and maintenance of remission in patients with CD and UC [24-26]. These biologic therapies are of proven benefit, reducing rates of hospitalization and surgery rates while improving quality of life [27,28]. Unfortunately, up to 50% of patients lose response to treatment (secondary nonresponders) and up to 30% do not respond at all (primary 3478 April 7, 2014 Volume 20 Issue 13

104 Yarur AJ et al. Therapeutic drug monitoring in IBD Patients not responding to AZA/MP Figure 2 Algorithm with recommendation on how to manage patients that fail therapy with Azathioprine/Mercaptopurine. AZA: Azathioprine; MP: Mercaptopurine; 6-TGN: 6-Thioguanine nucleotides; 6-MMP: 6-Methylmercaptopurine. Measure 6-TGN and 6-MMP Test results Low levels of 6-TGN and 6-MMP High levels of 6-TGN with or without high levels of 6-MMP Low levels of 6-TGN and high levels of 6-MMP Undetectable or minimal levels of 6-TGN and 6-MMP Possible explanation Receiving a low dose of AZA/6-MP Refractory to thiopurines Shunting 6-MP to 6-MMP Non-compliance with the treatment Intervention Increase the dose and re-assess metabolites in 4 wk Switch to another drug class Consider allopurinol, split dose or switch drug Re-assess causes and educate patient non-responders) [29]. The rationale for lack or loss of response is multi-factorial, but is likely related to the molecular structures and complex pharmacokinetics (PK) and pharmacodynamics of the medications, including the development of anti-drug antibodies. One of the proposed strategies when patients lose response to an anti-tnf is switching to another drug in the same or different class. Another strategy is to empirically increase the dose, hoping to overcome increased drug clearance. Unfortunately, this can lead to significant adverse events including hypersensitivity reactions. A third potential tactic is to measure drug levels and antibodies, trying to identify those patients who will benefit from dose escalation and those who will be best served by switching to an alternate drug class. This latter strategy has been proven to be more cost effective when compared to the former and highlights the importance and usefulness of measuring levels in these patients [30]. Most of our collective experience has been measuring IFX levels and antibodies to IFX (ATI), but the measurement of ADA and antibodies to ADA (ATA) has recently become available commercially. PHARMACOKINETICS OF ANTI-TNF Within an individual patient, a linear relationship exists between anti-tnf dose and serum medication levels [31]. However, inter-patient PK variability complicates the reliable prediction of medication levels among patients [32,33]. Some have theorized that this inconsistency occurs as a result of differences in body mass, but variable levels are observed with drugs with weight-based dosing like IFX. The clearance of TNF-inhibitors likely also plays a role. Anti-TNFs are monoclonal antibodies with a high molecular weight, and as is the case with other with IgGs, they are metabolized through many pathways. One of the primary mechanisms by which anti-tnfs are cleared involves the reticuloendothelial system (RES). The main component of IgG metabolism is the Brambell receptor (FcRn), which is responsible for internalizing an antibody intra-cellularly, degrading the antigen, and then recycling it by releasing it back into the circulation for excretion [31]. High circulating IgG levels may saturate the FcRn, translating into an inverse association between IgG levels and anti-tnf clearance. Many of the mechanisms underlying variation in drug levels and clearance of TNF-inhibitors are summarized in Figure 3. Anti-drug antibodies The human immune system recognizes these biologic drugs as foreign antigens, creating specific antibodies that neutralize their effect. This phenomenon, known as immunogenicity, increases drug clearance and ultimately may contribute to treatment failure. Immunogenicity was first described with IFX, a chimeric antibody. Originally, it was believed that ADA would demonstrate less immunogenicity as it is a fully human antibody, but this theory was not substantiated by clinical practice findings [33,34]. In patients with IBD, the negative effect that antibodies to IFX (ATI) and antibodies to ADA (ATA) have on clinical outcomes has been well established [32,35-38]. ATI and ATA bind to the medication, forming an immune complex that induces drug clearance via the RES. The ability to routinely measure drug levels and anti-drug antibodies has led to an understanding of why some patients lose response to therapy. Currently, there are several methods to measure IFX and ADA. Most studies have been performed using a solid-phase, double-antigen, enzyme-linked immunosorbent assay (ELISA). Unfortunately, this assay cannot measure 3479 April 7, 2014 Volume 20 Issue 13

105 Yarur AJ et al. Therapeutic drug monitoring in IBD Body mass Use of immunomodulators Anti-drug antibodies Anti-TNF levels Serum albumin Inflammatory cytokines Figure 3 Variables associated with Anti-tumor necrosis factor levels. TNF: Tumor necrosis factor. the presence of antibodies when there are detectable levels of anti-tnf. Another method is a fluid-phase radioimmunoassay, which can report antibodies irrespective of the presence of drug. A recently developed assay is a homogeneous mobility shift assay using size-exclusion high-performance liquid chromatography, which has a high sensitivity and specificity and can potentially detect all isotypes of immunoglobulin [39]. This modality permits the measurement of antibodies to anti-tnf medications, even in the presence of detectable drug. However, most clinical studies have used the ELISA assay, which could have implications in how we interpret the available data. Role of immunomodulators with biologic therapy Observations from randomized controlled trials have shown that the concomitant use of immunomodulators (AZA, MP or methotrexate) and biologic medications increases anti-tnf levels [36]. It is generally believed that this occurs by diminishing immunogenicity (decreasing the formation of anti-tnf antibodies) while simultaneously reducing the amount of systemic inflammation which, as we describe below, will negatively affect IgG levels. The presence of ATI has consistently been associated with lower IFX levels [36]. In an exploratory analysis of a randomized controlled trial looking at efficacy of IFX monotherapy, AZA monotherapy, and the two biologic drugs combined with immunomodulators, the authors found that at week 30 the IFX levels were 1.6 μg per milliliter for patients in the IFX group and 3.5 μg per milliliter for those in the combination therapy group (P < 0.001) [40]. They also found that only 1 of 116 patients (0.9%) receiving combination therapy (compared to 15 of 103 patients (14.6%) receiving IFX monotherapy) had ATI [40]. Studies looking at the effect of ATA on ADA levels have yielded similar results [33,37]. Even though most studies have shown a clear association between the presence of anti-tnf antibodies and drug levels, some data have failed to support this observation. Lichtenstein et al looked at the influence that concomitant immunomodulators and IFX therapy had on drug levels [41]. They reviewed the ACCENT Ⅰ and Ⅱ trials (effect on induction and maintenance of remission that IFX have in CD) as well as ACT 1 and 2 (effect on induction and maintenance of remission that IFX have in UC). In contrast to the previously mentioned studies, they found that IFX levels were similar when comparing patients who did and did not receive immunomodulators, even though those patients that received immunomodulators had a higher incidence of ATI antibodies to infliximab [41]. Of note, this report was based on a posthoc analysis and the results should be interpreted in that context. Disease activity and systemic inflammation Patients with severe disease have been found to have lower levels of anti-tnf. The exact mechanisms by which disease activity and systemic inflammation affect drug clearance are not completely understood. Systemic inflammation induces the RES to increase its catabolic activity, reducing levels of IgG and albumin. Direct and indirect markers of inflammation have been used to predict IFX efficacy. For example, higher levels of CRP correlate with poor response to IFX and may predict loss of response [32,42,43]. Also, low albumin levels are associated with lower IFX levels, which suggests that increased catabolic activity in systemic inflammation induces the Brambell receptor [44]. As with IFX, preliminary studies looking at ADA levels showed that higher CRP levels are associated with lower ADA levels [33,34]. In one study, a minimum ADA cut-point of 5 μg/ml best predicted elevation of CRP levels [33]. These studies cannot prove causation but do show an association between low drug levels, high CRP, and low albumin. Severe inflammation of the mucosa also induces a protein-losing enteropathy, with a loss of drug through the intestinal lumen. A recent study that included patients going through IFX induction therapy found that even though all patients had detectable IFX in the stool, those who did not respond to treatment had a significantly higher level of fecal IFX [45]. Body mass and composition may influence anti-tnf drug levels Many other variables have been proposed to affect the levels of anti-tnf. One study found that body mass and gender influence the central volume of distribution of IFX (increasing with weight and male gender) [46]. While one might suspect these results were due to the collinearity of gender and mass (as men weigh more than women), the authors tested each variable individually and concluded that each had independent influence on the central volume of distribution; this finding was attributed to the fact plasma volume is lower in women than in men. The influence of body mass on ADA levels in patients with IBD is unknown, but might play a key role as ADA does not have weight-based dosing; this also applies to other anti-tnfs approved for IBD (certolizumab pegol and golimumab). An important distinction must be made between total body mass and body composition. For example, Bultman et al [47] 3480 April 7, 2014 Volume 20 Issue 13

106 Yarur AJ et al. Therapeutic drug monitoring in IBD Loss of response to IFX Measure IFX and ATI Undetectable IFX and (-) ATI Detectable IFX and (-) ATI Detectable or undetectable IFX and (+) ATI Increase IFX dose IFX < 3 μg/ml IFX 3 μg/ml ATI 9 U/mL ATI < 9 U/mL Increase IFX dose Swith to another drug class Swith anti-tnf or drug class Drug escalation Switch anti-tnf or drug class No response Figure 4 Approaches to patients on infliximab in the setting of lose of response. ATI: Anti-infliximab antibody; IFX: Infliximab; TNF: Tumor necrosis factor. found that patients on ADA who required dose escalation had a higher body mass index (BMI) despite equivalent dosing of ADA given per kg of body weight. The component responsible for the increased BMI is unclear, but it may be related to mesenteric fat, an important contributor to the inflammatory response in CD [48]. CLINICAL IMPLICATIONS OF MEASURING ANTI-TNF LEVELS AND ANTIBODIES Many studies have shown that higher IFX levels are associated with better outcomes in patients with IBD. A detectable IFX trough level is associated with a higher rate of clinical remission, lower serum CRP levels, and an improvement in endoscopic disease activity [32,49]. The presence of ATI has been associated with treatment failure and lower IFX levels [36]. In a landmark study, Baert et al [35] studied the immunogenicity of a group of patients with CD. They found that 61% had detectable ATI after the 5 th infusion, which did not increase after subsequent infusions. This high prevalence of ATI can be explained by the fact those patients received episodic dosing (when they relapsed) and not scheduled (every 8 wk) infusions. They found that those patients with ATI >8.0 μg/ml before an IFX infusion had a shorter duration of response and a higher risk of infusion reactions [35]. Afif et al [50] looked at the clinical utility of measuring IFX levels and ATI in patients with loss of response or an incomplete response to IFX. They found that most patients with ATI did not respond to IFX dose escalation, but those with no ATIs and sub-therapeutic concentrations did benefit from a higher dose [50]. With ADA, the experience is more limited. In an observational study, Karmiris et al [37] found that lower ADA serum trough levels were associated with drug discontinuation and the presence of ATA was associated with lower ADA levels. Recent studies have found that detectable ATA and ADA levels < 5 μg/ml are associated with higher CRP serum levels, increased endoscopic inflammatory activity, and use of steroids [33,34]. Another recent study measuring ADA trough levels by ELISA assay showed similar results; an ADA trough of μg/ml was the optimal cut-off value for predicting clinical remission and mucosal healing [51]. ROLE OF ANTI-TNF DRUG MONITORING IN CLINICAL PRACTICE Commercial assays to measure anti-tnf drug and antibody levels have been available for a relatively short time; direct cost implications for patients and/or insurers have limited their widespread clinical use despite documented efficacy in directing patient management. Knowledge of drug levels and measurements of anti-drug antibodies can help identify those patients who will benefit from dose escalation versus those who are unlikely to respond to this strategy (high titers of anti-drug antibodies or those with high drug levels but persistent intestinal inflammation). A recent study modeled both strategies and found that testing for drug levels and antibodies in patients with secondary loss of response is more costeffective when compared to empiric drug escalation [52]. The titer of ATI may also predict response to dose escalation, as demonstrated by a cohort of 90 patients undergoing serial IFX and ATI levels with at least one ATI positive measurement throughout the follow-up period. In 15 of those patients (28%), ATI disappeared overtime [53]. Only 2 of those 15 patients (13%) required 3481 April 7, 2014 Volume 20 Issue 13

107 Yarur AJ et al. Therapeutic drug monitoring in IBD discontinuation of therapy, which is quite significant as ATI may be transient and do not necessarily lead to treatment failure. However, they also found that ATI > 7.95 U/mL was associated with IFX discontinuation, which may suggest that patients with high ATI titers would not benefit from dose adjustment [53]. Most of the available evidence includes patients on IFX, so further studies are needed with the other medications. A proposed algorithm to guide therapy in patients receiving IFX who lose response to treatment is shown in Figure 4. An unanswered question is the potential benefit of measuring drug levels in all patients and adjusting the dose accordingly to maintain adequate levels, thereby avoiding immunogenicity. Considering low trough IFX levels are associated with immunogenicity and the fact that the dose of IFX and serum levels are not always linear, measuring drug levels even in patients with clinical response could improve outcomes. The study cited above also showed that an IFX trough level at week 14 of < 2.2 μg/ml correlated with IFX discontinuation [53]. These results are based on a retrospective analysis and future prospective controlled studies are needed, especially considering anti-tnf levels are influenced by several variables that can fluctuate over time and among individuals. The repeated demonstration of clinical utility and evidence for cost-effective patient care have made routine measurement of anti-tnf drug and antibody levels a regular part of our group s clinical practice. For UC, when patients tend to have a lower starting albumin level and a markedly elevated CRP, our practice has been to give IFX at a higher dose (10 mg/kg) and then check levels after induction. While this practice has yet to be validated, we have had anecdotal success in achieving remission in patients who we feel clinically merit higher induction doses. Monitoring trough drug levels after induction allows for a tapered return to lower doses and a sustained remission. CONCLUSION The pharmacokinetic and pharmacodynamic properties of anti-tnfs and thiopurines are complex. Considerable inter-individual variability exists due not only to differences in pharmacogenetic factors but also disease phenotype and concomitant drug therapy. Measuring drug levels, metabolites, and anti-drug antibodies may help overcome treatment failures while mitigating toxic side effects, but further randomized studies to confirm these findings are needed. The treatment of IBD has evolved with the understanding of the pharmacologic profiles of immunomodulating and TNF-inhibiting medications, with good evidence for improvement in patient outcomes observed when measuring metabolic pathway indices. 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Split-dose administration of thiopurine drugs: a novel and effective strategy for managing preferential 6-MMP metabolism. Aliment Pharmacol Ther 2012; 36: [PMID: DOI: /j x] 22 Hanauer SB, Feagan BG, Lichtenstein GR, Mayer LF, Schreiber S, Colombel JF, Rachmilewitz D, Wolf DC, Olson A, Bao W, Rutgeerts P. Maintenance infliximab for Crohn s disease: the ACCENT I randomised trial. Lancet 2002; 359: [PMID: DOI: / S (02) ] 23 Rutgeerts P, Sandborn WJ, Feagan BG, Reinisch W, Olson A, Johanns J, Travers S, Rachmilewitz D, Hanauer SB, Lichtenstein GR, de Villiers WJ, Present D, Sands BE, Colombel JF. Infliximab for induction and maintenance therapy for ulcerative colitis. N Engl J Med 2005; 353: [PMID: DOI: /NEJMoa050516] 24 Hanauer SB, Sandborn WJ, Rutgeerts P, Fedorak RN, Lukas M, MacIntosh D, Panaccione R, Wolf D, Pollack P. Human anti-tumor necrosis factor monoclonal antibody (adalimumab) in Crohn s disease: the CLASSIC-I trial. Gastroenterology 2006; 130: ; quiz 591 [PMID: DOI: / j.gastro ] 25 Sandborn WJ, Hanauer SB, Rutgeerts P, Fedorak RN, Lukas M, MacIntosh DG, Panaccione R, Wolf D, Kent JD, Bittle B, Li J, Pollack PF. Adalimumab for maintenance treatment of Crohn s disease: results of the CLASSIC II trial. Gut 2007; 56: [PMID: DOI: /gut ] 26 Reinisch W, Sandborn WJ, Hommes DW, D Haens G, Hanauer S, Schreiber S, Panaccione R, Fedorak RN, Tighe MB, Huang B, Kampman W, Lazar A, Thakkar R. Adalimumab for induction of clinical remission in moderately to severely active ulcerative colitis: results of a randomised controlled trial. Gut 2011; 60: [PMID: DOI: /gut ] 27 Lichtenstein GR, Yan S, Bala M, Blank M, Sands BE. Infliximab maintenance treatment reduces hospitalizations, surgeries, and procedures in fistulizing Crohn s disease. Gastroenterology 2005; 128: [PMID: DOI: /j.gastro ] 28 Vogelaar L, Spijker AV, van der Woude CJ. The impact of biologics on health-related quality of life in patients with inflammatory bowel disease. Clin Exp Gastroenterol 2009; 2: [PMID: ] 29 Peyrin-Biroulet L, Deltenre P, de Suray N, Branche J, Sandborn WJ, Colombel JF. Efficacy and safety of tumor necrosis factor antagonists in Crohn s disease: meta-analysis of placebo-controlled trials. Clin Gastroenterol Hepatol 2008; 6: [PMID: DOI: /j.cgh ] 30 Steenholdt C, Brynskov J, Thomsen OO, Munck LK, Fallingborg J, Christensen LA, Pedersen G, Kjeldsen J, Jacobsen BA, Oxholm AS, Kjellberg J, Bendtzen K, Ainsworth MA. Individualised therapy is more cost-effective than dose intensification in patients with Crohn s disease who lose response to anti-tnf treatment: a randomised, controlled trial. Gut 2013; Epub ahead of print [PMID: DOI: / gutjnl ] 31 Mould DR, Green B. Pharmacokinetics and pharmacodynamics of monoclonal antibodies: concepts and lessons for drug development. BioDrugs 2010; 24: [PMID: DOI: / ] 32 Maser EA, Villela R, Silverberg MS, Greenberg GR. Association of trough serum infliximab to clinical outcome after scheduled maintenance treatment for Crohn s disease. Clin Gastroenterol Hepatol 2006; 4: [PMID: DOI: /j.cgh ] 33 Yarur AJ, Deshpande AR, Sussman DA, Hauenstein A, Lockton S, Barkin JS, Singh S, Abreu MT. Serum Adalimumab Levels and Antibodies Correlate With Endoscopic Intestinal Inflammation and Inflammatory Markers in Patients With Inflammatory Bowel Disease. Gastroenterology 2012; 144: S Velayos FS, Sheibani S, Lockton S, Hauenstein S, Singh S, Terdiman JP, Mahadevan U. Prevalence of Antibodies to Adalimumab (ATA) and Correlation Between Ata and Low Serum Drug Concentration on CRP and Clinical Symptoms in a Prospective Sample of IBD Patients. Gastroenterology 2012; 144: S Baert F, Noman M, Vermeire S, Van Assche G, D Haens G, Carbonez A, Rutgeerts P. Influence of immunogenicity on the long-term efficacy of infliximab in Crohn s disease. N Engl J Med 2003; 348: [PMID: DOI: / NEJMoa020888] 36 Nanda KS, Cheifetz AS, Moss AC. Impact of antibodies to infliximab on clinical outcomes and serum infliximab levels in patients with inflammatory bowel disease (IBD): a metaanalysis. Am J Gastroenterol 2013; 108: 40-47; quiz 48 [PMID: DOI: /ajg ] 37 Karmiris K, Paintaud G, Noman M, Magdelaine-Beuzelin C, Ferrante M, Degenne D, Claes K, Coopman T, Van Schuerbeek N, Van Assche G, Vermeire S, Rutgeerts P. Influence of trough serum levels and immunogenicity on long-term outcome of adalimumab therapy in Crohn s disease. Gastroenterology 2009; 137: [PMID: DOI: /j.gastro ] 38 West RL, Zelinkova Z, Wolbink GJ, Kuipers EJ, Stokkers PC, van der Woude CJ. Immunogenicity negatively influences the outcome of adalimumab treatment in Crohn s disease. Aliment Pharmacol Ther 2008; 28: [PMID: DOI: /j x] 39 Wang SL, Hauenstein S, Ohrmund L, Shringarpure R, Salbato J, Reddy R, McCowen K, Shah S, Lockton S, Chuang E, Singh S. Monitoring of adalimumab and antibodies-toadalimumab levels in patient serum by the homogeneous mobility shift assay. J Pharm Biomed Anal 2013; 78-79: [PMID: DOI: /j.jpba ] 3483 April 7, 2014 Volume 20 Issue 13

109 Yarur AJ et al. Therapeutic drug monitoring in IBD 40 Colombel JF, Sandborn WJ, Reinisch W, Mantzaris GJ, Kornbluth A, Rachmilewitz D, Lichtiger S, D Haens G, Diamond RH, Broussard DL, Tang KL, van der Woude CJ, Rutgeerts P. Infliximab, azathioprine, or combination therapy for Crohn s disease. N Engl J Med 2010; 362: [PMID: DOI: /NEJMoa ] 41 Lichtenstein GR, Diamond RH, Wagner CL, Fasanmade AA, Olson AD, Marano CW, Johanns J, Lang Y, Sandborn WJ. Clinical trial: benefits and risks of immunomodulators and maintenance infliximab for IBD-subgroup analyses across four randomized trials. Aliment Pharmacol Ther 2009; 30: [PMID: DOI: / j x] 42 Magro F, Rodrigues-Pinto E, Santos-Antunes J, Vilas-Boas F, Lopes S, Nunes A, Camila-Dias C, Macedo G. High C-reactive protein in Crohn s disease patients predicts nonresponse to infliximab treatment. J Crohns Colitis 2014; 8: [PMID: DOI: /j.crohns ] 43 Hibi T, Sakuraba A, Watanabe M, Motoya S, Ito H, Sato N, Yoshinari T, Motegi K, Kinouchi Y, Takazoe M, Suzuki Y, Matsumoto T, Kawakami K, Matsumoto T, Hirata I, Tanaka S, Ashida T, Matsui T. C-reactive protein is an indicator of serum infliximab level in predicting loss of response in patients with Crohn s disease. J Gastroenterol 2014; 49: [PMID: DOI: /s ] 44 Fasanmade AA, Adedokun OJ, Blank M, Zhou H, Davis HM. Pharmacokinetic properties of infliximab in children and adults with Crohn s disease: a retrospective analysis of data from 2 phase III clinical trials. Clin Ther 2011; 33: [PMID: DOI: /j.clinthera ] 45 Brandse JF, Wildenberg M, de Bruyn JR, Wolbink GJ, Lowenberg M, Posioen C, van den Brink GR, D Haens GR. Fecal Loss of Infliximab As a Cause of Lack of Response in Severe Inflammatory Bowel Disease. Gastroenterology 2012; 157: S-1 46 Ternant D, Aubourg A, Magdelaine-Beuzelin C, Degenne D, Watier H, Picon L, Paintaud G. Infliximab pharmacokinetics in inflammatory bowel disease patients. Ther Drug Monit 2008; 30: [PMID: DOI: / FTD.0b013e318180e300] 47 Bultman E, de Haar C, van Liere-Baron A, Verhoog H, West RL, Kuipers EJ, Zelinkova Z, van der Woude CJ. Predictors of dose escalation of adalimumab in a prospective cohort of Crohn s disease patients. Aliment Pharmacol Ther 2012; 35: [PMID: DOI: / j x] 48 Peyrin-Biroulet L, Gonzalez F, Dubuquoy L, Rousseaux C, Dubuquoy C, Decourcelle C, Saudemont A, Tachon M, Béclin E, Odou MF, Neut C, Colombel JF, Desreumaux P. Mesenteric fat as a source of C reactive protein and as a target for bacterial translocation in Crohn s disease. Gut 2012; 61: [PMID: DOI: /gutjnl ] 49 Seow CH, Newman A, Irwin SP, Steinhart AH, Silverberg MS, Greenberg GR. Trough serum infliximab: a predictive factor of clinical outcome for infliximab treatment in acute ulcerative colitis. Gut 2010; 59: [PMID: DOI: /gut ] 50 Afif W, Loftus EV, Faubion WA, Kane SV, Bruining DH, Hanson KA, Sandborn WJ. Clinical utility of measuring infliximab and human anti-chimeric antibody concentrations in patients with inflammatory bowel disease. Am J Gastroenterol 2010; 105: [PMID: DOI: / ajg ] 51 Roblin X, Marotte H, Rinaudo M, Del Tedesco E, Moreau A, Phelip JM, Genin C, Peyrin-Biroulet L, Paul S. Association between pharmacokinetics of adalimumab and mucosal healing in patients with inflammatory bowel diseases. Clin Gastroenterol Hepatol 2014; 12: e2 [PMID: DOI: /j.cgh ] 52 Velayos FS, Kahn JG, Sandborn WJ, Feagan BG. A test-based strategy is more cost effective than empiric dose escalation for patients with Crohn s disease who lose responsiveness to infliximab. Clin Gastroenterol Hepatol 2013; 11: [PMID: DOI: /j.cgh ] 53 Vande Casteele N, Gils A, Singh S, Ohrmund L, Hauenstein S, Rutgeerts P, Vermeire S. Antibody response to infliximab and its impact on pharmacokinetics can be transient. Am J Gastroenterol 2013; 108: [PMID: DOI: /ajg ] P- Reviewers: Desai ND, Kayadibi H, Lin JK S- Editor: Qi Y L- Editor: A E- Editor: Wu HL 3484 April 7, 2014 Volume 20 Issue 13

110 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (3): Inflammatory bowel disease TOPIC HIGHLIGHT Advanced therapeutic endoscopist and inflammatory bowel disease: Dawn of a new role Kunjam Modha, Udayakumar Navaneethan Kunjam Modha, Udayakumar Navaneethan, Digestive Disease Institute-Desk A31, The Cleveland Clinic, Cleveland, OH 44195, United States Author contributions: Modha K and Navaneethan U contributed to study concept and paper preparation; Navaneethan U contributed to design and critical revisions. Supported by Research grants from the Inflammatory Bowel Disease Working Group and the American College of Gastroenterology to Navaneethan U Correspondence to: Udayakumar Navaneethan, MD, FACP, Digestive disease Institute-Desk A31, The Cleveland Clinic, 9500 Euclid Ave., Cleveland, OH 44195, United States. navaneu@ccf.org Telephone: Fax: Received: October 11, 2013 Revised: January 7, 2014 Accepted: January 20, 2014 Published online: April 7, 2014 Abstract Endoscopy plays a key role in the diagnosis and treatment of patients with inflammatory bowel disease (IBD). Colonoscopy has been traditionally used in the diagnosis of IBD and helps in determination of an important end point in patient management, mucosal healing. However, the involvement of an advanced endoscopist has expanded with innovations in therapeutic and newer imaging techniques. Endoscopists are increasingly being involved in the management of anastomotic and small bowel strictures in these patients. The advent of balloon enteroscopy has helped us access areas not deemed possible in the past for dilations. An advanced endoscopist also plays an integral part in managing ileal pouchanal anastomosis complications including management of pouch strictures and sinuses. The use of rectal endoscopic ultrasound has been expanded for imaging of perianal fistulae in patients with Crohn s disease and appears much more sensitive than magnetic resonance imaging and exam under anesthesia. Advanced endoscopists also play an integral part in detection of dysplasia by employing advanced imaging techniques. In fact the paradigm for neoplasia surveillance in IBD is rapidly evolving with advancements in endoscopic imaging technology with pancolonic chromoendoscopy becoming the main imaging modality for neoplasia surveillance in IBD patients in most institutions. Advanced endoscopists are also called upon to diagnose primary sclerosing cholangitis (PSC) and also offer options for endoscopic management of strictures through endoscopic retrograde cholangiopancreatography (ERCP). In addition, PSC patients are at increased risk of developing cholangiocarcinoma with a 20% lifetime risk. Brush cytology obtained during ERCP and use of fluorescence in situ hybridization which assesses the presence of chromosomal aneuploidy (abnormality in chromosome number) are established initial diagnostic techniques in the investigation of patients with biliary strictures. Thus advanced endoscopists play an integral part in the management of IBD patients and our article aims to summarize the current evidence which supports this role and calls for developing and training a new breed of interventionalists who specialize in the management of IBD patients and complications specific to those patients Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Inflammatory bowel disease; Endoscopy; Therapeutic endoscopy; Primary sclerosing cholangitis Core tip: Endoscopy plays a key role in the diagnosis and treatment of patients with inflammatory bowel disease. The involvement of an advanced endoscopist has expanded with innovations in designs of endoscopes and newer imaging techniques. Our article aims to summarize the current evidence which supports the role of an advanced endoscopist in the management of colonic and ileal pouch strictures, biliary strictures in patients with primary sclerosing cholangitis, endoscopic 3485 April 7, 2014 Volume 20 Issue 13

111 Modha K et al. Endoscopy and inflammatory bowel disease diagnosis of colonic fistulae and surveillance of colon neoplasia and cholangiocarcinoma. Modha K, Navaneethan U. Advanced therapeutic endoscopist and inflammatory bowel disease: Dawn of a new role. World J Gastroenterol 2014; 20(13): Available from: URL: DOI: INTRODUCTION Crohn s disease (CD) and ulcerative colitis (UC) are a group of inflammatory bowel diseases (IBD) that have environmental, immunological and bacterial etiologies. The role of an endoscopist has been well defined in the initial diagnosis of these disorders, assessment of disease severity and differentiation between the two disease processes. Previous studies have reported that in 80% of CD patients, at last one surgical resection will be required within 10 years of CD diagnosis [1,2]. Although surgical treatment is effective for CD strictures, there is invariably a high risk of recurrence of CD which may result in repeat surgery in up to 34% of patients [1,3]. Repeated surgery can result in complications related to short bowel syndrome, requirement for total parenteral nutrition and its attendant complications. The advent of endoscopy in the management of complicated CD strictures has changed the approach to the management of anastomotic and small bowel strictures in these patients. The advent of balloon enteroscopy has helped us access areas not deemed possible in the past for dilations. An advanced endoscopist also plays an integral part in managing ileal pouch-anal anastomosis (IPAA) complications including management of pouch strictures and sinuses. Colorectal cancer (CRC) is a serious potential complication of IBD. Advanced endoscopists play an important role in detection of dysplasia by employing advanced imaging techniques to identify early and subtle neoplastic lesions [4]. In addition, primary sclerosing cholangitis (PSC) a chronic, cholestatic disorder is seen in 2.4%-7.5% of patients with UC [5] and about 3.4% of patients with CD [6]. Advanced endoscopists are called upon to diagnose PSC and also offer options for endoscopic management of strictures through endoscopic retrograde cholangiopancreatography (ERCP). In addition, PSC patients are at increased risk of developing cholangiocarcinoma (CCA) with a 20% lifetime risk [7,8]. Brush cytology obtained during ERCP and use of fluorescence in situ hybridization (FISH) which assesses the presence of abnormality in chromosome number are established initial diagnostic techniques in the investigation of patients with biliary strictures [9]. The aim of our review is to highlight the current evidence which supports the role of an advanced endoscopist in the management of IBD and complications specific to them. For the purpose of this article, we will discuss the role of an advanced endoscopist under various sections. Figure 1 illustrates the various roles of an advanced endoscopist in managing inflammatory bowel disease patient. Inflammatory bowel strictures The Vienna Classification describes three distinct groups of CD: inflammatory, stricturing and penetrating. It also demonstrated an association between location and disease behavior. Stricturing disease is predominant in the terminal ileum and ileocolonic locations [10]. Subsequent studies using this classification have confirmed that most patients over the course of time will develop a penetrating or stricturing complication with stricture formation being the second most common and that this change is governed by location of disease [11,12]. Data from the TREAT (the Crohn s therapy, resource, evaluation, and assessment tool) registry suggested factors associated with stricture formation. These were CD severity at the time of event onset (HR = 2.35, 95%CI: ); CD duration (HR = 1.02, 95%CI: ); ileal disease (HR = 1.56, 95%CI: ); and new corticosteroid use (HR = 2.85, 95%CI: ) [13]. Strictures in CD can occur de novo, at sites of bowel anastomosis or in the ileal pouch. Strictures are believed to be either inflammatory or fibrotic [13]. Inflammatory strictures have the option of being treated by medical therapy. Even then, when these patients are followed, 64% of these patients require surgery. CD patients with ileocolonic disease do worse with medical therapy (P = 0.026) and may require surgery sooner than patients with ileal disease (P = 0.023) [14]. The fibrotic strictures are largely treated surgically with either intestinal resection or strictureplasty [15]. Although the latter has the advantage of preserving bowel length, it has still been associated with a significant operative recurrence rate of 34% during a median follow up period of 7.5 years [16]. These results seem comparable to those from other review studies [17,18], one of them a meta-analysis [17], in which surgical recurrence rates have been cited as 24% (median surgical rate at 46 mo) and 23%. Younger patients tend to run an aggressive course with a shorter duration to reoperation [16]. Moreover, an 82% rate of second operation has been reported with upto 33% patients requiring more than 2 surgeries leading to risk of developing short bowel syndrome [19]. This means that avoidance of repeated surgeries is an important factor in considering alternative therapies. Endoscopic balloon dilation has been one such treatment alternative. Additionally, endoscopic balloon dilatation could be considered an adjunct to surgery given that it has been shown to add at least 50% efficacy to the initial surgery by prolonging the surgery free period. This was deduced after comparing interval of 6 years from surgery to first endoscopic dilation with the post dilatation surgery free period of 3 years [20]. It also has the advantages of reduced invasiveness and bowel preservation. Endoscopic balloon dilation There have been several studies aimed at reporting the clinical efficacy, technical feasibility and short and long 3486 April 7, 2014 Volume 20 Issue 13

112 Modha K et al. Endoscopy and inflammatory bowel disease Advanced endoscopist and IBD IBD stricture IBD and concomitant PSC Cholangiocarcinoma Colorectal neoplasia surveillance Perianal disease IPAA pouch sinuses and strictures Biliary stricture dilatation with or without stenting Cholangioscopy Standard colonoscopy Rectal endoscopic ultrasound Endoscopic dilatation ERCP with brushings and FISH Chromoendoscopy Self expanding metallic stents ERCP with pcle Virtual chromoendoscopy with NBI confocal laser endomicroscopy Figure 1 Figure illustrates the various roles of an advanced endoscopist in managing inflammatory bowel disease patient. IBD: Inflammatory bowel disease; IPAA: Ileo-pouch anal anastomosis; PSC: Primary sclerosing cholangitis; ERCP: Endoscopic retrograde cholangiopancreaticography; FISH: Fluorescence in situ hydribidization; pcle: Probe based confocal laser endomicroscopy; NBI: Narrowband imaging. term results of endoscopic balloon dilatation. Almost all studies have used resolution of symptoms and/or surgery free period as outcomes. A systemic review summarized the results of important studies [20]. Most studies included had less than 60 patients. Most of them included postsurgical strictures. The meta-analysis clearly highlighted the drawbacks with making clear conclusions from the literature because of varying caliber of endoscopic balloons used for dilation from 18 to 25 mm, different approaches that were used, number of dilatations in the same endoscopic sessions and heterogeneity concerning the duration of each single dilatation [20]. Overall, technical success was achieved in 86% patients. The complication rates were < 5% in all studies barring two which reported a higher complication rate [21,22]. The reasons for this have been unclear, aggressive dilatation in one of them [21] may have been a contributing factor. The only factor that significantly affected dilatation efficacy and surgery-free follow up was length of stricture. Naïve vs postsurgical, steroid injection, active vs inactive CD were all deemed to be non significant [20]. Importantly, endoscopic dilatation was successful in avoiding surgery at the end of the follow-up in 112 of the total 347 (67%) patients that were included in this review [20]. If the patients who had failed for technical reasons were excluded, the success rate measured by avoidance of surgery was up to 78% [20]. Subsequent to the meta-analysis, more studies have been published on the efficacy of endoscopic dilation and have shown similar short (51%-89%) and long (52%-89%) term success [23-28]. Most strictures included in these studies have been anastomotic with the exception of the Mueller et al [26] study, in which 69% had de novo strictures. The study by Gustavsson et al [27] is the largest study to date, including a total of 178 patients, and the one with the longest follow-up period (median 12 years). Most patients had either ileal or ileocolonic disease, and approximately 40% had stricturing disease at presentation. Most of the patients (80%) had anastomotic strictures. There was no difference in efficacy based on the etiology of their strictures whether anastomotic or de novo. Bowel perforation occurred in 1.4% of patients and use of 25 mm balloon was associated with a 9.3% complication rate, as compared to 3.5% for the other sizes (P < 0.01). Another prospective single center study from Germany of 55 patients, with 74 symptomatic strictures reported their efficacy [26]. Majority of patients in this study (69%) had de novo strictures. There was a 95% initial success rate, and 76% patients never required repeat treatment over the period of follow-up. 24% patients did eventually receive surgery over the follow-up period. Stricture length was the main factor that predicted the need for surgery. Interestingly, stricture location at ileocecal valve and stricture associated with fistula were significant predictors of 3487 April 7, 2014 Volume 20 Issue 13

113 Modha K et al. Endoscopy and inflammatory bowel disease a negative outcome [28]. Smoking has been reported to be a significant patient variable that negatively affects surgery free period [29,30] and doubles the risk of recurrent stricture formation requiring a new dilatation after the first one (P = 0.022) [31]. Given the high rate of stricture recurrence after dilation, intralesional injection of medications after dilation has been studied. A pilot study comparing intralesional steroid injection after balloon dilatation vs placebo did not find a reduction in time to redilatation [32]. This is in contrast to a pediatric study of 29 patients that reported a significant trend of patients who did not receive intralesional steroids towards redilation and surgery [33]. Effect of intralesional injection of infliximab has been studied in small number of patients but consensus is lacking. Thienpont et al [34] reported no significant effect of active disease at the time of dilatation or systemic medical therapy afterwards on redilatation or surgery. Thus, there is no clear evidence at this time to support the role of intralesional injection of medications following dilation. We routinely perform endoscopic dilation with a 16-, 17-, and 18-mm through the scope (TTS) balloon (Boston Scientific, Inc, Boston, MA) or a 18-, 19-, and 20-mm TTS balloon with guidewire assistance. If possible, retrograde dilation with passage of the endoscope beyond the stricture and introduction of the balloon and pulling the endoscope backward and dilating the stricture is preferred. Endoscopic balloon dilation with stenting Metal stents have been reported as an effective alternative to surgery for the palliation of patients with colorectal neoplastic obstruction. Data regarding their use in treating benign naïve or postsurgical strictures in CD is limited and controversial. Recent studies that have used self expanding metallic [35,36] and biodegradable [37-39] stents have reported a high incidence of migration [37,39]. On the other hand clinical success has ranged from 45% to 80%. The majority of stents in these studies were placed in postsurgical strictures. Overall, efficacy of endoscopic dilatation in the treatment of small and large bowel strictures is promising with an acceptable rate of complications. Length of stricture and location of stricture are important considerations. We have not employed stenting in the management of IBD related strictures. We prefer the use of needle-knife for strictures which are refractory to balloon dilation for management. Strictures in IPAA The role of an advanced endoscopist in managing IPAA complications mainly caters to strictures and sinuses. IPAA strictures can occur at the pouch inlet, outlet, afferent limb or pouch body. An 11-year-experience with 1005 patients after restorative proctocolectomy and IPAA reported stricture formation in 14% of patients of which the majority (97.9%) were successfully treated with digital or bougie dilatation and only 2.1% required surgery [40]. Another large retrospective study with 1884 patient re- ported a similar incidence (11.2%) of stricture formation but a much higher (12%) rate of surgery to salvage pouch function. Dilatation of non fibrotic strictures was more successful than fibrotic strictures (P = ) [41]. A more recent study in which a cumulative of 646 strictures were dilated reported that 87.3% patients over a median follow up of 9.6 years were able to retain their pouches. It concurred that endoscopic dilatation of pouch strictures is efficacious and safe with a low rate of complications when attempted by an experienced endoscopist [42]. Pouch sinus is typically a late presentation of an initial anastomotic leak. The most common location of a pouch sinus is the pouch-anal anastomotic site at the presacral space. Presenting symptoms include perianal pain, pelvic pressure/discomfort and/or evidence of pelvic sepsis or pouchitis, CD of the pouch, or refractory cuffitis; others may be asymptomatic. Sinus opening and sinus tract can be detected by a combined application of pouchoscopy, contrast pouchogram, examination under anesthesia and pelvic magnetic resonance imaging (MRI). Treatment usually includes incision and drainage of the chronically infected superficial sinuses [43]. Fibrin glue injection of the sinus may be attempted [44]. Patients with a long sinus track who fail to heal are suitable for redo pouch. An alternative strategy that has only recently been introduced and studied at our institution is endoscopic needle knife therapy for pouch sinuses. A total of 65 patients with pouch sinuses were treated with needle-knife therapy of which 84.6% achieved a complete or partial response. Duration from colectomy to needle knife treatment and complex nature of sinuses were inversely proportional to healing of sinuses [45]. Endoscopic ultrasonography in the evaluation of CD fistulae Approximately, 25% of all patients with CD develop a perianal fistula, with fistulae more frequent in patients with involvement of the rectum [46,47]. Identification of fistulae is difficult with digital rectal examination alone or even with exam under anesthesia (EUA) because perianal disease is associated with induration and scarring. Endoscopic ultrasonography (EUS) has been used in this setting to help in the evaluation of CD fistulae. Rectal EUS is performed by introducing a radial probe into the distal rectum and anal canal while the patient is in the left lateral position [48]. On EUS, the fistulae in the setting of CD appear as hypoechoic structures, but the presence of air or gas in the fistula tract may make the tract internally hyperechoic [48]. An abscess in the setting of CD fistulae can also be visualized as an anechoic or hypoechoic mass in the perianal region [48]. Some investigators have used hydrogen peroxide injection into the cutaneous fistula site to enhance visualization as it creates bubbles that appear hyperechoic and thus make the fistula easier to identify. The accuracy of rectal EUS in the evaluation of perianal disease has been demonstrated in 3 prospective, blinded studies [49-51]. One of these studies compared EUS to computerized tomography (CT) in 25 patients with 3488 April 7, 2014 Volume 20 Issue 13

114 Modha K et al. Endoscopy and inflammatory bowel disease suspected perianal CD [49]. Confirmation of fistulae was done at the time of surgery. EUS was found to be more accurate than CT in the evaluation of perianal fistulae (82% vs 24%) [49]. In another study, rectal EUS was compared with pelvic MRI and EUA in 22 patients with CD and perianal fistulae. Rectal EUS was found to be the most sensitive modality for imaging (82%) when compared with pelvic MRI (50%) [50]. Rectal EUS in this study was performed with only a 7-MHz linear scanning probe. Another study comparing EUS to MRI found both to be equally accurate in the assessment of CD perianal fistulae (91% vs 87%) [51]. Studies have also used EUS to monitor fistula healing and/or guide treatment. A small, randomized, prospective study showed a benefit to the use of EUS monitoring for fistula healing [52]. Thus, it seems that a team of colorectal surgeons and advanced endoscopists seem to be the future in the evaluation and management of perianal CD. IBD with concomitant primary sclerosing cholangitis PSC is a chronic, cholestatic liver disease characterized by inflammation and fibrosis of both intrahepatic and extrahepatic bile duct leading to the formation of bile duct strictures. An advanced endoscopist is integral in diagnosing PSC. Although magnetic resonance cholangiopancreatography (MRCP) is the initial preferred non-invasive technique to diagnose PSC, often ERCP is required to confirm diagnosis and rule out a dominant stricture. An advanced endoscopist is often called upon to manage patients with IBD and PSC who have concomitant dominant biliary stricture in the setting of PSC. A dominant stricture has been defined as a stenosis with a diameter of 1.5 mm in the common bile duct or of 1 mm in the hepatic duct [53,54]. It is a frequent finding and occurs in 36%-57% of patients during follow up [55]. It should always raise the suspicion of the presence of a CCA. The stricturing process may cause extrahepatic biliary obstruction leading to development of symptoms. Patient with jaundice, pruritis, right upper quadrant pain and abnormal biochemical studies have been deemed appropriate candidates for therapy. The goal of therapy is to relieve biliary obstruction. The optimal non surgical management of these dominant strictures is still debatable. Endoscopic balloon dilatation both with and without stenting has been studied. Long term stent therapy (3 mo) has been shown to have a high rate (close to 50%) of complications of cholangitis/ jaundice attributed to stent occlusion [56]. However, short term stenting (11 d) was associated with a lower rate (7%) of these complications, at the same time producing significant effects in symptom reduction and biochemical resolution of cholestasis. Additionally, 81% remained asymptomatic over a 19 mo follow up period and none of the patients had recurrence of clinical/biochemical cholestasis [57]. Another study reported similar positive results after short term stent therapy with significant effects on resolution of symptoms and biochemical cholestasis with 80% of patients remaining re-intervention free at the end of 1 year [58]. A randomized trial comparing balloon dilatation and stenting has not been performed. However, a retrospective study that compared at endoscopic dilatation with dilatation + stenting reported an increased number of complications and incidence of cholangitis in the group that received both without significant difference in improvement of cholestasis [59]. It has been suggested that the group that received both interventions may have been sicker as stenting was done only when dilatation alone did not improve biliary drainage [60]. Moreover, half of the stents were placed percutaneously and the authors reported a significantly higher rate of complications with percutaneous placement of stents as compared to the endoscopic approach. One study aimed at assessing a survival benefit of endoscopic treatment of strictures reported a 5 years survival that was significantly (P = 0.027) higher over that predicted by the Mayo risk score [61]. Indirect evidence from other studies has supported this finding [62,63]. Overall, endoscopic therapy of strictures has been has been proven to be a safe and efficacious mode of treating primary sclerosing cholangitis associated strictures that helps in amelioration of symptoms and cholestasis with a low rate of complications. If stenting is considered, short term therapy is deemed best. Surveillance for cholangiocarcinoma Patients with PSC are at risk of developing CCA. The risk after 10 years and 20 years is 9% and 19% respectively [64]. Patients with deterioration in functional status, worsening liver functions and/or weight loss should be evaluated for CCA. The distinction between a benign dominant stricture and CCA, however, has remained a challenge. A cut off value of CA19-9 (cancer antigen 19-9) of > 130 U/mL in symptomatic patients has a sensitivity and specificity of 79% and 98% respectively [65]. Its value as a screening tool in asymptomatic patients remains to be defined. CCA often presents in its advanced stage when identification of a mass lesion makes a diagnosis of CCA very likely. In early stages, however, diagnosis is difficult. A study that followed 230 patients over 6 years reported sensitivity of ultrasound, CT, MRI as 57%, 75% and 63% respectively when imaging alone was considered. The positive predictive value of ERCP, MRCP and MRCP + MRI was 23%, 21% and 23 % respectively [66]. Bile duct brushings are the most commonly used method for tissue sampling during ERCP are now routinely obtained at the time of ERCP [7-9]. The vast majority of extrahepatic CCA are periductal, cancers, and do not demonstrate mass lesions on imaging studies. Brush cytology obtained during ERCP is the usual diagnostic technique in the investigation of patients with biliary strictures. However the sensitivity of brushings is low for distinguishing benign strictures and CCA with a 43% sensitivity and a diagnostic accuracy of 50%-60% [7-9]. At the time of ERCP brushings of stricture for cytology, another set of brushings for FISH are obtained in our institution. FISH probes are used to target the centromeric regions of chromosomes 3, 7 and 17 and the 3489 April 7, 2014 Volume 20 Issue 13

115 Modha K et al. Endoscopy and inflammatory bowel disease 9p21 band (p16). Some studies have considered positive FISH based on polysomy only, while some have considered trisomy or tetrasomy as positive too. In a recent meta-analysis from our group, we pooled all the available evidence in order to better define the utility of FISH for detection of CCA. Our unpublished observations show that any FISH positivity has a pooled sensitivity and specificity of 68% (95%CI: 61%-74%) and 70% (95%CI: 66%-73%) respectively. Thus both brushings for cytology and FISH obtained at the time of ERCP contribute significantly in diagnosing CCA in PSC patients [9]. Recent studies have demonstrated promising results of cholangioscopy in the diagnosis of CCA. Tischendorf et al [67] prospectively studied 53 patients out of which 12 patients were found to have CCA based on tissue sampling. Patients underwent cholangioscopy in addition to ERCP, cholangioscopy was found to have higher sensitivity and significantly higher specificity, positive predictive value and negative predictive value than endoscopic retrograde cholangiography. A single center prospective study involving 36 patients and using peroral cholangioscopy and biopsy reported an overall accuracy of 89% in differentiating benign from malignant stenoses [68]. ERCP with probe-based confocal laser endomicroscopy is an emerging technology that enables high resolution assessment of gastrointestinal mucosal histology thereby allowing examination of optical biopsies and exponentially expanding the scope of imaging capabilities. The examination is done in vivo and images are displayed in real time. A single center small case series of 15 patients and 21 dominant stenoses reported 100% sensitivity and 100% negative predictive value in excluding neoplasia [69]. It concluded that if verified in large prospective studies, this technology could be used to risk stratify strictures in patients with PSC. Also, chromoendoscopy using methylene blue was studied for the first time in staining tissue of the bile duct [70]. It helped indentify normal, dysplastic and inflamed mucosa of the biliary tract as was subsequently proven by follow up or histology. A homogenous staining was suggestive normal tissue, absence of it on circumscribed lesions or diffuse staining predicted neoplastic or inflamed tissue [70]. We routinely send 2 sets of brushes with PSC-related dominant strictures; one for routine cytology and the other for FISH analysis. Thus an advanced endoscopist forms an integral part of the team managing IBD patients with associated PSC and its related complications and surveillance of these patients for CCA. IBD and colorectal neoplasia surveillance Patients with IBD are at increased risk of CRC. Incidence of cancer has been more extensively studied in relation to UC than Crohn s. The risk in UC is increased with the duration and extent of disease. A meta-analysis reported the risk is 2% at 10 years, 8% at 20 years, and 18% at 30 years of disease [71]. Patients with extensive colitis are at increased risk as compared to those with left sided colitis. Compared to age-matched controls, the risk begins to increase about 8-10 years after onset of symptoms. Patients with PSC are also more predisposed to developing CRC. Earlier studies on the risk of CRC with CD have been inconclusive. It is now believed that risk of developing CRC in UC and CD is nearly identical [72]. There have not been randomized controlled trials proving the effectiveness of surveillance colonoscopy. There have been three case series that have studied this [73-75]. A Cochrane analysis of these studies concluded that there was no clear evidence that surveillance improves survival. There is evidence that cancer tends to be detected earlier in patients who undergo surveillance and they are likely to have a better prognosis although lead time bias may affect this apparent benefit. It stated that there may be indirect evidence that surveillance is effective at reducing death associated with IBD and that it may be acceptably cost-effective [76]. Interval colonoscopies with random biopsies of abnormally appearing mucosa and targeted biopsies of suspicious lesions have been recommended. Newer methods aimed at detecting abnormal/dysplastic mucosa have been studied to help make this more effective. An advanced endoscopist with experience in complex mucosal imaging would play an important role in the surveillance of IBD patients for CRC. Newer endoscopic imaging modalities including high-definition endoscopy, chromoendoscopy, virtual chromoendoscopy and confocal laser endomicroscopy have the potential to significantly improve the detection and characterization of flat and subtle dysplasia, thereby setting the stage for a system of targeted neoplasia detection without random biopsies in IBD patients. Chromoendoscopy is a dye-spraying technique that highlights the borders and surface architecture of neoplastic lesions, thereby unmasking and delineating subtle lesions and aiding in the differentiation of neoplastic and non-neoplastic tissue [76]. Chromoendoscopy has been compared to standard-definition endoscopy for detection of neoplasia in both IBD and non-ibd patients and shown to be superior [77]. However, careful cleansing and inspection of the entire colonic mucosal surface needs to be done to detect neoplasia [76]. Importantly, the use of chromoendoscopy does not increase the mean procedure time and has been shown to be no better than whitelight endoscopy with random and targeted biopsies [78,79]. Chromoendoscopy described above using methylene blue has been studied in UC patients. The study included 102 patients, each of who had biopsies by different 3 techniques: standard colonoscopy with random biopsies, targeted biopsies and dye targeted biopsies in which methylene blue was segmentally applied throughout colon and abnormal mucosa that was made visible by dye spray was then biopsied. It concluded that dye targeted biopsies detected more dysplasia than random biopsies (P = 0.001) and more than targeted non dye biopsies. (P = 0.057) [78]. The disadvantages of this technique include the cost, time consuming nature and the fact the dye does not always coat the mucosa evenly and does not allow for a detailed analysis of the subepithelial network. Virtual chromoendoscopy technologies have also 3490 April 7, 2014 Volume 20 Issue 13

116 Modha K et al. Endoscopy and inflammatory bowel disease been employed including narrowband imaging (NBI; Olympus, Tokyo, Japan), i-scan (Pentax, Tokyo, Japan), Fuji Intelligent Chromo Endoscopy (FICE; Fujinon, Tokyo, Japan) with use of selective light filters or postimage processing techniques to improve visualization of vascular structures [80]. A recent randomized parallel group study with 112 patients randomized to get colonoscopy using either NBI or white lights reported no significant difference in detection of dysplasia [81]. Another study involving 60 patients concluded that NBI was a less time consuming and equally efficacious method compared to chromoendoscopy to detect intraepithelial neoplasia in long standing IBD patients but had a high lesion and patient miss rate and cannot be recommended as a standard technique [82]. Confocal laser endomicroscopy is another advanced imaging technique that permits direct histologic assessment of mucosa at the cellular and subcellular levels in vivo [83]. The potential application of technology in IBD patients would be in combination with white-light endoscopy and chromoendoscopy to more accurately predict lesion histology and thus aid in decision making regarding lesion resectability in the setting of chronic colitis [84]. Endoscopic mucosal resection (EMR) is being used for management of specific raised lesions in chronic UC patients getting surveillance colonoscopy for dysplasia. In a study, EMR was used to remove 79 flat Paris 0-Ⅱ lesions with a recurrence rate of 2.4% at 3 mo with no additional lesions detected in a 4-year follow-up period [85]. Thus management of dysplasia is emerging as a new intervention to develop in the area of IBD. 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119 Modha K et al. Endoscopy and inflammatory bowel disease Gastroenterology 2003; 124: [PMID: DOI: /gast ] 78 Marion JF, Waye JD, Present DH, Israel Y, Bodian C, Harpaz N, Chapman M, Itzkowitz S, Steinlauf AF, Abreu MT, Ullman TA, Aisenberg J, Mayer L. Chromoendoscopytargeted biopsies are superior to standard colonoscopic surveillance for detecting dysplasia in inflammatory bowel disease patients: a prospective endoscopic trial. Am J Gastroenterol 2008; 103: [PMID: DOI: / j x] 79 Rutter MD, Saunders BP, Schofield G, Forbes A, Price AB, Talbot IC. Pancolonic indigo carmine dye spraying for the detection of dysplasia in ulcerative colitis. Gut 2004; 53: [PMID: DOI: /gut ] 80 Wallace MB, Kiesslich R. Advances in endoscopic imaging of colorectal neoplasia. Gastroenterology 2010; 138: [PMID: DOI: /j.gastro ] 81 Ignjatovic A, East JE, Subramanian V, Suzuki N, Guenther T, Palmer N, Bassett P, Ragunath K, Saunders BP. Narrow band imaging for detection of dysplasia in colitis: a randomized controlled trial. Am J Gastroenterol 2012; 107: [PMID: DOI: /ajg ] 82 Pellisé M, López-Cerón M, Rodríguez de Miguel C, Jimeno M, Zabalza M, Ricart E, Aceituno M, Fernández-Esparrach G, Ginès A, Sendino O, Cuatrecasas M, Llach J, Panés J. Narrow-band imaging as an alternative to chromoendoscopy for the detection of dysplasia in long-standing inflammatory bowel disease: a prospective, randomized, crossover study. Gastrointest Endosc 2011; 74: [PMID: DOI: /j.gie ] 83 Neumann H, Kiesslich R, Wallace MB, Neurath MF. Confocal laser endomicroscopy: technical advances and clinical applications. Gastroenterology 2010; 139: , 392.e1-e2 [PMID: DOI: /j.gastro ] 84 Kiesslich R, Goetz M, Lammersdorf K, Schneider C, Burg J, Stolte M, Vieth M, Nafe B, Galle PR, Neurath MF. Chromoscopy-guided endomicroscopy increases the diagnostic yield of intraepithelial neoplasia in ulcerative colitis. Gastroenterology 2007; 132: [PMID: DOI: / j.gastro ] 85 Hurlstone DP, Sanders DS, Atkinson R, Hunter MD, McAlindon ME, Lobo AJ, Cross SS, Thomson M. Endoscopic mucosal resection for flat neoplasia in chronic ulcerative colitis: can we change the endoscopic management paradigm? Gut 2007; 56: [PMID: DOI: / gut ] P- Reviewers: Rerknimitr R, Vigsnaes LK S- Editor: Wen LL L- Editor: A E- Editor: Wu HL 3494 April 7, 2014 Volume 20 Issue 13

120 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (3): Inflammatory bowel disease From conception to delivery: Managing the pregnant inflammatory bowel disease patient TOPIC HIGHLIGHT Vivian W Huang, Flavio M Habal Vivian W Huang, Division of Gastroenterology, Department of Medicine, University of Alberta, Edmonton, Alberta T6G 2X8, Canada Flavio M Habal, Division of Gastroenterology, Department of Medicine, University Health Network, University of Toronto, Toronto ON M5G 2C4, Canada Author contributions: Huang VW reviewed the literature and drafted the manuscript; Habal FM reviewed the literature and revised the manuscript; Both authors contributed to conception and design of the review, and approved the final version for publication. Correspondence to: Flavio M Habal, MD, Division of Gastroenterology, Department of Medicine, University Health Network, University of Toronto, 200 Elizabeth street, Toronto ON M5G 2C4, Canada. flavio.habal@uhn.ca Telephone: Fax: Received: October 11, 2013 Revised: January 12, 2014 Accepted: February 26, 2014 Published online: April 7, 2014 Abstract Inflammatory bowel disease (IBD) typically affects patients during their adolescent and young adult years. As these are the reproductive years, patients and physicians often have concerns regarding the interaction between IBD, medications and surgery used to treat IBD, and reproduction, pregnancy outcomes, and neonatal outcomes. Studies have shown a lack of knowledge among both patients and physicians regarding reproductive issues in IBD. As the literature is constantly expanding regarding these very issues, with this review, we provide a comprehensive, updated overview of the literature on the management of the IBD patient from conception to delivery, and provide action tips to help guide the clinician in the management of the IBD patient during pregnancy Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Inflammatory bowel disease; Pregnancy; Biologics; Neonatal outcomes Core tip: Inflammatory bowel disease affects people during their reproductive years. Many patients and physicians have concerns about pregnancy in inflammatory bowel disease (IBD), and are unsure about management of IBD during pregnancy. Women with IBD have similar fertility as the general population, with the exception of certain prior surgeries, and actives disease. This review highlights the relative safety of medications used to treat IBD during pregnancy and breastfeeding, and summarizes the updated literature for immunosuppressants and biologics. Good control of disease and clinical remission at the time of conception increases the likelihood of having successful pregnancy outcomes, and quiescent disease during pregnancy. Huang VW, Habal FM. From conception to delivery: Managing the pregnant inflammatory bowel disease patient. World J Gastroenterol 2014; 20(13): Available from: URL: DOI: INTRODUCTION Inflammatory bowel disease (IBD) is a group of chronic bowel diseases, including Crohn s disease and ulcerative colitis, which can affect all aspects of a patient s life. The majority of patients with IBD are diagnosed in their adolescent to young adult years. The diagnosis of IBD and the medications and surgeries used to treat IBD come with many questions and concerns about how they can affect future education and work plans, and relationship and family plans. These questions and concerns not only affect the patient, but also the health care providers who are managing the patient. In particular, the complex interaction between IBD and pregnancy is important, es April 7, 2014 Volume 20 Issue 13

121 Huang VW et al. Managing the pregnant IBD patient pecially because any decisions regarding the management of the patient will not only affect her, but also her fetus. In addition, they will not only affect them currently, but may have long lasting effects. The concerns regarding IBD and reproduction certainly play a central role in any decisions made by the patient and the physician caring for her. Many women with IBD choose not to become pregnant and to remain childless. This concept of voluntary childlessness has been documented in early studies that showed up to 30% of women with IBD (compared to 7% of the general population) had voluntary infertility [1]. More recent studies confirm this voluntary childlessness continues to be an issue, with up to 18% of childless IBD patients indicating that the decision was influenced by IBD related factors [2-4]. The five major concerns of women with IBD that have been reported by previous studies include: fertility/conception, genetics, IBD-related congenital abnormalities, medication effects on pregnant and fetus, and effect of pregnancy on IBD [3-5]. Inadequate physician knowledge regarding reproduction in inflammatory bowel disease, and lack of comfort managing pregnant IBD patients may be contributing to the medical advice component of voluntary childlessness seen in IBD patients. In this review, we aim to address each of these major topics of concern, in the hopes that practitioners will be better equipped with up-to-date information to use in counseling their patients. CONCEPTION Fertility: what are the chances of becoming pregnant? Women with IBD have been shown to have similar fertility as the general population [1,2,6-9]. However, some studies report that Crohn s disease (CD) patients may have slightly decreased fertility [10,11], especially when their disease is active [9] or if they have adhesions from prior surgeries [10,12]. Ulcerative colitis (UC) patients can have normal fertility, however once UC patients have had a surgery, such as restorative proctocolectomy and ileal pouch anal anastomosis, they have an increased risk of infertility up to 3-4 fold [13-16]. It is hypothesized this increased infertility is due to tubal infertility from the adhesions and scarring [15,16], since UC patients who have had laparoscopic IPAA have been shown to have less adhesions [17], and lower infertility rates [18,19]. Although there have been variable reports on the infertility rates among women with Crohn s disease and ulcerative colitis, some of these differences may be attributed to voluntary childlessness. In a recent meta-analysis of eleven studies, the authors found that in women with CD, fertility was reduced 17%-44% compared to controls, but further analysis revealed this to be linked to voluntary childlessness; they did not find any reduction in fertility in women with UC [20]. Action point: In general, women with IBD have similar fertility rates as the general population. Previously reported infertility may be attributed to voluntary childlessness. Women with Crohn s disease may have decreased fertility rates when their disease is active, or if have had prior surgeries. Women with ulcerative colitis who have had pelvic surgery have decreased fertility rates. Genetics: what are the chances of offspring developing IBD? Earlier studies supported strong genetic risks of IBD in offspring of patients with IBD up to 13 times the general population [21,22]. When early twin studies were combined, results showed concordance ranging between 15.4% monozygote twin concordance for ulcerative colitis to 30.3% concordance for Crohn s disease [23]. Monozygote twins are born from the same zygote, so this low concordance rate suggests there are other non-genetic influences, such as environmental factors. A recent study re-ran the Swedish twin registry, which is one of the major data sources for twin studies, and found that previous twin studies overestimated the influence of genetics in Crohn s disease [24]. Although genetics does play an important role in the risk of developing IBD, they are not the only determinants. Action point: Genetics play an important role in the risk of developing IBD, but women should be counseled that there are other factors involved. Their offspring will not necessarily develop IBD. PREGNANCY Conception and beyond: what is the effect of IBD activity on the pregnancy? Some studies indicate that IBD, especially Crohn s disease, can be associated with an increase in adverse pregnancy outcomes, such as prematurity [2,10,14,25-33], low birth weight [14,25,26,28,31-32], small for gestational age [25,28,30,33,34], congenital abnormalities [28,31,35], miscarriages or spontaneous abortions [11,30]. Other studies report no significant association between IBD and adverse pregnancy outcomes [9,29,36-38]. However, the effect of IBD on pregnancy outcomes may be partially attributable to disease activity, and medications, rather than IBD alone. In addition, other demographic variables such as maternal age and smoking have been shown to be risk factors for congenital abnormalities and pregnancy outcomes such as preterm delivery, among women with IBD [38]. A few studies concluded that disease activity did not predict adverse pregnancy outcomes in women with IBD [1,30,32], but other studies found that active disease at the time of conception, and during pregnancy, increases the risk of adverse pregnancy outcomes, such as spontaneous abortion [1,6,7] and preterm delivery [37,39]. Action point: Women with IBD should be in remission before attempting to become pregnant. Conception and beyond: what is the effect of pregnancy on IBD course? Women with active disease at the time of conception, or 3496 April 7, 2014 Volume 20 Issue 13

122 Huang VW et al. Managing the pregnant IBD patient within 3 mo of conception, are more likely to have active disease during pregnancy than if their disease was in remission [40,41]. For both ulcerative colitis and Crohn s disease, the relative risk of a women having active disease during pregnancy if she had active disease at the beginning of pregnancy is about two-fold [41]. Conversely, women with disease in remission at the time of conception are more likely to have quiescent disease during pregnancy [6]. Pregnancy was reported to decrease activity of Crohn s disease in a study of 61 women [42], but a larger study including 92 women with Crohn s disease found no statistical significant difference in disease course during or after pregnancy [43]. Longer disease duration in Crohn s disease patients may increase the risk of relapse during pregnancy [43]. In ulcerative colitis patients, there was a higher risk of relapse during pregnancy and in the post partum period [43]. A large study on 543 women over 10 years reported that pregnancy was associated with a reduced number of flares in the years following pregnancy [44]. Action point: The disease activity at time of conception tends to predict disease course during pregnancy. Ideally, women should be in remission at the time of conception. Flares and remissions during pregnancy: which medications can be used during pregnancy? Women who require medications to achieve and maintain remission of their IBD, should continue these medications during pregnancy. With the exception of methotrexate, which should be stopped when attempting to conceive, and withheld during pregnancy, other medications used to manage IBD have not been associated with significant adverse fetal outcomes. Aminosalicylates [Food and Drug Administration (FDA) Class B, Asacol FDA Class C] and sulfasalazine (FDA Class B): Aminosalicylates and sulfasalazine are commonly used drugs for mild to moderate ulcerative colitis. A cohort study from Denmark found an increased risk of stillbirth and preterm birth in women prescribed 5-ASA drugs during pregnancy, but was unable to distinguish between effects of disease activity and 5-ASA [36]. Other studies found no significant association between 5-ASA drugs and poor pregnancy outcomes [45-51]. A recent meta- analysis reported slight but non-significant increases in congenital malformation (OR = 1.16), stillbirth (OR = 2.38), spontaneous abortion (OR = 1.14), and preterm delivery (OR = 1.35) and low birth weight (OR = 0.93) with 5-ASA medications [51]. However, again, the results were pooled, and they were unable to differentiate which 5-ASA drug, type of disease, or disease activity. Recently there has been concern about the dibutyl phthalate (DBP) coating on certain mesalamines, as animal studies found adverse effects on development and reproductive organs [52]. A recent study reported high mean urinary concentrations of the main DBP metabolite in a woman who used Asacol [52]. However, there have been no reports of adverse developmental or reproductive effects on humans. In addition, DBP can be found in many commonly used medications and dietary supplements [53]. Sulfasalazine inhibits folate synthesis, so women on sulfasalazine should be supplemented with folic acid (5 mg/d) to prevent neural tube defects [54,55]. Sulfasalazine can also displace bilirubin from albumin, which theoretically could lead to kernicterus in the newborn child, but no cases have been reported [55]. Action point: Amniosalicylates and sulfasalazine can be used during pregnancy, and are not significantly associated with adverse neonatal outcomes. Women on sulfasalazine should receive folic acid supplementation (5 mg/d). Azathioprine and 6-mercaptopurine/Purinethol (FDA Class D): Although thiopurines are classified as FDA Class D drugs, because of teratogenicities in animal studies, the use of azathioprine/6-mp during pregnancy in IBD is not associated with increased risk of preterm birth, low birth weight, neonatal adverse outcomes, or congenital abnormalities [33,49,56-60]. Disease activity rather than medication use can lead to neonatal adverse outcomes [57]. One study reported that thiopurines increased the risk for congenital malformations when compared to healthy women, but not when compared with IBD controls [61]. A large ongoing prospective registry of pregnancy outcomes in women with IBD exposed to immunomodulators and biologic therapy (PIANO study) has found no association with the use of immunosuppressants with congenital anomalies, abnormal newborn growth and development, or other complications [62]. In addition, a recent review found that thiopurine use during pregnancy was not associated with low birth weight or congenital abnormalities, but was associated with pre-term birth [63]. Infants may be exposed to a metabolite of Azathioprine, 6-TGN [64,65], and a recent study has found that up to 60% of infants exposed to thiopurines in utero are born with anemia [65]. In long-term (average 4 years) follow-up studies of babies exposed to azathioprine in utero, there was no increased risk of infection [66] or development and immune function [67]. Expert opinion is to continue thiopurine use during pregnancy to maintain remission of disease [54,68]. Action point: Thiopurines can be used during pregnancy, and are not significantly associated with adverse neonatal outcomes. Methotrexate (FDA Class X): Methotrexate is a teratogen and an abortifacient, and is therefore contraindicated during conception and pregnancy period. Methotrexate exposure during organogenesis (6-8 wk) may lead to congenital abnormalities, while exposure in the second and third trimesters can lead to fetal loss [55,69]. Since Methotrexate remains in the tissue for a period of time, patients should discontinue at least 3-6 mo prior to attempting to conceive [54,69]. Women who become pregnant while on methotrexate should seek medical attention immediately, for assessment of the fetus, and counseling 3497 April 7, 2014 Volume 20 Issue 13

123 Huang VW et al. Managing the pregnant IBD patient regarding options [54,55]. Action point: Methotrexate should be discontinued at least 3 to 6 mo before conception. Corticosteroids (FDA Class C): Glucocorticoids cross the placenta and can reach the fetus, but the placental enzymes convert corticosteroids to less active metabolites [54,55]. Prednisone, prednisolone, and methylprednisolone are the preferred agents during pregnancy, as they are more efficiently metabolized by the placenta than dexamethasone or betamethasone [54]. Most studies on glucocorticoid use during pregnancy have been in patients with various diseases, such as asthma. There has been a reported association of increased oral cleft in neonates exposed to glucocorticoids in utero in the first trimester, and this risk should be discussed with the patient [54,55,69]. Overall there is no increased risk of congenital abnormalities [50]. Budesonide has only been reported in one small study in Crohn s disease patients, and was not associated with adverse neonatal outcomes [70]. Action point: Corticosteroids may be used to treat flares of IBD during pregnancy. There is a small risk of oral cleft in neonates exposed to corticosteroids in the first trimester. Antibiotics: Metronidazole (FDA Class B) and Ciprofloxacin (FDA Class C) are commonly used to treat abscesses and fistulae in IBD. Animal studies showed carcinogenic effects from Metronidazole, and early studies suggested a risk of cleft lip [55], but this has not been reported in humans [71]. It was not associated with preterm birth (OR = 1.02, 95%CI: ), low birth weight (OR = 1.05, 95%CI: ), OR = congenital anomalies (OR = 0.86, 95%CI: ) in a large study of 2829 singleton/mother pairs [72]. In a small study (27 of 113 patients on Metronidazole) in female IBD patients, metronidazole was found to be safe in all trimesters of pregnancy [49]. Previously, there was concern that quinolones increase the risk of arthropathies in the offspring, Studies have reported no significant increase in major congenital anomalies, including musculoskeletal problems from the use of ciprofloxacin [73]. A meta-analysis of pregnancy outcomes after exposure to quinolones in the first trimester reported no increased risk of major malformations, stillbirths, preterm births, or low birth weight [74]. However, because of the known possible effect of ciprofloxacin on bone and cartilage, it has been recommended to avoid this medication during pregnancy [55]. Penicillins have not been shown to cause fetal malformations or adverse pregnancy outcomes, and are considered the first line therapy in pregnancy [71]. Amoxicillin (FDA Class B) can be used to treat abscesses and complications of IBD during pregnancy. Action point: Metronidazole can be used during pregnancy, preferably avoid use in first trimester. Ciprofloxa- cion should be avoided during pregnancy due to risk of arthropathy. Amoxicilin is safe to use during pregnancy. Biologics: Anti-tumour necrosis factor inhibitors (FDA Class B) such as Infliximab, Adalimumab, and Certolizumab, are commonly used to treat moderate to severe IBD, and fistulizing Crohn s disease. TNF-α is a proinflammatory cytokine that stimulates the production of prostaglandins, and increased levels are associated with preterm labor [75]. TNF levels increase during pregnancy, as it is mainly produced by the placenta [76]. TNF-α is important for the initial stages of pregnancy, and also for the development of the fetal immune system, and TNF deficient animals have been shown to have increased risk of immune developmental abnormalities [77]. However, increased levels of TNF-α have been associated with preeclampsia, gestational diabetes, obesity [76]. Initially Infliximab and Adalimumab were reported in a few cases of pregnant women with IBD [78-84] which did not show any adverse effects. Larger observational studies, registry studies, and systematic reviews have shown its safety for use during pregnancy [85-88]. The PIANO study has not found any increase in congenital anomalies, abnormal newborn growth and development, or other complications, among women receiving biologics [62]. Infliximab and Adalimumab are IgG1 monoclonal antibodies, and are actively transported across the placenta, while Certolizumab is a Fab fragment of IgG1, and has not been shown to have placental transportation [89]. This active transport of IgG1 antibodies occurs mainly in the third trimester [81,90]. Thus it has been recommended to stop Infliximab and Adalimumab at the onset of the third trimester [91,92]. However, in a recent study, in women with quiescent IBD, who discontinued anti-tnf therapy by week 30, Infliximab and Adalimumab were still detected in cord blood [93]. The exact time to hold anti-tnf is now debatable, especially with Adalimumab which is given weekly or biweekly, but in high risk patients, or patients with active disease, these biologics should be continued throughout the pregnancy [94]. Levels of Infliximab and Adalimumab have been detected in infants for as long as 6 mo [95]. At least for the short term, children exposed to intra-uterine Infliximab develop normally, without increased infections, allergic reactions, or decreased response to vaccinations [91]. However, infants exposed to combination of immunomodulators and biologics have been noted to have increase in infections from 9 to 12 mo of age [62]. Thus, it is still recommended that infants exposed to intra-uterine anti-tnf therapy delay live vaccinations for at least the first 6 mo. Action point: Anti-TNF therapies are safe to use during pregnancy. Infliximab and Adalimumab should be held after week 30, if not earlier, to decrease placental transport to the fetus. Neonates exposed to biologics during pregnancy should not have live vaccines during the first 6 mo post delivery April 7, 2014 Volume 20 Issue 13

124 Huang VW et al. Managing the pregnant IBD patient Cyclosporine (FDA Class C): Cyclosporine crosses the placenta, and has not been found to be teratogenic in animal models [96]. The majority of studies on cyclosporine in pregnancy involve post-transplant patients, which suggests an association with premature delivery and low birth weight infants [97]. In severe ulcerative colitis flares during pregnancy, cyclosporine has been used with successful control of the disease, avoidance of colectomy during pregnancy, and no significant adverse pregnancy outcome [98-104]. The most common side effect reported was hypertrichosis in the mother, however, in one case report, the patient developed severe hypertension and seizures 48 h post infusion [99]. Other adverse effects of cyclosporine include nephrotoxicity and hepatotoxicity [97]. Fulminant ulcerative colitis leading to colectomy has been associated with adverse pregnancy outcomes, with up to 49% fetal mortality and 22% maternal mortality rates in the literature [105]. Thus, in cases of severe fulminant ulcerative colitis, in order to avoid urgent colectomy, cyclosporine may be considered. Action point: Cyclosporine may be considered in cases of severe fulminant ulcerative colitis in pregnancy, in order to avoid colectomy during pregnancy. However, as biologics are FDA Class B, and there are more studies on Infliximab use during pregnancy, Infliximab may be the preferred first line option. Managing relapses during pregnancy: can we use induction medications? As already mentioned, active disease during pregnancy is associated with poor pregnancy outcomes. Since medications commonly used to treat IBD are not associated with significant adverse pregnancy outcomes, treating the mother to induce and maintain remission during the remainder of the pregnancy will lead to more beneficial outcomes. If hospitalization is required to manage an acute IBD flare, IV hydrocortisone [103] and IV infliximab [78,82,91,92,106] may be used for rescue therapy, as thus far, they have not been associated with significant adverse effects. One study has shown that IV cyclosporine can be used safely [103]. Action point: Active IBD is associated with adverse pregnancy outcomes. Management of flares of IBD during pregnancy may involve the use of steroids, biologics, and possibly cyclosporine. DELIVERY Women with IBD were initially reported to be more likely to have a caesarean section [2,14,25,33,34,107]. This has mainly been attributed to Crohn s disease patients, especially those with perianal disease [14,107,108] ; it has also been shown that vaginal delivery with episiotomy may be associated with subsequent perianal involvement [109]. Larger population studies found no significant difference in caesarean section rates among IBD patients [38], and no risk of progression of perianal disease in Crohn s disease with vaginal delivery [108]. Thus, the decision for caesarean section should not be made purely on the IBD diagnosis, but also obstetrical reasons. Action point: In women with Crohn s disease with active perianal disease, caesarean section should be considered on an individual basis. POST PARTUM What is the risk of IBD flaring after delivery? Some women may flare after delivery, while others fare well. A retrospective cohort study of 114 Crohn s disease patients reported more frequent disease progression after childbirth in patients who had active luminal disease prior to pregnancy [108]. A large multi-country prospective study found higher risk of relapse in the postpartum period in women with ulcerative colitis [43]. Action point: Ulcerative colitis patients have an increased risk of relapse after delivery. Crohn s disease patients with active luminal disease before pregnancy have a higher risk of relapse after delivery. BREASTFEEDING Breastfeeding physiologically may be associated with increased inflammation, as prolactin is associated with upregulation of TNF production [110] and increased levels are found in other autoimmune diseases such as lupus, rheumatoid arthritis [111]. Many women with IBD choose not to breastfeed their children [2,112]. This may be due to fears of medication effects, physician recommendation or personal choice [112]. The effect of breastfeeding on the development of IBD is thought to be related to the hygiene hypothesis, in which breastfeeding is thought to help the neonate develop tolerance to microflora and food antigens, thus preventing immune over activation to delayed antigen exposures [ ]. Does breastfeeding affect IBD? Studies investigating the effect of breastfeeding on developing IBD vary in methodology and conclusions. In one study, women who breastfeed were found to be more likely to have a postpartum flare of their disease, but this increased risk was non significant once adjusted for discontinuation of IBD medications during pregnancy [112]. A more recent population-based study found no increased rate of disease flare in the post partum year between those who breastfed (26%) vs those who did not (29.4%) [116]. Can breastfeeding affect the risk of IBD in the offspring? Some studies find no association between breast feeding and diagnosis of IBD [117]. However, some report that a lack of breastfeeding in infancy is associated with an increased risk of UC (OR = 1.5, 95%CI: ) and 3499 April 7, 2014 Volume 20 Issue 13

125 Huang VW et al. Managing the pregnant IBD patient CD (OR = 1.9, 95%CI: ) [118]. More recent studies reported a protective effect of breastfeeding to decrease the odds of developing IBD [119,120]. Two systematic reviews investigating the role of breastfeeding and the development of IBD found that breastfeeding is associated with lower risks of developing early-onset IBD [121,122]. Which medications can be used during breastfeeding? Aminosalicylates (FDA Class B, Asacol FDA Class C) and sulfasalazine (FDA Class B): Earlier studies have reported the rare occasion of infants exposed to 5-aminosalicylates via breast-milk developing watery diarrhea [123], however very small amounts of drug are excreted into breast milk, making risk of toxicity and reaction very unlikely [ ]. Sulphasalazine has also been reported to cause bloody diarrhea in the infant exposed via breastmilk [127], however, it has not been reported other than case reports. Sulfasalazine can have a bilirubin displacing effect leading to jaundice in the neonate, however the amount of drug transferred to the child via breast-milk is negligible to cause jaundice [128,129]. ACTION POINT: 5-aminosalicylates and sulphasalazine can be continued during breastfeeding. Azathioprine and 6-mercaptopurine/Purinethol (FDA Class D): Azathioprine and 6-MP can be continued during pregnancy, as described above, but there are concerns about the potential for tumorigenicity, and increased susceptibility to infections in neonates exposed during breastfeeding [66]. Recent studies show that only very small amounts of AZA/6-MP are measured in breast milk, and neglible amounts detected in the neonate [ ]. In addition, the highest concentration of AZA measured in the breast milk appears during the first 4 h after consumption [130], thus it has been recommended to pump and dump the first 4 h of breastmilk. Thus far, there has not been reported increase risk of infections among babies breastfed with exposure to azathioprine [66], and it is considered safe to continue these medications during breastfeeding [54,66, ]. Action point: Azathioprine and 6-MP are can be continued during breastfeeding; the first 4 h of breastmilk after consumption may be discarded to minimize the amount of drug transferred to the neonate. Methotrexate (FDA Class X): Methotrexate crosses into the breast milk [135] and because of its teratogenicity, it is contraindicated during breastfeeding [136,137]. Action point: Methotrexate is contraindicated during breastfeeding. Corticosteroids (FDA Class C): Corticosteroids do transfer to the breast milk, but in very low levels [138,139] and because the highest levels appear in the first 4 h [139], it is recommended to pump and dump [55,139] the first 4 h after medication consumption to minimize transfer of the drug to the neonate. Action point: Corticosteroids can be continued during breastfeeding if required to treat maternal IBD. Antibiotics: Metronidazole is transferred into the breast milk [139], but in minimal levels [140] and levels decline after h after maternal dose intake [141]. If metronidazole is required for the treatment of active IBD, it is recommended to wait h after metronidazole intake before breastfeeding [55], and long term use should be avoided [69]. Ciprofloxacin is also detectable in the breast milk in small amounts [142,143], but short term treatment can be used if indicated [55]. Action point: Metronidazole and ciprofloxacin can be continued for short term during breastfeeding if required to treat maternal IBD. Biologics: As mentioned, the biologic therapies can be continued during pregnancy, and held in the third trimester. Studies have shown nil to minimal levels of infliximab and adalimumab in the breast milk and no significant adverse events have been reported in the infant [81, ]. It is thought that any detectable levels in the neonate after delivery may be due to placental transfer during pregnancy [147]. Thus, although anti-tnf therapies are can be continued during breastfeeding, further studies are required to determine the effect of infant exposure to these biological therapies on the development of their gastrointestinal immunity and systemic immune system [89,146,148]. The preliminary results of the PIANO study have not found any association between breastfeeding and infection risk in the neonate exposed to biologic therapy [62]. Action point: Infliximab and adalimumab may be continued during breastfeeding. Cyclosporine (FDA Class C): Cyclosporine does cross into the breast milk, but if required for fulminant colitis, it can be used. Case reports and series of neonates exposed to cyclosporine during pregnancy and breastfeeding are mainly from the renal transplant literature, and have reported varying levels of cyclosporine in the breast milk, and relatively good outcomes in the mother and neonate [ ]. One case report of cyclosporine use in the management of severe ulcerative colitis while breastfeeding also reported short term good outcomes in the mother and neonate [154]. More studies are required to determine the long term effects of neonatal exposure to cyclosporine in the breast milk. Action point: Cyclosporine has been used to manage fulminant colitis during breastfeeding, however, infliximab is preferred due to the lack of studies for cyclosporine April 7, 2014 Volume 20 Issue 13

126 Huang VW et al. Managing the pregnant IBD patient CONCLUSION IBD affects people during an important time of their lives when they are considering family planning or are already pregnant. With the exception of Methotrexate, commonly used medications for the treatment of IBD are not associated with significant adverse pregnancy outcomes, and can be used throughout pregnancy. Good control of disease and clinical remission at the time of conception increases the likelihood of having successful pregnancy outcomes, and quiescent disease during pregnancy. There is no need to adjust medications during pregnancy, with the exception of biologics such as Infliximab and Adalimumab, which should be held during the third trimester in women who are in clinical remission. However, biologics may be continued throughout pregnancy if necessary to control disease. Induction of remission of IBD flares during pregnancy should be treated with appropriate medications, such as steroids, infliximab, and for severe fulminant colitis, cyclosporine, as active disease and fulminant colitis requiring surgery has increased risk of adverse fetal outcomes. The management of IBD in women during their reproductive years should include consideration of their family planning decisions, and education counseling regarding the overall safety of medications and the importance of medication adherence should occur prior to conception. REFERENCES 1 Hudson M, Flett G, Sinclair TS, Brunt PW, Templeton A, Mowat NA. Fertility and pregnancy in inflammatory bowel disease. Int J Gynaecol Obstet 1997; 58: [PMID: DOI: /S (97)00088-X] 2 Mañosa M, Navarro-Llavat M, Marín L, Zabana Y, Cabré E, Domènech E. Fecundity, pregnancy outcomes, and breastfeeding in patients with inflammatory bowel disease: a large cohort survey. 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130 Huang VW et al. Managing the pregnant IBD patient ture. World J Gastroenterol 2008; 14: [PMID: DOI: /wjg ] 114 Castiglione F, Diaferia M, Morace F, Labianca O, Meucci C, Cuomo A, Panarese A, Romano M, Sorrentini I, D Onofrio C, Caporaso N, Rispo A. Risk factors for inflammatory bowel diseases according to the hygiene hypothesis : a case-control, multi-centre, prospective study in Southern Italy. J Crohns Colitis 2012; 6: [PMID: DOI: /j.crohns ] 115 Frolkis A, Dieleman LA, Barkema H, Panaccione R, Ghosh S, Fedorak RN, Madsen K, Kaplan GG. Environment and the inflammatory bowel diseases. Can J Gastroenterol 2013; 27: e18-e24 [PMID: ] 116 Moffatt DC, Ilnyckyj A, Bernstein CN. A population-based study of breastfeeding in inflammatory bowel disease: initiation, duration, and effect on disease in the postpartum period. Am J Gastroenterol 2009; 104: [PMID: DOI: /ajg ] 117 Gilat T, Hacohen D, Lilos P, Langman MJ. Childhood factors in ulcerative colitis and Crohn s disease. 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Is infliximab safe to use while breastfeeding? World J Gastroenterol 2008; 14: [PMID: DOI: /wjg ] 145 Ben-Horin S, Yavzori M, Katz L, Picard O, Fudim E, Chowers Y, Lang A. Adalimumab level in breast milk of a nursing mother. Clin Gastroenterol Hepatol 2010; 8: [PMID: DOI: /j.cgh ] 146 Ben-Horin S, Yavzori M, Kopylov U, Picard O, Fudim E, Eliakim R, Chowers Y, Lang A. Detection of infliximab in breast milk of nursing mothers with inflammatory bowel disease. J Crohns Colitis 2011; 5: [PMID: DOI: /j.crohns ] 147 Kane S, Ford J, Cohen R, Wagner C. Absence of infliximab in infants and breast milk from nursing mothers receiving therapy for Crohn s disease before and after delivery. J Clin Gastroenterol 2009; 43: [PMID: DOI: 3505 April 7, 2014 Volume 20 Issue 13

131 Huang VW et al. Managing the pregnant IBD patient /MCG.0b013e31817f9367] 148 Grosen A, Julsgaard M, Kelsen J, Christensen LA. Infliximab concentrations in the milk of nursing mothers with inflammatory bowel disease. J Crohns Colitis 2014; 8: [PMID: ] 149 Thiru Y, Bateman DN, Coulthard MG. Successful breast feeding while mother was taking cyclosporin. BMJ 1997; 315: 463 [PMID: DOI: /bmj ] 150 Nyberg G, Haljamäe U, Frisenette-Fich C, Wennergren M, Kjellmer I. Breast-feeding during treatment with cyclosporine. Transplantation 1998; 65: [PMID: DOI: / ] 151 Munoz-Flores-Thiagarajan KD, Easterling T, Davis C, Bond EF. Breast-feeding by a cyclosporine-treated mother. Obstet Gynecol 2001; 97: [PMID: DOI: / S (01)01122-X] 152 Moretti ME, Sgro M, Johnson DW, Sauve RS, Woolgar MJ, Taddio A, Verjee Z, Giesbrecht E, Koren G, Ito S. Cyclosporine excretion into breast milk. Transplantation 2003; 75: [PMID: DOI: /01.TP D0] 153 Osadchy A, Koren G. Cyclosporine and lactation: when the mother is willing to breastfeed. Ther Drug Monit 2011; 33: [PMID: DOI: /FTD.0b013e318208e3a4] 154 Lahiff C, Moss AC. Cyclosporine in the management of severe ulcerative colitis while breast-feeding. Inflamm Bowel Dis 2011; 17: E78 [PMID: DOI: /ibd.21765] P- Reviewers: Hoffman A, Guangwen R, Kopylov U, Sonoda H S- Editor: Wen LL L- Editor: A E- Editor: Zhang DN 3506 April 7, 2014 Volume 20 Issue 13

132 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (3): Inflammatory bowel disease TOPIC HIGHLIGHT Glucose intolerance and diabetes mellitus in ulcerative colitis: Pathogenetic and therapeutic implications Giovanni Maconi, Federica Furfaro, Roberta Sciurti, Cristina Bezzio, Sandro Ardizzone, Roberto de Franchis Giovanni Maconi, Federica Furfaro, Roberta Sciurti, Cristina Bezzio, Sandro Ardizzone, Roberto de Franchis, Gastroenterology Unit, Department of Biomedical and Clinical Sciences, Luigi Sacco University Hospital, Milano, Italy Author contributions: Maconi G contributed to conception and design, literature search, manuscript preparation and editing; Furfaro F and Sciurti R literature search, manuscript preparation and editing; Bezzio C, Ardizzone S and de Franchis R contributed to critical revision and editing; all authors gave the final approval of the version; Maconi G is the guarantor of the article. Correspondence to: Giovanni Maconi, MD, Professor, Gastroenterology Unit, Department of Biomedical and Clinical Sciences, Luigi Sacco University Hospital, Via G.B. Grassi, 74, Milano, Italy. giovanni.maconi@unimi.it Telephone: Received: October 20, 2013 Revised: January 26, 2014 Accepted: March 6, 2014 Published online: April 7, 2014 Abstract Diabetes mellitus is one of the most frequent co-morbidities of ulcerative colitis patients. The epidemiological association of these diseases suggested a genetic sharing and has challenged gene identification. Diabetes co-morbidity in ulcerative colitis has also relevant clinical and therapeutic implications, with potential clinical impact on the follow up and outcome of patients. These diseases share specific complications, such as neuropathy, hepatic steatosis, osteoporosis and venous thrombosis. It is still unknown whether the coexistence of these diseases may increase their occurrence. Diabetes and hyperglycaemia represent relevant risk factors for postoperative complications and pouch failure in ulcerative colitis. Medical treatment of ulcerative colitis in patients with diabetes mellitus may be particularly challenging. Corticosteroids are the treatment of choice of active ulcerative colitis. Their use may be associated with the onset of glucose intolerance and diabetes, with difficult control of glucose levels and with complications in diabetic patients. Epidemiologic and genetic evidences about diabetes co-morbidity in ulcerative colitis patients and shared complications and treatment of patients with these diseases have been discussed in the present review Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Diabetes mellitus; Ulcerative colitis; Diabetes complications; Inflammatory bowel diseases; Glucose intolerance; Medical therapy; Corticosteroids Core tip: The relationship between ulcerative colitis and diabetes mellitus is intriguing, full of practical and speculative information, useful for clinical practice and basic research. Diabetes mellitus is one of the most frequent co-morbidities of ulcerative colitis and their epidemiological association suggests genetic sharing and stimulates studies for gene identification. Diabetes also shares specific complications with ulcerative colitis and represents a challenging condition in ulcerative colitis patients for the treatment of the disease, due to difficult control of glucose levels and for high risk of postoperative complications and pouch failure. All these issues have been discussed in the present review. Maconi G, Furfaro F, Sciurti R, Bezzio C, Ardizzone S, de Franchis R. Glucose intolerance and diabetes mellitus in ulcerative colitis: Pathogenetic and therapeutic implications. World J Gastroenterol 2014; 20(13): Available from: URL: DOI: INTRODUCTION Ulcerative colitis (UC) is a chronic disease characterised by mucosal inflammation, limited to the colon. Its inci April 7, 2014 Volume 20 Issue 13

133 Maconi G et al. Diabetes co-morbidity of ulcerative colitis dence and prevalence are increasing with time in different regions around the world, with the highest annual incidence of 24.3 per persons-year, and prevalence of 505 per persons in Europe [1]. The disease is associated with high costs, diseasespecific morbidity and decreased quality of life, mostly related to complications, surgery and co-morbid diseases. Co-morbid diseases in UC include several immune mediated diseases, such as rheumatoid arthritis, multiple sclerosis, lupus, psoriasis, hypothyroidism and diabetes mellitus [2-7]. Among these diseases, diabetes mellitus is the most frequent condition and its association with UC has epidemiological, pathogenetic, clinical and therapeutic implications. All these points represent the objects of the present review. An electronic literature search was conducted using PubMed and Medline as primary sources. No time limits were specified up to the date of the search (September 2013). A comprehensive search was performed using the following search terms: ulcerative colitis or inflammatory bowel diseases and diabetes mellitus or glucose intolerance. The search was restricted to articles involving humans and those in the English language (or with an English abstract) and following identification of relevant titles, the abstracts of these articles were read to decide if the study was eligible. The full text article was retrieved when the title and/ or abstract seemed to meet the pre-defined eligibility criteria. A manual cross-reference search of bibliographies was carried out to identify articles missed in the computerised search. DIABETES CO-MORBIDITY IN UC Diabetes mellitus, like other autoimmune disorders, is significantly associated with UC, both in children and in adult patients. A recent large case-control study, which included more than 1200 children with inflammatory bowel disease (IBD), 488 of whom with UC, showed that UC is associated with a higher prevalence of diabetes than in controls (OR = 2.7, 95%CI: ) with an overall prevalence of 2049 cases per children. Noteworthy, this association was confirmed excluding patient on treatment with anti-tnf alpha, and was the strongest among the other autoimmune conditions. It was also specific for UC, the association with Crohn s disease not being significant (OR = 1.4, 95%CI: 0.5-4) [7]. Regarding the association between UC and diabetes, the estimates in this paediatric study were only slightly higher than those in adult studies. Indeed, few other studies examined the concomitance of autoimmune diseases among adult IBD patients without showing a significant increased risk for type 1 diabetes mellitus [3,4] although this endocrine disorder represented the third most common co-morbid disease in UC patients (0.8%) after psoriasis (1.8%) and rheumatoid arthritis (1.1%). No study has assessed the prevalence of type 2 diabetes mellitus in IBD, so far. Interestingly, patients with both psoriasis and IBD showed significantly higher rates of diabetes (26.7% vs 11.0%) and autoimmune thyroiditis (2.1% vs 6.8%) and hepatitis (0.7% vs 6.2%) compared to individuals with psoriasis only [8]. On the other hand, a large study made possible by the availability of the Multigeneration Register in Sweden, estimated the associations between type 1 diabetes mellitus and 33 autoimmune and related diseases in parents, offspring, siblings and twins. This study showed that type 1 diabetes in offspring was associated with 13 diseases in parents, including UC (standardised incidence ratio 1.23) and few other gastrointestinal diseases such as primary biliary cirrhosis (3.63) and celiac disease (2.73) [9]. All these epidemiological findings suggest potential shared aetiological mechanisms for UC and type 1 diabetes mellitus. Although the aetiology for both diseases remains unclear, a large body of evidence supports the hypothesis that in genetically predisposed individuals, both host factors and environmental factors contribute to an uncontrolled immune function. Co-morbidity among these autoimmune disorders and familial associations with several autoimmune and related diseases suggest genetic sharing and represent a challenge for gene identification. On this regard, a recent genome-wide association study examined known susceptibility loci for IBD and type-1 diabetes mellitus in a cohort of 1689 Crohn s disease patients, 777 UC patients, 989 type-1 diabetes patients and 6197 control subjects, and identified multiple shared loci with opposite effects. In particular the study identified 1 diabetes mellitus locus (TNFAIP3) that confers UC risk and 2 UC loci (HERC2 and IL26) that confer type-1 diabetes mellitus risk [10]. The genetic association between UC and type-1 diabetes mellitus has been also suggested by the description of a monogenic form of diabetes with the typical features of type 1 diabetes (autoantibodies to β cells, lean and young at onset of hyperglycemia, rapid disappearance of C-peptide production and insulin dependence) together with insulin resistance, which appears as a consequence of an autosomal-dominant mutation in the SIRT1 gene. A recent case-report describes a family carrying a mutation in the SIRT1 gene, in which all five affected members developed an autoimmune disorder: four members developed type 1 diabetes and one developed UC [11]. It is particularly interesting to know that SIRT1 suppresses TNFα expression [12]. Importantly, both type-1 diabetes and UC are strongly associated with this cytokine, and TNF antagonism improves both conditions [13]. COMMON SHARED COMPLICATIONS IN DIABETES AND UC Diabetes mellitus and UC share a number of complications, namely neurological, hepatobiliary, osteoarticular, 3508 April 7, 2014 Volume 20 Issue 13

134 Maconi G et al. Diabetes co-morbidity of ulcerative colitis Table 1 Common shared complications in diabetes mellitus and ulcerative colitis Neurological Distal symmetric polyneuropathy (50% in DM and 0%-39% in UC) Hepatobiliary Cholelithiasis (20%-30% in DM, only after colectomy in UC) Hepatic steatosis Non alcoholic fatty liver disease Osteo-articular complications Osteoporosis Vascular complications Venous thrombosis (with ketoacidosis in DM, with active disease or surgery in UC) Post-operative complications Anastomotic dehiscence Infections Non-infectious complications DM: Diabetes mellitus; UC: Ulcerative colitis. vascular and post-operative. It is unknown whether concomitance of both diseases for a long time increases the risk of such complications. Although diabetic patients with mild or quiescent UC are likely to have a favourable outcome, UC patients with recurrent or steroid-refractory active disease, with consequent long term steroid treatment, could present hyperglycaemia and hyperinsulinemia and an increased risk of complications. Unfortunately, the outcome of UC in diabetic patients has not been investigated so far and no data are reported in the large therapeutic trials. Examples of the more frequently shared complications of diabetes mellitus and UC are presented hereafter (Table 1). Neurological complications Neuropathy is a well known complication of diabetes mellitus. In particular, distal symmetric polyneuropathy is the most frequent form of neurological involvement, occurring in up to 50% of diabetic patients [14]. Peripheral neuropathy is also a neurological complication of inflammatory bowel diseases [15,16] with an incidence ranging from 0% to 39%, depending on the features of the study populations and on the criteria adopted to define the neuropathy. A population-based study showed that incident at peripheral neuropathy in IBD patients occurs late in the course of the disease [17] and is likely due to nutritional (e.g., B12 deficiency), iatrogenic (e.g., metronidazole neurotoxicity) and immune-mediated causes. Hepatobiliary complications Cholelithiasis, a well known complication of 20%-30% of patients with diabetes mellitus probably due to impaired gallbladder contraction, obesity and hyperlipidemia [18], is also reported as a complication in UC patients, but only after colectomy, likely due to changes in bile composition and increase in cholesterol concentration in bile [19]. Hepatic steatosis is a frequent feature of both diabetes mellitus and UC. Nonalcoholic fatty liver disease (NAFLD) is characterized by insulin resistance and it is often associated with type 2 diabetes mellitus [20,21]. Up to 50% of these patients may have nonalcoholic steatohepatitis [22]. NAFLD is also frequently reported in UC patients, apart from classical risk factors such as obesity or insulin resistance [23]. Osteo-articular complications Diabetes mellitus-induced osteoporosis is often present in diabetic patients, probably due to changes in osteoblast fuction and bone formation, sustained by hyperglycemia [24]. A high prevalence of reduced bone density is also frequently reported in UC patients due to increased bone reasorption, not balanced by an appropriate bone formation. Long duration of disease, low body mass index, colectomy and in particular high doses and prolonged treatment with corticosteroids (cumulative use of steroids) have been recognised as risk factors for this complication and fractures [25-27]. Vascular complications Diabetes mellitus and UC share vascular complications, such as venous thrombosis. UC is characterised by a potential hypercoagulable state and an incidence of systemic thromboembolic events higher than in the general population, usually correlated with active disease and surgery [28-30]. Diabetes, although most commonly complicated by arterial thrombosis, may also be complicated by venous thromboembolism, in presence of ketoacidosis [31-33]. Post-operative complications Diabetes mellitus is a well known risk factor of poor outcome in colorectal surgery, mainly due to the occurrence of anastomotic dehiscence [34], infectious and noninfectious complications. Recently, it has been found that perioperative hyperglycemia, in diabetic patients and even in patients without a preoperative diagnosis of diabetes undergoing colorectal surgery, is associated with a high rate of infectious and noninfectious complications, reintervention and mortality [34]. Adverse outcomes may be associated with a single postoperative elevated glucose value and the risk of morbidity and mortality is related to the degree of hyperglycaemia [35,36]. Therefore, it is not surprising that the post surgical period is one of the most important time frames for morbidity and mortality in UC patients with co-morbid diabetes mellitus. Surgical-site infections is a major source of morbidity after colectomy for fulminant UC. A retrospective study including 59 patients operated for fulminant UC, showed that diabetes is one of the most frequent independent risk factors for surgical-site infections, along with white blood cell count, intraoperative blood loss and blood transfusion [37]. The poor outcome of the postopera April 7, 2014 Volume 20 Issue 13

135 Maconi G et al. Diabetes co-morbidity of ulcerative colitis Table 2 Predictive risk factors for the development of diabetes mellitus and hyperglycemia in ulcerative colitis patients treated with corticosteroids High dose of corticosteroids Long duration of corticosteroids therapy Advanced age High body mass index Family history of diabetes Previous gestational diabetes tive course in diabetic patients with UC has been also found in another large retrospective study that included 3754 patients undergoing ileoanal pouch, which showed that diabetes mellitus was an indipendent factor associated with the risk of pouch failure (HR = 2.31; 95%CI: ) [38]. CORTICOSTEROID-INDUCED DIABETES IN UC IBD are immune-mediated disorders which appear in genetically predisposed subjects. Corticosteroids are the main therapeutic agents in UC, because of multiple effects on the cellular and humoral immune system, including an inhibitory action on several pro-inflammatory cytokines and metabolites of arachidonic acid. For more than 50 years corticosteroids, such as prednisone and methyl-prednisolone, have been used to treat IBD during the acute phase. However, more than 50% of patients do not respond to the therapy (steroidresistance) or have a relapse after treatment discontinuation (steroid-dependence) and about half of them show side effects of variable severity [39,40]. In most cases, the appearance and the seriousness of side effects (except from osteonecrosis and idiosyncratic reactions) are related to duration and dose of therapy. Hyperglycemia and corticosteroid-induced diabetes are the most common systemic manifestations in IBD under steroid treatment and represent a real problem in the handling of UC patients with diabetes mellitus when relapses of the intestinal disease occur. To date, there are few data on the incidence of corticosteroid-induced hyperglycemia or diabetes in IBD and also about the onset of acute diabetic complications, such as ketoacidosis and hyperosmolar hyperglycemic state, in diabetic patient affected by IBD, under steroid treatment. Most of our knowledge is derived from nongastroenterological studies. A case-control study, conducted on 55 elderly patients with active Crohn s disease compared to 66 control subjects not treated with steroids, showed that treatment with high doses of corticosteroids may increase the risk of hyperglycemia, even if the difference was not statistically significant (RR = 1.53; 95%CI: ) [41]. However, another case-control study that included a large number of patients (11855 cases and controls) showed that corticosteroids (prednisone or analogues at a dose of 30 mg a day or more) confer a RR = 10 of hyperglycemia, compared to non treated patients [42]. A retrospective study, including 25 patients affected by neuropathy (median age: 50 years) showed that treatment with prednisolone at a dose of mg a day for at least 2 wk may result in a postprandial hyperglycemia, compatible with diabetes mellitus in 13 of 25 patients and demonstrated that advanced age is a risk factor for this complication [43]. The importance of age in the onset of corticosteroid-induced diabetes is confirmed also by a cohort study conducted on a large geriatric population (median age: 75 years) that shows an increased risk of diabetes induced by oral corticosteroids (RR = 2.31; 95%CI: ), compared to treatment with proton-pumps inhibitors (PPIs) [44]. Finally, a recent literature review has confirmed that corticosteroid-induced hyperglycemia is common both in patients with and without diabetes and it has estimated an OR = for the onset of diabetes mellitus in treated patients [45]. Overall, the available studies, mostly conducted on patients not affected by IBD, confirm that the total dose of corticosteroids and the long duration therapy are important predictive risk factors for the development of diabetes mellitus. In addition, these studies underline that other factors, such as advanced age, high BMI, family history of diabetes or previous gestational diabetes should be considered and recommend monitoring the blood glucose level during steroid therapy (Table 2). The possible underestimation of this condition in the handling of patient with IBD in clinical practice could be attributed to the short duration of steroids treatment and to the importance given to the fasting blood glucose only. High blood glucose levels in the short-term and in the postprandial period, should be considered for their prognostic value. Concerning the role of the corticosteroids dose on the onset of diabetes, high doses of steroids are associated with high values of blood glucose levels and can induce diabetic ketoacidosis in patients with type 1 diabetes mellitus or hyperosmolar hyperglycemic state [46,47]. The risk of these complications is particularly significant in diabetic patients with UC, in whom the first-line treatment is represented by corticosteroids. TREATMENT OF UC IN DIABETIC PA- TIENTS Treatment of patient with UC depends mainly on the activity and location of disease [48,49]. Severe UC Severe UC is characterized by bloody diarrhoea > 6 bowel movements/d and many signs of systemic toxicity (tachycardia > 90 bpm, fever > 37.8, Hb < 10.5 G/dL or ESR > 30 mm/h) [50]. Patients with severe UC should be admitted to hospi April 7, 2014 Volume 20 Issue 13

136 Maconi G et al. Diabetes co-morbidity of ulcerative colitis Table 3 Management of active severe ulcerative colitis in diabetic patients treated with corticosteroids Disease monitoring Disease treatment Regular monitoring of blood Rehydration with saline solution glucose level Correction of blood glucose levels Regular monitoring of disease Treatment of hypokaliemia activity (e.g., Disease Activity Treatment of hypomagnesaemia Index) Plain abdominal X-ray Dosage of: C-reactive protein Blood cell count Electrolytes Anion gap Osmolality Serum creatinine levels Ketones Urinalysis Blood gas analysis Consider alternative treatments Iv cyclosporine A (4 mg/d) Infliximab (5 mg/kg or 10 mg/kg) Adalimumab (160 mg/80 mg/40 mg eow) Tacrolimus Leucocytapheresis Other therapies (vedolizumab, visilizumab, abatacept, tofacitinib) tal for intensive treatment [49]. Corticosteroids, administered parenterally (e.g., Methylprednisolone 60 mg daily or hydrocortisone 100 mg four times daily), represent the first-line treatment of severe UC. Higher doses are not more effective, but lower doses are less effective [51]. Duration of treatment is 7-10 d, since further extension of therapy carries no additional benefit. Response to therapy reaches 67% and nonresponders UC patients require a second-line treatment, represented by cyclosporine, tacrolimus, infliximab or colectomy [52]. Steroid treatment of severe acute UC, in particular in diabetic patients, requires particular attention to hyperglycemia induced by therapy. The combination of dehydration, electrolyte imbalance (hypokalemia and hypomagnesemia), the possible presence of a septic condition and the need of total parenteral nutrition are important risk factors for hyperosmolar hyperglycemic state and diabetic ketoacidosis, the major complications of diabetes mellitus. Both these conditions are particularly dangerous and burdened by significant mortality, particularly in patient in which the state of diabetes was previously unknown. For this reason, in patients with diabetes mellitus with severe UC, in addition to a close monitoring of bowel disease which requires clinical evaluation, dosage of C-reactive protein, blood counts and abdominal X-ray studies, a careful evaluation of the diabetes is also required, through the regular monitoring of blood sugar and various blood parameters (electrolytes with evaluation of the anion gap and osmolality, phosphorus, magnesium, creatinine, urinalysis to evaluate ketones, blood-gas analysis to evaluate arterial or venous ph), in particular in patients with basal glycaemia exceeding 180 mg/dl (Table 3). Therapy of this condition is essentially based on rehydration with saline solution, correction of blood glucose by administering intravenous or subcutaneous insulin and treatment of hypokalemia by re-integration of potassium, bicarbonates, magnesium and phosphate (in diabetic ketoacidosis), in close collaboration with endocrinologist. Hypokalemia and hypomagnesemia are important risk factors for toxic megacolon [53], require a prompt correction and suggest careful radiographic and biochemical monitoring, particularly when a septic condition coexists (Table 3). Therefore, in diabetic patients with acute severe UC, close monitoring of the patient s clinical and biochemical condition is essential, in order to timely identify the opportunity of a second line medical treatment or the possible need for surgical treatment (Table 3). However, the outcome of severe UC in diabetics is still unknown. In diabetic patients with unstable blood glucose control, steroid treatment can be replaced by intravenous cyclosporine (4 mg/d), infliximab (5 mg/kg or 10 mg/kg at 0, 2, 6 wk and then every two months) or adalimumab 160 mg/80 mg/40 mg eow [49]. Efficacy and safety of cyclosporine and infliximab are comparable and, in clinical practive, the treatment choice should be guided by physician and centre experience [54]. A study by Moskovitz et al [55] showed that cyclosporine is less effective in patients treated with azathioprine and should be avoided in patients with low cholesterol or magnesium in view of the increased incidence of neurological side effects in this patient group. Furthermore, there is no scientific evidence on the effectiveness of cyclosporine in preventing colectomy [56], while other studies have shown that Infliximab can reduce the rate of colectomy compared with placebo [57]. So the choice should be placed on individual circumstances and the availability of drugs. The use of tacrolimus, as an alternative to steroid therapy in diabetic patients, is generally not recommended. The tacrolimus, infact, besides being unable to induce significant mucosal healing compared with placebo [58], can induce long-term hyperglycemia or even diabetes, as well as hypomagnesemia and it may promote the onset of opportunistic infections [49]. In contrast, leucocytapheresis, whose principle is based on the extracorporeal removal of leukocytes through an adsorptive system of cellulose acetate beads (Adacolumn, Otsuka Pharmaceuticals) or a polyester fibre filter (Cellsorba, Asahi Medical Company) represents a potential therapeutic remedy with a good safety profile, serious side effects being very rare. Leucocytapheresis can be associated with any medical treatment, but the real effectiveness of this device in acute severe UC remains to be determined [59]. Leucocytapheresis has a wide-spread acceptance in Japan, but its cost may limit its use and its future role in Europe will depend on the outcome of controlled trials [49]. On this regard, clinical efficacy outcomes are variable, being encouraging in some studies and disappointing in others, and the answer might ultimately lie in the patients disease status at entry. Patients with the first UC episode and short duration of disease or a fair level of intact mucosal tissue, seem to respond and can be spared from multiple drug therapy. Patients with extensive loss of mucosal tissue and those with a long history of exposure to multiple drugs, like corticosteroids, are 3511 April 7, 2014 Volume 20 Issue 13

