Gastroenterology

ISSN 1007-9327 CN 14-1219/R

World Journal of

Gastroenterology Indexed and Abstracted in: Current Contents®/Clinical Medicine, Science Citation Index Expanded (also known as SciSearch®) and Journal Citation Reports/Science Edition, Index Medicus, MEDLINE and PubMed, Chemical Abstracts, EMBASE/Excerpta Medica, Abstracts Journals, Nature Clinical Practice Gastroenterology and Hepatology, CAB Abstracts and Global Health. ISI JCR 2003-2000 IF: 3.318, 2.532, 1.445 and 0.993.

Volume 14 Number 13 April 7, 2008 World J Gastroenterol 2008 April 7; 14(13): 1969-2132 Online Submissions wjg.wjgnet.com www.wjgnet.com Printed on Acid-free Paper

A Weekly Journal of Gastroenterology and Hepatology

World Journal of

Gastroenterology Editorial Board 2007-2009 http://www.wjgnet.com E-mail: [email protected]

HONORARY EDITORS-IN-CHIEF Montgomery Bissell, San Francisco James L Boyer, New Haven Ke-Ji Chen, Beijing Li-Fang Chou, Taipei Jacques V Dam, Stanford Martin H Floch, New Haven Guadalupe Garcia-Tsao, New Haven Zhi-Qiang Huang, Beijing Shinn-Jang Hwang, Taipei Ira M Jacobson, New York Derek Jewell, Oxford Emmet B Keeffe, Palo Alto Min-Liang Kuo, Taipei Nicholas F LaRusso, Rochester Jie-Shou Li, Nanjing Geng-Tao Liu, Beijing Lein-Ray Mo, Tainan Bo-Rong Pan, Xi'an Fa-Zu Qiu, Wuhan Eamonn M Quigley, Cork David S Rampton, London Rafiq A Sheikh, Sacramento Rudi Schmid, Kentfield[1] Nicholas J Talley, Rochester Guido NJ Tytgat, Amsterdam Hsiu-Po Wang, Taipei Jaw-Ching Wu, Taipei Meng-Chao Wu, Shanghai Ming-Shiang Wu, Taipei Jia-Yu Xu, Shanghai Ta-Sen Yeh, Taoyuan EDITOR-IN-CHIEF Lian-Sheng Ma, Taiyuan ASSOCIATE EDITORS-IN-CHIEF Gianfranco D Alpini, Temple Bruno Annibale, Roma Roger W Chapman, Oxford Chi-Hin Cho, Hong Kong Hugh J Freeman, Vancouver Alexander L Gerbes, Munich Shou-Dong Lee, Taipei Walter E Longo, New Haven You-Yong Lu, Beijing Masao Omata, Tokyo Harry HX Xia, Hanover MEMBERS OF THE EDITORIAL BOARD Albania Bashkim Resuli, Tirana Argentina Julio H Carri, Córdoba Adriana M Torres, Rosario Australia Minoti V Apte, Liverpool Richard B Banati, Lidcombe Michael R Beard, Adelaide Patrick Bertolino, Sydney Filip Braet, Sydney

Andrew D Clouston, Sydney Graham Cooksley, Queensland Darrell HG Crawford, Brisbane Adrian G Cummins, Woodville South Guy D Eslick, Sydney Michael A Fink, Melbourne Robert JL Fraser, Daw Park Mark D Gorrell, Sydney Yik-Hong Ho, Townsville Gerald J Holtmann, Adelaide Michael Horowitz, Adelaide John E Kellow, Sydney Geoffrey W McCaughan, Sydney Finlay A Macrae, Victoria Daniel Markovich, Brisbane Phillip S Oates, Perth Stephen M Riordan, Sydney Ian C Roberts-Thomson, Adelaide Arthur Shulkes, Melbourne Ross C Smith, Sydney Kevin J Spring, Brisbane Nathan Subramaniam, Brisbane Herbert Tilg, Innsbruck Martin J Veysey, Gosford Daniel L Worthley, Bedford Austria Peter Ferenci, Vienna Valentin Fuhrmann, Vienna Alfred Gangl, Vienna Christoph Gasche, Vienna Kurt Lenz, Linz Markus Peck-Radosavljevic, Vienna Rudolf E Stauber, Auenbruggerplatz Michael Trauner, Graz Harald Vogelsang, Vienna Guenter Weiss, Innsbruck Belarus Yury K Marakhouski, Minsk Belgium Rudi Beyaert, Gent Bart Rik De Geest, Leuven Inge I Depoortere, Leuven Olivier Detry, Liège Benedicte Y De Winter, Antwerp Karel Geboes, Leuven Thierry Gustot, Brussels Yves J Horsmans, Brussels Geert G Leroux-Roels, Ghent Louis Libbrecht, Leuven Etienne M Sokal, Brussels Marc Peeters, De Pintelaan Gert A Van Assche, Leuven Yvan Vandenplas, Brussels Eddie Wisse, Keerbergen Brazil Heitor Rosa, Goiania Bulgaria Zahariy Krastev, Sofia www.wjgnet.com

Canada Fernando Alvarez, Québec David Armstrong, Ontario Jeffrey P Baker, Toronto Olivier Barbier, Québec Nancy Baxter, Toronto Matthew Bjerknes, Toronto Frank J Burczynski, Winnipeg Michael F Byrne, Vancouver Wang-Xue Chen, Ottawa Chantal Guillemette, Québec Samuel S Lee, Calgary Gary A Levy, Toronto Andrew L Mason, Alberta John K Marshall, Ontario Donna-Marie McCafferty, Calgary Thomas I Michalak, St. John's Gerald Y Minuk, Manitoba Paul Moayyedi, Hamilton William G Paterson, Kingston Eldon Shaffer, Calgary Morris Sherman, Toronto Martin Storr, Calgary Alan BR Thomson, Edmonton Elena F Verdu, Ontario John L Wallace, Calgary Eric M Yoshida, Vancouver Chile Silvana Zanlungo, Santiago China Henry LY Chan, Hongkong Xiao-Ping Chen, Wuhan Zong-Jie Cui, Beijing Da-Jun Deng, Beijing Er-Dan Dong, Beijing Sheung-Tat Fan, Hong Kong Jin Gu, Beijing De-Wu Han, Taiyuan Ming-Liang He, Hong Kong Wayne HC Hu, Hong Kong Chee-Kin Hui, Hong Kong Ching-Lung Lai, Hong Kong Kam Chuen Lai, Hong Kong James YW Lau, Hong Kong Yuk-Tong Lee, Hong Kong Suet-Yi Leung, Hong Kong Wai-Keung Leung, Hong Kong Chung-Mau Lo, Hong Kong Jing-Yun Ma, Beijing Lun-Xiu Qin, Shanghai Yu-Gang Song, Guangzhou Qin Su, Beijing Wai-Man Wong, Hong Kong Hong Xiao, Beijing Dong-Liang Yang, Wuhan Winnie Yeo, Hong Kong Yuan Yuan, Shenyang Man-Fung Yuen, Hong Kong Jian-Zhong Zhang, Beijing Xin-Xin Zhang, Shanghai Shu Zheng, Hangzhou Croatia Tamara Cacev, Zagreb Marko Duvnjak, Zagreb

I

Cuba Damian C Rodriguez, Havana Czech Milan Jirsa, Praha Denmark Peter Bytzer, Copenhagen Asbjørn M Drewes, Aalborg Hans Gregersen, Aalborg Jens H Henriksen, Hvidovre Claus P Hovendal, Odense Fin S Larsen, Copenhagen Søren Møller, Hvidovre Egypt Abdel-Rahman El-Zayadi, Giza Amr M Helmy, Cairo Sanaa M Kamal, Cairo Ayman Yosry, Cairo Estonia Riina Salupere, Tartu Finland Irma E Jarvela, Helsinki Katri M Kaukinen, Tampere Minna Nyström, Helsinki Pentti Sipponen, Espoo France Bettaieb Ali, Dijon Corlu Anne, Rennes Denis Ardid, Clermont-Ferrand Charles P Balabaud, Bordeaux Soumeya Bekri, Rouen Jacques Belghiti, Clichy Pierre Brissot, Rennes Patrice P Cacoub, Paris Franck Carbonnel, Besancon Laurent Castera, Pessac Bruno Clément, Rennes Benoit Coffin, Colombes Jacques Cosnes, Paris Thomas Decaens, Cedex Francoise L Fabiani, Angers Gérard Feldmann, Paris Jean Fioramonti, Toulouse Jean-Paul Galmiche, Nantes Catherine Guettier, Villejuif Chantal Housset, Paris Juan L Iovanna, Marseille Rene Lambert, Lyon Philippe Mathurin, Lille Patrick Marcellin, Paris Tamara Matysiak–Budnik, Paris Francis Mégraud, Bordeaux Richard Moreau, Clichy Thierry Piche, Nice Raoul Poupon, Paris Jean Rosenbaum, Bordeaux Jose Sahel, Marseille Jean-Philippe Salier, Rouen Jean-Yves Scoazec, Lyon Khalid A Tazi, Clichy Emmanuel Tiret, Paris Baumert F Thomas, Strasbourg Marie-Catherine Vozenin-brotons, Villejuif Jean-Pierre H Zarski, Grenoble Jessica Zucman-Rossi, Paris Germany Hans-Dieter Allescher, Garmisch-Partenkirchen Martin Anlauf, Kiel Rudolf Arnold, Marburg Max G Bachem, Ulm Thomas F Baumert, Freiburg Daniel C Baumgart, Berlin Hubert Blum, Freiburg Thomas Bock, Tuebingen

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Katja Breitkopf, Mannheim Dunja Bruder, Braunschweig Markus W Büchler, Heidelberg Christa Buechler, Regensburg Reinhard Buettner, Bonn Elke Cario, Essen Uta Dahmen, Essen Christoph F Dietrich, Bad Mergentheim Rainer J Duchmann, Berlin Volker F Eckardt, Wiesbaden Paul Enck, Tuebingen Fred Fändrich, Kiel Ulrich R Fölsch, Kiel Helmut Friess, Heidelberg Peter R Galle, Mainz Nikolaus Gassler, Aachen Andreas Geier, Aachen Markus Gerhard, Munich Dieter Glebe, Giessen Burkhard Göke, Munich Florian Graepler, Tuebingen Axel M Gressner, Aachen Veit Gülberg, Munich Rainer Haas, Munich Eckhart G Hahn, Erlangen Stephan Hellmig, Kiel Martin Hennenberg, Bonn Johannes Herkel, Hamburg Klaus R Herrlinger, Stuttgart Eberhard Hildt, Berlin Joerg C Hoffmann, Berlin Ferdinand Hofstaedter, Regensburg Werner Hohenberger, Erlangen Jörg C Kalff, Bonn Ralf Jakobs, Ludwigshafen Jutta Keller, Hamburg Andrej Khandoga, Munich Sibylle Koletzko, München Stefan Kubicka, Hannover Joachim Labenz, Siegen Frank Lammert, Bonn Thomas Langmann, Regensburg Christian Liedtke, Aachen Matthias Löhr, Mannheim Christian Maaser, Muenster Ahmed Madisch, Dresden Peter Malfertheiner, Magdeburg Michael P Manns, Hannover Helmut Messmann, Augsburg Stephan Miehlke, Dresden Sabine Mihm, Göttingen Silvio Nadalin, Essen Markus F Neurath, Mainz Johann Ockenga, Berlin Florian Obermeier, Regensburg Gustav Paumgartner, Munich Ulrich KS Peitz, Magdeburg Markus Reiser, Bochum Emil C Reisinger, Rostock Steffen Rickes, Magdeburg Tilman Sauerbruch, Bonn Dieter Saur, Munich Hans Scherubl, Berlin Joerg Schirra, Munich Roland M Schmid, München Volker Schmitz, Bonn Andreas G Schreyer, Regensburg Tobias Schroeder, Essen Hans Seifert, Oldenburg Manfred V Singer, Mannheim Gisela Sparmann, Rostock Jurgen M Stein, Frankfurt Ulrike S Stein, Berlin Manfred Stolte, Bayreuth Christian P Strassburg, Hannover Wolfgang R Stremmel, Heidelberg Harald F Teutsch, Ulm Robert Thimme, Freiburg Hans L Tillmann, Leipzig Tung-Yu Tsui, Regensburg Axel Ulsenheimer, Munich Patrick Veit-Haibach, Essen Claudia Veltkamp, Heidelberg Siegfried Wagner, Deggendorf Henning Walczak, Heidelberg www.wjgnet.com

Fritz von Weizsacker, Berlin Jens Werner, Heidelberg Bertram Wiedenmann, Berlin Reiner Wiest, Regensburg Stefan Wirth, Wuppertal Stefan JP Zeuzem, Homburg Greece Christos Dervenis, Athens Elias A Kouroumalis, Heraklion Ioannis E Koutroubakis, Heraklion Spiros Sgouros, Athens Hungary Peter L Lakatos, Budapest Zsuzsa Szondy, Debrecen Iceland Hallgrimur Gudjonsson, Reykjavik India Philip Abraham, Mumbai Kunissery A Balasubramanian, Vellore Sujit K Bhattacharya, Kolkata Yogesh K Chawla, Chandigarh Radha K Dhiman, Chandigarh Kalpesh Jani, Vadodara Sri Prakash Misra, Allahabad Nageshwar D Reddy, Hyderabad Iran Seyed-Moayed Alavian, Tehran Reza Malekzadeh, Tehran Seyed A Taghavi, Shiraz Ireland Billy Bourke, Dublin Ronan A Cahill, Cork Anthony P Moran, Galway Israel Simon Bar-Meir, Hashomer Abraham R Eliakim, Haifa Yaron Ilan, Jerusalem Avidan U Neumann, Ramat-Gan Yaron Niv, Pardesia Ran Oren, Tel Aviv Ami D Sperber, Beer-Sheva Italy Giovanni Addolorato, Roma Luigi E Adinolfi, Naples Domenico Alvaro, Rome Vito Annese, San Giovanni Rotond Adolfo F Attili, Roma Giovanni Barbara, Bologna Gabrio Bassotti, Perugia Pier M Battezzati, Milan Stefano Bellentani, Carpi Antomio Benedetti, Ancona Mauro Bernardi, Bologna Livia Biancone, Rome Luigi Bonavina, Milano Flavia Bortolotti, Padova Giuseppe Brisinda, Rome Giovanni Cammarota, Roma Antonino Cavallari, Bologna Giuseppe Chiarioni, Valeggio Michele Cicala, Rome Amedeo Columbano, Cagliari Massimo Conio, Sanremo Dario Conte, Milano Gino R Corazza, Pavia Francesco Costa, Pisa Antonio Craxi, Palermo Silvio Danese, Milan Roberto De Giorgio, Bologna Giovanni D De Palma, Naples Fabio Farinati, Padua

