World Journal of Gastroenterology

Transcription

1 ISSN (print) ISSN (online) World Journal of Gastroenterology World J Gastroenterol 2017 September 21; 23(35): Published by Baishideng Publishing Group Inc

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

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

4 Dian-Liang Zhang, Qingdao Ya-Ping Zhang, Shijiazhuang You-Cheng Zhang, Lanzhou Jian-Zhong Zhang, Beijing Ji-Yuan Zhang, Beijing Hai-Tao Zhao, Beijing Jian Zhao, Shanghai Jian-Hong Zhong, Nanning Ying-Qiang Zhong, Guangzhou Ping-Hong Zhou, Shanghai Yan-Ming Zhou, Xiamen Tong Zhou, Nanchong Li-Ming Zhou, Chengdu Guo-Xiong Zhou, Nantong Feng-Shang Zhu, Shanghai Jiang-Fan Zhu, Shanghai Zhao-Hui Zhu, Beijing Croatia Tajana Filipec Kanizaj, Zagreb Cuba Damian Casadesus, Havana Czech Jan Bures, Hradec Kralove Marcela Kopacova, Hradec Kralove Otto Kucera, Hradec Kralove Marek Minarik, Prague Pavel Soucek, Prague Miroslav Zavoral, Prague Denmark Vibeke Andersen, Odense E Michael Danielsen, Copenhagen Egypt Mohamed MM Abdel-Latif, Assiut Hussein Atta, Cairo Ashraf Elbahrawy, Cairo Mortada Hassan El-Shabrawi, Cairo Mona El Said El-Raziky, Cairo Elrashdy M Redwan, New Borg Alrab Zeinab Nabil Ahmed Said, Cairo Ragaa HM Salama, Assiut Maha Maher Shehata, Mansoura Mostafa Sira, Menofiya 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 23 Number 35 September 21, 2017 EDITORIAL 6371 Organization of future training in bariatric gastroenterology Koch TR, Shope TR, Gostout CJ 6379 Resistance to clarithromycin and gastroenterologist's persistence roles in nomination for Helicobacter pylori as high priority pathogen by World Health Organization Abadi ATB REVIEW 6385 Time to clinical response and remission for therapeutics in inflammatory bowel diseases: What should the clinician expect, what should patients be told? Vasudevan A, Gibson PR, van Langenberg DR ORIGINAL ARTICLE Basic Study 6403 Pharmacological inhibition of cannabinoid receptor 1 stimulates gastric release of nesfatin-1 via the mtor pathway Folgueira C, Barja-Fernandez S, Prado L, Al-Massadi O, Castelao C, Pena-Leon V, Gonzalez-Saenz P, Baltar J, Baamonde I, Leis R, Dieguez C, Pagotto U, Casanueva FF, Tovar SA, Nogueiras R, Seoane LM 6412 Effects of aspirin and enoxaparin in a rat model of liver fibrosis Li CJ, Yang ZH, Shi XL, Liu DL Retrospective Study 6420 Different clinical presentations of metachronous pulmonary metastases after resection of pancreatic ductal adenocarcinoma: Retrospective study and review of the literature Lovecek M, Skalicky P, Chudacek J, Szkorupa M, Svebisova H, Lemstrova R, Ehrmann J, Melichar B, Yogeswara T, Klos D, Vrba R, Havlik R, Mohelnikova-Duchonova B 6429 Evaluation of novel slim biopsy forceps for diagnosis of biliary strictures: Single-institutional study of consecutive 360 cases (with video) Yamamoto K, Tsuchiya T, Itoi T, Tsuji S, Tanaka R, Tonozuka R, Honjo M, Mukai S, Kamada K, Fujita M, Asai Y, Matsunami Y, Nagakawa Y, Yamaguchi H, Sofuni A 6437 Evaluation of the relationship between hepatocellular carcinoma location and transarterial chemoembolization efficacy Miki I, Murata S, Uchiyama F, Yasui D, Ueda T, Sugihara F, Saito H, Yamaguchi H, Murakami R, Kawamoto C, Uchida E, Kumita SI September 21, 2017 Volume 23 Issue 35

12 Contents World Journal of Gastroenterology Volume 23 Number 35 September 21, Evaluation of recurrence in gastric carcinoma: Comparison of contrast-enhanced computed tomography and positron emission tomography/computed tomography Kim JH, Heo SH, Kim JW, Shin SS, Min JJ, Kwon SY, Jeong YY, Kang HK 6457 Duodenum and ventral pancreas preserving subtotal pancreatectomy for low-grade malignant neoplasms of the pancreas: An alternative procedure to total pancreatectomy for low-grade pancreatic neoplasms Wang X, Tan CL, Song HY, Yao Q, Liu XB 6467 Validated preoperative computed tomography risk estimation for postoperative hepatocellular carcinoma recurrence Zhang W, Lai SL, Chen J, Xie D, Wu FX, Jin GQ, Su DK Observational Study 6474 Diagnostic delay in inflammatory bowel disease increases the risk of intestinal surgery Lee DW, Koo JS, Choe JW, Suh SJ, Kim SY, Hyun JJ, Jung SW, Jung YG, Yim HJ, Lee SW Prospective Study 6482 Fecal marker levels as predictors of need for endoscopic balloon dilation in Crohn s disease patients with anastomotic strictures Lopes S, Andrade P, Rodrigues-Pinto E, Afonso J, Macedo G, Magro F SYSTEMATIC REVIEWS 6491 Gastrosplenic fistula occurring in lymphoma patients: Systematic review with a new case of extranodal NK/ T-cell lymphoma Kang DH, Huh J, Lee JH, Jeong YK, Cha HJ META-ANALYSIS 6500 Proton pump inhibitors therapy and risk of Clostridium difficile infection: Systematic review and metaanalysis Trifan A, Stanciu C, Girleanu I, Stoica OC, Singeap AM, Maxim R, Chiriac SA, Ciobica A, Boiculese L 6516 Outcomes after liver transplantation in accordance with ABO compatibility: A systematic review and metaanalysis Lee EC, Kim SH, Park SJ CASE REPORT 6534 Case of colonic intussusception secondary to mobile cecum syndrome repaired by laparoscopic cecopexy using a barbed wound suture device Yamamoto T, Tajima Y, Hyakudomi R, Hirayama T, Taniura T, Ishitobi K, Hirahara N 6540 Acquired amegakaryocytic thrombocytopenia previously diagnosed as idiopathic thrombocytopenic purpura in a patient with hepatitis C virus infection Ichimata S, Kobayashi M, Honda K, Shibata S, Matsumoto A, Kanno H II September 21, 2017 Volume 23 Issue 35

13 Contents World Journal of Gastroenterology Volume 23 Number 35 September 21, 2017 LETTERS TO THE EDITOR 6546 Novel endoscopic management of buried bumper syndrome in percutaneous endoscopic gastrostomy: The Olympus HookKnife Wolpert LE, Summers DM, Tsang A III September 21, 2017 Volume 23 Issue 35

14 Contents World Journal of Gastroenterology Volume 23 Number 35 September 21, 2017 ABOUT COVER Editorial board member of World Journal of Gastroenterology, J Enrique Dominguez-Munoz, MD, PhD, Associate Professor, Chief Doctor, Director, Department of Gastroenterology and Hepatology, University Hospital of Santiago de Compostela, Santiago de Compostela, Spain 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 1375 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 (WJG) 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 and Directory of Open Access Journals. The 2017 edition of Journal Citation Reports cites the 2016 impact factor for WJG as (5-year impact factor: 3.176), ranking WJG as 29 th among 79 journals in gastroenterology and hepatology (quartile in category Q2). FLYLEAF I-IX Editorial Board EDITORS FOR THIS ISSUE Responsible Assistant Editor: Xiang Li Responsible Electronic Editor: Yu-Jie Ma Proofing Editor-in-Chief: Lian-Sheng Ma Responsible Science Editor: Ze-Mao Gong Proofing Editorial Office Director: Jin-Lei Wang NAME OF JOURNAL World Journal of Gastroenterology ISSN ISSN (print) ISSN (online) LAUNCH DATE October 1, 1995 FREQUENCY Weekly EDITORS-IN-CHIEF Damian Garcia-Olmo, MD, PhD, Doctor, Professor, Surgeon, Department of Surgery, Universidad Autonoma de Madrid; Department of General Surgery, Fundacion Jimenez Diaz University Hospital, Madrid 28040, Spain 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 BOARD MEMBERS All editorial board members resources online at EDITORIAL OFFICE Jin-Lei Wang, Director Yuan Qi, Vice Director Ze-Mao Gong, Vice Director World Journal of Gastroenterology Baishideng Publishing Group Inc 7901 Stoneridge Drive, Suite 501, Pleasanton, CA 94588, USA Telephone: Fax: editorialoffice@wjgnet.com Help Desk: PUBLISHER Baishideng Publishing Group Inc 7901 Stoneridge Drive, Suite 501, Pleasanton, CA 94588, USA Telephone: Fax: bpgoffice@wjgnet.com Help Desk: PUBLICATION DATE September 21, 2017 COPYRIGHT 2017 Baishideng Publishing Group Inc. Articles published by this Open-Access journal are distributed under the terms of the Creative Commons Attribution Noncommercial License, which permits use, distribution, and reproduction in any medium, provided the original work is properly cited, the use is non commercial and is otherwise in compliance with the license. SPECIAL STATEMENT All articles published in journals owned by the Baishideng Publishing Group (BPG) represent the views and opinions of their authors, and not the views, opinions or policies of the BPG, except where otherwise explicitly indicated. INSTRUCTIONS TO AUTHORS Full instructions are available online at wjgnet.com/bpg/gerinfo/204 ONLINE SUBMISSION IV September 21, 2017 Volume 23 Issue 35

15 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) EDITORIAL Organization of future training in bariatric gastroenterology Timothy R Koch, Timothy R Shope, Christopher J Gostout Timothy R Koch, Timothy R Shope, Center for Advanced Laparoscopic General and Bariatric Surgery, MedStar Washington Hospital Center and Georgetown University School of Medicine, Washington, DC 20010, United States Christopher J Gostout, Professor Emeritus, Division of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN 55905, United States ORCID number: Timothy R Koch ( ); Timothy R Shope ( ); Christopher J Gostout ( ). Author contributions: Koch TR, Shope TR and Gostout CJ outlined and wrote the manuscript. Conflict-of-interest statement: Koch TR has no conflict of interest to declare. Shope TR has received fees for serving as a Consultant for Ethicon Inc., Somerville, NJ, United States. Gostout CJ has received fees for serving as a Consultant for Olympus Medical Systems, Japan, as Chief Medical Officer is an employee of Apollo Endosurgery, Austin, TX, United States, and has equity in Apollo Endosurgery, Austin, TX, United States. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Invited manuscript Correspondence to: Timothy R Koch, MD, Professor of Medicine (Gastroenterology), Georgetown University School of Medicine, Center for Advanced Laparoscopic General and Bariatric Surgery, POB South, Suite 301, MedStar-Washington Hospital Center, 110 Irving Street, NW, Washington, DC 20010, United States. timothy.r.koch@medstar.net Telephone: Fax: Received: July 11, 2017 Peer-review started: July 12, 2017 First decision: August 10, 2017 Revised: August 18, 2017 Accepted: September 5, 2017 Article in press: September 5, 2017 Published online: September 21, 2017 Abstract A world-wide rise in the prevalence of obesity continues. This rise increases the occurrence of, risks of, and costs of treating obesity-related medical conditions. Diet and activity programs are largely inadequate for the long-term treatment of medically-complicated obesity. Physicians who deliver gastrointestinal care after completing traditional training programs, including gastroenterologists and general surgeons, are not uniformly trained in or familiar with available bariatric care. It is certain that gastrointestinal physicians will incorporate new endoscopic methods into their practice for the treatment of individuals with medically-complicated obesity, although the longterm impact of these endoscopic techniques remains under investigation. It is presently unclear whether gastrointestinal physicians will be able to provide or coordinate important allied services in bariatric surgery, endocrinology, nutrition, psychological evaluation and support, and social work. Obtaining longitudinal results examining the effectiveness of this ad hoc approach will likely be difficult, based on prior experience with other endoscopic measures, such as the adenoma detection rates from screening colonoscopy. As a longterm approach, development of a specific curriculum incorporating one year of subspecialty training in bariatrics to the present training of gastrointestinal fellows needs to be reconsidered. This approach should be facilitated by gastrointestinal trainees prior residency training in subspecialties that provide care for individuals with medical complications of obesity, including endocrinology, cardiology, nephrology, and neurology. Such training could incorporate additional rotations with collaborating providers in bariatric surgery, nutrition, and psychiatry. Since such training 6371 September 21, 2017 Volume 23 Issue 35

16 Koch TR et al. Training in GI bariatrics would be provided in accredited programs, longitudinal studies could be developed to examine the potential impact on accepted measures of care, such as complication rates, outcomes, and costs, in individuals with medically-complicated obesity. Key words: Obesity; Bariatrics; Bariatric surgery; Intragastric balloon; Endoscopic sleeve gastroplasty; Nutrition; Endoscopy; Training The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: A world-wide rise in the prevalence of obesity and obesity-related medical conditions needs to be addressed. Newer endoscopic methods will be incorporated into the clinical practice of gastrointestinal physicians, although the long-term impact of these endoscopic methods on medically-complicated obesity remains under investigation. As a potential long-term approach, development of a curriculum designed to incorporate one year of subspecialty training in bariatrics to the present training of gastrointestinal fellows needs to be reconsidered. Longitudinal studies could be performed to examine the impact of subspecialty training on medical complications, outcomes, and costs in treating individuals with medically-complicated obesity. Koch TR, Shope TR, Gostout CJ. Organization of future training in bariatric gastroenterology. World J Gastroenterol 2017; 23(35): Available from: URL: com/ /full/v23/i35/6371.htm DOI: org/ /wjg.v23.i INTRODUCTION The rising prevalence of obesity is a worldwide disorder, and therefore this problem will require a collaborative global approach. One commonly utilized definition of overweight and obesity is based upon the body mass index or BMI, which is calculated from an individual s height and weight (see Table 1). An extensive study has shown that the worldwide percentage of adults who are overweight (BMI kg/m 2 ) or obese (BMI 30.0 and higher) increased from 28.8% of men in 1980 to 36.9% of men in 2013, and increased from 29.8% of women in 1980 to 38.0% of women in 2013 [1]. Based on their data, the authors by extrapolation estimated that 2.1 billion individuals were overweight or obese by Similar results were recently reported in a study of 195 countries [2]. In more than 70 countries, the prevalence of obesity has doubled since 1980 [2]. Obesity-related medical conditions (see Table 2) include insulin-resistant diabetes mellitus, obstructive sleep apnea, mechanical or degenerative osteoarthritis, non-alcohol steatohepatitis, hypertension, dyslipidemia with secondary coronary artery disease, pseudotumor cerebri, and likely gastroesophageal reflux, deep vein thrombosis, and asthma [3]. Obesity has also been consistently associated with increased risk of kidney, breast, endometrial, colorectal, pancreatic, esophageal, and gall bladder carcinoma [4]. Excess body weight contributes to a significant percentage of cases of cancer [4]. With the increase in the incidence of obesity in Asia, The Gut and Obesity in Asia Workgroup has developed an experts consensus on obesity and gastrointestinal diseases in Asia [5]. In a worldwide systematic review, it was reported that obesity accounts for 0.7% to 2.8% of a country s total healthcare expenditures and that individuals with obesity have at least 30% higher healthcare costs [6]. In the United States, the medical care costs of obesity in 2008 were estimated to be $147 billion [7]. As one can ascertain from this rise in the worldwide prevalence of obesity, the previously suggested solutions with regards to this serious medical problem have not been effective. One suggested approach is education, with the inference that individuals will then make responsible choices when eating several times every day [8]. In the United States, the National Institutes of Health and the United States Preventive Services Task Force in 1998 and in 2012 suggested behavioral interventions, increased physical activity, and improved dietary intake [9,10]. Expectations of results from these recommendations however must be balanced against the findings of multiple metaanalyses [11-13] that have examined the potential benefits of behavioral interventions, diet and activity programs in individuals with excess weight. Reported long term results are for studies of only 12 to 18 mo. Unfortunately, the published weight loss results are modest with 1.5 to 5 kg of weight loss at 12 mo [11] after adopting behavioral interventions (which can include self-monitoring, setting weight loss goals, addressing barriers to change, and strategizing longterm change in lifestyle). These meta-analyses mention that there is higher weight loss reported for those individuals participating in dietary only programs compared to individuals involved in exercise or activity only programs. Combined dietary and physical activity programs tailored to individual metabolic and dietary response needs appear to be more beneficial than solely dietary programs. However, adding physical activity programs appear to increase overall weight loss by only an additional 0 to 3 kg. Our review of previous contributions in this field included specific searches on PubMed ( nlm.nih.gov/pubmed) on June 11, The terms gastroenterology fellow training yielded 111 abstracts; the terms gastroenterology fellow nutrition yielded 35 abstracts; the terms gastroenterology fellow obesity yielded 8 abstracts; the terms nutrition training gastrointestinal medicine yielded 251 abstracts; and 6372 September 21, 2017 Volume 23 Issue 35

17 Koch TR et al. Training in GI bariatrics Table 1 Obesity defined by body mass index BMI (kg/m 2 ) Definition Obesity class Normal Overweight Obesity Ⅰ Obesity Ⅱ Obesity Ⅲ BMI: Body mass index. Table 2 Obesity related medical disorders Diabetes mellitus Obstructive sleep apnea Degenerative osteoarthritis Non-alcohol steatohepatitis Hypertension Dyslipidemia with coronary artery disease Pseudo-tumor cerebri Gastroesophageal reflux Deep vein thrombosis Asthma Carcinomas (kidney, breast, endometrial, colorectal, pancreatic, esophageal, gallbladder) the terms obesity training gastrointestinal medicine yielded 97 abstracts. All abstracts and all manuscripts consistent with the topic of this present manuscript were reviewed by one of us (TRK). ONGOING ROLE OF GASTROINTESTINAL PHYSICIANS IN OBESITY CARE AND TRAINING Gastrointestinal physicians and general surgeons both deliver gastrointestinal care to individuals with medicallycomplicated obesity, including the performance of endoscopy. These physicians, after completing traditional training programs, are uniformly trained in or familiar with available bariatric care. This notion is not a new concern. A survey of gastroenterologists published in 1976 revealed that obesity was included as one of the five most common diagnostic areas [14]. The author suggested that his findings could be the basis for planning education, training, and research activities in gastroenterology. An editorial published 26 years later described a commitment of the American Gastroenterological Association to nutrition and obesity, including a summary of elements included in advanced or level 2 training in nutrition [15]. A 2006 study in Canada of gastrointestinal clinicians and gastrointestinal fellows reconfirmed the opinion that training in management of obesity was inadequate [16]. A follow-up survey of Canadian gastrointestinal fellows published in 2009 revealed that gastrointestinal fellows considered their knowledge of nutrition and obesity to be suboptimal [17]. A written examination completed by these gastrointestinal fellows confirmed that their knowledge base indeed was suboptimal, especially in the areas of nutritional support, knowledge of micronutrients and macronutrients, obesity, and nutrition in gastrointestinal diseases. Similar findings have been reported in studies of gastrointestinal fellows in Iran [18] and in the United States [19]. One response to these long-standing concerns and weaknesses in nutrition training has been the publication of a potentially extensive curriculum in nutrition as part of the core curriculum for fellows or trainees in gastroenterology [20]. Training of gastrointestinal fellows internationally has involved programs with a traditional pathway which include disease-related training and endoscopy [21], and programs that combine basic elements of gastrointestinal training with time for advanced training in a subspecialty of gastroenterology or hepatology. The traditional involvement of gastrointestinal clinicians in obesity has involved two levels of medical care: (1) direct care of gastrointestinal medical disorders related to obesity including non-alcohol steatohepatitis, gastroesophageal reflux, colorectal carcinoma, pancreatic carcinoma, esophageal carcinoma, and gall bladder carcinoma; and (2) consultants in collaboration with providers who treat patients for obesity, for example in the care of individuals who have undergone bariatric surgery. More recently, due to advances in therapeutic endoscopy, gastrointestinal physicians have begun to assume a role as the provider directly treating individuals for obesity. The question to now be considered is what pathways should be utilized to expand the role of gastrointestinal physicians in the care of individuals with medically-complicated obesity. INCORPORATION OF BARIATRIC MEDICINE INTO ONGOING GASTROINTESTINAL PRACTICE There are two major approaches for a process by which gastrointestinal physicians can evolve into physicians with primary responsibility for the treatment of individuals with medically-complicated obesity. A recent White Paper from the American Gastroenterology Association proposes incorporation of methods for obesity care and weight management into existing gastroenterological practices [22]. This approach immediately raises the question of how much weight loss is required for the appropriate treatment of medically-complicated obesity. An overview of treatment of individuals with non-alcohol steatohepatitis summarizes a proposed goal of loss of 10% of total body weight [23]. This weight loss goal can be difficult to reach in individuals with obesity through the use of dietary and activity programs. In agreement with recently published meta-analyses [11-13], a recent study from Cuba again demonstrated that only 10% of participants involved in dietary and activity 6373 September 21, 2017 Volume 23 Issue 35

18 Koch TR et al. Training in GI bariatrics Required weight loss (LBS) Overweight Class 1 obesity Class 2 obesity Class 3 obesity (Minimum loss) 5'2" 5'8" 6'2" Patient height Figure 1 The vertical bars depict weight loss required to reach the normal weight range in overweight and obese individuals of difference heights. The solid line approximates the best published weight loss obtained through diet and activity programs. Table 4 Potential endoscopic methods to treat medicallycomplicated obesity Method Delivery of a Weight Loss Device Intragastric Balloon Orbera 1 ReShape 1 Obalon 1 Duodeno-jejunal bypass sleeve Aspiration therapy Use of intraluminal suturing Endoscopic sleeve gastroplasty Transoral gastroplasty Potential physiological mechanisms Stomach distension; reduce volume required for satiety; delay gastric emptying Delay Gastric Emptying; Induce Malabsorption Reduce Intragastric Nutrients Induce early satiation; delay gastric emptying Reduce volume required for satiety 1 Listed in order of dates the devices were approved by United States Food Table 3 Weight loss medications approved by the United States Food and Drug Administration Medication Short term use Benzphetamine Diethylpropion Phendimetrazine Phentermine Long term use Orlistat Liraglutide Lorcaserin Naltrexone and bupropion Phentermine and topiramate Published weight loss Range from 3.6 to 8.1 kg 2.6 kg 4 to 6 kg 3.2 kg 5 kg 8.8 kg programs were able to lose 10% or more of their total body weight [24]. Again in agreement with ongoing experience [23], liver biopsies revealed that 90% of the participants who lost 10% or more of their total body weight had resolution of non-alcohol steatohepatitis. This study did not address the follow up concern which of course is long-term maintenance of weight loss, or an individual s weight regain. When considering treatment of individuals with medically-complicated obesity, Figure 1 summarizes weight loss (in pounds) required to reach the normal weight range in overweight individuals and in obese individuals of difference heights. The solid line approximates the best published weight loss obtained through dietary and activity programs. Figure 1 provides a graphic depiction of the extent of weight loss required to improve serious medical disorders in individuals with Type Ⅱ obesity and Type Ⅲ obesity. For this reason, we have previously suggested that bariatric surgery utilizing vertical sleeve gastrectomy will likely become a major consideration for the treatment of nonalcoholic steatohepatitis in those individuals with Type Ⅱ obesity and Type Ⅲ obesity [25]. As step-up therapy for the treatment of medicallycomplicated obesity, the United States Food and Drug and Drug Administration. Administration has approved nine medical therapies for weight loss (see Table 3) [26-29]. There are four sympathomimetic drugs, which increase adrenergic stimulus, that have been approved for short term (12 wk) of medical therapy. This group includes Benzphetamine, Diethylpropion, Phendimetrazine, and Phentermine. There are five medical therapies approved for long-term medical therapy, which includes Orlistat, Liraglutide, Lorcaserin, Naltrexone with Bupropion, and Phentermine with Topiramate. As shown in Table 3, the published weight loss results reported for these medications are modest, ranging from 2.6 to 8.8 kg. Prescribing providers should be familiar with the significant risks involved in using these medications. For example, Orlistat can lead to deficiencies of fat soluble vitamin A, vitamin D, vitamin E, and vitamin K. Lorcaserin should not be used with monoamine oxidase inhibitors or selective serotonin reuptake inhibitors. Topiramate is associated with oral clefts when taken by pregnant women. The next available step-up therapies for treatment of medically-complicated obesity involve the utilization of newer endoscopic techniques that are now available. These techniques can be divided into either the delivery of a weight loss device or the use of intralumenal suturing to promote weight loss [30] (see Table 4). Individualized care with scheduled post-procedural follow up is important for long-term optimization of an endoscopic weight loss program, as has been supported by studies of adjustable gastric banding [31]. Individuals might also benefit from transition to the use of a well selected long-term weight loss medication. Upper endoscopy has been utilized for placement of an intragastric balloon, a duodeno-jejunal bypass sleeve, or an aspiration tube. Other than The AspireAssist described below, all of these devices are meant to be removed at 6 to 12 mo after their placement. The United States Food and Drug Administration approved the 6374 September 21, 2017 Volume 23 Issue 35

19 Koch TR et al. Training in GI bariatrics first intragastric balloon, the Garren-Edwards bubble in Interest in intragastric balloon has grown due to the United States Food and Drug Administration s approval of 3 separate intragastric balloon systems in the past 2 years: the Orbera balloon, the ReShape balloon, and the Obalon balloon. All three balloon systems must be removed after 6 mo. Clinical research has examined factors important for weight loss with the use of an intragastric balloon [32]. The mechanism for weight loss from the fluid filled intra-gastric balloon, e.g., delayed gastric emptying, has also been identified [33]. A duodenojejunal bypass sleeve, EndoBarrier, (GI Dynamics, Inc., Lexington, MA, United States) is available in some countries. This bypass sleeve is a fluoroscopically assisted and endoscopically implanted, removable, and impermeable fluoropolymer sleeve with a nitinol anchor. At upper endoscopy, the 60 cm long device is deployed from the duodenal bulb and into the jejunum under fluoroscopic guidance. In 2016, the United States Food and Drug Administration approved The AspireAssist produced by Aspire Bariatrics, King of Prussia, PA, United States which provides a long term option for patients in the higher body mass index range. At upper endoscopy, a specialized aspiration tube is placed percutaneously into the stomach. This aspiration tube has both an intragastric portion with holes to permit aspiration as well as a skin port. Individuals with an aspiration tube are instructed to aspirate their gastric contents 20 min after a meal containing more than 200 kcal. The device is not meant to be placed in an individual with an eating disorder. The endoscopic sleeve gastroplasty (ESG), first reported from the Mayo Clinic, Rochester, MN, United States in 2013, has eclipsed all prior failed efforts at altering the gastric lumen endoscopically [34,35]. There have been over 2500 ESG procedures performed worldwide with patient follow-up now exceeding 2 years, and a recent multicenter study using this technique to produce an endoscopic sleeve gastroplasty reported 24-mo weight loss data [36]. Publications with individual and multicenter experiences are appearing and demonstrate not only sustained weight loss but improvement in co-morbidities. There is also published experience discussing mechanisms by which ESG is important a patients weight loss [37]. Others [38,39] are attempting to duplicate successful weight loss using technologies differing from the existing endoscopic suturing device (OverStitch, Apollo Endosurgery, Austin, TX, United States) presently used for the ESG. The advantages of development of an endoscopic procedure to mimic the popular and effective vertical sleeve gastrectomy are clear, e.g. reduced risk of anesthesia and reduced risk of a gastric leak or perforation. The importance of obtaining long term weight loss data to support durability is supported by results from the surgical literature. In the original report from the Mayo Clinic, Rochester, MN, United States of the surgical non-banded vertical gastroplasty, at 4 years of follow up, only 31% of patients were judged to have persistent excess weight lost [40]. In additional studies, risk factors important for treatment failures after vertical gastroplasty were examined [41]. Even with the availability of this information, a study (published 16 years later) involving seven years of follow up after vertical banded gastroplasty reported staple line disruption in 54% of patients [42]. Sixty percent of patients after vertical banded gastroplasty underwent a major re-operation during this seven year follow up period. After practicing gastrointestinal physicians incorporate these newer endoscopic techniques into their care of individuals with obesity, a major question will arise as to whether reliable follow up will be obtained. In examination of other endoscopic quality measures, the recommendation that endoscopists should track their adenoma detection rates from screening colonoscopy has been supported by results of interval cancers published in 2010 [43]. However, in a 2015 survey of training programs in the United States, 63.8% of programs did not assess adenoma detection rates in their trainees [44]. It is similarly likely that it will be difficult to obtain longitudinal results designed to examine the effectiveness of an ad hoc approach for incorporation into an established gastrointestinal practice of new endoscopic methods for treating individuals with medically-complicated obesity. COMPLICATIONS DURING BARIATRIC CARE It is expected [45] that physicians providing bariatric care will properly evaluate, diagnose, and treat individuals who develop complications from their weight loss therapy. Prevention of complications from weight loss therapy begins by screening patients for their suitability to undergo treatment of medically-complicated obesity. Gastrointestinal physicians will need to decide how they will integrate physiologic (e.g, low satiety or rapid gastric emptying), psychological, or psychiatric assessment of eating disorders and underlying psychiatric illness into their practice. For example, it is reported that the prevalence of obesity is higher individuals with schizophrenia [46,47]. Following bariatric surgery, a Danish study has described increased risks of self-harm, psychiatric service use, and the occurrence of mental disorders [48]. With regards to the potential for addiction, persistent opioid abuse can result from even minor surgical procedures [49]. A systematic review of postoperative bariatric patients suggested the presence of increased 6375 September 21, 2017 Volume 23 Issue 35

20 Koch TR et al. Training in GI bariatrics alcohol use and increased admissions to substance abuse treatment facilities [50]. Physicians involved in primary weight loss therapy will need a program to screen for these potential problems and regimens for appropriate and sufficient treatment. SUBSPECIALTY TRAINING OF GASTROINTESTINAL FELLOWS IN BARIATRICS A second, long-term approach for incorporation of bariatric care into gastrointestinal practice would involve adaptation of the training of gastrointestinal fellows for treatment of and long-term control of medically-complicated obesity. Such an approach is supported by studies demonstrating improved bariatric outcomes in hospitals with accredited bariatric fellowships [51]. The evolution of a field of medicine requires individuals who focus on the evaluation of alteration of proposed methods and treatments. This potential approach should be facilitated by the gastrointestinal trainees prior internal medicine residency training in subspecialties that provide care for individuals with medical complications of obesity, including endocrinology, cardiology, nephrology, and neurology. It is expected that gastrointestinal fellows trained in advanced endoscopy should be capable of learning the proper application of most endoscopic techniques for weight loss therapy in individuals with medically-complicated obesity. Gastrointestinal fellows will also need training to manage post-operative complications (e.g., leaks, fistulas, and endolumenal revision of the Roux en Y gastric bypass and the sleeve gastrectomy) in patients who have undergone bariatric surgery. The training time commitment would depend upon the specific bariatric care that the future gastrointestinal physician would plan to deliver. The issue of development of a curriculum incorporating one year of subspecialty training in bariatrics to the present training of gastrointestinal fellows needs to be revisited. A one year period of subspecialty training in bariatrics could incorporate additional rotations with collaborating providers in bariatric medicine, bariatric surgery, nutrition, and psychiatry. The trainees could then be instructed in postoperative care of patients who have undergone bariatric surgery. Trainees could be trained in the management of adjustable gastric bands, postoperative nutritional disorders [52], postoperative symptoms and, as mentioned above, complications after Roux-en-Y gastric bypass surgery [3], and the management of major complications of vertical sleeve gastrectomy including gastric perforation or leak [53] and vomiting/dysphagia [54]. As an advantage of this potential organization, since such training would be provided in accredited programs, longitudinal studies could be developed to examine the potential impact on accepted measures of care, such as complication rates, outcomes, and costs, in individuals with medically-complicated obesity. CONCLUSION The prevalence of obesity continues to rise worldwide. This rise increases the incidence of obesity-related diseases, and these obesity-related diseases are an important component of health care costs in countries across the world. Diet and activity programs are largely inadequate for the long-term treatment of medically-complicated obesity. Gastrointestinal physicians to date have not been extensively involved in weight loss programs in individuals with medicallycomplicated obesity. It is certain that gastrointestinal physicians will incorporate new methods into their endoscopic practice for treatment of individuals with medically-complicated obesity. This change will involve incorporation of new endoscopic techniques into present gastrointestinal practices. Improved bariatric care will likely require an organized curriculum in gastrointestinal training programs. Involvement in weight loss programs induces significant psycho-social disorders. Obtaining optimal results will require practice patterns that screen for recognized pre-disposing gastric physiological disorders and psycho-social disorders, and develop ongoing protocols for the management of complications of bariatric care. Optimizing strategies for the long-term maintenance of weight loss after the use of newer endoscopic techniques remain important ongoing issues. 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World J Gastroenterol 2016; 22: [PMID: DOI: /wjg.v22.i5.1729] 21 Hatanaka H, Yamamoto H, Lefor AK, Sugano K. Gastroenterology Training in Japan. Dig Dis Sci 2016; 61: [PMID: DOI: /s x] 22 Acosta A, Streett S, Kroh MD, Cheskin LJ, Saunders KH, Kurian M, Schofield M, Barlow SE, Aronne L. White Paper AGA: POWER - Practice Guide on Obesity and Weight Management, Education, and Resources. Clin Gastroenterol Hepatol 2017; 15: e10 [PMID: DOI: /j.cgh ] 23 Hsu CC, Ness E, Kowdley KV. Nutritional Approaches to Achieve Weight Loss in Nonalcoholic Fatty Liver Disease. Adv Nutr 2017; 8: [PMID: DOI: /an ] 24 Vilar-Gomez E, Martinez-Perez Y, Calzadilla-Bertot L, Torres- Gonzalez A, Gra-Oramas B, Gonzalez-Fabian L, Friedman SL, Diago M, Romero-Gomez M. Weight Loss Through Lifestyle Modification Significantly Reduces Features of Nonalcoholic Steatohepatitis. Gastroenterology 2015; 149: e5; quiz e14-15 [PMID: DOI: /j.gastro ] 25 Hsueh W, Shope TR, Koch TR, Smith CI. Bariatric surgery for the treatment of nonalcoholic fatty liver disease: Is vertical sleeve gastrectomy the best future option? J Gastroent Hepatol Res 2016; 5: [DOI: /j.issn ] 26 Mehta A, Marso SP, Neeland IJ. Liraglutide for weight management: a critical review of the evidence. Obes Sci Pract 2017; 3: 3-14 [PMID: DOI: /osp4.84] 27 Khera R, Murad MH, Chandar AK, Dulai PS, Wang Z, Prokop LJ, Loomba R, Camilleri M, Singh S. Association of Pharmacological Treatments for Obesity With Weight Loss and Adverse Events: A Systematic Review and Meta-analysis. JAMA 2016; 315: [PMID: DOI: /jama ] 28 Joo JK, Lee KS. Pharmacotherapy for obesity. J Menopausal 6377 September 21, 2017 Volume 23 Issue 35

22 Koch TR et al. Training in GI bariatrics Med 2014; 20: [PMID: DOI: / jmm ] 29 Velazquez A, Apovian CM. Pharmacological management of obesity. Minerva Endocrinol 2017; Epub ahead of print [PMID: DOI: /S ] 30 Rashti F, Gupta E, Ebrahimi S, Shope TR, Koch TR, Gostout CJ. Development of minimally invasive techniques for management of medically-complicated obesity. World J Gastroenterol 2014; 20: [PMID: DOI: /wjg.v20.i ] 31 Schouten R, van t Hof G, Feskens PB. Is there a relation between number of adjustments and results after gastric banding? Surg Obes Relat Dis 2013; 9: [PMID: DOI: / j.soard ] 32 Kotzampassi K, Shrewsbury AD, Papakostas P, Penna S, Tsaousi GG, Grosomanidis V. Looking into the profile of those who succeed in losing weight with an intragastric balloon. J Laparoendosc Adv Surg Tech A 2014; 24: [PMID: DOI: /lap ] 33 Gómez V, Woodman G, Abu Dayyeh BK. Delayed gastric emptying as a proposed mechanism of action during intragastric balloon therapy: Results of a prospective study. Obesity (Silver Spring) 2016; 24: [PMID: DOI: / oby.21555] 34 Abu Dayyeh BK, Rajan E, Gostout CJ. Endoscopic sleeve gastroplasty: a potential endoscopic alternative to surgical sleeve gastrectomy for treatment of obesity. Gastrointest Endosc 2013; 78: [PMID: DOI: /j.gie ] 35 Sharaiha RZ, Kumta NA, Saumoy M, Desai AP, Sarkisian AM, Benevenuto A, Tyberg A, Kumar R, Igel L, Verna EC, Schwartz R, Frissora C, Shukla A, Aronne LJ, Kahaleh M. Endoscopic Sleeve Gastroplasty Significantly Reduces Body Mass Index and Metabolic Complications in Obese Patients. Clin Gastroenterol Hepatol 2017; 15: [PMID: DOI: / j.cgh ] 36 Lopez-Nava G, Sharaiha RZ, Vargas EJ, Bazerbachi F, Manoel GN, Bautista-Castaño I, Acosta A, Topazian MD, Mundi MS, Kumta N, Kahaleh M, Herr AM, Shukla A, Aronne L, Gostout CJ, Abu Dayyeh BK. Endoscopic Sleeve Gastroplasty for Obesity: a Multicenter Study of 248 Patients with 24 Months Follow-Up. Obes Surg 2017; Epub ahead of print [PMID: DOI: /s ] 37 Abu Dayyeh BK, Acosta A, Camilleri M, Mundi MS, Rajan E, Topazian MD, Gostout CJ. Endoscopic Sleeve Gastroplasty Alters Gastric Physiology and Induces Loss of Body Weight in Obese Individuals. Clin Gastroenterol Hepatol 2017; 15: e1 [PMID: DOI: /j.cgh ] 38 Espinós JC, Turró R, Mata A, Cruz M, da Costa M, Villa V, Buchwald JN, Turró J. Early experience with the Incisionless Operating Platform (IOP) for the treatment of obesity: the Primary Obesity Surgery Endolumenal (POSE) procedure. Obes Surg 2013; 23: [PMID: DOI: / s ] 39 Miller K, Turró R, Greve JW, Bakker CM, Buchwald JN, Espinós JC. MILEPOST Multicenter Randomized Controlled Trial: 12-Month Weight Loss and Satiety Outcomes After pose SM vs. Medical Therapy. Obes Surg 2017; 27: [PMID: DOI: /s ] 40 Hocking MP, Kelly KA, Callaway CW. Vertical gastroplasty for morbid obesity: clinical experience. Mayo Clin Proc 1986; 61: [PMID: DOI: /S (12) ] 41 Pekkarinen T, Koskela K, Huikuri K, Mustajoki P. Long-term Results of Gastroplasty for Morbid Obesity: Binge-Eating as a Predictor of Poor Outcome. Obes Surg 1994; 4: [PMID: DOI: / ] 42 Schouten R, Wiryasaputra DC, van Dielen FM, van Gemert WG, Greve JW. Long-term results of bariatric restrictive procedures: a prospective study. Obes Surg 2010; 20: [PMID: DOI: /s ] 43 Kaminski MF, Regula J, Kraszewska E, Polkowski M, Wojciechowska U, Didkowska J, Zwierko M, Rupinski M, Nowacki MP, Butruk E. Quality indicators for colonoscopy and the risk of interval cancer. N Engl J Med 2010; 362: [PMID: DOI: /NEJMoa ] 44 Patel SG, Keswani R, Elta G, Saini S, Menard-Katcher P, Del Valle J, Hosford L, Myers A, Ahnen D, Schoenfeld P, Wani S. Status of Competency-Based Medical Education in Endoscopy Training: A Nationwide Survey of US ACGME-Accredited Gastroenterology Training Programs. Am J Gastroenterol 2015; 110: [PMID: DOI: /ajg ] 45 Tuchtan L, Kassir R, Sastre B, Gouillat C, Piercecchi-Marti MD, Bartoli C. Medico-legal analysis of legal complaints in bariatric surgery: a 15-year retrospective study. Surg Obes Relat Dis 2016; 12: [PMID: DOI: /j.soard ] 46 Li Q, Du X, Zhang Y, Yin G, Zhang G, Walss-Bass C, Quevedo J, Soares JC, Xia H, Li X, Zheng Y, Ning Y, Zhang XY. The prevalence, risk factors and clinical correlates of obesity in Chinese patients with schizophrenia. Psychiatry Res 2017; 251: [PMID: DOI: /j.psychres ] 47 Breland JY, Phibbs CS, Hoggatt KJ, Washington DL, Lee J, Haskell S, Uchendu US, Saechao FS, Zephyrin LC, Frayne SM. The Obesity Epidemic in the Veterans Health Administration: Prevalence Among Key Populations of Women and Men Veterans. J Gen Intern Med 2017; 32: [PMID: DOI: /s ] 48 Kovacs Z, Valentin JB, Nielsen RE. Risk of psychiatric disorders, self-harm behaviour and service use associated with bariatric surgery. Acta Psychiatr Scand 2017; 135: [PMID: DOI: /acps.12669] 49 Brummett CM, Waljee JF, Goesling J, Moser S, Lin P, Englesbe MJ, Bohnert ASB, Kheterpal S, Nallamothu BK. New Persistent Opioid Use After Minor and Major Surgical Procedures in US Adults. JAMA Surg 2017; 152: e [PMID: DOI: /jamasurg ] 50 Spadola CE, Wagner EF, Dillon FR, Trepka MJ, De La Cruz- Munoz N, Messiah SE. Alcohol and Drug Use Among Postoperative Bariatric Patients: A Systematic Review of the Emerging Research and Its Implications. Alcohol Clin Exp Res 2015; 39: [PMID: DOI: /acer.12805] 51 Kim PS, Telem DA, Altieri MS, Talamini M, Yang J, Zhang Q, Pryor AD. Bariatric outcomes are significantly improved in hospitals with fellowship council-accredited bariatric fellowships. J Gastrointest Surg 2015; 19: [PMID: DOI: /s ] 52 Bal BS, Finelli FC, Shope TR, Koch TR. Nutritional deficiencies after bariatric surgery. Nat Rev Endocrinol 2012; 8: [PMID: DOI: /nrendo ] 53 Joo MK. Endoscopic Approach for Major Complications of Bariatric Surgery. Clin Endosc 2017; 50: [PMID: DOI: /ce ] 54 Nath A, Yewale S, Tran T, Brebbia JS, Shope TR, Koch TR. Dysphagia after vertical sleeve gastrectomy: Evaluation of risk factors and assessment of endoscopic intervention. World J Gastroenterol 2016; 22: [PMID: DOI: /wjg.v22.i ] P- Reviewer: Fogli L, Milone M, Tarnawski AS S- Editor: Gong ZM L- Editor: A E- Editor: Ma YJ 6378 September 21, 2017 Volume 23 Issue 35

23 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) EDITORIAL Resistance to clarithromycin and gastroenterologist s persistence roles in nomination for Helicobacter pylori as high priority pathogen by World Health Organization Amin Talebi Bezmin Abadi Amin Talebi Bezmin Abadi, Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran 14115, Iran ORCID number: Amin Talebi Bezmin Abadi ( ). Author contributions: Amin TBA collected the primary data and designed the first draft and finally wrote the manuscript. Conflict-of-interest statement: No potential conflicts of interest relevant to this article were reported. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Invited manuscript Correspondence to: Amin Talebi Bezmin Abadi, PhD, Department of Bacteriology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box 111, Tehran 14115, Iran. amin.talebi@modares.ac.ir Telephone: Fax: Received: July 3, 2017 Peer-review started: July 4, 2017 First decision: July 28, 2017 Revised: August 6, 2017 Accepted: August 25, 2017 Article in press: August 25, 2017 Published online: September 21, 2017 Abstract Due to the increasing prevalence of clarithromycin resistance, future of management of Helicobacter pylori (H. pylori ) infections need to be recognized. To now, clarithromycin was the best effective, well-tolerated and safe antibiotic used in treatment of the bacterium, but, increasing trend of resistance reduced efficacy of recommended regimens. Indeed, gastroenterologists are mostly unable to start appropriate therapyaccording to the sensitivity profile-due to the certain difficulties in routine H. pylori culture procedure and being time consuming method. This announcement by World Health Organization (WHO) was an onset to reconsider current challenging dilemma about H. pylori clarithromycin resistant isolates. Therefore, investigating of various factors affecting this nomination by WHO is highly welcomed. In fact, WHO enumerated more than 16 pathogens which seriously threats human life and public health, thus better management or effective guidelines are necessary. Here for the first time, we nominated this phenomenon as gastroenterologist s persistence which should be equally investigated as antibiotic resistance. The ability of gastroenterologists to win the game against H. pylori infections is highly influenced by their collaboration with diagnostic laboratories to apply susceptibility patterns before any prescription. In conclusion, closer collaboration between two important partners (gastroenterologists and microbiologists) in management of H. pylori infection may hopefully trigger an era to remedy current crisis in clarithromycin resistance, a later gastric cancer can be practically preventable. Key words: Resistance; Clarithromycin; Helicobacter pylori ; World Health Organization 6379 September 21, 2017 Volume 23 Issue 35

24 Abadi ATB. Resistance to clarithromycin and gastroenterologist s persistence roles The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: Due to the increasing prevalence of clarithromycin resistance, future of management of Helicobacter pylori (H. pylori ) infections need to be rewritten. To now, clarithromycin was the best tolerated and safe antibiotic used in treatment of the bacterium, but, increasing trend of resistance reduced efficacy of therapeutic regimens recommended. In this paper, we discussed that why persistence of gastroenterologists is a critical item need to be considered if we really aim to increase efficacy of prescribed antibiotics against H. pylori. Abadi ATB. Resistance to clarithromycin and gastroenterologist's persistence roles in nomination for Helicobacter pylori as high priority pathogen by World Health Organization. World J Gastroenterol 2017; 23(35): Available from: URL: DOI: INTRODUCTION Helicobacter pylori (H. pylori) is an infectious agent colonizes half of the world population and must be eradicated with suitable antibiotics [1,2]. Of twenty years ago, H. pylori was recognized as a major cause of peptic ulcers, thus many of these clinicians attempted to treat those colonized individuals with antibiotic [3]. Later, the World Health Organization (WHO) recommended that bacterial elimination can be a useful strategy to decrease mortality of gastric cancer in the world. Subsequently, in the first half of 2017, WHO listed the clarithromycin resistant-h. pylori in the category of high priority which requires intense attention on the treatment. This announcement by WHO is an onset to reconsider current challenging dilemma about H. pylori clarithromycin resistant isolates. Therefore, investigating of various factors affecting this nomination by WHO is highly welcomed. In fact, WHO enumerated more than 16 pathogens which seriously threats human life and public health, thus better management or effective guidelines are necessary. New visions of H. pylori treatment as well as the better insight of uncertain prescription of antibiotics in the fight against this bacterium is guaranteeing the solution to address the dilemma. Until 2004, H. pylori standard triple therapy include proton pump inhibitor (PPI), amoxicillin and clarithromycin providing the acceptable eradication rate, however, increased prevalence of antibiotic resistance hampered continuing the triple therapy for next years. As such, newer formulations were in the focus of researches among the gastroenterologists and microbiologists. As first line treatment for H. pylori-positive subjects, bismuth quadruple therapy or concomitant therapy consisting of a PPI, clarithromycin, amoxicillin, and metronidazole are now highly recommended by many international guidelines [4-11]. Therefore, it seems that clarithromycin has an inevitable role in H. pylori treatment and even bacterial resistance seems not be able to remove its existence from therapeutic lines. In next section, we review the importance of clarithromycin and how the bacterium became resistant to this commonly used drug in treating the H. pylori. H. PYLORI THERAPY-CLARITHROMYCIN AS KEY ANTIBIOTIC Due to the traditional microbiologic rule, an infectious agent should be treated with appropriate antibiotics following the susceptibility tests. Despite many problematic infectious bacteria, the story is different for H. pylori. Notably, the success chance of regimen include clarithromycin, if a resistant bacterium exists, is less than 40%. This disappointing rate can apparently reflect the irrefutable role of clarithromycin as a core antibiotic in H. pylori therapy. The problem with this critical role is that antimicrobial resistance to this drug is sharply increasing and our hopes to have successful eradication regimens (i.e., consistent treatment success > 90%) including the clarithromycin is unfortunately falling. CLARITHROMYCIN MECHANISM OF ACTION AND RESISTANCE Clarithromycin is a bacteriostatic antibiotic mostly used in childhood to treat upper respiratory infections, but, its application to treat H. pylori is the most used indication [12,13]. The main action mode of clarithromycin as one of the wide-spectrum antibiotics used in H. pylori therapy is to prevent protein translation. Current knowledge about mechanisms of resistance to clarithromycin has shown the importance of several point mutations in the peptidyltransferase region encoded in domain V of 23s rrna [14-16]. In 1996, versalovic et al [14] has shown that responsible mutations in the peptidyltransferase region cause an inhibition in binding space between the ribosomal subunit dedicated to specific antibiotic-related protein synthesis and clarithromycin. Following the first exposure to the clarithromycin, spontaneously mutations (in both 23S rrna operons) confer H. pylori resistance phenotype and genotype. The direct impact of these mutations is emergence of H. pylori strains resistant to clarithromycin. To now, two major mutations A2142G and A2143G were listed as main cause of antibiotic resistance in clinical isolates [17]. Indeed, the problem is started since the first exposure is not occurred against H. pylori. Clarithromycin is usually used to treat upper respiratory infections, therefore, colonized H. pylori is exposed in childhood 6380 September 21, 2017 Volume 23 Issue 35

25 Abadi ATB. Resistance to clarithromycin and gastroenterologist s persistence roles A Prevalence of clarithromycin resistance (%) B Prevalence of clarithromycin resistance (%) C Prevalence of clarithromycin resistance (%) Before China Bulgaria Turkey Italy South Korea India Iran Italy Figure 1 Resistance to clarithromycin among the Helicobacter pylori isolates in various countries. A: Before 2005; B: ; C: already, while no gastroduodenal disorders happened! Another problematic side of this scenario is that antibiotic stress can easily increase the frequency of inducing mutations in cells. This fact in light of relevant usage of clarithromycin for upper respiratory infections in childhood can excuse why we need to be worried about wide-spread prescription of clarithromycin in clinical practice. In brief, our bacteria are almost resistant before we start to use clarithromycin against them in anti-h. pylori therapy. In other hands, efflux pump system is also a potential mechanism that confers clarithromycin resistance [18]. Current findings are insisting on at least four families of gene clusters of efflux pumps in H. pylori isolates [18,19]. Of them, the family that is found only in Gram-negative bacteria, is the resistance-nodulation-cell division (RND). Bina et al [20] was first reported this family of efflux pumps, but additional data are missing to prove exact role of these structures to induce antibiotic resistance in H. pylori. The present data show that existence of efflux pumps in H. pylori strains can have synergic effect to induce antibiotic resistance in parallel with 23S rrna mutations. The different angle of the problem China United States 9 67 France Bulgaria Crotia Italy Iran China Japan South Korea Brazil Iran Spain United States Bulgaria United Kindom China Italy South Korea Taiwan China Israel Iran Portugal Turkey Portugal is that while we still publishing the papers indicating on increased the emergence of clarithromycin resistance, our holistic view on the rationale for skyrocketing increase in resistance to clarithromycin is missing. Because of antibiotic resistance is a dynamic phenomenon, it may be found with outstandingly different rates even in a state of a country. In reality, the spread of clarithromycin resistant-h. pylori has another dimension which remains often undiscussed. Given a sharp increase in clarithromycin resistance during the last decade worldwide (P value < 0.011), H. pylori become difficult to eradicate even using various treatment guidelines (Figure 1). Within these three 5 years duration, we can understand the clarithromycin is getting more and more useless antibiotic against H. pylori in many countries. CLARITHROMYCIN RESISTANCE: MOLECULAR DETECTION METHODS Indeed, in absence of reliable culture-based method to produce data on susceptibility tests, molecular methods were developed rapidly in last decade [17]. Based on available molecular methods tracking 23S rrna gene, a long list of methods have been developed to quickly identify resistance in colonized H. pylori strains to the clarithromycin (Table 1). Apart from progress have been made, still we are lacking in a quick method with accurate findings. Taking together, real-time PCR can be proposed as best option to be used in hospitals and even smaller centers. During the last years, many companies started to produce this machine and now it is way cheaper than 10 years ago (almost 10 folds). Therefore, we are suggesting to apply this machine to see what kind of mutations are existed 23S rrna gene and forthcoming susceptibility profile will be easier to present. NEW DIMENSION OF PROBLEM: GASTROENTEROLOGIST'S PERSISTENCE Face to face with the problem, we definitely need gastroenterologists more actively involved. So far, due to the clinical and official settings, many of microbiologists do not have any access to the patients. The data for antibiotic susceptibility tests mainly come from laboratories which are dominated by microbiologists. Thus, as a historic conflict between clinicians and basic medical scientists, lack of connection can result in blind antibiotic prescription by the gastroenterologists. Another evidence for this conflict is that almost none of famous microbiologists are co-authored in recently published guidelines on treatment or diagnosis of the H. pylori. Moreover, the story is exacerbated when the microbiology journals have no scope for publishing such guidelines. This existed, but, unwanted gap deserves an intense attention to improve management of patients carrying H September 21, 2017 Volume 23 Issue 35

26 Abadi ATB. Resistance to clarithromycin and gastroenterologist s persistence roles Table 1 Molecular methods to identify mutations induce clarithromycin resistance in clinical Helicobacter pylori isolates Methods Advantages Disadvantages PCR-based method Ref. Real-time PCR Quick and reliable Relatively expensive Yes [21] High specificity, high sensitivity PCR-LiPA Fast and cheap Moderate specificity and sensitivity Yes [22] DNA sequencing Produce many information, Expensive, Yes [23] Time-consuming protocol 3 -mismatch PCR Fast and high specificity Produce limited data on the gene, not practically useful Yes [24] RFLP High specificity, high sensitivity Risk of contamination Yes [15,25] Low reproducibility FISH High specificity, high sensitivity Need invasive approach so not good for children No [26,27] FISH: Fluorescence in situ hybridisation; RFLP: Restriction fragment length polymorphism; PCR-LiPA: PCR line probe assay. pylori with resistance to clarithromycin. As we described above, in infectious diseases, treatment is highly recommended based on susceptibility pattern, a fact does not follow in the case of H. pylori. In other words, treatment of H. pylori in many of clinics are based on personal experiences by the gastroenterologists than antibiotic susceptibility tests determined in a microbiology laboratory. With this regard, an incoherent connection between those two populations is a major cause of inappropriate drug prescription ending in increased antibiotic resistance worldwide. Therefore, WHO announced that we are facing with the new generation of resistant H. pylori isolates to clarithromycin which endangers effectiveness of current chemotherapy against the bacterium. By continuing the current situation, the replacement of H. pylori by WHO in a critical level of priority in a ranking of threatening pathogen is not far from the mind. The next uninvestigated part of the problem is a natural persistence of clinicians on the application of susceptibility patterns reported by microbiologists! For example, metronidazole resistance is not under 60% in many of regions worldwide [28-31], but any trials can show intrinsic tendency among the clinicians who aim to use this ineffective drug for eradicating the H. pylori. Taking together, our knowledge about antibiotic resistance has evolved nicely during the relatively short time we treating the H. pylori, however, we need new insight on how to increase the reliability among the clinicians to use susceptibility pattern to efficiently eliminate the bacterium especially in patients at least with severe gastroduodenal disorders. Here for the first time, we called this phenomenon as gastroenterologist s persistence which should be equally investigated as antibiotic resistance. The ability of gastroenterologists to win the game against H. pylori infections is highly being influenced by their collaboration with diagnostic laboratories to apply susceptibility patterns before any prescription. Therefore, two major targets should be considered, (1) antibiotic resistance; and (2) clinician s persistence. Invasive nature of gastroscopy is a considerable factor that influence on rare access to culture-based information on susceptibility tests. Defeating most of the recommended guidelines to eliminate the H. pylori can be a major output of neglecting the clinician s persistence. CONCLUSION Nowadays, therapeutic failures in anti-h. pylori regimens has emerged as major concern for both gastroenterologists and microbiologists. The recent announcement by WHO should be considered as an alarming sound that indicates on emergence of clarithromycin-resistant strains causing global H. pylori treatment failure. Since clarithromycin has a critical role in the success of therapeutic regimens against the H. pylori, the spread of its resistant strains can easily hamper our attempts to eradicate this microbe. Conclusively, it is not the exaggeration to consider this antibiotic as the main drug in the treatment of H. pylori. We need to repeat that all records insisting on using the clarithromycin before recommending as anti-h. pylori agent should be considered as serious alarming item. Although the ideal and effective therapy to eliminate the H. pylori is not available, but optimizing the current formulations especially, cautious application of clarithromycin in regions with resistance rate > 20% is a likely successful approach. New research direction to produce and suggest novel antibiotics are welcomed to compensate the lacking in antimicrobial agents listed as therapeutic options. In future, application of MALDI-TOF as a novel method can be used directly to detect mutations from biopsy sample; a suggestion which is not far away. To my knowledge, this is one the first paper that pinpoints the gap between gastroenterologists and microbiologists following nomination for H. pylori as high priority pathogen by WHO. To be honest, although the persistence of gastroenterologists is a critical factor to increase antibiotic resistance among H. pylori strains, but, difficulties in bacterial culture, time consuming procedure of culture and high cost for rapid detection 6382 September 21, 2017 Volume 23 Issue 35

27 Abadi ATB. Resistance to clarithromycin and gastroenterologist s persistence roles of resistance are the list which belongs to another side of involving individuals. Finally, closer collaboration between two important partners (gastroenterologists and microbiologists) in management of H. pylori infection may hopefully trigger an era to remedy current crisis in clarithromycin resistance, a later gastric cancer can be practically preventable. REFERENCES 1 Abadi AT, Ierardi E, Lee YY. Why do we still have Helicobacter Pylori in our Stomachs. Malays J Med Sci 2015; 22: [PMID: ] 2 Yamaoka Y, Graham DY. Helicobacter pylori virulence and cancer pathogenesis. Future Oncol 2014; 10: [PMID: DOI: /fon.14.29] 3 Graham DY. History of Helicobacter pylori, duodenal ulcer, gastric ulcer and gastric cancer. 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Mutations in 23S rrna are associated with clarithromycin resistance in Helicobacter pylori. Antimicrob Agents Chemother 1996; 40: [PMID: ] 15 Stone GG, Shortridge D, Versalovic J, Beyer J, Flamm RK, Graham DY, Ghoneim AT, Tanaka SK. A PCR-oligonucleotide ligation assay to determine the prevalence of 23S rrna gene mutations in clarithromycin-resistant Helicobacter pylori. Antimicrob Agents Chemother 1997; 41: [PMID: ] 16 Abadi AT, Taghvaei T, Ghasemzadeh A, Mobarez AM. High frequency of A2143G mutation in clarithromycin-resistant Helicobacter pylori isolates recovered from dyspeptic patients in Iran. Saudi J Gastroenterol 2011; 17: [PMID: DOI: / ] 17 Mégraud F, Lehours P. Helicobacter pylori detection and antimicrobial susceptibility testing. Clin Microbiol Rev 2007; 20: [PMID: DOI: /CMR ] 18 Kutschke A, de Jonge BL. Compound efflux in Helicobacter pylori. Antimicrob Agents Chemother 2005; 49: [PMID: DOI: /AAC ] 19 Hirata K, Suzuki H, Nishizawa T, Tsugawa H, Muraoka H, Saito Y, Matsuzaki J, Hibi T. Contribution of efflux pumps to clarithromycin resistance in Helicobacter pylori. J Gastroenterol Hepatol 2010; 25 Suppl 1: S75-S79 [PMID: DOI: /j x] 20 Bina JE, Alm RA, Uria-Nickelsen M, Thomas SR, Trust TJ, Hancock RE. Helicobacter pylori uptake and efflux: basis for intrinsic susceptibility to antibiotics in vitro. Antimicrob Agents Chemother 2000; 44: [PMID: DOI: / AAC ] 21 Maurin M. Real-time PCR as a diagnostic tool for bacterial diseases. Expert Rev Mol Diagn 2012; 12: [PMID: DOI: /erm.12.53] 22 Owen RJ. Molecular testing for antibiotic resistance in Helicobacter pylori. Gut 2002; 50: [PMID: DOI: /gut ] 23 Binh TT, Shiota S, Suzuki R, Matsuda M, Trang TT, Kwon DH, Iwatani S, Yamaoka Y. Discovery of novel mutations for clarithromycin resistance in Helicobacter pylori by using nextgeneration sequencing. J Antimicrob Chemother 2014; 69: [PMID: DOI: /jac/dku050] 24 Oleastro M, Ménard A, Santos A, Lamouliatte H, Monteiro L, Barthélémy P, Mégraud F. Real-time PCR assay for rapid and accurate detection of point mutations conferring resistance to clarithromycin in Helicobacter pylori. J Clin Microbiol2003; 41: [PMID: DOI: /JCM ] 25 Ménard A, Santos A, Mégraud F, Oleastro M. PCR-restriction fragment length polymorphism can also detect point mutation A2142C in the 23S rrna gene, associated with Helicobacter pylori resistance to clarithromycin. Antimicrob Agents Chemother 2002; 46: [PMID: DOI: / AAC ] 26 Cerqueira L, Fernandes RM, Ferreira RM, Carneiro F, Dinis- Ribeiro M, Figueiredo C, Keevil CW, Azevedo NF, Vieira MJ. PNA-FISH as a new diagnostic method for the determination of clarithromycin resistance of Helicobacter pylori. BMC Microbiol 2011; 11: 101 [PMID: DOI: / ] 27 Vega AE, Alarcón T, Domingo D, López-Brea M. Detection of clarithromycin-resistant Helicobacter pylori in frozen gastric biopsies from pediatric patients by a commercially available fluorescent in situ hybridization. Diagn Microbiol Infect Dis 2007; 59: [PMID: DOI: /j.diagmicrobio ] 28 Shiota S, Reddy R, Alsarraj A, El-Serag HB, Graham DY September 21, 2017 Volume 23 Issue 35

28 Abadi ATB. Resistance to clarithromycin and gastroenterologist s persistence roles Antibiotic Resistance of Helicobacter pylori Among Male United States Veterans. Clin Gastroenterol Hepatol 2015; 13: [PMID: DOI: /j.cgh ] 29 Su P, Li Y, Li H, Zhang J, Lin L, Wang Q, Guo F, Ji Z, Mao J, Tang W, Shi Z, Shao W, Mao J, Zhu X, Zhang X, Tong Y, Tu H, Jiang M, Wang Z, Jin F, Yang N, Zhang J. Antibiotic resistance of Helicobacter pylori isolated in the Southeast Coastal Region of China. Helicobacter 2013; 18: [PMID: DOI: /hel.12046] 30 Lee JW, Kim N, Kim JM, Nam RH, Chang H, Kim JY, Shin CM, Park YS, Lee DH, Jung HC. Prevalence of primary and secondary antimicrobial resistance of Helicobacter pylori in Korea from 2003 through Helicobacter 2013; 18: [PMID: DOI: /hel.12031] 31 Abadi AT, Taghvaei T, Mobarez AM, Carpenter BM, Merrell DS. Frequency of antibiotic resistance in Helicobacter pylori strains isolated from the northern population of Iran. J Microbiol 2011; 49: [PMID: DOI: /s ] P- Reviewer: Adachi Y, Emara MH, Kocazeybek B, Kreisel W, Lei YC, Yamaoka Y S- Editor: Qi Y L- Editor: A E- Editor: Ma YJ 6384 September 21, 2017 Volume 23 Issue 35

29 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) REVIEW Time to clinical response and remission for therapeutics in inflammatory bowel diseases: what should the clinician expect, what should patients be told? Abhinav Vasudevan, Peter R Gibson, Daniel R van Langenberg Abhinav Vasudevan, Daniel R van Langenberg, Department of Gastroenterology and Hepatology, Eastern Health, Box Hill Hospital, Box Hill, Victoria 3128, Australia Abhinav Vasudevan, Daniel R van Langenberg, Monash University, Eastern Health Clinical School, Box Hill, Victoria 3128, Australia Peter R Gibson, Department of Gastroenterology, Alfred Health and Monash University, Victoria 3004, Australia ORCID number: Abhinav Vasudevan ( ); Peter R Gibson ( ); Daniel R van Langenberg ( ). Author contributions: Vasudevan A and van Langenberg DR were involved in the conception of the review, acquisition of data and analysis, drafting the article and final approval of the version to be submitted; Gibson PR was involved in drafting and the critical appraisal of the article. Conflict-of-interest statement: Vasudevan A has no conflict of interest to declare; Gibson PR has served as consultant or advisory board member for AbbVie, Ferring, Janssen, Merck, Nestle Health Science, Danone, Allergan, Pfizer, Celgene and Takeda. His institution has received speaking honoraria from AbbVie, Janssen, Ferring, Takeda, Fresenius Kabi, Mylan and Pfizer. He has received research grants for investigator-driven studies from AbbVie, Janssen, Falk Pharma, Danone and A2 Milk Company. His Department financially benefits from the sales of a digital application and booklets on the low FODMAP diet. He has published an educational/recipe book on diet. van Langenberg DR has served as a speaker and/or received travel support from Takeda, Ferring and Shire. He has consultancy agreements with Abbvie, Janssen and Pfizer. He received research funding grants for investigator-driven studies from Ferring, Shire and AbbVie. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Invited manuscript Correspondence to: Abhinav Vasudevan, B. Medicine, Gastroenterologist, Department of Gastroenterology and Hepatology, Eastern Health, Level 2, 5 Arnold Street, Box Hill Hospital, Box Hill, Victoria 3128, Australia. abhinav.vasudevan@monash.edu Telephone: Fax: Received: June 2, 2017 Peer-review started: June 6, 2017 First decision: June 22, 2017 Revised: July 3, 2017 Accepted: August 15, 2017 Article in press: August 15, 2017 Published online: September 21, 2017 Abstract An awareness of the expected time for therapies to induce symptomatic improvement and remission is necessary for determining the timing of follow-up, disease (re)assessment, and the duration to persist with therapies, yet this is seldom reported as an outcome in clinical trials. In this review, we explore the time to clinical response and remission of current therapies for inflammatory bowel disease (IBD) as well as medication, patient and disease related factors that may influence the time to clinical response. It appears that the time to therapeutic response varies depending on the indication for therapy (Crohn s disease or ulcerative colitis). Agents with the most rapid time to clinical response included corticosteroids, calcineurin inhibitors, exclusive enteral nutrition, aminosalicylates and anti-tumor necrosis factor therapy which will work in most patients within the first 2 mo. Vedolizumab, 6385 September 21, 2017 Volume 23 Issue 35

30 Vasudevan A et al. Time to response of therapies in IBD methotrexate and thiopurines had a longer time to clinical response and can take several months to achieve maximal efficacy. Factors affecting the time to clinical response of therapies included use of concomitant therapy, disease duration, smoking status, disease phenotype and advanced age. There appears to be marked variation in time to clinical response for therapies used in IBD which is further influenced by disease and patient related factors. Understanding the expected time to therapeutic response is integral to inform further decision making, maintain a patientcentered approach and ensure treatment is given an appropriate timeframe to achieve maximal benefit prior to cessation. Key words: Crohn s disease; clinical pharmacology; ulcerative colitis; thiopurines; Inflammatory bowel disease; biologics; nutrition The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: There appears to be marked variation in time to clinical response for therapies used in inflammatory bowel disease which is further influenced by disease and patient related factors. The most rapid response can be expected with corticosteroids, calcineurin inhibitors, exclusive enteral nutrition, aminosalicylates and anti-tumor necrosis factor therapy (within 2 mo), while methotrexate, thiopurines and vedolizumab can take several months to achieve maximal response. There is a lack of reporting of the time to response of therapies in clinical trials for inflammatory bowel disease and this remains an area that should be addressed in future studies. Vasudevan A, Gibson PR, van Langenberg DR. Time to clinical response and remission for therapeutics in inflammatory bowel diseases: What should the clinician expect, what should patients be told? World J Gastroenterol 2017; 23(35): Available from: URL: v23/i35/6385.htm DOI: i INTRODUCTION The therapeutic armamentarium for inflammatory bowel disease (IBD), comprising Crohn s disease (CD) and ulcerative colitis (UC), continues to expand, providing valuable additional opportunities to achieve optimal long term outcomes for patients. Equally, however, there is added complexity, commensurate with the number of options, an enhanced understanding of the risks and benefits, plus the differential effects of treatments on objective disease outcomes (e.g., mucosal healing), clinical remission and/or patient-reported outcomes. Yet when better outcomes are potentially achievable, there are higher expectations. It is increasingly important with current therapeutics that the physician plans ahead, given delays in escalating treatment are likely just as common and detrimental as delays in diagnosis are in IBD [1]. Hence an awareness of the approximate time expected to achieve a treatment goal is fundamental to making decisions such as whether to persist with a therapy or switch to an alternative. Equally, one does their patient a disservice by prematurely switching therapies before an agent is given an appropriate length of time to achieve efficacy. Achieving an optimal time to therapeutic response has further benefits in the doctor-patient relationship, as it allows the clinician to provide the patient a cogent framework of the expected period to see response to a new drug and hopefully empower the patient to persevere with, and maintain adherence to therapy. This is particularly relevant for therapies that have a longer time to therapeutic response, such as the immunomodulators, where it might take several months to reach maximal therapeutic efficacy without the patient necessarily experiencing any symptom benefit for a significant part of this. Hence, time-to-therapeutic response is an important yet underestimated factor in the day-today management of IBD and has not been a major focus of attention in the literature to date. This review attempts to address this unmet need by analyzing the available literature relating to the expected timeto-clinical-response for currently available therapies in IBD and measures that can assist the clinician in determining whether a medication has reached its therapeutic potential. We will also analyze disease and patient related factors that may impact on the time-toclinical-response of therapies. Therapies discussed in the review will include corticosteroids, aminosalicylates (5-ASA), thiopurines, methotrexate, anti-tumor necrosis factor (anti-tnf) therapies, vedolizumab, calcineurin inhibitors and exclusive enteral nutrition. DEFINING TIME TO CLINICAL RESPONSE AND REMISSION The concept of time-to-clinical-response is schematically represented in Figure 1. Given the lack of focus on time to response in previous literature, there is no broadly accepted definition. Table 1 provides a summary of some of the important components of time to response. These include the earliest time at which a patient can expect a response, the time at which most patients (i.e., greater than 50%) expecting to benefit from therapy will achieve a response and the time point where therapeutic benefit remains improbable, the so-called time to futility. For this review, time-to-clinical-response refers to the time from the initiation of therapy until the patient achieves a clinical response. It only pertains to patients who attain a clinical response and can thereby aid the 6386 September 21, 2017 Volume 23 Issue 35

31 Vasudevan A et al. Time to response of therapies in IBD Table 1 Expected time to clinical response for therapeutic agents used in the management of inflammatory bowel disease Agent Earliest published clinical response Earliest published Time to response objective response in most patients 1 Time to futility Use of therapeutic drug monitoring Comments Mesalazine (oral) 1 wk [21] 3 wk [148] 4 wk > 12 wk N A higher dose may lead to a more rapid response Prednisolone (oral) 2 wk [28] 2 wk [28] 3 to 7 wk 8 wk N May take longer for CD Corticosteroids (IV) 3 d [31] 1 wk [127] 3-5 d 7-10 d N Infliximab (IV) 1 wk [48] 8 wk [149] 2-8 wk > 6 mo Y Adalimumab (SC) 4 wk [59] 8 wk [59] 4-8 wk > 6 mo Y Response time better with 160/80 mg vs 40/40 mg induction dosing Certolizumab (SC) 2wk [61] 10 wk [64] 10 wk > 16 wk N Golimumab (SC) 6 wk [65] 6 wk [65] 6 wk > 14 wk Y Certolizumab (SC) 2 wk [61] 10 wk [64] 10 wk > 16 wk N Vedolizumab (IV) 6 wk [78] 6 wk [78] 19 wk 12 mo N 2 Response time may be better for UC vs CD Thiopurines (oral) 2 wk [80] 3 mo [88] 10 to 12 wk > 6-9 mo Y Endoscopic response may take much longer than clinical response Methotrexate (oral or SC) 9 wk [111] 12 wk [110] 9 wk > 6 mo N Response time and efficacy may be better in 1) CD vs UC, 2) SC vs oral Cyclosporin (IV then oral) 1 wk [127] 1 wk [127] 4 to 5 d > 14 d Y Tacrolimus (oral) 2 wk [122] 2 wk [122] 2 wk 4 wk Y EEN (oral) 10 d [131] 4 wk [139] 3 to 4 wk 8 wk N 1 Clinical response reported in at least 50% of patients who achieve a response to therapy; 2 Therapeutic drug monitoring is not yet widely available. EEN: Exclusive enteral nutrition; IV: Intravenous; SC: Subcutaneous; Y: Yes; N: No; CD: Crohn s disease; UC: Ulcerative colitis. clinician in judging the likelihood of a response being achieved based upon the elapsed time on therapy. Where available, estimates of timeframes in which the majority of patients who ultimately respond to therapy will be expected to respond to therapy will be reported. The time to futility of therapy is reported in Table 1, however, this will not be a primary focus of this review. The methods of determining when a response has occurred are heterogeneous and include both clinical symptoms and endoscopic (or objectively-assessed) findings. The correlation between symptomatic improvement and achievement of endoscopic remission differs between UC and CD, with improvement in symptoms correlating better with mucosal healing in UC than CD [2-5]. There are data to support early clinical remission, albeit not response, to be predictive of endoscopic improvement and healing at 12 mo [6]. The value of symptomatic improvement, however, cannot be discounted from a patient s perspective given the correlation with long-term steroid-free remission and the inherent part that alleviation of symptoms plays in improving quality of life [5,7]. Moreover, the complex interplay of patient symptoms and structural damage in IBD is being increasingly recognized with both symptoms and endoscopic findings important factors in determining overall disease severity and burden [8]. Thus, response is a multi-faceted concept and this review will primarily address the time-to-clinicalresponse and time-to-clinical-remission, given that the focus here is to engender a patient-centered approach when clinicians discuss therapeutic options with their patients. Time to endoscopic response and remission will also be reported where data are available, although this is a secondary focus. Given the heterogeneity across studies in defining clinical response, clinical remission and endoscopic remission, we have used broad outcome measures of clinical or endoscopic improvement or clinical or endoscopic remission or mucosal healing as defined by the authors of each study. For the purpose of this review, clinical response will consider symptom improvement only rather than an improvement in symptoms and laboratory indices. A summary of the relative time-to-therapeuticresponse of different therapies for IBD is presented in Table 1 and Figure 1. Medication related factors that influence the time to response of different therapies are discussed within each of the therapeutic classes. AMINOSALICYLATES Time to clinical response Aminosalicylates are more effective at inducing response and remission in patients with mild-moderate ulcerative colitis than placebo [9], but their evidence for efficacy in patients with Crohn s disease is poor [10,11]. Therefore, with regards to ulcerative colitis, available data indicate that it generally takes two to four weeks to achieve clinical response with oral and/or topical aminosalicylates. Mesalazine induces endoscopic remission in 67% of patients at 4 wk for active colitis for both 2 and 4 g preparations, while another study found higher endoscopic remission rates of 78% and 69% after 8 wk with multimatrix mesalazine 4.8 g and 2.4 g, respectively, suggesting some patients who will achieve endoscopic remission may take up to 8 wk [12,13]. Therapy-related factors affecting time-to-response Key issues for aminosalicylates include whether the formulation, the dose and/or the route(s) of delivery 6387 September 21, 2017 Volume 23 Issue 35

32 Vasudevan A et al. Time to response of therapies in IBD Infliximab Weeks since start of treatment Adalimumab Golimumab Certolizumab Vedolizumab Intravenous corticosteroids Oral corticosteroids Mesalazine Methotrexate Thiopurines Cyclosporin Tacrolimus Exclusive enteral nutrition Legend: Acute severe colitis Luminal Crohn's disease and ulcerative colitis 1 Perianal Crohn's disease Ulcerative colitis Luminal Crohn's disease Figure 1 Schematic representation of the range of expected time of clinical response for therapies based on indication. 1 This symbol is used to represent the expected time to response for therapies that can be used in either condition. influence the speed of onset of action. Formulation: Trials of sulfasalazine, olsalazine and balsalazide in mild to moderate UC demonstrated an improvement in clinical symptoms and endoscopic response with 2 to 3 wk of therapy in most patients [14-17]. In contrast according to published data, coated mesalazine preparations demonstrated a somewhat slower clinical and endoscopic improvement within 4 to 8 wk [18,19]. Two head-to-head studies suggested that an equimolar dose of balsalazide resulted in a more rapid clinical and endoscopic response than delayed-release mesalazine (using Eudragit S-coated tablets) therapy for patients with left-sided disease [17,20]. Dose: Time to therapeutic response for aminosalicylates may also be dose-dependent as demonstrated by Orchard et al [21] who found that 4.8 g daily of mesalazine (delayed release, Asacol MR ) improved and resolved symptoms more rapidly than 2.4 g daily (median 7 d vs 9 d, 19 d vs 29 d respectively). Another study found a numerically faster time-toclinical-response with mesalazine 4.5 g than 3 g or 1.5 g daily [22]. Kamm et al [13] described numerically higher endoscopic remission rates of 78% after 8 wk with mesalazine MMX 4.8 g vs 2.4 g daily (69%), but whether this equates to faster response in those who achieve remission was not reported [12,13]. Route of delivery: Combined oral and topical mesalazine was associated with more rapid resolution of rectal bleeding (mean 11.9 d) than with oral mesalazine only (25.5 d) for left-sided colitis in the only randomized study reporting this end-point [23]. Another randomized trial of sixty patients with distal UC comparing oral mesalazine with mesalazine enemas or combination topical and oral treatment found a median time to resolution of rectal bleeding of 8 d on combination therapy and bleeding were significantly lower after ten days with either topical or combination therapy compared to oral mesalazine only. The findings indicate that topical therapy alone or in combination with oral therapy achieves symptom resolution more rapidly than oral therapy [23,24]. The efficacy of topical 5-ASA does not appear dose-dependent in the single study where this was specifically examined, but rapidity of response was not addressed [25]. CORTICOSTEROIDS A clinical response to steroids should be expected within 1 to 4 wk of commencing therapy for both CD and UC (not applicable to acute severe colitis) with response occurring more rapidly with intravenous than oral therapy [26,27]. There are several types of corticosteroids available for the treatment of IBD and the influence of route of administration and type of corticosteroid are relevant to determining the time to response, as discussed below. Time to clinical response CD: Although most patients with CD can expect a response to high dose oral corticosteroids within 4 wk, some data suggest a more prolonged course 6388 September 21, 2017 Volume 23 Issue 35