137 Maconi G et al. Diabetes co-morbidity of ulcerative colitis unlikely to respond [60]. Other therapies such as vedolizumab, visilizumab, abatacept, tofacitinib, which are characterised by different mechanisms of action are currently under investigation and have no role in clinical practice to date [61]. Mild-moderate UC Treatment of mild-moderate UC depends on the site of disease. The first line therapy of mild-moderate distal colitis is topical mesalazine and/or oral mesalazine, in once daily administration, which is as effective as divided doses. Combining topical mesalazine and topical steroids, such as beclomethasone diproprionate, also helps. Patients who fail oral/topical mesalazine and topical steroids should be treated with the addition of oral prednisolone. In proctitis, it has been shown that topical mesalamine has a higher efficacy on symptoms and endoscopic resolution of the damage compared to topical steroids, generally using suppositories that are more appropriate than enemas. However, topical steroids may be used in substitution of mesalazine, if it is poorly tolerated, or in association with the latter. Indeed, the combination of beclomethasone dipropionate (3 mg) and mesalazine (2 mg) is able to induce an improvement in clinical, endoscopic and histologic proctitis, significantly superior to treatment with mesalazine alone [62]. In diabetic patients with mild-moderate distal UC, systemic steroid therapy should be avoided by resorting to rectal or oral administration of mesalazine and/or beclomethasone dipropionate or to new therapies such as budesonide Multi-Matrix System (MMX), fluticasone or prednisolone metasulphobenzoate. In treatment of extensive or distal colitis it is generally recommended, as first choice, a combination of oral and topical mesalazine [49,63,64]. In fact, the use of mesalazine alone, although at high doses or in controlled release formulation (MMX), gives a percentage of remission of at least 41% and a partial response, not exceeding 72%. Furthermore, in many patients the occurrence of relapse of intestinal disease is not uncommon, even during an appropriate maintenance therapy with mesalazine. In these circumstances the use of systemic steroid therapy is generally recommended, even in non severe forms of colitis. In the diabetic patient, this treatment requires the same level of attention already mentioned for patients with severe colitis. Controlled colonic release formulations of steroids and steroids equipped with low bioavailability, such as beclomethasone dipropionate, budesonide MMX, fluticasone or prednisolone metasulphobenzoate represent a potential therapeutic resource with the advantage of the absence or the minimisation of the side effects of other steroids, with respect to the suppression of the hypothalamic-pituitary-adrenal axis [65]. Although there are no specific studies on the undesirable effects of this kind of steroids in diabetic patients with UC, it should be noted that, in both normal subjects and elderly diabetic patients under dietary control, treatment with topical inhaled beclomethasone in high doses for 2 wk, did not produce significant changes in blood glucose and lipid metabolism [66]. In addition, in both adults and pediatric patients, for whom steroid treatment of UC is the remedy of choice, the oral administration of beclomethasone dipropionate is well tolerated and induces a rapid clinical and endoscopic remission in mild to moderate UC, comparable to mesalazine [67-69]. Budesonide MMX, 9 mg daily, is effective for the induction of remission of mild-moderate active UC. It is a novel oral formulation of budesonide that uses MMX technology to extend release to the colon, without notable increases in glucocorticosteroid-related side effects, probably due to low bioavailability and to targeted delivery of drug [70]. In patients with UC fluticasone has negative results, but prednisolone metasulphobenzoate, by oral or topical administration, appears to be effective in active distal UC and in mild-to-moderate UC with a lower incidence of systemic adverse effects in comparison with other glucocorticosteroids [71]. If systemic steroid treatment is necessary, the occurrence of hyperglycemia (particularly in patients with known diabetes) can be corrected or controlled by rapidacting insulin, usually administered in the preprandial period (generally at a dose of 0.1 U/kg) or by using biguanides, such as metformin, or thiazolidinediones (also known as glitazones), drugs just approved for the treatment of diabetes mellitus type 2. Metformin, and in particular the increase in dosage of this drug, is often burdened with gastrointestinal side effects such as nausea, vomiting, anorexia, diarrhea, abdominal pain. These symptoms are usually dose-related and occur especially at the beginning of therapy. In 3%-5% of cases, diarrhea may be persistent and cause discontinuation of the drug. In contrast, the thiazolidinediones are oral antidiabetics with anti-inflammatory properties, potentially useful in patient suffering from UC. In particular, these drugs work by binding to the gamma sub-unit of PPAR (peroxisome receptors that trigger proliferation), receptors located inside the cell nucleus, abundantly expressed in adipose tissue and in the colonic epithelium. Experimental evidence has shown that these molecules have anti-inflammatory activity, in particular in colon and that treatment with these drugs is able to attenuate the production of inflammatory cytokines and to reduce inflammation in animal models of colitis. One uncontrolled study showed that rosiglitazone (a drug used in the United States for treatment of diabetes mellitus type 2) is able to improve moderately active colitis, refractory to treatment with mesalazine. The same group later confirmed the efficacy of the drug, compared to placebo, in a double-blind controlled study [72,73]. Unfortunately, rosiglitazone was recently withdrawn from the market in some countries for the high risk of ischemic heart 3512 April 7, 2014 Volume 20 Issue 13

138 Maconi G et al. Diabetes co-morbidity of ulcerative colitis disease and myocardial infarction, but other thiazolidinediones are on the market (such as pioglitazone) or should soon enter in the pharmaceutical reference book and could be used in diabetic patients with UC, if their efficacy in UC will be confirmed. CONCLUSION Diabetes mellitus is a common disorder, significantly associated with UC. Co-morbidity among these autoimmune disorders and familial associations with several autoimmune and related diseases, suggest a genetic sharing but, although some shared loci at risk have been identified, the clinical implications of this findings are still unclear. Likewise, diabetes mellitus and UC share neurological, hepatobiliary, osteoarticular, vascular and post-operative complications. Their onset may be increased by the longstanding concomitance of both diseases. Although specific studies on this aspect have not yet been carried out, this deserves attention in clinical practice. In particular, the role of hyperglycaemia and the poor control of blood glucose level in diabetics deserve particular attention for the risk of morbidity of patients undergoing ileoanal pouch after proctocolectomy. One of the most common and challenging problems in diabetic patients with UC is the medical treatment. Corticosteroids, the treatment of choice of active UC, may be associated with the onset of glucose intolerance and diabetes, and with the difficult control of blood glucose levels and complications in diabetic patients. Advanced age, high body mass index, family history of diabetes or previous gestational diabetes should always suggest the need of monitoring blood glucose level during steroid therapy. Likewise, rehydration, correction of blood glucose and hypokalemia in close collaboration with endocrinologists, as well as the close monitoring of the patient s clinical and biochemical conditions are essential in diabetic patients with acute UC. 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Once-daily budesonide MMX in active, mildto-moderate ulcerative colitis: results from the randomised CORE II study. Gut 2014; 63: [PMID: ] 72 Lewis JD, Lichtenstein GR, Stein RB, Deren JJ, Judge TA, Fogt F, Furth EE, Demissie EJ, Hurd LB, Su CG, Keilbaugh SA, Lazar MA, Wu GD. An open-label trial of the PPARgamma ligand rosiglitazone for active ulcerative colitis. Am J Gastroenterol 2001; 96: [PMID: ] 73 Lewis JD, Lichtenstein GR, Deren JJ, Sands BE, Hanauer SB, Katz JA, Lashner B, Present DH, Chuai S, Ellenberg JH, Nessel L, Wu GD. Rosiglitazone for active ulcerative colitis: a randomized placebo-controlled trial. Gastroenterology 2008; 134: [PMID: DOI: / j.gastro ] P- Reviewers: Chang HY, Shi Z, Vetvicka V S- Editor: Wen LL L- Editor: A E- Editor: Liu XM 3515 April 7, 2014 Volume 20 Issue 13

141 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (3): Inflammatory bowel disease TOPIC HIGHLIGHT Hepatitis B and C virus reactivation in immunosuppressed patients with inflammatory bowel disease Stefano Sansone, Maria Guarino, Fabiana Castiglione, Antonio Rispo, Francesco Auriemma, Ilaria Loperto, Matilde Rea, Nicola Caporaso, Filomena Morisco Stefano Sansone, Maria Guarino, Fabiana Castiglione, Antonio Rispo, Francesco Auriemma, Ilaria Loperto, Matilde Rea, Nicola Caporaso, Filomena Morisco, Gastroenterology Unit, Department of Clinical Medicine and Surgery, University of Naples Federico II, Naples, Italy Author contributions: Sansone S, Guarino M and Morisco F wrote the paper; all authors made a substantial contribution to interpretation of the data, revised the manuscript critically for important intellectual content and approved the final version to be published. Correspondence to: Filomena Morisco, Professor, Gastroenterology Unit, Department of Clinical Medicine and Surgery, University of Naples Federico II, via Sergio Pansini, 5, Napoli, Italy. filomena.morisco@unina.it Telephone: Fax: Received: September 28, 2013 Revised: December 13, 2013 Accepted: March 6, 2014 Published online: April 7, 2014 Abstract In recent years, a number of case reports and clinical studies have highlighted the risk of hepatitis B and C virus reactivation in patients with inflammatory bowel disease who are treated with immunosuppressive drugs. The cases of viral hepatitis reactivation that have been reported are characterized by a wide range of clinical manifestations, from viremia without clinically relevant manifestations to fulminant life-threatening hepatitis. The development and dissemination of biological immunosuppressive drugs have led to a significant increase in the number of reports of interest to physicians in a variety of clinical settings. On this topic, there have been a number of published guidelines and reviews that have collected the available evidence, providing recommendations on prophylactic and therapeutic strategies and methods for monitoring patients at risk. However, it should be noted that, to date, very few clinical studies have been published, and most of the recommendations have been borrowed from other clinical settings. The published studies are mostly retrospective and are based on very heterogeneous populations, using different therapeutic and prophylactic regimens and obtaining conflicting results. Thus, it seems clear that it is desirable to concentrate our efforts on prospective studies, not conducting further reviews of the literature in the continued absence of new evidence Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Inflammatory bowel disease; Biological agents; Hepatitis B virus reactivation; Hepatitis C virus reactivation; Prophylaxis Core tip: Our review focused on the redundancy of papers on hepatitis B virus and hepatitis C virus reactivation in patients undergoing immunosuppressive therapy. However, we emphasize that, to date, very few clinical studies have been published, and most of them were retrospective with conflicting results. Thus, it is essential to conduct prospective studies before performing additional reviews of the literature. Sansone S, Guarino M, Castiglione F, Rispo A, Auriemma F, Loperto I, Rea M, Caporaso N, Morisco F. Hepatitis B and C virus reactivation in immunosuppressed patients with inflammatory bowel disease. World J Gastroenterol 2014; 20(13): Available from: URL: v20/i13/3516.htm DOI: i INTRODUCTION In recent years, a significant number of warnings have been given about the risk of hepatitis B and C reactiva April 7, 2014 Volume 20 Issue 13

142 Sansone S et al. HBV and HCV reactivation in IBD patients tion in patients receiving immunosuppressive therapy in different clinical settings (oncology, hematology, solid organ transplant, rheumatology, gastroenterology, and dermatology). In particular, a number of case reports and clinical studies have highlighted the risk of hepatitis virus B and C reactivation in patients with inflammatory bowel disease (IBD) treated with immunosuppressive drugs [1-7]. The cases of viral hepatitis reactivation that have been reported are characterized by a wide range of clinical manifestations, from viremia without clinical relevant manifestations to fulminant life threatening hepatitis. The risk and severity of clinical reactivation seems to be related to the type of immunosuppressive drug administered (steroids, traditional immunosuppressants, or biologics). The various reports and clinical studies on this topic describe and analyze patient populations subjected to different treatment regimens that are not comparable. Moreover, these studies have heterogeneous results and conclusions [8-13]. The magnitude of the reactivation problem seems much more relevant to the hepatitis B virus (HBV) than to the hepatitis C virus (HCV). To date, many reviews comparing few clinical trials have been published on this issue. Among the clinical trials, there was only one prospective study. All of the studies were conducted in different populations with different methods and drawing varying conclusions. Furthermore, the definitions of HBV and HCV reactivations used vary significantly among these studies. Some studies have considered only the increase of viral load, while others have considered the increased viral load in association with elevated levels of aminotransferases. While some recommendations may appear evidencebased, such as prophylaxis for hepatitis B surface antigen positive (HBsAg+) patients receiving immunosuppressive therapy, there are large gray areas, indicating the risk of reactivation in patients with hepatitis B core antibody (HBcAb) isolated or and HCV positivity. As mentioned above, there is currently not enough strong evidence regarding the prevalence and clinical impact of hepatitis B and C virus reactivation in patients with IBD who are receiving immunosuppressive therapy. This finding calls for the development of prospective clinical trials that can respond to the growing demand of evidence on this issue [14,15]. IMMUNOSUPPRESSIVE THERAPY IN IBD The ideal therapeutic approach to IBD should be aimed at inducing and maintaining long-term clinical remission with the minimal use of steroids and surgical interventions; this end-point is particularly important for patients affected by Crohn s disease (CD). However, about half of IBD cases show a steroid-dependent or steroid-refractory clinical course [16,17]. Currently, a traditional immunosuppressive therapeutic regimen is indicated for these patients and is mainly represented by the use of azathioprine and 6-mercaptopurine, even if this regimen produces a low percentage of clinical remission (50%) in the treated population [18,19]. Other potentially effective immune-suppressors are methotrexate, cyclosporine and tacrolimus. These drugs are rarely used in this setting due to the lack of significant evidence on their efficacy and are used in clinical practice for steroidrefractory ulcerative colitis (UC), as well as anti-tumor necrosis factor alpha (anti-tnfα) agents [20-30]. IBD patients who are refractory or intolerant to azathioprine are the main candidates for biological anti- TNFα drugs (i.e., adalimumab and infliximab). Infliximab has proven to be an effective drug in inducing and maintaining clinical remission in refractory luminal and fistulizing CD, with a remission rate of approximately 50% [29,31-36]. The efficacy of infliximab in treating UC is less impressive than in CD. However, the ACT-1 study [37] reported an efficacy of 39% and 20% for infliximab in inducing the remission of steroid-dependent UC at weeks 8 and 54, respectively. Furthermore, this drug can avoid the 3-mo need for colectomy in approximately 65% of patients who experience a severe attack of UC [38,39]. At present, adalimumab, a fully human recombinant anti-tnfα antibody, is approved for the treatment of CD and UC. This drug, administered by subcutaneous injection, has been efficient in inducing and maintaining remission in CD, with a 40% remission rate at week 56 [40,41]. Therefore, similar to infliximab, adalimumab has proven to be valuable in reducing the need for steroids and surgery [42]. In view of their high efficacy, it has also been suggested that biologics could be used in the early phases of IBD in accordance with a top-down therapeutic strategy [43], particularly for patients with a poor prognosis and factors implicating a potentially aggressive disease (i.e., young age, smoking, perianal/rectal disease in CD, extensive small bowel involvement, extra-intestinal manifestations, and steroid use at diagnosis) [42,44-47]. PREVALENCE OF HBV AND HCV INFECTION IN PATIENTS WITH IBD The prevalence of HBV infection varies greatly throughout the world, from the low rate of < 1% to the moderate rate of 1%-2% in Western countries, towards a much higher rate of > 8% in Asia and in most parts of Africa. The dramatic progress in controlling the spread of HBV in Western countries is prevalently due to the implementation of the vaccination and the adoption of satisfactory measures in preventing HBV transmission [48-53]. Patients with IBD are considered at risk of HBV and HCV infection because of the frequent need for surgical, endoscopic and transfusion procedures, suggesting the existence of nosocomial transmission. On the other hand, the magnitude of this risk is unknown because there are conflicting reviews and little information on this topic April 7, 2014 Volume 20 Issue 13

143 Sansone S et al. HBV and HCV reactivation in IBD patients Five studies from Italy [8,54], France [49], Spain [55] and China [51] have evaluated the prevalence of HBV and HCV infections in consecutive series of patients with IBD. Between 1997 and 1999, 332 patients affected by CD were enrolled in an Italian case-control study published in early The prevalence of HBsAg, HBcAb and HCV in this cohort was 2.1%, 10.9% and 7.4%, respectively. Ten years later, the prevalence of HBsAg and HCV was approximately 1% (Table 1) in 315, 2076 and 301 consecutive subjects in three series of French, Spanish and Italian IBD patients. HBcAb positivity was present in 8/315 (2.54%) of patients in the French study (3 HBsAg+ and HBcAb+; 2 isolated HBcAb+; and 3 HBcAb+ and anti- HBs+); in 154/2056 (7.49%) of patients in the Spanish study; and in 22/301 (7.31%) of patients in the Italian study. Therefore, the prevalence of HBV and HCV infection in IBD patients seems to be lower than expected, similar to the general population. These results indicate that IBD patients in Western European countries should no longer be considered as a risk group for HBV or HCV infection. However, a recent study conducted in China on 714 IBD patients showed that the prevalence of HBV infection in IBD patients was higher than reported in the control group. Indeed, the cumulative prevalence of HBsAg and HBcAb was 40.62% vs 27.58% in non-ibd patients. The prevalence of HCV infection was similar to that found in non-ibd patients (0.42% vs 0.36%). HBV INFECTION Mechanism of reactivation From an immunological point of view, a hepatitis flare rarely starts during the phase of maximal immunosuppression. The majority of reported HBV reactivations matured at the time of the withdrawing or tapering of immunosuppressive therapy, when the immune system has been able to react to the viral replication and to destroy infected hepatocytes [56]. In this perspective, any deficiency of the immune response to infections caused by immunosuppressive/chemotherapeutic drugs would play a crucial role in disease progression. In addition, several in vitro and in vivo animal studies have demonstrated that TNF-α plays a key role in clearing HBV from infected hepatocytes. Hepatitis B viral HBx proteins sensitize the cells to apoptotic killing by TNF, which is secreted by cytotoxic T lymphocytes, together with Interferon-γ (IFN-γ). Both TNF and IFN-γ clear HBV replicative intermediates from the cytoplasm and covalently close circular DNA from the nuclei of infected hepatocytes [57,58]. Based on these results, it is possible that the use of anti-tnfα drugs in patients with chronic HBV infections could cause an increase in viral replication, leading to liver immune-mediated damage when the inhibitory effect of therapy disappears [59]. According to this pathogenic model, the risk of HBV reactivation is closely related to the level of immunosuppression achieved, which changes significantly based on the immunomodulatory agents used during the therapy. Stronger immunosuppression may lead to increased viral replication; consequently, a more severe clinical reactivation may occur when immunosuppressive therapy is discontinued [56]. Immunosuppressive therapy and risk of reactivation in HBsAg positive patients The reactivation of HBV is an important concern for patients taking immunosuppressants because of the many ways it can affect the body, from a subtle shift in serum aminotransferase levels to fulminant hepatic failure and/ or death. The reactivation of HBV infection (with liver dysfunction, including fulminant hepatitis) is a welldescribed complication of immunosuppression in the setting of organ transplantation or cancer chemotherapy, occurring in approximately 50% of patients for whom concomitant anti-viral therapy is not used. Mortality from fulminant liver failure after the reactivation of HBV in patients receiving chemotherapy is reported in 4%-60% of cases. The traditional immunosuppressive drugs used in the management of IBD patients include the following: corticosteroids; thiopurines: azathioprine and 6-mercaptopurine; calcineurin inhibitors; cyclosporin and tacrolimus; and methotrexate. Moreover, in the past decade, these biological agents, especially TNFα inhibitors, have been used worldwide and are particularly beneficial in the management of complex and fistulizing diseases [19-23,31-36]. From a pathogenic perspective, the risk of HBV reactivation is closely related to the levels of immunosuppression. The use of conventional immunosuppressive drugs determines low levels of immunosuppression and does not seem to be associated with the risk of HBV reactivation; this claim is supported by the results of only one study [9]. In a series of 332 CD patients, only 4 HBsAg positive patients were treated with conventional immunosuppressive drugs (2 with azathioprine and 2 with corticosteroids) and followed-up for at least 1 year. No influence on the clinical course of HBV infection and no episodes of viral or biochemical reactivation were observed in this series of patients over the follow-up of 12 mo. Notably, only a few cases (4 cases) of HBV reactivation during conventional immunosuppressive therapy (prednisone and azathioprine) resulting in fulminant hepatic failure has been reported in the literature [11,60]. In recent years, a growing number of cases of HBV reactivation among patients with IBD treated with TNFα-inhibitors have been described. The available data are limited to a small number of single case reports and a very small series of consecutive patients [10,12-13,60-65]. Eight case reports describe the use of anti-tnfα drugs in patients with IBD infected with HBV (HBsAg April 7, 2014 Volume 20 Issue 13

144 Sansone S et al. HBV and HCV reactivation in IBD patients Table 1 Virologic and clinical outcomes of hepatitis B virus infection in patients with inflammatory bowel disease undergoing immunosuppressive therapy Ref. Disease Age/ sex HBsAg status HBV-DNA before therapy Anti- TNFα Contemporary drugs LAM prophylaxis HBV-DNA reactivation Biochemical reactivation Esteve et al [66] CD 34 M + IC NA IFX AZT No Yes ALT pg/ml AST 1561 CD 38 M + IC NA IFX AZT No Yes ALT pg/ml AST 2146 CD 26 M + CH Positive IFX AZT Yes No No del Valle et al [61] CD 40 M + CH Positive IFX AZT No No worsening No copies/mm 3 Ueno et al [64] CD 28 F + IC NA IFX AZT No Yes ALT LEG/mL AST 64 Millonig et al [12] CD 50 M + IC Positive IFX AZT No Yes ALT 983/50 20 IU/mL UI/mL AST 413/50 Colbert et al [10] CD 54 M + IC NA IFX AZT No Yes ALT pg/ml AST 143 Madonia et al [13] CD 41 F - OC NA IFX Steroids No Yes ALT 10 UNL AST 6 UNL Ojiro et al [63] CD 43 F + IC NA IFX AZT No Yes ALT LGE/mL AST 145 Zeitz et al [11] UC 43 M NA NA Steroids + AZT No Yes ALT UI/mL AST 2193 CD: Crohn s disease; UC: Ulcerative Colitis; IC: Inactive carrier; OC: Occult carrier; NA: Not available; CH: Chronic hepatitis; IFX: Infliximab; AZT: Azathioprine; LGE: Logarithm genome equivalent; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; HBsAg: Hepatitis B surface antigen; HBV: Hepatitis B virus; TNFα: Tumor necrosis factor alpha; LAM: Lipoarabinomannan. inactive carriers). All were affected by CD, and 7 out of 8 did not receive immunoprophylaxis. Only one patient received lamivudine as a prophylactic treatment. Six out of 7 showed HBV reactivation with a wide range of outcomes, ranging from a modest increase in the viral load and ALT levels to fatal hepatitis. Interestingly, the only patient who received prophylactic treatment did not experience viral, biochemical or clinical reactivation during the 6 mo follow-up [66]. The duration of anti-tnfα therapy before HBV relapse varied from a single infusion to many months of treatment, and all of the episodes of reactivation were observed in patients receiving infliximab and other immunosuppressive drugs at the same time (Table 1). No reports are available in patients using adalimumab. Recently, Loras et al [55] recorded all clinical information from IBD patients with HBV and HCV infections in 19 Spanish hospitals. HBV reactivation was observed in 9 out of 25 (36%) HBsAg+ patients. All but two cases were treated with simultaneous immunosuppressors [steroid + azathioprine (4 cases) and infliximab + azathioprine (3 cases)]. In contrast, the patients without reactivation were only treated with one immunosuppressant and/or received prophylactic antiviral treatment. In 6 out of the 9 cases (66%), HBV reactivation caused severe hepatic failure. On the contrary, Morisco et al [65] conducted a retrospective multicenter study, including 5096 patients with IBD. HBV reactivation was observed in only 1 out of 6 (16%) HBsAg+ patients undergoing immunosuppressive therapy. A combined immunosuppressive therapy had been administered in all cases of HBV reactivation (Table 2). Immunosuppressive therapy and risk of reactivation in HBsAg negative/hbcab positive carriers HBsAg negative and HBcAb positive IBD patients seem to have a low risk of HBsAg seroreversion and hepatitis flares. Indeed, in opposition to the results found in other clinical settings, only one case of HBV reactivation has been described in an IBD patient [13]. A female CD patient was treated with 25 mg/d of prednisone and infliximab for the relapse of disease after an unsatisfactory remission with conventional therapy (steroids, ciprofloxacin, metronidazole, and methotrexate). After a month of treatment with infliximab and steroids, the patient s aminotransferases increased (10 times over the upper normal limit), together with the emergence of HBsAg, hepatitis Be antibody, HBcAb, IgM HBcAb, and HBV-DNA positivity in the serum. HCV Immunosuppressive therapy and risk of reactivation To date, there is no conclusive information on the safety of immunosuppressive or immunomodulator drugs in HCV among IBD patients. These patients appear to be at low risk, although long-term safety studies are needed. There are several interesting concerns regarding immunosuppressive treatment for IBD in patients with HCV infection. For example, prednisone, which is frequently used to treat acute exacerbations of intestinal diseases, may negatively affect HCV infection by increasing the viral load [67-69]. On the other hand, anti-tnfα drugs seem to reduce inflammation through TNFα inhibition, playing a role in the pathogenesis of HCV [70] April 7, 2014 Volume 20 Issue 13

145 Sansone S et al. HBV and HCV reactivation in IBD patients Table 2 Reactivation in patients with inflammatory bowel disease undergoing immunosuppressive therapy Ref. HBsAg+ HBcAb+ HCV+ Loras et al [60] 9/25 0/65 8/51 Morisco et al [65] 1/6 1/4 1/10 Papa et al [54] 0/1 0/22 0/4 HBsAg: Hepatitis B surface antigen; HBcAb: Hepatitis B core antibody; HCV: Hepatitis C virus. Few studies have been performed to evaluate the safety of TNFα antagonist medications in IBD patients with HCV, and most of these are case reports or small case series [54,55,65]. The larger series available on this topic show a low risk of hepatitis flares in these patients with a mildmoderate clinical course [54,60,65]. Indeed, HCV reactivation was observed in 8/51 (15.7%) and in 1/10 (10%) HCV- RNA positive patients, respectively, by Loras et al [60] and Morisco et al [65]. All cases of reactivation had a very mild course, except for one patient, who died. The deceased patient described by Loras et al [60] and experienced severe liver failure. He died while receiving steroids. Importantly, he also had an occult HBV infection and was human immunodeficiency virus-positive. SCREENING AND VACCINATION Screening measures must be instituted in IBD patients. Recommendations are based on the potentially fatal consequences of HBV and HCV reactivation and the availability of safe and effective drugs to prevent these situations [11]. HBV The most recent guidelines of the European Crohn s and Colitis Organization (ECCO) on the management of opportunistic infections in IBD [17] recommend that all IBD patients be tested for HBsAg, HBcAb and HBsAb to assess their infection or vaccination status; vaccination is recommended in all seronegative patients. At present, only four studies have evaluated the HBV vaccination status of patients with IBD. Evidence of effective vaccination (positive anti-hbs and negative HBcAb) was only detected in 12%, 48.9%, 24% and 21.7% of the four cohorts of patients enrolled in Spain, France, Italy and China, respectively [49,51,54,55]. No information about the adherence to HBV vaccination is available in the literature for other series of European patients with IBD. HBV vaccination coverage significantly differs among European countries because the vaccination programs were started in different years and have been proposed for different target populations (newborns, adolescent and pre-adolescent subjects, only for high-risk groups, etc.) [71]. As consequence, the determination of the infectious or vaccination status at the time of a diagnosis of IBD seems to be appropriate. An administration of the vaccine in negative subjects (HBsAg, HBcAb and HBsAb negative) as soon as possible should progressively reduce the number of cases with problematic management. Improved adherence to the program of universal vaccination will reduce the need for screening in the future. HCV No recommendations have been proposed for HCV screening prior to starting immunomodulators by the ECCO guidelines [72]. However, we do believe that HCV screening (including HCV antibodies and HCV-RNA if anti-hcv+) should be routinely performed upon the completion of liver function tests before starting immunosuppressive therapy. If positive, these tests should be performed again every 3 mo to carefully monitor the patient s status during immunosuppressive treatment. With regard to the use of anti-tnfα agents, it is important to emphasize that their use should be evaluated on the basis of the clinical underlying condition of the patient. In particular, while the use of anti-tnfα in noncirrhotic patients appears safe, it is contraindicated in patients with decompensated cirrhosis and should be used with caution in patients with compensated cirrhosis on a case-by-case basis, according to the benefit/risk ratio. PROPHYLAXIS AND THERAPY Several studies have reported a preventive effect of antiviral agents on hepatitis B reactivation during immunosuppression therapy, and specific recommendations were elaborated by American Association for the Study of Liver Diseases, European Association for the Study of the Liver and Association for the Study of the Liver [59,73,74]. Nonetheless, significant questions regarding the optimal antiviral prophylaxis strategy have yet to be addressed. The effect of prophylactic antiviral therapy on the course of HBV infection in IBD patients undergoing immunosuppressive therapy has not been studied prospectively, and, until now, only a few studies have reported a good efficacy in a short period of follow-up [65,66]. As a result, the management strategy for these patients remains uncertain. Lamivudine prophylaxis was suggested on the basis of its well demonstrated efficacy; however, other nucleotides/nucleosides should be preferred, especially if immunosuppressive therapy is scheduled for more than 12 mo, due to their lower propensity to provoke drug resistance. Conversely, HBsAg- HBcAb+ patients (with or without HBsAb+), given their low risk for reactivation, do not require routine antiviral prophylaxis, but only periodic monitoring (about every 3 mo) for the elevation of aspartate aminotransferase/alanine aminotransferase and HBsAg, to prove the re-emergence of HBV-DNA [72]. Prophylactic therapy seems to be appropriate when biological therapy is scheduled, especially when combined with other immunosuppressive drugs. The role of antiviral drugs other than lamivudine, including entecavir, ad April 7, 2014 Volume 20 Issue 13

146 Sansone S et al. HBV and HCV reactivation in IBD patients efovir and tenofovir, is still unknown, and further studies are needed [75]. To date, there is no prophylaxis available for HCV reactivation. Interferon, which is currently the milestone of antiviral therapy for HCV, is contraindicated in IBD forms that require immunosuppressive therapy. The availability of interferon-free regimens could dramatically change this scenario. CONCLUSION The issue in question has increasingly gained interest over the past few years, with the emergence of a number of reports (case reports or case series) on the risk of reactivation of hepatitis in immunosuppressed patients with IBD. The first cases reported in literature date back to the 1990s and studied onco-hematological patients who demonstrated a fulminant HBV reactivation during or after chemotherapy treatments. In subsequent years, the development and dissemination of biological immunosuppressive drugs led to a significant increase in the number of reports aimed at reaching other clinical settings (dermatology, rheumatology, gastroenterology, etc.). In particular, the use of anti-tnfα is more frequent in the treatment of steroid-dependent and steroid-refractory UC and CD, as well as in the treatment of perianal fistulizing CD or extraintestinal manifestations associated with IBD (ankylosing spondylitis, pyoderma gangrenosum and uveitis). There have been a number of published guidelines and reviews on this topic that have collected the available evidence and provided recommendations on prophylactic and therapeutic strategies and methods for monitoring patients at risk. However, it should be noted that, to date, very few clinical studies have been published, and most of their recommendations have been borrowed from other clinical settings. The published studies are mostly retrospective and based on heterogeneous populations, using different therapeutic and prophylactic regimens to obtain conflicting results. In particular, the two studies with larger series are those of Loras et al [60] and Morisco et al [65]. The first group analyzed a population of 25 HBsAg+ and 51 HCV- RNA+ patients. Among HBsAg+ patients, 36% experienced a reactivation of the HBV, and 6 of them developed acute liver failure; however, no reactivation was observed in patients with HBsAg-HBcAb positivity. On the other hand, Morisco et al showed a different result in their study. Only 1/6 HBsAg+ (16%) and 1/4 HBcAb+ isolated (25%) had viral reactivations with mild clinical courses. The two groups of authors agreed on the low risk of HCV reactivation. 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148 Sansone S et al. HBV and HCV reactivation in IBD patients P, Grännö C, Vilien M, Ström M, Danielsson A, Verbaan H, Hellström PM, Magnuson A, Curman B. Infliximab as rescue therapy in severe to moderately severe ulcerative colitis: a randomized, placebo-controlled study. Gastroenterology 2005; 128: [PMID: DOI: / j.gastro ] 39 Kohn A, Daperno M, Armuzzi A, Cappello M, Biancone L, Orlando A, Viscido A, Annese V, Riegler G, Meucci G, Marrollo M, Sostegni R, Gasbarrini A, Peralta S, Prantera C. Infliximab in severe ulcerative colitis: short-term results of different infusion regimens and long-term follow-up. Aliment Pharmacol Ther 2007; 26: [PMID: DOI: /j x] 40 Hanauer SB, Sandborn WJ, Rutgeerts P, Fedorak RN, Lukas M, MacIntosh D, Panaccione R, Wolf D, Pollack P. Human anti-tumor necrosis factor monoclonal antibody (adalimumab) in Crohn s disease: the CLASSIC-I trial. 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Early combined immunosuppression or conventional management in patients with newly diagnosed Crohn s disease: an open randomised trial. Lancet 2008; 371: [PMID: DOI: /S (08) ] 44 Van Assche G, Vermeire S, Rutgeerts P. The potential for disease modification in Crohn s disease. Nat Rev Gastroenterol Hepatol 2010; 7: [PMID: DOI: /nrgastro ] 45 Bongartz T, Sutton AJ, Sweeting MJ, Buchan I, Matteson EL, Montori V. Anti-TNF antibody therapy in rheumatoid arthritis and the risk of serious infections and malignancies: systematic review and meta-analysis of rare harmful effects in randomized controlled trials. JAMA 2006; 295: [PMID: DOI: /jama ] 46 Siegel CA, Marden SM, Persing SM, Larson RJ, Sands BE. Risk of lymphoma associated with combination anti-tumor necrosis factor and immunomodulator therapy for the treatment of Crohn s disease: a meta-analysis. Clin Gastroenterol Hepatol 2009; 7: [PMID: DOI: / j.cgh ] 47 Peyrin-Biroulet L, Deltenre P, de Suray N, Branche J, Sandborn WJ, Colombel JF. Efficacy and safety of tumor necrosis factor antagonists in Crohn s disease: meta-analysis of placebo-controlled trials. Clin Gastroenterol Hepatol 2008; 6: [PMID: DOI: /j.cgh ] 48 Tolentino YF, Fogaca HS, Zaltman C, Ximenes LL, Coelho HS. Hepatitis B virus prevalence and transmission risk factors in inflammatory bowel disease patients at Clementino Fraga Filho university hospital. World J Gastroenterol 2008; 14: [PMID: DOI: /wjg ] 49 Chevaux JB, Nani A, Oussalah A, Venard V, Bensenane M, Belle A, Gueant JL, Bigard MA, Bronowicki JP, Peyrin-Biroulet L. Prevalence of hepatitis B and C and risk factors for nonvaccination in inflammatory bowel disease patients in Northeast France. Inflamm Bowel Dis 2010; 16: [PMID: DOI: /ibd.21147] 50 Di Stefano R, Stroffolini T, Ferraro D, Usticano A, Valenza LM, Montalbano L, Pomara G, Craxì A. Endemic hepatitis C virus infection in a Sicilian town: further evidence for iatrogenic transmission. J Med Virol 2002; 67: [PMID: DOI: /jmv.10081] 51 Huang ML, Xu XT, Shen J, Qiao YQ, Dai ZH, Ran ZH. Prevalence and factors related to hepatitis B and C infection in inflammatory bowel disease patients in China: A retrospective study. J Crohns Colitis 2014; 8: [PMID: ] 52 Stroffolini T, Menchinelli M, Taliani G, Dambruoso V, Poliandri G, Bozza A, Lecce R, Clementi C, Ippolito FM, Compagnoni A. High prevalence of hepatitis C virus infection in a small central Italian town: lack of evidence of parenteral exposure. Ital J Gastroenterol 1995; 27: [PMID: ] 53 Stroffolini T, Guadagnino V, Chionne P, Procopio B, Mazzuca EG, Quintieri F, Scerbo P, Giancotti A, Nisticò S, Focà A, Tosti ME, Rapicetta M. A population based survey of hepatitis B virus infection in a southern Italian town. Ital J Gastroenterol Hepatol 1997; 29: [PMID: ] 54 Papa A, Felice C, Marzo M, Andrisani G, Armuzzi A, Covino M, Mocci G, Pugliese D, De Vitis I, Gasbarrini A, Rapaccini GL, Guidi L. Prevalence and natural history of hepatitis B and C infections in a large population of IBD patients treated with anti-tumor necrosis factor-α agents. J Crohns Colitis 2013; 7: [PMID: DOI: / j.crohns ] 55 Loras C, Saro C, Gonzalez-Huix F, Mínguez M, Merino O, Gisbert JP, Barrio J, Bernal A, Gutiérrez A, Piqueras M, Calvet X, Andreu M, Abad A, Ginard D, Bujanda L, Panés J, Torres M, Fernández-Bañares F, Viver JM, Esteve M. Prevalence and factors related to hepatitis B and C in inflammatory bowel disease patients in Spain: a nationwide, multicenter study. Am J Gastroenterol 2009; 104: [PMID: DOI: /ajg ] 56 Perrillo RP. Acute flares in chronic hepatitis B: the natural and unnatural history of an immunologically mediated liver disease. Gastroenterology 2001; 120: [PMID: DOI: /gast ] 57 Herbein G, O Brien WA. Tumor necrosis factor (TNF)- alpha and TNF receptors in viral pathogenesis. Proc Soc Exp Biol Med 2000; 223: [PMID: DOI: / j x] 58 Kasahara S, Ando K, Saito K, Sekikawa K, Ito H, Ishikawa T, Ohnishi H, Seishima M, Kakumu S, Moriwaki H. Lack of tumor necrosis factor alpha induces impaired proliferation of hepatitis B virus-specific cytotoxic T lymphocytes. J Virol 2003; 77: [PMID: DOI: / JVI ] 59 Marzano A, Angelucci E, Andreone P, Brunetto M, Bruno R, Burra P, Caraceni P, Daniele B, Di Marco V, Fabrizi F, Fagiuoli S, Grossi P, Lampertico P, Meliconi R, Mangia A, Puoti M, Raimondo G, Smedile A. Prophylaxis and treatment of hepatitis B in immunocompromised patients. Dig Liver Dis 2007; 39: [PMID: DOI: / j.dld ] 60 Loras C, Gisbert JP, Mínguez M, Merino O, Bujanda L, Saro C, Domenech E, Barrio J, Andreu M, Ordás I, Vida L, Bastida G, González-Huix F, Piqueras M, Ginard D, Calvet X, Gutiérrez A, Abad A, Torres M, Panés J, Chaparro M, Pascual I, Rodriguez-Carballeira M, Fernández-Bañares F, Viver JM, Esteve M. Liver dysfunction related to hepatitis B and C in patients with inflammatory bowel disease treated with immunosuppressive therapy. Gut 2010; 59: [PMID: DOI: /gut ] 61 del Valle García-Sánchez M, Gómez-Camacho F, Poyato- González A, Iglesias-Flores EM, de Dios-Vega JF, Sancho- Zapatero R. Infliximab therapy in a patient with Crohn s disease and chronic hepatitis B virus infection. Inflamm Bowel 3523 April 7, 2014 Volume 20 Issue 13

149 Sansone S et al. HBV and HCV reactivation in IBD patients Dis 2004; 10: [PMID: DOI: / ] 62 Esteve M, Loras C, González-Huix F. Lamivudine resistance and exacerbation of hepatitis B in infliximab-treated Crohn s disease patient. Inflamm Bowel Dis 2007; 13: [PMID: DOI: /ibd.20202] 63 Ojiro K, Naganuma M, Ebinuma H, Kunimoto H, Tada S, Ogata H, Iwao Y, Saito H, Hibi T. Reactivation of hepatitis B in a patient with Crohn s disease treated using infliximab. J Gastroenterol 2008; 43: [PMID: DOI: /s x] 64 Ueno Y, Tanaka S, Shimamoto M, Miyanaka Y, Hiyama T, Ito M, Kitadai Y, Yoshihara M, Sumii M, Chayama K. Infliximab therapy for Crohn s disease in a patient with chronic hepatitis B. Dig Dis Sci 2005; 50: [PMID: DOI: /s ] 65 Morisco F, Castiglione F, Rispo A, Stroffolini T, Sansone S, Vitale R, Guarino M, Biancone L, Caruso A, D Inca R, Marmo R, Orlando A, Riegler G, Donnarumma L, Camera S, Zorzi F, Renna S, Bove V, Tontini G, Vecchi M, Caporaso N. Effect of immunosuppressive therapy on patients with inflammatory bowel diseases and hepatitis B or C virus infection. J Viral Hepat 2013; 20: [PMID: DOI: /j x] 66 Esteve M, Saro C, González-Huix F, Suarez F, Forné M, Viver JM. Chronic hepatitis B reactivation following infliximab therapy in Crohn s disease patients: need for primary prophylaxis. Gut 2004; 53: [PMID: DOI: /gut ] 67 Schiavon LL, Carvalho-Filho RJ, Narciso-Schiavon JL, Barbosa DV, Lanzoni VP, Ferraz ML, Silva AE. Impact of cyclosporine-based immunosuppressive therapy on liver histology of hepatitis C virus-infected renal transplant patients. Hepatology 2008; 48: [PMID: DOI: /hep.22331] 68 Samonakis DN, Triantos CK, Thalheimer U, Quaglia A, Leandro G, Teixeira R, Papatheodoridis GV, Sabin CA, Rolando N, Davies S, Dhillon AP, Griffiths P, Emery V, Patch DW, Davidson BR, Rolles K, Burroughs AK. Immunosuppression and donor age with respect to severity of HCV recurrence after liver transplantation. Liver Transpl 2005; 11: [PMID: DOI: /lt.20344] 69 Zekry A, Gleeson M, Guney S, McCaughan GW. A prospective cross-over study comparing the effect of mycophenolate versus azathioprine on allograft function and viral load in liver transplant recipients with recurrent chronic HCV infection. Liver Transpl 2004; 10: [PMID: DOI: /lt.20000] 70 Campbell S, Ghosh S. Infliximab therapy for Crohn s disease in the presence of chronic hepatitis C infection. Eur J Gastroenterol Hepatol 2001; 13: [PMID: DOI: / ] 71 Nardone A, Anastassopoulou CG, Theeten H, Kriz B, Davidkin I, Thierfelder W, O Flanagan D, Bruzzone B, Mossong J, Boot HJ, Butur D, Slaciková M, Panait ML, Hellenbrand W, DE Melker H, Sobotová Z, Icardi G, Andrews N, Pebody RG, VAN Damme P, Kafatos G, Miller E, Hatzakis A. A comparison of hepatitis B seroepidemiology in ten European countries. Epidemiol Infect 2009; 137: [PMID: DOI: /S ] 72 Rahier JF, Ben-Horin S, Chowers Y, Conlon C, De Munter P, D Haens G, Domènech E, Eliakim R, Eser A, Frater J, Gassull M, Giladi M, Kaser A, Lémann M, Moreels T, Moschen A, Pollok R, Reinisch W, Schunter M, Stange EF, Tilg H, Van Assche G, Viget N, Vucelic B, Walsh A, Weiss G, Yazdanpanah Y, Zabana Y, Travis SP, Colombel JF. European evidence-based Consensus on the prevention, diagnosis and management of opportunistic infections in inflammatory bowel disease. J Crohns Colitis 2009; 3: [PMID: DOI: /j.crohns ] 73 Lok AS, McMahon BJ. Chronic hepatitis B. Hepatology 2007; 45: [PMID: DOI: /hep.21513] 74 European Association For The Study Of The Liver. EASL Clinical Practice Guidelines: management of chronic hepatitis B. J Hepatol 2009; 50: [PMID: DOI: /j.jhep ] 75 López-Serrano P, Pérez-Calle JL, Sánchez-Tembleque MD. Hepatitis B and inflammatory bowel disease: role of antiviral prophylaxis. World J Gastroenterol 2013; 19: [PMID: DOI: /wjg.v19.i9.1342] P- Reviewers: Sung J, Yamamoto S S- Editor: Gou SX L- Editor: A E- Editor: Zhang DN 3524 April 7, 2014 Volume 20 Issue 13

150 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (3): Inflammatory bowel disease TOPIC HIGHLIGHT Clinical management of inflammatory bowel disease in the organ recipient Amedeo Indriolo, Paolo Ravelli Amedeo Indriolo, Paolo Ravelli, Digestive Endoscopy Unit, Department of Gastroenterology, Papa Giovanni XXIII Hospital, Bergamo, Italy Author contributions: Indriolo A and Ravelli P solely contributed to this paper. Correspondence to: Amedeo Indriolo, MD, Digestive Endoscopy Unit, Department of Gastroenterology, Papa Giovanni XXIII Hospital, Piazza OMS, 1, Bergamo, Italy. amedeo.indriolo@gmail.com Telephone: Fax: Received: September 28, 2013 Revised: November 6, 2013 Accepted: January 19, 2014 Published online: April 7, 2014 Abstract There was estimated a higher incidence of de novo inflammatory bowel disease (IBD) after solid organ transplantation than in the general population. The onset of IBD in the organ transplant recipient population is an important clinical situation which is associated to higher morbidity and difficulty in the medical therapeutic management because of possible interaction between anti-reject therapy and IBD therapy. IBD course after liver transplantation (LT) is variable, but about one third of patients may worsen, needing an increase in medical therapy or a colectomy. Active IBD at the time of LT, discontinuation of 5-aminosalicylic acid or azathioprine at the time of LT and use of tacrolimusbased immunosuppression may be associated with an unfavorable outcome of IBD after LT. Anti-tumor necrosis factor alpha (TNFα) therapy for refractory IBD may be an effective and safe therapeutic option after LT. The little experience of the use of biological therapy in transplanted patients, with concomitant anti-rejection therapy, suggests there be a higher more careful surveillance regarding the risk of infectious diseases, autoimmune diseases, and neoplasms. An increased risk of colorectal cancer (CRC) is present also after LT in IBD patients with primary sclerosing cholangitis (PSC). An annual program of endoscopic surveillance with serial biopsies for CRC is recommended. A prophylactic colectomy in selected IBD/PSC patients with CRC risk factors could be a good management strategy in the CRC prevention, but it is used infrequently in the majority of LT centers. About 30% of patients develop multiple IBD recurrence and 20% of patients require a colectomy after renal transplantation. Like in the liver transplantation, anti-tnfα therapy could be an effective treatment in IBD patients with conventional refractory therapy after renal or heart transplantation. A large number of patients are needed to confirm the preliminary observations. Regarding the higher clinical complexity of this subgroup of IBD patients, a close multidisciplinary approach between an IBD dedicated gastroenterologist and surgeon and an organ transplantation specialist is necessary in order to have the best clinical management of IBD after transplantation Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Inflammatory bowel disease; Ulcerative colitis; Crohn s disease; Primary sclerosing cholangitis; Liver transplantation; Heart transplantation; Renal transplantation; Anti-tumor necrosis factor alpha therapy Core tip: Inflammatory bowel disease (IBD) in the organ transplant recipient population is an important clinical situation which is associated to higher morbidity and difficulty in the medical therapeutic management because of possible interaction between anti-reject therapy and IBD therapy. IBD course after liver transplantation is variable, but about one third of patients may worsen, needing an increase in medical therapy or a colectomy. About 30% of patients develop multiple IBD recurrence and 20% of patients require colectomy after renal transplantation. Like in the liver transplantation, anti-tumor necrosis factor alpha therapy could be an effective treatment in IBD patients with conventional 3525 April 7, 2014 Volume 20 Issue 13

151 Indriolo A et al. Clinical management of IBD refractory therapy after renal or heart transplantation. Indriolo A, Ravelli P. Clinical management of inflammatory bowel disease in the organ recipient. World J Gastroenterol 2014; 20(13): Available from: URL: com/ /full/v20/i13/3525.htm DOI: org/ /wjg.v20.i INTRODUCTION Inflammatory bowel disease (IBD) is a complex chronic inflammatory intestinal disease with a prevalence steadily increasing during the recent years. The management of IBD is leaning towards more complex clinical situations, with possible interactions between the intestinal disease and others organ diseases. The organ recipient population is constantly increasing in the medical specialized centers in the world and it can happen that a patient with a solid organ transplantation has a recurrence of IBD. This is particularly important in the liver transplantation (LT) because there is a close pathophysiological correlation between primary sclerosing cholangitis (PSC) and ulcerative colitis (UC) before the transplant. De novo IBD has been reported after solid organ transplantation, with an incidence estimated ten times higher with respect to the expected incidence of IBD in the general population. Therefore, the organ recipient patient may be a new clinical scenario for IBD management. The first objective of this review was to examine the studies present in the English literature (PubMed) about the natural history in organ recipient patients.in particular, we have evaluated: (1) risk of recurrent IBD; (2) risk of de novo IBD; (3) need for colectomy; (4) risk of pouchitis; and (5) risk of colorectal cancer. The second objective was to examine the medical therapy for active IBD after organ transplantation. We have more knowledge of IBD clinical management after LT, but in this review we have evaluated the IBD studies after heart, renal, lung, and intestinal transplantation. EPIDEMIOLOGY AND CLINICAL FEATURE OF IBD AFTER LIVER TRANSPLANTATION Recurrent IBD Singh et al [1] has recently examined the studies on the natural history of IBD after LT for PSC, reporting 609 patients in 14 studies, followed approximately 4.8 years after LT (range, years), about 31% of patients have improvement of IBD activity, 39% of patients do not have a significant change in IBD activity, and 30% of patients develop worsening IBD, requiring intensification of medical therapy and/or surgery. The estimated risk of severe IBD flare up at 5 and 10 years after LT ranged from 39% to 63% and 39% to 98%, with a median time of a flare up around 1 year (range, years) [2-4]. The need of colectomy for acute IBD refractory to medical therapy is nearly 9% (range, 0%-21%) [2-6]. Dvorchik et al [7] observed a significant 3.1-fold increased risk of colectomy due to severe IBD flare up or medically refractory disease after LT compared to IBD patients who did not require LT. De novo IBD Wörns et al [8] evaluated 44 patients with IBD after solid organ transplant (SOT) and reported the de novo disease: 38 of 44 (86%) cases occurred following LT (23) or combined liver/kidney transplantation (15), 4 (9%) after heart transplantation, and 2 (5%) after kidney transplantation. Riley et al [9] identified 14 patients who developed de novo IBD in 6800 cases after liver and kidney transplantation. Twelve (86%) of the patients developed IBD post liver transplant and two (14%) were detected post kidney transplant. The authors estimated a higher incidence of de novo IBD after SOT than in the general population (206 vs 20 cases per persons-year. The higher prevalence of IBD post LT in the SOT patients can be related to the strong association between PSC and UC. In these patients, the 10-year risk of de novo IBD after LT is estimated to be 14%-30%, with median time to development of approximately 4 years (range, years) [2-4]. After a median follow-up of 4 years 18/86 (21%) patients who underwent LT for PSC developed IBD. Verdonk et al [3] observed that the de novo IBD patients tended to develop disease later in posttransplant period than the patients with pre-existing IBD. Moreover, the patients with de novo IBD after LT respond better to medical therapy and none required colectomy. RISK FACTORS FOR IBD AFTER LIVER TRANSPLANTATION Clinical activity of IBD The clinical activity of IBD at the time of LT may be a risk factor for worsening the intestinal disease after LT. In fact, a three-fold higher risk of IBD flare up after LT in patients with active IBD at the time of LT it was observed [3]. Smoking Joshi et al [4] evaluated 110 patients underwent LT for PSC. In the multivariate analysis, active smoking at the time of transplant was the only significant risk factor for flare up of IBD post-transplantation (HR = 17; 95%CI: 2-180). Cytomegalovirus Cytomegalovirus (CMV) mismatch (seropositive donor, seronegative recipient and CMV infection) have not been associated with the recurrence of IBD after LT [4,10,11]. CMV mismatch was associated with a 4.5-fold higher risk of de novo IBD after LT, but CMV infection is not related to de novo IBD post LT [11]. Therapy of IBD after LT The therapy with proven efficacy for active IBD may re April 7, 2014 Volume 20 Issue 13

152 Indriolo A et al. Clinical management of IBD Table 1 Interaction between inflammatory bowel disease therapy and anti-reject therapy in inflammatory bowel disease patients after liver transplantation Drug Efficacy Ref. IBD therapy Anti-reject therapy 5-ASA + [3] [12] Prednisone + + [12] [21] Azathioprine + + [2] Anti-TNFα + [15] [16] [13] [14] [18] Tacrolimus - + [2] [3] Cyclosporine + + [3] Mycophenolate - + [22] Mofetil [23] TNFα: Tumor necrosis factor alpha; IBD: Inflammatory bowel disease; 5-ASA: 5-aminosalicylates. duce the risk of disease exacerbation post-lt (Table 1). 5-aminosalicylates (5-ASA) therapy after LT appears to be protective against the worsening disease activity of IBD, decreasing the flare up and/or colectomy risk about 80% [3,12]. Azathioprine may reduce the risk of active IBD after transplant. In the study s Haagsma et al [2], a comparison between 55 patients who received azathioprine with the 23 patients did not receive azathioprine, was performed and this showed a significantly higher IBD-free survival for azathioprine group. In particular, at 1, 3 and 5 years after LT, the IBD-free survival rates in patients receiving azathioprine were 96%, 96% and 88%, respectively; while in patients not receiving azathioprine, these value were 87%, 63% and 54%, respectively. We have little knowledge about the use of anti-tumor necrosis factor alpha (TNFα) therapy for refractory IBD after transplant. To date, there have been only 22 patients treated with anti-tnf for relapsing IBD following LT; this number includes patients with UC, Crohn s disease, indeterminate colitis and pouchitis, treated with infliximab or adalimumab (Table 2) [13-18]. In our study, we evaluated the efficacy and safety of infliximabtherapy in a homogeneous series of four patients with refractory UC following LT, followed for a median time of 18 mo. At week 54, three patients (75%) experienced sustained improvement of IBD. Complete mucosal healing (defined as absence of lesions) was observed in one of three patients (33%). Steroid treatment was successfully withdrawn during infliximab therapy in all patients. Adverse events included only one infection by Molluscum contagiosum, which resolved without sequelae. No malignancies were observed in any patient following infliximab therapy. No cases of hepatic rejection were documented. Our results are in line with others studies about the efficacy and safety of anti-tnfα therapy in patients with refractory IBD following LT, but larger studies are needed to evaluate the safety profile of biological therapy combined with anti rejection treatment. Anti-reject therapy Tracrolimus is the principal immunosuppresive agent in SOT, but it has been observed, in retrospective studies, that it may be associated with a four-fold higher risk of post-lt IBD relapse [2,3]. In patients with tacrolimus for transplant-related immunosuppression, Dvorchik et al [7] found that the risk of relapse of IBD at 1- and 5-year was 13% and 64%, respectively; while, in patients with tacrolimus-free regimens, the risk of IBD was 4% and 10%, respectively. The cause of a possible relationship between tacrolimus and IBD flare up after transplant is not known. IBD results from inappropriate and ongoing activation of the mucosal immune system in the presence of normal luminal flora. Immunosuppression agents may promote infections which may lead to the change bacterial gut flora and decrease the intestinal barrier function [19]. Moreover, tacrolimus is a strong inhibitor of interleukin-2 production. Deficiency of interleukin-2 can result in T-cell dysregulation, leading to the development of intestinal chronic inflammation [2,4]. Cyclosporine, like anti-tnfα, is the chosen drug in severe steroid refractory UC. Cyclosporine does not seem to worsen the course of IBD after transplant [3,10]. In contrast with tacrolimus, the frequency of interleukin- 2-expressing T cells was significantly higher with cyclosporine in renal transplant patients [20]. This may explain in part the different effect of ciclosporine and tacrolimus on IBD course after transplant. Corticosteroids are effective in acute and chronic prevention of SOT rejection as well as in inducing clinical remission in IBD. Moncrief et al [21] and Navaneethan et al [12] observed that prednisone therapy may favorably modify the course of IBD after LT, but this therapeutic regime is associated to important side effects. Mycophenolate mofetil (MMF) has proven efficacy in SOT, but its role in IBD is not clear [22,23]. Moreover, MMF is associated with enterocolitis, which can mimic a IBD flare up. COLECTOMY POST-LIVER TRANSPLANTATION Ho et al [24] observed a prevalence of colectomy after LT about 35%. In this Scottish study 7 of 20 patients underwent colectomy with a median time of 3.4 years (range years) following LT [24]. The indication for colectomy was chronically active severe UC in three patients (43%), colonic dysplasia or colorectal cancer in three patients (43%) and benign stricture of colon in one patient (14%).The study in Cleveland compared 86 patients with UC and LT for PSC with 81 patients with UC and PSC who did not require LT [25]. The necessity of colectomy was significantly more frequently in the non-lt group 3527 April 7, 2014 Volume 20 Issue 13

153 Indriolo A et al. Clinical management of IBD Table 2 Anti-tumor necrosis factor alpha therapy for management of refractory inflammatory bowel disease after liver transplantation Author Number LT patients Clinical outcome Endoscopic outcome Adverse events Sandhu et al [13] 6 Response: 67% - Systemic lupus erythematosus Colorectal cancer Mohabbat et al [14] 8 Response: 87.5% Mucosal healing: 42.9% Oral candidiasis Clostridium difficile colitis Bacterial pmeumonia Cryptosporidiosis Post-tansplant lympho-proliferative disorder Lal et al [15] 1 Response: 100% Improvement: 100% No El-Nachef et al [16] 2 Response: 100% - No Indriolo et al [18] 4 Response: 75% Mucosal healing: 33% Molluscum contagiosum than the LT group (76.5% vs 34.9%). The percentage of patients which underwent colectomy for steroid dependent/refractory disease was lower in the LT group than patients in the non-lt group (39.9% vs 48.4%). Regarding the possible difficulties to surgically pack the J pouch in patients who underwent LT and Roux-en-Y biliaryjejunal reconstruction, Mathis et al [26] did not encounter any problems in the 13 patients operated on. POUCHITIS POST-LIVER TRANSPLANTATION The risk of acute pouchitis after LT for PSC ranged from 14% to 66% [26-28]. The risk of chronic pouchitis ranged from 9.1% to 73.7% [26-30]. Freeman et al [29] observed that the risk of chronic refractory pouchitis was comparable in patients who underwent LT and those who did not. Therefore, it seems that LT does not increase the risk of pouchitis. With regard to the therapy of pouchitis after LT, Mathis et al [26] evaluated 32 patients who underwent ileal pouch-anal anastomosis (IPAA) for UC and LT for PSC (in 13 patients IPAA followed by OLT). Two-thirds of the patients had pouchitis during follow-up, about half developed a chronic pouchitis that required daily antibiotic therapy. Only one patient needed a defunctioning ileostomy. Anti-TNFα therapy was used in only four IBD patients in three studies for refractory pouchitis after LT for PSC [13,14,18]. Even though a clinical improvement was observed, the little data available does not allow us to extrapolate any conclusions about the efficacy and safety of biological therapy in patients with chronic refractory pouchitis after LT. COLORECTAL CANCER POST-LIVER TRANSPLANT Epidemiology A large range with rates of risk of colorectal cancer after LT for PSC, from 0 to 31.5 per 1000 person/year, is present in literature in recent years [4,7,21,25,31-48]. Watt et al [31], reported a cumulative incidence of colorectal cancer (CRC) at 10-years post LT for PSC of 8.2% as compared to 2.6% after LT for non-psc patients. The CRC risk in PSC patients without IBD after LT is 2.8% at 10-years. There- fore, the combination of IBD and PSC after LT leads the patient to a higher risk in developing CRC. Moreover, the CRC risk is increased in patients with long-standing IBD, long-standing LT and extensive colonic involvement [32,35,45]. In fact, it was observed at 1-, 5- and 10-year, a cumulative incidence of CRC of 3.3%, 6.7%, and 11.8%, respectively, after LT. Regarding the colorectal cancer location, similarly to IBD/PSC patients before LT, the right-side colon is more frequently affected [39]. There is no clear evidence if the age of the patients at the time of LT is associated with CRC risk [7,33,35,45]. No significant association between clinical severity IBD and CRC risk after LT was observed [39,45]. Regarding the use of 5-ASA or ursodeoxycholic acid in the chemoprevention of CRC, it seems that they don t change the cancer risk [33,39]. Risk factors The risk of CRC may increase after LT because of errors in mucosa sampling during colonoscopy or perhaps due to the immunosuppression treatment of anti-reject therapy. Loftus et al [42] observed that the CRC rate was 4.4-fold higher after LT, as compared to a historical cohort patient with PSC/IBD who did not undergo LT. However, the role of LT in the risk of CRC in IBD/PSC patients still isn t clear. In fact, while Dvorchik found that LT did not significantly influence the risk of CRC, it was observed that LT may be an independent risk factor for CRC in other studies [7,35,42,45]. Surveillance A program of endoscopic surveillance with serial biopsies for CRC is recommended by the European Association for the Study of Liver [49]. A colonoscopy is suggested every year in IBD/PSC patients after LT. If dysplasia colonic mucosa is found, a colectomy is advised. It has been shown to be a relatively safe procedure in specialized surgical centers [45,46]. Management Adjuvant pharmacotherapy with drugs like oxaliplatin has also been shown to be well tolerated in patients post- LT and hepatic graft dysfunction was not documented [50]. Prophylactic colectomy in selected IBD/PSC patients with CRC risk factors could be a good management strategy in the CRC prevention, but it is used infrequently in 3528 April 7, 2014 Volume 20 Issue 13

154 Indriolo A et al. Clinical management of IBD IBD/PSC Before LT 1 Adequate treatment of IBD in active flare up and in remission period. 2 Annual colonoscopic surveillance program with serial biopsies for dysplasia/crc screening. Colectomy is indicated in patients with dysplasia/crc. 3 Evaluate indication for colectomy in selected patients with medically refractory IBD or frequent relapse before the PSC patient develops cirrhosis and liver disfunction. At the time of LT 1 Clinical remission of IBD patient is necessary at the time of LT. 2 Don't interrupt the 5-ASA and/or azathioprin therapy. 3 Don t smoke at the time of transplant because it may be a risk factor for flare up of IBD post-transplantation. After LT 1 Evaluate the possibility of substituting tacrolimus with other drugs, like ciclosporine for anti-reject immunosuppressive therapy. 2 Avoid mycophenolate mofetil in the anti-reject immunosuppressive therapy for its possible side effects (enterocolitis). 3 Exclude opportunistic intestinal infections (like CMV and clostridium difficile ) or consider the possibility that diarrhea is induced by the drugs. 4 Add azathioprine to therapy if the patient presents an IBD recurrence. 5 Anti-TNFα therapy seems effective and safe in refractory to conventional therapy IBD patients. The combination of biological therapy with anti-reject therapy needs a careful monitoring program for infections, autoimmune diseases and neoplasms. 6 An annual colonoscopic surveillance program with serial biopsies for dysplasia/crc screening is necessary. Colectomy is indicated in patients with dysplasia/crc or refractory medical therapy of recurrence/de novo IBD. 7 Adjuvant pharmacotherapy seems to be well tolerated in CRC patients. 8 Chronic refractory pouchitis is treated according to guidelines. Figure 1 Clinical management of inflammatory bowel disease/primary sclerosing cholangitis patients before and after liver transplantation. TNFα: Tumor necrosis factor alpha; IBD: Inflammatory bowel disease; LT: Liver transplantation; PSC: Primary sclerosing cholangitis; CRC: Colorectal cancer; CMV: Cytomegalovirus; 5-ASA: 5-aminosalicylates. the majority of LT centers [45,51]. CLINICAL MANAGEMENT OF ACTIVE IBD AFTER LT Patients who underwent LT can develop diarrhea and it is very important exclude an intestinal infection (CMV, Clostridium difficile), or consider the possibility that diarrhea may be caused by the drugs (Figure 1). If the symptoms of active IBD are confirmed from a colonoscopy and a histological exam, the patient starts the IBD therapy. The plan of IBD therapy in the patient who underwent transplant, is similar to the therapy plan before the transplant. 5-ASA at a dose of 2.4 g per day is indicated in induction and in maintenance in the mild-to-moderate UC patients. Topical 3529 April 7, 2014 Volume 20 Issue 13

155 Indriolo A et al. Clinical management of IBD therapy with 5-ASA and/or beclomethasone dipropionate can be useful in distal UC. Budesonide or systemic steroids are indicated in patients with mild Crohn s disease. In moderate-to-severe IBD patients the use of oral or iv corticosteroids are necessary. Prednisone at a dose of 50 mg per day or prednisolone at a dose of mg per day based on the patient s body weight (60-80 kg) may be used in clinical practice. A gradual tapering of corticosteroids is recommended, for example 5 mg every week in prednisone therapy. Maintenance immunosuppression therapy with azathioprine at a dose of mg/kg body weight per day is effective after the corticosteroid therapy. Anti- TNFα treatment could be effective and safe in refractory to conventional therapy IBD [13,14,18]. The little experience of the use of biological therapy in transplanted patients, with concomitant anti-rejection therapy, suggests there be a higher and more careful surveillance regarding the risk of infectious diseases, autoimmune diseases, and neoplasms. It is very important to consider the possibility of surgical treatment in patients with moderate-to-severe IBD after LT for PSC and even more so, because of the higher colorectal cancer risk. Proctocolectomy with IPAA is feasible and safe in dedicated surgical centers [26,27]. IBD AFTER HEART TRANSPLANTATION Three cases of de novo IBD after heart transplantation have been reported in 3 studies: two cases of Crohn s disease and one case of UC [52-54]. The onset of IBD has been observed in pediatric age in two of three patients. Rakhit et al [52] reported one case of Crohn s disease in 104 post-orthotopic heart transplant children. The patient developed diarrhea and rectal bleeding immediately after the transplant and IBD was diagnosed after one year. The patient continued to have flares despite immunosuppressive therapy. Harms et al [53] reported a 15-year-old girl developed IBD 10 years after cardiac transplantation and presented a severe growth failure and delayed onset of puberty. The patient was found to have pan-enteric Crohn s disease and has done remarkably well following a nutritional therapy. Jüngling et al [54] reported a 53-year-old patient who developed distal UC 2 years after heart transplantation. In spite of high-dose treatment with prednisolone the patient s clinical situation worsened with a progression of inflammation in the entire colon. Colectomy with ileostomy was necessary to obtain a good state of health. Three IBD cases have been observed after heart transplantation in spite of immunosuppresive therapy. Two of them have been treated with conventional medical therapy with success, whereas one IBD case required surgery with colectomy and ileostomy. No IBD patient was treated with anti-tnfα therapy. 11 studies were reported [9,16,54-63]. One patient presented erythema nodosum associated to IBD [60], one patient developed colonic cancer with liver metastasis 2 years later and died [60], thirteen (50%) patients were treated with conventional medical IBD therapy (mesalazine, corticosteroids, and azathioprine), achieving a clinical remission. Significant clinical improvement of IBD was observed with anti-tnfα therapy in three (11.5%) patients. No severe infections or graft reject were documented after biological therapy [22,61]. Five (19.2%) patients with Crohn s disease continued to have flare up despite treatment [9]. Five (19.2%) UC patients were refractory to therapy and required a colectomy [9,55,56,62]. IBD AFTER LUNG AND INTESTINAL TRANSPLANTATION The number of patients who underwent lung or intestinal transplantation is significantly lower than the patients who underwent liver, heart, and renal transplantation. This could probably be explained because no IBD cases were reported after lung and intestinal transplantation. CLINICAL MANAGEMENT OF IBD PATIENTS AFTER HEART AND RENAL TRANSPLANTATION The concomitant anti-reject immunosuppressive therapy can increase the risk of infectious diseases in transplanted patients. Therefore, in patients who develop diarrhea, it is very important to exclude an intestinal infection (CMV, Clostridium difficile). It is also necessary to exclude that diarrhea may be induced by the drugs. The onset of IBD after heart or renal transplantation could lead to a severe clinical situation for the transplanted patient. In fact, in about half of the patients, conventional IBD therapy combined with anti-reject therapy, can be non-effective. Nineteen percent of patients developed multiple recurrence on IBD in the renal transplantation group. Eleven percent of patients required anti-tnfα therapy in order to have clinical remission and in about 20% of patients required a colectomy. The LT model suggests that anti- TNFα therapy combined with anti-reject therapy could be useful in selected IBD patients with refractory to conventional therapy after heart or renal transplantation. Like in the LT, it is very important that there be a higher more careful surveillance regarding the risk of infectious diseases, autoimmune diseases, and neoplasms after biological therapy in heart and renal transplanted patients. The surgical option remains an essential treatment in complicated IBD cases which are refractory to the intensive medical therapy. IBD AFTER RENAL TRANSPLANTATION A total of about of twenty-seven de novo IBD patients (15 UC and 11 Crohn s disease) after renal transplantation in CONCLUSION There is estimated a higher incidence of de novo IBD after solid organ transplantation than in the general popula April 7, 2014 Volume 20 Issue 13

156 Indriolo A et al. Clinical management of IBD tion. The onset of IBD in the organ transplant recipient population is an important clinical situation which is associated with higher morbidity and difficulty in the medical therapeutic management because of the possible interaction between anti-reject therapy and IBD therapy. IBD course after LT is variable, but about one third of patients may worsen, needing increased medical therapy or a colectomy. Active IBD at the time of LT, discontinuation of 5-ASA or azathioprine at the time of LT and use of tacrolimus-based immunosuppression may be associated with an unfavorable outcome of IBD after LT. Anti-TNF therapy for refractory IBD may be an effective and safe therapeutic option after LT. The little experience of the use of biological therapy in transplanted patients, with concomitant anti-rejection therapy, suggests there be a higher more careful surveillance regarding the risk of infectious diseases, autoimmune diseases, and neoplasms. Therefore, it is very important to consider the possibility of a surgical treatment in refractory severe IBD after LT. An increased risk of colorectal cancer is also present after LT in IBD/PSC patient. An annual program of endoscopic surveillance with serial biopsies for CRC is recommended. If dysplasia colonic mucosa is found, a colectomy with IPAA is advised. It has been shown to be a relatively safe procedure in the specialized surgical centers. Adjuvant pharmacotherapy has been shown to be well tolerated in patients post-lt. Hepatic graft dysfunction has not been documented after adjuvant pharmacotherapy. A prophylactic colectomy in selected IBD/PSC patients with CRC risk factors could be a good management strategy in the CRC prevention, but it is used infrequently in the majority of LT centers. About 30% of patients developed multiple recurrences of IBD and 20% of patients required a colectomy after renal transplantation. Like in the LT, anti-tnfα therapy could be an effective treatment in IBD patients with conventional refractory therapy after renal or heart transplantation. A large number of patients are needed to confirm the preliminary observations. Regarding the higher clinical complexity of this subgroup of IBD patients, a close multidisciplinary approach between an IBD dedicated gastroenterologist and surgeon and an organ transplantation specialist is necessary in order to have the best clinical management of IBD after transplantation. REFERENCES 1 Singh S, Loftus EV, Talwalkar JA. Inflammatory bowel disease after liver transplantation for primary sclerosing cholangitis. 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Scand J Gastroenterol 2012; 47: [PMID: DOI: / ] 40 Kelly DM, Emre S, Guy SR, Miller CM, Schwartz ME, Sheiner PA. Liver transplant recipients are not at increased risk for nonlymphoid solid organ tumors. Cancer 1998; 83: [PMID: ] 41 Knechtle SJ, D Alessandro AM, Harms BA, Pirsch JD, Belzer FO, Kalayoglu M. Relationships between sclerosing cholangitis, inflammatory bowel disease, and cancer in patients undergoing liver transplantation. Surgery 1995; 118: ; discussion [PMID: ] 42 Loftus EV, Aguilar HI, Sandborn WJ, Tremaine WJ, Krom RA, Zinsmeister AR, Graziadei IW, Wiesner RH. Risk of colorectal neoplasia in patients with primary sclerosing cholangitis and ulcerative colitis following orthotopic liver transplantation. Hepatology 1998; 27: [PMID: ] 43 Narumi S, Roberts JP, Emond JC, Lake J, Ascher NL. Liver transplantation for sclerosing cholangitis. Hepatology 1995; 22: [PMID: ] 44 Sint Nicolaas J, Tjon AS, Metselaar HJ, Kuipers EJ, de Man RA, van Leerdam ME. Colorectal cancer in post-liver transplant recipients. Dis Colon Rectum 2010; 53: [PMID: DOI: /DCR.0b013e3181cc90c7] 45 Vera A, Gunson BK, Ussatoff V, Nightingale P, Candinas D, Radley S, Mayer A, Buckels JA, McMaster P, Neuberger J, Mirza DF. Colorectal cancer in patients with inflammatory bowel disease after liver transplantation for primary scleros April 7, 2014 Volume 20 Issue 13

158 Indriolo A et al. Clinical management of IBD ing cholangitis. Transplantation 2003; 75: [PMID: ] 46 Goss JA, Shackleton CR, Farmer DG, Arnaout WS, Seu P, Markowitz JS, Martin P, Stribling RJ, Goldstein LI, Busuttil RW. Orthotopic liver transplantation for primary sclerosing cholangitis. A 12-year single center experience. Ann Surg 1997; 225: ; discussion [PMID: ] 47 Jain AB, Yee LD, Nalesnik MA, Youk A, Marsh G, Reyes J, Zak M, Rakela J, Irish W, Fung JJ. Comparative incidence of de novo nonlymphoid malignancies after liver transplantation under tacrolimus using surveillance epidemiologic end result data. Transplantation 1998; 66: [PMID: ] 48 Koornstra JJ, Wesseling J, de Jong AE, Vasen HF, Kleibeuker JH, Haagsma EB. Increased risk of colorectal neoplasia in asymptomatic liver-transplant recipients. Gut 2007; 56: [PMID: DOI: /gut ] 49 European Association For The Study Of The Liver. EASL Clinical Practice Guidelines: management of cholestatic liver diseases. J Hepatol 2009; 51: [PMID: DOI: /j.jhep ] 50 Doroshow JH, Synold TW, Gandara D, Mani S, Remick SC, Mulkerin D, Hamilton A, Sharma S, Ramanathan RK, Lenz HJ, Graham M, Longmate J, Takimoto CH, Ivy P. Pharmacology of oxaliplatin in solid tumor patients with hepatic dysfunction: a preliminary report of the National Cancer Institute Organ Dysfunction Working Group. Semin Oncol 2003; 30: [PMID: ] 51 Levin B. Risk of cancer in ulcerative colitis. Gastrointest Endosc 1999; 49: S60-S62 [PMID: ] 52 Rakhit A, Nurko S, Gauvreau K, Mayer JE, Blume ED. Gastrointestinal complications after pediatric cardiac transplantation. J Heart Lung Transplant 2002; 21: [PMID: ] 53 Harms B, Bremner AR, Mulligan J, Fairhurst J, Griffiths DM, Salmon T, Beattie RM. Crohn s disease post-cardiac transplantation presenting with severe growth failure and delayed onset of puberty. Pediatr Allergy Immunol 2004; 15: [PMID: DOI: /j x] 54 Jüngling B, Kindermann I, Moser C, Püschel W, Ecker KW, Schäfers HJ, Böhm M, Zeuzem S, Giese T, Stallmach A. Development of ulcerative colitis after heart transplantation during immunosuppressive therapy. Z Gastroenterol 2005; 43: [PMID: DOI: /s ] 55 Hibbs AM, Bznik-Cizman B, Guttenberg M, Goldberg B, Meyers K. Ulcerative colitis in a renal transplant patient with previous Goodpasture disease. Pediatr Nephrol 2001; 16: [PMID: ] 56 Nagai H, Matsumaru K, Shiozawa K, Momiyama K, Wakui N, Shinohara M, Watanabe M, Ishii K, Nonaka H, Hasegawa A, Teramoto T, Yamamuro W, Sumino Y, Miki K. Disappearance of HCV after cessation of immunosuppression in a patient with ulcerative colitis and renal transplantation. J Gastroenterol 2005; 40: [PMID: ] 57 Halim MA, Said T, Nair P, Schmidt I, Hassan A, Johny KV, Al-Muzairai I, Samhan M, Nampoory MR, Al-Mousawi M. De novo Crohn s disease in a renal transplant recipient. Transplant Proc 2007; 39: [PMID: DOI: /j.transproceed ] 58 Stewart IJ, Gallagher JP, Dahms WJ. A case of new onset Crohn s disease after renal transplantation. Gastroenterol Hepatol (N Y) 2008; 4: [PMID: ] 59 Kourda N, Bettaïeb I, Blel A, Zoghlami A, Bedoui R, Najah N, Ben Jilani SB, Zermani R. An aggressive course of de novo ulcerative colitis after renal transplantation: colonic adenocarcinoma with choriocarcinomatous differentiation. Tunis Med 2009; 87: [PMID: ] 60 Gheith O, Al-Otaibi T, Tawab KA, Said T, Balaha MA, Halim MA, Nair MP, Nampoory MR. Erythema nodosum in renal transplant recipients: multiple cases and review of literature. Transpl Infect Dis 2010; 12: [PMID: DOI: /j x] 61 Temme J, Koziolek M, Bramlage C, Schaefer IM, Füzesi L, Ramadori G, Müller GA, Schwörer H. Infliximab as therapeutic option in steroid-refractory ulcerative colitis after kidney transplantation: case report. Transplant Proc 2010; 42: [PMID: DOI: / j.transproceed.2010] 62 Parameswaran S, Singh K, Nada R, Rathi M, Kohli H, Jha V, Gupta K, Sakhuja V. Ulcerative colitis after renal transplantation: A case report and review of literature. Indian J Nephrol 2011; 21: [DOI: / ] 63 Azevedo P, Freitas C, Aguiar P, Silva H, Santos T, Farrajota P, Almeida M, Pedroso S, Martins LS, Dias L, Vizcaíno R, Henriques AC, Cabrita A. A case series of de novo inflammatory bowel disease after kidney transplantation. Transplant Proc 2013; 45: [PMID: DOI: /j.transproceed ] P- Reviewers: Bonaz BL, Liu ZJ S- Editor: Gou SX L- Editor: A E- Editor: Wu HL 3533 April 7, 2014 Volume 20 Issue 13

159 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (3): Inflammatory bowel disease TOPIC HIGHLIGHT Pharmacogenetics of azathioprine in inflammatory bowel disease: A role for glutathione-s-transferase? Gabriele Stocco, Marco Pelin, Raffaella Franca, Sara De Iudicibus, Eva Cuzzoni, Diego Favretto, Stefano Martelossi, Alessandro Ventura, Giuliana Decorti Gabriele Stocco, Marco Pelin, Giuliana Decorti, Department of Life Sciences, University of Trieste, I Trieste, Italy Raffaella Franca, Sara De Iudicibus, Diego Favretto, Stefano Martelossi, Alessandro Ventura, Institute for Maternal and Child Health IRCCS Burlo Garofolo, I Trieste, Italy Eva Cuzzoni, Alessandro Ventura, Department of Medical, Surgical and Health Sciences, University of Trieste, I Trieste, Italy Author contributions: All authors participated in the preparation, writing and discussion of the manuscript. Correspondence to: Gabriele Stocco, PhD, Department of Life Sciences, University of Trieste, via A. Fleming 22, I Trieste, Italy. stoccog@units.it Telephone: Fax: Received: October 31, 2013 Revised: December 30, 2013 Accepted: January 19, 2014 Published online: April 7, 2014 Abstract Azathioprine is a purine antimetabolite drug commonly used to treat inflammatory bowel disease (IBD). In vivo it is active after reaction with reduced glutathione (GSH) and conversion to mercaptopurine. Although this reaction may occur spontaneously, the presence of isoforms M and A of the enzyme glutathione-s-transferase (GST) may increase its speed. Indeed, in pediatric patients with IBD, deletion of GST-M1, which determines reduced enzymatic activity, was recently associated with reduced sensitivity to azathioprine and reduced production of azathioprine active metabolites. In addition to increase the activation of azathioprine to mercaptopurine, GSTs may contribute to azathioprine effects even by modulating GSH consumption, oxidative stress and apoptosis. Therefore, genetic polymorphisms in genes for GSTs may be useful to predict response to azathioprine even if more in vitro and clinical validation studies are needed Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Inflammatory bowel disease; Azathioprine; Pharmacogenetics; Glutathione-S-transferase; Pediatric patients Core tip: Polymorphisms of glutathione-s-transferase-m1 may influence azathioprine effects in young patients with inflammatory bowel disease by increasing the drug activation and by modulating oxidative stress and apoptosis. Stocco G, Pelin M, Franca R, De Iudicibus S, Cuzzoni E, Favretto D, Martelossi S, Ventura A, Decorti G. Pharmacogenetics of azathioprine in inflammatory bowel disease: A role for glutathione- S-transferase? World J Gastroenterol 2014; 20(13): Available from: URL: v20/i13/3534.htm DOI: i INTRODUCTION Azathioprine, the 1-methyl-4-nitroimidazol-5-yl derivative of mercaptopurine, is a purine antimetabolite drug commonly used to treat inflammatory bowel disease (IBD). Despite the introduction of effective biological treatments, such as antibodies against tumor necrosis factor-α (TNF-α), azathioprine is still a mainstay for maintenance therapy of severe IBD. Azathioprine is a prodrug and requires complex conversion to active metabolites (Figure 1). The first step in this conversion is the reaction of azathioprine with reduced glutathione (GSH), to yield the prodrug mercaptopurine and a nitroimidazole derivative/conjugate of GSH. Even though this reaction can occur spontaneously [1] the presence of the enzyme glutathione-s-transferase (GST) may increase its speed, 3534 April 7, 2014 Volume 20 Issue 13

160 Stocco G et al. Effects of glutathione-s-transferase on azathioprine activation TPMT 6-Me-TITP AZA GST 6-TU acid 6-TITP XO ITPA 6-MP HPRT IMPDH GMPS k k 6-MP TPMT 6-TIMP TPMT 6-TXMP 6-TGMP 6-TGDP 6-TGTP 6-TGN 6-MMP 6-Me-TIMP Mercaptopurine Nitroimidazole Ribose Methyl Amino Hydroxy Phosphate Figure 1 Metabolism of azathioprine and mercaptopurine. 6-Me-TIMP: 6-methyl-thioinosine-monophosphate; 6-Me-TITP: 6-methyl-thioinosine-triphosphate; 6-MMP: 6-methyl-mercaptopurine; 6-MP: Mercaptopurine; 6-TGDP: 6-thioguanine-diphosphate; 6-TGMP: 6-thioguanine-monophopsphate; 6-TGN: 6-thioguanine nucleotide; 6-TGTP: 6-thioguanine-triphopshate; 6-TIMP: 6-thioinosine-monophosphate; 6-TITP: 6-thioinosine-triphosphate; 6-TU: 6-thiouric; 6-TXMP: 6-thioxanthosine-monophosphate; AZA: Azathioprine; GMPS: Guanosine-monophosphate-synthase; GST: glutathione-s-transferase; HPRT: Hypoxanthinephosphate-rybosiltransferase; IMPDH: Inosine-monophosphate-dehydrogenase; k: Kinase; TPMT: Thiopurine-S-methyl-transferase; XO: Xanthine-oxidase. as discussed later. Even mercaptopurine needs metabolic conversion to thioguanine nucleotides (TGNs), catalyzed by the enzymes of the purine salvage pathway. Moreover, mercaptopurine is inactivated in the liver mainly by xanthine oxidase (XO), while in the extra hepatic tissues mercaptopurine catabolism involves predominantly genes that display genetically determined polymorphic activity, such as thiopurine-s-methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA). After oral administration, intact azathioprine is undetectable in blood because of extensive first pass metabolism [2]. Mercaptopurine s cytotoxic effects are mainly related to incorporation of the active TGNs in the nucleic acids and to the consequent interference with the function of DNA processing enzymes and, to some extent, to inhibition of de novo purine synthesis, mainly operated by methylated precursors of TGNs. While mercaptopurine pharmacokinetics and pharmacodynamics have been characterized extensively, the mechanism of conversion of azathioprine to mercaptopurine and its clinical implications for therapy personalization have not been completely elucidated. EFFECTS OF GST POLYMORPHISMS ON AZATHIOPRINE EFFICACY AND METABOLISM IN PATIENTS WITH IBD The hypothesis that patients with reduced levels of specific GST isoforms, due to genetic polymorphisms, may present decreased sensitivity to azathioprine because of a reduced enzymatic conversion of azathioprine to mercaptopurine, was tested recently by our team in young patients with IBD. The deletion of GST-M1, GST-T1 and the coding non synonymous single nucleotide polymorphism rs1695 in GST-P1 were tested. An initial study considered a cohort of 70 young patients (median age 16.2 years, 36 females) with IBD (41 Crohn s disease, 29 ulcerative colitis). Among these, 15 patients developed adverse events during treatment with azathioprine: in particular, there were three cases of bone marrow suppression, three cases of liver toxicity, seven cases of pancreatic toxicity, one case of neuropathy and one case of arthralgia; all these side effects resolved completely after the reduction or interruption of azathioprine administration: azathioprine was therefore considered the main determinant of the adverse effects. Interestingly, the candidate genetic association analysis in these patients revealed that frequency of GST-M1 deletion was significantly lower in patients that developed an adverse event in comparison to patients that tolerated azathioprine treatment with no adverse event (frequency of deletion respectively 26.7% vs 67.3%, P = ). Moreover, the incidence of mild lymphopenia (lymphocytes count under 1000/mm 3 ), that was considered a marker of efficacy during azathioprine treatment, resulted associated with GST-M1 genotype: indeed, among patients tolerating azathioprine treatment 3535 April 7, 2014 Volume 20 Issue 13

161 Stocco G et al. Effects of glutathione-s-transferase on azathioprine activation and with lymphopenia, frequency of the deletion was 28.6% in comparison to 72.9% among patients tolerant to azathioprine but that did not present this drug effect (P = 0.032) [3]. Taken together, these results are in agreement with a model in which patients with GST-M1 deletion are less sensitive to the effects of azathioprine, putatively because of the contribution of this enzyme on the conversion of azathioprine to mercaptopurine. In a recent study, we evaluated the effects of GST polymorphisms on azathioprine metabolism in a cohort of 75 young patients (median age 15.2 years, 36 females) with IBD (46 Crohn s disease, 29 ulcerative colitis) tolerant to azathioprine therapy (taking azathioprine for more than 3 mo). Azathioprine metabolites were measured on samples collected from these patients using an high performance liquid chromatography assay (HPLC) [4] : 150 measurements of azathioprine metabolites were collected. Patients with the deletion of GST-M1 tolerated a dose of azathioprine significantly higher in comparison to patients with normal GST-M1 (mean dose of azathioprine 2.1 mg/kg per day vs 1.8 mg/kg per day, P = 0.022). Moreover, the amount of active TGNs generated in patients with the deletion of GST-M1 was significantly decreased in comparison to patients with a normal genotype (mean amount of TGNs metabolites concentration for mg/kg of azathioprine: 252 pmol/ erythrocytes vs 164 pmol/ erythrocytes, P = ). Multivariate analysis confirmed that this effect was independent from that of other genes with a significant effect, such as TPMT, the main gene known to influence mercaptopurine metabolism [5]. This study therefore supports a role of GST-M1 on azathioprine efficacy, mediated by an increased conversion of azathioprine to mercaptopurine. The reaction catalyzed by GST-M1 likely occurs after oral administration mainly in the intestine and the liver, modulating the amount of mercaptopurine and TGNs that are released in the main circulation. These studies considered even the effect of GST-P1 and GST-T1 polymorphisms on azathioprine effects and metabolism but did not detect any significant association. The lack of association may be due to the tissue distribution of GST-P1 and GST-T1, which are not highly expressed in the liver, but even to the lack of specific activity of these enzymes toward the catalysis of the reaction of azathioprine with glutathione [6]. Another study considered GST-M1 genetic polymorphisms as a candidate involved in azathioprine activation [7] : this report considered 51 Asian patients with systemic lupus erythematosus (SLE) and the effect of polymorphisms in the ITPA, TPMT, GST-M1 and GST-T1 genes on the response to a low dose of azathioprine (0.97 mg/kg per day). Response to therapy, evaluated as a change in disease activity index, was associated with ITPA genetic polymorphism but not with the other ones. A clear interpretation of this paper results may be difficult because of the lack of data on azathioprine metabolites concentrations. However, the lack of an effect of GST-M1 on azathioprine efficacy in this study may be due to the very low dose of drug used. This study indicated that in Asian patients with SLE the effect of ITPA might be predominant on those of TPMT and GST-M1 when azathioprine is used at very low doses; indeed, in this study even TPMT genetic polymorphism was not associated with azathioprine efficacy. It is known that in patients with Asian ancestry the frequency of variant TPMT is very low, while that of variant ITPA is increased in comparison to Caucasians [8,9]. INVOLVEMENT OF GST IN AZATHIOPRINE MOLECULAR AND CELLULAR EFFECTS GSTs are enzymes responsible for the inactivation of electrophilic substances, both endogenous and exogenous, by catalyzing their reaction with GSH. Human cytosolic GSTs are encoded by 17 genes and the proteins derived can be classified into 7 distinct classes based on their amino acid sequences. The most abundant GSTs in human cells are those of class P, M and A. GST-P1 is the principal isoform in most tissues, such as the small intestine and erythrocytes, but is not detectable in normal liver cells; on the other hand, GSTs M1, A1 and A2 are highly expressed in hepatocytes, while they are not expressed in erythrocytes. GST-T1 is expressed in the liver, intestine and erythrocytes even if its level of expression is lower compared to GSTs of the other classes [6]. All the genes for these GST classes display common genetic polymorphisms that influence the activity of the enzyme in some individuals. For GST-M1 and T1, a common deletion is present in humans, so that about 50% and 20% of caucasians lack activity of these enzymes because of this genetic variant. In patients of African and Asian ancestry, frequency of GST-T1 deletion is higher than Caucasian, reaching around 50%, while frequency of GST-M1 deletion is similar [10]. GST-P1 displays a common coding non-synonymous variant, an A-G transition at base 1578, resulting in the amino acid change I105V in the substrate binding site of the enzyme: the frequency of the variant genotype for this polymorphism in Caucasian and African is similar (13%-15%), while in Asian the percentage is lower (1%-2%) [11,12]. Even expression of GST-A is modulated by genetic polymorphism: GST-A1-69 C > T results in a reduced GST-A1 expression and related enzyme activity [13,14]. The GST-A1 polymorphism shows different frequencies for the mutated genotype between Caucasian and African (respectively 17% and 11%) and Asian (1%). For GST-A2, a C-T transition at base 328, resulting in the amino acid change P110S in the electrophilic substrate binding site, occurs only in heterozygosis, at a frequency of 11% in Caucasian and 23% in Asian population [15,16]. In the African population, the variant allele to date has not been found (Table 1). Genetic polymorphisms in GSTs, determining the interindividual variability in the activity of these important metabolic enzymes, have been related to the incidence of several pathologies, in particular oncological, and to altered sensitivity to medications, including azathioprine [17-20] April 7, 2014 Volume 20 Issue 13

162 Stocco G et al. Effects of glutathione-s-transferase on azathioprine activation Table 1 Glutathione-S-transferase polymorphisms and frequencies in different ethnic groups Gene Polymorphisms Caucasian African Asian GST-M1 Deletion 50% 50% 50% GST-T1 Deletion 20% 50% 50% GST-P1 rs1695 (A > G) 13% (GG) 15% (GG) 1%-2% (GG) GST-A1 rs (C > T) 17% (TT) 11% (TT) 1% (TT) GST-A2 rs (C > T) 11% (CT) 0% (CT) 23% (CT) GST: Glutathione-S-transferase. Enzymatic conversion Azathioprine conversion to mercaptopurine can occur spontaneously [1], even if in the presence of specific GST classes and at physiological ph values the reaction catalyzed by the enzyme may be prevalent. Kaplowitz described an initial report on the enzymatic contribution on the conversion of azathioprine to mercaptopurine in rat liver homogenates. While at relatively high ph levels (i.e., 8.0) the non-enzymatic reaction and the enzymatic one occur in similar proportions; at lower ph levels (i.e., ), closer to physiological values, the enzymatic reaction prevails [21]. The same reaction has been demonstrated in homogenates of human livers: in these samples, mainly from kidney transplant donors, conversion of azathioprine to mercaptopurine was inhibited by treatment with furosemide, a GST inhibitor [22]. Additional evidence obtained in animal models supports a significant contribution of the enzymatic conversion of azathioprine to mercaptopurine in vivo. Indeed, pretreatment of rats with probenecid, a GST inhibitor, determines a greater proportion of unmetabolized azathioprine in the liver and less hepatic GSH depletion. Bilirubin is also a GST inhibitor and, in a model of hyperbilirubinemic rat (Gunn rat), less hepatic GSH depletion was found during exposure to azathioprine [23]. These observations indicate that the conversion of azathioprine to mercaptopurine in vivo is mediated enzymatically by the GSTs. After oral administration of azathioprine this reaction likely occurs mostly in the liver: indeed after oral administration azathioprine is undetectable in blood, while mercaptopurine appears after either oral or iv azathioprine administration [2]. In addition it has been shown that after ip injection of azathioprine in rats, GSH was depleted rapidly in hepatocytes but not in other tissues (i.e., erythrocytes, kidneys and intestine) indicating that after administration of azathioprine, the hepatic contribution to total GSH consumption may be predominant [24]. Eklund et al [6] have shown that among 14 GSTs tested, GST-A1, GST-A2 and GST-M1 displayed the highest activity on the catalysis of azathioprine to mercaptopurine; these enzymes are all highly expressed in human hepatocytes and therefore in these cells the uncatalyzed reaction of azathioprine with GSH was estimated to be less than 1% of the GST-catalyzed biotransformation. Interestingly, GST-M1 and GST-A display genetically determined variable expression levels. These differences in GST activity may result in interindividual differences in the conversion of azathioprine to mercaptopurine. The authors suggest that individuals with high levels of GST could be particularly sensitive to azathioprine and potentially more prone to adverse effects during treatment with azathioprine, because of both increased concentrations of free mercaptopurine and of a more pronounced GSH depletion. Oxidative damage Considering the intracellular enzymes involved in thiopurines metabolism, it is reasonable to suggest that these agents are able to induce oxidative stress. Indeed, it has been demonstrated that thiopurines can generate directly reactive oxygen species (ROS) in cells exposed to ultra violet light [25,26]. Moreover, regarding azathioprine, some evidence suggest an indirect ability to induce oxidative stress, mediated primarily by GSH consumption during the metabolic conversion of azathioprine to mercaptopurine [27,28]. GSTs, influencing the reaction of azathioprine with GSH, may influence therefore the cellular effects of azathioprine mediated by oxidative damage. Moreover, even metabolism of thiopurines by XO may generate ROS, as assessed on primary cultures of rat hepatocytes [28]. XO, which metabolizes mercaptopurine, converting it to thiouric acid, is a well know producer of ROS, such as superoxide anion [29], whose accumulation could worsen the oxidative stress induced by GSH depletion. Allopurinol, a XO inhibitor, has been shown to restore response to thiopurines in patients with IBD unresponsive to thiopurines and with unfavorable metabolic ratio, increasing the concentration of active TGNs and decreasing those of the methylated nucleotides, likely because of inhibition of TPMT [30]. Cellular redox balance is largely determined by GSH. Depletion of such cellular antioxidant defenses allows the accumulation of significant amounts of ROS, as demonstrated in several systems [31,32], which, in turn, have been suggested to act as a signal for apoptosis induction [33]. Regarding azathioprine, exposure to this medication in vitro induces a rapid depletion of GSH in hepatocytes before any loss of viability. Addition of exogenous GSH or N-acetylcysteine protected against cell death. Lee et al [27] suggested that oxidative stress induced by GSH depletion is able to induce mitochondrial damage, opening of mitochondrial permeability transition pore (MPTP) and rapid consumption of adenosine triphosphate. Ultrastructural analysis showed occurrence of necrosis after azathioprine exposure. The fact that, under the same experimental conditions, mercaptopurine was not able to reduce hepatocyte viability, allows suggesting that the activating steps triggered by GST and the associated GSH depletion could be a crucial step in azathioprine cytotoxicity in vitro. Among the many secondary effects attributed to them, a role for ROS in mediating an anti-proliferative effect has been demonstrated. Indeed, the cellular redox balance fluctuates during cell cycle, so that the redox state modulates cell cycle progression from one phase to the next [34]. In this scenario, significantly higher GSH content 3537 April 7, 2014 Volume 20 Issue 13

163 Stocco G et al. Effects of glutathione-s-transferase on azathioprine activation Intestine/liver/lymphocytes Intestine/liver Inhibition of apoptosis Protein-protein interactions Immunosuppression Interference with DNA processing enzymes Therapeutic effects Inhibition of proliferation/cytotoxicity for lymphocytes Conversion of azathioprine to mercaptopurine Mercaptopurine metabolism Inhibition of small GTPases (Rac1) Glutathione-Stransferase Consumption of GSH Inhibition of de novo purine synthesis Adverse events Bone marrow suppression Hepatotoxicity Oxidative stress Pancreatic toxicity Figure 2 Schematic representation of different glutathione-s-transferase catalyzed phenomenons (blue boxes) that even through intermediate events (light blue) bring or contibute to the canonical mechanisms of action (yellow), determining the therapeutic effects (green) and/or adverse events (orange). GSH: Reduced glutathione. in the G2 and M phases compared with G1 were found [35]. Hence, it is reasonable to hypothesize a role for ROS in affecting the anti-proliferative effect of thiopurines, which are cell cycle phase specific agents and especially azathioprine, which consumes GSH during its conversion to mercaptopurine. Azathioprine half-life is very short and its therapeutic effects on lymphocytes are likely due to the metabolites produced after first pass metabolism in the intestinal and liver cells. However, azathioprine cytotoxic effects due to GSH consumption are likely to play an important role on the development of cytotoxicity in intestinal and liver cells, supporting a role in the induction of adverse events in these organ systems. However, the fact that on hepatocytes azathioprine is able to modulate a necrotic cell death through a cyclosporine-a sensitive MPTP opening, rise some doubts on the actual role of oxidative stress [27]. Studies are still required to demonstrate if ROS could represent an alternative mechanism of cytotoxicity induced by azathioprine and its clinical relevance. Modulation of apoptosis Azathioprine and mercaptopurine induce apoptosis in activated lymphocytes and these effects should be crucial in determining the efficacy of these medications as immunomodulators in young patients with IBD [36]. GSTs have been shown to modulate apoptosis and the incidence of lymphopenia during treatment of IBD patients and therefore modulation of apoptosis by GSTs may be of significant relevance as a mechanism for the role of these proteins on azathioprine effects. GST-P1 was the first isoenzyme found to play a role in signaling pathways that control cell survival, and so far is the most important one. This regulation role is achieved independently from the well-known conjugating activity and occurs through a physical protein-protein interaction with c-jun N-terminal Kinase (JNK), a member of the mitogen-activated protein kinases (MAPK), with the consequent inhibition of the downstream JNK-induced apoptotic pathway. GST-M1 plays a similar role of negative regulator by physically sequestering the apoptosis-signaling regulating kinase 1 (ASK-1), a MAPK kinase kinase that activates both JNK and stress responsive p-38 kinase (another MAPK) [37]. Stress triggers, such as heat shock or the pro-inflammatory cytokines TNF-α and interleukin 1-β, promotes the dissociation of GST-M1 from ASK1, resulting in the activation of ASK-1 and the phosphorylation-dependent activation of p38 [38]. The final cell fate (proliferation or apoptosis) depends on the strength and duration of the cellular stress. GST-A1 has been found to suppress activation of JNK signaling by a pro-inflammatory cytokine and oxidative stress in Caco-2 cells, suggesting also a protective role for GST-A1 in JNK-associated apoptosis [39]. The expression of GSTs of the M and A classes has been reported to drop under apoptotic conditions and their overexpression was able to block apoptosis in rat hepatocytes [40]. GST-M1 has been reported to bind to ASK1 and inhibit apoptosis [37]. Matsumaru et al [41] reported that depletion of cytosolic GSH could sensitize murine hepatocytes to apoptosis induced by TNF-α [41,42] ; clinical studies have shown that TNF-α protein and mrna levels are elevated in serum, intestinal tissue and stools of active IBD, in correlation with disease activity [43]. Therefore, even depletion of GSH catalyzed by GST could make cells of patients with IBD particularly sensitive to the cytotoxic effects of thiopurines, potentially leading to an increased incidence 3538 April 7, 2014 Volume 20 Issue 13

164 Stocco G et al. Effects of glutathione-s-transferase on azathioprine activation of adverse events. CONCLUSION It would be important that other studies validate the observation of the increased conversion of azathioprine to mercaptopurine in patients with normal GST-M1, resulting in increased sensitivity to the medication in patients with IBD, both clinically and using in vitro experiments. Moreover, since GSTs of the A class are highly expressed in the liver, have catalytic activity on the conversion of azathioprine to mercaptopurine and display genetically determined polymorphic activity, it would be important to evaluate the role even of variation in genes for the A class of GST on azathioprine pharmacokinetics and efficacy. Further insights on the role of genetic polymorphisms of GST and other enzymes on azathioprine pharmacogenetics could come from the use of innovative and more sensitive methods for the measurement of azathioprine metabolites. Indeed, most of the research published so far, including the papers mentioned in this report, have characterized thiopurine metabolites in erythrocytes from patients with IBD, using HPLC methods that group all thionucleosides as TGNs or methylated nucleotides [4,44], without distinguishing the degree of phosphorylation, which may be of relevance for thiopurines cellular effects [45]. Given the complexity of thiopurines metabolism, methods with increased sensitivity, allowing to assess the nucleotides degree of phosphorylation and potentially the identification of additional relevant species, such as those based on mass spectrometry are of great interest [46]. Moreover, these methods with increased sensitivity allow the use of very small volumes of patients samples and this is particularly relevant for pediatric patients [47,48]. The increasing complexity [49] of thiopurines pharmacogenetics has been consolidating: while TPMT is the strongest determinant of variability in the pharmacokinetics of these medications [50], currently used in several clinical protocols to adjust treatment with thiopurines, even other genes, such as ITPA, have been shown to be of relevance [51]. Based on the clinical and in vitro evidence highlighted in this paper (Figure 2), it seems that for azathioprine even GST-M1 genetic polymorphism could enter in a useful multi-locus genotype to predict patients response to this medication. However, the association of GST-M1 with azathioprine efficacy in patients with IBD still needs to be supported mechanistically by in vitro studies and validated by adequately sized prospective clinical trials. REFERENCES 1 Elion GB. The purine path to chemotherapy. Science 1989; 244: [PMID: ] 2 Lin SN, Jessup K, Floyd M, Wang TP, van Buren CT, Caprioli RM, Kahan BD. 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Clinical use and mechanisms of infliximab treatment on inflammatory bowel disease: a recent update. Biomed Res Int 2013; 2013: [PMID: ] 44 Cangemi G, Barabino A, Barco S, Parodi A, Arrigo S, Melioli G. A validated HPLC method for the monitoring of thiopurine metabolites in whole blood in paediatric patients with inflammatory bowel disease. Int J Immunopathol Pharmacol 2012; 25: [PMID: ] 45 Teichgräber U, Atreya I, Atreya R, Schwab M, Neurath MF. Infliximab treatment induces levels of the active azathioprine metabolite TGTP in Crohn s disease. Inflamm Bowel Dis 2013; 19: E54-E55 [PMID: DOI: /ibd.22998] 46 Hofmann U, Heinkele G, Angelberger S, Schaeffeler E, Lichtenberger C, Jaeger S, Reinisch W, Schwab M. Simultaneous quantification of eleven thiopurine nucleotides by liquid chromatography-tandem mass spectrometry. Anal Chem 2012; 84: [PMID: DOI: / ac ] 47 Kirchherr H, Shipkova M, von Ahsen N. 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166 Stocco G et al. Effects of glutathione-s-transferase on azathioprine activation tides and 6-methylmercaptopurine in whole-blood by LC/MSMS using isotope-labeled internal standards. Ther Drug Monit 2013; 35: [PMID: DOI: / FTD.0b013e318283ed5d] 48 Zhao W, Jacqz-Aigrain E. Principles of therapeutic drug monitoring. Handb Exp Pharmacol 2011; 205: [PMID: DOI: / _3] 49 Marsh S, Van Booven DJ. The increasing complexity of mercaptopurine pharmacogenomics. Clin Pharmacol Ther 2009; 85: [PMID: DOI: /clpt ] 50 Relling MV, Gardner EE, Sandborn WJ, Schmiegelow K, Pui CH, Yee SW, Stein CM, Carrillo M, Evans WE, Hicks JK, Schwab M, Klein TE. Clinical pharmacogenetics implementation consortium guidelines for thiopurine methyltransferase genotype and thiopurine dosing: 2013 update. Clin Pharmacol Ther 2013; 93: [PMID: ] 51 Stocco G, De Iudicibus S, Franca R, Addobbati R, Decorti G. Personalized therapies in pediatric inflammatory and autoimmune diseases. Curr Pharm Des 2012; 18: [PMID: DOI: / ] P- Reviewers: Cangemi G, Roberts RL, Sparrow MP S- Editor: Zhai HH L- Editor: A E- Editor: Wu HL 3541 April 7, 2014 Volume 20 Issue 13

167 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (3): Inflammatory bowel disease TOPIC HIGHLIGHT Anemia in inflammatory bowel disease: a neglected issue with relevant effects Danila Guagnozzi, Alfredo J Lucendo Danila Guagnozzi, Alfredo J Lucendo, Department of Gastroenterology, Hospital General de Tomelloso, Tomelloso, Ciudad Real, Spain Author contributions: Both authors contributed equally to this paper. Correspondence to: Danila Guagnozzi, MD, Department of Gastroenterology, Hospital General de Tomelloso, Vereda de Socuéllamos, s/n Tomelloso, Ciudad Real, Spain. danila_g@libero.it Telephone: Fax: Received: October 12, 2013 Revised: November 22, 2013 Accepted: March 5, 2014 Published online: April 7, 2014 Abstract Anemia, a common complication associated with inflammatory bowel disease (IBD), is frequently overlooked in the management of IBD patients. Unfortunately, it represents one of the major causes of both decreased quality of life and increased hospital admissions among this population. Anemia in IBD is pathogenically complex, with several factors contributing to its development. While iron deficiency is the most common cause, vitamin B12 and folic acid deficiencies, along with the effects of pro-inflammatory cytokines, hemolysis, drug therapies, and myelosuppression, have also been identified as the underlying etiology in a number of patients. Each of these etiological factors thus needs to be identified and corrected in order to effectively manage anemia in IBD. Because the diagnosis of anemia in IBD often presents a challenge, combinations of several hematimetric and biochemical parameters should be used. Recent studies underscore the importance of determining the ferritin index and hepcidin levels in order to distinguish between iron deficiency anemia, anemia due to chronic disease, or mixed anemia in IBD patients. With regard to treatment, the newly introduced intravenous iron formulations have several advantages over orally-administered iron compounds in treating iron deficiency in IBD. In special situations, erythropoietin supplementation and biological therapies should be considered. In conclusion, the management of anemia is a complex aspect of treating IBD patients, one that significantly influences the prognosis of the disease. As a consequence, its correction should be considered a specific, first-line therapeutic goal in the management of these patients Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Anemia; Inflammatory bowel disease; Iron deficiency; Anemia of chronic disease; Erythropoietin Core tip: Anemia represents one of the major causes of both decreased quality of life and increased hospital admissions among inflammatory bowel disease (IBD) patients. This paper analyses the complex etiological and pathophysiological mechanisms underlying anemia in IBD, including iron and micronutrients deficiency, effects of proinflammatory mediators and bone marrow insufficiency secondary to the disease by itself and IBD therapy. By a comprehensive review of the current diagnostic and therapeutic evidences on anemia in IBD, an state-of-the-art approach will be provided to effectively manage this challenging and common condition. Guagnozzi D, Lucendo AJ. Anemia in inflammatory bowel disease: a neglected issue with relevant effects. World J Gastroenterol 2014; 20(13): Available from: URL: wjgnet.com/ /full/v20/i13/3542.htm DOI: org/ /wjg.v20.i INTRODUCTION Anemia, a frequent systemic complication in patients 3542 April 7, 2014 Volume 20 Issue 13

