World Journal of Gastroenterology

Transcription

1 ISSN (print) ISSN (online) World Journal of Gastroenterology World J Gastroenterol 2014 April 21; 20(15):

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 15 April 21, 2014 TOPIC HIGHLIGHT 4115 Hepatocellular carcinoma review: Current treatment, and evidence-based medicine Raza A, Sood GK 4128 Role of the tissue microenvironment as a therapeutic target in hepatocellular carcinoma Rani B, Cao Y, Malfettone A, Tomuleasa C, Fabregat I, Giannelli G 4141 Staging systems of hepatocellular carcinoma: A review of literature Maida M, Orlando E, Cammà C, Cabibbo G 4151 Chemotherapy for advanced hepatocellular carcinoma in the sorafenib age Miyahara K, Nouso K, Yamamoto K 4160 Therapeutic response assessment of RFA for HCC: Contrast-enhanced US, CT and MRI Minami Y, Nishida N, Kudo M 4167 Genetic variations in colorectal cancer risk and clinical outcome Zhang K, Civan J, Mukherjee S, Patel F, Yang H 4178 Targeting mtor network in colorectal cancer therapy Wang XW, Zhang YJ 4189 Hallmarks in colorectal cancer: Angiogenesis and cancer stem-like cells Mathonnet M, Perraud A, Christou N, Akil H, Melin C, Battu S, Jauberteau MO, Denizot Y 4197 Circadian clock circuitry in colorectal cancer Mazzoccoli G, Vinciguerra M, Papa G, Piepoli A 4208 Role of cetuximab in first-line treatment of metastatic colorectal cancer Sotelo MJ, García-Paredes B, Aguado C, Sastre J, Díaz-Rubio E 4220 Role of stereotactic body radiotherapy for oligometastasis from colorectal cancer Takeda A, Sanuki N, Kunieda E 4230 Molecular and prognostic heterogeneity of microsatellite-unstable colorectal cancer Kim JH, Kang GH April 21, 2014 Volume 20 Issue 15

12 Contents World Journal of Gastroenterology Volume 20 Number 15 April 21, Multimodal imaging evaluation in staging of rectal cancer Heo SH, Kim JW, Shin SS, Jeong YY, Kang HK 4256 Interval to surgery after neoadjuvant treatment for colorectal cancer Wasserberg N 4263 Anti-EGFR and anti-vegf agents: Important targeted therapies of colorectal liver metastases Feng QY, Wei Y, Chen JW, Chang WJ, Ye LC, Zhu DX, Xu JM 4276 Advances in epigenetic biomarker research in colorectal cancer Wang X, Kuang YY, Hu XT 4288 MicroRNAs in colorectal cancer as markers and targets: Recent advances Ye JJ, Cao J REVIEW 4300 Invasive and non-invasive diagnosis of cirrhosis and portal hypertension Kim MY, Jeong WK, Baik SK ORIGINAL ARTICLE 4316 Non-transmissible Sendai virus vector encoding c-myc suppressor FBPinteracting repressor for cancer therapy Matsushita K, Shimada H, Ueda Y, Inoue M, Hasegawa M, Tomonaga T, Matsubara H, Nomura F RESEARCH REPORT 4329 Case-control study of factors that trigger inflammatory bowel disease flares Feagins LA, Iqbal R, Spechler SJ 4335 Pediatric non-alcoholic steatohepatitis: The first report on the ultrastructure of hepatocyte mitochondria Lotowska JM, Sobaniec-Lotowska ME, Bockowska SB, Lebensztejn DM 4341 Role of triamcinolone in radiation enteritis management Cetin E, Ozturk AS, Orhun H, Ulger S 4345 Hepcidin expression in colon during trinitrobenzene sulfonic acid-induced colitis in rats Gotardo EMF, Ribeiro GA, Clemente TRL, Moscato CH, Tomé RBG, Rocha T, Pedrazzoli Jr J, Ribeiro ML, Gambero A 4353 Risk factors associated with Barrett's epithelial dysplasia Fujita M, Nakamura Y, Kasashima S, Furukawa M, Misaka R, Nagahara H II April 21, 2014 Volume 20 Issue 15

13 Contents World Journal of Gastroenterology Volume 20 Number 15 April 21, Comparative study of esomeprazole and lansoprazole in triple therapy for eradication of Helicobacter pylori in Japan Nishida T, Tsujii M, Tanimura H, Tsutsui S, Tsuji S, Takeda A, Inoue A, Fukui H, Yoshio T, Kishida O, Ogawa H, Oshita M, Kobayashi I, Zushi S, Ichiba M, Uenoyama N, Yasunaga Y, Ishihara R, Yura M, Komori M, Egawa S, Iijima H, Takehara T 4370 Clinical meaning of BRAF mutation in Korean patients with advanced colorectal cancer Kim B, Park SJ, Cheon JH, Kim TI, Kim WH, Hong SP 4377 Unexpected FDG-PET uptake in the gastrointestinal tract: Endoscopic and histopathological correlations Goldin E, Mahamid M, Koslowsky B, Shteingart S, Dubner Y, Lalazar G, Wengrower D 4382 Effect of resistance training on non-alcoholic fatty-liver disease a randomizedclinical trial Zelber-Sagi S, Buch A, Yeshua H, Vaisman N, Webb M, Harari G, Kis O, Fliss-Isakov N, Izkhakov E, Halpern Z, Santo E, Oren R, Shibolet O 4393 Living-donor or deceased-donor liver transplantation for hepatic carcinoma: A case-matched comparison Wan P, Zhang JJ, Li QG, Xu N, Zhang M, Chen XS, Han LZ, Xia Q 4401 Clinicopathological features and trend changes of gastric carcinoma in Southern China Peng JJ, Xiao P, Xu JB, Song W, Liao B, He YL 4407 HBsAg levels in HBeAg-positive chronic hepatitis B patients with different immune conditions Zhang YM, Yang YD, Jia HY, Zeng LY, Yu W, Zhou N, Li LJ 4414 Caspase-3 expression in metastatic lymph nodes of esophageal squamous cell carcinoma is prognostic of survival Wang XS, Luo KJ, Bella AE, Bu SS, Wen J, Zhang SS, Hu Y 4421 Expression of monocyte chemotactic protein-1/ccl2 in gastric cancer and its relationship with tumor hypoxia Tao LL, Shi SJ, Chen LB, Huang GC 4428 Preliminary study correlating CX3CL1/CX3CR1 expression with gastric carcinoma and gastric carcinoma perineural invasion Lv CY, Zhou T, Chen W, Yin XD, Yao JH, Zhang YF III April 21, 2014 Volume 20 Issue 15

14 Contents World Journal of Gastroenterology Volume 20 Number 15 April 21, Anatomic resection of liver segments 6-8 for hepatocellular carcinoma Jia CK, Weng J, Chen YK, Fu Y 4440 Death decoy receptor overexpression and increased malignancy risk in colorectal cancer Zong L, Chen P, Wang DX 4446 Comparative study of intestinal tuberculosis and primary small intestinal lymphoma Zhu QQ, Zhu WR, Wu JT, Chen WX, Wang SA CASE REPORT 4453 Squamous cell carcinoma after radiofrequency ablation for Barrett's dysplasia Zeki SS, Haidry R, Justo-Rodriguez M, Lovat LB, Wright NA, McDonald SA 4457 Colonic perforation by a transmural and transvalvular migrated retained sponge: Multi-detector computed tomography findings Camera L, Sagnelli M, Guadagno P, Mainenti PP, Marra T, Scotto di Santolo M, Fei L, Salvatore M 4462 Failed stapled rectal resection in a constipated patient with rectal aganglionosis Pescatori LC, Villanacci V, Pescatori M IV April 21, 2014 Volume 20 Issue 15

15 Contents World Journal of Gastroenterology Volume 20 Number 15 April 21, 2014 APPENDIX I-VI Instructions to authors ABOUT COVER Editorial Board Member of World Journal of Gastroenterology, Romeo G Mihaila, MD, PhD, Associate Professor, Faculty of Medicine, "Lucian Blaga" University of Sibiu, Sibiu , Romania 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: Xiao-Mei Liu Proofing Editor-in-Chief: Lian-Sheng Ma Responsible Science Editor: Ya-Juan Ma Proofing Editorial Office Director: Xiu-Xia Song NAME OF JOURNAL World Journal of Gastroenterology ISSN ISSN (print) ISSN (online) LAUNCH DATE October 1, 1995 FREQUENCY Weekly EDITORS-IN-CHIEF Damian Garcia-Olmo, MD, PhD, Doctor, Professor, Surgeon, Department of Surgery, Universidad Autonoma de Madrid; Department of General Surgery, Fundacion Jimenez Diaz University Hospital, Madrid 28040, Spain Saleh A Naser, PhD, Professor, Burnett School of Biomedical Sciences, College of Medicine, University of Central Florida, Orlando, FL 32816, United States Stephen C Strom, PhD, Professor, Department of Laboratory Medicine, Division of Pathology, Karolinska Institutet, Stockholm , Sweden Andrzej S Tarnawski, MD, PhD, DSc (Med), Professor of Medicine, Chief Gastroenterology, VA Long Beach Health Care System, University of California, Irvine, CA, 5901 E. Seventh Str., Long Beach, CA 90822, United States EDITORIAL OFFICE Jin-Lei Wang, Director Xiu-Xia Song, Vice Director World Journal of Gastroenterology Room 903, Building D, Ocean International Center, No. 62 Dongsihuan Zhonglu, Chaoyang District, Beijing , China Telephone: Fax: 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 21, 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 21, 2014 Volume 20 Issue 15

16 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (1): Hepatocellular carcinoma TOPIC HIGHLIGHT Hepatocellular carcinoma review: Current treatment, and evidence-based medicine Ali Raza, Gagan K Sood Ali Raza, Department of Internal Medicine, St. Luke s Episcopal Hospital, Baylor College of Medicine, Houston, TX 77030, United States Gagan K Sood, Division of Gastroenterology and Hepatology, Baylor College of Medicine, St. Luke s Liver Center, Houston, TX 77030, United States Author contributions: Raza A performed the data research and helped writing the manuscript; Sood GK designed the manuscript; performed the research; helped writing the manuscript and critically analyzed the manuscript. Correspondence to: Gagan K Sood, MD, Division of Gastroenterology and Hepatology, Baylor College of Medicine, St. Luke s Liver Center, 6620 Main Street, Suite 1425, Baylor Clinic, Houston, TX 77030, United States. gksood@bcm.edu Telephone: Fax: Received: September 28, 2013 Revised: December 6, 2013 Accepted: March 6, 2014 Published online: April 21, 2014 Abstract Hepatocellular carcinoma (HCC) is the fifth most common tumor worldwide. Multiple treatment options are available for HCC including curative resection, liver transplantation, radiofrequency ablation, trans-arterial chemoembolization, radioembolization and systemic targeted agent like sorafenib. The treatment of HCC depends on the tumor stage, patient performance status and liver function reserve and requires a multidisciplinary approach. In the past few years with significant advances in surgical treatments and locoregional therapies, the short-term survival of HCC has improved but the recurrent disease remains a big problem. The pathogenesis of HCC is a multistep and complex process, wherein angiogenesis plays an important role. For patients with advanced disease, sorafenib is the only approved therapy, but novel systemic molecular targeted agents and their combinations are emerging. This article provides an overview of treatment of early and advanced stage HCC based on our extensive review of relevant literature Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Hepatocellular carcinoma; Trans-arterial chemoembolization; Drug-eluting beads; Radiofrequency ablation; Liver transplantation; Chemotherapy; Sorafenib; Radioembolization Core tip: The article discusses the current evidence based treatment of hepatocellular carcinoma. Specific focus is placed on emerging systemic molecular targeted therapies. Raza A, Sood GK. Hepatocellular carcinoma review: Current treatment, and evidence-based medicine. World J Gastroenterol 2014; 20(15): Available from: URL: wjgnet.com/ /full/v20/i15/4115.htm DOI: org/ /wjg.v20.i INTRODUCTION Hepatocellular carcinoma (HCC) is the fifth most common form of cancer worldwide and the third most common cause of cancer-related deaths. HCC often occurs in the background of a cirrhotic liver [1]. Orthotopic liver transplantation (OLT) is an effective treatment for both HCC and underlying cirrhosis, and is considered the best therapeutic option. Unfortunately, most cases of HCC present in an advanced stage and are not suitable candidates for OLT [2]. In recent years surveillance strategies in patients at a higher risk of HCC have led to the diagnosis of the disease at much earlier stages. Patients in early stages have a much higher chance of curative response with different treatment options [2,3]. Tumor staging plays an essential role in guiding the treatment decisions, but 4115 April 21, 2014 Volume 20 Issue 15

17 Raza A et al. HCC: Treatment and evidence-based medicine prognosis is affected by the severity of underlying liver dysfunction. A number of staging systems are available for use in HCC, and there is no worldwide consensus on a preferred system. The Child- Pugh classification system and the model for end-stage liver disease (MELD) score only assess the severity of liver disease and do not include the patient s performance status (PS) or cancerrelated symptoms. The only staging system currently in use that addresses each of these concerns is the Barcelona Clinic Liver Cancer (BCLC) classification. This classification links HCC staging with patient s PS and co-morbidities. This allows for an appropriate treatment strategy and defines the standard of care for each tumor stage. The major advantage of the BCLC system is that it can be used to identify the patients with early-stage HCC, who may benefit from curative therapies. This differentiates them from the patients with advanced-stage disease who would benefit more from palliative treatment. American Association for the Study of Liver Diseases (AASLD) and European Association for the Study of the Liver (EASL) have endorsed the BCLC system [4,5]. Several therapies have been proposed for these patients with proven survival benefits in the early-stage of HCC. These therapies comprise the surgical resection, various locoregional treatments including percutaneous ethanol injection (PEI), radiofrequency ablation (RFA), transarterial chemoembolization (TACE) and radioembolization [4-6]. LIVER TRANSPLANTATION Liver transplantation (LT) is a potentially curative treatment and the best treatment option for the patients with decompensated cirrhosis. Currently LT is recommended for the patients with HCC, whose tumor is within the Milan criteria for HCC (one lesion not larger than 5 cm, or up to 3 lesions with each 3 cm or smaller). This selection criterion results in a 5-year overall survival rate of 75% and a tumor recurrence rate of less than 15% [7-9]. This tumor burden is compatible with early-stage HCC in the BCLC staging system. Priority for assignment to the LT waiting list is based upon the MELD score, which is a good predictor of early mortality in patients with cirrhosis. However, MELD score is not able to predict mortality in the patient with HCC, therefore, a MELD exception has been developed to assign extra points to the HCC patients on the basis of the tumor burden. The exception criteria have resulted in an increased number of LTs being performed in the HCC patients; currently 30%-40% of the LTs are performed for HCC [10]. Some centers also consider the patients for LT who exceed the Milan criteria. Transplanting the patients with HCC beyond the established criteria falls into two categories; those whose tumors exceed the Milan criteria at presentation without any prior treatment (expanded criteria), and those who fulfill the Milan criteria after locoregional treatments (downstaging). Currently, however, there is no international consensus regarding these approaches in clinical practice [11,12]. Evidence for listing the patients for LT with tumor burden beyond Milan criteria is poor. Yet, it is clear that some patients with tumor burden beyond Milan criteria may benefit from transplantation. Similarly, studies looking at the LT outcomes in the patients with HCC after downstaging are very heterogeneous and no evidence-based recommendations can be made at this point. Few studies have shown that successful downstaging of HCC can be achieved in carefully selected patients and is associated with excellent post-transplantation outcomes [13]. Success in downstaging has been reported in many studies, although most of these are uncontrolled observational studies [9,14,15]. Multiple modalities including resection, RFA and TACE have been used for downsizing. The largest experience is with TACE and RFA. The two prospective studies showed that survival after liver transplantation in patients with large tumor burden successfully treated by downstaging was similar to survival in patients who initially met the criteria for LT [16,17]. It is essential to consider how expansion of criteria beyond the Milan criteria might affect the survival of candidates for liver transplantation who do not have HCC. In the European Liver Transplant Registry, Organ Procurement and Transplantation Network, and Australia and New Zealand Liver Transplant Registry, 5-year survival for non-hcc was 65%-87% [18]. According to studies based on Markov models using data from the United States, patients outside the Milan criteria would need to achieve 5-year survival of 60% or higher to prevent a substantial decrement to the life-years available to the entire population of candidates for liver transplantation [19,20]. International consensus conference on recommendations for liver transplantation in 2010 recommended that modest expansion of Milan criteria should be considered [18]. Among many proposals, only the University of California San Francisco criteria (one tumor 6.5 cm, three nodules at most with the largest 4.5 cm, and total tumor diameter 8 cm) have been prospectively validated by the proponent group, with outcome data comparable to those from other retrospective studies [17,21]. A minimum observational period of 3-6 mo after downstaging was required before the LT [17]. It has been recognized that tumor size and number are crude measures of prognosis. In future, studies with molecular markers or gene signatures will define tumor biology and these will be incorporated in the eligibility criteria for the transplant listing [22]. SURGICAL RESECTION Surgical resection is the treatment option for a small number of patients with single nodules, good liver function and no underlying cirrhosis. Surgical resection has an increased risk of hepatic decompensation in the patients with cirrhosis [23,24]. Thus, only patients with well-compensated cirrhosis, Child-Pugh class A, are considered the ideal candidates for surgical resection. Portal hypertension in cirrhotic patients is considered a relative contraindica April 21, 2014 Volume 20 Issue 15

18 Raza A et al. HCC: Treatment and evidence-based medicine tion for surgical resection according to EASL/AASLD guidelines. In earlier studies Bruix et al [4,25] reported that in Child-Pugh A cirrhotic patients undergoing hepatic resection, the presence of portal hypertension based on hepatic venous pressure gradient (HVPG) 10 mmhg, to be the best predictor of post-operative liver decompensation and poor long-term outcomes. However, measurement of HVPG is an invasive procedure and requires technical expertise. Some studies have used other surrogate markers of portal hypertension like the presence of esophageal varices or splenomegaly (major diameter > 12 cm) with a platelet count of < /mm 3. Few recent studies have reported comparable postoperative and long-term outcome in patients with and without portal hypertension using these surrogate markers of portal hypertension. These studies demonstrated that cirrhotic patients with both clinically significant portal hypertension and well-preserved liver function have similar short- and long-term outcomes compared with patients without portal hypertension. Overall surgical results depend not only on the presence of portal hypertension but also on the residual liver function, size of segmental resection and the remnant liver volume [26,27]. Moreover with improvement in anesthesia and surgical techniques, specifically laparoscopic resection, results of surgery are much superior [28]. Therefore, the prognostic relevance of clinically significant portal hypertension after hepatic resection in patients with HCC is still a matter for debate. The recent study by Santambrogio et al [29] reported that the presence of clinical portal hypertension alone does not influence the post-operative course of cirrhotic patients submitted to hepatic resection. If stringent preoperative selection criteria are met (i.e., Child-Pugh class A patients undergoing resection with a laparoscopic approach and limited segmental hepatic resection) the post-operative mortality rate is very low. Patients without portal hypertension or with clinically significant portal hypertension and preserved liver function (Child-Pugh A5 class) can undergo hepatic resection without hepatic decompensation and good long-term survival, if limited hepatic resection with enough remnant liver volume is done with laparoscopic approach. The patients who undergo surgical resection have nearly 70% five-year survival but have a high risk of recurrence. Recurrence rate correlates with the presence of microscopic vascular invasion, which is present in more than 30% of HCC patients without any evidence of macroscopic vascular invasion [30,31]. Early tumor recurrence within two years of surgery is mainly related to local invasion and intrahepatic metastasis. Late recurrence, occurring after two years of surgery, is mainly related to de novo tumor formation. Some studies have shown benefit of adjuvant therapies in decreasing the postoperative recurrence rate [32-34]. Though the hepatic resection is not often considered as an option in patients with multiple tumors, some centers have reported optimal results with hepatic resections even in patients with multiple tumors. Some of the biomarkers (gene signatures or molecular biomarkers) are promising in predicting the late recurrence [35]. These biomarkers are likely to improve selection of candidates for surgical resection with lower risk of recurrence. At present, surgical resection is recommended in the patients with early-stage disease and preserved liver function. But the surgical option should be weighed against the availability and the response rate of other local ablative therapies like radiofrequency ablation. RFA Surgical resection is currently considered the most curative strategy, but in the last decade highly satisfactory results have been obtained with local ablative therapies [36]. RFA is currently considered the most effective local ablative therapy. There has been considerable improvement in the RFA technique like the use of expandabletipped or cool-tip electrodes. A single electrode insertion can produce a necrotic area of up to 3.0 cm in diameter, thus allowing complete ablation of a 2 cm with necrosis of adjacent 0.5 cm to 1.0 cm margins, achieving tumor free margins like surgical resection. Local ablation with RFA is considered a standard of care for the patients with very early and early stage (BCLC 0-A) tumors not suitable for surgery. The best results were reported for RFA-treated patients with tumors < 2 cm in diameter who had 5-year survival rates ranging from 40% to 70%. A cohort study of RFA demonstrated that complete ablation of lesions smaller than 2 cm is possible in more than 90% of cases, with a local recurrence rate of less than 1% [37]. RFA has replaced percutaneous ethanol ablation as the locoregional therapy of choice. Three independent meta-analyses, including five randomized controlled trials, have provided evidence for better local control and increased survival benefits in patients treated with RFA compared to ablation with PEI. RFA has also been shown to provide a survival benefit in patients with tumors > 2 cm but < 5 cm, as compared to PEI [38-40]. Consequently, RFA has progressively replaced PEI for patients with small HCC who are not candidates for surgery. There is no consensus so far whether percutaneous RFA can replace surgical resection as first-line treatment for small tumors. Two RCTs provided conflicting evidence regarding the benefits of RFA vs surgical resection. The results from one RCT suggest a benefit for surgery over RFA in patients who met the Milan criteria followed for up to 5 years [41]. Another RCT did not identify a significant difference in survival between RFA and surgery in patients with solitary HCC and a diameter up to 4 cm [42]. At present strong evidence of the superiority or equality of RFA in comparison with surgical resection is lacking, but it is also true that there is no solid evidence that surgical resection is better than RFA for the treatment of small HCC. Studies addressing this issue 4117 April 21, 2014 Volume 20 Issue 15

19 Raza A et al. HCC: Treatment and evidence-based medicine Table 1 Surgical resection vs radiofrequency ablation Ref. 5-yr recurrence free survival 5-yr overall survival 1-yr recurrence-free survival 1-yr overall survival Tumor number n (SR, RFA) Mean tumor size (cm) Patients (n ) Study design Year Hasegawa et al [43] N/A SR: 71.1% RFA: 61.1% N/A N/A 1 (83, 73) 2 (13%, 20%) 3 (3%, 7%) SR: 2.3 RFA: 2.0 SR: 5361 RFA: 5548 Cohort study 2013 Tohme et al [44] SR: 34% RFA: 28% SR: 47% RFA: 35% SR: 66% RFA: 68% SR: 88% RFA: 86% 1 (78, 78) 2 (20%, 18%) 3 (2%, 3%) SR: 3.07 RFA: 2.36 SR: 50 RFA: 60 Cohort study 2013 Feng et al [42] N/A N/A SR: 90.6% RFA: 86.2% SR: 96% RFA: 93.1% 1 (62, 57) 2 (38%, 43%) SR: _ SR: 84 RFA: 84 RCT 2012 Huang et al [41] SR: 51.3% RFA: 28.6% SR: 75.6% RFA: 54.7% SR: 85.2% RFA: 81.7% SR: 98.2% RFA: 86.9% 1 (77, 73) 2 (20%, 26%) 3 (3%, 1%) 5 _ SR: 115 RFA: 115 RCT 2010 Hasegawa et al [45] N/A N/A N/A SR: 98.3% RFA: 98.5% 1 (84, 72) 2 (12%, 21%) 3 (3%, 7%) SR: 2.2 RFA: 2.0 SR: 2857 RFA: 3022 Cohort study 2008 Chen et al [46] N/A N/A SR: 86.6% RFA: 85.9% SR: 93.3% RFA: 95.8% 1 (100%, 100%) 5 _ SR: 90 RFA: 71 RCT 2006 Lü et al [47] N/A N/A SR: 82.4% RFA: 78.5% SR: 91.3% RFA: 93.5% 1 (96, 4) > 1 (88%, 12%) 3.2 _ SR: 54 RFA: 51 RCT 2006 Chen et al [48] N/A N/A N/A SR: 93.2% RFA: 92.8% 1 (100%, 100%) 5 _ SR: 65 RFA: 47 RCT 2005 N/A: Not applicable; RCT: Randomized controlled trial; RFA: Radiofrequency ablation; SR: Surgical resection. have been shown in Table 1 [41-48]. It is unlikely that any future RCT will address this issue, because it will require very large sample size to show significant differences between two modalities. RFA is less expensive, less invasive, with lower complication rates and shorter hospital stay than surgical resection. In patients with tumor > 3 cm but < 5 cm in size, the success rate of RFA alone is decreased. In these cases combination with TACE could be considered. Recently, the results of an RCT [49] aimed at evaluating the therapeutic efficacy of combining RFA with TACE for treating intermediate-size ( cm) HCCs have been published. Local tumor progression rate was significantly lower in the TACE and RFAtreated group than in the RFA-only group (6% vs 39%). RFA should be considered the first option for the treatment of small HCC. However, RFA is size-dependent. RFA can produce a necrotic area of about 4 cm, so it can be effective in HCC measuring up to 3 cm or smaller. In future technical developments allowing achieving ablation areas of 5 cm or more in diameter will make percutaneous thermal ablation an effective alternative to surgery even for tumors measuring 3 cm or more [50]. TACE TACE is currently considered a standard treatment for the patients with intermediate-stage HCC. Patients with compensated liver function (Child B up to 8 points), with large single nodule (< 5 cm) or multifocal HCC without evidence of vascular invasion or extra hepatic spread are considered candidates for TACE. The recommendation for TACE as the standard of care for intermediate-stage HCC is based on the demonstration of improved survival, as compared with the best supportive care or suboptimal therapies [51]. Llovet et al [52] reported a meta-analysis of six randomized controlled trials, comparing TACE with the best supportive care or suboptimal therapies. There was considerable heterogeneity between the individual study designs (including patient populations and TACE technique), as well as the study results, with only two of the six individual studies reporting 2-year survival with a statistically significant improvement [53,54]. TACE has been reported to achieve a partial response in 15%-62% patients, with significantly delayed tumor progression and improvement in median survival from mo [52]. Intermediate-stage HCC includes a heterogeneous population of the patients with variable tumor burden and liver function (Child-Pugh class A or B). The risk of TACEassociated complications may be greater in patients with more extensive disease requiring non-selective embolization, with portal vein thrombosis and with poor residual liver function. The patients with Child-Pugh class C and some with Child-Pugh class B should be excluded from TACE. In the studies reported in literature, MELD score has not been used to select patients. Patients with total bilirubin > 3 mg/dl were excluded. TACE benefits should be balanced with risk of treatment-induced liver failure. There is lack of standardized therapy regimen for TACE. The optimal schedule, choice of antineoplastic agents (e.g., mitomycin, cisplatin, and doxorubicin alone or in combination), embolizing agent (e.g., gelatin sponge particles or polyvinyl alcohol particles) use of iodized oil, or bland embolization vs chemoembolization, has not been fully established. From a technical point of view, while there is a general consensus about the fact that TACE should be as selective as possible, more standardization of TACE 4118 April 21, 2014 Volume 20 Issue 15

20 Raza A et al. HCC: Treatment and evidence-based medicine protocols is still needed. Selective TACE comprises the injection of chemotherapeutic agents into the segmental or sub segmental branches feeding the tumors. Golfieri et al [55] compared the effectiveness of selective or superselective TACE vs standard TACE in determining tumor necrosis in a prospective study of 67 consecutive patients (122 nodules, all < 5 cm). When compared with the standard TACE, selective/super-selective TACE was associated with higher mean levels of necrosis. A direct relationship was reported between the tumor diameter and the mean tumor necrosis level (59.6% for lesions < 2 cm, 68.4% for lesions cm and 76.2% for lesions > 3 cm). These findings suggest that selective/super selective TACE may determine a higher rate of tumor necrosis than the standard TACE; however, very small nodules (< 2 cm) may not respond as 3-4 cm nodules [56]. The ideal TACE procedure should allow maximum and sustained concentration of chemotherapeutic drug in the tumor, with minimal systemic exposure combined with calibrated tumor vessel obstruction. Lipiodol has been widely adopted in TACE protocols because HCC tumors have great avidity to lipiodol. However there is no data showing that lipiodol allows slow release of chemotherapeutic agents and achieves higher or sustained concentration of chemotherapeutic agents in tumor. One recent survey from multiple eastern and western centers in Europe showed that surgical resection is widely in practice among patients with multinodular, large, and macro-vascular invasive HCC, and provides acceptable short- and long-term results [57,58]. TACE with drug-eluting beads The recent introduction of embolic microspheres that have the ability to actively sequester doxorubicin hydrochloride from solution and release it in a controlled fashion has been shown to substantially diminish the amount of chemotherapeutic agent that reaches the systemic circulation, as compared with ethiodized oil-based regimens. This significantly increases the local concentration of the drug and the antitumor efficacy [59]. Recently published results from the PRECISION V trial indicate that TACE with drug-eluting beads is a valuable alternative to ethiodized oil-based conventional TACE. Compared with conventional TACE, the TACE with drug-eluting beads (DEB-TACE) with doxorubicineluted beads was associated with improved outcomes [60]. At 6 mo, the DEB-TACE group showed higher rates of complete response, objective response and disease control compared with the conventional TACE group. Although the predefined hypothesis of superiority was not met in the overall population, patients with Child- Pugh B, bi-lobar disease and recurrent disease showed a significant increase in objective response. In addition, DEB-TACE was associated with a reduction in serious liver toxicity and lower rate of doxorubicin-related side effects when compared with the standard TACE [60]. In some patients, there is a risk of systemic toxicity of chemotherapeutic agents used in conventional TACE or DEB-TACE. In these patients bland embolization can be performed. Trans-arterial bland embolization achieves tumor necrosis, but much less compared to DEB-TACE [61]. At present TACE is the standard of care for treating patients with intermediate-stage HCC, but due to heterogeneity of the patient population in this stage, all patients do not achieve the same response. DEB-TACE is preferred over conventional TACE. Repetition of TACE with aggressive schedule increases the adverse events. Repeat TACE should be considered based on objective evidence of tumor progression. Patient at risk of adverse outcome should be identified based on response to first TACE and effect on underlying liver disease. Recently describe ART score may help in identifying patients at high risk for poor outcome after repeated TACE [62,63]. RADIOEMBOLIZATION Radioembolization or selective internal radiation therapy (SIRT) has recently emerged as a therapeutic option for intermediate-stage HCC and its role in unresectable liver disease is still being refined [56,64-66]. In radioembolization, implantable radioactive microspheres are delivered into the arteries that feed the tumor so that tumor nodules are treated irrespective of their number, size or location. Radioembolization is different from the TACE. In TACE, the embolizing particles or drug eluting particles are usually μm in size, which cause ischemia of tumor; but in radioembolization the microspheres are usually smaller (35 μm) in diameter and deliver radiation to tumor without ischemia to the tumor or liver tissue. Currently, the most popular radioembolization technique uses microspheres coated with Y 90 b-emitting isotope (TheraSphere and SIR Sphere). The safety of Y 90 radioembolization has been documented in phase Ⅰ and phase Ⅱ clinical investigations [67]. A few observational studies and retrospective analyses have reported the efficacy of radioembolization in the treatment of HCC [68,69]. Median survivals for intermediate stage HCC, however, vary widely (between 7 and 27 mo) between phase Ⅱ studies, depending on the PS, extent of the disease and the degree of hepatic functional reserve. Salem et al [70] reported a large prospective study in 291 patients treated with glass-based Y 90 microspheres (TheraSphere) showing that liver function and portal vein thrombosis were main predictors of survival. Recently, a comparative analysis of radioembolization or TACE reported fewer side effects, better response rate and longer time to progression (13.3 mo vs 8.4 mo) in radioembolization group, but median survival time was not different (20.5 mo vs 17.5 mo) [68]. In another similar study by European Network on radioembolization with Y 90 resin microspheres. Sangro et al [69] reported similar safety profile and response rates. Results of RCTs would provide the highest level of evidence, but based on these studies, it has been estimated that more than 1000 patients would be required to confirm the statistical equivalence or superior April 21, 2014 Volume 20 Issue 15

21 Raza A et al. HCC: Treatment and evidence-based medicine ity of one treatment over other. Moreover, the relevant cost associated with radioembolization may limit a wide use of this technique. At present radioembolization appears to be safer in more advanced stage HCC including portal vein thrombosis and large tumor burden [69,71,72]. HCC TREATMENT AS A BRIDGE TO TRANSPLANT Patients with HCC receiving LT within Milan Criteria have a low rate of recurrence and excellent long-term survival. In recent years, waiting time for LT has progressively increased and despite priority for HCC within the Milan criteria, a significant rate of dropout from the waiting list occurs due to tumor progression. Hence treatment of HCC in patients awaiting LT has become routine, primarily in an effort to prevent tumor progression, reduce dropout rate and to decrease the post-transplant HCC recurrence. The risk of dropout for HCC within the Milan criteria correlates with the length of waiting time and initial tumor characteristics. In patients initially presenting with solitary HCC < 2 cm, risk of progression is low and only tumors > 2 cm receive priority on waiting list. Hence most transplant centers observe rather than treat these lesions until they grow to 2 cm. The cumulative dropout rates at 6 and 12 mo for patients with single HCC > 3 cm or with 2-3 nodules have been reported 12% and 56% vs 0% and 10% with solitary HCC 3 cm. These patients are often considered for treatment while awaiting LT. Chemoembolization, radiofrequency ablation and ethanol injection all are effective in controlling tumor growth; however, there is no high level evidence that these modalities are effective in stopping tumor progression in patients on the waiting list, reducing dropout rate or decreasing post-transplant recurrence. TACE has been widely used, as a bridge to transplant but there is no evidence-based data to support this practice. TACE has not been shown to decrease the dropout rates on waiting list [14,73], but most of the studies addressing this issue were heterogeneous in patient selection, TACE-related protocols and had variable waiting time on LT list. It is unlikely that well-designed RCTs will address this issue in the future. Nevertheless, particularly in the United States, where continued waiting list priority depends on maintaining HCC within Milan criteria, use of nonsurgical HCC treatment will likely continue in an effort to prevent tumor progression and waiting list dropout. TACE alone or combination with other treatments is recommended to bridge patients to transplant specifically when the waiting list time is more than six mo. TARGETED SYSTEMIC CHEMOTHERAPY Hepatocarcinogenesis is the result of genetic alterations affecting multiple signaling cascades resulting in uncontrolled growth of the hepatocytes. Systemic targeted therapies focus on the critical steps of the carcinogenic pathways, limiting widespread systemic toxicity. No single dominant or pathognomic pathway exists in the hepatocarcinogenesis. Overexpression of multiple signaling pathways have been implicated in the pathogenesis of HCC including Vascular endothelial growth factor (VEGF), epidermal growth factor, Ras mitogenactivated protein kinase (MAPK), insulin-like growth factor receptor, hepatocyte growth factor/c-met, PI3K/ PTEN/Akt/mammalian target of rapamycin (mtor) and Wnt/β-Catenin pathways [74-79]. Targeted molecular agents may block one or more steps in a targeted pathway or potentially more than one pathway to provide suitable results. Currently, sorafenib is approved for the treatment of HCC and represents a paradigm shift in the systemic treatment of HCC, and many new molecular therapies are under investigation. Sorafenib Multiple cellular kinases are involved in the development and progression of the HCC by promoting angiogenesis, cellular differentiation, proliferation and survival. Sorafenib is an oral bi-aryl urea, which inhibits multiple cell surface and downstream kinases involved in tumor progression. Cell surface tyrosine kinases inhibited by Sorafenib include VEGF receptor- (VEGFR-) 1, VEG- FR-2, VEGFR-3, platelet-derived growth factor receptor- (PDGFR-) β, RET, c-kit and FMS-like tyrosine kinase-3. Sorafenib also inhibits Ras/MAPK pathway, this pathway involves extracellular signal-regulated kinases and multiple intracellular serine/ threonine kinases including Raf-1 (C-Raf) and B-Raf (wild and mutanttypes). Ras/MAPK pathway activation could be due to the mutational activation of Ras oncogene or over expression of surface tyrosine kinases. Overexpression of these kinases is important in HCC proliferation and angiogenesis [80,81]. Two phase Ⅲ randomized placebocontrolled trials, the SHARP trial conducted mainly in America and Europe [82] and a similar trial conducted in Asia [83] reported improved overall survival with sorafenib. In the SHARP trial, the median overall survival was 10.7 mo with sorafenib and 7.9 mo with placebo. In the Asian study, the median overall survival was 6.5 mo with sorafenib and 4.2 mo with placebo. Sorafenib was generally well tolerated; toxicities were mild to moderate in severity, predominantly including diarrhea, fatigue, and hand-foot skin reaction. These two, phase Ⅲ trials have established sorafenib as the preferred systemic therapy for advanced HCC although the role of sorafenib in intermediate HCC is less clear. Moreover, only small numbers of patients with Child-Pugh B have been included in clinical trials, so it is not possible to assess efficacy and safety of sorafenib in this group of patients. Various phase Ⅲ trials reporting the overall survival in patients with advanced HCC treated with sorafenib, sunitinib, erlotinib, linifanib and brivanib are shown in Table April 21, 2014 Volume 20 Issue 15

22 Raza A et al. HCC: Treatment and evidence-based medicine Table 2 Phase Ⅲ clinical trials of systemic targeted agents Ref. Year Patients (n ) Overall survival (mo) Llovet et al [82] 2008 Sorafenib: 299 Sorafenib: 10.7 (SHARP trial) Placebo: 303 Placebo: 7.9 Cheng et al [83] 2009 Sorafenib: 150 Sorafenib: 6.5 (NCT ) Placebo: 76 Placebo: 4.2 Zhu et al [100] 2012 Sorafenib: 358 Sorafenib: 8.5 (SEARCH trial) Sorafenib + Sorafenib + Erlotinib: 362 Erlotinib: 9.5 Cheng et al [99] 2013 Sorafenib: 544 Sorafenib: 10.2 (SUN1170 trial) Sunitinib: 530 Sunitinib: 7.9 Cainap et al [101] 2013 Sorafenib: N/A Sorafenib: 9.8 (LIGHT trial) Linifanib: N/A Linifanib: 9.1 Johnson et al [95] 2013 Sorafenib: 578 Sorafenib: 9.9 (BRISK-FL trial) Brivanib: 577 Brivanib: 9.5 Llovet et al [94] 2013 Brivanib: 263 Brivanib: 9.4 (BRISK-PS trial) Placebo: 132 Placebo: 8.3 Sorafenib has also been used in combination with other systemic chemotherapeutic agents with a goal to improve efficacy. Sorafenib in combination with doxorubicin [84], octreotide [85] and oxaliplatin [86], tegafur/uracil [87], cisplatin and gemcitabine [88] and AVE 1642 (a human monoclonal antibody inhibiting the insulin-like growth factor-1 receptor) [89] has been used. All of these studies report some survival advantage over sorafenib alone. But most of the studies looking at the combination of sorafenib with other systemic therapies have small sample size. Large randomized double-blind studies are needed to establish the role and toxicity profile of these combination regimens. Other chemotherapeutic agents Sunitinib: Sunitinib is a multi-kinase blocker that targets VEGFR and PDGFR. Sunitinib was used in phase Ⅱ clinical trials for HCC treatment, which led to an openlabel phase Ⅲ trial comparing it with sorafenib [90]. A total of 1073 patients were randomized to receive either sorafenib (544) or sunitinib (529). This trial was terminated early due to increased side effects and futility concerns. Linifanib: Linifanib is a multi-kinase inhibitor targeting VEGFR and PDGFR along with other kinases. It was found to be effective in the treatment of the HCC with an acceptable safety profile in a single arm phase Ⅱ clinical trial [91]. Brivanib: Brivanib is a selective inhibitor of fibroblastic growth factor receptor and VEGFR. It showed somewhat promising results in the phase Ⅱ trials as first line (median overall survival: 10 mo) and second line (median overall survival: 9.5 mo) treatment agent for HCC [92,93]. Brivanib was tried in a phase Ⅲ BRISK-PS trial as a secondary treatment agent (failed prior systemic treatment due to side effects or progression of the disease) for the treatment of HCC. The median length of overall survival was 9.4 mo for brivanib recipients vs 8.2 mo in the placebo group, which was not statistically significant (P = 0.33) [94]. Another phase Ⅲ trial, BRISK-FL, compared brivanib with sorafenib as first line treatment agent for HCC [95]. Median survival was 9.5 mo in the brivanib group compared with 9.9 mo in the sorafenib group, which was not statistically significant. Sorafenib was better tolerated than brivanib leading to lesser discontinuation rate (33% vs 43% respectively). Tivantinib: Tivantinib is an oral MET receptor tyrosine kinase inhibitor. When added to sorafenib, it had synergistic effect against HCC as noted in a phase Ⅰ clinical trial [96]. In a randomized, placebo-controlled, doubleblind, phase Ⅱ trial, tivantinib was used as a second line agent for the treatment of HCC in previously unresectable HCC who progressed or could not tolerate the first line systemic therapy [97]. The patients were randomly assigned to receive tivantinib (n = 71) or placebo (n = 36). Time to progression of HCC was longer in tivantinib group (1.6 mo) than the placebo group (1.4 mo) (HR = 0.64; P = 0.04). The subgroup of patients who received tivantinib and expressed high tissue MET levels (n = 22) had even longer median time to progression of HCC (2.7 mo). A randomized, double-blinded, controlled phase Ⅲ study (METIV-HCC trial) is currently underway to determine the efficacy and safety of tivantinib plus sorafenib vs sorafenib alone in the patients with previously unresectable cancer as a first line treatment agent. Everolimus: Everolimus is an inhibitor of mtor. A phase Ⅰ/Ⅱ single arm trial using everolimus in advanced HCC patients (unresectable) with and without prior systemic therapy for HCC showed that the median progression free survival of 28 patients was 3.8 mo (95%CI: ) and overall survival was 8.4 mo (95%CI: ) [98]. And phase Ⅲ clinical trials of systemic targeted agents is shown in Table 2 [82,83,94,95,99-101]. A randomized, double blind, placebo control phase Ⅲ trial (EVOLVE-1) is underway to assess the role of everolimus in unresectable HCC patients who failed prior treatment with sorafenib. Sorafenib and TACE combination As previously discussed, TACE works by blocking the hyper-vascular arterial blood supply of the tumor with the help of an embolic agent and injecting the chemotherapeutic drug. As a result of TACE, a hypoxic environment is created around the surviving tumor cells. Hypoxia stimulates the expression of VEGF and hence the neovascularization of the surviving cells. Sorafenib appears to be a good choice to block the neovascularization at that stage. A phase Ⅲ trial comparing linifanib to sorafenib as a first line targeted agent has recently been reported [101]. Recently Gadani et al [102] presented their results of a retrospective analysis of 19 patients with Child-Pugh class A and B patients with HCC. Most of the patients were Child-Pugh class A (n = 16) and BCLC stage C (n = 13). Various studies have looked at the combination of 4121 April 21, 2014 Volume 20 Issue 15

23 Raza A et al. HCC: Treatment and evidence-based medicine TACE with sorafenib, where sorafenib was introduced few days to weeks after the first TACE (sequential introduction) or it was started prior to the planned TACE and only interrupted for few days around the procedure (interrupted scheduling). There has been reluctance to use combination of TACE and sorafenib due to fear of increased toxicity. In a prospective study patients with unresectable HCC received a combination of sorafenib (started 2-4 wk prior to TACE) and TACE with LC beads [103]. The authors reported safety of concurrent sorafenib and transarterial therapy but without clear benefit of survival. The efficacy of combination treatment has been assessed in few prospective studies. In a prospective, placebo controlled, randomized, double-blind study Sansonno et al [104] randomized 31 patients with Child-Pugh class A and BCLC-B HCC to receive conventional TACE plus sorafenib and similar number of patients to receive TACE plus placebo. Sorafenib was added 30 d after the first TACE procedure and the patients received more than one TACE procedures. The median time to progression was 9.2 and 4.9 mo in the TACE plus sorafenib and the TACE plus placebo groups respectively. In another study Kudo et al [105] did not find a difference in overall survival or time to progression benefit with TACE plus sorafenib combination compared with TACE plus placebo. But this effect was likely due to the fact that sorafenib was started late after the first TACE procedure (> 50% of the patients starting it more than 9 wk post-tace) and there were significant dose reductions and multiple dose interruptions. The START trial [106] was conducted to assess the combination of sorafenib with conventional TACE procedure. One subgroup analysis of the Chinese patients (n = 62) in the START trial was recently published [107]. Patients with unresectable HCC were enrolled and they received conventional TACE and sorafenib 400 mg twice a day. Sorafenib was continued until 4 d prior to the next TACE and was resumed 4 d after TACE procedure for safety reasons. The preliminary results of START indicate concurrent sorafenib and TACE therapy is safe and effective with no unexpected side effects. Similar results were produced in another subgroup analysis of the START trial in Asia-Pacific region, without any un-expected side effects [108]. Currently DEB-TACE has shown superiority over conventional TACE. DEB-TACE in combination with sorafenib has been studied in clinical trails (SPACE, and TACE-2 trials) [109,110]. Recently reported data from the randomized phase Ⅱ SPACE trial suggest that DEB-TACE in combination with sorafenib met the predefined primary endpoint of improving time to radiologic progression compared with DEB-TACE in combination with placebo [109]. The results of ongoing phase Ⅲ trials will determine whether there is a role to implement this combination in clinical practice. The results of concurrent TACE and sorafenib in intermediate stage appear promising but at present it is difficult to recommend combination therapy. There are uncertainties regarding dose, frequency and duration of sorafenib when used in combination with TACE. Sorafenib and radio-embolization Several on-going clinical trials are looking at the combination of radio-embolization and sorafenib in patients with HCC. Recently Gadani et al [102] presented their results of a retrospective analysis of patients with Child- Pugh class A and B patients with HCC. The patients were on sorafenib prior to yttrium-90 treatment, which was resumed post- treatment. The overall survival of the patients was higher than the previously reported studies that only used sorafenib. Further prospective studies are being conducted to evaluate the combination of radiation therapy and sorafenib. CONCLUSION Management of HCC depends on the tumor stage, liver function reserve, and patient performance status (BCLC stage), and requires a multidisciplinary approach for optimal treatment. LT and hepatic resection are the only curative options in early stage of disease. There have been significant advances in local ablative and transarterial therapies. In the early stage HCC, RFA is equivalent to surgical resection in well-selected patients. Drugeluting beads have improved the efficacy and safety of conventional TACE. Radioembolization with use of resin or glass sphere appear promising. 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26 Raza A et al. HCC: Treatment and evidence-based medicine son A, Pitton M, Sergent G, Pfammatter T, Terraz S, Benhamou Y, Avajon Y, Gruenberger T, Pomoni M, Langenberger H, Schuchmann M, Dumortier J, Mueller C, Chevallier P, Lencioni R. Prospective randomized study of doxorubicineluting-bead embolization in the treatment of hepatocellular carcinoma: results of the PRECISION V study. Cardiovasc Intervent Radiol 2010; 33: [PMID: DOI: / s ] 61 Nicolini D, Svegliati-Baroni G, Candelari R, Mincarelli C, Mandolesi A, Bearzi I, Mocchegiani F, Vecchi A, Montalti R, Benedetti A, Risaliti A, Vivarelli M. Doxorubicin-eluting bead vs conventional transcatheter arterial chemoembolization for hepatocellular carcinoma before liver transplantation. World J Gastroenterol 2013; 19: [PMID: DOI: /wjg.v19.i ] 62 Hucke F, Sieghart W, Pinter M, Graziadei I, Vogel W, Müller C, Heinzl H, Waneck F, Trauner M, Peck-Radosavljevic M. The ART-strategy: sequential assessment of the ART score predicts outcome of patients with hepatocellular carcinoma re-treated with TACE. J Hepatol 2014; 60: [PMID: DOI: /j.jhep ] 63 Sieghart W, Hucke F, Pinter M, Graziadei I, Vogel W, Müller C, Heinzl H, Trauner M, Peck-Radosavljevic M. The ART of decision making: retreatment with transarterial chemoembolization in patients with hepatocellular carcinoma. Hepatology 2013; 57: [PMID: DOI: / hep.26256] 64 Sangro B, Carpanese L, Cianni R, Golfieri R, Gasparini D, Ezziddin S, Paprottka PM, Fiore F, Van Buskirk M, Bilbao JI, Ettorre GM, Salvatori R, Giampalma E, Geatti O, Wilhelm K, Hoffmann RT, Izzo F, Iñarrairaegui M, Maini CL, Urigo C, Cappelli A, Vit A, Ahmadzadehfar H, Jakobs TF, Lastoria S. Survival after yttrium-90 resin microsphere radioembolization of hepatocellular carcinoma across Barcelona clinic liver cancer stages: a European evaluation. Hepatology 2011; 54: [PMID: DOI: /hep.24451] 65 Lau WY, Sangro B, Chen PJ, Cheng SQ, Chow P, Lee RC, Leung T, Han KH, Poon RT. Treatment for hepatocellular carcinoma with portal vein tumor thrombosis: the emerging role for radioembolization using yttrium-90. Oncology 2013; 84: [PMID: DOI: / ] 66 Shah RP, Brown KT, Sofocleous CT. Arterially directed therapies for hepatocellular carcinoma. AJR Am J Roentgenol 2011; 197: W590-W602 [PMID: DOI: / AJR ] 67 Mazzaferro V, Sposito C, Bhoori S, Romito R, Chiesa C, Morosi C, Maccauro M, Marchianò A, Bongini M, Lanocita R, Civelli E, Bombardieri E, Camerini T, Spreafico C. Yttrium-90 radioembolization for intermediate-advanced hepatocellular carcinoma: a phase 2 study. Hepatology 2013; 57: [PMID: DOI: /hep.26014] 68 Salem R, Lewandowski RJ, Kulik L, Wang E, Riaz A, Ryu RK, Sato KT, Gupta R, Nikolaidis P, Miller FH, Yaghmai V, Ibrahim SM, Senthilnathan S, Baker T, Gates VL, Atassi B, Newman S, Memon K, Chen R, Vogelzang RL, Nemcek AA, Resnick SA, Chrisman HB, Carr J, Omary RA, Abecassis M, Benson AB, Mulcahy MF. Radioembolization results in longer time-to-progression and reduced toxicity compared with chemoembolization in patients with hepatocellular carcinoma. Gastroenterology 2011; 140: e2 [PMID: DOI: /j.gastro ] 69 Sangro B, Iñarrairaegui M, Bilbao JI. Radioembolization for hepatocellular carcinoma. J Hepatol 2012; 56: [PMID: DOI: /j.jhep ] 70 Salem R, Lewandowski RJ, Mulcahy MF, Riaz A, Ryu RK, Ibrahim S, Atassi B, Baker T, Gates V, Miller FH, Sato KT, Wang E, Gupta R, Benson AB, Newman SB, Omary RA, Abecassis M, Kulik L. Radioembolization for hepatocellular carcinoma using Yttrium-90 microspheres: a comprehensive report of long-term outcomes. Gastroenterology 2010; 138: [PMID: DOI: /j.gastro ] 71 Salem R, Mazzaferro V, Sangro B. Yttrium 90 radioembolization for the treatment of hepatocellular carcinoma: biological lessons, current challenges, and clinical perspectives. Hepatology 2013; 58: [PMID: DOI: /hep.26382] 72 Kim YH, Kim do Y. Yttrium-90 radioembolization for hepatocellular carcinoma: what we know and what we need to know. Oncology 2013; 84 Suppl 1: [PMID: DOI: / ] 73 Lesurtel M, Müllhaupt B, Pestalozzi BC, Pfammatter T, Clavien PA. Transarterial chemoembolization as a bridge to liver transplantation for hepatocellular carcinoma: an evidencebased analysis. Am J Transplant 2006; 6: [PMID: DOI: /j x] 74 Chiang DY, Villanueva A, Hoshida Y, Peix J, Newell P, Minguez B, LeBlanc AC, Donovan DJ, Thung SN, Solé M, Tovar V, Alsinet C, Ramos AH, Barretina J, Roayaie S, Schwartz M, Waxman S, Bruix J, Mazzaferro V, Ligon AH, Najfeld V, Friedman SL, Sellers WR, Meyerson M, Llovet JM. Focal gains of VEGFA and molecular classification of hepatocellular carcinoma. Cancer Res 2008; 68: [PMID: DOI: / CAN ] 75 Ito Y, Takeda T, Sakon M, Tsujimoto M, Higashiyama S, Noda K, Miyoshi E, Monden M, Matsuura N. Expression and clinical significance of erb-b receptor family in hepatocellular carcinoma. Br J Cancer 2001; 84: [PMID: DOI: /bjoc ] 76 Calvisi DF, Ladu S, Gorden A, Farina M, Conner EA, Lee JS, Factor VM, Thorgeirsson SS. Ubiquitous activation of Ras and Jak/Stat pathways in human HCC. Gastroenterology 2006; 130: [PMID: DOI: / j.gastro ] 77 Sahin F, Kannangai R, Adegbola O, Wang J, Su G, Torbenson M. mtor and P70 S6 kinase expression in primary liver neoplasms. Clin Cancer Res 2004; 10: [PMID: DOI: / CCR ] 78 Takami T, Kaposi-Novak P, Uchida K, Gomez-Quiroz LE, Conner EA, Factor VM, Thorgeirsson SS. Loss of hepatocyte growth factor/c-met signaling pathway accelerates early stages of N-nitrosodiethylamine induced hepatocarcinogenesis. Cancer Res 2007; 67: [PMID: DOI: / CAN ] 79 Breuhahn K, Longerich T, Schirmacher P. Dysregulation of growth factor signaling in human hepatocellular carcinoma. Oncogene 2006; 25: [PMID: DOI: / sj.onc ] 80 Wilhelm S, Carter C, Lynch M, Lowinger T, Dumas J, Smith RA, Schwartz B, Simantov R, Kelley S. Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nat Rev Drug Discov 2006; 5: [PMID: DOI: /nrd2130] 81 Wilhelm SM, Carter C, Tang L, Wilkie D, McNabola A, Rong H, Chen C, Zhang X, Vincent P, McHugh M, Cao Y, Shujath J, Gawlak S, Eveleigh D, Rowley B, Liu L, Adnane L, Lynch M, Auclair D, Taylor I, Gedrich R, Voznesensky A, Riedl B, Post LE, Bollag G, Trail PA. BAY exhibits broad spectrum oral antitumor activity and targets the RAF/ MEK/ERK pathway and receptor tyrosine kinases involved in tumor progression and angiogenesis. Cancer Res 2004; 64: [PMID: DOI: / CAN ] 82 Llovet JM, Ricci S, Mazzaferro V, Hilgard P, Gane E, Blanc JF, de Oliveira AC, Santoro A, Raoul JL, Forner A, Schwartz M, Porta C, Zeuzem S, Bolondi L, Greten TF, Galle PR, Seitz JF, Borbath I, Häussinger D, Giannaris T, Shan M, Moscovici M, Voliotis D, Bruix J. Sorafenib in advanced hepatocellular carcinoma. N Engl J Med 2008; 359: [PMID: DOI: /NEJMoa ] 83 Cheng AL, Kang YK, Chen Z, Tsao CJ, Qin S, Kim JS, Luo 4125 April 21, 2014 Volume 20 Issue 15

27 Raza A et al. HCC: Treatment and evidence-based medicine R, Feng J, Ye S, Yang TS, Xu J, Sun Y, Liang H, Liu J, Wang J, Tak WY, Pan H, Burock K, Zou J, Voliotis D, Guan Z. Efficacy and safety of sorafenib in patients in the Asia-Pacific region with advanced hepatocellular carcinoma: a phase III randomised, double-blind, placebo-controlled trial. Lancet Oncol 2009; 10: [PMID: DOI: / S (08) ] 84 Abou-Alfa GK, Johnson P, Knox JJ, Capanu M, Davidenko I, Lacava J, Leung T, Gansukh B, Saltz LB. Doxorubicin plus sorafenib vs doxorubicin alone in patients with advanced hepatocellular carcinoma: a randomized trial. JAMA 2010; 304: [PMID: DOI: /jama ] 85 Prete SD, Montella L, Caraglia M, Maiorino L, Cennamo G, Montesarchio V, Piai G, Febbraro A, Tarantino L, Capasso E, Palmieri G, Guarrasi R, Bianco M, Mamone R, Savastano C, Pisano A, Vincenzi B, Sabia A, D Agostino A, Faiola V, Addeo R. Sorafenib plus octreotide is an effective and safe treatment in advanced hepatocellular carcinoma: multicenter phase II So.LAR. study. Cancer Chemother Pharmacol 2010; 66: [PMID: DOI: /s z] 86 Yau TCP, Chan P, Cheung FY, Lee AS, Yau TK, Choo SP, Lau J, Wong JS, Fan ST, Poon RT. Phase II trial of sorafenib with capecitabine and oxaliplatin (SECOX) in patients with locally advanced or metastatic hepatocellular carcinoma. EJC 2009; Suppl 7: Hsu CH, Shen YC, Lin ZZ, Chen PJ, Shao YY, Ding YH, Hsu C, Cheng AL. Phase II study of combining sorafenib with metronomic tegafur/uracil for advanced hepatocellular carcinoma. J Hepatol 2010; 53: [PMID: DOI: /j.jhep ] 88 Giuliana FAR, Addeo R, Febbraio A, Rizzi D, Macello E, del Prete S, Pisconti S, Fico M, Colucci G. Sorafenib plus cisplatin and gemcitabine in the treatment of advanced hepatocellular carcinoma: a phase II study by the Grupo Oncologico Dell Italia Meridonale. (PROT. GOIM 2705). Cancer Treat Rev 2010; 36 Suppl 4: S96 89 Faivre SFL, Fartoux L, Bumsel F, Bouattor M, Dreyer C, Raymond E, Rosmorduc O. Phase I safety, pharmacokinetic, and pharmacodynamic study of AVE 1642, a human monoclonal antibody inhibiting the insulin-like grouth factor-1 receptor (IGF-1R/CD221), administered as single agent and in combination with sorafinib as first line therapy in patients with advanced hepatocellular carcinoma. Hepatology 2010; 52 Suppl: 466A, Cheng A, Kang Y, Lin D, Park J, Kudo M, Qin S, Omata M, Lowenthal SW, Lanzalone S, Yang L, Lechuga M, Raymond E and SUN1170 HCC Study Group. Phase III trial of sunitinib (Su) versus sorafenib (So) in advanced hepatocellular carcinoma (HCC). J Clin Oncol 2011; 29 Suppl 15: Toh HC, Chen PJ, Carr BI, Knox JJ, Gill S, Ansell P, McKeegan EM, Dowell B, Pedersen M, Qin Q, Qian J, Scappaticci FA, Ricker JL, Carlson DM, Yong WP. Phase 2 trial of linifanib (ABT-869) in patients with unresectable or metastatic hepatocellular carcinoma. Cancer 2013; 119: [PMID: DOI: /cncr.27758] 92 Park JW, Finn RS, Kim JS, Karwal M, Li RK, Ismail F, Thomas M, Harris R, Baudelet C, Walters I, Raoul JL. Phase II, open-label study of brivanib as first-line therapy in patients with advanced hepatocellular carcinoma. Clin Cancer Res 2011; 17: [PMID: DOI: / CCR ] 93 Finn RS, Kang YK, Mulcahy M, Polite BN, Lim HY, Walters I, Baudelet C, Manekas D, Park JW. Phase II, open-label study of brivanib as second-line therapy in patients with advanced hepatocellular carcinoma. Clin Cancer Res 2012; 18: [PMID: DOI: / CCR ] 94 Llovet JM, Decaens T, Raoul JL, Boucher E, Kudo M, Chang C, Kang YK, Assenat E, Lim HY, Boige V, Mathurin P, Fartoux L, Lin DY, Bruix J, Poon RT, Sherman M, Blanc JF, Finn R, Tak WY, Chao Y, Ezzeddine R, Liu D, Walters I, Park JW. Brivanib versus placebo in patients with advanced hepatocellular carcinoma (HCC) who failed or were intolerant to sorafenib: results from the phase 3 BRISK-PS study. EASL 2012; Abstract Johnson PJ, Qin S, Park JW, Poon RT, Raoul JL, Philip PA, Hsu CH, Hu TH, Heo J, Xu J, Lu L, Chao Y, Boucher E, Han KH, Paik SW, Robles-Aviña J, Kudo M, Yan L, Sobhonslidsuk A, Komov D, Decaens T, Tak WY, Jeng LB, Liu D, Ezzeddine R, Walters I, Cheng AL. Brivanib versus sorafenib as first-line therapy in patients with unresectable, advanced hepatocellular carcinoma: results from the randomized phase III BRISK-FL study. J Clin Oncol 2013; 31: [PMID: DOI: /JCO ] 96 Martell RE, Puzanov I, Ma WW, Santoro A, Dy GK, Goff LW, Fetterly GJ, Michael SA, Means-Powell JA, Chai F, Lamar M, Simonelli M, Chiang WM, Jarboe J, Schwartz BE, Adjei AA, Nashville TN, Buffalo NY, Woburn MA. Safety and efficacy of MET inhibitor tivantinib (ARQ 197) combined with sorafenib in patients (pts) with hepatocellular carcinoma (HCC) from a phase I study. Proc Am Soc Clin Oncol 2012; 30 Suppl: abstract Santoro A, Rimassa L, Borbath I, Daniele B, Salvagni S, Van Laethem JL, Van Vlierberghe H, Trojan J, Kolligs FT, Weiss A, Miles S, Gasbarrini A, Lencioni M, Cicalese L, Sherman M, Gridelli C, Buggisch P, Gerken G, Schmid RM, Boni C, Personeni N, Hassoun Z, Abbadessa G, Schwartz B, Von Roemeling R, Lamar ME, Chen Y, Porta C. Tivantinib for second-line treatment of advanced hepatocellular carcinoma: a randomised, placebo-controlled phase 2 study. Lancet Oncol 2013; 14: [PMID: DOI: / S (12) ] 98 Zhu AX, Abrams TA, Miksad R, Blaszkowsky LS, Meyerhardt JA, Zheng H, Muzikansky A, Clark JW, Kwak EL, Schrag D, Jors KR, Fuchs CS, Iafrate AJ, Borger DR, Ryan DP. Phase 1/2 study of everolimus in advanced hepatocellular carcinoma. Cancer 2011; 117: [PMID: DOI: /cncr.26165] 99 Cheng AL, Kang YK, Lin DY, Park JW, Kudo M, Qin S, Chung HC, Song X, Xu J, Poggi G, Omata M, Pitman Lowenthal S, Lanzalone S, Yang L, Lechuga MJ, Raymond E. Sunitinib versus sorafenib in advanced hepatocellular cancer: results of a randomized phase III trial. J Clin Oncol 2013; 31: [PMID: DOI: /JCO ] 100 Zhu AX, Rosmorduc O, Evans J, Ross P, Santoro A, Carriho FJ, Leberre M, Jensen M, Meinhardt G, Kang Y. SEARCH: a phase III, randomized, double-blind, placebo-controlled trial of sorafenib plus erlotinib in patients with hepatocellular carcinoma (HCC). Ann Oncol 2012; 23 Suppl: LBA2 101 Cainap C, Qin S, Huang WT, Chung IJ, Pan H, Cheng Y, Kudo M, Kang YK, PChen PJ, Toh HC, Gorbunova V, Eskens F, Qian J, McKee MD, Ricker JL, Carlson DM, Nowiemet SE. Phase III trial of linifanib versus sorafenib in patients with advanced hepatocellular carcinoma (HCC). J Clin Oncol 2012; 30 (Suppl 34): abstr Gadani S, Mahvash A, Avritscher R, Chasen B, Kaseb A, Murthy R. Yttirum-90 resin microspheres as an adjunct to sorafenib in patients with unresectable HCC: A retrospective study for evaluation of survival benefit and adverse events. Society of Interventional Radiology Annual Scientific Meeting, Presented at: April 15, 2013: Abstract Cabrera R, Pannu DS, Caridi J, Firpi RJ, Soldevila-Pico C, Morelli G, Clark V, Suman A, George TJ, Nelson DR. The combination of sorafenib with transarterial chemoembolisation for hepatocellular carcinoma. Aliment Pharmacol Ther 2011; 34: [PMID: DOI: / j x] 104 Sansonno D, Lauletta G, Russi S, Conteduca V, Sansonno L, Dammacco F. Transarterial chemoembolization plus sorafenib: a sequential therapeutic scheme for HCV-related intermediate-stage hepatocellular carcinoma: a randomized 4126 April 21, 2014 Volume 20 Issue 15

28 Raza A et al. HCC: Treatment and evidence-based medicine clinical trial. Oncologist 2012; 17: [PMID: DOI: /theoncologist ] 105 Kudo M, Imanaka K, Chida N, Nakachi K, Tak WY, Takayama T, Yoon JH, Hori T, Kumada H, Hayashi N, Kaneko S, Tsubouchi H, Suh DJ, Furuse J, Okusaka T, Tanaka K, Matsui O, Wada M, Yamaguchi I, Ohya T, Meinhardt G, Okita K. Phase III study of sorafenib after transarterial chemoembolisation in Japanese and Korean patients with unresectable hepatocellular carcinoma. Eur J Cancer 2011; 47: [PMID: DOI: /j.ejca ] 106 Chao Y, Lee HC, Lee TY, Yoon J, Han G, Yang J, Wang J, Kim B, Shao G, Chung YH. START (Study in Asia of the Combination of TACE (transcatheter arterial chemoembolization) with Sorafenib in Patients with Hepatocellular Carcinoma) Trial. 5th International Liver Cancer Association (ILCA) Annual Conference; 2011, September 1-3; Hong Kong: Wanchai, 2011: Abstract 0-Abstract Han G, Yang J, Shao G, Teng G, Wang M, Yang J, Liu Z, Feng G, Yang R, Lu L, Chao Y, Wang J. Sorafenib in combination with transarterial chemoembolization in Chinese patients with hepatocellular carcinoma: a subgroup interim analysis of the START trial. Future Oncol 2013; 9: [PMID: DOI: /fon.13.11] 108 Chung YH, Han G, Yoon JH, Yang J, Wang J, Shao GL, Kim BI, Lee TY, Chao Y. Interim analysis of START: Study in Asia of the combination of TACE (transcatheter arterial chemoembolization) with sorafenib in patients with hepatocellular carcinoma trial. Int J Cancer 2013; 132: [PMID: DOI: /ijc.27925] 109 Pawlik TM, Reyes DK, Cosgrove D, Kamel IR, Bhagat N, Geschwind JF. Phase II trial of sorafenib combined with concurrent transarterial chemoembolization with drug-eluting beads for hepatocellular carcinoma. J Clin Oncol 2011; 29: [PMID: DOI: /JCO ] 110 Meyer T, Fox R, Bird D, Watkinson A, Hacking N, Stocken D, Johnson PJ, Palmer DH. TACE 2: A randomized placebocontrolled, double-blinded, phase III trial evaluating sorafenib in combination with transarterial chemoembolization (TACE) in patients with unresectable hepatocellular carcinoma (HCC). J Clin Oncol 2012; 30 suppl: TPS4150 P- Reviewers: Cong WM, Cucchetti A S- Editor: Gou SX L- Editor: A E- Editor: Liu XM 4127 April 21, 2014 Volume 20 Issue 15

29 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (1): Hepatocellular carcinoma TOPIC HIGHLIGHT Role of the tissue microenvironment as a therapeutic target in hepatocellular carcinoma Bhavna Rani, Yuan Cao, Andrea Malfettone, Ciprian Tomuleasa, Isabel Fabregat, Gianluigi Giannelli Bhavna Rani, Yuan Cao, Gianluigi Giannelli, Department of Medical Biosciences and Human Oncology, Padiglione Semeiotica Medica, Bari, Italy Andrea Malfettone, Isabel Fabregat, Bellvitge Biomedical Research Institute (IDIBELL), Barcelona, Spain Isabel Fabregat, Department of Physiological Sciences Ⅱ, University of Barcelona, Barcelona, Spain Ciprian Tomuleasa, Department of Hematology, Center for Genomics and Translational Medicine, Iuliu Hatieganu University of Medicine and Pharmacy, Cluj Napoca, Romania Author contributions: Rani B, Cao Y, Malfettone A and Tomuleasa C reviewed the literature; Fabregat I contributed to the work; Giannelli G organized the manuscript. Supported by EU-Marie Curie Initial Training Network (ITN), FP7-PEOPLE-2012-ITN 2012, Grant Agreement No Correspondence to: Gianluigi Giannelli, MD, Department of Medical Biosciences and Human Oncology, Padiglione Semeiotica Medica, Policlinico, Piazza G. Cesare 11, Bari, Italy. gianluigi.giannelli@uniba.it Telephone: Fax: Received: October 6, 2013 Revised: January 11, 2014 Accepted: February 16, 2014 Published online: April 21, Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Tissue microenvironment; Hepatocellular carcinoma; Transforming growth factor-beta; Laminin-5; Cancer stem cells; Therapy; Target therapy Core tip: We discuss a new hypothesis for therapeutic approaches in hepatocellular carcinoma (HCC). This novel idea is to regard the entire liver as responsible for the onset, growth and progression of HCC. In this scenario, we focus on the tissue microenvironment components as an ideal target for systemic therapies, taking into account the tumor/host interactions. Rani B, Cao Y, Malfettone A, Tomuleasa C, Fabregat I, Giannelli G. Role of the tissue microenvironment as a therapeutic target in hepatocellular carcinoma. World J Gastroenterol 2014; 20(15): Available from: URL: com/ /full/v20/i15/4128.htm DOI: org/ /wjg.v20.i Abstract Hepatocellular carcinoma is difficult to treat, primarily because the underlying molecular mechanisms driving clinical outcome are still poorly understood. Growing evidence suggests that the tissue microenvironment has a role in the biological behavior of the tumor. The main clinical issue is to identify the best target for therapeutic approaches. Here, we discuss the hypothesis that the entire tissue microenvironment might be considered as a biological target. However, the tissue microenvironment consists of several cellular and biochemical components, each of which displays a distinct biological activity. We discuss the major components of this environment and consider how they may interact to promote tumor/host crosstalk. INTRODUCTION The tissue microenvironment consists of a dynamic population of cellular and non-cellular components which form a multifaceted regulatory network that helps to maintain the homeostasis of an organ. The liver microenvironment consists of a heterogeneous multitude of several components including extracellular matrix (ECM) components (laminin, fibronectin, collagen and proteoglycans), immune cells, Kupffer cells, endothelial cells, cytokines, fibroblasts and various growth factors [1]. These normal cellular and non-cellular microenvironment components are not only essential for the normal physiological and biological behavior of an organ, but are critical in opposing resistance to malignant cell growth [2] (Figure 1A) April 21, 2014 Volume 20 Issue 15

30 Rani B et al. Tissue microenvironment in HCC Fundamentally, a tumor and its microenvironment have a mutual influence on each other s fate. Accumulations of mutations in normal cells bring them to a benign tumor state, where they stay dormant because they lack the main hallmark of cancer, namely the ability to invade, metastasize and form vasculature (angiogenesis) [3,4]. These mutual interactions between the mutated cells and the microenvironment modulate the ECM composition; they activate fibroblasts, recruit immune or inflammatory cells and pericytes, and stimulate endothelial cells to invoke angiogenesis, stirring the cascade of various cytokines, chemokines and growth factors [5,6]. This interactive, complex communication between the tumor and its microenvironment components favors cancer progression. During hepatocellular carcinoma (HCC) progression, as the microenvironment components continue to interact with each other as well as with HCC cells, they acquire an abnormal phenotype due to tissue remodeling, which contributes to modulate the biological behavior of the tumor and thus facilitates cancer progression and metastasis. The abrupt annual increase in HCC incidence in recent years of more than cases worldwide has constantly driven efforts to find new potential therapies for HCC [7,8]. Although surgical resection and liver transplantation are the so-called curative treatments, the limiting factors are a shortage of healthy donor livers as compared to the emerging cases of HCC, and the fact that in advanced stages of HCC, surgery is not possible. Therefore, many studies have been focused on tumordestructive approaches. The neovascularized nature of HCC is a potential target for the use of systemic therapies that can impede aberrant molecular pathways. The use of small molecule multi-kinase inhibitors such as Sorafenib has resulted in a significant improvement in overall survival of patients with advanced HCC [9,10]. Sorafenib targets the vascular endothelial growth factor receptor (VEGFR), and various other multi-kinase inhibitors targeting VEGFR are undergoing clinical trial for the treatment of HCC, such as sunitinib, axitinib, linifanib, pazopanib, vandetanib, cediranib and regorafenib, as well as monoclonal antibodies such as bevacizumab [11]. Another approach, namely transarterial chemoembolization (TACE), induces tumor hypoxia and this upregulates angiogenic factors such as VEGF [12,13]. TACE does not induce complete necrosis and after treatment the peripheral area of the tumor becomes viable again due to re-vascularization [14]. Combination therapy has been tried, but several studies failed to observe any survival benefits after the use of TACE with anti-angiogenic agents such as Sorafenib [15]. Cetuximab, a monoclonal antibody against the epidermal growth factor (EGFR), failed to show any significant activity against HCC in a phase Ⅱ study [16]. Clinical trials using small molecules targeting EGFR, such as erlotinib, gefitinib and lapatinib, were also ineffective against HCC [17-19]. A phase Ⅱ clinical trial of a multi-targeted agent, namely Dovitinib, which targets VEGFR, platelet-derived growth factor receptor and fibroblast growth factor receptor, is now under way [20]. Apart from genetic defects, epigenetics also play a pivotal role in hepatocarcinogenesis. In vitro and in vivo data have shown that the use of a histone deacetylase (HDAC) inhibitor (HDACi), along with dihydroartemisinin (DHA), elicited antitumor activity in liver cancer [21]. Recently, a phase Ⅰ dose escalation trial of an HDACi, CHR2845, against cancerassociated inflammation in HCC was started in the United Kingdom. A pre-clinical Phase Ⅰ/Ⅱ study of the HDACi, PXD101 (belinostat), has also been conducted and showed a good safety profile in HCC patients [22]. Due to the lack of early stage HCC diagnostic markers and efficient chemo-preventive strategies to limit HCC progression once cirrhosis is established, the survival rate of HCC patients is still poor and they mainly die of tumor progression and metastasis. Tumor heterogeneity is also a crucial barrier to HCC treatment, as tumor cells become resistant to chemotherapies [23]. In this scenario, it seems interesting to analyze the results of a potential therapeutic approach to HCC consisting of targeting the tumor tissue microenvironment. Tissue microenvironment components are genetically stable and are less likely to evolve into a drug-resistant phenotype, therefore it would be easier to target these components than tumor cells, which are genetically unstable and chemotherapy-resistant [24]. Additionally, tumor stroma exerts tumor-suppressing as well as tumor-promoting signals. A pancreatic cancer mouse model showed that inhibition of the Hedgehog signaling pathway reduced the level of tumor-associated stroma and improved the vascular delivery of gemcitabine [25]. Nevertheless, although several theories have been proposed to explain the role of stroma in carcinogen-induced tumors, the actual relationships are not yet proven. In addition, stromal cells may be a target for carcinogens, inducing either new cancers, or metastatic growth [26]. A better understanding of the complex network of interactions between tumor cells and their milieu could offer new insight into novel targets for HCC treatment. In this review, we have updated the literature and discussed the various issues with the aim of shedding further light on the role of the tissue microenvironment as a therapeutic target in HCC. CELLULAR COMPONENTS OF THE TISSUE MICROENVIRONMENT Cancer-associated fibroblasts Cancer-associated fibroblasts (CAFs) are the major component of the tumor microenvironment and play a crucial role in tumor-stromal interactions [27-29]. CAFs promote tumor progression, invasion and chemoresistance to clinical therapies [28,30,31]. The origin of CAFs in HCC is controversial and various studies have revealed multiple origins, including the trans-differentiation of hepatic stellate cells 4129 April 21, 2014 Volume 20 Issue 15

31 Rani B et al. Tissue microenvironment in HCC A Normal liver cell population Hepatocytes Space of disse HSC Endothelial cells Kupffer cells B Cancer injured hepatocytes TGF-β psmad2/3 CTGF. Vimentin. Fibronectin. α-smooth muscle actin. Cytokeratin. Loss of E-cadherin Fibrosis event TGF-β secretion from liver cancer cells HSC activation by TGF-β ECM deposition Sinusoid capillarization Recruitment of regulatory T cells C SDF-1α/CXCR4 EGF, HGF, TGF-β IL-6 HGF TGF-β HGF TNF-α PDGF IL-8 COX-2 VEGF VEGF PDGF Liver carcinoma progression Inflammation Recruitement of various inflammatory cells, release of various cytokines and growth factors 4130 April 21, 2014 Volume 20 Issue 15

32 Rani B et al. Tissue microenvironment in HCC D Wnt, notch, P13K/AKT/mTOR IL-6, IL-5 IL-22, IL-17 PDGF SOF CXCL 12 VEGF upa MMPs E-selectin Anglogenesis Metastasis upa MMPs Liver cancer progression Angiogenesis Invasion Metastasis Extracellular matrix Cytokines HSC Liver cancer cells Kupffer cells Quiescent HSC TGF-β Regulatory T cells TAM CAFs CSC Figure 1 Progression of hepatocellular carcinoma: Crosstalk between hepatocellular carcinoma and its milieu. A: Healthy liver cell population. The normal liver cell population consists of quiescent hepatic stellate cell (HSC), Kupffer cells, and fenestrated endothelial cells which allow the exchange of blood and substrates between the space of Disse and hepatocytes; B: Fibrotic liver and its microenvironment. Fibrotic liver features cancer-injured hepatocytes, which trigger the release of transforming growth factor-β (TGF-β), defenestration of endothelial cells and recruitment of regulatory T-cells. Binding of TGF-β to its receptor on HSC triggers phosphorylation of SMAD2/3 signaling, which activates HSC to secrete extracellular matrix (ECM) components such as vimentin, CTGF, cytokeratin and muscle actin; C: Progression of liver cancer and its interaction with the milieu. Malignant hepatocytes proliferate in an uncontrolled manner. Infiltration of immune cells causes inflammation. Malignant hepatocytes secrete TGF-β which binds to, and activates, HSC. Activated HSC deposit more ECM. Recruitment of immune cells and cancerassociated cells elicits a signaling cascade. Compressed air foam system (CAFs) secrete vascular endothelial growth factor (VEGF) to stimulate endothelial cells to induce angiogenesis. In turn, endothelial cells secrete VEGF and platelet-derived growth factor (PDGF), which triggers the release of hepatocyte growth factor (HGF) from HSC. HGF secreted by HSC promotes malignant hepatocyte proliferation. Also, PDGF induces the differentiation of HSC into myofibroblasts, which cause fibrosis and the development of HCC. Activated CAFs also secrete EGF, HGF, TGF-β and interleukin-6 (IL-6) to aid cancer cell proliferation. CAFs produce cyclo-oxygenase-2 (COX-2) and IL-6 to induce tumor-associated macrophages (TAMs) production. Activated TAMs release TNF-α and PDGF to reinforce CAFs activation. Stromal cell-derived factor-1α (SDF-1/CXCL12) and its receptor CXCR4 are crucial in cancer stem cell (CSC) interactions with their surroundings. TGF-β upregulates CXCR4 expression in liver cancer cells and allows them to migrate to SDF-1α enriched niches. D: Progression and growth of liver carcinoma. Angiogenesis, Invasion and Metastasis are the crucial hallmarks of cancer. In HCC, HSC secrete VEGF to promote angiogenesis. CAFs and TAMs secrete various upas and matrix metalloproteinases (MMPs) to induce metastasis. Cancer cells secrete E- selectin to induce metastasis. Cancer stem cells activate the Wnt, Notch, phosphoinositide 3-kinase/Protein Kinase B/mammalian target of rapamycin (PI3K/AKT/mTOR) pathway and thus contribute to the molecular heterogeneity of liver cancer. CAFs secrete SDF/CXCL12 to induce proliferation and invasion of liver tumor cells. Cytokines such as IL-6, IL-5, IL-22 and IL-17, produced by T regs or other immune cells, aid in liver cancer proliferation, angiogenesis and metastasis April 21, 2014 Volume 20 Issue 15

33 Rani B et al. Tissue microenvironment in HCC (HSC) during liver injury, activation of resting fibroblasts and a direct contribution of hepatocytes through the epithelial to mesenchymal transition (EMT) [32]. CAFs modulate the biological activity of HCC cells, as documented by the demonstration that lysophosphatidic acid induces HCC progression by recruiting peri-tumoral fibroblasts and promoting their trans-differentiation into myofibroblasts. In addition, activated CAFs create a favorable tumor environment by modulating immune cells such as NK cells. NK cells have anti-tumor activity, but this is significantly reduced in HCC [33]. Thus, activated CAFs remodel the ECM, which facilitates the release of various cytokines and growth factors and as a result magnifies HCC growth. Transforming growth factor-β (TGF-β) activates CAFs, which display α-smooth muscle actin (α-sma), fibroblast activation protein, fibroblast surface protein, and vimentin [27,29] expression. Because CAFs also enhance the metastatic potential of HCC cells, targeting TGF-β receptor type Ⅰ using LY can down-regulate CTGF production, which in turn interferes with the crosstalk between CAFS and HCC cells, and hence inhibits stromal growth and metastasis [34] (Figure 1B). These findings clearly demonstrate that CAFs have an important role in overall cancer progression and hence are the significant modifiers of cancer evolution. In certain types of cancers, CAFs regulate cancer stem cells (CSCs), and since CSCs are resistant to chemotherapies it seems possible that this is one reason why HCC is difficult to treat, also due to its high recurrence rate, CAFs may regulate the stemness of HCC. Further exploration of the interactions between CAFs and HCC cells may help to identify novel HCC targets. HSC HSC, also known as lipocytes or Ito cells, are multifunctional cells that perform several vital functions, including Vitamin A storage to maintain retinoid homeostasis [35], the production of matrix metalloproteinases (MMPs) and ECM components such as collagen to remodel the ECM [36], the production of various cytokines [37] (interleukin-6 (IL-6), interleukin-1β (IL-1β), chemotactic peptide-1, chemokines [38] (chemokine (C-C motif) ligands 5 and 21 (CCL5), (CCL21) and growth factors (such as TGF-α, TGF-β), epidermal growth factor (EGF), platelet derived growth factor and basic fibroblast growth factor [39]. HSCs display different phenotypes according to their morphology, functions and gene expression. The normal healthy liver stores the quiescent phenotypic state of HSCs, however, massive liver injury activates HSCs, leading to a cascade of various cytokines, ECM components and the up-regulation of cytoskeletal proteins such as α-sma [40]. Activated HSCs can proliferate through the action of potent inducers such as cathepsins B and D, hepatitis virus B and C, PDGF, TGF-β1, MMP-9, JNK, insulin-like growth factor binding protein 5, non-structural proteins that induce liver fibrosis and hepatocarcinogenesis, while adiponectin, for instance, suppresses HSC activation [41]. Apart from their active contribution in liver cirrhosis, these activated HSCs or myofibroblasts also infiltrate the stroma of liver tumors, and when confined around the tumor sinusoids, fibrous septa and capsule, they assist in HCC progression [42] (Figure 1C). In vivo studies further support these data and showed that HSCs and HCC cells implanted into nude mice promote tumor growth and invasiveness by activating nuclear factor-κb and extracellular regulated kinase (ERK) in HCC cells [42]. HSCs produce and secrete laminin-5 (Ln-5), which induces HCC migration by activating the mitogen-activated protein kinase (MEK/MAPK)/extracellular-signal-regulated kinase (ERK) pathway, but not the phosphoinositide 3-kinase (PI3K/Akt) pathway [43]. Being multifunctional in nature, HSCs also act as liver-specific pericytes and promote tumor vascularity [44]. Pericytes are characterized by the expression of PGDF receptors; similarly, HSC cells produce PGDF and express PGDF receptors during liver injury [45,46]. Tumors and endothelial cells secrete PGDF to stimulate and recruit pericytes and so induce angiogenesis; pericytes secrete VEGF to support neovascularization [47]. These multifunctional phenotypes of HSC display a pivotal role, suggesting that they may be a valid therapeutic target in HCC treatment. Tumor-associated macrophages Macrophages are circulatory immature monocytes released from bone marrow which travel through the blood circulation to reach their destined tissue, where they mature and undergo differentiation into resident macrophages, such as Kupffer cells in the liver. Tumorassociated macrophages (TAMs) are the major inflammatory cells that infiltrate tumors. Tumor-derived signals such as macrophage-colony stimulating factor (M-CSF or CSF-1), VEGF, macrophage inflammatory protein 1a (MIP-1a), CCL3, CCL4, CCL5, CCL8, and angiopoietin-2, attract TAMs into the tumor microenvironment [48]. Different microenvironment signals determine distinct polarized activation states of macrophages, namely the classically activated (M1) and the alternatively activated (M2) phenotypes. Lipopolysaccharides (LPS) and Th-1 cytokine interferon-γ (IFN-γ) exposure polarizes macrophages to the M1 phenotype, whereas Th-2 cytokines interleukin-4 (IL-4), IL-10, and IL-13 exposure polarizes macrophages to the M2 phenotype [48,49]. The M1 phenotype displays high levels of antigen-presenting cells with an increased expression of IL-12, whereas the M2 phenotype shows low levels of antigen-presenting cells with a distinctive expression of various cytokines such as IL-10 and TGF-β [50]. In the tumor microenvironment, TAMs are mostly polarized towards the M2 phenotype, with a high expression of IL-10, arginase I, IL-6 and low expression of IL-12, tumor necrosis factor (TNF) and proinflammatory cytokines such as nitric oxide (NO) and reactive oxygen species (ROS) [50-52]. An increased number of TAMs is correlated with tumor cell proliferation, angiogenesis, metastasis and a poor prognosis [53]. Indeed, depletion 4132 April 21, 2014 Volume 20 Issue 15

34 Rani B et al. Tissue microenvironment in HCC of macrophages by sorafenib significantly inhibits tumor progression, angiogenesis and metastasis [54]. Hence, TAMs are clearly essential for tumor growth. In human HCC, activated TAMs are localized within the peritumoral stroma and display strong expression of human leukocyte antigen (HLA-DR), IL-6, IL-1β and IL-23, whereas within the cancer niche, TAMs exhibit low expression of HLA-DR and IL-10 [55]. Additionally, HCC cells recruit TAMs by secreting VEGF, TGF-β, PDGF, CCL2 and M-CSF and by expressing glypican-3 [29,56,57]. TAMs secrete various cytokines, which contribute to the pathogenesis of HCC. For instance, in a diethylnitrosamine (DEN)-induced HCC mouse model system, DEN exposure promoted the release of IL-6 from Kupffer cells in response to IL-1β from damaged hepatocytes. Then, IL-6 promoted abnormal proliferation of the surviving hepatocytes by triggering the Signal Transducer and Activator of Transcription 3 (STAT-3) and Extracellular-signal-Regulated Kinases (ERK) pathways, which in turn control target genes involved in both cell proliferation and survival, thus contributing to HCC progression [58]. Moreover, TAMs-derived cytokines and growth factors induce immune suppression and contribute to tumor growth. IL-10 and TNF-α, secreted by TAMs in an autocrine manner, stimulate B7-H1 expression on macrophages and impair CD8 + T-cell activity, which allows immune escape of tumor cells [59]. Since TAMs are the main infiltrating cells in the tumor microenvironment, therefore, they may be a potential target for clinical therapy. A macrophage activation state within peritumoral and tumoral tissue is a potent prognostic factor. TAMs secrete numerous cytokines, chemokines and growth factors, which support HCC progression as these TAMs-derived signals modulate the interaction with tumor cells. Moreover, TAMs support angiogenesis, metastasis, and cancer progression. Therefore, the complex network of tumor-stroma crosstalk could be a promising target of therapy for HCC. Tumor-associated endothelial cells The morphology of tumor vasculature is different from that of normal vessels. Tumor vessels are irregular, incomplete and fenestrated blood vessels with an irregular blood flow and increased permeability; this is possibly due to the molecular and functional differences between tumoral and normal endothelial cells [60,61]. Tumorassociated endothelial cells (TAECs) are cytogenetically abnormal [62] with a rapid turnover rate, enhanced mobility, migration and high expression of the endoglin marker (also known as CD 105 or TGF-β receptor) [57]. TAECs also contain several secretory organelles such as Weibel-Palade bodies, tissue plasminogen activator (tpa) organelles and type-2 chemokine containing organelles. These organelles secrete tpa, cytokines IL-8 and IL-6, monocyte chemoattractant protein-1 (MCP-1), and growthregulated oncogene-α (GRO-α) [57,63]. Insufficient radiofrequency ablation (RFA) enhances TAEC migration and angiogenesis and promotes invasiveness of the residual HCC [64] (Figure 1D). NON-CELLULAR COMPONENTS IN THE TISSUE MICROENVIRONMENT TGF-β structure and receptor TGF-β is a cytokine and belongs to the TGF-β cytokines superfamily, which also includes activins, inhibins, Mullerian inhibitor substance (Mis) and bone morpho-genetic proteins (BMPs). TGF-β is a multifunctional cytokine that can control proliferation, cellular differentiation, adhesion, migration, apoptosis and other functions in most cells [65,66]. In mammals, TGF-β exists in three isoforms, TGF-β1, TGF-β2 and TGF-β3. TGF-β1 is the most abundant, showing the highest expression [65]. TGF-β can inhibit the proliferation of most cell types, especially epithelial cells. Furthermore, it enhances the proliferation of mesenchymal cells, producing the ECM, as well as inducing fibrosis [67]. There are two main receptors for TGF-β, namely TGF-β receptor Ⅰ or activin receptorlike kinase (ALK), and TGF-β receptor Ⅱ. In mammals, 7 types of receptor Ⅰ and 5 types of receptor Ⅱ have been found [68]. ECM ECM contains various proteoglycans, glycoproteins, polysaccharides and water [69]. Chemotaxis induces cell adhesion to the ECM, which is mediated by ECM receptors such as integrins, discoidin domain receptors and syndecans, as well as to various ECM components including fibronectin, laminin, collagens and elastin [70]. Ln-5 is a cell adhesion glycoprotein belonging to the Laminin family which forms a mesh-like structure to resist the tensile forces in the basal lamina. Each laminin is a heterodimer and consists of α3, β3 and γ2 chains encoded by three different genes, LAMA3, LAMB3 and LAMC2, respectively [71]. Ln-5 with the γ2 chain is a marker of invasiveness in several carcinomas, suggesting its role in tumor cell spread [72-74]. Ln-5 is not detected in normal liver, but shows high expression in HCC nodules, associated with a more proliferative and metastatic phenotype. The presence of the Ln-5 γ2 chain in metastatic HCC is correlated with poor prognosis and survival [75,76]. In HCC, invasive tumor cells secrete TGF-β1, which triggers invasiveness and motility in Ln-5 by inducing the expression of the transmembrane integrin receptor α3β1. Ln-5 also plays a crucial role in the epithelial to EMT, which is crucial in tumorigenesis. In HCC, Ln-5 upregulates the expression of the transcriptional repressors Snail and Slug, which induce the EMT [77,78]. Ln-5, together with TGF-β1, promotes the EMT in HCC by over-expressing Snail and Slug and downregulating E-cadherin, followed by translocation of β-catenin to the nucleus [78]. Gefitinib, a specific inhibitor of EGFR, suppresses HCC growth by inhibiting phosphorylation of the receptor and subsequently the Akt and Erk1/2 pathways in HCC cells in vitro. However, the presence of Ln-5 in HCC antagonises gefitinib s efficacy in a dose-dependent manner, suggesting a potential 4133 April 21, 2014 Volume 20 Issue 15

35 Rani B et al. Tissue microenvironment in HCC drug (gefitinib) failure in Ln-5 positive HCC cases [79]. CANCER STEM CELL NICHE AS A NEW PLAYER IN THE HCC MICROENVIRONMENT Cancer stem cells in liver tumors Constant proliferation of stem cells occurs in renewing liver tissues, where mutations expand the altered stem cells, perpetuating and increasing the likelihood of additional mutations and tumor progression. Overall, many data indicate that liver stem/progenitor cells follow their own rules and regulations. The same signals that are essential for their activation, expansion and differentiation are good candidates for the role of contributing, under suitable conditions, to the paradigm of transformation from a pro-regenerative to a pro-tumorigenic role [80] (Figure 2A, B). The fact that about 40% of HCCs are clonal suggests that these tumors may originate from stem celllike precursors. Recent evidence supports the existence of CSCs in HCC [81], which can be identified by several cell markers, including CD133, the epithelial cell-adhesion molecule EpCAM, as well as CD90, CD44, CD13, OV6, ALDH enzymatic activity, and the side population of cells (SP) revealed by Hoechst dye staining [82]. Indeed, the presence of EpCAM+ cells has been associated with a poorly differentiated morphology and high serum alpha-fetoprotein levels in HCC, whereas the presence of CD90+ cells was associated with a high incidence of distant organ metastasis [83]. Furthermore, circulating stem cell-like EpCAM+ tumor cells indicate a poor prognosis in HCC patients after curative resection. High CD44 standard isoform (CD44s) expression is associated with the EMT profile and with intrahepatic dissemination of HCC after local ablation therapy [84]. The expression of CD133 confers a malignant potential by regulating metalloproteinases in HCC cells. Importantly, CD13 is a marker for semiquiescent CSCs in human liver tumor cell lines and clinical samples. CD13+ cells predominate in the G0 phase of the cell cycle and typically form cellular clusters in cancer foci. Following treatment, these cells survived and were particularly abundant along the fibrous capsule where liver cancers usually relapse [85]. From the mechanistic standpoint, CD13 reduced reactive oxygen species (ROS)-induced DNA damage after genotoxic chemo/radiation stress and protected cells from apoptosis. In any case, these cells are likely resistant to targeted therapies such as sorafenib [86]. MECHANISM OF THE TGF-α SIGNALING PATHWAY The TGF-β superfamily plays a key role in a wide range of cellular processes and is therefore tightly regulated. The pathway is both positively and negatively modulated, for example, by extracellular antagonists, ligandbinding antagonists, and the regulation of receptor function and inhibition by I-SMADs. Positive regulation plays a key role in amplifying signaling through the TGF-β superfamily [87]. Negative regulation could be critical for the restriction and termination of signaling. In order to prevent the type Ⅰ receptor from becoming activated, BAMBI binds to it and serves as a negative regulator of the TGF-β signaling pathway. Similarly, FKBP12 can prevent phosphorylation by binding to the GS domain of type Ⅰ receptors. The most important negative regulation in the signaling pathway is inhibition by I-SMAD, which plays a critical role in transduction of the TGF-β signaling pathway. I-SMAD has different ways of regulating signaling both in the cytoplasm and nucleus. SMAD7 has been proved to form a complex with the type Ⅰ receptor to inhibit R-SMADs binding to the receptor and prevent their phosphorylation [88]. In the nucleus, SMAD6 binds to the transcription factor Hoxc-8 and acts as an antagonist of transduction. SMAD6 can also bind to DNA directly and recruit HDAC to inhibit transduction [89]. Non-SMAD signaling pathway Besides the SMAD pathway, TGF-β also activates other signaling cascades. For example, TGF-β can activate TGF-β-kinase 1 (TAK 1), (ErK), p38, mitogen-activated protein kinase (MAPK) and Akt [90]. These non-canonical or non-smad dependent means of activating the TGF-β pathway appear to involve signaling via jun N-terminal kinase (JNK), p38 MAPK, ERK or MEKK. The TGF-β inhibitor, LY , a surrogate compound of LY , has also shown inhibitory activity towards the non-canonical pathway that includes FAK, β1 integrin, MEK, Erk, Akt, mtor and PTEN, but not p-38-mak-kinase [91]. In rat intestine or mink lung epithelial cells, rapid activation of p21 (Ras) occurred after TGF-β treatment, which indicated that TGF-β could activate the Erk-MAPK signaling pathway [92,93]. In this case, TGF-β induced Erk activation and tyrosine phosphorylation. Similarly, TGF-β can rapidly activate JNK through MKK4 and p38 MAPK through MKK3/6 in several cell lines [94]. In human prostate cancer cells, SMAD7 triggers apoptosis by an association with TAK1, MKK3 and p38 MAPK, to help activate the TAK1-p38 MAPK signaling pathway [90]. It is known that TGF-β is involved in activation of the phosphoinositide 3-kinase (PI3K)-Akt signaling pathway, and recent studies also indicate that TGF-β utilizes the mtor pathway to regulate cell survival, metabolism, migration, and invasion [95,96]. Moreover, TGF-β induced the activation or inactivation of small GTPases and the phosphorylation of Par6, which may be critical events leading to the EMT [97]. Both the SMAD and non-smad signaling pathways determine the end results of the cellular response to TGF-β. ROLE OF TGF-α IN THE HCC MICROENVIRONMENT In human HCC, cells with stem cell markers show a 4134 April 21, 2014 Volume 20 Issue 15

36 Rani B et al. Tissue microenvironment in HCC A Vein Artery Central vein Portal triad Intralo bular blle duot Null cells Duotule Canal of hering Cholangiocytes N8Cs Blle oanalloulus Hepatocytes Central vein Hepatic lobule Normal stem cell-nlohe B Kupffer cells Genetic epigenetics changes C8Cs HSC Extraoellular matrix C TGF-β 8DF-1 CXCR4 + Tumor cells TGF-β EGF, HGF, IL-8 Cancer stem cell-nlohe 4135 April 21, 2014 Volume 20 Issue 15

37 Rani B et al. Tissue microenvironment in HCC D EMT TGF-β MET Primary tumor Meta stasis Extracellular matrix Cytokines HSC Liver cancer cells Kupffer cells Quiescent hepatic stellate cell (HSC) TGF-β Regulatory T cells TAM CAFs CSC Figure 2 Role of the stem cell niche in liver tumors. A: A hepatic lobule consists of one central vein and six surrounding portal triads, each of which has bile ducts, a hepatic artery and portal vein. BrdU-retaining cells (with black nuclei) represent putative stem cells (NSC) and are located in cholangiocytes of the intralobular bile duct, together with less-characterized null cells, small hepatocytes at the interface of cholangiocytes, and hepatocytes in the canals of Hering; B: A series of genetic and epigenetic changes in NSC (or committed progenitor cells) leads to the generation of cancer stem cell (CSC) and then to expansion of cells within the stem cell niche. The niche adapts to the presence of CSCs by recruiting cells that would not normally be present; C: The signaling pathways for the activation, expansion and differentiation of stem/progenitor cells are good candidates for contributing, under suitable conditions, to a pro-tumorigenic role. Migration and metastasis of CSCs is a multistep process, in which SDF-1 plays a crucial role by chemoattracting CXCR4+ tumor cells; D: Transforming growth factor-β (TGF-β)-induced transdifferentiation of hepatocytes and liver tumor cells from an epithelial to a mesenchymal phenotype (EMT) increases a population of cells with putative liver progenitor properties. The EMT plays a fundamental role in tumor progression and metastasis and the TGF-β-induced EMT can guide cancer cells to delaminate from primary tumors, migrate along the extracellular matrix network, and arrive at the site of metastasis via the peripheral blood. HSC: Hepatic stellate cell; TAM: Tumor-associated macrophage; CAFs: Compressed air foam system; EGF: Endothelial growth factor; HGF: Hepatocyte growth factor; IL: Interleukin. loss of TGF-β receptor Ⅱ and ELF (a β-spectrin that propagates TGF-β signal), and a marked activation of the IL-6 pathway, which is the major stem cell signaling pathway [98,99]. These data support the concept that the absence of TGF-β-driven epithelial differentiation favours carcinogenesis. Indeed, in addition to the wellknown transcriptional responses predominantly addressing tumor suppressor actions, TGF-β induces other Smad-dependent or independent effects that contribute to tumor progression [100] (Figure 2C). TGF-β induces trans-differentiation of hepatocytes and liver tumor cells from an epithelial to a mesenchymal phenotype, which results in a population of cells with putative liver progenitor properties [101,102]. Additionally, the TGF-β-induced EMT not only endows cells with migratory and invasive properties, but can also induce 4136 April 21, 2014 Volume 20 Issue 15

38 Rani B et al. Tissue microenvironment in HCC cancer cells to dedifferentiate and gain cancer stem-celllike properties [103]. Snail1 Snail2 and Twist may actively participate in this process [100,102,104] (Figure 2D). Furthermore, other studies have demonstrated the capacity of TGF-β to induce/maintain a stemness phenotype in other tumors [105]. CONCLUSION In conclusion, several components of the tissue microenvironment have been shown to participate in modulating the biological behavior of HCC. Biological redundancy complicates the understanding of the tumor/host crosstalk. Nevertheless, we hypothesize that HCC should be considered as a tumor of the whole liver, rather than just of few hepatocytes that grow in nodules. Taking into account this scenario, new therapeutic approaches targeting the TGF-β pathway seem promising. Directed against the microenvironment, they might have the effect of making this microenvironment more hostile to HCC progression. Up to now, targeting TGF-β in HCC has been considered a questionable approach in view of the tumor suppressor role that TGF-β exerts on cells. This apparent controversy has been addressed by examining the differences between the early and late TGF-β signature in patients with HCC [106]. The clinical role of the late signature has been further confirmed in a wider HCC molecular classification, suggesting a potential role for TGF-β as a possible therapeutic target [107]. Currently, a multicenter clinical trial is ongoing, evaluating the efficacy and effectiveness of a TGF-β receptor Ⅰ kinase inhibitor (LY ) in patients with HCC. However, it is not yet possible to predict at which stage of the disease such a drug would display the best therapeutic effect, as all the clinical trials so far have been restricted to patients with a more advanced disease stage of HCC, for ethical reasons. 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41 Rani B et al. Tissue microenvironment in HCC local ablation therapy. J Hepatobiliary Pancreat Sci 2013; 20: [PMID: DOI: /s ] 85 Haraguchi N, Ishii H, Mimori K, Tanaka F, Ohkuma M, Kim HM, Akita H, Takiuchi D, Hatano H, Nagano H, Barnard GF, Doki Y, Mori M. CD13 is a therapeutic target in human liver cancer stem cells. J Clin Invest 2010; 120: [PMID: DOI: /JCI42550] 86 Xin HW, Ambe CM, Hari DM, Wiegand GW, Miller TC, Chen JQ, Anderson AJ, Ray S, Mullinax JE, Koizumi T, Langan RC, Burka D, Herrmann MA, Goldsmith PK, Stojadinovic A, Rudloff U, Thorgeirsson SS, Avital I. Label-retaining liver cancer cells are relatively resistant to sorafenib. Gut 2013; 62: [PMID: DOI: /gutjnl ] 87 Miyazono K. Positive and negative regulation of TGF-beta signaling. J Cell Sci 2000; 113 (Pt 7): [PMID: ] 88 Hayashi H, Abdollah S, Qiu Y, Cai J, Xu YY, Grinnell BW, Richardson MA, Topper JN, Gimbrone MA, Wrana JL, Falb D. 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Hepatology 2008; 47: [PMID: DOI: / hep.22283] 107 Hoshida Y, Nijman SM, Kobayashi M, Chan JA, Brunet JP, Chiang DY, Villanueva A, Newell P, Ikeda K, Hashimoto M, Watanabe G, Gabriel S, Friedman SL, Kumada H, Llovet JM, Golub TR. Integrative transcriptome analysis reveals common molecular subclasses of human hepatocellular carcinoma. Cancer Res 2009; 69: [PMID: DOI: / CAN ] P- Reviewers: Emmanouil Z, Gupta DK, Hiasa Y, Li ZF, Vinciguerra M, Weng HL S- Editor: Qi Y L- Editor: Webster JR E- Editor: Liu XM 4140 April 21, 2014 Volume 20 Issue 15

42 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (1): Hepatocellular carcinoma TOPIC HIGHLIGHT Staging systems of hepatocellular carcinoma: A review of literature Marcello Maida, Emanuele Orlando, Calogero Cammà, Giuseppe Cabibbo Marcello Maida, Emanuele Orlando, Calogero Cammà, Giuseppe Cabibbo, Section of Gastroenterology, DIBIMIS, University of Palermo, Palermo, Italy Author contributions: All authors contributed to writing the paper and had full control over preparation of manuscript, and approved the final draft manuscript. Correspondence to: Calogero Cammà, MD, Section of Gastroenterology, DIBIMIS, University of Palermo, P.zza delle Cliniche 2, Palermo, Italy. calogero.camma@unipa.it Telephone: Fax: Received: November 1, 2013 Revised: January 17, 2014 Accepted: March 12, 2014 Published online: April 21, 2014 Abstract Hepatocellular carcinoma (HCC) is a major health problem with a high incidence and mortality all over the world. Natural history of HCC is severe and extremely variable, and prognostic factors influencing outcomes are incompletely defined. Over time, many staging and scoring systems have been proposed for the classification and prognosis of patients with HCC. Currently, the non-ideal predictive performance of existing prognostic systems is secondary to their inherent limitations, as well as to a non-universal reproducibility and transportability of the results in different populations. New serological and histological markers are still under evaluation with promising results, but they require further evaluation and external validation. The aim of this review is to highlight the main tools for assessing the prognosis of HCC and the main concerns, pitfalls and warnings regarding its staging systems currently in use Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Hepatocellular carcinoma; Natural history; Prognosis; Staging; Barcelona clinic liver cancer Core tip: Hepatocellular carcinoma is a major health problem, with a heterogeneous natural history that makes it difficult to identify accurate prognostic factors. The aim of this review is to highlight the main tools for assessing the prognosis of HCC and the main concerns, pitfalls and warnings regarding its staging systems currently in use. Maida M, Orlando E, Cammà C, Cabibbo G. Staging systems of hepatocellular carcinoma: A review of literature. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: INTRODUCTION Hepatocellular carcinoma (HCC) has an increasing incidence worldwide, and it is the leading cause of death in patients with cirrhosis [1]. It is the fifth most common cancer and the third most common cause of cancer-related death [2]. Despite intensive surveillance programs, considerable recent therapeutic advances and use of potentially radical treatments, prognosis and life expectancy remain poor in this setting [3]. Curative treatments are applicable for early stage tumors only, and include resection, liver transplantation and percutaneous ablation, while transarterial chemoembolization (TACE) and sorafenib are regarded as non-curative treatments able to improve survival in intermediate and advanced stages, respectively [4]. NATURAL HISTORY OF HCC According to the definition by Sackett et al [5], natural history is the course of a disease from its biological onset to its recovery or permanent disability or death April 21, 2014 Volume 20 Issue 15

43 Maida M et al. Staging systems and prognosis of HCC The natural history of HCC is extremely severe, as confirmed by mortality rates overlapping the incidence of the tumor [6]. In addition, it is extremely heterogeneous, due to the complex interplay between the biological characteristics of the tumor and the frequent presence of an underlying chronic liver disease, as showed by a recent meta-analysis which analyzed the survival rates of the placebo and untreated arms of several randomized controlled trials (RCTs) on HCC patients [7]. According to this study, the survival rates were 17.5% at 1 year and 7.3% at 2 years, with a significant heterogeneity among all studies (P < ) both for 1-year and 2-year survival. By meta-regression analysis, impaired performance status (PS), B and C Child-Pugh classes, and presence of portal vein thrombosis (PVT) were independently associated with a poor survival. The natural history of early HCC can not be evaluated by RCTs for ethical reasons, although a milestone paper published in 1989 showed that overall survival (OS) of asymptomatic patients with HCC and cirrhosis was 96% and 50% at 1- and 2-year, respectively [8]. A recent study analyzed a cohort of 320 patients affected by HCC and not suitable for curative or palliative treatments, confirming the heterogeneous behaviour of untreated HCC [9]. The overall median survival was 6.8 mo, and the 1-year survival was 32%. The 1-year survival according to barcelona clinic liver cancer (BCLC) classes was 100%, 79%, 12% and 0%, for BCLC A, B, C and D, respectively, with a significant difference in survival among each BCLC class. STAGING SYSTEMS AND PROGNOSTIC SCORES IN ONCOLOGY Staging systems assess and describe the extent of tumor burden in the originally primary organ and its spread throughout the body. They have a key role in the management of all cancers, allowing an accurate prognostic stratification of the tumor and the choice of the best therapeutic approach according to the stage. Furthermore, they are useful for grouping patients homogeneously in clinical trials and scientific research, and to make comparable patients of different clinical studies. Correct tools for the prognostic stratification of cancers An ideal staging system should be simple, easy and quick to be determined as soon as possible after diagnosis; it should provide information on prognosis and guide therapeutic decisions. In contrast to classical staging systems, which consider only the inherent characteristics of the tumor, prognostic scores include also all the variables that influence the patient s prognosis, over the tumor extension. Each staging system and prognostic score must be reproducible and externally validated, in order to be recommended and used on a large scale. Internal validation is an estimate of the internal reproducibility and answers the question asking if The score can be properly applied to the patient population from which it was derived. Using survival time as an outcome measure, important criteria for assessing the internal validation of a prognostic system are homogeneity, discrimination and monotonicity [10]. Homogeneity is the characteristic whereby the difference in survival time is small among patients classified into the same staging group, while discriminatory ability is the feature by which there are much greater differences in the survival times among patients classified into different groups. Finally, monotonicity is defined as the property of a staging system by which the mean survival time for a group classified as favourable by that system is always longer than the mean survival times experienced in less favourable groups (monotonicity of gradients). Besides, external validation is an estimate of transportability of the results, and answers the question asking if It is possible to apply the results of a prognostic study to any single patient. It is assessed by validation studies that are performed on populations other than, but related to the one from which the prognostic score was originally derived. STAGING OF HCC Generally, the extension of the tumour burden in the original primary organ and its spread throughout the body, is per se exhaustive for the staging of most solid tumors. Nevertheless, unlike other tumors, HCC usually occurs on a background of a liver disease, making the level of management complexity unique among all malignancies. It is well known that the functional impairment of the underlying liver disease has a significant impact on prognosis, irrespective of the tumour stage [4]. For this reason, systems that include the anatomical characteristics of the tumor only, such as the American Joint Committee on Cancer (AJCC)/International Union Against Cancer (UICC) staging system that stratifies patients using a Tumor-Node-Metastasis (TNM) classification, do not have per se a good predictive capability [11,12] (Table 1). Thus the TNM, along with all other systems that enclose it, represents a group of models useful for assessment of tumor extension only, but inadequate as prediction models. Therefore, many staging and scoring systems for the classification and prognosis of patients with HCC have been proposed over time (Figure 1). Okuda staging system The staging system proposed by Okuda et al [13] in 1984 (Table 1) is the first attempt to successfully combine the anatomical features of the tumor to the degree of the underlining liver disease. A distinction is made by three stages, considering the volume of the tumor (occupancy extended to or > 50% of the liver) together with the main indices of liver function (albumin, bilirubin, presence of ascites). This system has been widely adopted and used throughout the world for over two decades. However, the development of more advanced diagnostic 4142 April 21, 2014 Volume 20 Issue 15

44 Maida M et al. Staging systems and prognosis of HCC Table 1 Comparison of classifications Okuda staging system [13] Score 0 1 Tumor size 50% of the liver > 50% of the liver Albumin (g/dl) 3 < 3 Bilirubin (mg/dl) < 3 3 Ascites Absent Present CLIP score [14] Score Tumour morphology Uninodular and extension 50% Multinodular and extension 50% Massive or extension > 50% Child Pugh score A B C Alpha-fetoprotein (ng ml) < Portal vein thrombosis Absent Present - GRETCH score [21] Score Karnofsky index 80% < 80% Bilirubin (μmol L) < Alkaline phosphatase < 2 X ULN 2 X ULN Alpha-fetoprotein (μg L) < Portal vein thrombosis Absent Present BCLC [16] Stage 0 (very early) A (early) B (intermediate) C (advanced) D (end stage) ECOG Performance Status Liver function Child-Pugh A-B Child-Pugh A-B Child-Pugh A-B Child-Pugh A-B Child-Pugh C Tumor stage Single Single or 3 nodules < 3 cm Multinodular Vascular invasion or extrahepatic spread Any VARIABLE Weight TNM stage Ⅰ and Ⅱ -3 Ⅲ -1 Ⅳ 0 Total Bilirubin (μmol L) < Ascites 3 Alpha-fetoprotein > 500 ng/ml 2 Alkaline phosphatase > 200 IU/L 3 Asymptomatic disease on presentation -4 JIS [24] Score Child-Pugh score A B C TNM stage by LCSGJ Ⅰ Ⅱ Ⅲ Ⅳ Tokyo [22] Score Albumin (g/dl) > < 2.8 Bilirubin (mg/dl) < > 2 Tumor size (cm) < > 5 Numbers of nodules 3 - > 3 AJCC/UICC TNM staging system 7 th ed [12] Group Description Stage grouping T1 Single tumor without vascular invasion STAGE Ⅰ T1 N0 M0 T2 Single tumor with vascular invasion or multiple STAGE Ⅱ T2 N0 M0 tumors, none > 5 cm T3a Multiple tumors, any > 5 cm STAGE ⅢA T3a N0 M0 T3b Single tumor or multiple tumors of any size involving a major branch of portal or hepatic vein(s) STAGE ⅢB T3b N0 M April 21, 2014 Volume 20 Issue 15

45 Maida M et al. Staging systems and prognosis of HCC T4 Tumors with direct invasion of adjacent organs STAGE ⅢC T4 N0 M0 other than the gallbladder, or perforation of visceral peritoneum N1 Regional lymph node metastasis STAGE ⅣA Any T N1 M0 M1 Distant metastasis STAGE ⅣB Any T Any N M1 AJCC: American Joint Committee on Cancer; UICC: International Union Against Cancer; CLIP: Cancer of the Liver Italian Program; GRETCH: Groupe d Etude et de Traitement du Carcinome Hépatocellulaire; BCLC: Barcelona clinic liver cancer; CUPI: Chinese University Prognostic Index for hepatocellular carcinoma; JIS: Japan Integrated Staging Score; TNM: Tumor Node Metastasis; ECOG: Eastern Cooperative Oncology Group; LCSGJ: Liver Cancer Study Group of Japan Okuda CLIP GRETCH BCLC CUPI stnm JIS Tokyo AJCC/UICC 7 th ed Figure 1 Timeline of hepatocellular carcinoma staging system. AJCC: American Joint Committee on Cancer; UICC: International Union Against Cancer; CLIP: Cancer of the Liver Italian Program; GRETCH: Groupe d'etude et de Traitement du Carcinome Hépatocellulaire; BCLC: Barcelona Clinic Liver Cancer; CUPI: Chinese University Prognostic Index; JIS: Japan Integrated Staging Score; TNM: Tumor Node Metastasis. techniques over the years has permitted an increasing detection of small tumors, with an occupancy of the entire liver well below 50%, which are poorly discriminable within the two dimensional groups proposed by Okuda. In addition other prognostic variables not included in that model were identified, leading to the development of more accurate staging systems (Table 2). Cancer of the Liver Italian Program score Cancer of the Liver Italian Program (CLIP) is a simple scoring system designed by an Italian group with the aim of overcoming the main limitations of the TNM and Okuda. It has been derived from a retrospective cohort study of 435 patients [14] and then externally validated comparing its discriminatory ability and predictive power with the one of the Okuda staging system by a randomized trial that prospectively enrolled 196 patients with cirrhosis and HCC [15]. The CLIP includes the liver function according to Child-Pugh score, the morphology of the tumor (uninodular, multinodular, massive), its extension in the liver, the levels of Alpha-fetoprotein and the eventual presence of PVT. The combination of the different variables places all patients into 6 categories (Table 1). Although it was built with a correct methodology and externally validated, this score presents some limits, including the absence of general well-being assessment of the patient, and the inability to identify the early stages, which are susceptible to percutaneous or surgical therapies. BCLC staging classification The BCLC staging classification for HCC is currently the only staging system that includes an integrated assessment of liver disease, tumor extension, and presence of constitutional symptoms, providing in the meantime an indication of the first-line treatment. It classify stages of disease into five subgroups, from 0 to D, each associated with a specific therapy and prognosis (Table 1) [16]. Stage 0 is defined as very early stage disease, and is featured by a single nodule 2 cm without tumor invasion into surrounding tissues, in asymptomatic patients with preserved liver function. Stage A, or early disease, is classified as a solitary HCC of any size, or in maximum 3 nodules, each of them 3 cm, in asymptomatic patients with Child-Pugh A or B. Stage 0 and A can be effectively treated with curative therapies, such as surgical resection, liver transplantation, or by percutaneous ablation methods, including percutaneous ethanol injection (PEI) and radiofrequency ablation (RFA). These treatments allow to reach a complete response [17], with potential long-term curative effect and a 5-year survival better than 40%-70%. It must be emphasized that tumor size (< 5 cm) has been recently removed as a contraindication for radical therapy in single nodule HCC. However, in this particular case, the choice of the surgeon is finally decisive in defining a tumor as resectable. Stage B, or intermediate disease, consists of multinodular tumor, without macrovascular invasion or extrahepatic spread (ES), in asymptomatic patients with well-preserved liver function, and PS lower than 2. This subset of patients may be treated with TACE, which has proven a significant increase in survival compared with best supportive care (median survival, 20 mo vs 16 mo) [18]. Patients with mild related symptoms and/or macrovascular invasion or ES are classified as stage C (advanced stage). According to two pivotal RCTs [19,20], the standard of care in this group is Sorafenib, an inhibitor of Raf kinase and vascular endothelial growth factor receptor. Patients with cancer symptoms related to advanced liver failure, tumor growth with vascular involvement, ES, or physical impairment (PS > 2), are classified as stage D (end stage disease). They can not benefit from any specific cancer therapy and could only receive the best available supportive care. Although it is not an ideal staging system and has several gaps, the BCLC has been endorsed by EASL and AASLD as the standard staging system for 4144 April 21, 2014 Volume 20 Issue 15

46 Maida M et al. Staging systems and prognosis of HCC Table 2 Variables included in the main prognostic systems Variables Prognostic scores Okuda [13] CLIP [14] GRETCH [21] BCLC [16] CUPI [23] JIS [24] Tokyo [22] Child-Pugh score X X X Ascites X X Albumin X X Total Bilirubin X X X X Alkaline phosphatase X X Alpha-fetoprotein X X X Tumor size X X X X Numbers of nodules X X X TNM stage X X Portal vein thrombosis X X X Metastasis X Portal hypertension X Presence of symptoms and/or General Status X X X CLIP: Cancer of the Liver Italian Program; GRETCH: Groupe d Etude et de Traitement du Carcinome Hépatocellulaire; BCLC: Barcelona Clinic Liver Cancer; CUPI: Chinese University Prognostic Index; JIS: Japan Integrated Staging Score; TNM: Tumor Node Metastasis. patients with HCC, and it is currently the most used in Western countries. Other staging systems in western countries The GRETCH (Groupe d Etude et de Traitement du Carcinome Hépatocellulaire) score was derived by a prospective cohort of 761 patients with hepatocellular carcinoma from 24 medical centers in France, Belgium and Canada enrolled during a period of 30 mo (Table 1) [21]. Five prognostic factors were selected: Karnofsky index, serum bilirubin, serum alkaline phosphatase, serum alpha-fetoprotein > 35 pg/l, and ultrasonographic evidence of portal obstruction. Three risk groups with different 1-year survival rates were derived, and then independently validated in the test sample. However, it is not a well validated or a widely used staging system. Other staging systems in eastern countries The lower survival of Asian patients with HCC seems to be due not only to the different ethnic origin, but also to a different etiologic distribution and to a different and more severe natural history of liver disease. On this background, many prognostic scores have been built on Asian cohorts. The Tokyo score [22] was developed from a cohort of 403 consecutive Japanese patients with HCC treated by percutaneous ablation, and it is composed by four factors including serum albumin, bilirubin, size and number of tumors (Table 1). In the testing sample, the predictive power of this score resulted equal to CLIP and better than BCLC staging. Unfortunately, the score has been derived on a cohort of patients with early HCC, therefore its predictive ability can be transferred only to patients susceptible to radical therapies, whereas it poorly fits to patients with advanced HCC. The CUPI (Chinese University Prognostic Index for hepatocellular carcinoma) [23] was designed in 2002 by the analysis of a cohort of 926 Chinese patients with HCC, adding five prognostic factors (total bilirubin, presence of ascites, alkaline phosphatase, alpha fetoprotein, and asymptomatic disease on presentation) to the TNM, in order to set up 3 classes of risk with highly significant differences in survival (Table 1). This score was obtained from a monocentric and mono-ethnic cohort of patients, and most of the patients had a liver disease secondary to HBV infection. Therefore, the transportability of data of this score could be limited to this specific subset of patients. Finally, the JIS (Japan Integrated Staging Score) [24] is a score system that combines two existing classifications, named TNM and Child-Pugh (Table 1). It is widely used in Japan but it lacks external validation in Western countries. New prognostic markers To date, a growing attention is focused on the lookout for new prognostic markers able to increase the power of predictor models for HCC. Several years ago, the role of Estrogen Receptor (ER), defined as wild-type (wter) or variant (ver) [25], has been described for the biologic characterization of the tumor. A study by Villa et al [26] showed that the presence of wter is directly related with a good prognosis, and the survival is five-fold better in patients with HCC presenting with wter compared with the ones presenting with ver. Similarly to what happens in breast cancer, the presence of variant forms of ER seems to correlate with lack of hormonal control on the tumor growth, elevated proliferation rate and tumor aggressiveness. Thus, although ER characterization requires an invasive procedure - liver biopsy - it can be useful for an accurate prognosis and as a reliable assessment of sensitivity to treatment also for clinical decision making. More recently, some studies have focused on the impact of new serological markers in predicting prognosis of patients with HCC. For example, low serum vascular endothelial growth factor (VEGF) levels seems to be associated with a longer survival at each stage according to CLIP or BCLC, and the inclusion of baseline plasma VEGF levels increases the precision of the CLIP scoring system for predicting HCC prognosis ( V-CLIP staging) [27,28]. Likewise, high Insulin-like 4145 April 21, 2014 Volume 20 Issue 15

47 Maida M et al. Staging systems and prognosis of HCC Table 3 Comparison of different hepatocellular carcinoma staging system in the literature Ref. Country Year Case number Patient population The best Levy et al [58] Canada All CLIP Kudo et al [59] Japan All JIS Cillo et al [41] Italy All BCLC Grieco et al [39] Italy Early to intermediate BCLC Marrero et al [34] United States All BCLC Nanashima et al [60] Japan Surgery CLIP Huang et al [61] Taiwan Surgery TNM Toyoda et al [62] Japan All JIS Pascual et al [63] Spain All BCLC Georgiades et al [42] United States TACE Child-Pugh Cillo et al [64] Italy All BCLC Nanashima et al [65] Japan Surgery Modified JIS Kondo et al [66] Japan Surgery JIS Seong et al [67] South Korea Radiotherapy TNM Chen et al [40] Taiwan Surgery CLIP Huo et al [68] Taiwan All CLIP Cammà et al [37] Italy All CLIP Guglielmi et al [69] Italy RFA BCLC Collette et al [70] French Advanced CLIP Lu et al [71] China Surgery TNM Chung et al [72] Japan All JIS Lin et al [38] Taiwan All CLIP Hsu et al [36] Taiwan All CLIP Op den Winkel et al [73] German Non-surgical CLIP Kim et al [35] South Korea All BCLC CLIP: Cancer of the Liver Italian Program; GRETCH: Groupe d Etude et de Traitement du Carcinome Hépatocellulaire; BCLC: Barcelona Clinic Liver Cancer; CUPI: Chinese University Prognostic Index; JIS: Japan Integrated Staging Score; TNM: Tumor Node Metastasis. growth factor-1 (IGF-1) plasma levels seems to reflect time-to-recurrence, as well as overall survival [29], and the addition of plasma IGF-1 levels to CLIP ( I-CLIP staging) significantly improves prognostic stratification of patients with advanced HCC [30]. Finally, overexpression of the Forkhead box M1 (FOXM1) gene is associated with a poor outcome after OLT [31], and expression of the AKR1B10 (aldo-keto reductase enzyme) gene reflects a less aggressive tumour behaviour [32]. All these new markers have shown promising results, but require further evaluation and external validation. STAGING SYSTEMS FOR HCC: WHICH IS THE BEST ONE? To date several staging systems for HCC have been proposed, but currently none of these has been universally accepted, as pointed out by AASLD guidelines, which emphasize how there is not a worldwide consensus on the use of any given model for stadiation of HCC [33]. Several studies comparing the predictive power of different models have shown conflicting results, both in the general population and in the different subgroups of treatment (Table 3). According to the analysis performed by Marrero et al [34] on a cohort of 244 United States patients of any stage, the BCLC showed the best independent predictive power for survival when compared with the other 6 prognostic systems (TNM, CLIP, CUPI, JIS, GRETCH and Okuda). Similar results were found by an Asian study performed on 1717 treatment-naïve HCC patients, showing as BCLC was the best prognostic model if compared with other 5 systems (CLIP, CUPI, JIS, GRETCH and Tokyo score) [35]. Conversely, a study by Hsu et al [36] investigating the prognostic ability of the 5 staging systems (BCLC, CLIP, JIS, TNM and Tokyo score), showed the CLIP was the best long-term prognostic model in a cohort of patient with early to advanced stage HCC. Similarly, a recent study comparing the performance of BCLC, CLIP and GRETCH in a cohort of 406 consecutive patients with cirrhosis and HCC [37], showed the CLIP had the best discriminative capacity in the entire HCC cohort and in the advanced untreatable cases, while BCLC proved to be the best in predicting survival in treated patients. Finally, a subsequent study on a larger cohort of 3868 treated patients confirmed a modest discriminatory ability of CLIP for early HCC [38]. In addition, several studies have been also performed over time in order to weigh the performance of staging and prognostic systems in specific subsets of patients receiving different class of treatments (Table 3). An Italian retrospective study compared the performance of Okuda, CLIP and BCLC in a cohort of 268 patients treated with non-surgical therapy [39]. Both CLIP and BCLC scores were more effective than the Okuda score in stratifying patients into different risk groups of patients with early-intermediate HCC, even if BCLC showed a better prediction of prognosis in patients with very early stage HCC. Furthermore, a subsequent study compared Okuda, TNM, CLIP, BCLC, CUPI, JIS and 4146 April 21, 2014 Volume 20 Issue 15

48 Maida M et al. Staging systems and prognosis of HCC MELD in the prediction of survival among patients with HCC treated with major or minor hepatectomy [40]. Among all the seven staging systems, CLIP and JIS showed the best results. In particular, CLIP had a better discriminatory ability in the subset of patients treated with major hepatectomy, while JIS proved to be the most accurate in the minor hepatectomy group. Conversely, another retrospective analysis of 187 HCC Italian patients mainly treated with radical therapies (resection and percutaneous ablation) showed that BCLC had the greatest prognostic power among five systems (BCLC, CLIP, GRETCH, CUPI and Okuda) both for the whole study group and for the 2 subgroups of surgical and non-surgical patients [41]. These results do not seem to be confirmed in the group of patients treated with non-curative therapies. In this regard, the prognostic accuracy of 12 liver staging systems (nominal and categoric Child-Pugh, Okuda, CLIP, BCLC, MELD, CUPI, JIS, TNM, GRETCH, Liver Cancer Study Group of Japan, and Tokyo score) has been assessed in a cohort of 172 consecutive patients with unresectable HCC treated with TACE [42]. According to the results of this study, nominal Child-Pugh, CUPI, and Tokyo score provided the best prognostic accuracy, and the nominal Child-Pugh was the most accurate among them in predicting survival of patients with unresectable HCC treated with TACE. As already mentioned, currently there is not an ideal staging and prognostic system for HCC. Anyway, the BCLC seems to be the most comprehensive, since it integrates information about tumor extension, liver function and the presence of constitutional symptoms. It also provides prognostic information and guidance to the therapeutic choices, and it has been endorsed by EASL and AASLD as standard for patients with HCC. Since survival outcomes can be inevitably confounded by treatment strategies that may be quite different from one center to another [43], It must be noted that the external validation which uses the natural history of untreated HCC cohorts might be the most useful way to compare the prognostic value of each staging system [44,45]. In this regard, the previously mentioned meta-analysis by Cabibbo et al [7] which analyzed the survival rates of the untreated and placebo arms of several RCTs on HCC patients, confirmed that many of the prognostic variables of the BCLC (PS, Child-Pugh B-C class, and presence of PVT) are also robust predictors of death in untreated patients. This provides further evidence that the BCLC has a good discriminative capacity as prognostic score, regardless of the treatment strategy applied. Anyway, it does not represent a perfect model and still it has several unmet points. First, unlike CLIP [14], GRETCH [21] and CUPI [23], the BCLC was not derived from a cohort of HCC patients by a multivariate analysis, and therefore it is not a prognostic model able to predict the mortality of HCC patients, being internally and externally validated just as a staging system. Second, acting as classification model, it presents itself some inherent drawbacks. For example, the intermediate stage (BCLC B) includes an extremely heterogeneous population in terms of both liver function and tumor characteristics. In addition, according to the BCLC, any patient with a PS equal to 1 automatically falls in the advanced stage (BCLC C), even if this condition identifies a subject capable of performing all the normal daily activities according the original ECOG (Eastern Cooperative Oncology Group) definition. In addition, acting as treatment algorithm, the main limitation of the BCLC is represented by its rigidity. First, some prognostic factors, such as the presence of clinically significant portal hypertension, are outlined as contraindications that preclude a therapy, whereas evidences suggest that hepatic resection can be performed successfully, in highly selected cases, even in patients with portal hypertension and multiple hepatic lesions [46,47]. Second, it should be noted that not all patients defined by each stage of BCLC are ultimately candidates for the suggested treatment modality. For instance, TACE can be performed at earlier stages in patients not eligible to RFA or PEI because of tumor location (proximity to the gallbladder, biliary tree, or blood vessel), or failure of previous curative treatments and/or presence of medical comorbidities. Moreover, BCLC algorithm does not provide indications concerning second-line therapies, retreatment choices or combined treatments [48,49]. An important management problem is still represented by the indications for transplantation suggested by BCLC. For example, several lines of evidence show that transplant can get similar results in patients exceeding the Milan criteria, but conform to the up-to-seven [50] or the San Francisco criteria [51]. Furthermore, transplant is not indicated for end stage disease (BCLC D), which includes, among others, also patients with early tumor but with severe hepatic decompensation (Child-Pugh C). Despite the recommendations of the BCLC suggesting supportive care as the only available therapy, this subset of patients gets anyway the best benefit after transplantation [52,53]. As a result of its rigidity and unmet points, the BCLC is frequently difficult to apply, and its adherence in clinical practice is low [54]. Finally, to date, none of these staging systems have been analyzed or validated taking into account the prognosis of OLT, and therefore can not be recommended in the setting of liver transplantation [55]. CONCLUSION Currently, the non-ideal predictive performance of existing prognostic systems is secondary to their inherent limitations, as well as to a non-universal reproducibility and transportability of the results in different populations. In addition, other key factors must be considered. First, most of prognostic models are derived by a multiple regression analysis using time-fixed Cox model in order to identify independent factors for mortality. It is already well known as this kind of models may be unreli April 21, 2014 Volume 20 Issue 15

49 Maida M et al. Staging systems and prognosis of HCC able because of the potential interaction of time-varying predictors. In this context, compared with the time-fixed models, a time-dependent Cox model could have a better potential to estimate prognosis in HCC patients, as already demonstrated by a recent study [56]. Second, as already mentioned, the natural history of HCC is extremely heterogeneous. This is probably secondary to the existence of specific factors not accounted in the prognostic models that can have some impact on patient outcomes. In this regard, the evaluation of gene expression profiling may have an important role in the future to better understand the tumor biology and to improve the predictive power of the models. In conclusion, due to a non-perfect homogeneity and discrimination (internal validity) and a not absolute transportability of prognostic models in different populations (external validity), currently they are still far away from getting a good confidence in predicting outcome in the individual patient [57]. For these reasons, prognostic models should be used with caution, and staging systems that include integrated therapeutic algorithms should be considered as a general guide only. REFERENCES 1 Sherman M. Hepatocellular carcinoma: epidemiology, surveillance, and diagnosis. Semin Liver Dis 2010; 30: 3-16 [PMID: DOI: /s ] 2 Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBO- CAN Int J Cancer 2010; 127: [PMID: DOI: /ijc.25516] 3 Cabibbo G, Craxì A. Hepatocellular cancer: optimal strategies for screening and surveillance. Dig Dis 2009; 27: [PMID: DOI: / ] 4 Bruix J, Sherman M. Management of hepatocellular carcinoma. 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Predicting survival in early hepatocellular carcinoma. Gut 2005; 54: [PMID: ] 45 Llovet JM, Bustamante J, Castells A, Vilana R, Ayuso Mdel C, Sala M, Brú C, Rodés J, Bruix J. Natural history of untreated nonsurgical hepatocellular carcinoma: rationale for the design and evaluation of therapeutic trials. Hepatology 1999; 29: [PMID: ] 46 Cescon M, Cucchetti A, Grazi GL, Ferrero A, Viganò L, Ercolani G, Zanello M, Ravaioli M, Capussotti L, Pinna AD. Indication of the extent of hepatectomy for hepatocellular carcinoma on cirrhosis by a simple algorithm based on preoperative variables. Arch Surg 2009; 144: 57-63; discussion 63 [PMID: ] 47 Ishizawa T, Hasegawa K, Aoki T, Takahashi M, Inoue Y, Sano K, Imamura H, Sugawara Y, Kokudo N, Makuuchi M. Neither multiple tumors nor portal hypertension are surgical contraindications for hepatocellular carcinoma. Gastroenterology 2008; 134: [PMID: ] 48 Cabibbo G, Latteri F, Antonucci M, Craxì A. Multimodal approaches to the treatment of hepatocellular carcinoma. Nat Clin Pract Gastroenterol Hepatol 2009; 6: [PMID: DOI: /ncpgasthep1357] 49 Bolondi L, Cillo U, Colombo M, Craxì A, Farinati F, Giannini EG, Golfieri R, Levrero M, Pinna AD, Piscaglia F, Raimondo G, Trevisani F, Bruno R, Caraceni P, Ciancio A, Coco B, Fraquelli M, Rendina M, Squadrito G, Toniutto P. Position paper of the Italian Association for the Study of the Liver (AISF): the multidisciplinary clinical approach to hepatocellular carcinoma. Dig Liver Dis 2013; 45: [PMID: DOI: /j.dld ] 50 Mazzaferro V, Romito R, Schiavo M, Mariani L, Camerini T, Bhoori S, Capussotti L, Calise F, Pellicci R, Belli G, Tagger A, Colombo M, Bonino F, Majno P, Llovet JM. Prevention of hepatocellular carcinoma recurrence with alpha-interferon after liver resection in HCV cirrhosis. Hepatology 2006; 44: [PMID: ] 51 Yao FY, Xiao L, Bass NM, Kerlan R, Ascher NL, Roberts JP. Liver transplantation for hepatocellular carcinoma: validation of the UCSF-expanded criteria based on preoperative imaging. Am J Transplant 2007; 7: [PMID: ] 52 Cillo U, Vitale A, Volk ML, Frigo AC, Grigoletto F, Brolese A, Zanus G, D Amico F, Farinati F, Burra P, Russo F, Angeli P, D Amico DF. The survival benefit of liver transplantation in hepatocellular carcinoma patients. Dig Liver Dis 2010; 42: [PMID: ] 53 Vitale A, Morales RR, Zanus G, Farinati F, Burra P, Angeli 4149 April 21, 2014 Volume 20 Issue 15

51 Maida M et al. Staging systems and prognosis of HCC P, Frigo AC, Del Poggio P, Rapaccini G, Di Nolfo MA, Benvegnù L, Zoli M, Borzio F, Giannini EG, Caturelli E, Chiaramonte M, Trevisani F, Cillo U. Barcelona Clinic Liver Cancer staging and transplant survival benefit for patients with hepatocellular carcinoma: a multicentre, cohort study. Lancet Oncol 2011; 12: [PMID: ] 54 Borzio M, Sacco R. Nonadherence to guidelines in the management of hepatocellular carcinoma: an Italian or universal phenomenon? Future Oncol 2013; 9: [PMID: DOI: /fon.13.29] 55 Olthoff KM, Forner A, Hübscher S, Fung J. What is the best staging system for hepatocellular carcinoma in the setting of liver transplantation? Liver Transpl 2011; 17 Suppl 2: S26-S33 [PMID: DOI: /lt.22352] 56 Cabibbo G, Genco C, Di Marco V, Barbara M, Enea M, Parisi P, Brancatelli G, Romano P, Craxì A, Cammà C. Predicting survival in patients with hepatocellular carcinoma treated by transarterial chemoembolisation. Aliment Pharmacol Ther 2011; 34: [PMID: ] 57 Cammà C, Cabibbo G. Prognostic scores for hepatocellular carcinoma: none is the winner. Liver Int 2009; 29: [PMID: ] 58 Levy I, Sherman M. Staging of hepatocellular carcinoma: assessment of the CLIP, Okuda, and Child-Pugh staging systems in a cohort of 257 patients in Toronto. Gut 2002; 50: [PMID: ] 59 Kudo M, Chung H, Haji S, Osaki Y, Oka H, Seki T, Kasugai H, Sasaki Y, Matsunaga T. Validation of a new prognostic staging system for hepatocellular carcinoma: the JIS score compared with the CLIP score. Hepatology 2004; 40: [PMID: ] 60 Nanashima A, Omagari K, Tobinaga S, Shibata K, Sumida Y, Mine M, Morino S, Shibasaki S, Ide N, Shindou H, Nagayasu T. Comparative study of survival of patients with hepatocellular carcinoma predicted by different staging systems using multivariate analysis. 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Prospective validation of the Barcelona Clinic Liver Cancer staging system. J Hepatol 2006; 44: [PMID: ] 65 Nanashima A, Sumida Y, Abo T, Shindou H, Fukuoka H, Takeshita H, Hidaka S, Tanaka K, Sawai T, Yasutake T, Nagayasu T, Omagari K, Mine M. Modified Japan Integrated Staging is currently the best available staging system for hepatocellular carcinoma patients who have undergone hepatectomy. J Gastroenterol 2006; 41: [PMID: ] 66 Kondo K, Chijiiwa K, Nagano M, Hiyoshi M, Kai M, Maehara N, Ohuchida J, Nakao H, Ohkuwa Y. Comparison of seven prognostic staging systems in patients who undergo hepatectomy for hepatocellular carcinoma. Hepatogastroenterology 2007; 54: [PMID: ] 67 Seong J, Shim SJ, Lee IJ, Han KH, Chon CY, Ahn SH. Evaluation of the prognostic value of Okuda, Cancer of the Liver Italian Program, and Japan Integrated Staging systems for hepatocellular carcinoma patients undergoing radiotherapy. Int J Radiat Oncol Biol Phys 2007; 67: [PMID: ] 68 Huo TI, Lin HC, Hsia CY, Wu JC, Lee PC, Chi CW, Lee SD. The model for end-stage liver disease based cancer staging systems are better prognostic models for hepatocellular carcinoma: a prospective sequential survey. Am J Gastroenterol 2007; 102: [PMID: ] 69 Guglielmi A, Ruzzenente A, Pachera S, Valdegamberi A, Sandri M, D Onofrio M, Iacono C. Comparison of seven staging systems in cirrhotic patients with hepatocellular carcinoma in a cohort of patients who underwent radiofrequency ablation with complete response. Am J Gastroenterol 2008; 103: [PMID: ] 70 Collette S, Bonnetain F, Paoletti X, Doffoel M, Bouché O, Raoul JL, Rougier P, Masskouri F, Bedenne L, Barbare JC. Prognosis of advanced hepatocellular carcinoma: comparison of three staging systems in two French clinical trials. Ann Oncol 2008; 19: [PMID: DOI: /annonc/mdn030] 71 Lu W, Dong J, Huang Z, Guo D, Liu Y, Shi S. Comparison of four current staging systems for Chinese patients with hepatocellular carcinoma undergoing curative resection: Okuda, CLIP, TNM and CUPI. J Gastroenterol Hepatol 2008; 23: [PMID: DOI: /j x] 72 Chung H, Kudo M, Takahashi S, Hagiwara S, Sakaguchi Y, Inoue T, Minami Y, Ueshima K, Fukunaga T, Matsunaga T. Comparison of three current staging systems for hepatocellular carcinoma: Japan integrated staging score, new Barcelona Clinic Liver Cancer staging classification, and Tokyo score. J Gastroenterol Hepatol 2008; 23: [PMID: ] 73 op den Winkel M, Nagel D, Sappl J, op den Winkel P, Lamerz R, Zech CJ, Straub G, Nickel T, Rentsch M, Stieber P, Göke B, Kolligs FT. Prognosis of patients with hepatocellular carcinoma. Validation and ranking of established stagingsystems in a large western HCC-cohort. PLoS One 2012; 7: e45066 [PMID: DOI: /journal.pone ] P- Reviewers: Takagi H, Troncoso MF, Yoon JH S- Editor: Zhai HH L- Editor: A E- Editor: Zhang DN 4150 April 21, 2014 Volume 20 Issue 15

52 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (1): Hepatocellular carcinoma TOPIC HIGHLIGHT Chemotherapy for advanced hepatocellular carcinoma in the sorafenib age Koji Miyahara, Kazuhiro Nouso, Kazuhide Yamamoto Koji Miyahara, Kazuhiro Nouso, Kazuhide Yamamoto, Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama-city, Okayama , Japan Kazuhiro Nouso, Department of Molecular Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama-city, Okayama , Japan Author contributions: Miyahara K and Nouso K wrote the paper; Yamamoto K critically revised the manuscript for important intellectual content. Correspondence to: Koji Miyahara, MD, PhD, Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Shikata-cho, Kita-ku, Okayama-city, Okayama , Japan. mkojisup@yahoo.co.jp Telephone: Fax: Received: September 4, 2013 Revised: January 2, 2014 Accepted: February 26, 2014 Published online: April 21, 2014 Abstract The kinase inhibitor sorafenib is the only systemic therapy proven to have a positive effect on survival of patients with advanced hepatocellular carcinoma (HCC). After development of sorafenib and its introduction as a therapeutic agent used in the clinic, several critical questions have been raised. Clinical parameters and biomarkers predicting sorafenib efficacy are the most important issues that need to be elucidated. Although it is difficult to know the responders in advance using conventional characteristics of patients, there are specific serum cytokines and/or gene amplification in tumor tissues that have been reported to predict efficacy of sorafenib. Risk and benefits of continuation of sorafenib beyond radiological progression is another issue to consider because no other standard therapy for advanced HCC as yet exists. In addition, effectiveness of the expanded application of sorafenib is still controversial, although a few studies have shed some light on combinational treatment with sorafenib for intermediate-stage HCC. Recently, over 50 relevant drugs have been developed and are currently under investigation. The efficacy of some of these drugs has been extensively examined, but none have demonstrated any superiority over sorafenib, so far. However, there are several drugs that have shown efficacy for treatment after sorafenib failure, and these are proceeding to further studies. To address these issues and questions, we have done extensive literature review and summarize the most current status of therapeutic application of sorafenib Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Biomarker; Molecular targeted therapy; Clinical trial; Hepatic arterial infusion chemotherapy; Hepatocellular carcinoma; Sorafenib Core tip: Sorafenib is the only systemic therapy proven to have a positive effect on survival and to be well tolerated in patients with advanced hepatocellular carcinoma (HCC). We summarize the most current status of sorafenib therapy, focusing on (1) safety and efficacy of sorafenib for advanced HCC; (2) biomarkers predicting efficacy of sorafenib; (3) expanded application for the treatment of non-advanced HCC; (4) sorafenib efficacy beyond radiological progression; and (5) novel therapeutics and hepatic arterial infusion chemotherapy. Miyahara K, Nouso K, Yamamoto K. Chemotherapy for advanced hepatocellular carcinoma in the sorafenib age. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: April 21, 2014 Volume 20 Issue 15

53 Miyahara K et al. Chemotherapy for advanced hepatocellular carcinoma INTRODUCTION Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide and is associated with the second lowest 5-year survival rate of all tumor types [1]. For patients diagnosed at early stages, potentially curative treatments are available, such as radiofrequency ablation, resection, and liver transplantation; and patients at intermediate stages may be treated with transcatheter arterial chemoembolization (TACE). However, for disease that is diagnosed at an advanced stage or progresses after locoregional therapies, sorafenib is the choice of treatment. Sorafenib, which is an oral multi-kinase inhibitor, suppresses tumor angiogenesis and proliferation by inhibiting the activity of such targets as vascular endothelial growth factor (VEGF) receptor, platelet-derived growth factor (PDGF) receptor, mast/stem cell growth factor receptor (c-kit), rearranged during transfection, Fms-like tyrosine kinase 3, and the proto-oncoprotein, c-raf [2,3]. In addition, sorafenib also has been shown to induce apoptosis as direct effects on tumor cell [4]. The safety and efficacy of sorafenib in patients with advanced HCC was demonstrated in two phase Ⅲ randomized, double-blind, placebo-controlled trials, the Sorafenib HCC Assessment Randomized Protocol (SHARP) and Asia-Pacific (AP) trials [5,6], thereby establishing sorafenib as the standard systemic therapy for advanced HCC [7,8]. HCC is often resistant to chemotherapy, and the potential for liver failure accompanying this disease has complicated the therapies that are employed. However, there is currently no systemic therapy other than sorafenib, although many clinical trials are on-going. The aim of the present review is to summarize recent clinical evidence and present a current status of sorafenib in therapeutic treatment of HCC. STATUS OF SORAFENIB IN CLINICAL GUIDELINES Sorafenib is recommended as a treatment in patients with (1) extrahepatic lesions; (2) macrovascular invasion; or (3) those who do not response to TACE/arterial injection chemotherapy, when the liver function is Child- Pugh (CP)-A, in a consensus-based treatment algorithm for HCC (JSH Consensus 2010) [9]. The recommendation was based on the results demonstrated in SHARP and AP trials (Table 1). Patients recommended for sorafenib in this algorithm overlap with those recommended according to the European Association for the Study of the Liver (EASL), the European Organization for Research and Treatment of Cancer (EORTC) [10], the American Association for the Study of Liver Diseases (AASLD) [8], and the National Comprehensive Cancer Network (NCCN) Clinical Practice Guideline [11]. The EASL-EOTC, AASLD, and NCCN guidelines indicate sorafenib as an option for CP-B patients; whereas, no clear evidence, based on randomized controlled trials, has been presented on safety of sorafenib in CP-B patients. The recent report in Global investigation of therapeutic decisions in HCC and of its treatment with sorafenib (GIDEON), which is a global, non-interventional, surveillance study, has presented data from sorafenib treatment of patients with liver dysfunction [12]. In the study, CP subgroups showed similar incidence of all grade of adverse events (AEs) [84.0% (CP-A) vs 88.6% (CP-B)] and time to progression (TTP) [4.7 mo (CP-A) vs 4.4 mo (CP-B)]. In contrast, serious AEs were more common in CP-B (60.4%) than CP-A (36.0%) patients. The finding that severity of AEs is associated with poor liver function provides a certain warning to the use of sorafenib for the CP-B patient, even if the treatment efficacy is consistent irrespective of liver function. CLINICAL CHARACTERISTICS AND EFFICACY OF SORAFENIB Clinical characteristics at baseline that might affect responses to therapy have been examined. Subgroup analyses of SHARP and AP trials, in which patients with wellpreserved liver function had been enrolled, demonstrated the baseline status related to outcomes during sorafenib treatment [13-15]. In both analyses, the patients with Eastern Cooperative Oncology Group performance status (ECOG PS) 1 or 2, aspartate/alanine transaminase (AST/ALT) elevation, or macroscopic vascular invasion (MVI) had similar hazard ratios with the total population [hazard ratio (HR) of 0.69 in SHARP; 0.68 in AP]. These findings provide an opportunity for patients with these statuses to be treated with sorafenib, but it should be noted that high ECOG PS, AST/ALT elevation, or presence of MVI themselves were associated with short OS. Sorafenib treatment for patients with prior local therapy, prior TACE, or extrahepatic spread (EHS) also resulted in longer median OS than placebo, except for those with prior hepatectomy in the AP trial. However, careful interpretation of these results is needed because the studies did not aim to show the differences between these subgroups, and statistical confirmation had not yet been performed. BIOMARKERS FOR PREDICTING OUTCOMES OF SORAFENIB TREATMENT Predictive biomarkers are expected to advance the potential of personalized medicine in cancer treatment. Biomarker research for predicting the efficacy of sorafenib is a growing field, and a few candidate markers in plasma, serum, and tissue have been reported (Table 2). Llovet et al [16] reported results of sub-analysis in the SHARP trial, examining expression of 10 molecules in plasma of HCC patients. Plasma c-kit and hepatocyte growth factor were suggested as possible predictors of response to sorafenib, although the association was not statistically significant. In other preliminary studies, angiogenesis-related cytokines 4152 April 21, 2014 Volume 20 Issue 15

54 Miyahara K et al. Chemotherapy for advanced hepatocellular carcinoma Table 1 Overall survivals in sorafenib treatment Clinical trial Sorafenib Placebo P value n Median OS (mo) 95%CI n Median OS (mo) 95%CI SHARP [5] < AP [6] OS: Overall survival; AP: Asia-Pacific. Table 2 Biomarkers for predicting outcomes with sorafenib Ref. Year Obtained from Biomarker Llovet et al [16] 2012 Plasma HGF, c-kit Miyahara et al [17] 2011 Serum Angiogenesis-related cytokines 1 Arao et al [18] 2013 Tissue FGF3/FGF4 Huang et al [19] 2013 Tissue αb-crystallin Hagiwara et al [20] 2012 Tissue JNK Abou-Alfa et al [21] 2006 Tissue perk Shan et al [25] 2012 Cell line Nanog Blivet-Van Eggelpoël et al [26] 2012 Cell line EGFR, HER-3 Chen et al [27] 2012 Cell line SIRT1 Tai et al [28] 2011 Cell line STAT3 Liu et al [4] 2006 Cell line Mcl-1, eif4e 1 These include angiopoietin-2, follistatin, granulocyte colony-stimulating factor, hepatocyte growth factor, leptin, platelet-derived growth factor-bb, platelet endothelial cell adhesion molecule-1, and vascular endothelial growth factor. HGF: hepatocyte growth factor; c-kit: Also known as SCFR (mast/stem cell growth factor receptor); FGF: fibroblast growth factors; JNK: c-jun N-terminal kinase; perk: phosphorylated extracellular signal regulated kinase; EGFR: epidermal growth factor receptor; HER-3: Also known as ErbB3; STAT3: signal transducer and activator of transcription 3; Mcl-1: myeloid cell leukemia-1; eif4e: eukaryotic translation initiation factor 4E. in serum, including angiopoietin-2, were reported to correlate with treatment response [17]. Several candidates for tissue markers, such as FGF3/FGF4 [18], αb-crystallin [19], JNK [20], and perk [21], have been proposed. Amplification of FGF3/FGF4 was observed only in objective responders, but not in patients with stable or progressive disease. Frequency of FGF3/FGF4 amplification remains below a few percent in HCC [22-24] ; however, FGF3/FGF4 amplification might represent a promising therapeutic target, and it provides a novel insight for molecular-based therapy in HCC. Various molecules thought to have potential to be novel markers or therapeutic targets have been identified on the basis of basic research observations [4,25-28] (Table 2), but none of them has been verified in clinical studies. Candidate biomarkers should be validated in prospective clinical trials, in order to assess their potential to lead to personalized therapy. CONVENTIONAL TUMOR MARKERS DURING TREATMENT WITH SORAFENIB Conventional tumor markers for the diagnosis of HCC, i.e., α-fetoprotein (AFP) and des-gamma-carboxy prothrombin (DCP), have been reported to show contrasting behavior after administration of sorafenib. Early AFP decrease correlates with beneficial efficacy of sorafenib in patients with HCC [29-31], as observed in other therapies. However, DCP increases with sorafenib administration, regardless of the treatment response [29]. Interestingly, a few reports have shown that elevation of DCP [32,33] and NX-DCP, which is a vitamin K-specific phenotype of DCP [34], is associated with a highly therapeutic effect of sorafenib. These markers are expected to be used for monitoring HCC patients undergoing treatment, rather than as predictive biomarkers. ADVERSE EVENTS AND EFFICACY OF SORAFENIB Various adverse events were frequently observed during sorafenib therapy (Table 3). Adverse events of moleculartargeted agents sometimes associate with a favorable effect on prognosis [35-46]. Regarding sorafenib therapy, development of skin toxicities [47,48] and arterial hypertension [49] in some trials correlated with longer time to disease progression or longer survival in patients with HCC, and similar correlations were seen in those with renal cell carcinoma [50,51]. However, this correlation has not been identified in randomized controlled trials, and validation might be difficult if there are agents for preventive care being administrated, such as preventive and therapeutic skin care, systemic analgesics for pain, vitamin B6, etc., for hand-foot skin reaction [52]. Furthermore, haphazard continuation of chemotherapies with side effects requires careful management, as these may sometimes lead to dangerous conditions. EFFICACY OF SORAFENIB IN NON- ADVANCED HCC Sorafenib is recommended for treatment of advanced HCC in clinical algorithms, as described above, but the util April 21, 2014 Volume 20 Issue 15

55 Miyahara K et al. Chemotherapy for advanced hepatocellular carcinoma Table 3 Incidence of drug-related adverse events of sorafenib treatment Adverse event, % SHARP [5] AP [6] GIDEON (second interim analysis) [89] (n = 297) (n = 149) (n = 1571) Any adverse event Diarrhea Hand-foot skin reaction Fatigue Rash desquamation Anorexia Hypertension Alopecia Nausea Weight loss 9 NA NA: Not available; this adverse event was observed in < 10% of patients in AP trial. Incidence of all grades of adverse events was shown. Adverse events in 5% of the total population in GIDEON study were listed. ity for early or intermediate stages of HCC remain unclear. Intermediate-stage HCC with multiple nodules and without major vascular invasion or EHS is commonly treated by TACE. Ischemic injury after TACE induces up-regulation of VEGF [53], which is associated with poor prognosis [54,55]. However, combination with sorafenib, which inhibits angiogenic factors, including VEGF receptor, could theoretically reinforce the efficacy of TACE. Several clinical trials evaluating effects of TACE with sorafenib in treatment of intermediate-stage HCC are being conducted (Table 4). Two single-arm phase Ⅱ trials have shown promising efficacy [56,57]. A randomized placebo-controlled study of sorafenib or placebo in combination with TACE for intermediate-stage HCC (SPACE) successfully demonstrated that sorafenib prolongs TTP after TACE, although improvement of OS or time to untreatable progression (TTUP) was not observed [58]. In a randomized phase Ⅲ trial in patients who responded to TACE (post TACE study), sorafenib did not significantly prolong TTP after TACE, compared to placebo [59]. In this study, a long lag time of > 9 wk prior to administration of sorafenib may also have contributed to the absence of a positive effect of sorafenib. In response, a trial titled Transcatheter Arterial Chemoembolization Therapy in Combination with Sorafenib (TACTICS, NCT ) is currently being conducted, with a stipulated lag time (3-21 d). Furthermore, sorafenib is under evaluation as an adjuvant therapy for the prevention of recurrence following surgery or local ablation. The trial is a phase Ⅲ, randomized, double-blind, and placebo-controlled study, titled Sorafenib as Adjuvant Treatment in the Prevention of Recurrence of HCC (STORM trial; NCT ). The results from this study will provide more information about whether sorafenib has efficacy for HCC at early stages and reduces the risk of recurrence. CONTINUATION OF SORAFENIB AFTER RADIOLOGICAL PROGRESSION In general, tumor progression implies resistance to the therapy being employed, and it is thus a motivation to change therapy regimen. However, in contrast to typical cytotoxic agents, sorafenib seldom induces an objective response (2%-3%) [5,6] ; this modest response would make it difficult for physicians to decide whether to continue or discontinue sorafenib treatment. Several reports speculate about sorafenib efficacy beyond radiological progression. Sorafenib administration beyond first radiological progression was seen to continuously suppress tumor growth [60], and long-term treatment was associated with prolonged survival regardless of therapeutic effect [61]. Interestingly, the SHARP trial was designed to continue sorafenib after radiological progression, if symptomatic progression was absent [5]. Hence, it is reasonable to consider continuing sorafenib at the time of radiological progression when patients will tolerate the therapy and have no symptomatic disease progression or liver dysfunction in the present status, with no other effective therapy. HEPATIC ARTERIAL INFUSION CHEMOTHERAPY Before the development of molecular targeted therapies based on evidence from randomized controlled trials, hepatic arterial infusion chemotherapy (HAIC) had been used to treat advanced HCC with vascular invasion and/ or multiple intrahepatic lesions. The protocol of HAIC is not standardized. The most frequently used regimens in Japan are (1) continuous infusion of 5-fluorouracil plus low-dose cisplatin combination therapy (termed low-dose FP, for fluorouracil and platinum); (2) continuous intra-arterial infusion of 5-fluorouracil plus systemic interferon therapy (5-FU plus IFN), or one-shot infusion of cisplatin (one-shot CDDP). The response rates of HAIC were reported to be 24.5%-38.5% in low-dose FP [62-68] ; 21.5%-63% in 5-FU plus IFN [69-77] ; 3.6%-33.8% in one-shot CDDP [78-81], which were higher than that of sorafenib; but the survival benefit of these therapies are controversial. The lack of evidence based on randomized controlled trials in most of the regimens is a serious criticism of the importance of HAIC in HCC treatment. Trials evaluating the efficacy of HAIC in combination with sorafenib are currently on-going: Comparing Efficacy of Sorafenib vs Sorafenib in Combination with Low-dose FP in Patients with Advanced HCC (SILIUS Phase Ⅲ trial; NCT ); and Randomized Phase Ⅱ Study of Sorafenib and Hepatic Arterial Infusion Chemotherapy with Cisplatin vs Sorafenib for Advanced HCC (UMIN ). NOVEL THERAPEUTICS FOR ADVANCED HCC After the successful result of sorafenib in the SHARP and AP trials, more than 50 reagents are currently under evaluation in phase Ⅰ to Ⅳ trials ( Recently, phase Ⅲ studies have been reported to evalu April 21, 2014 Volume 20 Issue 15