33 Vasudevan A et al. Time to response of therapies in IBD may be necessary to capture response. For example, clinical response after three to seven weeks of 1 mg/kg per day oral prednisolone in a prospective cohort study of 146 patients with active ileocolonic or colonic CD increased from 63% to 92% between weeks 4 and 7 respectively, although only 29% achieved endoscopic remission [2]. These data suggest that a clinician should wait up to 7 wk before deciding that a response to high doses of prednisolone is unlikely if that approach is clinically acceptable. UC: Response to oral prednisolone is rapid in UC, with 17%-76% achieving clinical remission and 65%-78% endoscopic improvement after 2 wk of oral prednisone in two randomized studies, with the higher response rates noted by Truelove et al [28] who used both oral and rectal prednisolone in combination [28,29]. Other studies have also suggested a response within the first two weeks of a tapering dose of oral prednisolone beginning at 40 mg/d in the majority of patients with moderate UC [30]. Therapy related factors affecting time to response Route of administration: (1) CD: While direct comparisons between intravenous and oral corticosteroids are not available in CD, response appears rapid with intravenous corticosteroids, with 78% of patients having symptom resolution after five days of intravenous hydrocortisone (300 mg daily), which increased to 93% after 10 d in one randomized study comparing intravenous hydrocortisone to corticotrophin, to which response rates were also high (71% and 82% at days 5 and 10, respectively) [27,31]. (2) UC: Moderate UC has been shown to typically improve within five days of intravenous corticosteroids, including patients who failed to respond to high-dose oral prednisolone. Time frames for expected response are well described for acute severe colitis, where most patients appear to respond to therapy within 3 or 5 d of intravenous steroids (methylprednisolone 60 mg/d or hydrocortisone mg/d), although these are observational data only [32-35]. A lack of response within 5 d is associated with a higher rate of subsequent colectomies again in observational studies, and therapy beyond 7 d is unlikely to be beneficial [36]. Type of glucocorticoid: (1) CD: Several randomized studies have suggested the mean times to clinical response and remission with budesonide in CD were comparable to systemic corticosteroids, ranging from 22 to 27 d [37-40]. (2) UC: Induction of remission when using budesonide MMX 9 mg daily in mild to moderate UC should occur within 4 to 8 wk of commencing therapy, with 42%-47% of patients achieving an endoscopic or clinical improvement in randomized controlled trials (RCT) [41,42]. Dose: The effect of corticosteroid dose on time to response has not been evaluated. One randomized study assessed response rates with 20 mg, 40 mg and 60 mg daily of oral prednisolone for outpatient management of ulcerative colitis and suggested a higher response rate at both 2 wk and 3-5 wk of follow-up with 40 and 60 mg/d of therapy (both 50% at 2 wk, then 65% at 3-5 wk respectively) compared to 20 mg daily (20% then 30%), but did not specifically assess time to response [30]. Determining the appropriate dose of steroid has traditionally been either empiric or weight-based. Accordingly, corticosteroid dosing evaluated in clinical trials has varied; for instance, studies have used 1 mg/kg/d or mg/d of prednisolone, 9 mg of budesonide orally, while for intravenous therapy includes mg/d of hydrocortisone (divided doses) or 60 mg/d of methylprednisolone [26,43,44]. TUMOR NECROSIS FACTOR ALPHA INHIBITORS Pertinent issues relating to time to therapeutic response of anti-tnf therapy include the associations with serum drug levels and antibodies, plus concomitant therapy. Time to response Infliximab: (1) CD: Clinical response and remission after administration of infliximab appear to be rapid in luminal CD, taking 8 and 9 d respectively in one observational study of 129 patients [45]. Clinical response rates in RCTs of infliximab in CD were 61% and 81% for weeks 2 and 4 respectively after a single infusion of infliximab [46,47]. Clinical remission rates were reportedly 88% one week after a single infliximab dose for colonic CD although the data were observational only [48]. Rates of mucosal healing in Crohn s disease have ranged from 30%-67% after 6 mo of infliximab, with higher rates typically observed in real-world clinical cohorts than trials [49,50]. (2) UC: Clinical and endoscopic response to infliximab in patients with moderate to severe chronic active ulcerative colitis appears to take several weeks, although this may be due to a lack of reporting of early outcomes after initiation of therapy in the outpatient setting, given that response rates reported for acute severe colitis are generally more rapid than this. Nevertheless, about half of patients previously not responding to either intravenous or oral corticosteroids experienced a clinical response two weeks after the first infusion of infliximab in one prospective uncontrolled study [51]. Such early response rates have not been reported in RCTs, but data from such studies have shown a significantly higher rate of clinical response (69% vs 37%), remission (39% vs 15%) and mucosal healing (62% vs 33%) by week 8 with 5 mg/kg induction dosing versus placebo [52]. For infliximab 6389 September 21, 2017 Volume 23 Issue 35

34 Vasudevan A et al. Time to response of therapies in IBD use in ulcerative colitis, two large randomized studies of moderately severe ulcerative colitis showed a significantly higher rate of mucosal healing by week 8 with after both 5 mg/kg and 10 mg/kg induction therapy (62% vs 33% with placebo) [52]. For acute severe colitis, a clinical response to infliximab therapy should be expected within the first 7 d after therapy [53]. Achieving a higher serum infliximab level during induction has been associated with a higher rate of short term mucosal healing and an accelerated induction regimen of infliximab in acute severe colitis has been associated with a more prolonged time to colectomy than standard induction, although the rapidity of response has not been directly assessed [54,55]. Indeed, recent data suggest that a rebound of higher C-reactive protein, lower albumin and/or symptoms within a few days after the first dose of infliximab should prompt concerns of infliximab nonresponse and a potentially higher risk of colectomy [55]. A trial is currently underway to assess the utility of an accelerated induction regimen of infliximab in acute severe colitis and this may provide further information on the effect of dose and drug levels on time to response [56]. Adalimumab: (1) CD: An initial response to adalimumab typically occurs within the first few weeks of therapy, as inferred from a phase 2 RCT showing clinical remission rates of 36% at week 4 following induction treatment with 160/80 mg at week 0 and 2 for CD, compared to 12% with placebo [57]. While clinical remission rates were higher from week 1 than placebo in this study (16% vs 7% respectively), this only reached statistical significance at week 4. Moreover, the rate of mucosal healing for moderately severe ileocolonic CD with induction 160/80 mg adalimumab followed by 40 mg fortnightly was significantly higher than placebo at 12 wk (27% vs 13%) and sustained until 52 wk (24% vs 0%) in another RCT [58]. Endoscopic remission rates were 52% and 28% at weeks 12 and 52 respectively in this study, the latter likely reflecting secondary loss of response during maintenance therapy. (2) UC: Clinical remission and mucosal healing rates with adalimumab induction with 160/80 mg regimen in patients with moderate to severe UC after 8 wk was achieved in 19 and 47% respectively, with separation in clinical remission rates as early as week 4 compared to placebo in a RCT [59]. The lower remission rates in this study may relate to the high proportion (75%) of patients who had failed other therapies prior study enrolment [59]. In another RCT assessing long term remission rates with adalimumab in moderate to severe UC, mucosal healing rates were 41% at week 8 and 25% at week 52 with fortnightly adalimumab 40 mg, compared to 32% and 15% for placebo, respectively [60]. Mucosal healing rates following adalimumab induction for UC have varied between 32% and 47% in RCTs [59,60]. Certolizumab pegol: CD: Certolizumab pegol at a dose of 400 mg given subcutaneously at weeks 0, 2 and 4 wk, about a third of patients with CD will have a clinical response to therapy within 2 wk, increasing to 41% by week 6 based on RCT data [61]. One study found response rates peak at 10 wk of 400mg 4-weekly therapy [62], while another study found response rates, as per a reduction in CDAI of 100, peaked by week 16 and declined thereafter [63]. Endoscopic activity was assessed at week 10 in one prospective, open label clinical trial of patients on 400 mg certolizumab 4-weekly and showed endoscopic remission occurred in 37%, reducing to 27% by week 54 in CD [64]. Golimumab: UC: Golimumab is administered subcutaneously and has been approved for use in ulcerative colitis in many countries. Approximately half of patients will achieve a clinical response by 6 wk with regimens of 100/200 mg and 400/200 mg as induction at weeks 0 and 2 for moderate to severe ulcerative colitis from one large RCT [65]. Observational data suggest that response rates may continue to increase up until week 14, when reported to be between 69% and 86% [66,67]. Mucosal healing appears to be rapid, with 42 and 45% of patients achieving mucosal healing 6 wk after induction therapy with 100/200 mg and 400/200 mg, respectively [65]. Intravenous induction therapy for golimumab does not appear to confer any additional benefit in terms of response rate compared to subcutaneous induction, although time to response of this strategy has not been evaluated [68]. Since there are no data evaluating clinical response rates beyond week 14, the benefits of continuing therapy beyond this time point in patients who have not achieved a response remains uncertain. Factors affecting time to response Demographic factors: One study assessed baseline factors that were predictive of a more rapid attainment of clinical remission with induction certolizumab therapy for CD and found that younger age, nonsmokers, the absence of previous IBD surgery and a lower disease activity score were associated with a more rapid attainment of clinical remission [69]. Anti-TNF drug levels: Currently, data supporting a correlation between anti-tnf drug levels and time to therapeutic response are limited. For golimumab, drug levels at weeks 2 and 4 were shown to correlate with week 6 clinical response rates, but the effect on time to therapeutic response was not further assessed as response was only evaluated at a single time point in this study [70]. Higher certolizumab plasma concentrations at week 8 are associated with higher rates of clinical remission and endoscopic remission 6390 September 21, 2017 Volume 23 Issue 35

35 Vasudevan A et al. Time to response of therapies in IBD at week 10, but not a higher clinical response rate [71]. There is a lack of data concerning the relationship between time-to-response for infliximab and adalimumab in relation to drug levels. Concurrent immunomodulator therapy: While time to response has not been directly compared between anti-tnf monotherapy and in combination with immunomodulator therapy, there was a trend toward a higher rate of clinical remission at week 10 with combination infliximab and azathioprine compared to infliximab alone in the Study of Biologic and Immunomodulator Naive Patients in Crohn s Disease (SONIC) Study, and a significantly higher rate of endoscopic remission at 26 wk with adalimumab and azathioprine, suggesting combination therapy may work faster than either therapy alone [50,72]. Similar findings of more rapid clinical remission have also been found for certolizumab therapy when used with concomitant immunomodulator therapy in CD [69]. VEDOLIZUMAB Vedolizumab appears to have a generally slower time to response compared to other biologic agents. This may relate to the mechanism of anti-integrin therapy, with inhibition of lymphocyte gut migration taking more time to achieve therapeutic efficacy [73]. Time to response CD: Clinical remission with vedolizumab appears to take at least 10 to 14 wk in CD. This slower onset of action of vedolizumab, compared to anti-tnf therapies for instance, was evident in the RCTs GEMINI 2 and 3 registration trials of vedolizumab comparing therapy to placebo induction and maintenance therapy in CD [74]. Clinical remission rates increased from 15% to 27% between weeks 6 and 10 while remained stable in the placebo group (12%) at these time points in GEMINI 3 (i.e., those with prior anti-tnf failure), and in GEMINI 2 there was a significant increase in clinical remission after 6 wk compared to placebo [73]. Subsequent real-world observational data have also demonstrated that clinical remission rates tend to increase from week 6 to week 14 and in one study the median time to clinical response was 19 wk [75]. Mucosal healing rates of 30% were attained after a median of 22 wk in CD in one observational study [76]. Furthermore, GEMINI 2 found that almost 40% of initial vedolizumab responders remained in clinical remission to 52 wk although clinical remission rates only became superior to placebo after 30 wk [77]. UC: Response and remission rates appear more rapid in UC than CD. At week 6 after a 2-dose induction, clinical response, remission rates and mucosal healing were significantly higher with vedolizumab than placebo (47%, 17% and 41% vs 26%, 5% and 25% respectively) in one RCT [78]. Moreover, maintenance vedolizumab resulted in higher rates of clinical and endoscopic remission at week 52 than week 6 and mean partial Mayo scores continued to decline until week 52, suggesting that maximal response often takes several months. Therapy-related factors affecting time to response While immunomodulators appear to reduce the formation of antidrug antibodies to vedolizumab [78], the low proportion of patients who actually formed antidrug antibodies in trials might imply that combination therapy may be unnecessary and hence may not provide additional benefit to time to therapeutic response, in contrast to anti-tnf therapies [78]. THIOPURINES Thiopurines, including azathioprine and 6-mercaptopurine, appear to exert their effect via the metabolites, 6-thioguanine nucleotides (6-TGNs). Thiopurines inhibit the synthesis of DNA, RNA and proteins leading to inhibition of lymphocyte proliferation and apoptosis, thereby immunosuppression [79]. Time to response CD: Azathioprine or mercaptopurine take at least 4 to 8 wk to achieve clinical remission [80]. A small RCT suggested that a median of 10.7 wk was required to achieve clinical remission with oral azathioprine 2 mg/kg/d [81]. Time to clinical response with 1.5 mg/kg/d of mercaptopurine was assessed in an RCT by Present et al [82] in patients with CD failing corticosteroids or sulfasalazine. They demonstrated a wide-ranging time to therapeutic response from two weeks to nine months with a median of 3.1 mo; 19% of cases took more than 4 mo to achieve a sustained clinical response [82]. Similarly, in another RCT, Ardizzone et al [83] found a clinical remission rate gain from 33% to 63% from 3 to 6 mo after initiating azathioprine 2 mg/kg/d in patients with corticosteroid-dependent CD. Endoscopic remission rates also appear to be slow to achieve with only 17% of patients on azathioprine in the SONIC study achieving mucosal healing after 26 wk of therapy [50]. Yet another RCT comparing budesonide to azathioprine with 1 year follow up found mucosal healing occurred with thiopurines in 83% of steroiddependent CD by 12 mo, implying that endoscopic improvement continues to slowly accumulate over extended periods [84]. UC: Both clinical and endoscopic response to thiopurines in UC appear to take a minimum of one month but more typically 3-6 mo for steroid-dependent UC [85,86]. Ardizzone et al [87] found that more than half of patients on azathioprine were in steroid-free endoscopic and clinical remission after 6 mo for patients with steroid-dependent UC, which was significantly higher 6391 September 21, 2017 Volume 23 Issue 35

36 Vasudevan A et al. Time to response of therapies in IBD Table 2 Previously documented and potential/ novel methods of improving time to response to therapy in Crohn s disease and ulcerative colitis Clinical scenario Method Improves time to response Improves response rate Improves Published Comments tolerability data? Corticosteroids CD and UC Intravenous administration - - Yes [27,32,150] Anti-tumour necrosis factor-α Initial or for flare to recapture response (CD and UC) Addition of azathioprine - Yes [50] Thiopurine CD and UC Addition of allopurinol - - Yes [105,108] Split dosing of thiopurine - - Yes [151] Methotrexate CD High dose parenteral with corticosteroids if relapse on lower dose - - Yes Can recapture response Tacrolimus UC Target levels of ng/ml - - Yes [123] Aminosalicylates UC Maximize dose - Yes [21] Distal UC Choice of formulation (balsalazide) - - Yes [17,20] Ref. [152] CD: Crohn s disease; UC: Ulcerative colitis. than that with mesalazine (53% vs 21%) in an RCT. Another RCT showed a significant decrease in a composite disease activity score (including endoscopic, clinical and biochemical findings) at 3 and 6 mo after commencing azathioprine 2.5 mg/kg/d for steroiddependent UC, implying that time to therapeutic response is likely between 3 and 6 mo [88]. Factors affecting time to response Metabolite levels: Azathioprine undergoes rapid non-enzymatic conversion to 6-mercaptopurine which is then further metabolized to 6-TGNs in erythrocytes and leukocytes [89]. The therapeutic effect of thiopurines appears commensurate with 6-TGN concentrations, with steady state achieved at two to four weeks, so response can only be expected after this period [90]. Intravenous loading with azathioprine has been associated with a more rapid time to therapeutic response initially (1 wk for clinical response and 4 wk for endoscopic improvement). However, when compared to oral azathioprine at 8 wk, no difference in remission rates or 6-TGN levels had persisted [80,91]. Using dose titration guided by therapeutic drug monitoring, the mean time to therapeutic response decreased from 22 to 19 wk [92]. While prospective studies have not demonstrated a difference in outcomes between patients treated with weight-based and individualized, metaboliteguided dosing of thiopurines [93,94], robust retrospective data elucidated higher rates of clinical remission with 6-TGN levels above pmol/ RBCs [95-97]. Indeed, 78%-90% of patients had improved clinical outcomes from dose optimization after having a subtherapeutic 6-TGN level [98-100]. Addition of allopurinol: A combination of allopurinol 100 mg and 25%-50% of the standard thiopurine dose has been utilized to overcome a number of thiopurine related side effects and correct an unfavorable metabolite profile (so called hypermethylators), with high clinical efficacy [ ]. This has piqued interest as to whether combination allopurinol-thiopurine therapy might be able to achieve not only higher rates of, but quicker time to therapeutic response, especially compared to slow-titrating introductory dosing protocols (as advocated by treatment guidelines [26,106] and widely used to mitigate early side effects [107] ). The result of controlled trials addressing this issue are awaited [108]. Additional strategies that may affect time to response of therapies are summarized in Table 2. METHOTREXATE Methotrexate is a folic acid analogue used in the treatment of multiple autoimmune conditions, including IBD. Methotrexate has been shown effective as an induction and maintenance therapy in CD particularly when administered parenterally. The role of methotrexate in UC is less clear, although a placebocontrolled trial of subcutaneous methotrexate did suggest clinical efficacy in the induction of steroid-free remission [109]. Time to response CD: A clinical response should be expected within 12 wk on parenterally-administered methotrexate according to an open label, non-randomized trial by Kozarek et al [110]. An observational study suggested that the median clinical response time was 9 wk for both oral and parenteral therapy (although 86% were on parenteral therapy in this study) and clinical remission took 22 wk [111]. Despite most patients clinically responding within the first several weeks of therapy, a subgroup may take up to 6 mo to respond [83]. UC: While a placebo-controlled trial of oral methotrexate 12.5 mg in patients with steroid-refractory UC showed no significant difference in clinical remission between the groups, those who reached clinical remission with methotrexate took a mean of 4.1 mo 6392 September 21, 2017 Volume 23 Issue 35

37 Vasudevan A et al. Time to response of therapies in IBD to do so [112]. Subsequent uncontrolled observations by Kozarek et al [110] showed a rapid clinical response (71% within 12 wk) with high-dose intramuscular dosing in UC, albeit in only 7 patients. The METEOR RCT, assessed the efficacy of parenteral methotrexate 25 mg weekly in steroid-dependent UC and found almost a third of patients had corticosteroid-free clinical remission by week 16, which increased to 40% by week 24 [109]. Endoscopic remission was achieved in a numerically greater proportion then did the placebo group, but the difference did not reach statistical significance, perhaps due to lack of statistical power. Factors affecting time to response Route of administration: It is likely that parenteral methotrexate generally achieves a faster time to therapeutic response in a higher proportion of patients due to greater bioavailability, though supportive evidence is found only in the rheumatological literature [113]. Drug levels: Currently there is no reliable method to apply therapeutic drug monitoring of methotrexate in routine IBD care and this is therefore not useful in predicting time to therapeutic response. Serum methotrexate levels are only detectable for about 24 h post-dose and appear not to correlate with clinical response in IBD [114], adenosine levels do not correlate with efficacy [115], and methotrexate polyglutamates [active metabolite(s) of methotrexate] have displayed inconsistent results [ ]. Finally, although folic acid is an important adjunct to methotrexate use and may reduce gastrointestinal upset and hepatic dysfunction, it has no apparent impact on time to therapeutic response [119]. CALCINEURIN INHIBITORS Tacrolimus and cyclosporin are calcineurin inhibitors with evidence in IBD primarily for steroid-refractory UC. Small case series have suggested efficacy of tacrolimus for induction of remission in luminal CD [120]. Cyclosporin is an effective rescue therapy in acute severe colitis and has been used in moderate to severe chronic UC. Oral cyclosporine does not appear to be an effective induction therapy in CD [121]. Time to response Tacrolimus: (1) UC: When targeting a trough level of ng/ml, tacrolimus induces clinical response and mucosal healing rates of 50%-68% and 44%-79% respectively, in patients with UC within 2 wk based on results from two RCTs, with lower rates noted in the larger of these studies [122,123]. In contrast, clinical remission was reported in only 9%-20% after 2 wk in the aforementioned studies, but amongst those who continued tacrolimus, remission rates increased to 29% by week 12, with mucosal healing rates increasing from 67% to 86% over the same time period [122,123]. (2) CD: Small case series suggest a similarly rapid clinical response with tacrolimus in CD refractory to other therapies, occurring within d [124,125]. However, time to therapeutic response appears longer than UC, with one study finding that 36% achieved clinical remission by 20 d which increased to 64% at 120 d [125]. Cyclosporin: UC: Clinical response to cyclosporine in acute severe colitis failing to respond to intravenous corticosteroids is usually rapid, with a median response time reported to be 4-5 d, with the vast majority responding within 7 d in two randomized studies [53,126]. Clinical remission rates were approximately 65% with cyclosporine monotherapy and 93% in combination with steroids after 7 d, according to the results of a randomized study by D Haens et al [127] In the above studies, endoscopic response was described within 7 d of initiation, with a continued improvement in endoscopic activity noted between weeks 1 and 4 [126,127]. Factors affecting time to response Dose and drug levels: Targeting high levels (10-15 ng/ml) of tacrolimus for induction appears more effective than low levels (5-10 ng/ml) with possibly a more rapid time to therapeutic response [123]. For ongoing maintenance thereafter, targeting trough levels of 5-10 ng/ml appears sufficient [ ]. For cyclosporin, no additional benefit in clinical response was achieved with 4 mg/kg compared to 2 mg/kg intravenously in an RCT for acute severe colitis with a median time to response of 4 d in both groups, suggesting that doses above 2 mg/kg do not produce a more rapid time to response [126]. Route of administration: One observational study suggested that oral and intravenous tacrolimus achieved similar rates of clinical response by 14 d in steroid-refractory colitis, with comparable serum tacrolimus levels achieved with both strategies [128]. A retrospective study of oral and intravenous cyclosporin in ulcerative colitis actually found a higher early clinical response rate (exact timing not specified) with oral compared to intravenous cyclosporin (100% vs 65%) despite comparable serum drug levels, predominantly due to higher rates of side effects necessitating treatment cessation with intravenous cyclosporin [129]. Time to response was not directly assessed in this study and groups significantly differed with higher proportions of inpatients and intravenous corticosteroid failures in the intravenous cyclosporin group. EXCLUSIVE ENTERAL NUTRITION Exclusive enteral nutrition (EEN) involves the administration of a liquid nutrition formula to meet all 6393 September 21, 2017 Volume 23 Issue 35

38 Vasudevan A et al. Time to response of therapies in IBD Table 3 Factors affecting time to response and response rates of therapies in inflammatory bowel disease Variable Parameter Effect on time to response Effect on response rate Medications implicated Level of evidence 1 Ref. Age > 65 yr? Anti-tumour necrosis factor-α (anti-tnf) 2b Lobatón et al [143] Increased body mass BMI > 25 - Azathioprine 2b Holtmann et al [153] index Weight > 82 kg - Anti-TNF 1b Reinisch et al [59] Concomitant therapies Immunomodulators with anti-tnf 1b Colombel et al [50] Sandborn et al [69] Smoking status Current smoker Anti-TNF 1b, 2b Arnott et al [154] Sandborn et al [69] Disease duration > 2 yr - Anti-TNF 1b Colombel et al [155] Schreiber et al [156] D'Haens et al [157] 1 As per the Oxford level of evidence scoring; The available literature suggests a slower initial response but comparable long-term response rate; The use of - denotes an absence of published data addressing this issue. nutritional requirements, replacing normal diet, either orally or via nasoenteric tube. It is mostly used for induction of remission in CD typically over a duration of 6-8 wk. While most data emanates from pediatric studies, efficacy is also likely in adults [130]. Time to response with EEN seems rapid, with 75% of adult patients with active CD achieving clinical remission after 10 d of an elemental feeding in one small RCT [131]. Other small RCTs have demonstrated clinical remission rates of 25% to 80% within 3-4 wk of commencement [ ]. In two non-randomized cohort studies utilizing objective disease activity endpoints, 44% of patients achieved mucosal healing after 4 wk of EEN and, in a pediatric cohort, 36% had mucosal healing after 8 wk of EEN [139,140]. ADDITIONAL FACTORS AFFECTING TIME TO CLINICAL RESPONSE Disease related factors There are several patient and disease-related factors that appear important when predicting the likelihood of response and time to therapeutic response in individual patients (see Table 3). Agents tend to achieve a more rapid time to therapeutic response in UC than CD. This may relate to the transmural nature of CD thus treatment takes longer to achieve resolution of inflammatory changes. This difference in time to therapeutic response is exemplified by the vedolizumab registration studies, where induction therapy appeared to have higher response rates in UC than CD and the benefits in CD were predominantly observed later during the maintenance phase [73,78]. Patients who have not achieved an adequate response to prior therapies may have a slower response to subsequent therapies. This was noted with vedolizumab induction, where previous anti- TNF failures achieved clinical remission rates that only became significantly different to placebo after 10 wk, in contrast to the overall cohort where clinical remission rates were higher at week 6 compared to placebo [74]. Similar findings of a longer time to clinical remission were found in patients who had received infliximab previously and were subsequently treated with induction certolizumab for CD [69]. Patient related factors While several additional factors intuitively could affect time to therapeutic response such as nutritional status, age and the intestinal microbiome, published data are lacking. Advancing age is typically associated with a reduced glomerular filtration rate, greater oxidative stress, increased volume of distribution, comorbid conditions, decreased hepatic metabolism and frailty-any of which may affect treatment choice and a therapy s time to clinical response [141,142]. For instance, one retrospective study found that rates of clinical response to anti-tnf therapy for IBD in patients > 65 years was significantly less at week 10 but not significantly different at 6 mo than matched controls with similar co-morbidities aged < 65 years, suggesting a slower onset of response overall [143]. The limited available evidence also suggests that obesity can affect the rate of response, although the effect on time to response has not been studied. For instance, higher baseline weight was associated with a lower rate of clinical remission following induction therapy with adalimumab in the ULTRA-1 study and has been associated with an earlier loss of response to infliximab and adalimumab in IBD [59,144,145]. Although many of these factors are not modifiable, intervening early in the disease course and/or simple complementary measures such as improving nutrition may allow for a more rapid time to therapeutic response. DISCUSSION This review has elucidated multiple important principles 6394 September 21, 2017 Volume 23 Issue 35

39 Vasudevan A et al. Time to response of therapies in IBD of time to therapeutic response with direct applicability to clinical practice. First, patience is critical (for patients and clinicians alike) to reap the maximal benefits of some agents, particularly thiopurines, vedolizumab and methotrexate, with cumulative gains in response to these agents up to 12 mo after commencement. The clinical benefits indeed might lag significantly beyond the drug reaching therapeutic levels. For instance, this was reflected in a study assessing whether initial intravenous loading of azathioprine might hasten time to response. Indeed, therapeutic levels of 6-TGN were achieved rapidly. However, this did not improve the time to therapeutic response compared to standard oral administration [80]. Thus assessment of response should be delayed until sufficient time has lapsed to reach clinical efficacy. Whilst symptomatic improvement may occur relatively early following the initiation of many therapies, there often appears to be a lag in achieving mucosal healing, which should be considered when interpreting an early endoscopic assessment. This concept was demonstrated in a randomized controlled trial by Sutherland et al [146] comparing 4 g aminosalicylate enemas to placebo enemas for the management of distal UC. Clinical response rates and endoscopy were measured at 3 and 6 wk, with response rates reaching 60% by week 3 in the aminosalicylate arm, then plateauing to reach 68% by week 6. The mucosal healing rates over the same period increased from 25% to 42%, suggesting a later rise [24]. Additionally, patients kept a symptom diary for the first two weeks of this trial and rectal bleeding had ceased in over half of patients by day 6, so the symptom improvement may have occurred more rapidly than the time of outcome measurement. Hence, it may be appropriate to delay assessment until after the expected time to therapeutic response has passed. Secondly, it follows that a prolonged period of bridging therapy such as co-administration of EEN, corticosteroids or even perhaps tacrolimus is an important component of treatment planning, particularly in patients who are acutely unwell, so that relapse is avoided if possible, prior to the expected maximal response of a newly introduced therapy. Thirdly, in agents where a delayed time to therapeutic response is more likely, one must consider whether potential methods of hastening onset of action can be employed, such as thiopurine and allopurinol in combination or perhaps using EEN in combination with an anti-tnf agent in CD as induction therapy. Significantly, the lack of reporting of time-toresponse in clinical trials is a deficiency. Mostly, data must be extrapolated from studies rather than being directly reported. Given its utility in clinical practice and the relatively basic calculations required, this should be addressed in future studies. The expected time-to-response can potentially assist in trial design by providing an estimate of the necessary duration of a study to show maximal efficacy. For example, for vedolizumab for the management of CD, the CDAI-100 response rate was not significant at week 6 and only surpassed placebo around week 28 [73]. Similarly, modest early response rates were also noted with another anti-integrin therapy natalizumab at week 10 with response rates increasing during maintenance therapy [147]. The use of week 6 rather than week 10 or later as the response times for vedolizumab has been a criticism of the large randomized studies [77]. Hence, such information can potentially be reported in early studies and thus provide further insight into the rapidity of action of drugs or more widely, drug classes, which can be incorporated into future studies of that therapy, plus into clinical practice. Finally, the concept of time-to-response is a broad term and it is likely that it can be further divided based on the proportion of patients who have respond to therapy, as shown in Table 1. There is a time point at which most patients will respond and then following this there is progressively diminishing returns in the likelihood of further response, culminating in a point where response is unlikely; the time to futility. This concept remains clinically important, as the yield from persisting with therapy beyond this point is low and thus place patients at higher risk, for minimal or no benefit. Again, such information be ideally included in published data to better aid clinicians with therapeutic decisions. CONCLUSION In a chronic disease like IBD, where many therapies are effective yet of relatively slow onset of action, time-to-therapeutic response is of central importance both quantitatively to frame the patient s expectations and the clinician s decision making but also conceptually-encouraging the mindset of forward treatment planning, overlapping bridging therapies and accuracy in determining therapeutic failure. Furthermore, in contrast to the homogenized, groupbased reporting of results from the seminal RCTs as the reference standard for each new agent, time to therapeutic response is inherently patient-centric and individualized. This is not only because it has the potential to vary by indication, dosage, demographic, clinical factors and is dependent on which endpoint is chosen (i.e., clinical response, remission, or endoscopic remission) but one can argue that the time to therapeutic response is of far greater relevance to patients and perhaps even to clinicians in day-today practice, thus future studies should examine and incorporate this paradigm further. REFERENCES 1 Markowitz J. Early inflammatory bowel disease: different treatment response to specific or all medications? Dig Dis 2009; 27: [PMID: DOI: / ] 6395 September 21, 2017 Volume 23 Issue 35

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45 Vasudevan A et al. Time to response of therapies in IBD Fat composition may be a clue to explain the primary therapeutic effect of enteral nutrition in Crohn s disease: results of a double blind randomised multicentre European trial. Gut 2002; 51: [PMID: DOI: /gut ] 133 Bamba T, Shimoyama T, Sasaki M, Tsujikawa T, Fukuda Y, Koganei K, Hibi T, Iwao Y, Munakata A, Fukuda S, Matsumoto T, Oshitani N, Hiwatashi N, Oriuchi T, Kitahora T, Utsunomiya T, Saitoh Y, Suzuki Y, Nakajima M. Dietary fat attenuates the benefits of an elemental diet in active Crohn s disease: a randomized, controlled trial. Eur J Gastroenterol Hepatol 2003; 15: [PMID: DOI: /01.meg b3] 134 Mansfield JC, Giaffer MH, Holdsworth CD. Controlled trial of oligopeptide versus amino acid diet in treatment of active Crohn s disease. Gut 1995; 36: [PMID: DOI: / gut ] 135 Verma S, Brown S, Kirkwood B, Giaffer MH. Polymeric versus elemental diet as primary treatment in active Crohn s disease: a randomized, double-blind trial. 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Early mucosal healing with infliximab is associated with improved longterm clinical outcomes in ulcerative colitis. Gastroenterology 2011; 141: [PMID: DOI: /j.gastro ] 150 Llaó J, Naves JE, Ruiz-Cerulla A, Marín L, Mañosa M, Rodríguez- Alonso L, Cabré E, Garcia-Planella E, Guardiola J, Domènech E. Intravenous corticosteroids in moderately active ulcerative colitis refractory to oral corticosteroids. J Crohns Colitis 2014; 8: [PMID: DOI: /j.crohns ] 151 Shih DQ, Nguyen M, Zheng L, Ibanez P, Mei L, Kwan LY, Bradford K, Ting C, Targan SR, Vasiliauskas EA. Split-dose administration of thiopurine drugs: a novel and effective strategy for managing preferential 6-MMP metabolism. Aliment Pharmacol Ther 2012; 36: [PMID: DOI: / j x] 152 Feagan BG, Fedorak RN, Irvine EJ, Wild G, Sutherland L, Steinhart AH, Greenberg GR, Koval J, Wong CJ, Hopkins M, Hanauer SB, McDonald JW. A comparison of methotrexate with placebo for the maintenance of remission in Crohn s disease. North American Crohn s Study Group Investigators. N Engl J Med 2000; 342: [PMID: DOI: / NEJM ] 153 Holtmann MH, Krummenauer F, Claas C, Kremeyer K, Lorenz D, Rainer O, Vogel I, Böcker U, Böhm S, Büning C, Duchmann R, Gerken G, Herfarth H, Lügering N, Kruis W, Reinshagen M, Schmidt J, Stallmach A, Stein J, Sturm A, Galle PR, Hommes DW, D Haens G, Rutgeerts P, Neurath MF. Significant differences between Crohn s disease and ulcerative colitis regarding the impact of body mass index and initial disease activity on responsiveness to azathioprine: results from a European multicenter study in 1,176 patients. Dig Dis Sci 2010; 55: [PMID: DOI: /s ] 154 Arnott ID, McNeill G, Satsangi J. An analysis of factors influencing short-term and sustained response to infliximab treatment for Crohn s disease. Aliment Pharmacol Ther 2003; 17: [PMID: DOI: / j x] 155 Colombel JF, Sandborn WJ, Rutgeerts P, Enns R, Hanauer SB, Panaccione R, Schreiber S, Byczkowski D, Li J, Kent JD, Pollack PF. 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46 Vasudevan A et al. Time to response of therapies IBD 157 D'Haens G, Baert F, van Assche G, Caenepeel P, Vergauwe P, Tuynman H, De Vos M, van Deventer S, Stitt L, Donner A, Vermeire S, Van De Mierop FJ, Coche JR, van der Woude J, Ochsenkühn T, van Bodegraven AA, Van Hootegem PP, Lambrecht GL, Mana F, Rutgeerts P, Feagan BG, Hommes D; Belgian Inflammatory Bowel Disease Research Group; North-Holland Gut Club. Early combined immunosuppression or conventional management in patients with newly diagnosed Crohn s disease: an open randomised trial. Lancet 2008; 371: [PMID: DOI: /S (08) ] P- Reviewer: Gazouli M, Jadallah KA, Mitsui K S- Editor: Gong ZM L- Editor: A E- Editor: Huang Y 6402 September 21, 2017 Volume 23 Issue 35

47 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) Basic Study Pharmacological inhibition of cannabinoid receptor 1 stimulates gastric release of nesfatin-1 via the mtor pathway ORIGINAL ARTICLE Cintia Folgueira, Silvia Barja-Fernandez, Laura Prado, Omar Al-Massadi, Cecilia Castelao, Veronica Pena- Leon, Patricia Gonzalez-Saenz, Javier Baltar, Ivan Baamonde, Rosaura Leis, Carlos Dieguez, Uberto Pagotto, Felipe F Casanueva, Sulay A Tovar, Ruben Nogueiras, Luisa M Seoane Cintia Folgueira, Silvia Barja-Fernandez, Laura Prado, Cecilia Castelao, Veronica Pena-Leon, Patricia Gonzalez-Saenz, Ivan Baamonde, Luisa M Seoane, Grupo Fisiopatología Endocrina, Instituto de Investigación Sanitaria de Santiago de Compostela, Santiago de Compostela, Spain Cintia Folgueira, Silvia Barja-Fernandez, Omar Al-Massadi, Cecilia Castelao, Patricia Gonzalez-Saenz, Rosaura Leis, Carlos Dieguez, Felipe F Casanueva, Sulay A Tovar, Ruben Nogueiras, Luisa M Seoane, CIBER Fisiopatología Obesidad y Nutrición, Instituto de Salud Carlos III, Santiago de compostela, Spain Cintia Folgueira, Omar Al-Massadi, Carlos Dieguez, Sulay A Tovar, Ruben Nogueiras, Department of Physiology, Research Centre of Molecular Medicine and Chronic Diseases, Santiago de Compostela, Spain Silvia Barja-Fernandez, Rosaura Leis, Department of Pediatric, Universidad de Santiago de Compostela, Santiago de Compostela, Spain Javier Baltar, Ivan Baamonde, Servicio de Cirugía General, Complexo Hospitalario Universitario de Santiago, Santiago de Compostela, Spain Uberto Pagotto, Endocrinology Unit and Center for Applied Biomedical Research, Department of Medical and Surgical Sciences, Hospital S. Orsola-Malpighi, Alma Mater University of Bologna, Bologna, Italy Felipe F Casanueva, Laboratorio de Endocrinología Molecular y Celular. Universidad de Santiago de Compostela, Instituto de Investigación Sanitaria de Santiago de Compostela, Complexo Hospitalario Universitario de Santiago, Santiago de Compostela, Spain ORCID number: Cintia Folgueira ( ); Silvia Barja-Fernandez ( ); Laura Prado ( ); Omar Al-Massadi ( ); Cecilia Castelao ( ); Veronica Pena-Leon ( ); Patricia Gonzalez-Saenz ( ); Javier Baltar ( ); Ivan Baamonde ( ); Rosaura Leis ( ); Carlos Dieguez ( ); Uberto Pagotto ( ); Felipe F Casanueva ( ); Sulay A Tovar ( ); Ruben Nogueiras ( ); Luisa M Seoane ( ). Author contributions: Baltar J, Baamonde I, Leis R, Dieguez C, Casanueva FF, Tovar SA, Nogueiras R and Seoane LM substantially contributed to the conception and design of the study, acquisition, analysis and interpretation of data; Folgueira C, Barja-Fernandez S, Prado L, Al-Massadi O, Castelao C, Pena- Leon V and Gonzalez-Saenz P contributed to the experimental protocols, animal models development and data acquisition and analysis; all authors drafted the article and made critical revisions related to the intellectual content of the manuscript, and approved the final version of the article to be published; Folgueira C and Barja-Fernandez S contributed equally to this work. Supported by Instituto de Salud Carlos III, No. PI15/01272 cofounded by FEDER; Fondo de Investigaciones Sanitarias (LS: I3SNS-SERGAS/ISCIII). Centro de Investigacion Biomedica en Red Fisiopatología de la Obesidad y Nutrición (CIBERobn) is a iniciative of the Instituto de Salud Carlos III (ISCIII) of Spain which is supported by FEDER funds. Institutional review board statement: The study was reviewed and approved by the Comité Etico de Investigación Clinica de Galicia. Institutional animal care and use committee statement: The approaches in the present manuscript were performed under the procedure 15005/2015/003 reviewed and approved by the Faculty Animal Committee at the University of Santiago de Compostela. Conflict-of-interest statement: To the best of our knowledge, no conflict of interest exists September 21, 2017 Volume 23 Issue 35

48 Folgueira C et al. Pharmacological inhibition of CB1 stimulates gastric release Data sharing statement: No additional data are available. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Invited manuscript Correspondence to: Luisa M Seoane, PhD, Grupo Fisiopatología Endocrina, Laboratorio 14, Instituto de Investigación Sanitaria, Complexo Hospitalario Universitario de Santiago, Travesía da Choupana s/n, Santiago de Compostela, Spain. luisamaria.seoane@usc.es Telephone: Fax: Received: April 19, 2017 Peer-review started: April 21, 2017 First decision: July 28, 2017 Revised: July 31, 2017 Accepted: August 25, 2017 Article in press: August 25, 2017 Published online: September 21, 2017 Abstract AIM To determine whether Nucb2/nesfatin1 production is regulated by the cannabinoid system through the intracellular mtor pathway in the stomach. METHODS Sprague Dawley rats were treated with vehicle, rimonabant, rapamycin or rapamycin+rimonabant. Gastric tissue obtained from the animals was used for biochemical assays: Nucb2 mrna measurement by real time PCR, gastric Nucb2/nesfatin protein content by western blot, and gastric explants to obtain gastric secretomes. Nucb2/nesfatin levels were measured in gastric secretomes and plasma using enzyme-linked immunosorbent assay. RESULTS The inhibition of cannabinoid receptor 1 (CB1) by the peripheral injection of an inverse agonist, namely rimonabant, decreases food intake and increases the gastric secretion and circulating levels of Nucb2/ nesfatin-1. In addition, rimonabant treatment activates mtor pathway in the stomach as showed by the increase in pmtor/mtor expression in gastric tissue obtained from rimonabant treated animals. These effects were confirmed by the use of a CB1 antagonist, AM281. When the intracellular pathway mtor/s6k was inactivated by chronic treatment with rapamycin, rimonabant treatment was no longer able to stimulate the gastric secretion of Nucb2/nesfatin-1. CONCLUSION The peripheral cannabinoid system regulates food intake through a mechanism that implies gastric production and release of Nucb2/Nesfatin-1, which is mediated by the mtor/s6k pathway. Key words: NUCB2/nesfatin-1; Stomach; Food intake; Cannabinoid receptor 1; mtor The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: The peripheral pharmacological blockade of the cannabinoid receptor 1 (CB1) induces a decrease in food intake in rats by stimulating the gastric secretion, and consequently the circulating levels, of the anorexigenic peptide Nucb2/nesfatin-1. The present data show that gastric CB1 receptors modulate Nucb2/ Nesfatin-1 production in the stomach at the intracellular level, which is mediated by the mtor pathway. Folgueira C, Barja-Fernandez S, Prado L, Al-Massadi O, Castelao C, Pena-Leon V, Gonzalez-Saenz P, Baltar J, Baamonde I, Leis R, Dieguez C, Pagotto U, Casanueva FF, Tovar SA, Nogueiras R, Seoane LM. Pharmacological inhibition of cannabinoid receptor 1 stimulates gastric release of nesfatin-1 via the mtor pathway. World J Gastroenterol 2017; 23(35): Available from: URL: DOI: INTRODUCTION Nucb2/nesfatin-1 is a 42 kda peptide, first discovered as an anorexigenic factor that regulates energy balance. The product of the Nucb2 gene is a 396 amino acid secreted peptide that, after being processed by the enzyme prohormone convertase (PC-1/2), becomes the N-terminal fragment known as nesfatin-1 [1]. Nucb2 is mainly expressed in the gastric mucosa and adipose tissue, although its expression has also been detected elsewhere, such as hypothalamus, brain stem, pancreas and testis [1-5]. In the gastrointestinal tract, Nucb2 is most highly expressed in the X/A cells from the oxyntic mucosa of the stomach, which also synthesizes ghrelin, the main peptide hormone in the gastrointestinal tract [4,6,7]. Despite being secreted by the same cells, Nucb2 and ghrelin have opposing biological functions and regulation. Central and peripheral administration of Nucb2 suppresses feeding behavior [1,8], while ghrelin stimulates appetite [9,10]. Interestingly, the fact that Nucb2 and ghrelin are produced in the same gastric cells but in different vesicles suggests that both proteins might experience differential regulation at the gastric level to maintain energy balance. Supporting this theory, Nucb2/nesfatin-1 production and regulation by nutritional status decreases during 6404 September 21, 2017 Volume 23 Issue 35

49 Folgueira C et al. Pharmacological inhibition of CB1 stimulates gastric release fasting [1], while ghrelin levels are up-regulated [11]. Lastly, this decrease in Nucb2 production during fasting conditions is concomitant with inhibition of the mtor/ps6k1 pathway, a sensor of the metabolic status of organisms [12]. Ghrelin secretion, meanwhile, is negatively associated with mtor/ps6k1 pathway activity under fasting conditions [13]. The cannabinoid system plays an important role as an endogenous regulator of energy balance, acting at multiple levels. One of those mechanisms includes its interaction with gastric ghrelin production to regulate appetite and body weight [14]. More specifically, the blockade of the cannabinoid receptor CB1 decreases ghrelin expression in the stomach, and this effect was mediated by activation of the mtor/ps6k1 pathway [14]. Taking into account the opposing functions and regulation of ghrelin and Nucb2/nesfatin at the gastric level, as well as their opposite relationship with the mtor/s6k1 pathway, we sought to investigate whether the cannabinoid system might also regulate Nucb2/nesfatin-1 production in the stomach and whether the mtor/ps6k1 intracellular pathway mediates this effect. MATERIALS AND METHODS Ethics statement The authors of this manuscript declare that the animal work in this study was approved by the Animal Care Committee of Santiago de Compostela University (Santiago de Compostela, Spain) in accordance with our institutional guidelines and the European Union standards for the care and use of experimental animals. The approaches in the present manuscript were performed under the procedure 15005/2015/003 reviewed and approved by the Faculty Animal Committee at the University of Santiago de Compostela. Animal and experimental design Male Sprague-Dawley rats were housed in airconditioned rooms (22-24 ) under a controlled light/ dark cycle (12 h light, 12 h darkness) with free access to food and water. The animals were euthanatized, trunk blood was collected and immediately centrifuged, and the plasma was stored at -80 for biochemical measurements. Effects of pharmacological blockade of CB1 on food intake, gastric Nucb2 production, gastric Nucb2 secretion and plasma Nucb2/nesfatin-1 levels A group of overnight fasted adult male rats was treated with Rimonabant intraperitoneal (i.p.) at a dose of 3 mg/kg (drug or vehicle, a solution of saline-dmso 70%). To corroborate the data, a second set of animals was treated with AM-281 (Tocris Cookson Inc., Ellisville, MO), a CB1 antagonist. Animals were assigned to one of two experimental groups: the first group of rats was fasted for 36 h before receiving either an i.p. injection of Rimonabant or AM281 (3 mg/kg) 1 h prior to euthanasia (Rimonabant or AM281 group). The second group was fasted for 36 h prior to receiving an injection of vehicle (DMSO) 1 h before euthanasia (Control group). After sacrifice, the stomach was surgically excised and the plasma collected. An additional set of animals was utilized to perform the food intake study. A group of overnight fasted adult male rats was treated with Rimonabant (i.p.) at a dose of 3 mg/kg or vehicle (a solution of saline-dmso 70%) and immediately after treatment allowed free access to chow diet. One hour after the injection, the amount of food eaten was recorded for each rat. Effects of pharmacological blockade of CB1 on gastric Nucb2 production, gastric Nucb2 secretion and plasma Nucb2/Nesfatin-1 in animals with a pharmacological inhibition of the gastric mtor signaling by rapamycin treatment Adult male rats were assigned to one of two experimental groups: 36-hour fasted animals treated with rapamycin (1 mg/kg i.p. for 6 d) (Rapa group); 36-h fasted animals treated with rapamycin (1 mg/ Kg i.p. for 6 d) and rimonabant (3 mg/kg i.p.) 1 h before euthanasia (rapa+rimo group). Rapamycin was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, United States). Experimental techniques Tissue explant culture: Tissue explants were obtained from adult Sprague-Dawley rats. To obtain ex vivo tissue explants, after euthanasia the stomachs were rapidly excised and transported to the lab in Krebs-Ringer-HEPES buffer (NaCl, 125 mmol/l; KCl, 5 mmol/l; MgSO4, 1.2 mmol/l; KH2PO4, 1.3 mmol/l; CaCl2, 2 mmol/l; glucose, 6 mmol/l; HEPES, 25 mmol/l; ph 7.4). After removing the blood vessels and connective tissue, the stomach was washed with sterile Krebs-Ringer- HEPES buffer. The tissue explants were placed in sixwell dishes containing 2.5 ml of Dulbecco s modified Eagle s medium supplemented with L-glutamine (200 mmol/l), penicillin (1000 U/mL) and streptomycin (100 µg/ml). After a pre-incubation period of 1 h at 37 under a humidified atmosphere of 95% air-5% CO2, the media was aspirated, and 2.5 ml of fresh medium was added into each well. The culture medium was collected after 2 h and tissue was weighed [11]. The samples were stored at -80 until used for biochemical analysis or processed to sample concentration by ultracentrifugation units (Amicon Ultra 3-kDa cut off, Millipore, Billerica, United States) for Western blot analyses. Protein extraction and Western blotting analyses The fundus of the stomach was obtained after euthanasia from animal models and homogenizated using a TissueLyser II (Qiagen, Tokyo, Japan) in cold 6405 September 21, 2017 Volume 23 Issue 35

50 Folgueira C et al. Pharmacological inhibition of CB1 stimulates gastric release RIPA buffer [containing 200 mmol/l Tris/HCl (ph 7.4), 130 mmol/l NaCl, 10% (v/v) glycerol, 0.1%(v/v) SDS, 1%(v/v) Triton X-100, 10 mmol/l MgCl2] with anti-proteases and anti-phosphatases (Sigma-Aldrich; St. Louis, MO, United States). The tissue lysates were centrifuged for 10 min at g in a microfuge at 4. Then, 30 µg of stomach mucosa were separated in 10% sodium-dodecyl sulfate-polyacrylamide gels (SDS-PAGE) and transferred onto nitrocellulose membranes. Equal loading was confirmed by measuring the amount of β-actin in whole tissue protein extracts. The membranes were probed successively with primary antibodies and peroxide-conjugated secondary antibodies (Jackson Immunoresearch, Baltimore, United States). Specific antigen-antibody binding was visualized using a chemiluminescence method according to the manufacturer s instructions (Pierce ECL Western Blotting Sustrate, Thermo Scientific). Primary anti-nesfatin-1 (1-82) was purchased from Phoenix Pharmaceuticals, Inc. (Burlingame, United States) and anti- β-actin (A-5316) was purchased from Sigma Chemical Co., (St Louis, MO, United States). Western blots were performed using independent samples from different rats belonging to each group. β-actin detection was performed for all western blots as a loading control. The Western blot data are expressed as the mean ± SEM of percentages normalized to β-actin levels (arbitrary units). RNA isolation and real-time quantitative RT-PCR Total RNA was isolated from the stomach mucosa of animals using TRIzol (Invitrogen, CA, United States), according to the manufacturer s recommendations. The extracted total RNA was purified with DNase treatment using a DNA-free kit as a template (Ambion, United States) to generate first-strand cdnas using a High-capacity cdna Reverse Transcription kit (Applied Biosystems, United States). Quantitative real-time PCR was performed using a StepOne Plus Instrument (Applied Biosystems) with specific Taqman qrt-pcr primers and probes. For analyses, the NUCB2 gene expression levels (Assay ID: Rn _m1) were normalized to the mrna expression levels of the housekeeping gene hypoxanthine phosphoribosyltransferase 1 (HPRT1) (Assay ID: Rn _m1, TaqMan: Applied Biosystems) and are expressed relative to the average value of the control group. Biochemical analysis Variations in the Nucb2 mrna expression were measured in the rat gastric mucosa by real-time PCR and immunoblotting was performed to test the Nucb2 protein content in the mucosa. To measure plasma Nucb2/Nesfatin-1 levels, blood samples were collected in tubes containing EDTA (1 mg/ml blood) and immediately centrifuged. Gastric tissues explants obtained from the experimental animals were incubated as described below [11]. The secretome collected from the gastric explants were immediately processed for sample concentration (corrected per gram of tissue) by ultra-centrifugation units (Amicon Ultra UFC and UFC off, Millipore, Billerica, United States). Nucb2/Nesfatin-1 secretion was measured by ELISA in the secretome and plasma using reagents kits and methods provided by Phoenix Pharmaceuticals, Inc. (Burlingame, United States). The assay sensitivity limit was 0.1 ng/ml. The results are expressed as ng/ml of NUCB2/nesfatin-1. Statistical analysis The data are presented as the mean ± SEM. Statistical analyses were performed with GraphPad Prism 5 software. Analyses comparing different groups were performed using the non-parametric Mann-Whitney U test. The statistical significance is indicated as follows: a P < 0.05 and b P < RESULTS Effects of pharmacological blockade of CB1 on food intake, gastric Nucb2 production, gastric Nucb2 secretion and plasma Nucb2/nesfatin-1 levels Animals administered rimonabant (i.p.) showed a decrease in food intake after 1 h compared to control group (Figure 1A). Gastric mrna levels of Nucb2 (Control: 1.02 ± 0.06 vs Rimonabant: 0.74 ± 0.06 arbitrary units, a P < 0.05) (Figure 1B) and Nucb2 protein content in gastric mucosa (Control: 100 ± 6.81 vs Rimonabant: ± 4.76, a P < 0.05) (Figure 1C) were significantly reduced after the blockade of the peripheral CB1 receptors with rimonabant. The decreased content in Nucb2 in the gastric mucosa in the group of animals treated with rimonabant was associated with an increased secretion of Nucb2/Nesfatin-1 from the stomach, as found by Nucb2/Nesfatin-1 values measured in the secretome obtained from gastric explants (control 0.16 ± 0.06 vs rimonabant 0.71 ± 0.22, b P < 0.01 ) (Figure 1D). The circulating levels of Nucb2/Nesfatin-1 measured in plasma were accordingly increased after rimonabant treatment (control 5.83 ± 0.39 ng/ml vs rimonabant 7.37 ± 0.4 ng/ml, a P < 0.05) (Figure 1E). In keeping with data from the rimonabant treatment, the i.p. injection of a CB1 antagonist (AM281) caused a decrease in mrna levels of Nucb2 (Control: 1.02 ± 0.06 vs AM-281: 0.78 ± 0.06 arbitrary units, a P < 0.05) (Figure 2A). Moreover, the measurement of Nucb2 secretion from gastric explants from animals treated with AM-281 showed a tendency towards an increase in gastric Nucb2 secretion, although it was not statistically significant (Figure 2B). In addition, circulating Nucb2/ nesfatin-1 levels analyzed by ELISA showed a significant increase after AM281 treatment (Control: 3.39 ± September 21, 2017 Volume 23 Issue 35

51 Folgueira C et al. Pharmacological inhibition of CB1 stimulates gastric release A 8 B 1.2 Food intake (g) a NUCB2 mrna levels (arbitrary units) a 0 Control Rimonabant 0.0 Control Rimonabant C NUCB2 levels (% over control) Control NUCB2 β-actin a Rimonabant 50 kda 42 kda D Gastric NUCB2/Nesfatin-1 secretion (ng/ml) E Plasma NUCB2/Nesfatin-1 levels (ng/ml) Control b Rimonabant a 0 Control Rimonabant Figure 1 Effects of pharmacological blockade. Food Intake (A), mrna expression levels measured by real-time PCR for Nucb2 (B), Nucb2 protein levels and representative Western blot from the mucosa; β-actin was used as a loading control (C). Dividing lines indicate splicings within the same gel; D: Nucb2/Nesfatin-1 secretion from tissue explant analyzed by ELISA; E: Plasma Nucb2/Nesfatin-1 levels obtained from 36-h fasted animals that received i.p. treatment with Rimonabant (3 mg/kg i.p.) (n = 9) or vehicle (n = 9). a P < 0.05, b P < ng/ml vs AM281: 4.46 ± 0.38 ng/ml, a P < 0.05) (Figure 2C). Effects of pharmacological blockade of CB1 on gastric Nucb2 production, gastric Nucb2 secretion and plasma Nucb2/Nesfatin-1 in animals with inhibited gastric mtor signaling We found that gastric CB1 blockade with rimonabant treatment in the fasting state induces activation of the mtor/s6k1 pathway (vehicle: 100 ± vs rimonabant: ± 34.5; P < 0.05) (Figure 3A). Accordingly, in the present work we observed that AM281 administration to fasted animals induces activation of the mtor/ps6k1 pathway, as observed by increased expression of pmtor/mtor in the gastric mucosa of AM281 treated animals compared to controls (vehicle, 100 ± vs AM281, ± 15.31, P < 0.05) (Figure 3C). In light of mtor pathway activation in response to gastric CB1 blockade with rimonabant, the next step was to determine whether this pathway might be mediating the effect of cannabinoids on Nucb2/ nesfatin-1 production. To this end, we administered rimonabant to a group of animals subjected to chronic rapamycin treatment to establish mtor pathway inactivation. We find that, contrary to the effects found in control rats, i.p. administration of rimonabant to animals previously treated with rapamycin did not affect gastric Nucb2 mrna levels (Figure 4A), gastric Nucb2/Nesfatin-1 protein levels (Figure 4B), gastric secretion of Nucb2/Nesfatin-1 (Figure 4C) or circulating levels of Nucb2/Nesfatin-1 (Figure 4D). DISCUSSION Ghrelin and Nucb2/Nesfatin-1 are co-expressed in the same endocrine cells of the stomach, together with the cannabinoid receptor CB1 [4]. In the present study we show, for the first time to our knowledge, that Nucb2/ Nesfatin-1 regulation in the stomach is governed by the cannabinoid system in an inverse relation to the described in the bibliography for ghrelin [14]. First, we found that blockade of the endogenous CB1 receptor at peripheral level with two different antagonists, rimonabant and AM281, increases gastric Nucb2/ Nesfatin-1 secretion, which is reflected by an increase 6407 September 21, 2017 Volume 23 Issue 35

52 Folgueira C et al. Pharmacological inhibition of CB1 stimulates gastric release A 1.5 B 0.30 NUCB2 mrna levels (arbitrary units) Control a AM281 Gastric NUCB2/nesfatin-1 secretion (ng/ml) Control AM281 C 6 Plasma NUCB2/nesfatin--1 levels (ng/ml) a 0 Control AM281 Figure 2 mrna expression levels measured by real-time PCR for Nucb2 (A), nucb2/nesfatin-1 secretion from tissue explant analyzed by ELISA (B), plasma Nucb2/nesfatin-1 levels (C) obtained from 36-h fasted animals that received i.p. treatment with AM281 (3 mg/kg i.p.) (n = 6) or vehicle (n = 6). a P < 0.05 vs control. A pmtor/mtor (% over control) Control a Rimonabant B ps6k1/ms6k1 (% over control) Control Rimonabant C R C R pmtor 298 kda ps6k1 70 kda mtor 298 kda S6K1 70 kda C pmtor/mtor (% over control) Control a AM281 D ps6k1/ms6k1 (% over control) Control AM281 C AM281 C AM281 pmtor 298 kda ps6k1 70 kda mtor 298 kda S6K1 70 kda Figure 3 Measures of phospho-mtor/mtor levels (A) and phospho-s6k1/s6k1 (B) in gastric mucosa and representative Western blots from animals in the fasting state treated with i.p. rimonabant or vehicle (n = 9). Measures of phospho-mtor/mtor levels (C) and phospho-s6k1/s6k1 (D) in gastric mucosa and representative Western blots from animals in the fasting state treated with i.p. AM281 (n = 6) or vehicle (n = 6). β-actin was used as a loading control. Dividing lines indicate splicings within the same gel. The results are expressed as percentages over control, a P < September 21, 2017 Volume 23 Issue 35

53 Folgueira C et al. Pharmacological inhibition of CB1 stimulates gastric release A NUCB2 mrna levels (arbitrary units) Rapamycin Rapamycin + Rimonabant B NUCB2 protein levels (%) Rapamycin Rapamycin + Rimonabant NUCB2 50 kda β-actin 42 kda C Gastric NUCB2/nesfatin-1 secretion (ng/ml) D Plasma NUCB2/nesfatin--1 levels (ng/ml) Rapamycin Rapamycin + Rimonabant 0 Rapamycin Rapamycin + Rimonabant Figure 4 mrna expression levels measured by real-time PCR for Nucb2 (A), Nucb2 protein levels and representative Western blot from the mucosa. β-actin was used as a loading control. Dividing lines indicate splicings within the same gel (B), Nucb2/Nesfatin-1 secretion from tissue explant analyzed by ELISA (C), plasma Nucb2/Nesfatin-1 levels (D) obtained from 36-h fasted animals treated with rimonabant i.p. (n = 9) and/or rapamycin i.p. chronically for 1 wk (n = 9). in the circulating levels of this peptide. An anorexigenic effect of Nucb2/nesfatin-1 has been extensively described [4] animals treated with rimonabant showed a significant decrease in food intake. The anorexigenic effect after CB1 blockade might be attributed to the increased production of Nucb2/nesfatin-1 in the stomach. Moreover, it was previously shown that the peripheral blockade of CB1 with rimonabant resulted in decreased food intake that was mediated by a decrease in the production of the orexigenic ghrelin [14]. Altogether, the data in the present study shows that Nucb2/nesfatin-1 is regulated in the stomach in a way that is diametrically opposed to those previously described for ghrelin, suggesting an equilibrium at the gastric level between the anorexigenic Nucb2/ nesfatin-1 and the orexigenic ghrelin that regulates food intake to maintain body weight. The next step of the present work was to understand the intracellular mechanism mediating the effect of the cannabinoid system on gastric Nucb2/ nesfatin-1. It was previously demonstrated that the regulation of ghrelin production in the stomach by the cannabinoid system is driven by the mtor/ S6K1 pathway [14]. In this sense, it was also reported that Nucb2/nesfatin-1 production in the stomach is modulated by mtor signaling during nutritional changes [12]. Taking into account that ghrelin and Nucb2/nesfatin-1 are produced in the same cell type in the stomach, we wanted to determine whether the intracellular mtor/s6k1 pathway may also be involved in the gastric regulation of Nucb2/ nesfatin-1 by the cannabinoid system. As expected, pharmacological blockade of CB1 with rimonabant, or AM281, activated the mtor/s6k1 pathway, which is a sensor of nutritional status. In light of the present findings the blockade of the cannabinoid system by CB1 antagonism with rimonabant or AM281 exerts the activation of the mtor pathway and the increase of the anorexigenic signal Nucb2/nesfatin-1 that induces satiation. To further confirm our results and to more completely explore the mechanism mediating the effects of cannabinoid on Nucb2/nesfatin-1 production and release, we investigated whether injection of rimonabant would be effective in animals with mtor/ S6K1 pathway inactivation. In agreement with our hypothesis, peripheral injection of rimonabant did not affect gastric Nucb2/nesfatin-1 production or circulating levels in rats with an inactivated mtor/ S6K1 pathway by rapamycin treatment. In summary, the main findings of this present research are as follows: (1) peripheral CB1 receptors modulate food intake and the data might suggest that these regulation is drive through a mechanism that implies gastric Nucb2/nesfatin-1 production; (2) gastric CB1 receptors modulate Nucb2/nesfatin-1 production in the stomach at the intracellular level mediated by the mtor pathway; and (3) taking into account previous data with reference to cannabinoid regulation of ghrelin [14] and the present, we can conclude that Nucb2/nesfatin-1 and ghrelin are regulated at the gastric level by the cannabinoid system 6409 September 21, 2017 Volume 23 Issue 35

54 Folgueira C et al. Pharmacological inhibition of CB1 stimulates gastric release Food intake CB1 Rimonabant AM281 Ghrelin NUCB2/ nesfatin-1 Ghrelin MTOR/ S6K1 Rapamicin NUCB2/ nesfatin-1 Figure 5 Graphical abstract describing the pathways of ghrelin and nucb2/nefastatin-1 and association with cannabinoid system and mtor pathway regulating food intake. in a diametrically oppositional manner to regulate the food intake and maintain energy balance (Figure 5). Altogether, the data in the present paper show for the first time that the cannabinoid system, and more precisely CB1, regulates the nutrient sensor mtor, consequently controlling the production of the anorexigenic hormone Nucb2/Nesfatin-1. Moreover, this newly described mechanism also provides clues to the balance of anorexigenic and orexigenic signals in the stomach that maintain energy homeostasis. Further work should focus on testing whether this gastric mechanism is affected under pathological conditions such as obesity or anorexia, which lead to an unhealthy body weight. Moreover, the possibility of modulating a peripheral target, such as Nucb2/ Nesfatin-1, may represent a novel pharmacological therapy for obesity, thereby potentially avoiding the non-desirable off-target effects of many drugs. COMMENTS Background Nowadays the more effective treatment against obesity is gastric surgery which suggests that gastrointestinal tract signals are crucial factors in the regulation of energy homeostasis. Although several works in the last years has been focused in the role of different gastric signals with a relevant role in body weight regulation the full mechanisms governing this actions has not been elucidated. Research frontiers The discovery of new mechanisms at gastric level represents the possibility of new therapeutic targets to treat obesity avoiding the collateral effects at central level that suppose a serious problem in the pharmacological design of drugs. Innovations and breakthroughs The gastric mechanism described in the present regulating the production by the stomach of one of the newest gastrointestinal hormone related with a decreasing effect in appetite was has been previously reported with an inhibitory effect in the release of the only known gastric hormone with orexigenic effects. This data highlight the existence of a gastric mechanism controlling appetite through the differential regulation of anorexigenic and orexigenic gastric signals. Peer-review This is an interesting manuscript on the regulation of food intake through a mechanism that implies gastric production and release of Nucb2/Nesfatin-1. REFERENCES 1 Oh-I S, Shimizu H, Satoh T, Okada S, Adachi S, Inoue K, Eguchi H, Yamamoto M, Imaki T, Hashimoto K, Tsuchiya T, Monden T, Horiguchi K, Yamada M, Mori M. Identification of nesfatin-1 as a satiety molecule in the hypothalamus. Nature 2006; 443: [PMID: DOI: /nature05162] 2 Brailoiu GC, Dun SL, Brailoiu E, Inan S, Yang J, Chang JK, Dun NJ. Nesfatin-1: distribution and interaction with a G proteincoupled receptor in the rat brain. Endocrinology 2007; 148: [PMID: DOI: /en ] 3 García-Galiano D, Pineda R, Ilhan T, Castellano JM, Ruiz-Pino F, Sánchez-Garrido MA, Vazquez MJ, Sangiao-Alvarellos S, Romero-Ruiz A, Pinilla L, Diéguez C, Gaytán F, Tena-Sempere M. Cellular distribution, regulated expression, and functional role of the anorexigenic peptide, NUCB2/nesfatin-1, in the testis. Endocrinology 2012; 153: [PMID: DOI: /en ] 4 Stengel A, Goebel M, Yakubov I, Wang L, Witcher D, Coskun T, Taché Y, Sachs G, Lambrecht NW. Identification and characterization of nesfatin-1 immunoreactivity in endocrine cell types of the rat gastric oxyntic mucosa. Endocrinology 2009; 150: [PMID: DOI: /en ] 6410 September 21, 2017 Volume 23 Issue 35

55 Folgueira C et al. Pharmacological inhibition of CB1 stimulates gastric release 5 Kim J, Chung Y, Kim H, Im E, Lee H, Yang H. The Tissue Distribution of Nesfatin-1/NUCB2 in Mouse. Dev Reprod 2014; 18: [PMID: ] 6 Dornonville de la Cour C, Björkqvist M, Sandvik AK, Bakke I, Zhao CM, Chen D, Håkanson R. A-like cells in the rat stomach contain ghrelin and do not operate under gastrin control. Regul Pept 2001; 99: [PMID: DOI: / S (01) ] 7 Tschöp M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 2000; 407: [PMID: DOI: / ] 8 Shimizu H, Oh-I S, Hashimoto K, Nakata M, Yamamoto S, Yoshida N, Eguchi H, Kato I, Inoue K, Satoh T, Okada S, Yamada M, Yada T, Mori M. Peripheral administration of nesfatin-1 reduces food intake in mice: the leptin-independent mechanism. Endocrinology 2009; 150: [PMID: DOI: /en ] 9 Nakazato M, Murakami N, Date Y, Kojima M, Matsuo H, Kangawa K, Matsukura S. A role for ghrelin in the central regulation of feeding. Nature 2001; 409: [PMID: DOI: / ] 10 Al Massadi O, López M, Tschöp M, Diéguez C, Nogueiras R. Current Understanding of the Hypothalamic Ghrelin Pathways Inducing Appetite and Adiposity. Trends Neurosci 2017; 40: [PMID: DOI: /j.tins ] 11 Seoane LM, Al-Massadi O, Caminos JE, Tovar SA, Dieguez C, Casanueva FF. Sensory stimuli directly acting at the central nervous system regulate gastric ghrelin secretion. an ex vivo organ culture study. Endocrinology 2007; 148: [PMID: DOI: /en ] 12 Li Z, Xu G, Li Y, Zhao J, Mulholland MW, Zhang W. mtordependent modulation of gastric nesfatin-1/nucb2. Cell Physiol Biochem 2012; 29: [PMID: DOI: / ] 13 Xu G, Li Y, An W, Li S, Guan Y, Wang N, Tang C, Wang X, Zhu Y, Li X, Mulholland MW, Zhang W. Gastric mammalian target of rapamycin signaling regulates ghrelin production and food intake. Endocrinology 2009; 150: [PMID: DOI: /en ] 14 Senin LL, Al-Massadi O, Folgueira C, Castelao C, Pardo M, Barja-Fernandez S, Roca-Rivada A, Amil M, Crujeiras AB, Garcia-Caballero T, Gabellieri E, Leis R, Dieguez C, Pagotto U, Casanueva FF, Seoane LM. The gastric CB1 receptor modulates ghrelin production through the mtor pathway to regulate food intake. PLoS One 2013; 8: e80339 [PMID: DOI: /journal.pone ] P- Reviewer: Desiderio J, Schuller HM, Temraz S, Ulasoglu C S- Editor: Qi Y L- Editor: A E- Editor: Zhang FF 6411 September 21, 2017 Volume 23 Issue 35

56 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) ORIGINAL ARTICLE Basic Study Effects of aspirin and enoxaparin in a rat model of liver fibrosis Chen-Jie Li, Zhi-Hui Yang, Xiao-Liu Shi, De-Liang Liu Chen-Jie Li, Xiao-Liu Shi, De-Liang Liu, Department of Gastroenterology, Second Xiangya Hospital of Central South University, Changsha , Hunan Province, China Zhi-Hui Yang, Department of Medicine, Hospital of National University of Defense Technology, Changsha , Hunan Province, China Author contributions: All listed authors participated meaningfully in the study and have seen and approved the final manuscript; Li CJ and Shi XL designed the study; Li CJ and Yang ZH performed the animal experiments; Li CJ and Liu DL analyzed the data; Li CJ, Yang ZH, Shi XL and Liu DL wrote and revised the paper. Institutional review board statement: The study was reviewed and approved by the Institutional Review Board of the Second Xiangya Hospital of Central South University. Institutional animal care and use committee statement: All procedures involving animals were reviewed and approved by the Institutional Animal Care and Use Committee of the East China Normal University. Conflict-of-interest statement: To the best of our knowledge, no conflict of interest exists. Data sharing statement: No additional data are available. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Unsolicited manuscript Correspondence to: Chen-Jie Li, MD, PhD, Department of Gastroenterology, the Second Xiangya Hospital of Central South University, Changsha , Hunan Province, China. chenjielimao@126.com Telephone: Received: February 22, 2017 Peer-review started: February 24, 2017 First decision: June 5, 2017 Revised: June 21, 2017 Accepted: August 15, 2017 Article in press: August 15, 2017 Published online: September 21, 2017 Abstract AIM To examine the effects of aspirin and enoxaparin on liver function, coagulation index and histopathology in a rat model of liver fibrosis. METHODS Forty-five male Sprague-Dawley rats were randomly divided into the control group (n = 5) and model group (n = 40). Thioacetamide (TAA) was used to induce liver fibrosis in the model group. TAA-induced fibrotic rats received TAA continuously (n = 9), TAA + low-dose aspirin (n = 9), TAA + high-dose aspirin (n = 9) or TAA + enoxaparin (n = 9) for 4 wk. All rats were euthanized after 4 wk, and both hematoxylin-eosin and Masson staining were performed to observe pathological changes in liver tissue. RESULTS Liver fibrosis was assessed according to the METAVIR score. Compared with untreated cirrhotic controls, a significant improvement in fibrosis grade was observed in the low-dose aspirin, high-dose aspirin and enoxaparin treated groups, especially in the high-dose aspirin treated group. Alanine aminotransferase and total bilirubin were higher, albumin was lower and both prothrombin time and international normalized ratio 6412 September 21, 2017 Volume 23 Issue 35

57 Li CJ et al. Aspirin and enoxaparin for liver fibrosis were prolonged in the four treatment groups compared to controls. No significant differences among the four groups were observed. CONCLUSION Aspirin and enoxaparin can alleviate liver fibrosis in this rat model. Key words: Aspirin; Enoxaparin; Thioacetamide; Liver fibrosis; Rat The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: Several lines of evidence show that intrahepatic thrombosis is associated with liver fibrosis. Based on these observations, we hypothesized that routine use of antithrombotic drugs may prevent liver fibrosis. In our study, we successfully established a rat model of liver fibrosis after 8 wk and successfully established a rat model of liver cirrhosis after 12 wk through the thioacetamide-induced method. Our study clearly shows that aspirin and enoxaparin can reduce liver fibrosis in rats. However, reporting the results in humans is more complicated because other aspects, such as renal function, need to be considered. Li CJ, Yang ZH, Shi XL, Liu DL. Effects of aspirin and enoxaparin in a rat model of liver fibrosis. World J Gastroenterol 2017; 23(35): Available from: URL: wjgnet.com/ /full/v23/i35/6412.htm DOI: dx.doi.org/ /wjg.v23.i INTRODUCTION Liver fibrosis is a common liver disease posing a heavy burden on human health, and is a common cause of death. The mortality rate due to end-stage liver disease caused by fibrosis and subsequent severe liver cirrhosis is 20%-50%. In recent years, liver fibrosis has become a research hotspot, but its occurrence and development have not been fully elucidated. Liver fibrosis is closely related to the chronic inflammatory response of the liver, and inflammation leading to liver fibrosis has been studied in detail. In some cases, other pathways may also contribute to liver fibrosis in addition to inflammation. There is evidence showing that intra-hepatic thrombosis is associated with liver fibrosis. Wanless et al [1] found that microthrombosis in the intrahepatic portal vein and hepatic venous system was associated with hepatic atrophy and fibrosis in transplanted livers. Papatheodoridis et al [2] showed that, in patients with chronic viral hepatitis, risk factors for thrombosis were strongly associated with liver fibrosis and cirrhosis. Thrombin, a serine protease activated by proteolytic cleavage of prothrombin, can directly activate stellate cells through protease-activated receptors. Many antithrombotic therapies, such as standard heparin, low molecular weight heparin, vitamin K antagonists and antiplatelet agents, can reduce the proliferation and activation of stellate cells and reduce the fibrotic regions in various animal models with liver fibrosis. Based on these observations, we hypothesized that routine use of antithrombotic drugs may prevent liver fibrosis in humans. To test this hypothesis, we used thioacetamide (TAA) to establish a rat model of liver fibrosis and examined the effect of antithrombotic therapy with aspirin and enoxaparin in this model. MATERIALS AND METHODS Animals Forty-five healthy and pure Sprague-Dawley (SD) male rats weighing g were included in this study and were housed in a specific pathogenfree environment. The ambient temperature was maintained at and humidity was maintained at 50%-70%. The animals were acclimated to their new environment for 2 wk prior to experimentation. The rats were treated in accordance with ethical requirements for laboratory animal care, and were weighed weekly to adjust the dose of TAA and drugs. Drugs and reagents TAA was purchased from Sigma (St Louis, MO, United States). Aspirin enteric-coated tablets were produced by Bayer Healthcare Limited Corporation and enoxaparin was produced by Sanofi-Aventis (Beijing, China) Pharmaceutical Limited Corporation. Grouping and modeling Preparation period: SD rats were randomly divided into two groups: the control group (Group A, n = 5) and the model group (n = 40). Hepatic fibrosis was induced by intraperitoneal injection of TAA at a dosage of 200 mg/kg body weight, given twice weekly for 8 wk. The control group received an intraperitoneal injection of saline. After 8 wk, four rats in the model group were sacrificed at random and liver tissue was obtained to verify the model. Treatment period: The rats in the model group were subdivided into four groups: TAA group (Group B), TAA + low-dose aspirin group (Group C), TAA + high-dose aspirin group (Group D) and TAA + enoxaparin group (Group E), with 9 rats in each group. One milliliter of physiological saline was administered by gavage in Group A and B, 1 ml of aspirin (30 mg/kg) was administered by gavage in Group C, aspirin (300 mg/ kg) was administered by gavage in Group D, and 400 L of enoxaparin (2 mg/kg) was injected subcutaneously in Group E. The frequency of administration in each group was once a day over a period of 4 wk. During establishment of the model and the treatment 6413 September 21, 2017 Volume 23 Issue 35

58 Li CJ et al. Aspirin and enoxaparin for liver fibrosis period, the weight of the rats was measured twice a week and drug dosage was adjusted according to body weight. The dosage selected for each intervention was based on the results of previous studies using these agents in various fibrotic diseases [3,4]. To prevent spontaneous degeneration of liver cirrhosis, TAA (200 mg/kg) injections were continued twice a week during the treatment phase of the study in Groups B-E. General observation The main indices observed were activity, death and weight. Determination of liver biochemistry Serum and plasma were collected before the experiment, and at wk 8, 10 and 12 after treatment. Serum alanine aminotransferase (ALT), albumin (ALB), total bilirubin (TBIL) and plasma prothrombin time (PT) as well as international normalized ratio (INR) were measured. Determination of liver histopathology Liver histopathology was determined by nakedeye observation and photography and by staining, microscopic observation and photography. HE staining: Histopathological changes in the liver were observed, including necrosis of hepatocytes, the degree of fibrosis, the presence of regenerative nodules or pseudo-lobules, particularly the presence of thrombosis in sinusoidal or intrahepatic vessels, and the presence of luminal stenosis. Masson staining: Liver collagen fibers were stained using Masson stain. The degree of fibrotic hyperplasia in sections was classified using the METAVIR liver fibrosis scoring system [5], which was blindly applied to each liver biopsy sample. Statistical analysis All data were statistically analyzed using SPSS 18.0 statistical software. The data were expressed as median, and the overall variance analysis was carried out using nonparametric Kruskal-Wallis H and Mann- Whitney U tests, as the data obtained were not consistent with normal distribution and variance was not homogeneous. P-values of < 0.05 were considered statistically significant. RESULTS General observations of rat status At wk 12, healthy control rats had shiny hair and normal activities, with no mortality. The TAA group had disheveled hair and decreased activities; one rat died at wk 9 and another died at week 11, giving a mortality rate of 22.2%. Activity in the TAA + lowdose aspirin group, TAA + high-dose aspirin group and TAA + enoxaparin group decreased compared with the healthy control group, but was greater than that in the TAA group. The mortality rate in the TAA + low-dose aspirin group and TAA + high-dose aspirin group was 11.1%, with one death at wk 11 in each group. No deaths occurred in the TAA + enoxaparin group. Compared with the healthy control group, body weight in rats in the model groups was lower at wk 8, 10 and 12 (P < 0.05; Table 1), but there was no statistically significant difference between them. Biochemical changes in rats Biochemical changes in the rats are shown in Table 2. At wk 8, ALT and TBIL were higher in the model group than in the control group (P < 0.05). ALB decreased, and both PT and INR were prolonged, but were not statistically significant. Along with the formation of liver fibrosis, liver function became abnormal. At wk 8, 10 and 12, ALT and TBIL increased, ALB decreased, and both PT and INR were prolonged in the four model groups (P < 0.05). However, there were no significant differences between the groups. General observations of rat liver At wk 12, livers in the healthy control group were bright red, shiny and soft, with a sharp edge (Figure 1A). Livers in the TAA group were dark, dull and rough, and showed granular nodules, hard texture and a blunt edge (Figure 1B). Livers in the TAA + low-dose aspirin group (Figure 1C), TAA + high-dose aspirin group (Figure 1D) and TAA + enoxaparin group (Figure 1E) were slightly darker than in the healthy control group, but slightly softer than in the TAA group, and the granular nodules on the surface were not obvious compared to the TAA group. In particular, livers were improved in the TAA + high-dose aspirin group and TAA + enoxaparin group. TAA effectively induced liver fibrosis/cirrhosis in this rat model, while aspirin and enoxaparin reduced the degree of liver fibrosis/ cirrhosis. Rat liver histopathology using HE staining and Masson staining At wk 8, conventional HE staining of liver sections from the model group (Figure 2A) showed that hepatocytes around the central vein in lobules were disordered and both steatosis and fat vacuoles were visible in the cytoplasm. By Masson staining, collagen fibers around the central vein and portal area demonstrated significant proliferation, had a cord-like extension to the liver lobule and did not form pseudo-lobules. The rat model of liver fibrosis was successfully established. At wk 12, HE staining of liver sections from the healthy control group (Figure 2B) showed normal hepatocyte morphology, complete structure of liver lobules and a neat radiation-like arrangement of liver tissue around the central vein. Masson staining showed no obvious collagen fibrosis. HE staining of 6414 September 21, 2017 Volume 23 Issue 35