168 Guagnozzi D et al. Anemia in inflammatory bowel disease Vitamin B12 and folic acid deficiency Iron deficiency Hemolytic anemia Anemia Myelosuppression Inflammation (TNF-α, IL-1, IL-6) Absolute deficiency Iron homeostasis Functional deficiency Figure 1 Pathogenesis of multifactorial anemia associated to inflammatory bowel disease. TNF-α: Tumour necrosis factor-α; IL: interleukin. with inflammatory bowel disease (IBD), has a complex and multifactorial pathogenesis (Figure 1). It is considered a prototype of a combination of iron deficiency (IDA) and anemia of chronic disease (ACD), which is caused by the negative effects of an activated immune system at different levels of erythropoiesis [1,2]. Besides IDA and ACD, metabolic disturbances, vitamin deficiencies, and various drug therapies commonly used in IBD can aggravate anemia in IBD patients [3]. The study of anemia in these patients thus requires a specific diagnostic and therapeutic approach. It is important to highlight that anemia has a significant impact on the disease and is one of the most frequent comorbid conditions associated with mortality in IBD patients [4]. In addition, it also has a relevant effect on health related quality of life (QoL) and ability to work [5,6]. The fact that it is also a common cause of hospitalization and delay of discharge [7] only serves to underscore the need for prompt diagnosis and management of this condition. Although the correction of anemia in IBD patients can improve the QoL and the quality of patient management, the specific diagnosis and treatment of anemia is often a low priority for gastroenterologists and has thus received little attention. A recent study showed that further diagnostic tests were undertaken in only onethird of patients with proven anemia and that 54.3% of patients diagnosed with IDA receive no iron supplements [8,9]. This article reviews current data on the diagnosis and treatment of anemia in IBD patients. A search was conducted in the PubMed, Cochrane, MEDLINE, and Scopus libraries with the following individual and combined key words: Crohn s disease, ulcerative colitis, anemia, iron deficiency anemia, anemia of chronic disease, vitamin B12 deficiency, folic acid deficiency, myelodysplastic syndrome, refractory anemia, iron supplementation, intravenous iron therapy, erythropoietin, and inflammatory bowel disease. References cited in the articles retrieved were also searched in order to identify other potential sources of information. The results were limited to human studies available in English. PREVALENCE OF ANEMIA IN INFLAMMATORY BOWEL DISEASE The prevalence of anemia in IBD is markedly variable, ranging from 6% to 74% in two systematic reviews [10,11]. The more recent review calculated a mean prevalence of 17% (16% in outpatients and 68% in hospitalized patients), with anemia occurring more frequently in patients with Crohn s disease (CD) than in those with ulcerative colitis (UC) [3,12]. This variability in the prevalence of anemia depends on different factors. First, the definition of anemia is not homogenous in the various studies reviewed. In fact, because the widely accepted World Health Organization criterion for the diagnosis of anemia [hemoglobin (Hb) below 13 g/dl in men or 12 g/dl in non-pregnant women] [13] has been questioned due to racial differences, environmental conditions, and eating habits [14,15], its use cannot completely reflect the real prevalence of anemia in different IBD populations. Furthermore, estimations of the prevalence of anemia often depend on the specific groups of patients studied (for example, hospitalized patients vs outpatients). In this sense, a study from a Swedish cohort showed that the prevalence of anemia in hospitalized UC and CD patients was higher than among outpatient populations (5% vs 35% and 9% vs 50%, respectively) [16]. Furthermore, it is important to note that anemia has been poorly studied in pediatric IBD patients. One recent epidemiological study showed that while the prevalence of anemia was 72% at the time of diagnosis, the proportion of severely anemic pediatric patients decreased from 34% to 9% while the number of patients with mild anemia doubled after 1 year of follow-up [17]. Finally, because Hb levels form part of a widely used disease activity index, the presence of anemia correlates directly with disease activity, which means that the prevalence of anemia may change throughout the natural history of the disease. In fact, the prevalence of mild and moderate anemia in IBD has decreased over time, reflecting improved treatment and management of the disease. However, the prevalence of severe anemia in IBD patients over the last 10 years has not decreased in the same manner [18]. Pathogenesis of anemia in inflammatory bowel disease Iron deficiency anemia Iron deficiency is the most common cause of anemia in IBD patients, with a reported prevalence of up to 90% [11]. Iron deficiency may be related to absolute iron deficiency due to low dietary intake and blood loss from ulcerated intestinal mucosa (especially in UC patients) along with reduced iron absorption (especially in CD localized in the upper GI tract), or it may be related to functional iron deficiency. Iron is an essential mineral for the function of all 3543 April 7, 2014 Volume 20 Issue 13

169 Guagnozzi D et al. Anemia in inflammatory bowel disease body cells and is absorbed at the apical surface of enterocytes to be transported by ferroportin across the basolateral surface of the enterocyte into the circulation [19]. In the maintenance of iron homeostasis, the peptide hormone hepcidin is a master regulator that is produced in response to iron overload or upon induction by proinflammatory stimuli such as lipopolysaccharide or interleukin (IL)-6. In fact, inflammatory conditions can interfere with iron absorption by causing an increase in hepcidin that inhibits ferroportin activity [20], leading to its internalization and degradation [21]. The inhibition of ferroportin activity blocks the transfer of absorbed iron from the enterocyte into the circulation and causes iron retention in the macrophages and monocyte cells [22]. In addition, during inflammation other events contribute to the retention of iron in these cells, including the inhibition of ferroportin transcription by pro and antiinflammatory cytokine action and a reduction in the half-life of erythrocytes due to oxidative stress and lipidperoxidation, with iron recycling through erythrophagocytosis [23]. These mechanisms all lead to functional iron deficiency, which means that despite an abundance of iron in the body, it is not available for erythropoiesis. Anemia of chronic disease The exact prevalence of ACD in IBD patients is unknown [24], with its etiology being ascribed to altered erythropoiesis at different levels [25]. Firstly, chronic inflammation can decrease erythropoiesis by direct action of interferon (IFN)-γ, IFN-α, tumor necrosis factor (TNF)-α, and IL-1 in the bone marrow to exert pro-apoptotic effects on erythroid burst-forming units (BFU-E) and colony-forming units (CFU-E) [26]. Moreover, IL-1, IL-6, TNF-α, and hepcidin may decrease erythropoietin (EPO) synthesis and impair its biological activity [1,27]. In fact, EPO levels in ACD have been found to be inadequate in some chronic disease and IBD patients [11,28,29]. Low EPO production is due to direct inhibition of the activity of the promoter of the EPO gene by IL-1 and TNF-α, which in turn inhibits the synthesis of EPO in the kidney and acts indirectly on EPOproducer cells through cytokine-induced toxic radicals [30]. Impairment of the biological activity of EPO means that much higher amounts of EPO are needed to restore the formation of CFU-E in the bone marrow. Cytokines can also interfere with the signaling process mediated by the interaction of EPO and its receptor and can downregulate EPO receptors on erythroid progenitor cells [26], thus producing cell resistance to EPO activity. Finally, the limited availability of iron for heme biosynthesis induced by functional or absolute iron deficiency and the inhibition of iron uptake into erythroid progenitors due to the blocking of the transferrin receptor by alfa1- antitrypsin (an acute phase protein) negatively affect the biological functions of EPO along with cell growth and differentiation [1]. Other types of anemia Vitamin B12 deficiency has been observed in 48% of CD patients and in 5% of UC patients [31] while folic acid deficiency has been noted in 67% of CD patients and in 30%-40% of UC patients [31-33]. These types of deficiencies depend on low dietary intake as well as increased turnover of epithelial cells due to chronic inflammation in the intestinal mucosa and a reduced absorption in the intestinal tract [34-36]. In CD, several factors influence these deficiencies, including the inflammatory involvement of ileal mucosa, the presence of fistulas, secondary bacterial overgrowth with direct consumption of vitamin B12, and extensive surgical resections in small bowel segments with impaired absorption [37]. Deficiencies in patients with UC derive from proctocolectomy and ileo-pouch anastomosis, with the prevalence of vitamin B12 deficiency being affected more by surgical changes leading to impaired function of ileal receptors, reduced intestinal transit time, and secondary bacterial overgrowth than on the length of the ileal segment resected [38]. Autoimmune hemolytic anemia (AIHA) is a rare type of anemia observed in UC patients. It can be due either to the development of antibodies with crossreactivity with erythrocytes [39] or to the hemolytic effect of sulfasalazine in patients with glucose-6-phosphate dehydrogenase deficiency [40]. This association was first described in 1955 by Lorber et al [41] with the most recent studies calculating that the prevalence of AIHA in UC patients is between 0.2%-1.7%, as indicated by a positive Coombs test result in 1.8% of patients studied [40]. AIHA can occur before, after, or at the moment of diagnosing UC. Even when the potential relationship between disease activity and the occurrence of AIHA is not clear, a correlation with the extension of the disease has been demonstrated in several reports, which show a prevalence of AIHA of up to 28% in patients with extensive colitis [40]. Anemia can also represent a late manifestation of myelosuppression in IBD patients due to several factors. Firstly, myelosuppression can be associated with myelodysplastic syndrome (MDS), with ineffective erythropoiesis and a risk of progression to acute myeloid leukemia. Some studies have shown a frequent predominance of MDS in CD with colorectal involvement; however, it should be noted that the prognosis of IBD with concomitant MSD is determined by the MSD itself [42]. The prevalence of MDS in IBD patients has been estimated to be 0.17%, with a higher incidence in IBD patients than in the general population (170/ IBD patients/year vs 20-30/ of the general population over the age of 70/year) [43]. This is probably due to a undetermined common pathogenetic mechanism, the long-term use of immunosuppressive drugs, or chromosomal abnormalities in bone marrow cells that have been observed in 67% of patients with concomitant IBD and MSD [44,45]. These can induce the development of colitogenic monocytes, producing a large number of pro-inflammatory cytokines resistant to apoptosis upon stimulation with microbial antigens. Indeed, one of the first hypotheses about this association regarded IBD to be an extra-hematologic manifestation of MSD with a 3544 April 7, 2014 Volume 20 Issue 13

170 Guagnozzi D et al. Anemia in inflammatory bowel disease Table 1 Laboratory findings in anemia of inflammatory bowel disease patients Biomarkers IDA ACD Mixed anemia MCV (fl) < 80 Normal or reduced Normal or reduced MCH (pg) < 27 Normal Normal CHr (pg) < 28 Normal Normal C-RP (mg/dl) Vormal > 5 > 5 ferritin (ng/ml) < 30 > TfS (%) < 20 < 20 < 20 Ferritin index > 3.2 < 11 > 2 stfr Increased Normal Normal or increased Hepcidin (nmol/l) Reduced Increased (> 4) Increased (> 4) MCV: Mean corpuscular volume; MCH: Mean corpuscular hemoglobin; CHr: Reticulocyte hemoglobin content; C-RP: C-reactive protein; TfS: Transferrin saturation; stfr: Soluble transferrin receptor; IDA: Iron deficiency anemia; ACD: Anemia of chronic disease. vasculitic process at the level of the mesenteric arteries [46]. Alternatively, myelosuppression may represent a complication of severe UC with the development of a systemic inflammatory response syndrome, or even been a side effect of immunosuppressive drugs. There is an increasing concern about therapy-induced leukemias and myelodysplastic syndromes in patients treated with thiopurines, which are extensively used as immunosuppresants in IBD, particularly for maintenance therapy [47]. Data from a large French cohort of patients (19486) with inflammatory bowel disease identified a relative risk of developing lymphoproliferative disorders as 5.2 for patients who were treated with thiopurines compared to those who were not [48]. Finally, additional gastrointestinal diseases that do not usually cause bleeding should be also considered in case of iron deficiency anemia in those IBD patients who maintain disease into remission, including colon or gastric cancer o polyps, peptic ulcer, hiatal hernia with linear erosions, atrophic or Helicobacter pylori-associated gastritis, and celiac disease [49,50]. DIAGNOSIS OF ANEMIA IN INFLAMMATORY BOWEL DISEASE Basic laboratory screening for anemia in IBD should consist of hemoglobin and full blood counts (including reticulocytes to differentiate between regenerative or hypo-regenerative anemia), with a determination of erythrocyte mean corpuscular volume (MCV) to distinguish between microcytic, normocytic, and macrocytic anemia as well as a determination of mean corpuscular Hb (MCH) and reticulocyte Hb content (CHr), if available. Moreover, assessments of both the level of inflammation by means of C-reactive protein (CRP) and of iron status are required. There is no single biomarker to diagnose iron deficiency in IBD; a combination of different biomarkers is needed. In most cases, total store of body iron with serum-ferritin (or ferritin) and the iron available in the bone marrow with transferrin saturation (TfS) is sufficient to differentiate between IDA and ACD [51]. However, many of the laboratory measures of iron status may be unreliable in IBD patients because the inflammation influences all parameters of iron metabolism to produce functional iron deficiency [52,53]. For this reason, in some cases it is essential to use other, more specific biomarkers of iron status to allow for the differentiation between predominantly IDA, predominantly ACD, and ACD combined with iron deficiency in order to provide appropriate, more effective treatment [54] (Table 1). Further testing for causes of anemia in IBD may include tests for vitamin B12, folic acid (especially erythrocyte levels, which, when available, represent the best stable marker of folic acid deficiency), haptoglobin, lactate dehydrogenase, indirect bilirubin (with Coombs test if hemolytic anemia is confirmed), and serum creatinine in order to rule out potential hemolysis or renal failure, which in itself can cause macrocytic or normocytic anemia [51]. It should be noted that if the origin of anemia is not obvious, IBD patients should be tested for MDS, especially if normocytic and hypo-regenerative anemia are both present, carrying out a bone marrow study in selected patients. Once a diagnosis of IBD has been established, patients in clinical remission should be screened for anemia at least every 6 to 12 mo, whereas patients with active disease should be tested every 3 mo or at even shorter intervals, depending upon their iron status [55]. Iron deficiency anemia Patients are considered to suffer from IDA when they present with low Hb (men < 13 g/dl, non-pregnant women < 12 g/dl), TfS < 20%, and ferritin concentrations < 30 ng/ml without any biochemical or clinical signs of inflammation. A low MCH (< 27 pg) or even better a low CHr (< 28 pg) rather than MCV (< 80 fl) have became the most important red cell markers for detecting iron deficiency in circulating red blood cells. Although MCV is a reliable and widely available measurement, it tends to be a relatively late indicator in patients who are not actively bleeding [56]. A normal Hb level does not rule out iron deficiency and with an MCH in the lower limit of normal (normal range: pg) or an increased red cell distribution width (RDW, normal range: 11-15), one can suspect the presence of mild iron deficiency without anemia [57]. Although the main laboratory marker for iron deficiency with or without anemia is a low ferritin level (< 30 ng/ml) in the absence of inflammation, in the presence of inflammation a normal ferritin level (as an acute phase reactant) does not rule out iron deficiency; therefore, TfS should also be measured. Functional iron deficiency in inflammatory conditions should be defined by low TfS (< 20%) and normal ferritin concentration (> 100 ng/ml), whereas low TfS (< 20%) and intermediate ferritin values ( ng/ml) suggest absolute iron deficiency [57]. Some authors suggest a cut-off value of TfS < 16% combined with low iron value for the diagnosis of iron deficiency [51]. Iron deficiency can also be defined by a ferritin index > 3.2 (> 2 if CRP > 5 mg/l). The ferritin index, which reflects 3545 April 7, 2014 Volume 20 Issue 13

171 Guagnozzi D et al. Anemia in inflammatory bowel disease the iron supply for erythropoiesis, is calculated as the ratio between the soluble transferrin receptor (stfr) and the log of ferritin [58]. The stfr is a truncated fragment of the membrane receptor and its levels increase when the availability of iron for erythropoiesis is low, as occurs in IDA. CHr, which measures the Hb content of reticulocytes, reflects the direct measurement of available iron for erythropoiesis and is useful for differentiating IDA from ACD. In particular, CHr has a high sensitivity and specificity for diagnosing iron deficiency and is less affected by inflammation than TfS and ferritin, but no data are available for its use in IBD [58]. Anemia of chronic disease Patients are considered to suffer predominantly from ACD when they present evidence of inflammation (with increased levels of serum CRP and clinical signs), an Hb concentration < 13 g/dl for men and < 12 g/dl for non-pregnant women, and a low TfS < 20%, but normal or increased ferritin concentrations > 100 ng/ml. In the presence of intermediate ferritin concentrations ( ng/ml), a diagnosis of ACD combined with absolute iron deficiency is confirmed if the ferritin index has a value < 2 with normal CHr [54,58-60]. Still, some cases may require supplementary testing for the differential diagnosis between IDA and ACD. It has recently been shown that hepcidin levels may replace the ferritin index for the confirmation of combined IDA and ACD if the hepcidin levels are > 4 nmol/l with a CHr < 28 pg [61]. In fact, hepcidin levels have been found to be significantly higher in IBD patients compared with healthy controls, with a significant correlation with ferritin levels, CRP, and disease activity, whereas those of prohepcidin were observed to be significantly lower [62]. In addition, although other hematological indices may help in the diagnosis of iron deficiency in ACD, many of them are only available in specific hematology analyzers and their precise clinical usefulness has yet to be determined. In a recent study carried out with a Beckman-Coulter LH 780, high values of RDW and low values of blood cell Size Factor were the best markers for the diagnosis of IDA, whereas both Reticulocyte Distribution Width-Coefficient of Variation (RDWR-CV) and Reticulocyte Distribution Width- Standard Deviation (RDWR-SD) were significantly correlated to disease activity and CRP levels [63]. TREATMENT OF ANEMIA IN INFLAMMATORY BOWEL DISEASE Iron supplementation Iron supplementation should be considered in every patient presenting iron deficiency with or without anemia. In patients with mild to moderate anemia (Hb 10 g/ dl), the administration of oral iron at optimal low doses of mg/d is the conventional approach recommended by the Centers for Disease Control and Prevention [9,64]. Oral iron compounds are mostly available as inorganic ferrous salts, such as ferrous fumarate, ferrous sulphate, and ferrous gluconate containing 33%, 20%, and 12% of elemental iron, respectively. A single tablet of most of these ferrous salt preparations provides a sufficient dose for the treatment of iron deficiency [65,66]. In fact, there is no evidence to support the administration of high doses of iron in comparative trials [65-67] ; on the contrary, excessive doses may actually decrease tolerance and compliance while increasing gastrointestinal side effects, with a discontinuation of iron treatment in 20% of patients with or without IBD [68]. Nevertheless, there are several drawbacks associated with oral iron therapy that must be taken into account. In addition to the generally low bioavailability of oral iron, intestinal absorption is further compromised in IBD patients due to an inflammation-driven blockade caused by increased hepcidin levels. For this reason, in patients with active inflammation and combined ACD/iron deficiency, intravenous administration of iron may be preferable to oral iron therapy. Moreover, when achieved, the therapeutic effect of oral iron supplementation is slow, requiring two to three weeks to obtain increased Hb concentrations and up to two months to achieve normal values. At least six months are needed to replenish iron stores completely [69]. Moreover, non-absorbed iron salts can be toxic to the intestinal mucosa and oral iron has been shown to increase intestinal inflammation and possibly colon carcinogenesis in animal models through the production of reactive oxygen species that mediate intestinal damage and the alteration of the intestinal bacterial milieu in rodents [70-75]. Intravenous iron therapy is more effective, has a higher response rate, and is better tolerated by patients, with a lower discontinuation rate due to adverse events than oral iron supplementations in IBD patients, as demonstrated in a recent systematic review with metaanalysis [76]. However, it is important to highlight that of 757 articles identified, only three industry-funded articles met the inclusion criteria for this systematic review [69,77-83]. Nevertheless, intravenous iron therapy should be considered in patients with severe anemia (< 10 g/dl), with intolerance or inadequate response to oral iron, or with concomitant erythropoietin treatment and/or presence of active IBD. It should be noted that the new intravenous iron formulations (iron carboxymaltose, iron ferumoxytol, and iron isomaltoside) reduce both the risk of free iron reactions as well as that of immunogenicity without the need for administering a test dose before starting treatment. Treatment duration is also reduced because the new formulations are safer at higher doses than traditional intravenous iron formulations (iron sucrose, ferric gluconate, and low molecular weight iron dextran). Iron carboxymaltose is the only new intravenous iron formulation approved for use in Europe that has been studied in IBD patients. Its superiority at higher standardized doses over individually calculated doses of iron sucrose has been demonstrated along with its efficacy in reducing anemia recurrence as compared to a placebo [84-86]. After 12 wk of follow-up, 3546 April 7, 2014 Volume 20 Issue 13

172 Guagnozzi D et al. Anemia in inflammatory bowel disease ferric carboxymaltose led to higher response rates (66.1% vs 54.1%), a higher proportion of non-anemic patients (72.8% vs 61.8%), and better treatment adherence (92.5% vs 79.1%) than iron sucrose, with no difference in treatment-related adverse events (13.9% vs 11.3%). With regard to side effects, in the ferric carboxymaltose group there were more skin and subcutaneous tissue disorders (rash, dermatitis, pruritus) and more cases of hypophosphatemia, but fewer infusion site reactions than in the iron sucrose group. The superiority of high-dose intravenous iron supplementation in IBD probably depends on the iron overload produced in the macrophages of the reticulo-endothelial system. This induces an overexpression of ferroportin, which may, in turn, by-pass the hepcidin block in ACD [57]. Recently, an alternative dosage scheme to the traditional Ganzoni formula has been presented for ferric carboxymaltose treatment. In the new protocol, for a baseline Hb > 10 g/dl, the dose is 1.0 g for patients with a body weight < 70 kg and 1.5 g for patients > 70 kg; the corresponding total doses for serum Hb 10 g/dl are 1.5 g and 2.0 g [84]. Phase 3 clinical trials are currently underway to evaluate the use of ferumoxytol in patients with iron deficiency anemia, including a subgroup with IBD (ClinicalTrial.org identifier: NCT , NCT and NCT ). However, ferumoxytol may be problematic in IBD patients because it can interfere with MRI signals due to the paramagnetic nature of its iron core [19]. In addition, a Phase 3 clinical trial of iron isomaltoside (NCT ) and a Phase 4 study of iron sucrose (NCT ) are currently being carried out on IBD patients with IDA. All treatments being tested strive to achieve ferritin concentrations > 100 μg/l, measured as early as 8 wk after intravenous iron treatment to obtain a reliable result [55]. Considering that the recurrence of iron deficiency with or without anemia is frequent in IBD patients [87], regular controls at 12 wk intervals are advisable so that treatment can be restarted promptly if needed. Erythropoietin supplementation Several studies have shown that recombinant human erythropoietin may be effective for treating ACD in IBD patients [88-95]. In the anemia treatment algorithm, intravenous iron therapy should be considered as a firstline therapy in patients with severe anemia whereas erythropoietin treatment should be considered only in patients unresponsive to intravenous treatment, with low EPO levels, or who are unresponsive to aggressive management of IBD [29,53,96] since EPO can be used as an adjunct therapy to control the inflammation [97]. Recently, a prospective study on CD patients showed that EPO combined with enteral nutrition can improve the Hb levels in CD patients with a treatment success rate of 63.16% in the EPO group compared to none of the patients in the non-epo group [98]. When a decision has been made to administer EPO therapy, it should always be combined with intravenous iron supplementation to meet the increased demand caused by the functional iron deficiency typical in IBD patients [99]. Other treatments Intramuscular vitamin B12 continues to be the gold standard therapy for vitamin B12 deficiency, especially in symptomatic patients [100]. A dose of 1000 μg/wk for 8 wk, then 1000 μg once monthly for maintenance lifelong is recommended [101]. No therapeutic advantages have been demonstrated for either cyanocobalamine or hydroxycobalamine in terms or serum levels during maintenance therapy [102]. Effectiveness data for sublingual [103,104] and intranasal [105] routes for vitamin B12 administration have revealed as promising non-invasive alternatives. Specific treatment of IBD has been shown to gradually increase Hb levels over time, which indicates that the presence of anemia is positively associated with disease activity and disease-associated gut lesions. Some data suggest that anti-tnf-α treatment improves the anemia in a sub-group of patients with CD. In fact, patients who responded to treatment showed improvements in their anemia within 2 wk of the first infusion of Infliximab, with a parallel improvement in their CD activity index and an increase in endogenous EPO levels over time [12]. Infliximab seems to neutralize the inhibitory effects of TNF-α on EPO production, increasing the availability of iron for erythropoiesis and reducing anemia [30]. The drug produces these effects through various mechanisms, including reduced cytokine-induced formation of ferritin and hepcidin, with improvement of intestinal iron absorption and iron release from macrophages via ferroportin-mediated iron export [21,106,107]. Moreover, Infliximab improved the proliferation of cultured BFU-E, blocking the inhibitory effects of cytokines on erythroid progenitor cells [26]. Finally, it induced mucosal healing, thereby reducing the production of pro-inflammatory cytokines and the amount of blood loss through mucosal ulcers. Other therapies with potential for treating IBD associated with anemia include treatment with anti- IL-6, which is the major inflammation-driven inducer of hepcidin, and other new therapies that neutralize hepcidin, modify EPO and/or erythropoietin receptor sensitivity, or affect cytokines to effectively stimulate erythropoiesis. The multi-factorial origin of anemia in IBD implies that several leading mechanisms can be simultaneously identified in a single patient, including chronic intestinal blood loss, decreased absorption capabilities of the small bowel secondary to inflammation or resection, bacterial overgrowth, and an inability of many IBD patients to tolerate the side effects of oral ferrous sulfate, among others [108]. Each of these causative factors usually requires a specific therapeutic approach. Disease inflammatory activity and iron deficiency should be the first aspects to be restored in every patient [109] since they are the main causes of anemia and easily identified. Although more uncommon, vitamin B12 or folate deficiency, hemolytic and drug-induced anemia should also be born in mind. Effective treatment is only possible if the con April 7, 2014 Volume 20 Issue 13

173 Guagnozzi D et al. Anemia in inflammatory bowel disease tributing factors in a particular patient are clearly defined and corrected [110]. CONCLUSION Anemia is a common multifactorial complication in IBD that increases disease morbidity. Awareness on the part of gastroenterologists needs to be increased to improve the specific diagnosis and management of anemia in these patients. New generation Ⅳ iron compounds are currently available to treat iron deficiency effectively in IBD patients. Further studies are needed to establish standardized treatments to reduce the development and recurrence of anemia as well as to improve the clinical course of IBD. REFERENCES 1 Weiss G, Goodnough LT. Anemia of chronic disease. N Engl J Med 2005; 352: [PMID: DOI: / NEJMra041809] 2 Weiss G, Gasche C. Pathogenesis and treatment of anemia in inflammatory bowel disease. Haematologica 2010; 95: [PMID: DOI: /haematol ] 3 Gomollón F, Gisbert JP. Anemia and inflammatory bowel diseases. 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Rapid recurrence of IBD-associated anemia and iron deficiency after intravenous iron sucrose and erythropoietin treatment. Am J Gastroenterol 2009; 104: [PMID: 3550 April 7, 2014 Volume 20 Issue 13

176 Guagnozzi D et al. Anemia in inflammatory bowel disease DOI: /ajg ] 88 Gasché C, Reinisch W, Lochs H, Parsaei B, Bakos S, Wyatt J, Fueger GF, Gangl A. Anemia in Crohn s disease. Importance of inadequate erythropoietin production and iron deficiency. Dig Dis Sci 1994; 39: [PMID: ] 89 Horina JH, Petritsch W, Schmid CR, Reicht G, Wenzl H, Silly H, Krejs GJ. Treatment of anemia in inflammatory bowel disease with recombinant human erythropoietin: results in three patients. Gastroenterology 1993; 104: [PMID: ] 90 Schreiber S, Howaldt S, Schnoor M, Nikolaus S, Bauditz J, Gasché C, Lochs H, Raedler A. Recombinant erythropoietin for the treatment of anemia in inflammatory bowel disease. N Engl J Med 1996; 334: [PMID: DOI: /NEJM ] 91 Gasché C, Dejaco C, Waldhoer T, Tillinger W, Reinisch W, Fueger GF, Gangl A, Lochs H. Intravenous iron and erythropoietin for anemia associated with Crohn disease. A randomized, controlled trial. Ann Intern Med 1997; 126: [PMID: DOI: / ] 92 Gasche C, Dejaco C, Reinisch W, Tillinger W, Waldhoer T, Fueger GF, Lochs H, Gangl A. Sequential treatment of anemia in ulcerative colitis with intravenous iron and erythropoietin. Digestion 1999; 60: [PMID: DOI: / ] 93 Demirtürk L, Hülagü S, Yaylaci M, Altin M, Ozel M. Serum erythropoietin levels in patients with severe anemia secondary to inflammatory bowel disease and the use of recombinant human erythropoietin in patients with anemia refractory to treatment. Dis Colon Rectum 1995; 38: [PMID: DOI: /BF ] 94 Koutroubakis IE, Karmiris K, Makreas S, Xidakis C, Niniraki M, Kouroumalis EA. Effectiveness of darbepoetin-alfa in combination with intravenous iron sucrose in patients with inflammatory bowel disease and refractory anaemia: a pilot study. Eur J Gastroenterol Hepatol 2006; 18: [PMID: DOI: / ] 95 Dohil R, Hassall E, Wadsworth LD, Israel DM. Recombinant human erythropoietin for treatment of anemia of chronic disease in children with Crohn s disease. J Pediatr 1998; 132: [PMID: DOI: /S (98) ] 96 Katsanos KH, Tatsioni A, Natsi D, Sigounas D, Christodoulou DK, Tsianos EV. Recombinant human erythropoietin in patients with inflammatory bowel disease and refractory anemia: a 15-year single center experience. J Crohns Colitis 2012; 6: [PMID: DOI: /j.crohns ] 97 Cronin CC, Shanahan F. Anemia in patients with chronic inflammatory bowel disease. Am J Gastroenterol 2001; 96: [PMID: DOI: /j x] 98 Liu S, Ren J, Hong Z, Yan D, Gu G, Han G, Wang G, Ren H, Chen J, Li J. Efficacy of erythropoietin combined with enteral nutrition for the treatment of anemia in Crohn s disease: a prospective cohort study. Nutr Clin Pract 2013; 28: [PMID: DOI: / ] 99 Christodoulou DK, Tsianos EV. Anemia in inflammatory bowel disease - the role of recombinant human erythropoietin. Eur J Intern Med 2000; 11: [PMID: DOI: /S (00) ] 100 Majumder S, Soriano J, Louie Cruz A, Dasanu CA. Vitamin B12 deficiency in patients undergoing bariatric surgery: preventive strategies and key recommendations. Surg Obes Relat Dis 2013; 9: [PMID: DOI: / j.soard ] 101 Mullin GE. Micronutrients and inflammatory bowel disease. Nutr Clin Pract 2012; 27: [PMID: DOI: / ] 102 Hvas AM, Nexo E. Diagnosis and treatment of vitamin B12 deficiency--an update. Haematologica 2006; 91: [PMID: ] 103 Delpre G, Stark P, Niv Y. Sublingual therapy for cobalamin deficiency as an alternative to oral and parenteral cobalamin supplementation. Lancet 1999; 354: [PMID: DOI: /S (99) ] 104 Sharabi A, Cohen E, Sulkes J, Garty M. Replacement therapy for vitamin B12 deficiency: comparison between the sublingual and oral route. Br J Clin Pharmacol 2003; 56: [PMID: DOI: /j x] 105 Slot WB, Merkus FW, Van Deventer SJ, Tytgat GN. Normalization of plasma vitamin B12 concentration by intranasal hydroxocobalamin in vitamin B12-deficient patients. Gastroenterology 1997; 113: [PMID: DOI: / gast.1997.v113.pm ] 106 Johnson D, Bayele H, Johnston K, Tennant J, Srai SK, Sharp P. Tumour necrosis factor alpha regulates iron transport and transporter expression in human intestinal epithelial cells. FEBS Lett 2004; 573: [PMID: DOI: / j.febslet ] 107 Ludwiczek S, Aigner E, Theurl I, Weiss G. Cytokine-mediated regulation of iron transport in human monocytic cells. Blood 2003; 101: [PMID: DOI: / blood ] 108 Warsch S, Byrnes J. Emerging causes of iron deficiency anemia refractory to oral iron supplementation. World J Gastrointest Pharmacol Ther 2013; 4: [PMID: DOI: /wjgpt.v4.i3.49] 109 Gomollón F, Gisbert JP. Current management of iron deficiency anemia in inflammatory bowel diseases: a practical guide. Drugs 2013; 73: [PMID: DOI: /s ] 110 Gisbert JP, Gomollón F. Common misconceptions in the diagnosis and management of anemia in inflammatory bowel disease. Am J Gastroenterol 2008; 103: [PMID: DOI: /j x] P- Reviewers: Cheifetz A, Dickey W S- Editor: Ma YJ L- Editor: A E- Editor: Liu XM 3551 April 7, 2014 Volume 20 Issue 13

177 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (3): Inflammatory bowel disease TOPIC HIGHLIGHT Psychiatric comorbidity in the treatment of patients with inflammatory bowel disease Branislav R Filipovic, Branka F Filipovic Branislav R Filipovic, Faculty of Medicine, Institute of Anatomy Niko Miljanic, Belgrade, Serbia Branislav R Filipovic, Branka F Filipovic, Faculty of Medicine, University of Belgrade, Belgrade, Serbia Branka F Filipovic, Department of Gastroenterohepatology, Clinical and Hospital Center Bezanijska Kosa, Belgrade, Serbia Author contributions: Both authors contributed to the manuscript design, reference selection, interpretation of the results discussed in the review, and approved the manuscrip. Correspondence to: Branislav R Filipovic, Professor, Faculty of Medicine, Institute of Anatomy Niko Miljanic, 4/2 Dr Subotica Starijeg, Belgrade, Serbia. filipovic.branislav@gmail.com Telephone: Fax: Received: September 29, 2013 Revised: January 11, 2014 Accepted: January 20, 2014 Published online: April 7, 2014 Abstract Ulcerative colitis and Crohn s disease, commonly known as inflammatory bowel disease (IBD), draw attention from specialists of various disorders, including gastroenterology, psychiatry, and radiology. The involvement of a cortical influence in the brain-gut axis as well as the interaction of the hypothalamic-pituitary-adrenal axis and the peripheral nervous system provide an initial explanation of the psychological symptoms associated with IBD. The involvement of structures the limbic system, such as the anterior cingulate cortex, the prefrontal cortex, and the amygdala, paves the way for the discovery of the mechanisms underlying depression depression, anxiety, alexithymia, personality traits, and other psychological impairments following the onset of IBD. Psychiatric therapy in IBD patients is almost as important as the gastroenterological approach and consists of pharmacological treatment and psychotherapy. Neither of the available psychiatric treatment methods is considered the golden standard because both methods have side effects, and psychotropic medication can provoke the worsening of IBD symptoms. Thus, both approaches must be applied with awareness of the possibility of side effects. We suggest that psychiatrists and gastroenterologists work together to reach a consensus on IBD therapy to ensure success and to reduce side effects and relapse to the lowest possible rates Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Inflammatory bowel disease; Psychiatry; Treatment; Personality traits; Depression; Anxiety Core tip: The involvement of a dysfunction of brain-gut interactions in the pathogenesis of inflammatory bowel disease (IBD) is represented by a dysfunction of the autonomic nervous system, an abnormal hypothalamicpituitary-adrenal axis and cholinergic anti-inflammatory pathway, a deleterious effect of stress and depression, an abnormal coupling of the prefrontal cortex-amygdaloid complex, and an abnormal relation between the microbiota and the brain as pro-inflammatory factors. New investigations have provided a critical link between forebrain changes and abdominal pain independent of active disease and drug treatment, providing a potential basis for an explanation of the psychological symptoms and brain influence in the pathogenesis of IBD. Filipovic BR, Filipovic BF. Psychiatric comorbidity in the treatment of patients with inflammatory bowel disease. World J Gastroenterol 2014; 20(13): Available from: URL: DOI: ANATOMICAL BASIS FOR THE PSYCHIATRIC CHANGES IN PATIENTS WITH INFLAMMATORY BOWEL DISEASE Inflammatory bowel disease (IBD) results from an inap April 7, 2014 Volume 20 Issue 13

178 Filipovic BR et al. Psychiatric discomfort and IBD treatment propriate inflammatory response to intestinal microbes in a genetically susceptible host. In recent reports, authors [1,2] have discussed the involvement of a dysfunction of brain-gut interactions in the pathogenesis of IBD as represented by a dysfunction of the autonomic nervous system, an abnormal hypothalamic - pituitary - adrenal axis and cholinergic anti-inflammatory pathway, a deleterious effect of stress and depression as well as an abnormal coupling of the prefrontal cortex-amygdaloid complex and an abnormal relation between the microbiota and the brain as pro-inflammatory factors. The New investigations have provided a critical link between forebrain changes and abdominal pain independent of active disease and drug treatment because all patients examined in the morphometric evaluation were in remission and suffered from ongoing abdominal pain. Investigators observed decreased gray matter volumes in the dorsolateral prefrontal cortex and the anterior midcingulate cortex (amcc), and the disease duration was negatively correlated with volumes in the subgenual anterior cingulate (sacc), the posterior MCC (pmcc), the ventral posterior cingulate (vpcc), and the parahippocampal cortices. The amcc has a role in feedback-mediated decision making, and specific cognitive tasks that differentiate the amcc and pmcc can be used to evaluate defects in Crohn s disease (CD). The sacc is an important area because it has impaired functions in major depression. Because depressive symptoms are a feature in a subset of patients with active inflammatory diseases, including IBD, treatment targeting this subregion should prove efficacious. Finally, the vpcc has a role in ongoing self-monitoring of the personal relevance of sensory stimuli, including visceral signals via the sacc. This pathway may be interrupted by vpcc atrophy in CD. Cingulate atrophy in CD requires the targeting of chronic pain and psychiatric symptom therapies via neuronal circles involved in the cingulum. These therapies include psychotherapy, guided imagery and relaxation training, analgesic dosages of morphine or antidepressants, and hypnosis. Thus, a new generation of novel treatments may emerge from drug and nontraditional therapies for CD in this formative area of research [3,4]. Nevertheless, a certain level of caution should remain: the same areas have been found to be susceptible to changes in temporal epilepsy [5], and it remains unclear whether the volume alterations in these areas are specific to IBD or if they overlap with other diseases. The white matter is not spared from damage in IBD patients. The number of such lesions is significantly higher in IBD patients compared to controls (12.75 ± vs 3.20 ± 2.90, P < 0.05). However, there are no significant differences between UC and Crohn s disease patients with regard to magnetic resonance imaging (MRI) findings. In addition, the incidence of white matter lesions and other brain parenchymal lesions, sinusitis, and otitis-mastoiditis does not differ significantly with disease activity (P > 0.05 for all) [6]. Scheid et al [7] (2007) proposed the following three possible mechanisms for peripheral and central nervous system involvement in ulcerative colitis (UC): cerebrovascular conditions due to thromboembolic events, systemic and cerebral vasculitis, and neuropathy and cerebral demyelination due to immune-related mechanisms. In contrast, white matter lesion is a frequent finding in patients with IBD on MRI, and the development of these lesions has been attributed to ischemic mechanisms (atherosclerotic or vasculitic) or demyelination [8-10]. Thus, early identification of these lesions may be clinically helpful as an early indication of neurological involvement because they may represent another extra intestinal manifestation of the disease [10]. Studies performed by functional magnetic resonance imaging for both, patients and control subjects suffering from irritable bowel syndrome, which is also a psychosomatic disease, and control subjects, rectal distention stimulation increased the activity of the anterior cingulate cortex (number of positive answers to the stimulation/total number of patients: 35/37), the insular cortex (37/37), the prefrontal cortex (37/37), and the thalamus (35/37) in most cases. In patients with inflammatory bowel sydrome (IBS), the average percentage area of regions of interest increased in parallel with rectal distention volumes in the insular cortex, the prefrontal cortex, and the thalamic region. However, only the prefrontal cortex was statistically significant (P < 0.05). In controls, this tendency to increase only occurred in the anterior cingulate cortex. At 120 ml rectal distention, the average percentage area of regions of interest (ROI) and the average percentage change in MR signal intensity of ROIs in the insular cortex, the prefrontal cortex, and the thalamic region were significantly greater in patients with IBS than in control subjects [11,12]. PSYCHOLOGICAL SYMPTOMS IN IBD There is consistent evidence that psychological factors play a role in the pathophysiology and the course of IBD and in how patients cope with IBD [12]. One prospective study in a population-based cohort of individuals with IBD (n = 552) evaluated whether the presence of a stressful event and the perception of stress as well as other factors (i.e., nonsteroidal anti-inflammatory drugs, antibiotics, or infections) believed to contribute to triggering flares of IBD were, in fact, associated with symptomatic flares [13]. Subjects completed surveys on health issues every 3 mo for 1 year. In any 3-mo period, approximately 50% of subjects experienced some type of stress, and the majority of subjects reported the stresses were everyday life stresses. Family stress was the most commonly reported stress, followed by work or school and financial stress. Subjects were grouped by disease activity over time. Significantly more individuals in the persistently inactive disease group indicated they experienced no stressful events compared with individuals in the persistently active disease group. In terms of the association between variables experienced in one 3-mo period and a symptomatic flare in the next 3-mo period, only psychological factors, including the occurrence of a major life event, high perceived stress, and high negative mood during a previous 3-mo period, were significantly 3553 April 7, 2014 Volume 20 Issue 13

179 Filipovic BR et al. Psychiatric discomfort and IBD treatment associated with the subsequent occurrence of a flare. This study complements the growing evidence from experimental as well as clinical studies that stress exposure, including stressful events and perceived stress (the individual s view of his or her own level of demand relative to resources), may contribute to relapse risk in IBD [14-18]. In fact, using multivariate logistic regression analyses of these variables, only high perceived stress (adjusted OR = 2.40; 95%CI: ) was associated with an increased risk of flare. This finding speaks to the bidirectional relationship between stress and symptomatic disease. Being symptomatic may exacerbate or even incite stress, whereas being stressed may trigger symptomatic disease. Recent reports have shown that brain derived neurotrophic factor (BDNF) levels are highly dynamic in response to stress. BDNF levels not only vary across brain regions but also fluctuate rapidly, both immediately after a stressor and over the course of a chronic stress paradigm. However, BDNF alone is not sufficient to effect many of the changes observed after stress. Glucocorticoids and other molecules have been shown to act in conjunction with BDNF to facilitate both the morphological and molecular changes that occur, particularly changes in spine density and gene expression [19]. Although discrete personality traits have been studied in IBD patients, no specific personality type matches this disease. It is recommended that future research consider the discrete personality traits observed in these patients and integrate them in such a way that the traits will be addressed to include new personality types, such as types C and D [20,21], which are well matched with the unregulated immune and hormonal systems that are characteristics of IBD. Type C individuals are introverted, perfectionistic, sensitive, and thoughtful. Individuals with a type D personality have the tendency to experience increased negative emotions across time and situations and tend to not share these emotions with others because of a fear of rejection or disapproval. It is believed that depression and anxiety are dominant in patients with IBD [22]. These patients also have a higher prevalence of anxiety and depressive disorders than the general population but a lower prevalence of these disorders than patients with a functional bowel disorder. The prevalence (21%-35%) is similar to that found in other patients with chronic physical illness. Depressive disorder appears to be more common in older patients and individuals with a previous history of a psychiatric disorder [23,24]. Patients suffering from IBD take more medications than the healthy population: the use of antidepressants (OR = 1.44, 95%CI: ), anxiolytics (OR = 1.52, 95%CI: ), oral bisphosphonates (OR = 6.08, 95%CI: ), cardiovascular medications (OR = 1.38, 95%CI: ), antibiotics (OR = 4.01, 95%CI: ), proton pump inhibitors (OR = 3.90, 95%CI: ), and nonsteroidal antiinflammatory analgesics (OR = 1.17, 95%CI: ) is significantly more common in IBD patients than in controls. Individuals who use antidepressants, anxiolytics, or analgesics have significantly impaired health-rated quality of life (HRQOL) (P < 0.001) [25]. Both depression and anxiety precede ulcerative colitis significantly more often than would be predicted from the control population s experience [24]. The association is strongest when the two psychiatric disorders and ulcerative colitis are diagnosed in the same year, although the association between depression and ulcerative colitis is also significant when depression precedes ulcerative colitis by five or more years. Neither depression nor anxiety precedes Crohn s disease more often than expected by chance, although the study involved fewer cases with Crohn s disease than ulcerative colitis. Two prospective clinical studies of patients with IBD appear to produce conflicting results. During a 6-mo follow-up period, one study found a strong association between the change in disease activity and anxiety level and a weaker association with depressive symptoms. Changes in disease activity seemed to lead to changes in anxiety and depression. Beck Depression Inventory scores at baseline predicted the number and timing of relapses during an 18-mo follow-up period [26,27]. Nevertheless, the origin of depression and anxiety in patients with IBD remains at least insufficiently explained. In a review of psychotherapeutic approaches to IBD, Prasko et al [28] (2010) emphasized that higher scores of neuroticism, depression, inhibition, and emotional instability are typical for many patients with chronic diseases and nonspecific for chronic gastroenterological disorders. More directly, anxiety and depression are consequences of IBD symptoms, such as frequent stools with evidence of blood, pain in the abdominal region, and bloating. Because anxiety and mood disorders are the most common mental health concerns in the community, these disorders are usually the focus of screening efforts. The typical symptoms of anxiety disorders include high levels of physiological arousal, excessive worries about the future, avoidance of feared situations (including, in some cases, medical appointments and procedures) [29,30], and difficulty coping with unfamiliar situations. Depression typically presents with a constellation of affective, cognitive, and somatic symptoms, including a sad or depressed mood; a loss of interest in normal activities; feelings of guilt, worthlessness, or hopelessness; difficulties with concentration; reduced energy; changes in appetite and sleep; and withdrawal from usual activities. When these clusters of symptoms persist beyond a few weeks and begin to significantly interfere with daily functioning, they are typically considered to reach a clinical threshold [31]. The incidence of IBD in adolescents has been reported to be an increasing trend, especially in the Northern parts of Europe (Finland, Schotland) [32-34]. According to parent reports, adolescents with IBD have more emotional, social, and thought problems and lower competence than their healthy peers [35]. The disease disturbs adolescents quality of life [25,36,37], may have negative consequences for education and school functioning [38,39], and may be a cause of difficulties in employment, such as finding or maintaining a desired job [40-42]. Furthermore, adolescents with severe IBD have disturbed sleep and are overtired 3554 April 7, 2014 Volume 20 Issue 13

180 Filipovic BR et al. Psychiatric discomfort and IBD treatment more often than their healthy peers [43]. Some studies have shown alexithymia to be another common personality characteristic in IBD patients. Patients with alexithymia have difficulties in recognizing and verbalizing emotions, and their ability to regulate emotions and express them to others is usually reduced [44,45]. Numerous studies [46-50] have shown that IBD patients have higher scores for alexithymia than controls. In a study conducted by Jones et al [46] (2006), the scores of 74 IBS patients, 55 healthy control subjects, and 48 IBD patients were compared on the Toronto Alexithymia Scale. The results showed that IBS and IBD patients had higher scores on measures of alexithymia than the controls, but they did not differ from one another. In an epidemiological study, Porcelli et al [50] (1999) compared 121 functional gastrointestinal disorder patients, 116 IBD patients, and a group of 112 healthy subjects using the Toronto Alexithymia Scale. Their results showed that the FGID group was significantly more alexithymic than the IBD group, and the scores of the two gastrointestinal groups were higher than the normal healthy group. Even after controlling for the influence of education, gender, anxiety, depression, and gastrointestinal symptoms, these differences remained significant. Moreno-Jiménez et al [47] (2007) did not use a control group. In their sample comprising 60 UC and 60 CD patients, they attempted to address the question of which personality factors may predict HRQOL in IBD patients. They showed that difficulty in describing one s feelings was significant for predicting two dimensions of HRQOL, systemic symptoms and social functioning. Difficulty in describing one s feelings negatively predicted systemic symptoms and social functioning. Patients experiencing more difficulty in describing their feelings reported lower HRQOL. Although alexithymia may not be specific to IBD, it may lead patients to communicate their psychological distress through somatic and behavioral symptoms rather than verbal communication. This may occur particularly when patients have limited perceived social support or personality traits such as introversion. Regardless of whether alexithymia is specific to IBD, it has been reported that affected patients have greater difficulty in describing their feelings to others, poorer disease outcome, lower psychological functioning, and worse HRQOL [12,47,51]. Adolescents with IBD have mild cognitive problems compared to the same population with juvenile idiopathic arthritis, particularly in the acute phase. Adolescent patients with IBD produced more perseverative errors than patients with non-acute juvenile idiopathic arthritis. Perseveration in the California Verbal Learning Test may be related to a momentary loss of alertness in the tiresome and long verbal memory test. However, no other differences in cognitive functioning between the study groups were detected. These findings indicate that adolescents with active IBD may have some mild problems in verbal memory but no major cognitive deficits. Prior studies in adults with IBD found deficits, particularly in verbal functioning, suggesting that in the clinical evaluation of young patients with IBD, it may be relevant to pay atten- tion to even minor cognitive problems that may be aggravated during the growth process [43,52,53]. PSYCHIATRIC THERAPY IN IBD Psychiatric treatment of patients with IBD involves two types of approaches: (1) psychotropic medication; (2) psychotherapy; and (3) psychotropic medication. Anxiety and depression, the most common psychiatric symptoms in IBD patients, are highly treatable conditions. The interventions that are the most widely used and have been evaluated the most extensively for anxiety and depressive disorders are specific pharmacological agents [particularly selective serotonin reuptake inhibitors (SSRIs), such as citalopram, fluoxetine, fluvoxamine, paroxetine, and sertraline, and the serotonin norepinephrine reuptake inhibitor (SNRI) venlafaxine] and specific psychological treatments (particularly cognitive behavioral therapies). The SSRI and SNRI medications are secondgeneration antidepressants that have been well established in the literature as being similarly effective for anxiety and depression [54]. In humans, it has been observed that although antidepressants improve both the mental and somatic status of IBD patients, the low quality of available research provides significant barriers to making a definitive statement on their efficacy or lack thereof [55]. Animal models, however, have found a positive impact of desipramine and fluoxetine on inflammation in models of IBD. When doctors perspectives on antidepressants in IBD were examined, it was reported that gastroenterologists commonly treat IBD patients with antidepressants for pain, anxiety and/or depression, and insomnia. Gastroenterologists reported that antidepressants were successful in reducing pain, gut irritability, and urgency of defecation. In the most recent retrospective case-note audit of 287 patients, 83 (28.9%) patients had used an antidepressant at some time in their life [56-59]. Nonetheless, the design of the study does not allow a firm statement to be made about whether antidepressants improved the course of IBD. The recent study in this area conducted by Goodhand et al [60] (2012), which examined the disease course one year before and one year after the commencement of antidepressants, showed that patients reported fewer relapses and steroid treatment in the year after starting an antidepressant than in the year before. This effect was not observed in the control group. Although this report of decreased symptoms may simply reflect the report of fewer functional gastrointestinal symptoms when patients are in better psychological health [61,62], it may also indicate an inflammation-specific benefit from antidepressants. Thus, it is clear that randomized controlled trials are justified and needed to provide a definitive answer regarding the efficacy of antidepressants in IBD. Amitriptyline is an antidepressant drug that is widely used for the treatment of IBD and gastrointestinal disorders [55,63]. It is effective for treating psychological and somatic symptoms in patients suffering from IBD [55]. Other studies have shown the anti-inflammatory effects 3555 April 7, 2014 Volume 20 Issue 13

181 Filipovic BR et al. Psychiatric discomfort and IBD treatment of antidepressants by different mechanisms [64,65]. Amitriptyline also acts on α1-adrenoceptors to produce anti-inflammatory effects [64]. Due to its effects on the inhibitory cytokine interleukin-10, amitriptyline has been reported to suppress neuroinflammation [66]. Furthermore, antidepressants have anti-inflammatory effects by considerably decreasing the production of nitric oxide (NO) and umor necrosis factor-alpha (TNFα) in microglia and astrocyte cultures at mrna levels [65]. They can inhibit the degradation of IκB, the nuclear translocation of the p65 subunit of NF-κB. Therefore, NF-κB cannot transmigrate into the nucleus to bind with DNA to promote the expression of gene regions [66]. Antidepressants can also inhibit the phosphorylation of p38 mitogen-activated protein kinase in lipopolysaccharide-stimulated microglia cells [65]. This phosphorylation can induce the associated inflammatory gene expression to produce the proinflammatory cytokines and NO, which may be attenuated or inhibited by antidepressants [66]. NO can also induce ROS; therefore, it can increase intestinal damage and, with cytokine production, prolong the development of IBD. Based on these studies, the NF-κB pathway has been considered to play an important role in the inflammatory process. Therefore, investigators have hypothesized that the antidepressant-like effects of amitriptyline, via the modulation of this pathway, may be more effective for treating and suppressing the development of IBD through its anti-inflammatory actions [67]. Some authors have argued that antidepressants may, in fact, cause tolerance and present problems when tapering off medications [68,69]. Significant numbers of patients no longer need an antidepressant for their mental health problems, yet they suffer unbearable withdrawal effects when discontinuing the medication and thus remain on the treatment [58]. Patients participating in a study by Mikocka-Walus et al [58] (2012) reported antidepressants to be a medication worth recommending to fellow IBD sufferers as long as the decision for their use was taken into consideration. Although studies exploring attitudes toward antidepressant use in larger samples or in samples recruited in primary care (and thus with possibly bettercontrolled IBD) are not available, studies conducted in the general population in primary care have shown a less receptive attitude toward antidepressants. For example, a survey of 1054 primary care users showed that over 20% of individuals did not disclose depressive symptoms to their doctors due to fear that antidepressants would be prescribed [70]. Other studies have reported non-adherence to treatment with antidepressants due to patient beliefs or misconceptions about this type of medication [71,72]. In light of these findings, the positive attitudes toward antidepressants identified in previous studies should be interpreted with caution and confirmed by larger quantitative studies with more representative IBD samples. A systematic review of SSRIs indicated that although the medications were similar in efficacy, there were meaningful differences in their side effect profiles. These differences may guide decisions concerning the best choice for a particular patient [73]. Nevertheless, studies report serious adverse effects of selective serotonin reuptake drugs. Gastrointestinal side effects can be of particular concern to the IBD patient and have been reported for many antidepressant medications. These side effects are generally dose related and tend to decrease over the first weeks of treatment [74]. Nausea and vomiting are more frequent with the one SNRI evaluated (venlafaxine) compared to the SSRIs as a group (fluoxetine, fluvoxamine, sertraline, paroxetine, citalopram; 34% vs 22%). Diarrhea is reported more often with sertraline than with the other SSRIs. Other side effects that have been cited as problematic when patients decide to discontinue antidepressant medication early in the course of treatment include drowsiness/fatigue (10%), anxiety (6%), headache (6%), insomnia (2.7%), and dizziness (2.7%) [75]. Weight gain has been found to be a more significant problem with paroxetine and mirtazapine [74], but it remains a concern with all SSRI medications [76]. In some cases, there may be weight loss early in treatment and weight gain later [74]. Decreased sexual functioning is a relatively common dose-related side effect of antidepressant medications and may be a concern for IBD patients, given the disease-related difficulties with intimacy and sexual functioning [77]. It has been found that 60% to 70% of patients report reduced sexual functioning on SSRIs or SNRIs that does not improve with longer periods on the medication. Buproprion has the lowest rates of sexual dysfunction relative to other antidepressants [76], and it is usually recommended as a substitute for SSRIs in case of side effects. Kast et al [78] (2001) reported two patients who achieved long-lasting remission of Crohn s disease while using bupropion. These investigators hypothesized that this outcome may have resulted from decreased TNFα, which is known to play a vital role in Crohn s disease. Phenelzine and bupropion increase intracellular cyclic adenosine mono phosphate [79], which, in turn, decreases TNFα. Because phenelzine may cause a hypertensive crisis, bupropion is suggested to be a safer therapeutic option than phenelzine. Interestingly, phenelzine and other monoamine oxidase inhibitors have been noted to induce remission of rheumatoid arthritis, a disease in which, as in Crohn s disease, TNFα has a central role [80]. Kast [81] (2003) compared the use of bupropion and mirtazapine in patients with Crohn s disease. He speculated that both of these antidepressants have the potential to affect inflammatory responses: bupropion by lowering TNFα and mirtazapine by increasing its level. Therefore, according to the hypothesis of Kast [81] (2003), there are theoretical reasons for recommending bupropion and cautioning against mirtazapine when treating depression in patients with Crohn s disease. Although Kast s explanations appear logical and are supported by other investigators [82], their practical effectiveness needs to be experimentally confirmed in appropriate clinical studies [55]. With regard to the risk of severe side effects, recent reports have raised the possibility of a greater risk of upper gastrointestinal (GI) bleeds with the use of certain antidepressants [83-85]. Large-scale studies have found a moderately increased risk of GI bleeds with SSRIs as well as with 3556 April 7, 2014 Volume 20 Issue 13

182 Filipovic BR et al. Psychiatric discomfort and IBD treatment the SNRI venlafaxine [86-88]. The use of acid-suppressing agents mitigated the higher risk, whereas the use of nonsteroidal anti-inflammatory drugs (NSAIDs) increased the risk [86]. The absolute risk of taking SSRIs was low, however; 2000 patients per year would need to be treated with SSRIs for one case of upper GI tract bleeding to be attributed to such drugs. The risk is higher when SSRIs and NSAIDs are taken together, with one patient in 250 experiencing a GI bleed that could be attributed to that combination [86]. PSYCHOTHERAPEUTIC APPROACH The first study regarding the effectiveness of psychotherapy for ulcerative colitis was conducted half a century ago [89], but it was methodologically problematic. The effect of psychodynamic psychotherapy on patients with Crohn s disease was investigated in a randomized, multicenter study [90]. The psychotherapeutic intervention consisted of psychodynamic psychotherapy (26 sessions) and autogenic training (17 sessions). After 2 years, relapse was not experienced by 23% of the control group and 30% of the therapy group. Twenty-nine percent of the control group and 17% of the therapy group had to undergo surgery. The therapy group had better somatic data than the control group, but this was not significant. Nevertheless, a meta-analysis was necessary for this review article given that the authors analyzed a respectable number of investigations addressing the subject of interest, such as the use of psychotherapy in IBD patients [91,92]. Twentyone studies were included in the analysis. Four studies did not report results in detail and could not be included in any of the pooled analyses. These 4 studies included a controlled trial on hypnosis in ulcerative colitis [93], a trial on the effects of a multicomponent behavioral therapy package in 22 patients with IBD [94], and a trial examining the effects of support meetings [95]. Given its chronic nature and frequently reported poor quality of life for many patients, IBD is often associated with anxiety and depression. In addition to medical treatment, psychological intervention may be a crucial component of the treatment of IBD patients [96,97]. Prior research has found that the disease course is gender dependent and that females may have a higher risk of disease activity relapse than males [22,98]. Therefore, females may have more psychological symptoms. Furthermore, females more frequently become ill from Crohn s disease. Second, patients are stratified by treatment center. A distinction must be made between an academic setting and a peripheral setting. We expect that an academic hospital would treat more severe cases of IBD than a peripheral setting. Finally, the disease type is also used as a stratification factor. Previous research has found that CD patients undergo more surgical interventions and experience more disease exacerbations than ulcerative colitis patients [99]. This finding indicates that CD is a more complex disorder than UC. In addition, CD patients report poorer quality of life and more anxiety symptoms than UC patients [99,100]. Therefore, it is important that UC and CD patients are distributed evenly in the experimental group and the waiting list control group [101]. Of all psychotherapeutic interventions, cognitive behavioral therapy appears to be the most effective [102]. This therapy posits that an individual s biased information processing leads to restrictive thoughts, feelings, and behaviors that can culminate in anxiety and depressive symptoms and, eventually, in psychiatric disorders. Cognitive behavioral therapy offers a well-developed intervention protocol that has been found to enhance quality of life and to decrease psychological distress. Its positive effect has been emphasized in individuals with other chronic somatic illnesses, such as chronic obstructive pulmonary disease, diabetes, and cancer [103,104]. Cognitive behavior therapy was evaluated in an open trial of adolescents with major depression [105,106] and a randomized controlled trial (RCT) of adolescents with subsyndromal depression [107]. In all studies, treatment significantly reduced depression and improved global functioning. For individuals with a comorbid anxiety disorder, there was also a significant reduction in anxiety. The open trial did not find any change in illness severity posttreatment. The RCT reported a decrease in the number of individuals with moderate to severe disease posttreatment (29% pre-treatment vs 15% post-treatment), but the decrease was not significant. In an RCT of adults with IBD, a Spanish group reported clinically significant reductions in anxiety and depression following a structured cognitive-behavior therapy program that included components such as relaxation training, distraction, and cognitive restructuring [108]. Reviews considering the overall effectiveness of psychological therapies for IBD patients not selected for anxiety or depressive disorders have reached a more modest conclusion: there may be some clinical benefit related to psychological functioning, with little support at this point for a significant direct impact on disease parameters [ ]. These studies incorporated a broad range of treatments (e.g., psychodynamic therapy, supportive therapy, and cognitive behavioral therapies), some of which are not as well supported empirically. Further, the studies often involved unselected IBD patients or patients in remission with little elevated distress [112,113], resulting in the potential for floor effects. Thus, unsurprisingly, psychological treatment is not indicated for all patients with IBD [114]. The results of well-conducted studies imply that validated treatments should be targeted to high-risk subgroups, such as individuals with comorbid psychiatric conditions or elevated stress [110,111]. Certainly, IBD patients may be quite susceptible to psychological treatment. Among individuals reporting high distress, there is a strong level of interest in receiving support for these concerns [115]. A structured measure of desire for psychological care comparing patients with IBD and rheumatoid arthritis found that 2 to 3 times the number of IBD patients (31%) expressed an interest in receiving assistance compared to individuals with rheumatoid arthritis (13%) [116]. Other indicators of receptivity included positive evaluations of treatment [113] and low dropout rates despite the expectation of active participation [104,106] April 7, 2014 Volume 20 Issue 13

183 Filipovic BR et al. Psychiatric discomfort and IBD treatment The appropriate choice of medication depends on many factors that are best tailored to the individual patient. Different galenic preparations are released at different sites and may have local activity [such as mesalazine (5-ASA) preparations, budesonide, or types of enemas]. The choice is influenced by the balance between drug potency and side effects, previous response to treatment (especially when considering treatment for a relapse or treatment for corticosteroid-dependent or corticosteroidrefractory disease), and the presence of extraintestinal manifestations (indicating the need for systemic therapy) or complications. Despite general agreement that treatment decisions for active Crohn s disease should be based on the site as well as the activity and behavior of the disease, the sample size is too small for statistically valid conclusions to be drawn from therapeutic trials when patients are stratified according to the site of disease [117]. For mildly active IBD, budesonide 9 mg/d is favored because it is superior to both placebo (OR = 2.85, 95%CI: ) [118,119] and 5-ASA 4 g/d (OR = 2.8, 95%CI: ) [120], and it achieves remission in 51%-60% of individuals over 8-10 wk [119, ]. Budesonide is preferred to prednisolone for mildly active CD because it is associated with fewer side effects, although a Cochrane systematic review has shown budesonide to be somewhat less effective than prednisolone (pooled OR for the five trials 0.69, 95%CI: ) [119]. For corticosteroid-related adverse effects, budesonide showed no difference from the placebo (OR = 0.98, 95%CI: ) [118,119] but had fewer side effects than prednisone (pooled OR = 0.38, 95%CI: ). When IBD is estimated as moderately active, budesonide or prednisolone are appropriate. Prednisolone is associated with a good clinical response (92% remission within seven weeks at a high dose of 1 mg/kg), but it commonly causes more side effects than budesonide [117,124,125]. The dose of prednisolone is adjusted to the therapeutic response over a period of weeks. More rapid reduction is associated with early relapse. The consensus does not favor sole nutritional therapy, antibiotics (unless septic complications are suspected), infliximab (IFX) (until more data are available), or surgery for moderately active ileal CD as the first line therapy [122]. Prednisolone or intravenous hydrocortisone is appropriate for the initial treatment of severe ileal CD. Azathioprine (AZA) (or mercaptopurine) should be added for individuals who have relapsed because it has a corticosteroid-sparing effect (NNT 3) and is effective at maintaining remission [126,127]. Methotrexate should be considered an appropriate alternative if thiopurines cannot be tolerated, but it has specific contraindications, such as pregnancy. IFX is best reserved for patients who do not respond to initial therapy and for whom surgery is considered inappropriate. This does not mean that surgery takes precedence over IFX. IFX has emerged as a conservative option for cases with severe inflammatory activity, and it is in these cases that primary surgery will often be inappropriate. Surgical options should, however, be considered and discussed with the patient as part of an overall management strategy. The stage at which IFX is introduced may change if it can be established whether early therapy changes the pattern of disease. The threshold for surgery for localized ileocecal disease is lower than for disease elsewhere, and some experts advocate surgery over IFX for disease in this location. Other experts advocate resection if medical therapy is not effective within two to six weeks. It may sometimes be difficult to distinguish between active disease and a septic complication, but antibiotics should be reserved for patients with a fever or focal tenderness or in whom imaging has indicated an abscess. Adding ciprofloxacin and metronidazole to budesonide has been shown to have no advantage over budesonide alone in active CD [127,128]. BIOLOGICAL THERAPY Regular infusions of IFX 5 or 10 mg/kg every eight weeks are effective at maintaining an IFX-induced response in nonfistulating CD (EL1b). Patients in a scheduled treatment strategy with regular infusions of IFX seem to fare better in many (but not all) clinical end points compared with patients in an episodic (on-demand) strategy [129,130]. CONCLUSION The psychopathological impact in IBD patients is evident, and the most common symptoms of depression and anxiety may affect the success of the desired gastroenterological therapy. Personality traits, cognitive impairment, and sleep deprivation are the tip of the iceberg of psychological problems in patients with IBD. The worsening of psychological problems is often followed with the same trend in GI symptoms of IBD and vice versa; that is, relapse of IBD symptomatology, such as blood in the stool, bloating, and pain, may increase psychological problems. This vicious cycle could be broken by involving a trained psychiatrist in the IBD treatment team. Nevertheless, some measures of precaution should be taken, such as avoiding bias in the group selection for both psychotropic medication and psychotherapy and an awareness of the possible side effects of antidepressant therapy, especially SSRIs, such as more frequent liquid stools, addiction, or sexual dysfunction problems. Finally, we suggest that psychiatrists and gastroenterologists work together to determine the final consensus of the IBD therapy to ensure success and to reduce side effects and relapse to the lowest possible rates. REFERENCES 1 Bonaz B. Inflammatory bowel diseases: a dysfunction of brain-gut interactions? Minerva Gastroenterol Dietol 2013; 59: [PMID: ] 2 Bonaz BL, Bernstein CN. Brain-gut interactions in inflammatory bowel disease. Gastroenterology 2013; 144: [PMID: DOI: /j.gastro ] 3 Agostini A, Benuzzi F, Filippini N, Bertani A, Scarcelli A, Farinelli V, Marchetta C, Calabrese C, Rizzello F, Gionchetti 3558 April 7, 2014 Volume 20 Issue 13

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187 Filipovic BR et al. Psychiatric discomfort and IBD treatment terol 2007; 102: [PMID: DOI: / j x] 99 Larsson K, Lööf L, Rönnblom A, Nordin K. Quality of life for patients with exacerbation in inflammatory bowel disease and how they cope with disease activity. J Psychosom Res 2008; 64: [PMID: DOI: /j.jpsych ores ] 100 Jäghult S, Saboonchi F, Johansson UB, Wredling R, Kapraali M. Identifying predictors of low health-related quality of life among patients with inflammatory bowel disease: comparison between Crohn s disease and ulcerative colitis with disease duration. J Clin Nurs 2011; 20: [PMID: DOI: /j x] 101 Bennebroek Evertsz F, Thijssens NA, Stokkers PC, Grootenhuis MA, Bockting CL, Nieuwkerk PT, Sprangers MA. Do Inflammatory Bowel Disease patients with anxiety and depressive symptoms receive the care they need? J Crohns Colitis 2012; 6: [PMID: DOI: / j.crohns ] 102 Beck AT. The current state of cognitive therapy: a 40-year retrospective. Arch Gen Psychiatry 2005; 62: [PMID: DOI: /archpsyc ] 103 Osborn RL, Demoncada AC, Feuerstein M. Psychosocial interventions for depression, anxiety, and quality of life in cancer survivors: meta-analyses. Int J Psychiatry Med 2006; 36: [PMID: ] 104 Snoek FJ, Skinner TC. Psychological counselling in problematic diabetes: does it help? Diabet Med 2002; 19: [PMID: DOI: /j x] 105 Szigethy E, Carpenter J, Baum E, Kenney E, Baptista-Neto L, Beardslee WR, Demaso DR. Case study: longitudinal treatment of adolescents with depression and inflammatory bowel disease. J Am Acad Child Adolesc Psychiatry 2006; 45: [PMID: DOI: /01.chi a4] 106 Szigethy E, Whitton SW, Levy-Warren A, DeMaso DR, Weisz J, Beardslee WR. Cognitive-behavioral therapy for depression in adolescents with inflammatory bowel disease: a pilot study. J Am Acad Child Adolesc Psychiatry 2004; 43: [PMID: ] 107 Szigethy E, Kenney E, Carpenter J, Hardy DM, Fairclough D, Bousvaros A, Keljo D, Weisz J, Beardslee WR, Noll R, De- Maso DR. 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Effects of a comprehensive lifestyle modification program on quality-of-life in patients with ulcerative colitis: a twelve-month follow-up. Scand J Gastroenterol 2007; 42: [PMID: DOI: / ] 113 Oxelmark L, Magnusson A, Löfberg R, Hillerås P. Groupbased intervention program in inflammatory bowel disease patients: effects on quality of life. Inflamm Bowel Dis 2007; 13: [PMID: ] 114 Deter HC, Keller W, von Wietersheim J, Jantschek G, Duchmann R, Zeitz M. Psychological treatment may reduce the need for healthcare in patients with Crohn s disease. Inflamm Bowel Dis 2007; 13: [PMID: DOI: / ibd.20068] 115 Kunzendorf S, Jantschek G, Straubinger K, Heberlein I, Homann N, Ludwig D, Benninghoven D. The Luebeck interview for psychosocial screening in patients with inflammatory bowel disease. Inflamm Bowel Dis 2007; 13: [PMID: DOI: /ibd.20050] 116 Miehsler W, Weichselberger M, Offerlbauer-Ernst A, Dejaco C, Reinisch W, Vogelsang H, Machold K, Stamm T, Gangl A, Moser G. Which patients with IBD need psychological interventions? A controlled study. Inflamm Bowel Dis 2008; 14: [PMID: ] 117 Travis SP, Stange EF, Lémann M, Oresland T, Chowers Y, Forbes A, D Haens G, Kitis G, Cortot A, Prantera C, Marteau P, Colombel JF, Gionchetti P, Bouhnik Y, Tiret E, Kroesen J, Starlinger M, Mortensen NJ. European evidence based consensus on the diagnosis and management of Crohn s disease: current management. Gut 2006; 55 Suppl 1: i16-i35 [PMID: DOI: /gut b] 118 Greenberg GR, Feagan BG, Martin F, Sutherland LR, Thomson AB, Williams CN, Nilsson LG, Persson T. Oral budesonide for active Crohn s disease. Canadian Inflammatory Bowel Disease Study Group. N Engl J Med 1994; 331: [PMID: DOI: /NEJM ] 119 Otley A, Steinhart AH. Budesonide for induction of remission in Crohn s disease. Cochrane Database Syst Rev 2005; (4): CD [PMID: DOI: / CD pub2] 120 Thomsen OO, Cortot A, Jewell D, Wright JP, Winter T, Veloso FT, Vatn M, Persson T, Pettersson E. A comparison of budesonide and mesalamine for active Crohn s disease. International Budesonide-Mesalamine Study Group. N Engl J Med 1998; 339: [PMID: ] 121 Campieri M, Ferguson A, Doe W, Persson T, Nilsson LG. Oral budesonide is as effective as oral prednisolone in active Crohn s disease. The Global Budesonide Study Group. Gut 1997; 41: [PMID: ] 122 Rutgeerts P, Löfberg R, Malchow H, Lamers C, Olaison G, Jewell D, Danielsson A, Goebell H, Thomsen OO, Lorenz- Meyer H. A comparison of budesonide with prednisolone for active Crohn s disease. N Engl J Med 1994; 331: [PMID: DOI: /NEJM ] 123 Bar-Meir S, Chowers Y, Lavy A, Abramovitch D, Sternberg A, Leichtmann G, Reshef R, Odes S, Moshkovitz M, Bruck R, Eliakim R, Maoz E, Mittmann U. Budesonide versus prednisone in the treatment of active Crohn s disease. The Israeli Budesonide Study Group. Gastroenterology 1998; 115: [PMID: ] 124 Modigliani R, Mary JY, Simon JF, Cortot A, Soule JC, Gendre JP, Rene E. Clinical, biological, and endoscopic picture of attacks of Crohn s disease. Evolution on prednisolone. Groupe d Etude Thérapeutique des Affections Inflammatoires Digestives. Gastroenterology 1990; 98: [PMID: ] 125 Gross V, Andus T, Caesar I, Bischoff SC, Lochs H, Tromm A, Schulz HJ, Bär U, Weber A, Gierend M, Ewe K, Schölmerich J. Oral ph-modified release budesonide versus 6-methylprednisolone in active Crohn s disease. German/Austrian Budesonide Study Group. Eur J Gastroenterol Hepatol 1996; 8: [PMID: ] 126 Pearson DC, May GR, Fick G, Sutherland LR. Azathioprine for maintaining remission of Crohn s disease. Cochrane Database Syst Rev 2000; (2): CD [PMID: DOI: / CD000067] 127 Steinhart AH, Feagan BG, Wong CJ, Vandervoort M, Mikolainis S, Croitoru K, Seidman E, Leddin DJ, Bitton A, Drouin E, Cohen A, Greenberg GR. Combined budesonide and 3562 April 7, 2014 Volume 20 Issue 13

188 Filipovic BR et al. Psychiatric discomfort and IBD treatment antibiotic therapy for active Crohn s disease: a randomized controlled trial. Gastroenterology 2002; 123: [PMID: DOI: /gast ] 128 Fraser AG. Methotrexate: first-line or second-line immunomodulator? Eur J Gastroenterol Hepatol 2003; 15: [PMID: ] 129 Rutgeerts P, D Haens G, Targan S, Vasiliauskas E, Hanauer SB, Present DH, Mayer L, Van Hogezand RA, Braakman T, DeWoody KL, Schaible TF, Van Deventer SJ. Efficacy and safety of retreatment with anti-tumor necrosis factor antibody (infliximab) to maintain remission in Crohn s disease. Gastroenterology 1999; 117: [PMID: ] 130 Rutgeerts P, Feagan BG, Lichtenstein GR, Mayer LF, Schreiber S, Colombel JF, Rachmilewitz D, Wolf DC, Olson A, Bao W, Hanauer SB. Comparison of scheduled and episodic treatment strategies of infliximab in Crohn s disease. Gastroenterology 2004; 126: [PMID: DOI: / j.gastro ] P- Reviewers: Ho RCM, Tcheremissine OV S- Editor: Wen LL L- Editor: A E- Editor: Wu HL 3563 April 7, 2014 Volume 20 Issue 13

189 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (3): Inflammatory bowel disease Colon-specific prodrugs of 4-aminosalicylic acid for inflammatory bowel disease TOPIC HIGHLIGHT Suneela S Dhaneshwar Suneela S Dhaneshwar, Department of Pharmaceutical Chemistry, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Pune , Maharashtra, India Author contributions: Dhaneshwar SS has conceptualized, drafted and compiled this comprehensive review through extensive survey of the available literature and databases, adding a new perspective based on her expertise in this area. Correspondence to: Suneela S Dhaneshwar, M. Pharm, PhD, Professor of Pharmaceutical Chemistry, Department of Pharmaceutical Chemistry, Poona College of Pharmacy, Bharati Vidyapeeth Deemed University, Bharati Vidyapeeth Campus, Pune , Maharashtra, India. suneeladhaneshwar@rediffmail.com Telephone: Fax: Received: October 31, 2013 Revised: December 17, 2013 Accepted: January 8, 2014 Published online: April 7, 2014 Abstract Despite the advent of biological products, such as antitumor necrosis factor-α monoclonal antibodies (infliximab and adalimumab), for treatment of moderate to severe cases of inflammatory bowel disease (IBD), most patients depend upon aminosalicylates as the conventional treatment option. In recent years, the increased knowledge of complex pathophysiological processes underlying IBD has resulted in development of a number of newer pharmaceutical agents like low-molecular-weight heparin, omega-3 fatty acids, probiotics and innovative formulations such as high-dose, oncedaily multi-matrix mesalamine, which are designed to minimize the inflammatory process through inhibition of different targets. Optimization of delivery of existing drugs to the colon using the prodrug approach is another attractive alternative that has been utilized and commercialized for 5-aminosalicylic acid (ASA) in the form of sulfasalazine, balsalazide, olsalazine and ipsalazine, but rarely for its positional isomer 4-ASA - a wellestablished antitubercular drug that is twice as potent as 5-ASA against IBD, and more specifically, ulcerative colitis. The present review focuses on the complete profile of 4-ASA and its advantages over 5-ASA and colon-targeting prodrugs reported so far for the management of IBD. The review also emphasizes the need for reappraisal of this promising but unexplored entity as a potential treatment option for IBD Baishideng Publishing Group Co., Limited. All rights reserved. Key words: 4-Aminosalicylic acid; 5-Aminosalicylic acid; Sulfasalazine; Colon-specific prodrug; Inflammatory bowel disease; Ulcerative colitis; 2,4,6-trinitrobenzene sulphonic acid; Experimental colitis Core tip: Anti-inflammatory activity of antitubercular drug 4-aminosalicylic acid (ASA) was first described by Lover in Since then, numerous clinical trials were carried out to establish its efficacy in left-sided and active/quiescent ulcerative colitis. It is 50% more potent than 5-ASA against inflammation and does not produce 5-ASA-induced immunoallergic acute pancreatitis. 4-ASA is a stable and inexpensive alternative to 5-ASA in patients with acute pancreatitis. Despite all these positive findings, an extensive literature review surprisingly revealed few colon-targeting delivery systems for 4-ASA. The present review presents a complete profile of 4-ASA and its colon-specific prodrugs for inflammatory bowel disease. Dhaneshwar SS. Colon-specific prodrugs of 4-aminosalicylic acid for inflammatory bowel disease. World J Gastroenterol 2014; 20(13): Available from: URL: DOI: org/ /wjg.v20.i INTRODUCTION Anti-inflammatory activity of antitubercular drug 4-ami April 7, 2014 Volume 20 Issue 13

190 Dhaneshwar SS. Colon-targeting 4-ASA prodrugs for IBD nosalicylic acid (ASA) was first described by Lover in Since then, numerous clinical trials were carried out to establish its efficacy in left-sided and active/quiescent ulcerative colitis. It is 50% more potent than 5-aminosalicylic acid (5-ASA) against inflammation and does not produce 5-ASA-induced immunoallergic acute pancreatitis. 4-ASA is a stable and inexpensive alternative to 5-ASA in patients with acute pancreatitis. Despite all these positive findings, an extensive literature review surprisingly revealed few colon-targeting delivery systems for 4-ASA. The present review presents a complete profile of 4-ASA and its colon-specific prodrugs for inflammatory bowel disease (IBD). IBD Ulcerative colitis (UC) and Crohn s disease (CD) are two distinct idiopathic, chronic and relapsing inflammatory disorders of the gastrointestinal tract (GIT) that are grouped under the term IBD. Dysregulation of immune responses and upregulation of proinflammatory mediators are the two hallmarks of IBD that make it recurrent and nearly incurable [1]. UC is characterized by distinct episodes of active and inactive disease in 80%-90% of patients. Repetitive cycles of active and quiescent disease with varying clinical patterns are also observed in CD [2]. UC is one of the most common chronic inflammatory forms of IBD and is characterized by diffused mucosal inflammation, mainly limited to the large intestine and rectum. UC is characterized by diarrhea, rectal bleeding and abdominal pain. Inflammation of the rectum is common and generally extends proximally in a continuous fashion [3]. CD is a patchy transmural granulomatous inflammation that affects any part of the GIT and has a predilection for the terminal ileum and colon [4]. Despite the differences between UC and CD, both forms of IBD cause similar symptoms. UC is characterized by mild symptoms like progressive loosening of the stools, abdominal cramping, and diarrhea. In the severe form of the disease, patients may also experience weight loss, fatigue, and loss of appetite that may result in nutrient deficiencies, mucus in the stools, severe rectal bleeding, fever, and anemia. In CD, abdominal pain, diarrhea and weight loss are often the earliest signs; constitutional symptoms include malaise, lethargy, anorexia, nausea, vomiting and low-grade fever. CD can be complicated by the development of intestinal obstruction, fistulae and perianal disease. Fistulae develop in about one-third of patients. Perianal disease is a frequent complication of colonic and ileocolonic disease and is characterized by fissures, fistulae and abscesses [5,6]. The pathogenesis of IBD is obscure and is considered to involve multifactorial interactions amongst genetic, immunological and environmental triggers. The key factor differentiating individuals with IBD from normal ones is their inability to downregulate the uncontrolled and chronic inflammatory state. The pathological findings associated with IBD are: an increase in certain inflammatory mediators, signs of oxidative stress, a deranged colonic milieu, abnormal glycosaminoglycan content of the mucosa, decreased oxidation of short chain fatty acids, increased intestinal permeability, increased sulfide production, and decreased methylation. Although no single factor has been identified as the initial trigger for IBD, the etiological factors have been elucidated [7]. However, no guaranteed curative therapeutic regimen has been developed so far. IBD THERAPY Therapy for IBD has not seen major changes or breakthroughs during the past 4-5 decades and still revolves around the single nonsteroidal anti-inflammatory drug 5-ASA (Figure 1A) and its colon-targeting prodrug sulfasalazine (Figure 1B), followed by second-line treatment with steroids and immunomodulators, because reducing the extent and severity of colonic inflammation remains the primary focus of treatment. Antibiotics, probiotics, and nutritional supplements are used as supportive therapy. The use of anti-tumor necrosis factor monoclonal antibody (infliximab), recombinant anti-inflammatory cytokines, and related gene therapy are recent advances in the field [8,9]. High and repeated doses of 5-ASA are necessary for maintenance and preventive therapy of IBD relapse. The majority of orally administered 5-ASA is readily and extensively absorbed from the stomach and small intestine, leaving a small amount that is transported to the colon. 5-ASA is absorbed from the upper GIT, causes many systemic side effects, and at the same time, results in poor bioavailability at the site of action, namely, the colon. Bypassing absorption in the upper GIT and targeting the delivery of 5-ASA to the colon by designing colonspecific prodrugs is the rational approach to improve the bioavailability at the site of action. The earliest reported colon-specific prodrug of 5-ASA is sulfasalazine, which is extensively prescribed for the treatment of UC. It is an azo conjugate of 5-ASA with sulfapyridine. However, its sulfapyridine part is used as a carrier and is completely absorbed through the colon and produces adverse effects such as hypersensitivity, impotency, blood dyscrasia, hepatitis, abnormal liver function tests and hepatic failure, agranulocytosis, leukopenia, thrombocytopenia, hemolytic anemia, and cyanosis [10-12]. Although 5-ASA derivatives have an excellent safety profile, some undesirable effects may occur; the most prominent being immunoallergic acute pancreatitis [13,14]. These side effects contraindicate the further use of all 5-ASA-containing drugs and pose a problem, because they limit treatment of acute phases of the disease to corticosteroids and relapse to immunosuppressants. 4-ASA has also been shown to be effective in IBD; in enemas or in oral formulations [15-18]. 4-ASA 4-ASA (p-aminosalicylic acid, 4-amino-2-hydroxybenzoic acid) (Figure 1C), is a comparatively safe antitubercular agent that has been used for many years in resorbable high oral doses (ranging from 8 to 12 g/d) in multidrugresistant tuberculosis. It has a bacteriostatic effect on 3565 April 7, 2014 Volume 20 Issue 13

191 Dhaneshwar SS. Colon-targeting 4-ASA prodrugs for IBD A HOOC B HOOC O N HO NH2 HO N N S NH O C D E COOH OH CONHCH2COOH OH COOH OH COOH NH H2N C CH2 3 NH C NH2 H2N H NH2 NH2 Wherein: -NH2 is either at position 4 or 5 F O C R1 G HO R O R2 HOOC N N OH NH R = -H, -COOH, -CONHCH2COOH R3 Where, R1, R2, R3 = H2S releasing moiety H I OH HOOC N N OH H2N CONH H C COONa CH2 HO R2 R1 Where, R1 = -H, R2 = -CH3; R2 = -NO2 R2 = -H, R1 = -CONH2; R1 = -Cl, R1 = -CH3; R1 = -NO2 G OH K OH O HO H2N CONH H C COONa C HN O CH2 NH2 HO CH2OH OH HN 3566 April 7, 2014 Volume 20 Issue 13

192 Dhaneshwar SS. Colon-targeting 4-ASA prodrugs for IBD L CH3 M COOH H2C C n HO CO O R = NH2 N N CH2 O 3 HO NH2 OH COOH CO R Figure 1 Chemical structures of aminosalicylates and their colon-targeting prodrugs. A: 5-ASA; B: Sulfasalazine; colon-targeting prodrug of 5-ASA with sulfapyridine; C: 4-ASA; D: Amide prodrug of 4-ASA with glycine; E: L-arginine salts of 4-ASA and 5-ASA; F: H2S-releasing prodrugs of 5-ASA and 4-ASA; G: Prodrugs of 4-ASA with salicylic acid, hydroxybenzene and N-salicyloyl glycine; H: Prodrugs of 4-ASA with substituted phenols; I: amide conjugate of 4-ASA with D-phenylalanine; J: Amide conjugate of 4-ASA with L-tryptophan; K: Amide prodrug of 4-ASA with D-glucosamine; L: Polymeric prodrug of 4-ASA; M: Prodrug of 4-ASA with 4-ASA (4A- 4AAz). Mycobacterium tuberculosis and inhibits resistance against streptomycin and isoniazid. Chemically, it is distinguished from its position isomer 5-ASA by the position of the NH2 group, although they share the salicylic acid backbone. Therefore, their method of synthesis is also different. 4-ASA is prepared by heating 3-aminophenol with potassium bicarbonate or ammonium carbonate under pressure, while 5-ASA is prepared by zinc/hydrochloric acid-assisted reduction of m-nitrosalicylic acid [19]. 5-ASA is unstable and is degraded rapidly into a dark purple quinone containing tar but does not decarboxylate in the presence of moisture. However, 4-ASA is readily decarboxylated in the presence of moisture, but unlike 5-ASA, is slowly degraded into brown to purple material [20,21]. In view of the well-recognized chemical distinction between 4-ASA and 5-ASA, it is surprising to discover that not only does 4-ASA have anti-inflammatory activity, but it is also about 50% more potent than 5-ASA. Lover reported in animal models that 4-ASA may have anti-inflammatory properties comparable with those of 5-ASA [22]. Numerous clinical studies have shown that 4-ASA is highly effective in the topical treatment of active ulcerative proctitis or active left-sided UC [23]. Moreover, slow release tablets of 4-ASA are effective in active UC [17]. 4-ASA has been suggested as an effective treatment for both active and quiescent UC. 5-ASA is well established for maintenance treatment of inactive UC. Moreover, recent studies suggest that 5-ASA may also be effective in maintaining remission in Crohn s colitis. The efficacy of 1 year maintenance treatment with oral 4-ASA (1.5 g/d, slow release tablets, n = 19) and oral 5-ASA (1.5 g/d, slow release tablets, n = 21) was compared in a randomized double-blind trial in patients with quiescent Crohn s ileocolitis. That study concluded that 4-ASA may be as effective as 5-ASA for maintenance treatment of quiescent CD and there were no differences in the severity of relapse between both treatment groups [18]. In contrast to 5-ASA, no nephrotoxicity related to 4-ASA has been reported. The only other study that compared these two compounds examined the response to topical treatment in left-sided colitis and showed that 4-ASA and 5-ASA were equally effective [24]. Mechanism of action of 4-ASA The anti-inflammatory mechanism by which the aminosalicylates exert their therapeutic action is not well established but several hypotheses have been documented. 5-ASA is a potent inhibitor of arachidonic acid metabolism, decreasing the synthesis of both leukotrienes and prostaglandins. Moreover, 5-ASA is a potent scavenger of free radicals [25-27]. In contrast, 4-ASA does not seem to have an inhibitory effect on the lipoxygenation of arachidonic acid and is ineffective as a radical scavenger, but it is thought to act via nuclear factor (NF)-κB inhibition [28]. Both drugs, however, inhibit in vitro activation of T and B lymphocytes by pokeweed mitogen in a dose-dependent manner [29,30]. These findings suggest that the use of either drug in IBD may decrease the heightened state of lamina propria lymphocyte activation as a part of their therapeutic action [31]. Moreover, mechanisms not affecting arachidonic acid metabolism and superoxide release may contribute to the therapeutic potential of both drugs in IBD [32]. The most common side effects of 4-ASA include nausea, vomiting, and epigastric pain. Less frequent side effects are fever, joint pains, skin eruptions, and hepatitis; all of which may be attributed to hypersensitivity reactions [33]. More serious side effects are seen only rarely, such as leukopenia, thrombocytopenia, and agranulocytosis [33]. Because of the lower incidence of serious side effects, it was thought that delayed release formulation of 4-ASA at higher doses could be used in IBD with higher therapeutic efficacy than seen in previous studies. Schreiber et al [18] concluded that oral 4-ASA may be as effective as 5-ASA for maintenance treatment of remission in CD. Several other studies have suggested that 3567 April 7, 2014 Volume 20 Issue 13

193 Dhaneshwar SS. Colon-targeting 4-ASA prodrugs for IBD 4-ASA is effective in the treatment of active CD or UC; both as a topical formulation (enema) or as an oral slowrelease tablet [18]. The risk of cross intolerance reaction between 5-ASA and 4-ASA was evaluated by Gisbert et al [34]. They reported three cases of 5-ASA-induced pancreatitis, with no recurrence of pancreatitis during subsequent treatment with 4-ASA enemas. They concluded that 4-ASA enemas are a safe and well-tolerated therapeutic alternative whenever 5-ASA-induced pancreatitis occurs. 4-ASA has been used in the treatment of mild to moderate UC in patients intolerant of sulfasalazine, and in the treatment of CD [35]. It has been designated an orphan drug by the United States FDA for use in these conditions [36]. Pharmacokinetics of 4-ASA 4-ASA is readily absorbed from the GIT. It is distributed into various tissues and fluids including peritoneal fluid, pleural fluid and synovial fluid in concentrations approximately equal to plasma concentrations of the drug. Cerebrospinal fluid concentrations of 4-ASA are reported to be 10%-50% of concurrent plasma concentrations of the drug in patients with inflamed meninges. 4-ASA is 50%-73% bound to plasma proteins. The plasma halflife of 4-ASA is about 1 h. Plasma concentrations of the drug are not substantially affected by renal or hepatic insufficiency; however, the half-lives of the inactive metabolites may be prolonged in patients with impaired renal function. 4-ASA is inactivated in the intestinal mucosa and liver primarily by acetylation. The major metabolites are N-acetyl-p-aminosalicylic acid and p-aminosalicyluric acid. 4-ASA and its metabolites are excreted in urine by glomerular filtration and tubular secretion. After 2 h in simulated gastric acid, 10% of the dose of unprotected (nonenteric coated) aminosalicylic acid is decarboxylated to form m-aminophenol, a known hepatotoxin [37]. Dose of 4-ASA 4-ASA is about 50% more potent than 5-ASA, therefore, its therapeutic dose is about 60% of the effective dose of sulfasalazine or 5-ASA on a molar basis. The recommended daily dosage of sulfasalazine is 3-4 g (6-8 tablets) for adults and mg/kg/d (divided into 3-4 doses) for children (aged > 6 years). On this basis, a dosage regimen of g of 4-ASA divided into two or three doses can be used [38]. Contraindications of 4-ASA 4-ASA is contraindicated in patients who are hypersensitive to ASA or who have severe renal diseases, hepatic disease or gastric ulcers [39]. Interactions of 4-ASA 4-ASA impairs GI absorption of rifampin, reduces rate of acetylation of isoniazid, decreases the GI absorption of digoxin, and enhances the hypoprothrombinemic effect of oral anticoagulants. Concomitant probenecid increases the serum concentration of 4-ASA, ammonium chloride increases probability of crystalluria during therapy, and diphenhydramine impairs its absorption. Pregnancy 4-ASA is a FDA pregnancy category C drug. Adverse effects of 4-ASA The most frequent adverse effect of 4-ASA is GI disturbance including nausea, vomiting, abdominal pain and diarrhea. The acidic carboxylic group of 4-ASA is responsible for the gastrointestinal (GI) irritation. Occurrence of peptic ulcers and gastric hemorrhage is rare. Adverse GI effect can be minimized in patients by administration of the drug with food or discontinuation of the drug when symptoms are severe. Malabsorption of vitamin B12, folic acid, iron and lipids is also observed. Reported hypersensitive reactions include fever, skin eruptions of various types, pruritis, vasculitis, exfoliative dermatitis, joint pain, eosinophilia, leucopenia, agranulocytosis and thrombocytopenia [39,40]. COLON-SPECIFIC PRODRUGS OF 4-ASA Extensive clinical studies have shown the effectiveness and safety of 4-ASA for the topical treatment of active ulcerative proctitis or left-sided UC [23]. It presents many advantages over 5-ASA, such as: more stability, more potency, and absence of incidences of pancreatitis, but shares the same drawback of fast and extensive absorption in the upper GIT, before it reaches the colon. This is due to its weakly acidic nature (pka 3-4), which results in its ready absorption in the upper GIT [16]. Studies by Selby suggest that 4-ASA is a stable, inexpensive alternative to 5-ASA for the topical treatment of UC or for linking to carrier molecules for release in the colon [24]. Many colon-specific prodrugs of 5-ASA have been cited in the literature, while the prodrug approach has been only sporadically applied for colonic delivery of 4-ASA, for reasons that are not known. The possible reason could be the steric problem of 4-ASA, due to which it cannot be linked with same polymers (e.g., dextran, chitin, hydroxymethyl cellulose or synthetic polymers) as 5-ASA or to other polymers (e.g., cyclodextrins) using the same methods that have been used for 5-ASA [20]. Zhao et al [41] were the first to design a colon-targeting amide prodrug of 4-ASA (Figure 1D) with the nonessential amino acid glycine for the treatment of IBD. The amide prodrug was synthesized by reacting amino and hydroxyl protected 4-ASA acyl chloride with glycine, followed by deprotection. In vivo experiments on rats suggested that 4-aminosalicylglycine showed more curative effect than 4-ASA. Wallace et al [42] have patented L-arginine salts of 4-ASA and 5-ASA (Figure 1E) for inflammatory conditions of the GIT, and for irritable bowel syndrome (IBS). They claim that the salt is more effective in reducing the inflammation in the colon compared to individual drugs or L-arginine, as demonstrated from overall greater reduction in the colitis-associated edema, granulocyte infiltration, and body weight loss with enhanced free-radical scavenging and more potent antioxidant activity, thus 3568 April 7, 2014 Volume 20 Issue 13

194 Dhaneshwar SS. Colon-targeting 4-ASA prodrugs for IBD providing a synergistic effect. They ascribe the synergistic effect to the ability of arginine to act as a source of nitrogen for NO, which is a potent vasodilator that attenuates intestinal tissue damage [43,44]. A US Patent describes 5-ASA and 4-ASA prodrugs as having an H2S-releasing moiety linked via an azo, ester, anhydride, thioester or amide linkage (Figure 1F) for the treatment of IBD and IBS and chemoprevention of colon cancer [45]. The basis for using H2S-releasing carriers in this design is their encouraging and beneficial pharmacological profile. There is documented evidence for antinociceptive effect of H2S in the GIT by activating KATP channels and smooth muscle relaxant activity in intestinal tissue [46]. Based on the design of sulfasalazine and using azo bond chemistry, Zhao et al [47] have reported the synthesis of prodrugs of 4-ASA with salicylic acid, hydroxybenzene and N-salicyloyl glycine (Figure 1G), aiming to target 4-ASA to the colon for management of IBD with enhanced efficacy and fewer side effects. However, we could not find any reports of activity of these prodrugs in any of the experimental models of IBD. Oxidative stress is one of the key players in the pathogenesis of IBD causing cellular injury and colonic inflammation [48]. Keeping this in mind, Sheng et al [49] designed azo-linked colon-specific prodrugs of 4-ASA (Figure 1H) with several substituted phenols such as m-nitrophenol, salicylamide, o-chlorophenol, m-methylphenol, o-methylphenol, o-nitrophenol and o-dihydroxybenzene, possessing antioxidant properties. Their main aim was to achieve co-antioxidant effects of 4-ASA and substituted phenol that would result in higher reducibility and synergistic free-radical scavenging. These phenols were chosen on the basis of their reducibility, electronic and steric factors. 4-(3,4 -dihydroxyphenyl) azobenzene-2-acetylsalicylic acid, 4-(2 -methyl-4 -hydroxyphenyl) azobenzene salicylic acid and 4-(3 -methyl-4 -hydroxyphenyl) azobenzene salicylic acid were reported to be the compounds with maximum anti-inflammatory and antioxidant activities. Dhaneshwar et al [12] have explored mutual amide prodrug strategy for 5-ASA with several amino acids. Applying the same concept further to 4-ASA, they have reported its amide conjugates with D-phenylalanine (Figure 1I) and L-tryptophan (Figure 1G). The proposed mechanism of activation was enzymatic by the action of N-acyl amidases secreted by colonic bacteria. The amino acids used in the design were nontoxic carriers like D-phenylalanine and L-tryptophan, possessing wound healing and anti-inflammatory activities. The prodrugs furnished 86%-91% release of 4-ASA in rat fecal matter in 20 h. Their curative effect was investigated in a 2,4,6-trinitrobenzene-sulfonic-acid-induced experimental colitis model in rats. Pancreas, liver and stomach of rats were studied by histopathology for the assessment of any adverse reactions. The prodrugs were found to possess significantly superior safety profile than sulfasalazine, oral 4-ASA and 5-ASA, with comparable efficacy to sulfasalazine. In an innovative attempt, colon-specific 4-ASA-Dglucosamine amide prodrug (Figure 1K) was developed by Dhaneshwar et al [50] out of the urgent need to search for such alternate options that would not produce 5-ASAinduced pancreatitis and sulfapyridine-induced hepatitis. The hypothesis was to offer supplementation of D-glucosamine that would suppress activation of intestinal epithelial cells, thus helping to maintain the integrity of the colonic architecture. The protective effect of this prodrug was compared with 5-ASA-D-glucosamine conjugate as well as standard drugs like sulfasalazine, 4-ASA and 5-ASA on the course of 2,4,6-trinitrobenzene sulphonic acid (TNBS)-induced colitis. The mitigating effect of 5-ASA prodrug was comparable to that of sulfasalazine, while that for 4-ASA prodrug was moderate. However, hepatitis or pancreatitis was not seen with 4-ASA prodrug [50]. A polymeric prodrug of 4-ASA (Figure 1L) with an acrylic polymeric system and degradable ester bonds, such as hydroxy propyl methacrylate, was synthesized and evaluated for colon-targeted drug delivery by Yadav et al [51]. In the first 2 h, 40.01% release was observed in rat fecal matter at ph 7.4, which was followed by sustained release of 93% over a period of 12 h. The authors propose that the developed delivery system could be useful for controlled release, prolonged transit time, and colon-targeted delivery of 4-ASA. A macromolecular colon-targeting ester prodrug of 4-ASA with β-cyclodextrin as a promoiety was reported very recently by Vadnerkar et al [52]. Interestingly, 20%-23% release of 4-ASA was observed in the stomach and small intestinal homogenates, while the prodrug was found to be resistant to ph-dependent hydrolysis at ph = 1.2 and 7.4. Almost 68% and 92% release was obtained in rat cecal and fecal matter, respectively. The prodrug demonstrated a moderate ameliorating effect on TNBSinduced colitis as compared to sulfasalazine or 4/5-ASA administered rectally, but it was similar to that with orally administered aminosalicylates. Partial activation of the prodrug in the upper GIT homogenates causing incomplete transport of 4-ASA to the site of action could be responsible for its moderate effect. However, the prodrug was reported to be safe, producing no harmful effects on the stomach, liver or pancreas of rats [52]. In another innovation, Suneela et al [53] designed four azo prodrugs of 4- and 5-ASA using the same aminosalicylates as carriers. As known already, olsalazine is a dimer of 5-ASA, which is synthesized by coupling diazo salt of 5-ASA with salicylic acid, which upon reduction by azo reductase, generates two molecules of 5-ASA. However, the azo prodrugs reported by Suneela et al [53] are different in the sense that they are prepared by coupling diazo salt of either 4-ASA or 5-ASA with 4-ASA or 5-ASA as carriers in all possible permutations and combinations. This results in prodrugs that, upon activation by azo reductase reduction, generate only one molecule of either 4-ASA or 5-ASA, while the other molecule released is a diaminosalicylic acid and not another molecule of aminosalicylate. These prodrugs were designed to target the colon affected with IBD. Due to their improved hydrophilic nature, the prodrugs had minimum absorption in the 3569 April 7, 2014 Volume 20 Issue 13

195 Dhaneshwar SS. Colon-targeting 4-ASA prodrugs for IBD upper GIT. The prodrugs were considerably stable when incubated in the upper GIT. Approximately 68%-91% release was obtained on incubation with rat cecal matter. Amongst the series of four prodrugs, the prodrug of 4-ASA with 4-ASA (4A-4AAz) (Figure 1M) at a dose of 53 mg/kg was found to alleviate all the quantifying markers of TNBS-induced experimental colitis in Wistar rats. Due to absence of adverse effects in the stomach, liver and pancreas, it could be a promising alternative for IBD patients intolerant to 5-ASA-induced pancreatitis and sulfapyridine-induced adverse effects observed with sulfasalazine [53]. CONCLUSION Despite possessing more stability and potency than 5-ASA in treating UC, without the evident risk of 5-ASA-induced pancreatitis, 4-ASA or its colon-targeted prodrugs remain unexplored and neglected when it comes to the management of IBD patients intolerant to 5-ASA. 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196 Dhaneshwar SS. Colon-targeting 4-ASA prodrugs for IBD [PMID: DOI: /gut ] 28 Nielsen OH, Ahnfelt-Rønne I. 4-Aminosalicylic acid, in contrast to 5-aminosalicylic acid, has no effect on arachidonic acid metabolism in human neutrophils, or on the free radical 1,1-diphenyl-2-picrylhydrazyl. Pharmacol Toxicol 1988; 62: [PMID: DOI: /j tb01876.x] 29 Schreiber S, MacDermott RP, Raedler A, Pinnau R, Bertovich MJ, Nash GS. Increased activation of isolated intestinal lamina propria mononuclear cells in inflammatory bowel disease. Gastroenterology 1991; 101: [PMID: ] 30 Schreiber S, Raedler A, Stenson WF, MacDermott RP. The role of the mucosal immune system in inflammatory bowel disease. Gastroenterol Clin North Am 1992; 21: [PMID: ] 31 von Ritter C, Grisham MB, Granger DN. Sulfasalazine metabolites and dapsone attenuate formyl-methionyl-leucylphenylalanine-induced mucosal injury in rat ileum. Gastroenterology 1989; 96: [PMID: ] 32 Schless JM, Inglis RM, Hammond JH, Hale JM. Comparative evaluation of blood levels and tolerance of sodium PAS and conjugated PAS and ascorbic acid. Dis Chest 1966; 50: [PMID: DOI: /chest ] 33 Cannemeyer W, Thompson JR, Lichtenstein MR. Severe paraaminosalicylic acid hypersensitivity; blood and lymph node studies. Blood 1955; 10: [PMID: ] 34 Gisbert JP, Gomollón F, Maté J, Pajares JM. Role of 5-aminosalicylic acid (5-ASA) in treatment of inflammatory bowel disease: a systematic review. Dig Dis Sci 2002; 47: [PMID: ] 35 McEvoy GK. Aminosalicylic acid. AHFS Drug Information US: American Society of Health-System Pharmacists, 2007: Food and Drug Administration. Orphan designations pursuant to Section 526 of the Federal Food and Cosmetic Act as amended by the Orphan Drug Act (P.L ), to June 28, MD: Rockville, Aminosalicylic Acid. Available from: URL: drugs.com/monograph/aminosalicylic-acid.html. Accessed on 23rd Sep Drug information. Available from: URL: Accessed on 28th July Nagy F, Karacsony G, Varro V. Experience with topical administration of 4-aminosalicylic acid in ulcerative colitis. Dis Colon Rectum 1989; 32: [PMID: DOI: /BF ] 40 Mital OP, Singh G, Raj B. Case report of fatal reaction to paraaminosalicylic acid and streptomycin. Ind J Tub 1991; 2: Zhao ZB, Guo J, Wei YG, Liang TD. Synthesis of 4-aminosalicylglycine. Chinese Chem Lett 2005; 16: Wallace JL, Cirino G, Sparatore A, inventors. Antibe Therapeutics Inc, Calgary, CA, Assignee. Salts of 4- or 5-aminosalicylic acid. US patent A Jan 5 43 Akisu M, Ozmen D, Baka M, Habif S, Yalaz M, Arslanoglu S, Kultursay N, Bayindir O. Protective effect of dietary supplementation with L-arginine and L-carnitine on hypoxia/ reoxygenation-induced necrotizing enterocolitis in young mice. Biol Neonate 2002; 81: [PMID: DOI: / ] 44 Shah PS, Shah VS. Arginine supplementation for prevention of necrotising enterocolitis in preterm infants. Cochrane Database of Sys Rev 2007; (3): CD [PMID: ] 45 Wallace JL, Cirino G, Caheno G, Sparatore A, Santagada V, Fiorucci S, inventors. Derivatives of 4- and 5-aminosalicylic acids. US patent A Nov Distrutti E, Sediari L, Mencarelli A, Renga B, Orlandi S, Antonelli E, Roviezzo F, Morelli A, Cirino G, Wallace JL, Fiorucci S. Evidence that hydrogen sulfide exerts antinociceptive effects in the gastrointestinal tract by activating KATP channels. J Pharmacol Exp Ther 2006; 316: [PMID: DOI: /jpet ] 47 Zhao ZB, Zheng HX, Wei YG, Liu J. Synthesis of azo derivatives of 4-aminosalicylic acid. Chinese Chem Lett 2007; 18: [DOI: /j.cclet ] 48 Almenier HA, Al Menshawy HH, Maher MM, Al Gamal S. Oxidative stress and inflammatory bowel disease. Front Biosci (Elite Ed) 2012; 4: [PMID: DOI: /463] 49 Sheng SF, Zheng HX, Liu J, Zhao ZB. Synthesis of phenolclass azo derivatives of 4-aminosalicylic acid. Chinese Chem Lett 2008; 19: [DOI: /j.cclet ] 50 Dhaneshwar SS, Sharma M, Vadnerkar G. Co-drugs of aminosalicylates and nutraceutical amino sugar for ulcerative colitis. J Drug Del Sci Tech 2011; 21: Yadav R, Mahatma OP, Singhvi I. Preparation and evaluation of polymeric prodrug of 4-aminoslicylic acid: colonic drug delivery in inflammatory bowel disease. IJPI s J Pharmaceut Cosmetol 2011; 1: Vadnerkar G, Dhaneshwar S. Macromolecular prodrug of 4-aminosalicylic acid for targeted delivery to inflamed colon. Curr Drug Discov Technol 2013; 10: [PMID: DOI: / ] 53 Suneela D, Gaurav V, Himanshu R. Design and development of novel azo prodrugs using various permutations and combinations of 5- and 4-aminosalicylic acids for inflammatory bowel disease: a colon-targeted approach. Inflamm Allergy Drug Targets 2013; 12: [PMID: DOI: / ] P- Reviewers: Hernandez-Breijo B, Kiela PR S- Editor: Wen LL L- Editor: Kerr C E- Editor: Wu HL 3571 April 7, 2014 Volume 20 Issue 13