56 Miyahara K et al. Chemotherapy for advanced hepatocellular carcinoma Table 4 Clinical trials for evaluating the effect of transcatheter arterial chemoembolization with sorafenib in intermediate stage of hepatocellular carcinoma Ref. Acronym (NCT number) Reported year Trial phase Study design n TACE Outcomes Pawlik et al [56] Ⅱ Single-arm (TACE plus sorafenib) Park et al [57] Ⅱ Single-arm (TACE plus sorafenib) Kudo et al [59] Post TACE study 2011 Ⅲ TACE plus sorafenib vs TACE plus placebo Lencioni et al [58] SPACE 2012 Ⅱ TACE plus sorafenib vs TACE plus placebo Kudo et al 2 Meyer et al 2 TACTICS (NCT ) CRUK-TACE-2 (NCT ) Currently recruiting participants Currently recruiting participants Kauh et al 2 (NCT ) Currently recruiting participants Ⅱ Ⅲ Ⅲ TACE plus sorafenib vs TACE alone TACE plus sorafenib vs TACE plus placebo TACE plus sorafenib vs TACE plus placebo 33 DEB/scheduled Disease control rate = 100%, per lesion. Objective response = 58%, per lesion 50 Conventional 1 /on demand 458 Conventional 1 /1 or 2 sessions Median TTP = 7.1 mo 6-mo PFS rate = 52% Median TTP = 5.4 (sorafenib)/3.7 (placebo) mo. HR [sorafenib] = 0.87; 95%CI: ; P = DEB/scheduled Median TTP = 169 (sorafenib)/166 (placebo) d. HR [sorafenib] = 0.79; 95%CI: ; P = Conventional 1 /on demand Time to untreatable progression DEB/1 session PFS Conventional 1 or DEB/scheduled PFS 4 1 Conventional TACE indicated transcatheter arterial chemoembolization with gelatin foam and lipiodol; 2 The information of the trial can be accessed at ClinicalTrials.gov; 3 Estimated Enrollment; 4 Primary Outcome Measures. DBE: Drug-eluting beads; NCT: National clinical trial; PFS: Progression-free survival; TACE: Transcatheter arterial chemoembolization; TTP: Time to progression. ate the survival benefit of sunitinib, brivanib, linifanib, and the combination of sorafenib plus erlotinib over sorafenib monotherapy; however, there have so far been no agents showing survival improvement or alleviation of AEs [5,6,82-86]. Linifanib shows longer TTP and similar OS compared to sorafenib, but linifanib is inferior to sorafenib in safety [86]. Brivanib, as a second-line therapy after failure by, or intolerance to, sorafenib, shows longer TTP but similar OS relative to placebo [85]. The failures in these trials point to the difficulty of both improving the OS and alleviation of the AEs concurrently in advanced HCC. For effective use of molecular-targeted agents, clinical trials to investigate new agents in combination with predictive markers are on-going. These include c-met inhibitor (tivantinib; ARQ 197) and monoclonal antibody against glypican-3 (GC33). For patients with c-methigh tumors, TTP was found to be longer with tivantinib than for those with placebo in a randomized phase Ⅱ trial (2.7 vs 1.4 mo, p = 0.03) [87]. For patients treated with GC33, TTP was longer in patients with GPC3- high tumors than in those with GPC3-low tumors in a phase Ⅰ trial (26.0 vs 7.1 wk, p = 0.033) [88]. The efficacy of these surrogate markers are being evaluated in randomized, placebo-controlled phase Ⅲ (NCT ) and Ⅱ (NCT ) trials. CONCLUSION We have reviewed current status of chemotherapy for advanced HCC. Sorafenib has been established as a standard therapy prolonging survival in patients with advanced HCC, but it only provides a small treatment response. To compensate for the modest effect on tumor regression, new molecular-targeted drugs and their biomarkers for prediction of treatment efficacy are being investigated. 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60 Miyahara K et al. Chemotherapy for advanced hepatocellular carcinoma Yamashita T, Mizukoshi E, Sakai A, Nakamoto Y, Honda M, Kaneko S. Randomized, phase II study comparing interferon combined with hepatic arterial infusion of fluorouracil plus cisplatin and fluorouracil alone in patients with advanced hepatocellular carcinoma. Oncology 2011; 81: [PMID: DOI: / ] 77 Monden M, Sakon M, Sakata Y, Ueda Y, Hashimura E, FAIT Research Group. 5-fluorouracil arterial infusion + interferon therapy for highly advanced hepatocellular carcinoma: A multicenter, randomized, phase II study. Hepatol Res 2012; 42: [PMID: DOI: /j X x] 78 Yoshikawa M, Ono N, Yodono H, Ichida T, Nakamura H. Phase II study of hepatic arterial infusion of a fine-powder formulation of cisplatin for advanced hepatocellular carcinoma. Hepatol Res 2008; 38: [PMID: DOI: /j X x] 79 Kondo M, Morimoto M, Numata K, Nozaki A, Tanaka K. Hepatic arterial infusion therapy with a fine powder formulation of cisplatin for advanced hepatocellular carcinoma with portal vein tumor thrombosis. Jpn J Clin Oncol 2011; 41: [PMID: DOI: /jjco/hyq145] 80 Iwasa S, Ikeda M, Okusaka T, Ueno H, Morizane C, Nakachi K, Mitsunaga S, Kondo S, Hagihara A, Shimizu S, Satake M, Arai Y. Transcatheter arterial infusion chemotherapy with a fine-powder formulation of cisplatin for advanced hepatocellular carcinoma refractory to transcatheter arterial chemoembolization. Jpn J Clin Oncol 2011; 41: [PMID: DOI: /jjco/hyr037] 81 Kim BK, Park JY, Choi HJ, Kim do Y, Ahn SH, Kim JK, Lee do Y, Lee KH, Han KH. Long-term clinical outcomes of hepatic arterial infusion chemotherapy with cisplatin with or without 5-fluorouracil in locally advanced hepatocellular carcinoma. J Cancer Res Clin Oncol 2011; 137: [PMID: DOI: /s ] 82 Cheng A, Kang Y, Lin D, Park J, Kudo M, Qin S, Omata M, Pitman Lowenthal SW, Lanzalone S, Yang L, Lechuga M, Raymond E, SUN1170 HCC Study Group. Phase III trial of sunitinib (Su) versus sorafenib (So) in advanced hepatocellular carcinoma (HCC). J Clin Oncol 2011; Suppl; abstr Zhu AX, Rosmorduc O, Evans J, Ross P, Santoro A, Carrilho FJ, Leberre MA, Jensen M, Meinhardt G, Kang YK. SEARCH: a phase III, randomized, double-blind, placebo-controlled trial of sorafenib plus erlotinib in patients with hepatocellular carcinoma (HCC). 37th European Society of Medical Oncology Congress; Vienna: Austria, 2012: (abstr 917) 84 Johnson PJ, Qin S, Park JW, Poon RT, Raoul JL, Philip PA, Hsu CH, Hu TH, Heo J, Xu J, Lu L, Chao Y, Boucher E, Han KH, Paik SW, Robles-Aviña J, Kudo M, Yan L, Sobhonslidsuk A, Komov D, Decaens T, Tak WY, Jeng LB, Liu D, Ezzeddine R, Walters I, Cheng AL. Brivanib versus sorafenib as first-line therapy in patients with unresectable, advanced hepatocellular carcinoma: results from the randomized phase III BRISK-FL study. J Clin Oncol 2013; 31: [PMID: DOI: /JCO ] 85 Llovet JM, Decaens T, Raoul JL, Boucher E, Kudo M, Chang C, Kang YK, Assenat E, Lim HY, Boige V, Mathurin P, Fartoux L, Lin DY, Bruix J, Poon RT, Sherman M, Blanc JF, Finn RS, Tak WY, Chao Y, Ezzeddine R, Liu D, Walters I, Park JW. Brivanib in patients with advanced hepatocellular carcinoma who were intolerant to sorafenib or for whom sorafenib failed: results from the randomized phase III BRISK-PS study. J Clin Oncol 2013; 31: [PMID: DOI: /JCO ] 86 Cainap C, Qin S, Huang WT, Chung IJ, Pan H, Cheng Y, Kudo M, Kang YK, Chen PJ, Toh HC, Gorbunova V, Eskens F, Qian J, McKee MD, Ricker JL, Carlson DM, El Nowiem S. Phase III trial of linifanib versus sorafenib in patients with advanced hepatocellular carcinoma (HCC). J Clin Oncol 2012; Suppl 34: abstr Santoro A, Rimassa L, Borbath I, Daniele B, Salvagni S, Van Laethem JL, Van Vlierberghe H, Trojan J, Kolligs FT, Weiss A, Miles S, Gasbarrini A, Lencioni M, Cicalese L, Sherman M, Gridelli C, Buggisch P, Gerken G, Schmid RM, Boni C, Personeni N, Hassoun Z, Abbadessa G, Schwartz B, Von Roemeling R, Lamar ME, Chen Y, Porta C. Tivantinib for second-line treatment of advanced hepatocellular carcinoma: a randomised, placebo-controlled phase 2 study. Lancet Oncol 2013; 14: [PMID: DOI: / S (12) ] 88 Zhu AX, Gold PJ, El-Khoueiry AB, Abrams TA, Morikawa H, Ohishi N, Ohtomo T, Philip PA. First-in-man phase I study of GC33, a novel recombinant humanized antibody against glypican-3, in patients with advanced hepatocellular carcinoma. Clin Cancer Res 2013; 19: [PMID: DOI: / CCR ] 89 Lencioni R, Kudo M, Ye SL, Bronowicki JP, Chen XP, Dagher L, Furuse J, Geschwind JF, Ladrón de Guevara L, Papandreou C, Takayama T, Yoon SK, Nakajima K, Lehr R, Heldner S, Sanyal AJ. GIDEON (Global Investigation of therapeutic DEcisions in hepatocellular carcinoma and Of its treatment with sorafenib): second interim analysis. Int J Clin Pract 2013; Epub ahead of print [PMID: DOI: / ijcp.12352] P- Reviewers: Aghakhani A, Eghtesad B, Grassi G, Ishikawa T, Kakizaki S, Kim SH, Tsai JF S- Editor: Ma YJ L- Editor: A E- Editor: Liu XM 4159 April 21, 2014 Volume 20 Issue 15

61 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (1): Hepatocellular carcinoma Therapeutic response assessment of RFA for HCC: Contrast-enhanced US, CT and MRI TOPIC HIGHLIGHT Yasunori Minami, Naoshi Nishida, Masatoshi Kudo Yasunori Minami, Naoshi Nishida, Masatoshi Kudo, Department of Gastroenterology and Hepatology, Kinki University Faculty of Medicine, Osaka , Japan Author contributions: Minami Y drafted the manuscript and wrote the final version of the manuscript; Nishida N reviewed the manuscript; Kudo M approved the final version of the manuscript. Correspondence to: Masatoshi Kudo, MD, PhD, Department of Gastroenterology and Hepatology, Kinki University Faculty of Medicine, Ohno-Higashi Osaka-Sayama, Osaka , Japan. m-kudo@med.kindai.ac.jp Telephone: Fax: Received: October 12, 2013 Revised: January 22, 2014 Accepted: February 26, 2014 Published online: April 21, 2014 Abstract Radiofrequency ablation (RFA) is commonly applied for the treatment of hepatocellular carcinoma (HCC) because of the facile procedure, and the safety and effectiveness for the treatment of this type of tumor. On the other hand, it is believed that HCC cells should spread predominantly through the blood flow of the portal vein, which could lead to the formation of intrahepatic micrometastases. Therefore, monitoring tumor response after the treatment is quite important and accurate assessment of treatment response is critical to obtain the most favorable outcome after the RFA. Indeed, several reports suggested that even small HCCs of 3 cm in diameter might carry intrahepatic micrometastases and/or microvascular invasion. From this point of view, for preventing local recurrences, RFA should be performed ablating a main tumor as well as its surrounding non-tumorous liver tissue where micrometastases and microvascular invasion might exist. Recent advancement of imaging modalities such as contrast-enhanced ultrasonic, computed tomography, and magnetic resonance imaging are playing an important role on assessing the therapeutic effects of RFA. The local recurrence rate tends to be low in HCC patients who were proven to have adequate ablation margin after RFA; namely, not only disappearance of vascular enhancement of main tumor, but also an adequate ablation margin. Therefore, contrast enhancement gives important findings for the diagnosis of recurrent HCCs on each imaging. However, hyperemia of non-tumorous liver surrounding the ablated lesion, which could be attributed to an inflammation after RFA, may well obscure the findings of local recurrence of HCCs after RFA. Therefore, we need to carefully address to these imaging findings given the fact that diagnostic difficulties of local recurrence of HCC. Here, we give an overview of the current status of the imaging assessment of HCC response to RFA Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Hepatocellular carcinoma; Micrometastasis; Microvascular invasion; Radiofrequency ablation; Safety margin Core tip: Radiofrequency ablation (RFA) therapy is needed to ablate wider range of region than targeted tumor, including surrounding liver tissues that involve micrometastases and microvascular invasion. The local recurrence rate tends to be lower in hepatocellular carcinoma patients with an adequate ablation margin, and thus, it is essential to assess safety margin accurately to reduce local recurrence. From this point of view, we need to focus on the achievement of a sufficient ablation margin as well the lack of tumor vascular enhancement for the assessment of successful RFA. However, inflammatory hyperemia due to RFA which often appears as peripheral rim enhancement, and non-typical imaging features of tumor recurrence sometimes lead to the inappropriate diagnosis April 21, 2014 Volume 20 Issue 15

62 Minami Y et al. Imaging assessment of HCC response to RFA Minami Y, Nishida N, Kudo M. Therapeutic response assessment of RFA for HCC: Contrast-enhanced US, CT and MRI. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: INTRODUCTION Surgical resection is the first treatment of choice for hepatocellular carcinoma (HCC). Unfortunately, the majority of HCC patients are not suitable for curative resection at the time of diagnosis because of large tumor size, multifocal disease, vascular involvement, extrahepatic spread, poor liver function, etc. [1-6]. Therefore, there is a need to develop a simple and effective technique to treat unresectable HCC. Several local, minimally invasive hepatic therapies [percutaneous ethanol injection (PEI), acetic acid injection, microwave coagulation therapy, and radiofrequency ablation (RFA)] have been developed to prolong survival in unresectable HCC patients over the past few decades [7-13]. Especially, RFA is currently performed widely due to its ease of use, safety and effectiveness for managing HCC in patients with cirrhosis [14-17], while its high repeatability makes it particularly valuable for controlling intrahepatic recurrences [18]. Monitoring tumor response to therapy is an important part of the clinical management of cancer patients, and accurate assessment of tumor response is essential for favorable outcomes. Imaging techniques such as contrastenhanced ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI) are generally used to diagnose HCC or assess therapeutic effects [19-21]. However, these techniques naturally use different principles to generate images, and the type and dose of contrast agents are different. Contrast enhancement is an important finding on imaging; however, enhancement does not necessarily depict the same phenomenon between US, CT and MRI. Therefore, we need to be familiar with the findings on each modality after ablation to evaluate the success of treatment, detect residual or recurrent tumors, and diagnose new lesions. In this review, we focus our discussion on the imaging assessment of HCC response to RFA. CLINICOPATHOLOGICAL FEATURES OF HCC FOR ABLATION HCC cells spread mainly via the portal system and form intrahepatic micrometastases [22,23]. Among risk factors for recurrences, tumor size, portal vein invasion, and intrahepatic metastasis are generally considered the major causes of intrahepatic HCC recurrence after treatment. A previous pathologic study showed that intrahepatic metastasis occurs in 10% of cases even in early HCC (lesions < 2 cm in diameter) [22]. Okusaka et al [24] reported that 19% of HCC nodules of 3.0 cm or less in diameter had satellite lesions that were not detected during pretreatment evaluation. Nakashima et al [25] revealed that 59.1% of small HCC of 3.0 cm in diameter had micrometastases within 5 mm of each micrometastatic lesion and its primary HCC. Especially, among single nodular type HCC, micrometastases were shown in 77.8% within 5 mm. Several studies address the relation between microvascular invasion of HCC cells and tumor size. Kojiro et al [22] reported that the tumor invades the portal vein in 27% of cases even in early HCC (lesions < 2 cm in dimension). Esnaola et al [26] found the frequency of microvascular invasion to be 25% and 31% for tumors < 2 cm and 2-4 cm in the greatest dimension, respectively. On the other hand, microvascular invasion was shown in 17% of patients with tumors < 2 cm and 20% of patients with tumors 2-3 cm [24]. The reported frequency of microvascular invasion in patients with an HCC tumor of 2-3 cm ranges from approximately 20%-30%. Thus, it has reported that the risk factors for early local tumor recurrence were larger tumor size, poor pathologic differentiation of tumor cells and advanced tumor staging [27,28]. PRINCIPLE OF TUMOR RESPONSE ASSESSMENT TO RFA FOR HCC Efficacy of treatment is usually monitored radiologically. Effective treatment is indicated by not only lack of vascular enhancement of HCC, but also the safety margin. The safety margin is ablated peritumoral liver tissue that is located between a necrotic tumor and unablated liver tissue (Figure 1). For the RFA procedure to be considered technically successful, the tumor and at least a 5 mm safety margin must be included in the ablation zone [29]. The local recurrence rate differs markedly depending on whether or not a 5 mm safety margin is secured. Kudo et al [14] reported that the local recurrence rate was 2.6% in HCC patients with a 5 mm safety margin at 2 years after RFA, whereas it was 20.8% in HCC patients without safety margin (P = 0.01). Another report indicated that the significant risk factors for local recurrence of RFA were a tumor with a diameter 2.3 cm, an insufficient safety margin, and a multinodular tumor [30]. In addition, the safety margin has one more important role as security in avoiding limitations on CT assessment due to a partial volume effect. Alpha-fetoprotein (AFP), lens culinaris agglutininreactive fraction of AFP (AFP-L3), and des-γ-carboxy prothrombin (DCP) have been used as tumor markers for HCC [31-34]. Levels of tumor markers often fall to within the normal range after effective treatment and rise before tumor relapse is detected by imaging studies. However, sensitivity and specificity of tumor markers are insufficient to detect HCC in all patient samples, and the monitoring of tumor marker levels after therapy does not replace imaging [35]. Recurrence of tumors in the treated area or elsewhere is defined as re-appearance of vascular enhancement. The ideal imaging interval is unknown, but initially 4161 April 21, 2014 Volume 20 Issue 15

63 Minami Y et al. Imaging assessment of HCC response to RFA Micrometastases Ablation margin Microvascular invasion Figure 1 Ablation margin and micrometastases/microvascular invasion. Radiofrequency ablation (RFA) therapy is required to ablate the main tumor and its surrounding liver tissues involving micrometastases and microvascular invasion. However, as the tumor get bigger, micrometastases and microcascular invasion frequently occur. Unablated lesions lead to local recurrences after RFA. T: Tumor. a 3-4 mo interval is commonly used to monitor HCC lesions after initial treatment. After about 2 years of recurrence-free survival, the interval of follow-up imaging examinations can be at less frequent intervals [35]. Imaging CT: Contrast-enhanced CT has been most widely used for the evaluation of treatment response after RFA because of the advantages of CT: the rapid acquisition of images, clear and specific information, and the referring of a wide range of the abdomen including the liver. After a bolus dispense of contrast agent, tissue contrast enhancement depends on arterial blood flow, capillary permeability, rate of diffusion, and extravascular extracellular space volume. In clinical practice, evaluation of successful treatment was based on a visual comparison of the pre- and post-rfa CT images by referring specific landmarks such as hepatic vessels and the liver surface [14,36,37]. If the non-enhancing ablation zone included the original tumor and an adequate safety margin in all directions, the RFA should be regarded as technically successful [38]. Sala et al [39] revealed that the independent predictors of survival were Child-Pugh class (P = ) and initial complete response to percutaneous ablation (P = 0.006). Among patients classified as Child-Pugh grade A, a 20% difference of survival rate was achieved at 5 years (42% in responders vs 18% in non-responders), while among patients classified as Child-Pugh grade B, the difference of survival rate was observed at 3 years (42% in responders vs 16% in non-responders). Compared with RFA alone, the combination of RFA and transcatheter arterial chemoembolization (TACE) markedly increased the extent of induced coagulation of RFA [40]. Combined TACE and RFA have several advantages over RFA treatment alone. Theoretically, embolization along with the chemotherapy is synergic to thermal ablation by lowering the convection by vascular flow, decreasing the impedance in the tumor and facilitating heat distribution within the tumor. Moreover, satellite nodules, which are found more commonly around large HCCs can be depicted by Lipiodol spots. Thus, RFA combined with TACE has been reported to be promising for local control of medium-size of tumors [41-44]. In addition, Lipiodol-TACE could offer another advantage for assessment of following RFA procedure. When the ablated area could cover the whole HCC with a sufficient margin, the ablative margin can be shown as the boundary between the high density area as Lipiodol accumulation in HCC and the enhancing area as surrounding normal liver parenchyma. As the area of Lipiodol deposition is an ideal landmark of the tumor margin, the successful safety margin can be easily evaluated only by post-rfa dynamic CT images in HCC patients treated by RFA combined with TACE, without a comparison of the pre- and post-rfa CT images (Figure 2) [45]. Hyperemia in tissue surrounding the ablated lesion can represent an inflammatory reaction due to thermal injury. Peripheral rim enhancement resulting from reactive hyperemia is usually uniform in thickness and envelops the ablated lesion (Figure 2), whereas residual HCC demonstrates focal and irregular peripheral enhancement [46,47]. However, differentiation of reactive hyperemia from residual HCCs is sometimes difficult. Moreover, typical imaging features (arterial enhancement followed by delayed washout on dynamic contrast-enhanced CT) are not usually depicted for the diagnosis of recurrent HCCs. Mikami et al [48] reported that 17.5% of patients were diagnosed as local recurrent HCC with typical enhancement pattern, while 40.6% had arterial hypervascularity without washout in the portal venous phase and 11.9% showed washout in portal venous phase without arterial hypervascularity. A non-typical enhancement pattern of local HCC recurrence may reflect the fact that insufficient RFA therapy could lead to further malignant 4162 April 21, 2014 Volume 20 Issue 15

64 Minami Y et al. Imaging assessment of HCC response to RFA Figure 2 A 80-year-old woman with 2.5 cm hepatocellular carcinoma after radiofrequency ablation combined with transcatheter arterial chemoembolization. Early-phase dynamic computed tomography shows a high-density center indicating Lipiodol deposition in hepatocellular carcinoma (white arrow) and a surrounding low-density zone indicating radiofrequency ablation-induced coagulation necrosis of the liver. A microsatellite (black arrow) was depicted as a high-density spot in the low-density zone. Therefore, this ablation therapy achieved complete necrosis of chief tumor and micrometastasis. Moreover, hyperemia surrounding the ablated lesion is depicted as peripheral rim enhancement (arrowheads). transformation of HCC [49]. Therefore, careful comparison with imaging before ablation and close follow-up are necessary in in patients who showed unusual pattern of enhancement in the liver after RFA. US: US contrast agents consist of gas-cored microbubbles that are encapsulated by a shell constructed of a lipid monolayer or cross-linked albumin. Each bubble acts as a harmonic oscillator and contrast-enhanced echo signals contain significant energy components at higher harmonics, while tissue echoes do not. With the use of a contrast agent, contrast harmonic imaging possesses not only a very high sensitivity to contrast agents but also a high spatial resolution, and can depict signals from microbubbles with a very slow flow. Several researchers have reported that contrast-enhanced US is a useful tool for assessing the vascularity of local recurrence of HCCs [50-54]. The detectability of viable HCCs was 83.5% in B-mode US and increased to 93.2% in contrast-enhanced US, using contrast-enhanced CT was used as the reference standard [55]. As reported by Kim et al [56], the diagnostic concordance between the contrast-enhanced US just after the RFA and the CT after the 1-mo follow-up was 99% in terms of the assessment of the therapeutic response to RFA. The sensitivity, specificity, and diagnostic accuracy of contrast-enhanced US were 95.3%, 100%, and 98.1%, respectively [57]. Consequently, contrast-enhanced US may provide an alternative approach that shows high diagnostic concordance with dynamic CT in assessing the therapeutic response of RFA in hypervascular HCC (Figure 3). However, it is often difficult to identify the safety margin on US in the some cases. Zhou et al [58] found that contrast-enhanced US could not evaluate safety margin in 34.8% of HCC nodules because the tumor boundary could not be identified clearly by US after RFA. Therefore, contrast-enhanced US and contrast-enhanced CT should carry a complementary role for the evaluation of the treatment response after RFA. Perfluorocarbon microbubbles (Sonazoid) is classified as second-generation US-contrast agents. Unlike others, perfluorocarbon microbubbles are not trapped by Kupffer cells. A double contrast US technique using Sonazoid reinjection has been developed on the basis of two specific characteristics of Sonazoid: real-time blood flow images with low acoustic power and robust Kupffer images tolerable for repeated scanning in the Kupffer phase [59-61]. According to contrast-enhanced US using Sonazoid, peripheral hyperemia areas show hyper-echogenicity during the early vascular phase and iso-echogenicity as adjacent liver parenchyma during the Kupffer phase. On the other hand, residual HCC demonstrates a focal defect during the Kupffer phase and represents hypervascular enhancement by the reinjection of Sonazoid. Therefore, differentiation of reactive hyperemia from residual HCCs is not difficult. Dynamic contrast-enhanced US guidance in ablation therapy for locally recurrent HCCs should be an efficient approach [61]. MRI: MRI provides better contrast between the different soft tissues and higher spatial resolution with sensitivity than CT. Recent advances in MRI allow imaging of the liver with a high spatial resolution during a single breathhold. Khankan et al [62] reported that a hyperintense zone on non-enhanced T1-weighted MRI within 2 d after RFA reflected the extent of the ablated region. Evaluation of the safety margin also needs comparisons of the pre- and post-rfa images because of the blurriness of tumor boundary on non-enhanced MRI after RFA. Hepatocyte-specific MRI contrast agents were developed for detection and characterization of focal liver lesions. Gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acid (Gd-EOB-DTPA) is a contrast agent with combined properties of a conventional non-specific extracellular and hepatocyte-specific contrast agent [63]. It is recognized that hepatocyte phase images help to distinguish vascular pseudolesions (e.g., those due to arterioportal shunting, portal venule obstruction, nonportal splanchnic veins, or rib compression) from hypervascular tumors [64]. Meanwhile, a recent study [65] reported that more than 10% of vascular pseudolesions showed hypointensity on hepatocyte-phase images and that those pseudolesions occasionally mimicked the configurations and signal intensities of HCCs. Watanabe et al [66] analyzed the image of HCC tumors using the area under the receiver operating characteristic (ROC) curve, and concluded that the incorporation of hepatocyte phase images did not improve the diagnostic accuracy of Gd-EOB- DTPA-enhanced MRI for locally recurrent HCCs after RFA. On the other hand, Koda et al [67] reported the ablative margin grading assessment using superparamagnetic iron oxide (SPIO)-enhanced MRI. They intravenously injected ferucarbotran (0.016 ml/kg body weight) min before RFA, and performed MRI at 7 d after RFA. Because SPIO remained in ablated hepatic parenchyma, 4163 April 21, 2014 Volume 20 Issue 15

65 Minami Y et al. Imaging assessment of HCC response to RFA A B Figure 3 A 70-year-old man with 1.5 cm hepatocellular carcinoma after radiofrequency ablation. A: The ablated tumor is depicted as hyper echoic lesion (circle) on B-mode ultrasound (US). However, the boundary between ablated area and unablated liver tissue could not be identified clearly; B: Contrast-enhanced US using Sonazoid shows the defect (arrows) in the Kupffer phase. The ablation margin is shown as low echoic zone surrounding the ablated tumor. post-ablation MRI showed a high-intensity area of HCC surrounding by low-intensity area of ablative margin. CONCLUSION The prognosis of patients with small HCC is still unsatisfactory because of frequent recurrence even after complete ablation. The high recurrence rate may be attributed to the undefined satellite lesions or microvascular invasion before treatment, which are too small to be detected with the current imaging modality. For the procedure of local ablation therapies including RFA, we need to ablate wider range of region than targeted tumor, including surrounding non-tumorous liver tissues that could involve micrometastases and microvascular invasion. The local recurrence rate tends to be lower in HCC patients with an adequate ablation margin, and thus, it is essential to assess safety margin accurately to reduce local recurrence. From this point of view, we need to focus on the achievement of a sufficient ablation margin as well the lack of tumor vascular enhancement for the assessment of successful RFA. However, inflammatory hyperemia due to RFA which often appears as peripheral rim enhancement, and non-typical imaging features of tumor recurrence sometimes lead to the inappropriate diagnosis. Therefore, we need to be careful for the imaging findings given the fact that the diagnostic difficulties for local recurrence of HCC. 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Recurrent hepatocellular carcinoma treated with sequential transcatheter arterial chemoembolization and RF ablation versus RF ablation alone: a prospective randomized trial. Radiology 2012; 262: [PMID: DOI: / radiol ] 43 Lu Z, Wen F, Guo Q, Liang H, Mao X, Sun H. Radiofrequency ablation plus chemoembolization versus radiofrequency ablation alone for hepatocellular carcinoma: a meta-analysis of randomized-controlled trials. Eur J Gastroenterol Hepatol 2013; 25: [PMID: DOI: /MEG.0b013e32835a0a07] 44 Peng ZW, Zhang YJ, Chen MS, Xu L, Liang HH, Lin XJ, 4165 April 21, 2014 Volume 20 Issue 15

67 Minami Y et al. Imaging assessment of HCC response to RFA Guo RP, Zhang YQ, Lau WY. Radiofrequency ablation with or without transcatheter arterial chemoembolization in the treatment of hepatocellular carcinoma: a prospective randomized trial. J Clin Oncol 2013; 31: [PMID: DOI: /JCO ] 45 Nishikawa H, Inuzuka T, Takeda H, Nakajima J, Sakamoto A, Henmi S, Matsuda F, Eso Y, Ishikawa T, Saito S, Kita R, Kimura T, Osaki Y. Percutaneous radiofrequency ablation therapy for hepatocellular carcinoma: a proposed new grading system for the ablative margin and prediction of local tumor progression and its validation. J Gastroenterol 2011; 46: [PMID: DOI: /s ] 46 Limanond P, Zimmerman P, Raman SS, Kadell BM, Lu DS. Interpretation of CT and MRI after radiofrequency ablation of hepatic malignancies. AJR Am J Roentgenol 2003; 181: [PMID: DOI: /ajr ] 47 Kim SK, Lim HK, Kim YH, Lee WJ, Lee SJ, Kim SH, Lim JH, Kim SA. 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Eur J Radiol 2005; 56: [PMID: DOI: /j.ejrad ] 57 Wen YL, Kudo M, Zheng RQ, Minami Y, Chung H, Suetomi Y, Onda H, Kitano M, Kawasaki T, Maekawa K. Radiofrequency ablation of hepatocellular carcinoma: therapeutic response using contrast-enhanced coded phase-inversion harmonic sonography. AJR Am J Roentgenol 2003; 181: [PMID: DOI: /ajr ] 58 Zhou P, Kudo M, Minami Y, Chung H, Inoue T, Fukunaga T, Maekawa K. What is the best time to evaluate treatment response after radiofrequency ablation of hepatocellular carcinoma using contrast-enhanced sonography? Oncology 2007; 72 Suppl 1: [PMID: DOI: / ] 59 Kudo M. New sonographic techniques for the diagnosis and treatment of hepatocellular carcinoma. Hepatol Res 2007; 37 Suppl 2: S193-S199 [PMID: DOI: /j X x] 60 Kudo M, Hatanaka K, Maekawa K. Newly developed novel ultrasound technique, defect reperfusion ultrasound imaging, using sonazoid in the management of hepatocellular carcinoma. Oncology 2010; 78 Suppl 1: [PMID: DOI: / ] 61 Kudo M, Hatanaka K, Kumada T, Toyoda H, Tada T. Doublecontrast ultrasound: a novel surveillance tool for hepatocellular carcinoma. Am J Gastroenterol 2011; 106: [PMID: DOI: /ajg ] 62 Khankan AA, Murakami T, Onishi H, Matsushita M, Iannaccone R, Aoki Y, Tono T, Kim T, Hori M, Osuga K, Passariello R, Nakamura H. Hepatocellular carcinoma treated with radio frequency ablation: an early evaluation with magnetic resonance imaging. J Magn Reson Imaging 2008; 27: [PMID: DOI: /jmri.21050] 63 Campos JT, Sirlin CB, Choi JY. Focal hepatic lesions in Gd- EOB-DTPA enhanced MRI: the atlas. Insights Imaging 2012; 3: [PMID: DOI: /s ] 64 Yoon JH, Lee EJ, Cha SS, Han SS, Choi SJ, Juhn JR, Kim MH, Lee YJ, Park SJ. Comparison of gadoxetic acid-enhanced MR imaging versus four-phase multi-detector row computed tomography in assessing tumor regression after radiofrequency ablation in subjects with hepatocellular carcinomas. J Vasc Interv Radiol 2010; 21: [PMID: DOI: / j.jvir ] 65 Motosugi U, Ichikawa T, Sou H, Sano K, Tominaga L, Muhi A, Araki T. Distinguishing hypervascular pseudolesions of the liver from hypervascular hepatocellular carcinomas with gadoxetic acid-enhanced MR imaging. Radiology 2010; 256: [PMID: DOI: /radiol ] 66 Watanabe H, Kanematsu M, Goshima S, Yoshida M, Kawada H, Kondo H, Moriyama N. Is gadoxetate disodium-enhanced MRI useful for detecting local recurrence of hepatocellular carcinoma after radiofrequency ablation therapy? AJR Am J Roentgenol 2012; 198: [PMID: DOI: / AJR ] 67 Koda M, Tokunaga S, Miyoshi K, Kishina M, Fujise Y, Kato J, Matono T, Murawaki Y, Kakite S, Yamashita E. Ablative margin states by magnetic resonance imaging with ferucarbotran in radiofrequency ablation for hepatocellular carcinoma can predict local tumor progression. J Gastroenterol 2013; 48: [PMID: DOI: /s ] P- Reviewers: Beierle EA, Nagai H, Tanaka K S- Editor: Wen LL L- Editor: A E- Editor: Liu XM 4166 April 21, 2014 Volume 20 Issue 15

68 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (5): Colorectal cancer TOPIC HIGHLIGHT Genetic variations in colorectal cancer risk and clinical outcome Kejin Zhang, Jesse Civan, Sushmita Mukherjee, Fenil Patel, Hushan Yang Kejin Zhang, Sushmita Mukherjee, Fenil Patel, Hushan Yang, Division of Population Science, Department of Medical Oncology, Kimmel Cancer Center, Thomas Jefferson University, Philadelphia, PA 19107, United States Kejin Zhang, College of Life Science, Northwest University, Xi'an , Shaanxi Province, China Jesse Civan, Division of Gastroenterology and Hepatology, Department of Medicine, Thomas Jefferson University, Philadelphia, PA 19107, United States Author contributions: All the authors were involved in literature review and manuscript writing. Supported by A start-up grant from Thomas Jefferson University; and National Cancer Institute Grant, CA Correspondence to: Hushan Yang, PhD, Division of Population Science, Department of Medical Oncology, Kimmel Cancer Center, Thomas Jefferson University, 111 S 11 th St, Philadelphia, PA 19107, United States. hushan.yang@jefferson.edu Telephone: Fax: Received: September 28, 2013 Revised: January 8, 2014 Accepted: March 6, 2014 Published online: April 21, 2014 Abstract Colorectal cancer (CRC) has an apparent hereditary component, as evidenced by the well-characterized genetic syndromes and family history associated with the increased risk of this disease. However, in a large fraction of CRC cases, no known genetic syndrome or family history can be identified, suggesting the presence of missing heritability in CRC etiology. The genome-wide association study (GWAS) platform has led to the identification of multiple replicable common genetic variants associated with CRC risk. These newly discovered genetic variations might account for a portion of the missing heritability. Here, we summarize the recent GWASs related to newly identified genetic variants associated with CRC risk and clinical outcome. The findings from these studies suggest that there is a lack of understanding of the mechanism of many single nucleotide polymorphisms (SNPs) that are associated with CRC. In addition, the utility of SNPs as prognostic markers of CRC in clinical settings remains to be further assessed. Finally, the currently validated SNPs explain only a small fraction of total heritability in complex-trait diseases like CRC. Thus, the missing heritability still needs to be explored further. Future epidemiological and functional investigations of these variants will add to our understanding of CRC pathogenesis, and may ultimately lead to individualized strategies for prevention and treatment of CRC Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Colorectal cancer; Genome-wide association study; Single nucleotide polymorphism; Signal transduction pathways; Cell cycle control; Gene desert; Genome instability Core tip: This review covers the recent advances in genome-wide association studies (GWASs) that have identified genetic variants associated with an altered risk of colorectal cancer (CRC). In this review, we summarize single nucleotide polymorphisms (SNPs) located in or near genes that play crucial roles in signal transduction pathways, genome stability, cell cycle control, and gene expression and regulation. SNPs that are found in gene desert regions are also discussed. The relationship between genetic variations and clinical outcomes in CRC is presented from epidemiological studies that have identified SNPs with methods other than GWASs. Zhang K, Civan J, Mukherjee S, Patel F, Yang H. Genetic variations in colorectal cancer risk and clinical outcome. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: April 21, 2014 Volume 20 Issue 15

69 Zhang K et al. GWAS-identified CRC SNPs INTRODUCTION It is estimated that 35% of colorectal cancer (CRC) risk may be explained by heritable factors [1]. Heritable factors include well-characterized genetic syndromes inherited in a straightforward Mendelian manner, such as familial adenomatous polyposis and hereditary non-polyposis colorectal cancer, also known as Lynch Syndrome [2]. It is estimated that cumulatively, these and other wellcharacterized genetic syndromes with Mendelian mode of inheritance account for up to 10% of all CRC cases. In an estimated further 25% of cases, family history contributes to CRC risk in the absence of one of these identifiable genetic syndromes. The important role of family history in CRC risk is reflected in the guidelines published by the American College of Gastroenterology and the American Cancer Society, which recommend starting screening colonoscopies at an age cutoff that is a function of family history [3]. The combined effect of genetic syndromes and family history may explain up to 30% of CRC susceptibility, whereas the remaining genetic risk of CRC may be accounted for by a combination of high-prevalence and low-penetrance of common genetic variants. Recent advances using genome-wide association study (GWAS) have enabled the identification of multiple CRC-related single nucleotide polymorphisms (SNPs) [4-10]. These genetic variants can be broadly classified into two categories: those that affect the risk of developing CRC, and those that influence the clinical course of CRC once established. In this review, we summarize these GWASidentified genetic variants - including functional characterizations and implications for clinical applications - and discuss some of the limitations and challenges of these studies. GENETIC VARIANTS AND CRC RISK By comparing the distributions of millions of tagged SNPs between CRC patients (cases) and cancer-free populations (controls), a large number of common genetic variants have been identified under the common disease-common variant premise. To date, more than 40 chromosome regions harboring common variants conferring altered CRC risk have been identified by the GWAS approach. These variants are dispersed amongst almost every human chromosome and the vast majority of them exhibit a small effect size (Table 1). Most of these loci confer a modest increase in CRC risk, typically with an OR of less than Among the 48 SNPs listed in Table 1, eight had an OR of more than 1.20, of which only three exhibited an OR of more than A higher effect size (OR = 2.64) was reported for rs located upstream of the CSNK2A1 gene and validated in familial CRC populations, although only under a recessive genetic model with least statistical power [11]. The majority of GWAS-identified CRC risk variants are involved in known biological pathways; however, a few highly significant ones reside in gene desert regions, and the mechanism by which these variants contribute to colorectal carcinogenesis remains unclear. Here, we summarize these variants in relation to their implications in pathways of signal transduction, genome instability, cell cycle control, and gene expression and regulation (Table 1). These pathways and related significant SNPs identified by GWASs are also depicted in Figure 1. This figure was produced by combining pathways from various studies [12-15]. Genetic variants in signal transduction pathways CRC GWASs have identified significant variants in signal transduction pathways such as those mediated by WNT/ β-catenin, transforming growth factor (TGF)-β/bone morphogenetic protein (BMP), and mitogen-activated protein kinase (MAPK). Somatic mutations in the WNT/ β-catenin signaling pathway were discovered in more than 95% of CRC patient tissues [16], suggesting abnormalities of genes in this pathway may play an important role in colorectal carcinogenesis. The risk allele rs59336 located in the intron of TBX3 gene, a downstream target of WNT/β-catenin pathway, has been associated with a significantly higher risk of developing CRC [8]. Changes in β-catenin and SMAD7 expression can influence WNT/ β-catenin pathway signaling [17]. Moreover, perturbation of SMAD7 expression has been documented to affect CRC progression [18]. Three genetic variants of SMAD7 in chromosome 8q21 - rs , rs and rs confer an increased CRC risk [5]. These findings and other WNT/β-catenin variants were further independently identified and validated [6,19]. BMPs are closely related to signal transductions mediated by TGF-β. Two independent GWASs [9,20] identified 14 CRC risk loci, of which three were adjacent to genes involved in BMPmediated signaling transduction, including rs on BMP4, rs on BMP2, and rs on DNA family BMP antagonist GREM1. BMP-related variants were further confirmed in another independent CRC population [21]. The MAPK-mediated signaling pathway is known to be crucial for several cellular mechanisms such as cell proliferation, survival, and resistance to apoptosis. A GWAS using German familial CRC patients [9] observed that CRC risk increases significantly with an increase in the number of risk alleles in seven genes involved in MAPK signaling. The molecular basis of these observed associations remains undetermined. Genetic variants related to genome instability Genome instability is known to be both a contributor to, as well as a consequence of, colorectal carcinogenesis. There are several major genomic instability-related mechanisms in colorectal carcinogenesis, such as chromosomal instability, microsatellite instability, and CpG island methylator phenotypes [22]. Several loci involving these mechanisms were identified recently by GWASs. For example, Peters et al [10] identified rs , a significant SNP in an intergenic locus on chromosome 2q32.3, proximal to NABP1, which encodes human single-stranded DNA binding protein 2 and plays a role in a diverse array of 4168 April 21, 2014 Volume 20 Issue 15

70 Zhang K et al. GWAS-identified CRC SNPs Table 1 Genome-wide association study-identified common genetic variants associated with colorectal cancer risk SNP Loci Gene Full name of gene OR P value Pathway/function Ref. Method Common biological pathway-related rs p14.1 GLI3 and INHBA GLI family zinc finger 3 and inhibin, beta A E-05 MAPK signaling pathways [11] G rs p12.1 MYO3A Myosin ⅢA E-03 MAPK signaling pathways [11] G rs q24.21 TBX3 T-box E-06 Wnt pathway [10] G + M rs q22.2 BMP4 Bone morphogenetic E-11 BMP pathway [21] G protein 4 rs q22.2 BMP4 Bone morphogenetic E-09 BMP pathway [21] G protein 4 rs q13.3 GREM1 DAN family BMP E-08 BMP pathway [21] G antagonist rs q13.3 GREM1 DAN family BMP E-03 BMP pathway [21] G antagonist rs q13.3 GREM1 DAN family BMP E-10 BMP pathway [21] G antagonist rs q21 SMAD7 SMAD family member E-28 TGF-β1 pathway, cell arrest, [5,6,19] G cell proliferation rs q21 SMAD7 SMAD family member E-12 TGF-β and Wnt signaling [5] G rs q21 SMAD7 SMAD family member E-08 TGF-β and Wnt signaling [5] G rs p12.3 BMP2 Bone morphogenetic E-16 BMP pathway [21] G protein 2 rs p12.3 BMP2 Bone morphogenetic E-11 BMP pathway [21] G protein 2 rs p13 CSNK2A1 Casein kinase 2, alpha 1 polypeptide E-04 MAPK signaling pathways [11] G Genome instability-related rs q32.3 NABP1 Nucleic acid binding protein 1 rs q31.1 PITX1 Paired-like homeodomain transcription factor 1 rs p21 CDKN1A Cyclin-dependent kinase inhibitor 1A rs q13.4 POLD3 Polymerase DNA- directed δ3 rs p13 TP53 Promotor region of TP53 gene E-08 DNA repair, genomic stability [10] G + M E-10 RAS pathway; activate TP53; [29] G + M telomerase activity E-10 Microsatellite instability, [26] G + M DNA repair, genomic instability E-10 DNA mismatch and baseexcision [26] G + M repair E-04 TP53 [63] G Cell cycle control-related rs q25.3 LAMC1 Laminin gamma E-08 Gene transcription [10] G + M rs q41 DUSP10 Dual-specificity E-10 Inactivates p38 and SAPK [9] M phosphatase rs q41 DUSP10 Dual-specificity E-09 Inactivates p38 and SAPK [9] M phosphatase rs q31 LAMB1 Laminin β E-08 Anchoring the singlelayered epithelium, ulcerative colitis [33] G rs q23 POU2AF1 POU class 2 associating factor E-10 Growth of multiple myeloma cells [6] G rs p13.32 CCND2 Cyclin D E-08 Cell-cycle transition [29] G + M rs p13.32 CCND2 Cyclin D E-07 Cell-cycle transition [10] G + M rs p13.32 CCND2 Cyclin D2 1.1 < 5.0E-7 Cell-cycle transition [10] G + M rs q13.13 DIP2 Disco-interacting protein E-10 Cell morphogenesis [9] M 2B rs q22 CDH1 E-cadherin, E-08 Epithelial restitution, repair [33] G following mucosal damage, active colitis rs q13.33 RHPN2 Rho GTPase binding E-09 Actin cytoskeleton [20] G + M protein 2 rs q13.33 LAMA5 Large laminin A E-10 BMP pathway [9] M rs Xp22.2 SHR00M2 Shroom family member E-10 Cell morphogenesis [26] G + M Gene expression and regulation-related rs q23.3 EIF3H Eukaryotic translation E-18 Translation initiation [8] G initiation factor 3, subunit H rs q24 POU5FIP1 POU class 5 homeobox 1B E-26 Weak transcriptional activator [6] G 4169 April 21, 2014 Volume 20 Issue 15

71 Zhang K et al. GWAS-identified CRC SNPs rs q13.13 ATF1 Activating transcription E-08 Transcription [9] M factor 1 rs q13.12 HNF4A Transcription factor hepatocyte nuclear factor 4α E-17 Transcription [33] G Gene desert and others rs p E-07 - [33] G rs q31 Clorf E-08 - [11] G rs q12 PLGLA Plasminogen-like A, noncoding E-07 - [11] G RNA rs q26.2 MYNN Myoneurn gene E-08 Unknown [9] M rs p15.3 NCAPC Non-SMC E-07 - [11] G condensing Ⅰ complex, subunit G rs q26-27 SLC22A3 organic cation transporter E-09 Transport of cationic drugs, [19] G toxins, and endogenous metabolism rs q E-08 - [19] G rs q E-08 - [19] G rs q21.3 TLE4 Transducin-like enhancer E-08 - [11] G of spit 4 rs p E-13 - [8] G rs q E-07 - [33] G rs p12.3 PLCB1 Phospholipase C-beta E-09 Unknown [29] G + M SNP: Single nucleotide polymorphisms; G: Genome-wide association study; M: Meta-analysis; G + M: Combination of GWAS and meta-analysis; BMP: Bone morphogenetic protein; MAPK: Mitogen-activated protein kinase; TGF-β: Transforming growth factor; GWAS: Genome-wide association study. Genetic variants related to gene expression and regulation Thousands of transcription factors, cofactors, and chromatin regulators establish gene expression patterns and maintain specific cell stages in humans. Barrett et al [33] identified a significant association between CRC risk and SNP rs which maps to a recombination hot spot on chromosome 20q13 containing the 3 -untranslated region of the HNF4A gene. HNF4A encodes the transcription factor hepatocyte nuclear factor 4α, which regucellular processes such as DNA replication, recombination, transcription, and maintenance of genomic stability [23-25]. Another variant, rs , is in linkage disequilibrium with a region that encompasses the CDKN1A gene [26], which encodes the p21 protein that mediates p53-dependent growth arrest, and affects multiple tumor suppressor pathways. The p21 protein also interferes with the activity of proliferating cell nuclear antigen (PCNA)-dependent DNA polymerase, thereby regulating DNA replication and repair. It has been demonstrated that down-regulation of p21 inversely correlates with microsatellite instability status [27,28]. Two additional CRC risk variants - rs and rs could also potentially interact with p21 [26,29]. Other genome instability related SNPs include rs248999, located in an intron of the POLD3 gene which encodes a component of the DNA polymerase-δ complex of PCNA, and rs in a putative tumor suppressor homeodomain 1 gene PITX1, which has been reported to encode a protein that activates p53 protein and maintains genome stability [30,31]. Genetic variants related to cell cycle control Genetic pathways mediating cell-cycle control are commonly implicated in colorectal carcinogenesis. Polymorphisms of several cell cycle-related genes have been reported to be associated with CRC risk in recent GWASs, including two independent SNPs (rs and rs ) located in the introns of CCND2. Jia et al [29] identified another SNP, rs , located in 12q13.32, proximal to CCND2 in Asian populations. CCND2 encodes cyclin D2, a member of the D-type cyclin family which plays a critical role in cell cycle control, specifically at the G1/S boundary by activating cyclin-dependent kinases (CDKs), primarily CDK4 and CDK6 [32]. Two significant SNPs, rs and rs , lie about 275 kb apart within a large poorly-defined haplotype block covering the DIP2 gene, which encodes a protein with putative role in epithelial cell fate determination [9]. Another SNP, rs , is proximal to the promoter of encoding laminin gamma 1 (LAMC1) and confers a significantly increased CRC risk by virtue of influencing LAMC1 gene expression [10]. SNPs in two additional laminin genes (laminin beta1 in 7q31 and laminin alpha 5 in 20q13) were also identified in recent CRC GWASs [9,21,33]. Laminins are known to be involved in a variety of cellular mechanisms such as regulation of cell adhesion, differentiation, and migration [34,35]. Another important cell cycle-related SNP was reported by Dunlop et al [26] using five GWAS datasets. This SNP, rs , is on chromosome Xp22.2 and proximal to encoding shroom family member 2, a human homolog of the Xenopus laevis APX gene that is known to have broad functions in cell morphogenesis during endothelial and epithelial tissue development [36]. Missense mutations in this gene have been detected in a large-scale screening for recurrent mutations in cancer cell lines [37]. The relationship between Xp22.2 and CRC risk represents the first evidence for the role of X-chromosome variation in the predisposition to a nonsex-specific cancer April 21, 2014 Volume 20 Issue 15

72 Zhang K et al. GWAS-identified CRC SNPs WNT/β-Catenin Genomic instability Cell cycle (G2/M) TGF-β/BMP WIF WIF DKKs WNT CIN APC TP53 SMAD4 ATM/ATR p53 MDM4 ChR2 TGF-β ligands TGF-β Activins Nodals BMP2 BMP4 BMP7 BMP ligands Biomarkers Frizzled Dvl APC AXIN1 GSK3B CSNK1A1 β-catenin TCF Destruction box LRP Target genes MSI CIMP NABP1 CDKN1A MLH1 MSH2 MSH6 MINT1 MINT2 MINT3 PITX1 POLD3 p21 GADD45... BRCA1 cdc25 cdc2 CDK4 LAMC1 CCND2 SHROOM2 CDK2 G2-PHASE M-PHASE Smad2/3 Smad1/5/8 Smad4 AP-1 bzip RUNX bhlh Smad7 SMIF MSG1 ARC105 GREM1 Progression to carcinoma Normal Early adenoma Late adenoma Adenocarcinoma Figure 1 Major pathways with significant genetic variants implicated in the development of colorectal cancer. Several pathways and related genes involved in the progression of colorectal cancer are illustrated. Genes with significant single nucleotide polymorphisms that are associated with colorectal cancer risk are represented with gray color. TGF-β: Transforming growth factor-β; BMP: Bone morphogenetic protein; CIMP: CpG island methylator phenotype; MSI: Microsatellite instability; CIN: Chromosomal instability. lates the expression of multiple organ development-related genes. In addition, HNF4A has also shown to interact with β-catenin to regulate cell-cell adhesion and gene transcription [38]. Another significant SNP, rs , is close to activating transcription factor 1 (ATF1) [9], which belongs to the ATF subfamily and basic-region leucine zipper family. The protein product of ATF1 influences cellular processes by regulating the expression of many downstream target genes involved in cellular growth and survival. Previous studies have demonstrated that ATF1 protein interacts with EWSR1 to form a unique chimeric fusion protein complex which is important in the development of clear cell sarcoma [39,40] ; however, its role in colorectal carcinogenesis remains to be established. Moreover, ATF1 may also form cyclin-dependent kinase 3-mdiated activating transcription factor 1 complex that is critical in cellular proliferation and malignant transformation [41]. Genetic variants in the gene desert regions and others Although the majority CRC risk variants are related to well-established biological pathways, the functions of some reported loci remain elusive. Various independent studies have reported that multiple SNPs in chromosome region 8q24 are associated with altered risk of several solid tumor malignancies, including CRC. Three SNPs in this region, namely rs , rs , and rs , have been significantly associated with CRC risk in recent GWASs [4,6,19,42]. In addition, variants in the 8q24 region have also been associated with cancers of breast, prostate, ovarian, bladder, pancreas, and brain [6,42-49]. Nonetheless, majority of the significant SNPs identified in this region are not located in, or close to, any well annotated genes because the 8q24 region is largely a gene desert. Therefore, details of the molecular mechanisms underlying the observed effect of these SNPs remain largely unknown. It has been speculated that these SNPs may function through their long-range linkage with causal variants within other oncogenes or tumor suppressor genes. Others have conjectured that some SNPs may influence gene expression through long-range cis-regulatory elements. Wasserman et al [50] used an in vivo bacterial artificial chromosome enhancer-trapping strategy to scan the 8q24 gene desert region and found that a highly significant CRC risk variant, rs , resides within an in vivo prostate enhancer whose expression mimics that of the nearby MYC oncogene [51]. Another discovery illustrated a gene encoding a novel non-coding RNA, CCAT2, also mapped to the 8q24 gene desert region. Encompassing the rs SNP, this long non-coding RNA transcript is highly overexpressed in microsatellite-stable CRC, promoting tumor growth, metastasis, and chromosomal instability [52]. Another 8q24 locus, rs , in high linkage disequilibrium with rs , resides within 3 kb upstream 4171 April 21, 2014 Volume 20 Issue 15

73 Zhang K et al. GWAS-identified CRC SNPs Table 2 Common genetic variants associated with colorectal cancer clinical outcome Genes/loci SNP 1 Patient population Clinical outcome HR (95%CI) P value Ref. MTHFR glu429ala Mixed colorectal cancer (CRC) patients OS 1.71 ( ) [64] ESR2 rs Postmenopausal women with CRC OS 0.77 ( ) [65] SCN1A rs Stage Ⅱ/Ⅲ patients with adjuvant 5-fuorouracil (5-FU) TTR 2.26 ( ) [66] based chemotherapy SMAD7 rs Mixed CRC patients OS 1.16 ( ) [55] mir608 rs Stage Ⅲ patients with 5-FU based chemotherapy RE 1.65 ( ) 0.01 [67] rs OS 1.96 ( ) q13.3 rs10318 Stage Ⅱ patients with 5-FU based adjuvant chemotherapy ER 2.98 ( ) [56] 11q23.1 rs Stage Ⅲ patients with 5-FU based adjuvant chemotherapy ER 0.46 ( ) p12.3 rs ER 0.46 ( ) OS 0.24 ( ) p12.3 rs ER 0.48 ( ) OS 0.29 ( ) q21.1 rs OS 4.34 ( ) q24.21 rs OS 4.20 ( ) rs OS 4.20 ( ) q13 rs Chinese CRC patients OS 0.33 ( ) [57] 10p14 rs RE 0.55 ( ) 0.05 pre-mi-423 rs Mixed CRC patients OS 2.12 ( ) [68] rs RFS 1.59 ( ) pre-mi-608 rs RFS 0.61 ( ) CLOCK rs Resected CRC patients OS 0.55 ( ) [69] rs OS 0.31 ( ) 0.03 SCD rs7849 Stage Ⅱ patients with 5-FU based adjuvant chemotherapy RE 2.89 ( ) [70] VEGF Stage Ⅱ TTR 2.01 ( ) 0.02 [71] -460 TTR 0.50 ( ) 0.02 KDR rs Resected CRC patients RE 0.53 ( ) [72] CD44 rs8193 Stage Ⅲ and high risk stage Ⅱ patients with 5-FU based TTR 0.51 ( ) [73] ALCAM rs1157 chemotherapy TTR 0.56 ( ) LGR5 rs TTR 0.33 ( ) Only the most significant single nucleotide polymorphism (SNP) was shown. OS: Overall survival; RE: Recurrence; RFS: Recurrence free survival; TTR: Time to recurrence. of POU5F1P1, a pseudogene of POU5F1 that encodes an important stem cell-related protein regulating cellular pluripotency and self-renewal [53]. However, no functional implication of this SNP has been reported and it remains to be assessed whether it influences the development of CRC stem cells, a suspected small portion of cancer cells that are responsible for tumor progression and drug resistance [54]. In all, the identification of the large number of bona-fide risk variants in gene desert regions indicates that candidate-gene and pathway-based strategies may not be adequate to capture and understand the complete spectrum of common risk variants of CRC. Unbiased genome-wide interrogation in adequately powered studies, combined with meta-analysis and functional characterization is more likely to help us understand how common genetic variations play a role in CRC carcinogenesis. GENETIC VARIANTS AND CRC CLINICAL OUTCOME There have been reports of genetic variants associated with the clinical outcome of CRC patients which can be used to categorize patients with different survival patterns or responses to specific treatments. However, the majority of reported outcome-related SNPs are generated from candidate gene or pathway-based studies. As of yet, no GWAS has been reported to examine a direct relationship between genetic variations and CRC clinical outcome. The findings of some of recently published studies are summarized in Table 2. Three recent studies have examined the relationship between GWAS-identified CRC risk variants and the clinical outcome of the disease [55-57]. Based on the data from five GWAS populations of 2611 CRC patients, Phipps et al [55] assessed 16 SNPs and found rs , a SNP in the SMAD7 gene, to be significantly associated with reduced overall survival of patients (HR = 1.16, P = 0.002) and disease-specific survival (HR = 1.17, P = 0.005). Dai et al [56] used a Caucasian population of 285 stage Ⅱ or Ⅲ CRC patients receiving fluorouracil-based chemotherapy to evaluate 26 CRC risk variants derived from 10 GWASidentified chromosome loci. Although no SNP was found to be associated with the survival of all patients, they found that different SNPs might be associated with the clinical outcome of patients in specific stages. In another study of 380 Chinese CRC patients, Xing et al [57] reported two GWAS-identified CRC risk variants - rs on chromosome 15q13 and rs on 10p14 - were associated with reduced risk of both death and recurrence. Moreover, stratified analysis indicated that the beneficial effect of chemotherapy in this patient cohort was evident only in patients with the variant rs , but not 4172 April 21, 2014 Volume 20 Issue 15

74 Zhang K et al. GWAS-identified CRC SNPs in those with the wild-type genotype of this SNP. This indicates that rs may potentially be useful in selecting patients for chemotherapy treatments. Taken together, these findings suggest that genetic variants associated with CRC risk may also predict the clinical outcome of CRC patients. However, these studies are limited by small sample size and heterogeneous patient population and treatments. Therefore, their findings need to be interpreted with caution and warrant further validations. In addition to those GWAS-identified CRC risk loci, other epidemiological studies have also identified genetic variations associated with clinical outcome of CRC (Table 2). All of these studies are based on candidate gene or pathway-based approaches instead of GWAS. This is largely because compared to case-control studies, clinical outcome studies are generally based on cancer patients with highly heterogeneous characteristics and treatments that confound the very modest effect of genetic variants on patient outcomes. This issue could be partly resolved by the use of clinical trial patients that have more homogeneous characteristics and treatments, or consortium studies with much larger number of patients. CONCLUSION Findings from the first wave of GWASs seem to promise greater understanding of the genetic component of CRC pathogenesis on a molecular level. However, there are several major limitations in current GWAS approaches which may also pose challenges for future studies. First, the vast majority of currently identified SNPs lack known functional significance. Thus, whether they are causal variants or just surrogates that are in linkage disequilibrium with the functional loci remains largely unknown. Therefore, a major task ahead is to conduct fine-mapping in the immediate regions surrounding these loci, and narrow down the regions of association to pinpoint the causal variants [58]. Second, although the statistical significance of most GWAS-identified SNPs is high, the utility of these bona-fide variants in a clinical setting to predict the risk of developing cancer remains to be assessed. This is largely due to the modest effect size associated with most of the specific individual variants. Wacholder et al [59] reported a very modest increase in the power to predict breast cancer risk by adding 10 highly significant GWAS-identified breast cancer risk variants to the commonly recognized self-reported risk factors. Moreover, they found that the level of predicted breast cancer risk among most women barely changes by the addition of the GWAS-generated genetic information. Similarly, Park et al [60] reported that the combined use of all current genetic information derived from GWASs only has modest discriminative power (about 63.5% area under curve) in breast, prostate, and colorectal cancers. Therefore, further identification of additional low-penetrance common variants, especially the causal variants, is necessary to improve the clinical utility of GWAS-generated genetic information. Third, it is estimated that the currently validated SNPs in aggregate still explain only a small fraction of total heritability in most complex-trait diseases [61]. Several possible reasons may further account for this missing heritability. These include unidentified common variants, the unexplored territory of rare genetic variants that have high-penetrance but low-prevalence, and the largely un-assessed gene-gene and gene-environment interactions [21,62]. All of these issues could be partially addressed by increasing population size and thus statistical power. 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77 Zhang K et al. GWAS-identified CRC SNPs Genome-wide association scan identifies a colorectal cancer susceptibility locus on chromosome 8q24. Nat Genet 2007; 39: [PMID: ] 43 Turnbull C, Ahmed S, Morrison J, Pernet D, Renwick A, Maranian M, Seal S, Ghoussaini M, Hines S, Healey CS, Hughes D, Warren-Perry M, Tapper W, Eccles D, Evans DG, Hooning M, Schutte M, van den Ouweland A, Houlston R, Ross G, Langford C, Pharoah PD, Stratton MR, Dunning AM, Rahman N, Easton DF. Genome-wide association study identifies five new breast cancer susceptibility loci. 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Leveraging genetic variability across populations for the identification of causal variants. Am J Hum Genet 2010; 86: [PMID: DOI: /j.ajhg ] 59 Wacholder S, Hartge P, Prentice R, Garcia-Closas M, Feigelson HS, Diver WR, Thun MJ, Cox DG, Hankinson SE, Kraft P, Rosner B, Berg CD, Brinton LA, Lissowska J, Sherman ME, Chlebowski R, Kooperberg C, Jackson RD, Buckman DW, Hui P, Pfeiffer R, Jacobs KB, Thomas GD, Hoover RN, Gail MH, Chanock SJ, Hunter DJ. Performance of common genetic variants in breast-cancer risk models. N Engl J Med 2010; 362: [PMID: DOI: /NEJ- Moa ] 60 Park JH, Wacholder S, Gail MH, Peters U, Jacobs KB, Chanock SJ, Chatterjee N. Estimation of effect size distribution from genome-wide association studies and implications for future discoveries. Nat Genet 2010; 42: [PMID: DOI: /ng.610] 61 Visscher PM, Brown MA, McCarthy MI, Yang J. Five years of GWAS discovery. Am J Hum Genet 2012; 90: 7-24 [PMID: DOI: /j.ajhg ] 4176 April 21, 2014 Volume 20 Issue 15

78 Zhang K et al. GWAS-identified CRC SNPs 62 Zuk O, Hechter E, Sunyaev SR, Lander ES. The mystery of missing heritability: Genetic interactions create phantom heritability. Proc Natl Acad Sci USA 2012; 109: [PMID: DOI: /pnas ] 63 Stacey SN, Sulem P, Jonasdottir A, Masson G, Gudmundsson J, Gudbjartsson DF, Magnusson OT, Gudjonsson SA, Sigurgeirsson B, Thorisdottir K, Ragnarsson R, Benediktsdottir KR, Nexø BA, Tjønneland A, Overvad K, Rudnai P, Gurzau E, Koppova K, Hemminki K, Corredera C, Fuentelsaz V, Grasa P, Navarrete S, Fuertes F, García-Prats MD, Sanambrosio E, Panadero A, De Juan A, Garcia A, Rivera F, Planelles D, Soriano V, Requena C, Aben KK, van Rossum MM, Cremers RG, van Oort IM, van Spronsen DJ, Schalken JA, Peters WH, Helfand BT, Donovan JL, Hamdy FC, Badescu D, Codreanu O, Jinga M, Csiki IE, Constantinescu V, Badea P, Mates IN, Dinu DE, Constantin A, Mates D, Kristjansdottir S, Agnarsson BA, Jonsson E, Barkardottir RB, Einarsson GV, Sigurdsson F, Moller PH, Stefansson T, Valdimarsson T, Johannsson OT, Sigurdsson H, Jonsson T, Jonasson JG, Tryggvadottir L, Rice T, Hansen HM, Xiao Y, Lachance DH, O Neill BP, Kosel ML, Decker PA, Thorleifsson G, Johannsdottir H, Helgadottir HT, Sigurdsson A, Steinthorsdottir V, Lindblom A, Sandler RS, Keku TO, Banasik K, Jørgensen T, Witte DR, Hansen T, Pedersen O, Jinga V, Neal DE, Catalona WJ, Wrensch M, Wiencke J, Jenkins RB, Nagore E, Vogel U, Kiemeney LA, Kumar R, Mayordomo JI, Olafsson JH, Kong A, Thorsteinsdottir U, Rafnar T, Stefansson K. A germline variant in the TP53 polyadenylation signal confers cancer susceptibility. Nat Genet 2011; 43: [PMID: ] 64 Negandhi AA, Hyde A, Dicks E, Pollett W, Younghusband BH, Parfrey P, Green RC, Savas S. MTHFR Glu429Ala and ERCC5 His46His polymorphisms are associated with prognosis in colorectal cancer patients: analysis of two independent cohorts from Newfoundland. PLoS One 2013; 8: e61469 [PMID: ] 65 Passarelli MN, Phipps AI, Potter JD, Makar KW, Coghill AE, Wernli KJ, White E, Chan AT, Hutter CM, Peters U, Newcomb PA. Common single-nucleotide polymorphisms in the estrogen receptor β promoter are associated with colorectal cancer survival in postmenopausal women. Cancer Res 2013; 73: [PMID: ] 66 Benhaim L, Gerger A, Bohanes P, Paez D, Wakatsuki T, Yang D, Labonte MJ, Ning Y, El-Khoueiry R, Loupakis F, Zhang W, Laurent-Puig P, Lenz HJ. Gender-specific profiling in SCN1A polymorphisms and time-to-recurrence in patients with stage II/III colorectal cancer treated with adjuvant 5-fluoruracil chemotherapy. Pharmacogenomics J 2014; 14: [PMID: ] 67 Lin M, Gu J, Eng C, Ellis LM, Hildebrandt MA, Lin J, Huang M, Calin GA, Wang D, Dubois RN, Hawk ET, Wu X. Genetic polymorphisms in MicroRNA-related genes as predictors of clinical outcomes in colorectal adenocarcinoma patients. Clin Cancer Res 2012; 18: [PMID: ] 68 Xing J, Wan S, Zhou F, Qu F, Li B, Myers RE, Fu X, Palazzo JP, He X, Chen Z, Yang H. Genetic polymorphisms in premicrorna genes as prognostic markers of colorectal cancer. Cancer Epidemiol Biomarkers Prev 2012; 21: [PMID: ] 69 Zhou F, He X, Liu H, Zhu Y, Jin T, Chen C, Qu F, Li Y, Bao G, Chen Z, Xing J. Functional polymorphisms of circadian positive feedback regulation genes and clinical outcome of Chinese patients with resected colorectal cancer. Cancer 2012; 118: [PMID: ] 70 Lin M, Eng C, Hawk ET, Huang M, Lin J, Gu J, Ellis LM, Wu X. Identification of polymorphisms in ultraconserved elements associated with clinical outcomes in locally advanced colorectal adenocarcinoma. Cancer 2012; 118: [PMID: ] 71 Kjaer-Frifeldt S, Fredslund R, Lindebjerg J, Hansen TF, Spindler KL, Jakobsen A. Prognostic importance of VEGF-A haplotype combinations in a stage II colon cancer population. Pharmacogenomics 2012; 13: [PMID: ] 72 Dong G, Guo X, Fu X, Wan S, Zhou F, Myers RE, Bao G, Burkart A, Yang H, Xing J. Potentially functional genetic variants in KDR gene as prognostic markers in patients with resected colorectal cancer. Cancer Sci 2012; 103: [PMID: ] 73 Gerger A, Zhang W, Yang D, Bohanes P, Ning Y, Winder T, LaBonte MJ, Wilson PM, Benhaim L, Paez D, El-Khoueiry R, El-Khoueiry A, Kahn M, Lenz HJ. Common cancer stem cell gene variants predict colon cancer recurrence. Clin Cancer Res 2011; 17: [PMID: ] P- Reviewers: Camacho J, Tong WD, Yu B S- Editor: Gou SX L- Editor: A E- Editor: Ma S 4177 April 21, 2014 Volume 20 Issue 15

79 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (5): Colorectal cancer Targeting mtor network in colorectal cancer therapy TOPIC HIGHLIGHT Xiao-Wen Wang, Yan-Jie Zhang Xiao-Wen Wang, Yan-Jie Zhang, Rutgers Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08903, United States Yan-Jie Zhang, Department of Gastroenterology, No. 3 People s Hospital Affiliated to Shanghai Jiaotong University, School of Medicine, Shanghai , China Author contributions: Wang XW and Zhang YJ designed and wrote the review; Zhang YJ took responsibility for revising the manuscript and final approval of the version to be published. Supported by National Nature Science Foundation, No ; and International Cooperation Grant, No Correspondence to: Yan-Jie Zhang, MD, PhD, Rutgers Cancer Institute of New Jersey, 195 Little Albany Street, New Brunswick, NJ 08903, United States. zhang26@cinj.rutgers.edu Telephone: Fax: Received: October 28, 2013 Revised: December 28, 2013 Accepted: January 20, 2014 Published online: April 21, 2014 Abstract The mechanistic target of rapamycin (mtor) integrates growth factor signals with cellular nutrient and energy levels and coordinates cell growth, proliferation and survival. A regulatory network with multiple feedback loops has evolved to ensure the exquisite regulation of cell growth and division. Colorectal cancer is the most intensively studied cancer because of its high incidence and mortality rate. Multiple genetic alterations are involved in colorectal carcinogenesis, including oncogenic Ras activation, phosphatidylinositol 3-kinase pathway hyperactivation, p53 mutation, and dysregulation of wnt pathway. Many oncogenic pathways activate the mtor pathway. mtor has emerged as an effective target for colorectal cancer therapy. In vitro and preclinical studies targeting the mtor pathway for colorectal cancer chemotherapy have provided promising perspectives. However, the overall objective response rates in major solid tumors achieved with single-agent rapalog therapy have been modest, especially in advanced metastatic colorectal cancer. Combination regimens of mtor inhibitor with agents such as cytotoxic chemotherapy, inhibitors of vascular endothelial growth factor, epidermal growth factor receptor and Mitogen-activated protein kinase kinase (MEK) inhibitors are being intensively studied and appear to be promising. Further understanding of the molecular mechanism in mtor signaling network is needed to develop optimized therapeutic regimens. In this paper, oncogenic gene alterations in colorectal cancer, as well as their interaction with the mtor pathway, are systematically summarized. The most recent preclinical and clinical anticancer therapeutic endeavors are reviewed. New players in mtor signaling pathway, such as nonsteroidal anti-inflammatory drug and metformin with therapeutic potentials are also discussed here Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Mechanistic target of rapamycin pathway; Colorectal cancer; Mechanistic target of rapamycin inhibitor; Chemotherapy; Drug resistance Core tip: Mechanistic target of rapamycin (mtor) pathway serves as a central regulating axis that coordinates cell growth and proliferation. Single-agent mtor inhibition therapy, however, has provided only limited therapeutic efficacy towards colorectal cancer. Blocking compensatory pathways and multiple feedback loops is considered the challenge. Combination regimens are being intensively tested in clinic. This review summarizes extensive studies describing crosstalk between mtor pathway and major oncogenic pathways contributing to colorectal cancer development and novel combinational strategies targeting the mtor pathway in treating colorectal cancer are also introduced. Wang XW, Zhang YJ. Targeting mtor network in colorectal cancer therapy. World J Gastroenterol 2014; 20(15): Available from: URL: i15/4178.htm DOI: April 21, 2014 Volume 20 Issue 15

80 Wang XW et al. mtor signaling and human colorectal cancer INTRODUCTION The signaling pathway of mechanistic target of rapamycin (mtor), regulates cell growth and proliferation largely by promoting key anabolic processes, by sensing nutrition levels and growth factors, as well as various environmental cues [1,2]. The mtor pathway is conserved in organisms from yeast to human. The central protein, mtor, is an atypical serine/threonine protein kinase that belongs to the phosphoinositide 3-kinase (PI3K)- related kinase family. mtor interacts with several proteins to form two distinct complexes, known as mtor complex 1 (mtorc1) and complex 2 (mtorc2). Both complexes share a DEP domain-containing mtorinteracting protein (DEPTOR), a mammalian lethal with sec-13 protein 8 (mlst8, also known as GbL) [3] and Tti1/Tel2 [4]. Regulatory-associated protein of mammalian target of rapamycin (raptor) [5] and proline-rich Akt substrate 40 kda (PRAS40) [6] are unique to mtorc1 and rapamycin-insensitive companion of mtor (rictor) [7], mammalian stress-activated MAP kinase-interacting protein 1 (msin1) [8] and protein observed with rictor 1 and 2 (protor1/2) [9] are specific to mtorc2. mtor is the target of rapamycin (or sirolimus), but only mtorc1 is sensitive to rapamycin inhibition upon FK- BP12-rapamycin binding [10]. Rapamycin also inhibits the mtorc1 downstream targets differently [11]. mtorc1 plays a pivotal role in regulating protein and nucleotide synthesis by signaling through its main effectors, p70 ribosomal S6 kinase 1 (S6K1) and eif4e binding protein 1 (4E-BP1). S6 ribosomal protein, a component of the 40S ribosomal subunit, is the best characterized S6K1 substrate and a major effector of cell growth. Phosphorylated 4E-BP1 binds to eukaryotic translation initiation factor 4E (eif4e), which is an important component of the pre-initiation eif4f complex and prevents the complex from binding with the 5 end cap structure on messenger RNAs of proteins essential for the cell cycle progression, functioning as a rate-limiting factor in capdependent translation initiation. mtorc1 promotes de novo lipid synthesis by regulating Lipin-1 and SREBP1/2, and it promotes energy metabolism by positively regulating cellular metabolism and ATP production through activation of HIF1α and suppresses autophagy through ULK1 (unc-51-like kinase 1) and Atg13 (mammalian autophagy-related gene 13). mtorc2 phosphorylates protein kinase B (Akt/PKB), serum- and glucocorticoidinduced protein kinase 1 (SGK1), and protein kinase C-α (PKCα), regulating cell survival, metabolism, and cytoskeletal organization [12]. Multiple feedback loop mechanisms add to the complexity of the mtor signaling pathway [13]. mtorc1 integrates intracellular and extracellular signals--growth factors, stress, energy status, oxygen, and amino acids--mainly through the TSC1-TSC2 (hamartintuberin) complex. The TSC1/2 complex functions as a GAP (GTPase-activating protein) for the Ras homolog enriched in brain (Rheb), of which the GTP-bound form activates mtorc1. The TSC1/2 complex relays signals from upstream regulators that sense environmental growth signals and nutrition levels. TSC1 protects TSC2 from ubiquitin degradation [14]. In response to growth signals, multiple effectors phosphorylate TSC2, including Akt, extracellular-signal-regulated kinase1/2 (ERK1/2), and ribosomal S6 kinase (RSK1), thereby promoting mtor signaling activation. The TSC1/2 complex also responds to diverse stress signals. Upon hypoxia or low ATP state, adenosine monophosphate-activated protein kinase (AMPK) phosphorylates TSC2 and enhances its GAP activity toward Rheb [15]. Mitogen-activated protein kinase kinase kinase kinase 3 (MAP4K3) [16], mammalian vacuolar protein sorting 34 homolog (hvps34) [17] and inositol polyphosphate monokinase (IPMK) [18] are reported as amino acid sensing proteins. However, our understanding of the mechanisms by which mtor senses amino acids through the v-atpase (vacuolar H + -ATPase)-Ragulator (LAMTOR1-3)-Rag GTPase complex has evolved greatly in recent years. Four Rag proteins, RagA to RagD, form heterodimers: RagA with RagB, and RagC with RagD. When RagA/B is bound to GTP, RagC/D is bound to GDP, and vice versa. Amino acids promote GTP loading of RagA/B, thus enabling the heterodimer to interact with raptor. Ragulator binds with Rag GT- Pases and translocates to the lysosome surface, where mtorc1 interacts with GTP bound Rheb. v-atpase locates on the lysosomal membrane interacts with Ragulator to relay the amino acid level signals from the lysosomal lumen [19-22]. KEY COLORECTAL CARCINOGENESIS PATHWAYS AND THEIR INTERACTION WITH THE mtor PATHWAY Colorectal cancer (CRC) is the third most common cancer worldwide, with more than one million cases annually. CRC caused almost 0.7 million death in 2012 globally [23]. It is the second most deadly cancer among adults in the United States [24]. In approximately 75% of cases, the cancer are confined within the wall of the colon (stage Ⅰ and Ⅱ), or only spreads to regional lymph nodes (stage Ⅲ). These stages of cancer are mostly curable by surgical excision combined with chemotherapy. However, in about 20% of cases, the tumors metastasize to distant sites and are usually inoperable and incurable, with only a 12% 5-year relative survival rate [25,26]. Approximately 75%-80% of colorectal tumors develop in a sporadic manner [27]. An over-simplified model that generalizes the genetic cause of colorectal carcinogenesis is one where microsatellite instability (MSI) contributes to 85% of CRC, while the remaining 15% arise from chromosomal instability (CIN). However, some studies have shown that the MSI and CIN pathways are not mutually exclusive in CRC and considerable crosstalk exists between various pathways [28]. The canonical colorectal carcinogenesis model, that the carcinomas arise from pre-existing adenomas, was proposed in 1990 by Fearon and Vogelstein [29]. This 4179 April 21, 2014 Volume 20 Issue 15

81 Wang XW et al. mtor signaling and human colorectal cancer EGFP inhibitors EGFR Ras Anti-EGFR antibodies VEGFR inhibitors VEGFR Sorafenib IGF-1 IGF-BP3 Insulin IGF receptors IRS Metformin Glucose NSAIDs p 53 Rapamycin FKBP12 SREBP1 HIF1α Energy metabolism LKB1 RSK1 AMPK ATG13 Raf REDD1 MEK1/2 ULK Autophagy ERK1/2 Rheb mtorc1 TSC1 TSC2 p70s6k S6 p 53 PIP2 P13K PIP3 PTEN PI3K/mTOR Dual inhibitors PDK1 Wnt PRAS40 4E-BP1 eif4e Protein synthesis 473 AKT 308 mtorkis RHO mtorc2 RAC SGK1 PKCα Actin cytoskelecton dynamics Figure 1 Crosstalk between mechanistic target of rapamycin signaling pathway and colorectal oncogenic pathways. PI3K/AKT and Ras/MAPK pathways are the major upstream mediators of mechanistic target of rapamycin (mtor) signaling pathway in colorectal cancer. Therapeutic efforts for colorectal cancer targeting mtor signaling include Rapamycin (or Rapalogs) inhibition as well as mtor kinase inhibition, plus in combination with blockage of growth factor receptors, and components in upstream pathways such as Raf and PI3K, etc. New players regulating the mtor pathway such as non-steroidal anti-inflammatory drug (NSAIDs) and metformin merit further investigation. IGF: Insulin like growth factor; SGK1: Serum- and glucocorticoid-induced protein kinase; PRAS40: Proline-rich Akt substrate 40 kda; TSC1: Tuberous sclerosis-1; TSC2: Tuberous sclerosis-2; AMPK: Adenosine monophosphate-activated protein kinase; eif4e: Eukaryotic translation initiation factor 4E; RSK1: Ribosomal S6 kinase. model describes approximately 80%-90% of CRC and it is still accepted, despite a large body of new information on CRC that has emerged during the last two decades. In this model, the accumulation of genetic alterations, such as APC, p53, DCC, and K-ras, enable colorectal carcinogenesis, as well as histological malignancy progression [27]. Many of the genetic pathways involved lie upstream of mtor, and the oncogenes affected elicit part of their oncogenic effect through the mtor signaling pathway [30]. The interaction between mtor signaling and other important pathways involved in colorectal carcinogenesis are reviewed here (Figure 1). Wingless/wnt pathway Aberrant crypt foci (ACF) is considered the first identifiable precursor lesion in colorectal tissue [31]. ACF derives from epithelial cells in the lining of the colon and rectum and can develop into adenomatous polyps, which could potentially progress to adenocarcinoma [32]. Adenomatous polyposis coli (APC) tumor suppressor gene normally suppresses the Wnt pathway by actively degrading β-catenin and inhibits its nuclear localization [33]. A close link between β-catenin signaling and the regulation of VEGF-A expression was observed in human CRC, indicating the role of β-catenin in CRC angiogenesis [34]. β-catenin was also shown to induce cyclin D1 in CRC cells, which contributes to neoplastic transformation [35]. Aberrant, mutant APC or APC loss can cause constitutive activation of the Wnt pathway, which is considered the initiating event in colorectal cancer. APC mutation can cause more than 100 adenomatous polyps [36-38]. The Wnt signaling pathway stimulates the TSC-mTOR pathway [39]. mtor signaling, as well as the mtor protein level, was observed to be elevated in Apc Δ716 mice. Inhibition of the mtorc1 pathway by treating the APC mutant mice with RAD001 (everolimus) was reported to suppress intestinal polyp formation and reduce the mortality of the animals [15,39]. PI3K/AKT pathways Nutrient signals act mostly through insulin or insulin-like growth factor (IGF) signaling pathways. Growth factorsreceptors, such as epidermal growth factor receptor (EGFR), platelet-derived growth factor receptor (PDG- FR), insulin like growth factor-1 receptor (IGF-1R), and cell adhesion molecules, such as integrin and G-proteincoupled receptors, activate the PI3K pathway to promote cell survival, proliferation and cell growth [40]. The activated receptor tyrosine kinases interact with PI3K, where class Ⅰ PI3K family members convert phosphatidylinositol 4,5-bisphosphate (PIP2) to phosphatidylinositol 3,4,5-trisphosphate (PIP3), hence activating phosphoinositide-dependent kinase-1 (PDK1) and mtorc2. Specifically, phosphatase and tensin homolog (PTEN) reverses this process by dephosphorylating PIP3 to PIP2. IGF-BP3 binds to IGF-1 and prevents 4180 April 21, 2014 Volume 20 Issue 15

82 Wang XW et al. mtor signaling and human colorectal cancer over activation of IGF-1/AKT signaling. It is currently believed that PDK1 phosphorylates AKT on Thr308, whereas mtorc2 phosphorylates AKT at Ser473. Double phosphorylation fully activates AKT activity [41]. Hyperactivation of the PI3K/AKT pathway is associated with malignant behavior, including proliferation, adherence, transformation, and survival [42-45]. PI3K/ PTEN/AKT pathway mutations are found in a large number of CRC cell lines [46-49]. The PIK3CA mutation is found in 15% of metastatic colorectal cancer (mcrc) [50]. Germline PTEN ablation is associated with Cowden syndrome, which can cause an increased lifetime risk for CRC [51,52]. Elevated protein levels of PI3K subunit p85α and AKT1/2, and phosphorylation levels of mtor Ser2448 and phosphor-p70s6k Thr389 have been observed in CRC patients. Notably, p85α expression was considerably higher in stage Ⅳ tumors than in earlier stages [46]. Analyzing the mechanism of GP130-mediated mtorc1 activation in mice revealed a requirement for JAK and PI3K activity in the activation of mtorc1, leading to colorectal tumorigenesis [53]. mtor inhibition abolishes S6K phosphorylation and relieves the feedback suppression on RTK, leading to PI3K activation and, eventually, to AKT activation [54,55]. p53 pathway p53 is considered the guardian of the genome. p53 mediates diverse stress signals, such as DNA damage, energy and metabolic stress, hypoxia, oxidative stress, oncogene stress and ribosomal dysfunction. Functioning as a transcription factor, p53 regulates its downstream factors to elicit its tumor suppressive functions, which include cell cycle arrest, senescence, DNA repair, and programmed cell death [56]. Under normal conditions, p53 inhibits the mtor pathway by multiple routes. Deregulation of the p53 pathway by either mutation of the TP53 gene or by 17p chromosomal deletion is thought to be the second key step in tumorigenesis of CRC, marking the transition from adenoma to carcinoma [26,57]. p53 closely monitors the IGF-1/AKT pathways, which is an upstream regulation pathway of mtor [58,59]. p53 induces IGF-BP3 to inhibit mitogenic signaling [60] and directly regulates the transcription of PTEN [61]. In addition, p53 induces Sestrin1/2 upon DNA damage and oxidative stress, which negatively regulates mtor through activation of AMPK and TSC2 phosphorylation [62]. Furthermore, in colorectal cancer cell lines, p53 can suppress mtor activity by regulating AMPK-β1 and TSC2 directly. Notably, the increased mrna level of TSC2 by γ-irradiation-induced p53 activation can be cell type specific. However, data showed that p53-dependent induction of TSC2 exists in HCT116 cells and mouse colon tissue [63,64]. REDD1 is another p53 target gene that regulates the mtor pathway [65]. REDD1 is regulated by reactive oxygen species (ROS) and oxidative stress. REDD1 is necessary for hypoxia induced TSC1/2 activation [66]. RAS/RAF/MITOGEN-ACTIVATED PROTEIN KINASE PATHWAY Ras is the first identified oncogene and the most frequently mutated gene in human malignancy. Ras is a small GTPase that relays signals from a subset of growth factors responsive RTK to its effector pathways, which are responsible for growth, migration, adhesion, cytoskeletal integrity, survival and differentiation. The three true Ras proteins in the RAS family that have been most studied are H-Ras, N-Ras and K-RAS [67]. The K-RAS gene is the most mutated RAS pathway member in CRC, with a 35%-45% mutation rate in mcrc, compared with BRAF 8% and NRAS 4% [50]. K-RAS mutation is thought to be a relatively early event that correlates histologically with early to late adenomas. N-RAS mutations are also observed in a small percentage of CRC [31]. The major Ras downstream pathway is the Raf-mitogen-activated protein (MAP) kinase kinase-map kinase signal transduction pathway. Ras also indirectly signals to mtor through its other effector pathway, the PI3K/AKT pathway [68,69]. The p44/42 mitogen-activated protein kinase pathway (MAPK)-ERK1/2 directly phosphorylates and inactivates TSC2 [70,71]. ERK phosphorylates ribosomal protein S6, a direct effector of S6K1, stimulating capdependent translation [72,73]. The MAPK-activated kinase and RSK interact with and phosphorylate TSC2 at Ser-1798, thus inhibiting the tumor suppressor function of the TSC1/2 complex, resulting in increased mtor signaling to S6K1 [74]. Autophagy It is well established that mtorc1 negatively regulates autophagy. Atg1/ULK1 are central components in autophagy, and ULK1 as a direct target of TORC1 [75]. On the other hand, the role of autophagy in cancer, including colorectal cancer, can be complicated. Autophagy can contribute to cell death during chemotherapy, but could also serve as a survival mechanism for cancer cells. In fact, its function may vary in different types of tumors, as well as for various stages of cancer [76-78]. Autophagy is also reported to contribute to cancer cachexia. High level of HMGB1 was detected in the serum of CRC patients, and was associated with colorectal cancer progression. HMGBI was shown to induce autophagy in muscle tissue by reducing mtor phosphorylation in tumors bearing mice, therefore increasing plasma free amino acid levels, providing energy source to the cancer cells [79]. Other mechanisms The mtor signaling pathway may have a direct effect on carcinogenesis. Elevated mtor mrna and protein levels, as well as Raptor and Rictor levels, are observed in CRC patient tissues. Furthermore, a good correlation between a higher malignancy stage and higher expression level was observed [80,81] mtorc1/2 are critical for CRC 4181 April 21, 2014 Volume 20 Issue 15

83 Wang XW et al. mtor signaling and human colorectal cancer metastases via RhoA and Rac1 signaling [81]. Using a genetically engineered mouse model, mtor was proposed to contribute to tumorigenesis by causing chromosomal instability [82,83]. COLORECTAL CANCER THERAPIES TARGETING THE mtor PATHWAY mtor has a central role in the regulatory network sensing nutrition and growth signals, coordinating cell growth and proliferation. It has long been proposed that mtor inhibitors may be efficacious for treating and preventing tumor progression [84-86], particularly in CRCs [87]. Tremendous efforts have been made to develop potent and effective molecules to target the mtor pathway [88]. Efforts on targeting the mtor pathway for CRC treatment have been reviewed extensively in previously published reviews [89,90]. Nowadays, CRC chemotherapy consists mainly of oral fluoropyrimidines, with the addition of irinotecan and oxaliplatin. The emergence of targeted monoclonal antibodies (Mabs), such as bevacizumab (Bev) (anti-ve- FG-A), cetuximab and panitumumab (anti-egfr), has provided more treatment options to extend survival and improve clinical outcomes in mcrc [91,92]. However, less than 20% of patients with mcrc respond to clinically available targeted drugs when used as monotherapy [50]. This also suggests that a better understanding of the in depth molecular alterations in CRC is needed to discover more precise and effective therapeutic targets for those CRC cases that do not respond well to current treatment paradigms. First generation of mtor inhibitors-rapamycin and rapalogs Rapamycin is the first discovered natural inhibitor of mtor. The antitumor effect of rapamycin in colorectal cancer has been demonstrated in vitro and in various mouse models [39,53,93] Rapamycin inhibits mtorc1 with high specificity; however, its hydrophobicity and poor bioavailability has made it a less than optimal antitumor agent. Rapalogs, such as temsirolimus (CCI-779), everolimus (RAD001), and ridaforolimus (AP-23573, deforolimus) confer better potency, pharmacokinetic profiles and clinical activity than rapamycin, and are thus being used in the clinic or in developing treatments for many types of cancer, mostly solid tumors [90]. Temsirolimus and everolimus are approved for treating metastatic renal cell carcinoma and pancreatic neuroendocrine tumors. Everolimus is also approved for breast cancer therapy. However, multiple clinical trials have failed to demonstrate meaningful efficacy of everolimus in the treatment of CRC in the single agent setting [94,95]. Moreover, Rapalogs used alone are thought to be cytostatic in most tumor types and primarily stabilize clinical disease [83,86]. Deregulation of the PI3K and K-Ras signaling pathways determines therapeutic response to everolimus [96]. The central role of the PI3K/mTOR pathway in cancer biology suggests that other drug combinations showing mtor inhibition merit evaluation. Combinational regimens consisting of Rapalogs and other antitumor agents have shown promising results [97]. Both preclinical data and clinical trials have demonstrated that combined VEGF and mtor inhibition has greater anti-angiogenic and anti-tumor activity than either monotherapy. Bevacizumab and everolimus combined therapy was well tolerated, with prolonged stable disease in patients with refractory, metastatic colorectal cancer [98,99]. Reported side effects included risks related to mucosal damage and/or impaired wound healing [100]. The addition of a chemotherapy agent, such as doxorubicin, is also in development for advanced cancer therapy. Molecular analyses revealed an association between tumor response and a PIK3CA mutation and/or PTEN loss/mutation, suggesting further evaluation in patients with PI3K pathway dysregulation [100]. Using a xenograph tumor model, Lapatinib was shown to reduce tumor volume synergistically with everolimus, by reducing P-glycoprotein (P-gp) efflux of everolimus through inhibition of P-gp. This provided a new lead towards new chemotherapy in mcrc harboring K-RAS mutations [101]. The mosaic mutations in various oncogenic/tumor suppressive genes downstream of EGFRs undermines the therapeutic response of the anti-egfr antibodies. K-RAS and BRAF mutations are associated with poor prognosis in CRC [102]. Temsirolimus has limited efficacy in chemotherapy-resistant K-RAS mutant disease, and K-RAS mutation is a negative predictive prognostic factor during mcrc treatment with anti-egfr compounds [95,103]. Sorafenib, which is a multikinase inhibitor of Ras/MAPK signaling targeting Raf, also inhibits growth factor receptors, such as VEGFR and PDGFR. Sorafenib has been shown to enhance the therapeutic efficacy of rapamycin in CRC carrying oncogenic K-RAS and PIK3CA, in preclinical settings [104]. In a subgroup analysis of the Phase Ⅲ trial, the combination of everolimus and octreotide LAR demonstrated a significant prolonged median progression-free survival (PFS) in patients with advanced colorectal neuroendocrine tumors [105]. Everolimus combined with irinotecan proved to be well tolerated in a phase Ⅰ study as second-line therapy in mcrc; however, an in vitro study showed an additive effect in HT29 tumor xenografts, but not in HCT116, which both harbor BRAF/PIK3CA mutations [106]. Second generation of mtor inhibitors-dual PI3K/mTOR inhibitors and mtor kinase inhibitors Isolated inhibition of mtorc1 by rapamycin or Rapalogs proved that they were only partial inhibitors of mtorc1 and they do not have a meaningful contribution clinically in a single agent setting. Moreover, because of the release of feedback inhibition of AKT from S6K1 inhibition, a pro-survival effect derives from induced AKT activity. Inhibitors that block both the PI3K 4182 April 21, 2014 Volume 20 Issue 15

84 Wang XW et al. mtor signaling and human colorectal cancer signaling pathway and mtorc1/2 have been developed and have shown greater anticancer effects than Rapalogs [ ]. Dual PI3K/mTOR inhibitors are less likely to induce drug resistance than single-kinase inhibitors. mtor specific kinase inhibitors are expected to inhibit mtorc1 and mtorc2 simultaneously, although inhibiting mtorc1 may cause mtorc2 upstream AKT activation. Many dual PI3K/mTOR inhibitors and mtor kinase inhibitors are under preclinical study and some have entered the clinical phase [88]. Resistance arises from simultaneous mutation in parallel pathways related to the mtor pathway. Preclinical and clinical studies indicate that PIK3CA mutation in the absence of KRAS mutation is a predictive marker for the response to PI3K and mtor inhibitors [109,112]. However, CRC with the KRAS activation mutation is frequently observed, and it commonly coexists with PIK3CA mutations. Coexisting mutations of KRAS, BRAF and PIK3CA attenuate sensitivity to PI3K/mTOR inhibition in CRC cell lines [113,114]. Partial mtor inhibition from rapamycin and mtor kinase inhibitors indicates the existence of an unknown 4E-BP1 kinase that is potentially responsible for resistance in CRC [115]. The combination of a MEK inhibitor and PI3K/mTOR inhibitor was thus proposed to overcome the intrinsic resistance to MEK inhibition in CRCs [116,117]. Concomitant BRAF and PI3K/ mtor inhibition has been shown to be required for treatment of BRafV600E CRC [118]. Others Non-steroidal anti-inflammatory drugs, including aspirin and selective cyclooxygenase-2 (COX-2) inhibitors, have been investigated for protection against CRC development [119]. Aspirin was reported to lower the risk of, and improve the survival from, colorectal cancer [120,121]. PIK- 3CA mutation in colorectal cancer may serve as a predictive molecular biomarker for adjuvant aspirin therapy [122]. A study showed that aspirin reduced mtor signaling by activating AMPK; suppressed autophagy by mtor inhibition may contribute to the antitumor effect of aspirin [123]. Indomethacin and nimesulide are also reported to reduce mtor signaling and suppress CRC growth via a COX-2 independent pathway. These studies unveiled a novel mechanism through which COX-2 inhibitors exert their anticancer effects, as well showing protective effects against development of CRC, further emphasizing the validity of targeting mtor signaling in anticancer therapy [124]. Additive antitumor effects with low carbohydrate diets were observed with the mtor inhibitor CCI-779 and, especially, with the COX-2 inhibitor Celebrex [125]. A meta-analysis showed that diabetes mellitus increased risk of developing CRC [126], while metformin therapy appears to be associated with a significantly lower risk of colorectal cancer in patients with type 2 diabetes [127,128]. Metformin regulates glucose homeostasis by inhibiting liver glucose production and increasing muscle glucose uptake. A preclinical study showed that metformin inhibits insulin-independent growth and xenograft tumor growth of cells carrying the gain-of-function H1047R mutation of the PI3KCA gene, which has been shown to form diet restricted-refractory xenotumors [129], suggesting that metformin was not a bona fide diet restriction mimetic [130]. In both chemical carcinogen-induced and APC mutant colorectal carcinogenesis murine models, metformin activated AMPK and inhibited the mtor/s6k1 pathway, leading to suppressed colonic epithelial proliferation and reduced colonic polyp formation [131,132]. These data suggest that metformin might be a safe and promising candidate for the chemoprevention of CRC. Tandutinib inhibits several receptor tyrosine kinases, including the Fms-like tyrosine kinase 3 receptor, platelet-derived growth factor receptor (PDGFR), and c-kit receptor tyrosine kinase. Tandutinib inhibits the Akt/ mtor signaling pathway to inhibit CRC growth [133]. Curcumin, derived from the tropical plant Curcuma longa, has a long history in traditional Asian medicine. The preventive and therapeutic properties of curcumin are associated with its antioxidant, anti-inflammatory, and anticancer properties. Curcumin regulates the expression of inflammatory cytokines, growth factors, growth factor receptors, enzymes, adhesion molecules, apoptosis related proteins, and cell cycle proteins. Curcumin modulates the activity of several transcription factors and their signaling pathways [134]. The antitumor effect of curcumin towards CRC was mediated by modulation of Akt/mTOR signaling [135]. Another natural product, pomegranate polyphenolics, was shown to suppress azoxymethane-induced colorectal aberrant crypt foci and inflammation, possibly by suppressing the mir-126/vcam-1 and mir-126/pi3k/akt/mtor pathways [136]. PERSPECTIVE Epidemiological studies indicate that lifestyle factors throughout life influence CRC incidence and prognosis [137]. For example, a large waist circumference and body mass index has been associated with CRC risk [138,139]. A plausible mechanism was proposed that ample nutrition factors such as amino acids, insulin, glucose and IGF-1 circulating in the body, constantly activates the mtor pathway. Another study showed that hyperinsulinemia decreases IGFBP3 and consequently increases circulating IGF-1 and diabetes, both of which increase the risk of CRC [140]. CRC is a multifactorial disease. A new model: convergence of hormones, inflammation, and energy-related factors (CHIEF), proposes that various environmental agents (commensal bacteria, dietary antigens, mucosal irritants and pathogens) activate a basal, repetitive, mild subclinical inflammation, while additionally estrogen, androgens and insulin levels provoke the inflammation, which influences the CRC risk [141]. mtor appears to be in the hub of this network. The concept of the CHIEF model agrees with the contemporary therapeutic trend, recognizing multiple parallel pathways, and suggesting that combined inhibition of multiple pathways would provide more comprehensive tumor suppression 4183 April 21, 2014 Volume 20 Issue 15

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J Natl Cancer Inst 2005; 97: [PMID: DOI: /jnci/dji375] 127 Zhang ZJ, Zheng ZJ, Kan H, Song Y, Cui W, Zhao G, Kip KE. Reduced risk of colorectal cancer with metformin therapy in patients with type 2 diabetes: a meta-analysis. Diabetes Care 2011; 34: [PMID: DOI: / dc ] 128 Higurashi T, Takahashi H, Endo H, Hosono K, Yamada E, Ohkubo H, Sakai E, Uchiyama T, Hata Y, Fujisawa N, Uchiyama S, Ezuka A, Nagase H, Kessoku T, Matsuhashi N, Yamanaka S, Inayama Y, Morita S, Nakajima A. Metformin efficacy and safety for colorectal polyps: a double-blind randomized controlled trial. BMC Cancer 2012; 12: 118 [PMID: DOI: / ] 129 Kalaany NY, Sabatini DM. Tumours with PI3K activation are resistant to dietary restriction. Nature 2009; 458: [PMID: DOI: /nature07782] 130 Cufí S, Corominas-Faja B, Lopez-Bonet E, Bonavia R, Pernas S, López IÁ, Dorca J, Martínez S, López NB, Fernández SD, Cuyàs E, Visa J, Rodríguez-Gallego E, Quirantes-Piné R, Segura-Carretero A, Joven J, Martin-Castillo B, Menendez JA. Dietary restriction-resistant human tumors harboring the PIK3CA-activating mutation H1047R are sensitive to metformin. Oncotarget 2013; 4: [PMID: ] 131 Hosono K, Endo H, Takahashi H, Sugiyama M, Uchiyama T, Suzuki K, Nozaki Y, Yoneda K, Fujita K, Yoneda M, Inamori M, Tomatsu A, Chihara T, Shimpo K, Nakagama H, Nakajima A. Metformin suppresses azoxymethane-induced colorectal aberrant crypt foci by activating AMP-activated protein kinase. Mol Carcinog 2010; 49: [PMID: DOI: / mc.20637] 132 Tomimoto A, Endo H, Sugiyama M, Fujisawa T, Hosono K, Takahashi H, Nakajima N, Nagashima Y, Wada K, Nakagama H, Nakajima A. Metformin suppresses intestinal polyp growth in ApcMin/+ mice. Cancer Sci 2008; 99: [PMID: DOI: /j x] 133 Ponnurangam S, Standing D, Rangarajan P, Subramaniam D. Tandutinib inhibits the Akt/mTOR signaling pathway to inhibit colon cancer growth. Mol Cancer Ther 2013; 12: [PMID: DOI: / MCT ] 134 Shishodia S. Molecular mechanisms of curcumin action: gene expression. Biofactors 2013; 39: [PMID: DOI: /biof.1041] 135 Johnson SM, Gulhati P, Arrieta I, Wang X, Uchida T, Gao T, Evers BM. Curcumin inhibits proliferation of colorectal carcinoma by modulating Akt/mTOR signaling. Anticancer Res 2009; 29: [PMID: ] 136 Banerjee N, Kim H, Talcott S, Mertens-Talcott S. Pomegranate polyphenolics suppressed azoxymethane-induced colorectal aberrant crypt foci and inflammation: possible role of mir-126/vcam-1 and mir-126/pi3k/akt/mtor. Carcinogenesis 2013; 34: [PMID: DOI: /carcin/bgt295] 137 Weijenberg MP, Hughes LA, Bours MJ, Simons CC, van Engeland M, van den Brandt PA. The mtor Pathway and the Role of Energy Balance Throughout Life in Colorectal Cancer Etiology and Prognosis: Unravelling Mechanisms Through a Multidimensional Molecular Epidemiologic Approach. Curr Nutr Rep 2013; 2: [PMID: DOI: /s ] 138 Hughes LA, Simons CC, van den Brandt PA, Goldbohm RA, van Engeland M, Weijenberg MP. Body size and colorectal cancer risk after 16.3 years of follow-up: an analysis from the Netherlands Cohort Study. Am J Epidemiol 2011; 174: [PMID: DOI: /aje/kwr247] 139 Pischon T, Lahmann PH, Boeing H, Friedenreich C, Norat T, Tjønneland A, Halkjaer J, Overvad K, Clavel-Chapelon F, Boutron-Ruault MC, Guernec G, Bergmann MM, Linseisen J, Becker N, Trichopoulou A, Trichopoulos D, Sieri S, Palli D, Tumino R, Vineis P, Panico S, Peeters PH, Bueno-de-Mesquita HB, Boshuizen HC, Van Guelpen B, Palmqvist R, Berglund G, Gonzalez CA, Dorronsoro M, Barricarte A, Navarro C, Martinez C, Quirós JR, Roddam A, Allen N, Bingham S, Khaw KT, Ferrari P, Kaaks R, Slimani N, Riboli E. Body size and risk of colon and rectal cancer in the European Prospective Investigation Into Cancer and Nutrition (EPIC). J Natl Cancer Inst 2006; 98: [PMID: DOI: /jnci/djj246] 140 Kaaks R, Lukanova A. Energy balance and cancer: the role of insulin and insulin-like growth factor-i. Proc Nutr Soc 2001; 60: [PMID: DOI: /PNS200070] 141 Slattery ML, Fitzpatrick FA. Convergence of hormones, inflammation, and energy-related factors: a novel pathway of cancer etiology. Cancer Prev Res (Phila) 2009; 2: [PMID: DOI: / capr ] P- Reviewers: Hong J, Pan CC S- Editor: Qi Y L- Editor: A E- Editor: Liu XM 4188 April 21, 2014 Volume 20 Issue 15

90 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (5): Colorectal cancer TOPIC HIGHLIGHT Hallmarks in colorectal cancer: Angiogenesis and cancer stem-like cells Muriel Mathonnet, Aurelie Perraud, Niki Christou, Hussein Akil, Carole Melin, Serge Battu, Marie-Odile Jauberteau, Yves Denizot Muriel Mathonnet, Aurelie Perraud, Niki Christou, Carole Melin, Department of Digestive Surgery, Limoges University Hospital, Limoges, France Muriel Mathonnet, Aurelie Perraud, Hussein Akil, Carole Melin, Serge Battu, Marie-Odile Jauberteau, Homéostasie Cellulaire at Pathologies, Faculty of Medicine, University of Limoges, Limoges, France Yves Denizot, Institut 145 GEIST, UMR CNRS 7276, Faculty of Medicine, University of Limoges, Limoges, France Author contributions: Mathonnet M, Christou N, Akil H and Battu S analyzed the literature; Perraud A and Mathonnet M wrote the manuscript; Jauberteau MO and Denizot Y revised the manuscript; Melin C contributed to the bibliography. Supported by Grants from the University of Limoges, Limoges University Hospital, La Ligue Contre le Cancer and the Région Limousin, which was given financial by the Comité Orientation Recherche Cancer (to Perraud A, Christou N and Akil H) Correspondence to: Muriel Mathonnet, MD, PhD, Professor of Digestive Surgery, Department of Digestive Surgery, Limoges University Hospital, 2 Ave Martin Luther King, Limoges, France. mathonnet@unilim.fr Telephone: Fax: Received: October 3, 2013 Revised: January 26, 2014 Accepted: March 19, 2014 Published online: April 21, 2014 Abstract Carcinogenesis is a multistep process that requires the accumulation of various genetic and epigenetic aberrations to drive the progressive malignant transformation of normal human cells. Two major hallmarks of carcinogenesis that have been described are angiogenesis and the stem cell characteristic of limitless replicative potential. These properties have been targeted over the past decade in the development of therapeutic treatments for colorectal cancer (CRC), one of the most commonly diagnosed and lethal cancers worldwide. The treatment of solid tumor cancers such as CRC has been challenging due to the heterogeneity of the tumor itself and the chemoresistance of the malignant cells. Furthermore, the same microenvironment that maintains the pool of intestinal stem cells that contribute to the continuous renewal of the intestinal epithelia also provides the necessary conditions for proliferative growth of cancer stem-like cells. These cancer stem-like cells are responsible for the resistance to therapy and cancer recurrence, though they represent less than 2.5% of the tumor mass. The stromal environment surrounding the tumor cells, referred to as the tumor niche, also supports angiogenesis, which supplies the oxygen and nutrients needed for tumor development. Anti-angiogenic therapy, such as with bevacizumab, a monoclonal antibody against vascular-endothelial growth factor, significantly prolongs the survival of metastatic CRC patients. However, such treatments are not completely curative, and a large proportion of patient tumors retain chemoresistance or show recurrence. This article reviews the current knowledge regarding the molecular phenotype of CRC cancer cells, as well as discusses the mechanisms contributing to their maintenance. Future personalized therapeutic approaches that are based on the interaction of the carcinogenic hallmarks, namely angiogenic and proliferative attributes, could improve survival and decrease adverse effects induced by unnecessary chemotherapy Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Colon cancer; Stem cell; Cancer stem-like cell; Tumor-initiating cell; Microenvironment Core tip: Recent progress in the therapeutic treatment of colorectal cancer has resulted from targeting the hallmark stem cell-like properties of tumor cells. The survival of colorectal cancer patients has been significantly prolonged with bevacizumab, which inhibits angiogenesis providing the proliferative conditions for tu April 21, 2014 Volume 20 Issue 15

91 Mathonnet M et al. Colorectal cancer angiogenesis and stem cells mor cell growth. Personalized therapeutic approaches, centered on the angiogenic and proliferative properties of cancerous cells, could improve patient survival and decrease adverse effects induced by unnecessary chemotherapy. Mathonnet M, Perraud A, Christou N, Akil H, Melin C, Battu S, Jauberteau MO, Denizot Y. Hallmarks in colorectal cancer: Angiogenesis and cancer stem-like cells. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: org/ /wjg.v20.i INTRODUCTION Carcinogenesis is a multistep process reflecting a series of genetic and epigenetic alterations in normal human cells that drives their progressive transformation into highly malignant derivatives. The successful growth of metastatic cells depends on the interactions and the properties of the cancer cells and the potential target organs, proposed as the seed and soil hypothesis by Paget [1]. In 2000, Hanahan and Weinberg suggested that the malignant growth of nearly all types of tumors is a result of six essential alterations in cell physiology: self-sufficiency in growth factors, insensitivity to growth-inhibitory signals, evasion of apoptosis, limitless replicative potential, sustained angiogenesis, and the ability to invade and metastasize [2]. Since then, two additional hallmarks of carcinogenesis have been described: the reprogramming of energy metabolism and the evasion of immune destruction, which have contributed to the reconceptualization of cancer cell biology. The notion of a tumor microenvironment [3] has led to profound changes in the study of cancer and in the therapeutic approach. Furthermore, the study of angiogenesis and stem cell-like limitless replicative potential of cancer cells in the tumor microenvironment has facilitated progress in cancer treatment, especially for colorectal cancer (CRC). The formation of new vasculature, or angiogenesis, is actively involved in tumor development, progression, and metastasis. Although the initial step of tumor angiogenesis is not well understood, the recruitment of perivascular support cells is necessary for blood vessel formation [4]. Diverse tissue-specific tumor-associated stromal stem cell types contribute to the formation of the tumor niche, including carcinoma-associated fibroblasts (CAFs), tumor-associated macrophages, lymphocytes, pericyte cells, inflammatory cells, normal epithelial cells, and mesenchymal stem cells (MSCs). Recently, it was shown than MSCs migrate to tumors and can transition into CAFs [5,6]. These processes appear at the earliest stage of tumor development. The tumor niche provides conditions favorable for cell proliferation and protection against conventional therapies [7], and contains cells that possess stem cell-like properties such as self-renewal and multipotentiality [8]. These cells have been termed tumor-initiating cells, or cancer stem-like cells (CSCs). Although CSCs represent only a small proportion of cell types within the tumor, they may be responsible for the resistance to cancer therapies and tumor recurrence in solid cancers such as glioblastoma [8] and CRC [9,10], which is composed of a heterogeneous population of dormant (or quiescent) and active cells [11]. CSCs are intrinsically resistant to apoptosis and express several members of the ATP-binding cassette (ABC) transporter family, which are overexpressed in the multidrug-resistant phenotype [12]. Microenvironment stimuli, such as hypoxia, also contribute to chemoresistance by inducing a stem cell-like phenotype in cancer cells [13]. The relationship between these cells and the angiogenic microenvironment is fundamental for understanding tumor progression and therapeutic resistance. INTESTINAL STEM CELLS Normal intestinal epithelial cells have a lifetime of around five days and are continuously renewed by stem cells (SCs) under microenvironmental influence [14]. The intestinal SCs are located at the crypt base and are involved in tissue homeostasis and repair. These cells undergo asymmetric division, giving rise to one identical daughter cell, and one cell with the potential to migrate to the top of the crypt and fully differentiate into an intestinal cell. Intestinal crypts contain two pools of SCs. At the lowest part of the crypt base is an active pool of SCs that express Lgr-5 (leucine-rich repeat containing G proteincoupled receptor 5). A second reserve pool of quiescent SCs resides at the +4 position, i.e., the fourth cell above the lowermost cell of the intestinal crypt. These cells express Bmi-1 and TERT (telomerase reverse transcriptase) and have the ability to replace Lgr-5-expressing cells [15,16]. However, the identification of intestinal SCs remains an issue of debate. Many molecules have been proposed as putative markers (Table 1), such as the cell surface proteins Lgr-5, aldehyde dehydrogenase-1 (ALDH-1), and integrin-β1 (CD29), but none have been widely accepted as specific molecular markers. CANCER STEM-LIKE CELLS Any normal cell can accumulate mutations and become a cancer origin cell, such as a CSC. CSCs are multipotent cells that give rise to progenitors and differentiated cells causing tumor heterogeneity, and whose migration results in metastasis. The existence of CSCs was first hypothesized in 1994 by Lapidot et al [17] and later confirmed when Ricci-Vitiani et al [14] isolated CD133+ cells from colon cancer tumors and characterized them as CSCs. CSCs express markers common to stem and progenitor cells, and are similarly capable of unlimited growth in vitro. While only a small portion of cells within a tumor (< 2.5%) are endowed with tumor propagation [18], CSCs have the ability to reproduce the parental tumor in vivo April 21, 2014 Volume 20 Issue 15

92 Mathonnet M et al. Colorectal cancer angiogenesis and stem cells Table 1 Markers used to identify normal colonic stem cells and colonic cancer stem-like cells Marker Function Normal stem cell Integrin-β1 (CD29) Cell surface receptor - cell adhesion molecule Hes-1 Transcriptional repressor - transactivated by Msi-1 Msi-1 RNA binding protein - maintenance of undifferentiated state Bmi-1 Polycomb receptor - maintenance of chromatin silencing Lgr-5 Wnt target gene - potential of self renewal ALDH-1 Detoxifying enzyme DCAMKL-1 Kinase - radioresistance abilities TERT Quiescent stem cells and radio resistant Ascl-2 Transcription factor - target of Wnt and Notch pathways Cancer stem-like cell CD133 Pentaspan transmembrane glycoprotein CD44 Hyaluronic acid receptor CD166 Cell adhesion molecule ALDH1 Enzyme 4-Oct POU-domain transcription factor SOX2 Transcription factor c-myc Transcription factor Integrin-β1 (CD29) Cell surface receptor-cell adhesion molecule Clonal evolution of proliferating CSCs may lead to a gain or loss of stem cell-like attributes through individual responses to microenvironmental stimuli, including epigenetic modifications and additional genetic aberrations [16]. The Wingless/Int (Wnt) signaling pathway plays a pivotal role in the regulation of SC self-renewal [19]. In normal cells, Wnt signals are transduced through the Frizzled/LRP5/6 complex to inhibit the phosphorylation-dependent degradation of β-catenin. In colon cancer cells, constant but heterogeneous mutations in adenomatous polyposis coli (APC) and β-catenin genes result in high Wnt signaling activity. Moreover, extrinsic signals given by neighboring or matrix cells, such as stromal myofibroblasts, can regulate Wnt activity and SC attributes of CSCs [20]. Four other major signaling pathways are also altered in CSCs. In normal colon tissue, TGF-β and Notch signaling pathways regulate cell proliferation, differentiation, migration, apoptosis, and SC maintenance and function. Altered TGF-β signaling in more advanced and metastatic CRCs results in an inhibition of its tumor suppressive activity. Notch-1 is abundantly expressed in the stem cell zone in normal colon tissue, and is upregulated in CRC [21]. Moreover, Notch signaling is 10 to 30 fold higher in CRC-CSCs compared to commonly used colon cancer cell lines [22]. This upregulation prevents CSC apoptosis through p27, a cell-kinase inhibitor, which maintains CSC renewal and represses cell lineage differentiation genes. Neighboring myofibroblasts produce bone morphogenetic protein antagonists Gremlin 1 and Gremlin 2 in addition to Wnt signaling ligands, which can also modulate Notch signaling [23]. The Hedgehog signaling pathway is one of the key regulators of animal embryogenesis, and is also involved in the proliferation, migration, and differentiation of cells. Hedgehog signaling has been implicated in tumor growth and CD133+ stem cells in CRC [20]. Recently, a role for neurotrophins was highlighted in CRC both in vitro and in tumors, where enhanced brain-derived neurotrophic factor signaling as a result of increased expression of tropomyosin-related kinase B (TrkB) receptors, was associated with advanced disease and a worse prognosis [24]. Moreover, some studies suggest that TrkB regulates epithelial-mesenchymal transition (EMT) in solid cancers [25], especially in CRC [26]. CSC IDENTIFICATION Identification of CSCs is based on SC markers (Table 1), especially Lgr-5 and Bmi-1, the only markers rigorously evaluated in vivo [27,28]. CD133 and CD44 are two classical markers that have also been used, though they are not specific. Two transcription factors, Oct-4 and Sox2, are more promising CSC markers as they are involved in cell renewal. Levels of Oct-4 and Sox2 are elevated in CRC and correlate with increased CSC proliferation and poor prognosis [29,30]. Several methods have been developed to isolate CSCs based on the expression of these markers [16,31] using flow cytometry (fluorescence-activated cell sorting or FACS) or magnetic-activated cell sorting [32] (Table 2), though tumor heterogeneity as well as the low abundance and lack of differentiation has made the isolation of CSCs from patient tumors and in vitro cultures difficult. These methods rely on specific antigen recognition and thus are restricted by the availability of highly specific antibodies. In addition, labeling of cell-surface markers by antibodies could trigger signaling pathways and induce cell modification and differentiation. Therefore, the development of methods that do not rely on marker labeling is greatly needed. Tools based on intrinsic biophysical properties such as size or density may be of benefit. Counterflow centrifugal elutriation, which separates cells by weight, has been a valuable tool for obtaining homogeneous populations [33], though experiments to isolate CRC-CSCs have not yet been attempted. More recently, CSCs have been sorted from a panel of CRC cell lines using sedimentation field flow fractionation technology, in which sorting is based on cell size and density [34] April 21, 2014 Volume 20 Issue 15

93 Mathonnet M et al. Colorectal cancer angiogenesis and stem cells Table 2 Advantages and disadvantages of the cell sorting methods Method Advantages Disadvantages MACS Fast, easy to make Cell labeling indispensable FACS Fast Cell labeling indispensable, flux cytometry indispensable CCE Cell labeling not necessary, cell weight based method Time consuming, specific instrumentation indispensable SdFFF Cell labeling not necessary, cell size and density based method Time consuming, specific instrumentation indispensable MACS: Magnetic-activated cell sorting; FACS: Fluorescence-activated cell sorting; CCE: Counterflow centrifugal elutriation; SdFFF: Sedimentation flux force fractionation. TUMOR NICHE AND MICROENVIRONMENT The non-cancerous niche is a dynamic milieu, consisting of stem cells, neural cells, lymphocytes, macrophages, endothelial cells, fibroblasts, smooth muscle cells, and myofibroblasts surrounded by a stromal microenvironment. The niche adapts in response to environmental cues to ensure the optimal conditions for SC proliferation and differentiation, even in the absence of SCs [19]. Intestinal SCs can also be affected by the components of the crypt lumen, such as bacteria or epithelial cells [16]. One of the most extensively studied niche components is intestinal subepithelial myofibroblasts, which regulate intestinal SCs by secreting growth factors and cytokines. CSCs can secure the niche microenvironment by displacing normal SCs and interact with it to generate vascular precursors [35]. The tumorigenic niche is composed of recruited myeloid cells, vascular and lymphovascular endothelial cells, macrophages, and transformed myofibroblasts surrounded by stromal tissue. Stromal fibroblasts secrete various cytokines and growth factors that act in an autocrine or paracrine fashion on tumor cells, such as tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), and hepatocyte growth factor, which is an enhancer of Wnt activity [36,37]. CAFs that are present in the tumorigenic niche secrete the cytokines CXCL1 and CXCL2, as well as IL-1β and IL-6 to enhance angiogenesis and tumor progression [38]. These cells are able to modulate the expression of oncogenic genes in cancer cells, such as Her2, EGFR, and Ras and thereby contribute to chemotherapeutic resistance [39]. CAFs are a major contributor to the tumor-prone microenvironment, and thus promote tumor growth. Targeting of these cells remains a challenge due to the presence of distinct CAF populations that do not express tumor-specific markers [40]. MSCs are non-hematopoietic precursor cells residing in the bone marrow that also contribute to the tumor microenvironment. MSCs have been shown to influence tumor development, progression, metastatic diffusion, and resistance to chemotherapy in many solid cancers, including colon cancer [41]. The interaction of MSCs and cancer occurs early in tumor formation via numerous pathways. MSCs in the colon express a high level of vascular endothelial growth factor (VEGF) when stimulated by interferon-gamma and TNF-α, thus leading to colon cancer growth [42]. They can recruit endothelial cells by se- creting CXCL12 [43], and their secretion of IL-6 can induce non-cancer stem cells to express CSC markers and cause tumor formation in vivo [44]. IL-6 induces the secretion of endothelin-1 (ET-1) from cancer cells and promotes tumor development by recruiting endothelial cells and activating signaling pathways that regulate protein synthesis. This was demonstrated in a study by Huang et al [45] showing that angiogenesis as a result of mixing non-tumorigenic MSCs and HT-29 cells, a colorectal cancer cell lineage, was blocked by IL-6 or ET-1 antibodies. Moreover, patients with CRC have significantly higher VEGF and IL-6 serum levels, which correlate with advanced stages and metastatic disease [46]. Additional studies have also implicated IL-6 in tumor development [47-50]. ANGIOGENESIS AND HYPOXIA In normal adult tissues, angiogenesis is only transiently turned-on. Tumor angiogenesis generates neovascularization in response to the need for nutrients and oxygen and for the elimination of metabolic wastes and carbon dioxide. During tumor progression, an angiogenic switch is activated causing normally quiescent vasculature to continually sprout new vessels that sustain expanding neoplastic growth [3]. The angiogenic switch is governed by countervailing factors that either induce or oppose angiogenesis, such as VEGF and thrombospondin, respectively. VEGF signaling is complex with alternative splice variants that are regulated at multiple levels, and VEGF gene expression can be upregulated by hypoxia, through activation of the HIF1 transcription factor, and by integrin or oncogene signaling. HIF1 is known to induce self-destruction or autophagy of the tumor in order to preserve nutrients in hypoxic conditions [51]. VEGF is secreted through a K-ras/PI3K/Rho/ROCK/c-Myc axis in CRC [13]. VEGF ligands signal through tyrosine receptor kinases, two of which are implicated in angiogenesis, namely VEGFR1 and VEGFR2, and a third receptor, VEGFR3, which is implicated in lymphangiogenesis. VEGFRs are not only expressed in vascular endothelial cells, but also in other cell types, including macrophages and monocytes, suggesting they play a role in EMT [52]. Other signaling pathways that have been implicated in angiogenesis have crosstalk with VEGF signaling, such as the Ang/Tie or Notch signaling pathways [53]. Other factors, such as fibroblast growth factor, or platelet-derived growth factor, are involved in the maintenance of the 4192 April 21, 2014 Volume 20 Issue 15

94 Mathonnet M et al. Colorectal cancer angiogenesis and stem cells angiogenic process. The blood vessels produced within the tumor are typically aberrant with abnormal endothelial proliferation and apoptosis [54]. Numerous cells originating from bone marrow play crucial roles in pathological angiogenesis and in the formation of primary tumor and metastatic sites, notably macrophages, neutrophils, mast cells, myeloid progenitors, and endothelial progenitor cells (EPC). EPCs account for 12% of the total number of endothelial cells in tumor vessels, and play a critical role in the metastatic angiogenic switch. Most of these cells can migrate into neoplastic lesions and become intercalated into the neovasculature as pericytes or endothelial cells [55]. CSCs AND MICROENVIRONMENT INTERACTIONS: IMPLICATIONS FOR PHYSICIANS An understanding of angiogenic pathways has progressed the development of cancer therapeutics over the last decade, especially for treatment of CRC. In 2004, the first treatment with an anti-angiogenic compound, a monoclonal antibody against VEGF named bevacizumab, was recommended for use with first and second line adjuvant chemotherapy, FOLFOX (5-flourouracil, leucovorin, and oxaliplatin) or FOLFIRI (5-fluorourcil, leucovorin, and irinotecan). A meta-analysis conducted in 2009 including more than 3000 patients concluded that the addition of bevacizumab to chemotherapy for metastatic CRC prolonged both specific free survival and overall survival despite a higher incidence in grade Ⅲ/Ⅳ hypertension, arterial thromboembolic events, and gastrointestinal perforations [56]. However, a phase 3 randomized trial assessing the use of bevacizumab in combination with oxaliplatin-based therapy in adjuvant treatment of patients with resected stage Ⅲ or high-risk stage Ⅱ colon carcinoma (the AVANT study), suggested a detrimental effect of bevacizumab that involved more serious adverse effects without an improvement in disease-free survival [57]. Thus, anti-angiogenic therapy may only benefit CRC patients with liver metastasis, though further evaluation is needed. Other anti-angiogenic therapies, such as aflibercept, a VEGFA, VEGFB and placenta growth factor decoy receptor, or ramucirumab, a VEGFR1/2/3 and Tie2 tyrosine kinase inhibitor, have been validated by clinical trials [13]. Due to the increase in plasma VEGF levels and EPCs after partial hepatectomy in CRC metastatic patients, Pocard and Eveno [58] claim the following: (1) the primary cancer should be resected rapidly to minimize metastatic niche activation; (2) systemic chemotherapy associated with anti-angiogenic drugs should be administered after surgery; (3) liver metastases should be resected; and (4) immunomodulatory and anti-angiogenic treatments should be administered to minimize recurrence. In practice, neither anti-angiogenic drugs nor adjuvant chemotherapy can completely eliminate recurrence or resistance events. It is now acknowledged that CSCs and EMT can induce chemoresistance through intrinsic and indirect mechanisms. Intrinsic mechanisms involve proficient DNA repair machinery, high expression of ABC transporters, and altered cell cycle kinetics. For example, the blockade of ABC transporters improves patient response to neoadjuvant radiotherapy [59]. Alterations in cell cycle leave some CSCs in a state of quiescence where they are protected from chemotherapeutic toxicity, thus enabling tumor regrowth [60,61]. In addition, the overexpression of IL-4 that occurs in CRC amplifies the expression of anti-apoptotic mediators, thus the in vivo efficacy of cytotoxic therapy is increased with IL-4 blockade [62]. Indirect contributions to chemoresistance come from the microenvironment [12]. The dynamic interactions between CSCs and the microenvironment result in a continuous remodeling of both compartments. These epitheliomesenchymal interactions occur in the EMT, which in addition to promoting metastasis development, plays a role in chemoresistance. Furthermore, the chaotic and dysfunctional vasculature of the tumor, which inhibits supply of oxygen and nutrients, prohibits the accrual of optimal concentrations of chemotherapeutic agents within the tumor [12]. Therefore, targeting of both the intrinsic and indirect mechanisms with anti-angiogenic agents or inhibitors of EMT/hypoxia-associated effectors will more effectively deplete the CSC pool and contribute to increased chemotherapeutic response. A main prognostic indicator for CRC is the identification of the predominant cell type. Traditionally, the basis for prognosis and care has come from the classification of CRC as outlined by the American Joint Committee on Cancer [63]. Surgery is curative for stages 1-3, adjuvant chemotherapy is indicated for high-risk stages 2 and 3 CRC, and anti-angiogenic drugs are recommended for metastatic patients. Unfortunately, it is still difficult to predict disease progression or treatment response. Studies of CRC have attempted to determine a signature capable of identifying the patient populations with high-risk for recurrence that need adjuvant therapy. Currently, patients are screened for mutations in the KRAS or BRAF genes that indicate resistance to therapy, though a large proportion of patients with wild-type KRAS are also chemoresistant [64]. Multiple molecular subtypes of CRC have been identified, and whereas the most differentiated CRC subtypes, named transit-amplified and goblet-like subtypes, have a good prognosis and do not need adjuvant therapy, the stem cell-like subtype has the poorest prognosis and requires adjuvant chemotherapy (FOLFIRI), even in cases of metastasis [65,66]. A new classification system has been proposed based on the cellular phenotype and therapeutic response. Sadanandam et al [67] combined gene expression analyses with differential responses to cetuximab to define six CRC subtypes in cultured cell lines and from patient tissues. The CRC subtypes were associated with cellular differentiation state and Wnt signaling activity from distinct anatomical regions of the colon crypts. The heterogeneity of CRC indicates that a change to 4193 April 21, 2014 Volume 20 Issue 15

95 Mathonnet M et al. Colorectal cancer angiogenesis and stem cells therapeutic schemas is needed. Most importantly, therapeutic approaches should include multiple targets, aimed at disrupting the cooperative interaction between the tumor cell and its microenvironment. Advancement of personalized therapeutic approaches will help to improve patient survival, not only by increasing specific survival, but also by decreasing the adverse effects induced by unnecessary chemotherapy. REFERENCES 1 Paget S. The distribution of secondary growths in cancer of the breast Cancer Metastasis Rev 1989; 8: [PMID: ] 2 Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100: [PMID: DOI: /S (00) ] 3 Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 144: [PMID: DOI: /j.cell ] 4 Carmeliet P. Angiogenesis in life, disease and medicine. Nature 2005; 438: [PMID: DOI: /nature04478] 5 Spaeth EL, Dembinski JL, Sasser AK, Watson K, Klopp A, Hall B, Andreeff M, Marini F. 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96 Mathonnet M et al. Colorectal cancer angiogenesis and stem cells CS, Chang CH, Chen KH, Chang YL, Tseng LM, Song WS, Wang JJ, Lin JK, Huang PI, Lan YT. Oct4-related cytokine effects regulate tumorigenic properties of colorectal cancer cells. Biochem Biophys Res Commun 2011; 415: [PMID: DOI: /j.bbrc ] 31 Tomlinson MJ, Tomlinson S, Yang XB, Kirkham J. Cell separation: Terminology and practical considerations. J Tissue Eng 2013; 4: [PMID: DOI: / ] 32 Cammareri P, Lombardo Y, Francipane MG, Bonventre S, Todaro M, Stassi G. Isolation and culture of colon cancer stem cells. Methods Cell Biol 2008; 86: [PMID: DOI: /S X(08) ] 33 Bauer J. Advances in cell separation: recent developments in counterflow centrifugal elutriation and continuous flow cell separation. J Chromatogr B Biomed Sci Appl 1999; 722: [PMID: DOI: /SO (98) ] 34 Mélin C, Perraud A, Akil H, Jauberteau MO, Cardot P, Mathonnet M, Battu S. 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97 Mathonnet M et al. Colorectal cancer angiogenesis and stem cells [PMID: DOI: / CAN ] 63 AJCC Cancer Staging Manual. 7th ed. New York: Springer, 2010: Zhou SW, Huang YY, Wei Y, Jiang ZM, Zhang YD, Yang Q, Xie DR. No survival benefit from adding cetuximab or panitumumab to oxaliplatin-based chemotherapy in the first-line treatment of metastatic colorectal cancer in KRAS wild type patients: a meta-analysis. PLoS One 2012; 7: e50925 [PMID: DOI: /journal.pone ] 65 Perez-Villamil B, Romera-Lopez A, Hernandez-Prieto S, Lopez-Campos G, Calles A, Lopez-Asenjo JA, Sanz-Ortega J, Fernandez-Perez C, Sastre J, Alfonso R, Caldes T, Martin- Sanchez F, Diaz-Rubio E. Colon cancer molecular subtypes identified by expression profiling and associated to stroma, mucinous type and different clinical behavior. BMC Cancer 2012; 12: 260 [PMID: DOI: / ] 66 Schlicker A, Beran G, Chresta CM, McWalter G, Pritchard A, Weston S, Runswick S, Davenport S, Heathcote K, Castro DA, Orphanides G, French T, Wessels LF. Subtypes of primary colorectal tumors correlate with response to targeted treatment in colorectal cell lines. BMC Med Genomics 2012; 5: 66 [PMID: DOI: / ] 67 Sadanandam A, Lyssiotis CA, Homicsko K, Collisson EA, Gibb WJ, Wullschleger S, Ostos LC, Lannon WA, Grotzinger C, Del Rio M, Lhermitte B, Olshen AB, Wiedenmann B, Cantley LC, Gray JW, Hanahan D. A colorectal cancer classification system that associates cellular phenotype and responses to therapy. Nat Med 2013; 19: [PMID: DOI: /nm.3175] P- Reviewers: Feng ZL, Huang J S- Editor: Qi Y L- Editor: A E- Editor: Ma S 4196 April 21, 2014 Volume 20 Issue 15

98 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. TOPIC HIGHLIGHT WJG 20 th Anniversary Special Issues (5): Colorectal cancer circadian clock circuitry in colorectal cancer Gianluigi Mazzoccoli, Manlio Vinciguerra, Gennaro Papa, Ada Piepoli Gianluigi Mazzoccoli, Manlio Vinciguerra, Department of Medical Sciences, Division of Internal Medicine and Chronobiology Unit, IRCCS Scientific Institute and Regional General Hospital Casa Sollievo della Sofferenza, San Giovanni Rotondo Foggia, Italy Manlio Vinciguerra, Istituto EuroMEditerraneo di Scienza e Tecnologia (IEMEST), Palermo, Italy Manlio Vinciguerra, Division of Medicine, Royal Free Campus, Institute for Liver and Digestive Health, University College London, London NW3 2PF, United Kingdom Gennaro Papa, Department of Surgical Sciences, Division of Urology, University of Chieti, Chieti, Italy Ada Piepoli, Department of Medical Sciences, Research Laboratory and Division of Gastroenterology, IRCCS Scientific Institute and Regional General Hospital Casa Sollievo della Sofferenza, San Giovanni Rotondo Foggia, Italy Author contributions: Mazzoccoli G and Vinciguerra M contributed equally to this work; Mazzoccoli G and Piepoli A conceived the review article; Papa G performed the literature mining; Mazzoccoli G, Vinciguerra M, Papa G and Piepoli A wrote the paper. Supported by the 5x1000 voluntary contribution and by a grant to GM from the Italian Ministry of Health through Department of Medical Sciences, Division of Internal Medicine and Chronobiology Unit, IRCCS Scientific Institute and Regional General Hospital Casa Sollievo della Sofferenza, Opera di Padre Pio da Pietrelcina, San Giovanni Rotondo (FG), Italy, Nos. RC1203ME46 and RC1302ME31; by a grant to AP from the Italian Ministry of Health through Department of Medical Sciences, Division of Gastroenterology and Research Laboratory, Nos. RC1203GA55 and RC1203GA56; and by a grant to MV from AIRC, No. MFAG-AIRC Correspondence to: Gianluigi Mazzoccoli, MD, Department of Medical Sciences, Division of Internal Medicine and Chronobiology Unit, IRCCS Scientific Institute and Regional General Hospital Casa Sollievo della Sofferenza, Cappuccini Avenue, San Giovanni Rotondo Foggia, Italy. g.mazzoccoli@operapadrepio.it Telephone: Fax: Received: September 9, 2013 Revised: January 6, 2014 Accepted: January 19, 2014 Published online: April 21, 2014 system cancers. Carcinogenesis relies on disrupted control of cellular processes, such as metabolism, proliferation, DNA damage recognition and repair, and apoptosis. Cell, tissue, organ and body physiology is characterized by periodic fluctuations driven by biological clocks operating through the clock gene machinery. Dysfunction of molecular clockworks and cellular oscillators is involved in tumorigenesis, and altered expression of clock genes has been found in cancer patients. Epidemiological studies have shown that circadian disruption, that is, alteration of bodily temporal organization, is a cancer risk factor, and an increased incidence of colorectal neoplastic disease is reported in shift workers. In this review we describe the involvement of the circadian clock circuitry in colorectal carcinogenesis and the therapeutic strategies addressing temporal deregulation in colorectal cancer Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Colorectal cancer; Circadian rhythm; Clock gene Core tip: The biological clock drives crucial cell processes, such as growth, proliferation, differentiation and apoptosis, controls metabolic pathways, and regulates tissue functions and behavioral cycles. Derangement of these phenomena is involved in colorectal carcinogenesis. The circadian clock circuitry is a leading actor in physiological regulation, a drawn in bystander in colorectal tumorigenesis, and a possible therapeutic target. Mazzoccoli G, Vinciguerra M, Papa G, Piepoli A. circadian clock circuitry in colorectal cancer. World J Gastroenterol 2014; 20(15): Available from: URL: com/ /full/v20/i15/4197.htm DOI: org/ /wjg.v20.i Abstract Colorectal cancer is the most prevalent among digestive INTRODUCTION Digestive system cancers account for approximation 4197 April 21, 2014 Volume 20 Issue 15

99 Mazzoccoli G et al. Colorectal cancer and circadian rhythmicity 20% of neoplastic disease incidence, and colorectal cancer (CRC) is the most prevalent, representing > 50% of all these cancers. CRC is the third most commonly diagnosed cancer, representing approximation 10% of all newly diagnosed cancers apart from skin cancers, with 19.4 new cases per of the male population and 17 new cases per of the female population. CRC is the fourth leading cause of cancer deaths, accounting for 8.1% of all cancer-related deaths worldwide. Epidemiological data report a 5-year CRC prevalence of 66.3 cancer survivors per population worldwide, and a cumulative CRC risk in persons aged < 75 years of 1.96% worldwide; 2.35% in men and 1.62% in women [1,2]. At present, the first-choice treatment is surgical excision with or without adjuvant chemotherapy, and the identification of novel biomarkers for prognosis and molecular targets for therapeutic intervention is urgently needed. CRC onset and progression are related to mutational pathways, such as chromosomal instability and microsatellite instability (MSI). MSI is distinguished as H (high frequency or probability), L (low frequency or probability) and S (stable); is determined by defects in the normal DNA damage response and mismatch repair process, leading to the loss or gain of repeated units on the daughter strand and resulting in length variation [3,4] ; and influences cancer-specific survival and time to recurrence in CRC patients [5,6]. An early step in colorectal carcinogenesis is represented by mutations in the gene adenomatous polyposis coli (APC), causing large deletions in the C terminus of the proteins that are found in > 80% of human colon cancers. This region is involved in the binding of β-catenin and axin, and its deletion leads to post-translational stabilization and accumulation of β-catenin. In turn, this transcriptional factor translocates from the cytoplasm to the nucleus, where it relieves T-cell factor (TCF)/lymphocyte enhancer factor (LEF)1-mediated transcriptional repression, to activate several oncogenes and other gene targets, such as c-myc and CCND1, which stimulate target-gene expression and eventually tumor phenotype development [7,8]. Circadian clock circuitry and the bowel Colorectal carcinogenesis is related to the progressive loss of homeostatic control of cell proliferation, differentiation and apoptosis. Cellular processes and functions in living organisms show time-related variations [9-11]. The patterns of oscillation may be rhythmic with a period of approximation 24 h and are called circadian [12]. The circadian timing system responsible for the generation of these rhythmic variations is composed of central and peripheral oscillators [13]. The central pacemaker is located in the suprachiasmatic nuclei of the brain, which are entrained to the environmental light-dark cycle by photic inputs conveyed by the retino-hypothalamic tract, and synchronize self-sustained oscillators in peripheral tissues through autonomic nervous system fibers and hormone output; mainly represented by cortisol and melatonin [14,15]. The molecular clock responsible for the generation of circadian rhythmicity consists of a set of interlocking transcription-translation feedback loops that complete one cycle each day and are driven by the core clock genes encoding the circadian proteins brain and muscle aryl-hydrocarbon receptor nuclear translocatorlike (ARNT)l (BMAL1/2 also called ARNTL/2), circadian locomotor output cycles kaput (CLOCK) or its paralog neuronal PAS domain protein (NPAS)2, PE- RIOD (PER) 1-2, CRYPTOCHROME (CRY) 1-2 [16]. Other proteins that in some way are related to the clock gene machinery are represented by PER3, TIMELESS, timeless-interacting protein (TIPIN), and protein kinases. The genes PER1-2 and CRY1-2 are transcriptionally activated by the basic helix-loop-helix/pas (period, aryl-hydrocarbon-receptor, single minded) transcription factors CLOCK and BMAL1, which heterodimerize and bind to E-box enhancer elements in the promoters of these genes [16]. In turn, PER and CRY proteins form a repression complex that translocates back into the nucleus and interacts with CLOCK and BMAL1, hindering their activity. PER3 is believed to be the product of one of the output genes, more than a core clock gene, considering that Per3 knockout mice do not show any circadian phenotype, whereas PER1 and PER2 play an essential role in the molecular clockwork [17]. TIMELESS is the homolog of a core circadian gene of Drosophila melanogaster and is maintained in mammals, but its role in the function of the mammalian molecular clock is still unclear. TIMELESS and its partner TIPIN interact with components of the DNA replication system to regulate DNA replication processes under both normal and stress conditions and are essential for ataxia telangiectasia and Rad3-related (ATR)-checkpoint kinase (Chk)1 and ataxia telangiectasia mutated (ATM)-Chk 2-mediated signaling and S-phase arrest [18,19]. CLOCK-BMAL1 heterodimer activates an assisting loop that promotes expression of the nuclear receptors reverse transcript of erythroblastosis gene (REV-ERB) α/β (encoded by NR1D1 and NR1D2, respectively), and retinoic acid-related (RAR) orphan receptor (ROR)α/γ, which in turn compete for ROR-responsive elements (ROREs) of the BMAL1 promoter, and control negatively and positively the rhythmic transcription of BMAL1, as the REV-ERBs repress BMAL1 transcription, while RORs activate it. This stabilizing negative loop is important for precise control of the circadian pacemaker, and these nuclear receptors regulate a number of physiological functions, including circadian rhythmicity, lipid metabolism, and cellular differentiation [20-23]. The correct functioning of the clock gene machinery relies on post-translational modifications of circadian proteins, represented by phosphorylation, O-GlcNAcylation, SUMOylation, acetylation, and deacetylation. Phosphorylation is operated by protein kinases, such as casein kinase (CK)1-ε (encoded by CSNK1Ε), which targets the proteins BMAL1, PER1, PER2, and CRY1; CK2, which targets BMAL1 and PER2; AMP-activated kinase (AMPK), which targets the CRY proteins; and glycogen synthase kinase (GSK) April 21, 2014 Volume 20 Issue 15

100 Mazzoccoli G et al. Colorectal cancer and circadian rhythmicity 3β, which targets the proteins CLOCK, BMAL1, PER2 and CRY2 [24]. In the absence of GSK-3β-mediated phosphorylation BMAL1 becomes stabilized, decreasing the dependent circadian gene expression [25]. In turn, GSK-3β regulates the activity of O-GlcNAc transferase, which works as a metabolic sensor gauging the nutrient flux into the hexosamine biosynthesis pathway, and finetunes the regulation of the circadian clock through reversible change of structure and transcriptional activity of the circadian proteins CLOCK and PER, as well as stabilization of BMAL1 and CLOCK as a result of inhibition of their ubiquitination [26,27]. Furthermore, BMAL1 is SUMOylated in vivo on Lys259; CLOCK is necessary to stimulate this post-translational modification; and BMAL1 SUMOylation and activation oscillate with circadian rhythmicity in the mouse liver [28,29]. Acetylation is operated by CLOCK, whereas deacetylation is operated by SIRT1, a type Ⅲ NAD + -dependent histone/protein deacetylase that is required for high-magnitude circadian transcription of several proteins encoded by core clock genes, including BMAL1, PER2, and CRY1, which counterbalances the histone acetyltransferase activity of CLOCK, and promotes the deacetylation and degradation of PER2. The interaction of SIRT1 with CLOCK: BMAL1 is not time dependent, whereas the NAD + - dependent SIRT1 activity changes in a circadian manner, and the circadian regulation of SIRT1 activity depends on CLOCK:BMAL1-mediated regulation of expression of nicotinamide phosphoribosyltransferase (NAMPT); the rate-limiting enzyme involved in NAD + synthesis [30-34]. The clock gene machinery controls the expression of hundreds of genes (clock-controlled genes) that drive the expression of proline-and acidic amino acidrich domain basic leucine zipper (PAR bzip), including DBP (albumin D-site binding protein), TEF (thyrotroph embryonic factor), HLF (hepatic leukemia factor), and the nuclear factor, interleukin-3-regulated protein (NFIL3, also known as E4BP4), whose promoter contains a RORE and is transcriptionally regulated by REV- ERBs [35]. Many physiological processes in the gastrointestinal tract, such as motility of gut sections, activity of mucosal enzymes, function of mucosal transporters, and proliferation rate of different cell types, exhibit diurnal rhythms [36,37]. Studies in animal models and observations in humans have demonstrated that a circadian biological clock operates in the gastrointestinal tract, and the core clock genes drive the circadian expression of clockcontrolled genes and tissue-specific output genes [38,39], coding for proteins involved in gut functions, such as NHE3, encoding an electroneutral Na + /H + exchanger, and Atp1a1, ENaCg, Dra, AE1 and NHERF1, involved in colonic NaCl absorption [40]. In addition, the clock gene machinery drives the circadian rhythmicity of gut physiology and motility [41,42], suggesting possible links between disruptions of circadian biology and diseases of the gastrointestinal tract, such as motility disorders, inflammation, and cancer [43,44]. The biological clock in colorectal carcinogenesis The clock genes drive the circadian rhythmicity of expression of so-called clock-controlled genes, which represent 5%-20% of the genome, comprising key cell-cycle regulators, tumor suppressor genes and oncogenes, and their expression regulates timing of basic cell functions, such as metabolism, xenobiotic detoxification, DNA damage repair, and autophagy [45,46]. Cell-cycle progression is tightly regulated and the circadian system is involved in the control of cell proliferation and apoptosis, driving the transcription and/or post-translational modification of key proteins that are essential for DNA replication such as thymidylate synthase, and molecules that gate cell division in mitosis such as WEE-1 [47-49]. Disruption of the circadian clock may lead to deregulated cell proliferation and deregulation of the circadian clock has been implicated in CRC [49-52]. The alteration of bodily temporal organization, defined as circadian disruption, is considered a cancer risk factor, and an increased incidence of CRC has been reported in shift workers by epidemiological studies [53-55]. The role of the disruption of the molecular clockwork in colorectal carcinogenesis and CRC progression is hinted by experimental data showing distortion of circadian regulation in colonic neoplastic tissue and genespecific disruption in the matched nontumorous tissues. Overexpression of PER1 in human cancer cell lines results in reduced colony formation and clonogenic expansion, in sensitization to radiation-induced apoptosis, and in altered expression of transcriptional target genes such as c-myc and p21. In contrast, Per2-null mice show an increase in hyperplasia and neoplasia in response to γ radiation, and Per2 mutation has been shown to accelerate intestinal polyp formation in Apc Min/+ mice [56-58]. PER1 and PER2 are involved in ATM-Chk1/Chk2 DNA damage response pathways and modulate β-catenin, encoded by the clock-controlled gene CTNNB1, whose promoter shows BMAL1 occupancy, indicating direct circadian regulation by this transcription factor [59,60], and that can function as an oncogene, influencing cell proliferation in colon cancer cells. In turn, intestinal tumorigenesis may alter clock function as a result of increased β-catenin destabilization of PER2 protein and impairing circadian clock gene expression in intestinal mucosa of Apc Min/+ mice [61]. In mice, mutation in the Period genes leads to altered temporal expression of genes involved in cell-cycle regulation and tumor suppression, such as c-myc, Cyclin D1, Cyclin A, Mdm-2 and GADD45Α, deregulation of DNAdamage response, accelerated intestinal polyp formation in Apc Min/+ mice, and increased neoplastic growth [62]. Moreover, the BMAL1 gene is a fundamental hinge in the clock gene machinery and plays a key role in the regulation of tumor cell apoptosis, cell-cycle progression, and DNA damage response. In in vitro and in vivo experiments the knockdown of BMAL1 by RNA interference in murine colon cancer cells (C26) reduced the expression of Per1, Per2, Per3, Wee1 and Tp53; decreased apop April 21, 2014 Volume 20 Issue 15

101 Mazzoccoli G et al. Colorectal cancer and circadian rhythmicity tosis induced by etoposide; reduced the distribution in the G2/M phases of cells treated by docetaxel; and decreased DNA damage induced by cisplatin, accelerating cell proliferation in vitro and promoting tumor growth in mice [63]. A role in the early stages of colorectal carcinogenesis is played also by CK1ε, as demonstrated by in vitro and in vivo studies, and knock down of CSNK1E or use of a kinase inhibitor specific to CK1ε induced tumor-cell-selective cytotoxicity, because tumor cells depend more on the kinase activity of CK1ε than normal cells do [64,65]. The function of the circadian clock during neoplastic transformation was evaluated in a mouse model of chemically induced primary CRC, and the daily profiles of the core clock genes Per1, Per2, Rev-Erbα and Bmal1, the clock-controlled gene Dbp and the clock-controlled cell cycle genes Wee1, c-myc and p21 were assessed in the tumor, matched nontumorous tissue, and the liver [66]. The circadian rhythmicity of Per1, Per2, Rev-Erbα and Dbp was significantly decreased in CRC compared with nontumorous tissue, and the expression of Bmal1 was not rhythmic. Besides, the circadian expression of Per1, Per2, Rev-Erbα and Dbp was present in the nontumorous matched colonic tissue, but the expression of Bmal1 did not show a circadian rhythm [66]. The expression patterns of Wee1, c-myc and p21 did not show circadian rhythmicity in tumors or the colon of healthy animals. A phase shift was evidenced for the rhythmic expression of the clock genes in the liver of tumor-bearing mice [66]. clock genes in human colorectal cancer There has been much interest in the alteration of expression of clock genes and clock-controlled genes in humans affected by CRC [67,68]. CLOCK is reported to be mutated in cancer, and may be involved in carcinogenesis through changed response to DNA damage. Microarray gene expression data combined with public gene sequence information have identified CLOCK as a potential target of somatic mutations in microsatellite unstable CRCs, and CLOCK mutations occurred in 53% of MSI CRCs [69]. Restoring CLOCK expression in a human colon adenocarcinoma cell line derived from a primary colon cancer lacking wild-type CLOCK, and testing the effects of UV-induced apoptosis and radiation by DNA content analysis using flow cytometry, demonstrated protection against UV-induced apoptosis and decreased G2/M arrest in response to ionizing radiation. Novel CLOCK-binding elements were identified near DNA damage genes p21, NBR1, BRCA1 and RAD50 by means of chromatin immunoprecipitation with parallel DNA sequencing [69]. Furthermore, changes in the expression of PER2 were demonstrated by immunohistochemical staining and real-time polymerase chain reaction in tumor tissue of CRC patients, with heterogeneous, and negative or weak staining patterns in cancerous cells, with higher PER2 expression in welldifferentiated cancer cells when compared to poorly differentiated ones, and associations of decreased PER2 levels with patient age, histological grade, TNM stage and expression of nuclear proliferation-related antigen Ki67 [70]. Besides, downregulation of PER3 was seen in tumor tissues of CRC patients, associated with various clinicopathological factors, comprising tumor location, differentiation, and stage, and to poorer survival, suggesting an important role in CRC progression [71]. Among the clock-controlled genes that drive intestinal physiology, an important role is played by PPARG, which codes for the transcription factor peroxisome proliferatoractivated receptor (PPAR)γ, which is involved in several physiological processes, and an important interplay has been demonstrated between PPARγ and β-catenin [72]. β-catenin is crucial in the WNT signaling pathway, and plays an essential role in the regulation of gene expression interacting with several molecular partners, such as APC and the TCF/LEF1 family transcription factors, and in cell adhesion, bridging between cadherins and the actin cytoskeleton at the cell-cell adherens junctions. Cytoplasmic but not membrane-bound β-catenin forms a complex with APC, the associated protein axin/conductin, CK2 and GSK-3β, which phosphorylate β-catenin and other elements of the molecular proteolytic complex. Activation of WNT signaling involves the inhibition of GSK-3β through mechanisms that may involve axin binding to the proteins Dishevelled or low-density lipoprotein receptor-related protein (LRP)-5, determining nuclear β-catenin accumulation, binding to TCF/ LEF1 transcription factors, and expression of target genes including c-myc and CCND1 [73]. The protein kinase CK1ε is involved in cell proliferation by stabilizing β-catenin, and CK1ε overexpression mimics the effect of WNT signalling, resulting in cytoplasmic accumulation of β-catenin and its subsequent nuclear localization, to control transcription and support tumorigenesis [74-76]. The role of CK1ε in cell proliferation and in circadian clock function in the context of colorectal carcinogenesis might be not crucial. In the absence of WNT signaling, β-catenin is destabilized by GSK-3β, which plays a role in mammalian circadian clock function, counteracts the ability of CK1ε to promote β-catenin stability, and is involved in circadian control of cell-cycle progression [77]. PPARγ is a ligand-activated transcription factor belonging to the large superfamily of nuclear receptors, regulates cellular proliferation/differentiation, and plays a role in colorectal carcinogenesis [78]. In the normal mucosa of the colon and rectum there is high PPARγ expression, and in mouse small intestine and colon, decreased intestinal PPARγ levels are associated with enhanced tumorigenicity [79]. In CRC patients, PPARγ expression levels are not uniformly changed, suggesting that PPARG deregulation plays different and context-dependent roles in CRC onset and progression, and that this nuclear receptor inhibits tumor growth only in the presence of functional APC [80]. The functioning of the clock gene machinery is severely altered in patients affected by CRC, with downregulation of BMAL1, PER1, PER2, PER3 and CRY2, 4200 April 21, 2014 Volume 20 Issue 15

102 Mazzoccoli G et al. Colorectal cancer and circadian rhythmicity upregulation of BMAL2 and TIMELESS (in particular in MSI-H and MSI-L tumors), and significant differences in survival related to differential expression of clock genes [44,81]. The altered expression of clock genes influences also phenotypic characteristics of colon cancer cells and disease outcome, and a correlation between high expression of the BMAL1 gene and low expression of the PER1 gene and liver metastasis, and between high expression of the PER2 gene and significantly better outcomes of CRC have been observed [82]. The upregulation of BMAL2 is accompanied by high expression of SERPINE1 in CRC patients [81]. Invasion by cancer cells requires proteases to degrade the cellular matrix. Cathepsins, which are cysteine proteases, play a crucial role in this process through the destruction of the cellsurrounding extracellular matrix [83]. Plasmin is formed from its zymogen, plasminogen; a reaction catalyzed by the serine protease urokinase-type plasminogen activator (upa) and partially regulated by plasminogen activator inhibitors, such as plasminogen activator inhibitor (PAI)-1 [84]. A high concentration of PAI1 in tumor biopsy specimens, the major physiological regulator of the pericellular plasmin-generating cascade, is associated with poor prognosis, and high preoperative plasma concentrations of PAI-1 are associated with shorter survival in CRC patients [85]. Both CLOCK:BMAL1 and CLOCK: BMAL2 heterodimers powerfully activate the promoter of the gene encoding PAI-1, officially called SERPINE1 and located on the seventh chromosome (7q21.3-q22), underlying the circadian variation in circulating PAI-1 [86,87]. CLOCK:BMAL2, binding two E-box enhancers in the promoter, is more potent than CLOCK:BMAL1 in its ability to activate SERPINE1 [88-90]. Colorectal tumorigenesis is influenced also by SIRT1 that may behave as a tumor suppressor or as an oncogene in different in vitro and in vivo conditions. The involvement of SIRT1 in onset and progression of CRC is corroborated by the observation that transgenic mice overexpressing SIRT1 in the intestine show reduced development of tumors caused by Apc Min/+ mutation, and that SIRT1 / mice show increased tumor incidence when crossed to a p53 / background. In CRC patients, the expression levels are unevenly modified, reflecting the different settings that characterize the experimental conditions, and different colon cancer cell lines show dissimilar levels of SIRT1 expression and variable changes in clock gene expression after ectopic upregulation [91]. In normal colon, significant levels of nuclear SIRT1 are expressed in proliferating colon epithelial cells at the base of the crypt, and expression gradually decreases in cells migrating toward the lumen. In contrast, in samples of benign adenomas (polyps), SIRT1 is detected in all cells with an adenomatous morphology of benign adenomas (polyps), but not in the adjacent normal mucosa. Colonic adenocarcinoma shows a heterogeneous SIRT1 expression profile, suggesting that in normal colon epithelium, SIRT1 levels correlate with active cell proliferation. SIRT1 is expressed at high levels in normal colon and benign lesions, and high SIRT1 expression is an intrinsic response to cell proliferation in untransformed mucosa and premalignant adenoma. In CRC tissue, SIRT1 expression is downregulated in a subset of tumors to facilitate tumor growth, and in particular in high grade tumors, where the growth-inhibitory activity of SIRT1 becomes a rate-limiting factor for tumor progression, whereas some low- grade tumors may overexpress SIRT1 to benefit from its antiapoptotic effects [92]. Circadian rhythmicity and CRC treatment The body systems involved in xenobiotic detoxification show circadian variations of function and activity driven by the clock gene machinery, determining time-dependent toxicity of xenobiotics and drugs [93-95]. The product of the multidrug resistance (mdr1a) gene, P-glycoprotein, works as a xenobiotic transporter contributing to the intestinal barrier, and intestinal expression of the mdr1a gene and its efflux pump function show oscillation with 24-h periodicity driven by the circadian clock through the opposing action of HLF and E4BP4, connecting the circadian timekeeping system to xenobiotic detoxification [96]. The time-of-day-dependent variations in cancer cell proliferation, as well as drug metabolism, toxicity and effectiveness represent the rationale for the timing of drug administration over a 24-h period [97-100], and they form the basis for chronomodulated chemotherapy of advanced-stage CRC, characterized by an opposite phase of delivery of oxaliplatin in the afternoon, with respect to 5-fluorouracil and leucovorin that are delivered late at night (chronoflo4) [101]. Chronomodulated delivery of chemotherapy has shown better tolerability and antitumor activity compared with conventional chemotherapy, with less myelosuppression and more gastrointestinal toxicity for the chronoschedule, but with a favorable difference between treatments in median survival time only in male patients. Indeed, sex dependency in the effects of this schedule has been demonstrated with shorter survival and greater toxicity reported in female CRC patients in the European Organisation for Research and Treatment of Cancer (EORTC) Chronotherapy Group trial [102]. From these studies, sex emerges as the single predictor of survival, conditioning outcome of chronoflo4 and determining genetic variation of metabolic responses that influence time-related variables and hinder administration of maximal effective dosing. A different genotypic profile between men and women could characterize CRC patients, and the higher burden of toxicity reported in women treated with 5-fluorouracil-based chemotherapy may be also due to the lower expression of dihydropyrimidine dehydrogenase in the tumors in female patients and/or to sex dependency of circadian pharmacology [68]. Xenobiotic detoxification and metabolic pathways differ between male and female mammals including humans. In double mutant Cry1 / Cry2 / male mice, sex dimorphism in hepatic drug metabolism is altered. Upon inactivation of the Cryptochrome genes, the levels of sex-specific liver products, including several cytochrome P450 enzymes, 4201 April 21, 2014 Volume 20 Issue 15

103 Mazzoccoli G et al. Colorectal cancer and circadian rhythmicity CK P E4BP4 P Pn REV-ERB Pn Pn ROR HSP90 XAP2 AHR PPAR DEC DBP HLF TEF P C E4BP4 Ppor Dec Ror Dbp Hlf Tef Clock controlled genes Per 1-3 Cry 1-3 P23 CK P Pn C ARNT XRE REV-ERB ROR Genes CK P Pn C Clock BMAL1 SIRT1 P C Pn P TIM REV-ERB ROR CHK1/ATR CHK2/ATM P21 WEE1 β-catenin c-myc Cell cycle checkpoints Proliferation/death CK P AMPK Cyclin A, D1, D3, E1, B1, Cdk2, Cdc2 Pn P C C Pn Cell cycle progression Degradation P53, p63, Mdm2, Bcl2 Apoptosis Figure 1 biological clock and the cell processes involved in colorectal carcinogenesis. The scheme renders the transcriptional/translational feedback loop through which the molecular clockwork operates, and depicts its interplay with the aryl hydrocarbon receptor (AHR)/ARNT system in the control of the cell cycle and apoptosis, and ultimately in the regulation of the processes of cell proliferation and death, whose deregulation brings about colorectal cancer (CRC) onset and progression. CK: Casein kinase; AHR: Aryl hydrocarbon receptor; PPAR: Peroxisome proliferator-activated receptor; CHK: Checkpoint kinase; ATR: Ataxia telangiectasia and rad3-related; ATM: Ataxia telangiectasia mutated; ARNT: Aryl hydrocarbon receptor nuclear translocator; HLF: Hepatic leukaemia factor; TEF: Thyrotroph embryonic factor. expressed by male mice are similar to those expressed by female mice [103]. In humans, the decreased expression level of Cryptochrome genes observed in female patients affected by CRC [44] might explain the different median survival and increased toxicity observed after chronomodulated chemotherapy administration in the EORTC Chronotherapy Group trial [104]. High Cryptochrome gene expression and protein levels in tumor tissue are correlated with tumor progression and poorer overall and disease-free survival in CRC patients [104]. Many cytotoxic anticancer drugs damage DNA and may activate DNA checkpoints permitting attempted DNA repair. This process is essential for cell survival, but may reduce the cytotoxicity of anticancer drugs. TIME- LESS is required for ATM-dependent Chk2-mediated signaling of doxorubicin-induced DNA double-strand breaks, and downregulation of TIMELESS by sirna significantly attenuates doxorubicin-induced G2/M cell cycle arrest and sensitizes cancer cells to doxorubicin-induced cytotoxicity. Therefore, upregulation of TIMELESS in CRC may predict altered drug sensitivity, and TIMELESS inhibition is a potential novel anticancer drug target to enhance the cytotoxic effectiveness of chemotherapeutic drugs known to activate DNA response pathways within cancer cells [105]. TIMELESS expression is also significantly associated with MSI status in CRC patients, and higher expression is found in patients with MSI-H and MSI-L [44]. Approximately 15% of CRC is characterized by defects in the DNA mismatch repair system, leading to MSI that generates a large number of substitution, as well as insertion and deletion mutations. These mutations typically target microsatellite sequences and lead to shifts in the reading frame, resulting in truncation or other alterations of the protein product. The presence of premature stop codons decreases the expression of mrnas with such frameshift mutations and results in degradation of some of the mutant mrna through the nonsense-mediated decay pathway. Response to adjuvant chemotherapy may be influenced by MSI status in CRC patients, because MSI-H tumors show better outcome with irinotecan-containing than 5-fluorouracil-containing regimens [106]. The increased expression of TIMELESS in MSI-H and MSI-L CRC may be related to the process of tumorigenesis and might cause reduced response to possible adiuvant chemotherapy. The circadian CLOCK-BMAL1 transactivation complex may be directly involved in the response to stress at the individual cell level, because Clock mutant and Bmal1 knockout mice are highly sensitive to cyclophosphamide treatment in a time-of-day-independent manner, suggesting that CLOCK and BMAL1 directly regulate 4202 April 21, 2014 Volume 20 Issue 15