Giammarco Fava, Ancona Francesco Feo, Sassari Stefano Fiorucci, Perugia Andrea Galli, Firenze Valeria Ghisett, Turin Gianluigi Giannelli, Bari Edoardo G Giannini, Genoa Paolo Gionchetti, Bologna Mario Guslandi, Milano Pietro Invernizzi, Milan Giacomo Laffi, Firenze Giovanni Maconi, Milan Lucia Malaguarnera, Catania Emanuele D Mangoni, Napoli Paolo Manzoni, Torino Giulio Marchesini, Bologna Fabio Marra, Florence Marco Marzioni, Ancona Giuseppe Montalto, Palermo Giovanni Monteleone, Rome Giovanni Musso, Torino Gerardo Nardone, Napoli Valerio Nobili, Rome Luisi Pagliaro, Palermo Francesco Pallone, Rome Fabrizio R Parente, Milan Francesco Perri, San Giovanni Rotondo Raffaele Pezzilli, Bologna Alberto Pilotto, San Giovanni Rotondo Mario Pirisi, Novara Anna C Piscaglia, Roma Paolo Del Poggio, Treviglio Gabriele B Porro, Milano Piero Portincasa, Bari Bernardino Rampone, Siena Cosimo Prantera, Roma Claudio Romano, Messina Marco Romano, Napoli Gerardo Rosati, Potenza Mario Del Tacca, Pisa Pier A Testoni, Milan Enrico Roda, Bologna Domenico Sansonno, Bari Vincenzo Savarino, Genova Vincenzo Stanghellini, Bologna Giovanni Tarantino, Naples Roberto Testa, Genoa Dino Vaira, Bologna Japan Kyoichi Adachi, Izumo Yasushi Adachi, Sapporo Taiji Akamatsu, Matsumoto Sk Md Fazle Akbar, Ehime Takafumi Ando, Nagoya Akira Andoh, Otsu Taku Aoki, Tokyo Masahiro Arai, Tokyo Tetsuo Arakawa, Osaka Yasuji Arase, Tokyo Masahiro Asaka, Sapporo Hitoshi Asakura, Tokyo Takeshi Azuma, Fukui Yoichi Chida, Fukuoka Takahiro Fujimori, Tochigi Jiro Fujimoto, Hyogo Kazuma Fujimoto, Saga Mitsuhiro Fujishiro, Tokyo Yoshihide Fujiyama, Otsu Hirokazu Fukui, Tochigi Hiroyuki Hanai, Hamamatsu Kazuhiro Hanazaki, Kochi Naohiko Harada, Fukuoka Makoto Hashizume, Fukuoka Tetsuo Hayakawa, Nagoya Kazuhide Higuchi, Osaka Keisuke Hino, Ube Keiji Hirata, Kitakyushu Yuji Iimuro, Nishinomiya Kenji Ikeda, Tokyo Fumio Imazeki, Chiba Yutaka Inagaki, Kanagawa Yasuhiro Inokuchi, Yokohama Haruhiro Inoue, Yokohama Masayasu Inoue, Osaka

Akio Inui, Kagoshima Hiromi Ishibashi, Nagasaki Shunji Ishihara, Izumo Toru Ishikawa, Niigata Kei Ito, Sendai Masayoshi Ito, Tokyo Hiroaki Itoh, Akita Ryuichi Iwakiri, Saga Yoshiaki Iwasaki, Okayama Terumi Kamisawa, Tokyo Hiroshi Kaneko, Aichi-Gun Shuichi Kaneko, Kanazawa Takashi Kanematsu, Nagasaki Mitsuo Katano, Fukuoka Junji Kato, Sapporo Mototsugu Kato, Sapporo Shinzo Kato, Tokyo Norifumi Kawada, Osaka Sunao Kawano, Osaka Mitsuhiro Kida, Kanagawa Yoshikazu Kinoshita, Izumo Tsuneo Kitamura, Chiba Seigo Kitano, Oita Kazuhiko Koike, Tokyo Norihiro Kokudo, Tokyo Satoshi Kondo, Sapporo Shoji Kubo, Osaka Masato Kusunoki, Tsu Mie Shigeki Kuriyama, Kagawa[2] Katsunori Iijima, Sendai Shin Maeda, Tokyo Masatoshi Makuuchi, Tokyo Osamu Matsui, Kanazawa Yasuhiro Matsumura, Chiba Yasushi Matsuzaki, Tsukuba Kiyoshi Migita, Omura Tetsuya Mine, Kanagawa Hiroto Miwa, Hyogo Masashi Mizokami, Nagoya Yoshiaki Mizuguchi, Tokyo Motowo Mizuno, Hiroshima Morito Monden, Suita Hisataka S Moriwaki, Gifu Yasuaki Motomura, Iizuka Yoshiharu Motoo, Kanazawa Kazunari Murakami, Oita Kunihiko Murase, Tusima Masahito Nagaki, Gifu Masaki Nagaya, Kawasaki Yujl Naito, Kyoto Hisato Nakajima, Tokyo Hiroki Nakamura, Yamaguchi Shotaro Nakamura, Fukuoka Mikio Nishioka, Niihama Shuji Nomoto, Nagoya Susumu Ohmada, Maebashi Masayuki Ohta, Oita Tetsuo Ohta, Kanazawa Kazuichi Okazaki, Osaka Katsuhisa Omagari, Nagasaki Saburo Onishi, Nankoku Morikazu Onji, Ehime Satoshi Osawa, Hamamatsu Masanobu Oshima, Kanazawa Hiromitsu Saisho, Chiba Hidetsugu Saito, Tokyo Yutaka Saito, Tokyo Isao Sakaida, Yamaguchi Michiie Sakamoto, Tokyo Yasushi Sano, Chiba Hiroki Sasaki, Tokyo Iwao Sasaki, Sendai Motoko Sasaki, Kanazawa Chifumi Sato, Tokyo Shuichi Seki, Osaka Hiroshi Shimada, Yokohama Mitsuo Shimada, Tokushima Tomohiko Shimatan, Hiroshima Hiroaki Shimizu, Chiba Ichiro Shimizu, Tokushima Yukihiro Shimizu, Kyoto Shinji Shimoda, Fukuoka Tooru Shimosegawa, Sendai Tadashi Shimoyama, Hirosaki Ken Shirabe, Iizuka www.wjgnet.com

Yoshio Shirai, Niigata Katsuya Shiraki, Mie Yasushi Shiratori, Okayama Masayuki Sho, Nara Yasuhiko Sugawara, Tokyo Hidekazu Suzuki, Tokyo Minoru Tada, Tokyo Tadatoshi Takayama, Tokyo Tadashi Takeda, Osaka Koji Takeuchi, Kyoto Kiichi Tamada, Tochigi Akira Tanaka, Kyoto Eiji Tanaka, Matsumoto Noriaki Tanaka, Okayama Shinji Tanaka, Hiroshima Wei Tang, Tokyo Hideki Taniguchi, Yokohama Kyuichi Tanikawa, Kurume Akira Terano, Shimotsugagun Hitoshi Togash, Yamagata Kazunari Tominaga, Osaka Takuji Torimura, Fukuoka Minoru Toyota, Sapporo Akihito Tsubota, Chiba Shingo Tsuji, Osaka Takato Ueno, Kurume Naomi Uemura, Tokyo Shinichi Wada, Tochigi Hiroyuki Watanabe, Kanazawa Toshio Watanabe, Osaka Yuji Watanabe, Ehime Toshiaki Watanabe, Tokyo Chun-Yang Wen, Nagasaki Koji Yamaguchi, Fukuoka Takayuki Yamamoto, Yokkaichi Takashi Yao, Fukuoka Masashi Yoneda, Tochigi Hiroshi Yoshida, Tokyo Masashi Yoshida, Tokyo Norimasa Yoshida, Kyoto Kentaro Yoshika, Toyoake Masahide Yoshikawa, Kashihara Lebanon Bassam N Abboud, Beirut Ala I Sharara, Beirut Joseph D Boujaoude, Beirut Lithuania Limas Kupcinskas, Kaunas Macedonia Vladimir C Serafimoski, Skopje Malaysia Andrew Seng Boon Chua, Ipoh Khean-Lee Goh, Kuala Lumpur Jayaram Menon, Sabah Mexico Diego Garcia-Compean, Monterrey Eduardo R Marin-Lopez, Jesús García Saúl Villa-Treviño, México Jesus K Yamamoto-Furusho, México Monaco Patrick Rampal, Monaco Morocco Abdellah Essaid, Rabat Netherlands Ulrich Beuers, Amsterdam Gerd Bouma, Amsterdam Lee Bouwman, Leiden J Bart A Crusius, Amsterdam Janine K Kruit, Groningen Ernst J Kuipers, Rotterdam CBHW Lamers, Leiden Ton Lisman, Utrecht

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Yi Liu, Amsterdam Servaas Morré, Amsterdam Chris JJ Mulder, Amsterdam Michael Müller, Wageningen Amado S Peña, Amsterdam Robert J Porte, Groningen Ingrid B Renes, Rotterdam Andreas Smout, Utrecht Reinhold W Stockbrugger, Maastricht Luc JW van der Laan, Rotterdam Karel van Erpecum, Utrecht Gerard P VanBerge-Henegouwen,Utrecht New Zealand Ian D Wallace, Auckland Nigeria Samuel B Olaleye, Ibadan Norway Trond Berg, Oslo Tom H Karlsen, Oslo Helge L Waldum, Trondheim Pakistan Muhammad S Khokhar, Lahore Syed MW Jafri, Karachi Peru Hector H Garcia, Lima Poland Tomasz Brzozowski, Cracow Robert Flisiak, Bialystok Hanna Gregorek, Warsaw Dariusz M Lebensztejn, Bialystok Wojciech G Polak, Wroclaw Marek Hartleb, Katowice Portugal Rodrigues MP Cecília, Lisbon Miguel C De Moura, Lisbon Russia Vladimir T Ivashkin, Moscow Leonid Lazebnik, Moscow Vasiliy I Reshetnyak, Moscow Saudi Arabia Ibrahim A Al Mofleh, Riyadh Serbia Dusan M Jovanovic, Sremska Kamenica

Spain Juan G Abraldes, Barcelona Agustin Albillos, Madrid Raul J Andrade, Málaga Luis Aparisi, Valencia Fernando Azpiroz, Barcelona Ramon Bataller, Barcelona Josep M Bordas, Barcelona Xavier Calvet, Sabadell Andres Cardenas, Barcelona Vicente Carreño, Madrid Jose Castellote, Barcelona Antoni Castells, Barcelona Vicente Felipo, Valencia Juan C Garcia-Pagán, Barcelona Jaime B Genover, Barcelona Javier P Gisbert, Madrid Jaime Guardia, Barcelona Mercedes Fernandez, Barcelona Angel Lanas, Zaragoza María IT López, Jaén José M Mato, Derio Juan F Medina, Pamplona Miguel A Muñoz-Navas, Pamplona Julian Panes, Barcelona Miguel M Perez, Valencia Miguel Perez-Mateo, Alicante Josep M Pique, Barcelona Jesús M Prieto, Pamplona Sabino Riestra, Pola De Siero Luis Rodrigo, Oviedo Manuel Romero-Gómez, Sevilla Sweden Einar S Björnsson, Gothenburg Curt Einarsson, Huddinge Per M Hellström, Stockholm Ulf Hindorf, Lund Hanns-Ulrich Marschall, Stockholm Lars C Olbe, Molndal Lars A Pahlman, Uppsala Matti Sallberg, Stockholm Magnus Simrén, Göteborg Xiao-Feng Sun, Linköping Ervin Tóth, Malmö Weimin Ye, Stockholm Christer S von Holstein, Lund

South Africa Michael C Kew, Parktown

Switzerland Chrish Beglinger, Basel Pierre A Clavien, Zurich Jean-Francois Dufour, Bern Franco Fortunato, Zürich Jean L Frossard, Geneva Gerd A Kullak-Ublick, Zurich Pierre Michetti, Lausanne Francesco Negro, Genève Bruno Stieger, Zurich Radu Tutuian, Zurich Stephan R Vavricka, Zurich Gerhard Rogler, Zurich Arthur Zimmermann, Berne

South Korea Byung Ihn Choi, Seoul Ho Soon Choi, Seoul Marie Yeo, Suwon Sun Pyo Hong, Gyeonggi-do Jae J Kim, Seoul Jin-Hong Kim, Suwon Myung-Hwan Kim, Seoul Chang Hong Lee, Seoul

Turkey Yusuf Bayraktar, Ankara Figen Gurakan, Ankara Aydin Karabacakoglu, Konya Serdar Karakose, Konya Hizir Kurtel, Istanbul Osman C Ozdogan, Istanbul Özlem Yilmaz, Izmir Cihan Yurdaydin, Ankara

Singapore Bow Ho, Singapore Khek-Yu Ho, Singapore Francis Seow-Choen, Singapore Slovakia Anton Vavrecka, Bratislava Slovenia Sasa Markovic, Ljubljana

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Jong Kyun Lee, Seoul Eun-Yi Moon, Seoul Jae-Gahb Park, Seoul Dong Wan Seo, Seoul Dong Jin Suh, Seoul

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United Arab Emirates Sherif M Karam, Al-Ain United Kingdom David H Adams, Birmingham Navneet K Ahluwalia, Stockport Ahmed Alzaraa, Manchester Charalambos G Antoniades, London Anthony TR Axon, Leeds Qasim Aziz, Manchester Nicholas M Barnes, Birmingham Jim D Bell, London Mairi Brittan, London Alastair D Burt, Newcastle Simon S Campbell, Manchester Simon R Carding, Leeds Paul J Ciclitira, London Eithne Costello, Liverpool Tatjana Crnogorac-Jurcevic, London Amar P Dhillon, London William Dickey, Londonderry Emad M El-Omar, Aberdeen Annette Fristscher-Ravens, London Elizabeth Furrie, Dundee Daniel R Gaya, Edinburgh Subrata Ghosh, London William Greenhalf, Liverpool Indra N Guha, Southampton Peter C Hayes, Edinburgh Gwo-Tzer Ho, Edinburgh Anthony R Hobson, Salford Stefan G Hübscher, Birmingham Robin Hughes, London Pali Hungin, Stockton David P Hurlstone, Sheffield Rajiv Jalan, London Janusz AZ Jankowski, Oxford Brian T Johnston, Belfast David EJ Jones, Newcastle Roger Jones, London Michael A Kamm, Harrow Peter Karayiannis, London Laurens Kruidenier, Harlow Patricia F Lalor, Birmingham Hong-Xiang Liu, Cambridge Kenneth E L McColl, Glasgow Stuart AC McDonald, London Dermot P Mcgovern, Oxford Giorgina Mieli-Vergani, London Nikolai V Naoumov, London John P Neoptolemos, Liverpool James Neuberger, Birmingham Mark S Pearce, Newcastle Upon Tyne Stephen P Pereira, London D Mark Pritchard, Liverpool Stephen E Roberts, Swansea Marco Senzolo, Padova Soraya Shirazi-Beechey, Liverpool Robert Sutton, Liverpool Simon D Taylor-Robinson, London Paris P Tekkis, London Ulrich Thalheimer, London Nick P Thompson, Newcastle David Tosh, Bath Frank I Tovey, London Chris Tselepis, Birmingham Diego Vergani, London Geoffrey Warhurst, Salford Peter J Whorwell, Manchester Roger Williams, London Karen L Wright, Bath Min Zhao, Foresterhill United States Gary A Abrams, Birmingham Maria T Abreu, New York Reid B Adams, Virginia Golo Ahlenstiel, Bethesda BS Anand, Houston Frank A Anania, Atlanta Meenakshisundaram Ananthanarayanan, New York Gavin E Arteel, Louisville