197 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. REVIEW Remote ischemic preconditioning as treatment for non-ischemic gastrointestinal disorders: Beyond ischemia-reperfusion injury Carlos Rodrigo Camara-Lemarroy Carlos Rodrigo Camara-Lemarroy, Departamento de Medicina Interna, Hospital Universitario Dr. José E. González, Universidad Autónoma de Nuevo León, Monterrey, NL 64460, México Correspondence to: Carlos Rodrigo Camara-Lemarroy, MD, Departamento de Medicina Interna, Hospital Universitario Dr. José E. González, Universidad Autónoma de Nuevo León, Madero y Gonzalitos S/N, Monterrey, NL 64460, México. crcamara83@hotmail.com Telephone: Fax: Received: July 19, 2013 Revised: October 23, 2013 Accepted: January 2, 2014 Published online: April 7, 2014 Abstract Common gastrointestinal diseases such as radiation enteritis (RE), acute pancreatitis, inflammatory bowel diseases (IBD) and drug-induced hepatotoxicity share pathophysiological mechanisms at the molecular level, mostly involving the activation of many pathways of the immune response, ultimately leading to tissue injury. Increased oxidative stress, inflammatory cytokine release, inflammatory cell infiltration and activation and the up-regulation of inflammatory transcription factors participate in the pathophysiology of these complex entities. Treatment varies in each specific disease, but at least in the cases of RE and IBD immunosuppressors are effective. However, full therapeutic responses are not always achieved. The pathophysiology of ischemiareperfusion (IR) injury shares many of these mechanisms. Brief and repetitive periods of ischemia in an organ or limb have been shown to protect against subsequent major IR injury in distant organs, a phenomenon called remote ischemic preconditioning (RIP). This procedure has been shown to protect the gut, pancreas and liver by modulating many of the same inflammatory mechanisms. Since RIP is safe and tolerable, and has shown to be effective in some recent clinical trials, I suggest that RIP could be used as a physiologically relevant adjunct treatment for non-ischemic gastrointestinal inflammatory conditions Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Ischemia reperfusion; Ischemic preconditioning; Remote ischemic preconditioning; Acute pancreatitis; Radiation enteritis; Inflammatory bowel diseases; Hepatotoxicity; Inflammation Core tip: Ischemia-reperfusioin injury, which commonly occurs during transplant procedures, is associated with inflammatory cytokine release, endothelial dysfunction and oxidative stress. Remote ischemic-preconditioning is a procedure that can attenuate all of these alterations. The pathophysiology of many non-ischemic gastrointestinal disorders, such as radiation-induced enteritis and inflammatory bowel disease, among others, involves many of these same mechanisms. Future research should address the question of whether remote ischemic-preconditioning could also favorably alter the course of these diseases. Camara-Lemarroy CR. Remote ischemic preconditioning as treatment for non-ischemic gastrointestinal disorders: Beyond ischemia-reperfusion injury. World J Gastroenterol 2014; 20(13): Available from: URL: com/ /full/v20/i13/3572.htm DOI: org/ /wjg.v20.i INTRODUCTION Ischemia-reperfusion (IR) injury occurs when blood flow to an organ is interrupted, such as in arterial embolism or thrombosis, during transplant procedures or 3572 April 7, 2014 Volume 20 Issue 13

198 Camara-Lemarroy CR. Remote ischemic preconditioning in gastroenterology shock, followed by restoration of blood flow [1,2]. The initial injury (ischemia) causes depletion of tissue energy resources, activation of proteases and increased calcium influx into ischemic cells, and reperfusion exacerbates the extent of injury through the stimulation of an intense systemic inflammatory response. This leads to tissue microcirculatory failure, necrosis and apoptosis. Marked pro-inflammatory cytokine release, inflammatory cell infiltration, production of reactive oxygen species (ROS), increased expression of nitric oxide (NO), Toll-like receptor (TLR)-4 signaling and activation of inflammatory transcription factors, among other complex pathways, are centrally involved in the events leading to IR-injury in various organs [2-4]. The gut and the liver are among the most ischemia-sensitive tissues. Treatments aimed at attenuating IR-injury often involve the inhibition of one or many of these inflammatory pathways. Experimental success has been achieved with single cytokine blockers as well as with pleiotropic compounds with various immunomodulatory effects in both intestinal and liver IR-injury [5,6]. Recently, another strategy has proven useful in reducing IR-injury, ischemic preconditioning (IP). IP consists of a brief episode of ischemia that precedes the major ischemic event, and leads to the modulation of the innate organ defenses and inflammatory response, leading to decreased resulting injury [7]. The molecular mechanisms of IP are complex, and involve various pathways. IP leads to increased natural antioxidants such as glutathione, superoxide dismutase, heme-oxygenase-1 (HO-1), and reduced levels of lipid peroxidation [8]. Nuclear transcription factors such as nuclear factor-kappab (NF-KappaB) are also regulated by IP, leading to reduced pro-inflammatory cytokines production and release [7]. Additionally, NO production is modulated by IP, leading to preserved sinusoidal blood flow, oxygenation and mitochondrial function [8]. These events prepare tissues to withstand IR injury, with impressive efficacy. Remote ischemic-preconditioning (RIP) has emerged as an interesting alternative to direct IP. RIP involves the induction of protection against IR injury in an organ by brief episodes of ischemia and reperfusion in an entirely different organ or limb [9]. These protective effects begin few hours after preconditioning and may persist for up to 72 h. Circulating mediators and/or signals are hypothesized to mediate this effect, and some mechanisms involved so far are similar to those that applied in direct IP, including modulation of inflammatory cells, cytokines and oxidative stress [9,10]. Specific mechanisms involved in RIP-induced protection are also being uncovered. The induction of intestinal or hepatic RIP was shown to protect against subsequent renal IR by the attenuation of oxidative stress, lipid peroxidation, cytokine release, NO production and the upregulation of endogenous antioxidant mechanisms [11,12], and further studies have established that NO is an important mediator of RIP as well [13,14]. RIP normalizes NO production in mitochondria, and attenuates oxidative stress-mediated mitochondrial injury (24). HO-1, heat shock proteins (hsp), antioxidant and cytoprotective defense systems, are also upregulated by RIP and also attenuate tissue injury [9,10,15]. RIP also leads to reduced levels of tumor necrosis factor alpha (TNF-α) and inhibits crosstalk between TNF-α receptors and the induction of NF-KappaB [9,10,16]. RIP leads to reduced production and release of other proinflammatory cytokines and suppression of NF- KappaB-induced inflammation, and RIP has been shown to reduce long term transforming growth factor-beta (TGF-β) expression and fibrosis in kidneys damaged by IR injury [17,18]. Signal transduction and activator of transcription (STAT) pathways have also been involved in the pathophysiology of IR injury, and RIP is known to modulate STAT activity, leading to regulation of inflammation and cell survival [19,20]. Rats that are deficient in TLR-4 do not show RIP liver protection, suggesting a role for TLRs in the induction of preconditioning-associated ischemic tolerance [21,22]. In experimental studies, both IP and RIP have been shown to be beneficial in IR-injury of the gut, liver and pancreas [23-25]. The traditional view is that both IP and RIP protect tissues from a subsequent episode of full blown IR-injury. However, the variety of the inflammatory pathways regulated by these interventions suggests that they have wide-ranging immunomodulatory properties (Figure 1). Experimental studies have shown that IP and RIP are able to attenuate the inflammatory response and the severity of injury in various animal models of non-ischemic insults such as lipopolysaccharide-induced sepsis [26], traumatic brain injury [27] and cerulein-induced pancreatitis [28]. An interesting question that arises is whether these properties could be beneficial in non-ischemic but inflammatory conditions of the gastrointestinal system. In this review, I propose that given the similarity in the physiopathology of IR-injury, RIP-induced protection and some gastrointestinal inflammatory conditions, RIP could show beneficial effects in their clinical course. That is, the idea of RIP as a general immunomodulatory strategy, outside of its application solely to limit IR-injury. I focus on drug-induced hepatotoxicity, inflammatory bowel diseases (IBD), radiation enteritis (RE) and acute pancreatitis (AP). Although it is outside the scope of this paper to review in detail the pathophysiology of these diseases, in the following sections I discuss relevant evidence in support for the current proposal. HUMAN STUDIES OF REMOTE ISCHEMIC PRECONDITIONING RIP is less invasive than IP and has led to great clinical interest for its application [10]. RIP is safe, tolerable, with no clear side effects, and has been used effectively in several clinical trials [29]. The induction of forearm or leg IR by the inflation of a blood-pressure cuff for brief 3573 April 7, 2014 Volume 20 Issue 13

199 Camara-Lemarroy CR. Remote ischemic preconditioning in gastroenterology Ischemia-reperfusion ATP depletion Protease activation Increased Ca++ influx Target organ and systemic inflammation (1) Activated lymphocyte, myeloid cell and neutrophil infiltration STAT NF-KappaB (2) Mediator release Cytokines Chemokines Others (3) Oxidative stress Nitric oxide ROS Depletion of EA (4) HO-1 TLRs hsp Impaired mitochondrial function (5) Tissue injury Microcirculatory failure Necrosis Apoptosis Figure 1 Pathophysiology of ischemia-reperfusioin injury and mechanisms modulated by remote ischemic preconditioning. There are multiple pathways involved in the pathophysiology of ischemia-reperfusion (IR) injury. This leads to end organ damage as discussed in the text. Inflammatory cell infiltration and activation during IR is driven by multiple signaling pathways and lead to tissue inflammation. (1) Remote ischemic preconditioning (RIP) can modulate inflammatory cell infiltration and activity. Inflammatory cells and resident cells produce inflammatory cytokines and chemokines, partly in response to signal transducers and activators of transcription factor (STAT) and nuclear factor kappa B (NF-Kappa B) signaling; (2) RIP can reduce cytokine and chemokine production and modulate STAT activity. Local and infiltrating inflammatory cells produce reactive oxygen species (ROS) that deplete endogenous antioxidants (EA), resulting in tissue distruction; (3) RIP reduces production of ROS and increases levels of EA. During IR, heme-oxygenase-1 (HO-1) and heat shock proteins (hsp) are upregulated, Toll-like receptors (TLRs) are activated and mitochondrial function is disrupted; (4) RIP modulates TLR signaling, protects mitochondria and regulates HO-1 production. All of these events lead to microcirculatory failure and finally cellular apoptosis; and (5) RIP preserves microcirculation and reduces pro-apoptotic signals. ATP: Adenosine triphosphate. episodes (5 min) is the preferred method in human trials, that has shown promising results in the setting of myocardial ischemia [10]. The possibility of using it on multiple occasions or intermittently remains open. A recent meta-analysis of 23 clinical trials including 1878 patients undergoing RIP, mostly for cardiac surgery, was recently published [30]. Although it found no mortality benefit, it did find reduced levels of myocardial infarction and confirmed the procedure s safety. RIP has no known interaction with drug treatment, so it could be safely used as an adjunct to standard therapy. A recent small randomized controlled trial showed that arm RIP provided protection against contrast-medium induced nephrotoxicity [31]. This is the first clinical example of a study that used RIP with the aim of providing protection against a non-ischemic insult (contrast nephropathy). RIP is associated with few side effects apart from slight discomfort, and published protocols can be easily replicated. One of the most common protocols involves performing 4 cycles of alternating 5-min inflation and 5-min deflation of a standard upper-arm blood pressure cuff to the individual s systolic blood pressure plus 50 mmhg to induce transient and repetitive arm ischemia and reperfusion [31]. Number of cycles, ischemia time, and selected limb (arm or leg) can vary. DRUG-INDUCED HEPATOTOXICITY Drug induced acute liver failure (ALF) is one of the most severe and feared adverse reactions associated with drugs. Drug induced hepatotoxicity in general population has been estimated to be around 14/ inhabitants in Western countries, and contributes to 10%-50% of all causes of ALF [32]. Acetaminophen (ACP) intoxication, either accidental or intentional, is by far the most common, due to the universal access to this analgesic, and has a mortality rate of 32%-50% [32]. The specific treatment consist of n-acetylcysteine administration, an antioxidant that also affects ACP metabolism [33]. However, it requires complex dosing regimens, and the search for alternative therapeutic strategies is an active field of research. The pathophysiology of ACP induced hepatotoxicity is well understood. ACP is metabolized by cytochrome P450 into N-acetyl-p-benzoquinone imine (NAPQI), a toxic metabolite, which is then conjugated to glutathione and detoxified under physiologic circumstances to mercapturic acid [34]. With ACP overdose, glutathione is depleted and excess NAPQI binds to intracellular proteins causing mitochondrial dysfunction and permeability, leading to increased oxidative stress and the induction of a sterile inflammatory response [35]. Hepatocytes, Kupffer cells, and other circulating non parenchymal cells resi April 7, 2014 Volume 20 Issue 13

200 Camara-Lemarroy CR. Remote ischemic preconditioning in gastroenterology Table 1 Pathways participating in the physiopathology of some gastrointestinal diseases and effects of remote ischemic preconditioning Some mechanisms of ACP-induced hepatotoxicity, RE, IBD and AP Effects of RIP Inflammatory cytokine production Reduced inflammatory cytokines Increased oxidative stress Inhibits oxidative stress Depletion of glutathione Increased glutathione synthesis NO expression and release Modulation of NO function inos expression Modulation of inos HO-1 and hsp70 production Promotes HO-1 and hsp70 production TLR signaling Modulates TLR signaling Th1 and Th2 cytokine imbalance Reduced inflammatory cytokines STAT and NF-kappaB signaling Modulation of STAT and NF-KappaB TGF-β production Regulation of TGF-β production Note: Adapted from text. ACP: Acetaminophen; RE: Radiation-induced enteritis; IBD: Inflammatory bowel disease; AP: Acute pancreatitis; RIP: Remote ischemic preconditioning; HO-1: Heme oxygenase-1; hsp70: Heat shock protein-70; TLR: Toll-like receptor; NO: Nitric oxide; inos: Inducible nitric oxide synthase; STAT: Signal transduction and activator of transcription; NF-kappaB: Nuclear factor-kappab; TGF-β: Transforming growth factor-beta. dent synthesized other pro-inflammatory and injurious substances in response to these events. Among these, NO has been shown to play a major role in controlling lipid peroxidation and oxidative stress [36,37]. Induction of proteins that act as natural antioxidant defenses, such as HO-1 and heat shock protein 70 (hsp70), are increased by ACP in hepatocytes [38,39] in an attempt to reduce oxidative stress injury. Pro-inflammatory cytokines such as TNF-α are produced and other mediators such as TLR-4 are activated by ACP intoxication and are responsible for further damage and the perpetuation of the inflammatory response [40,41]. All of these pathways have been shown to be modulated by RIP in the liver. In a rodent model of hindlimb RIP, RIP led to the suppression of pro-inflammatory genes and the expression of antioxidant genes, leading to increased levels of glutathione, HO-1 and reduced TNF-α release in hepatocytes [22,24]. NO induced in the liver by RIP leads to preservation of the sinusoidal structure and maintenance of blood flow through the hepatic microcirculation, as well as improved mitochondrial oxygenation and reduced acidosis [4,42]. Finally, TLR-4 knockout mice have failed to induce hepatoprotection in RIP, suggesting a role for TLR-4 as well [22]. The overlap between the mechanisms responsible for ACP hepatotoxicity and those of RIP is striking (Table 1). Hypothetically, upon receiving a patient with suspected ACP intoxication, besides conventional treatment, forearm RIP could be initiated, which would hypothetically lead to reduced pro-inflammatory mediators production and increased antioxidant defense in the liver. This could partially mitigate the hepatotoxic effects of ACP, a common entity with high morbidity and mortality. Thus RIP could function as a physiologically reasonable adjunct treatment for ACP intoxication. INFLAMMATORY BOWEL DISEASE IBD encompass a wide variety of conditions, of which ulcerative colitis and Crohn s disease are the best understood. Their combined incidence and prevalence is approximately 5 cases per and 100 cases per population, respectively [43]. They constitute chronic inflammatory disorders of the gastrointestinal tract that share clinical and pathophysiological similarities, and can present with diarrhea, bloody stools, abdominal pain, weight loss and systemic signs of inflammation (depending on specific disease type). Alterations in innate immunity and in resident and infiltrating inflammatory cells such as neutrophils, CD4 lymphocytes, macrophages and mast cells lead to increased production of inflammatory cytokines of both the Th1 and Th2 response are the main effectors in these diseases [44]. Although initiator cytokines such as interleukin (IL)-17 and IL-13 dominate the initial response, the downstream mediators IL-1, IL-6 and TNF-α are the central regulators of tissue damage in IBD [45]. Besides immunosuppressive therapy, TNF-α antagonist therapy has shown tremendous effectiveness in the management of IBD. However, definitive treatment only exists in the form of surgery (in some cases), and disease can progress even under optimal pharmacologic therapy. It is known that RIP is effective in reducing intestinal IR injury [23], and I have previously reviewed evidence that suggests RIP can modulate inflammatory cytokine production, including robust effects over TNF-α. Intestinal IP in particular has been shown to reduce the mrna levels of many pro-inflammatory cytokines, including TNF-α and IL-1 [46]. STAT proteins participate in cytokine-induced T-cell activation in IBD, and activation of STAT-4 and STAT-3 in T cells is an essential step in the physiopathology of Crohn s disease [47]. The transcription factor NF-kappaB actively participates in the excessive inflammatory response observed in IBD, using TLR pathways, both in intestinal mucosal and inflammatory cells [48]. As I discussed, RIP is also able to modulate both of these transcription factors. Additionally, during IBD, endothelial cells are modified by intestinal inflammation, leading to altered responses to leukocyte-cell interactions and inflammatory cytokines, contributing to the initiation and propagation of IBDrelated pathology [49]. RIP is known to protect endothelial cells from various insults. Intestinal IP can directly reduce NF-KappaB activation after IR-injury [46], and can also modulate leukocyte-endothelial interactions [50]. Recent evidence has shown that in response to inflammatory cytokine production, ROS are produced during disease activity in IBD, and that oxidative stress could be a triggering factor, rather than a concomitant occurrence during the pathogenesis of these diseases [51]. Not only are oxidants overproduced but antioxidants also are overexpressed as a natural defense mechanism against oxidative injury during IBD activity [52]. HO-1 is also a main endogenous defense mechanism against intestinal 3575 April 7, 2014 Volume 20 Issue 13

201 Camara-Lemarroy CR. Remote ischemic preconditioning in gastroenterology inflammation that has been hypothesized to participate in the pathophysiology of IBD [53]. Experimental studies have also established a protective role for Hsp70 against colitis trough suppression of inflammatory cytokines and apoptosis [54]. Constitutive and inducible NO synthetase (inos) participates in the initiation and regulation of inflammation during IBD [55], and has complex immunemodulating effects in normal intestinal homeostasis. Of course, RIP is associated with antioxidant effects, HO-1 and Hsp70 expression and modulation of NO responses. In rat models of intestinal IR, both IP and RIP were able to increase serum levels of HO-1 conferring cytoprotection [23,50], and RIP has been shown to modulate NO production and decrease lipid peroxidation in the colon [56]. Intestinal inflammatory diseases such as IBD also share many pathophysiological mechanisms with IR injury (Table 1), and there is direct experimental evidence suggesting that the mediators discussed above are capable of being regulated by RIP in intestinal tissues, making IBD another important candidate for RIP therapy. Of course, the ideal phase of intervention would be an acute flare of IBD, since these conditions have clear chronic courses. Some form of pulse therapy or periprocedural (in endoscopic procedures) use of RIP could also be promising. RADIATION-INDUCED ENTERITIS Radiotherapy for pelvic and abdominal tumors causes severe gastrointestinal complications. The intestines are very sensitive to radiation injury and they constitute a dose-limiting organ. As many as 16% of patients receiving abdominal or pelvic radiotherapy will develop chronic RE, and even more will suffer from the acute phase of this entity [57]. Acute symptoms include diarrhea, nausea and vomiting, and chronic manifestations include stricture formation, obstruction, inflammation, bleeding and fibrosis. Both phases of RE are thought to occur due to an imbalance of inflammatory and anti-inflammatory mediators. There is scarce evidence favoring an effective therapeutic regimen for RE, and the search for novel treatment or preventive strategies is ongoing [58] Experimental evidence points towards an important role for oxidative stress in the physiopathology of RE. After radiation exposure, there is an important imbalance in the oxidative status of the intestine [59]. Increased myeloperoxidase, malonaldehide and decreased levels of glutathione have been associated with the acute inflammatory phase of RE [60]. RE also induces HO-1 expression, and the over-expression of HO-1 leads to tissue protection in acute RE [61]. The administration of antioxidants, like vitamin-e, diminish experimental RE by decreasing lipid peroxidation and increasing glutathione levels in the gut [62,63]. Increased induction of inos has also been observed in the colon after acute RE, and inos inhibitors diminish some of the alterations associated to RE [64,57]. The coordinated activity of several adhesion molecules participate in the recruitment of leukocytes into inflamed tissue in the setting of RE [65]. RE also induces the expression of adhesion molecules and other inflammatory mediators, such as cytokines and chemokines, both locally and systemically [66]. There are many lines of evidence that implicate cytokine imbalances directly in the pathogenesis of RE. In a rodent model of RE, the ileal expression of IL-1, IL-6, TNF-α, TGF-β was markedly increased, while the levels of the anti-inflammatory cytokine IL-10 were found to be diminished, all in association with activation of NF-KappaB [67]. NF-KappaB inhibition, in turn, was able to diminish the severity of RE, as well as the associated inflammatory response [68]. The shift towards a Th-2 like response, with release of inflammatory cytokines IL-1 and TNF-α, was shown to persist for up to 6 mo after RE, suggesting a role for this mechanisms in the chronic phase as well [64]. This same study also showed the induction of the IL-10/STAT 3 pathway by RE. At least one study has confirmed elevated levels of inflammatory cytokines in the mucosa of patients with radiation-induced proctitis [69]. The chronic phase of RE, characterized by fibrosis, is also associated to an inflammatory response. In rodent models, TGF-β immunoreactivity was found to be increased in the intestines after RE and to correlate well with both acute and chronic histopathologic lesions [70]. TGF-β and its pathways are central mediators of fibrogenesis and currently constitute an important therapeutic target for RE [71]. In experimental studies, IP could reduce the chronic elevations of TGF-β after renal IR-injury [17], but these results have not been replicated in models of liver IR [72]. RE constitutes another pathology that shares many pathophysiological mechanisms with IR-injury (Table 1). It is therefore unsurprising that many of these mechanism are affected by RIP as well, including regulation of cytokine balance, attenuation of oxidative stress and the modulation of transcription factors such as NF-KappaB. There is another important aspect to RE which makes it ideal for intervention with RIP: the fact that it is an expected complication, a side effect. This allows for the use of RIP as pre-treatment, or as a preventive measure. It is in this setting that RIP has shown greater clinical benefit in human trials. ACUTE PANCREATITIS AP is one of the most common emergency department admission gastrointestinal diagnoses in the world, with increasing incidences of up to 30 cases per population, with an overall mortality of around 2% [73]. Many etiologies account for AP, but gallstones and alcohol constitute for nearly 70% of cases. Irrespective of cause, a common pathogenesis seems to involve pancreatic hyperstimulation, enzyme (including trypsin) activity upregulation in acinar cells and enzyme reflux, leading to auto-digestion of the gland and local inflammation [74,75]. Although the full details of these pathogenic processes 3576 April 7, 2014 Volume 20 Issue 13

202 Camara-Lemarroy CR. Remote ischemic preconditioning in gastroenterology remain unknown, an intense local and systemic inflammatory response is characteristic of AP and accounts for most aspects of disease severity, systemic complications and clinical outcomes. Treatment is mostly supportive, and no specific pharmacologic treatment is currently available. Unsurprisingly, many of the inflammatory mechanisms I have discussed in previous sections again turn out to play essential roles in the pathophysiology of AP. NF- KappaB is upregulated in AP, leading to the activation of many inflammatory genes and the upregulation of various pro-inflammatory mediators [76]. Monocytes form patients with AP show impaired NF-KappaB and STAT3 regulation [77]. The STAT signal transducers also promote the expression of TNF-α, IL-1 and IL-6 in acute pancreatitis [78]. Human and experimental studies have confirmed the central role of these pro-inflammatory cytokines in the regulation and perpetuation of a systemic inflammatory response in AP [79]. Experimental inhibition of these molecules leads to attenuation of the severity AP, and their serum levels have even been useful as predictors of severity in the clinical setting. Interactions between pro-inflammatory cytokines and oxidative stress occurs in the development of the inflammatory response in AP, activating common signal transduction pathways that lead to amplification of the inflammatory cascade such as NF-KappaB [80]. Increased oxidative stress, lipid peroxidation and production of free radicals are important steps in the development of pancreatic tissue injury during AP [80,81]. HO-1 is upregulated in pancreatic tissue after experimental AP, contributing to intercellular defenses against increased oxidative stress [82], and neutrophils and monocytes derived from patients with AP have shown to be primed for HO-1 production, suggesting that HO-1 upregulation could be a viable therapeutic target [83]. Hsp s and TLRs also participate in an interplay between pancreas tissues and etiological agents, modulating the local and systemic inflammatory cascade [84,85]. Although the role of NO in the pathophysiology of AP remains controversial, it can regulate many molecular targets and crucial steps of the inflammatory response [86]. Despite advances in the understanding of these processes, there is no evidence of a clinical benefit of antioxidant or anti-cytokine therapies in AP. We have already seen that both IP and RIP modulate all of these mechanisms in general, but particular evidence concerning the pancreas is available. Pancreatic IP is effective in reducing the severity of IR-induced pancreatitis, partly through the modulation of IL-1 signaling [87-89]. IP also reduces the severity of caerulein-induced pancreatitis and can also accelerate pancreatic repair, also partly through a reduction of the inflammatory process and induction of hsp70 [28,90]. IP can also preserve microcirculation, reduce neutrophil infiltration and necrosis after IR-induced pancreatitis [91]. Some of these findings have been extended to RIP. RIP via hepatic IP also reduced the severity of IR-induced pancreatitis, in part through reduced IL-1 production [92], but these findings could not be replicated by renal RIP [93]. Considering that a systemic inflammatory response is the hallmark of severe acute pancreatitis and its associated systemic complications, RIP again emerges as an intriguing therapeutic possibility (Table 1). Its wide-ranging immunomodulatory effects, including those that target essential pathways known to participate in the physiopathology of AP, could turn out to be beneficial. Prevention of systemic complications and subsequent repair could also be achieved by RIP. RIP could also be specially effective in post-endoscopic retrograde cholangiopancreatography pancreatitis, as a periprocedural preventive measure. CONCLUSION The clinical application of RIP is an active field of research. It is a safe, tolerable and simple procedure, with wide-ranging immunomodulatory effects. Most clinical studies concern the utility of RIP in preventing IRinjury in the setting of vascular surgery or myocardial infarction, but at least one study has found a beneficial effect of RIP in reducing a non-ischemic injury, that of contrast nephropathy [31]. Recent developments also suggest that gastrointestinal protection via RIP might be clinically effective. Serum from human subjects subjected to RIP was able to protect human cultured intestinal cells from hypoxia-induced damage [94]. And in a recent randomized control trial, RIP was shown to protect the intestine against IR-injury associated to open abdominal aneurism repair, as evidenced by decreased levels of intestinal injury markers such as serum intestinal fatty acidbinding protein, endotoxin levels and diamine oxidase activity [95]. Considering that the protective effects of RIP are thought to depend on its modulation of the inflammatory response and oxidative status, its therapeutic potential could be expanded to other non-ischemic inflammatory conditions. I have shown that inflammatory gastrointestinal conditions such as IBD, RE, AP and drug-induced hepatotoxicity could be ideal candidates for RIP treatment, considering that their pathophysiological processes mirror to an extent those of IR-injury, and have themselves been shown to be modulated by RIP. RIP is most often thought of as a preventive strategy, since it is usually applied before the development of injury or disease. This makes RE and post-endoscopic retrograde colangiopancreatography pancreatitis the most logical candidates. However, the immunomodulatory effects of ischemic conditioning are known to persist even after an initial ischemic injury is established, a phenomenon known as ischemic post-conditioning [96]. Of course, clinical applicability would have to be determined following extensive pre-clinical experimentation and appropriate clinical trials. Precise timing, preconditioning technique and route would have to be determined as well. However, the current evidence suggests RIP has a great potential in treating these and other pathologic conditions April 7, 2014 Volume 20 Issue 13

203 Camara-Lemarroy CR. Remote ischemic preconditioning in gastroenterology REFERENCES 1 Berland T, Oldenburg WA. Acute mesenteric ischemia. Curr Treat Options Gastroenterol 2008; 11: 3-10 [PMID: DOI: /s ] 2 de Groot H, Rauen U. Ischemia-reperfusion injury: processes in pathogenetic networks: a review. Transplant Proc 2007; 39: [PMID: DOI: /j.transproceed ] 3 Montalvo-Jave EE, Escalante-Tattersfield T, Ortega-Salgado JA, Piña E, Geller DA. Factors in the pathophysiology of the liver ischemia-reperfusion injury. J Surg Res 2008; 147: [PMID: DOI: /j.jss ] 4 Abu-Amara M, Yang SY, Tapuria N, Fuller B, Davidson B, Seifalian A. Liver ischemia/reperfusion injury: processes in inflammatory networks--a review. Liver Transpl 2010; 16: [PMID: DOI: /lt.22117] 5 Iñiguez M, Dotor J, Feijoo E, Goñi S, Prieto J, Berasain C, Avila MA. Novel pharmacologic strategies to protect the liver from ischemia-reperfusion injury. 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206 Camara-Lemarroy CR. Remote ischemic preconditioning in gastroenterology ] 93 Warzecha Z, Dembiński A, Ceranowicz P, Cieszkowski J, Konturek SJ, Dembiński M, Kuśnierz-Cabala B, Tomaszewska R, Pawlik WW. Ischemic preconditioning of the hindlimb or kidney does not attenuate the severity of acute ischemia/ reperfusion-induced pancreatitis in rats. J Physiol Pharmacol 2008; 59: [PMID: ] 94 Zitta K, Meybohm P, Bein B, Heinrich C, Renner J, Cremer J, Steinfath M, Scholz J, Albrecht M. Serum from patients undergoing remote ischemic preconditioning protects cultured human intestinal cells from hypoxia-induced damage: involvement of matrixmetalloproteinase-2 and -9. Mol Med 2012; 18: [PMID: DOI: /molmed ] 95 Li C, Li YS, Xu M, Wen SH, Yao X, Wu Y, Huang CY, Huang WQ, Liu KX. Limb remote ischemic preconditioning for intestinal and pulmonary protection during elective open infrarenal abdominal aortic aneurysm repair: a randomized controlled trial. Anesthesiology 2013; 118: [PMID: DOI: /ALN.0b013e da5] 96 Vinten-Johansen J, Shi W. The science and clinical translation of remote postconditioning. J Cardiovasc Med (Hagerstown) 2013; 14: [PMID: DOI: / JCM.0b013e32835cecc6] P- Reviewers: Krishnan T, Rakonczay Z S- Editor: Wen LL L- Editor: A E- Editor: Wang CH 3581 April 7, 2014 Volume 20 Issue 13

207 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. BRIEF ARTICLE REVIEW What physicians should know about the management of chronic hepatitis B in children: East side story Hun-Jee Choe, Byung-Ho Choe Hun-Jee Choe, Seoul National University College of Medicine, Seoul , South Korea Byung-Ho Choe, Department of Pediatrics, Kyungpook National University School of Medicine, Daegu , South Korea Author contributions: All authors contributed to this work. Supported by Kyungpook National University Research Fund, 2012 Correspondence to: Byung-Ho Choe, MD, PhD, Professor of Pediatrics, Department of Pediatrics, Kyungpook National University School of Medicine, Dongduk-Ro 200, Jung-Gu, Daegu , South Korea. bhchoe@knu.ac.kr Telephone: Fax: Received: September 28, 2013 Revised: December 11, 2013 Accepted: January 8, 2014 Published online: April 7, 2014 Abstract Understanding the natural course of chronic hepatitis B virus (HBV) infection is very important for the management and treatment of chronic hepatitis B in children. Based on treatment guidelines, the management of HBV carriers and treatment of active hepatitis have been advancing and resulted in increased survival, as well as decreased risks of complications such as liver cirrhosis and hepatocellular carcinoma. Development of a continuing medical education (CME) program for primary physicians becomes an important responsibility of pediatric hepatologists. CME could prevent misdiagnosis and unnecessary treatment that could lead to liver complications or antiviral resistance. In addition, education of patients and their parents is necessary to achieve better therapeutic outcomes Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Hepatitis B virus; Natural course; Diagnosis; Treatment; Lamivudine; Resistance Core tip: Chronic hepatitis B virus infection is very important for the management and treatment of chronic hepatitis B in children. Education of patients and their parents is necessary to achieve better therapeutic outcomes. Choe HJ, Choe BH. What physicians should know about the management of chronic hepatitis B in children: East side story. World J Gastroenterol 2014; 20(13): Available from: URL: DOI: INTRODUCTION Chronic hepatitis B is defined as the presence of HBsAg and elevated serum alanine aminotransferase (ALT) levels for more than 6 mo, along with distinctive necroinflammation in the liver [1]. More than 240 million people have chronic hepatitis B in the world, with the highest incidence in sub-saharan African and East Asian regions [2]. In China, approximately 120 million people are hepatitis B virus (HBV) carriers [3]. Most people in HBV prevalent countries become infected with HBV during childhood, which becomes a financial burden of governments, especially in East Asia. The universal vaccination program has reduced the prevalence rate of hepatitis B, especially in children. The seroprevalence of HBsAg in Korean children decreased to 0.2% of preschool children and 0.44% of early teenage students in 2007 owing to the universal vaccination and hepatitis B perinatal transmission prevention program [1]. In Taiwanese children (< 15 years of age), the rate of HBsAg positivity dropped to 0.6% after the successful implementation of universal vaccination for 20 years, compared to 10% in the pre-vaccination era [4]. However, treatment is not always easy because of inattention to the natural course of chronic hepatitis B in children and emerging antiviral drug resistance. It is essential to minimize the severity of active hepatitis and 3582 April 7, 2014 Volume 20 Issue 13

208 Choe HJ et al. Chronic hepatitis B in children reduce the length of the immune clearance phase, and avoid inappropriate treatment or negligence. Spontaneous HBeAg seroconversion does not indicate histologic remission of chronic hepatitis. Currently, medications for the treatment of chronic hepatitis B infections are conventional interferon (IFN), lamivudine and adefovir in children. Global studies on potent antiviral agents such as entecavir and tenofovir in children are ongoing; however, these drugs need approved as the primary treatment option in children. In the meanwhile, there are still a lot of misconception about the natural course, diagnosis and treatment of chronic hepatitis B, especially in children. Children are not little adults is a true sentence for chronic hepatitis B. In the East where vertical infection is a major transmission mode, it is difficult to determine the facts on the basis of western journal articles. The authors have reviewed published articles comparing Asian children to adults as well as to western children, especially in the management and treatment of chronic hepatitis B in children. NATURAL COURSE OF CHRONIC HEPATITIS B After acute HBV exposure, 90% of infants of HBeAg seropositive mothers become chronic HBV carriers, whereas 25%-70% of children aged < 3-5 years and 5% of adults become chronic carriers [5-7]. The immature immune system in young children may be accountable for this. Chronic hepatitis B in children with HbsAg positivity for at least six months is transmitted predominantly through vertical infection [7]. In this case, the initial phase is the immune tolerance phase, in which HBeAg is positive and the serum HBV DNA level is over 10 5 copies/ml (over copies/ml in practice) due to a high level of HBV replication; however HBV carriers are generally asymptomatic with normal aspartate aminotransferase (AST)/ALT values. Liver damage is minimal regardless of the active HBV replication rate during this phase, for HBV is a virus that does not attack hepatocytes directly [8]. As the stage progresses onto the immune clearance phase (immune active phase), accompanied by necrosis of liver tissues, ALT rises due to the transaminase that comes from the damaged hepatocytes. This results in chronic active hepatitis with HBeAg positivity and fluctuation of serum HBV DNA level, complicated by necrosis and fibrosis of liver tissues. The majority of patients who have been vertically transmitted progress into chronic active hepatitis before the age of 35. Though liver damage is usually mild in children, liver cirrhosis or hepatocellular carcinoma may even develop as complications 2-7 years after vertical infection [9,10]. At the end of the immune clearance phase and transition to the non/low replicative phase, removal of infected hepatocytes results in low HBV DNA and HBeAg seroconversion [11]. Also known as the inactive HBV carrier state, serum aminotransferase normalizes and serum HBV DNA also declines to the level of less than 2000 IU/mL (10 4 copies/ml). Whilst 2/3 of untreated inactive carriers stay in the non (low) replicative phase for a prolonged period, this is important to note since approximately 20% of untreated inactive carriers may go through the reactivation phase, some through repetitive hepatitis flares, and 4% may revert to HBeAg positive chronic hepatitis B [12,13]. However, these kinds of acute exacerbation are unusual in children after HBeAg seroconversion [14]. SPONTANEOUS HBEAG/ANTI-HBE SEROCONVERSION It is rare for HBeAg to spontaneously seroconvert during the immune tolerance phase. Spontaneous HBeAg seroconversion occurs during the immune clearance phase. It takes place at different periods of life from childhood to the 5 th decade, but most commonly between the ages of 15 and 30 years. Ages ranging from thirties to forties are the most common in Asian countries where vertical transmission is the major route, due to the slower development of the immune clearance phase [12]. The rate of spontaneous seroconversion is lower in children than in adults. Spontaneous HBeAg seroconversion has been shown to be less than 2% annually in children aged < 3 years, which increases to 4%-5% over the age of 3, and 10%-14% for the year age group in Taiwan [15]. In South Korea, HBeAg clearance occurs in around one third of HBV infected children by 19 years of age [16]. In Asia, the major mode of HBV infection is usually via vertical transmission which leads to a prolonged immune-tolerant phase. That may explain why the spontaneous seroconversion rate is low in Asia compared to western countries. Higher HBeAg seroconversion rates have been reported in children infected horizontally than in those infected perinatally [17,18]. Although HBeAg seroconversion during treatment is the primary therapeutic target, spontaneous HBeAg seroconversion occurs through the immune clearance phase, after necroinflammation of the liver and subsequent removal of infected hepatocytes. It is during this phase that the plausibility of liver complications may increase, according to the severity and duration of active hepatitis. Even normalization of ALT and disappearance of HBeAg would not guarantee the prevention of liver cirrhosis or hepatocellular carcinoma, had it been untreated or neglected after a series of inappropriate herbal remedies which may have deteriorated the disease for a prolonged period (Figure 1A). In general, spontaneous HBeAg seroconversion usually leads to good prognosis in adults; however, one third of active hepatitis (HBeAg positive or negative) may recur and some of these patients may even develop liver cirrhosis or hepatocellular carcinoma [12]. A study of European children with horizontally transmitted chronic hepatitis B, with an average of 5.2 ± 4.0 years of follow up, showed that most of 3583 April 7, 2014 Volume 20 Issue 13

209 Choe HJ et al. Chronic hepatitis B in children A ALT Delayed or No Tx B ALT Early Tx LC, HCC LC, HCC 1 ULN 1 ULN Carrier Active hepatitis Inactive carrier Age Carrier Active hepatitis Inactive carrier Age HBeAg (+) High HBV DNA HBeAg (-) Low HBV DNA HBeAg (+) High HBV DNA HBeAg (-) Low HBV DNA Figure 1 Altered course of chronic hepatitis B according to timing of starting treatment (Tx). A: Delayed or no treatment results in higher incidence of liver cirrhosis (LC) or hepatocellular carcinoma (HCC); B: Treatment in early immune-clearance phase results in lower incidence of LC or HCC. ALT should be higher than 2 times of upper limit of normal values (> 2 ULN); HBV: Hepatitis B virus. them lost HBeAg and had a good prognosis, but some made the transition to HBeAg negative chronic hepatitis or progressed into hepatocellular carcinoma [19]. It is extremely rare for HBsAg to spontaneously disappear in South Korea unlike western countries where the major route of infection is through horizontal transmission. In Taiwan, a country where the majority of hepatitis B is transmitted vertically as in Korea, the spontaneous HBsAg loss rate is also very low, 0.56%, compared to western countries [20,21]. Korean reports also show that HBsAg spontaneously disappears only in 1.5% of patients under the age of nineteen [16]. MANAGEMENT OF CHILDHOOD CHRONIC HEPATITIS B CARRIERS Most chronic hepatitis B patients are asymptomatic. Childhood chronic hepatitis B generally goes through the immune tolerance phase where ALT is normal and there is little or no inflammation in the liver tissue. This period persists for about years but may also progress into the immune clearance phase in children [8]. In two follow up studies in Korea and Taiwan, spontaneous HBeAg seroconversion occurred in 11% and 24% of children under the age of 10, respectively [16,20]. In addition, these studies showed that 32% of those under the age of 19 turned HBeAg negative in South Korea [16]. Since spontaneous HbeAg seroconversion occurs at the end of the immune clearance phase, the long-lasting immune clearance phase could lead to the development of liver cirrhosis or even hepatocellular carcinoma. Therefore, early management to shorten the duration of active hepatitis (immune clearance phase) is important (Figure 1B). Monitoring ALT levels at intervals of at least six months is necessary to avoid patients going through unrecognized active hepatitis in their childhood, as well as to detect the earliest transition time to the immune clearance phase for patients in the immune tolerant phase. In a Korean study of children with chronic hepatitis B, the estimated rate of entry to the immune clearance phase was 4.6% for those under the age of 6, 7.1% for those between 6 and 12 years, and 28.0% for patients between 12 and 18 years, which was significantly higher than that observed for children under the age of 12 [22]. If left untreated or treated inappropriately during the immune clearance phase, liver damage is unavoidable as it has already occurred and progressed, even though spontaneous HBeAg seroconversion has occurred, because the rate of HBeAg seroconversion is not significantly increased by long-term follow-up, treated or not [23]. The risk is known to increase proportionally with time until the point of HBeAg seroconversion [24-27]. DIAGNOSIS OF CHRONIC HEPATITIS B Laboratory interpretation Positive HBeAg does not mean active hepatitis in children. HBeAg is positive in both the immune tolerance phase and the immune clearance phase. Serum HBV DNA, ALT, and HBeAg titer are the parameters for distinguishing the two phases. Active hepatitis is defined when serum transaminase AST/ALT is persistently increased and serum HBV DNA level is over copies/ml, regardless of HBeAg status. If the HBV DNA level is high despite negative HBeAg, it may indicate the possibility of HBeAg negative chronic hepatitis B by a precore mutant virus or core promoter mutation [28]. These types of hepatitis are also classified as active hepatitis B if they accompany high levels of serum ALT and serum HBV DNA over copies/ml, and also need active treatment [29]. It is not prevalent but not negligible during childhood years and needs special attention during treatment and monitoring [28]. Quantitative serum HBV DNA tests are especially important so as to analyze viral response (VR), for it is impossible to assay the level of viral replication with HBeAg alone in HBeAg negative chronic hepatitis patients April 7, 2014 Volume 20 Issue 13

210 Choe HJ et al. Chronic hepatitis B in children Liver biopsy Liver biopsy is imperative for assessing the necroinflammatory grade and fibrosis stage of liver damage, and helps as a guide for making treatment decisions [30]. However, histological assessment is not essential for the initiation of treatment, especially in children [31]. Biopsy results are usually near normal in HBV carrier children; however, that does not mean that they are always normal. Most liver biopsy samples from children with chronic hepatitis B show minor inflammation and fibrosis; nevertheless, cases exist with severity up to liver cirrhosis or hepatocellular carcinoma [32]. An HBV carrier whose ALT level has been normal since they were young would be in the immune tolerance phase and the liver tissue would be favorable without significant necroinflammation or fibrosis [33]. However, prognosis and liver status would be different from those of HBeAg negative carriers in the low or nonreplicative phase who have gone through active hepatitis. Histology of the liver in the nonreplicative phase may vary from minimal necroinflammation and fibrosis to liver cirrhosis depending on the period and severity of liver damage during the immune clearance phase. The fact that even HBeAg positive healthy carriers show signs of chronic hepatitis in 40% of cases indicates that the majority of Korean adult carriers go through chronic active hepatitis B without realizing it [34]. These findings are similar to those studied in western countries, where extended fibrosis and even liver cirrhosis have been found in more than half of liver biopsy samples of children (ages 1-19, mean age 9.8 years) with HBeAg positivity and increased ALT [35]. As a non-invasive alternative test to liver biopsy, liver stiffness measurement (Fibroscan ) is frequently used for the evaluation of liver fibrosis, which is valuable in fibrosis staging in Asian patients with chronic hepatitis B [36]. OPTIMAL TIMING OF STARTING TREATMENT OF CHRONIC HEPATITIS B While most children with chronic hepatitis B remain asymptomatic, it may progress into liver cirrhosis or hepatocellular carcinoma if left untreated during the immune clearance phase. A keen attention should be paid to the fact that a predominant portion of patients with chronic hepatitis B that progress into liver cirrhosis or hepatocellular carcinoma get infected during the childhood period. Therefore, persistent follow up should be mandatory in order not to miss the appropriate period for treatment. Active treatment should be considered with the onset of the immune clearance phase. Wait and see Treatment is not needed for children with normal ALT for it is not active hepatitis, although there are very high serum HBV levels along with positive HBeAg. HBeAg positivity does not indicate active hepatitis during the immune tolerance phase. Treatment during this period does not ameliorate near-normal liver tissues nor does it bring elimination of HBeAg. On the other hand, such unnecessary treatment would bring tolerance to the medication and may lead to treatment failure in the future when active hepatitis may flare. Treatment is therefore not given to HBV carriers with normal AST/ALT values in children [8]. Consider treatment Treatment should be considered, but not initiated immediately when HBeAg is positive and HBV DNA and ALT begin to increase. Treatment is considered in patients who have had serological evidence of HBV infection for at least 6 mo: that is, when HBsAg, HBeAg, HBV DNA are positive and ALT is consistently elevated; this would be regarded as the immune clearance phase of chronic hepatitis B which is appropriate for the initiation of treatment. Special caution is needed before starting treatment immediately for HBV carriers with elevated liver enzymes. ALT elevation may not be due to chronic hepatitis B, but rather due to other systematic infections such as respiratory tract infection and urinary tract infection in infants and toddlers [8]. Moreover, as obesity is becoming a social problem nowadays, there is an increasing number of obese children and adolescents who may also have nonalcoholic fatty liver disease (NAFLD). Weight monitoring should be the priority in this case, and if ALT does not normalize after weight reduction, liver biopsy should be done to determine whether the severity of liver damage is associated with hepatitis B or NAFLD. HBV carriers with concomitant Wilson s disease or muscular dystrophy are some of the other plausible reasons that may account for high levels of ALT, and need to be included in the differential diagnosis in children. Even with the apparent increase in ALT, starting treatment at less than twice the level of the upper limit of ALT may lead to a higher chance of drug resistance, which could result in higher treatment failure [37]. Good predictor of treatment Therapeutic response is better when serum ALT is high and HBV DNA low; that does not mean initiation of treatment should be left until this stage. Optimal treatment time should not be delayed until the end of the immune clearance phase when serum ALT is high and HBV DNA low, overlapping the period of HBeAg seroconversion. If left untreated until this period, the HBeAg seroconversion rate may increase by initiation of treatment at this moment because of the additional effect of spontaneous HBeAg seroconversion; however, it would bring the consequence of neglecting the chance to treat at the optimal time during the early immune clearance phase. Our ultimate goal does not lie in HBeAg seroconversion only. It lies in halting the replication of HBV, normalizing ALT and stopping the progression to liver cirrhosis or hepatocellular carcinoma so as to reduce complications and mortality before the liver damage becomes irrevers April 7, 2014 Volume 20 Issue 13

211 Choe HJ et al. Chronic hepatitis B in children ible [38]. The immune clearance phase is when liver tissue necrosis results in fibrosis by active inflammation, the severity and prolonged length relates to higher rates of complications such as liver cirrhosis (Figure 1). Children and adolescents are of no exception. Delayed treatment and liver complication Complications of chronic hepatitis B (liver cirrhosis, hepatocellular carcinoma) can develop even in teenage carriers. It is known that the longer the immune clearance phase and the more frequently the ALT flare-up occurs, the more likely liver cirrhosis or hepatocellular carcinoma is to occur. If active hepatitis is left untreated during the immune clearance phase, there is no guarantee that it will not progress into liver cirrhosis or hepatocellular carcinoma even if spontaneous seroconversion of HBeAg or HBsAg occurs [18,19]. Progression rate into liver cirrhosis in HBeAg positive patients is known to be related to the length of HBeAg positive period and the reactivation rate of hepatitis [27]. If the serum HBV DNA level is persistently over 10 4 copies/ml, it then becomes a risk factor for hepatocellular carcinoma [39]. Therefore, the longer the immune tolerance phase and the immune clearance, the more likely complications may occur from chronic hepatitis; and of the two, the immune clearance phase plays a key role. In the natural course of chronic hepatitis B, it may seem as if the severity of liver complications increases from the point where HBeAg is lost. However, when HBeAg is lost during the initial phase of the immune clearance phase, it leads to a decrease of the HBeAg positive period and high HBV load period, meaning a reduction in liver complications as well. Meanwhile, it is necessary at all costs to check serum HBV DNA after the loss of HBeAg to confirm whether or not it is HBeAg negative chronic hepatitis. TREATING CHRONIC HEPATITIS B WITH NUCLEOS(T)IDE ANALOGUE The therapeutic efficacy of IFN-alpha in a multinational randomized controlled study was 33% at 48 wk after the initiation of 24 wk of treatment for children [40]. IFN therapy may have more chance of cure in the aspect of HBsAg clearance, especially in younger children < 5 years of age [41]. Currently, clinical trials are undergoing in children using pegylated interferon-α alone or combined with nucleos(t)ide analogue which may have promising results, especially in Western countries. The predominant genotypes of HBV are B and C in China and South East Asian countries [23]. In adult studies, the therapeutic response to IFN was better for genotype B than C [42]. However, IFN is not very effective in genotype C predominant regions, where most children are vertically infected [43,44]. Children treated with lamivudine showed a significantly higher HBeAg and HBsAg seroconversion rate compared to IFN-treated children in a long-term follow-up study in Korea, where genotype C was predominant [45]. Antiviral resistance Drug resistance increases with the prolonged use of the nucleos(t)ide analogues. The resistance rate seen in children after two years is 23% [45]. Liver function may deteriorate after antiviral resistance to medication due to the YMDD mutation and may require substitution of the drug. A successful therapeutic response could be achieved when HBeAg seroconversion occurs before the advent of drug tolerance. At present, entecavir and tenofovir are registered for use in children over 16 years and 12 years, respectively, though they are the first-line anti-viral agents according to most HBV treatment guidelines. In children, more effective virologic responses have been demonstrated with the use of either add-on adefovir or switching to entecavir monotherapy compared with those with lamivudineresistant chronic hepatitis B who switched to adefovir monotherapy [46]. However, resistance to lamivudine is a risk factor for entecavir resistance and adefovir is no longer the first line option. How long to use The nucleos(t)ide analogue is not a drug that should be stopped after a certain period of time, but rather a medication to be continued until HBeAg seroconversion occurs. If stopped prior to seroconversion, hepatitis is most likely to reactivate. The therapeutic goals for antiviral treatment in HBeAg positive patients are as follows: undetected HBV DNA in PCR (< 10 3 copies/ml), normalized serum ALT, at least one year of undetected HBeAg before discontinuing the medication, and persistence of anti-hbe [47,48]. HBV DNA suppression has a close correlation with recovery of liver tissues and HBeAg seroconversion [49]. It is true that up to 10% of infected hepatocytes may survive after one year of treatment with lamivudine since lamivudine does not affect the covalently closed circular DNA (cccdna) of HBV inside the nucleus of hepatocytes [50]. HBV DNA will decrease, however, to the level where it would be impossible for HBV DNA to be resistant to antiviral agents, if treated and suppressed for a sufficient period. Complete viral eradication means removal of remaining cccdna from the hepatocyte, and this is made possible by the normal turnover of hepatocytes and reactivation of the host immune response [28]. This supports the 2011 KASL and 2012 EASL guidelines for continuing the nucleos(t)ide analogue for one year or more after disappearance of HBeAg [48,51]. Special caution is needed in HBeAg negative chronic hepatitis patients when using the nucleos(t)ide analogue so that the medication is not halted shortly after HBV DNA clearance. HBeAg/anti-HBe seroconversion cannot be effectively measured in HbeAg negative patients for anti-hbe positivity from the initial time point. In my opinion, at least two or three additional years of antiviral treatment is required for such children after the occur April 7, 2014 Volume 20 Issue 13

212 Choe HJ et al. Chronic hepatitis B in children rence of HBV DNA clearance [52]. HBsAg clearance In adults, treatment of hepatitis rarely results in a loss of HBsAg, which is the ultimate goal of treating chronic hepatitis B. However, in a study conducted by Choe et al [45], loss of HBsAg occurred in 42% in patients of preschool age after two years of lamivudine treatment. HBsAg disappeared in 13 of the 49 (26.5%) patients who experienced lamivudine-induced HBeAg seroconversion [53]. However, HBsAg loss in school children rarely occurred, similar to those in adults. PREVENTION OF NUCLEOS(T)IDE ANALOGUE RESISTANCE Pretreatment baseline ALT > 2 ULN The resistance rates to lamivudine were reported in children (pretreatment baseline ALT > 2 ULN) to be 10% at 1 year of treatment and 23%-26% at 2 years after the initiation of treatment [37,45]. Breakthrough was only seen in 5.9% of the 2-year treatment group in preschool children with higher pretreatment baseline ALT [37]. Similar results have been shown by other studies in Korean children [54,55]. The results in children are lower than those in adult studies [56,57]. On the other hand, in a western multicenter study, antiviral resistance analysis showed that the YMDD mutation rate was as high as 49% in 2-year treatment and 64% in 3-year treatment, in that order [58]. These outcomes were even worse than those of adults. That study included an inappropriate portion of patients with active hepatitis. Only 51% and 11% of children with pretreatment ALT > 2 ULN were enrolled for the 2 and 3-year treatment groups, respectively. Enrolling significant numbers of patients with a pretreatment ALT level < 2 ULN explains such a high resistance rate in western children. Therefore, an elevated pretreatment ALT level (> 2 ULN) is key to decreasing antiviral resistance. Confirm the immune clearance phase Primary treatment with the nucleos(t)ide analogue should be considered if patients have persistently elevated ALT levels > 2 ULN for more than 6 mo, nonetheless it should be confirmed that patients are in the immune clearance phase. Prior to starting therapy, a comprehensive assessment of the patient s status is important to exclude other causes of abnormal liver function tests, such as reactive hepatitis in infants and nonalcoholic steatohepatitis in obese children. Compliance One of the most important predictive factors for the therapeutic response to the nucleos(t)ide analogue is good compliance [59]. A significant portion of viral breakthrough is due to poor adherence to the medication [60]. Education of children and their parents (guardian) is required to continue good compliance leading to ideal therapeutic outcome, based on trust between doctors and patients. In addition, the optimal dosage according to body weight should be adjusted by the clinicians as children grow rapidly. On treatment monitoring and keep up-to-date by the most current guidelines Treatment response at 24 wk is important to take which treatment strategy to use. Patients with a complete virologic response generally have a very low possibility of antiviral resistance [61]. Proper monitoring for virologic breakthrough is needed, because early detection is critical to decide the most ideal intervention. If the nucleos(t)ide analogue is stopped before or immediately after HBeAg seroconversion, the likelihood of recurrence increases. Therefore, the 2011 KASL (Korean Association for Study of Liver) guidelines and 2012 EASL guidelines advise continuing the nucleos(t)ide analogue for one year or more after the disappearance of HBeAg to sustain a serological and/or virological response [48,51]. CONCLUSION In most children with chronic hepatitis B, treatment indications should be very carefully evaluated. Treatment should concentrate on suppressing viral replication in the early period of immune active hepatitis. High potent nucleos(t)ide analogues such as tenofovir and entecavir are anticipated to be available for use in younger children, which also have a high genetic barrier. REFERENCES 1 Choe BH. The epidemiology and present status of chronic hepatitis B in Korean children. Korean J Pediatr 2008; 51: [DOI: /kjp ] 2 World Health Organization. Hepatitis B Fact Sheet No. 204 (Updated July 2013), Available from: URL: Liu J, Fan D. Hepatitis B in China. Lancet 2007; 369: [PMID: DOI: /S (07) ] 4 Ni YH, Chang MH, Wu JF, Hsu HY, Chen HL, Chen DS. Minimization of hepatitis B infection by a 25-year universal vaccination program. J Hepatol 2012; 57: [PMID: DOI: /j.jhep ] 5 McMahon BJ, Alward WL, Hall DB, Heyward WL, Bender TR, Francis DP, Maynard JE. 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Korean J Pediatr Gastroenterol Nutr 1998; 1: Hui CK, Leung N, Yuen ST, Zhang HY, Leung KW, Lu L, Cheung SK, Wong WM, Lau GK. Natural history and disease progression in Chinese chronic hepatitis B patients in immune-tolerant phase. Hepatology 2007; 46: [PMID: DOI: /hep.21724] 34 Chung WK, Han JY. Pattern of histologic progression from acute and chronic hepatitis B to cirrhosis. Korean J Hepatol 1996; 2: Godra A, Jonas M, Perez-Atayde A. Histopathology of the liver in children with chronic hepatitis B. Mod Pathol 2005; 18: 346A 36 Kim BK, Kim HS, Park JY, Kim do Y, Ahn SH, Chon CY, Park YN, Han KH, Kim SU. Prospective validation of ELF test in comparison with Fibroscan and FibroTest to predict liver fibrosis in Asian subjects with chronic hepatitis B. PLoS One 2012; 7: e41964 [PMID: DOI: /journal. pone ] 37 Hong SJ, Kim YH, Choe BH, Park HJ, Tak WY, Kweon YO. Current role of Lamivudine regarding therapeutic response and resistance in children with chronic hepatitis B. 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214 Choe HJ et al. Chronic hepatitis B in children is associated with better response to interferon therapy in HBeAg(+) chronic hepatitis than genotype C. Hepatology 2002; 36: [PMID: DOI: / jhep ] 43 Choe BH, Ko C. Combined Therapy of Alfa-Interferon and Thymodulin on Children with Chronic Active Hepatitis B. Korean J Pediatr Gastroenterol Nutr 1998; 1: Jang CH, Lee KH, Hwang WK, Oh KW, Park WS, Lee JH, Ko CW, Choe BH. The Comparison of Interferon-alpha Treatment by Dosages and Retreatment for Chronic Hepatitis B in Children. Korean J Pediatr Gastroenterol Nutr 2003; 6: Choe BH, Lee JH, Jang YC, Jang CH, Oh KW, Kwon S, Hyun MC, Ko CW, Lee KS, Lee WK. Long-term therapeutic efficacy of lamivudine compared with interferon-alpha in children with chronic hepatitis B: the younger the better. J Pediatr Gastroenterol Nutr 2007; 44: [PMID: DOI: /01.mpg e] 46 Chu M, Cho SM, Choe BH, Cho MH, Kwon S, Lee WK. Virologic responses to add-on adefovir dipivoxil treatment versus entecavir monotherapy in children with lamivudine-resistant chronic hepatitis B. J Pediatr Gastroenterol Nutr 2012; 55: [PMID: DOI: / MPG.0b013e318262a737] 47 Liaw YF, Leung N, Guan R, Lau GK, Merican I, McCaughan G, Gane E, Kao JH, Omata M. Asian-Pacific consensus statement on the management of chronic hepatitis B: a 2005 update. Liver Int 2005; 25: [PMID: ] 48 Korean Association for the Study of the Liver. KASL Clinical Practice Guidelines: Management of chronic hepatitis B. Clin Mol Hepatol 2012; 18: [PMID: DOI: /cmh ] 49 Mommeja-Marin H, Mondou E, Blum MR, Rousseau F. Serum HBV DNA as a marker of efficacy during therapy for chronic HBV infection: analysis and review of the literature. Hepatology 2003; 37: [PMID: DOI: /jhep ] 50 Moraleda G, Saputelli J, Aldrich CE, Averett D, Condreay L, Mason WS. Lack of effect of antiviral therapy in nondividing hepatocyte cultures on the closed circular DNA of woodchuck hepatitis virus. J Virol 1997; 71: [PMID: ] 51 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 ] 52 Kim JM, Hong SJ, Choi BH, Chu MA, Cho SM, Choe BH. Clinical Experience with Long-term Lamivudine Therapy to- Determine the Adequate Duration of Treatment in Childrenand Adolescents with HBeAg-Negative Chronic Hepatitis B. Korean J Pediatr Gastroenterol Nutr 2009; 12: Kim JM, Choe BH, Chu MA, Cho SM. [Comparison of lamivudine-induced HBsAg loss rate according to age in children with chronic hepatitis B]. Korean J Hepatol 2009; 15: [PMID: DOI: /kjhep ] 54 Lee EH, Jang JY, Kim KM. Efficacy of lamivudine therapy for chronic hepatitis B in children. Korean J Pediatr Gastroenterol Nutr 2008; 11: Yoe S, Na SY, Yang HR, Chang JY, Ko JS, Kim BS, Kang GH, Seo JK. Incidence and risk factors of the YMDD mutation during lamivudine therapy in children with chronic hepatitis B. Proceedings of the 11th Congress of the Asian Pan-Pacific Society of Pediatric Gastroenterology, Hepatology and Nutrition (APPSPGHAN); 2009 Sep 25-28; South Korea. Seoul, South Korea: APPSPGHAN, 2009: Allen MI, Deslauriers M, Andrews CW, Tipples GA, Walters KA, Tyrrell DL, Brown N, Condreay LD. Identification and characterization of mutations in hepatitis B virus resistant to lamivudine. Lamivudine Clinical Investigation Group. Hepatology 1998; 27: [PMID: ] 57 Lok AS, Lai CL, Leung N, Yao GB, Cui ZY, Schiff ER, Dienstag JL, Heathcote EJ, Little NR, Griffiths DA, Gardner SD, Castiglia M. Long-term safety of lamivudine treatment in patients with chronic hepatitis B. Gastroenterology 2003; 125: [PMID: ] 58 Sokal EM, Kelly DA, Mizerski J, Badia IB, Areias JA, Schwarz KB, Vegnente A, Little NR, Gardener SD, Jonas MM. Long-term lamivudine therapy for children with HBeAg-positive chronic hepatitis B. Hepatology 2006; 43: [PMID: DOI: /hep.21020] 59 Hong SJ, Choe BH. Strategy to Overcome Drug Resistance That Develops during Treatment of Chronic Hepatitis B in Children. Pediatr Gastroenterol Hepatol Nutr 2012; 15: [DOI: /pghn ] 60 Kamezaki H, Kanda T, Arai M, Wu S, Nakamoto S, Chiba T, Maruyama H, Fujiwara K, Kanai F, Imazeki F, Nomura F, Yokosuka O. Adherence to medication is a more important contributor to viral breakthrough in chronic hepatitis B patients treated with entecavir than in those with Lamivudine. Int J Med Sci 2013; 10: [PMID: DOI: /ijms.5795] 61 Yuen MF, Sablon E, Hui CK, Yuan HJ, Decraemer H, Lai CL. Factors associated with hepatitis B virus DNA breakthrough in patients receiving prolonged lamivudine therapy. Hepatology 2001; 34: [PMID: ] P- Reviewer: Marignani M S- Editor: Wen LL L- Editor: O Neill M E- Editor: Zhang DN 3589 April 7, 2014 Volume 20 Issue 13

215 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. MINIREVIEWS Contrast-enhanced ultrasound in the diagnosis of nodules in liver cirrhosis Tae Kyoung Kim, Hyun-Jung Jang Tae Kyoung Kim, Hyun-Jung Jang, Department of Medical Imaging, Toronto General Hospital, University of Toronto, Toronto ON M5G 2N2, Canada Author contributions: All authors contributed to this work. Correspondence to: Tae Kyoung Kim, MD, Professor, Department of Medical Imaging, Toronto General Hospital, University of Toronto, 585 University Avenue, Toronto ON M5G 2N2, Canada. taekyoung.kim@uhn.ca Telephone: Fax: Received: November 29, 2013 Revised: January 4, 2014 Accepted: February 26, 2014 Published online: April 7, Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Hepatocellular carcinoma; Liver cirrhosis; Dysplastic nodule; Contrast ultrasound; Imaging Core tip: Contrast-enhanced ultrasound is a relatively new imaging technique that can be effectively used in the diagnostic algorithms for liver nodules detected in hepatocellular carcinoma surveillance. There are several unique advantages of this technique that make this imaging technique very useful. Abstract Contrast-enhanced ultrasound (CEUS) using microbubble contrast agents are useful for the diagnosis of the nodules in liver cirrhosis. CEUS can be used as a problem-solving method for indeterminate nodules on computed tomography (CT) or magnetic resonance imaging (MRI) or as an initial diagnostic test for small newly detected liver nodules. CEUS has unique advantages over CT and MRI including no renal excretion of contrast, real-time imaging capability, and purely intravascular contrast. Hepatocellular carcinoma (HCC) is characterized by arterial-phase hypervascularity and later washout (negative enhancement). Benign nodules such as regenerative nodules or dysplastic nodules are usually isoechoic or slightly hypoechoic in the arterial phase and isoechoic in the late phase. However, there are occasional HCC lesions with atypical enhancement including hypovascular HCC and hypervascular HCC without washout. Cholangiocarcinomas are infrequently detected during HCC surveillance and mostly show rimlike or diffuse hypervascularity followed by rapid washout. Hemangiomas are often found at HCC surveillance and are easily diagnosed by CEUS. CEUS can be effectively used in the diagnostic work-up of small nodules detected at HCC surveillance. CEUS is also useful to differentiate malignant and benign venous thrombosis and to guide and monitor the local ablation therapy for HCC. Kim TK, Jang HJ. Contrast-enhanced ultrasound in the diagnosis of nodules in liver cirrhosis. World J Gastroenterol 2014; 20(13): Available from: URL: com/ /full/v20/i13/3590.htm DOI: org/ /wjg.v20.i INTRODUCTION Ultrasound (US) is most commonly used for imaging surveillance for hepatocellular carcinoma (HCC) in high-risk patients [1]. Once a focal hepatic nodule is detected during HCC surveillance, a diagnostic imaging test is performed. The assessment of tumor vascularity is most important in the differential diagnosis of the nodules. Doppler US techniques have been used to detect vascularity in liver tumors; however, these techniques lack sensitivity to detect tumor vascularity. Contrast-enhanced computed tomography (CT) or magnetic resonance imaging (MRI) is most commonly used to characterize liver masses. Contrastenhanced ultrasound (CEUS) using a microbubble contrast agent is a relatively new imaging technique and has been proved to be useful for characterizing liver tumors. CEUS enables a real-time demonstration of continuous hemodynamic changes of liver tumors after injection of the contrast material April 7, 2014 Volume 20 Issue 13

216 Kim TK et al. CEUS in diagnosis of nodules A B C Figure 1 A 61-year-old man with hepatocellular carcinoma and renal failure. A: Gray-scale ultrasound shows a hypoechoic mass (arrow) within the cirrhotic liver. There is a large amount of ascites surrounding the liver. Contrast-enhanced computed tomography or magnetic resonance scan could not be performed because of renal failure; B: Contrast-enhanced ultrasound (CEUS) image in the arterial phase shows hypervascularity of the mass (arrow) relative to the liver; C: The mass (arrow) shows washout in the portal venous phase. The diagnosis of hepatocellular carcinoma was made based on imaging findings of CEUS without biopsy. Recent practice guidelines for HCC provided recommendations for the diagnostic algorithm for newly detected nodules at HCC surveillance [1-3]. The application of the imaging test varies depending on the size of the nodules. For very small lesions (< 1 cm in size), followup with US scan is usually recommended in 3 mo as further imaging tests may not be reliable for the diagnosis. For lesions same or larger than 1 cm, the performance of multiphasic contrast-enhanced CT, MRI or CEUS is a reasonable next step. There has been a controversy on the use of CEUS because intrahepatic cholangiocarcinoma can be misdiagnosed as HCC and CEUS has been subsequently excluded from the diagnostic tests for HCC in updated AASLD practice guidelines [1]. However, it has been argued that intrahepatic cholangiocarcinoma is relatively rare in liver cirrhosis and CEUS can depict suggestive findings of cholangiocarcinoma [4]. As the imaging diagnosis of small nodules with 1-2 cm in size can be particularly challenging, a multi-modality approach is often needed. Biopsy is performed only when imaging findings are inconclusive. In this article, we explain the technical aspects of CEUS and its differences from CT and MRI. Then CEUS imaging findings of HCC and other cirrhosisrelated nodules and the role of CEUS in diagnosing tumor thrombosis and monitoring local ablation therapy for HCC are reviewed. WHAT IS CEUS? CEUS uses intravenous microbubble contrast agents that are small (3-5 μm) enough to pass through the pulmonary circulation. Microbubble contrast agents are approved for radiologic use in more than 50 countries and have excellent safety record with lower rate severe adverse reactions than CT contrast [5]. There are a few different types of microbubble contrast agents that are commercially available. Presently Definity (Lantheus Medical Imaging) and SonuVue (Bracco) are most widely used. These are purely intravascular contrast agents that show strong vascular enhancement after bolus injection and slowly diffuse into the blood over about 5 min. Therefore, the contrast agent can be repeatedly injected with about 5-min intervals as needed. Sonazoid (Daiichi), which is most actively used in Japan, shows similar vascular enhancement and is taken up by Kupffer cells in the late phase. CEUS requires a contrast specific imaging technique that is now widely available in most commercially available ultrasound systems. Low mechanical index (MI) contrast-specific mode is used to visualize the microbubbles continuously while suppressing signals from tissue. A dual-imaging mode, which enables simultaneous real-time display of gray-scale and contrast-specific mode, is essential for scanning small liver lesions. High MI frames can be used to disrupt microbubbles and the enhancement pattern of refilling the scanning plane can be evaluated. This is called disruption-replenishment technique and is useful to visualize vascular morphology within the tumor of enhancement pattern of rapidly-enhancing lesions, especially when it is used with maximum-intensity projection method [6]. WHY DO WE NEED CEUS? CEUS has several advantages over CT or MRI. Firstly, microbubbles can be safely injected in patients with renal failure as there is no renal excretion of the contrast [5]. CEUS is therefore a useful problem-solving method for characterizing liver masses when CT or MRI is contraindicated due to renal failure (Figure 1). There is no need of blood test for renal function before contrast injection. Secondly, CEUS allows real-time assessment of arterialphase enhancement, eliminating the issue of appropriate arterial-phase timing. CEUS often detects arterial-phase hypervascularity when CT or MRI fails to show it because of incorrect arterial-phase timing (Figure 2) [7]. Real-time evaluation also enables a detailed assessment of arterialphase filling pattern and vascular morphology within the liver lesion which are often critical for differential diagnosis of rapidly enhancing hypervascular liver lesions [8]. Thirdly, washout phenomenon (negative enhancement of liver lesion relative to the liver in the late phase) in ma April 7, 2014 Volume 20 Issue 13

217 Kim TK et al. CEUS in diagnosis of nodules A B Figure 2 A 63-year-old man with moderately-differentiated hepatocellular carcinoma. A: Computed tomography scan in the arterial phase shows a small exophytic nodule (arrow) which shows similar attenuation to the liver; B: The nodule (arrow) is slightly hypoattenuating to the liver in the delayed phase; C: Contrast-enhanced ultrasound (CEUS) in the arterial phase clearly demonstrates hypervascularity of the nodule (arrow); D: CEUS in the portal venous phase shows washout of the nodule (arrow). C D lignant liver lesions is more consistently seen on CEUS than CT/MRI. This is due to the different properties of the contrast material. Microbubbles in CEUS are purely intravascular and show washout in malignant tumors in the late phase; however, CT or MRI may not show washout in malignant tumors with high vascular permeability and large extracellular interstitial space because the contrast agent can leak into the interstitium [7,9]. Fourth, CEUS is relatively inexpensive and very well tolerated by the patients who are claustrophobic. CEUS can be easily repeated in short intervals if needed without the risk of ionizing radiation. Lastly, CEUS can be performed immediately when a new liver nodule is identified, allowing an immediate characterization of benign liver nodules such as hemangiomas and avoiding additional imaging tests [10]. On the other hand, CEUS is operator-dependent and requires extensive hands-on experience. There are sonographically difficult regions in the liver such as high right subphrenic area. CEUS is not an appropriate staging modality for HCC as it only images some part of the liver with each contrast injection. CT or MRI is always needed for proper tumor staging once HCC diagnosis is made. However, CEUS can be effectively used as a useful problemsolving method utilizing its unique advantages when CT or MRI is indeterminate or contraindicated (Figures 1 and 2) [10]. WHEN DO WE USE CEUS? Image findings of liver nodules HCC is characterized by arterial-phase hypervascularity and later washout on CEUS (Figures 1 and 2). The arterial-phase enhancement is often diffuse or heterogeneous. Peripheral rim-like enhancement is unusual in HCC and is commonly seen in metastasis or intrahepatic cholangiocarcinoma [11]. Large HCCs often show non-enhancing areas due to necrosis or internal hemorrhage. Maximum intensity projection images can visualize vascular morphology in HCC which is typically very irregular and dysmorphic [6]. Washout in HCC tends to be late and often begins later than 90 s after injection whereas metastases or intrahepatic cholangiocarcinomas consistently show rapid washout (< 60 s) [9]. Most of small cholangiocarcinomas that are infrequently detected during HCC surveillance can be characterized by CEUS by demonstrating rim-like arterial phase enhancement and rapid washout in our experience (Figure 3). Biopsy should be performed when these unusual enhancement patterns for HCC are observed on CEUS. Washout may not be seen in occasional cases of welldifferentiated HCC. Washout timing is related to the pathologic differentiation of HCC: well-differentiated HCC tends to show later washout or no washout whereas poorly-differentiated HCC tends to show rapid washout [12]. It is important to understand that most new hypervascular nodules on CEUS detected during HCC surveillance are HCC regardless of washout if the nodules do not show the appearance of hemangioma [13]. However, a biopsy is needed to confirm HCC for hypervascular nodules without washout. CEUS is superior to CT or MRI for detecting hypervascularity of HCC because of real-time evaluation of arterial-phase enhancement [14-16]. Therefore, CEUS can be effectively used for characterizing non-hypervascular, indeterminate lesions on CT and MRI (Figure 2). There is a small subset of hypovascular HCC which is mostly of well-differentiated pathology. These lesions usually show a transient hypovascularity 3592 April 7, 2014 Volume 20 Issue 13

218 Kim TK et al. CEUS in diagnosis of nodules A B Figure 3 A 70-year-old man with intrahepatic cholangiocarcinoma detected during surveillance for hepatocellular carcinoma. A: Gray-scale ultrasound shows a hypoechoic mass in the liver; B: Contrast-enhanced ultrasound (CEUS) in the arterial phase obtained 14 s after contrast injection shows mild hypervascularity in the periphery of the mass (arrows); C: CEUS obtained at 19 s after contrast injection shows diffuse hypervascularity of the mass (arrows); D: The mass (arrows) shows rapid washout at 28 s after contrast injection. Biopsy revealed cholangiocarcinoma. C D A B C Figure 4 A 69-year-old man with well-differentiated hypovascular hepatocellular carcinoma. A: Gray-scale ultrasound shows an exophytic hypoechoic mass in the liver; B: The mass (arrows) is hypoechoic relative to the adjacent liver in the arterial phase of contrast-enhanced ultrasound; C: The mass (arrows) is isoechoic to the liver in the portal venous phase. Biopsy revealed well-differentiated hepatocellular carcinoma. followed by gradual enhancement and the lesions become isoechoic relative to the normal in the portal venous phase and late phase (Figure 4). Regenerative nodules or dysplastic nodules (DNs) are usually non-hypervascular. CEUS may show a transient hypoenhancement relative to the liver in the arterial phase, reflecting the step-wise changes of nodule perfusion during the hepatocarcinogenesis (Figure 5). Therefore, there is an overlap of imaging findings on CEUS between DNs and well-differentiated HCCs [17]. The differentiation is difficult not only on imaging but also on histological examination. Recent practice guidelines recommend a biopsy for all new liver nodules 1 cm that are indeterminate on imaging [18] ; however, many of these nodules are borderline lesions, in which the differential diagnosis in small needle biopsy specimens is challenging due to histological heterogeneity within the nodules. In the setting of a competing potentially fatal disease (i.e., cirrhosis), the intensive identification, biopsy, and treatment of early HCCs has yet to be justified. Khalili et al [19], in their study of 93 indeterminate 1-2-cm nodules on dynamic CT, MRI, and CEUS, suggested a strategy of close imaging follow-up for most indeterminate 1-2-cm nodules, with selective application of biopsy for nodules with arterial hyperenhancement or in the presence of a synchronous typical HCC. Hemangiomas are frequently detected during HCC surveillance. Gray-scale ultrasound findings, such as diffuse hyperechogenicity or hyperechoic peripheral rim, can help suggest the diagnosis of hemangioma. In fatty liver, hemangiomas are often hypoechoic relative to the echogenic liver parenchyma. In our recent study [20], 43/ April 7, 2014 Volume 20 Issue 13

219 Kim TK et al. CEUS in diagnosis of nodules A B C Figure 5 A 69-year-old man with dysplastic nodule. A: Gray-scale ultrasound shows a small hypoechoic nodule (arrows) in the subcapsular portion of the liver; B: The nodule (arrows) is hypoechoic to the liver in the arterial phase of contrast-enhanced ultrasound; C: The nodule is isoechoic to the liver in the portal venous phase. Biopsy revealed low-grade dysplastic nodule. A B Figure 6 A 53-year-old woman with hemangioma. A: Grayscale ultrasound shows a hypoechoic nodule (arrow) in the subcapsular portion of the liver; B: There is a peripheral strong hyperenhancement (arrows) in the arterial phase of contrastenhanced ultrasound; C: The nodule (arrow) is homogeneously hyperechoic to the liver in the portal venous phase; D: The nodule (arrow) is homogeneously hyperintense to the liver without the appearance of peripheral enhancement in the arterial phase of contrast-enhanced T1-weighed magnetic resonance image. C D (23%) of newly detected nodules at HCC surveillance were hemangiomas. Immediate performance of CEUS can achieve a confident diagnosis by demonstrating the characteristic enhancement pattern that includes peripheral nodular enhancement, gradual central fill-in, and sustained enhancement and avoid further imaging tests such as CT or MRI. CEUS is also useful to demonstrate the characteristic enhancement pattern in fast filling hemangioma which often shows homogeneous enhancement in the arterial phase of CT or MRI (Figure 6) [21]. Nontumorous arterioportal shunting is a common mimicker of malignancy in cirrhotic liver. It is typically wedge-shaped, peripherally located, and homogeneous hypervascular in the arterial phase. The lesion becomes isointense to the liver in the venous phase and never shows washout (Figure 7). Focal fat deposits or focal fat sparing areas with nodular appearance can mimic the appearance of focal liver masses. There lesions are mostly isoechoic to the liver in the arterial and late phases on CEUS. The presence of normal portal veins within the lesion can confirm the benign lesion [21]. CEUS as guidance or post-radiofrequency ablation monitoring for HCC US is most commonly used for imaging guidance for radiofrequency ablation (RFA) of HCC because of its realtime imaging capability and no ionizing radiation. It is occasionally difficult to visualize the HCC lesion on US when the lesion is isoechoic to the liver or the underlying liver is severely cirrhotic with markedly heterogeneous echotexture. CEUS can help localize the lesion before RFA procedure by demonstrating a focal hypervascular 3594 April 7, 2014 Volume 20 Issue 13

220 Kim TK et al. CEUS in diagnosis of nodules A B Figure 7 A 65-year-old man with nontumorous arterioportal shunt. A: Contrast-enhanced ultrasound in the arterial phase shows a wedge-shaped hypervascular lesion (arrows) in the subcapsular portion of the liver; B: The lesion is not visualized in the portal venous phase due to isoechogenicity to the liver. A B Figure 8 A 70-year-old man with recurrent hepatocellular carcinoma after radiofrequency ablation. A: There is a focal nodular hypervascular lesion (arrows) adjacent to an ablation zone (*) in the arterial phase of contrast-enhanced ultrasound; B: The lesion (arrows) shows washout in the portal venous phase. * * A B C Figure 9 A 73-year-old man with malignant portal vein thrombosis associated with hepatocellular carcinoma. A: Gray-scale ultrasound shows a hypoechoic thrombus (arrow) within the anterior segmental branch of the right portal vein; B: The thrombus (arrows) is heterogeneously enhancing in the arterial phase of contrast-enhanced ultrasound; C: The thrombus (arrows) is hypoechoic relative to the liver in the portal venous phase. lesion. Marginal recurrence adjacent to the ablation zone may not be easily located on gray-scale US because of the pre-existing abnormality. CEUS can easily identify the exact location of the recurrent HCC that can provide excellent guidance for RFA needle positioning (Figure 8) [22]. For post-rfa monitoring for HCC, CT or MRI is primarily used in our institution. CEUS is often performed when there is an indeterminate lesion on CT or MRI and often clarifies the abnormality with high confidence. CEUS for determining malignant or benign venous thrombosis Malignant thrombosis in the portal veins or hepatic veins is a critical determinant of HCC staging as it directly influences treatment strategy. CEUS is highly accurate to differentiate malignant and benign venous thrombosis. CEUS demonstrates enhancement within the malignant thrombosis in the arterial phase and subsequent washout similar to the parenchyma HCC masses (Figure 9) [23]. The presence of formed vessels within the thrombus is another useful feature to diagnose malignant thrombosis. However, care should be taken in assessing formed vessels within the thrombus on CEUS as recanalized portion within chronic benign thrombosis may mimic the appearance of formed vessels in malignant thrombus. Occlusive thrombosis and expansion of the venous lumen are more frequently seen in malignant than benign thrombosis, but these are not specific features of malignancy April 7, 2014 Volume 20 Issue 13

221 Kim TK et al. CEUS in diagnosis of nodules CONCLUSION CEUS is an excellent imaging technique with several unique advantages over CT or MRI, including safe usage in renal failure patients, better detection of arterial-phase hypervascularity due to real-time imaging capability, consistent demonstration of washout in malignant lesions, and excellent patient s compliance. Therefore CEUS can be effectively used in the diagnostic algorithms of new liver nodules detected during HCC surveillance and preor post-rfa evaluation for HCC. REFERENCES 1 Bruix J, Sherman M. Management of hepatocellular carcinoma: an update. Hepatology 2011; 53: [PMID: DOI: /hep.24199] 2 Omata M, Lesmana LA, Tateishi R, Chen PJ, Lin SM, Yoshida H, Kudo M, Lee JM, Choi BI, Poon RT, Shiina S, Cheng AL, Jia JD, Obi S, Han KH, Jafri W, Chow P, Lim SG, Chawla YK, Budihusodo U, Gani RA, Lesmana CR, Putranto TA, Liaw YF, Sarin SK. 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Assessment of arterial hypervascularity of hepatocellular carcinoma: comparison of contrast-enhanced US and gadoxetate disodium-enhanced MR imaging. Eur Radiol 2012; 22: [PMID: DOI: / s ] 17 Kim TK, Lee KH, Khalili K, Jang HJ. Hepatocellular nodules in liver cirrhosis: contrast-enhanced ultrasound. Abdom Imaging 2011; 36: [PMID: DOI: / s ] 18 Bruix J, Sherman M, Llovet JM, Beaugrand M, Lencioni R, Burroughs AK, Christensen E, Pagliaro L, Colombo M, Rodés J. Clinical management of hepatocellular carcinoma. Conclusions of the Barcelona-2000 EASL conference. European Association for the Study of the Liver. J Hepatol 2001; 35: [PMID: DOI: / S (01) ] 19 Khalili K, Kim TK, Jang HJ, Yazdi LK, Guindi M, Sherman M. Indeterminate 1-2-cm nodules found on hepatocellular carcinoma surveillance: biopsy for all, some, or none? Hepatology 2011; 54: [PMID: DOI: / hep.24638] 20 Kim TK, Lee KH, Jang HJ, Haider MA, Jacks LM, Menezes RJ, Park SH, Yazdi L, Sherman M, Khalili K. Analysis of gadobenate dimeglumine-enhanced MR findings for characterizing small (1-2-cm) hepatic nodules in patients at high risk for hepatocellular carcinoma. Radiology 2011; 259: [PMID: DOI: /radiol ] 21 Kim TK, Jang HJ, Wilson SR. Benign liver masses: imaging with microbubble contrast agents. Ultrasound Q 2006; 22: [PMID: ] 22 Kim YJ, Lee MW, Park HS. Small hepatocellular carcinomas: ultrasonography guided percutaneous radiofrequency ablation. Abdom Imaging 2013; 38: [PMID: DOI: /s ] 23 Raza SA, Jang HJ, Kim TK. Differentiating malignant from benign thrombosis in hepatocellular carcinoma: contrast-enhanced ultrasound. Abdom Imaging 2014; 39: [PMID: DOI: /s ] P- Reviewers: Chan KM, Lin ZY, Wang K S- Editor: Wen LL L- Editor: A E- Editor: Wang CH 3596 April 7, 2014 Volume 20 Issue 13

222 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. ORIGINAL BRIEF ARTICLE Clinicopathological study of primary biliary cirrhosis with interface hepatitis compared to autoimmune hepatitis Mio Kobayashi, Yuko Kakuda, Kenichi Harada, Yasunori Sato, Motoko Sasaki, Hiroko Ikeda, Mitsuhiro Terada, Munenori Mukai, Shuichi Kaneko, Yasuni Nakanuma Mio Kobayashi, Yuko Kakuda, Kenichi Harada, Yasunori Sato, Motoko Sasaki, Yasuni Nakanuma, Department of Human Pathology, Kanazawa University Graduate School of Medicine, Kanazawa , Japan Hiroko Ikeda, Division of Pathology, Kanazawa University Hospital, Kanazawa , Japan Mitsuhiro Terada, Department of Gastroenterology, Kouseiren Takaoka Hospital, Takaoka , Japan Munenori Mukai, Department of Pathology, Kouseiren Takaoka Hospital, Takaoka , Japan Shuichi Kaneko, Department of Gastroenterology, Kanazawa University Graduate School of Medicine, Kanazawa , Japan Author contributions: Kobayashi M performed the majority of experiments and wrote the manuscript; Kakuda Y analyzed the data; Harada K, Sato Y and Sasaki M contributed to the design of the study; Ikeda H, Terada M, Mukai M and Kaneko S provided the experimental materials; Nakanuma Y organized the study. Supported by Primary Biliary Cirrhosis Subdivision of Intractable Hepatobiliary Diseases Study Group of Japan (Chairman, Hirohito Tsubouchi; Department of Human and Environmental Sciences, Graduate School of Medical and Dental Sciences, Kagoshima University, Kagoshima, Japan) Correspondence to: Yasuni Nakanuma, MD, Department of Human Pathology, Kanazawa University Graduate School of Medicine, Takaramachi 13-1, Kanazawa , Japan. nakanuma@staff.kanazawa-u.ac.jp Telephone: Fax: Received: May 28, 2013 Revised: July 26, 2013 Accepted: August 17, 2013 Published online: April 7, 2014 Abstract AIM: To investigate histological and immunohistochemical differences in hepatitis between autoimmune hepatitis (AIH) and primary biliary cirrhosis (PBC) with AIH features. METHODS: Liver needle biopsies of 41 PBC with AIH features and 43 AIH patients were examined. The activity of periportal and lobular inflammation was scored 0 (none or minimal activity) to 4 (severe), and the degree of hepatitic rosette formation and emperipolesis was semiquantatively scored 0-3. The infiltration of mononuclear cells positive for CD20, CD38, CD3, CD4, and CD8 and positive for immunoglobulins (IgG, IgM, and IgA) at the periportal areas (interface hepatitis) and in the hepatic lobules (lobular hepatitis) were semiquantitatively scored in immunostained liver sections (score 0-6). Serum aspartate aminotransferase (AST), immunoglobulins, and autoantibodies at the time of liver biopsy were correlated with the histological and immunohistochemical scores of individual lesions. RESULTS: Lobular hepatitis, hepatitic rosette formation, and emperipolesis were more extensive and frequent in AIH than in PBC. CD3+, CD4+, and CD8+ cell infiltration scores were higher in the hepatic lobules and at the interface in AIH but were also found in PBC. The degree of mononuclear cell infiltration correlated well with the degree of interface and lobular hepatitis in PBC, but to a lesser degree in AIH. CD20+ cells were mainly found in the portal tracts and, occasionally, at the interface in both diseases. Elevated AST correlated well with the hepatocyte necroinflammation and mononuclear cell infiltration, specifically CD38+ cells in PBC. No correlation existed between autoantibodies and inflammatory cell infiltration in PBC or AIH. While most AIH cases were IgG-predominant at the interface, PBC cases were divided into IgM-predominant, IgM/IgGequal, and IgG-predominant types, with the latter sharing several features with AIH. CONCLUSION: These results suggest that the hepatocellular injuries associated with interface and lobular hepatitis in AIH and PBC with interface hepatitis may not be identical Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Primary biliary cirrhosis; Autoimmune hepa April 7, 2014 Volume 20 Issue 13

223 Kobayashi M et al. AIH and PBC with interface hepatitis titis; Interface hepatitis; Lobular hepatitis; Plasma cell subclass Core tip: Primary biliary cirrhosis (PBC) can sometimes present with features of autoimmune hepatitis (AIH). Therefore, the clinicopathological features and immunophenotypes of infiltrated mononuclear cells of AIH and PBC with AIH features were examined. Lobular hepatitis, hepatitic rosette formation, and emperipolesis were more frequent in AIH than PBC. The degree of mononuclear cell infiltration correlated well with hepatitis. Furthermore, elevation of aspartate aminotransferase levels correlated well with necroinflammation and infiltration of mononuclear cells in PBC, which tended to be IgM-predominant. These results suggest that hepatocellular injuries in AIH and PBC may not be identical. Kobayashi M, Kakuda Y, Harada K, Sato Y, Sasaki M, Ikeda H, Terada M, Mukai M, Kaneko S, Nakanuma Y. Clinicopathological study of primary biliary cirrhosis with interface hepatitis compared to autoimmune hepatitis. World J Gastroenterol 2014; 20(13): Available from: URL: com/ /full/v20/i13/3597.htm DOI: org/ /wjg.v20.i INTRODUCTION Primary biliary cirrhosis (PBC) is characterized by the progressive destruction of interlobular bile ducts (chronic nonsuppurative destructive cholangitis) and frequent antimitochondrial antibodies (AMAs) in the serum [1-8]. While some PBC patients may remain in the initial stage with chronic cholangitis, a considerable number exhibit histological progression suggested by interface changes, such as chronic cholestatic changes (biliary piecemeal necrosis) and hepatitic features (lymphocytic piecemeal necrosis or interface hepatitis) [9-11]. Rarely, PBC patients with considerable interface hepatitis will also have circulating antinuclear antibodies (ANAs), a characteristic feature of autoimmune hepatitis (AIH). These cases are referred to as PBC with AIH features or PBC-AIH overlapping syndrome [12-17]. These patients may have a more severe disease course with the earlier development of liver fibrosis and liver failure, though steroid or immunosuppressive therapies may be effective [1,3,11,12,15-18]. Histologically, AIH exhibits the features of chronic hepatitis with interface hepatitis and plasma cells are reportedly predominant in the portal tract and at the interface [19-21]. Entrapment of hepatocytes in the enlarged portal tract, called hepatitic rosette formation, is relatively frequent and lobular hepatitis is often severe in AIH. Furthermore, engulfment of lymphocytes by hepatocytes, termed emperipolesis, is often found and has been adopted as a useful histological feature for the diagnosis of AIH [19,22]. The combination of these lesions has been regarded as a typical feature of AIH [22]. Histologically, the hepatitic features of PBC resemble those of AIH. Plasma cells are relatively prominent at the interface of PBC with interface hepatitis, similar to AIH [3,11]. However, several histopathological studies have distinguished PBC from AIH. Several reports have shown that the density and proportion of IgM+ and IgG+ plasma cells in portal tracts can be used to differentiate AIH from PBC by liver biopsies [9,10,23,24]. In addition, Graham et al [25] reported that CD1a infiltration favors a diagnosis of PBC. However, differences and similarities in the hepatitic lesions at the interface and in the hepatic lobules between these two groups have not been studied in detail. Whether the hepatitic features in PBC reflect the overlap of AIH in PBC or an inherent feature of PBC is yet to be determined [3,11,12,14,26]. In this study, we examined similarities and differences in interface hepatitis and the lobular changes in AIH and PBC with interface hepatitis by needle liver biopsies and immunohistochemistry. The subtypes of infiltrating mononuclear cells in interface and lobular hepatitis were also compared. The histological and immunohistochemical features of hepatic necroinflammation were then correlated with the clinical and laboratory features of the two groups. MATERIALS AND METHODS Selection of patients with PBC and AIH and tissue preparation In this study, we tried to compare PBC with interface hepatitis with AIH, and we selected PBC patients with interface hepatitis by adopting Batts and Ludwig s scoring system [27]. In this system, the activity of periportal (interface) and lobular inflammation was scored semiquantitatively in routinely processed sections: portal/periportal activity score 0 (none or minimal activity), 1 (portal inflammation only), 2 (mild interface hepatitis), 3 (moderate interface hepatitis), and 4 (severe interface hepatitis); and lobular activity score 0 (none), 1 (inflammatory cells, but no hepatocellular death), 2 (focal cell death), 3 (severe focal cell death, confluent necrosis without bridging), and 4 (damage including bridging necrosis). We collected liver specimens of PBC with portal/periportal activity scores 2 (Figure 1A and B). Patient collection: A total of 84 liver needle biopsies were used in this study. They consisted of specimens from 41 patients with PBC with more than a 2+ portal/periportal activity score (above-mentioned) and 43 patients with AIH, obtained at Kanazawa University hospital and affiliated hospitals ( ). All AIH cases were associated with a portal/periportal activity score of 2. All of these patients were negative for hepatitis B and hepatitis C viral screening and had no history of alcohol abuse. All PBC and AIH cases fulfilled the clinical and pathological diagnostic features of PBC [1] and AIH type Ⅰ [10,12], respectively. Regarding a diagnosis of PBC, when histological changes compatible with PBC such as chronic cholangitis and/or bile duct loss were 3598 April 7, 2014 Volume 20 Issue 13

224 Kobayashi M et al. AIH and PBC with interface hepatitis A B C D Figure 1 Histological features of primary biliary cirrhosis with interface hepatitis and autoimmune hepatitis. A: Moderate interface hepatitis (score 3) in autoimmune hepatitis (AIH); B: Moderate interface hepatitis (score 3) in primary biliary cirrhosis (PBC); C: Rosette formation; regenerative hepatocytes arranged around a bile canaliculus (arrow), found in AIH; D: Emperipolesis, the engulfment of lymphocytes within hepatocytes (arrowheads), was found in areas of interface hepatitis. Hematoxylin and eosin stain, original magnifications 200 ( A, B); 600 (C, D). Table 1 Main clinicopathological features of primary biliary cirrhosis with interface hepatitis and autoimmune hepatitis PBC 1 (n = 41) AIH (n = 43) Gender (male:female) 5:36 4:39 Age (yr, mean ± SD) 59.0 ± ± 10.8 Laboratory data Ratio of AMA(+) or M2(+) and titer of AMA Ratio of ANA(+) and titer 2 75% and ± % and ± % and 0 ± 0 91% and ± AST (IU/L) 51.6 ± ± ALT (IU/L) 54.0 ± ± ALP (IU/L) ± ± IgG (mg/dl) ± ± IgM (mg/dl) ± ± IgA (mg/dl) ± ± Laboratory data is shown as the mean ± SD; anti-mitochondrial antibodies (AMA)(+) 40; M2(+) 5; antinuclear antibodies (ANA)(+) With interface hepatitis; 2 The average titer of ANA was higher in primary biliary cirrhosis (PBC) than in autoimmune hepatitis (AIH) because two PBC cases had a titer of 5120, which influenced the average. AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; ALP: Alkaline phosphatase. found in the patients with elevation of biliary enzymes and serological data compatible with PBC, such cases were diagnosed as PBC and included in this study. Other hepatobiliary diseases were excluded in these cases. No patient had a history of treatment with ursodeoxycholic acid (PBC) or steroid or immunosuppressive drugs (AIH) at the time of liver biopsy. The main clinicopathological and laboratory findings of these cases are shown in Table 1. As for AMA positivity, AMA and/or M2 were evaluated in a combination in individual cases. Tissue preparation: All liver specimens were fixed in 10% formalin and embedded in paraffin. Routinely processed hematoxylin and eosin staining was available in one PBC and 19 AIH cases. Routine histological stains and immunohistochemistry for the remaining cases were perfumed on 4 μm-thick serial sections. Histological features The activity of periportal (interface) and lobular inflammation was scored semiquantitatively in routinely processed sections by Batts and Ludwig s scoring system as described above [27]. Hepatitic rosette formation was defined as the entrapment of several hepatocytes with dilated canaliculi and clear cytoplasm in the enlarged portal tract (Figure 1C), and emperipolesis was defined as engulfed lymphocyte(s) in the cytoplasm of hepatocytes in the periportal regions (Figure 1D). The lesions were surveyed and scored as follows: 0 (no lesion), 1 (one to several lesions), 2 (intermediate), and 3 (many lesions). Immunohistochemistry The antibodies used in this study and their sources are 3599 April 7, 2014 Volume 20 Issue 13

225 Kobayashi M et al. AIH and PBC with interface hepatitis Table 2 Primary antibodies used in this study Primary antibody Source Clone Animal Type Pretreatment Dilution CD3 Nichirei PS1 Mouse Mono MW 3 1:1 CD4 Nichirei 4B12 Mouse Mono MW 3 1:1 CD8 Dako C8/144B Mouse Mono MW 1 1:20 CD20 Dako L26 Mouse Mono - 1:400 CD38 Epitomics EPR4106 Rabbit Mono AC 2 1:800 CD138 Dako ML15 Mouse Mono MW 3 1:100 IgG Dako A57H Mouse Mono - 1:200 IgM Nichirei R1/69 Mouse Mono MW 3 1:1 IgA Novocastra N1CLA Mouse Mono PK 1:300 1 Citrate, ph = 6.0; 2 Ethylenediaminetetraacetic acid (EDTA) buffer, ph8.0; 3 Tris EDTA buffer, ph = 9.0. MW: Microwave; AC: Autoclave; PK: Proteinase K, ph = 7.5 (Dako). shown in Table 2. Deparaffinized sections were pretreated by methods suitable for each antibody, if necessary, followed by pretreatment with 1% H2O2 in methanol for 20 min to block endogenous peroxidase activity, and then with normal goat serum for 20 min to block nonspecific reactions. The sections were then incubated overnight at 4 with each primary antibody at optimal dilutions and then at room temperature for 1 h with anti-mouse (except for CD38) or anti-rabbit (for CD38) immunoglobulins conjugated to a peroxidase-labeled dextran polymer (Simple Staining Kit; Nichirei). The reaction products were developed by immersing the sections in a solution of 3,3 -diaminobenzidine tetrahydrochloride solution containing 0.03% hydrogen peroxide. Nuclei were lightly counterstained with hematoxylin. Double immunofluorescence staining Double-color immunostaining of CD38 and CD138, IgG, or IgM was performed as follows: deparaffinized sections were pretreated and incubated overnight at 4 with a mixture of primary antibodies for CD38 and CD138, CD38 and IgG, or CD38 and IgM (Table 2). The sections were firstly incubated with the Alexa Fluor 488 (Invitrogen, Carlsbad, CA) secondary antibody against rabbit IgG for the detection of CD38 and with Alexa Fluor 594 (Invitrogen) secondary antibody against mouse IgG for the detection of CD138 and IgM or the secondary antibody against mouse IgM for the detection of IgG. Semiquantitative immunohistochemical evaluation of inflammatory cells The infiltration of mononuclear cells positive for CD20, CD38, CD3, CD4, and CD8 and for immunoglobulin classes (IgG, IgM, and IgA) at the periportal areas (interface hepatitis) and in the hepatic lobules (lobular hepatitis) were semiquantitatively scored twice in immunostained liver sections (0, no positive cells; 1, minimal, a few positive cells in several regions; 2, moderate, considerable amount of positive cells in about half of the regions; and 3, many positive cells in almost all regions). The sum of the two evaluations was used as the individual score of individual cases (score 0-6). CD20 is known to be expressed in immature B cells, CD38 in mature plasma cells and also in other T or cell series, CD3 in T cells, CD4 in helper T cells, and CD8 in cytotoxic T cells [28,29]. IgG, IgM, and IgA are expressed in mature plasma cells. CD138 is more specifically expressed in plasma cells [28], while it is also expressed variably in bile ductules and bile ducts as well as in the hepatocyte membranes in our preliminary study. The expressions of CD38 and CD138, shown by double immunostaining, were similar in the infiltrating mononuclear cells (data not shown). There were no double positive cells when double immunostaining was performed for CD38 and CD3 in AIH and PBC (data not shown); therefore, T cells were not included in the pool of CD38+ cells in the liver tissue specimens in this study. Thus, CD38 was used to survey mature plasma cells. Comparison of histopathological and immunohistochemical data with laboratory data Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), IgG, IgM, IgA, AMAs, and ANAs at the time of liver biopsy were correlated with the scores of individual lesions (degree of interface hepatitis, lobular hepatitis, emperipolesis, and hepatitic rosette formation) and scores of infiltrating mononuclear cells positive for individual phenotypes (CD20, CD38, CD3, CD4, CD8, IgG, IgM, and IgA). Statistical analysis All statistical analyses were performed using JMP software (version 8.0, SAS Institute Japan, Tokyo, Japan). Values were expressed graphically as the mean ± SD and median. Statistical differences between groups were determined using a two tailed Mann-Whitney unpaired test and Fisher s exact probability test with 95%CI. The correlation coefficient of 2 factors was evaluated using Spearman s rank correlation test. P values of < 0.05 were considered significant. RESULTS Histopathologies of the interfaces and hepatic lobules of PBC and AIH Interface hepatitis and lobular hepatitis were more or less diffuse in the needle liver biopsies in AIH than in PBC April 7, 2014 Volume 20 Issue 13

226 Kobayashi M et al. AIH and PBC with interface hepatitis A B b IFH 3 LH PBC AIH PBC AIH C a D b 3 3 Rosette 2 1 Emperipolesis PBC AIH PBC AIH Figure 2 Comparison of necroinflammation in autoimmune hepatitis and primary biliary cirrhosis with interface hepatitis. A: The scores of interface hepatitis (IFH) were not significantly different between primary biliary cirrhosis (PBC) (2.49 ± 0.64) and AIH (2.74 ± 0.66) (P = ) because PBC cases with IFH were chosen for this comparative study with autoimmune hepatitis (AIH); B: The scores of lobular hepatitis (LH) were higher in AIH (2.58 ± 0.70) than in PBC (2.22 ± 0.61) (P = ); C: The scores of hepatitic rosette formation were higher in AIH (0.55 ± 0.83) than in PBC (0.17 ± 0.44) (P = ); D: The scores of emperipolesis were higher in AIH (1.00 ± 0.96) than in PBC (0.32 ± 0.52) (P = ). Horizontal bars of the graph show the median scores; a P < 0.05 vs control, b P < 0.01 vs control in the Mann-Whitney test. Table 3 Degree of mononuclear cell infiltration with respect to immunological phenotypes at the interface and in the hepatic lobules PBC 1 AIH P value At the interface; scores of infiltrating mononuclear cells positive for CD3 2.8 ± 1.2 (3) ± 1.1 (4) CD4 1.5 ± 1.0 (1) 2.0 ± 1.2 (2) CD8 1.5 ± 0.9 (1) 2.7 ± 1.7 (3) CD ± 0.6 (1) 1.0 ± 1.0 (1) CD ± 1.5 (3) 3.7 ± 1.3 (4) IgG 1.3 ± 1.0 (1) 1.8 ± 1.3 (2) IgM 1.0 ± 0.8 (1) 0.6 ± 0.8 (0.5) IgA 0.5 ± 0.6 (0) 0.6 ± 0.7 (1) In the hepatic lobules; scores of infiltrating mononuclear cells positive for CD3 2.2 ± 0.9 (2) 3.3 ± 1.3 (3) CD4 0.8 ± 0.9 (0.5) 1.8 ± 1.1 (2) CD8 0.8 ± 0.8 (1) 2.5 ± 1.5 (2) < CD ± 0.6 (0) 0.7 ± 0.8 (0.5) CD ± 1.0 (1) 2.0 ± 1.2 (2) IgG 0.6 ± 0.8 (0) 1.4 ± 1.3 (1) IgM 0.4 ± 0.5 (0) 0.6 ± 0.7 (0.5) IgA 0.3 ± 0.7 (0) 0.6 ± 0.7 (0.5) With interface hepatitis; 2 The scores were graded from 0-6 for each case, and are shown as the mean ± SD and (median). PBC: Primary biliary cirrhosis; AIH: Autoimmune hepatitis. Lobular hepatitis was more or less irregularly distributed in the liver specimens of PBC. There was no significant difference in the scores of interface hepatitis between AIH and PBC (Figure 2A), which was expected because PBC cases with interface hepatitis were selected in this study. The scores of lobular hepatitis were significantly higher in AIH than in PBC (Figure 2B), and the former frequently showed zonal or even bridging necrosis in addition to focal hepatocellular necrosis. The scores of hepatitic rosette formation (Figure 1C) and emperipolesis (Figure 1D) were significantly higher in AIH than in PBC (Figure 2C and D). Immunophenotypes of infiltrating inflammatory cells at the interfaces and in the hepatic lobules The degree of infiltration (scores) of mononuclear cells with individual immunophenotypes at the interfaces and in the hepatic lobules are shown in Table 3. CD3, CD4, and CD8: CD3+ cells were frequently observed in the portal tracts, at the interface, and also within the hepatic lobules in both diseases (Figures 3A and 4A). Their scores at the interface and within hepatic lobules were higher in AIH than in PBC. The scores of CD April 7, 2014 Volume 20 Issue 13

227 Kobayashi M et al. AIH and PBC with interface hepatitis A B C D E BD * Figure 3 Immunohistochemical findings of primary biliary cirrhosis with interface hepatitis. Many CD3+ cells were found within the portal tract and at the interface (A), whereas CD38+ cells heavily infiltrated the interface (B), IgG+ cells were mainly found in the portal tract and also at the interface (C), and IgM+ cell infiltration was predominant at the interface (D). Double staining for IgM (red) and CD38 (green) (E) showed that IgM-CD38 double positive cells, IgM-producing plasma cells, were frequently seen in the periportal area (star) (original magnifications: 200). IgM+ plasma cells were also found around the intralobular bile duct. Asterisk: Hepatic lobule, BD: Bile duct. A B C D E * Figure 4 Immunohistochemical findings of autoimmune hepatitis. CD3+ cells were found within the portal tract and in the periportal area (A), whereas CD38+ cells infiltrated prominently in the periportal area (B). IgG+ cells (C) and IgM+ cells (D) were also found in the periportal area, and IgG+ cell infiltration was predominant in the majority of autoimmune hepatitis cases. The distribution of these cells was similar to primary biliary cirrhosis (Figure 3); E: Double staining for IgG (red) and CD38 (green) showed that IgG-CD38 double positive cells, which may be IgG-producing plasma cells, were frequently seen in the periportal area (star). Original magnifications: 200. Asterisk: Hepatic lobule. cell infiltration at the interface and the hepatic lobules increased with the degree of interface hepatitis and lobular hepatitis in PBC (Figure 5A). The scores of CD3+ cell infiltration at the interface also increased with the degree of interface hepatitis in AIH, but such a correlation was not seen in lobular hepatitis (Figure 5B). The distribution of CD4 and CD8 was similar to that of CD3, though their number was smaller than that of 3602 April 7, 2014 Volume 20 Issue 13