104 Mazzoccoli G et al. Colorectal cancer and circadian rhythmicity the expression of cell-cycle- and apoptosis-related genes, and that time-related and allelic-dependent variations in response to chemotherapy correlate with the functional status of the CLOCK-BMAL1 heterodimer [93-95,107]. The frequencies of the 311T>C CLOCK gene CC genotype and C allele were significantly higher among CRC patients compared to controls, increasing the risk of CRC by and 1.78-fold, respectively [108]. Furthermore, higher levels of CLOCK gene and protein expression were observed in human CRC tissues, particularly in poorly differentiated, advanced Dukes stage tumors and in cases with lymph node involvement, and a strong positive linear correlation was found with ARNT, hypoxia-inducible factor- 1α and vascular endothelial growth factor expression in tumor tissue [109,110]. BMAL1 protein suppresses the protooncogene c-myc, and stimulates the tumor suppressor WEE1, confirming the important role played by the altered expression of this core clock gene in the process of carcinogenesis [50]. In addition, a tight interplay has been demonstrated between aryl hydrocarbon receptor (AHR)/ ARNT system and circadian pathways, and this interconnection might be critical to influence onset, promotion, and progression of colorectal malignant neoplasms [46]. An important role is played in carcinogenesis by the transcription factors of the AHR/ARNT system, which bind to a xenobiotic response element and control the expression of genes encoding enzymes involved in the metabolism and detoxification of exogenous and endogenous compounds, but they are antagonized by the AHR repressor [111] (Figure 1). Upon ligand-mediated activation, the AHR/ARNT system modulates metabolic pathways and cell processes, such as survival, cell cycle, proliferation, apoptosis, differentiation, epithelial to mesenchymal transition, angiogenesis, inflammation, cell contact inhibition, cell-matrix interaction, and extracellular matrix remodeling, which are crucial in the initiation and evolution of neoplastic disease [ ]. CONCLUSION The biological clock plays an important role in bowel physiology, and alteration of the molecular clockwork is involved in colorectal carcinogenesis. The circadian genes and proteins are variably altered in colorectal malignant neoplasm and influence phenotypic characteristics of colon cancer cells, disease progression, patient survival, and response to chemotherapy. 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107 Mazzoccoli G et al. Colorectal cancer and circadian rhythmicity 2012: [PMID: DOI: /2012/560846] 81 Mazzoccoli G, Pazienza V, Panza A, Valvano MR, Benegiamo G, Vinciguerra M, Andriulli A, Piepoli A. ARNTL2 and SERPINE1: potential biomarkers for tumor aggressiveness in colorectal cancer. J Cancer Res Clin Oncol 2012; 138: [PMID: DOI: /s ] 82 Oshima T, Takenoshita S, Akaike M, Kunisaki C, Fujii S, Nozaki A, Numata K, Shiozawa M, Rino Y, Tanaka K, Masuda M, Imada T. Expression of circadian genes correlates with liver metastasis and outcomes in colorectal cancer. Oncol Rep 2011; 25: [PMID: DOI: / or ] 83 Talieri M, Papadopoulou S, Scorilas A, Xynopoulos D, Arnogianaki N, Plataniotis G, Yotis J, Agnanti N. Cathepsin B and cathepsin D expression in the progression of colorectal adenoma to carcinoma. Cancer Lett 2004; 205: [PMID: DOI: /j.canlet ] 84 Troy AM, Sheahan K, Mulcahy HE, Duffy MJ, Hyland JM, O Donoghue DP. 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J Biol Chem 2010; 285: [PMID: DOI: /jbc.M ] 106 Bertagnolli MM, Niedzwiecki D, Compton CC, Hahn HP, Hall M, Damas B, Jewell SD, Mayer RJ, Goldberg RM, Saltz LB, Warren RS, Redston M. Microsatellite instability predicts improved response to adjuvant therapy with irinotecan, fluorouracil, and leucovorin in stage III colon cancer: Cancer and Leukemia Group B Protocol J Clin Oncol 2009; 27: [PMID: DOI: /JCO ] 107 Gorbacheva VY, Kondratov RV, Zhang R, Cherukuri S, Gudkov AV, Takahashi JS, Antoch MP. Circadian sensitivity to the chemotherapeutic agent cyclophosphamide depends on the functional status of the CLOCK/BMAL1 transactivation complex. Proc Natl Acad Sci USA 2005; 102: [PMID: DOI: /pnas ] 108 Karantanos T, Theodoropoulos G, Gazouli M, Vaiopoulou A, Karantanou C, Stravopodis DJ, Bramis K, Lymperi M, Pektasidis D. Association of the clock genes polymorphisms with colorectal cancer susceptibility. J Surg Oncol 2013; 108: 4206 April 21, 2014 Volume 20 Issue 15

108 Mazzoccoli G et al. Colorectal cancer and circadian rhythmicity [PMID: DOI: /jso.23434] 109 Karantanos T, Theodoropoulos G, Gazouli M, Vaiopoulou A, Karantanou C, Lymberi M, Pektasides D. Expression of clock genes in patients with colorectal cancer. Int J Biol Markers 2013; 28: [PMID: DOI: /jbm ] 110 Wang L, Chen B, Wang Y, Sun N, Lu C, Qian R, Hua L. hclock gene expression in human colorectal carcinoma. Mol Med Rep 2013; 8: [PMID: DOI: / mmr ] 111 Zudaire E, Cuesta N, Murty V, Woodson K, Adams L, Gonzalez N, Martínez A, Narayan G, Kirsch I, Franklin W, Hirsch F, Birrer M, Cuttitta F. The aryl hydrocarbon receptor repressor is a putative tumor suppressor gene in multiple human cancers. J Clin Invest 2008; 118: [PMID: DOI: /JCI30024] 112 Feng S, Cao Z, Wang X. Role of aryl hydrocarbon receptor in cancer. Biochim Biophys Acta 2013; 1836: [PMID: DOI: /j.bbcan ] 113 Niestroy J, Barbara A, Herbst K, Rode S, van Liempt M, Roos PH. Single and concerted effects of benzo[a]pyrene and flavonoids on the AhR and Nrf2-pathway in the human colon carcinoma cell line Caco-2. Toxicol In Vitro 2011; 25: [PMID: DOI: /j.tiv ] 114 Xie G, Peng Z, Raufman JP. Src-mediated aryl hydrocarbon and epidermal growth factor receptor cross talk stimulates colon cancer cell proliferation. Am J Physiol Gastrointest Liver Physiol 2012; 302: G1006-G1015 [PMID: DOI: /ajpgi ] P- Reviewers: Hirayama J, Shi Q, Xiong XJ S- Editor: Ma YJ L- Editor: Kerr C E- Editor: Liu XM 4207 April 21, 2014 Volume 20 Issue 15

109 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (5): Colorectal cancer TOPIC HIGHLIGHT Role of cetuximab in first-line treatment of metastatic colorectal cancer Miguel Jhonatan Sotelo, Beatriz García-Paredes, Carlos Aguado, Javier Sastre, Eduardo Díaz-Rubio Miguel Jhonatan Sotelo, Beatriz García-Paredes, Carlos Aguado, Javier Sastre, Eduardo Díaz-Rubio, Center affiliate to the Red Temática de Investigación Cooperativa (RD12/0036/006), Instituto Carlos Ⅲ, Spanish Ministry of Economy and Competitivity. Oncology Department, Hospital Clínico San Carlos, Madrid, Spain Eduardo Díaz-Rubio García, Department of Medicine, School of Medicine, Universidad Complutense de Madrid, Madrid, Spain Author contributions: Sotelo MJ, García-Paredes B, Aguado C, Sastre J and Díaz-Rubio E designed, wrote and approved the final version of the manuscript. Correspondence to: Eduardo Díaz-Rubio, MD, PhD, Department of Medicine, School of Medicine, Universidad Complutense de Madrid, Oncology Department, Hospital Clínico San Carlos. Prof. Martín Lagos, s/n, Madrid, Spain. ediazrubio.hcsc@salud.madrid.org Telephone: Fax: Received: September 20, 2013 Revised: January 1, 2014 Accepted: February 17, 2014 Published online: April 21, 2014 Abstract The treatment of metastatic colorectal cancer (mcrc) has evolved considerably in the last decade, currently allowing most mcrc patients to live more than two years. Monoclonal antibodies targeting the epidermal growth factor receptor (EGFR) and vascular endothelial growth factor play an important role in the current treatment of these patients. However, only antibodies directed against EGFR have a predictive marker of response, which is the mutation status of v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS). Cetuximab has been shown to be effective in patients with KRAS wild-type mcrc. The CRYSTAL study showed that adding cetuximab to FOLFIRI (regimen of irinotecan, infusional fluorouracil and leucovorin) significantly improved results in the first-line treatment of KRAS wildtype mcrc. However, results that evaluate the efficacy of cetuximab in combination with oxaliplatin-based chemotherapy in this setting are contradictory. On the other hand, recent advances in the management of colorectal liver metastases have improved survival in these patients. Adding cetuximab to standard chemotherapy increases the response rate in patients with wild-type KRAS and can thus increase the resectability rate of liver metastases in this group of patients. In this paper we review the different studies assessing the efficacy of cetuximab in the first-line treatment of mcrc Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Cetuximab; First-line; Metastatic colorectal cancer; Colorectal liver metastases; Elderly patients Core tip: This article contains updated data regarding biomarkers of response to epidermal growth factor receptor-targeted therapy and reviews the major studies that have evaluated the efficacy of cetuximab in the first-line treatment of metastatic colorectal cancer (mcrc). We have also compiled the most important data supporting the use of cetuximab in the neoadjuvant treatment of colorectal liver metastases. Finally, we review the current evidence regarding the efficacy and safety of cetuximab in the treatment of elderly patients with mcrc. Sotelo MJ, García-Paredes B, Aguado C, Sastre J, Díaz-Rubio E. Role of cetuximab in first-line treatment of metastatic colorectal cancer. World J Gastroenterol 2014; 20(15): Available from: URL: i15/4208.htm DOI: INTRODUCTION Colorectal cancer is the third most common malignancy in men and the second most common in women worldwide. It is the fourth leading cause of cancer death, ac April 21, 2014 Volume 20 Issue 15

110 Sotelo MJ et al. Cetuximab in first-line metastatic colorectal cancer counting for 8% of global cancer deaths [1]. Approximately 35% of patients have metastatic disease at diagnosis and 20%-50% of patients with stages Ⅱ-Ⅲ develop metastases in the course of the disease, the liver being the most common site of metastatic spread [2]. There is a small subset of patients with metastatic disease isolated in the liver or lungs that can be offered potentially curative surgical treatment. Hepatic resection has become the treatment of choice for patients who have liver metastases metastases only and neoadjuvant chemotherapy currently plays a major role in this setting, increasing the resection rate. However, the vast majority of patients with metastatic colorectal cancer (mcrc) are incurable and can only be offered systemic treatment with palliative intent [3]. In the last decade there have been significant advances in the treatment of mcrc that have changed the natural history of the disease in these patients. Median survival in the era of 5-fluorouracil (5FU) was 6-12 mo [4]. The introduction of oxaliplatin, irinotecan and subsequently biological agents (bevacizumab, cetuximab, panitumumab, aflibercept and regorafenib) made it possible to prolong the survival of patients with mcrc, reaching median survival times in excess of 20 mo [5], so that a significant percentage of these patients now live longer than two years. One of the great challenges of modern oncology is to identify biomarkers to select subsets of patients who will benefit from a particular drug, and thus to develop personalised medicine. In mcrc, the mutation status of v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) predicts response to drugs targeting epidermal growth factor receptor (EGFR) [6-13]. EGFR is a receptor tyrosine kinase that belongs to the ErbB receptor family, which plays an important role in colorectal cancer progression. The binding of ligands will activate the EGFR, resulting in homodimerization and heterodimerization with other members of the ErbB family, which in turn trigger the activation of downstream signalling pathways, finally resulting in cell growth and proliferation. KRAS is a protein that is an essential component of the EGFR signalling cascade [5,8]. There are reports that activating mutations in the KRAS gene, which encode this effector protein involved in EGFR-driven signalling, are present in 35%-40% of colorectal cancers [14,15]. Cetuximab is a chimeric monoclonal antibody that competitively binds to the extracellular domain of EGFR with a higher affinity than its endogenous ligands, blocking EGFR-driven signalling, resulting in inhibition of cell growth and induction of apoptosis [16-18]. There are also reports that cetuximab can mediate antibody-dependent cellular cytotoxicity against tumour cells [16,19]. This monoclonal antibody has been proven effective in patients with KRAS wild-type mcrc [6,7,20,21]. In addition, there is evidence that not only do patients with KRAS mutations not benefit from treatment with cetuximab, but that it could actually have a detrimental effect on them [7,21]. In this paper we review the most relevant clinical trials that have assessed the role of cetuximab in the firstline treatment of mcrc. KRAS AND OTHER BIOMARKERS IN COLORECTAL CANCER KRAS mutation status is a predictor of response to cetuximab that has been validated in several clinical trials. However, the KRAS wild-type status does not guarantee response to cetuximab in all patients [6-8,10,20,21]. This has led to intensive investigation of mechanisms of primary resistance to anti-egfr drugs, which has identified potential molecular markers in the complex signalling pathway driven by the EGFR. While the prognostic value of V-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations in colorectal cancer has been clearly demonstrated [20,22-26], its role as a predictor of response to EGFR-targeted agents is still controversial [12,20,22,24]. On the other hand, there is evidence that neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS) mutations are associated with a lack of response to anti-egfr agents [23,27,28]. In a recent analysis of the PRIME trial, it was found that KRAS and NRAS wild-type mcrc patients showed a significant improvement in progression-free survival (PFS) and overall survival (OS) when treated with FOLFOX-panitumumab compared with FOLFOX alone [hazard ratio (HR) for PFS 0.72, 95%CI: , P < 0.01; HR for OS 0.78, 95%CI: , P = 0.04], whereas this benefit was not observed in patients with KRAS or NRAS mutations (HR for PFS 1.28, 95%CI: , P = 0.32; HR for OS 1.29, 95%CI: , P = 0.31). In this analysis, the BRAF mutation status was not found to be a predictive marker of response [28]. Similarly, recent data from the FIRE-3 study showed that in patients with both wild-type KRAS and NRAS, cetuximab-regimen of irinotecan, infusional fluorouracil and leucovorin (FOLFIRI) significantly improved OS compared with bevacizumab-folfiri (33.1 mo vs 25.9 mo, HR = 0.69; 95%CI: , P = 0.01) [29]. In view of these results, the mutational status of NRAS seems to position itself as a new biomarker that could help us to select responders to anti-egfr therapies. Currently, there are data suggesting that in addition to analysis of KRAS mutation status, the evaluation of EGFR gene copy number, levels of EGFR ligands, BRAF, NRAS, PIK3CA mutations and PTEN loss could also help us achieve a more accurate selection of patients who may benefit from anti-egfr targeted drugs [22,27,30-32], although this needs to be validated in large prospective studies. CETUXIMAB-BASED THERAPY FOR FIRST-LINE TREATMENT OF MCRC Cetuximab in combination with irinotecan-based chemotherapy Data from previous studies that showed an apparent ability of cetuximab to reverse resistance to irinotecan and obtain responses in patients who had previously progressed on irinotecan suggested a potential increased efficiency by combining both drugs [33-36]. This possible 4209 April 21, 2014 Volume 20 Issue 15

111 Sotelo MJ et al. Cetuximab in first-line metastatic colorectal cancer Table 1 Clinical trials of cetuximab in combination with irinotecan-based chemotherapy Clinical trial Type of study KRAS analysis Treatment Response rate R0 resection rate PFS (mo) OS (mo) 67% 19% Folprecht et al [37] Phase Ⅰ/Ⅱ No Cetuximab-Irinotecan/ 5FU/FA 1 Raoul et al [38] Phase Ⅰ/Ⅱ No Cetuximab-FOLFIRI 48% 19.20% CRYSTAL, Van Cutsem et al [6,20] Phase Ⅲ Yes Cetuximab-FOLFIRI vs FOLFIRI 57.3% vs 39.7% (OR = 2.069, P < 0.001) 2 5.1% vs 2% (OR = 2.65, P = ) vs 8.4 (HR = 0.696, P = ) vs 20 (HR = 0.796, P = ) 2 FIRE-3, Phase Ⅲ Yes Cetuximab-FOLFIRI vs Heinemann et al [41] Bevacizumab-FOLFIRI 62 vs 57 (OR = 1.18, P = 0.183) 10 vs 10.3 (HR = 1.06, P = 0.547) 28.7 vs 25 (HR = 0.77, P = 0.017) 1 AIO group regimen of irinotecan, folinic acid and infusional 5FU; 2 Results in wild-type KRAS population. PFS: Progression-free survival; OS: Overall survival; FA: Folinic acid; 5FU: 5-fluorouracil; FOLFIRI: Regimen of irinotecan, infusional fluorouracil and leucovorin; AIO: Arbeitgemeinschaft internistische onkologie; CRYSTAL: CRYSTAL trial (cetuximab combined with irinotecan in first-line therapy for metastatic colorectal cancer); FIRE-3: AIO KRK-0306 trial; KRAS: v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog. synergistic activity led to combinations of cetuximab and irinotecan-based regimens being assessed in first-line treatment of mcrc (Table 1). In a small phase Ⅰ/Ⅱ German study that included 21 patients with EGFR-expressing mcrc who were treated with cetuximab and a combination of irinotecan, infusional 5FU and folinic acid [Arbeitgemeinschaft internistische onkologie (AIO) group regimen], the response rate (RR) was 67% (two patients achieved complete response), median time to progression (TTP) was 9.9 mo and median OS was 33 mo. It is noteworthy that in this study five of the 21 patients (24%) initially had unresectable liver metastases, which became resectable. Four patients underwent surgery with curative intent, achieving R0 resection, and the fifth patient refused surgery [37]. Another phase Ⅰ/Ⅱ study recruited a total of 52 patients with EGFR-expressing mcrc who were treated with cetuximab in combination with FOLFIRI, showing a RR of 48%, a median PFS of 8.6 mo and a median OS of 22.4 mo. In this study 14 of the 52 patients (27%) with initially unresectable metastatic disease (21% with liver metastases, 2% with lung involvement and 4% with metastasis to other sites) were able to undergo curative surgery, achieving R0 resections in 10 cases [38]. Based on these results, the CRYSTAL study (Cetuximab Combined with Irinotecan in First-Line Therapy for Metastatic Colorectal Cancer) was conducted, a randomised phase Ⅲ trial evaluating the efficacy of combining cetuximab-folfiri as compared to FOLFIRI alone in the first-line treatment of mcrc. A total of 1198 patients with EGFR-expressing mcrc were randomised to both arms of the study (599 subjects in each arm). This study conducted a retrospective analysis of KRAS mutation status. Knowing the KRAS mutation status in 89% of the patients, it was observed that the addition of cetuximab to FOLFIRI significantly increased RR (57.3% vs 39.7%, OR = 2.069; 95%CI: , P < 0.001) PFS (9.9 mo vs 8.4 mo, HR = 0.696; 95%CI: , P = ) and OS (23.5 mo vs 20 mo, HR = 0.796; 95%CI: , P = ) in the group of patients with wild-type KRAS. It was also noted that in this group there was a significant increase in the R0 resection rate for cetuximab-folfiri (5.1% vs 2.0%, OR = 2.650; 95%CI: , P = ) [6,20]. In an analysis of data from the CRYSTAL study, Piessevaux et al [39] observed that in wild-type KRAS patients treated with cetuximab-folfiri early tumour response > 20% was significantly associated with better OS (28.3 mo vs 19.6 mo, HR = 0.643; 95%CI: , P = 0.003) than in patients with early tumour response < 20%; however, this was not observed in patients treated with FOLFIRI alone (21.2 mo vs 20.2 mo, HR = 0.814; 95%CI: , P = 0.125) [39]. These findings were confirmed in an analysis of data from the CRYSTAL and OPUS studies, which showed a strong association between early tumour response and long-term outcomes in patients with KRAS wild-type mcrc treated with chemotherapy and cetuximab, compared with patients treated with chemotherapy alone [40]. These results suggest that early tumour response could be useful as a predictor of outcome, although this needs to be confirmed in prospective studies. Despite the proven benefit of cetuximab in combination with FOLFIRI in the first-line treatment of KRAS wild-type mcrc, the optimal sequence of treatment in these patients has not been established. Whether we should start with an anti-egfr and upon progression continue with an anti- vascular endothelial growth factor or vice versa is a question that has yet to be answered in randomised clinical trials. In this sense, preliminary results of the randomised FIRE-3 trial have recently been reported. This study conducted by the AIO group in Germany randomised patients in first-line therapy to cetuximab vs bevacizumab in combination with FOLFIRI. Of 592 patients with wild-type KRAS, 297 were treated with cetuximab-folfiri and 295 with bevacizumab- FOLFIRI. In the ITT population, the RR was comparable in both arms of the study: 62% with cetuximab- FOLFIRI vs 57% with bevacizumab-folfiri (OR = 1.18; 95%CI: , P = 0.183); however, in the population evaluable for response, there was a significant benefit in favour of the cetuximab arm (72.2% vs 63.1%, OR = 1.52; 95%CI: , P = 0.017). There were no statistically significant differences in PFS (10 mo vs 10.3 mo, HR = 1.06; 95%CI: , P = 0.547); however, cetuximab-folfiri significantly prolonged overall sur April 21, 2014 Volume 20 Issue 15

112 Sotelo MJ et al. Cetuximab in first-line metastatic colorectal cancer Table 2 Clinical trials of cetuximab in combination with oxaliplatin-based chemotherapy Clinical trial Type of study KRAS analysis Treatment Response rate (%) R0 resection rate (%) PFS (mo) Arnold et al [48] Phase Ⅰ No Cetuximab-FUFOX 1 57% 4% b/Ⅱ Tabernero et al [49] Phase Ⅱ No Cetuximab-FOLFOX4 72% 21% OPUS, Bokemeyer et al [7,21] Phase Ⅱ Yes Cetuximab-FOLFOX4 vs FOLFOX4 COIN, Maughan et al [23] Phase Ⅲ Yes CetuximabmFOLFOX6/XELOX vs mfolfox6/ XELOX NORDIC Ⅶ, Tveit et al [50] Phase Ⅲ Yes Cetuximab-Nordic FLOX vs Nordic FLOX 3 OS (mo) 57% vs 34% (OR = 2.551, P = ) 2 12% vs 3% (P = ) vs 7.2 (HR = 0.567, P = ) vs 18.5 (HR = 0.855, P = 0.39) 2 64% vs 57% (OR = 15% vs 13 % (P 8.6 vs 8.6 (HR = 0.96, 17 vs 17.9 (HR = 1.04, 1.35, P = 0.049) 2 = 0.74) 2 P = 0.60) 2 P = 0.67) 2 46% vs 47% (OR = 13.4% vs 14.4% vs 8.7 (HR = 1.07, 20.1 vs 22 (HR = 1.14, 0.96, P = 0.89) 2 P = 0.66) 2 P = 0.48) 2 1 FUFOX: AIO group weekly regimen of oxaliplatin, folinic acid and infusional 5FU; 2 Results in wild-type KRAS population; 3 Nordic FLOX: Biweekly regimen of oxaliplatin, bolus 5FU and folinic acid. KRAS: v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog; PFS: Progression-free survival; OS: Overall survival; 5FU: 5-fluorouracil; XELOX: Capecitabine plus oxaliplatin. vival as compared to bevacizumab-folfiri (28.7 mo vs 25 mo, HR = 0.77; 95%CI: 0.62 to 0.96, P = 0.017) [41]. This difference may be explained by various hypotheses. The deepness of response is the percentage of tumour shrinkage observed at the nadir compared with baseline. It was noted that the deepness of response could be a predictor for OS. A recent analysis of data from the CRYSTAL and OPUS trials observed that the median deepness of response was significantly greater in patients treated with chemotherapy and cetuximab as compared to chemotherapy alone (50.9% with FOLFIRIcetuximab vs 33.3% with FOLFIRI alone, P < , and 57.9% with FOLFOX4-cetuximab vs 30.7% with FOLFOX4 alone, P = ); in addition, a statistically significant association was identified between deepness of response and OS in the CRYSTAL study (P < ) and the OPUS study (P < 0.005) [42,43]. It would be interesting to know about this particular point in the FIRE-3 study as well. The data available to date show that the presence of BRAF mutations confers a poor prognosis in patients with mcrc [20,22-26]. A difference in the percentage of patients with mutated BRAF between the two groups in this study may also have contributed to this difference in OS, although this seems unlikely, since the incidence of BRAF mutations is generally low (less than 10%) and not sufficient to explain a difference of four mo in median overall survival. In addition, an imbalance in second-line treatments between the two arms could explain the OS results observed in this study. However, recent data presented by Modest et al [44] at the ESMO 15 th World Congress on Gastrointestinal Cancer reject this possible explanation. The percentages of patients receiving oxaliplatin, bevacizumab and anti-egfr were 63.7%, 46.6% and 15.2% in the cetuximab arm and 62.8%, 17.3% and 41.4% in the bevacizumab arm. Despite the results of the FIRE-3 trial, there are still doubts about when is the right time to use cetuximab or bevacizumab in the treatment of patients with KRAS wild-type mcrc. We must wait for the results of the phase Ⅲ CALGB trial, which compares chemotherapy (FOLFOX or FOLFIRI) with cetuximab or bevacizumab as first-line treatment in this patient group (clinicaltrials.gov/nct ). It is expected to provide us with definitive data on the efficacy of cetuximab and its role in this setting. Cetuximab in combination with oxaliplatin-based chemotherapy Although studies comparing oxaliplatin-based regimens to irinotecan-based regimens in first-line treatment of mcrc have shown that both appear to be equivalent in terms of efficacy [45-47], there are data suggesting that oxaliplatin-based regimens could produce a greater reduction in the size of metastatic lesions and thereby increase the chances of curative surgery [47]. These data coupled with the proven benefit of cetuximab alone or in combination with irinotecan [33-36] make it possible to assess the efficacy of cetuximab in combination with oxaliplatin-based regimens (Table 2). An Ⅰb/Ⅱ phase German study assessed the feasibility of treatment with cetuximab in combination with FUFOX regimen (weekly schedule of oxaliplatin, infusional 5FU and folinic acid) in 49 patients with previously untreated EGFRexpressing mcrc, observing an RR of 57%, a median PFS of 8.1 mo and a median OS of 28.2 mo. In this study, four patients (8%) with initially unresectable liver metastases were able to undergo curative surgery, achieving an R0 resection in 2 cases (4%) [48]. Tabernero et al [49] reported encouraging results from a phase Ⅱ study which included 43 patients with EGFRexpressing mcrc who were treated with cetuximab in combination with FOLFOX4 as first-line treatment. An RR of 72%, a median PFS of 12.3 mo and a median OS of 30 mo were observed. The most important data from this study include the fact that 10 patients (23%) were able to undergo resection with curative intent of previously unresectable metastatic lesions, achieving an R0 resection in 9 of them (21%) April 21, 2014 Volume 20 Issue 15

113 Sotelo MJ et al. Cetuximab in first-line metastatic colorectal cancer The randomised phase Ⅱ OPUS trial (Oxaliplatin and Cetuximab in First-Line Treatment of Metastatic Colorectal Cancer) assessed the efficacy of cetuximab in combination with FOLFOX4 as first-line treatment of mcrc [7,21]. This study included a total of 337 patients who were treated with FOLFOX4 with or without cetuximab. The KRAS mutation status was analysed in 315 patients (93%), of which 179 (57%) were wild-type KRAS. Patients with wild-type KRAS treated with FOLFOX4- cetuximab had a significant increase in RR (57% vs 34%, OR = 2.551, 95%CI: , P = ), PFS (8.3 mo vs 7.2 mo, HR = 0.567, 95%CI: , P = ) and R0 resection rate (12% vs 3%, P = ) as compared to patients with wild-type KRAS treated with FOLFOX4 alone. The OS was higher in the cetuximab- FOLFOX4 arm, although this difference was not statistically significant (22.8 mo vs 18.5 mo, HR = 0.855, 95%CI: , P = 0.39) [21]. One of the possible causes for this lack of statistical significance is the limited sample size. There was a difference of four mo in median OS in favour of the experimental arm, which is clinically relevant, and it is likely that this difference would have reached statistical significance with a larger sample size. Also, it is noteworthy that the OS for first-line treatment can be affected by therapy received after the study (in this study 23% of patients in the control arm received anti- EGFR treatment after the study). While both the CRYSTAL and the OPUS studies demonstrate the clinical efficacy of adding cetuximab to standard chemotherapy in first-line treatment of KRAS wild-type mcrc, two phase Ⅲ randomised trials of cetuximab and oxaliplatin-based chemotherapy did not confirm these results and raise questions about the efficacy of cetuximab in this setting. In the COIN trial (Continuous Chemotherapy plus Cetuximab or Intermittent Chemotherapy), 2445 patients with mcrc were randomised to the 3 treatment arms (continuous chemotherapy, continuous chemotherapy with cetuximab and intermittent chemotherapy), of which 815 patients were assigned to receive continuous chemotherapy and 815 patients to continuous chemotherapy in combination with cetuximab. Of these 1630 patients, the KRAS mutation status was analysed in tumour samples of 1316 (81%) patients, and 565 (43%) showed KRAS mutations. The cetuximab arm included 357 patients with wild-type KRAS, of whom 117 received mfolfox6 and 240 received capecitabine plus oxaliplatin (XELOX). In the KRAS wild-type population, although there was a significant increase in RR in the cetuximab group (64% vs 57%, OR = 1.35, 95%CI: , P = 0.049), no significant differences were found in either resection rate (13% in the control group vs 15% in the cetuximab group, P = 0.74) or PFS (8.6 mo in the control group vs 8.6 mo in the cetuximab group, HR = 0.96; 95%CI: , P = 0.60) or OS (17.9 mo in the control arm vs 17.0 mo in the cetuximab arm, HR = 1.04; 95%CI: , P = 0.67). However, an exploratory analysis in the KRAS wildtype population showed an improvement in PFS with cetuximab in patients receiving mfolfox6 (HR = 0.72, 95%CI: , P = 0.037), but not in patients receiving XELOX (HR = 1.02, 95%CI: , P = 0.88) [23]. The NORDIC Ⅶ trial evaluated the efficacy of cetuximab in combination with a bolus 5FU, folinic acid and oxaliplatin regimen (Nordic FLOX regimen). A total of 566 patients were recruited, who were randomised to 3 treatment arms: Nordic FLOX (arm A), Nordic FLOX with cetuximab (arm B) and Nordic FLOX intermittently with continuous cetuximab (arm C). The KRAS mutation status was evaluated in 498 (88%) patients, of whom 303 were wild-type KRAS (97, 97 and 109 patients in arms A, B and C, respectively). The main comparison to evaluate the efficacy of cetuximab was conducted between arms A and B, showing that adding cetuximab to Nordic FLOX did not provide any benefit in the KRAS wildtype population, the RR being 46% vs 47% (OR = 0.96; 95%CI: , P = 0.89), median PFS 7.9 mo vs 8.7 mo (HR = 1.07; 95%CI: , P = 0.66) and median OS 20.1 mo vs 22 mo (HR = 1.14; 95%CI: , P = 0.48), with Nordic biweekly regimen of oxaliplatin, bolus 5FU and folinic acid (FLOX)-cetuximab and Nordic FLOX, respectively [50]. It has been suggested that a possible explanation for these discrepant results could be that the chemotherapeutic agents combined with fluoropyrimidines may affect the response to cetuximab in different ways and that irinotecan-based regimens may be more effective than oxaliplatin-based regimens in combination with cetuximab. However, the OPUS study results and preclinical data support the synergistic activity of cetuximab and oxaliplatin [51-54]. In addition, a phase Ⅱ study performed by the Central European Cooperative Oncology Group (CECOG) found no significant differences in RR (43% vs 45%, OR = 0.93; 95%CI: ), PFS (8.6 mo vs 8.3 mo, HR = 1.06; 95%CI: , P = ) or OS (17.4 mo vs 18.9 mo, HR = 0.98; 95%CI: , P = ) when comparing combinations of cetuximab with FOLFOX6 or FOLFIRI [55]. Another important observation that can be drawn from these studies is that cetuximab may be more effective with infusional 5FU-based regimens than with bolus 5FU- or capecitabine-based regimens [54]. In the cetuximab arm of the COIN study, the number of patients receiving XELOX exceeded the number of patients receiving mfolfox6, while in the NORDIC Ⅶ study all patients were treated with 5FU bolus-based chemotherapy, which may also have contributed to the negative results of both studies. Furthermore, the COIN study showed increased toxicity when cetuximab was added to XELOX, and the capecitabine dose therefore had to be reduced in that arm of the study. The combination of cetuximab and capecitabine appears to increase the gastrointestinal and dermatological toxicity observed with each of these drugs separately. The reduced intensity of the chemotherapy dose may have contributed to the lack of benefit seen in this study [23] April 21, 2014 Volume 20 Issue 15

114 Sotelo MJ et al. Cetuximab in first-line metastatic colorectal cancer Finally, an analysis of combined data from the CRYS- TAL and OPUS studies confirms the benefit in all efficacy parameters of adding cetuximab to first-line chemotherapy in patients with KRAS wild-type mcrc. In this combined analysis of 845 patients with KRAS wild-type tumours, it was observed that adding cetuximab to standard chemotherapy in this setting significantly improves the RR (OR = 2.16; P < ), PFS (HR = 0.66; P < 0.001) and OS (HR = 0.81; P = ) [24]. CETUXIMAB-BASED THERAPY FOR SPECIAL SITUATIONS Cetuximab plus chemotherapy as neoadjuvant treatment of liver-only metastases In patients with metastatic disease limited to the liver, surgical resection of these metastases is the only potentially curative option, with 5-year survival rates of 45% -55% reported in recent publications [56-58]. In this setting, there is evidence suggesting that treatment with standard chemotherapy regimens based on oxaliplatin or irinotecan in patients with unresectable liver metastases increases the number of candidates for surgical rescue [59-66]. As we described previously, resection rates of 8%-23% for initially unresectable liver metastases were reported in phase Ⅱ studies of patients with an unknown KRAS mutation status, treated with cetuximab and irinotecanor oxaliplatin-based chemotherapy [37,38,48,49]. Moreover, adding cetuximab to standard chemotherapy has proven to increase the likelihood of response in patients with wild-type KRAS, significantly increasing R0 resection rates [20,21]. Despite this, no definitive conclusions can be drawn from these studies, since they were not formally designed to assess resection rate. However, these encouraging results have led to studies assessing the role of neoadjuvant cetuximab in the treatment of liver metastases, showing high rates of R0 resections (Table 3). In the CELIM phase Ⅱ study, 111 patients with unresectable colorectal liver metastases were randomised to cetuximab in combination with FOLFIRI or FOLFOX6. It was observed that for cetuximab-folfox6 the RR was 68%, while for cetuximab-folfiri the RR was 57%; however, this difference was not statistically significant (OR = 1.62; 95%CI: , P = 0.23). The R0 resection rate was 34% in the overall population, 38% in the cetuximab-folfox6 group and 30% in the cetuximab-folfiri group. In a retrospective analysis based on KRAS mutation status, a greater RR was observed in patients with wild-type KRAS (70%) as compared to patients with mutated KRAS (41%); this difference was statistically significant (OR = 3.42; 95%CI: , P = ), with an R0 resection rate of 33% reported in the KRAS wild-type population. In this study, a team of expert surgeons conducted a retrospective review of resectability, observing that the resectability rate increased significantly, from 32% at baseline to 60% after chemotherapy (P < ) [67,68]. In the KRAS wildtype population, there were no significant differences between cetuximab-folfox6 and cetuximab-folfiri in terms of PFS (12.1 mo vs 11.5 mo, HR = 1.09; 95%CI: ) or OS (35.8 mo vs 41.6 mo, HR = 1.01; 95%CI: ) [68]. It should be noted that patients who underwent R0 liver resection showed a significant improvement in OS as compared to patients without R0 resection (53.9 mo vs 27.3 mo, P = 0.002) [69]. In a recent publication by Ye et al [70], 138 patients with KRAS wild-type mcrc and unresectable synchronous liver metastases after resection of the primary tumour were randomised to receive cetuximab plus chemotherapy (mfolfox6 or FOLFIRI) or chemotherapy alone. The addition of cetuximab to chemotherapy was associated with a significant increase in RR (57.1% vs 29.4%, P < 0.01) and R0 resection rate (25.7% vs 7.4%, OR = 4.37; P < 0.01). Treatment with cetuximab plus chemotherapy significantly prolonged PFS (10.2 mo vs 5.8 mo, HR = 0.60; P = 0.004) and OS (30.9 mo vs 21.0 mo, HR = 0.54; P = 0.013) as compared to chemotherapy alone. Furthermore, in the group of patients treated with cetuximab plus chemotherapy, no significant differences were observed between mfolfox6 and FOLFIRI in terms of RR (52.8% vs 59.1%, P = 0.31), PFS (10.1 mo vs 9.1 mo, P = 0.28) or OS (34.8 mo vs 23.1 mo, P = 0.24). The study also found that patients who underwent a liver resection had a significant improvement in OS vs patients who did not undergo surgery, both in the cetuximab plus chemotherapy group (46.4 mo vs 25.7 mo, P = 0007) and in the chemotherapy alone group (36.0 mo vs 19.6 mo, P = 0.016). A higher response rate has been shown to be associated with a higher probability of resection with curative intent [71], and it has been seen that triple- vs doubleagent chemotherapy significantly increases the RR and R0 resection rate [72]. Based on these assumptions, the association of cetuximab with a triple combination of chemotherapeutic agents (5FU, irinotecan and oxaliplatin) was also assessed in order to increase the resectability of colorectal liver metastases. Garufi et al [73] conducted the POCHER phase Ⅱ trial, which assessed the combination of cetuximab with chronomodulated infusion of irinotecan, 5FU, leucovorin and oxaliplatin (chrono-iflo regimen) as neoadjuvant treatment in 43 patients with unresectable colorectal liver metastases. KRAS mutation analysis was performed in 37 patients, showing a high incidence of wild-type KRAS (80%). In the overall population, the RR was 79.1%, reaching a rate of 60% for R0 resections. With a median followup of 22 mo, the estimated median OS was 37 mo, with a 2-year survival rate of 68.2% in the overall population, 80.6% in patients who underwent R0 resection and 47.1% in patients who did not undergo resection of metastases (P = 0.01). In a phase Ⅱ study, 42 patients with mcrc received cetuximab in combination with 5FU, leucovorin, irinotecan and oxaliplatin (FOLFIRINOX regimen) as first-line treatment. Of 40 patients evaluated for KRAS mutation status, 60% were KRAS wild-type. The RR was 80.9% in the overall population, 83.3% in patients with wild April 21, 2014 Volume 20 Issue 15

115 Sotelo MJ et al. Cetuximab in first-line metastatic colorectal cancer Table 3 Clinical trials of neoadjuvant cetuximab in the treatment of liver metastases Clinical trial Type of study KRAS analysis Treatment Response rate R0 resection rate PFS (mo) OS (mo) CELIM, Folprecht et al [67,68] Phase Ⅱ Yes Cetuximab-FOLFOX6 vs cetuximab-folfiri Wild-type KRAS vs mutated KRAS Ye et al [70] Phase Ⅳ Yes Cetuximab-mFOLFOX6/ FOLFIRI vs mfolfox6/ FOLFIRI Cetuximab-mFOLFOX6 vs Cetuximab-FOLFIRI 68% vs 57% (OR = 1.62, P = 0.23) 70% vs 41% (OR = 3.42, P = 0.008) 57.1% vs 29.4% (P < 0.01) 52.8% vs 59.1% (P = 0.31) 38% vs 30% 11.2 vs 10.5 (HR = 1.15, NS) 33% vs 30% 11.9 vs 9.9 (HR = 1.31, NS) 25.7% vs 7.4% (P < 0.01) 10.2 vs 5.8 (HR = 0.6, P = 0.004) 35.7 vs 29.0 (HR = 1.09, NS) 36.1 vs 27.4 (HR = 1.48, NS) 30.9 vs 21 (HR = 0.54, P = 0.013) 10.1 vs 9.1 (P = 0.28) 34.8 vs 23.1 (P = 0.24) POCHER, Garufi et al [73] Phase Ⅱ Yes Cetuximab-Chrono-IFLO Saridaki et al [75] Phase Ⅱ Yes Cetuximab-FOLFIRINOX Chronomodulated infusion of irinotecan, 5FU, leucovorin and oxaliplatin; 2 R0 resection rate in overall population; 3 R0 resection rate in the subgroup of patients with metastatic disease limited to the liver. KRAS: v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog; PFS: Progression-free survival; OS: Overall survival; NS: Not significant; 5FU: 5-fluorouracil; FOLFIRI: Regimen of irinotecan, infusional fluorouracil and leucovorin. type KRAS and 81.3% in patients with mutated KRAS, with no statistical differences between groups. In the overall population, median PFS was 9.5 mo and median OS was 24.7 mo. In the KRAS population, median PFS was slightly higher in the wild-type KRAS group as compared to mutated KRAS group (9.9 mo vs 7.8 mo); the median OS in the mutated KRAS group was 23.1 mo, while it was not reached in the wild-type KRAS group [74]. These promising results have led to the commencement of the PRODIGE 14 randomised phase Ⅱ trial, which is currently ongoing. It will assess resectability in patients with unresectable colorectal liver metastases who will be treated with targeted therapy in combination with a triple-agent (FOLFIRINOX) or double-agent (FOLFOX or FOLFIRI) chemotherapy. The targeted therapy will be chosen according to KRAS mutation status: cetuximab in patients with wild-type KRAS and bevacizumab in mutated KRAS patients (clinicaltrials.gov/nct ). Another small phase Ⅱ study also assessed the firstline effectiveness of cetuximab in combination with FOLFIRINOX in 30 unselected patients with KRAS wild-type mcrc, showing an RR of 70% (with 13.3% complete responses), median TTP of 10.2 mo and a median OS of 30.3 mo. Of all the patients, 11 (37%) underwent R0 secondary resection (10 patients with liver lesions and one patient with metastatic lung disease). In this study, 16 patients had metastatic disease limited to the liver, with a rate of R0 secondary resection in this subgroup of 62% (10/16) [75]. While triple-agent chemotherapy in combination with cetuximab could generate a high response rate and consequently increase the resectability rate, it also appears to be associated with a high incidence of grade 3-4 toxicity [73-75], so we must wait for results of randomised studies with a larger sample size, such as the PRODIGE 14 study, to confirm the efficacy of this combination and obtain further details about its toxicity profile and tolerability. Treatment with cetuximab in elderly patients The goals of chemotherapy in elderly patients with unresectable mcrc are the same as in younger patients, namely to control symptoms and prolong survival. However, elderly patients often have comorbidities and may have impaired organ function associated with age, so we must carefully evaluate the risks and benefits expected from chemotherapy [76,77]. In several clinical trials the number of elderly patients included was small and few have been conducted specifically in this population, so the best available evidence on the efficacy and toxicity of treatment in this patient group mainly derives from subgroup analysis of large phase Ⅲ trials and extrapolating data from the non-elderly population. Studies carried out in the elderly and analysis of combined data and subgroups of phase Ⅲ studies suggest that the benefits of elderly patients with mcrc being treated with combination therapy are similar to those observed in younger patients [78-83]. Data also show that the efficacy of cetuximab-based therapy in elderly patients with previously treated mcrc appears to be similar to that of younger patients, with acceptable tolerability [84,85]. The role of cetuximab in first-line treatment of mcrc in this population has been evaluated in two phase Ⅱ clinical trials conducted by the Spanish Cooperative Group for the Treatment of Digestive Tumours. The first phase Ⅱ study evaluated the efficacy and safety of cetuximab monotherapy as first-line treatment for elderly patients with mcrc. There were a total of 41 patients 70 years, with the KRAS mutation analysis only being possible in 23 of them, showing five KRAS mutated patients and 18 wild-type KRAS. In the overall population, there was a low RR of 14.6%, with a median TTP of 2.5 mo and a median OS of 11.1 mo. In the KRAS population, five KRAS wild-type patients had an objective response, whereas KRAS mutated patients showed no objective response; in addition, seven patients with wild-type KRAS were progression-free at week 12, whereas only one patient with mutated KRAS was progression-free at the same time point; however, these differences were not statistically significant, probably be April 21, 2014 Volume 20 Issue 15

116 Sotelo MJ et al. Cetuximab in first-line metastatic colorectal cancer cause of the small sample size [86]. In another phase Ⅱ study, Sastre et al [87] evaluated the combination of cetuximab with capecitabine in the same setting with the aim of increasing RR. A total of 66 patients 70 years were included, performing KRAS mutation analysis in 58 (88%) of them, which showed an incidence of 50% for both wild-type and mutated KRAS. After 27 patients were included the protocol had to be amended for safety reasons, reducing the dose of capecitabine from 1250 to 1000 mg/m 2 per 12 h. In the overall population, the RR was 31.8%, median PFS was 7.1 mo and median OS was 16.1 mo. The RR and median PFS were significantly higher in the KRAS wild-type as compared to the KRAS mutated group (48.3% vs 20.7%, P = 0.027, and 8.4 mo vs 6.0 mo, P = 0.024), while only a non-significant trend toward a longer OS was observed for KRAS wild-type patients (18.8 mo vs 13.5 mo, P = 0.107). It is important to mention that before dose reduction, the incidence of grade 3-4 toxicity was high, mainly paronychia (29.6%), acneiform rash (29.6%), hand-foot syndrome (22.2%) and diarrhoea (18.5%); however, after dose reduction, the incidence of paronychia and diarrhoea decreased (7.7% and 12.8%, respectively), although the incidence of acneiform rash and hand-foot syndrome was similar (28.2% and 20.5%, respectively). As described previously, the combination of cetuximab and capecitabine appears to increase the toxicity associated with each of these drugs; thus, the high incidence of grade 3-4 toxicity observed in this study may be explained by the additive toxic effect of the combination, rather than the age of patients. Although no definitive conclusions can be drawn from these studies, in elderly patients with KRAS wildtype mcrc, the results appear to be comparable to those observed in younger patients. Therefore, it seems advisable to use cetuximab in combination with chemotherapy, choosing the regimen based on the toxicity profile and assessing each patient individually. CONCLUSION KRAS is a biomarker that has proved useful in selecting patients eligible to receive cetuximab. Currently, NRAS is presented as a new biomarker that could help identify responders to anti-egfr drugs. However, further studies are still needed to identify new biomarkers that, in combination with KRAS, can help us make a more precise selection of suitable patients for EGFR-targeted therapy. The benefit of cetuximab in combination with FOL- FIRI as first-line treatment in patients with KRAS wildtype mcrc has been clearly demonstrated in the CRYS- TAL study, so this combination could be considered as standard. While there is evidence supporting the use of cetuximab in combination with FOLFOX in patients with KRAS wild-type mcrc, given the conflicting results observed in the COIN and NORDIC Ⅶ trials, the use of cetuximab combined with oxaliplatin plus capecitabine or bolus 5FU cannot be recommended. Given the high response rates and high rates of R0 resection observed in selected population studies, cetuximab in combination with standard chemotherapy doublets should be a therapeutic option to consider in patients with unresectable liver-only metastases from KRAS wild-type colorectal cancer. In this setting, the use of triple-agent chemotherapy in combination with cetuximab has shown encouraging results; however, we expect results from studies with a larger sample size to provide more data on efficacy and toxicity. 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Phase III trial of infusional fluorouracil, leucovorin, oxaliplatin, and irinotecan (FOLFOXIRI) compared with infusional fluorouracil, leucovorin, and irinotecan (FOLFIRI) as first-line treatment for metastatic colorectal cancer: the Gruppo Oncologico Nord Ovest. J Clin Oncol 2007; 25: [PMID: DOI: /JCO ] 73 Garufi C, Torsello A, Tumolo S, Ettorre GM, Zeuli M, Campanella C, Vennarecci G, Mottolese M, Sperduti I, Cognetti F. Cetuximab plus chronomodulated irinotecan, 5-fluorouracil, leucovorin and oxaliplatin as neoadjuvant chemotherapy in colorectal liver metastases: POCHER trial. Br J Cancer 2010; 103: [PMID: DOI: /sj.bjc ] 74 Assenat E, Desseigne F, Thezenas S, Viret F, Mineur L, 4218 April 21, 2014 Volume 20 Issue 15

120 Sotelo MJ et al. Cetuximab in first-line metastatic colorectal cancer Kramar A, Samalin E, Portales F, Bibeau F, Crapez-Lopez E, Bleuse JP, Ychou M. Cetuximab plus FOLFIRINOX (ERBIRINOX) as first-line treatment for unresectable metastatic colorectal cancer: a phase II trial. Oncologist 2011; 16: [PMID: DOI: /theoncologist ] 75 Saridaki Z, Androulakis N, Vardakis N, Vamvakas L, Kabouraki E, Kalbakis K, Hatzidaki D, Voutsina A, Mavroudis D, Georgoulias V, Souglakos J. A triplet combination with irinotecan (CPT-11), oxaliplatin (LOHP), continuous infusion 5-fluorouracil and leucovorin (FOLFOXIRI) plus cetuximab as first-line treatment in KRAS wt, metastatic colorectal cancer: a pilot phase II trial. Br J Cancer 2012; 107: [PMID: DOI: /bjc ] 76 Balducci L, Extermann M. Management of cancer in the older person: a practical approach. Oncologist 2000; 5: [PMID: DOI: /theoncologist ] 77 Honecker F, Köhne CH, Bokemeyer C. Colorectal cancer in the elderly: is palliative chemotherapy of value? Drugs Aging 2003; 20: 1-11 [PMID: ] 78 Sastre J, Marcuello E, Masutti B, Navarro M, Gil S, Antón A, Abad A, Aranda E, Maurel J, Valladares M, Maestu I, Carrato A, Vicent JM, Díaz-Rubio E. Irinotecan in combination with fluorouracil in a 48-hour continuous infusion as first-line chemotherapy for elderly patients with metastatic colorectal cancer: a Spanish Cooperative Group for the Treatment of Digestive Tumors study. J Clin Oncol 2005; 23: [PMID: DOI: /JCO ] 79 Feliu J, Salud A, Escudero P, Lopez-Gómez L, Bolaños M, Galán A, Vicent JM, Yubero A, Losa F, De Castro J, de Mon MA, Casado E, González-Barón M. XELOX (capecitabine plus oxaliplatin) as first-line treatment for elderly patients over 70 years of age with advanced colorectal cancer. Br J Cancer 2006; 94: [PMID: DOI: /sj.bjc ] 80 Mitry E, Douillard JY, Van Cutsem E, Cunningham D, Magherini E, Mery-Mignard D, Awad L, Rougier P. Predictive factors of survival in patients with advanced colorectal cancer: an individual data analysis of 602 patients included in irinotecan phase III trials. Ann Oncol 2004; 15: [PMID: DOI: /annonc/mdh267] 81 Goldberg RM, Tabah-Fisch I, Bleiberg H, de Gramont A, Tournigand C, Andre T, Rothenberg ML, Green E, Sargent DJ. Pooled analysis of safety and efficacy of oxaliplatin plus fluorouracil/leucovorin administered bimonthly in elderly patients with colorectal cancer. J Clin Oncol 2006; 24: [PMID: DOI: /JCO ] 82 Sastre J, Aranda E, Massutí B, Tabernero J, Chaves M, Abad A, Carrato A, Reina JJ, Queralt B, Gómez-España A, González-Flores E, Rivera F, Losa F, García T, Sanchez-Rovira P, Maestu I, Díaz-Rubio E. Elderly patients with advanced colorectal cancer derive similar benefit without excessive toxicity after first-line chemotherapy with oxaliplatin-based combinations: comparative outcomes from the 03-TTD-01 phase III study. Crit Rev Oncol Hematol 2009; 70: [PMID: DOI: /j.critrevonc ] 83 Jackson NA, Barrueco J, Soufi-Mahjoubi R, Marshall J, Mitchell E, Zhang X, Meyerhardt J. Comparing safety and efficacy of first-line irinotecan/fluoropyrimidine combinations in elderly versus nonelderly patients with metastatic colorectal cancer: findings from the bolus, infusional, or capecitabine with camptostar-celecoxib study. Cancer 2009; 115: [PMID: DOI: /cncr.24305] 84 Bouchahda M, Macarulla T, Spano JP, Bachet JB, Lledo G, Andre T, Landi B, Tabernero J, Karaboué A, Domont J, Levi F, Rougier P. Cetuximab efficacy and safety in a retrospective cohort of elderly patients with heavily pretreated metastatic colorectal cancer. Crit Rev Oncol Hematol 2008; 67: [PMID: DOI: /j.critrevonc ] 85 Jehn CF, Böning L, Kröning H, Possinger K, Lüftner D. Cetuximab-based therapy in elderly comorbid patients with metastatic colorectal cancer. Br J Cancer 2012; 106: [PMID: DOI: /bjc ] 86 Sastre J, Aranda E, Grávalos C, Massutí B, Varella-Garcia M, Rivera F, Soler G, Carrato A, Manzano JL, Díaz-Rubio E, Hidalgo M. First-line single-agent cetuximab in elderly patients with metastatic colorectal cancer. A phase II clinical and molecular study of the Spanish group for digestive tumor therapy (TTD). Crit Rev Oncol Hematol 2011; 77: [PMID: DOI: /j.critrevonc ] 87 Folprecht G, Gruenberger T, Bechstein W, Lordick T, Lang H, Weitz J, Suedhoff T, Hartmann J, Liersch T, Koehne C. Progression free and overall survival after neoadjuvant treatment of colorectal liver metastases with cetuximab plus FOLFOX or FOLFIRI - Results of the CELIM study. Oncologist 2012; 17: [PMID: DOI: /theoncologist ] P- Reviewers: Cejas P, Improta G S- Editor: Zhai HH L- Editor: A E- Editor: Wu HL 4219 April 21, 2014 Volume 20 Issue 15

121 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (5): Colorectal cancer TOPIC HIGHLIGHT Role of stereotactic body radiotherapy for oligometastasis from colorectal cancer Atsuya Takeda, Naoko Sanuki, Etsuo Kunieda Atsuya Takeda, Naoko Sanuki, Radiation Oncology Center, Ofuna Chuo Hospital, Kanagawa , Japan Etsuo Kunieda, Department of Radiation Oncology, Tokai University, Kanagawa , Japan Author contributions: Takeda A contributed to the manuscript idea, literature search, manuscript writing and final revision of the article; Sanuki N contributed to the manuscript idea and the final revision of the article; Kunieda E contributed to the final revision of the article. Correspondence to: Atsuya Takeda, MD, PhD, Radiation Oncology Center, Ofuna Chuo Hospital, , Ofuna, Kamakura, Kanagawa , Japan. takeda@1994.jukuin.keio.ac.jp Telephone: Fax: Received: September 27, 2013 Revised: December 24, 2013 Accepted: February 20, 2014 Published online: April 21, 2014 Abstract Systemic chemotherapy has enabled prolongation of survival in patients with stage Ⅳ colorectal cancer. This has subsequently increased the relative significance of local therapy for patients with oligometastases because they can be cured by removal of oligometastatic lesions. One of the most frequently reported tumor histologies for oligometastases is colorectal cancer. Resection is the standard therapy in most settings of oligometastases. Recently, studies have shown that stereotactic body radiotherapy (SBRT) may become a treatment option that provides high local control with minimal morbidity. Two-year local control rates following SBRT for hepatic and pulmonary oligometastases are almost over 80% and are even higher for patients treated with high-dose regimens. The indications of SBRT for other metastatic sites or conditions include isolated lymph nodes, spinal and adrenal metastasis, and post-surgical pelvic recurrence. Many retrospective studies have indicated that SBRT for various lesions results in good outcomes with low morbidity, both in the curative and palliative setting. However, few reports with a high level of evidence have indicated the efficacy of SBRT compared to standard therapy. Hereafter, the optimal indication of SBRT needs to be prospectively investigated to obtain convincing evidence Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Oligometastasis; Colorectal cancer; Radiation therapy; Stereotactic ablation body radiation therapy; Local therapy Core tip: Systemic chemotherapy has enabled prolongation of survival in patients with stage Ⅳ colorectal cancer. This has subsequently increased the relative significance of local therapy. Resection is the standard therapy in most settings. Recently, stereotactic body radiotherapy (SBRT) provides high local control with minimal morbidity, both in the curative and palliative setting. The indications of SBRT include liver, lung, isolated lymph nodes, spinal and adrenal metastasis, and postsurgical pelvic recurrence. However, few reports with a high level of evidence have indicated the efficacy of SBRT. Hereafter, the optimal indication of SBRT needs to be prospectively investigated to obtain convincing evidence. Takeda A, Sanuki N, Kunieda E. Role of stereotactic body radiotherapy for oligometastasis from colorectal cancer. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: INTRODUCTION Colorectal cancer (CRC) is currently the second- or thirdleading cause of death from cancer in both genders, and its frequency continues to increase [1,2]. Although there ex April 21, 2014 Volume 20 Issue 15

122 Takeda A et al. SBRT for oligometastasis from CRC ist somewhat different characteristics between colon cancer and rectal cancer [3,4], most articles investigated them together. Among patients with CRC, 20% have metastases at initial presentation, and an additional 25%-50% develop metastases after treatment of early-stage disease. Among patients with an initial finding of metastasis, 50% will have disease limited to the liver, and at the time of death, 20% have liver metastasis only [5]. Rates of metastases are 60%-71% in the liver, 25%-40% in the lung, 5%-10% in bones, 3%-5% in the ovary, 1% in the adrenal gland, and 1% in the central nervous system [6]. Since the late 1990s, development of new agents such as irinotecan, oxaliplatin, and biologic agents targeting either epidermal growth factor receptor or vascular endothelial growth factor has greatly prolonged progression-free survival and overall survival in stage Ⅳ CRC patients [7,8]. Such prolongation of survival attributed to chemotherapy has increased the relative significance of local therapy for patients with limited metastases. An increasing amount of data suggests that curative resection of isolated metastases yields a survival benefit regardless of whether the metastatic site is the liver [9,10], lung [11,12], peritoneum [13,14], ovary [15,16], or extra-regional lymph nodes [17,18]. Although complete surgical resection of these metastases does not result in long-term survival in all patients, select patients can survive for a relatively long period without recurrence. Stereotactic body radiotherapy (SBRT) is a high-precision conformal external-beam radiation technique that ablates the target at extracranial sites with hypofractionated high-dose radiation while sparing the surrounding normal tissue. SBRT results in minimal morbidity and provides high local control rates for medically inoperable stage Ⅰ non-small-cell lung cancer (NSCLC) [19]. Currently, SBRT is considered to be a treatment option for patients with medically inoperable, early-stage NSCLC [20]. However, few adequate studies have evaluated SBRT for pulmonary oligometastases from CRC [21]. In this article, we review the clinical outcomes of SBRT for oligometastases from CRC and discuss the role of SBRT in oligometastasis treatment in general. CONCEPT OF OLIGOMETASTASIS Hellman et al [22] first proposed the idea of an oligometastatic state in These authors suggested that initially, a few metastases from various cancers exist, before the malignant cells acquire widespread metastatic potential. The term oligometastases indicates an intermediate state of cancer that lies between localized disease and widespread metastases. Metastases from solid tumors are regarded as being representative of disseminated cancer and are not considered to be curable. In contrast, evidence has emerged that patients with oligometastases can be cured by resection of these lesions. The most frequently reported tumor histologies in one surgical series of oligometastases were CRC and sarcoma [23]. Resection of liver metastases from CRC patients resulted in 5-year survival rates of 25%-50% [24-26], and a large series of more than 1000 patients reported a 10-year overall survival rate of 22% [27]. Among resection survivors who lived for 10 years, Tomlinson et al [28] demonstrated high disease-specific survival, with only 1 cancer death among 102 patients, reinforcing the concept that this group was truly cured of cancer. For patients with oligometastases from CRC, noninvasive local therapy is appropriate because they are already receiving systematic chemotherapy, are often frail, and may survive only a short time. In addition to resection, local ablation therapies such as cryoablation and radiofrequency ablation (RFA) have been reported to be feasible treatment options. SBRT may become a more suitable treatment option for these patients, because less morbidity occurs following SBRT than that following RFA [29] and resection. CONVENTIONAL RADIOTHERAPY AND SBRT Conventional radiotherapy (i.e., Gy per fraction) results in a tumoricidal effect by means of mitotic death of cancer cells, allowing simultaneous recovery of late sublethal damage of normal tissues. In contrast, SBRT may provide a novel mechanism of radiation-induced damage: data with higher doses per fraction (i.e., Gy per fraction) suggest that, in addition to direct cytotoxicity, a different mechanism involving microvascular damage begins to have a substantial effect on the tumor cell kill [30,31]. Endothelial apoptosis results in microvascular disruption and death of the tissue supplied by that vasculature [30]. Over 1 decade ago, conventional radiotherapy was offered to medically inoperable patients with stage I NSCLC. The prescribed doses of Gy per Gy fractions were administered to these patients. However, those doses were determined based on critical doses of serious radiation pneumonitis rather than sufficient doses to achieve high local control of primary lesions. Therefore, the 5-year local control and overall survival rates were insufficient (40% and 21%, respectively) [32]. In contrast, SBRT enables delivery of sufficiently high doses to a target volume and omission of unnecessary doses to surrounding normal lung tissue. Therefore, 5-year local control and overall survival rates associated with SBRT are relatively high (> 90% and 42%, respectively) [33]. Currently, SBRT is an established treatment option for medically inoperable patients with stage Ⅰ NSCLC. Furthermore, a propensity score-matched analysis revealed superior local control and comparable overall survival of SBRT compared to video-assisted thoracoscopic surgery [34]. In addition, SBRT appears to be less costly than surgery [35]. Quality of life also appears to favor SBRT, because no statistically or clinically significant worsening of any quality of life functioning or symptom scores has been observed in patients with stage Ⅰ NSCLC treated with SBRT [36]. In contrast, quality of life has been shown to be significantly impaired after surgery. Randomized, 4221 April 21, 2014 Volume 20 Issue 15

123 Takeda A et al. SBRT for oligometastasis from CRC controlled trials to compare SBRT to resection are therefore needed. The liver is thought to be a relatively radiosensitive organ. It was difficult to irradiate with a sufficient dose to eradicate tumors without causing lethal radiation-induced liver disease. Therefore, conventional radiotherapy has played a very limited role in the treatment of hepatocellular carcinoma [37]. In contrast, reports of SBRT for hepatocellular carcinoma have been steadily increasing since Although the SBRT literature primarily consists of retrospective, small, single-institution series, SBRT has been associated with high local control rates, most in the range of 70%-90% at 1-2 years [38-40]. In a retrospective analysis of previously untreated hepatocellular carcinoma, SBRT yielded a 3-year overall survival rate of 73%, which is comparable to that of a series treated with surgery or RFA [41]. As for NSCLC and hepatocellular carcinoma, SBRT is expected to play a role in the treatment of oligometastases from CRC. However, negative factors also exist: for example, CRC metastases contain larger proportions of hypoxic cells compared to other tumor types [42], and hypoxia leads to a decrease in radiosensitivity; another is that microscopic extension of oligometastases from CRC may compromise local control [43]. In fact, the local control rates of SBRT in CRC oligometastases are significantly worse than those of oligometastases from other cancers, including NSCLC. Thus, dose escalation should be considered to achieve better local control [44]. SBRT FOR OLIGOMETASTASES BY SITE Several studies have investigated SBRT for oligometastases. However, these have been retrospective and included small sample sizes. Furthermore, patients in these studies were affected by a variety of primary cancers in addition to CRC. Therefore, it is difficult to refer to the outcome of SBRT for oligometastases from CRC exclusively. LIVER METASTASIS Combination chemotherapy and resection of liver metastases has been used to manage patients with confined liver metastasis from CRC. According to the Clinical Practice Guidelines in Oncology by the National Comprehensive Cancer Network (NCCN), resection is the mainstay of treatment for CRC patients with liver metastases only [45]. This combined therapy has resulted in 5-year survival rates of 25%-50% [24-26]. The cumulative 3-year local recurrence-free survival rate following resection of solitary liver metastases was reported to be 88%-95% [46,47]. However, hepatic metastases are resectable in only about 20% of patients [48]. For the remaining 80% of patients, resection is contraindicated due to the presence of diffuse hepatic metastases, non-resectable extrahepatic disease, or impaired liver function. Several technical improvements have been made in diagnostic assessment and treatment strategies for CRC hepatic metastases. For example, modern computed tomography (CT), magnetic resonance imaging, and positronemission tomography-ct techniques enable accurate diagnosis and staging. Furthermore, surgical dissection techniques and potent systemic chemotherapy protocols have been optimized. As a result, even patients with > 3 metastases or with metastases > 5 cm in diameter can be cured with appropriate surgical treatment [28]. Resectability is often limited by an unfavorable anatomical metastatic site, poor function of the remaining hepatic parenchyma, and/or poor general patient condition. Postoperative hepatic function can be predicted more precisely with the aid of CT volumetry. This technique enables prediction of the remaining volume of hepatic tissue after surgery to within 10% of the actual value. Metastases are considered resectable [49] when the following criteria are met: (1) exclusion of a non-resectable extrahepatic tumor manifestation; (2) parenchymal involvement < 75%; (3) < 3 hepatic veins and < 7 hepatic segments involved; (4) no hepatic insufficiency, i.e., no Child B or C cirrhosis; and (5) no severe accompanying diseases. Retrospective studies of SBRT in patients with medically or technically unresectable liver metastases have been performed. Table 1 shows the outcomes of SBRT for liver metastasis from CRC and other origins [50-58]. Various prescribed doses of SBRT were used in these studies. The 2-year local control rates were almost over 80% and were higher for patients treated with high-dose regimens in two studies [51,57]. The 2-year overall survival rates varied from 32%-83%. It is important to note that these outcomes may depend on biased patient selection. And yet, little toxicity was observed. As a local treatment, the use of RFA is controversial. Most centers only ablate tumors in those patients who are deemed unresectable. Patients with large, poorly placed tumors have the highest likelihood of recurrence, regardless of therapy. Retrospective analyses of RFA for liver metastases from CRC have shown broad variability in 2-year local control rates, ranging from 32%-76% [46,59-61], and in 5-year overall survival rates, ranging from 14%-55% [46,61]. A meta-analysis revealed that RFA yielded a higher rate of local intrahepatic recurrence compared to resection (OR = 4.89), although a selection bias was noted [62]. PULMONARY METASTASIS As chemotherapeutic and biological agents have considerably improved outcomes in patients with stage Ⅳ CRC, resection of pulmonary oligometastases is increasingly performed with curative intent. In the NCCN Clinical Practice Guidelines, pulmonary resection is recommended as well as hepatic oligometastatic resection [45]. However, no prospective randomized studies have been performed to validate the efficacy of resection in this setting. Therefore, it is currently not possible to identify which CRC patients may benefit most from this surgical strategy [63]. A meta-analysis revealed that factors correlated with 4222 April 21, 2014 Volume 20 Issue 15

124 Takeda A et al. SBRT for oligometastasis from CRC Table 1 Summary of stereotactic body radiotherapy for liver metastasis Ref. Study Patients (n ) (primary sites) Herfarth et al [50] P Ⅰ CRC (n = 18) others (n = 14) Meta (n ) Wulf et al [51] Retro 39 CRC (n = 23) others (n = 28) Institution 60- Heidelberg Univ MFU (mo) Dose (Gy)/fr Time (d) Prescription specification /1 1 Isocenter, PTV surrounded by 80% isodose LC (mo) OS (mo) Toxicity P value 0% (24) 32% (24) N MT P < 0.01 P Ⅱ 26/1 1 81% (24) 83% (24) Wuerzburg / PTV 58% (24) 81% Univ /3 or interval periphery: 82% (24) (24 for all) 26/1 65% isodose of maximum Katz et al [52] Retro CRC (n = 20) others (n = 49) Rusthoven et al [53] P Ⅰ/Ⅱ CRC (n = 20) others (n = 49) Lee et al [54] P Ⅰ CRC (n = 40) others (n = 28) 174 Rochester Univ - Princess Margaret Hospital 15 50/5f preferred 14 Maximum, PTV surrounded by the 80% isodose /3 < 14 Isocenter, PTV surrounded by 80%-90% isodose /6 (median: 41.8) van der Pool et al [55] Retro CRC (n = 20) 31 Erasmus Univ /3f preferred Rule et al [56] P Ⅰ CRC (n = 12) others (n = 15) 63 Multiinstitution Vautravers- Retro CRC (n = 30) Dewas et al [57] others (n = 15) Scorsetti et al [58] P Ⅱ CRC (n = 29) others (n = 32) 57% (20) 37% (20) N MT N MT P = % (24) 30% (24) Grade 3: 2% > 14 PTV periphery: 71% isodose of maximum 71% (12) 47% (18) N MT 5-6 D95 of PTV 74% (24) 83% (24) N MT 36 Texas 20 3/30 < 14 PTV 59% (24) 56% (24) N MT Southwestern 50/5 17 periphery, 89% (24) 67% (24) Univ 60/ %-85% isodose of maximum 100% (24) 50% (24) 62 Centre Oscar 14 40/ PTV 86% (12) 48% N MT P = 0.07 Lambret 45/3 (mean: periphery, 80% 100% (12) (24 for all) 9) isodose of the CRC (n = 30) maximum 86% (12) P = 0.07 others (n = 15) 100% (12) 76 Humanitas /3 3 Mean dose to 90.6% (24) 37% (24) N MT Cancer PTV Center MFU: Median follow up duration; LC: Local control rate; OS: Overall survival rate; P X: Phase X; retro: Retrospective; CRC: Colorectal cancer; fr: Fractions; PTV: Planning target volume; Dx: The dose delivered to x%; N MT: No major toxicity. better survival included a prolonged disease-free interval between primary tumor and metastatic spread, normal pre-thoracotomy carcinoembryonic antigen levels, absence of thoracic node involvement, and a single pulmonary lesion [64]. Of 44 patients with 3 lesions and a < 1-year disease-free interval, 0 were cured by surgery. In contrast, recurrence-free survival was 49% at 3 years for patients with 1 lesion and a disease-free interval > 1 year. Therefore, medical management alone should be considered standard for patients who have 3 pulmonary metastases and a < 1-year disease-free interval [65]. Crude local recurrence rates following resection of oligometastasis from CRC have been reported to be 19.5%-28% [66-68]. Local recurrence may occur even in cases with pathologically negative margins [69]. A wide surgical margin around oligometastasis from CRC is required to prevent local recurrence, because satellite tumor cells are often present [68]. For medically inoperable patients with limited pulmonary metastases from CRC, SBRT may be administered. Table 2 shows the outcomes of SBRT for pulmonary metastases from CRC and other origins [44,70-80]. SBRT was given at various prescribed doses. The local control rates were almost over 80%, and the 2-year overall survival rates ranged from 33%-86%. These outcomes may have depended on patient selection. With respect to toxicities, grade 3 radiation pneumonitis was observed in only 0%-8% of patients. No other toxicities were observed. Widder et al [78] compared outcomes after SBRT with those after pulmonary metastasectomy in patients with pulmonary oligometastasis. In their institution, patients were offered pulmonary metastasectomy as the first choice and SBRT in cases that they considered to be less suitable surgical candidates. Patients treated with SBRT had more unfavorable prognostic factors: they were significantly older, had a shorter metastasis-free interval, and a different distribution of primary tumor origins; thus, they were regarded as having a worse prognosis overall. Despite this selection bias, survival after SBRT was no worse than that after pulmonary metastasectomy. Prospective comparative studies are therefore required to define the role of both SBRT and pulmonary metastasectomy in oligometastatic disease. Among minimal ablation techniques, RFA is the most 4223 April 21, 2014 Volume 20 Issue 15

125 Takeda A et al. SBRT for oligometastasis from CRC Table 2 Summary of stereotactic body radiotherapy for pulmonary metastasis Ref. Study Patients (n ) (primary sites) Wulf et al [70] Retro CRC (n = 4) others (n = 37) Okunieff et al [71] Retro CRC (n = 14) others (n = 35) Norihisa et al [72] Retro CRC (n = 14) others (n = 35) Meta (n ) Institution MFU (mo) 51 Wuerzburg Univ 125 Rochester Univ. Kim et al [73] Retro CRC (n = 13) 18 Korea Cancer Center Rusthoven et al [74] P I/II CRC (n = 9) others (n = 29) Takeda et al [44] Retro CRC (n = 15) others (n = 19) CRC (n = 21) others (n = 23) Dose (Gy)/ /3 or 26/1 Time (d) 2-3 interval 19 Oct times per week 43 Kyoto Univ / (med: 12) 63 multiinstitution Ofuna Chuo Hospital Oh et al [75] Retro Samsung Medical Center CRC, HCC (n = 16) others (n = 51) Ricardi et al [76] Retro Giovanni Battista Univ Inoue et al [77] Retro Hokkaido Univ. Widder et al [78] Retro CRC (n = 31) others (n = 11) Inoue et al [79] Retro CRC (n = 37) others (n = 50) Groningen Univ Miyakojima IGRT Clinic Prescription specification PTV periphery: 65% isodose of maximum LC (mo) OS (mo) Toxicity P value 80% (24) 33% (24) N MT Isocenter 91% (24) 38% (24) Grade 3 pleural effusion: 2% Isocenter 90% (24) 84.3% Grade 3 RP: 3% (24) /3 3 PTV periphery: 75%-80% isodose of maximum 53% (24) 76% (24) N MT /3 < 14 Isocenter, PTV 96% (24) 39% (24) Grade 3 RP: 8% surrounded by 80%-90% isodose 29 May-50 5 PTV 72% (24) - N MT P < 0.05 periphery: 15 75%-80% 94% (24) - isodose of maximum /4-5 - PTV 92% (24) 57% (24) Grade 5 RP: 2% periphery: 75%-80% isodose of 81% (24) P = 0.01 maximum 100% (24) 20 26/1 or 3 PTV 89% (24) 66.5% (24) Grade 3 RP: 2% 36-45/3 periphery: 25 Apr % isodose 100% (24) 80% (24) N MT of maximum isocenter 43 3/8/ PTV 94% (24) 86% (24) - periphery: adapted risk of toxicity 15 48/4, 52-60/4 or 50/ % (24) 47% (24) Grade 3 RP: 6% Grade4 RP: 1% MFU: Median follow up duration; LC: Local control rate; OS: Overall survival rate; P X: Phase X; retro: Retrospective; CRC: Colorectal cancer; fr: Fractions; PTV: Planning target volume; Dx: The dose delivered to x%; N MT: No major toxicity; RP: Radiation pneumonitis. frequently used method for pulmonary oligometastasis. In a prospective multicenter trial [81], RFA yielded a confirmed complete response rate of 88% (in both primary and metastatic lesions) and promising overall and cancerspecific survival outcomes. Retrospective analyses of RFA for pulmonary metastases from CRC have shown that the 2-year local control rates in all tumors, tumors < 3 cm, and tumors > 3 cm were 56%-80%, 69%-87%, and 19%-32% [82,83], respectively, and that the 2-year overall survival rate was 34%-68% [82-84]. ISOLATED LYMPH NODE METASTASIS Retroperitoneal recurrence occurs in 15% of colon cancer cases and 5% of rectal cancer cases [85], and isolated retroperitoneal recurrence occurs in approximately 1% of all CRC patients following curative surgery [13,18]. Favorable results have been reported for curative surgical resection for isolated retroperitoneal lymph node recurrence of CRC [13,18]. However the indication for resection is limited. Surgery for metastatic retroperitoneal lymph nodes is not feasible when (1) a recurrent retroperitoneal tumor is encased in or involves major vascular structures such as the superior mesenteric artery, celiac axis, and aorta; (2) the tumor invades adjacent organs such as the pancreas, bile duct, and duodenum; or (3) the patient has a poor performance status or comorbid disease [13,18]. Even when lesions are localized, surgical resection is not widely accepted due to their relative rarity, high associated postoperative morbidity, and poor prognosis. In addition, the operative morbidity rate is high at 30%, which includes abscess, phlebitis, pneumonia, intestinal obstruction, and bladder 4224 April 21, 2014 Volume 20 Issue 15

126 Takeda A et al. SBRT for oligometastasis from CRC leakage [13,18]. The role of curative radiotherapy for isolated lymph node metastasis is also controversial. In a study that evaluated SBRT for isolated lymph node metastasis from CRC, preliminary results from 7 patients who received doses of Gy in 3 fractions indicated local recurrence and Grade 4 intestinal obstruction in 1 patient each [86]. Furthermore, the 3-year overall survival rate was 71%. The indication of SBRT in this setting is also limited because the lesions are often adjacent to the gastrointestinal tract. Considering the risk of gastrointestinal toxicities, conventional fractionated radiotherapy may be a better choice. Chemoradiotherapy with prescribed doses of 55.8bed Gy in 2020 escribed doses of 55.8bed doses of 55.8 was 71% results, with a 3-year overall survival rate of 65% and no gastrointestinal toxicity Grade 3 [87]. METASTASIS OF OTHER REGIONS SBRT has played a limited palliative role in the treatment of the sites described above, and may not contribute to improved survival. The indications of SBRT remain to be determined and should be considered based on the condition of each individual patient. Among patients treated with radical surgery for rectal cancer, 20%-50% develop loco-regional recurrence [88,89]. Most patients are not candidates for curative resection of recurrent pelvic disease, and even when radical surgery is possible, the 5-year survival rate after reoperation is < 35%. When no treatment is given, patients with locally recurrent rectal cancer have a median survival of 8 mo and suffer from severe symptoms, particularly pain, resulting in an extremely poor quality of life [89-91]. In most patients, radiotherapy and chemotherapy provide only temporary symptom relief. Kim et al [92] reported that 23 patients with recurrent rectal cancer were treated with SBRT at a median total dose of 39 (range, 30-51) Gy in 3 fractions. The 4-year overall survival and local control rates were 25% and 74%, respectively. Grade 4 rectal perforation was reported in 1 patient. Abusaris et al [93] reported that symptom relief was observed in 96% in 27 patients who were re-irradiated with SBRT after conventional radiotherapy. In patients with spinal metastasis, conventional radiotherapy is standard palliative therapy. In contrast, SBRT enables irradiation using a higher biologically effective dose compared to conventional radiotherapy. The goal of SBRT is, therefore, aimed at maximizing both local tumor and pain control. Local control appears to be excellent, with crude rates of 81%-94% [94], although the prescribed doses vary significantly among series. A multi-institutional study revealed that caution must be used when treating with 20 Gy/fraction, particularly for patients with lytic tumors, spinal misalignment, and baseline vertebral compression fracture [95]. The incidence of adrenal metastases from CRC is approximately 1% [6]. With continuing progress in imaging techniques, an increasing number of adrenal metastases can be detected incidentally during follow-up or at the time of initial presentation. Open surgery represents the standard approach. Radiotherapy has been limited to palliation of painful adrenal metastases from lung cancer [96]. Radiotherapy contributes to prolonged survival in these patients [97]. Results of SBRT for adrenal metastasis from various origins have been reported: the 2-year local control rate was 32% with a median total dose of 32 Gy in 4 fractions [98] and 90% with a median total dose of 36 Gy in 3 fractions [99], without any Grade 3 toxicities. FUTURE PERSPECTIVE Many studies of SBRT for oligometastasis were retrospective and consisted of small sample sizes. In addition, patients in these studies were often affected by various types of primary cancer. We do not think the reasons for the small sample sizes and heterogeneous characteristics are uncommon indications or negative outcomes of SBRT for oligometastasis. In fact, these studies showed an improvement of survival and quality of life with low morbidity. In the future, we should evaluate whether SBRT is a valid treatment modality with a higher evidence level. For curative intent, dose escalation should be attempted for patients with pulmonary and hepatic oligometastasis to achieve better local control and subsequently to improve survival. SBRT outcomes may conceivably be comparable to those of surgery, with less morbidity in some patients. For palliative intent, optimal indications need to be defined. SBRT may have nearly equivalent efficacy compared to resection, with a shorter treatment duration and hospital stay, as well as a better quality of life. These questions should be addressed in future studies. CONCLUSION Many retrospective studies indicate that SBRT for various lesions achieves good outcomes with low morbidity, both in curative and palliative settings. However, few reports with a high evidence level have compared the efficacy of SBRT to that of standard therapy. Moving forward, we should prospectively investigate the indications for SBRT in robust studies. REFERENCES 1 Jemal A, Siegel R, Ward E, Murray T, Xu J, Thun MJ. Cancer statistics, CA Cancer J Clin 2007; 57: [PMID: ] 2 Verdecchia A, Francisci S, Brenner H, Gatta G, Micheli A, Mangone L, Kunkler I. Recent cancer survival in Europe: a period analysis of EUROCARE-4 data. Lancet Oncol 2007; 8: [PMID: DOI: / S (07) ] 3 Kornmann M, Staib L, Wiegel T, Kron M, Henne-Bruns D, Link KH, Formentini A. 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130 Takeda A et al. SBRT for oligometastasis from CRC ly curative surgery. Cancer 1983; 52: [PMID: ] 89 Pilipshen SJ, Heilweil M, Quan SH, Sternberg SS, Enker WE. Patterns of pelvic recurrence following definitive resections of rectal cancer. Cancer 1984; 53: [PMID: ] 90 McCall JL, Cox MR, Wattchow DA. Analysis of local recurrence rates after surgery alone for rectal cancer. Int J Colorectal Dis 1995; 10: [PMID: ] 91 McDermott FT, Hughes ES, Pihl E, Johnson WR, Price AB. Local recurrence after potentially curative resection for rectal cancer in a series of 1008 patients. Br J Surg 1985; 72: [PMID: ] 92 Kim MS, Choi C, Yoo S, Cho C, Seo Y, Ji Y, Lee D, Hwang D, Moon S, Kim MS, Kang H. Stereotactic body radiation therapy in patients with pelvic recurrence from rectal carcinoma. Jpn J Clin Oncol 2008; 38: [PMID: DOI: / jjco/hyn083] 93 Abusaris H, Hoogeman M, Nuyttens JJ. Re-irradiation: outcome, cumulative dose and toxicity in patients retreated with stereotactic radiotherapy in the abdominal or pelvic region. Technol Cancer Res Treat 2012; 11: [PMID: ] 94 Sahgal A, Bilsky M, Chang EL, Ma L, Yamada Y, Rhines LD, Létourneau D, Foote M, Yu E, Larson DA, Fehlings MG. Stereotactic body radiotherapy for spinal metastases: current status, with a focus on its application in the postoperative patient. J Neurosurg Spine 2011; 14: [PMID: DOI: / SPINE091005] 95 Sahgal A, Atenafu EG, Chao S, Al-Omair A, Boehling N, Balagamwala EH, Cunha M, Thibault I, Angelov L, Brown P, Suh J, Rhines LD, Fehlings MG, Chang E. Vertebral compression fracture after spine stereotactic body radiotherapy: a multiinstitutional analysis with a focus on radiation dose and the spinal instability neoplastic score. J Clin Oncol 2013; 31: [PMID: DOI: /JCO ] 96 Short S, Chaturvedi A, Leslie MD. Palliation of symptomatic adrenal gland metastases by radiotherapy. Clin Oncol (R Coll Radiol) 1996; 8: [PMID: ] 97 Oshiro Y, Takeda Y, Hirano S, Ito H, Aruga T. Role of radiotherapy for local control of asymptomatic adrenal metastasis from lung cancer. Am J Clin Oncol 2011; 34: [PMID: DOI: /COC.0b013e3181dbb727] 98 Scorsetti M, Alongi F, Filippi AR, Pentimalli S, Navarria P, Clerici E, Castiglioni S, Tozzi A, Reggiori G, Mancosu P, Ricardi U. Long-term local control achieved after hypofractionated stereotactic body radiotherapy for adrenal gland metastases: a retrospective analysis of 34 patients. Acta Oncol 2012; 51: [PMID: DOI: / X ] 99 Casamassima F, Livi L, Masciullo S, Menichelli C, Masi L, Meattini I, Bonucci I, Agresti B, Simontacchi G, Doro R. Stereotactic radiotherapy for adrenal gland metastases: university of Florence experience. Int J Radiat Oncol Biol Phys 2012; 82: [PMID: DOI: /j.ijrobp ] P- Reviewers: Chow J, Tang D, Vinh-Hung V S- Editor: Gou SX L- Editor: A E- Editor: Wu HL 4229 April 21, 2014 Volume 20 Issue 15

131 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. TOPIC HIGHLIGHT WJG 20 th Anniversary Special Issues (5): Colorectal cancer Molecular and prognostic heterogeneity of microsatellite-unstable colorectal cancer Jung Ho Kim, Gyeong Hoon Kang Jung Ho Kim, Department of Pathology, SMG-SNU Boramae Medical Center, Seoul , South Korea Gyeong Hoon Kang, Department of Pathology, Seoul National University College of Medicine, Seoul , South Korea Gyeong Hoon Kang, Laboratory of Epigenetics, Cancer Research Institute, Seoul National University College of Medicine, Seoul , South Korea Author contributions: Kim JH performed the literature review and drafted the article; Kang GH revised and edited the article; all of the authors have read and approved the final version of the article. Supported by The National R&D Program for Cancer Control funded by the Ministry of Health and Welfare, South Korea, No ; the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIP), No ; Priority Research Centers Program through the NRF grant funded by the Ministry of Education, Science and Technology (MEST), South Korea, No ; and the Mid-career Researcher Program through the NRF grant funded by MEST, No Correspondence to: Gyeong Hoon Kang, MD, PhD, Professor, Department of Pathology, Seoul National University College of Medicine, 103 Daehak-ro, Jongno-gu, Seoul , South Korea. ghkang@snu.ac.kr Telephone: Fax: Received: September 25, 2013 Revised: January 30, 2014 Accepted: February 20, 2014 Published online: April 21, 2014 Abstract Colorectal cancers (CRCs) with a high level of microsatellite instability (MSI-H) are clinicopathologically distinct tumors characterized by predominance in females, proximal colonic localization, poor differentiation, mucinous histology, tumor-infiltrating lymphocytes, a Crohn s-like lymphoid reaction and a favorable prognosis. In terms of their molecular features, MSI-H CRCs are heterogeneous tumors associated with various genetic and epigenetic alterations, including DNA mismatch repair deficiency, target microsatellite mutations, BRAF mutations, a CpG island methylator phenotype-high (CIMP-H) status, and a low level of genomic hypomethylation. The molecular heterogeneity of MSI-H CRCs also depends on ethnic differences; for example, in Eastern Asian countries, relatively low frequencies of CIMP-H and BRAF mutations have been observed in MSI-H CRCs compared to Western countries. Although the prognostic features of MSI-H CRCs include a favorable survival of patients and low benefit of adjuvant chemotherapy, there may be prognostic differences based on the molecular heterogeneity of MSI-H CRCs. Here, we have reviewed and discussed the molecular and prognostic features of MSI-H CRCs, as well as several putative prognostic or predictive molecular markers, including HSP110 expression, beta2-microglobulin mutations, myosin 1a expression, CDX2/CK20 expression, SMAD4 expression, CIMP status and LINE-1 methylation levels Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Colorectal cancer; Microsatellite instability; DNA mismatch repair; DNA methylation; CpG islands; Prognosis; Adjuvant chemotherapy Core tip: A high level of microsatellite instability (MSI-H) is a known molecular indicator of a favorable prognosis and low benefit of 5-fluorouracil-based adjuvant chemotherapy in patients with colorectal cancer (CRC). However, MSI-H CRCs are molecularly heterogeneous tumors, which are characterized by DNA mismatch repair deficiency and various genetic and epigenetic alterations. Therefore, we hypothesized that MSI-H CRCs can be divided into prognostic subgroups based on the molecular heterogeneity. This article provides an up-todate review concerning the underlying molecular features of MSI-H CRCs and potential prognostic or predictive molecular markers for MSI-H CRCs. Kim JH, Kang GH. Molecular and prognostic heterogeneity of 4230 April 21, 2014 Volume 20 Issue 15

132 Kim JH et al. Microsatellite-unstable CRC microsatellite-unstable colorectal cancer. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: org/ /wjg.v20.i INTRODUCTION Microsatellite instability (MSI) is a unique molecular alteration induced by deficiencies in the DNA mismatch repair (MMR) system and is characterized by unstable (length-changeable) microsatellites, a type of simple DNA sequence repeat. The MSI phenotype has been regarded as one of the main molecular subtypes of colorectal cancers (CRCs) and accounts for 12%-20% and 6%-13% of CRCs in Western and Eastern countries, respectively [1-6]. Hereditary CRCs with a high level of MSI (MSI-H) constitute approximately 3%-5% of all CRCs and arise exclusively in patients with Lynch syndrome. Lynch syndrome was formerly called hereditary nonpolyposis colorectal cancer and is caused by a germline mutation in at least one of the MMR genes (MLH1, MSH2, PMS2, and MSH6), frequently resulting in the development of early-onset malignancies, including CRC and endometrial cancer [7,8]. Sporadic MSI-H CRCs account for approximately 3%-15% of all CRCs and develop mainly as a result of inactivation of the MLH1 gene via promoter CpG island hypermethylation [9]. MSI status in CRCs can be determined by DNA testing using microsatellite markers, and five microsatellite markers recommended by the National Cancer Institute (NCI) workshop have been officially used for MSI analysis: BAT25, BAT26, D2S123, D5S346 and D17S250 [10]. In DNA analysis using these NCI markers, instability observed in two or more of the five markers corresponds to MSI-H. MSI-H can be interpreted as the presence of MSI. In contrast, a low level of MSI (MSI-L), which is assigned when only one unstable marker is detected, is not regarded as a true MSI-positive status. Microsatellite-stable (MSS) status can be assigned when all of the markers show stability. Immunohistochemistry (IHC) for MMR proteins can be applied as a screening test or a supportive test for MSI analysis. MSI-H CRC is known to have distinct clinicopathological and molecular features, including preferential localization in the proximal colon, a less advanced cancer stage, extracellular mucin production, medullary carcinoma and poorly differentiated carcinoma, tumorinfiltrating lymphocytes, a Crohn s-like lymphoid reaction, and a BRAF V600E mutation [4,11-14]. In addition, and more importantly, it has been consistently reported that MSI-H CRC is associated with favorable survival and chemotherapy resistance. In a considerable number of previous studies, patients with MSI-H CRC demonstrated a significantly better survival than MSS/MSI-L CRC patients [15,16], whereas the beneficial effect of 5-fluorouracil (5-FU)-based adjuvant chemotherapy in patients with MSI-H CRC has been controversial [15-19]. These prognostic features of MSI-H CRCs have been increasingly reported, and MSI is currently regarded as a molecular marker indicating favorable prognosis for CRCs [20]. However, because MSI-H CRCs are characterized by various underlying molecular changes, including a defective MMR (dmmr) system and genetic and epigenetic alterations, it is likely that molecular factors for the stratification of patient prognosis and prediction of chemotherapy response in MSI-H CRCs could be identified. On the basis of this hypothesis, we reviewed the literature and provide information about several putative prognostic molecular factors for MSI-H CRCs. MOLECULAR HETEROGENEITY OF MSI-H CRC DNA MMR deficiency Germline mutation or sporadic methylation of MMR genes: As described above, MSI is caused by the inactivation of at least one of the MMR genes. The inactivation of MMR genes can be induced by a germline mutation or by promoter CpG island hypermethylation. Germline mutation of MMR genes, including MLH1, MSH2, PMS2, or MSH6, represents a major cause of hereditary MSI-H CRCs in Lynch syndrome. Among these MMR genes, germline mutations in the MLH1 or MSH2 genes account for the majority of Lynch syndrome CRCs [21]. Promoter methylation of MMR genes is a major cause of sporadic MSI-H CRCs and exclusively involves the MLH1 gene. MLH1 promoter methylation is closely associated with the CpG island methylator phenotype (CIMP) in sporadic CRCs and has been used as one of the major molecular markers for CIMP determination in CRCs [4,22-24]. It is also generally expected that all of the MLH1-methylated MSI-H CRCs are CIMP-high (CIMP-H) tumors, although discordance between MLH1 methylation and CIMP status can be observed in a small subset of MSI-H CRCs [25]. A detailed review and discussion concerning the molecular basis and prognostic implication of CIMP in MSI-H CRCs will be presented in the following sections. Constitutional (germline) epimutation of MMR genes: Promoter methylation of MSH2 owing to germline deletion of 3 exons in the EPCAM gene (also known as the TACSTD1 gene), which is located in the region immediately upstream of MSH2, was recently identified as a cause of MSI-H CRC in Lynch syndrome [26,27]. This molecular alteration is associated with a small subset of patients with Lynch syndrome and is also called the MSH2 epimutation because of its unique feature of heritable constitutional epigenetic change [28,29]. In addition, constitutional epimutation of the MLH1 gene has also been found in a few patients with Lynch syndrome [30-34]. Although it has been reported that an association between MLH1 epimutation and a family history of CRC is not evident [30], the clinical and pathological significance of epimutations in MMR genes in hereditary MSI-H CRCs 4231 April 21, 2014 Volume 20 Issue 15

133 Kim JH et al. Microsatellite-unstable CRC remains unclear. According to our data, MLH1 methylation-positive/cimp-negative tumors account for 7.3% of MSI-H CRCs [25], and these cases were associated with an early age of onset and favorable survival. It cannot be excluded that MLH1 epimutation-associated MSI-H CRCs may be included in these MLH1 methylation-positive/cimp-negative MSI-H CRCs, and the predominance of young patients can imply the presence of MLH1 epimutation carriers. Thus, additional studies should be performed to investigate the detailed epidemiology and clinical implications of MMR gene epimutations in MSI-H CRC. Expression profile of MMR proteins: Normal DNA MMR function is executed by MMR protein complexes composed of heterodimers of MutL homologues (the MLH or PMS series) or MutS homologues (the MSH series). The major role of the human DNA MMR system is performed by the MutSα and MutLα complexes. The MutSα complex comprises a MSH2-MSH6 heterodimer, whereas the dimer of MLH1 and PMS2 forms the MutLα complex [21,35]. These complexes are essential components of the human DNA MMR machinery, and defects in any one of these four MMR proteins lead to a dysfunctional MMR system and ultimately result in MSI. Therefore, loss of expression of MMR proteins can serve as a molecular hallmark of a dmmr system and the MSI-H status in tumors. IHC for MMR proteins is a simple and valuable tool for the detection of dmmr CRC and can be helpful for investigating the underlying molecular alteration and hereditary/sporadic status of MSI-H CRCs. The immunohistochemical profile of four MMR proteins in MSI-H CRCs can be summarized as four expression phenotypes: MLH1-negative/PMS2- negative, PMS2-negative only, MSH2-negative/MSH6- negative, and MSH6-negative only [8,36]. These four phenotypes most likely represent inactivation of MLH1, PMS2, MSH2, and MSH6, respectively. The majority of MSI-H CRCs are induced by inactivation of MLH1 or MSH2, whereas inactivation of PMS2 or MSH6 causes only a minor portion of MSI-H CRCs. Interestingly, an inactivating mutation or methylation of MLH1 is accompanied by PMS2 loss, and inactivation of MSH2 is combined with the loss of MSH6 expression due to their heterodimer structures. However, inactivation (mainly through germline mutations) of PMS2 or MSH6 does not accompany a loss of MLH1 or MSH2 expression, respectively [8,36]. Although IHC for MLH1 and MSH2 has generally been used for the screening of MMR status in CRCs, the inclusion of screening for PMS2 and MSH6 would compensate for the equivocal results of MLH1 or MSH2 IHC and aid in the detection of rare MSI-H CRCs with germline mutations of PMS2 or MSH6. Genetic alterations Microsatellite mutations: A recent study elucidating the molecular landscape of CRC by The Cancer Genome Atlas Project reported that the hypermutated phenotype mainly overlaps with MSI-H status in CRCs [37]. This finding is not surprising because MSI-H status tumors are highly vulnerable to insertions or deletions in microsatellite sequences, as described above. In CRCs with MSI, many types of genetic mutations can occur, but the majority of mutations that develop under MSI-H status are frameshift mutations because the instability of microsatellite sequences in coding regions can alter entire reading frames adjacent to the insertion or deletion point. Previous investigations have reported frameshift mutations of various genes caused by the instability of microsatellites in MSI-H CRCs. The target genes for microsatellite mutations in MSI-H CRCs include TGFBR2, BAX, ACVR2, IGF2R, BLM, MSH3, MSH6, E2F4, PTEN, AIM2, CASPASE5, MBD4, TCF4, STK11, RAD50, CHK1, AXIN2, WISP3, B2M, MYO1A and CDX2 [38-54]. These genes are known to be associated with important biological functions such as signal transduction, apoptosis regulation, cell cycle regulation, cell proliferation, cell differentiation, and DNA MMR; therefore, loss-offunction mutations in these genes can critically contribute to tumorigenesis. However, although the biological and clinical implications of mutations in these genes in CRCs have been explored, their potential use as prognostic markers or therapeutic targets has not been established. For instance, although mutations in TGFBR2 and BAX, which are representative target tumor suppressor genes for microsatellite mutations in MSI-H CRCs, have been previously analyzed to determine their prognostic implications, conflicting results have been reported. A few groups have reported that TGFBR2 and BAX mutations were associated with a favorable prognosis in MSI-H CRCs [55,56], whereas other investigators did not find a prognostic significance of these mutations in MSI-H CRCs [57,58]. Interestingly, microsatellite alterations in intronic regions could induce mutations in genes such as HSP110 and MRE11 in MSI-H CRCs [59,60]. In the recent study by Dorard et al [59], deletions of the T17 mononucleotide repeat located in intron 8 of the HSP110 gene were shown to lead to exon 9 skipping and the production of truncated mutant proteins of HSP110. The HSP110 mutation as a potential prognostic and predictive marker in MSI CRC-H will be discussed briefly in the Putative prognostic or predictive molecular markers for MSI-H CRC section. BRAF /KRAS mutations: BRAF is a member of the RAF kinase family of genes and is a downstream effector of the KRAS gene, whereas KRAS is a member of the RAS family of genes and is a downstream effector of the EGFR gene in the Ras-Raf-MEK-ERK signaling pathway. The Ras-Raf-MEK-ERK signaling pathway is commonly involved in cell cycle progression and cell proliferation, and thus, activating mutations of key component genes in this pathway, including mutations in the BRAF or KRAS gene, can bring about uncontrolled cell growth and increased cell survival and may play an important role in tumorigenesis. BRAF and KRAS mutations are mutually exclusive in cancers, and the majority of mutations of BRAF and KRAS in human tumors are 4232 April 21, 2014 Volume 20 Issue 15

134 Kim JH et al. Microsatellite-unstable CRC Table 1 Frequency of CpG island methylator phenotype-high in colorectal cancers: A review of the literature Ref. Country CIMP-H CRCs/tested CRCs n (%) In all CRCs In MSI-H CRCs Western countries Samowitz et al [22], 2005 United States 250/859 (29.1) 64/78 (82.1) Weisenberger et al [13], United States 33/187 (17.6) NA 2006 Samowitz et al [84], 2006 United States 313/1271 (24.6) 105/170 (61.8) Barault et al [85], 2008 France 95/578 (16.4) 58/80 (72.5) Ogino et al [4], 2009 United States 123/631 (19.5) 86/118 (72.9) Dahlin et al [86], 2010 Sweden 46/411 (11.2) 34/61 (55.7) Zlobec et al [24], 2011 Switzerland 22/314 (7) NA Lochhead et al [63], United States 205/1173 (17.5) 140/184 (76.1) 2013 Eastern countries Koinuma et al [101], 2004 Japan NA 16 1 /28 (57.1) Nagasaka et al [102], 2008 Japan and Germany NA 15 2 /36 (41.7) Kim et al [98], 2009 South Korea 29/271 (10.7) 8/33 (24.2) Min et al [99], 2011 South Korea 34/245 (13.9) NA Bae et al [97], 2013 South Korea 47/734 (6.4) 18/65 (27.7) Kim et al [25], 2013 South Korea NA 64/220 (29.1) 1 MLH1-methylated colorectal cancers (CRCs) instead of CpG island methylator phenotype-high (CIMP-H) CRCs; 2 Sporadic microsatellite instability-high (MSI-H) CRCs instead of CIMP-H CRCs. NA: Not applicable. hot spot mutations in codon 600 (V600E) and codons 12 or 13, respectively [61,62]. According to previous studies, BRAF mutations were found in 5%-15% of overall CRCs [61,63-66], whereas 32%-40% of CRCs have KRAS mutations [63,64,67-72]. However, it has been revealed that BRAF V600E mutations are highly associated with MSI-H CRCs, although the incidence of KRAS mutations is inversely correlated with MSI-H status in CRCs [36]. The frequencies of BRAF and KRAS mutations in MSI-H CRCs have been reported to be 16%-52% and 12%-20%, respectively, in Western countries [63,73-76]. Notably, because BRAF V600E mutations have been found exclusively in sporadic tumors among MSI-H CRCs, it has been suggested that detection of BRAF mutations in CRCs may be a useful supportive tool for distinguishing sporadic CRCs from Lynch syndrome CRCs [14,73,77]. In fact, it is thought that this observed correlation between BRAF mutations and sporadic MSI-H CRCs is mostly based on the more significant association between BRAF mutations and CIMP-H status in CRCs [13,23]. Regarding the implications for prognosis, several previous studies have reported that BRAF mutations indicate poor survival in patients with CRC [4,78]. However, it has been suggested that the contribution of BRAF mutations to an adverse prognosis is significant for MSI-L/MSS CRCs but not MSI-H CRCs [65,79]. Therefore, the clinical and prognostic significance of BRAF mutations in MSI-H CRCs should be carefully explored in larger samples. Epigenetic alterations CIMP: CIMP represents a distinct subset of CRCs that show extensive promoter CpG island methylation and are characterized by transcriptional repression of many tumor suppressor genes as a result of promoter methylation [80]. The CIMP status of CRCs can be classified into three subtypes: CIMP-H, CIMP-low (CIMP-L), and CIMP-zero (CIMP-0) [24,81,82]. Among these subtypes, CIMP-H is generally regarded as the true CIMPpositive status. Previous investigations have revealed that CIMP-H is highly associated with sporadic MSI-H owing to the high frequency of MLH1 promoter methylation in CIMP-H CRCs [4,13,83]. According to data from Western countries, CIMP-H CRCs account for 7%-29% of all CRCs, and 56%-82% of MSI-H CRCs have the CIMP-H subtype (Table 1) [4,13,22,24,63,84-86]. In our previous study investigating differential clinicopathological features of MSI-H CRCs depending on CIMP status, we found that the CIMP-H subtype was significantly associated with older age, frequent BRAF V600E mutations, poor differentiation, medullary carcinoma components, and signet ring cell carcinoma components in MSI-H CRCs [87]. The effect of CIMP status on MSI-H CRC prognosis will be discussed in the Putative prognostic or predictive molecular markers for MSI CRC section. Genome-wide DNA methylation: The genomic methylation levels of specific tissues can be estimated by measuring the methylation levels of repetitive DNA elements, such as long interspersed nucleotide element-1 (LINE-1) and Alu, because these repetitive elements are globally distributed and occupy considerable portions of the human genome [88,89]. Of these repetitive elements, the methylation level of LINE-1 has been generally used as a reliable surrogate marker for the global DNA methylation level. In particular, LINE-1 hypomethylation has been regarded as one of the molecular characteristics that distinguishes CRC tumors from normal tissue [90]. Interestingly, several previous investigations have reported that LINE-1 hypomethylation is inversely correlated with MSI-H status in CRCs [91-93]. In a study by Ogino et al [93], a relatively high level of LINE-1 methylation was significantly associated with both MSI-H and CIMP-H statuses in CRCs. Notably, in this study, a correlation between the LINE-1 methylation level and MSI status was significant regardless of CIMP status; furthermore, a low LINE-1 methylation level was associated with 18q loss of heterozygosity (LOH) in CRCs. These findings suggest that genomic hypomethylation may be a characteristic phenomenon of the chromosomal instability (CIN) pathway, rather than the MSI pathway, in colorectal carcinogenesis. However, the mechanistic correlation between MSI status and resistance to genomic hypomethylation in CRCs should be further evaluated. The prognostic value of the LINE-1 methylation level in MSI-H CRCs will be described below, in the Putative prognostic or predictive molecular markers for MSI-H CRC section. Molecular heterogeneity among ethnic groups As mentioned above, MSI-H CRCs constitute approximately 15% of all CRCs in Western countries [8,9,35]. However, according to previous studies reported by our 4233 April 21, 2014 Volume 20 Issue 15

135 Kim JH et al. Microsatellite-unstable CRC Table 2 Frequency of BRAF V600E mutations in colorectal cancers: A review of the literature Ref. Country BRAF -mutant CRCs/tested CRCs n (%) In all CRCs In MSI-H CRCs Western countries Samowitz et al [22], United States 86/859 (10) 43/78 (55.1) 2005 Weisenberger et al [13], 2006 United States 26/187 (13.9) NA Samowitz et al [84], 2006 United States 123/1271 (9.7) 67/170 (39.4) Goel et al [103], 2007 United States 26/126 (20.6) 17/24 (70.8) Maestro et al [104], 2007 Spain 12/324 (3.7) 9/49 (18.4) Barault et al [85], 2008 France 76/578 (13.1) 51/80 (63.8) French et al [105], 2008 United States 77/490 (15.7) 35/58 (60.3) Ogino et al [4], 2009 United States 105/631 (16.6) 53/118 (44.9) Richman et al [106], 2009 United Kingdom 56/710 (7.9) NA Kumar et al [107], 2009 United States 7/98 (7.1) 7/30 (23.3) (African) Vilkin et al [108], 2009 Israel 24/128 (18.8) 6/13 (46.2) Roth et al [79], 2010 Europe 103/1307 (7.9) 45/188 (23.9) Dahlin et al [86], 2010 Sweden 55/411 (13.4) 34/61 (55.7) Zlobec et al [24], 2011 Switzerland 42/314 (13.4) NA Tie et al [109], 2011 Australia 52/525 (9.9) 24/75 (32) Yamauchi et al [110], 2012 United States 183/1276 (14.3) NA Kalady et al [111], 2012 United States 56/475 (11.8) 29/76 (38.2) Tian et al [112], 2013 The Netherland and Spain 42/381 (11) NA Lochhead et al [63], 2013 United States 182/1253 (14.5) 101/193 (52.3) Eastern countries Koinuma et al [101], 2004 Japan 16/140 (11.4) 12/28 (42.9) Nagasaka et al [113], 2004 Japan and Australia 21/234 (9) 16/35 (45.7) Chang et al [114], 2006 Taiwan 9/213 (4.2) 7/19 (36.8) Nagasaka et al [102], 2008 Japan and Germany 20/243 (8.2) 10/36 (27.8) Kim et al [98], 2009 South Korea 13/271 (4.8) 3/33 (9.1) Yagi et al [115], 2010 Japan 13/149 (8.7) NA Shen et al [116], 2011 China 2/118 (1.7) NA Liou et al [117], 2011 Taiwan 12/314 (3.8) NA Yokota et al [118], 2011 Japan 15/229 (6.6) NA Aoyagi et al [119], 2011 Japan 1/134 (0.7) NA Kwon et al [100], 2011 South Korea 4/92 (4.3) NA Min et al [99], 2011 South Korea 11/245 (4.5) 6/49 (12.2) Hsieh et al [120], 2012 Taiwan 2/182 (1.1) NA Nakanishi et al [121], 2012 Japan 17/254 (6.7) 11/31 (35.5) Bae et al [97], 2013 South Korea 39/728 (5.4) 4/65 (6.2) Kim et al [25], 2013 South Korea NA 26/219 (11.9) CRC: Colorectal cancer; MSI: Microsatellite instability; NA: Not applicable. group and others, a relatively low frequency of MSI-H (5.5%-9.4%) has been consistently observed in Korean patients with CRC, regardless of the institutions at which the study samples were collected [6,25,87,94-97]. Furthermore, the frequencies of CIMP-H and BRAF V600E mutations in CRCs are lower in Koreans (6.4%-13.9% and 4.3%-5.4%, respectively) than in Western populations [97-100]. We hypothesized that the low frequency of MSI-H CRCs in Korea is mainly based on the low prevalence of CIMP-H CRCs and that there are ethnic differences in the major molecular alterations associated with CRCs. Therefore, we performed a literature review to assess the frequencies of CIMP-H and BRAF mutations in CRCs, and the results are summarized in Tables 1 and 2, respectively. CIMP-H CRCs account for 7%-29.1% of all CRCs and 55.7%-82.1% of MSI-H CRCs in Western countries (Table 1) [4,13,22,24,63,84-86], whereas CIMP-H CRCs constitute 6.4%-13.9% of all CRCs and 24.2%-57.1% of MSI-H CRCs in Eastern Asian countries (Table 1) [25,97-99,101,102]. In Western countries, the BRAF V600E mutations were present in 3.7%-20.6% of all CRCs and in 18.4%-70.8% of MSI-H CRCs (Table 2) [4,13,22,24,63,79,84-86, ]. These proportions were lower, at 0.7%-11.4% of all CRCs and 6.2%-45.7% of MSI-H CRCs, in Eastern Asian countries (Table 2) [25,97-102, ]. These findings suggest that the Eastern Asian ethnicity is associated with a relatively low prevalence of CIMP-H and BRAF mutations in CRCs; consequently, these epidemiologic features may also result in a low frequency of MSI-H status in CRCs because it is thought that the majority of sporadic MSI-H CRCs are derived from CIMP-H CRCs. Thus, the low incidence of CIMP-H and BRAF mutations in Eastern Asian patients with CRC may be due to genetic or environmental differences between Eastern and Western ethnic groups. However, the detailed epidemiology and causal factors of the molecular heterogeneity of CRCs between ethnic groups should be elucidated in further investigations. PROGNOSTIC HETEROGENEITY OF MSI-H CRC Prognostic features and chemotherapy responses of MSI-H CRC Although several previous investigations have failed to identify prognostic significance of MSI status in CRCs [18,122], it has been consistently reported that patients with MSI-H CRC show a better survival than MSI-L/ MSS CRC patients [15,16]. However, there has been controversy regarding the predictive value of MSI status for the response to adjuvant chemotherapy in patients with CRC [ ]. Regardless of the controversy, it is generally agreed that there are fewer or no beneficial effects of adjuvant chemotherapy, especially 5-FU-based chemotherapy, for patients with MSI-H CRC compared to patients with MSI-L/MSS CRC [17,19, ]. According to previous in vitro experiments, the preservation of MMR function in cancer cells is likely important for inducing the apoptotic effect of 5-FU [ ], and this finding could explain the molecular basis of resistance to 5-FU-based chemotherapy in MSI-H CRCs. In contrast to the tendencies towards a poor response to 5-FU, and although the findings remain controversial [132], several studies supporting MSI-H as a predictive factor for improved response to irinotecan or irinotecan-based chemotherapy in CRC patients have been reported [133,134]. In previous experiments, it has also been suggested that mutations of MRE11 and/or RAD50 found in MSI-H CRC cells could account for increased sensitivity to irinotecan [135,136]. Concerning the response to the leucovorin/5-fu/oxaliplatin (FOLFOX) regimen in CRC patients, a few investigations have suggested that MSI is associated with improved 4234 April 21, 2014 Volume 20 Issue 15

136 Kim JH et al. Microsatellite-unstable CRC Table 3 Potential prognostic molecular factors for microsatellite instability-high colorectal cancer: A review of the literature Molecular factors HSP110 HSP110: Dorard et al [59] recently reported that the expression level of mutant HSP110 (heat shock protein 110 kda) is significantly associated with prognosis and chemotherapy response in MSI-H CRCs. According to this study, the T17 mononucleotide repeat located within intron 8 of HSP110 is vulnerable to deletions under the dmmr condition in CRCs, and these deletions can lead to exon 9 skipping and the generation of a truncated mu- Beta2- microglobulin Myosin 1a CDX2/CK20 SMAD4 Prognostic implication in MSI-H CRC (molecular alteration) Favorable (high expression of mutant HSP110/low expression of wild-type HSP110) Favorable (mutation of beta2- microglobulin) Unfavorable (low expression of myosin 1a) Unfavorable (loss of CDX2/ CK20 expression) Favorable (high expression of SMAD4) Ref. Dorard et al [59], 2011 Kim et al [96], 2014 Kloor et al [54], 2007 Tikidzhieva et al [143], 2012 Mazzolini et al [53], 2012 Kim et al [149], 2013 Isaksson-Mettavainio et al [150], 2012 CIMP Unfavorable (CIMP-H) Bae et al [87], 2011 LINE-1 Unfavorable (low LINE-1 Rhee et al [162], 2012 methylation level) CIMP: CpG island methylator phenotype; CIMP-H: CIMP-high; CRC: Colorectal cancer; LINE-1: Long interspersed nucleotide element-1; MSI-H: Microsatellite instability-high; HSP110: Heat shock protein 110 kda; CDX2: Caudal-type homeobox 2; CK20: Cytokeratin 20; SMAD4: SMAD family member 4. survival in CRC patients who are treated with adjuvant FOLFOX [137], whereas other studies have reported that MSI status was not significantly associated with a survival benefit of CRC patients after adjuvant FOLFOX treatment [ ]. Collectively, in patients with CRC, although MSI-H can indicate a better prognosis than MSI-L/MSS status, whether there is an association between MSI status and response to adjuvant chemotherapy remains controversial. Thus, further investigation is needed to confirm the predictive value of MSI status regarding responses to various chemotherapy regimens for CRCs. Putative prognostic or predictive molecular markers for MSI-H CRC Although MSI is known to be a molecular factor indicating a favorable prognosis in CRCs, we hypothesized that prognostic heterogeneity based on molecular heterogeneity might be present in MSI-H CRCs. Therefore, we expected that molecular markers stratifying patient prognosis or predicting chemotherapy response among MSI-H CRCs could be identified (Figure 1). By performing a review of the literature, potential prognostic or predictive molecular markers in MSI-H CRC were identified and are summarized in Table 3. These markers are introduced and discussed in following sections. tant HSP110 (HSP110ΔE9). MSI-H CRC patients with a high mrna expression level of HSP110ΔE9 survived longer, and this improved survival was maintained in both stage Ⅲ and adjuvant chemotherapy-treated subgroups [59]. In our recent study, we evaluated the expression status of wild-type HSP110 (HSP110wt) by IHC in MSI-H CRCs [96] and found that reduced expression of HSP110wt was correlated with a large deletion in the HSP110 T17 repeat and favorable prognosis in MSI-H CRCs, which is reasonable because the HSP110wt expression level is expected to be inversely correlated with the HSP110ΔE9 expression level. Mutation of HSP110 and variation in HSP110 expression are representative of the molecular heterogeneity associated with the prognostic heterogeneity of MSI-H CRCs, and it is expected that these molecular alterations could be used as predictive markers and therapeutic targets in MSI-H CRCs [142]. Beta2-microglobulin: According to recent investigations, coding microsatellite mutations in the beta2- microglobulin (B2M) gene occur in approximately 30% of MSI-H CRCs and are significantly associated with a low risk of disease relapse and a low frequency of distant metastasis in MSI-H CRCs [54,143]. Although the molecular mechanism underlying how B2M mutations affect prognosis in MSI-H CRCs is not fully understood, the biological functions of B2M may be associated with the metastatic potential of cancer cells, based on results demonstrating that B2M can induce epithelial-mesenchymal transition in cancer cells and may mediate bone metastasis of cancers [144]. As a putative prognostic marker and potential therapeutic target, the functional and prognostic significance of B2M mutations in MSI-H CRCs should be further evaluated. Myosin 1a: A recent study by Mazzolini et al [53] reported that the brush border protein myosin 1a (MYO1A) could act as a tumor suppressor in the intestine, and frameshift mutations in the MYO1A gene were detected in 32% of MSI-H CRCs. Interestingly, according to this study, a low expression level of MYO1A was associated with worse survival in patients with MSI-H CRCs, and MYO1A expression was identified as an independent prognostic factor in MSI-H CRCs. However, there is a lack of data elucidating the biological and clinicopathological significance of reduced MYO1A expression in CRCs; additional experimental and clinical studies are therefore needed. CDX2/CK20: CDX2 (caudal-type homeobox 2) and CK20 (cytokeratin 20) are proteins associated with intestinal differentiation and are also important markers of the normal intestinal epithelium and CRCs. Several previous studies identified that a loss of CDX2 and/or CK20 expression in CRCs was associated with MSI-H or CIMP-H status [24, ]. In a recent investigation, we found that a loss of CDX2/CK20 expression was significantly associated with poor differentiation, CIMP-H status, and an unfavorable prognosis in MSI-H CRCs [149]. According to our study, CRC patients with simultaneous loss of CDX April 21, 2014 Volume 20 Issue 15

137 Kim JH et al. Microsatellite-unstable CRC Prognostic or predictive molecular markers Good prognosis Chemo-sensitive MSI-H CRC patients DNA MMR deficiency Stratification of patients prognosis Genetic alterations Epigenetic alterations Prediction of chemotherapy response Ethnic/environmental differences Poor prognosis Chemo-resistant Figure 1 Graphical summary of this review, consisting of a conceptual model for prognostic heterogeneity based on the molecular heterogeneity of microsatellite-unstable colorectal cancers. CRC: Colorectal cancer; MSI-H: Microsatellite instability-high; MMR: Mismatch repair. and CK20 expression in tumor tissue constituted a highly aggressive subgroup of MSI-H CRC patients, with early death or recurrence occurring in this subgroup. Although CDX2/CK20 loss is not a specific molecular alteration associated with MSI-H CRC, these molecular factors can likely be used as markers to classify patients with MSI-H CRCs into prognostic subgroups. SMAD4: In a recent investigation by Isaksson-Mettävainio et al [150], high SMAD4 (SMAD family member 4) expression was significantly correlated with a favorable prognosis in MSI-H CRCs. Previous studies have also revealed that a loss of SMAD4 expression is associated with advanced stage, metastatic potential and an adverse prognosis in CRCs [ ]. In fact, loss of SMAD4 has been shown to be associated with 18q LOH [154,155] and may not be correlated with MSI status in CRCs because 18q LOH is a characteristic molecular alteration in the CIN pathway. Although it is thought that the prognostic implication of SMAD4 expression can be applied to all CRCs, a variation in SMAD4 expression and its significance for the prognosis of MSI-H CRCs remains an interesting field of study. Therefore, the underlying mechanism and prognostic value of variations in SMAD4 expression in MSI-H CRCs should be further explored. CIMP: The prognostic value of CIMP status in CRCs remains unclear. A few previous studies reported that the CIMP-H subtype was associated with poor prognosis in patients with CRC; however, this adverse effect of CIMP-H on CRC prognosis was significant only in the MSS patient subgroup and not in the MSI-H patient subgroup [86,98]. However, Ogino et al [4] provided contrasting data showing that CIMP-H was associated with a low cancer-specific mortality in CRC patients, regardless of both MSI status and BRAF mutations. In addition, although there is a lack of data elucidating the prognostic significance of CIMP-L in CRCs, a study by Dahlin et al [86] found that the CIMP-L subtype was associated with an unfavorable prognosis for CRCs, regardless of MSI status. The dependence of a chemotherapeutic response on CIMP status in CRCs is also controversial. Some investigators have suggested that CIMP-H is associated with a survival benefit in CRC patients receiving 5-FU-based chemotherapy [99,156], whereas the opposite results have also been reported [157,158]. Focusing on the prognostic implication of CIMP for MSI-H CRCs, we previously reported that for MSI-H CRC patients, those with CIMP-H tumors had worse survival than those with CIMP-L/0 tumors [87]. In fact, it is suspected that the differences in survival according to CIMP status in patients with MSI-H CRC may reflect differences in age distribution; in particular, patients with sporadic MSI-H CRC, who are expected to have CIMP-H status, are older and may have various comorbidities. In contrast, those with Lynch syndrome CRC, who have a CIMP-L/0 status, are younger and may be relatively healthy. This age distribution may critically affect patient prognosis, and thus, the prognostic significance of CIMP status in MSI-H CRC may be partly influenced by this age effect. Currently, it is debated whether CIMP status can serve as a true prognostic factor for MSI-H CRCs; therefore, more attention must be paid to analyzing the prognostic and predictive effect of CIMP status in CRCs. LINE-1 methylation: Several recent investigations have revealed that a low LINE-1 methylation level is independently associated with an adverse prognosis for CRCs [ ]. According to one of our studies [162], this prognostic significance of LINE-1 methylation was also 4236 April 21, 2014 Volume 20 Issue 15

138 Kim JH et al. Microsatellite-unstable CRC maintained in MSI-H CRCs. A low LINE-1 methylation level was an independent factor indicating poor prognosis in MSI-H CRC. These findings indicate that the LINE-1 methylation level can be a useful molecular factor for selecting the poor prognostic subgroup among patients with MSI-H CRC, which is known to be associated with a favorable prognosis, despite the level of LINE-1 hypomethylation being mild in MSI-H CRCs, as described above [92,93]. CONCLUSION MSI-H CRCs have been characterized as demonstrating a favorable prognosis and low benefit of adjuvant chemotherapy. However, MSI-H CRCs are molecularly heterogeneous tumors; thus, it is strongly suspected that prognostic and predictive molecular factors may be present. To date, several molecular factors, including HSP110, B2M, MYO1A, CDX2/CK20, SMAD4, CIMP, and LINE-1, have been explored as potential prognostic markers for MSI-H CRCs. 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144 Kim JH et al. Microsatellite-unstable CRC 147 Lugli A, Tzankov A, Zlobec I, Terracciano LM. Differential diagnostic and functional role of the multi-marker phenotype CDX2/CK20/CK7 in colorectal cancer stratified by mismatch repair status. Mod Pathol 2008; 21: [PMID: DOI: /modpathol ] 148 Baba Y, Nosho K, Shima K, Freed E, Irahara N, Philips J, Meyerhardt JA, Hornick JL, Shivdasani RA, Fuchs CS, Ogino S. Relationship of CDX2 loss with molecular features and prognosis in colorectal cancer. Clin Cancer Res 2009; 15: [PMID: DOI: / CCR ] 149 Kim JH, Rhee YY, Bae JM, Cho NY, Kang GH. Loss of CDX2/CK20 expression is associated with poorly differentiated carcinoma, the CpG island methylator phenotype, and adverse prognosis in microsatellite-unstable colorectal cancer. Am J Surg Pathol 2013; 37: [PMID: DOI: /PAS.0b013e31829ab1c1] 150 Isaksson-Mettävainio M, Palmqvist R, Dahlin AM, Van Guelpen B, Rutegård J, Oberg A, Henriksson ML. High SMAD4 levels appear in microsatellite instability and hypermethylated colon cancers, and indicate a better prognosis. Int J Cancer 2012; 131: [PMID: DOI: /ijc.26473] 151 Alazzouzi H, Alhopuro P, Salovaara R, Sammalkorpi H, Järvinen H, Mecklin JP, Hemminki A, Schwartz S, Aaltonen LA, Arango D. SMAD4 as a prognostic marker in colorectal cancer. Clin Cancer Res 2005; 11: [PMID: DOI: / CCR ] 152 Tanaka T, Watanabe T, Kazama Y, Tanaka J, Kanazawa T, Kazama S, Nagawa H. Loss of Smad4 protein expression and 18qLOH as molecular markers indicating lymph node metastasis in colorectal cancer--a study matched for tumor depth and pathology. J Surg Oncol 2008; 97: [PMID: DOI: /jso.20896] 153 Tanaka T, Watanabe T, Kazama Y, Tanaka J, Kanazawa T, Kazama S, Nagawa H. Chromosome 18q deletion and Smad4 protein inactivation correlate with liver metastasis: A study matched for T- and N- classification. Br J Cancer 2006; 95: [PMID: DOI: /sj.bjc ] 154 Miyaki M, Iijima T, Konishi M, Sakai K, Ishii A, Yasuno M, Hishima T, Koike M, Shitara N, Iwama T, Utsunomiya J, Kuroki T, Mori T. Higher frequency of Smad4 gene mutation in human colorectal cancer with distant metastasis. Oncogene 1999; 18: [PMID: DOI: / sj.onc ] 155 Thiagalingam S, Lengauer C, Leach FS, Schutte M, Hahn SA, Overhauser J, Willson JK, Markowitz S, Hamilton SR, Kern SE, Kinzler KW, Vogelstein B. Evaluation of candidate tumour suppressor genes on chromosome 18 in colorectal cancers. Nat Genet 1996; 13: [PMID: DOI: /ng ] 156 Van Rijnsoever M, Elsaleh H, Joseph D, McCaul K, Iacopetta B. CpG island methylator phenotype is an independent predictor of survival benefit from 5-fluorouracil in stage III colorectal cancer. Clin Cancer Res 2003; 9: [PMID: ] 157 Jover R, Nguyen TP, Pérez-Carbonell L, Zapater P, Payá A, Alenda C, Rojas E, Cubiella J, Balaguer F, Morillas JD, Clofent J, Bujanda L, Reñé JM, Bessa X, Xicola RM, Nicolás- Pérez D, Castells A, Andreu M, Llor X, Boland CR, Goel A. 5-Fluorouracil adjuvant chemotherapy does not increase survival in patients with CpG island methylator phenotype colorectal cancer. Gastroenterology 2011; 140: [PMID: DOI: /j.gastro ] 158 Shen L, Catalano PJ, Benson AB, O Dwyer P, Hamilton SR, Issa JP. Association between DNA methylation and shortened survival in patients with advanced colorectal cancer treated with 5-fluorouracil based chemotherapy. Clin Cancer Res 2007; 13: [PMID: DOI: / CCR ] 159 Ahn JB, Chung WB, Maeda O, Shin SJ, Kim HS, Chung HC, Kim NK, Issa JP. DNA methylation predicts recurrence from resected stage III proximal colon cancer. Cancer 2011; 117: [PMID: DOI: /cncr.25737] 160 Baba Y, Huttenhower C, Nosho K, Tanaka N, Shima K, Hazra A, Schernhammer ES, Hunter DJ, Giovannucci EL, Fuchs CS, Ogino S. Epigenomic diversity of colorectal cancer indicated by LINE-1 methylation in a database of 869 tumors. Mol Cancer 2010; 9: 125 [PMID: DOI: / ] 161 Ogino S, Nosho K, Kirkner GJ, Kawasaki T, Chan AT, Schernhammer ES, Giovannucci EL, Fuchs CS. A cohort study of tumoral LINE-1 hypomethylation and prognosis in colon cancer. J Natl Cancer Inst 2008; 100: [PMID: ] 162 Rhee YY, Kim MJ, Bae JM, Koh JM, Cho NY, Juhnn YS, Kim D, Kang GH. Clinical outcomes of patients with microsatellite-unstable colorectal carcinomas depend on L1 methylation level. Ann Surg Oncol 2012; 19: [PMID: DOI: /s ] P- Reviewers: Sipos F, Yu B S- Editor: Gou SX L- Editor: A E- Editor: Wu HL 4243 April 21, 2014 Volume 20 Issue 15

145 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (5): Colorectal cancer TOPIC HIGHLIGHT Multimodal imaging evaluation in staging of rectal cancer Suk Hee Heo, Jin Woong Kim, Sang Soo Shin, Yong Yeon Jeong, Heoung-Keun Kang Suk Hee Heo, Jin Woong Kim, Yong Yeon Jeong, Heoung- Keun Kang, Department of Radiology, Chonnam National University Hwasun Hospital, Chonnam National University Medical School, Jeollanam-do , South Korea Sang Soo Shin, Department of Radiology, Chonnam National University Hospital, Chonnam National University Medical School, Gwang-ju , South Korea Author contributions: Heo SH and Jeong YY contributed equally to conception, design, drafting, revision and final approval of the article; Kim JW, Shin SS and Kang HK contributed to editing, drafting, revision and final approval of the article. Supported by A grant (CRI ) of the Chonnam National University Hospital Research Institute of Clinical Medicine Correspondence to: Yong Yeon Jeong, MD, Department of Radiology, Chonnam National University Hwasun Hospital, Chonnam National University Medical School, 322 Seoyang-ro, Hwasun-eup, Hwasun-gun, Jeollanam-do , South Korea. yjeong@jnu.ac.kr Telephone: Fax: Received: September 27, 2013 Revised: December 20, 2013 Accepted: February 20, 2014 Published online: April 21, 2014 Abstract Rectal cancer is a common cancer and a major cause of mortality in Western countries. Accurate staging is essential for determining the optimal treatment strategies and planning appropriate surgical procedures to control rectal cancer. Endorectal ultrasonography (EUS) is suitable for assessing the extent of tumor invasion, particularly in early-stage or superficial rectal cancer cases. In advanced cases with distant metastases, computed tomography (CT) is the primary approach used to evaluate the disease. Magnetic resonance imaging (MRI) is often used to assess preoperative staging and the circumferential resection margin involvement, which assists in evaluating a patient s risk of recurrence and their optimal therapeutic strategy. Positron emission tomography (PET)-CT may be useful in detecting occult synchronous tumors or metastases at the time of initial presentation. Restaging after neoadjuvant chemoradiotherapy (CRT) remains a challenge with all modalities because it is difficult to reliably differentiate between the tumor mass and other radiation-induced changes in the images. EUS does not appear to have a useful role in post-therapeutic response assessments. Although CT is most commonly used to evaluate treatment responses, its utility for identifying and following-up metastatic lesions is limited. Preoperative high-resolution MRI in combination with diffusion-weighted imaging, and/or PET-CT could provide valuable prognostic information for rectal cancer patients with locally advanced disease receiving preoperative CRT. Based on these results, we conclude that a combination of multimodal imaging methods should be used to precisely assess the restaging of rectal cancer following CRT Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Rectal cancer; Multimodality; Imaging; Staging; Restaging Core tip: In rectal cancer, accurate staging and circumferential resection margin assessment are essential for stratifying the risks of recurrence and determining the optimal therapeutic strategy for individual patients. In the preoperative setting, a combination of multimodal imaging methods, including endorectal ultrasonography, computed tomography (CT) and magnetic resonance imaging (MRI), can be used to precisely assess the preoperative staging of rectal cancer. However, restaging after neoadjuvant therapy remains a challenge with all of these modalities. Recently, high-resolution MRI with diffusion-weighted imaging and/or positron emission tomography-ct imaging methods have been developed to precisely assess the restaging of rectal cancer following neoadjuvant chemoradiation therapy. Heo SH, Kim JW, Shin SS, Jeong YY, Kang HK. Multimodal imaging evaluation in staging of rectal cancer. World J Gastroenterol 2014; 20(15): Available from: URL: wjgnet.com/ /full/v20/i15/4244.htm DOI: April 21, 2014 Volume 20 Issue 15

146 Heo SH et al. Multimodal imaging evaluation of rectal cancer org/ /wjg.v20.i INTRODUCTION Rectal cancer is a tumor that arises from the rectum within 15 cm of the anal verge [1]. In 2013, there were an estimated new cases of rectal cancer in the United States, and rectal cancer is a major cause of cancer-related deaths in the developed world [2]. The prognosis of rectal cancer patients depends on the disease stage at the time of diagnosis [3-5] ; thus, accurate disease evaluation is necessary to properly treat rectal cancer. Rectal cancer prognosis is largely determined by tumor-node-metastasis staging, which evaluates the depth of tumor invasion into the rectal wall as well as the presence of lymph node (LN) and other distant metastases (Table 1) [3,4]. Again, accurate staging is crucial to determine the optimal treatment strategy, including selecting the appropriate surgical procedure. For example, earlystage (T1 and some T2) tumors may be suitable for local excision or trans-anal endoscopic microsurgery, whereas more advanced lesions (T3-4 and/or N1-2) may merit neoadjuvant chemoradiation therapy (CRT) followed by total mesorectal excision [TME; the surgical removal of the rectum and mesorectum enveloped by the mesorectal fascia (MRF)] [5,6]. In cases requiring TME, the status of the circumferential resection margin (CRM; defined by the spreading of the tumor to the MRF) is an important prognostic factor for predicting survival and local recurrence [1,5,7-9]. Because neoadjuvant CRT is the standard treatment for locally advanced rectal cancer, assessing tumor response using conventional imaging methods has been challenging [10-15]. The goal of neoadjuvant CRT is to preserve sphincter function while improving tumor resectability and reducing tumor mass prior to surgery [5]. Therefore, a more reliable imaging method would facilitate the design of more effective pre- and post-operative treatment strategies. Moreover, an effective assessment method would allow stratification of patients according to the risk of recurrence using preoperative and post- CRT assessments of the depth of tumor invasion and nodal status as well as of CRM involvement [16,17]. Currently, several imaging modalities, including endorectal ultrasound (EUS), computed tomography (CT), magnetic resonance imaging (MRI), and positron emission tomography (PET) or PET-CT, are used to assess preoperative staging and therapeutic response. This review will address the advantages and limitations for each of these imaging modalities in the context of staging and assessing therapeutic response in rectal cancer patients. IMAGING MODALITIES FOR PREOPERATIVE STAGING The preoperative staging of a rectal cancer tumor has been correlated with the patient s long-term prognosis [3-5]. Table 1 Tumor nodes metastasis staging of rectal cancer based on the American Joint Committee on Cancer 7 th edition Primary tumor (T) Tx Primary tumor cannot be assessed T0 No evidence of primary tumor Tis Carcinoma in situ T1 Tumor invades submucosa T2 Tumor invades muscularis propria T3 Tumor invades through the muscularis propria and into perirectal tissues T4a Tumor penetrates to the surface of the visceral peritoneum T4b Tumor directly invades or is adherent to other organs or structures Regional lymph nodes (N) Nx Regional lymph nodes cannot be assessed N0 No regional lymph node metastasis N1 Metastases in 1-3 regional lymph nodes N1a Metastasis in 1 regional lymph node N1b Metastases in 2-3 regional lymph nodes N1c Tumor deposit(s) in the subserosa, mesentery, pericolic, or perirectal tissues without regional nodal metastasis N2 Metastases in 4 or more regional lymph nodes N2a Metastases in 4-6 regional lymph nodes N2b Metastases in 7 or more regional lymph nodes Distant metastasis (M) M0 No distant metastasis M1 Distant metastasis M1a Metastasis confined to 1 organ or site (e.g., liver, lung, ovary, non-regional node, external iliac lymph node) M1b Metastases in > 1 organ/site or the peritoneum Note-Adapted from the American Joint Committee on Cancer staging system [3]. In particular, tumor staging facilitates the formulation of a structured multidisciplinary approach to manage the disease and assess a patient s prognosis over the course of treatment. Preoperative staging of rectal cancer can be divided into either local or distant staging. Local staging primarily involves assessment of mural wall invasion and nodal status for metastasis as well as CRM involvement, while distant staging assesses the extent of metastatic disease. Currently, several modalities are used for the preoperative staging of rectal cancer (Table 2). EUS EUS is performed by introducing a water-filled balloon containing a high-frequency transducer and trans-anal probe into the rectum. This approach can delineate the five layers of the rectal wall as alternating hyper-echoic and hypo-echoic bands [18,19]. EUS is particularly effective for assessing the depth of tumor invasion into the rectal wall, with 69%-94% accuracy (Figure 1) [20-23]. EUS can accurately distinguish between early- and advancedstage rectal lesions with sensitivity, specificity and accuracy rates of 96%, 85% and 94% respectively [6]. EUS is more specific in evaluating local tumor invasion when compared to MRI imaging (86% vs 69% specificity rates respectively), although both methods have similar high sensitivities for evaluating the depth of tumor penetration into the muscularis propria (94%) [24]. A recent meta-analysis of 5039 patients from 42 studies carried out between 1980 and 2008 supports 4245 April 21, 2014 Volume 20 Issue 15

147 Heo SH et al. Multimodal imaging evaluation of rectal cancer Table 2 Advantages and disadvantages of imaging modalities in preoperative staging Imaging modalities Advantages Disadvantages Preoperative staging EUS Depth of tumor invasion in early rectal cancer CT Wide availability Fast scanning time MRI High soft tissue resolution Local staging and CRM involvement Limited field of view Perirectal invasion and LN metastasis Local staging and CRM involvement in lower rectal cancer Distant metastasis PET/CT Distant metastasis Poor spatial resolution Perirectal invasion and CRM involvement Post-therapeutic restaging EUS None Low accuracy of T staging and LN staging CT MRI Relatively high accuracy of CRM status High specificity of therapeutic response assessment High accuracy of CRM involvement Relatively low accuracy of T staging and LN staging Relatively poor sensitivity PET/CT Distant metastasis No standard parameter and follow-up protocol MRI: Magnetic resonance image; EUS: Endorectal ultrasonography; CT: Computed tomography; PET: Positron emission tomography; CRM: Circumferential resection margin; LN: Lymph node. the accuracy of EUS for determining T stages, with pooled sensitivity and specificity rates of approximately 81%-96% and 91%-98%, respectively [25]. However, the accuracy of EUS in assessing the depth of invasion into the rectal wall appears to vary with the tumor stage, with lower accuracy in T2 lesions, compared with that in early-(t1) and advanced-(t3-t4) stage lesions [25]. Furthermore, EUS cannot reliably distinguish between peritumoral inflammation or transmural tumor infiltration, which may lead to over-staging of T2 tumors as T3 tumors and subsequent overtreatment [21,26,27]. The staging of bulky, distal and/or stenotic lesions with EUS can also be challenging due to the limited field of view and the inability of probes (especially rigid probes) to traverse the lesion [19,23]. Lymph node (LN) staging with EUS remains difficult and is less accurate than T staging, with reported accuracy rates of 64%-83% [20,21,23,28]. Although EUS-detected morphological characteristics, including a round shape, peritumoral location, size, and hypo-echogenecity) could be associated with malignant LNs, these features are neither sensitive nor specific [10,19,29]. Moreover, EUS can evaluate only perirectal or mesorectal LNs, thereby limiting the screening capacity of this method. In contrast, other imaging modalities such as CT, MRI and PET/CT can visualize the iliac and mesenteric or retroperitoneal nodes, allowing for more comprehensive LN staging. Figure 1 Endorectal ultrasound image of T3 rectal cancer. Endorectal ultrasonography image shows a hypo-echoic mass (arrows) that extends into the echogenic mesorectal fat (arrowheads). Recent developments in 3-dimensional EUS technology, with enhanced resolution and a multi-planar display, have resulted in improved T- and LN-staging accuracy rates compared to 2-dimensional EUS [30]. Nevertheless, an assessment of the MRF remains impossible and the accuracy of the procedure is largely dependent on the experience of the operator [19,21,30]. Thus, the major role of EUS in rectal cancer staging is for assessment of tumor invasion depth, particularly in early-stage rectal tumors, for which EUS can be used to evaluate whether tumors are suitable for treatment by trans-anal or local excision [6,17,25]. CT CT is commonly used as the initial staging modality for rectal cancer because of its wide availability and fast scanning times. In a single examination, CT can also assess the entire abdomen, pelvis and chest, allowing for both local staging and distant metastases evaluations [17,31]. Initial studies using conventional CT to assess locally advanced rectal cancers (i.e., T3) reported T staging accuracy rates of 79%-94% [32-34]. The technical advances in multidetector CT (MDCT) have provided enhanced spatial resolution though thin-collimation scanning and multiplanar reformation, and have improved the accuracy rates to greater than 90% [35]. Nevertheless, MDCT has shown limited value for early-stage lesions confined to the rectal wall (e.g., differentiation between T1 and T2 tumors), for which EUS may be more effective. Furthermore, the lower resolution of MDCT is unable to reliably distinguish the layers of the rectal wall and to differentiate between desmoplastic or peritumoral inflammatory reactions and tumor infiltration into the perirectal fat [24,36]. These limitations lead to a tendency to over-stage T1 or T2 tumors as T3 tumors when using MDCT. LN staging is predominantly based on the size criterion and, to a lesser extent, on morphology [17]. One study suggests that a LN with axis above 4.5 mm in diameter is usually considered malignant, but such size criteria are not generally considered to be accurate [37]. The lack of a clear cut-off diameter to determine whether a LN is metastatic 4246 April 21, 2014 Volume 20 Issue 15

148 Heo SH et al. Multimodal imaging evaluation of rectal cancer A B Figure 2 Stage T2 and T3 rectal cancer detected by magnetic resonance imaging. A: T2-weighted magnetic resonance image shows an intraluminal polypoid mass (arrows) confined to the intact, hypo-intense muscularis propria (arrowheads), suggestive of a T2 cancer; B: T3 lesion is seen as a tumor (arrows) that extends through the hypo-intense muscle layer into the perirectal fat (arrowheads). has led to wide variability in LN-staging sensitivity and accuracy using CT, with respective rates of 25%-86% and 35%-84% [17,37]. Moreover, even with the enhanced resolution of MDCT, accurate assessment of the nodal status remains challenging because microscopic metastases in normal-sized LNs cannot be depicted on CT. Regarding assessments of CRM involvement, in a multicenter study of 250 patients [38], MDCT showed overall sensitivity and specificity rates of 76% and 96%, respectively for mid to upper rectal cancer patients. This result suggests that CT may be an alternative to MRI to predict CRM involvement in such patients. In lower rectal cancer, however, CT is less accurate and inconsistently predicts CRM involvement [39,40]. Thus, in a preoperative setting, CT is best suited for evaluations of distant metastases, as this modality is limited with regard to local staging and CRM status determinations. MRI Since 1986, MRI has been used to delineate rectal tumors and locally stage rectal cancers. Initial MRI studies using a body coil have reported overall accuracy rates ranging from 59% to 88%, similar to the accuracy range for CT [41-43]. Recently, rectal MRI has been performed with either an endorectal coil or a phased-array surface coil. Several studies with phased-array MRI showed accuracy rates of 65%-86%, and studies with an endorectal coil reported slightly improved T staging rates (71%-91%) [43,44]. Although the endorectal coil permits a more detailed differentiation of the rectal wall, its routine use in clinical practice is controversial due to some limitations of the technology. Specifically, it is difficult to clearly differentiate between the mucosa and submucosa, to access high and/or stenotic rectal lesions, and to completely evaluate the mesorectum and surrounding structures due to the limited field of view [10,17,45]. However, in a comparative study on endorectal and phased-array coils, both techniques yielded similar diagnostic accuracy rates (80%) for the depth of tumor invasion [46]. In addition, recent studies have shown that the use of 3.0-T MRI improves the overall T staging accuracy for rectal cancer with accuracy rates of 86%-95% [47-49]. This represents a significant improvement over 1.5-T MRI, which has T staging accuracy rates of 67%-86% [50-52]. Moreover, improvements in scanner gradients and dedicated external coils have allowed rectal MRI to be successfully performed with phased-array surface coils; allowing high-resolution imaging with thin-sections and an enhanced field of view, and providing a more comfortable patient experience [43]. The use of rectal MRI techniques for clinical decisions regarding bowel preparation, the use of antispasmodic agents, and the use of rectal gel can vary according to the preference of the radiologists [53]. Although the use of gadolinium contrast enhancement rarely improves rectal cancer staging via MRI, many centers still acquire images both before and after gadolinium administration [54,55]. To identify and stage rectal cancers with MRI, highresolution T2-weighted image with thin-sections is the primary approach used to differentiate between the tumor, mucosal and submucosal layers, muscular layer, perirectal fat, and MRF [56]. However, because the submucosal layer is not always efficiently visualized on MRI [16], the ability to differentiate between T1 and T2 lesions may be limited. Using MRI, a T3 lesion is defined as a tumor that extends through the hypo-intense muscle layer into the hyper-intense perirectal fat with a bulging or nodular appearance, whereas a T2 tumor is confined to the hypointense muscle layer (Figure 2) [56]. In prospective comparative studies with histopathology, MRI-based staging strongly correlated with the depth of tumor invasion (82%) [56] and agreed with the extramural depth of invasion (95%) [57]. However, staging failures commonly occur when differentiating T2 from borderline T3 tumors, as it is difficult to distinguish direct tumor infiltration (T3) from speculated structures in the perirectal fat due to fibrosis or peritumoral inflammation (T2) [58]. In contrast, MRI is particularly accurate when identifying T3 and T4 tumors, with a sensitivity of 80%-86% and specificity of 71%-76% for T3 tumors [59]. Regarding LN assessments with MRI, a size cut-off of 5 mm in diameter is the most commonly used criterion. However, 15%-42% of patients with rectal cancer have metastatic perirectal LNs that are smaller than April 21, 2014 Volume 20 Issue 15

149 Heo SH et al. Multimodal imaging evaluation of rectal cancer Figure 3 Mesorectal fascia invasion detected by magnetic resonance imaging. T2-weighted magnetic resonance image shows a T3 tumor (arrows) with involvement of the mesorectal fascia (arrowheads). mm [60,61], indicating that this criterion is not accurate enough for reliable assessment. Using a combination of other criteria, including an irregular border and/or a mixed signal intensity, MRI for nodal staging shows an accuracy of 85%, with a moderate sensitivity of 75% and high specificity of 98% [61]. However, a recent metaanalysis of 1249 patients from 23 data sets showed that the ability to assess LN involvement was consistently poor with a pooled sensitivity of 77% and specificity of 71% [62]. Similarly, in another meta-analysis study, MRI showed poor accuracy when compared with EUS and CT [24]. With improvements to MRI scanners and techniques, the nodal staging accuracy has increased to 91%, with a high sensitivity of 89% [47]. High-resolution MRI with thin-sections has enabled evaluations of the detailed characteristics of LNs. Nevertheless, it remains challenging to detect micro-metastases in small or normal-sized LNs using MRI. MRI is a reliable imaging modality for CRM status assessments, making the technique an important element of surgical planning and prognosis. Most commonly, MRI is used to measure the distance of a tumor from the MRF, as the MRF is observed as a thin hypo-intense line that envelops the perirectal fat and rectum. In a prospective study by the Magnetic Resonance Imaging and Rectal Cancer European Equivalence (MERCURY) study group, which defines CRM involvement as a tumor within 1 mm of the MRF, MRI showed 92% specificity for preoperative predictions of a clear CRM (Figure 3) [63]. This 1 mm threshold is the most commonly used criterion to assess CRM involvement, although Beets-Tan et al [58] reported that a 5 mm cut-off on MRI could also predict CRM involvement with a high degree of certainty. Published data have shown that MRI has a high accuracy rate of 86% for predicting CRM involvement, with good intra- and inter-observer agreement [17,58,64]. Thus, MRI is a powerful method for preoperatively assessing both local staging and CRM involvement with high accuracy and reproducibility, effectively identifying patients who require preoperative CRT in order to minimize incomplete tumor resections and local recurrences [63]. PET-CT PET enables the measurement and visualization of metabolic changes within cancer cells. Fluorodeoxyglucose (FDG) is the most common PET tracer used, as its uptake is increased in tumors due to increased metabolic activity in the tumor cells, which facilitates efficient radiolabelling. Studies have shown that PET-CT can reliably detect colorectal cancer but not its depth of tumor invasion [65,66]. The poor spatial resolution and lack of detailed anatomy provided by FDG-PET make the determination of the degree of local tumor spread, LN involvement, CRM status, or relationship to the sphincter complex difficult. The utility of PET-CT scanning for initial staging of rectal cancer remains unclear [14]. While PET-CT is capable of detecting occult synchronous tumors or metastases at the time of initial presentation, the detection rate is too low to justify the costs and radiation exposure for its routine use. In addition, PET is limited to the identification of non-mucinous tumors because FDG uptake is hampered by the presence of mucin [67]. Currently, PET- CT is reserved for the staging of patients prior to surgical removal of recurrent lesions or distant metastases and to identify recurrent lesions in patients with an unexplained rise in serum carcinoembryonic antigen. PET-CT may be particularly useful for the staging of distant metastatic spread, particularly if radical surgery is being considered but no local staging information is available using other imaging methods [14]. One recent study demonstrated that preoperative PET altered the treatment strategy in 17% of patients, including 13% for whom surgery was cancelled and 4% for whom the radiotherapy field was changed [68]. One explanation for this finding is that PET-CT frequently yields additional staging information in patients with low rectal cancer. Thus, PET-CT could be used to improve the accuracy of pretreatment imaging, thereby allowing for more appropriate stage-specific therapy [69]. IMAGING MODALITIES FOR EVALUATION OF POST-TREATMENT RESPONSES Preoperative CRT has been widely adopted for the management of patients with locally advanced rectal cancers because it can facilitate tumor downsizing and downstaging, leading to increased rates of sphincter-sparing surgeries and pathologic complete response [5]. If tumor responses to neoadjuvant CRT could be assesses before surgery, patients could receive appropriately tailored treatments. For example, patients who respond well the CRT could be offered less radical treatments, whereas those who fail to respond could be identified as candidates for more radical surgeries or second-line therapies [14]. Traditionally, tumor response assessments are achieved by measuring the percentage of the tumor size reduction according to the Response Evaluation Criteria in Solid Tumors criteria, as the change in tumor size is generally thought to correlate with treatment efficacy [70]. However, 4248 April 21, 2014 Volume 20 Issue 15

150 Heo SH et al. Multimodal imaging evaluation of rectal cancer Figure 4 Over-staging due to post-chemoradiotherapy changes. T2- weighted image obtained after chemoradiotherapy shows hypo-intense infiltration (arrows) to the perirectal fat and a spiculated lymph node (arrowheads), which was misinterpreted as a remnant T3 lesion and metastatic node. However, there was neither a residual tumor nor metastatic lymph node on the pathological examinations after surgery. this assessment approach is insensitive to early treatment changes, and it can be difficult to distinguish between viable tumors and CRT-associated non-tumoral masses (e.g., inflammation, necrosis, or fibrosis) [11]. Given these limitations, the response of locally advanced rectal cancers should be addressed not only by tumor size, but also by additional prognostic factors, including the extramural depth of invasion, nodal status, and CRM involvement, as well as the relationship with pelvic structures (Table 2). Thus, it is important to determine which imaging modalities can provide accurate and reliable information regarding these criteria to accurately assess tumor responses. Efficacy of EUS for assessing post-crt rectal cancer response EUS does not appear to play a role in post-therapeutic response assessments because it cannot reliably differentiate between post-radiation edema, inflammation, fibrosis, and viable tumors [19]. Due to this limitation, the accuracy of EUS for rectal cancer restaging after radiation therapy is markedly low (47%) [71]. In locally advanced rectal cancers, the T staging accuracy rates for EUS, when performed at 4-6 wk after the completion of CRT, were 29% in responsive patients and 82% in nonresponsive patients, with a high misinterpretation rate (71%) in responsive patients [72]. In the same study, the overall accuracy of EUS for LN involvement after CRT was 57% [72]. Thus, EUS is not recommended for restaging after radiotherapy or CRT. Efficacy of CT for assessing post-crt rectal cancer response CT is most commonly used to evaluate treatment responses in solid tumors. However, in CRT-treated rectal cancer, the utility of CT is limited for identifying and following-up metastatic lesions. In a recent study to correlate tumor responses measured by CT in accordance with the histopathology, the overall accuracy rates of T and N restaging were 65% and 67% [73] respectively, which represents an improvement over a previous study (reporting T restaging of 37% and N restaging of 62%) [13]. However, the tumor over-staging and under-staging rates after CRT were 23% and 12% respectively, because rectal wall thickening caused by CRT-induced fibrosis can be misidentified as residual tumors on CT [73]. In CRM status assessments after CRT, CT accurately predicted a negative CRM in 71% of cases [13]. Nevertheless, it remains unclear whether CT would have a clinical impact on the therapeutic outcome of local staging in patients with locally advanced, CRT-treated rectal cancer because of its poor resolution, further studies are necessary to evaluate the efficacy of this approach [14]. With the recent advances aimed at reducing radiation exposure, perfusion CT is an emerging and noninvasive technique that permits measurements of tumor vascularity and perfusion changes, allowing measurements of CRT response. In rectal cancers, perfusion CT has been shown to differentiate rectal cancer from the normal rectal wall [74,75]. In addition, perfusion CT could be useful for predicting tumor responses and clinical outcomes of CRT, as it can differentiate between tumors with normal vs low blood flow, as low blood flow before CRT is associated with a poor response [74,75]. Efficacy of MRI for assessing post-crt rectal cancer response As in preoperative evaluation, T2-weighted MRI plays a major role in rectal cancer restaging after CRT. The concordance between the post-crt MRI and the histological T stage was only fair to moderate (κ = 0.40), suggesting rather low accuracy [76]. However, when a morphological response assessment (e.g., responder vs nonresponder) was used, the overall accuracy rate improved to 79% [77]. High-resolution MRI with thin-sections yielded an improved T staging accuracy rate after CRT, with good concordance (κ = 0.64) [78]. Despite these improvements, a recent meta-analysis showed that MRImediated rectal cancer restaging after preoperative CRT remains challenging, with poor sensitivity (50.4%) due to the difficulty of differentiating tumor changes from the residual tumor (Figure 4) using MRI [79]. In cases of mucinous tumors, which are hyper-intense on T2-weighted images, stage prediction errors can also arise, making it difficult to distinguish between a remnant tumor and a mucin pool [76,80]. However, the pooled specificity rate was as high as 91.2%, which suggests that positive MRI results can accurately identify CRT-responsive patients; however, negative MRI results may not similarly identify non-responsive patients [79]. To determine the LN status after CRT, the same pre-crt morphological criteria (i.e., size, irregular borders, and signal homogeneity) are applied. This assessment method has a reported overall accuracy rate of 64%-68% [76,81,82]. Moreover, recent studies of MRI assessment of post-crt LN status reported high accuracy rates of 87%-88% [77,78]. However, changes in morphological appearances after CRT, including high-signal interfer April 21, 2014 Volume 20 Issue 15

151 Heo SH et al. Multimodal imaging evaluation of rectal cancer A B C D E F Figure 5 Combination of high-resolution magnetic resonance imaging with diffusion-weighted imaging and positron emission tomography/computed tomography for assessing tumor response after neoadjuvant chemoradiotherapy. A-C: T2-weighted magnetic resonance image (MRI) (A), diffusion-weighted imaging (DWI) (B), and positron emission tomography (PET)/computed tomography (CT) (C) obtained before chemoradiotherapy (CRT) shows a T3 tumor (arrows) with the restricted diffusion and intense hyper-metabolism; D-F: After CRT, T2-weighted MRI (D) shows a marked decrease in the tumor size. DWI (E) shows the focal diffusion restriction confined to the rectal wall, corresponding to histological proven T2 lesion. PET/CT (F) also demonstrates the good tumor response to CRT. ence from mucin pools or spiculated LN margins, can make MRI assessments prone to over-staging (Figure 4) [12,77,78]. According to a prospective study by the MERCURY group, the accuracy rate and negative predictive value for identifying CRM involvement in 97 CRT-treated patients were 77% and 98% respectively, whereas the same values were 91% and 93% in 311 patients who underwent primary surgery [63]. A recent meta-analysis study reported a similar result, with a mean sensitivity of 76.3% and mean specificity of 85.9% [79]. However, it should be noted that CRM involvement errors frequently occur in MRI assessments because of diffuse hypo-intense infiltration into the perirectal fat or MRF after CRT [63,83]. Nevertheless, MRI assessment is considered effective for restaging tumors after CRT, particularly with respect to potential CRM involvement. Use of diffusion-weighted imaging in MRI for assessing post-crt rectal cancer response Diffusion-weighted imaging (DWI) is a newly emerging, functional MRI technique that can supplement conventional MRI assessment and enable the noninvasive characterization of biological tissues based on the properties of water diffusion. Thus, DWI can provide microstructural information, including changes in cellularity and integrity of cellular membranes, which often precede conventional morphological alterations. DWI has been used to detect and characterize tumors and distinguish between necrotic and viable solid portions within tumors by quantifying apparent diffusion coefficient (ADC) values [84,85]. The ADC value decreases with increasing cell 4250 April 21, 2014 Volume 20 Issue 15