Jasmohan S Bajaj, Milwaukee Subhas Banerjee, Palo Alto Peter A Banks, Boston Jamie S Barkin, Miami Kim E Barrett, San Diego Marc D Basson, Detroit Wallace F Berman, Durham Timothy R Billiar, Pittsburgh Edmund J Bini, New York Jennifer D Black, Buffalo Herbert L Bonkovsky, Charlotte Andrea D Branch, New York Robert S Bresalier, Houston Alan L Buchman, Chicago Ronald W Busuttil, Los Angeles Alan Cahill, Philadelphia John M Carethers, San Diego David L Carr-Locke, Boston Maurice A Cerulli, New York Ravi S Chari, Nashville Jiande Chen, Galveston Xian-Ming Chen, Omaha Ramsey Chi-man Cheung, Palo Alto William D Chey, Ann Arbor John Y Chiang, Rootstown Parimal Chowdhury, Arkansas Raymond T Chung, Boston James M Church, Cleveland Ram Chuttani, Boston Mark G Clemens, Charlotte Vincent Coghlan, Beaverton David Cronin II, New Haven John Cuppoletti, Cincinnati Mark J Czaja, New York Peter V Danenberg, Los Angeles Kiron M Das, New Brunswick Conor P Delaney, Cleveland Sharon DeMorrow, Temple Deborah L Diamond, Seattle Peter Draganov, Florida Douglas A Drossman, Chapel Hill Katerina Dvorak, Tucson Bijan Eghtesad, Cleveland Hala El-Zimaity, Houston Michelle Embree-Ku, Providence Alessio Fasano, Baltimore Ronnie Fass, Tucson Mark A Feitelson, Philadelphia Ariel E Feldstein, Cleveland Alessandro Fichera, Chicago Robert L Fine, New York Chris E Forsmark, Gainesville Chandrashekhar R Gandhi, Pittsburgh Susan L Gearhart, Baltimore Xupeng Ge, Boston John P Geibel, New Haven Xin Geng, New Brunswick Jean-Francois Geschwind, Baltimore Ignacio Gil-Bazo, New York Shannon S Glaser, Temple Ajay Goel, Dallas Richard M Green, Chicago Julia B Greer, Pittsburgh James H Grendell, New York David R Gretch, Seattle Stefano Guandalini, Chicago Anna S Gukovskaya, Los Angeles Sanjeev Gupta, Bronx David J Hackam, Pittsburgh Stephen B Hanauer, Chicago Gavin Harewood, Rochester Margaret M Heitkemper, Washington Alan W Hemming, Gainesville Samuel B Ho, San Diego Colin W Howden, Chicago Hongjin Huang, Alameda Jamal A Ibdah, Columbia Atif Iqbal, Omaha Hajime Isomoto, Rochester Hartmut Jaeschke, Tucson Dennis M Jensen, Los Angeles Leonard R Johnson, Memphis Michael P Jones, Chicago Peter J Kahrilas, Chicago

Anthony N Kalloo, Baltimore Marshall M Kaplan, Boston Neil Kaplowitz, Los Angeles Serhan Karvar, Los Angeles Rashmi Kaul, Tulsa Jonathan D Kaunitz, Los Angeles Ali Keshavarzian, Chicago Miran Kim, Providence Joseph B Kirsner, Chicago Leonidas G Koniaris, Miami Burton I Korelitz, New York Robert J Korst, New York Richard A Kozarek, Seattle Michael Kremer, Chapel Hill Shiu-Ming Kuo, Buffalo Paul Y Kwo, Indianapolis Daryl Tan Yeung Lau, Galvesto Stephen J Lanspa, Omaha Joel E Lavine, San Diego Dirk J van Leeuwen, Lebanon Glen A Lehman, Indianapolis Alex B Lentsch, Cincinnati Andreas Leodolter, La Jolla Gene LeSage, Houston Cynthia Levy, Gainesville Ming Li, New Orleans Zhiping Li, Baltimore Lenard M Lichtenberger, Houston Gary R Lichtenstein, Philadelphia Otto Schiueh-Tzang Lin, Seattle Martin Lipkin, New York Edward V Loftus, Rocheste Robin G Lorenz, Birmingham Michael R Lucey, Madison James D Luketich, Pittsburgh Henry T Lynch, Omaha Patrick M Lynch, Houston John S Macdonald, New York Bruce V MacFadyen, Augusta Willis C Maddrey, Dallas Ashok Malani, Los Angeles Peter J Mannon, Bethesda Charles M Mansbach, Tennessee John F Di Mari, Texas John M Mariadason, Bronx Jorge A Marrero, Ann Arbor Paul Martin, New York Wendy M Mars, Pittsburgh Laura E Matarese, Pittsburgh Lynne V McFarland, Washington Kevin McGrath, Pittsburgh Harihara Mehendale, Monroe Stephan Menne, New York Howard Mertz, Nashville George W Meyer, Sacramento George Michalopoulos, Pittsburgh James M Millis, Chicago Fabrizio Michelassi, New York Albert D Min, New York Pramod K Mistry, New Haven Smruti R Mohanty, Chicago Satdarshan S Monga, Pittsburgh Timothy H Moran, Baltimore Steven F Moss, Providence Andrew J Muir, Durham Milton G Mutchnick, Detroit Masaki Nagaya, Boston Victor Navarro, Philadelphia Laura E Nagy, Cleveland Hiroshi Nakagawa, Philadelphia Douglas B Nelson, Minneapolis Patrick G Northup, Charlottesville Brant K Oelschlager, Washington Curtis T Okamoto, Los Angeles Stephen JD O’Keefe, Pittsburgh Dimitry Oleynikov, Omaha Natalia A Osna, Omaha Stephen J Pandol, Los Angeles Pankaj J Pasricha, Galveston Zhiheng Pei, New York Michael A Pezzone, Pittsburgh CS Pitchumoni, New Brunswiuc Paul J Pockros, La Jolla Jay Pravda, Gainesville Massimo Raimondo, Jacksonville www.wjgnet.com

GS Raju, Galveston Murray B Resnick, Providence Adrian Reuben, Charleston Douglas K Rex, Indianapolis Victor E Reyes, Galveston Basil Rigas, New York Richard A Rippe, Chapel Hill Marcos Rojkind, Washington Philip Rosenthal, San Francisco Hemant K Roy, Evanston Shawn D Safford, Norfolk Bruce E Sands, Boston James M Scheiman, Ann Arbor Eugene R Schiff, Miami Nicholas J Shaheen, Chapel Hill Vanessa M Shami, Charlottesville Prateek Sharma, Kansas City Harvey L Sharp, Minneapolis Stuart Sherman, Indianapolis Shivendra Shukla, Columbia Alphonse E Sirica, Virginia Shanthi V Sitaraman, Atlanta Stuart J Spechler, Dallas Shanthi Srinivasan, Atlanta Michael Steer, Boston Peter D Stevens, New York Gary D Stoner, Columbus Liping Su, Chicago Christina Surawicz, Seattle Ned Snyder, Galveston Robert W Summers, Iowa City Gyongyi Szabo, Worcester Yvette Taché, Los Angeles Seng-Lai Tan, Seattle Andrzej S Tarnawski, Orange K-M Tchou-Wong, New York Neil D Theise, New York Christopher C Thompson, Boston Paul J Thuluvath, Baltimore Swan N Thung, New York Natalie J Torok, Sacramento RA Travagli, Baton Rouge George Triadafilopoulos, Stanford James F Trotter, Denver Chung-Jyi Tsai, Lexington Andrew Ukleja, Florida Michael F Vaezi, Nashville Hugo E Vargas, Scottsdale Arnold Wald, Wisconsin Scott A Waldman, Philadelphia Jian-Ying Wang, Baltimore Timothy C Wang, New York Irving Waxman, Chicago Steven A Weinman, Galveston Steven D Wexner, Weston Keith T Wilson, Baltimore Jacqueline L Wolf, Boston Jackie Wood, Ohio George Y Wu, Farmington Jian Wu, Sacramento Samuel Wyllie, Houston Wen Xie, Pittsburgh Vijay Yajnik, Boston Yoshio Yamaoka, Houston Vincent W Yang, Atlanta Francis Y Yao, San Francisco Hal F Yee, San Francisco Min You, Tampa Zobair M Younossi, Virginia Liqing Yu, Winston-Salem David Yule, Rochester Ruben Zamora, Pittsburgh Michael E Zenilman, New York Zhi Zhong, Chapel Hill Stephen D Zucker, Cincinnati Uruguay Henry Cohen, Montevideo Javier S Martin, Punta del Este Passed away on October 20, 2007 Passed away on June 11, 2007 Total of 1050 editorial members from 60 countries world wide have been active in peer review and editing

[1] [2]

Ⅴ

World Journal of ®

Gastroenterology Weekly Established in October 1995 National Journal Award 2005

Volume 14 Number 13 April 7, 2008

™©

Contents EDITORIAL

1969

Wireless capsule endoscopy Mata A, Llach J, Bordas JM

1972

Current view of the immunopathogenesis in inflammatory bowel disease and its implications for therapy Torres MI, Ríos A

REVIEW

1981

Role of endoscopy in the management of acute diverticular bleeding Philichos C, Bobotis E

TOPIC HIGHLIGHT

1984

Endoscopic mucosal resection in the upper gastrointestinal tract Ahmadi A, Draganov P

GASTRIC CANCER

1990

Relationship between cell adhesion molecules expression and the biological behavior of gastric carcinoma Chu YQ, Ye ZY, Tao HQ, Wang YY, Zhao ZS

LIVER CANCER

1997

Long-term outcome of percutaneous ethanol injection therapy for minimum-sized hepatocellular carcinoma Taniguchi M, Kim SR, Imoto S, Ikawa H, Ando K, Mita K, Fuki S, Sasase N, Matsuoka T, Kudo M, Hayashi Y

2003

Anti-cancer and anti-angiogenic effects of curcumin and tetrahydrocurcumin on implanted hepatocellular carcinoma in nude mice Yoysungnoen P, Wirachwong P, Changtam C, Suksamrarn A, Patumraj S

VIRAL HEPATITIS

2010

Serial changes in expression of functionally clustered genes in progression of liver fibrosis in hepatitis C patients Takahara Y, Takahashi M, Zhang QW, Wagatsuma H, Mori M, Tamori A, Shiomi S, Nishiguchi S

BASIC RESEARCH

2023

Pancreatic stellate cells promote proliferation and invasiveness of human pancreatic cancer cells via galectin-3 Jiang HB, Xu M, Wang XP

CLINICAL RESEARCH

2029

Probiotic intervention has strain-specific anti-inflammatory effects in healthy adults Kekkonen RA, Lummela N, Karjalainen H, Latvala S, Tynkkynen S, Järvenpää S, Kautiainen H, Julkunen I, Vapaatalo H, Korpela R

RAPID COMMUNICATION 2037

A combination therapy of ethanol injection and radiofrequency ablation under general anesthesia for the treatment of hepatocellular carcinoma Kurokohchi K, Watanabe S, Yoneyama H, Deguchi A, Masaki T, Himoto T, Miyoshi H, Mohammad HS, Kitanaka A, Taminato T, Kuriyama S www.wjgnet.com

World Journal of Gastroenterology

Contents

Volume 14 Number 13 April 7, 2008 2044

Serum type Ⅳ collagen level is predictive for esophageal varices in patients with severe alcoholic disease Mamori S, Searashi Y, Matsushima M, Hashimoto K, Uetake S, Matsudaira H, Ito S, Nakajima H, Tajiri H

2049

Early effects of Lansoprazole orally disintegrating tablets on intragastric pH in CYP2C19 extensive metabolizers Yamagishi H, Koike T, Ohara S, Horii T, Kikuchi R, Kobayashi S, Abe Y, Iijima K, Imatani A, Suzuki K, Hishinuma T, Goto J, Shimosegawa T

2055

p16 promoter hypermethylation: A useful serum marker for early detection of gastric cancer Abbaszadegan MR, Moaven O, Sima HR, Ghafarzadegan K, A'rabi A, Forghani MN, Raziee HR, Mashhadinejad A, Jafarzadeh M, Esmaili-Shandiz E, Dadkhah E

2061

Prospective evaluation of small bowel preparation with bisacodyl and sodium phosphate for capsule endoscopy Franke A, Hummel F, Knebel P, Antoni C, Böcker U, Singer MV, Löhr M

2065

Effect of Prometheus liver assist system on systemic hemodynamics in patients with cirrhosis: A randomized controlled trial study Dethloff T, Tofteng F, Frederiksen HJ, Hojskov M, Hansen BA, Larsen FS

2072

Ultrasonography in differentiation between chronic viral hepatitis and compensated early stage cirrhosis Iliopoulos P, Vlychou M, Karatza C, Yarmenitis SD, Repanti M, Tsamis I, Tepetes K

2080

Endoscopic band ligation and endoscopic hemoclip placement for patients with Mallory-Weiss syndrome and active bleeding Cho YS, Chae HS, Kim HK, Kim JS, Kim BW, Kim SS, Han SW, Choi KY

2085

Effects of honey as a scolicidal agent on the hepatobiliary system Kilicoglu B, Kismet K, Kilicoglu SS, Erel S, Gencay O, Sorkun K, Erdemli E, Akhan O, Akkus MA, Sayek I

2089

Predictive factors for early aspiration in liver abscess Khan R, Hamid S, Abid S, Jafri W, Abbas Z, Islam M, Shah H, Beg S