228 Kobayashi M et al. AIH and PBC with interface hepatitis A 6 5 r = P < PBC 5 4 r = P = B 6 5 r = P = AIH 6 5 r = P = CD3 interface CD3 lobular 3 2 CD3 interface CD3 lobular IFH LH IFH LH C 5 4 r = P < r = P = D 5 4 r = P = r = P = CD4 interface CD4 lobular 2 1 CD4 interface CD4 lobular IFH LH IFH LH E CD8 interface r = P < CD8 lobular r = P = F CD8 interface r = P = CD8 lobular r = P = IFH LH IFH LH G 6 5 r = P < r = P = H 6 5 r = P = r = P = CD38 interface CD38 lobular 3 2 CD38 interface CD38 lobular IFH LH IFH LH Figure 5 Correlation between scores for necroinflammation and infiltrating mononuclear cells positive for CD3, CD4, CD8, and CD38 in primary biliary cirrhosis with interface hepatitis and autoimmune hepatitis. A: In primary biliary cirrhosis (PBC), the degree of CD3+ mononuclear cell infiltration and the scores of interface hepatitis and lobular hepatitis showed positive correlations; B: In autoimmune hepatitis (AIH), the degree of CD3+ mononuclear cell infiltration and the scores of interface hepatitis showed positive correlation, whereas that of CD3+ mononuclear cell infiltration and lobular hepatitis failed to correlate positively; C: In PBC, the degree of CD4+ mononuclear cell infiltration and the scores of interface hepatitis and lobular hepatitis showed positive correlations; D: In AIH, the degree of CD4+ mononuclear cell infiltration and the scores of interface hepatitis showed a positive correlation, whereas that of CD4+ mononuclear cell infiltration and lobular hepatitis failed to correlate positively; E: In PBC, the degree of CD8+ mononuclear cell infiltration and the scores of interface hepatitis and lobular hepatitis showed positive correlations; F: In AIH, the degree of CD8+ mononuclear cell infiltration and the scores of interface hepatitis and lobular hepatitis showed positive correlations; G: In PBC, the degree of CD38+ mononuclear cell infiltration and the scores of interface hepatitis and lobular hepatitis showed positive correlations; H: In AIH, the degree of CD38+ mononuclear cell infiltration and the scores of interface hepatitis showed positive correlation, whereas that of CD38+ mononuclear cell infiltration and lobular hepatitis failed to correlate positively. Spearman s rank correlation test in A-H. CD3 in both diseases. The scores of CD4+ and CD8+ T cells at the interface and in the hepatic lobules were higher in AIH than in PBC, and their scores increased with the degree of interface hepatitis and lobular hepatitis, respectively, in PBC (Figure 5C and E). The scores of CD4+ and CD8+ cells in AIH increased with the degree of interface hepatitis. CD8+ cells were associated with the scores of lobular hepatitis, whereas CD4+ cells were not (Figure 5D and F). CD20: CD20+ cells were mainly found in the portal tracts, and occasionally at the interface in both diseases. While the number of CD20+ cells was smaller than that of CD3+, CD4+, and CD8+ cells, they were relatively 3603 April 7, 2014 Volume 20 Issue 13

229 Kobayashi M et al. AIH and PBC with interface hepatitis % PBC 16 more numerous at the interface and hepatic lobules in AIH than in PBC. CD38: CD38+ cells with a plump cytoplasm, corresponding to mature plasma cells, were the most predominant at the interface in both diseases (Figure 3B and 4B). The number of these cells was higher in AIH than in PBC both at the interface and in hepatic lobules. The scores of CD38+ cell infiltration at the interface and within the hepatic lobules increased with the degree of interface hepatitis and lobular hepatitis in PBC (Figure 5G). Their scores at the interface increased with the degree of interface hepatitis in AIH, but such correlations were not seen within the hepatic lobules (Figure 5H). Analysis of infiltrating plasma cells with respect to immunoglobulin classes Infiltration of IgG+, IgM+, and IgA+ plasma cells: IgG+ plasma cells were frequently observed at the interfaces in PBC and AIH (Figures 3C and 4C), and their scores were significantly higher in AIH (Table 3). IgM+ plasma cells were found in interface hepatitis in both diseases (Figures 3D and 4D), and the scores were significantly higher in PBC. These cells were not prominent in the hepatic lobules in both diseases. Double immunostaining showed that IgG+ and IgM+ cells were also CD38+ (Figures 3E and 4E). Clinicopathological features of the IgG- or IgMpredominant type in PBC: By comparing the density of IgG+ and IgM+ plasma cells at the interface, 16 cases of AIH were revealed to be IgG-predominant, 8 cases to be IgM/IgG-equal, and none IgM-predominant. In contrast, 5 cases of PBC were IgM-predominant, 23 were IgM/IgG-equal, and 12 were IgG-predominant (Figure 6). Then each PBC case was classified into one of 2 groups: group A (IgM/IgG-equal and IgM-predominant types) 0 8 AIH IgG < IgM IgG = IgM IgG > IgM Figure 6 Classification of primary biliary cirrhosis with interface hepatitis and autoimmune hepatitis based on the predominance of the subclass of plasma cells infiltrating the interface. The majority of autoimmune hepatitis (AIH) cases (16 cases) were IgG-predominant, whereas 8 cases were IgG/IgMequal. There are no IgM-predominant AIH cases. In primary biliary cirrhosis (PBC), 23 cases were IgG/IgM-equal, whereas 12 cases were IgG-predominant and 5 cases were IgM-predominant. Table 4 Clinicopathological features of primary biliary cirrhosis based on the predominant immunoglobulin class of the plasma cell No. of cases (female:male) Group A 28 (25:3) Group B 12 (10:2) P value 1.3 (3) Age (yr) 56.1 ± 14.5 (56) 66.0 ± 7.2 (66) Laboratory data AST (IU/L) 50.5 ± 31.2 (43.5) 55.0 ± 28.3 (42.5) ALT (IU/L) 54.2 ± 43.6 (45.5) 55.0 ± 35.4 (46.5) ALP (IU/L) ± (446) ± (358.5) IgG (mg/dl) ± (1600) ± (2062.5) IgM (mg/dl) ± ± (133.5) (280.5) IgA (mg/dl) ± (281) ± (268.5) AMA, M % 55.60% (positive ratio) ANA (positive ratio) 65.40% 80.00% Histological findings (scores) Interface hepatitis ± 0.6 (2) 2.8 ± 0.6 (3) Lobular hepatitis ± 0.6 (2) 2.3 ± 0.6 (2) Infiltration of mononuclear cells positive for (scores) CD3 at the interface 2.7 ± 1.2 (2.5) 2.8 ± 3 CD38 at the interface 2.4 ± 1.5 (2) 3.3 ± 1.3 (4) CD3 in the hepatic 2.1 ± 0.8 (2) 2.4 ± 1.2 (2) lobules 3 CD38 in the hepatic lobules ± 0.9 (1) 1.6 ± 1.1 (2) The score was graded from 2 to 4 for each case; 2 the score was graded from 0 to 4 for each case; 3 The score was graded from 0 to 6 for each case. Group A, IgM-predominant cases and IgM/IgG-equal cases; group B, IgG-predominant cases. Age, laboratory data expect autoantibodies and scores are shown as the mean ± SD and (median); anti-mitochondrial antibodies (AMA)(+) 40; M2(+) 5; antinuclear antibodies (ANA)(+) 40. AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; ALP: Alkaline phosphatase. and group B (IgG-predominant type). The laboratory data, histological features, and degree of inflammatory cell infiltration were compared between the two groups (Table 4). Serum IgM levels were significantly higher in group A, and interface hepatitis activities were slightly higher in group B. Interestingly, the degree of CD38+ cell infiltration at the interface and in the hepatic lobules was significantly higher in group B, which suggests that PBC in group B may share more features with AIH. There was no correlation in the positive ratio of AMA or M2 and that of ANA between these two groups. Laboratory data and immunophenotypic features of interface hepatitis and lobular hepatitis The laboratory data and immunophenotypes of infiltrating mononuclear cells are summarized in Tables 5 and 6. As a marker of hepatocellular damage, the serum level of AST was evaluated. Serum level of AST and interface hepatitis, lobular hepatitis, and mononuclear cell infiltration: The degree of scores for CD3+, CD4+, CD38+, IgG+, IgM+, 3604 April 7, 2014 Volume 20 Issue 13

230 Kobayashi M et al. AIH and PBC with interface hepatitis Table 5 Correlation between the elevations in aspartate aminotransferase and the scores of hepatitis and mononuclear cell infiltration of primary biliary cirrhosis and autoimmune hepatitis Scores of inflammatory cells at the interface vs elevation of aspartate aminotransferase 1 With interface hepatitis. and IgA+ cell infiltration at the interface and within the hepatic lobules and that of CD8+ cell infiltration at the interface correlated well with the elevation in AST (Table 5), suggesting that these mononuclear cells play a role in immune-mediated hepatocellular injuries at the interfaces and within the hepatic lobules. In AIH, only the degree of lobular hepatitis, CD3+ cell and CD8+ cell infiltration within the hepatic lobules was correlated with the elevation in AST, whereas the other correlations found in PBC were not evident. Correlation between AMA or M2 and ANA positivity and the degree of interface hepatitis and infiltration of mononuclear cells positive for CD3, CD38, IgG, and IgM: There was no difference in the degree of interface hepatitis between AMA or M2 and ANA positive and negative groups in both AIH and PBC (Table 6). There was also no difference in the degree of infiltrated mononuclear cells expressing CD3, CD38, IgG, or IgM at the interface between AMA or M2 positive and negative groups in PBC or between the ANA positive and negative groups in AIH. DISCUSSION Primary biliary cirrhosis 1 Autoimmune hepatitis r P value r P value Scores of interface hepatitis Scores of lobular hepatitis Scores of CD3+ cells at the interface Scores of CD3+ in the lobules Scores of CD4+ at the interface Scores of CD4+ in the lobules Scores of CD8+ at the interface Scores of CD8+ in the lobules Scores of CD38+ at the interface Scores of CD38+ in the lobules Scores of IgG+ at the interface Scores of IgM+ at the interface The findings obtained in this study can be summarized as follows: (1) AIH exhibited more intense lobular hepatitis and more frequent hepatitic rosette formation and emperipolesis than that seen in PBC with interface hepatitis; (2) CD3+ and CD38+ cells were predominant in interface and lobular hepatitis in PBC, and increased with the degree of interface and lobular hepatitis in PBC. CD4+ and CD8+ showed a similar distribution to CD3+ cells, and the degree of infiltration of these cells correlated well with the degree of necroinflammation and elevations in AST. In AIH, the infiltration of these cells was more intense, while the degree of mononuclear cells did not correlate well with the degree of necroinflammation and elevations in AST seen in PBC; and (3) IgG+ plasma cells at the interface were predominant in AIH, while IgM+ predominant and IgG+/IgM+ equal cases were common in PBC. Thus, it seems likely that immunopathological processes appear to be similar at the hepatitic lesions in PBC and AIH, whereas hepatocellular injuries may not occur by the same mechanisms. PBC and AIH are relatively well-defined clinicopathological entities; nevertheless, the histological distinction between PBC and AIH is sometimes challenging because interface hepatitis, which is characteristic of AIH, may also be encountered in PBC [9]. This study found that lobular hepatitis was more extensive in liver biopsies in AIH than in PBC with interface hepatitis. In the latter, such changes were more or less irregularly distributed in the liver specimens. Hepatitic rosette formation and emperipolesis have reportedly been significantly correlated with autoimmune liver disease [30] and are regarded as the typical features of AIH in the simplified AIH scoring system [22]. In this study, both of these features were also found in PBC, though their incidence and scores were much lower. These hepatitic features may be closely related to the progression of these diseases and may eventually be followed by the development of fibrosis and cirrhosis in PBC and AIH [3,19]. However, it is unclear whether the mechanism(s) of hepatocellular injuries are the same in these two diseases. CD3+, CD4+, and CD8+ T cells were found in interface and lobular hepatitis in PBC and their scores at the interface and in the hepatic lobules increased with the degree of necroinflammation and correlated well with the elevation in AST. These cells have been suggested to be involved in immune-mediated hepatocellular injuries in PBC, and CD8+ cells particularly may be cytotoxic against hepatocytes. Interestingly, mononuclear cells with similar phenotypes were also found in AIH and were more prominent in the hepatitic lesions than in PBC. While the infiltration scores of CD3+ and CD8+ cells were also correlated with the degree of lobular hepatitis in AIH, other correlations between the infiltration of these cells and the degree of necroinflammation and elevations in AST shown in PBC were not evident in AIH. Thus, it appears possible that the immunopathological features in interface and lobular hepatitis may be similar or shared in AIH and PBC; however, the precise hepatocellular injury mechanisms may not be the same. More detailed phenotypic analysis of the immunopathologies including antigen recognition and immunomodulation such as Th1 and Th2 cells, regulatory T cells, and NK cells [29] appear to be necessary to evaluate the mechanisms of hepatocellular injuries in PBC and AIH. As for the B cell series, a small number of CD20+ B cells were only occasionally seen in interface hepatitis in PBC. In contrast, CD38+ cells, the majority being mature plasma cells, were numerous in interface and lobular hepatitis. Their scores increased with the degree of interface hepatitis and lobular hepatitis, suggesting that they are in April 7, 2014 Volume 20 Issue 13

231 Kobayashi M et al. AIH and PBC with interface hepatitis Table 6 Comparison of the degree of hepatitis and mononuclear cell infiltration in autoantibody positive and negative groups in primary biliary cirrhosis and autoimmune hepatitis PBC AMA(+) or M2(+) AMA(-) and M2(-) P value Comparison between AMA, M2 positive and negative group in PBC Scores of interface hepatitis ± 0.64 (2) 2.33 ± 0.50 (2) Scores of CD3+ cells at interface ± 1.13 (2) 2.44 ± 1.24 (3) Scores of CD38+ cells at interface ± 1.39 (2.5) 2.33 ± 1.22 (2) Scores of IgG+ cells at interface ± 0.59 (1) 1.22 ± 1.64 (1) Scores of IgM+ cells at interface ± 0.63 (1) 0.89 ± 0.93 (1) Comparison between ANA positive and negative group in PBC Scores of interface hepatitis ± 0.65 (2) 2.30 ± 0.48 (2) Scores of CD3+ cells at interface ± 1.16 (2) 2.80 ± 1.23 (2) Scores of CD38+ cells at interface ± 1.16 (2) 2.50 ± 1.18 (3) Scores of IgG+ cells at interface ± 1.08 (1) 1.30 ± 0.67 (1) Scores of IgM+ cells at interface ± 0.65 (1) 1.30 ± 0.82 (1) Comparison between ANA positive and negative group in AIH Scores of interface hepatitis ± 0.64 (3) 2.33 ± 0.58 (2) Scores of CD3+ cells at interface ± 1.13 (4) 3.33 ± 1.53 (3) Scores of CD38+ cells at interface ± 1.18 (4) 2.33 ± 1.15 (3) Scores of IgG+ cells at interface ± 1.39 (2) 1.00 ± 1.00 (1) Scores of IgM+ cells at interface ± 0.76 (1) 0.00 ± 0.00 (0) The scores were graded from 2 to 4 for each case, and are shown as the mean ± SD and (median); 2 The scores were graded from 0 to 6 for each case, and are shown as the mean ± SD and (median). Anti-mitochondrial antibodies (AMA)(+) 40; M2(+) 5; antinuclear antibodies (ANA)(+) 40. PBC: Primary biliary cirrhosis; AIH: Autoimmune hepatitis. volved in the pathogenesis of necroinflammation at these areas in PBC. In fact, the scores of CD38+ and IgG+ cells at the interface and in the hepatic lobules and those of IgM+ cells at the interface were correlated with the elevations in AST in PBC. While the scores of CD38+, IgG+, and IgM+ cells in interface hepatitis and lobular hepatitis were higher in AIH than in PBC, no correlation existed between these scores and the elevation in serum AST in AIH. The elevation in serum IgM level is typical of PBC, and serum IgG elevations are common in AIH [23]. Some reports have shown that IgG and IgM immunostaining of the liver helped to differentiate AIH from PBC [9,10,23,24]. Daniels et al [23] reported that PBC showed equal or more IgM staining than IgG staining, and that AIH always showed more IgG staining than IgM staining. Moreira et al [10] reported that an IgM/IgG ratio of 1 accurately distinguished PBC from AIH or PSC. However, Lee et al [9] reported that although IgM predominance seemed specific for PBC, a significant number of PBC cases also demonstrated IgG predominance. This study found that the average IgG+ cell infiltration score was significantly higher in AIH, whereas the IgM+ cell infiltration score was significantly higher in PBC. The number of IgA+ cells was small at the interface in both diseases. The majority of AIH cases were IgG-predominant, whereas the majority of PBC cases were IgM-predominant or IgM/IgG-equal and some cases were IgG-predominant. When PBC cases were largely divided into IgM-predominant or an equivalent group (group A) and IgG-predominant group (group B), serum IgM levels were significantly higher in group A. Interestingly, the severity of interface hepatitis was slightly higher and the degree of CD38+ cell infiltration at the interface and in the hepatic lobules was significantly higher in group B, suggesting that the PBC in group B may share more features with AIH. More than 90% of PBC patients are AMA-positive, and more than 90% of AIH patients are ANA-positive [20,31,32]. These antibodies have been considered to be important for the diagnosis of PBC and AIH, though their pathogenic roles in hepatocellular injuries are speculative. This study found no difference in the degree of interface and lobular hepatitis or mononuclear cell infiltration at the interface irrespective of an AMA and ANA positivity in PBC and AIH. Therefore, it appears likely that ANA and AMA have no direct influence on interface and lobular hepatitis. While ANA and AMA may play a role in hepatocyte or cholangiocyte injury [31,33], these autoantibodies are not necessarily linked to the severity and pathogenesis of PBC and AIH [32-37], which was supported by the results of this study. While PBC with interface hepatitis has been regarded as a hepatitic form of PBC or PBC with AIH features, some reports insist that PBC and AIH overlap in such cases and could preferably be called PBC-AIH syndrome [1,2,13,14,26]. It remains unclear whether the hepatitic features of these cases indicate an overlap of AIH in PBC or an inherent feature of PBC. The present study suggests that the immunopathogenesis of the hepatitic lesions in AIH and in PBC with interface hepatitis appears to be similar, whereas the precise hepatocellular injury mechanisms may not be the same in these two diseases, indicating that these hepatitic features in PBC do not reflect an overlap of AIH in PBC. Coss Adame et al [13] reported that so-called PBC-AIH overlapping cases showed different HLA phenotypes from type I AIH, suggesting that the former does not reflect an overlap of AIH in PBC, but a different disease from AIH, which supports the abovementioned suggestion. In conclusion, while the immunophenotypes of infil April 7, 2014 Volume 20 Issue 13

232 Kobayashi M et al. AIH and PBC with interface hepatitis trating cells at the interface and lobular hepatitis in AIH and PBC with interface hepatitis appear to be similar, the precise mechanisms of hepatocellular injuries may not be identical. More studies on the mechanisms of hepatocellular injuries in PBC are necessary to distinguish PBC with interface hepatitis from AIH. COMMENTS Background Primary biliary cirrhosis (PBC) infrequently presents with the features of autoimmune hepatitis (AIH), such as interface hepatitis histologically, in addition to the classical features of PBC. Such cases have been called PBC with AIH features, and may respond well to steroid therapy. Research frontiers Previous studies have shown that the density and proportion of IgM+ and IgG+ plasma cells in portal tracts can be used to differentiate AIH from PBC by liver biopsies: IgM+ plasma cells tended to be predominant in portal tracts of PBC. However, the differences and similarities in the hepatitic lesions at the interface and in the hepatic lobules between PBC with interface hepatitis and AIH have not been studied pathologically and immunohistochemically in detail till now. Innovations and breakthroughs Immunohistochemical analysis showed that AIH exhibited more intense lobular hepatitis and more frequent hepatitic rosette formation and emperipolesis than that seen in PBC with interface hepatitis. In PBC, CD3+ and CD38+ cells were predominant in interface and lobular hepatitis, and the degree of infiltration of these cells correlated well with the degree of necroinflammation and elevations in AST. In AIH, the infiltration of these cells was more intense than in PBC, while the degree of mononuclear cell infiltration did not correlate well with the degree of necroinflammation and elevations in AST seen in PBC. IgG+ plasma cells at the interface were predominant in AIH, while IgM+ predominant and IgG+/IgM+ equal cases were common in PBC. Applications This study implicates that while the histological features of interface hepatitis in AIH and PBC with interface hepatitis appear to be similar, the precise pathogenetic mechanisms of hepatocellular injuries may not be identical. These results guarantee further studies on the pathogenesis of PBC with interface hepatitis in comparison with AIH. Terminology Interface hepatitis is characterized by dense lymphoplasmacytic infiltration and destruction of hepatocytes at the limiting plates. This lesion is consistently found in chronic progressive diseases such as AIH and chronic viral hepatitis. Hepatitic rosette is entrapped hepatocyte clusters arranged in glandular structures in the enlarged portal tracts and reflects ongoing hepatocellular loss at the limiting plates. 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233 Kobayashi M et al. AIH and PBC with interface hepatitis Burt AD, Portmann BC, Ferrell LD, editors. MacSween s pathology of the liver. London: Churchill Livingstone, 2011: Alvarez F, Berg PA, Bianchi FB, Bianchi L, Burroughs AK, Cancado EL, Chapman RW, Cooksley WG, Czaja AJ, Desmet VJ, Donaldson PT, Eddleston AL, Fainboim L, Heathcote J, Homberg JC, Hoofnagle JH, Kakumu S, Krawitt EL, Mackay IR, MacSween RN, Maddrey WC, Manns MP, McFarlane IG, Meyer zum Büschenfelde KH, Zeniya M. International Autoimmune Hepatitis Group Report: review of criteria for diagnosis of autoimmune hepatitis. J Hepatol 1999; 31: [PMID: ] 21 Lohse AW, Mieli-Vergani G. Autoimmune hepatitis. J Hepatol 2011; 55: [PMID: DOI: / j.jhep ] 22 Hennes EM, Zeniya M, Czaja AJ, Parés A, Dalekos GN, Krawitt EL, Bittencourt PL, Porta G, Boberg KM, Hofer H, Bianchi FB, Shibata M, Schramm C, Eisenmann de Torres B, Galle PR, McFarlane I, Dienes HP, Lohse AW. Simplified criteria for the diagnosis of autoimmune hepatitis. Hepatology 2008; 48: [PMID: DOI: /hep.22322] 23 Daniels JA, Torbenson M, Anders RA, Boitnott JK. Immunostaining of plasma cells in primary biliary cirrhosis. Am J Clin Pathol 2009; 131: [PMID: DOI: / AJCP8WHR0IEVUUOJ] 24 Cabibi D, Tarantino G, Barbaria F, Campione M, Craxì A, Di Marco V. Intrahepatic IgG/IgM plasma cells ratio helps in classifying autoimmune liver diseases. Dig Liver Dis 2010; 42: [PMID: DOI: /j.dld ] 25 Graham RP, Smyrk TC, Zhang L. Evaluation of langerhans cell infiltrate by CD1a immunostain in liver biopsy for the diagnosis of primary biliary cirrhosis. Am J Surg Pathol 2012; 36: [PMID: DOI: / PAS.0b013e31824b1dff] 26 Nakanuma Y, Zen Y, Harada K, Sasaki M, Nonomura A, Uehara T, Sano K, Kondo F, Fukusato T, Tsuneyama K, Ito M, Wakasa K, Nomoto M, Minato H, Haga H, Kage M, Yano H, Haratake J, Aishima S, Masuda T, Aoyama H, Miyakawa- Hayashino A, Matsumoto T, Sanefuji H, Ojima H, Chen TC, Yu E, Kim JH, Park YN, Tsui W. Application of a new histological staging and grading system for primary biliary cirrhosis to liver biopsy specimens: Interobserver agreement. Pathol Int 2010; 60: [PMID: DOI: / j x] 27 Theise ND, Bodenheimer Jr HC, Ferrell LD. Acute and chronic viral hepatitis. In: Burt AD, Portman BC, Ferrell LD, editors. MacSween's Pathology of the Liver. 6th ed. Edinburg, London: Churchill Livingstone, 2011: Takahashi T, Miura T, Nakamura J, Yamada S, Miura T, Yanagi M, Matsuda Y, Usuda H, Emura I, Tsuneyama K, He XS, Gershwin ME. Plasma cells and the chronic nonsuppurative destructive cholangitis of primary biliary cirrhosis. Hepatology 2012; 55: [PMID: DOI: /hep.24757] 29 Longhi MS, Ma Y, Mieli-Vergani G, Vergani D. Aetiopathogenesis of autoimmune hepatitis. J Autoimmun 2010; 34: 7-14 [PMID: DOI: /j.jaut ] 30 Kumari N, Kathuria R, Srivastav A, Krishnani N, Poddar U, Yachha SK. Significance of histopathological features in differentiating autoimmune liver disease from nonautoimmune chronic liver disease in children. Eur J Gastroenterol Hepatol 2013; 25: [PMID: DOI: / MEG.0b013e32835a68a1] 31 Moritoki Y, Lian ZX, Ohsugi Y, Ueno Y, Gershwin ME. B cells and autoimmune liver diseases. Autoimmun Rev 2006; 5: [PMID: ] 32 O Brien C, Joshi S, Feld JJ, Guindi M, Dienes HP, Heathcote EJ. Long-term follow-up of antimitochondrial antibodypositive autoimmune hepatitis. Hepatology 2008; 48: [PMID: DOI: /hep.22380] 33 Czaja AJ. Behavior and significance of autoantibodies in type 1 autoimmune hepatitis. J Hepatol 1999; 30: [PMID: ] 34 Goodman ZD, McNally PR, Davis DR, Ishak KG. Autoimmune cholangitis: a variant of primary biliary cirrhosis. Clinicopathologic and serologic correlations in 200 cases. Dig Dis Sci 1995; 40: [PMID: ] 35 Invernizzi P, Crosignani A, Battezzati PM, Covini G, De Valle G, Larghi A, Zuin M, Podda M. Comparison of the clinical features and clinical course of antimitochondrial antibodypositive and -negative primary biliary cirrhosis. Hepatology 1997; 25: [PMID: ] 36 Silveira MG, Talwalkar JA, Lindor KD, Wiesner RH. Recurrent primary biliary cirrhosis after liver transplantation. Am J Transplant 2010; 10: [PMID: DOI: / j x] 37 Mehendiratta V, Mitroo P, Bombonati A, Navarro VJ, Rossi S, Rubin R, Herrine SK. Serologic markers do not predict histologic severity or response to treatment in patients with autoimmune hepatitis. Clin Gastroenterol Hepatol 2009; 7: [PMID: DOI: /j.cgh ] P- Reviewers: Takahashi T, Umemura T S- Editor: Zhai HH L- Editor: Logan S E- Editor: Zhang DN 3608 April 7, 2014 Volume 20 Issue 13

234 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH BRIEF ARTICLE REPORT Association of rs , rs and rs polymorphisms with anti-tnf medication response in Greek patients with Crohn s disease Diamantis Thomas, Maria Gazouli, Theodoros Karantanos, Stella Rigoglou, Georgios Karamanolis, Konstantinos Bramis, George Zografos, George E Theodoropoulos Diamantis Thomas, Theodoros Karantanos, Konstantinos Bramis, George Zografos, George E Theodoropoulos, Colorectal Unit, 1 st Department of Propaedeutic Surgery, Hippokrateio Hospital, School of Medicine, University of Athens, Athens, Greece Maria Gazouli, Stella Rigoglou, Department of Basic Medical Science, Laboratory of Biology, School of Medicine, University of Athens, Athens, Greece Georgios Karamanolis, Gastroenterology Unit, 2 nd Department of Surgery, Aretaieio Hospital, University of Athens, Athens 11527, Greece Author contributions: All authors contributed to the manuscript. Correspondence to: Maria Gazouli, Assistant Professor, Department of Basic Medical Science, Laboratory of Biology, School of Medicine, University of Athens, Michalakopoulou 176, Athens, Greece. mgazouli@med.uoa.gr Telephone: Fax: Received: November 19, 2013 Revised: December 23, 2013 Accepted: January 20, 2014 Published online: April 7, 2014 Abstract AIM: To investigate the correlation between rs , rs and rs polymorphisms and response to infliximab in a cohort of Greek patients with Crohn s disease (CD). METHODS: One hundred and twenty-six patients diagnosed with CD based on standard clinical, endoscopic, radiological, and pathological criteria were enrolled in this study at the Gastroenterology Unit of the 2 nd Department of Surgery and at the Colorectal Unit of the 1st Department of Propaedeutic Surgery. Infliximab at a dose of 5 mg/kg was administered intravenously at weeks 0, 2, 6 and then every 8 wk. Clinical and serological responses were assessed using the Harvey- Bradshaw Index and serum C-reactive protein (CRP) levels, respectively, and the endoscopic response was evaluated by ileocolonoscopy performed at baseline and after wk of therapy. The changes in endoscopic appearance compared to baseline were classified into four categories, and patients were classified as responders and non-responders. Genomic DNA from whole peripheral blood was extracted and genotyping was performed by allele-specific polymerase chain reactions. χ 2 test with Yate s correction based on the S-Plus was used to compare the genotype frequencies. RESULTS: Eighty patients (63.49%) were classified as complete and 32 (25.39%) as partial responders to infliximab, while 14 (11.11%) were primary non-responders. No correlation was found between response to infliximab and patients characteristics such as age, gender and disease duration. There was consistency between Harvey-Bradshaw index scores and serum CRP levels. The TT genotype of the rs polymorphism was significantly related to partial response (P = 0.024) and resistance to infliximab (P = 0.007) while the AT genotype was more frequent in partial responders (P = 0.035) and in primary non-responders (P = 0.032). Regarding rs , the CC genotype was found to be associated with partial response (P = 0.005) and primary resistance (P = 0.002) to infliximab while no association was found between the rs polymorphism and the clinical response to infliximab. CONCLUSION: Based on our results, the rs and rs polymorphisms are associated with clinical and biochemical response to infliximab in Greek patients with Crohn s disease Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Crohn s disease; Treatment response; Inf April 7, 2014 Volume 20 Issue 13

235 Thomas D et al. Anti-TNF treatment response in Greek CD patients liximab; Polymorphisms Core tip: A common treatment for inflammatory bowel disease is the use of tumor necrosis factor (TNF)-a inhibitors such as Infliximab (IFX). The discovery of novel markers of response to anti-tnf agents will provide valuable information for better stratification of these patients which will eventually further improve their clinical course and quality of life. Our results suggest that the rs and rs polymorphisms are associated with clinical and biochemical response to IFX in patients with Crohn s disease. Thomas D, Gazouli M, Karantanos T, Rigoglou S, Karamanolis G, Bramis K, Zografos G, Theodoropoulos GE. Association of rs , rs and rs polymorphisms with anti- TNF medication response in Greek patients with Crohn s disease. World J Gastroenterol 2014; 20(13): Available from: URL: DOI: INTRODUCTION The use of anti-tumor necrosis factor (TNF) agents such as infliximab (IFX), certolizumab and adalimumab has impressively improved the clinical course of patients with Crohn s disease (CD) during the last decades [1]. Across these agents, in the pivotal clinical trials, the initial response rate was approximately 60%, while only 30% of these responders maintained remission for as long as one year [2,3]. Moreover, these medications have numerous reported side effects rendering the benefit to risk ratio narrower [4]. These conclusions have led to the identification of multiple clinical parameters such as duration of treatment [5] and disease phenotype [6], and biological factors such as cytokines [7] and C-reactive protein (CRP) levels [8], as predictive markers of response to anti-tnf agents. Despite their utility and easy estimation in the clinical setting, these factors fail to fully predict the response of CD patients to the anti-tnf agents. Therefore, the discovery of novel markers of response to anti-tnf agents will provide valuable information for better stratification of these patients which will eventually further improve their clinical course and quality of life. Recent studies highlight the potential effect of the individual s genetic background on the response to anti-tnf treatment. Taylor et al [9] demonstrated that patients homozygous for a TNF-polymorphism (LTA NcoI-TNFc-aa13L-aa haplotype) were poor responders to IFX while Pierik et al [10] found that the biological response to IFX was lower in patients carrying the TNFR1 36G mutation in the TNFR1 gene. Additionally, López-Hernández et al [11] recently also maintained that particular TNF-α genotypes may be involved in the different responses to TNF-α inhibitor treatment in Spanish patients with inflammatory bowel diseases (IBD). However, other reports have failed to confirm the correlation between polymorphisms in the TNF genes and clinical response to this agent [8,12]. Moreover, according to Niess et al [13], p.arg702trp, p.gly908arg and p.leu1007fsx1008 polymorphisms in the NOD2/CARD15 gene are related to poorer response to anti-tnf agents, while Weiss et al [14] found that NOD2/CARD15 mutations did not have any impact on the response to IFX which was consistent with previous reports [15]. Finally, the rs C allele was found to be correlated with higher serum IL1β concentrations and lower response to IFX treatment in CD patients [16]. Umicevic-Mirkov et al [17], in a recent report, performed a genome-wide association analysis in a cohort of 882 patients with rheumatoid arthritis and evaluated the association between single nucleotide polymorphisms and response to anti-tnf therapy. Three genetic loci (rs , rs and rs ) with improved P value in the overall meta-analysis showed directional consistency over all four cohorts studied by the authors. The rs polymorphism is located in the Loc , encoding a hypothetical protein, while the rs maps in the intronic region of contactin 5 (CNTN5) which is a member of the immunoglobulin superfamily and contactin family and mediates cell surface interactions during nervous system development [18]. According to our knowledge, these genes have not been yet implicated in the development and progression of IBD. However, the correlation of these polymorphisms with the response to anti-tnf in patients with a systemic inflammatory disease such as rheumatoid arthritis suggests that they can be evaluated as potential biomarkers of the response of patients with CD to an anti-tnf agent such as IFX. The aim of this study was to determine whether these reported loci (rs , rs and rs ) reflect an association with response to IFX in patients with CD. MATERIALS AND METHODS Patients One hundred and twenty six patients diagnosed with CD attending the IBD Clinic at the Gastroenterology Unit of the 2 nd Department of Surgery, Aretaieio Hospital, and at the Colorectal Unit of the 1 st Department of Propaedeutic Surgery, Hippokrateio Hospital, Athens, Greece, were enrolled in this case-control study. The diagnosis of CD was based on standard clinical, endoscopic, radiological, and pathological criteria [19,20]. Patients with inflammatory (luminal) disease who were naïve to IFX were eligible for the study. IFX was administered intravenously at a dose of 5 mg/kg at week 0, 2, 6 and then every 8 wk. Clinical and serological responses were assessed using the Harvey- Bradshaw Index (HBI) [21] and serum levels of CRP, respectively, at baseline (before the 1 st infusion of IFX), the day before each subsequent IFX infusion, and after 12 wk 3610 April 7, 2014 Volume 20 Issue 13

236 Thomas D et al. Anti-TNF treatment response in Greek CD patients Table 1 Demographic, clinical and biological characteristics of the study population n (%) Characteristics Complete responders Partial responders Primary non-responders P value 80 (63.49) 32 (25.39) 14 (11.11) Age (yr, mean ± SD) ± ± ± Gender (%) Male 62 (77.5) 19 (58.37) 8 (57.14) Female 18 (22.5) 13 (40.63) 6 (42.86) CRP levels (mg/dl, mean ± SD) Pre-treatment (0 wk) 3.47 ± ± ± 2.15 < Post-treatment (12 wk) 1.07 ± ± ± 1.4 < δcrp levels (%) ± ± ± Disease years 8 ± ± ± Infliximab dosing (mg/kg) Localization (%) Colitis 26 (32.5) 4 (12.5) 2 (14.28) Ileocolitis 50 (62.5) 27 (84.75) 12 (85.72) Upper gastroenteric 4 (5) 1 (2.75) 0 Behaviour (%) Inflammatory 34 (42.5) 10 (31.25) 5 (35.71) Stricturing 14 (17.5) 9 (28.13) 2 (14.29) Penetrating 32 (40) 13 (40.62) 7 (50) of treatment. Ileocolonoscopy was performed by a single endoscopist at baseline and after wk of therapy to evaluate the presence of mucosal bleeding. The changes in endoscopic appearance compared to baseline were classified in four categories and patients were classified based on their response to IFX therapy with standard criteria as previously described [22-24]. The ethical committee of the participating hospitals approved the study, and written inform consent was obtained in advance from each patient. Genotyping Genomic DNA from whole peripheral blood containing EDTA was extracted using validated techniques (NucleoSpin Blood kit; Macherey-Nagel, Germany). The genotyping was performed by allele-specific polymerase chain reactions (PCRs). Primer sequences for the rs polymorphism were forward 5 -TA- AAATACCAAGAAGCATGA-3, reverse Τ 5 -CTGAT- CAATCCTTTTTTAA-3, and reverse A 5 -CTGAT- CAATCCTTTTTTAT-3 ; for the rs , reverse 5 -CATTAATCTCACTGTCCTTTGC-3, forward G 5 -TTTCTCCAGCTGTGTTTAACTG-3 and forward C 5 -TTTCTCCAGCTGTGTTTAACTC-3 ; for the rs , reverse 5 -GACTCCATCTCCCTCATC- CA-3, forward G 5 -CACTCAACTCCAGTCCA- CAAG-3 and forward C 5 -CACTCAACTCCAGTCCA- CAAC-3. The PCR products were then subjected to 3% agarose-gel electrophoresis. Statistical analysis Genotype frequencies were compared with the χ 2 test with Yate s correction using S-Plus (v.6.2insightful, Seattle, WA). Odds ratios (OR) and 95%CI were obtained with GraphPad (v.300, GraphPad Software, San Diego, CA). The P values are all two-sided. P values of < 0.05 were considered to be significant. RESULTS Patients demographic and clinical characteristics are presented in Table 1. Eighty patients (63.49%) were classified as complete, and 32 (25.39%) as partial responders to IFX, while 14 (11.11%) patients were primary nonresponders in this study. There were no statistically significant differences between complete or partial responders and primary non-responders in terms of mean age, gender, disease duration, location and behavior and smoking habits. There was consistency between HBI scores and serum CRP levels classifying patients as complete or partial responders and primary non-responders. Regarding the rs polymorphism, the AT genotype was more frequent in partial responders (P = 0.035) and in primary non-responders (P = 0.032) (Table 2). The TT genotype was also more frequent in partial responders (P = 0.024) and primary non-responders (P = 0.007) (Table 2). Based on these data, patients with AT genotype have 2.71 and 4.75 times increased risk of presenting partial response and primary resistance, respectively, compared to patients with AA genotype. Finally, patients with TT genotype have 8.14 and times increased risk of presenting partial response and primary resistance, respectively, compared to patients with AA genotype. These results suggest that the carriers of the rs t allele are more likely to fail the IFX treatment. As far as the rs polymorphism is concerned, partial responders (P = 0.005) and primary non-responders (P = 0.002) present higher frequency of the CC genotype (Table 2). This result can be translated to the conclusion that the presence of the CC genotype is associated with 6.13 times increased risk of partial response and 11.5 times increased risk of primary resistance to IFX compared to the presence of GG genotype. Finally, the evaluation of the rs polymorphism in our cohort showed that there was no associa April 7, 2014 Volume 20 Issue 13

237 Thomas D et al. Anti-TNF treatment response in Greek CD patients Table 2 Distribution of genotypes in patients and controls n (%) Genotype Complete responders (n = 80) Partial responders (n = 32) P value; OR (95%CI) Non-responders (n = 14) P value; OR (95%CI) rs AA 57 (71.25) 14 (43.75) 1.0 (reference) 4 (28.57) 1.0 (reference) AT 21 (26.25) 14 (43.75) 0.035; 2.71 ( ) 7 (50) 0.032; 4.75 ( ) TT 2 (2.5) 4 (12.5) 0.024; 8.14 ( ) 3 (21.43) 0.007; ( ) rs GG 46 (57.5) 10 (31.25) 1.0 (reference) 4 (28.57) 1.0 (reference) GC 28 (35) 14 (43.75) 0.09; 2.3 ( ) 4 (28.57) 0.7; 1.64 ( ) CC 6 (7.5) 8 (25) 0.005; 6.13 ( ) 6 (42.86) 0.002; 11.5 ( ) rs GG 54 (67.5) 17 (53.12) 1.0 (reference) 10 (71.43) 1.0 (reference) GA 24 (30) 12 (37.5) 0.34; 1.58 ( ) 4 (28.57) 1; 0.9 ( ) AA 2 (2.5) 3 (9.37) 0.11; 4.76 ( ) 0 1; 1.04 ( ) tion between any of the genotypes and response to IFX (Table 2). DISCUSSION IFX has been widely adopted for the treatment of Crohn s disease that is refractory to corticosteroids and immunosuppressive therapy, such as azathioprine and methotrexate. Recently, IFX has been used in combination with immunosuppression earlier during disease progression showing better initial control and improved long-term mucosal healing [25]. Despite the obvious benefits in patients quality of life [26], the response rates differ between different studies and different populations [2,3,27]. Data from induction trials in patients with moderate-to-severe CD resistant to conventional therapies showed that between 21% and 44% more patients achieved remission with IFX than with placebo. Moreover, according to two large maintenance trials, between 14% and 24% more patients achieved remission with IFX [28]. The variability of the response rates between different individuals clearly decreases the efficacy of this agent in the treated patients, suggesting that the identification of predictive biomarkers is critical to improve patient outcomes. The widely used clinical parameters such as disease location and biological factors such as cytokines can only partially predict the response to IFX [29], while recent reports have evaluated the predictive value of gene polymorphisms [26,30], fecal markers [31] and gene expression profiles [32]. According to a recent study, genome-wide association analysis in patients with rheumatoid arthritis discovered that three genetic loci (rs , rs and rs ) showed directional consistency over all cohorts studied [17]. Rheumatoid arthritis, as a systemic inflammatory disease with significant implication of T cell induced immunity, shares important characteristics with CD and it is reasonable to suggest that these polymorphisms may serve as good candidates for prediction of response. To our knowledge, this is the first study evaluating the predictive value of these polymorphisms regarding the response to IFX in patients with CD. The efficacy of IFX was assessed with clinical, serological and endoscopic parameters. Clinical response to IFX was evaluated using the HBI, which has shown good correlation with the CD activity index in clinical trials [33]. Serologic evaluation of response to IFX was based on serum CRP alterations, which have been shown to be correlated with clinical course and inflammatory activity [34,35]. Finally, the patients underwent ileocolonoscopy wk after the initiation of therapy to obtain an objective view of the intestinal mucosa. According to our results, the TT and AT genotypes of the rs polymorphism are significantly associated with partial and non-response to IFX. Interestingly, Umiċeviċ Mirkov et al [17] showed that the presence of the A allele is related to good response to anti-tnf agents in patients with rheumatoid arthritis, supporting our data, despite the absence of any known pathophysiological mechanism connecting this genetic locus with the inflammatory pathways. This particular polymorphism has not yet been associated with the pathogenesis and progression of CD, and further studies are needed to evaluate its role in the development and progression of inflammatory bowel diseases. Furthermore, only the CC genotype of the rs polymorphism was associated with partial response and initial resistance to IFX based on the above presented clinical, serologic and endoscopic criteria. Umiċeviċ Mirkov et al [17] found that the presence of the C allele predicts for poor response to anti-tnf in patients with rheumatoid arthritis, which is in agreement with our results. This polymorphism, as mentioned above, is located in the intronic region of CNTN5 which is implicated in the development of the nervous system [18]. Interestingly, antibodies against the CNTN1/CASPR1 complex occur in a subset of patients with chronic inflammatory demyelinating polyradiculoneuropathy who share common clinical features [34] and CNTN2 is known to be targeted by T cells and autoantibodies during the development of the inflammatory process in multiple sclerosis [35]. These results suggest that members of the contactin superfamily have already been implicated in T cell mediated autoimmune diseases of the nervous system, supporting their potential role in the development of autoimmune inflammatory processes in other organs such as joints and intestine. Finally, our data suggest that different genotypes of 3612 April 7, 2014 Volume 20 Issue 13

238 Thomas D et al. Anti-TNF treatment response in Greek CD patients the rs genetic locus do not have any impact on the response to IFX. The marker rs maps the Loc , encoding a hypothetical protein and, according to Umiċeviċ Mirkov et al [17], the presence of the C allele is associated with good response to anti-tnf agents in patients with rheumatoid arthritis. The absence of any correlation in our study may be attributed to the relatively small population of patients, and further studies are needed to evaluate any potential impact of this genetic locus on the response of CD patients to IFX. It is known that the basic mechanisms of inflammation are similar among different autoimmune diseases. The efficacy of IFX is associated with the role of TNF in the development and progression of inflammation within a particular genetic background for each individual. It could be hypothesized that genetic alterations and variations may alter the response to an anti-tnf agent such as IFX in various autoimmune diseases. According to our data, two genetic loci which are known predictive biomarkers for response to anti-tnf agents in patients with rheumatoid arthritis do predict the response to IFX in a cohort of patients with CD. This conclusion supports the concept of a strong genetic implication in autoimmune inflammation which may be similar among different autoimmune diseases, especially when they share common features such as the critical role of T cells. Finally, the predictive value of these polymorphisms regarding the response to IFX provides us with novel tools for patient stratification to improve the efficacy of this widely used anti-tnf agent. COMMENTS Background A common treatment for inflammatory bowel diseases is the use of tumor necrosis factor (TNF)-α inhibitors such as Infliximab (IFX). The discovery of novel markers of response to anti-tnf agents will provide valuable information for better stratification of these patients which will eventually further improve their clinical course and quality of life. Research frontiers Recent studies highlight the potential effect of the individual s genetic background on the response to anti-tnf treatment. Innovations and breakthroughs Umiċeviċ Mirkov et al recently reported identifying genetic factors predicting anti-tnf treatment outcome in patients with RA using a genome-wide association approach. Eight genetic loci showed improved p value in the overall metaanalysis compared with the first stage, three of which (rs , rs and rs ) showed directional consistency over all four cohorts studied. In accordance with our results, they suggest genetic loci are associated with response to anti-tnf treatment. Applications The predictive value of genetic polymorphisms regarding the response to IFX provides us with novel tools for patient stratification to improve the efficacy of this widely used anti-tnf agent. Peer review This is a good descriptive study in which authors analyze the predictive effect of different genetic loci regarding response to anti-tnf treatment. REFERENCES 1 Peyrin-Biroulet L, Deltenre P, de Suray N, Branche J, Sandborn WJ, Colombel JF. Efficacy and safety of tumor necrosis factor antagonists in Crohn s disease: meta-analysis of placebo-controlled trials. Clin Gastroenterol Hepatol 2008; 6: [PMID: DOI: /j.cgh ] 2 Hanauer SB, Feagan BG, Lichtenstein GR, Mayer LF, Schreiber S, Colombel JF, Rachmilewitz D, Wolf DC, Olson A, Bao W, Rutgeerts P. Maintenance infliximab for Crohn s disease: the ACCENT I randomised trial. Lancet 2002; 359: [PMID: ] 3 Colombel JF, Sandborn WJ, Rutgeerts P, Enns R, Hanauer SB, Panaccione R, Schreiber S, Byczkowski D, Li J, Kent JD, Pollack PF. 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239 Thomas D et al. Anti-TNF treatment response in Greek CD patients ] 15 Vermeire S, Louis E, Rutgeerts P, De Vos M, Van Gossum A, Belaiche J, Pescatore P, Fiasse R, Pelckmans P, Vlietinck R, Merlin F, Zouali H, Thomas G, Colombel JF, Hugot JP. NOD2/CARD15 does not influence response to infliximab in Crohn s disease. Gastroenterology 2002; 123: [PMID: ] 16 Lacruz-Guzmán D, Torres-Moreno D, Pedrero F, Romero- Cara P, García-Tercero I, Trujillo-Santos J, Conesa-Zamora P. Influence of polymorphisms and TNF and IL1β serum concentration on the infliximab response in Crohn s disease and ulcerative colitis. Eur J Clin Pharmacol 2013; 69: [PMID: DOI: /s ] 17 Umiċeviċ Mirkov M, Cui J, Vermeulen SH, Stahl EA, Toonen EJ, Makkinje RR, Lee AT, Huizinga TW, Allaart R, Barton A, Mariette X, Miceli CR, Criswell LA, Tak PP, de Vries N, Saevarsdottir S, Padyukov L, Bridges SL, van Schaardenburg DJ, Jansen TL, Dutmer EA, van de Laar MA, Barrera P, Radstake TR, van Riel PL, Scheffer H, Franke B, Brunner HG, Plenge RM, Gregersen PK, Guchelaar HJ, Coenen MJ. Genome-wide association analysis of anti-tnf drug response in patients with rheumatoid arthritis. Ann Rheum Dis 2013; 72: [PMID: DOI: /annrheumdis ] 18 Ogawa J, Kaneko H, Masuda T, Nagata S, Hosoya H, Watanabe K. Novel neural adhesion molecules in the Contactin/F3 subgroup of the immunoglobulin superfamily: isolation and characterization of cdnas from rat brain. Neurosci Lett 1996; 218: [PMID: ] 19 Podolsky DK. Inflammatory bowel disease. N Engl J Med 2002; 347: [PMID: ] 20 Lennard-Jones JE. Classification of inflammatory bowel disease. 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Lancet 2008; 371: [PMID: DOI: /S (08) ] 25 Costa J, Magro F, Caldeira D, Alarcão J, Sousa R, Vaz- Carneiro A. Infliximab reduces hospitalizations and surgery interventions in patients with inflammatory bowel disease: a systematic review and meta-analysis. Inflamm Bowel Dis 2013; 19: [PMID: DOI: / MIB.0b013e c2] 26 Nogueira IM, Miszputen SJ, Ambrogini Jr O, Artigiani-Neto R, Carvente CT, Zanon MI. Assessment of the response of patients with Crohn s disease to biological therapy using new non-invasive markers: lactoferrin and calprotectin. Arq Gastroenterol 2013; 50: [PMID: ] 27 Dretzke J, Edlin R, Round J, Connock M, Hulme C, Czeczot J, Fry-Smith A, McCabe C, Meads C. A systematic review and economic evaluation of the use of tumour necrosis factoralpha (TNF-α) inhibitors, adalimumab and infliximab, for Crohn s disease. Health Technol Assess 2011; 15: [PMID: DOI: /hta15060] 28 Magro F, Rodrigues-Pinto E, Santos-Antunes J, Vilas-Boas F, Lopes S, Nunes A, Camila-Dias C, Macedo G. High C-reactive protein in Crohn s disease patients predicts nonresponse to infliximab treatment. J Crohns Colitis 2014; 8: [PMID: DOI: /j.crohns ] 29 Medrano LM, Taxonera C, Márquez A, Barreiro-de Acosta M, Gómez-García M, González-Artacho C, Pérez-Calle JL, Bermejo F, Lopez-Sanromán A, Martín Arranz MD, Gisbert JP, Mendoza JL, Martín J, Urcelay E, Núñez C. Role of TN- FRSF1B polymorphisms in the response of Crohn s disease patients to infliximab. Hum Immunol 2014; 75: [PMID: DOI: /j.humimm ] 30 Moroi R, Endo K, Kinouchi Y, Shiga H, Kakuta Y, Kuroha M, Kanazawa Y, Shimodaira Y, Horiuchi T, Takahashi S, Shimosegawa T. FCGR3A-158 polymorphism influences the biological response to infliximab in Crohn s disease through affecting the ADCC activity. Immunogenetics 2013; 65: [PMID: DOI: /s ] 31 Mesko B, Poliska S, Váncsa A, Szekanecz Z, Palatka K, Hollo Z, Horvath A, Steiner L, Zahuczky G, Podani J, Nagy AL. Peripheral blood derived gene panels predict response to infliximab in rheumatoid arthritis and Crohn s disease. Genome Med 2013; 5: 59 [PMID: ] 32 Denis MA, Reenaers C, Fontaine F, Belaïche J, Louis E. Assessment of endoscopic activity index and biological inflammatory markers in clinically active Crohn s disease with normal C-reactive protein serum level. Inflamm Bowel Dis 2007; 13: [PMID: ] 33 Colombel JF, Sandborn WJ, Reinisch W, Mantzaris GJ, Kornbluth A, Rachmilewitz D, Lichtiger S, D Haens G, Diamond RH, Broussard DL, Tang KL, van der Woude CJ, Rutgeerts P. Infliximab, azathioprine, or combination therapy for Crohn s disease. N Engl J Med 2010; 362: [PMID: DOI: /NEJMoa ] 34 Querol L, Nogales-Gadea G, Rojas-Garcia R, Martinez-Hernandez E, Diaz-Manera J, Suárez-Calvet X, Navas M, Araque J, Gallardo E, Illa I. Antibodies to contactin-1 in chronic inflammatory demyelinating polyneuropathy. Ann Neurol 2013; 73: [PMID: DOI: /ana.23794] 35 Derfuss T, Parikh K, Velhin S, Braun M, Mathey E, Krumbholz M, Kümpfel T, Moldenhauer A, Rader C, Sonderegger P, Pöllmann W, Tiefenthaller C, Bauer J, Lassmann H, Wekerle H, Karagogeos D, Hohlfeld R, Linington C, Meinl E. Contactin-2/TAG-1-directed autoimmunity is identified in multiple sclerosis patients and mediates gray matter pathology in animals. Proc Natl Acad Sci USA 2009; 106: [PMID: DOI: /pnas ] P- Reviewers: Iebba V, Mazzoccoli G, Muro M S- Editor: Qi Y L- Editor: Logan S E- Editor: Zhang DN 3614 April 7, 2014 Volume 20 Issue 13

240 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH REPORT Rabeprazole, clarithromycin, and amoxicillin Helicobacter pylori eradication therapy: Report of an efficacy study Charles Asabamaka Onyekwere, Joan Nwabuaku Odiagah, Rufina Igetei, Amancia Olufunmilayo Duro Emanuel, Francis Ekere, Stella Smith Charles Asabamaka Onyekwere, Joan Nwabuaku Odiagah, Rufina Igetei, Amancia Olufunmilayo Duro Emanuel, Francis Ekere, Gastroenterology unit, Department of Medicine, Lagos State University Teaching Hospital, Ikeja, Lagos , Nigeria Stella Smith, Molecular Biology Department, NIMR Yaba, Lagos , Nigeria Author contributions: Onyekwere CA and Odiagah JN designed trial; Onyekwere CA, Odiagah JN, Duro Emanuel AO, Igetei R, Ekere F and Smith S collected data; Onyekwere CA, Odiagah JN contributed data analysis and manuscript writing; all approved final manuscript. Correspondence to: Dr. Charles Asabamaka Onyekwere, Department of Medicine, Lagos State University Teaching Hospital, Mobolaji Bank Anthony Way, Ikeja, lagos , Nigeria. ifymobi@yahoo.com Telephone: Fax: Received: October 10, 2013 Revised: December 19, 2013 Accepted: January 3, 2014 Published online: April 7, 2014 Abstract AIM: To investigate the efficacy of a standard triple therapy (comprising rabeprazole, clarithromycin, and amoxicillin) for Helicobacter pylori (H. pylori ) eradication, noting factors that influence the outcome and documenting any adverse events. METHODS: Following institutional ethical approval, fifty consecutive and consenting symptomatic patients with evidence of H. pylori infection by either a positive urea breath test (UBT) and/or a campylobacter-like organism test who presented to the Gastroenterology clinic of Lagos State University Teaching Hospital between 2012 and 2013 were recruited into the study. Patients were openly randomized to either a 7-d or a 10-d regimen of amoxicillin 1 g, clarithromycin 500 mg and rabeprazole 20 mg twice daily. The extent of symptom resolution was noted following the treatment, and at the end of one month after the completion of treatment, a repeat UBT was performed in each patient to document the eradication of the infection. All data (demographics, symptoms, and eradication rates) were collated and analyzed with SPSS version 18. RESULTS: Forty-seven patients completed the study (three were excluded from the analysis for breaching the study protocol). The patients included 18 males and 29 females within the age range of years (mean 43.7, SD 16.8). The clinical features of the study subjects were dyspepsia, reflux symptoms and features of gastrointestinal bleeding. The average eradication rate was 87.2%. Eighteen subjects were enrolled in the 7-d arm, while 29 were in the 10-d arm. There was no statistically significant difference in the age or sex distributions of the two arms. There was no significant advantage of the 10-d treatment duration over the 7-d duration (P = 0.78), and the eradication outcomes were not influenced by the gender or age of the subjects. No adverse effects were reported in either arm. CONCLUSION: The triple therapy regime, employing a combination of amoxicillin, clarithromycin and rabeprazole, showed great efficacy and safety in the eradication of H. pylori, and this outcome was not influenced by gender or age. No difference was observed between the 7-d and 10-d regimens Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Helicobacter pylori ; Eradication therapy; Amoxicillin clarithromycin; Rabeprazole triple therapy Core tip: Helicobacter pylori (H. pylori ) infection is widespread in Nigeria, along with the associated risk of serious gastroduodenal diseases, including gastric cancer. The use of different eradication therapies for H. pylori is commonplace, as no consensus exists regarding the optimal treatment regimen, despite guidelines 3615 April 7, 2014 Volume 20 Issue 13

241 Onyekwere CA et al. Rabeprazole, clarithromycin and amoxicillin triple therapy and recommendations. This lack of consensus is most likely due to the lack of evidence-based data, and the findings in this report may help to fill this gap. Onyekwere CA, Odiagah JN, Igetei R, Duro Emanuel AO, Ekere F, Smith S. Rabeprazole, clarithromycin, and amoxicillin Helicobacter pylori eradication therapy: Report of an efficacy study. World J Gastroenterol 2014; 20(13): Available from: URL: DOI: INTRODUCTION Helicobacter pylori (H. Pylori) are gram-negative spiral bacteria that reside within the gastric mucosa and are implicated in the etiopathogenesis of a number of gastroduodenal diseases [1]. Although many infected persons do not experience clinically meaningful sequelae, many others develop serious gastrointestinal-related conditions, including gastric cancer. There is a high prevalence of this infection in developing countries, including Nigeria, where reports indicate a prevalence ranging from 45% to 64% among adult dyspeptics, depending on the diagnostic criteria employed [2,3]. Several guidelines for the eradication of this infection have been published by different professional groups in different geographical settings, including the European consensus report and the World Gastroenterology Organization [4,5]. The aim of H. pylori eradication is to reduce the lifetime risk of peptic ulcer disease and possibly of gastric cancer. The test and treat strategy has been advocated in a setting of high H. Pylori prevalence (> 20%) and low gastric cancer risk [6]. The urea breath test (UBT), which uses essentially ( 13 C) urea, remains the best test to diagnose an H. pylori infection due to its high accuracy and ease of use [7]. When endoscopy is performed, biopsy-based tests, such as the rapid urease test, histology and culture, can be carried out. Hitherto, the UBT as a diagnostic tool has not been widely employed in Nigerian settings; poor accessibility, which includes costs and availability, is partially responsible for this scenario. The current H. Pylori treatment guidelines suggest the use of triple therapy [usually a proton-pump inhibitor (PPI), amoxicillin, and clarithromycin (standard triple therapy)] as the first-line therapy for a duration ranging from 7 to 14 d [6]. Other combinations involving the use of metronidazole may be used, depending on the local resistance pattern. Extending the duration of treatment from 7 to d has been shown to increase the success rate of eradication [8]. Other first-line therapies include the quadruple therapy, which involves the addition of a bismuth compound; although this approach is cheaper while having a similar efficacy, more side effects are associated with poor compliance [5] when compared with the PPI. These side effects were later associated with a low rate of transient and asymptomatic serum aminotransferase elevations and represent a rare cause of clinically apparent liver injury [9]. The choice of an eradication regimen is often influenced by such factors as efficacy, adverse effects, cost, compliance, and local resistance patterns. Earlier studies indicated a high rate of metronidazole and amoxicillin resistance in our setting [10]. The empirical use of eradication therapy has become commonplace in our setting, following the National Institutes of Health consensus statement [11] on testing and treatment strategies for H. pylori. Often, no retesting is performed to ensure that the eradication of the infection has been accomplished following treatment. Presently, no consensus exists on the choice of an optimal eradication regime in Nigeria, despite the guidelines [5] and recommendations made in view of differing antimicrobial sensitivity patterns across and within different geographical settings. We set out to investigate the efficacy of the standard triple therapy regimen (comprising rabeprazole, clarithromycin and amoxicillin). MATERIALS AND METHODS From June 2012 to August 2013, we recruited subjects into this randomized, controlled trial, carried out at the Gastroenterology Unit of The Lagos State University Teaching Hospital, Ikeja Nigeria. Inclusion criteria were the following: (1) persons who presented to the unit with upper gastrointestinal (GI) symptoms, such as dyspepsia, upper GI bleeding, or gastroesophageal reflux symptoms (heartburn); and (2) persons with evidence of H. pylori infection. H. pylori infection was said to be present based on either a positive UBT or campylobacter-like organism test. The UBT test was carried out using the Kibion heliprobe machine and involved the ingestion of 13 C labeled urea (helicap) after six hours of fasting and the subsequent exhalation into a breath card. The amount of exhaled 13 C is measured by the heliprobe breath analyzer. The details of measurement are given according to the manufacturer s protocol [12]. The exclusion criteria are stated below: (1) persons with upper GI symptoms who were already on antisecretory drugs, including PPI or antibiotics; (2) persons with upper GI symptoms who were acutely ill and required hospitalization; and (3) persons who refused consent. Using an estimated eradication rate of 85% based on previous reports [13] of triple therapy and a degree of accuracy of 0.1, a sample size of 49 was calculated using the formula N = Z 2 pq/d 2, where N = the required sample size, Z = the standard normal deviation (usually set at 1.96 to correspond to the 95%CI), p = the proportion in the target population estimated to have a particular characteristic, q = 1 - p, d = the degree of accuracy desired set at 0.1. Following institutional ethical approval and written informed consent, the study s participants were consecutively recruited. Alternate patients were openly randomized to either a 7- or 10-d course of the eradication regimen. They were instructed not to use any antibiotic or other antisecretory drugs, including proton pump inhibitors, during the study period and to report any adverse reactions to the principal investigator, whose contact information was shared with all of the participating subjects. The 10-d regimen arm involved use of rabeprazole 20 mg, amoxicillin 1 g and Clarithromycin 3616 April 7, 2014 Volume 20 Issue 13

242 Onyekwere CA et al. Rabeprazole, clarithromycin and amoxicillin triple therapy 79% Pattern of clinical presentation of the study subjects Table 1 Relationship between the patients profiles and their outcomes from Helicobacter pylori eradication Successful eradication Yes No Total number of subjects Dyspepsia GI bleeding GERD Multiple symptoms 500 mg twice daily for 10 d, while the 7-d treatment arm used the same combination of drugs and dosages but for 7 d. At the end of 1 mo following the completion of the treatment, a repeat UBT was performed on each patient to document the eradication of the infection. The outcome parameters for this study are symptomatic improvement and successful eradication. Symptomatic improvement refers to the resolution of the initial presenting symptoms. Successful eradication was said to be present if the repeat UBT at one month following the completion of treatment was negative. Statistical analyses This study were performed with SPP version 18. The test statistics used included Student s t test for the comparison of quantitative data and the χ 2 test for the comparison of qualitative variables. A P value of 0.05 was deemed significant. RESULTS 4.30% 8.50% 8.50% Figure 1 Clinical features of the study subjects. GI: Gastrointestinal; GERD: Gastroesophageal reflux disease. Forty-seven patients completed the study, as three of the patients (2 from the 7-d arm and 1 from the 10-d arm) dropped out of the study (2 for using other proton pump inhibitors and 1 for not adhering to the study protocol). Thus, the non-compliant patients were excluded from the analysis. The study group was made up of 18 males and 29 females within the age range years (mean 43.7 SD 16.8). The clinical features of the study subjects are given in Figure 1. The average eradication rate was 87.2%. Adverse effects. No adverse effects were reported in either arm. Table 1 illustrates the relationship between the outcome of the H. pylori eradication and the patients demographics (age and sex) and clinical presentations. There was no significant relationship between the age, sex or pattern of clinical presentation among those subjects with successful H. Pylori eradication. Eighteen subjects were in the seven-day arm, while 29 were in the 10-d arm. The age and sex distributions of the subjects in the two arms are given in Table 2. There was no statistically significant difference in the age and sex distribution or in the rate of successful H. Pylori eradication between the two arms. Age of the patient vs eradication outcome Age grouping of patient DISCUSSION 0-39 yr yr yr Total 41 (mean age 41 ± 17) 6 (mean age 48 ± 16) P value (χ 2 = 0.986) Gender vs outcome of eradication therapy Gender Male Female Total P value (χ 2 = 0.072) Patients clinical presentation vs outcomes of eradication treatment Clinical presentation Dyspepsia GERD GI bleeding Multiple presentation Total P value (χ 2 = 6.531) GI: Gastrointestinal; GERD: Gastroesophageal reflux disease. Infection with H. pylori continues to be a cause for concern, and the search for an optimal therapy continues due to the changing antibiotic sensitivity patterns in different geographic settings. Triple therapy with an antisecretory drug and two antibiotics (amoxicillin, metronidazole or clarithromycin) has often been advocated as the first-line therapy; the choice of the antibiotics varies, depending on local sensitivity patterns [6]. Our study has shown an overall symptom improvement of over 95% and a H. pylori eradication rate of over 87%, which is a little less than the 90% per protocol recommended by World Gastroenterology Organisation guidelines [5]. This rate is also slightly lower than the rates obtained by Sokwala et al [13] in a Kenyan population employing same antibiotics but another PPI (Esomeprazole). However, our rate is better than the 85% reported by Wong et al [14] in their multi-center study of a triple therapy, employing the same antibiotics but with omeprazole in Asians and Africans. Still, it has been reported that, in clinical trials, failure to eradicate H. pylori is approximately 20% after first-line eradication therapy [15]. Antibiotic resistance patterns [10], due most likely to the abuse of drugs as antibiotics, are sold without prescriptions in our setting; this may partly explain our lower efficacy. The current report indicates a resistance of < 5% to tetracycline, < 30% to amoxicillin, < 16% to clarithromycin and 100% to metronidazole [16] in Lagos, Nigeria. This observed eradication rate, in any case, gives us some confidence that eradication was achieved in the majority of our treated patients through the use of this triple therapy combination. Still, in routine practice, post-treatment evaluation is hardly ever performed, contrary to the guidelines and 3617 April 7, 2014 Volume 20 Issue 13

243 Onyekwere CA et al. Rabeprazole, clarithromycin and amoxicillin triple therapy Table 2 Demographics, clinical features and response patterns of the subjects to the Helicobacter pylori eradication regimes n (%) Factor Seven-day arm Ten-day arm P value Age range (41.56 ± 16.94) (44.93 ± 16.94) (mean ± SD) Sex Male 6 (33.3) 12 (41.4) Female 12 (66.7) 17 (58.6) Symptomatic improvement Yes 17 (94.4) 29 (100.0) No 1 (5.6) 0 (0.0) Successful eradication Yes 16 (88.9) 25 (86.2) No 2 (11.1) 4 (13.8) recommendations [6]. The few (13%) patients who failed to respond to this regime were treated successfully with either a sequential therapy [17] or quadruple therapy [5]. Further, the eradication rates did not vary with the age, despite that previous reports [18] have suggested worse outcomes at younger ages. Equally, some reports have indicated that the failure of eradication is more common in the female sex [19] ; however our findings did not suggest any gender predisposition. The pattern of clinical manifestation of the subjects had no relation to their outcome, although some reports suggested an association with the subjects gastric histology features [20]. We did not find any statistically significant differences in the eradication rates between those treated for 7 d (88.9%) and those treated for 10 d (86.2%), contrary to earlier reports [7]. Similar observations were made in two other reports involving African populations [12,14]. Although no consensus exists for the optimal duration of triple therapy in Africa, it is noteworthy that a number of European guidelines [4] favor 7-d therapies, as opposed to the extended 10 to 14 d treatment in America [21]. We were, however, constrained by our modest subject sample size due to the limited resources available to our investigators. Additionally, we did not investigate the effects of the subjects habits, such as smoking or alcohol consumption, on the eradication rate, although some reports [13] have indicated that these habits could affect the outcomes of the eradication treatment. The cost benefit analysis of this triple therapy regime was also not investigated, as this was not included into the study design. The triple therapy regime, employing a combination of amoxicillin, clarithromycin and rabeprazole, has shown great efficacy and safety in the eradication of H. pylori, demonstrating an average eradication rate of 87.2%. The 10-d treatment duration showed no significant advantage over the 7-d duration, and the outcome was not influenced by the gender or age of the subjects. ACKNOWLEDGMENTS We appreciate the support of Biofem pharmaceuticals and Solidum pharmaceuticals in carrying out this project, as well as the support of Dr. O. Ogbera for helping to proofread the manuscript. COMMENTS Background Helicobacter pylori (H. pylori) infection is common in Sub-Saharan Africa, including Nigeria, along with its attendant consequences, including gastric cancer. Several guidelines have been recommended regarding an eradication therapy using a number of regimes. No consensus on such therapies exists in the face of changing anti-microbial sensitivity patterns. Research frontiers Although post-treatment tests are recommended to ensure that H. pylori eradication is achieved, this is not often performed by practitioners. There is a need for an evidence-based, safe, and effective treatment regime in a setting with the widespread use of empirical H. pylori eradication. Innovations and breakthroughs This randomized trial has demonstrated the efficacy of a rabeprazole, amoxicillin and ciarithromycin triple regime in achieving a high eradication rate; additionally, a 7-d regime was shown to be just as effective as a 10-d regime. The outcomes of the treatment were not influenced by age or gender. Applications The study results suggest that the triple therapy, employing rabeprazole, amoxicillin and clarithromycin for seven days, can be used to treat H. pylori infections in Lagos, Nigeria. Peer review The article is aim to investigate the efficacy of a triple therapy (comprising of Rabeprazole, Clarithromycin and Amoxicillin) and note factors influencing the outcome as well as document any adverse event. The manuscript is very wellwritten. REFERENCES 1 Marshall BJ. Helicobacter Foundation. Available from: URL: viewed 2/12/13 2 Smith SI, Oyedeji KS, Arigbagbu A, Anomneze EE, Chibututu CC, Atimomo EA, Atoyebi A, Adesanya, A, Coker AO. Prevalence of H. pylori in patients with gastritis and peptic ulcer in Western, Nigeria. Biomed Lett 1999; 60: Jemilohun AC, Otegbayo JA, Ola SO, Oluwasola OA, Akere A. Prevalence of Helicobacter pylori among Nigerian patients with dyspepsia in Ibadan. 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Prolonged treatment duration is required for successful Helicobacter pylori eradication with proton pump inhibitor triple therapy in Canada. Can J Gastroenterol 2013; 27: [PMID: ] 9 Smith SI, Oyedeji KS, Arigbabu AO, Atimomo C, Coker 3618 April 7, 2014 Volume 20 Issue 13

244 Onyekwere CA et al. Rabeprazole, clarithromycin and amoxicillin triple therapy AO. High amoxycillin resistance in Helicobacter pylori isolated from gastritis and peptic ulcer patients in western Nigeria. J Gastroenterol 2001; 36: [PMID: DOI: /s ] 10 Sandig C, Flechtenmacher C, Stremmel W, Eisenbach C. Pantoprazole induces severe acute hepatitis. Z Gastroenterol 2011; 49: [PMID: DOI: /s ] 11 US dept of Health and Human services. Helicobacter pylori in peptic ulcer disease. NIH consensus statement 1994; 12: Oztürk E, Yeşilova Z, Ilgan S, Arslan N, Erdil A, Celasun B, Ozgüven M, Dağalp K, Ovali O, Bayhan H. A new, practical, low-dose 14C-urea breath test for the diagnosis of Helicobacter pylori infection: clinical validation and comparison with the standard method. Eur J Nucl Med Mol Imaging 2003; 30: [PMID: DOI: /s ] 13 Sokwala A, Shah MV, Devani S, Yonga G. Helicobacter pylori eradication: A randomised comparative trial of 7-day versus 14-day triple therapy. S Afr Med J 2012; 102: [PMID: ] 14 Wong BC, Chang FY, Abid S, Abbas Z, Lin BR, Van Rensburg C, Chen PC, Schneider H, Simjee AE, Hamid SS, Seebaran A, Zhang J, Destefano M, Lam SK. Triple therapy with clarithromycin, omeprazole, and amoxicillin for eradication of Helicobacter pylori in duodenal ulcer patients in Asia and Africa. Aliment Pharmacol Ther 2000; 14: [PMID: ] 15 Gisbert JP, Calvet X. Review article: the effectiveness of standard triple therapy for Helicobacter pylori has not changed over the last decade, but it is not good enough. Aliment Pharmacol Ther 2011; 34: [PMID: DOI: /j x] 16 Smith Stella. Nigerian Institute of Medical Research. Antibiotic Sensitivity patterns of helicobacter isolates from South West Nigeria. Personal communication 17 Chung JW, Jung YK, Kim YJ, Kwon KA, Kim JH, Lee JJ, Lee SM, Hahm KB, Lee SM, Jeong JY, Yun SC. Ten-day sequential versus triple therapy for Helicobacter pylori eradication: a prospective, open-label, randomized trial. J Gastroenterol Hepatol 2012; 27: [PMID: DOI: / j x] 18 Treiber G, Wittig J, Ammon S, Walker S, van Doorn LJ, Klotz U. Clinical outcome and influencing factors of a new short-term quadruple therapy for Helicobacter pylori eradication: a randomized controlled trial (MACLOR study). Arch Intern Med 2002; 162: [PMID: DOI: /archinte ] 19 Cai W, Zhou L, Ren W, Deng L, Yu M. Variables influencing outcome of Helicobacter pylori eradication therapy in South China. Helicobacter 2009; 14: [PMID: DOI: /j x] 20 Georgopoulos SD, Ladas SD, Karatapanis S, Mentis A, Spiliadi C, Artikis V, Raptis SA. Factors that may affect treatment outcome of triple Helicobacter pylori eradication therapy with omeprazole, amoxicillin, and clarithromycin. Dig Dis Sci 2000; 45: [PMID: ] 21 Chey WD, Wong BC. American College of Gastroenterology guideline on the management of Helicobacter pylori infection. Am J Gastroenterol 2007; 102: [PMID: ] P- Reviewers: Amornyotin S, Ibrahim M, Pereira RD, Takeno S S- Editor: Cui XM L- Editor: A E- Editor: Ma S 3619 April 7, 2014 Volume 20 Issue 13

245 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH REPORT Influence of the safety and diagnostic accuracy of preoperative endoscopic ultrasound-guided fine-needle aspiration for resectable pancreatic cancer on clinical performance Taiki Kudo, Hiroshi Kawakami, Masaki Kuwatani, Kazunori Eto, Shuhei Kawahata, Yoko Abe, Manabu Onodera, Nobuyuki Ehira, Hiroaki Yamato, Shin Haba, Kazumichi Kawakubo, Naoya Sakamoto Taiki Kudo, Hiroshi Kawakami, Masaki Kuwatani, Kazunori Eto, Shuhei Kawahata, Yoko Abe, Manabu Onodera, Nobuyuki Ehira, Hiroaki Yamato, Shin Haba, Kazumichi Kawakubo, Naoya Sakamoto, Department of Gastroenterology and Hepatology, Hokkaido University Graduate School of Medicine, Sapporo , Japan Author contributions: Kudo T and Kawakami H contributed equally to this work; Kawakami H managed the patient, performed the endoscopic examination, designed the research and provided discussion; Kudo T and Kawakami H analysed the data; Kuwatani M, Eto K, Kawahata S, Abe Y, Onodera M, Ehira N, Yamato H, Haba S and Kawakubo K collected the data and provided clinical advice; Kudo T and Kawakami H collected the data and wrote the paper; Kawakami H and Kuwatani M revised the paper; Sakamoto N supervised the research; all authors approved the final manuscript for publication. Correspondence to: Hiroshi Kawakami, MD, PhD, Department of Gastroenterology and Hepatology, Hokkaido University Graduate School of Medicine, Kita 15, Nishi 7, Kita-ku, Sapporo , Japan. hiropon@med.hokudai.ac.jp Telephone: Fax: Received: October 3, 2013 Revised: December 1, 2013 Accepted: January 3, 2014 Published online: April 7, 2014 Abstract AIM: To evaluate the safety and diagnostic accuracy of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) in a cohort of pancreatic cancer patients. METHODS: Of 213 patients with pancreatic cancer evaluated between April 2007 and August 2011, 82 were thought to have resectable pancreatic cancer on the basis of cross-sectional imaging findings. Of these, 54 underwent EUS-FNA before surgery (FNA+ group) and 28 underwent surgery without preoperative EUS- FNA (FNA- group). RESULTS: All 54 lesions were visible on EUS, and all 54 attempts at FNA were technically successful. The diagnostic accuracy according to cytology and histology findings was 98.1% (53/54) and 77.8% (42/54), respectively, and the total accuracy was 98.1% (53/54). One patient developed mild pancreatitis after EUS-FNA but was successfully treated by conservative therapy. No severe complications occurred after EUS-FNA. In the FNA+ and FNA- groups, the median relapse-free survival (RFS) was 742 and 265 d, respectively (P = ), and the median overall survival (OS) was 1042 and 557 d, respectively (P = ). RFS and OS were therefore not inferior in the FNA+ group. These data indicate that the use of EUS-FNA did not influence RFS or OS, nor did it increase the risk of peritoneal recurrence. CONCLUSION: In patients with resectable pancreatic cancer, preoperative EUS-FNA is a safe and accurate diagnostic method Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Pancreatic cancer; Diagnosis; Biopsy; Endoscopic ultrasound-guided fine-needle aspiration; Preoperative diagnosis Core tip: Whether preoperative endoscopic ultrasoundguided fine-needle aspiration (EUS-FNA) is safe and effective for resectable pancreatic cancer has not yet been established. In the present study, patients who underwent EUS-FNA had better relapse-free survival 3620 April 7, 2014 Volume 20 Issue 13

246 Kudo T et al. EUS-FNA for resectable pancreatic cancer and overall survival than did those who did not, although it should be noted that more patients in the FNA before surgery group received adjuvant chemotherapy. Our findings suggest that preoperative EUS-FNA does not adversely affect surgery or prognosis in patients with resectable pancreatic cancer. EUS-FNA can also potentially reduce the inappropriate performance of pancreatic surgery by facilitating an accurate diagnosis. These findings are important because the use of preoperative EUS-FNA is becoming more widespread. Kudo T, Kawakami H, Kuwatani M, Eto K, Kawahata S, Abe Y, Onodera M, Ehira N, Yamato H, Haba S, Kawakubo K, Sakamoto N. Influence of the safety and diagnostic accuracy of preoperative endoscopic ultrasound-guided fine-needle aspiration for resectable pancreatic cancer on clinical performance. World J Gastroenterol 2014; 20(13): Available from: URL: DOI: INTRODUCTION Pancreatic cancer is the fourth and fifth leading cause of cancer-related deaths in the United States and Japan, respectively, with deaths per year worldwide [1,2]. Patients with unresectable pancreatic cancer have a much worse prognosis than do those with resectable disease [2], making a sensitive screening examination and early diagnosis essential. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) was first reported by Vilmann et al [3] in 1992 and has been increasingly used worldwide to diagnose pancreatic tumors, because it can be difficult to distinguish between benign and malignant tumors using conventional imaging modalities. EUS-FNA can be used to make a pathological diagnosis of pancreatic tumors and has several advantages over computed tomography (CT)- or ultrasound (US)-guided biopsy with respect to its success rate and safety [1]. However, whether the use of preoperative EUS-FNA for diagnosing pancreatic tumors is safe, given the risk of complications such as bleeding, perforations, pancreatitis, and tumor seeding, is still a matter of debate [4-13]. Previous studies have found that EUS-FNA used for pancreatic cancer is associated with only a very low risk of complications [13] and that there was no significant increase in pancreatic adenocarcinoma seeding, suggesting that the risk associated with EUS- FNA is outweighed by the likely benefit of making an accurate and early pathological diagnosis [6,8,11,12]. The utility and safety of EUS-FNA for the diagnosis of cancer in the body and tail of the pancreas has also been reported recently [14]. However, the safety and efficacy of preoperative EUS-FNA in diagnosing pancreatic cancer and the long-term prognoses of patients who have undergone preoperative EUS-FNA have not yet been reported [4]. The need for a more accurate diagnostic test is em- phasized by cases in which benign pancreatic disease has been misdiagnosed as cancer and resected, increasing the associated risk of morbidity and mortality. Preoperative EUS-FNA may also reduce the misdiagnosis of benign pancreatic diseases [15]. The purpose of this study was to evaluate the efficacy and safety of preoperative EUS- FNA for diagnosing pancreatic cancer and the long-term prognosis of patients after surgery. MATERIALS AND METHODS Patients We evaluated 213 consecutive patients with pancreatic cancer between April 2007 and August Among them, 91 patients were diagnosed with resectable pancreatic cancer, 9 of whom underwent neoadjuvant chemotherapy or chemoradiotherapy to treat local invasion. After excluding these 9 cases, 82 patients were enrolled: 54 patients underwent EUS-FNA before surgery (FNA+ group) and 28 patients underwent surgery without preoperative EUS-FNA (FNA- group) (Figure 1). We performed EUS-FNA when requested by the surgeon or if patients were hospitalized at our department. The preoperative levels of tumor markers such as carcinoembryonic antigen (CEA), carbohydrate antigen 19-9 (CA19-9), SPan-1, and DU-PAN- 2, were examined in all cases. US or CT was performed in all cases. EUS-FNA procedure Preoperative EUS-FNA was performed by a single experienced endoscopist (H.K.) using a curvilinear echoendoscope (GF-UCT240-AL5; Olympus Medical Systems Co., Tokyo, Japan) and 19, 22 and 25-gauge needles (Echotip ultra; Cook Japan, Tokyo, Japan) under conscious sedation. Briefly, the lesions were visualized by EUS, after which, the needle was advanced into the lesion through the gastric or duodenal wall. The central stylet was removed, and a syringe was attached to the needle hub to apply negative suction pressure. The needle was then moved back and forth within the lesion at least 10 times, it was removed from the lesion through the scope, and the stylet was inserted back into the needle. The specimen obtained by aspiration was placed on a slide, air-dried, alcohol-fixed, and used to prepare smears. These were then stained using the rapid Romanowsky technique allowing them to be quickly interpreted and assessed for sample adequacy (Diff-Quik stain; Kokusai Shiyaku, Kobe, Japan). Diff-Quik staining was performed on all specimens by a cytotechnologist. Cytological and histological diagnoses were made for the specimens obtained by EUS-FNA. Outcome measurements The characteristics of the patients, operative procedures, pathological stage according to the Union Internationale Contre le Cancer (UICC) classification, microscopic margin, the use of adjuvant chemotherapy, and the diagnostic accuracy and complications of EUS-FNA were investigated. An EUS-FNA diagnosis was considered to be 3621 April 7, 2014 Volume 20 Issue 13

247 Kudo T et al. EUS-FNA for resectable pancreatic cancer Pancreatic cancer n = 213 Table 1 Patient characteristics Resectability Yes n = 91 No n = 122 Patients with NAC were excluded NAC n = 9 FNA+ FNA- Number of patients Median age (range), yr 68 (43-82) 70 (45-84) NS 1 Gender (M/F) 34/20 16/12 NS 2 Location (Ph/Pb/Pt) 33/17/4 20/6/2 NS 2 Median CEA (95%CI), 4.89 ( ) 5.18 ( ) NS 1 ng/ml Median CA19-9 (95%CI), U/mL 46.1 ( ) 96.7 ( ,661.9) NS 1 Median SPan-1 (95%CI), ( ) 64.5 ( ) NS 1 U/mL Median DU-PAN-2 (95%CI), U/mL 129 ( ) 303 ( ) NS 1 EUS-FNA Yes n = 54 No n = 28 Figure 1 Study participants. This flowchart explains how the final sample size was arrived at and which patients were included. Ninety-one patients with pancreatic cancer underwent radical surgery. Nine patients were treated with chemotherapy or chemoradiotherapy preoperatively and were therefore excluded. The remaining 82 patients were divided into 2 groups. One group consisted of patients who underwent endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) before the operation (FNA+ group; n = 54), and the other group included patients who did not undergo EUS-FNA before the operation (FNA- group; n = 28). The decision to use preoperative EUS-FNA was made by a surgeon. NAC: Neoadjuvant chemotherapy or chemoradiotherapy; EUS-FNA: Endoscopic ultrasound-guided fine-needle aspiration. accurate if it matched the pathological diagnosis of the corresponding resected specimens. Diagnostic accuracy was assessed by comparing biopsy results with those of the final pathological diagnosis. Complications arising from the use of EUS-FNA (as described by Eloubeidi et al [5] ) were monitored until surgery was performed. Pancreatitis and its severity were defined according to the criteria proposed by Cotton et al [16]. We referred to the Standards of Practice Committee of the American Society for Gastrointestinal Endoscopy workshop [17] for the definition of other complications. All the procedures were performed on an inpatient basis. Our institute s review board approved the study. All patients provided written, informed consent. Statistical analysis Statistical analyses were performed using JMP software version 8 (SAS Institute, Cary, NC, United States). Patient characteristics were compared using the Fisher s exact test and chi-square test. The median relapse-free survival (RFS) and overall survival (OS) time were calculated in October 2011 and were estimated using the Kaplan-Meier method and the log-rank test. The Cox proportional hazard model was used to analyze the prognostic factors for OS, including age ( 65 years vs < 65 years), serum CEA and CA19-9 levels prior to surgery, tumor size (> 20 mm vs 20 mm), portal vein invasion (yes vs no), pathological stage according to the UICC classification (IIB-4 vs 0-IIA), microscopic margin (positive vs negative), the use of adjuvant chemotherapy (yes vs no), and 1 Mann-Whitney U test; 2 Fisher s exact test. FNA+: The patient group who underwent endoscopic ultrasound-guided fine-needle aspiration before surgery; FNA-: The patient group who did not undergo endoscopic ultrasound-guided fine-needle aspiration before surgery; NS: No significant difference; Ph: Pancreas head; Pb: Pancreas body; Pt: Pancreas tail; CEA: Carcinoembryonic antigen; CA 19-9: Carbohydrate antigen EUS-FNA before surgery (yes vs no). CEA and CA19-9 were categorized into two groups according to the median value of the total study population. All reported P values are the results of two-sided tests, with P < 0.05 considered statistically significant. RESULTS Patient characteristics and the locations of the lesions are shown in Table 1. Patient characteristics did not differ significantly between the FNA+ and FNA- groups. The preoperative levels of tumor markers such as CEA, CA19-9, SPan-1, and DU-PAN-2, did not differ significantly between the 2 groups (Table 1). All lesions could be visualized using EUS, and all 54 procedures to puncture the lesions were successful. Among them, 25 procedures were performed via the gastric wall and 29 procedures were performed via the duodenal wall. The mean number of needle passes was 2.6 (range, 1-5). We used a 22-gauge, 25-gauge, and 19-gauge needle in 43, 9, and 5 procedures, respectively (in 4 cases, we used both a 22-gauge and a 25-gauge needle). The mean duration from EUS-FNA to surgery was 22.3 d (range, 5-57 d). All procedures yielded specimens for diagnosis by cytology or histology. The accuracy of diagnoses based on cytology and histology findings was 98.1% (53/54) and 77.8% (42/54), respectively (Table 2), and the overall accuracy was 98.1% (53/54). One patient developed mild pancreatitis after EUS-FNA, but this was successfully treated by conservative therapy. In that particular case, a 22-mm lesion was found in the head of the pancreas. This was assessed using EUS-FNA with a 22-gauge needle and by making 2 punctures through the duodenal wall. All patients underwent curative surgical resection. One patient in the FNA+ group was found to have 3622 April 7, 2014 Volume 20 Issue 13

248 Kudo T et al. EUS-FNA for resectable pancreatic cancer Table 2 Endoscopic ultrasound-guided fine-needle aspiration procedure and its diagnostic accuracy 1.0 FNA+ FNA- Log-rank P < 0.05 Puncture position Stomach/duodenum 25/29 Needle size 19-gauge 5 22-gauge gauge 9 Puncture number, range (mean) 1-5 (2.6) Mean duration from EUS-FNA to surgery (d) 22.3 Accuracy of cytology diagnosis 98.1% (53/54) Accuracy of histology diagnosis 77.8% (42/54) Relapse free survival 0.5 EUS-FNA: Endoscopic ultrasound-guided fine-needle aspiration Time from surgery/d Table 3 Operative method, outcome, tumor size, histological type, pathological stage, and adjuvant chemotherapy FNA+ FNA- Number of patients Operative method (%) PD 59.3 (32/54) 75 (21/28) NS 1 DP 38.9 (21/54) 17.9 (5/28) TP 1.9 (1/54) 3.6 (1/28) Partial resection 1.9 (1/54) 3.6 (1/28) Outcome (%) R (52/54) 96.4 (27/28) NS 1 R1 3.7 (2/54) 3.6 (1/28) R2 0 (0/54) 0 (0/28) Tumor size (mm) 30.0 ± 1.9 SD 29.5 ± 2.5 SD NS 1 Histological type Adenocarcinoma NS 1 Adenosquamous carcinoma 1 0 IPMC 0 2 UICC (%) 0 0 (0/54) 3.6 (1/28) NS 1 ⅠA 3.7 (2/54) 7.1 (2/28) ⅠB 1.9 (1/54) 0 (0/28) ⅡA 48.1 (26/54) 21.4 (6/28) ⅡB 38.9 (21/54) 53.6 (15/28) Ⅲ 3.7 (2/54) 3.6 (1/28) Ⅳ 3.7 (2/54) 10.7 (3/28) AC administration (%) 74.1 (40/54) 50 (14/28) P < Pearson s χ 2 test. FNA+: The patient group who underwent endoscopic ultrasound-guided fine-needle aspiration before surgery; FNA-: The patient group who did not undergo endoscopic ultrasound-guided fineneedle aspiration before surgery; PD: Pancreatoduodenectomy; DP: Distal pancreatectomy; TP: Total pancreatectomy; SD: Standard deviation; IPMC: Intraductal papillary mucinous carcinoma of the pancreas; UICC: Pathological stage of the Union Internationale Contre le Cancer; AC: Adjuvant chemotherapy. malignant cells in a peritoneal lavage cytology sample. However, there was no sign of peritoneal dissemination, for example, omental inflammation or a nodule in the peritoneum. Table 3 summarizes the operative methods, surgical outcome, tumor size, histological type of the resected specimen, UICC stage of resected specimens, and the use of adjuvant chemotherapy. We pathologically checked for lymph node metastasis, perineural and lymphovascular invasion, histological type of the lesion, and transfusion rates. No significant differences were found with respect to any of these factors between the 2 groups (data not shown). However, adjuvant chemotherapy was Figure 2 Kaplan-Meier curves for relapse-free survival in pancreatic cancer patients. Kaplan-Meier curves for relapse-free survival in patients who underwent resection for pancreatic cancer. The solid line indicates the Kaplan- Meyer curve for the FNA+ group and the dotted line represents that for the FNA- group. The median relapse-free survival time of the FNA+ and FNAgroups was 742 and 265 d, respectively (Log-rank test; P < 0.05). FNA+: The group of patients who underwent endoscopic ultrasound-guided fine-needle aspiration before surgery; FNA-: The group of patients who did not undergo endoscopic ultrasound-guided fine-needle aspiration before surgery. Overall survival FNA+ FNA Time from surgery/d Log-rank P < 0.05 Figure 3 Kaplan-Meier curves for overall survival in pancreatic cancer patients. Kaplan-Meier curves for overall survival in patients who underwent resection for pancreatic cancer. The solid line indicates the Kaplan-Meyer curve for the FNA+ group and the dotted line represents that for the FNA- group. The median survival time of the FNA+ and FNA- groups was 1042 and 557 d, respectively (Log-rank test; P < 0.05). FNA+: The group of patients who underwent endoscopic ultrasound-guided fine-needle aspiration before surgery; FNA-: The group of patients who did not undergo endoscopic ultrasound-guided fine-needle aspiration before surgery. significantly more common among patients in the FNA+ group than among those in the FNA- group (P < 0.05). The median RFS times in the FNA+ and FNAgroups were 742 d (range, d) and 265 d (range, d), respectively (P < 0.05) (Figure 2). The median OS times in the FNA+ and FNA- groups were 1042 d (range, d) and 557 d (range, d), respectively (P < 0.05) (Figure 3). Recurrent lesions occurred in the liver (14 in the FNA+ group and 11 in the FNA- group), peripancreatic soft tissue (7 in the FNA+ group and 6 in the FNA- group), peritoneum (7 in the FNA+ and 5 in the FNA- group), lymph nodes, lungs, bone, and adrenal body. RFS and OS were also analyzed 3623 April 7, 2014 Volume 20 Issue 13

249 Kudo T et al. EUS-FNA for resectable pancreatic cancer A 1.0 FNA+ FNA- Log-rank P = 0.30 A 1.0 FNA+ FNA- Log-rank P < 0.05 Relapse free survival 0.5 Relapse free survival Time from surgery/d Time from surgery/d B 1.0 FNA+ FNA- Log-rank P < 0.05 B 1.0 FNA+ FNA- Log-rank P = 0.08 Overall survival 0.5 Overall survival Time from surgery/d Time from surgery/d Figure 4 Kaplan-Meier curves for relapse-free survival and overall survival in patients who received adjuvant chemotherapy. A: Kaplan-Meier curves for relapse-free survival in patients who underwent resection for pancreatic cancer and who received adjuvant chemotherapy. The solid line indicates the Kaplan-Meyer curve for the FNA+ group and the dotted line represents that for the FNA- group. The median relapse-free survival time of the FNA+ and FNA- groups was 596 and 332 d, respectively (Log-rank test; P = 0.30); B: Kaplan-Meier curves for overall survival in patients who underwent resection for pancreatic cancer and who received adjuvant chemotherapy. The solid line indicates the Kaplan-Meyer curve for the FNA+ group and the dotted line represents that for the FNA- group. The median overall survival time of the FNA+ and FNA- groups was 1042 and 636 d, respectively (Log-rank test; P < 0.05). FNA+: The group of patients who underwent endoscopic ultrasound-guided fine-needle aspiration before surgery; FNA-: The group of patients who did not undergo endoscopic ultrasound-guided fine-needle aspiration before surgery. Figure 5 Kaplan-Meier curves for relapse-free survival and overall survival in patients who did not receive adjuvant chemotherapy. A: Kaplan- Meier curves for relapse-free survival in patients who underwent resection for pancreatic cancer without adjuvant chemotherapy. The solid line indicates the Kaplan-Meyer curve for the FNA+ group and the dotted line represents that for the FNA- group. The median relapse-free survival time of the FNA+ and FNA- groups was 852 and 158 d, respectively (Log-rank test; P = 0.04); B: Kaplan-Meier curves for overall survival in patients who underwent resection for pancreatic cancer without adjuvant chemotherapy. The solid line indicates the Kaplan-Meyer curve for the FNA+ group and the dotted line represents that for the FNA- group. The median overall survival time of the FNA+ and FNA- groups was 829 and 400 d, respectively (Log-rank test; P = 0.08). FNA+: The group of patients who underwent endoscopic ultrasound-guided fine-needle aspiration before surgery; FNA-: The group of patients who did not undergo endoscopic ultrasound-guided fine-needle aspiration before surgery. according to the administration of adjuvant chemotherapy. The RFS of patients in the FNA+ (n = 40) and FNA- (n = 14) groups who were treated with adjuvant chemotherapy was 596 d and 332 d, respectively (P = 0.30, log-rank test) (Figure 4A). The median OS of patients treated with adjuvant chemotherapy in the FNA+ and FNA- groups was 1042 d and 636 d, respectively (P < 0.05, log-rank test) (Figure 4B). For patients who did not receive adjuvant chemotherapy the RFS of the FNA+ (n = 14) and FNA- (n = 14) groups was 852 d and 158 d, respectively (P = 0.04, log-rank test) (Figure 5A). However, the median OS of patients not treated with adjuvant chemotherapy in the FNA+ and FNA- groups was 829 and 400 d, respectively (P = 0.08, log-rank test) (Figure 5B). In addition, we performed univariate and multivariate analyses for OS (Table 4) and RFS (Table 5). The hazard ratios of EUS-FNA for OS and RFS were 0.46 (P < 0.05) and 0.46 (P = 0.060), respectively, indicating that EUS-FNA was not an adverse prognostic factor for pancreatic surgery. These data indicate that the use of EUS- FNA did not influence RFS and OS, nor did it increase the risk of peritoneal recurrence. DISCUSSION The sensitivity and specificity of diagnostic tests for pancreatic neoplasms are gradually improving with the technological progress of imaging modalities. However, the diagnostic accuracy of pancreatic neoplasms based on imaging studies alone remains unsatisfactory. Approximately 10% of resected specimens that are preoperatively diagnosed as malignant pancreatic neoplasms [18] are subsequently found to be benign lesions, including focal autoimmune pancreatitis or chronic pancreatitis [19-21]. The 3624 April 7, 2014 Volume 20 Issue 13

250 Kudo T et al. EUS-FNA for resectable pancreatic cancer Table 4 Univariate and multivariate analyses of factors affecting overall survival Table 5 Univariate and multivariate analyses of factors affecting relapse-free survival after surgery Univariate Multivariate HR (95%CI) P value HR (95%CI) P value Age (over 65 years old) ( ) ( ) CEA 3.85 ng/ml ( ) ( ) CA U/mL ( ) ( ) Tumor size (> 20 mm) ( ) ( ) Portal vein invasion ( ) ( ) UICC ⅡB ( ) ( ) R1/R ( ) ( ) Adjuvant chemotherapy ( ) ( ) EUS-FNA ( ) ( ) Univariate Multivariate HR (95%CI) P value HR (95%CI) P value Age (over 65 years old) ( ) ( ) CEA 3.85 ng/ml ( ) ( ) CA U/mL ( ) ( ) Tumor size (> 20 mm) ( ) ( ) Portal vein invasion ( ) ( ) UICC ⅡB ( ) ( ) R1/R ( ) ( ) Adjuvant chemotherapy ( ) ( ) EUS-FNA ( ) ( ) UICC: Pathological stage of the Union Internationale Contre le Cancer; EUS-FNA: Endoscopic-ultrasound-guided fine-needle aspiration. UICC: Pathological stage of the Union Internationale Contre le Cancer; EUS-FNA: Endoscopic ultrasound-guided fine-needle aspiration. overall mortality rate after pancreatic surgery generally ranges from 0% to 10% [22,23]. Pancreatoduodenectomy is associated with relatively high mortality and morbidity rates, ranging from 0% to 7.1% and 20.8% to 59%, respectively [24], as is distal pancreatectomy, with mortality and morbidity rates ranging from 0% to 6% and 10% to 47%, respectively [25]. Thus, surgery for patients with benign pancreatic lesions must be avoided. EUS-FNA provides an accurate preoperative diagnosis of pancreatic solid tumors, compared to other imaging modalities, with a diagnostic accuracy [14] of 75%-95% [26-30]. The performance of EUS-FNA depends to a large extent on the ability of the endoscopist, and indeed, the diagnostic accuracy of EUS-FNA for adenocarcinoma of the pancreas has been shown to increase with operator experience [31]. It is noteworthy that the reported specificity of EUS-FNA for pancreatic solid neoplasms is almost 100% [32] and that the associated complication rate is < 1% [13]. Major complications of EUS-FNA are rare, but can include pancreatitis, bleeding, and post-procedural pain [13], although tumor seeding following EUS-FNA has been reported in 7 cases, 4 of which involved adenocarcinoma [6-12]. Preoperative EUS-FNA is avoided by some surgeons and physicians because of the risk of these complications, and it remains controversial as to whether preoperative EUS-FNA for pancreatic solid masses is always necessary [33]. Therefore, we reviewed the prognosis of postsurgical patients with pancreatic cancer and examined whether EUS-FNA adversely affected survival after pancreatic surgery. The results revealed no significant differences in complications or sites of recurrent lesions between patients who underwent FNA before surgery and those who did not. In our study, patients who underwent EUS-FNA had better RFS and OS than did those who did not, although it should be noted that more patients in the FNA+ group underwent adjuvant chemotherapy. Multivariate analysis revealed that tumor size and adjuvant chemotherapy were both prognostic factors for OS and RFS in this study. EUS-FNA, however, was not a prognostic factor for RFS. Thus, it is possible that patients in the FNA+ group benefited from the chemotherapy administered immediately after surgery. Furthermore, neither RFS nor OS were significantly different between the 2 groups when the administration of adjuvant chemotherapy was accounted for. These data indicate that preoperative EUS- FNA does not adversely affect surgery or prognosis in patients with resectable pancreatic cancer. EUS-FNA can also potentially improve the outcomes of pancreatic surgery by providing a more accurate diagnosis. These findings are important because the use of preoperative EUS- FNA is becoming more widespread. This study had some limitations. First, it was conducted in a single center with a small sample size, and a population bias is possible because our institute is a pancreatobiliary cancer referral center. Second, this was a retrospective study and some selection bias was observed between the 2 groups as described above. A randomized controlled trial in a multicenter setting is needed to confirm our results. ACKNOWLEDGMENTS We thank Mr. Katsuji Marukawa, CT IAC and Mr. Jun Moriya, CT IAC of the Department of Surgical Pathology, Hokkaido University Hospital for their technical assistance. We also thank Satoshi Hirano, MD, PhD and 3625 April 7, 2014 Volume 20 Issue 13

251 Kudo T et al. EUS-FNA for resectable pancreatic cancer Eiichi Tanaka, MD, PhD of the Department of Gastroenterological Surgery Ⅱ, Hokkaido University Graduate School of Medicine for their assistance with surgical treatment. COMMENTS Background Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) has been developed as a practical method for obtaining specimens for the definitive diagnosis of pancreatic lesions, with a low risk of adverse events. However, it is not yet fully established whether preoperative EUS-FNA is safe and effective for resectable pancreatic cancer, and there have been very few studies to address this. Research frontiers As it is now possible to resect some pancreatic cancers, the key research question is whether preoperative EUS-FNA is associated with an increased risk of adverse surgical events and whether preoperative EUS-FNA affects relapsefree survival (RFS) and overall survival (OS). Innovations and breakthroughs The diagnostic accuracy of EUS-FNA based on cytology and histology findings was 98.1% (53/54) and 77.8% (42/54), respectively, and the overall accuracy was 98.1% (53/54). No severe complications occurred after EUS-FNA. In the EUS-FNA and non-eus-fna groups, the median RFS was 742 and 265 d, respectively (P = ), and the median OS was 1042 and 557 d, respectively (P = ). Applications The study results suggest that preoperative EUS-FNA does not adversely affect surgery or prognosis in patients with resectable pancreatic lesions. Terminology Relapse-free survival: In cancer cases, the length of time after the end of primary treatment for a cancer for which the patient survives without any signs or symptoms of that cancer. In a clinical trial, measuring relapse-free survival is one way to determine the efficacy of a new treatment. Peer review This is a good descriptive study in which preoperative EUS-FNA is shown to be a practical and safe technique for acquiring pancreatic specimens. These results are interesting and suggest that preoperative EUS-FNA does not adversely affect surgery or prognosis in patients with resectable pancreatic lesions. REFERENCES 1 Raimondi S, Maisonneuve P, Lowenfels AB. Epidemiology of pancreatic cancer: an overview. Nat Rev Gastroenterol Hepatol 2009; 6: [PMID: DOI: /nrgastro ] 2 Vincent A, Herman J, Schulick R, Hruban RH, Goggins M. Pancreatic cancer. Lancet 2011; 378: [PMID: DOI: /S (10) ] 3 Vilmann P, Jacobsen GK, Henriksen FW, Hancke S. Endoscopic ultrasonography with guided fine needle aspiration biopsy in pancreatic disease. Gastrointest Endosc 1992; 38: [PMID: DOI: /S (92)70385-X] 4 Mizuno N, Hara K, Hijioka S, Bhatia V, Shimizu Y, Yatabe Y, Yamao K. Current concept of endoscopic ultrasoundguided fine needle aspiration for pancreatic cancer. Pancreatology 2011; 11 Suppl 2: [PMID: DOI: / ] 5 Eloubeidi MA, Gress FG, Savides TJ, Wiersema MJ, Kochman ML, Ahmad NA, Ginsberg GG, Erickson RA, Dewitt J, Van Dam J, Nickl NJ, Levy MJ, Clain JE, Chak A, Sivak MV, Wong R, Isenberg G, Scheiman JM, Bounds B, Kimmey MB, Saunders MD, Chang KJ, Sharma A, Nguyen P, Lee JG, Edmundowicz SA, Early D, Azar R, Etemad B, Chen YK, Waxman I, Shami V, Catalano MF, Wilcox CM. Acute pancreatitis after EUS-guided FNA of solid pancreatic masses: a pooled analysis from EUS centers in the United States. Gastrointest Endosc 2004; 60: [PMID: DOI: /S (04) ] 6 Ahmed K, Sussman JJ, Wang J, Schmulewitz N. A case of EUS-guided FNA-related pancreatic cancer metastasis to the stomach. Gastrointest Endosc 2011; 74: [PMID: DOI: /j.gie ] 7 Hirooka Y, Goto H, Itoh A, Hashimoto S, Niwa K, Ishikawa H, Okada N, Itoh T, Kawashima H. Case of intraductal papillary mucinous tumor in which endosonography-guided fine-needle aspiration biopsy caused dissemination. J Gastroenterol Hepatol 2003; 18: [PMID: DOI: /j x] 8 Paquin SC, Gariépy G, Lepanto L, Bourdages R, Raymond G, Sahai AV. A first report of tumor seeding because of EUSguided FNA of a pancreatic adenocarcinoma. Gastrointest Endosc 2005; 61: [PMID: DOI: / S (05) ] 9 Shah JN, Fraker D, Guerry D, Feldman M, Kochman ML. Melanoma seeding of an EUS-guided fine needle track. Gastrointest Endosc 2004; 59: [PMID: DOI: /S (04) ] 10 Doi S, Yasuda I, Iwashita T, Ibuka T, Fukushima H, Araki H, Hirose Y, Moriwaki H. Needle tract implantation on the esophageal wall after EUS-guided FNA of metastatic mediastinal lymphadenopathy. Gastrointest Endosc 2008; 67: [PMID: DOI: /j.gie ] 11 Katanuma A, Maguchi H, Hashigo S, Kaneko M, Kin T, Yane K, Kato R, Kato S, Harada R, Osanai M, Takahashi K, Shinohara T, Itoi T. Tumor seeding after endoscopic ultrasound-guided fine-needle aspiration of cancer in the body of the pancreas. Endoscopy 2012; 44 Suppl 2 UCTN: E160-E161 [PMID: DOI: /s ] 12 Chong A, Venugopal K, Segarajasingam D, Lisewski D. Tumor seeding after EUS-guided FNA of pancreatic tail neoplasia. Gastrointest Endosc 2011; 74: [PMID: DOI: /j.gie ] 13 Wang KX, Ben QW, Jin ZD, Du YQ, Zou DW, Liao Z, Li ZS. Assessment of morbidity and mortality associated with EUSguided FNA: a systematic review. Gastrointest Endosc 2011; 73: [PMID: DOI: /j.gie ] 14 Beane JD, House MG, Coté GA, DeWitt JM, Al-Haddad M, LeBlanc JK, McHenry L, Sherman S, Schmidt CM, Zyromski NJ, Nakeeb A, Pitt HA, Lillemoe KD. Outcomes after preoperative endoscopic ultrasonography and biopsy in patients undergoing distal pancreatectomy. Surgery 2011; 150: [PMID: DOI: /j.surg ] 15 Klapman JB, Chang KJ, Lee JG, Nguyen P. Negative predictive value of endoscopic ultrasound in a large series of patients with a clinical suspicion of pancreatic cancer. Am J Gastroenterol 2005; 100: [PMID: DOI: /j x] 16 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) ] 17 Davila RE, Rajan E, Adler DG, Egan J, Hirota WK, Leighton JA, Qureshi W, Zuckerman MJ, Fanelli R, Wheeler- Harbaugh J, Baron TH, Faigel DO. ASGE Guideline: the role of endoscopy in the patient with lower-gi bleeding. Gastrointest Endosc 2005; 62: [PMID: DOI: /j.gie ] 18 Wolfson D, Barkin JS, Chari ST, Clain JE, Bell RH, Alexakis N, Neoptolemos JP. Management of pancreatic masses. Pancreas 2005; 31: [PMID: DOI: /01. mpa ca] 19 Smith CD, Behrns KE, van Heerden JA, Sarr MG. Radical pancreatoduodenectomy for misdiagnosed pancreatic mass. Br J Surg 1994; 81: [PMID: DOI: / bjs ] 3626 April 7, 2014 Volume 20 Issue 13