59 Li CJ et al. Aspirin and enoxaparin for liver fibrosis A B C D E Figure 1 General observations of the rat model of liver fibrosis/cirrhosis. A: Healthy control group; B: TAA group; C: TAA + low-dose aspirin group; D: TAA + high-dose aspirin group; and E: TAA + enoxaparin group. TAA: Thioacetamide. liver sections from the TAA group (Figure 2C) showed damaged liver lobule structure and abnormal structure of pseudo-lobules due to regeneration of hepatocytes. Masson staining revealed significant proliferation of collagen tissue and the formation of pseudolobules. HE staining of liver sections in the remaining 6415 September 21, 2017 Volume 23 Issue 35

60 Li CJ et al. Aspirin and enoxaparin for liver fibrosis Table 1 Body weight in each group at wk 8, 10 and 12 Group Body weighting 8 wk 10 wk 12 wk Healthy control group, A TAA group, B TAA + low-dose aspirin group, C TAA + high-dose aspirin group, D TAA + enoxaparin group, E TAA: Thioacetamide. Table 3 Staging and scoring of liver fibrosis in each group at wk 12 Group n Liver fibrosis stage Liver fibrosis score F0 F1 F2 F3 F4 Healthy control group, A TAA group, B b 4 TAA + low-dose b,c,e 2 aspirin group, C TAA + high-dose b,c 1 aspirin group, D TAA + enoxaparin group, E b,c,e 2 b P < 0.01 vs the healthy control group; c P < 0.05 vs the TAA group; e P < 0.05 Table 2 Changes in alanine aminotransferase, albumin, total bilirubin, prothrombin time and International normalized ratio in each group Group Wk ALT ALB TBIL PT INR Healthy control group, A groups (Figure 2D-F) showed disordered hepatocyte arrangement, but no obvious formation of pseudolobules. Masson staining exhibited different degrees of collagen fiber proliferation. The staging and scoring of liver fibrosis in each group at wk 12 are shown in Table 3. The difference in fibrosis grading between the healthy control group and the other model groups was statistically significant (P < 0.01), indicating that the model was successfully established. The difference between the TAA group and the other drug-intervention groups was statistically significant (P < 0.05), indicating that the drug intervention was effective. DISCUSSION TAA group, B TAA + low-dose aspirin group, C TAA + high-dose aspirin group, D TAA + enoxaparin group, E ALB: Albumin; ALT: Alanine aminotransferase; INR: International normalized ratio; PT: Prothrombin time; TBIL: Total bilirubin; TAA: Thioacetamide. Liver fibrosis/cirrhosis is a common outcome of chronic liver disease due to various etiologies, with high morbidity and mortality. Treatment of advanced liver fibrosis/cirrhosis is very unsatisfactory and is currently limited to the treatment of its underlying etiologies and remission of its complications. Therefore, there is an urgent need to develop new antifibrotic treatments that can prevent, halt or even reverse liver fibrosis. Recent, vs the TAA + high-dose aspirin group. TAA: Thioacetamide. evidence has shown that intrahepatic thrombosis is associated with liver fibrosis. Mancuso [6] proposed that chronic microvascular ischemia of the liver may be a milestone in the development of liver cirrhosis. Chronic liver damage can eventually lead to endothelial injury and microvascular thrombosis, triggering inappropriate hepatocyte proliferation and fibrosis. In order to validate this theory, we established a rat model of liver fibrosis induced by TAA, determined the presence or absence of thrombosis in rat fibrotic livers, and examined whether antithrombotic therapy with aspirin and enoxaparin could reverse liver fibrosis. The establishment of this animal model of liver fibrosis is of great significance in understanding the pathogenesis and treatment strategy of liver fibrosis and cirrhosis. To date, animal models have been developed using mice, rats, rabbits and pigs to mimic the complex processes of liver fibrosis and cirrhosis. Liver fibrosis induced by TAA is a widely used model and TAA can be administered by oral intake or peritoneal injection; however, peritoneal injection provides more consistent results. TAA-induced liver fibrosis is very similar to human liver fibrosis in terms of changes in hemodynamics, morphological and biochemical metabolism changes [7]. TAA damages DNA, RNA and protein synthetase in hepatocytes, leading to disorders of metabolism and necrosis of hepatocytes. An obvious feature of this model relative to the carbon tetrachloride model is that its fibrosis remains stable for several weeks after TAA withdrawal. However, TAA is a weak carcinogen and is both toxic and volatile. Large nodular cirrhosis and portal hypertension developed in the present model at 12 wk. A significantly highly powered cycle may be required to induce fibrosis for longer periods. Approximately 40% of rats develop ascites. After 18 wk of TAA administration, rats may develop cholangiocarcinoma. In the present study, the rat liver fibrosis model was successfully induced by TAA in the 8th week. ALT and TBIL in the model group were significantly higher than those in the control group (P < 0.05). ALB 6416 September 21, 2017 Volume 23 Issue 35

61 Li CJ et al. Aspirin and enoxaparin for liver fibrosis A B C D E F Figure 2 Liver histopathology of rat liver fibrosis/cirrhosis. A: Liver histopathology in the model group at wk 8; B: Liver histopathology in the healthy control group at wk 12; C: Liver histopathology in the TAA group at wk 12; D: Liver histopathology in the TAA + low-dose aspirin group at wk 12; E: Liver histopathology in the TAA + high-dose aspirin group at wk 12; and F: Liver histopathology in the TAA + enoxaparin group at wk 12. Left: HE 100; Right: Masson 100. TAA: Thioacetamide. decreased, and both PT and INR were prolonged, but prolongation was not statistically significant. These findings indicated that following the formation of liver fibrosis, liver function became abnormal. At the 12th week, the rat model of liver cirrhosis was successfully established. In 1995, Wanless et al [8] showed that both congestion and non-congestion in liver cirrhosis were present in the hepatic circulation during microthrombosis. By studying liver biopsy specimens from patients with chronic viral hepatitis, Papatheodoridis et al [2] in 2003 found that thrombosis-related factors were closely related to hepatitis staging and the degree of liver fibrosis, and antithrombosis drugs seemed to reverse liver fibrosis. It was suggested that microthrombosis may be involved in the formation of liver fibrosis in chronic liver disease, rather than the result of cirrhosis. In the present study, both HE staining and Masson staining showed that liver fibrosis in model rats was significant, and progressed to liver cirrhosis at 12 wk. However, no apparent microthrombosis was found, probably because HE staining and Masson staining were not sensitive enough to detect microthrombi. It is necessary to identify a more sensitive experimental method to observe microthrombosis in the liver. Aspirin is regarded as an agent for preventing arterial thrombosis, but can also reduce the risk of venous thromboembolism [9]. The mechanism underlying the antifibrotic properties of aspirin may be related to its antithrombotic properties. Sinusoidal endothelial cell injury can also lead to increased platelet adhesion and a coagulation state. Indeed, such mechanisms have been implicated in injury repair and hepatic sinus obstruction syndrome after liver transplantation, and aspirin has been shown to reduce the risk of chemotherapy-associated sinusoidal lesions. Aspirin also exhibits anti-inflammatory activity and inhibits nuclear factor kappa B (NFκB) transcript adhesion molecules in endothelial cells and vascular smooth muscle cells in vitro, thereby affecting macrophage and T lymphocyte adherence to these cells. Aspirin also reduces the levels of interleukin-6, monocyte chemoattractant protein-1, transforming growth factor-beta (TGF-β), tumor necrosis factoralpha and platelet-derived growth factor. Chavez et al [10] showed that in the liver fibrosis model induced by 6417 September 21, 2017 Volume 23 Issue 35

62 Li CJ et al. Aspirin and enoxaparin for liver fibrosis carbon tetrachloride, aspirin partially inhibited the expression of TGF-β and prevented translocation of NFκB into the nucleus. The anti-inflammatory effects of aspirin are achieved by endogenous anti-inflammatory lipids known as aspirin-triggering lipoxins (ATLs). ATLs are derived from arachidonic acid (converted by COX-2 acetylation), and even low-dose aspirin can induce ATLs [11]. Interestingly, ATL analogues reduced the level of TGF-β and reversed the fibrosis process in a mouse model of pulmonary fibrosis induced by bleomycin [12]. Thus, the effect of aspirin in reducing liver fibrosis is mediated by its antithrombotic and/or anti-inflammatory properties. Some studies have attempted to determine whether aspirin or other antiplatelet drugs play a role in the progression of liver fibrosis. In a rat model of nonalcoholic fatty liver disease, Fujita et al [13] reported that antiplatelet drugs, such as aspirin, ticlopidine and cilostazol, significantly reduced liver fibrosis. Wanless et al [14] also proved that in a rabbit model of liver sinus fibrosis induced by cholesterol and diethylstilbestrol, dipyridamole inhibited platelet aggregation and reduced fibrosis in vitro. Yoshida et al [15] recently reported that platelet activation directly promoted liver fibrosis, and aspirin prevented the progression of liver fibrosis in animal models. In addition to animal studies, Poujol-Robert et al [16] reported that daily low-dose aspirin administration reduced the progression of liver fibrosis in patients with recurrent hepatitis C after liver transplantation. Our study found that aspirin reduced liver fibrosis in rats and that high-dose aspirin (300 mg/kg) was superior to low-dose aspirin (30 mg/kg) in terms of antifibrotic effects. Current experimental evidence suggests that coagulation factors, particularly thrombin, are closely related to liver fibrosis. Thrombin, the major effector of the coagulation cascade, causes a series of cellular effects through a family of G protein-coupled receptors known as protease-activated receptors. Through these receptors, thrombin stimulates the proliferation of hepatic stellate cells, which are important mediators of liver fibrogenesis. Thrombin receptors are expressed in the liver, and during the process of acute and chronic liver injury, the expression of thrombin receptors is up-regulated. Heparin, through its interaction with antithrombin, induces a conformational change in the antithrombin molecule, which greatly facilitates the interaction between antithrombin and targets of serine proteases such as thrombin, as well as the factors Xa, IXa, XIa and XIIa [17]. Low molecular weight heparin induces inhibition of the coagulation cascade, and its potential beneficial effects in liver fibrosis have been evaluated [18]. In our study, enoxaparin was shown to reduce liver fibrosis in rats. In conclusion, we successfully established a rat model of liver fibrosis after 8 wk and successfully established a rat model of liver cirrhosis after 12 wk induced by TAA. Our study clearly shows that aspirin and enoxaparin can reduce liver fibrosis in rats. However, it is unclear whether the antifibrotic mechanisms of the two drugs are directly related to their anticoagulant effects in preventing thrombosis, or to other mechanisms, Further investigations are needed using anticoagulants as potential treatment for liver fibrosis/cirrhosis. ACKNOWLEDGMENTS The authors would like to thank the members of the East China Normal University for their technical support. COMMENTS Background Liver fibrosis is a hotspot of research. It is closely related to the chronic inflammatory response of the liver, and inflammation leading to liver fibrosis has been studied in detail. At present, several lines of evidence show that intrahepatic thrombosis is associated with liver fibrosis. A variety of chronic liver damage can eventually lead to endothelial injury and microvascular thrombosis, triggering inappropriate hepatocyte proliferation and fibrosis. Based on these observations, they hypothesized that routine use of antithrombotic drugs may prevent liver fibrosis. Research frontiers In the past, there was widespread belief that patients with liver cirrhosis experienced hypocoagulability. In recent years, however, this belief has been challenged. Studies found that patients with cirrhosis had hypercoagulability and even had a thrombus in some cases. There is evidence that coagulation is an important factor in the progression of cirrhosis. It is important to discuss the relationship among coagulation, anticoagulant therapy and liver fibrosis/ cirrhosis. Innovations and breakthroughs Reports of studies on antithrombotic therapies and antiplatelet agents reducing liver fibrosis in animals are rare. In this study, the authors evaluated if aspirin and enoxaparin are beneficial in a thioacetamide-induced rat model of liver fibrosis. Applications Aspirin and enoxaparin can reduce liver fibrosis in a thioacetamide-induced rat model. They are beneficial in humans with liver fibrosis as well. Terminology Liver fibrosis and subsequent liver cirrhosis are common outcomes of chronic liver disease caused by various etiologies, with high morbidity and mortality. Peer-review The manuscript is very interesting, describing a study using a rat model, but reporting the results in humans is more complicated because we should consider other aspects, such as renal function. The manuscript is well written. REFERENCES 1 Wanless IR, Wong F, Blendis LM, Greig P, Heathcote EJ, Levy G. Hepatic and portal vein thrombosis in cirrhosis: possible role in development of parenchymal extinction and portal hypertension. Hepatology 1995; 21: [PMID: ] 2 Papatheodoridis GV, Papakonstantinou E, Andrioti E, Cholongitas E, Petraki K, Kontopoulou I, Hadziyannis SJ. Thrombotic risk factors and extent of liver fibrosis in chronic viral 6418 September 21, 2017 Volume 23 Issue 35

63 Li CJ et al. Aspirin and enoxaparin for liver fibrosis hepatitis. Gut 2003; 52: [PMID: DOI: / gut ] 3 Denda A, Tang Q, Endoh T, Tsujiuchi T, Horiguchi K, Noguchi O, Mizumoto Y, Nakae D, Konishi Y. Prevention by acetylsalicylic acid of liver cirrhosis and carcinogenesis as well as generations of 8-hydroxydeoxyguanosine and thiobarbituric acid-reactive substances caused by a choline-deficient, L-amino acid-defined diet in rats. Carcinogenesis 1994; 15: [PMID: DOI: /carcin/ ] 4 Frizelle S, Schwarz J, Huber SA, Leslie K. Evaluation of the effects of low molecular weight heparin on inflammation and collagen deposition in chronic coxsackievirus B3-induced myocarditis in A/J mice. Am J Pathol 1992; 141: [PMID: ] 5 Goodman ZD. Grading and staging systems for inflammation and fibrosis in chronic liver diseases. J Hepatol 2007; 47: [PMID: DOI: /j.jhep ] 6 Mancuso A. Cirrhosis development probably arises from chronic micro-vascular ischemia. Med Hypotheses 2014; 82: 244 [PMID: DOI: /j.mehy ] 7 Yang MC, Chang CP, Lei HY. Induction of liver fibrosis in a murine hepatoma model by thioacetamide is associated with enhanced tumor growth and suppressed antitumor immunity. Lab Invest 2010; 90: [PMID: DOI: / labinvest ] 8 Wanless IR, Liu JJ, Butany J. Role of thrombosis in the pathogenesis of congestive hepatic fibrosis (cardiac cirrhosis). Hepatology 1995; 21: [PMID: ] 9 Becattini C, Agnelli G, Schenone A, Eichinger S, Bucherini E, Silingardi M, Bianchi M, Moia M, Ageno W, Vandelli MR, Grandone E, Prandoni P; WARFASA Investigators. Aspirin for preventing the recurrence of venous thromboembolism. N Engl J Med 2012; 366: [PMID: DOI: / NEJMoa ] 10 Chávez E, Castro-Sánchez L, Shibayama M, Tsutsumi V, Pérez Salazar E, Moreno MG, Muriel P. Effects of acetyl salycilic acid and ibuprofen in chronic liver damage induced by CCl4. J Appl Toxicol 2012; 32: [PMID: DOI: /jat.1638] 11 Chiang N, Bermudez EA, Ridker PM, Hurwitz S, Serhan CN. Aspirin triggers antiinflammatory 15-epi-lipoxin A4 and inhibits thromboxane in a randomized human trial. Proc Natl Acad Sci USA 2004; 101: [PMID: DOI: / pnas ] 12 Martins V, Valença SS, Farias-Filho FA, Molinaro R, Simões RL, Ferreira TP, e Silva PM, Hogaboam CM, Kunkel SL, Fierro IM, Canetti C, Benjamim CF. ATLa, an aspirin-triggered lipoxin A4 synthetic analog, prevents the inflammatory and fibrotic effects of bleomycin-induced pulmonary fibrosis. J Immunol 2009; 182: [PMID: DOI: /jimmunol ] 13 Fujita K, Nozaki Y, Wada K, Yoneda M, Endo H, Takahashi H, Iwasaki T, Inamori M, Abe Y, Kobayashi N, Kirikoshi H, Kubota K, Saito S, Nagashima Y, Nakajima A. Effectiveness of antiplatelet drugs against experimental non-alcoholic fatty liver disease. Gut 2008; 57: [PMID: DOI: / gut ] 14 Wanless IR, Belgiorno J, Huet PM. Hepatic sinusoidal fibrosis induced by cholesterol and stilbestrol in the rabbit: 1. Morphology and inhibition of fibrogenesis by dipyridamole. Hepatology 1996; 24: [PMID: DOI: /hep ] 15 Yoshida S, Ikenaga N, Liu SB, Peng ZW, Chung J, Sverdlov DY, Miyamoto M, Kim YO, Ogawa S, Arch RH, Schuppan D, Popov Y. Extrahepatic platelet-derived growth factor-β, delivered by platelets, promotes activation of hepatic stellate cells and biliary fibrosis in mice. Gastroenterology 2014; 147: [PMID: DOI: /j.gastro ] 16 Poujol-Robert A, Boëlle PY, Conti F, Durand F, Duvoux C, Wendum D, Paradis V, Mackiewicz V, Chazouillères O, Corpechot C, Poupon R. Aspirin may reduce liver fibrosis progression: Evidence from a multicenter retrospective study of recurrent hepatitis C after liver transplantation. Clin Res Hepatol Gastroenterol 2014; 38: [PMID: DOI: / j.clinre ] 17 Laux V, Perzborn E, Heitmeier S, von Degenfeld G, Dittrich- Wengenroth E, Buchmüller A, Gerdes C, Misselwitz F. Direct inhibitors of coagulation proteins-the end of the heparin and lowmolecular-weight heparin era for anticoagulant therapy? Thromb Haemost 2009; 102: [PMID: DOI: / TH ] 18 Abdel-Salam OM, Baiuomy AR, Ameen A, Hassan NS. A study of unfractionated and low molecular weight heparins in a model of cholestatic liver injury in the rat. Pharmacol Res 2005; 51: [PMID: DOI: /j.phrs ] P- Reviewer: Lai S S-Editor S- Editor: Gong ZM L- Editor: Filipodia E- Editor: Ma YJ 6419 September 21, 2017 Volume 23 Issue 35

64 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) ORIGINAL ARTICLE Retrospective Study Different clinical presentations of metachronous pulmonary metastases after resection of pancreatic ductal adenocarcinoma: Retrospective study and review of the literature Martin Lovecek, Pavel Skalicky, Josef Chudacek, Marek Szkorupa, Hana Svebisova, Radmila Lemstrova, Jiri Ehrmann, Bohuslav Melichar, Tharani Yogeswara, Dusan Klos, Radek Vrba, Roman Havlik, Beatrice Mohelnikova-Duchonova Martin Lovecek, Pavel Skalicky, Josef Chudacek, Marek Szkorupa, Dusan Klos, Radek Vrba, Roman Havlik, Department of Surgery I, University Hospital Olomouc and Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic Hana Svebisova, Radmila Lemstrova, Department of Oncology, University Hospital Olomouc and Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic Jiri Ehrmann, Department of Molecular Pathology, Faculty of Medicine and Dentistry, Palacky University Olomouc, University Hospital Olomouc, Olomouc, Czech Republic Jiri Ehrmann, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic Bohuslav Melichar, Beatrice Mohelnikova-Duchonova, Department of Oncology, Faculty of Medicine and Dentistry, Palacky University Olomouc, University Hospital Olomouc, Olomouc, Czech Republic Bohuslav Melichar, Beatrice Mohelnikova-Duchonova, Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic Tharani Yogeswara, Department of Surgery I, Faculty of Medicine and Dentistry, Palacky University, Olomouc, Czech Republic Author contributions: Lovecek M and Mohelnikova-Duchonova B designed the research; Lovecek M, Skalicky P, Chudacek J, Szkorupa M, Svebisova H, Lemstrova R, Ehrmann J and Havlik R performed the research; Lovecek M, Klos D, Vrba R and Mohelnikova-Duchonova B analyzed the data; Lovecek M, Melichar B, Yogeswara T and Mohelnikova-Duchonova B wrote the paper; all authors critically revised the manuscript for important intellectual content. Supported by the Ministry of Health of the Czech Republic, no A to Mohelnikova-Duchonova B. Institutional review board statement: This study has been approved by the institutional Review Board at the University Hospital Olomouc. Conflict-of-interest statement: The authors declare that they have no conflict of interest. Data sharing statement: No additional data are available. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Unsolicited manuscript Correspondence to: Beatrice Mohelnikova-Duchonova, MD, PhD, associate professor, Department of Oncology, Faculty of Medicine and Dentistry, Palacky University Olomouc, University Hospital Olomouc, I.P. Pavlova 6, Olomouc, Czech Republic. d.beatrice@seznam.cz Telephone: Fax: Received: May 12, 2017 Peer-review started: May 16, September 21, 2017 Volume 23 Issue 35

65 Lovecek M et al. Metachronous pulmonary metastases of pancreatic cancer First decision: June 5, 2017 Revised: June 25, 2017 Accepted: August 1, 2017 Article in press: August 2, 2017 Published online: September 21, 2017 Abstract AIM To analyze pancreatic cancer patients who developed metachronous pulmonary metastases (MPM) as a first site of recurrence after the curative-intent surgery. METHODS One-hundred-fifty-nine consecutive pancreatic ductal adenocarcinoma (PDAC) patients who underwent radical pancreatic surgery between 2006 and 2013 were included in this retrospective analysis. The clinical data including age, sex, grade, primary tumor location, ptnm stage, lymph node infiltration, microangioinvasion, perineural invasion, lymphovascular invasion, the therapy administered, and follow-up were all obtained from medical records. Further analysis covered only patients with metachronous metastases. Clinical and histopathological data (age, sex, grade, primary tumor location, ptnm stage, lymph node infiltration, microangioinvasion, perineural invasion, lymphovascular invasion, the therapy administered and follow-up) of patients with metachronous non-pulmonary metastases and patients with metachronous pulmonary metastases were statistically assessed. disease-free survival (DFS) from pancreas resection until metastases onset and overall survival (OS) were calculated. Wilcoxon test, χ 2 test and survival functions computed by the Kaplan- Meier method were used. Statistical significance was evaluated by the log-rank test using SPSS. A P -value of less than 0.05 was considered statistically significant. RESULTS Metachronous pulmonary metastases were observed in 20 (16.9%) and were operable in 3 (2.5%) of PDAC patients after a prior curative-intent surgery. Patients with isolated pulmonary metastases (oligometastases and multiple metastases) had estimated prior DFS and OS of 35.4 and 81.4 mo, respectively, and those with metachronous pulmonary metastases accompanied by other metastases had prior DFS and OS of 17.3 and 23.4 mo, respectively. Patients with non-pulmonary metastases had prior DFS and OS of 9.4 and 15.8 mo, respectively. Different clinical scenarios according to the presentation of MPM were observed and patients could be divided to three subgroups with different prognosis which could be used for the selection of treatment strategy: isolated pulmonary oligometastases, isolated multiple pulmonary metastases and pulmonary metastases accompanied by other metastases. CONCLUSION Surgery should be considered for all patients with isolated pulmonary oligometastases, but the risk of intervention has to be individually weighted for each patient. Key words: pancreatic cancer; pulmonary metastases; metachronous metastases; surgical resection; overall survival; chemotherapy The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: Metachronous pulmonary metastases represent the most frequent extraabdominal site of recurrence following radical resection for pancreas cancer. Three different clinical scenarios according to the presentations of metachronous pulmonary metastases were observed in our cohort: isolated oligometastases, multiple pulmonary metastases and pulmonary metastases accompanied by other metastases. Patients with isolated pulmonary metastases have the longest disease-free survival and overall survival. Surgery should be considered for all patients with isolated oligometastases. Lovecek M, Skalicky P, Chudacek J, Szkorupa M, Svebisova H, Lemstrova R, Ehrmann J, Melichar B, Yogeswara T, Klos D, Vrba R, Havlik R, Mohelnikova-Duchonova B. Different clinical presentations of metachronous pulmonary metastases after resection of pancreatic ductal adenocarcinoma: Retrospective study and review of the literature. World J Gastroenterol 2017; 23(35): Available from: URL: com/ /full/v23/i35/6420.htm DOI: org/ /wjg.v23.i INTRODUCTION Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive malignant disease with an increasing incidence [1]. Even though there is a long latency period between the emergence of a malignant cell clone and the death from metastatic disease, the majority of patients still present with either locally advanced or metastatic disease [2]. Only 15%-20% of patients have localized disease which may be considered surgically resectable at the time of diagnosis. An additional 20%-30% of patients have border-line resectable or locally advanced disease among whom one to two thirds could be potential candidates for curative surgery following a neoadjuvant therapy [3,4]. Finally, around 50% of patients have a metastatic disease at the time of diagnosis. The prognosis of PDAC patients after curative surgery is still relatively poor with the 5-year survival rate ranging between 15%-35% [5-7]. Up to 30% of PDAC patients die within the first year after radical surgery due to the disease progression and the mortality rate increases to around 60% by the end of the second year [8,9]. Although the most frequent site of metastases 6421 September 21, 2017 Volume 23 Issue 35

66 Lovecek M et al. Metachronous pulmonary metastases of pancreatic cancer in PDAC is the liver [10], among long-term PDAC survivors the most common site of metastases is the lung [5]. Since the pulmonary metastases usually occur in the context of general disease progression, chemotherapy is considered to represent the standard treatment option. However, with regard of PDAC longterm survivors, there is a subgroup of patients with metachronous pulmonary metastases being the only site of the disease recurrence, presenting an option for surgical treatment. The literature concerning the use of surgical therapy in the treatment of metachronous isolated pulmonary PDAC metastases is still very limited. Isolated late pulmonary metastases in PDAC have been reported as single cases [11-14], case series [15], and retrospective cohort studies [7,16-21]. Katz et al [5] in a study focusing on long-term survivors reported the highest rate (22%) of pulmonary metastases. Most recently, some retrospective analyses tried to define criteria based on the report derived of Thomford et al [22], including a disease-free survival (DFS) of more than 20 mo after the primary surgery, pulmonary metastases 16 mm, primary stage Ⅰ, good performance status and a technically operable tumor, for patients in whom the surgical resection of isolated metastases would be meaningful [18,22,23]. However, due to the low prevalence of metachronous pulmonary metastases reliable data regarding the clinical outcome and indications for the optimal surgical or medical treatment are currently limited. The aim of the present study was to retrospectively analyze a homogeneous cohort of PDAC patients who underwent a surgery with curative intent between the years 2006 and 2013 in our center. The primary objective was the identification of patients with metachronous pulmonary metastases as the first site of a disease recurrence after surgery and the assessment of prior DFS, and an overall survival (OS) in this subgroup of PDAC patients compared to the patients with non-pulmonary metastases. Different clinical scenarios according to a type of pulmonary metastases could also be discerned. MATERIALS AND METHODS Patients A cohort of 159 consecutive PDAC patients who underwent radical surgery between the years 2006 and 2013 in our center was included in this retrospective analysis. Histopathological diagnosis of PDAC was performed according to the previous standard classification, and all specimens were evaluated before the introduction of the Leeds protocol. The clinical data including age, sex, grade, primary tumor location, ptnm stage, lymph node infiltration, microangioinvasion, perineural invasion, lymphovascular invasion, the therapy administered, and follow-up were all obtained from medical records or from the Czech National Oncological Registry. Disease free survival (DFS) was defined as the time between the date of surgery and the date of diagnosis of new disease recurrence. Criteria for new recurrence were applied according to previous studies [5,19]. The patients were followed in 3-mo intervals with clinical examination including CA 19-9 level assessment, and 6-mo intervals with crosssectioning computed tomography (CT), or positron emission tomography (PET/CT) in case elevation CA 19-9 was noted [24,25]. Oligometastatic disease was defined as limited systemic metastatic tumors in which local ablative therapy could be curative [26-30]. As the study was focused to metachronous metastatic PDAC, patients without recurrence, who died in postoperative period (90 d), had R2 resections and those with incomplete data were excluded from DFS analysis. The Thomford criteria for pulmonary metastasectomy including controlled primary lesion, no extrapulmonary metastases present, metastases limited to one lung, patient being able to tolerate surgery were applied [22]. Consensus from a multidisciplinary team had to be obtained before surgery. OS was defined as the time between the date of primary surgery and death. Surviving patients were censored at the last follow-up on 31 st December Further analysis covered only patients with metachronous metastases. Clinical and histopathological data (age, sex, grade, primary tumor location, ptnm stage, lymph node infiltration, microangioinvasion, perineural invasion, lymphovascular invasion, the therapy administered and follow-up) of patients with metachronous non-pulmonary metastases and patients with metachronous pulmonary metastases (MPM) were statistically assessed. Patients with MPM were initially divided into two groups; metachronous isolated pulmonary metastases, and metachronous pulmonary metastases with metastases to other organs. Statistical analysis Wilcoxon test and χ 2 test were used for analysis of age and other parameters. The survival functions were computed by the Kaplan-Meier method and statistical significance was evaluated by the log-rank test using SPSS. A P-value of less than 0.05 was considered statistically significant. This study has been approved by the institutional Review Board at the University Hospital Olomouc. RESULTS Study population The study population consisted of 159 patients with resectable PDAC, i.e., no evidence of clinically detectable metastases, cm0 (Supplementary Table 1). R0 and R1 resection rate was not formally assessed as the samples have been evaluated before the introduction of the Leeds protocol into clinical practice at our institution, and the results concerning 6422 September 21, 2017 Volume 23 Issue 35

67 Lovecek M et al. Metachronous pulmonary metastases of pancreatic cancer A B 1.0 MPM MNPM 1.0 MPM MNPM 0.8 Log-rank test P = Log-rank test P = Cumulative survival Cumulative survival DFS (mo) OS (mo) Figure 1 Prior disease-free survival (A) and overall survival (B) in patients with metachronous pulmonary metastases and metachronous non-pulmonary metastases. MPM: Metachronous pulmonary metastases; MNPM: Metachronous non-pulmonary metastases; DFS: Disease-free survival; OS: Overall survival. Table 1 Prior disease-free survival and overall survival in patients with metachronous metastases n DFS OS Non-pulmonary Metachronous pulmonary metastases Isolated pulmonary Pulmonary and other metastases Metastatic cohort Entire cohort DFS: Disease-free survival; OS: Overall survival. R0 resection could have been overestimated.two patients (1.3%) had neoadjuvant therapy consisting of concomitant radiotherapy with continuous 5-fluorouracil administration. Adjuvant chemotherapy consisting of nucleoside analogues (gemcitabine and 5-fluorouracil) was administered in 111 (69.8%) patients. Forty-one patients (25.8%) were excluded from further analysis due to no recurrence (n = 24), incomplete data (n = 14), postoperative death (n = 2) or R2 resection (n = 1). In total 1-, 2-, 3- and 5-years survival rates in entire cohort were 66.7%, 36.6%, 28.1% and 18.7%, respectively. The perineural invasion and lymph node infiltration were frequently identified (66.7% and 53.5%), while lymphovascular and microangioinvasion were identified less often (32.7% and 18.2%). The median DFS was 10 mo, and the median OS in entire cohort reached 17.6 mo. The most common sites of metastases within the first year included liver (57%), locoregional recurrence (37%), lymph nodes (22%), peritoneum (18%), and lung (10%), with multiple sites involved in 51%. In the second year, most frequent were locoregional (45%), lymph nodes (40%), liver (20%), and lung (20%), with involvement of multiple sites in 40%. In most patients who subsequently developed pulmonary metastases the primary procedures were head resections or total pancreatectomies with the predominant tumor location in pancreatic head (88.7%). Splenic vein obstructions were not identified. Further analysis was focused on patients who developed metachronous metastases. Data of patients with metachronous metastases included in the further analysis are summarized in Supplementary Table 2. Most patients with isolated pulmonary metastases were diagnosed using PET/CT scan triggered by CA 19-9 elevation during the follow up. In total 20 patients (17%) had metachronous pulmonary metastases, almost all diagnosed with PET/CT scans. Adjuvant chemotherapy after primary pancreatic resection was used in 85% of MPM patients compared to 74% of non-pulmonary metastases group. Perineural invasion was higher in MPM patients, but the difference was not statistically significant (p = 0.057). Statistically significant differences between patients with metachronous pulmonary and non-pulmonary metastases were evident in the sex (p = 0.012) and survival rate (p = 0.004). Figure 1 displays prior DFS and OS in both subgroups, with statistically significant differences (p = and 0.004, respectively). Metachronous pulmonary metastases patients In five patients (25%), all females, metastases were isolated to the lung. Pulmonary metastases along with multiple metastases to other organs were detected in 15 cases (75%). Figure 2 displays DFS and OS in both groups. Analysis is difficult due to very small numbers, but OS prolongation is of borderline statistical significance (p = 0.054). While the estimated median OS is similar in patients with non-pulmonary metastases and the entire cohort, the median OS is double in MPM (Table 1). In the subgroup of metachronous isolated metastases (n = 5) estimated medians of DFS and OS are 35.4 and 81.4 mo, respectively (Table 1). There were two patients with multiple bilateral lesions and three patients with isolated oligometastatic recurrence.the patients with multiple bilateral lesions were treated with systemic chemotherapy (5-fluorouracil combined 6423 September 21, 2017 Volume 23 Issue 35

68 Lovecek M et al. Metachronous pulmonary metastases of pancreatic cancer Table 2 Previous reports of metachronous pulmonary metastases Study IPM DFS MPMR SMT SMU OSPMD Van den Broeck et al [16] RA nr nr nr nr 1 Katz et al [5] nr nr nr nr nr nr 2 Arnaoutakis et al [17] RCCS nr 1 Thomas et al [18] RA nr nr nr 2 Wangjam et al [7] RA nr nr nr 8.5 Downs-Canner et al [20] RA nr Yamashita et al [19] RCCS nr 2 70 nr nr Kruger et al [21] RA nr nr nr 25.5 Decoster et al [36] RCCS nr 3 nr nr nr Deeb et al [15] Cserie 5 0 Brieau et al [14] CR 2 2 Kotoulas et al [12] CR 1 1 Moon et al [11] CR 1 1 Sanjeevi et al [13] CR 1 1 Kitasato et al [33] CR + national cases Nakajima et al [23] CR + national cases 17 3 Present study Cserie nr 12+ 1,2 Same institution; 3 Mostly same cases. IPM: Isolated pulmonary metastases; DFS: Median of disease free survival; MPMR: Resected metachronous pulmonary metastases; SMT: Median survival of MPM treated; SMU: Median survival of MPM untreated; OSPMD: OS after diagnose of MPM; RA: Retrospective analysis; RCCD: Retrospective case control study; Cserie: Case serie; CR: Case report. A B 1.0 MPM MIPM 1.0 MPM MIPM Cumulative survival Cumulative survival Log-rank test P = Log-rank test P = DFS (mo) OS (mo) Figure 2 Prior disease-free survival (A) and overall survival (B) in patients with metachronous isolated pulmonary vs metachronous pulmonary and other metastases patients. MPM: Metachronous pulmonary metastases; MIPM: Metachronous isolated pulmonary metastases; DFS: Disease-free survival; OS: Overall survival. with oxaliplatin or gemcitabine). In one patient thoracoscopic biopsy identified PDAC metastases 22 mo after initial pancreatic resection. The lesions were relatively stable and progressed slowly during the sequential lines of systemic chemotherapy, but brain metastases developed 80 mo after primary resection and the patient died 82.5 mo after resection. No intraabdominal recurrence was detected. The second patient was treated with gemcitabine and died of progressive disease 50.5 mo after the initial pancreatic resection. Patients with metachronous isolated oligometastases (n = 3) could all be considered candidates for surgery. One patient was treated outside of our institution with gemcitabine and subsequently developed liver metastases and died 12.5 mo after diagnosis of MPM. Two patients in this group underwent uncomplicated pulmonary resections for isolated oligometastatic recurrence that manifested 34 and 52 mo after primary pancreatic resections and PDAC metastases were histologically verified. As of December 2016, these patients are still alive without recurrence with a second disease free interval after pulmonary resection of 11+ and 13+ mo, respectively. In further analysis of patients with pulmonary metastases along with metastases to other organs (n = 15) there were 3 patients with pulmonary metastases and extrapulmonary metastases restricted to retroperitoneal paraaortal lymph nodes. These patients had DFS similar to patients with isolated pulmonary metastases (median 35 mo) and significantly longer OS (median 45 mo) compared to 6424 September 21, 2017 Volume 23 Issue 35

69 Lovecek M et al. Metachronous pulmonary metastases of pancreatic cancer the patients with pulmonary metastases accompanied by metastases to other organs (median DFS and OS of 13.5 mo and 19.6 mo, respectively). However, the data are very limited because of small numbers of patients. The shortest DFS (9.5 mo) and OS (16.2 mo) were observed in patients with metachronous nonpulmonary metastases where the first location of metastatic disease were liver, peritoneum, locoregional, or lymph nodes. DISCUSSION PDAC may metastasize to any organ [10,26,31-34] by hematogenous, lymphogenic, and perineural route or by direct intracavitary spread [10,26]. Pulmonary metastases as the first site of disease recurrence after resections for PDAC can present as isolated oligometastases, isolated multiple metastases or in association to metastatic spread to other organs. The incidence of PDAC in Central Europe is high, and, in fact, the incidence in the Czech Republic is currently the highest in the world. To the best of our knowledge, the present study is the first large homogenous cohort of PDAC describing these differences on Central European population. Patients with PDAC after primary radical multidisciplinary treatment (surgical resection followed by adjuvant therapy) have a very high risk of distant recurrence. One-hundred-thirty-two patients (83.1%) in the present cohort developed metastases. Even though metachronous pulmonary metastases after pancreatic resection are the most frequent extraabdominal site of PDAC recurrence, this presentation of recurrent disease is relatively rare, with the reported incidence of MPM as first site being between 3%-22% [5,7,16-19,21]. The highest rate of 22% was reported by Katz et al [5] in a highly selected cohort of patients of whom 77% had neoadjuvant therapy administered before primary resections. In the present study isolated pulmonary recurrence was observed in 4.2% of all cases of recurrent PDAC and 3.1% of entire cohort, in accordance with 3% rate of MPM reported previously by Arnaoutakis et al [17]. The present study indicates a low rate in unselected patients after radical primary treatment not exceeding 5%. Interestingly, isolated metachronous pulmonary metastases developed only in women, and female predominance was statistically significant in comparison of MPM and non-pulmonary metastatic patients. Kamisawa et al [10] in an autopsy series in an unselected cohort of patients treated with chemotherapy, radiation, palliative or radical procedures focusing on hematogenous PDAC metastases reported a prevalence of clinically detectable pulmonary metastases in an absence of liver metastases in 12.3%. Hematogenous spread to the lungs was considered mostly for primary location in the tail and splenic vein obstruction. In the present cohort the location of primary tumor in the tail was only in 5% and no case with splenic vein obstruction was noted. In addition, remarkably high rate of perineural invasion in tumor analysis was reported in patients with MPM, but the difference was not statistically significant in comparison with non-pulmonary metastatic patients and, therefore, thus needs to be evaluated in larger cohort or metaanalysis. Due to the low prevalence of MPM, the clinical course and optimal surgical or medical therapy are currently poorly defined. Early diagnosis of MPM in long-term survivors can bring the best therapeutic result and the highest chance for prolonged OS or even salvage therapy with curative intent. The rate of postoperative systemic therapy after primary resection in patients with pulmonary metastases varies between 85% in the present study to 100% [21]. MPM develop later than intraabdominal metastases and are the most frequent extraabdominal site across the studies [17-20]. In case of isolated pulmonary metastases the age at primary diagnosis is lower than in other groups (44-66 years, median 57). MPM can present as three different clinical scenarios, in contrast to patients with non-pulmonary metastases as evidenced in the present cohort. Metachronous isolated pulmonary oligometastases with up to 10 small lesions in one lung are eligible for surgical therapy [7,22,23,26,29]. If the patient is fit for pulmonary resection, this represents the best therapeutic option with the potential of cure and longest OS (Table 1). In the present cohort only three cases were operable according to the previously established criteria [7,22,23]. One patient was managed with gemcitabine chemotherapy without surgery. Subsequent liver metastases developed and patient died 12.5 mo after diagnosis of MPM. Only two patients were radically operated and both are currently alive more than one year without recurrence. In cases of metachronous isolated multiple pulmonary metastases local therapy including surgery or radiotherapy has no rationale and systemic therapy is only potential option. Long-term survival was observed in both patients (50.5 and 82.5 mo), but these patients ultimately died of progressive disease or development of subsequent metastases typical for primary pulmonary carcinomas, in one case the brain metastases. The third scenario included metachronous pulmonary with metastases to other organs. In this group a very small subgroup of patients with MPM and retroperitoneal paraaortal lymph node metastases with DFS comparable with group of isolated MPM was identified. Prior DFS and OS in those patients were longer than in patients with local or regional recurrence. In accordance with prior reports MPM patients in the present study had significantly longer DFS and OS compared to patients with non-pulmonary metastases. In patients with isolated pulmonary metastases median DFS ranges between 10.5 and 52.4 mo and OS between 23 and 92.3 mo [16-18,20,21]. The longest DFS of 156 mo after distal pancreatectomy was reported in 6425 September 21, 2017 Volume 23 Issue 35

70 Lovecek M et al. Metachronous pulmonary metastases of pancreatic cancer one case by Kitasato et al [35]. In the present study we observed median DFS of 34 mo in isolated pulmonary metastatic patients with an estimated median OS of 81.4 mo. Arnaoutakis et al [17], Thomas et al [18] and Downs-Canner et al [20] demonstrated in a total of 21 patients the feasibility and safety of surgical resection for MPM with significantly improved survival in comparison with non-surgical therapy. Reports from Japan include at least 23 cases with resected MPM of PDAC with long-term survival [19,23,35]. The most recent European study by Decoster et al [36] reported 22 MPM patients, 3 of whom were treated surgically, and indicated a female predominance. Our study also showed massive predominance of women in isolated pulmonary metastases. Female predominance in both studies is interesting, but there are currently no evidence based facts to explain this association. An effect of sex hormone could be one of the potential mechanisms, but without support by in vitro or in vivo data remains only speculative. Future studies should investigate whether MPM cases are characterized by specific molecular pathogenesis and biology that could explain this particular metastatic pattern. The most obvious limitation of the present study is the low number of patients with MPM, but due to the rarity of this presentation, our study is comparable to the earlier published retrospective analyses (Table 2). Even though some of the results cannot be statistically significant because of the small number of patients, together with the previously published data on other small cohorts the findings seem to be highly relevant for clinical practice. Another limitation is the evaluation of resection margins. Because the present study is of retrospective nature, resection margins could not be reevaluated according to the currently used Leeds protocol due to a technically completely different method of resection specimen evaluation. On the other hand, the present study is unique because of the origin of the study population. To date, most studies were published on American PDAC population [16-20], with two studies originating from Japanese PDAC centers (with most of the patients overlapping between both studies) [23,35]. The present study on Central European population together with three similar retrospective analyses from Western Europe [16,21,36] published during last year are important to confirm the results previously reported elsewhere. MPM, especially cases with isolated pulmonary metastases, are rare, and, obviously, prospective trials addressing the optimal management of these patients would be difficult to organize. Thus, for the foreseeable future retrospective analyses like the present report would remain the only source of information to guide the management of these patients. Most importantly, in patients with metastatic PDAC only systemic therapy is considered in general practice. Despite some improvements resulting from the introduction of new regimens such as the combination of oxaliplatin, irinotecan, folinic acid and 5-fluorouracil or the combination of gemcitabine with nab-paclitaxel, the efficacy of systemic therapy is still very limited. The results of trials with targeted agents so far have been disappointing [37-39]. Contrary to the prevalent opinion in the medical and surgical oncology community, present data indicate that there is a subgroup of patients with metastatic PDAC who would benefit from surgical therapy. Future studies should identify clinical parameters and molecular biomarkers that would select this population and help with therapeutic decisions. In conclusion, metachronous pulmonary metastases as first and only site of recurrence after resection for PDAC develop later and have better prognosis than other presentations of metastatic disease. OS is longer than in patients with other sites of metastases. Three different clinical scenarios according to the type of pulmonary oligometases with different prognosis and distinct therapeutic strategy were noted. The resection of pulmonary metastases was safe, with zero mortality and low morbidity. Patients who underwent pulmonary metastasectomy had longer survival compared to patients treated with chemotherapy or symptomatic therapy. Thus, surgery should be considered for all patients with solitary pulmonary metastases, but this has to be carefully weighted for each individual patient. Early diagnosis of recurrence may be viewed as precondition for successful management in these cases. Careful follow up with short (quarterly) interval and flexible PET/CT scanning can lead to early identification of recurrence. COMMENTS Background Metachronous pulmonary metastases (MPM) represent the most frequent extraabdominal site of pancreatic ductal adenocarcinoma (PDAC) recurrence. The aim of this study was to analyze PDAC patients who underwent a curativeintent surgery with focus on MPM as a first site of recurrence and to assess disease free survival prior to the diagnosis of metastases and the overall survival. Research frontiers The manuscript addresses the very rare and clinically important entity of metachronous pulmonary metastases as a first site of pancreatic cancer recurrence with the need of an individualized therapeutic approach as the surgery significantly prolongs overall survival of patients. In particular, the data suggest that surgery should be considered for all patients with isolated pulmonary metastases, but the risk of intervention has to be individually weighted for each patient. Innovations and breakthroughs Metachronous pulmonary metastases as first and only site of recurrence after resection for PDAC develop later and have better prognosis than other presentations of metastatic disease. Overall survival is longer than in patients with other sites of metastases. Three different clinical scenarios according to the type of pulmonary oligometases with different prognosis and distinct therapeutic strategy were noted. The resection of pulmonary metastases was safe, with zero mortality and low morbidity. Patients who underwent pulmonary metastasectomy had longer survival compared to patients treated with chemotherapy or symptomatic therapy. Thus, surgery should be considered 6426 September 21, 2017 Volume 23 Issue 35

71 Lovecek M et al. Metachronous pulmonary metastases of pancreatic cancer for all patients with solitary pulmonary metastases, but this has to be carefully weighted for each individual patient. Applications The data suggested that metachronous pulmonary metastases of pancreatic cancer need an individualized therapeutic approach Terminology Metachronous metastases were defined as those cases in which lesions were observed later on or within 3 mo after the after curative-intent surgery. Peer-review The authors studied a special subgroup of PDAC patients who suffered from metachronous pulmonary metastasis after surgery in a Central European population. They identified three different patterns of metachronous pulmonary metastasis and found much better prognosis in such subgroup compared to non-pulmonary metastatic patients. This clinical finding is important and valuable to clinical practice. REFERENCES 1 Globocan Web portal for International Cancer Research. 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72 Lovecek M et al. Metachronous pulmonary metastases of pancreatic cancer 25 Kysucan J, Lovecek M, Klos D, Tozzi I, Koranda P, Buriánková E, Neoral C, Havlík R. [Benefit of PET/CT in the preoperative staging in pancreatic carcinomas]. Rozhl Chir 2010; 89: [PMID: ] 26 Lu F, Poruk KE, Weiss MJ. Surgery for oligometastasis of pancreatic cancer. Chin J Cancer Res 2015; 27: [PMID: DOI: /j.issn ] 27 Hellman S, Weichselbaum RR. Oligometastases. J Clin Oncol 1995; 13: 8-10 [PMID: DOI: /JCO ] 28 Kaneda H, Saito Y. Oligometastases: Defined by prognosis and evauated by cure. Cancer Treat Commun 2015; 3: 1-6 [DOI: /j.ctrc ] 29 Niibe Y, Hayakawa K. Oligometastases and oligo-recurrence: the new era of cancer therapy. Jpn J Clin Oncol 2010; 40: [PMID: DOI: /jjco/hyp167] 30 Huang F, Wu G, Yang K. Oligometastasis and oligo-recurrence: more than a mirage. Radiat Oncol 2014; 9: 230 [PMID: DOI: /s ] 31 Yachida S, Iacobuzio-Donahue CA. The pathology and genetics of metastatic pancreatic cancer. Arch Pathol Lab Med 2009; 133: [PMID: DOI: / ] 32 Embuscado EE, Laheru D, Ricci F, Yun KJ, de Boom Witzel S, Seigel A, Flickinger K, Hidalgo M, Bova GS, Iacobuzio-Donahue CA. Immortalizing the complexity of cancer metastasis: genetic features of lethal metastatic pancreatic cancer obtained from rapid autopsy. Cancer Biol Ther 2005; 4: [PMID: DOI: /cbt ] 33 Disibio G, French SW. Metastatic patterns of cancers: results from a large autopsy study. Arch Pathol Lab Med 2008; 132: [PMID: DOI: / (2008)132] 34 Mao C, Domenico DR, Kim K, Hanson DJ, Howard JM. Observations on the developmental patterns and the consequences of pancreatic exocrine adenocarcinoma. Findings of 154 autopsies. Arch Surg 1995; 130: [PMID: DOI: / archsurg ] 35 Kitasato Y, Nakayama M, Akasu G, Yoshitomi M, Mikagi K, Maruyama Y, Kawahara R, Ishikawa H, Hisaka T, Yasunaga M, Horiuchi H, Saito N, Takamori S, Okabe Y, Kage M, Kinoshita H, Tanaka H. Metastatic pulmonary adenocarcinoma 13 years after curative resection for pancreatic cancer: report of a case and review of Japanese literature. JOP 2012; 13: [PMID: ] 36 Decoster C, Gilabert M, Autret A, Turrini O, Oziel-Taieb S, Poizat F, Giovannini M, Viens P, Iovanna J, Raoul JL. Heterogeneity of metastatic pancreatic adenocarcinoma: Lung metastasis show better prognosis than liver metastasis-a case control study. Oncotarget 2016; 7: [PMID: DOI: / oncotarget.9861] 37 Van Laethem JL, Riess H, Jassem J, Haas M, Martens UM, Weekes C, Peeters M, Ross P, Bridgewater J, Melichar B, Cascinu S, Saramak P, Michl P, Van Brummelen D, Zaniboni A, Schmiegel W, Dueland S, Giurescu M, Garosi VL, Roth K, Schulz A, Seidel H, Rajagopalan P, Teufel M, Childs BH. Phase I/II Study of Refametinib (BAY ) in Combination with Gemcitabine in Advanced Pancreatic cancer. Target Oncol 2017; 12: [PMID: DOI: /s y] 38 Deplanque G, Demarchi M, Hebbar M, Flynn P, Melichar B, Atkins J, Nowara E, Moyé L, Piquemal D, Ritter D, Dubreuil P, Mansfield CD, Acin Y, Moussy A, Hermine O, Hammel P. A randomized, placebo-controlled phase III trial of masitinib plus gemcitabine in the treatment of advanced pancreatic cancer. Ann Oncol 2015; 26: [PMID: DOI: / annonc/mdv133] 39 Fuchs CS, Azevedo S, Okusaka T, Van Laethem JL, Lipton LR, Riess H, Szczylik C, Moore MJ, Peeters M, Bodoky G, Ikeda M, Melichar B, Nemecek R, Ohkawa S, Świeboda-Sadlej A, Tjulandin SA, Van Cutsem E, Loberg R, Haddad V, Gansert JL, Bach BA, Carrato A. A phase 3 randomized, double-blind, placebo-controlled trial of ganitumab or placebo in combination with gemcitabine as first-line therapy for metastatic adenocarcinoma of the pancreas: the GAMMA trial. Ann Oncol 2015; 26: [PMID: DOI: /annonc/mdv027] P- Reviewer: Kleeff J, Luyer M, Wei DY, Zhang Q S- Editor: Gong ZM L- Editor: A E- Editor: Huang Y 6428 September 21, 2017 Volume 23 Issue 35

73 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) ORIGINAL ARTICLE Retrospective Study Evaluation of novel slim biopsy forceps for diagnosis of biliary strictures: Single-institutional study of consecutive 360 cases (with video) Kenjiro Yamamoto, Takayoshi Tsuchiya, Takao Itoi, Shujiro Tsuji, Reina Tanaka, Ryosuke Tonozuka, Mitsuyoshi Honjo, Shuntaro Mukai, Kentaro Kamada, Mitsuru Fujita, Yasutsugu Asai, Yukitoshi Matsunami, Yuichi Nagakawa, Hiroshi Yamaguchi, Atsushi Sofuni Kenjiro Yamamoto, Takayoshi Tsuchiya, Takao Itoi, Shujiro Tsuji, Reina Tanaka, Ryosuke Tonozuka, Mitsuyoshi Honjo, Shuntaro Mukai, Kentaro Kamada, Mitsuru Fujita, Yasutsugu Asai, Yukitoshi Matsunami, Atsushi Sofuni, Department of Gastroenterology and Hepatology, Tokyo Medical University, Shinjuku-ku, Tokyo , Japan Yuichi Nagakawa, Third Department of Surgery, Tokyo Medical University, Shinjuku-ku, Tokyo , Japan Hiroshi Yamaguchi, Department of Pathology, Tokyo Medical University, Shinjuku-ku, Tokyo , Japan Author contributions: Yamamoto K, Tsuchiya T and Itoi T contributed equally to this work; Yamamoto K, Tsuchiya T, Itoi T, Tsuji S, Tanaka R, Tonozuka R, Honjo M, Mukai S, Kamada K, Fujita M, Asai Y, Matsunami Y, Nagakawa Y and Sofuni A performed the research and collected the data; Yamamoto K, Tsuchiya T, Itoi T and Sofuni A reviewed the data analysis; Yamaguchi H reviewed all pathological specimens. Institutional review board statement: This study was reviewed and approved by our institutional review board (Tokyo Medical University No. 3516). Informed consent statement: Patients were not required to give informed consent to the study because the analysis used anonymous clinical data that were obtained after each patient agreed to treatment by written consent. For full disclosure, the details of the study are published on the home page of Tokyo Medical University. Conflict-of-interest statement: We have no financial relationships to disclose. Data sharing statement: No additional data are available. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Unsolicited manuscript Correspondence to: Takao Itoi, MD, PhD, FACG, FASGE, Department of Gastroenterology and Hepatology, Tokyo Medical University, Nishishinjuku, Shinjuku-ku, Tokyo , Japan. itoi@tokyo-med.ac.jp Telephone: Fax: Received: March 18, 2017 Peer-review started: March 20, 2017 First decision: June 8, 2017 Revised: July 16, 2017 Accepted: August 2, 2017 Article in press: August 2, 2017 Published online: September 21, 2017 Abstract AIM To evaluate the feasibility and reliability of endoscopic transpapillary bile duct biopsy for the diagnosis of biliary strictures. METHODS A total of 360 patients (241 men) who underwent endoscopic retrograde cholangiopancreatography for biliary strictures with biopsy from April 2012 to 6429 September 21, 2017 Volume 23 Issue 35

74 Yamamoto K et al. Biopsy forceps for diagnosing biliary strictures March 2016 at Tokyo Medical University Hospital were retrospectively reviewed. This study was approved by our Institutional Review Board (No. 3516). Informed consent was obtained from all individual participants included in this study. The biopsy specimens were obtained using a novel slim biopsy forceps (Radial Jaw 4P, Boston Scientific, Boston, MA, United States). RESULTS The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy were 69.6%, 100%, 100%, 59.1%, and 78.8%, respectively. The sensitivity was 75.6% in bile duct cancer, 64% in pancreatic cancer, 61.1% in gallbladder cancer, and 57.1% in metastasis. In bile duct cancer, a lower sensitivity was observed for perihilar bile duct stricture (68.7%) than for distal bile duct stricture (83.1%). In terms of the stricture lengths of pancreatic cancer, gallbladder cancer, and metastasis, a longer stenosis resulted in a better sensitivity. In particular, there was a significant difference between pancreatic cancer and gallbladder cancer (P < 0.05). One major complication was perforation of the extrahepatic bile duct with bile leakage. CONCLUSION Endoscopic transpapillary biopsy alone using novel slim biopsy forceps is feasible and reliable, but restrictive. Biopsy should be performed in consideration of the stricture level, stricture length, and cancer type. Key words: endoscopic transpapillary biopsy; biopsy forceps; biliary stricture; bile duct cancer; endoscopic retrograde cholangiography The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: Various radiological imaging procedures have been established as first-line modalities for detecting biliary strictures. However, a definitive diagnosis of biliary strictures can only be established by histocytological examination. At present, there are several histocytological sampling techniques such as aspiration cytology, brush cytology, aspiration needle biopsy, and forceps biopsy. However, the optimal sampling technique remains controversial. In this study, we found that transpapillary biopsy was feasible and reliable for diagnosing biliary strictures and should be performed in consideration of the stricture level, stricture length, and cancer type. Yamamoto K, Tsuchiya T, Itoi T, Tsuji S, Tanaka R, Tonozuka R, Honjo M, Mukai S, Kamada K, Fujita M, Asai Y, Matsunami Y, Nagakawa Y, Yamaguchi H, Sofuni A. Evaluation of novel slim biopsy forceps for diagnosis of biliary strictures: Singleinstitutional study of consecutive 360 cases (with video). World J Gastroenterol 2017; 23(35): Available from: URL: DOI: INTRODUCTION To date, transabdominal ultrasonography (TAUS), computed tomography (CT), magnetic resonance imaging (MRI), and magnetic resonance cholangiopancreatography (MRCP) have been established as the first-line modalities for detecting biliary strictures, being minimally invasive modalities. However, a definitive diagnosis of biliary strictures can only be established by histocytological examination, which is crucial for making a decision regarding the further management of this disease entity. Endoscopic retrograde cholangiopancreatography (ERCP) is the most widely used close-up examination method using direct cholangiography for evaluating biliary strictures which allows transpapillary histocytological sampling from the strictures by aspiration cytology, brush cytology, and endobiliary forceps biopsy. Several studies have examined whether combining tissue sampling techniques can improve the diagnostic accuracy for biliary strictures during ERCP [1-4]. Theoretically, core tissue sampling using biopsy forceps can provide more sufficient information for diagnosing biliary strictures than cytology. However, as conventional biopsy forceps has been used in gastrointestinal tract diseases and has a thick and hard shaft, transpapillary insertion via the accessory channel of the duodenoscope is difficult. Recently, a novel biopsy forceps which has a thin and soft shaft has been developed. Herein, we evaluated this novel slim biopsy forceps for the diagnosis of biliary strictures. MATERIALS AND METHODS A total of 360 patients (241 men) who underwent ERCP for biliary strictures with biopsy from April 2012 to March 2016 at Tokyo Medical University Hospital were retrospectively reviewed. Patients were excluded in the following cases: (1) prior histological confirmation of malignancy; (2) postoperative biliary strictures; (3) ampullary tumor; and (4) less than 6 mo of follow-up in patients with negative malignant results. The final diagnosis was established by histopathological examination of tissues obtained endoscopically or surgically. If the histopathological diagnosis was negative for carcinoma, a clinical diagnosis was made from the clinical course for over 6 mo or more and several radiological image findings from various imaging modalities such as TAUS, CT, MRI, MRCP, and endoscopic ultrasonography (EUS). If biopsy forceps failed to achieve a definitive diagnosis and biliary strictures or filling defects were still indeterminate even with the use of various radiological modalities, peroral cholangioscopy was performed for a definitive diagnosis. ERCP was performed using the standard technique with a duodenoscope. When biliary strictures were 6430 September 21, 2017 Volume 23 Issue 35

75 Yamamoto K et al. Biopsy forceps for diagnosing biliary strictures A B Figure 1 Biopsy forceps. A: Radial Jaw 4P; B: Swing function. Table 1 Patient characteristics Patient characteristics Number of patients 360 Age [mean ± SD (range), yr] 71 ± 11.8 (17-95) Sex, male/female 241/119 Disease (number of patients) Bile duct cancer 132 Pancreatic cancer 86 Gallbladder cancer 18 Metastasis 14 Benign stricture 110 identified under radiographic guidance, endoscopic sphincterotomy (EST) was performed before tissue sampling. The biopsy specimens were obtained using a novel slim biopsy forceps (Radial Jaw 4P, Boston Scientific, Boston, MA, United States) (Figure 1A, video 1). The forceps was transpapillary advanced to the distal end of the stricture alongside the guidewire. The obtained specimen was immediately fixed in 10% formalin. All procedures were performed by operators with experience of more than 100 ERCP cases per year. This study was approved by our Institutional Review Board (No. 3516) and informed consent was obtained from all individual participants included in this study. Statistical analysis Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and overall accuracy were calculated. Statistical analyses were performed using StatMate Ⅲ (ATMS, Tokyo, Japan). A P-value < 0.05 was considered to indicate a statistically significant difference. RESULTS The characteristics of the patients are shown in Table 1. Their median age was 71 ± 11.8 years (range: years). The diseases were bile duct cancer in 132 patients, pancreatic cancer in 86, gallbladder cancer in 18, metastasis of other cancers in 14, and benign biliary stricture in 110. Among the patients with metastases, the primary sites were as follows: colon cancer in 3 patients, gastric cancer in 2, pancreatic cancer in 2, lung cancer in 2, breast cancer in 2, ovarian cancer in 2, and cholangiocellular carcinoma in 1. Sample collection rate The biopsy forceps could be inserted via the papilla after EST, and the specimens evaluated were collected from all 360 patients (technical success: 100%), with 820 bites in total (range: 1-6 bites). The mean number of biopsies was 2.28 ± 0.91 (Table 2). Overall results The sensitivity, specificity, PPV, NPV, and accuracy were 69.6%, 100%, 100%, 59.1%, and 78.8%, respectively (Table 2). The sensitivity was 75.6% in bile duct cancer, 64% in pancreatic cancer, 61.1% in gallbladder cancer, and 57.1% in metastasis (Table 3). Type of cancers The examination results for bile duct cancer are shown in Table 4. According to the stricture level of bile duct cancer, the sensitivity was 68.7% in the perihilar bile duct and 83.1% in the distal bile duct; 95% in the middle and 76.7% in the lower. Furthermore, comparing the type of stricture with space that the biopsy forceps could open with the type of stricture without space in the lower bile duct (Figure 2), the sensitivity with an open space was 84.6% and that without an open space was 64.7%. The examination results for extrinsic neoplasms are shown in Table 5. As for pancreatic cancer according to the stricture level, the sensitivity was 45.5% in the middle bile duct and 66.7% in the lower bile duct. In addition, according to the localization of the primary tumor, the sensitivity was 68.4% in pancreatic head cancer and 30% in pancreatic body cancer. As for gallbladder cancer according to the stricture level, 6431 September 21, 2017 Volume 23 Issue 35

76 Yamamoto K et al. Biopsy forceps for diagnosing biliary strictures Table 2 Overall diagnostic ability in all cases (n = 360) Sample collection rate Total number of biopsies 820 Mean number of biopsies, ± SD (range) 2.28 ± 0.91 (1-6) Overall results Sensitivity 69.6% Specificity 100% PPV 100% NPV 59.1% Accuracy 78.8% PPV: Positive predictive value; NPV: Negative predictive value. Table 4 Sensitivity of biopsy forceps in bile duct cancer n Sensitivity Total 100/ % Site of stricture Perihilar bile duct 46/ % Distal bile duct 54/ % Middle 21/22 95% Lower 33/ % (Open space + 22/ % (Open space 11/ % Table 3 Overall sensitivity for malignancy Diagnosis n Sensitivity Bile duct cancer 100/ % Pancreatic cancer 55/ % Gallbladder cancer 11/ % Metastasis 8/ % Table 5 Sensitivity of biopsy forceps in extrinsic neoplasms n Sensitivity Pancreatic cancer 55/ % Site of stricture Middle bile duct 5/ % Lower bile duct 50/ % Localization of tumor Head 52/ % Body 3/ % Gallbladder cancer 11/ % Site of stricture Perihilar bile duct 6/ % Distal bile duct 5/7 71.4% Metastasis 8/ % Site of stricture Perihilar bile duct 4/6 66.7% Distal bile duct 4/8 50.0% Table 6 Mean stricture lengths in extrinsic neoplasms Positive (mm) Negative (mm) P value Pancreatic cancer < 0.05 Gallbladder cancer < 0.05 Metastasis NS NS: Not significant. the sensitivity was 54.5% in the perihilar bile duct and 71.4% in the distal bile duct. As for metastasis according to the stricture level, the sensitivity was 66.7% in the perihilar bile duct and 50% in the distal bile duct. Length of stricture in cancers Apart from the stricture level, the mean stricture lengths were 16.9 mm, 22 mm, and 17.1 mm in the biopsypositive group and 13.5 mm, 13.4 mm, and 13.2 mm in the biopsy-negative group for pancreatic cancer, gallbladder cancer, and metastasis, respectively (Table 6). The biopsy-positive group tended to have a longer stricture length than the biopsy-negative group. In particular, there was a significant difference between pancreatic cancer and gallbladder cancer (P < 0.05). Procedure-related adverse events There were no procedure-related adverse events, although there was 1 patient with perihilar bile duct cancer in whom perforation of the distal bile duct with bile leakage was observed. The condition was improved only by temporary endoscopic nasobiliary drainage tube placement. DISCUSSION In this study, we found that transpapillary biopsy using a novel slim biopsy forceps could be easily and safely performed and was effective for diagnosing biliary strictures. At present, there are several cytological and histological sampling techniques such as aspiration cytology, brush cytology, aspiration needle biopsy, and forceps biopsy. However, the optimal sampling technique remains controversial. The ideal technique for obtaining tissue for a definitive diagnosis of malignant biliary strictures should be simple, safe, and effective with a high sensitivity and specificity. Although bile cytology is easy, its accuracy range is very low (6%-32%) [5,6]. Mohandas et al [7]. described that bile cytology with dilation of biliary strictures up to 10-Fr during ERCP enhanced its sensitivity by 63% compared with the 27% sensitivity of bile cytology without dilation. Regarding brush cytology, although this can be undertaken relatively easily and safely in a short time, its sensitivity is still considered to be low (30%-57%) [1-3]. Farrell et al [8] reported that the sensitivity of brush cytology increased by 85% after stricture dilation and endoscopic needle aspiration compared with the 57% sensitivity of brush cytology alone. In contrast, de Bellis et al [9] suggested that 6432 September 21, 2017 Volume 23 Issue 35

77 Yamamoto K et al. Biopsy forceps for diagnosing biliary strictures A B Figure 2 Fluoroscopic images during endoscopic retrograde cholangiopancreatography. A: Stricture with an open space; B: Stricture without an open space. Table 7 Characteristics of studies evaluating bile duct biopsies Study Year Sample size (n ) Sensitivity, % Pugliese et al [1] Ponchon et al [3] Howell et al [10] Sugiyama et al [11] Jailwala et al [2] Rösch et al [12] Kitajima et al [13] Weber et al [14] Our case stricture dilation did not improve the sensitivity of brush cytology for cancer detection. Hence, the efficacy of stricture dilation for diagnosis is still controversial. On the other hand, transpapillary forceps biopsy has not been widely used because it is thought to be technically difficult, resulting in a waste of time. Moreover, its sensitivity is very variable (15%-81%) [1-3,10-14] (Table 7). In 2002, de Bellis et al [9] summarized 502 previously reported cases and found that the sensitivity and specificity of transpapillary forceps biopsy were 56% (43%-81%) and 97% (90%-100%), respectively. Their report showed that the diagnostic performance of forceps biopsy was higher than that of other tissue sampling methods [15,16]. Furthermore, forceps biopsy is useful not only for the diagnosis of malignant/benign tumors but also for the selection of therapeutic methods (e.g., chemotherapy) based on the histological types of cancer, and the judgment of the superficial intraductal spread of bile duct cancer. In the present study, we used a novel slim biopsy forceps (Radial Jaw 4P, Boston Scientific) (Figure 1A). The Radial Jaw series are the world s first disposable biopsy forceps released in The first type of Radial Jaw had a diameter of 2.2 mm and had tiny teeth around the cup. After repeated improvements, the present Radial Jaw 4P was released in Radial Jaw 4P has a thinner shaft of 1.8 mm, a wire with a sheath to pass through the scope smoothly, and an open width/angle of 5.4 mm/150. In addition, its thinner cup increases sampling capacity and its swing function helps achieve tangential biopsy (Figure 1B). Sufficient tissue samples for a definitive diagnosis, which include the mucosal cancer or invasive cancer below the bile duct epithelium, can be obtained using this forceps (Figure 3). Hence, the present Radial Jaw 4P may be more appropriate for performing optimal biopsy. In fact, in the present study, this novel forceps could collect adequate specimens for evaluation from all of the patients. Thus, our data showed a higher sensitivity than previous studies of bile duct biopsies (Table 7), suggesting that this novel slim biopsy forceps appears suitable as a tissue sampling device for the diagnosis of biliary strictures. Regarding bile duct cancer, a lower sensitivity was observed for the perihilar bile duct stricture than for the distal bile duct stricture. This may be due to the narrow and smooth stricture and the distance from the papilla, preventing the biopsy forceps from being able to sufficiently open the stricture and precisely hitting the tumor. In contrast, a better sensitivity was observed for the distal bile duct stricture, particularly in the middle bile duct. For the lower bile duct, a better sensitivity was observed for the stricture with an open space than for the stricture without an open space. In the stricture without an open space for a biopsy forceps to open sufficiently, adequate and accurate biopsy tissue samplings from the target site may be slightly difficult. Thus far, some reports have shown that the combination of brush cytology and forceps biopsy improved sensitivity [3,8,10]. However, a recent systematic review and a meta-analysis have suggested that the combination of brush cytology and forceps biopsy only modestly increased sensitivity [17]. These combination techniques may be ideal because the tumor is usually exposed to the bile duct surface, allowing brush cytology to obtain cells from the entire lumen surface in bile duct cancer, particularly in severe strictures of the perihilar bile duct and lower bile duct without an open space. On the other hand, the sensitivity of biopsy in pancreatic cancer was lower than that in 6433 September 21, 2017 Volume 23 Issue 35

78 Yamamoto K et al. Biopsy forceps for diagnosing biliary strictures A B Figure 3 Biopsy specimens obtained using Radial Jaw 4P. The biopsy specimen obtained was sufficient and included adenocarcinoma. A: Bile duct cancer; B: Pancreatic cancer (HE. stain, 10). bile duct cancer similarly to previous reports [1,3,11,18]. Theoretically, tumor cells from the surface of the bile duct in pancreatic cancer appear to be fewer than tumor cells in bile duct cancer. Thus, sensitivity in pancreatic cancer as well as in gallbladder cancer and metastases, namely, extrinsic neoplasms, appears to be limited even with the use of additional brush cytology in combination with biopsy forceps. Interestingly, some reports [3,19] have also demonstrated that improvement of sensitivity cannot be expected with the combination of aspiration cytology and brush cytology for cancers with a bile duct stricture by extrinsic exclusion. Furthermore, the sensitivity for a stricture in pancreatic body cancer was lower than that for a stricture in pancreatic head cancer. This is the reason why the bile duct stricture in pancreatic body cancer, which even shows a biliary stricture that is far from the bile duct, occurs not by direct invasive reaction but as a secondary fibrous cicatricial stricture by stromal reaction. In addition, many pancreatic body cancers occur in the middle bile duct stricture, resulting in a lower sensitivity for the middle bile duct stricture. In the present study, we showed that a longer stricture had a better sensitivity for diagnosis. This suggests an increase in the positive diagnostic rate of forceps biopsy because the contact area with the bile duct of extrinsic cancer becomes wider according to the stricture length. Hence, to increase the diagnostic accuracy, multiple biopsies appear necessary for the diagnosis of a shorter biliary stricture due to extrinsic cancers. Recently, EUS-FNA has been introduced for evaluating biliary strictures [12,20]. EUS-FNA may be a preferable method for tissue sampling of neoplasms of extrinsic origin such as pancreatic cancer, gallbladder cancer, and metastasis after obtaining negative or nondiagnostic ERCP tissue sampling results. On the other hand, ERCP-based tissue sampling may be better for bile duct cancer of biliary epithelial origin. Pancreatitis and cholangitis are generally observed, although their incidence is low and their severity is mild in most cases requiring only conservative treatment [2,21]. Although rare, bile duct perforation can occur during forceps biopsy [1]. Repeated biopsies at the same site may account for the perforation. In the present study, 1 case of perforation of the extrahepatic bile duct with bile leakage occurred during forceps biopsy in a patient with perihilar bile duct cancer. Although Radial Jaw 4P is a soft biopsy forceps, care should be taken during its usage. Various tips in performing forceps biopsy have been reported. Of interest is the study of Tamada et al [22] which suggested factors for a successful bile duct biopsy under percutaneous transhepatic cholangioscopy (PTCS) from the viewpoint of the growth type of bile duct cancers. They recommended that 2 biopsy samples should be obtained from the mass in polypoid-type bile duct cancers and from the margin in stenotic-type bile duct cancers with a dilated vessel. Moreover, 3 biopsy samples should be obtained from the margin of the stricture in stenotictype bile duct cancers without a dilated vessel, and biopsy specimens should be obtained from within the stricture in pancreatic cancer. Hence, transpapillary bile duct biopsy is performed under fluoroscopic guidance and is not similar to the biopsy performed under direct vision by PTCS. Thus, more than 3 biopsy specimens may be necessary and the biopsy sites, such as the margin of the stricture or within the stricture, should be considered according to the types of cancers such as cancers of biliary epithelial or extrinsic origin. The limitations of this study were its retrospective nature, the lack of a control group, and the inclusion of a single-center experience. In conclusion, endoscopic transpapillary biopsy using a novel slim biopsy forceps is feasible and reliable, but is a restrictive method for tissue sampling of malignant biliary strictures. When performing the biopsy, it is necessary to consider the stricture levels, stricture lengths, and types of cancers such as cancers of biliary epithelial or extrinsic origin to improve the diagnosis of malignant biliary strictures September 21, 2017 Volume 23 Issue 35

79 Yamamoto K et al. Biopsy forceps for diagnosing biliary strictures COMMENTS Background There are several cytological and histological sampling techniques for a definitive diagnosis of malignant biliary strictures. However, the optimal sampling technique remains controversial. The ideal technique should be simple, safe, and effective with a high sensitivity and specificity. Research frontiers Transpapillary forceps biopsy has not been widely used because it is thought to be technically difficult, resulting in a waste of time. Moreover, its sensitivity is very variable (15%-81%). In the present study, a novel slim biopsy forceps could collect adequate specimens for evaluation from all of the patients and our data showed a higher sensitivity (70%) than previous studies of bile duct biopsies. Innovations and breakthroughs It is necessary to consider the stricture levels, stricture lengths, and types of cancers such as cancers of biliary epithelial or extrinsic origin to improve the diagnosis of malignant biliary strictures. Applications Endoscopic transpapillary biopsy using a novel slim biopsy forceps is feasible and reliable. Terminology Endoscopic transpapillary biopsy for diagnosis of biliary strictures should be performed in consideration of the stricture level, stricture length, and cancer type. Peer-review The paper deals with the problem of obtaining reliable biopsies from lesions in the biliary tree. Evidently the methods described here yields a progress in this field. The paper is well written. This is good study exploring the efficacy and diagnostic value of a new slim forceps by Boston. The manuscript is generally interesting and offers useful clinical information to those who carry out endoscopic retrograde cholangiopancreatography. 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A prospective study of biliary cytology in 100 patients with bile duct strictures. Hepatology 1993; 18: [PMID: ] 7 Mohandas KM, Swaroop VS, Gullar SU, Dave UR, Jagannath P, DeSouza LJ. Diagnosis of malignant obstructive jaundice by bile cytology: results improved by dilating the bile duct strictures. Gastrointest Endosc 1994; 40: [PMID: ] 8 Farrell RJ, Jain AK, Brandwein SL, Wang H, Chuttani R, Pleskow DK. The combination of stricture dilation, endoscopic needle aspiration, and biliary brushings significantly improves diagnostic yield from malignant bile duct strictures. Gastrointest Endosc 2001; 54: [PMID: ] 9 de Bellis M, Fogel EL, Sherman S, Watkins JL, Chappo J, Younger C, Cramer H, Lehman GA. Influence of stricture dilation and repeat brushing on the cancer detection rate of brush cytology in the evaluation of malignant biliary obstruction. Gastrointest Endosc 2003; 58: [PMID: DOI: / mge ] 10 Howell DA, Parsons WG, Jones MA, Bosco JJ, Hanson BL. Complete tissue sampling of biliary strictures at ERCP using a new device. Gastrointest Endosc 1996; 43: [PMID: ] 11 Sugiyama M, Atomi Y, Wada N, Kuroda A, Muto T. Endoscopic transpapillary bile duct biopsy without sphincterotomy for diagnosing biliary strictures: a prospective comparative study with bile and brush cytology. Am J Gastroenterol 1996; 91: [PMID: ] 12 Rösch T, Hofrichter K, Frimberger E, Meining A, Born P, Weigert N, Allescher HD, Classen M, Barbur M, Schenck U, Werner M. ERCP or EUS for tissue diagnosis of biliary strictures? A prospective comparative study. Gastrointest Endosc 2004; 60: [PMID: ] 13 Kitajima Y, Ohara H, Nakazawa T, Ando T, Hayashi K, Takada H, Tanaka H, Ogawa K, Sano H, Togawa S, Naito I, Hirai M, Ueno K, Ban T, Miyabe K, Yamashita H, Yoshimura N, Akita S, Gotoh K, Joh T. Usefulness of transpapillary bile duct brushing cytology and forceps biopsy for improved diagnosis in patients with biliary strictures. J Gastroenterol Hepatol 2007; 22: [PMID: DOI: /j x] 14 Weber A, von Weyhern C, Fend F, Schneider J, Neu B, Meining A, Weidenbach H, Schmid RM, Prinz C. Endoscopic transpapillary brush cytology and forceps biopsy in patients with hilar cholangiocarcinoma. World J Gastroenterol 2008; 14: [PMID: DOI: /wjg ] 15 De Bellis M, Sherman S, Fogel EL, Cramer H, Chappo J, McHenry L Jr, Watkins JL, Lehman GA. Tissue sampling at ERCP in suspected malignant biliary strictures (Part 1). Gastrointest Endosc 2002; 56: [PMID: DOI: / mge ] 16 de Bellis M, Sherman S, Fogel EL, Cramer H, Chappo J, McHenry L Jr, Watkins JL, Lehman GA. Tissue sampling at ERCP in suspected malignant biliary strictures (Part 2). Gastrointest Endosc 2002; 56: [PMID: DOI: / mge ] 17 Navaneethan U, Njei B, Lourdusamy V, Konjeti R, Vargo JJ, Parsi MA. Comparative effectiveness of biliary brush cytology and intraductal biopsy for detection of malignant biliary strictures: a systematic review and meta-analysis. Gastrointest Endosc 2015; 81: [PMID: DOI: /j.gie ] 18 Kubota Y, Takaoka M, Tani K, Ogura M, Kin H, Fujimura K, Mizuno T, Inoue K. Endoscopic transpapillary biopsy for diagnosis of patients with pancreaticobiliary ductal strictures. Am J Gastroenterol 1993; 88: [PMID: ] 19 Lee SJ, Lee YS, Lee MG, Lee SH, Shin E, Hwang JH. Triple-tissue sampling during endoscopic retrograde cholangiopancreatography increases the overall diagnostic sensitivity for cholangiocarcinoma. Gut Liver 2014; 8: [PMID: DOI: / gnl13292] 20 DeWitt J, Misra VL, Leblanc JK, McHenry L, Sherman S. EUSguided FNA of proximal biliary strictures after negative ERCP brush cytology results. Gastrointest Endosc 2006; 64: [PMID: DOI: /j.gie ] 21 Ryan ME. Cytologic brushings of ductal lesions during ERCP September 21, 2017 Volume 23 Issue 35