2094

Enhancement of CD4+ T cell activities and modulation of Th1/Th2 lineage development in radiated tumor-bearing rats treated with male zooid of Antheraea pernyi extracts Zhao WH, Li L, Zhang B, Zhang WD, Zong M, Tang JD, Zhang HY, Li S

2100

Effect of Oxymatrine on the TGFbeta-Smad signaling pathway in rats with CCl4-induced hepatic fibrosis Wu XL, Zeng WZ, Jiang MD, Qin JP, Xu H

2106

Intraperitoneal administration of gonadotropin-releasing hormone-PE40 induces castration in male rats Yu L, Zhang ZF, Jing CX, Wu FL

2110

Expression of connective tissue growth factor in tumor tissues is an independent predictor of poor prognosis in patients with gastric cancer Liu LY, Han YC, Wu SH, Lv ZH

CASE REPORT

2115

Endoscopic ultrasonography-guided trucut biopsy for the preoperative diagnosis of peripancreatic castleman’s disease: A case report Rhee KH, Lee SS, Huh JR www.wjgnet.com

World Journal of Gastroenterology

Contents

Volume 14 Number 13 April 7, 2008 2118

Primary rectal signet ring cell carcinoma with peritoneal dissemination and gastric secondaries Sim HL, Tan KY, Poon PL, Cheng A

2121

Diagnosis and treatment of Gardner syndrome with gastric polyposis: A case report and review of the literature Gu GL, Wang SL, Wei XM, Bai L

BOOK REVIEW

2124

Rome Ⅲ: The functional gastrointestinal disorders, third edition, 2006 Mostafa R

LETTERS TO THE EDITOR 2126

Role of ABCC2 common variants in intrahepatic cholestasis of pregnancy Sookoian S, Castaño G, Pirola CJ

ACKNOWLEDGMENTS

2128

Acknowledgments to Reviewers of World Journal of Gastroenterology

APPENDIX

2129

Meetings

2130

Instructions to authors

I-V

Editorial Board

FLYLEAF INSIDE BACK COVER

Online Submissions

INSIDE FRONT COVER

Online Submissions

RESPONSIBLE EDITORS FOR THIS ISSUE

Assistant Editor: Yan Jiang Review Editor: Jing Zhu Electronic Page Editor: Yong Liu Editor-in-Charge: Jian-Xia Cheng Copy Editor: Pietro Invernizzi, MD, PhD Associate Senior Editor: Ye Liu Layout Editor: Lian-Sheng Ma

NAME OF JOURNAL World Journal of Gastroenterology RESPONSIBLE INSTITUTION Department of Science and Technology of Shanxi Province SPONSOR Taiyuan Research and Treatment Center for Digestive Diseases, 77 Shuangta Xijie, Taiyuan 030001, Shanxi Province, China EDITING Editorial Board of World Journal of Gastroenterology, 77 Shuangta Xijie, Taiyuan 030001, Shanxi Province, China Telephone: +86-351-4078656 E-mail: [email protected] PUBLISHING Editorial Department of World Journal of Gastroenterology, 77 Shuangta Xijie, Taiyuan 030001, Shanxi Province, China Telephone: +86-351-4078656 E-mail: [email protected] http://www.wjgnet.com PRINTING Beijing Kexin Printing House

SUBSCRIPTION RMB 50 Yuan for each issue, RMB 2400 Yuan for one year CSSN ISSN 1007-9327 CN 14-1219/R HONORARY EDITORS-IN-CHIEF Ke-Ji Chen, Beijing Li-Fang Chou, Taipei Zhi-Qiang Huang, Beijing Shinn-Jang Hwang, Taipei Min-Liang Kuo, Taipei Nicholas F LaRusso, Rochester Jie-Shou Li, Nanjing Geng-Tao Liu, Beijing Lein-Ray Mo, Tainan Bo-Rong Pan, Xi'an Fa-Zu Qiu, Wuhan Eamonn M Quigley, Cork David S Rampton, London Rudi Schmid, kentfield Nicholas J Talley, Rochester Guido NJ Tytgat, Amsterdam H-P Wang, Taipei Jaw-Ching Wu, Taipei Meng-Chao Wu, Shanghai Ming-Shiang Wu, Taipei Jia-Yu Xu, Shanghai Ta-Sen Yeh, Taoyuan

PUBLICATION DATE April 7, 2008

ASSOCIATE EDITORS-IN-CHIEF Gianfranco D Alpini, Temple Bruno Annibale, Roma Roger William Chapman, Oxford Chi-Hin Cho, Hong Kong Alexander L Gerbes, Munich Shou-Dong Lee, Taipei Walter Edwin Longo, New Haven You-Yong Lu, Beijing Masao Omata, Tokyo Harry HX Xia, Hanover

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DEPUTY DIRECTOR Jian-Zhong Zhang, Beijing LANGUAGE EDITORS Director: Jing-Yun Ma, Beijing Deputy Director: Xian-Lin Wang, Beijing MEMBERS Gianfranco D Alpini, Temple BS Anand, Houston Richard B Banati, Lidcombe Giuseppe Chiarioni, Valeggio John Frank Di Mari, Texas Shannon S Glaser, Temple Mario Guslandi, Milano Martin Hennenberg, Bonn Atif Iqbal, Omaha Manoj Kumar, Nepal Patricia F Lalor, Birmingham Ming Li, New Orleans Margaret Lutze, Chicago Jing-Yun Ma, Beijing Daniel Markovich, Brisbane Sabine Mihm, Göttingen Francesco Negro, Genève Bernardino Rampone, Siena Richard A Rippe, Chapel Hill Stephen E Roberts, Swansea Ross C Smith, Sydney Seng-Lai Tan, Seattle Xian-Lin Wang, Beijing Eddie Wisse, Keerbergen Daniel Lindsay Worthley, Bedford NEWS EDITOR Lixin Zhu, Berkeley COPY EDITORS Gianfranco D Alpini, Temple Sujit Kumar Bhattacharya, Kolkata Filip Braet, Sydney Kirsteen N Browning, Baton Rouge Radha K Dhiman, Chandigarh John Frank Di Mari, Texas Shannon S Glaser, Temple

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Martin Hennenberg, Bonn Eberhard Hildt, Berlin Patricia F Lalor, Birmingham Ming Li, New Orleans Margaret Lutze, Chicago MI Torrs, Jaén Sri Prakash Misra, Allahabad Giovanni Monteleone, Rome Giovanni Musso, Torino Valerio Nobili, Rome Osman Cavit Ozdogan, Istanbul Francesco Perri, San Giovanni Rotondo Thierry Piche, Nice Bernardino Rampone, Siena Richard A Rippe, Chapel Hill Ross C Smith, Sydney Daniel Lindsay Worthley, Bedford George Y Wu, Farmington Jian Wu, Sacramento COPYRIGHT © 2008 Published by WJG. All rights reserved; no part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise without the prior permission of WJG. Authors are required to grant WJG an exclusive licence to publish. SPECIAL STATEMENT All articles published in this journal represent the viewpoints of the authors except where indicated otherwise. INSTRUCTIONS TO AUTHORS Full instructions are available online at http://www.wjgnet.com/wjg/help/ instructions.jsp. If you do not have web access please contact the editorial office. ONLINE SUBMISSION http://wjg.wjgnet.com

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World J Gastroenterol 2008 April 7; 14(13): 1969-1971 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

EDITORIAL

Wireless capsule endoscopy A Mata, J Llach, JM Bordas A Mata, J Llach, JM Bordas, Digestive Endoscopy Unit, Gastroenterology Service, Imd, Hospital Clinic Barcelona, Barcelona 08036, Spain Correspondence to: Dr. Josep M Bordas, Digestive Endoscopy Unit, IMD, Hospital Clinic of Barcelona, c/Villarroel 170, Barcelona 08036, Spain. [email protected] Telephone: +34-93-2275513 Fax: +34-93-2279859 Received: August 11, 2006 Revised: December 12, 2006

Abstract Wireless capsule endoscopy is a new technique that allows complete exploration of the small bowel without external wires. Its role has been analyzed in many small bowel diseases such as obscure gastrointestinal bleeding, Crohn’s disease and gastrointestinal polyposis syndromes with promising results. Studies on other pathologies (i.e. small bowel tumour, celiac disease) are under evaluation to define the role of this technique. © 2008 WJG . All rights reserved.

Key words: Wireless capsule endoscopy; Small bowel; Obscure gastrointestinal bleeding; Crohn’s disease; Gastrointestinal polyposis syndrome Peer reviewer: Joerg C Hoffmann, Dr, Medizinische Klinik I, Charité-Universitätsmedizin Berlin, Campus Benjamin Franklin, Hindenburgdamm 30, Berlin D12200, Germany

Mata A, Llach J, Bordas JM. Wireless capsule endoscopy. World J Gastroenterol 2008; 14(13): 1969-1971 Available from: URL: http://www.wjgnet.com/1007-9327/14/1969.asp DOI: http://dx.doi. org/10.3748/wjg.14.1969

The need and wish to perform endoscopic examination of the small bowel have led to the development of an ingestible miniature camera device capable of obtaining images of the whole small intestine. Wireless capsule endoscopy is a new type of radiotelemetry video system which is small enough to be swallowed and has no external wires, fiberoptic bundles or cables. It measures 11 mm × 26 mm and weighs 3.7 g. By using a lens of a short focal length, images are obtained as the optical window of the capsule sweeps past the gut wall, without requiring air insufflation of the gut lumen. The capsule is propelled by peristalsis through the gastrointestinal tract and does not require a pushing force to propel it

through the bowel. Up to 2002, more than 250 000 capsule explorations had been performed [1], and nowadays this number has increased significantly. The M2A capsule [Mouth to (2) Anus] initially, and Pillcam SB (Small Bowel) later, from GIVEN (GastroIntestinal Video Endoscopy, Given Imaging Limited, Yoqneam, Israel), and endocapsule from Olympus are the capsules that have been approved for use in the clinical setting. Each capsule contains a lens, light emitting diodes (LEDs), a color camera, 2 batteries, a radio frequency transmitter and an antenna. The camera takes 2 images per second and transmits these by means of radio frequency to a sensor array in a belt placed around the patient’s abdomen and from there to a recording device in the belt. Once the study is completed (between 6 and 8 h), the recording device is removed and the images are downloaded to a computer workstation with software that displays the video images on a computer monitor. Capsule endoscopy can be performed as an outpatient procedure. Small bowel preparation is still a controversial issue. Some groups used fasting or clear liquids for 10 to 12 h (or even for 24) before the study, although some studies suggest that bowel preparation (with 2 or 4 litres of polyethylene glycol based electrolyte solution or oral sodium phosphate preparation) improves the visualization of the small intestine[2,3]. A recent Spanish prospective multicenter trial published in abstract form, has shown that all three strategies have similar results[4]. The role of wireless capsule endoscopy has been analyzed in patients with obscure gastrointestinal bleeding and in comparative studies with endoscopic[5] or radiographic methods[6]. Capsule endoscopy has shown a diagnostic yield of 71% compared to 29% of push enteroscopy, in a recent analysis of 7 prospective studies[7]. Another study has shown that the detection rate of capsule endoscopy is higher in patients with ongoing overt bleeding than in those with anemia or prior overt bleeding[8]. In a comparative study with intraoperative enteroscopy, the sensitivity, specificity, positive and negative predictive value of capsule endoscopy were 95%, 75%, 95% and 86%, respectively[9]. For obscure gastrointestinal bleeding, capsule endoscopy has shown better results than radiographic studies, which have a low diagnostic yield in detecting small bowel lesions[6,10]. Capsule endoscopy has also shown its usefulness in the evaluation of the small intestine in patients with suspected or known Crohn’s disease[11], and is superior to small bowel follow-through[12-14], enteroclysis[15,16], push enteroscopy[16] and CT enteroclysis[17] for identifying small intestinal dis-

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ease. The sensitivity and specificity of capsule endoscopy have recently been estimated to be 89.6% and 100%, respectively[18]. However, the diagnostic criteria of capsule endoscopy for Crohn’s disease have not yet been defined. Mucosal breaks and aphthous ulcers or erosions are also seen in asymptomatic healthy volunteers, and small bowel ulcers and strictures have been associated with the use of nonsteroidal anti-inflammatory agents, making it, at times, difficult to differentiate these findings with the presence of a Crohn’s disease[9]. Capsule endoscopy has been performed in patients with gastrointestinal polyposis syndrome, and several studies have suggested that it may be useful in the detection of small bowel polyps[19,20]. A comparative prospective study showed that capsule endoscopy can detect more polypoid lesions than small-bowel follow through in these patients[21]. Nevertheless, more prospective studies with longer follow-up are required, to define the role of capsule endoscopy findings in the outcome of patients with gastrointestinal polyposis syndrome. Capsule endoscopy in the pediatric population and esophageal capsule endoscopy (Pillcam ESO) have shown promising results but larger prospective trials are needed to define their role in these patients. Other possible indications for capsule endoscopy, such as celiac disease, HIV positive patients with gastrointestinal symptoms, mal-absorption or small bowel transplantation, have not been defined so far, and more prospective trials assessing the use of capsule endoscopy in these groups of patients are still needed. The main contraindication of performing the capsule endoscopy procedure is the suspicion or knowledge of a gastrointestinal obstruction, stricture or fistula. Other former contraindications such as implanted cardiac pacemakers or other electro-medical devices and patients with swallowing disorders have been excluded since some studies showed no interference between capsule endoscopy and pacemaker or implantable defibrillators functioning[22,23] and endoscopic placement of the capsule into the gut[24]. The capsule retention rate varies with the indication of the examination, being reported of 1.5% in patients with obscure gastrointestinal bleeding[25] and 5% in patients with suspected Crohn’s disease[26], who are usually asymptomatic[10,25] and may require endoscopic removal or surgery. How to prevent capsule retention has yet o be defined since neither radiologic studies nor the “patency capsule” has shown conclusive results so far. The clinical setting of each patient, as well as some features related to intestinal strictures (previous small bowel surgery, NSAIDs, suspected small bowel Crohn’s disease), have to be analyzed prior to the study. Patients should be informed about the possibility of capsule retention and further treatment.