152 Heo SH et al. Multimodal imaging evaluation of rectal cancer density due the restricted diffusion of water molecules, which tends to increase in necrotic regions [84,86]. The ADC values of tumors with good or complete responses were significantly higher than those of tumors with poor responses, and the presence of higher pre-crt ADC values reflected necrotic CRT-resistant tumors [86,87], suggesting that this approach is useful for predicting CRT response ahead of treatment. In contrast, the post-crt ADC values of rectal cancers were lower than those of the pre-crt tumors; a difference that was attributed to fibrosis or scar tissue formation and was not correlated with tumor response. These results indicate that the addition of DWI to standard MRI may facilitate the detection of viable tumors and identify CRTresponsive patients (Figure 5) [88-90]. Moreover, in a recent meta-analysis, restaging with DWI showed good pooled sensitivity (83.6%) and specificity rates (84.8%) [79]. Thus, DWI can provide a useful biomarker to assess and monitor treatment responses to CRT [86,91]. Efficacy of PET-CT for assessing post-crt rectal cancer response PET-CT is a promising modality for identifying recurrent rectal cancer and distant metastasis. A significant reduction of standardized uptake values on post-crt PET of responders compared with non-responders has been noted by a number of studies (Figure 5), suggesting that this approach can effectively assess CRT response [15,92,93]. However, the efficacy of PET-CT for the prediction of tumor response remains controversial, especially for assessing pathological complete response to CRT in rectal cancer due to several limitations of the technology. One limitation is the variation in definitions and parameters used for defining a good vs a poor response. Another limitation is the uncertainty regarding the timing of PET imaging after the completion of radiotherapy. The optimal time to carry out PET assessment after the completion of CRT remains unclear, with initial studies suggesting an interval of only 6 wk being too soon to confirm metabolic response [93]. Therefore, at present, the role of PET-CT in post-crt restaging is not well established. It is possible that PET-CT may provide additional information in assessing response; however, PET-CT needs to be formally compared with existing methods to properly evaluate its efficacy. FUTURE DIRECTIONS FOR MULTIMODAL IMAGING EVALUATION IN RECTAL CANCER STAGING Multimodal imaging assessments in rectal cancer facilitate the design of treatment strategies and predict patient prognosis. Continued improvement of imaging techniques will provide superior image resolution, threedimensional viewing, and decreased image acquisition times, and may provide new functional qualities. The use of EUS is likely to increase in future staging investigations, which will be complementary to the management of rectal cancer. DWI has been applied as a useful biomarker, which could be used to assess and monitor treatment responses to CRT, although protocol standardization and experienced radiologists are required to maximize the efficacy of this approach. Although the role of PET-CT needs to be formally compared with existing methods, PET-CT could provide additional valuable information for the assessment of post-crt response. Restaging using a combination of imaging methods including high-resolution MRI, DWI and PET-CT will provide valuable prognostic information before definitive surgery. Despite these improvements, determination of the LN metastasis remains challenging in rectal cancer staging. LN research is further needed. CONCLUSION In preoperative settings, EUS is suitable for assessing the depth of tumor invasion, particularly in superficial and early-stage rectal tumors. CT imaging is a common method for evaluation of distant metastases. MRI is a useful and reliable modality for assessing preoperative staging and CRM involvement, allowing physicians to stratify the risks of recurrence and determine the optimal therapeutic planning for individual patients. PET or PET- CT assessment is recommended when there is clinical, biochemical or radiological suspicion of systemic disease. Restaging after neoadjuvant therapy, particularly CRT, is challenging with all modalities because it is difficult to reliably differentiate between radiation-induced changes and the tumor itself. 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Ann Surg Oncol 2006; 13: [PMID: DOI: / ASO ] 70 Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan RS, Rubinstein L, Verweij J, Van Glabbeke M, van Oosterom AT, Christian MC, Gwyther SG. New guidelines to evaluate the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 2000; 92: [PMID: ] 71 Napoleon B, Pujol B, Berger F, Valette PJ, Gerard JP, Souquet JC. Accuracy of endosonography in the staging of rectal cancer treated by radiotherapy. Br J Surg 1991; 78: [PMID: ] 72 Rau B, Hünerbein M, Barth C, Wust P, Haensch W, Riess H, Felix R, Schlag PM. Accuracy of endorectal ultrasound after preoperative radiochemotherapy in locally advanced rectal cancer. Surg Endosc 1999; 13: [PMID: ] 73 Lee CT, Chow NH, Liu YS, Lin SC, Lin PC, Wu YH, Lee JC, Tsai HM. Computed tomography with histological correlation for evaluating tumor regression of rectal carcinoma after preoperative chemoradiation therapy. Hepatogastroenterology 2012; 59: [PMID: DOI: /hge12165] 74 Sahani DV, Kalva SP, Hamberg LM, Hahn PF, Willett CG, Saini S, Mueller PR, Lee TY. Assessing tumor perfusion and treatment response in rectal cancer with multisection CT: initial observations. Radiology 2005; 234: [PMID: DOI: /radiol ] 75 Bellomi M, Petralia G, Sonzogni A, Zampino MG, Rocca A. CT perfusion for the monitoring of neoadjuvant chemotherapy and radiation therapy in rectal carcinoma: initial experience. Radiology 2007; 244: [PMID: DOI: /radiol ] 76 Allen SD, Padhani AR, Dzik-Jurasz AS, Glynne-Jones R. Rectal carcinoma: MRI with histologic correlation before and after chemoradiation therapy. AJR Am J Roentgenol 2007; 188: [PMID: DOI: /AJR ] 77 Barbaro B, Fiorucci C, Tebala C, Valentini V, Gambacorta MA, Vecchio FM, Rizzo G, Coco C, Crucitti A, Ratto C, Bonomo L. Locally advanced rectal cancer: MR imaging in prediction of response after preoperative chemotherapy and radiation therapy. Radiology 2009; 250: [PMID: DOI: /radiol ] 78 Koh DM, Chau I, Tait D, Wotherspoon A, Cunningham D, Brown G. Evaluating mesorectal lymph nodes in rectal cancer before and after neoadjuvant chemoradiation using thin-section T2-weighted magnetic resonance imaging. Int J Radiat Oncol Biol Phys 2008; 71: [PMID: DOI: /j.ijrobp ] 79 van der Paardt MP, Zagers MB, Beets-Tan RG, Stoker J, Bipat S. Patients who undergo preoperative chemoradiotherapy for locally advanced rectal cancer restaged by using diagnostic MR imaging: a systematic review and metaanalysis. Radiology 2013; 269: [PMID: DOI: /radiol ] 80 Kim DJ, Kim JH, Lim JS, Yu JS, Chung JJ, Kim MJ, Kim KW. Restaging of Rectal Cancer with MR Imaging after Concurrent Chemotherapy and Radiation Therapy. Radiographics 2010; 30: [PMID: DOI: / rg ] 81 Kuo LJ, Chern MC, Tsou MH, Liu MC, Jian JJ, Chen CM, Chung YL, Fang WT. Interpretation of magnetic resonance imaging for locally advanced rectal carcinoma after preoperative chemoradiation therapy. Dis Colon Rectum 2005; 48: [PMID: ] 82 Chen CC, Lee RC, Lin JK, Wang LW, Yang SH. How accurate is magnetic resonance imaging in restaging rectal cancer in patients receiving preoperative combined chemoradiotherapy? Dis Colon Rectum 2005; 48: [PMID: DOI: /s ] 83 Vliegen RF, Beets GL, Lammering G, Dresen RC, Rutten HJ, Kessels AG, Oei TK, de Bruïne AP, van Engelshoven JM, Beets-Tan RG. Mesorectal fascia invasion after neoadjuvant chemotherapy and radiation therapy for locally advanced rectal cancer: accuracy of MR imaging for prediction. Radiology 2008; 246: [PMID: DOI: /radiol ] 84 Koh DM, Collins DJ. Diffusion-weighted MRI in the body: applications and challenges in oncology. AJR Am J Roentgenol 2007; 188: [PMID: DOI: / AJR ] 85 Heo SH, Shin SS, Kim JW, Lim HS, Jeong YY, Kang WD, Kim SM, Kang HK. Pre-treatment diffusion-weighted MR imaging for predicting tumor recurrence in uterine cervical cancer treated with concurrent chemoradiation: value of histogram analysis of apparent diffusion coefficients. Korean J Radiol 2013; 14: [PMID: DOI: / kjr ] 86 Thoeny HC, Ross BD. Predicting and monitoring cancer treatment response with diffusion-weighted MRI. J Magn Reson Imaging 2010; 32: 2-16 [PMID: DOI: / jmri.22167] 87 Jung SH, Heo SH, Kim JW, Jeong YY, Shin SS, Soung MG, Kim HR, Kang HK. Predicting response to neoadjuvant chemoradiation therapy in locally advanced rectal cancer: diffusion-weighted 3 Tesla MR imaging. J Magn Reson Imaging 2012; 35: [PMID: DOI: / jmri.22749] 88 Song I, Kim SH, Lee SJ, Choi JY, Kim MJ, Rhim H. Value of diffusion-weighted imaging in the detection of viable tumour after neoadjuvant chemoradiation therapy in patients with locally advanced rectal cancer: comparison with T2 weighted and PET/CT imaging. Br J Radiol 2012; 85: [PMID: DOI: /bjr/ ] 89 Lambregts DM, Vandecaveye V, Barbaro B, Bakers FC, Lambrecht M, Maas M, Haustermans K, Valentini V, Beets GL, Beets-Tan RG. Diffusion-weighted MRI for selection of complete responders after chemoradiation for locally advanced rectal cancer: a multicenter study. Ann Surg Oncol 2011; 18: [PMID: DOI: / s ] 90 Sassen S, de Booij M, Sosef M, Berendsen R, Lammering G, Clarijs R, Bakker M, Beets-Tan R, Warmerdam F, Vliegen R. Locally advanced rectal cancer: is diffusion weighted MRI helpful for the identification of complete responders (ypt0n0) after neoadjuvant chemoradiation therapy? Eur Radiol 2013; 23: [PMID: DOI: / s ] 91 Harry VN, Semple SI, Parkin DE, Gilbert FJ. Use of new imaging techniques to predict tumour response to therapy. Lancet Oncol 2010; 11: [PMID: DOI: / S (09) ] 92 Kim JW, Kim HC, Park JW, Park SC, Sohn DK, Choi HS, Kim DY, Chang HJ, Baek JY, Kim SY, Kim SK, Oh JH. 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156 Heo SH et al. Multimodal imaging evaluation of rectal cancer patients with locally advanced rectal cancer treated by preoperative chemoradiotherapy. Int J Colorectal Dis 2013; 28: [PMID: DOI: /s ] 93 Guillem JG, Ruby JA, Leibold T, Akhurst TJ, Yeung HW, Gollub MJ, Ginsberg MS, Shia J, Suriawinata AA, Riedel ER, Mazumdar M, Saltz LB, Minsky BD, Nash GM, Paty PB, Temple LK, Weiser MR, Larson SM. Neither FDG-PET Nor CT can distinguish between a pathological complete response and an incomplete response after neoadjuvant chemoradiation in locally advanced rectal cancer: a prospective study. Ann Surg 2013; 258: [PMID: DOI: /SLA.0b013e318277b625] P- Reviewers: Bujanda L, Cai SJ, Riss S, Triantafyllou K S- Editor: Gou SX L- Editor: A E- Editor: Wu HL 4255 April 21, 2014 Volume 20 Issue 15

157 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (5): Colorectal cancer Interval to surgery after neoadjuvant treatment for colorectal cancer TOPIC HIGHLIGHT Nir Wasserberg Nir Wasserberg, Department of Surgery B, Rabin Medical Center, Beilinson Campus, Petach Tikva and Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel Author contributions: Wasserberg N solely contributed to this paper. Correspondence to: Nir Wasserberg, MD, Department of Surgery B, Rabin Medical Center, Beilinson Campus, Petach Tikva and Sackler School of Medicine, Tel Aviv University, Haim Levanon 55, Tel Aviv, Israel. nirw@clalit.org.il Telephone: Fax: Received: September 28, 2013 Revised: November 11, 2013 Accepted: January 14, 2014 Published online: April 21, 2014 Abstract The current standard treatment of low-lying locally advanced rectal cancer consists of chemoradiation followed by radical surgery. The interval between chemoradiation and surgery varied for many years until the 1999 Lyon R90-01 trial which compared the effects of a short (2-wk) and long (6-wk) interval. Results showed a better clinical tumor response (71.7% vs 53.1%) and higher rate of positive and pathologic tumor regression (26% vs 10.3%) after the longer interval. Accordingly, a 6-wk interval between chemoradiation and surgery was set to balance the oncological results with the surgical complexity. However, several recent retrospective studies reported that prolonging the interval beyond 8 or even 12 wk may lead to significantly higher rates of tumor downstaging and pathologic complete response. This in turn, according to some reports, may improve overall and disease-free survival, without increasing the surgical difficulty or complications. This work reviews the data on the effect of different intervals, derived mostly from retrospective analyses using a wide variation of treatment protocols. Prospective randomized trials are currently ongoing Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Rectal cancer; Chemoradiation therapy; Neoadjuvant; Surgery; Interval to surgery; Colorectal cancer Core tip: The traditional 6-wk interval between chemoradiation and surgery in the treatment of rectal cancer was based primarily on a single publication. There has been a trend in recent years to prolong this interval based on studies showing that it may be advantageous in terms of tumor downstaging and pathologic complete response, without increasing surgical difficulty or complications. The data so far are derived largely from retrospective studies using a wide variation of treatments. Further investigations with a higher level of evidence are required to definitively resolve this issue. Wasserberg N. Interval to surgery after neoadjuvant treatment for colorectal cancer. World J Gastroenterol 2014; 20(15): Available from: URL: v20/i15/4256.htm DOI: i INTRODUCTION Colorectal cancer is the fourth most common malignancy in the United States and the second most frequent cause of cancer-related death [1]. Approximately 50% of rectal cancers are diagnosed at the locally advanced stage, with metastatic spread to the lymph nodes in two-thirds of these cases [2]. The standard treatment of rectal cancer is curative surgical resection, combined with preoperative chemoradiation therapy (CRT) for T3 and/or node-positive tumors of the mid/low rectum (located 12 cm from the anal verge), and additional adjuvant therapy if indicated [3-7]. Local recurrence rates following CRT and surgery 4256 April 21, 2014 Volume 20 Issue 15

158 Wasserberg N. CRT-surgery interval in rectal cancer are less than 10%, especially when modern surgical techniques such as total mesorectal excision are used [8]. Studies have shown that the addition of CRT has a beneficial effect on tumor downstaging and pathologic complete response (pcr) [9,10], with improved tumor resectability, possibly increased sphincter preservation rates [11,12], and maybe even increased overall survival rates [13]. Recent evidence suggests that CRT-induced tumor regression may allow for a watch and wait approach that avoids surgery altogether in selected patients [14]. Several strategies have been suggested to improve the response to CRT, including better patient selection [15], case-personalized approach with specific genetic fingerprinting [16,17], variations in the radiation and chemotherapy regimens [18-20], and additional chemotherapy during a rest interval after CRT [21]. In recent years, researchers have directed attention to optimizing the CRT-surgery interval [22], which remains controversial [22]. Ideally, the most favorable interval should facilitate maximal tumor regression, defined by maximal tumor downstaging and downsizing, with minimal risk of deterioration in the surgical results, defined by low short- term morbidity in mainly relation to perineal and anastomotic complications and better long-term oncological and functional outcomes. Early trials conducted from the 1970s to the late 1990s used a broad range of CRT-surgery intervals with widely varying results [23-27]. The 1999 Lyon R90-91 trial [28] was the first to specifically address this issue. A cohort of 201 patients with rectal cancer were prospectively randomized to undergo surgery at 2 or 6 wk after completion of radiotherapy. The longer-interval group was found to have a better tumor response and improved pathological downstaging, with similar rates of complications and survival to the shorter-interval group. The authors concluded that 2 wk may be too short a time to achieve maximum benefits of radiation-induced tumor regression. As a consequence of this study, surgery at 6 wk after completion of radiotherapy became the standard of care. However, later data suggested that the response to CRT in patients with rectal cancer is time-dependent, and complete tumor regression may take months [29]. Thus, the interval between CRT and surgery should be sufficient to attain greater tumor regression and to permit the acute radiation effects of tissue swelling and local inflammation to dissipate before surgery. At the same time, a too-long interval poses a risk of tumor progression during the wait for surgery, with a higher rate of distant metastasis. Furthermore, the accelerated repopulation of tumor cells that are not completely eradicated could lead to multidrug resistance. These drawbacks may explain the reported lack of change in survival in patients with rectal cancer despite the improvements in local control [30]. Others have raised concerns that delaying surgery beyond 6 to 8 wk from CRT could also increase the technical operative risk due to radiation-induced pelvic fibrosis, thereby increasing the rate of surgical complications [31] and locoregional recurrence [32,33]. The purpose of the present review was to summarize the current data on the optimal timing of surgical resection after CRT for rectal cancer. EFFECT OF PROLONGED CRT-SURGERY INTERVAL ON ONCOLOGICAL OUTCOME Tumor regression and rate of pcr The time elapsed from after preoperative CRT is one of the factors affecting the process of T or N stage reduction. Foster et al [22] systematically reviewed 15 studies, each based on different neoadjuvant treatment indications and regimens and different CRT-surgery intervals. Four of the 9 studies that evaluated the effect of a prolonged interval on tumor regression reported a significant improvement. Most of the studies did not apply a histologically based tumor regression grade to estimate the degree of postoperative tumor regression and fibrosis [34], although proven to be of prognostic significance [35]. The only randomized study among these publications was the Lyon R90-01 trial [28] in which the longer-interval group had significantly higher rates of a positive clinical tumor response (71.7% vs 53.1%) and pathologic tumor regression (26% vs 10.3%) than the shorter-interval group (P < 0.05 for both factors). They also had a nonsignificantly higher rate of pcr (13.8% vs 7.1%). The Dutch surgical colorectal audit is the most recent published study to address the CRT-surgery interval in terms of tumor regression [36]. A total of 1593 patients with rectal cancer were evaluated. The results showed that delaying surgery by wk from the end of CRT was associated with the highest chance of a pcr. Accordingly, in a study of predictive factors of pcr, Kalady et al [37] concluded that an extended interval between completion of neoadjuvant therapy and surgery was the single most important determinant. This finding was in line with the study of Garcia-Aguilar et al [38] which analyzed the impact of both prolonging the CRT-surgery interval and adding resting-period chemotherapy. Rates of tumor downstaging and pcr significantly increased after longer intervals to surgery (18% vs 25%). Using another approach, Perez and co-workers [39] investigated changes in labeled fluoro-2-deoxy-d-glucose uptake on positron emission tomography/computed tomography (PET/CT) imaging, at baseline and 6 and 12 wk after CRT. The maximal standard uptake value (SUVmax) was measured at 1 and 3 h at each time point. The authors found that patients with an increase in early SUVmax were less likely to have significant tumor downstaging, suggesting that the variation in PET/CT SUVmax at 6 wk might serve as a criterion for selecting patients who may be expected to benefit from a longer CRT-surgery interval. A few studies of the impact of the CRT-surgery interval included an analysis of nodal regression [40-44]. No significant impact of a longer interval was found. The Lyon R90-01 trial, however, yielded a significant effect of a longer interval on nodal regression in patients with N2 disease [28]. Similar results were noted in the Dutch colorectal surgery [36] audit in which surgery was per April 21, 2014 Volume 20 Issue 15

159 Wasserberg N. CRT-surgery interval in rectal cancer formed wk from the start of CRT. Others found that nodal retrieval is time-dependent, with a negative correlation after longer post-crt time [45]. Thus, it is possible that lymph nodes have a more rapid response to CRT which may override the effect of prolonging the CRT-surgery interval [46]. Surgical margins The status of the resection margins, including the distal mucosal and mesorectal margins, and specifically, the circumferential margins, is one of the most important factors determining disease recurrence after surgery [47,48]. Neoadjuvant CRT has been associated with reduced rates of local recurrence and tapering of the recommended margins [49]. Among the studies that examined the effect of a prolonged CRT-surgery interval on resection margin clearance [41,44], one found microscopically involved margins (R1) in 2% of patients who underwent surgery before 44 d from CRT and in 1% of patients who underwent surgery later [44]. Another reported a similar rate of positive circumferential resection margins (2.8%) with short (< 41 d) or longer intervals [41]. In neither was the effect of a prolonged interval on resection margins significant. This was true of other studies as well [22]. Prognosis Both tumor downstaging and pcr are correlated with a better oncological outcome after CRT for rectal cancer [50,51]. Some studies reported an improved prognosis after a longer CRT-surgery interval [42,52]. Tulchinsky et al [52] compared patients operated on at an interval of more or less than 7 wk after CRT. The longer-interval group had a significantly higher overall survival rate (93% vs 81%) and significantly lower distant metastasis rate (6% vs 19%) than the shorter-interval group. However, there was no between-group difference in local recurrence rate. Similarly, Coucke et al [42] demonstrated that delaying surgery for more than 5 d after hyperfractionated accelerated radiotherapy (41.6 Gy/26 Fx bid) led to a significantly higher rate of overall survival (69% vs 47% for < 5 d, P = 0.002), disease-free survival (62% vs 41%, P = ), and cancer-specific survival (82% vs 57%, P = ) at a median follow-up of 39 mo. In this study, too, there was no difference in local control rate between the groups. de Campos-Lobato et al [53] found a significant 3-year local recurrence benefit for delaying surgery for more than 8 wk after CRT (10.5% vs 1.2%), and Wolthuis et al [54] reported significantly improved 5-year cancer-specific survival (91% vs 83%) and recurrence-free survival (73% vs 83%) when CRT-surgery intervals were prolonged. Pach et al [55] randomized 154 patients to receive preoperative shortcourse radiation and surgery after 7 d vs surgery after 4-6 wk. Long CRT-surgery interval was associated with more tumor downstaging and tumor regression. Nevertheless, survival was similar in the two groups. On analysis of the oncological results of the Lyon R90-01 trial after a median follow-up of 6.3 years (range years), Glehen et al [56] failed to find any significant between-group differences. These results were supported by a Korean study in which 397 patients were randomized to undergo surgery or d after long-course CRT [41]. Rates of local and distal recurrence and of overall survival were similar in the two groups. By contrast, a retrospective multivariate analysis of 102 patients with low rectal cancer demonstrated that delaying surgery beyond 16 wk from rectal cancer diagnosis had a negative impact on overall and metastasis-free survival (OR = 2.59; 95%CI: , P = 0.005) [57]. A long interval between radiation therapy and surgery (6-8 wk) was not recommended for patients who may not benefit from tumor downstaging by sphincter preservation. Table 1 reviews the literature on the effect of a prolonged CRT-surgery interval on oncological outcome [28,36,38,40-44,49-52,54-62]. EFFECT OF A PROLONGED CRT-SURGERY INTERVAL ON SURGICAL OUTCOME Sphincter preservation The benefit of preoperative CRT in increasing the sphincter preservation rate in patients with low-lying rectal cancer is controversial. The German CAO/ARO/ AIO 94 Preoperative vs Postoperative Rectal Trial [11] reported that the preoperative administration of CRT led to a higher rate of sphincter preservation in clinical candidates for abdominoperineal resection. By contrast, a systematic review and meta-analysis of trials comparing preoperative radiation with preoperative chemoradiation showed that although preoperative CRT significantly increased the rate of pcr (P < 0.001), this did not translate into a higher rate of sphincter preservation (P = 0.29) [63]. The original Lyon R90-01 trial [28] suggested that extending the interval from CRT to surgery from 2 to 6 wk led to a trend of reduced rates of abdominoperineal resection in the longer-interval group. Yet in neither this study nor others that investigated sphincter preservation rates by CRT-surgery interval were the findings statistically significant [22,28,36]. Surgical difficulty and complications Neoadjuvant radiotherapy for rectal cancer increases postoperative complications, predominantly because of an increased risk of anastomotic leaks and delayed perineal wound healing after abdominoperineal resection [31,64]. Delaying surgery after CRT is based on the rationale that it will allow more time for resolution of the acute inflammatory response to radiotherapy. At the same time, however, it could make dissection in the narrow pelvis more complex owing to the establishment of postradiation fibrosis [58]. Garcia-Aguilar et al [38] examined the surgical difficulty and complication rate in 144 patients who underwent total mesorectal excision at 6 or 11 wk after CRT. The longer-interval group also received 2 cycles of modified FOLFOX-6 during the late resting period. There were no significant between-group differences in operative time, blood loss, or rates of diverting 4258 April 21, 2014 Volume 20 Issue 15

160 Wasserberg N. CRT-surgery interval in rectal cancer Table 1 Effect of chemoradiotherapy-surgery intervals on oncological outcome Ref. Year CRT-surgery interval Pts (n ) pcr LR OS Francois et al [28] + Glehen et al [56] /6-8 wk 201 7%/14% NS 13%/10% 69%/66% Stein et al [43] / %/14% NA NA Moore et al [44] d < %/19% NA NA Coucke et al [42] d < 250 NA 10%/7% 47%/69% 2 Supiot et al [57] wk < 102 NA 16% NA Tran et al [40] wk < 48 6%/9% 0%/9% NA Dolinsky et al [60] wk < 107 Same (P = 0.8) 11% NA Veenhof et al [62] /6-8 wk %/12% 7%/2% 64%/77% Habr-Gama et al [61] wk < %/6% 4 NA 3 NA Lim et al [41] /42-56 d %/15% 8.2% 3 NA Tulchinsky et al [52] wk < %/35% 2 6%/4% 81%/93% Kerr et al [59] 2008 Median 76 d (6-215 d) % 21% NA de Campos-Lobato et al [53] wk < %/31% %/1.2% NA Garcia-Aguilar et al [38] /11 wk %/25% 2 NA NA Evans et al [58] wk < 95 5%/12%/17% 2 NA NA Wolthuis et al [54] wk < %/28% 2 6%/3% NA Pach et al [55] d/4-5 wk %/10.4% 2 1.5%/7% 63%/73% Sloothaak et al [36] wk/8-9/10-11/11 < %/13%/18% 2 /11% NA NA 1 Short-course chemoradiotherapy (CRT); 2 Statistically significant difference; 3 Local and systemic recurrence; 4 Pts with complete clinical response who were not operated were excluded. pcr: Pathological complete response; LR: Local recurrence; OS: Overall survival; NA: Not available. stoma, sphincter preservation, and R0 resection. Additionally, the proportion of patients who acquired any postoperative complications was similar in both groups (40%), with no significant difference in rate of anastomotic leaks. Surgeons participating in that study reported more widespread fibrosis in the longer-interval group. However, they were not blinded to the treatment protocol and ultimately rated surgical difficulty similarly (on a scale of 1-10) for both groups. Other authors, however, reported a longer operative time when the CRT-surgery interval was longer, which may reflect increased surgical difficulty [40,52]. Nonetheless, extending the CRT-surgery interval did not increase the complication rate [22] ; indeed, one study noted significantly higher rates of anastomotic leak and perineal wound complications in patients in the shorter-interval (< 44 d) arm [59]. CONCLUSION The traditional 6-wk CRT-surgery interval used for years in the treatment of rectal cancer was based primarily on a single study showing its oncological benefit compared to a 2-wk interval, which was apparently too short to yield significant post-radiation changes. Recent studies that sought to further improve outcome in patients with locally advanced, low-lying rectal tumors found that prolonging the interval beyond 6 wk was advantageous, mainly in terms of tumor downstaging and pcr rates, without increasing surgical difficulty or complications. These findings appear to have prompted a recent trend towards increasing the time from neoadjuvant CRT to surgery. However, the data available so far derive largely from retrospective studies that applied different CRT protocols and different CRT-surgery intervals, with no consideration of the effect of variability in preoperative staging. Further investigations with a higher level of evidence are required to definitively resolve this issue. Several centers are currently conducting prospective randomized control studies to determine the optimal interval between CRT and surgery. The multicenter Swedish Stockholm Ⅲ trial [65] that is testing different regimens of radiotherapy will be completed in Participants are divided into 3 groups: short-course CRT followed by surgery one week later; short-course CRT followed by surgery 4-8 wk later; and long-course CRT followed by surgery 4-8 wk later. The study will include an estimated 840 patients. An interim analysis of 303 patients showed that short-course CRT and surgery at 7-11 d was associated with a trend for more complications [66]. In another study begun in 2009 in the United Kingdom, patients are randomized to undergo CRT and surgery after 6 or 12 wk. The final cohort will include 218 patients at the end of recruitment [67]. 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Preoperative radiation with concurrent chemotherapy for resectable rectal cancer: effect of dose escalation on pathologic complete response, local recurrencefree survival, disease-free survival, and overall survival. Int J Radiat Oncol Biol Phys 2006; 64: [PMID: DOI: /j.ijrobp ] 20 Wong SJ, Winter K, Meropol NJ, Anne PR, Kachnic L, Rashid A, Watson JC, Mitchell E, Pollock J, Lee RJ, Haddock M, Erickson BA, Willett CG. Radiation Therapy Oncology Group 0247: a randomized Phase II study of neoadjuvant capecitabine and irinotecan or capecitabine and oxaliplatin with concurrent radiotherapy for patients with locally advanced rectal cancer. Int J Radiat Oncol Biol Phys 2012; 82: [PMID: DOI: /j.ijrobp ] 21 Habr-Gama A, Perez RO, Sabbaga J, Nadalin W, São Julião GP, Gama-Rodrigues J. Increasing the rates of complete response to neoadjuvant chemoradiotherapy for distal rectal cancer: results of a prospective study using additional chemotherapy during the resting period. Dis Colon Rectum 2009; 52: [PMID: DOI: /DCR.0b013e3181ba14ed] 22 Foster JD, Jones EL, Falk S, Cooper EJ, Francis NK. Timing of surgery after long-course neoadjuvant chemoradiotherapy for rectal cancer: a systematic review of the literature. Dis Colon Rectum 2013; 56: [PMID: DOI: / DCR.0b013e31828aedcb] 23 Kligerman MM, Urdaneta N, Knowlton A, Vidone R, Hartman PV, Vera R. Preoperative irradiation of rectosigmoid carcinoma including its regional lymph nodes. Am J Roentgenol Radium Ther Nucl Med 1972; 114: [PMID: DOI: /ajr ] 24 Rider WD, Palmer JA, Mahoney LJ, Robertson CT. Preoperative irradiation in operable cancer of the rectum: report of the Toronto trial. Can J Surg 1977; 20: [PMID: ] 25 Higgins GA, Humphrey EW, Dwight RW, Roswit B, Lee LE, Keehn RJ. Preoperative radiation and surgery for cancer of the rectum. Veterans Administration Surgical Oncology Group Trial II. 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162 Wasserberg N. CRT-surgery interval in rectal cancer radiotherapy and curative surgery for the management of localized rectal carcinoma. Cochrane Database Syst Rev 2007; (2): CD [PMID: ] 32 Nozue M, Isaka N, Fukao K. Over-expression of vascular endothelial growth factor after preoperative radiation therapy for rectal cancer. Oncol Rep 2001; 8: [PMID: ] 33 Baeten CI, Castermans K, Lammering G, Hillen F, Wouters BG, Hillen HF, Griffioen AW, Baeten CG. Effects of radiotherapy and chemotherapy on angiogenesis and leukocyte infiltration in rectal cancer. Int J Radiat Oncol Biol Phys 2006; 66: [PMID: DOI: / j.ijrobp ] 34 Dworak O, Keilholz L, Hoffmann A. Pathological features of rectal cancer after preoperative radiochemotherapy. Int J Colorectal Dis 1997; 12: [PMID: DOI: / s ] 35 Rödel C, Martus P, Papadoupolos T, Füzesi L, Klimpfinger M, Fietkau R, Liersch T, Hohenberger W, Raab R, Sauer R, Wittekind C. Prognostic significance of tumor regression after preoperative chemoradiotherapy for rectal cancer. J Clin Oncol 2005; 23: [PMID: DOI: / JCO ] 36 Sloothaak DA, Geijsen DE, van Leersum NJ, Punt CJ, Buskens CJ, Bemelman WA, Tanis PJ. Optimal time interval between neoadjuvant chemoradiotherapy and surgery for rectal cancer. Br J Surg 2013; 100: [PMID: DOI: /bjs.9112] 37 Kalady MF, de Campos-Lobato LF, Stocchi L, Geisler DP, Dietz D, Lavery IC, Fazio VW. Predictive factors of pathologic complete response after neoadjuvant chemoradiation for rectal cancer. Ann Surg 2009; 250: [PMID: DOI: /SLA.0b013e3181b91e63] 38 Garcia-Aguilar J, Smith DD, Avila K, Bergsland EK, Chu P, Krieg RM. Optimal timing of surgery after chemoradiation for advanced rectal cancer: preliminary results of a multicenter, nonrandomized phase II prospective trial. Ann Surg 2011; 254: [PMID: DOI: /SLA.0b013e e1f] 39 Perez RO, Habr-Gama A, São Julião GP, Gama-Rodrigues J, Sousa AH, Campos FG, Imperiale AR, Lynn PB, Proscurshim I, Nahas SC, Ono CR, Buchpiguel CA. Optimal timing for assessment of tumor response to neoadjuvant chemoradiation in patients with rectal cancer: do all patients benefit from waiting longer than 6 weeks? Int J Radiat Oncol Biol Phys 2012; 84: [PMID: DOI: / j.ijrobp ] 40 Tran CL, Udani S, Holt A, Arnell T, Kumar R, Stamos MJ. Evaluation of safety of increased time interval between chemoradiation and resection for rectal cancer. Am J Surg 2006; 192: [PMID: DOI: /j.amjsurg ] 41 Lim SB, Choi HS, Jeong SY, Kim DY, Jung KH, Hong YS, Chang HJ, Park JG. Optimal surgery time after preoperative chemoradiotherapy for locally advanced rectal cancers. Ann Surg 2008; 248: [PMID: DOI: / SLA.0b013e31817fc2a0] 42 Coucke PA, Notter M, Matter M, Fasolini F, Calmes JM, Schlumpf R, Schwegler N, Stamm B, Phuoc Do H, Bouzourene H. Effect of timing of surgery on survival after preoperative hyperfractionated accelerated radiotherapy (HART) for locally advanced rectal cancer (LARC): is it a matter of days? Acta Oncol 2006; 45: [PMID: DOI: / ] 43 Stein DE, Mahmoud NN, Anné PR, Rose DG, Isenberg GA, Goldstein SD, Mitchell E, Fry RD. Longer time interval between completion of neoadjuvant chemoradiation and surgical resection does not improve downstaging of rectal carcinoma. Dis Colon Rectum 2003; 46: [PMID: DOI: /s ] 44 Moore HG, Gittleman AE, Minsky BD, Wong D, Paty PB, Weiser M, Temple L, Saltz L, Shia J, Guillem JG. Rate of pathologic complete response with increased interval between preoperative combined modality therapy and rectal cancer resection. Dis Colon Rectum 2004; 47: [PMID: DOI: /s ] 45 Sermier A, Gervaz P, Egger JF, Dao M, Allal AS, Bonet M, Morel P. Lymph node retrieval in abdominoperineal surgical specimen is radiation time-dependent. World J Surg Oncol 2006; 4: 29 [PMID: DOI: / ] 46 Bipat S, Glas AS, Slors FJ, Zwinderman AH, Bossuyt PM, Stoker J. Rectal cancer: local staging and assessment of lymph node involvement with endoluminal US, CT, and MR imaging--a meta-analysis. Radiology 2004; 232: [PMID: DOI: /radiol ] 47 Quirke P, Durdey P, Dixon MF, Williams NS. Local recurrence of rectal adenocarcinoma due to inadequate surgical resection. Histopathological study of lateral tumour spread and surgical excision. Lancet 1986; 2: [PMID: DOI: /S (86) ] 48 Wibe A, Rendedal PR, Svensson E, Norstein J, Eide TJ, Myrvold HE, Søreide O. Prognostic significance of the circumferential resection margin following total mesorectal excision for rectal cancer. Br J Surg 2002; 89: [PMID: DOI: /j x] 49 Wasserberg N, Gutman H. Resection margins in modern rectal cancer surgery. J Surg Oncol 2008; 98: [PMID: DOI: /jso.21036] 50 de Campos-Lobato LF, Stocchi L, da Luz Moreira A, Kalady MF, Geisler D, Dietz D, Lavery IC, Remzi FH, Fazio VW. Downstaging without complete pathologic response after neoadjuvant treatment improves cancer outcomes for ciii but not cii rectal cancers. Ann Surg Oncol 2010; 17: [PMID: DOI: /s ] 51 Maas M, Nelemans PJ, Valentini V, Das P, Rödel C, Kuo LJ, Calvo FA, García-Aguilar J, Glynne-Jones R, Haustermans K, Mohiuddin M, Pucciarelli S, Small W, Suárez J, Theodoropoulos G, Biondo S, Beets-Tan RG, Beets GL. Long-term outcome in patients with a pathological complete response after chemoradiation for rectal cancer: a pooled analysis of individual patient data. Lancet Oncol 2010; 11: [PMID: DOI: /S (10) ] 52 Tulchinsky H, Shmueli E, Figer A, Klausner JM, Rabau M. An interval & gt; 7 weeks between neoadjuvant therapy and surgery improves pathologic complete response and diseasefree survival in patients with locally advanced rectal cancer. Ann Surg Oncol 2008; 15: [PMID: DOI: /s ] 53 de Campos-Lobato LF, Geisler DP, da Luz Moreira A, Stocchi L, Dietz D, Kalady MF. Neoadjuvant therapy for rectal cancer: the impact of longer interval between chemoradiation and surgery. J Gastrointest Surg 2011; 15: [PMID: DOI: /s ] 54 Wolthuis AM, Penninckx F, Haustermans K, De Hertogh G, Fieuws S, Van Cutsem E, D Hoore A. Impact of interval between neoadjuvant chemoradiotherapy and TME for locally advanced rectal cancer on pathologic response and oncologic outcome. Ann Surg Oncol 2012; 19: [PMID: DOI: /s ] 55 Pach R, Kulig J, Richter P, Gach T, Szura M, Kowalska T. Randomized clinical trial on preoperative radiotherapy 25 Gy in rectal cancer--treatment results at 5-year follow-up. Langenbecks Arch Surg 2012; 397: [PMID: DOI: /s ] 56 Glehen O, Chapet O, Adham M, Nemoz JC, Gerard JP. Long-term results of the Lyons R90-01 randomized trial of preoperative radiotherapy with delayed surgery and its effect on sphincter-saving surgery in rectal cancer. Br J Surg 2003; 90: [PMID: DOI: /bjs.4162] 57 Supiot S, Bennouna J, Rio E, Meurette G, Bardet E, Buecher B, Dravet F, Le Neel JC, Douillard JY, Mahé MA, Lehur PA. Negative influence of delayed surgery on survival after preoperative 4261 April 21, 2014 Volume 20 Issue 15

163 Wasserberg N. CRT-surgery interval in rectal cancer radiotherapy in rectal cancer. Colorectal Dis 2006; 8: [PMID: DOI: /j x] 58 Evans J, Patel U, Brown G. Rectal cancer: primary staging and assessment after chemoradiotherapy. Semin Radiat Oncol 2011; 21: [PMID: DOI: / j.semradonc ] 59 Kerr SF, Norton S, Glynne-Jones R. Delaying surgery after neoadjuvant chemoradiotherapy for rectal cancer may reduce postoperative morbidity without compromising prognosis. Br J Surg 2008; 95: [PMID: DOI: /bjs.6377] 60 Dolinsky CM, Mahmoud NN, Mick R, Sun W, Whittington RW, Solin LJ, Haller DG, Giantonio BJ, O Dwyer PJ, Rosato EF, Fry RD, Metz JM. Effect of time interval between surgery and preoperative chemoradiotherapy with 5-fluorouracil or 5-fluorouracil and oxaliplatin on outcomes in rectal cancer. J Surg Oncol 2007; 96: [PMID: DOI: / jso.20815] 61 Habr-Gama A, Perez RO, Proscurshim I, Nunes Dos Santos RM, Kiss D, Gama-Rodrigues J, Cecconello I. Interval between surgery and neoadjuvant chemoradiation therapy for distal rectal cancer: does delayed surgery have an impact on outcome? Int J Radiat Oncol Biol Phys 2008; 71: [PMID: DOI: /j.ijrobp ] 62 Veenhof AA, Bloemena E, Engel AF, van der Peet DL, Meijer OW, Cuesta MA. The relationship of histological tumor regression grade (TRG) and two different time intervals to surgery following radiation therapy for locally advanced rectal cancer. Int J Colorectal Dis 2009; 24: [PMID: DOI: /s ] 63 Ceelen W, Fierens K, Van Nieuwenhove Y, Pattyn P. Preoperative chemoradiation versus radiation alone for stage II and III resectable rectal cancer: a systematic review and meta-analysis. Int J Cancer 2009; 124: [PMID: DOI: /ijc.24247] 64 Påhlman L, Glimelius B. Pre- or postoperative radiotherapy in rectal and rectosigmoid carcinoma. Report from a randomized multicenter trial. Ann Surg 1990; 211: [PMID: DOI: / ] 65 The Stockholm III Trial on Different Preoperative Radiotherapy Regimens in Rectal Cancer. Accessed: September, 26, Available from: URL: NCT Pettersson D, Cedermark B, Holm T, Radu C, Påhlman L, Glimelius B, Martling A. Interim analysis of the Stockholm III trial of preoperative radiotherapy regimens for rectal cancer. Br J Surg 2010; 97: [PMID: DOI: / bjs.6914] 67 Cancer Research United Kingdom. Treatment for rectal cancer. Accessed: September, 26, Available from: URL: 68 STARRCAT Trial: Surgical timing after radiotherapy for rectal cancer. Accessed: September, 26, Available at: P- Reviewers: Chen YJ, Kita H, Meshikhes AN, Stephane S, Wang XS S- Editor: Gou SX L- Editor: A E- Editor: Wu HL 4262 April 21, 2014 Volume 20 Issue 15

164 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (5): Colorectal cancer Anti-EGFR and anti-vegf agents: Important targeted therapies of colorectal liver metastases TOPIC HIGHLIGHT Qing-Yang Feng, Ye Wei, Jing-Wen Chen, Wen-Ju Chang, Le-Chi Ye, De-Xiang Zhu, Jian-Min Xu Qing-Yang Feng, Ye Wei, Jing-Wen Chen, Wen-Ju Chang, Le-Chi Ye, De-Xiang Zhu, Jian-Min Xu, Department of General Surgery, Zhongshan Hospital, Fudan University, Shanghai , China Author contributions: Feng QY and Wei Y wrote the paper; Chen JW, Chang WJ, Ye LC and Zhu DX assisted with data collection, proofreading and in other ways; Xu JM was the invited author and established the theme of this paper; all authors discussed the opinions expressed in this article. Correspondence to: Jian-Min Xu, MD, PhD, Department of General Surgery, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai , China. xujmin@aliyun.com Telephone: Fax: Received: September 26, 2013 Revised: February 7, 2014 Accepted: February 20, 2014 Published online: April 21, 2014 Abstract Colorectal liver metastasis (CLM) is common worldwide. Targeted therapies with monoclonal antibodies have been proven effective in numerous clinical trials, and are now becoming standards for patients with CLM. The development and application of anti-epidermal growth factor receptor (anti-egfr) and anti-vascular endothelial growth factor (anti-vegf) antibodies represents significant advances in the treatment of this disease. However, new findings continue to emerge casting doubt on the efficacy of this approach. The Kirsten rat sarcoma viral oncogene (KRAS) has been proven to be a crucial predictor of the success of anti-egfr treatment in CLM. Whereas a recent study summarized several randomized controlled trials, and showed that patients with the KRAS G13D mutation significantly benefited from the addition of cetuximab in terms of progress-free survival (PFS, 4.0 mo vs 1.9 mo, HR = 0.51, P = 0.004) and overall survival (OS, 7.6 mo vs 5.7 mo, HR = 0.50, P = 0.005). Some other studies also reported that the KRAS G13D mutation might not be absolutely predictive of non-responsiveness to anti- EGFR therapy. At the same time, new RAS mutations, including mutations in neuroblastoma RAS viral (vras) oncogene homolog (NRAS) and exons 3 and 4 of KRAS, have been suggested to be predictors of a poor treatment response. This finding was first reported by the update of the PRIME trial. The update showed that for patients with non-mutated KRAS exon 2 but other RAS mutations, panitumumab-fluorouracil, leucovorin, and oxaliplatin (FOLFOX)4 treatment led to inferior PFS (HR = 1.28, 95%CI: ) and OS (HR = 1.29, 95%CI: ), which was consistent with the findings in patients with KRAS mutations in exon 2. Then, the update of the PEAK trial and the FIRE-Ⅲ trial also supported this finding, which would reduce candidates for anti-egfr therapy but enhance the efficacy. In firstline targeted combination therapy, the regimens of cetuximab plus FOLFOX was called into question because of the inferior prognosis in the COIN trial and the NOR- DIC-Ⅶ trial. Also, bevacizumab plus oxaliplatin-based chemotherapy was questioned because of the NO16966 trial. By the update and further analysis of the COIN trial and the NORDIC-Ⅶ trial, cetuximab plus FOLFOX was reported to be reliable again. But bevacizumab plus oxaliplatin-based chemotherapy was still controversial. In addition, some trials have reported that bevacizumab is not suitable for conversion therapy. The results of the FIRE-Ⅲ trial showed that cetuximab led to a significant advantage over bevacizumab in response rate (72% vs 63%, P = 0.017) for evaluable population. With the balanced allocation of second-line treatment, the FIRE- Ⅲ trial was expected to provide evidence for selecting following regimens after first-line progression. There is still no strong evidence for the efficacy of targeted therapy as a preoperative treatment for resectable CLM or postoperative treatment for resected CLM, although the combined regimen is often administered based on experience. Combination therapy with more than one targeted agent has been proven to provide no benefit, and even was reported to be harmful as first-line treatment by four large clinical trials. However, recent studies reported positive results of erlotinib plus bevacizumab for 4263 April 21, 2014 Volume 20 Issue 15

165 Feng QY et al. Targeted therapy of colorectal liver metastases maintenance treatment. The mechanism of antagonism between different targeted agents deserves further study, and may also provide greater understanding of the development of resistance to targeted agents Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Oncology; Colorectal cancer; Liver metastases; Chemotherapy; Targeted therapy Core tip: Targeted therapy is becoming standard for patients with colorectal liver metastases, but new findings continue to improve our understanding of these therapies. New RAS mutations, rather than the Kirsten rat sarcoma viral oncogene G13D mutation, may be predictive of non-responsiveness to anti-epidermal growth factor receptor therapy. The regimen of cetuximab plus FOLFOX is likely effective, but bevacizumab plus oxaliplatin-based chemotherapy remains controversial. Bevacizumab was suggested to be unsuitable for conversion therapy. Further confirmation is required to demonstrate the effectiveness of targeted therapy as a pre- or postoperative treatment. Combination therapy with more than one targeted agent is not recommended. Feng QY, Wei Y, Chen JW, Chang WJ, Ye LC, Zhu DX, Xu JM. Anti-EGFR and anti-vegf agents: Important targeted therapies of colorectal liver metastases. World J Gastroenterol 2014; 20(15): Available from: URL: com/ /full/v20/i15/4263.htm DOI: org/ /wjg.v20.i INTRODUCTION Colorectal cancer is one of the most common malignant tumors throughout the world [1,2]. It has the third highest incidence and death rate among all cancers in the United States [3], with new cases and deaths in In China, colorectal cancer was the third most common cancer and had the fifth highest death rate in 2009 [4]. Liver metastases are particularly common in patients with colorectal cancer. Almost 50% of patients will eventually develop liver metastases during the natural course of the disease [5,6], and 25% of patients present with liver metastases at diagnosis [7-9]. Autopsies have shown that more than half of patients who died of colorectal cancer had liver metastases, with metastatic liver disease as the cause of death in most patients. However, over the last two decades, the long-term survival of patients with colorectal liver metastases (CLM) has significantly improved. From 1990 to 1997, the median overall survival (OS) was 14.2 mo (95%CI: ), significantly increased to 29.2 mo (95%CI: ) from 2004 to 2006 (P < 0.05). The 5-year survival rate also rose to 32% (95%CI: 27-38) [10]. These achievements should be attributed to the popularity of early diagnostic techniques, the expansion of indications for liver resection, the introduction of new chemotherapeutic agents (oxaliplatin, irinotecan, etc.), and the application of novel targeted agents (cetuximab, panitumumab, bevacizumab, etc.). TARGETED THERAPY AND MOLECULAR PREDICTORS Targeted biological therapy is becoming a standard personalized medical treatment for patients with CLM. Two options are currently available in routine clinical practice for patients with CLM: anti-epidermal growth factor receptor (anti-egfr) antibodies and anti-vascular endothelial growth factor (anti-vegf) antibodies. The anti-eg- FR antibodies include cetuximab, a chimeric monoclonal antibody, and panitumumab, a fully human monoclonal antibody. These antibodies are directed against EGFR and inhibit downstream signaling pathways, leading to the inhibition of both cell proliferation and angiogenesis. The humanized monoclonal anti-vegf antibody bevacizumab exerts its anti-tumor effects by binding VEGF and inhibiting VEGF from binding to its functional receptor, leading to the inhibition of tumor vessel growth and neovascularization and to a decreased permeability in the surviving vasculature. Currently, no effective predictor of treatment success with angiogenesis inhibitors has been established, although the presence of the Kirsten rat sarcoma viral oncogene (KRAS) gene mutation is known to play a crucial role in predicting the success of treatment with anti-eg- FR antibodies [11,12]. Three genes in the RAS gene family are associated with human tumorigenesis, v-ha-ras Harvey rat sarcoma viral oncogene homolog (HRAS), KRAS and neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS). Every RAS gene has four exons. Past research suggested that RAS mutations were mainly concentrated in KRAS exon 2, codon 12 and 13. Approximately 40% of colorectal cancers are characterized by KRAS gene mutations in these two coding regions [13,14]. Many studies have confirmed that KRAS exon 2 mutations are predictive of no benefit from anti-egfr treatment. The CRYSTAL trial [15] proved that for patients with KRAS mutations, the addition of cetuximab to FOLFIRI (irinotecan, fluorouracil and leucovorin) as a first-line therapy did not lead to a significant improvement in median progress-free survival (PFS; median PFS, 7.4 mo vs 7.7 mo, hazard ratio, HR = 1.171, P = 0.260) and median overall survival (OS; median OS, 16.2 mo vs 16.7 mo, HR = 1.035, P = 0.75). Another clinical trial [16] of panitumumab also proved that for patients with KRAS mutations, no PFS benefit was associated with panitumumab (median PFS, 7.3 wk vs 7.4 wk, HR = 0.99, 95%CI: ). The subsequent quantitative interaction test compared the magnitude of the relative treatment effect on PFS between wild type and mutant KRAS groups, and showed a statistically significant difference (P < ). A recent meta-analysis [17] also demonstrated the predictive role of the KRAS gene in anti-egfr therapy April 21, 2014 Volume 20 Issue 15

166 Feng QY et al. Targeted therapy of colorectal liver metastases However, some recent studies raised the possibility that KRAS exon 2, codon 13 mutations, mainly G13D, might not be absolutely predictive of non-responsiveness to anti- EGFR therapy. A retrospective study by De Roock et al [18] pooled the clinical trials CO.17, BOND, MABEL, EMR202600, EVEREST, BABEL and SALVAGE. The results indicated that in comparison with other KRAS mutation, patients with the KRAS G13D mutation significantly benefited from the addition of cetuximab in OS (median OS, 7.6 mo vs 5.7 mo, HR = 0.50, 95%CI: , P = 0.005) and PFS (median PFS, 4.0 mo vs 1.9 mo, HR = 0.51, 95%CI: , P = 0.004). There was also a significant interaction between KRAS mutation status (G13D vs other KRAS mutations) and overall survival benefit with cetuximab treatment (adjusted HR = 0.30, 95%CI: , P = 0.003). No difference was observed between patients with the KRAS G13D mutation and wild type KRAS in OS (median OS, P = 0.79; median PFS, P = 0.66). However, for patients with the KRAS G13D mutation, the difference in OS or PFS between cetuximab plus chemotherapy and chemotherapy alone became non-significant in the multivariate analysis (adjusted OS, HR = 0.40, 95%CI: , P = 0.12; PFS, HR = 0.53, 95%CI: , P = 0.29). Soon afterwards, Tejpar et al [19] pooled the clinical trials CRYSTAL and OPUS, finding that in patients with G13D mutant tumors, the addition of cetuximab to chemotherapy significantly improved tumor response (40.5% vs 22.0%, odds ratio, OR = 3.38, P = 0.042) and PFS (median PFS, 7.4 mo vs 6.0 mo, HR = 0.47, P = 0.039), but not OS (median OS, 15.4 mo vs 14.7 mo, HR = 0.89, P = 0.68) after adjusting for differences in baseline variables. Otherwise, in comparison with patients with the KRAS wild type allele, patients with the KRAS G13D mutation still had poorer tumor response (OR = 0.50, 95%CI: , P = 0.040) and OS (HR = 1.61, 95%CI: , P = ) with cetuximab. Another meta-analysis by Mao et al [20] included 10 studies and 1487 patients, concluding that cetuximab led to significantly higher response rates (relative risk, RR = 1.642, 95%CI: ) in patients with the KRAS G13D mutation than in those with the KRAS G12 mutation. These studies showed benefits from cetuximab in patients with the KRAS G13D mutation, and not worse than KRAS wild type patients. The authors suggested that the KRAS G13D mutation should be distinguished from other KRAS mutations. At the same time, other studies supported opposing views. Peeters et al [21] conducted a retrospective study assessing the prognostic and predictive impact of individual KRAS codon 12 and 13 mutations on panitumumab combined with chemotherapy. They pooled three randomized phase Ⅲ studies including a total of 1053 patients and came to a negative conclusion. The results showed that out of all types of KRAS mutations (including G12D, G12V, G12C, G12A, G12S, G12D), only G12A was associated with a negative predictive effect on OS. No other significant differences were observed between panitumumab plus chemotherapy and chemotherapy alone in OS or PFS. Considering the common mechanism of cetuximab and panitumumab, the efficacy of anti-egfr therapy for patients with KRAS G13D mutations remained in doubt. In addition, the above studies are all retrospective and only include a limited number of cases. Thus, the function of the KRAS G13D test should be interpreted and applied conservatively. Further randomized controlled clinical trials should be conducted for confirmation. In addition to exon 2 mutations, other activating RAS mutations may also be negative predictive biomarkers for anti-egfr therapy, including those in NRAS and exons 3 and 4 of KRAS, which together are called new RAS. Douillard et al [22] tested the new RAS status of the KRAS wild type patients from the PRIME trial. The results showed that in 512 patients without any RAS mutations, panitumumab plus FOLFOX4 led to a 2.2 mo improvement in PFS (median PFS, 10.1 mo vs 7.9 mo, HR = 0.72, P = 0.004) and a 5.8 mo improvement in OS (median OS, 26.0 mo vs 20.2 mo, HR = 0.78, P = 0.04), a greater improvement than that observed in patients with only wild type KRAS exon 2. A total of 108 patients (17%) with non-mutated KRAS exon 2 had other RAS mutations. These mutations were associated with inferior PFS (HR = 1.28, 95%CI: ) and OS (HR = 1.29, 95%CI: ) with panitumumab-folfox4 treatment, which was consistent with the findings in patients with KRAS mutations in exon 2. At the same time, the head-to-head phase Ⅱ trial PEAK evaluated panitumumab plus mfolfox6 and bevacizumab plus mfolfox6 in patients with previously untreated KRAS exon 2 wild type CLM. The findings from this trial were reported at the 2013 ASCO Annual Meeting [23], with no significant differences observed between panitumumab and bevacizumab in PFS (median PFS, 10.9 mo vs 10.1 mo, HR = 0.97, 95%CI: ) or OS (median OS, not reached vs 25.4 mo, HR = 0.72, 95%CI: ). However, in subgroup analysis of new RAS wild type variants, panitumumab led to a significant improvement in PFS (median PFS, 13.1 mo vs 9.5 mo, HR = 0.63, P = 0.02) and OS (median OS, not reached vs 29.0 mo, HR = 0.55, P = 0.06). These results indicated that the new RAS biomarkers should also be tested when receiving anti-egfr treatment. This increasing number of predictors is leading to the decreasing number of patients suitable for anti-egfr therapy, but is also resulting in an enhanced efficacy of the treatment. As a gene downstream of KRAS, v-raf murine sarcoma viral oncogene homolog B1 (BRAF) is also a hotspot. Approximately 5% to 9% of colorectal cancers are characterized by a specific mutation in the BRAF gene, mainly V600E [14,15]. For all practical purposes, BRAF mutations are limited to tumors that do not have KRAS mutations. Several studies have confirmed BRAF as a strong prognostic marker [13,15,24,25], but the utility of BRAF status as a predictive marker remains unclear. The subgroup analysis of the CRYSTAL trial [15] showed that in patients with wild type KRAS and mutated BRAF, improvements in PFS (median PFS, 8.0 mo vs 5.6 mo, HR = 0.934, P = 0.87) and OS (median OS, 14.1 mo vs 10.3 mo, HR = 4265 April 21, 2014 Volume 20 Issue 15

167 Feng QY et al. Targeted therapy of colorectal liver metastases 0.908, P = 0.74) associated with the addition of cetuximab to FOLFIRI were not statistically significant. Although no evidence was reported of an independent interaction between the treatment approach and the tumor BRAF mutation status, BRAF, like KRAS, is expected to become a new predictive factor for treatment efficacy. TARGETED THERAPY FOR UNRESECTABLE METASTATIC COLORECTAL CANCER First-line treatment Anti-EGFR antibody as first-line treatment: Currently, cetuximab plays a major role in the clinical application of anti-egfr therapeutics. Many clinical trials of cetuximab combined with chemotherapy have demonstrated the significant utility of this agent in treating metastatic colorectal cancer (details in Table 1). The CRYSTAL trial [15,26] involved 1198 patients with unresectable CLM, comparing the addition of cetuximab to irinotecan, fluorouracil, and leucovorin (FOLFIRI) with FOLFIRI alone as the first-line treatment. The results showed that for the intent-to-treat (ITT) population, the cetuximab combined group exhibited a significant increase in the primary end point, PFS (median PFS, 8.9 mo vs 8.0 mo, HR = 0.85, P = 0.048), but not significant in the secondary end point, OS (median OS, 19.9 mo vs 18.6 mo, HR = 0.93, P = 0.31). For patients with wild type KRAS, the cetuximab combined group exhibited a greater benefit in terms of PFS (median PFS, 9.9 mo vs 8.7 mo, HR = 0.68, 95%CI: ) and OS (median OS, 23.5 mo vs 20.0 mo, HR = 0.796, P = ). The CRYSTAL study thus became a pivotal study in obtaining European Medicines Agency approval of the use of cetuximab as a first-line treatment for metastatic colorectal cancer. Another sizable clinical trial, OPUS [27,28], also proved that for patients with wild type KRAS, cetuximab plus FOLFOX (fluorouracil, leucovorin, and oxaliplatin) significantly improved PFS (median PFS, 8.3 mo vs 7.2 mo, HR = 0.567, P = ) but not OS (median OS, 22.8 mo vs 18.5 mo, HR = 0.855, P = 0.39). Therefore, cetuximab combined with chemotherapy is considered an important part of first-line treatment for unresectable CLM. However, the efficacy of cetuximab combined with FOLFOX was called into question by recent studies. In the COIN trial [24], the addition of cetuximab to oxaliplatin-based chemotherapy did not result in a significant improvement in the primary end point OS (median OS, 17.0 mo vs 17.9 mo, P = 0.68), or the secondary end point PFS (median PFS, 8.6 mo in both arms, P = 0.60). Only the response rate was significantly increased by the addition of cetuximab (64% vs 57%, P = 0.049). Then in the NORDIC-Ⅶ trial [29], patients with wild type KRAS derived no benefit from cetuximab plus FLOX (fluorouracil, leucovorin, and oxaliplatin) in the primary end point PFS (median PFS, 8.7 mo vs 7.9 mo, P = 0.66), or in the secondary end point OS (median OS, 22.0 mo vs 20.1 mo, P = 0.48). Considering these two clinical trials, the national comprehensive cancer network (NCCN) guidelines have removed the regimen of cetuximab plus FOLFOX from first-line treatment since the 2012 version. However, the 2012 European Society for Medical Oncology (ESMO) guidelines retained this regimen and assigned its highest level of recommendation (Recommendation +++) because of defects in the COIN trial and the NORDIC- Ⅶ trial. In the COIN trial [24], cetuximab plus capecitabine and oxaliplatin (XELOX) induced more severe diarrhea, and the dose of capecitabine had to be reduced by 15%. Additionally, in recent subgroup analysis, a significant improvement in PFS with cetuximab was observed in allwild-type (KRAS, NRAS, BRAF) patients treated with fluorouracil-based therapy (HR = 0.72, 95%CI: , P = 0.037) but not in those treated with capecitabinebased therapy (HR = 1.02, 95%CI: , P = 0.88). Patients with no more than one metastatic site also exhibited improved PFS with cetuximab (HR = 0.73, 95%CI: , P = 0.03). In the NORDIC-Ⅶ trial [29], fluorouracil was given as a bolus infusion instead of a continued infusion, which was not exactly the same as the FOLFOX regimen. In conclusion, the regimen of cetuximab combined with FOLFOX has been proven to be reliable as a first-line treatment for CLM. Panitumumab combined with chemotherapy is also an important first-line treatment for CLM (details in Table 1). The PRIME trial showed that panitumumab plus FOLFOX4 significantly improved PFS in KRAS exon 2 wild type patients [30] (median PFS, 9.6 mo vs 8.0 mo, HR = 0.80, P = 0.02) and all RAS wild type patients (mentioned above). However, the regimen of panitumumab plus FOLFIRI was not analyzed in a reliable randomized controlled clinical trial as a first-line treatment, and its efficacy was mainly inferred from the results of clinical trials where it was used as a second-line treatment. Therefore, panitumumab plus FOLFIRI was only listed as an alternative in the NCCN guidelines (V. 2013) and was not recommended in ESMO guidelines (V. 2012). Taking into account the differences between populations receiving first-line and second-line treatment, the combination of chemotherapy with panitumumab should be carefully chosen when used in first-line treatment. Anti-VEGF antibody as a first-line treatment: The anti-vegf antibody bevacizumab has been widely used as a first-line treatment for CLM, and achieves good effects (details in Table 1). Kabbinavar et al [31] pooled data from patients receiving bevacizumab plus fluorouracil/ leucovorin from three clinical trials, and reported significant improvements in response rate (34.1% vs 24.5%, P = 0.019), PFS (median PFS, 8.8 mo vs 5.6 mo, HR = 0.63, P < ) and OS (median OS, 17.9 mo vs 14.6 mo, HR = 0.74, P = 0.008). The pivotal AVF2107g trial [32] proved that the addition of bevacizumab to IFL (irinotecan, fluorouracil, and leucovorin) resulted in a statistically significant and clinically meaningful improvement in the primary end point, OS (median OS, 20.3 mo vs 15.6 mo, HR = 0.66, P < 0.001), and the secondary end point, PFS (median PFS, 10.6 mo vs 6.2 mo, HR = 0.54, P < 0.001) April 21, 2014 Volume 20 Issue 15

168 Feng QY et al. Targeted therapy of colorectal liver metastases Table 1 Phase Ⅱ/Ⅲ clinical trials of targeted agents in combination with chemotherapy as first-line treatments for metastatic colorectal cancer Clinical trial (reporters) Year Primary Population Regimen Patient Median end point number PFS (mo) HR P value (95%CI) Median OS (mo) HR P value (95%CI) Response rates OR P value (95%CI) CRYSTAL [26] 2009 PFS ITT FOLFIRI + Cet HR = HR = % OR = FOLFIRI a P = P = % a P = KRAS WT FOLFIRI + Cet HR = HR = % OR = subgroup FOLFIRI a P = a P = % a P < KRAS MT FOLFIRI + Cet HR = HR = % OR = subgroup FOLFIRI P = P = % P = 0.35 OPUS [27] 2009 Response ITT FOLFOX4 + Cet HR = HR = % OR = rates FOLFOX P = P = % P = KRAS WT FOLFOX4 + Cet HR = HR = % OR = subgroup FOLFOX a P = P = % a P = KRAS MT FOLFOX4 + Cet HR = HR = % OR = subgroup FOLFOX a P = P = % a P = COIN [24] 2011 OS KRAS WT FOLFOX/XELOX + Cet NORDIC-Ⅶ [29] HR = 0.96 P = HR = 1.04 P = % OR = 1.35 a P = FOLFOX/XELOX % KRAS WT FOLFOX + Cet HR = FOLFOX P = KRAS WT XELOX + Cet HR = XELOX P = 0.56 KRAS MT FOLFOX/XELOX HR = Cet P = 0.80 FOLFOX/XELOX PFS ITT FLOX + Cet HR = 0.89 P = 0.31 KRAS WT subgroup KRAS MT subgroup Intermittent FLOX + Cet 19.7 HR = 1.06 P = Not reported 20.3 HR = 1.03 P = % OR = 1.35 P = % Not report Nordic FLOX Control 20.4 Control 41% Control FLOX + Cet HR = 1.07 P = 0.66 Intermittent FLOX + Cet 20.1 HR = 1.14 P = Not reported 21.4 HR = 1.08 P = % OR = 0.96 P = % OR = 0.96 P = 0.89 Nordic FLOX Control 22.0 Control 47% Control FLOX + Cet HR = 0.71 P = 0.07 Intermittent FLOX + Cet 21.1 HR = 1.03 P = Not reported 20.5 HR = 1.04 P = % OR = 1.44 P = % Not report Nordic FLOX Control 20.4 Control 40% Control PRIME [30] 2010 PFS KRAS WT FOLFOX4 + Pan HR = HR = % OR = 1.35 FOLFOX a P = P = % P = KRAS MT FOLFOX4 + Pan HR = HR = % - FOLFOX a P = P = % AVF2107g [32] 2004 OS ITT IFL + Bev HR = HR = % a P = IFL a P < a P < % NO16966 [33] 2008 PFS ITT FOLFOX4/ XELOX + Bev FOLFOX4/ XELOX CAIRO-2 [67] 2009 PFS ITT XELOX + Bev + Cet HR = 0.83 a P = HR = 0.89 P = % Subgroup FOLFOX4 + Bev FOLFOX HR = %CI: HR = %CI: Subgroup XELOX + Bev HR = HR = 0.84 XELOX %CI: %CI: KRAS WT subgroup KRAS MT subgroup HR = 1.22 a P = HR = 1.15 P = % OR = 1.00 P = % P = 0.49 XELOX + Bev % XELOX + Bev P = P = % P = 0.06 Cet XELOX + Bev % XELOX + Bev a P = a P = % a P = 0.03 Cet XELOX + Bev % 4267 April 21, 2014 Volume 20 Issue 15

169 Feng QY et al. Targeted therapy of colorectal liver metastases PACCE [68] 2009 PFS ITT Ox-CT + Bev + Pan KRAS WT subgroup KRAS MT subgroup HR = %CI: HR = %CI: HR = %CI: a P = HR = %CI: HR = %CI: NE HR = %CI: P = HR = %CI: Ox-CT + Bev % Ox-CT + Bev + Pan HR = %CI: Ox-CT + Bev % Ox-CT + Bev + Pan HR = %CI: % OR = %CI: % - 47% - Ox-CT + Bev % ITT Iri-CT + Bev + Pan HR = % OR = 1.11 Iri-CT + Bev %CI: % 95%CI: KRAS WT subgroup KRAS MT subgroup Iri-CT + Bev + Pan HR = % - Iri-CT + Bev %CI: % Iri-CT + Bev + Pan HR = % - Iri-CT + Bev %CI: % a P < PFS: Progress-free survival; OS: Overall survival; ITT: Intent to treat; WT: Wild type; MT: Mutant type; NE: Not estimatable; KRAS: KRAS exon 2, codons 12 and 13; FOLFOX: Fluorouracil, leucovorin, and oxaliplatin; XELOX: Capecitabine and oxaliplatin; FLOX: Fluorouracil, leucovorin, and oxaliplatin; Ox-CT: Oxaliplatin-based chemotherapy; FOLFIRI: Irinotecan, fluorouracil, and leucovorin; IFL: Irinotecan, fluorouracil, and leucovorin; Iri-CT: Irinotecan-based chemotherapy; Cet: Cetuximab; Pan: Panitumumab; Bev: Bevacizumab. However, the regimen of bevacizumab combined with oxaliplatin-based chemotherapy remains controversial. The large, double-blind, placebo- controlled clinical trial NO16966 [33] involved 1401 patients with unresectable CLM, and compared bevacizumab-plus-oxaliplatin-based chemotherapy (FOLFOX, XELOX) with chemotherapy alone. The addition of bevacizumab was associated with a more modest increase in the primary end point, PFS, of 1.4 mo (median PFS, 9.4 mo vs 8.0 mo, HR = 0.83, P = ). But no benefit was observed in the secondary end point OS (median OS, 21.3 mo vs 19.9 mo, HR = 0.89, P = 0.077). More attention should be paid on the results of the subgroup analysis, which indicated that bevacizumab was only associated with significant improvements in PFS when added to XELOX (HR = 0.77, P = ) but not FOLFOX (HR = 0.89, P = ). Another recent meta-analysis [34] came to a similar conclusion. This meta-analysis included six randomized clinical trials with a total of 3060 patients, and found that bevacizumab resulted in significant improvements in PFS (HR = 0.72, 95%CI: , P < ) and OS (HR = 0.84, 95%CI: , P < ). However, the advantage in OS was limited to irinotecan-based regimens (HR = 0.78, 95%CI: , P = ). Neither fluorouracil nor oxaliplatin-based treatments presented statistically significant data. Thus, bevacizumab combined with oxaliplatin-based chemotherapy is not currently the best choice as a first-line treatment. Conversion therapy Approximately 80%-90% of patients with CLM have unresectable metastatic liver disease at presentation [35]. However, some of these patients might become resectable after response to conversion chemotherapy. Such patients are referred to as Group 1 patients in the ESMO guidelines. For these patients, the treatment aim should be achieving maximum tumor shrinkage to create an opportunity for radical surgery [36]. Therefore, short-term indicators should be set as the end points of conversion therapy, such as resection rate, response rate, and rate of early tumor shrinkage (ETS). ETS is a novel short-term indicator suitable for conversion therapy. The shrinkage of tumor size is directly associated with the ability to operate, and has also been proven to be associated with long-term survival. Giessen et al [37] defined ETS as a 20% decrease in maximum tumor diameter between baseline and seven weeks of treatment, and found that patients with ETS had a more favorable outcome in terms of PFS (9.9 mo vs 6.1 mo, P = 0.029) and OS (27.5 mo vs 17.8 mo, P = 0.002). Modest et al [38] observed ETS 20% in 59% of patients with KRAS wild type tumors and indicated that patients with ETS 20% exhibited increases in their overall response rate (82% vs 19%, P < 0.001), PFS (8.9 mo vs 4.7 mo, P < 0.001) and OS (31.6 mo vs 15.8 mo, P = 0.005). These studies suggested that ETS was an effective predictor of the success of conversion therapy. In conversion therapy, the clinical application of anti- EGFR treatment is also common and effective. In the CRYSTAL trial [15,26], patients in ITT population had a higher response rate (46.9% vs 38.7%, P = 0.004) and a higher R0 resection rate (4.8% vs 1.7%) with the addition of cetuximab. For patients with hepatic-only metastases, cetuximab plus FOLFIRI was associated with a 77% response rate. The OPUS trial [27,28] also showed that for patients with wild type KRAS, cetuximab plus FOLFOX4 was associated with significantly higher best overall response (57% vs 34%, OR = 2.551, P = ). A recent study by Ye et al [39] compared cetuximab plus chemotherapy with chemotherapy alone as a first-line treatment for patients with initially unresectable CLM. In this trial, the primary end point was set as the rate of patient conver April 21, 2014 Volume 20 Issue 15

170 Feng QY et al. Targeted therapy of colorectal liver metastases sion to resection for liver metastases. The results showed that for ITT population, cetuximab plus chemotherapy significantly improved the rate of patient conversion to resection (28.6% vs 13.2%, P = 0.027), R0 resection rate (25.7% vs 7.4%, P = 0.004) and response rate (57.1% vs 29.4%, P = 0.001). Long-term outcomes were also improved, such as PFS (median PFS, 10.2 mo vs 5.8 mo, P = 0.004) and OS (median OS, 30.9 mo vs 21.0 mo, P = 0.013). For anti-vegf treatment, the AVF2107g trial [32] showed a significant improvement in response rate (44.8% vs 34.8%, P = 0.004) with bevacizumab plus IFL. But the NO16966 trial [33] came to a negative conclusion that bevacizumab plus oxaliplatin-based chemotherapy did not improve the response rate (47% vs 49%, OR = 0.90, 97.5%CI: 0.71 to 1.14, P = 0.31). The meta-analysis [34] mentioned previously also suggested no improvement in response rate (OR = 1.12, 95%CI: , P = 0.21) with the addition of bevacizumab as a first-line treatment. Based on these results, some researchers held that bevacizumab was not suitable for conversion therapy. To investigate this suspicion, a phase Ⅲ clinical trial FIRE-Ⅲ comparing FOLFIRI plus cetuximab with FOLFIRI plus bevacizumab as first-line treatment was reported at the 2013 ASCO Annual Meeting [40]. For ITT population, there was no significant difference between the cetuximab group and the bevacizumab group in primary endpoint response rate (62% vs 58%, P = 0.183). But for the evaluable population, cetuximab led to a significant advantage over bevacizumab (72% vs 63%, P = 0.017). At the secondary end point no difference was observed in PFS, but a 3.7 mo improvement in OS was observed in the cetuximab group (28.7 mo vs 25 mo, P < 0.017). In this clinical trial, cetuximab exhibited an advantage over bevacizumab in conversion therapy. However, the evaluable population decreased by 66 patients (11.1%) compared with the ITT population. The final results on exit bias should be emphasized. And in an update presented at the 2013 ESMO Annual Meeting [41], the new RAS mutations were tested. The results showed that all RAS wild-type patients had a longer median OS with FOLFIRI plus cetuximab, leading to a more significant improvement of 7.5 mo (33.1 mo vs 25.6 mo, HR = 0.70, P = 0.011), compared with 3.7 mo in patients with only KRAS exon 2 wild type. This study again confirmed the predictive role of new RAS. All previous clinical trials came to a conclusion that bevacizumab was not preferred for conversion therapy. However, bevacizumab is still considered acceptable because it cannot be known in advance whether resectability will be achieved, and bevacizumab provided a benefit in terms of long-term survival. Targeted therapy after progression Numerous clinical trials have proven the benefits of second-line targeted therapy for patients who had first-line progression and did not receive targeted agents as firstline treatment [42-46]. However, for patients who received first-line targeted therapy, it is unclear whether and how to use targeted agents continuously. Some studies have suggested that the continuation of bevacizumab following progression on first-line bevacizumab could provide benefits. In the TML trial [47], patients with CLM who progressed on regimens containing bevacizumab received second-line therapy consisting of a different chemotherapy with or without bevacizumab. The results showed that continuing on bevacizumab led to a modest improvement in OS (median OS, 11.2 mo vs 9.8 mo, HR = 0.81, P = ). PFS was also improved by 1.6 mo (median PFS, 5.7 mo vs 4.1 mo, HR = 0.68, P < ). Another study [48] retrospectively analyzed 573 patients from the US Oncology iknowmed electronic medical record system and showed that continuous bevacizumab after progression was associated with longer OS (HR = 0.76, 95%CI: ) and longer post-progression OS (HR = 0.74, 95%CI: ) in multivariate analysis. Some single-arm studies [49,50] also showed a median post-progression PFS of 5 to 5.6 mo and a median OS of 13.9 to 15.4 mo with continuous bevacizumab. For anti-egfr antibodies, NCCN guidelines (V. 2013) concluded that if cetuximab or panitumumab were used as the initial therapy, then neither cetuximab nor panitumumab should be used in second or subsequent lines of therapy. This conclusion might be hasty because of the lack of evidence from related clinical trials. The ongoing clinical trial CAPRI was the first study to investigate a treatment strategy of continuing cetuximab after first-line progress. KRAS wild type patients with CLM were given first-line cetuximab plus FOLFIRI and were randomized at progression (1:1) to receive FOLFOX alone or in combination with cetuximab. The results of this trial were expected to resolve this controversy. The FIRE-Ⅲ trial also provided evidence for selecting second-line treatments. After first-line progression, 48.2% patients in cetuximab group received bevacizumab instead, and 14.4% patients continued on cetuximab. In bevacizumab group, 42.9% patients changed into cetuximab, and 17.6% patients continued on bevacizumab. The allocation of second-line treatment was balanced (P = 0.347). As previously mentioned, cetuximab plus FOLFIRI had advantages in terms of OS (28.7 mo vs 25 mo, P < 0.017), but not PFS. This anomaly might be explained that cetuximab and bevacizumab were similar as first-line treatment in PFS and OS. The benefits in terms of OS in cetuximab group mainly came from the secondline bevacizumab treatment, which didn t prolong PFS. This explanation suggested that first-line cetuximab followed by second-line bevacizumab was better than firstline bevacizumab followed by second-line cetuximab. Considering that the FIRE-Ⅲ trial was not over, the final results were expected. For patients failed with targeted therapy as first-line treatment, the major difficulty lies in identifying the resistance arises from whether cytotoxic chemotherapy or targeted monoclonal antibodies. Although it is reasonable to replace only part of the regimen, delays in treatment and unnecessary economic waste must be taken into account. The selection of second-line or subsequent treatment is a severe test of the experience and judgment of physicians April 21, 2014 Volume 20 Issue 15

171 Feng QY et al. Targeted therapy of colorectal liver metastases PREOPERATIVE CHEMOTHERAPY COMBINED WITH TARGETED AGENTS FOR RESECTABLE METASTATIC COLORECTAL CANCER Preoperative chemotherapy is believed to be advantageous because of the earlier treatment of micro metastatic disease, the determination of responsiveness to chemotherapy, and the avoidance of local therapy for those patients with early disease progression. However, only a few large randomized clinical trial provided evidence on neoadjuvant chemotherapy for patients with upfront resectable CLM, aside from additional reports from some small-sample and/or single-arm studies [51-53]. The large randomized clinical trial EORTC [54] involved 364 potentially resectable patients with up to four liver metastases and compared FOLFOX4 as perioperative chemotherapy with surgery alone. The results showed a significantly higher 3-year PFS rate (35.4% vs 28.1%, HR = 0.79, P = 0.058) in patients receiving perioperative chemotherapy. However, a recent update of this trial [55] reported that the difference of OS after a median follow up of 8.5 years was not significant, with a 5-year OS of approximately 50% in both groups. In addition to the lack of benefit for long-term survival, this trial was not designed to compare preoperative chemotherapy alone, which made it difficult to prove the effect of preoperative chemotherapy. Adam et al [56] also reported the negative result that preoperative chemotherapy for metachronous CLM provided no benefit in long-term OS and PFS. In terms of preoperative chemotherapy combined with targeted agents, the NCCN and ESMO guidelines gave no strong evidence. Only a few small-sample, singlearm studies were available. Gruenberger et al [57] reported a non-randomized clinical trial evaluating bevacizumab plus XELOX as a preoperative therapy for patients with potential resectable CLM. After preoperative therapy with bevacizumab, they observed a complete response rate of 8.9%, a partial response rate of 64.3%, and a progressive disease rate of only 5.4%. No interference in liver regeneration was found after resection. There are also a number of studies concentrating on locally advanced rectal cancer [58-62], but these results were not suitable for patients with common colon cancer, because of the differences between colon and rectal cancer in route of metastasis, surgical strategy, application of radiotherapy and so on. The risks of preoperative targeted therapy should be taken into account. These risks mainly lie in missing the window of opportunity for resection; the achievement of a complete response, making it difficult to identify areas for resection; and potential liver toxicity, making resection impossible. It is also difficult to determine the best cycles of preoperative therapy. The results of EORTC and a number of other trials suggested that preoperative chemotherapy plus targeted agents might be useful. More detailed evidences are needed to enable physicians to weigh the advantages and disadvantages of this approach. CHEMOTHERAPY COMBINED WITH TARGETED AGENTS AS POSTOPERATIVE ADJUVANT TREATMENT For patients with resected stage Ⅱ/Ⅲ colorectal cancer, postoperative adjuvant treatment with targeted agents has been proven to be useless or even harmful. In the N0147 trial [63], cetuximab plus FOLFOX significantly increased grade 3/4 adverse events (72.5% vs 52.3%, odds ratio, OR = 2.4, P < 0.001), providing no benefit in primary end point 3-year PFS rate (65.0% vs 67.1%, HR = 1.12, P = 0.38). In the interim analysis of the ongoing PETACC-8 trial reported at the 2012 ESMO Annual Meeting, cetuximab plus FOLFOX provided no benefit for KRAS wild-type patients in disease-free survival (DFS, HR = 1.047, P = ) or OS (HR = 1.092, P = ). Worse DFS was observed with cetuximab in patients aged > 70 years (n = 149, HR = 1.97, P = 0.051), in females (n = 666, HR = 1.45, P = 0.031) and in patients with right-sided colon cancer (n = 570, HR = 1.40, P = 0.043). The NSABP C-08 trial [64] reported negative results with the addition of bevacizumab to FOLFOX in 3-year DFS (77.4% vs 75.5%, HR = 0.89, P = 0.15). The results of the AVANT trial [65] also failed to show benefits from bevacizumab plus oxaliplatin-based chemotherapy in DFS, with an even worse outcome for the FOLFOXcombined regimen in terms of OS (HR = 1.27, 95%CI: , P = 0.02). The four large randomized clinical trials above came to a conclusion that targeted agents had no role in the adjuvant treatment of resected stage Ⅱ/Ⅲ colorectal cancer. For resected CLM, the role of postoperative adjuvant chemotherapy remains unclear [36]. However, postoperative chemotherapy with FOLFOX plus bevacizumab is often administered despite the lack of data supporting this regimen. In the interim analysis of the Dutch HEPATICA trial [66] reported at the 2011 ASCO Annual Meeting, bevacizumab plus capecitabine and oxaliplatin (CAPOX) as postoperative adjuvant chemotherapy provided no significant benefit compared with CAPOX alone in 2-year DFS rate (70% vs 52%, P = 0.074), and exhibited no significant differences in toxicity. For patients who received targeted therapy and responded before surgery, it is reasonable to continue targeted therapy as a postoperative adjuvant treatment. However, for upfront resected CLM without preoperative targeted therapy, postoperative targeted therapy is not recommended, based on the negative results of trials N0147, PETACC-8, NSABP C-08 and AVANT. COMBINATION THERAPY WITH MORE THAN ONE TARGETED AGENT As anti-egfr antibodies and anti-vegf antibodies exert their anti-tumor effects in different mechanisms, their combination should theoretically lead to greater effects. Preclinical trials also demonstrated that the com April 21, 2014 Volume 20 Issue 15

172 Feng QY et al. Targeted therapy of colorectal liver metastases bination of more than one targeted agent led to stronger inhibition of the downstream signaling pathways compared with single treatment. However, most clinical trials showed no advantages and in some cases even showed disadvantages of the combination of anti-egfr and anti-vegf antibodies as first-line treatment. The CAI- RO-2 trial [67] compared cetuximab plus bevacizumab with bevacizumab alone based on the XELOX regimen. For the ITT population, the combination of cetuximab with bevacizumab led to shorter PFS (median PFS, 9.4 mo vs 10.7 mo, P = 0.01). There was no differences in OS (median OS, 19.4 mo vs 20.3 mo, P = 0.16) or response rates (52.7% vs 50.0%, P = 0.49). Subgroup analysis also showed no difference for KRAS wild type patients in response rate (61.4% vs 50%, P = 0.06), PFS (median PFS, 10.5 mo vs 10.6 mo, P = 0.30) or OS (median OS, 21.8 mo vs 22.4 mo, P = 0.64). Similar results were observed in the PACCE trial [68] with the addition of panitumumab to regimens containing bevacizumab and oxaliplatin/irinotecan. In patients receiving panitumumab plus bevacizumab and oxaliplatin-based chemotherapy, the addition of panitumumab significantly decreased the PFS (median PFS, 10.0 mo vs 11.4 mo, HR = 1.27, 95%CI: ) and OS (median OS, 19.4 mo vs 24.5 mo, HR = 1.43, 95%CI: ). In patients receiving irinotecan-based chemotherapy, no differences were observed between the two groups in their PFS or OS. The combination of more than one targeted agent as a maintenance treatment is also controversial. The GERCOR-DREAM trial [69], reported at the 2012 ASCO Annual Meeting, provided positive results that the addition of erlotinib to bevacizumab as a maintenance treatment significantly improved the duration of maintenance PFS (median MT-PFS, 5.8 mo vs 4.6 mo, HR = 0.73, P = 0.005) after first-line treatment in CLM. Although the OS data were not mature, they were promising and warranted continuation of the trial. However, the similar Nordic ACT trial [70] reported opposite results that the addition of erlotinib to bevacizumab as a maintenance treatment did not significantly improve PFS (median PFS, 5.73 mo vs 4.23 mo, HR = 0.79, P = 0.19) or OS (median OS, 21.5 mo vs 22.8 mo, HR = 0.88, P = 0.51). Despite their conflicting results, these two trials provided novel research ideas. The clinical trials described above raise the questions that whether cytotoxic agents interfere with targeted agents, in which phase combinations of targeted agents should be used, and what are the differences between experiments in vivo and in vitro. Although it is not presently recommended to combine more than one targeted agent, more mechanistic research is meaningful and valuable. This could also lead to a breakthrough in understanding the development of resistance to targeted agents. CONCLUSION Surgical resection undoubtedly remains the gold standard for the treatment of resectable CLM. However, a wellcoordinated multidisciplinary approach is more important to achieving optimal outcomes for patients with CLM. Targeted therapy with monoclonal antibodies is becoming increasingly important for patients with CLM, with new findings and doubts continuing to emerge regarding these relatively new agents. For patients with the KRAS G13D mutation, anti-egfr therapy might have positive effects. The new RAS mutations were considered to be predictors of poor responsiveness to anti-egfr therapy. The efficacy of cetuximab plus FOLFOX is likely to be reliable, but bevacizumab plus oxaliplatin-based chemotherapy remains controversial. Studies suggest that bevacizumab is not suitable for conversion therapy. More evidence is needed to confirm the utility of targeted therapy for neoadjuvant and adjuvant treatment. Combination therapy with more than one targeted agent is not currently recommended. In the era of personalized cancer medicine, current research goals should be focused on further defining the roles of targeted agents at different stages of disease and treatment, as well as optimizing their sequencing with other treatments. More clinical trials are necessary to answer the remaining questions. Treatment regimens should be more accurate and personalized based on novel molecular markers. The growing number of molecular predictors has led to the conjecture that tumor classification will soon be based on molecular markers rather than localization or histology. In the future, every patient will be genotyped for several markers and treated with appropriate targeted agents. This may be the best way to approach the tumor heterogeneity among patients. 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176 Feng QY et al. Targeted therapy of colorectal liver metastases DOI: /NEJMoa ] 68 Hecht JR, Mitchell E, Chidiac T, Scroggin C, Hagenstad C, Spigel D, Marshall J, Cohn A, McCollum D, Stella P, Deeter R, Shahin S, Amado RG. A randomized phase IIIB trial of chemotherapy, bevacizumab, and panitumumab compared with chemotherapy and bevacizumab alone for metastatic colorectal cancer. J Clin Oncol 2009; 27: [PMID: DOI: /jco ] 69 Tournigand C, Samson B, Scheithauer W, al. e. Bevacizumab (Bev) with or without erlotinib as maintenance therapy, following induction first-line chemotherapy plus Bev, in patients (pts) with metastatic colorectal cancer (mcrc): efficacy and safety results of the International GERCOR DREAM Phase III trial. J Clin Oncol 2012; 30 Suppl 18: Abstract LBA Johnsson A, Hagman H, Frödin JE, Berglund A, Keldsen N, Fernebro E, Sundberg J, De Pont Christensen R, Garm Spindler KL, Bergström D, Jakobsen A. A randomized phase III trial on maintenance treatment with bevacizumab alone or in combination with erlotinib after chemotherapy and bevacizumab in metastatic colorectal cancer: the Nordic ACT Trial. Ann Oncol 2013; 24: [PMID: DOI: /annonc/mdt236] 71 Gerlinger M, Rowan AJ, Horswell S, Larkin J, Endesfelder D, Gronroos E, Martinez P, Matthews N, Stewart A, Tarpey P, Varela I, Phillimore B, Begum S, McDonald NQ, Butler A, Jones D, Raine K, Latimer C, Santos CR, Nohadani M, Eklund AC, Spencer-Dene B, Clark G, Pickering L, Stamp G, Gore M, Szallasi Z, Downward J, Futreal PA, Swanton C. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 2012; 366: [PMID: DOI: /NEJMoa ] P- Reviewers: Deutsch JC, Mocellin S, Ramia JM S- Editor: Gou SX L- Editor: A E- Editor: Wu HL 4275 April 21, 2014 Volume 20 Issue 15

177 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (5): Colorectal cancer TOPIC HIGHLIGHT Advances in epigenetic biomarker research in colorectal cancer Xi Wang, Ye-Ye Kuang, Xiao-Tong Hu Xi Wang, Ye-Ye Kuang, Xiao-Tong Hu, Biomedical Research Center, Sir Run Run Shaw Hospital, Zhejiang University and Key Laboratory of Biotherapy of Zhejiang Province, Hangzhou , Zhejiang Province, China Author contributions: Wang X and Kuang YY drafted the manuscript; Hu XT approved the final version for publication. Supported by National Natural Science Foundation of China, No and No ; the Program for Zhejiang Leading Team of ST innovation, No. 2012R ; Major State Basic Research Development Program, No. 2010CB834303; National High Technology Research and Development Program of China, No. 2012AA02A601; Major Projects in Zhejiang Province, No. 2012C ; and the Fundamental Research Funds for the Central Universities, No. 2012FZA7020 Correspondence to: Xiao-Tong Hu, MD, PhD, Biomedical Research Center, Sir Run Run Shaw Hospital, Zhejiang University and Key Laboratory of Biotherapy of Zhejiang Province, Qing Chun East Road 3, Hangzhou , Zhejiang Province, China. hxt_hangzhou@sina.com Telephone: Fax: Received: September 23, 2013 Revised: November 5, 2013 Accepted: January 14, 2014 Published online: April 21, 2014 Abstract Colorectal cancer (CRC) causes approximately deaths annually and is the third leading cause of cancer mortality worldwide. Despite significant advancements in treatment options, CRC patient survival is still poor owing to a lack of effective tools for early diagnosis and a limited capacity for optimal therapeutic decision making. Since there exists a need to find new biomarkers to improve diagnosis of CRC, the research on epigenetic biomarkers for molecular diagnostics encourages the translation of this field from the bench to clinical practice. Epigenetic alterations are thought to hold great promise as tumor biomarkers. In this review, we will primarily focus on recent advances in the study of epigenetic biomarkers for colorectal cancer and discuss epigenetic biomarkers, including DNA methylation, microrna expression and histone modification, in cancer tissue, stool, plasma, serum, cell lines and xenografts. These studies have improved the chances that epigenetic biomarkers will find a place in the clinical practices of screening, early diagnosis, prognosis, therapy choice and recurrence surveillance for CRC patients. However, these studies have typically been small in size, and evaluation at a larger scale of well-controlled randomized clinical trials is the next step that is necessary to increase the quality of epigenetic biomarkers and ensure their widespread clinical use Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Colorectal cancer; Epigenetic biomarker; DNA methylation; MicroRNA biomarker; Histone modification Core tip: Epigenetic biomarkers, including DNA methylation, microrna expression and histone modification, may have the potential for the screening, diagnosis, prognosis and recurrence surveillance in colorectal cancer patients. Wang X, Kuang YY, Hu XT. Advances in epigenetic biomarker research in colorectal cancer. World J Gastroenterol 2014; 20(15): Available from: URL: com/ /full/v20/i15/4276.htm DOI: org/ /wjg.v20.i INTRODUCTION Colorectal cancer (CRC) is one of the most widespread cancers in the world, accounting for over 1 million new diagnoses each year and over half of a million deaths [1,2]. Among all CRC cases, approximately 95% are adenocarcinoma. Less common types include lymphoma and 4276 April 21, 2014 Volume 20 Issue 15

178 Wang X et al. Epigenetic biomarker research in colorectal cancer squamous cell carcinoma. CRC patients are characterized by a lack of clinical manifestations until the late stages of cancer, leading to poor prognosis and a high mortality rate. Adenomas are the primary precursor lesion of colon cancer and often develop into colorectal carcinomas, but the process is slow, localized and asymptomatic, which is the primary factor contributing to late diagnosis. At the time of primary diagnosis, 80% of the patients are offered resection and potentially are cured by that. However, 40%-45% of these patients experience a later recurrence and was therefore not cured by resection [3]. Therefore, the identification of useful screening tools for CRC is a high priority. Currently, the detection of trace blood in stool using the fecal occult blood test (FOBT) and subsequent internal imaging of the colon by flexible sigmoidoscopy or colonoscopy represent the gold standard for CRC detection. Although widespread, these techniques suffer from several shortcomings. For example, the FOBT lacks specificity, often needs to be repeated, and is easily interfered by the contents of the bowel. Colonoscopy, on the other hand, is invasive, expensive, and has a high risk of complications, which often leads to poor patient compliance. As a result, the identification of biomarkers that are simple, noninvasive, cost-efficient and reasonably sensitive/specific is urgently needed. Over the past decade, the rapidly expanding field of epigenetics has shown great promise for the detection of CRC at earlier stages and the identification of resectable CRC lesions prior to metastasis, thereby providing patients with the highest chance of survival. Epigenetic alterations are widely known to play an important role in tumorigenesis and are prevalent in CRC. Epigenetic changes in colorectal tumor tissues and CRC cell lines have been widely reported, and a substantial amount of information has been accumulated [4,5]. These alterations include aberrant DNA methylation of promoter CpG islands, changes in microrna (mirna) expression profiles and various histone modifications. The exploration of epigenetic biomarkers in cancer for clinical use is a relatively new but rapidly developing field. Applications include screening, diagnosis, classification, surveillance and targeted therapies. If epigenetic factors are to be effective biomarkers in clinical practice, they must be detectable by noninvasive means and outperform the current gold standard. It should be emphasized that sample collection methods are a crucial factor. For example, mirnas extracted from tissues should be evaluated separately from mirnas isolated from serum and stool because their clinical potential is quite different. Serum and stool biomarkers are ideal for patient screening, but biomarkers from postoperative tissue may be more effective for prognosis, including the prediction of mean survival, resectability of the primary tumor and the administration of targeted therapies. DNA METHYLATION In vitro and preclinical studies Generally speaking, in vitro studies are the first step in the discovery of new epigenetic biomarkers. Researchers often compare profiles of CRC cell lines with normal colorectal cells and then compile a list of candidate biomarkers for further study. Similarly, identifying valuable prediction biomarkers in CRC patients often begins with preclinical studies using xenograft tumors, which allow one to observe tumor growth and how it responds to different therapies. Nevertheless, a significant shortcoming of this approach is that the tumor and vasculature are of mouse origin rather than human. Studying tumors in a different growth environment makes it difficult to explain the results accurately and translate them into clinical application. However, in vitro and preclinical studies are still the foundations on which most clinical studies are built. In the human genome, DNA methylation typically occurs on the cytosine of the sequence 5 -CpG-3, which is found in promoter regions of approximately 70% of genes [6]. In this biochemical process, a methyl group (-CH3) is added to cytosine nucleotides by a DNA methyltransferase (DNMT). A large body of evidence has demonstrated that promoter hypermethylation is associated with gene silencing, while hypomethylation results in gene-product upregulation. In this section, in vitro studies will be discussed first, followed by clinical studies that utilize blood or stool to identify DNA methylation in CRC patients. Khamas et al [7] conducted a genome-wide screen of 15 CRC cell lines and 23 paired tumor and normal samples from CRC patients to identify a set of methylationsilenced genes in CRC. Gene expression studies were then used to confirm whether the methylated genes were really regulated by their methylation status. The results of this study revealed that 139 genes showed greater than 1.5-fold up-regulation in at least one 5-aza-2 -deoxycytidine-treated cell line and no less than a 1.2-fold change in other treated CRC cell lines. Among them, eight genes, DCAF4L1, DDX43, ICAM1, MSX1, PGF, PTPRO, ZFP42 and the cancer-germline antigen families, had previously been reported to be up-regulated by demethylation in CRC and were thus excluded from the analysis. Twenty genes with poor annotation, 20 genes located on the X chromosome, 16 genes with duplicated probes, two genes with no CpG islands, 8 genes with unknown function, 23 without a relevant function in tumorigenesis and 22 genes with potential oncogenic activity were also excluded, leaving 20 candidates (CAMK2B, CHAC1, THSD1, CSTA, COL1A1, GADD45B, DMRTB1, COL6A1, GAS5, GPRC5A, GPSM1, KLHL35, LTBP2, NAA11, RBP4, SEMA7A, SYCP3, TBRG1, TNFSF9 and TXNIP) that had not been previously reported to be affected by epigenetic mechanisms in CRC. Therefore, from the genes analyzed, a much smaller set of genes was isolated as potential biomarkers for CRC. In this study, two genes, THSD1 and GADD45B, were selected for further analysis. THSD1 methylation appeared to have the potential for diagnostic, prognostic or therapeutic use. Thrombospondin type-1 domaincontaining protein 1 (THSD1) is located in a region that is strongly associated with the progression of colorectal 4277 April 21, 2014 Volume 20 Issue 15

179 Wang X et al. Epigenetic biomarker research in colorectal cancer Table 1 Biomarkers of DNA methylation in blood of colorectal cancer patients, n (%) Markers Sensitivity Specificity Ref. APC 3 (6) 0 (100) [20] hmlh1 21 (43) 1 (98) [20] HLTF 17 (34) 1 (98) [20] HLTF 22 (21) 0 (100) [21] ALX4 25 (83) 9 (70) [22] TMEFF2 87 (65) 56 (69) [23] NGFR 68 (51) 29 (84) [23] 9-Sep 92 (69) 25 (86) [23] 9-Sep 90 (72) 19 (90) [24] 9-Sep 24 (72) 3 (90) [25] 9-Sep 18 (60) 5 (89) [26] 9-Sep 45 (90) 11 (89) [27] NEUROG1 14 (52)-stage Ⅰ 4 (91) [28] 45 (64)-stage Ⅱ SFRP2 113 (67) 4 (94) [29] CDKN2A/P16 12 (71) 0 (100) [30] RUNX3 11 (65) 0 (100) [30] TPEF/HPP1 13 (13) 0 (100) [21] APC: Adenomatosis polyposis coli; hmlh1: Homo mutl homolog 1; HLTF: Helicase-like transcription factor; ALX4: ALX homeobox 4; TMEFF2: Transmembrane protein with EGF-like and two follistatin-like domains 2; NGFR: Nerve growth factor receptor; NEUROG1: Neurogenin 1; SFRP2: Secreted frizzled-related protein 2; CDKN2A: Cyclin-dependent kinase inhibitor 2A; RUNX3: Runt-related transcription factor 3; HPP1: Hyperpigmentation, progressive, 1; TPEF: Transmembrane protein endothalial factor. adenoma to carcinoma and encodes a transmembrane molecule containing a thrombospondin type 1 repeat that might be involved in cell adhesion and angiogenesis. High THSD1 expression positively correlated with better distant metastasis survival in breast cancer. Therefore, its loss may be associated with metastatic tumor spread. Additionally, as one of the consensus radiation-response genes in primary human fibroblasts, THSD1 may play a role in radiation response in cancer stem cell. Moreover, a recent study has shown that THSD1 was expressed in CRC classified as D in Duke s classification scheme for CRC and thus may be relevant to tumor progression [8]. GADD45B functions as a tumor suppressor in many cancers, can inhibit cell proliferation at different stages and induce cell apoptosis, but its function in CRC is unknown. In addition, Schuebel et al [9] described another genomewide, expression array-based approach for the identification of genes silenced by promoter hypermethylation in human CRC, and approximately 500 hypermethylated genes were identified. They analyzed the top-tier hypermethylome of each cell line (HCT116 and SW480) and then made a comparison of hypermethylation frequencies in cell lines, normal human tissues and human tumor samples. They found that BOLL, DKK3, CABYR, EFEMP1, GNB4, GSTM3, FOXL2, HOXD1, JPH3, NEF3, NEURL, PPP1R14A, RAB32, TLR2, SALL4, TP53AP1 and ZFP42 were hypermethylated and underexpressed in both CRC cell lines and in colon cancers, but not in normal tissues. These genes possess great promise as useful biomarkers for molecular diagnostics, early detection and CRC therapy. Recently, Yi et al [10] also reported that hypermethylation of promoter DNA in the FBN2 and TCERG1L genes might provide excellent biomarkers for early detection of CRC. Both genes showed a high frequency of methylation in colon cancer cell lines, adenomas and carcinomas. In addition, methylation of the hmlh1, p16ink4a, APC, MGMT, sfrp1, GATA-5, sfrp4, sfrp5, GATA-4, B4GALT1 TFPI2, SOX17 and TMEM25 genes has been described in several studies [11-17]. These genes are hypermethylated and downregulated in CRC and thus may serve as excellent candidate biomarkers. In addition, insulin-like growth factor-binding protein 3 and Enah/Vasp-like have been validated as prognostic biomarkers for CRC and found to be useful in stratifying high-risk CRC patients who would benefit from adjuvant chemotherapy [18]. PPP2R2B was also found to be hypermethylated in CRC and was connected to therapeutic resistance [19]. These genes could serve as candidate biomarkers for prognosis. However, clinical studies are required to confirm these results, and it remains to be seen if these alterations can be detected in blood or stool. Biomarkers of DNA methylation in blood Biomarkers detected in patient blood samples would provide the most practical screening tool for CRC because of the ease with which these samples can be acquired. It has been well documented that genetic material can shed from tumor cells, and aberrant DNA methylation can be specifically quantified in blood despite the large amounts of normal DNA in circulation. Bisulfite treatment and methylation specific polymerase chain reaction (PCR) are the two most commonly used techniques. A blood biomarker with a high sensitivity and specificity for CRC can not only be used to segregate high-risk patients for further clinical tests but also be an excellent tool for monitoring CRC recurrence in patients who have undergone tumor resection (Table 1) [20-30]. The SEPT9 gene, encoding a guanosine triphosphate enzyme involved in cytokinesis and cell cycle control, has been reported to be associated with several cancers. The v2 region of the Septin 9 (SEPT9) promoter has been shown to be methylated in CRC tissue compared with normal colonic mucosa. Using highly sensitive realtime PCR assays, methylated SEPT9 was first detected in the plasma of CRC patients with an overall sensitivity of 72% and a specificity of 90% [24]. Significant validation has been performed for this methylation biomarker, and Warren et al [27] have confirmed a sensitivity of up to 90% and a specificity of up to 88% for SEPT9. Based on these results, SEPT9 methylation appears to have the highest probability of correctly distinguishing between the blood of cancerous and non-cancerous persons for CRC detection. Currently, two CRC detection kits using plasma SEPT9 methylation analysis are marketed for clinical application. Combining SEPT9 with other methylation biomarkers would improve the detection rate [31]. Further studies are needed to compare these panels and kits and discover their advantages and limitations. Ultimately, the 4278 April 21, 2014 Volume 20 Issue 15

180 Wang X et al. Epigenetic biomarker research in colorectal cancer most effective ones should be chosen for clinical use. Other genes, such as APC, hmlh1, ALX4, TMEFF2, NGFR, NEUROG1, SFRP2, CDKN2A/P16, TPEF/ HPP1 and RUNX3, have also emerged as serum methylation markers for CRC, with sensitivities ranging from 6% to 83% and specificities ranging from 69% to 100% (Table 1). Among them, ALX4, TMEFF2 and NEUROG1 showed better performance relative to the others, and the use of these markers in combination can improve detection accuracy [25,31]. In addition to the successful identification of DNA methylation-based blood biomarkers, it is important to find genes that have prognostic value in the blood of patients with CRC. Methylation of helicase-like transcription factor (HLTF) has shown a strong correlation with tumor size, metastatic disease and tumor stage and is also associated with an increased risk of disease recurrence in CRC patients. Therefore, the methylation of this gene can serve as an independent biomarker for the identification of CRC with an increased risk of death. These results indicate that detection of HLTF methylation in the blood of CRC patients has the potential as a pretherapeutic predictor of patient outcome [32]. Deafness, autosomal dominant 5 (DFNA5) is another candidate biomarker for the noninvasive screening and monitoring of CRC. DFNA5 methylation has been observed in DNA from the peripheral blood (PB) of CRC patients at a high frequency (48% or 12/25) relative to healthy controls (only 12% or 3/25). Moreover, the methylation of DFNA5 in PB samples from CRC patients was significantly correlated with lymph node metastasis and distant metastasis (P = 0.027) [5], which suggests that DFNA5 could potentially be an independent prognostic serum biomarker for CRC patients. It is clear, however, that further validation in large-scale prospective trials is necessary before these biomarkers are ready for use in the clinic [26]. DNA methylation biomarkers in stool As a more attractive alternative to tissue sampling, biomarkers from feces could be of great clinical value because sampling is noninvasive and has much higher specificity. These properties offer a distinct advantage over endoscope- and FOBT-based screening strategies for the detection of both CRC and critical precursor lesions. Over the past decade, numerous studies have engaged in the development of methylation-based detection assays for stool biomarkers of CRC (Table 2) [33-66], though the fecal biomarker detection can only be performed in only less than 50% of patients due to very limited compliance. The best-studied and top-performing methylation biomarkers are secreted frizzled-related protein 2 (SFRP2) and vimentin. SFRP2 was the first reported DNA methylation marker in stool, has shown a sensitivity of 77%-90% and specificity of 77% [66] and has since been studied extensively. SFRP2 methylation has been shown to be the most sensitive biomarker for CRC, with detection rates ranging from 77% to 94% (Table 2). When SFRP2 methylation was used in a multigene, fecal methylation panel, detec- tion of CRC and a small number of advanced adenomas reached a sensitivity and specificity of 96% [58]. A followup study found that SFRP2 methylation was detectable in the stool of almost half of all patients with hyperplastic polyps or colorectal adenomas [53], further supporting its use in the detection of premalignant lesions. Fecal SFRP2 methylation also drops dramatically after surgery [postoperative: 8.7% (6/69) vs preoperative: 87% (60/69)] [52], suggesting its possible utility as a biomarker for recurrence. The vimentin gene, which encodes an intermediate filament protein involved in cell attachment, migration, and signaling, was identified in the stool of 83% of CRC patients with a specificity of 90% [62]. Since then, many studies have been devoted to vimentin methylation. Follow-up studies have obtained similar results and thus have reinforced the utility of vimentin as a standalone biomarker [47,54,55,59,61]. This has led to the commercialization of a single-gene stool kit for CRC detection based on vimentin methylation. More recently, vimentin methylation has been used in combination with other methylation markers to further increase detection rates, and vimentin has also been found in urine, suggesting an alternative method of detection [34,48,67]. Vimentin has a low detection rate in serum, however, and is thus most likely not suitable for use as a serum biomarker for CRC. Recently, Ahlquist et al [26] reported that a panel of methylation markers from stool that includes vimentin has shown a significantly higher sensitivity for CRC, primarily because of higher detection rates in stage Ⅰ-Ⅲ CRC (91% vs 50%). In addition to SFRP2 and vimentin, several other methylation biomarkers have been identified; these include GATA4, HIC1, ITG4, NDRG4, OSMR, TFPI2, ESR1, SLIT2, PHACTR3, SPG20, 3OST2 and MGMT. These genes have sensitivities for CRC ranging from 38% to 89% and specificities ranging from 79% to 100%. The combination of different methylation biomarkers (combinations of 2 to 7 genes including APC, ATM, CDKN2A, GSTP1, HLTF, hmlh1, HPP1, MGMT, RASSF1, SFRP2, MAL, P16, or vimentin) increased sensitivity from 55% to 100% and increased specificity from 87% to 100% (Table 2). However, more clinical studies are required to confirm these results. MIRNA BIOMARKERS mirna and cancer In recent years, mirna has been a relatively new but rapidly expanding field, as is evidenced by the increasing number of assays in development. mirnas are small non-coding RNA molecules that function in transcriptional and post-transcriptional regulation of gene expression and control various cellular functions. Currently, more than 1000 mirnas have been discovered in the human genome, and their activities and regulatory mechanisms are being intensively investigated. mirnas typically function via base pairing with complementary sequences in mrna molecules, resulting in gene silencing via translational repression or target degradation. It 4279 April 21, 2014 Volume 20 Issue 15

181 Wang X et al. Epigenetic biomarker research in colorectal cancer Table 2 Biomarkers of DNA methylation in the stool of colorectal cancer patients Markers Sample Sensitivity Specificity Ref. AGTR1/WNT2/ SLIT2/VIM/SEPT9 Vimentin/EYA4/ BMP3/NDRG4 214 CRC 20%-78% 86%-100% [33] 25 IBD 39 controls CRC 67%-100% 89% [34] ESR1 19 CRC 65% 81% [35] 38 controls SLIT2 60 CRC 100% [36] 32 IBD associated CRC 20 HR-IBD 25% 65 LR-IBD 28 controls PHACTR3 64 CRC 66% 100% [37] 71 A 32% 34 controls TFPI2 60 CRC 68.3% 100% [38] 20 A 30 controls CNRIP1/FBN/INA/ MAL/SNCA/SPG20 78 CRC 65%-94% 95%-100% [39] 61 A 35%-91% 48 controls SPG20 9 CRC 67% Unknown [40] MAL/CDKN2A/ MGMT 69 CRC 55.1%-78.3% 96.2%-100% [41] 24 A 37.5%-58.3% 19 HP 10.5%-26.3% 24 controls 3OST2 21 CRC 72.7% 90% [42] ITGA4/SFRP2/p16 30 CRC 70% 96.8% [43] 25 A 72% 21 controls RARB2/p16/ INK4a/MGMT/APC 26 CRC 62% 100% [44] 20 A 40% 16 IBD 13% 20 controls RASSF1/SFRP2 84 CRC 75% 89% [45] 27 advanced 44% A 29 nonadvanced 28% A 12 HP 25% 4 IC 25% 2 UC 100% 113 controls OSMR 69 CRC 38% 95% [46] 81 controls Vimentin 22 CRC 41% 95% [47] 20 advanced A 45% 38 controls MGMT/hMLH1/ Vimentin 60 CRC 75% 87% [48] 22 advanced A 46% 30 nonadvanced A 70% 37 controls ITGA4 13 A 69% 79% [49] NDRG4 75 CRC 53%-61% 93%-100% [50] 75 controls GATA4 75 CRC 51%-71% 84%-93% [51] 75 controls TFPI2 26 CRC 76%-89% 79%-93% [17] 45 controls SFRP2 69 CRC 87.0% 93% [52] 34 A 61.8% 26 HP 42.3% 30 controls SFRP2 13 A 46% 100% [53] 6 HP 33% 6 controls Vimentin 103 A 46% 84% [54] 75 controls Vimentin/DIA 42 CRC 86% 73% [55] 241 controls CDKN2A/p16/ MSI/long DNA 25 CRC 64% 95% [56] 20 controls SFRP2 52 CRC 94% 93% [57] 10 advanced 70% A 11:00 AM 36% 8 HP 38% 6 UC 17% 24 controls SFRP2/HPP1/ 52 CRC 96% 96% [58] MGMT 10 advanced 80% A 11 nonadvanced 64% A 8 HP 38% 6 UC 17% 24 controls Vimentin/DIA 40 CRC 88% 82% [59] 122 controls ATM/APC/ MGMT/hMLH1/ HLTF/SFRP2/GSTP1 20 CRC 75% 90% [60] 30 A 68% 30 controls 241 controls Vimentin 74 CRC 72% 89% [61] 62 A 84% 70 controls Vimentin 94 CRC 46% 90% [62] 198 controls HIC1 26 CRC 42% 98% [63] 13 A 31% 9 HP 0% 41 controls CDKN2A/MGMT/ hmlh1 ATM/APC/ MGMT/hMLH1/ HLTF 29 A 55% 72% [64] 10 HP 40% 25 controls 20 CRC 70% 100% [65] 20 controls SFRP2 23 CRC 77%-90% 77% [66] 26 controls 10 HP 40% 25 controls A: Adenoma; HP: Hyperplastic polyp; HR: High risk; LR: Low risk; IBD: Inflammatory bowel disease; IC: Ischemic colitis; UC: Ulcerative colitis; CRC: Colorectal cancer; AGTR1: Angiotensin Ⅱ receptor, type 1; WNT2: Wingless-type MMTV integration site family member 2; SLIT2: Slit homolog 2; VIM: Vimentin; EYA4: Eyes absent homolog 4; BMP3: Bone morphogenetic protein 3; NDRG4: NDRG family member 4; ESR1: Estrogen receptor 1; PHACTR3: Phosphatase and actin regulator 3; TFPI2: Tissue factor pathway inhibitor 2; CNRIP1: Cannabinoid receptor interacting protein 1; FBN: Fibrillin; INA: Internexin neuronal intermediate filament protein, alpha; MAL: Mal, T-cell differentiation protein; SNCA: Synuclein, alpha; SPG20: Spastic paraplegia 20; MGMT: O-6-methylguanine-DNA methyltransferase; 3OST2: Heparan sulfate (glucosamine) 3-O-sulfotransferase 2; ITGA4: Integrin, alpha 4; RARB2: Retinoic acid receptor, beta 2; RASSF1: Ras association (RalGDS/AF-6) domain family member 1; OSMR: Oncostatin M receptor; GATA4: GATA binding protein 4; TFPI2: Tissue factor pathway inhibitor 2; CDKN2A: Cyclin-dependent kinase inhibitor 2; ADIA: DNA integrity assay; MSI: Microsatellite instability; GSTP1: Glutathione S-transferase pi 1; HIC1: Hypermethylated in cancer 1; ATM: Ataxia telangiectasia mutated; APC: Adenomatosis polyposis coli; hmlh1: Homo mutl homolog 1; HLTF: Helicase-like transcription factor; SFRP2: Secreted frizzled-related protein April 21, 2014 Volume 20 Issue 15

182 Wang X et al. Epigenetic biomarker research in colorectal cancer Table 3 MicroRNAs found in colorectal cancer but not in normal tissue Table 4 MicroRNA biomarkers in the blood and stool of colorectal cancer patients Up-regulated mir-7, mir-17, mir-18a, mir-19a, mir-20a, mir-20, mir-21, mir-25, mir-29a, mir-29b, mir-32, mir-33a, mir-34a, mir-34b, mir-92a, mir-93, mir-95, mir-96, mir-101, mir-106a, mir-106b, mir-130, mir-135a, mir- 135b, mir-181b, mir-182, mir-183, mir-191, mir-200c, mir-203, mir-222, mir-223, mir-224, mir-378, mir-155 Down-regulated mir-30a, mir-30c, mir-34a, mir-125a, mir-126, mir- 133a, mir-133b, mir-143, mir-145, mir-191, mir-192, mir-195, mir-215, mir-342, mir-497, mir-375, mir-378, mir-1, mir-9, mir-129, mir-137, mir-139 The underlined genes mir-34a, mir-191 and mir-378 were reported to be up-regulated in some studies [71-73] and down-regulated in others [74-76]. has been well documented that many mirnas are regulated by the methylation of their promoter region, and some mirnas target epigenetic activity. For example, mir-29b has been reported to induce DNA hypomethylation and the re-expression of tumor suppressor genes in acute myeloid leukemia by targeting DNMT [68]. These results suggest that there is a strong relationship between mirna expression and epigenetic mechanisms. Notably, many mirnas have been found in CRC, and researchers have quantified specific mirnas for the purpose of CRC diagnosis and prognosis in patient blood, stool and tissue samples. In vitro studies have also been conducted to identify any correlation between epigenetic aberrations and therapy response. In vitro studies Currently, 54 mirnas have been identified that are regulated either up or down in CRC cells relative to nontumor cells (Table 3) [69,70]. Of these, mir-17, mir-20, mir-21, mir-31, mir-92a, mir-93, mir-183 and mir-203 were upregulated in CRC cells, while mir-30a, mir-30c, mir-133a, mir-143, mir-145 were downregulated. These observations have been validated in subsequent studies. The upregulated mirnas were associated with chromosomal regions that are often amplified in CRC, and the downregulated mirnas often associated with chromosomal regions that were typically deleted. These changes may be closely related to genetic alterations as well as epigenetic modification. However, there are some discrepancies between studies. For example, mir-34a, mir-191 and mir-378 were reported to be upregulated in some studies [71-73] and yet were down regulated in others [74-76]. This may have been caused by heterogeneity between the different studies with regards to tumor stage, tumor location, genetic background and technical issues. We believe that the accumulation of further studies will allow us to determine which mirnas will be the most effective biomarkers and also better understand their role in colorectal cancer. mirna biomarkers in blood It is widely believed that mirnas can shed from tumor Markers Sample Sensitivity Specificity Ref. Blood mir CRC 76.8%-82.8% 81.1%-90.6% [77] 43 A 91.9% 81.1% 53 controls mir-601/ mir CRC 83.3% 69.1% [78] 48 AA 72.1% 62.1% 58 controls mir CRC 90.0% 90.0% [79] 30 controls mir-29a 258 plasma 75.0% 75.0% [80] sample mir CRC 77.1%-90.9% 77.1%-89.7% [81] mir-92a 120 CRC 84.0% 71.2% [82] 37 AA 64.9% 81.4% 59 controls mir-29a 120 CRC 69.0% 89.1% [82] 37 AA 62.2% 84.70% 59 controls mir CRC 86.0% 41.0% [83] 37 controls mir-17-3p 90 CRC 64.0% 70.0% [84] 50 controls mir-92a 90 CRC 89.0% 70.0% [84] 50 controls Stool mir CRC 55.7% 73.3% [85] 57 polyps 43.9% 101 controls mir-92a 88 CRC 71.6% 73.3% [85] 57 polyps 56.1% 101 controls mir-144* 75 CRC 74.0% 87.0% [86] mir cluster 197 CRC 69.5% 81.5% [87] 119 controls mir CRC 46.2% 95.0% [87] 119 controls mir CRC 14.7% 91.6% [87] 119controls mir-92a 59 CRC 50.0% 80.0% [88] 74 controls mir CRC 50.0% 83.0% [88] 74 controls A: Adenoma; CRC: Colorectal cancer. cells via exosomes and survive in a stable form in the circulation. Many studies have been performed to quantify mirnas in the blood for use as a biomarker (Table 4) [77-88]. mir-92a, located on chromosome 13q13, is a member of the mir gene cluster. This cluster promotes cell proliferation, suppresses apoptosis, induces angiogenesis and accelerates tumor progression. mir-92a was first identified by Ng et al [84] as a potential noninvasive biomarker for CRC detection with a sensitivity of 89% and specificity of 70%. mir-17-3p, another member of the mir gene cluster, was also evaluated in this study as a detection biomarker. This mirna produced a sensitivity of 64% and a specificity of 70%. To follow this study, Huang et al [82] performed a receiver-operating characteristic (ROC) analysis on 120 CRC patients, 37 patients with advanced adenomas and April 21, 2014 Volume 20 Issue 15

183 Wang X et al. Epigenetic biomarker research in colorectal cancer Table 5 Relationship between micrornas and screening, diagnosis and prognosis in colorectal cancer Screening Diagnosis Prognosis Treatment mir-17-92, mir-20a, mir-21, mir-92, mir-96, mir-106a, mir-135, mir-144, mir-203, mir-326, mir-181b, mir-601, mir-760 mir-133b, mir-143, mir-145, mir-17-92, mir-18a, mir- 20a, mir-21, mir-31, mir-92, mir-96, mir-135b, mir-183 mir-18a, mir-21, mir-20a, mir-31, mir-143, mir-145, mir-155, mir-181b, mir-200c, mir-203, mir-106a, mir-17-92, mir-135a, mir-335, mir-206, mir-10b, mir- 146a/b, let7a/b mir-21, mir-17, mir-215, mir-125b, mir-137, mir-143, mir-145, mir-192, mir-622, mir-630 healthy controls. In this analysis, the researchers found that they could not only discriminate CRC from controls (mir-29a yielded an area under the curve (AUC) of 0.844, and mir-92a yielded an AUC of 0.838), but also discriminate advanced adenomas from controls (the AUC was for mir-29a and for mir-92a). Furthermore, combined ROC analyses using these two mirnas revealed an increased AUC with an 83.0% sensitivity and 84.7% specificity in discriminating CRC, and an AUC demonstrating 73.0% sensitivity and 79.7% specificity in discriminating advanced adenomas. These results suggested that plasma mir-29a and mir-92a have potential as novel noninvasive biomarkers for CRC detection and that a combination of different mirnas may provide a higher sensitivity and specificity than a single mirna. More recently, mir-21, mir-601, mir-760 and mir-221 from plasma were also reported to be potential CRC biomarkers. In these studies, mir-221 and mir-21 were upregulated in the plasma of CRC patients compared to healthy controls [79,83], while mir-601 and mir-760 were down-regulated [78]. Moreover, a study conducted in two independent CRC cohorts suggested that high levels of plasma mir-141 could predict poor survival, and thus mir-141 may serve as an independent prognostic factor for advanced CRC patients [81]. mirna biomarkers in stool Stool-based mirna detection has been widely studied as a noninvasive screening method for CRC (Table 4). Koga et al [87] conducted an mirna expression analysis of exfoliated colonocytes isolated from the feces of 197 CRC patients and 119 healthy controls. They analyzed the mirna expression of the mir cluster (including mir-17, mir-18a, mir-19a, mir-19b, mir-20a and mir-92a), mir-21, and mir-135 by quantitative real-time PCR and found that expression of the mir cluster and mir-135 was much higher in CRC patients than in healthy controls (P < ). mir-21, on the other hand, could not discriminate between the two groups. The mir cluster detected distal tumors better than proximal tumors, as the sensitivity of mirna expression for these tumors was 81.5% and 52.9%, respectively. In another study, Wu et al [85] evaluated the feasibility of mir-21 and mir-92a detection in stool samples from 88 patients with CRC, 57 patients with colorectal polyps and 101 healthy controls. These results showed that patients with CRC had significantly higher levels of mir-21 (P < 0.01) and mir-92a (P < ) in their stool compared with normal controls. mir-92a levels provided a higher sensitivity for distal rather than proximal CRC (P < 0.05). In addition, stool mir-21 and mir-92a levels decreased significantly (P < 0.01) after surgical resection of tumor, which suggests that mir-92a and mir-21 from stool samples could serve as screening biomarkers for colorectal cancer. In addition, mir-144* and mir-106a were found to be significantly overexpressed in adenomas and in the stool of CRC patients compared with healthy individuals [86,89]. These studies have confirmed that mirnas from stool samples require validation as diagnostic biomarkers for CRC. Brief summary mirnas have been closely linked to colorectal cancer development. They can serve as screening and diagnosis markers for CRC and also as potential prognostic and predictive markers. As a rough outline for the reader, we provide here a table to display the relationship between currently identified mirnas and screening, diagnosis, prognosis and treatment in colorectal cancer (Table 5). As research continues, more mirnas correlated with CRC will be discovered, and the mechanism of mirna regulation will be deciphered. Therefore, it is highly likely that more effective mirna biomarkers for CRC patients will be found in the future. HISTONE MODIFICATION Although DNA methylation has been the most extensively studied epigenetic alteration in CRC, increasing numbers of studies have also explored how histone modifications in tumor cells compared to normal colorectal cells. Only tissue samples can be used for histone profiling, so these biomarkers are most useful for the postoperative prognosis of CRC patients. Thus far, the best studies on histone modification have addressed post-translational methylation and acetylation by multiple enzymes. Tamagawa et al [90] created duplicate 2-mm-core tissue microarrays from 54 paraffin-embedded samples of primary colorectal adenocarcinomas and corresponding liver metastases to evaluate the methylation patterns of histone H3 lysine 27 (H3K27), H3 lysine 36 (H3K36) and the expression of H3K27 methylase EZH2. These microarrays were then probed in immunohistochemical assays to search for biomarkers that could identify these patients. These results revealed that H3K27me2 levels were lower in liver metastases than in the corresponding primary tumors, and these levels correlated with tumor size and poorer survival rates. H3K36me2 levels were higher in liver metastases than in the corresponding primary tumors and correlated with histological type and lymph node metastasis. In addition, this study conducted a multivariate survival analysis and suggested that the methylation level of H3K27me2 detected by immuno April 21, 2014 Volume 20 Issue 15

184 Wang X et al. Epigenetic biomarker research in colorectal cancer histochemistry may be an independent prognostic factor for metachronous liver metastasis in colorectal cancer patients. In fact, prior to this study, this group used the same method to validate other histone patterns, including histone H3 lysine 4 (H3K4) dimethylation, histone H3 lysine 9 (H3K9) dimethylation and histone H3 lysine 9 (H3K9) acetylation. They found that dimethylation of H3K4 and acetylation of H3K9 correlated with tumor histological type, and lower levels of H3K4 dimethylation correlated with a poor survival rate. Multivariate survival analysis showed that H3K4 dimethylation status is an independent prognostic factor for colorectal cancer patients [91]. Using chromatin immunoprecipitation (ChIP) coupled with quantitative PCR and high-throughput sequencing, Gezer et al [92] observed reduced plasma levels for two histone methylation biomarkers, H3K9me3 and H4K20me3, in patients with CRC and characterized these modifications in the circulation. They found that lower H3K9me3 levels had potential as biomarkers for CRC. These studies have provided a good start for the examination of histone modification for the prognosis of CRC. Research is ongoing to find histone biomarkers useful for colorectal cancer patients. DISCUSSION As we have discussed above, a variety of DNA methylation, mirna and histone biomarkers from stool, blood and tissue have been reported for CRC detection. Some of the markers identified are derived from tumor cells and others are derived from non-tumor cells in the tumor microenvironment or blood. DNA and mirna biomarkers mostly shed from tumor cells, and so, theoretically, these molecules should be more specific than protein biomarkers such as carcinoembryonic antigen (CEA), carbohydrate antigen (CA) 19-9, CA242 and CA724, which are currently applied widely in the clinic. This is because nucleic acid-based markers can be amplified and thus produce a stronger signal, thereby permitting a greater sensitivity. In addition to the superior assay performance of DNA and mirna, these samples are easier to store because effective preservation buffers that can prevent DNA and mirna degradation in stool samples are available. In contrast, no preservation buffer for proteins in stool samples has been reported so far. In comparison with DNA and mirna, protein biomarkers show lower specificity because tumors often induce inflammatory reactions, and some of the biomarkers that initially showed promise for cancer detection now appear to also detect a wide range of bowel diseases, such as ulcerative colitis and Crohn s disease. Moreover, protein biomarkers have often been altered in more than one type of cancer. For example, CEA has been reported as a biomarker for various malignancies, including colorectal, pancreatic, lung, renal and breast cancers [93-96]. In spite of these issues, protein biomarkers may still be useful for large-scale screening for CRC because proteins can be observed through assays in small sample volumes with relatively simple and cheap assays. Regarding the comparison between stool and blood biomarkers, we know that both of these sample types have been under investigation and improvements continue to be made. In a biomarker search, sample collection, storage and handling have a significant impact on the performance of a specific test. Indeed, using stool samples to detect new biomarkers is not standardized; for example, the buffers used to collect and store stool samples were different in each study, and the methods of DNA or RNA isolation also varied. Therefore, it is difficult to compare the performance of different biomarkers based on the current research. By contrast, blood detection is more standardized and readily accepted by the general population. Moreover, biomarkers in blood are more stable than in stool because of the absence of microflora. DNA, mirna and proteins have all been shown to be stable in unprocessed EDTA tubes or noncentrifuged clotted blood for 24 h or longer at room temperature. This is particularly true for mirna, which, as a result of its short length, is more stable in blood than other types of nucleic acids. So, at a practical level, degradation problems for biomarkers during storage and transport should be taken into account, and it is important to standardize detection procedures. For example, the bias will be enormous and the results will not be interpretable if comparing the data from newly collected samples of CRC patients with those from archived samples of adenoma patients or healthy persons. Thus, various samples should be collected in the very same manner at the very same time according to the REMARK guidelines to improve the comparability between various results. Studies specifically addressing these questions are highly desirable CONCLUSION Epigenetic biomarkers and the use of blood and stool samples each have their own advantages and disadvantages for clinical screening, diagnosis and prognosis. Although many studies on these biomarkers are preliminary, some markers have demonstrated better performance than the current FOBT test. No biomarker-based assay is ready for large-scale population screening, however. The standardization of sample preparation and testing protocols is very important for the widespread deployment of techniques and the comparison of results from different studies. Moreover, large well-controlled studies are urgently needed to identify the accuracy of epigenetic biomarkers for CRC detection in asymptomatic populations. Much work remains before such observations can be translated into routine clinical practice. REFERENCES 1 Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBO- CAN Int J Cancer 2010; 127: [PMID: DOI: /ijc.25516] 4283 April 21, 2014 Volume 20 Issue 15

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187 Wang X et al. Epigenetic biomarker research in colorectal cancer [PMID: ] 53 Oberwalder M, Zitt M, Wöntner C, Fiegl H, Goebel G, Zitt M, Köhle O, Mühlmann G, Ofner D, Margreiter R, Müller HM. SFRP2 methylation in fecal DNA--a marker for colorectal polyps. Int J Colorectal Dis 2008; 23: [PMID: ] 54 Ahlquist DA, Sargent DJ, Loprinzi CL, Levin TR, Rex DK, Ahnen DJ, Knigge K, Lance MP, Burgart LJ, Hamilton SR, Allison JE, Lawson MJ, Devens ME, Harrington JJ, Hillman SL. Stool DNA and occult blood testing for screen detection of colorectal neoplasia. Ann Intern Med 2008; 149: , W81 [PMID: ] 55 Itzkowitz S, Brand R, Jandorf L, Durkee K, Millholland J, Rabeneck L, Schroy PC, Sontag S, Johnson D, Markowitz S, Paszat L, Berger BM. A simplified, noninvasive stool DNA test for colorectal cancer detection. Am J Gastroenterol 2008; 103: [PMID: DOI: / j x] 56 Abbaszadegan MR, Tavasoli A, Velayati A, Sima HR, Vosooghinia H, Farzadnia M, Asadzedeh H, Gholamin M, Dadkhah E, Aarabi A. 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Plasma micrornas are promising novel biomarkers for early detection of colorectal cancer. Int J Cancer 2010; 127: [PMID: 4286 April 21, 2014 Volume 20 Issue 15

188 Wang X et al. Epigenetic biomarker research in colorectal cancer DOI: /ijc.25007] 83 Pu XX, Huang GL, Guo HQ, Guo CC, Li H, Ye S, Ling S, Jiang L, Tian Y, Lin TY. Circulating mir-221 directly amplified from plasma is a potential diagnostic and prognostic marker of colorectal cancer and is correlated with p53 expression. J Gastroenterol Hepatol 2010; 25: [PMID: DOI: /j x] 84 Ng EK, Chong WW, Jin H, Lam EK, Shin VY, Yu J, Poon TC, Ng SS, Sung JJ. Differential expression of micrornas in plasma of patients with colorectal cancer: a potential marker for colorectal cancer screening. Gut 2009; 58: [PMID: DOI: /gut ] 85 Wu CW, Ng SS, Dong YJ, Ng SC, Leung WW, Lee CW, Wong YN, Chan FK, Yu J, Sung JJ. Detection of mir-92a and mir-21 in stool samples as potential screening biomarkers for colorectal cancer and polyps. Gut 2012; 61: [PMID: DOI: /gut ] 86 Kalimutho M, Del Vecchio Blanco G, Di Cecilia S, Sileri P, Cretella M, Pallone F, Federici G, Bernardini S. Differential expression of mir-144* as a novel fecal-based diagnostic marker for colorectal cancer. J Gastroenterol 2011; 46: [PMID: DOI: /s ] 87 Koga Y, Yasunaga M, Takahashi A, Kuroda J, Moriya Y, Akasu T, Fujita S, Yamamoto S, Baba H, Matsumura Y. MicroRNA expression profiling of exfoliated colonocytes isolated from feces for colorectal cancer screening. Cancer Prev Res (Phila) 2010; 3: [PMID: DOI: / CAPR ] 88 Wu CW, Ng SS, Leung WW, Lee CW, Wong CY, Yu J, Sung JJ. S1164 MicroRNAs in stool as screening marker for colorectal cancer. Gastroenterology 2010; 138: S-194 [DOI: /S (10) ] 89 Link A, Balaguer F, Shen Y, Nagasaka T, Lozano JJ, Boland CR, Goel A. Fecal MicroRNAs as novel biomarkers for colon cancer screening. Cancer Epidemiol Biomarkers Prev 2010; 19: [PMID: DOI: / EPI ] 90 Tamagawa H, Oshima T, Numata M, Yamamoto N, Shiozawa M, Morinaga S, Nakamura Y, Yoshihara M, Sakuma Y, Kameda Y, Akaike M, Yukawa N, Rino Y, Masuda M, Miyagi Y. Global histone modification of H3K27 correlates with the outcomes in patients with metachronous liver metastasis of colorectal cancer. Eur J Surg Oncol 2013; 39: [PMID: DOI: /j.ejso ] 91 Tamagawa H, Oshima T, Shiozawa M, Morinaga S, Nakamura Y, Yoshihara M, Sakuma Y, Kameda Y, Akaike M, Masuda M, Imada T, Miyagi Y. The global histone modification pattern correlates with overall survival in metachronous liver metastasis of colorectal cancer. Oncol Rep 2012; 27: [PMID: DOI: /or ] 92 Gezer U, Ustek D, Yörüker EE, Cakiris A, Abaci N, Leszinski G, Dalay N, Holdenrieder S. Characterization of H3K9me3- and H4K20me3-associated circulating nucleosomal DNA by high-throughput sequencing in colorectal cancer. Tumour Biol 2013; 34: [PMID: DOI: / s ] 93 Chung HW, Lim JB, Jang S, Lee KJ, Park KH, Song SY. Serum high mobility group box-1 is a powerful diagnostic and prognostic biomarker for pancreatic ductal adenocarcinoma. Cancer Sci 2012; 103: [PMID: DOI: / j x] 94 Grunnet M, Sorensen JB. Carcinoembryonic antigen (CEA) as tumor marker in lung cancer. Lung Cancer 2012; 76: [PMID: DOI: /j.lungcan ] 95 Ye YL, Bian J, Huang YP, Guo Y, Li ZX, Deng CH, Dai YP, Sun XZ. Primary mucinous adenocarcinoma of the renal pelvis with elevated CEA and CA19-9. Urol Int 2011; 87: [PMID: DOI: / ] 96 Marić P, Ozretić P, Levanat S, Oresković S, Antunac K, Beketić- Oresković L. Tumor markers in breast cancer--evaluation of their clinical usefulness. Coll Antropol 2011; 35: [PMID: ] P- Reviewers: Nielson HJ, Tiberio GAM, Yu B S- Editor: Gou SX L- Editor: Wang TQ E- Editor: Wu HL 4287 April 21, 2014 Volume 20 Issue 15

189 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. WJG 20 th Anniversary Special Issues (5): Colorectal cancer TOPIC HIGHLIGHT MicroRNAs in colorectal cancer as markers and targets: Recent advances Jing-Jia Ye, Jiang Cao Jing-Jia Ye, Jiang Cao, Clinical Research Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou , Zhejiang Province, China Author contributions: Ye JJ and Cao J contributed to this paper. Supported by National Natural Science Foundation of China, No , No , No and No Correspondence to: Jiang Cao, PhD, Clinical Research Center, The Second Affiliated Hospital, Zhejiang University School of Medicine, 88 Jie-fang Road, Hangzhou , Zhejiang Province, China. caoj@zju.edu.cn Telephone: Fax: Received: September 27, 2013 Revised: January 2, 2014 Accepted: January 19, 2014 Published online: April 21, 2014 Abstract MicroRNAs are evolutionarily conserved small noncoding RNA molecules encoded by eukaryotic genomic DNA, and function in post-transcriptional regulation of gene expression via base-pairing with complementary sequences in target mrnas, resulting in translational repression or degradation of target mrnas. They represent one of the major types of epigenetic modification and play important roles in all aspects of cellular activities. Altered expression of micrornas has been found in various human diseases including cancer. Many efforts have been made to discover the characteristic microrna expression profiles, to understand the roles of aberrantly expressed micrornas and underlying mechanisms in different cancers. With the application of DNA microarray, real-time quantitative polymerase chain reaction and other molecular biology techniques, increasing evidence has been accumulated which reveal that aberrant micrornas can be detected not only intracellularly within the cancer cells, but also extracellularly in plasma of patients, postulating the potential of aberrant micrornas as promising diagnostic/prognostic markers and attracting therapeutic targets. This review is intended to provide the most recent advances in microrna studies in one of the most common cancers, colorectal cancer, especially the identification of those specifically altered micrornas in colorectal cancer, validation for their relevance to clinical pathological parameters of patients, functional analyses and potential applications of these micrornas Baishideng Publishing Group Co., Limited. All rights reserved. Key words: MicroRNA; Epigenetic modification; Colorectal cancer; Marker; Therapy Core tip: MicroRNAs represent one of the important epigenetic modifications for gene expression and play important roles in all aspects of cellular activities. Altered expression of micrornas has been found in various human diseases including cancer. Aberrant micrornas can be detected not only intracellularly within the cancer cells, but also extracellularly in plasma of patients, postulating the potential of aberrant micrornas as promising diagnostic/prognostic markers and attracting therapeutic targets. This review focuses on recent advances in identification, validation and functional analyses for such micrornas in colorectal cancer, and potential applications of these altered micrornas. Ye JJ, Cao J. MicroRNAs in colorectal cancer as markers and targets: Recent advances. World J Gastroenterol 2014; 20(15): Available from: URL: com/ /full/v20/i15/4288.htm DOI: org/ /wjg.v20.i INTRODUCTION Colorectal cancer is one of the most common malignancies worldwide, with new cases and deaths estimated in 2008 [1]. Prevention, early diagnosis 4288 April 21, 2014 Volume 20 Issue 15

190 Ye JJ et al. MicroRNAs in colorectal cancer and treatment can greatly reduce the incidence and mortality. All these depend on the achievements in a comprehensive understanding of every aspect of colorectal cancer at molecular and cellular levels. More and more evidence shows that epigenetic modifications, such as hyper- or hypo-methylation at specific sites in DNAs or proteins, acetylation or de-acetylation of nucleosome histones and conditionally specifically expressed non-coding RNAs like micrornas, are involved in the carcinogenesis of many types of cancer including colorectal cancer [2-4], and some of the epigenetic modifications can serve as markers for diagnosis, treatment efficacy monitoring and prognosis, or be developed into targets for therapeutic interventions. MicroRNAs are evolutionarily conserved small noncoding RNA molecules that are encoded by eukaryotic genomic DNA. Located in the spacer regions between protein-coding genes or in the introns of protein-encoding genes, microrna coding sequences have their own promoters or utilizes the same promoters as proteincoding genes, and are transcribed as primary micrornas (pri-micrornas) in the same manner as the messenger RNAs of the protein-coding genes do. Pri-microRNAs are processed into microrna precursors (pre-micror- NAs) in the nucleus and transported to the cytoplasm and further processed into mature micrornas, and function in post-transcriptional regulation of gene expression via base-pairing with complementary sequences in target mrnas, resulting in translational suppression of imperfectly matched mrnas or degradation of perfectly matched mrnas [5]. Both strands of a pre-microrna may be processed into two mature micrornas, with similar efficiencies which are discriminated by -5p and -3p, or with one dominantly processed and the recessive one star-labeled (*), which function differently against different target genes. This post-transcriptional regulation of expression of multiple genes represents one of the major types of epigenetic modification and exhibits important impacts in all aspects of cellular activities, under both physiological and pathological conditions. Many of the known micrornas appear in clusters on a single polycistronic transcript [6], which may modulate the expression of genes whose products work together to fulfill the same task. It is now well documented that micrornas play important roles in the pathogenesis of many human diseases including cancer. Aberrant expression of micrornas has been observed in cancers of various tissues such as lung, breast, liver, colon and rectum, and prostate. Upregulation of certain specific micrornas may suppress genes responsible for growth/proliferation inhibition, down-regulation of other specific micrornas may augment genes responsible for growth/proliferation promotion, and either may result in the development and progression of cancer. The specifically altered microrna expression patterns may serve as diagnostic/prognostic markers, and correction of these aberrant micrornas may reverse the malignant phenotypes of cancer cells and therefore provide means for cancer treatment [7-9]. Numerous investigations on screening for altered expression of micrornas in various types of cancer have been conducted during the past decade, with more and more functional validations in recent years. The aberrantly expressed micrornas exert their functions by modulating oncogenic or tumor-suppressive genes and play important roles in the development and progression of cancers, therefore exhibit their potentials as oncogenic or tumor-suppressive micrornas. Some of the alterations are common among different cancers, while others are type-specific. MicroRNAs function in a multitarget manner that one microrna may modulate the expression of multiple genes, and one target gene may also be modulated by multiple micrornas. While the microrna-modulated gene expression is one kind of epigenetic modification, the expression of microrna itself is modulated by other epigenetic modifications such as hyper- or hypo-methylation. This review focuses on the most recent advances in studies on some extensively investigated micrornas in colorectal cancer, especially with regards to the potentials as bio-markers or therapeutic targets. ONCOGENIC MICRORNAS AS POTENTIAL MARKERS AND TARGETS IN COLORECTAL CANCER MiR-21 MiR-21 is one of the most extensively investigated oncogenic micrornas whose expression is frequently upregulated in colorectal cancer. The identified target genes regulated by mir-21 include programmed cell death 4, RhoB and transforming growth factor beta receptor 2 (TGFBR2). MiR-21 regulates cell proliferation, invasion and apoptosis, and induces stemness. Through its pro-metastatic effect, ectopic stromal mir-21 expression associates with increased epithelial invasiveness. The expression level of mir-21 correlates with clinical stage, and increases with advanced disease, decreased recurrence-free cancer-specific survival and shorter overall survival (OS). There is higher stool level of mir-21 in patients with colorectal cancer but not polyps. Plasma/ serum mir-21 can be served as a potential diagnostic and prognostic marker. The post-therapeutic mir-21 level in colorectal cancer is lower and can predict the pathological tumor response to chemotherapy. Down-regulation of mir-21 reduces cell proliferation, migration and invasion, induces apoptosis and inhibits cell cycle progression, upregulates Spry2 and phosphatase and tensin homologue deleted on chromosome 10 and enhances the cytotoxic effects of 5-fluorouracil and metformin, and also leads to differentiation of chemoresistant cells, therefore inhibition of mir-21 may serve as a novel therapeutic approach [10-30]. MiR-155 Up-regulation of oncogenic mir-155 in colorectal cancer cells promotes cell proliferation, migration and inva April 21, 2014 Volume 20 Issue 15

191 Ye JJ et al. MicroRNAs in colorectal cancer sion, increases chemoresistance and correlates with poor prognosis. Claudin-1, a member of integral membrane proteins that constitute tight junctions, is the identified target gene modulated by mir-155 [30-33]. Low expression of claudin-1 is associated with lymphatic involvement, histological differentiation, extent of poorly differentiated component, reduced disease-free and overall survival of colorectal cancer patients [34]. MiR-31 With rat sarcoma viral oncogene homolog (RAS) p21 Guanosine-5 -triphosphatase (GTPase) activating protein 1 (RASA1) gene as the target gene, mir-31 overexpression activates oncogene RAS by repressing RASA1, and elevated expression of mir-31 [35-38] is associated with aggressive mucinous phenotype. For metastatic colorectal cancer patients with wild-type kirsten rat sarcoma viral oncogene homolog/v-raf murine sarcoma viral oncogene homolog B (KRAS/BRAF) who received anti-epidermal growth factor receptor (EGFR) monoclonal antibody (mab) treatment, significant mir-31* up-regulation appeared in progressive disease vs disease control, and can be used to predict the benefits of anti-egfr mab treatment. MiR-92a The overexpression of mir-92a [14,26,39-43] correlates with tumor metastasis and poor prognosis. Higher mir-92a level in stool in patients with colorectal cancer and polyps can be detected. BCL-2-interacting mediator of Cell Death (BIM) is the target gene of mir-92a. As the downregulation of BIM gene by over-expressed mir-92a in colon cancer cells may lead to the evasion of apoptosis, anti-mir-92a strategy effectively induces apoptosis of colorectal cancer cells, which suggests a potential new therapeutic approach. MiR-17 Elevated in colon cancer, mir-17 [18,42-45] expression is associated with poor survival and is an independent prognostic marker. By targeting tumor suppressor gene Rho family GTPase 3 (RND3), mir-17 promotes proliferation, growth and cell cycle progression. Moreover, elevated oncofoetal mir-17-5p expression resulted in shorter overall survival rates by repressing its target gene retinoblastoma-like protein 2 (P130), but caused a better response to adjuvant chemotherapy. MiR-106a and mir-106b MiR-106a [46,47] is highly expressed in metastatic colorectal cancer cells and regulates migration and invasion. Tumor suppressor Rb1 is one of the target genes of mir-106a, and the regulatory role for Rb1 may happen in sporadic colorectal cancer. Similar to mir-21, mir-106a also inhibits the expression of TGFBR2, leading to increased colorectal cancercell migration and invasion. Interestingly, mir-106b [42] is upregulated in cancer stromal tissues compared with normal stroma, and the stromal mir-106b expression level is associated with clinicopathologic factors, suggesting the possibility that mirnas in cancer stroma are crucially involved in cancer progression, a similar phenomenon observed for stromal mir-21 [10]. MiR-135a and mir-135b Oncogenic mir-135a [24,48] promotes the growth and invasion of colorectal cancer cells in vitro by repressing its target gene metastasis suppressor 1. The level of mir- 135b [17,20,49] is also elevated in colorectal cancer, which correlates with clinical stage, liver metastasis, and both disease-free survival (DFS) and cancer-specific survival of patients, and inhibition of mir-135b leads to decreased viability of colorectal cancer cells in vitro. TUMOR SUPPRESSIVE MICRORNAS AS POTENTIAL MARKERS AND TARGETS IN COLORECTAL CANCER Let-7 The let-7 family is one of the most ancient and conserved micrornas, which functions as a well-recognized tumor suppressor targeting oncogenic KRAS and whose expression is deregulated in many types of cancer including colorectal cancer [50,51]. Recent studies on let-7 family members showed that: let-7a expression is elevated in metastatic colorectal cancer with KRAS mutation compared to normal mucosa or non-metastatic disease [24], and the high level of let-7a in KRAS-mutated colorectal cancer may rescue anti-egfr therapy effects [52] ; decreased expression of let-7b at tumor invasion front is an adverse prognostic marker for recurrence and OS of colorectal cancer patients [53] ; let-7c is a metastasis suppressor in colorectal cancer by targeting matrix metalloproteinase 11 and pre-b-cell leukemia homeobox 3 [54] ; and let-7e is overexpressed in responders to neoadjuvant chemoradiotherapy [55]. Another current research focus is the correlation between a functional polymorphism in let-7 complementary site within the 3 untranslated region (3 -UTR) of KRAS (rs ) and the risk for development of colorectal cancer, pathological and clinical parameters, clinical outcome, progression-free survival (PFS) or OS in metastatic colorectal cancer patients [52,56-58]. All these novel findings for the Let-7 family member micrornas provide us further predictive/prognostic markers in the management of colorectal cancer patients. MiR-143 MiR-143 [28,37,53,59-65] targets hexokinase 2 (HK2) gene and metastasis-associated in colon cancer-1 gene, and inhibit invasion/migration of colorectal cancer cells. Loss of mir-143-mediated repression of HK2 can promote glucose metabolism in cancer cells. The complementary strand mir-143* is down-regulated in colon cancer cells and forced expression significantly abrogated invasive potential. MiR-143 is often down-regulated in colorectal cancer, especially at liver invasion front, and the reduced ex April 21, 2014 Volume 20 Issue 15

192 Ye JJ et al. MicroRNAs in colorectal cancer Table 1 Colorectal cancer-associated micrornas with identified targets MiR (family) Role/ potential Identified targets Ref. 9 ON E-cadherin [36,81,82] 16 TS COX-2, cyclin D1, [84-86] survivin, CDK6 17 ON RND3, P130 [18,42-45] 21 ON PDCD4, RhoB, TGFBR2 [10-30] 22 TS p21 [90-93] 31 ON RASA1 [14,17,35-38] 33a TS Pim-1 [67] 34 family TS Axin2 [33,70, ] 92a ON BIM [14,26,39,40-43] 95 ON Nexin 1 [106] 139 TS RAP1B, IGFR-1 [37,121,122] 143 TS HK2, MACC1 [28,37,53,59-65] 145 family TS PAK4, NRAS, IRS1 [14,28,53,60,64,66-68] 148 family TS Bcl-2, CCK-2 receptor [69-72] 155 ON Claudin-1 [30-33] 215 TS DTL [20,55,73-75] 320a TS Neuropilin 1, β-catenin [147,148] 339-5p TS PRL-1 [151] 342 TS DNMT1 [152] 365 TS Cyclin D1, Bcl-2 [155] 373 TS RAB22A [157] 499-5p ON FOXO4, PDCD4 [162] 506 ON PPARα [164] 1915 TS Bcl-2 [170] Let-7 family TS MMP11, PBX3 [24,38,52-57] TS: Tumor suppressor; ON: Oncogene; COX-2: Cyclooxygenase-2; CDK6: Cyclin-dependent kinase 6; RND3: Rho family GTPase 3; PDCD4: Programmed cell death 4; TGFBR2: Transforming growth factor beta receptor 2; RASA1: RAS p21 protein activator 1; BIM: Bcl-2 interacting mediator of cell death; RAP1B: RAS related protein 1b; IGFR-1: Insulin-like growth factor receptor-1; HK2: Hexokinase 2; MACC1: Metastasis associated in colon cancer 1; PAK4: p21 protein-activated kinase 4; NRAS: Neuroblastoma RAS viral oncogene homolog; IRS1: Insulin receptor substrate 1; CCK-2: Cholecystokinin-2; DTL: Denticleless E3 ubiquitin protein ligase homolog; PRL-1: Phosphatases of regenerating liver-1; DNMT1: DNA methyltransferase 1; FOXO4: Forkhead box O4; PDCD4: Programmed cell death 4; PPARα: Peroxisome proliferator-activated receptor alpha; Bcl-2: B-cell leukemia/lymphoma 2; MMP11: Matrix metallopeptidase 11; PBX3: Pre-B-cell leukemia homeobox 3. pression correlates with aggressive mucinous phenotype. MiR-143 also inhibits tumor growth and angiogenesis and sensitizes chemosensitivity to oxaliplatin. The posttherapeutic level of mir-143 increases and can be used for predicting response to treatment and prognosis. Down-regulation of fecal mir-143 is a potentional marker for colorectal cancer, and the mir-143 level in blood and tissue can be used as a new diagnostic marker and therapeutic target as well. MiR-145 MiR-145 [14,28,53,60,64,66-68] exerts its tumor suppressive function by modulating several target genes: it can block the activation of AKT and ERK1/2 pathways and the expression of HIF-1 and vascular endothelial growth factor via directly targeting neuroblastoma RAS viral oncogene homolog and insulin receptor substrate 1, down-regulate phosphorylated-extracellular signal-regulated kinase 1 level and lead to inhibition of tumor growth by targeting p21 protein-activated kinase 4. MiR-145 is down-regulated in plasma and cancer tissues and liver invasion front of colorectal cancer patients. Down-regulation of fecal mir-145 is proposed as a potential marker for colorectal cancer. Decreased expression of mir-145 occurs before the mutation of APC gene and is involved in the initiation step of colorectal cancer. Similar to mir-143, the post-therapeutic level of mir-145 increases, predicting the response to treatment and prognosis. Overexpression of mir-145 inhibits cell proliferation, migration and invasion due to its proapoptotic and antiproliferative role, showing its potential in mirna-replacement therapy of colorectal cancer. MiR-148a and 148b Tumor suppressive mir-148a [69-71] promotes apoptosis via repressing anti-apoptotic Bcl-2 expression. Hypermethylation leads to down-regulation of mir-148a in advanced CRC. Low mir-148a expression is associated with significantly shorter DFS, a worse therapeutic response, and poor OS. The mir-148a level can serve as a disease progression follow-up marker, and has prognostic/predictive value in chemotherapy as well. MiR-148b [72] also acts as a tumor suppressor in colorectal cancer by targeting the choecystokinin-2 receptor which functions depending on the gastrin in colorectal cancer, and suppresses the growth of cancer cells. The expression of mir-148b is significantly down-regulated in human colorectal cancer tissues and correlates with tumor size, and is important in the cancer transformation process. Forced expression of mir-148b in colorectal cancer cells inhibits cell proliferation in vitro and suppresses tumorigenicity in vivo. mir-148b can be further evaluated as a biomarker and therapeutic tool against colorectal cancer. MiR-215 As a tumor suppressor candidate, mir-215 [20,73-75] level is decreased in cancer tissues of colorectal cancer patients, especially those relapsed patients. The expression level of mir-215 is an independent predictive marker for relapse and associated with poor OS. However, overexpressed mir-215 can be observed in non-responders to neoadjuvant chemoradiotherapy, and the high mir-215 level confers chemoresistance due to cell cycle arrest and reduced proliferation by targeted inhibition of thymidylate synthase, dihydrofolate reductase and denticleless protein homolog, genes that play essential roles in DNA synthesis, cell cycle progression, proliferation, and differentiation. OTHER COLORECTAL CANCER-ASSOCIATED MICRORNAS In addition to the above micrornas, there are other micrornas that have been identified to be involved in the development/progression of colorectal cancer. A 4291 April 21, 2014 Volume 20 Issue 15

193 Ye JJ et al. MicroRNAs in colorectal cancer Table 2 Colorectal cancer-associated micrornas without identified target MiR (family) Role/potential Ref. 1 TS [76-78] 7 TS [79,80] 10b Marker, +, chemo [83] 15a TS [84] 18a ON [87,88] 19 family Marker, +, chemo [18,53,89] 23a ON [94,95] 27b TS [96] 29 family Marker, + [55,87,97,98] 30a-5p TS [99] 32 ON [100] 93 TS [79,104,105] 96 Marker, + [17] 101 TS [14,107,108] 103/107 ON [109] 106 family ON [14,42,46,47] 122 ON [110] 124 TS [ ] 125 family TS [114] 126 Marker, + [115] 127-3p Marker, + [40] 129 TS [116] 130a/301a/454 ON [117] 133 family Marker, - [78,118] 135 family ON [17,20,24,48,49] 137 TS [112,119,120] 140 Marker, + [38,123] 141 Circ, Marker, + [124] 144 Marker, - [125,126] 146a SNP [127,128] 149 Marker, - [129] 150 Marker, - [130] 181a ON [131] 182 Marker, + [36,132] 185 Marker, + [118] 186, 216b, 337-3p TS [133] 190b Marker, +, chemo [55] 192 Marker, - [74] 193a-3p, 338-5p Circ, Marker, + [94] 194 Marker [53,74] 19, 512, 801, 246 Marker, + [123] 196a2 SNP [ ] 199a-5p TS [139] 206 Marker, + [24] 211 ON [140] 212 TS [141] 218 TS [142] 221*, 224 TS [143] 222 TS [144] 223 TS [145] 297 TS [146] 328 TS [149] 330 TS [150] 340 TS [112] 345 TS [153] 362-3p TS [154] 367 SNP [156] 372 Marker, + [21] 375, 422a TS [20] 378 Marker, - [20,40] 409-3p Cirl, Marker, + [79] 424* TS [65] 429 ON [158] 450 family, 99a* Marker, -, chemo [55] 451 TS [159] 486-3p Marker, + [40] 493, 493* TS [160] 497 TS [161] 502 TS [163] 574-5p ON [165] 592 Marker, + [38] 601 Marker, + [166] 608 SNP [167] 625-3p Marker, +, chemo [168] 627 TS [169] 638 Marker, - [53] 760 Marker, + [133, 66] p Marker, - [38] 1275 Marker, - [53] TS: Tumor suppressor; ON: Oncogene; Marker, +: Up-regulated marker; Marker, -: Down-regulated marker; Chemo: Chemosensitivity-associated; SNP: Single nucleotide polymorphism-associated. collection of the most recently investigated/concluded colorectal cancer-associated micrornas are listed in Tables 1 and 2 for reference. CONCLUSION As discussed above, a number of alterations of micror- NAs play important roles in the development and progression of colorectal cancer, and even alterations of the microrna processing machinery components are of prognostic values [171,172]. The expression of micrornas is regulated not only by other epigenetic modifications such as hyper- or hypomethylation, but also by other interacting molecules, i.e., LIN28 and let-7 [173], and in a clustered manner. Moreover, the polymorphisms of either micrornas or targeted genes have a significant impact on colorectal cancer risk [174,175], even in population-based studies [ ], and the responses to chemotherapy and prognosis [179,180] as well. We can expect that the altered expression of micrornas detection will serve as effective biomarkers for screening, diagnosis, monitoring therapy and prognosis of colorectal cancer in the future, as they can be detected from various kinds of samples including cell-free plasma/serum [ ], circulating tumor cells [185], mucosal wash fluid [186], feces [187,188] and formalin-fixed paraffinembedded tissues [189]. Based on the achievements in this field, we can also expect that novel therapeutics be developed to re-normalize the altered micrornas in colorectal cancer [190], not only by directly restoring down-regulated micrornas or knocking down the up-regulated micrornas, but also by epigenetic therapy [191]. REFERENCES 1 Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011; 61: [PMID: DOI: /caac.20107] 2 Goel A, Boland CR. Epigenetics of colorectal cancer. Gastroenterology 2012; 143: e1 [PMID: DOI: /j.gastro ] 3 Li BQ, Yu H, Wang Z, Ding GH, Liu L. MicroRNA mediated network and DNA methylation in colorectal cancer. Protein Pept Lett 2013; 20: [PMID: ] 4292 April 21, 2014 Volume 20 Issue 15

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197 Ye JJ et al. MicroRNAs in colorectal cancer 91 Yamakuchi M, Yagi S, Ito T, Lowenstein CJ. MicroRNA-22 regulates hypoxia signaling in colon cancer cells. PLoS One 2011; 6: e20291 [PMID: DOI: /journal. pone ] 92 Li J, Zhang Y, Zhao J, Kong F, Chen Y. Overexpression of mir-22 reverses paclitaxel-induced chemoresistance through activation of PTEN signaling in p53-mutated colon cancer cells. Mol Cell Biochem 2011; 357: [PMID: DOI: /s ] 93 Tsuchiya N, Izumiya M, Ogata-Kawata H, Okamoto K, Fujiwara Y, Nakai M, Okabe A, Schetter AJ, Bowman ED, Midorikawa Y, Sugiyama Y, Aburatani H, Harris CC, Nakagama H. Tumor suppressor mir-22 determines p53-dependent cellular fate through post-transcriptional regulation of p21. Cancer Res 2011; 71: [PMID: DOI: / CAN ] 94 Yong FL, Law CW, Wang CW. Potentiality of a triple microrna classifier: mir-193a-3p, mir-23a and mir-338-5p for early detection of colorectal cancer. 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200 Ye JJ et al. MicroRNAs in colorectal cancer rs and mir-196a2 rs polymorphisms with cancer risk: a meta-analysis of 32 studies. Mutagenesis 2012; 27: [PMID: DOI: /mutage/ges052] 176 Lv M, Dong W, Li L, Zhang L, Su X, Wang L, Gao L, Zhang L. Association between genetic variants in pre-mirna and colorectal cancer risk in a Chinese population. J Cancer Res Clin Oncol 2013; 139: [PMID: ] 177 He B, Pan Y, Cho WC, Xu Y, Gu L, Nie Z, Chen L, Song G, Gao T, Li R, Wang S. The association between four genetic variants in micrornas (rs , rs , rs , rs ) and cancer risk: evidence from published studies. PLoS One 2012; 7: e49032 [PMID: DOI: /journal. pone ] 178 Azimzadeh P, Romani S, Mohebbi SR, Mahmoudi T, Vahedi M, Fatemi SR, Zali N, Zali MR. Association of polymorphisms in microrna-binding sites and colorectal cancer in an Iranian population. Cancer Genet 2012; 205: [PMID: DOI: /j.cancergen ] 179 Bovell LC, Shanmugam C, Putcha BD, Katkoori VR, Zhang B, Bae S, Singh KP, Grizzle WE, Manne U. The prognostic value of micrornas varies with patient race/ethnicity and stage of colorectal cancer. Clin Cancer Res 2013; 19: [PMID: ] 180 Xing J, Wan S, Zhou F, Qu F, Li B, Myers RE, Fu X, Palazzo JP, He X, Chen Z, Yang H. Genetic polymorphisms in premicrorna genes as prognostic markers of colorectal cancer. Cancer Epidemiol Biomarkers Prev 2012; 21: [PMID: DOI: / EPI ] 181 Luo X, Stock C, Burwinkel B, Brenner H. Identification and evaluation of plasma micrornas for early detection of colorectal cancer. PLoS One 2013; 8: e62880 [PMID: DOI: /journal.pone ] 182 Blanco-Calvo M, Calvo L, Figueroa A, Haz-Conde M, Antón-Aparicio L, Valladares-Ayerbes M. Circulating micrornas: molecular microsensors in gastrointestinal cancer. Sensors (Basel) 2012; 12: [PMID: ] 183 Menéndez P, Villarejo P, Padilla D, Menéndez JM, Montes JA. Diagnostic and prognostic significance of serum micror- NAs in colorectal cancer. J Surg Oncol 2013; 107: [PMID: DOI: /jso.23245] 184 Luo X, Burwinkel B, Tao S, Brenner H. MicroRNA signatures: novel biomarker for colorectal cancer? Cancer Epidemiol Biomarkers Prev 2011; 20: [PMID: DOI: / EPI ] 185 Mostert B, Sieuwerts AM, Martens JW, Sleijfer S. Diagnostic applications of cell-free and circulating tumor cell-associated mirnas in cancer patients. Expert Rev Mol Diagn 2011; 11: [PMID: DOI: /erm.11.11] 186 Kamimae S, Yamamoto E, Yamano HO, Nojima M, Suzuki H, Ashida M, Hatahira T, Sato A, Kimura T, Yoshikawa K, Harada T, Hayashi S, Takamaru H, Maruyama R, Kai M, Nishiwaki M, Sugai T, Sasaki Y, Tokino T, Shinomura Y, Imai K, Toyota M. Epigenetic alteration of DNA in mucosal wash fluid predicts invasiveness of colorectal tumors. Cancer Prev Res (Phila) 2011; 4: [PMID: DOI: / CAPR ] 187 Yamazaki N, Koga Y, Yamamoto S, Kakugawa Y, Otake Y, Hayashi R, Saito N, Matsumura Y. Application of the fecal microrna test to the residuum from the fecal occult blood test. Jpn J Clin Oncol 2013; 43: [PMID: ] 188 Cho WC. Epigenetic alteration of micrornas in feces of colorectal cancer and its clinical significance. Expert Rev Mol Diagn 2011; 11: [PMID: DOI: /erm.11.57] 189 Bovell L, Shanmugam C, Katkoori VR, Zhang B, Vogtmann E, Grizzle WE, Manne U. mirnas are stable in colorectal cancer archival tissue blocks. Front Biosci (Elite Ed) 2012; 4: [PMID: ] 190 Dassow H, Aigner A. MicroRNAs (mirnas) in colorectal cancer: from aberrant expression towards therapy. Curr Pharm Des 2013; 19: [PMID: ] 191 Suzuki H, Takatsuka S, Akashi H, Yamamoto E, Nojima M, Maruyama R, Kai M, Yamano HO, Sasaki Y, Tokino T, Shinomura Y, Imai K, Toyota M. Genome-wide profiling of chromatin signatures reveals epigenetic regulation of MicroRNA genes in colorectal cancer. Cancer Res 2011; 71: [PMID: DOI: / CAN ] P- Reviewers: Lakatos PL, Wig JD S- Editor: Gou SX L- Editor: Wang TQ E- Editor: Wu HL 4299 April 21, 2014 Volume 20 Issue 15