80 Yamamoto K et al. Biopsy forceps for diagnosing biliary strictures Gastrointest Endosc 1991; 37: [PMID: ] 22 Tamada K, Kurihara K, Tomiyama T, Ohashi A, Wada S, Satoh Y, Miyata T, Ido K, Sugano K. How many biopsies should be performed during percutaneous transhepatic cholangioscopy to diagnose biliary tract cancer? Gastrointest Endosc 1999; 50: [PMID: ] P- Reviewer: Bramhall S, Hussain A, Kreisel W, Lee HC, Neri V, Garcia-Olmo D S- Editor: Ma YJ L- Editor: A E- Editor: Huang Y 6436 September 21, 2017 Volume 23 Issue 35

81 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) ORIGINAL ARTICLE Retrospective Study Evaluation of the relationship between hepatocellular carcinoma location and transarterial chemoembolization efficacy Izumi Miki, Satoru Murata, Fumio Uchiyama, Daisuke Yasui, Tatsuo Ueda, Fumie Sugihara, Hidemasa Saito, Hidenori Yamaguchi, Ryusuke Murakami, Chiaki Kawamoto, Eiji Uchida, Shin-ichiro Kumita Izumi Miki, Satoru Murata, Daisuke Yasui, Tatsuo Ueda, Fumie Sugihara, Hidemasa Saito, Ryusuke Murakami, Shinichiro Kumita, Department of Radiology, Center of Advanced Medicine, Nippon Medical School, Bunkyo-ku, Tokyo , Japan Fumio Uchiyama, Department of Radiology, Ebina-Sogo Hospital, Ebina-shi , Japan Hidenori Yamaguchi, Department of Radiology, Tamanagayama Hospital, Nippon Medical School, Tama-shi, Tokyo , Japan Chiaki Kawamoto, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Nippon Medical School, Bunkyo-ku, Tokyo , Japan Eiji Uchida, Department of Gastrointestinal and Hepato-Biliary- Pancreatic Surgery, Nippon Medical School, Bunkyo-ku, Tokyo , Japan Author contributions: Miki I and Murata S contributed equally to this work; Murata S designed the research; Miki I, Uchida F, Yasui D, Ueda T, Sugihara F and Saito H and Yamaguchi H, Murakami R performed the research; Miki I and Uchiyama F, Yasui D analyzed the data; Miki I and Murata S wrote the paper; Kawamoto C, Uchida E provided resources; Murata SI and Kumita S oversaw the project. Institutional review board statement: The study was reviewed and approved by the Nippon Medical School Institutional Review Board. Informed consent statement: Patients were not required to give informed consent for this study because the analysis used anonymous clinical data that were obtained after each patient agreed to treatment by written consent. For full disclosure, the details of the study are published on the home page of Nippon Medical School. Conflict-of-interest statement: There are no conflicts of interest to declare. Data sharing statement: The technical appendix, statistical code, and dataset are available from the corresponding author at genji@nms.ac.jp. Participants gave informed consent for data sharing. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Unsolicited manuscript Correspondence to: Satoru Murata, MD, PhD, Department of Radiology, Center of Advanced Medicine, Nippon Medical School, 1-1-5, Sendagi, Bunkyo-ku, Tokyo , Japan. genji@nms.ac.jp Telephone: Fax: Received: June 6, 2017 Peer-review started: June 7, 2017 First decision: July 13, 2017 Revised: July 25, 2017 Accepted: August 15, 2017 Article in press: August 15, 2017 Published online: September 21, September 21, 2017 Volume 23 Issue 35

82 Miki I et al. HCC location and TACE efficacy Abstract AIM To evaluate the relationship between the location of hepatocellular carcinoma (HCC) and the efficacy of transarterial chemoembolization (TACE). METHODS We evaluated 115 patients (127 nodules), excluding recurrent nodules, treated with TACE between January 2011 and June TACE efficacy was evaluated according to mrecist. The HCC location coefficient was calculated as the distance from the central portal portion to the HCC center (mm)/liver diameter (mm) on multiplanar reconstruction images rendered (MPR) to visualize bifurcation of the right and left branches of the portal vein and HCC center. The HCC location coefficient was compared between complete response (CR) and non-cr groups in Child-Pugh grade A and B patients. RESULTS The median location coefficient of HCC among all nodules, the right lobe, and the medial segment was significantly higher in the CR group than in the non-cr group in the Child-Pugh grade A patients (0.82 vs 0.62, P < 0.001; 0.71 vs 0.59, P < 0.01; 0.81 vs 0.49, P < 0.05, respectively). However, there was no significant difference in the median location coefficient of the HCC in the lateral segment between in the CR and in the non-cr groups (0.67 vs 0.65, P > 0.05). On the other hand, in the Child-Pugh grade B patients, the HCC median location coefficient in each lobe and segment was not significantly different between in the CR and in the non-cr groups. CONCLUSION Improved TACE efficacy may be obtained for HCC in the peripheral zone of the right lobe and the medial segment in Child-Pugh grade A patients. Key words: Hepatocellular carcinoma location; Transarterial chemoembolization efficacy; Child-Pugh; Modified response evaluation criteria in solid tumors; Central zone; Peripheral zone The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: The relationship between hepatocellular carcinoma (HCC) location and transcatheter arterial chemoembolization (TACE) efficacy was evaluated. In Child-Pugh A, the median location coefficient of HCC among all nodules, right lobe, and medial segment was significantly higher in the complete response (CR) group than in the non-cr group, with no significant differences in the lateral segment. In Child-Pugh B, the median location coefficient of HCC in each lobe and segment was not significantly different between the two groups. Therefore, improved TACE efficacy may be obtained for HCC in the peripheral zone of the right lobe and medial segment in Child-Pugh A patients. Miki I, Murata S, Uchiyama F, Yasui D, Ueda T, Sugihara F, Saito H, Yamaguchi H, Murakami R, Kawamoto C, Uchida E, Kumita SI. Evaluation of the relationship between hepatocellular carcinoma location and transarterial chemoembolization efficacy. World J Gastroenterol 2017; 23(35): Available from: URL: DOI: INTRODUCTION Hepatocellular carcinoma (HCC) is one of the most common malignant diseases worldwide, and it is a lethal tumor whose prognosis largely depends on tumor stage at diagnosis and patient access to radical treatment [1,2]. Management of HCC has been standardized according to clinical staging systems, such as the Barcelona Clinical Liver Cancer (BCLC) classification, in which patients are stratified by tumor stage and underlying liver disease. According to the BCLC staging system, transarterial chemoembolization (TACE) is the current standard of care for BCLC stage B patients. Stage B is defined as an intermediate-stage disease [3] that involves highly heterogeneous patients who have Child-Pugh grade A or B liver function with four or more tumors irrespective of size or two to three tumors larger than 3 cm in diameter, in the absence of cancer-related symptoms, macrovascular invasion, or extrahepatic spread [4]. Several studies and well-designed randomized trials have shown that TACE has a positive effect on patient outcome and survival [5-7]. Although many studies have reported on several prognostic factors for stage-b HCC patients, tumor number, maximal tumor diameter, Child-Pugh score, and tumor response to TACE are fundamentally strong predictors of survival in patients with initially unresectable HCC [8,9]. Complete necrosis following repeated TACE was a significant independent predictor of favorable survival after the first TACE [10]. Most HCC patients have some degree of cirrhosis. Regardless of etiology, compared with noncirrhotic livers, cirrhotic livers show right-lobe and medialsegment atrophy and lateral-segment and caudatelobe hypertrophy [11]. This pattern suggests that microcirculatory changes play a role in the development of cirrhosis within segments. Indeed, an in vivo microscopic study [12] demonstrated differences in the blood flow in central and peripheral regions, indicating a direct and indirect reduction in the blood flow to the periphery of the main lobe. Histopathological investigations of HCCs that were resected after TACE have shown that the most viable tissue is located at the periphery of the tumor [13]. The efficacy of TACE is limited by the dual blood supply (artery and portal vein) of liver tumors and collateral 6438 September 21, 2017 Volume 23 Issue 35

83 Miki I et al. HCC location and TACE efficacy arterial supply after TACE, which make it difficult to achieve complete necrosis of tumor tissue because of insufficient tumor ischemia. However, complete necrosis is sometimes obtained in the long-term. Therefore, before performing this procedure, it is very important for the physician to predict whether TACE will be effective, particularly while considering other therapeutic modalities. Thus, the aim of this retrospective study was to evaluate the relationship between the location of the HCC, in particular, the location close to the peripheral or hepatic portal portion of the liver, and the efficacy of TACE. MATERIALS AND METHODS This retrospective study was conducted with the approval of our institutional review board. The requirement for informed consent was waived. Patients Between January 2011 and June 2014, 777 patients with HCC were treated with TACE in our institution. The eligibility criteria for the study were as follows: (1) diagnosis of HCC based on histologic findings, findings on dynamic contrast-enhanced multidetector computed tomography (MDCT), or magnetic resonance imaging (MRI) performed in our institution in accordance with the American Association for the Study of Liver Disease [14] and/or findings of cirrhosis; (2) no indications for hepatic resection, percutaneous ethanol injection therapy, liver transplantation, or thermal ablation therapy; (3) presence of a bidimensionally measurable hepatic lesion; (4) adequate liver function (serum total bilirubin level < 2.0 mg/dl and Child- Pugh grade A or B); (5) adequate bone marrow function (leukocyte count > 3000 cells/mm 3 and platelet count > cells/mm 3 ); (6) adequate renal function (serum creatinine concentration 1.2 mg/ dl); (7) no ascites; (8) absence of vascular invasion; (9) technical success of TACE; and (10) MDCT or MRI performed before TACE and at least 6 mo after TACE. The exclusion criteria were as follows: (1) recurrent HCC; (2) tumor size > 7 cm; (3) inadequate iodized oil accumulation in the target lesion because of large tumor size or anastomosis with vital vessels; (4) balloon-occluded TACE; and (5) drug-eluting bead TACE. Overall, 115 patients with 127 HCC nodules were included in our study. TACE procedure Experienced interventional radiologists performed all TACE procedures. After administering local anesthesia in all patients, we punctured the femoral artery using the Seldinger technique. A 4-French sheath (Super Sheath; Medikit, Miyazaki, Japan) was inserted via the femoral artery. After performing arterial portography and hepatic arteriography to confirm feeding arteries to HCC, we inserted a 2.0-French microcatheter (Gold Crest-MRT; Koshin Medical, Tokyo, Japan) into the feeding arteries. Then, an emulsion of anticancer drugs [cisplatin (50 mg/m 2, up to 100 mg) or epirubicin (30 mg/m 2 )] and iodized oil [Lipiodol (the sum of the major axis of the tumor up to 15 ml) Ultra Fluid, Laboratoires Guerber, Aulnay-sous-Bois, France] was injected slowly under fluoroscopic guidance until the vascular bed of the target nodule was filled with the emulsion. We performed embolization with 2 mm pieces of gelatin sponge (Gelpart; Nippon Kayaku, Tokyo, Japan) following TACE. Imaging evaluation of TACE efficacy Dynamic contrast-enhanced CT or MRI was used to evaluate the effect of TACE 6 mo after and on a per nodule basis, according to the Modified Response Evaluation Criteria in Solid Tumors (mrecist) [15] : complete response (CR) = disappearance of any intratumoral arterial enhancement in all target lesions; partial response (PR) = at least a 30% decrease in the sum of the diameter of viable target lesions; stable disease (SD) = any cases that did not qualify as either PR or progressive disease; progressive disease (PD) = an increase by at least 20% of the sum of the diameters of the viable target lesions. The evaluation was performed by two independent and blinded observers. After individual evaluation, the findings were disclosed, and any discrepancy in the findings was discussed by both the observers. Measurement of HCC location The location of the HCC was determined using the most recent dynamic contrast-enhanced MDCT (within 1 mo) before TACE. The MDCT row data of the arterial or portal phase of pre-tace were transferred to a workstation (OsiriX 7.0; Pixmeo, Bernex, Switzerland). The location coefficient of HCC was defined as follows: multi-planar reconstructed (MPR) images were rendered to visualize the bifurcation of the right and left branches of the portal vein and the center of the HCC, which was defined as the intersection of the long axes with the short axes in the maximum cross-section of the target nodule. Next, we traced the straight line through the bifurcation of the right and left branches of the portal vein to the center of the target nodule and the peripheral surface of the liver using MPR images (Figure 1). The location coefficient of the HCC was determined as a ratio of the distance from an internal surface of the liver to the center of the HCC/diameter of the liver on the traced line. An increased location coefficient indicates a more peripheral nodule. The bifurcation was the baseline point of measurement of the HCC location because the bifurcation had few 6439 September 21, 2017 Volume 23 Issue 35

84 Miki I et al. HCC location and TACE efficacy portal venous variations. Evaluation of efficacy due to HCC location The location coefficient of the HCC was compared between the CR group and non-cr group, including the PR, SD, and PD subgroups, among all nodules and each lobe or segment in Child Pugh A and B patients. The relationship between the efficacy of TACE and the location of the HCC was assessed. Statistical analysis All statistical analysis was performed using R (CRAN: the Comprehensive R Archive Network at R-project.org/). We compared the location coefficient of the HCC between the CR group and non- CR group using the Mann-Whitney U test. Moreover, receiver operating characteristic (ROC) curve analyses were used to determine the appropriate cut-off points of the location coefficient to predict the efficacy of TACE. In all comparisons, the results of the statistical analyses were considered significant at a P-value < RESULTS A B Figure 1 A 73-year-old man with hepatocellular carcinoma. An MPR image of the arterial phase shows a well-defined enhanced mass in segment 7 (arrowhead) and the bifurcation of the right and left branches of the portal vein. HCC location coefficient = distance from the medial surface of liver to the central HCC tumor (B)/diameter of the liver (A). MPR: Multi-planar reconstructed images; HCC: Hepatocellular carcinoma. The median patient age was 73 year (range, year; 88 men and 27 women). The tumor size ranged from 5.0 mm to 56.7 mm (mean ± standard deviation: ± 10.30) (Table 1). Other patient characteristics are presented in Table 1. Among the 127 HCC nodules, 81 achieved CR 6 mo after TACE according to the mrecist criteria. Local recurrences were classified into the PR, SD, or PD subgroup in 46 nodules. Twenty-six nodules were classified as PR, 13 as SD, and 7 as PD (Table 2). The median recurrence time was 2 mo. Table 2 summarizes the efficacy of TACE for each segment of the liver where the HCC nodule presented in Child-Pugh grade A and B Table 1 Baseline characteristics (n = 115 patients, 127 nodules) n (%) Characteristics patients. Values Age Median (range) 73 (49-92) Sex Male/Female 88 (76.5)/27 (23.5) Etiology Hepatitis B/C/B + C 13 (11.3)/85 (73.9)/2 (1.7) Alcohol 10 (8.7) NASH 2 (1.7) PBC 3 (2.6) Child-Pugh class A/B 99 (86.1)/16 (13.9) Location of target nodules Anterior segment 39 (30.7) Posterior segment 37 (29.1) Medial segment 27 (21.3) Lateral segment 24 (18.9) Size of target nodules (mm) ± ( ) Chemotherapeutic drug Cisplatin/Epirubicin 87 (68.5)/40 (31.5) 1 Values are continuous data and are presented as the means ± SD. Numbers in parentheses are the ranges. NASH: Non-alcoholic steatohepatitis; PBC: Primary biliary cirrhosis. Correlation between HCC location and efficacy The median location coefficient of HCC was significantly higher in the CR group than the non-cr group among all 127 nodules (0.80 vs 0.63, P < 0.001). Among HCCs that presented in the right lobe and medial segment, the median location coefficient of HCC was also significantly higher in the CR group than in the non-cr group (0.81 vs 0.68, P < 0.05, 0.80 vs 0.49, P < 0.01, respectively). However, among HCCs in the lateral segment, there was no significant difference in the location coefficient of HCC between the CR group and non-cr group (0.74 vs 0.73, P > 0.05) (Figure 2 and Table 3). HCC location and efficacy according to Child-Pugh grades In the group classified as Child-Pugh grade A, the median location coefficient of HCC among all nodules in the right lobe and of those in the medial segment was also significantly higher in the CR group than in the non-cr group (0.82 vs 0.62, P < 0.001; 0.71 vs 0.59, P < 0.01; 0.81 vs 0.49, P < 0.05, respectively). However, there was no significant difference in the median location coefficient of HCC in the lateral segment between the CR group and non-cr group (0.67 vs 0.65, P > 0.05) (Table 3 and Figure 3). On the other hand, in the group classified as Child-Pugh grade B, the median location coefficient of HCC was not significantly different between the CR and non-cr 6440 September 21, 2017 Volume 23 Issue 35

85 Miki I et al. HCC location and TACE efficacy A All lobe P < B Right lobe P < 0.05 Location ration of HCC Location ration of HCC Non CR Efficacy of TACE CR 0.2 Non CR CR Efficacy of TACE C Medial segment D Lateral segment P < 0.01 P > 0.05 Location ration of HCC Location ration of HCC Non CR CR Efficacy of TACE Non CR CR Efficacy of TACE Figure 2 Relationship between transcatheter arterial chemoembolization efficacy and hepatocellular carcinoma location. A: All lobes; B: Right lobe; C: Medial segment; D: Lateral segment. The relationship between TACE efficacy and HCC location among all nodules and nodules that are present in the right lobe, medial segment, and lateral segment. TACE: Transcatheter arterial chemoembolization; HCC: Hepatocellular carcinoma; CR: complete response. Table 2 Evaluation of efficacy 6 mo after transcatheter arterial chemoembolization according to the modified response evaluation criteria in solid tumors criteria CR PR SD PD Total All nodules (63.8) (20.5) (10.2) (5.5) Anterior segment Posterior segment Medial segment Lateral segment Patient with Child-Pugh grade A (65.1) (19.8) (11.6) (3.5) Anterior segment Posterior segment Medial segment Lateral segment Patients with Child-Pugh grade B (61.0) (22.0) (7.3) (9.7) Anterior segment Posterior segment Medial segment Lateral segment TACE: Transarterial chemoembolization; CR: Complete response; PR ± Partial response; SD: Stable disease; PD: Progressive disease. groups for nodules in any lobe or segment (Figure 4). Boundary line of HCC location When we performed a ROC curve analysis for the location coefficient of HCC in all nodules, we found that the area under the curve (AUC) was and that the cut-off value was 0.643, with a sensitivity of 56.5% and specificity of 85.2%. In the right lobe and medial segment, the ROC curve AUC was and 0.852, respectively, and the cut-off value was with a sensitivity of 50.0% and specificity of 89.6% and with a sensitivity of 84.6% and specificity of 78.6%, respectively. In the group classified as Child- Pugh grade A, similar results were also obtained in all nodules, the right lobe, and medial segment; the AUC was 0.767, 0.753, and 0.827, respectively, and the cutoff value was 0.743, with a sensitivity of 80.0% and specificity of 69.6%, with a sensitivity of 61.1% and specificity of 84.8%, and with sensitivity of 88.9% and specificity of 77.8%, respectively (Figure 5). DISCUSSION In this retrospective study, we evaluated the relationship between the location of the HCC (in particular, the location close to the peripheral or hepatic portal portion of the liver) and the efficacy of TACE. Among all 127 nodules, the median location coefficient of the HCC was significantly higher in the CR group than in the non-cr group. Although the median location coefficient of HCC in the right lobe and medial segment was also significantly higher in the CR group than in the non- CR group, there was no significant difference in the location coefficient of HCC in the lateral segment September 21, 2017 Volume 23 Issue 35

86 Miki I et al. HCC location and TACE efficacy A All lobe P < B Right lobe P < 0.01 Location ration of HCC Location ration of HCC Non CR CR 0.2 Non CR CR Efficacy of TACE Efficacy of TACE C Medial segment P < 0.05 D Lateral segment P > 0.05 Location ration of HCC Location ration of HCC Non CR CR Efficacy of TACE Non CR CR Efficacy of TACE Figure 3 Results in the group classified as Child-Pugh grade A. A: All lobes; B: Right lobe; C: Medial segment; D: Lateral segment. Relationship between TACE efficacy and HCC location in the group classified as Child-Pugh grade A. TACE: Transcatheter arterial chemoembolization; HCC: Hepatocellular carcinoma; CR: Complete response. Table 3 Hepatocellular carcinoma location coefficient in the complete response and non-complete response groups Non-CR group CR group P value All lobes < Right lobe < 0.05 Medial segment < 0.01 Lateral segment > 0.05 Patients with Child-Pugh grade A All lobes < Right lobe < 0.01 Medial segment < 0.05 Lateral segment > 0.05 Patients with Child-Pugh grade B All lobes > 0.05 Right lobe > 0.05 Medial segment > 0.05 Lateral segment > 0.05 Values are expressed as the medians. HCC: Hepatocellular carcinoma; CR: Complete response. These results suggested that the location of HCC may influence the efficacy of TACE for the treatment of HCC in the right lobe and medial segment. Moreover, considering the liver function represented by the Child- Pugh grade, a significant difference was observed between the location of HCC and the efficacy of TACE only in patients classified as Child-Pugh grade A. In the patients classified as Child-Pugh grade B, the median location coefficient of HCC was not significantly different between the CR group and the non-cr group in each lobe and segment. These results may represent conditions that have hepatic circulatory, anatomical, or biological significance. TACE is a useful palliative treatment for HCC because the hepatic artery delivers > 99% of the blood supply to hepatic tumors. Compact lipiodol uptake results in complete necrosis in the resected tissues [16-19], and complete deposition of iodized oil in HCC lesions correlates with near-total necrosis [20-22]. However, HCCs showing a high uptake of iodized oil may continue to be partially fed by the collateral arteries or the portal vein [23]. Chan et al [24] reported that TACE induces a posttreatment surge of angiogenic factors, such as vascular endothelial growth factor (VEGF), which can occur as early as a few hours after TACE. This process may contribute to revascularization of the tumor, thus leading to the recurrence of HCC and reduced efficacy of TACE [25,26]. Generally, residual HCC tumor cells are observed in the peripheral region of the HCC. The reasons for 6442 September 21, 2017 Volume 23 Issue 35

87 Miki I et al. HCC location and TACE efficacy A 0.9 All lobe P > 0.05 B 0.9 Right lobe P > 0.05 Location ration of HCC Location ration of HCC Non CR Efficacy of TACE CR Non CR Efficacy of TACE CR C 0.8 Medial segment P > 0.05 D 0.75 Lateral segment P > 0.05 Location ration of HCC Non CR Efficacy of TACE CR Location ration of HCC Non CR Efficacy of TACE CR Figure 4 Results in the group classified as Child-Pugh grade B. A: All lobes; B: Right lobe; C: Medial segment; D: Lateral segment. Results of the relationship between TACE efficacy and HCC location in the group classified as Child-Pugh grade B. TACE: Transcatheter arterial chemoembolization; HCC: Hepatocellular carcinoma CR: Complete response. A 1.0 All lobe B 1.0 Right lobe C 1.0 Medical lobe Sensivity (0.800, 0.696) Sensivity (0.611, 0.848) Sensivity (0.889, 0.778) Specificity Specificity Specificity Figure 5 Receiver operating characteristic curve for hepatocellular carcinoma location. A: All lobes; B: Right lobe; C: Medial segment. ROC curve for HCC location as a predictor of TACE efficacy in all lobes, right lobe, and the medial segment in the group classed as Child-Pugh A. Values are the cut-off point (sensitivity and specificity). ROC: Receiver operating characteristic curve; HCC: Hepatocellular carcinoma; TACE: Transcatheter arterial chemoembolization. this are complex and may be partially explained by the following: (1) incomplete embolization or partial recanalization of the hepatic artery after embolization or (2) formation of collateral circulation or the opening of communicating vessels as a microcirculatory reaction. Notably, residual viable tumor cells after TACE are exposed to an extremely hypoxic or even anoxic environment. Ischemic hypoxia is considered a physiological stimulus for angiogenesis, including endothelial cell proliferation [22,23]. Tumor cells induce neovascularization by producing VEGF, which in turn can nourish more tumor cells. Thus, neovascularization and VEGF expression may be important reasons for the survival of residual tumor cells after TACE. There are several reasons why the median location coefficient of HCC in the right lobe and medial segment 6443 September 21, 2017 Volume 23 Issue 35

88 Miki I et al. HCC location and TACE efficacy was significantly higher in the CR group than in the non-cr group. An in vivo microscopic study [12] indicated a direct and indirect reduction in the blood flow to the periphery of the main lobe. Moreover, several reports have described functional and hemodynamic differences between the right and left lobes or between the peripheral (near the subcapsular area) and the central areas (near the porta hepatic) in the liver [27-30]. Okubo et al [31], in a study with gadoliniumethoxybenzyl-diethylenetriamine pentaacetic acidenhanced MRI, reported that the right lobe or central zone showed significantly more enhancement than the left lobe or peripheral zone. The hemodynamic differences between the peripheral and central zones and between the right and left lobes could cause the differences in hepatic enhancement. These reports suggested that vascularity around the HCC in the peripheral area is less than that in the central area, and TACE may induce much more ischemia in the HCC tissue in the peripheral area than in the central area because of protecting the formation of collateral circulation or the opening of communicating vessels. We also evaluated why a significant difference was found only in the right lobe and medial segment and not in the lateral segment. The abovementioned reports suggested that there were differences between the right and left lobes. However, the medial segment is in the left lobe. We do not have a definitive answer to this question, but several phenomena may provide clues. First, thin anastomosis branches often present between the right hepatic and the middle hepatic arteries [32]. Second, cirrhotic livers associated with all etiologies demonstrate atrophy of the right lobe and medial segment as well as hypertrophy of the caudate lobe and lateral segment [11]. These phenomena suggest that differences in the frequency of revascularization between the central and peripheral areas also exist in the medial segment. Our study demonstrated a significant difference between the location of HCC and the efficacy of TACE only in the Child-Pugh grade A patients. In Child- Pugh grade B patients, there was no relationship between any nodules, even in the right lobe and medial segment. These results suggested that hepatic hemodynamics may change depending on cirrhotic liver status. Indeed, qualitative analysis indicated that the parenchymal enhancement in the cirrhotic liver was significantly heterogeneous compared with that in the normal liver [33]. Moreover, the quantitative liver-spleen contrast ratio was significantly decreased with an elevation of the Child-Pugh score [31]. These reports suggest that hemodynamic features are different depending on cirrhotic liver status. In our study, considering a cut-off value of the location coefficient of HCC on the ROC curve, a boundary line that demonstrated a significant, effective difference in patients with Child-Pugh grade A was expected in the range of in the right lobe and median segment. Considering these results and the changing hepatic hemodynamics that depend on cirrhotic liver status, we concluded that livers with advanced cirrhosis had atrophy of the peripheral area in the right lobe and medial segment. Consequently, there was no significant difference between the location of the HCC and the efficacy of TACE in Child-Pugh grade B patients. A novel finding in our study was that the median location coefficient of HCC in the right lobe and medial segment was significantly higher in the CR group than in the non-cr group. Therefore, the coefficient could be used as an index when choosing an HCC treatment strategy. When HCC is located in the peripheral area in the right lobe or medial segment, TACE is much more suitable for treatment. However, when HCC is located in the central area in the right lobe or medial segment, an early combination of local ablation therapy (such as radiofrequency ablation and percutaneous ethanol injection) with TACE may be efficacious. Indeed, the synergistic effect of such an approach has already been demonstrated in both nonsurgical and recurrent HCC patients [34-36]. Therefore, the combination of local ablation therapy with TACE may be considered as a treatment of HCC in the central area. There are several limitations in our study. First, there were very few HCC nodules in the caudate lobe in our study. If there were more HCCs in the caudate lobe, further studies could evaluate the difference in the efficacy of TACE in each lobe or segment in detail. Second, the baseline point of the center when measuring the HCC location was the bifurcation of the portal vein because of few portal vein variations in this study. The HCC location coefficient and the boundary line, demonstrating the difference in efficacy of TACE between the peripheral area and central area, may change when other baseline points, such as the bifurcation of the right and left branches of the hepatic artery, are defined. Therefore, further studies are necessary to confirm our findings. In conclusion, our study suggested that improved efficacy of TACE may be obtained for treatment of HCC in the peripheral area of the right lobe and medial segment in Child-Pugh grade A patients. ACKNOWLEDGMENTS We appreciate the contributions of Dr. Nobuhiko Taniai (Gastrointestinal and Hepato-Biliary-Pancreatic Surgery), Dr. Keiko Kaneko, and Dr. Takeshi Fukuda (Gastroenterology and Hepatology Medicine, Nippon Medical School) to this work. COMMENTS Background Transarterial chemoembolization (TACE) is the current standard of care for patients with Barcelona 6444 September 21, 2017 Volume 23 Issue 35

89 Miki I et al. HCC location and TACE efficacy clinical liver cancer stage B, which includes a highly heterogeneous group of patients. TACE has a positive effect on patient outcome and survival. Studies have shown that tumor number, maximal tumor diameter, Child-Pugh score, and tumor response to TACE are fundamentally strong predictors of survival in patients with initially unresectable hepatocellular carcinoma (HCC). Most HCC patients have some degree of cirrhosis. Microcirculatory changes play a role in the development of cirrhosis within segments. Histopathological investigations of HCCs that were resected after TACE have shown that the most viable tissue is located at the tumor periphery. Considering all these factors, it is very important for the physician to distinguish effective and ineffective TACE before performing this procedure. In this study, we evaluated the relationship between HCC location-in particular, a location close to the peripheral or hepatic portal portion of the liver-and TACE efficacy. Research frontiers Predicting whether TACE will be effective before performing this procedure is very important for the physician, especially while considering other therapeutic modalities. The results of this study evaluating the relationship between HCC location and TACE efficacy may prove to be an index when determining a HCC treatment strategy. Innovations and breakthroughs Several studies and well-designed randomized trials have shown that TACE has a positive effect on patient outcome and survival. However, no previous study has evaluated the relationship between the efficacy of this procedure and HCC. This retrospective study evaluated the relationship between the location of HCC and the efficacy of TACE and suggested that an improved efficacy of TACE may be obtained for treatment of HCC in the peripheral area of the right lobe and medial segment in patients with Child-Pugh grade A tumors. For patients with Child-Pugh grade B or HCC located in the central area or in the lateral segment, it might be difficult to obtain complete necrosis with TACE only. Applications This study suggests that improved TACE efficacy may be obtained for HCC in the peripheral zone of the right lobe and the medial segment in Child-Pugh grade A patients. When HCC is located in the central area in the right lobe or the medial segment, an early combination of local ablation therapy with TACE may be efficacious. Terminology TACE: A minimally invasive procedure performed in interventional radiology to reduce a tumor s blood supply. Child-Pugh score: A score to assess the prognosis of liver cirrhosis. Peer-review In this retrospective study, the authors evaluated the relationship between the location of the HCC (in particular, the location close to the peripheral or hepatic portal portion of the liver) and the efficacy of TACE. It is an interesting study on TACE for HCC. The results demonstrated a significant difference between the location of HCC and the efficacy of TACE only in the Child-Pugh grade A patients. When HCC is located in the peripheral area in the right lobe or medial segment, TACE is much more suitable for treatment. When HCC is located in the central area in the right lobe or medial segment, an early combination of local ablation therapy (such as radiofrequency ablation and percutaneous ethanol injection) with TACE may be efficacious. These findings will provide very important clinical guidance for the selection of TACE for HCC. REFERENCES 1 Parkin DM, Bray F, Ferlay J, Pisani P. 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Liver function test by gadolinium-ethoxybenzyl-diethylenetriamine pentaacetic acidenhanced magnetic resonance imaging with consideration of intrahepatic regional differences. Hepatogastroenterology 2013; 60: [PMID: ] 32 Hiramatsu K, Koda E, Mori M, Isobe Y. X-ray Anatomy of the Abdominal Vascular System. Tokyo: Igaku-Shoin; 1982: [PMID: ] 33 Tamada T, Ito K, Higaki A, Yoshida K, Kanki A, Sato T, Higashi H, Sone T. Gd-EOB-DTPA-enhanced MR imaging: evaluation of hepatic enhancement effects in normal and cirrhotic livers. Eur J Radiol 2011; 80: e311-e316 [PMID: DOI: / j.ejrad ] 34 Cheng BQ, Jia CQ, Liu CT, Fan W, Wang QL, Zhang ZL, Yi CH. Chemoembolization combined with radiofrequency ablation for patients with hepatocellular carcinoma larger than 3 cm: a randomized controlled trial. JAMA 2008; 299: [PMID: DOI: /jama ] 35 Ishii H, Okada S, Sato T, Nose H, Okusaka T, Yoshimori M, Takayasu K, Takayama T, Kosuge T, Yamasaki S. 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91 Miki I et al. HCC location and TACE efficacy Hepatogastroenterology 1996; 43: [PMID: ] 36 Sato M, Watanabe Y, Iseki N, Ueda S, Kawach K, Kimura S, Itoh Y, Ohkubo K, Onji M. Chemoembolization and percutaneous ethanol injection for intrahepatic recurrence of hepatocellular carcinoma after hepatic resection. Hepatogastroenterology 1996; 43: [PMID: ] P- Reviewer: El-Bendary MM, Qin JM, Rodriguez-Peralvarez ML S- Editor: Ma YJ L- Editor: A E- Editor: Ma YJ 6447 September 21, 2017 Volume 23 Issue 35

92 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) ORIGINAL ARTICLE Retrospective Study Evaluation of recurrence in gastric carcinoma: Comparison of contrast-enhanced computed tomography and positron emission tomography/computed tomography Jong Hyeon Kim, Suk Hee Heo, Jin Woong Kim, Sang Soo Shin, Jung Jun Min, Seong Young Kwon, Yong Yeon Jeong, Heoung Keun Kang Jong Hyeon Kim, Suk Hee Heo, Jin Woong Kim, Yong Yeon Jeong, Heoung Keun Kang, Department of Radiology, Chonnam National University Medical School, Chonnam National University Hwasun Hospital, Jeonnam , South Korea Sang Soo Shin, Department of Radiology, Chonnam National University Medical School, Chonnam National University Hospital, Gwangju , South Korea Jung Jun Min, Seong Young Kwon, Department of Nuclear Medicine, Chonnam National University Medical School, Chonnam National University Hwasun Hostpital, Jeonnam , South Korea ORCID number: Jong Hyeon Kim ( ); Suk Hee Heo ( ); Jin Woong Kim ( ); Sang Soo Shin ( ); Jung Jun Min ( ); Seong Young Kwon ( ); Yong Yeon Jeong ( ); Heoung Keun Kang ( ). Author contributions: Kim JH and Jeong YY made substantial contributions to conception and design and analysis of data; Heo SS, Kim JW, Min JJ and Kwon SY contributed to acquisition of data; Kang HK and Shin SS reviewed the literature. Institutional review board statement: This study was reviewed and approved by the Chonnam National University Hwasun Hospital Institutional Review Board. Informed consent statement: Informed consent was waived because of the retrospective nature of the study and the analysis used anonymous clinical data. Conflict-of-interest statement: The authors declare that there are no conflicts of interest in publishing this article. Data sharing statement: No additional data are available. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Unsolicited manuscript Correspondence to: Yong-Yeon Jeong, MD, Chonnam National University Hwasun Hospital, 322 Seoyang-ro, Hwasuneup, Hwasun-gun, Jeonnam , South Korea. yjeong@jnu.ac.kr Telephone: Fax: Received: June 12, 2017 Peer-review started: June 13, 2017 First decision: July 17, 2017 Revised: July 26, 2017 Accepted: August 25, 2017 Article in press: August 25, 2017 Published online: September 21, 2017 Abstract AIM To compare the value of contrast-enhanced abdominal computed tomography (CT) and fluorodeoxyglucose 6448 September 21, 2017 Volume 23 Issue 35

93 Kim JH et al. Gastric carcinoma follow-up: CT and PET/CT (FDG) positron emission tomography/computed tomography (PET/CT) for detecting gastric carcinoma recurrence. METHODS We retrospectively examined data from 2475 patients who underwent both contrast-enhanced abdominal CT and FDG PET/CT for the surveillance of gastric carcinoma curative resection. Patients had an interval of less than 1 mo between their CT and PET/CT scans. Sixty patients who had recurrence were enrolled. Among 1896 patients who did not have recurrence, 60 were selected by simple random sampling. All CT and PET/CT images were reviewed retrospectively by two reviewers blinded to all clinical and pathologic information except curative resection due to gastric carcinoma. RESULTS The pathological stage of the recurrence group was statistically significantly higher than that of the control group (P < 0.001). In the 60 patients who had recurrence, there were 79 recurrent lesions. Fortyfour patients had only one location of recurrence, 13 patients had two locations, and 3 patients had three. In the detection of patient-based overall recurrence, no statistically significant differences existed between the two modalities (P = 0.096). However, for peritoneal carcinomatosis, CT had a statistically significantly higher sensitivity compared to PET/CT (96% vs 50%, P = 0.001). Adenocarcinoma was the most common type of gastric carcinoma. On the pathology-based analysis, CT also had a statistically significantly higher sensitivity compared to PET/CT (98% vs 80%, P = 0.035). CONCLUSION Contrast-enhanced CT was superior to PET/CT in the detection of peritoneal carcinomatosis and pathologic type of adenocarcinoma. Key words: Gastric carcinoma; Surgery; Contrastenhanced abdominal computed tomography; Fluorodeoxyglucose positron emission tomograph/ computed tomography; Surveillance; Recurrence The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: Contrast-enhanced abdominal computed tomography (CT) and positron emission tomography/ computed tomography (PET/CT) are commonly used imaging methods for surveillance of recurrence after gastric cancer surgery. However, the ideal method for early detection of gastric carcinoma recurrence remains controversial. In this study, we compared the value of contrast-enhanced abdominal CT and PET/CT for detecting the recurrence of gastric carcinoma after curative resection. We found that contrast-enhanced CT was superior to PET/CT in the detection of peritoneal carcinomatosis and pathologic type of adenocarcinoma. Kim JH, Heo SH, Kim JW, Shin SS, Min JJ, Kwon SY, Jeong YY, Kang HK. Evaluation of recurrence in gastric carcinoma: Comparison of contrast-enhanced computed tomography and positron emission tomography/computed tomography. World J Gastroenterol 2017; 23(35): Available from: URL: DOI: INTRODUCTION Gastric carcinoma is a leading cause of cancer death worldwide [1]. In Asia, the incidence of gastric carcinoma is high. Its 5-year survival rate is 55%-66%, and a major cause of death after curative surgery for gastric carcinoma is recurrence with a substantial number of patients experiencing such recurrence [2-4]. Although the recurrence rate of early gastric cancer after curative resection is very low, advanced gastric cancer cases show a high recurrence rate after curative resection [5,6]. Several methods exist to detect the recurrence of gastric carcinoma after surgery, including tumor markers, endoscopy, and imaging studies [7]. Elevated tumor marker levels such as those of carcinoembryonic antigen (CEA) and cancer antigen 19-9 (CA 19-9) can be associated with tumor recurrence [8]. Non-systemically, endoscopy can be used to detect locoregional tumor recurrence [9]. Contrast-enhanced abdominal computed tomography (CT) and fluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) are cross-sectional imaging modalities that can be used to detect locoregional and distant recurrence. However there is no specific guideline or indication for using CT or PET/CT in surveillance for recurrence after gastric cancer surgery. Contrast-enhanced abdominal CT is a commonly used imaging method because it is widely available and relatively inexpensive [10]. Despite its widespread use, however, there have been controversies about the role of CT, compared to PET/CT, in evaluating the recurrence of gastric carcinoma after curative surgery. Previous reports showed that PET or PET/CT might be useful in the evaluation of the recurrent gastric cancer after gastric cancer surgery [10-12]. Additional use of PET/ CT in contrast-enhanced CT studies could improve the detection of recurrence and provide other information such as an unexpected secondary malignancy [7]. On the other hand, PET or PET/CT might not be suitable for the detection and confirmation of recurrence after gastric cancer surgery [13,14]. Some reports have shown that contrast-enhanced CT and PET/CT have no substantial differences in their ability to detect gastric cancer recurrence after curative resection, except for detecting peritoneal carcinomatosis [7]. Thus, the ideal method for early detection of gastric carcinoma recurrence remains controversial [10,11,13-15]. Generally, previous studies reviewed images without 6449 September 21, 2017 Volume 23 Issue 35

94 Kim JH et al. Gastric carcinoma follow-up: CT and PET/CT being blinded to the clinical findings and results of the imaging studies, or only evaluated PET or PET/CT for detecting recurrence after gastric surgery [10-12,14,16,17]. The aim of this study was to compare the efficacy of CT and PET/CT in detecting recurrence after curative gastric surgery, when using blinded clinical and imaging findings. MATERIALS AND METHODS Patient selection This retrospective study was approved by the Institutional Review Board. Informed consent was waived because of the retrospective nature of the study. The data of 2475 patients who subsequently underwent both contrast-enhanced abdominal CT and PET/CT for the surveillance of gastric carcinoma recurrence after curative resection, from December 2011 to June 2015, were analyzed. Patients all had an interval of less than 1 mo between their CT and PET/ CT scans. Of the 2475 patients, 519 who did not have a diagnosis validated by histopathological confirmation or subsequent imaging study least 6 mo were excluded. Sixty patients had recurrence confirmed by histopathologic evaluation or subsequent imaging study least 6 mo. Twenty eight patients had pathologic confirmation and 32 patients had imaging confirmation by serial contrast-enhanced CT, making a total of 60 patients. Radiologically, recurrence was defined to be present when a suspicious lesion showed the interval increment in size during serial imaging studies. In the remaining 1896 patients without recurrence, a control group of 60 patients was selected by simple random sampling (Figure 1). In case of patients who had recurrence and underwent both CT and PET/CT many times with intervals of less than 1 mo, the first CT or PET/CT images suspected to show recurrence were analyzed. In case of patients who had no recurrence and underwent both CT and PET/CT many times with intervals of less than 1 mo, the first CT and PET/CT images were analyzed. Contrast-enhanced CT and FDG PET/CT scanning Contrast-enhanced abdominal CT was performed with either 16 or 64-detector row CT scanner (LightSpeed 16 or LightSpeed VCT; GE Healthcare, Milwaukee, WI, United States) with the following parameters; 120 kv, ma, slice thickness: 3.75 mm, slice increments: 3.75 mm, and pitch: 0.984:1. Automated tube current modulation (AutomA; GE Healthcaere, Milwaukee, WI, United States) was routinely used for all patients. AutomA was set between 100 and 300 ma with a noise index of 15. Single-phase (portal venous phase) contrast-enhanced CT was performed after intravenous injection of ml of iopromide (Ultravist ; Schering, Berlin, Germany) at a rate of 2-3 ml/s. The total amount of injected contrast material was adjusted according to the body weight of the patients (2 ml/kg). Scanning was started 90 s after the intravenous injection, spanning from the liver dome to the symphysis pubis. 18 F-FDG PET/CT was performed with a Discovery ST PET/CT system (GE Healthcare), consisting of a bismuthgermanate full scanner and a 16-detectorrow CT scanner. The patients fasted for at least 6 h prior to the intravenous administration of 18 F-FDG (7.4 MBq per body weight). At 60 min after 18 F-FDG administration, transmission data were acquired using low-dose CT (120 kv, automated from 10 to 130 ma, 50 cm field of view (FOV), a scan length of s and a slice thickness of 3.75 mm, and a rotation time of 0.7 s), extending from the base of the skull to the proximal thighs. Immediately after CT acquisition, PET emission scans were acquired in the same anatomic locations with a 15.7 cm axial FOV acquired in the twodimensional mode with 3 min/bed position. The CT data were used for attenuation correction. The images were reconstructed using a conventional iterative algorithm. A workstation (Xeleris) providing multiplanar reformatted images was also used for image display and analysis. Image analysis All CT and PET/CT images were reviewed retrospectively, and recurrence was defined by the consensus of two experienced abdominal radiologists (22 and 12 years of experience) and two experienced nuclear medicine physicians (20 and 12 years of experience). Reviewers were blinded to all clinical and pathological information about the patients except for their curative resection of gastric carcinoma. We classified recurrent lesions into 5 categories: (1) Locoregional recurrence; (2) Lymph node recurrence; (3) Liver metastasis; (4) Peritoneal carcinomatosis; and (5) Other recurrence, for recurrence not included in categories 1-4. In CT images, locoregional recurrence was defined as showing wall thickening with contrast enhancement of the anastomotic site and adjacent remnant stomach [18]. Lymph nodes more than 10 mm long in the short axis were considered lymph node recurrence. A nearly round shape, spiculated or indistinct borders, central necrosis, and marked or heterogeneous enhancement were integrated with the CT size criteria [19]. Liver metastasis was defined as a hypoattenuating lesion with peripheral rim enhancement [20]. Nodular-plaque or infiltrative soft tissue stranding in the mesentery or omentum, parietal peritoneal thickening and contrast enhancement with or without ascites was considered peritoneal carcinomatosis [21]. Focal hypermetabolic activity (> 3.0 SUV) in the anastomotic site and adjacent remnant stomach was considered locoregional recurrence [7]. Lymph nodes were graded as malignant or benign based on functional criteria (increased metabolism relative to the surrounding lymph nodes) independent of their size [22] September 21, 2017 Volume 23 Issue 35

95 Kim JH et al. Gastric carcinoma follow-up: CT and PET/CT Castric carcinoma patients undergone CT and PET/CT after curative resection The interval of CT and PET/CT was less than 1 mo (n = 2475) Excluded patients - Recurrence did not validated by histopathologic confirmation nor subsequent imaging study follow-up at least 6 mo (n = 519) Patients without recurrence (n = 1896) Patients with recurrence (n = 60) Randomly selected patients without recurrence (n = 60) Figure 1 Flow diagram of study patients. CT: Computed tomography; PET/CT: Positron emission tomography/computed tomography. Table 1 Patient characteristics n (%) Characteristics Recurrence group (n = 60) Control group (n = 60) P value Age (yr) Median Range Sex Male 37 (62) 16 (27) Female 23 (38) 44 (73) Operation Total 17 (28) 12 (20) gastrectomy Subtotal 43 (72) 48 (80) gastrectomy AJCC stage ⅠA 0 (0) 33 (55) < ⅠB 5 (8) 10 (17) ⅡA 7 (12) 9 (15) ⅡB 9 (15) 3 (5) ⅢA 7 (12) 1 (2) ⅢB 10 (16) 2 (3) ⅢC 15 (25) 2 (3) Ⅳ 7 (12) 0 (0) Pathology Adenocarcinoma 51 (85) 55 (92) Signet ring cell 5 (8) 2 (3) carcinoma Mucinous adenocarcinoma 4 (7) 3 (5) Interval between CT and PET/CT (d) Median < Range AJCC: American Joint Committee on Cancer (7 th edition); CT: Computed tomography; PET/CT: Positron emission tomography/computed tomography. Focal hypermetabolic activity greater than adjacent normal liver was considered liver metastasis [23]. Diffuse hypermetabolism spreading throughout the abdominopelvic cavity, obscuring visceral outlines and randomly located discrete foci of uptake anteriorly within the abdomen or dependently within the pelvis and unrelated to solid viscera or nodal stations was considered peritoneal carcinomatosis [19]. Statistical analysis Data from the reviewers were analyzed separately. The characteristics of patients with and without recurrence were compared using Student s t test for noncategorical variables and the χ 2 test for categorical variables. Sensitivity, specificity, and accuracy were calculated. McNemar s test was conducted for comparing the diagnostic efficacy of contrast-enhanced abdominal CT and PET/CT in patients-, lesion- and pathological type-based analysis. A P-value of < 0.05 was considered to indicate a significant difference. RESULTS Patient characteristics Table 1 details the patient characteristics. The pathological stage of the recurrence group was statistically significantly higher than that of the control group (P < 0.001). In the control group, the stage of 33 patients (55%) was Ia and none of the patients was IV. In the recurrence group, none of the patients was Ia and seven patients were. These patients did not have distant metastasis in their preoperative clinical stage. After the curative surgery, five patients had distant lymph node metastasis and two patients had peritoneal metastasis as revealed by peritoneal washing cytology. The median interval between contrast-enhanced CT and PET/CT was statistically significantly longer in the recurrence group than in the control group (P < 0.001). Other patient characteristics did not differ. Characteristics of recurrence In the 60 patients who had recurrence, there were 79 recurrent lesions (Table 2). Forty-four patients had only one location of recurrence, 13 patients had two locations, and 3 patients had three. Locations of the other recurrence sites (n = 18) were intestines (n = 6451 September 21, 2017 Volume 23 Issue 35

96 Kim JH et al. Gastric carcinoma follow-up: CT and PET/CT A B Figure 2 Peritoneal carcinomatosis detected only on contrast-enhanced computed tomography in a 46-year-old woman who underwent gastrectomy for advanced gastric cancer 17 mo prior. A: Contrast-enhanced abdominal computed tomography shows enhanced peritoneal thickening (arrow) with ascites. Peritoneal metastasis was confirmed by histological analysis of ascitic fluid. B: Positron emission tomography/computed tomography image at the same level without evident hypermetabolism in the peritoneum. A B Figure 3 Locoregional recurrence detected on both contrast-enhanced computed tomography and positron emission tomography/computed tomography in a 76-year-old woman who underwent gastrectomy for advanced gastric cancer 21 mo prior. A: Contrast-enhanced abdominal computed tomography shows enhanced wall thickening (arrow) at the anastomotic site; B: Positron emission tomography/computed tomography image at the same level shows hypermetabolism at the anastomotic site. Table 2 Locations of recurrent lesions Location Number of lesions n (%) Locoregional recurrence 10 (13) Lymph node recurrence 24 (30) Liver metastasis 3 (4) Peritoneal carcinomatosis 24 (30) Other recurrence 1 18 (23) Total 79 (100) 1 Other recurrence included intestine, bone, ovary, etc. 7), bone (n = 5), ovaries (n = 3), spleen (n = 2), and pancreas (n = 1). Detection of recurrence in contrast-enhanced CT and PET/CT For the detection of patient-based overall recurrence, CT had a sensitivity of 97%, a specificity of 97%, and an accuracy of 97%, whereas PET/CT had a sensitivity of 82%, a specificity of 95%, and an accuracy of 88%. These differences were not statistically significant (P = 0.096; Table 3). Two patients with recurrence did not have it detected by CT, and 12 patients who had recurrence did not have it detected by PET/CT. For the detection of lesion-based overall recurrence, CT had a sensitivity of 86%, a specificity of 98%, and an accuracy of 97%, whereas PET/CT had a sensitivity of 76%, a specificity of 98%, and an accuracy of 96%; these differences were not statistically significant (P = 0.089). For peritoneal carcinomatosis, CT had a sensitivity of 96%, a specificity of 100%, and an accuracy of 99%, whereas PET/CT had a sensitivity of 50%, a specificity of 100%, and an accuracy of 90%, a statistically significant difference (P = 0.001; Figure 2). For detecting locoregional, and lymph node recurrences, and liver metastasis, there were no statistically significant differences between CT and PET/CT (Table 3, Figures 3 and 4). For detecting bone metastasis, CT had a sensitivity of 20%, a specificity of 100%, and an accuracy of 97%, whereas PET/CT had a sensitivity of 100%, a specificity of 100%, and an accuracy of 100%, these differences were not statistically significant (P = 0.125; Figure 5).Two patients who had locoregional recurrence with inadequate bowel distension were missed on CT. Another two patients with locoregional recurrence were missed on PET/CT, and their 6452 September 21, 2017 Volume 23 Issue 35

97 Kim JH et al. Gastric carcinoma follow-up: CT and PET/CT Table 3 Comparison of contrast-enhanced computed tomography and positron emission tomography/computed tomography in the detection of recurrence Site Imaging Sensitivity (%) Specificity (%) Positive predictive value (%) Negative predictive value (%) Accuracy (%) Overall CT 97 (58/60) 97 (58/60) 97 (58/60) 97 (58/60) 97 (114/120) PET/CT 82 (49/60) 95 (57/60) 94 (49/52) 84 (57/68) 88 (106/120) Locoregional CT 80 (8/10) 100 (110/110) 100 (8/8) 98 (112/110) 98 (118/120) PET/CT 80 (8/10) 99 (109/110) 89 (8/9) 98 (109/111) 98 (117/120) Lymph node CT 92 (22/24) 99 (95/96) 96 (22/23) 98 (95/97) 98 (117/120) PET/CT 88 (21/24) 99 (95/96) 95 (21/22) 97 (95/98) 97 (116/120) Liver CT 67 (2/3) 96 (112/117) 29 (2/7) 99 (112/113) 95 (114/120) PET/CT 100 (3/3) 98 (115/117) 60 (3/5) 100 (115/115) 98 (118/120) Peritoneal carcinomatosis P value CT 96 (23/24) 100 (96/96) 100 (23/23) 99 (96/97) 99 (119/120) PET/CT 50 (12/24) 100 (96/96) 100 (12/12) 89 (96/108) 90 (108/120) Total lesion CT 86 (68/79) 98 (511/521) 87 (68/78) 98 (511/522) 97 (579/600) PET/CT 76 (60/79) 98 (513/521) 88 (60/68) 96 (513/532) 96 (573/600) Numbers in parentheses are raw data. CT: Computed tomography; PET/CT: Positron emission tomography/computed tomography. A B Figure 4 Lymph node recurrence detected on both contrast-enhanced computed tomography and positron emission tomography/computed tomography in a 73-year-old man who underwent gastrectomy 18 mo prior. A: Contrast-enhanced abdominal computed tomography shows enlarged lymph nodes at the aortocaval and para-aortic spaces; B: Positron emission tomography/computed tomography image at the same level shows hypermetabolism at the aortocaval and para-aortic spaces. recurrences were concealed by physiological uptake of remnant stomach. Two patients who had lymph node recurrence of less than 10 mm in the short axis were missed on CT, but were detected on PET/ CT. Three patients who had lymph node recurrence without significant hypermetabolism were missed on PET/CT. One hepatic metastasis (about 1.5 cm) was not detected on CT and the metastatic tumor had no attenuation difference compared to the adjacent liver. One patient with peritoneal carcinomatosis was considered as having peritonitis on CT, because he also had a very large abdominal abscess. Twelve patients who had peritoneal carcinomatosis without significant hypermetabolism were missed on PET/CT. Adenocarcinoma was the most common type of gastric carcinoma (Table 1). On the pathological type-based analysis, CT had a sensitivity of 98%, a specificity of 95%, and an accuracy of 96% for detecting recurrence of adenocarcinoma, whereas PET/CT had a sensitivity of 80%, a specificity of 95%, and an accuracy of 88%; these differences were not statistically significant (P = 0.035; Table 4). For detecting the recurrence of signet ring cell carcinoma and mucinous adenocarcinoma patients, CT and PET/ CT had no significant differences. DISCUSSION The recurrence rate of early gastric cancer after curative resection is very low and PET/CT is not commonly used for these patients. However advanced gastric cancer cases show a high recurrence rate. Contrast-enhanced abdominal CT and PET/CT are commonly used imaging methods, but the ideal method for early detection of gastric carcinoma recurrence remains controversial. In terms of the patient characteristics, the pathological stages of the recurrence group were statistically significantly higher than those of the control group. In the recurrence group, the stages of 33 patients (55%) were Ia, none was Ia, and seven patients were IV. The median time interval between the contrast-enhanced CT and PET/CT was also statistically significantly longer in the recurrence group than in the control group. One of the inclusion criteria for our study was an interval shorter than 1 mo between the CT and PET/CT examination. Most of the patients in the control group were prescribed both CT and PET/CT around the same day for their regular follow ups. All 6453 September 21, 2017 Volume 23 Issue 35

98 Kim JH et al. Gastric carcinoma follow-up: CT and PET/CT Table 4 Comparison of contrast-enhanced computed tomography and positron emission tomography/computed tomography in the detection of recurrence according to pathological type Type Imaging Sensitivity (%) Specificity (%) Positive predictive value (%) Negative predictive value (%) Accuracy (%) P value Overall CT 97 (58/60) 97 (58/60) 97 (58/60) 97 (58/60) 97 (114/120) PET/CT 82 (49/60) 95 (57/60) 94 (49/52) 84 (57/68) 88 (106/120) Adenocarcinoma CT 98 (50/51) 95 (52/55) 94 (50/53) 98 (52/53) 96 (102/106) PET/CT 80 (41/51) 95 (52/55) 93 (41/44) 84 (52/62) 88 (93/106) Signet ring cell carcinoma Mucinous adenocarcinoma CT 100 (5/5) 100 (2/2) 100 (5/5) 100 (2/2) 100 (7/7) 1 PET/CT 80 (4/5) 100 (2/2) 100 (4/4) 67 (2/3) 86 (6/7) CT 75 (3/4) 100 (3/3) 100 (3/3) 75 (3/4) 86 (6/7) 1 PET/CT 100 (4/4) 100 (3/3) 100 (4/4) 100 (3/3) 100 (7/7) Numbers in parentheses are raw data. CT: Computed tomography; PET/CT: Positron emission tomography/computed tomography. A B Figure 5 Bone metastasis detected only on positron emission tomography/computed tomography in a 47-year-old man who underwent gastrectomy for advanced gastric cancer 16 mo prior. A: Contrast-enhanced abdominal computed tomography showing the absence of bone lesions; B: Positron emission tomography/computed tomography image at the same level showing multiple hypermetabolic lesions in the pelvic bones and sacrum. patients underwent only CT, and were only sometimes prescribed both CT and PET/CT for regular follow up. However, when recurrence was suspected in a regular follow-up CT in the recurrence group, an additional PET/CT was prescribed to increase confidence or evaluate the recurrent lesion. Our institution is a tertiary referral hospital, and appointments for imaging studies were therefore delayed. For detecting locoregional and lymph node recurrence, the diagnostic performance of CT and PET/CT was not significantly different in our study, which is in agreement with previous studies [7,13]. Locoregional recurrence manifests as localized bowel wall thickening of the anastomotic site or remnant stomach on CT. However, inadequate bowel distension, surgical plication, bowel adhesion, and stomal polypoid hypertrophic gastritis can lower the specificity of the CT [23]. In PET/CT, focal hypermetabolism of the anastomotic site or remnant stomach was considered locoregional recurrence; however, physiological uptake in the bowel can compromise the diagnostic performance of PET-CT [7]. These disadvantages of both contrast-enhanced CT and PET/CT contribute to decreased diagnostic performance in the detection of recurrent gastric cancer. In our study, some locoregional recurrences were missed on CT with inadequate bowel distension and some were missed on PET/CT with physiological uptake in the stomach. In this study, PET/CT was more sensitive than CT in detecting hepatic metastasis (100% vs 66%), but the number of patients who had hepatic metastases was too small to show statistical significance. Kinkel et al [16] reported that PET was the most sensitive method for the detection of hepatic metastases from gastrointestinal-tract cancer. However, other studies showed no significant difference between CT and PET/ CT in detecting hepatic metastases [7,13]. CT was more sensitive for peritoneal carcinomatosis compared with PET/CT in this study. Previous studies showed similar results [7,11,13]. The detection of peritoneal carcinomatosis is difficult in cross-sectional images because the lesions are usually small (below 1 cm) and often flat. On PET/CT, physiological peritoneal FDG uptake due to involuntary muscle activity during scanning can hide underlying peritoneal metastases [14]. Therefore, patients who suspected to have peritoneal carcinomatosis in contrast-enhanced abdominal CT may not see any benefit in any additional PET/CT due to its moderate sensitivity. For bone metastasis, CT had a detection sensitivity of 20% compared to 100% for PET/CT in our study. However, this difference was not statistically significant. Previous research showed that, PET/CT was more sensitive in detecting bone metastasis than CT mostly because it provides functional information [24]. However, the number of patients with bone metastasis enrolled in this study was too small to show statistical significance September 21, 2017 Volume 23 Issue 35

99 Kim JH et al. Gastric carcinoma follow-up: CT and PET/CT In our study, CT was more sensitive than PET/CT in detecting the recurrence of adenocarcinoma, contrary to the findings of previous studies [7,25]. Considering the high proportion of patients with a pathological type of adenocarcinoma was 88.6% in this study, CT efficacy may be superior to that of PET/CT. There have been reports that FDG avidity depends on histological type. Mucinous adenocarcinoma and signet ring cell carcinoma have low FDG avidity [25]. Although the number of mucinous and signet right cell carcinoma cases was not large, there was no statistically significant difference in detecting recurrence. This study had two limitations. The first was its retrospective study design; thus, the indications for PET/CT were not defined and it was not possible to standardize the methods of follow-up imaging studies. This could cause selection bias, because patients who had not undergone CT or PET/CT were excluded. Secondly, not all the recurrent lesions were diagnosed histopathologically. Deeply located lymph node metastasis is difficult to histopathologically confirm in clinical practice. Practically, however, a clinical follow-up is a useful diagnostic method to confirm recurrence. It was regarded as a negative lesion when there was no change between images obtained with at least a 6 mo interval, as in a previous study [13]. But 6 mo of imaging follow up may not be enough to confirm the absence of recurrence. In conclusion, the overall diagnostic performance of contrast-enhanced CT and PET/CT in patient- and lesion-based analysis was not significantly different in our study. However, contrast-enhanced CT was superior to PET/CT in the detection of peritoneal carcinomatosis. In patients with the most common pathological type of adenocarcinoma, contrastenhanced CT might be more sensitive in detecting recurrence than PET/CT. Contrast-enhanced CT could thus be the primary method of surveillance for the recurrence of gastric carcinoma after curative resection. COMMENTS Background Gastric carcinoma is still a leading cause of cancer death worldwide. Surgical resection is the potentially curative treatment for gastric cancer. Contrastenhanced abdominal computed tomography (CT) and positron emission tomography/computed tomography (PET/CT) are commonly used imaging methods for surveillance of recurrence after gastric cancer surgery. But ideal method for early detection of gastric carcinoma recurrence remains controversial. Research frontiers Contrast-enhanced CT was superior to PET/CT in the detection of peritoneal carcinomatosis and pathologic type of adenocarcinoma. Contrast-enhanced CT could thus be the primary method of surveillance for the recurrence of gastric carcinoma after curative resection. Innovations and breakthroughs Contrast-enhanced abdominal CT and PET/CT are commonly used imaging methods for surveillance of recurrence after gastric cancer surgery. But there have been controversies about the role of CT, compared to PET/CT, in evaluating the recurrence of gastric carcinoma after curative surgery. We found contrast-enhanced CT was superior to PET/CT in the detection of peritoneal carcinomatosis and pathologic type of adenocarcinoma Applications Contrast-enhanced CT could be the primary method of surveillance for the recurrence of gastric carcinoma after curative resection. Peer-review This manuscript is very interesting and should be published in priority after minimal revision has been made. It is a retrospective study comparing the value of contrast enhanced abdominal CT and the fluorodeoxyglucose. Positron emission tomography/computed tomography for detecting recurrence of gastric carcinoma after curative resection. REFERENCES 1 Siegel R, Ma J, Zou Z, Jemal A. Cancer statistics, CA Cancer J Clin 2014; 64: 9-29 [PMID: DOI: / caac.21208] 2 Park JM, Kim YH. Current approaches to gastric cancer in Korea. Gastrointest Cancer Res 2008; 2: [PMID: ] 3 D Angelica M, Gonen M, Brennan MF, Turnbull AD, Bains M, Karpeh MS. Patterns of initial recurrence in completely resected gastric adenocarcinoma. Ann Surg2004; 240: [PMID: DOI: /01.sla ] 4 Markar SR, Karthikesalingam A, Jackson D, Hanna GB. Longterm survival after gastrectomy for cancer in randomized, controlled oncological trials: comparison between West and East. Ann Surg Oncol 2013; 20: [PMID: DOI: /s ] 5 Lai JF, Xu WN, Noh SH, Lu WQ. Effect of World Health Organization (WHO) Histological Classification on Predicting Lymph Node Metastasis and Recurrence in Early Gastric Cancer. Med Sci Monit 2016; 22: [PMID: DOI: /msm ] 6 Moriguchi S, Maehara Y, Korenaga D, Sugimachi K, Nose Y. Risk factors which predict pattern of recurrence after curative surgery for patients with advanced gastric cancer. Surg Oncol 1992; 1: [PMID: DOI: / (92)90034-i] 7 Kim DW, Park SA, Kim CG. Detecting the recurrence of gastric cancer after curative resection: comparison of FDG PET/CT and contrast-enhanced abdominal CT. J Korean Med Sci 2011; 26: [PMID: DOI: /jkms ] 8 Shimada H, Noie T, Ohashi M, Oba K, Takahashi Y. Clinical significance of serum tumor markers for gastric cancer: a systematic review of literature by the Task Force of the Japanese Gastric Cancer Association. Gastric Cancer 2014; 17: [PMID: DOI: /s ] 9 Lee SY, Lee JH, Hwang NC, Kim YH, Rhee PL, Kim JJ, Paik SW, Rhee JC, Sohn TS, Kim S. The role of follow-up endoscopy after total gastrectomy for gastric cancer. Eur J Surg Oncol 2005; 31: [PMID: DOI: /j.ejso ] 10 Jadvar H, Tatlidil R, Garcia AA, Conti PS. Evaluation of recurrent gastric malignancy with [F-18]-FDG positron emission tomography. Clin Radiol 2003; 58: [PMID: DOI: /s (02) ] 11 Park MJ, Lee WJ, Lim HK, Park KW, Choi JY, Kim BT. Detecting recurrence of gastric cancer: the value of FDG PET/ CT. Abdom Imaging 2009; 34: [PMID: DOI: /s ] 12 Lee JW, Lee SM, Son MW, Lee MS. Diagnostic performance of FDG PET/CT for surveillance in asymptomatic gastric cancer patients after curative surgical resection. Eur J Nucl Med Mol Imaging 2016; 43: [PMID: DOI: / s ] 6455 September 21, 2017 Volume 23 Issue 35

100 Kim JH et al. Gastric carcinoma follow-up: CT and PET/CT 13 Sim SH, Kim YJ, Oh DY, Lee SH, Kim DW, Kang WJ, Im SA, Kim TY, Kim WH, Heo DS, Bang YJ. The role of PET/CT in detection of gastric cancer recurrence. BMC Cancer 2009; 9: 73 [PMID: DOI: / ] 14 De Potter T, Flamen P, Van Cutsem E, Penninckx F, Filez L, Bormans G, Maes A, Mortelmans L. Whole-body PET with FDG for the diagnosis of recurrent gastric cancer. Eur J Nucl Med Mol Imaging 2002; 29: [PMID: DOI: / s ] 15 Cayvarlı H, Bekiş R, Akman T, Altun D. The Role of 18F-FDG PET/CT in the Evaluation of Gastric Cancer Recurrence. Mol Imaging Radionucl Ther 2014; 23: [PMID: DOI: /mirt.83803] 16 Kinkel K, Lu Y, Both M, Warren RS, Thoeni RF. Detection of hepatic metastases from cancers of the gastrointestinal tract by using noninvasive imaging methods (US, CT, MR imaging, PET): a meta-analysis. Radiology 2002; 224: [PMID: DOI: /radiol ] 17 Baiocchi GL, Marrelli D, Verlato G, Morgagni P, Giacopuzzi S, Coniglio A, Marchet A, Rosa F, Capponi MG, Di Leo A, Saragoni L, Ansaloni L, Pacelli F, Nitti D, D Ugo D, Roviello F, Tiberio GA, Giulini SM, De Manzoni G. Follow-up after gastrectomy for cancer: an appraisal of the Italian research group for gastric cancer. Ann Surg Oncol 2014; 21: [PMID: DOI: /s ] 18 Ha HK, Kim HH, Kim HS, Lee MH, Kim KT, Shinn KS. Local recurrence after surgery for gastric carcinoma: CT findings. AJR Am J Roentgenol 1993; 161: [PMID: DOI: /ajr ] 19 Lim JS, Yun MJ, Kim MJ, Hyung WJ, Park MS, Choi JY, Kim TS, Lee JD, Noh SH, Kim KW. CT and PET in stomach cancer: preoperative staging and monitoring of response to therapy. Radiographics 2006; 26: [PMID: DOI: /rg ] 20 Federle MP, Raman SP. Diagnostic Imaging: Gastrointestinal: Elsevier Health Sciences, Available from: URL: federle/ Kim KA, Park CM, Park SW, Cha SH, Seol HY, Cha IH, Lee KY. CT findings in the abdomen and pelvis after gastric carcinoma resection. AJR Am J Roentgenol 2002; 179: [PMID: DOI: /ajr ] 22 Kwak JY, Kim JS, Kim HJ, Ha HK, Yu CS, Kim JC. Diagnostic value of FDG-PET/CT for lymph node metastasis of colorectal cancer. World J Surg 2012; 36: [PMID: DOI: /s ] 23 Kim KW, Choi BI, Han JK, Kim TK, Kim AY, Lee HJ, Kim YH, Choi JI, Do KH, Kim HC, Lee MW. Postoperative anatomic and pathologic findings at CT following gastrectomy. Radiographics 2002; 22: [PMID: DOI: /radiographics g02mr23323] 24 Liu NB, Zhu L, Li MH, Sun XR, Hu M, Huo ZW, Xu WG, Yu JM. Diagnostic value of 18F-FDG PET/CT in comparison to bone scintigraphy, CT and 18F-FDG PET for the detection of bone metastasis. Asian Pac J Cancer Prev 2013; 14: [PMID: DOI: /APJCP ] 25 Kim SK, Kang KW, Lee JS, Kim HK, Chang HJ, Choi JY, Lee JH, Ryu KW, Kim YW, Bae JM. Assessment of lymph node metastases using 18F-FDG PET in patients with advanced gastric cancer. Eur J Nucl Med Mol Imaging 2006; 33: [PMID: DOI: /s ] P- Reviewer: Misiakos EP, Garcia-Olmo D S- Editor: Qi Y L- Editor: A E- Editor: Ma YJ 6456 September 21, 2017 Volume 23 Issue 35

101 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) ORIGINAL ARTICLE Retrospective Study Duodenum and ventral pancreas preserving subtotal pancreatectomy for low-grade malignant neoplasms of the pancreas: An alternative procedure to total pancreatectomy for low-grade pancreatic neoplasms Xing Wang, Chun-Lu Tan, Hai-Yu Song, Qiang Yao, Xu-Bao Liu Xing Wang, Chun-Lu Tan, Hai-Yu Song, Xu-Bao Liu, Department of Pancreatic Surgery, West China Hospital, Sichuan University, Chengdu , Sichuan Province, China Qiang Yao, West China School of Public Health, Sichuan University, Chengdu , Sichuan Province, China ORCID number: Xing Wang ( ); Chun-Lu Tan ( ); Hai-Yu Song ( ); Qiang Yao ( X); Xu-Bao Liu( ). Author contributions: Wang X and Liu XB designed the research; Wang X, Tan CL and Yao Q performed the research and analyzed the data; Wang X, Tan CL and Song HY wrote the paper; Liu XB proofread and revised the manuscript; all authors approved the version to be published. Conflict-of-interest statement: No potential conflicts of interest relevant to this article were reported. Data sharing statement: No additional data are available. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Unsolicited manuscript Correspondence to: Xu-Bao Liu, MD, PhD, Department of Pancreatic Surgery, West China Hospital, Sichuan University, No. 37, Guo Xue Alley, Chengdu , Sichuan Province, China. xbliu@medmail.com.cn Telephone: Fax: Received: June 13, 2017 Peer-review started: June 16, 2017 First decision: July 17, 2017 Revised: July 27, 2017 Accepted: August 15, 2017 Article in press: August 15, 2017 Published online: September 21, 2017 Abstract AIM To describe the indications, technique and outcomes of the novel surgical procedure of duodenum and ventral pancreas preserving subtotal pancreatectomy (DVPPSP). METHODS Data collected retrospectively from 43 patients who underwent DVPPSP and TP between 2009 and 2015 in our single centre were analysed. For enrolment, only patients with low-grade pancreatic neoplasms, such as pancreatic neuroendocrine tumors, intraductal papillary mucinous neoplasms (IPMNs), and solid pseudopapillary tumors, were included. Ten DVPPSP (group 1) and 13 TP (group 2) patients were selected in this study. RESULTS There were no significant differences in age, gender, comorbidities, preoperative symptoms, American Society of Anesthesiologists score or indications for surgery between the two groups. The most common indication was IPMN for DVPPSP and TP (60% vs 85%, P = 0.411). Compared with the TP group, the DVPPSP group had 6457 September 21, 2017 Volume 23 Issue 35

102 Wang X et al. DVPPSP are safe and effective comparable postoperative morbidities (P = 0.405) and mortalities (both nil), but significantly shorter operative time (232 ± 19.6 min vs 335 ± 32.3 min, P < 0.001). DVPPSP preserved better long-term pancreatic function with less supplementary therapy (P < 0.001) and better quality of life (QoL) after surgery, including better scores in social (P = 0.042) and global health (P = 0.047) on functional scales and less appetite loss (P = 0.049) on the symptom scale. CONCLUSION DVPPSP is a feasible and safe procedure that could be an alternative to TP for low-grade neoplasms arising from the body and tail region but across the neck region of the pancreas; DVPPSP had better metabolic function and QoL after surgery. Key words: Low-grade malignant neoplasm; Ventral pancreas preserving; Subtotal pancreatectomy; Quality of life The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: Although total pancreatectomy (TP) can be performed safely, the long-term metabolic complications prevent its application. Herein, we report our experience of duodenum and ventral pancreas preserving subtotal pancreatectomy (DVPPSP), which could be an alternative procedure to TP for low-grade pancreatic neoplasms, with a focus on the surgical indications, techniques and outcomes. Our results indicate that DVPPSP is a feasible and safe procedure that could be an alternative to TP for low-grade neoplasms arising from the body and tail region but across the neck region of the pancreas, with a better metabolic function and quality of life after surgery. Wang X, Tan CL, Song HY, Yao Q, Liu XB. Duodenum and ventral pancreas preserving subtotal pancreatectomy for lowgrade malignant neoplasms of the pancreas: An alternative procedure to total pancreatectomy for low-grade pancreatic neoplasms. World J Gastroenterol 2017; 23(35): Available from: URL: v23/i35/6457.htm DOI: i INTRODUCTION Parenchyma-sparing procedures are increasingly performed for low-grade pancreatic neoplasms, such as intraductal papillary mucinous neoplasms (IPMNs), neuroendocrine tumors (NETs), and pseudo-papillary tumors (SPTs) [1,2]. These procedures preserve the pancreatic parenchyma and decrease the risk of longterm endocrine and exocrine dysfunction [3,4]. However, according to different reports, in these low-grade diseases, the percentages of total pancreatectomy (TP) range from 5% to 28% because these diseases frequently involve the entire gland [5-7]. The indications for primary elective TP are typically these pancreatic diseases. Although TP could be performed safely in many centres, the long-term metabolic complications that include brittle diabetes and unmanageable steatorrhea prevent its application. Additionally, wide resection of the total pancreas and duodenum after TP could lead to a reduced quality of life (QoL), which makes patients anguished. The conventional parenchyma-sparing procedures, such as enucleation and central pancreatectomy, are unable to substitute TP for lesions involving the head, body and tail of the pancreas. As a function preserving procedure, Thayer et al [8] reported a complete dorsal pancreatectomy with preservation of the ventral pancreas (the proximal duodenum was resected for ischemia) in Duodenum-preserving pancreatic head resection was introduced into surgical practice in This surgical method has been well developed by Doctor Beger et al [9] in past decades. The rationale to apply a duodenumpreserving pancreatic resection is to avoid resection of the gastric antrum, to preserve the duodenum and the extrahepatic biliary duct for low-grade lesions. This procedure has low short-term postoperative morbidity and mortality, and maintenance of the endocrine and exocrine functions in the long-term after surgery. A significant superiority of the duodenum-preserving procedure regarding overall improvement of QoL was reported [10]. Herein, we report our experience of duodenum and ventral pancreas preserving subtotal pancreatectomy (DVPPSP), which could be an alternative procedure to TP for selected patients with low-grade pancreatic neoplasms. The aim of our present study was mainly to describe the indications, operative technique and outcomes of the novel surgical procedure compared with TP. MATERIALS AND METHODS From January 2009 to January 2015, patients who underwent DVPPSP for low-grade pancreatic neoplasms (such as NETs, IPMNs and SPTs) were identified in our institution. To evaluate the outcomes of DVPPSP compared with TP, patients who underwent TP for low-grade pancreatic neoplasms were also identified during this period. Surgeries for pancreatic ductal adenocarcinoma and chronic pancreatitis determined on postoperative pathology were excluded. The selected patients were divided into two groups as follows: DVPPSP group (group 1) and TP group (group 2). The groups were analysed regarding age, gender, type of comorbidities, body mass index (BMI), American Society of Anesthesiologists score (ASA), operative time, blood transfusion and postoperative data (histology of pancreatic disease, postoperative 6458 September 21, 2017 Volume 23 Issue 35

103 Wang X et al. DVPPSP are safe and effective mortality and morbidity, postoperative length of hospital stay). The postoperative mortality rate included all deaths within 30 d after surgery. The morbidity rate included all complications following surgery until discharge. Major postoperative morbidities were defined and graded using the criteria recommended by the International Study Group of Pancreatic Surgery, including postoperative pancreatic fistula, delayed gastric emptying, and postpancreatectomy haemorrhage [11-13]. Biliary leak was defined as drainage of any volume of fluid from percutaneous drains or a wound consistent with bile, which was defined by a bilirubin concentration greater than the serum concentration. Enteric leaks were identified using radiographic contrast imaging. Fluid collection in the intra-abdominal or pleural regions was determined via radiographic imaging, which was differentiated from abscess by positive microbial cultures. Pneumonia was diagnosed based on chest X-ray changes following antibiotic therapy. Follow-up examinations were collected from the store of medical information centre in the hospital and from the results of follow-up by letters and telephone at the time of this study. The endocrine and exocrine controls were assessed by episodes of hyperglycaemia/day, daily dosage of insulin, pancreatic enzyme replacement. The QoL of patients were evaluated with the European Organization for Research and Treatment of Cancer Quality of life Questionnaire (EORTC QLQ-C30) version 3.0. The questionnaires were requested to be filled in by the patients themselves via mailing or completing in the clinic. The patients who had been dead or had tumor recurrence during the study were excluded from the analysis of QoL. This study was approved by the Ethics Committee of Sichuan University. Surgical indication DVPPSP was an alternative operative technique to TP of low-grade pancreatic tumor arising from the body and tail region but across the neck region of the pancreas (Figure 1). Contrast-enhanced computed tomography (CT), magnetic resonance imaging (MRI), or/and endoscopic ultrasound (EUS) were routinely performed to evaluate the lesions. Furthermore, DVPPSP was finally considered after intraoperative evaluation of the lesion, which confirmed negative for neoplasm of ventral pancreas based on palpation and intraoperative ultrasonography. Frozen sectioning was routinely performed to achieve negative margins in the present study. If invasive carcinoma was found in the frozen section of the stump, the DVPPSP was immediately converted to TP with formal lymphadenectomy. Surgical technique The surgical procedures were performed by one group of experienced surgeons in our department of pancreatic surgery. We performed a complete Kocher Figure 1 Preoperative computed tomography view of a 26-year-old female patient with a diagnosis of pseudo-papillary tumors. The figure shows that the lesion occupied the body and neck region with necrosis of the tail on preoperative computed tomography imaging. Normal pancreatic tissue is observed in the ventral pancreas region. manoeuvre to mobilize the duodenum, the pancreatic head and uncinate process from the posterior connective tissue membrane. There was a call for intraoperative ultrasound to confirm that the ventral pancreas was negative for neoplasm. Then, the neck and body of the pancreas was freed from the common hepatic artery, portal vein, and superior mesenteric vein. The gastrocolic trunk was divided and ligated at its confluence. Then, we dissected and taped the gastroduodenal artery (GDA). The anterior superior pancreaticoduodenal artery (ASPD) and other small branches of GDA in front of the cephalic pancreas could be resected to expose the common bile duct (CBD) at the upper edge of the pancreas and to completely resect the neoplasm. We preserved the GDA and the posterior superior pancreaticoduodenal artery (PSPD) (Figure 2). The transection of the cephalic side of the pancreas was begun at the edge of the neoplasm and was continued caudally. The posterior transection plane was along the anterior side of the CBD (Figure 2). Inferiorly, we dissected and exposed the CBD from the resecting pancreas toward the major papilla to achieve complete resection of the neoplasm. If the neoplasm involved the entire dorsal pancreas, such as IPMN that involved the whole dorsal duct (including the Santorini duct) but ventral duct (Figure 3), a complete dorsal pancreatectomy was needed to achieve radical resection of the tumor. The embryological fusion plane between the ventral and the dorsal pancreas was confirmed by autopsy [14] and was described as a clear fibrous septum [8]. These anatomical characteristics made the complete dorsal pancreatectomy procedure theoretically feasible. However, the plane was difficult to identify in our study during surgery. Instead, we continued pancreatic transection and exposed the whole distal CBD (except its last centimetre) to achieve a complete dorsal pancreatectomy and we the confluence of the CBD with the ventral duct. The dorsal pancreatic duct was 6459 September 21, 2017 Volume 23 Issue 35