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REFERENCES 1

Ginsberg GG, Barkun AN, Bosco JJ, Isenberg GA, Nguyen CC, Petersen BT, Silverman WB, Slivka A, Taitelbaum G. Wireless capsule endoscopy: August 2002. Gastrointest Endosc 2002; 56: 621-624

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Dai N, Gubler C, Hengstler P, Meyenberger C, Bauerfeind P. Improved capsule endoscopy after bowel preparation. Gastrointest Endosc 2005; 61: 28-31 de Franchis R, Avgerinos A, Barkin J, Cave D, Filoche B. ICCE consensus for bowel preparation and prokinetics. Endoscopy 2005; 37: 1040-1045 Pons V, Gonzalez B, Gonzalez C, Perez-Cuadrado E, Fernandez S, Fernandez-Urien I, Mata A, Espinos J, Perez Grueso MJ, Arguello L. Evaluation of different bowel preparations for study with capsule endoscopy: a prospective randomized controlled study. Abstract presented at the ICCE Paris, France, 2006 Mata A, Bordas JM, Feu F, Gines A, Pellise M, FernandezEsparrach G, Balaguer F, Pique JM, Llach J. Wireless capsule endoscopy in patients with obscure gastrointestinal bleeding: a comparative study with push enteroscopy. Aliment Pharmacol Ther 2004; 20: 189-194 Costamagna G, Shah SK, Riccioni ME, Foschia F, Mutignani M, Perri V, Vecchioli A, Brizi MG, Picciocchi A, Marano P. A prospective trial comparing small bowel radiographs and video capsule endoscopy for suspected small bowel disease. Gastroenterology 2002; 123: 999-1005 Melmed GY, Lo SK. Capsule endoscopy: practical applications. Clin Gastroenterol Hepatol 2005; 3: 411-422 Pennazio M, Santucci R, Rondonotti E, Abbiati C, Beccari G, Rossini FP, De Franchis R. Outcome of patients with obscure gastrointestinal bleeding after capsule endoscopy: report of 100 consecutive cases. Gastroenterology 2004; 126: 643-653 Hartmann D, Schmidt H, Bolz G, Schilling D, Kinzel F, Eickhoff A, Huschner W, Moller K, Jakobs R, Reitzig P, Weickert U, Gellert K, Schultz H, Guenther K, Hollerbuhl H, Schoenleben K, Schulz HJ, Riemann JF. A prospective two-center study comparing wireless capsule endoscopy with intraoperative enteroscopy in patients with obscure GI bleeding. Gastrointest Endosc 2005; 61: 826-832 Hara AK, Leighton JA, Sharma VK, Fleischer DE. Small bowel: preliminary comparison of capsule endoscopy with barium study and CT. Radiology 2004; 230: 260-265 Papadakis KA, Lo SK, Fireman Z, Hollerbach S. Wireless capsule endoscopy in the evaluation of patients with suspected or known Crohn’s disease. Endoscopy 2005; 37: 1018-1022 Mow WS, Lo SK, Targan SR, Dubinsky MC, Treyzon L, Abreu-Martin MT, Papadakis KA, Vasiliauskas EA. Initial experience with wireless capsule enteroscopy in the diagnosis and management of inflammatory bowel disease. Clin Gastroenterol Hepatol 2004; 2: 31-40 Herrerias JM, Caunedo A, Rodriguez-Tellez M, Pellicer F, Herrerias JM Jr. Capsule endoscopy in patients with suspected Crohn's disease and negative endoscopy. Endoscopy 2003; 35: 564-568 Fireman Z, Mahajna E, Broide E, Shapiro M, Fich L, Sternberg A, Kopelman Y, Scapa E. Diagnosing small bowel Crohn’s disease with wireless capsule endoscopy. Gut 2003; 52: 390-392 Liangpunsakul S, Chadalawada V, Rex DK, Maglinte D, Lappas J. Wireless capsule endoscopy detects small bowel ulcers in patients with normal results from state of the art enteroclysis. Am J Gastroenterol 2003; 98: 1295-1298 Chong AK, Taylor A, Miller A, Hennessy O, Connell W, Desmond P. Capsule endoscopy vs. push enteroscopy and enteroclysis in suspected small-bowel Crohn’s disease. Gastrointest Endosc 2005; 61: 255-261 Voderholzer WA, Beinhoelzl J, Rogalla P, Murrer S, Schachschal G, Lochs H, Ortner MA. Small bowel involvement in Crohn’s disease: a prospective comparison of wireless capsule endoscopy and computed tomography enteroclysis. Gut 2005; 54: 369-373 Dubcenco E, Jeejeebhoy KN, Petroniene R, Tang SJ, Zalev AH, Gardiner GW, Baker JP. Capsule endoscopy findings in patients with established and suspected small-bowel Crohn’s disease: correlation with radiologic, endoscopic, and histologic findings. Gastrointest Endosc 2005; 62: 538-544 Schulmann K, Hollerbach S, Kraus K, Willert J, Vogel T, Moslein G, Pox C, Reiser M, Reinacher-Schick A, Schmiegel W.

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Feasibility and diagnostic utility of video capsule endoscopy for the detection of small bowel polyps in patients with hereditary polyposis syndromes. Am J Gastroenterol 2005; 100: 27-37 Soares J, Lopes L, Vilas Boas G, Pinho C. Wireless capsule endoscopy for evaluation of phenotypic expression of smallbowel polyps in patients with Peutz-Jeghers syndrome and in symptomatic first-degree relatives. Endoscopy 2004; 36: 1060-1066 Mata A, Llach J, Castells A, Rovira JM, Pellise M, Gines A, Fernandez-Esparrach G, Andreu M, Bordas JM, Pique JM. A prospective trial comparing wireless capsule endoscopy and barium contrast series for small-bowel surveillance in hereditary GI polyposis syndromes. Gastrointest Endosc 2005; 61: 721-725 Leighton JA, Sharma VK, Srivathsan K, Heigh RI, McWane



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TL, Post JK, Robinson SR, Bazzell JL, Fleischer DE. Safety of capsule endoscopy in patients with pacemakers. Gastrointest Endosc 2004; 59: 567-569 23 Leighton JA, Srivathsan K, Carey EJ, Sharma VK, Heigh RI, Post JK, Erickson PJ, Robinson SR, Bazzell JL, Fleischer DE. Safety of wireless capsule endoscopy in patients with implantable cardiac defibrillators. Am J Gastroenterol 2005; 100: 1728-1731 24 Leung WK, Sung JJ. Endoscopically assisted video capsule endoscopy. Endoscopy 2004; 36: 562-563; author reply 563-564 25 Sears DM, Avots-Avotins A, Culp K, Gavin MW. Frequency and clinical outcome of capsule retention during capsule endoscopy for GI bleeding of obscure origin. Gastrointest Endosc 2004; 60: 822-827 26 Lewis B. How to prevent endoscopic capsule retention. Endoscopy 2005; 37: 852-856 S- Editor Liu Y L- Editor Wang XL

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World J Gastroenterol 2008 April 7; 14(13): 1972-1980 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

EDITORIAL

Current view of the immunopathogenesis in inflammatory bowel disease and its implications for therapy MI Torres, A Ríos

MI Torres, Department of Experimental Biology, University of Jaén, Jaén 23071, Spain A Ríos, Department of Cell Biology, University of Granada, Granada 18071, Spain Correspondence to: Dr. MI Torres, Department of Experimental Biology, Faculty of Experimental Sciences, University of Jaén, Paraje de las Lagunillas s/n. 23071 Jaén, Spain. [email protected] Telephone: +34-953-212762  Fax: +34-953-211875 Received: November 16, 2007 Revised: January 15, 2008

Abstract Although the aetiology of inflammatory bowel disease (IBD) remains unknown, the pathogenesis is gradually being unravelled, seeming to be the result of a combination of environmental, genetic, and immunological factors in which an uncontrolled immune response within the intestinal lumen leads to inflammation in genetically predisposed individuals. Multifactorial evidence suggests that a defect of innate immune response to microbial agents is involved in IBD. This editorial outlines the immunopathogenesis of IBD and their current and future therapy. We present IBD as a result of dysregulated mucosal response in the intestinal wall facilitated by defects in epithelial barrier function and the mucosal immune system with excessive production of cytokines growth factors, adhesion molecules, and reactive oxygen metabolites, resulting in tissue injury. Established and evolving therapies are discussed in the second part of this editorial and at the end of this section we review new therapies to modulate the immune system in patients with IBD. © 2008 WJG . All rights reserved.

Key words: Inflammatory bowel disease; Ulcerative colitis; Crohn’s disease; Tolerance; Cytokines; Mucosal inflammation Peer reviewer: J Bart A Crusius, PhD, Laboratory of Immunogenetics,

Department of Pathology, VU University Medical Center, Room 3E48, De Boelelaan 1117, PO Box 7057, 1007 MB, Amsterdam, The Netherlands Torres MI, Ríos A. Current view of the immunopathogenesis in inflammatory bowel disease and its implications for therapy. World J Gastroenterol 2008; 14(13): 1972-1980 Available from: URL: http://www.wjgnet.com/1007-9327/14/1972.asp DOI: http://dx.doi.org/10.3748/wjg.14.1972

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INTRODUCTION The intestinal mucosa constitutes an immunologic system in which oral tolerance and defence against harmful organisms develop[1]. The mucosal immune system, which plays a pivotal role in host defence, is continuously exposed to large amounts of exogenous (i.e., dietary) and endogenous (e.g., bacterial) antigens[2]. Activation of lamina propia (LP) T cells by lumen antigens may lead to the production of inflammatory cytokines and subsequent mucosal inflammation and tissue damage[3,4]. Dysfunctions of the intestinal immune system and cross-reactivity against host epithelial cells have been implicated as major mechanisms by which inflammation occurs[5]. In addition to immune factors as leukocytes, macrophages, polymorphonuclear cells, mast cells, B and T lymphocytes, other potential mediators of inflammation are involved in the disease: eicosanoids, biological amines, cytokines, nitrogen- and oxygen-reactive metabolites, platelet-activating factor have been noted to influence inflammatory processes[6]. Among environmental factors, antigens to intestinal bacteria play an important role. The loss of tolerance to intestinal bacterial flora is manifested as an abnormal immune response[7]. Accordingly, inflammatory bowel disease (IBD) could be considered to be an imbalance between pro-inflammatory and anti-inflammatory mediators[3]. The following editorial summarizes the current view of the immunopathogenesis basis of IBD in relation to barrier disruption, immune deregulation genetic epidemiology and susceptibility to environmental dysregulation, triggers and its implications for therapies.

EPIDEMIOLOGY AND CLINICAL FEATURES IBD like ulcerative colitis (UC) and Crohn’ disease (CD), comprises a group of multifactorial intestinal disorders with high incidence throughout the human population[8]. The highest incidence rates and prevalence have been reported in northern Europe and North America. The disease is characterized by cycles of clinical exacerbation and remission, with periods of improvement followed by relapse, and appears to be immunologically mediated[12]. UC and CD show some overlapping clinical features, and in 10%-15% of cases it is not possible to differentiate between the two diseases. But also, differences exist in the nature and location of the lesion damage between UC and CD. While UC is restricted to the large intestine and is associated with continuous mucosal inflammation, including crypt abscesses as well as ulcers, which typically

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Table 1 IBD immunopathogenesis Immune dysregulation Cytokines Epiteliol barrier function Oral tolerance Reactive oxygen species (ROS) Antioxidant defence system

spread from the most caudal part of the rectum, CD can affect any part of the gastrointestinal tract and is characterized by segmental and transmural inflammation, fistulas, oedema and granulomas in whole intestinal wall[13]. At the histological level, various abnormalities have been observed in patients with IBD and in experimental models of intestinal inflammation including the presence of a significant number of neutrophils within the lamina propria and the crypts, where micro-abscesses can form, the depletion of goblet cell mucin, induced by disturbed transformation of undifferentiated cells in an environment exposed to inflammatory cytokines, damage to the nervous system[13,14] and hypertrophy of smooth muscle cells[15]. Intestinal inflammation alters the contractile activity of intestinal smooth muscle, and the motility disorders induce abnormal growth of the intestinal flora, resulting in disturbance of the intestinal flora that can aggravate the pathogenesis of mucosal inflammation[16].

GENETIC FACTORS The discovery of susceptibility genes to IBD have shown the importance of (1) dysregulation of the innate immune response to enteric microflora or pathogens; (2) increased permeability across the epithelial barrier, and (3) defective regulation of the adaptive immune system of epithelial barrier function in disease pathogenesis. IBD is considered a complex polygenic disease and its susceptibility genes could interfere with the disease and with the response to different therapies. Strong evidence for genetic factors has been reported contributing to IBD susceptibility[17]. IBD is thought to be caused by the mutual reactions among host susceptibility genes (CARD15/NOD2, CARD4/NOD1, HLA, TLR4, DLG5, NF-κB1), environmental factors including enteric flora and food antigens, and abnormal immune balance[9-11]. The genetic contribution may be more important in CD than UC with multiple gene products contributing to risk[18]. Identifying NOD2 as a susceptibility gene in CD and additional susceptibility loci have been implicated in IBD such as IBD5, IL23R and ATG16L1 among other loci[19-21]. The number of potential gene continues to increase, additional novel loci map to chromosomes 16q24.1, TNFSF15, NKX2-3 and the intergenic region on chromosome 10q21.1 might contribute to the IBD[22]. Future genetic research may include focus on phenotypes, control for environmental variables and gene-gene interactions.

PATHOGENESIS OF IBD There is compelling evidence that dysregulation of the

  

 

  

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mucosal immune system is a major factor contributing to the pathogenesis of IBD[13]. The pathogenesis of these diseases is understood to represent the outcome of three essential, interactive cofactors: host susceptibility, enteric microflora and mucosal immunity[12,13]. The basis of IBD is the presence of genetically determined alterations that result in a mucosal immune system that overreacts to normal intestinal microflora. These immune responses may be induced by defects in the epithelial barrier, and increased intestinal permeability, adherence of bacteria, and decreased expression of defensins[12]. The mucosal immune system senses and interprets the local microenvironment, recognizing and avoiding reactions to commensal flora (tolerance), whilst retaining its capacity to respond to episodic challenge from pathogens [1,2]. Increased synthesis and release of pro-inflammatory mediators such as cytokines, chemokines, eicosanoids, platelet activating factor, reactive oxygen and nitrogen metabolites, as well as other abnormalities have been in IBD[6]. We described IBD as a result of dysregulated mucosal response in the intestinal wall facilitated by defects in epithelial barrier function and mucosal immune system with excessive production of cytokines growth factors, adhesion molecules, and reactive oxygen metabolites, resulting in tissue injury (Table 1).