201 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. REVIEW Invasive and non-invasive diagnosis of cirrhosis and portal hypertension Moon Young Kim, Woo Kyoung Jeong, Soon Koo Baik Moon Young Kim, Soon Koo Baik, Department of Internal Medicine, Yonsei University Wonju College of Medcine, Wonju Severance Christan Hospital, Wonju , South Korea Woo Kyoung Jeong, Department of Radiology and Center for Imaging Science, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul , South Korea Author contributions: Baik SK manuscript drafting or manuscript revision for important intellectual conten; all authors contributed to the manuscript. Correspondence to: Soon Koo Baik, MD, PhD, Professor Department of Internal Medicine, Yonsei University Wonju College of Medicine, Wonju Severance Christan Hospital, 20, Ilsanro, Wonju , South Korea. baiksk@yonsei.ac.kr Telephone: Fax: Received: October 2, 2013 Revised: January 20, 2014 Accepted: February 17, 2014 Published online: April 21, 2014 Abstract With advances in the management and treatment of advanced liver disease, including the use of antiviral therapy, a simple, one stage description for advanced fibrotic liver disease has become inadequate. Although refining the diagnosis of cirrhosis to reflect disease heterogeneity is essential, current diagnostic tests have not kept pace with the progression of this new paradigm. Liver biopsy and hepatic venous pressure gradient measurement are the gold standards for the estimation of hepatic fibrosis and portal hypertension (PHT), respectively, and they have diagnostic and prognostic value. However, they are invasive and, as such, cannot be used repeatedly in clinical practice. The ideal noninvasive test should be safe, easy to perform, inexpensive, reproducible as well as to give numerical and accurate results in real time. It should be predictive of long term outcomes related with fibrosis and PHT to allow prognostic stratification. Recently, many types of noninvasive alternative tests have been developed and are under investigation. In particular, imaging and ultrasound based tests, such as transient elastography, have shown promising results. Although most of these noninvasive tests effectively identify severe fibrosis and PHT, the methods available for diagnosing moderate disease status are still insufficient, and further investigation is essential to predict outcomes and individualize therapy in this field Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Hepatic fibrosis; Portal hypertension; Liver biopsy; Hepatic venous pressure gradient; Non-invasive test; Transient elastography Core tip: Chronic liver disease is a heterogeneous and dynamic condition. So, noninvasive exact estimations of the status and changes in hepatic fibrosis and portal hypertension are essential in the management of this disease. Recently, a few tests, such as liver stiffness measurement based on transient elastography (TE) or magnetic resonance have shown promising results in this field. However, the reproducibility of these noninvasive tests needs to be validated in diverse clinical situations and etiologies. Especially, it is important to study about the long term prognostic value of non-invasive tests in hepatic fibrosis and portal hypertension that can leads to new paradigm in the tailored management of chronic liver disease. Notably, the integration of serologic tests with other non-invasive tests, such as TE, allows the progression in the management of patients with chronic liver disease in the future. Kim MY, Jeong WK, Baik SK. Invasive and non-invasive diagnosis of cirrhosis and portal hypertension. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: org/ /wjg.v20.i INTRODUCTION Regardless of its underlying etiology, fibrosis is the main 4300 April 21, 2014 Volume 20 Issue 15

202 Kim MY et al. Diagnosis of cirrhosis and portal hypertension component of chronic liver damage that directly relates to the severity and prognosis of the disease. Hepatic fibrosis and its secondary result, portal hypertension (PHT) are currently viewed as a dynamic process that can be reversible in some situation, if the underlying insult that has caused the fibrosis and cirrhosis has been removed [1]. Over time, the excess fibrous tissue of cirrhotic liver may also regress. Therefore, an accurate estimation of the severity of fibrosis and PHT is essential to evaluate the disease state and prognosis and is the first step towards the optimization of the treatment and estimation of its response so, the diagnosis of the heterogeneity of cirrhosis and PHT has become even more important for effective treatment [2,3]. Liver biopsy is the gold standard for assessing fibrosis. However, standard liver biopsy procedures have several limitations, including sampling errors, inter- and intraobserver variability, and invasiveness [4]. PHT, a frequently presenting clinical syndrome, is defined as a pathological increase in portal venous pressure between the portal vein and the inferior vena cava to higher than the normal range ( 5 mmhg). Increased portal pressure is the main factor determining the clinical course of cirrhosis. Presently, the favored method for determining portal venous pressure involves the catheterization of the hepatic vein and the measurement of the hepatic venous pressure gradient (HVPG). HVPG is one of the best surrogate markers in chronic liver disease; however, the measurement of the HVPG also has limitations, such as its invasiveness [5]. The ideal noninvasive test for diagnosing fibrosis and PHT should be simple and reproducible, readily available, less expensive than a biopsy, and able to predict the full spectrum of fibrosis and reflect any changes induced by therapy [6]. Moreover, recent advances in knowledge and treatment have led to proposals for more detailed histological diagnoses of fibrosis and these have made it even more difficult to find an ideal noninvasive substitute for liver biopsy [7,8]. In addition, it should be predictive of long-term outcomes related with fibrosis and PHT to allow prognostic stratification. Recently, many trials aimed at identifying such a test have been conducted, but no test has yet satisfied all of the aforementioned requirements. In this report, we review the role of the gold-standard methods for the estimation of hepatic fibrosis and PHT and also recently studied alternative, non-invasive methods with suggestion of future directions. DIRECT ESTIMATION OF HEPATIC FIBROSIS AND PHT Liver biopsy and histological analysis Cirrhosis is defined by anatomical changes within the liver parenchyma, including fibrosis and the development of regenerating nodules, and its assessment is based on histologic examination. A liver biopsy is considered the gold standard for the diagnosis of cirrhosis. In general, histologic scoring systems assess the grade and stage of chronic hepatitis. The grade is used as a measure of necro-inflammatory activity, and the stage is used as a measure of fibrosis and architectural changes. These scoring systems use scales of four (1-4), five (0-4), or seven (0-6) stages for chronic hepatitis [7,9-12]. For biopsies, an adequate specimen needs to have a length of at least 1.5 cm or the presence of six to eight portal tracts [4]. However, the width of the biopsy core is also important because the hepatic architecture is difficult to appreciate in thin biopsies [13], and biopsy specimens obtained from subcapsular locations generally contain more fibrous tissue, which should be considered. However, in clinical practice, liver biopsies have important limitations, such as invasiveness, potential for complications, sampling error, and inter- and intraobserver variability. Another important limitation is that a biopsy only represents approximately 1/50000 of the liver parenchyma [14]. Furthermore, the histologic diagnosis of cirrhosis maybe impossible in cases with large parenchymal nodules or so-called incomplete septal cirrhosis. However, despite these limitations, liver biopsy and histologic analysis have revolutionized the assessment of disease severity in chronic liver disease. Hepatic fibrosis is currently viewed as a dynamic process, in some cases, even the excess fibrous tissue of cirrhotic liver may regress over time. Distinguishing between the amount of hepatic fibrosis and the disease stage, which is related to both fibrosis and architectural changes, is important for the assessment of the effects of antifibrotic treatments. Nevertheless, a one-stage description of cirrhosis is inadequate, and subclassification has potential clinical utility [15,16]. From the aspect of histology, such subclassifications might allow better the prediction of prognosis and be helpful clinically. A few recent studies have shown the possibility of the clinical application of histological subclassification. In these studies, fibrosis scores, derived from nodule size and septal thickness, showed a significant correlation with the clinical stage [8] and prognosis [17] as well as the HVPG (Figure 1) [18,19]. Currently, the proportion of the liver biopsy specimen occupied by collagen (collagen proportion area, CPA) on Picro-Sirius stained histologic sections, as assessed by computer-assisted digital image analysis, has emerged as a useful continuous variable for the quantitative assessment of fibrosis in various clinical situations. The CPA measurement is based on the segmentation of digital images to measure the area occupied by collagen compared with the entire area of liver tissue [20,21]. The CPA has been reported to correlate with the HVPG in liver transplant recipients with hepatitis C virus infections, with or without cirrhosis, and the CPA measured at 1-year post-transplantation was found to be a predictor of clinical decompensation in later years [21]. This parameter has also been found to correlate with the liver stiffness (LS) measurements, as assessed by transient elastography (TE), in patients with chronic viral hepatitis [22,23]. Therefore, the quantitative assessment of hepatic fibrosis in liver biopsy 4301 April 21, 2014 Volume 20 Issue 15

203 Kim MY et al. Diagnosis of cirrhosis and portal hypertension A B n n n n C n n Figure 1 Heterogeneity of cirrhosis in histology. All pictures are histological finding of cirrhosis however, they show different feature in thickness of sepata and the size of nodules. A: Shows mild cirrhosis with thin septa (MTC stain, 100); B: Shows moderate cirrhosis with at least two broad septa; C: Shows severe cirrhosis with at least one very broad septa (MTC stain, respectively, 200). A, B, C are correspond to subclass of Laennec fibrosis scoring system F4A, F4B and F4C respectively, The widths between two arrows show the significant difference among subclass of cirrhosis. n: Regenerating nodule; MTC: Masson trichrome stain [8]. specimens is promising as a prognostic marker and as a means to validate other noninvasive markers of fibrosis. HVPG measurement for PHT The measurement of the HVPG is the gold standard technique for the evaluation of PHT in liver disease. The HVPG is the difference between the wedged hepatic venous pressure (WHVP) and the free hepatic venous pressure (FHVP). The WHVP is measured by occluding the hepatic vein as stopping the blood flow causes a static column of blood to be formed, which is equal in pressure to the hepatic sinusoids. FHVP is a measure of the pressure of the non-occluded hepatic vein. In cirrhosis, WHVP provides an accurate estimate of portal pressure, as has been demonstrated in both alcoholic and viral cirrhosis patients [5,24]. In patients with cirrhosis, HVPG measurements provide independent prognostic information on survival and the risk of decompensation. Clinically significant PHT (CSPH, HVPG 10 mmhg) is necessary for the formation of esophageal varices, its bleeding, and the development of decompensation [15,25-27]. In patients who have already developed decompensation, HVPG provides predictive information about the risk of mortality in the future [28]. HVPG can also reflect liver parenchymal function [29], and correlates with the degree of histological liver fibrosis [8,24,30]. In addition, in some studies on HCVrelated fibrosis and cirrhosis, HVPG showed a good correlation with the therapeutic response which suggested that the repeated HVPG measurements could be helpful in estimating the progression or regression of cirrhosis in patients with advanced hepatitis C virus (HCV)-related chronic liver disease [31,32]. HVPG also showed usefulness in identifying those patients who were at highest risk of decompensation by the recurrence of severe hepatitis C among those who had undergone post-liver transplantation for HCV-related cirrhosis [33-35]. CSPH is an independent predictor of the risk of developing hepatocellular carcinoma (HCC) [36], and it also increases the risk of liver failure and death after liver resection in patients with compensated chronic liver disease or hepatocellular carcinoma [37,38]. However, in case of non-cirrhotic portal fibrosis (NCPF) and extra-hepatic PV obstruction (EHPVO) which present only with features of PHT without any evidence of significant parenchymal dysfunction, HVPG is normal in EHPVO, whereas it is normal or slightly elevated in NCPF (median 7 mmhg). So, HVPG is not useful in the estimation of the severity of PHT and EV bleeding risk in EHPVO or NCPF [39]. The HVPG response to pharmacological therapy enables the identification of those patients with PHT who are most likely to benefit from treatment. The development of esophageal varices is less likely if the HVPG is < 10 mmhg. The development of variceal bleeding and the presence of ascites have been known to occur when the HVPG is 12 mmhg [40]. If the HVPG falls to April 21, 2014 Volume 20 Issue 15

204 Kim MY et al. Diagnosis of cirrhosis and portal hypertension mmhg with drug therapy, such as nonselective β blockers, the development of variceal bleeding can be prevented, and varices that are already present may decrease in size [41,42]. However, even if this target is not achieved, a decrease in the HVPG of 20% from baseline levels has shown similar effects to having an HVPG of < 12 mmhg [41]. In addition, this response markedly decreases the rebleeding risk in secondary prevention [42]. In addition, a good hemodynamic response was independently associated with a decreased risk of ascites and spontaneous bacterial peritonitis on follow-up and was an independent predictor of survival [43,44]. The evaluation of the acute HVPG response to intravenous propranolol therapy has been known to be useful for predicting the efficacy of nonselective β blockers at preventing the first bleed or rebleeding [45,46]. Furthermore, the acute HVPG response to propranolol is independently associated with survival in patients with cirrhosis and PHT [45]. However, although the HVPG measurement procedure is very well tolerated, its invasiveness and limited availability at hospitals has led to attempts to develop noninvasive alternatives, such as laboratory tests, imaging techniques, and LS measurements [47,48]. However, there has been no noninvasive alternative that can replace the HVPG measurement until now. NON-INVASIVE DIAGNOSIS OF HEPATIC FIBROSIS AND PHT Laboratory tests Because of the attractiveness, such as offering a sampling that reflects the whole liver, allowing repeated testing, reducing invasiveness, and increasing simplicity, many hematological and biochemical serum markers of fibrosis have been studied. In general, serum markers of fibrosis can be divided into two groups: indirect markers, which reflect the degree of fibrosis indirectly, and direct markers, which directly measure liver matrix components or enzymes that participate in matrix regulation [49]. Indirect markers in blood test include parameters related to cell lysis or inflammation (AST and ALT), cholestasis (γgt and bilirubin), hepatocyte synthetic function (INR, cholesterol, ApoA1, haptoglobin, and N-glycans), and hypersplenism due to PHT (platelet count) [49]. Many scores or indices of indirect parameters have been studied and the Fibrotest [α2-macroglobulin (α2m), ApoA1, bilirubin, γgt, and haptoglobin combination [50] and AST to platelet ratio index (APRI)] [51] have been the most widely validated. In some studies, these indices have been shown to be good predictors of fibrosis and cirrhosis (AUROC = 0.92, 0.80, respectively) [52,53], but in a metaanalysis of chronic hepatitis C [54], they lacked reliability in the discrimination of the fibrosis stage. Direct markers allow the quantitative assessment of the total amount of the hepatic extracellular matrix and its deposition or removal [51]. To improve accuracy, the use of a combination of markers is preferable to the use of a single marker [55], and various panels of direct markers have been described. The original European Liver Fibrosis panel is an example of such a panel of biomarkers shown to be accurate in diagnosing significant fibrosis in a large, mixed liver disease population [56]. This panel incorporates hyaluronic acid, tissue inhibitor of matrix metalloproteinases-1, procollagen type Ⅲ propeptide and age. The panel has since been simplified by removing age while maintaining diagnostic accuracy, as the enhanced liver fibrosis (ELF) test, which has been shown to be accurate in predicting significant liver fibrosis in independent populations [57-60]. ELF score predicts liver outcomes, with people having the highest ELF scores being significantly more likely to have clinical outcomes than those in lower-score groups with hazard ratios of 75 (ELF score ), 20 ( ) and 5 ( ) compared with patients with ELF < 8.34 [61]. In non-alcoholic fatty liver disease (NAFLD), one study reported favorable results of AUROC = 0.90, sensitivity 80%, and specificity 90% for advanced liver fibrosis, but this test needs to be further investigated [58]. A Cytokeratin-18 fragment in plasma indicates the apoptosis of liver cells and is the marker of NASH [62-64]. Plasma cytokeratin-18 fragment levels increased significantly in patients with steatohepatitis compare to normal or simple fatty liver patients. CK-18 fragment level showed a possibility as a screening test for NASH by showing favorable results (sensitivity 78%, specificity 87%, AUROC = 0.82) in meta-analysis [62]. However, this marker is currently unavailable in the clinical practice yet and the standard cut-off value for diagnosis also has to be established. NAFLD Fibrosis Score (NFS) is one of the most widely-studied biochemical panels for the diagnosis of fibrosis in NAFLD, and it is composed of 6 markers (age, BMI, diabetes/impaired glucose tolerance, platelet count, albumin level, AST/ALT ratio) which are clinically or biochemically measured and calculated easily in the website ( NFS has two cutoff values, [< (low probability) and > (high probability)] in evaluating liver fibrosis. According to a meta-analysis of 13 studies performed on 3064 patients, NFS showed a high AUROC value of 0.85 in diagnosing liver fibrosis greater than stage F3. By taking < as cut-off, sensitivity was 90% and specificity was 60% in excluding advanced liver fibrosis. By taking > as cut-off, sensitivity was 67% and specificity 97% in diagnosing advanced liver fibrosis. However, about 20%-58% of patients showed middle values between those two cutoff values (indeterminate probability), so in these case, liver biopsy has to be considered [62,65]. About the noninvasive estimation of PHT using laboratory test, just a few studies have been reported. Direct markers such as serum laminin levels, serum hyaluronic acid and procollagen type Ⅲ propeptide were evaluated in an old small population studies and laminin and hyaluronic acid showed correlation with HVPG, 4303 April 21, 2014 Volume 20 Issue 15

205 Kim MY et al. Diagnosis of cirrhosis and portal hypertension A B HVPG: 16 mmhg HVPG: 11 mmhg Figure 2 Doppler ultrasonography showing change in the hepatic vein waveform and damping index (A) before and (B) 3 mo after propranolol treatment in a patient with liver cirrhosis. Hepatic vein waveform changed from biphasic to near triphasic, quantitative measurement of damping index (DI) decreased from 0.61 to 0.33 in association with a hepatic venous pressure gradient (HVPG) decrease from 18 to 11 mmhg [92]. HVPG: Hepatic venous pressure gradient. however these markers has limitations in clinical application because of low predictive values for the presence of severe PHT and EVs [66-68]. In case of Fibrotest, it has a significant correlation with HVPG (Pearson correlation coefficient = 0.58), however, the correlation was weaker in cirrhosis patients (Pearson correlation coefficient = 0.24) and the AUROC for the diagnosis of severe PHT (HVPG > 12 mmhg) was only 0.79 [69]. Additional study is needed to estimate the clinical usefulness of Fibrotest for the diagnosis of PHT. The platelet count, and the ratio of the platelet count to spleen diameter have shown to be able to exclude the existence of EVs, however, in the other studies, the results were not constant [70-72]. In case of APRI score also showed low AUROC in recent study in the prediction of the presence of EVs (EVs 0.62, large EVs 0.71) [73,74]. In summary, noninvasive laboratory markers are still insufficient for evaluating the dynamic changes in fibrosis and especially in PHT, and wider validations in various clinical situations are needed (Table 1). DIAGNOSTIC IMAGING MODALITIES In chronic liver disease, the imaging diagnosis plays several significant roles in patient management, both in terms of diagnosing hepatocellular carcinoma and predicting its progression to cirrhosis. The basic diagnostic imaging modalities consist of ultrasound (US), computed tomography (CT), and magnetic resonance (MR) based methods, and many specific techniques derived from these basic methods are currently being developed to achieve convenient, non-invasive, and accurate diagnoses. US based approaches Gray scale and Doppler US are noninvasive, relatively simple, and inexpensive tests that are used to study and follow-up patients with chronic liver disease and cirrhosis. Various factors, including the liver size, bluntness of the liver edge, coarseness of the liver parenchyma, nodularity of the liver surface, portal vein (PV) velocity, and spleen size, have been suggested as useful parameters for the US-based evaluation of hepatic fibrosis or PHT in chronic liver disease [75-82]. However, although US can provide a qualitative assessment of the composition of the hepatic parenchyma, it is both subjective and operator dependent. In addition, some studies have shown that the sensitivity and specificity of US for hepatic fibrosis are unacceptably low and that there is no correlation between US findings and the histological stage of fibrosis on liver biopsy [83,84]. Regional hepatic and systemic hemodynamic changes are essential findings in liver fibrosis [85]. Therefore, Doppler US has been used to detect the hemodynamic changes that are known to be present from the pre-cirrhotic stages of hepatic fibrogenesis. Doppler US indices include the PV blood volume, mean or maximum PV velocity, portal blood flow, congestion index of the PV, effective portal liver perfusion, and resistance indices of arteries in the liver and spleen [77,85-90]. Furthermore, pulsed wave Doppler can be applied to determine the changes in the waveforms of the proper hepatic arteries, PV, and hepatic vein (HV). Although the normal flow pattern in the right HV is triphasic, patients with biphasic or monophasic flow patterns tend to have advanced fibrosis (Figure 2) [88,91-93]. In addition, HV waveform also showed promising data for the prediction of severe PHT (HVPG 12 mmhg). Especially, damping index (DI) that was calculated by the minimum velocity/maximum velocity of downward HV flow showed high accuracy with AUROC = for the prediction of severe PHT with sensitivity 75.9% and specificity 81.8% and positive and negative predictive values 91.1% and 58.1% respectively in the value of 0.6 of DI [93]. In addition, it showed a parallel change to the change of HVPG after nonselective β blocker [93], so more studies for the ability in estimation of portal hemodynamic change are needs. However, Doppler measurement is influenced by many patient-related factors, such as respiration and the timing of meals, as well as observer variability and equipment differences. Furthermore, collateral pathways, hepatic steatosis, and inflammation further contribute to the variability in the Doppler measurements [94-96] April 21, 2014 Volume 20 Issue 15

206 Kim MY et al. Diagnosis of cirrhosis and portal hypertension Table 1 Diagnostic performance of serum biomarkers of fibrosis for significant fibrosis (F 2) and cirrhosis (F4) in patients With hepatitis B or C Biomarkers Component Etiologies Patients (n ) F 2 (%) F4 (%) AUROC F2 AUROC F4 FibroTest [52] GGT, haptoglobin, bilirubin, HCV apolipoprotein A1, alpha-2-macroglobulin Forns Index [162] Age, GGT, cholesterol, platelets HCV APRI [53] AST, platelets HCV ELF [56] N-terminal propeptide of collagen type Ⅲ, Mixed 1021/ hyaluronic acid, TIMP-1, age Hepascore [163] Age, sex, alpha-2-macroglobulin, HCV hyaluronate, bilirubin, GGT Fibrometer [164] Platelets, prothrombin time, macroglobulin, Mixed 598/ AST, hyaluronate, age, urea FIB-4 [165] Age, ALT, AST, platelets HCV Fibroscan [110] HCV Fibroscan [115] HBV ARFI imaging [134] Mixed 146/ MRE [161] Mixed Number of HCV patients; 2 Number of patients with viral hepatitis. AUROC: Area under ROC curve; CC: Correctly classified: true positive and negative; HBV: Chronic hepatitis B; HCV: Chronic hepatitis C; NA: Not available; Se: Sensitivity; Sp: Specificity. Taken together, gray scale and Doppler US are safe, inexpensive and simple to use at the bedside or for outpatients, and combining multiple US indices can improve the diagnostic accuracy of cirrhosis under some conditions. Contrast-enhanced ultrasound (CEUS) imaging represents a new US modality for the assessment of chronic liver disease. CEUS involves the intravenous administration of minute, gas-filled microbubbles that strongly enhance the intensity of signals from the intravascular flow. The various commercially available contrast agents differ in their designs and kinetics, and can therefore yield different results [97]. Bolus injections of microbubble agents can be used for first-pass kinetics studies and to assess transit times. Hepatic vein transit times (HVTT) have been shown to be reduced with worsening liver disease [98-102]. In theory, shorter hepatic vein transit times in patients with chronic liver disease mainly present secondary to arteriovenous shunting, sinusoid remodeling and arterialization of the capillary beds in the liver and, to a lesser degree, shunting in the pulmonary and gastrointestinal capillaries. In our study for compensated cirrhosis, HVTT showed a significantly strong correlation with PHT and the AUROC of HVTT for the diagnosis of CSPH was In addition, a shorter HVTT was associated with worse Child-Pugh score (P < 0.001) and esophageal varices (P = 0.018) (Figure 3) [102]. In addition, the time intensity curve differs significantly between the normal liver parenchyma and the cirrhotic liver, and the level of decrease is related to the degree of liver damage present or PHT [102,103]. Berzigotti et al [104] reported that the regional hepatic perfusion (RHP, which was calculated as microbubbles velocity X microbubble) increased in patients with cirrhosis and correlated with the degree of liver failure. RHP increased along with liver functional reserve decrease which was measured by indocyanin green clearance and they suggested CEUS as a feasible novel, objective, quantitative, non-invasive tool for the estimation of RHP. Especially in this study, RHP showed slight positive correlation with HVPG (R = 0.279, P = 0.041) and a tendency to decrease after intravenous propranolol administration (P = 0.08). In other study [104], the signal intensity of a region of interest within the liver parenchyma have also revealed that the accumulation of microbubbles in the liver parenchyma is decreased in nonalcoholic steatohepatitis, but not in nonalcoholic fatty liver disease or chronic viral hepatitis. CEUS-based tests for hepatic fibrosis and PHT could be simple and noninvasive tests based on contrast agent transit or the parenchymal enhancement pattern for reliably excluding cirrhosis. In particular, CEUS-based tests could be useful in the assessment of intrahepatic or systemic hemodynamic changes which are essential in advanced chronic liver disease. However, this method also has some limitations, such as the requirements for the injection of a contrast agent, considerable operator skill, and access to the relevant technology. More intensive studies and validation are needed US based ls measurement: TE, acoustic radiation force impulse, supersonic shear-wave elastography, and realtime elastography The increase in LS associated with chronic liver disease is primarily due to the presence of fibrosis [105]. Tissue elastography, which was introduced in 1992, is used to visualize differences in the mechanical properties among tissues [106]. The most attractive advantage of tissue elastography is its ability to quantify the viscoelasticity of the tissue, which means that it can be used to measure hepatic fibrosis in the liver. To date, two US elastography techniques have been used to measure LS: shear-wave based elastography and real-time elastography. Shearwave based elastography includes TE (Fibroscan ), which is the most widely evaluated and used, acoustic radiation force impulse (ARFI) imaging, and supersonic shearwave elastography (SSWE). Shear-wave based elastog April 21, 2014 Volume 20 Issue 15

207 Kim MY et al. Diagnosis of cirrhosis and portal hypertension A B Hepatic vein C D 10 s injection 10 s injection 21.0 s 37.0 s 10% 10% E 25.0 r 2 = P < HVAT (s) HVPG (mmhg) Figure 3 Hepatic vein enhancement with microbubble contrast-enhanced ultrasound and measurement of Hepatic vein arrival time and the correlation between Hepatic Venous Pressure Gradient and Hepatic vein arrival time. A: Ultrasound images showing the HV (white arrows) before the contrast injection; B: Arrival of microbubble contrast agent in the HV after contrast enhancement; HVAT was calculated as the time (in seconds) from injection to a sustained increase in signal in the TIC to more than 10% above baseline; C: Recorded TIC profile shows early HVAT (11.0 s; the 10-s lead time was subtracted from 21.0 s) in a patient with cirrhosis with HVPG of 20 mmhg; D: Recorded TIC profile shows an HVAT of 27.0 s (37.0 min 10 s) in a healthy control; E: HVAT was significantly linearly correlated with HVPG in the patients with compensated cirrhosis (r 2 = 0.545; P < 0.001). HV: Hepatic vein; CEUS: Contrast-enhanced ultrasonography; HVAT: Hepatic vein arrival time; HVPG: Hepatic venous pressure gradient; TIC: Time intensity curve [102]. raphy involves using an ultrasonic beam to measure the propagation velocity of a shear wave through the soft tissue under investigation, and LS is displayed in kilopascals (kpa) or centimeters per second (cm/s). TE is the first commercialized elastography protocol that was developed to noninvasively assess the stiffness of deep soft tissues such as the liver. A mechanical vibrator generates a low-frequency elastic wave at 50 Hz to produce a shear stress in the target tissue at a distance of 4 cm, after which, as mentioned above, the velocity of the shear wave is measured using an ultrasound signal. TE has been validated extensively by numerous investigations targeted at patients with chronic liver disease and cirrhosis, and TE findings are generally accepted to be strongly correlated with the stage of liver fibrosis. However, it does not provide a B-mode image which is very helpful for targeting, and it has a high measurement failure rate (15%-20%), mainly due to limiting factors such as obesity and ascites [107]. ARFI imaging and SSWE use focused high-intensity, short-duration acoustic pulses instead of the mechanical vibration used in TE to produce a shear wave in the target tissue [108]. Similar to TE, the shear-wave velocity is observed by aiming repeated ultrasound beam pulses across the region of interest (ROI). SSWE is a new type of the shear-wave based US elastography technique and uses multiple acoustic 4306 April 21, 2014 Volume 20 Issue 15

208 Kim MY et al. Diagnosis of cirrhosis and portal hypertension radiation force impulses [109]. It can assess the viscoelastic properties in all areas within the ROI, and the results are displayed on a color-coded lookup table. ARFI imaging and SSWE are expected to overcome the limitations of TE, such as measurement failures due to severe obesity, thick subcutaneous fat, and ascites. Moreover, they are able to display a gray scale US image on the background of the elastography and are thus more reliable and familiar to physicians who use conventional US. However, a high level of clinical experience and evidence are needed to apply either ARFI imaging or SSWE for the diagnosis of liver fibrosis. Most clinical studies about TE have focused on its ability to identify significant fibrosis and cirrhosis, because of an indication for antiviral treatment and initiating surveillance program for the early detection of hepatocellular carcinoma (HCC) development. A number of studies have demonstrated that TE values are significantly correlated with histological fibrosis stage and have high diagnostic accuracy. In chronic hepatitis C (CHC), the AUROC of TE ranged from 0.77 to 0.90, with a cutoff value of kpa for assessment of significant fibrosis (F 2), and an AUROC of and cutoff value of kpa for assessment of cirrhosis [22, ]. For chronic hepatitis B (CHB), AUROC and cutoff value of TE for predicting significant fibrosis and cirrhosis are , kpa and kpa, kpa respectively [ ]. In a recent meta-analysis, TE also showed high sensitivity and specificity in hepatic fibrosis assessment of NAFLD [ ]. However, in case of obesity [(body mass index (BMI) 30 kg/m 2 ] which is commonly associated in patients with NAFLD, the accuracy of TE decreases, and test may not be performable in some cases (5%-13%) [62]. Recently introduced CAP is known to relatively accurately evaluate the degree of fatty infiltration, and a large number of clinical studies are anticipated to report meaningful results [ ]. TE also reflects a progressive rise in PHT due to increased hepatic vascular resistance related with hepatic fibrosis. TE has a good performance in discriminating between patients with and without CSPH (AUROC = ) [127]. In addition, TE value < 13 kpa exclude reliably CSPH, while values > 21 kpa had an accuracy equal to that of HVPG 10 mmhg for the prediction of first clinical decompensation in patients with compensated cirrhosis [128]. As variceal bleeding is the most important complication of PHT, the relationship between TE values and the presence of EVs has been also investigated in several studies [73,129,130], and these studies showed that there is a significant correlations between TE values and the presence of EVs. However, TE still has limitations in clinical application because of the cutoff values (range, kpa) and performance of TE variability among the studies (AUROC: ) [73,129,130]. Recently a few studies have shown that the combination of TE, platelet count and spleen size by ultrasound have superior diagnostic values in the identification of the presence of CSPH and esophageal varices than single methods in patients with compensated cirrhosis of different etiologies [127]. Kim et al [131] recently proposed a novel prediction model, LS-spleen diameter to platelet ratio score (LSPS), using TE values and spleen diameter to platelet ratio that reflect PHT in patients with CHB. This model showed excellent diagnostic accuracy for prediction of high risk esophageal varices (AUROC = 0.953; negative predictive value 94.7%, positive predictive value 93.3%). Another prospective study showed that LSPS can be a reliable predictor of the development of variceal bleeding. In this prospective, CHB patients with LSPS 5.5 had higher cumulative incidence rates of esophageal variceal bleeding during the follow-up period and LSPS score 6.5 was an independent risk factor of variceal bleeding from high risk esophageal varices, indicating that prophylactic treatment should be considered in these high risk patients [132]. A recent validation study for LSPS showed that more than 80% of patients were accurately classified using LSPS. Additionally, in this study, a new modified LSPS, varices risk score were suggested and it was superior to all other noninvasive tests for identifying patients with EVs (AUROC = 0.909) with 85% correct classification of patients [133]. ARFI imaging has similar accuracy with TE or ELF for significant fibrosis with AUROC = (0.861 and 0.764, for TE and ELF, respectively) and cirrhosis (0.936, 0.918, and 0.841) and the combination of ELF with ARFI imaging or TE increased the negative and positive predictive values of single tests for the diagnosis of significant fibrosis and cirrhosis [134]. A recent meta-analysis included 13 studies in which 1163 patients with chronic liver diseases showed similar sensitivity and specificity between ARFI imaging and TE for detection of significant fibrosis ( F2) (Se and Sp were 0.74 and 0.83 for ARFI imaging, 0.78 and 0.84 for TE) and cirrhosis (0.87 and 0.87 for ARFI imaging, 0.89 and 0.87 for TE) [135]. For the relationship with PHT, just a little data can be found and ARFI imaging also showed a good correlation with HVPG (R = 0.709) and AUROC = for predicting CSPH [136]. However, in the prediction of EVs, ARFI imaging just has shown low AUROC = 0.58 and more data is needed for establishment of its clinical significance [137]. In case of SSWE, one study evaluated 133 patients with chronic hepatitis C by means of SSWE, TE and, in a subgroup of patients, with liver biopsy. The AUROCs of SSEW were for F2, for F3 and for F4 cirrhosis [138]. Because just a little data has been reported, more data is needed for establishment of its clinical usefulness. Another new trial in this field is to measurement of spleen stiffness (SS). As well known, spleen congestion is a specific feature of PHT; SS using TE or ARFI imaging has been recently applied to diagnose of CSPH and esophageal varices, and had a higher accuracy than other noninvasive parameters such as LSPS or platelet/spleen ratio and MR elastography (MRE) [132,133,139]. In a recent study, SS using TE showed AUROC = 0.941, and for the prediction of EV, CSPH and severe PHT 4307 April 21, 2014 Volume 20 Issue 15

209 Kim MY et al. Diagnosis of cirrhosis and portal hypertension (HVPG 12 mmhg) respectively and these were similar with LS using TE [139]. In case of SS using ARFI, SS showed better accuracy in the prediction of EV than LS (AUROC = vs 0.746) [140]. In addition, in a study for 65 patients with EHPVO, LS and SS were higher in patients with EHPVO (6.7 ± 2.3 kpa and 51.7 ± 21.5 kpa, respectively) than in control subjects (4.6 ± 0.7 kpa and 16.0 ± 3.0 kpa, respectively) and patients with a history of a bleed had a higher SS than did those without a bleed(60.4 ± 5.4 kpa vs 30.3 ± 14.2 kpa, P = 0.01). So, SS showed a possibility as a predictor for variceal hemorrhage in this disease population [141]. However, measurement of SS with TE could have substantial limitations in real performance and reproducibility, more validation has to be followed. Because advanced liver fibrosis and cirrhosis are the most important risk factors of HCC development, several studies have evaluated the clinical role of TE for predicting HCC development [142,143]. In CHC patients study, patients with higher TE values had a significantly higher risk of HCC development according to the increase of TE values (hazard ratio of 16.7 with kpa, 20.9 with kpa, 25.6 with kpa, 45.5 with over 25 kpa) as compared to under 10 kpa [142]. Similarly, in populations with CHB, TE value was also identified as an independent risk factor for HCC development, with relative risks of 3.07, 4.68, 5.55, and 6.60 for respective TE values of 8-13 kpa, kpa, kpa, and > 23 kpa compared TE value to less than 8 kpa [143]. In summary, the development of various promising techniques for measuring LS have helped overcome the limitations not only for evaluating hepatic fibrosis or CSPH but also for predicting the development of fatal complications related to chronic liver disease and the patient s prognosis. Therefore, they can be considered a useful tool to reduce the need of HVPG measurement and endoscopy, and to improve the selection of patients requiring further risk stratification. However, despite the above mentioned advances in non-invasive assessment of PHT, HVPG measurement cannot be fully replaced. In addition, they cannot accurately predict the hemodynamic response to non-selective β blocker [144]. Further investigations in this field have to be followed. CT- and MRI-based approaches Cross-sectional imaging studies such as CT and MRI are useful imaging modalities for the diagnosis of cirrhosis. These modalities are considered to be standard methods for the diagnosis of HCC on the background of chronic liver disease, including cirrhosis [139,140,145]. The radiologic features of advanced cirrhosis are normally obvious and include surface nodularity, prominent fibrous septa, shrinkage of liver volume, and an enlarged portal venous system including varices and splenomegaly due to PHT. However, it is difficult to diagnose the early stage of cirrhosis. As such, various functional techniques using CT and MRI have been developed recently and described in many hepatology and radiology journals. Morphologic changes: In the advanced stages of cirrhosis, the morphologic changes in the liver can be clearly demonstrated by both CT and MRI. Although the imaging features are highly specific for cirrhosis, the sensitivity is very low for early cirrhosis. As cross-sectional imaging can be used for the diagnosis of cirrhosis, several imaging signs related to morphologic changes of the liver have been investigated. A modified caudate: right-lobe ratio is one of these morphologic CT signs [146,147], and is defined by using the bifurcation of the right PV as a landmark, which is considered to be a lateral boundary for the caudate lobe and a medial boundary for the right lobe. According to that study, the sensitivity and accuracy for diagnosing cirrhosis was approximately 72% and 74%, respectively, when using a modified caudate: right-lobe ratio cut-off of greater than The use of these signs produces a very high specificity and positive predictive value for diagnosing cirrhosis, but usually a low sensitivity. In other words, the morphologic changes associated with cirrhosis that is visible on CT and MRI is usually only observed in advanced cases. As such, CT and MRI are not suitable for the early diagnosis of cirrhosis. Hemodynamic changes: Hemodynamically, the cirrhotic liver is highly resistant to portal inflow, and the systemic response that increase the splanchnic circulation aggravates the situation, creating a vicious cycle that may result in the development of PHT, hyperdynamic circulation and its complications [148]. Because conventional CT and MRI are not functional imaging modalities, they are not appropriate for evaluating the hemodynamic changes in the liver. However, they are useful for observing the secondary changes of PHT in a cirrhotic patient, such as portosystemic collateral formation, splenomegaly, portal hypertensive gastropathy, portal hypertensive colopathy, and ascites. Esophageal and paraesophageal varices are common complications of cirrhosis, and arise from the impaired venous drainage of the esophageal vein due to increased portal venous pressure. A gastric varix is less common than an esophageal varix and occurs in approximately 20% of patients with PHT [149]. In general, the estimation of the risk of variceal bleeding is made by using endoscopic findings such as the size, color, and location of varices [150]. However, in the era of multidetector CT, which enables CT scanning at a submillimeter thickness, CT can be used to obtain information not only about the cirrhotic liver itself but also about the PHT caused by cirrhosis. Various portosystemic collateral veins can also be depicted in the CT scan, and physicians can plan a strategy for the treatment of varices, including the insertion of a transjugular intrahepatic portosystemic shunt and balloon-occluded retrograde transvenous obliteration. Moreover, as with endoscopy, demonstrating the presence of esophageal and gastric varices is now possible using CT [151]. The sensitivity and specificity of CT were found to be 96% and 55%, respectively, to detect esophageal varices and 93% and 80%, respectively 4308 April 21, 2014 Volume 20 Issue 15

210 Kim MY et al. Diagnosis of cirrhosis and portal hypertension to detect high-risk esophageal varices [152]. Using the 1- to 3-mm multiplanar reformat or surface-shaded display can also increase the specificity of CT for the risk stratification of esophageal varices 101. With regard to gastric varices, the sensitivity and specificity were 83%-89% and 75%-79%, respectively [153,154]. Although these results are not bad, the accuracy for small varices remains low. MRE: MRE is an emerging technology that is used to quantitatively assess the elasticity of the liver [86]. This modality can improve the contrast between different tissues in the body compared with other imaging modalities, such as ultrasound, CT, and conventional MRI. MRE has been used in clinical practice for the assessment of hepatic fibrosis since 2006 [155,156]. Several clinical studies have established the feasibility of MRE for the evaluation of hepatic fibrosis [ ], and in a recent meta-analysis, the overall sensitivity, specificity, and AUROC of MRE for histological grade F2 were 0.94, 0.95, and 0.98 and for F4 were 0.99, 0.94, and 0.99 respectively [161]. There are several advantages of MRE over US based elastography [158,166], such as not being influenced by body habitus, not being operator dependent, and its potential ability to assess the entire liver. However, this technique is expensive and cannot yet be regularly used in all medical institutions and further research is required to validate liver MRE against long-term clinical outcomes. CONCLUSION Chronic liver disease is a heterogeneous and dynamic condition. The exact estimations of the stage and the changes in hepatic fibrosis and PHT are essential in the management of patients with this disease. Until now, no non-invasive diagnostic tests have satisfied such clinical needs. 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A liver stiffness measurementbased, noninvasive prediction model for high-risk esophageal varices in B-viral liver cirrhosis. Am J Gastroenterol 2010; 105: [PMID: DOI: /ajg ] 132 Kim BK, Kim do Y, Han KH, Park JY, Kim JK, Paik YH, Lee KS, Chon CY, Ahn SH. Risk assessment of esophageal variceal bleeding in B-viral liver cirrhosis by a liver stiffness measurement-based model. Am J Gastroenterol 2011; 106: , 1730 [PMID: DOI: /ajg ] 133 Berzigotti A, Seijo S, Arena U, Abraldes JG, Vizzutti F, García- Pagán JC, Pinzani M, Bosch J. Elastography, spleen size, and platelet count identify portal hypertension in patients with compensated cirrhosis. Gastroenterology 2013; 144: e April 21, 2014 Volume 20 Issue 15

215 Kim MY et al. Diagnosis of cirrhosis and portal hypertension [PMID: DOI: /j.gastro ] 134 Crespo G, Fernández-Varo G, Mariño Z, Casals G, Miquel R, Martínez SM, Gilabert R, Forns X, Jiménez W, Navasa M. ARFI, FibroScan, ELF, and their combinations in the assessment of liver fibrosis: a prospective study. J Hepatol 2012; 57: [PMID: DOI: /j.jhep ] 135 Bota S, Herkner H, Sporea I, Salzl P, Sirli R, Neghina AM, Peck-Radosavljevic M. Meta-analysis: ARFI elastography versus transient elastography for the evaluation of liver fibrosis. Liver Int 2013; 33: [PMID: DOI: /liv.12240] 136 Billiau A. The mode of action of interferons in viral infections and their possible role in the control of hepatitis B. J Hepatol 1986; 3 Suppl 2: S171-S179 [PMID: ] 137 Vermehren J, Polta A, Zimmermann O, Herrmann E, Poynard T, Hofmann WP, Bojunga J, Sarrazin C, Zeuzem S, Friedrich-Rust M. 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Cirrhosis: modified caudate-right lobe ratio. Radiology 2002; 224: [PMID: ] 149 Ahn JH, Yu JS, Hwang SH, Chung JJ, Kim JH, Kim KW. Nontumorous arterioportal shunts in the liver: CT and MRI findings considering mechanisms and fate. Eur Radiol 2010; 20: [PMID: DOI: /s z] 150 Sarin SK, Lahoti D, Saxena SP, Murthy NS, Makwana UK. Prevalence, classification and natural history of gastric varices: a long-term follow-up study in 568 portal hypertension patients. Hepatology 1992; 16: [PMID: ] 151 Dell'era A, Bosch J. Review article: the relevance of portal pressure and other risk factors in acute gastro-oesophageal variceal bleeding. Aliment Pharmacol Ther 2004; 20 Suppl 3: 8-15; discussion [PMID: ] 152 Kim H, Choi D, Gwak GY, Lee JH, Park MK, Lee HIe, Kim SH, Nam S, Yoo EY, Do YS. Evaluation of esophageal varices on liver computed tomography: receiver operating characteristic analyses of the performance of radiologists and endoscopists. 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Clin Gastroenterol Hepatol 2007; 5: e2 [PMID: ] 157 Huwart L, Sempoux C, Salameh N, Jamart J, Annet L, Sinkus R, Peeters F, ter Beek LC, Horsmans Y, Van Beers BE. Liver fibrosis: noninvasive assessment with MR elastography versus aspartate aminotransferase-to-platelet ratio index. Radiology 2007; 245: [PMID: ] 158 Huwart L, Sempoux C, Vicaut E, Salameh N, Annet L, Danse E, Peeters F, ter Beek LC, Rahier J, Sinkus R, Horsmans Y, Van Beers BE. Magnetic resonance elastography for the noninvasive staging of liver fibrosis. Gastroenterology 2008; 135: [PMID: DOI: /j.gastro ] 159 Asbach P, Klatt D, Schlosser B, Biermer M, Muche M, Rieger A, Loddenkemper C, Somasundaram R, Berg T, Hamm B, Braun J, Sack I. Viscoelasticity-based staging of hepatic fibrosis with multifrequency MR elastography. Radiology 2010; 257: [PMID: DOI: /radiol ] 160 Wang Y, Ganger DR, Levitsky J, Sternick LA, McCarthy RJ, Chen ZE, Fasanati CW, Bolster B, Shah S, Zuehlsdorff S, Omary RA, Ehman RL, Miller FH. Assessment of chronic hepatitis and fibrosis: comparison of MR elastography and diffusion-weighted imaging. AJR Am J Roentgenol 2011; 196: [PMID: DOI: /AJR ] 161 Wang QB, Zhu H, Liu HL, Zhang B. Performance of magnetic resonance elastography and diffusion-weighted imaging for the staging of hepatic fibrosis: A meta-analysis. Hepatology 2012; 56: [PMID: DOI: / hep.25610] 4314 April 21, 2014 Volume 20 Issue 15

216 Kim MY et al. Diagnosis of cirrhosis and portal hypertension 162 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: ] 163 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: ] 164 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: ] 165 Sterling RK, Lissen E, Clumeck N, Sola R, Correa MC, Montaner J, S Sulkowski M, Torriani FJ, Dieterich DT, Thomas DL, Messinger D, Nelson M. Development of a simple noninvasive index to predict significant fibrosis in patients with HIV/HCV coinfection. Hepatology 2006; 43: [PMID: ] 166 Hong WK, Kim MY, Baik SK, Shin SY, Kim JM, Kang YS, Lim YL, Kim YJ, Cho YZ, Hwang HW, Lee JH, Chae MH, Kim HA, Kang HW, Kwon SO. The usefulness of non-invasive liver stiffness measurements in predicting clinically significant portal hypertension in cirrhotic patients: Korean data. Clin Mol Hepatol 2013; 19: [PMID: DOI: /cmh ] 167 Moon KM, Kim G, Baik SK, Choi E, Kim MY, Kim HA, Cho MY, Shin SY, Kim JM, Park HJ, Kwon SO, Eom YW. Ultrasonographic scoring system score versus liver stiffness measurement in prediction of cirrhosis. Clin Mol Hepatol 2013; 19: [PMID: DOI: /cmh ] P- Reviewers: Berzigotti A, Procopet B, Sharma V S- Editor: Qi Y L- Editor: A E- Editor: Ma S 4315 April 21, 2014 Volume 20 Issue 15

217 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. ORIGINAL BRIEF ARTICLE Non-transmissible Sendai virus vector encoding c-myc suppressor FBP-interacting repressor for cancer therapy Kazuyuki Matsushita, Hideaki Shimada, Yasuji Ueda, Makoto Inoue, Mamoru Hasegawa, Takeshi Tomonaga, Hisahiro Matsubara, Fumio Nomura Kazuyuki Matsushita, Fumio Nomura, Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Chiba , Japan Hideaki Shimada, Department of Surgery, School of Medicine, Toho University, Tokyo , Japan Yasuji Ueda, Makoto Inoue, Mamoru Hasegawa, DNAVEC Corporation, 6 Ohkubo, Tsukuba City, Ibaraki , Japan Takeshi Tomonaga, Proteome Research Center, Proteome Research Project, National Institute of Biomedical Innovation, Osaka , Japan Hisahiro Matsubara, Department of Frontier Surgery, Chiba University, Graduate School of Medicine, Chiba , Japan Author contributions: Matsushita K designed the research; Matsushita K, Ueda Y and Inoue M performed the research; Shimada H, Tomonaga T, Hasegawa M, Matsubara H and Nomura F contributed new reagents/analytic tools and scientific discussions; Matsushita K analyzed the data and wrote the paper. Supported by in part by the 21 st Century COE (Center Of Excellence) Programs to Dr. Takenori Ochiai and by a Grant-in- Aid to K.M from the Ministry of Education, Science, Sports and Culture of Japan Correspondence to: Kazuyuki Matsushita, MD, PhD, Associate Professor, Department of Molecular Diagnosis, Graduate School of Medicine, Chiba University, Inohana, Chuo-Ku, Chiba , Japan. kmatsu@faculty.chiba-u.jp Telephone: Fax: Received: October 22, 2013 Revised: December 14, 2013 Accepted: January 19, 2014 Published online: April 21, 2014 Abstract AIM: To investigate a novel therapeutic strategy to target and suppress c-myc in human cancers using far up stream element (FUSE)-binding protein-interacting repressor (FIR). METHODS: Endogenous c-myc suppression and apoptosis induction by a transient FIR-expressing vector was examined in vivo via a HA-tagged FIR (HA-FIR) expression vector. A fusion gene-deficient, non-transmissible, Sendai virus (SeV) vector encoding FIR cdna, SeV/dF/FIR, was prepared. SeV/dF/FIR was examined for its gene transduction efficiency, viral dose dependency of antitumor effect and apoptosis induction in HeLa (cervical squamous cell carcinoma) cells and SW480 (colon adenocarcinoma) cells. Antitumor efficacy in a mouse xenograft model was also examined. The molecular mechanism of the anti-tumor effect and c-myc suppression by SeV/dF/FIR was examined using Spliceostatin A (SSA), a SAP155 inhibitor, or SAP155 sirna which induce c-myc by increasing FIR exon2 in HeLa cells. RESULTS: FIR was found to repress c-myc transcription and in turn the overexpression of FIR drove apoptosis through c-myc suppression. Thus, FIR expressing vectors are potentially applicable for cancer therapy. FIR is alternatively spliced by SAP155 in cancer cells lacking the transcriptional repression domain within exon 2 (FIR exon2), counteracting FIR for c-myc protein expression. Furthermore, FIR forms a complex with SAP155 and inhibits mutual well-established functions. Thus, both the valuable effects and side effects of exogenous FIR stimuli should be tested for future clinical application. SeV/dF/FIR, a cytoplasmic RNA virus, was successfully prepared and showed highly efficient gene transduction in in vivo experiments. Furthermore, in nude mouse tumor xenograft models, SeV/dF/FIR displayed high antitumor efficiency against human cancer cells. SeV/dF/FIR suppressed SSA-activated c-myc. SAP155 sirna, potentially produces FIR exon2, and led to c-myc overexpression with phosphorylation at Ser62. HA-FIR suppressed endogenous c-myc expression and induced apoptosis in HeLa and SW480 cells. A c-myc transcriptional suppressor FIR expressing SeV/dF/FIR showed high gene transduction efficiency with significant antitumor effects and apoptosis induction in HeLa and SW480 cells. CONCLUSION: SeV/dF/FIR showed strong tumor growth 4316 April 21, 2014 Volume 20 Issue 21

218 Matsushita K et al. FIR-encoding Sendai virus vector for cancer gene therapy suppression with no significant side effects in an animal xenograft model, thus SeV/dF/FIR is potentially applicable for future clinical cancer treatment Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Cancer gene therapy; c-myc suppressor; Far up stream element-binding protein-interacting repressor; Sendai virus vector Core tip: The authors performed in vivo experiments and included an animal model to examine the Sendai virus/df/far Up Stream Element-Binding Protein-Interacting Repressor for cancer gene therapy to minimize side effects for clinical use. Matsushita K, Shimada H, Ueda Y, Inoue M, Hasegawa M, Tomonaga T, Matsubara H, Nomura F. Non-transmissible Sendai virus vector encoding c-myc suppressor FBP-interacting repressor for cancer therapy. World J Gastroenterol 2014; 20(15): Available from: URL: com/ /full/v20/i15/4316.htm DOI: org/ /wjg.v20.i INTRODUCTION c-myc plays an essential role in cell proliferation and tumorigenesis. c-myc activation was also shown to be required for skin epidermal and pancreatic beta-cell tumor maintenance in c-myc-er TAM transgenic mice [1]. High c-myc expression level in colorectal cancer tissues was associated with poor long-term survival of colorectal cancer patients [2]. The far up stream element (FUSE) is a sequence required for correct expression of the human c-myc gene [3]. The FUSE is located at 1.5 kb upstream of c-myc promoter P1, and binds the FUSE binding protein (FBP), a transcription factor which stimulates c-myc expression in a FUSE-dependent manner [4]. Yeast twohybrid analysis revealed that FBP binds to a protein that has transcriptional inhibitory activity termed the FBP interacting repressor (FIR). FIR interacts with the central DNA binding domain of FBP [5]. Recently, FIR was found to engage the TFIIH/p89/XPB helicase and repress c-myc transcription by delaying promoter escape [5,6]. Furthermore, exogenous FIR expression represses endogenous c-myc transcription, and drives apoptosis due to the decrease in c-myc [7]. Although these observations indicate that cancer therapies targeting c-myc suppression by FIR may be a useful strategy, the mechanism of the antitumor effect of FIR should be determined in detail prior to clinical testing. For example, first, FIR is alternatively spliced in colorectal cancer lacking the transcriptional repression domain within exon 2 (FIR exon2) [7]. Second, FIR and FIR exon2 form a homo- or hetero-dimer, which complexes with SAP155, a subunit of the essential splicing factor 3b (SF3b) subcomplex in the spliceosome, and is required for correct P27Kip1 (P27) pre-mrna splicing, after which P27 arrests cells in G1 [8]. Third, SAP155 is required for correct FIR pre-mrna splicing and thus the FIR/FIR exon2/sap155 interaction bridged c-myc and p27 expression [9]. Accordingly, SAP155-mediated alternative splicing of FIR serves as a molecular switch for c-myc expression [9]. Finally, spliceostatin A (SSA), a natural SF3b inhibitor, markedly inhibited P27 expression by disrupting its pre-mrna splicing and reducing cdk2/cycline expression [10]. Taken together, these findings suggest that exogenous FIR stimuli potentially affect the FIR/FIR exon2/sap155 interaction which is pivotal for the cell cycle, cancer development and differentiation. In this study, a fusion gene-deficient human FIR-expressing Sendai virus vector (SeV/dF/FIR) was prepared for future cancer therapy for the following reasons; Sendai virus (SeV), a member of the Paramyxoviridae family, has envelopes and a nonsegmented negative-strand RNA genome. The SeV genome contains six major genes in tandem on a single negative-strand RNA. Three proteins, the nucleoprotein (NP), phosphoprotein (P) and large protein (L; the catalytic subunit of the polymerase) form a ribonucleoprotein complex (RNP) with the SeV RNA. Matrix proteins (M) contribute to the assembly of viral particles, hemagglutinin-neuraminidase (HN) and fusion proteins (F) engage in the attachment of viral particles and infiltration of RNPs into infected cells. Importantly, SeV does not transform cells by integrating its genome into the cellular genome [11]. Therefore, SeV can mediate gene transfer and expression to a cytoplasmic location using cellular tubulin [12], thereby avoiding possible malignant transformation due to the genetic alteration of host cells. These are the safety advantages of SeV. Recently, a novel SeV vector was established where an enhanced green fluorescent protein (EGFP) reporter gene was inserted at the 3 -end of fusion gene-deficient SeV genomic RNA (SeV/dF/EGFP) [13]. This SeV/dF/EGFP is incapable of self-replication, but capable of infecting various cells, including human smooth muscle cells, hepatocytes, and endothelial cells, thus the SeV/dF/EGFP has a broad spectrum of gene transfer activity [9,10]. In this study, SeV/dF/FIR was prepared following the method for SeV/dF/EGFP [12,13]. The validity of SeV/dF/FIR for cancer therapy was examined in animal xenograft models as SeV/dF vectors have been shown to be applicable for clinical use [14-18]. The clinical use of SeV/dF/FIR for cancer therapy is also discussed. MATERIALS AND METHODS Plasmids Full-length FIR cdna (HA-FIR) was cloned into the pcgnm2 vector plasmid to introduce the hemagglutinin (HA)-tag at the amino termini [7]. Full-length FIR cdna was cloned into the p3xflag-cmv-14 vector (Sigma, MO, United States) to introduce the Flag-tag at the amino termini for the selection of FIR-Flag in 293T cells (performed by Dr. T.N.). Plasmids were prepared by 4317 April 21, 2014 Volume 20 Issue 21

219 Matsushita K et al. FIR-encoding Sendai virus vector for cancer gene therapy CsCl ultra-centrifugation or the Endofree Plasmid Maxi Kit (Qiagen, MD, United States) and the DNA sequences were verified. Tumor cell lines HeLa cells (human cervical squamous cell carcinoma cells), LoVo and SW480 cells (human colon cancer cell lines) and LLC-MK2 a rhesus monkey kidney cell line were purchased from the American Type Culture Collection (Manassas, VA, United States). Yes-5, a human esophageal squamous cell carcinoma cell line was established by Dr Takuo Murakami (Yamaguchi University, Yamaguchi, Japan). All cell lines were cultured at 37 in a humidified atmosphere containing 5% CO2. All tumor cell lines, except LLC-MK2 cells [which were maintained in DMEM; Dulbecco s Modified Eagle s Medium (Gibco BRL, NY, United States)] were cultured in tissue flasks or Petri dishes containing RPMI-1640 (Gibco, NY, United States) supplemented with 10% heat-inactivated FBS and penicillin (100 units/ml), streptomycin (0.1 mg/ml), and 2 mmol/l glutamine. Immunocytochemistry, protein extraction and immunoblotting Immunocytochemistry was performed as described previously [7]. Protein extraction and immunoblotting are described elsewhere [8,9]. sirna against FIR or SAP155 SAP155 sirna duplexes were purchased from Sigma Aldrich. The target sequences for SAP155 sirna oligonucleotides were listed previously [8]. Luciferase GL2 duplex was used as a negative control for sirna targeting 5 -CGTACGCGGAATACTTCGA-3. Transient transfection of sirna was carried out using Lipofectamine 2000 (Invitrogen) according to the manufacturer s instructions. The transfected cells were cultured for 72 h at 37 in a 5% CO2 incubator. Apoptosis detection Apoptotic cells were detected by terminal deoxynucleotidyl transferase dutp nick end labeling (TUNEL) assay according to the manufacturer s instructions (Apoptosis Detection System, Fluorescein, Promega, WI, United States) as described previously [7]. Apoptosis detection by APOPercentage apoptosis assay (Funakoshi Co., Ltd., Tokyo, Japan) was performed according to the manufacturer s instructions [9]. Construction of SeV vector Human FIR cdna was amplified with a pair of NotI site-tagged primers containing SeV-specific transcriptional regulatory signal sequences, (End and Start, italicized below) 5 -ATTGCGGCCGCCAAGGTTCAATGGC- GACGGCGACCATAGC-3 and 5 -ATTGCGGCCGC- GATGAACTTTCACCCTAAGTTTTTCTTACTACG- GTCACGCAGAGAGGTCACTGTTATCAAAACGC-3. The amplified fragment was introduced into the NotI site of the parental SeV vector cdna, psev 18+ b(+)/df [15], to generate psev 18+ hfir/df. psev 18+ hfir/df was transfected to LLC-MK2 cells which were preliminarily infected with psoralen- and long-wave UV-treated vaccinia virus vtf7-3, expressing T7 polymerase. The cells were then washed twice with DMEM, and cultured for 24 h in DMEM containing cytosine β-d-arabinofuranoside (AraC; 40 μg/ml) and trypsin (7.5 μg/ml). LLC-MK2/F7/A cells expressing the F protein were suspended in DMEM containing AraC and trypsin, and layered onto the transfected cells, and cultured at 37 for an additional 48 h. The recovered vector in the culture supernatants was propagated using the LLC-MK2/F7/A cells. A GFP expression vector (SeV/ df/gfp) was prepared as previously described [8]. The viral vectors were further amplified by several rounds of propagation. The virus titers of the recovered vectors were determined by their infectivity and expressed using cell-infectious units (CIU). These vectors were frozen at -80 until use. SeV/dF/GFP-mediated green fluorescent protein transduction efficiency One million LLC-MK2 cells and HeLa cells were seeded in six-well plates and transduced with SeV/dF/GFP when monolayers reached 60%-80% confluence. As the standard inoculation procedure for vaccination, the monolayers were washed twice with PBS and overlaid with serumfree medium containing SeV/dF/GFP at a multiplicity of infection (MOI) of 0, 1, 10, 50, 100, or 300. After incubation at 37 for 90 min, non-adsorbed virus was removed, medium containing 10% FBS was added, and the cells were incubated for more than 48 h at 37. The transduction studies were carried out in triplicate for each MOI. Microscopy was used to detect transduced cells by GFP fluorescence. At 72 h after transduction, the GFPtransduced cells were analyzed for GFP expression using a FACS Calibrator (BD Pharmingen, Franklin Lakes, NJ, United States). MTS assay to assess cell viability The inhibitory effects of viruses on the proliferation of cultured cells were examined using the CellTiter96 AQueous One Solution Proliferation Assay (Promega, Madison, WI, United States). In brief, five thousand cells were plated in each well on day 0. On day 1, 24 h later, HeLa cells were infected with SeV/dF/FIR or SeV/dF/GFP as the control at 0.1 to 10 MOI, and cultured for 2 d. On day 3, cell viability was quantified by measuring the absorbance at 570 nm after incubation with the tetrazolium compound [3-(4,5-dimethylthiozol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS), and an electron coupling agent, phenazine ethosulfate (PES)] (Promega, Madison, WI, United States) for 4 h. The absorbance at 570 nm was measured by a Mutiabel Counter, ARVOSX WAIIAC (Perkin Elmer, MA, United States). The results are shown as percentages of the uninfected control cells. Mice Six- to eight-week male immuno-competent Balbc/nu/ 4318 April 21, 2014 Volume 20 Issue 21

220 Matsushita K et al. FIR-encoding Sendai virus vector for cancer gene therapy nu mice were purchased from Clea Japan (Tokyo, Japan) and housed in the Animal Maintenance Facility at Chiba University under specific pathogen-free conditions. All animal experiments were approved by the Committee of the Ethics on Animal Experiments in the Faculty of Medicine, Chiba University and carried out following the Guidelines for Animal Experiments in the Faculty of Medicine, Chiba University, Chiba, Japan and The Law and Notification of the Government. Mouse experiments were carried out at least twice to confirm the results. Tumor xenograft experiments The in vivo inhibition of tumorigenicity of HeLa cells (human cervical squamous cell carcinoma cells) was examined by SeV/dF/FIR or SeV/dF/GFP injection (as the control) cells/50 μl PBS of HeLa cells were injected under the skin in the right thigh of nude mice (6-wk old males). Tumor growth was observed and the long and short diameter measured for tumor volume calculation. Thirty days after inoculation, tumors grew up to 5-8 mm in diameter in 18 of 18 mice (100%). Tumor size was calculated using the formula, (a b 2 )/2, where a and b represent the larger and smaller diameters, respectively, and was monitored every 3 d. FIR-binding protein identification The methods for the direct nanoflow liquid chromatography-tandem mass spectrometry system with FIR-FLAG transiently transfected 293T nuclear extracts have been described previously [19-23]. RESULTS HA-FIR suppresses endogenous c-myc expression with apoptosis induction in vivo To examine endogenous c-myc gene suppression by FIR, HA-FIR expression plasmids were transfected into HeLa cells, and c-myc expression was visualized by immunostaining with anti-c-myc antibodies (Figure 1A; upper panels: HA-FIR is red; c-myc is green). c-myc levels were greatly diminished in HA-FIR expressing cells (arrows), demonstrating that FIR represses endogenous c-myc expression in SW480 (Figure 1A; middle panels) and LoVo cells (Figure 1A; lower panels). After HA-FIR expression plasmids were transfected into HeLa, SW480 or LoVo cells, apoptotic cells were visualized by TUNEL assay. HA-FIR transfected cells were definitively associated with apoptosis (Figure 1B). FIR protein expression by fusion gene-deficient SeV/dF/ FIR SeV/dF/FIR and SeV/dF/GFP were prepared as described in Materials and Methods (Figure 2A, B). SeV/ df/fir vectors were infected into LLC-MK2 or HeLa cells. FIR protein expression level was examined by western blot with anti-fir antibody (6B4) (Figure 2C). At least CIU of fusion gene-deficient SeV/dF/FIR virus particles were prepared at amplification for use in the experiments. Transduction efficiency of the SeV/dF/GFP vector in various human tumor cell lines Nine human and five murine tumor cell lines plus nontumor cells, propagated in vitro were collected, transduced by SeV/dF/GFP, and examined for gene transduction efficiency. Flow cytometric analyses showed dose-dependent GFP expression, and optimal expression was obtained at a MOI of ; > 90% GFP-positive tumor cell lines were detected at a MOI over 10 (Figure 3A and data not shown). Furthermore, SeV/dF/FIR, but not SeV/dF/GFP significantly suppressed HeLa cell (human cervical squamous carcinoma) growth as shown by the APOPercentage assay (Figure 3B), indicating SeV/dF/ FIR suppresses tumor cell growth by apoptosis in vivo. SeV/dF/FIR vector showed anti-tumor activity in the mouse xenograft model SeV/dF/FIR, but not SeV/dF/GFP, significantly suppressed cell growth in HeLa cells (Figure 4A) and SW480 cells when analyzed by Dunnet s test for multiple comparisons (Figure 4B). Of note, xenograft tumors 2 cm in diameter disappeared completely following SeV/dF/FIR treatment, indicating that SeV/dF/FIR has immunological effects (Figure 5) [24,25]. FIR was co-immunoprecipitated with SAP155 If SeV/dF/FIR is to be tested clinically, endogenous FIR-interacting proteins should be identified to avoid unexpected side effects. For this purpose, the FIR-FLAG tag vector was transiently expressed in 293T cells and co-immunoprecipitated with anti-flag conjugated beads to detect FIR-binding proteins [19-23] (Table 1). As reported previously, FBP (Far upstream element-binding protein) [26,27], SAP155 [28], and SRp54 (splicing factor, arginine/serine rich-12) [28] were identified as candidate FIRbinding proteins. To date, no significant side effects have been observed following SeV/dF/FIR treatment including our study [29]. SeV/dF/FIR suppressed SSA-activated c-myc We previously reported that the adenovirus vector encoding FIR exon2 (Ad-FIR exon2) activates not only c-myc transcription, but also c-myc protein expression in HeLa cells [8]. However, the extent of c-myc protein activation by Ad-FIR exon2, evaluated by western blot analysis, could not be explained solely by c-myc transcription activation [8]. Therefore, we hypothesized that c-myc protein should be modified by Ad-FIR exon2 to be more stable. Ad-FIR exon2 expression leads to increased levels of c-myc phosphorylated at Ser62 (data not shown), indicating that stable c-myc protein accumulates in cells [30,31]. As reported previously, SAP155 sirna inhibited FIR premrna splicing and generated FIR exon2 [8,9]. In fact, SAP155 sirna increased levels of c-myc phosphorylated at Ser62 and Ad-FIR exon2 (Figure 6A). In other words, Ad-FIR exon2, which lacks the transcriptional repressor 4319 April 21, 2014 Volume 20 Issue 21

221 Matsushita K et al. FIR-encoding Sendai virus vector for cancer gene therapy A c-myc HA-FIR DAPI LoVo SW480 HeLa B TUNEL DAPI Merge LoVo SW480 HeLa Figure 1 Far up stream element-binding protein-interacting repressor suppresses endogenous c-myc in SW480 and LoVo cells as well as HeLa cells. A: 100 fmol of HA-FIR was transfected into the colon cancer cell lines, SW480 and LoVo cells, as well as HeLa cells (cervical squamous cell carcinoma cells) in a 6-well plate. After 48 h transfection, cells were fixed and immunostained against c-myc (left, green) or HA (middle, red) antibodies. Arrowheads (HeLa), thick arrows (SW480) and thin arrows (LoVo) show the cells in which HA-FIR plasmids were expressed. c-myc expression (left, green) was markedly reduced in most HA-FIR-expressing cells (middle, red) (indicated by arrowheads and arrows); B: HA-FIR transfected cells were definitively associated with apoptosis as revealed by TUNEL assay in HeLa, SW480 and LoVo cells. Nuclear DNA was stained with DAPI (right, blue). FIR: FBP Interacting Repressor; FBP: FUSE-Binding protein; FUSE: Far Upstream Element April 21, 2014 Volume 20 Issue 21

222 Matsushita K et al. FIR-encoding Sendai virus vector for cancer gene therapy A SeV wild type 3' NP P M F HN L 5' T7 Rbz SeV/dF non-transmissible 3' 5' df hfir or GFP B NP P L SeV T7 T7 T7 T7 Rbz psev 18+ hfir/df or psev 18+ GFP/dF UV-vTF7-3 C Mock SeV/dF/GFP SeV/dF/FIR FIR dimer T7 FIR FIR monomer RNP b-actin SeV/dF/FIR or SeV/dF/GFP LLC-MK2 Figure 2 Structures and procedures for generating fusion gene-deficient Sendai virus/df/far up stream element-binding protein-interacting repressor (SeV/ df/fir) or Sendai virus/df/green fluorescent protein vectors (SeV/dF/GFP) from Sendai virus genome RNA. A: Schematic genome structures of wild-type (SeV) and fusion gene-deficient (SeV/dF; non-transmissible) vector carrying the human FIR (hfir) gene or jellyfish green fluorescent protein (GFP). The open reading frame or the FIR or GFP gene was inserted with the SeV-specific transcriptional regulatory signal sequences. T7; T7 promoter, Rbz; hepatitis delta virus ribozyme sequence; B: Schematic representation of the procedure for generating the fusion gene-deficient SeV/dF/FIR or SeV/dF/GFP. SeV/dF/FIR or SeV/dF/GFP virus particles were propagated using fusion protein-expressing packaging cells (LLC-MK2/F7/A) after preparation in LLC-MK2 cells using the four plasmids driven by a recombinant vaccinia virus expressing T7 RNA polymerase which had been inactivated with psoralen and long-wave UV light (UV-vTF7-3); C: SeV/dF/FIR virus vectors were infected into HeLa cells and whole cell proteins were extracted for western blot analysis. SeV/dF/FIR expresses FIR proteins. FIR: FBP Interacting Repressor; FBP: FUSE- Binding protein; FUSE: Far Upstream Element; SeV: Sendai virus; NP: nucleoprotein; P: phosphoprotein; L: the catalytic subunit of the polymerase large protein forms a ribonucleoprotein complex (RNP) vector and was transfected separately with the SeV RNA. See Materials and Methods. domain, directly or indirectly activated c-myc expression not only through transcription, but also through protein level, suggesting that FIR exon2 acts in opposition to the repressor function by FIR [8]. In this study, the effect of SeV/dF/FIR was examined to determine whether it suppresses the increase in c-myc after SSA treatment. SeV/dF/FIR suppressed SSA-induced c-myc activation (Figure 6B, compare lane 2 with lane 1), but not basal c-myc expression (Figure 6B, compare lanes 4 to 3 and 6 to 5, respectively). These results were consistent with previous reports that FIR suppresses activated, but not basal, c-myc transcription [6]. These observations suggest that the increase in c-myc by either SAP155 sirna or SSA treatment is due to reduced FIR activity, or an increase in the ratio of FIR exon2/ FIR in HeLa cells. Taken together, these results suggest that SeV/dF/FIR is potentially clinically applicable for cancer therapy as it counteracts SSA-activated c-myc (Figure 6B, compare lane 2 with lane 1) as well as endogenous c-myc (Figure 1A). SeV/dF vector transduction F gene-deficient SeV vectors (SeV/dF) can transduce cells in a wide range of tissues such as respiratory, nervous, muscular, epithelial and immune tissues [11,32-35]. Transduction efficiency to cell lines from various tissues was examined and compared to the adenovirus vector expressing LacZ (Ad5/LacZ) at the same MOI. The transduction efficiency of SeV/dF to those cells was even higher than that of adenovirus vector (Figure 7). DISCUSSION Overexpression of c-myc has been known to promote 4321 April 21, 2014 Volume 20 Issue 21

223 Matsushita K et al. FIR-encoding Sendai virus vector for cancer gene therapy A SeV/dF/FIR 10 MOI 3 d Mock SeV/dF/GFP 10 MOI 3 d SeV/dF/GFP 10 MOI 3 d B SeV/dF/FIR 10 MOI 3 d Mock SeV/dF/GFP 5 mmol/l H2O2 Figure 3 High efficiency of Sendai virus/df/green fluorescent protein vectors to HeLa cells and Sendai virus/df/green fluorescent protein vectors indicates significant cell growth inhibition with apoptosis. A: HeLa cells were infected with a 10 MOI of SeV/dF/FIR virus vector and cultured in DMEM for 3 d (72 h). The same amount of 10 MOI SeV/dF/GFP was also used to infect the control. As shown in the left lower panel, SeV/dF/GFP infected almost 100% cells (green). Under these conditions, SeV/dF/FIR significantly inhibited cell growth as shown in the left upper panel. Mock and 10 MOI SeV/dF/GFP showed no cell growth inhibition as seen in the right upper and lower panels; B: HeLa cells infected by 10 MOI SeV/dF/FIR for 3 d showed significant cell damage revealed by the APO Percentage apoptosis assay, compared to mock or the same conditions of SeV/dF/GFP virus infected HeLa cells. 5 mmol/l H2O2 was used as a positive control. FIR: FBP Interacting Repressor; FBP: FUSE-Binding protein; FUSE: Far Upstream Element; SeV: Sendai virus; GFP: green fluorescent protein; MOI: multiplicity of infection. cell growth, proliferation and immortalization, whereas a reduction in c-myc induces apoptosis. The recent genetic construction of the mouse in which the expression of c-myc can be switched on or off in vivo has emphasized the significance of c-myc expression in tumorigenesis. Ectopic c-myc expression in hematopoietic cells using the tetracycline regulatory system caused malignant T cell lymphomas and acute myeloid leukemia; subsequent inactivation of the transgene caused regression of established tumors [36]. These observations have provided encourage April 21, 2014 Volume 20 Issue 21

224 Matsushita K et al. FIR-encoding Sendai virus vector for cancer gene therapy A 100 SeV/dF/GFP B 3.5 The relative suppression ratio SeV/dF/FIR: SeV/dF/GFP (%) SeV/dF/FIR P = Tumor volume ratio Control (n = 5) SeV/dF/GFP (n = 5) SeV/dF/FIR (n = 6) P = MOI t/d Figure 4 Sendai virus/df/far up stream element-binding protein-interacting repressor decreased HeLa cell growth in vitro and in a xenograft animal model. HeLa cells (A) and SW480 cells (B) were infected with 0, 0.1, 1, and 10 MOI of SeV/dF/FIR or SeV/dF/GFP vectors and cell growth was measured by MTS assay (see Materials and Methods). Results are shown as the percentage of cell number at day 0. Points, mean of three separate experiments; bars, SD. Statistical significance was analyzed by Dunnett s test for multiple comparisons (SeV/dF/FIR vs SeV/dF/GFP, P < 0.007). Two weeks after HeLa cells were xenografted into the right thigh of Balbc/nu/nu mice, the tumor size was approximately 7-8 mm CIU of SeV/dF/FIR or SeV/dF/GFP vector were injected directly around the tumor, and the tumor growth was observed and measured every three days as described in Materials and Methods. Results are shown as the ratio of tumor volume compared to the size at day 0. The tumor volume at day 0 of SeV/dF/FIR (n = 6), SeV/dF/GFP (n = 5), and control (n = 5) were , , and (average ± SD) mm 3, respectively. The average tumor volume at day 0 was estimated as 1 in each experiment. Arrows indicate the injection of SeV/dF/FIR or SeV/dF/ GFP vectors. FIR: FBP Interacting Repressor; FBP: FUSE-Binding protein; FUSE: Far Upstream Element; SeV: Sendai virus; GFP: green fluorescent protein; MOI: multiplicity of infection. Day 0 Day 14 Day 90 Day CIU each of SeV/dF/FIR injection for total even times Tumor volume 10 3 (mm 3 ) (Yes-5 cells) Day after Yes-5 cells xenograft (d) Figure 5 Sendai virus/df/far up stream element-binding protein-interacting repressor vector showed anti-tumor activity in a mouse xenograft model Yes-5 cells were xenografted into the right thigh of Balbc/nu/nu mice, and the tumor size was approximately 15 mm in diameter at Day CIU of SeV/dF/FIR vectors were injected directly around the tumor every two days, seven times in total. Tumor growth was observed and measured every two days as described in Materials and Methods. Ulcer formation was observed in the center of the tumor (day 14 after SeV/dF/FIR injection). Tumor size was significantly diminished with ulcer formation (day 90) and disappeared completely during surveillance (day 140). Thick arrows in the images indicate the tumor margin. Thin arrows indicate the injection of SeV/dF/FIR vectors into the tumor. FIR: FBP Interacting Repressor; FBP: FUSE-Binding protein; FUSE: Far Upstream Element; SeV: Sendai virus; CIU: cellinfectious units April 21, 2014 Volume 20 Issue 21

225 Matsushita K et al. FIR-encoding Sendai virus vector for cancer gene therapy Table 1 Co-immunoprecipitaed proteins with far up stream element-binding protein-interacting repressor in 293T cells Hit preys CDKN2AIP CDKN2A interacting protein CDYL Chromodomain protein, Y chromosome-like DAZAP1 DAZ associated protein 1 DDX17 DEAD box polypeptide 17 DDX5 DEAD (Asp-Glu-Ala-Asp) box polypeptide 5 ELAVL1 ELAV-like 1 FAM120A Oxidative stress-associated Src activator FUBP1 FUBP3 KHSRP Far upstream element-binding protein family FUBP1 KHSRP Far upstream element-binding protein family HNRNPA1 Heterogeneous nuclear ribonucleoprotein A1 HNRNPA1 HNRNPA1L2 Heterogeneous nuclear ribonucleoprotein; A1 or A1-like HNRNPA1 HNRNPA1L2 LOC Nuclear ribonucleoprotein A1 family HNRNPA2B1 Heterogeneous nuclear ribonucleoprotein A2/B1 HNRNPA3 Heterogeneous nuclear ribonucleoprotein A3 HNRNPAB Heterogeneous nuclear ribonucleoprotein A/B; isoform a HNRNPAB HNRNPD Heterogeneous nuclear ribonucleoprotein; A/B or D HNRNPD Heterogeneous nuclear ribonucleoprotein D; isoform c HNRNPH1 Heterogeneous nuclear ribonucleoprotein H1 HNRNPK Heterogeneous nuclear ribonucleoprotein K HNRNPL Heterogeneous nuclear ribonucleoprotein L HNRNPM Heterogeneous nuclear ribonucleoprotein M; isoform a HNRNPR Heterogeneous nuclear ribonucleoprotein R HNRNPU Heterogeneous nuclear ribonucleoprotein U; isoform a HNRNPUL1 Heterogeneous nuclear ribonucleoprotein U-like 1 HNRPDL Heterogeneous nuclear ribonucleoprotein D-like IFIT5 Interferon-induced protein with tetratricopeptide repeats 5 IGF2BP1 Insulin-like growth factor 2 mrna binding protein 1 IGF2BP1 IGF2BP3 Insulin-like growth factor 2 mrna binding protein; 1 or 3 IGF2BP2 Insulin-like growth factor 2 mrna binding protein 2 IGF2BP3 Insulin-like growth factor 2 mrna binding protein 3 ILF2 Interleukin enhancer binding factor 2 KHDRBS1 KH domain containing, RNA binding, signal transduction associated 1 KHDRBS1 KHDRBS2 KHDRBS3 KH; domain containing, RNA binding, signal transduction associated 1 Or domain-containing, RNA-binding, signal transduction- KHSRP KH-type splicing regulatory protein KIAA1967 p30 DBC protein LARP1 La related protein LRPPRC Leucine-rich PPR motif-containing protein LSM12 LSM12 homolog MAGOH MAGOHB Mago-nashi homolog; or B MATR3 Matrin 3 MOV10 Mov10, Moloney leukemia virus 10, homolog MSI2 Musashi 2; isoform a PABP3 PABPC1 PABPC3 Poly (A) binding protein, cytoplasmic 1 Or poly (A) binding protein, cytoplasmic 3 PABPC1 Poly (A) binding protein, cytoplasmic 1 PABPC1 PABPC1L PABPC5 Poly (A) binding protein, cytoplasmic 1 or poly(a) -binding protein, Cytoplasmic 1-like or poly (A) binding protein, cytoplasmic 5 PABPC1 PABPC4 Poly (A) binding protein, cytoplasmic 1 Or poly A binding protein, cytoplasmic 4 PABPC1L2B PABPC4 Poly (A) binding protein, cytoplasmic 1-like 2B Or poly A binding protein, cytoplasmic 4 PABPC4 Poly A binding protein, cytoplasmic 4 PABPC4 PABPC4L Poly A binding protein, cytoplasmic 4 PABPN1 Poly (A) binding protein, nuclear 1 PCBP1 PCBP2 PCBP3 PCBP4 Poly (rc) binding protein; 1 or 2 or 3 or 4 PCBP2 PCBP3 Poly (rc) binding protein; 2 or 3 PTBP1 Polypyrimidine tract-binding protein 1; isoform d SF3B1 Splicing factor 3b, subunit 1 SF3B14 Splicing factor 3B, 14 kda subunit SF3B3 Splicing factor 3b, subunit 3 SF3B4 Splicing factor 3b, subunit 4 SFPQ Splicing factor proline/glutamine rich (polypyrimidine tract binding SFRS11 Splicing factor, arginine/serine-rich 11 (SRp54) SFRS12 Splicing factor, arginine/serine-rich 12 SNRPD1 Small nuclear ribonucleoprotein D1 polypeptide 16 kda SSB Autoantigen La SYNCRIP Synaptotagmin binding, cytoplasmic RNA interacting protein 4324 April 21, 2014 Volume 20 Issue 21

226 Matsushita K et al. FIR-encoding Sendai virus vector for cancer gene therapy THADA Thyroid adenoma associated TRMT1 trna methyltransferase 1 TROVE2 TROVE domain family, member 2; isoform 2 XRN2 5'-3' exoribonuclease 2 Total hit preys: 67 A SAP155 GL2 72 h SAP155 sirna B SSA 50 ng/ml 48 h SeV/dF/GFP SeV/dF/FIR SeV/dF/GFP MetOH SeV/dF/FIR SeV/dF/GFP H2O SeV/dF/FIR c-myc pser62-c-myc c-myc Total ERK c-myc-pser62 cycline p-cdk2 FIR Dimer Monomer cycline cdk2 b-actin b-actin Figure 6 SAP155 sirna induces c-myc activation with ErK phosphorylation, but suppresses phosphorylated-cdk2/cycline expression. HeLa cells were treated with SAP155 sirna for three days (72 h). A: SAP155 sirna, as well as SSA treatment, increased not only c-myc expression level, but also c-myc phosphorylation at both Ser62, but suppressed phosphorylated-cdk2 and cycline in a dose-dependent manner. Thus, SAP155 sirna activates c-myc potentially via inhibiting endogenous FIR pre-mrna splicing; B: FIR Sendai virus (SeV/dF/FIR) reversed the cytotoxicity of SSA by suppressing activated endogenous c-myc. HeLa cells were treated with 50 ng/ml SSA for 48 h with control (MetOH and H2O). 10 MOI of SeV/dF/FIR apparently suppressed activated c-myc expression, whereas SeV/dF/ FIR did not influence basal expression (MetOH or H2O). FIR: FBP Interacting Repressor; FBP: FUSE-Binding protein; FUSE: Far Upstream Element; SeV: Sendai virus; GFP: green fluorescent protein; MOI: multiplicity of infection; SSA: Spliceostatin. ment for the future development of cancer therapies based on targeting individual oncogenes such as c-myc. We have previously reported that FIR strongly represses endogenous c-myc transcription, and induces apoptosis [7] and is thus applicable for cancer treatment. In this study, first, we demonstrated that c-myc suppressor FBP-interacting repressor (FIR) strongly repressed endogenous c-myc transcription and induced apoptosis in SW480, LoVo (human colon cancer cell lines) as well as HeLa cells (human cervical squamous cancer cell line). Second, SeV/dF/FIR showed strong anti-tumor effects in both cultured cells and xenograft tumor growth in an animal model. These results provide new insight into a new therapeutic target for tumor treatment. What type of suitable vector should be selected and how should FIR expressing vectors be conveyed to cancers? Sendai virus is an RNA virus and exists only in the cytoplasm, hence it is relatively safe as it does not affect chromosomes. In addition, SeV does not transform cells by integrating its genome into the cellular genome, thereby avoiding possible malignant transformation due to the genetic alteration of host cells; this is a safety advantage of SeV. For this reason, we chose SeV and prepared a fusion gene-deficient SeV/dF/FIR vector. The fusion gene-deficient SeV vector cannot transmit to F protein-non-expressing cells as F protein is essential for viral infection. The fusion gene-deficient SeV vector in this study does not require helper virus for reproduction, but is self-replicable in infected cells. Thus, the fusion gene-deficient SeV vector has several advantages over expressing vectors as a gene delivery system for human disease including cancer treatment. First, the fusion gene-deficient SeV vector is not pathogenic in humans. Second, the virus replicates only in the cytoplasm, therefore does not affect chromosome DNA in host cells. Third, SeV vector shows highly efficient gene transfer to a wide spectrum of cells, even to smooth muscle cells, nerve cells, or endothelial cells which are generally difficult to infect. Fourth, the SeV vector shows highly efficient gene transfer to a wide spectrum of cells, even to smooth muscle cells and does not generate wild-type virus in packaging cells. Recently, a gene-deficient SeV (SeV/dF) vector alone demonstrated tumor suppression by activating dendritic cells (DCs) [24], or if granulocyte 4325 April 21, 2014 Volume 20 Issue 21

227 Matsushita K et al. FIR-encoding Sendai virus vector for cancer gene therapy SeV/dF/LacZ Ad2/LacZ MOI 0.1 MOI 3 MOI 0.1 MOI 3 A549 MDCK HeLa LLC-MK2 Figure 7 Sendai virus/df/lacz transduction efficiency was examined in some human and animal cell lines. Confluent culture of LLC-MK2 (macaque kidney fibroblasts), HeLa (human adenocarcinoma cells), MDCK (canine kidney cells), and A549 (human lung carcinoma cells) were infected with LacZ expressing SeV vector (SeV/dF/LacZ) at a MOI of 0.1 or 3.0. LacZ expressing Adenovirus vector (Ad5/LacZ) was used as a control. Two days after infection, the cells were stained with X Gal. SeV: Sendai virus. macrophage colony-stimulating factor was encoded, it produced autologous tumor vaccines [25]. Therefore, the SeV/dF/FIR vector in this study may suppress tumor growth by a dual function through c-myc suppression of tumor cells and DC activation. Furthermore, SeV/dF/ FIR showed a synergistic effect with cisplatin in the treatment of malignant pleural mesothelioma [29]. FIR-binding proteins are basically classified into four categories (Table 1); (1) RNA binding proteins and splicing factors; (2) transcription factors and chromatin remodeling proteins; (3) actin-binding proteins; and (4) signal transduction and protein kinase families. This suggests that FIR potentially engages in some different intracellular events, such as RNA transport, DNA damage repair and pre-mrna splicing. Accordingly, the side effects of SeV/dF/FIR need to be considered before clinical use, such as premrna splicing disturbance [8,9], DNA damage repair [37] or intracellular protein transport interference. For clinical safety, SeV/dF/FIR is preferable for local tumor growth control rather than systemic cancer therapy. Taken together, these findings show that SeV/dF/ FIR is a promising approach for cancer gene therapy, although further clinical and basic research are required to explain the precise mechanism of tumor suppression by FIR expressing vectors. ACKNOWLEDGMENTS The authors thank to Dr. David Levens (NCI, NIH, United States) for scientific discussions, Dr. Tohru Natsume (Biomedicinal Information Research Center, National Institute of Advanced Industrial Science and Technology, Tokyo, Japan) for FIR-binding proteins analysis, and Dr. Minoru Yoshida (Chemical Genetics Laboratory, RIKEN Advanced Science Institute, Saitama, Japan) for the kind gift of Spliceostatin A (SSA). COMMENTS Background Far Up Stream Element-Binding Protein-Interacting Repressor (FIR) is a c-myc transcriptional repressor. Thus, FIR expressing vectors are applicable for cancer therapy. In this study, the authors examined a novel therapeutic strategy to suppress c-myc in human cancers using a fusion gene-deficient Sendai virus (SeV/dF/FIR) which is inherently non-transmissible April 21, 2014 Volume 20 Issue 21

228 Matsushita K et al. FIR-encoding Sendai virus vector for cancer gene therapy Research frontiers As c-myc transcriptional control is largely unknown, modulation of c-myc regulation by SeV/dF/FIR for cancer therapy should be monitored, strictly and skeptically, from several aspects. This study revealed that SeV/dF/FIR is effective for cancer gene therapy without significant side effects in a xenograft model. Innovations and breakthroughs SeV/dF/FIR showed high gene transduction efficiency with significant antitumor effects and apoptosis induction in HeLa and SW480 cells. In the xenograft model, SeV/dF/FIR showed strong suppression of tumor growth with no significant side effects. Applications SeV/dF/FIR is potentially applicable for future clinical cancer treatment as SeV/dF/FIR suppresses endogenous c-myc as well as Spliceostatin A (SSA)- activated c-myc. Terminology FUSE: Far Upstream Element which is required for correct c-myc transcription. FBP: FUSE-Binding protein which has strong transcriptional activity. FIR: FBP interacting repressor which is a critical transcriptional repressor of c-myc gene. SeV: Sendai virus, a member of the Paramyxoviridae family, has envelopes and a nonsegmented negative-strand RNA genome. The SeV genome contains six major genes in tandem on a single negative-strand RNA. DC: Dendritic cell. The gene-deficient SeV (SeV/dF) vector alone demonstrates tumor suppression by activating dendritic cells (DCs). Peer review The authors performed the enthusiastic experiments in vivo and animal model to examine the SeV/dF/FIR for cancer gene therapy to minimize the side effect for the clinical use. REFERENCES 1 Pelengaris S, Khan M, Evan GI. Suppression of Myc-induced apoptosis in beta cells exposes multiple oncogenic properties of Myc and triggers carcinogenic progression. 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229 Matsushita K et al. FIR-encoding Sendai virus vector for cancer gene therapy tography-tandem mass spectrometry system for interaction proteomics. Anal Chem 2002; 74: [PMID: DOI: /ac020018n] 23 Komatsu M, Chiba T, Tatsumi K, Iemura S, Tanida I, Okazaki N, Ueno T, Kominami E, Natsume T, Tanaka K. A novel protein-conjugating system for Ufm1, a ubiquitin-fold modifier. EMBO J 2004; 23: [PMID: DOI: /sj.emboj ] 24 Komaru A, Ueda Y, Furuya A, Tanaka S, Yoshida K, Kato T, Kinoh H, Harada Y, Suzuki H, Inoue M, Hasegawa M, Ichikawa T, Yonemitsu Y. Sustained and NK/CD4+ T cell-dependent efficient prevention of lung metastasis induced by dendritic cells harboring recombinant Sendai virus. J Immunol 2009; 183: [PMID: DOI: /jimmunol ] 25 Inoue H, Iga M, Nabeta H, Yokoo T, Suehiro Y, Okano S, Inoue M, Kinoh H, Katagiri T, Takayama K, Yonemitsu Y, Hasegawa M, Nakamura Y, Nakanishi Y, Tani K. Non-transmissible Sendai virus encoding granulocyte macrophage colony-stimulating factor is a novel and potent vector system for producing autologous tumor vaccines. Cancer Sci 2008; 99: [PMID: DOI: /j x] 26 Liu J, He L, Collins I, Ge H, Libutti D, Li J, Egly JM, Levens D. The FBP interacting repressor targets TFIIH to inhibit activated transcription. Mol Cell 2000; 5: [PMID: DOI: /S (00) ] 27 Corsini L, Hothorn M, Stier G, Rybin V, Scheffzek K, Gibson TJ, Sattler M. Dimerization and protein binding specificity of the U2AF homology motif of the splicing factor Puf60. J Biol Chem 2009; 284: [PMID: DOI: /jbc. M ] 28 Page-McCaw PS, Amonlirdviman K, Sharp PA. PUF60: a novel U2AF65-related splicing activity. RNA 1999; 5: [PMID: DOI: /S ] 29 Kitamura A, Matsushita K, Takiguchi Y, Shimada H, Tada Y, Yamanaka M, Hiroshima K, Tagawa M, Tomonaga T, Matsubara H, Inoue M, Hasegawa M, Sato Y, Levens D, Tatsumi K, Nomura F. Synergistic effect of non-transmissible Sendai virus vector encoding the c-myc suppressor FUSE-binding proteininteracting repressor plus cisplatin in the treatment of malignant pleural mesothelioma. Cancer Sci 2011; 102: [PMID: DOI: /j x] 30 Junttila MR, Westermarck J. Mechanisms of MYC stabilization in human malignancies. Cell Cycle 2008; 7: [PMID: DOI: /cc ] 31 Lee T, Yao G, Nevins J, You L. Sensing and integration of Erk and PI3K signals by Myc. PLoS Comput Biol 2008; 4: e [PMID: DOI: /journal.pcbi ] 32 Yonemitsu Y, Kitson C, Ferrari S, Farley R, Griesenbach U, Judd D, Steel R, Scheid P, Zhu J, Jeffery PK, Kato A, Hasan MK, Nagai Y, Masaki I, Fukumura M, Hasegawa M, Geddes DM, Alton EW. Efficient gene transfer to airway epithelium using recombinant Sendai virus. Nat Biotechnol 2000; 18: [PMID: DOI: /79463] 33 Inoue M, Tokusumi Y, Ban H, Shirakura M, Kanaya T, Yoshizaki M, Hironaka T, Nagai Y, Iida A, Hasegawa M. Recombinant Sendai virus vectors deleted in both the matrix and the fusion genes: efficient gene transfer with preferable properties. J Gene Med 2004; 6: [PMID: DOI: / jgm.597] 34 Shirakura M, Inoue M, Fujikawa S, Washizawa K, Komaba S, Maeda M, Watabe K, Yoshikawa Y, Hasegawa M. Postischemic administration of Sendai virus vector carrying neurotrophic factor genes prevents delayed neuronal death in gerbils. Gene Ther 2004; 11: [PMID: DOI: /sj.gt ] 35 Okano S, Yonemitsu Y, Nagata S, Sata S, Onimaru M, Nakagawa K, Tomita Y, Kishihara K, Hashimoto S, Nakashima Y, Sugimachi K, Hasegawa M, Sueishi K. Recombinant Sendai virus vectors for activated T lymphocytes. Gene Ther 2003; 10: [PMID: ] 36 Felsher DW, Bishop JM. Reversible tumorigenesis by MYC in hematopoietic lineages. Mol Cell 1999; 4: [PMID: DOI: /S (00) ] 37 Rahmutulla B, Matsushita K, Satoh M, Seimiya M, Tsuchida S, Kubo S, Shimada H, Otsuka M, Miyazaki M, Nomura F. Alternative splicing of FBP-interacting repressor coordinates c-myc, P27Kip1/cyclinE and Ku86/XRCC5 expression as a molecular sensor for bleomycin-induced DNA damage pathway. Oncotarget 2013; December 21 P- Reviewer: Takenaga K S- Editor: Ma YJ L- Editor: Webster JR E- Editor: Zhang DN 4328 April 21, 2014 Volume 20 Issue 21

230 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH BRIEF ARTICLE REPORT Case-control study of factors that trigger inflammatory bowel disease flares Linda A Feagins, Ramiz Iqbal, Stuart J Spechler Linda A Feagins, Ramiz Iqbal, Stuart J Spechler, VA North Texas Healthcare System, Dallas, TX 75216, United States Linda A Feagins, Stuart J Spechler, Division of Gastroenterology, University of Texas Southwestern Medical Center, Dallas, TX 75216, United States Author contributions: Feagins LA designed the study, enrolled study patients, analyzed and interpreted the data, and drafted and approved the article; Iqbal R played a critical role in patient recruitment and data acquisition; Spechler SJ played a critical role in data interpretation and revising the manuscript for important intellectual content. Supported by The Office of Medical Research, Department of Veteran s Affairs, and in part, by Janssen, Inc. Correspondence to: Linda A Feagins, MD, Division of Gastroenterology, University of Texas Southwestern Medical Center, Dallas VA Medical Center, 4500 S Lancaster Road, Dallas, TX 75216, United States. linda.feagins@va.gov Telephone: Fax: Received: August 1, 2013 Revised: September 24, 2013 Accepted: October 19, 2013 Published online: April 21, 2014 Abstract AIM: To explore the association between inflammatory bowel diseases (IBD) flares and potential triggers. METHODS: Patients evaluated for an acute flare of IBD by a gastroenterologist at the Dallas VA Medical Center were invited to participate, as were a control group of patients with IBD in remission. Patients were systematically queried about nonsteroidal anti-inflammatory drug use, antibiotic use, stressful life events, cigarette smoking, medication adherence, infections, and travel in the preceding 3 mo. Disease activity scores were calculated for each patient at the time of enrollment and each patient s chart was reviewed. Multivariate regression analysis was performed. RESULTS: A total of 134 patients with IBD (63 with Crohn s disease, 70 with ulcerative colitis, and 1 with indeterminate colitis) were enrolled; 66 patients had flares of their IBD and 68 were controls with IBD in remission (for Crohn s patients, average Crohn s disease activity index was 350 for flares vs 69 in the controls; for UC patients, Mayo score was 7.6 for flares vs 1 for controls in those with full Mayo available and 5.4p for flares vs 0.1p for controls in those with partial Mayo score). Only medication non-adherence was significantly more frequent in the flare group than in the control group (48.5% vs 29.4%, P = 0.03) and remained significant on multivariate analysis (OR = 2.86, 95%CI: ). On multivariate regression analysis, immunomodulator use was found to be associated with significantly lower rates of flare (OR = 0.40, 95%CI: ). CONCLUSION: In a study of potential triggers for IBD flares, medication non-adherence was significantly associated with flares. These findings are incentive to improve medication adherence Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Inflammatory bowel diseases; Flare; Nonadherence; Crohn s disease; Ulcerative colitis Core tip: Flares of Crohn s disease and colitis are often unpredictable and physicians and patients alike search for triggers for these flares in an attempt to prevent future flares. In this study, we prospectively enrolled and queried patients with flares of inflammatory bowel diseases (IBD) and compared their responses to IBD patients in remission. We found that medication nonadherence was the only significant trigger for flares of IBD, while nonsteroidal anti-inflammatory drug use, antibiotic use, infections, smoking, travel and emotional stress were not associated with flares. Clinicians should be aware the significant role that non-adherence plays in flare-ups of IBD in order to counsel their patients appropriately April 21, 2014 Volume 20 Issue 15

231 Feagins LA et al. Triggers in inflammatory bowel disease Feagins LA, Iqbal R, Spechler SJ. Case-control study of factors that trigger inflammatory bowel disease flares. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: INTRODUCTION The inflammatory bowel diseases (IBDs), Crohn s disease (CD) and ulcerative colitis (UC), are lifelong ailments whose courses are characterized by recurring cycles of exacerbation and remission. These times of disease exacerbation, or flares, can be debilitating to patients causing significant pain and discomfort, need for hospitalization and/or surgery, and time lost from work and normal activities of daily life. A number of disparate factors (e.g., certain drugs, infections, tobacco, stress, and poor treatment adherence) have been proposed as triggers for flares of IBD. If physicians and patients were better able to identify modifiable triggers for these flares, these times of disease exacerbation might be fewer. In some retrospective studies, the use of nonsteroidal anti-inflammatory drugs (NSAIDs) and antibiotics has been associated with flares of IBD [1,2]. It has been proposed that NSAIDs might contribute to those flares by altering prostaglandin production, by increasing leukotriene production, or by altering nuclear factor (NF)-κB signaling in the gut [1]. Antibiotics can alter bowel flora, which might promote flares by decreasing gut levels of protective bacteria or by increasing levels of potentially harmful microorganisms. Enteric infections associated with antibiotic use, such as Clostridium difficile, are now well known to be associated with IBD flares [3]. However, antibiotics also are used to treat active IBD (specifically CD), and there are some data to suggest that antibiotic use, at least in CD, actually might protect against flares [2]. Thus, the frequency with which NSAIDs and antibiotics contribute to IBD exacerbations remains unclear. Tobacco usage, specifically active smoking in CD patients and smoking cessation in UC patients, has been linked to flares of IBD for reasons that are not clear [4,5]. Proposed mechanisms for the effects of tobacco use in IBD are numerous and include modulation of cellular and humoral immunity, changes in cytokine levels, modification of eicosanoid-mediated inflammation, reduction of antioxidant capacity, release of endogenous glucocorticoids, colonic mucus effects, alterations of mucosal blood flow, prothrombotic effects and promotion of microvascular thrombosis, alteration of gut permeability, and modification of gut motility [6]. Many patients blame emotional stress as the trigger for exacerbation of their IBD [7]. Some studies have demonstrated that experimental stress plays a role in activating gut inflammation in animal models of colitis [8,9]. Stress has been suggested to modulate the release of proinflammatory cytokines, to alter the activity of immune and inflammatory cells, and to increase gut permeability, all of which might exacerbate IBD [10]. Moreover, a survey of a population-based registry found that high-perceived stress was associated with an increased risk of flare [11]. Lastly, poor adherence with prescribed medications for IBD has been reported to increase the risk of flares. A systematic review on the use of 5-aminosalicylates in UC found the relative risk for a flare in nonadherent patients to be at least 3.65 [12]. The veterans affairs (VA) health system offers a unique system in which to study patients in that the patients who served in the military simply have to enroll in the VA system and can receive free or low cost health care. No prior studies have addressed the question of triggers for flares in this unique population. We performed a case-control study to explore systematically the association between IBD flares and potential triggering factors in our veteran population. MATERIALS AND METHODS This study was approved by the Institutional Review Board of the Dallas VA Medical Center. Informed consent was obtained from each patient included in the study. Between July 2009 and April 2012, all patients with flares of UC or CD who were evaluated by the Division of Gastroenterology during the course of their clinical care at the Dallas VA Medical Center, either in clinics or as inpatients, were invited to participate in this study. A control group of IBD patients followed in our IBD clinic who were in clinical remission for at least 6 mo also were invited to participate. Disease activity was measured at the time of enrollment using the Crohn s Disease Activity Index (CDAI) score (for CD patients) or the Mayo score (for UC patients) with CDAI > 150 or Mayo > 2 (or partial Mayo 2) defining a flare [13,14]. This was calculated by the study team at enrollment. Control patients were required to have CDAI < 150 or Mayo score 2 (or < 2 for partial Mayo). Patients were systematically queried about recent (within the previous 3 mo) infections (including upper respiratory tract, gastrointestinal, urinary tract and skin infections), NSAID use, antibiotic use, stressful life events (divorce/separation, death in the family, birth of a child, loss or change of job, and other perceived stressful situations), pertinent tobacco use (discontinuation for UC, ongoing or starting smoking for CD patients), adherence with IBD-specific medications, and travel away from home. A written list of all potential NSAIDs, prescription and over-the-counter, was reviewed with the patient. Patients were queried with regard to how often they missed a dose of their IBD medications (on aggregate) and categorized as never, once per month, 2-4 times per month, 1-3 times per week, four or five times per week, or at least once a day. Patients were considered to be noncompliant with their medications if they missed a dose of medication at least 2-4 times per month. In addition to the data collected during the study enrollment, as detailed above, each patient s chart was reviewed and data were collected with regard to recent laboratory work (within 1 mo), dis April 21, 2014 Volume 20 Issue 15

232 Feagins LA et al. Triggers in inflammatory bowel disease Table 1 Patient characteristics Flares (n = 66) Controls (n = 68) P value Age (yr, mean ± SD) 49.0 ± ± Age at diagnosis 37.7 ± ± (yr, mean ± SD) Disease duration 12.1 ± ± (yr, mean ± SD) Sex (male/female) 59/7 63/ Race White 49 (74.2) 54 (79.4) 0.54 Black 15 (22.7) 12 (17.6) 0.52 Hispanic 2 (3.0) 2 (2.9) 1.00 Medication use 5-ASAs 50 (75.8) 54 (79.4) 0.68 Immunomodulators 17 (25.8) 30 (44.1) 0.03 Biologics 12 (18.2) 15 (22.1) 0.67 Antibiotics 0 (0) 0 (0) - Steroids 3 (4.5) 0 (0) 0.12 CD Colonic disease 11 (16.7) 8 (11.8) 0.47 Ileal disease 9 (13.6) 7 (10.3) 0.60 Ileocolonic 12 (18.2) 15 (21.1) 0.67 UC Extensive 18 (27.3) 19 (27.9) 1.00 Left-sided 15 (22.7) 13 (19.1) 0.67 Proctitis 1 (1.5) 5 (7.4) 0.21 Indeterminate colitis CD: Crohn s disease; UC: Ulcerative colitis. ease characteristics, and demographics. Repeat laboratory work was not mandated by study participation and data were only available if these tests were clinically indicated by their treating provider. Therefore, stool tests were typically collected only in patients with flares who were willing to produce a stool sample. Statistical analysis Analyses were performed using Fisher s exact test for categorical variables, and the unpaired t test (normally distributed) or Mann-Whitney test (not normally distributed) for continuous variables. Numbers are expressed as mean ± SD. Multivariate logistic regression analysis was performed using SAS and the macro by Bursac and colleagues (2008) Purposeful Selection of Analyses [15]. The macro by Bursac and colleagues is a SAS algorithm that automates the variable selection process for logistic regression analysis. Any variable having a significant univariate test at P = 0.25 is selected as a candidate for the multivariate analysis. In the iterative process of variable selection, covariates are removed from the model if they are nonsignificant and not a confounder. Significance is evaluated at the 0.1 α level and confounding as a change in any parameter estimate > 20%. The macro allows the user to specify all decision criteria. At the end of this iterative process, the model contains significant covariates and confounders. At this point any variable not selected for the original multivariate model is added back one at a time, with significant covariates and confounders retained earlier. Any that are significant at the 0.1 level are put in the model, and the model is iteratively reduced as before but only for the variables that are additionally added. For the analysis of risk factors for flares, the variables evaluated were immunomodulator use, NSAID use, smoking, antibiotics, stress, non-adherence, infections, and travel. For the analysis of risk factors for non-adherence, the variables evaluated were type of medication used for IBD (5-ASA, immunomodulator, and biologics; too few patients were taking steroids or antibiotics to be included in the analysis), number of medications taken, age, disease type (UC or CD), disease duration, and race. Age was evaluated as a continuous variable. The final model for flares contained three retained predictors: immunomodulator use, non-compliance, and infection. The final model for non-compliance contained one retained predictor, age. This study is registered at ClinicalTrials.gov, # NCT RESULTS A total of 134 patients with IBD (63 CD, 70 UC, and 1 indeterminate colitis) were enrolled in the study; 66 patients had flares of IBD and 68 were controls with IBD in remission. The two groups (flares and controls) had no significant differences with regard to age, sex, race, medication use, and disease type and location (Table 1). However, the use of immunomodulators (6-mercaptopurine, azathioprine or methotrexate) was significantly higher in the control group. As would be expected, the mean CDAI score of the patients with flares of CD was significantly higher than that of control patients with CD in remission (356 vs 69, P < ) (Table 2). Similarly, the mean Mayo scores of patients with flares of UC were significantly higher than those of control patients with UC in remission (7.55 vs 1 in patients with a full Mayo score, and 5.41 vs 0.1 in those with a partial Mayo score, both P < ). In addition, the flare group had significantly lower hemoglobin and albumin levels, as well as significantly higher levels of C-reactive protein (CRP) and higher erythrocyte sedimentation rates than the controls. Although routine stool studies were not done in our patients in remission, most those with exacerbation of their disease did have their stools analyzed. Of 40 assessed, two were positive for Clostridium difficile toxin (5%), none had a positive stool culture, and none had a positive stool evaluation for ova and parasites. Potential factors for exacerbation of IBD No significant differences between the flare and control groups were observed (Figure 1) for recent NSAID use (62.1% flares vs 61.8% controls, P > 0.99, OR = 1.02, 95%CI: ), pertinent tobacco usage (9.1% flares vs 8.8% controls, P > 0.99, OR = 1.03, 95%CI: ), recent antibiotic use (25.8% flares vs 23.5% controls, P = 0.84, OR = 1.13, 95%CI: ), major life stressors (47.0% flares vs 39.7% controls, P = 0.49, OR = 1.35, 95%CI: ), recent infections (33.3% flares vs 45.6% controls, P = 0.16, OR = 0.60, 95%CI: ), and recent travel (31.8% flares vs 29.4% controls, P = 0.85, OR = 1.12, 95%CI: ). Of 4331 April 21, 2014 Volume 20 Issue 15

233 Feagins LA et al. Triggers in inflammatory bowel disease Table 2 Disease indices in patients with a disease exacerbation (flares) and those in remission (controls) Disease indices Flares Controls P value Average CDAI score (CD) ± ± 52 < Average full Mayo score (UC) ± ± 1.1 < Average partial Mayo score 5.41p ± p ± 0.3 < (UC) 1 C-reactive protein 3.48 ± ± (mg/dl) (normal 0-1.0) ESR (MM/HR) (normal 0-30) 29.9 ± ± Albumin (g/dl) 3.40 ± ± 0.4 < (normal ) Hemoglobin (g/dl) (normal ) 13.1 ± ± One of two values for Mayo score per patient, patients had either full score (n = 19) or partial score (n = 51) depending on whether the patient had sigmoidoscopy. ESR: Erythrocyte sedimentation rate. CDAI: Crohn's Disease Activity Index; CD: Crohn s disease; UC: Ulcerative colitis. In this case-control study in which we systematically searched for a number of IBD triggers in our veteran patients with CD and UC, only medication non-adherence was significantly associated with flares of IBD. We found no significant differences between flare patients and controls in the frequency of other proposed triggers including recent infections, NSAID use, antibiotic use, stressful life events, travel away from home, and cigarette smoking. This is an interesting finding, particularly in a veteran population in which access to medications should not be a limiting factor. The two most common reasons reported for medication non-adherence were failure to refill medication prescriptions and forgetting to take the medications as prescribed. When the groups were subdivided by type of IBD (UC vs CD), non-adherence to medications remained a significant predictor of flare only for patients with UC. Multivariate logistic regression analysis confirmed that non-adherence was significantly associated with flares, increasing the risk of flare almost threefold. Multivariate logistic regression analysis for predictors of non-adherence revealed only age to be a significant factor, with younger patients being less compliant. Specifall the potential triggers, only non-adherence with prescribed medication was significantly associated with IBD flares (48.5% flares vs 29.4% controls, P = 0.03, OR = 2.26, 95%CI: ). We performed separate subgroup analyses comparing the frequency of potential triggers in patients who had CD in the flare group with that in patients who had CD in the control group, and comparing the frequency of potential triggers in patients who had UC in the flare group with that in patients who had UC in the control group. There was no significant difference between flares and controls with CD in the frequency of medication nonadherence (39.4% flares vs 26.7% controls, P = 0.42). For patients with UC, however, medication non-adherence was significantly more frequent in the flare group (57.6% flares vs 32.4% controls, P = 0.05). Subgroup analyses for all other potential triggers revealed no significant differences between the flare and control groups. Multivariate logistic regression analysis revealed that medication non-adherence and immunomodulator use were significantly associated with flares. The adjusted OR for medication non-adherence was 2.86 (95%CI: ), indicating that patients who were not compliant were almost three times more likely to have a flare than those who took their medications as prescribed. The adjusted OR for immunomodulator use was 0.40 (95%CI: ), indicating that patients using immunomodulators were 60% less likely to have a flare. Patients were questioned regarding the frequency with which they missed doses of their medications. Approximately half of them admitted to missing a dose at least once a month or more, and 28.4% missed a dose at least 2-4 times per month or more. The most common reasons given for missing a dose was running out of medication or having difficulty remembering to take the medication (Figures 2 and 3). We performed multivariate logistic regression analyses to identify predictors of non-adherence. We evaluated several factors that could contribute to non-adherence including: number of medications being prescribed for Patients reporting exacerbating factor (%) P > 0.99 OR = 1.02 NSAID use IBD, type of medications prescribed to treat IBD (5-ASA, immunomodulator, or biologics), age, disease type (CD or UC), disease duration, and race. Of these, only age was a significant predictor for non-adherence, with younger patients more likely to be noncompliant. When age was evaluated as a continuous variable, the OR for age was 0.96 (95%CI: ), indicating that for every 1 year older, the patient was 4% more likely to be compliant. When evaluated on a yearly basis, for every 5 years older, the patient was 18.5% more likely to be compliant and for every decade older, the patient was 33.5% more likely to be compliant. DISCUSSION P > 0.99 OR = 1.03 Tobacco P = 0.84 OR = 1.13 Antibiotic use P = 0.49 OR = 1.35 Stress P = 0.03 OR = 2.26 Non-compliance P = 0.16 OR = 0.60 Infections Controls Flares P = 0.85 OR = 1.12 Travel Figure 1 Frequency of potential triggers in patients with a flare of inflammatory bowel disease and in control patients in remission for at least 6 mo. NSAID: Nonsteroidal anti-inflammatory drug April 21, 2014 Volume 20 Issue 15

234 Feagins LA et al. Triggers in inflammatory bowel disease 40 Flares Controls Flares Controls No. of patients 20 No. of patients Never 1x/mo 2-4x/mo 1-3x/wk 4-5x/wk Frequency of medication non-compliance 1x/d 0 Ran out of medications Difficulty remembering Not tolerating Just not taking Figure 2 Frequencies of missing a dose of medication to treat inflammatory bowel disease. ic medications (5-ASAs vs immunomodulators vs biologics), number of medications prescribed, disease type (UC vs CD), disease duration, and race did not significantly affect adherence. Non-adherence to prescribed medication regimens is a common problem in patients with chronic diseases that require lifelong maintenance therapies. One study of patients with UC found that those who did not take their mesalamines as prescribed had a fivefold higher risk of flare than patients who were compliant with mesalamine therapy [16]. Another study explored factors associated with adherence in 106 IBD patients, and found lower adherence with medications dosed three or more times per day [17]. In our study, our patients taking mesalamines were dosed two or three times per day, because once-daily doses of mesalamine were not available at our institution during the time of this study. We assessed type of medication being taken (with the assumption that mesalamines are dosed several times per day), but found no increased risk of non-adherence for treatment with mesalamines as compared to other IBD treatments. The only factor that we identified as associated with non-adherence was younger age. These findings, in our unique study population of veterans, in which access to or cost of medications should not be a limiting factor, highlights the notion that other factors apart from cost or access are involved in non-adherence. On multivariate regression analysis, immunomodulator use was found to be significantly associated with lower rates of flares. This did not seem to be related to adherence because we evaluated specific medications (5-ASAs vs immunomodulators vs biologics) in a multivariate analysis for predictors of non-adherence and did not find these to be a significant predictor. These findings suggest that using an immunomodulator lowers the risk of flares, at least in our population of patients. The major limitation of our study was recall bias, because patients were queried and asked to remember possible triggers or exposures occurring in the preceding Figure 3 Reasons for medication non-adherence. 3 mo. To minimize this potential bias regarding medication triggers, we reviewed the medical records to identify recent medications (NSAIDs and antibiotics) that were dispensed by the VA pharmacy. However, this review did not identify the use of over-the-counter drugs. NSAID use recall during patient interviews was assisted by reviewing with the patients a list of available proprietary NSAID medications that included their commercial and generic names. Medication adherence was also assessed by patient recall, as a written drug log was not possible with our study design of enrolling patients when they presented to the hospital or clinic with an IBD flare (i.e., patients could not be identified 3 mo prior to the start of a log). To minimize recall bias regarding medication adherence, we asked multiple questions about adherence including queries about the frequency of missing medications, of running out of medications, of problems in tolerating medications, and of failure to take the medication. In conclusion, our study suggests that medication non-adherence is the most important of the proposed triggers for IBD flares. We found no association between flares and other putative triggers including NSAIDS, antibiotics, recent infections, tobacco use, travel and emotional stress. Our findings suggest that these factors either are not triggers at all, or are only weak triggers for IBD flares that might be identified only in much larger studies. Our report highlights the importance of medication adherence for patients with IBD. COMMENTS Background Several factors have been proposed as triggers for flares of inflammatory bowel diseases (IBD), including medications [nonsteroidal anti-inflammatory drugs (NSAIDs) and antibiotics], infections, travel, emotional stress, tobacco use, and poor adherence with prescribed medications. Data implicating these factors have come largely from retrospective, uncontrolled studies. Research frontiers A number of disparate factors (e.g., certain drugs, infections, tobacco, stress, 4333 April 21, 2014 Volume 20 Issue 15

235 Feagins LA et al. Triggers in inflammatory bowel disease and poor adherence) have been proposed as triggers for flares of IBD. Flares of IBD decrease quality of life greatly and carry a high burden of disease. If physicians and patients were better able to identify modifiable triggers for these flares, these times of disease exacerbation might be fewer. Innovations and breakthroughs The authors enrolled patients with IBD and active flares of their disease and queried them about exposure to potential triggers for their flares (NSAIDs, antibiotics, stress, tobacco use, medication adherence, infections, and travel). Disease activity indices were calculated and data on any available laboratory studies and endoscopy studies were collected. The authors also enrolled patients with IBD in remission and collected the same data. Applications The results suggest that medication non-adherence is the most important of the proposed triggers for flares of IBD. Moreover, use of an immunomodulator, such as azathioprine, mercaptopurine or methotrexate, was associated with a decreased risk of flares. Physicians should be aware of these findings to assist in counseling their patients on the importance of medication adherence. Peer review This is a well written report of a case-control study that studied the association between IBD flares and potential triggering factors. The research question studied was an important one given the physical, emotional, and economic costs of disease relapse. The results are interesting and suggest that medication nonadherence is an important trigger for flares. REFERENCES 1 Feagins LA, Cryer BL. Do non-steroidal anti-inflammatory drugs cause exacerbations of inflammatory bowel disease? Dig Dis Sci 2010; 55: [PMID: DOI: / s ] 2 Aberra FN, Brensinger CM, Bilker WB, Lichtenstein GR, Lewis JD. Antibiotic use and the risk of flare of inflammatory bowel disease. Clin Gastroenterol Hepatol 2005; 3: [PMID: ] 3 Issa M, Ananthakrishnan AN, Binion DG. Clostridium difficile and inflammatory bowel disease. Inflamm Bowel Dis 2008; 14: [PMID: DOI: /ibd.20500] 4 Cosnes J, Carbonnel F, Carrat F, Beaugerie L, Cattan S, Gendre J. Effects of current and former cigarette smoking on the clinical course of Crohn s disease. Aliment Pharmacol Ther 1999; 13: [PMID: ] 5 Beaugerie L, Massot N, Carbonnel F, Cattan S, Gendre JP, Cosnes J. Impact of cessation of smoking on the course of ulcerative colitis. Am J Gastroenterol 2001; 96: [PMID: ] 6 Birrenbach T, Böcker U. Inflammatory bowel disease and smoking: a review of epidemiology, pathophysiology, and therapeutic implications. Inflamm Bowel Dis 2004; 10: [PMID: ] 7 Vidal A, Gómez-Gil E, Sans M, Portella MJ, Salamero M, Piqué JM, Panés J. Life events and inflammatory bowel disease relapse: a prospective study of patients enrolled in remission. Am J Gastroenterol 2006; 101: [PMID: ] 8 Melgar S, Engström K, Jägervall A, Martinez V. Psychological stress reactivates dextran sulfate sodium-induced chronic colitis in mice. Stress 2008; 11: [PMID: DOI: / ] 9 Qiu BS, Vallance BA, Blennerhassett PA, Collins SM. The role of CD4+ lymphocytes in the susceptibility of mice to stress-induced reactivation of experimental colitis. Nat Med 1999; 5: [PMID: ] 10 Singh S, Graff LA, Bernstein CN. Do NSAIDs, antibiotics, infections, or stress trigger flares in IBD? Am J Gastroenterol 2009; 104: ; quiz 1314 [PMID: DOI: /ajg ] 11 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 ] 12 Jackson CA, Clatworthy J, Robinson A, Horne R. Factors associated with non-adherence to oral medication for inflammatory bowel disease: a systematic review. Am J Gastroenterol 2010; 105: [PMID: DOI: / ajg ] 13 Best WR, Becktel JM, Singleton JW, Kern F. Development of a Crohn s disease activity index. National Cooperative Crohn s Disease Study. Gastroenterology 1976; 70: [PMID: ] 14 Schroeder KW, Tremaine WJ, Ilstrup DM. Coated oral 5-aminosalicylic acid therapy for mildly to moderately active ulcerative colitis. A randomized study. N Engl J Med 1987; 317: [PMID: ] 15 Bursac Z, Gauss CH, Williams DK, Hosmer DW. Purposeful selection of variables in logistic regression. Source Code Biol Med 2008; 3: 17 [PMID: DOI: / ] 16 Kane S, Huo D, Aikens J, Hanauer S. Medication nonadherence and the outcomes of patients with quiescent ulcerative colitis. Am J Med 2003; 114: [PMID: ] 17 Bermejo F, López-San Román A, Algaba A, Guerra I, Valer P, García-Garzón S, Piqueras B, Villa C, Bermejo A, Rodríguez-Agulló JL. Factors that modify therapy adherence in patients with inflammatory bowel disease. J Crohns Colitis 2010; 4: [PMID: DOI: / j.crohns ] P- Reviewers: Adams SV, Chen JL, Fellermann K, Reigada LC, Vetvicka V S- Editor: Zhai HH L- Editor: Kerr C E- Editor: Zhang DN 4334 April 21, 2014 Volume 20 Issue 15

236 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH BRIEF ARTICLE REPORT Pediatric non-alcoholic steatohepatitis: The first report on the ultrastructure of hepatocyte mitochondria Joanna M Lotowska, Maria E Sobaniec-Lotowska, Sylwia B Bockowska, Dariusz M Lebensztejn Joanna M Lotowska, Department of General Pathomorphology, Medical University of Bialystok, Bialystok, Poland Maria E Sobaniec-Lotowska, Department of Medical Pathomorphology, Medical University of Bialystok, Bialystok, Poland Sylwia B Bockowska, Department of Laboratory Diagnostics, Maria Sklodowska-Curie Memorial Bialystok Oncology Center, Bialystok, Poland Dariusz M Lebensztejn, Department of Pediatrics, Gastroenterology and Allergology, Medical University of Bialystok, Bialystok, Poland Author contributions: Sobaniec-Lotowska ME and Lotowska JM contributed equally to this work; Sobaniec-Lotowska ME, Lotowska JM, Bockowska SB and Lebensztejn DM designed the research and wrote the paper. Correspondence to: Maria E Sobaniec-Lotowska, MD, PhD, Professor of Medicine, Department of Medical Pathomorphology, Medical University of Bialystok, Waszyngtona Street 13, Bialystok, Poland. maria.sobaniec-lotowska@umb.edu.pl Telephone: Fax: Received: August 21, 2013 Revised: December 2, 2013 Accepted: January 2, 2014 Published online: April 21, 2014 Abstract AIM: To investigate the ultrastructure of abnormal hepatocyte mitochondria, including their cellular and hepatic zonal distribution, in bioptates in pediatric nonalcoholic steatohepatitis (NASH). METHODS: Ultrastructural investigations were conducted on biopsy liver specimens obtained from 10 children (6 boys and 4 girls) aged 2-14 years with previously clinicopathologically diagnosed NASH. The disease was diagnosed if liver biopsy revealed steatosis, inflammation, ballooned hepatocytes, Mallory hyaline, or focal necrosis, varying degrees of fibrosis in the absence of clinical, serological, or histological findings of infectious liver diseases, autoimmune hepatitis, metabolic liver diseases, or celiac disease. For ultrastructural analysis, fresh small liver blocks (1 mm 3 volume) were fixed in a solution containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 mol/l cacodylate buffer. The specimens were postfixed in osmium tetroxide, subsequently dehydrated through a graded series of ethanols and propylene oxide, and embedded in Epon 812. The material was sectioned on a Reichert ultramicrotome to obtain semithin sections, which were stained with methylene blue in sodium borate. Ultrathin sections were contrasted with uranyl acetate and lead citrate, and examined using an Opton EM 900 transmission electron microscope. RESULTS: Ultrastructural analysis of bioptates obtained from children with non-alcoholic steatohepatitis revealed characteristic repetitive mitochondrial abnormalities within hepatocytes; mainly mitochondrial polymorphisms such as megamitochondria, loss of mitochondrial cristae, and the presence of linear crystalline inclusions within the mitochondrial matrix of an increased electron density. The crystalline inclusions were particularly evident within megamitochondria (MMC), which seemed to be distributed randomly both within the hepatic parenchymal cell and the zones of hepatic lobule, without special variations in abundance. The inclusions appeared as bundles viewed longitudinally, or as an evenly spaced matrix in cross section, and frequently caused mitochondrial deformation. The average diameter of these linear structures was 10 nm and the average space between them 20 nm. Sometimes enlarged intramitochondrial granules were seen in their vicinity. Foamy cytoplasm of hepatocytes was found, resulting from the proliferation of smooth endoplasmic reticulum and glycogen accumulation. The perivascular space of Disse was frequently dilated, and contained transitional hepatic stellate cells, as well as mature and/or newly forming collagen fiber bundles. CONCLUSION: Marked ultrastructural abnormalities observed in hepatocyte mitochondria, especially their polymorphism in the form of MMC and loss of mitochondrial cristae, accompanied by foamy cytoplasm, 4335 April 21, 2014 Volume 20 Issue 15

237 Lotowska JM et al. Mitochondrial ultrastructure in pediatric NASH clearly indicate a major role of these organelles in the morphogenesis of pediatric NASH. Our findings seem to prove the high effectiveness of electron microscopy in the diagnosis of the disease Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Pediatric non-alcoholic steatohepatitis; Hepatocyte ultrastructure; Megamitochondria with crystalline inclusions; Foamy cytoplasm of hepatocytes Core tip: Our electron-microscopic analysis of liver bioptates, being the first known investigation into pediatric non-alcoholic steatohepatitis (NASH), revealed characteristic repetitive mitochondrial abnormalities within hepatocytes, mainly mitochondrial polymorphism in the form of megamitochondria, loss of cristae, and the presence of linear crystalline inclusions within the mitochondrial matrix of increased electron density, frequently accompanied by foamy cytoplasm. As the discovery of these morphological indices of hepatocyte injury may be very useful in the diagnosis of NASH in pediatric patients, the ultrastructural analysis of liver biopsy can be recommended as an effective diagnostic method. Lotowska JM, Sobaniec-Lotowska ME, Bockowska SB, Lebensztejn DM. Pediatric non-alcoholic steatohepatitis: The first report on the ultrastructure of hepatocyte mitochondria. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: INTRODUCTION Non-alcoholic fatty liver disease (NAFLD), regarded as a hepatic manifestation of metabolic syndrome, is a growing health problem worldwide both among children and adults. Importantly, the more severe form of this condition, non-alcoholic steatohepatitis (NASH), may progress to fibrosis, as well as cryptogenic cirrhosis and its complications, including hepatocellular carcinoma [1-6]. Unfortunately, the pathogenesis of NASH still remains unknown. The last decade has brought increasing evidence that mitochondrial dysfunction, more specifically respiratory chain deficiency, plays a causative role in the pathophysiology of NASH, whatever its initial cause [2,7-14]. Basaranoglu et al [2] have noted that the pathogenesis of NASH is multifactorial, and that excess intracellular fatty acids, oxidant stress, ATP depletion, and mitochondrial dysfunction are essential for hepatocyte injury in this condition. Despite clinical symptoms, laboratory data, and imaging findings, the microscopic analysis of liver biopsies still remains the golden standard for the diagnosis of non-alcoholic steatohepatitis [4,7]. Histopathological investigations of liver biopsy specimens from NASH patients have revealed, aside from steatosis accompanied by lobular inflammatory infiltration, evidence of hepatocyte injury in the absence of clinical, serological, or histological findings of infectious liver diseases, autoimmune hepatitis, metabolic liver disease, or celiac disease [1,4,7,8,15]. Damage to liver parenchymal cells have been demonstrated in the form of hydropic change (ballooning cell injury), Mallory s hyaline bodies, and focal lytic necrosis causing sinusoidal distortion, and reduced intrasinusoidal volume and microvascular blood flow [1,3,4,7,8,15-17]. It has been reported that in the course of non-alcoholic steatohepatitis, cell types other than hepatocytes are involved, i.e., non-parenchymal hepatic cells: hepatic stellate cells (HSCs), Kupffer cells, sinusoidal endothelial cells, and inflammatory cells and platelets. This leads to deregulation of microvascular blood flow and may result in pericellular/perisinusoidal fibrosis and cirrhosis [5,6,17-19]. However, the diagnostic criteria of NASH have not yet been established. In spite of increasing interest in the morphogenesis of non-alcoholic steatohepatitis, there are only a few reports, mainly limited to adult patients, on the ultrastructure of hepatocytes in this pathology [20-24]. We could not find any other publications to compare to the current findings, apart from our Letter to the Editor of the American Journal of Gastroenterology [25] on the ultrastructure of hepatocyte mitochondria in five children with NASH published 10 years ago. Thus, the study objective was to analyze the image of abnormal electron microscopic findings observed in the hepatocytes of liver biopsy material collected from a larger group of children with clinicopathologically diagnosed NASH. The search for repetitive specific ultrastructural indices of this disease in liver bioptates would facilitate its early morphological diagnosis, which may result in undertaking targeted therapy. MATERIALS AND METHODS Patients This study was carried out in accordance with the Declaration of Helsinki (2000) of the World Medical Association. Ultrastructural investigations were conducted on liver specimens obtained by needle biopsy from 10 children (6 boys and 4 girls) aged 2-14 years (mean 5 years) with previously clinicopathologically diagnosed NASH. Laboratory tests revealed increased serum alanine transaminase activity, and ultrasound examination showed liver brightness in all study patients. NASH was diagnosed if the liver biopsy revealed steatosis, inflammation, ballooned hepatocytes, Mallory hyaline, focal necrosis, or varying degrees of fibrosis in the absence of clinical, serological, or histological findings of infectious liver diseases (hepatitis B, hepatitis C, cytomegalovirus, or Toxoplasma gondii), autoimmune hepatitis, metabolic liver diseases (Wilson s disease, cystic fibrosis, galactosemia, or 4336 April 21, 2014 Volume 20 Issue 15

238 Lotowska JM et al. Mitochondrial ultrastructure in pediatric NASH alpha 1-antitrypsin deficiency), and celiac disease. Alcohol consumption was excluded in all the children. The study was carried out in the Department of Medical Pathomorphology, Medical University of Bialystok, by an electron microscopist blinded to the clinical information. Ultrastructural analysis For transmission electron microscopic investigations, fresh small tissue blocks (1 mm 3 volume) from the liver bioptates were fixed in a solution containing 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 mol/l cacodylate buffer (ph 7.4) at room temperature. Subsequently, the specimens were postfixed in 2% osmium tetroxide (OsO4) in 0.1 mol/l cacodylate buffer (ph 7.4) for 1 h. The material was then dehydrated through a graded series of ethanols and propylene oxide, embedded in Epon 812, and sectioned on a Reichert ultramicrotome (Reichert Ultracut S) to obtain semithin sections. Next, the sections were stained with 1% methylene blue in 1% sodium borate and preliminarily examined under a light microscope to select Epon blocks. The blocks were then used to prepare ultrathin sections ca. 75 nm thick, which were placed on grids, double stained with uranyl acetate and lead citrate, examined, and photographed via an Opton EM 900 transmission electron microscope (Zeiss, Oberkochen, Germany). This processing procedure had been used in our earlier ultrastructural investigations of the liver in children [18,25,26]. RESULTS Out of 10 clinicopathologically diagnosed cases of pediatric NASH, nine presented with repetitive mitochondrial abnormalities observed within hepatic parenchymal cells under transmission electron microscope. In 7 patients, the majority of mitochondria were affected, especially within ballooned hepatocytes; in 2 cases the abnormalities were found in a smaller number of hepatic parenchymal cells. The mitochondrial lesions showed no particular differences in quality or topography. They were similarly pronounced in the organelles located both in the vicinity of the cell nucleus, at a certain distance from the nucleus, and on the cell periphery (Figure 1). The major submicroscopical exponent was the polymorphism of these organelles, with the presence of the so-called megamitochondria, accompanied by variously pronounced defects of the cristae, including their complete loss and increased electron-density of the matrix to its condensation (Figure 1). Frequently, remnants of such cristae, resembling bristles, were found on the periphery of enlarged organelles. The characteristic feature of the ultrastructure of the altered mitochondria, especially megamitochondria, was the presence of intramitochondrial linear crystalline inclusions within the changed matrix, seen as long parallel strands that often caused deformation of these organelles. They appeared as bundles when viewed longitudinally, or as an evenly-spaced matrix in cross section (Figure 1). The average diameter of these linear structures was 10 nm and the average space between them 20 nm. Sometimes in their vicinity there were enlarged intramitochondrial dense granules (Figure 1C, E, F). The megamitochondria with characteristic crystalline inclusions (MMC) seemed to be distributed randomly, both within the hepatic parenchymal cell and the zones of the hepatic lobule, without special variations in abundance. They were found in close vicinity to lipid material (Figure 1C, E, F), and also in hepatocytes with no distinct features of steatosis. We also observed a wide range of changes in hepatocellular ultrastructure, including the foamy cytoplasmic appearance resulting from smooth endoplasmic reticulum proliferation (Figure 1A, B, D) and glycogen accumulation (Figure 1C, E, F), mainly in the nuclear regions (Figure 1C). Heterogeneous lipofuscin pigment granules of various size and shapes were occasionally found. The sinusoids with the adherent vascular pole of hepatocytes that showed distinct mitochondrial lesions had markedly swollen endothelial linings (Figure 1A). Kupffer cells in the sinusoidal lumen were considerably activated, sometimes showing features of erythrophagocytosis (their morphology in children with NASH was described in our earlier paper) [18]. The perivascular space of Disse was frequently dilated and contained transitional (i.e., activated) hepatic stellate cells (t-hscs) (Figure 1B), deposits of electron-dense material, as well as mature and/or collagen forming fiber bundles (Figure 1A, B). DISCUSSION Although light microscopy is routinely used to evaluate liver biopsy specimens from NASH patients, the ultrastructural investigations that make the diagnosis more profound are conducted in a limited number of cases, especially with respect to children. No reports have been published, apart from those in our center, on the electron-microscopic picture of liver bioptates from pediatric patients with NASH. We focused not only on hepatocytes [25,27], but also on the changes concerning non-parenchymal liver cells, especially Kupffer and liver progenitor/oval cells [18,26,28]. The current ultrastructural observations of hepatocytes obtained from pediatric NASH biopsies were consistent with our preliminary findings presented recently at the 186 th Falk Symposium [27], as well as in the previously mentioned Letter to the Editor [25]. The studies revealed numerous mitochondrial abnormalities similar to those found in adults [20,24], including polymorphisms such as megamitochondria, loss of cristae, increased electrondensity of the matrix, and the presence of characteristic linear crystalline inclusions within the matrix. The megamitochondria with crystalline inclusions were seen as parallel strands, and reduced cristae were the major repetitive ultrastructural abnormalities in pediatric NASH cases 4337 April 21, 2014 Volume 20 Issue 15

239 Lotowska JM et al. Mitochondrial ultrastructure in pediatric NASH A En B * C Li * * N t-hsc D E F Li bc Li Li Figure 1 Ultrastructural appearance of polymorphic hepatic mitochondria, with megamitochondria at the foreground, containing linear crystalline inclusions and reduced cristae in bioptates from different pediatric patients with non-alcoholic steatohepatitis. Abnormal mitochondria distributed randomly in topographically varied parts of hepatocytes. A-F: Linear inclusions - cut longitudinally (arrows) and in cross section (arrowheads) in the matrix of moderately or markedly increased electron density in the majority of altered mitochondria, especially elongated and rounded megamitochondria. A, B: Megamitochondria (MMC) located in the vascular pole of hepatocytes; the mitochondrial lesions are accompanied by the proliferation of smooth endoplasmic reticulum; considerably swollen endothelial cell (En) of the sinusoidal vessel; transitional hepatic stellate cells (t-hsc), accumulation of electron-dense material, and collagen formed (asterisk) in the dilated space of Disse (original magnification 7000); C: The MMC located in the nuclear region of hepatocyte is accompanied by glycogen accumulation; the megamitochondrium shows enlarged intramitochondrial dense granules in the vicinity of linear crystalline inclusions; hepatocyte nucleus (N); lipid material (Li) within hepatocyte cytoplasm (original magnification 12000); D: MMC distributed randomly within the biliary pole of hepatocytes accompanied by the proliferation of smooth endoplasmic reticulum; bc - biliary canaliculus (original magnification 7000); E, F: MMC located at a certain distance from the nucleus of hepatocyte; mitochondrial abnormalities are accompanied by glycogen accumulation; some MMC show enlarged intramitochondrial dense granules in the vicinity of linear crystalline inclusions; lipid material (Li) (original magnification 12000, 20000, respectively). analyzed in the current study. These MMC seemed to be distributed randomly, both within the hepatocytes and the zones of the hepatic lobule. In addition to mitochondrial alterations, we quite frequently observed foamy cytoplasm of hepatocytes resulting from the proliferation of smooth endoplasmic reticulum and glycogen accumulation, which was also reported by Ahishai et al [20] in adult patients with NASH. The space of Disse was frequently dilated and contained t-hscs, which were accompanied by electron-dense material and collagen accumulation. However, the sinusoids were lined with distinctly swollen endothelium and markedly activated Kupffer cells. The mitochondrial abnormalities showing varying degrees of cristae loss and increased matrix density evidently indicate decreased intra-mitochondrial protein synthesis and respiratory chain dysfunction, which was also emphasized by Ahishali et al [20] with respect to adult patients with NASH. However, according to Caldwell et al [21] and Le et al [22], the formation of megamitochondria with crystalline inclusions of unknown composition may, in patients with NASH, represent an adaptive process to oxidative stress rather than a secondary injury. The formation of crystalline inclusions in the mitochondrial matrix has been noted in a variety of other con- ditions. Similar crystal-like structures have been reported mainly in the early phase of alcoholic steatohepatitis or in copper metabolism disorders such as Wilson s disease [29,30]. As already mentioned, they have been recently described in non-alcoholic fatty liver in adult patients, where their presence is correlated indirectly with oxidative stress [20,24], and their absence is noted when cirrhosis develops [21,24]. According to Le et al [22], as well as in our own observations concerning children, in NASH the MMC seem to be distributed randomly within the hepatic zones and without variation in abundance, regardless of the stage of fibrosis. In contrast, rodent models of NASH do not develop mitochondrial crystalline inclusions. The major electron-microscopic changes in rodents include severe mitochondrial swelling with coexisting proliferation of the smooth endoplasmic reticulum and abnormal granular endoplasmic reticulum [31]. Considering the role of mitochondria in the pathophysiology of NASH, it has been emphasized that hepatic oxidative stress increases the release of lipid peroxidation products, as reflected by increased fatty acid beta-oxidation and cytokines, which together trigger liver lesions [2,7,9,10,12,13,19,24]. In conclusion, this is the first full report on the hepatocyte ultrastructure in pediatric NASH. In our study, the 4338 April 21, 2014 Volume 20 Issue 15

240 Lotowska JM et al. Mitochondrial ultrastructure in pediatric NASH most significant repetitive electron microscopic alterations in this pathology involved the appearance of randomly distributed megamitochondria within hepatocytes, with characteristic crystalline inclusions seen as parallel strands and reduced mitochondrial cristae, including their complete loss. Additionally, foamy cytoplasm of liver parenchymal cells was found, resulting from the proliferation of smooth endoplasmic reticulum and glycogen accumulation. The submicroscopic changes observed in hepatocytes, especially mitochondrial abnormalities, may play a major role in the morphogenesis of pediatric nonalcoholic steatohepatitis. We hope that similar reports will appear in other centers to provide comparative material for our current study in children. The discovery of submicroscopic repetitive indices of damage to liver parenchymal cells, specific to this pathology, may have both diagnostic and therapeutic implications for the treatment of fatty liver disease in pediatric patients. Considering the great usefulness of transmission electron microscopy in the diagnosis of NASH in children, ultrastructural analysis of liver bioptates should be recommended as an effective diagnostic tool, at least in diagnostically difficult cases, despite its high costs and effort. COMMENTS Background Despite increasing interest in non-alcoholic steatohepatitis (NASH), there are only a few reports on the hepatocyte ultrastructure in this pathology. This manuscript is the first full original paper known to be conducted on biopsy material in the course of this pathology in children. Innovations and breakthroughs The main objective of the study was ultrastructural analysis of the image of abnormal hepatocyte mitochondria, including their cellular and hepatic zonal distribution in biopsy material in clinicopathologically diagnosed NASH in children. The electron microscopic investigations revealed characteristic mitochondrial structural defects within hepatocytes, mainly mitochondrial polymorphism (megamitochondria), loss of mitochondrial cristae, and the presence of linear crystalline inclusions within the mitochondrial matrix of increased electron density. The crystalline inclusions were particularly evident within megamitochondria. The megamitochondria with characteristic inclusions (MMC) seemed to be distributed randomly, both within the cell and in the hepatic zones, without any special variation in abundance. In addition to mitochondrial alterations, the authors could quite frequently observe foamy cytoplasm of hepatocytes resulting from the proliferation of smooth endoplasmic reticulum and glycogen accumulation. Applications The authors ultrastructural investigations indicate that pediatric NASH is associated with marked abnormalities in hepatocyte mitochondria, especially their polymorphism in the form of MMC, as well as the loss of mitochondrial cristae accompanied by foamy cytoplasm of hepatocytes. These findings seem to prove the high effectiveness of electron microscopy in the diagnosis of the disease. Peer review This study appears to be properly conducted form a technical view-point. The conclusions of the authors are supported by the data reported. REFERENCES 1 Alexander J, Torbenson M, Wu TT, Yeh MM. Non-alcoholic fatty liver disease contributes to hepatocarcinogenesis in non-cirrhotic liver: a clinical and pathological study. J Gastroenterol Hepatol 2013; 28: [PMID: DOI: /jgh.12116] 2 Basaranoglu M, Basaranoglu G, Sentürk H. From fatty liver to fibrosis: a tale of second hit. World J Gastroenterol 2013; 19: [PMID: DOI: /wjg.v19.i8.1158] 3 Caldwell SH, Lee VD, Kleiner DE, Al-Osaimi AM, Argo CK, Northup PG, Berg CL. NASH and cryptogenic cirrhosis: a histological analysis. Ann Hepatol 2009; 8: [PMID: ] 4 Brunt EM, Tiniakos DG. Histopathology of nonalcoholic fatty liver disease. World J Gastroenterol 2010; 16: [PMID: ] 5 Jeen YM, Jin SY. [Pathology of nonalcoholic steatohepatitis]. Korean J Hepatol 2009; 15: [PMID: DOI: /kjhep ] 6 Farrell GC, Teoh NC, McCuskey RS. Hepatic microcirculation in fatty liver disease. Anat Rec (Hoboken) 2008; 291: [PMID: DOI: /ar.20715] 7 Takahashi Y, Fukusato T. Pediatric nonalcoholic fatty liver disease: overview with emphasis on histology. World J Gastroenterol 2010; 16: [PMID: DOI: / wjg.v16.i ] 8 Basaranoglu M, Turhan N, Sonsuz A, Basaranoglu G. Mallory-Denk Bodies in chronic hepatitis. World J Gastroenterol 2011; 17: [PMID: DOI: /wjg.v17. i ] 9 Fromenty B, Robin MA, Igoudjil A, Mansouri A, Pessayre D. The ins and outs of mitochondrial dysfunction in NASH. Diabetes Metab 2004; 30: [PMID: ] 10 Henkel J, Frede K, Schanze N, Vogel H, Schürmann A, Spruss A, Bergheim I, Püschel GP. Stimulation of fat accumulation in hepatocytes by PGE2-dependent repression of hepatic lipolysis, β-oxidation and VLDL-synthesis. Lab Invest 2012; 92: [PMID: DOI: /labinvest ] 11 Jiang Y, Zhao M, An W. Increased hepatic apoptosis in highfat diet-induced NASH in rats may be associated with downregulation of hepatic stimulator substance. J Mol Med (Berl) 2011; 89: [PMID: DOI: /s y] 12 Pessayre D. Role of mitochondria in non-alcoholic fatty liver disease. J Gastroenterol Hepatol 2007; 22 Suppl 1: S20-S27 [PMID: ] 13 Rolo AP, Teodoro JS, Palmeira CM. Role of oxidative stress in the pathogenesis of nonalcoholic steatohepatitis. Free Radic Biol Med 2012; 52: [PMID: DOI: /j.free radbiomed ] 14 Thomas A, Klein MS, Stevens AP, Reinders Y, Hellerbrand C, Dettmer K, Gronwald W, Oefner PJ, Reinders J. Changes in the hepatic mitochondrial and membrane proteome in mice fed a non-alcoholic steatohepatitis inducing diet. J Proteomics 2013; 80C: [PMID: DOI: /j.jprot ] 15 Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander- Tetri BA, Bacon BR. Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol 1999; 94: [PMID: DOI: / j x] 16 Tandra S, Yeh MM, Brunt EM, Vuppalanchi R, Cummings OW, Unalp-Arida A, Wilson LA, Chalasani N. Presence and significance of microvesicular steatosis in nonalcoholic fatty liver disease. J Hepatol 2011; 55: [PMID: DOI: /j.jhep ] 17 Tiniakos DG. Liver biopsy in alcoholic and non-alcoholic steatohepatitis patients. Gastroenterol Clin Biol 2009; 33: [PMID: DOI: /j.gcb ] 18 Lotowska JM, Sobaniec-Lotowska ME, Lebensztejn DM. The role of Kupffer cells in the morphogenesis of nonalcoholic steatohepatitis - ultrastructural findings. The first report in pediatric patients. Scand J Gastroenterol 2013; 48: [PMID: DOI: / ] 4339 April 21, 2014 Volume 20 Issue 15

241 Lotowska JM et al. Mitochondrial ultrastructure in pediatric NASH 19 Rombouts K, Marra F. Molecular mechanisms of hepatic fibrosis in non-alcoholic steatohepatitis. Dig Dis 2010; 28: [PMID: DOI: / ] 20 Ahishali E, Demir K, Ahishali B, Akyuz F, Pinarbasi B, Poturoglu S, Ibrisim D, Gulluoglu M, Ozdil S, Besisik F, Kaymakoglu S, Boztas G, Cakaloglu Y, Mungan Z, Canberk Y, Okten A. Electron microscopic findings in non-alcoholic fatty liver disease: is there a difference between hepatosteatosis and steatohepatitis? J Gastroenterol Hepatol 2010; 25: [PMID: DOI: /j x] 21 Caldwell SH, Swerdlow RH, Khan EM, Iezzoni JC, Hespenheide EE, Parks JK, Parker WD. Mitochondrial abnormalities in non-alcoholic steatohepatitis. J Hepatol 1999; 31: [PMID: DOI: /S (99) ] 22 Le TH, Caldwell SH, Redick JA, Sheppard BL, Davis CA, Arseneau KO, Iezzoni JC, Hespenheide EE, Al-Osaimi A, Peterson TC. The zonal distribution of megamitochondria with crystalline inclusions in nonalcoholic steatohepatitis. Hepatology 2004; 39: [PMID: DOI: / hep.20202] 23 Park KS, Jang BK, Chung WJ, Cho KB, Hwang JS, Ahn SH, Kang YN, Hwang JB, Keum DY. [Abnormal electron microscopic findings of nonalcoholic steatohepatitis and related factors]. Korean J Gastroenterol 2005; 45: [PMID: ] 24 Sanyal AJ, Campbell-Sargent C, Mirshahi F, Rizzo WB, Contos MJ, Sterling RK, Luketic VA, Shiffman ML, Clore JN. Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities. Gastroenterology 2001; 120: [PMID: DOI: /gast ] 25 Sobaniec-Lotowska ME, Lebensztejn DM. Ultrastructure of hepatocyte mitochondria in nonalcoholic steatohepatitis in pediatric patients: usefulness of electron microscopy in the diagnosis of the disease. Am J Gastroenterol 2003; 98: [PMID: DOI: /j x] 26 Sobaniec-Łotowska ME, Lebensztejn DM, Lotowska JM, Kańczuga-Koda L, Sulkowski S. Ultrastructure of liver progenitor/oval cells in children with nonalcoholic steatohepatitis. Adv Med Sci 2011; 56: [PMID: DOI: /v ] 27 Lotowska JM, Sobaniec-Lotowska ME, Sulkowska U, Lebensztejn DM. Mitochondrial abnormalities in nonalcoholic steatohepatitis in pediatric patients. Falk Symposium 186. Challenges of Liver Cirrhosis and Tumors: Prevent it, Treat it, Manage Consequences; October 5-6, 2012, Mainz, Germany, 46. Available from: URL: de/uploads/tx_tocfpshoperw/fs186_program.pdf 28 Sobaniec-Lotowska ME, Lotowska JM, Bockowska SB, Lebensztejn DM. The role of Kupffer cells in the pathogenesis of non-alcoholic steatohepatitis: immunohistochemical and ultrastructural findings in pediatric patients. Falk Symposium 181. Innate Immunity in Gastrointestinal Disorders: Basic and Therapeutic Concepts; February 8-9, 2012, Munich, Germany, 28. Available from: URL: de/uploads/tx_tocfpshoperw/fs181_program.pdf 29 Chedid A, Mendenhall CL, Tosch T, Chen T, Rabin L, Garcia-Pont P, Goldberg SJ, Kiernan T, Seeff LB, Sorrell M. Significance of megamitochondria in alcoholic liver disease. Gastroenterology 1986; 90: [PMID: ] 30 Phillips MJ, Poucell S, Patterson J, Valencia P. The Liver: An Atlas and Text of Ultrastructural Pathology. New York: Raven Press, 1987: Kirsch R, Clarkson V, Shephard EG, Marais DA, Jaffer MA, Woodburne VE, Kirsch RE, Hall Pde L. Rodent nutritional model of non-alcoholic steatohepatitis: species, strain and sex difference studies. J Gastroenterol Hepatol 2003; 18: [PMID: ] P- Reviewers: Romani A, Tanaka N, Williams GM S- Editor: Wen LL L- Editor: Rutherford A E- Editor: Zhang DN 4340 April 21, 2014 Volume 20 Issue 15

242 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH REPORT Role of triamcinolone in radiation enteritis management Eren Cetin, Aysen Sevgi Ozturk, Haluk Orhun, Sukran Ulger Eren Cetin, Sukran Ulger, Department of Radiation Oncology, Faculty of Medicine, Gazi University, Ankara 06500, Turkey Aysen Sevgi Ozturk, Haluk Orhun, Division of Radiation Oncology, Medicana International Hospital, Ankara 06500, Turkey Author contributions: Cetin E and Ozturk AS contributed equally to this work; Cetin E designed the research; Cetin E, Ozturk AS and Orhun H performed the research; Cetin E analyzed data; Cetin E and Ulger S wrote the paper. Correspondence to: Dr. Eren Cetin, Department of Radiation Oncology, Faculty of Medicine, Gazi University, Besevler, Ankara 06500, Turkey. erencetin@gazi.edu.tr Telephone: Received: July 23, 2013 Revised: November 25, 2013 Accepted: January 2, 2014 Published online: April 21, 2014 Abstract AIM: To investigate the role of triamcinolone in the management of acute and chronic enteritis caused by pelvic radiotherapy. METHODS: Twenty-eight patients with rectum adenocarcinoma or endometrium adenocarcinoma were studied. We compared the results of 14 patients treated with injected triamcinolone acetonide (TA) with those of 14 patients who were not treated with TA. For the TA group, 40 mg of TA was injected intramuscularly on the 1 st, 11 th and 21 st d of radiotherapy; the control group received no injections. All of the study participants had a median age of 65 years, had undergone postoperative radiotherapy and were evaluated weekly using Radiation Therapy Oncology Group and the European Organization for Research and Treatment of Cancer Acute Morbidity Score Criteria, and complete blood counts for every 10 d. RESULTS: Triamcinolone was found to effectively prevent and treat radiation-induced acute gastrointestinal (enteritis) and genitourinary (cystitis) side effects (P = and P = 0.023). For the lower GI side effect follow up, 11 patients in the control group had Grade 2 toxicity and 3 patients had Grade 1 toxicity. In the TA group, 5 patients had Grade 2 toxicity and 9 patients had Grade 1 toxicity. For the genitourinary system side effect follow up, 4 patients had Grade 2 toxicity and 6 patients had Grade 1 toxicity. Additionally, 2 patients had Grade 2 toxicity and 2 patients had Grade 1 toxicity. The neutrophil counts did not differ between the TA group and the control group. There was no meaningful difference between age groups and primary cancers. At the 12 th mo of follow up, there were no differences between groups for chronic side effects. CONCLUSION: Triamcinolone is a moderately potent steroid, that is inexpensive and has a good safety profile. It would be beneficial for reducing medical expenses related to treatment of radiation induced enteritis Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Radiotherapy; Enteritis; Cystitis; Triamcinolone Core tip: Radiation enteritis, proctitis and cystitis are acute side effects of radiotherapy of the pelvic region that change patients performance status and can interrupt the radiotherapy program. An important effort should be made to treat the side effects and to provide patient follow-up. Many drugs are available to treat the side effects, and although supportive digestive supplements may be used, the medical expenses associated with tratment are increasing, and the patients are suffering. In this study, we evaluated the role of triamcinolone acetonide, which is moderately effective, lowpriced and easy to use, in the management of acute gastrointestinal and genitourinary system side effects. Cetin E, Ozturk AS, Orhun H, Ulger S. Role of triamcinolone in radiation enteritis management. World J Gastroenterol 2014; 20(15): Available from: URL: com/ /full/v20/i15/4341.htm DOI: org/ /wjg.v20.i April 21, 2014 Volume 20 Issue 15

243 Cetin E et al. Triamcinolone for radiation enteritis INTRODUCTION Radiotherapy of the pelvic region is commonly used for a significant percentage of patients with rectosigmoid and genitourinary system tumors to target the lymphatic region and the primary tumor bed. Radiation enteritis, proctitis and cystitis are acute side effects that change patients performance status and may interrupt the radiotherapy program. Intestinal mucosa with a high mitotic rate, is affected by radiotherapy, and tenesmus, diarrhea and hematochezia may be seen. To treat these side effects, antispasmotics, antidiarrhetics, analgesics, antiinflammatory drugs and intravenous serum replacement are often used in certain cases. Chemotherapy, older age, pelvic inflammatory disease, peritoneal adhesion after operation, diabetes mellitus, hypertension and collagen vascular diseases are also risk factors for radiation induced side effects. The total radiation dose, dose of fractions and scheme of radiotherapy affect the situation directly. Many studies have examined reducing the rate of radiation enteritis by treatment positioning or by providing palliative medical treatments. Doctors make as an important efforts to treat the side effects and provide patient follow-up; however, the associated medical expenses are increasing, and patients are suffering as a result. Although the use of steroids for radiation-induced proctitis is reported in the literature, in this study, we evaluated the role of triamcinolone asetonide (TA), which is moderately effective and easy to use, for the management of acute gastrointestinal system (GIS) and genitourinary system (GUS) side effects of pelvic radiotherapy. MATERIALS AND METHODS Patients Patients with diabetes mellitus, pelvic inflammatory disease, hypertension, collagen vascular disease, and fungalviral infections were excluded. From December 2008 to December 2010, 28 patients had pelvic conformal radiotherapy with conventional fraction sizes of 4 fields for a total of 50.0 to 50.4 Gy that was planned postoperatively. The upper limit of the treatment field was the L5-S1 intervertebral space, and the lower limit of V40 of the intestines within the treatment volume was 90%. Six of the patients had endometrial cancer, and 22 had rectal cancer. The median age of all the patients was 65. Chemotherapy was applied for the rectal cancer patients concomitantly as 5-fluorouracil (425 mg/m 2 twice every 3 wk) and folinic acid (25 mg/m 2 ). The patients were divided into two equal groups. The patients characteristics are summarized in Table 1. For the TA group patients, 40 mg of TA was injected intramuscularly on the 1 st, 11 th and 21 st d of radiotherapy; the patients in the control group received no injections. For both groups, medical support was also planned if needed. Evaluations were performed weekly during radiotherapy using the Radiation Therapy Oncology Group and the European Organization for Research and Treatment of Cancer (RTOG/EORTC) Acute Radiation Morbidity Scoring Criteria, and complete blood counts were performed every 10 d ( Statistical analysis Statistical assessment was conducted using SPSS V13 program. Normality was tested using Shapiro-Wilk W test (P < 0.05) and Pearson s χ 2 test was used to test for meaning (P < 0.05). RESULTS For the lower GI side effect follow-up, 11 patients in the control group had Grade 2 toxicity and 3 patients had Grade 1 toxicity. In the TA group, 5 patients had Grade 2 toxicity and 9 patients had Grade 1 toxicity. For the GUS side effect follow-up, 4 patients in the control group had Grade 2 toxicity and 6 patients had Grade 1 toxicity. In the TA group, 2 patients had Grade 2 toxicity and 2 patients had Grade 1 toxicity. For GIS and GUS, Grade 2 toxicities occured on the 21 st (median) day and Grade 1 toxicities occured on the 14 th (median) day. The neutrophil numbers decreased with Grade 1 toxicity in 3 patients from each group on the 21 st day. The patient score frequencies are summarized in Table 2. The statistical analysis showed that TA was effective for preventing and treating acute radiation-induced GIS side effects from pelvic radiotherapy when Grade 1 and Grade 2 toxicities were compared (P = 0.022). TA was also effective for preventing and treating acute radiationinduced GUS side effects from pelvic radiotherapy when Grade 0 and Grade 1-2 toxicities were compared (P = 0.023). The neutrophil counts did not differ between the groups. There was no meaningful difference between age groups and primary cancers. At the 12 mo follow up, there were no differences between groups in terms of chronic side effects. DISCUSSION Small bowels are highly sensitive, dose-limiting organs for pelvic or abdominal radiotherapy [1]. The fraction size, fractionation schema, dose and volume of the treatment site are important factors in radiation-induced enteropathy. Additionally, patient-dependent factors, such as genetic factors, are minimally defined, as the methods for this treatment are not highly sensitive [2]. During radiation, matrix metalloproteinase2 (MMP2) and tumor necrosis factor (TNF) are secreted by the cells at the site [3,4]. After their induction by radiation, transient mucosal atrophy and plasma cells and polymorphonuclear leucocytes are accommodated. Inflammatory reactions and endothelium loss, microabcesses, mucosal ulcers, water-electrolyte loss and bacterial infiltration are seen as a cascade [5-7]. Typically, 8 to 12 mo after radiotherapy, obliterative endarteritis occures as a vascular occlusion, followed by tissue growth factor (TGF)-induced tissue ischemia, necrosis and fibrosis [8,9]. Additionally, angiogenesis and chronic 4342 April 21, 2014 Volume 20 Issue 15