104 Wang X et al. DVPPSP are safe and effective B A Drain amylase analysis was performed from postoperative day one to three and then every other day. For patients without a fistula, we progressively withdrew drainage starting on postoperative day three and finally removed the drain on postoperative day five. Postoperative octreotide was routinely used for seven days to decrease the secretion of the pancreas. We routinely performed abdominal EUS before finally removing the drain. The long-term follow-up should be performed after DVPPSP, even if the stump is negative. Figure 2 Intraoperative view of duodenum and ventral pancreas preserving subtotal pancreatectomy. A: Superior mesenteric vein; B: Ventral pancreas. Black arrow: intrapancreatic bile duct; White arrow: gastroduodenal artery. Statistical analysis The Pearson χ 2 test was used for categorical variables. Fisher s exact test was used in cases with a variable count < 5. For continuous variables, Student s t test was used for normally distributed variables and the Wilcoxon rank-sum test was used for non-normally distributed variables. P < 0.05 was considered statistically significant. All analyses were performed using SPSS version 18.0 software on a personal computer (SPSS Inc., Chicago, IL, United States). Figure 3 Preoperative magnetic resonance imaging view of a 71-yearold female patient with diagnosis of MD-IPMN. The figure shows that the lesion involves the whole dorsal duct (including Santorini duct). The diameter of ventral pancreatic duct is normal on the MRCP image. divided and ligated 3 mm away from the duodenal lumen and was free of neoplasia on frozen section examination. The anterior inferior pancreaticoduodenal artery (AIPD) could be included in the resection site if necessary. Then, we identified the main pancreatic duct (Wirsung duct) in the stump of the residual pancreas and ligated this duct. The stump was negative for tumor on frozen sectioning, which was routinely performed in the present study. Parenchyma sutures and tissue sealant were used in selected cases. Bipolar cautery, ultrasound scalpel and the suture technique using 4-0 absorbable sutures (Ethicon, Inc) were used for haemostasis and pancreatostasis. It was worth noting that bipolar cautery was not performed in the deep areas near the Wirsung duct or other important structures (CBD or major vessels) due to presumed heat injury. Parenchymal sutures and tissue sealant were used for cases in which haemostasis and pancreatostasis were not satisfied exclusively using bipolar cautery and ultrasound scalpel. Finally, we routinely placed at least one drain adjacent to the cut surface. RESULTS DVPPSP was performed for 10 patients, and TP was performed for 33 patients between January 2009 and January 2015 in our department. Patients with chronic pancreatitis, pancreatic cancers (PC) and pancreatic metastases were excluded in this study. Of these, twenty patients who underwent TP, including 2 with chronic pancreatitis, 16 with PC, and 2 with pancreatic metastases from renal cell cancer, were excluded in our present study. Finally, ten DVPPSP (group 1) and 13 TP (group 2) patients were selected in this study. The preoperative characteristics of the patients undergoing DVPPSP and TP are summarized in Table 1. There were no significant differences in age, gender, comorbidities, preoperative symptoms, ASA score or indications for surgery between the two groups. The median age was 49 in the DVPPSP group and 56 in the TP group. Four male patients (40%) were in the DVPPSP group, and 7 males (54%) were in the TP group. In the DVPPSP and TP groups, the majority of patients were symptomatic (100% vs 85%), and abdominal pain (40% vs 55%) was the most common symptom. The ASA Ⅲ was given to 3 patients in DVPPSP and TP group. The pathologic features of the lesions in the DVPPSP and TP groups are shown in Table 1. The most common indication for DVPPSP or TP was IPMN (6/10, 60% vs 11/13, 85%). All the 6 IPMNs who underwent DVPPSP were main duct type (MD-IPMN). The 11 IPMNs in the TP group comprised 9 MD-IPMN, 1 mixed-type (MT-IPMN) and 1 multifocal type. According to the 2010 WHO classification, one out of 6 IPMN in the DVPPSP group and 5 out of 11 IPMN in the TP group were detected as invasive carcinoma (16.7% vs 45.5%, P = 0.333). The 2 NETs in the DVPPSP group and the 1 NET in the TP group 6460 September 21, 2017 Volume 23 Issue 35

105 Wang X et al. DVPPSP are safe and effective Table 1 Preoperative characteristics and final pathologic features of 23 patients in the duodenum and ventral pancreas preserving subtotal pancreatectomy and total pancreatectomy groups n (%) Patient characteristics DVPPST (n = 10) TP (n = 13) P vaule Age (range, yr) 49 ± 16.9 (24-71) 56 ± 10.1 (40-70) Gender (%) Male 4 (40) 7 (54) Female 6 (60) 6 (46) Major comorbidity CAD COPD DM Hypertension Preoperative symptoms Incidental 0 2 (15) Symptomatic (%) 10 (100) 11 (85) Abdominal pain 4 (40) 6 (55) Pancreatitis Weight loss Gastrointestinal symptoms ASA score Ⅰ/Ⅱ 7 10 Ⅲ 3 3 Pathology (%) IPMN 6 (60) 11 (85) Low-grade moderate-grade dysplasia High-grade dysplasia Invasive carcinoma 1 (16.7) 5 (45.5) Neuroendocrine 2 1 tumor G SPT 2 1 Total invasive malignancy 1 (10) 5 (38.5) CAD: Coronary artery disease; COPD: Chronic obstructive pulmonary disease; DM: Diabetes mellitus; IPMN: Intraductal papillary mucinous neoplasm; SPT: Pseudo-papillary tumor; DVPPSP: Duodenum and ventral pancreas preserving subtotal pancreatectomy. were classified as G2. The intra-operative and postoperative short-term outcomes of the two groups are summarized in Table 2. None of the parameters listed were significantly different between the two groups except the operative time, which was significantly shorter in the DVPPSP group compared with the TP group (232 min vs 335 min, P < 0.001). The total morbidities were 20% (2/10) and 38% (5/13) in the DVPPSP and TP group, respectively. There was no significant difference in the post-operative morbidity between the two groups, including delay gastric emptying (DGE) and biliary fistula. Clinical pancreatic fistula (grade B or C) was not found in either group. There were no reoperations or readmissions in either group. The mortalities were nil in both groups. Although there was no significant difference, the post-operative hospital stay days (LOS) Table 2 Intraoperative and postoperative outcomes of 23 patients after duodenum and ventral pancreas preserving subtotal pancreatectomy and total pancreatectomy Operative characteristics Intraoperative transfusion (U) DVPPSP (n = 10) Operative time (min) 232 ± 19.6 ( ) TP (n = 13) 0.20 ± 0.63 (0-2) 0.54 ± 1.05 (0-3) 335 ± 32.3 ( ) P vaule < Postoperative characteristics DGE Biliary fistula Pancreatic fistula 0 0 (B and C) Overall morbidity Major morbidity ( grade 3) Reoperation 0 0 Readmission (30 d) 0 0 LOS 9.7 ± 3.8 (7-20) 11.7 ± (8-26) Mortality 0 0 Recurrence 1 (%) 2 (20%) 5 (38.5%) IPMN 2/6 5/ The recurrences of 2 patients in the DVPPSP group and 5 patients in the TP group occurred after surgery for IPMN. DGE: Delayed gastric emptying; LOS: Length of postoperative stay; DVPPSP: Duodenum and ventral pancreas preserving subtotal pancreatectomy. appeared to be slightly shorter in the DVPPSP group (9.7 d vs 11.7 d, P = 0.318). The mean follow-up lengths were 26.6 and 33.9 months in the DVPPSP and TP groups, respectively. There were 2 out of 10 patients in the DVPPSP group and 5 out of 13 patients in the TP group proven to have recurrences (20% vs 38.5%, P = 0.405) (Table 2). Two out of 6 IPMNs in the DVPPSP group were shown to have recurrence at 28 and 36 mo after surgery separately, although negative margins were detected by intra-operative frozen sections. A total of 5 out of 11 IPMNs in the TP group were shown to have recurrence at 16, 26, 30, 34, 50 mo after surgery separately (2/6, 33.3% vs 5/11, 45.5%, P = 1.000). The differences in replacement therapy are summarized in Table 3. The mean follow-up lengths were 26.6 and 33.9 mo in the DVPPSP and TP groups, respectively. There were significant differences in replacement therapy between the two groups. The mean enzyme replacements were 0.9 cps/d vs 9.2 cps/d (P < 0.001) in the DVPPSP and TP groups, respectively. The total insulin doses were 6.4 U/d vs 29.7 U/d (P < 0.001). No patients had hyperglycaemia after insulin treatment in the DVPPSP group, whereas five patients (38%) in the TP group still had hyperglycaemia occasionally (P = 0.046). There were still 2 patients (20%) having diarrhea and steatorrhea occasionally after replacement treatment in the DVPPSP group, compared with 4 patients (31%) in the TP group (P = 0.660). The long term 6461 September 21, 2017 Volume 23 Issue 35

106 Wang X et al. DVPPSP are safe and effective Table 3 Postoperative condition of supplementary therapy of 23 patients after duodenum and ventral pancreas preserving subtotal pancreatectomy and total pancreatectomy Parameters DPPSP (n = 10) TP (n = 13) P vaule Enzymes replacement 0.9 ± ± 2.2 < (cps/d) Total insulin dose 6.4 ± ± 9.3 < (U/d) Diarrhea or steatorrhea Hyperglycemia TP: Total pancreatectomy; DVPPSP: Duodenum and ventral pancreas preserving subtotal pancreatectomy. Table 4 Postoperative condition of diabetes of 10 patients in the duodenum and ventral pancreas preserving subtotal pancreatectomy group Parameters DPPSP (n = 10) New-onset diabetes 0 / 7 Worsen diabetes 1 1/3 Diet to oral medication Diet to insulin Oral medication to insulin 1 1/3 1 There was 3 out of 10 patients in DVPPSP group who had diabetes before surgery and were treated by oral medicine. DVPPSP: Duodenum and ventral pancreas preserving subtotal pancreatectomy. Table 5 Quality of lives after duodenum and ventral pancreas preserving subtotal pancreatectomy and total pancreatectomy Parameters DVPPSP TP (n = 8) P vaule (n = 8) Functional scale Physical 95.0 ± ± 7.1 (80-100) (80-100) Working ability Cognitive Emotional 98.4 ± 2.9 ( ) 92.2 ± 8.0 (75-100) Social 95.8 ± 7.7 ( ) Global QoL 96.9 ± 6.2 ( ) 85.4 ± ( ) 88.5 ± 8.9 (75-100) Symptom scale Fatigue 4.2 ± 8.3 (0-22.2) 11.1 ± 11.7 (0-33.3) Nausea and ± 12.4 (0-33.3) vomiting Pain 0 0 Appetite loss ± 24.8 (0-66.7) Dyspnea 0 0 Sleep disturbance 4.2 ± ± 17.2 (0-33.3) (0-33.3) Constipation ± 11.8 (0-33.3) Diarrhea 4.2 ± ± 24.8 (0-66.7) (0-33.3) Financial strain ± 15.4 (0-33.3) TP: Total pancreatectomy; QoL: Quality of life; DVPPSP: Duodenum and ventral pancreas preserving subtotal pancreatectomy. results regarding pancreatic endocrine function after DVPPSP are summarized in Table 4. Of the 10 patients who had DVPPSP, seven patients had no diabetes before surgery. There were no patients with newonset diabetes. Of the 3 patients who had diabetes before surgery in the DVPPSP group, only one patient (1/3, 33%) had the diabetes worsen after surgery. This patient changed treatment with oral medication to treatment with insulin. The quality of life (QoL) according the EORTC QLQQ-C30 is summarized in Table 5. A total 16 patients were qualified to complete questions about the EORTC QLQ-C30 at the time of this study. Each group included 8 patients. Due to the recurrence of IPMNs, two patients in the DVPPSP group and 5 patients in the TP group mentioned above were excluded from the inquiry. As presented in Table 5, patients in the DVPPSP group could have a higher quality of life status after surgery compared with the TP group. A high scale score in the global health, physical, working ability, emotional, cognitive and social functioning represented a high quality of life status. Regarding the symptoms and financial impact of the disease, the low scale score indicated no important problems. There were significant differences existing in social (P = 0.042) and global health (P = 0.047) on functional scale. There was a significant difference existing in appetite loss (P = 0.049) on the symptom scale. DISCUSSION Although TP could be performed safely as reported by different authors in the literature, patients would suffer from the pain of replacement therapy throughout their lives [15-17]. When faced with benign or lowgrade malignant disease with long life expectancy such as pnets, IPMNs and SPTs, the need for a proper treatment has to be balanced with the risk of long term impairment of exocrine/endocrine function of the gland. Herein, we described the outcomes after 10 DVPPSPs performed for low-grade malignant disease compared with 13 TPs performed for the same spectrum of disease, showing that DVPPSP is safe and effective for preserving the exocrine/endocrine function of the gland. This procedure could be an alternative to TP for low-grade neoplasms arising from the body and tail region but across the neck region of the pancreas. It is a difficult question when we find the lowgrade malignant neoplasm originating from the distal pancreas across the pancreatic neck. TP might bring negative margins but be accompanied by a loss of exocrine/endocrine function. When choosing distal pancreatectomy, positive margins might be inevitable. We used to choose primary or complete TP for this type of neoplasm in our department 8 years ago. After the first DVPPSP was performed successfully, this procedure 6462 September 21, 2017 Volume 23 Issue 35

107 Wang X et al. DVPPSP are safe and effective became our first choice when negative margins could be achieved at the pancreatic head. Intraoperative EUS and frozen sectioning are indispensable to decide where to perform limited pancreatic resection and to achieve negative margins. It is worth noting that only patients with benign or low-grade pancreatic neoplasms are selected as candidates for DVPPSP. The patients with such neoplasms could obtain long disease-free life expectancy if negative margins are achieved [18-20]. If invasive features or any doubts about the low-grade character of the stump were found in the frozen section, DVPPSP was immediately converted to TP with formal lymphadenectomy. Moreover, diseases such as IPMN might predispose the entire gland, especially for the type of main duct, so detailed examination (such as endoscopic ultrasonography) could be needed before DVPPSP to detect the condition of the main duct [21]. DVVPSP is suitable for disease with a normal Wirsung duct in the ventral pancreas. If the entire duct is involved, the ventral pancreas should not be preserved. In this case, duodenum preserving total/subtotal pancreatic resection might be the best choice [22]. Due to the biological features of low-grade malignant neoplasms, normal interstitial space exists between the neoplasm and the peri-pancreatic tissue. This space is the key prerequisite to perform DVPPSP successfully. According to Yoshihiro s research [14] based on autopsy specimens, the dorsal pancreas makes up the pancreatic neck, body, and tail, as well as the anterior segment of the head, whereas the ventral pancreas comprises the posterior segment of the head and uncinate process. Moreover, Thayer et al [8] described the embryonic fusion plane of the two segments as a clear fibrous septum. These anatomic features make the duodenum and ventral pancreas preserving subtotal pancreatectomy (DVPPSP) a theoretically feasible procedure. As this plane was difficult to identify by some researchers [23] and by us in practice, we performed the pancreatic transection along the anterior side of the CBD and exposed the entire distal CBD (except 1-2 cm from the major papilla) to achieve negative margins. The confluence of the CBD with the ventral duct and ventral pancreas was carefully preserved. A case of complete dorsal pancreatectomy was presented previously [8], with the first part of the duodenum resected due to ischemic damage. The gastroduodenal artery (GDA) was ligated in that procedure, while both the gastroduodenal artery and the posterior pancreaticoduodenal arterial arcade were preserved in our series. Additionally, the Kocher manoeuvre was suggested to increase the risk of duodenal ischemia in another previous case report [23]. However, we performed a complete Kocher manoeuvre to make the potential haemorrhage easier to control and to make the surgical procedure smoother during resection of the cephalic side of the pancreas. The duodenum was not found to be cyanotic in our present study with 10 DVPPSP cases. Careful dissection and reservation of the GDA and the posterior pancreaticoduodenal arterial arcade appeared to be essential for preservation of the duodenum in DVPPSP. The successful preservation of the duodenum prevents reconstruction in the upper digestive tract. Our results (Table 2) also demonstrated that, compared with the TP group, DVPPSP had comparable short-term postoperative morbidities. Postoperative pancreatic fistula (POPF) is the most common complication after pancreatectomy [24,25]. However, clinical POPF (Grade B or C) was not detected in the 10 patients who underwent DVPPSP according to the revised definition by the International Study Group on Pancreatic Fistula (ISGPF) [26]. DVPPSP appears to be a safe and feasible procedure for selected lowgrade pancreatic neoplasms based on our results. As shown in Table 2, 33.3% IPMN in the DVPPSP group and 45.5% IPMN in the TP group were shown to have recurrences (P = 1.000). The rate of postoperative recurrence of IPMN appeared slightly high compared with previous reports (0%-30%) [27-29]. The malignant component was suggested to be associated with tumor recurrence [29,30]. The high rate of malignancy (highgrade dysplasia or invasive carcinoma, 13/17, 76.5%; Table 1) of IPMN in our study might play a role in this recurrence, which was reported by others [29,31,32] to range from 35.7% to 68%. Furthermore, the small number of patients prevented us from making strong conclusions. Given the long life expectancy after surgery for these diseases, the postoperative long-term pancreatic function and QoL become a major concern. Compared with the TP group, we found that DVPPSP did preserve better long-term pancreatic function with less supplementary therapy and resulted in better QoL after surgery in the follow-up of our study. No newonset diabetes occurred in the DVPPSP group in the follow-up. Only one out of three patients had diabetes worsen, as shown in Table 4. This reservation could be attributed to less loss of the pancreatic parenchyma in DVPPSP. In recent years, several investigators have advocated that postoperative QoL is as significant a clinical outcome as morbidity and mortality. The QoL of DVPPSP compared with TP in this study appeared to be better no matter replacement therapy or survey of quality of life. The high dependence of pancreatic enzyme replacement and insulin mainly negatively impacted the social and global quality of life in TP patients. The diabetes after TP could cause a negative impact on QoL [33]. More patients in the TP group felt a loss of appetite compared with patients in the DVPPSP group, perhaps because of manual changes of the digestive tract. The enterogastric reflux was considered an 6463 September 21, 2017 Volume 23 Issue 35

108 Wang X et al. DVPPSP are safe and effective unavoidable consequence after reconstruction in the upper digestive tract [34]. The great advantage of DVPPSP is preservation of the natural digestive channel. Other reports have demonstrated better QoL in duodenum preserving pancreatic surgery [35-37]. Our study is limited by the small number of cases because of the infrequent indications for DVPPSP. Some questions need to be researched further. A major limitation of the present study was its retrospective nature, which prevented us from making strong conclusions. Quality of life metrics were administered only once and were not compared with preoperative baseline measurements. In conclusion, DVPPSP is a feasible and safe procedure that could be an alternative to TP for low-grade neoplasms arising from the body and tail region but across the neck region of the pancreas, with a better metabolic function and QoL after surgery. COMMENTS Background Parenchyma-sparing procedures are increasingly performed for low-grade pancreatic neoplasms, such as intraductal papillary mucinous neoplasms (IPMNs), neuroendocrine tumors, and pseudo-papillary tumors. These procedures preserve the pancreatic parenchyma and decrease the risk of long-term endocrine and exocrine dysfunction. The conventional parenchymasparing procedures, such as enucleation and central pancreatectomy, are unable to substitute for total pancreatectomy (TP) for lesions involving the head, body and tail of the pancreas. Herein, we report our experience of duodenum and ventral pancreas preserving subtotal pancreatectomy (DVPPSP), which could be an alternative procedure to TP for low-grade pancreatic neoplasms. Research frontiers Although TP could be performed safely as reported by different authors in studies, patients would suffer from the pain of replacement therapy throughout their lives. These results showed that DVPPSP is safe and effective for preserving the exocrine/endocrine function of the gland, with a better metabolic function and QoL after surgery. DVPPSP could be an alternative to TP for lowgrade neoplasms arising from the body and tail region but across the neck region of the pancreas. Innovations and breakthroughs This study described the indications, operative technique and outcomes of the novel surgical procedure DVPPSP. Furthermore, comparisons between DVPPSP with TP were presented in the present study. The findings indicate that DVPPSP is a feasible and safe procedure and could be an alternative to TP for low-grade neoplasms arising from the body and tail region but across the neck region of the pancreas, with a better metabolic function and QoL after surgery. Applications DVPPSP is a feasible and safe procedure. It could be an alternative to TP for low-grade neoplasms arising from the body and tail region but across the neck region of the pancreas, with a better metabolic function and QoL after surgery. Terminology The DVPPSP is a parenchyma-sparing procedure and could be an alternative to TP for low-grade neoplasms arising from the body and tail region but across the neck region of the pancreas. Peer-review The present study explored a new surgical technique so called duodenum and ventral pancreas preserving subtotal pancreatectomy as a choice of treatment for low-grade pancreatic neoplasms, such as IPMN, neuroendocrine tumors and pseudo-papillary tumors. The results showed DVPPSP was superior to total pancreatectomy by shorter operative time, better postoperative pancreatic function and better quality of life. But there was no detail pathological examination information of surgical specimens and follow-up data about tumor recurrence in the study. 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110 Wang X et al. DVPPSP are safe and effective pancreatic head resection for the treatment of chronic pancreatitis. Pancreas 2012; 41: [PMID: DOI: / MPA.0b013e318221c91b] 37 Lü WP, Shi Q, Zhang WZ, Cai SW, Jiang K, Dong JH. A metaanalysis of the long-term effects of chronic pancreatitis surgical treatments: duodenum-preserving pancreatic head resection versus pancreatoduodenectomy. Chin Med J (Engl) 2013; 126: [PMID: ] P- Reviewer: Zhang CW S- Editor: Qi Y L- Editor: A E- Editor: Ma YJ 6466 September 21, 2017 Volume 23 Issue 35

111 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) ORIGINAL ARTICLE Retrospective Study Validated preoperative computed tomography risk estimation for postoperative hepatocellular carcinoma recurrence Wei Zhang, Shao-Lv Lai, Jie Chen, Dong Xie, Fei-Xiang Wu, Guan-Qiao Jin, Dan-Ke Su Wei Zhang, Shao-Lv Lai, Dong Xie, Guan-Qiao Jin, Dan- Ke Su, Departments of Radiology, Affiliated Tumor Hospital of Guangxi Medical University, Nanning , Guangxi Zhuang Autonomous Region, China Jie Chen, Fei-Xiang Wu, Hepatobiliary Surgery, Affiliated Tumor Hospital of Guangxi Medical University, Nanning , Guangxi Zhuang Autonomous Region, China ORCID number: Wei Zhang ( X); Sha-Lv Lai ( ); Jie Chen ( ); Dong Xie ( ); Fei-Xiang Wu ( X); Dan-Ke Su ( ). Author contributions: Zhang W, Lai SL, Chen J and Xie D contributed equally to this work; Su DK designed the research; Zhang W wrote the manuscript; Zhang W, Lai SL, Chen J and Xie D collected the data; Wu FX and Jin GQ recorded the data; Zhang W, Lai SL, Chen J and Xie D performed the research; Zhang W analyzed the data; Su DK modified the manuscript; All authors gave final approval of this submission. Institutional review board statement:: The study was reviewed and approved by the Affiliated Tumor Hospital of Guangxi Medical University Institutional Review Board. Informed consent statement: Patients were not required to give informed consent to the study because the analysis used anonymized clinical data that were obtained after each patient agreed by written consent to undergo computed tomography and curative resection. Conflict-of-interest statement: The authors declared that there is no conflict of interest related to this study. Data sharing statement: No additional data are available. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Unsolicited manuscript Correspondence to: Dr. Dan-Ke Su, Professor, Department of Radiology, Affiliated Tumor Hospital of Guangxi Medical University, 71 Hedi Road, Nanning , Guangxi Zhuang Autonomous Region, China. sudanke@gxmu.edu.cn Telephone: Fax: Received: June 14, 2017 Peer-review started: June 15, 2017 First decision: July 17, 2017 Revised: July 26, 2017 Accepted: August 15, 2017 Article in press: August 15, 2017 Published online: September 21, 2017 Abstract AIM To develop and validate a risk estimation of tumor recurrence following curative resection of operable hepatocellular carcinoma (HCC). METHODS Data for 128 patients with operable HCC (according to Barcelona Clinic Liver Cancer imaging criteria) who underwent preoperative computed tomography (CT) evaluation at our hospital from May 1, 2013 through May 30, 2014 were included in this study. Follow-up data were obtained from hospital medical records. Follow-up data through May 30, 2016 were used to retrospectively analyze preoperative multiphasic CT 6467 September 21, 2017 Volume 23 Issue 35

112 Zhang W et al. Preoperative prediction of HCC recurrence findings, surgical histopathology results, and serum α-fetoprotein and thymidine kinase-1 levels. The χ 2 test, independent t -test, and Mann-Whitney U test were used to analyze data. A P -value of < 0.05 was considered statistically significant. RESULTS During the follow-up period, 38 of 128 patients (29.7%) had a postoperative HCC recurrence. Microvascular invasion (MVI) was associated with HCC recurrence (χ 2 = , P < 0.001). Despite postoperative antiviral therapy and chemotherapy, 22 of 44 patients with MVI experienced recurrence after surgical resection. The presence of MVI was 57.9% sensitive, 75.6% specific and 70.3% accurate in predicting postoperative recurrence. Of 84 tumors without MVI, univariate analysis confirmed that tumor margins, tumor margin grade, and tumor capsule detection on multiphasic CT were associated with HCC recurrence (P < 0.05). Univariate analyses showed no difference between groups with respect to hepatic capsular invasion, Ki-67 proliferation marker value, Edmondson-Steiner grade, largest tumor diameter, necrosis, arterial phase enhanced ratio, portovenous phase enhanced ratio, peritumoral enhancement, or serum α-fetoprotein level. CONCLUSION Non-smooth tumor margins, incomplete tumor capsules and missing tumor capsules correlated with postoperative HCC recurrence. HCC recurrence following curative resection may be predicted using CT. Key words: Hepatocellular carcinoma; Microvascular invasion; Computed tomography; Recurrence; Tumor margin; Tumor capsule The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: We discuss risk estimation for recurrence following curative resection of operable hepatocellular carcinoma that meets Barcelona Clinic Liver Cancer imaging criteria. Preoperative multiphasic computed tomography findings, including non-smooth tumor margins, incomplete tumor capsule and missing tumor capsule, can predict hepatocellular carcinoma recurrence following curative resection. Treatment should include wide margins during curative resection, followed by antiviral therapy and chemotherapy. Zhang W, Lai SL, Chen J, Xie D, Wu FX, Jin GQ, Su DK. Validated preoperative computed tomography risk estimation for postoperative hepatocellular carcinoma recurrence. World J Gastroenterol 2017; 23(35): Available from: URL: DOI: INTRODUCTION Hepatocellular carcinoma (HCC) is the fifth most common malignancy and the third most common cause of cancer-related death worldwide [1-3]. Surgical resection and liver transplantation are potentially curative treatment options for patients with HCC [4]. However, the 5-year HCC recurrence rate may be as high as 70% [5]. Recent studies have shown that tumor size and number [6], microvascular invasion (MVI) [5,7-9] and α-fetoprotein (AFP) levels [10,11] are associated with recurrence following surgical resection. Although many potential risk factors for HCC postoperative recurrence have been described, a reliable preoperative method to estimate this risk has not been established. Kanai et al [12] first described nodular HCC subclassification based on microscopic, clinical and prognostic features; these being single nodular type, single nodular type with extra-nodular growth, and contiguous multinodular type. Studies have shown that non-smooth tumor margins are associated with increased MVI [13-16]. Although computed tomography (CT) examination is part of the standard of care for HCC evaluation, no study has demonstrated the validity of preoperative CT assessment to predict postoperative recurrence of solitary HCC. Thymidine kinase 1 (TK1), a DNA synthesis enzyme, is an important serum proliferation marker that correlates with clinical stage and is an independent prognostic factor for recurrence-free survival in HCC [17-20]. However, to our knowledge, there are no reports about serum TK1 levels and postoperative HCC recurrence. In this study, we retrospectively assessed whether preoperative CT findings could predict postoperative HCC recurrence. We specifically evaluated largest tumor diameter, tumor margins, tumor capsule, necrosis, peritumoral enhancement, tumor enhanced ratio and hepatic capsular invasion. Furthermore, we assessed whether MVI, serum AFP, and serum TK1 levels were correlated with postoperative recurrence of solitary HCC. MATERIALS AND METHODS Patients This retrospective study included 128 patients (105 males and 23 females), aged 24 to 79 years (mean, 47.7 years), with operable HCC (according to the Barcelona Clinic Liver Cancer imaging criteria) who underwent preoperative CT evaluation at our hospital from May 1, 2013 through May 30, Follow-up data were obtained from hospital medical records. HCC recurrence was defined as a pathologic or radiologic diagnosis of recurrent HCC during the followup period that ended on May 30, All patients 6468 September 21, 2017 Volume 23 Issue 35

113 Zhang W et al. P reoperative prediction of HCC recurrence in this study: (1) had no cancer-related treatment or liver biopsy prior to CT imaging; (2) underwent partial hepatectomy and had HCC diagnosis confirmed by histopathology; (3) had no macrovascular invasion or metastasis on CT imaging; and (4) had livers with a Child-Pugh classification of A or B. This study was approved by our hospital institutional review board and all patients provided written informed consent. CT imaging protocol Hepatic CT images were obtained using a 64-MDCT scanner (SOMATOM Sensation 64; Siemens, Forchheim, Germany) with Z-axis modulation, a spiral pitch of 1, a 5 mm section thickness, a 2 mm reconstruction gap, a field of view of 311 mm, 120 kvp, 230 ma, and a standard reconstruction algorithm. Nonionic contrast medium (300 mg I/mL iopromide) was administered at an injection rate of 3 ml/s for a total dose of 100 ml. For the hepatic arterial and portovenous phases, scanning was begun approximately 25 and 60 s after contrast media injection, respectively. Equilibrium phase images were acquired approximately 180 s after contrast media injection. The scanning range was the whole-liver zone while patients held their breath. Coronary and sagittal images were reconstructed with 5 mm section thickness. Image evaluation Tumor margins on the liver map (i.e. transverse, coronary and sagittal planes) were defined as three subtypes (smooth margin, non-smooth margin focal extranodular and non-smooth margin multinodular), based on previous reports [12-16,21,22]. The tumor margins were categorized into one of the following three grades: Grade 0: smooth margin; Grade 1: focal extranodular type; and Grade 2: multinodular type. The tumor capsule was defined as a linear and enhanced structure surrounding the tumor detected by equilibrium phase CT imaging. Tumor capsules were categorized into the following three groups: Grade 0: complete tumor encapsulation; Grade 1: incomplete tumor encapsulation; and Grade 2: no tumor encapsulation. Negative or positive peritumoral enhancement was determined by multiphase CT imaging. Positive peritumoral enhancement was defined in comparison to liver parenchyma as a hyperdense area proximal to the tumor border during arterial phase imaging that changed to an isodense area during equilibrium phase imaging. The tumor enhanced ratio was calculated as (CTenhanced- CTunenhanced)/CTunenhanced All CT findings were reviewed by two radiologists experienced in liver CT evaluation: Lai SL (22 years of experience) and Xie D (21 years of experience). In the case of disagreements over CT findings, a third radiologist helped resolve discrepancies to achieve consensus. Serum samples and histopathologic analysis The serum TK1 assay was performed using a commercial kit, based on an enhanced chemiluminescence dot blot assay. Serum AFP detection was carried out using enzyme immunoassay. Ki67 expression, hepatic capsular invasion and MVI were confirmed by immunohistochemistry. The Edmondson-Steiner (E-S) grade was determined using the WHO Liver Cancer Study Group guidelines [23]. Statical analysis Statistical analysis was performed using SPSS version 16.0 statistical software (SPSS, Chicago, IL, United States). An independent t-test was used to compare recurrence and non-recurrence groups with respect to largest tumor diameter, arterial phase enhanced ratio, portovenous phase enhanced ratio, serum AFP, serum TK1 and Ki67. Receiver operating characteristic curve analyses were used to determine the optimal cut-off value and diagnostic performance for P < The chi-square test was used to compare tumor margins, necrosis, peritumoral enhancement, MVI and hepatic capsular invasion between groups. The tumor margin grade, E-S grade and tumor capsule were analyzed using the Mann-Whitney U test. A P-value of less than 0.05 was considered statistically significant. RESULTS Quantitative and qualitative findings are summarized in Table 1. MVI was higher in the recurrence group (22 of 38 patients) compared to the non-recurrence group (22 of 90 patients) (χ 2 = , P < 0.001). Despite antiviral therapy and chemotherapy during follow-up, 22 of 44 patients with MVI experienced recurrence after surgical resection. The presence of MVI was 57.9% sensitive, 75.6% specific and 70.3% accurate in predicting postoperative recurrence. Of 84 recurrences without MVI, univariate analyses showed no difference between groups with respect to hepatic capsular invasion (χ 2 = 1.691, P = 0.193), Ki67 value (P = 0.686), E-S grade (Z = , P = 0.646), largest tumor diameter (P = 0.366), necrosis (χ 2 = 0.412, P = 0.521), arterial phase enhanced ratio (P = 0.725), portovenous phase enhanced ratio (P = 0.685), peritumoral enhancement (P = 0.240) or serum AFP level (P = 0.882). In 27 of 84 recurrences without MVI, serum TK1 did not differ between groups (P = 0.377). Univariate analyses showed that nonsmooth tumor margins (χ 2 = 5.042, P = 0.025), tumor margin grade (Z = , P = 0.019) and absence of tumor capsule on CT (Z = , P = 0.023) were 6469 September 21, 2017 Volume 23 Issue 35

114 Zhang W et al. P reoperative prediction of HCC recurrence A B C Table 1 Quantitative and qualitative factors associated with recurrence of hepatocellular carcinoma following curative resection Risk factor Follow-up recurrence P value MVI Positive b MVI-negative cases Hepatic capsular invasion Yes No Negative Positive Negative Ki ± ± E-S grade 1 Ⅰ Ⅱ 8 36 Ⅲ 7 26 Largest tumor diameter, cm 5.5 ± ± Arterial phase enhanced ratio 0.62 ± ± Portovenous phase enhanced ratio 0.77 ± ± Tumor capsule Complete a Incomplete 7 19 Negative 6 14 Necrosis Positive Negative 8 40 Tumor margins Peritumoral enhancement Smooth a Focal extranodular 5 11 Multinodular 2 1 Positive Negative AFP, ng/ml ± ± TK1, U/L 5.7 ± ± E-S classification rubric as in ref. 23. b P < 0.01 and a P < 0.05, statistically significant; Statistics are presented as mean ± SD. AFP: α-fetoprotein; E-S: Edmondson-Steiner; HCC: Hepatocellular carcinoma; MVI: Microvascular invasion; TK1: Thymidine kinase-1. Figure 1 Tumor margins on the liver computed tomography map. A: Portovenous phase CT showing smooth margin at liver segment VI in a 36-year-old male patient without postoperative recurrence (dashed box); B: Portovenous phase CT showing focal extranodular type at liver segments V and VIII in a 38-year-old female patient with postoperative recurrence (dashed box); C: Portovenous phase CT showing multinodular type at liver segments V and VI in a 35-year-old male patient with postoperative recurrence (dashed box). CT: Computed tomography. correlated with tumor recurrence (Figure 1A-C and Figure 2A-C). DISCUSSION Surgical resection of HCC suffers from high rates of tumor recurrence [5]. A reliable and validated prognostic method to estimate individual HCC recurrence risk would help guide future treatment strategies. Factors commonly associated with high risk for postoperative HCC recurrence include tumor vascular invasion and poorly differentiated tumor grade [24]. In this study, however, E-S grade was not associated with tumor recurrence. We did confirm, however, that MVI may be a powerful independent prognostic factor for postoperative recurrence and metastasis [5,7,14,25-27]. Moreover, in this retrospective study, we further validated MVI as an independent prognostic factor correlated with HCC recurrence. A 50% HCC recurrence rate was found following surgical resection for tumors with MVI, despite antiviral therapy and chemotherapy. Thus, the possibility of recurrence for HCC with MVI is substantial. However, MVI can rarely be confirmed preoperatively and, consequently, it is difficult to choose an appropriate procedure (e.g., wide resection margins) to prevent HCC recurrence [28]. Thus, it is important to identify preoperative imaging markers that could predict HCC recurrence. It has been reported that conventional CT could be used to reconstruct global HCC gene expression patterns [29]. When Zhou et al [30] investigated the correlation between CT-based radiomics signature and early HCC recurrence, they identified internal arteries and necrosis as independent risk factors. Their results, which were similar to those of previous studies [5,29], led to their hypothesis that internal arteries may be correlated with MVI. The finding that necrosis is a marker of early HCC recurrence [30], however, is inconsistent with our study findings. We suspect that different follow-up times (1 year in their study vs 2 years in ours), was responsible for this difference September 21, 2017 Volume 23 Issue 35

115 Zhang W et al. P reoperative prediction of HCC recurrence A B C Figure 2 Tumor capsules on the liver computed tomography map. A: Equilibrium phase CT showing complete tumor capsule (red arrow) involvement at liver segment V in a 33-year-old male patient without postoperative recurrence (dashed box); B: Equilibrium phase CT showing incomplete tumor capsule (red arrow) at liver segments V and VI in a 57-year-old female patient with postoperative recurrence (dashed box); C: Equilibrium phase CT showing missing tumor capsule at liver segment VI in a 41-year-old male patient with postoperative recurrence (dashed box). CT: Computed tomography. In this study, we further investigated whether preoperative CT findings correlate with HCC recurrence after surgical resection. Our finding that non-smooth tumor margins on CT could predict tumor recurrence has not been evaluated in previous studies. However, a previous study did show that non-smooth tumor margins on CT were predictive of MVI, and that MVI is an independent predictor of poor outcomes following surgical resection [13]. In our stratified analysis of tumors without MVI, tumor margins were significantly associated with postoperative recurrence during the follow-up period. This finding may be related to HCC multi-centricity being an important independent prognostic factor [31], and tumor aggressiveness should be considered [32]. We believe that non-smooth tumor margins can be considered an independent predictor of HCC recurrence. Adachi et al [33] reported that vessels of the fibrous liver capsule were often invaded by tumor cells and that complete liver capsule fibrosis was a predictor of portal venous invasion. However, complete liver capsule fibrosis has been reported as a favorable prognostic marker because it can prevent HCC invasion into adjacent liver parenchyma [13]. Our results also showed that CT evidence of complete tumor capsules were associated with a low risk of postoperative recurrence. Peritumoral enhancement on magnetic resonance imaging (MRI) has been associated with a high risk of MVI [34]. In our study, peritumoral enhancement on CT was not related to HCC recurrence. The discrepancy in findings may be due to differences in imaging modalities used in various studies. Lu et al [35] reported that, when HCC was larger than 3 cm in diameter, the tumor tended to be more aggressive and was associated with a poor prognosis [36]. However, large tumor diameter was not a significant risk factor for postoperative recurrence in our study. At present, it is clear that HCC nodule size is not the only prognostic factor for recurrence [32]. Additionally, the relationship between postoperative HCC recurrence and AFP level remains controversial [5,10,11]. This study has two limitations. One potential limitation is the use of CT instead of MRI. Although CT is commonly used for HCC evaluation, MRI may provide additional information. A second limitation is the relatively small number of postoperative recurrences, except among those with preoperative evidence of MVI on CT. However, we further analyzed all cases and found similar results for the MVI-positive and MVI-negative groups. Thus, the study sample may reflect true population postoperative recurrences. In conclusion, evaluation of patients with HCC should include preoperative CT findings of non-smooth tumor margins, incomplete tumor capsules and missing tumor capsules, which indicate possible postoperative recurrence. The treatment plan should include wide resection margins during curative resection, followed by antiviral therapy and chemotherapy. COMMENTS Background Surgical resection of hepatocellular carcinoma (HCC) suffers from high rates of tumor recurrence. Although many potential risk factors for HCC postoperative recurrence have been described, a reliable preoperative method to estimate this risk has not been established. It has been reported that conventional computed tomography (CT) could be used to reconstruct global HCC gene expression patterns. Therefore, preoperative CT risk estimation for postoperative HCC recurrence should be clarified. Research frontiers A reliable and validated prognostic method to estimate individual HCC recurrence risk would help guide future treatment strategies. The results of this study contribute to clarifying the correlation of CT signature with postoperative HCC recurrence September 21, 2017 Volume 23 Issue 35

116 Zhang W et al. P reoperative prediction of HCC recurrence Innovations and breakthroughs In the study, preoperative CT provides evidence of non-smooth tumor margins, incomplete tumor capsules and missing tumor capsules, which had been clarified as independent risk factors of postoperative HCC recurrence. Applications This study suggests that preoperative CT is useful for predicting postoperative HCC recurrence. If the preoperative CT findings of a patient are non-smooth tumor margins, incomplete tumor capsules and missing tumor capsules, the treatment plan should include wide resection margins during curative resection, followed by antiviral therapy and chemotherapy. Peer-review The manuscript from Zhang W et al reported the correlation of preoperative CT findings with postoperative HCC recurrence. HCC recurrence following curative resection may be predicted using CT. The entire sets of data are nicely presented, and highly supportive of the conclusion. 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Exp Ther Med 2011; 2: [PMID: DOI: /etm ] 19 Nisman B, Allweis T, Kaduri L, Maly B, Gronowitz S, Hamburger T, Peretz T. Serum thymidine kinase 1 activity in breast cancer. Cancer Biomark 2010; 7: [PMID: DOI: / CBM ] 20 Nisman B, Yutkin V, Nechushtan H, Gofrit ON, Peretz T, Gronowitz S, Pode D. Circulating tumor M2 pyruvate kinase and thymidine kinase 1 are potential predictors for disease recurrence in renal cell carcinoma after nephrectomy. Urology 2010; 76: 513. e1-513.e6 [PMID: DOI: /j.urology ] 21 Eguchi S, Takatsuki M, Hidaka M, Soyama A, Tomonaga T, Muraoka I, Kanematsu T. Predictor for histological microvascular invasion of hepatocellular carcinoma: a lesson from 229 consecutive cases of curative liver resection. World J Surg 2010; 34: [PMID: DOI: / s ] 22 Nakashima Y, Nakashima O, Tanaka M, Okuda K, Nakashima M, Kojiro M. Portal vein invasion and intrahepatic micrometastasis in small hepatocellular carcinoma by gross type. 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117 Zhang W et al. P reoperative prediction of HCC recurrence 462::AID-CNCR >3.0.CO;2-E] 24 Mehta N, Heimbach J, Harnois DM, Sapisochin G, Dodge JL, Lee D, Burns JM, Sanchez W, Greig PD, Grant DR, Roberts JP, Yao FY. Validation of a Risk Estimation of Tumor Recurrence After Transplant (RETREAT) Score for Hepatocellular Carcinoma Recurrence After Liver Transplant. JAMA Oncol 2017; 3: [PMID: DOI: /jamaoncol ] 25 Lim KC, Chow PK, Allen JC, Chia GS, Lim M, Cheow PC, Chung AY, Ooi LL, Tan SB. Microvascular invasion is a better predictor of tumor recurrence and overall survival following surgical resection for hepatocellular carcinoma compared to the Milan criteria. Ann Surg 2011; 254: [PMID: DOI: /SLA.0b013e31821ad884] 26 Kaibori M, Ishizaki M, Matsui K, Kwon AH. Predictors of microvascular invasion before hepatectomy for hepatocellular carcinoma. J Surg Oncol 2010; 102: [PMID: DOI: /jso.21631] 27 Imamura H, Matsuyama Y, Tanaka E, Ohkubo T, Hasegawa K, Miyagawa S, Sugawara Y, Minagawa M, Takayama T, Kawasaki S, Makuuchi M. Risk factors contributing to early and late phase intrahepatic recurrence of hepatocellular carcinoma after hepatectomy. J Hepatol 2003; 38: [PMID: DOI: /S (02) ] 28 Hirokawa F, Hayashi M, Miyamoto Y, Asakuma M, Shimizu T, Komeda K, Inoue Y, Uchiyama K. Outcomes and predictors of microvascular invasion of solitary hepatocellular carcinoma. Hepatol Res 2014; 44: [PMID: DOI: / hepr.12196] 29 Segal E, Sirlin CB, Ooi C, Adler AS, Gollub J, Chen X, Chan BK, Matcuk GR, Barry CT, Chang HY, Kuo MD. Decoding global gene expression programs in liver cancer by noninvasive imaging. Nat Biotechnol 2007; 25: [PMID: DOI: / nbt1306] 30 Zhou Y, He L, Huang Y, Chen S, Wu P, Ye W, Liu Z, Liang C. CTbased radiomics signature: a potential biomarker for preoperative prediction of early recurrence in hepatocellular carcinoma. Abdom Radiol (NY) 2017; 42: [PMID: DOI: /s ] 31 Shimada M, Hamatsu T, Yamashita Y, Rikimaru T, Taguchi K, Utsunomiya T, Shirabe K, Sugimachi K. Characteristics of multicentric hepatocellular carcinomas: comparison with intrahepatic metastasis. World J Surg 2001; 25: [PMID: DOI: /s ] 32 Detry O, Govaerts L, Deroover A, Vandermeulen M, Meurisse N, Malenga S, Bletard N, Mbendi C, Lamproye A, Honoré P, Meunier P, Delwaide J, Hustinx R. Prognostic value of (18)F-FDG PET/CT in liver transplantation for hepatocarcinoma. World J Gastroenterol 2015; 21: [PMID: DOI: /wjg.v21. i ] 33 Adachi E, Maeda T, Kajiyama K, Kinukawa N, Matsumata T, Sugimachi K, Tsuneyoshi M. Factors correlated with portal venous invasion by hepatocellular carcinoma: univariate and multivariate analyses of 232 resected cases without preoperative treatments. Cancer 1996; 77: [PMID: DOI: /(SIC I) ( )77:10<2022::AID-CNCR9>3.0.CO;2-S] 34 Lee S, Kim SH, Lee JE, Sinn DH, Park CK. Preoperative gadoxetic acid-enhanced MRI for predicting microvascular invasion in patients with single hepatocellular carcinoma. J Hepatol 2017; Epub ahead of print [PMID: DOI: /j.jhep ] 35 Lu XY, Xi T, Lau WY, Dong H, Xian ZH, Yu H, Zhu Z, Shen F, Wu MC, Cong WM. Pathobiological features of small hepatocellular carcinoma: correlation between tumor size and biological behavior. J Cancer Res Clin Oncol 2011; 137: [PMID: DOI: /s ] 36 Moribe T, Iizuka N, Miura T, Kimura N, Tamatsukuri S, Ishitsuka H, Hamamoto Y, Sakamoto K, Tamesa T, Oka M. Methylation of multiple genes as molecular markers for diagnosis of a small, welldifferentiated hepatocellular carcinoma. Int J Cancer 2009; 125: [PMID: DOI: /ijc.24394] P- Reviewer: Wan YL S- Editor: Qi Y L- Editor: Filipodia E- Editor: Ma YJ 6473 September 21, 2017 Volume 23 Issue 35

118 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) ORIGINAL ARTICLE Observational Study Diagnostic delay in inflammatory bowel disease increases the risk of intestinal surgery Dong-won Lee, Ja Seol Koo, Jung Wan Choe, Sang Jun Suh, Seung Young Kim, Jong Jin Hyun, Sung Woo Jung, Young Kul Jung, Hyung Joon Yim, Sang Woo Lee Dong-won Lee, Ja Seol Koo, Jung Wan Choe, Sang Jun Suh, Seung Young Kim, Jong Jin Hyun, Sung Woo Jung, Young Kul Jung, Hyung Joon Yim, Sang Woo Lee, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Korea University Ansan Hospital, Ansan-si, Gyeonggido 15355, South Korea ORCID number: Dong-won Lee ( X); Ja Seol Koo ( X); Jung Wan Choe ( ); Sang Jun Suh ( ); Seoung Young Kim ( ); Jong Jin Hyun ( ); Sung Woo Jung ( ); Young Kul Jung ( ); Hyung Joon Yim ( ); Sang Woo Lee ( ). Author contributions: Koo JS contributed to study conception and design; Lee D, Koo JS, Choe JW, Suh SJ, Kim SY, Hyun JJ, Jung SW, Jung YG, Yim HJ and Lee SW contributed to patient recruitment and data collection; Lee D and Koo JS contributed to data generation and analysis; Lee D and Koo JS contributed to manuscript drafting; all authors reviewed, commented upon, and approved the final submission. Institutional review board statement: This study was approved by the Institutional Review Board of Korea University Ansan Hospital (IRB number AS16206). Informed consent statement: The requirement for obtaining informed patient consent was waived because the present study has been based on the retrospective analysis of existing medical records. Conflict-of-interest statement: The authors do not have any disclosures to report. Data sharing statement: No additional data are available. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Unsolicited manuscript Correspondence to: Ja Seol Koo, MD, PhD, Division of Gastroenterology and Hepatology, Department of Internal Medicine, Korea University Ansan Hospital, Ansan-si, Gyeonggi-do 15355, South Korea. jskoo@korea.ac.kr Telephone: Fax: Received: June 14, 2017 Peer-review started: June 15, 2017 First decision: July 17, 2017 Revised: July 28, 2017 Accepted: July 28, 2017 Article in press: August 15, 2017 Published online: September 21, 2017 Abstract AIM To investigate the factors affecting diagnostic delay and outcomes of diagnostic delay in inflammatory bowel disease (IBD) METHODS We retrospectively studied 165 patients with Crohn s disease (CD) and 130 patients with ulcerative colitis (UC) who were diagnosed and had follow up durations > 6 mo at Korea University Ansan Hospital from January 2000 to December A diagnostic delay was 6474 September 21, 2017 Volume 23 Issue 35

119 Lee DW et al. Diagnostic delay in IBD defined as the time interval between the first symptom onset and IBD diagnosis in which the 76 th to 100 th percentiles of patients were diagnosed. RESULTS The median diagnostic time interval was 6.2 and 2.4 mo in the patients with CD and UC, respectively. Among the initial symptoms, perianal discomfort before diagnosis (OR = 10.2, 95%CI: , P = 0.006) was associated with diagnostic delays in patients with CD; however, no clinical factor was associated with diagnostic delays in patients with UC. Diagnostic delays, stricturing type, and penetrating type were associated with increased intestinal surgery risks in CD (OR = 2.54, 95%CI: ; OR = 4.44, 95%CI: ; OR = 3.79, 95%CI: , respectively). In UC, a diagnostic delay was the only factor associated increased intestinal surgery risks (OR = 6.81, 95%CI: ). CONCLUSION A diagnostic delay was associated with poor outcomes, such as increased intestinal surgery risks in patients with CD and UC. Key words: Diagnostic delay; Intestinal surgery; Inflammatory bowel disease; Crohn s disease; Ulcerative colitis The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: As the manifestations of inflammatory bowel disease (IBD) were nonspecific, the diagnosis is often established following considerable delay. There have been few reports about diagnostic delay associated with poor outcomes in Asian IBD patients. We aimed to investigate the factor affecting diagnostic delay and its effect in Korean IBD patients. In present study, a diagnostic delay was significantly associated with poor outcomes, such as increased IBD related intestinal surgery risks in patients with Crohn s disease and ulcerative colitis. Therefore, it is important for the improvement of clinical outcomes in IBD patients to early diagnose and manage adequately. Lee DW, Koo JS, Choe JW, Suh SJ, Kim SY, Hyun JJ, Jung SW, Jung YG, Yim HJ, Lee SW. Diagnostic delay in inflammatory bowel disease increases the risk of intestinal surgery. World J Gastroenterol 2017; 23(35): Available from: URL: DOI: INTRODUCTION Inflammatory bowel disease (IBD) represented by Crohn s disease (CD) and ulcerative colitis (UC) is characterized by chronic inflammation in all or part of digestive tract without definite cause [1]. Persisting and relapsing inflammation causes not only functional dysfunction in the gut, but also structural destruction of bowel such as stenosis, fistula formation and perforation [2]. Recent studies have reported that an early intensive control of inflammation with immunosuppressive agents or biological agents improved the prognosis of newly diagnosed patients with IBD, which emphasized the importance of early treatment following early diagnosis [3-5]. IBD has a waxing and waning course with asymptomatic remission period and with episodes of disease where patients present with symptoms, such as hematochezia, fever, and abdominal pain [6]. Because symptoms were not specific and not constant over time and the findings of diagnostic studies overlapped with those of other disease, such as tuberculosis and connective tissue disease, the diagnosis is often established following a considerable delay [7-9]. Indeed, in the Swiss IBD cohort study, the median duration of diagnostic delay period in CD patients was 9 mo, and about 25% of them had a duration of > 24 mo from symptom to diagnosis [9]. The median diagnostic delay period in patients with UC was 4 mo, which was shorter than that in patients with CD. Further, in the French CD cohort with 364 patients, about 40% of patients had a duration of > 12 mo from symptom to diagnosis [10]. A diagnostic delay was associated with poor clinical outcomes in patients with CD, such as an increased risk of bowel stenosis, fistula and abscess formation, and intestinal surgery [11,12]. However, no study has reported the association between the clinical outcomes and diagnostic delays in patients with UC. Unlike in Western countries, there are few studies on diagnostic delays associated with IBD in Asia. Considering the significant differences in the epidemiological and clinical features of IBD according to ethnicities and environmental factors, diagnostic delays and the associated factors may differ according to countries [13,14]. Therefore, we aimed to investigate the clinical factors and outcomes associated with diagnostic delays in Korean patients with CD and UC. MATERIALS AND METHODS Subjects This study included 177 patients with CD and 143 patients with UC who were definitely diagnosed at Korea University Ansan Hospital from January 1, 2000 to December 31, The CD and UC diagnoses were confirmed based on the clinical, endoscopic, radiological and histological findings. The patients younger than 18 years at the time of diagnosis, those who had a followup duration of < 6 mo, and those who had incomplete medical records were excluded from this study. In the present study, 25 patients with IBD were excluded because they were under 18 years old (CD 3), had insufficient medical records (CD 3, UC 7), or had a 6475 September 21, 2017 Volume 23 Issue 35

120 Lee DW et al. Diagnostic delay in IBD follow up duration time of < 6 mo (CD 5, UC 6). The remaining 165 patients with CD and 130 patients with UC were analyzed in the present study This study was approved by the Institutional Review Board of Korea University Ansan Hospital (AS16206) and conducted in accordance with the Declaration of Helsinki. The need for informed consent was waived in view of the retrospective observational design of the study. Methods The clinical and demographic data such as age at diagnosis, sex, smoking status, the first IBD related symptom, family history of IBD and prescribed medications were collected from each patient s medical records. The diagnostic time interval was the duration from the first symptom to the diagnosis of IBD. This diagnostic time interval included patient-dependent delay (time interval from first symptoms to the physician visit) and physician-dependent delay (time interval from first physician visit to IBD diagnosis). Because there are no established criteria to define diagnostic delays, it was defined based on the time interval in which the 76 th to 100 th percentile of the patients were diagnosed. Followup duration was defined as the time from the date of first diagnosis of IBD to the date of the last follow up. Disease extent and behavior at the initial diagnosis in CD and UC were classified in accordance with the Montreal classification [15]. Disease severity was evaluated using the C-reactive protein level at diagnosis. In UC, the Mayo score was additionally used for severity assessment. Data on the prescribed medications for IBD, including 5-aminosalicylic acid, systemic corticosteroids, immunomodulator and antitumor necrosis factor alpha (anti-tnfα) agents were also obtained from medical records. To assess the clinical outcomes associated with a diagnostic delay, the following outcomes were measured: the number and type of intra-abdominal surgery, number and type of any surgery including perianal surgical procedures, number and date of hospitalization, and time duration from diagnosis to immunomodulator or anti-tnfα administration. Frequent admission was defined as two or more hospitalizations during the follow-up period. Statistical analysis The values for the continuous variables are expressed as mean ± (SD) or medians ± (SE). Categorical or discrete variables are presented as percentages. The groups were compared using the Student t-test or χ 2 test. Logistic regression was used to calculate the odds ratios (ORs) with 95% (CIs) for evaluating the risk factors for a long diagnostic delay. The covariates used in our multivariate analyses included variables with a significant result on the univariate analysis in addition to variables associated with diagnostic delays in previous studies and clinical experience. The Kaplan- Meier method was used to evaluate the association between diagnostic delays and clinical outcomes, such as IBD-related surgery and admission. The difference between the groups according to diagnostic time interval was analyzed using the log-rank test. The Cox proportional hazard regression analysis was performed to evaluate the association of long diagnostic delays with the clinical outcomes; the result was expressed as hazards ratio with 95%CI. The statistical methods of this study were reviewed by biostatistician from Korea University Ansan Hospital. And all statistical analyses were performed using the Statistical Package for the Social Science statistical software (version 18; SPSS- IBM, Chicago, IL, United States) or R version 3.02 (R Foundation for Statistical Computing, Vienna, Austria). All P < 0.05 was considered statistically significant. RESULTS The mean age at diagnosis was 28.2 ± 13.8 years in patients with CD and 38.8 ± 15.6 years in patients with UC. Male sex was predominant in both patients with CD and UC. Abdominal pain (51.5%) was the most prevalent symptom among the chief complaints in patients with CD and hematochezia (66.9%) in patients with UC. Regarding the disease location at diagnosis, the ileocolic area (L3, 51.5%) was the most common in CD and proctitis (40.3%) was the most prevalent in UC. The median diagnostic time interval was 6.2 mo in the patients with CD and 2.4 mo in the patients with UC. In the present study, the diagnostic time interval of 21.4 mo and 6.2 mo were defined as long diagnostic delays in CD and UC, respectively, based on the highest quartile cut off limit. Tables 1 and 2 showed the baseline characteristics of the patients with CD and UC stratified by presence of a long diagnostic delay. Although age, sex, smoking status, IBD family history, and disease location were not different between the diagnostic delayed group and non-delayed group, the disease behavior type was significantly different; the stenosis type was especially more prevalent in the CD delayed group (P = 0.021). However, there was no significant baseline characteristic difference, except for the follow-up duration in UC. The factors associated with a diagnostic delay in the patients with CD and UC are summarized in Tables 3 and 4. In the patients with CD, perianal discomfort was the only clinical factor associated with diagnostic delays (OR 10.23, 95%CI: ). However, there was no clinical factor associated with significant diagnostic delays in the patients with UC. Medication history during the follow-up period in the patients with IBD is shown in Supplementary Table 1. There was no difference in the types and frequencies of prescribed medications between the diagnostic delay and non-diagnostic delay groups in 6476 September 21, 2017 Volume 23 Issue 35

121 Table 2 Baseline characteristics of ulcerative colitis patients Lee DW et al. Diagnostic delay in IBD Table 1 Baseline characteristics of Crohn s disease patients n (%) Table 2 Baseline characteristics of ulcerative colitis patients n (%) Total (n = 165) Delayed (n = 41) Non-delayed (n = 124) P value Total (n = 130) Delayed (n = 32) Non-delayed (n = 98) P value Age, yr (± SD) 28.2 ( ± 1.1) 29.2 ( ± 12.7) 27.9 ( ± 14.2) Age (16.4) 5 (12.2) 22 (17.7) Male, 126 (76.4) 33 (80.5) 93 (75.0) Family history, 6 (3.6) 0 (0) 6 (4.8) Symptom to diagnosis 1, d Symptom to visit 1, d Visit to diagnosis 1, d 185 ( ) 57.0 ( ) 20.0 ( ) 1108 ( ) 739 ( ) 150 ( ) 79.5 ( ) 33.5 ( ) 14.0 ( ) Smoking, 41 (24.8) 12 (29.3) 29 (23.4) Chief complain Diarrhea 29 (17.6) 4 (9.8) 25 (20.2) GI bleeding 20 (12.1) 4 (9.8) 16 (12.9) Perianal 25 (15.2) 10 (24.4) 15 (12.1) discomfort Abdominal 85 (51.5) 22 (53.7) 63 (50.8) pain Others 6 (3.6) 1 (2.4) 5 (4.0) Location, L1 39 (23.6) 12 (29.3) 27 (21.8) L2 18 (10.9) 3 (7.3) 15 (12.1) L3 85 (51.5) 20 (48.8) 65 (52.4) L4 23 (13.9) 6 (14.6) 17 (13.7) Behavior, B1 116 (70.3) 23 (56.1) 93 (75.0) B2 32 (19.4) 14 (34.1) 18 (14.5) B3 17 (10.3) 4 (9.8) 13 (10.5) Perianal disease 31 (18.8) 5 (12.2) 26 (21.0) CRP at diagnosis, (± SD) 4.17 (± 7.76) 3.73 (± 4.35) 4.33 (± 8.68) Age at diagnosis 38.9 ± ± ± (± SD) Male, 71 (54.6) 17 (53.1) 54 (55.1) Family history 7 (5.4) 2 (6.3) 5 (5.1) Symptom to diagnosis 1, d Symptom to visit 1, d Visit to diagnosis 1, d 73.0 ( ) 635 ( ) 50.5 ( ) ( ) ( ) ( ) 7.00 ( ) 10.0 ( ) 6.00 ( ) Smoking 26 (20.2) 6 (18.8) 20 (20.6) Chief complaints 0.55 Hematochezia 87 (66.9) 20 (62.5) 67 (68.4) Diarrhea 31 (23.8) 10 (31.3) 21 (21.4) Abdominal 9 (6.9) 2 (6.3) 7 (7.1) pain Others 3 (2.3) 0 (0) 3 (3.1) Location Proctitis 52 (40.3) 12 (38.7) 40 (40.8) Left sided 37 (28.7) 11 (35.5) 26 (26.5) Pancolitis 40 (31.0) 8 (25.8) 32 (32.7) Mayo score at 5.60 ± ± ± diagnosis Severity Remission (0-2) 2 (1.5) 1 (3.1) 1 (1.0) Mild (3-5) 53 (40.8) 14 (43.8) 39 (39.8) Moderate 74 (56.9) 17 (53.1) 57 (58.2) (6-10) Severe (11-12) 1 (0.8) 0 (0) 1 (1.0) CRP at diagnosis, (± SD) 1.82 ± ± ± The duration was expressed as median (interquartile range); CRP: C-reactive protein. 1 The duration was expressed as median (interquartile range); 2 The severity was classified according to the Mayo score. patients with CD and UC. However, the median time interval from diagnosis to anti-tnfα administration was significantly shorter in the non-diagnostic delay group of the patients with CD than in the diagnostic delay group. In the patients with UC, the time interval from diagnosis to systemic steroid, immunomodulator and anti-tnfα administration was not different between groups. After diagnosis, 28 (17.0%) patients with CD underwent intestinal surgeries due to CD related problems, such as uncontrolled internal fistula, stenosis, or abdominal abscess and 43 (26.1%) underwent any surgical treatments including perianal surgery due to perianal abscess or fistula. Among the patients with UC, 6 (4.6%) underwent intestinal surgeries due to uncontrolled inflammation and UC-related colon cancer. Figure 1 show that a long diagnostic delay was associated with an increased risk of intestinal surgery in the patients with CD and UC, which was analyzed using Kaplan-Meier method. Tables 5 and 6 show the clinical risk factors associated with intestinal or any surgery in the patients with IBD. In patients with CD, a long diagnostic delay (OR = 2.54, 95%CI: ) was a significant risk factor for intestinal surgery, but not for any surgery. Further, stricturing and penetrating types were significantly associated with intestinal and any surgery. However, the diagnostic delay was the only clinical risk factor associated with intestinal surgery in UC (OR = 6.81, 95%CI: ). Kaplan Meier analysis showed that the diagnostic delay group of the patients with CD had an increased risk of admission and frequent admission as shown in Figure 2. However, after adjusting for covariates, such as age and sex, a long diagnostic delay was not associated with admission and frequent admission in the patients with CD (OR = 1.60, 95%CI: and OR 2.05; 95%CI: , respectively), as shown in Supplementary Table 2. In the patients with UC, pancolitis was the only risk factor of admission (OR = 3.94; 95%CI: ), as shown in supplementary Table 3. A diagnostic delay was not associated with admission and frequent admission in patients with UC (OR = 1.04; 95%CI: and OR = 2.05; 95%CI: , respectively) September 21, 2017 Volume 23 Issue 35

122 Lee DW et al. Diagnostic delay in IBD A 1.0 Blue: Non-delayed diagnosis group Green: Delayed diagnosis group B 1.0 P = Blue: Non-delayed diagnosis group Green: Delayed diagnosis group Probability Probability P = Follow-up duration (d) Follow-up duration (d) Figure 1 Surgery-free survival according to the presence of a long diagnostic delay in the patients with inflammatory bowel disease. A: Intestinal surgery in the patients with CD; B: Intestinal surgery in the patients with UC. IBD: Inflammatory bowel disease; CD: Crohn s disease; UC: Ulcerative colitis. Table 3 Factors associated with a long diagnostic delay in Crohn s disease Table 4 Factors associated with a long diagnostic delay in ulcerative colitis Variable n OR (95%CI) P value Age ( ) Male ( ) Smoking ( ) Chief complaint Diarrhea 29 Ref. - GI bleeding ( ) Perianal discomfort ( ) Abdominal pain ( ) Others ( ) Location L1 39 Ref. - L ( ) L ( ) L ( ) Behavior B1 116 Ref. B ( ) B ( ) Perianal disease ( ) CRP at diagnosis ( ) GI: Gastrointestinal; OR: Odds ratio; CRP: C-reactive protein; CI: Confidence interval. DISCUSSION This study demonstrated a considerable diagnostic delay in patients with IBD, especially in patients with CD. Further, a long diagnostic delay was significantly associated with a higher risk of IBD-related intestinal surgery in both the patients with CD and UC. In the present study, the diagnostic period for CD was longer than that for UC; this finding is consistent with previous studies [9,12]. In western IBD registries, such as the Swiss IBD cohort and the Romanian national registry, about 25% of patients with CD have a considerable diagnostic delay period of about Variable n OR (95%CI) P value Age ( ) Male ( ) Smoking ( ) IBD family history ( ) Chief complaint Hematochezia 87 Ref. - Diarrhea ( ) Abdominal pain ( ) Others 3 Not assessed - Location Proctitis 39 Ref. - Left sided ( ) Left sided ( ) Severity 1 Mild 53 Ref. Moderate to severe ( ) The severity was classified according to the Mayo score. IBD: Inflammatory bowel disease; OR: Odds ratio; CI: Confidence interval. 1.5 years. The diagnostic period of UC ranged from 1 mo to 4 mo. The significant gap in the diagnostic period between CD and UC might be because of initial symptoms [16]. Abdominal pain is the most prevalent initial chief complaint in patients with CD; however, it is also a common symptom of other digestive disorders, such as irritable bowel syndrome [17]. Furthermore, when the pain is not severe, it is easily regarded as trivial, and the diagnosis may be missed because the tests needed for the diagnosis of IBD are not performed. In contrast, hematochezia, which is a major symptom of UC, is not only a rare symptom but also causes great fear to the public [18]. This may cause patients to visit the hospital relatively quickly, leading to early diagnosis. Among the initial chief complaints, perianal discomfort was significant clinical factor associated with long 6478 September 21, 2017 Volume 23 Issue 35

123 Lee DW et al. Diagnostic delay in IBD A 1.0 Blue: Non-delayed diagnosis group Green: Delayed diagnosis group P value: B P value: Blue: Non-delayed diagnosis group Green: Delayed diagnosis group Probability C Follow-up duration (d) Blue: Non-delayed diagnosis group Green: Delayed diagnosis group P value: D Probability Follow-up duration (d) Blue: Non-delayed diagnosis group Green: Delayed diagnosis group P value: Probability Follow-up duration (d) Probability Follow-up duration (d) Figure 2 Admission-free survival according to the presence of a long diagnostic delay in the patients with inflammatory bowel disease. A: Admission in the patients with CD; B: Frequent admission in the patients with CD; C: Admission in the patients with UC; D: Frequent admission in the patients with UC. IBD: Inflammatory bowel disease; CD: Crohn s disease; UC: Ulcerative colitis. diagnostic delay in our study. The patients with perianal discomfort were significantly younger than those with other symptoms and tended to have longer duration from symptom to diagnosis (Supplementary Table 4). Interestingly, the time from the visit to the diagnosis (physician-dependent delay) was significantly longer in the patients with perianal discomfort, although there was no difference in the time from symptom to the hospital visit (patient-dependent delay) in this study. This is related to the tendency of CD patients with perianal discomfort to visit the colorectal/anus surgery clinic or general doctor s clinic for the first time in South Korea. However, they might tend to overlook and miss the diagnosis of CD because anal disorders, such as hemorrhoids, are relatively common and IBD is rare in the East than in the West [19]. Therefore, strengthening IBD education for general doctors and general surgeons is considered to be a good way to reduce the delay of diagnosis of IBD disease, especially CD patients with perianal disease. In our analyses, a long diagnostic delay was an independent risk factor of CD-related intestinal surgery in addition to stricturing and penetrating behavior types at diagnosis, which was similar to those of other studies [11,12,20]. Further, a long diagnostic delay in patients with UC was an independent risk factor of intestinal surgery. Considering that, there was no difference in treatments between the diagnostic delay group and the non-diagnostic delay group after the diagnosis in the present study, greater irreversible damage to the intestines might occur and accumulated, as the exposure period to the disease before the diagnosis is extended. Such damages may reduce the responsiveness to medical treatments and increase the risks of intestinal surgeries. To our knowledge, this is the first study to demonstrate the association between long diagnostic delays and poor clinical outcomes, such as an increased risk of intestinal surgery in patients with UC. This result is in part owing to the relatively long diagnostic delay compared with those of other UC diagnostic delay studies and to ethnic differences [9,12]. In present study, the duration from symptom onset to first hospital visit is significantly different between the delayed and nondelayed groups. This difference is caused by various factors such as patient s perception, attitude toward the disease and sensitivity to symptoms, and these factors are thought to influence patient s prognosis. However, a large multicenter study is needed to reveal the exact association between the diagnostic delay and 6479 September 21, 2017 Volume 23 Issue 35

124 Lee DW et al. Diagnostic delay in IBD Table 5 Risk factors associated with Crohn s disease related surgery Table 6 Risk factors associated with ulcerative colitis related surgery Intestinal surgery Any surgery Variables OR 95%CI P value OR 95%CI P value Age Male Smoking Location L1 1 (Ref.) (Ref.) - - L L L Behavior B1 1 (Ref.) (Ref.) - - B B Perianal disease CRP at diagnosis Delayed diagnosis CD: Crohn s disease; CRP: C-reactive protein; CI: Confidence interval; OR: Odds ratio. prognosis in patients with UC. A diagnostic delay was not directly associated with an increased risk of IBD-related admission in our analyses. However, stricturing behavior and penetrating type at diagnosis were significantly associated with admission and frequent admission in patients with CD. Among the patients with UC, those with pancolitis at diagnosis had an increased risk of admission. Considering that the disease behavior of CD tends to change from an inflammatory type to either stricturing or penetrating type and the disease extent progresses proximally from the rectum in UC, an early diagnosis might be associated with a better prognosis, such as reduced hospitalization [18,21-23]. Our study has a few limitations. First, as a limitation of retrospective study, important clinical information, such as the onset of IBD-related symptoms and first hospital visit date, may be inaccurate owing to a recall bias. Second, this study was conducted at a single center, and its results might not fully reflect the overall patients with IBD in South Korea. Therefore, a large multicenter prospective study is needed for to a better understanding of the association between the diagnostic delay and prognosis of IBD. Our study demonstrated a considerable diagnostic delay in patients with IBD. In addition, a long diagnostic delay was significantly associated with an increased risk of IBD-related intestinal surgeries in patients with CD and UC. Based on these results, efforts should be made to reduce diagnostic delays, such as increasing awareness for physicians and the public, to improve the prognosis of patients with IBD. COMMENTS Background The diagnosis of inflammatory bowel diseases (IBD) is often established following considerable delay due to nonspecific and inconsistent symptoms. In previous western studies, the delayed diagnosis was associated with poor outcome in patients with Crohn s disease (CD). Research frontiers There are few studies on diagnostic delays associated with IBD in Asia. Because there were significant differences in the epidemiological and clinical features of IBD according to ethnicities and environmental factors, the authors aimed to investigate the clinical factors and outcomes associated with diagnostic delays in Korean patients with IBD. Innovations and breakthroughs In present study, a long diagnostic delay was defined based on the time interval in which the 76 th to 100 th percentile of the patients was diagnosed due to no established criteria. This study demonstrated a considerable diagnostic delay in patients with Asian patients with IBD. Further, a long diagnostic delay was significantly associated with poor outcome such as a higher risk of IBD-related intestinal surgery in both the patients with CD and ulcerative colitis. Applications Efforts should be made to reduce diagnostic delays, such as increasing awareness and strengthening educations about IBD for physicians and the public, to improve the prognosis of patients with IBD. Peer-review The manuscript has convincing data and is publishable. REFERENCES Intestinal surgery Variables OR 95%CI P value Age Male Smoking IBD family history Location Proctitis 1 (Ref.) - - Left sided Pancolitis Severity 1 Mild 1 (Ref.) - - Moderate to severe Delayed diagnosis The severity was classified according to the Mayo score. UC: Ulcerative colitis; IBD: Inflammatory bowel disease; CI: Confidence interval; OR: Odds ratio. 1 Hanauer SB. Inflammatory bowel disease: epidemiology, pathogenesis, and therapeutic opportunities. Inflamm Bowel Dis 2006; 12 Suppl 1: S3-S9 [PMID: DOI: /01. MIB ] 2 Cosnes J, Gower-Rousseau C, Seksik P, Cortot A. Epidemiology and natural history of inflammatory bowel diseases. Gastroenterology 2011; 140: [PMID: DOI: /j.gastro ] 6480 September 21, 2017 Volume 23 Issue 35

125 Lee DW et al. Diagnostic delay in IBD 3 D Haens G, Baert F, van Assche G, Caenepeel P, Vergauwe P, Tuynman H, De Vos M, van Deventer S, Stitt L, Donner A, Vermeire S, Van De Mierop FJ, Coche JR, van der Woude J, Ochsenkühn T, van Bodegraven AA, Van Hootegem PP, Lambrecht GL, Mana F, Rutgeerts P, Feagan BG, Hommes D; Belgian Inflammatory Bowel Disease Research Group; North-Holland Gut Club. Early combined immunosuppression or conventional management in patients with newly diagnosed Crohn s disease: an open randomised trial. Lancet 2008; 371: [PMID: DOI: /S (08) ] 4 Colombel JF, Rutgeerts P, Reinisch W, Esser D, Wang Y, Lang Y, Marano CW, Strauss R, Oddens BJ, Feagan BG, Hanauer SB, Lichtenstein GR, Present D, Sands BE, Sandborn WJ. Early mucosal healing with infliximab is associated with improved long-term clinical outcomes in ulcerative colitis. Gastroenterology 2011; 141: [PMID: DOI: /j.gastro ] 5 Oldenburg B, Hommes D. Biological therapies in inflammatory bowel disease: top-down or bottom-up? Curr Opin Gastroenterol 2007; 23: [ PMID: DOI: / MOG.0b013e32815b601b] 6 Baumgart DC, Sandborn WJ. Inflammatory bowel disease: clinical aspects and established and evolving therapies. Lancet 2007; 369: [PMID: DOI: /S (07)60751-X] 7 Huang X, Liao WD, Yu C, Tu Y, Pan XL, Chen YX, Lv NH, Zhu X. Differences in clinical features of Crohn s disease and intestinal tuberculosis. World J Gastroenterol 2015; 21: [PMID: DOI: /wjg.v21.i ] 8 Lee SK, Kim BK, Kim TI, Kim WH. Differential diagnosis of intestinal Behçet s disease and Crohn s disease by colonoscopic findings. Endoscopy 2009; 41: 9-16 [PMID: DOI: /s ] 9 Vavricka SR, Spigaglia SM, Rogler G, Pittet V, Michetti P, Felley C, Mottet C, Braegger CP, Rogler D, Straumann A, Bauerfeind P, Fried M, Schoepfer AM; Swiss IBD Cohort Study Group. Systematic evaluation of risk factors for diagnostic delay in inflammatory bowel disease. Inflamm Bowel Dis 2012; 18: [PMID: DOI: /ibd.21719] 10 Nahon S, Lahmek P, Lesgourgues B, Poupardin C, Chaussade S, Peyrin-Biroulet L, Abitbol V. Diagnostic delay in a French cohort of Crohn s disease patients. J Crohns Colitis 2014; 8: [PMID: DOI: /j.crohns ] 212 Zaharie R, Tantau A, Zaharie F, Tantau M, Gheorghe L, Gheorghe C, Gologan S, Cijevschi C, Trifan A, Dobru D, Goldis A, Constantinescu G, Iacob R, Diculescu M; IBDPROSPECT Study Group. Diagnostic Delay in Romanian Patients with Inflammatory Bowel Disease: Risk Factors and Impact on the Disease Course and Need for Surgery. J Crohns Colitis 2016; 10: [PMID: DOI: /ecco-jcc/jjv215] 13 Ng SC. Epidemiology of inflammatory bowel disease: focus on Asia. Best Pract Res Clin Gastroenterol 2014; 28: [PMID: DOI: /j.bpg ] 14 Ng SC, Tang W, Ching JY, Wong M, Chow CM, Hui AJ, Wong TC, Leung VK, Tsang SW, Yu HH, Li MF, Ng KK, Kamm MA, Studd C, Bell S, Leong R, de Silva HJ, Kasturiratne A, Mufeena MN, Ling KL, Ooi CJ, Tan PS, Ong D, Goh KL, Hilmi I, Pisespongsa P, Manatsathit S, Rerknimitr R, Aniwan S, Wang YF, Ouyang Q, Zeng Z, Zhu Z, Chen MH, Hu PJ, Wu K, Wang X, Simadibrata M, Abdullah M, Wu JC, Sung JJ, Chan FK; Asia Pacific Crohn s and Colitis Epidemiologic Study (ACCESS) Study Group. Incidence and phenotype of inflammatory bowel disease based on results from the Asia-pacific Crohn s and colitis epidemiology study. Gastroenterology 2013; 145: e2 [PMID: DOI: /j.gastro ] 15 Satsangi J, Silverberg MS, Vermeire S, Colombel JF. The Montreal classification of inflammatory bowel disease: controversies, consensus, and implications. Gut 2006; 55: [PMID: DOI: /gut ] 16 Heikenen JB, Werlin SL, Brown CW, Balint JP. Presenting symptoms and diagnostic lag in children with inflammatory bowel disease. Inflamm Bowel Dis 1999; 5: [PMID: DOI: / ] 17 Burgmann T, Clara I, Graff L, Walker J, Lix L, Rawsthorne P, McPhail C, Rogala L, Miller N, Bernstein CN. The Manitoba Inflammatory Bowel Disease Cohort Study: prolonged symptoms before diagnosis--how much is irritable bowel syndrome? Clin Gastroenterol Hepatol 2006; 4: [PMID: DOI: /j.cgh ] 18 Farmer RG, Easley KA, Rankin GB. Clinical patterns, natural history, and progression of ulcerative colitis. A long-term followup of 1116 patients. Dig Dis Sci 1993; 38: [PMID: DOI: /BF ] 19 Wang YF, Zhang H, Ouyang Q. Clinical manifestations of inflammatory bowel disease: East and West differences. J Dig Dis 2007; 8: [PMID: DOI: / j x] 20 Moon CM, Jung SA, Kim SE, Song HJ, Jung Y, Ye BD, Cheon JH, Kim YS, Kim YH, Kim JS, Han DS; CONNECT study group. Clinical Factors and Disease Course Related to Diagnostic Delay in Korean Crohn s Disease Patients: Results from the CONNECT Study. PLoS One 2015; 10: e [PMID: DOI: / journal.pone ] 21 Cosnes J, Cattan S, Blain A, Beaugerie L, Carbonnel F, Parc R, Gendre JP. Long-term evolution of disease behavior of Crohn s disease. Inflamm Bowel Dis 2002; 8: [PMID: DOI: / ] 22 Louis E, Collard A, Oger AF, Degroote E, Aboul Nasr El Yafi FA, Belaiche J. Behaviour of Crohn s disease according to the Vienna classification: changing pattern over the course of the disease. Gut 2001; 49: [PMID: DOI: /gut ] 23 Meucci G, Vecchi M, Astegiano M, Beretta L, Cesari P, Dizioli P, Ferraris L, Panelli MR, Prada A, Sostegni R, de Franchis R. The natural history of ulcerative proctitis: a multicenter, retrospective study. Gruppo di Studio per le Malattie Infiammatorie Intestinali (GSMII). Am J Gastroenterol 2000; 95: [PMID: DOI: /j t x] P- Reviewer: Desai DC, Lakatos PL, M'Koma AE S- Editor: Qi Y L- Editor: A E- Editor: Ma YJ 6481 September 21, 2017 Volume 23 Issue 35