252 Kudo T et al. EUS-FNA for resectable pancreatic cancer 20 Abraham SC, Wilentz RE, Yeo CJ, Sohn TA, Cameron JL, Boitnott JK, Hruban RH. Pancreaticoduodenectomy (Whipple resections) in patients without malignancy: are they all chronic pancreatitis? Am J Surg Pathol 2003; 27: [PMID: DOI: / ] 21 van Gulik TM, Reeders JW, Bosma A, Moojen TM, Smits NJ, Allema JH, Rauws EA, Offerhaus GJ, Obertop H, Gouma DJ. Incidence and clinical findings of benign, inflammatory disease in patients resected for presumed pancreatic head cancer. Gastrointest Endosc 1997; 46: [PMID: DOI: /S (97) ] 22 Gooiker GA, van Gijn W, Wouters MW, Post PN, van de Velde CJ, Tollenaar RA. Systematic review and meta-analysis of the volume-outcome relationship in pancreatic surgery. Br J Surg 2011; 98: [PMID: DOI: / bjs.7413] 23 Nimura Y, Nagino M, Takao S, Takada T, Miyazaki K, Kawarada Y, Miyagawa S, Yamaguchi A, Ishiyama S, Takeda Y, Sakoda K, Kinoshita T, Yasui K, Shimada H, Katoh H. Standard versus extended lymphadenectomy in radical pancreatoduodenectomy for ductal adenocarcinoma of the head of the pancreas: long-term results of a Japanese multicenter randomized controlled trial. J Hepatobiliary Pancreat Sci 2012; 19: [PMID: DOI: /s ] 24 Diener MK, Knaebel HP, Heukaufer C, Antes G, Büchler MW, Seiler CM. A systematic review and meta-analysis of pylorus-preserving versus classical pancreaticoduodenectomy for surgical treatment of periampullary and pancreatic carcinoma. Ann Surg 2007; 245: [PMID: DOI: /01.sla a9] 25 Knaebel HP, Diener MK, Wente MN, Büchler MW, Seiler CM. Systematic review and meta-analysis of technique for closure of the pancreatic remnant after distal pancreatectomy. Br J Surg 2005; 92: [PMID: DOI: /bjs.5000] 26 Hartwig W, Hackert T, Hinz U, Gluth A, Bergmann F, Strobel O, Büchler MW, Werner J. Pancreatic cancer surgery in the new millennium: better prediction of outcome. Ann Surg 2011; 254: [PMID: DOI: / SLA.0b013e31821fd334] 27 Wiersema MJ, Vilmann P, Giovannini M, Chang KJ, Wiersema LM. Endosonography-guided fine-needle aspiration biopsy: diagnostic accuracy and complication assessment. Gastroenterology 1997; 112: [PMID: DOI: /S (97) ] 28 Yamao K, Ohashi K, Mizutani S, Furukawa T, Watanabe Y, Nakamura T, Suzuki T, Takeda K. Endoscopic ultrasoundguided fine-needle aspiration (EUS-FNA) for the diagnosis of digestive diseases. Endoscopy 1998; 30 Suppl 1: A176-A178 [PMID: DOI: /s ] 29 Itoi T, Sofuni A, Itokawa F, Irisawa A, Khor CJ, Rerknimitr R. Current status of diagnostic endoscopic ultrasonography in the evaluation of pancreatic mass lesions. Dig Endosc 2011; 23 Suppl 1: [PMID: DOI: / j x] 30 Haba S, Yamao K, Bhatia V, Mizuno N, Hara K, Hijioka S, Imaoka H, Niwa Y, Tajika M, Kondo S, Tanaka T, Shimizu Y, Yatabe Y, Hosoda W, Kawakami H, Sakamoto N. Diagnostic ability and factors affecting accuracy of endoscopic ultrasoundguided fine needle aspiration for pancreatic solid lesions: Japanese large single center experience. J Gastroenterol 2013; 48: [PMID: DOI: /s ] 31 Mertz H, Gautam S. The learning curve for EUS-guided FNA of pancreatic cancer. Gastrointest Endosc 2004; 59: [PMID: DOI: /S (03) ] 32 Hewitt MJ, McPhail MJ, Possamai L, Dhar A, Vlavianos P, Monahan KJ. EUS-guided FNA for diagnosis of solid pancreatic neoplasms: a meta-analysis. Gastrointest Endosc 2012; 75: [PMID: DOI: /j.gie ] 33 Lachter J, Rosenthal Y, Kluger Y. A multidisciplinary survey on controversies in the use of EUS-guided FNA: assessing perspectives of surgeons, oncologists and gastroenterologists. BMC Gastroenterol 2011; 11: 117 [PMID: DOI: / X ] P- Reviewers: Shah OJ, Swan Michael, Teoh AYB S- Editor: Qi Y L- Editor: A E- Editor: Ma S 3627 April 7, 2014 Volume 20 Issue 13

253 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH REPORT Tumors of the angle of Treitz: A single-center experience Yi-Bin Xie, Hao Liu, Liang Cui, Gu-Sheng Xing, Lin Yang, Yue-Min Sun, Xiao-Feng Bai, Dong-Bing Zhao, Cheng-Feng Wang, Yan-Tao Tian Yi-Bin Xie, Hao Liu, Liang Cui, Yue-Min Sun, Xiao-Feng Bai, Dong-Bing Zhao, Cheng-Feng Wang, Yan-Tao Tian, Department of Abdominal Surgical Oncology, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing , China Gu-Sheng Xing, Department of Diagnostic Radiology, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing , China Lin Yang, Department of Pathology, Cancer Hospital, Chinese Academy of Medical Sciences, Beijing , China Author contributions: Xie YB, Liu H, Cui L, Xing GS, Yang L, Sun YM, Bai XF, Zhao DB and Wang CF performed the research; Xie YB and Tian YT were involved in designing the study and editing the manuscript. Supported by Beijing Municipal Science Technology Commission Fund No. Z and the Beijing Hope Run Special Fund LC2012A09 Correspondence to: Yan-Tao Tian, MD, Department of Abdominal Surgical Oncology, Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, 17 Pan Jia Yuan Nan Li, Beijing , China. tyt67@163.com Telephone: Fax: Received: January 2, 2014 Revised: February 10, 2014 Accepted: March 7, 2014 Published online: April 7, 2014 The mean tumor size was 8.1 cm (range, cm). Histologic examination showed 11 gastrointestinal stromal tumors (GISTs) and 2 adenocarcinomas. Five of the GIST patients presented with potential low risk, and 6 presented with intermediate and high risk, according to the National Institutes of Health criteria. One potentially high-risk patient showed tumor progression at 46 mo and died 52 mo after surgery. One patient with locally advanced adenocarcinoma received neoadjuvant chemotherapy and adjuvant radiotherapy, but the disease progressed, and the patient died 9 mo after surgery. One GIST patient without progression died 16 mo after surgery because of a postoperative intestinal obstruction. The median overall survival rate was 84.6 mo, and the median disease-free survival rate was 94.5 mo. CONCLUSION: The overall survival of patients with tumors of the angle of Treitz was encouraging even when the tumor size was relatively large. A segmental resection on the left side of the mesenteric vessels is considered to be a reliable and curative option for tumors of the angle of Treitz Baishideng Publishing Group Co., Limited. All rights reserved. Abstract AIM: To explore the feasibility and oncologic outcomes of segmental jejunal resection on the left side of the mesenteric vessels in patients with tumors of the angle of Treitz using data from a single center. METHODS: Thirteen patients with tumors of the angle of Treitz who underwent surgery at our institution were prospectively followed. A segmental jejunal resection on the left side of the mesenteric vessels was performed in all patients. Formalin-fixed and paraffin-embedded tumor samples were examined. The primary end point of this analysis was disease-free survival. RESULTS: In this study, there were 8 males and 5 females (mean age, 50.1 years; range, years). Key words: Gastrointestinal stromal tumor; Adenocarcinoma; Angle of Treitz; Surgical treatment; Prognosis Core tip: This single-center study investigated a type of rare tumor originating from the angel of Treitz. The symptoms, diagnosis, surgical procedure, histology, and prognosis were evaluated. Although the tumors tended to be large, segmental jejunal resection on the left side of the mesenteric vessels was the treatment of choice. Xie YB, Liu H, Cui L, Xing GS, Yang L, Sun YM, Bai XF, Zhao DB, Wang CF, Tian YT. Tumors of the angle of Treitz: A single-center experience. World J Gastroenterol 2014; 20(13): Available from: URL: com/ /full/v20/i13/3628.htm DOI: April 7, 2014 Volume 20 Issue 13

254 Xie YB et al. Tumors of the angle of Treitz Table 1 Clinical and pathologic characteristics of patients with tumors of the angle of Treitz Patient Age (y) Gender Size (cm) Presentation Pathology Surgical procedure Lymph node positive/total Adjuvant therapy Site of progression Cause of death 1 41 Male 15 Health examination GIST Segmentectomy 0/0 Gleevec 3 yr 2 74 Male 4 Health examination GIST Segmentectomy 0/ Male 5 Health examination GIST Segmentectomy 0/ Male 5 Health examination GIST Segmentectomy 0/ Female 7.5 Pain GIST Segmentectomy 0/0 Obstruction 6 51 Female 6.7 Health examination GIST Segmentectomy 0/0 Gleevec 1 yr 7 61 Male 13 Health examination GIST Segmentectomy 0/0 Liver Liver metastasis 8 36 Male 4.5 Bleeding GIST Segmentectomy 0/ Female 3.2 Pain GIST Segmentectomy 0/ Male 14 Pain GIST Multi-visceral 0/7 Gleevec 3 yr Male 8.5 Bleeding, pain, GIST Segmentectomy 0/1 anemia Female 3.3 Pain, vomiting Cancer Segmentectomy 0/ Female 15 Vomiting, pain, anemia Cancer Segmentectomy 2/7 Radiotherapy Local Tumor progression GIST: Gastrointestinal stromal tumors. org/ /wjg.v20.i INTRODUCTION It is known that tumors of the small intestine, which account for less than 5% of digestive tract tumors [1], rarely occur. Tumors occurring in the angle of Treitz are even rarer. As a result, tumors of the angle of Treitz have never been reported on a large scale. Most published studies are case reports [2-5]. Most cases occur sporadically, but some may occur in the context of a familial syndrome (i.e., Peutz-Jeghers syndrome or Crohn s disease) [6]. These patients usually present with abdominal pain, iron-deficiency anemia, gastrointestinal bleeding, or vomiting caused by an obstruction [7]. A tumor of the angle of Treitz may also be an incidental finding during clinical examination. Although segmental resection is the treatment of choice, this is not apparent from the literature. Moreover, data from our center suggest that most tumors of the angle of Treitz are gastrointestinal stromal tumors (GISTs), and only a few are adenocarcinomas. In this study, we report a series of patients with tumors of the angle of Treitz to clarify the diagnosis and treatment method. MATERIALS AND METHODS Patients and tumor specimens This study examined 13 patients (8 men and 5 women; mean age, 50.1 years; range, years). All patients in this study underwent surgical resection of tumors of the angle of Treitz (Table 1). Formalin-fixed and paraffinembedded tumor samples were examined. The pathological categories for the 13 patients were GIST (11 cases) and adenocarcinoma (2 cases). For the patients with GISTs, immunohistochemical evaluations of CD117, CD34, S-100, smooth muscle actin (SMA), desmin, and Ki67 were performed in all cases (Table 2). Assessments of the maximal tumor size, histologic growth pattern, and mitotic count in 50 high-power fields (HPFs, for GISTs) were independently performed by two senior pathologists. The malignant potential of the GISTs was estimated based on tumor size, mitotic count, rupture, and tumor location, according to the criteria published in 2008 by the National Institutes of Health (NIH) [8]. The survival data were obtained either by reviewing the clinical records or at follow-up. In addition, preoperative and postoperative therapies with tyrosine kinase inhibitors or chemotherapy were assessed. Surgical procedure The surgical procedure included segmental jejunal resection and multivisceral resection. The decision regarding which of the two procedures to use was made intraoperatively based on tumor size and site. Segmental jejunal resection was performed on the left side of the mesenteric blood vessels (Figure 1). When the tumor infiltrated into adjacent organs, multivisceral resection was performed. Statistical analysis All statistical analyses were performed with the software program IBM SPSS Statistics 21 (IBM Corp., Armonk, NY, United States). The disease-free survival and overall survival were estimated using the Kaplan-Meier method. The nonparametric log-rank test was used to compare the groups. RESULTS Clinicopathologic characteristics Table 1 summarizes the clinicopathologic characteristics of the 13 patients with tumors of the angle of Treitz. Mean patient age was 50.1 years, and there was no significant correlation between age and tumor recurrence (95%CI: ; P = 0.686). Mean tumor size was 8.1 cm (range, cm). There was no significant 3629 April 7, 2014 Volume 20 Issue 13

255 Xie YB et al. Tumors of the angle of Treitz Table 2 Immunohistochemical examination and clinical data of 11 gastrointestinal stromal tumors Patient CD117 CD34 Desmin SMA S-100 Ki67 Mitotic count (/50HPF) Other malignancy NIH risk scale %+ 3 No High < 5% < 2 No Low % < 5 No Low %+ 1 No Low < 2% 1 No Medium < 2% 3 No Medium % 1 Colon cancer High < 5% 1 No Low < 2% 1 Gastric cancer Low % > 10 No High % 2 Intestinal GIST 16 years ago Medium SMA: Smooth muscle actin; NIH: National Institutes of Health. A B Figure 1 An intraoperative photograph of the mesenteric vessels (A), and of anastomosis on the left side of the mesenteric vessels (B). A B Figure 2 A well-encapsulated gastrointestinal stromal tumor with a normal mucous membrane(a), and an ulcerative carcinoma that has infiltrated into the adjacent tissue (B). correlation between tumor size and tumor recurrence (95%CI: ; P = 0.284). Six patients were diagnosed without symptoms in clinical examinations. Six of the remaining patients presented with abdominal pain: 2 presented with vomiting; 2 presented with anemia; and 2 presented with gastrointestinal bleeding. Both of the patients with adenocarcinoma presented with vomiting, whereas none of the GIST patients had such symptoms. The GISTs tended to be sharply demarcated without infiltrative growth, which was in contrast to the adenocarcinomas (Figure 2). Based on tumor histology, 11 tumors were diagnosed as GISTs with spindle-cell differentiation, and the other 2 as adenocarcinomas (Figure 3). The immunohistochemical analysis showed that all GISTs expressed CD117. Five tumors expressed S-100, 8 tumors expressed CD34, and 5 tumors expressed SMA. No desmin-positive tumors were identified. The mitotic rate per 50 HPFs ranged from 1 to more than 10 (median, 2 per 50 HPFs). The potential risk of malignancy (according to NIH criteria) was rated as low in 5 cases, medium 3630 April 7, 2014 Volume 20 Issue 13

256 Xie YB et al. Tumors of the angle of Treitz A B Figure 3 Gastrointestinal stromal tumors with spindle cell differentiation (A) ( 20), and carcinoma with moderate-topoor differentiation (B) ( 20). A B C D Figure 4 Computed tomography scan. A, B: Computed tomography (CT) scan and upper gastrointestinal radiography for gastrointestinal stromal tumors of the angle of Treitz (arrow); C, D: CT scan of adenocarcinoma of the angle of Treitz before (C) and after (D) chemotherapy (arrow). in 3 cases, and high in 3 cases. All 13 patients underwent computed tomography (CT) examination. The CT results indicated that 5 tumors originated from the angle of Treitz, and 8 tumors were detected in the left upper abdomen (Figure 4). Only one patient was diagnosed using traditional endoscopy. Follow-up Survival data for all patients were obtained. One of the two patients with adenocarcinoma had tumor progression and died 9 mo after surgery. The other patient with adenocarcinoma survived and showed no recurrence at the 18-mo follow-up. For the 11 patients with GISTs, although only one showed tumor progression (high-risk potential), 2 patients ultimately died: one 16 mo postoperatively from postoperative obstruction, and the other 52 mo postoperatively from tumor progression. Overall survival and disease-free survival rates are shown in Figure 5. The median overall survival rate was 84.6 mo, and the median disease-free survival rate was 94.5 mo. The surgical procedures included segmental jejunal resection in 11 patients and multivisceral resection in one patient. The multivisceral resection comprised a segmental jejunal resection combined with segmental colon resection because of tumor infiltration into the middle colic artery. One of the patients who underwent segmental jejunal resection was treated with secondary descending duodenum jejunal side-to-side anastomosis 10 d after the initial operation because of postoperative obstruction by greater omental adhesion, which constricted the fourth part of the duodenum. The patient died 16 mo later of intractable intestinal obstruction. Three of the 11 GIST patients received imatinib after surgery, including one patient with a medium risk of 3631 April 7, 2014 Volume 20 Issue 13

257 Xie YB et al. Tumors of the angle of Treitz A 1.0 B Delete 1.0 Delete Overall survival Disease-free survival t /mo t /mo Figure 5 Kaplan-Meier analysis demonstrates the overall (A) and disease-free survival (B) rates for the 13 patients with tumors of the angle of Treitz. progression and 2 high-risk patients. None of these patients had any evidence of relapse. The other 8 patients did not receive adjuvant imatinib therapy. One high-risk patient (patient 7) had a synchronous carcinoma of the sigmoid colon (pt3n0m0), which was resected simultaneously, and received systemic chemotherapy for 6 cycles (FOLFOX4 regimen). After 46 mo, the patient developed liver metastasis and died 6 mo later. One of the two adenocarcinoma patients (patient 12) did not receive any adjuvant therapy and remained alive at the 18-mo followup. Another patient (patient 13) received neoadjuvant chemotherapy (oxaliplatin + capecitabine) for 3 cycles, but the response was mild, and the tumor did not shrink. After palliative surgery, the patient underwent subsequent radiotherapy (2 Gy, 25 times, 5 wk). However, the disease progressed, and the patient died 9 mo after resection. DISCUSSION Tumors of the angle of Treitz are difficult to diagnose because of their rarity and nonspecific symptoms [9]. In this group, 6 patients had no presentation and were diagnosed incidentally (by clinical examination); the average tumor size was 8.1 cm. Interestingly, these patients all had GISTs. In symptomatic patients, only those with acute gastrointestinal bleeding or vomiting were diagnosed within 2 weeks, whereas the diagnosis of patients with vague abdominal pain was delayed beyond 2 mo (range, 2 mo to 5 years). Similar to our findings, the study of Cunningham et al [10] showed that the mean time from symptom onset to diagnosis was approximately 4 mo. Therefore, the treatment of these tumors often occurs late. According to the literature [11], barium meal radiography of the digestive tract is useful for detecting endoluminal diseases. However, the optimal examination method is capsule endoscopy or endoscopy using a tube capable of reaching the angle of Treitz [12]. Moreover, under exophytic growth conditions, as in most GISTs, CT may be useful for localizing and assessing resectability [13]. Before the use of capsule endoscopy and modern helical CT, an upper intestinal barium study was the most frequently used radiographic diagnostic method. This approach has a diagnostic rate as low as 22% [14]. Recent studies [15] have shown that CT enteroclysis using spiral and multidetector-row CT with an enteral contrast agent has become the radiographic diagnostic tool of choice for suspected small bowel neoplasms because the sensitivity and specificity are 100% and 95%, respectively. Regarding the resection margin and lymphadenectomy of GISTs, investigators have reached a consensus that R0 resection is sufficient and that there is no need for lymphadenectomy [16]. However, the surgical procedure for adenocarcinoma of the angle of Treitz remains controversial. The particular vascularization of this area is technically challenging for surgeons. To increase the resection margin, some investigators [17] recommend resection with anastomosis on the right side of the mesenteric vessels even if there is a risk of damaging the papilla. Conversely, other investigators [5] suggest a segmental resection on the left side of the mesenteric vessels to protect the blood supply of the duodenal stump. These investigators identified radical resection of this disease as having a proximal resection margin of > 2 cm instead of > 5 cm. Additionally, the extent of lymphadenectomy is also disputed. Although extended lymphadenectomy for adenocarcinoma is recommended [18], total removal of all cancer cells for tumors in this area makes lymphatic drainage difficult [19]. From a technical perspective, we believe that only lymph nodes visible to the naked eye or involved in the area of resection should be removed [20]. In this patient group, we performed resections only on the left side of the mesenteric vessels, and all patients recovered uneventfully. The prognoses and disease-free survival rates of GIST patients depend on tumor biology, which is determined by tumor size, localization, rupture, and mitotic index (using the NIH scale). This finding has been affirmed by Miettinen et al [21] for duodenal GISTs. This result was also verified by our patient who developed liver metastasis. In our series, 10 cases (90.9%) had a mitotic count of no more than 5 mitoses per 50 HPFs. This result is consistent with the findings of Winfield et al [22]. The use of adjuvant imatinib after radical resection is widely accepted and is recommended at a dose of 400 mg/d for 1 year for medium-risk patients and for 3 years for high April 7, 2014 Volume 20 Issue 13

258 Xie YB et al. Tumors of the angle of Treitz risk patients [23]. In our group, only 3 of the 6 patients who were potentially at medium or high risk received adjuvant therapy for economic reasons. None of these patients developed recurrence. Early studies showed that the 5-year survival rate of patients with carcinomas confined to duodenal segments D3, D4, and the angle of Treitz was approximately 75% [19]. However, recent studies revealed a 5-year survival rate of only 23%-30% [10,14], which might be attributed to the different disease stages among the patients who underwent resection. The factors influencing long-term survival include R1 or palliative resection, a locally advanced tumor, positive regional lymph nodes, and poor response to adjuvant chemotherapy [18]. Consistent with the results of previous studies [14,18], two adenocarcinoma patients in our series had markedly different outcomes. One patient (with a relatively early-stage tumor) who underwent radical resection survived 18 mo and showed no tumor progression. The other patient with a locally advanced tumor underwent palliative resection but died 9 mo after surgery despite undergoing neoadjuvant chemotherapy and adjuvant radiotherapy. Several studies have shown that adjuvant therapy has no benefit after radical resection [24]. In conclusion, the overall survival of patients with tumors of the angle of Treitz was encouraging even though the tumor size was often relatively large. Segmental resection on the left side of the mesenteric vessels is considered to be a reliable and curative option for tumors of the angle of Treitz. For GIST patients, the prognosis depends on the risk scale. The use of adjuvant imatinib is recommended for patients at medium or high risk. Surgery remains the treatment of choice for adenocarcinoma patients. The role of neoadjuvant or adjuvant chemotherapy requires further investigation. COMMENTS Background It is known that tumors of the small intestine rarely occur, and tumors of the angle of Treitz are even rarer. The majority of the published literature consists of case reports. Patients usually present with abdominal pain, iron-deficiency anemia, gastrointestinal bleeding, or vomiting caused by obstruction. Tumors of the angle of Treitz may also be incidental findings during a clinical examination. The unique vascularization of this area is technically challenging for surgeons. Although segmental resection is the treatment of choice, this is not apparent from the literature. Research frontiers Some authors recommend resection with anastomosis on the right side of the mesenteric vessels, whereas others suggest segmental resection on the left side. Surgeons debate over the best approach. Innovations and breakthroughs Most previous studies involved case reports of adenocarcinoma of the angle of Treitz. According to our study, tumors of the angle of Treitz include more gastrointestinal stromal tumor cases than adenocarcinoma cases. As previously reported, patients usually present with abdominal pain, iron-deficiency anemia, gastrointestinal bleeding, or vomiting caused by obstruction. Tumors of the angle of Treitz may also be incidental findings during a clinical examination. Based on experiences in our center, we performed all resections on the left side of the mesenteric vessels. Applications The results of our study suggest that resection with anastomosis on the left side of the mesenteric vessels is a safe and effective treatment for tumors of the angle of Treitz. Terminology The angle of Treitz is also known as the ligament of Treitz and is the junction of the duodenum and jejunum adjacent to the mesenteric vessels. Peer review In this manuscript, the authors describe clinicopathologic characteristics and surgical outcomes of tumors of the angle of Treitz, which is a rare condition, experienced at a single center. The results are of great value for further clinical study. REFERENCES 1 Moglia A, Menciassi A, Dario P, Cuschieri A. Clinical update: endoscopy for small-bowel tumours. Lancet 2007; 370: [PMID: DOI: /S (07) ] 2 Maglio R, Valabrega S, Ramacciato G. Duodenal carcinoma at ligament of Treitz. Case report and review of the literature. G Chir 2012; 33: [PMID: ] 3 Kalogerinis PT, Poulos JE, Morfesis A, Daniels A, Georgakila S, Daignualt T, Georgakilas AG. Duodenal carcinoma at the ligament of Treitz. A molecular and clinical perspective. BMC Gastroenterol 2010; 10: 109 [PMID: DOI: / X ] 4 Nabeshima K, Machimura T, Wasada M, Takayasu H, Ogoshi K, Makuuchi H. A case of primary jejunal cancer diagnosed by preoperative small intestinal endoscopy. 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Surgically treated primary malignant tumor of small bowel: a clinical analysis. World J Gastroenterol 2010; 16: [PMID: DOI: /wjg.v16.i ] 10 Cunningham JD, Aleali R, Aleali M, Brower ST, Aufses AH. Malignant small bowel neoplasms: histopathologic determinants of recurrence and survival. Ann Surg 1997; 225: [PMID: DOI: / ] 11 Zhan J, Xia ZS, Zhong YQ, Zhang SN, Wang LY, Shu H, Zhu ZH. Clinical analysis of primary small intestinal disease: A report of 309 cases. World J Gastroenterol 2004; 10: [PMID: ] 12 He Q, Bai Y, Zhi FC, Gong W, Gu HX, Xu ZM, Cai JQ, Pan DS, Jiang B. Double-balloon enteroscopy for mesenchymal tumors of small bowel: nine years experience. World J Gastroenterol 2013; 19: [PMID: DOI: / wjg.v19.i ] 13 Landi B. Gastrointestinal stromal tumors: clinical features and diagnosis. Bull Acad Natl Med 2012; 196: ; discussion [PMID: ] 14 Dabaja BS, Suki D, Pro B, Bonnen M, Ajani J. Adenocarcinoma of the small bowel: presentation, prognostic factors, and outcome of 217 patients. 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259 Xie YB et al. Tumors of the angle of Treitz 15 Schmidt S, Felley C, Meuwly JY, Schnyder P, Denys A. CT enteroclysis: technique and clinical applications. Eur Radiol 2006; 16: [PMID: DOI: / s ] 16 Siu J, Lim M, Fischer J, Dobbs B, Wakeman C, Ing A, Frizelle F. Ten-year review of gastrointestinal stromal tumours at a tertiary referral hospital in New Zealand. ANZ J Surg 2013; Epub ahead of print [PMID: DOI: / ans.12429] 17 Sabiani P, Le Treut YP, Maillet B, Bozon-Verduraz E, Bricot R. Adenocarcinoma of the duodenojejunal angle. Diagnostic and therapeutic problems. J Chir (Paris) 1987; 124: [PMID: ] 18 Agrawal S, McCarron EC, Gibbs JF, Nava HR, Wilding GE, Rajput A. Surgical management and outcome in primary adenocarcinoma of the small bowel. Ann Surg Oncol 2007; 14: [PMID: DOI: /s ] 19 Lowell JA, Rossi RL, Munson JL, Braasch JW. Primary adenocarcinoma of third and fourth portions of duodenum. Favorable prognosis after resection. Arch Surg 1992; 127: [PMID: DOI: /archsurg ] 20 Barnes G, Romero L, Hess KR, Curley SA. Primary adenocarcinoma of the duodenum: management and survival in 67 patients. Ann Surg Oncol 1994; 1: [PMID: DOI: /BF ] 21 Miettinen M, Kopczynski J, Makhlouf HR, Sarlomo-Rikala M, Gyorffy H, Burke A, Sobin LH, Lasota J. Gastrointestinal stromal tumors, intramural leiomyomas, and leiomyosarcomas in the duodenum: a clinicopathologic, immunohistochemical, and molecular genetic study of 167 cases. Am J Surg Pathol 2003; 27: [PMID: DOI: / ] 22 Winfield RD, Hochwald SN, Vogel SB, Hemming AW, Liu C, Cance WG, Grobmyer SR. Presentation and management of gastrointestinal stromal tumors of the duodenum. Am Surg 2006; 72: ; discussion [PMID: ] 23 Dematteo RP, Ballman KV, Antonescu CR, Maki RG, Pisters PW, Demetri GD, Blackstein ME, Blanke CD, von Mehren M, Brennan MF, Patel S, McCarter MD, Polikoff JA, Tan BR, Owzar K. Adjuvant imatinib mesylate after resection of localised, primary gastrointestinal stromal tumour: a randomised, double-blind, placebo-controlled trial. Lancet 2009; 373: [PMID: DOI: / S (09) ] 24 Scott-Coombes DM, Williamson RC. Surgical treatment of primary duodenal carcinoma: a personal series. Br J Surg 1994; 81: [PMID: DOI: / bjs ] P- Reviewers: Bar H, Takahashi T S- Editor: Qi Y L- Editor: Cant MR E- Editor: Wang CH 3634 April 7, 2014 Volume 20 Issue 13

260 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH REPORT Seroepidemiology of Helicobacter pylori infection in elderly people in the Beijing region, China Mei Zhang, Ying-Zhi Zhou, Xiao-Ying Li, Zhe Tang, Hong-Ming Zhu, Yan Yang, Jagadish K Chhetri Mei Zhang, Ying-Zhi Zhou, Xiao-Ying Li, Zhe Tang, Hong- Ming Zhu, Yan Yang, Jagadish K Chhetri, Department of Gastroenterology, XuanWu Hospital, Capital Medical University, Beijing , China Author contributions: Zhang M conceived the study and drafted the paper; Li XY approved and supervised the paper; Zhou YZ and Chhetri JK critically reviewed and revised the paper; Tang Z collected source material and analyzed the preliminary data; Zhu HM and Yang Y analyzed the data and performed the statistics. Correspondence to: Mei Zhang, Professor, Department of Gastroenterology, Xuanwu Hospital, Capital Medical University, No. 45 Changchun Street, Xuanwu District, Beijing100053, China. zhang2955@sina.com Telephone: Fax: Received: February 13, 2014 Revised: February 25, 2014 Accepted: March 5, 2014 Published online: April 7, 2014 Abstract AIM: To investigate seroepidemiology of caga + and vaca + strains of Helicobacter pylori (H. pylori ) in an elderly population in Beijing and to determine risk factors for seropositivity. METHODS: A total of 2006 elderly persons (> 60 years) were selected using a random cluster sampling method in different parts of the Beijing area (urban, suburban and mountainous districts). Structured questionnaires were completed during home visits, including history of H. pylori infection, history of gastrointestinal diseases, diet types, hygiene habits, occupation and economic status. Blood samples (2 ml) were collected from each participant, and serum IgG antibodies to caga, vaca and H. pylori urease antigens were measured by immunodetection. RESULTS: The prevalence of H. pylori infection in elderly subjects was 83.4% and the type Ⅰ H. pylori strain infection rate was 56%. The seroprevalence for type Ⅰ H. pylori strain infection in urban and suburban districts was higher than that in the mountainous areas (P < 0.001). Elderly subjects who had previously performed manual labor or were in the young-old age group (age < 75 years) had a higher seroprevalence of H. pylori infection than those who had previously performed mental labor or were in the oldest-old age group (age 75 year) (P < 0.05). The type Ⅰ H. pylori strain infection rate in the elderly with vegetarian diets was higher than in those eating high-protein foods (P < 0.001). There was no significant difference in the prevalence of H. pylori strains between male and female elderly participants (P > 0.05). CONCLUSION: Type Ⅰ H. pylori seroprevalence is higher in elderly people. The distribution of strains of H. pylori is significantly affected by age, area and dietary habits Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Elderly; Epidemiology; Helicobacter pylori ; Virulence factors; Immunoblotting Core tip: As society ages, a considerable proportion of the elderly population will suffer from digestive diseases combined with Helicobacter pylori (H. pylori ) infection. In Beijing, there are no data regarding the pattern of H. pylori genotypes in the elderly. Our study investigated the seroepidemiology of the cytotoxin-associated gene product caga + and vacuolating cytotoxin vaca + strains of H. pylori in elderly people in Beijing, and risk factors for seropositivity. Interesting associations between H. pylori seropositivity and subjects habits were found in this population. This is the first prospective study conducted in China to investigate the genotype profiles of H. pylori. Zhang M, Zhou YZ, Li XY, Tang Z, Zhu HM, Yang Y, Chhetri JK. Seroepidemiology of Helicobacter pylori infection in elderly 3635 April 7, 2014 Volume 20 Issue 13

261 Zhang M et al. Epidemiology of H. pylori in elderly Chinese people in the Beijing region, China. World J Gastroenterol 2014; 20(13): Available from: URL: com/ /full/v20/i13/3635.htm DOI: org/ /wjg.v20.i INTRODUCTION Evidence shows that Helicobacter pylori (H. pylori) infection is associated with chronic gastritis, peptic ulcers, gastric mucosa-associated lymphoid tissue lymphoma and gastric cancer [1-5]. It has been estimated that half of the world s population is infected by H. pylori [6,7], but with different clinical outcomes. The role of H. pylori as an obligate pathogen remains questionable. One of the challenges of H. pylori research is to ascertain why so many people carry it (approximately 50%), but only approximately 20% of those become sick. Recent research has shown that H. pylori virulence factors, the cytotoxinassociated gene product A (caga) and vacuolating cytotoxin A (vaca), in addition to host and environmental factors, may be very important [8,9]. It is clear that there is a paucity of well-designed studies of asymptomatic populations. With an aging society, elderly people suffering from digestive diseases combined with H. pylori infection account for a considerable proportion. In Beijing, there are no data regarding the pattern of H. pylori genotypes in elderly people; therefore, this is the first prospective study conducted in our country to investigate the genotype profiles (vaca and caga) of H. pylori. Until we can obtain a better understanding of the nature of H. pylori and its relation to the human host in asymptomatic individuals, indiscriminate eradication of this infection may likely do more harm than good at the community level, and may represent a waste of medical and economic resources. To ascertain the prevalence of type Ⅰ H. pylori strain infection in the elderly population in the Beijing area, we chose elderly persons in the general community in different parts of Beijing (urban, suburban and mountainous districts), to study the seroepidemiology of the type Ⅰ H. pylori strain and risk factors for its seropositivity. MATERIALS AND METHODS Research subjects The cluster sampling was based on a random sample of the elderly population of three Beijing districts [Xuanwu (urban), Daxing (suburban) and Huairou (mountainous)], stratified by living conditions, education and age in A total of 2006 elderly persons aged more than 60 years were randomly selected. Their average age was 70.9 years. Of these, 1005 (50.1%) were male and 1001 (49.9%) female, with 966 (48.2%), 522 (26.0%) and 518 (25.8%) from urban, suburban and mountainous areas, respectively. Methods The survey was comprised questionnaires that were provided to the residents. Contents of the survey included history of H. pylori infection, occupation and diet types, hygiene habits, economic status and history of gastrointestinal diseases. A blood sample (2 ml) was collected after the survey. The specimen was centrifuged to separate the serum, which was then stored at low temperature (-20 ) for later analysis. An immunodetection method was used to test for H. pylori IgG antibodies (H. pylori-blot kit 1.0 reagent; Shenzhen Blot Biotech, Shenzhen, China ). Study protocol An immunodetection method was used to test H. pylori IgG antibodies as reported previously [10]. H. pylori antigen was put into the reaction wells of a microtiter plate and inactivated sample serum was added. H. pylori antibody recognition of protein caga (116 kda), protein vaca (87 kda) and urine enzyme subunit (66 kda) was measured using a plate reader (Special Assay Kit for Helicobacter Pylori Tester, Shenzhen Blot Biotech, Shenzhen, China). Positive, negative and blank controls were included in each test. Result criteria If the samples were seropositive for caga and/or vaca (i.e., vaca + caga +, vaca + caga - or vaca - caga + ) it was considered as a type Ⅰ H. pylori strain infection. If antibodies only positive for urease and negative for caga and vaca (i.e., vaca - caga - ) were present, the sample was considered as type Ⅱ H. pylori strain infection. Negativity for all three antibodies was considered as no H. pylori infection. Statistical analysis The χ 2 test was used to analyze the data. P < 0.05 was considered statistically significant. All calculations were carried out with SPSS ver statistical software (SPSS, Chicago, IL, United States). RESULTS Prevalence of different strains of H. pylori infection and distribution characteristics of the H. pylori strains with respect to sex Of the 2006 elderly persons, 1673 (83.4%) were seropositive for H. pylori infection: 1124 (56%) were seropositive for the type Ⅰ H. pylori strain, and 549 (27.4%) were seropositive for type Ⅱ H. pylori strain infection. Of the 1673 patients seropositive for H. pylori infection, 67% were seropositive for the type Ⅰ H. pylori strain and 33% were seropositive for the type Ⅱ H. pylori strain. Overall, 851 (84.7%) of male persons were seropositive and 822 (82.1%) of females were seropositive for H. pylori infection. There was no significant difference in the prevalence of H. pylori strains between male and female participants (Table 1). Characteristics and distribution of H. pylori strains in elderly people of different ages H. pylori infection rates were not different among differ April 7, 2014 Volume 20 Issue 13

262 Zhang M et al. Epidemiology of H. pylori in elderly Chinese Table 1 Seropositivity for Helicobacter pylori strains affected by age, sex and area n (%) Table 3 Prevalence of Helicobacter pylori strains affected by previous occupation n (%) n H. pylori infection rate H. pylori strains Ⅰ Ⅱ n H. pylori infection rate H. pylori strains Ⅰ Ⅱ Sex Male (84.7) 578 (57.5) 273 (27.2) Female (82.1) 546 (54.5) 276 (27.6) Age < 75 yr (83.3) 792 (57.9) 348 (25.4) 75 yr (83.5) 332 (52.0) a 201 (31.5) b Area Mountainous (91.1) 258 (49.8) 214 (41.3) (huairou) Suburban (77.2) d 303 (58) d 100 (19.2) d (daxing) Urban (82.6) d 563 (58.3) d 235 (24.3) d (xuanwu) Total (83.4) 1124 (56) 549 (27.4) a P < 0.05, b P < 0.01 vs young-old persons. d P < 0.01 vs among urban, suburban and mountainous areas. H. pylori: Helicobacter pylori. Mental work (79.5) 351 (54.3) 163 (25.2) Manual work (84.9) b 519 (55.9) 269 (29.0) Total (82.7) 870 (55.2) 432 (27.4) b P < 0.01 vs mental workers in the total Helicobacter pylori infection rate. H. pylori: Helicobacter pylori. Table 4 Helicobacter pylori infection rate affected by diets n (%) n H. pylori infection rate H. pylori strains Ⅰ Ⅱ Vegetarian diet (88.4) 345 (58.2) 179 (30.2) High-protein diet (81.3) b 779 (55.1) b 370 (26.2) Total (83.4) 1124 (56) 549 (27.4) b P < 0.01 vs elderly persons on a vegetarian diet. H. pylori: Helicobacter Table 2 Seropositivity of Helicobacter pylori strains affected by age Age (yr) n H. pylori infection rate H. pylori cases % Total H. pylori: Helicobacter pylori. ent ages. However, young-old persons had a significantly higher seropositivity for the type Ⅰ H. pylori strain than older-old persons in the selected group (Tables 1 and 2). Distribution of H. pylori strains in different areas The overall H. pylori infection rate was significantly higher in the mountainous district than in the urban and suburban districts, while type Ⅰ H. pylori strain seropositivity was the reverse (Table 1). Prevalence of H. pylori strains in different occupations Of the 2006 elderly persons, 1575 persons reported a definite occupation, among whom 1302 persons were seropositive, with a corresponding H. pylori infection rate of 82.7%. There was a significant difference in the total H. pylori infection rate between subjects who had been mental and manual workers; however, no difference of type Ⅰ H. pylori strain infection rate was found between them (Table 3). pylori. Prevalence of H. pylori strains affected by different diet types According to the structured questionnaire, a diet containing protein (e.g., chicken, duck, fish, meat, egg) more than 3 d a week for at least 1-5 years was considered a highprotein diet; a diet containing vegetables everyday and meat occasionally for at least 1-5 years was considered a vegetarian diet. The prevalence of H. pylori infection and type Ⅰ H. pylori strain infection was higher in elderly persons on a vegetarian diet than those on a high-protein diet (Table 4). DISCUSSION Studies have shown that the virulence of CagA and VacA protein is immuno-independent, but they are strongly associated with each other in terms of pathogenicity. The molecule size of VacA is 87 kda [11].VacA is the major virulence factor of H. pylori. It is encoded by the vaca gene, and regulates the virulence of the vacuolating cell, inducing epithelial cell vacuolating proliferation. The molecule size of CagA is 128 kda, and it is encoded by the caga gene [12]. There are two main virulent strains of H. pylori [13] : the type Ⅰ strain H. pylori expressing both the CagA and VacA proteins, and the type Ⅱ strain expressing neither of them. The type Ⅰ strain of H. pylori shows higher pathogenicity than the type Ⅱ strain, which was closely correlated to gastric cancer and peptic ulceration [14-16]. There have been many epidemiological reports on H. pylori infection. Some reports indicate that the Beijing area adult infection rate was approximately 40%-50% [17] ; however, there have been fewer investigations concerning the elderly generation. As the elderly population is increasing globally, we should pay more attention to geriatric infection rates. Therefore, we conducted a seroepidemiological study of type Ⅰ H. pylori strain infection in elderly persons in different parts of the Beijing area. The results showed that 83.4% of the elderly people were seropositive for H. pylori infection (56% for type Ⅰ H. pylori strain and 27.4% for the type 3637 April 7, 2014 Volume 20 Issue 13

263 Zhang M et al. Epidemiology of H. pylori in elderly Chinese Ⅱ strain). In H. pylori seropositive persons, type Ⅰ H. pylori strain infection had higher prevalence (67%) than the type Ⅱ strain. There was no significant difference in the prevalence of H. pylori infection and no difference in the distribution of H. pylori strains between men and women, which was consistent with reports from other countries [18]. The type Ⅰ H. pylori strain infection rate was 60%-80% in Western countries [19,20], and more than 90% in Asian countries [21], indicating that the infection rate differed between populations and areas [22]. Our study aimed to investigate the elderly group in different parts of the Beijing area [Xuanwu (urban), Daxing (suburban) and Huairou (mountainous)]. The results showed that the H. pylori infection rate of the mountainous district was the highest (91.1%) and that the type Ⅰ H. pylori infection rate was 49.8% overall, representing 54.6% of the infected persons. The prevalence of H. pylori infection was relatively lower in the suburban area (77.2%) and its type Ⅰ strain H. pylori infection rate was 58% overall, representing 75.1% among H. pylori-infected persons. These results indicated that the type Ⅰ H. pylori strain infection rate was significantly higher in the suburban area compared with the mountainous area. This suggested that in different parts of a country or in different geographical areas of one province, the H. pylori infection rate may be different. Although the suburban population had a lower H. pylori infection rate than the mountainous area, its major infected strain of H. pylori was type Ⅰ. Whether this phenomenon was related to host factors, environmental factors or others, or if this correlated with some high occurrence of diseases, requires further epidemiological studies. On the other hand, in the study group, persons in the young-old group had a higher type Ⅰ H. pylori strain infection rate. H. pylori infection also varied for different occupations among the elderly population. Those who performed manual or technical labor had a higher infection rate than those with other non-manual occupations; however, there was no significant difference when comparing the sub-strains between these two groups. The type Ⅰ H. pylori infection rate was significantly higher in those with a mainly vegetarian diet than those whose diet was higher in protein. Studies have shown an association of H. pylori seropositivity with a low frequency of eating meat [23]. Other studies have shown that food antioxidant factors inhibit H. pylori infection [24-26]. Some scholars have reported that eating soy foods may reduce male H. pylori risk of infection (especially beans containing the antioxidant factors, isoflavones). This requires further study. There have been published reports that H. pylori infection was positively correlated with age, according to the statistics displayed in age groups. In the elderly population, the total infection rate of H. pylori was not significantly difference in the elderly of different ages, while the H. pylori subtype (caga, vaca) infection rate in the younger elderly and elderly was different. The World Health Organization and the Chinese Committee on Aging define the younger elderly as persons under the age of 75 years, and oldestold as persons aged 75 year and older. The type Ⅰ H. pylori infection rate was also significantly lower among the oldest portion of the elderly population compared to the youngest. The decline may be related to social activity, with social factors reducing the influence of H. pylori infection. CONCLUSION The prevalence of H. pylori infection was higher in the elderly population in the Beijing area, and the type Ⅰ H. pylori strain was the major infection sub-strain. The type Ⅰ H. pylori strain infection rate was different by area, age and diet. Whether we can decrease the type Ⅰ H. pylori strain infection rate by changing some of the factors mentioned, so as to decrease the incidence of the correlated digestive diseases, still requires further research. COMMENTS Background With the ageing of society, more elderly people will suffer from digestive diseases combined with Helicobacter pylori (H. pylori). In Beijing, there are no data regarding the pattern of H. pylori genotypes in elderly people. Research frontiers Recent studies have shown that H. pylori virulence factors, the cytotoxin-associated gene product A (caga) and vacuolating cytotoxin A (vaca), in addition to host and environmental factors, may be very important. Innovations and breakthroughs This study investigated the seroepidemiology of the cytotoxin-associated gene product caga + and vacuolating cytotoxin vaca + strains of H. pylori in elderly people in Beijing, and assessed risk factors for seropositivity. This is the first prospective study conducted in China to investigate the genotype profiles of H. pylori. Peer review In this epidemiologic study entitled Seroepidemiology of Helicobacter pylori infection in elderly people in the Beijing region, China, the authors considered a large elderly population and reported interesting associations between H. pylori seropositivity and subjects habits. REFERENCES 1 Dixon MF. Helicobacter pylori and peptic ulceration: histopathological aspects. J Gastroenterol Hepatol 1991; 6: [PMID: DOI: /j tb01451.x] 2 Tytgat GN, Noach LA, Rauws EA. Helicobacter pylori infection and duodenal ulcer disease. Gastroenterol Clin North Am 1993; 22: [PMID: ] 3 Parsonnet J, Friedman GD, Vandersteen DP, Chang Y, Vogelman JH, Orentreich N, Sibley RK. Helicobacter pylori infection and the risk of gastric carcinoma. N Engl J Med 1991; 325: [PMID: DOI: / NEJM ] 4 Wotherspoon AC, Ortiz-Hidalgo C, Falzon MR, Isaacson PG. 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264 Zhang M et al. Epidemiology of H. pylori in elderly Chinese 8 Liu YE, Gong YH, Sun LP, Xu Q, Yuan Y. The relationship between H. pylori virulence genotypes and gastric diseases. Pol J Microbiol 2012; 61: [PMID: ] 9 Shimoyama T, Crabtree JE. Bacterial factors and immune pathogenesis in Helicobacter pylori infection. Gut 1998; 43 Suppl 1: S2-S5 [PMID: ] 10 Laheij RJ, Straatman H, Jansen JB, Verbeek AL. Evaluation of commercially available Helicobacter pylori serology kits: a review. J Clin Microbiol 1998; 36: [PMID: ] 11 Cover TL, Blaser MJ. Purification and characterization of the vacuolating toxin from Helicobacter pylori. J Biol Chem 1992; 267: [PMID: ] 12 Covacci A, Censini S, Bugnoli M, Petracca R, Burroni D, Macchia G, Massone A, Papini E, Xiang Z, Figura N. Molecular characterization of the 128-kDa immunodominant antigen of Helicobacter pylori associated with cytotoxicity and duodenal ulcer. Proc Natl Acad Sci USA 1993; 90: [PMID: DOI: /pnas ] 13 Xiang Z, Censini S, Bayeli PF, Telford JL, Figura N, Rappuoli R, Covacci A. Analysis of expression of CagA and VacA virulence factors in 43 strains of Helicobacter pylori reveals that clinical isolates can be divided into two major types and that CagA is not necessary for expression of the vacuolating cytotoxin. Infect Immun 1995; 63: [PMID: ] 14 Antonio-Rincón F, López-Vidal Y, Castillo-Rojas G, Lazcano-Ponce EC, Ponce-de-León S, Tabche-Barrera ML, Aguilar- Gutiérrez GR. Pathogenicity island cag, vaca and IS605 genotypes in Mexican strains of Helicobacter pylori associated with peptic ulcers. Ann Clin Microbiol Antimicrob 2011; 10: 18 [PMID: DOI: / ] 15 Wu AH, Crabtree JE, Bernstein L, Hawtin P, Cockburn M, Tseng CC, Forman D. Role of Helicobacter pylori CagA+ strains and risk of adenocarcinoma of the stomach and esophagus. Int J Cancer 2003; 103: [PMID: ] 16 Nakamura S, Matsumoto T. Helicobacter pylori and gastric mucosa-associated lymphoid tissue lymphoma: recent progress in pathogenesis and management. World J Gastroenterol 2013; 19: [PMID: DOI: /wjg.v19. i ] 17 Zhang DH, Zhou LY, Lin SR, Ding SG, Huang YH, Gu F, Zhang L, Li Y, Cui RL, Meng LM, Yan XE, Zhang J. Epidemiology of Helicobacter pylori infection in Shandong and Beijing areas. Zhonghua Nei Ke Za Zhi 2009; 48: [PMID: ] 18 Graham DY, Malaty HM, Evans DG, Evans DJ, Klein PD, Adam E. Epidemiology of Helicobacter pylori in an asymptomatic population in the United States. Effect of age, race, and socioeconomic status. Gastroenterology 1991; 100: [PMID: ] 19 Crabtree JE, Taylor JD, Wyatt JI, Heatley RV, Shallcross TM, Tompkins DS, Rathbone BJ. Mucosal IgA recognition of Helicobacter pylori 120 kda protein, peptic ulceration, and gastric pathology. Lancet 1991; 338: [PMID: DOI: / (91) ] 20 Audibert C, Burucoa C, Janvier B, Fauchère JL. Implication of the structure of the Helicobacter pylori cag pathogenicity island in induction of interleukin-8 secretion. Infect Immun 2001; 69: [PMID: DOI: / IAI ] 21 Maeda S, Ogura K, Yoshida H, Kanai F, Ikenoue T, Kato N, Shiratori Y, Omata M. Major virulence factors, VacA and CagA, are commonly positive in Helicobacter pylori isolates in Japan. Gut 1998; 42: [PMID: DOI: /gut ] 22 Zhang WD, Hu FL, Xiao SD, Xu ZM. The Team of Collaboration of Helicobacter pylori research in China. Prevalence of Helicobacter pylori infection in China. Xiandai Xiaohua ji Jieru Zhenliao 2010; 15: Alvarado-Esquivel C. Seroepidemiology of Helicobacter pylori infection in a Mennonite community in Durango State, Mexico. Helicobacter 2013; 18: [PMID: DOI: /hel.12032] 24 Guo Z, Li Y, Xu Z, Ji F, Wang L, Chen K. A case-control study on risk factors of helicobacter pylori infection in outpatients with stomach diseases. Zhonghua Yu Fang Yi Xue Za Zhi 2002; 36: [PMID: ] 25 Zhang YW, Eom SY, Yim DH, Song YJ, Yun HY, Park JS, Youn SJ, Kim BS, Kim YD, Kim H. Evaluation of the relationship between dietary factors, CagA-positive Helicobacter pylori infection, and RUNX3 promoter hypermethylation in gastric cancer tissue. World J Gastroenterol 2013; 19: [PMID: DOI: /wjg.v19.i ] 26 Wang XQ, Yan H, Terry PD, Wang JS, Cheng L, Wu WA, Hu SK. Interaction between dietary factors and Helicobacter pylori infection in noncardia gastric cancer: a population-based case-control study in China. J Am Coll Nutr 2012; 31: [PMID: DOI: / ] P- Reviewer: Tsikas D S- Editor: Song XX L- Editor: Stewart G E- Editor: Wang CH 3639 April 7, 2014 Volume 20 Issue 13

265 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH REPORT Prognostic value of number of examined lymph nodes in patients with node-negative gastric cancer Xu-Guang Jiao, Jing-Yu Deng, Ru-Peng Zhang, Liang-Liang Wu, Li Wang, Hong-Gen Liu, Xi-Shan Hao, Han Liang Xu-Guang Jiao, Jing-Yu Deng, Ru-Peng Zhang, Liang-Liang Wu, Li Wang, Hong-Gen Liu, Xi-Shan Hao, Han Liang, Key Laboratory of Cancer Prevention and Therapy, Department of Gastrointestinal Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Tianjin , China Author contributions: Jiao XY, Deng JY, Liang H performed the majority of the study; Jiao XG, Deng JY, Zhang RP, Wu LL, Wang L and Liu HG designed the study and analyzed data; Jiao XG, Deng JY wrote the manuscript; Hao XS and Liang H revised the manuscript. Supported by A grant from the National Basic Research Program of China (973 Program), No. 2010CB Correspondence to: Han Liang, MD, Key Laboratory of Cancer Prevention and Therapy, Department of Gastrointestinal Oncology, Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center for Cancer, Huanhuxi Road, Hexi District, Tianjin , China. tjlianghan022@sohu.com Telephone: Fax: Received: August 7, 2013 Revised: November 9, 2013 Accepted: December 12, 2013 Published online: April 7, 2014 Abstract AIM: To elucidate the potential impact of examined lymph nodes (elns) on long-term survival of nodenegative gastric cancer patients after curative surgery. METHODS: A total of 497 node-negative gastric cancer patients who underwent curative gastrectomy between January 2000 and December 2008 in our center were enrolled in this study. Patients were divided into 4 groups according to elns through cut-point analysis. Clinicopathological features were compared between 15 elns group and > 15 elns group and potential prognostic factors were analyzed. The Log-rank test was used to assess statistical differences between the groups. Independent prognostic factors were identified using the Cox proportional hazards regression model. Stratified analysis was performed to investigate the impact of elns on patient survival in each stage. Overall survival was also compared among the four groups. Finally, we explored the recurrent sites associated with elns. RESULTS: Patients with elns > 15 had a better survival compared with those with elns 15 for the entire cohort. By the multivariate survival analysis, we found that the depth of invasion and the number of elns were the independent predictors of overall survival (OS) of patients with node-negative gastric cancer. According to the cut-point analysis, T2-T4 patients with elns had a significantly longer mean OS than those with 4-10 elns or 1-3 elns. Patients with 15 elns were more likely to experience locoregional and peritoneal recurrence than those with > 15eLNs. CONCLUSION: Number of elns could predict the prognosis of node-negative gastric cancer, and dissection of > 15 elns is recommended during lymphadenectomy so as to improve the long-term survival Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Gastric carcinoma; Examined lymph nodes; Node-negative; Prognosis Core tip: The number of metastatic lymph nodes has been shown to be associated with prognosis in gastric cancer patients. In this study, we found that the overall survival of node-negative gastric cancer patients was significantly affected by examined lymph nodes (elns), and number of elns was an independent prognostic factor in multivariate analysis. Dissection of > 15 elns could reduce locoregional and peritoneal recurrence. We suggest that over 15 lymph nodes should be examined to improve the long-term outcome of node April 7, 2014 Volume 20 Issue 13

266 Jiao XG et al. Lymph node count for gastric cancer prognosis negative gastric cancer patients following curative gastrectomy. Jiao XG, Deng JY, Zhang RP, Wu LL, Wang L, Liu HG, Hao XS, Liang H. Prognostic value of number of examined lymph nodes in patients with node-negative gastric cancer. World J Gastroenterol 2014; 20(13): Available from: URL: DOI: INTRODUCTION Lymph node metastasis is one of the most important prognostic factors for gastric cancer patients following curative resection [1,2]. It is generally accepted that patients could benefit from a standardized approach of lymphadenectomy in terms of a higher overall survival (OS) [3-5]. At present, the category based on the absolute number of metastatic lymph nodes has been adopted by the Union for International Cancer Control (UICC) and American Joint Commission for Cancer as the N stage of the tumour, node, metastasis (TNM) classification since 1997 and the examination of no less than 15 regional lymph nodes was recommended for nodal metastatic status determination according to the latest version [6-8]. The 7 th edition of the TNM classification designates node-negative (N0) disease as any gastric cancer with all examined lymph nodes (elns) negative, regardless of the total number of elns. Several studies have found that the prognosis of node-negative gastric cancer patients was associated with the number of dissected lymph nodes [9,10]. However, there is still controversy over how many nodes should be removed and examined when performing a radical gastrectomy for node-negative gastric cancer patients [9-11]. Therefore, the aim of the present study was to determine the optimal number of elns required for curative surgery. Furthermore, we intend to explore an appropriate classification based on the count of elns for precise prediction of the prognosis of gastric cancer patients with node-negative metastasis after curative surgery. MATERIALS AND METHODS Patients A total of 2824 gastric cancer patients who underwent curative resection at the Gastric Cancer Surgery Department of Tianjin Medical University Cancer Hospital from January 2000 through December 2008 were selected for this study. The inclusion criteria for this study included: (1) patients with histologically proven primary adenocarcinoma of the stomach; (2) patients who underwent a lymphadenectomy (limited or extended); and (3) patients with all dissected lymph nodes proven to be negative by pathological examination. The exclusion criteria were: (1) patients who underwent palliative surgery; (2) patients with gastroesophageal junction tumor or cardia tumor; (3) patients who had distant metastasis or peritoneal dissemination that was confirmed during the operation; (4) patients who received neoadjuvant chemotherapy or radiotherapy before surgery; (5) patients with remnant gastric carcinoma; (6) patients who died during the initial hospital stay or within 1 mo after surgery; and (7) patients who were lost to follow-up. Based on these inclusion and exclusion criteria, a total of 497 gastric cancer patients were enrolled in this study. Surgical treatment and pathological examination All patients were operated on according to the potentially curative gastrectomy and lymphadenectomy. Curative resection was defined as a complete lack of grossly visible tumor tissue and metastatic lymph nodes remaining after resection, with pathologically negative resection margins [12]. Primary tumors were resected en bloc with limited or extended lymphadenectomy according to the Japanese Gastric Cancer Association [13]. The choice of surgical procedure of gastrectomy was made by the attending surgeon, s preference, and based mainly on the gastric cancer treatment guidelines in Japan [14]. Lymph nodes were meticulously dissected from the en bloc specimens by a specialist surgeon. Dissected lymph nodes were fixed in 10% formalin, embedded in paraffin, and stained with hematorylin-eosin. Each lymph node was re-examined microscopically and the status was determined by experienced pathologists. Adjuvant therapy Most of the patients received the adjuvant chemotherapy based on fluorouracil and leucovorin calcium after curative gastrectomy. Radiotherapy was not routinely administrated. Statistical analysis To determine the appropriate cutoffs for the number of examined negative lymph nodes, the cut-point survival analysis was adopted. The χ 2 or Fisher s exact test and t test were used to test differences in distributions between different elns subgroups. OS was calculated from the date of surgery to death from any causes. Survival curves were estimated using the Kaplan-Meier method and compared by the Log-rank test. Factors deemed to be of potential importance on univariate analyses (P < 0.05) were included in the multivariate analyses. Multivariate analysis of OS was performed by means of the Cox proportional hazards model, using the forward stepwise logistic regression procedure for variable selection. Hazard ratios and 95%CIs were generated. In all statistical analyses, significance was defined as P < 0.05 (two-sided). All analyses were performed using the statistical analysis program package SPSS version 17.0 (SPSS, Chicago, IL, United States). Follow-up After curative surgery, all patients were followed every 6 mo for 2 years, then every year or until death. The 3641 April 7, 2014 Volume 20 Issue 13

267 Jiao XG et al. Lymph node count for gastric cancer prognosis 8.0% 6.0% Percentage 4.0% 2.0% 0.0% Number of examined lymph nodes Figure 1 Percentage of the number of examined lymph nodes for the entire cohort. follow-up of all patients who were included in this study was completed in October The routine examination during follow-up included a physical examination, blood chemistry, B-mode ultrasonography, computed tonography scans, chest X-ray, and endoscopy. RESULTS Clinicopathological outcomes The mean ± SD number of pathologically proved elns for the entire cohort of 497 patients was 13.8 ± 10.5 (ranging from 1 to 67). The 5-year survival rate (5-YSR) of all enrolled patients was 67.2%, and 332 patients were alive when the follow-up was completed. The median OS of all patients after surgery was 58.3 mo. The percentage of the number of elns for the entire cohort is shown in Figure 1. Cut-point survival analysis for detection of best cutoffs of negative node counts A cut-point survival analysis was performed to determine the negative lymph node counts that determine the greatest actuarial survival difference among the resulting subgroups. We selected the ability to detect differences among the subgroups on the basis of the magnitude of the log rank test χ 2 statistic. Results for all relevant cutpoints and stage subgroups are listed in Table 1. The cut-point analysis yielded the greatest survival difference at the level of 15 in pt1-2, pt3-4, and entire cohort. Patients with 15 elns were divided into 3 subgroups: 1-3 elns, 4-10 elns, and elns according to the result of cut-point analysis. Comparison of clinicopathologic characteristics in patients of different elns groups is shown in Table 2. There were significant differences between the two groups of patients in the distribution of gender, age at surgery, tumor location, type of gastrectomy, histological type, and the extent of elns. Univariate survival analysis and multivariate survival analysis By univariate survival analysis, we found that five clinicopathological variables were significantly associated with the OS of node-negative gastric cancer patients after curative resection. They are as follows: extent of elns, extent of lymphadenectomy, depth of invasion, number of elns, and the 7 th UICC TNM classification of gastric caner (Table 3). All of the five variables considered significant on univariate analysis were included in the Cox proportional hazards model (forward stepwise procedure) to calculate the independent prognostic factors. As a result, there were two independent, statistically significant prognostic parameters: depth of invasion (P < 0.001) and number of elns (P < 0.001). The hazard ratios and their 95%CIs are listed in Table April 7, 2014 Volume 20 Issue 13

268 Jiao XG et al. Lymph node count for gastric cancer prognosis Table 1 Overall survival by total examined lymph nodes and cut-point analysis in each subgroup Depth of invasion 3 vs 4 10 vs vs vs 26 χ 2 P value χ 2 P value χ 2 P value χ 2 P value T1 + T T3 + T < Total < < Table 2 Comparison of clinicopathologic characteristics between patients with 15 and >15 examined lymph nodes n (%) Variables Number of examined nodes χ 2 P value 15 > 15 Total 331 (66.6) 166 (33.4) Gender Male 259 (78.2) 106 (63.9) Female 72 (21.8) 60 (36.1) Age at surgery (yr) (46.8) 96 (57.8) > (53.2) 70 (42.2) Tumor size (cm) (49.2) 89 (53.6) > (50.8) 77 (46.4) Tumor location Lower 158 (47.7) 98 (59.0) Middle 31 (9.4) 16 (9.6) Upper 111 (33.5) 35 (21.1) Diffuse 31 (9.4) 17 (7.7) Type of gastrectomy Subtotal 284 (85.8) 130 (78.3) Total 47 (14.2) 36 (21.7) Histological type Undifferentiated 177 (53.5) 113 (68.1) Differentiated 154 (46.5) 53 (31.9) Extent of lymphadenectomy < Limited 242 (73.6) 39 (23.6) Extended 87 (26.4) 126 (76.4) Extent of examined lymph nodes < Perigastric 189 (57.1) 23 (13.9) Extragastric 142 (42.9) 143 (86.1) Depth of invasion T1 21 (6.3) 13 (7.8) T2 59 (17.8) 35 (21.1) T3 47 (14.2) 24 (14.5) T4 204 (61.6) 94 (56.7) TNM Classification 7 th Ⅰa 21 (6.3) 13 (7.8) Ⅰb 59 (17.8) 35 (21.1) Ⅱa 47 (14.2) 24 (14.5) Ⅱb 196 (59.2) 89 (53.6) Ⅲb 8 (2.4) 5 (3.0) Survival analysis and subgroup analysis As shown in Figure 2A, the 5-YSR of patients with > 15 elns was significantly higher than those with 15 elns (84.3% vs 58.6%, P < 0.001). As shown in Table 4 and Figure 2B, the 5-YSR was 43.3% for patients with 1-3 elns compared with 56.6%, 71.8%, and 84.3% for those with 4-10 elns, elns, and > 15 elns, respectively. Subgroup analysis was then conducted to evaluate the survival of the patients in different pt categories (Table 5). For patients with stage T2 disease, the 5-YSR differences were statistically significant among 1-3, 4-10, 11-15, and > 15 elns groups, and patients with > 15 elns had the best OS, so were patients with stage T3 or T4 disease (Figure 2C-E). The OS in patients with T1 tumors showed no statistical difference (Table 5). Recurrent sites of node-negative gastric cancer Among all the 497 patients, 85 (17.1%) had tumor recurrences during follow-up. The recurrence patterns observed in the 85 patients were locoregional recurrence (n = 46), hematogenous spread (n = 31), and peritoneal seeding (n = 28). A single recurrence pattern was noted in 65 patients: 29 had locoregional recurrence, 19 had hematogenous spread, and 17 had peritoneal seeding. More than 1 recurrence pattern was observed in 20 patients. Table 5 shows the distribution of recurrent sites in terms of 15 elns and > 15 elns. Patients with 15 elns had a high rate of total recurrence (19.6% vs 12.0%, P = 0.043), locoregional recurrence (11.2% vs 5.4%, P = 0.048) and peritoneal seeding (7.3% vs 2.4%, P = 0.037). There was no statistical difference in hematogenous spread rate between the two groups (7.3% vs 4.2%, P = 0.239). DISCUSSION Although several new staging methods have been proposed to evaluate the prognosis of gastric cancer, the TNM staging system based on the count of positive lymph nodes is still widely used, for the number of positive lymph nodes reflects well the prognosis [15-17]. It was reported that gastric cancer patients might be staged incorrectly because of an insufficient number of elns [18]. Large population studies from various institutions and countries demonstrated that there was a significant association between the number of elns and the OS of patients, so the latest TNM staging system recommends that no less than 15 lymph nodes should be examined for the accuracy of N staging [6-8]. Meanwhile, the same edition staging system designates node-negative disease as any gastric cancer with all elns negative, regardless of the total number of elns, thus the present study was designed to determine the optimal number of lymph nodes required for curative surgery for node-negative gastric cancer. Adachi et al [19] pointed out that the clinicopathologic characteristics and prognosis of node-negative gastric cancer patients were similar to those of patients with early gastric cancer. Several studies showed that in patients with node-negative gastric cancer, the 5- and 10-year OS rates varied from 72% to 92% and 88% to 3643 April 7, 2014 Volume 20 Issue 13

269 Jiao XG et al. Lymph node count for gastric cancer prognosis Table 3 Univariate and multivariate survival analyses of 497 node-negative gastric cancer patients Variables 5-YSR Univariate analysis Multivariate analysis χ 2 P value HR (95%CI) P value Gender Male 66.0 Female 70.4 Age at surgery (yr) > Tumor size (cm) > Tumor location Upper 69.9 Middle 53.2 Lower 69.5 Diffuse 60.4 Type of gastrectomy Subtotal 68.3 Total 61.4 Histological type Undifferentiated 67.9 Differentiated 66.2 Extent of lymphadenectomy Limited 63 Extended 73.2 Extent of examined lymph nodes (LNs) Perigastric 58.9 Extragastric 73.3 Depth of invasion < < T T ( ) T ( ) T ( ) Number of examined LNs < ( ) < > ( ) < TNM Classification 7 th < Ⅰa 88.2 Ⅰb 79.8 Ⅱa 74.6 Ⅱb 59.3 Ⅲb YSR: 5-year survival rate; TNM: Tumour, node, metastasis. Table 4 Five-year overall survival by T stage subgroups and total number of examined lymph nodes Variables Number of examined nodes (n ) χ 2 P value Depth of invasion T1 60.0% 91.7% % T2 58.3% 68.2% 84.0% 91.4% T3 50.0% 63.3% 81.8% 91.7% T4 35.1% 48.6% 62.5% 78.7% < Total 43.3% 56.6% 71.8% 84.3% < %, respectively, and these patients had a significantly better survival than the patients with node-positive gastric cancer [20-24]. In this study, the 5-YSR was 67.2%, which was lower than that observed in studies in Japan and Korea due largely to the lower proportion of patients with early stage disease (6.8%) in our study. Our previous study found that the node-positive gastric cancer patients had a better OS with an increased count of negative lymph nodes retrieved [17]. This phenomenon was also observed in the studies about node-negative gastric cancer. There are several possible explanations for this observation. First, there may have been lymph node metastases that were missed during dissection or pathological examination in node-negative patients who had 15 elns, which may cause inappropriate understaging. More elns should increase the opportunity to 3644 April 7, 2014 Volume 20 Issue 13

270 Jiao XG et al. Lymph node count for gastric cancer prognosis A Cumulatively overall survival probability 1.0 > 15 elns elns 0.4 (P < 0.001) B Cumulatively overall survival probability > 15 elns elns elns 1-3 elns Time after surgery (mo) Time after surgery (mo) C Cumulatively overall survival probability 1.0 > 15 elns elns elns elns D Cumulatively overall survival probability 1.0 > 15 elns elns 4-10 elns elns Time after surgery (mo) Time after surgery (mo) E Cumulatively overall survival probability > 15 elns elns 4-10 elns elns Time after surgery (mo) Figure 2 Survival curves. A: For 15 elns group and > 15 elns group in total node-negative gastric cancer patients; B: For 4 categories in total node-negative gastric cancer patients according to the number of examined negative lymph nodes (P < 0.001); C: For 4 categories in pathological T2 patients according to the number of examined negative lymph nodes (P = 0.022); D: For 4 categories in pathological T3 patients according to the number of examined negative lymph nodes (P = 0.033); E: For 4 categories in pathological T4 patients according to the number of examined negative lymph nodes (P < 0.001). elns: Examined lymph nodes. find metastatic lymph nodes, which could reduce the influence of inappropriate understaging [25-27]. Second, micrometastasis was considered to play an important role in prognosis of gastric cancer patients. Patients with micrometastasis were reported to have an especially high risk of recurrence [10]. It was reported that 17%-32% of node-negative gastric cancer patients were identified by routine histologic examination, and micrometastasis was detected by immunohistochemical and molecular examinations. Therefore, dissection of adequate negative lymph nodes could improve the prognosis of node-negative gastric cancer patients by reducing micrometastasis. Third, several studies have confirmed that lymphovascular invasion is associated with poor survival in gastric cancer patients [28,29]. More elns means dissection of potential lymphovascular invasion, which may 3645 April 7, 2014 Volume 20 Issue 13

271 Jiao XG et al. Lymph node count for gastric cancer prognosis Table 5 Recurrent sites of node-negative gastric cancer in terms of 15 and > 15 examined lymph nodes n (%) Recurrent sites Number of examined nodes χ 2 P value 15 > 15 Recurrence Yes 65 (19.6) 20 (12.0) No 266 (80.4) 146 (88.0) Locoregional recurrence Yes 37 (11.2) 9 (5.4) No 294 (88.8) 157 (94.6) Peritoneal seeding Yes 24 (7.3) 4 (2.4) No 307 (92.7) 162 (97.6) Hematogenous spread Yes 24 (7.3) 7 (4.2) No 307 (92.7) 159 (95.8) improve prognosis by reducing recurrence. In addition, it is speculated that patients with more lymph nodes in the specimen have better immune response against the tumor, and smaller LNs were correlated with decreased immune response in gastrointestinal cancer [32]. Strong immune status also contributes to good prognosis. Son et al [33] investigated patients with gastric cancer, and the results showed that the majority of patients with 15 elns had T1 tumors (64.2%) or pn0 disease (81.3%). Consequently, the patients with no more than 15 elns had a significantly worse prognosis after they underwent standard curative lymphadenectomy, compared with patients with > 15 elns. Our data revealed that there were significant survival differences between the patients who had > 15 elns and 15 elns by cut-point analysis. By the multivariate analyses, we found that the number of elns is an independent prognostic factor that was associated with a worse prognosis. In this study, 66.6% of patients had 15 elns, which contributed to the lower OS of the entire cohort. The main reasons for examination of 15 elns after curative gastrectomy were deemed to be the inaccurate lymph node dissection or examination [34-36]. These inaccuracies most likely occur by inexperienced surgeons or pathologists [37]. In addition, the inaccurate lymph node dissection was considered to be closely related to host clinicopathologic status and immunologic response [38,39]. Besides, patient, s comorbidities, the intrinsic number of LNs, and lymphovascular invasion status may play a role in inadequate LN dissection and assessment. Therefore, it is important to evaluate the prognostic value for patients with 15 elns. In this study, we performed cutpoint analysis and determined 3 cut-off points to classify all patients into 4 subgroups as 1-3, 4-10, 11-15, and 16 elns, which can not only reflect the distribution of elns, but also demonstrate the correlation between the prognosis of patients and the number of elns intuitively. Gastrectomy with lymphadenectomy remains the standard treatment option for gastric cancer, while it is often performed without an accurate knowledge of nodal status. Sentinel lymph node (SLN) is defined as the first LN to receive drainage by a primary tumor. Therefore, the status of SLN could predict the overall status of LN metastasis. As extensive lymphadenectomies are not without complications, limited gastric resections without an extended lymphadenectomy have been applied to the selected patients with early gastric cancer according to the status of SLN [40]. For early N0 gastric cancer, SLN should be used in selected patients for less invasive surgical approaches, while it is not easy to predict accurately the status of LN metastasis through SLN because lymphatic drainage is complex for advanced N0 disease, so the extended lymphadenectomy is still essential for advanced gastric cancer. In addition to the number of elns, the depth of tumor invasion was identified as one of the most important prognostic indicators of node-negative gastric cancer in the present study, which was similar to other studies [9,20-23,33]. The number of elns was reported to be associated with depth of invasion. Compared with the patients who had less than 6 elns, patients who had 6-10 elns, elns, and 16 elns had a significantly lower chance of death in T2-4 disease, while the gastric cancer specific survival among different elns groups had no statistical difference in T1 stage, because patients with stage T1 disease have a small number of cases and a higher 5-YSR [9]. Based on our results, patients with > 15 elns had better prognosis than those with 15 elns in T2-T4 stages. Patients with 15 elns should be divided into 3 groups as 1-3 elns, 4-10 elns, and elns for acquisition of significant statistical differences among the subgroups of patients in T2-T4 stages. Recently, Huang et al [22] suggested that the D2 lymphadenectomy should involve 15 nodes for pt1-t2 disease and 20 nodes for pt3-t4 disease. However, it is difficult to determine accurately the T stage before surgery. From our results, we suggested that patients with node-negative gastric cancer following curative dissection should have more than 15 elns. It should be considered as a necessary supplement to the TNM staging system. COMMENTS Background Count of metastatic lymph nodes has been shown to be associated with prognosis of gastric cancer. Several studies have found that the prognosis of nodenegative gastric cancer was associated with the number of dissected lymph node. However, there is still controversy over how many nodes should be removed and examined when performing a radical gastrectomy for node-negative gastric cancer patients. Research frontiers Radical gastrectomy with regional lymph node dissection is the only possible curative treatment for gastric cancer. A significant portion of patients with gastric cancer have been staged incorrectly because of an insufficient number of examined lymph nodes (elns). Research has demonstrated an association between an adequate number of elns and improved overall survival. Lymph node stage can be determined appropriately when the number of total elns is > 15 according to the latest tumour, node, metastasis (TNM) staging system, while it is still controversial over how many nodes should be removed and examined when performing a radical gastrectomy for node-negative gastric cancer patients. In this study, the authors demonstrated that number of elns was an independent prognostic factor for node-negative gastric cancer after curative 3646 April 7, 2014 Volume 20 Issue 13

272 Jiao XG et al. Lymph node count for gastric cancer prognosis resection and more than 15 elns should be obtained. Innovations and breakthroughs This study reported that patients with node-negative gastric cancer following curative dissection should have more than 15 examined lymph nodes in a large sample analysis. It should be considered as a necessary supplement to the TNM staging system. Patients with 15 examined lymph nodes should be divided into 3 groups as 1-3 elns, 4-10 elns, and elns for acquisition of significant statistical differences among the subgroups of patients in T2-T4 stages. Applications By understanding the positive association between the number of elns and prognosis of gastric cancer, this study may stimulate surgeons and pathologists to acquire more than 15 elns during curative gastrectomy. Terminology elns are the number of dissected lymph nodes during surgery, which is visually estimated by surgeons and pathologists immediately after surgery. Sentinel lymph node is defined as the first lymph node to receive drainage by a primary tumor. Peer review The most appropriate number of elns required during a radical gastrectomy for node-negative gastric cancer patients is still controversial. This study shows that patients with node-negative gastric cancer following curative dissection should have more than 15 examined lymph nodes in a large sample analysis. This conclusion has some significance for guiding clinical work. REFERENCES 1 Sasako M, Saka M, Fukagawa T, Katai H, Sano T. Surgical treatment of advanced gastric cancer: Japanese perspective. Dig Surg 2007; 24: [PMID: ] 2 Hartgrink HH, Jansen EP, van Grieken NC, van de Velde CJ. Gastric cancer. Lancet 2009; 374: [PMID: DOI: /S (09) ] 3 Kim JP, Lee JH, Kim SJ, Yu HJ, Yang HK. Clinicopathologic characteristics and prognostic factors in patients with gastric cancer. 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273 Jiao XG et al. Lymph node count for gastric cancer prognosis 26 Baxter NN, Tuttle TM. Inadequacy of lymph node staging in gastric cancer patients: a population-based study. Ann Surg Oncol 2005; 12: [PMID: DOI: / ASO ] 27 Smith DD, Schwarz RR, Schwarz RE. Impact of total lymph node count on staging and survival after gastrectomy for gastric cancer: data from a large US-population database. J Clin Oncol 2005; 23: [PMID: DOI: /JCO ] 28 Kim JH, Park JM, Jung CW, Park SS, Kim SJ, Mok YJ, Kim CS, Chae YS, Bae JW. The significances of lymph node micrometastasis and its correlation with E-cadherin expression in pt1-t3n0 gastric adenocarcinoma. J Surg Oncol 2008; 97: [PMID: DOI: /jso.20937] 29 Yasuda K, Adachi Y, Shiraishi N, Inomata M, Takeuchi H, Kitano S. Prognostic effect of lymph node micrometastasis in patients with histologically node-negative gastric cancer. Ann Surg Oncol 2002; 9: [PMID: ] 30 Kunisaki C, Makino H, Kimura J, Takagawa R, Kosaka T, Ono HA, Akiyama H, Fukushima T, Nagahori Y, Takahashi M. Impact of lymphovascular invasion in patients with stage I gastric cancer. Surgery 2010; 147: [PMID: DOI: /j.surg ] 31 Kim J, Huynh R, Abraham I, Kim E, Kumar RR. Number of lymph nodes examined and its impact on colorectal cancer staging. Am Surg 2006; 72: [PMID: ] 32 Wong SL, Ji H, Hollenbeck BK, Morris AM, Baser O, Birkmeyer JD. Hospital lymph node examination rates and survival after resection for colon cancer. JAMA 2007; 298: [PMID: DOI: /jama ] 33 Son T, Hyung WJ, Lee JH, Kim YM, Kim HI, An JY, Cheong JH, Noh SH. Clinical implication of an insufficient number of examined lymph nodes after curative resection for gastric cancer. Cancer 2012; 118: [PMID: DOI: /cncr.27426] 34 Roviello F, Rossi S, Marrelli D, Pedrazzani C, Corso G, Vindigni C, Morgagni P, Saragoni L, de Manzoni G, Tomezzoli A. Number of lymph node metastases and its prognostic significance in early gastric cancer: a multicenter Italian study. J Surg Oncol 2006; 94: ; discussion 274 [PMID: DOI: /jso.20566] 35 Mahar AL, Qureshi AP, Ottensmeyer CA, Pollett A, Wright FC, Coburn NG, Chetty R. A descriptive analysis of gastric cancer specimen processing techniques. J Surg Oncol 2011; 103: [PMID: DOI: /jso.21827] 36 Lemmens VE, van Lijnschoten I, Janssen-Heijnen ML, Rutten HJ, Verheij CD, Coebergh JW. Pathology practice patterns affect lymph node evaluation and outcome of colon cancer: a population-based study. Ann Oncol 2006; 17: [PMID: DOI: /annonc/mdl312] 37 Callahan MA, Christos PJ, Gold HT, Mushlin AI, Daly JM. Influence of surgical subspecialty training on in-hospital mortality for gastrectomy and colectomy patients. Ann Surg 2003; 238: ; discussion [PMID: DOI: /01.sla a] 38 Dhar DK, Kubota H, Tachibana M, Kotoh T, Tabara H, Masunaga R, Kohno H, Nagasue N. Body mass index determines the success of lymph node dissection and predicts the outcome of gastric carcinoma patients. Oncology 2000; 59: [PMID: ] 39 Dudeja V, Habermann EB, Zhong W, Tuttle TM, Vickers SM, Jensen EH, Al-Refaie WB. Guideline recommended gastric cancer care in the elderly: insights into the applicability of cancer trials to real world. Ann Surg Oncol 2011; 18: [PMID: DOI: /s ] 40 Kitagawa Y, Takeuchi H, Takagi Y, Natsugoe S, Terashima M, Murakami N, Fujimura T, Tsujimoto H, Hayashi H, Yoshimizu N, Takagane A, Mohri Y, Nabeshima K, Uenosono Y, Kinami S, Sakamoto J, Morita S, Aikou T, Miwa K, Kitajima M. Sentinel node mapping for gastric cancer: a prospective multicenter trial in Japan. J Clin Oncol 2013; 31: [PMID: ] P- Reviewers: Stanojevic GZ, Takao S S- Editor: Gou SX L- Editor: Wang TQ E- Editor: Liu XM 3648 April 7, 2014 Volume 20 Issue 13

274 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH REPORT Prevalence and characteristics of dyspepsia among college students in Zhejiang Province Meng Li, Bin Lu, Li Chu, Hong Zhou, Ming-Yan Chen Meng Li, Bin Lu, Li Chu, Hong Zhou, Ming-Yan Chen, Department of Gastroenterology, First Affiliated Hospital of Zhejiang Chinese Medical University, Hangzhou , Zhejiang Province, China Author contributions: Lu B designed and supervised the study; Li M, Chu L, Zhou H and Chen MY conducted the survey; Li M analyzed and interpreted the data and wrote the manuscript; all authors read and approved the final version for publication. Supported by The National Natural Science Foundation of China Correspondence to: Bin Lu, MD, Department of Gastroenterology, First Affiliated Hospital of Zhejiang Chinese Medical University, 54 Youdian Road, Hangzhou , Zhejiang Province, China. lvbin@medmail.com.cn Telephone: Fax: Received: July 17, 2013 Revised: November 19, 2013 Accepted: January 2, 2014 Published online: April 7, 2014 Abstract AIM: To investigate the prevalence and characteristics of uninvestigated dyspepsia among college students in Zhejiang Province. METHODS: Young adult students attending undergraduate (within the 4-year program) and graduate (only first-year students) colleges in Zhejiang Province were recruited between November 2010 and March 2011 to participate in the self-report survey study. The questionnaire was designed to collect data regarding demographics (sex and age), general health [weight and height, to calculate body mass index (BMI)], and physical episodes related to gastrointestinal disorders. Diagnosis of dyspepsia and irritable bowel syndrome (IBS) was made according to the Rome Ⅲ criteria. Gastroesophageal reflux disease (GERD) was defined by episodes of heartburn and/or acid reflux that occurred at least once a week, according to the Montreal definition. RESULTS: Of 2520 students recruited for survey participation, only 1870 (males: 967; age range: years, mean age: 21.3 years) returned a completed questionnaire. One hundred and eight (5.67%) of the student participants fit the criteria for dyspepsia diagnosis. Stratification analysis of dyspepsia and non-dyspepsia cases showed no statistically significant differences in age or BMI; however, the prevalence of dyspepsia was significantly higher in women than in men (7.53% vs 4.14%, P < 0.05). Stratification analysis of dyspepsia by grade level showed that year 4 undergraduate students had a significantly higher prevalence of dyspepsia (10.00% vs undergraduate year 1: 5.87%, year 2: 3.53% and year 3: 7.24%, and graduate year 1: 3.32%). Nearly all (95.37%) students with dyspepsia reported symptoms of postprandial distress syndrome, but only a small portion (4.63%) reported symptoms suggestive of abdominal pain syndrome. The students with dyspepsia also showed significantly higher rates of IBS (16.67% vs non-dyspepsia students: 6.30%, P < 0.05) and GERD (11.11% vs 0.28%, P < 0.05). CONCLUSION: Although the prevalence of dyspepsia among Zhejiang college students is low, the significantly higher rates of concomitant IBS and GERD suggest common pathophysiological disturbances Baishideng Publishing Group Co., Limited. All rights reserved. Key words: College student; Dyspepsia; Gastroesophageal; Irritable bowel syndrome Core tip: This college-based population survey aimed to determine the prevalence and investigate the characteristics of uninvestigated dyspepsia (UD, according to Rome Ⅲ criteria) in Zhejiang Province, China. The overall prevalence of UD was relatively low (5.67% in 1870 students), but female sex and senior (year 4) undergraduate status were represented more frequently among the UD cases. In addition, UD cases were more likely to have concomitant irritable bowel syndrome or gastroesophageal reflux disease, suggesting the exis April 7, 2014 Volume 20 Issue 13

275 Li M et al. Dyspepsia among Chinese college students tence of common etiologies or molecular mechanisms among these gastrointestinal disorders. Li M, Lu B, Chu L, Zhou H, Chen MY. Prevalence and characteristics of dyspepsia among college students in Zhejiang Province. World J Gastroenterol 2014; 20(13): Available from: URL: i13/3649.htm DOI: INTRODUCTION The gastroduodenal symptoms of dyspepsia are relatively non-specific, and include general abdominal discomfort, early satiety, and bloating. Patients often ignore the condition and not present to clinic until more severe symptoms manifest, such as belching, heartburn, nausea, vomiting, and pain. While untreated dyspepsia is not associated with an increased risk of death, its pathology can put the individual at greater risk of secondary tissue injury, such as mucosal erosion by gastric reflux leading to a chronic inflammation state, or may mask symptoms of other gastrointestinal diseases. Epidemiological surveys have estimated the global prevalence of uninvestigated dyspepsia (UD) to be as low as 7% and as high as 45% [1]. Results from clinical investigations are used to classify the dyspepsia diagnosis as functional dyspepsia (FD) or organic dyspepsia. For both dyspepsia types, some readilymodifiable factors have been identified as potential etiologies, including diet and gastrointestinal infection [mainly Helicobacter pylori (H. pylori)]; in addition, clinical management of a co-existing organic disease may help to reduce or eliminate the dyspeptic condition [2]. The lack of an effective targeted therapy, however, means that many patients suffer from chronic dyspepsia and an accompanying detrimental impact on quality of life [3]. Demographic and national factors may play a role in the development and progression of dyspepsia. Epidemiologic studies of dyspepsia in Asian countries have reported incidence rates between 8% and 30% [2], and have suggested that variable symptom profiles of the evaluated subjects and inconsistent diagnostic criteria used by the researchers and healthcare providers may explain this wide range. One survey in Malaysia demonstrated that the economic impact of dyspepsia is greater in urban settings, compared to rural settings [4], in conjunction with the greater disease prevalence in the former. A key differential feature of urban and rural settings is the socioeconomic status, which affects nearly every aspect of life from living conditions and diet to working conditions and stress levels. Within Asian urban settings, university students represent a unique population of advanced and productive society members, living in a high stress environment with relatively better access to and understanding of healthcare than their rural counterparts. Adolescents and young adults, between the ages of 14 and 29 years, account for roughly one-fourth of China s population and a large portion of city dwellers (State Statistical Bureau of the People s Republic of China, 2009). The Zhejiang Province, located at the heart of the Yangtze River Delta, is one of the fastest developing urban areas in China and is facing the challenge of establishing a healthcare system adequately equipped and staffed to address the healthcare needs of its expanding population. Considerations for such an endeavor include the particularly high rates of gastric diseases in China, such as gastric cancer and H. pylori infection (although the reported correlation with dyspepsia was relatively weak) [5] ; however, the epidemiological characteristics of dyspepsia among urban-dwelling populations of Chinese young adults remain largely unknown. The current study was designed as a self-report survey of college students in the Zhejiang Province to determine the prevalence of UD and investigate its relation to various demographic and physical parameters as well as other common gastrointestinal disorders. MATERIALS AND METHODS Study population Study recruitment was conducted at two college campuses in the Zhejiang Province between November 2010 and March Matriculated students in the undergraduate (within the 4-year program) and graduate (only first-year students) programs were included in the study. A total of 2520 students (48% males; mean age: 22.6 ± 3.0 years) were interviewed and offered the survey questionnaire for self-completion. Unreturned or incomplete questionnaires were excluded from the study. Survey The questionnaire was designed to collect data regarding demographics (sex and age), general health [weight and height, to calculate body mass index (BMI)], and physical episodes related to gastrointestinal disorders. Data related to dyspepsia and irritable bowel syndrome (IBS) were chosen according to the ROME Ⅲ criteria [9]. An individual was classified as having UD if they reported experiencing at least one of the following symptoms during the previous 3 mo, with the first instance occurring at least 6 mo earlier: (1) bothersome postprandial fullness; (2) early satiety; (3) epigastric pain; or (4) epigastric burning. Classification of IBS was made according to report of abdominal pain or discomfort persisting for at least 3 d at least once a month during the previous 3 mo and accompanied by at least two of the following symptoms: (1) relief of pain or discomfort attained after defecation; (2) onset of pain or discomfort associated with a change in stool frequency; or (3) onset of pain or discomfort associated with a change in stool appearance. Data related to gastroesophageal reflux disease (GERD) were chosen according to the Montreal definition for the diagnosis of GERD in population-based studies [10]. Individuals were classified as having GERD if they reported experiencing 3650 April 7, 2014 Volume 20 Issue 13

276 Li M et al. Dyspepsia among Chinese college students Table 1 Prevalence of uninvestigated dyspepsia in relation to student characteristics UD Non-UD Total P (n = 108) (n = 1762) (n = 1870) Sex < Male 40 (4.14) Female 68 (7.53) Age, yr ± ± ± 2.56 BMI ± ± ± 4.27 College program and year < Undergraduate Year 1 16 (5.67) Year 2 16 (3.53) Year 3 56 (7.24) Year 4 12 (10.00) Graduate Year 1 8 (3.32) Females vs Males; 2 Year 4 undergraduate students vs all other programs and attendance year. UD: Uninvestigated dyspepsia; Non-UD: Students without dyspepsia; BMI: Body mass index. GERD 0.15% 0.48% 0.12% 0.16% 0.80% 4.23% 5.81% UD Figure 1 Overlaps in the prevalence of uninvestigated dyspepsia, irritable bowel syndrome and gastroesophageal reflux disease among the 1870 students surveyed from the Zhejiang Province. GERD: Gastroesophageal reflux disease; UD: Uninvestigated dyspepsia; IBS: Irritable bowel syndrome. IBS frequent (at least once weekly) episodes of heartburn or acid regurgitation. For each of the gastrointestinal disorders, the survey questions were translated from the original source into Chinese language by the authors. Any individual classified with two or more of the above disorders was categorized as GI symptom complex overlap. Individuals not meeting the criteria for any of the above disorders were categorized as no functional GI disorder. Finally, individuals were categorized as having postprandial distress syndrome if they reported bothersome postprandial fullness experienced after an ordinary-sized meal and/or early satiety that prevented from finishing the meal. Statistical analysis Data are expressed as frequency or mean with SD. Normally distributed data were analyzed by the t test, and categorical data were analyzed by the χ 2 test. A P value < 0.05 was considered to indicate statistical significance. All statistical analyses were carried out with the Statistical Package for Social Sciences v15 (SPSS Inc., Chicago, IL, United States). RESULTS Of the 2520 students recruited into the study, 315 declined participation and 335 failed to return or returned an incomplete questionnaire. Therefore, the study population consisted of 1870 students in total; the demographic data are summarized in Table 1. The male to female ratio was nearly 1:1. The mean age was 21.3 ± 2.6 years, and the average BMI was ± Only 5.67% of the total students met the criteria for UD diagnosis. The UD group had a statistically significant predominance of females (P < 0.05) and students in the final year of the 4-year undergraduate program (vs undergraduate years 1, 2 and 3, and graduate year 1, P < 0.05 for all). The mean age and BMI of the UD group were statistically similar to those of the non-ud group. Among the total study population, there were 17 (0.9%) and 129 (6.9%) students who met the criteria for GERD and IBS diagnoses, respectively. Nearly all (95.4%) of the students in the UD group had postprandial distress syndrome. The least frequently reported dyspepsiarelated symptoms were intermittent pain or burning localized to the epigastric area (4.6% in the UD group). Twenty-nine of the students had GI symptom complex overlap, with UD + IBS in 18 students (1.0%) and UD + GERD in 12 (0.6%) (Figure 1). The rates of both IBS and GERD were significantly higher in the UD group than in the non-ud group (IBS: 16.7% vs 6.3% and GERD: 11.1% vs 0.3%, P < 0.05 for both). Conversely, the rate of UD was significantly lower in non-ibs students (6.4% vs IBS: 14.0%, P < 0.05) and non-gerd students (0.3% vs GERD: 70.6%, P < 0.05). DISCUSSION Studies of UD prevalence in Asian populations have yielded varied results. Using the Rome I criteria, a study from Hong Kong found a rate of 18.4% [11], while a crosssectional community study from Singapore that defined UD by the presence of upper abdominal pain found a rate of 7.9% [12] ; however, studies from Korea and Malaysia that both used the Rome Ⅱ criteria found similar rates of UD (approximately 14%) [13,14]. The limited numbers of these studies, though, precludes the ability to definitively conclude if the true rates of UD are associated with national/ethnic differences or the different criteria used to diagnose the condition. Nonetheless, these studies have provided insights into possible etiologic factors of UD in Asians, including age (predominance in older (> 40 years) subjects) [15]. In addition, several Asian studies have evaluated the rate of UD among college students, who represent a unique stress-heavy lifestyle model consisting of long hours of focused physical and cognitive activity under significant social-competitive pressure. As with the studies conducted in the general population, the rates of 3651 April 7, 2014 Volume 20 Issue 13

277 Li M et al. Dyspepsia among Chinese college students UD among college students have varied widely by country and study design. A study of college students in Japan found a UD rate of 6.7% using the Rome Ⅱ criteria [16], while the frequency of dyspepsia found in students from Beijing using Rome Ⅲ and Anhui using Rome Ⅱ was 1.6% and 8.3%, respectively [17,18]. The general clinical observation of gastrointestinal symptoms increasing with age has been confirmed by population-based studies [19,20] and associated with diminished sensory responses of the gut tissues [21]. In addition, socioeconomic factors, such as gross domestic product (GDP), have been shown to be inversely associated with dyspepsia [22]. The college students assessed in the current survey resided in an economically developed region; the relatively younger age, higher GDP, and features of the modern urban lifestyle (including high levels of internet connectivity) that characterize college life in Zhejiang Province may contribute to the relatively low rate of dyspepsia detected in our survey (5.8%). In addition, regular smoking was identified as a risk factor of dyspepsia in North Americans (i.e., United States and Canada) [23,24], and regular alcohol intake as a risk factor of UD in Asia- Pacific populations [15,25]. It is possible that the college students surveyed in the current study may have a generally healthier lifestyle, with lower rates of smoking and alcohol consumption than the general population. Another potential explanation for the lower rate detected in the current study, compared to other studies, may be the ROME Ⅲ diagnostic criteria applied to UD diagnosis [26]. The college students in the current study also showed a predominance of UD in females. In general, functional gastrointestinal disorders have a higher prevalence in women [27], possibly because women are more likely to present for diagnosis and undertake clinical management of such functional disorders, as was shown by a study of IBS and globus hystericus [28] ; however, that same study showed no difference in the prevalence of FD between men and women [28]. Studies of individual dyspeptic symptoms have demonstrated sex-related differences in prevalence, as well as in gastric emptying and visceral sensitivity [1,29]. The female-predominant prevalence of UD observed in the current survey may be related to sex-related symptom profiles that were not evaluated in our study. Another key finding of the current study was the significantly higher levels of dyspeptic symptoms in senior level undergraduates (in year 4 of the program); it is possible that this result was related to an increased stressful environment, as these students are facing a rapidly approaching significant lifestyle change (from college matriculation to employment). Dyspepsia, GERD, and IBS are common gastrointestinal conditions both in China and worldwide. The different prevalence rates of these three disorders observed in the current population of college students may also reflect the different strengths of associations with stress-related factors. For example, a study of the relation between psychological disorders and IBS, FD and non-erosive reflux disorder showed that anxiety was significantly more common in patients with IBS [30]. College students are at a higher risk of psychological disorders, such as anxiety [31]. However, the current study population showed a remarkably lower prevalence of GERD (< 1%) compared to the most recent worldwide estimates, which range between 10% and 20% [10]. The lower rate may be associated with the particularly younger age of the current study population and/or the lower BMI [22]. The rate of dyspepsia overlapping with GERD was higher, and closer to that previously reported from a Japanese cohort of college students [16]. Several case reports of dyspepsia, IBS and GERD overlap exist [32-34]. In the same Japanese cohort, the prevalence of dyspepsia + IBS was significantly higher than that of dyspepsia + GERD [16], which was similar to the findings in the current study of Chinese college students (1.0% and 0.6%, respectively). Collected data in the literature suggest that GERD is a common occurrence in dyspepsia, with rates of 22% to 50% being reported [35-38]. Similarly, some studies have suggested that the occurrence of IBS in dyspeptic patients is not uncommon; the current study population showed a rate of dyspepsia + IBS that fell within the range of prevalence rates previously reported for Asian populations [16,39-40]. While the precise mechanisms underlying such overlap in gastrointestinal disorders remain unknown, it is likely that pathophysiological factors shared between the three, such as altered motility, visceral hypersensitivity, brain-gut dysfunction or psychological distress, play key roles [41-44]. In addition, the significant overlap of symptoms among dyspepsia, IBS and GERD raises the question of whether the functional gastrointestinal disorders actually represent multiple separate disorders or a single clinical entity. Though dyspepsia is not fatal, it is associated with substantial impairment of quality of life and poses a significant burden on society, with sufferers experiencing higher rates of work absenteeism, reduced productivity, and higher reliance on healthcare resources than the general population [45,46]. For college students, the symptoms associated with dyspepsia may have a marked impact on the individual s study habits, ability to concentrate or attend classes, and performance on tests. To the best of our knowledge, no Chinese universities currently have a student outreach program to enhance their students knowledge of digestive health; by providing campuswide health lectures, which would include symptoms and management of common stress-related psychological and physical disorders, the rates of gastrointestinal disorders, such as dyspepsia, may be reduced. Although this investigation provides new insights into the prevalence and characteristics of UD in Chinese college students, it relied solely on data gathered from a self-report questionnaire; thus, the physical- and diseaserelated data may have been underreported or exaggerated. In addition, the questionnaire was newly developed according to the objectives of the current study. Since the questionnaire has not been validated, its test-retest reliability and validity are unknown. Moreover, the focus 3652 April 7, 2014 Volume 20 Issue 13

278 Li M et al. Dyspepsia among Chinese college students of questions to determine specific symptoms of dyspepsia, GERD and IBS precludes the ability of this study to analyze organic gastrointestinal diseases. In conclusion, the prevalence of UD in college students from the Zhejiang Province was low (5.7%), possibly due to the young age and low BMI of this population [47]. The UD rates were highest in students who were female and in the senior year of a 4-year undergraduate program. Nearly all of the students who fit the criteria for UD diagnosis also reported symptoms suggestive of postprandial distress syndrome and there was a remarkable portion of dyspepsia cases with concomitant IBS or GERD, suggesting the existence of common etiologies or molecular mechanisms among these gastrointestinal disorders. COMMENTS Background Epidemiological studies suggest that uninvestigated dyspepsia (UD) is a common gastrointestinal disorder worldwide. However, there is little data on the prevalence of UD and its overlap with other gastrointestinal diseases in China, particularly among college students who represent a unique population of young adults living in urban settings under particularly high stress conditions. Research frontiers Using a questionnaire with validated Chinese language Rome Ⅲ diagnostic criteria included, this study surveyed undergraduate (4-year program) and graduate (in year 1) students residing in the Zhejiang Province to determine prevalence and characteristics of UD. Innovations and breakthroughs According to Rome Ⅲ criteria, the prevalence of UD in college students from the Zhejiang Province was 5.7%. The symptoms of dyspepsia were most frequently reported by females and senior level (year 4) undergraduate students, suggesting sex- and/or stress-related components. There was a remarkable portion of dyspepsia cases with concomitant irritable bowel syndrome (IBS) or gastroesophageal reflux disease (GERD), suggesting the existence of common etiologies or molecular mechanisms among these gastrointestinal disorders. Applications While the reasons for the relatively low prevalence rate of UD among college students from the Zhejiang Province remain to be fully elucidated, the data indicate that this condition is not infrequent among this population and deserves attention to promote both diagnosis and clinical management that may prevent potential damage caused by a chronic condition. Peer review This is a cross-sectional survey of 2520 Chinese college students in the Zhejiang Province of China that examined the prevalence and characteristics of UD, including overlap with IBS and GERD. 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Scand J Gastroenterol Suppl 1987; 130: [PMID: DOI: / ] 42 Corsetti M, Caenepeel P, Fischler B, Janssens J, Tack J. Impact of coexisting irritable bowel syndrome on symptoms and pathophysiological mechanisms in functional dyspepsia. Am J Gastroenterol 2004; 99: [PMID: DOI: /j x] 43 Iovino P, Azpiroz F, Domingo E, Malagelada JR. The sympathetic nervous system modulates perception and reflex responses to gut distention in humans. Gastroenterology 1995; 108: [PMID: DOI: / (95) ] 44 Kennedy TM, Jones RH, Hungin AP, O flanagan H, Kelly P. Irritable bowel syndrome, gastro-oesophageal reflux, and bronchial hyper-responsiveness in the general population. Gut 1998; 43: [PMID: DOI: /gut ] 45 Lu CL, Lang HC, Chang FY, Chen CY, Luo JC, Wang SS, Lee SD. Prevalence and health/social impacts of functional dyspepsia in Taiwan: a study based on the Rome criteria questionnaire survey assisted by endoscopic exclusion among a physical check-up population. 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280 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH BRIEF ARTICLE REPORT Lack of association between apolipoprotein C3 gene polymorphisms and risk of nonalcoholic fatty liver disease in a Chinese Han population Tong-Hong Niu, Man Jiang, Yong-Ning Xin, Xiang-Jun Jiang, Zhong-Hua Lin, Shi-Ying Xuan Tong-Hong Niu, Man Jiang, Medical College of Qingdao University, Qingdao , Shandong Province, China Tong-Hong Niu, Man Jiang, Yong-Ning Xin, Xiang-Jun Jiang, Zhong-Hua Lin, Shi-Ying Xuan, Department of Gastroenterology, Qingdao Municipal Hospital, Qingdao , Shandong Province, China Author contributions: Niu TH and Jiang M contributed equally to the work, both drafted and wrote the article; Xin YN, Jiang XJ and Lin ZH revised the paper; and Xuan SY approved the final version. Supported by National Natural Science Foundation of China, No /H0304 Correspondence to: Shi-Ying Xuan, PhD, Professor, Department of Gastroenterology, Qingdao Municipal Hospital, Qingdao , Shandong Province, China. xsy_emuch@126.com Telephone: Fax: Received: August 22, 2013 Revised: November 17, 2013 Accepted: January 2, 2014 Published online: April 7, 2014 Abstract AIM: To investigate the association between two polymorphisms of apolipoprotein C3 (APOC3 ) and risk of nonalcoholic fatty liver disease (NAFLD) in a Chinese Han population. METHODS: Genotypes for rs and rs in APOC3 and the known rs in patatin-like phospholipase domain-containing protein 3 (PNPLA3) in 390 patients with NAFLD and 409 control subjects were determined by sequencing and polymerase chain reaction analysis. Serum lipid profiles were determined using biochemical methods, and an index of insulin resistance (IR, HOMA-IR), serum APOC3 concentrations and total antioxidant status (TAS) were also assessed. RESULTS: No significant differences in genotype and allele frequencies of rs and rs were found between the NAFLD population and the controls (P > 0.05). The OR for the association between -455C and -482T allele carriers and the risk of NAFLD were 1.06 (95%CI: , P > 0.05) and 1.00 (95%CI: , P > 0.05), respectively. The variant carriers did not have a significantly increased risk of NAFLD or elevated clinical and biochemical parameters such as APOC3 concentrations, IR (1.42 ± 0.43 vs 1.48 ± 0.52, P > 0.05), liver enzymes and TAS (13.94 ± 2.01 vs ± 1.92, P > 0.05) compared with the controls. Moreover, the results were similar when testing was carried out independent of the genetic variation in PNPLA3. CONCLUSION: The two polymorphisms of the APOC3 gene are not associated with a risk of NAFLD, or with lipid profiles, IR and oxidative stress in the Chinese Han population Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Polymorphism; Single nucleotide; Nonalcoholic fatty liver disease; Apolipoprotein C3; Insulin resistance; Oxidative stress Core tip: In this study, we determined the relationship between two polymorphisms in apolipoprotein C3 (APOC3 ) and susceptibility to nonalcoholic fatty liver disease (NAFLD) in a Han population in China. We also examined the influence of APOC3 genotypes on insulin resistance, obesity, fasting TG levels and total antioxidant status. We found that the two polymorphisms of the APOC3 gene are not associated with a risk of NAFLD, or with lipid profiles, IR and oxidative stress in the Chinese Han population. Niu TH, Jiang M, Xin YN, Jiang XJ, Lin ZH, Xuan SY. Lack of association between apolipoprotein C3 gene polymorphisms and risk of nonalcoholic fatty liver disease in a Chinese Han population. World J Gastroenterol 2014; 20(13): Available from: URL: April 7, 2014 Volume 20 Issue 13

281 Niu TH et al. APOC3 and nonalcoholic fatty liver disease i13/3655.htm DOI: INTRODUCTION Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and is now recognized as a major public health problem worldwide [1,2]. It is estimated that the prevalence of NAFLD ranges from 6.3% to 33% with a median of 20% in the general population, based on a variety of assessment methods [3]. It is not a Western disease, it is recognized as the main cause of chronic liver disease in the developing world, and the prevalence of NAFLD in the Chinese population is approximately 15% [4-6]. NAFLD encompasses a wide spectrum of hepatic disorders, ranging from simple fatty liver NAFL to nonalcoholic steatohepatitis and hepatic cirrhosis, which may progress to hepatocellular carcinoma. NAFLD is a multifactorial disorder arising from the interplay between genetic and environmental influences. Genetic association is a powerful tool for determining the mechanism of NAFLD [7,8], in particular, patatinlike phospholipase-3 (PNPLA3) and apolipoprotein C3 (APOC3) have been suggested as potential genes related to NAFLD susceptibility or disease progression. The association between the PNPLA3 gene I148M variant and NAFLD has been demonstrated in a number of studies, confirming that this gene is an important genetic determinant of disease development [9-11]. APOC3 is involved in triacylglycerol metabolism, which inhibits lipoprotein lipase (LPL) and mediates lipoprotein uptake by the liver [12]. Recently, Petersen et al [13] carried out a candidate gene study and reported that two variants (rs and rs ) of the promoter region in the APOC3 gene located in an insulin response element (IRE) [14] led to increased uptake of chylomicron remnants by the liver, and this resulted in NAFLD and hepatic insulin resistance in Asian-Indian men, however, subsequent studies obtained different results in American or European subjects [15-18]. No research has been carried out on the association between the two polymorphisms of APOC3 and NAFLD in the Chinese population. In this study, we determined the relationship between the two polymorphisms in APOC3 and susceptibility to NAFLD in a Han population in China. In addition, we examined the influence of APOC3 genotypes on insulin resistance, obesity, fasting TG levels and total antioxidant status. MATERIALS AND METHODS Subjects We selected a total of 799 unrelated adult subjects of both sexes, including 390 unrelated Chinese patients (175 males, 215 females, mean age ± years) diagnosed with NAFLD by B-type ultrasonography between April 2012 and April 2013, and 409 healthy controls matched for sex and age (198 males, 211 females, mean age ± years). The volunteers were recruited from the Department of Gastroenterology and the Medical Center of Qingdao Municipal Hospital. All subjects were Northern Han Chinese in origin. The diagnosis of NAFLD was performed under standard clinical evaluation conditions according to the AASID criteria. Other causes of liver disease were excluded, including increased alcohol intake (> 210/140 g/wk for males/females), as confirmed by at least one family member or friend and carboxydesialylated transferrin determination, viral and autoimmune hepatitis, hereditary hemochromatosis, and alphal-antitrypsin deficiency. We also excluded subjects with type 1 diabetes mellitus. The controls were confirmed as healthy by medical history, general examinations and laboratory examinations at the same hospital. We excluded subjects as previously described [19]. Basic information (names, ages, etc.) was obtained by questionnaire. All subjects were of Chinese Han ethnicity. Informed consent was obtained from subjects who agreed to participate in the study, and the study protocol was approved by the Ethical Committee of Qingdao Municipal Hospital. Biochemical analyses Blood samples for biochemical analyses were obtained after an overnight fast. Glucose, insulin, cholesterol, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, and triglycerides (TG) were measured using routine enzymatic methods. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) and γ-glutamyltransferase (GGT) concentrations were measured as previously described [20]. Plasma APOC3 concentrations were measured enzymatically using an Assay Max human Apolipoprotein C-Ⅲ enzyme linked immunosorbent assay (ELISA) Kit (n = 180) (BlueGene, China). Insulin sensitivity was assessed using the homeostasis model assessment of insulin resistance (HOMA-IR) and a 2-h oral glucose-tolerance test (OGTT). The HOMA-IR was calculated as fasting insulin concentration (μu/ml) fasting glucose concentration (mmol/l)/22.5 [21]. Ninety-eight subjects were selected to complete a standard 75 g OGTT and measurements of glucose and insulin were recorded at baseline and after 30, 60, 90, and 120 min. Total antioxidant status (TAS), independent of the genetic variation in PNPLA3 (n = 231), was measured colorimetrically using the Total Antioxidant Capacity Assay Kit (Sciencell, United States) and the ABTS method. The color change was measured using a spectrophotometer. The reaction was calibrated with Trolox (a water-soluble analogue of vitamin E). Genetic analysis Genomic DNA was extracted from peripheral blood using the Genomic DNA Purification Kit (BioTeke, Biotechnology, Beijing, China) following the supplier s instructions. The success rate for DNA extraction was 100% and the DNA samples were stored in a freezer at -20 until 3656 April 7, 2014 Volume 20 Issue 13