244 Cetin E et al. Triamcinolone for radiation enteritis Table 1 Patient properties Table 2 Score frequencies for acute radiation morbidity Rectum Ca Endometrium Ca Total Patient number Median age Female 12 6 Male 10 TA group 11 (6 male) 3 14 Control group 11 (4 male) 3 14 GIS GUS Neutrophils TA Grade Grade Grade Control Grade Grade Grade TA: Triamcinolone acetonide. inflammation are seen after vascular endothelial growth factor (VEGF) secretion [10]. There are many studies using patient positioning and medical palliation to decrease the rate of radiationinduced enteritis. There are varying results for the prone positions during radiotherapy. A 16% decrease in the small bowel median dose has been reported with the use of belly board, but its applicability for patients has been criticized [11,12]. No wide-range study has evaluated the treatment effects of aminosalicylates. For the treatment of radiation-induced enteropathy, there are preliminary results for amifostine, but additional data are needed [13]. According to a double-blind Phase Ⅲ study of pelvic radiotherapy in 120 patients, glutamine is not effective for the treatment of acute radiation enteritis [14]. In contrast, glucocorticoids are used for a vast number of inflammatory diseases. Long term use cause side effects. These drugs inhibit MMP2 secretion and prevent leucocytes infiltrations and TNF-induced inflammatory and angiogenic steps. Steroids are effective agents for preventing leucocyte infiltration, mucosal inflammation and edema [15,16]. Based on these molecular aspects, steroids would be appropriate agents to prevent or attenuate the tissue reaction to radiation. In a study of 24 patients receiving radiation therapy, the steroid treatment methylprednisolone was reported to improve parenteral nutrition and clinical effects in 8 wk [17]. The rectal use of beclamethasone was also reported to be effective for preventing radiation-induced mucosal changes and rectal bleeding in a randomized study of 120 patients [18]. Periocular injections of TA have been reported to be effective for treating symptomatic macular edema and retinopathy after radiotherapy. The same group designed a prospective randomized study of 163 patients and reported that TA is effective for preventing macular edema [19,20]. In our study, the acute GIS and GUS side effects of pelvic radiotherapy patients were evaluated. These patients often require relevant medication and follow up. It is likely that the acute side effects of radiation arise from patient-dependent factors in the first weeks of conventional fractions rather than the total radiotherapy dose itself. A steroid drug would be preferable because it might prevent the tissue reactions to radiation at some molecular steps. The long-term use of oral or intravenous steroids GIS: Gastrointestinal system; GUS: Genitourinary system; TA: Triamcinolone acetonide. may have many side effects; consequently, doctors usually avoid such use. Nevertheless, TA has a good safety profile and is a member of a moderately effective steroid group. In our study, TA was used every 10 d intramuscularly, which is an easy pattern of administration. As a result, TA was found to be effective for preventing and treating radiation-induced acute GIS and GUS side effects. Compared with other drugs and support products, TA would be beneficial for reducing the medical expenses associated with treating radiation-induced enteritis. To precisely assess the role of TA in preventing and treating radiation-induced side effects, a double-blind randomized study and laboratory confirmation are needed. We think that, our study contributes additional data about preventing and treating radiation-induced side effects and the safety of steroid use in radiation oncology. ACKNOWLEDGMENTS The study plan was controlled by Gazi University Noninvasive Clinical Studies Committee ( , 127). COMMENTS Background Many drugs are available to treat the side effects of radiation-induced enteritis. Supportive digestive supplements are used; however, the associated medical expenses are increasing, and patients are suffering as a result. In this study, the authors evaluated the role of triamcinolone acetonide, which is moderately effective, low-cost and easy to use, to manage acute gastrointestinal and genitourinary system side effects. Research frontiers Steroids are effective agents to prevent leucocyte infiltration, mucosal inflammation and edema. Based on these molecular aspects, steroids should be appropriate agents to prevent or attenuate the tissue reaction to radiation. In a study of 24 patients undergoing radiation therapy, the steroid treatment methylprednisolone was reported to improve parenteral nutrition and clinical effects in 8 wk. The rectal use of beclamethasone is also reported to be effective for preventing radiation-induced mucosal changes and rectal bleeding in a randomized study of 120 patients. Innovations and breakthroughs Periocular injection of triamcinolone acetonide (TA) has been reported to be effective for treating symptomatic macular edema and retinopathy after radiotherapy. The same researchers designed a prospective randomized study of 163 patients and reported that TA is effective for preventing macular edema. Applications The authors evaluated the effects of injected TA in 14 patients compared the results with those of 14 untreated patients. For the TA group, 40 mg of TA was injected intramuscularly on the 1st, 11 th and 21 st d of radiotherapy; the control 4343 April 21, 2014 Volume 20 Issue 15

245 Cetin E et al. Triamcinolone for radiation enteritis group received no injections. Terminology RTOG/EORTC Acute Morbidity Score Criteria: Toxicity criteria of the Radiation Therapy Oncology Group and the European Organization for Research and Treatment of Cancer. Peer review Triamcinolone was found to be effective for preventing and treating radiationinduced acute gastrointestinal system (enteritis) and genitourinary system (cystitis) side effects. Triamcinolone is a moderately potent steroid with a low price and a good safety profile. It would be beneficial for reducing medical expenses associated with the treatment of radiation-induced enteritis. REFERENCES 1 Zheng H, Wang J, Koteliansky VE, Gotwals PJ, Hauer-Jensen M. Recombinant soluble transforming growth factor beta type II receptor ameliorates radiation enteropathy in mice. Gastroenterology 2000; 119: [PMID: DOI: /gast ] 2 Andreyev HJ. Gastrointestinal problems after pelvic radiotherapy: the past, the present and the future. Clin Oncol (R Coll Radiol) 2007; 19: [PMID: DOI: /j.clon ] 3 Burger A, Löffler H, Bamberg M, Rodemann HP. Molecular and cellular basis of radiation fibrosis. Int J Radiat Biol 1998; 73: [PMID: DOI: / ] 4 Pons I, Gras G, Courberand S, Benveniste O, Dormont D. Consequences of gamma-irradiation on inflammatory cytokine regulation in human monocytes/macrophages. Int J Radiat Biol 1997; 71: [PMID: DOI: / ] 5 MacNaughton WK. Review article: new insights into the pathogenesis of radiation-induced intestinal dysfunction. Aliment Pharmacol Ther 2000; 14: [PMID: DOI: /j x] 6 Yeoh EK, Horowitz M. Radiation enteritis. Surg Gynecol Obstet 1987; 165: [PMID: ] 7 Rodier JF. Radiation enteropathy--incidence, aetiology, risk factors, pathology and symptoms. Tumori 1995; 81: [PMID: ] 8 Kwitko AO, Pieterse AS, Hecker R, Rowland R, Wigg DR. Chronic radiation injury to the intestine: a clinico-pathological study. Aust N Z J Med 1982; 12: [PMID: DOI: /j tb02476.x] 9 Haydont V, Mathé D, Bourgier C, Abdelali J, Aigueperse J, Bourhis J, Vozenin-Brotons MC. Induction of CTGF by TGF-beta1 in normal and radiation enteritis human smooth muscle cells: Smad/Rho balance and therapeutic perspectives. Radiother Oncol 2005; 76: [PMID: DOI: /j.radonc ] 10 Carmeliet P. Angiogenesis in life, disease and medicine. Nature 2005; 438: [PMID: DOI: /nature04478] 11 Koelbl O, Richter S, Flentje M. Influence of patient positioning on dose-volume histogram and normal tissue complication probability for small bowel and bladder in patients receiving pelvic irradiation: a prospective study using a 3D planning system and a radiobiological model. Int J Radiat Oncol Biol Phys 1999; 45: [PMID: DOI: /S (99) ] 12 Allal AS, Bischof S, Nouet P. Impact of the belly board device on treatment reproducibility in preoperative radiotherapy for rectal cancer. Strahlenther Onkol 2002; 178: [PMID: DOI: /s ] 13 Athanassiou H, Antonadou D, Coliarakis N, Kouveli A, Synodinou M, Paraskevaidis M, Sarris G, Georgakopoulos GR, Panousaki K, Karageorgis P, Throuvalas N. Protective effect of amifostine during fractionated radiotherapy in patients with pelvic carcinomas: results of a randomized trial. Int J Radiat Oncol Biol Phys 2003; 56: [PMID: DOI: /S (03)00187] 14 Kozelsky TF, Meyers GE, Sloan JA, Shanahan TG, Dick SJ, Moore RL, Engeler GP, Frank AR, McKone TK, Urias RE, Pilepich MV, Novotny PJ, Martenson JA. Phase III doubleblind study of glutamine versus placebo for the prevention of acute diarrhea in patients receiving pelvic radiation therapy. J Clin Oncol 2003; 21: [PMID: DOI: /JCO ] 15 Araya J, Maruyama M, Sassa K, Fujita T, Hayashi R, Matsui S, Kashii T, Yamashita N, Sugiyama E, Kobayashi M. Ionizing radiation enhances matrix metalloproteinase-2 production in human lung epithelial cells. Am J Physiol Lung Cell Mol Physiol 2001; 280: L30-L38 [PMID: ] 16 Nehmé A, Edelman J. Dexamethasone inhibits high glucose-, TNF-alpha-, and IL-1beta-induced secretion of inflammatory and angiogenic mediators from retinal microvascular pericytes. Invest Ophthalmol Vis Sci 2008; 49: [PMID: DOI: /iovs ] 17 Loiudice TA, Lang JA. Treatment of radiation enteritis: a comparison study. Am J Gastroenterol 1983; 78: [PMID: ] 18 Fuccio L, Guido A, Laterza L, Eusebi LH, Busutti L, Bunkheila F, Barbieri E, Bazzoli F. Randomised clinical trial: preventive treatment with topical rectal beclomethasone dipropionate reduces post-radiation risk of bleeding in patients irradiated for prostate cancer. Aliment Pharmacol Ther 2011; 34: [PMID: DOI: /j x] 19 Shields CL, Demirci H, Marr BP, Mashayekhi A, Dai VV, Materin MA, Shields JA. Intravitreal triamcinolone acetonide for acute radiation papillopathy. 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246 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH REPORT Hepcidin expression in colon during trinitrobenzene sulfonic acid-induced colitis in rats Érica Martins Ferreira Gotardo, Gilberto de Almeida Ribeiro, Thayane Rodrigues Leite Clemente, Camila Henrique Moscato, Renata Bortolin Guerra Tomé, Thalita Rocha, José Pedrazzoli Jr, Marcelo Lima Ribeiro, Alessandra Gambero Érica Martins Ferreira Gotardo, Gilberto de Almeida Ribeiro, Thayane Rodrigues Leite Clemente, Camila Henrique Moscato, Renata Bortolin Guerra Tomé, Thalita Rocha, José Pedrazzoli Jr, Marcelo Lima Ribeiro, Alessandra Gambero, Clinical Pharmacology and Gastroenterology Unit, São Francisco University Medical School, Bragança Paulista, SP , Brazil Author contributions: Gotardo EMF and Ribeiro GA contributed equally to this work; Gotardo EMF, Ribeiro GA, Clemente TRL, Moscato CH and Tomé RBG performed the experiments; Rocha T and Pedrazzoli Jr J analyzed the data and wrote the manuscript; Ribeiro ML and Gambero A designed the experiments, analyzed the data and wrote the manuscript. Supported by the Fundação de Amparo à Pesquisa do Estado de São Paulo, No. FAPESP 2010/ Correspondence to: Alessandra Gambero, PhD, Clinical Pharmacology and Gastroenterology Unit, São Francisco University Medical School, Av. São Francisco de Assis 218, Bragança Paulista, SP , Brazil. alessandra.gambero@usf.edu.br Telephone: Fax: Received: October 22, 2013 Revised: December 18, 2013 Accepted: January 19, 2014 Published online: April 21, 2014 Abstract AIM: To investigate hepcidin expression, interleukin-6 (IL-6) production and iron levels in the rat colon in the presence of trinitrobenzene sulfonic acid (TNBS)- induced colitis. METHODS: In rats, we evaluated the severity of colitis induced by repeated TNBS administration using macroscopic and microscopic scoring systems and myeloperoxidase activity measurements. The colonic levels of hepcidin, tumor necrosis factor alpha (TNF-α), IL-10 and IL-6 were measured by Enzyme-Linked Immunosorbent Assay, and hepcidin-25 expression and iron deposition were analyzed by immunohistochemistry and the Prussian blue reaction, respectively. Stat-3 phosphorylation was assessed by Western blot analysis. Hematological parameters, iron and transferrin levels, and transferrin saturation were also measured. Additionally, the ability of iron, pathogen-derived molecules and IL-6 to induce hepcidin expression in HT-29 cells was evaluated. RESULTS: Repeated TNBS administration to rats resulted in macroscopically and microscopically detectable colon lesions and elevated colonic myeloperoxidase activity. Hepcidin-25 protein levels were increased in colonic surface epithelia in colitic rats (10.2 ± 4.0 pg/mg protein vs 71.0 ± 8.4 pg/mg protein, P < 0.01). Elevated IL-6 levels (8.2 ± 1.7 pg/mg protein vs 14.7 ± 0.7 pg/mg protein, P < 0.05), TNF-α levels (1.8 ± 1.2 pg/mg protein vs 7.4 ± 2.1 pg/mg protein, P < 0.05) and Stat-3 phosphorylation were also observed. Systemic alterations in iron homeostasis, hepcidin levels and anemia were not detected in colitic rats. Iron deposition in the colon was only observed during colitis. Hepcidin gene expression was increased in HT-29 cells after IL-6 and lipopolysaccharide [a toll-like receptor 4 (TLR-4) ligand] treatment. Deferoxamine, ferric citrate and peptidoglycan (a TLR-2 ligand) were unable to alter the in vitro expression of hepcidin in HT-29 cells. CONCLUSION: Colitis increased local hepcidin-25 expression, which was associated with the IL-6/Stat-3 signaling pathway. An increase in local iron sequestration was also observed, but additional studies are needed to determine whether this sequestration is a defensive or pathological response to intestinal inflammation Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Hepcidin-25; interleukin-6; iron; Stat-3; anemia; toll-like receptor April 21, 2014 Volume 20 Issue 15

247 Gotardo EMF et al. Hepcidin expression in colitis Core tip: Hepcidin is an endogenous peptide with weak antimicrobial properties that regulates changes in iron metabolism during inflammation. However, infectionassociated cytokines, pathogen-derived molecules or whole pathogens can induce hepcidin synthesis as part of the host response to infection. This is the first study to describe that colitis induces hepcidin expression in colons associated with the interleukin-6/stat-3 signaling pathways and local iron sequestration. This finding suggests a host response to infection because reducing the iron available to pathogens is an important antimicrobial mechanism. However, we could not exclude the possibility that hepcidin expression contributes to increased local inflammation by stimulating pro-inflammatory macrophages. Gotardo EMF, Ribeiro GA, Clemente TRL, Moscato CH, Tomé RBG, Rocha T, Pedrazzoli Jr J, Ribeiro ML, Gambero A. Hepcidin expression in colon during trinitrobenzene sulfonic acid-induced colitis in rats. World J Gastroenterol 2014; 20(15): Available from: URL: com/ /full/v20/i15/4345.htm DOI: org/ /wjg.v20.i INTRODUCTION In addition to intestinal inflammation, Crohn s disease (CD) and ulcerative colitis (UC) patients present with several extraintestinal symptoms, including systemic iron deficiency. Iron deficiency is the main cause of anemia in inflammatory bowel disease (IBD) patients, which occurs as result of several factors, such as intestinal blood loss, absorptive deficiencies and/or undertreatment of anemia [1]. Anemia of chronic disease (ACD), also known as anemia of inflammation, is the second most common cause of anemia in IBD patients; it frequently occurs with iron deficiency [2,3]. ACD is normally associated with low serum iron levels and low transferrin saturation. However, in this case, hypoferremia is a consequence of macrophage iron sequestration; therefore, the total body iron content may be increased [4]. Inflammatory cytokines produced during inflammation, most notably interleukin-6 (IL-6), have been hypothesized to contribute to systemic iron deficiency through their effects on hepcidin synthesis [5]. Hepcidin is a 25-amino acid cysteine-rich peptide with weak antimicrobial properties that is regulated by a number of factors such as liver iron levels, inflammation, hypoxia and anemia. Hepcidin is mainly expressed and released by the liver in response to increased circulating iron levels, but hepcidin synthesis also occurs to a lesser degree in adipose tissue, the heart, placenta and kidneys. However, the functions of hepcidin in these tissues are currently unknown [6]. Furthermore, hepcidin decreases intestinal iron absorption and blocks macrophage iron efflux, thus protecting the body against excessive iron levels [7]. Infection-associated cytokines, pathogen-derived molecules and whole pathogens can also induce hepcidin synthesis as part of the host response to infection because reducing available iron is an important antimicrobial mechanism [8]. Hepcidin has been proposed to be directly involved in IBD pathogenesis because in Hfe knockout mice, which have low hepcidin expression, the severity of lipopolysaccharide (LPS)- and Salmonella-induced enterocolitis is attenuated [9]. On the other hand, antimicrobial peptides, such as hepcidin, are induced in response to gut microbe invasion during colonic inflammation and affect colitis progression [10,11]. Furthermore, hepcidin has been found to be expressed in human gastric parietal cells and colorectal cancer tissue [12,13], but no data are currently available regarding the expression of hepcidin in the colon during IBD. Therefore, we investigated rat colonic hepcidin expression in trinitrobenzene sulfonic acid (TNBS)-induced colitis [14] and correlated hepcidin expression with local IL-6 production and iron levels. This experimental model included periods of relapse and remission that resembled the colonic inflammation present in human IBD [14]. The ability of HT-29 cells [15] to express hepcidin was also assessed. MATERIALS AND METHODS Animals Specific pathogen-free male Wistar rats ( g, 6-8 wk old) were obtained from CEMIB (State University of Campinas, Campinas, SP, Brazil). All experiments were performed in accordance with the principles outlined by the Brazilian College for Animal Experimentation and received approval from the Ethics Committee of São Francisco University, Bragança Paulista, SP, Brazil (Protocol ). Rats were maintained in a room with controlled humidity and temperature in collective cages and were exposed to 12 h light-dark cycles. Twelve hours prior to experimental procedures, the animals were deprived of food (standard chow) but not water. Each study used 5-7 rats per group. TNBS-induced colitis Animals were anesthetized with ketamine/xylazine (1:1, v/v), and colitis was induced by the intracolonic administration of 3 mg TNBS dissolved in 0.3 ml of 50% ethanol (Sigma, St. Louis, MO, United States). The solution was injected into the colon 8 cm proximal to the anus using a catheter. TNBS administration lasted only a few seconds, and the rats were maintained in a vertical position until they recovered from anesthesia. The same procedure was repeated 14 and 28 d after the first TNBS administration, and the rats were sacrificed on 35 th day. Control rats received saline through the same route. An additional colitis induction protocol was performed, and the rats were evaluated 24 h after the first TNBS administration. Serum and hematological parameters Blood samples were collected from the rat vena cava un April 21, 2014 Volume 20 Issue 15

248 Gotardo EMF et al. Hepcidin expression in colitis der anesthesia (1:1, v/v of xylazine 2%-ketamine 10%). Blood/ethylenediamine tetraacetic acid (EDTA) was used to analyze the hematological parameters (ABX Pentra 120, Horiba Instruments Brazil, Jundiai, SP, Brazil), and serum samples were obtained from collected blood lacking anticoagulant solution for the enzymatic assay (EIA) quantification of transferrin (GenWay, San Diego, CA, United States) and hepcidin (USCN Life Science, Wuhan, China) levels. Iron levels and the iron binding capacity were also measured in serum samples using colorimetric commercial kits (Bioclin, Belo Horizonte, MG, Brazil). Colitis characterization by macroscopic damage and myeloperoxidase activity Colons were immediately removed from the animals, opened lengthwise and evaluated for macroscopically visible damage by two observers who were blinded to the experimental groups. The criteria for macroscopic colonic damage were as follows: no damage (0 points), hyperemia without ulcers (1 point), linear ulcer with no significant inflammation (2 points), linear ulcer with inflammation at one site (3 points), two or more sites of ulceration/inflammation (4 points), and two or more major sites of ulceration and inflammation or one site of ulceration/inflammation extending 1 cm along the length of the colon (5 points). If the damage extended at least a 2 cm length of colon tissue, the score was increased by 1 point for each additional centimeter of involvement (6-10 points). Lengthwise colon samples obtained from a site of macroscopically detectable inflammation (or a corresponding tissue site with no macroscopically detectable inflammation) were homogenized in 0.5% (w/v) hexadecyltrimethylammonium bromide in 50 mmol/l potassium phosphate buffer, ph 6.0. For the myeloperoxidase assay, 50 µl of each sample was added to 200 µl of o-dianisidine solution (0.167 mg/ml o-dianisidine dihydrochloride and % hydrogen peroxide in 50 mmol/l phosphate buffer, ph 6.0) immediately prior to measuring the absorbance at 460 nm over a 5-min period using a microplate reader (Multiscan MS, Labsystems). Cytokine measurements and Western blot analysis To measure cytokine concentrations by EIA, colon tissue samples were collected as described above, excised and immediately homogenized in solubilization buffer at 4 (1% Triton X-100, 100 mmol/l Tris-HCl, 100 mmol/l sodium pyrophosphate, 100 mmol/l sodium fluoride, 10 mmol/l EDTA, 10 mmol/l sodium orthovanadate, 2.0 mmol/l phenylmethylsulfonyl fluoride, and 0.1 mg/ ml aprotinin). The insoluble fraction was removed by centrifugation at 9000 g for 20 min at 4. The protein supernatant concentrations were determined using the Biuret method. Hepcidin, tumor necrosis factor alpha (TNF-α), IL-10 and IL-6 levels were quantified using commercial kits (hepcidin, USCN Life Science; TNF-α and IL-10, GE Healthcare, United Kingdom; and IL-6, R and D Systems, Minneapolis, MN, United States). Liver biopsies were also used for hepcidin quantification as described above for colon samples. For protein analysis by Western blot (WB) assay, supernatant aliquots were treated with Laemmli sample buffer containing 100 mmol/l dithiothreitol, and the samples were then heated in a boiling water bath for 5 min. The samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in a Bio-Rad miniature slab gel apparatus (Mini-Protean). For WB experiments, 0.15 mg of protein extracted from tissue samples was separated by SDS-PAGE, transferred to nitrocellulose membranes and blocked for 2 h with 5% blocking agent (GE Healthcare, United Kingdom) in Tris buffer solution. The membranes were then incubated overnight with anti-stat-3 and anti-phospho-stat-3 antibodies (1:1000; Santa Cruz Biotechnology, CA, United States) and developed using a commercial chemiluminescent kit (GE Healthcare, United Kingdom). The band intensities were quantified by optical densitometry (Scion Image software, ScionCorp, Frederick, MD) from the developed autoradiography. Immunohistochemistry, Prussian blue reaction and Microscopic damage assessments in the colon Hydrated, 5.0 µm sections of paraformaldehyde-fixed, paraffin-embedded colon tissue were used for immunohistochemistry (IHC) and the Prussian blue reaction. Sections stained with hematoxylin and eosin (HE) were used to microscopically score determination. IHC sections were probed with an anti-rabbit hepcidin-25 antibody (1:100; Abcam, Cambridge, MA, United States) and biotinylated anti-rabbit secondary antibody, and developed with DAB (ImmunoCruz ABC Staining System, Santa Cruz Biotechnology, Santa Cruz, CA, United States). Additionally, hematoxylin staining was used to reveal the nuclear morphology. For the Prussian blue reaction, slides were incubated with a 5% potassium ferrocyanide aqueous solution and a 5% hydrochloric acid aqueous solution. After washing in distilled water, sections were counterstained with Nuclear Fast Red. Microscopic tissue damage was scored on a scale from 0 to 20 according to the criteria previously described by Rodríguez-Cabezas et al [16]. Six parameters were scored: neutrophil infiltration into the epithelium (0-2), lamina propria (0-2), submucosa (0-2), muscularis propria (0-2) and serosa (0-2); fibrin deposition into the mucosa (0-1) and submucosa (0-1); submucosa neutrophil margination (0-1); submucosal edema (0-2); epithelial necrosis (0-2); and epithelial ulceration (0-1). In vitro hepcidin expression HT-29 cells (Rio de Janeiro Cell Bank, RJ, Brazil) were incubated with 20 µmol/l ferric citrate (Sigma) for 72 h, 30 mmol/l deferoxamine (Sigma) for 4 h, 40 ng/ml IL-6 (Peprotech, Rocky Hill, NJ, United States) [12], 1 µg/ml of the toll-like receptor 2 (TLR-2) ligand peptidoglycan [peptidoglycan (PGN); Sigma] or 10 µg/ml of the TLR-4 ligand lipopolysaccharide (LPS; Sigma) for 6 h [12]. Then, cells were harvested, and total RNA was isolated according to the manufacturer s instruc April 21, 2014 Volume 20 Issue 15

249 Gotardo EMF et al. Hepcidin expression in colitis Table 1 Macroscopic evaluation, myeloperoxidase activity, body weight and cytokine production in colon tissues Table 2 Hematological parameters in the control and colitis groups IL-6 and TLR-4 regulate HT-29 hepcidin expression The human colon adenocarcinoma cell line HT-29 was used to model colonic hepcidin expression. Upon stimutions (RNeasy Mini Kit; Qiagen Valencia, CA, United States). cdna was synthesized with a High-capacity cdna archive kit (Applied Biosystems, Foster City, CA, United States) according to the manufacturer s protocol. Quantitative polymerase chain reaction and melting curve analyses were performed as previously described [17]. The primer sequences were as follows: hepcidin sense, 5 -CAGGGCAGGTAGGTTCTACG-3 ; hepcidin antisense, 5 -CACTTCCCCATCTGCATTTT-3 ; β-actin sense, 5 -ACACTGGCTCGTGTGACAAGG-3 ; and β-actin antisense, 5 -CGGCTAATACACACTC- CAAGGCG-3. Statistical analysis All data are expressed as means ± SE. Non-parametric data (histological scores) are expressed as the median (range) and were analyzed using the Mann-Whitney test. Comparisons between groups were performed using the unpaired Student s test. Statistical analyses were performed using GraphPad InStat (GraphPad Software, La Jolla, CA, United States). An associated probability (P value) of less than 0.05 was considered significant. RESULTS Control Colitis Damage score 0 (0-1) 5 (4-9) a Myeloperoxidase activity (U/g tissue) 2.2 ± ± 4.8 b Initial body weight (g) 299 ± ± 3 Final body weight (g) 405 ± ± 4 a Microscopic score 2 (0-3) 11 (6-14) a TNF-a (pg/mg protein) 1.8 ± ± 2.1 a IL-6 (pg/mg protein) 8.2 ± ± 0.7 a IL-10 (pg/mg protein) 23.5 ± ± 1.7 b a P < 0.05 and b P < 0.01 compared with the control group (n = 5-7). TNF-a: Tumor necrosis factor-alpha; IL: Interleukin. Colitis and hematological assessments Repeated intracolonic TNBS administration to Wistar rats resulted in macroscopic lesions characterized by hyperemia and small ulcers in the colon. In association with these observed macroscopic lesions, the colonic myeloperoxidase levels, a marker of neutrophil infiltration, were increased (Table 1). Histological assessment of colon tissues revealed the presence of ulcers and extensive neutrophil infiltration in the mucosa and submucosa. Severe edema and, in some cases, transmural inflammation were observed. The median score of the colitis group was elevated compared with the control group (Table 1). Additionally, colitis development corresponded with a low body weight at the end of the experimental protocol (Table 1). The red blood cell count, hemoglobin and hematocrit levels in colitic rats were similar to the levels observed in control rats. Leukocytosis was observed in colitic rats (Table 2). Control Colitis Red blood cells (10 6 /μl) 7.6 ± ± 0.2 Hemoglobin (g/dl) 15.2 ± ± 0.1 Hematocrit (%) 44.1 ± ± 0.3 White blood cells (cells/μl) 5.2 ± ± 0.5 b b P < 0.01 compared with the control group (n = 5). Table 3 Serum parameters in the control and colitis groups Control Colitis Hepcidin (pg/ml) ± ± 65.8 Transferrin (ng/ml) 18.0 ± ± 1.5 Iron (μg/dl) 69.0 ± ± 10.0 Transferrin saturation (%) 26.1 ± ± 1.2 Colonic hepcidin and cytokine levels In colon extracts, hepcidin levels were increased in colitic animals compared with control animals (Figure 1G), and hepcidin was predominantly expressed in the surface epithelium of colitic animals (Figure 1). In the colon, hepcidin levels were correlated with increased levels of IL-6 and TNF-α and decreased levels of IL-10 (Table 1). In an additional group, rats were evaluated 24 h after the first TNBS administration, and the level of IL-6 was significantly higher in the colitic group compared with the control group (9.1 ± 0.8 pg/mg protein and 39.8 ± 7.1 pg/mg protein for the control and colitis groups, respectively; P < 0.01, n = 5), but the hepcidin levels were not significantly different (11.8 ± 4.2 pg/mg protein and 30.9 ± 11.8 pg/mg protein for the control and colitis groups, respectively; P = 0.22, n = 5). Serum and liver hepcidin, iron and transferrin levels The serum hepcidin levels were similar between the colitic and control rats, and the iron and transferrin levels and iron saturation were also unchanged (Table 3). Furthermore, the liver hepcidin levels were not modified by colitis (653 ± 27 pg/mg protein and 675 ± 27 pg/mg protein for the control and colitis groups, respectively, n = 4). Colonic Stat-3 phosphorylation and iron levels In colitic rats, Stat-3 phosphorylation was increased compared to the controls (Figure 2). Iron deposition in the colons of colitic animals was observed using the Prussian blue reaction, and no reaction was evident in control colon tissues (Figure 3). In colitic rats, the observed blue precipitate was not localized to the surface or crypt epithelium, which suggested that iron might have accumulated in immune cells April 21, 2014 Volume 20 Issue 15

250 Gotardo EMF et al. Hepcidin expression in colitis A B C D E F 50 μm G Hepcidin (pg/mg protein) b 0 Control Colitis Figure 1 Colonic hepcidin expression in control and colitis rats. A: Immunohistochemistry of control rat tissue (x 100); B: Immunohistochemistry of an adjacent colitic area (x 100); C: Immunohistochemistry of a colitic area (x 100); D: Immunohistochemistry of control rat tissue (x 400); E: Immunohistochemistry of an adjacent colitic area (x 400); F: Immunohistochemistry of a colitic area (x 400); G: Enzymatic assay detection of hepcidin expression levels. Data are expressed as the means ± SE (n = 4 per group), and b P < 0.01 compared with control. lation with IL-6 and LPS (a TLR-4 ligand), hepcidin expression significantly increased (Figure 4). Deferoxamine, ferric citrate and PGN (a TLR-2 ligand) were unable to modify hepcidin expression in these human colon cells (Figure 4). DISCUSSION Iron is an important factor in the competition for nutritional resources between microbial pathogens and their hosts. In humans, host defense responses to infectious agents modulate local and systemic iron availability, interfering with infections such as malaria and tuberculosis [8]. Hepcidin is the major regulator of iron homeostasis in humans and other mammals. Increased iron levels and innate immunity (most likely through Toll-like receptor activation/il-6 induction) can induce hepcidin expression [8,18], Conversely, macrophages also produce hepcidin, and this autocrine production is related to the downregulation of ferroportin expression and iron sequestration in these cells [19]. Macrophage iron accumulation correlates with a more pro-inflammatory phenotype and consequent increased cytokine release [20]. In this study, we used a well-established experimental colitis model, induced by repeated TNBS administration, to examine hepcidin expression in the infected/inflamed colon. In this experimental model, moderate colonic inflammation and alterations in mesenteric adipose tissue are observed, simulating some aspects of CD [14]. Although increased hepcidin expression has been described in adipose tissue in obese patients [21] and in subcutaneous adipose tissue during cardiac surgery [22], no alterations in hepcidin expression were observed in the mesenteric adipose tissue in our study (data not shown). We demonstrated that colonic hepcidin expression was 4349 April 21, 2014 Volume 20 Issue 15

251 Gotardo EMF et al. Hepcidin expression in colitis A Control Colitis 15 b p-stat (86 KDa) Stat (86 KDa) Hepcidin/β-actin 10 5 a B p-stat-3/stat Control a Colitis 0 Control IL-6 Iron citrate Deferoxamine Figure 4 Analysis of hepcidin mrna expression (hepcidin to β-actin ratio) in HT-29 cells. Untreated control cells were compared with cells treated with interleukin-6 (IL-6) (40 ng/ml), iron citrate (20 μmol/l), deferoxamine (30 mmol/l), lipopolysaccharide (LPS) (1 μg/ml) or peptidoglycan (PGN) (10 μg/ml). Data are expressed as the means ± SE of 2 experiments performed in triplicate. a P < 0.05 and b P < 0.01 compared with control. PGN LPS Figure 2 Stat-3 phosphorylation levels in control and colitic rats. A: Representative Western blot images (from one individual experiment); B: Bar graph densitometry quantifications of phospho-stat-3 expression levels that were normalized to colonic Stat-3 expression levels (n = 4 per group). Data are expressed as the means ± SE, and a P < 0.05 compared with control. A Figure 3 Prussian blue reaction assessing the presence of ferric iron and ferritin in colons. A: Control ( 200); B: Colitis ( 200). Arrows denote areas of Prussian blue reactivity. increased in experimental inflammation. Previously, in two experimental models of the acute-phase response, increased hepcidin gene expression was observed in both the small intestine and colon [23]. Hepcidin immunoreactivity in colorectal tissue and increased urinary hepcidin levels have also been described in cancer patients [13]. Here, the increased colonic hepcidin expression observed during inflammation was associated with elevated levels of pro-inflammatory cytokines such as IL-6 and a significant increase in Stat-3 phosphorylation. Increased IL-6 levels were observed 24 h after the first TNBS administration, in the absence of increased hepcidin levels, suggesting that B pro-inflammatory cytokines may induce hepcidin expression. Our in vitro experiments demonstrating that IL-6 induced hepcidin expression in HT-29 cells support this hypothesis. A direct activation of TLR-4 but not TLR-2 also induced hepcidin expression, suggesting that PAMPs and cytokines could stimulate colonic hepcidin expression. Several pro-inflammatory cytokines are candidates for this effect, but the most well-characterized is IL-6, which signals through the Jak/Stat-3 pathway [24]. Colonic hepcidin production did not alter serum or liver hepcidin, iron or transferrin levels. Additionally, our experimental rats were not anemic, suggesting a localized role for hepcidin in intestinal inflammation. In an initial report, low serum hepcidin-25 levels were found in IBD patients with or without iron deficiency anemia [25]. A subsequent report demonstrated that serum hepcidin-25 levels are increased in UC and CD patients compared with healthy control patients, and in UC patients, this increase was related to disease activity but not iron deficiency [26]. Furthermore, in a recent report, serum hepcidin-25 levels were implicated in the differential diagnosis of IBD-associated anemia, but hepcidin-20 levels were independently regulated by inflammation [27]. Moreover, increased IL-6 and hepcidin serum levels were measured in CD patients with ACD [28], but no studies have examined colonic hepcidin expression in human IBD. The main site of dietary iron absorption in adults is the proximal small intestine, whereas the distal small intestine and colon are absorb only small amounts of iron [29]. Curiously, the Prussian blue reaction in the colons of the control group did not reveal the presence of iron, as observed in inflamed tissue. Hepcidin acts by triggering ferroportin internalization and degradation through a mechanism most likely involving ferroportin ubiquitination [30]. Ferroportin downregulation results in intracellular 4350 April 21, 2014 Volume 20 Issue 15

252 Gotardo EMF et al. Hepcidin expression in colitis iron accumulation and unavailable local or plasma iron. As previously discussed, local iron sequestration could participate in an antimicrobial response associated with intestinal inflammation, but we could not exclude the possibility that it contributes to increased local inflammation by recruiting macrophages with a pro-inflammatory phenotype. In the treatment of iron deficiency anemia in patients with IBD, oral iron supplementation induced adverse gastrointestinal events that resulted in drug discontinuation in 20.8% of patients [31]. A recent study also demonstrated that dietary iron intake was negatively associated with quality of life in mildly active IBD patients [32]. In conclusion, we are the first to demonstrate that colonic inflammation can increase local hepcidin-25 expression in association with IL-6 production and Stat-3 activation. Although local iron deposition was also observed, additional studies are necessary to understand whether this observation constitutes a defensive or pathological response to intestinal inflammation. COMMENTS Background Hepcidin is an endogenous peptide with weak antimicrobial properties that regulates iron metabolism during inflammation. Hepcidin expression in intestinal inflammation has not been previously studied and could aid in the understanding of the pathophysiology of inflammatory bowel diseases. Research frontiers Hepcidin expression appears to be induced by pathogen-associated molecular patterns, likely through interleukin-6 (IL-6) induction, as part of the host immune response. In this study, authors described colitis-induced colonic hepcidin expression that was associated with the IL-6/Stat-3 signaling pathway and local iron sequestration. These observations suggest a host response to infection that reduces the iron available for pathogen use as an important antimicrobial mechanism. However, authors could not exclude the possibility that hepcidin expression contributed to increased local inflammation by recruiting pro-inflammatory macrophages. Innovations and breakthroughs Recent reports have highlighted the importance of hepcidin as the major regulator of iron homeostasis in humans and other mammals. Iron is an important factor in the competition for nutritional resources between microbial pathogens and their hosts. Here, authors described colitis-induced colonic hepcidin expression associated with the IL-6/Stat-3 signaling pathway and local iron sequestration. Local iron sequestration could participate in an antimicrobial response associated with intestinal inflammation. Furthermore, human colon adenocarcinoma HT-29 cells increased hepcidin expression after IL-6 and lipopolysaccharide (a toll-like receptor 4 ligand) treatment, suggesting that inflammation and microbial pathogens could participate in authors in vivo observations. Applications By demonstrating that intestinal inflammation induced hepcidin expression and that hepcidin could function in a protective role, this study contributes to a better understanding of the pathophysiology of inflammatory bowel disease. Terminology Hepcidin is a peptide that is mainly expressed and released by the liver in response to increased circulating iron levels. Inflammation and infections are also able to induce hepcidin synthesis resulting in hypoferremia. Hepcidin acts by triggering ferroportin internalization and degradation by a mechanism that most likely involves ferroportin ubiquitination. Downregulation of ferroportin results in the intracellular accumulation of iron and its unavailability locally and in plasma. Peer review This manuscript focuses on hepcidin in an animal model of gut inflammation. The management of iron, and the mechanisms leading to iron deficiency in inflammatory bowel disease are important issues. This work adds further to this area. REFERENCES 1 Mullin GE. Micronutrients and inflammatory bowel disease. Nutr Clin Pract 2012; 27: [PMID: DOI: / ] 2 Kent AJ, Blackwell VJ, Travis SP. What is the optimal treatment for anemia in inflammatory bowel disease? Curr Drug Deliv 2012; 9: [PMID: ] 3 Sun CC, Vaja V, Babitt JL, Lin HY. Targeting the hepcidinferroportin axis to develop new treatment strategies for anemia of chronic disease and anemia of inflammation. Am J Hematol 2012; 87: [PMID: DOI: / ajh.23110] 4 Ganz T, Nemeth E. Iron sequestration and anemia of inflammation. Semin Hematol 2009; 46: [PMID: DOI: /j.seminhematol ] 5 Finberg KE. 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Cathelicidin signaling via the Toll-like receptor protects against colitis in mice. Gastroenterology 2011; 141: e1-3 [PMID: DOI: /j.gastro ] 11 Ho S, Pothoulakis C, Koon HW. Antimicrobial peptides and colitis. Curr Pharm Des 2013; 19: [PMID: ] 12 Schwarz P, Kübler JA, Strnad P, Müller K, Barth TF, Gerloff A, Feick P, Peyssonnaux C, Vaulont S, Adler G, Kulaksiz H. Hepcidin is localised in gastric parietal cells, regulates acid secretion and is induced by Helicobacter pylori infection. Gut 2012; 61: [PMID: DOI: /gut ] 13 Ward DG, Roberts K, Brookes MJ, Joy H, Martin A, Ismail T, Spychal R, Iqbal T, Tselepis C. Increased hepcidin expression in colorectal carcinogenesis. World J Gastroenterol 2008; 14: [PMID: ] 14 Gambero A, Maróstica M, Abdalla Saad MJ, Pedrazzoli J. Mesenteric adipose tissue alterations resulting from experimental reactivated colitis. Inflamm Bowel Dis 2007; 13: [PMID: DOI: /ibd.20222] 15 Funakoshi T, Yamashita K, Ichikawa N, Fukai M, Suzuki T, Goto R, Oura T, Kobayashi N, Katsurada T, Ichihara S, Ozaki M, Umezawa K, Todo S. A novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin, ameliorates inflammatory colonic injury in mice. J Crohns Colitis 2012; 6: [PMID: DOI: /j.crohns ] 16 Rodríguez-Cabezas ME, Gálvez J, Lorente MD, Concha A, Camuesco D, Azzouz S, Osuna A, Redondo L, Zarzuelo A. Dietary fiber down-regulates colonic tumor necrosis factor alpha and nitric oxide production in trinitrobenzenesulfonic acid-induced colitic rats. J Nutr 2002; 132: [PMID: ] 17 Clemente TR, Dos Santos AN, Sturaro JN, Gotardo EM, de Oliveira CC, Acedo SC, Caria CR, Pedrazzoli J, Ribeiro ML, 4351 April 21, 2014 Volume 20 Issue 15

253 Gotardo EMF et al. Hepcidin expression in colitis Gambero A. Infliximab modifies mesenteric adipose tissue alterations and intestinal inflammation in rats with TNBSinduced colitis. Scand J Gastroenterol 2012; 47: [PMID: DOI: / ] 18 Layoun A, Santos MM. Bacterial cell wall constituents induce hepcidin expression in macrophages through MyD88 signaling. Inflammation 2012; 35: [PMID: DOI: /s ] 19 Theurl I, Theurl M, Seifert M, Mair S, Nairz M, Rumpold H, Zoller H, Bellmann-Weiler R, Niederegger H, Talasz H, Weiss G. Autocrine formation of hepcidin induces iron retention in human monocytes. Blood 2008; 111: [PMID: DOI: /blood ] 20 Recalcati S, Locati M, Gammella E, Invernizzi P, Cairo G. Iron levels in polarized macrophages: regulation of immunity and autoimmunity. Autoimmun Rev 2012; 11: [PMID: DOI: /j.autrev ] 21 Bekri S, Gual P, Anty R, Luciani N, Dahman M, Ramesh B, Iannelli A, Staccini-Myx A, Casanova D, Ben Amor I, Saint- Paul MC, Huet PM, Sadoul JL, Gugenheim J, Srai SK, Tran A, Le Marchand-Brustel Y. Increased adipose tissue expression of hepcidin in severe obesity is independent from diabetes and NASH. Gastroenterology 2006; 131: [PMID: DOI: /j.gastro ] 22 Vokurka M, Lacinová Z, Kremen J, Kopecký P, Bláha J, Pelinková K, Haluzík M, Necas E. Hepcidin expression in adipose tissue increases during cardiac surgery. Physiol Res 2010; 59: [PMID: ] 23 Sheikh N, Dudas J, Ramadori G. Changes of gene expression of iron regulatory proteins during turpentine oil-induced acute-phase response in the rat. Lab Invest 2007; 87: [PMID: DOI: /labinvest ] 24 Gaffney-Stomberg E, McClung JP. Inflammation and diminished iron status: mechanisms and functional outcomes. Curr Opin Clin Nutr Metab Care 2012; 15: [PMID: DOI: /MCO.0b013e328357f63b] 25 Arnold J, Sangwaiya A, Bhatkal B, Geoghegan F, Busbridge M. Hepcidin and inflammatory bowel disease: dual role in host defence and iron homoeostasis. Eur J Gastroenterol Hepatol 2009; 21: [PMID: DOI: / MEG.0b013e32830e2885] 26 Oustamanolakis P, Koutroubakis IE, Messaritakis I, Malliaraki N, Sfiridaki A, Kouroumalis EA. Serum hepcidin and prohepcidin concentrations in inflammatory bowel disease. Eur J Gastroenterol Hepatol 2011; 23: [PMID: DOI: /MEG.0b013e328343b885] 27 Bergamaschi G, Di Sabatino A, Albertini R, Costanzo F, Guerci M, Masotti M, Pasini A, Massari A, Campostrini N, Corbella M, Girelli D, Corazza GR. Serum hepcidin in inflammatory bowel diseases: biological and clinical significance. Inflamm Bowel Dis 2013; 19: [PMID: DOI: /MIB.0b013e31829a6e43] 28 Basseri RJ, Nemeth E, Vassilaki ME, Basseri B, Enayati P, Shaye O, Bourikas LA, Ganz T, Papadakis KA. Hepcidin is a key mediator of anemia of inflammation in Crohn s disease. J Crohns Colitis 2013; 7: e286-e291 [PMID: DOI: /j.crohns ] 29 Frazer DM, Wilkins SJ, Anderson GJ. Elevated iron absorption in the neonatal rat reflects high expression of iron transport genes in the distal alimentary tract. Am J Physiol Gastrointest Liver Physiol 2007; 293: G525-G531 [PMID: DOI: /ajpgi ] 30 Qiao B, Sugianto P, Fung E, Del-Castillo-Rueda A, Moran- Jimenez MJ, Ganz T, Nemeth E. Hepcidin-induced endocytosis of ferroportin is dependent on ferroportin ubiquitination. Cell Metab 2012; 15: [PMID: DOI: / j.cmet ] 31 Schröder O, Mickisch O, Seidler U, de Weerth A, Dignass AU, Herfarth H, Reinshagen M, Schreiber S, Junge U, Schrott M, Stein J. Intravenous iron sucrose versus oral iron supplementation for the treatment of iron deficiency anemia in patients with inflammatory bowel disease--a randomized, controlled, open-label, multicenter study. Am J Gastroenterol 2005; 100: [PMID: DOI: / j x] 32 Powell JJ, Cook WB, Hutchinson C, Tolkien Z, Chatfield M, Pereira DIa, Lomer MC. Dietary fortificant iron intake is negatively associated with quality of life in patients with mildly active inflammatory bowel disease. Nutr Metab (Lond) 2013; 10: 9 [PMID: DOI: / ] P- Reviewers: Day AS, Horiguchi H, Pantopoulos K, Yamakawa M S- Editor: Ma YJ L- Editor: A E- Editor: Wu HL 4352 April 21, 2014 Volume 20 Issue 15

254 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH REPORT Risk factors associated with Barrett s epithelial dysplasia Mikiko Fujita, Yuri Nakamura, Saeko Kasashima, Maiko Furukawa, Ryoichi Misaka, Hikaru Nagahara Mikiko Fujita, Yuri Nakamura, Saeko Kasashima, Maiko Furukawa, Ryoichi Misaka, Hikaru Nagahara, Department of Gastroenterology, Aoyama Hospital, Tokyo Women s Medical University, Tokyo , Japan Author contributions: Fujita M, Misaka R and Nagahara H designed the study and wrote the manuscript; Fujita M, Nakamura Y, Kasashima S and and Furukawa M performed the endoscopic diagnosis and acquired the data; Misaka R and Nagahara H analyzed and interpreted the data; Nagahara H gave final approval to the version to be published. Correspondence to: Hikaru Nagahara, Professor, Department of Gastroenterology, Aoyama Hospital, Tokyo Women s Medical University, Kitaaoyama, Minato-ku, Tokyo , Japan. nagahara.hikaru@twmu.ac.jp Telephone: Fax: Received: September 5, 2013 Revised: October 27, 2013 Accepted: November 18, 2013 Published online: April 21, 2014 Abstract AIM: to elucidate risk factors associated with dysplasia of short-segment Barrett s esophagus (BE). METHODS: A total of 151 BE patients who underwent endoscopic examination from 2004 to 2008 in Aoyama Hospital, Tokyo Women s Medical University, Japan and whose diagnosis was confirmed from biopsy specimens were enrolled in the study. BE was diagnosed based on endoscopic findings of gastric-appearing mucosa or apparent columnar-lined esophagus proximal to the esophagogastric junction. Dysplasia was classified into three grades - mild, moderate and severe - according to the guidelines of the Vienna Classification System for gastrointestinal epithelial neoplasia. Anthropometric and biochemical data were analyzed to identify risk factors for BE dysplasia. The prevalence of Helicobacter pylori (H. pylori ) infection and the expression of p53 by immunohistological staining were also investigated. RESULTS: Histological examination classified patients into three types: specialized columnar epithelium (SCE) (n = 65); junctional (n = 38); and gastric fundic (n = 48). The incidence of dysplasia or adenocarcinoma from BE of the SCE type was significantly higher than that of the other two types (p < 0.01). The univariate analysis revealed that sex, H. pylori infection, body weight, p53 overexpression, and low diastolic blood pressure (BP) were associated with BE dysplasia. In contrast, body mass index, waist circumference, metabolic syndrome complications, and variables related to glucose or lipid metabolism were not associated with dysplasia. Multivariate logistic analysis showed that overexpression of p53 [odds ratio (OR) = 13.1, p = 0.004], H. pylori infection (OR = 0.19, p = 0.066), and diastolic BP (OR = 0.87, p = 0.021) were independent risk factors for epithelial dysplasia in BE patients with the SCE type. CONCLUSION: Overexpression of p53 is a risk factor for dysplasia of BE, however, H. pylori infection and diastolic BP inversely associated with BE dysplasia might be protective Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Barrett s esophagus; dysplasia; Helicobacter pylori; p53; risk factors Core tip: Barrett s esophagus (BE) is known to be a precancerous state of adenocarcinoma will become common in Asian countries, therefore, it is important to establish a high-risk group or strategy for screening or follow-up of BE. We present here the results of univariate and multivariate analysis to identify variables associated with dysplasia of BE. p53 expression in immunohistochemistry was associated with dysplasia, and Helicobacter pylori infection and high diastolic blood pressure may act as protective factors against dysplastic change of BE. These three factors may be candidates to establish a high-risk group for esophageal adenocarcinoma. Fujita M, Nakamura Y, Kasashima S, Furukawa M, Misaka R, 4353 April 21, 2014 Volume 20 Issue 15

255 Fujita M et al. Risk factors for Barrett s epithelial dysplasia Nagahara H. Risk factors associated with Barrett s epithelial dysplasia. World J Gastroenterol 2014; 20(15): Available from: URL: i15/4353.htm DOI: INTRODUCTION Barrett s esophagus (BE) is defined as a condition in which normal squamous mucosa is replaced by columnar epithelium. This intestinal metaplasia of the distal esophagus is considered to be a premalignant condition where metaplasia may progress to dysplasia and subsequently to adenocarcinoma [1-6]. BE is generally regarded as a complication of chronic and severe gastroesophageal reflux disease (GERD). Elevation of the intraabdominal pressure by obesity is a factor contributing to GERD, suggesting that obesity is a risk factor for BE [7-10]. GERD and BE appear to be metabolic syndrome (MS)- related complications, given that waist circumference, obesity, and body mass index (BMI) are associated with GERD [11-16]. Moreover, Helicobacter pylori (H. pylori) infection may play a key role in suppression of BE. Two main inhibiting roles for development of BE have been postulated in H. pylori infection: H. pylori-induced atrophic gastritis resulting in less gastric acid secretion; and neutralization of the gastric acid by ammonia produced by H. pylori independently of gastric atrophy [17-23]. Cag-A positive H. pylori infection is strongly associated with a reduced risk of esophageal adenocarcinoma, and the association is independent of gastric atrophy, suggesting the involvement of a mechanism other than reduced acidic gastric reflux [24-26]. In Japan, the prevalence of H. pylori is declining and it can be easily eradicated; however, it remains uncertain whether the incidence of BE will increase or decrease as a consequence of the low prevalence of H. pylori infection [27,28]. BE is characterized by three types of columnar epithelium, namely, cardiac type (junctional type), fundic type, and intestinal metaplasia type [specialized columnar epithelium type (SCE) type]. It has been shown that there is a high incidence of adenocarcinoma in the distal esophagus arising from SCE in patients with BE [29,30]. BE is classified as either long-segment type (length 3 cm) or short-segment type (length < 3 cm). In western countries, long-segment Barrett s esophagus (LSBE) is more prevalent, while short-segment Barrett s esophagus (SSBE) is more common, and the incidence of adenocarcinoma arising from SSBE is steadily increasing in Japan [27,28,31-33]. Several studies have shown that most patients with BE do not progress to cancer, although some do [1-3,29,30]. Thus, it is important to determine how BE progresses to dysplasia and adenocarcinoma and to identify the type of BE patients who may have a possibility of malignant transformation in SCE. It has been reported that central adiposity, metabolic syndrome, and high BMI are associated with BE and adenocarcinoma [10-16]. In this study, we investigated the risk factors associated with BE dysplasia. MATERIALS AND METHODS Study population A total of 151 patients (105 male, 46 female) with histologically diagnosed BE were enrolled. All cases were incident cases and enrolled in a consecutive series from April 2004 to March Patients who had received antibiotics, steroids, or nonsteroidal anti-inflammatory drugs were excluded from the study. Patients were also excluded if they had peptic ulcer, underwent partial gastrectomy, consumed alcohol excessively, or had comorbid diseases such as liver cirrhosis and uremia. Written informed consent was obtained from all patients. Endoscopic examination BE was diagnosed based on endoscopic findings of gastric-appearing mucosa or apparent columnar lined esophagus proximal to the esophagogastric junction. The esophagogastric junction was defined as the pinch at the end of the tubular esophagus coinciding with the proximal margin of the gastric folds of the hiatal hernia. BE was defined as columnar mucosa proximal to the distal ends of esophageal longitudinal palisading vessels according to the Japanese Society of Esophageal Disease [34,35]. These veins are visible endoscopically when a conscious patient takes a deep breath. SSBE was defined as an epithelium length < 3 cm and LSBE as an epithelium length > 3 cm, as described previously [1,2,29,30] The length of BE were measured by measuring forceps (Olympus, Tokyo, Japan). When abnormal columnar mucosa characteristics such as erosions, red flares, elevated regions, or mucosal breaks were observed between the proximal limit of the gastric folds and squamous epithelium, we detected metaplastic change by chromoendoscopy and staining mucosa with crystal violet (Figure 1a, b). For chromoendoscopy U pronase (Pronase MS; Kaken Pharmaceutical Co., Matsumoto, Japan) dissolved in 300 ml warm water was sprayed around the esophagogastric junction area with a spray tube, and a 0.03% solution of crystal violet was applied to the same area. A few minutes later, the sprayed area was washed thoroughly with water. When mucosa showing a tubular or villous pit pattern, which is typical of SCE in BE, was observed in the esophagogastric junction, we performed a targeted biopsy in that area. BE was confirmed by histological findings from biopsy specimens in all patients. Histology All biopsy specimens were fixed in formalin, embedded in paraffin, sectioned, mounted on slides and then stained with hematoxylin and eosin using standard techniques. Dysplasia was classified into three grades: mild, moderate and severe, according to the guidelines of the Vienna Classification System for gastrointestinal epithe April 21, 2014 Volume 20 Issue 15

256 Fujita M et al. Risk factors for Barrett s epithelial dysplasia A B Figure 1 Barrett s esophagus stained by crystal violet. A: Regular observation of Barrett s esophagus; B: Staining with crystal violet in the same region. lial neoplasia [17,36]. To perform immunohistological staining of p53, an anti-human p53 antibody (DO-7 mouse monoclonal antibody, IR616; Dako, Denmark) was used according to the manufacturer s protocol. The expression level of p53 protein was determined and graded based on the intensity of nuclear staining in columnar cells as follows: no staining (-), positive nuclear staining in 5%-10% of cells (+), and positive nuclear staining in > 10% of cells (++), according to the work of Keswani et al [37] (Figure 2). All biopsy specimens were examined by an experienced gastrointestinal pathologist. H. pylori infection The presence of gastric H. pylori was determined based on the results of Giemsa and/or Steiner s silver staining in a minimum of three gastric surveillance biopsies (one each obtained from the antral greater curvature, greater curvature of the mid to distal body, and lesser curvature in the proximal body). H. pylori colonization was assessed by an experienced pathologist blinded to the clinical data. Patients who were not confirmed H. pylori infection by using above histological analysis were retested by other methods. We confirmed H. pylori negativity by combination of serum HP-specific antibody test with 13Curea breath test or H. pylori antigen test in the stools. Anthropometry and blood pressure The body weights of patients, while not wearing heavy outdoor clothing or shoes, was measured to the nearest 0.1 kg using a digital scale. Height (barefoot) was measured using a portable stadiometer. Waist circumference was measured to the nearest 0.1 cm using a plastic tape just above the umbilical portion while standing in a relaxed position after gentle expiration. BMI was calculated as weight in kilograms divided by the square of height in meters (kg/m 2 ). Blood pressure was measured with a mercury sphygmomanometer on each arm after at least 10 min of rest. Definition of MS and biochemical analysis MS was diagnosed according to the criteria set out by the Japanese Committee for the Diagnostic Criteria of Metabolic Syndrome [38] : central obesity (waist circumference 85 cm Japanese men, 90 cm Japanese women) plus any two of the following; raised triglycerides 150 mg/dl or specific treatment for this lipid abnormality; reduced high density lipoprotein (HDL)-cholesterol < 40 mg/dl in men and women; raised blood pressure (systolic 130 mmhg or diastolic 85 mmhg) or treatment of hypertension; fasting glucose 110 mg/dl or previously diagnosed type 2 diabetes mellitus. After a 12-h overnight fast, venous blood samples were taken for the standard biochemical data. Statistical analysis Statistical analysis was performed using SPSS for Windows version 17.0 (SPSS, Tokyo, Japan). Results for continuous variables were expressed as mean ± SD for each subject group. The statistical difference was determined by two-sided Student s t test (for equal variance cases) or Welch s t test (for unequal variance cases). Non-normally distributed variables were compared by the Mann-Whitney U test. Variables given as proportions were compared using the c 2 test. The relationships between risk factors and dysplasia including adenocarcinoma of BE were examined by multivariate logistic regression analysis. p < 0.05 was considered to be statistically significant. Differences in mean laboratory data and anthropometric data across three categories were evaluated using oneway analysis of variance (ANOVA). RESULTS Endoscopic findings of BE by crystal violet staining Crystal violet staining was performed when we recognized BE during routine endoscopic examination. The intestinal metaplastic lesion was stained with a violet color, resulting in easy recognition of the targeted biopsy (Figure 1a, b). Dysplasia in SCE-type BE The average age of the 151 BE patients was 62.9 ± 10.6 years and there were 105 men to 46 women (ratio 2.3:1). The demographic characteristics of the patients according to pathological classification are shown in Table 1. BE patients were classified into three categories: SCE type (n = 65), junctional type (n = 38), and gastric fundic 4355 April 21, 2014 Volume 20 Issue 15

257 Fujita M et al. Risk factors for Barrett s epithelial dysplasia A No dysplasia, p53 (-) 40, HE 40, p53 B Moderate dysplasia, p53 (+) 40, HE 40, p53 C Adenoca., p53 (++) 40, HE 40, p53 Figure 2 Immunostaining of p53. The upper panel shows hematoxylin and eosin staining and the lower panel shows immunostaining of p53 using the identical sample. A: (-), no p53 expression; B: (+), moderate p53 expression characterized by positive nuclear staining in 5%-10% of cells; C: (++), high p53 expression characterized by positive nuclear staining in > 10% of cells. Table 1 Characterization of the three types of Barrett s esophagus Specialized columnar P value Epithelium type Junctional type Gastric fundic type (n = 65) (n = 38) (n = 48) Sex 52/13 10/28 25/ Age (yr) 62.9 ± ± ± Dysplasia 20/65 (30.8%) 3/38 (7.9%) 2/48 (4.2%) type (n = 48), and the incidence of dysplasia in these three groups was 30.8% (20/65), 7.9% (3/38) and 4.2% (2/48), respectively. The ratio of dysplasia in patients with SCE-type BE was significantly higher than in patients with junctional- and gastric-fundic-type BE (p = 0.02 and p = 0.002, respectively). Variables associated with dysplasia in SCE-type BE We focused on the SCE type of BE because of the high rate of dysplastic change associated with this condition, as shown in Table 1. We compared variables between SCE-type BE patients with and without dysplasia (Table 2). H. pylori infection, p53 overexpression (Figure 2), body weight, and diastolic BP were identified as risk factors strongly associated with dysplastic change. In contrast, BMI, waist circumference, MS complications, and variables related to glucose or lipid metabolism were not associated with dysplasia. We then conducted multivariate logistic analysis of those variables that showed low P 4356 April 21, 2014 Volume 20 Issue 15

258 Fujita M et al. Risk factors for Barrett s epithelial dysplasia Table 2 Univariate analysis of variables associated with Barrett s epithelial dysplasia Normal (n = 45) Dysplasia (n = 20) P value OR 95%CI Sex (male/female) Dec-33 19/ Age (yr) 65.0 ± ± Length of BE (SSBE/LSBE) Feb-43 18/ H. pylori infection rate 34/9 (2) 7/12 (1) p53 positive rate 4/41 13/7 < Body weight (kg) 61.0 ± ± BMI (kg/m 2 ) 23.1 ± ± Waist circumference (cm) 86.9 ± ± GERD 19/26 9/ Hypertension (BP > 130/85) 29/14 (2) 9/10 (1) Systolic BP (mmhg) 127 ± ± Diastolic BP (mmhg) 76 ± ± Diabetes 14/25 (6) 10/9 (1) Fasting blood glucose (mg/dl) 118 ± ± HbA1c (%) 5.6 ± ± Dyslipidemia 24/19 (2) 11/8 (1) TG (mg/dl) 144 ± ± TC (mg/dl) 202 ± ± HDL-C (mg/dl) 55 ± ± LDL-C (mg/dl) 118 ± ± Fatty liver 17/13 (15) 6/8 (6) γ-gt (U/L) 55 ± ± AST (IU/L) 24 ± 8 25 ± ALT (IU/L) 24 ± ± hs-crp (mg/dl) ± ± MS 15/14 (16) 6/8 (6) ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; BP: Blood pressure; GT: Glutamyl transpeptidase; HbA1c: Hemoglobin A1c; HDL-C: High-density lipoprotein cholesterol; hs-crp: High sensitivity C-reactive protein; LDL-C: Low-density lipoprotein cholesterol; TG: Triglyceride; TC: Total cholesterol; H. pylori: Helicobacter pylori. Table 3 Multivariate analysis of variables associated with Barrett s epithelial dysplasia Risk factor P value OR 95%CI p H. pylori Diastolic BP H. pylori: Helicobacter pylori; BP: Blood pressure. values in the univariate analysis shown in Table 2; namely, sex, H. pylori infection, body weight, p53 overexpression, and low diastolic BP. Among these these variables, p53 overexpression, H. pylori infection, and low diastolic BP were independent risk factors associated with dysplasia complicated in patients with BE of the SCE type (Table 3). The p value of H. pylori infection was (not less than 0.05), but the odds ratio was 0.187, which seemed to give a relatively strong effect on the association with Barrett epithelial dysplasia. Thus, we included it in Table 3. Risk factors associated with progression of SCE from nondysplastic epithelium to low-grade and high-grade dysplasia We assesed the linearity of the relationship between risk factors and progression. We classified dysplasia into three groups: no dysplasia (n = 45), low-grade dysplasia (n = 14), and high-grade dysplasia (n = 6) including adenocarcinoma (Table 4). Based on ANOVA, six variables were significantly associated with alteration of SCE from nondysplasia to high-grade dysplasia: length of BE, H. pylori infection, p53 overexpression, body weight, GERD, and low diastolic BP. Only two of these six variables showed a linear correlation with alteration to high-grade dysplasia; that is, H. pylori infection and p53 overexpression (Table 4). Correlation between p53 expression and progression of SCE from non-dysplasia to low- and high-grade dysplasia Given the strong association observed between p53 overexpression and dysplasia seen in the multivariate logistic analysis (Table 3), we analyzed the level of p53 expression and its association with progression of nondysplastic SCE to low- and high-grade dysplasia including adenocarcinoma. The expression of p53 was categorized as no expression (-), moderate expression characterized by positive nuclear staining in 5-10% of cells (+), and high expression, characterized by positive nuclear staining in > 10% of cells (++) (Figure 2). As shown in Table 5, only 10% of patients in the nondysplastic SCE group expressed p53 at a low level, whereas expression was high in the group with high-grade dysplasia (p < 0.01). DISCUSSION Several studies, based on endoscopic, biochemical and anthropometric data, have identified GERD, absence of H. pylori infection, MS, waist circumference, and body 4357 April 21, 2014 Volume 20 Issue 15

259 Fujita M et al. Risk factors for Barrett s epithelial dysplasia Table 4 Analysis of variance for the three categories of Barrett s epithelium: non-dysplasia, low-grade dysplasia, and high-grade dysplasia No dysplasia (n = 45) Low grade (n = 14) High grade (n = 6) P value Sex (male/female) Dec-33 13/1 6/ Age (yr) 65.0 ± ± ± Length of BE (SSBE/LSBE) Feb-43 14/0 4/ H. pylori infection rate Sep-34 6/7 1/ p53 positive rate 4/41 7/7 6/0 < Body weight (kg) 61.0 ± ± ± BMI (kg/m 2 ) 23.1 ± ± ± Waist circumference (cm) 86.9 ± ± ± GERD 19/26 9/5 0/ Hypertension (BP > 130/85) 29/14 6/7 3/ Systolic BP (mmhg) 127 ± ± ± Diastolic BP (mmhg) 76 ± ± 9 71 ± Diabetes 14/25 7/6 3/ Fasting blood glucose (mg/dl) 118 ± ± ± HbA1c (%) 5.6 ± ± ± Dyslipidemia 24/19 5/8 3/ TG (mg/dl) 144 ± ± ± TC (mg/dl) 202 ± ± ± HDL-C (mg/dl) 55 ± ± 9 48 ± LDL-C (mg/dl) 118 ± ± ± Fatty liver 17/13 5/7 1/ γ-gtp (U/L) 55 ± ± ± AST (IU/L) 24 ± 8 26 ± ± ALT (IU/L) 24 ± ± ± Hs-CRP (mg/dl) ± ± ± Metabolic syndrome 15/14 4/7 2/ SSBE: Short segment Barrett esophagus; BMI: Body mass index; LSBE: Long-segment Barrett s esophagus; GERD: Gastroesophageal reflux disease; BP: Blood pressure; TG: Triglyceride; TC: Total cholesterol; HDL-C: High-density lipoprotein cholesterol; LDL-C: Low-density lipoprotein cholesterol; ALT: Alanine aminotransferase; AST: Aspartate aminotransferase; hs-crp: High sensitivity C-reactive protein; GTP: Guanosinetriphosphate. Table 5 Relationship between the level of p53 expression and grade of dysplasia Normal (n = 45) Low grade (n = 14) High grade and Ca (n = 6) p53 (-) p53 (+) p53 (++) Pearson χ 2 test, P < weight as risk factors associated with the presence of BE [7-16]. One of the most notable findings from epidemiological reports has been a strong inverse association between H. pylori infection and dysplasia of Barrett s epithelium [17,18,21-26]. Esophageal adenocarcinoma derived from BE is not common in Japan as compared with western countries, whereas gastric carcinoma is more prevalent in Japan. This inverse relationship may reflect the high prevalence of H. pylori infection in Japan and the low prevalence in western countries [24,39,40]. Another notable epidemiological difference between these regions is the length of BE; that is, SSBE is common in Japan but LSBE is more prevalent in western countries [27,28,30,31]. The underlying reasons for this difference are not currently known. Here, we identified risk factors associated with the presence of BE dysplasia by comparison of patients with non-dysplasia to those with low- to high-grade dysplasia including adenocarcinoma. In our cohort, 94% of BE cases were the SSBE type (Table 2). Overexpression of p53 was the most important risk factor associated with dysplasia and adenocarcinoma (Table 3), and the level of p53 expression was strongly related to the grade of dysplasia (Table 5). Several studies have shown that p53 overexpression is increased in parallel with progression of histological changes from metaplasia to high-grade dysplasia and adenocarcinoma [41-44]. In specimens obtained from surgical resection, expression of p53 has been observed in the region of adenocarcinoma as well as in adjacent dysplastic epithelia. In addition, in many cases, p53 gene mutations are found at the specific position resulting in a change in a specific amino acid residue in both adenocarcinoma and adjacent dysplastic epithelia [41,42]. These results suggest that p53 mutation, which is relatively uncommon in nondysplastic BE, is an important step in the progression to adenocarcinoma. Galipeau et al [45] have shown that inactivation of p53 by mutation is strongly associated with progression to aneuploidy, possibly through the loss of p53-mediated apoptosis and cell-cycle arrest. The accumulation of these aneuploid cell populations has been shown to increase the risk of developing adenocarcinoma [46]. The possible causal role of p53 in tumorigenesis as well as tumor progression in BE has been postulated, based on histological evidence showing that p53 mutations are more frequent in advanced stages in histology. Thus, it is important to address the hypothesis that p April 21, 2014 Volume 20 Issue 15

260 Fujita M et al. Risk factors for Barrett s epithelial dysplasia overexpression could predict progression of nondysplastic BE to adenocarcinoma. Younes et al [47] studied p53 accumulation via immunohistochemistry in 54 patients with Barrett s metaplasia, dysplasia, or adenocarcinoma; p53 accumulation increased in parallel with histological progression from metaplasia to adenocarcinoma. Follow-up biopsies were available in 23 of 54 patients who had dysplasia in at least one biopsy specimen. They showed that only one of 21 (4.8%) patients who were all p53 negative in multiple biopsies had histological progression. In contrast, two of three patients with p53- positive biopsies progressed to high-grade dysplasia or intramucosal carcinoma (one patient was lost to followup). These retrospective data suggest that p53 accumulation increases the risk of progression from low- to highgrade dysplasia. These data are also consistent with our results showing that the level of p53 expression was correlated with the grade of dysplasia (Table 5), suggesting that mutated p53, which is expressed at an early stage, may stimulate the tumor progression in the metaplasia dysplasia-adenocarcinoma sequence of BE. We found a strong inverse association between H. pylori infection and dysplasia in BE (Table 2); the lowest prevalence of H. pylori was observed in the high-grade dysplasia group (Table 4). Many studies have reported that the absence of H. pylori colonization is associated with a greater likelihood of developing esophageal dysplasia and adenocarcinoma [18,20-23]. Hence, H. pylori infection appears to have a protective effect against the development of dysplasia and adenocarcinoma in BE. The mechanism through which the absence of H. pylori colonization is associated with dysplasia in BE is unknown, but there are several possibilities. First, H. pylori infection, in particular the more virulent Cag-A-positive strain, may lead to gastric atrophy resulting in suppression of acid production; this may lower the risk of BE and esophageal adenocarcinoma [24,25]. We analyzed the degree of gastric atrophy according to the Kimura-Takemoto Classification [48] to confirm the association of low acidity and BE dysplasia. We could not find any significant difference in the ratio of atropy and distribution of degree of atrophy in normal individuals and patients with dysplasia (data not shown). With regard to the possible outcome of H. pylori eradication in BE, only a few studies have reported that the SSBE developed at 24 mo after H. pylori eradication, suggesting induction of SSBE by the eradication [49]. In Japan, the prevalence of H. pylori infection has been decreasing recently and the use of H. pylori eradication therapy has flourished. For this reason, the incidence of BE and adenocarcinoma is likely to increase, and it is therefore important to determine risk factors for malignant changes associated with the development of dysplasia and adenocarcinoma in BE after H. pylori eradication. In our multivariate logistic analysis, diastolic blood pressure was an independent risk factor associated with progression of BE from nondysplastic to dysplastic epithelium (Table 3). When we evaluated users of antihypertensive drugs, especially Ca antagonists, in normal individuals and patients with dysplasia, we could not find any significant difference (data not shown). Although this is believed to be the first report of a relationship between diastolic blood pressure and dysplasia in BE, the underlying mechanisms are unclear. When we analyzed the relationship variables among three categories such as patients with no dysplasia, lowor high- grade dysplasia by ANOVA, we detected no difference in diastolic BP (Table 4). We hypothesized the nonlinear relationship of diastolic BP among the three groups shown in Table 4 as follows: if the variables were associated with malignant potential of Barrett s epithelium, it may show the linear relationship among the three groups, normal, low-grade and high-grade dysplasia. The diastolic BP, however, may be associated with the step such as columnar epithelial metaplasia, which is a relatively early step of transformation. In our univariate analysis, body weight was extracted as a risk factor for dysplasia in BE, but BMI and waist circumference were not. In a Swedish study of 189 cases of newly diagnosed esophageal adenocarcinoma, a strong positive association was found between BMI and esophageal adenocarcinoma when controlling for GERD symptoms [11]. A study from the Veterans Association in the United States found that individuals with a BMI > 30 had a fourfold greater risk for BE as compared with controls with a BMI < 25 [7]. More recently, several studies have revealed that waist circumference, but not BMI, has a modest independent associations with the incidence BE, dysplasia and adenocarcinoma. Other studies have reported that a higher waist-to-hip ratio is associated with BE when data are adjusted for GERD symptoms and BMI [14,50]. In our multivariate logistic analysis, anthropometric variables were not extracted as risk factors. In conclusion we demonstrated that p53 overexpression, absence of H. pylori infection, and low diastolic BP are independent risk factors associated with the dysplastic changes of BE. Future studies such as welldesigned prospective studies are needed to elucidate the mechanisms underlying the association of these risk factors with the sequence of progression of dysplasia to adenocarcinoma in BE and establish a high-rsk group progressing to adenocarcinoma. COMMENTS Background Barrett s esophagus (BE) is defined as a condition in which normal squamous mucosa is replaced by columnar epithelium. This intestinal metaplasia of the distal esophagus is considered to be a premalignant condition where metaplasia may progress to dysplasia and subsequently to adenocarcinoma. It has been reported that central adiposity, metabolic syndrome, and high body mass index (BMI) are associated with BE and adenocarcinoma. Research frontiers In order to identify variables associated with BE dysplasia, many variables taken from biochemical and anthropometric data, p53 expression examined by immunostaining and Helicobacter pylori (H. pylori) infection were analyzed. Consequently, the authors analyzed many different types of variables April 21, 2014 Volume 20 Issue 15

261 Fujita M et al. Risk factors for Barrett s epithelial dysplasia Innovations and breakthroughs Only three variables were selected by multivariate analysis. p53 stainig was positively associated with dysplasia, but H. pylori infection and diastolic blood pressure were inversely associated with BE dysplasia. Applications Using these three variables, a prospective study to determine the high-risk group for BE dysplasia or factors related to progression of dysplasia may be the next step. Terminology BE is defined as intestinal metaplasia in which normal squamous mucosa is replaced by columnar epithelium. BE is characterized by three types of columnar epithelium, namely, cardiac type (junctional type), fundic type, and intestinal metaplasia type [specialized columnar epithelium type (SCE) type]. In these three type of BE, SCE type is especially considered to be a precancerous lesion of esophageal adenocarcinoma. Peer review The authors showed the risk factors associated with Barrett s epithelial dysplasia. They enrolled 151 patients with BE in a single hospital. This study was well organized and well investigated. They clearly showed that p53 expression, absence of H. pylori infection and low diastolic blood pressure are risk factors associated with dysplasia of BE. REFERENCES 1 Milind R, Attwood SE. Natural history of Barrett s esophagus. World J Gastroenterol 2012; 18: [PMID: DOI: /wjg.v18.i ] 2 Wiseman EF, Ang YS. Risk factors for neoplastic progression in Barrett s esophagus. World J Gastroenterol 2011; 17: [PMID: DOI: /wjg.v17.i ] 3 den Hoed CM, van Blankenstein M, Dees J, Kuipers EJ. The minimal incubation period from the onset of Barrett s oesophagus to symptomatic adenocarcinoma. Br J Cancer 2011; 105: [PMID: DOI: /bjc ] 4 Ronkainen J, Aro P, Storskrubb T, Johansson SE, Lind T, Bolling-Sternevald E, Vieth M, Stolte M, Talley NJ, Agréus L. 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Role of Helicobacter pylori CagA+ strains and risk of adenocarcinoma of the stomach and esophagus. Int J Cancer 2003; 103: [PMID: DOI: /ijc.10887] 4360 April 21, 2014 Volume 20 Issue 15

262 Fujita M et al. Risk factors for Barrett s epithelial dysplasia 26 Huang JQ, Zheng GF, Sumanac K, Irvine EJ, Hunt RH. Metaanalysis of the relationship between caga seropositivity and gastric cancer. Gastroenterology 2003; 125: [PMID: DOI: /j.gastro ] 27 Hongo M. Review article: Barrett s oesophagus and carcinoma in Japan. Aliment Pharmacol Ther 2004; 20 Suppl 8: [PMID: DOI: /j x] 28 Okita K, Amano Y, Takahashi Y, Mishima Y, Moriyama N, Ishimura N, Ishihara S, Kinoshita Y. Barrett s esophagus in Japanese patients: its prevalence, form, and elongation. J Gastroenterol 2008; 43: [PMID: DOI: / s y] 29 Moons LM, Bax DA, Kuipers EJ, Van Dekken H, Haringsma J, Van Vliet AH, Siersema PD, Kusters JG. The homeodomain protein CDX2 is an early marker of Barrett s oesophagus. J Clin Pathol 2004; 57: [PMID: DOI: /jcp ] 30 Wang KK, Sampliner RE. Updated guidelines 2008 for the diagnosis, surveillance and therapy of Barrett s esophagus. Am J Gastroenterol 2008; 103: [PMID: DOI: /j x] 31 Nobukawa B, Abraham SC, Gill J, Heitmiller RF, Wu TT. Clinicopathologic and molecular analysis of high-grade dysplasia and early adenocarcinoma in short- versus longsegment Barrett esophagus. Hum Pathol 2001; 32: [PMID: DOI: /hupa ] 32 Vahabzadeh B, Seetharam AB, Cook MB, Wani S, Rastogi A, Bansal A, Early DS, Sharma P. Validation of the Prague C & amp; M criteria for the endoscopic grading of Barrett s esophagus by gastroenterology trainees: a multicenter study. Gastrointest Endosc 2012; 75: [PMID: DOI: /j.gie ] 33 Goh KL. Gastroesophageal reflux disease in Asia: A historical perspective and present challenges. J Gastroenterol Hepatol 2011; 26 Suppl 1: 2-10 [PMID: DOI: / j x] 34 Takubo K, Aida J, Sawabe M, Arai T, Kato H, Pech O, Arima M. The normal anatomy around the oesophagogastric junction: a histopathologic view and its correlation with endoscopy. Best Pract Res Clin Gastroenterol 2008; 22: [PMID: DOI: /j.bpg ] 35 Takubo K, Vieth M, Aida J, Sawabe M, Kumagai Y, Hoshihara Y, Arai T. Differences in the definitions used for esophageal and gastric diseases in different countries: endoscopic definition of the esophagogastric junction, the precursor of Barrett s adenocarcinoma, the definition of Barrett s esophagus, and histologic criteria for mucosal adenocarcinoma or high-grade dysplasia. Digestion 2009; 80: [PMID: DOI: / ] 36 Schlemper RJ, Riddell RH, Kato Y, Borchard F, Cooper HS, Dawsey SM, Dixon MF, Fenoglio-Preiser CM, Fléjou JF, Geboes K, Hattori T, Hirota T, Itabashi M, Iwafuchi M, Iwashita A, Kim YI, Kirchner T, Klimpfinger M, Koike M, Lauwers GY, Lewin KJ, Oberhuber G, Offner F, Price AB, Rubio CA, Shimizu M, Shimoda T, Sipponen P, Solcia E, Stolte M, Watanabe H, Yamabe H. The Vienna classification of gastrointestinal epithelial neoplasia. Gut 2000; 47: [PMID: DOI: /gut ] 37 Keswani RN, Noffsinger A, Waxman I, Bissonnette M. Clinical use of p53 in Barrett s esophagus. Cancer Epidemiol Biomarkers Prev 2006; 15: [PMID: DOI: / EPI ] 38 Shimamoto K. [Metabolic syndrome: epidemiology]. Nihon Naika Gakkai Zasshi 2004; 93: [PMID: DOI: /naika ] 39 Clark GW. Effect of Helicobacter pylori infection in Barrett s esophagus and the genesis of esophageal adenocarcinoma. World J Surg 2003; 27: [PMID: DOI: /s ] 40 Thrift AP, Pandeya N, Smith KJ, Green AC, Hayward NK, Webb PM, Whiteman DC. Helicobacter pylori infection and the risks of Barrett s oesophagus: a population-based case-control study. Int J Cancer 2012; 130: [PMID: DOI: /ijc.26242] 41 Neshat K, Sanchez CA, Galipeau PC, Blount PL, Levine DS, Joslyn G, Reid BJ. p53 mutations in Barrett s adenocarcinoma and high-grade dysplasia. Gastroenterology 1994; 106: [PMID: ] 42 Bian YS, Osterheld MC, Bosman FT, Benhattar J, Fontolliet C. p53 gene mutation and protein accumulation during neoplastic progression in Barrett s esophagus. Mod Pathol 2001; 14: [PMID: DOI: /modpathol ] 43 Novotna K, Trkova M, Pazdro A, Smejkal M, Soukupova A, Kodetova D, Smejkal P, Sedlacek Z. TP53 gene mutations are rare in nondysplastic Barrett s esophagus. Dig Dis Sci 2006; 51: [PMID: DOI: /s ] 44 Ramel S, Reid BJ, Sanchez CA, Blount PL, Levine DS, Neshat K, Haggitt RC, Dean PJ, Thor K, Rabinovitch PS. Evaluation of p53 protein expression in Barrett s esophagus by two-parameter flow cytometry. Gastroenterology 1992; 102: [PMID: ] 45 Galipeau PC, Cowan DS, Sanchez CA, Barrett MT, Emond MJ, Levine DS, Rabinovitch PS, Reid BJ. 17p (p53) allelic losses, 4N (G2/tetraploid) populations, and progression to aneuploidy in Barrett s esophagus. Proc Natl Acad Sci USA 1996; 93: [PMID: DOI: /pnas ] 46 Giaretti W. Aneuploidy mechanisms in human colorectal preneoplastic lesions and Barrett s esophagus. Is there a role for K-ras and p53 mutations? Anal Cell Pathol 1997; 15: [PMID: ] 47 Younes M, Lebovitz RM, Lechago LV, Lechago J. p53 protein accumulation in Barrett s metaplasia, dysplasia, and carcinoma: a follow-up study. Gastroenterology 1993; 105: [PMID: ] 48 Kimura K, Takemoto T. An endoscopic recognition of the atrophic border and its significance in chronic gastritis. Endoscopy 1969; 3: [DOI: /s ] 49 Yachida S, Saito D, Kozu T, Gotoda T, Inui T, Fujishiro M, Oda I, Okabayashi T, Kakugawa Y, Ono H, Kondo H. Endoscopically demonstrable esophageal changes after Helicobacter pylori eradication in patients with gastric disease. J Gastroenterol Hepatol 2001; 16: [PMID: DOI: /j x] 50 Nelsen EM, Kirihara Y, Takahashi N, Shi Q, Lewis JT, Namasivayam V, Buttar NS, Dunagan KT, Prasad GA. Distribution of body fat and its influence on esophageal inflammation and dysplasia in patients with Barrett s esophagus. Clin Gastroenterol Hepatol 2012; 10: ; quiz e61-62 [PMID: ] P- Reviewers: Goll R, Naito Y, Seow-Choen F S- Editor: Ma YJ L- Editor: Kerr C E- Editor: Liu XM 4361 April 21, 2014 Volume 20 Issue 15

263 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH REPORT Comparative study of esomeprazole and lansoprazole in triple therapy for eradication of Helicobacter pylori in Japan Tsutomu Nishida, Masahiko Tsujii, Hirohisa Tanimura, Shusaku Tsutsui, Shingo Tsuji, Akira Takeda, Atsuo Inoue, Hiroyuki Fukui, Toshiyuki Yoshio, Osamu Kishida, Hiroyuki Ogawa, Masahide Oshita, Ichizo Kobayashi, Shinichiro Zushi, Makoto Ichiba, Naoto Uenoyama, Yuichi Yasunaga, Ryu Ishihara, Mamoru Yura, Masato Komori, Satoshi Egawa, Hideki Iijima, Tetsuo Takehara Tsutomu Nishida, Masahiko Tsujii, Hideki Iijima, Tetsuo Takehara, Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, Suita, Osaka , Japan Hirohisa Tanimura, Department of Gastroenterology, Osaka Kaisei Hospital, Osaka , Japan Shusaku Tsutsui, Department of Gastroenterology, Itami City Hospital, Itami , Japan Shingo Tsuji, Department of Gastroenterology, Osaka Seamen s Insurance Hospital, Osaka , Japan Akira Takeda, Department of Gastroenterology, Ashiya Municipal Hospital, Ashiya , Japan Atsuo Inoue, Department of Gastroenterology, Osaka General Medical Center, Osaka , Japan Hiroyuki Fukui, Department of Gastroenterology, Yao Municipal Hospital, Yao , Japan Toshiyuki Yoshio, Department of Gastroenterology, Osaka National Hospital, Osaka , Japan Osamu Kishida, Department of Gastroenterology, Sumitomo Hospital, Osaka , Japan Hiroyuki Ogawa, Department of Gastroenterology, Nishinomiya Municipal Central Hospital, Nishinomiya , Japan Masahide Oshita, Department of Internal Medicine, Osaka Police Hospital, Osaka , Japan Ichizo Kobayashi, Department of Gastroenterology, Higashiosaka City General Hospital, Higashiosaka , Japan Shinichiro Zushi, Department of Gastroenterology, Ikeda Municipal Hospital, Ikeda , Japan Makoto Ichiba, Department of Gastroenterology, Toyonaka Municipal Hospital, Toyonaka , Japan Naoto Uenoyama, Department of Gastroenterology, Otemae Hospital, Osaka , Japan Yuichi Yasunaga, Department of Gastroenterology, Hyogo Prefectural Nishinomiya Hospital, Nishinomiya , Japan Ryu Ishihara, Department of Gastrointestinal Oncology, Osaka Medical Center for Cancer and Cardiovascular Disease, Osaka , Japan Mamoru Yura, Department of Gastroenterology, Minoh City Hospital, Minoh , Japan Masato Komori, Department of Gastroenterology, Osaka Rosai Hospital, Sakai , Japan Satoshi Egawa, Department of Gastroenterology, Kansai Rosai Hospital, Amagasaki , Japan Author contributions: Nishida T wrote the manuscript; Nishida T, Tsujii M and Tsuji S concept and designed this study; Nishida T, Tsujii M, Tanimura H, Tsutsui S, Tsuji S, Takeda A, Inoue A, Fukui H, Yoshio T, Kishida O, Ogawa H, Oshita M, Kobayashi I, Zushi S, Ichiba M, Uenoyama N, Yasunaga Y, Ishihara R, Yura M, Komori M, Egawa S and Iijima H collected the data; Takehara T provided scientific editing and assisted with writing the manuscript. Correspondence to: Tetsuo Takehara, MD, PhD, Department of Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka , Japan. takehara@gh.med.osaka-u.ac.jp Telephone: Fax: Received: November 19, 2013 Revised: December 31, 2013 Accepted: January 20, 2014 Published online: April 21, 2014 Abstract AIM: To evaluate the efficacy and safety of esomeprazole-based triple therapy compared with lansoprazole therapy as first-line eradication therapy for patients with Helicobacter pylori (H. pylori ) in usual post-marketing use in Japan, where the clarithromycin (CAM) resistance rate is 30%. METHODS: For this multicenter, randomized, openlabel, non-inferiority trial, we recruited patients ( 20 years of age) with H. pylori infection from 20 hospitals in Japan. We randomly allocated patients to esomeprazole therapy (esomeprazole 20 mg, CAM 400 mg, amoxicillin (AC) 750 mg for the first 7 d, with all drugs given twice daily) or lansoprazole therapy (lansoprazole 30 mg, CAM 400 mg, AC 750 mg for the first 7 d, with all drugs given twice daily) using a minimization meth April 21, 2014 Volume 20 Issue 15

264 Nishida T et al. Comparison of Nexium and Takepron for H. pylori od with age, sex, and institution as adjustment factors. Our primary outcome was the eradication rate by intention-to-treat (ITT) and per-protocol (PP) analyses. H. pylori eradication was confirmed by a urea breath test from 4 to 8 wk after cessation of therapy. RESULTS: ITT analysis revealed the eradication rates of 69.4% (95%CI: 61.2%-76.6%) for esomeprazole therapy and 73.9% (95%CI: 65.9%-80.6%) for lansoprazole therapy (P = ). PP analysis showed eradication rate of 76.9% (95%CI: 68.6%-83.5%) for esomeprazole therapy and 79.8% (95%CI: 71.9%-86.0%) for lansoprazole therapy (P = ). There were no differences in adverse effects between the two therapies. CONCLUSION: Esomeprazole showed non-inferiority and safety in a 7 day-triple therapy for eradication of H. pylori compared with lansoprazole Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Helicobacter pylori ; Eradication; Esomeprazole; Lansoprazole; Proton pomp inhibitor Core tip: Amoxicillin is a standard regime for Helicobacter pylori (H. pylori ) eradication in Japan. Esomeprazole is a second-generation PPI that became available in 2011 in Japan. Several studies have reported eradication data comparing esomeprazole to first-generation proton pump inhibitors (PPIs), but it is not known whether the H. pylori eradication rates of esomeprazole are equal to those of lansoprazole, a first-generation PPI, under circumstances of increased resistance to clarithromycin in Japan. Nishida T, Tsujii M, Tanimura H, Tsutsui S, Tsuji S, Takeda A, Inoue A, Fukui H, Yoshio T, Kishida O, Ogawa H, Oshita M, Kobayashi I, Zushi S, Ichiba M, Uenoyama N, Yasunaga Y, Ishihara R, Yura M, Komori M, Egawa S, Iijima H, Takehara T. Comparative study of esomeprazole and lansoprazole in triple therapy for eradication of Helicobacter pylori in Japan. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: INTRODUCTION Proton pump inhibitors (PPIs) are widely used for the treatment of acid-peptic diseases. Their mechanism of action involves inhibition of the H-K-adenosine triphosphatase enzyme in the parietal cells of the gastric mucosa. Helicobacter pylori (H. pylori) eradication therapy is effective for accelerating healing and inhibiting the recurrence of gastric or duodenal ulcers, and is strongly recommended in various guidelines as a first-line therapy for H. pyloripositive gastric or duodenal ulcers [1,2]. Treatment with PPIs in combination with antibiotics has increased the eradication rate of H. pylori [3,4]. Acid inhibition increases eradication efficacy because antibiotics are more stable in a less acidic gastric environment and induces a higher antibiotic sensitivity in the bacteria [5]. PPI-containing triple therapy with clarithromycin (CAM) and amoxicillin (AC) is a standard regime for H. pylori eradication in Japan. This triple therapy in patients with gastric or duodenal ulcers has been covered under Japan s national health insurance scheme since 2000, and in 2013 its indication was expanded for H. pylori eradication, including H. pylori-positive atrophic gastritis. In 2007, a second-line eradication therapy, substituting metronidazole (MTZ) for CAM was also approved. The recent subsequent increase in bacterial resistance to CAM in Japan, however, led to a decline in the eradication rate by first-line therapy [6]. The Japanese Society for Helicobacter Research conducted a CAM sensitivity study to examine the prevalence of bacterial resistance from 2002 to 2006 in Japan. Surveillance revealed that the CAM resistance rate was 18.9% from 2002 to 2003, and then gradually increased to 21.1% from 2003 to 2004, to 27.7% from 2004 to 2005 [7]. Due to the increased resistance to CAM, it is recommended that drug sensitivity tests be performed before initiating eradication therapy. Sensitivity tests are not common, however, because other antibiotics are not approved for first-line eradication therapy. Esomeprazole, the S-isomer of omeprazole, is a second-generation PPI that became available in 2011 in Japan. Esomeprazole is considered to induce greater acid secretion suppression than first-generation PPIs because of pharmacologic advantages over the racemic compound [8], and is less likely to be affected by CYP2C19 polymorphisms [9]. Several studies have reported eradication data comparing esomeprazole to first-generation PPIs [10], but it is not known whether the H. pylori eradication rates of esomeprazole are equal to those of lansoprazole, a first-generation PPI, under circumstances of increased resistance to CAM. Therefore, in this study, we compared 7 d triple therapy with esomeprazole or lansoprazole containing AC and CAM. MATERIALS AND METHODS Patients and study design This was a prospective, randomized, controlled study. At baseline, patients were evaluated for inclusion and exclusion criteria and written informed consent was obtained. Patients aged at least 20 years, diagnosed with gastric ulcers, duodenal ulcers, or gastric mucosa associated lymphoid tissue lymphoma, idiopathic thrombocytopenic purpura, or early gastric cancer with H. pylori infections who met the inclusion criteria and who wished to receive eradication therapy for H. pylori were enrolled into the study. Those diseases are covered for H. pylori eradication by Japanese health insurance. In patients with gastric ulcers, H. pylori eradication therapy was administered after the active gastric ulcer healed because 1 wk of H. pylori eradication therapy is insufficient to heal gastric ulcers [11] April 21, 2014 Volume 20 Issue 15

265 Nishida T et al. Comparison of Nexium and Takepron for H. pylori Table 1 Participating hospitals attending the osaka gut forum Hospital Patients (esomeprazole/ lansoprazole) 1 Osaka Kaisei Hospital 52 (28/24) Itami City Hospital 39 (20/19) Osaka University Hospital 26 (12/14) Osaka Seamen s Insurance Hospital 24 (11/13) Ashiya Municipal Hospital 17 (6/11) Osaka General Medical Center 17 (7/10) Yao Municipal Hospital 16 (9/7) Osaka National Hospital 13 (7/6) Sumitomo Hospital 11 (6/5) Nishinomiya Municipal Central Hospital 9 (4/5) Osaka Police Hospital 7 (3/4) Higashiosaka City General Hospital 6 (2/4) Ikeda Municipal Hospital 6 (3/3) Toyonaka Municipal Hospital 6 (3/3) Otemae Hospital 5 (3/2) Hyogo Prefectural Nishinomiya Hospital 4 (2/2) Osaka Medical Center for Cancer and 4 (1/3) 1.5 Cardiovascular Disease 18 Minoh City Hospital 3 (1/2) Osaka Rosai Hospital 2 (1/1) Kansai Rosai Hospital 1 (0/1) 0.4 Total 268 (134/134) 100 In patients with active duodenal ulcers, however, 1 wk of H. pylori eradication therapy is considered sufficient [12]. At entry, a patient was diagnosed as H. pylori-positive if at least one of the following tests was positive histochemically-detectable H. pylori (hematoxylin and eosin staining), rapid urease test, urea breath test, or stool antigen test. Exclusion criteria were as follows: past history of drug allergy to PPIs, AC or CAM; previous therapy for H. pylori; clinically significant renal or hepatic disease, or any other clinically significant medical condition that could increase risk; pregnancy; alcohol abuse; drug addiction; and previous partial or total gastrectomy. Patients were enrolled by a gastroenterologist at each participating institute after assessment of the appropriate indications and ruling out any contraindications to the therapies. Patients were then randomly allocated to the esomeprazole group (esomeprazole 20 mg, CAM 400 mg, AC 750 mg for the first 7 d; with all drugs given twice daily) or lansoprazole therapy (lansoprazole 30 mg, CAM 400 mg, AC 750 mg for the first 7 d; with all drugs given twice daily) by the minimization method with age, sex, and institution as adjustment factors, using an allocation system, Waritsuke-kun (Mebix, Inc., Tokyo, Japan). The dose of CAM could be reduced to 200 mg twice daily at the attending doctor s discretion according to patients age or physical size. Adverse events (AE) were evaluated using the Common Terminology Criteria version 4.0. Compliance was measured based on patient s medication diary at each visit. Compliance was calculated using the following formula: compliance = actual number of internal use/14 (total number of internal use/7 d) 100 (%). Determination of eradication was made from 4 to 8 wk after the completion of eradication therapy. Determination of H. pylori eradication was made using a urea % breath test but measurement of H. pylori antigen in the feces could be used instead of the urea breath test according to institution availability. The eradication rate was defined as the number of successfully treated patients divided by the number of all treated patients. The study was performed according to the Declaration of Helsinki and was approved by each institution s ethics committee. This trial is registered with UMIN Clinical trials: number UMIN Signed informed consent was obtained from each patient before study enrollment. Statistical analysis The trial was designed as a non-inferiority trial to compare a 7 d triple therapy with esomeprazole vs lansoprazole, AC, and CAM for H. pylori infection in patients naïve to therapy. The eradication rate was evaluated by intention-to-treat (ITT) and per protocol (PP). In the ITT analysis, all enrolled patients that were lost during follow-up or did not get the breath test or stool antigen test to evaluate eradication or withdrew due to AE were classified as failed to eradicate. Our primary outcome was the eradication rate by ITT and PP analyses of the two therapies. The secondary outcomes were drug adherence and adverse events. In the PP analysis, patients who were lost during follow-up or did not follow the protocol were excluded from the analyses. We calculated the sample size based on a non-inferiority margin of 10%, a successful eradication rate of at least 70%, a two-sided test at the 5% level, and a power of 80%. Based on this, a sample size of 119 patients per therapy group was calculated to be sufficient. We decided to increase the number to 130 patients per therapy group, however, to compensate for a potential 10% loss at follow-up. The significance level was set at P < The 95%CI were constructed by normal approximation. Univariate logistic regressions were performed to predict successful eradication. Statistical analysis was performed using JMP software (ver d1, SAS Institute Inc., Cary, NC). RESULTS Patients In total, 268 patients evaluated at 20 hospitals attending the Osaka Gut Forum were enrolled from May 2012 to February 2013 (Table 1). We randomly assigned the patients to receive esomeprazole therapy (n = 134) or lansoprazole therapy (n = 134) (Figure 1). Patients were diagnosed with gastric ulcers (n = 163), duodenal ulcers (n = 59), both gastric and duodenal ulcers (n = 7), gastric mucosa associated lymphoid tissue lymphoma (n = 2), idiopathic thrombocytopenic purpura (n = 1), or early gastric cancer after endoscopic therapy (n = 36) with H. pylori infection at entry. The demographic and clinical characteristics of the patients in the two groups were comparable (Table 2). Mean age ± SD was 61 ± 14 years. There were 186 men (69%). There were no significant differences found in age, sex, body mass index, smoking habit, alcohol use, underlying diseases, or CAM dose be April 21, 2014 Volume 20 Issue 15

266 Nishida T et al. Comparison of Nexium and Takepron for H. pylori Enrolled patients n = 268 Full intention-to-treat population Figure 1 Study flow chart. H. pylori: Helicobacter pylori. Esomeprazole R Lansoprazole n = 134 n = 134 Excluded n = 1 Withdraw consent n = 1 Ceased due to adverse events n = 2 Lost to follow-up n = 5 7-d triple therapy with esomeprazole vs lansoprazole, amoxicillin and clarithromycin for H. pylori eradication Excluded n = 3 Withdraw consent n = 1 Ceased due to adverse events n = 1 Lost to follow-up n = 3 Protocol violation n = 4 Protocol violation n = 2 n = 121 Per protocol population n = 124 Table 2 Demographic and baseline characteristics of the study population Characteristics Age (yr) (mean ± SD) range Total patients n = ± Esomeprazole therapy n = ± Lansoprazole therapy n = ± Sex, male, % 186, 69% 92, 69% 94, 70% BMI (mean ± SD) 22.9 ± ± ± 3.5 Alcohol 1, n, (%) 119, 44% 54, 42% 65, 49% Smoking (%) 75, 28% 37, 28% 38, 28% Underlying diseases Hypertension, n, % 69, 26% 35, 26% 34, 25% Diabetes mellitus, n, % 35, 13% 18, 13% 17, 13% Lipid disorder, n, % 31, 12% 12, 9% 19, 14% Chronic lung disease, n, % 7, 3% 4, 3% 3, 2% Disease for H. pylori eradication Gastric ulcer, n, % 163, 61% 82, 61% 81, 61% Duodenal ulcer, n, % 59, 22% 29, 22% 30, 22% Gastroduodenal ulcer, n, % 7, 3% 5, 4% 2, 1.5% Gastric MALT lymphoma, n, % 2, 0.7% 0, 0% 2, 1.5% ITP, n, % 1, 0.4% 1, 0.8% 0, 0% EGC after endoscopic therapy, n, % 36, 13% 17, 13% 19, 14% Therapy and test for H. pylori eradication Clarithromycin 28/233 12/118 16/ mg/800 mg per day Urea breath test/stool antigen 230/17/4 113/8/3 117/9/1 test/others Compliance 2 Good, n, % 226 (84%) 110 (82%) 116 (87%) 1 Alcohol habit was defined as drinking more than twice a week, consumption of more than 15 g of ethanol on each occasion in this study, 100 (%) If compliance was more than 75%, it graded as good; 2 Compliance = actual number of internal use/14 (total number of internal use/7 d). BMI: Body mass index; MALT: Mucosa associated lymphoid tissue; ITP: Idiopathic thrombocytopenic purpura; EGC: Early gastric cancer; H. pylori: Helicobacter pylori. tween the two groups. For the initial determination of H. pylori infection status, a rapid urease test was performed in 113 patients (42%), blood antibody test in 110 patients (41%), stool antigen test in 7 patients (2.6%), histopathology of biopsy specimens in 25 (9.3%) patients, and urea breath test in 13 patients (4.9%). Figure 1 shows the flow chart for this study. After randomization, 4 patients were excluded 2 patients in the lansoprazole group had a history of gastrectomy, 1 patient in the lansoprazole group had comorbid gastric cancer, and 1 patient in the esomeprazole group had a history of penicillin allergy. One patient in each group withdrew their informed consent before therapy. Two patients in the esomeprazole and 1 patient in the lansoprazole group withdrew due to adverse effects (Grade 3 vomiting and Grade 1 diarrhea in the esomeprazole group and Grade 1 vomiting in the lansoprazole group). Three patients in the lansoprazole group and 5 patients in the esomeprazole group were lost to followup. Two patients in the lansoprazole group and four in the esomeprazole group were excluded due to a protocol violation about judging method for H. pylori eradication or internal method (Figure 1). Eradication of H. pylori infection and adverse events The efficacy of the two eradication therapies is shown in Figure 2. ITT analysis showed eradication rates 69.4% (95%CI: 61.2%-76.6%) for esomeprazole therapy and 73.9% (95%CI: 65.9%-80.6%) for lansoprazole therapy. The PP eradication rate was 76.9% (95%CI: 68.6%-83.5%) for esomeprazole therapy and 79.8% (95%CI: 71.9%-86.0%) for lansoprazole therapy. Comparisons of the two therapies, both in the ITT and PP analyses, showed non-inferiority (ITT, P = ; PP, P = ). Univariate logistic regression analysis showed that there were no predicting factors (sex, age, habit of smoking or alcohol, body mass index, dose of CAM, and underling diseases) for successful eradication (Table 3). Figure 3 shows eradication rates in the PP analysis stratified by age. The incidence of AE was comparable between the 4365 April 21, 2014 Volume 20 Issue 15

267 Nishida T et al. Comparison of Nexium and Takepron for H. pylori Eradication rates of H. pylori (%) two therapies (Table 4). The incidence of AE incidence of all grades was 56% (75/134) in the esomeprazole group and 53% (71/134) in the lansoprazole group. Most AEs were Grade 1 (85% in both groups). In both groups, diarrhea was the AE with highest incidence (27% in the esomeprazole group and 31% in the lansoprazole group). AEs were severe enough to discontinue therapy in 2 patients (1.4%) in the esomeprazole group and 1 (0.7%) in the lansoprazole group. One patient stopped therapy due to Grade 1 diarrhea. One patient in the lansoprazole group had Grade 3 diarrhea, but discontinued to take the medicine every day or every other day based on his own judgment. He was excluded from the PP analysis due to a protocol violation. Compliance of all patients except for 3 patients (50%, 79%, 86%) was 100% in the PP population and excellent based on all the patients medication diaries. DISCUSSION 69.4 Esomezorazole Lansoprazole Figure 2 Eradication rates of esomeprazole and lansoprazole and noninferiority comparisons. PP: Per protocol; ITT: Intention to treat; H. pylori: Helicobacter pylori. Age (yr) Eradication rate in the PP analysis (%) No of H. pylori eradicated pts Total number The present study compared esomeprazole and lansoprazole therapy in H. pylori eradication. Generally, PPIs are used for H. pylori eradication. Two mechanisms are assumed to underlie the increased eradication rate: enhancement of antibiotic efficacy induced by acid control [10], and direct antibacterial effect of PPIs. PP ITT < Figure 3 Eradication rate in the per protocol analysis stratified by age. H. pylori: Helicobacter pylori; PP: Per-protocol. Omeprazole and lansoprazole (first-generation PPIs) have anti-acid properties and demonstrated efficacy for H. pylori eradication. The second-generation PPIs (rabeprazole and esomeprazole) have demonstrated even higher acid inhibition [13,14]. Kirchheiner et al [13] reported that the relative potencies of the PPIs compared to omeprazole based on mean 24 h gastric ph were 0.9 and 1.6 for lansoprazole and esomeprazole, respectively. Even though considering the difference of dose of each PPI in this study, second-generation PPIs, esomeprazole are considered to more effectively suppress acid secretion than first-generation PPIs, lansoprazole. One of the reasons is considered that esomeprazole is less likely to be affected than omeprazole by CYP2C19 polymorphisms [9]. More acid inhibition is considered to increase eradication efficacy because antibiotics are more stable in a less acidic gastric environment and induce higher antibiotic sensitivity in the bacteria [5]. Moreover, PPIs themselves may have direct antibacterial properties. Benzimidazole PPIs inhibit the growth of H. pylori [15]. Omeprazole competitively inhibits the extracellular urease enzyme of H. pylori in a dose-dependent manner [16]. Miwa et al [17] reported that rabeprazole is equivalent to omeprazole and lansoprazole in the PPI/AC triple therapy in Japan Differential inhibitory effects of esomeprazole and lansoprazole on urease, however, have not been demonstrated. Esomeprazole, the S-isomer of omeprazole (a racemic mixture of S- and R-optical isomers), is the first PPI to be developed as a single optical isomer. Esomeprazole has been available in Japan since Esomeprazole is effective for H. pylori eradication. Our findings indicated non-inferiority and safety in 7 day-triple therapy based on esomeprazole compared with lansoprazole. A number of studies have compared esomeprazole with other firstgeneration PPIs. Wang et al [18] reported that the mean H. pylori eradication rate (ITT) with esomeprazole plus antibiotics was 86%, a rate comparable to that of other PPI therapies, 81% (ITT); the odds ratio (OR) was 1.38 (95%CI: ) in 11 randomized controlled trials including 2159 subjects in a meta-analysis. The OR by a focused meta-analysis of six selected high-quality studies was 1.17 (95%CI: ) and sub-analysis that included only studies comparing different doses of esomeprazole with omeprazole or pantoprazole revealed no significant differences [18]. Another recently meta-analysis (35 studies, 5998 patients) showed higher eradication rates for esomeprazole than for first-generation PPIs [82.3% vs 77.6%; OR = 1.32 (95%CI: )] [10]. After excluding some studies due to methodological differences, the OR was increased to 1.52 (95%CI: ). In subanalyses based on esomeprazole dose, esomeprazole 40 mg twice daily had an OR of 2.27 (95%CI: ), while esomeprazole 20 mg twice daily had an OR of 1.04 (95%CI: ). Thus, it seemed that the newgeneration PPIs esomeprazole or rabeprazole are more effective than first-generation PPIs, omeprazole or lansoprazole, for H. pylori eradication when using a higher dose of esomeprazole. The most common causes for failed eradication are 4366 April 21, 2014 Volume 20 Issue 15

268 Nishida T et al. Comparison of Nexium and Takepron for H. pylori Table 3 Univariate analysis of predictors for successful H.pylori eradication in per-protocol analysis OR 95%CI P value Sex Male 1 Female Age (yr) < Smoking No 1 Yes Drinking No 1 Yes BMI < Clarithromycin 400 mg/d mg/d Diabetes Mellitus No 1 Yes Hypertension No 1 Yes Lipid disorder No 1 Yes Chronic lung disease No 1 Yes BMI: Body mass index. the presence of H. pylori resistance against antibiotics or compliance of therapy, or both. Recently, a subsequent increase in bacterial resistance to CAM was reported in Japan, leading to a decline in the eradication rate of firstline therapy [6]. Generally, the best approach for H. pylori eradication is the use of regimens that have proven to be reliably excellent locally [19]. Unfortunately, sensitivity tests are not common in Japan because antibiotics other than the PPI/AC/CAM triple therapy have not been approved for first-line eradication. The eradication rate of PPI/ AC/MTZ triple therapy, which was approved as a second- line therapy has remained consistently high, about 90% [20], and eradication is successful in most patients who are positive for H. pylori infection. The history of the patient s prior antibiotic use and any prior therapies will also help to identify which antibiotics are likely to be successful and those for which resistance is probable. In this study, we excluded patients with prior H. pylori eradication therapy. To evaluate the patient s antibiotic use history, we examined patients with chronic lung disease because those patients often used CAM. In the present study, however, patients background was not significantly different between groups. All but 3 patients were 100% compliant in per protocol population and compliance was excellent. Therefore, drug compliance might not have influenced the eradication rate in this study. On the other hand, actual compliance with therapy Table 4 Adverse effects in each therapy Adverse events Esomeprazole therapy Lansoprazole therapy to eradicate H. pylori can be problematic because patients often need to take as many as three different medications. Sasaki et al [21] suggested that packs of eradication medicine are useful for increasing eradication success. A drug pack, Lansap (Takeda Chemical Industries, Ltd, Osaka, Japan) to simplify dosing has been available since 2002 in Japan. Lansap provides daily dose cards for a 7 d therapy cycle. Each dose card contains three different prescription drugs for 1-d therapy. Nagahara et al [22] reported PP analysis using data from 57 patients in the Lansap group and 67 patients in a separate tablets group whose compliance was equal to or greater than 80%. The cure rates for the groups receiving Lansap and the separate tablets were 86.0% (95%CI: 74%-94%) and 76.1% (95%CI: 64%-86%), respectively, in the PP analysis. The eradication rates did not differ significantly between these two groups, although the eradication rate was about 10% higher with the Lansap. Kawai et al [23] also reported that a medication package might be useful to prevent mistakes regarding medicine dosage. In the present study, patients in the lansoprazole group could use Lansap at the attending doctor s discretion, whereas patients in the esomeprazole group were used separate tablets, suggesting that the packaging did not have a significant influence on the eradication efficiency. Drug compliance was suggested to be positively related to age over 60 years [24]. In the present study, however, eradication rate stratified by age was not significantly different among ages (Figure 3). In conclusion, esomeprazole was not inferior and safe in 7 d triple therapy for eradication of H. pylori compared with lansoprazole. ACKNOWLEDGMENTS G1 G2 G3 Total, % G1 G2 G3 Total, % Diarrhea , 27% , 31% Bitter taste , 12% , 12% Nausea , 0.7% , 0.7% Vomiting , 2.2% , 0% Eruption , 2.2% , 2.2% Headache , 0% , 1.5% Fatigue , 0.7% , 0% Appetite loss , 2.2% , 0.7% Stomatitis , 0% , 0.7% Cheilitis , 0% , 1.5% Thirst , 1.5% , 0% Belch , 0.7% , 0% Bad breath , 0.7% , 0% Sore throat , 0.7% , 0% Joint paint , 0.7% , 0% Leg edema , 0% , 0.7% Chest discomfort , 1.5% , 0% Floating , 0.7% , 0% Abdominal wind , 0.7% , 0% Constipation , 1.5% , 1.5% Pruritus ani , 0% , 0.7% Total , 56% , 53% Study investigators: Hideharu Ogiyama, Eriko Nakamura, 4367 April 21, 2014 Volume 20 Issue 15

269 Nishida T et al. Comparison of Nexium and Takepron for H. pylori Yukako Taniguchi, Yoshiko Sugimoto, Koichiro Watabe (Osaka Kaisei Hospital); Yoshito Hayashi, Tomofumi Akasaka, Motohiko Kato, Shinichiro Shinzaki (Osaka University Hospital); Yoko Murayama (Itami City Hospital); Tadaharu Takemura (Ashiya Municipal Hospital); Kosaku Ohnishi, Yasutoshi Nozaki, Osamu Nishiyama (Osaka General Medical Center); Naoki Kawai, Akiyoshi Okada (Osaka Police Hospital); Tadashi Kegasawa, Fumitaka Terabe (Yao Municipal Hospital); Tetsuya Iwasaki, Yuko Sakakibara, Takuya Yamada (Osaka National Hospital); Kazuo Kinoshita (Sumitomo Hospital); Hirofumi Kashihara (Nishinomiya Municipal Central Hospital); Katsumi Yamamoto, Shiro Hayashi, Mitsuhiko Shibuya (Toyonaka Municipal Hospital); Akihiro Nishihara(Minoh City Hospital); Daisuke Utsunomiya, Rui Mizumoto, Yasushi Matsumoto (Ikeda Municipal Hospital); Masako Sato (Osaka Rosai Hospital). COMMENTS Background Esomeprazole is a second-generation proton pump inhibitors (PPIs) that became available in 2011 in Japan. Esomeprazole is considered to induce greater acid secretion suppression than first-generation PPIs because of pharmacologic advantages over the racemic compound. Several studies have reported eradication data comparing esomeprazole to first-generation PPIs, but it is not known whether the Helicobacter pylori (H. pylori) eradication rates of esomeprazole are equal to those of lansoprazole, a first-generation PPI, under circumstances of increased resistance to clarithromycin. Research frontiers Esomeprazole, the S-isomer of omeprazole, is the first PPI to be developed as a single optical isomer. Esomeprazole is considered to more effectively suppress acid secretion than first-generation PPIs. More acid inhibition is considered to increase eradication efficacy because antibiotics are more stable in a less acidic gastric environment and induce higher antibiotic sensitivity in the bacteria. Innovations and breakthroughs Recently meta-analysis showed higher eradication rates for esomeprazole than for first-generation PPIs. In sub-analyses based on esomeprazole dose, esomeprazole 40 mg twice daily had higher eradication rates, while esomeprazole 20 mg twice daily did not show the significant eradication rates. Applications The study results showed that esomeprazole was not inferior and safe in 7-d triple therapy for eradication of H. pylori compared with lansoprazole under circumstances of increased resistance to clarithromycin in Japan. Terminology Determination of eradication was made from 4 to 8 wk after the completion of eradication therapy. Determination of H. pylori eradication was made using a urea breath test but measurement of H. pylori antigen in the feces could be used instead of the urea breath test according to institution availability. The eradication rate was defined as the number of successfully treated patients divided by the number of all treated patients. Peer review Generally clarithromycin-containing eradication therapy should not be considered as first-line therapy for H. pylori eradication if clarithromycin resistance rate is over 15%-20%. Unfortunately, Japanese health insurance dose not allowed other regimens but clarithromycin containing triple therapy. Therefore the regimens containing clarithromycin in the present study are standard regimens as first-line therapy for H. pylori eradication in Japan. REFERENCES 1 Asaka M, Kato M, Takahashi S, Fukuda Y, Sugiyama T, Ota H, Uemura N, Murakami K, Satoh K, Sugano K. Guidelines for the management of Helicobacter pylori infection in Japan: 2009 revised edition. Helicobacter 2010; 15: 1-20 [PMID: DOI: /j x] 2 Malfertheiner P, Megraud F, O Morain CA, Atherton J, Axon AT, Bazzoli F, Gensini GF, Gisbert JP, Graham DY, Rokkas T, El-Omar EM, Kuipers EJ. Management of Helicobacter pylori infection--the Maastricht IV/ Florence Consensus Report. Gut 2012; 61: [PMID: DOI: /gutjnl ] 3 Gomollón F, Calvet X. Optimising acid inhibition treatment. Drugs 2005; 65 Suppl 1: [PMID: ] 4 Boparai V, Rajagopalan J, Triadafilopoulos G. Guide to the use of proton pump inhibitors in adult patients. Drugs 2008; 68: [PMID: ] 5 Calvet X, Gomollón F. What is potent acid inhibition, and how can it be achieved? Drugs 2005; 65 Suppl 1: [PMID: ] 6 Rimbara E, Noguchi N, Tanabe M, Kawai T, Matsumoto Y, Sasatsu M. Susceptibilities to clarithromycin, amoxycillin and metronidazole of Helicobacter pylori isolates from the antrum and corpus in Tokyo, Japan, Clin Microbiol Infect 2005; 11: [PMID: DOI: / j x] 7 Kobayashi I, Murakami K, Kato M, Kato S, Azuma T, Takahashi S, Uemura N, Katsuyama T, Fukuda Y, Haruma K, Nasu M, Fujioka T. Changing antimicrobial susceptibility epidemiology of Helicobacter pylori strains in Japan between 2002 and J Clin Microbiol 2007; 45: [PMID: DOI: /JCM ] 8 Miner P, Katz PO, Chen Y, Sostek M. Gastric acid control with esomeprazole, lansoprazole, omeprazole, pantoprazole, and rabeprazole: a five-way crossover study. Am J Gastroenterol 2003; 98: [PMID: DOI: / j x] 9 Hassan-Alin M, Andersson T, Niazi M, Röhss K. A pharmacokinetic study comparing single and repeated oral doses of 20 mg and 40 mg omeprazole and its two optical isomers, S-omeprazole (esomeprazole) and R-omeprazole, in healthy subjects. Eur J Clin Pharmacol 2005; 60: [PMID: DOI: /s ] 10 McNicholl AG, Linares PM, Nyssen OP, Calvet X, Gisbert JP. Meta-analysis: esomeprazole or rabeprazole vs. firstgeneration pump inhibitors in the treatment of Helicobacter pylori infection. Aliment Pharmacol Ther 2012; 36: [PMID: DOI: /j x] 11 Terano A, Arakawa T, Sugiyama T, Suzuki H, Joh T, Yoshikawa T, Higuchi K, Haruma K, Murakami K, Kobayashi K. Rebamipide, a gastro-protective and anti-inflammatory drug, promotes gastric ulcer healing following eradication therapy for Helicobacter pylori in a Japanese population: a randomized, double-blind, placebo-controlled trial. J Gastroenterol 2007; 42: [PMID: DOI: / s ] 12 Gisbert JP, Pajares JM. Systematic review and meta-analysis: is 1-week proton pump inhibitor-based triple therapy sufficient to heal peptic ulcer? Aliment Pharmacol Ther 2005; 21: [PMID: DOI: /j x] 13 Kirchheiner J, Glatt S, Fuhr U, Klotz U, Meineke I, Seufferlein T, Brockmöller J. Relative potency of proton-pump inhibitors-comparison of effects on intragastric ph. Eur J Clin Pharmacol 2009; 65: [PMID: DOI: / s ] 14 McKeage K, Blick SK, Croxtall JD, Lyseng-Williamson KA, Keating GM. Esomeprazole: a review of its use in the management of gastric acid-related diseases in adults. Drugs 2008; 68: [PMID: ] 15 Sjøstrøm JE, Kühler T, Larsson H. Basis for the selective antibacterial activity in vitro of proton pump inhibitors against 4368 April 21, 2014 Volume 20 Issue 15

270 Nishida T et al. Comparison of Nexium and Takepron for H. pylori Helicobacter spp. Antimicrob Agents Chemother 1997; 41: [PMID: ] 16 Mirshahi F, Fowler G, Patel A, Shaw G. Omeprazole may exert both a bacteriostatic and a bacteriocidal effect on the growth of Helicobacter pylori (NCTC 11637) in vitro by inhibiting bacterial urease activity. J Clin Pathol 1998; 51: [PMID: ] 17 Miwa H, Ohkura R, Murai T, Sato K, Nagahara A, Hirai S, Watanabe S, Sato N. Impact of rabeprazole, a new proton pump inhibitor, in triple therapy for Helicobacter pylori infection-comparison with omeprazole and lansoprazole. Aliment Pharmacol Ther 1999; 13: [PMID: ] 18 Wang X, Fang JY, Lu R, Sun DF. A meta-analysis: comparison of esomeprazole and other proton pump inhibitors in eradicating Helicobacter pylori. Digestion 2006; 73: [PMID: DOI: / ] 19 Graham DY, Shiotani A. New concepts of resistance in the treatment of Helicobacter pylori infections. Nat Clin Pract Gastroenterol Hepatol 2008; 5: [PMID: DOI: /ncpgasthep1138] 20 Asaoka D, Nagahara A, Matsuhisa T, Takahashi S, Tokunaga K, Kawai T, Kawakami K, Suzuki H, Suzuki M, Nishizawa T, Kurihara N, Ito M, Sasaki H, Omata F, Mizuno S, Torii A, Ohkusa T, Mine T, Sakaki N. Trends of second-line eradication therapy for Helicobacter pylori in Japan: a multicenter study in the Tokyo metropolitan area. Helicobacter 2013; 18: [PMID: DOI: /hel.12063] 21 Sasaki M, Ogasawara N, Utsumi K, Kamiya T, Kataoka H, Tanida S, Mizoshita T, Shimura T, Hirata Y, Kasugai K, Joh T. The effectiveness of packed therapy with three drugs in Helicobacter pylori eradication in Japan. Methods Find Exp Clin Pharmacol 2010; 32: [PMID: DOI: / mf ] 22 Nagahara A, Miwa H, Hojo M, Yoshizawa T, Kawabe M, Osada T, Kurosawa A, Ohkusa T, Watanabe S. Efficacy of Lansap combination therapy for eradication of H. pylori. Helicobacter 2007; 12: [PMID: DOI: / j x] 23 Kawai T, Kawakami K, Kataoka M, Takei K, Taira S, Itoi T, Moriyasu F, Takagi Y, Aoki T, Matsubayasiu J, Mukai K, Rimbara E, Noguchi N, Sasatsu M. The Effectiveness of Packaged Medicine in Eradication Therapy of Helicobacter pylori in Japan. J clinl biochem and nutr 2006; 38: Sirey JA, Bruce ML, Alexopoulos GS, Perlick DA, Friedman SJ, Meyers BS. Stigma as a barrier to recovery: Perceived stigma and patient-rated severity of illness as predictors of antidepressant drug adherence. Psychiatr Serv 2001; 52: [PMID: ] P- Reviewers: Imaeda H, Kuo FC, Matsukawa J S- Editor: Qi Y L- Editor: A E- Editor: Wu HL 4369 April 21, 2014 Volume 20 Issue 15

271 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH REPORT Clinical meaning of BRAF mutation in Korean patients with advanced colorectal cancer Bun Kim, Soo Jung Park, Jae Hee Cheon, Tae Il Kim, Won Ho Kim, Sung Pil Hong Bun Kim, Soo Jung Park, Jae Hee Cheon, Tae Il Kim, Won Ho Kim, Sung Pil Hong, Department of Internal Medicine, Institute of Gastroenterology, Yonsei University College of Medicine, Seoul , South Korea Author contributions: Hong SP designed the study; Kim B acquired clinical data and performed the statistical analysis; Park SJ, Cheon JH, Kim TI and Kim WH contributed equally to this study by performing data interpretation and offering important intellectual content; Hong SP and Kim B drafted the manuscript. Correspondence to: Sung Pil Hong, MD, PhD, Department of Internal Medicine, Institute of Gastroenterology, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul , South Korea. sphong@yuhs.ac Telephone: Fax: Received: September 2, 2013 Revised: November 4, 2013 Accepted: January 8, 2014 Published online: April 21, 2014 Abstract AIM: To evaluate the clinicopathological features of colorectal cancer (CRC) with a v-raf murine sarcoma viral oncogene homolog B1 (BRAF ) mutation and its molecular interaction with microsatellite instability (MSI) and v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS ) in patients with advanced CRCs. METHODS: From October 2009 to December 2011, 141 patients with stage Ⅲ (n = 51) or Ⅳ (n = 90) CRCs who were tested for the BRAF mutation at Severance Hospital were included. Among 141 patients, five were excluded due to follow-up loss. Therefore, 136 patients were included in the study. The clinicopathological data, MSI status, and KRAS /BRAF mutation status were reviewed retrospectively. In addition, to evaluating the value of BRAF mutation status, progressionfree survival and overall survival in all patients were collected and compared between the BRAF wild-type group and BRAF mutation group. RESULTS: Of 136 patients, 80 (58.8%) were male and the mean age was 59 years. BRAF and KRAS mutations were detected in 9.6% and 35.3% of patients, respectively. Only 4.3% of patients had MSIhigh tumors and there were no MSI-high in tumors with a BRAF mutation. BRAF mutations tended to be more frequent in stage Ⅳ than in stage Ⅲ (11.76% vs 5.88%, p = 0.370). Patients with a BRAF mutation had a lower incidence of KRAS mutation than those without (7.69% vs 38.21%, p = 0.033). Overall survival was significantly shorter in the BRAF mutation group than in the BRAF wild-type group both by univariate analysis (p = 0.041) and multivariate analysis (HR = 2.195; 95%CI: ; p = 0.039), while progression-free survival was not different according to BRAF mutation status. CONCLUSION: CRCs with a BRAF mutation have distinct molecular features and resulted in a poor prognosis in Korean patients with advanced CRC Baishideng Publishing Group Co., Limited. All rights reserved. Key words: BRAF ; Colorectal cancer; Molecular features; Chemotherapy response; Prognosis Core tip: This study identified the clinicopathological features of colorectal cancer (CRC) with BRAF mutation and its molecular interaction with microsatellite instability and KRAS targeting only to stage Ⅲ/Ⅳ CRCs. These molecular markers enable the classification of CRCs into meaningful subtypes for prognosis. Our data strongly support the prognostic role of BRAF mutation in Korean patients with advanced CRC. Kim B, Park SJ, Cheon JH, Kim TI, Kim WH, Hong SP. Clinical meaning of BRAF mutation in Korean patients with advanced colorectal cancer. World J Gastroenterol 2014; 20(15): April 21, 2014 Volume 20 Issue 15

272 Kim B et al. BRAF mutation in advanced colorectal cancer Available from: URL: v20/i15/4370.htm DOI: i INTRODUCTION Colorectal cancer (CRC) is the third most common cancer in Western countries. In Korea, CRC is the second most common cancer in males and the third in females, with an estimated new cases and 7645 deaths each year [1]. TNM staging of CRC has become a promising tool in determining treatment and prognosis, but it is evident that CRC has a significant clinical heterogeneity even within the same pathologic stage [2-4]. Recent advances in molecular genetics enable the classification of CRC using molecular markers, including microsatellite instability (MSI) and v-ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) and v-raf murine sarcoma viral oncogene homolog B1 (BRAF) mutations, to predict prognosis and treatment response [5-8]. BRAF is a part of the Ras/Raf/mitogen-activated protein kinase kinase (MAP2K)/mitogen-activated protein kinases (MAPK) signaling pathway. Many of the transcription factors activated by the Ras/Raf/MAP2K/ MAPK pathway are involved in cell proliferation and differentiation and many growth factor genes have binding sites for transcription factors activated by the Ras ERK pathway, located in their promoter regions. Thus, aberrant activation of this pathway may provoke selfsufficiency in proliferative signals and continuous stimulation of cell growth [9]. Mutations of KRAS or BRAF activate this pathway and are an established mechanism that drives colorectal carcinogenesis [10]. The most common mutation of BRAF is the classic GTG-GAG substitution at position 1799 of exon 15, which results in the V600E amino acid change and the subsequent constitutive activation of the EGFR signaling pathway. Generally, 10% to 20% of CRCs have BRAF mutations, and the incidence of BRAF mutation varies according to the status of the MSI. It has been reported that 34% to 70% of CRCs classified as MSI-high have BRAF mutations [11-13]. Compared to KRAS mutations, patients with CRC and BRAF mutations show some different clinical manifestations. It is well known that CRCs with KRAS mutations respond poorly to cetuximab [14]. However, the predictive role of BRAF mutations in response to cetuximab is not yet clear. As for prognostic markers, CRC with BRAF mutations have been shown to have significantly poor prognosis compared to those with BRAF wild-type [15-17], while KRAS mutations have no prognostic role [18]. Although CRCs have rapidly increased in Korea, the incidence of BRAF mutation and its clinical meaning have not yet been explored in Korean patients with CRC. The aim of this study was to evaluate the clinicopathological features of CRC with a BRAF mutation and its molecular interaction with MSI and KRAS mutation status in patients with stage Ⅲ or Ⅳ CRC. MATERIALS AND METHODS From October 2009 to December 2011, 141 patients in stage Ⅲ (n = 51) and Ⅳ (n = 90) CRC underwent molecular testing, including MSI analysis and determination of KRAS and BRAF mutation status in Severance Hospital. Among the 141 patients, five were excluded due to follow-up loss. In total, 136 patients were included in the present study. After an initial staging work-up including a CT scan, patients without metastasis or with resectable liver or lung metastasis received surgery and adjuvant chemotherapy with FOLFOX. Patients with unresectable metastatic disease received palliative chemotherapy with FOLFOX or FOLFIRI [19,20]. The clinicopathological data, including age, sex, family history of CRC, BMI (body mass index), Eastern Cooperative Oncology Group (ECOG) performance status tumor stage, tumor location, tumor grade, initial CEA and chemotherapeutic regimen were reviewed retrospectively. This study was approved by the Institutional Review Boards of Yonsei University College of Medicine. DNA extract, MSI analysis, BRAF and KRAS sequencing Before obtaining tissue samples, written informed consent was obtained from all patients. Tissue samples from the tumor and normal colonic mucosa were obtained from each patient after resection. DNA extracted from each tumor was amplified by a standard polymerase chain reaction using five Bethesda guidelines panel loci (BAT25, BAT26, MFD15, D2S123, and D5S346) [21]. In accordance with the consensus definitions of the National Cancer Institute, tumor samples were classified as displaying high-degree microsatellite instability (MSI-H, instability at 30% or more of the markers tested), lowdegree microsatellite instability (MSI-L, instability at less than 30% of the markers tested), or microsatellite stability (MSS, stability at all of the markers tested). Due to the similar biological properties between MSI-L and MSS, these two molecular phenotypes were grouped together in all analyses. KRAS and BRAF were charged at ISU ABXI (Seoul, South Korea). Genomic DNA was extracted from 10-μm-thick paraffin sections containing a portion of tumor tissue by the QIAamp DNA Mini kit (Qiagen, Hilden, Germany). Fifty nanograms of DNA were amplified in a 20 μl reaction solution containing 10 μl of 2 X concentrated HotStarTaq Master Mix (Qiagen, Hilden, Germany), including polymerase chain reaction (PCR) buffer with 3 mmol/l MgCl2, 400 μmol/l of each dntp, and 0.3 μmol/l of each primer pair (KRAS F: 5 -ttatgtgtgacatgttctaat, R: 5 -agaatggtcctgcaccagtaa/braf, F: 5 -atgcttgctctgataggaaaatga, R: 5 -agcagcatctcagggcca). Amplifications were performed using a 15-min initial denaturation at 95, followed by 35 cycles of 30 s at 94, 30 s at 59, and 30 s at 72, and a 10-min final extension at 72. PCR 4371 April 21, 2014 Volume 20 Issue 15

273 Kim B et al. BRAF mutation in advanced colorectal cancer Table 1 Baseline characteristics of enrolled patients n (%) BRAF wild-type (n = 123) BRAF Mutant (n = 13) P value Sex Male/Female 74 (60.2)/49 (39.8) 6 (46.2)/7 (53.8) Age (mean ± SD, age) ± ± Age (< 60 yr) 60 (48.8) 5 (38.5) BMI (mean ± SD, kg/m 2 ) ± ± Family history of colorectal 25 (20.3) 1 (7.7) cancer ECOG performance status (94.3) 13 (100.0) (5.7) 0 (0.0) Tumor type Colon 97 (78.9) 10 (76.9) Rectum 26 (21.1) 3 (23.1) Tumor location Proximal 35 (28.5) 6 (46.2) Distal 88 (71.5) 7 (53.8) Histology WD and MD 113 (91.9) 10 (76.9) PD and UD 10 (8.1) 3 (23.1) AJCC tumor stage Stage Ⅲ 48 (39.0) 3 (23.1) ⅢA 5 (4.1) 1 (7.7) ⅢB 30 (24.4) 1 (7.7) ⅢC 13 (10.6) 1 (7.7) Stage Ⅳ 75 (61.0) 10 (76.9) ⅣA 27 (22.0) 3 (23.1) ⅣB 48 (39.0) 7 (53.8) MSI MSS and MS-low 110 (89.4) 12 (92.3) MSI-high 5 (4.1) 0 Unchecked 8 (6.5) 1 (7.7) K-ras Wild 76 (61.8) 12 (92.3) Mutant 47 (38.2) 1 (7.7) Initial CEA (mean ± SD, ng/ml) ± ± SD: Standard deviation; BMI: Body mass index; ECOG: Eastern Cooperative Oncology Group; WD: Well differentiated; MD: Moderately differentiated; PD: Poorly differentiated; UD: Undifferentiated; AJCC: American Joint Committee on Cancer; MSI: Microsatellite instability; MSS: Microsatellite stable; CEA: Carcinoembryonic antigen. products separated in 2% gel were purified with a QIAgen gel extraction kit (Qiagen). DNA templates were processed for the DNA sequencing reaction using the ABI-PRISM BigDye Terminator version 3.1 (Applied Biosystems, Foster City, CA, United States) with both forward and reverse sequence-specific primers. Twenty nanograms of purified PCR products were used in a 10 μl sequencing reaction solution containing 1 μl of BigDye Terminator v3.1 and 0.1 μmol/l of the same PCR primer. Sequencing reactions were performed using 25 cycles of 10 s at 96, 5 s at 50, and 4 min at 60. Sequence data were generated with the ABI PRISM 3730 DNA Analyzer (Applied Biosystems) and analyzed by Sequencing Analysis software (Applied Biosystems) to compare variations. Statistical analysis The primary outcome was to compare overall survival (OS) and progression-free survival (PFS) of patients with BRAF mutation to those with BRAF wild-type. The secondary outcome was to evaluate the gene-gene interaction between BRAF mutation and KRAS mutation or MSI. Continuous variables were expressed as mean ± SD. Each patient s baseline characteristics were analyzed by descriptive statistics. OS was calculated from the time of diagnosis until death or the last follow-up visit, and PFS was calculated from the time of diagnosis until disease recurrence or progression. OS and PFS were analyzed using the Kaplan-Meier method, and survival curves were compared using the log-rank method. Cox proportional hazards modeling was used to control multiple risk factors that have been shown to influence CRC survival by computing 95% confidence intervals (CIs). A P-value < 0.05 was considered statistically significant. All statistical analyses were performed using SPSS version 18.0 (SPSS Inc., Chicago, IL, United States). RESULTS Patient characteristics Of 136 patients, 107 (78.7%) patients were diagnosed with colon cancer and 29 (21.3%) with rectal cancer. Fifty-one (37.5%) patients were stage Ⅲ and 85 (62.5%) were stage Ⅳ. Thirteen patients (9.6%) had BRAF mutations, and 48 (35.3%) patients had KRAS mutations. The most frequent mutation at KRAS was G12D, which accounted for 29.2% of KRAS mutations (14/48). The second most frequent mutation was G13D (12/48), and the remainder occurred in the following order: G12V (9/48), G12C (6/48), G12S (4/48), G13C (2/48) and G12A (1/48). Five (4.3%) patients had MSI-high tumors. Molecular characteristics such as MSI status and KRAS and BRAF mutation status were not significantly different according to the tumor location. The clinicopathological characteristics of patients according to BRAF mutation are summarized in Table 1. Tumors with BRAF wild-type tended to exhibit more differentiated histology (91.9% vs 76.9%, P = 0.211) and an earlier stage (stage Ⅲ, 39.0% vs 23.1%; P = 0.370) than tumors with BRAF mutation. There was no MSI-H in tumors with BRAF mutation. Tumors with BRAF wild-type had significantly more KRAS mutations than tumors with BRAF mutation (38.2% vs 7.7%, P = 0.033). Only one (7.7%) tumor with BRAF mutation had KRAS mutation. The other clinicopathological findings, such as sex, age, BMI, family history of CRC, ECOG performance status, tumor location and initial CEA level, were not different between tumors with BRAF wild-type and mutation. Treatment modalities in enrolled patients are summarized in Table 2. Ninety-three (75.6%) patients with BRAF wildtype and nine (69.2%) with BRAF mutation received surgery (P = 0.737). One hundred nineteen (96.7%) patients with BRAF wild-type and 11 (84.6%) with BRAF mutation received chemotherapy (P = 0.182). Chemotherapy regimens and targeted agents were not significantly different between the two groups. The mean ± SD follow-up duration in all patients was 21.5 ± 13.3 mo April 21, 2014 Volume 20 Issue 15

274 Kim B et al. BRAF mutation in advanced colorectal cancer Table 2 Treatment modality of the patients n (%) Tumor response and survival according to BRAF mutation status To determine the value of BRAF mutation status as a prognostic marker, PFS and OS were compared between patients with BRAF wild-type and BRAF mutation (Figure 1). PFS was not statistically different between the two groups (BRAF wild type vs BRAF mutation, 10.1 ± 7.8 mo vs 7.2 ± 5.0 mo; P = 0.135). The OS was significantly shorter in patients with BRAF mutation than those with BRAF wild-type (BRAF wild type vs BRAF mutation, 22.2 ± 13.2 mo vs 18.8 ± 13.6 mo; P = 0.041). In contrast to BRAF mutation, KRAS mutation status was not associated with PFS or OS in patients with stage Ⅲ/Ⅳ CRCs. Univariate and multivariate analyses were performed to validate the prognostic factors for survival in patients with stage Ⅲ/Ⅳ CRC. Univariate analysis revealed that histology, tumor stage, surgery and BRAF status were significant prognostic factors for survival (p = 0.001, p < 0.001, p < and p = 0.041, respectively). Multivariate analysis showed that TNM stage Ⅳ (HR = 3.183; 95%CI: ; p = 0.002), poor differentiation and lack of differentiation in histology (HR = 2.821; 95%CI: ; p = 0.005), no surgery (HR = 3.694; 95%CI: ; p < 0.001) and BRAF mutation (HR = 2.195; 95%CI: ; p = 0.039) were significant poor prognostic factors for survival in patients with stage Ⅲ/Ⅳ CRC (Table 3). DISCUSSION BRAF wild-type (n = 123) BRAF Mutant (n = 13) P value Surgery Yes 93 (75.6) 9 (69.2) No 30 (24.4) 4 (30.8) Chemotherapy Yes 119 (96.7) 11 (84.6) No 4 (3.3) 2 (15.4) Chemotherapy regimen FOLFOX 97 (82.2) 9 (81.8) FOLFIRI 5 (4.2) 0 FL 7 (5.9) 2 (15.4) Xeloda 8 (6.8) 0 SOX 1 (0.8) 0 Target agent use No 91 (74.0) 8 (61.5) Bevacizumab 19 (15.4) 5 (38.5) Cetuximab 13 (10.6) 0 This retrospective study demonstrated that stage Ⅲ or Ⅳ CRC in Korean patients have distinct molecular characteristics, including exclusive mutations between KRAS and BRAF genes and a low incidence of MSI-H. BRAF mutant tumors showed significantly shorter survival than BRAF wild-type tumors, while the KRAS mutation had no prognostic impact. It has been reported that 10% to 20% of CRCs have Table 3 Prognostic factors in colorectal cancer patients in multivariate analysis HR (95%CI) P value Age (older than 60 yr vs younger) ( ) Sex (male vs female) ( ) Tumor type (rectum vs colon) ( ) Initial stage (stage Ⅳ vs Ⅲ) ( ) Histology (PD and UD vs WD and MD) ( ) Surgical treatment (no vs yes) ( ) < BRAF mutation (mutant vs wild-type) ( ) KRAS mutation (mutant vs wild-type) ( ) CI: Confidence interval; PD: Poorly differentiated; UD: Undifferentiated; WD: Well differentiated; MD: Moderately differentiated; CEA: Carcinoembryonic antigen. a BRAF mutation [3,22]. The BRAF mutation is associated with MSI-H through hmlh1 promoter hypermethylation, which is known to be associated with a high level of CpG island methylator phenotype (CIMP) [11]. Several reports have revealed a low incidence of BRAF mutations in CRC. Although it is not clear if ethnicity affects the status of the BRAF mutation, previous studies with small sample sizes of Koreans reported only 3.8%-7% of BRAF mutant CRCs in Korean patients [23,24]. A few studies showed that BRAF mutations were extremely uncommon in rectal cancer, with an incidence of 0%-2% [25,26]. However, the present study indicated that the incidence of BRAF mutation was not affected by ethnicity or tumor location. Although it is known that BRAF genes are exclusively mutated with KRAS genes, coincident mutations of KRAS and BRAF rarely occur in CRC with an incidence of 0.001% [27]. Herein, we found that one case had both KRAS (G12V) and BRAF mutations. This patient was a 49-year-old male and had rectosigmoid junction cancer of MSS type in MSI. It is not clear whether these tumors have a different biology and natural history than KRAS or BRAF only mutant tumors or which of the two mutations is the dominant oncogene driving tumor proliferation because coincident KRAS and BRAF mutation were infrequently observed [27]. The present study showed a small number of MSI-H tumors compared to previous studies [5,28] and thus failed to demonstrate the relationship between MSI status and BRAF mutation. This may have been due to the advanced stage of CRCs in the present study. Our previous study demonstrated that MSI-H tumors were strongly associated with early tumor stage, and only 6% of stage Ⅲ/Ⅳ CRC had MSI-H tumors [29]. Compared with colon cancer, rectal cancer has a low incidence of MSI-H tumors and this may have affected the results of the present study [29]. CRC has significant clinical heterogeneity based on several molecular markers such as MSI, KRAS and BRAF [2-4]. It has been well documented that MSI-H tumors have a better prognosis than MSS/MSI-L tumors [5,29]. In contrast, BRAF mutant tumors have a poor prognosis compared to BRAF wild-type tumors [6,7]. The 4373 April 21, 2014 Volume 20 Issue 15

275 Kim B et al. BRAF mutation in advanced colorectal cancer A 1.0 B Log rank P = Log rank P = Probability of PFS Probability of OS BRAF wild type BRAF mutant type 0.0 BRAF wild type BRAF mutant type Progression free survival (mo) Overall survival (mo) C 1.0 Log rank P = D 1.0 Log rank P = Probability of PFS Probability of OS KRAS wild type KRAS mutant type 0.0 KRAS wild type KRAS mutant type Progression free survival (mo) Overall survival (mo) Figure 1 Progression-free survival and overall survival according to BRAF or KRAS mutation status. A: PFS was not significantly different by the BRAF mutation status (p = 0.135); B: OS was significantly poorer in the BRAF mutant group than in the BRAF wild-type group (p = 0.041); PFS (C) and OS (D) were not significantly different by the KRAS mutation status (p = and p = 0.536, respectively). PFS: Progression-free survival; OS: overall survival. BRAF mutation has been used as a strong prognostic factor for overall survival in patients with CRCs, which was also confirmed in the present study. Thus, CRCs can be classified into four subtypes by these two distinct prognostic markers of MSI status and BRAF mutation [7]. MSS/BRAF mutant tumors are known to exhibit the worst prognosis, while MSI-H/BRAF wild-type tumors have the best prognosis. MSI-H/BRAF mutant or MSS/BRAF wild-type tumors have been suggested as intermediate subtypes [7]. However, several recent studies suggested that the association of BRAF mutation with poor prognosis is limited to MSS tumors [16,30]. Thus, further studies are necessary to adapt this molecular classification for clinical practice. This study has several limitations. First, a retrospective study design has inherent limitations. Second, because of the small number of MSI-H tumors, the present study failed to evaluate the association of BRAF mutation with MSI status. Finally, even for advanced cancer, the follow-up period was relatively insufficient at a mean of 21 mo. In conclusion, CRCs have distinct molecular features, including MSI status and mutations of KRAS and BRAF. These molecular markers enable the classification of CRCs into meaningful subtypes for prognosis. Our data strongly support the prognostic role of BRAF mutation in Korean patients with advanced CRC. COMMENTS Background Approximately 10% of colorectal cancers (CRCs) have BRAF mutations and these CRCs have a worse prognosis than those with BRAF wild-type. In addition, the BRAF gene is thought to be closely associated with several molecular markers such as microsatellite instability (MSI) and KRAS in CRCs. However, although the prevalence of CRCs has rapidly increased in South Korea, the incidence of BRAF mutation and its clinical meaning are unknown in Korean CRC patients. Research frontiers Recent advances in molecular genetics enable the classification of CRC by molecular markers, including MSI and KRAS and BRAF mutations, to predict prognosis and treatment response. It is well known that CRCs with KRAS mutations respond poorly to cetuximab. However, the predictive role of BRAF mutations in cetuximab response is not clear. As for prognostic markers, MSI-high tumors have a better prognosis than MSS/MSI-low tumors. In contrast, CRC with BRAF mutations have a worse prognosis than those with BRAF wild-type, while KRAS mutations have no prognostic role. Future research should aim to uncover the role and purpose of molecular markers in CRC and to identify their potential usage clinically. Innovations and breakthroughs This study identified the clinicopathological features of CRC with a BRAF mutation and its molecular interaction with MSI and KRAS targeting for stage Ⅲ/Ⅳ CRC. Applications Molecular markers such as BRAF, KRAS, MSI may be used to classify cases of colorectal carcinoma into subtypes for prognosis. Terminology BRAF: BRAF is a human gene that makes a protein called B-Raf. The gene is also referred to as proto-oncogene B-Raf and v-raf murine sarcoma viral onco April 21, 2014 Volume 20 Issue 15

276 Kim B et al. BRAF mutation in advanced colorectal cancer gene homolog B1, while the protein is more formally known as serine/threonineprotein kinase B-Raf. The B-Raf protein is involved in sending signals inside cells, which are involved in directing cell growth. KRAS: KRAS is a human gene that makes a protein called KRAS. Like other members of the Ras family, the KRAS protein is a GTPase and is an early player in many signal transduction pathways. The protein product of the normal KRAS gene performs an essential function in normal tissue signaling, and the mutation of a KRAS gene is an essential step in the development of many cancers. Peer review The BRAF mutant tumors had a significantly shorter survival than that of BRAF wild-type tumors, while the KRAS mutation had no prognostic impact. These results are interesting and this finding allows these molecular markers to be used to classify cases of colorectal carcinoma into subtypes for prognosis. 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Prognostic role of EGFR gene copy number and KRAS mutation in patients with locally advanced rectal cancer treated with preoperative chemoradiotherapy. Br J Cancer 2010; 103: [PMID: DOI: / sj.bjc ] 19 Douillard JY, Cunningham D, Roth AD, Navarro M, James RD, Karasek P, Jandik P, Iveson T, Carmichael J, Alakl M, Gruia G, Awad L, Rougier P. Irinotecan combined with fluorouracil compared with fluorouracil alone as first-line treatment for metastatic colorectal cancer: a multicentre randomised trial. Lancet 2000; 355: [PMID: ] 20 Giacchetti S, Perpoint B, Zidani R, Le Bail N, Faggiuolo R, Focan C, Chollet P, Llory JF, Letourneau Y, Coudert B, Bertheaut-Cvitkovic F, Larregain-Fournier D, Le Rol A, Walter S, Adam R, Misset JL, Lévi F. Phase III multicenter randomized trial of oxaliplatin added to chronomodulated fluorouracil-leucovorin as first-line treatment of metastatic colorectal cancer. 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277 Kim B et al. BRAF mutation in advanced colorectal cancer [PMID: DOI: /gcc.20854] 23 Kang GH. Four molecular subtypes of colorectal cancer and their precursor lesions. Arch Pathol Lab Med 2011; 135: [PMID: DOI: / ra.1] 24 Rhee YY, Kim MJ, Bae JM, Koh JM, Cho NY, Juhnn YS, Kim D, Kang GH. Clinical outcomes of patients with microsatelliteunstable colorectal carcinomas depend on L1 methylation level. Ann Surg Oncol 2012; 19: [PMID: DOI: /s ] 25 Slattery ML, Curtin K, Wolff RK, Boucher KM, Sweeney C, Edwards S, Caan BJ, Samowitz W. A comparison of colon and rectal somatic DNA alterations. Dis Colon Rectum 2009; 52: [PMID: DOI: /DCR.0b013e3181a0e5df] 26 Yamauchi M, Morikawa T, Kuchiba A, Imamura Y, Qian ZR, Nishihara R, Liao X, Waldron L, Hoshida Y, Huttenhower C, Chan AT, Giovannucci E, Fuchs C, Ogino S. Assessment of colorectal cancer molecular features along bowel subsites challenges the conception of distinct dichotomy of proximal versus distal colorectum. Gut 2012; 61: [PMID: DOI: /gutjnl ] 27 Sahin IH, Kazmi SM, Yorio JT, Bhadkamkar NA, Kee BK, Garrett CR. Rare Though Not Mutually Exclusive: A Report of Three Cases of Concomitant KRAS and BRAF Mutation and a Review of the Literature. J Cancer 2013; 4: [PMID: DOI: /jca.3619] 28 Brim H, Mokarram P, Naghibalhossaini F, Saberi-Firoozi M, Al-Mandhari M, Al-Mawaly K, Al-Mjeni R, Al-Sayegh A, Raeburn S, Lee E, Giardiello F, Smoot DT, Vilkin A, Boland CR, Goel A, Hafezi M, Nouraie M, Ashktorab H. Impact of BRAF, MLH1 on the incidence of microsatellite instability high colorectal cancer in populations based study. Mol Cancer 2008; 7: 68 [PMID: DOI: / ] 29 Hong SP, Min BS, Kim TI, Cheon JH, Kim NK, Kim H, Kim WH. The differential impact of microsatellite instability as a marker of prognosis and tumour response between colon cancer and rectal cancer. Eur J Cancer 2012; 48: [PMID: DOI: /j.ejca ] 30 Samowitz WS, Sweeney C, Herrick J, Albertsen H, Levin TR, Murtaugh MA, Wolff RK, Slattery ML. Poor survival associated with the BRAF V600E mutation in microsatellitestable colon cancers. Cancer Res 2005; 65: [PMID: DOI: / can ] P- Reviewers: Nath G, Vorobjova T S- Editor: Ma YJ L- Editor: Webster JR E- Editor: Liu XM 4376 April 21, 2014 Volume 20 Issue 15

278 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH BRIEF ARTICLE REPORT Unexpected FDG-PET uptake in the gastrointestinal tract: Endoscopic and histopathological correlations Eran Goldin, Mahmud Mahamid, Benjamin Koslowsky, Shimon Shteingart, Yael Dubner, Gadi Lalazar, Dov Wengrower Eran Goldin, Mahmud Mahamid, Benjamin Koslowsky, Shimon Shteingart, Dov Wengrower, Digestive Diseases Institute, Shaare Zedek Medical Center, Affiliated with the Hebrew University School of Medicine, Jerusaleml 91031, Israel Yael Dubner, Gadi Lalazar, Department of Gastroenterology, Hadassah University Hospital, Jerusaleml 91031, Israel Author contributions: Goldin E and Mahamid M contributed equally to this article; Goldin E, Wengrower D and Mahamid M developed the concept and design of the study; Dubner Y and Lalazar G performed the acquisition of the data; Shteingart S contributed to the statistical analysis of the data, and final manuscript revisions; Koslowsky B contributed to the statistical analysis of the data and performed major article revisions including the final approval of the manuscript. Correspondence to: Benjamin Koslowsky, MD, Digestive Diseases Institute, Shaare Zedek Medical Center, Affiliated with the Hebrew University School of Medicine, 12 Bayit street, Jerusaleml 91031, Israel. binyaminkos@szmc.org.il Telephone: Fax: Received: April 8, 2013 Revised: August 6, 2013 Accepted: September 13, 2013 Published online: April 21, 2014 Abstract AIM: To investigate the nature and significance of unexpected positron emission tomography with fluorodeoxyglucose (FDG-PET) uptake within the gastrointestinal tract (GIT). METHODS: Patients with unexpected FDG-PET findings in the GIT were evaluated. All patients had a previous confirmed malignancy, either solid or lymphoproliferative. The radiologic reports were performed by experienced radiologists with an exclusive PET expertise. Endoscopy, i.e., esophagogastroduodenoscopy (EGD) and colonoscopy, and histopathological evaluation of all findings was performed in all patients in accordance to the FDG-PET results. The findings from each of these modalities were compared to each other. Both clinically significant and insignificant findings were assessed. RESULTS: Seventy-two patients were endoscopically evaluated. Twenty-seven patients (37.5%) had primarily a lymphoproliferative tumor and 45 (62.5%) had solid tumors. In 50 patients (69.4%) the endoscopic examination revealed lesions in the same anatomical areas as the FDG-PET findings. Among these 50 patients, malignant and premalignant lesions i.e., adenomatous polyps were found in 16 (32%) and 9 (18%) patients, respectively. Inflammation was noted in an additional 20 patients (40%). Compared to primary solid tumors, a background of primary lymphoproliferative malignancy was more likely to reveal an additional primary malignancy (15.6% vs 33.3%, respectively, P < 0.01). EGD compared to colonoscopy, revealed altogether 11 (25.6%) new malignancies compared to 5 (17.2%), respectively, P = No GIT clinically significant findings were overseen by the FDG-PET. CONCLUSION: Unexpected FDG uptake in the GIT is commonly encountered and may contain significant findings. Endoscopy evaluation is justified in order to detect these additional findings Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Positron emission tomography with fluorodeoxyglucose; Gastrointestinal tract; Malignancy; Endoscopy; Unexpected findings Core tip: Positron emission tomography with fluorodeoxyglucose (FDG-PET) is gradually gaining acceptance as a first line radiological modality for both solid and hematological malignancies. While both technology and expertise is improving, incidental findings are more frequently encountered. The gastrointestinal tract is a common source for these unexpected findings. In this manuscript we assessed the significance of these 4377 April 21, 2014 Volume 20 Issue 15

279 Goldin E et al. The importance of incidental PET-CT findings findings and correlated them with upper and lower endoscopy findings. Surprisingly, we encountered much clinical significant information. More so, the FDG-PET was considerably sensitive to gastrointestinal findings. These results suggest that gastrointestinal incidental findings should be evaluated. Goldin E, Mahamid M, Koslowsky B, Shteingart S, Dubner Y, Lalazar G, Wengrower D. Unexpected FDG-PET Uptake in the Gastrointestinal Tract: Endoscopic and Histopathological correlations. World J Gastroenterol 2014; 20(15): Available from: URL: i15/4377.htm DOI: INTRODUCTION Positron emission tomography (PET) with fluorodeoxyglucose (FDG) has recently been established as a diagnostic tool for the evaluation of patients with suspected or confirmed malignancy [1]. The normal distribution of 18-F FDG uptake has been described in both children and adults [2-4]. A number of physiologic variants are commonly encountered, including normal physiologic uptake in the head and neck, heart, breast, liver, spleen, gastrointestinal tract (GIT), genito-urinary system, bone marrow, muscles, and brown adipose tissue. Benign lesions with increased 18-F FDG uptake are commonly seen and are frequently misinterpreted as malignancies [5-7]. 18-F FDG is glucose analog labeled with a positron-emitting isotope, F (fluorine)-18. It is transported into the cells by glucose transporters, subsequently phosphorylated by hexokinase and trapped within the cell [8]. Malignant tissues accumulate 18-FDG more rapidly than normal tissues due to their increased glucose metabolism rate, increased expression of glucose transporters, and highly active hexokinase bound to tumor mitochondrial compared to normal tissue [9]. 18-F FDG uptake is also known to occur in nonmalignant conditions, mainly inflammation and infection [10]. Increased uptake in inflammation is due to an increased number of glucose transporters [11]. Imaging of inflammation with 18-F FDG PET is also based on the fact that infiltrated granulocytes and tissue macrophages use glucose as an energy source [12]. 18-F FDG uptake in the normal GIT is highly variable and can range from mild to intense with a focal, diffuse, or segmental distribution. The origin of 18-F FDG uptake in the GIT is not fully understood and is most likely multifactorial. Many nonmalignant conditions in the GIT can influence 18-F FDG uptake. Patients with esophagitis caused by gastroesophageal reflux disease or due to radiation therapy may reveal marked uptake in the esophagus. Barrett s esophagus, a premalignant esophageal finding, may also demonstrate increased uptake in the distal esophagus. Gastric uptake may be associated with Helicobacter pylori infection [13]. Differentiating malignancy or inflammation from a normal variant is quite challenging in colonic uptake. 18-F FDG uptake is also associated with Crohn s disease (CD), autoimmune pancreatitis, liver and gastrointestinal neoplasms [14-19]. Long-segment diffuse uptake in the GIT is most often associated with physiological uptake, whereas focal uptake in the GIT is more likely to be considered a pathological finding [20]. PET imaging using 18-F FDG (FDG-PET) is well accepted in the imaging work-up or follow-up of many malignancies [1,20]. Incidental or unexpected FDG uptake in the GIT is not rare [21-23], and the significance of such uptake in patients with suspected or confirmed malignancy is yet to be established [24]. The aim of this study was to retrospectively assess the significance of FDG uptake in the gastrointestinal tract in patients who underwent PETcomputer tomography (CT) due to a preexisting malignancy. MATERIALS AND METHODS A dual center retrospective study conducted between October 2009 to December 2011, included 72 patients who underwent a FDG PET-CT scan due to a confirmed malignancy. Patients who were found to have incidental uptake in the GIT were included in the study. Primary malignancy in any solid tumor or hematologic malignancies were included. Patients whose primary malignancy was in the GIT, i.e., colorectal, gastric or esophageal cancer who demonstrated increased uptake in the area of the primary malignancy were excluded. All participants underwent FDG PET-CT following a 6 h fast. In selected cases intravenous furosemide was administered to enhance urinary clearance of excreted FDG. These cases were chosen according to the discretion of the radiologist performing the examination. Intravenous FDG was given at doses that ranged between 185 to 444 MBq. Unenhanced CT scan was routinely obtained for attenuation correction and for diagnostic purposes. PET and PET-CT scans were reviewed on a computer workstation using a 3D-volume and obtaining coronal, sagittal, and trans-axial views by a PET-accredited nuclear physician. The radiographs were all interpreted by a doctor with a minimum of five year experience. Additionally, all cases were presented at a forum which included very experienced radiology experts in PET-CT. The clinical data of all participants were available to the nuclear physician performer. Incidental GIT uptake was the primary inclusion criteria into the study. Esophagogastroduodenoscopy (EGD) was conducted in all patients with any PET finding that correlated with the upper GIT. A full colonoscopy was performed when findings suggested a lower GIT lesion. All procedures were carried out by an accredited gastroenterologist. The examination was directed to correspond with the FDG uptake in the GIT; if any endoscopic irregularity was detected, multiple biopsies were taken for histopathological analysis. The histopathologic diagnoses were grouped as malignant lesion, premalignant lesion (polyps with any degree of dysplasia), or as inflammation. The outcome 4378 April 21, 2014 Volume 20 Issue 15

280 Goldin E et al. The importance of incidental PET-CT findings Number of patients Lymphoma Esophageal Colon Gastric Lung Thyroid Pancreas Bladder Primary malignancy Ovary Prostate Breast ENT Figure 1 Primary malignancy origin. ENT: Ear, nose and throat. was measured as the correlation between the imaging data compared to the endoscopic and histopathology findings. Statistical analysis Case details were computerized and analyzed using SPSS version 17.0 software (Chicago, IL, United States). Data was analyzed using a standard unpaired two-tailed Student s t-test. All P values were two-sided and considered statistically significant if < RESULTS The study population comprised of 72 patients (38 male, 34 female) with a mean age of 62 ± 7 years (range years) who were diagnosed with various types of malignancies. The origin of the primary malignancy is listed in Figure 1. Twenty-seven patients (37.5%) had a lymphoproliferative tumor and 45 (62.5%) had solid tumors. Forty-seven patients had undergone surgical intervention and 34 were receiving chemotherapy and/or radiotherapy. Forty-three patients underwent EGD and 29 patients underwent colonoscopy. No participant underwent both examinations. Fifty patients (69.4%) had a positive endoscopic finding corresponding to the PET-FDG uptake. Among them, 16 (32%) were found to have a malignant lesion and 9 (18%) had a premalignant lesion (i.e., polyps). 19 (38%) patients were found to have an inflammation other than inflammatory bowel disease such as gastritis, duodenitis or nonspecific colitis, and 1 patient (2%) had findings compatible with CD. Five patients (10%) had an endoscopic impression of an irregularity but the histological findings were normal. Excluding these 5 patients with a normal biopsy a total of 45 patients (62.5%) had a significant endoscopic finding corresponding to the FDG uptake. Among the 16 patients with new malignancies, 4 had adenocarcinoma of the stomach, 5 were diagnosed with stomach lymphoma, 2 were found to have adenocarcinoma of the esophagus, and 4 were identified with colorectal cancer. Carcinoma in-situ was found in another patient. Premalignant lesions, i.e., polyps in the colon were found in 9 patients, all corresponding to the PET- FDG uptake. A total of 25/72 patients (34.7%) were diagnosed with malignant or premalignant lesions in areas corresponding to the FDG uptake. 12 (18%) patients had additional endoscopic findings which were not detected by the PET-FDG examination. All of these findings were small polyps less than 5 mm. No malignant lesions were unidentified by the PET-FDG uptake. Among the 72 participants, 27 (37.5%) had a primary lymphoproliferative malignancy, while 45 (62.5%) had a primary solid tumor. Comparing the primary lymphoproliferative group to the primary solid tumor group, a new malignancy was found in 9/27 (33%) and 7/45 (15.5%), respectively, (P < 0.01) (Figure 2A). When comparing the diagnostic yield of EGD vs colonoscopy, 43/72 (59.7%) underwent EGD and 29/72 (40.3%) underwent a colonoscopy. Eleven malignancies were found by EGD, with a diagnostic yield of 25.6%. Colonoscopy revealed 5 malignancies achieving a diagnostic yield of 17.2% (P = 0.12) (Figure 2B). The difference was not statistically significant. DISCUSSION FDG-PET has been successfully used to aid in the diagnosis, staging, and monitoring of variable malignancies [1,3]. Unfortunately increased FDG uptake is not specific to cancerous cells [5-7]. Accordingly, FDG-PET has a low specificity. FDG is accumulated in several types of inflammatory cells such as lymphocytes, neutrophils, and macrophages due to their increased glucose metabolism [10]. Thus in addition to identification of malignant tumors, the possibility of false-positive findings must be kept in mind to prevent over diagnosis and therapeutic mistakes. Histological analysis is used to distinguish between malignancies and other causes April 21, 2014 Volume 20 Issue 15