DYSREGULATED IMMUNITY Cytokines Alterations in the production of many cytokines have been described in patients with active IBD[23]. The significance of these findings in the pathogenesis of IBD remains poorly understood and controversy has continued as to whether these changes really represent a primary defect in the regulation of the immune system or are a secondary consequence of immune activation[6]. The nature of the immune response and the cytokine profile generated are under genetic control and determine the features of the inflammatory process in IBD. In active IBD the balance of regulatory and effector cells is disturbed, predominating effector T cells (Th1, Th2) over regulatory T cells (Th3, Tr). CD has been associated with type 1 helper T-cell cytokines (Th1), such as interferon-γ, TNF-α and IL-12[24,25]. In ulcerative colitis, the pattern of cytokine is less clear; there is a modified Th2 response associated with cytokines such as IL-15 and IL-10[25,27]. Among the cytokines involved, Interleukin-10 is a regulatory cytokine which plays a crucial role in the balance of the mucosal immune system, promoting physiological activation and preventing the pathological inflammation that characterizes the inflammatory bowel diseases [26]. The immuno-regulatory activity of IL-10 is based upon its ability to inhibit both cytokine synthesis and antigen presentation, with maintenance of intestinal immunoregulation and tolerance to components of intestinal flora, controlling the inflammatory responses to intestinal antigens[26]. This pathophysiological concept for IBD is changing as a result of recent advances with the description of another type of effector immunologic response CD4+ Th pathway, namely interleukin-23/interleukin-17 axis. IL-23 www.wjgnet.com

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is produced by antigen-presentation cells and promotes a population of T lymphocytes that produce IL-17, IL-6 and TNF-alpha (Th17 cells)[27,28]. These IL-23-driven CD4+ T cells are highly inflammatory and elicit IL-17-dependent autoimmunity. IL-17 is an inflammatory cytokine expressed by activated CD4+ T cells and triggers the NF-kB signalling cascade and MAP kinase pathway, leading to T cells proliferation and up-regulation of inflammatory molecules[29]. Recent publications have reported that the IL-23/IL-17 pathway may have a pivotal role in intestinal inflammation[30,31]. These findings demonstrate that IL-23, but not IL-12, is essential for the development of intestinal inflammation in IBD. In addition, levels of expression of IL-23 and IL-17 are increased in patients with CD[32]. Recently, the study of Duerr et al[33] reported a significant association between CD and a gene on chromosome 1p31 that encodes a receptor for IL-23 (IL-23R) that is highly expressed by memory T cells. Also, the interesting works of Bamias et al [34] and Prehn et al[35], respectively, have described a novel TNFlike cytokine co-stimulator of IFN-gamma, namely TL1A that play an important role in mucosal inflammation and in experimental ileitis. TL1A is expressed in dendritic cells and acts on CD4+ cells and provides co-stimulation for proliferation of IFN-gamma and is up-regulated in patients with active IBD. A single nucleotide polymorphism in the gene encoding TL1A (TNFSF15) confers susceptibility to CD[36]. Mucosal epithelial barrier function The polarized epithelial cells provide a crucial barrier function, with high concentrations of dietary and bacterial antigens at the apical surfaces in the mucosa and with high concentrations of lymphoid cells at the basolateral surface [37] . The intestinal epithelium is considered a constitutive component of the mucosal immune system. Intestinal epithelial cells (IECs) are connected by tight junctions, which are dynamically regulated in response to cytokines and are down-regulated by the junctional complexes in human IBD (E-cadherin and β-catenin)[38]. The epithelium is in constant communication with luminal flora and the underlying network of innate and adaptive immune cells, and IECs constitutively express or can be induced to express costimulatory molecules[39] and components of the human major histocompatibility complex (MHC) [40] , toll-like receptors (TLRs) [41] , NOD proteins[42], inflammatory cytokines[43], as well as antimicrobial peptides[44]. IECs contribute to the initiation and regulation of innate and adaptive defence mechanisms by interacting with lamina propria dendritic cells (DC), lamina propria lymphocytes (LPL), intraepithelial lymphocytes (IEL) and mediators of the immune and the enteric nerve system[45]. IECs such as non-professional antigen-presenting cells might interact with naïve T cells (Th0) through MHC Ⅱ receptors, and produce co-stimulatory signals suppressing or inducing anergy in mucosal T cells[46]. The epithelial barrier is leaky in IBD and has a lower epithelial resistance and increased permeability that enables the proliferation of non-pathologic organisms (normal microflora) in close juxtaposition to elements of the mucosal immune system. www.wjgnet.com

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Also, IBD have disturbed innate immune mechanisms of the epithelial layer and mucosal epithelial cells have a different pattern of TLR expression [47]. Also, an upregulation of NOD2 in IECs and disturb in antigen recognition and processing by antigen-presenting cells are present in patients with IBD[48]. Paneth cells secrete antimicrobial peptides, including α-defensins that play an important role in innate intestinal defences. The Paneth cell deficiency in α-defensins increases the risk of CD and polymorphism in the defensin gene associated with CD[49]. Goblet cells are also an important component of the epithelium and are responsible for defence and epithelial mucosal repair in colitis. Defects in mucus production have been reported in IBD[50,51]. The adaptive immune response in IBD failure to balance, and the DCs might be responsible for a dysregulated innate immune response [52]. DCs are the primordial cells in controlling immunity against pathogens and tolerance towards commensals and are dominant subsets of antigen-presenting cells in the intestinal lamina propria[53]. DCs penetrate their dendrites between epithelial cells to sample luminal antigens without altering the mucosal barrier and they contain components of commensal bacteria. Depending upon the nature of the antigen and the activation state of DCs, the end result may be immune activation or tolerogenic action[54]. DCs also have an important role in mucosal inflammation through the production of cytokines, resulting in persistent activation of effector T cells[55]. An increase in intraepithelial lymphocytes (IEL) appeared in IBD and the activation of these IELs could be responsible for: (1) defending the mucosal barrier against intraluminal microorganisms, (2) modulation on epithelial cells of the expression of MHC antigens, or (3) non-MHC as well as MHC-restricted cytotoxicity[56]. The increase of IELs is known to be associated with an abnormal expression of class Ⅱ MHC molecules on surface and crypt colon epithelial cells. Helper T cells could trigger an MHC-restricted immune mechanism. Oral tolerance In an antigenic environment like intestine, the mucosal immune system must maintain tolerance to commensal bacteria, food and self antigens and must be able to initiate defence responses to pathogens. Studies have suggested that IBD is a consequence of the breakdown of mucosal tolerance and tolerance to normal flora is broken [57]. DCs in conjunction with antigen-specific T lymphocytes trigger the maintenance of immune tolerance. Activation of intestinal NKT cells by CD1d-expressing IECs and professional antigen presenting cells (DC cells) may contribute to induction of oral tolerance[58,59]. In IBD, atypical antigen-presenting cells become potent effector-T-cell activators, and the IECs can act as antigen-presenting cells capable of stimulating primed T cells which acquire an activated phenotype with increased histocompatibility molecule expression in the presence of inflammatory cytokines[60]. IECs might also activate T cells via non-classical MHC. Our research group has previously shown that intestinal epithelial cells expressing HLA-G at

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Table 2 Medical management based on pathogenesis Dietary nutrients Polyunsaturated fatty acids (PUFAs) Fiber Probiotics Prebiotics Flavonoids Antagonist platelet-activating factor (PAFs) Biological therapies Anti-inflammatory/Immunosuppressive Immunomodulators Generation regulatory T cells/Activation effector T cells Inhibition of recruitment, migration, adhesion molecules Epithelial repair/Restitution barrier function Induction apoptosis Autologous hematopoietic stem cell transplant

the apical surface of the epithelial layer and at crypt level in ulcerative colitis, but not in CD[61]. HLA-G is a nonclassical major histocompatibility complex class Ⅰ, which is selectively expressed at the maternal-foetal interface of cytotrophoblast cells and protects the foetus from maternal rejection and creating a general state of tolerance[62]. Non-typical antigen-presenting cells, as epithelial cells, might acquire an activated phenotype with increased expression of HLA-G in the presence of inflammatory cytokines as IFN-gamma and TNF-alpha and might activate T cells via non-classical MHC molecules as HLA-G[61]. In UC the specific expression of HLA-G, considered as a molecule implicated in tolerance immunity will be of great importance in the maintenance of the tolerance to fed antigens and of gut microflora and support the notion that it may serve as a regulator of mucosal immune responses to antigens of undefined origin.

REACTIVE OXYGEN SPECIES (ROS) The intestinal mucosa is vulnerable to oxidative stress from constant exposure to reactive oxygen species (ROS) generated by the lumen contents such as oxidized food debris, transition metals such as iron and copper, bacterial metabolites, bile acids and salivary oxidants[63]. A balance is maintained between oxidant and antioxidant systems under physiological conditions, but it is impaired in pathological success. Oxidant-mediated injury plays an important role in the pathophysiology of IBD[64]. It has been suggested that intestinal damage in IBD is related to increased free radical production and to impaired antioxidant defence systems[65]. There are an increased number of activated inflammatory cells in lamina propria of IBD releasing reactive oxygen radicals that are highly produced by neutrophils, macrophages and DCs [63], and exceeds the limited intestinal antioxidant defence system, contributing to intestinal oxidative injury in IBD[64]. Our research group has undertaken to determine whether the small intestine is subjected to oxidative damage during experimental IBD induced by administration of TNBS, as well as, to examine the accompanying changes in antioxidant status, in order to understand its role in the pathogenesis of disease.

  

 

  

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TNBS may be metabolized to yield superoxide anion (O 2-), and hydrogen peroxide (H 2O 2), suggesting that TNBS-induced intestinal inflammation may be partially mediated by cytotoxic reactive oxygen metabolites generated by the oxidative metabolism of TNBS [66] . We evaluated the activity of the antioxidant enzymes catalase (CAT), glutathione peroxidase (GPX), glutathione reductase (GR), glutathione transferase (GT) and superoxide dismutase (SOD), as well as the levels of total glutathione (GSH). We found that GSH levels and SOD activity decreased in animals administered TNBS. These result are in agreement with studies in human IBD, that have been reported a decreased superoxide dismutase activity, as well as low total glutathione levels.[67]. Decreased GSH in gut epithelial cells may increase susceptibility to oxidative injury and exacerbate degeneration of the intestinal mucosa[28]. Therefore, the elevated activities of glutathione peroxidase (GPX) and catalase (CAT) enzymes observed suggest that TNBS led to the formation of high levels of peroxides, including H2O2, increasing tissue injury[68]. Nutritional deficiencies have been reported in IBD, such as lower levels of vitamins A, E and C, important natural antioxidants for the organism, as well as a decrease in trace elements such as zinc and selenium, which are crucial components of several antioxidant enzymes such as SOD[69].

MEDICAL MANAGEMENT BASED ON PATHOGENESIS The main goal of therapy for IBD is to induce a clinical remission and then maintain it for a long period of time, in order to realize the best attainable quality of life. Choice of therapy depends on the severity and location of disease, as well as side effects and other adverse events. Although 5-aminosalicylates, corticosteroids and immunosuppressive drugs are generally used in the treatment of IBD, there are an important numbers of the patients who are not controlled by these conventional therapies[70], indicating a considerable need for develop new and more effective therapy. Treatment with anti-diarrhoeal agents, proper nutrition, antagonistics of activation platelets factors, flavonoids, probiotics and prebiotics can be efficient, but also, a growing number of new biological agents are under investigation, as monoclonal antibodies to antisense mRNA products, peptides and vaccines among others. In Table 2 we summarised medical management based on pathogenesis. Dietary nutrients Dietary management of IBD may be an interesting alternative to drug therapy if it proves to be effective without side effects and can be used as a remission induction and maintenance therapy[71]. Nutrients may be involved in the modulation of the immune response, thus, as components of cell membranes, nutrients can mediate the expression of proteins involved in the immune response, such as cytokines, adhesion molecules, etc[71]. A potential relationship between components of the www.wjgnet.com

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diet and disease pathophysiology has been considered, and immunologic mechanisms have been postulated to link food antigens and the development of intestinal inflammation. Some reports have suggested that consumption of refined sugar might be a risk factor for CD, but not UC[72]. Fat intake has been reported to be positively associated with UC, whereas fruit, vegetables, and fiber consumption seem to decrease the risk of IBD[73]. Polyunsaturated fatty acids (PUFAs) The composition of lipids in the cell membrane is modified by dietary changes and can influence cellular responses. Dietary lipids are one of the most active nutritional substrates modulating the immune response. The link between fatty acids and inflammation is that the eicosanoid family of inflammatory mediators is generated from PUFAs [74]. Long chain n-3 PUFAs decrease the production of inflammatory eicosanoids, cytokines and adhesion molecules, inhibiting the arachidonic acid metabolism and altering the expression of inflammatory genes across effects on transcription factor activation and intraluminal bacterial content[75]. Several studies have shown that patients with IBD had an abnormal plasma phospholipids fatty acid profile[76,77]. The loss of the omega-3 fatty acids which increases the n-6. n-3 ratio, would lead to a predominance of proinflammatory eicosanoids. In an experimental model of IBD induced by rectal administration of TNBS when the animals received dietary n-3 PUFAs, they showed beneficial effect by competing with n-6 PUFAs for the production of lipid inflammatory mediators such as leukotrienes (LTB4), thromboxane (TXA2), Prostaglandins (PGE2) and cytokines[77]. The restoration of GSH levels after administration of the n-3 PUFAs demonstrated the decrease in oxidative stress. It was suggested that dietary PUFAs could affect mucosal adhesion sites for gastrointestinal bacteria by modifying the composition of the intestinal wall and as a result, the dietary PUFAs could modulate the probiotics action[78]. Although LC-PUFA may be of interest in the dietary management of IBD, these fatty acids are highly susceptible to peroxidation, and indeed they may influence the antioxidant defence system. To overcome lipid peroxidation by products and cell damage from diets supplemented with PUFAs, appropriate antioxidants should be provided. Dietary fiber Several studies have shown that dietary fiber actively contributes to the intestinal anti-inflammatory effect, supporting its potential role in the treatment of IBD. Their therapeutic effect is associated with an increased production of short-chain fatty acids, mainly acetate, propionate and butyrate in the colonic lumen and with the promotion the use of these fatty acids, specifically butyrate by colonic epithelial cells[79]. The result is the restoration of the metabolic function of the intestinal cells by aerobic ATP production after butyrate oxidation by epithelial cells that accelerate the intestinal repair preserving the integrity of the intestinal mucosa and downregulating the exacerbated immune response presented in IBD[80]. www.wjgnet.com