282 Niu TH et al. APOC3 and nonalcoholic fatty liver disease Table 1 Demographic and clinical features of patients with nonalcoholic fatty liver disease and controls n (%) NAFLD patients (n = 390) Controls (n = 409) P value BMI (kg/m 2 ) ± ± a Waist (cm) ± ± a Hypertension 89 (22.8) 42 (10.3) c T2DM 87 (22.3) 36 (8.8) c ALT (U/L) ± ± a AST (U/L) ± ± GGT (U/L) ± ± a FPG (mmol/l) 6.03 ± ± UA (mmol/l) ± ± a TG (mmol/l) 2.91 ± ± a TC (mmol/l) 5.17 ± ± a HDL (mmol/l) 1.71 ± ± a LDL (mmol/l) 4.28 ± ± a FINS (μu/ml) 6.27 ± ± a HOMA-IR (U) 1.67 ± ± a Data are expressed as mean ± SD, a P < 0.05 vs controls, by unpaired Student s t test; c P < 0.05 vs controls, by χ 2 test. Body mass index (BMI) was calculated as body weight (kg)/height (m 2 ); ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; GGT: γ-glutamyltransferase; FPG: Fasting blood glucose; UA: uric Acid; TC: Total cholesterol; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; FINS: Fasting blood insulin; NAFLD: Nonalcoholic fatty liver disease; TG: Triglyceride; HOMA-IR: Homeostatic model assessment of insulin resistance. use. The primers for rs and rs used in the reaction were: 5 -GAAGGTGAACGAGAGAAT- CAGTCCTG-3 (forward); 5 -GCCTCGGGCCCATCT- CAGCCTTTCACACTG-3 (reverse). Amplification was performed in a total volume of 25 μl containing 12.5 μl Taq polymerase chain reaction (PCR) MasterMix, 2 μl genomic DNA, 1 μl forward primer, 1 μl reverse primer and 8 μl purified water, on PCR amplification equipment (Labnet, United States) as follows: initial step of 95 for 10 min, followed by 35 cycles: denaturation at 95 for 40 s, annealing at 56 for 40 s, and elongation at 72 for 50 s. Amplicon size was 511 bp. The primers for rs were 5 -CTGAAGTCCGAGGGT- GTATG-3 (forward); 5 -AGCTGTGGCTACTCT- GTCTG-3 (reverse). The PCR amplification profile was as follows: initial step of 94 for 5 min, followed by 30 cycles: denaturation at 94 for 30 s, annealing at 57.5 for 30 s, and elongation at 72 for 1 min. Amplicon size was 396 bp. All PCR products were resolved using 2% agarose gel electrophoresis at 110 V for 30 min and stained with ethidium bromide. The genotypes of the two genes underwent direct sequencing using the ABI Prism Sequence Detection System ABI3730 (Genscript). The genotyping success rate was > 95%. The two single nucleotide polymorphisms (SNPs) in APOC3 are in strong linkage disequilibrium [13-22], thus, we divided the study subjects into two groups for analysis: no variant allele expression (noncarriers) at both SNPs and one or more alleles expressed (Carriers) at either SNP. Statistical analysis Statistical analyses were performed using SPSS statistical software, version 17.0 for Windows. Genotypes and alleles were estimated by counting and the distributions between NAFLD patients and controls were analyzed by Pearson s χ 2 test and Fisher s exact test when appropriate. Hardy-Weinberg equilibrium between expected and observed genotype distributions was assessed using the χ 2 test. Baseline characteristics are shown as mean ± SD. Differences in the characteristics of the study population between different groups were examined using the Student s t test, paired samples t test or the χ 2 test. The strength of the association between the polymorphisms and NAFLD was evaluated by logistic regression analysis adjusted for confounders [gender, age, body mass index (BMI), weight and waistline, which were considered as continuous variables], estimated by the OR with 95%CI. Linear regression analysis was performed to determine the correlation between plasma APOC3 concentrations and serum TG levels. Linkage disequilibrium between the rs and rs SNPs was calculated using Haploview software and was shown as D. The present sample size of the study revealed 90% power to detect a significant association (P < 0.05) with an effect size index of 0.1. Levels of significance were defined as P < RESULTS Patients in the NAFLD group and control group were matched for sex (P = 0.316) and age (P = 0.068). The clinical characteristics of the two groups are shown in Table 1, and patients with NAFLD had higher levels of ALT, GGT, FPG, TG, TC and LDL than controls. As NAFLD is a metabolic disease, it showed a strong interaction with hypertension and diabetes (Table 1). Distributions of the genotypes of these two polymorphisms were in accordance with the Hardy-Weinberg equilibrium in the NAFLD and control groups (PNAFLD = 0.130, 0.054; Pcontrol = 0.350, 0.882, respectively). The variant allele frequencies (-455C and -482T) were and in NAFLD patients, and and in controls, respectively. To ensure genotyping accuracy, we randomly selected 150 subjects for reverse sequencing. The success rate of duplicated genotyping was > 100%. There were no significant differences in the genotype and allele distribution between the two groups (P > 0.05) (Table 2). Presence of the -455C allele and the -482T allele did not increase the risk of developing NAFLD (OR = 1.00, 95%CI: , and OR = 0.99, 95%CI: , respectively). In contrast, the rs738409g allele of PNPLA3 was associated with a significantly increased risk of NAFLD in the study population (OR = 2.00, 95%CI: ) (Table 3). There was strong linkage disequilibrium between T-455C and C-482T with D = Compared with the noncarriers, none of the lipid variables (total cholesterol, HDL, LDL, and circulating TG) and indices of liver damage severity (serum ALT and AST levels) showed statistically relevant differences according to the variant APOC3 promoters in both the overall series and in the subgroups (Table 4). In the same 3657 April 7, 2014 Volume 20 Issue 13

283 Niu TH et al. APOC3 and nonalcoholic fatty liver disease Table 2 Distribution of genotype and allele frequencies of apolipoprotein C3 (-455, -482) and patatin-like phospholipase domain-containing protein 3 (rs738409) in nonalcoholic fatty liver disease patients and controls n (%) PNPLA3 gene I148M variant plays a role in NAFLD, which has been demonstrated in a number of studies [9-11]. Our research also demonstrated similar results. In the Petersen s study, APOC3 genotypes were found to be associated with hepatic triglyceride content measured by H-MRS [13], while different results were obtained in the Dallas Heart Study [15]. The mechanisms associated with the accumulation of TG in the liver and subsequent hepatocellular damage are not fully understood [23]. We evaluated the changes in ALT and AST, which were used as markers of liver fat accumulation [24-26] and are commonly used in clinical practice [27]. We did not observe significant differences in plasma concentrations of these transaminases between carriers of the variants and noncarrier subjects, even when independent of the genetic variation in PNPLA3. Similar findings were reported in a Caucasian population [17]. Although the pathogenesis of NAFLD is unknown, it is conceivable that a network of interactions between multiple factors is involved in both the development and progression of the disease, such as insulin resistance, obesity and oxidative stress [28]. Insulin resistance is recognized as an essential pathophysiological factor or first hit in the development of NAFLD, and may play a fundamental role in the pathogenesis of this disorder [29,30]. We are in agreement with other researchers who have reported that NAFLD has an important role in the etiology of hepatic insulin resistance [31,32]. Our data were consistent with previous findings that genetic variation in APOC3 expression has little effect on the insulin sensitivity index (HOMA-IR) in humans [15,16,33]. In addition, insulin sensitivity did not change the results of oral glucose-tolerance testing in carriers compared to non-carriers. APOC3 expression in the liver is physiologically inhibited by insulin, and no obvious increase in serum insulin or decrease in plasma APOC3 concentration was observed. However, plasma TG concentrations were significantly positively correlated with plasma APOC3 concentrations (r = 0.706), which was consistent with earlier observations [34,35]. APOC3 was the first lipid-associated gene to be linked to hystudy subjects, the PNPLA3 genotype was shown to predispose to NAFLD. We next evaluated whether these results were similar when controlling for the PNPLA3 mutation. Similar results were obtained (Table 5). We assessed the relationship between APOC3 variants and fasting blood insulin (FINS), HOMA-IR and plasma TG. No differences between wild-type homozygotes and variant allele carriers were observed (Tables 4 and 5). APOC3 concentrations showed a consistent relationship with plasma TG concentrations (r = 0.706, P = 0.000) (Figure 1), as indicated by linear regression analysis. No associations between the variants and fasting levels of plasma glucose, insulin or HOMA-IR were found. Glucose and insulin levels were similar in the groups throughout the OGTT (Figure 2). The control group had significantly higher TAS than the NAFLD group (P = 0.000). However, mean TAS in the noncarrier and carrier groups was similar at admission (P = 0.098) (Figure 3). DISCUSSION NAFLD Controls χ 2 P value -455 genotype TT 102 (26.2) 104 (25.4) TC 180 (46.2) 195 (47.7) CC 108 (27.7) 110 (26.9) Allele T 384 (49.2) 403 (49.3) C 396 (50.8) 415 (51.2) genotype CC 107 (27.4) 104 (25.4) CT 176 (45.1) 203 (49.6) TT 107 (27.4) 102 (51.2) Allele C 390 (50.0) 411 (50.2) T 390 (50.0) 407 (49.8) rs genotype CC 48 (12.3) 183 (44.7) CG 153 (39.2) 176 (43.0) GG 189 (48.5) 50 (12.2) Allele C 249 (31.9) 542 (66.3) G 531 (68.1) 276 (33.7) NAFLD: Nonalcoholic fatty liver disease. We read with great interest the article by Petersen who reported a higher prevalence of NAFLD in APOC3 variant allele carriers compared with wild-type carriers in Indian men [13]. In apparent disagreement with these results, recent findings in the Dallas Heart Study demonstrated different results in the three major ethnic groups included in that study [15]. To highlight the role of APOC3 in NAFLD progression, we enrolled a total of 799 unrelated Chinese adults in a case-control study to investigate the relationship between two APOC3 polymorphisms (rs and rs ) and the risk of NAFLD. We did not find significant associations between these polymorphisms and the risk of NAFLD in the Chinese Han population. Table 3 Odds ratios for nonalcoholic fatty liver disease according to apolipoprotein C3 and patatin-like phospholipase domain-containing protein 3 genotypes and alleles in the study population Unadjusted model Adjusted model OR (95%CI) P value OR (95%CI) P value APOC3 gene -455TT TC + CC 0.96 ( ) ( ) CC CT + TT 0.90 ( ) ( ) 1.00 PNPLA3 gene CC 1 1 CG + GG 5.77 ( ) ( ) C vs T: OR (95%CI) = 1.00 ( ): -482C vs T: OR (95%CI) = 0.99 ( ); PNPLA3G vs C: OR (95%CI) = 2.00 ( ). The multiple logistic regression model was adjusted for age, gender, body mass index, waist, weight and presence of hypertension and T2DM April 7, 2014 Volume 20 Issue 13

284 Niu TH et al. APOC3 and nonalcoholic fatty liver disease Table 4 Clinical characteristics of apolipoprotein C3 wild-type homozygotes (rs C/C and rs T/T) and carriers of one or more variant alleles (rs T and rs C) in the study population Characteristic Overall series NAFLD patients Controls Noncarriers Carriers P value Noncarriers Carriers P value Noncarriers Carriers P value n Female/male 110/87 306/ /48 157/ /39 149/ Age (yr) ± ± ± ± ± ± BMI (kg/m 2 ) ± ± ± ± ± ± Waist (cm) ± ± ± ± ± ± ALT (U/L) ± ± ± ± ± ± AST (U/L) ± ± ± ± ± ± FPG (U/L) 5.46 ± ± ± ± ± ± TG (mmol/l) 2.67 ± ± ± ± ± ± TC (mmol/l) 4.97 ± ± ± ± ± ± HDL (mmol/l) 2.25 ± ± ± ± ± ± LDL (mmol/l) 3.84 ± ± ± ± ± ± FINS (μu/ml ) 5.83 ± ± ± ± ± ± HOMA-IR (U) 1.42 ± ± ± ± ± ± APOC3 (mg/dl) ± 2.01 (n = 34) ± 1.92 (n = 146) ± 1.90 (n = 19) ± 1.98 (n = 80) ± 1.79 (n = 15) ± 1.89 (n = 66) Data are shown as mean ± SD. Statistically significant differences were tested using the Student s unpaired t test or by χ 2 test when appropriate. A P value < 0.05 was considered significant. ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; GGT: γ-glutamyltransferase; FPG: Fasting blood glucose; UA: uric Acid; TC: Total cholesterol; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; FINS: Fasting blood insulin; NAFLD: Nonalcoholic fatty liver disease; TG: Triglyceride; HOMA-IR: Homeostatic model assessment of insulin resistance; BMI: Body mass index. Table 5 Clinical characteristics of patatin-like phospholipase domain-containing protein 3 and apolipoprotein C3 (independently of patatin-like phospholipase domain-containing protein 3 I148M) wild-type homozygotes and carriers of one or more variant alleles in the study population Characteristic PNPLA3 APOC3 (independently of PNPLA3 I148M) Noncarriers Carriers P value Noncarriers Carriers P value n Female/male 124/ / /70 109/ Age (yr) ± ± ± ± BMI (kg/m 2 ) ± ± ± ± Waist (cm) ± ± ± ± ALT (U/L) ± ± ± ± AST (U/L) ± ± ± ± FPG (U/L) 5.30 ± ± ± ± TG (mmol/l) 2.34 ± ± ± ± TC (mmol/l) 4.79 ± ± ± ± HDL (mmol/l) 2.64 ± ± ± ± LDL (mmol/l) 3.28 ± ± ± ± FINS (μu/ml) 5.67 ± ± ± ± HOMA-IR (U) 1.34 ± ± ± ± ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; GGT: γ-glutamyltransferase; FPG: Fasting blood glucose; UA: uric Acid; TC: Total cholesterol; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; FINS: Fasting blood insulin; NAFLD: Nonalcoholic fatty liver disease; TG: Triglyceride; HOMA-IR: Homeostatic model assessment of insulin resistance; PNPLA3: Patatin-like phospholipase domain-containing protein 3; APOC3: Apolipoprotein C3. pertriglyceridemia [36]. It was reported that the APOC3 nonsense allele (R19X) caused a 45% reduction in plasma TG levels [37]. However, results for the polymorphic SstI site in the 3 untranslated region of APOC3 varied. Most studies found a strong interaction between them [38], but the research by Russo resulted in a different conclusion [39]. We confirmed the lack of association between APOC3 promoter polymorphisms and plasma TG levels in NAFLD patients, whereas Petersen et al [13] found that fasting plasma TG levels were approximately 60% higher in male Asian Indian carriers of the variant alleles. These differences may be due to linkage disequilibrium with causal variants in different races. Obesity is a common and well documented risk factor for NAFLD. The prevalence of NAFLD can exceed 90% in patients with severe obesity undergoing surgery [40]. The classification of obesity is based on BMI, and for Chinese subjects a BMI of 28 kg/m 2 or more is an index of obesity, whereas a BMI greater than 24 kg/m 2 is an index of overweight. In this study, BMI was higher in the NAFLD group (24.83 ± 2.82 kg/m 2 ) than in controls (22.18 ± 4.02 kg/m 2 ). However, the two APOC3 polymorphisms did not influ April 7, 2014 Volume 20 Issue 13

285 Niu TH et al. APOC3 and nonalcoholic fatty liver disease b P = a P = TG (mmol/l) TAS (mmol/l) APOC3 (mg/dl) Controls NAFLD Carriers Noncarriers Groups Figure 1 Relationship between serum triglycerides level and plasma apolipoprotein C3 concentration. TG: Triglycerides. r and P values were calculated using a linear regression model (r = 0.706, P = 0.000). Figure 3 Comparison of total antioxidant status in different groups. a P and b P were tested by Student s unpaired t test, a P = vs carriers, b P < 0.01 vs controls, respectively. TAS: Total antioxidant status. A Mean (glucose mg/dl) B Mean (insulin mu/dl) t /min t /min Noncarriers Carriers Noncarriers Carriers Noncarriers Carriers Noncarriers Carriers Figure 2 Comparison of glucose (A) and insulin (B) levels between noncarrier and carrier groups in the oral glucose tolerance test. P values were calculated by paired samples t test (P = 0.137, 0.537). ence BMI (P > 0.05). The first metabolic abnormality leading to liver steatosis is a lipotoxic reaction with an oxidative stress component [41-43]. Antioxidants in the body can interrupt oxidative stress-induced reactive oxygen species (ROS). Therefore, plasma TAS should be ranked on the basis of methods currently used to evaluate oxidative stress [43]. We found that TAS was lower in NAFLD patients [41], however, TAS was not reduced in the variant allele expression group. Limitations of our study include the lack of direct measurement of hepatic fat content by liver biopsy. Liver biopsy or elastography (Fibroscan) is ideal for measuring hepatic fat, but is probably not achievable in a study of this size. We diagnosed NAFLD using routine blood testing and liver ultrasonography, which can reduce the related cost and risk, but are not very accurate. The number of subjects in our study may have been too small to detect a significant association. We think that ethnic origin influences the prevalence of NAFLD, and our study was carried out on non-asian patients. To resolve this issue, larger studies which should include Asian Indian populations across multiple ethnic groups are required. We need genome-wide association studies to determine the association between the two variant alleles of APOC3 and NAFLD, similar to the detection of PNPLA3I148M which strongly influences NAFLD [9]. In summary, compared with PNPLA3, the two genetic variants (T-455C at rs and C-482T at rs ) in the APOC3 gene were not associated with NAFLD risk. Even when independent of the PNPLA3 I148M genotype, the two gene polymorphisms of APOC3 did not contribute to the inter-individual differences in lipid profiles, insulin resistance, obesity, oxidative stress and susceptibility to NAFLD in the Chinese Han population. COMMENTS Background Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease and is now recognized as a major public health problem worldwide. It is a multifactorial disorder arising from the interplay between genetic and environmental influences. Genetic association is a powerful tool for determining the mechanism of NAFLD. Patatin-like phospholipase domain-containing protein 3 (PNPLA3) and apolipoprotein C3 (APOC3) have been suggested as potential genes related to NAFLD susceptibility or disease progression. However, since China is the most populated country in the world, a study of the Han population is insufficient to determine the association between these polymorphisms and NAFLD. Research frontiers The association between the PNPLA3 gene I148M variant and NAFLD has been demonstrated in a number of studies. A recent study suggested that pro April 7, 2014 Volume 20 Issue 13

286 Niu TH et al. APOC3 and nonalcoholic fatty liver disease moter region polymorphisms in APOC3 (T-455C and C-482T) may contribute to NAFLD, TG concentrations and IR in Indian subjects. However, subsequent studies obtained different results in American or European populations. Further studies are required to confirm the association between the two polymorphisms of APOC3 and NAFLD in different ethnic groups. Innovations and breakthroughs This is the first study to systemically determine the relationship between the two polymorphisms (T-455C and C-482T) in APOC3 and susceptibility to NAFLD in the Han population in China. The findings suggested that the two genetic variants in the APOC3 gene were not associated with a risk of NAFLD, even when independent of PNPLA3 I148M genotypes. As the pathogenesis of NAFLD is unknown, the authors studied, for the first time, the two-hit theory of NAFLD to support their results. Applications NAFLD is genetically influenced and the number of common genetic variants associated with this disease is expanding. This study confirmed that PNPLA3I148M plays an important role in NAFLD and related diseases, providing new insights into the biology and genetics of NAFLD and opening up avenues for biological, diagnostic, and therapeutic research into this condition in humans. Although this research found that the two polymorphisms of APOC3 were not associated with NAFLD risk in Chinese patients, it provides a new direction for further studies on the genetics of NAFLD. Peer review The authors collected a total of 799 unrelated Chinese adults in a case-control study to investigate the relationship between the two APOC3 polymorphisms and the risk of NAFLD, and demonstrated that the two polymorphisms of APOC3 gene were not associated with risk of NAFLD, or with the lipid profiles, insulin resistance and oxidative stress in the Han population in China. 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287 Niu TH et al. APOC3 and nonalcoholic fatty liver disease DOI: / X ] 23 Adams LA, Angulo P. Recent concepts in non-alcoholic fatty liver disease. Diabet Med 2005; 22: [PMID: ] 24 Schindhelm RK, Diamant M, Dekker JM, Tushuizen ME, Teerlink T, Heine RJ. Alanine aminotransferase as a marker of non-alcoholic fatty liver disease in relation to type 2 diabetes mellitus and cardiovascular disease. Diabetes Metab Res Rev 2006; 22: [PMID: ] 25 Prati D, Taioli E, Zanella A, Della Torre E, Butelli S, Del Vecchio E, Vianello L, Zanuso F, Mozzi F, Milani S, Conte D, Colombo M, Sirchia G. Updated definitions of healthy ranges for serum alanine aminotransferase levels. Ann Intern Med 2002; 137: 1-10 [PMID: ] 26 Pratt DS, Kaplan MM. Evaluation of abnormal liver-enzyme results in asymptomatic patients. N Engl J Med 2000; 342: [PMID: ] 27 Marchesini G, Bugianesi E, Forlani G, Cerrelli F, Lenzi M, Manini R, Natale S, Vanni E, Villanova N, Melchionda N, Rizzetto M. Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome. Hepatology 2003; 37: [PMID: ] 28 Malhi H, Gores GJ. Molecular mechanisms of lipotoxicity in nonalcoholic fatty liver disease. Semin Liver Dis 2008; 28: [PMID: DOI: /s ] 29 Cortez-Pinto H, Camilo ME, Baptista A, De Oliveira AG, De Moura MC. Non-alcoholic fatty liver: another feature of the metabolic syndrome? Clin Nutr 1999; 18: [PMID: ] 30 Marceau P, Biron S, Hould FS, Marceau S, Simard S, Thung SN, Kral JG. Liver pathology and the metabolic syndrome X in severe obesity. J Clin Endocrinol Metab 1999; 84: [PMID: ] 31 Samuel VT, Liu ZX, Wang A, Beddow SA, Geisler JG, Kahn M, Zhang XM, Monia BP, Bhanot S, Shulman GI. Inhibition of protein kinase Cepsilon prevents hepatic insulin resistance in nonalcoholic fatty liver disease. J Clin Invest 2007; 117: [PMID: ] 32 Zhang D, Liu ZX, Choi CS, Tian L, Kibbey R, Dong J, Cline GW, Wood PA, Shulman GI. Mitochondrial dysfunction due to long-chain Acyl-CoA dehydrogenase deficiency causes hepatic steatosis and hepatic insulin resistance. Proc Natl Acad Sci USA 2007; 104: [PMID: ] 33 Reaven GM, Mondon CE, Chen YD, Breslow JL. Hypertriglyceridemic mice transgenic for the human apolipoprotein C-III gene are neither insulin resistant nor hyperinsulinemic. J Lipid Res 1994; 35: [PMID: ] 34 Mauger JF, Couture P, Bergeron N, Lamarche B. Apolipoprotein C-III isoforms: kinetics and relative implication in lipid metabolism. J Lipid Res 2006; 47: [PMID: ] 35 Zheng C, Khoo C, Furtado J, Sacks FM. Apolipoprotein C-III and the metabolic basis for hypertriglyceridemia and the dense low-density lipoprotein phenotype. Circulation 2010; 121: [PMID: DOI: /CIRCULA- TIONAHA ] 36 Rees A, Stocks J, Sharpe CR, Vella MA, Shoulders CC, Katz J, Jowett NI, Baralle FE, Galton DJ. Deoxyribonucleic acid polymorphism in the apolipoprotein A-1-C-III gene cluster. Association with hypertriglyceridemia. J Clin Invest 1985; 76: [PMID: ] 37 Pollin TI, Damcott CM, Shen H, Ott SH, Shelton J, Horenstein RB, Post W, McLenithan JC, Bielak LF, Peyser PA, Mitchell BD, Miller M, O Connell JR, Shuldiner AR. A null mutation in human APOC3 confers a favorable plasma lipid profile and apparent cardioprotection. Science 2008; 322: [PMID: DOI: /science ] 38 Dammerman M, Sandkuijl LA, Halaas JL, Chung W, Breslow JL. An apolipoprotein CIII haplotype protective against hypertriglyceridemia is specified by promoter and 3 untranslated region polymorphisms. Proc Natl Acad Sci USA 1993; 90: [PMID: ] 39 Russo GT, Meigs JB, Cupples LA, Demissie S, Otvos JD, Wilson PW, Lahoz C, Cucinotta D, Couture P, Mallory T, Schaefer EJ, Ordovas JM. Association of the Sst-I polymorphism at the APOC3 gene locus with variations in lipid levels, lipoprotein subclass profiles and coronary heart disease risk: the Framingham offspring study. Atherosclerosis 2001; 158: [PMID: ] 40 Beymer C, Kowdley KV, Larson A, Edmonson P, Dellinger EP, Flum DR. Prevalence and predictors of asymptomatic liver disease in patients undergoing gastric bypass surgery. Arch Surg 2003; 138: [PMID: ] 41 Pirgon Ö, Bilgin H, Çekmez F, Kurku H, Dündar BN. Association between insulin resistance and oxidative stress parameters in obese adolescents with non-alcoholic fatty liver disease. J Clin Res Pediatr Endocrinol 2013; 5: [PMID: ] 42 Musso G, Gambino R, Cassader M. Recent insights into hepatic lipid metabolism in non-alcoholic fatty liver disease (NAFLD). Prog Lipid Res 2009; 48: 1-26 [PMID: DOI: /j.plipres ] 43 Pinchuk I, Shoval H, Dotan Y, Lichtenberg D. Evaluation of antioxidants: scope, limitations and relevance of assays. Chem Phys Lipids 2012; 165: [PMID: DOI: /j.chemphyslip ] P- Reviewers: Assy N, Maleki I, Pirola CJ, Sookoian S, Zhang SJ S- Editor: Wen LL L- Editor: Wang TQ E- Editor: Zhang DN 3662 April 7, 2014 Volume 20 Issue 13

288 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. EVIDENCE-BASED MEDICINE Psychosocial issues in evidence-based guidelines on inflammatory bowel diseases: A review Winfried Häuser, Gabriele Moser, Petra Klose, Antonina Mikocka-Walus Winfried Häuser, Department of Internal Medicine Ⅰ, Klinikum Saarbrücken, D Saarbrücken, Germany Winfried Häuser, Department Psychosomatic Medicine and Psychotherapy, Technische Universität München, D München, Germany Gabriele Moser, Department of Internal Medicine Ⅲ, Division of Gastroenterology and Hepatology, Medical University of Vienna, 1090 Vienna, Austria Petra Klose, Department Internal Medicine Ⅴ (Integrative Medicine), Kliniken Essen-Mitte, Essen, D Essen, Germany Antonina Mikocka-Walus, Department of Health Sciences, University of York, Heslington, York, YO105DD, United Kingdom Author contributions: Häuser W and Klose P performed the search of the literature; Häuser W designed the study and prepared the first draft of the manuscript; all authors were involved in extracting data and in editing the manuscript; all authors provided feedback on drafts and approved the final version of the manuscript. Correspondence to: Winfried Häuser, MD, Department of Internal Medicine Ⅰ, Klinikum Saarbrücken, Winterberg 1, D Saarbrücken, Germany. whaeuser@klinikum-saarbruecken.de Telephone: Fax: Received: August 26, 2013 Revised: January 27, 2014 Accepted: February 26, 2014 Published online: April 7, 2014 Abstract AIM: To study statements and recommendations on psychosocial issues as presented in international evidence-based guidelines on the management of inflammatory bowel diseases (IBD). METHODS: MEDLINE, guidelines International Network, National Guideline Clearing House and National Institute for Health and Care Excellence were searched from January 2006 to June 30, 2013 for evidencebased guidelines on the management of IBD. RESULTS: The search yielded 364 hits. Thirteen guidelines were included in the review, of which three were prepared in Asia, eight in Europe and two in the United States. Eleven guidelines made statements and recommendations on psychosocial issues. The guidelines were concordant in that mental health disorders and stress do not contribute to the aetiology of IBD, but that they can influence its course. It was recommended that IBD-patients should be screened for psychological distress. If indicated, psychotherapy and/or psychopharmacological therapy should be recommended. IBDcentres should collaborate with mental health care specialists. Tobacco smoking patients with Crohn s disease should be advised to quit. CONCLUSION: Patients and mental health specialists should be able to participate in future guideline groups to contribute to establishing recommendations on psychosocial issues in IBD. Future guidelines should acknowledge the presence of psychosocial problems in IBD-patients and encourage screening for psychological distress Baishideng Publishing Group Co., Limited. All rights reserved. Key words: anxiety; depression; guidelines; inflammatory bowel diseases; Psychological; Psychotherapy; Smoking; Stress Core tip: A search of the literature found 13 evidencebased guidelines on the management of inflammatory bowel diseases (IBD). Three guidelines were prepared in Asia, eight in Europe and two in the United States. Mental health care specialists participated in establishing six guidelines and representatives of patient support groups participated in establishing seven guidelines. Eleven guidelines made statements and recommendations on psychosocial issues. The guidelines were concordant in that mental health disorders and stress do not contribute to the aetiology of IBD, but they can influence its course. IBD-patients should be screened for psychological distress. If indicated, 3663 April 7, 2014 Volume 20 Issue 13

289 Häuser W et al. Psychosocial issues in IBD-guidelines psychotherapy and/or psychopharmacological therapy should be recommended. Häuser W, Moser G, Klose P, Mikocka-Walus A. Psychosocial issues in evidence-based guidelines on inflammatory bowel diseases: A review. World J Gastroenterol 2014; 20(13): Available from: URL: v20/i13/3663.htm DOI: i INTRODUCTION Psychosocial issues are a significant dimension of inflammatory bowel diseases (IBD), chronic conditions of the gastrointestinal tract with an unknown aetiology and unpredictable course. Two main subtypes of IBD are Crohn s disease (CD) and ulcerative colitis (UC). A systematic review of qualitative studies exploring the phenomenon of living with IBD demonstrated a highly negative impact of the disease on the overall quality of life [1]. In the European Federation of Crohn s and Ulcerative Colitis Associations (EFCCA) patient survey (n = 5576 participants), 75% of patients reported that symptoms affected their ability to enjoy leisure activities and 69% felt that symptoms affected their ability to perform at work [2]. In a survey of the French association of IBD patients (n = 1663), 12% of IBD patients reported depression and 41% reported anxiety [3]. Other studies reported that as many as 75% of IBD patients believe that stress is a major contributor to the development of their disease and up to 90% of patients believe that stress triggers flares of their disease [4-6]. In the recent study, 30% of patients (of n = 302) expressed the need for psychological interventions [7] while in another paper it was demonstrated that 60% of IBD patients did not receive adequate help for their psychological problems [8]. The importance of psychosocial issues as perceived by individual patients is also supported by cohort studies with IBD populations. There is a strong evidence for the high comorbidity of anxiety and depression with IBD, indicating also that the symptoms of these mental health conditions are more severe during periods of active IBD and that the course of the disease worsens in depressed patients [9]. Health-related quality life in IBD-patients is not only negatively affected by disease activity but also by depression [10] and psychological distress has been found to be associated with non-adherence to medical treatment [11]. A recent systematic review demonstrated that 13 of 18 prospective studies reported significant relationships between stress and adverse outcomes in IBD [12]. Evidence-based guidelines gain growing recognition in providing patients and physicians with recommendations on diagnosis and treatment of the most frequent clinical situations based on the best available knowledge. Given the importance of psychosocial issues in IBD for the disease prognosis and management, this systematic review aims to assess the role of psychosocial issues in the course of IBD and explore the recommendations on the diagnosis and treatment of psychosocial problems as presented in the recent international evidence-based guidelines on the management of IBD. MATERIALS AND METHODS Protocol Methods of the analysis and the inclusion criteria for the present study were specified in advance. The review followed the recommendations of the Cochrane Collaboration for systematic reviews of systematic reviews [13]. Eligibility criteria Types of guidelines: We selected evidence-based guidelines on the management of IBD (UC and/or CD) which included: (1) a systematic search and analysis of the literature; and (2) a structured consensus (informal and/or Delphi process and/or consensus development conference and/or nominal group technique) to build recommendations. We excluded evidence-based guidelines which: (1) focussed on single diagnostic (e.g., screening for colorectal cancer) or therapeutic procedures (e.g., biologicals) or special situations (e.g., severe ulcerative colitis); and (2) were only based on expert consensus and/or a narrative search of the literature. Types of outcome measures: We included any statements and recommendations on psychosocial issues in IBD. The following psychosocial issues were considered to be relevant: psychological distress (anxiety, depression), stress (daily hassles, major life events), coping, healthrelated quality of life, psychological diagnostics, psychological therapies, psychotherapy, psychopharmacological therapies. We assessed whether the evidence level (EL) of statements or recommendations was reported by the guidelines. Data sources and searches The electronic bibliographic databases screened included MEDLINE, guidelines International Network, National Guideline Clearing House and National Institute for Health and Care Excellence (NICE) from January 2006 to June 30, We did not search for guidelines published before 2006, because evidence-based guidelines need to be updated regularly and this review was concerned with the most up-to-date evidence. The search strategy for MEDLINE followed the recommendation of the Association of the Medical Scientific Societies in Germany to identify evidence-based guidelines [14]. The search strategy for PubMed is outlined in Table 1. The search strategy was adapted for each database if necessary. No language restrictions were made. In addition, reference sections of evidence-based guidelines on the management of IBD were screened manually April 7, 2014 Volume 20 Issue 13

290 Häuser W et al. Psychosocial issues in IBD-guidelines Identification Records identified through database searching (n = 364) G-I-N: 19 NGC: 45 NICE: 40 PubMed: 260 Additional records identified through other sources (n = 0) Screening Records after duplicates removed (n = 150) Records screened (n = 150) Records excluded (n = 137) Eligibility Full-text articles assessed for eligibility (n = 13) Full-text articles excluded, with reasons (n = 0) Included Studies included in qualitative synthesis (n =13) Figure 1 Preferred reporting items for systematic reviews and meta-analyses flow diagram. G-I-N: Guidelines International Network; NGC: National Guideline Clearing House; NICE: National Institute for Health and Care Excellence. Table 1 Search strategy used for PubMed To locate guidelines 1 "Practice guidelines as topic"[mesh] 2 "Practice guideline"[publication type] 3 "Consensus development conferences as topic"[mesh] 4 "Consensus development conferences, NIH as topic"[mesh] 5 "Consensus development conference, NIH "[Publication type] 6 "Consensus development conference "[Publication type] 7 or 1-6 To locate IBD 8 Inflammatory bowel disease [MESH] 9 Colitis, ulcerative [MESH] 10 Crohn Disease [MESH] 11 or , 7 and 11 IBD: Inflammatory bowel diseases. Guidelines selection Two authors independently screened the titles and abstracts of potentially eligible studies identified by the search strategy detailed above (WH, AMW). The full text articles were then examined independently by two authors to determine if they met the inclusion criteria (WH, AMW). Discrepancies were rechecked and consensus achieved by discussion. If needed, a third author reviewed the data to reach a consensus (GM). Data collection process Two authors independently extracted the data using standard extraction forms (WH, AMW). Discrepancies were rechecked and consensus achieved by discussion. If needed, a third author reviewed the data to reach a consensus (GM). RESULTS The search of the literature yielded 364 hits. One hit (guideline of the Finnish Society of Gastroenterology) was not available by libraries or the website of the Finnish Society of Gastroenterology. Thirteen guidelines were included into the review after screening of full papers (Figure 1). Three guidelines were prepared in Asia, eight in Europe and two in the United States. Three guidelines were on IBD and five each on UC and CD. The composition of the guidelines groups are outlined in Table 2. The methods used in the guideline process are outlined in Table 3. We present the statements and recommendations of the guidelines in the chronological order. NICE guidelines on UC published in 2013 gave two recommendations on information provided to patients which should include impact of IBD on sexual functions, lifestyle, psychological wellbeing and sources of support and advice to be given before and after surgery (no EL reported). The NICE guidelines did not comment on psychological therapies in the summary of the topics which the guideline does not cover [15]. European Crohn s Colitis Organization (ECCO) guidelines on UC published in 2013 included three statements on the impact of psychosocial factors on the disease, three recommendations on psychosocial diagnostics and two statements on psychological treatment (Table 4) [16] April 7, 2014 Volume 20 Issue 13

291 Häuser W et al. Psychosocial issues in IBD-guidelines Table 2 Overview of the methods of guidelines on the management of inflammatory bowel diseases: composition of guideline panel Organization Country/continent Year Type of IBD Guideline group (number of persons, specialties) Asia Pacific Working Group on inflammatory bowel diseases Asia 2013 IBD Number not reported: Gastroenterologist, pathologist, colorectal surgeon, pharmacist, nurse, patient support group representative NICE United Kingdom 2013 UC 13: Gastroenterologist, surgeon, pharmacist, nurse, general practitioner, psychiatrist, patient support group representative ECCO Europe 2013 UC 35: Gastroenterologist, surgeon, psychosomatic medicine Japanese Society of Gastroenterology Japan 2013 CD 18: Gastroenterologist, surgeon, general internal medicine NICE United Kingdom 2012 CD 19: Gastroenterologist, colorectal surgeon, pharmacist, nurse, dietician, health economist, patient support group representative British Society of Gastroenterology United Kingdom 2011 IBD 11: Gastroenterologist, surgeon, pediatrist Asia Pacific Working Group on IBD Asia and Australia 2010 UC 28: Gastroenterologist, colorectal surgeon, pathologist, pharmacist, nurse practitioners, patient support group representatives German Society of Gastroenterology Germany 2011 UC 71: Gastroenterologist, colorectal surgeon, pathologist, dietician, psychosomatic medicine, patient support group representative Dutch Society for Gastroenteroolgy 2010 Number not reported: Gastroenterologist, general practitioners, psychologist, internist, dietician, pharmacist, occupational medicine, surgery, gynecologist, pathologist, radiologist, nurses, patients American College of Gastroenterology USA 2010 UC No detailed information American College of Gastroenterology USA 2010 CD 30: No detailed information German Society of Gastroenterology Germany 2011 CD 49: Gastroenterologist, colorectal surgeon, pathologist, dietician, psychosomatic medicine, patient support group representative ECCO Europe 2007 CD 25: Gastroenterologist, surgeon, psychosomatic medicine IBD: Inflammatory bowel diseases; ECCO: European Crohn s Colitis Organization; NICE: National Institute for Health and Care Excellence; CD: Crohn s disease; UC: Ulcerative colitis. The Japanese guidelines on CD published in 2013 made one statement on HRQOL and gave three recommendations on life style modification: (1) in the active phase of the disease, patients face restrictions of social activities, such as school or work, due to treatment or hospitalization (no EL reported); (2) upon diagnosis, the patient with CD should quit smoking (EL3); (3) patients should be advised to avoid irregular lifestyle and eating habits, and to refrain from excessive alcohol drinking (no EL reported); and (4) patients should be advised to adopt a lifestyle without excessive mental stress, to have as little stress as possible (no EL reported) [17]. NICE guidelines on CD published in 2012 gave two recommendations on treatment decisions including psychosocial issues. If appropriate, age-appropriate multidisciplinary (gastroenterologist, surgeon, IBD-nurse specialist) support to deal with any concerns about the disease and its treatment, including concerns about body image, living with a chronic illness, and attending school and higher education should be offered. If appropriate, information, advice and support in line with published NICE guidance on smoking cessation and patient experience should be given. No levels of evidence were reported for these recommendations. The guidelines did mention psychological therapies in the summary of the topics which the guideline does not cover. However, mental health specialists were not mentioned in the context of the multidisciplinary team [18]. British Society of Gastroenterology Guidelines for the management of inflammatory bowel disease in adults published in 2011 included two statements and three recommendations on psychosocial issues (table 5) [19]. The German Society of Gastroenterology guidelines on UC published in 2011 included three statements on the impact of psychosocial factors on the disease, three recommendations on psychosocial diagnostics and two statements on psychological treatment (table 6) [20]. The Asia-Pacific consensus statements on UC published in 2010 did not include any statement or recommendations on psychosocial issues [21]. The American College of Gastroenterology ulcerative colitis practice guidelines published in 2010 did not include any statements or recommendations on psychosocial issues [22]. The Dutch practice guidelines on Diagnostics and treatment of inflammatory bowel diseases for adults published in 2010 gave three statements on the impact of psychosocial factors on the disease and five statements on psychological treatment (table 7) [23]. ECCO guidelines on CD published in 2010 included three statements on the impact of psychosocial factors on the disease, three recommendations on psychosocial diagnostics and two statements on psychological treatment (table 8) [24]. The American College of Gastroenterology Crohn s disease practice guidelines published in 2009 included one statement on psychosocial issues: Although many patients (and family members) are convinced that stress is an important factor in the onset or course of illness, it has not been possible to correlate the development of disease with any psychological predisposition to or exacerbations of stressful life events. No levels of evidence were reported for this statement [25]. The German Society of Gastroenterology guidelines on CD published in 2008 included three statements on the impact of psychosocial factors on the disease, three recommendations on psychosocial diagnostics and three statements on psychological treatment (table 9) [26] April 7, 2014 Volume 20 Issue 13

292 Häuser W et al. Psychosocial issues in IBD-guidelines Table 3 Overview of the methods of guidelines on the management of inflammatory bowel diseases: databases, search and type of consensus Organization Country/ continent Year Type of IBD Databases used Searches until Type of consensus Asia Pacific Working Group on inflammatory bowel diseases Asia 2013 IBD English language publications in MEDLINE, EMBASE and the Cochrane Trials Register in human subjects. All national and international guidelines on Ulcerative Colitis were solicited NICE United Kingdom 2013 UC MEDLINE, Embase, Cinahl and the Cochrane Library Not reported 15 th November 2012 Modified Delphi process Canadian task force Grade A-E Meta-analyses, grading the quality of evidence Evidence tables Formal consensus ECCO Europe 2013 UC Medline, Central December, 2010 Delphi process and structured consensus Japanese Society of Gastroenterology Japan 2013 CD MEDLINE, the Cochrane Library, and the Japan Medical Abstracts Society NICE United Kingdom 2012 CD MEDLINE, Embase, Cinahl and The Cochrane Library British Society of Gastroenterology German Society of Gastroenterology Asia Pacific Working Group on IBD Dutch Society for Gastroenterology American College of Gastroenterology American College of Gastroenterology German Society of Gastroenterology United Kingdom 2011 IBD PubMed, Medline and the Cochrane database Up to 2007 Delphi process and structured consensus 13 th March 2012 Meta-analyses, grading the quality of evidence Evidence tables Formal consensus No details reported Informal committee consensus Germany 2011 UC Medline, Central, PsycInfo Until May 2009 Structured consensus process Asia and Australia 2010 UC English language publications in MEDLINE, EMBASE and the Cochrane Trials Register in human subjects. All national and international guidelines on Ulcerative Colitis were solicited No details reported Modified Delphi process 2010 Central, Medline, PsychInfo 2007 Modified Delphi process United States 2010 UC Medline No details reported Informal consensus United States 2010 CD Medline No details reported Informal consensus Germany 2011 CD Medline, Central, PsycInfo Until April 2007 Structured consensus process ECCO Europe 2007 CD Medline, Central No details reported Delphi process and structured consensus IBD: Inflammatory bowel diseases; ECCO: European Crohn s Colitis Organization; NICE: National Institute for Health and Care Excellence; CD: Crohn s disease; UC: Ulcerative colitis. Table 4 European Crohn s Colitis Organization guideline statements and recommendations on psychosocial issues in ulcerative colitis [15] 1 There is no conclusive evidence for anxiety, depression and psychosocial stress contributing to risk for UC onset (EL2) 2 Psychological factors may have an impact on the course of UC. Perceived psychological stress (EL2) and depression (EL2) are risk factors for relapse of the disease. Depression is associated with low health-related quality of life (EL3). Anxiety is associated with non adherence with treatment (EL4) 3 Psychological distress and mental disorder are more common in patients with active ulcerative colitis than in population-based controls, but not in patients in remission (EL3) 4 Clinicians should particularly assess depression among their patients with active disease and those with abdominal pain in remission (EL2) 5 The psychosocial consequences and health- related quality of life of patients should be taken into account in clinical practice at regular visits (EL3). Patients' disease control can be improved by combining selfmanagement and patient-centred consultations (EL1b) 6 Physicians should screen patients for anxiety, depression and need for additional psychological care and recommend psychotherapy if indicated (EL2). Patients should be informed of the existence of patient associations (EL 5) 7 Psychotherapeutic interventions are indicated for psychological disorders and low quality of life (EL1) 8 The choice of psychotherapeutic method depends on the psychological disturbance and should best be made by specialists (Psychotherapist, Specialist for Psychosomatic Medicine, Psychiatrist). Psychopharmaceuticals should be prescribed for defined indications (EL5) UC: Ulcerative colitis; EL: Evidence level April 7, 2014 Volume 20 Issue 13

293 Häuser W et al. Psychosocial issues in IBD-guidelines Table 5 British society of gastroenterology guidelines for the management of inflammatory bowel disease in adults [19] 1 The essential supporting services to which the IBD team should have access should include a psychologist/counsellor (no EL reported) 2 Stress and adverse life events do not appear to trigger the onset of Crohn s disease or ulcerative colitis, but most reports indicate that they may be involved in triggering relapse of IBD. Furthermore, behaviour limiting exposure to stressful situations is associated with reduced symptomatic relapse, at least in Crohn s disease (no EL reported) 3 Evidence indicates that psychosocial support is useful, particularly in adolescents. There is no definitive evidence that psychological interventions improve the course of IBD itself but they do usually improve patients quality of life and wellbeing (no EL reported) 4 Psychological support should be available to patients with IBD (no EL reported) IBD: Inflammatory bowel diseases; EL: Evidence level. Table 6 German guideline guideline statements and recommendations on psychosocial issues in the management of ulcerative colitis [20] 1 Adverse life events, psychological stress and mental health disorders are not aetiologically linked to the onset of UC (EL2) 2 Subjective stress and affective disorders may have a negative impact on the course of UC 3 High disease activity is associated with high psychological symptom burden EL2) 4 Mental health disorders have a negative impact on the course of the disease and quality of life EL2) 5 Patients with persistent abdominal pain or diarrhea which cannot be explained by disease activity or complications of the disease should be assessed for irritable bowel syndrome (IBS) or depressive disorder. If IBS or depressive disorder is diagnosed, these disorders should be treated according to guideline recommendations (EL2) 6 Psychosocial co-morbidities and health- related quality of life (accounting for gender differences) should be taken into account in clinical practice at regular visits (EL2) 7 Care should involve cooperations with specialists in psychotherapy or psychosomatic medicine (EL2) 8 Physicians should inform patients about IBD self-help organisations (EL5) 9 In the case of a mental health disorder, psychotherapy is recommended (EL2) 10 Psychosocial support should be offered to children and adolescents (EL1) UC: Ulcerative colitis; EL: Evidence level. Table 7 Summary of the dutch national practice guidelines on the management of inflammatory bowel diseases [23] 1 Personality traits do not contribute to the aetiology of IBD 1 2 Psychosocial factors such as stress, depression/anxiety and coping have an impact on the course of IBD 1 3 Health-related quality of life is influenced by disease activity but also by stress, anxiety/depression, social support and quality of treatment 1 4 A positive relationship between patients and health care professionals characterized by mutual respect, communication, education and emotional support for patients and families is recommended 1 5 Psychosocial problems associated with the disease should be treated by psychological interventions, e.g., stress management training, selfempowerment, cognitive-behavioural therapy 1 6 Anxiety and depression should be treated according to appropriate guidelines 7 IBD-patients who smoke should be advised to quit smoking 1 1 No level of evidence reported. IBD: Inflammatory bowel diseases. Table 8 European crohn s colitis organization guideline statements and recommendations on psychosocial issues in Crohn s disease [24] 1 Psychological disturbances seem to be a consequence of the illness rather than the cause or specific to Crohn s disease (EL1). The degree of psychological distress correlates with the disease severity (EL2) 2 An association between psychological factors and the aetiology of Crohn s disease is unproven (EL3), but there is a moderate influence on the course of the disease (EL1) 3 Depression and perceived chronic distress seem to represent further risk factors for relapse of the disease (EL1). It remains unclear whether acute life events trigger relapses (EL1). Most patients consider stress to have an influence on their illness (EL2) 4 Physicians should assess the patient s psychosocial status and request additional psychological care and psychotherapy if indicated. Integrated psychosomatic care should be provided in IBD centres (EL2) 5 Patients should be informed of the existence of patient associations (EL5) 6 The psychosocial consequences and health related quality of life of patients should be taken into account in clinical practice at regular visits (EL1) 7 Psychotherapeutic interventions are indicated for psychological disorders, such as depression, anxiety, reduced quality of life with psychological distress, as well as maladaptive coping with the illness (EL1) 8 The choice of psychotherapeutic method depends on the psychological disturbance and should best be made by specialists (psychotherapist, specialist in psychosomatic medicine, psychiatrist). Psycho-pharmaceuticals should be prescribed for defined indications (EL5) EL: Evidence level; IBD: Inflammatory bowel diseases April 7, 2014 Volume 20 Issue 13

294 Häuser W et al. Psychosocial issues in IBD-guidelines Table 9 German guideline statements and recommendations on psychosocial issues in the management of Crohn s disease [26] 1 Mental health disorders are the sequelae rather than the cause of CD. Psychological distress is correlated with disease activity and has an impact on health-related quality of life and course of the disease (EL2) 2 Psychosocial factors (personality traits, adverse life events, daily hassles) do not contribute to the etiology of CD (EL4) 3 Depression, anxiety and perceived stress are risk factors for flares. It is not certain whether acute adverse life events trigger flares (EL3). Most patients believe that stress has an impact on the disease (EL4) 4 The psychosocial consequences and health- related quality of life of patients (accounting for gender differences) should be taken into account in clinical practice at regular visits (EL5) 5 Physcians should assess the psychosocial status of patients and their need for psychotherapy. If indicated, psychotherapy should be provided. IBDcenters should provide an integrated psychosomatic care EL2) 6 Patients should be informed on the existence of patient organisations and selfhelp groups (EL5) 7 Psychotherapy is indicated in the case of mental health disorders such as anxiety and depression, reduced health-related quality of life with psychological distress and maladaptive coping (EL2) 8 The choice of a psychotherapeutic method depends on the psychological disturbance and should best be made by a psychotherapist. The choice of psychopharmacotherapy should be at the discretion of a psychiatrist specialist in the case of comorbid mental health disorders (e.g., depression and anxiety) (EL5) 9 The increased risk of a mental health disorder is associated with an early manifestation of the disease. If needed, psychosocial support should be offered to the patient and their families (EL5) 10 Patients using tobacco products should be encouraged to quit smoking (EL2) CD: Crohn s disease; EL: Evidence level. DISCUSSION There is a growing body of evidence demonstrating the role of stress, anxiety and depression in IBD presentation and progression [9,12,27,28]. Evidence-based guidelines are increasingly becoming the major source of recommendations on diagnosis and treatment not only in IBD but also in other conditions and that is why it is important that problems of clinical significance (such as the role of psychosocial issues in IBD management) are included and commented on in the guidelines. This is the first review which has summarised current guidelines on the management of IBD with respect to psychosocial issues. The most striking observation arising from this review was that as many as 10 out of 13 guidelines provided comments on the psychosocial issues demonstrating the recognition of their importance in IBD management. However, the published guidelines differed in the inclusion of psychosocial topics, particularly between Asia/ Pacific, Europe and the United States, with European guidelines more likely to discuss the issue. In the cases where psychosocial issues were included in the guidelines, the statements and recommendations were similar between the countries. ECCO [16,24], Dutch [23] and German guidelines [20,26] made the most extensive statements and recommendations on psychosocial issues. In contrast, three guidelines [15,21,22] did not include any statements or recommendations on psychosocial issues. Seven guidelines included patient representatives [15,17,18,20,21,26]. However, the composition of the guidelines group (i.e., types of professionals involved and/or inclusion of patients) did not affect the role of psychosocial issues in the guideline. Importantly, only six guidelines included comments regarding mental health specialists [15,16,20,23,24,26]. The major statements and recommendations on psychosocial issues were as follows: (1) psychosocial factors such as mental disorders or stress do not play a role in the aetiology of IBD [15,19,20,23-26] ; (2) psychosocial factors such as mental health disorders or stress may have a negative impact on the course of IBD [15,19,20,23,24,26] ; (3) physicians should screen IBD-patients for psychological distress and the psychological care recommended when required [15,19,24,26] ; (4) if indicated, psychosocial care (psychotherapy, psychopharmacotherapy) should be offered [15,19,20,23,24,26] ; (5) IBD-centres should collaborate with mental health specialists [15,19,20,24,26] ; and (6) smoking CDpatients should be advised to quit [17,18,23,26]. The major statements and recommendations included in the guidelines were in line with the recent systematic reviews demonstrating that the role of psychological distress and personality as predisposing factors for the development of IBD remains controversial. Attempts to investigate the role of psychological factors in IBD exhibited rather conflicting results. Among the studies concerning the effects of stress or depression on the course of IBD, the majority suggest that stress worsens IBD, the rest produce either negative or inconclusive results [29]. Eighteen randomized controlled studies (19 papers) were included in a review on psychological therapies in IBD. Psychotherapy was found to have minimal effect on measures of anxiety, depression, quality of life and disease progression although showed promise in reducing pain, fatigue, relapse rate and hospitalisation, and improving medication adherence. It may also be cost effective [30]. Smoking remains the most important environmental factor in IBD. Active smoking increases the risk of developing CD. Moreover, CD patients who start or continue smoking after disease diagnosis are at risk for poorer outcomes such as higher therapeutic requirements and disease-related complications, as compared to those patients who quit smoking or who never smoked [31]. Psychosocial issues are discussed in the majority (10/13) of recently published evidence-based guidelines, supporting their important role in IBD management. There is a need for interdisciplinary evidence-based guidelines in Asia and the United States to provide spe April 7, 2014 Volume 20 Issue 13

295 Häuser W et al. Psychosocial issues in IBD-guidelines cific recommendations on the aetiology and management of psychosocial issues in IBD. Patients and mental health specialists should be able to participate in guideline groups to contribute perspectives on psychosocial issues to the guidelines. There is strong evidence on co-morbidity of IBD with mental disorders which have been shown to impact the disease course. Future guidelines should acknowledge the presence of psychosocial problems in IBD-patients and encourage screening for psychological distress. The best treatment of psychosocial problems of IBD-patients is yet to be determined. REFERENCES 1 Kemp K, Griffiths J, Campbell S, Lovell K. An exploration of the follow-up up needs of patients with inflammatory bowel disease. J Crohns Colitis 2013; 7: e386-e395 [PMID: DOI: /j.crohns ] 2 Ghosh S, Mitchell R. 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296 Häuser W et al. Psychosocial issues in IBD-guidelines cal care]. Ned Tijdschr Geneeskd 2010; 154: A1900 [PMID: ] 24 Dignass A, Van Assche G, Lindsay JO, Lémann M, Söderholm J, Colombel JF, Danese S, D Hoore A, Gassull M, Gomollón F, Hommes DW, Michetti P, O Morain C, Oresland T, Windsor A, Stange EF, Travis SP. The second European evidence-based Consensus on the diagnosis and management of Crohn s disease: Current management. J Crohns Colitis 2010; 4: [PMID: DOI: / j.crohns ] 25 Lichtenstein GR, Hanauer SB, Sandborn WJ, Practice Parameters Committee of American College of Gastroenterology. Management of Crohn's disease in adults. Am J Gastroenterol 2009; 104: ; quiz 464, 484 [PMID: DOI: /ajg ] 26 Hoffmann JC, Preiss JC, Autschbach F, Buhr HJ, Häuser W, Herrlinger K, Höhne W, Koletzko S, Krieglstein CF, Kruis W, Matthes H, Moser G, Reinshagen M, Rogler G, Schreiber S, Schreyer AG, Sido B, Siegmund B, Stallmach A, Bokemeyer B, Stange EF, Zeitz M. [Clinical practice guideline on diagnosis and treatment of Crohn s disease]. Z Gastroenterol 2008; 46: [PMID: DOI: / s ] 27 Bernstein CN, Singh S, Graff LA, Walker JR, Miller N, Cheang M. A prospective population-based study of triggers of symptomatic flares in IBD. Am J Gastroenterol 2010; 105: [PMID: DOI: /ajg ] 28 Langhorst J, Hofstetter A, Wolfe F, Häuser W. Short-term stress, but not mucosal healing nor depression was predictive for the risk of relapse in patients with ulcerative colitis: a prospective 12-month follow-up study. Inflamm Bowel Dis 2013; 19: [PMID: ] 29 Triantafillidis JK, Merikas E, Gikas A. Psychological factors and stress in inflammatory bowel disease. Expert Rev Gastroenterol Hepatol 2013; 7: [PMID: DOI: /egh.13.4] 30 McCombie AM, Mulder RT, Gearry RB. Psychotherapy for inflammatory bowel disease: a review and update. J Crohns Colitis 2013; 7: [PMID: DOI: / j.crohns ] 31 Nos P, Domènech E. Management of Crohn s disease in smokers: is an alternative approach necessary? World J Gastroenterol 2011; 17: [PMID: DOI: / wjg.v17.i ] P- Reviewers: Lakatos PL, Ohkusa T, Rocha R, Torres MI, Zippi M S- Editor: Ma YJ L- Editor: A E- Editor: Ma S 3671 April 7, 2014 Volume 20 Issue 13

297 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. Transcription factor ERG is a specific and sensitive diagnostic marker for hepatic angiosarcoma RETROSPECTIVE STUDY Zhan-Bo Wang, Jing Yuan, Wei Chen, Li-Xin Wei Zhan-Bo Wang, Jing Yuan, Wei Chen, Li-Xin Wei, Department of Pathology, Chinese People s Liberation Army General Hospital, Beijing , China Author contributions: Wang ZB and Yuan J contributed equally to this work and are co-first authors on this study; Wang ZB, Yuan J and Wei LX designed the research; Wang ZB, Yuan J and Chen W performed the research; Wang ZB and Yuan J analyzed the data; Wang ZB and Yuan J wrote the paper; Wang ZB, Yuan J and Wei LX revised the manuscript; all authors read and approved the final manuscript. Correspondence to: Li-Xin Wei, MD, Professor, Department of Pathology, Chinese People s Liberation Army General Hospital, 28 Fuxing Road, Haidian, Beijing , China. weilx301@126.com Telephone: Fax: Received: December 4, 2013 Revised: February 9, 2014 Accepted: March 8, 2014 Published online: April 7, 2014 Abstract AIM: To investigate the expression of ERG, CD34, CD31 (PECAM-1, platelet/endothelial cell adhesion molecule 1) and factor Ⅷ-related antigen (FⅧRAg) in the diagnosis of hepatic angiosarcoma patients. METHODS: Patient samples were collected from January 1986 to December 2012 from the People s Liberation Army General Hospital in Beijing, China. We obtained twenty-four samples of hepatic angiosarcoma (HAS) that were confirmed by two pathologist. The samples were the result of three autopsy cases, eight biopsy cases and 13 patients who underwent surgical tumor removal. The HAS cases accounted for 2.23% (24/1075) of all hepatic vascular tumors at the hospital during the same time period. Patient histories including age, gender, clinical manifestations, medical treatments, laboratory tests, radiological images, histological observations and outcomes for each case were analyzed in detail. All samples were evaluated histologically with hematoxylin and eosin staining. Using immunohistochemistry, the expression and localization of ERG was examined in all HAS specimens and compared to the known endothelial markers CD34, CD31 and FⅧRAg. The endothelial markers were also evaluated in a panel of non-has tumors. RESULTS: This cohort of 24 HAS cases is, to the best of our knowledge, currently the largest cohort in the world in the publicly available literature. Hepatic angiosarcoma tissue samples were obtained from 14 males and 10 females with a mean age of 50.6 years (range: 7-86 years). The patients presented with the following clinical manifestations: abdominal pain (16/24), back pain (3/24), heart palpitations (1/20), cough (1/24) or no clinical symptoms (3/24). Tumors were predominantly localized in the right hepatic lobe (15/24) or left hepatic lobe (6/24), or a diffuse growth on the right and left hepatic lobes (3/24). Eleven patients underwent surgical resection (45.8%), two patients received a liver transplant (8.3%), eight patients received interventional therapy (33.3%) and three patients received no treatment (lesions discovered at autopsy, 12.5%). Postoperative follow-up of patients revealed that 87.5% (21/24) of patients had died and three cases were not able to be tracked. In all cases, the mean survival time was 12.1 mo. While 100% of the HAS samples were positive for ERG expression, expression of the other markers was more variable. CD31 was expressed in 79.2% (19/24) of samples, CD34 was expressed in 87.5% (21/24) of samples and FⅧRAg was expressed in 41.7% (10/24) of samples. CONCLUSION: ERG is a more sensitive and specific diagnostic marker for hepatic angiosarcoma in comparison to CD31, CD34 and FⅧRAg Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Liver; Angiosarcoma; ERG; Immunohisto April 7, 2014 Volume 20 Issue 13

298 Wang ZB et al. ERG and hepatic angiosarcoma diagnosis chemistry; Diagnosis Core tip: Hepatic angiosarcoma (HAS) is a rare disease and formal therapeutic guidelines have not been established. A method to detect HAS early using molecular markers would improve patient survival. Here, we have assembled the largest cohort to date of 24 HAS cases to determine if ERG, CD31, CD34 or factor Ⅷ-related antigen (FⅧRAg) represent a diagnostic marker for the disease. Expression of CD31, CD34 and FⅧRAg was variable across the 24 samples, while ERG was consistently expressed in all HAS samples. ERG showed increased sensitivity and specificity in comparison to the other markers examined and represents a novel diagnostic marker for HAS. Wang ZB, Yuan J, Chen W, Wei LX. Transcription factor ERG is a specific and sensitive diagnostic marker for hepatic angiosarcoma. World J Gastroenterol 2014; 20(13): Available from: URL: i13/3672.htm DOI: INTRODUCTION Angiosarcoma is a malignant neoplasm that affects endothelial cells and can occur in any location throughout the body [1,2]. The location of tumor origin often permits metastases to distant sites. Although hepatic angiosarcoma (HAS) is a very rare disease and accounts for only 2% of primary liver malignancies [3], it is still the third most common primary liver malignancy [4,5]. Case-control studies have shown that approximately one-third of HAS cases appear to be caused by environmental carcinogens via exposure to thorium dioxide, arsenical insecticide or polyvinyl chloride [6]. The diagnosis of HAS is difficult, especially if the patient has no history of carcinogen exposure. Long-term survival in HAS patients is poor due to its rapid progression, high recurrence rate and resistance to traditional chemo- and radiotherapies [7-9]. Due to a high recurrence rate and poor surgical outcomes, liver transplantation as a form of therapy for HAS is no longer performed [5]. No formal guidelines exist for the treatment of HAS. Currently, the best treatment option for HAS is partial surgical resection of the liver to remove the tumor. Thus, a means to detect HAS early using specific molecular markers will provide a critical time window during which surgical resection can be performed, which will ultimately improve patient survival. CD31, CD34 and factor Ⅷ-related antigen (FⅧRAg) are expressed in endothelial cells and have been hypothesized to serve as diagnostic markers of angiosarcoma [10]. CD31, a transmembrane glycoprotein adhesion molecule, is expressed by platelets, megakaryocytes, and endothelial cells [11]. CD34 is a cell-surface marker expressed in both endothelial cells and hematopoietic stem cells [12]. FⅧRAg is expressed in Weibel-Palade bodies of endothelial cells and in megakaryocytes [13]. Although these markers are touted as diagnostic for angiosarcoma, the precise role of each of these markers in HAS remains unclear. ERG (avian v-ets erythroblastosis virus E26 oncogene homolog), a member of the ETS family of transcription factors, is expressed in endothelial cells [14]. ERG plays essential roles in the regulation of angiogenesis and apoptosis of endothelial cells [15]. Chromosomal translocations affecting ERG have been implicated in the development of several cancers [16,17]. Recent reports have demonstrated that ERG is a specific and sensitive biological marker in the diagnosis of angiosarcoma [18-20]. The diagnostic value of ERG in HAS is largely unknown and only one previous publication has documented ERG expression in a HAS patient [18]. To this end, we assembled the largest cohort to date of HAS tissues to evaluate the role of ERG expression in HAS. Moreover, we analyzed how ERG expression compares to CD31, CD34 and FⅧRAg endothelial markers. MATERIALS AND METHODS Patients All patient specimens were collected from January 1986 to December 2012 at the Chinese People s Liberation Army General Hospital in Beijing, China. We confirmed 24 cases of HAS by pathology after 13 patients underwent surgical tumor removal, eight patients were biopsied and three patients were autopsied. These cases accounted for 2.23% (24/1075) of hepatic vascular tumors during the same time period. Detailed clinical histories, including gender, age, clinical symptoms, tumor location, size, imaging data, and laboratory test results were collected from each patient s record. Follow-up information on each case was obtained through telephone conversations and hospital records to evaluate patient prognosis in November As controls, we obtained other types of liver spindle cell tumors, including hepatic epithelioid hemangioendotheliomas (EHE; n = 3), undifferentiated sarcomas (n = 3), leiomyosarcoma (n = 1), sarcomatoid carcinomas (n = 3) and gastrointestinal stromal tumor liver metastases (n = 2). Histological observations All hematoxylin and eosin-stained slides were reviewed for each case. Histological changes were evaluated by experienced pathologists using a light microscope. Immunohistochemistry Tissues were fixed in 4% formalin and embedded in paraffin for immunohistochemistry using the EnVision kit (DAKO; Denmark). The antibodies used in this study are listed in Table 1. Statistical analysis Statistical analysis was performed using the SPSS 17.0 statistical software package (SPSS Inc., Chicago, IL, United States). Cumulative survival curves were generated us April 7, 2014 Volume 20 Issue 13

299 Wang ZB et al. ERG and hepatic angiosarcoma diagnosis Table 1 Antibodies used for expression analysis in hepatic angiosarcoma samples Protein name Antibody clone Source Concentration Location Vimentin V9 Invitrogen 1:200 C CD31 Jc70A Gene Tech 1:100 M CD34 QBEnd-10 Dako 1:50 M FVIIIRAg F8/86 Dako 1:50 C ERG Ep111 Epitomics 1:100 N CK Polyclonal Dako 1:100 C GPC-3 1G12 Cell marque 1:150 C;M Hepatocyte OCH1E5 Zeta 1:100 C CD117 Polyclonal Dako 1:400 M Desmin D33 Dako 1:100 C Ki-67 MIB-1 Dako 1:200 N Cum survival Survival function Factor censored C: Cytoplasm; M: Membrane; N: Nucleus; FⅧRAg: Factor Ⅷ-related antigen; CK: Pan-cytokeratin; GPC-3: Glypican-3; CD117: C-kit protooncogene. ing the Kaplan-Meier method. The hazard ratio and 95% confidence interval were also calculated. RESULTS Clinical data of the HAS cohort The HAS tissues were obtained from 14 males and 10 females, with a mean age of 50.6 (range: 7-86) years (Table 2). The patients presented with the following clinical manifestations: abdominal pain (16/24), back pain (3/24), heart palpitations (1/20), cough (1/24) or no clinical symptoms (3/24). Tumors were located in the right lobe (15/24), left hepatic lobe (6/24) or as a diffuse growth on the right and left hepatic lobes (3/24) (Table 2). Patients underwent surgical resection (11/24), liver transplantation (2/24), interventional therapy (8/24) or they received no treatment (lesions discovered at autopsy; 3/24). Postoperative follow-up of each case showed that 18 patients had died and three cases could not be accounted for. Kaplan- Meier survival curves demonstrated that the prognosis of HAS cases was poor (Figure 1, Table 3). Laboratory tests for alpha-fetoprotein, carcinoembryonic antigen, CA19-9, CA125 and other tumor markers were normal. Two patients were positive for the hepatitis B surface antigen and one patient had abnormal hemoglobin levels (90 g/l). Radiological imaging, which included ultrasound, CT and magnetic resonance imaging, showed cystic or solid masses with varying textures and ill-defined borders (Figure 2A). Sixteen of the 24 samples were obtained via surgery or autopsy and of these, 10 were classified as single huge nodules (62.5%), two with multiple nodules (12.5%), three with single large nodules with peripheral small nodules (18.8%) and one with diffuse micronodules (6.3%). The mean tumor diameter was 9.58 cm (range: 3-27 cm). Tumor tissues were dark red in color and had a honeycomb appearance (Figure 2B). Tumor histology When tumor histology was examined, one layer or multiple layers of tumor cells lined a vascular lumen and gave t /mo Figure 1 Survival curve of hepatic angiosarcoma patients. A cumulative survival curve generated using the Kaplan-Meier method. rise to the lumen-like structure (Figure 3A and B). The tumor cells grew along the sinusoids, with the liver serving as a scaffold for tumor growth (Figure 3C). Three of the samples had overt compartments that were filled with red blood cells. The tumors predominantly contained atypical spindle cells and mitotic cells were frequently observed. Moreover, we observed significant necrosis and calcification within the tumors. Areas of epithelial differentiation were apparent in some tumors. In patients with recurrent tumors following liver transplantation, the tumors contained a large blood chamber that differed from the morphology observed in the primary lesion. Expression of ERG, CD31, CD34 and FVIIIRAg in hepatic angiosarcoma tumors To evaluate ERG, CD31, CD34 and FⅧRAg as diagnostic markers of HAS, we evaluated their expression by immunohistochemistry. ERG expression was observed in all HAS samples examined (24/24; Figure 4A). In contrast, the other three endothelial markers examined were not uniformly expressed across HAS samples. CD34, CD31, and FⅧRAg were expressed in 21/24, 19/24 and 10/24 cases, respectively (Figure 4B-D). Moreover, only one case was positive for pan-cytokeratin (CK) expression (1/24). The cell proliferation index was assayed by Ki-67 staining. In all samples, the cell proliferation index was greater than 10% and up to 60%. Desmin, c-kit protooncogene (CD117) and Glypican-3 (GPC-3) expression was absent in all samples. When control samples were evaluated for expression of the endothelial markers, all three cases of EHE tumors were positive for ERG, CD34 and CD31 expression, while only one was positive for FⅧRAg (33.3%). The undifferentiated sarcoma samples (n = 3) lacked ERG, CD31 and FⅧRAg expression and only one sample (33.3%) was positive for CD34 expression. The leiomyosarcoma sample was positive for CD34 expression, but lacked ERG, CD31 and FⅧRAg expression. Simi April 7, 2014 Volume 20 Issue 13

300 Wang ZB et al. ERG and hepatic angiosarcoma diagnosis Table 2 Clinical and pathological features of the hepatic angiosarcoma cohort No. Gender Age/yr Clinical symptoms Tumor location Maximum tumor size/cm Treatment methods Follow-up 1 1 Male 42 Abdominal discomfort Left lateral lobe of the liver 2 1 Female 30 Abdominal discomfort Left inner hepatic lobe Adjuvant therapy Spleen metastasis at treatment and died 10 mo later Adjuvant therapy Unable to follow-up after 9 mo 3 Male 60 Upper abdominal pain Right hepatic lobe Surgery Died after 12 mo 4 1 Female 58 Liver pain Left lobe of liver Adjuvant therapy Unable to follow-up after 6 mo 5 1 Female 52 Back pain Left and right hepatic lobes Adjuvant therapy Thoracic metastasis at treatment and died 8 mo later 6 Male 7 Abdominal pain, fever Right hepatic lobe Surgery Died 1 mo later 7 Female 52 Upper abdominal pain Left hepatic lobe Surgery Post-operative thoracic and lung metastases and died 31 mo later 8 Female 62 Abdominal discomfort Left hepatic lobe Surgery Post-operative lung metastases and died 34 mo later 9 1 Male 68 Upper abdominal pain Right hepatic lobe Adjuvant therapy Portal vein cancer thrombosis at diagnosis and died 9 mo later 10 Female 29 Hypo-proteinemia Left and right hepatic lobes Liver transplant Spleen metastasis at diagnosis and died during surgery 11 Male 49 Asymptomatic Right hepatic lobe Surgery Bone metastases and died 43 mo later 12 Female 62 Upper abdominal pain Right hepatic lobe Liver Transplant Recurrence of tumor in the transplanted liver and died 4 mo later 13 Female 32 Asymptomatic Right hepatic lobe Surgery Died 3 mo later 14 Female 51 Upper abdominal pain Right hepatic lobe Surgery Died 8 mo later 15 Male 53 Upper abdominal pain Left hepatic lobe Surgery Recurrence after 4 mo and died 7 mo later 16 Male 49 Upper abdominal pain Right hepatic lobe Surgery Died 10 mo later 17 1 Male 66 Back pain Right hepatic lobe Adjuvant therapy Thoracic metastasis at diagnosis and died 5 mo later 18 Male 86 Upper abdominal pain Left and right hepatic lobes Autopsy findings without treatment 19 Male 86 Upper abdominal pain Right hepatic lobe Autopsy findings without treatment 20 Male 70 Palpitations Right hepatic lobe Autopsy findings without treatment Died due to tumor rupture Tumor metastasis and died due to heart failure Tumor metastasis and died due to heart failure 21 1 Female 45 Cough Right hepatic lobe Adjuvant therapy Lung metastasis at diagnosis and died 6 mo later 22 Male 21 Right shoulder pain Right hepatic lobe Surgery Tumor recurrence and died 8 mo later 23 1 Male 21 Upper abdominal pain Right hepatic lobe Adjuvant therapy Unable to follow-up after 10 mo 24 Male 63 Upper abdominal pain Right hepatic lobe Surgery Tumor recurrence after 18 mo and died 21 mo later 1 Patients who received percutaneous biopsies. A B Figure 2 Magnetic resonance imaging and whole-mount images of hepatic angiosarcoma. A: Magnetic resonance imaging of a hepatic angiosarcoma (HAS) patient showed a significantly increased volume in the left lobe of the liver and an abnormal signal shadow with heterogeneous enhancement; B: The HAS tumor showed honeycomb morphology with an ill-defined border. larly, only one sarcomatoid carcinoma (33.3%) showed CD34 expression while ERG, CD31 and FⅧRAg were absent. Both gastrointestinal stromal tumor liver metastases were positive for CD34, but lacked ERG, CD31 and 3675 April 7, 2014 Volume 20 Issue 13

301 Wang ZB et al. ERG and hepatic angiosarcoma diagnosis A B C Figure 3 Analysis of hepatic angiosarcoma histology with hematoxylin and eosin staining. A: The tumor was similar in appearance to a cavernous hemangioma on the inside and was lined by endothelial cells of similar shapes ( 200); B: Endothelial cells are distended with a spindle or polygonal morphology. Frequent mitotic events (indicated by arrow) and a pale eosinophilic cytoplasm were also observed ( 300); C: Tumor cells were observed growing around the vascular lumen and formed a vascular lumen-like structure ( 300). A B C D Figure 4 Expression of transcription factor ERG, CD31 (platelet/endothelial cell adhesion molecule 1), CD34 and factor Ⅷ-related antigen in hepatic angiosarcoma tissues. A: Positive expression was observed for nuclear ERG expression in hepatic angiosarcoma (HAS) ( 300); B: Membranous CD34 staining in HAS ( 300); C: Membranous CD31 staining in HAS; D: Cytoplasmic factor Ⅷ-related antigen in hepatic angiosarcoma staining in HAS ( 300) April 7, 2014 Volume 20 Issue 13

302 Wang ZB et al. ERG and hepatic angiosarcoma diagnosis Table 3 Means for survival time Factor 1 Mean 1 FⅧRAg expression. DISCUSSION Estimate Std. error 95%CI Lower bound Upper bound Estimation was limited to the largest survival time if it is censored. Primary sarcomas of the liver are rare. The most common hepatic sarcomas are HAS, embryonal sarcoma, leiomyosarcoma, EHE, fibrosarcoma and malignant fibrous histiocytoma [21]. HAS is primarily observed in the elderly and often presents with nonspecific symptoms such discomfort or distension of the abdomen, weight loss or fatigue, which makes the diagnosis difficult [3]. HAS is frequently caused by exposure to carcinogens such as thorium dioxide, arsenical insecticide or polyvinyl chloride [6,22-26]. However, in all the Chinese patients that comprised our cohort, carcinogen exposure was not an important factor in disease onset. Our data are consistent with reports on HAS development from Taiwan and South Korea [27,28]. To better address the role of carcinogens and HAS in China, an approach involving a survey of multiple medical centers across the country would be beneficial. Infection from hepatitis B virus (HBV) has also been implicated as a risk factor for HAS. In our study, the frequency of HBV infection was no different to that in the normal, healthy Chinese population, indicating that HBV infection did not play a role in HAS onset [29,30]. HAS often appears as a single mass when viewed histologically, however, multiple masses have also been observed and HAS may affect the whole liver. In the clinic, four primary patterns of disease have been observed: (1) multiple nodules; (2) a large dominant mass; (3) a dominant mass combined with multiple nodules; and (4) diffuse micronodular infiltration of the liver, although this is observed less frequently [31]. In the HAS patient cohort, a large dominant mass was most frequently observed (62.5%). The remaining patients had a dominant mass and multiple nodules (18.8%), multiple nodules (12.5%) or diffuse micronodular infiltration of the liver (6.3%). When HAS samples are viewed by microscopy, the tumor cells are often mitotic, have a spindle or polygonal morphology and may be in a single layer or multi-layered surrounding a vascular source, connected with thick hepatic cables. HAS tumor cells have the ability to form papillary structures and may show epithelial differentiation. HAS patient survival is currently very poor, with a median survival of 6 mo without treatment. Even with treatment, only 3% of patients are reported to survive longer than 2 years [5]. Poor prognosis of HAS patients is primarily due to early metastases and an extended time between disease onset and correct diagnosis. Surgical resection is currently the standard treatment for HAS. Early diagnosis of HAS is required for surgical resection to be beneficial to the patient. Other treatments such as chemotherapy or radiotherapy have not shown a conclusive survival benefit in HAS patients [7-9]. Thus, the identification of specific and sensitive biomarkers to diagnose HAS would significantly improve patient outcome. Here, we have assembled the largest HAS cohort to date and provide evidence that ERG is a specific diagnostic marker for HAS. ERG regulates angiogenesis and differentiation of embryonic stem cells into endothelial cells. Chromosomal translocations affecting ERG expression have been shown to play a role in the development of several cancers, including prostate cancer [16,17]. The diagnostic value of ERG in HAS, however, has not been evaluated. Here, we have shown that ERG expression was detected in all HAS cases examined. While three cases of EHE, one of our controls, also showed positive expression of ERG, the remaining controls-undifferentiated sarcoma, sarcomatoid carcinoma and gastrointestinal stromal tumor liver metastases-were negative for ERG expression. These results suggest that ERG expression is relatively specific to HAS tumors. In comparison to the other endothelial markers (i.e., CD31, CD34 and FⅧRAg) examined, ERG was more sensitive and had increased specificity for HAS. Thus, ERG may represent a novel diagnostic marker for HAS. Because ERG expression shows a similar pattern in both benign and malignant vascular populations, histological examination is required to differentiate angiosarcomas from other endothelial neoplasms [18]. Tumor cell mitotic activities, atypical mitotic features and necrosis have been fundamentally recognized as probable factors determining propensity for HAS. In conclusion, ERG is a new biomarker for the diagnosis of HAS. While CD31, CD34 and FVIIIRAg were variably expressed across our HAS cohort, ERG was expressed in all tumors examined. Because HAS is a relatively rare malignancy, our cohort (n = 24) is, to the best of our knowledge, the largest in the world to date. The data provided from this patient cohort will help to establish a standard for symptoms, diagnosis and treatment of HAS. Moreover, our data implicate ERG as a potent marker in the diagnosis of HAS tumors. COMMENTS Background Hepatic angiosarcoma (HAS) is a rare disease characterized by poor prognosis due to its rapid progression, high recurrence rate and resistance to traditional chemo- and radiotherapies. Surgical resection, which requires a prompt diagnosis, is currently the best treatment option for HAS. Because the initial symptoms of HAS can be nonspecific, it is difficult to diagnose. Thus, a specific molecular marker for the early diagnosis of HAS would provide a critical time window for surgical resection and greatly improve patient survival. ERG (V-Ets Erythroblastosis Virus E26 Oncogene Homolog) is a marker of endothelial cells that has been implicated in other cancers, but its value as a diagnostic marker in HAS has not yet been evaluated April 7, 2014 Volume 20 Issue 13

303 Wang ZB et al. ERG and hepatic angiosarcoma diagnosis Research frontiers ERG is a member of the ETS family of transcription factors and is expressed in endothelial cells. Angiogenesis, endothelial cell differentiation and endothelial homeostasis are regulated by ERG. Previous reports have shown that ERG is a specific and sensitive biological marker for the diagnosis of angiosarcoma. The diagnostic value of ERG expression in HAS has not been evaluated. In addition, because HAS is quite rare, it has been difficult to evaluate diagnostic criteria in a large patient cohort. Innovations and breakthroughs This study has assembled the largest HAS cohort to date (n = 24 cases) to evaluate the role of ERG expression in liver angiosarcoma. Moreover, this study compares ERG expression to other known endothelial markers such as CD31, CD34 and factor Ⅷ-related antigen (FⅧRAg) as a means of HAS diagnosis. While CD31, CD34 and FⅧRAg had variable expression across the cohort samples, ERG expression was found in 100% of the cases examined. The data provided from this patient cohort will help to establish a standard for symptoms, diagnosis and treatment of HAS. This is the first study to identify ERG expression as a marker for HAS. Applications The findings in this study highlight ERG expression as a potent and novel marker for HAS tumors, which will aid in developing diagnostic tests for the disease. Terminology HAS is a rare type of cancer that affects the endothelial cells of the liver and is difficult to diagnose until the disease has spread. ERG, CD31, CD34 and FVIIIRAg are genes expressed in endothelial cells, the cell population affected in HAS, and may therefore represent a way of identifying HAS tumors. Peer review This is a good original research article that demonstrates that ERG is a specific and sensitive diagnostic biomarker for hepatic angiosarcoma. 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Expression of ERG, an Ets family transcription factor, distinguishes cutaneous angiosarcoma from histological mimics. Histopathology 2012; 61: [PMID: DOI: / j x] 21 Weitz J, Klimstra DS, Cymes K, Jarnagin WR, D Angelica M, La Quaglia MP, Fong Y, Brennan MF, Blumgart LH, Dematteo RP. Management of primary liver sarcomas. Cancer 2007; 109: [PMID: DOI: /cncr.22530] 22 Ito Y, Kojiro M, Nakashima T, Mori T. Pathomorphologic characteristics of 102 cases of thorotrast-related hepatocellular carcinoma, cholangiocarcinoma, and hepatic angiosarcoma. Cancer 1988; 62: [PMID: ] 23 Wagoner JK. Toxicity of vinyl chloride and poly(vinyl chloride): a critical review. Environ Health Perspect 1983; 52: [PMID: DOI: /ehp ] 24 Makarov IA, Fedotova IV. [Mechanisms of the carcinogenic effects of vinyl chloride (literature review)]. Gig Tr Prof Zabol 1983; (6): [PMID: ] 25 Zaeva GN. [Carcinogenic properties of vinyl chloride (a review of the literature)]. 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304 Wang ZB et al. ERG and hepatic angiosarcoma diagnosis 27 Huang NC, Wann SR, Chang HT, Lin SL, Wang JS, Guo HR. Arsenic, vinyl chloride, viral hepatitis, and hepatic angiosarcoma: a hospital-based study and review of literature in Taiwan. BMC Gastroenterol 2011; 11: 142 [PMID: DOI: / X ] 28 Kim HR, Rha SY, Cheon SH, Roh JK, Park YN, Yoo NC. Clinical features and treatment outcomes of advanced stage primary hepatic angiosarcoma. Ann Oncol 2009; 20: [PMID: DOI: /annonc/mdn702] 29 Liang X, Bi S, Yang W, Wang L, Cui G, Cui F, Zhang Y, Liu J, Gong X, Chen Y, Wang F, Zheng H, Wang F, Guo J, Jia Z, Ma J, Wang H, Luo H, Li L, Jin S, Hadler SC, Wang Y. Epidemiological serosurvey of hepatitis B in China- -declining HBV prevalence due to hepatitis B vaccination. Vaccine 2009; 27: [PMID: DOI: / j.vaccine ] 30 Zhou YM, Li B, Yin ZM, Xu F, Wang B, Xu W, Liu P, Yang JM. Results of hepatic resection for primary hepatic angiosarcoma in adults. Med Sci Monit 2010; 16: CR61-CR66 [PMID: ] 31 Koyama T, Fletcher JG, Johnson CD, Kuo MS, Notohara K, Burgart LJ. Primary hepatic angiosarcoma: findings at CT and MR imaging. Radiology 2002; 222: [PMID: DOI: /radiol ] P- Reviewer: Munoz M S- Editor: Ma YJ L- Editor: Webster JR E- Editor: Ma S 3679 April 7, 2014 Volume 20 Issue 13

305 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. META-ANALYSIS BRIEF ARTICLE Evidence based medicine and surgical approaches for colon cancer: Evidences, benefits and limitations of the laparoscopic vs open resection Laura Lorenzon, Marco La Torre, Vincenzo Ziparo, Francesco Montebelli, Paolo Mercantini, Genoveffa Balducci, Mario Ferri Laura Lorenzon, Marco La Torre, Vincenzo Ziparo, Francesco Montebelli, Paolo Mercantini, Genoveffa Balducci, Mario Ferri, Surgical and Medical Department of Translational Medicine, Sant Andrea Hospital, Faculty of Medicine and Psychology University of Rome La Sapienza, Rome, Italy Author contributions: Lorenzon L, La Torre M, Ziparo V and Ferri M designed the manuscript; Lorenzon L, Montebelli F and Mercantini P performed the database search; Montebelli F, Mercantini P and Balducci G assessed manuscripts for inclusion/exclusion; Lorenzon L, Ziparo V, Mercantini P and Balducci G performed the systematic review; Lorenzon L, La Torre M and Ferri M performed the meta-analysis; Lorenzon L, La Torre M and Ferri M wrote the paper; all authors have reviewed and approved the manuscript in its final form. Supported by The PhD University Grant program Clinical and Experimental Research Methodologies in Oncology provided by the Faculty of Medicine and Psychology University of Rome La Sapienza to La Torre M Correspondence to: Laura Lorenzon, MD, PhD, Surgical and Medical Department of Translational Medicine, Sant Andrea Hospital, Faculty of Medicine and Psychology University of Rome La Sapienza, Via di Grottarossa , Rome, Italy. laura.lorenzon@uniroma1.it Telephone: Fax: Received: September 29, 2013 Revised: December 26, 2013 Accepted: February 17, 2014 Published online: April 7, 2014 Abstract AIM: To report a meta-analysis of the studies that compared the laparoscopic with the open approach for colon cancer resection. METHODS: Forty-seven manuscripts were reviewed, 33 of which employed for meta-analysis according to the PRISMA guidelines. The results were differentiated according to the study design (prospective randomized trials vs case-control series) and according to the tumor s location. Outcome measures included: (1) shortterm results (operating times, blood losses, bowel function recovery, post-operative pain, return to the oral intake, complications and hospital stay); (2) oncological adequateness (number of nodes harvested in the surgical specimens); and (3) long-term results (including the survivals rates and incidence of incisional hernias) and (4) costs. RESULTS: Meta-analysis of trials provided evidences in support of the laparoscopic procedures for a several short-term outcomes including: a lower blood loss, an earlier recovery of the bowel function, an earlier return to the oral intake, a shorter hospital stay and a lower morbidity rate. Opposite the operating time has been confirmed shorter in open surgery. The same trend has been reported investigating case-control series and cancer by sites, even though there are some concerns regarding the power of the studies in this latter field due to the small number of trials and the small sample of patients enrolled. The two approaches were comparable regarding the mean number of nodes harvested and long-term results, even though these variables were documented reviewing the literature but were not computable for meta-analysis. The analysis of the costs documented lower costs for the open surgery, however just few studies investigated the incidence of postoperative hernias. CONCLUSION: Laparoscopy is superior for the majority of short-term results. Future studies should better differentiate these approaches on the basis of tumors location and the post-operative hernias Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Laparoscopy; Colon resection; Colon can April 7, 2014 Volume 20 Issue 13

306 Lorenzon L et al. EBM and surgical resection for colon cancer cer; Meta-analysis; Evidence-based medicine Core tip: This is a comprehensive meta-analysis of studies investigating laparoscopic resection in comparison with the open surgery for colon cancer, with the aim of evidencing short term and long term results of the surgical approaches. Results were provided according to the study designs (randomized trials, case control series) and according to the tumor s location. Lorenzon L, La Torre M, Ziparo V, Montebelli F, Mercantini P, Balducci G, Ferri M. Evidence based medicine and surgical approaches for colon cancer: Evidences, benefits and limitations of the laparoscopic vs open resection. World J Gastroenterol 2014; 20(13): Available from: URL: com/ /full/v20/i13/3680.htm DOI: org/ /wjg.v20.i INTRODUCTION Colon cancer remains a major health and social issue affecting in the US more than new patients/year [1] ; the surgical resection remains the standard of care for treating and staging non-metastatic colon cancer. During the last twenty years several progresses were made for improving the treatments, the survivals and the quality of life of cancer patients; the main innovation in the surgical technique was that outbreak of the laparoscopy. The use of laparoscopy for colon resection has been introduced in 1991 [2,3]. Initial concerns (including e.g., a long training/learning curve, the possible development of port-site metastasis and an inadequate oncologic resection [4-7] ) were subsequently surmounted; indeed in recent years a number of studies recognized the adequateness of the laparoscopic approach along with a number of short-term functional benefits and equivalent long-term results. Nevertheless, the scientific literature in this field is quite heterogeneous regarding the study design adopted (randomized/non-randomized studies), objectives and outcome measures (short-term and logterm results, costs analysis), population enrolled (colon and/or rectal cancer patients), thus it might be difficult for clinicians and surgeons to summarize results and take at home univocal messages. The aim of this manuscript is to review the studies comparing the laparoscopic and the open approach for colon cancer differentiating results by: (1) prospective randomized trials; (2) case-control series (including prospective and retrospective studies); and (3) the comparison of these techniques according to the tumor s location. This type of differentiation is seldom conducted and might implement the analysis and help the readers in understanding the results. Moreover we focused our investigation on the costs analyses provided in the series and trials that were herein reviewed. Indeed, the goal of this paper is to divulgate a comprehensible meta-analysis of the evidences by each category of investigation and to provide a message of clinical use for clinicians and surgeons committed in the care of colon cancer patients. MATERIALS AND METHODS Data source and search strategies This investigation has been conducted adhering at the PRISMA Statements for review and meta-analysis (Figure 1, Table 1). We conducted a systematic review of the literature by searching PubMed database for all the published series and trials comparing the laparoscopic and open surgical approach for colon cancer from 1995 to December Keywords: laparoscopic vs open colectomy AND colon cancer, languages: English, limit to human including clinical trials and comparative studies. We also included references from the retrieved publications. Duplicate references were removed by manual search. Authors of this study were blinded to authors and journals name while reviewing the series, and did not have any contacts with the authors of the included papers. We did not consider any journal s scores (e.g., journal s Impact Factors) of the published series as exclusion criteria for this review. Study design and selection of papers Each paper retrieved was assessed for inclusion or exclusion for this manuscript, by revision of the titles and the abstracts. Published series with the aim to investigate exclusively rectal carcinomas and/or non cancer-diseases (i.e., laparoscopic proctocolectomies for ulcerative colitis or diverticulitis) were excluded (Figure 1). Conversely manuscripts including few rectal cancers (or few nonmalignant diseases) into the series, along with the other colon cancer localizations were included into this review. Hand-assisted and totally laparoscopy procedures were considered altogether into the laparoscopic group, whereas all the open procedures (midline or transverse incisions) were considered as open resections. All selected papers were categorized into the following sub-groups: (1) randomized studies; and (2) non-randomized studies (including prospective and retrospective case-control series). Furthermore we identified those researches that investigated the comparison of these two approaches according to the tumor s location (i.e., right side colectomies, left side colectomies and transverse resections) and highlighted within these groups the comparison of the costs derived by these two approaches. Outcome measures Whenever possible we collected data regarding: study design, population and power of the study, types of surgical procedure. We considered as short-term outcome measures: operating times (measured in minutes), blood 3681 April 7, 2014 Volume 20 Issue 13

307 Lorenzon L et al. EBM and surgical resection for colon cancer PubMed search Laparoscopy vs open colectomy and colon cancer Limits: English; Humans; Range from to articles Excluded 39 articles (see exclusion list) 41 articles Manual search: 6 articles 47 articles 47 articles for qualitative analysis 33 articles for meta-analysis Figure 1 Study design. Study design according to the PRISMA statement for systematic reviews and meta-analysis. Table 1 Exclusion list of the manuscripts No. Author Ref. Cause of exclusion 1 Sasaki J J Nippon Med Sch 2012; 79: Different outcome measure 2 Turagava J N Z Med J 2012; 125: Only laparoscopy group 3 Poulsen M J Gastrointest Surg 2012; 16: Only laparoscopy group 4 Roscio F Int J Surg 2012; 10: Only laparoscopy group 5 Wang G Hepatogastroenterology 2012; 59: Only laparoscopy group 6 Rottoli M Surg Endosc 2012; 26: Different outcome measure 7 Campos FG Surg Laparosc Endosc Percutan Tech 2011; 21: Exclusively familial polyposis patients 8 Panait L Chirurgia (Bocur) 2011; 106: Only laparoscopy group 9 Hendren S Dis Colon Rectum 2011; 54: Different outcome measure 10 Issa N J Gastrointest Surg 2011; 15: Only laparoscopy group 11 McNicol FJ Colorect Dis 2012; 14: Rectal cancers 12 Fujii S Hepatogastroenterology 2011; 58: Only laparoscopy group 13 Akmal Y Surg Endosc 2011; 25: Only laparoscopy group 14 Senthil M Arch Surg 2010; 145: Different outcome measure 15 Han SA Int J Colorect Dis 2010; 25: Different outcome measure 16 Park IJ J Gastrointest Surg 2009; 13: Emergency resections 17 Heise CP Dis Colon Rectum 2008; 51: Rectal cancers 18 Strhölein MA Dis Colon Rectum 2008; 51: Rectal cancers 19 Moloo H Dis Colon Rectum 2008; 51: Only laparoscopy group 20 Zhang H Minim Invasiv Ther allied Technol 2007; 16: Rectal cancers 21 Polle SW Surg Endosc 2007; 21: Rectal cancers 22 Hasegawa H Surg Endosc 2007; 21: Rectal cancers 23 Del Rio P Minerva Chir 2006; 61: Only laparoscopy group 24 Schlachta CM Surg Endosc 2007; 21: Only laparoscopy group 25 Wong DC Tech Coloproctol 2006; 10: Rectal cancers 26 Moloo H Dis Colon Rectum 2006; 49: Only laparoscopy group 27 Larson DW Dis Colon Rectum 2005; 48: Rectal cancers 28 Kuhry E Surg Endosc 2005; 19: Different outcome measure 29 Adaki Y Hepatogastroenterology 2003; 50: Different outcome measure 30 Dunker MS Dis Colon Rectum 2003; 46: Different outcome measure 31 Pasupathy S Tech Coloproctol 2001; 5: Rectal cancers 32 Weeks JC JAMA 2002; 287: Different outcome measure 33 Nelson H Swiss Surg 2001; 7: Different outcome measure 34 Delgrado S Dis Colon Rectum 2001; 44: Different outcome measure 35 Brown SR Dis Colon Rectum 2001; 44: Rectal cancers 36 Marcello PW Dis Colon Rectum 2000; 43: Rectal cancers 37 Hewitt PM Dis Colon Rectum 1998; 41: Different outcome measure 38 Fukushima R Dis Colon Rectum 1996; 39 (Suppl): S29-34 Different outcome measure 39 Bokey EL Dis Colon Rectum 1996; 39 (Suppl): S29-34 Only laparoscopy group 3682 April 7, 2014 Volume 20 Issue 13

308 Lorenzon L et al. EBM and surgical resection for colon cancer loss (measured in milliliter), bowel function recovery (defined by the passage of the first flatus/stool; measured in days), post-operative pain (defined as the usage of analgesic -measured in days- and/or the score obtained by the visual analogue scale), return to the oral intake (usually liquid diet; measured in days), morbidity defined by peri-operative complications and hospital stay (measured in days). Of note, since complications were often reported using different modalities (e.g., major vs minor complications, rate of adverse events etc.), we extrapolated the overall morbidity rate in each category (laparoscopic and open surgery) reported in all the investigated series. The oncologic adequateness was recorded whenever considered by the authors as the mean number of lymph nodes harvested in the surgical specimen. Mean followup (months) was recorded and long-term outcome measures were considered as the rate of relapses, the survivals and the incidence of incisional hernias. The costs analysis has been conducted recording the overall hospital costs for both procedures, providing results in US dollars, in order to analyze a single currency. We did not considered conversion surgery, since it might bias the results of the open surgery procedures. On the same extent we did not consider the hospital volume, since it is seldom reported, even though the learning curves and the volume of patients might vary shortterm results and costs. A first analysis has been conducted reviewing papers in each category (prospective-randomized trials; case control series; studies investigating cancer by sites) and highlighting in each article were the evidences stand for (e.g., significant statistical analyses supporting laparoscopy or open surgery) for the different outcome measures (e.g., considering the operating time, blood losses etc.). Moreover and whenever computable we provided a meta-analysis of the results. Statistical analysis Continuous variables were analysed using means, medians and standard deviations, whereas categorical variables were analysed using frequencies and percents. Statistical analyses and Meta-analysis were performed using Med- Calc for Windows, version (MedCalc Software, MariaKerke, Belgium). In order to provide significant results, a meta-analysis has been conducted for all variables in different categories (prospective-randomized trials; case control series; studies investigating cancer by sites) whenever at least 3 studies provided data computable. The Mantel-Haenszel method was used for calculating the weighted summary Odds ratio under the fixed effects model. Next the heterogeneity statistic is incorporated to calculate the summary odds ratio under the random effects model. The total odds ratio with 95%CI is given both for the Fixed effects model and the Random effects model. If the value 1 is not within the 95%CI, then the Odds ratio is statistically significant at the 5% level (P < 0.05). For meta-analysis of studies with a continuous measure (comparison of means between treated cases and controls), the Hedges g statistic was used as a formulation for the standardized mean difference (SMD) under the fixed effects model. Next the heterogeneity statistic is incorporated to calculate the summary standardized mean difference under the random effects model. If the value 0 is not within the 95%CI, then the SMD is statistically significant at the 5% level (P < 0.05). Statistical heterogeneity of the results of the trials was assessed on the basis of a test of heterogeneity (standard chi-squared test on N degrees of freedom where N equals the number of trials contributing data minus one). Three possible causes for heterogeneity were pre-specified: (1) differing response according to difference in the quality of the trial; (2) differing response according to sample size; and (3) differing response according to clinical heterogeneity. If the test of heterogeneity is statistically significant (P < 0.05) then more emphasis should be placed on the random effects model. RESULTS Figure 1 outlines the study design. PubMed search provided 80 results, however 39 studies were excluded due to different outcome measures (e.g., investigations aimed to outline the quality of life or the immunological response), due to the evaluation of the laparoscopy procedures per se (missing the open surgery group) or evaluating rectal cancer patients (see exclusion list). All the 47 articles retrieved were included in the systematic review, however only 33 articles provided data computable for meta-analysis. Randomized controlled trials Figure 2 outlines results of this analysis. 11 studies were included for review [8-18] from 1995 to 2012, including overall 2992 patients in the laparoscopy group and 2717 in the open surgery group. Of note 4 studies enrolled less than 50 patients/arm [8,10-11,13]. The systematic review of the manuscripts documented a number of benefits of the laparoscopic procedure for the vast majority of the short-term outcome measures (bowel function recovery, return to the oral intake, post-operative pain, blood loss and hospital stay), whereas it was documented a longer operating time comparing the open approach. Of note, all the studies - with the exclusion of the first trial conducted by Lacy and co-authors in 1995 [8] - reported a comparable morbidity rate within the two approaches. Similarly the mean count of the nodes harvested (LNH) in the surgical specimens were similar in the 3 studies investigating this variable [8-9,15], and the survivals were reported homogeneous in the vast majority of the studies [11,12,14,17,18], with the sole exception of the trial conducted by Lacy in 2002 [9]. It seems important to highlight that only two randomized trials investigated 3683 April 7, 2014 Volume 20 Issue 13

309 Lorenzon L et al. EBM and surgical resection for colon cancer A Author Year Lap arm OS arm FU (mo) OT BF OI PP BL HOSP Morbidity LNH Survival/ relapse Hernia n n Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lacy et al [8] X X X NS X X NS Lacy et al [9] X X X X X NS NS X Winslow et al [10] X X NS NS Kaiser et al [11] X X X NS X NS NS COST Trial X X X NS NS Kang et al [13] NS X X X X X NS Braga et al [14] X NS NS NS COLOR Trial X X X X X X NS NS CLASiCC Trial NS NS NS NS NS COLOR Trial X X NS Bagshaw et al [18] NS Total B Variable Laparoscopy Open surgery Total SMD/OR 95%CI P value Operative time Bowel function (-0.371) Oral intake (-0.469) 0.07 Blood loss (-0.425) 0.06 Hospital stay (-0.352) Morbidity 302/ / C Meta-analysis SMD Standardized mean difference Figure 2 Randomized studies. A: Randomized trails comparing laparoscopy and open surgery. X in the table refers to a statistical association provided in the studies; B: Meta-analysis of the out-come measures; C: Forest plot graph regarding studies investigating blood loss. Lap arm: Laparoscopy arm; OS arm: Open surgery arm; FU: Mean follow-up; OT: Operative time; BF: Bowel function; OI: Oral intake; PP: Post-operative Pain; BL: Blood loss; HOSP: Hospital stay; LNH: Lymph node harvest; NS: Not significant. the incidence of post-operative hernias [10,14], reporting a comparable rate of events (Figure 2A). Meta-analysis conducted on 9 studies in this group [8-16] confirmed the evidences of the short-term outcome measures in favour of the laparoscopy (bowel function recovery, return to the oral intake, blood loss and hospital stay), plus it documented a better morbidity rate for the laparoscopy group (OR = 0.609; 95%CI: ). Conversely the operating time has been confirmed shortly in the open procedure group (Figure 2B and C). Case-control studies Figure 3 reports data from this analysis. 16 case-control studies were included, counting overall 3819 patients in the laparoscopy group and 6990 in the open surgery group [19-34]. The vast majority of these studies agree in reporting shorter operating time in the open surgery group comparing with the laparoscopic procedures, with the exceptions of the studies conducted by Saba, Bilimoira and de Campos Lobato that provided homogeneous results [19,27,30]. All studies investigating the bowel function recovery recognized a benefit for the laparoscopy procedure [19,21-22,24,26] ; similarly the investigation of the return to the oral intake provided homogeneous results in the 2 studies that investigated this outcome measure [19,29]. Even if Shabbir reported a similar use of analgesic in the post-operatory recovery [22], the other studies reported significant benefits associated with the laparoscopic procedure in this field [21,22,24]. A significant reduction in the blood loss for the patients undergone laparoscopy has been reported by all the authors [20,26,30,32,33], with the sole exception of Slow [24]. The hospital stay has been reported in favour of the laparoscopy group in all the studies, with the exclusion of the series investigated by 3684 April 7, 2014 Volume 20 Issue 13

310 Lorenzon L et al. EBM and surgical resection for colon cancer A Author Year Lap group OS group FU (mo) OT BF OI PP BL HOSP Morbidity LNH Survival/ relapse Hernia n n Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Saba et al [19] NS X X X NS 1 Franklin et al [20] X 1 X 1 X 1 NS 1 NS 1 NS X NS Lezoche et al [21] X X X X NS X Stocchi et al [22] X X X X X Feliciotti et al [23] NS Sklow et al [24] X X X NS X NS Patankar et al [25] NS NS NS Vignali et al [26] X X X X NS NS Bilimoira et al [27] NS X X Cermak et al [28] NS NS NS Shabbir et al [29] X X NS X NS de Campos X Lobato et al [30] Gouvas et al [31] NS McKay et al [32] X X X X NS SCOAP Study X X X X Cianchi et al [34] X Total B C Variable Laparoscopy Open surgery Total SMD/OR 95%CI P value Operative time < Hospital stay (-0.349) < Morbidity 389/ / LNH < Meta-analysis SMD Standardized mean difference Figure 3 Non-randomized studies. A: Case-control studies comparing laparoscopy and open surgery. X in the table refers to a statistical association provided in the studies; B: Meta-analysis of the out-come measures; C: Forest plot graph regarding studies investigating hospital stay. 1 Statistic analysis not performed; 2 Significant difference of follow-up according to the stage of the disease. Lap: Laparoscopy; OS: Open surgery; FU: Mean follow-up; OT: Operative time; BF: Bowel function; OI: Oral intake; PP: Post-operative Pain; BL: Blood loss; HOSP: Hospital stay; LNH: Lymph node harvest; NS: Not significant. Cermak et al [28] in The study of the morbidity rate provided dis-homogeneous results: even though some studies documented some benefits in the laparoscopy group [20,22,27,32,33], others documented comparable results between the 2 approaches [21,24-26,28,30]. Notably, Cianchi in 2012 highlighted a better LNH in the laparoscopy group [34], whereas the others reported homogeneous results. Similarly, with the exception of Lezoche et al [21], the authors reported comparable survival rates within these 2 approaches. Seems important to highlight that the vast majority of the studies did not investigate the rate of post-operative hernias, with the sole exception of Pantankar in 2008 [25] that reported similar results in the 2 groups. A meta-analysis has been conducted for 11 studies in this category [20-22,24-28,30,32,33], Figure 3B. The operative time has been reported longer in the laparoscopy group, whereas this procedure showed a shorter hospital stay, Figure 3C. Interestingly the meta-analysis provided also in this category of studies some evidences regarding a lower rate of morbidity in the post-operative period following a laparoscopy operation (OR = 0.644; 95%CI: ). The analysis of the LNH variable was not 3685 April 7, 2014 Volume 20 Issue 13

311 Lorenzon L et al. EBM and surgical resection for colon cancer A Author Year Lap group OS Study design; group FU (mo) OT BF OI PP BL HOSP Morbidity LNH Survival/ relapse Hernia n n Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lezoche et al [35] CC; 42.2 X X X NS NS NS Sklow et al [24] CC X 2 X 3 X 3 X 3 Zheng et al [36] CC; 26.6 NS X NS X X X NS NS NS Lohsiriwat et al [37] CC X NS NS NS NS NS NS Tong et al [38] CC; 21.5 X NS X NS X NS NS NS Chung et al [39] M R; 29.0 X X X X X X NS NS Braga et al [40] M R; 48.0 X X X X NS NS 1 Nakamura et al [41] CC; NS X X X NS Veenhof et al [42] T CC; 19.0 X X X NS NS NS NS Tanis et al [43] M CC X X NS NS X NS NS 23-T Total B Variable Laparoscopy Open surgery Total SMD/OR 95%CI P value Operative time < Bowel function (-0.426) < Post-operative pain (-0.307) < Blood loss < Hospital stay (-0.508) < Morbidity 57/ / LNH C Meta-analysis SMD Standardized mean difference Figure 4 Right sided colectomies. A: Studies comparing right-side laparoscopy colectomy and open surgery. X in the table refers to a statistical association provided in the studies; B: Meta-analysis of the out-come measures; C: Forest plot graph regarding studies investigating operative time. 1 Statistic analysis not performed; 2 Exclusively if patients > 75 years old; 3 Exclusively if patients < 75 years old. Lap: Laparoscopy; OS: Open surgery; M: Midline incision; T: transverse incision R: Randomized, CC: Case-control; FU: Mean follow-up; OT: Operative time; BF: Bowel function; OI: Oral intake; PP: Post-operative Pain; BL: Blood loss; HOSP: Hospital stay; LNH: Lymph node harvest; NS: Not significant. of statistical value (95%CI: ). Right-sided colectomies We identified 10 studies in this category [24,35-43], involving 427 laparoscopy patients and 485 open colectomy patients, Figure 4. Overall we included 8 case-control series [24,35-38,41-43] and 2 randomized trials [39,40]. Of note the open surgical approach (midline vs transverse incisions) has been categorized exclusively in 4 studies [39,40,42,43]. All the studies analysed documented homogeneous results regarding the hospital stay (statistically better associated with the laparoscopy approach), LNH and survival rate (comparable results between the 2 procedures). Discordant data were documented for the operating time: even though the vast majority of the authors reported a shorter operation in the open group, the studies conducted by Zheng in 2005 and by Nakamura in 2009 documented comparable results [36,41]. The bowel function recovery has been reported shorter in the laparoscopy group in the studies conducted by Lezoche, Slow, Zeng, Chung and Tanis [24,35,36,39,43], whereas other 2 articles provided similar recoveries for both groups [37,38]. Three studies reported comparable results within the 2 surgical approaches regarding the return to the oral intake [36,37,43] ; opposite Tong, Chung and Braga [38-40] reported results in favour of the laparoscopy group. Zeng, Chung and Slow (exclusively in patients < 75 years old) [24,36,39] reported a lower use of analgesics in the laparoscopy group, 3686 April 7, 2014 Volume 20 Issue 13