281 Goldin E et al. The importance of incidental PET-CT findings A Number of patients B Number of patients No malignancy Malignancy % Lymphoproliferative No malignancy Malignancy Gastroscopy 25.6% Solid tumor Several large sample size studies have shown that FDG- PET is a useful tool for detecting premalignant and malignant lesions. Gutman et al [25] reviewed a series of 1716 PET- CT scans of patients with various malignant diseases other than colorectal cancer and found focal FDG uptake in the colon in 20 patients. These foci were found to be correspondent to three carcinomas and 10 adenomas found during colonoscopy. Israel et al [26] found 58 foci or intense FDG uptake in the GIT in a series of PET-CT scans of 4390 patients. Among the 34 patients who underwent GIT studies, 24 (71%) had abnormal findings, including malignant and premalignant lesions. This study shows a higher rate of FDG uptake in the GIT than found in previous studies. A partial explanation lies in the advances in the technology and equipment combined with a higher level of interpretation expertise. Additional multifactor explanations are: age, primary malignancy and indication for the exam, which among others may influence GIT uptake. Our results indicate that patients with a primary lymphoproliferative or solid malignancy with a focal FDG uptake are at a higher risk for an additional primary malignancy. As many as one third of these findings may have a clinical significance and up to one fifth may provide a complete new diagnosis. Primary lymphoproliferative disease adds a considerable risk. As this tool is increasingly Colonoscopy 15.6% 17.2% Figure 2 Risk of additional malignancy. A: Primary was lymphoproliferative (33%) vs solid tumor (15.5%), P < 0.01; B: Primary endoscopic modality was gastroscopy (25.6%) vs colonoscopy (17.2%), P = being accepted as a primary work up method for both diagnostic and follow up purposes, the likelihood for incidental findings rises. Upper GIT lesions were found to be slightly more specific for malignancy than lower GIT lesions. Premalignant findings were more prevalent in colonoscopy examinations, as expected in the general population. Interestingly, no malignant or significant premalignant findings were found at endoscopy at areas other than those detected by PET-PDG. These findings emphasize the importance and significance of PET-CT findings in the GIT. Technological advances may lead to better differentiation between physiological and significant pathological findings. Currently the recommendation for patients with an incidental FDG uptake in the GIT should be a complete GIT workup. In summary, unexpected FDG uptake in the GIT is commonly encountered. Significant findings may be exposed by further evaluation. Endoscopy and pathology evaluation is justified in order to detect these additional findings. COMMENTS Background Positron emission tomography with fluorodeoxyglucose (FDG-PET) has been successfully used to aid in the diagnosis, staging, and monitoring of variable malignancies. Unfortunately increased FDG uptake is not specific to cancerous cells. Research frontiers Several large sample size studies have shown that FDG-PET is a useful tool for detecting premalignant and malignant lesions. These foci were found to be correspondent to three carcinomas and 10 adenomas found during colonoscopy. Innovations and breakthroughs This study shows a higher rate of FDG uptake in the gastrointestinal tract than found in previous studies. A partial explanation lies in the advances in the technology and equipment combined with a higher level of interpretation expertise. Applications The results indicate that patients with a primary lymphoproliferative or solid malignancy with a focal FDG uptake are at a higher risk for an additional primary malignancy. Peer review This paper a very interesting manuscript, well done and well written and of potential interest for the readers. The high sensitivity and specificity in finding new pathologies during follow up is impressive. REFERENCES 1 Gambhir SS, Czernin J, Schwimmer J, Silverman DH, Coleman RE, Phelps ME. A tabulated summary of the FDG PET literature. J Nucl Med 2001; 42: 1S-93S [PMID: ] 2 Hillner BE, Siegel BA, Liu D, Shields AF, Gareen IF, Hanna L, Stine SH, Coleman RE. Impact of positron emission tomography/computed tomography and positron emission tomography (PET) alone on expected management of patients with cancer: initial results from the National Oncologic PET Registry. J Clin Oncol 2008; 26: [PMID: DOI: /JCO ] 3 Tatsumi M, Miller JH, Wahl RL. 18F-FDG PET/CT in evaluating non-cns pediatric malignancies. J Nucl Med 2007; 48: [PMID: DOI: /jnumed ] 4 Storto G, Nicolai E, Salvatore M. [18F]FDG-PET-CT for early monitoring of tumor response: when and why. Q J Nucl Med 4380 April 21, 2014 Volume 20 Issue 15

282 Goldin E et al. The importance of incidental PET-CT findings Mol Imaging 2009; 53: [PMID: ] 5 Ahmad Sarji S. Physiological uptake in FDG PET simulating disease. Biomed Imaging Interv J 2006; 2: e59 [PMID: DOI: /biij.2.4.e59] 6 Nakamoto Y, Tatsumi M, Hammoud D, Cohade C, Osman MM, Wahl RL. Normal FDG distribution patterns in the head and neck: PET/CT evaluation. Radiology 2005; 234: [PMID: DOI: /radiol ] 7 Cook GJ, Wegner EA, Fogelman I. Pitfalls and artifacts in 18FDG PET and PET/CT oncologic imaging. Semin Nucl Med 2004; 34: [PMID: ] 8 Timmers HJ, Chen CC, Carrasquillo JA, Whatley M, Ling A, Eisenhofer G, King KS, Rao JU, Wesley RA, Adams KT, Pacak K. Staging and functional characterization of pheochromocytoma and paraganglioma by 18F-fluorodeoxyglucose (18F-FDG) positron emission tomography. 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283 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH REPORT Effect of resistance training on non-alcoholic fatty-liver disease a randomized-clinical trial Shira Zelber-Sagi, Assaf Buch, Hanny Yeshua, Nahum Vaisman, Muriel Webb, Gil Harari, Ofer Kis, Naomi Fliss-Isakov, Elena Izkhakov, Zamir Halpern, Erwin Santo, Ran Oren, Oren Shibolet Shira Zelber-Sagi, Assaf Buch, Hanny Yeshua, Nahum Vaisman, Muriel Webb, Naomi Fliss-Isakov, Elena Izkhakov, Zamir Halpern, Erwin Santo, Ran Oren, Oren Shibolet, Department of Gastroenterology, Tel Aviv Medical Center, Tel-Aviv, Israel Shira Zelber-Sagi, Gil Harari, School of Public Health, University of Haifa, Haifa, Israel Assaf Buch, Hanny Yeshua, Nahum Vaisman, Naomi Fliss- Isakov, Zamir Halpern, Erwin Santo, Ran Oren, Oren Shibolet, The Sackler Faculty of Medicine, Tel-Aviv University, Tel Aviv, Israel Ofer Kis, Wingate Institute for Physical Education and Sport, Netanya, Israel Author contributions: Zelber-Sagi S conceived the study; Zelber-Sagi S and Buch A designed the study; Zelber-Sagi S, Buch A and Harari G analyzed the data; Zelber-Sagi S, Buch A and Yeshua H performed the data collection; Vaisman N, Webb M, Kis O, Fliss-Isakov N, Izkhakov E helped in data collection; Halpern Z, Santo E, Oren R and Shibolet O conducted on data collection; Zelber-Sagi S, Buch A and Shibolet O wrote the manuscript; all authors critically reviewed the manuscript and approved it. Correspondence to: Shira Zelber-Sagi, PhD, Department of Gastroenterology, Tel Aviv Medical Center, 6 Weizman st., Tel-Aviv, Israel. zelbersagi@bezeqint.net Telephone: Fax: Received: September 25, 2013 Revised: January 6, 2014 Accepted: January 19, 2014 Published online: April 21, 2014 Abstract AIM: To evaluate the effect of resistance training (RT) on non alcoholic liver disease (NAFLD) patients. METHODS: A randomized clinical trial enrolling NAFLD patients without secondary liver disease (e.g., without hepatitis B virus, hepatitis C virus or excessive alcohol consumption). Patients were randomly allocated either to RT, three times weekly, for 3 mo or a control arm consisting of home stretching. The RT included leg press, chest press, seated rowing, latissimus pull down etc. with 8-12 repetitions, 3 sets for each exercise, for a total duration of 40 min. Hepatic ultrasound, fasting blood tests, anthropometrics and body composition by dual energy X-ray absorptiometry were assessed. At baseline and follow-up, patients filled out a detailed semi-quantitative food frequency questionnaire reporting their habitual nutritional intake. Steatosis was quantified by the hepatorenal-ultrasound index (HRI) representing the ratio between the brightness level of the liver and the right kidney. The HRI has been previously demonstrated to be highly reproducible and was validated against liver biopsy and proton magnetic resonance spectroscopy. RESULTS: Eighty two patients with primary NAFLD were randomized to receive 3 mo of either RT or stretching. After dropout or exclusion from analysis because of protocol violation (weight change > 3 kg), thirty three patients in the RT arm and 31 in the stretching arm completed the study per protocol. All baseline characteristics were similar for the two treatment groups with respect to demographics, anthropometrics and body composition, blood tests and liver steatosis on imaging. HRI score was reduced significantly in the RT arm as compared to the stretching arm (-0.25 ± 0.37 vs ± 0.28, P = 0.017). The RT arm had a significantly higher reduction in total, trunk and android fat with increase in lean body mass. There was no correlation between the reduction in HRI in the RT arm and weight change during the study, but it was positively correlated with the change in trunk fat (r = 0.37, P = 0.048). The RT arm had a significant reduction in serum ferritin and total cholesterol. There was no significant difference between arms in dietary changes and these did not correlate with HRI change. CONCLUSION: Three months RT improves hepatic fat content accompanied by favorable changes in body composition and ferritin. RT may serve as a complement to treatment of NAFLD April 21, 2014 Volume 20 Issue 15

284 Zelber-Sagi S et al. Resistance training in NAFLD 2014 Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Resistance exercise; Obesity; Nutrition; Physical activity; Abdominal fat Core tip: Resistance training is viewed as a complement to aerobic training. However, data on the effect of resistance training on non alcoholic liver disease (NAFLD) is scant. A three month resistance training in NAFLD patients exerted a significant reduction in liver fat as well as reduction in total body and trunk fat with increase in lean body mass. Furthermore, resistance training led to reduction in serum ferritin and cholesterol. In NAFLD patients, compliance to aerobic training may be low due to fatigue. Therefore, resistance training can serve as an easier alternative or a complement form of exercise in these patients. Zelber-Sagi S, Buch A,Yeshua H, Vaisman N, Webb M, Harari G, Kis O, Fliss-Isakov N, Izkhakov E, Halpern Z, Santo E, Oren R, Shibolet O. Effect of resistance training on non-alcoholic fattyliver disease a randomized-clinical trial. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: org/ /wjg.v20.i INTRODUCTION Lifestyle modifications, including weight reduction and physical activity, improve many of the risk factors for non alcoholic liver disease (NAFLD) [1] and have become the primary treatment modalities for the disease [2]. The role of physical activity (PA) as a potential treatment for NAFLD has been tested in several observational studies and a few clinical trials, mostly testing the effect of aerobic training. Resistance training (strength training) is a means for developing and maintaining muscular strength, endurance, power, and muscle mass that has grown in popularity over the past two decades [3,4]. In a study of the general population, resistance training (RT) was inversely associated with NAFLD. This association remained significant after adjusting to multiple confounders including body mass index (BMI), homeostasis model assessment (HOMA), nutritional factors, adiponectin, and resistin [5]. Two small trials found beneficial effects for RT as a single treatment in NAFLD patients, but results regarding reduction in steatosis were conflicting [6,7]. In a randomized clinical trial (RCT) in 19 sedentary adult NAFLD patients, 8 wk of RT, consisting of 45 min sessions trice weekly, led to a reduction in liver fat without weight loss [7]. In an uncontrolled clinical trial in 12 obese adolescents, a three-month RT program consisting of 1 h sessions twice weekly did not change hepatic fat content but improved hepatic insulin sensitivity [6]. In a recent RCT among type-2 diabetic patients, it was demonstrated that resistance training and aerobic training are equally effective in reducing hepatic fat content [8]. NAFLD patients report a poorer health-related quality of life compared with healthy United States population both on physical and mental health scores [9]. Furthermore, fatigue is a common symptom in NAFLD patients [10], and they report low scores for vitality [9]. Although NAFLD patients understand the benefits of exercise, they lack the confidence to perform it and express a fear of falling [11]. The potential benefits of RT are not only to cardiovascular health and to weight management but it also improves balance and reduces the risk of falls [12-14]. RT improves several components of physical function, contributes to health-related quality of life [15,16], and is well tolerated even among patients with coronary heart disease or the elderly [15,17]. In recent years, increasing attention has been paid to RT as a useful adjunctive tool of exercise in various metabolic diseases including diabetes and heart disease [4,18,19]. Indeed, the American Heart association and American College of Sport Medicine (ACSM) recommend RT at least twice a week in addition to aerobic training [4]. For those patients who may have physical limitation or low motivation that prevents them from performing aerobic PA, RT can serve as an alternative option. Therefore we conducted a randomized controlled trial of RT vs stretching. Our main aim was to evaluate the effect of 3 mo RT on the presence of fatty liver measured by abdominal ultrasound and on the hepatorenal index (HRI) as a quantitative objective measurement of steatosis. We also evaluated the effect of the RT program on liver enzymes, metabolic parameters and body composition. MATERIALS AND METHODS We conducted a RCT (sealed envelopes randomization stratified by gender) in consecutive patients with ultrasound diagnosed fatty liver attending the liver clinic at the Tel-Aviv Medical center during and community regional HMO s clinics. Inclusion criteria were age between years and a diagnosis of fatty liver by ultrosound in the past 6 mo and on the baseline US examination. Exclusion criteria were any known secondary liver disease including the presence of hepatitis B surface antigen or anti-hepatitis C virus antibodies, excessive alcohol consumption defined as 30 g/d in men or 20 g/d in women, administration of medical treatment that may elevate alanine aminotransferase (ALT) or lead to hepatic steatosis, known diabetes, major chronic diseases including: renal, cardiovascular, lung, uncontrolled hypertension, inflammatory bowel disease, active cancer, autoimmune disorders and orthopedic contraindications for RT. Adults with diabetes were excluded to avoid a confounding effect, since it is unclear whether they would have the same response to physical training and since changes in antidiabetic medications during the trial might occur. We also excluded patients regularly performing RT in the 3 mo or 6 mo prior to 4383 April 21, 2014 Volume 20 Issue 15

285 Zelber-Sagi S et al. Resistance training in NAFLD study enrolment for novice and progressive trainee respectively (novice-trained continuously less than 2 mo, progressive-trained continuously more than 2 mo). Patients performing vigorous aerobic PA in the 3 mo prior to the study, defined as aerobic exercise 5 d a week 30 min at moderate pace or 3 d a week 20 min at vigorous pace or 4 times a week with combination of both and patients with recent weight reduction (more than 3 kg in the last 3 mo) were also excluded. A sample size of 32 patients in each group was calculated to be needed for a 90% power to detect a difference of 0.25 with a standard deviation of 0.30, based on previously published data on HRI change following weight change [20], with a two-sided significance level. Additional 20% patients were recruited taking into consideration attrition or protocol violation. The study was approved by the Tel-Aviv Medical center ethics committee and all patients signed an informed consent. The study was pre-registered in the NIH registration website (TRIAL no. NCT ). Lifestyle and medical evaluation Each patient underwent at baseline a face-to-face interview by the same trained interviewer. The questionnaire was assembled by the Israeli center for disease control and was used in the first Israeli National Health Survey. It consists of structured questions about alcohol consumption, medications and medical history. PA evaluation was performed by a questionnaire that was tailored and validated for the Israeli population [21], according to which a summarized index of all questions was calculated and used as an indicator of PA levels. At baseline, patients filled out a detailed semi-quantitative food frequency questionnaire (FFQ) reporting their habitual nutritional intake in the past year. The FFQ was assembled by the Food and Nutrition Administration, Ministry of Health and was previously described in detail [5,22,23]. The FFQ is composed of 120 food items with specified serving sizes or standard weight and volume measures of the servings commonly consumed in this study population. The nutrient components of each food item were taken from the Israeli National Nutrient Database. At the end of the trial patients filled out the same FFQ but reported their nutritional intake in the past 3 mo in order to evaluate changes in nutritional habits during the trial. Blood pressure was measured by an experienced nurse following a uniform protocol. Each participant underwent biochemical testing, following a 12 h fast, for liver enzymes, serum lipid profile, and fasting serum glucose and insulin levels. The HOMA was calculated as fasting [serum insulin (μu/ml) fasting plasma glucose (mmol/ L)]/22.5. Ultrasonographic examination for determination of NAFLD and quantification of steatosis Fatty liver was assessed by abdominal ultrasonography using standardized criteria [24]. Ultrasonography was performed in all subjects both at baseline and at follow up with the same equipment (EUB-8500 scanner Hitachi Medical Corporation, Tokyo, Japan) and by the same experienced radiologist (Webb M) as described previously [22,23,25]. The radiologist was blinded to patient allocation and to laboratory values and medical history of the participants. During the ultrasonography, a histogram of brightness levels, i.e., a graphical representation of echo intensity within a region of interest (ROI) was obtained. In the liver, the ROI was measured in the 7 th or 8 th intercostal space in the mid or anterior axillary line in the superficial aspect of the liver. In the right kidney, the ROI was determined as the cortical area between the pyramids. The brightness level for each organ was recorded and the ratio between the median brightness level of the liver and the right kidney cortex was calculated to determine the HRI. The HRI has been previously demonstrated to be highly reproducible (r = 0.77, P < 0.001, kappa = 0.86) and was validated against liver biopsy [26]. HRI 1.5 indicates fatty liver. Anthropometric and body composition evaluation Height, weight and waist circumference were measured following a uniform protocol and BMI was calculated. Lean body mass (LBM) and fat mass (FM) were evaluated by the dual energy X-ray absorptiometry (DEXA) method [27,28] by a blinded technician at the Metabolic Nutrition Clinic. All participants were instructed to maintain their pretrial PA habits, regular nutritional intake, medications and nutritional supplements. A weight change during the study of more than 3 kg in either direction, which is clinically significant for NAFLD [20] and other metabolic parameters [29,30], was pre-defined as a protocol violation. Intervention Patients were randomly allocated either to RT, three times weekly, for 3 mo or a control arm consisting of home stretching. RT training: The RT program was according to the ACSM 2009 position paper on Progression Models in Resistance Training for Healthy Adults [31]. Exercises included: leg press, leg extension, leg curl, seated chest press, seated rowing, latissimus pull down, biceps curl and shoulder press with 8-12 repetitions, 3 sets for each exercise with 1-2 min rest between sets, for a total duration of about 40 min. Participants performed the training in a community setting in one of the hosting gyms closest to their house or place of work. On the first training meeting, the researchers performed a personal training session and provided explanation on the RT equipment using a comfortable load (determined by volitional fatigue reached with repetitions). The load was gradually increased by 2%-10% in the following training sessions, according to the individual ability of the patient (when the patient felt he can perform 1-2 extra repetitions) and with consultation of the profes April 21, 2014 Volume 20 Issue 15

286 Zelber-Sagi S et al. Resistance training in NAFLD sional trainers of the hosting gym. All changes were routinely documented. Standardization of the RT for all participants was ensured by the highly controlled environment at the gyms and a uniform protocol including: a uniform and meticulous familiarization with the training, all participants (treatment and control) received a comprehensive booklet graphically illustrating by pictures all exercises. Participating gyms (all belonging to a single regional chain) have uniform standard equipment and all the gyms instructors, certified by the sports ministry, were given detailed and comprehensive instructions regarding the training protocol. Using equipment that was not included in the study protocol was not allowed. Every 2 wk phone calls were made to ensure adherence to the training protocol and participants were repeatedly instructed not to perform aerobic training (cycling, treadmill, etc.) during the sessions. All patients were observed for an entire exercise session at least twice during the trial by the researchers. Active control arm: The home stretching routine followed the ACSM s guidelines for a general stretching program [32]. The program included 8 stretching exercises for the major muscle/tendon groups using the static stretching technique. The participants performed 4 repetitions of these static stretches each lasting 20 s. Each session was performed on 3 non-consecutive days a week [32]. Participants received a booklet with instructions on the stretching training illustrated by pictures. Statistical analysis Statistical analyses were performed using SPSS version 19 (SPSS Inc., Chicago, IL, United States) software and SAS version 9.1 (SAS Institute, Cary North Carolina). Continuous variables are presented as mean ± SD. Paired t-tests were used to evaluate within group changes from baseline to end of treatment. To test baseline differences in continuous variables between the two groups the independent samples t-test was performed. The Wilcoxon signed ranks test or the Mann-Whitney test were used if non-parametric tests were required based on data distribution. Analysis of variance using repeated measurements model was applied for testing the group X time interactions and exact F statistics was performed. Associations between nominal variables were performed with the Pearson Chi-Square test. Pearson correlation was used to test the correlation between change in HRI and change in other parameters. P < 0.05 was considered statistically significant for all analyses. RESULTS Trial participants and compliance Eighty two patients with primary NAFLD were randomized to receive 3 mo of either RT or stretching. Forty four were randomized to the RT group of which 36 (82%) completed the 3 mo follow up period. Thirty eight were randomized to the stretching arm and 33 (87%) completed the study. Two patients dropped out of the RT group due to adverse events (knee pain, shoulder pain), and one from the stretching arm (back pain). Five patients were excluded from analysis because of protocol violation reaching a weight change of more than the pre-defined 3 kg. Hence, thirty three patients in the RT arm and 31 in the stretching arm completed the study per protocol. A flow chart of trial participation is described in Figure 1. The average age was ± years, with 34 (53%) males. Average BMI was ± 4.32 kg/m 2. The average fasting insulin levels were elevated, but in accordance with the exclusion criteria fasting glucose levels were within the normal range. All baseline characteristics were similar for the two treatment groups with respect to demographics, anthropometrics and body composition, blood tests including liver enzymes and liver steatosis on imaging as assessed by the HRI (Table 1). Furthermore, no difference in dietary intake at baseline was observed between arms (P 0.40 for all comparisons, data not shown). The average number of training sessions at the gym, that was automatically recorded every time a patient entered the gym with his personal chip, was 2.2 ± 0.65 times a week, representing 73% ± 20.5% of the recommended number of sessions of 3 times a week during the 12 wk trial. There was a significant increment in the weight lifted during the trial: leg press increased from ± to ± kg (P < 0.001), and chest press increased from ± to ± kg (P < 0.001). Primary outcome HRI score was significantly reduced in the RT arm as compared to the stretching arm (-0.25 ± 0.37 vs ± 0.28, P = 0.017), representing an 11% vs 3.5% relative reduction from the baseline in the two groups respectively (Figure 2). The RT arm had a significant but small reduction in weight (-0.39 ± 1.43 kg vs 0.33 ± 1.21 kg) and BMI (Table 2) compared to the stretching arm. The RT arm had a significant reduction in total FM, trunk fat and android fat and increase in LBM compared to the stretching arm (Figure 3). There was no correlation between the reduction in HRI in the RT arm and weight change or BMI change during the study (r = 0.25, P = 0.17) nor with total FM change (r = 0.29, P = 0.13) as observed by DEXA. However, the change in HRI was positively correlated with the change in trunk fat (r = 0.37, P = 0.048). In contrast, in the stretching arm the change in HRI was positively correlated with weight change (r = 0.35, P = 0.055) and BMI change (r = 0.36, P = 0.049). There was no significant difference between arms in dietary change during the study in total calories, carbohydrates, protein, fat and different types of fat (P for all comparisons) (Table 2). Furthermore, there was no correlation between the reduction in HRI in the RT arm and change in dietary intake of total calories and different dietary components (P 0.42 for all correlations) April 21, 2014 Volume 20 Issue 15

287 Zelber-Sagi S et al. Resistance training in NAFLD Consecutive NAFLD patients from: Tel-Aviv hospital liver clinic and referred from regional HMO'S and Approaching following advertising in the hospital's website 82 met the inclusion criteria and were randomized 44 in the RT arm 38 in the control arm 2 withdrew from study 4 lost to follow up 2 withdrew from study 2 lost to follow up 2 quit due to adverse events (knee pain, shoulder pain) 1 quit due to adverse events (back pain) 36 completed the trial 33 completed the trial 3 excluded from analysis 1 2 excluded 33 in analysis 31 in analysis from analysis 1 Figure 1 Flow chart of trial participants. 1 Excluded from analysis due to > 3 kg weight change. RT: Resistance training; NAFLD: Non alcoholic liver disease. Table 1 Comparison between the two treatment arms (mean ± SD) Parameter Normal range Total population Resistance training Stretching P (n = 64) (n = 33) (n = 31) Gender (males) 53.1% 48.5% 58.1% Age (yr) ± ± ± HRI (score) 2.04 ± ± ± BMI (kg/m 2 ) ± ± ± Waist circumference (cm) ± ± ± Systolic BP (mmhg) ± ± ± Diastolic BP (mmhg) ± ± ± Trunk fat 43.45% ± 6.21% 44.28% ± 5.99% 42.55% ± 6.42% Android fat 47.49% ± 6.41% 48.10% ± 6.30% 46.88% ± 6.57% 0.47 Total fat 39.81% ± 7.89% 40.64% ± 7.29% 38.92% ± 8.52% Lean body mass 58.20% ± 7.50% 57.34% ± 6.89% 59.11% ± 8.12% Physical activity (index) 5.18 ± ± ± Glucose (mg/dl) ± ± ± Insulin (mcu/ml) ± ± ± HOMA (score) 5.80 ± ± ± HbA1C 5.59 ± ± ± Cholesterol (mg/dl) ± ± ± Triglycerides (mg/dl) ± ± ± HDL (mg/dl) ± ± ± LDL (mg/dl) ± ± ± ALT (U/L) ± ± ± AST (U/L) ± ± ± GGT (U/L) ± ± ± Ferritin (ng/ml) ± ± ± HRI: Hepatorenal-ultrasound index; BMI: Body mass index; BP: Blood pressure; HOMA: Homeostasis model assessment; HbA1C: Hemoglobin A1c; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; ALT: Alanine transaminase; AST: Aspartate aminotransferase; GGT: Gamma-glutamyltransferase April 21, 2014 Volume 20 Issue 15

288 Zelber-Sagi S et al. Resistance training in NAFLD A Resistance arm B Stretching arm 3.5 P = vs the stretching arm 3.5 No significant change 3 3 HRI index 2.5 HRI index Baseline 3 mo Baseline 3 mo Figure 2 Change (absolute) in hepatorenal-ultrasound index values between baseline and end of trial by treatment arm. A: Resistance arm; B: Stretching arm. Each line represents a single patient. HRI: Hepatorenal-ultrasound index. Table 2 Between group comparisons of changes from baseline to end of treatment and within group comparisons (mean ± SD) Parameter Normal range Resistance training (n = 33) Stretching (n = 31) P value between groups HRI (score) ± 0.37 b ± Weight (kg) ± ± BMI (kg/m 2 ) ± ± Waist circumference (cm) ± 2.00 a 0.70 ± Systolic BP (mmhg) 1.27 ± ± Diastolic BP (mmhg) ± 5.61 a ± Glucose (mg/dl) ± ± Insulin (mcu/ml) ± ± HOMA (score) 0.37 ± ± HbA1C % ± 0.13% 0.04% ± 0.14% Cholesterol (mg/dl) ± ± Triglycerides (mg/dl) ± ± HDL (mg/dl) 0.13 ± ± LDL (mg/dl) ± ± ALT (U/L) ± 9.65 b ± AST (U/L) ± 7.75 a ± 6.95 a GGT (U/L) ± ± Ferritin (ng/ml) ± a 8.25 ± Total calories (kcal) ± b ± Total fat (g) ± b ± b Carbohydrates (g) ± b ± a Protein (g) ± b ± b Saturated fat (g) ± b ± MUFA (g) ± ± PUFA (g) ± ± For paired samples t-test, a P < 0.05, b P < 0.01 within group comparisons. HRI: Hepatorenal-ultrasound index; BMI: Body mass index; BP: Blood pressure; HOMA: Homeostasis model assessment; HbA1C: Hemoglobin A1c; HDL: High-density lipoprotein; LDL: Low-density lipoprotein; ALT: Alanine transaminase; AST: Aspartate aminotransferase; GGT: Gamma-glutamyltransferase; MUFA: Monounsaturated fat; PUFA: Polyunsaturated fat April 21, 2014 Volume 20 Issue 15

289 Zelber-Sagi S et al. Resistance training in NAFLD Mean change Android fat % Trunk fat % Total fat % Total LBM % Stretching arm P = P = P = P = Resistance arm Secondary outcomes The RT arm had significantly higher reduction in serum ferritin and total cholesterol. There was no significant difference in reduction of liver enzymes between arms. ALT was significantly reduced only in the RT arm in within group comparison. RT had no significant impact on serum glucose, insulin, glycosylated hemoglobin and triglycerides (Table 2). DISCUSSION Error bars: +/-1 SE Figure 3 Change (absolute) in body composition parameters between baseline and end of trial by treatment arm. P represents the significance of difference between resistance and stretching arm for each parameter (n = 55). LBM: Lean body mass. In this randomized controlled trial, NAFLD patients without diabetes underwent either RT trice weekly or static stretching. The results suggest that RT exerts beneficial effects on several clinical and biochemical parameters including liver fat and body composition. The 2007 update of the American Heart Association dealing with resistance exercise concludes that RT should be viewed as a complement to aerobic exercise [14]. However, the beneficial effect of RT for patients with steatosis was so far not supported by strong evidence. The present trial is one of the first to test this question on a large group of patients. We have demonstrated a significant reduction in steatosis as measured by an objective ultrasonographic tool, the HRI. The modest relative reduction of about 10% is similar to the 13% relative reduction in steatosis previously demonstrated [7]. Although the RT arm had a nonsignificant reduction in caloric intake and a small but significant weight reduction of less than half a kg, these changes did not correlate with HRI change. Our results suggest that the reduction in steatosis in the RT arm cannot be explained by weight loss or dietary change. RT also improved body composition, most importantly trunk fat mass that was positively correlated with the change in HRI. The weight-reduction independent beneficial effect of aerobic exercise in NAFLD is supported by clinical trials demonstrating a relative reduction of hepatic triglyceride concentration by 21%-35% [33-35] following supervised training such as cycling. However, in a trial of a more modest activity that included brisk walk, there was a relative reduction of 10.3% in liver fat [36], similar to the one observed by the present study. In previous published trials about the effect of RT in adult NAFLD patients, there was a significant improvement in glycemic control and no improvement in liver enzymes [7,8]. Our study did not demonstrate improved glucose metabolism, this discrepancy may stem from the exclusion of diabetic patients from our study. It was previously shown that RT improves hyperglycemia, only in patients with disturbed glucose metabolism or diabetes [37-41]. Our study showed significant improvement in the liver enzymes (ALT and aspartate aminotransferase-ast) within group but with no difference between arms. Interestingly, there seems to be a limited correlation between exercise and liver enzyme reduction. Aerobic PA led to a significant reduction in liver enzymes in some trials [42] while no reduction was seen in other trials despite reduced steatosis [8,34,35]. With specific reference for resistance training, it has been shown that weightlifting exercise resulted in increases in liver enzymes; AST and ALT, though the underlying mechanisms are unknown [43]. Thus, it may be in our study that the reduction of liver enzymes in the RT arm was masked and is underestimated. Serum ferritin was significantly reduced only in the RT group. This novel effect of RT in NAFLD patients, that has been previously demonstrated with aerobic training [42] and lifestyle intervention [44], is of importance due to the strong association of ferritin with fibrosis and inflammation [45,46] and with insulin resistance (IR) [23,47] in NAFLD patients. The mechanisms by which PA, and specifically RT, reduces serum ferrtin are unknown. Serum ferritin is associated with insulin resistance [23,48] and is an acute phase protein that can be induced in the setting of systemic or hepatic inflammation [49-52]. Serum ferritin was demonstrated to be a predictor of histologic severity including steatosis [46]. Thus, we can assume that a reduction in liver fat demonstrated in our study or improved hepatic insulin sensitivity by RT, that was demonstrated in another study [6], led to a reduction of serum ferritin. Furthermore, RT may have an anti-inflammatory effect, as demonstrated by increasing adiponectin levels [53], and serum ferritin was found to be inversely correlated with serum adiponectin [54]. However, it is unclear if hyperferritinemia in NAFLD is simply a consequence of disease severity or actively contributes to disease progression [46]. Ferritin was found to inhibit the secretion of apolipoprotein B and in this way may alter cholesterol and triglyceride transport in the liver [55]. Interestingly, some studies reported that PA plays an important role in reducing serum ferritin concentration [56,57] and this may be another explanation for the reduction in liver fat in our study. Another beneficial effect of RT in our study was a significant reduction of serum cholesterol. Although data regarding the effect of RT on lipid metabolism are 4388 April 21, 2014 Volume 20 Issue 15

290 Zelber-Sagi S et al. Resistance training in NAFLD equivocal, reduction of serum total cholesterol and LDL by resistance training has been previously demonstrated in a meta-analysis of randomized controlled trials [58]. It is well established that liver steatosis is associated with IR and lipid abnormalities including alteration in cholesterol metabolism [59-62]. Recent data show that increased IR contributes to the shift in cholesterol metabolism to increased synthesis and decreased absorption, independent of body weight [63-65]. Several studies have demonstrated that resistance training improves IR, including hepatic IR [6,66], and therefore may contribute to decreased synthesis of hepatic cholesterol. However, the precise mechanisms involved still need to be clarified [67]. Professional societies recommend 30 min of moderate-intensity aerobic PA on most, and preferably all, days of the week, or vigorous-intensity PA 3 times per week for 20 min each time. However, only 27.7% United States adults meet recommended levels of either moderate or vigorous physical activity, whereas 29.2% report no regular PA outside of their work [68,69]. Moreover, the prevalence of physically active adults among patients with diabetes is lower than in those without diabetes [70] and they are less likely to meet PA recommendations [71]. In NAFLD patients, compliance may be even lower because fatigue has been demonstrated to be markedly higher in NAFLD patients compared to controls, and is associated with inactivity and excessive daytime sleepiness [10]. Therefore, an alternative or a complement form of exercise that may be easier to perform or to adhere to, such as RT, may be helpful in the treatment of NAFLD patients. The major limitation of this study is that due to ethical and practical considerations we were unable to perform repeated liver biopsies in our short term study preventing any inference regarding the effect of RT on inflammation and fibrosis. Ideally, quantification of liver fat is performed by liver biopsy or magnetic resonance spectroscopy ( 1 H-MRS) that is an accepted noninvasive method to reliably quantify steatosis [72,73]. HRI has been validated vs liver biopsy and provides a highly sensitive, objective and quantitative tool for liver fat evaluation with a high correlation (r = 0.82, P < 0.001) and a kappa of 0.75 as compared with histological steatosis [26]. Another group compared HRI to 1 H-MRS as a reference standard, demonstrating high correlation (r 2 = 0.92, P < ) and therefore confirming that HRI can be a valuable analytic tool in clinical investigation [72]. It was also recently shown that HRI highly correlates with biochemical surrogate markers of liver steatosis: the fatty liver index (FLI) (r = 0.55, P < 0.001) and the SteatoTest (r = 0.52, P < 0.001) [74]. Furthermore, since a baseline biopsy wasn t performed, we were unable to distinguish between patients with simple steatosis or NASH. NASH is the NAFLD variant that needs to be treated more urgently due to a worse natural history of progression to cirrhosis, hepatocellular carcinoma [75] and increased liver related mortality [76]. Recently, ultrasonographic FLI, another semi-quantitative evaluation tool of hepatic steatosis, was demonstrated to successfully predict biopsy diagnosed NASH [77]. In conclusion, this relatively large randomized clinical trial demonstrated a significant reduction in steatosis, as assessed by HRI, during 3 mo RT accompanied by favorable changes in body composition and reduction of serum ferritin. ACKNOWLEDGMENTS We thank the "Great Shape" gyms for their cooperation and contribution during the study. COMMENTS Background Lifestyle modifications, including weight reduction and physical activity, improve many of the risk factors for non alcoholic liver disease (NAFLD) and have become the primary treatment modalities for the disease. The role of physical activity as a potential treatment for NAFLD has been tested in several observational studies and a few clinical trials, mostly testing the effect of aerobic training. Research frontiers Resistance training (RT) (strength training) is a means for developing and maintaining muscular strength, endurance, power, and muscle mass that has grown in popularity over the past two decades. In a study of the general population, resistance training was inversely associated with NAFLD. This association remained significant after adjusting to multiple confounders including body mass index, homeostasis model assessment, nutritional factors, adiponectin, and resistin. Small trials found beneficial effects for RT as a single treatment in NAFLD patients, but results regarding reduction in steatosis were conflicting. Innovations and breakthroughs Aerobic training is a recommended treatment for NAFLD. Resistance training is viewed as a complement to aerobic training. However, data on the effect of resistance training on NAFLD is scant. In this study, three months of resistance training in NAFLD patients exerted reduction in liver fat as well as reduction in total body and trunk fat with increase in lean body mass. Furthermore, resistance training led to reduction in serum ferritin. Applications In NAFLD patients, compliance to aerobic training may be low due to fatigue. Therefore, resistance training can serve as an easier alternative or a complement form of exercise in these patients. Terminology Hepato-renal index (HRI): During the ultrasonography, a histogram of brightness levels, i.e., a graphical representation of echo intensity within a region of interest (ROI) is obtained in the liver and in the right kidney. The brightness level for each organ is recorded and the ratio between the median brightness level of the liver and the right kidney cortex is calculated to determine the HRI. Resistance training (strength training) is a means for developing and maintaining muscular strength, endurance, power, and muscle mass. Peer review The study is an independent study focusing on the physical exercise pattern and the improvement of vital parameters in NAFLD patients. The study demonstrates an important aspect of resistance training that can be added to the therapeutic approach benefiting patients with NAFLD. This randomized trial is of potential interest given the paucity of data on effective NAFLD treatment. REFERENCES 1 Duncan GE, Perri MG, Theriaque DW, Hutson AD, Eckel RH, Stacpoole PW. Exercise training, without weight loss, increases insulin sensitivity and postheparin plasma lipase activity in previously sedentary adults. Diabetes Care 2003; 26: [PMID: ] 2 Bellentani S, Dalle Grave R, Suppini A, Marchesini G. Behavior therapy for nonalcoholic fatty liver disease: The need 4389 April 21, 2014 Volume 20 Issue 15

291 Zelber-Sagi S et al. Resistance training in NAFLD for a multidisciplinary approach. Hepatology 2008; 47: [PMID: ] 3 Kraemer WJ, Ratamess NA. Fundamentals of resistance training: progression and exercise prescription. Med Sci Sports Exerc 2004; 36: [PMID: ] 4 Pollock ML, Franklin BA, Balady GJ, Chaitman BL, Fleg JL, Fletcher B, Limacher M, Piña IL, Stein RA, Williams M, Bazzarre T. AHA Science Advisory. Resistance exercise in individuals with and without cardiovascular disease: benefits, rationale, safety, and prescription: An advisory from the Committee on Exercise, Rehabilitation, and Prevention, Council on Clinical Cardiology, American Heart Association; Position paper endorsed by the American College of Sports Medicine. Circulation 2000; 101: [PMID: ] 5 Zelber-Sagi S, Nitzan-Kaluski D, Goldsmith R, Webb M, Zvibel I, Goldiner I, Blendis L, Halpern Z, Oren R. Role of leisure-time physical activity in nonalcoholic fatty liver disease: a population-based study. 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292 Zelber-Sagi S et al. Resistance training in NAFLD and flexibility in healthy adults. Med Sci Sports Exerc 1998; 30: [PMID: ] 33 Finucane FM, Sharp SJ, Purslow LR, Horton K, Horton J, Savage DB, Brage S, Besson H, De Lucia Rolfe E, Sleigh A, Martin HJ, Aihie Sayer A, Cooper C, Ekelund U, Griffin SJ, Wareham NJ. The effects of aerobic exercise on metabolic risk, insulin sensitivity and intrahepatic lipid in healthy older people from the Hertfordshire Cohort Study: a randomised controlled trial. Diabetologia 2010; 53: [PMID: DOI: /s z] 34 van der Heijden GJ, Wang ZJ, Chu ZD, Sauer PJ, Haymond MW, Rodriguez LM, Sunehag AL. A 12-week aerobic exercise program reduces hepatic fat accumulation and insulin resistance in obese, Hispanic adolescents. Obesity (Silver Spring) 2010; 18: [PMID: DOI: /oby ] 35 Johnson NA, Sachinwalla T, Walton DW, Smith K, Armstrong A, Thompson MW, George J. Aerobic exercise training reduces hepatic and visceral lipids in obese individuals without weight loss. 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Proc Natl Acad Sci USA 1991; 88: [PMID: ] 52 Pham CG, Bubici C, Zazzeroni F, Papa S, Jones J, Alvarez K, Jayawardena S, De Smaele E, Cong R, Beaumont C, Torti FM, Torti SV, Franzoso G. Ferritin heavy chain upregulation by NF-kappaB inhibits TNFalpha-induced apoptosis by suppressing reactive oxygen species. Cell 2004; 119: [PMID: DOI: /j.cell ] 53 de Piano A, de Mello MT, Sanches Pde L, da Silva PL, Campos RM, Carnier J, Corgosinho F, Foschini D, Masquio DL, Tock L, Oyama LM, do Nascimento CM, Tufik S, Dâmaso AR. Long-term effects of aerobic plus resistance training on the adipokines and neuropeptides in nonalcoholic fatty liver disease obese adolescents. Eur J Gastroenterol Hepatol 2012; 24: [PMID: DOI: / MEG.0b013e ac] 54 Ku BJ, Kim SY, Lee TY, Park KS. Serum ferritin is inversely correlated with serum adiponectin level: population-based cross-sectional study. Dis Markers 2009; 27: [PMID: DOI: /DMA ] 55 Hevi S, Chuck SL. Ferritins can regulate the secretion of apolipoprotein B. 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293 Zelber-Sagi S et al. Resistance training in NAFLD Koster A, Chaves PH, Jonsdottir B, Garcia M, Gudnason V, Harris TB. Associations of visceral and liver fat with the metabolic syndrome across the spectrum of obesity: the AGES- Reykjavik study. Obesity (Silver Spring) 2011; 19: [PMID: DOI: /oby ] 62 Bugianesi E, Moscatiello S, Ciaravella MF, Marchesini G. Insulin resistance in nonalcoholic fatty liver disease. Curr Pharm Des 2010; 16: [PMID: ] 63 Simonen P, Kotronen A, Hallikainen M, Sevastianova K, Makkonen J, Hakkarainen A, Lundbom N, Miettinen TA, Gylling H, Yki-Järvinen H. Cholesterol synthesis is increased and absorption decreased in non-alcoholic fatty liver disease independent of obesity. J Hepatol 2011; 54: [PMID: DOI: /j.jhep ] 64 Hoenig MR, Sellke FW. Insulin resistance is associated with increased cholesterol synthesis, decreased cholesterol absorption and enhanced lipid response to statin therapy. Atherosclerosis 2010; 211: [PMID: DOI: / j.atherosclerosis ] 65 Flannery C, Dufour S, Rabøl R, Shulman GI, Petersen KF. Skeletal muscle insulin resistance promotes increased hepatic de novo lipogenesis, hyperlipidemia, and hepatic steatosis in the elderly. Diabetes 2012; 61: [PMID: DOI: /db ] 66 Brooks N, Layne JE, Gordon PL, Roubenoff R, Nelson ME, Castaneda-Sceppa C. Strength training improves muscle quality and insulin sensitivity in Hispanic older adults with type 2 diabetes. Int J Med Sci 2007; 4: [PMID: ] 67 Mann S, Beedie C, Jimenez A. Differential effects of aerobic exercise, resistance training and combined exercise modalities on cholesterol and the lipid profile: review, synthesis and recommendations. Sports Med 2014; 44: [PMID: DOI: /s ] 68 Pratt M, Macera CA, Blanton C. Levels of physical activity and inactivity in children and adults in the United States: current evidence and research issues. Med Sci Sports Exerc 1999; 31: S526-S533 [PMID: ] 69 Peterson JA. Get moving! Physical activity counseling in primary care. J Am Acad Nurse Pract 2007; 19: [PMID: ] 70 Morrato EH, Hill JO, Wyatt HR, Ghushchyan V, Sullivan PW. Physical activity in U.S. adults with diabetes and at risk for developing diabetes, Diabetes Care 2007; 30: [PMID: DOI: /dc ] 71 Zhao G, Ford ES, Li C, Mokdad AH. Compliance with physical activity recommendations in US adults with diabetes. Diabet Med 2008; 25: [PMID: ] 72 Mancini M, Prinster A, Annuzzi G, Liuzzi R, Giacco R, Medagli C, Cremone M, Clemente G, Maurea S, Riccardi G, Rivellese AA, Salvatore M. Sonographic hepatic-renal ratio as indicator of hepatic steatosis: comparison with (1)H magnetic resonance spectroscopy. Metabolism 2009; 58: [PMID: DOI: /j.metabol ] 73 Ratziu V, Bellentani S, Cortez-Pinto H, Day C, Marchesini G. A position statement on NAFLD/NASH based on the EASL 2009 special conference. J Hepatol 2010; 53: [PMID: DOI: /j.jhep ] 74 Zelber-Sagi S, Webb M, Assy N, Blendis L, Yeshua H, Leshno M, Ratziu V, Halpern Z, Oren R, Santo E. Comparison of fatty liver index with noninvasive methods for steatosis detection and quantification. World J Gastroenterol 2013; 19: [PMID: DOI: /wjg.v19.i1.57] 75 Vernon G, Baranova A, Younossi ZM. Systematic review: the epidemiology and natural history of non-alcoholic fatty liver disease and non-alcoholic steatohepatitis in adults. Aliment Pharmacol Ther 2011; 34: [PMID: DOI: /j x] 76 Angulo P. Diagnosing steatohepatitis and predicting liverrelated mortality in patients with NAFLD: two distinct concepts. Hepatology 2011; 53: [PMID: DOI: /hep.24403] 77 Ballestri S, Lonardo A, Romagnoli D, Carulli L, Losi L, Day CP, Loria P. Ultrasonographic fatty liver indicator, a novel score which rules out NASH and is correlated with metabolic parameters in NAFLD. Liver Int 2012; 32: [PMID: DOI: /j x] P- Reviewers: Assy N, Lonardo A, Saleh J, Shivshankar P, Wang CX S- Editor: Gou SX L- Editor: A E- Editor: Liu XM 4392 April 21, 2014 Volume 20 Issue 15

294 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH REPORT Living-donor or deceased-donor liver transplantation for hepatic carcinoma: A case-matched comparison Ping Wan, Jian-Jun Zhang, Qi-Gen Li, Ning Xu, Ming Zhang, Xiao-Song Chen, Long-Zhi Han, Qiang Xia Ping Wan, Jian-Jun Zhang, Qi-Gen Li, Ning Xu, Ming Zhang, Xiao-Song Chen, Long-Zhi Han, Qiang Xia, Department of Liver Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai , China Author contributions: Xia Q is the guarantor and designed the study; Wan P conducted the majority of the study and wrote the manuscript; Xia Q, Zhang JJ, Li QG, Xu N, Zhang M, Chen XS and Han LZ offered suggestions for this work and revised the manuscript. Supported by Key Discipline and Specialty Foundation of Shanghai Municipal Commission of Health and Family Planning; Training Program for Superb Academic Leaders in Shanghai Health System, No. XBR ; Special Fund for Building of Leading Talent Teams in Shanghai Correspondence to: Qiang Xia, Professor, Department of Liver Surgery, Ren Ji Hospital, School of Medicine, Shanghai Jiao Tong University, No Dongfang Road, Shanghai , China. xiaqiang@medmail.com.cn Telephone: Fax: Received: September 29, 2013 Revised: December 30, 2013 Accepted: February 17, 2014 Published online: April 21, 2014 Abstract AIM: To compare the surgical outcomes between living-donor and deceased-donor liver transplantation in patients with hepatic carcinoma. METHODS: From January 2007 to December 2010, 257 patients with pathologically confirmed hepatic carcinoma met the eligibility criteria of the study. Forty patients who underwent living-donor liver transplantation (LDLT) constituted the LDLT group, and deceaseddonor liver transplantation (DDLT) was performed in 217 patients. Patients in the LDLT group were randomly matched (1:2) to patients who underwent DDLT using a multivariate case-matched method, so 40 patients in the LDLT group and 80 patients in the DDLT group were enrolled into the study. We compared the two groups in terms of clinicopathological characteristics, postoperative complications, long-term cumulative survival and relapse-free survival outcomes. The modified Clavien-Dindo classification system of surgical complications was used to evaluate the severity of perioperative complications. Furthermore, we determined the difference in the overall biliary complication rates in the perioperative and follow-up periods between the LDLT and DDLT groups. RESULTS: The clinicopathological characteristics of the enrolled patients were comparable between the two groups. The duration of operation was significantly longer (553 min vs 445 min, P < 0.001) in the LDLT group than in the DDLT group. Estimated blood loss (1188 ml vs 1035 ml, P = 0.055) and the proportion of patients with intraoperative transfusion (60.0% vs 43.8%, P = 0.093) were slightly but not significantly greater in the LDLT group. In contrast to DDLT, LDLT was associated with a lower rate of perioperative grade Ⅱ complications (45.0% vs 65.0%, P = 0.036) but a higher risk of overall biliary complications (27.5% vs 7.5%, P = 0.003). Nonetheless, 21 patients (52.5%) in the LDLT group and 46 patients (57.5%) in the DDLT group experienced perioperative complications, and overall perioperative complication rates were similar between the two groups (P = 0.603). No significant difference was observed in 5-year overall survival (74.1% vs 66.6%, P = 0.372) or relapse-free survival (72.9% vs 70.9%, P = 0.749) between the LDLT and DDLT groups. CONCLUSION: Although biliary complications were more common in the LDLT group, this group did not show any inferiority in long-term overall survival or relapse-free survival compared with DDLT Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Liver cancer; Hepatocellular carcinoma; Liver transplantation; Living donor; Survival; Recurrence; Complication Core tip: Several retrospective studies from single cen April 21, 2014 Volume 20 Issue 15

295 Wan P et al. Liver transplantation for hepatic carcinoma ters have shown that living-donor liver transplantation (LDLT) might be associated with higher rates of tumor recurrence than deceased-donor liver transplantation (DDLT). In this study, we compared the surgical outcomes between LDLT and DDLT in patients with hepatic carcinoma using a multivariate case-matched method, which minimized the disparity from case selection and thus made our results statistically more persuasive than others. Our results suggest that LDLT did not show any inferiority in long-term overall survival or relapse-free survival in comparison with DDLT. Wan P, Zhang JJ, Li QG, Xu N, Zhang M, Chen XS, Han LZ, Xia Q. Living-donor or deceased-donor liver transplantation for hepatic carcinoma: A case-matched comparison. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: INTRODUCTION Liver cancer is the sixth most common cancer and the third leading cause of cancer-related death, accounting for 7% of all cancers worldwide [1]. In the United States and Japan, the incidence of liver cancer is highest in the elderly population (approximately 70 years), with a strong male preponderance [2,3], while the mean age of patients with liver cancer in the Chinese population is appreciably younger. Patients who develop liver cirrhosis after hepatitis B virus (HBV) infection have a high risk of developing liver cancer. Owing to the high incidence of HBV infection, China has a large population of patients with HBV-related cirrhosis, which makes Chinese cases of and deaths from liver cancer constitute 55% of the global total [4]. A significant proportion of patients with liver cancer are accompanied by serious liver cirrhosis or liver dysfunction, so radical liver resection is limited in such patients. Liver transplantation (LT) is the best option for patients with a hepatic tumor 5 cm in diameter and concurrent decompensated cirrhosis (Child-Pugh class B or C) [5]. However, due to the severe shortage of donor organs, a considerable number of patients with liver cancer die from a lack of donors every year. As a solution, living-donor liver transplantation (LDLT) is currently an effective alternative. Currently, approximately 70% of patients undergoing LDLT are from Japan, South Korea, Hong Kong and Taiwan, but worries exist that LDLT may be associated with higher rates of tumor recurrence [6-10] and biliary complications [11,12] than deceaseddonor liver transplantation (DDLT). Which group has better postoperative results is not certain, as results from various centers differ from one another [13-16]. Whether LDLT can have more benefits on perioperative and longterm outcomes than DDLT is still disputable. Based on our long-term follow-up experience, we carried out this study to compare the two approaches of LT for primary liver cancer patients in terms of perioperative and longterm survival outcomes. MATERIALS AND METHODS Patients and case-matching procedure From January 2007 to December 2010, 281 patients underwent either LDLT or DDLT for clinically diagnosed primary liver cancer in the Department of Liver Surgery, Ren Ji Hospital, Shanghai, China. Twenty-four patients were excluded for the following reasons: (1) 11 patients who received preoperative downstaging treatment had complete tumor necrosis; (2) 7 patients had possible metastatic disease before LT; (3) coexistence of gallbladder carcinoma was pathologically found with hepatocellular carcinoma (HCC) in 2 patients after LT; (4) one patient underwent additional left nephrectomy for concurrent renal carcinoma; (5) one patient underwent combined liver-kidney transplantation; (6) one patient was pathologically diagnosed with hepatic diffuse large B cell lymphoma after LT; and (7) one patient was pathologically diagnosed with hepatic sarcomatoid carcinoma after surgery. A total of 257 patients with pathologically confirmed hepatic carcinoma met the eligibility criteria of the study, of whom 40 consecutive patients who underwent LDLT constituted the LDLT group, leaving 217 patients in the DDLT pool from which the case-matching was performed. To minimize any disparity in case selection between the two groups, we used a 1:2 multivariate matching analysis of similar patient cohorts. Each case in the LDLT group was exactly matched to 2 cases from the DDLT pool. We set 4 exact matching variables and 3 relative matching variables. Gender, age (± 5 years), within or outside the Milan criteria (the Pittsburgh staging system was used to classify the cases outside the Milan criteria [17] ) and history of hepatic surgery were selected as exact matching variables by which each case in the LDLT group should have been absolutely matched to the corresponding 2 cases in the DDLT pool. After more than two matches were identified, we used relative matching variables to select the best candidates for the DDLT group. Child-Pugh classification, MELD score (± 5 points), and serum alpha-fetoprotein (< 400 or 400 ng/ml) were defined as relative matching variables. After two rounds of matching, a random selection through the use of computer software was performed to determine the final members of the DDLT group. Finally, 40 patients in the LDLT group and 80 patients in the DDLT group were enrolled into the study. Methods and clinical data collection The clinicopathological characteristics and surgical data of the two groups were retrospectively reviewed from our prospectively collected database of LT. All the surgeons involved in the study were from the same surgical team. Preoperative down-staging treatment for size reduction of the tumors included transcatheter arterial 4394 April 21, 2014 Volume 20 Issue 15

296 Wan P et al. Liver transplantation for hepatic carcinoma chemoembolization, radiofrequency ablation, percutaneous ethanol injection and stereotactic body radiation therapy (gamma knife), thereby facilitating LT. Data of oncological characteristics (tumor within or outside the Milan criteria, tumor size, tumor number, vascular invasion and tumor pathological type) were based on the intraoperative findings and confirmed pathologically after LT. Tumor size was measured with the maximal tumor diameter in the resected specimens. The duration of the operation was collected from operation or anesthesia records. Follow-up data were obtained through outpatient visits or telephone inquiries. All of the living organs were donated with informed consent. Cadaveric donors involved in the study were obtained from brain-dead or no-heart beating donors. All the surgical procedures were performed by specialists with experience with the LT technique in the Department of Liver Surgery, Ren Ji Hospital, Shanghai, China. Surgery was performed using standard techniques. Classic orthotopic LT was the only surgery type in the DDLT group. All patients undergoing LDLT were operated on using right liver grafts without the middle hepatic vein. Biliary tract reconstruction was performed using a duct-to-duct anastomosis. The posterior anastomosis line was sutured continuously with 7-0 Prolene, while an interrupted suture was applied to the anterior anastomosis line. Evaluation of complications The definition of postoperative complication introduced by Copeland et al [18] does not cover many common complications after LT, so complications that were confirmed clinically were also included. Perioperative complications occurring during the initial hospital stay for LT were compared between the two groups. The modified Clavien-Dindo classification system of surgical complications was used to evaluate the severity of perioperative complications [19]. As for biliary complications, a bile leak was diagnosed through abdominal drains or reexploration findings, while an anastomotic stenosis was detected on the basis of an overt dilatation of the intrahepatic duct according to computed tomography or ultrasonography, or on the basis of direct visualization using endoscopic retrograde cholangiopancreatography (ERCP). Wound infection not requiring pharmacological treatment was defined as a grade Ⅰ complication, so it was not included in postoperative bacterial infection of grade Ⅱ. Suspected cases of acute rejection were confirmed by liver biopsy. Postoperative hypertension was defined as hypertension lasting longer than 3 d after surgery in the absence of a history of hypertension. If a patient died of a grade Ⅳ complication, the reason of death was not listed in the complications, but other coexistent complications were still recorded. Furthermore, we compared the overall biliary complication rates in the perioperative and long-term follow-up periods between the LDLT and DDLT groups. Immunosuppressive regimens After LT, a triple drug regimen of tacrolimus or cyclosporine (CsA) combined with methylprednisolone and/or mycophenolate mofetil (MMF) was used. Immunosuppression was started during surgery with 500 mg methylprednisolone, followed by tapering from 240 mg on postoperative day 1 to 40 mg on postoperative day 6. Maintenance prednisone at an initial dose of 20 mg daily was gradually reduced every week and was withdrawn 3 mo post-transplantation. The initial dose of tacrolimus was mg/kg every 24 h with a target trough level of 8-10 ng/ml during the first 30 d. MMF was administered orally after LT at g twice a day. If tacrolimus did not reach the target level, it was replaced with CsA at 6-10 mg/kg per day. The target C0 and C2 levels for CsA were ng/ml and ng/ ml, respectively. Statistical analysis Statistical analyses were performed with SPSS for Windows version 13.0 to compare the differences between the LDLT group and DDLT group. Categorical data were analyzed with the χ 2 test. The test of normality for all related variables was checked by the Shapiro-Wilk method, and a nonparametric statistical method (Mann- Whitney test) was applied to the variables without a normal distribution. The Kaplan-Meier method was used to estimate the cumulative overall survival (OS) and relapse-free survival (RFS). A log-rank method was used to test the equality of survival distributions between the two groups. P values < 0.05 were considered statistically significant. RESULTS Patient characteristics The clinical characteristics of the LDLT and DDLT groups are summarized in Table 1. Matching in terms of age, sex distribution, MELD score, Child-Pugh score and proportion of patients with previous hepatic resection was well achieved in all cases. Twelve patients (30.0%) in the LDLT group and 29 patients (36.3%) in the DDLT group received down-staging treatment before LT, with no significant difference between the two groups. One hundred and sixteen patients (96.7%) suffered cirrhosis due to HBV infection. One patient in the LDLT group had no cirrhosis, and the DDLT group included one case with alcoholic cirrhosis, one case with autoimmune cirrhosis and a third case without cirrhosis. No significant difference was seen in the overall distribution of different causes of liver cirrhosis. The proportions of patients who had particular comorbidities, including hypertension, diabetes mellitus, cardiac diseases, cerebrovascular diseases and previous history of abdominal surgery were also similar between the two groups. Table 2 shows the outcomes of preoperative serological detection in the two groups; there was no significant dif April 21, 2014 Volume 20 Issue 15

297 Wan P et al. Liver transplantation for hepatic carcinoma Table 1 Clinical characteristics n (%) Variable LDLT (n = 40) DDLT ( n = 80) P value Age (yr) 48.6 ± ± Gender > Male 34 (85.0) 68 (85.0) Female 6 (15.0) 12 (15.0) MELD score (17.5) 22 (27.5) (70.0) 49 (61.2) (7.5) 8 (10.0) (5.0) 1 (1.3) Child-Pugh score A 12 (30.0) 25 (31.2) B 18 (45.0) 38 (47.5) C 10 (25.0) 17 (21.3) Previous hepatic resection 3 (7.5) 6 (7.5) > Preoperative downstaging 12 (30.0) 29 (36.3) treatment Liver cirrhosis > Hepatitis B cirrhosis 39 (97.5) 77 (96.3) Alcoholic cirrhosis 0 1 (1.3) Autoimmune cirrhosis 0 1 (1.3) No cirrhosis 1 (2.5) 1 (1.3) Comorbidities Hypertension 4 (10.0) 8 (10.0) > Diabetes mellitus 4 (10.0) 8 (10.0) > Cardiac disease 0 1 (1.3) > Cerebrovascular disease 1 (2.5) 4 (5.0) Previous abdominal surgery 11 (27.5) 13 (16.3) LDLT: Living-donor liver transplantation; DDLT: Deceased-donor liver transplantation; MELD: Model for end-stage liver disease. Table 2 Preoperative serological detection outcomes n (%) Variable LDLT (n = 40) DDLT (n = 80) P value Serum alpha-fetoprotein < 400 ng/ml 25 (62.5) 57 (71.2) 400 ng/ml 15 (37.5) 23 (28.8) Blood group A 10 (25.0) 25 (31.3) B 11 (27.5) 28 (35.0) AB 2 (5.0) 9 (11.3) O 17 (42.5) 18 (22.5) HBVsAg positive 40 (100.0) 76 (95.0) HBV DNA < 1000 copies/ml 20 (50.0) 32 (40.0) 1000 copies/ml 20 (50.0) 48 (60.0) HCV antibody positive 0 0 > LDLT: Living-donor liver transplantation; DDLT: Deceased-donor liver transplantation; HBVsAg: Hepatitis B virus surface antigen; HCV: Hepatitis C virus. ference in serum alpha-fetoprotein, blood group or the proportion of patients with positive hepatitis B virus surface antigen (HBVsAg) or positive hepatitis B virus DNA (HBV DNA). No patient with positive hepatitis C virus antibody was found in either group. Table 3 Perioperative outcomes and oncological characteristics n (%) Variable LDLT ( n = 40) DDLT (n = 80) P value Duration of operation (min) 553 ± ± 75 < Estimated blood loss (ml) 1188 ± ± Intraoperative transfusion 24 (60.0) 35 (43.8) Tumor pathological type HCC 39 (97.5) 79 (98.8) ICC 0 1 (1.3) chcc-cc 1 (2.5) 0 Tumor size (cm) 3.8 ± ± Tumor number Single 27 (67.5) 62 (77.5) Multiple 13 (32.5) 18 (22.5) Vascular invasion 3 (7.5) 4 (5.0) Within Milan criteria 24 (60.0) 48 (60.0) > Outside Milan criteria > (Pittsburgh staging) Ⅰ 12 (30.0) 24 (30.0) Ⅳa 2 (5.0) 4 (5.0) Ⅳb 2 (5.0) 4 (5.0) Data are expressed as absolute mean ± SD or numbers (percentage). LDLT: Living-donor liver transplantation; DDLT: Deceased-donor liver transplantation; HCC: Hepatocellular carcinoma; ICC: Intrahepatic cholangiocarcinoma; chcc-cc: Combined hepatocellular carcinoma and cholangiocarcinoma. Perioperative and oncological outcomes As shown in Table 3, the duration of operation was significantly longer (P < 0.001) in the LDLT group than in the DDLT group. Estimated blood loss (P = 0.055) and the proportion of patients with intraoperative transfusion (P = 0.093) were slightly but not significantly greater in the LDLT group. Tumor pathological type, tumor size, tumor number and the proportion of patients with vascular invasion were similar between the two groups. The mean tumor sizes in the LDLT and DDLT groups were 3.8 ± 1.9 cm and 4.1 ± 2.3 cm, respectively. The proportions of patients who met the Milan criteria and of patients staged by the Pittsburgh system who did not meet the Milan criteria were exactly matched; 60% of the entire cohort of patients with hepatic carcinoma met the Milan criteria, and 10% of the enrolled patients fell within stage Ⅳ according to the Pittsburgh staging system [17]. Postoperative complications Details of all perioperative complications are listed in Table 4. Multiple complications were possible in a single patient, so the sum of the individual complications was not equal to the total number of patients with complications. Twenty-one patients (52.5%) in the LDLT group and 46 patients (57.5%) in the DDLT group experienced perioperative complications. No significant difference was observed between the 2 groups in terms of overall number of patients with complications, whereas a significantly higher incidence of grade Ⅱ complications was noted in the DDLT group than in the LDLT group, and postoperative bacterial infection accounted for the majority of grade Ⅱ complications. The 3 most common complications during the initial hospital stay for LT were postoperative bacterial infection, intra-abdominal 4396 April 21, 2014 Volume 20 Issue 15

298 Wan P et al. Liver transplantation for hepatic carcinoma Table 4 Perioperative complications (modified Clavien-Dindo classification) n (%) Grade LDLT ( n = 40) DDLT (n = 80) P value Grade Ⅰ 3 (7.5) 5 (6.3) > Wound infection 3 4 Mental symptom 0 1 Grade Ⅱ 18 (45.0) 52 (65.0) Postoperative bacterial infection Virus infection 2 2 Bile leak 2 0 Hypertension 1 2 Mental symptom 1 4 Acute rejection 0 5 Peripheral nerve injury 0 1 Intra-abdominal bleeding 1 2 Subdural hematoma 0 1 Grade Ⅲa 3 (7.5) Biliary stricture 1 0 Hepatic artery thrombosis 1 0 Wound dehiscence 1 0 Grade Ⅲb 2 (5.0) 10 (12.5) Intra-abdominal bleeding 0 5 Bile leak 2 1 Wound dehiscence 0 2 Biliary stricture 0 1 Hepatic artery thrombosis 0 1 Grade Ⅳa 0 2 (2.5) Renal insufficiency 0 2 Grade Ⅳb 1 (2.5) 1 (1.3) > MODS 1 1 Grade Ⅴ 1 (2.5) 4 (5.0) Death 1 4 Overall 21 (52.5) 46 (57.5) LDLT: Living-donor liver transplantation; DDLT: Deceased-donor liver transplantation; MODS: Multiple organ dysfunction syndrome. Table 5 Overall biliary complications n (%) Biliary complications LDLT (n = 40) DDLT (n = 80) P value Perioperative period 5 (12.5) 2 (2.5) Bile leak 4 1 Biliary stricture 1 1 Long-term follow-up period 6 (15.0) 4 (5.0) Biliary stricture 6 4 Overall 11 (27.5) 6 (7.5) LDLT: Living-donor liver transplantation; DDLT: Deceased-donor liver transplantation. bleeding and biliary complications. Most intra-abdominal bleeding (5 out of 8) and biliary complications (5 out of 7) were solved through reoperation or biliary stent placement by ERCP. It is worth highlighting here that during the perioperative period, the LDLT group had more biliary complications (12.5% vs 2.5%, P = 0.073), while postoperative bacterial infection (27.5% vs 43.8%, P = 0.084) and intraabdominal bleeding (2.5% vs 8.8%, P = 0.365) seemed to be more common in the DDLT group. However, only a slight difference existed in the 3 kinds of complications between the two groups. In the long-term followup period after the first hospital stay for LT, 6 patients Table 6 Survival outcomes n (%) Variable in the LDLT group (15.0%) and 4 patients in the DDLT group (5.0%) experienced biliary stricture, so the LDLT group had a higher proportion of patients suffering biliary stricture (P = 0.129). Consequently, total biliary complications in the LDLT group reached a rate of 27.5%, which was significantly higher than that in the DDLT group (27.5% vs 7.5%, P = 0.003) (Table 5). Survival and recurrence after LT The median follow-up period of this cohort of patients was 50 mo (range, 1-76 mo) from the surgery day. Table 6 shows the details of survival outcomes between the two groups. With a median follow-up period of 57 mo (range, 1-75 mo) in the LDLT group and 48 mo (range, 1-76 mo) in the DDLT group, the two groups were comparable in the follow-up period (P = 0.236). The 1- and 5-year OS rates in the two groups were, respectively, 89.8% vs 84.9% and 74.1% vs 66.6% (Figure 1A, P = 0.372). Moreover, the RFS rate also did not differ between the two groups. The 1- and 5-year RFS rates were 81.2% vs 80.3% and 72.9% vs 70.9% in the LDLT vs DDLT group, respectively (Figure 1B, P = 0.749). In the LDLT group, recurrence occurred in 10 patients (25.0%) and caused 5 deaths (12.5%). In contrast, 22 recurrences (27.5%) occurred and 16 patients (20%) died of recurrence in the DDLT group. Three patients with relapse in the LDLT group (7.5%) and 3 patients with that in the DDLT group (3.8%) were still alive by the end of follow-up. In patients treated with LDLT, 3 deaths were caused by biliary complications and 2 deaths were caused by severe bacterial infection. On the other hand, 10 deaths from non-recurrent causes in the DDLT group included the following causes: severe bacterial infection (n = 4), cerebral hemorrhage (n = 2), biliary complication (n = 1), gastrointestinal tract hemorrhage (n = 1), severe coagulation disorder (n = 1) and hemothorax after thoracentesis (n = 1). DISCUSSION LDLT (n = 40) DDLT (n = 80) P value Survival status Alive without relapse 27 (67.5) 51 (63.8) Alive with disease 3 (7.5) 3 (3.8) Died of relapse 5 (12.5) 16 (20.0) Died of other causes 5 (12.5) 10 (12.5) Overall survival yr 89.8% 84.9% 5-yr 74.1% 66.6% Relapse-free survival yr 81.2% 80.3% 5-yr 72.9% 70.9% LDLT: Living-donor liver transplantation; DDLT: Deceased-donor liver transplantation. In recent decades, more and more patients with endstage liver diseases have been saved thanks to the rapid 4397 April 21, 2014 Volume 20 Issue 15

299 Wan P et al. Liver transplantation for hepatic carcinoma A 100 B 100 Overall survival (%) LDLT DDLT Relapse-free survival (%) LDLT DDLT Months after surgery Months after surgery Figure 1 Long-term survival outcomes. A: Overall survival rate (P = 0.372); B: Relapse-free survival rate (P = 0.749). LDLT: Living-donor liver transplantation; DDLT: Deceased-donor liver transplantation. progress in LT technology. Mazzaferro et al [5] put forward the Milan criteria for LT in HCC patients in Nevertheless, patients chance of cure has been restricted by such a strict indication, and whether the criteria could be expanded to enable more patients to qualify as transplant candidates has been a moot point. The EASL-EORTC clinical practice guidelines have mentioned that the criteria of up-to-seven in patients without microvascular invasion could be considered for LT for HCC [1,20]. Additionally, extended indications for LT in patients with hepatic carcinoma having similar survival outcomes with the Milan criteria have been reported by some transplant centers [21-24]. Theoretically speaking, LDLT calls for preservation of the inferior vena cava and a long internal structure of the hepatoduodenal ligament, which seems to render it less radical and thus less effective than DDLT. Moreover, it may expose the living donors to the risk of surgery. Therefore, most reported expanded indications are currently based on DDLT. However, several studies from Japan have shown encouraging outcomes of extended criteria for LDLT [25-27]. In the current study, tumors were staged according to the intraoperative findings and pathological outcomes of the resected specimens. Patients with tumor invasion were confirmed after LT. The Pittsburgh staging system seemed to have a direct correlation with the tumor-free survival rate based on the clinical data in our medical center, so we used it to match the patients of the two groups who did not meet the Milan criteria. Although 40% of the enrolled patients did not meet the Milan criteria, the OS and RFS turned out to be favorable on the whole. Organs from donation after cardiac death (DCD) cannot meet the needs of LT. Consequently, a large number of patients die of a shortage of liver donors every year, making living donors another important source of organs. However, animal experiments have suggested that the regenerating liver might have a potential effect on the growth of HCC [28,29], and LDLT has been criticized for its higher HCC recurrence rate than DDLT. Based on our experience, the long-term follow-up results do not support such a conclusion. Therefore, we carried out this multivariate case-matched comparison to maximize the comparability of the LDLT and DDLT groups. Our results show similar overall and relapse-free survival rates between the two groups, and LDLT conferred a 7.5% survival benefit compared with DDLT, so LDLT did not compromise the survival of patients or increase tumor recurrence. In addition, for emergency patients with fulminant hepatic failure, LDLT is often an optimal choice and can provide a timely graft to save their lives. Patients with HCC, now widely accepted as legitimate transplant candidates, require special consideration to achieve timely transplantation. A study by Mizuno et al [30] showed that the median period between the registration for LDLT and the occurrence of extrahepatic metastasis, macroscopic vascular invasion or rupture of HCC was 12.2 mo (range, mo), indicating that the waiting period suitable for a liver transplant is quite limited for HCC patients. Many patients with hepatic carcinoma lose their chance of LT or die of tumor progression during the waiting period, whereas the waiting period has greatly shortened and the survival has greatly improved with the advent of LDLT [31]. In the present study, the median preoperative waiting time for LT in the LDLT group was 14 d (range, 5-63 d), which was significantly less than the 45 d (range, d) in the DDLT group. However, LDLT can only be feasible when a suitable volunteer is available, even though it is in theory a good alternative to DDLT. In Hong Kong, the policy of a 6-mo wait for cadaveric liver allocation benefits the HCC patients who have practically no chance of undergoing LDLT. These modifications of the cadaveric liver allocation policy could result in transplants for as many HCC patients as possible but would not deprive non-hcc patients of a fair chance of undergoing LT [32]. Another important criticism of LDLT is that biliary complications in patients who undergo LDLT are more common than in DDLT patients [11,12]. Our data showed a similar pattern that LDLT was associated with a significantly increased rate of biliary complications compared with DDLT. In the 5 patients of the LDLT group who died of non-recurrent causes, 3 deaths (60%) were caused by biliary complications, which may be a weak point of LDLT. There were 5 bile leak cases in this study, all of them diagnosed within the first month, of whom 2 underwent reexploration. Continuous abdominal drainage was the most important treatment modality for bile leaks April 21, 2014 Volume 20 Issue 15

300 Wan P et al. Liver transplantation for hepatic carcinoma Unlike bile leaks, the stenoses (10 out of 12 cases) were mostly detected in the outpatient clinic after worsening of liver function, symptoms of cholangitis, or intrahepatic duct dilatation on computed tomography scans or magnetic resonance cholangiopancreatography. However, they could be successfully controlled by placement of a biliary stent through ERCP in most cases. By and large, the LDLT and DDLT groups were comparable in the total number of patients with perioperative complications, but it should be noted that complications occurring in the DDLT group seemed to be more serious than those in the LDLT group. For example, in the DDLT group 10 patients (12.5%) underwent surgical or endoscopic interventions under general anesthesia due to grade Ⅲb complications, and 7 patients (8.8%) suffered complications higher than grade Ⅲb. Our data of perioperative complications and followup results revealed that DDLT tended to result in more bleedings after surgery, including intra-abdominal bleeding, cerebral hemorrhage and gastrointestinal tract hemorrhage. In our comparison, 5 intra-abdominal bleedings needed a second operation, and another 4 patients died of hemorrhage in the DDLT group. A slight but not significant difference was observed in these findings, which prompts the question whether a difference exists in the coagulation function among liver grafts from different sources. Further large-scale, prospective, randomized trials are needed to address the question. Postoperative bacterial infection is always the most common complication in the field of LT. In our comparative study the DDLT group showed a significantly higher proportion of patients with grade Ⅱ complications, which were mainly composed of postoperative bacterial infections. At times, bacterial infections after LT are not accompanied by fever, so identifying and preventing the development of infections after LT is particularly important. In conclusion, grade Ⅱ complications occurred more frequently in the DDLT group than in the LDLT group, but the overall proportions of patients with perioperative complications were comparable between the two groups. Although LDLT led to a higher rate of biliary complications, it did not show any inferiority in longterm OS or RFS. Large-scale, prospective, randomized controlled trials are needed to reveal the inherent characteristics of complications between the two groups. COMMENTS Background Liver cancer is the sixth most common cancer and the third cause of cancerrelated death, accounting for 7% of all cancers worldwide. Liver transplantation (LT) is the best option for patients with a hepatic tumor 5 cm in diameter and concurrent decompensated cirrhosis. Living-donor liver transplantation (LDLT) is currently an effective alternative to alleviate the organ shortage, but worries exist that LDLT may be associated with higher rates of tumor recurrence and biliary complications than deceased-donor liver transplantation (DDLT). Whether LDLT can have more benefits on perioperative and long-term outcomes than DDLT is still disputable. Research frontiers Due to a severe shortage of cadaveric donor organs, LDLT is currently an effective alternative to alleviate organ shortage, but it has been criticized for its underlying higher cancer recurrence rate than DDLT. As for the surgical selection for patients with hepatic carcinoma, the research hotspot is whether LDLT will result in more postoperative complications or tumor recurrences than DDLT. Innovations and breakthroughs It remains controversial whether LDLT can achieve similar or better long-term survival than DDLT. In the present study, the authors used a multivariate casematched method to compare the results of LDLT and DDLT in patients with hepatic carcinoma. In spite of a higher rate of biliary complications in the LDLT group, DDLT was associated with more grade Ⅱ complications, while the overall perioperative complication rates were similar between the two groups. Furthermore, compared with the DDLT group, the LDLT group had comparable long-term follow-up survival. The present data do not suggest that LDLT results in more tumor recurrences than DDLT. Applications The study results suggest that LDLT not only could greatly shorten the waiting period of patients with hepatic carcinoma but also would not compromise longterm survival or increase tumor recurrence, so LDLT is a favorable alternative to solve the problem of organ shortage. Terminology Liver transplantation is a surgical technique that removes a diseased liver and replaces it with a healthy donor liver in patients with end-stage liver disease. Case-matched comparison is a comparative study between groups in which each case is matched by one or more comparable cases (1:1 or 1:N) in terms of several measurable parameters. Peer review The study used a special statistical method to compare the surgical outcomes of LDLT and DDLT in patients with hepatic carcinoma, which is a good way to establish greater parity between the two surgery groups. The results suggested that LDLT could have similar survival outcomes as DDLT. This study has significant merits. REFERENCES 1 European Association For The Study Of The Liver, European Organisation For Research And Treatment Of Cancer. EASL- EORTC clinical practice guidelines: management of hepatocellular carcinoma. J Hepatol 2012; 56: [PMID: DOI: /j.jhep ] 2 El-Serag HB, Mason AC. 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Expansion of selection criteria for patients with hepatocellular carcinoma in living donor liver transplantation. Liver Transpl 2007; 13: [PMID: DOI: /lt.21281] 26 Soejima Y, Taketomi A, Yoshizumi T, Uchiyama H, Aishima S, Terashi T, Shimada M, Maehara Y. Extended indication for living donor liver transplantation in patients with hepatocellular carcinoma. Transplantation 2007; 83: [PMID: DOI: /01.tp ec] 27 Sugawara Y, Tamura S, Makuuchi M. Living donor liver transplantation for hepatocellular carcinoma: Tokyo University series. Dig Dis 2007; 25: [PMID: DOI: / ] 28 Picardo A, Karpoff HM, Ng B, Lee J, Brennan MF, Fong Y. Partial hepatectomy accelerates local tumor growth: potential roles of local cytokine activation. Surgery 1998; 124: [PMID: DOI: /S (98) ] 29 Shi JH, Huitfeldt HS, Suo ZH, Line PD. Growth of hepatocellular carcinoma in the regenerating liver. Liver Transpl 2011; 17: [PMID: DOI: /lt.22325] 30 Mizuno S, Yokoi H, Shiraki K, Usui M, Sakurai H, Tabata M, Sugimoto K, Takei Y, Yamakado K, Takeda K, Uemoto S, Isaji S. Prospective study on the outcome of patients with hepatocellular carcinoma registered for living donor liver transplantation: how long can they wait? Transplantation 2010; 89: [PMID: DOI: /TP.0b013e3181cd4ae9] 31 Berg CL, Gillespie BW, Merion RM, Brown RS, Abecassis MM, Trotter JF, Fisher RA, Freise CE, Ghobrial RM, Shaked A, Fair JH, Everhart JE. Improvement in survival associated with adult-to-adult living donor liver transplantation. Gastroenterology 2007; 133: [PMID: DOI: /j.gastro ] 32 Chan SC, Sharr WW, Chok KS, Chan AC, Lo CM. Wait and transplant for stage 2 hepatocellular carcinoma with deceased-donor liver grafts. Transplantation 2013; 96: [PMID: DOI: /TP.0b013e3182a339a7] P- Reviewer: Mizuno S S- Editor: Zhai HH L- Editor: Wang TQ E- Editor: Liu XM 4400 April 21, 2014 Volume 20 Issue 15

302 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH REPORT Clinicopathological features and trend changes of gastric carcinoma in Southern China Jian-Jun Peng, Ping Xiao, Jian-Bo Xu, Wu Song, Bing Liao, Yu-Long He Jian-Jun Peng, Jian-Bo Xu, Wu Song, Yu-Long He, Department of Gastrointestinal Surgery, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 51080, Guangdong Province, China Jian-Jun Peng, Jian-Bo Xu, Wu Song, Yu-Long He, Gastric cancer center, Sun Yat-sen University, Guangzhou 51080, Guangdong Province, China Ping Xiao, Bing Liao, Department of Pathology, the First Affiliated Hospital, Sun Yat-sen University, Guangzhou 51080, Guangdong Province, China Author contributions: Peng JJ designed the study and wrote the manuscript; Xu JB, Song W and Liao B contributed to data collection and selection of patients; Xiao P contributed to data analysis; He YL contributed to supervision and revision; all authors approved the version to be published. Correspondence to: Yu-Long He, Professor, Department of Gastrointestinal Surgery, the First Affiliated Hospital, Sun Yatsen University, No.58, Zhongshan 2 nd Road, Guangzhou 51080, Guangdong Province, China. yhl@medmail.com.cn Telephone: Fax: Received: October 30, 2013 Revised: February 7, 2014 Accepted: March 5, 2014 Published online: April 21, 2014 Abstract AIM: To investigate the clinicopathological features of gastric carcinoma in southern China and disease trends changes over the last 18 years. METHODS: We designed a retrospective study in the Department of Gastrointestinal Surgery, the first affiliated hospital, Sun Yat-sen University. A total of 2100 adult patients with definitely diagnosed, histologically proven gastric carcinomas treated with radical gastrectomy from 1994 to 2013 were examined retrospectively. In all cases patient age, gender, tumor location, Borrmann type, histopathological type and grade, and ptnm stage were identified and recorded. The information was obtained from hospital records. The data were analyzed with Stata12.0 software. RESULTS: In this study, the mean age of patients was 57 years with a range from years. A higher incidence was found in patients over 60 years of age. In the study population, 67.38% of patients were male and 32.62% were female. Women had a higher disease incidence than men in patients less than 40 years of age (P < 0.001). No obvious change of patient age and gender was observed in the last 18 years. The rates of disease by location were the following: antrum (44.57%), followed by fundus/ body (24.95%) and cardia/gastroesophageal junction (23.00%). The mean tumor diameter was 5.57 cm, and advanced gross type Borrmann Ⅲ was most common. Most patients were at advanced stages when first diagnosed, and patients with early stage disease were relatively rare. More early stage patients were detected in recent years, especially after 2000 (P < 0.001). Gastric carcinoma has different features in young and old patients. The young patients had the following features: more frequently female, tumors in the antrum, larger tumor size, poorly differentiated carcinoma, high rate of metastasis to other sites and advanced stages (P < 0.05). CONCLUSION: In southern China, gastric carcinoma was more frequent in old men and young women. Young and old patients should be treated differently for having different features Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Gastric carcinoma; Retrospective study; Clinicopathological features; Southern China; Youth Core tip: Gastric carcinoma is one of the most common malignant tumors in the digestive tract and is the second leading cause of cancer-related death. This study retrospectively examined 2100 adult patients from southern China. Gastric carcinomas were found frequently in old men and young women. No obvious changes of patient age and gender were observed in 4401 April 21, 2014 Volume 20 Issue 15

303 Peng JJ et al. Features of GC in Southern China the last 18 years. Although most patients were at advanced stages when first diagnosed, early detection rates have increased. The young and the old patients differed in gender composition, tumor size, predilection site, pathological type and prognosis. Therefore, these patients should be treated differently in the clinic. Peng JJ, Xiao P, Xu JB, Song W, Liao B, He YL. Clinicopathological features and trend changes of gastric carcinoma in southern china. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: INTRODUCTION Gastric carcinoma (GC) is one of the most common malignant tumors of the digestive tract and is the second leading cause of cancer related death [1]. The geographical distribution of GC is characterized by wide international variations. More than 70% of cases occur in developing countries, and half of the world s cases occur in Eastern Asia [2]. China has a high incidence of gastric cancer, and it accounts for over 40% of all new gastric cancer cases [3]. This study retrospectively examined 2100 adult patients from southern China and enrolled in the first affiliated hospital, Sun Yet-sen University from We have summarized the clinicopathological features of GC in Southern China and evaluated the changes over the last 18 years. MATERIALS AND METHODS Data collection The data were collected from 2100 adult patients with definitely diagnosed, histologically proven gastric carcinomas treated at the Department of Gastrointestinal Surgery, the First Affiliated Hospital, Sun Yet-sen University between 1994 and The data included the following information: gender, age, tumor location, Borrmann type, pathological type (based on the WHO classification) [4], and ptnm stage (based on the criterion of the American Joint Committee on Cancer, AJCC) [5]. All information was obtained retrospectively from hospital records (including pathologic and operative reports). Statistical analysis The data were examined and confirmed by a controller after entering and were then analyzed by using Stata12.0 software. The χ 2 test, Fisher s exact test, ANOVA test and Student s t-test were used to analyze data. A P < 0.05 was considered statistically significant. RESULTS Age and gender This study evaluated 2100 cases with GC identified from 1994 to Within the study population, 67.38% of Table 1 Age and sex n (%) Age Male Female Total P value Age Group (4.33) 122 (5.81) 213 (10.14) (9.33) 136 (6.48) 332 (15.81) (19.62) 181 (8.62) 593 (28.24) (34.10) 246 (11.71) 962 (45.81) < Number of periods (13.24) 106 (5.05) 384 (18.29) (19.38) 216 (10.29) 623 (29.67) (34.76) 363 (17.29) 1093 (52.05) Total 1415 (67.38) 685 (32.62) 2100 (100) Mean ± SD age (yr) ± ± ± Mean age of periods ± ± ± ± ± ± ± ± ± patients were male and 32.62% were female. The patient ages ranged from 19 to 89 years, and the mean age was ± years. The data indicated that 45.81% of cases were diagnosed in patients over 60 years of age, and 25.95% of cases occurred in patients under the age of 50. Women had a higher disease incidence than men in cases under 40 years old. There were significantly more male patients over age 40. We also analyzed the changes in patient age and gender for GC over the last 18 years. The results indicated there was no change of mean age in the population or in the frequency of male and female patients. The incidence of female cases showed a trend of increasing frequency after However, this trend was not statistically significant. These data are listed in Table 1. Morphologic features Table 2 summarizes the morphological features of GC. The mean tumor diameter was 5.57 ± 3.43 cm. Tumors with diameters between 2.1 and 5 cm were most common, and accounted for nearly half of all cases. The tumor sites was most common in the antrum (44.57%), followed by fundus/body (24.95%) and cardia/gastroesophageal junction (23.00%) (GEJ). As for macroscopic type, Borrmann Ⅲ was more common (59.62%) than Borrmann Ⅱ. WHO pathologic classifications showed tubular/papillary adenocarcinoma was the most common histopathological type (79.57%). Most of the tumors were poorly differentiated. ptnm stage Tumor stages and the changes over the last 18 years are illustrated in Table 3. Most patients were at advanced stages (ptnm stage Ⅱ, Ⅲ and Ⅳ), when they were first diagnosed. Only a few tumors were detected at early stages (ptnm stage 0 and Ⅰ). However, the detection rates of early patients substantially improved after The results are listed in Table 3. Clinicopathological features of different ages Table 4 shows that patients of different ages had differ April 21, 2014 Volume 20 Issue 15

304 Peng JJ et al. Features of GC in Southern China Table 2 Morphologic features n (%) Size (cm) Mean ± SD 5.57 ± (12.81) (46.14) (34.43) > (6.62) Location Cardia/EGJ 483 (23.00) Fundus/body 524 (24.95) Antrum 936 (44.57) Diffuse 75 (3.57) Remnant 74 (3.52) Multiple 8 (0.38) Borrmann type Ⅰ 97 (4.62) Ⅱ 510 (24.29) Ⅲ 1252 (59.62) Ⅳ 241 (11.48) Pathologic type Tubular/papillary adenocarcinoma 1617 (79.57) Mucinous carcinoma 127 (6.05) Signet cell carcinoma 255 (12.14) Neuroendocrine neoplasm 9 (0.43) Squamous carcinoma 32 (1.52) Others 6 (0.29) Differentiation Well 87 (4.14) Moderate 613 (29.19) Poor 1400 (66.67) Table 3 ptnm stage and time periods Stage Total P value (0.57%) Ⅰa (8.14%) Ⅰb (7.57%) Ⅱa (12.24%) Ⅱb (18.38%) Ⅲa (14.24%) Ⅲb (10.05%) Ⅲc (5.62%) Ⅳ (23.19%) Total < ent clinicopathological features. With respect to gross features, patients older than 50 years of age had a smaller mean tumor size and fewer patients had tumors larger than 10 cm. The frequency of Borrmann Ⅳ (diffuse gastric cancer) tumors decreased significantly and the incidence of cardia/gej carcinoma increased significantly with age. Microscopically, signet ring cell carcinoma decreased significantly after 60 years of age. Increased age was associated with fewer poorly differentiated carcinomas and significantly more well-differentiated carcinomas. The proportion of ptnm stage Ⅳ and distant metastasis gradually declined with age. All of these results were statistically significant. DISCUSSION Gastric carcinoma is one of the most prevalent malignant tumors in China. According to the latest GLOBO- CAN statistics on Chinese gastric cancer for 2008, the total incidence rate in Chinese patients has increased from 42% to 47% since Thus, gastric cancer now accounts for approximately 1/5 of all cancer deaths [6,7]. We investigated the clinicopathological features of GC in southern China. Consistent with previous research, this study showed GC was prevalent in men. The overall male:female ratio was 2.07:1. The average patient age is 57 years, and the highest morbidity occurs in patients over 60 years [8,9]. A major cause of GC in middleaged and older people may be the long-term repeated action of carcinogenic substances that cause cellular damage and malignant transformation. In addition, a declining immune system and a reduced ability to eliminate mutant cells may also be involved in disease development in the elderly. We analyzed the age changes of the past 18 years, but found no obvious differences of mean age. This result differs from reports in South Korea and Japan, which showed that GC patients are getting older and the proportion of elderly patients continues to increase [10,11]. Additionally, the mean age of GC patients in China is younger than that reported in South Korea, Japan and the West. In the study, women had a higher incidence of GC than men before 40 years of age, while GC in male patients was more frequent than in females after age 40. This result may be related with sex hormone levels. Both estrogen and androgen may be involved in disease pathogenesis. Studies have shown high estrogen receptor expression in many GC patients, including male patients [12]. Estrogen levels are especially high in women with diffusely infiltrative GC and poorly differentiated adenocarcinoma [13]. Previously published studies have shown the influences of estrogens on GC are controversial. Whether estrogen increases or decreases the risk of GC is still unclear [14]. The androgen receptor was also found in GC, and it was an independent unfavorable prognostic factor [15,16]. Independent of sex hormones, male incidence increases significantly after 40 years of age. This result may also be related to the use of cigarettes and alcohol, irregular life, heavy social pressure and other factors [17]. Chung et al [18] thought hormonal factors were more commonly associated with females, whereas environmental factors were more commonly associated with males in young GC patients. Although there was no significant difference over the last 18 years for incidence of disease by gender, there is a trend of increasing female incidence after This research showed the mean tumor diameter was 5.57 cm. Most tumor diameters were between 2.1 and 5 cm. Only 12.81% tumors were less than 2.0 cm. Advanced gross type Borrmann Ⅲ was the most common tumor type. Moreover, most patients were at advanced stage (ptnm stage Ⅱ, Ⅲ and Ⅳ) when first diagnosed, and cases of early stage disease (ptnm stage 0 and Ⅰ) were relatively rare. According to our results, the mean tumor size is larger and the detection rates of early gastric carcinoma are lower in China than in Japan and South Korea [10]. Fortunately, the detection rates of early tumors have increased, especially after This may be related to gradually increased use of gastroscopy in 4403 April 21, 2014 Volume 20 Issue 15

305 Peng JJ et al. Features of GC in Southern China Table 4 Clinicopathological features of different ages n (%) 40 yr yr yr 60 yr Size (cm) Mean ± SD 5.83 ± ± ± ± 3.17 < (1.62) 44 (2.10) 80 (3.81) 111 (5.29) (4.19) 140 (6.67) 290 (13.81) 451 (21.48) (3.29) 119 (5.67) 183 (8.71) 352 (16.76) > (1.05) 29 (1.38) 40 (1.90) 48 (2.29) Location Cardia/GEJ 21 (1.00) 47 (2.24) 146 (6.95) 269 (12.81) Fundus/body 77 (3.67) 101 (4.81) 148 (7.05) 198 (9.43) Antrum 103 (4.9) 162 (7.71) 258 (12.29) 413 (19.67) Diffuse 9 (0.43) 18 (0.86) 25 (1.19) 23 (1.10) Remnant 2 (0.10) 3 (0.14) 15 (0.71) 54 (2.57) Multiple 1 (0.05) 1 (0.05) 1 (0.05) 5 (0.24) < Borrmann type Ⅰ 10 (0.48) 8 (0.38) 37 (1.76) 59 (2.81) Ⅱ 48 (2.29) 84 (4.00) 146 (6.95) 229 (10.90) Ⅲ 116 (5.52) 179 (8.52) 335 (15.95) 605 (27.86) Ⅳ 39 (1.86) 61 (2.90) 75 (3.57) 89 (4.24) < Pathologic type Tubular or papillary adenocarcinoma 164 (7.81) 265 (12.62) 456 (21.71) 786 (37.43) Mucinous carcinoma 9 (0.43) 12 (0.57) 36 (1.71) 70 (3.33) Signet cell carcinoma 37 (1.76) 48 (2.29) 91 (4.33) 79 (3.76) Neuroendocrine neoplasm 0 (0.00) 2 (0.10) 2 (0.10) 5 (0.24) Squamous carcinoma 3 (0.14) 5 (0.24) 6 (0.29) 18 (0.86) Others 0 (0.00) 0 (0.00) 2 (0.10) 4 (0.19) Differentiation Well 5 (0.24) 8 (0.38) 23 (1.10) 51 (2.43) Moderate 15 (0.71) 66 (3.14) 169 (8.05) 363 (17.29) Poor 193 (9.19) 258 (12.29) 401 (19.10) 548 (26.10) < ptnm stage 0 1 (0.05) 1 (0.05) 1 (0.05) 9 (0.43) Ⅰa 21 (1.00) 27 (1.29) 48 (2.29) 75 (3.57) Ⅰb 14 (0.67) 31 (1.48) 56 (2.67) 58 (2.76) Ⅱa 25 (1.19) 33 (1.57) 82 (3.90) 117 (5.57) Ⅱb 29 (1.38) 53 (2.52) 99 (4.71) 205 (9.76) Ⅲa 29 (1.38) 35 (1.67) 80 (3.81) 155 (7.38) Ⅲb 18 (0.86) 36 (1.71) 65 ( 3.10) 92 (4.38) Ⅲc 11 (0.52) 23 (1.10) 29 (1.38) 55 (2.62) Ⅳ 65 (3.10) 93 (4.43) 133 (6.33) 196 (9.33) Metastasis M0 148 (7.05) 239 (11.38) 460 (21.90) 766 (36.48) M1 65 (3.10) 93 (4.43) 133 (6.33) 196 (9.33) China, particularly in the east and south of China where there are better economic and health conditions. Earlier diagnosis leads to better effect of treatment for gastric carcinoma. However, there is no nationwide screening program for GC. Therefore, the early detection of GC relies on opportunistic screening [19]. In our research, GC predominantly occurred in the gastric antrum, followed by body and cardia, which is similar with most statistics. Recently, scholars found the incidence of proximal GC was increasing. The Chinese and European/American literature report the incidence of distal GC was significantly declining and cardia/gej carcinoma was significantly increasing [20,21]. In the United States, male cardia carcinoma incidence is increasing annually, and it is becoming one of the fastest growing tumors. The incidence of this tumor type accounts for nearly half of GCs. Smoking, drinking, obesity, and esophageal reflux are risk factors of cardia carcinoma. Additionally, cardia carcinoma is related to diet, especially reduced cereal fiber intake and high protein, fat and salt consumption. These dietary problems may increase the incidence of cardia carcinoma. Increasing cereal fiber intake can reduce the risk of cardia carcinoma. In addition, the eradication of Helicobacter pylori (H. pylori) reduces distal GC and relatively increases the incidence of cardia carcinoma [22]. Reports from Turkey, Iran and Korea demonstrated the antrum is the most common site of GC [10,23,24]. The incidence of cardia has not increased and this result may be related to the local environment, habits and customs. In southern China the people have a bland diet, and less smoking and drinking are popular. We found young patients differed from the older patients not only in the sex ratio but also in the morphologic features and clinical stages. In the young, the tumors were larger and the proportion of Borrmann Ⅳ (diffuse gastric cancer) was higher than in the older patients. The incidence of cardia/gej carcinoma increased significantly with age. Microscopically, poorly differentiated and signet ring type carcinoma decreased significantly after 60 years of age. Clinically, the proportion of ptnm stage Ⅳ and distant metastasis gradually declined with age. The above data suggest that GC in young adults has more aggressive infiltration ability, higher malignancy, faster progress, and worse prognosis than GC in older patients. Although the predominant tumor site is the antrum in China, we found the risk of occurrence of cardia/ GEJ carcinoma significantly increased in patients more than 50 years old. The following may be the possible reasons: (1) the degeneration of esophageal smooth 4404 April 21, 2014 Volume 20 Issue 15

306 Peng JJ et al. Features of GC in Southern China muscle in older people, the lower esophageal sphincter muscle tension, disorders of nervous regulation mechanism are commonly seen simultaneously, which causes gastroesophageal reflux and repeated reflux results in inflammatory injury, repair and hyperplasia, and finally cancerous changes of the membrane of cardia area; (2) the fundic glands atrophy, the junction of fundus and body moves up in the old, which results in weakening of the mucosal barrier; and (3) as the patients age, the gastric mucosa epithelium degenerates and intestinal metaplasia develops to the fundus from the antrum [25]. The difference of predilection site between the young and the old may mainly relate to the above, but the specific mechanism is still unclear. In southern China, GC is predominant in old men and young women and is mostly located at the gastric antrum. Although most patients have advanced stage disease at diagnosis, more patients are being detected at early stages. The detection and treatment of early GC needs further improvement. Patients of different ages have different clinicopathological features. Young and old patients differ in the gender composition, predilection site, pathological type and disease prognosis. Thus, these diseases should be treated differently in clinical settings. COMMENTS Background Despite the decreasing global incidence, gastric carcinoma remains one of the most common forms of malignancy around the world. The geographical distribution of gastric carcinoma is characterized by wide international variations. According to WHO data, more than 70% of cases occur in developing countries and half of the world s cases occur in Eastern Asia. China has highest rates of morbidity and mortality of gastric cancer. Research frontiers The available data in the literature regarding gastric carcinoma in Southern China are limited. Therefore, the aim was to investigate the clinicopathological features of gastric carcinoma in Southern China and changes in trends over the last 18 years. Innovations and breakthroughs In the present study, the authors found in Southern China gastric carcinoma had a predilection in old men and young women. There were no changes in patient age and gender observed over the last 18 years. Most patients were diagnosed at advanced stages and early stage patients were relatively rare. More patients with early disease have been diagnosed since Applications The study results suggest early detection rates of gastric carcinoma are increasing, but still lag behind developed countries and need further improvement. The young and the old patients had different clinicopathologic features and should be treated differently in the clinic. Terminology Southern China is one of the seven major Chinese geographical divisions and is located in China s southernmost region. Southern China includes the provinces of Guangdong, Guangxi, Hainan, Hong Kong and Macao. Peer review This paper retrospectively studied 2100 adult gastric cancer patients from southern China from This is a well-written article with interesting data. REFERENCES 1 Jemal A, Center MM, DeSantis C, Ward EM. Global patterns of cancer incidence and mortality rates and trends. Cancer Epidemiol Biomarkers Prev 2010; 19: [PMID: DOI: / EPI ] 2 Jemal A, Bray F, Center MM, Ferlay J, Ward E, Forman D. Global cancer statistics. CA Cancer J Clin 2011; 61: [PMID: DOI: /caac.20107] 3 Bu ZD, Ji JF. A current view of gastric cancer in China. Transl Gastrointest Cancer 2013; 2 (S1): Bosman F, Cameiro F, Hmban R. WHO. Classification of tumours: of the digestive system, 2010; Rausei S, Dionigi G, Boni L. 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307 Peng JJ et al. Features of GC in Southern China Wu KC, Wu DC, Sollano J, Kachintorn U, Gotoda T, Lin JT, You WC, Ng EK, Sung JJ. Screening for gastric cancer in Asia: current evidence and practice. Lancet Oncol 2008; 9: [PMID: DOI: /S (08)70072-X] 20 Cai B, Chen X, Guo Y. Analysis of clinical features of 1517 cases of gastric cancer. Shanxi Yike Daxue Xuebao 2009; 40: Ireland AP, Clark GWB, DeMeester TR. Carcinoma of the cardia: Role of short-segment Barrett s esophagus and columnar metaplasia. Dis esophagus 1996; 9: González CA, Sala N, Rokkas T. Gastric cancer: epidemiologic aspects. Helicobacter 2013; 18 Suppl 1: [PMID: DOI: /hel.12082] 23 Norouzinia M, Asadzadeh H, Shalmani HM, Al Dulaimi D, Zali MR. Clinical and histological indicators of proximal and distal gastric cancer in eight provinces of Iran. Asian Pac J Cancer Prev 2012; 13: [PMID: ] 24 Selcukbiricik F, Tural D, Bilici A, Uzel EK, Ozguroglu M, Demirelli F, Buyukunal E, Serdengecti S. Clinicopathological features and localization of gastric cancers and their effects on survival in Turkey. Asian Pac J Cancer Prev 2013; 14: [PMID: ] 25 Li C, Zhang B, Wang F. Contrastive clinical analysis of 750 cases of gastric cancer in the young and the old. Zhongguo Shiyan Zhenduanxue 2009; 13: P- Reviewers: Fekete GL, Yang JH, Zhang YJ S- Editor: Qi Y L- Editor: O Neill M E- Editor: Liu XM 4406 April 21, 2014 Volume 20 Issue 15

308 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH BRIEF ARTICLE REPORT HBsAg levels in HBeAg-positive chronic hepatitis B patients with different immune conditions Yi-Min Zhang, Yi-Da Yang, Hong-Yu Jia, Lin-Yan Zeng, Wei Yu, Ning Zhou, Lan-Juan Li Yi-Min Zhang, Yi-Da Yang, Hong-Yu Jia, Lin-Yan Zeng, Wei Yu, Ning Zhou, Lan-Juan Li, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou , Zhejiang Province, China Author contributions: Zhang YM and Yang YD contributed equally to this paper; Li LJ designed the study; Jia HY, Zeng LY, Yu W and Zhou N collected the data; Zhang YM and Yang YD collected and analyzed the data and wrote the paper. Supported by China National Science and Technology Major Project, No. 2012ZX and No. 2013ZX ; the Chinese High Tech Research and Development (863) Program, No. 2011AA020104; Zhejiang CTM Science and Technology Project, No. 2011ZB061 Correspondence to: Lan-Juan Li, MD, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, School of Medicine, Zhejiang University, No. 79 Qingchun Road, Shangcheng District, Hangzhou , Zhejiang Province, China. ljli@zju.edu.cn Telephone: Fax: Received: July 6, 2013 Revised: December 31, 2013 Accepted: February 17, 2014 Published online: April 21, 2014 Abstract AIM: To investigate hepatitis B surface antigen (HBsAg) levels in patients with HBeAg-positive chronic hepatitis B (CHB) and different immune conditions. METHODS: HBeAg-positive CHB patients with different immune conditions were enrolled in this cross-sectional study. These patients were grouped according to the following criteria: immune-tolerant patients, IT group; patients with a mild immune response in the immune clearance phase, IC-Mild group; and patients with a dramatic immune response in the immune clearance phase and exhibiting acute on chronic liver failure (ACLF), ACLF group. All these patients had not previously received antiviral therapy and were enrolled at a pre-settled ratio of 2:2:1. Serum HBsAg levels and the correlation between serum HBsAg level and serum hepatitis B virus (HBV) DNA level were evaluated in these groups. RESULTS: In total, 180 HBeAg-positive CHB patients [IT group (n = 72), IC-Mild group (n = 72), and ACLF group (n = 36)] were enrolled in this study. The median serum HBsAg levels varied among the groups (P < 0.001): IT, 4.86 log10 IU/mL; IC-Mild, 3.97 log10 IU/mL; and ACLF, 3.57 log10 IU/mL. Serum HBsAg level showed a moderate positive correlation with serum HBV-DNA level in the IC-Mild group (r = 0.60, P < 0.001), but exhibited a weaker correlation in the IT (r = 0.52, P < 0.001) and ACLF groups (r = 0.51, P = 0.001). The ratio of HBsAg/HBV DNA did not differ significantly among the IT, IC-Mild, and ACLF groups (medians: 0.56, 0.55, and 0.56, respectively; P = 0.179). CONCLUSION: Serum HBsAg levels varied significantly in HBeAg-positive patients with different immune conditions. These findings may have important implications for understanding the immune clearance of HBV in HBeAg-positive CHB patients Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Chronic hepatitis B; Hepatitis B surface antigen; Hepatitis B e antigen; Immune tolerance; Immune clearance; Acute on chronic liver failure Core tip: This study showed that a dramatic immune response demonstrated that acute on chronic liver failure results in lower serum hepatitis B surface antigen levels compared with a mild immune response in patients in the immune clearance phase. These findings may have important implications for understanding the immune clearance of hepatitis B virus in these patients. Zhang YM, Yang YD, Jia HY, Zeng LY, Yu W, Zhou N, Li LJ. HBsAg levels in HBeAg-positive chronic hepatitis B patients 4407 April 21, 2014 Volume 20 Issue 15

309 Zhang YM et al. HBsAg levels in different HBeAg-positive patients with different immune conditions. World J Gastroenterol 2014; 20(15): Available from: URL: com/ /full/v20/i15/4407.htm DOI: org/ /wjg.v20.i INTRODUCTION Hepatitis B surface antigen (HBsAg) is considered a milestone in the research history of hepatitis B virus (HBV) and has been used for more than 40 years to confirm HBV infections [1]. With the second generation of standard HBsAg samples introduced by the World Health Organization, quantitative HBsAg tests have been performed [2,3]. Studies have shown the potential application of HBsAg quantification in predicting responses to interferonbased therapy and nucleos(t)ide therapy [4-7]. In addition, the quantified serum HBsAg level has been used as a potential surrogate or supplemental marker of HBV DNA to identify the phase of the natural history of the disease and to predict seroconversion in CHB [8,9]. In the HBeAg-positive condition, the natural course of CHB patients is divided into the immune tolerance and immune clearance phases. The serum HBsAg level is higher in the immune tolerance phase and declines in the immune clearance phase [10-12], indicating different immune conditions associated with various HBsAg levels. In addition to the different immune conditions between the immune tolerance and immune clearance phases, the degree of host immune response is different when immune clearance begins [13,14]. In general, patients show moderately increased alanine aminotransferase (ALT) levels without hepatic decompensation, indicating a mild immune response. However, when a dramatic immune response occurs in the immune clearance phase, liver function decompensates or deteriorates to acute on chronic liver failure (ACLF), which is usually described as acute hepatic injury manifested as jaundice and coagulopathy [15,16]. Although all patients in the IC phase have been evaluated as a whole, the serum HBsAg levels in these patients with different immune responses in the immune clearance phase have not been evaluated individually. Lee et al [17] showed that baseline serum HBsAg levels can be used as a prognostic factor of the anti-hbv treatment response in HBeAg-positive CHB patients during the immune clearance phase. Thus, studies should be conducted to investigate whether a dramatic immune response exhibiting ACLF will yield different HBsAg levels compared with a mild immune response in patients in the immune clearance phase, which may indicate antiviral therapy results. In addition, the HBsAg levels should be compared with those in immune-tolerant patients to determine whether different characteristics are observed when patients in the immune clearance phase are subdivided according to different immune clearance conditions. Therefore, three groups of HBeAg-positive CHB patients were enrolled in this study: immune-tolerant patients, IT group; patients with a mild immune response in the immune clearance phase, IC-Mild group; and patients with a dramatic immune response in the immune clearance phase and exhibiting ACLF, ACLF group. The HBsAg levels were compared among these three groups. In different phases of the HBV infection, the connection between HBsAg production and HBV replication is not stable [2]. This study also aimed to evaluate the correlation between serum HBsAg levels and serum HBV DNA levels among these three groups. MATERIALS AND METHODS Patients and study design Three groups of HBeAg-positive CHB patients with different immune conditions were recruited into this crosssectional study. Each group was classified as follows: IT group, high HBV DNA levels and ALT < upper limit of normal (ULN); IC-Mild group, increased HBV DNA levels, 2 ULN < ALT < 10 ULN, total bilirubin (TB) < 2 ULN (42 μmol/l), international normalized ratio (INR) (a laboratory test representing coagulopathy) < 1.27, and approximate prothrombin time activity percentage (PTA) > 60%; ACLF group, increased HBV DNA levels, ALT > 2 ULN, extreme fatigue with severe digestive symptoms, serum TB level > 171 μmol/l, INR > 1.50, and approximate PTA < 40%. Patient history showed that all patients had not received antiviral therapy and were HBsAg positive for longer than six months. The patients had no clinical or ultrasonographic findings of cirrhosis. The patients were recruited from the First Affiliated Hospital, College of Medicine, Zhejiang University with a pre-settled ratio of 2:2:1 in the IT, IC-Mild, and ACLF groups, respectively, from October 2011 to May The target number of patients in each group was estimated according to the serum HBsAg levels of the first 50 patients enrolled (data not shown). In this study, the ULN of ALT was 40 U/L, whereas the ULN of TB was 21 μmol/l. Patients in the IT group met the criteria of the IT phase according to the Asian-Pacific Consensus Statement on the Management of Chronic Hepatitis B: A 2008 Update [14]. Patients in the IC-Mild group represented patients without liver function decompensation in the IC phase, and this definition is consistent with the same consensus statement regarding CHB. Patients in the ACLF group met the criteria according to the ACLF consensus recommendations of the Asian Pacific Association for the Study of the Liver [15]. None of the enrolled patients suffered from coinfection with viruses, such as hepatitis A, hepatitis C, hepatitis D, hepatitis E, or human immunodeficiency viruses. Patients with cirrhosis, drug-induced liver injury, autoimmune hepatitis, alcoholic liver diseases, genetic and other liver diseases, chemo/immunotherapy, or cancer were excluded. All pediatric patients were also excluded April 21, 2014 Volume 20 Issue 15

310 Zhang YM et al. HBsAg levels in different HBeAg-positive patients Table 1 Baseline characteristics of three groups of hepatitis B e antigen -positive chronic hepatitis B patients Variables IT group (n = 72) IC-Mild group (n = 72) ACLF group (n = 36) Kruskal-Wallis test P value Age (yr) 25 (22-29) 30 (24-37) 34 (28-41) < Gender (male/female) 44/28 55/17 32/ HBV DNA (log10 IU/mL) 8.39 ( ) 7.58 ( ) 6.33 ( ) < HBsAg (log10 IU/mL) 4.86 ( ) 3.97 ( ) 3.57 ( ) < ALT (IU/mL) 25 (19-32) 220 ( ) 468 ( ) < TB (μmol/l) 11 (9-15) 16 (10-29) 314 ( ) < INR 1.01 ( ) 1.08 ( ) 2.24 ( ) < PLT (10 9 /L) 210 ( ) 172 ( ) 124 ( ) < Data are expressed as the median (interquartile range) or as a ratio. CHB: Chronic hepatitis B; IT: Immune-tolerant patients; IC-Mild: Patients with a mild immune response in the immune clearance phase; ACLF: Patients with a dramatic immune response in the immune clearance phase and exhibiting acute on chronic liver failure; ALT: Alanine aminotransferase; WBC: White blood cell; PLT: Platelet; TB: Total bilirubin; INR: International normalized ratio. For the IT and ACLF patients, serum HBsAg and HBV DNA levels were determined once at the time of enrollment. IC-Mild patients had a median follow-up of three weeks (minimum 2 wk, maximum 8 wk); serum HBsAg and HBV DNA levels were determined once at the time of enrollment and repeated at least once during the follow-up period. Serum samples for the HBsAg and HBV DNA tests were collected at the same time points during enrollment or follow-up. The patients who experienced liver function decompensation, but did not meet the criteria of the IC-Mild group were excluded. Serum HBsAg levels were compared among the three groups in this study. The correlation between serum HBsAg level and serum HBV DNA level was determined. This study was conducted according to the guidelines of the Declaration of Helsinki and was approved by the Review Board of the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University. Written informed consent was obtained from the patients or their relatives. Laboratory tests Biochemical parameters were analyzed by routine automated methods (Hitachi 7600, Japan). HBeAg was qualitatively analyzed, and HBsAg levels were quantitatively analyzed using the Abbott ARCHITECT assay (Abbott Diagnostics, Germany) according to the manufacturer s instructions. The dynamic range of HBsAg levels was IU/mL. The samples were diluted to 1:500 or 1:1000 using ARCHITECT HBsAg Manual Diluent (Abbott Diagnostics) if the HBsAg levels were greater than 250 IU/mL. Serum HBV DNA was quantified using a determination kit for HBV DNA (Life River, China) at a detection range of IU/mL; for the samples with > 10 8 IU/mL HBV DNA levels, the HBV DNA assay was repeated after dilution to 1:1000. Statistical analysis The data are presented as medians (interquartile range), except when stated otherwise. The Kruskal-Wallis test was performed for multivariate comparisons. The Mann- Whitney test and Fisher s exact test were performed for univariate comparisons, as appropriate. The Spearman rank test was conducted for correlation analyses. Multivariate linear regression with the HBsAg concentration as an independent factor was performed. Statistical calculations were performed using GraphPad Prism (Version 5.0, GraphPad Software, San Diego, United States). A P value of 0.05 (two-tailed) was considered significant. RESULTS Patient characteristics In total, 180 HBeAg-positive CHB patients were enrolled in this cross-sectional study (72 in the IT group, 72 in the IC-Mild group, and 36 in the ACLF group). A predominance of males (131/180, 72.8%) was observed in the study population. The patient characteristics, including age, gender, serum HBV DNA levels, serum HBsAg levels, ALT, TB, INR, and platelets, are described in detail in Table 1. All these parameters were significantly different among the IT, IC-Mild, and ACLF groups. Distribution of serum HBsAg levels The distribution of serum HBsAg levels in the three groups was evaluated as described in Figure 1. The median serum HBsAg levels in each group were as follows: IT group, 4.86 log10 IU/mL; IC-Mild group, 3.97 log10 IU/mL; and ACLF group, 3.57 log10 IU/mL. The serum HBsAg levels were significantly different in each group (IT vs IC-Mild, P < 0.001; IT vs ACLF, P < 0.001; IC- Mild vs ACLF, P = 0.001). Correlation between serum HBsAg levels and serum HBV DNA levels The HBsAg/HBV DNA ratio was evaluated (Figure 2). The median levels of the HBsAg/HBV DNA ratio in the IT, IC-Mild, and ACLF groups were 0.56, 0.55, and 0.56, respectively. The results showed no significant differences among these three groups (P = 0.179). The correlations between serum HBsAg levels and HBV DNA levels in each group are shown in Figure 3. A modest correlation was found between HBsAg and HBV DNA in the IC-Mild group (r = 0.60, P < 0.001). Serum HBsAg level was weakly correlated with HBV DNA in the IT group (r = 0.52, P < 0.001) and the ACLF group (r 4409 April 21, 2014 Volume 20 Issue 15

311 Zhang YM et al. HBsAg levels in different HBeAg-positive patients P < A 10 IT group 6 P < P = HBsAg (log10 IU/mL) 4 2 HBV DNA (log10 IU/mL) r = 0.52 P < Figure 1 Distribution of serum hepatitis B surface antigen levels in the three groups of hepatitis B e antigen-positive chronic hepatitis B patients. Median values are presented. There was a significant difference between these three groups (IT vs IC-Mild, P < 0.001; IC-Mild vs ACLF P = 0.001; IT vs ACLF, P < 0.001). CHB: Chronic hepatitis B; IT: Immune-tolerant patients; IC-Mild: Patients with a mild immune response in the immune clearance phase; ACLF: Patients with a dramatic immune response in the immune clearance phase and exhibiting acute on chronic liver failure; HBsAg: Hepatitis B surface antigen; HBV: Hepatitis B virus. HBsAg/HBV DNA (log10 IU/mL) = 0.51, P < 0.001) compared with the IC-Mild group. DISCUSSION IT IC-Mild ACLF P > 0.05 IT IC-Mild ACLF Figure 2 Ratio of hepatitis B surface antigen/hepatitis B virus DNA in the three groups of hepatitis B e antigen-positive chronic hepatitis B patients. Median values are presented. There was no significant difference among these three groups (P > 0.05). CHB: Chronic hepatitis B; IT: Immune-tolerant patients; IC-Mild: Patients with a mild immune response in the immune clearance phase; ACLF: Patients with a dramatic immune response in the immune clearance phase and exhibiting acute on chronic liver failure; HBsAg: Hepatitis B surface antigen; HBV: Hepatitis B virus. Several research programs have been established to investigate HBsAg levels in the natural history of CHB in Asian and European ethnicities [10-12]. With regard to HBeAg-positive status, a higher and more stable titer of serum HBsAg (median log10 IU/mL) is observed in the IT phase when the host s immune response B HBV DNA (log10 IU/mL) C HBV DNA (log10 IU/mL) HBsAg (log10 IU/mL) IC-Mild group r = 0.60 P < HBsAg (log10 IU/mL) ACLF group r = 0.51 P = HBsAg (log10 IU/mL) Figure 3 Correlation between serum hepatitis B surface antigen levels and hepatitis B virus DNA levels in the three groups of Hepatitis B e antigen-positive chronic hepatitis B patients. A: Immune-tolerant patients (IT) group; B: Patients with a mild immune response in the immune clearance phase (IC-Mild) group; C: Patients with a dramatic immune response in the immune clearance phase and exhibiting acute on chronic liver failure (ACLF) group. HBsAg: Hepatitis B surface antigen; HBV: Hepatitis B virus. against HBV-infected hepatocytes has not been triggered [10-12]. When immune clearance has been triggered and a fraction of the HBV-infected hepatocytes has been destroyed, decreased serum HBsAg levels have been found in the IC phase (median log10 IU/mL) [10-12], which was proven in our study. The degree of a host s 4410 April 21, 2014 Volume 20 Issue 15

312 Zhang YM et al. HBsAg levels in different HBeAg-positive patients immune response has been shown to be different when immune clearance begins. However, scientists have often considered all patients in the IC phase as a whole or have excluded ACLF patients from their studies [10-12]. This study is the first to subdivide HBeAg-positive CHB patients into groups according to the different immune responses. The results revealed that the median level of serum HBsAg was significantly lower in the ACLF group than in the IC-Mild group (3.57 log10 IU/mL vs 3.97 log10 IU/mL). The explanation for this difference may be the dramatic immune response in the ACLF group compared with the mild immune response in the IC-Mild group. Although the HBsAg clearance mechanism is complex in HBeAg-positive ACLF patients, several clues have revealed that the mechanism appears related to the remarkable immune response in which pro-inflammatory cytokines play an important role [18]. Changes in serum HBsAg levels in HBeAg-positive CHB patients with different immune conditions may provide useful information regarding the HBV life cycle. Chan et al [10] performed a longitudinal study and showed that the median serum HBsAg level in IC patients was similar before HBeAg seroconversion was achieved. Another study demonstrated that the early decrease in the amount of HBsAg in HBeAg-positive patients can predict sustained response to pegylated interferon. Pegylation enhances the half-life of interferon compared with its native form [5]. In this study, a dramatic immune response in the ACLF group caused a significant reduction in HBsAg levels compared with the IC-Mild group. The decreased HBsAg levels in the ACLF group suggest that HBeAg seroconversion may be achieved after this dramatic immune response. For these reasons, longitudinal follow-up is recommended. HBsAg synthesis during persistent HBV infection is complex and differs from HBV DNA synthesis. HBsAg is synthesized in the endoplasmic reticulum and from HBV DNA integrated into a host s genome [19]. Three forms of HBsAg are found in serum HBsAg: part of an HBV virion, the filament, and spheroid particles; the amount of HBsAg produced is usually greater [19] than the value required for virion assembly [2]. The methods used to quantify serum HBsAg cannot differentiate these three forms. Furthermore, the synthesis ratio of these three forms of HBsAg is complex in the natural history of CHB. Previous studies showed a strong correlation between HBsAg production and HBV DNA replication in the early phase of immune clearance, such as in acute hepatitis B [11] or in patients responding to Peg- IFN treatments [20]. In addition, a positive association was observed between serum HBsAg and HBV DNA in the IC phase in patients with genotypes B, C, and D according to previous studies [2]. Patients in China predominantly exhibit genotype B or C, and thus, we also found a positive correlation between serum HBsAg and HBV DNA in the IC-Mild group (r = 0.60, P < 0.001). Studies demonstrating the relationship between HBsAg and HBV DNA in the IT phase are limited. For example, Nguyen et al [12] reported that HBsAg production is poorly correlated with viral replication (r = 0.30, P = 0.09). Our study showed a stronger correlation between HBsAg and HBV DNA in the IT patients (r = 0.52, P < 0.001). This result may be attributed to the unavailable HBV genotype composition of the patients and the relatively large number of patients enrolled in the IT group compared with those in previous studies. Early studies described rapid viral clearance with undetectable serum HBsAg and HBV DNA in HBV-related liver failure [21,22]. However, the test methods for HBsAg and HBV DNA are qualitative. A recent study noted a consequential, rapid decrease in the HBV viral load within four days in chronic hepatitis B-induced liver failure [23]. A positive correlation between HBsAg and HBV DNA in ACLF patients was found (r = 0.51, P = 0.001) in our study. This result is possible evidence that the serum HBsAg level rapidly decreases in ACLF as the HBV DNA rapidly declines. The ratio of HBsAg/HBV DNA is stable in HBeAgpositive patients before HBeAg seroconversion, indicating a stable correlation between virus replication and HBsAg secretion [24]. This stable correlation was proven in our study. The HBsAg/HBV DNA ratios were approximately 0.55 in the three groups in this study. This study also has certain limitations. First, it is a cross-sectional study, whereas a longitudinal investigation would be more useful. However, long-term follow-up without the influence of anti-hbv therapy is difficult because antiviral therapy was administered to almost all patients in the IC-Mild or ACLF groups. Second, the HBV genotype cannot be used in this study. Investigations should also be conducted to determine the influence of genotypes on the production of HBsAg under different immune conditions. In addition, the distribution of serum HBsAg levels does not show significant differences in genotype B or C HBeAg-positive patients [12]. This finding partly resolves such limitations. In conclusion, serum HBsAg levels are significantly different in HBeAg-positive CHB patients with different immune conditions. In particular, the highest, moderately high, and lowest HBsAg levels were found in the IT, IC- Mild, and ACLF groups, respectively. These findings may have important implications in elucidating the immune clearance and sequential immune control of HBV infection in HBeAg-positive CHB. COMMENTS Background Quantitative serum Hepatitis B surface antigen (HBsAg) levels have been characterized in four typical phases of the natural history of chronic hepatitis B (CHB), indicating the dynamic interaction between hepatitis B virus and its host s immune system. However, the severity of the immune response is different in the immune clearance phase, and HBsAg levels have not been evaluated in these subdivided patients and compared with those in immune-tolerant patients. Research frontiers Studies on HBsAg quantification should focus on predicting the responses to antiviral therapy according to the baseline HBsAg levels or according to changes in the HBsAg levels during therapy. Therefore, studies should be conducted 4411 April 21, 2014 Volume 20 Issue 15

313 Zhang YM et al. HBsAg levels in different HBeAg-positive patients to investigate the HBsAg levels under different immune conditions during the natural history of CHB. In this way, a link between the immune reaction and antiviral efficacy might be revealed. Innovations and breakthroughs In this study, HBsAg levels in HBeAg-positive patients who met the criteria of acute on chronic liver failure were evaluated for the first time. The HBsAg levels were lower in these patients than in patients without evident liver decompensation in the immune clearance phase. Applications These findings may have important implications in elucidating the immune clearance and sequential immune control of HBV infection in HBeAg-positive CHB. Terminology HBsAg is a surface antigen of the hepatitis B virus. HBsAg has been used as a hallmark of hepatitis B infection for more than 40 years. 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314 Zhang YM et al. HBsAg levels in different HBeAg-positive patients hepatitis B virus-related acute liver failure are separable by quantitative serum immunoglobulin M anti-hepatitis B core antibody and hepatitis B virus DNA levels. Hepatology 2012; 55: [PMID: DOI: / hep.24732] 24 Chan HL, Wong VW, Tse AM, Tse CH, Chim AM, Chan HY, Wong GL, Sung JJ. Serum hepatitis B surface antigen quantitation can reflect hepatitis B virus in the liver and predict treatment response. Clin Gastroenterol Hepatol 2007; 5: [PMID: DOI: /j.cgh ] P- Reviewers: Marcos M, Nagata T, Sanai FM S- Editor: Zhai HH L- Editor: Webster JR E- Editor: Zhang DN 4413 April 21, 2014 Volume 20 Issue 15

315 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. Caspase-3 expression in metastatic lymph nodes of esophageal squamous cell carcinoma is prognostic of survival RESEARCH REPORT Xiu-Shen Wang, Kong-Jia Luo, Amos Ela Bella, Shan-Shan Bu, Jing Wen, Shui-Shen Zhang, Yi Hu Xiu-Shen Wang, Shan-Shan Bu, Department of Radiation Oncology, Cancer Hospital of Zhengzhou University, Zhengzhou , Henan Province, China Kong-Jia Luo, Amos Ela Bella, Jing Wen, Shui-Shen Zhang, Yi Hu, State Key Laboratory of Oncology in South China, Cancer Center, Sun Yat-sen University, Guangzhou , Guangdong Province, China Kong-Jia Luo, Amos Ela Bella, Jing Wen, Shui-Shen Zhang, Yi Hu, Department of Thoracic Oncology, Cancer Center, Sun Yatsen University, Guangzhou , Guangdong Province, China Kong-Jia Luo, Amos Ela Bella, Jing Wen, Shui-Shen Zhang, Yi Hu, Guangdong Esophageal Cancer Research Institute, Guangzhou , Guangdong Province, China Author contributions: Luo KJ and Bella AE drafted the article; Bu SS revised the manuscript; Weng J and Zhang SS provided the raw data; Wang xs and Hu Y designed the study. Wang XS, Luo KJ and Bella AE contributed equally to this work and share first authorship. Correspondence to: Yi Hu, MD, PhD, Department of Thoracic Oncology, Cancer Center, Sun Yat-sen University, 651 Dong Feng Road East, Guangzhou , Guangdong Province, China. huyi@sysucc.org.cn Telephone: Fax: Received: September 5, 2013 Revised: October 27, 2013 Accepted: January 8, 2014 Published online: April 21, 2014 Abstract AIM: To assess whether differential expression of caspase-3 in paired metastatic lymph nodes (LNs) is prognostic of survival in patients with resectable esophageal squamous cell carcinoma (ESCC). METHODS: Capases-3 expression was evaluated immunohistochemically in 122 pairs of primary ESCCs and regional metastatic LNs assembled on tissue microarrays. The impact of caspase-3 expression on survival outcomes was analyzed by the Kaplan-Meier method and Cox proportional hazards regression model. RESULTS: The level of caspase-3 expression was significantly higher in LN metastases than in primary tumors (P < 0.001). Caspase-3 expression in the primary tumors was associated with longer median survival (23 mo vs 21 mo, P = 0.033), whereas higher expression in paired metastatic LNs was associated with shorter median survival (20 mo vs 22 mo, P = 0.043). Multivariate analysis showed that both were independent prognostic factors. CONCLUSION: Caspase-3 expression in metastatic LNs may be a potential independent predictor of poorer overall survival in patients with resected ESCC and LN metastasis. Protein expression in metastatic tumors may be a biomarker prognostic of survival Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Esophageal squamous cell carcinoma; Caspase-3 protein; prognosis; Lymph node Core tip: Reduced caspase-3 expression in primary esophageal squamous cell carcinoma (ESCC) is associated with poorer outcomes, but the prognostic value of caspase-3 in metastatic lymph nodes (MLNs) is unclear. Analysis of 122 patients with primary ESCCs and paired MLNs showed that higher caspase-3 expression in MLNs from ESCC was associated with poorer prognosis. To the best of our knowledge, this study is the first to report the potential prognostic value of caspase-3 expression in MLNs of ESCC. Additional studies are needed to assess the prognostic importance of this biomarker. Wang XS, Luo KJ, Bella AE, Bu SS, Wen J, Zhang SS, Hu Y. Caspase-3 expression in metastatic lymph nodes of esophageal squamous cell carcinoma is prognostic of survival. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: April 21, 2014 Volume 20 Issue 15

316 Wang XS et al. Caspase-3 expression in LN metastases of ESCC INTRODUCTION Esophageal carcinoma is the eighth most common malignancy worldwide [1]. In China, esophageal squamous cell carcinoma (ESCC) accounts for most esophageal malignant tumors, where it bears more than half of the global burden [2]. Despite incorporation of new therapeutic approaches, it remains an aggressive disease with a dismal prognosis [3]. Biomarkers predictive of patient prognosis may help design more effective and targeted therapies for ESCC. Caspase-3 (also known as CPP32, YAMA, and apopain) is a cysteine protease related to interleukin-1betaconverting enzyme (ICE) and the human homologue of Ced-3, a protein in Caenorhabditis elegans required for programmed apoptosis [4]. The normal apoptotic process can be initiated by a cascade of specific death-inducing signals with the activation of caspase-3 acknowledged as a penultimate step generally. The dysregulation of apoptotic pathways in many malignances can extend cell life span and may support anchorage-independent survival during metastasis [5-7]. Immunohistochemical studies have shown that caspase-3 is expressed in 55.4% to 79.7% of primary ESCCs [8-11], with reduced expression of caspase-3 associated with enhanced malignant potential and decreased survival. Genomic instability is a hallmark of cancer caused by constant selection pressure. Specific populations of tumor cells may be more prone to metastasis than others, which is likely to result in an enrichment of the former and maintenance of their genetic aberrations in metastases. Alternatively, tumor cells may acquire new genetic modifications after spreading to metastatic sites [12]. Substantial genetic differences may therefore exist between primary tumors (PTs) and their metastases. Although the association between caspase-3 expression and clinical outcomes has been analyzed in PTs, it is unclear whether caspase-3 expression in lymphatic metastases is prognostic of patient outcomes. Therefore, the purposes of our study were to assess possible changes in caspase-3 expression between PTs and paired metastatic lymph nodes (PMLNs) and analyze whether capase-3 expression in the latter is associated with clinical outcomes. MATERIALS AND METHODS Patient selection Between June 1997 and December 2004, 1120 consecutive patients with ESCC underwent esophagectomy in the Department of Thoracic Surgery at Sun Yat-sen University Cancer Center. Patients were included with the following eligibility criteria: (1) histological proof of thoracic ESCC; (2) pathological evaluation of lymph node metastasis; (3) no neoadjuvant therapy; and (4) complete surgical resection (R0). Patients were excluded with the following criteria: history of other cancer or death during the perioperative period. The study protocol was approved by the Institutional Review Board of the Cancer Center of Sun Yat-sen University. All patients provided written informed consent before surgery and all had undergone transthoracic esophagectomy (the Sweet or Mckeown procedure) with standard or total dissection of thoracic and abdominal lymph nodes. Metastatic lymph node selection Of the 1120 patients who had undergone esophagectomy during the study period, 288 were deemed eligible for this study. We obtained 288 PT and 3720 regional lymph node samples from these patients. All samples were collected in the operating room and were routinely fixed immediately after collection in 10% neutral buffered formalin for approximately 24 h at room temperature. After fixation, the samples were dehydrated, incubated in xylene, infiltrated with paraffin, and finally embedded in paraffin (Oxford Labware, St Louis, MO). Each tissue sample was identified on hematoxylin-and-eosin stained slides, and the corresponding paraffin-embedded tissue blocks were obtained. Two trained pathologists, blindly to clinical data, selected those lymph node samples according to the following eligibility criteria: histologic proof of squamous cell carcinoma of the metastatic lymph node, and the diameter of metastatic lesion more than 3 mm. Forty-two patients had multiple lymph nodes satisfying these criteria. We randomly selected one lymph node from each. Finally, 164 pairs of surgically resected ESCC PTs and corresponding metastatic lymph nodes were selected. Tissue microarray construction Tissue microarrays (TMA) were constructed using a tissue microarrayer (Beecher Instruments, Sun Prairie, WI). During sample selection, the pathologists marked areas containing viable tumor on the paraffin wax tissue blocks. For each case, three 1-mm tissue cores from marked areas of the same tissue block were selected (three cores per case) [13] and transferred to a TMA. Hematoxylinand eosin-stained sections from each TMA block were checked by the pathologists to ensure that adequate targeted tissues were included. Immunohistochemical staining Immunohistochemical (IHC) staining was performed on 4 μm sections obtained from tissue microarray blocks. Rabbit polyclonal caspase-3 antibody (CPP32 Ab-4, Lab Vision Corporation, Cheshire, Unite Kingdom) was applied at a 1:100 dilution and incubated overnight at 4 C. Immunoperoxidase staining was carried out using EliVison plus Kit (Maxim Bioscience, Fuzhou, China) according to the manufacturer s instructions. Briefly, the slides were incubated with polymer enhancer for 15 min, washed three times in PBS, and incubated with secondary antibody for 30 min at room temperature. As a negative control, the slides were incubated with nonimmune rabbit immunoglobulin G. After staining, the pathologists checked the sections again to ensure that interpretable results were obtained from each pair of PT and matched metastatic lymph node samples. Interpretable results could not be obtained from 42 pairs of specimens due 4415 April 21, 2014 Volume 20 Issue 15

317 Wang XS et al. Caspase-3 expression in LN metastases of ESCC Table 1 Demographic and clinicopathologic characteristics of the patient cohort Characteristic n (%) Age at diagnosis (yr) (50) > (50) Gender Male 102 (83.6) Female 20 (16.4) Surgical approach 1 Sweet 58 (47.5) McKeown 64 (52.5) Tumor location Upper 13 (10.6) Middle 69 (56.6) Lower 40 (32.8) Tumor grade G1 28 (22.9) G2 59 (48.4) G3 35 (28.7) pt category T2 12 (9.8) T3 103 (84.4) T4a 2 7 (5.7) pn category N1 64 (52.5) N2 45 (36.9) N3 13 (10.6) 1 The sweet procedure consists of esophagectomy with standard dissection of thoracic and abdominal lymph nodes through left thoracotomy; the McKeown procedure consists of esophagectomy with extended dissection of thoracic and abdominal lymph nodes through thoracic-abdominal-cervical incision; 2 T4a, resectable tumor invading pleura (n = 4), pericardium (n = 1), and diaphragm (n = 2). to secondary technical issues, including sectioning artifacts and obscuring debris. Staining evaluation Two trained pathologists, blindly to clinical data, evaluated all results independently at first and then reviewed together. The intensity (I) and proportion (Prop) of caspase-3 cytoplasmic staining in tumor cells were recorded individually for each core, with I defined as: none = 0, weak = 1, moderate = 2, strong = 3, Prop as the percentage of positive cells with at least 200 nuclei counted and the total score for each core calculated as I Prop [14]. For statistical analysis, the minimum score was set at 1.00 with scores ranging from 1 to 300. The final score for each tumor was defined as the average of the score of three cores in the same case. Based on previous studies [9,11], we defined positive expression as a final score 80 and high level expression as a final score 160. Statistical methods All statistical analyses were performed using SPSS 13.0 for Windows software (SPSS Inc, Chicago, IL). Differences in caspase-3 expression between group of PTs and PMLNs were analyzed by McNemar s test, with intra-patient paired comparisons analyzed by Wilcoxon signedrank paired tests. The correlation between caspase-3 expression and clinicopathologic parameters was analyzed by Mann-Whitney U-test or χ 2 test where applicable. For graphical representations, the log10 of the ratio of PMLNs to PTs was calculated, with log10 (ratios) of < 0 and > 0 indicating higher and lower levels of expression, respectively, in PMLNs. Disease-specific survival (DSS) was calculated from the time of surgery to the time of death from ESCC. If the patient was lost to follow-up or died of a cause other than ESCC, data were censored at the time of last follow-up or death. Survival curves were constructed by the Kaplan-Meier method and analyzed by log-rank test. Multivariate analysis using Cox proportional hazards regression model with a forward stepwise procedure was performed to determine factors independently predictive of survival. A significant difference was defined as a two-tailed P value less than RESULTS Clinical and pathological data Following selection, 122 patients with paired PTs and MLNs were included. They consisted of 102 men and 20 women, ranging in age from 34 to 78 years (mean of 58.2 years). Other clinical and pathological characteristics are shown in Table 1. Eighteen patients received postoperative chemotherapy [cis-diaminedichloroplatinum (CDDP) plus fluorouracil]. Follow-up data were obtained from all patients, with a median survival of 19 mo (range, mo). Discordant caspase-3 expression in primary ESCCs and metastatic lymph nodes Representative tissue samples of IHC are depicted in Figure 1. Most ESCC specimens showed cytoplasmic expression, with nuclear staining being rare. Eighty-eight of 122 PTs (72.1%) and 116 of 122 PMLNs (95.1%) showed positive expression of caspase-3 (P < 0.001, McNemar s test). High level expression was observed in 54 of 122 PTs (44.3%) and 94 of 122 PMLNs (77.0%). This discordance was statistically significant (P < 0.001, McNemar s test). As shown in Figure 2, the log10 ratio of caspase-3 expression in PLMNs to PTs was > 0 in 94 of the 122 patients (77.0%), indicating that, in individual patients the level of caspase-3 expression was higher in PMLNs than in PTs from the same patient (P < 0.001, Wilcoxon signed-rank test). Correlation of caspase-3 expression with clinicopathologic factors Based on the 7 th edition AJCC staging system [15] and previously assessed prognostic factors, patients clinicopathologic features were dichotomized for statistical analysis (Table 2). The levels of caspase-3 expression in PT and PMLN differed significantly in patients with pt2 (P < 0.001, Mann-Whitney U-test) and pt3/4 (P = 0.016, Mann-Whitney U-test) tumors. There was no association between caspase-3 expression and other clinicopathologic features April 21, 2014 Volume 20 Issue 15

318 Wang XS et al. Caspase-3 expression in LN metastases of ESCC A B Figure 1 Representative immunohistochemical staining for caspase-3 in a primary tumor (A) and a paired metastatic lymph node (B) resected from the same patient (original magnification 200) Log10 (ratio) Patients Figure 2 Log10 ratio 1 of caspase-3 expression in a metastatic lymph node and primary tumor for each patient. 1 Log10 ratio > 0 and < 0 indicate higher and lower expression, respectively, in metastatic lymph nodes. Table 2 Correlations between caspase-3 protein expression and clinicopathologic features Characteristic Number of patients Caspase-3 expression in PT Caspase-3 expression in PMLN Mean rank P value 1 Mean rank P value 1 Gender Male Female Age (yr) > Tumor location Upper/middle Lower Tumor grade G G2/G pt category T < T3/T4a LN metastases 2 N N2/N Mann-Whitney U-test; 2 Number of involved lymph nodes. PT: Primary tumors; PMLN: Paired metastatic lymph nodes. Caspase-3 expression as a prognostic factor in ESCC Kaplan-Meier analysis (Table 3) showed that, median DSS was significantly longer in patients with PTs positive than negative for caspase-3 expression (23 mo vs 21 mo; P = 0.033, log-rank test; Figure 3A). In contrast, median DSS was significantly shorter in patients with PMLNs showing high level than low level caspase-3 expression (20 mo vs 22 mo; P = 0.043, log-rank test; Figure 3B). Cox multivariate analysis of demographic and clinicopathologic characteristics, including gender, age, tu April 21, 2014 Volume 20 Issue 15

319 Wang XS et al. Caspase-3 expression in LN metastases of ESCC Table 3 Disease-specific survival analyses according to caspase-3 expression in primary esophageal squamous cell carcinoma and paired metastatic lymph nodes Characteristic mor location, differentiation grade, surgery approach, adjuvant therapy, pt category, pn category, caspase-3 expressions in PT, and high level caspase-3 expression in PMLN, showed that only age, lymph node involvement, caspase-3 expression in PT, and high level caspase-3 expression in PMLN were independent factors for longterm survival (Table 4). DISCUSSION Number of patients Disease-specific survival (mo) P value 1 Mean Median Expression in PT Negative Positive Expression in PT Low level High level Expression in PMLN Negative 6 81 NR Positive Expression in PMLN Low level High level Kaplan-Meier method (log-rank test); 2 The results were uncertain, due to few patients in the negative subgroup. PT: Primary tumors; NR: Not reached; PMLN: Paired metastatic lymph nodes. A Cumulative survival (%) B Cumulative survival (%) P = Disease-specific survival (mo) P = Disease-specific survival (mo) Caspase-3 expression in PT Negative expression Positive expression Negative expression-censored Positive expression-censored Caspase-3 expression in PMLN Low level expression High level expression Low level expression-censored High level expression-censored Figure 3 Disease-specific survival curves relative to caspase-3 expression in (A) primary tumor and (B) paired metastatic lymph node. PT: primary tumor; PMLN: paired metastatic lymph node. Numerous studies have been performed to identify useful markers associated with cancer progression and clinical outcomes. Most of these studies are performed on PTs, whereas others have not mentioned the origin of tumor tissue. The parallel progression model hypothesizes that tumor cells depart the primary lesion before the acquisition of fully malignant phenotypes undergoing somatic progression and metastatic growth at distant sites [16,17]. Thus, early dissemination and divergent progression of PT and disseminated tumor cells, the latter toward metastasis, suggest that assays of PTs may not be predictive of clinical outcomes. We therefore evaluated the patterns of caspase-3 expression in paired tissue samples from PTs and metastatic lymph nodes obtained from patients with ESCC in a single institution. This enabled an investigation of possible differences in caspase-3 expression in metastatic lymph nodes. High level expression of caspase-3 was observed in a significantly higher fraction of metastatic lymph nodes than in PTs. In general, there was a trend toward increased expression of caspase-3 in metastases, a finding confirmed by comparisons of individual patients, which showed that caspase-3 expression was upregulated in metastatic lymph nodes compared with PTs. Neoplasms are biologically heterogeneous and contain subpopulations of cells with different malignant properties. By IHC method, the patterns of biomarker expression were previously analyzed in samples of PTs and corresponding metastatic lesions. Conflicting results on some biomarkers, such as epidermal growth factor receptor (EGFR), have been observed. For example, differential expression of EGFR was observed in 33% of primary lung cancers and metastases, with EGFR down-regulated in the latter [14,18]. In contrast, high degrees of concordant EGFR expression were observed in breast and colon cancers and their respective metastases [19,20]. According to limited previous studies on ESCC, the patterns of some biomarkers, including E-cadherin, VEGF, MMP-9, and CD44V6, have been shown to differ between ES- CCs and their metastases [21-23]. The present findings were novel, showing that the level of caspase-3 protein expression was higher in metastatic lymph nodes of ESCC than in PTs. After correlation analysis of protein expression and clinical outcomes, we found that there was no association between caspase-3 expression and any clinicopathological factors, although high caspase-3 expression in PT was associated with a favorable prognosis. Moreover, multivariate analysis of clinical factors and biomarkers indicated that caspase-3 expression in PT was an independent prognostic factor of improved survival, consistent with previous immunohistochemical [9,11] and flow cytometry [24] findings in ESCC. Caspase-3 is the main effecting caspase 4418 April 21, 2014 Volume 20 Issue 15

320 Wang XS et al. Caspase-3 expression in LN metastases of ESCC Table 4 Univariate and multivariate analyses1 of factors associated with disease-specific survival in 122 patients with esophageal squamous cell carcinoma Prognostic factor P value RR 95%CI Univariate survival analysis Gender (male vs female) Age ( 58 vs > 58) Location(lower vs upper/middle) Differentiation (grade 1 vs grade 2/3) Surgical approach (Sweet vs McKeown) Adjuvant therapy (No vs Yes) pt category (T2 vs T3/T4a) pn category (N1 vs N2/N3) < Caspase-3 expression in PT (negative vs positive) Caspase-3 expression in PMLN (low vs high level expression) Multivariate survival analysis Age ( 58 vs > 58) < pn category (N1 vs N2/N3) < Caspase-3 expression in PT (negative vs positive) Caspase-3 expression in PMLN (low vs high level) 1 Cox proportional hazards regression analysis (Forward stepwise); RR: Relative risk; PT: Primary tumor; PMLN: Paired metastatic lymph node. in apoptotic process. The active form of caspase-3 is generated by proteolytic cleavage of procaspase-3 into two subunits with molecular masses of 17 kda and 12 kda, respectively. The antibody we used to detect caspase-3 protein in paraffin sections recognizes the inactive form of caspase-3, i.e. procaspase-3 or full length human caspase-3 protein. A previous study using an antibody against pro-caspase-3 showed a concordance between caspase-3 expression in ESCC and apoptotic index, as assessed by TUNEL technique [8]. Another study, in which caspase-3 activation and procaspase-3 expression were determined using specific antibodies, reported that the absence of caspase-3 activation was significantly correlated with loss of procaspase-3 expression [25]. Thus, immunohistochemical analysis of caspase-3 protein expression could be considered as a feasible method for detecting apoptosis in our cohort. The high level of caspase-3 expression in primary ESCC could therefore reflect increased apoptosis and may suppress the progression of ESCC. Interestingly, we found that caspase-3 expression in metastases of ESCC was associated with an unfavorable outcome. DSS was significantly poorer in patients with higher than lower level caspase-3 expression in PMLN. Further, Cox multivariate analysis showed that caspase-3 expression in PMLN was independently prognostic of poor prognosis. Apoptosis in esophageal lesions has been reported to increase gradually during the progression of esophageal carcinogenesis [26]. Caspase-3 expression in metastatic lesions does not accord with increased apoptosis, implying that this protein has a function other than the execution of cell death programs. Recently, Fujita et al [27] and Janzen et al [28] conducted a series of elegant studies to explore the unexpected role of caspase-3 which mediates the differentiation of embryonic stem cells (ESCs) and hematopoietic stem cells (HSCs), respectively. These findings suggested that caspase-3 protein is involved in regulating stem cell development and differentiation. Tissue development and maintenance that affect tumor metastasis are dependent on a complex interplay of stem cell self-renewal, differentiation, and apoptosis [29]. Further studies assessing the biological mechanisms by which caspases affect cancer stem cells may enhance our understanding of metastasis in ESCC. In conclusion, our study revealed a novel phenomenon that the level of caspase-3 protein expression differed substantially between primary ESCCs and metastatic lymph nodes. In contrast to PTs, in which caspase-3 expression was associated with better survival, caspase-3 expression in metastatic lymph nodes was independently prognostic of poorer survival in patients resected for ESCC and lymph node metastasis. Based on our findings, it finally underlined that protein expression in metastatic tumors could be considered for prognostic prediction and further studies on caspase-3 will provide a new visual angle to make a better understanding of this familiar biomarker in ESCC. ACKNOWLEDGMENTS The authors thank Ms Miao-Qing Lin for patient followup and Maxim Ltd. for technical assistance. COMMENTS Background Esophageal squamous cell carcinoma (ESCC) remains an aggressive disease with poor prognosis despite incorporation of new therapeutic approaches. Recent research focused on biomarkers, including caspase-3, may help to find out more effective therapies for patients with ESCC. Previous studies suggest that reduced caspase-3 expression in primary tumors (PTs) is associated with poorer outcomes, but the prognostic value of caspase-3 expression in metastatic lymph nodes (MLNs) is unclear. Based on the question of tumor heterogeneity, additional studies are needed to assess the prognostic importance of this biomarker in MLNs. Research frontiers Primary tumors and regional metastatic lymph nodes assembled on tissue microarrays in pairs were evaluated by immunohistochemical analysis for caspase-3. The impact of caspase-3 expression on survival outcome was analyzed by the Kaplan-Meier method and Cox proportional hazards regression model. Innovations and breakthroughs In analyzing 122 patients with primary ESCC tumors and paired MLNs, we found that reduced caspase-3 expression in ESCC from different lesions was associated with different prognoses. To the best of our knowledge, this study is the first to report the potential prognostic value of caspase-3 expression in MLNs. It suggested that ongoing studies should be needed to make a better understanding of this familiar biomarker. Applications Our results suggested that the level of caspase-3 expression was significantly increased in lymphatic metastases compared with primary tumors. Positive caspase-3 expression in PTs was associated with a favorable prognosis, but high level expression in paired MLNs was associated with a poor outcome. Multivariate analysis demonstrated that they were both independent prognostic factors. Terminology Tissue microarrays consist of paraffin blocks in which up to 1000 separate cores from different tissue blocks are assembled in a new paraffin block. They can enable the high throughput analysis of a large number of tissue samples. Peer review This is an interesting study analyzing relations between caspase-3 expression 4419 April 21, 2014 Volume 20 Issue 15

321 Wang XS et al. Caspase-3 expression in LN metastases of ESCC in MLNs and clinical outcomes of ESCC. The main results in this study show that the level of caspase-3 expression in MLNs is higher than in PTs, implying the existence of tumor heterogeneity, and caspase-3 expression in MLNs is also a biomarker independently predictive of survival. Further study is needed to indentify the different role of caspase-3 during the progression of ESCC. REFERENCES 1 Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBO- CAN Int J Cancer 2010; 127: [PMID: DOI: /ijc.25516] 2 Parkin DM, Bray F, Ferlay J, Pisani P. Global cancer statistics, CA Cancer J Clin 2005; 55: [PMID: ] 3 Jemal A, Siegel R, Xu J, Ward E. Cancer statistics, CA Cancer J Clin 2010; 60: [PMID: ] 4 Nicholson DW, Ali A, Thornberry NA, Vaillancourt JP, Ding CK, Gallant M, Gareau Y, Griffin PR, Labelle M, Lazebnik YA. 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Absence of caspase 3 activation in neoplastic cells of nasopharyngeal carcinoma biopsies predicts rapid fatal outcome. Mod Pathol 2005; 18: [PMID: ] 26 Wang LD, Zhou Q, Yang WC, Yang CS. Apoptosis and cell proliferation in esophageal precancerous and cancerous lesions: study of a high-risk population in northern China. Anticancer Res 1999; 19: [PMID: ] 27 Fujita J, Crane AM, Souza MK, Dejosez M, Kyba M, Flavell RA, Thomson JA, Zwaka TP. Caspase activity mediates the differentiation of embryonic stem cells. Cell Stem Cell 2008; 2: [PMID: ] 28 Janzen V, Fleming HE, Riedt T, Karlsson G, Riese MJ, Lo Celso C, Reynolds G, Milne CD, Paige CJ, Karlsson S, Woo M, Scadden DT. Hematopoietic stem cell responsiveness to exogenous signals is limited by caspase-3. Cell Stem Cell 2008; 2: [PMID: ] 29 Abdul-Ghani M, Megeney LA. Rehabilitation of a contract killer: caspase-3 directs stem cell differentiation. Cell Stem Cell 2008; 2: [PMID: ] P- Reviewers: Chen LF, Chi SG, Gonzalez-Aseguinolaza G, Mishra PK, Scarpa M S- Editor: Ma YJ L- Editor: Wang TQ E- Editor: Liu XM 4420 April 21, 2014 Volume 20 Issue 15

322 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH BRIEF ARTICLE REPORT Expression of monocyte chemotactic protein-1/ccl2 in gastric cancer and its relationship with tumor hypoxia Lei-Lei Tao, Shu-Jing Shi, Long-Bang Chen, Gui-Chun Huang Lei-Lei Tao, Shu-Jing Shi, Long-Bang Chen, Gui-Chun Huang, Medical Oncology Department of Jinling Hospital, Medical School of Nanjing University, Nanjing , Jiangsu Province, China Author contributions: Tao LL participated in the design of the study, performed the research and wrote the paper; Shi SJ participated in the design of the study and helped to draft the manuscript; Chen LB designed the study and helped to draft the manuscript; Huang GC participated in the design of the study, performed immunohistochemistry and helped to write the paper; All authors read and approved the final manuscript. Supported by National Natural Science Foundation of China, No ; China International Medical Foundation, No. CIMF-F-H Correspondence to: Gui-Chun Huang, MD, PhD, Medical Oncology Department of Jinling Hospital, Medical School of Nanjing University, 305 ZhongShan Eastern Road, Nanjing , Jiangsu Province, China. huangguichun@nju.edu.cn Telephone: Fax: Received: September 9, 2013 Revised: January 4, 2014 Accepted: January 14, 2014 Published online: April 21, 2014 Abstract AIM: To investigate the expression and prognostic value of CCL2 in gastric cancer, as well as its relationship with tumor hypoxia. METHODS: Tumor tissues from 68 gastric cancer patients (GC) were analyzed, and the expression of CCL2 and hypoxia-inducible factor 1 alpha (HIF-1α) in tumor tissues was detected by immunohistochemistry. Statistical evaluations that were used included univariate logrank tests of Kaplan-Meier curves and multivariate Cox regression model analysis. RESULTS: CCL2 was highly expressed in 66.2% (45/68) of gastric cancer specimens. The distribution of CCL2 expression in tumor tissue was consistent with that of HIF-1α. Patients with high CCL2 expression in GC had a lower overall survival rate [50.6 mo (95%CI: ) vs 64.6 mo (95%CI: ), P = 0.013]. CONCLUSION: CCL2 expression correlates closely with HIF-1α expression in gastric cancer. CCL2 may be an independent prognostic marker for GC Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Chemokines; Monocyte chemotactic protein-1/ccl2; Gastric cancer; Prognostic marker; Immunohistochemistry Core tip: The authors have performed immunohistochemical analysis of CCL2 and hypoxia-inducible factor 1 alpha (HIF-1α) in consecutive formalin-fixed paraffin-embedded sections of 68 gastric tumor samples taken from gastric cancer patients. The expression of the monocyte chemotactic protein-1/ccl2 is associated with the expression of HIF-1α. The research results showed a correlation between the expression of both proteins in gastric carcinoma. The authors have statistically analyzed the relationship of CCL2 expression with the clinicopathological characteristics of the patients and their survival time. The expression of CCL2 could be used as a prognostic biomarker for gastric cancer. Tao LL, Shi SJ, Chen LB, Huang GC. Expression of monocyte chemotactic protein-1/ccl2 in gastric cancer and its relationship with tumor hypoxia. World J Gastroenterol 2014; 20(15): Available from: URL: com/ /full/v20/i15/4421.htm DOI: org/ /wjg.v20.i INTRODUCTION Gastric cancer (GC) is one of the most prevalent malig April 21, 2014 Volume 20 Issue 15

323 Tao LL et al. Expression of CCL2 in gastric cancer nancies in the world [1], and it is the fourth most common cancer in men and the fifth in women around the world. Approximately 8% of the total diagnosed cases and 10% of annual cancer deaths are attributed to gastric cancer worldwide [2]. Presently, surgical resection remains the only curative treatment option. However, most patients have advanced cancer in stages Ⅲ or Ⅳ and show lymphatic metastasis at the time of diagnosis. Currently, there is no specific biomarker that has been detected in GC for clinically diagnostic and prognostic purposes [3]. Therefore, a set of prognostic molecular biomarkers is needed for gastric cancer to improve the design and evaluation of individualized therapeutic strategies for this lethal disease. Hypoxia is a characteristic of the tumor microenvironment that may play a critical role in tumor angiogenesis, survival response, invasion and metastasis [4]. Accumulating evidence suggests that tumor hypoxia is an independent marker of poor prognosis in patients with various types of cancer, including cervical cancer, breast cancer, head and neck cancer, soft-tissue sarcoma, cutaneous melanoma and prostatic adenocarcinoma [5]. Hypoxia can activate relevant gene expression through hypoxiainducible factors (HIF), an important transcription factor family that includes HIF-1, HIF-2 and HIF-3. HIF is a heterodimer composed of an alpha and a beta subunit, in which the HIF-1α protein is a master regulator of the hypoxic response [6]. Hypoxic regions of solid tumors are often characterized by the large accumulation of macrophages, which contribute to tumor angiogenesis and development [7]. The trophic effect of tumor hypoxia on tumor-associated macrophages (TAMs) is clinically relevant, as a high TAM number is considered to be a negative prognostic marker in various human malignancies, including Hodgkin s disease, glioma, cholangiocarcinoma and breast carcinoma [8]. Monocyte chemotactic protein-1 (MCP-1/CCL2), a member of the CC family of chemokines, is known to recruit monocytes and macrophages to inflammation sites and tumors [9]. The elevated expression of a number of monocyte chemoattractants, including CCL2, by both cancer and stromal cells has been shown to positively correlate with increased TAM numbers in several human tumors [10]. CCL2 has been demonstrated to regulate monocyte and macrophage infiltration and reported to be present in tumor sites, suggesting a role for this chemokine in TAM recruitment. To better characterize the clinicopathological significance of CCL2 in gastric cancer, we hypothesized that CCL2 might be a potential prognostic biomarker for gastric cancer, and its expression would be associated with HIF-1α expression. MATERIALS AND METHODS Patients and tumor samples Formaldehyde-fixed and paraffin-embedded gastric carcinoma samples were obtained from the Pathology Department of Jinling Hospital. All of the pathological diagnoses were confirmed by two independent pathologists. None of the patients had received any preoperative treatments. Of all the patients, 73.5% were male, 26.5% were female, and the mean age was years. All patients were followed from the date of surgery to the date of their death or the end of the study in December The data of patients who were lost to follow-up or who died of causes other than GC were censored in the survival analysis. The study was approved by the Ethics Committee of Jinling Hospital. Immunohistochemistry To evaluate the CCL2 protein expression in clinical samples that had been embedded in paraffin blocks, we stained the sections as follows. Three micrometer continuous sections were cut on slides for immunohistochemical analysis. Antigen retrieval was achieved by pressure cooking the slides with citric acid buffer at ph 6.0 for 1 min. Endogenous peroxidase activity was blocked by immersing the slides in 3% H2O2 for 10 min, and the background, nonspecific binding was reduced by incubating the slides with 5% bovine serum albumin (BSA) in PBS for 5 min. The continuous slides were incubated overnight at 4 with rabbit monoclonal CCL2 antibody (1:500 dilution, Santa Cruz) and mouse monoclonal HIF- 1α antibody (1:100 dilution, MAB1935, R and D), respectively. Finally, the slides were washed five times in PBS 1, ph 7.4, for 5 min. To reduce variability, all samples were processed at the same time in a single experiment using a single batch of antibody diluted in PBS with BSA. Slides were then washed in PBS and incubated sequentially with biotinylated goat anti-rabbit/mouse IgG at a dilution of 1:500 for 30 min at 37. The reaction product was developed using diaminobenzidine tetrahydrochloride. Finally, the slides were counterstained with hematoxylin. Subsequently, the tissues were washed in distilled water for 5 min, dehydrated sequentially and mounted in resinous mountant. Evaluation of immunohistochemical analysis The evaluation of CCL2 expression was performed independently by two experienced pathologists, who were blinded to the clinical data. The staining results for CCL2 were scored semi-quantitatively by calculating the immunostaining intensity and the distribution of the percentage of positive cells. The percentage of positive tumor cells was determined in at least 5 areas under 400 magnification and averaged. The mean percentage was then divided into five categories: 0, < 5%; 1, 5%-25%; 2, 26%-50%; 3, 51%-75%; and 4, > 75%. The staining intensity was calculated by assigning no coloring, slightly yellow, brown yellow and tan stains to values of 0, 1, 2 and 3, respectively. Finally, we calculated the product of staining intensity and positive cell percentage: 5 was defined as low expression, and 6 as high expression. To further study the relationship between CCL2 and 4422 April 21, 2014 Volume 20 Issue 15