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The dietary fiber associated with the production of short-chain fatty acids, also can contribute to the inhibition of the production and release of proinflammatory cytokines, including IL-6, IL-8, TNF-alpha, and other mediators of inflammation as reactive oxygen and nitrogen metabolites (NO production)[81]. Probiotics and prebiotics IBD represent a malfunction of tolerance to the commensal microbiota, for this reason attention is focused on this relationship as a potential therapeutic action. Therapies such as prebiotic and probiotics that selectively manipulate the gastrointestinal microbiota present an attractive treatment in IBD with maintenance of remission and without major side effects. Some evidences support that the use of probiotics and prebiotics in IBD need to use standardized methodology to confirm their utilization as therapy[82]. Probiotics The human intestine contains different bacterial species, named intestinal microflora that plays a critical role in maintaining intestinal health. This intestinal microflora protects against pathogens and maintains the epithelial barrier integrity[83]. The functions of these bacteria include control of proliferation and differentiation of IECs and development and balance of the immune system. Probiotics are living microorganisms that upon ingestion in specific numbers have beneficial effect and exert their therapeutic effects to modulate the barrier function, the inhibition of pathogenic bacteria, the intestinal production of cytokines, with anti-inflammatory properties and enhancement of the digestion and absorption of food[83,84]. Prebiotics Prebiotics are selectively fer mented shor t-chain carbohydrates that allow specific changes in the composition and activity of the microbiota in the gastrointestinal tract and confers health benefits, these include fructooligosaccharides and galacto-oligosaccharide[85,86]. Prebiotics have been shown to enhance the immunoregulatory bacteria of lumen, to reduce the activity of pro-inflammatory transcription factors and attenuate the inflammation. In this sense prebiotics produce butyrate and acetate that inhibit, mucosal inflammation, acting on epithelial and DCs function[87]. Flavonoids Flavonoids are polyphenolic compounds that occur ubiquitously in foods of plant origin and exer t antimicrobial, antiviral, antineoplastic, antihepatotoxic, hypolipidemic, antiallergic and anti-inflammatory features[88]. Biochemical investigations of flavonoid mechanisms have demonstrated that these compounds inhibit a wide variety of enzymatic systems. Variations in the heterocyclic ring C give rise to flavonols, such as morin. Morin (2’,3,4’,5,7-pentahydroxyflavone, flavonol) is a yellowish pigment extractable from the wood of Chlorophora tinctoria and acts as a broad-spectrum and non-toxic antioxidant [89]. Our laboratory analyzed the effects of morin on experimental TNBS-induced IBD

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in rats. Oral administration of morin, at doses ranging from 10 to 200 mg/kg, significantly reduced the mucosal damage by 20%-40% induced by the TNBS experimental model of IBD, although these beneficial effects were not dose-related. Morin reduced the enzymatic activity of myeloperoxidase (MPO) (marker of neutrophil infiltration) and can be interpreted as a manifestation of this antiinflammatory property [89]. Also, significantly inhibited LTB4 synthesis, and this inhibition was maximal at the highest dose of morin assayed (200 mg/kg). Also, morin reduces colon oxidative stress induced in the TNBS model. This reduction may be explained on the basis of its ability both to inhibit free-radical production and to scavenge free radicals once they have been released and by inhibition of colon LTB4 synthesis. Antagonistic platelet-activating factor Sulphasalazine, a diazo compound with 5-aminosalicylic acid (5-ASA) linked to sulphapyridine that acts as a carrier, has been used as therapy for IBD. This agent is useful in maintaining remission by prevention of relapses in patients with IBD[90]. Unfortunately, long-term administration of sulphasalazine is accompanied by a considerable number of side-effects, either dose-dependent such as nausea, vomiting, headache, etc, or allergic such as cutaneous rashes, exanthema, fever, etc. Several mechanisms have been postulated as being involved in the intestinal anti-inflammatory effect exerted by 5-ASA derivatives, including antioxidant and/or radical scavenging properties, inhibition of leukocyte chemotaxis and inhibition of IL-1 synthesis [91]. It has been shown that colon mucosa from patients with IBD produces high levels of platelet-activating factor (PAF) with an important role in the pathogenesis of IBD, its inhibition by specific antagonists may have a potential therapeutic benefit in the treatment and management of these inflammatory diseases[91]. UR-12746 is a compound which combines 5-ASA and UR-12715 through an azo link, and possesses antagonist PAF activity. We demonstrate the therapeutic efficacy of UR-12746 when administered orally at doses of 50 and 100 mg/kg in acute and chronic stages of the TNBS model of IBD[92]. The intestinal anti-inflammatory effect of UR-12746 was associated with a decrease in leukocyte infiltration in the colon mucosa and with a reduction in myeloperoxidase activity. This effect was higher than that seen with sulphasalazine, when assayed at the same doses and in the same experimental conditions. This result suggests that the intestinal anti-inflammatory activity of UR-12746 by inhibition of leukotriene B4 synthesis in the inflamed colon, improvement of the altered colon oxidative status, and reduction of colon IL-1β production. Treatment with UR-12746 was able to ameliorate the altered colon oxidative status by significantly increasing glutathione content and by reducing the colon malonyldialdehyde levels[92]. Future in therapy: Modulation of the immune response and biologic therapies Advances in the understanding of IBD pathogenesis have allowed the development of novel therapies, which at least

  

 

  

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theoretically represent a more specific management of the disease with fewer side effects. The real future in therapy should be to develop an approach to prevention of the initiation and perpetuation of the inflammatory status before tissue damage success, involving the induction of tolerance, commensal flora, generation of regulatory T cells and gene transfer, among others. Between these therapeutics factors, the immunosuppressors like calcineurin inhibitor (tacrolimus and cyclosporine), that suppresses pro-inflammatory cytokine production and T-cell activation, have been used to treat inflammatory bowel disease, especially in refractory ulcerative colitis, and to treat an extra-intestinal manifestation of IBD[93]. Understanding the role of cytokines has been an important advance in IBD therapies, along with the monoclonal antibody technology, which made possible the targeted inhibition of specific disease related cytokines. Also, it has been possible to give inhibitory cytokines as therapeutic agents [94]. The blockage of proinflammatory cytokine TNF-alpha (anti-TNF-α monoclonal antibody) serves as a model for development of new therapy[94]. In this sense, the use of infliximab inhibits the bioactivity of TNF-alpha by directly binding to the cytokine and also modulates the function of TNF-alpha-producing cells[95]. Many case reports have been published on the use of infliximab for treating patients with extraintestinal manifestations of IBD[96]. Other potential modes of therapeutic actions in IBD include the induction of the anti-inflammatory cytokines IL-10 or TGF-beta via retrograde signalling or induction of a certain subset of regulatory T cells[97]. The IL-23 pathway has been a target of antibody blockade[40]. Further research is needed to know how the IL-23R gene and the IL-23/ IL-17-mediated inflammatory axis contribute to disease susceptibility and will lead to therapeutic interventions. The process of T-cell activation, by enhancing regulatory T cells as opposed to inhibiting effector T cells could be considered to be effective for the treatment of IBD. Various populations of T cells exert a downregulatory effect on immune responses, including Tr1 cells (IL-10 secretion), Th3 cells (TGF-beta) and CD4+CD25+ T regulatory cells, in which inhibition is through direct cell contact. The earliest work of this therapy may involve the selection and engineering of T cells delivering IL-10[98]. Lymphocyte-endothelial interactions mediated by adhesion molecules are important in leukocyte migration and recruitment to sites of inflammation in IBD, a selective inhibition of these adhesion molecules offers many potential targets for specific intervention against inflammation[99] to treat CD. In the next years, the role of anti-CD3 drugs (visilizumab), which induces apoptosis in activated T cells, the epithelial repair, and autologous hematopoietic stem cell transplants[100] will be established. Ultimately, the future therapy for IBD should be individualized and directed at induction of remission over a long period of time with the avoidance of important side-effects and maintaining the patient’s quality of life.

CONCLUSION Relevant advances in the understanding of the molecular www.wjgnet.com

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pathogenesis of IBD have been made, discovering susceptibility genes, identification of environmental factors implicated, and dysregulation of immunity in disease pathogenesis. Although the precise mechanisms underlying the development of IBD are not known, sufficient data have been collected to suggest interplay between genetic predispositions, accompanied by the importance of epithelial barrier function, and innate and adaptive immunity. Current therapies generally involve combinations of phar macologic agents such as aminosalicylates, azathioprine, steroids, with dietary manipulation. Newer agents including monoclonal antibodies targeted to specific proinflammatory cytokines, to adhesion molecules, and the induction of anti-inflammatory cytokines and T-cell activation, have emerged and provided clinical benefit in the treatment and relapse of IBD.

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Cho JH. A frameshift mutation in NOD2 associated with susceptibility to Crohn's disease. Nature 2001; 411: 603-606 Duerr RH, Taylor KD, Brant SR, Rioux JD, Silverberg MS, Daly MJ, Steinhart AH, Abraham C, Regueiro M, Griffiths A, Dassopoulos T, Bitton A, Yang H, Targan S, Datta LW, Kistner EO, Schumm LP, Lee AT, Gregersen PK, Barmada MM, Rotter JI, Nicolae DL, Cho JH. A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science 2006; 314: 1461-1463 Hampe J, Franke A, Rosenstiel P, Till A, Teuber M, Huse K, Albrecht M, Mayr G, De La Vega FM, Briggs J, Gunther S, Prescott NJ, Onnie CM, Hasler R, Sipos B, Folsch UR, Lengauer T, Platzer M, Mathew CG, Krawczak M, Schreiber S. A genome-wide association scan of nonsynonymous SNPs identifies a susceptibility variant for Crohn disease in ATG16L1. Nat Genet 2007; 39: 207-211 Silverberg MS, Duerr RH, Brant SR, Bromfield G, Datta LW, Jani N, Kane SV, Rotter JI, Philip Schumm L, Hillary Steinhart A, Taylor KD, Yang H, Cho JH, Rioux JD, Daly MJ. Refined genomic localization and ethnic differences observed for the IBD5 association with Crohn's disease. Eur J Hum Genet 2007; 15: 328-335 Walters TD, Silverberg MS. Genetics of inflammatory bowel disease: current status and future directions. Can J Gastroenterol 2006; 20: 633-639 Rogler G, Andus T. Cytokines in inflammatory bowel disease. World J Surg 1998; 22: 382-389 Papadakis KA, Targan SR. Role of cytokines in the pathogenesis of inflammatory bowel disease. Annu Rev Med 2000; 51: 289-298 Brown SJ, Mayer L. The immune response in inflammatory bowel disease. Am J Gastroenterol 2007; 102: 2058-2069 Schreiber S, Heinig T, Thiele HG, Raedler A. Immunoregulatory role of interleukin 10 in patients with inflammatory bowel disease. Gastroenterology 1995; 108: 1434-1444 Yen D, Cheung J, Scheerens H, Poulet F, McClanahan T, McKenzie B, Kleinschek MA, Owyang A, Mattson J, Blumenschein W, Murphy E, Sathe M, Cua DJ, Kastelein RA, Rennick D. IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6. J Clin Invest 2006; 116: 1310-1316 Iwakura Y, Ishigame H. The IL-23/IL-17 axis in inflammation. J Clin Invest 2006; 116: 1218-1222 Hata K, Andoh A, Shimada M, Fujino S, Bamba S, Araki Y, Okuno T, Fujiyama Y, Bamba T. IL-17 stimulates inflammatory responses via NF-kappaB and MAP kinase pathways in human colonic myofibroblasts. Am J Physiol Gastrointest Liver Physiol 2002; 282: G1035-G1044 Hue S, Ahern P, Buonocore S, Kullberg MC, Cua DJ, McKenzie BS, Powrie F, Maloy KJ. Interleukin-23 drives innate and T cell-mediated intestinal inflammation. J Exp Med 2006; 203: 2473-2483 Kullberg MC, Jankovic D, Feng CG, Hue S, Gorelick PL, McKenzie BS, Cua DJ, Powrie F, Cheever AW, Maloy KJ, Sher A. IL-23 plays a key role in Helicobacter hepaticus-induced T cell-dependent colitis. J Exp Med 2006; 203: 2485-2494 Fujino S, Andoh A, Bamba S, Ogawa A, Hata K, Araki Y, Bamba T, Fujiyama Y. Increased expression of interleukin 17 in inflammatory bowel disease. Gut 2003; 52: 65-70 Duerr RH, Taylor KD, Brant SR, Rioux JD, Silverberg MS, Daly MJ, Steinhart AH, Abraham C, Regueiro M, Griffiths A, Dassopoulos T, Bitton A, Yang H, Targan S, Datta LW, Kistner EO, Schumm LP, Lee AT, Gregersen PK, Barmada MM, Rotter JI, Nicolae DL, Cho JH. A genome-wide association study identifies IL23R as an inflammatory bowel disease gene. Science 2006; 314: 1461-1463 Bamias G, Martin C 3rd, Marini M, Hoang S, Mishina M, Ross WG, Sachedina MA, Friel CM, Mize J, Bickston SJ, Pizarro TT, Wei P, Cominelli F. Expression, localization, and functional activity of TL1A, a novel Th1-polarizing cytokine in inflammatory bowel disease. J Immunol 2003; 171: 4868-4874 Prehn JL, Mehdizadeh S, Landers CJ, Luo X, Cha SC, Wei P, Targan SR. Potential role for TL1A, the new TNF-family member and potent costimulator of IFN-gamma, in mucosal