312 Lorenzon L et al. EBM and surgical resection for colon cancer A Author Year Lap group OS group Study design; OT BF OI PP BL HOSP Morbidity LNH Survival/ relapse Hernia n n FU (mo) Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lezoche et al [35] CC; 42.3 X X X NS NS NS Sklow et al [24] CC X X 1 X 1 X 1 Leung et al [44] R; 50.9 X X X X NS X NS NS NS Liang et al [45] R; 40.0 X X X X X NS NS NS Hinojosa et al [46] CC X X Cheung et al [47] R NS X X NS X X Nakashima et al [48] CC X X X X X X X Total B Variable Laparoscopy Open surgery Total SMD/OR 95%CI P value Hospital stay (-0.300) 1262 Morbidity 293/ / C Meta-analysis OR Odds ratio Figure 5 Left sided colectomies. A: Studies comparing left-side laparoscopy colectomy and open surgery. X in the table refers to a statistical association provided in the studies; B: Meta-analysis of the out-come measures; C: Forest plot graph regarding studies investigating morbidity rate. 1 Exclusively if patients > 75 years old. Lap: Laparoscopy; OS: Open surgery; M: Midline incision; T: transverse incision R: Randomized, CC: Case-control; FU: Mean follow-up; OT: Operative time; BF: Bowel function; OI: Oral intake; PP: Post-operative Pain; BL: Blood loss; HOSP: Hospital stay; LNH: Lymph node harvest; NS: Not significant. whereas Lohsiriwat and Tanis failed in reporting a statistical association [37,43]. Within the laparoscopy procedures, all the authors with the exceptions of Lohsiriwat and Tong [37,38], documented lower blood losses. Also in this category, the investigation of the rate of post-operative hernias has been conducted in a single study [42]. A meta-analysis has been conducted or 9 manuscripts [24,35-42] within this category, Figure 4B. Significant evidences were highlighted for the laparoscopy procedures regarding the bowel function recovery, post-operative pain, blood losses, hospital stay and interestingly for the morbidity rate (OR = 0.524; 95%CI: ). Also in this category the investigation of the LNH variable was not of statistical value (95%CI: ). Conversely the operating time has been confirmed shorter in the open surgery group, Figure 4C. Left-sided colon cancers Seven studies were included in this category [24,35,44-48], encompassing 1608 patients undergone laparoscopy and 9981 patients undergone open surgical resections, Figure 5. Three studies were randomized [44,45,47], the remaining were case-control studies. It seems important to highlight that 3 studies enrolled less than 50 patients/ arm [24,47,48]. The analyses of the bowel function recovery, the return to the oral intake, the post-operative pain and the survival rates provided homogeneous results among studies (benefits for the laparoscopy patients for the short-term outcome measures, comparable results between the 2 approaches for long term survivals). The operating time has been reported in favour of the open approach in all the studies with the exception of Cheung et al [47]. Conversely the analysis of the blood losses was in favour of the laparoscopy procedures in all the researches, excluding the article by Leung et al [44]. A shorter hospital stay has been reported statistically associated to the laparoscopy procedure in all the studies with the sole exclusion of the report by Cheung et al [47]. Interestingly the analysis of the morbidity rate provided discordant data: 3 studies reported benefits for the laparoscopy procedure [46-48], whereas other 3 studies reported comparable results [35,44,45]. Notably the analysis of the LNH reported homogeneous results among different studies, with the exceptions of Cheung and Nakashima [47,48]. A meta-analysis has been provided including all the 3687 April 7, 2014 Volume 20 Issue 13

313 Lorenzon L et al. EBM and surgical resection for colon cancer Author Year Lap group n OS group n Study design OT BF OI PP BL HOSP Morbidity LNH Survival/ relapse Hernia Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS Lap OS NS NS NS NS Kim et al [49] CC NS X X X NS NS NS Akiyoshi et al [50] CC X X X X X NS X Fernández CC X X NS Cebrián et al [51] Total Figure 6 Transverse colectomies. Studies comparing transverse laparoscopy colectomy and open surgery. X in the table refers to a statistical association provided in the studies. Lap: Laparoscopy; OS: Open surgery; M: Midline incision; T: transverse incision; R: Randomized; CC: Case-control; OT: Operative time; BF: Bowel function; OI: Oral intake; PP: Post-operative Pain; BL: Blood loss; HOSP: Hospital stay; LNH: Lymph node harvest; NS: Not significant. 7 studies, but it was computable for only two variables (Figure 5B) and interestingly both the hospital stay and the morbidity rate were confirmed in favour of the laparoscopy group, Figure 5C. Transverse colon cancers Three studies were included in this category of investigation [49-51], including 124 patients in the laparoscopy group and 141 in the open surgery group, Figure 6. All the studies were case-controls series with some concerns regarding the power of the analyses due to the small samples enrolled. These studies reported homogeneous results in favour of the laparoscopy procedures for the bowel function recovery and the intra-operative blood loss. Notably all the studies documented similar morbidity rates for both surgical approaches. The studies conducted by Kim et al [49] and by Fernández-Cebrián et al [51] documented comparable operating times, whereas Akiyoshi et al [50] reported benefits in this field in the open surgery group. Fernández-Cebrián et al [51] documented comparable results within the 2 procedures regarding the return to the oral intake, whereas the others documented a statistical correlation with the laparoscopy procedure [49,50]. The hospital stay has been reported comparable by Kim and by Fernández-Cebrián [49,51], whereas Akiyoshi et al [50] reported a shorter hospitalization in the laparoscopy group. The analysis of the LNH reported similar results between the surgical procedures in the studies conducted by Kim et al [49] and by Fernández- Cebrián et al [51], whereas Akiyoshi et al [50] documented a higher mean number of nodes harvested in the open surgery group. None of these studies investigated the survivals or the incidence of post-operative hernias. It was not possible to computable data for metaanalysis within this category of investigation. Costs analysis Nine studies were included in this investigation [19,29,36,44-46,52-54], including 3 randomized trials [44,45,52], Figure 7. The overall number of patients pooled in the laparoscopy group has been of ; otherwise patients were included in the open surgery group. Costs were expressed or converted whenever necessary in United States dollars. Overall laparoscopy procedures provided costs ranging from $4000 to $41000; conversely the open surgery expenses were ranging from $1800 to $ studies were meta-analysed [36,44-46]. Results of the meta-analysis confirmed a significant reduction of the costs in the open surgery group comparing with laparoscopy (SMD = 4.843; 95%CI: ), Figure 7B and C. DISCUSSION The primary goal of this manuscript was to divulgate the evidences obtained reviewing 47 manuscripts in this field, 33 of which provided data computable for metaanalysis; the results were categorized on the basis of the study design (randomized trials, case-control series) and on the basis of the tumor locations. Moreover we conducted an analysis of the costs derived by the two surgical procedures. Our results are in agreement with that of other meta-analyses in this field documenting better short-term results in the laparoscopy groups comparing with the open surgery procedures [55,56], even though it is associated with a significant longer operative time. We considered any complications provided by different studies in the morbidity rate outcome measure, since it was very difficult to provide homogeneous results reviewing studies in this field; indeed authors considered different complications in the analyzed studies (infections, bleeding etc.). The interpretation of results, however, might be implemented if future studies could stratify the severity of adverse events using standard classifications (e.g., the Clavien s classification) [57]. Nevertheless, the laparoscopy procedure for colon cancer resection has been reported oncologically safe [58,59]. The same results were reporting analysing exclusively right-sided colectomies [60]. In the field of the evaluation of resections by cancers site, we noted that still to-date few studies compared the right-side colectomy by transverse incisions with the laparoscopy procedures; indeed as highlighted by Tanis et al [43] the shortterm results obtained by the transverse incisions are often between laparoscopy and the midline approach. It is our opinion that this field of studies should be implemented. Moreover in the field of left-side colectomies the some series pool left-sided and recto-sigmoid cancers [45,46], thus it is often difficult to differentiate pure colectomies from the recto-sigmoid resections. Of note just 4 studies out of the 47 reviewed, investigated the rate of post-operative hernias, providing homogeneous 3688 April 7, 2014 Volume 20 Issue 13

314 Lorenzon L et al. EBM and surgical resection for colon cancer A Author Year Lap group OS group Study design Cost P value n n Laparoscopy (mean ± SD) Open surgery (mean ± SD) Saba et al [19] CC < 0.05 Leung et al [44] R 9297 ± ± 2164 < Zheng et al [36] CC ± ± NS Liang et al [45] R ± ± 86.0 <0.001 Hinojosa et al [46] CC ± ± Franks et al [52] R Steele et al [53] CC NS Shabbir et al [29] CC NS Vaid et al [54] CC < Total B Variable Laparoscopy Open surgery Total SMD/OR 95%CI P value Costs < C Meta-analysis SMD Standardized mean difference Figure 7 Costs analysis. A: Studies comparing laparoscopy and open surgery: analysis of the costs; B: Meta-analysis; C: Forest plot graph. Lap: Laparoscopy; OS: Open surgery; NS: Not significant; R: Randomized; CC: Case-control. results comparing the 2 methodologies [11,14,25,42], thus also this field of investigation should be implemented by incoming studies. Take at home messages Short term outcome measures including: a lower blood loss, an earlier recovery of the bowel function, an earlier return to the oral intake, a shorter hospital stay and a lower morbidity rate were statistically associated to the laparoscopic procedures in randomized trials. Opposite the operating time has been confirmed shorter in the open surgery group. Even though the majority of the trials reported a statistical association with less post-operative pain in the laparoscopic group, this data was not computable on meta-analysis, similarly the comparable results of the LNH within the 2 procedures was documented at the review but not computable at the meta-analysis. This trend has been confirmed analyzing case-control series and cancer by sites, even though there are some concerns regarding the power of the studies in this latter field due to the small number of trials and the small sample of patients often enrolled. The analysis of the costs documented lower costs for the open surgery procedures, however seems important to highlight that just few studies investigated the incidence of post-operative hernia. The analysis of the postoperative hernia could add important information; indeed a re-intervention might substantially implement the costs and might put into question the cost-effectiveness of the procedure. COMMENTS Background Colon cancer is a major health issue. Over the last twenty years several progresses were made for improving the treatment and the quality of life of cancer patients, and the main innovation in the field of colon cancer surgical technique was that outbreak of the laparoscopy procedures (minimally-invasive treatments). Research frontiers Several studies recognized the adequateness of the laparoscopic approach along with a number of short-term functional benefits and equivalent long-term results comparing to open approach. Nevertheless, the scientific literature in this field is quite heterogeneous, thus it might be difficult for clinicians and surgeons to summarize results and take at home univocal messages. Innovations and breakthroughs The goal of this paper is to divulgate a comprehensible meta-analysis of the evidences and to provide a message of clinical use for clinicians and surgeons committed in the care of colon cancer patients April 7, 2014 Volume 20 Issue 13

315 Lorenzon L et al. EBM and surgical resection for colon cancer Applications The results are in agreement with that of other meta-analyses in this field documenting better short-term results in the laparoscopy groups comparing with the open surgery procedures, even though it is associated with a significant longer operative time. Peer review This is a comprehensive review and meta-analysis of laparoscopic vs open colectomy for colon cancer. It s well-written with a solid analysis. REFERENCES 1 Siegel R, Naishadham D, Jemal A. Cancer statistics, CA Cancer J Clin 2012; 62: [PMID: DOI: /caac.20138] 2 Fowler DL, White SA. Laparoscopy-assisted sigmoid resection. Surg Laparosc Endosc 1991; 1: [PMID: ] 3 Jacobs M, Verdeja JC, Goldstein HS. Minimally invasive colon resection (laparoscopic colectomy). Surg Laparosc Endosc 1991; 1: [PMID: ] 4 Jacquet P, Averbach AM, Jacquet N. Abdominal wall metastasis and peritoneal carcinomatosis after laparoscopic-assisted colectomy for colon cancer. 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316 Lorenzon L et al. EBM and surgical resection for colon cancer da Costa PM. Surgical resection for colon cancer: laparoscopic assisted vs. open colectomy. Hepatogastroenterology 2008; 55: [PMID: ] 29 Shabbir A, Roslani AC, Wong KS, Tsang CB, Wong HB, Cheong WK. Is laparoscopic colectomy as cost beneficial as open colectomy? ANZ J Surg 2009; 79: [PMID: DOI: /j x] 30 de Campos-Lobato LF, Alves-Ferreira PC, Geisler DP, Kiran RP. Benefits of laparoscopy: does the disease condition that indicated colectomy matter? Am Surg 2011; 77: [PMID: ] 31 Gouvas N, Pechlivanides G, Zervakis N, Kafousi M, Xynos E. Complete mesocolic excision in colon cancer surgery: a comparison between open and laparoscopic approach. Colorectal Dis 2012; 14: [PMID: DOI: /j x] 32 McKay GD, Morgan MJ, Wong SK, Gatenby AH, Fulham SB, Ahmed KW, Toh JW, Hanna M, Hitos K. Improved short-term outcomes of laparoscopic versus open resection for colon and rectal cancer in an area health service: a multicenter study. 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Br J Cancer 2006; 95: 6-12 [PMID: ] 53 Steele SR, Brown TA, Rush RM, Martin MJ. Laparoscopic vs open colectomy for colon cancer: results from a large nationwide population-based analysis. J Gastrointest Surg 2008; 12: [PMID: ] 54 Vaid S, Tucker J, Bell T, Grim R, Ahuja V. Cost analysis of laparoscopic versus open colectomy in patients with colon cancer: results from a large nationwide population database. Am Surg 2012; 78: [PMID: ] 55 Abraham NS, Young JM, Solomon MJ. Meta-analysis of short-term outcomes after laparoscopic resection for colorectal cancer. Br J Surg 2004; 91: [PMID: ] 56 Tilney HS, Lovegrove RE, Purkayastha S, Heriot AG, Darzi AW, Tekkis PP. Laparoscopic vs open subtotal colectomy 3691 April 7, 2014 Volume 20 Issue 13

317 Lorenzon L et al. EBM and surgical resection for colon cancer for benign and malignant disease. Colorectal Dis 2006; 8: [PMID: ] 57 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: ] 58 Bonjer HJ, Hop WC, Nelson H, Sargent DJ, Lacy AM, Castells A, Guillou PJ, Thorpe H, Brown J, Delgado S, Kuhrij E, Haglind E, Påhlman L. Laparoscopically assisted vs open colectomy for colon cancer: a meta-analysis. Arch Surg 2007; 142: [PMID: ] 59 Korolija D, Tadić S, Simić D. Extent of oncological resection in laparoscopic vs. open colorectal surgery: meta-analysis. Langenbecks Arch Surg 2003; 387: [PMID: ] 60 Rondelli F, Trastulli S, Avenia N, Schillaci G, Cirocchi R, Gullà N, Mariani E, Bistoni G, Noya G. Is laparoscopic right colectomy more effective than open resection? A meta-analysis of randomized and nonrandomized studies. Colorectal Dis 2012; 14: e447-e469 [PMID: DOI: / j x] P- Reviewers: Gayet B, Sing RF S- Editor: Zhai HH L- Editor: A E- Editor: Zhang DN 3692 April 7, 2014 Volume 20 Issue 13

318 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. CASE REPORT Colonic and anal metastases from pancreato-biliary malignancies Farshid Ejtehadi, Nikolaos A Chatzizacharias, Rebecca J Brais, Nigel R Hall, Edmund M Godfrey, Emmanuel Huguet, Raaj K Praseedom, Asif Jah Farshid Ejtehadi, Nikolaos A Chatzizacharias, Emmanuel Huguet, Raaj K Praseedom, Asif Jah, Department of HPB and Transplant Surgery, Addenbrooke s Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridgeshire CB2 0QQ, United Kingdom Rebecca J Brais, Department of Histopathology, Addenbrooke s Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridgeshire CB2 0QQ, United Kingdom Nigel R Hall, Cambridge Colorectal Unit, Addenbrooke s Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridgeshire, CB2 0QQ, United Kingdom Edmund M Godfrey, Department of Radiology, Addenbrooke s Hospital, Cambridge University Hospitals NHS Foundation Trust, Cambridgeshire CB2 0QQ, United Kingdom Author contributions: All authors contributed in the writing of the manuscript; Brais RJ also conducted the immunohistochemical analysis. Correspondence to: Dr. Asif Jah, Consultant surgeon, Department of HPB and Transplant Surgery, Addenbrooke s Hospital, Cambridge University Hospitals NHS Foundation Trust, Hills Road, Cambridgeshire CB2 0QQ, United Kingdom. asif.jah@addenbrookes.nhs.uk Telephone: Fax: Received: October 28, 2013 Revised: December 27, 2013 Accepted: January 20, 2014 Published online: April 7, 2014 Abstract Pancreato-biliary malignancies often present with locally advanced or metastatic disease. Surgery is the mainstay of treatment although less than 20% of tumours are suitable for resection at presentation. Common sites for metastases are liver, lungs, lymph nodes and peritoneal cavity. Metastatic disease carries poor prognosis, with median survival of less than 3 mo. We report two cases where metastases from pancreato-biliary cancers were identified in the colon and anal canal. In both cases specific immunohistochemical staining was utilised in the diagnosis. In the first case, the pre- senting complaint was obstructive jaundice due to an ampullary tumour for which a pancreato-duodenectomy was carried out. However, the patient re-presented 4 wk later with an atypical anal fissure which was found to be metastatic deposit from the primary ampullary adenocarcinoma. In the second case, the patient presented with obstructive jaundice due to a biliary stricture. Subsequent imaging revealed sigmoid thickening, which was confirmed to be a metastatic deposit. Distal colonic and anorectal metastases from pancreatobiliary cancers are rare and can masquerade as primary colorectal tumours. The key to the diagnosis is the specific immunohistochemical profile of the intestinal lesion biopsies Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Pancreatobiliary cancer; Rare metastatic sites; Colonic metastasis; Anal metastasis; Immunohistochemistry Core tip: Pancreato-biliary malignancies often present with locally advanced or metastatic disease. Surgery is the mainstay of treatment although less than 20% are suitable for resection at presentation. Common sites for metastases are liver, lungs, lymph nodes and peritoneal cavity and carry poor prognosis, with median survival of less than 3 mo. Distal colonic and anorectal metastases from pancreato-biliary cancers are rare and can masquerade as primary colorectal tumours. The key to the diagnosis is the specific immunohistochemical profile of the intestinal lesion biopsies. Ejtehadi F, Chatzizacharias NA, Brais RJ, Hall NR, Godfrey EM, Huguet E, Praseedom RK, Jah A. Colonic and anal metastases from pancreato-biliary malignancies. World J Gastroenterol 2014; 20(13): Available from: URL: April 7, 2014 Volume 20 Issue 13

319 Ejtehadi F et al. Coloanal metastases from pancreato-biliary cancers net.com/ /full/v20/i13/3693.htm DOI: org/ /wjg.v20.i INTRODUCTION Pancreato-biliary malignancies often present with locally advanced or metastatic disease [1,2]. The commonest tumour type is adenocarcinoma followed by rarer varieties such as neuroendocrine and adeno-squamous cell carcinomas [3-5]. Surgery is the mainstay of treatment although less than 20% are deemed resectable at the time of presentation [1,5-8]. Metastatic disease carries a poor prognosis with median survival of less than 3 mo [5-12]. The common sites for metastases are the liver, lungs, lymph nodes and peritoneal cavity [2]. Unusual metastatic sites such as kidney [13], colon [14-17] and skin [18], have also been reported. We report two cases where metastases from pancreato-biliary cancers were identified in the colon and anal canal. CASE REPORT Case 1 A 79-year-old lady presented with obstructive jaundice. A computed tomography (CT) scan and a subsequent endoscopic ultrasound scan identified a resectable ampullary mass. Staging CT did not reveal any evidence of metastases and the patient underwent a Whipple s resection following which she made an uncomplicated recovery. The histopathology of the resected specimen confirmed poorly differentiated ampullary adenocarcinoma. The tumour demonstrated a pancreato-bilary immunophenotype being cytokeratin-7/cytokeratin-17/mucin-1 (CK7/ CK17/MUC1) positive and cytokeratin-20/homeobox protein CDX2/mucin-2 (CK20/CDX2/MUC2) negative (Figure 1A). Approximately 4 wk after discharge, the patient re-presented with perianal pain exacerbated by defecation. Examination under anaesthesia (EUA) confirmed the presence of an atypical anal fissure, biopsies of which revealed anal mucosa extensively infiltrated by a poorly differentiated carcinoma, demonstrating similar morphological appearance to the original ampullary adenocarcinoma. Subsequent, immunohistochemistry also demonstrated an identical pancreato-biliary phenotype as seen previously (Figure 1B), suggesting that this was a metastatic deposit from the ampullary cancer. The patient underwent two cycles of palliative radiotherapy to the anal canal for control of pain and was discharged with full palliative support. She died approximately 3 wk after discharge. Case 2 A 63-year-old lady presented with obstructive jaundice due to a biliary stricture involving the hilum of the bile ducts and extending longitudinally into the distal common bile duct. Endoscopic retrograde cholangiopancreatographic brushings were inconclusive. She underwent further staging with a CT scan and laparoscopy. The laparoscopy showed no evidence of peritoneal disease, however the CT scan revealed thickening of the sigmoid colon. Colonoscopic biopsies confirmed a submucosal infiltrating moderately differentiated adenocarcinoma with no associated mucosal dysplasia. This was not supportive of a colorectal primary and favoured an extrinsic origin. Subsequent immunohistochemical analysis showed strong staining for CK7, with occasional positive staining for CK20; and negative CDX2, CK17 and Estrogen Receptor (ER) immunoreactivity. This profile, although not specific, was consistent with metastatic deposit from a hilar cholagiocarcinoma. She underwent palliative biliary stenting and was referred for palliative chemotherapy. DISCUSSION The colon, rectum and anal canal are rare sites for metastases for any type of malignancy. However, such cases have been reported from primary carcinomas of breast [19-22], lung [23,24], colon [25,26] and prostate [27]. Additionally, a small number of such metastases have been reported to occur in relation to cholangiocarcinomas [14,15] and pancreatic adenocarcinomas [13,16,17]. To the best of our knowledge metastasis to the anal canal from pancreatobiliary malignancies has not been reported before in the literature. The extrinsic nature of these metastatic deposits may not be apparent in superficial mucosal biopsies. Immunohistochemical studies may be warranted in such cases where there is a suspicion of metastases or if there are unusual features such as adenocarcinoma undermining an intact, non-dysplastic mucosal surface. In the two cases that we have presented (Table 1) the immunohistochemical profiles of the tumours were more consistent with pancreato-bilary origin than lower gastrointestinal tract origin although are not definitive and final diagnosis requires correlation with the clinical and radiological parameters. Ampullary adenocarcinomas can demonstrate either an intestinal (CK20/CDX2/MUC2 positive) or pancreaticobilary immunophenotype (CK7/CK17/ MUC1 positive) [28-31], as in case 1. The tumours demonstrating a pancreato-bilary phenotype can also co-express CK20, but this is usually only focal in distribution. On the contrary, the vast majority of colorectal adenocarcinomas demonstrate a typical lower gastrointestinal immunophenotype, being CK20 and CDX2 positive (CDX2 is an intestine-specific nuclear transcription factor, which can be used as a marker for intestinal-type differentiation of colorectal adenocarcinomas) [29,30]. We acknowledge that we do not have direct corroborative histology of the pancreato-biliary primary in the second case. However, unequivocal evidence of a locally advanced malignant pancreatic mass noted on staging CT. Pancreatic biopsy was not attempted due to poor performance status of the patient. In this case there was sufficient radiological and immunohistochemical evidence to support the diagnosis of metastatic deposit in the large 3694 April 7, 2014 Volume 20 Issue 13

320 Ejtehadi F et al. Coloanal metastases from pancreato-biliary cancers A CK7 CK17 MUC1 B CK7 MUC1 CK17 Figure 1 Immunohistochemical staining. A: Immunohistochemical staining of the surgical specimen demonstrating pancreato-biliary phenotype: cytokeratin-7/cytokeratin-17/mucin-1 (CK7/CK17/MUC1) positive and cytokeratin-20/homeobox protein CDX2/mucin-2 (CK20/CDX2/MUC2) negative; B: Immunohistochemical staining of the anal fissure biopsy demonstrating pancreato-biliary phenotype similar to the ampullary adenocarcinoma. intestine from the primary biliary cancer. Although pancreato-biliary malignancies commonly lead to metastatic deposits on the peritoneal surfaces, the cases described here did not have any evidence of diffuse peritoneal involvement. There was no evidence of perito- neal nodules, omental infiltration or ascites on any of the CT scans or staging laparoscopy. Therefore we believe that these are focal metastases to the colon and anal canal rather than a part of diffuse peritoneal involvement. Distal colonic and anorectal metastases are rare. They 3695 April 7, 2014 Volume 20 Issue 13

321 Ejtehadi F et al. Coloanal metastases from pancreato-biliary cancers Table 1 Summarising characteristics of the 2 cases Demographic information Mode of presentation Initial investigations and findings Site of primary lesion Site of metastases Histology of metastasis Outcome 79, female Presented with obstructive jaundice 63, female Presented with obstructive jaundice EUS, CT, ERCP, tissue diagnosis obtained after Whipple's resection Colonoscopy and biopsy, staging CT Ampullary Anal canal Adenocarcinoma, PB type. CK7/ CK17/MUC1 positive, (negative for K20/CDX2/MUC2) Hilum of bile ducts Sigmoid colon Palliation Microscopy Palliative Immunohistochemistry percutaneous strong CK7 staining, with occasional biliary stenting positive staining for CK20; and chemotherapy (negative CDX2, CK17 and ER immunoreactivity) CT: Computed tomography; EUS: Endoscopic ultrasound scan; ERCP: Endoscopic retrograde cholangiopancreatography. may present simultaneously with or in isolation from the primary, with symptoms identical to a primary colo-rectal tumour. The key to diagnosis is a high index of suspicion if the clinical picture is atypical coupled with specific immunohistochemical staining. Atypical immunohistochemical pattern that does not fit with a colorectal primary should raise suspicion regarding metastases from an extrinsic source. COMMENTS Case characteristics Case 1, a 79-year-old lady presented with peri-anal pain approximately 4 wk after discharge following a Whipple's procedure for an ampullary mass; case 2, a 63-year-old lady presented with obstructive jaundice. Clinical diagnosis Perianal pain, with an atypical anal fissure on examination under anaesthesia; obstructive jaundice, nil else. Differential diagnosis Benign anal fissure, anal cancer, metastatic disease; gallstone disease, benign biliary stricture, pancreatic cancer, cholangiocarcinoma, ampullary cancer, metastatic disease from unknown primary. Laboratory diagnosis Nortmal blood indices; liver function tests of obstructive picture. Imaging diagnosis Not applicable; endoscopic retrograde cholangiopancreatographic showed a proximal biliary stricture and staging computed tomography scan revealed thickening of the sigmoid colon. Pathological diagnosis Histopathological analysis revealed anal mucosa extensively infiltrated by a poorly differentiated carcinoma, demonstrating similar morphological appearance to the original ampullary adenocarcinoma [cytokeratin-7/cytokeratin-17/ mucin-1 (CK7/CK17/MUC1) positive CK20/CDX2/MUC2 negative]; colonoscopic biopsies confirmed a submucosal infiltrating moderately differentiated adenocarcinoma with strong positivity for CK7, occasional positive staining for CK20 and negative CDX2, CK17 and ER immunoreactivity, profile consistent with metastatic deposit from a hilar cholagiocarcinoma. Treatment Palliative chemoradiotherapy; palliative palliative biliary stenting and chemotherapy. Related reports Metastases of pancreatobiliary malignancies to the colon are extremely rare and metastasis to the anal canal has not been reported before in the literature to the best of our knowledge. Term explanation Immunohistochemistry is refers to the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues. Experiences and lessons Symptoms from distal colonic and anorectal metastases may be identical to a primary colorectal tumour and the key to diagnosis is a high index of suspicion if the clinical picture is atypical coupled with specific immunohistochemical staining. Peer review This is a report of two unusual metastases of pancreato-biliary malignancies to the sigmoid colon and anal canal. The cases stress the importance of complete physical examination, including rectodigital examination, and surveillance of the lower gastro-intestinal tract in upper gastro-intestinal and pancreato-biliary malignancies, even though the incidence of such metastasis is rare. REFERENCES 1 Patel T. Cholangiocarcinoma. 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322 Ejtehadi F et al. Coloanal metastases from pancreato-biliary cancers 10 Young JL Jr, Roffers SD, Ries LAG, Fritz AG, Hurlburt AA, editors. SEER summary staging manual-2000: Codes and coding instructions. Bethesda, MD: National Cancer Institute, Lee JH, Lee KG, Ha TK, Jun YJ, Paik SS, Park HK, Lee KS. Pattern analysis of lymph node metastasis and the prognostic importance of number of metastatic nodes in ampullary adenocarcinoma. Am Surg 2011; 77: [PMID: ] 12 Yeh CC, Jeng YM, Ho CM, Hu RH, Chang HP, Tien YW. Survival after pancreaticoduodenectomy for ampullary cancer is not affected by age. World J Surg 2010; 34: [PMID: DOI: /s y] 13 Bellows C, Gage T, Stark M, McCarty C, Haque S. Metastatic pancreatic carcinoma presenting as colon carcinoma. South Med J 2009; 102: [PMID: DOI: /SMJ.0b013e3181a8fad7] 14 Tokodai K, Kawagishi N, Miyagi S, Takeda I, Sato K, Akamatsu Y, Sekiguchi S, Ishida K, Satomi S. Intestinal obstruction caused by colonic metastasis from intrahepatic cholangiocarcinoma 6 years after removal of the primary tumor: report of a case. Surg Today 2012; 42: [PMID: DOI: /s ] 15 Fujii K, Goto A, Yoshida Y, Suzuki K, Matunaga Y, Shinomura Y. Education and imaging. Gastrointestinal: Transmural colonic metastasis arising from primary cholangiocarcinoma. J Gastroenterol Hepatol 2010; 25: 1329 [PMID: DOI: /j x] 16 Ogu US, Bloch R, Park G. A rare case of metachronous skip metastasis of pancreatic cancer to the colon. Am Surg 2012; 78: E342-E343 [PMID: ] 17 Fukatsu H, Nagahara Y, Ishiki K, Iwamura M, Hamada F. Pancreatic cancer metastasis to the rectum detected on colonoscopy. Endoscopy 2009; 41 Suppl 2: E167-E168 [PMID: DOI: /s ] 18 Hafez H. Cutaneous pancreatic metastasis: a case report and review of literature. Indian J Cancer 2007; 44: [PMID: ] 19 Bochicchio A, Tartarone A, Ignomirelli O, Latorre G, Cangiano R, Gallucci G, Coccaro M, Feudale E, Aieta M. Anal metastasis from breast cancer: a case report and review of the literature. Future Oncol 2012; 8: [PMID: DOI: /fon.12.9] 20 Puglisi M, Varaldo E, Assalino M, Ansaldo G, Torre G, Borgonovo G. Anal metastasis from recurrent breast lobular carcinoma: a case report. World J Gastroenterol 2009; 15: [PMID: ] 21 Efthimiadis C, Kosmidis C, Fotiadis P, Anthimidis G, Vasiliadou K, Mekras A, Ioannidou G, Basdanis G. Breast cancer metastatic to the rectum: a case report. Tech Coloproctol 2011; 15 Suppl 1: S91-S93 [PMID: DOI: / s ] 22 Matsuda I, Matsubara N, Aoyama N, Hamanaka M, Yamagishi D, Kuno T, Tsukamoto K, Yamano T, Noda M, Ikeuchi H, Tomita N, Hirota S. Metastatic lobular carcinoma of the breast masquerading as a primary rectal cancer. World J Surg Oncol 2012; 10: 231 [PMID: DOI: / ] 23 Kawahara K, Akamine S, Takahashi T, Nakamura A, Kusano H, Nakagoe T, Nakazaki T, Ayabe H, Tomita M. Anal metastasis from carcinoma of the lung: report of a case. Surg Today 1994; 24: [PMID: ] 24 Sakai H, Egi H, Hinoi T, Tokunaga M, Kawaguchi Y, Shinomura M, Adachi T, Arihiro K, Ohdan H. Primary lung cancer presenting with metastasis to the colon: a case report. World J Surg Oncol 2012; 10: 127 [PMID: DOI: / ] 25 Takahashi H, Ikeda M, Takemasa I, Mizushima T, Yamamoto H, Sekimoto M, Doki Y, Mori M. Anal metastasis of colorectal carcinoma origin: implications for diagnosis and treatment strategy. Dis Colon Rectum 2011; 54: [PMID: DOI: /DCR.0b013e318205e116] 26 Smiley D, Goldberg RI, Phillips RS, Barkin JS. Anal metastasis from colorectal carcinoma. Am J Gastroenterol 1988; 83: [PMID: ] 27 Abbas TO, Al-Naimi AR, Yakoob RA, Al-Bozom IA, Alobaidly AM. Prostate cancer metastases to the rectum: a case report. World J Surg Oncol 2011; 9: 56 [PMID: DOI: / ] 28 Duval JV, Savas L, Banner BF. Expression of cytokeratins 7 and 20 in carcinomas of the extrahepatic biliary tract, pancreas, and gallbladder. Arch Pathol Lab Med 2000; 124: [PMID: DOI: / (2000)12 4<1196:EOCAIC>2.0.CO;2] 29 Groisman GM, Bernheim J, Halpern M, Brazowsky E, Meir A. Expression of the intestinal marker Cdx2 in secondary adenocarcinomas of the colorectum. Arch Pathol Lab Med 2005; 129: [PMID: DOI: / (2005)129[920:EOTIMC]2.0.CO;2] 30 De Lott LB, Morrison C, Suster S, Cohn DE, Frankel WL. CDX2 is a useful marker of intestinal-type differentiation: a tissue microarray-based study of 629 tumors from various sites. Arch Pathol Lab Med 2005; 129: [PMID: DOI: / (2005)129[1100:CIAUMO]2.0.CO;2] 31 Moriya T, Kimura W, Hirai I, Takasu N, Mizutani M. Expression of MUC1 and MUC2 in ampullary cancer. Int J Surg Pathol 2011; 19: [PMID: DOI: / ] P- Reviewers: Lin JK, Redondo-Cerezo E S- Editor: Song XX L- Editor: A E- Editor: Ma S 3697 April 7, 2014 Volume 20 Issue 13

323 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. CASE REPORT Colonoscopy-induced ischemic colitis in patients without risk factors Sang Ok Lee, Sae Hee Kim, Sung Hee Jung, Chan Woong Park, Min Ji Lee, Jin A Lee, Hyun Cheol Koo, Anna Kim, Hyun-Young Han, Dong-Wook Kang Sang Ok Lee, Sae Hee Kim, Sung Hee Jung, Chan Woong Park, Min Ji Lee, Jin A Lee, Hyun Cheol Koo, Anna Kim, Division of Gastroenterology, Department of Internal Medicine, Eulji University College of Medicine, Eulji University Hospital, Daejeon , South Korea Hyun-Young Han, Department of radiology, Eulji University College of Medicine, Eulji University Hospital, Daejeon , South Korea Dong-Wook Kang, Department of pathology, Eulji University College of Medicine, Eulji University Hospital, Daejeon , South Korea Author contributions: Lee SO interpreted the data and wrote the manuscript; all authors contributed to analyzing the patient s data and revising the article; Kim SH approved the final manuscript. Correspondence to: Sae Hee Kim, MD, Division of Gastroenterology, Department of Internal Medicine, Eulji University College of Medicine, Eulji University Hospital, 1306 Dunsan-dong, Seo-Gu, Daejeon , South Korea. cozy129@eulji.ac.kr Telephone: Fax: Received: October 30, 2013 Revised: December 10, 2013 Accepted: January 14, 2014 Published online: April 7, 2014 Abstract Ischemic colitis is the most common form of intestinal ischemia. It is a condition that is commonly seen in the elderly and among individuals with risk factors for ischemia. Common predisposing conditions for ischemic colitis are major vascular occlusion, small vessel disorder, shock, some medications, colonic obstructions and hematologic disorders. Ischemic colitis following colonoscopy is rare. Here, we report two cases of ischemic colitis after a routine screening colonoscopy in patients without risk factors for ischemia Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Ischemic colitis; Colonoscopy; Hematochezia Core tip: Ischemic colitis following colonoscopy is rare and is predominantly involved at the sigmoid colon and splenic flexure. However, our two cases were found on the right side of the colon after a routine screening colonoscopy in patients without risk factors for ischemia. We believe these cases are interesting and instructive for gastroenterologists. Lee SO, Kim SH, Jung SH, Park CW, Lee MJ, Lee JA, Koo HC, Kim A, Han HY, Kang DW. Colonoscopy-induced ischemic colitis in patients without risk factors. World J Gastroenterol 2014; 20(13): Available from: URL: DOI: org/ /wjg.v20.i INTRODUCTION Ischemic colitis is the most common form of intestinal ischemia and accounts for 1 in 1000 hospitalizations [1]. Although more frequent in the elderly, younger patients may also be affected [2]. Clinically, ischemic colitis manifests as a spectrum of injuries from transient self-limited ischemia involving the mucosa and submucosa, which has a good prognosis, to acute fulminant ischemia with transluminal infarction, which may progress to necrosis and death. Colon ischemia was first described as being caused by the ligation of the inferior mesenteric artery during aortic reconstruction or colon resection [3,4], but it is now recognized to have many potential causes. Common predisposing conditions for ischemic colitis are major vascular occlusion, small vessel disorder, shock, some medications, colonic obstructions and hematologic disorders. However, very few cases have been reported following endoscopic examinations thus far [5,6]. Here, we present two cases of ischemic colitis following a routine screening colonoscopy in young patients with no risk 3698 April 7, 2014 Volume 20 Issue 13

324 Lee SO et al. Colonoscopy-induced ischemic colitis A B C D E F Figure 1 Case 1 findings. A: Initial colonoscopic findings, Normal colonoscopic findings in ascending colon; B, C Follow up colonoscopic findings; B: The results showed severe petechial hemorrhages with edematous mucosal inflammation in the ascending colon; C: The results showed longitudinal ulcers and hemorrhages in the ascending colon; D: Microscopic findings of affected ascending colon (H and E stain, 100). Biopsy was compatible with the findings of ischemic colitis, showing erosions and hemorrhages; E: Abdominal computed tomography findings, the results shows diffuse wall thickening of the ascending colon and fluid collection around the ascending colon; F: Follow up colonoscopic findings in 6 mo, the results showed totally normal colonoscopic findings in the ascending colon. factors. CASE REPORT Case 1 A 47-year-old woman presented to the emergency room complaining of abdominal pain with hematochezia 7 h after a routine screening colonoscopy in the primary clinic. She had no history of coagulopathy or embolic risk factors and a body mass index (BMI) of 22.3 kg/m 2. The screening colonoscopic findings were normal (Figure 1A) except for a single colonic polyp in the transverse colon, which was removed by a forceps biopsy. The patient was prepared for the colonoscopy with a 4 L splitdose of polyethylene glycol, and the procedure itself had been uneventful with insufflations of room air during On admission, her initial blood pressure was 130/80 mmhg, and her heart rate was 70 beats per minute. Her abdomen was soft to palpation, with some tenderness on the right lower quadrant but no sign of peritonitis. The initial laboratory data showed elevated WBC 14.0 k/μl, but all other levels were normal, including HGB 13.2 g/dl and CRP 0.15 mg/dl. Stool cultures were negative, as was the hypercoagulable work-up. We believed minor bleeding had occurred at the biopsy site, but the bleeding quickly stopped. Therefore, we closely observed her conditions and vital signs with bowel rest, intravenous fluids and empirical antibiotics. Despite these measures, her abdominal pain and hematochezia persisted. On the next day, another colonoscopy showed severe submucosal hemorrhages with mucosal edema and longitudinal ulcers in the ascending colon (Figure 1B and C). The biopsy was compatible with findings of ischemic colitis and showed erosions and hemorrhages (Figure 1D). The possibility of bleeding from the forcep biopsy site was excluded by a complete colonoscopic examination. Abdominal com April 7, 2014 Volume 20 Issue 13

325 Lee SO et al. Colonoscopy-induced ischemic colitis A B C D Figure 2 Case 2 findings. A: The results showed diffuse edematous mucosal inflammations, submucosal hemorrhages, erosions, ulcers and friability; B: The results showed longitudinal ulcers and hemorrhages; C, D: Abdominal computed tomography findings. The results shows submucosal edematous changes and decreased enhancement of thickened colonic wall from the ascending colon to the distal transverse colon. puted tomography (CT) scans showed diffuse wall thickening of the ascending colon and fluid collection around the ascending colon (Figure 1E). The patient was managed supportively and resumed her diet on the 5 th hospital day. She recovered uneventfully and was discharged on the 9 th hospital day without complications. We performed a follow up colonoscopy after 6 mo and observed totally normal colonoscopic findings (Figure 1F). Case 2 A 40-year-old man presented to the emergency room complaining of hematochezia with diffuse abdominal pain 18 hours after a routine screening colonoscopy in the primary clinic. He had no history of coagulopathy or embolic risk factors and a BMI of 19.8 kg/m 2. The screening colonoscopic findings were normal. The patient was prepared for the colonoscopy with a 4 L splitdose of polyethylene glycol, and the procedure itself had been uneventful with insufflations of room air during On admission, his blood pressure was 130/80 mm/hg, and his heart rate was 75 beats per minute. His abdomen was soft to palpation with moderate tenderness on the whole abdomen; however, there was no sign of peritonitis. The initial laboratory data showed an elevated WBC k/μl and CRP 1.61 mg/dl, but all other data were normal, including HGB 14.8 g/dl. The stool cultures were negative, and a hypercoagulable work-up was not performed. We decided to perform a colonoscopy, but not above the distal transverse colon due to the patient s pain and very friable colonic mucosa. Therefore, no biopsies were performed either. The colonoscopy showed diffuse edematous mucosal inflammations, submucosal hemorrhages, erosions and longitudinal ulcers with friability in the distal transverse colon and proximal sigmoid colon (Figure 2A and B). Abdomen CT scans showed submucosal edematous changes and decreased enhancement of thickened colonic wall from the ascending colon to the distal transverse colon (Figure 2C and D). The patient was also treated supportively with bowel rest, intravenous fluids and empirical antibiotics. He recovered without complications and was discharged on the 9 th hospital day. We followed up in the outpatient department, and he complained of no symptoms. DISCUSSION Ischemic colitis is the most common form of intestinal ischemia. Although the incidence of ischemic colitis is increased in the elderly and among those with many with risk factors for vascular disease, an index lesion on angiography is unusual [2]. When present, abnormalities may include the narrowing of the small vessels and tortuosity of the long colic arteries [7]. Rather than a specific vascular lesion, there appears to be an acute, self-limited compromise in intestinal blood flow, which is inadequate for meeting the metabolic demands of the colon [8]. The colon is predisposed to ischemia by its relatively low blood flow compared with the rest of the gastrointestinal tract [9,10]. Experimental distension has been found to increase intraluminal pressure, reduce total blood and reduce the arteriovenous oxygen gradient in the colonic wall [11]. This most likely resembles the mechanism for the 3700 April 7, 2014 Volume 20 Issue 13

326 Lee SO et al. Colonoscopy-induced ischemic colitis rare occurrence of ischemic colitis during colonoscopy or barium enema [6,12]. There are numerous conditions that predispose patients to ischemic colitis. The most common mechanism is hypotension from sepsis or impaired left ventricular function and hypovolemia from dehydration or hemorrhage, producing a compromise in systemic perfusion and triggering a reflex mesenteric vasoconstriction [2]. Numerous medications may produce colonic ischemia by a similar mechanism. The most common drugs are antihypertensive agents, diuretics, non-steroidal anti-inflammatory drugs, digoxin, oral contraceptives, pseudoephedrine, cocaine and alosetron [2]. Predisposing factors for colonic ischemia in young adults include vasculitis, abdominal surgery, use of cocaine, oral contraceptive pills and nonsteroidal anti-inflammatory drugs, hypercoagulable states, colonic obstruction and marathon running [13]. None of these risk factors were present in our cases. Most patients present with the acute onset of mild, crampy abdominal pain and tenderness over the affected bowel. Within 24 h, there is usually passage of bright red or maroon blood often mixed with stool. Usually blood loss is minimal, without hemodynamic deterioration or the need for transfusion [2,9]. Any part of the colon may be affected, but (different pont size) the left colon is the predominant location in approximately 75% of patients [10]. Splenic flexure may be the most common site, as it is involved in one-quarter of patients [10], and isolated right colon ischemia occurs in approximately 10% of cases [13]. Because the rectosigmoid junction and splenic flexure may be vulnerable in systemic low-flow states due to the watershed area called Sudeck s point, where the sigmoidal artery meets the superior colic artery, and Griffith s point, where the mid-colic artery meets the left colic artery [14]. In our two cases, right colon ischemia developed. This incidence may be explained by the potentially increased vulnerability of the right colon to a systemic low-flow state, as the marginal artery of Drummond is poorly developed at this location in 50% of the population [15]. The diagnosis of ischemic colitis depends on characteristic findings in the appropriate clinical setting. Laboratory markers for ischemia, such as serum lactate, lactate dehydrogenase, alkaline phosphatase and metabolic acidosis, may be present, but they are uncommon. Plain abdominal films are insensitive and nonspecific, but they are important when excluding other disorders. A barium enema may show findings suggestive of ischemic colitis, such as thumbprinting, but this procedure is nonspecific and is present in many other infective or inflammatory colitis [16]. Abdominal computed tomography may show suggestive findings of ischemic colitis in up to 89% of patients. The most common finding is segmental circumferential wall thickening of affected colon [2]. Colonoscopy has largely supplanted the barium enema as the diagnostic modality of choice because of its higher sensitivity for detecting mucosal changes and its ability to obtain biopsy specimens if necessary. However, as there are no specific endoscopic findings for ischemic colitis, the clinical situations must be considered. Findings that favor ischemic colitis rather than inflammatory bowel disease are in segmental areas of injury, abrupt transition between normal and affected mucosa, rectal sparing and rapid resolution of mucosal changes on serial colonoscopy [2]. Treatment varies with the severity of the ischemia. In the absence of gangrene or perforation, supportive care is sufficient. Patients need bowel rest, intravenous fluid and often empirical broad-spectrum antibiotics to minimize bacterial translocation and sepsis. Ischemic colitis following colonoscopy is comparatively rare. There have been 5 domestic cases of ischemic colitis after colonoscopy [14]. However, relatively young patients with no risk factors or those who prepare their bowels with polyethylene glycol was first noticed in this report. The causes of developing ischemic colitis following colonoscopy are damage to microvasculatures, mechanical compression, increased intraluminal pressure and hyperextension [17]. If the colonic intraluminal pressure rises up to mmhg, reversible circulatory compromises may occur; however, if the intraluminal pressure rises above 50 mmhg, irreversible damages may occur [18]. In these cases, approximately 15 min encompassed the whole colonoscopic procedure, and no hyperextension or hyperinflation were performed. Ischemic colitis as a complication of colonoscopy is rare, but the incidence can increase when a prior history of intra-abdominal surgery, tortuous colon, longer procedure time or other risk factors of ischemia is present. Thus, we need to pay more attention to reducing colonoscopic procedure time, hyperinflation and hyperextension. Further, we should check the risk factors for ischemia and supply sufficient fluid to prevent the risk of ischemia before bowel preparation. COMMENTS Case characteristics A 47-year-old woman and 40-year-old man without prior medical history presented with abdominal pain with hematochezia. Clinical diagnosis Their abdomen were soft to palpation, with tenderness on right lower quadrant (case 1) and whole abdomen (case 2) but no sign of peritonitis. Differential diagnosis Infectious colitis, Inflammatory bowel disease, Diverticulitis, Colon cancer. Laboratory diagnosis In the case 1, WBC 14.0 k/μl; HGB 13.2 g/dl; CRP 0.15 mg/dl and In case 2, WBC k/μl; HGB 14.8 g/dl ; CRP 1.61 mg/dl; stool cultures were negative. Imaging diagnosis Abdominal computed tomography scans showed diffuse wall thickening of ascending colon and fluid collection around ascending colon (case 1) and submucosal edematous changes and decreased enhancement of thickened colonic wall from ascending colon to distal transverse colon (case 2). Pathological diagnosis Colonoscopic biopsy in case 1 was compatible with findings of ischemic colitis, showing erosions and hemorrhages. Treatment The patients were treated supportively with bowel rest, intravenous fluids and empirical antibiotics. Related reports It is unclear how colonoscopy induces ischemic colitis and our cases have right sided ischemic colitis and they do not have ischemic risk factors April 7, 2014 Volume 20 Issue 13

327 Lee SO et al. Colonoscopy-induced ischemic colitis Experiences and lessons It is rare that ischemic colitis following colonoscopy is occurred in patients without risk factors of ischemia. Peer review Awareness of the risk of this potential complication following colonoscopy in patients without risk factors of ischemia may lead to prompt diagnosis and effective treatment with improved outcome. REFERENCES 1 Theodoropoulou A, Koutroubakis IE. Ischemic colitis: clinical practice in diagnosis and treatment. World J Gastroenterol 2008; 14: [PMID: DOI: / wjg ] 2 Green BT, Tendler DA. Ischemic colitis: a clinical review. South Med J 2005; 98: [PMID: DOI: /01.SMJ ] 3 SHAW RS, GREEN TH. Massive mesenteric infarction following inferior mesenteric-artery ligation in resection of the colon for carcinoma. N Engl J Med 1953; 248: [PMID: DOI: /NEJM ] 4 SMITH RF, SZILAGYI DE. Ischemia of the colon as a complication in the surgery of the abdominal aorta. Arch Surg 1960; 80: [PMID: DOI: /archsurg ] 5 Church JM. Ischemic colitis complicating flexible endoscopy in a patient with connective tissue disease. Gastrointest Endosc 1995; 41: [PMID: DOI: / S (05) ] 6 Cremers MI, Oliveira AP, Freitas J. Ischemic colitis as a complication of colonoscopy. Endoscopy 1998; 30: S54 [PMID: DOI: /s ] 7 Binns JC, Isaacson P. Age-related changes in the colonic blood supply: their relevance to ischaemic colitis. Gut 1978; 19: [PMID: DOI: /gut ] 8 Tendler DA. Acute intestinal ischemia and infarction. Semin Gastrointest Dis 2003; 14: [PMID: ] 9 Reinus JF, Brandt LJ, Boley SJ. Ischemic diseases of the bowel. Gastroenterol Clin North Am 1990; 19: [PMID: ] 10 Gandhi SK, Hanson MM, Vernava AM, Kaminski DL, Longo WE. Ischemic colitis. Dis Colon Rectum 1996; 39: [PMID: DOI: /BF ] 11 Boley SJ, Agrawal GP, Warren AR, Veith FJ, Levowitz BS, Treiber W, Dougherty J, Schwartz SS, Gliedman ML. Pathophysiologic effects of bowel distention on intestinal blood flow. Am J Surg 1969; 117: [PMID: DOI: / (69) ] 12 Versaci A, Macrì A, Scuderi G, Bartolone S, Familiari L, Lupattelli T, Famulari C. Ischemic colitis following colonoscopy in a systemic lupus erythematosus patient: report of a case. Dis Colon Rectum 2005; 48: [PMID: DOI: /s z] 13 Brandt LJ, Boley SJ. Colonic ischemia. Surg Clin North Am 1992; 72: [PMID: ] 14 Lee KW, Han KH, Kang JW, Lee JH, Jang KH, Kim YD, Kang GH, Cheon GJ. Two cases of ischemic colitis after colonoscopy. Korean J Gastrointest Endosc 2010; 41: Elder K, Lashner BA, Al Solaiman F. Clinical approach to colonic ischemia. Cleve Clin J Med 2009; 76: [PMID: DOI: /ccjm.76a.08089] 16 Iida M, Matsui T, Fuchigami T, Iwashita A, Yao T, Fujishima M. Ischemic colitis: serial changes in double-contrast barium enema examination. Radiology 1986; 159: [PMID: ] 17 Lee BO, Choi H, Kang IJ, Kim KY, Lee HI, Kim JP, Lee BI, Kim BW, Cho SH, Choi KY, Chung IS, Cha SB. A case of ischemic colitis following colonoscopy. Korean J Gastrointest Endosc 2003; 27: Park JE, Moon W, Nam JH, Kim NH, Kim SH, Park MI, Park SJ, Kim KJ. [A case of ischemic colitis presenting as bloody diarrhea after normal saline enema]. Korean J Gastroenterol 2007; 50: [PMID: ] P- Reviewers: Iijima H, Meucci G, Naito Y S- Editor: Qi Y L- Editor: A E- Editor: Wang CH 3702 April 7, 2014 Volume 20 Issue 13

328 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. CASE REPORT Successful treatment of liver abscess secondary to foreign body penetration of the alimentary tract: A case report and literature review Lee-Won Chong, Cheuk-Kwan Sun, Chin-Chu Wu, Cheuk-Kay Sun Lee-Won Chong, Cheuk-Kay Sun, Division of Hepatology and Gastroenterology, Department of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital, Taipei 111, Taiwan Lee-Won Chong, Institute of Clinical Medicine, School of Medicine, National Yang Ming University, Taipei 112, Taiwan Cheuk-Kwan Sun, Department of Emergency Medicine, E-Da Hospital, I-Shou University, Kaohsiung 824, Taiwan Cheuk-Kwan Sun, School of Medicine, Chung Shan Medical University, Taichung City 40201, Taiwan Chin-Chu Wu, Department of Radiology, Shin Kong Wu Ho-Su Memorial Hospital, Taipei 111, Taiwan Cheuk-Kay Sun, School of Medicine, College of Medicine, Fu Jen Catholic University, Taipei 242, Taiwan Cheuk-Kay Sun, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan Author contributions: Chong LW and Sun CK designed the study and wrote the manuscript; Chong LW, Wu CC, Sun CK collected and analyzed the data; all authors have read and approved the final version to be published. Correspondence to: Cheuk-Kay Sun, MD, Division of Hepatology and Gastroenterology, Department of Internal Medicine, Shin Kong Wu Ho-Su Memorial Hospital, No. 95, Wen Chang Road, Shih Lin District, Taipei 111, Taiwan. M000651@ms.skh.org.tw Telephone: Fax: Received: November 5, 2013 Revised: December 24, 2013 Accepted: January 19, 2014 Published online: April 7, 2014 Abstract Hepatic abscess caused by foreign body penetration of the alimentary tract is rare. We report a case of gastric antrum penetration due to a toothpick complicated by liver abscess formation. A 41-year-old man was admitted to our hospital with the chief complaint of upper abdominal pain for 2 mo. Esophagogastroduodenoscopy performed at a local clinic revealed a toothpick penetrating the gastric antrum. Computed tomography (CT) of the abdomen at our hospital revealed a gastric foreign body embedded in the posterior wall of gastric antrum with regional phlegmon over the lesser sac and adhesion to the pancreatic body without notable vascular injury, and a hepatic abscess seven cm in diameter over the left liver lobe. Endoscopic removal of the foreign body was successfully performed without complication. The liver abscess was treated with parenteral antibiotics without drainage. The patient s recovery was uneventful. Abdominal ultrasonography demonstrated complete resolution of the hepatic abscess six months after discharge. Relevant literature from the PubMed database was reviewed and the clinical presentations, diagnostic modalities, treatment strategies and outcomes of 88 reported cases were analyzed. The results showed that only 6 patients received conservative treatment with parenteral antibiotics, while the majority underwent either image-guided abscess drainage or laparotomy. Patients receiving abscess drainage via laparotomy had a significantly shorter length of hospitalization compared with those undergoing imageguided drainage. There was no significant difference in age between those who survived and those who died, however, the latter presented to hospitals in a more critical condition than the former. The overall mortality rate was 7.95% Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Hepatic abscess; Foreign body; Endoscopy; Laparotomy; Drainage Core tip: Hepatic abscess caused by foreign body penetration of the alimentary tract is rare and most cases are treated surgically. We demonstrate the successful conservative treatment of a patient by combining endoscopic removal of the foreign body and broadspectrum antibiotic coverage. A review of the literature highlighted the importance of ruling out the possibility 3703 April 7, 2014 Volume 20 Issue 13

329 Chong LW et al. Penetrating gastrointestinal foreign body-induced hepatic abscess of foreign body penetration of the alimentary tract in patients with liver abscesses and no identifiable underlying condition, particularly those refractory to conventional treatment. Appropriate diagnostic strategies, including imaging studies and surgical exploration, may be indicated for early diagnosis and timely treatment of this potentially lethal condition. Chong LW, Sun CK, Wu CC, Sun CK. Successful treatment of liver abscess secondary to foreign body penetration of the alimentary tract: A case report and literature review. World J Gastroenterol 2014; 20(13): Available from: URL: DOI: INTRODUCTION Accidental ingestion of foreign bodies is not uncommon and about 80%-90% of ingested foreign bodies pass through the gut uneventfully within 1 wk [1]. In fact, less than 1% of patients who ingested a foreign body developed complications such as perforation or penetration of the gastrointestinal tract [2]. The development of a hepatic abscess secondary to a foreign body penetrating the gastrointestinal tract is even more unusual. We report a case of liver abscess induced by an ingested foreign body which penetrated the gastric antrum. The abscess was successfully treated by endoscopic retrieval of the foreign body and broad-spectrum antibiotic coverage. We performed a search of relevant English literature in the PubMed database, reviewed the cases reported, and analyzed the data. Crucial information regarding this disease entity was then collected, reviewed, and summarized in this study. CASE REPORT A 41-year-old man with no previous history of medical illness presented to our outpatient clinic with intermittent upper abdominal pain and acid regurgitation for two months. The patient did not experience nausea, vomiting, melena, passage of bloody stool or change in bowel habit. However, he recalled that fever occurred when the abdominal pain first started. Although he denied foreign body ingestion, esophagogastroduodenoscopy performed at a local clinic revealed a toothpick penetrating the posterior wall of the gastric antrum. He was then referred to our emergency department for further treatment. On arrival at the emergency department, his vital signs were stable with a body temperature of 36.9, pulse rate of 95 breaths/min, respiratory rate of 16 breaths/min, and blood pressure of 140/71 mmhg. Physical examination was essentially normal except for mild epigastric tenderness. Laboratory studies showed a white blood cell count of 14600/mm 3 with 66.4% neutrophils, a hemoglobin level of 13.4 mg/dl, and a C-reactive protein concentration of mg/dl. Serum biochemical analyses includ- ing glucose level, renal function, liver enzymes, amylase, lipase, and alpha-fetoprotein, were all within normal limits. Blood culture yielded no bacterial growth. Chest and abdominal radiographs were unremarkable. Computed tomography (CT) of the abdomen revealed a hyperdense, linear foreign body within the gastric antrum with transgastric penetration through the posterior wall of the antrum and close contact with the pancreatic body (Figure 1). Significant edematous wall thickening of the gastric antrum and lower body with regional phlegmon over the lesser sac extending to the porta hepatis was also found. There was no apparent vascular injury. In addition, a 7-cm hypodense mass was found in the left liver lobe (Figure 2). The initial diagnosis was hepatic abscess complicating foreign body penetration of the gastric antrum. The foreign body, a 7-cm wooden toothpick, was successfully removed endoscopically using a pair of grasping forceps (FG-44NR-1, Olympus, Tokyo, Japan) (Figure 3) without notable complications. His upper abdominal pain subsided dramatically after the foreign body was removed. A parenteral proton pump inhibitor was prescribed for the small gastric ulcer resulting from removal of the foreign body. Percutaneous drainage of the liver abscess was not performed due to the absence of liquefaction. Intravenous antibiotics including ceftriaxone 2 g/d and metronidazole 500 mg every eight hours were administered. The patient was discharged after 10 d of parenteral antibiotic treatment. He remained symptom-free and treated with an oral antibiotic (levofloxacin 500 mg/d) for a further two weeks during follow-up at the outpatient clinic until C-reactive protein decreased to the normal limits. After discharge, the patient underwent serial abdominal ultrasonographic examinations every two weeks for one month and then monthly to confirm resolution of the liver abscess. Complete resolution of the hepatic abscess was noted six months after discharge. DISCUSSION Review of literature We searched the PubMed database for relevant English literature from 1955 to 2013 using the key words liver abscess, hepatic abscess, and foreign body. The clinical presentations, diagnostic modalities, treatment strategies and outcomes of the reported cases were reviewed and analyzed. All statistical analyses were performed using commercially available software (SPSS, version 15.0 for Windows). Data were expressed as mean ± SD. The Student t test was used for comparison of two different sets of continuous values. Differences were considered statistically significant when P < Background review The first case of hepatic abscess secondary to gastrointestinal tract perforation caused by a foreign body was reported by Lambert in 1898 [3]. Since then, migrated foreign bodies, albeit uncommon, have been increasingly recognized as a potential cause of failure in the treatment 3704 April 7, 2014 Volume 20 Issue 13

330 Chong LW et al. Penetrating gastrointestinal foreign body-induced hepatic abscess A A B B Figure 1 Imaging studies with contrast-enhanced computed tomography scan. A: A hyperdense linear foreign body embedded in the posterior wall of the antrum with transgastric penetration and attachment to the upper pancreatic border (arrow); B: Sagittal multiplanar reformation image demonstrating close contact of the foreign body to the pancreas without evidence of vascular injury (arrow). Figure 2 Liver abscess demonstrated in imaging study. Contrast-enhanced computed tomography scan showing a 7-cm hypodense mass in the lateral segment of the left hepatic lobe (outlined by arrows). of hepatic abscesses [4]. Demography and clinical manifestations In the present study, a total of 79 journal articles were identified with 88 patients reviewed [4-82], including 64 (73%) males and 24 (27%) females. The mean age of the patients was 50.4 ± 18.6 years (ranging from 11 mo to 86 years). Although the signs and symptoms of hepatic abscesses resulting from gastrointestinal perforation by foreign bodies were often subtle and non-specific [65], Figure 3 Endoscopic retrieval of the foreign body penetrating the stomach. A: Removal of the toothpick penetrating the posterior wall of the gastric antrum by a pair of grasping forceps; B: The foreign body removed: a 7-cm long wooden toothpick with two sharp ends. abdominal pain (77.3%) and fever (58%) were the most common, followed by vomiting (19.3%) and nausea (13.6%). The frequencies of clinical manifestations in this study were consistent with those of a previous study [65]. However, weight loss, which was considered a feature of the systemic response to an ongoing infection such as abscess formation, was not apparent in the present study. Moreover, the value of a history of foreign body ingestion as a guide for diagnosis was disappointing as only 5% of patients reported a positive history [4]. In addition, our patient did not recall any episode of foreign body ingestion even after the diagnosis was made. Foreign bodies Of the foreign bodies reported, the frequency of occurrence in decreasing order was fish bone (33%), toothpick (27.3%), chicken bone (12.5%), and needle (9.1%), while other objects including clothespin [75], toothbrush [38,40], rosemary twig [36], pacemaker [34], rabbit bone [70], pen [59], lobster shell [77], metal wire [43], and dental plate [67] have also been sporadically documented. Similar findings were observed in a previous study [4]. The literature review in the present study demonstrated that the size of the foreign bodies ranged from as small as 1 cm (fish bone) [72] to over 19 cm (toothbrush) [38]. Site of perforation, location and size of liver abscess The review of available literature showed that the stom April 7, 2014 Volume 20 Issue 13

331 Chong LW et al. Penetrating gastrointestinal foreign body-induced hepatic abscess Table 1 Diagnostic tools adopted for the diagnosis of foreign body penetration of the alimentary tract from the literature review Diagnostic tools n (%) Computed tomography 53 (60.20) Ultrasonography 21 (23.90) Radiographs 14 (15.90) Laparotomy 13 (14.80) Autopsy 7 (7.95) Esophagogastroduodenoscopy 4 (4.55) Colonoscopy 3 (3.41) Endoscopic ultrasonography 1 (1.14) ach was the most common site of perforation (40.9%), followed by the duodenum (20.5%) and colon (11.4%). These results are similar to those of a previous study which reported that the stomach and duodenum were the most common sites of foreign body-induced perforation [65]. With regard to the location of the hepatic abscess, the left lobe was most commonly affected (65.9%), followed by the right lobe (29.5%), and bilobar involvement (4.5%). The size of the liver abscesses ranged from 2 to 16 cm (mean, 6.82 ± 3.09 cm). In contrast to cryptogenic hepatic abscesses which often affect the right liver lobe, a predominance of left lobe involvement was noted in liver abscesses induced by foreign body penetration [4]. Furthermore, liver abscess secondary to foreign body penetration of the gastrointestinal tract should be ruled out when encountering a lesion without an identifiable underlying condition combined with treatment failure [4]. Establishment of diagnosis Diagnosis was made by CT in the majority of patients (53/88, 60.2%), followed by ultrasonography (21/88, 23.9%), radiographs (14/88, 15.9%), and laparotomy (13/88, 14.8%). Combined diagnostic modalities were also adopted, including the combination of ultrasonography and CT (13/88, 14.8%), radiographs and CT (3/88, 3.4%) as well as the combination of radiographs, ultrasonography and CT (6/88, 6.8%). Initial diagnosis was not established in two patients who presented to hospitals with persistent symptoms during their second visit when correct diagnoses were made [49,63]. Diagnosis was not established in seven cases until autopsy [13,20,22,26,27,71]. The frequencies of use of different diagnostic modalities are summarized in Table 1. The choice of diagnostic modality depends on the nature and size of the ingested object. For instance, although plain abdominal radiography is usually the initial screening imaging study of choice for patients with a complaint of abdominal pain, the ingested foreign body is usually not identified on plain radiography unless it is radiopaque. On the other hand, a metallic object is easily detected by plain radiography, despite poor definition of the exact location. In such cases, abdominal ultrasonography may be a convenient and radiation-free screening tool for identifying the abscess and possibly the foreign body. CT, on the other hand, is the preferred imaging study for diagnosis due to its high resolution and accuracy in the identification of foreign bodies [44]. It is also useful in evaluating the depth and complication of the penetration. Indeed, the literature review in the present study showed that although there is no unanimous gold standard for the diagnosis of small-sized foreign bodies that have penetrated the alimentary tract, CT was the most commonly used diagnostic tool (60.2%). However, the accuracy of CT is limited by the lack of observer awareness. A high index of suspicion must be maintained for the correct diagnosis. It is also noteworthy that diagnosis was established only via laparotomy in up to 14.8% of all reported patients whose recovery was uneventful. Therefore, timely surgical exploration is recommended for patients with diagnostic uncertainties. Endoscopy may aid in diagnosis when it is performed at an early stage, before complete migration of the foreign body and mucosal healing [51]. In our patient, a toothpick penetrating the gastric antrum was disclosed by esophagogastroduodenoscopy as his initial presentations were upper abdominal pain and acid regurgitation. CT later revealed a 7-cm left hepatic lobe abscess. Hepatic abscesses may result from hematogenous dissemination of pathogens via either the portal venous system from the gastrointestinal tract or the hepatic artery from sepsis, ascending cholangitis, or local spread of infection. Development of the hepatic abscess in our case was probably caused by the spread of phlegmon along the lesser sac. The clinical manifestations of hepatic abscess secondary to foreign body perforation vary and are usually subtle. In our case, classic symptoms of pyogenic hepatic abscess such as high fever and severe abdominal pain were absent. Adhesions from slow progressive inflammatory or fibrotic reactions (Figure 1) may have prevented free intra-peritoneal spillage of gastrointestinal contents and the resulting full-blown peritonitis which usually presents with high fever and severe abdominal pain. In the current study, the lack of significant differences in demographic, clinical manifestations, and pathogens between the survivors and the patients who died, except for the correct diagnosis in the former, highlighted the importance of timely diagnosis of this condition. Pathogens With regard to the identification of pathogens, a review of the relevant literature showed a single bacterial strain in the majority of cases (54.5%) with an incidence of two bacterial flora and multi-flora (i.e., three or more) being 18.2% and 12.7%, respectively. Negative culture results were noted in 14.5% of all reported cases, whereas bacterial culture was positive in only one of six (16.7%) patients in the literature with undrainable abscesses. Of all the identified pathogens, the most commonly isolated was Streptococcus species (72.3%), followed by Escherichia coli (17%) and Klebsiella pneumoniae (10.6%) (Table 2). One patient with identified candidal infection was found to have received a local steroid injection for long-standing lumbar 3706 April 7, 2014 Volume 20 Issue 13

332 Chong LW et al. Penetrating gastrointestinal foreign body-induced hepatic abscess Table 2 Prevalence of bacterial flora identified in patients with foreign body penetration of the alimentary tract with liver abscess formation Table 4 Management strategies for liver abscesses caused by foreign body penetration of the alimentary tract in the 88 reported patients Bacterial flora Prevalence No. of patients Streptococcus sp % 34 Escherichia coli 17.00% 8 Klebsiella pneumoniae 10.60% 5 Gram (+) cocci 6.38% 3 Bacteroids sp. 4.26% 2 Eikenella corrodens 4.26% 2 Enterobacter cloacae 4.26% 2 Mixed anaerobes 4.26% 2 Staphylococcus aureus 4.26% 2 Anaerobic Gram (+) cocci 2.13% 1 Candida sp. 2.13% 1 Enterococcus sp. 2.13% 1 Gram (+) bacilli 2.13% 1 Gram (-) bacilli 2.13% 1 Proteus sp. 2.13% 1 Table 3 Procedures for foreign body removal in 88 reported patients Procedures n (%) Surgery 62 (70.50) Laparotomy 54 (61.40) Laparoscopy 8 (9.10) Endoscopic intervention 10 (11.36) Esophagogastroduodenoscopy 4 (4.55) Colonoscopy 4 (4.55) Sigmoidoscopy 1 (1.14) Single balloon enteroscopy 1 (1.14) Autopsy 7 (7.95) Foreign body not removed 3 (3.41) Spontaneous passage 2 (2.27) Not mentioned 2 (2.27) Percutaneous interventional radiological approach 1 (1.14) Ultrasound guided fluoroscopy 1 (1.14) and leg pain. An immunocompromised status due to prolonged steroid use, therefore, may have contributed to the infection. As Streptococcus species, Escherichia coli and Klebsiella pneumoniae were the most commonly identified pathogens, empirical antibiotic therapy with ampicillin/sulbactam or second-generation cephalosporins (e.g., cefoxitin) may be included in the initial treatment regimen. Treatment strategy and hospital course The recommended treatment protocol for this clinical entity comprises removal of the foreign body and drainage of the hepatic abscess. In cases of hepatic abscesses related to foreign body migration, the overall rate of cure without foreign body removal is low (9.5%) [4]. Therefore, removal of the foreign body is critical for resolving the abscess and closure of the fistulous tract. Strategies for the removal of a foreign body penetrating the gastrointestinal tract include laparotomy as well as laparoscopic, endoscopic, or percutaneous interventional radiological approaches [23,31,51,62,63]. The literature review showed that the foreign bodies were removed by surgery (laparotomy Procedures n (%) Drainage via laparotomy 43 (48.86) Image guided drainage 18 (20.45) Hepatectomy 8 (9.10) Laparotomy 6 (6.82) Laparoscopy 2 (2.27) Autopsy 7 (7.95) Antibiotics alone 6 (6.82) Laparoscopic drainage 5 (5.68) Not mentioned 1 (1.14) or laparoscopic approach) in 62 (70.5%) patients, and by endoscopy in 10 (11.4%) patients. Other approaches included percutaneous radiological intervention [62] (n = 1, 1.1%) and ultrasound-guided fluoroscopy [29] (n = 1, 1.1%). The foreign bodies were allowed to remain in place in three (3.4%) patients [17,53,80], whereas spontaneous passage was noted in two (2.3%) cases [10,46] (Table 3). On the other hand, liver abscesses were treated by drainage via laparotomy in the majority of patients (n = 43, 48.9%), followed by image-guided drainage (n = 18, 20.5%), hepatectomy (n = 8, 9.1%), and laparoscopic drainage (n = 5, 5.7%). Six (6.8%) patients received only antibiotic treatment [17,34,51,53,69,81] (Table 4). The mean length of hospitalization in the documented cases was 17.5 ± 17.3 d (ranging from 2 h to 64 d). The hospital stay was significantly shorter for patients receiving laparotomy (13.2 ± 4.6 d), compared with those undergoing abscess drainage under either CT- (47 ± 24 d) or echo-guidance (29.8 ± 22.2 d) (P < 0.05). Moreover, the hospitalization in patients receiving image-guided drainage was longer than those receiving only antibiotic treatment (11 ± 4.2 d), although the difference did not reach statistical significance (P = ). One possible explanation for this finding is that the drainage group generally had severe disease and a more complicated clinical course with larger abscesses and liquefaction compared with the antibiotics-only group. No statistically significant difference in the length of hospitalization between the open drainage and antibiotics-only groups was observed (P = ). Although there is no established guideline regarding the choice of treatment strategy for this rare disease entity, the current study showed that abscess drainage via laparotomy in suitable candidates may shorten the length of hospital stay compared with those receiving image-guided drainage. On the other hand, the choice of conservative treatment with parenteral antibiotics may be a therapeutic option for patients with liver abscesses without evidence of liquefaction. Indeed, a liver abscess up to 7 cm was resolved after antibiotic treatment in our patient. Although the literature review in the present study showed no bacterial resistance in this clinical setting, broad-spectrum antibiotics (e.g., carbapenems) may be included in the empirical treatment regimen against drug-resistant organisms April 7, 2014 Volume 20 Issue 13

333 Chong LW et al. Penetrating gastrointestinal foreign body-induced hepatic abscess Most patients previously reported to have hepatic foreign bodies were treated with laparotomy which is effective, but invasive [65]. On the other hand, although endoscopy can be performed via the gastrointestinal tract, this procedure can be technically demanding [31,51]. In our case, as most of the toothpick was within the stomach and apparent intra-peritoneal free air or vascular injury was absent, endoscopic removal was the preferred treatment strategy. This was performed successfully without complication. Treatment of hepatic abscess includes drainage and antibiotic therapy. As liquefaction of the hepatic abscess was not evident in our case, conservative treatment rather than drainage was chosen. Unlike other reported cases of hepatic abscesses secondary to foreign body penetration with delayed diagnosis and complicated clinical courses [4,20,33], our patient recovered uneventfully and was discharged after 10 d of antibiotic treatment. Possible reasons for the non-complicated clinical course may be the relatively small perforation without gross intra-peritoneal spillage of gastrointestinal contents, effective antibiotic treatment against relatively less virulent bacterial flora of the upper compared with those of the lower gastrointestinal tract, and prompt removal of the foreign body before its complete migration which may have caused further damage to the surrounding structures such as the pancreatic ductal system and vasculature. Prognosis All patients with a diagnosis established during hospitalization eventually recovered regardless of the treatment strategy chosen. On the other hand, seven (four males and three females) died due to the originally undetected condition that was evident only after autopsy [13,20,22,26,27,71], giving an overall mortality rate of 7.95% (7/88). The major cause of death was septic shock. The age of the patients who died ranged from 43 to 86 years (mean 56.1 ± 15.8 years). There was no significant difference in age between the expired patients and the survivors (mean 50.4 ± 18.5 years; range 1-80 years). The overall survival rate of patients with timely diagnosis was 100% (81/81), whereas the mortality rate of those without definite diagnosis until autopsy was also 100% (7/7). In summary, we report an unusual case of hepatic abscess caused by toothpick penetration of the gastric antrum. Although most cases of foreign body penetration of the gastrointestinal tract complicated by liver abscess are treated surgically, we demonstrated that a conservative management strategy combining endoscopic removal of the foreign body and broad-spectrum antibiotic coverage may be a feasible therapeutic option. Abscess drainage in suitable cases and identification of causative organisms are also necessary for successful conservative treatment. A review of the literature highlighted the importance of ruling out the possibility of foreign body-induced penetration of the alimentary tract in patients with liver abscesses and no identifiable underlying condition, particularly those refractory to conventional treatment. Appropriate diagnostic strategies, including imaging studies and surgical exploration, may be indicated for early diagnosis and timely treatment of this potentially lethal condition. COMMENTS Case characteristics A 41-year-old man with no previous history of medical illness presented with intermittent upper abdominal pain for 2 mo. Clinical diagnosis Physical examination demonstrated mild epigastric tenderness on palpation. Differential diagnosis The presence of pyogenic abscess may be assessed using abdominal sonography, whereas peptic ulcer disease and reflux esophagitis may be ruled out by esophagogastroduodenoscopy. Laboratory diagnosis Not only did a hematological study show no remarkable anemia, leukocytosis or left-shift (i.e., hemoglobin concentration: 13.4 mg/dl; white blood cell count: 14600/mm 3 ; neutrophils: 66.4%), but serum biochemical analyses also demonstrated no notable findings in C-reactive protein concentration (0.535 mg/dl), renal function, liver enzymes as well as levels of glucose, amylase, lipase, and alpha-fetoprotein. Imaging diagnosis Although chest and abdominal radiographs were unremarkable, computed tomography of the abdomen revealed a hyperdense, linear foreign body within the gastric antrum with transgastric penetration through the posterior wall of the antrum and close contact with the pancreatic body as well as a 7-cm hypodense mass in the left liver lobe. Pathological diagnosis As the patient received neither laparotomy nor drainage, no specimen was available for pathological diagnosis. Treatment The patient was successfully treated conservatively by a combination of endoscopic foreign body removal and parenteral/oral broad-spectrum antibiotic coverage (i.e., intravenous ceftriaxone two g/d and metronidazole 500 mg every eight hours for 10 d during hospitalization; oral levofloxacin 500 mg/d for two weeks after discharge). Related reports A review of 79 journal articles and 88 reported cases in the present study showed that most liver abscesses caused by foreign body penetration of the gastrointestinal tract were treated surgically or by image-guided drainage. Experiences and lessons The literature review highlighted the importance of ruling out the possibility of foreign body-induced penetration of the alimentary tract in patients with liver abscesses and no identifiable underlying condition, particularly those refractory to conventional treatment and abscesses in the left liver lobe. Peer review The strengths of the article include the comprehensiveness of review and detailed analysis of the reported cases, including demography and clinical manifestations, foreign bodies involved sites of perforation, location and size of liver abscess, tools for establishment of diagnosis, most common pathogens, treatment strategy and hospital course as well as prognosis. On the other hand, the weakness of the current study is that literature review identified only a small number of patients with undrainable abscesses who showed a high rate of negative bacterial culture so that limited insight can be gained regarding the appropriate antibiotic regimen for this patient population. REFERENCES 1 McCanse DE, Kurchin A, Hinshaw JR. Gastrointestinal foreign bodies. Am J Surg 1981; 142: [PMID: DOI: / (81) ] 2 Maleki M, Evans WE. Foreign-body perforation of the intestinal tract. Report of 12 cases and review of the literature. Arch Surg 1970; 101: [PMID: DOI: / archsurg ] 3 Lambert A. Abscess of the liver of unusual origin. NY Med J 3708 April 7, 2014 Volume 20 Issue 13

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335 Chong LW et al. Penetrating gastrointestinal foreign body-induced hepatic abscess fish bone and had a fistula with the ascending colon. Dig Dis Sci 2007; 52: [PMID: DOI: / s x] 40 Kumar S, Gupta NM. Foreign bodies migrating from gut to liver. Indian J Gastroenterol 2000; 19: 42 [PMID: ] 41 Lanthaler M, Grissmann T, Schwentner L, Nehoda H. Unusual differential diagnosis of upper abdominal pain. Diagn Ther Endosc 2009; 2009: [PMID: DOI: /2009/817052] 42 Lawhorne TW, Schaff HV. Occult liver abscess and foreign body perforation of the bowel. Am Surg 1979; 45: [PMID: ] 43 Lee KF, Chu W, Wong SW, Lai PB. Hepatic abscess secondary to foreign body perforation of the stomach. Asian J Surg 2005; 28: [PMID: DOI: / S (09) ] 44 Liu HJ, Liang CH, Huang B, Xie SF, Wang GY. Migration of a swallowed toothpick into the liver: the value of multiplanar CT. Br J Radiol 2009; 82: e79-e81 [PMID: DOI: /bjr/ ] 45 Liu YY, Tseng JH, Yeh CN, Fang JT, Lee HL, Jan YY. 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336 Chong LW et al. Penetrating gastrointestinal foreign body-induced hepatic abscess abscess caused by an ingested foreign body. J Gastroenterol Hepatol 2009; 24: 1575 [PMID: DOI: / j x] 77 Wasserman E, Lee R, Song J, Ehrlich T. Gastric perforation with an associated subhepatic liver abscess related to an accidentally ingested lobster shell. J Clin Gastroenterol 2008; 42: [PMID: DOI: /MCG.0b013e b3] 78 Wood MK, Harrison MR. A-pin-dicitis and liver abscess. JAMA 1981; 246: 940 [PMID: DOI: /jama ] 79 Yamaguchi N, Suzuki A. Electronic clinical challenges and images in GI. Shivering and right upper quadrant pain. Gastroenterology 2010; 138: e1-e2 [PMID: DOI: / j.gastro ] 80 Yang CY, Kao JH, Liu KL, Chen SJ. Medical treatment of fish bone-related liver abscess. Clin Infect Dis 2005; 41: [PMID: DOI: /498034] 81 Yen HH, Hsu YC. Education and Imaging: Gastrointestinal: pyogenic liver abscess associated with a penetrating fish bone. J Gastroenterol Hepatol 2010; 25: 1900 [PMID: DOI: /j x] 82 Zambrana JL, García-Gutiérrez JA, Díez F. Subphrenic abscess related to the ingestion of a toothpick. N Engl J Med 1998; 338: [PMID: DOI: /NEJM ] P- Reviewers: Cerwenka HR, Ferraioli G, Kirby R, Yaita K S- Editor: Zhai HH L- Editor: A E- Editor: Ma S 3711 April 7, 2014 Volume 20 Issue 13

337 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. CASE REPORT Hemobilia and other complications caused by percutaneous ultrasound-guided liver biopsy Hai-Bo Zhou Hai-Bo Zhou, Department of Gastroenterology, Second Hospital of Zhejiang University, Hangzhou , Zhejiang Province, China Author contributions: Zhou HB contributed solely to this work. Correspondence to: Hai-Bo Zhou, MD, Department of Gastroenterology, Second Hospital of Zhejiang University, Jiefang Rd 88, Hangzhou , Zhejiang Province, China. zhouhaibohz@163.com Telephone: Fax: Received: November 14, 2013 Revised: February 12, 2014 Accepted: March 6, 2014 Published online: April 7, 2014 with doctors, and showed obvious arteriovenous fistula of the right liver. The hepatic artery was selected and embolized by spring orbs. The active bleeding was stopped after embolization of the hepatic artery. Zhou HB. Hemobilia and other complications caused by percutaneous ultrasound-guided liver biopsy. World J Gastroenterol 2014; 20(13): Available from: URL: DOI: org/ /wjg.v20.i Abstract Hemobilia accounts for approximately 3% of all major percutaneous liver biopsy complications, and rarely results from arterioportal fistula. We report a patient who suffered from four complications over 11 d after ultrasound-guided percutaneous liver biopsy: hemobilia, acute pancreatitis, acute cholecystitis, and multiple stomach ulcers. Digital subtraction angiography was done after consultation with doctors, and showed obvious arteriovenous fistula of the right liver. The hepatic artery was selected and embolized by spring orbs. The active bleeding was stopped after embolization of the hepatic artery. The patient was discharged home on day 12 after embolization and remained well Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Hemobilia; Acute pancreatitis; Acute cholecystitis; Stomach ulcers; Ultrasound-guided liver biopsy Core tip: We report a patient who suffered from four complications over 11 d after ultrasound-guided percutaneous liver biopsy: hemobilia, acute pancreatitis, acute cholecystitis, and multiple stomach ulcers. Digital subtraction angiography was done after consultation INTRODUCTION Percutaneous ultrasound-guided liver biopsy is a common practice in the differential diagnosis and treatment of chronic liver disease. The rates of major complications and mortality are 2%-4% and 0.01%-0.33%, respectively. Arterioportal fistula as a complication of percutaneous liver biopsy is infrequently seen and normally asymptomatic. Hemobilia accounts for approximately 3% of overall major percutaneous liver biopsy complications, and rarely results from arterioportal fistula. We report a patient who suffered from four complications over 11 d after ultrasound-guided percutaneous liver biopsy: hemobilia, acute pancreatitis, acute cholecystitis, and multiple stomach ulcers. CASE REPORT A 57-year-old woman underwent ultrasound-guided liver biopsy because of abnormal liver function for 4 years. She experienced acute epigastric pain and melena without hematemesis 7 d after the procedure. Type-B ultrasound showed cholecystitis, cholangitis, and siltation of biliary mud in the gallbladder. Enhanced computed tomography showed cholangitis, cholecystolithiasis, high-density reflection in the common bile duct, and mild cholangiec April 7, 2014 Volume 20 Issue 13

338 Zhou HB. Hemobilia by percutaneous ultrasound-guided liver biopsy A B Figure 1 Magnetic resonance cholangiopancreatography. A: Magnetic resonance cholangiopancreatography (MRCP) revealed pancreatitis, cholecystolithiasis, cholecystitis, and cholangiectasis and abnormal signals that were considered to indicate muddy stone or hematocele in the common bile duct and hepatic duct; B: After treatment, MRCP showed there was little exudation in the gallbladder fossa, and bile ducts in the left liver were thickened. A B Figure 2 Gastroscopy. A: Gastroscopy showed multiple gastric ulcers; B: After treatment, gastroscopy revealed chronic superficial gastritis, and no gastric ulcers. tasis. After antibiotics, proton pump inhibitors and analgesics, the patient had no obvious improvement and had severe abdominal pain, hematemesis, and bloody stools. After fasting, gastrointestinal decompression and fluid replacement, the patient was hospitalized. In the following days, she developed worsening right epigastric pain and 1500 ml red bloody stools. Her hemoglobin level decreased from 134 to 73 g/l (normal range: g/l). Serum amylase was 614 U/L (normal range: U/L), and total bilirubin was 65 mg/dl (normal range: mg/dl). Ultrasound examination demonstrated enlargement of the gallbladder and the possibility of empyema. There was a low echo-level mass (hematocele) in the common bile duct, and distension of the pancreatic duct. Magnetic resonance cholangiopancreatography (MRCP) revealed pancreatitis, cholecystolithiasis, cholecystitis, cholangiectasis, and abnormal signals indicating muddy stone or hematocele in the common bile duct and hepatic duct (Figure 1). The gastroscope showed multiple gastric ulcers and bleeding duodenal papilla (Figure 2). The epigastric pain was decreased after percutaneous ultrasound, which was guided by the tube drainage of the tumescent gallbladder. About ml of red bile was drained within 1 d. Her hemoglobin level decreased to 52 g/l after 4 d in hospital. She received 4 U packed red blood cells. Digital subtraction angiography (DSA) was performed, which showed obvious arteriovenous fistula of the right liver. The hepatic artery was selected and embolized by spring orbs. The active bleeding was stopped after embolization of the hepatic artery. The patient was discharged home on day 12 after embolization and remained well. After 2 mo, her hemoglobin level increased to 140 g/l. Serum amylase was 68 U/L, and total bilirubin was 0.75 mg/dl. MRCP showed little exudation in the gallbladder fossa and the bile ducts in the left liver were thickened. Gastroscopy revealed chronic superficial gastritis (Figure 3). DISCUSSION Hemobilia is often considered to occur in liver injury and after calculus removal from the bile duct. With the application of traumatic examination of the liver and endoscopic technology, the traumatic intrahepatic biliary bleeding that is induced by iatrogenic trauma such as percutaneous transhepatic cholangial drainage has gradually increased [1]. Traumatic intrahepatic biliary bleeding may not occur during the procedure, but possibly a few days later. One needs to be extra vigilant in the clinic. Due to the close relationship of the intrahepatic bile duct and hepatic artery and portal vein, a puncture needle can easily injury all of these structures to form arteriovenous fistula, arterial bile duct fistula, and venous bile duct fistula. Venous bile duct fistula bleeding can often stop spontaneously because of the low venous pressure. When patients have biliary obstruction and infection, the hepatic arterial blood flow increases, and hyperplasia and distention of the arterial peribiliary vascular plexus and the branch of the hepatic artery in periportal areas can lead to bile duct fistula hemorrhage. This is the patholog April 7, 2014 Volume 20 Issue 13

339 Zhou HB. Hemobilia by percutaneous ultrasound-guided liver biopsy A B Figure 3 Digital subtraction angiography. A: Digital subtraction angiography showed obvious arteriovenous fistula of the right liver; B: There was no obvious arteriovenous fistula of the right liver after the hepatic artery was selected and embolized by spring orbs. ical basis of biliary bleeding. Blood clots formed in the bile duct can cause obstructive jaundice, leading to acute cholangitis and acute pancreatitis [2,3]. The present case had a tumescent gallbladder because of a hematocele. A blood clot in the biliary tract could have caused the increased pressure. That could have provoked acute pancreatitis and increasing hemodiastase. Pancreatitis caused by hemobilia and increased pressure can be improved by reducing the biliary tract pressure. Patients with hemobilia and increased pressure in the biliary tract should undergo puncture gallbladder to reduce pressure. This could strive the time of treatment. Reducing the biliary tract pressure can prevent pancreatitis. In the present case, traumatic arteriovenous fistula of the liver was detected by DSA. Lim et al [4] have reported that percutaneous liver biopsy can lead to liver arteriovenous fistula. Lee et al [5] reported that arteriovenous fistula formed in 38% (8/21) of cases after liver biopsy. Arteriovenous fistula bleeding in the liver often results from intrahepatic hematoma and portal hypertension. Although DSA did not directly show arteriobiliary fistula, and the contrast agent was not obviously leaking into the bile, traumatic arteriovenous fistula guided us to find the possible bleeding artery. In our case, selective arterial embolization was performed and gastrointestinal bleeding stopped. Usually, the best opportunity for DSA is acute sustained bleeding with at a rate > 0.5 ml/min. Tillotson et al [6] have suggested that patients with unexplained gastrointestinal bleeding should receive immediate access to DSA and interventional treatment. Interventional treatment includes arterial embolization and drug infusion. Embolic materials often used are gelatin sponge, autoallergy-sludged blood, spring orb and polyvinyl alcohol particles. Hypophysin is also commonly used. Several studies have suggested that superselective embolization is a safer and more effective treatment for active bleeding, but it is not a substitute for emergency surgery [7,8]. In patients with hematemesis and/or melena, right upper quadrant pain, and iatrogenic jaundice occurring after invasive surgery of the liver, care is needed to guard against the presence of tardive hemobilia. It is important to request the correlated case history. It is necessary to perform DSA and give interventional treatment when the treatment of gastrointestinal tract hemorrhage is ineffective. COMMENTS Case characteristics The patient experienced acute epigastric pain and red bloody stools after ultrasound-guided liver biopsy. Clinical diagnosis We report a rare case of a 57-year-old woman who suffered from four complications over 11 d after ultrasound-guided percutaneous liver biopsy: hemobilia, acute pancreatitis, acute cholecystitis, and multiple stomach ulcers. Differential diagnosis Acute epigastric pain and red bloody stools hinted at differential diagnosis between hemobilia and bleeding peptic ulcer. Laboratory diagnosis Serological testing revealed anemia, hyperamylasemia and hyperbilirubinemia. Imaging diagnosis Magnetic resonance cholangiopancreatography (MRCP) revealed pancreatitis, cholecystolithiasis, cholecystitis, cholangiectasis, and digital subtraction angiography (DSA) showed obvious arteriovenous fistula of the right liver. Treatment The hepatic artery was selected and embolized by spring orbs. Term explanation MRCP is an evolving technique for noninvasive imaging of diseases of the biliary tree and pancreatic duct. DSA is a widely used fluoroscopy technique for vascular imaging, which produces a sequence of projection X-ray images of blood vessels that is used in diagnosis and treatment. Experiences and lessons The author reports a rare case of a patient who suffered from four complications over 11 d after ultrasound-guided percutaneous liver biopsy. This rare clinical condition has seldom been reported in the literature. Peer review In this manuscript, the author reported an interesting case of a patient who suffered from four complications over 11 d after ultrasound-guided percutaneous liver biopsy. This case is rare and seldom reported in the literature. REFERENCES 1 Oh HC, Lee SK, Lee TY, Kwon S, Lee SS, Seo DW, Kim MH. Analysis of percutaneous transhepatic cholangioscopyrelated complications and the risk factors for those complications. Endoscopy 2007; 39: [PMID: DOI: /s ] 2 Edden Y, St Hilaire H, Benkov K, Harris MT. Percutaneous liver biopsy complicated by hemobilia-associated acute cholecystitis. World J Gastroenterol 2006; 12: [PMID: ] 3 Albuquerque W, Arantes V, de Paula Farah K, Lambertucci JR. Acute pancreatitis and acute cholecystitis caused by hemobilia after percutaneous ultrasound-guided liver biopsy. Endoscopy 2005; 37: [PMID: DOI: /s ] 4 Lim JH, Lee SJ, Lee WJ, Lim HK, Choo SW, Choo IW. Iodized 3714 April 7, 2014 Volume 20 Issue 13

340 Zhou HB. Hemobilia by percutaneous ultrasound-guided liver biopsy oil retention due to postbiopsy arterioportal shunt: a false positive lesion in the investigation of hepatocellular carcinoma. Abdom Imaging 1999; 24: [PMID: DOI: /s ] 5 Lee SJ, Lim JH, Lee WJ, Lim HK, Choo SW, Choo IW. Transient subsegmental hepatic parenchymal enhancement on dynamic CT: a sign of postbiopsy arterioportal shunt. J Comput Assist Tomogr 1997; 21: [PMID: DOI: / ] 6 Tillotson CL, Geller SC, Kantrowitz L, Eckstein MR, Waltman AC, Athanasoulis CA. Small bowel hemorrhage: an- giographic localization and intervention. Gastrointest Radiol 1988; 13: [PMID: DOI: /BF ] 7 Burgess AN, Evans PM. Lower gastrointestinal haemorrhage and superselective angiographic embolization. ANZ J Surg 2004; 74: [PMID: DOI: / j x] 8 Horiguchi J, Naito A, Fukuda H, Nakashige A, Ito K, Kiso T, Mori M. Morphologic and histopathologic changes in the bowel after super-selective transcatheter embolization for focal lower gastrointestinal hemorrhage. Acta Radiol 2003; 44: [PMID: DOI: /j x] P- Reviewers: Casciaro S, Markic D, Streba CT S- Editor: Gou SX L- Editor: A E- Editor: Wang CH 3715 April 7, 2014 Volume 20 Issue 13

341 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 7; 20(13): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. CASE REPORT Ulcerative colitis flair induced by mesalamine suppositories hypersensitivity Hao Ding, Xiao-Chang Liu, Qiao Mei, Jian-Ming Xu, Xiang-Yang Hu, Jing Hu Hao Ding, Xiao-Chang Liu, Qiao Mei, Jian-Ming Xu, Jing Hu, Department of Gastroenterology, First Affiliated Hospital of Anhui Medical University, Hefei , Anhui Province, China Xiang-Yang Hu, Department of Pathology, First Affiliated Hospital of Anhui Medical University, Hefei , Anhui Province, China Author contributions: Ding H and Liu XC contributed equally to this work; Ding H and Liu XC drafted the paper under the close supervision of Mei Q; Xu JM and Hu J participated in the design and coordination of the study; Hu XY performed the pathological examination. Correspondence to: Qiao Mei, MD, Department of Gastroenterology, First Affiliated Hospital of Anhui Medical University, No. 218, Jixi Road, Hefei , Anhui Province, China. meiqiao@hotmail.com Telephone: Fax: Received: September 15, 2013 Revised: November 5, 2013 Accepted: December 12, 2013 Published online: April 7, 2014 the suppositories Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Mesalamine suppositories; Hypersensitivity; Ulcerative colitis; Surgery; Sulfasalazine Core tip: Mesalamine suppository, a kind of newly designed 5-aminosalicylic acid formulations, is considered safe and effective and is widely used in the treatment of distal ulcerative colitis. Few cases concerning its side effect of hypersensitivity have been reported. Herein, we report a case of ulcerative colitis flare induced by mesalamine suppository hypersensitivity in an attempt to improve our understandings of the rare side effect of mesalamine suppositories. Abstract Mesalamine suppositories have been used widely for the treatment of distal ulcerative colitis and considered to be safer than systemic administration for its limited systemic absorption. However, previous studies have shown that mesalamine suppository occasionally causes severe hypersensitivity reactions including fever, rashes, colitis exacerbation and acute eosinophilic pneumonia. Here we present a 25-year-old woman with ulcerative colitis with bloody diarrhea accompanied by abdominal pain and fever which were aggravated after introduction of mesalamine suppositories. In light of symptom exacerbation of ulcerative colitis, increased inflammatory injury of colon mucosa shown by colonoscopy and elevated peripheral eosinophil count after mesalamine suppositories administration, and the Naranjo algorithm score of 10, the possibility of hypersensitivity reaction to mesalamine suppositories should be considered, warning us to be aware of this potential reaction after administration of mesalamine formulations even if it is Ding H, Liu XC, Mei Q, Xu JM, Hu XY, Hu J. Ulcerative colitis flair induced by mesalamine suppositories hypersensitivity. World J Gastroenterol 2014; 20(13): Available from: URL: DOI: INTRODUCTION Mesalamine suppository is a kind of newly designed 5-aminosalicylic acid formulations, it is supposed to be safe for its limited systemic absorption and lack of sulphapyridine moiety that has been implicated in many of the adverse reactions associated with sulfasalazine (SASP) [1], thus is widely used in the treatment of distal ulcerative colitis (UC). Hypersensitivity to mesalamine suppositories is very rare, there are only 3 cases documented worldwide so far. Here, we present a first case of symptomatic exacerbation of UC induced by mesalamine suppositories in China April 7, 2014 Volume 20 Issue 13

342 Ding H et al. Mesalamine suppositories induced UC flair CASE REPORT A 25-year-old woman experienced abdominal discomfort and diarrhea (3-5 per day) with no blood or purulence 8 months ago. Because of continuous bloody stools (1-2 per day) accompanied with crampy abdominal pain for 2 mo, she presented to the local health clinic where a pancolonoscopy was obtained which revealed erythema, edema, and shallow ulcers in rectum, she was diagnosed with UC of proctitis and treated with SASP 0.5 g tid for 20 d which seemed to aggravate bloody diarrhea (7-8 per day containing purulence), abdominal pain and high fever (up to 39 ) and generalized erythematous rashes. She was immediately admitted to the gastroenterology department of a local hospital, symptoms were improved with reduction of bloody purulent diarrhea (1-2 per day with reduced blood) and partial regression of skin lesions by comprehensive treatment including hydrocortisone, the patient then was referred to our hospital for further treatment. On admission, physical examination revealed high temperature (37.8 ) and erythematous rashes all over the body with partial desquamation, a repeat pancolonoscopy confirmed previous endoscopic findings, laboratory examinations showed abnormal liver biochemical changes [alanine aminotransferase (ALT) 245 U/L, normal range: 5-40 U/L; aspartate aminotransferase (AST) 336 U/L, normal range: 8-40 U/L; alkaline phosphatase (ALP) 216 U/L, normal range: U/L; gamma glutamyl transferase (GGT) 217 U/L, normal range: 0-50 U/L; lactate dehydrogenase (LDH) 806 U/L, normal range: U/L]. Hepatitis serology was negative. C-reactive protein (CRP) (4.96 mg/l, normal range: 0-3 mg/l) and erythrocyte sedimentation rate (ESR) (23 mmhg, normal range: 0-20 mmhg) were slightly elevated. Peripheral white blood cell count ( /μl, normal range: /μl) and neutrophil count ( / μl, normal range: /μl) was significantly elevated, and hemoglobin was decreased slightly (9.8 g/dl, normal range: g/dl). She was diagnosed with moderately active UC based on her symptoms and endoscopic findings, for her severe systemic symptoms and ineffectiveness of SASP treatment. Corticosteroids therapy (intravenous hydrocortisone 200 mg/d for the first 5 d and oral prednisone 40 mg/d for subsequent 3 d) was applied, resulting in significant improvement of her fever (36-37 ), skin rashes (basically subsided) and liver biochemical abnormalities (ALT 47 U/L, AST 18 U/L, ALP 111 U/L, GGT 101 U/L, LDH 347 U/L). While the obvious rectal irritation symptoms were still bothering her, mesalamine suppositories (0.5 g QN) were prescribed. After taking only 3 suppositories, the patient developed a temperature of 37.6, and experienced new rash in the face and aggravation of bloody purulent diarrhea (up to 10 per day with evident blood and mucosal exfoliation). Blood tests showed an increase of peripheral white blood cell count ( /μl) with normal neutrophil count, of particular note, the eosinophil count was increased to /μl (normal range: /μl). ESR and CRP were in the normal range. Stool studies were negative for infection. A proctosigmoidoscopy revealed diffuse erythema, edema, shallow ulcers and contact hemorrhage with purulent exudate beyond the view of scope, mesalamine suppositories were immediately discontinued and a 5-d steroid treatment was administered. The patient had remained symptomatic (bloody purulent diarrhea and fever), a rescue therapy of infliximab or cyclosporine A was proposed, but she refused it for her poor economic condition and severe illness at that time. She had to accept surgery eventually which removed the whole colon and the upper part of rectum. Postoperative pathology showed chronic inflammation cell infiltration with local ulceration and scattered distribution of eosinophils. For her persistent rectal irritation symptoms, a mesalamine suppository was prescribed, resulting in the recurrent bloody stool, abdominal pain and fever which were improved as soon as mesalamine suppository was discontinued, thus by an unintentional repeated medication, her intolerance of mesalamine suppositories was confirmed objectively. DISCUSSION SASP has been the first-line therapy to induce and maintain remission in mild to moderately active UC [2,3]. However, up to 30% of patients cannot take it due to intolerance or hypersensitivity reactions which are often attributed to the sulphapyridine moiety [4]. Mesalamine formulations, due to their less frequent causation of sulfa-related adverse events, have replaced SASP as the first-choice treatment of UC. Adverse events from hypersensitivity and intolerance can occur. Skin rash, fever, and systemic reactions have been described [5]. More severe reactions associated with use of mesalamine occur infrequently, such as pancreatic, renal, myo-pericardial and pulmonary toxicity, as well as hematological disorders [6]. In this case report, it was surprising and skeptical when the patient reported fever and rash after mesalamine suppository administration, for at least 80% of patients intolerant of SASP can tolerate mesalamine preparations [7]. However, in light of twice symptom exacerbation of UC and increased peripheral eosinophil count after mesalamine suppository administration, and the Naranjo algorithm score [6] of 10, the patient s hypersensitivity to mesalamine suppositories was confirmed. Intolerance to SASP is not rare which are often attributed to the sulphapyridine moiety. Bousseaden et al [8] reported a patient intolerant to SASP (fever and skin rash), who developed a severe hypersensitivity reaction to oral mesalamine characterized by abdominal pain and bloodstained diarrhea and had to receive coloproctectomy eventually. In this case, we speculated that SASP hypersensitivity was responsible for the first symptom aggravation of UC, rashes and abnormalities of liver function. The similar adverse event after both SASP and mesalamine suppository administration in this patient suggests that this pattern of SASP intolerance may in fact be caused by the 5-aminosalicylic acid moiety of the molecule [8]. The precise mechanism of colitis exacerbation induced 3717 April 7, 2014 Volume 20 Issue 13

343 Ding H et al. Mesalamine suppositories induced UC flair by mesalamine is not clear. Scheurlen et al [9] mentioned that the mechanism of diarrhea caused by mesalamine was attributed to a secretory mechanism secondary to the inhibition of ileal and colonic Na + K + ATPase. Another proposed mechanism was an alteration of arachidonic acid metabolism which was manifested mainly with secretory diarrhea and malabsorption [10]. Both mechanisms, however, can not explain the bloody diarrhea or the endoscopic and pathological evidence of inflammation seen in this case. Mesalamine suppositories are considered to be safer than systemic administration by bypassing the threat of small bowel absorption [11]. Three cases concerning the hypersensitivity reactions after mesalazine suppositories administration were well documented [12-14], and it is demonstrated that small amounts of enema or suppository can be absorbed when administered topically [15]. Therefore, in this case, we speculated that the hypersensitivity reactions were probably due to hematogenous spread of mesalazine absorbed by the rectal mucosa. Further investigation with blood, luminal fluid and tissue cytokine determination is required to elucidate the mechanism for this sensitivity reaction. Clinicians must keep in mind the possibility of salicylate sensitivity and colitis exacerbation, especially in atopic individuals and in patients who failed to respond appropriately to SASP. COMMENTS Case characteristics This report a case of ulcerative colitis flare induced by mesalamine suppository hypersensitivity. Clinical diagnosis She was diagnosed with moderately active ulcerative colitis (UC) based on her severe systemic symptoms, endoscopic findings and ineffectiveness of sulfasalazine (SASP) treatment. Differential diagnosis In light of twice symptom exacerbation of UC and increased peripheral eosinophil count after mesalamine suppository administration, and the Naranjo algorithm score of 10, the patient s hypersensitivity to mesalamine suppositories was confirmed. Laboratory diagnosis Laboratory tests including peripheral neutrophil count, erythrocyte sedimentation rate, C-reactive protein and fecal pathogenic examination were nearly normal in addition to a significant increased peripheral eosinophil count. Antibiotic and intravenous steroid treatment was ineffective, and the Naranjo algorithm score was 10, the patient eventually underwent surgery. The postoperative pathology showed chronic inflammation cell infiltration with local ulceration and scattered distribution of eosinophils, and hypersensitivity to mesalamine suppositories was confirmed finally. Experiences and lessons Only three similar cases have been documented worldwide so far, not including this one. Clinicians must keep in mind the possibility of salicylate sensitivity and colitis exacerbation, especially in atopic individuals and in patients who failed to respond appropriately to SASP. Peer review Authors described a case of systemic hypersensitivity reaction on mesalamine suppository in ulcerative colitis. The description is clear and concise. REFERENCES 1 Das KM, Eastwood MA, McManus JP, Sircus W. Adverse reactions during salicylazosulfapyridine therapy and the relation with drug metabolism and acetylator phenotype. N Engl J Med 1973; 289: [PMID: DOI: / NEJM ] 2 Dick AP, Grayson MJ, Carpenter RG, Petrie A. Controlled trial of sulphasalazine in the treatment of ulcerative colitis. Gut 1964; 5: [PMID: DOI: / gut ] 3 Dissanayake AS, Truelove SC. A controlled therapeutic trial of long-term maintenance treatment of ulcerative colitis with sulphazalazine (Salazopyrin). Gut 1973; 14: [PMID: DOI: /gut ] 4 Sturgeon JB, Bhatia P, Hermens D, Miner PB. Exacerbation of chronic ulcerative colitis with mesalamine. Gastroenterology 1995; 108: [PMID: DOI: / (95)90154-X] 5 le Gros V, Saveuse H, Lesur G, Brion N. Lung and skin hypersensitivity to 5-aminosalicylic acid. BMJ 1991; 302: 970 [PMID: DOI: /bmj a] 6 Sposato B, Allegri MP, Riccardi MP, Chigiotti S, Nencioni C, Ricciardi B, Carli T, Cresti A, Perari MG, Migliorini MG, Toti M. Mesalazine-induced multi-organ hypersensitivity. Clin Drug Investig 2010; 30: [PMID: DOI: /BF ] 7 Schroeder KW. Is mesalamine safe? Gastroenterol Hepatol (N Y) 2007; 3: [PMID: ] 8 Bousseaden A, Ajana FZ, Essamri W, Benelbarhdadi I, Afifi R, Benazzouz M, Essaid A. Mesalamine enema-induced exacerbation of ulcerative colitis. Int J Colorectal Dis 2009; 24: [PMID: DOI: /s x] 9 Scheurlen C, Allgayer H, Kruis W, Erdmann E, Sauerbruch T. Effect of olsalazine and mesalazine on human ileal and colonic (Na+ + K+)-ATPase. A possible diarrhogenic factor? Clin Investig 1993; 71: [PMID: DOI: / BF ] 10 Fine KD, Sarles HE, Cryer B. Diarrhea associated with mesalamine in a patient with chronic nongranulomatous enterocolitis. N Engl J Med 1998; 338: [PMID: DOI: /NEJM ] 11 Qureshi AI, Cohen RD. Mesalamine delivery systems: do they really make much difference? Adv Drug Deliv Rev 2005; 57: [PMID: DOI: /j.addr ] 12 Weidenhiller M, Raithel M, Hahn EG. Hypersensitivity to 5-ASA suppositories. Dig Dis Sci 1999; 44: [PMID: DOI: /A: ] 13 Kapur KC, Williams GT, Allison MC. Mesalazine induced exacerbation of ulcerative colitis. Gut 1995; 37: [PMID: DOI: /gut ] 14 Kim JH, Lee JH, Koh ES, Park SW, Jang AS, Kim D, Park CS. Acute eosinophilic pneumonia related to a mesalazine suppository. Asia Pac Allergy 2013; 3: [PMID: DOI: /apallergy ] 15 Campieri M, Lanfranchi GA, Boschi S, Brignola C, Bazzocchi G, Gionchetti P, Minguzzi MR, Belluzzi A, Labò G. Topical administration of 5-aminosalicylic acid enemas in patients with ulcerative colitis. Studies on rectal absorption and excretion. Gut 1985; 26: [PMID: DOI: / gut ] P- Reviewers: Andus T, Bassotti G, De Silva AP S- Editor: Gou SX L- Editor: Ma JY E- Editor: Wang CH 3718 April 7, 2014 Volume 20 Issue 13

344 Online Submissions: World J Gastroenterol 2014 April 7; 20(13): I-VI ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. 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. 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347 Instructions to authors 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 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. 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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 IV April 7, 2014 Volume 20 Issue 13

348 Instructions to authors 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: 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. V April 7, 2014 Volume 20 Issue 13

349 Instructions to authors 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. 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 April 7, 2014 Volume 20 Issue 13

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