126 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) ORIGINAL ARTICLE Prospective Study Fecal marker levels as predictors of need for endoscopic balloon dilation in Crohn s disease patients with anastomotic strictures Susana Lopes, Patrícia Andrade, Eduardo Rodrigues-Pinto, Joana Afonso, Guilherme Macedo, Fernando Magro Susana Lopes, Patrícia Andrade, Eduardo Rodrigues-Pinto, Guilherme Macedo, Fernando Magro, Gastroenterology Department, Faculty of Medicine, Hospital de São João, Porto, Portugal Joana Afonso, Fernando Magro, Department of Pharmacology and Therapeutics, University of Porto, Porto, Portugal Author contributions: Lopes S and Magro F were responsible for study design; Lopes S and Andrade P were responsible for data collection; Afonso J performed all laboratorial procedures; Rodrigues-Pinto E and Andrade P were responsible for statistical analysis; Lopes S and Andrade P wrote the manuscript; Magro F and Macedo G revised and approved the final manuscript; All the named authors, contributing directly to the work described, have agreed on the respective roles of each author, read the manuscript and approved submission. Institutional review board statement: The study was approved by the Ethics Committee of Centro Hospitalar São João, Porto, Portugal. Informed consent statement: All patients gave informed written consent to participate in the study. Conflict-of-interest statement: The authors of this manuscript have no conflict of interest to declare. Data sharing statement: There is no additional data available. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Unsolicited manuscript Correspondence to: Guilherme Macedo, PhD, Department of Gastroenterology, Centro Hospitalar São João, Alameda Professor, Hernani Monteiro, Porto, Portugal. guilherme.macedo@hsjoao.min-saude.pt Telephone: Fax: Received: March 1, 2017 Peer-review started: March 3, 2017 First decision: June 3, 2017 Revised: June 22, 2017 Accepted: July 12, 2017 Article in press: July 12, 2017 Published online: September 21, 2017 Abstract AIM To evaluate the accuracy and best cut-off value of fecal calprotectin (FC) and fecal lactoferrin (FL) to predict disease recurrence in asymptomatic patients presenting with anastomotic strictures. METHODS This was a longitudinal single tertiary center study based on prospectively collected data (recorded in a clinical database created for this purpose) performed between March 2010 and November Crohn s disease (CD) patients with anastomotic stricture who submitted to postoperative endoscopic evaluation were included. Stools were collected on the day before bowel cleaning for FC and FL. Endoscopic balloon dilation (EBD) was performed if the patient presented an anastomotic stricture not traversed by the colonoscope, regardless of patients symptoms. Successful dilation was defined as passage of the colonoscope through the dilated stricture into the neotermimal ileum September 21, 2017 Volume 23 Issue 35

127 Lopes S et al. Fecal markers in CD anastomotic strictures Postoperative recurrence was defined as a modified Rutgeerts score of i2b. RESULTS In a total of 178 patients who underwent colonoscopy, 58 presented an anastomotic stricture, 86% were asymptomatic, and 48 (54% male; median age of 46.5 years) were successfully dilated. Immediate success rate was 92% and no complications were recorded. FC and FL levels correlated significantly with endoscopic recurrence (p < 0.001) with an optimal cut-off value of µg/g (sensitivity of 95.5%, specificity of 69.2%, positive predictive value (PPV) of 72.4%, negative predictive value (NPV) of 94.7% and accuracy of 81%] for FC and of 5.6 µg/g (sensitivity of 77.3%, specificity of 69.2%, PPV of 68%, NPV of 78.4% and accuracy of 72.9%) for FL. CONCLUSION Fecal markers are good predictors of CD endoscopic recurrence in patients with asymptomatic anastomotic stricture. FC and FL may guide the need for EBD in this context. Key words: Crohn s disease; anastomotic strictures; endoscopic balloon dilation; fecal markers; postoperative recurrence The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: This longitudinal study evaluated the accuracy of fecal calprotectin (FC) and fecal lactoferrin (FL) to predict disease recurrence in postoperative Crohn s disease asymptomatic patients with an anastomotic stricture. FC and FL levels accurately predicted endoscopic recurrence in the presence of anastomotic stricture and thus may guide the need for endoscopic balloon dilation (EBD) in this context. A normal value of fecal markers can reassure clinicians and be safely used to avoid balloon dilation if we only aim to diagnose recurrence. A high value of fecal markers has a high likelihood of recurrence so EBD should be performed in order to provide adequate endoscopic therapy and adjust or optimize medical therapy. Lopes S, Andrade P, Rodrigues-Pinto E, Afonso J, Macedo G, Magro F. Fecal marker levels as predictors of need for endoscopic balloon dilation in Crohn s disease patients with anastomotic strictures. World J Gastroenterol 2017; 23(35): Available from: URL: v23/i35/6482.htm DOI: i INTRODUCTION Crohn s disease (CD) is a chronic inflammatory disorder with progression to penetrating or stricturing phenotype as part of the natural history. More than half of CD patients experience these complications during disease lifetime and will need surgery [1,2]. It is well known that disease recurrence proximal or at the anastomosis is almost universal, with progression to luminal narrowing and stricturing behavior [3]. It is known that despite the permanent reduction of luminal caliber and disease progression, the majority of patients remain asymptomatic, with normal biomarkers. As the primary therapeutic goal of CD has shifted from clinical remission to achieving mucosal healing [4,5], it may be important to access the mucosa proximal to strictures to evaluate disease recurrence and escalate therapy if needed. Over the last decade there is increasing evidence for endoscopic balloon dilation (EBD) as a safe and minimally invasive effective method for the treatment of stricturing disease [6-14]. Median technical success has been reported as 90%, with major complication rate around 3%-10% [6,9,10]. Symptomatic recurrence is common, with a reported frequency ranging from 13% to 100% [12]. However, it has been shown that repeated dilations do not reduce the efficacy of the procedure and may prevent surgery in compliant patients [8-12]. Fecal markers, namely fecal calprotectin (FC) and fecal lactoferrin (FL), have proved to be useful and accurate non-invasive tools in evaluating disease activity in CD. Recent works have also demonstrated their validity in diagnosing recurrence in the postoperative setting, suggesting that normal values of fecal biomarkers can obviate the need for endoscopic evaluation [15-18]. In patients with elevated levels of fecal biomarkers, endoscopy should be performed in order to confirm recurrence and escalate therapy if indicated [19]. To our knowledge there are no studies evaluating the performance of FC and FL in patients with asymptomatic anastomotic CD strictures and limited data is available on the long-term effect of medical therapy escalation after balloon dilation of anastomotic strictures. The aims of this study were, therefore, to evaluate the accuracy of FC and FL in the diagnosis of recurrent disease in the neoterminal ileum in asymptomatic/mild disease patients that had undergone bowel resection for CD and present with stricture of the anastomosis, to evaluate the best cut-off value of FC and FL to diagnose recurrence, and to evaluate the immediate technical success and safety rate of EBD. MATERIALS AND METHODS A longitudinal single tertiary center study based on prospectively collected data (recorded in a clinical database created for this purpose), was performed between March 2010 and November All patients gave informed written consent to participate in the study that was approved by the Ethics Committee of our Institution. From a cohort of consecutive CD 6483 September 21, 2017 Volume 23 Issue 35

128 Lopes S et al. Fecal markers in CD anastomotic strictures patients who submitted to postoperative endoscopic evaluation after ileocolectomy, we selected the group of patients with anastomotic stricture. All patients were followed at our inflammatory bowel disease (IBD) outpatient clinic and were referred for endoscopic evaluation at our institution. Inclusion criteria were definitive diagnosis of CD established by standard clinical, radiographic, endoscopic and histological criteria [20], previous ileocolectomy, and existence of an anastomotic stricture. Exclusion criteria were: age less than 18-years-old; strictures length greater than 6 cm; fistulae or deep ulceration of the strictured segment; technical impossibility of passing the catheter/balloon through the strictures; and active disease in the colon or upper digestive tract. Clinical disease activity was assessed on the day of endoscopic examination, according to the clinical criteria of the Harvey-Bradshaw index (HBI) [21,22]. Clinically inactive disease was defined as HBI < 5. All procedures were performed under propofol sedation, with CO2 insufflation, by a single senior endoscopist (SL) and on an outpatient basis. Mechanical intestinal bowel preparation was done the day before colonoscopy with polyethylene glycol bowel preparation solution (Klean Prep ; Helsinn Birex Pharmaceuticals, Dublin, Ireland). Stool samples were collected the day before beginning bowel preparation (preferably from the first stool in the morning) and then kept in the refrigerator until being brought to the hospital. For FC, within a maximum 7 d after collection, stools were extracted in accordance with the manufacturer s instructions, using the Fecal Sample Preparation Kit (Roche Diagnostics, Mannheim, Germany) and analyzed using immunoassay (EliA Calprotectin ; Thermo Fisher Scientific, Freiburg, Germany). For FL evaluation, stools were stored at -80 upon arrival at the laboratory, and samples were thawed and analyzed using a commercially available quantitative enzyme-linked immunoassay test (IBD-Scan ; Tech- Lab, Blacksburg, VA, United States). The techniques for measurement of fecal markers followed the manufacturer s guidelines. Postoperative disease activity of the neoterminal ileum was evaluated according to the modified Rutgeerts score [23] (i0: no lesions in the distal ileum; i1: < 5 aphthous lesions in the distal ileum; i2a: lesions confined to the ileocolonic anastomosis, including anastomotic strictures; i2b: > 5 aphthous ulcers or larger lesions, with normal mucosa in between, in the neoterminal ileum, with or without anastomotic lesions; i3: diffuse aphthous ileitis with diffusely inflamed mucosa; i4: large ulcers with diffuse mucosal inflammation or nodules or strictures in the neoterminal ileum). Postoperative recurrence was defined as a modified Rutgeerts score of i2b. EBD was performed if the patient presented an anastomotic stricture not traversed by the colonoscope, regardless of the patient s symptoms. All dilations were performed with the same type of colonoscope (Olympus CF type H180AL; Tokyo, Japan) under fluoroscopic control to allow the endoscopist to characterize the strictures, exclude peristricture fistulae, evaluate the optimal diameter of the balloon to use and to prompt identify any complication during the procedure. Dilations were performed with a guidewire (Boston Scientific Jagwire in; Marlborough, MA, United States) placed through the strictures [after contrast instillation through a catheter (Olympus Ball Tip/6Fr) over which a through-thescope balloon (Cook Medical, Bloomington, IN, United States) was placed]. The balloon was inflated using contrast agent and the pressure maintained for 2 min, to a maximum diameter of 18 mm. The procedure could be repeated at the discretion of the endoscopist. Successful dilation was defined as passage of the colonoscope through the dilated stricture into the neotermimal ileum. Only patients with a successful dilation were analyzed, as progression to the neoterminal ileum was mandatory to evaluate disease recurrence. Major complications were defined as major bleeding requiring surgery, blood transfusion or hospital admission and perforation. Minor, selflimited bleeding was not registered as a complication. Statistical analysis SPSS 20.0 for Windows (SPSS, Chicago, IL, United States) was used for statistical analysis. Categorical variables were described as absolute frequencies (n) and relative frequencies (%); continuous variables were described as mean ± sd (parametric distributions) or as median and percentiles (non-parametric distributions). The normality of the continuous variables was tested using the Kolmogorov-Smirnov test and the respective histogram. Student s t-test was used to compare quantitative variables with a normal distribution, and the Mann-Whitney U test was used to compare the quantitative variables without a normal distribution. Any groups with more than two quantitative variables were compared using the Kruskal-Wallis test. A Pearson χ 2 test was used to compare categorical variables. Kaplan-Meier analysis with log rank statistics was used to estimate event-free interval. A logistic regression was performed to assess predictors of disease recurrence and need for dilation. Statistical significance was set at p < RESULTS Patient characteristics One hundred and seventy-eight consecutive CD patients (51.7% male; median age 46.4 years) who previously submitted to right ileocolectomy were evaluated by colonoscopy. At the time of endoscopic evaluation, 31 (17.4%) patients complained of subocclusive symptoms, and 66.3% of patients were being treated with immunomodulators and 38.2% with 6484 September 21, 2017 Volume 23 Issue 35

129 Lopes S et al. Fecal markers in CD anastomotic strictures 178 CD patients submitted to ileocolectomy 58 patients with anastomotic stricture Dilation not performed (n = 6) 3 deep ulceration, 2 strictures > 6 cm, 1 technical inability 52 dilated patients Inability to passe into the neoileum (n = 4) 48 patients successfully dilated in a total of 69 procedures Success rate 92% 1 dilation n = 34 2 dilations n = 9 1 dilations n = 3 1 dilations n = 2 Figure 1 Study flowchart. CD: Crohn s disease. biologics. Of the 178 evaluated patients, 58 (32.6%) presented with an anastomotic stricture. The majority were asymptomatic, with only 8 (13.8%) patients presenting with subocclusive symptoms. All patients were in clinical remission (HBI < 5 in 83.3%) or with mild clinical disease (HBI 5-7 in 16.7%). Among the 58 patients presenting with anastomotic stricture, 52 were dilated (6 were excluded due to deep ulceration, stricture size > 6 cm or technical inability). Of the total 52 dilated patients, 4 were excluded as it was not possible to evaluate the neoterminal ileum (figure 1). Baseline characteristics of the 48 successfully dilated patients are summarized in table 1. The majority were men (54%) with a median age of 46.5 years. At the time of dilation, 42% of patients were being treated with biologics and 54% with immunomodulators. EBD Overall, technical success rate was 92% (48/52) and no major complications were recorded. The 18-mm balloon diameter was used in 69.2% of the patients and the 15-mm diameter in 25.0%. Redilation was required in 14 patients (29.2%) during a median follow-up time of 34 mo ( mo). During the follow-up period, only 2 patients needed surgery, both cases due to long strictures that did not allow EBD (Table 2). Fecal markers and endoscopic recurrence Of the 48 successfully dilated patients, 22 presented with endoscopic recurrence defined as modified Rutgeerts score of i2b. Of these, 16 patients presented with severe disease (i3 = 3 and i4 = 13). Recurrence was diagnosed only after dilation of the anastomotic stricture and intubation of the neoterminal ileum. Comparing FC and FL levels in patients with endoscopic recurrence and in patients with endoscopic remission we found a significantly higher level in patients with endoscopic recurrence [FC: μg/g, interquartile range (IQR): μg/g) vs 53.9 μg/g, IQR: μg/g; p < and FL: September 21, 2017 Volume 23 Issue 35

130 Lopes S et al. Fecal markers in CD anastomotic strictures Table 1 Patient characteristics, n = 48 Characterization n (%) Women, 22 (45.8) Median time between diagnosis and surgery, mo 36.0 ( ) (IQR) Median time between surgery and endoscopic ( ) evaluation, mo (IQR) Median age at endoscopic evaluation, yr (IQR) 46.5 ( ) Montreal classification Age at diagnosis A1, 16 yr 6 (12.5) A2, yr 33 (68.8) A3, > 40 yr 9 (18.8) Location L1: ileal 30 (62.5) L3: ileocolonic 17 (35.4) L4: upper gastrointestinal tract 1 (2.1) Behavior B1: non-stricturing, non-penetrating 1 (2.1) B2: stricturing 27 (56.2) B3: penetrating 20 (41.7) Perianal disease 14 (29.2) Smoking Never 26 (54.2) Current 12 (25.0) Past 10 (20.8) Concomitant treatment Corticosteroids 9 (18.8) Immunomodulators (Azathioprine/6MP/ 26 (54.2) Methotrexate) Biologics (Infliximab, adalimumab) 20 (41.7) 5-ASA 17 (35.4) Median fecal calprotectin, μg/g (IQR) ( ) Median, fecal lactoferrin, μg/g (IQR) 6.2 ( ) Modified Rutgeerts score i0, i1, i2a 26 (54.2) i2b, i3, i4 22 (45.8) Subocclusive symptoms 8 (16.7) Harvey-Bradshaw index Remission (HBI < 5) 40 (83.3) Mild disease (HBI 5-7) 8 (16.7) Need of redilation 14 (29.2) Surgery after dilation 2 (1.4) Median follow up, mo (IQR) 34.0 ( ) HBI: Harvey-Bradshaw index; IQR: Interquartile range. μg/g, IQR: μg/g vs 3.9 μg/g, IQR: μg/g; p = 0.042] (Figure 2). No other clinical variable or biomarker reached statistical difference between the two groups (Table 3). The best calculated cut-off value for FC to predict recurrent disease was µg/g, with a sensitivity of 95.5%, a specificity of 69.2%, a positive predictive value (PPV) of 72.4%, a negative predictive value (NPV) of 94.7% and an accuracy of 81%. The area under the ROC curve for diagnosing endoscopic recurrence was (95%CI: , p < 0.05) for FC. Concerning FL, the best calculated cut-off was 5.6 µg/ g, with sensitivity of 77.3%, specificity of 69.2%, PPV of 68%, NPV of 78.4% and accuracy of 72.9%. The area under the ROC curve for diagnosing endoscopic recurrence was (95%CI: , p = 0.042) (figure 3). Table 2 Characterization of the dilation procedure Characterization n (%) Patients with stricture 58 (32.6) Stricture type Anastomotic 58 (100) Dilated patients 52 (29.2) Causes for non-dilation Length of stenosis 2 (33.3) Ulceration of mucosa 3 (50.0) Technical inability 1 (16.7) Successful dilated patients Balloon size, n = (92.3) 15 mm 13 (25.0) 16.5 mm 3 (5.8) 18 mm 36 (69.2) Complications 0 (0.0) DISCUSSION One of the major drawbacks of CD surgical therapy is the high recurrence rate of the disease at the anastomotic site or in the neoterminal ileum, with the development of stricture and bowel obstruction [8]. As the primary therapeutic goal of CD has shifted from clinical remission to achieving mucosal healing [4,5], it may be important to access the mucosa proximal to strictures to evaluate disease recurrence and escalate therapy if needed. The recently published POCER trial [16] demonstrated that initial postoperative treatment according to clinical risk stratification and early endoscopic evaluation and step-up therapy if there was recurrent disease was superior to standard medical therapy. In this study, endoscopy was the gold standard method to define recurrent disease. With respect to non-invasive methods, both serum biomarkers and clinical activity indexes have demonstrated a poor correlation with disease activity [24-26]. The use of fecal markers in the postoperative period has been studied in small groups of patients with variable results. Recently, however, some studies evaluated the value of fecal biomarker measurement after surgery for CD. In 2015, Wright et al [15] demonstrated that FC was sensitive enough to diagnosis CD recurrence in 135 patients submitted to bowel resection with a high enough NPV to reassure clinicians that few patients with recurrence would be missed. More recently, Lopes et al [27] showed that both FC and FL strongly correlated with endoscopic findings in the evaluation of CD after surgery and accurately predicted endoscopic recurrence in 99 CD patients who submitted to ileocolonic resection. The results of both of these studies suggests that fecal biomarkers may be incorporated in the postoperative management algorithm, both to diagnose recurrence and to assess response to therapy. In our study, we evaluated if in asymptomatic CD patients with anastomotic strictures not traversed 6486 September 21, 2017 Volume 23 Issue 35

131 Lopes S et al. Fecal markers in CD anastomotic strictures A P < B Calprotectin Lactcferrin P = i0, i1, i2a i2b, i3, i4 Modified rutgeerts 0.00 i0, i1, i2a i2b, i3, i4 Modified rutgeerts Figure 2 Box plots of fecal markers according to endoscopic activity defined by the modified Rutgeerts score, in asymptomatic patients presenting with anastomotic strictures. A: Fecal calprotectin; B: Fecal lactoferrin. A 1.0 ROC curve B 1.0 ROC curve Sensitivity Sensitivity FU (AUC = 0.786, 95%CI: , P < 0.001) Specifity Diagonal segments are produced by ties 0.2 FU (AUC = 0.672, 95%CI: , P = 0.042) Specifity Diagonal segments are produced by ties Figure 3 Receiver operating characteristic curves for fecal markers for discriminating between endoscopic recurrence and remission in asymptomatic patients presenting with anastomotic strictures. A: Fecal calprotectin; B: Fecal lactoferrin. by the colonoscope, fecal markers perform as well as a predictor of disease recurrence, defining groups of patients that will need more invasive methods. In this series of 48 patients with strictures, only 18% complained of subocclusive symptoms. Similarly to what has been published [28-30], we also did not find any correlation between patient symptoms, serum biomarkers and HBI and endoscopic or radiographic findings, supporting the belief that using only symptoms or C-reactive protein levels to inform treatment decisions may increase the risk of disease progression and complications. It is controversial whether or not asymptomatic strictures should be endoscopically treated. Despite being a safe and minimally invasive technique, there is a 3%-10% [6,9,10] described risk of major complications, especially in centers with limited procedural volume per year. Our data confirmed the safety and efficacy of EBD in the context of CD anastomotic strictures, with a technical success rate of 92%. We had no serious complications, which may be explained by several factors: careful patient selection; use of fluoroscopic image to evaluate, in real time, stricture characteristics and the therapeutic procedure indicated; maximum diameter of the balloon used (18 mm); use of carbon dioxide as type of insufflation; and experience of the endoscopist performing the technique with a uniform technical approach. The dilation of the anastomosis allowed the diagnosis of recurrence in 22 patients, that otherwise would have been missed if we only relied on symptoms or biochemical markers. We used the modified Rutgeerts score to diagnose endoscopic recurrence, although it is not yet validated. Despite being used for several decades in clinical trials and clinical practice, the Rutgeerts score is also an invalidated score. In this study, we chose to use the modified Rutgeerts score, as demonstrated in a previous work [27] as having a better correlation between the modified score and fecal markers to diagnose recurrent disease. Recurrence was defined by a modified Rutgeerts score of i2b, not considering the presence of only anastomotic stricture as a criterion, as many other factors may be implicated in stricture development [1,2]. In that paper [27] the calculated best cut-off level for FC for predicting recurrence was 100 µg/g, with a sensitivity of 74% and a NPV of 91% September 21, 2017 Volume 23 Issue 35

132 Lopes S et al. Fecal markers in CD anastomotic strictures Table 3 Comparison between patients with and without endoscopic recurrence Endoscopic recurrence, n = 22 No endoscopic recurrence, n = 26 P value Sex, M:F 11:11 15: Median age, yr (IQR) 47.0 ( ) 46.5 ( ) Median hemoglobin, 13.8 ( ) 13.9 ( ) g/dl (IQR) Median albumin, g/l 42.4 ( ) 42.1 ( ) (IQR) Median C-reactive protein, mg/l (IQR) 5.6 ( ) 2.4 ( ) Median fecal calprotectin, μg/g (IQR) Median fecal lactoferrin, μg/g (IQR) ( ) < ( ) 9.1 ( ) 3.9 ( ) Smoking, yes/no/past 3/13/6 9/13/ Subocclusive symptoms, 2/20 6/ yes/no HBI, remission/mild 17/5 24/ Concomitant treatment Anti-TNF- α agents, 12/10 14/ yes/no Immunomodulators, 9/13 11/ yes/no Steroids, yes/no 4/18 5/ HBI: Harvey-Bradshaw index; IQR: Interquartile range. This is in accordance with other studies [15] that defined in the post-operative setting a higher cut-off than that of 50 μg/g used to diagnose inflammatory bowel disease [31]. In the present study, both FC and FL were also significantly higher in patients with endoscopic recurrence, with an area under the receiver operating characteristic curve for detection of endoscopic recurrence of for FC and of for FL. The best cut-off value of FC as predictor of recurrence was μg/g, with sensitivity of 95.5%, NPV of 94.7% and accuracy of 81%. If we adopted the commonly used cut-off value of 7.25 μg/g for lactoferrin [32-34], we would have missed patients with recurrence (false negative results). The cut-off value of 5.6 μg/g had sensitivity of 77.3%, NPV of 78.4% and accuracy of 72.9%. Our findings support the potential value of these two noninvasive markers in the monitoring of patients submitted to bowel resection and presenting with an anastomotic stricture. Indeed, a FC and/or FL concentration lower than μg/g and 5.6 μg/g respectively, have high accuracy to exclude disease recurrence, with no need to further therapeutic intervention. This may be of particular interest in centers with low expertise in EBD and/or with a lower procedural volume per year, in order to avoid complications and/or be used as an indication for referring patients to tertiary centers. If these results are reproduced and validated by others, in a large number of patients, this conservative strategy could be adopted, reserving balloon dilation for symptomatic patients and those with high levels of fecal markers, in order to facilitate step-up therapy. To our knowledge this is the first report on the performance of FC and FL in the context of anastomotic strictures in CD. Our results suggest that low values of fecal markers can predict, with a great amount of certainty, disease remission. This may avoid application of endoscopic dilation in asymptomatic patients in centers with less endoscopic expertise, reassuring physicians that the use of fecal markers serves as a good indicator to monitor disease recurrence. The serial monitoring of FC and FL can help to make decisions in indeterminate results and a persistently elevated value may serve as another useful indicator when considering therapy intensification. In conclusion, postoperative FC and FL levels accurately predicted endoscopic recurrence in the presence of anastomotic stricture. Considering that a significant number of patients remain asymptomatic, with normal serum biomarkers, despite the permanent reduction of luminal caliber, a normal value of fecal markers can reassure clinicians and be safely used to avoid balloon dilation if we only aim to diagnose recurrence. In asymptomatic patients with a high FC and/or FL level, there is a great chance of having disease activity proximal to the stricture, so EBD should be performed in order to provide adequate endoscopic therapy and adjust or optimize medical therapy. COMMENTS Background Recurrent disease in the neoterminal ileum and anastomotic strictures are frequent complications of Crohn s disease (CD). Despite the permanent reduction of luminal caliber and disease progression, the majority of patients remain asymptomatic. Fecal calprotectin (FC) and lactoferrin have been suggested as surrogate non-invasive markers for diagnosing postoperative disease recurrence. There are no studies evaluating the performance of fecal markers as predictors of disease recurrence in asymptomatic patients with an anastomotic stricture. Research frontiers The results demonstrated that FC and lactoferrin are good predictors of CD endoscopic recurrence in patients with asymptomatic anastomotic stricture and may guide the need for endoscopic balloon dilation in this context. Innovations and breakthroughs A normal value of fecal markers can reassure clinicians and be safely used to avoid balloon dilation if we only aim to diagnose recurrence. A high value of fecal markers has a high likelihood of recurrence, so endoscopic balloon dilation should be performed in order to provide adequate endoscopic therapy and adjust or optimize medical therapy. Applications Fecal markers may avoid the need of endoscopic balloon dilation in asymptomatic patients with anastomotic stricture if we only aim to diagnose disease recurrence. Terminology Calprotectin is a protein complex, constituting up to 60% of neutrophil cytosol 6488 September 21, 2017 Volume 23 Issue 35

133 Lopes S et al. Fecal markers in CD anastomotic strictures protein that is released upon neutrophil activation. Lactoferrin, an iron-binding protein, is the main component of secondary granules that degranulate during inflammatory process. Both these proteins are remarkably stable and resistant to degradation, easily detected and have been proved to reflect endoscopic disease activity in CD, predicting endoscopic inflammation and being a surrogate marker of mucosal healing. Peer-review The authors have performed a very interesting and important study. They concluded that postoperative FC and FL levels accurately predicted endoscopic recurrence in the presence of anastomotic stricture. REFERENCES 1 Parkes GC, Whelan K, Lindsay JO. Smoking in inflammatory bowel disease: impact on disease course and insights into the aetiology of its effect. J Crohns Colitis 2014; 8: [PMID: DOI: /j.crohns ] 2 De Cruz P, Kamm MA, Prideaux L, Allen PB, Desmond PV. 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134 Lopes S et al. Fecal markers in CD anastomotic strictures Modified Rutgeerts Score. Inflamm Bowel Dis 2016; 22: [PMID: DOI: /MIB ] 28 Regueiro M, Kip KE, Schraut W, Baidoo L, Sepulveda AR, Pesci M, El-Hachem S, Harrison J, Binion D. Crohn s disease activity index does not correlate with endoscopic recurrence one year after ileocolonic resection. Inflamm Bowel Dis 2011; 17: [PMID: DOI: /ibd.21355] 29 Cellier C, Sahmoud T, Froguel E, Adenis A, Belaiche J, Bretagne JF, Florent C, Bouvry M, Mary JY, Modigliani R. Correlations between clinical activity, endoscopic severity, and biological parameters in colonic or ileocolonic Crohn s disease. A prospective multicentre study of 121 cases. The Groupe d Etudes Thérapeutiques des Affections Inflammatoires Digestives. Gut 1994; 35: [PMID: DOI: /gut ] 30 Landi B, Anh TN, Cortot A, Soule JC, Rene E, Gendre JP, Bories P, See A, Metman EH, Florent C. Endoscopic monitoring of Crohn s disease treatment: a prospective, ran-domized clinical trial. The Groupe d Etudes Therapeutiques des Affections Inflammatoires Digestives. Gastroenterology 1992; 102: [PMID: DOI: / (92)91725-J] 31 van Rheenen PF, Van de Vijver E, Fidler V. Faecal calprotectin for screening of patients with suspected inflammatory bowel disease: diagnostic meta-analysis. BMJ 2010; 341: c3369 [PMID: DOI: /bmj.c3369] 32 Lamb CA, Mansfield JC. Measurement of faecal calprotectin and lactoferrin in inflammatory bowel disease. Frontline Gastroenterol 2011; 2: [PMID: DOI: /fg ] 33 Zhou XL, Xu W, Tang XX, Luo LS, Tu JF, Zhang CJ, Xu X, Wu QD, Pan WS. Fecal lactoferrin in discriminating inflammatory bowel disease from irritable bowel syndrome: a diagnostic metaanalysis. BMC Gastroenterol 2014; 14: 121 [PMID: DOI: / X ] 34 Wang Y, Pei F, Wang X, Sun Z, Hu C, Dou H. Diagnostic accuracy of fecal lactoferrin for inflammatory bowel disease: a meta-analysis. Int J Clin Exp Pathol 2015; 8: [PMID: ] P- Reviewer: Lankarani KB, Sun sy S- Editor: Ma YJ L- Editor: A E- Editor: Ma YJ 6490 September 21, 2017 Volume 23 Issue 35

135 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) SYSTEMATIC REVIEWS Gastrosplenic fistula occurring in lymphoma patients: Systematic review with a new case of extranodal NK/T-cell lymphoma Dong Hyeok Kang, Jimi Huh, Jong Hwa Lee, Yoong Ki Jeong, Hee Jeong Cha Dong Hyeok Kang, Jimi Huh, Jong Hwa Lee, Yoong Ki Jeong, Department of Radiology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan , South Korea Hee Jeong Cha, Department of Pathology, Ulsan University Hospital, University of Ulsan College of Medicine, Ulsan , South Korea Author contributions: Kang DH and Huh J performed the literature selection, data extraction, and initial drafting of the manuscript; Lee JH and Jeong YK conceptualized and designed the review and performed critical revision/editing of the manuscript; Cha HJ provided pathology data and performed critical revision/editing of the manuscript; all authors reviewed and approved the final manuscript as submitted. Conflict-of-interest statement: No authors have conflict-ofinterest. Data sharing statement: No additional data are available. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Unsolicited manuscript Correspondence to: Jimi Huh, MD, Department of Radiology, Ulsan University Hospital, University of Ulsan College of Medicine, 877 Bangeojinsunhwando-ro, Dong-gu, , Ulsan , South Korea. jimihuh@mail.ulsan.ac.kr Telephone: Fax: Received: April 6, 2017 Peer-review started: May 4, 2017 First decision: June 7, 2017 Revised: June 20, 2017 Accepted: July 24, 2017 Article in press: July 24, 2017 Published online: September 21, 2017 Abstract AIM To provide the overall spectrum of gastrosplenic fistula (GSF) occurring in lymphomas through a systematic review including a patient at our hospital. METHODS A comprehensive literature search was performed in the MEDLINE database to identify studies of GSF occurring in lymphomas. A computerized search of our institutional database was also performed. In all cases, we analyzed the clinicopathologic/radiologic features, treatment and outcome of GSF occurring in lymphomas. RESULTS A literature search identified 25 relevant studies with 26 patients. Our institutional data search added 1 patient. Systematic review of the total 27 cases revealed that GSF occurred mainly in diffuse, large B-cell lymphoma (n = 23), but also in diffuse, histiocytic lymphoma (n = 1), Hodgkin s lymphoma (n = 2), and NK/T-cell lymphoma (n = 1, our patient). The common clinical presentations are constitutional symptoms (n = 20) and abdominal pain (n = 17), although acute gastrointestinal bleeding (n = 6) and infection symptoms due to splenic abscess (n = 3) are also noted. In all patients, computed tomography scanning was very helpful for diagnosing GSF and for evaluating the lymphoma extent. GSF could occur either post-chemotherapy (n = 10) or spontaneously (n = 17). Surgical resection has been 6491 September 21, 2017 Volume 23 Issue 35

136 Kang DH et al. Gastrosplenic fistula occurring in the lymphoma the most common treatment. Once patients have recovered from the acute illness status after undergoing surgery, their long-term outcome has been favorable. CONCLUSION This systematic review provides an overview of GSF occurring in lymphomas, and will be helpful in making physicians aware of this rare disease entity. Key words: Gastrosplenic fistula; Lymphoma; NK/T-cell lymphoma; Systematic review The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: Gastrosplenic fistula (GSF) is a very rare complication occurring in lymphomas. Due to its rarity, GSF had not been well-investigated. Therefore, we intend to provide an overall spectrum of GSF occurring in lymphomas through a systematic review. GSF occurred mainly in diffuse, large B-cell lymphoma, but also in various kinds of lymphoma. Common clinical presentations are constitutional symptoms and abdominal pain. Occasionally, acute gastrointestinal bleeding and splenic abscess have occurred. Computed tomography was helpful for diagnosing GSF and for evaluating the lymphoma extent. GSF could occur either post-chemotherapy or spontaneously. Surgical resection has been the most common treatment. Kang DH, Huh J, Lee JH, Jeong YK, Cha HJ. Gastrosplenic fistula occurring in lymphoma patients: Systematic review with a new case of extranodal NK/T-cell lymphoma. World J Gastroenterol 2017; 23(35): Available from: URL: DOI: INTRODUCTION Gastrosplenic fistula (GSF) is a rare and potentially fatal complication of various diseases, including lymphoma, gastric adenocarcinoma, Crohn s disease, splenic abscess, and trauma [1]. Lymphoma is the most common cause of GSF. Of these diseases, the majority have occurred in patients with diffuse, large, B-cell lymphoma (DLBCL), and there has been no patient with NK/T-cell lymphoma. In our hospital, a tertiary cancer center, we recently encountered a patient with GSF developed in NK/T-cell lymphoma. A multidisciplinary team was organized in our hospital for management of this patient. Our team performed an extensive literature search, however, and determined that the characteristics of GSF occurring in lymphoma patients have not been thoroughly investigated due to its extreme rarity. In contrast, the number of case reports regarding these patients has increased over the last 10 years [1-24]. Despite the increasing number of these patients, there has been no systematic review to summarize the variable presentations of GSF occurring in lymphomas. Therefore, we performed the current, systematic review with the addition of our single patient in order to provide a perspective regarding this rare disease entity. MATERIALS AND METHODS Systematic literature search A computerized search of the MEDLINE database was conducted to find relevant studies published prior to February 10, Studies were eligible for inclusion if they described the clinicopathologic features, imaging findings, treatment and outcome of the cases with regard to GSF occurring in lymphoma. The following search terms were used: GSF and lymphoma. To expand the search, the bibliographies of articles that remained after the selection process were screened for other potentially suitable articles. We did not limit the language of the articles. Institutional data search and case presentation Our institutional review board approved the search of the electronic medical records for this study. Informed consent was waived from our institutional review board. We performed a systematic computerized search of our institutional database from January 2000 to January 2017 using the diagnostic codes of GSF, splenic fistula, and lymphoma. Using these search terms, we identified only one case which we recently encountered, as described below. In our institutional database, the patient s record was anonymized and provided to us. We present here the clinical course, pathologic findings, and imaging features of this case. We also included this case in the systematic review of GSF of lymphoma. Analysis of clinicopathologic and radiologic features For patients with GSF occurring in lymphoma identified in the literature and at our institution, we analyzed their clinicopathologic features, imaging findings, treatment and outcome. RESULTS Literature selection Our study selection process is shown in Figure 1. The literature search in the MEDLINE database generated 24, initial candidate articles. After reviewing the titles and the abstracts, all 23 articles were included. One study was subsequently excluded due to the lack of a full-text manuscript. Therefore, we reviewed the full text of all 23 articles, including 2 written in Spanish and 1 written in Korean. Search of the bibliographies of these articles found two, additional, eligible studies [8,14]. Therefore, a total of 25 articles were included in our 6492 September 21, 2017 Volume 23 Issue 35

137 Kang DH et al. Gastrosplenic fistula occurring in the lymphoma Included Eligibility Screening Identification Records identified through MEDLINE database (n = 24) Records screened (n = 24) Full-text articles assessed for eligibility (n = 23) Studies included in qualitative synthesis (n = 25) Studies included in qualitative synthesis (n = 25) Figure 1 Flow diagram for the selection of studies. Records excluded (n = 1) Bibliography search (n = 2) Figure 2 On a coronal computed tomography image taken 2 mo after autologous stem-cell transplantation, the spleen was enlarged, measuring 17 cm in the longest dimension, and indicative of recurred lymphoma. The enlarged spleen abutted to the gastric fundus. systematic review. Of these articles, 24 described one case, while 1 described two cases [3]. Therefore, a total of 27 cases were retrieved from the systematic literature search. Presentation of our case A 50-year-old man was admitted to our hospital for a biopsy-proven NK/T-cell lymphoma of the right anterior nasal cavity. On contrast-enhanced computed tomography (CT), there was an ill-defined, homogeneous, enhancing, soft-tissue mass in the right anterior nasal cavity. There was no significantly enlarged lymph node in his neck, chest or abdomenpelvis. However, hepatosplenomegaly was noted with numerous, ill-defined, low, attenuating nodules in the liver. His spleen measured 15.5 cm in the longest dimension. Hypermetabolic enlargement of the liver and spleen was revealed on positron emission tomography scanning. These findings were indicative Figure 3 On an axial computed tomography image taken after chemotherapy, a huge fistula was shown between the gastric lumen and the spleen. The spleen was totally infarcted. of lymphoma involvement in the liver and spleen. The patient was subsequently started on the combination of a SMILE chemotherapy regime consisting of methotrexate, leucovorin, ifosfamide, etoposide, and L-asparaginase followed by autologous stem-cell transplantation (ASCT). After completing the ASCT, complete remission was confirmed by a bone marrow biopsy and CT scans with resolution of the nasal cavity mass, hepatosplenomegaly, and focal liver lesions. Two months later, follow-up CT showed a huge splenomegaly measuring 17.5-cm in its longest diameter, which was indicative of lymphoma recurrence (Figure 2). Chemotherapy was started for the lymphoma recurrence. Tumor lysis syndrome occurred, and the patient underwent dialysis treatment. He then soon experienced left, upper quadrant, abdominal pain and nausea/vomiting. Follow-up abdominal CT showed a diffusely enlarged spleen with nearly total splenic infarction. A huge fistula track was also seen between the gastric lumen and the infarcted spleen (Figure 3). He underwent emergency surgery for gastric wedge resection and splenectomy. During the surgery, a large GSF was observed and there was adhesion between the infarcted spleen and the stomach. These surgical findings suggested that the extensive lymphoma lysis due to chemotherapy resulted in disruption of the splenic capsule, adhesion to the adjacent gastric fundus, and eventually led to the large GSF. In the gross and microscopic specimen, there was extensive hemorrhagic infarction in the spleen with a large GSF. The atypical lymphoma cells, which were positive for CD3 on immunohistochemistry stain and positive for Epstein-Barr virus (EBV) on EBV RNA stain, were found in the stomach wall near the GSF as well as in the whole spleen (Figure 4). The histopathological diagnosis was NK/T-cell lymphoma and these findings suggest that lymphoma cells may infiltrate from the spleen to the stomach wall through the adhesion/ perforation site September 21, 2017 Volume 23 Issue 35

138 Kang DH et al. Gastrosplenic fistula occurring in the lymphoma A H&E CD3 EBV B H&E CD3 EBV Figure 4 Microscopic specimen of the spleen and stomach. A: Atypical lymphoma cells were found in the spleen on hematoxylin-eosin stain (left). These cells showed positivity for CD3 on immunohistochemistry stain (middle) and EBV on EBV RNA stain (right). There was also extensive coagulative necrosis indicative of splenic infarction; B: Lymphoma cells were found in the stomach wall near the gastrosplenic fistula on hematoxylin-eosin stain (left), and which also showed positivity for CD3 (middle) and EBV (right). NK/T-cell lymphoma was diagnosed. These findings suggested that lymphoma cells may have infiltrated from the spleen to the stomach wall through the perforation site. EBV: Epstein-Barr virus. Systematic review The characteristics of the 27 included cases (26 literature cases and 1 presented case) of GSF occurring in lymphomas are summarized in Table 1. Although there was a substantial diversity in the clinicopathologic and radiologic features, the systematic review allowed us to summarize the characteristic features of GSF occurring in lymphomas. Clinicopathologic features The mean age of these patients was 50.6 ± 16.8 years with a range of years. The male to female ratio was 4.4 (22 males, 5 females). Regarding the presenting symptoms or signs, constitutional symptoms such as weight loss, fever and fatigue (n = 20, 74.1%), were followed by abdominal/flank pain (n = 17, 63.0%), acute gastrointestinal bleeding such as hematemesis (n = 6, 22.2%), and infection due to splenic abscess (n = 3, 11.1%). Among the different histological types, GSF occurred most commonly in DLBCL (n = 23, 85.2%). The other types of lymphoma associated with GSF include diffuse histiocytic lymphoma (n = 1, 3.7%), Hodgkin s lymphoma (n = 2, 7.4%), and extranodal NK/T-cell lymphoma (n = 1, 3.7%). Of these, GSF occurred due to splenic lymphoma involvement (n = 8, 29.6%, including the present case), gastric lymphoma involvement (n = 6, 22.2%), extensive lymphoma including both gastric and splenic involvement (n = 1, 3.7%), and not specified (n = 12, 44.4%). GSF occurred either post-chemotherapy (n = 12, 44.4%) or spontaneously (n = 17, 63.0%). Radiologic features In all 27 cases, the GSF was depicted or suspected on CT scans, as there was a defect in the gastric wall and splenic capsule where the stomach and spleen were closely attached. Of these, 10 cases described the size of the GSF which ranged from 0.25 cm to 6 cm (mean size: 2.87 cm). In 21 cases, endoscopy was performed to confirm the diagnosis of GSF and to evaluate the bleeding or other concomitant disease, such as a gastric ulcer. The CT scan was excellent for identifying the extent of lymphoma involvement. The splenic involvement was most commonly noted as a moderate to massive splenomegaly (n = 13). Five of seven cases specifically noted the gastric lymphoma as a diffuse/segmental, gastric wall thickening or mass. There were five cases with extensive lymphoma masses in the left upper quadrant, including diaphragm involvement. The CT scan was also very helpful for identifying any associated GSF complications. Indeed, a splenic abscess was diagnosed on CT in three cases. Acute bleeding was seen as leakage of contrast agent in the gastric lumen. Even when there was no leakage of contrast agent seen on CT, the presence of a hematoma in the stomach and/or spleen could suggest a recent hemorrhagic event. Treatment and outcome Regarding the treatment, surgical resection was performed in 23 patients (88.9%). Two patients underwent chemotherapy without surgery, and one patient refused any treatment September 21, 2017 Volume 23 Issue 35

139 Kang DH et al. Gastrosplenic fistula occurring in the lymphoma Table 1 Summary of the 27 cases of gastrosplenic fistula occurring in lymphomas Ref. Diagnosis Sex/age Size of lymphoma Disease status Presentation Diagnostic modality Intervention/therapy Outcome Bubenik et al [1] (1983) Hiltunen et al [2] (1991) Blanchi et al [3] (1995) case 1 Blanchi et al [3] (1995) case 2 Carolin et al [4] (1997) Bird et al [5] (2002) Choi et al [6] (2002) Yang et al [7] (2002) Puppala et al [8] (2005) Kerem et al [9] (2006) Al-Ashgar et al [10] (2007) Aribaş et al [11] (2008) Palmowski et al [12] (2008) Seib et al [13] (2009) Diffuse histiocytic lymphoma Gastric DLBCL Splenic DLBCL Splenic DLBCL Gastric DLBCL Splenic DLBCL Splenic DLBCL Gastric and splenic DLBCL Male/58 Not available Post-CTx Nonspecific LUQ discomfort CT abdomen followed by Splenectomy, gastric greater Uneventful post-operative period; endoscopy of upper GI tract curvature resection, distal no further details pancreatectomy Male/36 Not available Post-CTx Hematemesis, splenomegaly CT abdomen followed by endoscopy Male/62 Not available Initial presentation Male/45 Not available Initial presentation Male/46 Not available Initial presentation Male/36 Not available Initial presentation Male/24 Not available Initial presentation Male/21 Not available Initial presentation DLBCL Female/66 Not available Initial presentation DLBCL Male/57 10 cm 7 cm 2 cm in the stomach and 8 cm 5 cm 4 cm in the spleen Hodgkin s lymphoma- (nodular sclerosis)-iiis Initial presentation Female/16 Not available Initial presentation Left abdominal pain and fever Endoscopy of upper GI tract followed by CT abdomen Epigastric pain and weight loss Endoscopy of upper GI tract followed by CT abdomen Epigastric pain, fatigue, weight loss and splenomegaly Hematemesis, melena, fatigue, weight loss and splenomegaly LUQ pain and constitutionals symptoms (splenic mass) LUQ pain, fatigue, weight loss, fever, and splenomegaly Endoscopy of upper GI tract followed by CT abdomen Endoscopy of upper GI tract followed by CT abdomen CT abdomen followed by endoscopy of upper GI tract/ biopsy CT abdomen followed by endoscopy of upper GI tract Laparotomy without details Followed-up over 3 yr Resection of spleen, tail of pancreas, and involved stomach At 6 mo after the operation, the patient was in complete remission after CTx No further details. No further details Laparotomy, but no further details. No further details Splenic artery embolization, near total gastrectomy and splenectomy CTx followed by splenectomy, gastric wedge resection, and distal pancreatectomy Splenectomy, gastric wedge resection, and distal Disease-free after three cycles of CTx; no further details Not available After surgery, the patient underwent CTx pancreatectomy LUQ pain CT abdomen oral contrast CTx Died after 2 mo of Ctx Abdominal pain, epigastric tenderness and splenomegaly LUQ pain, constitutional symptoms and splenomegaly DLBCL Male/25 Not available Post-CTx Abdominal pain, weight loss, fever, chill and splenomegaly DLBCL Male/56 15 cm of spleen After three cycles of CTx Hodgkin s lymphoma Male / cm splenic mass Relapsed post-ctx Fever and signs of acute infection (splenic mass) LUQ pain and constitutional symptoms (splenic mass) CT abdomen followed by PETCT and endoscopy of upper GI tract Endoscopy of upper GI tract, barium swallow, CT abdomen Splenectomy, proximal gastrectomy, esophagojejunostomy, proximal pyroloplasty followed by CTx Laparoscopic surgical repair followed by seven cycles CTx CT cystography followed by USG Gastric wedge resection, fistulectomy and splenectomy CT abdomen Splenectomy with partial gastric resection CT abdomen Partial gastrectomy and fistulectomy Uneventful post-op period; underwent chemotherapy. Alive and in remission after 1 yr Discharged after a month and died 2 mo later due to progression of lymphoma and infection due to pancreatic and gastric fistulas Finished six cycles of CTx Died after 5 mo 6495 September 21, 2017 Volume 23 Issue 35

140 Kang DH et al. Gastrosplenic fistula occurring in the lymphoma Moran et al [14] (2009) Maillo et al [15] (2009) García et al [16] (2009) Khan et al [17] (2010) Rothermel et al [18] (2010) Dellaportas et al [19] (2011) DLBCL Male/ cm 5.3 cm of gastrosplenic mass Splenic DLBCL Gastric DLBCL Gastric DLBCL Splenic DLBCL Splenic DLBCL Initial presentation Female/76 Not available Initial presentation Male/76 Not available Initial presentation Female/ cm 10 cm 8.6 cm of splenic mass Initial presentation Male/74 Not available Initial presentation Male/68 Not available Initial presentation LUQ pain and constitutional symptoms Massive hematemesis, fever and fatigue (splenic abscess) Epigastric pain, weight loss and splenomegaly Upper abdominal pain and constitutional symptoms (splenic mass) Jain et al [20] (2011) DLBCL Male/55 Not available Post-CTx Progressive weakness, fatigue, melena and splenomegaly Ding et al [21] (2012) Favre Rizzo et al [22] (2013) Senapati et al [29] (2014) Gentilli et al [23] (2016) Sousa et al [24] (2016) DLBCL Male/62 7 cm of splenic segment Gastric DLBCL Initial presentation Male/55 Not available Initial presentation DLBCL Male/57 Splenomegaly of 15 cm Gastric DLBCL Gastric DLBCL Present case NK/T cell lymphoma Female/ cm 3 cm of splenic mass CT abdomen followed by endoscopy of upper GI tract CT abdomen followed by endoscopy of upper GI tract CT abdomen followed by endoscopy of upper GI tract Endoscopy of upper GI tract followed by CT Fever, chill and weight loss Endoscopy of upper GI tract followed by CT Hematemesis (splenic mass) Endoscopy of upper GI tract followed by CT abdomen LUQ pain with constitutional symptoms and splenomegaly Hematemesis, epigastric pain, weight loss and splenomegaly CT abdomen followed by endoscopy of upper GI tract CT abdomen followed by endoscopy of upper GI tract Post-CTx No symptom but splenomegaly PET/CT followed by endoscopy of upper GI tract Post-CTx Weakness, fatigue, weight loss and splenomegaly Abscess drainage; splenectomy, total gastrectomy, Roux-en-Y esophagojejunostomy followed by CTx splenectomy, partial gastrectomy, diaphragmatic primary repair, drainage chest tube and a feeding tube jejunostomy Total gastrectomy, splenectomy and distal pancreatectomy Received CTx after surgery; no further details available At 2 mo later the patient developed a pulmonary infection and died because of multi-organic failure Remained asymptomatic at the 36-mo follow-up, no further details CTx Complete remission after two cycles of CTx; no further details Splenectomy, stapled gastric-sleeve resection Surgical en bloc resection followed by chemotherapy Splenectomy and partial gastrectomy Splenectomy, gastric wedge resection, and distal pancreatectomy followed by CTx and RT CT abdomen Partial gastrectomy, splenectomy and distal pancreatectomy Endoscopy of upper GI tract followed by CT After surgery, the patient underwent CTx; good prognosis for long-term survival Post-CTx on follow up No details available Received CTx after surgery; no further details available Well at follow up; no further details available After surgery; no further details available Refused any surgical intervention Lost to follow-up Gastric wedge resection, splenectomy Male/52 Not available Post-CTx Hematemesis Endoscopy of upper GI tract Total gastrectomy, splenectomy, distal pancreatectomy Male/50 11 cm 5 cm 13 cm of spleen Post-CTx LUQ pain, nausea, vomiting and splenomegaly CT abdomen Gastric wedge resection and splenectomy Discharged after surgery; no further details Patient was lost to follow-up after discharge At 3 mo later, gastric perforation occurred and the patient expired due to sepsis CT: Computed tomography; CTx: Chemotherapy; DLBCL: Diffuse large B-cell lymphoma; GI: Gastrointestinal; LUQ: Left upper quadrant; PET: Positron emission tomography; RT: Radiation therapy; USG: Ultrasonography. Among the 27 cases, death was reported in 5 patients due to a gastric perforation (n = 1), progression of lymphoma and infection (n = 1), pulmonary infection with multi-organic failure (n = 1), and no further details (n = 2). The time to death ranged from 2-5 mo after the initial surgery or diagnosis. In 18 cases, the patients were alive at that time or were discharged. Patient outcomes were not reported for three patients, and two patients were lost to follow-up. DISCUSSION GSF is a very rare complication of lymphoma, as only 27 cases have been reported including the present case. It is very difficult to identify the spectrum of presentation, 6496 September 21, 2017 Volume 23 Issue 35

141 Kang DH et al. Gastrosplenic fistula occurring in the lymphoma clinicopathologic/radiologic features, and outcome in this kind of extremely rare disease entity. Systematic review of pre-existing cases might be the best way to evaluate the overview of this rare disease [25]. From that perspective, our systematic review may provide a systematic summary and overview of GSF occurring in lymphomas. As our systematic review includes all of the available articles in PubMed without the limitation of language, it is the largest reported series of GSF cases occurring in lymphomas. Although there was a diversity in the clinicopathologic features, imaging features and outcomes, we can summarize that GSF occurs mainly in DLBCL (85.2%) and it can occur either post-chemotherapy (37%) or spontaneously (63%). GSF can also cause fatal complications such as acute bleeding and splenic abscess. Once these fatal complications in the acute illness stage are controlled, the long-term outcome is good. Regarding the pathologic type, DLBCL is the most common type associated with GSF. The present case of GSF occurred in extranodal NK/T-cell lymphoma of the nasal type and, to our knowledge, is the first reported case. In general, NK/T-cell lymphoma occurs in the nasal cavity. If NK/T-cell lymphoma involves an area outside the nasal cavity, the gastrointestinal tracts and skin are common and preferential extranodal organs of lymphoma involvement [26]. Rare sites of involvement include the spleen, prostate, pancreas and adrenal glands [27]. From this perspective, our case demonstrated a very rare complication of GSF which occurred in a rarely involved site of NK/T-cell lymphoma of the nasal type. In general, NK/T-cell lymphoma is characterized by its aggressive behavior and with an angiocentric/ angiodestructive growth pattern. In our case, we at first postulated that the GSF might occur due to these aggressive behaviors. Thereafter, a multidisciplinary discussion at our medical center indicated that extensive tumor lysis from chemotherapy might also be an important causative factor of GSF. The spleen parenchyma and capsule were largely infiltrated by NK/T-cell lymphoma cells, and thus caused splenomegaly. When the enlarged spleen was attached to the adjacent gastric fundus, tumor cells might infiltrate into the gastric wall. When chemotherapy caused tumor lysis and necrosis, the defect in the spleen capsule and gastric wall occurred, eventually leading to the GSF. When we systematically reviewed the literature reports, we discovered that there has been a commonly proposed theory of GSF development, i.e., necrosis of lymphoma tissue involving the spleen or stomach may cause GSF. According to this theory, invasion of the gastric wall and splenic capsule is required. If the lymphoma predominantly involves the spleen, the tumor cells should also infiltrate the gastric wall, and vice versa. The rapid necrosis of infiltrated lymphoma cells in the gastric wall and splenic capsule may result in the formation of GSF which may occur either spontaneously or post-chemotherapy [6,28]. Most of the GSFs described in the published literature are small. However, if a larger part of the stomach is infiltrated, it is possible for a large opening in the stomach wall to arise, as was reported in our case. As the initial presentations of GSF occurring in lymphomas are somewhat non-specific, such as constitutional symptoms and abdominal pain, the diagnosis of GSF may be difficult and delayed. CT scanning can have an important role in the diagnosis of GSF, can identify GSF-associated complications such as abscess or bleeding, and can evaluate the disease extent and surgical planning. If the diagnosis and treatment of GSF are delayed, the clinical outcome might not be favorable, as GSF in lymphomas recover spontaneously. Therefore, radiologists should be aware of this rare disease entity and should notify physicians when GSF is suspected in patients with lymphoma involving the spleen or stomach. There are various therapeutic options for managing GSF. Surgery is regarded as the most important and curative treatment in the vast majority of literature reports. The surgical method can be determined based on the tumor extent and the surgeon s preference. The most common surgical method in the literature reports is partial gastrectomy (mostly laparoscopic) with/without splenectomy. However, in some cases with a large tumor extent, near-total gastrectomy and splenectomy has also been performed. When patients were too ill to have surgery or refused it, the outcome was not good. Once patients had surgery and overcame the acute illness status, the long-term outcome was generally good. Considering all of these cases, we propose that aggressive surgery at the early stage of disease might be the best way to save patients with GSF occurring in lymphomas. In conclusion, this systematic review provides an overview and spectrum of GSF occurring in lymphomas and covering the clinicopathologic features, radiologic features, treatment and outcome. We also included a rare case of GSF occurring in extranodal NK/T-cell lymphoma. This information will be helpful for physicians so that they can become aware of this rare disease entity. COMMENTS Background Gastrosplenic fistula (GSF) is a very rare complication occurring in lymphomas. Due to its rarity, GSF have not been well-investigated. Therefore, the intent of our study was to provide the overall spectrum of GSF occurring in lymphomas through a systematic review including a patient at our hospital who had extranodal NK/T-cell lymphoma. Research frontiers A systematic review of 27, published studies was conducted. The clinicopathologic/radiologic features, treatment and outcome of GSF occurring 6497 September 21, 2017 Volume 23 Issue 35

142 Kang DH et al. Gastrosplenic fistula occurring in the lymphoma in lymphomas were analyzed. Innovations and breakthroughs This systematic review includes all of the available articles in PubMed without the limitation of language and it is the largest reported series of GSF cases occurring in lymphomas. It provides a systematic summary and overview of GSF occurring in lymphomas in order to understand the spectrum of presentation, clinicopathologic/radiologic features and outcome in this type of extremely rare disease entity. The authors also added our present case of GSF occurring in extranodal NK/T-cell lymphoma of the nasal type and, to our knowledge, this is the first reported case of this type. Applications When lymphoma patients present symptoms or signs of constitutional symptoms, such as weight loss, fever and fatigue, abdominal/flank pain, acute gastrointestinal bleeding, such as hematemesis or any infection signs with or without history of chemotherapy, computed tomography scanning was very helpful for diagnosing GSF and evaluating the lymphoma extent. GSF could occur either post-chemotherapy or spontaneously. Surgical resection has been the most common treatment. Once patients have recovered from their acute illness status after undergoing surgery, their long-term outcome has been good. The authors should be aware of GSF, which is a rare disease entity in patients with lymphoma, especially involving the spleen or stomach. Terminology GSF is a communication between the stomach lumen and spleen parenchyma. Peer-review This manuscript systematically reviewed a very rare complication of GSF occurring in lymphomas. The review analyzed the current literature regarding the clinicopathologic/radiologic features, treatment and outcome of GSF. It therefore provides useful recording of the disease. REFERENCES 1 Bubenik O, Lopez MJ, Greco AO, Kraybill WG, Cherwitz DL. Gastrosplenic fistula following successful chemotherapy for disseminated histiocytic lymphoma. Cancer 1983; 52: [PMID: ] 2 Hiltunen KM, Airo I, Mattila J, Helve O. Massively bleeding gastrosplenic fistula following cytostatic chemotherapy of a malignant lymphoma. J Clin Gastroenterol 1991; 13: [PMID: DOI: / ] 3 Blanchi A, Bour B, Alami O. Spontaneous gastrosplenic fistula revealing high-grade centroblastic lymphoma: endoscopic findings. Gastrointest Endosc 1995; 42: [PMID: DOI: /S (95)70017-X] 4 Carolin KA, Prakash SH, Silva YJ. Gastrosplenic fistulas: a case report and review of the literature. Am Surg 1997; 63: [PMID: ] 5 Bird MA, Amjadi D, Behrns KE. Primary splenic lymphoma complicated by hematemesis and gastric erosion. South Med J 2002; 95: [PMID: DOI: / ] 6 Choi JE, Chung HJ, Lee HG. Spontaneous gastrosplenic fistula: a rare complication of splenic diffuse large cell lymphoma. Abdom Imaging 2002; 27: [PMID: DOI: / s ] 7 Yang SE, Jin JY, Song CW, Park JC, Lee JI, Kim W, Kim J, Lee HG. Gastrosplenic Fistula Complicated in a Patient with Non- Hodgkin s Lymphoma. Cancer Res Treat 2002; 34: [PMID: DOI: /crt ] 8 Puppala S, Williams R, Harvey J, Crane M. Spontaneous gastrosplenic fistula in primary gastric lymphoma: case report and review of literature. Clinical Radiology Extra 2005; 60: Kerem M, Sakrak O, Yilmaz TU, Gultekin FA, Dursun A, Bedirli A. Spontaneous gastrosplenic fistula in primary gastric lymphoma: Surgical management. Asian J Surg 2006; 29: [PMID: DOI: /S (09) ] 10 Al-Ashgar HI, Khan MQ, Ghamdi AM, Bamehriz FY, Maghfoor I. Gastrosplenic fistula in Hodgkin s lymphoma treated successfully by laparoscopic surgery and chemotherapy. Saudi Med J 2007; 28: [PMID: ] 11 Aribaş BK, Başkan E, Altinyollar H, Ungül U, Cengiz A, Erdil HF. Gastrosplenic fistula due to splenic large cell lymphoma diagnosed by percutaneous drainage before surgical treatment. Turk J Gastroenterol 2008; 19: [PMID: ] 12 Palmowski M, Zechmann C, Satzl S, Bartling S, Hallscheidt P. Large gastrosplenic fistula after effective treatment of abdominal diffuse large-b-cell lymphoma. Ann Hematol 2008; 87: [PMID: DOI: /s ] 13 Seib CD, Rocha FG, Hwang DG, Shoji BT. Gastrosplenic fistula from Hodgkin s lymphoma. J Clin Oncol 2009; 27: e15-e17 [PMID: DOI: /JCO ] 14 Moran M, Bilgiç İ, Dizen H, Dilektaşlı E, Köseoğlu T, Özmen MM. Spontaneous gastrosplenic fistula resulting from primary gastric lymphoma: case report and review of the literature. Available from: URL: neous_gastrosplenic_fistula_resulting_from_primary_gastric_l ymphoma_case_report_and_review_of_the_literature 15 Maillo C, Bau J. [Gastrosplenic and thoracosplenic fistula due to primary untreated splenic lymphoma]. Rev Esp Enferm Dig 2009; 101: [PMID: ] 16 García MA, Bernardos GL, Vaquero RA, Menchén VL, Turégano FF. Spontaneous gastrosplenic fistula secondary to primary gastric lymphoma. Revista espanola de enfermedades digestivas: organo oficial de la Sociedad Espanola de Patologia Digestiva 2009; 101: Khan F, Vessal S, McKimm E, D Souza R. Spontaneous gastrosplenic fistula secondary to primary splenic lymphoma. BMJ Case Rep 2010; 2010: pii: bcr [PMID: DOI: /bcr ] 18 Rothermel LD, Chadwick CL, Thambi-Pillai T. Gastrosplenic fistula: etiologies, diagnostic studies, and surgical management. Int Surg 2010; 95: [PMID: ] 19 Dellaportas D, Vezakis A, Fragulidis G, Tasoulis M, Karamitopoulou E, Polydorou A. Gastrosplenic fistula secondary to lymphoma, manifesting as upper gastrointestinal bleeding. Endoscopy 2011; 43 Suppl 2 UCTN: E395 [PMID: ] 20 Jain V, Pauli E, Sharzehi K, Moyer M. Spontaneous gastrosplenic fistula secondary to diffuse large B-cell lymphoma. Gastrointest Endosc 2011; 73: [PMID: DOI: / j.gie ] 21 Ding YL, Wang SY. Gastrosplenic fistula due to splenic large B-cell lymphoma. J Res Med Sci 2012; 17: [PMID: ] 22 Favre Rizzo J, López-Tomassetti Fernández E, Ceballos Esparragón J, Santana Cabrera L, Hernández Hernández JR. Massive upper gastrointestinal bleeding secondary to gastrosplenic fistula. Rev Esp Enferm Dig 2013; 105: [PMID: ] 23 Gentilli S, Oldani A, Zanni M, Ferreri E, Terrone A, Valente G, Occhipinti P. Gastro-splenic fistula as a complication of chemotherapy for large B cell lymphoma. Ann Ital Chir 2016; 87: pii: S X [PMID: ] 24 Sousa M, Gomes A, Pignatelli N, Nunes V. Massive gastrointestinal bleeding after chemotherapy for gastric lymphoma. Int J Surg Case Rep 2016; 21: [PMID: DOI: /j.ijscr ] 25 Huh J, Byun JH, Hong SM, Kim KW, Kim JH, Lee SS, Kim HJ, Lee MG. Malignant pancreatic serous cystic neoplasms: systematic review with a new case. BMC Gastroenterol 2016; 16: 97 [PMID: DOI: /s ] 26 Haverkos BM, Pan Z, Gru AA, Freud AG, Rabinovitch R, Xu- Welliver M, Otto B, Barrionuevo C, Baiocchi RA, Rochford R, Porcu P. Extranodal NK/T Cell Lymphoma, Nasal Type (ENKTL- NT): An Update on Epidemiology, Clinical Presentation, and Natural History in North American and European Cases. Curr 6498 September 21, 2017 Volume 23 Issue 35

143 Kang DH et al. Gastrosplenic fistula occurring in the lymphoma Hematol Malig Rep 2016; 11: [PMID: DOI: /s ] 27 Cao Q, Huang Y, Ye Z, Liu N, Li S, Peng T. Primary spleen extranodal NK/T cell lymphoma, nasal type, with bone marrow involvement and CD30 positive expression: a case report and literature review. Diagn Pathol 2014; 9: 169 [PMID: DOI: /s ] 28 de Scoville A, Bovy P, Demeester P. [Radiologic aerosplenomegaly caused by necrotizing splenic lymphosarcoma with double fistulization into the digestive tract]. Acta Gastroenterol Belg 1967; 30: [PMID: ] 29 Senapati J, Devasia AJ, Sudhakar S, Viswabandya A. Asymptomatic gastrosplenic fistula in a patient with marginal zonal lymphoma transformed to diffuse large B cell lymphoma--a case report and review of literature. Ann Hematol 2014; 93: [PMID: DOI: /s ] P- Reviewer: Chen F, Li YZ S- Editor: Qi Y L- Editor: Filipodia E- Editor: Zhang FF 6499 September 21, 2017 Volume 23 Issue 35

144 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) META-ANALYSIS Proton pump inhibitors therapy and risk of Clostridium difficile infection: Systematic review and meta-analysis Anca Trifan, Carol Stanciu, Irina Girleanu, Oana Cristina Stoica, Ana Maria Singeap, Roxana Maxim, Stefan Andrei Chiriac, Alin Ciobica, Lucian Boiculese Anca Trifan, Irina Girleanu, Oana Cristina Stoica, Ana Maria Singeap, Roxana Maxim, Stefan Andrei Chiriac, Institute of Gastroenterology and Hepatology, St. Spiridon Hospital, Grigore T. Popa University of Medicine and Pharmacy, Iasi, Romania Carol Stanciu, Institute of Gastroenterology and Hepatology, St. Spiridon Hospital, Iasi, Romania Alin Ciobica, Department of Research, Faculty of Biology, Alexandru Ioan Cuza University of Iasi, Iasi, Romania Lucian Boiculese, Department of Preventive Medicine and Interdisciplinarity, Grigore. T. Popa University of Medicine and Pharmacy, Iasi, Romania ORCID number: Anca Trifan ( ); Carol Stanciu ( ); Irina Girleanu ( ); Oana Cristina Stoica ( ); Ana Maria Singeap ( X); Roxana Maxim ( ); Stefan Andrei Chiriac ( ); Alin Ciobica ( ); Lucian Boiculese ( ). Author contributions: Trifan A and Stanciu C designed the study, contributed to the selection of studies, analyzed the data and wrote the manuscript; Stoica OC, Maxim R, Singeap AM, Girleanu I, Chiriac SA and Ciobica A were involved in the acquisition of data and contributed to the analysis and interpretation of data; Girleanu I, Stoica OC, Maxim R and Singeap AM drafted the manuscript; Boiculese L and Girleanu I contributed to the statistical analysis; all authors have read and approved the final version of the manuscript; all authors accept responsibility for its content. Conflict-of-interest statement: The authors deny any conflict of interest. Data sharing statement: No additional data are available. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Unsolicited manuscript Correspondence to: Carol Stanciu, MD, FRCP, Professor, Institute of Gastroenterology and Hepatology, St. Spiridon Hospital, Independentei Street No. 1, Iasi, Romania. stanciucarol@yahoo.com Telephone: Fax: Received: July 13, 2017 Peer-review started: July 17, 2017 First decision: August 10, 2017 Revised: August 11, 2017 Accepted: August 25, 2017 Article in press: August 25, 2017 Published online: September 21, 2017 Abstract AIM To perform a systematic review and meta-analysis on proton pump inhibitors (PPIs) therapy and the risk of Clostridium difficile infection (CDI). METHODS We conducted a systematic search of MEDLINE/ PubMed and seven other databases through January 1990 to March 2017 for published studies that evaluated the association between PPIs and CDI. Adult case-control and cohort studies providing information on the association between PPI therapy and the development of CDI were included. Pooled odds ratios (ORs) estimates with 95% confidence intervals (CIs) were calculated using the random effect. Heterogeneity was assessed by I 2 test and Cochran s Q statistic September 21, 2017 Volume 23 Issue 35

145 Trifan A et al. PPIs and Clostridium difficile Potential publication bias was evaluated via funnel plot, and quality of studies by the Newcastle-Otawa Quality Assessment Scale (NOS). RESULTS Fifty-six studies (40 case-control and 16 cohort) involving patients met the inclusion criteria and were analyzed. Both the overall pooled estimates and subgroup analyses showed increased risk for CDI despite substantial statistical heterogeneity among studies. Meta-analysis of all studies combined showed a significant association between PPI users and the risk of CDI (pooled OR = 1.99, CI: , P < 0.001) as compared with non-users. The association remained significant in subgroup analyses: by design-case-control (OR = 2.00, CI: , P < ), and cohort (OR = 1.98, CI: , P < ); adjusted (OR = 1.95, CI: , P < ) and unadjusted (OR = 2.02, CI: , P < ); unicenter (OR = 2.18, CI: , P < ) and multicenter (OR = 1.82, CI: , P < ); age 65 years (OR = 1.93, CI: , P < ) and < 65 years (OR = 2.06, CI: , P < 0.01). No significant differences were found in subgroup analyses (test for heterogeneity): P = 0.93 for case-control vs cohort, P = 0.85 for adjusted vs unadjusted, P = 0.24 for unicenter vs multicenter, P = 0.86 for age 65 years and < 65 years. There was significant heterogeneity across studies (I 2 = 85.4%, P < 0.001) as well as evidence of publication bias (funnel plot asymmetry test, P = 0.002). CONCLUSION This meta-analysis provides further evidence that PPI use is associated with an increased risk for development of CDI. Further high-quality, prospective studies are needed to assess whether this association is causal. Key words: Proton pump inhibitors; Clostridium difficile infection; Risk; Systematic review; Meta-analysis The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: A possible association between the use of proton pump inhibitors (PPIs) and the risk of Clostridium difficile infection (CDI) have been suggested by several studies. This meta-analysis, including the largest number of studies published to date found the risk of CDI almost two-times higher in PPIs users than in nonusers. Because all the studies analyzed were observational, the causality could not be confirmed. Nevertheless, clinicians should be aware of such potential association and prescribe the PPIs only where they are clearly indicated. Trifan A, Stanciu C, Girleanu I, Stoica OC, Singeap AM, Maxim R, Chiriac SA, Ciobica A, Boiculese L. Proton pump inhibitors therapy and risk of Clostridium difficile infection: Systematic review and meta-analysis. World J Gastroenterol 2017; 23(35): Available from: URL: com/ /full/v23/i35/6500.htm DOI: org/ /wjg.v23.i INTRODUCTION Over the past two decades Clostridium difficile (C. difficile) infection (CDI) has registered an increasing trend worldwide both in incidence and severity [1-5], with healthcare costs varying between 1.2 and 4.7 billion dollars each year in the United States alone [6-9]. In addition to the broad-spectrum antimicrobial therapy which has been the most prominent causative factor for CDI [10,11], other potential risk factors have been identified such as: advanced age, hospitalization [particularly in intensive care units (ICU)], immunosuppression, renal insufficiency, hypoalbuminemia, lengthy hospital stay, the use of nasogastric tubes, invasive gastrointestinal procedures, chemotherapy, the presence of comorbidities, environment-related factors, and the emergence of a hypervirulent strain of the bacterium known as North American pulso-type 1 in some areas [12-21]. However, there might be some other risk factors for the CDI epidemic in the recent years despite tighter control on the use of antibiotics and stricter control policies on hospital-related infections [17]. A possible association between the use of proton pump inhibitors (PPIs) and the development of CDI has been suggested and numerous studies have examined it, reporting conflicting results [22-43]. Since their release in the late 1980s, PPIs have become some of the most widely prescribed agents both in outpatient and inpatient settings throughout the world [44-53], with sales totalling billions dollars worldwide [54,55]. These drugs have proven effective in the treatment of ulcer disease (including bleeding peptic ulcer), gastroesophageal reflux disease, Helicobacter pylori (in combination with antibiotics), Zollinger-Ellison syndrome, in the prophylaxis of upper gastrointestinal complications with nonsteroidal anti-inflammatory drugs (NSAIDs) therapy, stress ulcer prophylaxis in ICU patients, and functional dyspepsia [50,53,56-60]. The widespread use of PPIs during the last 25 years in clinical practice is the result not only of their high efficacy but also of their excellent safety profile, proving to be one of the safest class of medication used in gastroenterology [57,61-64]. Nevertheless, like in the case of other drugs, PPIs are not as safe as it has been thought and more recently, concerns have been raised about their potential association with pneumonia [65-67], bone fractures [68-70], interstitial nephritis and acute kidney injury [71]. More recently, reports of other potential PPIs adverse events such as risk for chronic kidney disease [72,73], dementia [74], spontaneous bacterial peritonitis [75,76], acute myocardial infarction [77,78], micronutrient 6501 September 21, 2017 Volume 23 Issue 35

146 Trifan A et al. PPIs and Clostridium difficile deficiency (magnesium, calcium, iron) [79,80] were published, although the quality of evidence for these is consistently low to very low [81]. An association between PPIs use and CDI is, at least theoretically, rational. Thus, intestinal homeostasis is maintained by host defense mechanisms in which gastric acid plays an important role as a barrier to ingested bacteria and bacterial overgrowth [82]. PPIs therapy profoundly inhibits gastric acid production leading to the proliferation of spores and their ability to convert to a vegetative form of C. difficile [83]. Moreover, PPIs impair leukocyte function by inhibiting phagocytosis and acidification of phagolysosome [84]. Several systematic reviews and meta-analyses have reported conflicting results regarding the association between PPIs use and increased risk of CDI. Thus, no less than six meta-analyses [85-90] found a significant association between PPIs therapy and increased risk of CDI. These findings were also supported by several studies [19,22-26,39,91-114] which reported a risk for CDI two or three times higher in PPIs users than in nonusers. Moreover, the United States Food and Drug Administration (FDA) informed the public about a possible correlation between PPIs use and CDI [115]. Still, other studies and meta-analyses have failed to associate PPIs use with the development of CDI [11,27,34,3 8,40-43, ]. It should be mentioned that PPIs continue to be among the most used drugs despite the above mentioned concerns about long-term side effects. Furthermore, beside a marked overuse of PPIs, over half of prescriptions are for non-indicated reasons [29]. One study reported that 60.7% of patients with CDI used PPIs, of whom only 47.1% had an evidence-based indication [30]. The aim of this systematic review and meta-analysis is to summarize data on the association between PPIs use and the risk of CDI as presented in the published studies. MATERIALS AND METHODS Information sources A systematic literature search was independently conducted by four study investigators (Girleanu I, Stoica OC, Singeap AM and Chiriac SA) using a variety of databases including MEDLINE/PubMed, Web of Science (ISI Web of Knowledge), Scopus, EMBASE, Science Direct, Cumulative Index to Nursing and Allied Health Literature (CINAHL), Excerpta Medica Database, and Cochrane Library, from January 1990 (the first PPI received FDA approval in 1989) to March The database searches were performed using the following medical subject heading (MeSH) terms: proton pump inhibitors, acid suppressive therapy, omeprazole, pantoprazole, lansoprazole, rabeprazole, esomeprazole, combined with C. difficile infection, C. difficile-associated diarrhea, pseudomembranous colitis. Reference lists of all retrieved papers were hand-searched to identify any additional studies that may have been missed in the computed-assisted literature search. The investigation was limited to studies performed in adult human beings, written and published in English, French, and German, in any geographic region. Inclusion and exclusion criteria Selection of the studies.inclusion and exclusion criteria were established a priori by two authors (Trifan A and Stanciu C). First, duplicate citations were identified and removed, then three of us (Ciobica A, Maxim R and Singeap AM) independently reviewed the titles and abstracts of the studies and excluded those which did not answer the search question. Adult case-control and cohort studies providing information on the association between PPI therapy and the development of CDI were included. Studies conducted on pediatric patients, systematic reviews and meta-analyses, consensus documents, studies using PPIs simultaneously with H2 receptor antagonists (H2RA) or reporting exclusively on H2RA, case reports, editorials, protocols, and studies presented only as abstracts were excluded. There was no restriction related to the type of PPI regimen or diagnostic methods of CDI. Any disagreements about study inclusion were resolved in consensus with a third author (Stanciu C or Trifan A) after the full-text of the potential study had been reviewed; all eligible studies were assessed in full. They were subsequently included in this meta-analysis only if reported odds ratios (ORs) or risk ratios (RRs) for (adjusted or unadjusted) casecontrol and cohort studies, respectively, or data for their calculation were available. Data extraction Extracted data were cross-checked independently by four authors (Girleanu I, Stoica OC, Chiriac SA and Ciobica A) from each included study using a standardized data extracting sheet which included the last name of first author, journal and year of publication, country where the study was carried out, study design, sample size, age (mean or median) and gender distribution of patients, duration of the PPI treatment, effect estimates ORs or RRs, and 95% confidence intervals (CIs) of PPI exposure with and without adjustment for confounding variables. Any disagreement between reviewers was resolved in consensus with a third reviewer (Stanciu C or Trifan A). Study quality assessment Assessment of study quality was made independently by two authors (Boiculese L and Girleanu I) using the Newcastle-Ottawa Quality Assessment Scale (NOS; ranging 0-9) [124] as recommended by the Cochrane Handbook for Systematic Reviews of Interventions [125]. The NOS comprises three domains: selection, comparability, and outcome for cohort studies or exposure for case-control studies. A maximum of 6502 September 21, 2017 Volume 23 Issue 35