324 Tao LL et al. Expression of CCL2 in gastric cancer Table 1 Relationship between clinicopathological characteristics and CCL2 expression in gastric cancer Clinical characteristic CCL2 expression level P value 1 HIF-1α in gastric cancer, the spatial distributions of CCL2 and HIF-1α expression were analyzed in pairs. Statistical analysis Differences between clinicopathological variables and the expression of CCL2 were examined by the χ 2 test. Survival curves were calculated using the Kaplan-Meier method and compared by the log-rank test. Multivariate Cox regression model analysis was applied to assess the prognostic values of protein expression. The confidence level for statistical inference was 95% (P < 0.05). Statistical analysis was performed using SPSS software (Version 16.0, Chicago, IL, United States). RESULTS High Low Gender Male Female 9 9 Age (yr) < Tumor diameter (cm) > Differentiation grade Well 2 2 Moderate Poor Histopathology Tubular 14 6 Poorly Signet-ring 7 1 Mucinous 5 5 Lymph node metastasis Yes 14 7 No AJCC stage Ⅰ/Ⅱ Ⅲ/Ⅳ χ 2 or Fisher exact test; 2 AJCC: American Joint Committee on Cancer. Follow-up visits Patients with gastric cancer were interviewed by telephone, and additional data were collected from the medical records. The clinicopathological characteristics of the 68 patients are shown in Table 1. The ages of the patients ranged from 24 to 59 years, with a mean age of years. According to the American Joint Committee on Cancer (AJCC, 2010) classification, there were 33 stage Ⅲ/Ⅳ patients and 35 stage Ⅰ/Ⅱ patients. The patients were followed for a period of 1-84 mo. Two patients did not receive a full follow-up, and 37 patients died during the follow-up period. Expression pattern of CCL2 in human gastric cancer The expression levels of MCP-1 protein in GC were detected by immunostaining. Typical immunohistochemical findings of CCL2 in gastric tumor tissue are shown in Figures 1 and 2. CCL2 protein was found to be located in the cytoplasm of the malignant cells, and we repeated the experiment twice to exclude false positive results. Immunohistochemical analysis showed that, in gastric carcinoma, 45 (66.2%) of 68 tumor samples had high CCL2 expression. Correlation between the expression of CCL2 and HIF-1α To evaluate whether the expression of the inflammatory cytokine CCL2 was associated with HIF-1α expression, the expression levels of CCL2 and HIF-1α were measured in consecutive sections. The expression of HIF-1α was consistent with the spatial distribution of CCL2 in tumor cells (Figure 1). The consistency of spatial distribution of the expression of these two proteins also suggests that CCL2 expression is related to tumor hypoxia. Statistical analysis Survival curves were calculated using the Kaplan-Meier method and compared using the log-rank test. The follow-up time was censored if the patient died from another cause rather than gastric cancer or was lost during the follow-up period. Patients with high levels of CCL2 expression in gastric cancer (P < 0.005) had a statistically significant correlation with poor overall survival. (Figure 3; 50.6 mo (95%CI: ) vs 64.6 mo (95%CI: ), P = 0.013)). The independent effects of all significant factors were evaluated in a multivariate Cox regression model. The result demonstrated that tumor CCL2 expression level (P = 0.045, HR = 2.311, 95%CI: ) and AJCC stage (P = 0.004, HR = 3.242, 95%CI: ) were independent prognostic factors for gastric cancer patients, and other factors showed no statistical significance with prognosis (Table 2). DISCUSSION Advanced gastric cancer is characterized by the rapid emergence of systemic metastasis, resulting in poor prognosis due to a lack of curative treatment options. However, there is no single marker for GC. Therefore, research to identify specific markers for this malignant tumor is important and necessary. Solid tumors need new blood vessels to support their growth by providing enough nutrients and oxygen. As tumors grow, the diffusion distance from the existing vascular supply increases, which results in hypoxia [11]. Hypoxia is a common feature of the microenvironment of a diverse range of solid tumors including gastric cancer. Hypoxia plays a critical role in various cellular and physiologic events, including cell proliferation, survival, angiogenesis, immunosurveillance, metabolism, and tumor invasion and metastasis, and it is often associated with poor prognosis [4,12] April 21, 2014 Volume 20 Issue 15

325 Tao LL et al. Expression of CCL2 in gastric cancer A A-1 A mm 20 mm 20 mm B 1 2 B-1 B mm 20 mm 20 mm Figure 1 The expression patterns of CCL2 and hypoxia-inducible factor 1α were detected in consecutive human gastric cancer tissues sections. A: Expression pattern of hypoxia-inducible factor 1 alpha (HIF-1α) in human gastric cancer tissues (100 ); B: Expression pattern of CCL2 in the human gastric cancer tissues (400 ). The spatial distribution of HIF-1α expression was consistent with that of CCL2 in tumor sections. (A-1 vs B-1, A-2 vs B-2). A C D B E 100 mm Figure 2 Immunohistochemical detection of the expression pattern of CCL2 in human gastric cancer tissue. A: Expression pattern of CCL2 in gastric cancer (100 ); C, E: Positive staining for CCL2; D, E: Expression pattern of CCL2 in gastric cancer (400 ); The images in D and E are the amplified sections corresponding to B and C. B, D: Negative staining for CCL2. HIF-1α is one of the principal mediators of homeostasis in response to hypoxia in human tissues. Under hypoxic conditions, HIF-1α produces an active heterodimer and drives the transcription of a number of genes important for cell survival, immune reaction and chemokine production [6]. Hypoxic tumors secrete higher amounts of chemoattractants and other factors that enhance monocyte/macrophage attachment to and migration through the tumor vasculature. Recent evidence has shown that TAM may accumulate in high numbers in hypoxic areas of breast, prostate and ovarian carcinoma due to the hypoxic release of macrophage chemoattractants [13]. CCL2, also known as MCP-1, is a small cytokine and a highly potent chemokine that attracts and activates monocytes/macrophages to sites of tissue injury and inflammation, as well as to tumor sites. CCL2 acts as an immune inhibitor in tumors. CCL2 is produced by endothelial cells, fibroblasts, epithelial cells, smooth muscle cells, 4424 April 21, 2014 Volume 20 Issue 15

326 Tao LL et al. Expression of CCL2 in gastric cancer Cum survival High CCL2 expression Low CCL2 expression Survival time (mo) Figure 3 Relationship between the expression of CCL2 and overall survival time in gastric cancer patients. Patients with high CCL2 expression had a poor overall survival [50.6 mo (95%CI: ) vs 64.6 mo (95%CI: ), P = 0.013]. astrocytes, macrophages, microglial cells, and even certain tumor cells themselves [14]. Furthermore, several recent investigations have shown that CCL2 gene expression was upregulated in hypoxic regions. CCL2 expression levels were dependent on the O2 concentration and duration of the hypoxic exposure [15]. The CCL2 gene contains several binding sites for HIF in the promoter, which is believed to account for increased CCL2 expression in response to hypoxia-induced HIF stabilization [16]. However, the biological significance of CCL2 in the tumor microenvironment appears particularly complex, and the relationship between HIF-1α and CCL2 in gastric tumors has not been clarified. In our study, we analyzed the expression of CCL2 and HIF-1α in gastric cancer samples and found high expression levels of CCL2 in primary gastric cancer by immunohistochemical analysis. Both the tumor cells and stromal cells were observed to express CCL2, and the distribution of HIF-1α expression was consistent with that of CCL2 expression in tumor sections. The consistency of the spatial distribution of the two proteins indicated that CCL2 expression was related to tumor hypoxia. Studies have demonstrated that CCL2 is expressed in several tumors including glioma [17], melanoma [18], ovarian carcinoma [19], and uterine cervical tumors [20]. Significantly higher levels of CCL2 expression were found in the epithelial region of various tumors, including breast cancer [21]. It has also been shown that CCL2, both at the mrna and protein levels, is expressed mainly in the epithelial regions of prostate cancer tissues [22]. Because tumor cells produce CCL2, it is considered to have an important role in the progression and invasion of cancer. This role has been confirmed in many types of cancer, such as prostate cancer [23] and breast cancer [24]. In addition, interrupting the CCR2/CCL2 interaction using a CCL2 specific antibody or CCR2 sirna markedly reduced the recruitment of monocytes/macrophages and delayed tumor progression and metastasis [25]. This Table 2 Evaluation of all significant factors in the multivariate Cox regression model suggests that CCL2 expression within tumors may be a critical determinant of monocyte recruitment, as CCR2 is highly expressed in classical monocytes. Based on the expression patterns of CCL2 and CCR2, we hypothesize that this ligand/receptor pair is a critical determinant of the recruitment of classical monocytes. It has also been reported that CCL2 may be a candidate molecular tumor marker, which could be targeted for cancer immunotherapy [26]. Consistent with the clinicopathological significance, gastric cancer patients with high expression levels of CCL2 in tumor cells exhibited poor overall survival in our study. Thus, high expression of CCL2 was an indicator of poor clinical prognosis. Therefore, we propose that CCL2 might be a valuable predictive marker of gastric carcinoma, as it is correlated with cancer stage according to the AJCC tumor staging system. Because CCL2 is involved in many aspects of the tumor microenvironment, it may be a potential molecular target for malignant tumor therapy. ACKNOWLEDGMENTS The authors wish to thank Dr. Xiaoxia Lu for her help in data collection. COMMENTS HR (95%CI) Survival time P value Gender ( ) Male vs female Tumor volume ( ) > 5 cm vs < 5 cm Differentiation ( ) Mid + High vs Low Lymph node ( ) Yes vs No AJCC stage ( ) Ⅰ/Ⅱ vs Ⅲ/Ⅳ CCL2 expression ( ) High vs Low AJCC: American Joint Committee on Cancer. Background Gastric carcinoma is the fourth most common cancer in men and the fifth in women around the world. Tumor specific biomarkers in gastric carcinoma may be helpful for clarifying histological heterogeneity and the underlying molecular mechanisms. A set of better molecular biomarkers for the prognosis of gastric cancer is needed to design and evaluate individualized therapeutic strategies for this malignancy. Research frontiers Elevated expression of CCL2 had been showed in various cancers. However, few studies have investigated the correlation between CCL2 and gastric carcinoma. Previous studies have indicated that CCL2 gene expression is regulated in hypoxic regions, but the correlation between CCL2 expression and hypoxiainducible factor-1α (HIF-1α) expression has not been observed. In this study, the authors demonstrated that CCL2 protein was regulated in gastric carcinoma 4425 April 21, 2014 Volume 20 Issue 15

327 Tao LL et al. Expression of CCL2 in gastric cancer and showed a close correlation with HIF-1α expression. Innovations and breakthroughs The research data showed the correlation between the expression of these two proteins in gastric carcinoma and found that CCL2 may be a prognostic marker of gastric carcinoma. Moreover, high expression of CCL2 could be an indicator of poor clinical prognosis in patients with gastric carcinoma. Applications The study suggested that examination of CCL2 and HIF-1α expression by immunohistochemistry analysis could be used as an effective way to identify patients at high risk of tumor progression, thus allowing the optimization of the individual treatment of patients with gastric carcinoma. Terminology Monocyte chemotactic protein-1 (MCP-1/CCL2), a member of the CC family of chemokines, is known to recruit monocytes and macrophages to sites of inflammation and tumor. HIF is an important transcription factor family that includes HIF-1, HIF-2 and HIF-3. 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328 Tao LL et al. Expression of CCL2 in gastric cancer 25 Qian BZ, Li J, Zhang H, Kitamura T, Zhang J, Campion LR, Kaiser EA, Snyder LA, Pollard JW. CCL2 recruits inflammatory monocytes to facilitate breast-tumour metastasis. Nature 2011; 475: [PMID: DOI: /nature10138] 26 Fridlender ZG, Buchlis G, Kapoor V, Cheng G, Sun J, Singhal S, Crisanti MC, Wang LC, Heitjan D, Snyder LA, Albelda SM. CCL2 blockade augments cancer immunotherapy. Cancer Res 2010; 70: [PMID: DOI: / CAN ] P- Reviewers: Kim H, Kim SY, Lin CY, Sala N S- Editor: Cui XM L- Editor: Wang TQ E- Editor: Zhang DN 4427 April 21, 2014 Volume 20 Issue 15

329 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH REPORT Preliminary study correlating CX3CL1/CX3CR1 expression with gastric carcinoma and gastric carcinoma perineural invasion Cheng-Yu Lv, Tao Zhou, Wei Chen, Xin-Dao Yin, Jian-Hong Yao, Yi-Fan Zhang Cheng-Yu Lv, Tao Zhou, Wei Chen, Xin-Dao Yin, Jian-Hong Yao, Yi-Fan Zhang, Department of General Surgery, Nanjing Hospital Affiliated to Nanjing Medical University, Nanjing , Jiangsu Province, China Author contributions: Lv CY designed the study; Zhang YF and Zhou T performed the majority of experiments; Chen W and Yin XD carried out the data collection and analysis; Yao JH drafted the manuscript; Lv CY revised the manuscript critically for important intellectual content. Supported by Nanjing Science and Technology Project, No Correspondence to: Cheng-Yu Lv, Professor, Department of General Surgery, Nanjing Hospital Affiliated to Nanjing Medical University, 68#Changle Road, Nanjing , Jiangsu Province, China. lcy_1234@aliyun.com Telephone: Fax: Received: September 15, 2013 Revised: January 18, 2014 Accepted: March 5, 2014 Published online: April 21, 2014 Abstract AIM: To study the relationship between the CX3CL1 chemokine, its receptor CX3CR1, and gastric carcinoma/gastric carcinoma perineural invasion (PNI). METHODS: Thirty cases of gastric carcinoma were surgically resected (radical resection or palliative resection) between February 2012 and July Tumour and tumour-adjacent tissues were evaluated for the presence of CX3CL1 (ELISA) and CX3CR1 (immunohistochemistry and Western blotting) in an effort to analyse the relationship between CX3CL1/CX3CR1 and gastric carcinoma/gastric carcinoma PNI. RESULTS: Of these 30 cases, 14 were PNI-positive (46.7%). No significant differences in CX3CL and CX3CR1 expression in tumour-adjacent tissues were found between the PNI positive and negative groups. Expression levels of CX3CL and CX3CR1 in tumour tissues were significantly higher than those in adjacent tissues (P < 0.01), and were significantly higher in tumour tissues from the PNI-positive group compared to the PNI-negative group (P < 0.01). CONCLUSION: CX3CL1/CX3CR1 expression may be associated with the occurrence and development of gastric carcinoma as well as gastric carcinoma PNI Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Gastric carcinoma, Perineural invasion, CX- 3CL1, CX3CR1 Core tip: This preliminary study determined the relationship between the CX3CL1/CX3CR1 system and gastric carcinoma/gastric carcinoma perineural invasion (PNI). The results revealed that the CX3CL1/CX3CR1 system may be associated with the occurrence and development of gastric carcinoma, and had an obvious relationship with gastric carcinoma PNI. Lv CY, Zhou T, Chen W, Yin XD, Yao JH, Zhang YF. Preliminary study correlating CX3CL1/CX3CR1 expression with gastric carcinoma and gastric carcinoma perineural invasion. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: INTRODUCTION Gastric carcinomas are one of the most common malignant tumours, and often undergo metastasis. Studies have increasingly reported tumour perineural invasion, 4428 April 21, 2014 Volume 20 Issue 15

330 Lv CY et al. CX3CL1/CX3CR1 and gastric carcinoma/gastric carcinoma perineural invasion Table 1 Comparison of CX3CL1 levels (pg/mg) in gastric carcinoma perineural invasion-positive and negative groups PNI positive group (mean ± SD) PNI negative group (mean ± SD) Tumor tissue 674 ± ± (P < 0.05) Adjacent tissue 479 ± ± (P > 0.05) t (P < 0.01) (P < 0.02) PNI: Perineural invasion. although this phenomenon has not been explored. In 1835, Cruveilhier [1] reported perineural invasion (PNI), a particular biological behaviour in tumour cells, in which the tumour approaches nerves and covers approximately 33% (or more) of the nerve perimeter or penetrates any of the 3 nerve-sheath layers [2]. The role of chemokines in the pathogenesis of gastric carcinoma and gastric carcinoma PNI is of interest. However, relative to other subfamilies, the current research on CX3CL1 is relatively sparse. The aim of this study was to study the expression of CX3CL1 and CX3CR1 in gastric carcinoma and explore their relationship with gastric carcinoma and gastric carcinoma PNI. MATERIALS AND METHODS Specimens Thirty patients underwent gastric carcinoma surgery in the Department of General Surgery, Nanjing Affiliated Hospital of Nanjing Medical University, between February 2012 and July Tumour tissue and 5 cm of adjacent tissue samples from the tumour boundary (control) were collected. CX3CL1 ELISA Proteins were extracted from the samples and the protein content was quantified using the Bradford method. Samples were evaluated using the Chemokine C-X3-Cbasic ligand 1 (CX3CL1) ELISA kit (USCN Co., Ltd.) according to the instructions provided with the kit, and read at 450 nm. The CX3CL1 concentration in each sample was determined by comparison to appropriatelydiluted standards. t Immunohistochemical detection of CX3CR1 (LP method) Paraffin sections were dewaxed in water, treated with 3% H2O2 to inactivate endogenous peroxidase and incubated with CX3CR1 rabbit anti-human monoclonal antibody (Abcam Co., Ltd.). Bound antibody was detected with the Universal One-Step Hypersensitive Detection Kit (Xiya Golden Bridge Biotechnology Co. Ltd.) using DAB as the peroxidase substrate (brown precipitate). Slides were counterstained with hematoxylin, dehydrated and coverslipped. Ten randomly-selected high-power fields (400 ) were evaluated in a double-blind manner and the following metric used: If the cytoplasm of the tumour cells contained stained granules and the tumour cell staining was > 10%, it was considered positive; if the tumour was unstained or the staining was less than 10%, the result was considered negative. Western blot assay for CX3CR1 protein expression Tissues were extracted using the whole protein extraction kit KGP250 (Nanjing KGI Biological Technology Development Co. Ltd.). Extracted proteins (X amount) were mixed with loading buffer, denatured, and polyacrylamide gel electrophoresis performed to separate the proteins. The gel was blotted to a nitrocellulose membrane, the membrane was stained with Ponceau red, and the band(s) of interest excised and blocked with 8% skim milk. Rabbit anti-human CX3CR1 antibody (Abcam Co. Ltd.) was added and incubated overnight at 4. The membrane was washed and goat anti-rabbit IgG (Jackson Co. Ltd.) added and incubated for X time at Y temperature. The membrane was washed and electrochemiluminescence (ECL) was used to detect the labelled protein(s). Statistical analysis SPSS17.0 was used for statistical analysis of the data, and the t-test was used where needed to compare data sets. P < 0.05 was considered statistically significant. RESULTS CX3CL1 expression in PNI-positive and negative groups (ELISA) Samples were divided into PNI-positive and PNI-negative groups based on CX3CL1 expression in the tumour and adjacent tissues detected by ELISA (Table 1). Of the 30 cases of gastric carcinoma, 14 cases were PNI-positive (46.7%). In tumour tissues, CX3CL1 levels in the PNIpositive group were significantly higher than those in the PNI-negative group (P < 0.05). When the PNI-negative and PNI-positive groups were compared, CX3CL1 expression in the tumour tissue was significantly higher (P < 0.05) than that in the adjacent tissue. CX3CR1 expression in the PNI-positive and negative groups (immunohistochemistry) CX3CR1 expression was evaluated in the tumour and adjacent tissues (Figure 1). Tumour tissues from the PNInegative group contained CX3CR1 positive cells and nerve cells were unstained. In the PNI-positive group, CX3CR1 expression was seen in both tumour and nerve cells. CX3CR1 expression levels in the tumour tissues and adjacent tissues of PNI positive/negative groups by Western blot assay CX3CR1 expression in tumour tissues from the PNI positive/negative groups was higher than that in adjacent tissues (Figure 2) and the expression of CX3CR April 21, 2014 Volume 20 Issue 15

331 Lv CY et al. CX3CL1/CX3CR1 and gastric carcinoma/gastric carcinoma perineural invasion A B Figure 1 CX3CR1 expression. A: CX3CR1 expression ( 200) in perineural invasion (PNI)-positive tumour cells and nerve cells; B: CX3CR1 expression was present in tumour cells and absent in nerve cells from the PNI-negative group ( 200). A B a b c d a b c d Figure 2 CX3CR1 expression levels in the perineural invasion-positive group (A) and perineural invasion-negative groups (B). a and b are from tissues adjacent to the tumour, while c and d are tumour tissues, and β-actin is shown below. Table 2 Comparison of CX3CR1 levels in gastric carcinoma perineural invasion positive/negative groups in the tumour tissues from the PNI-positive group was significantly higher than that in the PNI-negative group (P < 0.01) (Table 2). No statistically significant differences were seen in CX3CR1 levels in the adjacent tissues of the PNI-positive and negative groups (P > 0.05). Mean CX3CR1-positive values in the PNI positive/negative groups were significantly higher than those in the adjacent tissues, and the difference was statistically significant (P < 0.01). DISCUSSION PNI positive group (mean ± SD) PNI negative group (mean ± SD) Tumor tissues 1.01 ± ± (P < 0.01) Adjacent tissues 0.34 ± ± (P > 0.05) t (P < 0.01) (P < 0.01) PNI: Perineural invasion. PNI is significantly correlated with a poor prognosis in prostate, pancreatic and especially head and neck cancer patients, and is an important pathological index, indicating a shorter survival period, increased local recurrence rate, and shorter recurrence time [3]. While PNI is an important factor which affects survival, according to the t reports of the Association of American Pathologists [4], head and neck cancers are the only tumours which require the assessment of the perineural invasion state. The relationship between PNI and the prognosis of gastric carcinoma patients has attracted more attention in recent years, and although no consensus has been reached, some scholars have indicated that PNI is not an independent factor affecting prognosis [5]. Clinical pathology data [6] from 178 cases of gastric carcinoma revealed that overall survival time and disease-free survival time in PNI-positive patients were statistically different when compared with PNI-negative patients. Multi-factor analysis also revealed that PNI was an independent risk factor affecting the prognosis of gastric carcinoma patients, which was consistent with data reported by Bilici et al [7] and Tianhang et al [8]. Although there was significant correlation between PNI and the prognosis of tumour patients, the pathogenesis is still unclear. Among the 4 chemokine subfamilies, relatively little work has been carried out on CX3CL1. CX3CL1 is reported [9] to have a dual function, acting as a chemical inducer to stimulate the anti-tumour effect of leukocytes, while the combination of CX3CL1 and CX3CR1 can induce the aggregation/adherence of NK cells, CD8+T and CD4+T on/near tumour cells, where they can exert their anti-tumoural effect. However, little has been published on the role of CX3CL1 in clinical assessments. Ohta et al [10] found that colorectal cancer patients with high expression of CX3CL1 had a higher density 4430 April 21, 2014 Volume 20 Issue 15

332 Lv CY et al. CX3CL1/CX3CR1 and gastric carcinoma/gastric carcinoma perineural invasion of tumour-infiltrating immune cells, and therefore better prognosis than patients with low CX3CL expression. Others [11] detected CX3CL1 expression by immunohistochemistry, and combined with the prognosis, suggested patients with high CX3CL1 expression had a longer survival time than those with low expression. Vitale et al [12] reported that invasion and metastasis of colon cancer cells which expressed CX3CL1 was reduced, and the growth of metastases slowed, the membrane type and secreting type performed different roles in tumour development. For gastric carcinoma, few reports exploring the relationship between CX3CL1 and gastric carcinoma have been published. Hyakudomi [13] evaluated CX3CL1 expression in 158 cases of T2/T3 stage gastric carcinoma and used two antibodies (anti-cd57 and anti-cd8) to assess the infiltration of NK cells and CD8+T cells, respectively. These data, combined with clinical pathology and follow-up, suggested that patients in the CX3CL1- high-expression group had a better prognosis and disease-free survival period compared to the CX3CL1- low-expression group. Multivariate analysis showed the expression of CX3CL1 was the independent factor which affected the disease-free survival period of gastric carcinoma patients. In this study, ELISA data indicated that CX3CL1 content in the tumour tissues, irrespective of PNI-negative or PNI-positive, was higher than in adjacent tissues. Western blotting revealed that expression of the receptor CX3CR1 in PNI-positive and negative groups was significantly higher in the tumour tissues than in the adjacent tissues. Taken together, these data suggested that CX3CL1/ CX3CR1 may be associated with the occurrence and development of gastric carcinoma. Another function of CX3CL1 is to act as an adhesion molecule, and combined with the receptors expressed by tumour cells, may be the key factor leading to the clinical differences seen in the CX3CL1/CX3CR1 system response against tumours [9]. In certain tumours, the potential anticancer effect of CX3CL1 was overcome by its actions promoting tumour development. In prostate cancer, CX3CL1 expressed by bone marrow endothelial cells could induce the aggregation of CX3CR1 inside tumour cells, playing an important role in the bone metastasis of prostate cancer [14]. In breast cancer, although there was no significant correlation between CX3CL1/ CX3CR1 and the overall survival rate and disease-free survival rate of patients, expression did indicate the occurrence of brain metastases [15]. Recently, the CX3CL1/ CX3CR1 system has been reported to play an important role in the bone metastasis of neuroblastoma [16]. The adhesion effect of CX3CL1 in the nervous system also suggests a role in nerves peripheral to other tumours, which may be an underrated source of metastases. In addition to the traditional routes of tumour metastasis (blood, lymphatic and implantation metastasis), cancer cells could also move along the nerve (PNI). Literature addressing the relationship between CX- 3CL1 and PNI in digestive tract tumours primarily studied pro-nerve tumours, such as pancreatic ductal adenocarcinoma (PDA) which accounted for the majority, while no relevant literature on tumours in the gastrointestinal tract have been found. In PDA, PNI occurs in the early stage of the disease [17-19], and when biopsies are evaluated, tumour cells show an elevated expression of CX3CR1 compared to normal pancreatic cells. When examined in vitro, the CX3CR1-positive PDA cells directionally migrate towards the CX3CL1 expressed on other cells and specifically adhere to the nerve cells, where pathogenesis involved the activation of a Gi protein and adhesion molecules (β1 integrin and focal adhesion kinase) [17]. Postoperative pathology confirmed that for PNI-positive pancreatic cancer, 90% were CX3CR1 positive and 56% exhibited a strong positive expression, while systematic assessment of the relationship between tumour CX- 3CR1 expression and PNI revealed that elevated levels of CX3CR1 were closely related to the early localized recurrence of the tumour. In this study, the results of immunohistochemistry revealed that when PNI occurred, tumour and nerve cells expressed CX3CR1, whereas when PNI was absent, CX3CR1 expression was seen in tumour cells only. ELISA and Western blotting of CX3CL1/CX3CR1 revealed that CX3CL1 and CX3CR1 expression in tumour tissues was significantly higher in the PNI-positive group compared to the PNI-negative group. No statistical significance was seen for CX3CL1 and CX3CR1 in the adjacent tissues, suggesting that high expression of CX3CL1/CX3CR1 may be correlated with gastric carcinoma PNI. However, whether expression of the CX- 3CL1/CX3CR1 system was involved in the stimulation of gastric carcinoma-associated PNI could not be clearly determined in this study, and will be further explored. In summary, this research determined the relationship between the CX3CL1/CX3CR1 system and gastric carcinoma/gastric carcinoma PNI, and indicated that PNI may be related to the occurrence and development of gastric carcinoma. Although overexpression could be significantly associated with gastric carcinoma PNI, how the combination of CX3CL1 and CX3CR1 complete signal transduction inside the intracellular pathway, whether or not it is involved in the occurrence of PNI, and the specific relationship between CX3CL1/CX3CR1 and the prognosis of gastric carcinoma should be studied further. COMMENTS Background Gastric carcinoma is one of the most common malignant tumours, and easily undergoes metastasis. Perineural invasion (PNI) occurs when the tumour approaches nerves and encompasses at least 33% of the nerve perimeter or tumour cells penetrate any of the 3 nerve sheath layers. Research frontiers With respect to the pathogenesis of gastric carcinoma and gastric carcinoma PNI, the role of chemokines has attracted the attention of researchers. However, relative to other subfamilies, the current research on CX3CL1 and its 4431 April 21, 2014 Volume 20 Issue 15

333 Lv CY et al. CX3CL1/CX3CR1 and gastric carcinoma/gastric carcinoma perineural invasion receptor, CX3CR1, is minimal. Innovations and breakthroughs This research determined the relationship between the CX3CL1/CX3CR1 system and gastric carcinoma/gastric carcinoma PNI. The data suggested that CX3CL1/CX3CR1 expression may be associated with the occurrence and development of gastric carcinoma and also plays a role in gastric carcinoma PNI. Applications The CX3CL1/CX3CR1 system plays an important role in a series of physiological and pathological processes. The dynamic changes, regulatory mechanisms and clinical significance of the CX3CL1/CX3CR1 system and its interaction with other chemokines and cytokines have significant potential application in preventing and treating cancer and PNI. The development of a CX3CL1 gene knockout/transgenic or monoclonal antibody technology, and directing them toward clinical applications, will be the focus of future research. Peer review In this manuscript, the authors performed a preliminary study on the correlation between CX3CL1/CX3CR1 and gastric carcinoma/gastric carcinoma PNI. The manuscript is interesting in that it explores the potential of using an assessment of the CX3CL1/CX3CR1 system to the prevention and treatment of cancer and PNI. REFERENCES 1 Cruveilhier J. Maladies des Nerfs: anatomie Pathologique du Corp Humain. 2nd ed. Paris: Bailliere JB, 1835: Liebig C, Ayala G, Wilks JA, Berger DH, Albo D. Perineural invasion in cancer: a review of the literature. Cancer 2009; 115: [PMID: DOI: /cncr.24396] 3 Rodin AE, Larson DL, Roberts DK. Nature of the perineural space invaded by prostatic carcinoma. Cancer 1967; 20: [PMID: ] 4 Pilch BZ, Gillies E, Houck JR Jr, Min KW, Novis D, Shah J, Zarbo RJ, Wenig MB. Upper aerodigestic tract: Head and Neck. New York: College of American Pathologists, 2005: Johnston M, Yu E, Kim J. Perineural invasion and spread in head and neck cancer. 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Nature 1997; 387: [PMID: DOI: /42491] 10 Ohta M, Tanaka F, Yamaguchi H, Sadanaga N, Inoue H, Mori M. The high expression of Fractalkine results in a better prognosis for colorectal cancer patients. Int J Oncol 2005; 26: [PMID: ] 11 Zeng SQ, Cao J, Zhang XY, Zeng J, Chen JP, Zhang WJ, Liu HJ. Increased expression of CX3CL1 is correlated with a better prognosis in colon carcinoma patients. Redai Yixue Zazhi 2010; 10: Vitale S, Cambien B, Karimdjee BF, Barthel R, Staccini P, Luci C, Breittmayer V, Anjuère F, Schmid-Alliana A, Schmid- Antomarchi H. Tissue-specific differential antitumour effect of molecular forms of fractalkine in a mouse model of metastatic colon cancer. Gut 2007; 56: [PMID: DOI: /gut ] 13 Hyakudomi M, Matsubara T, Hyakudomi R, Yamamoto T, Kinugasa S, Yamanoi A, Maruyama R, Tanaka T. Increased expression of fractalkine is correlated with a better prognosis and an increased number of both CD8+ T cells and natural killer cells in gastric adenocarcinoma. Ann Surg Oncol 2008; 15: [PMID: DOI: / s ] 14 Shulby SA, Dolloff NG, Stearns ME, Meucci O, Fatatis A. CX3CR1-fractalkine expression regulates cellular mechanisms involved in adhesion, migration, and survival of human prostate cancer cells. Cancer Res 2004; 64: [PMID: DOI: / CAN ] 15 Andre F, Cabioglu N, Assi H, Sabourin JC, Delaloge S, Sahin A, Broglio K, Spano JP, Combadiere C, Bucana C, Soria JC, Cristofanilli M. Expression of chemokine receptors predicts the site of metastatic relapse in patients with axillary node positive primary breast cancer. Ann Oncol 2006; 17: [PMID: DOI: /annonc/mdl053] 16 Nevo I, Sagi-Assif O, Meshel T, Ben-Baruch A, Jöhrer K, Greil R, Trejo LE, Kharenko O, Feinmesser M, Yron I, Witz IP. The involvement of the fractalkine receptor in the transmigration of neuroblastoma cells through bone-marrow endothelial cells. 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334 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH REPORT Anatomic resection of liver segments 6-8 for hepatocellular carcinoma Chang-Ku Jia, Jie Weng, You-Ke Chen, Yu Fu Chang-Ku Jia, Jie Weng, You-Ke Chen, Yu Fu, Department of Hepatobiliary Pancreatic Surgery, the Affiliated Hospital of Hainan Medical College, Haikou , Hainan Province, China Author contributions: Jia CK, as a chief surgeon, performed the operation; Jia CK and Weng J designed the study; Weng J wrote the manuscript; Chen YK and Fu Y revised the manuscript. Supported by A grant from the Science and Technology Projects of Education Department of Hainan Province, China, No. Hjkj Correspondence to: Chang-Ku Jia, MD, Department of Hepatobiliary Pancreatic Surgery, the Affiliated Hospital of Hainan Medical University, 31 Longhua Road, Haikou , Hainan Province, China. jiachk@126.com Telephone: Fax: Received: November 3, 2013 Revised: December 25, 2013 Accepted: February 20, 2014 Published online: April 21, 2014 Abstract AIM: To report the devised anatomic liver resection of segments 6, 7 and 8 to improve the resection rate for patients with right liver tumors. METHODS: We performed anatomic liver resection of segments 6, 7 and 8 to guarantee the maximum preservation of the remaining normal liver tissue. Segment 5 was determined by two steps of Glissonean pedicle occlusion. And a shaped broken resection line was marked upon the diaphragmatic surface of the liver. Selective right hemihepatic inflow occlusion was used to reduce blood loss during parenchymal transection between segments 6 and 5 and between segments 8 and 5. If needed, total hepatic Glissonean pedicle occlusion was used during parenchymal transection between segment 8 and the left liver. RESULTS: Compared to right hemihepatectomy, the percentage of future liver remnant volume was increased by an average of 13.9% if resection of segments 6, 7 and 8 was performed. Resection of segments 6, 7 and 8 was completed uneventfully. After hepatectomy, the inflow and outflow of segment 5 were maintained. There was no perioperative mortality, postoperative abdominal bleeding or bile leakage in this group. Alpha-fetoprotein (AFP) returned to the normal range within 2 mo after the operation in all the patients. One patient died 383 d postoperatively due to obstructive suppurative cholangitis. One patient suffered from severe liver dysfunction shortly after surgery and had intrahepatic recurrence 4 mo postoperatively. Postoperative lung metastasis was found in one patient. No tumor recurrence was found in the other patients and the parameters including liver function and AFP level were in the normal range. CONCLUSION: Anatomic liver resection of segments 6, 7 and 8 can be a conventional operation to improve the overall resection rate for hepatocellular carcinoma Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Anatomic hepatectomy; Hepatocellular carcinoma; Selective occlusion; Alpha-fetoprotein; Liver tumor Core tip: Hepatic resection is the only curative treatment for patients with huge and multifocal tumors. However, patients with huge or multifocal tumors in the right liver and with a small volume of left liver cannot undergo right hemihepatectomy because of the possibility of postoperative liver failure, thus leading to a low overall resection rate for hepatocellular carcinoma. To increase the number of resectable patients and improve the overall resection rate, we devised anatomic liver resection of segments 6, 7 and 8 in patients with right liver tumors. Jia CK, Weng J, Chen YK, Fu Y. Anatomic resection of liver segments 6-8 for hepatocellular carcinoma. World J Gastroenterol 2014; 20(15): Available from: URL: April 21, 2014 Volume 20 Issue 15

335 Jia CK et al. Anatomic segmentectomy for HCC wjgnet.com/ /full/v20/i15/4433.htm DOI: dx.doi.org/ /wjg.v20.i INTRODUCTION Hepatic resection is the only curative treatment for patients with huge and multifocal tumors. These patients are less likely to benefit from liver transplantation or local ablative therapy [1-4]. In many such cases, complete resection of hepatocellular carcinoma (HCC) can only be achieved by hepatectomy that removes a significant proportion of the liver parenchyma. Recent advances in preoperative imaging, understanding of liver anatomy, surgical techniques, and anesthetic monitoring have increased the safety of liver resection [5-7]. Unfortunately, in some cases, hepatic failure occurs after major liver resection and carries poor prognosis due to the lower remnant liver volume [8]. So it restrains those patients who have much less remnant liver volume from radical operations. For example, patients with huge or multifocal tumors in the right liver and with a small volume of left liver cannot undergo right hemihepatectomy because of possible postoperative liver failure. To increase the number of resectable patients, we devised anatomic liver resection of segments 6, 7 and 8 using the technique of selective occlusion of hepatic inflow in patients with right liver tumors. This is a radical operation and benefits patients with huge or multifocal tumors in the right lobe. MATERIALS AND METHODS Patients Six patients undergoing anatomic liver resection of segments 6, 7 and 8 from December 2011 to November 2013 in our department were included in this study. All these patients were male with a mean age of 55 years (range: years), and had a history of hepatitis B virus infection. All the tumors were located in the right liver with multifocal tumors in two patients and huge tumors in four (Table 1). Preoperative imaging showed that segment 5 was free of tumor in all patients (Figure 1). Serum α-fetoprotein (AFP) level was elevated in all patients. Conventional follow-up was done, ranging from 6 to 23 mo. Preoperative assessment Liver function, hepatic functional reserve and hepatic imaging, including ultrasound B and computed tomography (CT), were carried out preoperatively. Liver function was classified by Child-Pugh score and hepatic functional reserve was evaluated by the indocyanine green retention at 15 min (ICG-R15) test. Contrast-enhanced CT scans were generated with a helical scanner. Manual 3D reconstructions of the liver were made using reconstructed 5-mm-thick axial slices from 2-3-mm original slices. The total liver, left liver and segment 5, as well as the tumors were manually outlined using portal and hepatic veins as landmarks for segmental division [9]. The volumes of the total liver, left liver, segment 5 and tumor were calculated. The percentage of future liver remnant volume (%FLRV) was expressed using the formula: %FLRV = (remnant liver volume 100)/(total liver volume - tumor volume) [10]. If right hemihepatectomy was performed, %FLRV would be < 40% in all of the patients. The risk of postoperative liver failure would be high due to insufficient remnant functional liver because all these cases had liver cirrhosis. However, if resection of segments 6, 7 and 8 was performed, %FLRV would increase by an average of 13.9% (Table 1). The risk of postoperative liver failure would be low due to sufficient remnant functional liver. Therefore, we designed anatomic resection of liver segments 6, 7 and 8, with retention of segment 5, to guarantee the maximum preservation of remnant functional liver tissue. Surgical procedures Selective occlusion of hepatic inflow was the key to complete anatomic liver resection of segments 6, 7 and 8. Segment 5 was determined by two steps of Glissonean pedicle occlusion. After cholecystectomy, the porta hepatis was dissected based on Glissonean pedicle anatomy [11-13]. The right hemihepatic Glissonean pedicle and the segment 6 and 7 Glissonean pedicle were sequentially divided (Figure 2A). After occlusion of the right hemihepatic Glissonean pedicle, the right liver developed obvious ischemia. So, the demarcation between the right and left liver could be easily determined (Figure 2B). After demarcation, the right hemihepatic Glissonean pedicle was left unoccluded. Then the segment 6 and 7 Glissonean pedicle was dissected and ligated, and segments 6 and 7 showed obvious ischemia (Figure 2C). The interface between segments 6 and 5 could be easily demarcated. Intraoperative ultrasound B was used to form the demarcation between segments 8 and 5, with a transverse marked line upon the diaphragmatic surface of the liver. This line was located 1-2 cm below the tumor in segment 8. Finally, segment 5 was determined and a shaped broken resection line was marked upon the diaphragmatic surface of the liver (Figure 2D). Short hepatic veins were dissected, isolated and ligated. Parenchymal transection was performed along the broken resection line using an ultrasonic scalpel and cavitron ultrasonic surgical aspirator. Selective right hemihepatic inflow occlusion was used to reduce blood loss during parenchymal transection between segments 6 and 5 and between segments 8 and 5. If needed, total hepatic Glissonean pedicle occlusion was used during parenchymal transection between segment 8 and the left liver. Postoperative treatment Postoperative follow-up was done with conventional liver function tests and measurement of serum AFP level. Abdominal ultrasonography and CT were performed at intervals of 30 d in the first 3 mo postoperatively, and at intervals of 2-3 mo thereafter. Chest X-ray, whole-body 4434 April 21, 2014 Volume 20 Issue 15

336 Jia CK et al. Anatomic segmentectomy for HCC Table 1 Clinical features and postoperative outcomes No. Sex Age (yr) Diagnosis ICG-R15 Child-Pugh liver function grade %FLRV for right hemihepatectomy %FLRV for 6-8 segmentectomy Survival period (d) 1 Male 61 Multifocal tumors 6.8% A , DFS 2 Male 43 Huge tumor, 12.2 cm 9.3 cm 5.5% A , dead 3 Male 59 Huge tumor, 11.5 cm 10.7 cm 13.8% B , intrahepatic recurrence, alive 4 Male 54 Huge tumor, 13.2 cm 10.6 cm 8.3% A , lung metastasis, alive 5 Male 53 Multifocal tumors 2.6% A , DFS 6 Male 60 Huge tumor, 13.5 cm 11.6 cm 7.9% A , DFS DFS: Disease-free survival; FLRV: Future liver remnant volume. A B Figure 1 Preoperative imaging showed that segment 5 was free of tumor in all patients. A: Multiple tumors were found in segments 6, 7 and 8 by contrastenhanced computed tomography scans (arrows) in Case 1; B: Huge tumor located in the right liver in Case 4. Arrow: Middle hepatic vein; Arrowhead: Right anterior portal vein; Triangular arrow: Right posterior portal vein; Curved arrow: Gallbladder April 21, 2014 Volume 20 Issue 15

337 Jia CK et al. Anatomic segmentectomy for HCC A B C1 D C2 Figure 2 Surgical procedures. A: Right hemihepatic Glissonean pedicle and segment 6 and 7 Glissonean pedicle in Case 4 were sequentially divided. Arrow: Segment 6 and 7 Glissonean pedicle; arrowhead: Segment 5 and 8 Glissonean pedicle; curved arrow: Right hemihepatic Glissonean pedicle; B: After occlusion of the right hemihepatic Glissonean pedicle in Case 4, the right liver showed obvious ischemia. The demarcation between the right and left liver could be easily determined (arrowheads); C: After the segment 6 and 7 Glissonean pedicle of Case 4 was ligated, segments 6 and 7 showed obvious ischemia. The interface between segments 6 and 5 could be easily demarcated. Arrow: Demarcation between segments 6 and 5 upon the diaphragmatic surface of the liver; Arrowheads: Demarcation between the right and left liver; Bold arrows: Demarcation between segments 6 and 5 on the visceral surface of the liver; Curved arrow: Fossa of gallbladder (C1, C2); D: Segment 5 was determined and a shaped broken resection line was marked upon the diaphragmatic surface of the liver in Case 4. Arrowheads: Demarcation between segments 5 and 8; Arrow: Segment 5. bone scan, or other imaging studies were undertaken in patients who had suspected metastasis. All the patients received three sessions of postoperative transcatheter arterial chemoembolization (TACE). If affordable, sorafenib, a multi-targeted tyrosine kinase receptor inhibitor was used for metastatic or recurrent cases. RESULTS All hepatectomies were uneventfully completed with an average operation time of 326 min (range: min) and average blood loss of 758 ml (range: ml). After hepatectomy, the inflow and outflow of segment 5 were maintained (Figure 3A). Gross specimens showed that tumors were completely resected and postoperative pathology verified a diagnosis of HCC (Figure 3B). There was no perioperative mortality, postoperative abdominal bleeding, or bile leakage. Serum AFP decreased to the normal range within 2 mo postoperatively. Case 2 survived well with normal AFP level and liver function for about 12 mo postoperatively. However, he was hospitalized 381 d postoperatively due to fever and 15 d progressive elevation of jaundice at home without any treatment. CT and ultrasound B showed no tumor recurrence and metastasis inside or outside the liver. Serum total bilirubin was μmol/l (normal range: μmol/l) 4436 April 21, 2014 Volume 20 Issue 15

338 Jia CK et al. Anatomic segmentectomy for HCC A B Figure 3 Surgical results. A: After hepatectomy, the inflow and outflow of segment 5 were maintained. Triangular arrow: remnant segment 5 in Case 1; arrow: Remnant segment 5 in Case 4; B: Gross specimen showed that tumors in Case 4 were completely resected. Segments 6, 7 and 8 are indicated. at admission with direct bilirubin being μmol/l (normal range: 0-6 μmol/l). Two days after admission, this patient died from obstructive suppurative cholangitis. Case 3 had Child-Pugh score 7 and 13.8% ICG, which indicated severe postoperative liver dysfunction. His serum total bilirubin increased constantly and peaked at μmol/l on postoperative day 8. Ascites and hepatoencephalopathy occurred consecutively due to liver dysfunction. These symptoms resolved gradually as liver dysfunction improved. However, intrahepatic recurrence was found by CT and digital substraction angiography 4 mo postoperatively. Although AFP level increased to ng/ml at that time, it decreased to the normal range after two sessions of TACE. To date, this tumorbearing patient has had > 6 mo survival. Lung metastasis was found by CT 5 mo postoperatively in Case 4. Sorafenib was administered to this patient. To date, this patient has had > 4 mo tumor-bearing survival with AFP in the normal range. No tumor recurrence was found in the other patients and the parameters including liver function and AFP level were in the normal range. Postoperative outcome and quality of life are summarized in Table 1. DISCUSSION Inadequate future liver remnant may not meet the hepatic metabolic demands after major hepatectomy [14,15]. Therefore, patients with huge or multifocal tumors in the right liver and a small volume of left liver cannot undergo right hemihepatectomy because of possible postoperative liver failure. These patients are excluded from radical operations. In the present study, all %FLRV would have been < 40% if right hemihepatectomy was performed. The risk of postoperative liver failure would have been high because of liver cirrhosis and insufficient remnant functional liver. However, if resection of segments 6, 7 and 8 was performed, %FLRV would increase by an average of 13.9%. The risk of postoperative liver failure would be low because of sufficient remnant functional liver. Therefore, all patients underwent anatomic liver resection of segments 6, 7 and 8, with retention of segment 5. The patients underwent radical surgery with tumor-free margins and maximum preservation of remnant functional liver tissue [16-19]. This technique could improve the resection rate in patients with huge or multifocal tumors in the right liver. No remnant tumor was found on imaging shortly after the operation and AFP decreased to the normal range within 2 mo postoperatively. The tumors were completely resected by anatomic liver resection of segments 6, 7 and 8. These six patients have survived > 6 mo with satisfactory quality of life postoperatively. We demonstrated that this operation had a good therapeutic efficacy April 21, 2014 Volume 20 Issue 15

339 Jia CK et al. Anatomic segmentectomy for HCC It is worth mentioning that the hepatectomies were performed with a shaped broken resection line. This naturally increased the resection length, area, operation time and blood loss. However, the blood loss in our study (mean: 758 ml) was no more than that previously reported for huge HCC (e.g., 1015 [20] and 780 ml [21] ). There were no severe complications such as perioperative mortality, postoperative abdominal bleeding, and bile leakage. We demonstrated that liver resection of segments 6, 7 and 8 is safe and effective for the treatment of HCC. Anatomic liver resection of segments 6, 7 and 8 could become a conventional operation for patients whose tumors do not involve segment 5, to enable maximum preservation of remaining normal liver tissue. It may be an alternative modality for maximal hepatectomy in the treatment of HCC. Finally, selective occlusion of right hemihepatic inflow was applied during parenchymal transection between segments 6 and 5 and between segments 8 and 5. If needed, total hepatic inflow occlusion was used during parenchymal transection between segment 8 and the left liver. Selective right hemihepatic Glissonean pedicle occlusion enables blood inflow to the left liver and avoids splanchnic stasis during hepatectomy [22-24]. Thus, liver ischemia-reperfusion injury and hemodynamic instability were reduced during the operation. It particularly benefits patients with cirrhosis [25-27]. COMMENTS Background Hepatic resection is the only curative treatment for patients with huge and multifocal tumors. However, patients with huge or multifocal tumors in the right liver and a small volume of left liver cannot undergo right hemihepatectomy because of possible postoperative liver failure, thus making the overall resection rate for hepatocellular carcinoma (HCC) low. To increase the number of resectable patients, we devised anatomic liver resection of segments 6, 7 and 8 in patients with right liver tumors. Research frontiers Anatomic liver resection of segments 6, 7 and 8 has not been reported previously. By the techniques of selective occlusion of hepatic inflow and anatomic liver resection, complicated hepatectomy can be performed. Anatomic liver resection of segments 6, 7 and 8 could be a conventional technique for patients whose tumors do not involves segment 5, to enable maximum preservation of remaining normal liver tissue, thus improving the overall resection rate for HCC. Innovations and breakthroughs Compared to right hemihepatectomy, the percentage of future liver remnant volume (%FLRV) was increased by an average of 13.9% when resection of segments 6, 7 and 8 was performed. Segmentectomy was completed uneventfully in this group. Segment 5 was determined by two steps of hepatic inflow occlusion. A shaped broken resection line was marked upon the diaphragmatic surface of the liver. Selective right hemihepatic inflow occlusion was used to reduce blood loss during parenchymal transection between segments 6 and 5 and between segments 8 and 5. If needed, total hepatic Glissonean pedicle occlusion was used during parenchymal transection between segment 8 and the left liver. Thus, liver ischemia-reperfusion injury and hemodynamic instability were maximally reduced during the operation. It particularly benefits patients with cirrhosis. Applications Patients with huge or multifocal tumors in the right liver and with a small volume of left liver cannot undergo right hemihepatectomy because of the possibility of postoperative liver failure. To improve the overall resection rate for HCC, anatomic resection of segments 6, 7 and 8 can be performed in these cases. This operation is radical and benefits these patients because it avoids postoperative liver failure. Terminology %FLRV is an important measure for patients undergoing major liver resection. Selective occlusion of hepatic inflow was the key procedure for anatomic liver resection. Peer review Partial hepatectomy is a powerful tool for HCC patients. Selective resection of right anterior/posterior segments often requires an experienced surgeon. The procedures may involve a unique technique. Surgical technique of parenchymasparing liver resection for right liver HCC (huge or multiple) developed on liver cirrhosis. This is a description of an atypical technique of anatomic liver resection. REFERENCES 1 Livraghi T, Meloni F, Di Stasi M, Rolle E, Solbiati L, Tinelli C, Rossi S. Sustained complete response and complications rates after radiofrequency ablation of very early hepatocellular carcinoma in cirrhosis: Is resection still the treatment of choice? 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340 Jia CK et al. Anatomic segmentectomy for HCC Masuda T, Imai K, Mima K, Nakagawa S, Kuroki H, Nitta H, Ishiko T, Hashimoto D, Yamashita Y, Baba H. Remnant liver volume-based predictors of postoperative liver dysfunction after hepatectomy: analysis of 625 consecutive patients from a single institution. Int J Clin Oncol 2013; Epub ahead of print [PMID: DOI: /s ] 11 Takasaki K. Glissonean pedicle transection method for hepatic resection: a new concept of liver segmentation. J Hepatobiliary Pancreat Surg 1998; 5: [PMID: DOI: /s ] 12 Karamarković A, Doklestić K, Milić N, Djukić V, Bumbasirević V, Sijački A, Gregorić P, Bajec D. Glissonean pedicle approach in major liver resections. Hepatogastroenterology 2012; 59: [PMID: DOI: /hge12198] 13 Katagiri S, Ariizumi S, Kotera Y, Takahashi Y, Yamamoto M. Right hepatectomy using Glissonean pedicle transection method with anterior approach (with video). J Hepatobiliary Pancreat Sci 2012; 19: [PMID: DOI: / s y] 14 Chun YS, Ribero D, Abdalla EK, Madoff DC, Mortenson MM, Wei SH, Vauthey JN. Comparison of two methods of future liver remnant volume measurement. J Gastrointest Surg 2008; 12: [PMID: DOI: /s ] 15 Di Domenico S, Santori G, Balbis E, Traverso N, Gentile R, Bocca B, Gelli M, Andorno E, Cottalasso D, Valente U. Biochemical and morphologic effects after extended liver resection in rats: preliminary results. Transplant Proc 2010; 42: [PMID: DOI: /j.transproceed ] 16 Chen J, Huang K, Wu J, Zhu H, Shi Y, Wang Y, Zhao G. Survival after anatomic resection versus nonanatomic resection for hepatocellular carcinoma: a meta-analysis. Dig Dis Sci 2011; 56: [PMID: DOI: /s ] 17 Hasegawa K, Kokudo N, Imamura H, Matsuyama Y, Aoki T, Minagawa M, Sano K, Sugawara Y, Takayama T, Makuuchi M. Prognostic impact of anatomic resection for hepatocellular carcinoma. Ann Surg 2005; 242: [PMID: DOI: /01.sla ] 18 Agrawal S, Belghiti J. Oncologic resection for malignant tumors of the liver. Ann Surg 2011; 253: [PMID: DOI: /SLA.0b013e3181fc08ca] 19 Nanashima A, Sumida Y, Abo T, Nagasaki T, Tobinaga S, Fukuoka H, Takeshita H, Hidaka S, Tanaka K, Sawai T, Yasutake T, Nagayasu T. Comparison of survival between anatomic and non-anatomic liver resection in patients with hepatocellular carcinoma: significance of surgical margin in non-anatomic resection. Acta Chir Belg 2008; 108: [PMID: ] 20 Liau KH, Ruo L, Shia J, Padela A, Gonen M, Jarnagin WR, Fong Y, D Angelica MI, Blumgart LH, DeMatteo RP. Outcome of partial hepatectomy for large (& gt; 10 cm) hepatocellular carcinoma. Cancer 2005; 104: [PMID: DOI: /cncr.21415] 21 Chen X, Wu Z, Qiu F. Hepatectomy for huge primary liver cancer: report of 171 patients. Zhonghua Waike Zazhi 2000; 38: 6-9 [PMID: ] 22 Giordano M, Lopez-Ben S, Codina-Barreras A, Pardina B, Falgueras L, Torres-Bahi S, Albiol M, Castro E, Figueras J. Extra- Glissonian approach in liver resection. HPB (Oxford) 2010; 12: [PMID: DOI: /j x] 23 Dello SA, Reisinger KW, van Dam RM, Bemelmans MH, van Kuppevelt TH, van den Broek MA, Olde Damink SW, Poeze M, Buurman WA, Dejong CH. Total intermittent Pringle maneuver during liver resection can induce intestinal epithelial cell damage and endotoxemia. PLoS One 2012; 7: e30539 [PMID: DOI: /journal.pone ] 24 Yanaga K, Matsumata T, Nishizaki T, Shimada M, Sugimachi K. Alternate hemihepatic vascular control technique for hepatic resection. Am J Surg 1993; 165: [PMID: ] 25 Rahbari NN, Wente MN, Schemmer P, Diener MK, Hoffmann K, Motschall E, Schmidt J, Weitz J, Büchler MW. Systematic review and meta-analysis of the effect of portal triad clamping on outcome after hepatic resection. Br J Surg 2008; 95: [PMID: DOI: /bjs.6141] 26 Wang HQ, Yang JY, Yan LN. Hemihepatic versus total hepatic inflow occlusion during hepatectomy: a systematic review and meta-analysis. World J Gastroenterol 2011; 17: [PMID: ] 27 Arkadopoulos N, Kyriazi MA, Theodoraki K, Vassiliou P, Perelas A, Vassiliou I, Smyrniotis V. Central hepatectomy under sequential hemihepatic control. Langenbecks Arch Surg 2012; 397: [PMID: DOI: /s y] P- Reviewers: Hori T, Regimbeau JM, Wang DS S- Editor: Gou SX L- Editor: Wang TQ E- Editor: Liu XM 4439 April 21, 2014 Volume 20 Issue 15

341 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. Death decoy receptor overexpression and increased malignancy risk in colorectal cancer RESEARCH BRIEF ARTICLE REPORT Liang Zong, Ping Chen, Da-Xin Wang Liang Zong, Ping Chen, Department of Gastrointestinal Surgery, Su Bei People s Hospital of Jiangsu Province, Yangzhou University, Yangzhou , Jiangsu Province, China Liang Zong, Department of Gastrointestinal Surgery, Graduate School of Medicine, University of Tokyo, Tokyo , Japan Da-Xin Wang, Institute of Biomedical Engineering, Yangzhou University, Yangzhou , Jiangsu Province, China Author contributions: Zong L, Chen P and Wang DX contributed equally to study conception, data collection and analysis, and writing of the manuscript. Correspondence to: Liang Zong, MD, PhD, Department of Gastrointestinal Surgery, Su Bei People s Hospital of Jiangsu Province, Yangzhou University, No. 98 Nantong West Road, Yangzhou , Jiangsu Province, China @qq.com Telephone: Fax: Received: September 2, 2013 Revised: December 6, 2013 Accepted: January 8, 2014 Published online: April 21, 2014 Abstract AIM: To evaluate human epidermal growth factor receptor 2 (HER2) and death decoy receptor (DcR3) as colorectal cancer prognostic indicators. METHODS: Colorectal carcinoma specimens from 300 patients were analyzed by immunohistochemistry to detect the staining patterns of HER2 and DcR3. Classification of HER2 staining was carried out using the United States Food and Drug Administration semi-quantitative scoring system, with scores of 0 or 1+ indicating a tumor-negative (normal expression) status and scores of 2+ and 3+ indicating a tumor-positive (overexpression) status. Classification of DcR3 was carried out by quantitating the percentage of positive cells within the stained section, with < 10% indicating a tumor-negative status and 10% indicating a tumor-positive status. Correlation of the HER2 and DcR3 staining status with clinicopathological parameters [age, sex, tumor size, differentiation, and the tumor, node, metastasis (ptnm) classification] and survival was statistically assessed. RESULTS: Tumor-positive status for HER2 and DcR3 was found in 18.33% and 58.33% of the 300 colorectal carcinoma specimens, respectively. HER2 tumorpositive status showed a significant correlation with tumor size (P = 0.003) but not with other clinicopathological parameters. DcR3 tumor-positive status showed a significant correlation with tumor differentiation (P < 0.001), ptnm stage (P < 0.001), and lymph node metastasis (P < 0.001). However, correlation coefficient analysis did not indicate that a statistically significant correlation exists between tumor-positive status for the HER2 and DcR3 overexpression (P = 0.236). Patients with specimens classified as DcR3-overexpressing had a significantly worse overall survival (OS) rate than those without DcR3 overexpression (median OS: vs mo; HR = 50.27, 95%CI: , P < 0.001). HER2 overexpression had no significant impact on median OS (35.10 mo vs mo; HR = 44.40, 95%CI: , P = 0.344). However, patients with specimens classified as both HER2- and DcR3- overexpressing had a significantly poorer median OS than those with only HER2 overexpression (31.80 mo vs mo; HR = 35.10, 95%CI: , P = 0.006). CONCLUSION: HER2 overexpression is not an independent prognostic marker of colorectal cancer, but DcR3 overexpression is highly correlated with lymph node metastasis and poor OS Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Colorectal carcinoma; human epidermal growth factor receptor 2; death decoy receptor; Immunohistochemistry; Prognosis Core tip: Overexpression of the human epidermal growth factor receptor 2 (HER2) and death decoy re April 21, 2014 Volume 20 Issue 15

342 Zong L et al. DcR3 overexpression in colorectal cancer ceptor (DcR3) has been observed in clinical specimens of colorectal cancer, but their roles in prognosis remain unknown. In this systematic investigation of the immunohistochemistry staining patterns of HER2 and DcR3 in 300 clinical specimens, only DcR3 overexpression was identified as a potential prognostic marker of colorectal cancer. Specifically, DcR3 tumor-positive staining showed a strong statistical correlation with lymph node metastasis and poor overall survival. Moreover, HER2- positive patients with DcR3 overexpression had poorer overall survival than their DcR3-negative counterparts. Zong L, Chen P, Wang DX. Death decoy receptor overexpression and increased malignancy risk in colorectal cancer. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: INTRODUCTION Colorectal cancer remains one of the most common malignancies diagnosed worldwide. Extensive research effort has been put forth to elucidate the molecular mechanisms underlying progression to metastatic colorectal carcinoma, yet there remains a notable absence of accurate and convenient prognostic biomarkers [1]. Disease management of both non-metastatic and metastatic cases consists of surgical resection and chemotherapeutic drug delivery (systemic, as well as cancer cell-targeted). However, cases of resistance to the cytotoxic agents are not infrequent, and this condition represents a particular clinical challenge as no other treatment options are available. The multistep carcinogenic process of colorectal cancer is known to involve perturbed activation and signaling of inflammation and oxidative stress pathways. Two key factors of these pathways are the pro-inflammatory human epidermal growth factor receptor 2 (HER2) and the anti-inflammatory death decoy receptor 3 (DcR3), both of which have been observed as overexpressed in clinical samples of colorectal cancer [2,3]. Moreover, response to adjuvant chemotherapy with 5-fluorouracil is associated with the DcR3 chromosomal locus [4]. HER2 is a transmembrane tyrosine kinase receptor that regulates cell growth and differentiation. As such, abnormal HER2 expression and/or activity can promote growth and progression of malignant cells [5,6], and its carcinogenic role has been defined in several human cancers [5-11]. In breast cancer, chemotherapy treatment based on herceptin, an anti-her2 monoclonal antibody, has been shown to significantly improve the overall survival (OS) rate [12,13]. DcR3 is a tumor necrosis factor (TNF) receptor that counteracts activation of signals of the inflammation pathway by competitively binding to the TNF ligands [Fas ligand (FasL), LIGHT, and TL1A] [14-17]. The DcR3- mediated inhibition of TNF signaling has been shown to protect tumor cells from apoptosis and to promote invasiveness and metastasis. Abnormal DcR3 expression has been reported in clinical specimens of lung, gastrointestinal, renal and ovarian cancers, as well as colon cancer [18-22]. Thus, the protein expression levels of HER2 and DcR3 may represent prognostic markers of colorectal cancer and may reflect malignant potential of resected tumor specimens. This study was designed to investigate the correlations between immunohistochemical staining patterns of HER2 and DcR3, individually and in combination, and clinicopathological parameters of colorectal cancer in order to evaluate their potential prognostic value. MATERIALS AND METHODS Patient information This retrospective study involved 300 treatment-naïve patients who had undergone elective surgical resection for colorectal cancer at the Department of General Surgery of Su Bei People s Hospital of Yangzhou University (China) between January 2003 and December The study population was composed of 177 males and 123 females, ranging in age from 21 to 85 years (mean age: 56.3 years). For each patient, data on the clinicopathological parameters were collected from the medical records database and included patient age and sex, and tumor size, differentiation, and pathological classification according to the tumor, node, and metastasis (ptnm) scoring system. All patients provided informed consent for storage and future use of their resected tumor specimens for research purposes, and this study was authorized by the Ethics Committee of Su Bei People s Hospital of Jiangsu Province. Immunohistochemical staining of HER2 and DcR3 Formalin-fixed paraffin-embedded sections of surgical specimens on glass slides were deparaffinized in xylene and rehydrated through an ethanol gradient. Endogenous peroxidase was blocked by a 10-min incubation with 0.3% H2O2 in methanol, after which antigen retrieval was performed by 15 min of heating in 10 mmol/l sodium citrate (ph 6.0). After the sample was cooled for 20 min to room temperature and washed with phosphate-buffered saline (PBS), non-specific binding sites were blocked by a 10 min pre-incubation with 10% fetal calf serum in PBS with 0.01% sodium azide. The primary antibody against HER2 (1:100 dilution; HercepTest kit from Dako, Glostrup, Denmark) or DcR3 (1:200; Dako) was added and the slide incubated for 1 h in a humidified chamber. Following a triplicate wash with PBS, the appropriate secondary antibody (undiluted; Envision-HRP Complex from Dako) was added and the slide incubated for an additional 60 min in the humidified chamber. Immunoreactivity was visualized by incubation with the chromagen 3-3 -diaminobenzidine. All sections were counterstained with hematoxylin. For each specimen, a negative control 4441 April 21, 2014 Volume 20 Issue 15

343 Zong L et al. DcR3 overexpression in colorectal cancer A B C Figure 1 Immunohistochemical detection of human epidermal growth factor receptor 2 and death decoy receptor expression in colorectal cancer. Serialsection specimens from one representative case are shown ( 200). A: human epidermal growth factor receptor 2 (HER2)-positive tumor section, scored as 2+ (arrow); B: HER2-positive tumor section, scored as 3+ (arrow); C: death decoy receptor-positive tumor section showing staining in > 10% of the tumor cells (arrow). was generated by replacing the primary antibody with PBS alone. Scoring of HER2 and DcR3 immunostaining Each processed slide was assessed by two pathologists working independently. Instances of interobserver variability were resolved by consensus discussion during simultaneous dual re-evaluation. Scoring of the HER2 staining was carried out according to the semi-quantitative strategy recommended by the United States Food and Drug Administration [23], as follows: no staining or membrane staining in < 10% of the tumor cells = 0; incomplete membrane staining in > 10% of tumor cells = 1+; weak-to-moderate complete membrane staining in > 10% of tumor cells = 2+; moderate-to-strong complete membrane staining in > 10% of tumor cells = 3+. Specimens with scores of 0 or 1+ were classified as tumor-negative for HER2 (normal expression), and specimens with scores of 2+ or 3+ were classified as tumor-positive for HER2 (overexpression). Classification of DcR3 was carried out by quantitating the percentage of positive cells within the stained section, with < 10% indicating a tumor-negative status and 10% indicating a tumor-positive status. Follow-up Patients were followed to December 2010 or until death. The median follow-up interval was 50.3 mo (2-93 mo). Follow-up examinations included imaging analysis to detect any signs of recurrence. Statistical analysis All statistical analyses were carried out with the SPSS statistical software for Windows, version 17 (SPSS Inc., Chicago, IL, United States). The significance of associations between HER2 and DcR3 immunostaining and the various clinicopathological parameters was analyzed by χ 2 test. OS curves were generated using the Kaplan-Meier method, and the significance of differences between the curves was analyzed by log-rank testing. The threshold for statistical significance was set at a p value < RESULTS Immunohistochemical staining patterns of HER2 and DcR3 in human colorectal cancer specimens Of the total 300 colorectal cancer specimens examined, 55 (18.33%) showed HER2 overexpression, with the immunostaining scores being mostly moderate (2+, n = 45) (Figure 1A) and only 10 having strong scores (3+) (Figure 1B). In contrast, a remarkably larger percentage of the specimens showed DcR3 overexpression (58.33%) (Figure 1C). Correlation of HER2 or DcR3 overexpression with clinicopathological features of colorectal cancer The correlation of HER2 and DcR3 immunostaining patterns with clinicopathological features is presented in Table 1. HER2 overexpression was found to be significantly correlated only with tumor size. In contrast, DcR3 overexpression was found to be significantly correlated with tumor differentiation, lymph node metastases, and ptnm stage (p < 0.05). Association between HER2 and DcR3 overexpression in colorectal cancer specimens A little more than half (65.45%) of the specimens showing HER2 overexpression also showed DcR3 overexpression, which was not significantly different from the percentage of specimens showing normal HER2 expression and DcR3 overexpression (56.73%) (p = 0.236). Correlation of HER2 or DcR3 overexpression with survival HER2 immunostaining status had no impact on OS; the median OS of patients with HER2-positive tumor specimens was mo, compared to mo for the patients with HER2-negative tumor specimens (HR = 44.40, 95%CI: , p = 0.344) (Figure 2A). In contrast, DcR3-positive status was associated with a poorer OS (median OS of mo vs DcR3-negative status: mo; HR = 50.27, 95%CI: , p < 0.001) (Figure 2B). Moreover, patients whose tu April 21, 2014 Volume 20 Issue 15

344 Zong L et al. DcR3 overexpression in colorectal cancer Table 1 Correlation of colorectal cancer clinicopathological variables with immunohistochemical staining patterns of human epidermal growth factor receptor 2 and death decoy receptor expression Variable n HER2 P DcR3 P value 1 value 1 Positive Negative Positive Negative Age < Sex Male Female Tumor size (cm) < Differentiation grade Well Moderate < Poor Lymph node status Negative < Positive Distant metastasis M M Perineural invasion Negative Positive ptnm stage Ⅰ < Ⅱ Ⅲ Ⅳ Pearson s χ 2 test; 2 Adjusted χ 2 test. HER2: human epidermal growth factor receptor 2; DcR3: death decoy receptor; ptnm: the tumor, node, metastasis classification. mor specimens showed double positivity [HER-2(+)/ DcR3(+)] had worse OS than their counterparts without DcR3 overexpression [HER-2(+)/DcR3(-)] (median OS: mo vs mo, respectively; HR = 35.10, 95%CI: , p = 0.006) (Figure 2C). A Overall survival probability B Overall survival probability C Overall survival probability Month of follow-up Month of follow-up DISCUSSION In this investigation of 300 human colorectal cancer specimens, immunohistochemical detection of DcR3, and not HER2, overexpression showed promise as a clinical marker of disease prognosis. Similar to the findings from a previous study of growth factor expression in Chinese patient specimens (i.e., the pro-angiogenic vascular endothelial growth factor (VEGF) in relation to the HER2 growth factor receptor) [24], immunohistochemical detection of HER2 overexpression showed a correlation with only tumor size and had no significant relation with survival. However, in the previous study, HER2 overexpression showed a statistical correlation with distant metastasis; this inconsistency could be explained by a variance due to limited sample size. In the present study, immunohistochemically detected DcR3 overexpression reflected features of colorectal cancer progression. Specifically, the specimens showing Month of follow-up Figure 2 Kaplan-Meier overall survival curves. A: Kaplan-Meier overall survival curves for the 300 colorectal cancer patients, according to immunohistochemical detection of human epidermal growth factor receptor 2 normal (green) and overexpression (blue). The difference between the curves did not reach statistical significance (p = 0.344); B: Kaplan-Meier overall survival curves for the 300 colorectal cancer patients, according to immunohistochemical detection of death decoy receptor normal (green) and overexpression (blue). The difference between the curves was significant (p < 0.001); C: Kaplan-Meier overall survival curves for the 300 colorectal cancer patients, according to immunohistochemical detection of human epidermal growth factor receptor 2 overexpression in isolation (blue) or coincident with (green) death decoy receptor overexpression. The difference between the curves was significant (p = 0.006). DcR3-positive immunostaining were less differentiated and more invasive. Moreover, the patients with DcR3- positive tumors were more likely to have lymph node metastases at the time of resection and poorer OS. Thus, 4443 April 21, 2014 Volume 20 Issue 15

345 Zong L et al. DcR3 overexpression in colorectal cancer DcR3 may represent a useful prognostic marker of colorectal cancer malignancy and risk of death. Coincident overexpression of DcR3 significantly worsened the OS in patients with HER2 overexpression. Malignant transformation of a tumor cell requires molecular signals that promote growth and inhibit apoptosis, as well as those that allow a cell to migrate and invade surrounding tissues. While HER2 overexpression may contribute to the unrestricted growth of a cancer cell, DcR3 may play a key role in promoting metastasis, which is related to higher risk of cancer-related death and complicates the clinical management. In summary, colorectal cancer specimens from Chinese patients show both isolated and coincident overexpression of HER2 and DcR3. Isolated DcR3 overexpression may be a clinical prognostic marker of malignancy and survival, and coincident DcR3/HER2 overexpression may indicate more robust malignancy risk. Future studies need to elucidate the molecular mechanism of DcR3-mediated carcinogenesis to further define its relation to patient prognosis (in various ethnicities and disease conditions) and its potential as a target of molecularbased therapies. COMMENTS Background Overexpression of the pro-inflammatory human epidermal growth factor receptor 2 (HER2) and anti-inflammatory death decoy receptor 3 (DcR3) has been observed in various human cancers, including colorectal carcinoma. Yet, their roles in malignant transformation of colorectal cancer cells and potential as clinical markers of disease prognosis remain unknown. Research frontiers Immunohistochemical detection of DcR3 overexpression in human colorectal cancer specimens resected from Chinese patients is correlated with malignancy and overall survival. DcR3 may represent a useful prognostic biomarker of colorectal cancer in this patient population. Moreover, while overexpression of HER2 may promote the unrestricted growth of colorectal tumor cells, the overexpression of DcR3 may promote cell invasiveness and migration to increase the risk of metastasis and cancer-related death. Innovations and breakthroughs To date, no study in the publicly available literature has reported data on the coincident overexpression of HER2 and DcR3 in human colorectal cancer specimens. Clinical detection of the immunohistochemical staining patterns of these two cancer-related proteins may represent a convenient and accurate approach for determining the prognosis of Chinese patients with colorectal cancer. Applications Immunohistochemical detection of DcR3 overexpression, in isolation or coincident with HER2 overexpression, may be a useful clinical approach for predicting risk of malignancy in and survival of colorectal cancer patients. Peer review This study investigated the potential association of two cancer-related proteins, DcR3 and HER2, with colorectal cancer, and evaluated whether isolated or coincident overexpression may be indicative of increased risk of malignancy and/or overall survival. According to statistical correlation analysis of the HER2 and DcR3 immunohistochemical staining patterns and clinicopathological parameters, DcR3 overexpression, in isolation or coincident with HER2 overexpression, was highly associated with increased malignancy and poorer survival. Specifically, DcR3 overexpression was shown to be significantly correlated with less differentiated tumors, worse ptnm stage, and presence of lymph node metastasis. Thus, the study provides novel evidence supporting the potential utility of DcR3 as a prognostic marker for clinical evaluation of colorectal cancer patients, and provides a foundation for future studies to explore the role of this receptor in carcinogenesis and its potential as a novel target of anticancer drugs. REFERENCES 1 Ferlay J, Shin HR, Bray F, Forman D, Mathers C, Parkin DM. Estimates of worldwide burden of cancer in 2008: GLOBO- CAN Int J Cancer 2010; 127: [PMID: DOI: /ijc.25516] 2 Nathanson DR, Culliford AT, Shia J, Chen B, D Alessio M, Zeng ZS, Nash GM, Gerald W, Barany F, Paty PB. HER 2/ neu expression and gene amplification in colon cancer. Int J Cancer 2003; 105: [PMID: DOI: / ijc.11137] 3 Wu Y, Han B, Sheng H, Lin M, Moore PA, Zhang J, Wu J. Clinical significance of detecting elevated serum DcR3/ TR6/M68 in malignant tumor patients. 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Hum Pathol 2002; 33: [PMID: DOI: / HUPA ] 8 Bang YJ, Van Cutsem E, Feyereislova A, Chung HC, Shen L, Sawaki A, Lordick F, Ohtsu A, Omuro Y, Satoh T, Aprile G, Kulikov E, Hill J, Lehle M, Rüschoff J, Kang YK. Trastuzumab in combination with chemotherapy versus chemotherapy alone for treatment of HER2-positive advanced gastric or gastro-oesophageal junction cancer (ToGA): a phase 3, openlabel, randomised controlled trial. Lancet 2010; 376: [PMID: DOI: /S (10)61121-X] 9 Caruso ML, Valentini AM. Immunohistochemical p53 overexpression correlated to c-erbb-2 and cathepsin D proteins in colorectal cancer. Anticancer Res 1996; 16: [PMID: ] 10 Osako T, Miyahara M, Uchino S, Inomata M, Kitano S, Kobayashi M. Immunohistochemical study of c-erbb-2 protein in colorectal cancer and the correlation with patient survival. Oncology 1998; 55: [PMID: DOI: / ] 11 Yang JL, Ow KT, Russell PJ, Ham JM, Crowe PJ. Higher expression of oncoproteins c-myc, c-erb B-2/neu, PCNA, and p53 in metastasizing colorectal cancer than in nonmetastasizing tumors. Ann Surg Oncol 1996; 3: [PMID: DOI: /BF ] 12 Slamon DJ, Leyland-Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M, Baselga J, Norton L. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that overexpresses HER2. N Engl J Med 2001; 344: [PMID: DOI: /NEJM ] 13 Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, Fehrenbacher L, Slamon DJ, Murphy M, Novotny WF, Burchmore M, Shak S, Stewart SJ, Press M. Efficacy and safety of trastuzumab as a single agent in first-line treatment of HER April 21, 2014 Volume 20 Issue 15

346 Zong L et al. DcR3 overexpression in colorectal cancer overexpressing metastatic breast cancer. J Clin Oncol 2002; 20: [PMID: DOI: /JCO ] 14 Houghton JA, Harwood FG, Tillman DM. Thymineless death in colon carcinoma cells is mediated via fas signaling. Proc Natl Acad Sci USA 1997; 94: [PMID: DOI: /pnas ] 15 Roth W, Isenmann S, Nakamura M, Platten M, Wick W, Kleihues P, Bähr M, Ohgaki H, Ashkenazi A, Weller M. Soluble decoy receptor 3 is expressed by malignant gliomas and suppresses CD95 ligand-induced apoptosis and chemotaxis. Cancer Res 2001; 61: [PMID: ] 16 Chen PH, Yang CR. Decoy receptor 3 expression in AsPC-1 human pancreatic adenocarcinoma cells via the phosphatidylinositol 3-kinase-, Akt-, and NF-kappa B-dependent pathway. J Immunol 2008; 181: [PMID: ] 17 Elnemr A, Ohta T, Yachie A, Kayahara M, Kitagawa H, Fujimura T, Ninomiya I, Fushida S, Nishimura GI, Shimizu K, Miwa K. Human pancreatic cancer cells disable function of Fas receptors at several levels in Fas signal transduction pathway. Int J Oncol 2001; 18: [PMID: ] 18 Pitti RM, Marsters SA, Lawrence DA, Roy M, Kischkel FC, Dowd P, Huang A, Donahue CJ, Sherwood SW, Baldwin DT, Godowski PJ, Wood WI, Gurney AL, Hillan KJ, Cohen RL, Goddard AD, Botstein D, Ashkenazi A. Genomic amplification of a decoy receptor for Fas ligand in lung and colon cancer. Nature 1998; 396: [PMID: ] 19 Bai C, Connolly B, Metzker ML, Hilliard CA, Liu X, Sandig V, Soderman A, Galloway SM, Liu Q, Austin CP, Caskey CT. Overexpression of M68/DcR3 in human gastrointestinal tract tumors independent of gene amplification and its location in a four-gene cluster. Proc Natl Acad Sci USA 2000; 97: [PMID: DOI: /PNAS ] 20 Takahama Y, Yamada Y, Emoto K, Fujimoto H, Takayama T, Ueno M, Uchida H, Hirao S, Mizuno T, Nakajima Y. The prognostic significance of overexpression of the decoy receptor for Fas ligand (DcR3) in patients with gastric carcinomas. Gastric Cancer 2002; 5: [PMID: DOI: / S ] 21 Macher-Goeppinger S, Aulmann S, Wagener N, Funke B, Tagscherer KE, Haferkamp A, Hohenfellner M, Kim S, Autschbach F, Schirmacher P, Roth W. Decoy receptor 3 is a prognostic factor in renal cell cancer. Neoplasia 2008; 10: [PMID: ] 22 Connor JP, Felder M. Ascites from epithelial ovarian cancer contain high levels of functional decoy receptor 3 (DcR3) and is associated with platinum resistance. Gynecol Oncol 2008; 111: [PMID: DOI: /J.YGYNO ] 23 Jacobs TW, Gown AM, Yaziji H, Barnes MJ, Schnitt SJ. Specificity of HercepTest in determining HER-2/neu status of breast cancers using the United States Food and Drug Administration-approved scoring system. J Clin Oncol 1999; 17: [PMID: ] 24 Li Q, Wang D, Li J, Chen P. Clinicopathological and prognostic significance of HER-2/neu and VEGF expression in colon carcinomas. BMC Cancer 2011; 11: 277 [PMID: DOI: / ] P- Reviewers: Michael L, Harry HXX S- Editor: Ma YJ L- Editor: Wang TQ E- Editor: Zhang DN 4445 April 21, 2014 Volume 20 Issue 15

347 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. RESEARCH REPORT Comparative study of intestinal tuberculosis and primary small intestinal lymphoma Qing-Qiang Zhu, Wen-Rong Zhu, Jing-Tao Wu, Wen-Xin Chen, Shou-An Wang Qing-Qiang Zhu, Wen-Rong Zhu, Jing-Tao Wu, Wen-Xin Chen, Shou-An Wang, Department of Medical Imaging, Subei People s Hospital, Medical School of Yangzhou University, Yangzhou , Jiangsu Province, China Author contributions: Zhu QQ and Zhu WR contributed equally to this work; Zhu QQ, Zhu WR, Wu JT and Chen WX designed the research; Zhu QQ, Zhu WR, Wu JT and Wang SA performed the research; Zhu QQ, Zhu WR and Wu JT analyzed the data; Zhu QQ and Zhu WR wrote the paper. Supported by Fundamental Research Funds, Yangzhou, China, No. SGG ; and College fund No. yzucms Correspondence to: Jing-Tao Wu, MD, Department of Medical Imaging, Subei People s Hospital, Medical School of Yangzhou University, No. 98, West Nantong Road, Yangzhou , Jiangsu Province, China. wujingtaodoctor@163.com Telephone: Fax: Received: September 12, 2013 Revised: December 22, 2013 Accepted: January 20, 2014 Published online: April 21, 2014 Abstract AIM: To characterize the clinical, radiological, endoscopic and pathological features of intestinal tuberculosis (ITB) and primary small intestinal lymphoma (PSIL). METHODS: This was a retrospective study from February 2005 to October 2012 of patients with a diagnosis of ITB (n = 41) or PSIL (n = 37). All patients with ITB or PSIL underwent computed tomography (CT) and pathological examination. Thirty-five patients with ITB and 32 patients with PSIL underwent endoscopy. These patients were followed for a further 18 mo to ascertain that the diagnosis had not changed. Clinical, endoscopic, CT and pathological features were compared between ITB and PSIL patients. RESULTS: Night sweating, fever, pulmonary TB and ascites were discovered significantly more often in ITB than in PSIL patients (P < 0.05), however, abdominal mass, hematochezia and intestinal perforation were found significantly more frequently in PSIL than in ITB patients (P < 0.05). Ring-like and rodent-like ulcers occurred significantly more often in ITB than in PSIL patients (P < 0.05), however, enterorrhagia and raised lesions were significantly more frequent in PSIL than in ITB patients (P < 0.05). The rate of granuloma was significantly higher in ITB than in PSIL patients (87.8% vs 13.5%, χ 2 = , P < 0.05), and the incidence of confluent granulomas with caseous necrosis was significantly higher in ITB than in PSIL patients (47.2% vs 0.0%, χ 2 = 4.034, P < 0.05). Multi-segmental lesions, mural stratification, mural gas sign, and intestinal stricture were more frequent in ITB than in PSIL patients (P < 0.05), however, a single-layer thickening of bowel wall, single segmental lesions, and intussusception were more common in PSIL than in ITB patients (P < 0.05). Necrotic lymph nodes, comb sign and inflammatory mass were more frequent in ITB than in PSIL patients (P < 0.05). The bowel wall enhancement in ITB patients was greater than that in PSIL patients (P < 0.05), while the thickening and lymph node enlargement in PSIL patients were higher than those in ITB patients (P < 0.05). CONCLUSION: Combined evaluation of clinical, radiological, endoscopic and pathological features is the key to differentiation between ITB and PSIL Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Intestinal tuberculosis; Primary small intestinal lymphoma; Clinical features; Endoscopic features; Computed tomography Core tip: Treatment for intestinal tuberculosis (ITB) differs completely from that for primary small intestinal lymphoma (PSIL). Differentiating ITB from PSIL continues to be a challenge. Combined evaluation of clinical, radiological, endoscopic and pathological features is the key to differentiation between ITB and PSIL. For 4446 April 21, 2014 Volume 20 Issue 15

348 Zhu QQ et al. Intestinal tuberculosis and lymphoma example, night sweating, ascites, ring-like and rodentlike ulcers, granuloma, multi-segmental lesions, mural stratification, necrotic lymph nodes, comb sign, and inflammatory mass are more suggestive of ITB. However, abdominal mass, hematochezia, enterorrhagia, raised lesions, single-layer thickening of bowel wall, single segmental lesions, and intussusception are more suggestive of PSIL. Zhu QQ, Zhu WR, Wu JT, Chen WX, Wang SA. Comparative study of intestinal tuberculosis and primary small intestinal lymphoma. World J Gastroenterol 2014; 20(15): Available from: URL: v20/i15/4446.htm DOI: i INTRODUCTION Intestinal tuberculosis (ITB) is a specific chronic intestinal disease caused by Mycobacterium tuberculosis (M. tuberculosis) infection [1]. In recent decades, with improvement of economics, quality of life, and sanitary conditions, the incidence of TB has declined and the prevalence of ITB has gradually decreased [2]. However, there is still no sensitive, accurate, convenient and specific marker to diagnose ITB. Therefore, clinicians still need to pay much attention to ITB. The clinical manifestations of primary small intestinal lymphoma (PSIL) are nonspecific, such as abdominal pain, vomiting, weight loss and intestinal perforation [3]. Although the incidence is not high, it is similar to ITB in clinical manifestations and still needs to be distinguished [4]. Many studies have reported that ITB is similar to PSIL with regard to clinical, endoscopic, pathological and computed tomography (CT) features [5,6]. Treatment for ITB is completely different from that for PSIL. The first-line therapy for ITB is the combined anti-tb medication, while the major therapies for PSIL patients include surgery and radiotherapy [7]. It is clear that misdiagnosis between ITB and PSIL leads to severe clinical events, such as M. tuberculosis diffusion and delaying the medical management of PSIL [8]. An accurate diagnosis is important for appropriate treatment. Therefore, the aim of this study was to investigate the clinical, CT, endoscopic and pathological features in 41 cases of ITB and 37 of PSIL. MATERIALS AND METHODS Patients Upon searching our hospital pathology and image archiving and communications system, we found 41 patients with ITB and 37 with PSIL who were admitted to our hospital from February 2005 to October All patients with ITB or PSIL underwent CT and pathological examination. Thirty-five patients with ITB and 32 with PSIL underwent upper GI endoscopy. Methods The diagnosis of ITB complied with the established clinical, CT, histological and microbiological criteria. The diagnosis of PSIL conformed with the 1961 Dawson standards. All patients with ITB or PSIL were followed for a further 18 mo to ascertain that the diagnosis had not changed. Statistical analysis Two gastrointestinal radiologists analyzed the images together, which resulted in a consensus interpretation. Statistical analysis was undertaken using SPSS version 17.0 (SPSS, Chicago, IL, United States). Numerical data are expressed as mean and standard deviation, and categorical data are expressed as percentages. Evaluated characteristics were compared using the χ 2 test or independent-samples t test. P < 0.05 was considered statistically significant. RESULTS Comparative study of clinical features in ITB and PSIL Night sweating, fever, pulmonary TB, and ascites were discovered significantly more often in ITB than in PSIL patients (P < 0.05). However, abdominal mass, hematochezia and intestinal perforation were significantly more frequent in PSIL than in ITB patients (P < 0.05) (Table 1). Comparative study of endoscopic and pathological features in ITB and PSIL Rodent-like (Figure 1) and ring-like (Figure 2) ulcers were found significantly more often in ITB than in PSIL patients (P < 0.05). However, enterorrhagia (Figure 3) and raised lesions (Figure 4) were found significantly more frequently in PSIL than in ITB patients (P < 0.05). The rate of granuloma was significantly higher in ITB than in PSIL patients (87.8% vs 13.5%, χ 2 = , P < 0.05), and the incidence of confluent granulomas with caseous necrosis was significantly more frequent in ITB than in PSIL patients (47.2% vs 0.0%, χ 2 = 4.034, P < 0.05) (Table 2). Comparative study of CT features in ITB and PSIL Multisegmental lesions, mural stratification (Figure 1), mural gas sign, and intestinal stricture (Figure 1) were seen significantly more often in ITB than in PSIL patients (P < 0.05). Single-layer thickening of the bowel wall, single segmental lesions, and intussusception were significantly more frequent in PSIL than in ITB patients (P < 0.05). Necrotic lymph nodes (Figure 2) and comb sign were discovered significantly more often in ITB than in PSIL patients (P < 0.05). Bowel wall enhancement in ITB patients was significantly greater than that in PSIL patients (83.3 ± 7.6 HU vs 55.9 ± 4.2 HU, P < 0.05), while lymph node enlargement (Figure 3) (19.6 ± 3.2 mm vs 9.8 ± 2.7 mm) and bowel thickening (Figure 4) (18.6 ± 3.3 mm vs 11.1 ± 3.7 mm) were more common 4447 April 21, 2014 Volume 20 Issue 15

349 Zhu QQ et al. Intestinal tuberculosis and lymphoma Table 1 Comparative study of clinical features of intestinal tuberculosis and primary small intestinal lymphoma, n (%) Diarrhea Ascites Febrility Night sweating Hematochezia Pulmonary TB ITB 12 (29.2) 22 (53.6) 23 (56.1) 25 (60.9) 3 (7.3) 26 (63.4) PSIL 10 (27.0) 8 (21.6) 2 (5.4) 5 (13.5) 19 (51.3) 2 (5.4) χ 2 value P value ITB: Intestinal tuberculosis; PSIL: Primary small intestinal lymphoma. A B C D Figure 1 Computed tomography, endoscopic and pathological changes of intestinal tuberculosis in a 38-year-old man. A: Plain computed tomography (CT) scan showed bowel-wall thickening (7.2 mm) and intestinal stricture in the ileocecum; B: During the arterial phase, contrast-enhanced CT demonstrated moderately stratified enhancement; C: Endoscopic examination showed rodent-like ulcer, ring-like ulcer and intestinal stricture in the ileocecum; D: Microscopic findings showed granulomas with caseous necrosis (hematoxylin and eosin staining; original magnification, 400 ). Table 2 Comparative study of endoscopic features of intestinal tuberculosis and primary small intestinal lymphoma, n (%) Ring-like ulcer Rodent-like ulcer Enterorrhagia Raised lesions Stricture ITB 13 (37.1) 12 (34.3) 3 (8.6) 0 (0) 22 (62.8) PSIL 0 (0) 0 (0) 17 (53.1) 21 (65.6) 6 (18.7) χ 2 value P value ITB: Intestinal tuberculosis; PSIL: Primary small intestinal lymphoma. in PSIL than in ITB patients (P < 0.05) (Tables 3 and 4). DISCUSSION The differential diagnosis between ITB and PSIL is still a challenge because of the lack of an economic, simple and reliable diagnostic method. Current clinical research demonstrates that ITB and PSIL have marked overlap in clinical, CT and endoscopic features, thus, differentiating between ITB and PSIL can be a major diagnostic challenge, particularly in developing countries where ITB remains common [9]. The misdiagnosis of ITB and PSIL 4448 April 21, 2014 Volume 20 Issue 15

350 Zhu QQ et al. Intestinal tuberculosis and lymphoma A B C D Figure 2 Computed tomography and endoscopic changes of intestinal tuberculosis in a 43-year-old man. A: Plain computed tomography (CT) scan showed bowel-wall thickening in the ileocecum; B: During the arterial phase, contrast-enhanced CT scan demonstrated moderate homogeneous enhancement; C: Mesenteric necrotic lymph nodes and comb sign were noted on coronal CT imaging; D: Endoscopic examination showed a ring-like ulcer in the ileocecum. A B Figure 3 Computed tomography and endoscopic changes of primary small intestinal lymphoma in a 37-year-old man. A: Plain computed tomography scan showed mesenteric lymphadenectasis (19 mm); B: Endoscopic examination showed intestinal hemorrhage in the ileocecum. Table 3 Comparative study of computed tomography imaging features of intestinal tuberculosis and primary small intestinal lymphoma, n (%) Mural stratification Mural single layer Bowel gas sign Multi segmental lesions ITB 24 (58.5) 6 (14.6) 13 (31.7) 35 (85.4) PSIL 4 (10.8) 27 (73.0) 0 (0) 8 (21.6) χ 2 value P value ITB: Intestinal tuberculosis; PSIL: Primary small intestinal lymphoma. can lead to serious problems in the subsequent treatment of these two conditions [10]. Therefore, it is particularly important to distinguish ITB from PSIL. In our study, we found that the first symptom of ITB was abdominal discomfort or pain, while that of PSIL tended to be hematochezia or intestinal perforation. This 4449 April 21, 2014 Volume 20 Issue 15

351 Zhu QQ et al. Intestinal tuberculosis and lymphoma A B C Figure 4 Computed tomography and endoscopic changes of primary small intestinal lymphoma in a 39-year-old man. A: Plain computed tomography (CT) scan showed bowel thickening in the distal ileum; B: During the arterial phase, contrast-enhanced CT scan demonstrated mild enhancement; C: Endoscopic examination showed a raised lesion in the ileum. Table 4 Comparative study of mesenteric findings and complications of intestinal tuberculosis and primary small intestinal lymphoma, n (%) Inflammatory mass Comb sign Peritoneal abscess Necrotic lymph nodes Intussusception ITB 5 (12.2) 26 (63.4) 3 (7.3) 19 (46.3) 0 (0) PSIL 0 (0) 4 (10.8) 1 (2.7) 2 (5.4) 21 (56.7) χ 2 value P value ITB: Intestinal tuberculosis; PSIL: Primary small intestinal lymphoma. differed from diarrhea as the first symptom of Crohn s disease (CD). We speculated that ITB lesions were not only inflammatory ulcers [11], but also proliferative lesions, whereas in PSIL, inflammation and ulcers were both involved in intestinal wall thickening and damage [12]. Therefore, when symptoms in patients are complex and lack specificity, the first symptom plays a role in differentiating ITB and PSIL. Both ITB and PSIL are chronic granulomatous conditions and show an overlap in their histological features. PSIL lesions are located in the ileocecum and more limited than those of ITB [13]. PSIL endoscopic mucosal biopsies are mainly taken from a single lesion, whereas for ITB, there are multiple biopsy sites due to the wide range of lesions. This may have an impact on the efficiency of endoscopic biopsy [14]. Mucosal hallmarks of CD, such as ulcer shape, also contribute to the differential diagnosis between CD and ITB [15]. For example, ring-like and rodent-like ulcers suggest a diagnosis of ITB, while enterorrhagia and raised lesions suggest PSIL. However, longitudinal and gridlike ulcers and cobblestone pattern suggest a diagnosis of CD. In our study, granuloma detection rate in the ITB and PSIL groups was 87.8% (n = 36) and 13.5% (n = 5), respectively. Among these lesions, the incidence of confluent granulomas with caseous necrosis in the ITB group was 47.2% (n = 17), while that in the CD group was zero. Caseous granuloma remains a specific diagnostic marker for ITB. Therefore, if pathological examination only finds noncaseating granuloma, it is not immediate evidence of PSIL, which requires a combination of other pathological changes [16]. If pathological examination finds both noncaseating granuloma and submucosal lymphocyte aggregation, the patient is more likely to have a diagnosis of PSIL [17]. Abdominal CT has a certain value for the differential diagnosis between ITB and PSIL [18]. These two diseases have their own characteristic distribution of lesions, so it is important to master lesions by perfecting checks for the differential diagnosis of the diseases. Bowel wall thickness normally measures 1-3 mm in distended small bowel, and generally ranges from 5 to 10 mm in bowel affected by ITB. Wall thickening is the most consistent imaging finding of ITB and has been shown to correlate with the presence and severity of disease [19]. However, wall thickness generally ranges from 15 to 20 mm in bowel affected by PSIL. We noted significant differences in bowel wall thickness in patients with ITB and PSIL (P < 0.05). Bowel wall enhancement plays an important role in determining disease severity and may be one of the earliest signs of disease [20]. Enhancement can be assessed during several phases based on the timing of the scan relative to contrast injection. The optimal scan time has still not been determined. Peak wall enhancement in normal volunteers was s (portal venous phase). However, Zappa et al [21] have found that differentiation is best achieved by the level of enhancement in delayed phase images. In our study, the enhancement was lower in PSIL than in ITB patients in the portal venous phase (P < 0.05) April 21, 2014 Volume 20 Issue 15

352 Zhu QQ et al. Intestinal tuberculosis and lymphoma Increased mesenteric blood flow resulting in vascular engorgement, known as the comb sign, has mostly been reported in active ITB disease [22,23]. There was a significant difference in the comb sign in patients with ITB and PSIL. Mesenteric necrotic lymph nodes on CT scanning are suggestive of ITB, while lymph node enlargement in PSIL was more frequent than in ITB. Lymph node enlargement and the percentage of necrotic mesenteric lymph nodes were greater in ITB than in CD. In our study, PSIL patients with hematochezia and intestinal perforation were common, however, these manifestations are rare in ITB patients [24,25]. These differences may be due to mild progression of ITB in China, but the exact cause needs to be further studied. Besides, emergency surgery is more common in PSIL patients because of the serious complications [26], whereas medicinal treatment is more common in ITB because complications of ITB are less severe and the disease course is chronic [27]. This phenomenon indicates that complications are more frequent in PSIL than in ITB patients and their progression is faster. This indicates that patients with serious complications and surgical procedures are more likely to have a diagnosis of PSIL [28]. In conclusion, differentiating ITB from PSIL continues to be a challenge. At present, combination of clinical, endoscopic, radiological and pathological features continues to be the key to differentiation between the two conditions. We need to continue to develop new differential diagnostic tests. Our study was limited by the relatively small number of patients with these two diseases. Further research is needed to verify these findings in larger patient populations. COMMENTS Background Many studies have reported that intestinal tuberculosis (ITB) is similar to primary small intestinal lymphoma (PSIL) with regard to clinical, endoscopic, pathological and computed tomography (CT) features. Treatment for ITB is completely different from that for PSIL. Misdiagnosis between ITB and PSIL leads to severe clinical events, such as Mycobacterium tuberculosis diffusion and delaying the medical management of PSIL. Accurate diagnosis is important for appropriate treatment. Previously published reports on ITB and PSIL have documented the pathological and clinical features. However, there are only relatively small-sample reports focusing on a comparative study of CT imaging findings. Research frontiers Combined evaluation of clinical, radiological, endoscopic and pathological features is the key to differentiation between ITB and PSIL. Innovations and breakthroughs The authors used a multimodal method to characterize the differences between ITB and PSIL. The results showed that combined evaluation of clinical, radiological, endoscopic and pathological features was the key to differentiation between ITB and PSIL. Applications The results showed that radiological and endoscopic features were the key to differentiation between ITB and PSIL. Terminology Intestinal tuberculosis (ITB) is a specific chronic intestinal disease caused by Mycobacterium tuberculosis infection. The clinical manifestations of primary small intestinal lymphoma (PSIL) are nonspecific, such as abdominal pain, vomiting, weight loss and intestinal perforation. Peer review The paper has novel information on comparison between ITB and PSIL. This article presents useful information about differential diagnosis of intestinal diseases. The study was well designed and the experimental and statistical methods used are described in detail. REFERENCES 1 Pulimood AB, Peter S, Ramakrishna B, Chacko A, Jeyamani R, Jeyaseelan L, Kurian G. Segmental colonoscopic biopsies in the differentiation of ileocolic tuberculosis from Crohn s disease. J Gastroenterol Hepatol 2005; 20: [PMID: DOI: /j x] 2 Oostenbrug LE, van Dullemen HM, te Meerman GJ, Jansen PL, Kleibeuker JH. Clinical outcome of Crohn s disease according to the Vienna classification: disease location is a useful predictor of disease course. Eur J Gastroenterol Hepatol 2006; 18: [PMID: DOI: / ] 3 Gasche C, Scholmerich J, Brynskov J, D Haens G, Hanauer SB, Irvine EJ, Jewell DP, Rachmilewitz D, Sachar DB, Sandborn WJ, Sutherland LR. A simple classification of Crohn s disease: report of the Working Party for the World Congresses of Gastroenterology, Vienna Inflamm Bowel Dis 2000; 6: 8-15 [PMID: DOI: /ibd ] 4 Epstein D, Watermeyer G, Kirsch R. Review article: the diagnosis and management of Crohn s disease in populations with high-risk rates for tuberculosis. Aliment Pharmacol Ther 2007; 25: [PMID: DOI: / j x] 5 Jayanthi V, Robinson RJ, Malathi S, Rani B, Balambal R, Chari S, Taghuram K, Madanagopalan N, Mayberry JF. Does Crohn s disease need differentiation from tuberculosis? J Gastroenterol Hepatol 1996; 11: [PMID: DOI: /j tb00058.x] 6 Carter MJ, Lobo AJ, Travis SP. Guidelines for the management of inflammatory bowel disease in adults. Gut 2004; 53 Suppl 5: V1-16 [PMID: DOI: /gut ] 7 Baumgart DC, Carding SR. Inflammatory bowel disease: cause and immunobiology. Lancet 2007; 369: [PMID: DOI: /S (07) ] 8 Marra F, Cox VC, FitzGerald JM, Moadebi S, Elwood RK. Successful treatment of multidrug-resistant tuberculosis following drug-induced hepatic necrosis requiring liver transplant. Int J Tuberc Lung Dis 2004; 8: [PMID: ] 9 Nikolaus S, Schreiber S. Diagnostics of inflammatory bowel disease. Gastroenterology 2007; 133: [PMID: DOI: /j.gastro ] 10 Bruining DH, Loftus EV, Ehman EC, Siddiki HA, Nguyen DL, Fidler JL, Huprich JE, Mandrekar JN, Harmsen WS, Sandborn WJ, Fletcher JG. Computed tomography enterography detects intestinal wall changes and effects of treatment in patients with Crohn s disease. Clin Gastroenterol Hepatol 2011; 9: e1 [PMID: DOI: / j.cgh ] 11 Choi D, Jin Lee S, Ah Cho Y, Lim HK, Hoon Kim S, Jae Lee W, Hoon Lim J, Park H, Rae Lee Y. Bowel wall thickening in patients with Crohn s disease: CT patterns and correlation with inflammatory activity. Clin Radiol 2003; 58: [PMID: ] 12 Baker ME, Walter J, Obuchowski NA, Achkar JP, Einstein D, Veniero JC, Vogel J, Stocchi L. Mural attenuation in normal small bowel and active inflammatory Crohn s disease on CT enterography: location, absolute attenuation, relative attenuation, and the effect of wall thickness. AJR Am J Roentgenol 2009; 192: [PMID: DOI: /AJR ] 13 Fiorino G, Bonifacio C, Peyrin-Biroulet L, Minuti F, Repici A, Spinelli A, Fries W, Balzarini L, Montorsi M, Malesci A, Danese S. Prospective comparison of computed tomography 4451 April 21, 2014 Volume 20 Issue 15

353 Zhu QQ et al. Intestinal tuberculosis and lymphoma enterography and magnetic resonance enterography for assessment of disease activity and complications in ileocolonic Crohn s disease. Inflamm Bowel Dis 2011; 17: [PMID: DOI: /ibd.21533] 14 Knollmann FD, Dietrich T, Bleckmann T, Böck J, Mäurer J, Radtke C, Felix R. Magnetic resonance imaging of inflammatory bowel disease: evaluation in a rabbit model. J Magn Reson Imaging 2002; 15: [PMID: DOI: / jmri.10054] 15 Allen BC, Baker ME, Einstein DM, Remer EM, Herts BR, Achkar JP, Davros WJ, Novak E, Obuchowski NA. Effect of altering automatic exposure control settings and quality reference mas on radiation dose, image quality, and diagnostic efficacy in MDCT enterography of active inflammatory Crohn s disease. AJR Am J Roentgenol 2010; 195: [PMID: DOI: /AJR ] 16 Lee SS, Kim AY, Yang SK, Chung JW, Kim SY, Park SH, Ha HK. Crohn disease of the small bowel: comparison of CT enterography, MR enterography, and small-bowel followthrough as diagnostic techniques. Radiology 2009; 251: [PMID: DOI: /radiol ] 17 De Backer AI, Mortelé KJ, Deeren D, Vanschoubroeck IJ, De Keulenaer BL. Abdominal tuberculous lymphadenopathy: MRI features. Eur Radiol 2005; 15: [PMID: ] 18 Danese S, Semeraro S, Papa A, Roberto I, Scaldaferri F, Fedeli G, Gasbarrini G, Gasbarrini A. Extraintestinal manifestations in inflammatory bowel disease. World J Gastroenterol 2005; 11: [PMID: ] 19 Mackalski BA, Bernstein CN. New diagnostic imaging tools for inflammatory bowel disease. Gut 2006; 55: [PMID: ] 20 Kam KM, Yip CW. Surveillance of Mycobacterium tuberculosis susceptibility to second-line drugs in Hong Kong, , after the implementation of DOTS-plus. Int J Tuberc Lung Dis 2004; 8: [PMID: ] 21 Zappa M, Stefanescu C, Cazals-Hatem D, Bretagnol F, Deschamps L, Attar A, Larroque B, Tréton X, Panis Y, Vilgrain V, Bouhnik Y. Which magnetic resonance imaging findings accurately evaluate inflammation in small bowel Crohn s disease? A retrospective comparison with surgical pathologic analysis. Inflamm Bowel Dis 2011; 17: [PMID: ] 22 Wong WM, Lai KC, Yiu WC, Wong BC, Chan FL, Lai CL. Intestinal tuberculosis mimicking fistulizing Crohn s disease. J Gastroenterol Hepatol 2007; 22: [PMID: DOI: /j x] 23 Kataoka H, Mizuno K, Hayashi N, Tanaka M, Nishiwaki H, Ebi M, Mizoshita T, Mori Y, Kubota E, Tanida S, Kamiya T, Joh T. Diagnostic utility of small-caliber and conventional endoscopes for gastric cancer and analysis of endoscopic falsenegative gastric cancers. World J Gastrointest Endosc 2013; 5: [PMID: DOI: /wjge.v5.i9.440] 24 Xu W, Liu Y, Lu Z, Jin ZD, Hu YH, Yu JG, Li ZS. A new endoscopic ultrasonography image processing method to evaluate the prognosis for pancreatic cancer treated with interstitial brachytherapy. World J Gastroenterol 2013; 19: [PMID: DOI: /wjg.v19.i ] 25 Winther KV, Føgh P, Thomsen OØ, Brynskov J. Inflammatory bowel disease (ulcerative colitis and Crohn s disease): diagnostic criteria and differential diagnosis. Drugs Today (Barc) 1998; 34: [PMID: ] 26 Zhu QQ, Wu JT, Chen WX, Wang SA, Zheng J. [Differential diagnosis of intestinal tuberculosis and primary small intestinal lymphoma using endoscopy and computerized tomography]. Zhonghua Weichang Waike Zazhi 2012; 15: [PMID: ] 27 Almadi MA, Ghosh S, Aljebreen AM. Differentiating intestinal tuberculosis from Crohn s disease: a diagnostic challenge. Am J Gastroenterol 2009; 104: [PMID: DOI: /ajg ] 28 Simpson P, Papadakis KA. Endoscopic evaluation of patients with inflammatory bowel disease. Inflamm Bowel Dis 2008; 14: [PMID: DOI: /ibd.20398] P- Reviewers: Bugaj AM, Benjamin P, Ray G, Subhada PP S- Editor: Song XX L- Editor: Wang TQ E- Editor: Liu XM 4452 April 21, 2014 Volume 20 Issue 15

354 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. CASE REPORT Squamous cell carcinoma after radiofrequency ablation for Barrett's dysplasia Sebastian S Zeki, Rehan Haidry, Manuel Justo-Rodriguez, Laurence B Lovat, Nicholas A Wright, Stuart A McDonald Sebastian S Zeki, Nicholas A Wright, Stuart A McDonald, Centre for Tumour Biology, Bart s Cancer Institute, London EC1M 6BQ, United Kingdom Rehan Haidry, Laurence B Lovat, Research Department of General Surgery, University College London, London WC1E 6BT, United Kingdom Manuel Justo-Rodriguez, Department of Pathology, University College London, London WC1E 6BT, United Kingdom Author contributions: Zeki SS designed and performed research; Haidry R, Justo-Rodriguez M, Lovat LB, Wright NA and McDonald SA contributed new reagents or analytic tools; Zeki SS analyzed data and wrote the paper. Correspondence to: Dr. Sebastian S Zeki, Centre for Tumor Biology, Bart s Cancer Institute, Charterhouse Square, London, EC1M 6BQ, United Kingdom. sebastiz@hotmail.com Telephone: Fax: Received: July 9, 2013 Revised: August 23, 2013 Accepted: September 4, 2013 Published online: April 21, 2014 Abstract Barrett s oesophagus (BO) is a usually indolent condition that occasionally requires endoscopic therapy. Radiofrequency ablation (RFA) is an effective endoscopic treatment for high grade dysplasia (HGD) and intramucosal cancer in BO. It has a good efficacy, durability and safety profile although complications can occur. Here we describe a case of RFA in a patient with high grade dysplasia. Although the response to treatment was initially very good with the development of neosquamous epithelium, the patient very rapidly developed a squamous cell cancer of the oesophagus confirmed on radiology, histology and immunohistochemistry. Sanger sequencing confirmed that the original HGD and the squamous cell cancer (SCC) were derived from separate clonal origins. The report highlights the fact that SCC of the oesophagus has been noted after endoscopic ablation for BO previously and suggest that ablation of BO may encourage the clonal expansion of cells carrying carcinogenic mutations once a dominant clonal population has been eradicated Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Squamous carcinoma oesophagus; Barrett s oesophagus; Radiofrequency ablation; Squamous cell cancer; High grade dysplasia Core tip: The development of squamous cell cancer of the oesophagus after endoscopic ablation can happen and may add further weight to the argument for continued surveillance after radiofrequency ablation for Barrett s related pathologies. Zeki SS, Haidry R, Justo-Rodriguez M, Lovat LB, Wright NA, McDonald SA. Squamous cell carcinoma after radiofrequency ablation for Barrett s dysplasia. World J Gastroenterol 2014; 20(15): Available from: URL: com/ /full/v20/i15/4453.htm DOI: org/ /wjg.v20.i INTRODUCTION Radiofrequency ablation (RFA) is an effective and safe treatment for the eradication of Barrett s oesophagus (BO) related high grade dysplasia (HGD) and intramucosal cancer [1]. Recent evidence suggests that Barrett s related pathology can still recur after RFA [2,3]. There are also case reports on the occurrence of squamous carcinomas occurring after endoscopic ablation. Some evidence suggests that the a stem cell origin of Barrett s mucosa may be common between squamous and columnar lined epithelium [4] although this relationship has not been demonstrated either with synchronous or metachronous Barrett s and squamous carcinoma. Here we describe the first 4453 April 21, 2014 Volume 20 Issue 15

355 Zeki SS et al. SCC after RFA for Barrett s oesophagus A (1) T G G T G G T G C (2) T G G T G A T G C * B C D Figure 1 Endoscopic, genetic and histological figures of the patient before and immediately after radiofrequency ablation for Barrett s related high grade dysplasia. A: Sanger sequencing from columnar lined oesophagus taken in June 2010 (2) showing a mutation in CDKN2A c.286 G > A (p.v100m) (starred). A control sequence is also shown (1); B: Haematoxylin and eosin stain at 10 magnification from a biopsy taken in June This demonstrates high grade dysplasia with nuclear pleiomorphism, increased nuclear: cytoplasmic ratio and disordered nuclei (arrow); C: Endoscopic view of the patient s columnar-lined oesophagus (April 2009) showing classical salmon pink mucosa between 28cm and 36cm from the incisors; D: Endoscopic view of the patient s re-epithelialized oesophagus (September 2010) after radiofrequency ablation. documented case of squamous carcinoma occurring after radiofrequency ablation and investigate its clonal relationship to the pre-treatment Barrett related HGD. CASE REPORT A 52-year-old man was admitted to the endoscopy unit in November 2008 for routine surveillance of Barrett s oesophagus which he had undergone since The Barrett s segment extended between 26 and 38 cm. Quadrantic biopsies demonstrated columnar-lined non-dysplastic epithelium throughout the segment apart from biopsies taken at 38 cm which demonstrated a high grade dysplasia. This was confirmed on subsequent endoscopies in December 2008 and February In April 2009 the patient underwent focussed radiofrequency ablation [RFA (HALO 90)] with a further session of circumferential RFA in June Further focal RFA was applied to several Barrett s islands in December 2009 and June A follow up gastroscopy in September 2010 with quadrantic biopsies showed normal, non-dysplastic squamous re-epithelialisation in all biopsies between 28 to 38 cm apart from two isolated areas at 35 and 38 cm which showed intestinal metaplasia only on histology. The patient underwent a further surveillance gastroscopy in August A large, ulcerating mass was found between 35 to 39 cm involving the GOJ. No intestinal metaplasia was seen. Apart from two short tongues of Barrett s that were separate to the ulcerating lesion the entire oesophagus was otherwise squamous-lined (Figure 1). The ulcerating lesion was characterized histologically as a squamous carcinoma (Figure 2) and confirmed immunophenotypically by p63 positivity (Figure 2B). A staging computed tomography scan confirmed the squamous cell cancer to involve loco-regional lymph nodes without distant metastases. The patient died prior to undergoing chemoradiotherapy due to an unrelated pneumonia. In order to further characterise the genotypic features of the Barrett s related HGD and the squamous carcinoma, the samples underwent laser microdissection and sequencing of exons 5-9 of TP53 and exon 2 of CDKN2A as previously described [5]. These are known to be commonly mutated in both squamous cell cancer and Barrett s HGD [6]. The biopsy samples of HGD demonstrated a mutation of CDKN2A c.286 G>A (p.v100m) (Figure 1A) only. However the squamous cell cancer did not contain the mutation detected in HGD, but demonstrated a different mutation of TP53 (c.817 C>T p. R273C) (Figure 2D). DISCUSSION A number of therapeutic modalities are now available 4454 April 21, 2014 Volume 20 Issue 15

356 Zeki SS et al. SCC after RFA for Barrett s oesophagus A B D T G C G T * C T G C G T Figure 2 Endoscopic, genetic and histological figures of the patient after radiofrequency ablation. A: Endoscopic view of a 4cm ulcerating mass between 35 to 39 cm; B: Example of positive p63 stain of the biopsies taken from the ulcerating mass (arrow); C: Haematoxylin and eosin stain of biopsies taken from the ulcer crater demonstrating invasive squamous cell cancer (arrow); D: Sanger sequencing of a mutation in TP53 c.817 C>T p. R273C (upper panel, starred) Wild type Sanger sequence of TP53 (lower panel). for the treatment of Barrett s oesophagus. Endoscopic therapies are now widely used and robust analyses have demonstrated efficacy in the treatment of HGD and intramucosal cancer [3,7]. Radiofrequency ablation of Barrett s mucosa is a relatively safe technique with perforation and stricturing occurring less commonly than other endoscopic ablation techniques [1]. Our case is unique in that it shows the first demonstration of a squamous carcinoma occurring rapidly in an oesophageal segment recently treated with radiofrequency ablation for high-grade dysplasia. There is a literature on the development of subsquamous Barrett s after RFA with case reports demonstrating the rare development of adenocarcinoma in these areas [8]. There are also reports of squamous carcinoma occurring after ablation [9] although we have described the first case after RFA. The presence of squamous carcinoma in this and other reports so soon after a normal endoscopy subsequent to a successful ablation of Barrett s related pathology indicates that patients may still warrant surveillance of neo-squamous epithelium after RFA. We have previously found somatic mutations in one case of normal squamous epithelium after radiofrequency ablation (accepted for publication by Am J Gastro). The fact that this may be a complication of RFA itself is indicated firstly by the absence of risk factors for squamous carcinoma development in this patient and secondly by the rapidity of its growth after RFA thermal injury. Thermal injury is already implicated as a causative factor in squamous carcinoma development [10,11] albeit over longer time courses. Several studies have recently demonstrated a potential common progenitor for squamous and columnar epithelium by using mitochondrial and DNA mutations [4,5,12]. The fact that the two cancers are not clonally related on targeted sequencing of genes commonly mutated in both adenocarcinoma and squamous cell cancer indicates a different clonal origin of the two neoplasias. Given the squamous tumour was found at the same location as the previous Barrett s HGD and morphologically was not a mixed adenosquamous type tumour, an alternative and interesting conjecture is that the eradication of Barrett s dysplasia has allowed a protumorigenic clonal squamous population to proliferate. Such clonal competition has been suggested to occur in a number of different neoplasias including Barrett s oesophagus [5]. An interesting and related issue also pertains to why metachronous squamous carcinoma and Barrett s is so uncommon given the number of shared risk factors [13] and adds further weight to the idea that the clonal populations that characterise BO, or the phenotype of the tissue itself may protect against the development of squamous carcinoma. REFERENCES 1 Bulsiewicz WJ, Kim HP, Dellon ES, Cotton CC, Pasricha S, Madanick RD, Spacek MB, Bream SE, Chen X, Orlando RC, Shaheen NJ. Safety and efficacy of endoscopic mucosal therapy with radiofrequency ablation for patients with neoplastic Barrett s esophagus. Clin Gastroenterol Hepatol 2013; 11: [PMID: DOI: /j.cgh ] 2 Korst RJ, Santana-Joseph S, Rutledge JR, Antler A, Bethala V, DeLillo A, Kutner D, Lee BE, Pazwash H, Pittman RH, Rahmin M, Rubinoff M. Patterns of recurrent and persistent intestinal metaplasia after successful radiofrequency ablation of Barrett s esophagus. J Thorac Cardiovasc Surg 2013; 145: [PMID: DOI: /j.jtcvs ] 3 Shaheen NJ, Overholt BF, Sampliner RE, Wolfsen HC, Wang KK, Fleischer DE, Sharma VK, Eisen GM, Fennerty MB, Hunter JG, Bronner MP, Goldblum JR, Bennett AE, Mashimo H, Rothstein RI, Gordon SR, Edmundowicz SA, Madanick RD, Peery AF, Muthusamy VR, Chang KJ, Kimmey MB, Spechler SJ, Siddiqui AA, Souza RF, Infantolino A, Dumot JA, Falk GW, Galanko JA, Jobe BA, Hawes RH, Hoffman BJ, Sharma P, Chak A, Lightdale CJ. Durability of 4455 April 21, 2014 Volume 20 Issue 15

357 Zeki SS et al. SCC after RFA for Barrett s oesophagus radiofrequency ablation in Barrett s esophagus with dysplasia. Gastroenterology 2011; 141: [PMID: DOI: /j.gastro ] 4 Nicholson AM, Graham TA, Simpson A, Humphries A, Burch N, Rodriguez-Justo M, Novelli M, Harrison R, Wright NA, McDonald SA, Jankowski JA. Barrett s metaplasia glands are clonal, contain multiple stem cells and share a common squamous progenitor. Gut 2012; 61: [PMID: DOI: /gutjnl ] 5 Leedham SJ, Preston SL, McDonald SA, Elia G, Bhandari P, Poller D, Harrison R, Novelli MR, Jankowski JA, Wright NA. Individual crypt genetic heterogeneity and the origin of metaplastic glandular epithelium in human Barrett s oesophagus. Gut 2008; 57: [PMID: DOI: /gut ] 6 Catalogue of Somatic Mutations in Cancer. Available from: URL: cosmic 7 Shaheen NJ, Sharma P, Overholt BF, Wolfsen HC, Sampliner RE, Wang KK, Galanko JA, Bronner MP, Goldblum JR, Bennett AE, Jobe BA, Eisen GM, Fennerty MB, Hunter JG, Fleischer DE, Sharma VK, Hawes RH, Hoffman BJ, Rothstein RI, Gordon SR, Mashimo H, Chang KJ, Muthusamy VR, Edmundowicz SA, Spechler SJ, Siddiqui AA, Souza RF, Infantolino A, Falk GW, Kimmey MB, Madanick RD, Chak A, Lightdale CJ. Radiofrequency ablation in Barrett s esophagus with dysplasia. N Engl J Med 2009; 360: [PMID: DOI: /NEJMoa ] 8 Titi M, Overhiser A, Ulusarac O, Falk GW, Chak A, Wang K, Sharma P. Development of subsquamous high-grade dysplasia and adenocarcinoma after successful radiofrequency ablation of Barrett s esophagus. Gastroenterology 2012; 143: e1 [PMID: DOI: /j.gastro ] 9 Allende D, Dumot J, Yerian L. Esophageal squamous cell carcinoma arising after endoscopic ablation therapy of Barrett s esophagus with high-grade dysplasia. Report of a case. Dis Esophagus 2013; 26: [PMID: DOI: /j x] 10 Ribeiro U, Posner MC, Safatle-Ribeiro AV, Reynolds JC. Risk factors for squamous cell carcinoma of the oesophagus. Br J Surg 1996; 83: [PMID: ] 11 Islami F, Boffetta P, Ren JS, Pedoeim L, Khatib D, Kamangar F. High-temperature beverages and foods and esophageal cancer risk--a systematic review. Int J Cancer 2009; 125: [PMID: DOI: /ijc.24445] 12 Paulson TG, Xu L, Sanchez C, Blount PL, Ayub K, Odze RD, Reid BJ. Neosquamous epithelium does not typically arise from Barrett s epithelium. Clin Cancer Res 2006; 12: [PMID: DOI: / CCR ] 13 Streppel MM, Siersema PD, de Leng WW, Morsink FH, Vleggaar FP, Maitra A, Montgomery EA, Offerhaus GJ. Squamous cell carcinoma in Barrett s esophagus: field effect versus metastasis. Dis Esophagus 2012; 25: [PMID: DOI: /j x] P- Reviewers: Chai JY, Nagahara H, Tarnawski AS, Redondo-Cerezo E S- Editor: Gou SX L- Editor: A E- Editor: Wang CH 4456 April 21, 2014 Volume 20 Issue 15

358 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. CASE REPORT Colonic perforation by a transmural and transvalvular migrated retained sponge: Multi-detector computed tomography findings Luigi Camera, Marco Sagnelli, Paolo Guadagno, Pier Paolo Mainenti, Teresa Marra, Maria Scotto di Santolo, Landino Fei, Marco Salvatore Luigi Camera, Marco Sagnelli, Maria Scotto di Santolo, Marco Salvatore, Department of Radiology, University Federico Ⅱ, Naples, Italy Luigi Camera, Pier Paolo Mainenti, Institute of Biostructures and Bioimaging, National Research Council (CNR) - Via Tommaso De Amicis 95, Naples, Italy Paolo Guadagno, Teresa Marra, Landino Fei, Gastrointestinal Surgery Unit-Second University School, Naples, Italy Author contributions: Camera L designed the report and revised the manuscript; Sagnelli M drafted the manuscript; Guadagno P and Marra T performed the laparotomy; Mainenti PP and Scotto di Santolo M performed the literature search; Fei L and Salvatore M edited the manuscript. Correspondence to: Luigi Camera, MD, Department of Radiology, University Federico Ⅱ, Via S. Pansini, Naples, Italy. camera@unina.it Telephone: Fax: Received: June 4, 2013 Revised: September 6, 2013 Accepted: September 15, 2013 Published online: April 21, 2014 Abstract Transmural migrated retained sponges usually impact at the level of the ileo-cecal valve leading to a small bowel obstruction. Once passed through the ileo-cecal valve, a retained sponge can be propelled forward by peristaltic activity and eliminated with feces. We report the case of a 52-year-old female with a past surgical history and recurrent episodes of abdominal pain and constipation. On physical examination, a generalized resistance was observed with tenderness in the right flank. Contrast-enhanced multi-detector computed tomography findings were consistent with a perforated right colonic diverticulitis with several out-pouchings at the level of the ascending colon and evidence of free air in the right parieto-colic gutter along with an air-fluid collection within the mesentery. In addition, a ring-shaped hyperdense intraluminal material was also noted. At surgery, the ascending colon appeared irregularly thickened and folded with a focal wall interruption and a peri-visceral abscess at the level of the hepatic flexure, but no diverticula were found. A right hemi-colectomy was performed and on dissection of the surgical specimen a retained laparotomy sponge was found in the bowel lumen Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Retained sponge; Transmural migration; Multi-detector computed tomography; Colonic perforation; Acute abdomen Core tip: Transmural migrated retained sponges usually impact at the level of the ileo-cecal valve leading to a small bowel obstruction. We report the case of a 52-year-old female with a past surgical history and recurrent episodes of abdominal pain. Contrast-enhanced multi-detector computed tomography findings were consistent with a perforated right colonic diverticulitis with additional evidence of hyperdense intraluminal material. At surgery, a right hemi-colectomy was performed and on dissection of the surgical specimen a retained laparotomy sponge was found in the bowel lumen. Camera L, Sagnelli M, Guadagno P, Mainenti PP, Marra T, Scotto di Santolo M, Fei L, Salvatore M. Colonic perforation by a transmural and transvalvular migrated retained sponge: Multidetector computed tomography findings. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: org/ /wjg.v20.i April 21, 2014 Volume 20 Issue 15

359 Camera L et al. Colonic perforation by a retained sponge INTRODUCTION Retained foreign bodies represent a relatively uncommon complication of abdominal or pelvic surgery with a reported incidence of 1 every operations. However, the real frequency of retained foreign bodies may be higher because of underreporting due to medicolegal concerns [1]. The commonest retained foreign bodies are surgical sponges left behind during abdominal or pelvic surgery, mainly after emergency or gynaecological interventions [2]. Clinical manifestations of retained surgical sponges can be extremely variable and often non-specific with patients being either asymptomatic or complaining of recurrent and/or intermittent episodes of abdominal pain occurring from the immediate post-operative period to several years after surgery [3]. This is largely based on the type of reaction, fibrinous or exudative, induced by the retained sponge in the surrounding tissues. In the former, the retained sponge is also referred to as gossypiboma [2]. Imaging characteristics of gossypibomas have been well described on both abdominal plain film [4] and cross-sectional imaging [5,6]. More rarely, the retained sponge may migrate into the gut lumen as a result of an exudative reaction leading to a fistula within the bowel wall [7-9]. In this instance, the patient will manifest signs and symptoms of small bowel obstruction as the retained sponge impacts at the level of the ileo-cecal valve [10]. Herein, we report a case of a 52-year old woman with multi-detector computed tomography (CT) findings consistent with a perforated right colonic diverticulitis who was found at surgery to have a retained sponge migrated into both the ascending and proximal transverse colon through the ileo-cecal valve. CASE REPORT A 52-year-old female was admitted to our hospital with a 5-d history of abdominal pain and constipation with tenderness in the right flank. Her past medical history included a hysterectomy performed 5 years before for uterine fibromatosis with several episodes of intermittent abdominal pain and alternate alvus thereafter. Abdominal ultrasonography showed a concentric wall thickening at the level of both the terminal ileum and the ascending colon. Based on these clinical and instrumental findings, a presumed diagnosis of inflammatory bowel disease was formulated and the patient received treatment with mesalazine (500 mg 3/d). An abdominal magnetic resonance imaging (MRI) scan was also performed and no remarkable findings were noted. On physical examination, a pale abdomen was observed with a generalized resistance, more evident in the right flank, with no rebound. Laboratory tests were unremarkable except for mild leucocytosis (WBC / ml) and increased levels (17.2 mg/dl; n.v. < 1.0 mg/dl) of C-reactive protein. At this time, a contrast-enhanced CT scan was performed after oral administration of 1 L of 2% diluted Diatrizoate-Dimeglumine (Gastrografin, Bayer, Berlin, Germany). Multi-detector row CT (Aquilion 64, Toshiba, Japan) was performed using a detector configuration of 2 mm 16 mm, section thickness 5 mm, table speed of 36 mm/s and a gantry rotation time of 0.75 (pitch factor = 0.844), 120 kvp and automatic dose modulation. A mono-phasic caudo-cranial acquisition was performed 80 s. after iv bolus injection of 150 ml of non-ionic iodinated contrast medium (Ultravist 370; Bayer, Berlin, Germany) at a rate of 2 ml/s. On contrast-enhanced CT, concentric wall thickening was seen at the level of both the ascending and proximal transverse colon with several out-pouchings consistent with colonic diverticula (Figure 1A) and associated hyperplasic ileo-colic lymph nodes (Figure 1B). A small airfluid collection was also observed within the mesentery with associated fat-stranding (Figure 1B). Extra-luminal air was also evident at a more caudal level (Figure 1C) as best shown on the pulmonary window setting (Figure 1D). CT findings were considered to be consistent with a perforated right colonic diverticulitis. However, a ringshaped hyperdense intra-luminal material was also noted in both the ascending colon and the proximal transverse colon (Figure 1A-D). The patient underwent urgent laparotomy. At surgery, there was free air discharge on opening the peritoneum with evidence of multiple adhesions between the terminal ileum, the adjacent small bowel loops and the right colon. The latter appeared irregularly thickened and folded with evidence of a focal wall interruption and a peri-visceral abscess at the level of the hepatic flexure. A right hemicolectomy was performed and on dissection of the surgical specimen a retained laparotomy sponge was found within the bowel lumen (Figure 2). Retrospective Maximum Intensity Projection reconstructed images clearly showed the transmural and transvalvular migrated sponge extending from the ileo-cecal valve to the proximal transverse colon (Figure 3). The post-operative period was uneventful and the patient was discharged the following week. DISCUSSION Surgical sponges are the commonest foreign bodies left behind after abdominal or pelvic operations. However, while a retained sponge certainly represents an unusual complication, its reported incidence is underestimated due to medico-legal concerns [1]. As the cotton matrix is inert, the fate of a retained surgical sponge is largely dependent on the type of reaction induced by the foreign material in the surrounding tissues [3]. The first type is an aseptic fibrinous reaction which generates adhesions and encapsulation of the cotton matrix. This usually results in a foreign body granu April 21, 2014 Volume 20 Issue 15

360 Camera L et al. Colonic perforation by a retained sponge A B C D * * Figure 1 Multi-detector contrast-enhanced computed tomography. Transverse images of the lower abdomen are shown (A-D). A-B: Concentric thickening of the bowel wall can be seen at the level of both the ascending and proximal transverse colon (arrow-heads) with evidence of several out-pouchings and associated hyperplasic ileo-colic lymph-nodes; In B a small air-fluid collection is also depicted within the mesenteric fat with associated fat-stranding (arrow); C: At a more caudal level, extra-luminal air can also be seen, as best shown with pulmonary window settings (D). Retrospectively, a cluster of small bowel loops (arrow) is noted proximal to the cecum. There is evidence of hyperdense ring-shaped intra-luminal material in all images; Orally administered iodinated contrast medium (Gastrografin) can be seen in the small bowel loops which appear mildly dilated. * sb Figure 2 Right hemi-colectomy. A huge laparotomy sponge (arrow-heads) can be seen on dissection of the surgical specimen which included a cluster of small bowel loops (sb) and the appendix (arrow). Figure 3 Multi-detector contrast-enhanced computed tomography. An 80 mm thick maximum intensity projection (MIP) reconstructed image is shown; The laparotomy sponge (arrow-heads) is clearly depicted in the right flank extending from the ileo-cecal valve to the proximal transverse colon where the perforation occurred; Stomach (*) and jejunal loops ( ) appear opacified by orally administered iodinated contrast medium (Gastrografin). loma often referred to as gossypiboma, from the Latin word Gossypium, meaning cotton, and the Swahili word boma, meaning place of concealment [6]. This type of reaction is the most common and often clinically asymptomatic. As a result, gossypibomas are usually incidentally detected on both abdominal plain films as well as crosssectional imaging and exhibit characteristic features [4-6]. More rarely, a surgical sponge may induce an exudative reaction leading to a phlegmon or abscess formation resulting in the erosion and perforation of a hollow viscus followed by migration of the foreign body into the gut lumen [7]. This process can take several years in which the patient most often manifests unspecific symptoms such as intermittent abdominal pain, weight loss, nausea, anorexia and even mild fever [7-9]. Most commonly, the sponge migrates into the small bowel lumen where it is pushed forward by peristaltic activity and, as long as it does not encounter adhesions or 4459 April 21, 2014 Volume 20 Issue 15

361 Camera L et al. Colonic perforation by a retained sponge strictures, it reaches the ileo-cecal valve where it usually becomes impacted leading to a small bowel obstruction [10]. Once the sponge passes through the ileo-cecal valve it can be propelled forward and eliminated with feces [11]. To the best of our knowledge, there is no reported case in which a transmural and transvalvular migrated surgical sponge manifested clinically with a colonic perforation. Our patient had long been complaining of recurrent episodes of abdominal pain which had been erroneously thought to be consistent with an inflammatory bowel disease. As we do not have any explanation as to how such a diagnosis could only be based on clinical and ultrasonographic findings, the patient was under treatment with mesalazine (500 mg 3/d) and underwent an abdominal MRI scan 3 mo prior to admission at our Institution. As the MRI report did not mention any pathological signal alteration within the peritoneal cavity, we can reasonably assume that the perforation found at surgery was recent. As far as the missed MRI diagnosis of an intra-luminal foreign body is concerned, it is well known that retained surgical sponges can be difficult to detect on MR images due to the diamagnetic properties of the radiopaque filaments and the very few free protons contained in the cotton matrix [12]. In our case, we can reasonably argue that the surgical sponge was likely mistaken for normal fecal content given its intraluminal localization, whereas an intra-abdominal gossypiboma may appear as a well-defined mass of mixed signal intensities on MR images [13]. Surgical findings accounted for the concentric wall thickening observed at the level of both the ascending and proximal transverse colon (Figure 1A) as well as the fluid collection and the nearby fat stranding depicted within the mesentery (Figure 1B). At surgery, however, no diverticula were found. Following dissection of the surgical specimen (Figure 2), it was assumed that the retained sponge eroded the colonic wall from the inside. As it is unknown why the perforation occurred at the level of the proximal transverse rather than the ascending colon, it can be argued that it involved the more distal portion of the retained intraluminal sponge because it had been lying there for a longer period of time. As far as the oral administration of water-soluble iodinated contrast medium is concerned, this is still a controversial practice in the clinical setting of an adhesive small bowel obstruction. As the patient had a past surgical history with recurrent episodes of abdominal pain and constipation, it was wrongly assumed to be an adhesive small bowel obstruction by the on-call radiologist. Indeed, a recent meta-analysis showed a beneficial effect of orally administered Gastrografin in terms of both reducing the need for surgery and shortening hospital stay for patients with adhesive small bowel obstruction [14]. However, in the present case, the presence of intraluminal contrast agent did not affect the correct interpretation of CT images as it did not reach the ileo-cecal valve as shown by the coronal Maximum Intensity Projection (MIP) reconstructed image (Figure 3). As the latter was not obtained in the prospective image analysis, the present case underscores the added diagnostic value of the coronal reformatted or reconstructed multidetector CT (MDCT) images in the setting of acute abdomen [15]. If it had been generated prospectively, the coronal MIP image (Figure 3) would have pointed to the correct diagnosis of an iatrogenic perforation as assessed by laparotomy (Figure 2). We report the case of a transmural and transvalvular migration of a retained surgical sponge which manifested with a colonic perforation. To the best of our knowledge, no such cases have been reported in the literature. REFERENCES 1 Lincourt AE, Harrell A, Cristiano J, Sechrist C, Kercher K, Heniford BT. Retained foreign bodies after surgery. J Surg Res 2007; 138: [PMID: DOI: / j.jss ] 2 McIntyre LK, Jurkovich GJ, Gunn ML, Maier RV. Gossypiboma: tales of lost sponges and lessons learned. Arch Surg 2010; 145: [PMID: DOI: /archsurg ] 3 Kaiser CW, Friedman S, Spurling KP, Slowick T, Kaiser HA. The retained surgical sponge. Ann Surg 1996; 224: [PMID: ] 4 O Connor AR, Coakley FV, Meng MV, Eberhardt SC. Imaging of retained surgical sponges in the abdomen and pelvis. AJR Am J Roentgenol 2003; 180: [PMID: DOI: /ajr ] 5 Kalovidouris A, Kehagias D, Moulopoulos L, Gouliamos A, Pentea S, Vlahos L. Abdominal retained surgical sponges: CT appearance. Eur Radiol 1999; 9: [PMID: DOI: /s ] 6 Manzella A, Filho PB, Albuquerque E, Farias F, Kaercher J. Imaging of gossypibomas: pictorial review. AJR Am J Roentgenol 2009; 193: S94-S101 [PMID: DOI: / AJR ] 7 Düx M, Ganten M, Lubienski A, Grenacher L. Retained surgical sponge with migration into the duodenum and persistent duodenal fistula. Eur Radiol 2002; 12 Suppl 3: S74-S77 [PMID: DOI: /s ] 8 Dhillon JS, Park A. Transmural migration of a retained laparotomy sponge. Am Surg 2002; 68: [PMID: ] 9 Godara R, Marwah S, Karwasra RK, Goel R, Sen J, Singh R. Spontaneous transmural migration of surgical sponges. Asian J Surg 2006; 29: [PMID: DOI: / S (09) ] 10 Tandon A, Bhargava SK, Gupta A, Bhatt S. Spontaneous transmural migration of retained surgical textile into both small and large bowel: a rare cause of intestinal obstruction. Br J Radiol 2009; 82: e72-e75 [PMID: DOI: / bjr/ ] 11 Alis H, Soylu A, Dolay K, Kalayci M, Ciltas A. Surgical intervention may not always be required in gossypiboma with intraluminal migration. World J Gastroenterol 2007; 13: [PMID: DOI: /wjg ] 12 Kim HS, Chung TS, Suh SH, Kim SY. MR imaging findings of paravertebral gossypiboma. AJNR Am J Neuroradiol 2007; 28: [PMID: ] 13 Kim CK, Park BK, Ha H. Gossypiboma in abdomen and pelvis: MRI findings in four patients. AJR Am J Roentgenol 2007; 189: [PMID: ] 14 Branco BC, Barmparas G, Schnüriger B, Inaba K, Chan LS, Demetriades D. Systematic review and meta-analysis of the 4460 April 21, 2014 Volume 20 Issue 15

362 Camera L et al. Colonic perforation by a retained sponge diagnostic and therapeutic role of water-soluble contrast agent in adhesive small bowel obstruction. Br J Surg 2010; 97: [PMID: DOI: /bjs.7019] 15 Zangos S, Steenburg SD, Phillips KD, Kerl JM, Nguyen SA, Herzog C, Schoepf UJ, Vogl TJ, Costello P. Acute abdomen: Added diagnostic value of coronal reformations with 64-slice multidetector row computed tomography. Acad Radiol 2007; 14: [PMID: DOI: /j.acra ] P- Reviewers: KeeseM, Sun ZH S- Editor: Qi Y L- Editor: Webster JR E- Editor: Wang CH 4461 April 21, 2014 Volume 20 Issue 15

363 Online Submissions: doi: /wjg.v20.i World J Gastroenterol 2014 April 21; 20(15): ISSN (print) ISSN (online) 2014 Baishideng Publishing Group Co., Limited. All rights reserved. BRIEF CASE ARTICLE REPORT Failed stapled rectal resection in a constipated patient with rectal aganglionosis Lorenzo C Pescatori, Vincenzo Villanacci, Mario Pescatori Lorenzo C Pescatori, Mario Pescatori, Coloproctology Unit, Clinica Parioli, Rome, Italy Vincenzo Villanacci, Patology Unit, Spedali Civili, Brescia, Italy Author contributions: Pescatori M and Pescatori LC designed the report; Pescatori M collected the patient s clinical data and performed the biopsies; Villanacci V performed the histological examination; Pescatori LC, Pescatori M and Villanacci V analyzed the data, wrote and approved the paper. Correspondence to: Lorenzo C Pescatori, MD, Coloproctology Unit, Parioli Clinic, Via Felice Giordano 8, Rome, Italy. lorenzo.carlo.pescatori@gmail.com Telephone: Fax: Received: November 18, 2013 Revised: January 7, 2014 Accepted: February 17, 2014 Published online: April 21, 2014 Abstract A rare case of a severely constipated patient with rectal aganglionosis is herein reported. The patient, who had no megacolon/megarectum, underwent a STARR, i.e., stapled transanal rectal resection, for obstructed defecation, but her symptoms were not relieved. She started suffering from severe chronic proctalgia possibly due to peri-retained staples fibrosis. Intestinal transit times were normal and no megarectum/megacolon was found at barium enema. A diverting sigmoidostomy was then carried out, which was complicated by an early parastomal hernia, which affected stoma emptying. She also had a severe diverting proctitis, causing rectal bleeding, and still complained of both proctalgia and tenesmus. A deep rectal biopsy under anesthesia showed no ganglia in the rectum, whereas ganglia were present and normal in the sigmoid at the stoma site. As she refused a Duhamel procedure, an intersphincteric rectal resection and a refashioning of the stoma was scheduled. This case report shows that a complete assessment of the potential causes of constipation should be carried out prior to any surgical procedure Baishideng Publishing Group Co., Limited. All rights reserved. Key words: Constipation; Rectal agangliosis; Obstructed defecation; Stapled rectal resection; Parastomal hernia Core tip: A patient with persisting constipation following STARR or transanal stapled rectal resection, carried out for rectal internal prolapse, needed a diverting sigmoidostomy. She also had proctalgia due to retained staples. Despite normal manometry and intestinal transit times, a deep rectal biopsy showed marked alterations of the intrinsic plexus, which was the main cause of symptoms. Both morphology and function of the anorectum should be carefully investigated prior to indicate surgery. Obstructed defecation may be considered an Iceberg syndrome : the rectal internal prolapse is just the tip of the iceberg, and occult underlying lesions should be properly diagnosed and cured. Pescatori LC, Villanacci V, Pescatori M. Failed stapled rectal resection in a constipated patient with rectal aganglionosis. World J Gastroenterol 2014; 20(15): Available from: URL: DOI: INTRODUCTION Chronic constipation may present in form of reduced frequency or evacuation or as obstructed defecation, which is usually characterized by frequent attempts to evacuate with a lot of straining and without the sensation to have the rectum emptied. It may also require selfdigitation and it is frequently associated with a rectal internal mucosal prolapse. Constipation may also be present since the childhood and may be due to a defect of rectal ganglia and to an uneffective peristalsis, usually showed by abnormal anorectal manometry and intestinal 4462 April 21, 2014 Volume 20 Issue 15

364 Pescatori LC et al. Failed stapled rectal resection transit time [1]. To find both conditions causing constipation in the same patient is rather unusual. Even more unusual, the first time to our knowledge, is that, despite normal manometry and transit times, the patient had a rectal aganglionosis. Therefore we thought it was worth publishing this rare case. CASE REPORT A 56 years-old nulliparous female patient presented at our Unit complaining of severe proctalgia, tenesmus and rectal bleeding two years after a STARR procedure for obstructed defecation and six months after a diverting sigmoidostomy. She also had a parastomal hernia. The patient looked anxious, but was in good general conditions. Routine blood tests, EKG and Chest X-ray were normal. The study of large bowel transit, carried out eight months before by means of radiopaque markers was also normal, as 80% of the markers had been expelled within 72 h. Nevertheless she complained of constipation, i.e., infrequent and difficult emptying of the stoma. At the digital exploration a tender painful mass was felt at the level of the lower rectum, just above the anal canal, on both the posterior and the anterior aspects, in correspondence of retained staples, two of which could be detected by palpation. Proctoscopy showed a marked inflamed rectum, with bleeding ulcerations and a hyperemic fragile mucosa. As the patient reported that her constipation started in the childhood an adult hypoganglionosis was suspected and a deep rectal biopsy under anesthesia was carried out, despite the anorectal manometry, performed one year before, had shown a normal recto-anal inhibitory reflex. A deep colonic biopsy at the site of the sigmoidostomy was also carried out, as shown in Figure 1. The G.I. pathologist (V.V.) diagnosed a diversion proctitis and a marked rectal alteration of the ganglion cells, whereas the intrinsic innervations at the deep colonic biopsy looked normal (Figures 2-4). As the patient s symptoms were worsening, a surgical operation was advised consisting of rectosigmoid resection and Duhamel anastomosis at the anal canal. The patient, once informed that this major operation had to be scheduled in two stages (recto-sigmoid resection and diverting ileostomy, plus closure of the stoma after six months), refused to undergo a restorative procedure as was afraid to become incontinent. An intersphincteric resection of the rectum and the lower sigmoid with a refashioning of the stoma, i.e., correction of the parastomal hernia with a mesh and fashioning of a terminal sigmoidostomy was then scheduled. DISCUSSION Constipation may be due to several causes, among them rectal aganglionosis, which may also occur in the adult. In this case a complete absence of the ganglia is unlikely, whereas either an hypoganglionosis or a ganglion dysplasia is much more frequent [2]. However, patients with Figure 1 Details of the lesions and the procedures carried out in our patient. The drawing shows the staple line anastomosis of the STARR with the peristaples fibrosis (grey) triggering the nerve spindles (black dots) located above the puborectalis muscle and the levator ani. The blue circles indicate the deep rectal and colonic biopsies, aimed at evaluating the intrinsic nerves. The red spots in the rectum indicate the diversion proctitis and the asterix on the small bowel loop indicates the parastomal hernia, due to the diastasis of the abdominal wall muscle. Figure 2 Diversion proctitis. Presence of minimal architectural distortion of the crypts and lymphoyd follicular hyperplasia in the lamina propria (arrow): hematoxylin and eosin 20. rectal aganglionosis, or Hirschprung s disease, usually have a megarectum-megacolon, an absence or alteration of the recto-anal inhibitory reflex at anorectal manometry and slowered intestinal transit times [3], whereas rectoanal reflex and transit time were normal in our patient. Moreover, patients with rectal a- or hypoganglionosis and those with neuronal dysplasia are likely to have a slow transit constipation with an empty rectum and a reduced frequency of the evacuations, whereas our patient more often suffered from obstructed defecation, i.e., various ineffective attempts to empty the bowel, requiring straining and sometime self-digitation, accompanied by pelviperineal heaviness and sense of incomplete evacuation. Due to these symptoms, she underwent her first operation, i.e., a STARR procedure, which is a double stapled resection of the anterior and posterior wall of the lower rectum, aimed at correcting the recto-rectal or recto-anal intussusception. Boccasanta et al [4] achieved good results on the short-term, but also reported 20% of painful defecation and proctalgia in 20% of their patient at one 4463 April 21, 2014 Volume 20 Issue 15

365 Pescatori LC et al. Failed stapled rectal resection A B C D Figure 3 Microscopic findings (deep rectal biopsy). A: Rectal mucosa hematoxylin and eosin (HE) 20; B: Nervous plexus in the muscular layer; absence of mature gangliar cells HE 40; C: S-100 immunostain: nervous intramuscular plexus. Absence of gangliar cells, positivity of glial cells 40; D: NSE immunostain: Nervous intramuscular plexus. Absence of gangliar cells, 40. year, which was the symptom reported by our patient. Moreover, pain may arise at the site of staple line due to the peristaple fibrosis, triggering the nervous network on the puborectalis muscle [5], exactly the same area where our patient felt the maximum pain at the digital exploration. Recurrence of symptoms, i.e., constipation, following STARR is widely reported by the literature: according Gagliardi et al [6] 55% of the patients still are constipated 18 mo after surgery. This was the case of our patient. Symptoms are likely to have persisted because a complete assessment for the potential causes for constipation was not carried pre-operatively. According to our theory of obstructed defecation as an iceberg syndrome, based on a prospective study carried out on 100 patients, the recto-anal intussusception is just the tip of the iceberg and a number of potential both functional and organic underwater rocks, i.e., underlying occult lesions, should be investigated prior to indicate surgery, e.g., altered psychological pattern [7]. Should we have seen the patients first, we would have performed a deep rectal biopsy, which would have revealed the disordered ganglia, and a psychological assessment, which would have probably shown a high level of anxiety. Then, in case of unsuccessful biofeed-back, colonic irrigation and psychological support, we would have carried first a non-invasive Lynn rectal myectomy, a procedure often effective in case of short rectal aganglionosis and then, in case of failure, a Duhamel procedure. Due to both proctalgia and recurrent severe constipation after STARR, she underwent a stoma creation in another hospital. Stoma creation is an alternative treatment to restorative surgeries for persisting constipation not responding to conservative and other surgical treatment [8]. Finally, parastomal hernia is the most frequent complication after stoma formation [9]. Diversion proctitis, which affected our patient, may arise after fecal diversion due to the alteration of the trophism of the rectal mucosa, which is due to the passage of the stool [10]. Bleeding is one of the more common symptoms in patient s with diverting proctitis. The fact that our patient refused a major restorative operation is comprehensible, as Duhamel procedure may be followed by fecal incontinence in a number of cases [11]. Therefore an intersphincteric resection of the rectum and the lower sigmoid, which may be carried out by a combined transanal and abdominal route with a cosmetic Pfannestiel incision seemed to us a valid alternative. The excision of the peristapled fibrosis, aimed at curing the proctalgia, was also planned. In conclusion, this clinical case seemed to us worth to be reported as it seems quite unusual, due to the absence of rectal ganglia in an adult with normal anorectal manometry and intestinal transit time study. This discrepancy might be due to the fact that the pathological disorders of the intrinsic nerves was not severe enough to completely impair large bowel motility. Moreover, the present report may offer a warning against the abuse of a novel high-cost procedure, such as the STARR opera April 21, 2014 Volume 20 Issue 15

366 Pescatori LC et al. Failed stapled rectal resection A B C D Figure 4 Microscopic findings (deep sigmoid biopsy). A: Afferent loop of the sigmoidostomy HE 20; B: Nervous plexus in the muscular layer; presence of mature gangliar cells (arrows) HE 40; C: S-100 immunostain: nervous intramuscular plexus. Presence of gangliar cells (arrows), positivity of glial cells 40; D: NSE immunostain: nervous intramuscular plexus. Presence of gangliar cells positive at immunostain (arrows), Camilleri M, Szarka L. Dysmotility of the small intestine and colon. In: Yamada T. Textbook of Gastroenterology. 5th ed. Oxford: Wiley-Blackwell, 2009: Pescatori M, Mattana C, Castiglioni GC. Adult megacolon due to total hypoganglionosis. Br J Surg 1986; 73: 765 [PMID: ] 3 Morais MB, Sdepanian VL, Tahan S, Goshima S, Soares AC, Motta ME, Fagundes Neto U. [Effectiveness of anorectal manometry using the balloon method to identify the inhibitory recto-anal reflex for diagnosis of Hirschsprung s disease]. Rev Assoc Med Bras 2005; 51: ; discussion 312 [PMID: DOI: /S ] 4 Boccasanta P, Venturi M, Stuto A, Bottini C, Caviglia A, Carrition, which may carry high recurrence ad reintervention rate, without a proper complete assessment of the patient prior to surgery. COMMENTS Case characteristics The patient suffered from anxiety, persisting constipation, i.e., difficulties to empty her stoma, and severe proctalgia following STARR and sigmoidostomy. Differential diagnosis This study suspected that constipation was also due to other reasons rather than simply the rectal internal mucosal prolapse causing obstructed defecation, for which the STARR had been carried out, and we wanted to investigate if the patient had a pathology of the ganglia in the intrinsic plexus, as she started to suffer constipation from her infancy. Laboratory diagnosis Blood test were normal and anorectal manometry, carried out elsewhere, was normal. Imaging diagnosis Intestinal transit times, also performed elsewhere after the ingestion of 20 radiopaque markers, were normals. Pathological diagnosis The deep rectal biopsy showed diversion proctitis and absence of ganglion cells, whereas the intrinsic nervous system was normal at the deep sigmoid biopsy and the biopsies taken close to the STARR staple line showed a fibrosis triggering the nerve spindles above the puborectalis muscle. Treatment This study suggested an intersphincteric resection of the rectum plus agrapphectomy, as the patient had refused a Duhamel procedure for fear of incontinence, and steroid enemas which improved the diversion proctitis. Related reports The patient also had a parastomal hernia causing troubled stool evacuation through the stoma, and her anxiety, which might have worsened constipation prior to the STARR, was mainly due to the loss of an adoptive son. Term explanation STARR is a transanal stapled resection of the rectum, aimed at excising rectocele and rectal internal prolapse, which carries 20% of chronic proctalgia and occasional life-threatening complications. Experiences and lessons This case report demonstrates that normal anorectal manometry and intestinal transit time cannot exclude rectal aganglionosis, rectal biopsy being the goldstandard for the diagnosis, and supports our theory of constipation as an Iceberg syndrome, in which lesions such rectal internal mucosal prolapse, often targeted by the surgeons, are just the tip of the iceberg, the main causes of symptoms being underlying occult lesions, such as, in this case, rectal aganglionosis and, possibly, anxiety. Peer review They could not explain with sound data the discordance between rectal aganglionosis and normal manometry and transit times study, this is perhaps the weakness of the study. REFERENCES 4465 April 21, 2014 Volume 20 Issue 15

367 Pescatori LC et al. Failed stapled rectal resection ero A, Mascagni D, Mauri R, Sofo L, Landolfi V. Stapled transanal rectal resection for outlet obstruction: a prospective, multicenter trial. Dis Colon Rectum 2004; 47: ; discussion [PMID: DOI: /s ] 5 De Nardi P, Bottini C, Faticanti Scucchi L, Palazzi A, Pescatori M. Proctalgia in a patient with staples retained in the puborectalis muscle after STARR operation. Tech Coloproctol 2007; 11: [PMID: ] 6 Gagliardi G, Pescatori M, Altomare DF, Binda GA, Bottini C, Dodi G, Filingeri V, Milito G, Rinaldi M, Romano G, Spazzafumo L, Trompetto M. Results, outcome predictors, and complications after stapled transanal rectal resection for obstructed defecation. Dis Colon Rectum 2008; 51: ; discussion 195 [PMID: DOI: /s ] 7 Pescatori M, Spyrou M, Pulvirenti d Urso A. A prospective evaluation of occult disorders in obstructed defecation using the iceberg diagram. Colorectal Dis 2006; 8: [PMID: DOI: /j x] 8 Pfeifer J. Surgery for constipation. Acta Chir Iugosl 2006; 53: [PMID: DOI: /ACI P] 9 Hansson BM. Parastomal hernia: treatment and prevention 2013; where do we go from here? Colorectal Dis 2013; 15: [PMID: DOI: /codi.12420] 10 Winslet MC, Poxon V, Youngs DJ, Thompson H, Keighley MR. A pathophysiologic study of diversion proctitis. Surg Gynecol Obstet 1993; 177: [PMID: ] 11 Keshtgar AS, Ward HC, Clayden GS, de Sousa NM. Investigations for incontinence and constipation after surgery for Hirschsprung s disease in children. Pediatr Surg Int 2003; 19: 4-8 [PMID: ] P- Reviewers: Furnari M, Hassan M S- Editor: Qi Y L- Editor: A E- Editor: Zhang DN 4466 April 21, 2014 Volume 20 Issue 15

368 Online Submissions: World J Gastroenterol 2014 April 21; 20(15): 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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371 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. For example, Montgomery Bissell, MD, Professor of Medicine, Chief, Liver Center, Gastroenterology Division, University of California, Box 0538, San Francisco, CA 94143, United States. montgomery.bissell@ucsf.edu Telephone and fax: Telephone and fax should consist of +, country number, district number and telephone or fax number, e.g., Telephone: Fax: Peer reviewers: All articles received are subject to peer review. Normally, three experts are invited for each article. Decision on acceptance is made only when at least two experts recommend publication of an article. All peer-reviewers are acknowledged on Express Submission and Peer-review System website. Abstract There are unstructured abstracts (no less than 200 words) and structured abstracts. The specific requirements for structured abstracts are as follows: An informative, structured abstract should accompany each manuscript. Abstracts of original contributions should be structured into the following sections: AIM (no more than 20 words; Only the purpose of the study should be included. Please write the Aim in the form of To investigate/study/ ), METH- ODS (no less than 140 words for Original Articles; and no less than 80 words for Brief Articles), RESULTS (no less than 150 words for Original Articles and no less than 120 words for Brief Articles; You should present P values where appropriate and must provide relevant data to illustrate how they were obtained, e.g., 6.92 ± 3.86 vs 3.61 ± 1.67, P < 0.001), and CONCLUSION (no more than 26 words). Key words Please list 5-10 key words, selected mainly from Index Medicus, which reflect the content of the study. Core tip Please write a summary of less than 100 words to outline the most innovative and important arguments and core contents in your paper to attract readers. Text For articles of these sections, original articles and brief articles, the main text should be structured into the following sections: INTRODUCTION, MATERIALS AND METHODS, RE- SULTS and DISCUSSION, and should include appropriate Figures and Tables. Data should be presented in the main text or in Figures and Tables, but not in both. Illustrations Figures should be numbered as 1, 2, 3, etc., and mentioned clearly in the main text. Provide a brief title for each figure on a separate page. Detailed legends should not be provided under the figures. This part should be added into the text where the figures are applicable. Keeping all elements compiled is necessary in line-art image. Scale bars should be used rather than magnification factors, with the length of the bar defined in the legend rather than on the bar itself. File names should identify the figure and panel. Avoid layering type directly over shaded or textured areas. Please use uniform legends for the same subjects. For example: Figure 1 Pathological changes in atrophic gastritis after treatment. A:...; B:...; C:...; D:...; E:...; F:...; G: etc. It is our principle to publish high resolution-figures for the E-versions. Tables Three-line tables should be numbered 1, 2, 3, etc., and mentioned clearly in the main text. Provide a brief title for each table. Detailed legends should not be included under tables, but rather added into the text where applicable. The information should complement, but not duplicate the text. Use one horizontal line under the title, a second under column heads, and a third below the Table, above any footnotes. Vertical and italic lines should be omitted. Notes in tables and illustrations Data that are not statistically significant should not be noted. a P < 0.05, b P < 0.01 should be noted (P > 0.05 should not be noted). If there are other series of P values, c P < 0.05 and d P < 0.01 are used. A third series of P values can be expressed as e P < 0.05 and f P < Other notes in tables or under illustrations should be expressed as 1 F, 2 F, 3 F; or sometimes as other symbols with a superscript (Arabic numerals) in the upper left corner. In a multi-curve illustration, each curve should be labeled with,,,,,, etc., in a certain sequence. Acknowledgments Brief acknowledgments of persons who have made genuine contributions to the manuscript and who endorse the data and conclusions should be included. Authors are responsible for obtaining written permission to use any copyrighted text and/or illustrations. REFERENCES Coding system The author should number the references in Arabic numerals according to the citation order in the text. Put reference numbers in square brackets in superscript at the end of citation content or after the cited author s name. For citation content which is part of the narration, the coding number and square brackets should be typeset normally. For example, Crohn s disease (CD) is associated with increased intestinal permeability [1,2]. If references are cited directly in the text, they should be put together within the text, for example, From references [19,22-24], we know that.... When the authors write the references, please ensure that the order in text is the same as in the references section, and also ensure the spelling accuracy of the first author s name. Do not list the same citation twice. PMID and DOI Pleased provide PubMed citation numbers to the reference list, e.g., PMID and DOI, which can be found at nlm.nihgov/sites/entrez?db=pubmed and org/simpletextquery/, respectively. The numbers will be used IV April 21, 2014 Volume 20 Issue 15

372 Instructions to authors 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 21, 2014 Volume 20 Issue 15

373 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 21, 2014 Volume 20 Issue 15

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