147 Trifan A et al. PPIs and Clostridium difficile four stars were awarded for selection, two stars for comparability, and three stars for exposure/outcome. Studies with cumulative score 7 were considered high quality, 6 stars to be of moderate quality, and less than 6 stars to be of low quality. When disagreement, after discussion with the third author (Trifan A or Stanciu C) a consensus was reached. The final analysis included 56 high and moderate quality studies. As none of the studies was randomized, and all were observational (case-control and cohort), the methods used in our systematic review and metaanalysis followed the MOOSE (Meta-Analysis of Observational Studies in Epidemiology) criteria [126]. Statistical analysis Meta-analyses were performed both for all studies together and separately for case-control and cohort studies using DerSimonian and Laird [127] random effects model due to expected heterogeneity between studies. Our primary analysis focused on the association between PPIs therapy and the risk for developing CDI and because all of PPIs have similar efficacy we have not performed meta-analyses stratified by type of PPIs. The results are reported as pooled ORs with 95%CIs for primary and subgroup analyses. Heterogeneity between studies was assessed by I 2 statistic and Cochran s Q-statistic. The level of heterogeneity was considered as high when I 2 > 75% or P < 0.10 for the Q statistic [128]. I 2 values between 61%-75%, 30%-60%, and < 30% were considered to represent substantial, moderate and low level of heterogeneity, respectively [129]. Seven potential confounders were considered: study design, effect estimate (adjusted vs unadjusted), setting (community vs inpatient), number of centers (single center vs multicenter), age, study quality, and geographical region. Publication bias was assessed quantitatively using Egger s regression asymmetry test [130] and a P < 0.1 was considered statistically significant for asymmetry, and qualitatively by visual inspection of funnel plots of the logarithmic OR vs their standard errors [131]. Asymmetrical funnel plots were regarded to indicate high risk of publication bias. Number needed to harm (NNH) estimates the number of patients needed to be treated with PPI for one additional person to have a CDI, and was calculated using the pooled OR (95%CI) from the meta-analysis and Patient Expected Event Rate (1.67%) [120]. All statistical tests were two tailed, and results associated with P < 0.05 (except for heterogeneity and publication bias) were considered significant. All analyses were performed using R version software for the metaphor package 1.9-8, which provides a comprehensive collection of validated functions [132]. The statistical analyses of this study were performed by an expert in biostatistics from Grigore T. Popa University of Medicine and Pharmacy, Department of Medical Informatics and Biostatistics. RESULTS Search results The initial online databases search identified 944 studies and 12 more were found from the reference lists of the articles retrieved. After reviewing all titles and abstracts, 216 studies were selected for full-text review, from which 56 studies were found to fulfill the inclusion criteria and were included in meta-analysis. Five of the 56 studies were published after the last meta-analysis (Figure 1). Characteristics of included studies The characteristics of the included studies are shown in Table 1. Of the included 56 studies, 40 (71.4%) were case-control, and 16 (28.6%) cohort studies, addressed to hospital-acquired (n = 43), communityacquired (n = 6), and both hospital and communityacquired CDI (n = 7). Most of the studies (n = 31) were single-center. The size of the study population ranged from 40 to In total, subjects were included, most of them from North-American and European studies (n = 46). Quality assessment The median value of NOS quality assessment was 7, with a mean 6.67 ± 0.74, range 6-8. In studies reporting gender, the proportion of men ranged from 47% to 67%, and from those that reported the age, the average age ranged between 18 and 82.2 years. Thirty-eight studies identified confounding factors (sex, age, antibiotic use, comorbidities) used for adjustment of the association between PPI therapy and risk of CDI. The majority of the studies were retrospective (85.7%) and only 8 were prospective (14.3%). None of the studies was randomized. Meta-analysis Meta-analysis of all studies combined. The results of pooled analysis for all 56 studies showed a significant association between PPI therapy and the risk of CDI as compared with non-ppi users (OR = 1.99, CI: , P < 0.001) (Figure 2). There was significant heterogeneity of effects across studies (I 2 = 85.41%; P < 0.001). Subgroup analyses of case-control and cohort studies also showed a significant higher risk of CDI with PPI use (Table 2). There was no significant difference of effects between cohort and case-control studies (P = 0.931). The pooled OR for the cohort studies was 1.98 similar to OR for case-control that was 2.0. The association remained also significant after limiting meta-analysis to studies with both adjusted (OR = 1.95, CI: , P < 0.001) and unadjusted 6503 September 21, 2017 Volume 23 Issue 35

148 Trifan A et al. PPIs and Clostridium difficile Included Eligibility Screening Identification Records identified through database searching (n = 944) Additional records identified through other sources (n = 12) Records after duplicates removed (n = 946) Records after screened (n = 755) Full-text articles assessed for eligibility (n = 216) Studies included in qualitative synthesis (n = 56) Studies included in quantitative synthesis (meta-analysis) (n = 56) Records excluded (n = 191) Full-text articles excluded with reasons (n = 160) Figure 1 Study selection process. data (OR = 2.02, CI: , P < 0.001). There was also no significant difference of effects between adjusted and unadjusted studies (P = 0.856). PPIs use was found to be associated with an increased risk of CDI in both single-center studies (OR = 2.18, 95%CI: ) and multicenter studies (OR = 1.82, 95%CI: ). There was no significant difference between inpatients and outpatients regarding CDI risk (P = 0.868). For both inpatients and outpatients the PPIs use almost doubled the risk of CDI (OR = 1.95, OR = 2.10, respectively). When grouped by region, a direct association was found in the European group (OR = 1.78, 95%CI: ), the North American group (OR = 2.00, 95%CI: ), while the highest risk of CDI after PPI treatment was demonstrated in the Asian group (OR = 2.31, 95%CI: ). The subgroup of high-quality studies (NOS 7) showed a direct association (OR = 1.88, 95%CI: ) between PPIs and risk of CDI, and this association was also significant in the medium-quality group (OR = 2.11, 95%CI: ), with no difference between the two groups (P = 0.441). There was no statistical difference regarding the risk for CDI for elderly ( 65 years) compared with younger group (< 65 years) (P = 0.860). Publication bias We have drawn the funnel plot for 3 levels of confidence interval (90%, 95% and 99% corresponding to shades white, gray and dark gray) (Figure 3). The Egger s test of asymmetry proved no significance (Z = , P = 0.711). Number needed to harm Based on reported incidence of CDI (at 14 d after hospital admission) of 1.67% in patients who have not used PPI, we estimate a NNH of 63 (95%CI: 48-78), if these patients will receive PPIs September 21, 2017 Volume 23 Issue 35

149 Trifan A et al. PPIs and Clostridium difficile Table 1 Characteristics of studies included in the meta-analysis Author, yr Region Study design Centers Setting Sample size, n Mean age, yr Identified confounders OR (95%CI) Akhtar et al Shaheen [91], 2007 Al-Tureihi et al [19], 2005 America Case-control Unicenter Inpatient 1290 Adjusted for age, sex, comorbidities 2.1 ( ), antibiotics, chemotherapy America Case-control Unicenter Inpatient 53 Adjusted for age, antibiotics 3.1 ( ) Aseeri et al [23], 2008 America Case-control Unicenter Inpatient 188 Adjusted for admission date, sex, 4.4 ( ) age group, antibiotic use, patient location, and room type Bajaj et al [133], 2010 America Case-control Multicenter Mixt 162 Adjusted for antibiotics, PPI 37.6 ( ) Barletta et al [92], 2014 Asia Case-control Unicenter Inpatient 408 Adjusted for PPI exposure, 2.1 ( ) antibiotics, immunosuppression Baxter et al [93], 2008 America Case-control Multicenter Inpatient 4493 Adjusted for antibiotics, age, 1.2 ( ) hospital stay, other infections Beaulieu et al [27], 2007 Cohort Unicenter Inpatient 827 Adjusted for age, sex, length of stay, 1.3 ( ) comorbidities, APACHE score, NGT feeding, tracheal tube placement, antibiotics Branch et al [94], 2007 America Case-control Unicenter Inpatient No 13.0 ( ) Buendgens et al [95], 2014 Europe Case-control Multicenter Inpatient 3286 Adjusted for age, sex, antibiotics, comorbidities, other treatment Campbell et al [38], 2013 America Case-control Unicenter Inpatient 96 Adjusted for antibiotics, hospitalization Cunningham et al [96], 2003 Europe Case-control Unicenter Inpatient 320 Adjusted for antibiotics and chemotherapy 3.1 ( ) 2.2 ( ) 2.5 ( ) Dalton et al [22], 2009 America Cohort Multicenter Inpatient Adjusted for number of medication 1.9 ( ) groups, antibiotic days, age, length of stay, medical service, PPI days Debast et al [116], 2009 Europe Case-control Unicenter Inpatient 154 Adjusted for age, hospital stay, 1.1 ( ) comorbidities, antibiotics Dial et al [26], 2004 America Case-control Multicenter Inpatient 188 Adjusted for age, antibiotics 2.6 ( ) (case-control) Dial et al [26], 2004 America Cohort Multicenter Inpatient 1187 Adjusted for age, antibiotics 2.1 ( ) (cohort) Dial et al [98], 2005 Europe Case-control Multicenter Outpatient Adjusted for age, sex, antibiotics 2.9 ( ) Dial et al [97], 2006 Europe Case-control Multicenter Outpatient 3484 Adjusted for PPI and antibiotics 3.5 ( ) Dial et al [134], 2008 America Case-control Multicenter Outpatient Adjusted for age, sex, antibiotics, comorbidities, physician visits, hospital admissions, length of stay 1.6 ( ) Dubberke et al [99], 2007 America Cohort Unicenter Inpatient Adjusted for age, admissions, antibiotics, albumin level, leukemia/lymphoma, mechanical ventilation, antimotility agents 1.6 ( ) Elseviers et al [100], 2015 Europe Case-control Multicenter Inpatient Adjusted for age, co-morbidity, 1.9 ( ) endoscopic procedures Faleck et al [42], 2016 America Cohort Unicenter Inpatient Adjusted for age, sex, antibiotics, 0.6 ( ) comorbidities, length of stay Garzotto et al [43], 2015 Europe Case-control Multicenter Inpatient 225 No 0.4 ( ) Hebbard et al [135], 2017 Asia Case-control Unicenter Inpatient Adjusted for age, chemotherapy, 2.4 ( ) abdominal surgery, antibiotics Hensgens et al [117], Europe Case-control Unicenter Inpatient 169 Adjusted for age, co-morbidity, 1.1 ( ) 2011 antibiotics, ICU stay Howell et al [136], 2010 America Cohort Unicenter Inpatient Adjusted for age, comorbidities, antibiotics 1.7 ( ) Ingle et al [40], 2011 Asia Cohort Unicenter Mixt Adjusted for immunosuppression 1.8 ( ) Ingle et al [118], 2013 Asia Case-control Unicenter Community no 2.3 ( ) Jayatilaka et al [101], 2007 America Case-control Unicenter Inpatient 366 Adjusted for PPI 2.7 ( ) 6505 September 21, 2017 Volume 23 Issue 35

150 Trifan A et al. PPIs and Clostridium difficile Kazakova et al [102], 2006 America Case-control Unicenter Mixt 195 Adjusted for antibiotics, PPI, length of stay, 5.0 ( ) psychosis, depression Khan et al [39], 2012 Asia Cohort Unicenter Inpatient 123 Adjusted for surgery, PPI, 3.2 ( ) antibiotics, hospitalization, Underlying debilitating conditions Khanafer et al [119], 2013 Europe Cohort Unicenter Inpatient ( ) Kuntz et al [2], 2011 America Case-control Unicenter Mixt 3344 no 2.3 ( ) Kurti et al [3], 2015 Europe Case-control Multicenter Inpatient Adjusted for antibiotics, PPI, length 1.6 ( ) of stay, Kutty et al [41], 2010 America Case-control Multicenter Outpatient No 1.7 ( ) Lewis et al [103], 2016 America Cohort Unicenter Inpatient No 6.4 ( ) Lin et al [137], 2013 Asia Case-control Multicenter Inpatient Age, sex, unit, 10.1 ( ) antibiotics, length of stay Linney et al [24], 2010 America Case-control Unicenter Inpatient 284 Age, sex, discharge date and 2.4 ( ) hospital unit, antibiotics, diabetes mellitus, IBD, cancer, enteral feeding, length of stay Loo et al [120], 2005 America Case-control Multicenter Inpatient 474 no 1.0 ( ) Loo et al [138], 2011 America Cohort Multicenter Inpatient Adjusted for age, PPI, antibiotics, 2.6 ( ) chemotherapy Lowe et al [121], 2006 America Case-control Multicenter Inpatient Adjusted for antibiotics, other 0.9 ( ) medications, and comorbidities McFarland et al [122], America Case-control Multicenter Mixt 368 No 0.8 ( ) 2007 Mizui et al [104], 2013 Asia Case-control Multicenter Inpatient No 3.2 ( ) Modena et al [105], 2005 America Case-control Unicenter Inpatient 250 Adjusted for macrolides, ICU, length of stay, infections 3.3 ( ) Mori et al [123], 2015 Asia Case-control Unicenter Outpatient No 0.4 ( ) Muto et al [106], 2005 America Case-control Multicenter Inpatient 406 Adjusted for PPI, antibiotics, 2.4 ( ) diabetes mellitus, organ transplantation Pakyz et al [107], 2014 America Case-control Multicenter Inpatient No 1.4 ( ) Peled et al [108], 2007 America Cohort Unicenter Inpatient 217 Adjusted for PPI, low albumin level, 3.7 ( ) Pepin et al [11], 2005 America Cohort Unicenter Inpatient 5619 Adjusted for age, length of stay, 1.0 ( ) antibiotics Ro et al [139], 2016 Asia Cohort Unicenter Inpatient Adjusted for age, antibiotics, comorbidities 3.3 ( ) Roughead et al [109], 2016 Asia Cohort Multicenter Mixt Adjusted for antibiotics, PPI, length of stay, 2.4 ( ) Shah et al [34], 2000 Europe Case-control Unicenter Inpatient 252 No 0.8 ( ) Southern et al [110], 2010 Europe Cohort Multicenter Inpatient No 2.3 ( ) Vesteinsdottir et al [111], Europe Case-control Multicenter Mixt 333 No 1.6 ( ) 2012 Yang et al [112], 2011 Asia Case-control Multicenter Inpatient No 1.9 ( ) Yearsley et al [25], 2006 Europe Case-control Unicenter Inpatient Adjusted for PPI, antibiotics, female sex 1.9 ( ) Yip et al [140], 2001 America Case-control Unicenter Inpatient 54 No 3.0 ( ) CI: Confidence interval; IBD: Inflammatory bowel disease; ICU: Intensive care unit; PPI: Proton pump inhibitor; NGT: Naso-gastric tube; OR: Odds ratio. DISCUSSION This systematic review and meta-analysis w h i c h i n c l u d e s 5 6 s t u d i e s a n d subjects [2,3,11,19,22-27,34,38-43,91-112, , ] found a significant association between PPI therapy and the risk for CDI development. Both the overall pooled estimates (OR = 1.99, CI: , P < 0.001) and subgroup analyses showed a significant increased risk for CDI in patients on PPI therapy compared to nonusers, despite substantial statistical heterogeneity among studies and evidence of publication bias. Thus, in line with previous meta-analyses, our results add further evidence to PPIs use as a risk factor for development of CDI [85-89]. Since 2001, when Yip et al [140] first suggested a possible association between PPIs use and the risk of CDI, other studies, systematic reviews, and metaanalyses have reported such an association. It should be mentioned that a decade earlier (1993), Walker et al [141] suggested that the H2RAs therapy was a potential risk factor for CDI. In an earlier systematic review which included 11 studies with patients, Leonard et al [113] reported a significant association between PPI therapy and CDI (OR = 2.05, 95%CI: ) although there was significant heterogeneity among the studies (χ 2 = 50.9, P < 6506 September 21, 2017 Volume 23 Issue 35

151 Trifan A et al. PPIs and Clostridium difficile RE Model Study Participants Weight OR [ 95%CI] 1. Akntar and Shaheen, % 2.13 [1.68, 2.70] 2. AI-Tureihi et al, % 3.17 [1.03, 9.76] 3. Aseeri et al, % 4.42 [2.36, 8.28] 4. Bajaj et al, % [6.22, ] 5. Barletta et al, % 2.19 [1.26, 3.81] 6. Baxter et al, % 1.23 [1.03, 1.47] 7. Beaulieu et al, % 1.39 [0.94, 2.06] 8. Branch et al, % [7.53, 22.72] 9. Buendgens et al, % 3.11 [1.11, 8.71] 10. Campbell et al, % 2.20 [0.60, 8.07] 11.Cunningham et al, % 2.50 [1.50, 4.17] 12. Dalton et al, % 1.96 [1.42, 2.17] 13. Debast et al, % 1.10 [0.50, 2.42] 14. Dial et al, 2004 (case-control) % 2.60 [1.33, 5.08] 15.Dial et al, 2004 (cohort) % 2.10 [1.23, 3.59] 16. Dial et al, % 2.90 [2.40, 3.50] 17. Dial et al, % 3.50 [2.30, 5.33] 18. Dial et al, % 1.60 [1.30, 1.97] 19. Dubberke et al, % 1.98 [1.15, 3.41] 20. Elseviers et al, % 1.98 [1.15, 3.41] 21. Faleck D et al, % 0.64 [0.48, 0.85] 22. Garzotto et al, % 0.43 [0.20, 0.92] 23. Hebbard et al, % 2.46 [1.05, 5.76] 24. Hensgens et al, % 1.14 [0.51, 2.55] 25. Howell et al, % 1.74 [1.39, 2.18] 26. Ingle et al, % 1.84 [0.45, 7.52] 27. Ingle et al, % 2.37 [0.60, 9.36] 28. Jay atilaka et al, % 2.75 [1.65, 4.58] 29. Kazakova et al, % 5.02 [1.30, 19.38] 30. Khan et al, % 3.22 [1.21, 8.57] 31. Khanafer et al, % 2.55 [0.67, 9.71] 32. Kuntz et al, % 2.30 [1.56, 3.39] 33. Kurti et al, % 1.62 [1.15, 2.28] 34. Kutty et al, % 1.70 [0.70, 4.13] 35. Lewis PO et al, % 6.46 [3.63, 11.50] 36. Lin et al, % [1.20, 85.01] 37. Linney et al, % 2.40 [1.40, 4.11] 38. Loo et al, % 1.02 [0.71, 1.47] 39. Loo et al, % 2.64 [1.71, 4.08] 40. Lowe et al, % 0.90 [0.77, 1.05] 41. McFarland et al, % 0.84 [0.50, 1.41] 42. Mizui et al, % 3.22 [1.42, 7.32] 43. Modena et al, % 3.36 [1.66, 6.80] 44. Mori et al, % 0.49 [0.12, 2.00] 45. Muto et al, % 2.40 [1.30, 4.43] 46. Pakyz et al, % 1.43 [1.30, 1.57] 47. Peled et al, % 3.76 [1.52, 9.30] 48. Pepin et al, % 1.00 [0.79, 1.27] 49. Ro Y et al, % 3.30 [1.50, 7.26] 50. Roughead E et al, % 2.40 [1.90, 3.03] 51. Shah et al, % 0.86 [0.47, 1.57] 52 Southern et al, % 2.32 [1.18, 4.56] 53. Vestinsdottir et al, % 1.69 [1.07, 2.67] 54 Yang et al, % 1.92 [1.34, 2.75] 55. Yearsley et al, % 1.90 [1.10, 3.28] 56. Yip C et al, % 3.00 [0.81, 11.11] RE model % 1.99 [1.73, 2.30] Figure 2 Forest plot of the meta-analysis Odd ratio (log axis) ). During the last years, six meta-analyses have been published on this topic, and all reported a positive association between PPIs use and the risk for CDI. Thus, Janarthanan et al [88] in a meta-analysis including 6507 September 21, 2017 Volume 23 Issue 35

152 Trifan A et al. PPIs and Clostridium difficile Table 2 Subgroup analysis Subgroup analysis No. of studies (n = 56) ORs 95%CI Heterogeneity, I 2, % Heterogeneity between groups, P value Study design Case-control Cohort Study type Adjusted Unadjusted Centers Unicentric Multicentric Type Inpatient Outpatient Mixt Region Europe America Asia Age Age < 65 yr Age 65 yr NOS NOS NOS < CI: Confidence interval; NOS: Newcastle-Ottawa Quality Assessment Scale; ORs: Odds ratio. 23 observational studies with nearly patients found a 65% increase in the incidence of CDI among PPIs users with an estimated risk of 1.69 and 95%CI from to In another meta-analysis (30 studies, patients), Desphande et al [85] reported that PPI therapy was associated with a 2-fold increase in risk for CDI, but their study is limited by unadjusted risk estimates. Recently, the same team [90] performed a meta-analysis examining the relationship between PPI therapy and the risk for recurrent CDI, and found a positive association with the pooled risk ratio of 1.58 (95%CI: ). A third meta-analysis by Kwok et al [87] including 42 studies ( participants) also found a statistically significant association between PPIs use and the risk for CDI compared with nonusers (OR = 2.51; 95%CI: ; P = 0.05). Tleyjeh et al [86] in a systematic review and meta-analysis including 51 observational studies (37 case-control and 14 cohort) examining healthcare and communityassociated CDI, found a very low quality evidence for an association between PPI therapy and CDI not supporting a cause-effect relationship. Authors reported a pooled OR of 1.65 (95%CI: ) with evidence of publication bias and significant statistical heterogeneity among the studies (I 2 = 89.9%). More recently, Arriola et al [89] suggest, in a meta-analysis including only inpatients, that PPIs use significantly increases the risk of hospital-acquired CDI (OR = 1.81). Bavishi et al [114] in a systematic review regarding the use of PPI and increased susceptibility to enteric infection found 27 studies evaluating an association between PPI therapy and the risk of CDI, 17 of which reported a significant association. Based on an analysis of 28 studies, US FDA issued a warning on the risk of CDI with PPIs use [115], and similar warnings are found in CDI treatment guidelines [142]. Several studies reported that PPIs use is also a risk factor for community-acquired CDI. Dial et al [26], in a study including over 1000 cases of communityacquired CDI, found that patients who had received PPIs within the previous 90 d had a nearly 3-fold increased risk for CDI. A similar result was reported by Kutty et al [41] who found a 2-3-fold increased for community-acquired CDI in patients treated with PPIs within the previous 6 mo. Marwick et al [143] in a study including patients aged 65 years or older identified all cases of community-acquired CDI and found that patients prescribed PPIs within the previous 6 mo had a 1.7-fold increased risk for CDI compared to matched controls. A study assessing the epidemiology of community-acquired CDI found rates of PPI use of nearly 30% among patients with this infection compared to less than 3% in the general population [144]. These results indicate a similar degree of association between PPIs use and CDI risk, be it community-acquired CDI or hospital-acquired CDI [145]. Nevertheless, the association between PPIs use and the risk for CDI remains to a certain extent controversial despite the results reported above, as several studies failed to find such an association [11,27,34,94,122]. Beaulieu et al [27] found that the use of gastric acid-suppression therapy does not predispose to development of CDI, while McFarland et al [122] reported no relation between CDI and the use of PPIs. Branch et al [94] found that 6508 September 21, 2017 Volume 23 Issue 35

153 Trifan A et al. PPIs and Clostridium difficile Standard error Publication bias OR (log axis) Figure 3 Funnel plot with 95% confidence limits. PPI use did not increase the incidence of CDI in hospitalized patients. The mechanism by which PPI therapy contributes to an increased risk of CDI is unclear. It has been proposed that a vegetative form of C. difficile survives in conditions of gastric ph greater than 4 [114] -the threshold for enteric infections acquisition, including C. difficile. Howell et al [136] reported that the risk of nosocomial CDI rose with increasing levels of acid suppression. Hegarty et al [146] reported that PPI therapy decreased the expression of genes holding an important role in colonocyte integrity, thus favoring the development of CDI. Other studies show that long-term use of PPIs decreases microbial diversity, a condition found in patients with CDI [147]. As we have already mentioned, our subgroup analyses also showed an increased risk for CDI. There were no significant differences of effects between cohort and case-control studies, adjusted and unadjusted data, single-center and multicenter studies, hospitalized-and community-acquired CDI or among geographic regions. Advanced age is a wellknown risk factor for CDI. To our surprise, we found no increased risk of CDI in elderly patients ( 65 years) using PPIs compared with youngers (OR = 1.93 vs OR = 2.06, P = 0.860). A possible explanation is that many of such patients may have atrophic gastritis with low gastric acid output [148] and PPIs use cannot further lower gastric acid secretion, without any additional risk of CDI [32]. As data regarding the association between PPI therapy and risk of CDI are supported only by observational studies, a final estimation of the real risk is not possible. It should be mentioned that randomized placebo-controlled clinical trials evaluating the association of PPIs use and the risk for CDI are ethically unfeasible and therefore, such studies could not be performed in the future. Thus, a weak association between PPI therapy and CDI does not confirm causality and could be the result of bias and uncontrolled confounding (e.g., comorbidities, comedication use, etc.) which were lacking in most studies. Our meta-analysis has some strengths such as the largest number of studies published to date, adjusted effect estimates concerning the association between PPI use and the risk of CDI, and subgroup analyses based on age, region, type, design and quality of the study. However, it also has several limitations: the included studies were observational, influenced by confounding variables despite statistical adjustment, the significant heterogeneity among most of them and lack of information regarding the dose and duration of PPI use as well as patient compliance to PPI therapy. Although the above presented data from several meta-analyses and many studies demonstrated an association between PPI therapy and the risk for development of CDI, PPIs continue to be overused even in patients who are at high risk of CDI, because they are still considered safe drugs by most physicians. There is evidence that over half of PPI users who developed CDI had no valid indications for such therapy [25]. While in many countries PPIs are now totally available as over-the-counter medication, clinicians should inform their patients about the risk of CDI when PPIs are used on the long-term and without valid indication. In spite of the aforementioned limitations of our and several other meta-analyses, clinicians should be aware of the risk of CDI when prescribing longterm PPI therapy, particularly in patients at high risk (e.g., hospitalized patients on antibiotics). It should be underlined that PPIs remain, on the whole, a safe group of drugs [149], providing enormous benefits when prescribed for well-established indications. Unfortunately, many prescriptions fall outside accepted indications [90]. In conclusion, this systematic review and metaanalysis provides further evidence that PPI use significantly increases the risk for developing CDI, despite the substantial heterogeneity and publication bias present among studies. Due to the fact that all the studies included in our analysis are observational and cannot confirm causality, further large, high quality, prospective studies are needed to assess the association between PPI use and the risk of CDI. COMMENTS Background Proton pump inhibitors (PPIs) are among the most widely prescribed agents by gastroenterologists because of their high efficacy and excellent safety profile. However, more recently, concerns have been raised about association between PPI therapy and several potentially serious adverse events including Clostridium difficile (C. difficile) infection (CDI). This systematic review and meta-analysis explored the existing evidence regarding the association of PPI therapy and CDI. Research frontiers Many observational studies and meta-analyses have reported conflicting results regarding the association between PPI therapy and the risk for CDI. Innovations and breakthroughs This systematic review and meta-analysis, including the largest number 6509 September 21, 2017 Volume 23 Issue 35

154 Trifan A et al. PPIs and Clostridium difficile of studies published to date, provides further evidence that PPI therapy is associated with an increased risk for development of CDI. Because all the studies analyzed were observational, with inherent limitations, the causality could not be confirmed. Applications Although our systematic review and meta-analysis, in line with previous studies and meta-analyses, reported an association between PPI therapy and the risk for development of CDI, such association remains controversial and a final estimation of the real risk has not been established. Further high-quality, prospective studies are needed to assess whether this association is causal. Until then, clinicians should be aware that long-term PPI therapy may be associated with the risk of CDI, and prescribe the PPIs in the lowest effective dose only to patients with a clear indication. Terminology PPIs are a group of potent inhibitors of gastric acid secretion. CDI is a symptomatic infection due to the spore-forming bacterium C. difficile. Peer-review This manuscript is an interesting, informative and well-presented meta-analysis on PPI therapy and risk of C. difficile infection. REFERENCES 1 Khanna S, Pardi DS. The growing incidence and severity of Clostridium difficile infection in inpatient and outpatient settings. Expert Rev Gastroenterol Hepatol 2010; 4: [PMID: DOI: /egh.10.48] 2 Kuntz JL, Chrischilles EA, Pendergast JF, Herwaldt LA, Polgreen PM. Incidence of and risk factors for community-associated Clostridium difficile infection: a nested case-control study. BMC Infect Dis 2011; 11: 194 [PMID: DOI: / ] 3 Kurti Z, Lovasz BD, Mandel MD, Csima Z, Golovics PA, Csako BD, Mohas A, Gönczi L, Gecse KB, Kiss LS, Szathmari M, Lakatos PL. Burden of Clostridium difficile infection between 2010 and 2013: Trends and outcomes from an academic center in Eastern Europe. World J Gastroenterol 2015; 21: [PMID: DOI: /wjg.v21.i ] 4 Honda H, Dubberke ER. 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155 Trifan A et al. PPIs and Clostridium difficile 25 Yearsley KA, Gilby LJ, Ramadas AV, Kubiak EM, Fone DL, Allison MC. Proton pump inhibitor therapy is a risk factor for Clostridium difficile-associated diarrhoea. Aliment Pharmacol Ther 2006; 24: [PMID: DOI: / j x] 26 Dial S, Alrasadi K, Manoukian C, Huang A, Menzies D. Risk of Clostridium difficile diarrhea among hospital inpatients prescribed proton pump inhibitors: cohort and case-control studies. CMAJ 2004; 171: [PMID: DOI: /cmaj ] 27 Beaulieu M, Williamson D, Pichette G, Lachaine J. Risk of Clostridium difficile-associated disease among patients receiving proton-pump inhibitors in a Quebec medical intensive care unit. Infect Control Hosp Epidemiol 2007; 28: [PMID: DOI: /521664] 28 Pant C, Madonia P, Minocha A. Does PPI therapy predispose to Clostridium difficile infection? Nat Rev Gastroenterol Hepatol 2009; 6: [PMID: DOI: / nrgastro ] 29 Rashid S, Rajan D, Iqbal J, Lipka S, Jacob R, Zilberman V, Shah M, Mustacchia P. 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159 Trifan A et al. PPIs and Clostridium difficile inhibitors induce changes in colonocyte gene expression that may affect Clostridium difficile infection. Surgery 2014; 156: [PMID: DOI: /j.surg ] 147 Seto CT, Jeraldo P, Orenstein R, Chia N, DiBaise JK. Prolonged use of a proton pump inhibitor reduces microbial diversity: implications for Clostridium difficile susceptibility. Microbiome 2014; 2: 42 [PMID: DOI: / ] 148 Husebye E, Skar V, Høverstad T, Melby K. Fasting hypochlorhydria with gram positive gastric flora is highly prevalent in healthy old people. Gut 1992; 33: [PMID: DOI: / gut ] 149 Scarpignato C, Gatta L, Zullo A, Blandizzi C; SIF-AIGO-FIMMG Group; Italian Society of Pharmacology, the Italian Association of Hospital Gastroenterologists, and the Italian Federation of General Practitioners. Effective and safe proton pump inhibitor therapy in acid-related diseases - A position paper addressing benefits and potential harms of acid suppression. BMC Med 2016; 14: 179 [PMID: DOI: /s z] P- Reviewer: Chiba T, Garcia-Olmo D, Mohammad RN S-Editor: Wei LJ L- Editor: A E- Editor: Ma YJ 6515 September 21, 2017 Volume 23 Issue 35

160 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) META-ANALYSIS Outcomes after liver transplantation in accordance with ABO compatibility: A systematic review and meta-analysis Eung Chang Lee, Seong Hoon Kim, Sang-Jae Park Eung Chang Lee, Seong Hoon Kim, Sang-Jae Park, Center for Liver Cancer, National Cancer Center, Goyang-si, Gyeonggido , South Korea ORCID number: Eung Chang Lee ( ); Seong Hoon Kim ( ); Sang-Jae Park ( ). Author contributions: Lee EC, Kim SH, Park SJ designed the research; Lee EC performed the research and wrote the paper; Kim SH supervised the study and revised the paper. Conflict-of-interest statement: No conflicts of interest were declared for all authors. Data sharing statement: Technical appendix, statistical code, and dataset available from the corresponding author at kshlj@ ncc.re.kr. No additional data are available. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Unsolicited manuscript Correspondence to: Seong Hoon Kim, MD, PhD, Center for Liver Cancer, National Cancer Center, 323 Ilsan-ro, Ilsandonggu, Goyang-si, Gyeonggi-do , South Korea. kshlj@ncc.re.kr Telephone: Fax: Received: May 26, 2017 Peer-review started: May 26, 2017 First decision: June 23, 2017 Revised: July 7, 2017 Accepted: August 15, 2017 Article in press: August 15, 2017 Published online: September 21, 2017 Abstract AIM To evaluate the differences in outcomes between ABOincompatible (ABO-I) liver transplantation (LT) and ABO-compatible (ABO-C) LT. METHODS A systematic review and meta-analysis were performed by searching eligible articles published before November 28, 2016 on MEDLINE (PubMed), EMBASE, and Cochrane databases. The primary endpoints were graft survival, patient survival, and ABO-I-related complications. RESULTS Twenty-one retrospective observational studies with a total of 8247 patients were included in this metaanalysis. Pooled results of patient survival for ABO-I LT were comparable to those for ABO-C LT. However, ABO-I LT showed a poorer graft survival than ABO-C LT (1-year: OR = 0.66, 95%CI: , P < 0.001; 3-year: OR = 0.74, 95% CI , P < 0.001; 5-yearr: OR =0.75, 95%CI: , P < 0.001). Furthermore, ABO-I LT was associated with more incidences of antibody-mediated rejection (OR = 74.21, 95%CI: , P < 0.001), chronic rejection (OR =2.28, 95%CI: , P = 0.05), cytomegalovirus infection (OR = 2.64, 95%CI: , P < 0.001), overall biliary complication (OR = 1.52, 95%CI: , P = 0.04), and hepatic artery complication (OR = 4.17, 95%CI: , P < 0.001) than ABO-C LT. In subgroup analyses, ABO-I LT and ABO-C LT showed a comparable graft survival in pediatric patients and those using rituximab, and ABO-I LT showed an 6516 September 21, 2017 Volume 23 Issue 35

161 Lee EC et al. ABO incompatability in liver transplantation increased acute cellular rejection in cases involving deceased donor grafts. CONCLUSION Although patient survival in ABO-I LT was comparable to that in ABO-C LT, ABO-I LT was inferior to ABO-C LT in graft survival and several complications. Graft survival of ABO-I LT could be comparable to that of ABO-C LT in pediatric patients and those using rituximab. Key words: ABO-incompatibility; Liver transplantation; Graft survival; Patient survival; Complications The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: This meta-analysis analyzed more than 8000 cases of ABO-incompatible (ABO-I) and ABOcompatible (ABO-C) liver transplantation (LT). Although patient survival was similar, ABO-I LT was inferior to ABO-C LT in graft survival and several ABO-Irelated complications. Graft survival of ABO-I LT was comparable to that of ABO-C LT in pediatric patients and those using rituximab. Lee EC, Kim SH, Park SJ. Outcomes after liver transplantation in accordance with ABO compatibility: A systematic review and meta-analysis. World J Gastroenterol 2017; 23(35): Available from: URL: v23/i35/6516.htm DOI: i INTRODUCTION In an animal experiment in 1969, Starzl et al [1] reported that the liver is a privileged organ that could be transplanted with relatively lower prevalence of acute rejection than those associated with the kidney or heart. Furthermore, in 1979, Starzl et al [2] reported 11 cases of successful ABO-incompatible (ABO-I) liver transplantation (LT) without graft rejections [2]. Since then, however, there has been a series of reports of heightened prevalence of antibody-mediated rejection (AMR), lower graft survival, hepatic artery thrombosis (HAT), and cholangitis in ABO-I LT compared to ABOcompatible (ABO-C) LT [3-6]. Nevertheless, the application of various desensitization strategies, such as plasma exchange (PE) (or plasmapheresis), splenectomy, graft local infusion (GLI), mycophenolate mofetil (MMF), rituximab, and intravenous immunoglobulin (IVIG) to ABO-I LT highlight the potential of ABO-I LT as a promising alternative to ABO-C LT, and the introduction of rituximab has brought about substantial improvements in the outcomes of ABO-I LT [7-12]. Currently, its importance is expanding in the East, where the proportion of uses of ABO-I allografts for living donor liver transplantation (LDLT) is higher than that in the West, and particularly in Korea and Japantwo countries that show notably higher proportions of interfamilial organ donation. However, there are still heated debates with regard to the prevalence of graft survival, patient survival, ABO-I-related complications, such as rejection, infection, biliary stricture, and HAT associated with ABO-I LT and ABO-C LT, with much heterogeneity in different reports. Therefore, considering the fact that cases of ABO-I LT would inevitably rise due to demands for donor organs far outnumbering the supply and increased difficulty of matching appropriate ABO-C liver allografts, a comprehensive analysis of the results from previous reports of LT across the ABO blood group barrier is needed. MATERIALS AND METHODS Study selection Systematic review and meta-analysis were performed in compliance with the Preferred Reporting Items for Systematic Reviews and Meta-Analysis (PRISMA) guidelines [13]. Databases of Medline (Pubmed), EMBASE, and Cochrane library were used to search for relevant articles among publications dated November 28, Publication year and language were not specified or limited for the search. The following keywords for the database search were used: (ABO OR = blood group OR = blood type) AND (incompatibility OR = mismatch OR = barrier) AND liver transplantation. Title and abstracts of identified articles were screened independently by two investigators (Lee EC, Shim JR ), and full-text articles with potential relevance were obtained. Eligibility criteria The included studies were articles that compared ABO-I LT and ABO-C LT with a minimum of one outcome of interest. The following types of articles were excluded: abstracts, meeting papers, case reports/series, reviews, meta-analyses, letters, editorial comments, animal studies, single-arm studies, and studies unable to extract data. When there were overlapping cohorts examined by the same institutions, data from the most recent studies were used. Assessment of methodological quality Because there was no randomized controlled trial study included in this review, methodological quality was assessed based on a maximum score of 9 for selection of patients, comparability, and outcome of study as per the Newcastle-Ottawa Quality Assessment Scale for Cohort Studies [14]. All of the studies included in this meta-analysis were assessed by two investigators (Lee EC, Shim JR), and disagreements were resolved by a consensus September 21, 2017 Volume 23 Issue 35

162 Lee EC et al. ABO incompatability in liver transplantation Data extraction Data for the following items were extracted: first author, publication year, study periods, region, sample size, population, recipient age, donor age, urgent indication, donor type, prescription for ABO-I LT, immunosuppression, graft survival, patient survival, AMR, acute cellular rejection (ACR), CR (chronic rejection), bacterial infection, fungal infection, cytomegalovirus (CMV) infection, overall biliary complication, bile leak, biliary stricture, hepatic artery (HA) complication, hepatic vein (HV) complication, and portal vein (PV) complication. For studies that divided ABO-C LT into ABO-C non-identical LT and ABO- Identical (ABO-Id) LT and reported separate outcomes for each [15-20], the outcomes were combined for the purpose of this meta-analysis. If the required data were not clearly articulated in the selected articles, we requested the original data via an to the corresponding authors. All data were independently investigated and cross-checked by two investigators (Lee EC, Shim JR), and another investigator (Kim SH) provided the final confirmation. Statistical analysis This meta-analysis was performed in compliance with the Cochrane guidelines for systematic revie [21]. Categorical variables were analyzed with odds ratios (OR) with 95% confidence interval (CI) using the Mantel-Haenszel method, and continuous variables were analyzed with weighted mean differences (WMD) with 95%CI using an inverse variance method. Heterogeneity among studies was assessed with Higgin s I 2 index [22] or Cochran s Q test [21,23]. The random-effects model was used when I 2 was > 50% or P-value (Cochran s Q test) was < 0.10, and the fixed-effects model was used in all other cases. Heterogeneous results were further examined with a sensitivity analysis using the leave-one-out method and subgroup analyses. Possible publication bias was assessed using funnel plots and Egger s regression test, which evaluates a funnel plot asymmetry [24,25]. If a publication bias with P-value < 0.10 (Egger s regression test) was detected, the impact on the outcomes of the meta-analysis was assessed after enhancing the symmetry using the trim-andfill method of Duvall and Tweedie [26]. The presence of publication bias in fewer than 10 studies was considered unreliable as per the Cochrane Handbook for Systematic Reviews [27]. A P-value of < 0.05 was considered statistically significant. Statistical analyses were performed using the Review Manager (RevMan) software version 5.3 ( revman) and R package meta ( org/web/packages/meta). RESULTS Search results A total of 986 citations were found in the primary search using combinations of keywords for database search, of which 762 irrelevant or duplicated citations were excluded. After retrieving the titles and abstracts from the remaining 224 articles, another 186 articles were excluded. From the 38 potentially relevant studies, additional studies were excluded for the following reasons: single-arm studies (n = 9), overlapping cohorts from the same institutions (n = 4), studies investigating multiple desensitization protocols (n = 1), timing of rituximab administration (n = 1), and pathology (n = 1), and unable data extraction (n = 1). As a result, 21 studies were included in this meta-analysis [5,15-20,28-41]. The flow diagram of study selection is shown in Figure 1. Characteristics of included studies This meta-analysis included 21 retrospective observational studies that were conducted on a total of 8247 patients [5,15-20,28-41]. Of these patients, 1494 underwent ABO-I LT, while 6753 underwent ABO-C LT. There has been no randomized clinical trial conducted on this topic. Study periods ranged from For studies involving less than 10% heterogeneity in study population (adult vs pediatric), donor type (deceased vs living), urgent indication [i.e., fulminant hepatic failure (FHF), acute liver failure (ALF), retransplantation, and critically ill patients in the intensive care unit], and use of rituximab for ABO-I LT, these parameters were classified according to the majority. One registry study was included in this metaanalysis [35]. Characteristics of the included studies are summarized in Table 1. Methodological quality assessment All studies included in this meta-analysis showed a Newcastle-Ottawa Scale (NOS) score 6. Four case or propensity score matched studies were included [29,30,35,41]. Quality assessments of included cohort studies are presented in Table 2. Graft and patient survival Graft survival and patient survival were reported by 16 and 15 studies, respectively. ABO-I LT showed poorer outcomes than those of ABO-C LT in the pooled results of graft survival (1-year: OR = 0.66, 95%CI: , P < 0.001; 3-year: OR = 0.74, 95%CI: , P < 0.001; 5-year: OR = 0.75, 95%CI: , P < 0.001; 10-year: OR = 0.80, 95%CI: , P = 0.004; Figure 2). There were no differences in 1-, 3-, and 5-year patient survival in accordance with ABO compatibility (1-year: OR = 0.88, 95%CI: , P = 0.38; 3-year: OR = 0.89, 95%CI: , P = 0.47; 5-year: OR = 0.89, 95%CI: , P = 0.45; Figure 3). However, there was a significant difference in the 10-year patient survival between ABO-I and ABO-C groups (10-year: OR = 0.46, 95%CI: , P = 0.004; Figure 3). There was no significant 6518 September 21, 2017 Volume 23 Issue 35

163 Lee EC et al. ABO incompatability in liver transplantation Citations identified in primary search (n = 986) Irrelevant studies (n = 415) Duplicated studies (n = 347) Titles and abstracts screening (n = 224) Abstract/meeting (n = 104) Case reports/series (n = 50) Reviews/meta-analysis (n = 15) Letters/editorial comments (n = 9) Animal studies (n = 1) Others (n = 7) Potentially relevant studies (n = 38) Single-arm studies (n = 9) Overlapping cohorts (same institution) (n = 4) Studies investigating multiple desensitization protocols (n = 1), timing of rituximab administration (n = 1), and pathology (n = 1) Unable data extracting (n = 1) Eligible studies included in the meta-analysis (n = 21) Figure 1 Flow diagram showing the selection of articles for meta-analysis. heterogeneity in graft and patient survival. Complications Rejection: AMR, ACR, and CR were reported by 4, 14, and 4 studies, respectively. Pooled results showed that the risks for AMR (OR = 74.21, 95%CI , P < 0.001; Figure 4A) and CR (OR = 2.28, 95%CI: , P = 0.05; Figure 4C) were significantly higher in ABO-I LT than in ABO-C LT, but there was no statistically significant difference in the risk for ACR (OR = 1.23, 95%CI: , P = 0.15; Figure 4B). There was no significant heterogeneity in the results for rejection. Infection: Bacterial and fungal infections were each reported by 4 studies, while CMV infection was reported by 5 studies. Although there were no differences in bacterial infection (OR = 0.72, 95%CI: , P = 0.16; Figure 4D) and fungal infection (OR = 0.66, 95%CI: , P = 0.16; Figure 4E) in accordance with ABO-compatibility, CMV infection was more prevalent in ABO-I LT than in ABO-C LT (OR = 2.64, 95%CI: , P < 0.001; Figure 4F). There was no significant heterogeneity in the results for infection. Biliary: There were no statistically significant differences in overall biliary complication (OR = 1.75, 95%CI: , P = 0.10; Figure 5A), bile leak (OR = 1.85, 95%CI: , P = 0.39; Figure 5B), and biliary stricture (OR = 1.37, 95%CI: , P = 0.36; Figure 5C) in accordance with ABO compatibility. However, there were high heterogeneities in overall biliary complication (χ 2 = 15.22, degree of freedom [d.f.] = 7, P = 0.03; I 2 = 54%), bile leak (χ 2 = 4.25, d.f. = 2, P = 0.12; I 2 = 53%), and biliary stricture (χ 2 = 9.36, d.f. = 5, P = 0.10; I 2 = 47%). Vascular: HA complication (OR = 4.17, 95%CI: , P < 0.001; Figure 5D) was significantly more prevalent in ABO-I LT than in ABO-C LT. However, there were no significant differences between ABO-I LT and ABO-C LT in HV complication (OR = 1.60, 95%CI: , P = 0.32; Figure 5E) and PV complication (OR = 1.83, 95%CI: , P = 0.22; Figure 5F). There was no severe heterogeneity in vascular complications. Sensitivity and subgroup analyses In this meta-analysis, there were high heterogeneities in overall biliary complication, bile leak, and biliary stricture. We performed a sensitivity analysis on these variables using the leave-one-out method and found that omitting the study of Sanchez et al [41] with a wide range of CI (overall biliary complication: OR = 85.00, 95%CI: ; Bile leak: OR = 10.82, 95%CI: ) eliminated the heterogeneity in overall biliary complication and bile leak. Particularly, postsensitive analysis results showed that ABO-I LT was associated with higher prevalence of overall biliary complications than ABO-C LT (OR = 1.52, 95%CI: , P = 0.04; Table 3) September 21, 2017 Volume 23 Issue 35

164 Lee EC et al. ABO incompatability in liver transplantation Table 1 Characteristics of the included studies Ref. Publication (yr) Study periods (yr) Region Arms Sample size Population 3 Recipient age Donor age Urgent Donor type 3 Prescription for ABO-I LT Immunosuppression Indications 3,4 Song et al[ 28] South Korea ABO-I 235 Adult 52.8 ± ± 9.1 No Living Rituximab, PE, GLI (±), Splenectomy Steroids, Basiliximab,Tac, MMF ABO-C ± ± 7.6 (±), Cyclophosphamide Kim et al [29] South Korea ABO-I 472 Adult 50 (22-65) 32 (18-68) No Living Rituximab, PE, GLI (±) Steroids, Basiliximab, Tac, MMF ABO-C (20-68) 30 (18-62) Kim et al [30] South Korea ABO-I 252 Adult 51.3 ± ± 11.2 No Living Rituximab, PE, IVIG, Preoperative Steroids, Basiliximab, Tac, MMF ABO-C ± ± 11.3 MMF Ikegami et al [31] Japan ABO-I 19 Adult 47.7 ± ± 11.3 No Living Rituximab3, IVIG (±), PE, GLI (±), Steroids, Tac (or CsA), MMF ABO-C ± ± 10.5 Splenectomy (±), Preoperative MMF (±) Lee et al [32] Taiwan ABO-I 46 Adult 53.5 (19-67) NA No Living Rituximab, Plasmapheresis (or PE) Steroids, Tac, MMF ABO-C (18-70) NA Shen et al [33] China ABO-I 35 Adult 46.7 ± 12.1 NA Yes Deceased Rituximab, IVIG Steroids, Basiliximab, Tac, MMF ABO-C ± 10.2 NA Heffron et al [34] ABO-C 21 NA NA United States ABO-I 12 Pediatric NA NA Yes Deceased - Steroids, Daclizumab, Tac, MMF Stewart et al [35] United States ABO-I 1302 Infant No Deceased NA NA ABO-C ABO-I 1162 Pediatric ABO-C ABO-I 5852 Adult ABO-C Iwamoto Japan ABO-I 15 Adult NA NA No Living NA NA et al [36] ABO-C 37 NA NA Toso et al [20] Canada ABO-I 14 Adult 42 (17-61) NA Yes Deceased Lymphocyte-depleting antibodies5, Plasmapheresis (±) ABO-C (16-62) NA ABO-Id (17-66) NA Steroids, Daclizumab, CsA (or Tac), AZA (or MMF, Sirolimus) Saito et al [37] Japan ABO-I 10 All ages NA NA No Deceased, NA NA ABO-C 81 NA NA Living Koukoutsis United et al [19] Kingdom ABO-I 4 Adult NA NA Yes Deceased NA NA ABO-C1 73 NA NA ABO-Id 203 NA NA Ueda et al [18] United States ABO-I 16 Pediatric 6.5 ± 6.2 NA No Deceased Plasmapheresis (±) Steroids, Daclizumab, Tac, MMF Japan ABO-I 74 Pediatric NA NA No Living Steroids pulse weekly, PGE1, CsA - > Steroids, Tac ABO-C1 114 NA NA AZA (1 mo after LT) ABO-Id 380 NA NA Heffron et al [38] ABO-C ± 6.2 NA Bjøro et al [17] Nordic countries ABO-I 10 All ages NA 44.8 (22-55) Yes Deceased NA NA ABO-C 76 NA 42.3 (12-85) ABO-Id 143 NA 41.0 (2-75) Chui et al [39] Australia ABO-I 7 All ages 13 (6-32) NA Yes Deceased Plasmapheresis (±), Splenectomy (±) Steroids, CsA, AZA ABO-C 36 NA NA Cacciarelli ABO-C ± 1.0 NA ABO-Id ± 0.5 NA et al [16] Tac United States ABO-I 14 Pediatric 2.2 ± 1.1 NA No Deceased OKT3 (or ATG, CsA) Steroids, ATG (or OKT3, CsA), 6520 September 21, 2017 Volume 23 Issue 35

165 Lee EC et al. ABO incompatability in liver transplantation Lo et al [40] United States ABO-I 29 All ages NA NA Yes Deceased ATG (±) Steroids, CsA (or OKT3), AZA ABO-C 196 NA NA Sanchez et al [41] ABO-C (17-59) NA United States ABO-I 182 Adult 45 (16-61) NA No Deceased NA NA 30 Reding Belgium ABO-I 16 All ages NA NA Yes Deceased OKT3 (±) Steroids, CsA, AZA(±) et al [15] ABO-C 16 NA NA ABO-Id 38 NA NA Gugenheim France ABO-I 17 All ages (12-49) NA Yes Deceased - Steroids, CsA, AZA et al [5] ABO-C 217 NA NA 1 Compatible, but not identical; 2 Propensity or case matched patients; 3 If there are minority groups that make up less than about 10%, the article is categorized as covering the majority; 4 Such as FHF, ALF, retransplantation, and critically ill patients in the intensive care unit; 5 Documented as "lymphocyte-depleting antibodies", but not clarified exactly. ABO-C: ABO-compatible; ABO-I: ABO-incompatible; ABO-Id: ABO-identical; ATG: anti-thymocyte globulin; AZA: Azathioprine; CsA: Cyclosporin A; GLI: Graft local infusion; IVIG: Intravenous immunoglobulin; LT: Liver transplantation; MMF: Mycophenolate mofetil; NA: Not applicable; OKT3: Muromonab-CD3; PE: Plasma exchange; Tac: Tacrolimus. Although there were no significant heterogeneities in most comparisons, such as in graft survival, patient survival, and complications, we attempted to minimize potential heterogeneities and detail the subgroup-specific differences through subgroup analyses. Possible confounding factors-the parameters that were speculated to impact the outcomes of this meta-analysis-included study population (adult vs pediatric), use of rituximab for ABO-I LT, urgent indication, and donor type (deceased vs living). There were no significant differences in most subgroup comparisons. However, studies that involved pedia-tric patients [16,18,34,35,38] showed better 1-year (OR = 0.88, 95%CI: vs OR = 0.59, 95%CI: ; P = 0.01) and 3-year graft survivals (OR = 0.95, 95%CI: vs OR = 0.67, 95%CI: ; P = 0.04) after ABO-I LT than those involving adult patients. Furthermore, in such studies, there were no significant differences between ABO-I LT and ABO-C LT in 1-, 3-, 5-, and 10-year graft survivals (1-year: OR=0.88, 95%CI: , P = 0.35; 3-year: OR = 0.95, 95%CI: , P = 0.71; 5-year: OR = 0.82, 95%CI: , P = 0.14; 10-year: OR = 0.71, 95%CI: , P = 0.22). Meanwhile, using rituximab in ABO-I LT patients [28-33] showed better 1-year graft survival (OR = 0.88, 95%CI: vs OR = 0.44, 95%CI: ; P = 0.02) after ABO-I LT compared to cases not using rituximab [15,16,18, 34,38-40, 42]. Moreover, in such cases, 1-, 3-, and 5-year graft survival of ABO-I LT were not significantly different from those of ABO-C LT (1-year: OR = 0.88, 95%CI: , P = 0.55; 3-year: OR = 0.81, 95%CI: , P = 0.28; 5-year: OR = 0.96, 95%CI: , P = 0.83). On the other hand, when rituximab was not used, incidence of biliary stricture (OR = 1.00, 95%CI: vs OR=4.32, 95%CI: ; P = 0.06) and ACR (OR = 0.86, 95%CI: vs OR = 1.61, 95%CI: ; P = 0.048) tended to be higher in ABO-I LT than in ABO-C LT. However, there was no difference in AMR incidence (OR=48.32, 95%CI: vs OR = , 95%CI: ; P = 0.45) and patient survival (1-year: OR = 0.88, 95% CI vs OR = 0.67, 95%CI: , P = 0.38; 3-year: OR = 0.90, 95%CI: vs OR = 0.80, 95%CI: , P = 0.82; 5-year: OR = 0.90, 95%CI: vs OR = 1.00, 95%CI: , P = 0.89) in accordance with the use of rituximab. Studies that involved urgent indica-tions [15,17,19,20,33,34,39,40,42] had worse 1-year graft survival (OR = 0.37, 95%CI: vs OR=0.70, 95%CI: ; P = 0.01) but better 5-year patient survival (OR = 2.38, 95%CI: vs OR = 0.79, 95%CI: , P = 0.043) for ABO-I LT than those for ABO-CT when compared to studies without urgent indications. In addition, compared to cases that involved the use of deceased donor liver allografts, those that involved the use of living donor liver allografts [18,28-32,36] showed lower prevalence of ACR (OR = 0.87, 95%CI: vs OR = 1.69, 95%CI: ; P = 0.02) and biliary stricture (OR = 1.00, 95%CI: vs OR = 4.49, 95%CI: ; P = 0.04) in ABO-I LT. Results of sensitivity analysis and subgroup analysis are summarized in Table 3. Publication bias In this meta-analysis, 1-year patient survival was found to have a potential publication bias with a funnel plot asymmetry (Figure 6A) and a P = 0.06 calculated by the Egger s regression test. Other variables did not show a significant publication bias. After adjusting for funnel plot asymmetry using the trim-and-fill method [26] (Figure 6B), ABO-I LT showed a significantly poorer 1-year patient survival than ABO-C LT (OR=0.73, 95%CI: , P = 0.02; I 2 = 25.8%) September 21, 2017 Volume 23 Issue 35

166 Lee EC et al. ABO incompatability in liver transplantation ABO-I ABO-C Odds ratio Odds ratio Study or Subgroup Events Total Events Total Weight M-H.Fixed. 95%CI M-H. Fixed. 95%CI A 1-yr graft survival Song % 0.75 [0.43, 1.33] Ikegami % 2.35 [0.31, 18.06] Lee % 1.12 [0.50, 2.52] Shen % 0.63 [0.21,1.87] Heffron % 0.55 [0.03, 9.68] Stewart % 0.69 [0.58, 0.81] Toso % 0.93 [0.29, 3.00] Koukoutsis % 0.59 [0.08, 4.23] Ueda % 0.58 [0.32, 1.06] Heffron % 2.94 [0.37, 23.55] Bjøro % 0.28 [0.08, 1.03] Chui % 0.42 [0.08, 2.19] Cacciarelli % 0.48 [0.14, 1.68] Sanchez % 0.23 [0.05, 0.97] Reding % 0.29 [0.07, 1.13] Gugenheim % 0.10 [0.03, 0.29] Subtotal(95% CI) % 0.66 [0.57, 0.76] Total events Heterogeneity: χ 2 = 23.51, df = 15 (P = 0.07); I 2 = 36% Test for overall effect: Z = 5.82 (P < ) B 3-yr graft survival Song % 0.76 [0.48, 1.22] Lee % 1.04 [0.51, 2.15] Shen % 0.63 [0.21, 1.87] Heffron % 0.71 [0.13, 3.87] Stewart % 0.73 [0.62, 0.85] Chui % 0.42 [0.08, 2.19] Cacciarelli % 0.69 [0.20, 2.35] Subtotal (95% CI) % 0.74 [0.64, 0.85] Total events Heterogeneity: χ 2 = 1.48, df = 6 (P = 0.96); I 2 = 0% Test for overall effect: Z = 4.17 (P < ) C 5-yr graft survival Song % 0.84 [0.53, 1.34] Ikegami % 2.10 [0.47, 9.27] Lee % 1.01 [0.51, 2.01] Stewart % 0.74 [063, 0.87] Toso % 0.95 [0.30, 2.94] Koukoutsis % 0.22 [0.02, 2.18] Ueda % 0.59 [0.33, 1.03] Chui % 0.42 [0.08, 2.19] Subtotal(95% CI) % 0.75 [0.66, 0.86] Total events Heterogeneity: χ 2 = 5.32, df = 7 (P = 0.62); I 2 = 0% Test for overall effect: Z = 4.02 (P < ) D 10-yr graft survival Stewart % 0.84 [0.71, 0.98] Ueda % 0.51 [0.31, 0.85] Subtotal(95%CI) % 0.80 [0.69, 0.93] Total events Heterogeneity: χ 2 = 3.25, df = 1 (P = 0.07); I 2 = 69% Test for overall effect: Z = 2.85 (P = 0.004) Total (95%CI) % 0.73 [0.68, 0.79] Total events Heterogeneity: χ 2 = 37.26, df = 32 (P = 0.24); I 2 = 14% Test for overall effect: Z = 8.44 (P < ); Test for subgroup differences: Not applicable Favours ABO-C Favours ABO-I Figure 2 Comparison of graft survival between ABO-incompatible and ABO-compatible liver transplantation. ABO-C: ABO-compatible; ABO-I: ABOincompatible; LT: Liver transplantation September 21, 2017 Volume 23 Issue 35

167 Lee EC et al. ABO incompatability in liver transplantation Table 2 Modified Newcastle-Ottawa quality assessment scale for cohort studies included in the meta-analysis Ref. Selection Comparability 1 Outcome Overall Representativeness Selection Ascertainment Incident disease Assessment Length of follow-up Adequacy of follow-up Quality Score (Maximum 9) Song et al [28] 6 Kim et al [29] 8 Kim et al [30] 8 Ikegami 6 et al [31] Lee et al [32] 6 Shen et al [33] 6 Heffron 6 et al [34] Stewart 8 et al [35] Iwamoto 6 et al [36] Toso et al [20] 6 Saito et al [37] 6 Koukoutsis 6 et al [19] Ueda et al [18] 6 Heffron 6 et al [38] Bjøro et al [17] 6 Chui et al [39] 6 Cacciarelli 6 et al [16] Lo et al [40] 6 Sanchez 8 et al [41] Reding 6 et al [15] Gugenheim et al [5] 6 1 A maximum of two downward arrows ( ) can be given for comparability. : Consistent with criteria and low risk of bias; : Not consistent with criteria and high risk of bias. DISCUSSION Since the first attempt of ABO-I LT by Starzl et al [1,2], poor outcomes after ABO-I LT, including AMR, lower graft survival, HAT, and cholangitis, were insurmountable barriers for expanding the application of transplantation across the ABO blood group barrier, from a few urgent cases to cases of chronic liver disease and liver cancer [3-6]. Extraordinary improvements have been made in the outcomes of ABO-I LT with the introduction of multiple desensitization strategies, such as PE (or plasmapheresis), splenectomy, GLI, rituximab, MMF, and IVIG, as well as with advances of immunosuppression agents [7-12]. However, whether ABO-I LT is comparable to ABO-C LT remains a topic of debate. This meta-analysis revealed that pooled results of graft survival were poorer in ABO-I LT than in ABO-C LT. However, patient survival did not significantly vary in accordance with ABO compatibility in most cases. The data on 10-year patient survival had low reliability, as they were only reported by one study-although produced based on a long-term follow-up [18]. Meanwhile, the cumulative meta-analysis in order by the median year of study period showed that the cumulative results of graft and patient survival remained consistent since the early 2000s (Supplementary Figures 1 and 2). This is mainly due to the stabilization of the desensitization protocol through the application of PE (or plasmapheresis) [42-44], muromonab-cd3 (OKT3) [42-44], splenectomy [9], PV infusion [10,45], HA infusion [46,47], rituximab [48-50], and IVIG [7,8,51] in ABO-I LT. However, from a different perspective, the fact that ABO-I LT patients showed a poorer graft survival than ABO-C LT patients and that cumulative meta-analysis of graft survival remained mostly unchanged since the early 2000s implies that the current desensitization protocol for ABO-I LT still requires an improvement. With regard to ABO-I-related complications, the prevalence of AMR, CR, CMV infection, and HA complication was higher in ABO-I LT than in ABO-C LT. Overall biliary complication-after omitting a study of Sanchez et al [41] with a wide range of CI in the 6523 September 21, 2017 Volume 23 Issue 35

168 Lee EC et al. ABO incompatability in liver transplantation ABO-I ABO-C Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H. Fixed. 95%CI M-H. Fixed. 95%CI A 1-yr patient survival Song % 0.72 [0.41, 1.27] Kim % 1.23 [0.41, 3.72] Lee % 1.12 [0.50, 2.52] Shen % 0.76 [0.25, 2.35] Heffron % 0.55 [0.03, 9.68] Toso % 2.81 [0.59, 13.36] Koukoutsis % 1.50 [0.15, 14.62] Ueda % 0.61 [0.33, 1.12] Heffron % 2.76 [0.15, 49.72] Bjøro % 0.80 [0.20, 3.21] Chui % 3.39 [0.37, 31.34] Cacciarelli % 0.48 [0.12, 1.91] Sanchez % 4.00 [0.85, 18.84] Reding % 0.67 [0.22, 2.06] Gugenheim % 0.43 [0.14, 1.30] Subtotal (95% CI) % 0.88 [0.67, 1.16] Total events Heterogeneity: χ 2 = 13.41, df = 14 (P = 0.49); I 2 = 0% Test for overall effect: Z = 0.88 (P = 0.38) B 3-yr patient survival Song % 0.74 [0.46, 1.18] Kim % 1.54 [0.60, 3.96] Lee % 1.04 [0.51, 2.15] Shen % 0.76 [0.25, 2.35] Heffron % 0.15 [0.01, 1.65] Chui % 3.39 [0.37, 31.34] Cacciarelli % 0.71 [0.18, 2.75] Subtotal (95% CI) % 0.89 [0.64, 1.23] Total events Heterogeneity: χ 2 = 5.79, df = 6 (P = 0.45); I 2 = 0% Test for overall effect: Z = 0.73 (P = 0.47) C 5-yr patient survival Song % 0.84 [0.53, 1.34] Lee % 1.01 [0.51, 2.01] Toso % 2.28 [0.59, 8.71] Koukoutsis % 1.79 [0.18, 17.40] Ueda % 0.58 [0.32, 1.02] Chui % 3.39 [0.37, 31.34] Subtotal (95% CI) % 0.89 [0.66, 1.20] Total events Heterogeneity: χ 2 = 6.03, df = 5 (P = 0.30); I 2 = 17% Test for overall effect: Z = 0.73 (P = 0.45) D10-yr patient survival Ueda % 0.46 [0.28, 0.78] Subtotal (95% CI) % 0.46 [0.28, 0.78] Total events Heterogeneity: Not applicable Test for overall effect: Z = 2.89 (P = 0.004) Total (95% CI) % 0.84 [0.71, 0.98] Total events Heterogeneity: χ 2 = 29.99, df = 28 (P = 0.36); I 2 = 7% Test for overall effect: Z = 2.17 (P = 0.03) Test for subgroup differences: Not applicable Favours ABO-C Favours ABO-I Figure 3 Comparison of patient survival between ABO-incompatible and ABO-compatible liver transplantation. ABO-C: ABO-compatible; ABO-i: ABOincompatible; LT: Liver transplantation. sensitivity analysis-was more prevalent in ABO-I LT than in ABO-C LT. In the subgroup analyses, studies that only involved pediatric patients [16,18,34,35,38], compared to those that did not, showed better 1-year and 3-year graft survivals after ABO-I LT than those after ABO-C LT. Furthermore, in such cases, 1-, 3-, 5-, and 10-year graft survivals after ABO-I LT were comparable to those after ABO-C LT. There were several reports that pediatric ABO-I LT was more successful than 6524 September 21, 2017 Volume 23 Issue 35

169 Lee EC et al. ABO incompatability in liver transplantation Table 3 Sensitivity analysis and subgroup analysis Variables No. of studies OR=[95%CI] P 1 value I 2 (%) Sensitivity analysis Overall biliary complication 1.75 [ ] Omitting Sanchez et al [41] [ ] Omitting Cacciarelli et al [16] [ ] Omitting Heffron et al [38] [ ] Omitting Toso et al [20] [ ] Omitting Iwamoto et al [36] [ ] Omitting Shen et al [33] [ ] Omitting Lee et al [32] [ ] Omitting Kim et al [29] [ ] Bile leak 1.85 [ ] Omitting Sanchez et al [41] [ ] Omitting Kim et al [30] [ ] Omitting Kim et al [29] [ ] Biliary stricture 1.37 [ ] Omitting Sanchez et al [41] [ ] Omitting Lo et al [40] [ ] Omitting Ikegami et al [31] [ ] Omitting Kim et al [30] [ ] Omitting Kim et al [29] [ ] Omitting Song et al [28] [ ] Subgroup analysis 1-yr graft survival 0.66 [ ] < Rituximab for ABO-I LT (yes/no) 4 vs [ ] vs 0.44 [ ] vs 49 Living donor (yes/no) 4 vs [ ] vs 0.64 [ ] vs 44 Urgent indication (yes/no) 8 vs [ ] vs 0.70 [ ] vs 8 Pediatric2 (yes/no) 5 vs [ ] vs 0.59 [ ] vs 45 3-yr graft survival 0.74 [ ] < Rituximab for ABO-I LT (yes/no) 3 vs [ ] vs 0.60 [ ] vs 0 Living donor (yes/no) 2 vs [ ] vs 0.72 [ ] vs 0 Urgent indication (yes/no) 3 vs [ ] vs 0.74 [ ] vs 0 Pediatric2 (yes/no) 3 vs [ ] vs 0.67 [ ] vs 0 5-yr graft survival 0.75 [ ] < Rituximab for ABO-I LT (yes/no) 3 vs [ ] vs 0.56 [ ] vs 0 Living donor (yes/no) 4 vs [ ] vs 0.73 [ ] vs 0 Urgent indication (yes/no) 3 vs [ ] vs 0.76 [ ] vs 0 Pediatric 2 (yes/no) 2 vs [ ] vs 0.73 [ ] vs 0 10-yr graft survival 0.80 [ ] Rituximab for ABO-I LT (yes/no) 0 vs 1 NA vs 0.51 [ ] NA NA Living donor (yes/no) 1 vs [ ] vs 0.84 [ ] 0.07 NA vs NA Urgent indication (yes/no) 0 vs 2 NA vs 0.70 [ ] NA NA vs 69 Pediatric 2 (yes/no) 2 vs [ ] vs 0.81 [ ] vs NA 1-yr patient survival 0.88 [ ] Rituximab for ABO-I LT (yes/no) 4 vs [ ] vs 0.67 [ ] vs 0 Living donor (yes/no) 4 vs [ ] vs 1.04 [ ] vs 9 Urgent indication (yes/no) 8 vs [ ] vs 0.87 [ ] vs 21 Pediatric (yes/no) 4 vs [ ] vs 0.98 [ ] vs 5 3-yr patient survival 0.89 [ ] Rituximab for ABO-I LT (yes/no) 4 vs [ ] vs 0.80 [ ] vs 43 Living donor (yes/no) 3 vs [ ] vs 0.78 [ ] vs 15 Urgent indication (yes/no) 3 vs [ ] vs 0.90 [ ] vs 0 Pediatric (yes/no) 2 vs [ ] vs 0.94 [ ] vs 0 5-yr patient survival 0.89 [ ] Rituximab for ABO-I LT (yes/no) 2 vs [ ] vs 1.00 [ ] vs 57 Living donor (yes/no) 3 vs [ ] vs 2.38 [ ] vs 0 Urgent indication (yes/no) 3 vs [ ] vs 0.79 [ ] vs 0 Pediatric (yes/no) 1 vs [ ] vs 1.04 [ ] 0.09 NA vs 0 10-yr patient survival 0.46 [ ] Rituximab for ABO-I LT (yes/no) 0 vs 1 NA vs 0.46 [ ] NA NA Living donor (yes/no) 1 vs [ ] vs NA NA NA Urgent indication (yes/no) 0 vs 1 NA vs 0.46 [ ] NA NA Pediatric (yes/no) 1 vs [ ] vs NA NA NA ACR 1.23 [ ] Rituximab for ABO-I LT (yes/no) 5 vs [ ] vs 1.61 [ ] vs 0 Living donor (yes/no) 4 vs [ ] vs 1.69 [ ] vs 0 Urgent indication (yes/no) 6 vs [ ] vs 1.08 [ ] vs September 21, 2017 Volume 23 Issue 35

170 Lee EC et al. ABO incompatability in liver transplantation Pediatric (yes/no) 3 vs [ ] vs 1.16 [ ] vs 0 CR 2.28 [ ] Rituximab for ABO-I LT (yes/no) 2 vs [ ] vs 2.64 [ ] vs 0 Living donor (yes/no) 2 vs [ ] vs 2.64 [ ] vs 0 Urgent indication (yes/no) 2 vs [ ] vs 6.45 [ ] vs 80 Pediatric (yes/no) 0 vs 4 NA vs 2.28 [ ] NA NA vs 42 AMR [ ] < Rituximab for ABO-I LT (yes/no) 2 vs [ ] vs [ ] vs NA Living donor (yes/no) 1 vs [ ] vs [ ] 0.3 NA vs 18 Urgent indication (yes/no) 3 vs [ ] vs [ ] vs NA Pediatric (yes/no) 0 vs 4 NA vs [ ] NA NA vs 12 Bacterial infection 0.72 [ ] Rituximab for ABO-I LT (yes/no) 4 vs [ ] vs NA NA 0 vs NA Living donor (yes/no) 3 vs [ ] vs 0.84 [ ] vs NA Urgent indication (yes/no) 1 vs [ ] vs 0.70 [ ] 0.75 NA vs 0 Pediatric (yes/no) 0 vs 4 NA vs 0.72 [ ] NA NA vs 0 Fungal infection 0.66 [ ] Rituximab for ABO-I LT (yes/no) 3 vs [ ] vs NA NA 0 vs NA Living donor (yes/no) 2 vs [ ] vs 0.63 [ ] vs 62 Urgent indication (yes/no) 2 vs [ ] vs 0.65 [ ] vs 0 Pediatric (yes/no) 0 vs 4 NA vs 0.71 [ ] NA NA vs 0 CMV infection 2.64 [ ] < Rituximab for ABO-I LT (yes/no) 3 vs [ ] vs NA NA 4 vs NA Living donor (yes/no) 3 vs [ ] vs 6.43 [ ] vs 82 Urgent indication (yes/no) 1 vs [ ] vs 2.77 [ ] 0.4 NA vs 53 Pediatric (yes/no) 0 vs 5 NA vs 2.64 [ ] NA NA vs 43 Overall Biliary complication 1.75 [ ] Rituximab for ABO-I LT (yes/no) 3 vs [ ] vs 1.25 [ ] vs 0 Living donor (yes/no) 3 vs [ ] vs 2.36 [ ] vs 66 Urgent indication (yes/no) 2 vs [ ] vs 2.08 [ ] vs 66 Pediatric (yes/no) 2 vs [ ] vs 1.95 [ ] vs 67 Bile leak 1.85 [ ] Rituximab for ABO-I LT (yes/no) 2 vs [ ] vs NA NA 0 vs NA Living donor (yes/no) 2 vs [ ] vs [ ] vs NA Urgent indication (yes/no) 0 vs 3 NA vs 1.85 [ ] NA NA vs 53 Pediatric (yes/no) 0 vs 3 NA vs 1.85 [ ] NA NA vs 53 Biliary stricture 1.37 [ ] Rituximab for ABO-I LT (yes/no) 4 vs [ ] vs 4.32 [ ] vs NA Living donor (yes/no) 4 vs [ ] vs 4.49 [ ] vs 0 Urgent indication (yes/no) 1 vs [ ] vs 1.44 [ ] 0.11 NA vs 42 Pediatric (yes/no) 0 vs 6 NA vs 1.52 [ ] NA NA vs 47 HV complication 1.60 [ ] 0.32 NA Rituximab for ABO-I LT (yes/no) 1 vs [ ] vs NA NA NA Living donor (yes/no) 1 vs [ ] vs NA NA NA Urgent indication (yes/no) 0 vs 1 NA vs 1.60 [ ] NA NA Pediatric (yes/no) 0 vs 1 NA vs 1.60 [ ] NA NA PV complication 1.83 [ ] Rituximab for ABO-I LT (yes/no) 3 vs [ ] vs 2.65 [ ] vs NA Living donor (yes/no) 3 vs [ ] vs 3.27 [ ] vs 0 Urgent indication (yes/no) 2 vs [ ] vs 1.19 [ ] vs 0 Pediatric (yes/no) 0 vs 5 NA vs 1.83 [ ] NA NA vs 0 HA complication 4.17 [ ] < Rituximab for ABO-I LT (yes/no) 3 vs [ ] vs 4.43 [ ] vs 53 Living donor (yes/no) 4 vs [ ] vs 4.44 [ ] vs 10 Urgent indication (yes/no) 3 vs [ ] vs 3.30 [ ] vs 0 Pediatric (yes/no) 1 vs [ ] vs 5.26 [ ] 0.11 NA vs 0 1 P value for overall effect or test for differences in subgroup analysis; 2 Stewart et al [35] reported graft survival rates of pediatric and adults, respectively. ABO-I: ABO-Incompatible; ACR: Acute cellular rejection; AMR: Antibody-mediated rejection; CMV: Cytomegalovirus; CR: Chronic rejection; HA: Hepatic artery; HV: Hepatic vein; LT: Liver transplantation; PV: Portal vein. adult ABO-I LT [52,53]. Egawa et al [52] reported that an advanced recipient age for ABO-I LDLT is associated with poor outcomes, including graft and patient survivals, intrahepatic biliary complications, and hepatic necrosis. Maternal anti-abo antibodies (Ab) begin to disappear from week two after birth, and neonates begin to produce their own reservoir of anti- ABO Ab from weeks 8-12 after birth, which reaches a level similar to that of adults by age 5-10 [54,55]. Thus, younger pediatric patients may be immunologically immature, showing lower anti-abo Ab levels and immature complement system [56,57], which could be a possible explanation for our result of pediatric graft survival September 21, 2017 Volume 23 Issue 35

171 Lee EC et al. ABO incompatability in liver transplantation A ABO-I ABO-C Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H. Fixed. 95%CI M-H. Fixed. 95%CI Song % [12.49, ] Shen % 9.93 [0.46,272.67] Toso % [0.81, ] Gugenhenim % [13.04, ] Total (95% CI) % [16.32, ] Total events 26 0 Heterogenity χ 2 = 3.39, df = 3 (P = 0.34); I 2 = 12% Test for overall effect: Z = 5.57 (P < ) B Favours ABO-I Favours ABO-C ABO-I ABO-C Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H. Fixed. 95%CI M-H. Fixed. 95%CI Song % 0.85 [0.51, 1.41] Kim % 0.66 [0.24, 1.81] Kim % 0.97 [0.04, 24.67] lkegami % 1.52 [0.49, 4.75] Shen % 0.62 [0.06, 6.17] Heffron % 2.00 [0.47, 8.53] Toso % 1.72 [0.55, 5.36] Koukoutsis % 1.28 [0.18, 9.23] Heffron % 0.96 [0.33, 2.81] Cacciarelli % 2.86 [0.76, 10.75] Lo % 1.97 [0.85, 4.53] Sanchez % 4.09 [1.01, 16.58] Gugenheim % 1.15 [0.42, 3.15] Total (95%CI) % 1.23 [0.93, 1.62] Heterogenity χ 2 = 10.61, df = 12 (P = 0.68); I 2 = 0% Total events Test for overall effect: Z = 1.44 (P = 0.15) Favours ABO-I Favours ABO-C C ABO-I ABO-C Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H. Fixed. 95%CI M-H. Fixed. 95%CI Song % 1.11 [0.24, 5.09] lkegami % [2.49, ] Lo % 1.71 [0.18, 15.89] Gugenheim % 3.11 [0.80, 12.10] Total (95% CI) % 2.28 [1.00, 5.22] Total events 7 28 Heterogenity χ 2 = 5.17, df = 3 (P = 0.16); I 2 = 42% Test for overall effect: Z = 1.96 (P = 0.05) D Favours ABO-I Favours ABO-C ABO-I ABO-C Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H. Fixed. 95%CI M-H. Fixed. 95%CI Song % 0.81 [0.40, 1.65] Kim % 0.64 [0.30, 1.38] lkegami % 0.40 [0.50, 3.10] Shen % 0.84 [0.29, 2.45] Total (95% CI) % 0.72 [0.46, 1.14] Total events Heterogenity χ 2 = 0.58, df = 3 (P = 0.90); I 2 = 0% Test for overall effect: Z = 1.42 (P = 0.16) E Favours ABO-I Favours ABO-C ABO-I ABO-C Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H. Fixed. 95%CI M-H. Fixed. 95%CI Song % 0.73 [0.26, 2.10] Kim % 0.58 [0.21, 1.56] Shen % 1.40 [0.41, 4.80] Toso % 0.20 [0.03, 1.62] Total (95%CI) % 0.66 [0.37, 1.18] Total events Heterogenity χ 2 = 2.81, df = 3 (P = 0.42); I 2 = 0% Test for overall effect: Z = 1.40 (P = 0.16) Favours ABO-I Favours ABO-C 6527 September 21, 2017 Volume 23 Issue 35

172 Lee EC et al. ABO incompatability in liver transplantation F ABO-I ABO-C Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H. Fixed. 95%CI M-H. Fixed. 95%CI Song % 1.28 [0.36, 4.53] Kim % 1.85 [0.73, 4.69] lkegami % 3.75 [1.48, 9.50] Toso % 1.45 [0.45, 4.74] Saito % [2.48, ] Total (95% CI) % 2.64 [1.63, 4.29] Total events Heterogenity χ 2 = 7.02, df = 4 (P = 0.13); I 2 = 43% Test for overall effect: Z = 3.95 (P < ) Favours ABO-I Favours ABO-C Figure 4 Comparison of rejection and infection between ABO-incompatible and ABO-compatible liver transplantation. A: AMR; B: ACR; C: CR; D: Bacterial infection; E: Fungal infection; F: CMV infection. ABO-C: ABO-Compatible; ABO-I: ABO-incompatible; ACR: Acute cellular rejection; AMR: Antibody-mediated rejection; CMV: Cytomegalovirus; CR: Chronic rejection; LT: Liver transplantation. In this meta-analysis, cases that used rituximab in ABO-I LT patients [28-33] showed better 1-year graft survival after ABO-I LT than those that did not use rituximab [5,15,16,18,34,38-40]. Furthermore, in such cases, 1-, 3-, and 5-year graft survivals of ABO-I LT were comparable to those of ABO-C LT. On the other hand, biliary stricture and ACR tended to be more prevalent after ABO-I LT when rituximab was not used. There were no differences in AMR and patient survival in accordance with the use of rituximab. Rituximab is a chimeric human anti-cd20 monoclonal antibody, which destroys B cells via antibodydependent cell-mediated cytotoxicity, direct antigenantibody reaction, and complement-dependent cytotoxicity [58,59]. Since its first introduction as a prophylactic in Japan in 2002, multiple centers have used rituximab during ABO-I LT, which is considered to have contributed to the dramatic improvements in the outcomes of ABO-I LT [9,11,60,61]. In this meta-analysis, it was noted that using rituximab improved graft survival while reducing incidences of biliary stricture and ACR after ABO-I LT. However, its effects on AMR and patient survival were rather unclear, and we speculate this to be a result of excluding some studies from the subgroup analyses for lack of clear descriptions of desensitization methods in ABO-I LT [17,19,20, 35-37,41]. Meanwhile, studies that involved urgent indications, such as FHF, ALF, re-transplantation, and critically ill patients in the intensive care unit [5,15,17,19,20,33,34,39,40], showed worse 1-year graft survival but better 5-year patient survival in ABO-I LT than in ABO-C LT when compared to studies that did not involve urgent indications. Further, there were no significant differences in 3- and 5-year graft survivals between the two types of LT. This may be due to the fact that studies that only examined recipients with urgent indications mostly involved relatively lower prevalences of chronic liver disease and liver cancer but more advanced disease severity and inadequate desensitizations before ABO-I LT. Shaked et al [62] showed that there were no differences in biopsy-proven ACR and graft loss by rejection between LDLT and deceased donor liver transplantation (DDLT). However, our subgroup analysis showed that ACR was less prevalent after ABO-I LT in cases that only used living donor liver grafts [18,28-32,36] than in cases that did not. In other words, there were no differences in ACR in accordance with ABO compatibility in cases of LDLT, but incidences of ACR increased in cases of ABO-I LT using deceased donor liver grafts. It could be assumed that compared to DDLT, LDLT has immunological advantages resulting from high genetic similarities between organ donor and recipient and short cold ischemic times [63,64]. Further, biliary complications, such as bile leak and biliary stricture, are known to be more prevalent in LDLT with inherent weakness arising from a small duct size, possible multiplicity of bile duct, and cutting liver parenchyma, compared to those in DDLT [65-68]. However, our analysis revealed that using only living donor liver grafts [18,28-32,36] resulted in fewer cases of biliary stricture in ABO-I LT. One of the possible reasons is that studies only involving deceased donor grafts in our meta-analysis of biliary stricture [40,41] were published at least 20 years earlier than the studies involving living donor allografts [28-31]. Further, some of them involved urgent indications [40], which would have resulted in the use of markedly different desensitization and immunosuppression methods and surgical techniques from those employed today. Meanwhile, in this meta-analysis, a potential publication bias was detected in the 1-year patient survival. Possible sources of asymmetry in the funnel plot would most definitely include small study effects, but poor methodological quality, true heterogeneity, artifactual, and chance could be other sources as well [21,25,69-72]. This review has some limitations. First, it was based on non-randomized controlled trials because it is practically impossible to randomly allocate patients into either ABO-C LT or ABO-I LT group. Second, some articles lacked clear descriptions about patient demographics and study design, such as age, enrollment criteria, graft type, and desensitization and immunosuppression methods. Third, some results showed heterogeneity and potential publication bias September 21, 2017 Volume 23 Issue 35

173 Lee EC et al. ABO incompatability in liver transplantation A ABO-I ABO-C Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H. Random. 95%CI M-H. Random. 95%CI Kim % 0.82 [0.34, 1.97] Lee % 2.37 [1.27, 4.41] Shen % 0.94 [0.16, 5.40] Iwamoto % 1.56 [0.38, 6.38] Toso % 1.39 [0.44, 4.34] Heffron % 1.20 [0.24, 5.87] Cacciarelli % 1.35 [0.15, 11.86] Sanchez % [7.97, ] Total (95% CI) % 1.75 [0.89, 3.43] Total events Heterogenity Tau 2 = 0.45 χ 2 = 15.22, df = 7 (P = 0.03); I 2 = 54% Test for overall effect: Z = 1.63 (P = 0.10) B Favours ABO-I Favours ABO-C ABO-I ABO-C Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H. Random. 95%CI M-H. Random. 95%CI Kim % 0.81 [0.27, 2.46] Kim % 1.54 [0.27, 8.98] Sanchez % [1.17, ] Total (95% CI) % 1.85 [0.46, 7.39] Total events Heterogenity Tau 2 = 0.80 χ 2 = 4.25, df = 2 (P = 0.12); I 2 = 53% Test for overall effect: Z = 0.87 (P = 0.39) Favours ABO-I Favours ABO-C C ABO-I ABO-C Odds ratio Odds ratio Odds Ratio Study or subgroup Events Total Events Total Weight M-H. Random. 95%CI M-H. Fixed. 95%CI Song % 1.79 [1.24, 2.57] Kim % 0.58 [0.20, 1.70] Kim % 0.31 [0.02, 5.99] lkegami % 0.75 [0.21, 2.63] Lo % 4.32 [1.18, 15.81] Sanchez % 5.61 [0.25, ] Total (95% CI) % 1.37 [0.70, 2.70] Total events Heterogenity Tau 2 = 0.29 χ 2 = 9.36, df = 5 (P = 0.10); I 2 = 47% Test for overall effect: Z = 0.91 (P = 0.36) D Favours ABO-I Favours ABO-C ABO-I ABO-C Odds rati0 Odds ratio Study or subgroup Events Total Events Total Weight M-H. Fixed. 95%CI M-H. Fixed. 95%CI Song % 2.78 [0.51, 15.28] Kim % 3.08 [0.19, 51.20] lkegami % 1.51 [0.08, 27.83] Iwamoto % [0.63, ] Toso % 3.78 [0.98, 14.54] Heffron % 0.47 [0.03, 8.56] Lo % 5.54[ 1.17, 26.15] Sanchez % [0.77, ] Gugenheim % [2.32, 56.05] Total (95% CI) % 4.17 [2.26, 7.67] Total events Heterogenity: χ 2 = 5.88, df = 8 (P = 0.66); I 2 = 0% Test for overall effect: Z = 4.58 (P < ) Favours ABO-I Favours ABO-C E ABO-I ABO-C Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H. Fixed. 95%CI M-H. Fixed. 95%CI Song % 1.60 [0.64, 4.00] Total (95%CI) % 1.60 [0.64, 4.00] Total events 6 21 Heterogenity: Not applicable Test for overall effect: Z = 1.00 (P < 0.32) Favours ABO-I Favours ABO-C 6529 September 21, 2017 Volume 23 Issue 35

174 Lee EC et al. ABO incompatability in liver transplantation F ABO-I ABO-C Odds ratio Odds ratio Study or subgroup Events Total Events Total Weight M-H. Fixed. 95%CI M-H. Fixed. 95%CI Song % 1.39 [0.15, 12.45] Kim % 0.41 [0.02, 8.14] lkegami % 3.03 [0.35, 25.97] Toso % 3.75 [0.62, 22.71] Gugenheim % 2.65 [0.29, 24.07] Total (95% CI) % 1.83 [0.70, 4.76] Total events 5 23 Heterogenity: χ 2 = 1.96, df = 4 (P = 0.74); I 2 = 0% Test for overall effect: Z = 1.23 (P = 0.22) Favours ABO-I Favours ABO-C Figure 5 Comparison of biliary and vascular complications between ABO-Incompatible and ABO-Compatible liver transplantation. A: Overall biliary complication; B: Bile leak; C: Biliary stricture; D: HA complication; E: HV complication; F: PV complication. ABO-C: ABO-compatible; ABO-I: ABO-incompatible; HA: Hepatic artery; HV: Hepatic vein; LT: Liver transplantation; PV: Portal vein. A Standard error yr patient survival B Standard error yr patient survival Figure 6 (A) Funnel plot and (B) Adjusted funnel plot using the Trim-and-Fill method of studies reporting on 1-yr patient survival after ABO-incompatible liver transplantation vs ABO-Compatible liver transplantation. Closed circles represent observed published studies; open circles represent imputed unpublished studies. ABO-C: ABO-compatible; ABO-I: ABO-incompatible; LT: Liver transplantation. This meta-analysis is the largest review that integrating more than 8000 cases of ABO-I LT and ABO-C LT. It revealed that ABO-I LT is associated with poorer graft survival and higher prevalence of AMR, CR, CMV infection, overall biliary complication, and HA complication than those of ABO-C LT. There were no significant differences in patient survival, ACR, bacterial infection, fungal infection, bile leak, biliary stricture, and HV and PV complications in accordance with ABO compatibility. In our subgroup analysis, graft survival in ABO-I LT was found to be comparable to that in ABO-C LT in pediatric patients. Use of rituximab was associated with better graft survival in ABO-I LT. In cases of DDLT, there was a higher incidence of ACR after ABO-I LT. Although substantial improvements and advances have been made in liver transplantations across the ABO blood group barrier, persistent limitations call for further endeavors to achieve better outcomes. ACKNOWLEDGMENTS The authors wish to thank Jae Ryong Shim, MD for his contribution to this article. COMMENTS Background Increased ABO-incompatible (ABO-I) liver transplantation (LT) is inevitable due to reduced organ donation and difficulty in finding suitable ABO-compatible (ABO-C) allografts. In particular, the importance of ABO-I LT is increasing in Asian countries where the use rate of ABO-I liver allograft is higher than that of Western countries due to the large number of organ donations in the family in living donor liver transplantation. Research frontiers Outcomes after LT in accordance with ABO compatibility is still controversial. Therefore, it is necessary to evaluate the possibilities and the limitations of ABO-I LT by meta-analysis integrating outcomes of previous reports comparing ABO-I and ABO-C LT. Innovations and breakthroughs ABO-I LT is comparable to ABO-C LT in terms of patient survival, but is inferior in graft survival, antibody-mediated rejection, chronic rejection, cytomegalovirus infection, overall biliary complication, and hepatic artery complication. However, in pediatric patients and those using rituximab, the graft survival of ABO-I LT was comparable to that of ABO-C LT. Applications The authors performed a meta-analysis of outcomes after liver transplantation in accordance with ABO compatibility. In this way, the possibilities and the limitations of ABO-I LT can be clarified September 21, 2017 Volume 23 Issue 35

175 Lee EC et al. ABO incompatability in liver transplantation Terminology ABO-I transplantation is an assignment method for organ transplantation, which allows the use of available organs more efficiently regardless of the ABO blood type, which cannot otherwise be used due to hyperacute rejection. Peer-review This meta-analysis is the largest review article of more than 8000 cases of ABO-I and ABO-C LT. The authors concluded that ABO-I LT, although patient survival was similar, was inferior to ABO-C LT in graft survival and several ABO-I-related complications. The article is well written and of highly clinical implications. REFERENCES 1 Starzl TE, Putnam CW. Experience in hepatic transplantation: Saunders Philadelphia, Starzl TE, Koep LJ, Halgrimson CG, Hood J, Schroter GP, Porter KA, Weil R 3 rd. Fifteen years of clinical liver transplantation. Gastroenterology 1979; 77: [PMID: ] 3 Rego J, Prevost F, Rumeau JL, Modesto A, Fourtanier G, Durand D, Suc JM, Ohayon E, Ducos J. Hyperacute rejection after ABOincompatible orthotopic liver transplantation. Transplant Proc 1987; 19: [PMID: ] 4 Gugenheim J, Samuel D, Fabiani B, Saliba F, Castaing D, Reynes M, Bismuth H. Rejection of ABO incompatible liver allografts in man. Transplant Proc 1989; 21: [PMID: ] 5 Gugenheim J, Samuel D, Reynes M, Bismuth H. Liver transplantation across ABO blood group barriers. Lancet 1990; 336 : [PMID: DOI: / (90)92082-S] 6 Sanchez-Urdazpal L, Sterioff S, Janes C, Schwerman L, Rosen C, Krom RA. Increased bile duct complications in ABO incompatible liver transplant recipients. Transplant Proc 1991; 23: [PMID: ] 7 Urbani L, Mazzoni A, Bianco I, Grazzini T, De Simone P, Catalano G, Montin U, Petruccelli S, Morelli L, Campani D, Pollina L, Biancofiore G, Bindi L, Tascini C, Menichetti F, Scatena F, Filipponi F. The role of immunomodulation in ABOincompatible adult liver transplant recipients. 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176 Lee EC et al. ABO incompatability in liver transplantation DOI: /bjs.10048] 30 Kim JD, Choi DL, Kim SG, Lee AJ. Single-Center Experience of ABO-Incompatible Living-Donor Liver Transplantation With a New Simplified Intravenous Immunoglobulin Protocol: A Propensity Score-Matching Analysis. Transplant Proc 2016; 48: [PMID: DOI: /j.transproceed ] 31 Ikegami T, Yoshizumi T, Soejima Y, Uchiyama H, Shirabe K, Maehara Y. Feasible usage of ABO incompatible grafts in living donor liver transplantation. Hepatobiliary Surg Nutr 2016; 5: [PMID: DOI: /j.issn ] 32 Lee CF, Cheng CH, Wang YC, Soong RS, Wu TH, Chou HS, Wu TJ, Chan KM, Lee CS, Lee WC. Adult living donor liver transplantation across ABO-incompatibility. Medicine (United States) 2015; 94: e1796 [DOI: /MD ] 33 Shen T, Lin BY, Jia JJ, Wang ZY, Wang L, Ling Q, Geng L, Yan S, Zheng SS. A modified protocol with rituximab and intravenous immunoglobulin in emergent ABO-incompatible liver transplantation for acute liver failure. Hepatobiliary Pancreat Dis Int 2014; 13: [PMID: DOI: /S (14)60268-X] 34 Heffron TG, Pillen T, Smallwood G, Rodriguez J, Sekar S, Henry S, Vos M, Casper K, Gupta NA, Fasola CG, Romero R. Pediatric liver transplantation for acute liver failure at a single center: a 10-year experience. Pediatr Transplant 2010; 14: [PMID: DOI: /j x] 35 Stewart ZA, Locke JE, Montgomery RA, Singer AL, Cameron AM, Segev DL. ABO-incompatible deceased donor liver transplantation in the United States: a national registry analysis. Liver Transpl 2009; 15: [PMID: DOI: / lt.21723] 36 Iwamoto H, Hama K, Nakamura Y, Osamu K, Yokoyama T, Kihara Y, Ashizawa T, Niido T, Matsuno N, Nagao T. Biliary complications after 52 adult living donor liver transplantations: a single-center experience. Transplant Proc 2008; 40: [PMID: DOI: /j.transproceed ] 37 Saito T, Egawa H, Kudo T, Takakura S, Fujihara N, Iinuma Y, Ichiyama S. Pre-transplant risk factors predicting posttransplant cytomegalovirus infection in liver transplant recipients. Transpl Int 2007; 20: [PMID: DOI: / j x] 38 Heffron T, Welch D, Pillen T, Asolati M, Smallwood G, Hagedorn P, Nam C, Duncan A, Guy M, Martinez E, Spivey J, Douglas P, Fasola C, De Paolo J, Rodriguez J, Romero R. Successful ABOincompatible pediatric liver transplantation utilizing standard immunosuppression with selective postoperative plasmapheresis. Liver Transpl 2006; 12: [PMID: DOI: / lt.20760] 39 Chui AKK, Ling J, McCaughan GW, Painter D, Shun A, Dorney SFA, Mears DC, Sheil AGR. ABO blood group incompatibility in liver transplantation: A single- centre experience. Australian and New Zealand J Surg 1997; 67: [DOI: / j tb01962.x] 40 Lo CM, Shaked A, Busuttil RW. Risk factors for liver transplantation across the ABO barrier. Transplantation 1994; 58: [PMID: DOI: / ] 41 Sanchez-Urdazpal L, Batts KP, Gores GJ, Moore SB, Sterioff S, Wiesner RH, Krom RA. Increased bile duct complications in liver transplantation across the ABO barrier. Ann Surg 1993; 218: [PMID: DOI: / ] 42 Tokunaga Y, Tanaka K, Fujita S, Yamaguchi T, Sawada H, Kato H, Uemoto S, Yamaoka Y, Ozawa K. Living related liver transplantation across ABO blood groups with FK506 and OKT3. Transpl Int 1993; 6: [PMID: DOI: / j tb00673.x] 43 Dunn SP, Halligan GE, Billmire DF, Vinocur CD, Lawrence J, Falkenstein K, Weintraub W, Meyers R. ABO-incompatible liver transplantation: a risk worth taking. Transplant Proc 1993; 25: 3109 [PMID: ] 44 Renard TH, Andrews WS. An approach to ABO-incompatible liver transplantation in children. Transplantation 1992; 53: [PMID: DOI: / ] 45 Demetris AJ, Jaffe R, Tzakis A, Ramsey G, Todo S, Belle S, Esquivel C, Shapiro R, Markus B, Mroczek E. Antibody-mediated rejection of human orthotopic liver allografts. A study of liver transplantation across ABO blood group barriers. Am J Pathol 1988; 132: [PMID: ] 46 Nakamura Y, Matsuno N, Iwamoto H, Yokoyama T, Kuzuoka K, Kihara Y, Taira S, Sagara T, Jojima Y, Konno O. Tashiro J, Akashi I, Hama K, Narumi K, Iwahori T, Uchiyama M, Tanaka K, Nagao T. Successful case of adult ABO-incompatible liver transplantation: beneficial effects of intrahepatic artery infusion therapy: a case report. Transplant Proc 2004; 36: [PMID: DOI: /j.transproceed ] 47 Taenaka N, Shimada Y, Hirata T, Nishijima MK, Takezawa J, Yoshiya I, Kambayashi J. Gabexate mesilate (FOY) therapy of disseminated intravascular coagulation due to sepsis. Crit Care Med 1983; 11: [PMID: DOI: / ] 48 Monteiro I, McLoughlin LM, Fisher A, de la Torre AN, Koneru B. Rituximab with plasmapheresis and splenectomy in abo-incompatible liver transplantation. Transplantation 2003; 76: [PMID: DOI: /01. TP ] 49 Kawagishi N, Satoh K, Enomoto Y, Akamatsu Y, Sekiguchi S, Fukumori T, Fujimori K, Satomi S. New strategy for ABOincompatible living donor liver transplantation with anti- CD20 antibody (rituximab) and plasma exchange. Transplant Proc 2005; 37: [PMID: DOI: /j.tra nsproceed ] 50 Usuda M, Fujimori K, Koyamada N, Fukumori T, Sekiguchi S, Kawagishi N, Akamatsu Y, Enomoto Y, Satoh K, Satoh A, Ishida K, Moriya T, Satomi S. Successful use of anti-cd20 monoclonal antibody (rituximab) for ABO-incompatible living-related liver transplantation. Transplantation 2005; 79: [PMID: DOI: /01. TP E4] 51 Jordan S, Cunningham Rundles C, McEwan R. Utility of intravenous immune globulin in kidney transplantation: efficacy, safety, and cost implications. Am J Transplant 2003; 3: [ PMID: DOI: / j x] 52 Egawa H, Oike F, Buhler L, Shapiro AM, Minamiguchi S, Haga H, Uryuhara K, Kiuchi T, Kaihara S, Tanaka K. Impact of recipient age on outcome of ABO-incompatible livingdonor liver transplantation. Transplantation 2004; 77: [PMID: DOI: /01.TP C] 53 Varela-Fascinetto G, Treacy SJ, Lillehei CW, Jonas MM, Lund DP, Kevy SV, Pérez A, Zurakowski D, Vacanti JP. Long-term results in pediatric ABO-incompatible liver transplantation. Transplant Proc 1999; 31: [PMID: DOI: /S (98) ] 54 Wuttke NJ, Macardle PJ, Zola H. Blood group antibodies are made by CD5+ and by CD5- B cells. Immunol Cell Biol 1997; 75: [PMID: DOI: /icb ] 55 Eastlund T. The histo-blood group ABO system and tissue transplantation. Transfusion 1998; 38: [PMID: DOI: /j x] 56 Yandza T, Lambert T, Alvarez F, Gauthier F, Jacolot D, Huault G, Fabre M, Valayer J. Outcome of ABO-incompatible liver transplantation in children with no specific alloantibodies at the time of transplantation. Transplantation 1994; 58: [PMID: DOI: / ] 57 Ferriani VP, Barbosa JE, de Carvalho IF. Serum haemolytic classical and alternative pathways of complement in infancy: age-related changes. 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177 Lee EC et al. ABO incompatability in liver transplantation 58 Boross P, Leusen JH. Mechanisms of action of CD20 antibodies. Am J Cancer Res 2012; 2: [PMID: ] 59 Weiner GJ. Rituximab: mechanism of action. Semin Hematol 2010; 47: [PMID: DOI: /j.seminhem atol ] 60 Egawa H, Ohmori K, Haga H, Tsuji H, Yurugi K, Miyagawa- Hayashino A, Oike F, Fukuda A, Yoshizawa J, Takada Y, Tanaka K, Maekawa T, Ozawa K, Uemoto S. B-cell surface marker analysis for improvement of rituximab prophylaxis in ABO-incompatible adult living donor liver transplantation. Liver Transpl 2007; 13: [PMID: DOI: /lt.21092] 61 Fujii Y, Shibata Y, Miyata S, Inaba S, Asai T, Hoshi Y, Takamatsu J, Takahashi K, Ohto H, Juji T, Sagawa K. Consecutive national surveys of ABO-incompatible blood transfusion in Japan. Vox Sang 2009; 97: [PMID: DOI: /j x] 62 Shaked A, Ghobrial RM, Merion RM, Shearon TH, Emond JC, Fair JH, Fisher RA, Kulik LM, Pruett TL, Terrault NA; A2ALL Study Group. Incidence and severity of acute cellular rejection in recipients undergoing adult living donor or deceased donor liver transplantation. Am J Transplant 2009; 9: [PMID: DOI: / j x] 63 Maluf DG, Stravitz RT, Cotterell AH, Posner MP, Nakatsuka M, Sterling RK, Luketic VA, Shiffman ML, Ham JM, Marcos A, Behnke MK, Fisher RA. Adult living donor versus deceased donor liver transplantation: a 6-year single center experience. Am J Transplant 2005; 5: [PMID: DOI: /j x] 64 Liu LU, Bodian CA, Gondolesi GE, Schwartz ME, Emre S, Roayaie S, Schiano TD. Marked Differences in acute cellular rejection rates between living-donor and deceased-donor liver transplant recipients. Transplantation 2005; 80: [PMID: DOI: /01.tp a] 65 Seehofer D, Eurich D, Veltzke-Schlieker W, Neuhaus P. Biliary complications after liver transplantation: old problems and new challenges. Am J Transplant 2013; 13: [PMID: DOI: /ajt.12034] 66 Wang SF, Huang ZY, Chen XP. Biliary complications after living donor liver transplantation. Liver Transpl 2011; 17: [PMID: DOI: /lt.22381] 67 Zimmerman MA, Baker T, Goodrich NP, Freise C, Hong JC, Kumer S, Abt P, Cotterell AH, Samstein B, Everhart JE, Merion RM. Development, management, and resolution of biliary complications after living and deceased donor liver transplantation: a report from the adult-to-adult living donor liver transplantation cohort study consortium. Liver Transpl 2013; 19: [PMID: DOI: /lt.23595] 68 Simoes P, Kesar V, Ahmad J. Spectrum of biliary complications following live donor liver transplantation. World J Hepatol 2015; 7: [PMID: DOI: /wjh.v7.i ] 69 Zhang Z, Xu X, Ni H. Small studies may overestimate the effect sizes in critical care meta-analyses: a metaepidemiological study. Crit Care 2013; 17: R2 [PMID: DOI: /cc11919] 70 Nüesch E, Trelle S, Reichenbach S, Rutjes AW, Tschannen B, Altman DG, Egger M, Jüni P. Small study effects in meta-analyses of osteoarthritis trials: meta-epidemiological study. BMJ 2010; 341: c3515 [PMID: DOI: /bmj.c3515] 71 Sterne JA, Gavaghan D, Egger M. Publication and related bias in meta-analysis: power of statistical tests and prevalence in the literature. J Clin Epidemiol 2000; 53: [PMID: DOI: /S (00) ] 72 Sterne JA, Sutton AJ, Ioannidis JP, Terrin N, Jones DR, Lau J, Carpenter J, Rücker G, Harbord RM, Schmid CH, Tetzlaff J, Deeks JJ, Peters J, Macaskill P, Schwarzer G, Duval S, Altman DG, Moher D, Higgins JP. Recommendations for examining and interpreting funnel plot asymmetry in meta-analyses of randomised controlled trials. BMJ 2011; 343: d4002 [PMID: DOI: /bmj.d4002] P- Reviewer: Cerwenka H, Ramsay MA, Topaloglu S, ZS- Editor: Qi Y L- Editor: A E- Editor: Ma YJ 6533 September 21, 2017 Volume 23 Issue 35

178 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) CASE REPORT Case of colonic intussusception secondary to mobile cecum syndrome repaired by laparoscopic cecopexy using a barbed wound suture device Tetsu Yamamoto, Yoshitsugu Tajima, Ryoji Hyakudomi, Takanori Hirayama, Takahito Taniura, Kazunari Ishitobi, Noriyuki Hirahara Tetsu Yamamoto, Yoshitsugu Tajima, Ryoji Hyakudomi, Takanori Hirayama, Takahito Taniura, Kazunari Ishitobi, Noriyuki Hirahara, Department of Digestive and General Surgery, Shimane University Faculty of Medicine, Izumo, Shimane , Japan Author contributions: All authors contributed equally to the conception, design, literature review and analysis of this study, and the drafting, critical revision, editing, and approval of the final version of this manuscript. Institutional review board statement: The study was reviewed and approved by the Shimane University Institutional Review Board. Informed consent statement: Informed consent was obtained from the patient. Conflict-of-interest statement: The authors declare that there are no conflicts of interest. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Unsolicited manuscript Correspondence to: Tetsu Yamamoto, MD, PhD, Department of Digestive and General Surgery, Shimane University Faculty of Medicine, 89-1 Enya, Izumo, Shimane , Japan. t2t2@med.shimane-u.ac.jp Telephone: Fax: Received: February 19, 2017 First decision: February 21, 2017 Revised: June 1, 2017 Accepted: July 12, 2017 Article in press: July 12, 2017 Published online: September 21, 2017 Abstract A 27-year-old man with recurrent right lower quadrant pain was admitted to our hospital. Ultrasonography and computed tomography examination of the abdomen revealed a target sign in the ascending colon, which was compatible with the diagnosis of cecal intussusception. The intussusception was spontaneously resolved at that time, but it relapsed 6 mo later. The patient underwent a successful colonoscopic disinvagination; there was no evidence of neoplastic or inflammatory lesions in the colon and terminal ileum. The patient underwent laparoscopic surgery for recurring cecal intussusception. During laparoscopy, we observed an unfixed cecum on the posterior peritoneum (i.e. a mobile cecum). Thus, we performed laparoscopic appendectomy and cecopexy with a lateral peritoneal flap using a barbed wound suture device. The patient s post-operative course was uneventful, and he continued to do well without recurrence at 10 mo after surgery. Laparoscopic cecopexy using a barbed wound suture device is a simple and reliable procedure that can be the treatment of choice for recurrent cecal intussusception associated with a mobile cecum. Key words: Colonic intussusception; Adult; Mobile cecum; Cecopexy; Barbed wound suture device The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved September 21, 2017 Volume 23 Issue 35

179 Yamamoto T et al. Colonic intussusception due to mobile cecum syndrome Core tip: Colonic intussusception secondary to mobile cecum is uncommon, and the surgical procedure is controversial. As the main cause of intussusception comprises neoplasms, most patients with colonic intussusception undergo intestinal resection. In our case, the cause of colonic intussusception was only the mobile cecum. Thus, we performed laparoscopic cecopexy using a barbed wound suture device, after which the patient made steady progress. This surgical procedure benefits from its good cosmetic outcomes and reduced invasiveness. In addition, a barbed wound suture device is useful for laparoscopic cecopexy, as it is easy to handle and results in a shorter operation time. Yamamoto T, Tajima Y, Hyakudomi R, Hirayama T, Taniura T, Ishitobi K, Hirahara N. Case of colonic intussusception secondary to mobile cecum syndrome repaired by laparoscopic cecopexy using a barbed wound suture device. World J Gastroenterol 2017; 23(35): Available from: URL: wjgnet.com/ /full/v23/i35/6534.htm DOI: org/ /wjg.v23.i INTRODUCTION Mobile cecum syndrome is characterized by chronic right lower quadrant pain without evidence of appendicitis or other etiological factors. Although abnormal mobility of the cecum and ascending colon has been estimated to occur in 10%-20% of the population, intussusception or volvulus of the cecum secondary to a mobile cecum is uncommon [1,2]. Approximately one-third of the intussusceptions of the intestinal tract occur as a result of surgery-induced changes, such as adhesion, submucosal edema, and intestinal motility disorders; colonic intussusceptions in adults are usually caused by malignant or benign tumors [3]. Therefore, surgical interventions, such as intestinal resection through either an open or laparoscopic approach, have been performed for the majority of adult patients with colonic intussusception [4]. We describe herein a rare case of colonic intussusception secondary to mobile cecum syndrome, which was successfully treated with laparoscopic cecopexy using a barbed wound suture device. CASE REPORT A 27-year-old man with recurring abdominal pain in the right lower quadrant was re-admitted to our hospital. The patient had a 3-year history of mild ulcerative colitis, but he had been free from abdominal symptoms and continued to do well without medications. On admission, abdominal ultrasonography (Figure 1A) and contrast-enhanced computed tomography (CT) (Figure 1B) showed a target sign in the ascending colon with dilatation of the cecum and ileum, which was compatible with the diagnosis of cecal intussusception. The patient had no previous surgical history, and there was no evidence of intraabdominal tumors or inflammatory conditions in these imaging evaluations. Thereafter, the intussusception spontaneously resolved. The patient was discharged a few days after admission, but the abdominal pain recurred 6 mo later. Abdominal CT scan again revealed cecal intussusception. An emergency colonoscopy was performed to reduce the intussusception, in which the cecum had advanced into the ascending colon. An edematous colonic mucosa restricted to the lead point of intussusception was identified (Figure 1C). No other inflammatory changes or neoplastic lesions were detected in the colon and terminal ileum, even after reduction of the intussusception. The patient was thus diagnosed with recurrent cecal intussusception that was likely due to a mobile cecum, and underwent an elective laparoscopic surgery. The patient was placed in the supine position under general anesthesia. A 20-mm long skin incision was made on the umbilicus, and a 12-mm trocar was inserted. After creating a pneumoperitoneum with carbon dioxide at an intra-abdominal pressure of 10 mmhg, additional 5-mm and 12-mm trocars were placed at the left upper quadrant and the middle of the lower abdomen, respectively. Upon laparoscopy, no evidence of ischemia, inflammation, or caliber change in the gastrointestinal tract, including the cecum, appendix and terminal ileum, was found. A linear indentation running along the minor axis of the large bowel was identified in the middle portion of the ascending colon, which left a trace of intussusception (Figure 2A). The cecum was easily mobilized from the right lower quadrant to the upper abdominal cavity, as it was not fixed to the posterior parietal peritoneum (Figure 2B). The findings confirmed that the recurrent cecal intussusception was associated with an abnormal fixation of the cecum and ascending colon to the parietal peritoneum (i.e. a mobile cecum). Laparoscopic appendectomy and cecopexy were performed. After the appendectomy, an approximately 10-cm long incision was made in the right parietal peritoneum along the ascending colon for the cecopexy (Figure 3A); then, the cecum and ascending colon were fixed to the incision line of the parietal peritoneum with a continuous suture technique, using an absorbable barbed wound suture device (V-Loc 180; Medtronic, Tokyo, Japan) (Figure 3B). The operation time was 87 min, with less than 10 g of blood loss. The patient s postoperative course was uneventful, and he was discharged 4 d after surgery. The patient continued to do well without recurrence at 10 mo after surgery. DISCUSSION Intestinal intussusception is the leading cause of gastrointestinal obstruction in children. However, it is 6535 September 21, 2017 Volume 23 Issue 35

180 Yamamoto T et al. Colonic intussusception due to mobile cecum syndrome A B C Figure 1 Target sign on abdominal ultrasonography (A) and contrast-enhanced computed tomography scan (B). The so-called bowel-within-bowel configuration, in which the layers of the bowel are duplicated, thereby forming concentric rings, is seen (white arrows). Dilatation of the terminal ileum is observed (black arrows). C: A colonoscopic view of the intussusception. Edematous colonic mucosa was identified on the lead point of intussusception. No neoplastic lesion was detected, even after reduction of intussusception. A B Appendix Cecum Ascending colon Cecum Ileum Appendix Figure 2 Laparoscopic view. A: A linear indentation leaving a trace of intussusception was found in the middle portion of the ascending colon (white arrows). The cecum was not attached to the retroperitoneum (black arrows); B: The cecum was easily moved to the upper abdomen. No fixation of the cecum was observed (black arrows). A B Ascending colon Cecum Ileum Figure 3 Laparoscopic cecopexy. A: An approximately 10-cm long incision (black arrows) was made on the right parietal peritoneum along the ascending colon; B: The cecum and ascending colon were stitched to the parietal peritoneum with continuous sutures using an absorbable barbed wound suture device (black arrows). rarely encountered in adults, accounting for 1%-5% of all cases of intestinal obstruction [5]. Although the exact mechanism of bowel intussusception remains unclear, one leading theory is that intestinal motility disorder due to intraluminal lesions or inflammation induces invagination [6,7]. The common site of intussusception is the junction between the free intestine and the portion fixed to the retroperitoneum or at postoperative adhesions [7,8]. Non-malignant lesions, such as benign or inflammatory neoplasms, Meckel s diverticulum and appendix, are the most common cause of intussusception in the small bowel, with only 30% of cases due to malignant neoplasms [6,9]. In contrast, malignant lesions are responsible for most cases of colonic intussusceptions [7,10]. In the present case, neither malignant or benign 6536 September 21, 2017 Volume 23 Issue 35

181 Yamamoto T et al. Colonic intussusception due to mobile cecum syndrome Table 1 Colonic intussusception associated with mobile cecum in adults: reported cases from 2005 to 2016 Ref. Age Sex Clinical symptoms Repeated symptoms Episode duration Diagnostic modalities Characteristic finding Etiology of intussusception / location Operative procedure Hamdi et al [16] 85 F Abdominal pain, diarrhea Drnovsek et al [15] 65 M Abdominal pain, rectal bleeding Kuzmich et al [14] 62 M Abdominal pain, weight loss Yes 3 mo Barium enema, CT Target mass Tumor /cecum No 12 h CT Target sign Tubulovillous adenoma /cecum Yes 2 mo US Target sign Submucosal lipoma /ileocecal valve Frydman et al [13] 22 F Rectal prolapse No 1 d CT Target sign Villous adenoma /cecum Present case 27 M Right lower quadrant pain Resection Right hemicolectomy Right hemicolectomy Right hemicolectomy Yes 7 mo US, CT Target sign None Laparoscopic cecopexy CT: Computed tomography; US: Ultrasonography. tumors nor inflammatory conditions were identified upon colonoscopic, ultrasonographic and CT examinations. Laparoscopically, other than the unfixed cecum and ascending colon on the posterior peritoneum, no notable finding (e.g., abdominal adhesions or mesenteric lymph node swelling) was observed, thereby indicating that the cecal intussusception occurred secondary to the mobile cecum in our patient. Mobile cecum is not uncommon, with up to 25% to 64% of cadavers exhibiting a mobile cecum. Nevertheless, colonic intussusception related to mobile cecum is rare in adults [11,12]. We performed a review of the literature from 1995 to 2016 using the PubMed database with the search terms intussusception, mobile cecum and adult, which yielded only 4 cases of colonic intussusception associated with mobile cecum [13-16]. Moreover, all of these cases had some lead lesions as a cause of intussusception, such as villous adenoma and submucosal lipoma in the cecum or ileocecal valve (Table 1). Meanwhile, our case showed no evidence of neoplastic lesions or inflammatory conditions in the colon and terminal ileum, except edematous colonic mucosa identified at the lead point of intussusception. In terms of the cause of intussusception, our patient represented a rare case of colonic intussusception associated with a mobile cecum. The mucosal edema identified on the lead point of intussusception might be a cause or an effect of the intussusception in our patient. It is undeniable that the mucosal edema might have been induced by ulcerative colitis because the patient had a history of mild ulcerative colitis; however, he had been free from ulcerative colitis-related specific symptoms and required no medications for the treatment. The presenting symptoms of colonic intussusception are nonspecific, such as abdominal pain, nausea and emesis, and a definitive diagnosis can be made only in 33% of patients prior to surgery [17]. The typical target sign or a sausage-shaped mass on a CT scan is used to confirm the diagnosis of intussusception [17, 18]. In our patient, cecal intussusception was diagnosed on the basis of the target sign on abdominal ultrasonography and CT examination. Ultrasonography is a noninvasive and readily available imaging modality and easy to perform in routine clinical practice, thus, it was useful in the diagnostic workup of colonic intussusception in our patient. In the treatment of colonic intussusception, colonoscopic reduction is worth considering because it is less invasive than surgery, although its efficacy remains controversial [9]. In adult patients, surgical resection is generally required because most cases have intraluminal neoplasms as a cause of intussusception [3,7,19]. In the present case, we performed an elective laparoscopic cecopexy after endoscopic reduction of cecal intussusception because the patient had neither intraluminal lesions nor mesenteric lymphadenopathy. Cecopexy or right hemicolectomy can be performed to treat mobile cecum syndrome with abdominal symptoms. Some studies have recommended only colectomy because there is a possibility of recurrence of the abdominal pain due to volvulus or intussusception after performing a cecopexy [20,21]. Meanwhile, other reports have described cecopexy as a good treatment option for patients with a mobile cecum, which is associated with a low rate of disease and symptom recurrence [22,23]. Given the absence of any recurrence in our case, laparoscopic cecopexy seems to be a useful treatment of choice for cecal intussusception secondary to mobile cecum without neoplastic lesions. Open and laparoscopic surgery can be performed for mobile cecum syndrome, and the benefits of laparoscopic surgery, in terms of cosmetic outcomes and less invasiveness, have been reported [24,25]. In the present case, we performed an elective laparoscopic cecopexy after colonoscopic reduction of cecal intussusception because the patient was generally stable and had no neoplastic lesions that could have possibly caused cecal intussusception. In addition, a barbed wound suture device was used to fix the cecum to the posterior peritoneum. Previous studies have reported various benefits of using barbed sutures, including knotless suturing, shorter stitching time and increased convenience for the surgeon [26,27]. In our case, a barbed suture device was useful for laparoscopic cecopexy, as it was easy to handle and resulted in a shorter operation 6537 September 21, 2017 Volume 23 Issue 35

182 Yamamoto T et al. Colonic intussusception due to mobile cecum syndrome time. Laparoscopic cecopexy is a useful strategy to treat colonic intussusception secondary to a mobile cecum without any other pathologic disorders. A barbed suture device is also useful in performing a laparoscopic cecopexy in terms of its ease in handling and shorter stitching time. COMMENTS Case characteristics A 27-year-old man with recurring abdominal pain in the right lower quadrant. Clinical diagnosis The patient s abdomen was slightly distended and hard. Tenderness of the right lower quadrant was detected with a palpable mass when intussusception occurred. Differential diagnosis Cecal volvulus or intra-abdominal tumors. Laboratory diagnosis Laboratory data were within normal limits. Imaging diagnosis Abdominal ultrasonography and computed tomography scan showed a target sign in the ascending colon with dilatation of the cecum and ileum, which was compatible with the diagnosis of cecal intussusception. Pathological diagnosis There was no evidence of malignancy or any other inflammatory disease. Treatment Laparoscopic cecopexy using a barbed wound suture device. Related reports Past studies have recommended only colectomy to treat the mobile cecum, given the high rate of recurrence after cecopexy. Recently, some case reports have described a cosmetic benefit and reduced invasiveness of cecopexy without recurrence of cecal intussusception. Term explanation A mobile cecum, which is defined as the unfixed cecum and ascending colon on the posterior peritoneum, is not uncommon. However, colonic intussusception related to mobile cecum is rare in adults. Experiences and lessons Colonic intussusception secondary to a mobile cecum is uncommon, and the surgical procedure for treatment is controversial. In this case, we performed laparoscopic cecopexy using a barbed wound suture device. This surgical procedure produces a good cosmetic outcome, while a barbed wound suture device is useful for laparoscopic cecopexy, as it is easy to handle and results in a shorter operation time. Peer-review The paper is an interesting Case Report of colonic intussusception secondary to mobile cecum syndrome and is suitable for publication. REFERENCES 1 Katoh T, Shigemori T, Fukaya R, Suzuki H. Cecal volvulus: report of a case and review of Japanese literature. World J Gastroenterol 2009; 15: [PMID: DOI: /wjg ] 2 Rogers RL, Harford FJ. Mobile cecum syndrome. Dis Colon Rectum 1984; 27: [PMID: DOI: / BF ] 3 Agha FP. Intussusception in adults. AJR Am J Roentgenol 1986; 146: [PMID: DOI: /ajr ] 4 de Clerck F, Vanderstraeten E, De Vos M, Van Steenkiste C. Adult intussusception: 10-year experience in two Belgian centres. Acta Gastroenterol Belg 2016; 79: [PMID: ] 5 Yakan S, Caliskan C, Makay O, Denecli AG, Korkut MA. Intussusception in adults: clinical characteristics, diagnosis and operative strategies. World J Gastroenterol 2009; 15: [PMID: DOI: /wjg ] 6 Begos DG, Sandor A, Modlin IM. The diagnosis and management of adult intussusception. Am J Surg 1997; 173: [PMID: DOI: /S (96) ] 7 Wang LT, Wu CC, Yu JC, Hsiao CW, Hsu CC, Jao SW. Clinical entity and treatment strategies for adult intussusceptions: 20 years experience. Dis Colon Rectum 2007; 50: [PMID: DOI: /s ] 8 Sachs M, Encke A. [Entero-enteral invagination of the small intestine in adults. A rare cause of uncertain abdomen ]. Langenbecks Arch Chir 1993; 378: [PMID: DOI: /BF ] 9 Tan KY, Tan SM, Tan AG, Chen CY, Chng HC, Hoe MN. Adult intussusception: experience in Singapore. ANZ J Surg 2003; 73: [PMID: DOI: /j t x] 10 Eisen LK, Cunningham JD, Aufses AH Jr. Intussusception in adults: institutional review. J Am Coll Surg 1999; 188: [PMID: DOI: /S (98) ] 11 Consorti ET, Liu TH. Diagnosis and treatment of caecal volvulus. Postgrad Med J 2005; 81: [PMID: DOI: /pgmj ] 12 Solanke TF. Intestinal obstruction in Ibadan. West Afr Med J Niger Pract 1968; 17: [PMID: ] 13 Frydman J, Ben-Ishay O, Kluger Y. Total ileocolic intussusception with rectal prolapse presenting in an adult: a case report and review of the literature. World J Emerg Surg 2013; 8: 37 [PMID: DOI: / ] 14 Kuzmich S, Connelly JP, Howlett DC, Kuzmich T, Basit R, Doctor C. Ileocolocolic intussusception secondary to a submucosal lipoma: an unusual cause of intermittent abdominal pain in a 62-year-old woman. J Clin Ultrasound 2010; 38: [PMID: DOI: /jcu.20620] 15 Drnovsek V, Ruff MB, Riehl PA, Plavsic BM. Gastrointestinal case of the day. Chronic ileocolocolic intussusception secondary to a mobile cecum and a benign fibrovascular mass. Radiographics 1999; 19: [PMID: DOI: /radiographics g99jl211102] 16 Hamdi M, Blondio JV, Algaba R, Van Gysel JP. Adult intussusception. A case report. Acta Chir Belg 1996; 96: [PMID: ] 17 Azar T, Berger DL. Adult intussusception. Ann Surg 1997; 226: [PMID: DOI: / ] 18 Gayer G, Zissin R, Apter S, Papa M, Hertz M. Pictorial review: adult intussusception--a CT diagnosis. Br J Radiol 2002; 75: [PMID: DOI: /bjr ] 19 Ajao OG. Non-infantile intussusception. J Natl Med Assoc 1979; 71: [PMID: ] 20 Remes-Troche JM, Pérez-Martínez C, Rembis V, Arch Ferrer J, Ayala González M, Takahashi T. [Surgical treatment of colonic volvulus. 10-year experience at the Instituto Nacional de la Nutrición Salvador Zubirán]. Rev Gastroenterol Mex 1997; 62: [PMID: ] 21 Meyers JR, Heifetz CJ, Baue AE. Cecal volvulus: a lesion requiring resection. Arch Surg 1972; 104: [PMID: DOI: /archsurg ] 22 Howard RS, Catto J. Cecal volvulus. A case for nonresectional therapy. Arch Surg 1980; 115: [PMID: DOI: /archsurg ] 6538 September 21, 2017 Volume 23 Issue 35

183 Yamamoto T et al. Colonic intussusception due to mobile cecum syndrome 23 Rabinovici R, Simansky DA, Kaplan O, Mavor E, Manny J. Cecal volvulus. Dis Colon Rectum 1990; 33: [PMID: DOI: /BF ] 24 Gomes CA, Soares C Jr, Catena F, Di Saverio S, Sartelli M, Gomes CC, Gomes FC. Laparoscopic Management of Mobile Cecum. JSLS 2016; 20: [PMID: DOI: / JSLS ] 25 Kakizoe S, Kakizoe Y, Kakizoe H, Kakizoe T, Kakizoe K. Experience of laparoscopic cecoplication for mobile cecum. Endoscopy 2000; 32: S4-S5 [PMID: ] 26 Greenberg JA, Goldman RH. Barbed suture: a review of the technology and clinical uses in obstetrics and gynecology. Rev Obstet Gynecol 2013; 6: [PMID: ] 27 Facy O, De Blasi V, Goergen M, Arru L, De Magistris L, Azagra JS. Laparoscopic gastrointestinal anastomoses using knotless barbed sutures are safe and reproducible: a single-center experience with 201 patients. Surg Endosc 2013; 27: [PMID: DOI: /s ] P- Reviewer: Tovey FI S- Editor: Qi Y L- Editor: Filipodia E- Editor: Xu XR 6539 September 21, 2017 Volume 23 Issue 35

184 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) CASE REPORT Acquired amegakaryocytic thrombocytopenia previously diagnosed as idiopathic thrombocytopenic purpura in a patient with hepatitis C virus infection Shojiro Ichimata, Mikiko Kobayashi, Kohei Honda, Soichiro Shibata, Akihiro Matsumoto, Hiroyuki Kanno Shojiro Ichimata, Mikiko Kobayashi, Kohei Honda, Hiroyuki Kanno, Department of Pathology, Shinshu University School of Medicine, Matsumoto, Nagano , Japan Soichiro Shibata, Akihiro Matsumoto, Department of Medicine, Shinshu University School of Medicine, Matsumoto, Nagano , Japan ORCID number: Shojiro Ichimata ( ); Mikiko Kobayashi ( ); Kohei Honda ( ); Soichiro Shibata ( ); Akihiro Matsumoto ( ); Hiroyuki Kanno ( ). Author contributions: Ichimata S and Kobayashi M performed the histological evaluation, analyzed the serum sample, and drafted the manuscript; Honda K analyzed the serum sample; Shibata S and Matsumoto A evaluated clinical data; Kanno H performed the histological evaluation, analyzed the serum sample, and critically reviewed the manuscript. Institutional review board statement: This case report was exempt from the Institutional Review Board standard at Shinshu University School of Medicine. Informed consent statement: All study participants, or their legal guardian, provided informed consent prior to study enrollment. Conflict-of-interest statement: The authors have no conflict of interest. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Unsolicited manuscript Correspondence to: Mikiko Kobayashi, MD, PhD, Department of Pathology, Shinshu University School of Medicine, Asahi, Matsumoto , Japan. mikko@shinshu-u.ac.jp Telephone: Fax: Received: May 18, 2017 Peer-review started: May 18, 2017 First decision: June 22, 2017 Revised: July 6, 2017 Accepted: August 8, 2017 Article in press: August 8, 2017 Published online: September 21, 2017 Abstract We report the first case of a patient with hepatitis C virus (HCV) infection and idiopathic thrombocytopenic purpura (ITP), who later developed acquired amegakaryocytic thrombocytopenia (AAMT), with autoantibodies to the thrombopoietin (TPO) receptor (c-mpl). A 64-year-old woman, with chronic hepatitis C, developed severe thrombocytopenia and was diagnosed with ITP. She died of liver failure. Autopsy revealed cirrhosis and liver carcinoma. In the bone marrow, a marked reduction in the number of megakaryocytes was observed, while other cell lineages were preserved. Therefore, she was diagnosed with AAMT. Additionally, autoantibodies to c-mpl were detected in her serum. Autoantibodies to c-mpl are one of the causes of AAMT, acting through inhibition of TPO function, megakaryocytic maturation, and platelet formation. HCV infection induces several autoantibodies. HCV infection might also induce autoantibodies to c-mpl, resulting in the development of AAMT. This mechanism may be one 6540 September 21, 2017 Volume 23 Issue 35

185 Ichimata S et al. AAMT associated with HCV infection of the causes of thrombocytopenia in patients with HCV infection. A Key words: Hepatitis C virus; Acquired amegakaryocytic thrombocytopenia; Anti-thrombopoietin receptor (c-mpl) autoantibodies; Idiopathic thrombocytopenic purpura; Thrombocytopenia The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: Thrombocytopenia occurs frequently in patients with hepatitis C virus (HCV) infection. Acquired amegakaryocytic thrombocytopenia (AAMT) is one of the causes of severe thrombocytopenia. The exact mechanisms of AAMT have not been fully elucidated. However, patients with autoantibodies to thrombopoietin receptor (c-mpl) develop AAMT. Similarly, autoantibodies are sometimes generated in patients with HCV infection. Here, we report the first case of a patient with HCV infection who later developed AAMT with autoantibodies to c-mpl. AAMT with autoantibodies to c-mpl may be one of the causes of thrombocytopenia in patients with HCV infection. B Ichimata S, Kobayashi M, Honda K, Shibata S, Matsumoto A, Kanno H. Acquired amegakaryocytic thrombocytopenia previously diagnosed as idiopathic thrombocytopenic purpura in a patient with hepatitis C virus infection. World J Gastroenterol 2017; 23(35): Available from: URL: wjgnet.com/ /full/v23/i35/6540.htm DOI: org/ /wjg.v23.i C INTRODUCTION Thrombocytopenia occurs frequently in patients with chronic hepatitis C. The causes of thrombocytopenia in patients with chronic hepatitis C are multiple, such as hypersplenism, immunological processes, and decreased thrombopoietin (TPO) level [1,2]. Acquired amegakaryocytic thrombocytopenia (AAMT) is one of the causes of severe thrombocytopenia and is characterized by a marked reduction in the number of megakaryocytes, with preserved hematopoiesis of the other lineages in the bone marrow [3]. The exact mechanisms of AAMT have not been fully elucidated. However, the defect of TPO receptor (c-mpl) expression due to c-mpl gene mutation is the major cause of congenital amegakaryocytic thrombocytopenia [4]. Furthermore, patients with systemic lupus erythematosus (SLE) and systemic sclerosis who have autoantibodies to c-mpl develop AAMT [5,6]. TPO, produced mainly by hepatocytes, binds to c-mpl on hematopoietic stem cells and megakaryocytes, and promotes all stages of platelet production, from the proliferation and differentiation of megakaryocytes to megakaryocytic maturation and Figure 1 Histopathological features of liver biopsy specimen and clot section of bone marrow aspirate. A: The liver biopsy specimen shows fibrous portal expansion. There is no fibrous bridging (Elastica-Goldner staining, scale bar; 500 μm); B: Mild piecemeal necrosis, mild intralobular degeneration and focal necrosis, and moderate portal inflammation are observed (H and E staining, scale bar; 200 μm); C: The clot section of bone marrow aspirate shows normal numbers of megakaryocytes and other cell lineages are preserved (Periodic Acid Schiff staining, scale bar; 100 μm). platelet formation [1]. Thus, autoantibodies to c-mpl may be one of the causes of AAMT, through inhibiting TPO function. AAMT with autoantibodies to c-mpl has not previously been reported in patients with hepatitis C virus (HCV) infection. Here, we report the first case of a patient with HCV infection and idiopathic thrombocytopenic purpura (ITP), who later developed AAMT with autoantibodies to c-mpl. CASE REPORT A 64-year-old woman was admitted with the chief 6541 September 21, 2017 Volume 23 Issue 35

186 Ichimata S et al. AAMT associated with HCV infection Table 1 Laboratory data on last admission CBC Chemistry WBC /μl TP 6.4 g/dl Na 129 meq/l Neutrophils 89% Alb 2.4 g/dl K 4.8 meq/l Lymphocytes 7% BUN 29.5 mg/dl Cl 96 meq/l RBC /μl Cre 1.13 mg/dl Glu 178 mg/dl Hemoglobin 10 g/dl AST 78 U/L CRP 1.08 mg/dl HCT 30% ALT 55 U/L NH3 63 μg/dl Platelets /μl γ-gt 88 U/L HCV-Ab 12.8 COI T-bil 3.88 mg/dl HCV (RT-PCR) 5.2 L.IU/mL Coagulation D-bil 2.72 mg/dl T-AFP ng/ml PT 17.2 s ALP 402 U/L AFP L ng/ml APTT 39.3 s LD 273 U/L PIVKA2 15 mau/ml Fibrinogen 123 mg/dl AMY 63 U/L D-dimer 5 μg/ml ChE 27 U/L AFP L3: Alpha-fetoprotein L3 isoform; ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; AMY: Amylase; AST: Aspartate aminotransferase; BUN: Blood urea nitrogen; CBC: Complete blood count; ChE: Cholinesterase; Cre: Creatinine; CRP: C-reactive protein; D-bil: Direct bilirubin; Glu: glucose; HCV-Ab: Hepatitis C antibody; HCT: Hematocrit; HCV (RT-PCR): Hepatitis C RNA (reverse transcriptase polymerase chain reaction); LDH: Lactate dehydrogenase; PIVKA2: Protein induced be vitamin K absence 2; PT: Prothrombin time; RBC: Red blood cells; T-AFP: Total alpha-fetoprotein; T-Bi: Total bilirubin; TP: Total protein; WBC: White blood cell; γ-gt: γ-glutamyltransferase. complaint of dyspnea. She had a past history of post-transfusion hepatitis approximately forty years beforehand, and subsequently she was diagnosed with HCV infection (genotype 1b, high). At the age of 41, she developed thrombocytopenia (platelets count: /μl). At that time, she received interferon therapy, but the HCV infection persisted. At the age of 51 and 52, liver biopsy and bone marrow aspirations were performed, respectively. Liver biopsy specimens revealed periportal mild inflammatory cell infiltration and fibrosis (Modified Histological Activity Index: activity was 5/18, fibrosis was 1/6; Figure 1A and B). The clot section of the bone marrow aspirate showed no significant change, and the number of megakaryocytes was within the normal range. Although the platelet-associated IgG (PA IgG) was not measured, she was diagnosed with ITP (Figure 1C). At the age of 61, liver cancer was detected, using computed tomography and magnetic resonance imaging, and she received transcatheter arterial chemoembolization (TACE) on several occasions. On the most recent admission, her liver cancer was found to be enlarged and ascites and pleural effusion had increased. Laboratory data are shown in Table 1. Her laboratory data indicated hepatic dysfunction, remnants of liver cancer and thrombocytopenia. On day 15 of her admission, her general condition deteriorated, and she died of liver failure. An autopsy was performed. At autopsy, she showed generalized jaundice and purpura in the anterior chest wall. Ascites (2600 ml) and pleural effusion (left: 100 ml, right: 3400 ml) were observed. Liver weight was 660 g, indicating severe atrophy. The cut surface of the liver showed diffuse micronodular cirrhosis with a dark green nodule (15 mm 15 mm) in the left lobe, and a yellow, partly reddish or green, lesion with an irregular margin (70 mm 50 mm) in the right lobe (Figure 2A). Spleen weight was 240 g, indicating mild enlargement. Varicose veins were observed in the lower esophagus, stomach, and rectum. Histopathologically, liver specimens showed diffuse small regenerative nodules with fibrous septum and septal mild mononuclear cell infiltration (Figure 2B). The right lobe lesion was mainly composed of two components: hepatocellular carcinoma with bile production (Figure 2C) and adenocarcinoma with mucin production (Figure 2D). Therefore, the diagnosis of combined hepatocellular-cholangiocarcinoma was made. There were no viable tumor cells in the left lobe lesion, compatible following TACE treatment for liver carcinoma. Microscopic metastases were observed in both lungs. Bone marrow specimens showed slight hypocellularity (30%-40%), with a myeloid to erythroid ratio: 3 to 1, and a marked reduction in the number of megakaryocytes, < 1 megakaryocyte/10 highpower fields (Figure 2E). Immunostaining for CD41 revealed scattered small megakaryocytes (Figure 2F). Other lineages of hematopoietic cells were preserved, and myelofibrosis, dysplasia, and metastatic lesions were not observed. Spleen specimens showed mild congestion without extramedullary hematopoiesis. Characteristic histopathological findings of the spleen in patients with ITP, such as an increase of secondary follicles with well-delineated germinal centers, an expansion of a follicular marginal zone of the white pulp and a diffuse proliferation of foamy histiocytes, were not obvious in this patient. There was no definite lesion in the thyroid. Next, we evaluated serum TPO levels at the time of her last admission using an enzyme-linked immunosorbent assay (ELISA) kit (Quantikine, R&D Systems, Minneapolis, United States) according to the manufacturer s protocol. The serum TPO level of the patient was 54 pg/ml, and the serum TPO levels of two healthy individuals without HCV infection were September 21, 2017 Volume 23 Issue 35

187 Ichimata S et al. AAMT associated with HCV infection A B C D E F Figure 2 Macroscopic and histopathological features of autopsy specimens. A: The cut surface of the liver shows diffuse micronodular cirrhosis with a yellowgreen lesion in the right lobe; B: The non-tumorous liver shows diffuse small regenerative nodules with fibrous septum (Elastica-Goldner staining, scale bar; 1000 μm); C and D: Histopathological findings of combined hepatocellular-cholangiocarcinoma; C: hepatocellular carcinoma component (H and E staining) and D: adenocarcinoma component (Alcian Blue-Periodic Acid Schiff staining) (C and D, scale bar; 200 μm); E: In the bone marrow, no megakaryocytes are observed (H and E staining); F: A small megakaryocyte is identified through immunostaining for CD41 (E and F, scale bar; 100 μm). pg/ml and 37 pg/ml (mean 32 pg/ml). In addition, the presence or absence of anti-tpo receptor (c-mpl) autoantibodies was determined, using Human antithrombopoietin receptor (C-MPL) autoantibodies IgG ELISA kit (CUSABIO, Wuhan, China), according to the manufacturer s protocol. The serum sample of the patient at the time of her last admission was positive for anti-c-mpl antibodies, compared to the negative results of the sera of two healthy individuals without HCV infection. DISCUSSION In the current case, AAMT associated with autoantibodies to c-mpl was considered to be one of the major causes of her severe thrombocytopenia. Hypersplenism and paraneoplastic autoimmunity were considered to be restrictive causes of her thrombocytopenia because she showed thrombocytopenia before development of her liver cirrhosis and liver cancer. In addition, her serum TPO level was preserved at the time of her last admission. Yet ITP was one of the causes of her thrombocytopenia. However, characteristic histopathological findings of the spleen in patients with ITP were not obvious in this patient at the time of autopsy. It is unclear when autoantibodies to c-mpl started to be produced in this patient. The patient showed a normal number of megakaryocytes at least ten years before her death, that is, ten years after the onset of thrombocytopenia. This finding is compatible with ITP. Therefore, autoantibodies to c-mpl might have 6543 September 21, 2017 Volume 23 Issue 35

188 Ichimata S et al. AAMT associated with HCV infection HCV infection (1) Anti-c-Mpl antibodies (2) Early stage Platelet (3) Destruction of platelets ITP-like thrombocytopenia Advanced stage (4) Megakaryocyte Progenitor cell (5) Inhibition of development and proliferation Severe thrombocytopenia (AAMT) Stem cell Figure 3 A schema of the pathogenesis of the current case. Hepatitis C virus infection may cause the generation of anti-c-mpl antibodies (1). At first, most of the generated antibodies would be absorbed with the c-mpl on platelets because platelets are the largest component in the megakaryocyte lineage (2). These antibody-attached platelets are destroyed in the spleen (3), therefore, idiopathic thrombocytopenic purpura (ITP)-like clinical manifestations are observed (Early stage). Following a sufficient reduction of platelets, these antibodies begin to bind to the c-mpl on the megakaryocytes and its progenitor cells in the bone marrow (4). Attached antibodies block the functions of thrombopoietin, causing inhibition in the development and proliferation of the megakaryocyte lineage (5). Thus, severe reduction of megakaryocytes in the bone marrow occurs, that is, acquired amegakaryocytic thrombocytopenia (AAMT) (Advanced stage). developed after the diagnosis of ITP and the patient subsequently developed AAMT. It is possible that autoantibodies to c-mpl had already been produced at the time of the ITP diagnosis. Kuwana et al [5] reported that autoantibodies to c-mpl are detected in approximately 8% of ITP patients. Thus, patients with autoantibodies to c-mpl might develop ITP at an early stage, and then develop AAMT during the course of the disease. This hypothesis is summarized in Figure 3. The receptor c-mpl is expressed in the megakaryocytic lineage from late progenitors to platelets, and platelets display high-affinity receptors for TPO [7]. Therefore, even if autoantibodies to c-mpl had been produced at an early stage, most of the autoantibodies would have been absorbed with c-mpl on platelets, and the proliferation and differentiation of megakaryocytes would not have been severely impaired. Thus, the number of megakaryocytes in bone marrow would be relatively well preserved and the patient s bone marrow may show a histopathology compatible with ITP. After a sufficient reduction in the number of platelets, AAMT then could then develop because autoantibodies to c-mpl start to bind to c-mpl on megakaryocytes and progenitor cells, inhibiting their development and maturation. Hoffman et al [8]. described a patient with ITP who later developed AAMT associated with antibodies that suppressed the colony formation of megakaryocytes [8]. HCV infection might have induced autoantibodies to c-mpl in the current patient. Autoantibodies to c-mpl are not detected in healthy controls [5]. In patients with HCV infection, several autoantibodies are produced, such as anti-nuclear antibodies, anti-smooth muscle antibodies, organ-specific autoantibodies, and antiplatelet antibodies [1]. In addition, anti-platelet IgG antibodies are detected in 26.3% of patients with HCV infection, showing a higher prevalence compared to healthy controls [9]. These mechanisms may play a role in the development of AAMT, with autoantibodies to c-mpl in patients with HCV infection. However, interferon therapy induces several autoantibodies to multiple organ systems, such as anti-thyroid antibodies, auto-antibodies indicative of autoimmune hepatitis, and anti-platelet autoantibodies [10], and exhibits side effects of developing autoimmune diseases. Autoimmune thrombocytopenia sometimes occurs both during and after interferon therapy [11]. Thus, interferon therapy might have induced autoantibodies to c-mpl in the current patient. We consider that interferon therapy did not play a significant role in the induction of autoantibodies to c-mpl here because the patient s bone marrow had shown a normal number of megakaryocytes for ten years at least, following interferon therapy. The current case provides a new perspective on thrombocytopenia in patients with HCV infection. AAMT with autoantibodies to c-mpl may be one of the causes of thrombocytopenia in these patients. Some patients with HCV infection-associated thrombocytopenia, for whom thrombopoietin receptor agonists have a weak effect, might have this condition. Further investigation will be necessary, especially concerning the relationship between AAMT with autoantibodies to c-mpl and HCV infection. AAMT with autoantibodies to c-mpl can be one of the causes of thrombocytopenia in patients with chronic HCV infection. COMMENTS Case characteristics A 64-year-old woman with hepatitis C virus (HCV) infection and idiopathic thrombocytopenic purpura presented with dyspnea. Clinical diagnosis Liver failure due to chronic hepatitis C September 21, 2017 Volume 23 Issue 35

189 Ichimata S et al. AAMT associated with HCV infection Differential diagnosis Heart failure and renal failure. Laboratory diagnosis Anemia, thrombocytopenia, decreased albumin, elevated bilirubin, liver dysfunction, elevated alpha-fetoprotein. Imaging diagnosis Computed tomography revealed liver cirrhosis with a right lobe mass, bilateral pleural effusions and ascites. Pathological diagnosis Liver cirrhosis, combined hepatocellular-cholangiocarcinoma in the liver and microscopic metastases in both lungs, and acquired amegakaryocytic thrombocytopenia (AAMT) in the bone marrow. Related reports There are a limited number of reports describing AAMT with autoantibodies to thrombopoietin receptor (c-mpl) in patients with systemic lupus erythematosus and systemic sclerosis. Term explanation AAMT is characterized by a marked reduction in the number of bone marrow megakaryocytes and occurs, in part, through autoantibodies to c-mpl. Experiences and lessons In patients with HCV infection, several autoantibodies are produced. Autoantibodies to c-mpl may also be produced and AAMT may occur in patients with HCV infection. Thus, AAMT with autoantibodies to c-mpl may be one of the causes of thrombocytopenia in patients with HCV infection. Peer-review The case record is correctly described and documented. The authors describe the first case of AAMT associated with HCV infection. REFERENCES 1 Weksler BB. Review article: the pathophysiology of thrombocytopenia in hepatitis C virus infection and chronic liver disease. Aliment Pharmacol Ther 2007; 26 Suppl 1: [PMID: DOI: /j x] 2 Hayashi H, Beppu T, Shirabe K, Maehara Y, Baba H. Management of thrombocytopenia due to liver cirrhosis: a review. World J Gastroenterol 2014; 20: [PMID: DOI: /wjg.v20.i ] 3 Agarwal N, Spahr JE, Werner TL, Newton DL, Rodgers GM. Acquired amegakaryocytic thrombocytopenic purpura. Am J Hematol 2006; 81: [PMID: DOI: / ajh.20510] 4 Ballmaier M, Germeshausen M, Schulze H, Cherkaoui K, Lang S, Gaudig A, Krukemeier S, Eilers M, Strauss G, Welte K. c-mpl mutations are the cause of congenital amegakaryocytic thrombocytopenia. Blood 2001; 97: [PMID: ] 5 Kuwana M, Okazaki Y, Kajihara M, Kaburaki J, Miyazaki H, Kawakami Y, Ikeda Y. Autoantibody to c-mpl (thrombopoietin receptor) in systemic lupus erythematosus: relationship to thrombocytopenia with megakaryocytic hypoplasia. Arthritis Rheum 2002; 46: [PMID: DOI: / art.10420] 6 Katsumata Y, Suzuki T, Kuwana M, Hattori Y, Akizuki S, Sugiura H, Matsuoka Y. Anti-c-Mpl (thrombopoietin receptor) autoantibody-induced amegakaryocytic thrombocytopenia in a patient with systemic sclerosis. Arthritis Rheum 2003; 48: [PMID: DOI: /art.10965] 7 Broudy VC, Lin NL, Sabath DF, Papayannopoulou T, Kaushansky K. Human platelets display high-affinity receptors for thrombopoietin. Blood 1997; 89: [PMID: ] 8 Hoffman R, Zaknoen S, Yang HH, Bruno E, LoBuglio AF, Arrowsmith JB, Prchal JT. An antibody cytotoxic to megakaryocyte progenitor cells in a patient with immune thrombocytopenic purpura. N Engl J Med 1985; 312: [PMID: DOI: /NEJM ] 9 Christodoulou D, Katsanos K, Zervou E, Theopistos V, Papathanasopoulos A, Christou L, Tsianos EV. Platelet IgG antibodies are significantly increased in chronic liver disease. Ann Gastroenterol 2011; 24: [PMID: ] 10 Sleijfer S, Bannink M, Van Gool AR, Kruit WH, Stoter G. Side effects of interferon-alpha therapy. Pharm World Sci 2005; 27: [PMID: DOI: /s ] 11 Arena R, Cecinato P, Lisotti A, Buonfiglioli F, Calvanese C, Grande G, Montagnani M, Azzaroli F, Mazzella G. Severe immune thrombocytopenia after peg-interferon-alpha2a, ribavirin and telaprevir treatment completion: A case report and systematic review of literature. World J Hepatol 2015; 7: [PMID: DOI: /wjh.v7.i ] P- Reviewer: Gonzalez-Reimers E, Larrubia JR, Lee HC, Sergi CM S- Editor: Ma YJ L- Editor: A E- Editor: Huang Y 6545 September 21, 2017 Volume 23 Issue 35

190 Submit a Manuscript: DOI: /wjg.v23.i World J Gastroenterol 2017 September 21; 23(35): ISSN (print) ISSN (online) LETTERS TO THE EDITOR Novel endoscopic management of buried bumper syndrome in percutaneous endoscopic gastrostomy: The Olympus HookKnife Laura E Wolpert, Dominic M Summers, Andrew Tsang Laura E Wolpert, Dominic M Summers, Andrew Tsang, Department of General Surgery, Peterborough City Hospital, Edith Cavell Campus, Bretton Gate, Peterborough PE3 9GZ, United Kingdom ORCID number: Laura E Wolpert ( ); Dominic M Summers ( ); Andrew Tsang ( ). Author contributions: Wolpert LE wrote the letter; Summers DM and Tsang A revised the letter. Conflict-of-interest statement: The authors declare no conflict of interest. Open-Access: This article is an open-access article which was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: licenses/by-nc/4.0/ Manuscript source: Unsolicited manuscript Correspondence to: Laura E Wolpert, BM BCh, Doctor, Department of General Surgery, Peterborough City Hospital, Edith Cavell Campus, Bretton Gate, Peterborough PE3 9GZ, United Kingdom. laura@wolperts.com Telephone: Fax: Received: June 16, 2017 Peer-review started: June 19, 2017 First decision: July 17, 2017 Revised: July 31, 2017 Accepted: August 25, 2017 Article in press: August 25, 2017 Published online: September 21, 2017 Abstract Buried bumper syndrome (BBS) is an uncommon but serious complication of percutaneous endoscopic gastrostomy. It involves the internal fixation device, or bumper, migrating into the gastric wall and subsequent mucosal overgrowth. We described a case series of four patients with BBS treated with a novel endoscopic technique using a HookKnife between June 2016 and February The HookKnife is a rotating L-shaped cutting wire designed for hooking tissue and pulling it away from the gastric wall towards the lumen. The technique was successful in all four cases with no complications. Each patient was discharged on the day of treatment. The HookKnife is a manoeuvrable, safe and effective device for endoscopic removal of buried bumpers and could avoid surgery in a high risk group of patients. To our knowledge this technique has not been described previously. We suggest that this technique should be added to the treatment algorithms for managing BBS. Key words: Buried bumper syndrome; Percutaneous endoscopic gastrostomy; HookKnife The Author(s) Published by Baishideng Publishing Group Inc. All rights reserved. Core tip: This letter to the editor describes a case series of four patients who underwent a novel endoscopic 6546 September 21, 2017 Volume 23 Issue 35

191 Wolpert LE et al. Endoscopic management of BBS technique for managing buried bumper syndrome using the Olympus HookKnife. This technique was successful in all four patients and no complications were recorded. We propose that this technique may be a safe and effective treatment for buried bumper syndrome in percutaneous endoscopic gastrostomy feeding. Wolpert LE, Summers DM, Tsang A. Novel endoscopic management of buried bumper syndrome in percutaneous endoscopic gastrostomy: The Olympus HookKnife. World J Gastroenterol 2017; 23(35): Available from: URL: DOI: TO THE EDITOR We enjoyed Cyrany et al [1] article on Buried bumper syndrome : A complication of percutaneous endoscopic gastrostomy. Buried bumper syndrome (BBS) is an uncommon but serious complication of percutaneous endoscopic gastrostomy (PEG) with an incidence of 0.3%-2.4% per PEG patient year [1]. Symptoms include inability to insert the PEG tube, loss of patency and leakage from the PEG. Here, we describe a case series of four patients with BBS treated with a novel endoscopic technique using a HookKnife (Olympus EndoTherapy) (Figure 1). The HookKnife is a rotating L-shaped cutting wire designed for hooking tissue and pulling it away from the gastric wall towards the lumen [2] and intended for use in colonic polypectomies. Other applications have been described in the literature; Oyama et al [3] describe a technique whereby the HookKnife was used to endoscopically resect lymph node metastases of gastric and oesophageal mucosal cancer. Three patients were referred to our centre by their general practitioner due to inflammation around the PEG site and an inability to advance the PEG tube. The fourth patient was referred after their original PEG had stopped functioning and a new PEG had been inserted by the medical team. Patients were selected for treatment following referral for suspected BBS which was then evaluated by endoscopy. The patients ranged in age from 28 to 61 and had PEG placement for multiple sclerosis, Huntington s disease, cerebral palsy and tetraplegia. Two patients underwent the procedure in theatre under general anaesthetic whilst the other two underwent the procedure in endoscopy under sedation. In each case the external PEG tubing was cut short to around 5 cm from the abdominal wall. A 15 mm through-the-scope dilation balloon was passed externally via the PEG tubing and inflated to dilate the mucosal orifice. A mucosal orifice was visible in all the patients in this case series. If a mucosal orifice cannot be clearly visualised then it may be found using the balloon catheter tip inserted externally. If there is no Figure 1 The Olympus HookKnife [2] -A rotating L-shaped cutting wire designed to hook mucosal tissue and pull it towards the lumen. It was originally designed for use in colonic polypectomies. In Oyama et al [3] the HookKnife was deployed to resect lymph node metastases of gastric and oeseophageal mucosal cancers. orifice then the mucosa can be cut over the bumper to create an orifice. After dilating the mucosal orifice using the TTS balloon the HookKnife was deployed to incise the gastric mucosa over the buried bumper by hooking the tissue, pulling it towards the lumen and then cutting it using diathermy under direct vision. The balloon was then re-inflated inside the tubing at the level of the PEG bumper to stiffen the PEG system (Figure 2), and both the tubing and balloon catheter clamped together using artery forceps. Short, but firm, controlled pressure was applied towards the gastric lumen to force the bumper into the stomach. The PEG was then removed using a standard technique. In one case a new PEG had already been placed prior to the procedure. In the other 3 cases, a new PEG was placed during the procedure. There were no complications and all four patients were discharged on the day of treatment. The HookKnife is a manoeuvrable, safe and effective device for endoscopic removal of buried PEG bumpers. There are several benefits of the HookKnife technique. First, compared with techniques that rely on incising the mucosa towards the gastric wall, such as the needle knife technique and papillotome technique discussed in Cyrany et al [1],the HookKnife allows elevation of the gastric mucosa away from the bumper as the mucosal tissue can be hooked using the knife and pulled towards the lumen [2]. This additional level of control allows a more precise incision, more accurate evaluation of the depth of the bumper migration and reduces the risk of inadvertent gastric perforation as the mucosa is cut by pulling it towards the lumen as opposed to cutting towards the gastric wall. Second, the HookKnife could avoid surgery and could therefore be suitable for high risk patients. As this technique can be performed as a day case it avoids the need for an overnight stay in hospital. Finally, none of the patients in this case series experienced bleeding, which was a frequent complication in patients who 6547 September 21, 2017 Volume 23 Issue 35

192 Wolpert LE et al. Endoscopic management of BBS A B C Figure 2 Endoscopic removal of a buried percutaneous endoscopic gastrostomy bumper using the HookKnife technique. A: Visualisation of the buried bumper. The gastric mucosa has overgrown the internal percutaneous endoscopic gastrostomy (PEG) fixation device leaving a small mucosal orifice. A through-thescope balloon can be used to dilate the mucosal orifice; B: View of the Olympus HookKnife being deployed. Radial incisions are made by hooking the gastric mucosa and pulling it towards the lumen; C: A through-the-scope balloon has been inserted externally to stiffen the PEG system. Pressure is applied to the balloon to force the bumper into the lumen. underwent the needle knife technique [1]. Adequate haemostasis was achieved by using the knife on a coagulation setting of watts and by not pulling the hook too quickly. To our knowledge, this technique has not been described previously and we suggest that this technique should be added to the treatment algorithms for the management of BBS [4]. However, as only a small number of cases are included in this series larger numbers of patients may need to be included to establish whether the HookKnife is a safe and effective method of endoscopic removal of buried bumpers. A possible limitation of this method of BBS removal is that it relies on a subjective assessment of the depth that the bumper is buried. REFERENCES 1 Cyrany J, Rejchrt S, Kopacova M, Bures J. Buried bumper syndrome: A complication of percutaneous endoscopic gastrostomy. World J Gastroenterol 2016; 22: [PMID: DOI: /wjg.v22.i2.618] 2 Olympus. HookKnife Electrosurgical Knife. Available from: URL: 3 Oyama T, Kikuchi Y. Aggressive endoscopic mucosal resection in the upper GI tract-hook knife EMR method. Minim Invasive Ther Allied Technol 2002; 11: [PMID: DOI: / ] 4 British Association for Parenteral and Enteral Nutrition. Percutaneous endoscopic gastrostomy management of buried bumper syndrome-decision tree. BAPEN P- Reviewer: Imaeda H, Kawahara H, Kim GH, Konishi H, Trevisani L, Velayos B, Wong K S- Editor: Qi Y L- Editor: A E- Editor: Ma YJ 6548 September 21, 2017 Volume 23 Issue 35

193 Published by Baishideng Publishing Group Inc 7901 Stoneridge Drive, Suite 501, Pleasanton, CA 94588, USA Telephone: Fax: Help Desk: I S S N Baishideng Publishing Group Inc. All rights reserved.