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of oral tolerance. J Immunol 1998; 160: 5815-5825 55 Rescigno M, Urbano M, Valzasina B, Francolini M, Rotta G, Bonasio R, Granucci F, Kraehenbuhl JP, Ricciardi-Castagnoli P. Dendritic cells express tight junction proteins and penetrate gut epithelial monolayers to sample bacteria. Nat Immunol 2001; 2: 361-367 56 Watanabe M, Takaishi H, Hosoda Y, Ezaki T, Yajima T, Inoue N, Ueno Y, Iwao Y, Ishii H, Ishikawa H. CD4+ intestinal mucosal lymphocytes in the pathogenesis of Crohn's disease. J Gastroenterol 1995; 30 Suppl 8: 73-75 57 Kraus TA, Mayer L. Oral tolerance and inflammatory bowel disease. Curr Opin Gastroenterol 2005; 21: 692-696 58 Steinman RM, Hawiger D, Nussenzweig MC. Tolerogenic dendritic cells. Annu Rev Immunol 2003; 21: 685-711 59 Baumgart DC, Metzke D, Schmitz J, Scheffold A, Sturm A, Wiedenmann B, Dignass AU. Patients with active inflammatory bowel disease lack immature peripheral blood plasmacytoid and myeloid dendritic cells. Gut 2005; 54: 228-236 60 Mayer L. Epithelial cell antigen presentation. Curr Opin Gastroenterol 2000; 16: 531-535 61 Torres MI, Le Discorde M, Lorite P, Rios A, Gassull MA, Gil A, Maldonado J, Dausset J, Carosella ED. Expression of HLA-G in inflammatory bowel disease provides a potential way to distinguish between ulcerative colitis and Crohn's disease. Int Immunol 2004; 16: 579-583 62 Rouas-Freiss N, Goncalves RM, Menier C, Dausset J, Carosella ED. Direct evidence to support the role of HLA-G in protecting the fetus from maternal uterine natural killer cytolysis. Proc Natl Acad Sci USA 1997; 94: 11520-11525 63 Rezaie A, Parker RD, Abdollahi M. Oxidative stress and pathogenesis of inflammatory bowel disease: an epiphenomenon or the cause? Dig Dis Sci 2007; 52: 2015-2021 64 Keshavarzian A, Morgan G, Sedghi S, Gordon JH, Doria M. Role of reactive oxygen metabolites in experimental colitis. Gut 1990; 31: 786-790 65 Babbs CF. Oxygen radicals in ulcerative colitis. Free Radic Biol Med 1992; 13: 169-181 66 Grisham MB, Volkmer C, Tso P, Yamada T. Metabolism of trinitrobenzene sulfonic acid by the rat colon produces reactive oxygen species. Gastroenterology 1991; 101: 540-547 67 Nieto N, Torres MI, Fernandez MI, Giron MD, Rios A, Suarez MD, Gil A. Experimental ulcerative colitis impairs antioxidant defense system in rat intestine. Dig Dis Sci 2000; 45: 1820-1827 68 Sido B, Hack V, Hochlehnert A, Lipps H, Herfarth C, Droge W. Impairment of intestinal glutathione synthesis in patients with inflammatory bowel disease. Gut 1998; 42: 485-492 69 Lih-Brody L, Powell SR, Collier KP, Reddy GM, Cerchia R, Kahn E, Weissman GS, Katz S, Floyd RA, McKinley MJ, Fisher SE, Mullin GE. Increased oxidative stress and decreased antioxidant defenses in mucosa of inflammatory bowel disease. Dig Dis Sci 1996; 41: 2078-2086 70 Baumgart DC, Sandborn WJ. Inflammatory bowel disease: clinical aspects and established and evolving therapies. Lancet 2007; 369: 1641-1657 71 Grimble RF. Interaction between nutrients, pro-inflammatory cytokines and inflammation. Clin Sci (Lond) 1996; 91: 121-130 72 Riordan AM, Ruxton CH, Hunter JO. A review of associations between Crohn's disease and consumption of sugars. Eur J Clin Nutr 1998; 52: 229-238 73 Hunter JO. Nutritional factors in inflammatory bowel disease. Eur J Gastroenterol Hepatol 1998; 10: 235-237 74 Calder PC. Polyunsaturated fatty acids and inflammation. Prostaglandins Leukot Essent Fatty Acids 2006; 75: 197-202 75 Belluzzi A. N-3 fatty acids for the treatment of inflammatory bowel diseases. Proc Nutr Soc 2002; 61: 391-395 76 Fernandez-Banares F, Esteve-Comas M, Mane J, Navarro E, Bertran X, Cabre E, Bartoli R, Boix J, Pastor C, Gassull MA. Changes in mucosal fatty acid profile in inflammatorybowel disease and in experimental colitis: a common response to bowel inflammation. Clin Nutr 1997; 16: 177-183 77 Nieto N, Fernandez MI, Torres MI, Rios A, Suarez MD, Gil A. Dietary monounsaturated n-3 and n-6 long-chain polyunsaturated fatty acids affect cellular antioxidant defense www.wjgnet.com

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delivery of 5-aminosalicylic acid - new evidence. Aliment Pharmacol Ther 2006; 24 Suppl 1: 2-9 91 Meenan J, Grool TA, Hommes DW, Dijkhuizen S, ten Kate FJ, Wood M, Whittaker M, Tytgat GN, van Deventer SJ. Lexipafant (BB-882), a platelet activating factor receptor antagonist, ameliorates mucosal inflammation in an animal model of colitis. Eur J Gastroenterol Hepatol 1996; 8: 569-573 92 Galvez J, Garrido M, Merlos M, Torres MI, Zarzuelo A. Intestinal anti-inflammatory activity of UR-12746, a novel 5-ASA conjugate, on acute and chronic experimental colitis in the rat. Br J Pharmacol 2000; 130: 1949-1959 93 Chow DK, Leong RW. The use of tacrolimus in the treatment of inflammatory bowel disease. Expert Opin Drug Saf 2007; 6: 479-485 94 Pizarro TT, Cominelli F. Cytokine therapy for Crohn's disease: advances in translational research. Annu Rev Med 2007; 58: 433-444 95 Rutgeerts P, Van Assche G, Vermeire S. Optimizing anti-TNF treatment in inflammatory bowel disease. Gastroenterology 2004; 126: 1593-1610 96 de Ridder L, Benninga MA, Taminiau JA, Hommes DW, van Deventer SJ. Infliximab use in children and adolescents with inflammatory bowel disease. J Pediatr Gastroenterol Nutr 2007; 45: 3-14 97 Van Montfrans C, Hooijberg E, Rodriguez Pena MS, De Jong EC, Spits H, Te Velde AA, Van Deventer SJ. Generation of regulatory gut-homing human T lymphocytes using ex vivo interleukin 10 gene transfer. Gastroenterology 2002; 123: 1877-1888 98 Uhlig HH, Coombes J, Mottet C, Izcue A, Thompson C, Fanger A, Tannapfel A, Fontenot JD, Ramsdell F, Powrie F. Characterization of Foxp3+CD4+CD25+ and IL-10-secreting CD4+CD25+ T cells during cure of colitis. J Immunol 2006; 177: 5852-5860 99 Plevy S, Salzberg B, Van Assche G, Regueiro M, Hommes D, Sandborn W, Hanauer S, Targan S, Mayer L, Mahadevan U, Frankel M, Lowder J. A phase I study of visilizumab, a humanized anti-CD3 monoclonal antibody, in severe steroidrefractory ulcerative colitis. Gastroenterology 2007; 133: 1414-1422 100 Oyama Y, Craig RM, Traynor AE, Quigley K, Statkute L, Halverson A, Brush M, Verda L, Kowalska B, Krosnjar N, Kletzel M, Whitington PF, Burt RK. Autologous hematopoietic stem cell transplantation in patients with refractory Crohn's disease. Gastroenterology 2005; 128: 552-563 S- Editor Ma L L- Editor Alpini GD

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World J Gastroenterol 2008 April 7; 14(13): 1981-1983 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

REVIEW

Role of endoscopy in the management of acute diverticular bleeding Charalampos Pilichos, Emmanouil Bobotis Charalampos Pilichos, Department of Digestive Endoscopy, Sismanogleion General Hospital, Athens 15126, Greece Emmanouil Bobotis, Department of Surgery, Sismanogleion General Hospital, Athens 15126, Greece Author contributions: Pilichos C and Bobotis E contributed equally to this work. Correspondence to: Pilichos Charalampos, Department of Digestive Endoscopy, Sismanogleion General Hospital, Athens 15126, Greece. [email protected] Telephone: +30-210-8234515 Received: December 4, 2007 Revised: January 27, 2008

Abstract Colonic diverticulosis is one of the most common causes of lower gastrointestinal bleeding. Endoscopy is not only a useful diagnostic tool for localizing the bleeding site, but also a therapeutic modality for its management. To date, haemostatic methods have included adrenaline injection, mechanical clipping, thermal and electrical coagulation or combinations of them. The results of all published data are herein reviewed. © 2008 WJG . All rights reserved.

Key words: Diverticulosis; Colon; Bleeding; Endoscopy; Therapy Peer reviewer: Shingo Tsuji, Professor, Department of Internal

Medicine and Therapeutics, Osaka University Graduate School of Medicine (A8), 2-2 Yamadaoka, Suita, Osaka 5650871, Japan Pilichos C, Bobotis E. Role of endoscopy in the management of acute diverticular bleeding. World J Gastroenterol 2008; 14(13): 1981-1983 Available from: URL: http://www.wjgnet. com/1007-9327/14/1981.asp DOI: http://dx.doi.org/10.3748/ wjg.14.1981

Forty percent of lower gastrointestinal bleeding (LGB) is due to colonic diverticulae. Inversely, bleeding complicates only 5% of all cases of colonic diverticulosis[1]. In most of cases, hemorrhage ceases spontaneously; however, in 20% it persists, thus imposing an emergency treatment[2]. Endoscopic hemostasis is a proven therapy in some indications of LGB, such as in the post-polypectomy bleeding and the post-radiation rectitis[3,4]. Nevertheless, any attempt of endoscopic treatment presupposes either a certain diagnosis or a strong presumption of the bleeding

lesion, which may not always be evident. Since the end of 1980’s, the benefit of an emergency colonoscopy in this setting has been investigated, but yielded contradictory conclusions (Table 1). After adequate bowel preparation urgent colonoscopy is incomplete in 0%-45% of cases and the risk of complications does not exceed 11%. The method allows positive diagnosis in approximately two thirds of cases and hemostasis in one third and may result in a shorter duration of hospitalization[5-13]. The timing of colonoscopy has little or no impact on the diagnostic yield, but when it is performed early (within 24 h following admission) it seems associated with a better clinical outcome[12-14]. The criteria proposed by Zuckerman for attributing LGB to diverticulosis are inspired by the Forrest classification of bleeding gastro duodenal ulcers[15]. They include typical endoscopic stigmata such as active bleeding, visible vessel and adherent clot and presumptive findings, such as presence of fresh blood within one or more bowel segments and diverticular erosions. Using these criteria, a lower bleeding could undoubtedly be attributed to diverticulosis in only 20% of cases. In all other cases, the diverticular origin of bleeding is either presumed indirectly by the absence of any other lesion or considered as incidental[16]. Endoscopic treatment aims to stop active bleeding, reduce the risk of recurrence, diminish transfusion needs and avoid surgery. To date, haemostatic methods have included adrenaline injection, mechanical clipping, thermal and electrical coagulation or associations of them. All of them are of similar efficacy, but mechanical clipping also offers the theoretical advantage of marking the bleeding site, which might be useful in case of relapse[17]. Beyond these traditional techniques, Farrell, based on the previous work of Witte et al, has reported 4 cases of diverticular bleeding treated by elastic band ligation and suggested that this might be a promising method not only for the hemostasis, but for diverticular reversion as well [18,19]. Concerning APC, despite its excellent results in vascular malformations, it has never been tested in the setting of diverticular hemorrhage, to our knowledge. Apart of three original studies including only one prospective trial, all other published data are limited to some case reports. In the first study, Jensen et al compared 10 endoscopically managed patients with certain diverticular bleeding to 17 non-treated historical controls. Endoscopic treatment included diluted adrenaline injections in cases of active bleeding, bipolar coagulation in cases of visible vessel and association of www.wjgnet.com

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Table 1 Summarized data of the largest series evaluating the efficacy of endoscopy in the diagnosis and the management of lower gastrointestinal bleeding Author

Publication year Number of patients Incomplete colonoscopy (%) Positive diagnosis (%) Endoscopic hemostasis (%) Complications (%)

Jensen[5] Richter[6] Geller[7] Kok[8] Prakash[9] Chaudhry[10] Ohyama[11] Schmulewitz[12] Strate[13]

1988 1995 1997 1998 1998 1998 2000 2003 2003

80 78 524 190 30 85 345 415 144

0 11.5 2 16 0 2 45 NS 5

74 90 42 78 60 97 89 89 90

39 13 17 5 6 31 14 10 10

5 NS 0.5 0 0 3.5 11 0.002 NS

NS: Not stated.

Table 2 Summarized data of all studies evaluating the efficacy of therapeutic endoscopy in the management of diverticular bleeding Publication year

Type of study

n

Therapy

Early recurrence

Complications

Follow-up period (mo)

Johnston[21] Bertoni[22] Kim[23] Savides[24] Foutch[25] Ramirez[26] Hokama[27] Prakash[28] Jensen[16]

1986 1990 1993 1994 1996 1996 1997 1999 2000

Case Report Case Report Case Report Case Report Case Report Case Report Case Report Case Report Prospective

4 1 1 3 4 4 3 3 10

0 0 0 0 1 0 0 0 0

0 0 0 0 0 0 0 0 0

NS NS NS NS NS NS NS NS NS

Ohyama[11] Bloomfeld[20] Smoot[14]

2000 2001 2003

Retrospective Retrospective Retrospective

6 13 7

0 5 0

0 0 0

NS 35 NS

Cuillerier[29] Simpson[30] Total

2003 2004

Case Report Case Report

2 2 63

Coagulation ADR ADR Coagulation Coagulation ADR Clips Coagulation ADR, Coagulation, ADR + Coagulation ADR ± Coagulation ± Clips ADR ± Coagulation ADR (4), Clips (2), Thermocoagulation (1) ADR ADR + Clips

0 0 6 (9.5%)

0 0 0 (0%)

3-18 4-30

Author

NS: Not stated.

both methods in cases of an adherent clot. None of the treated patients relapsed during a 30 mo observation period, whereas 9 of the 17 controls (52%) presented a recurrence within 3 years and 6 of them (35%) failed to avoid surgery[16]. In the study of Bloomfeld, although the haemostatic methodology was similar to the previous study, results were less encouraging. Of 13 patients with active diverticular bleeding endoscopically treated, 5 and 3 presented an early or a late recurrence (up to 35 mo), respectively, thus a success rate of 46%[20]. Finally, in the most recent study of Smoot et al, endoscopic haemostasis carried out in 7 patients actively bleeding, was proven both effective and free of complications[14]. The methodology and the results of all relevant studies and reports are presented in Table 2. Summarizing the above data, one could say that out of 63 patients endoscopically managed, 6 (9.5%) presented an early relapse of bleeding and none a method-related complication. However, the long-term efficacy of endoscopic therapies is not demonstrated and randomized trials including larger number of patients are needed to make safe conclusions. Thus, based on the above data one can conclude that in cases of lower GI bleeding, urgent colonoscopy after an adequate bowel preparation should be attempted. Endoscopic hemostasis might help patients to avoid at www.wjgnet.com

least emergency surgery, if a diverticular origin seems certain or highly probable.

REFERENCES 1 2 3 4

5 6

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Tudor RG, Farmakis N, Keighley MR. National audit of complicated diverticular disease: analysis of index cases. Br J Surg 1994; 81: 730-732 McGuire HH Jr. Bleeding colonic diverticula. A reappraisal of natural history and management. Ann Surg 1994; 220: 653-656 Sorbi D, Norton I, Conio M, Balm R, Zinsmeister A, Gostout CJ. Postpolypectomy lower GI bleeding: descriptive analysis. Gastrointest Endosc 2000; 51: 690-696 Canard JM, Vedrenne B, Bors G, Claude P, Bader R, Sondag D. Long term results of treatment of hemorrhagic radiation proctitis by argon plasma coagulation. Gastroenterol Clin Biol 2003; 27: 455-459 Jensen DM, Machicado GA. Diagnosis and treatment of severe hematochezia. The role of urgent colonoscopy after purge. Gastroenterology 1988; 95: 1569-1574 Richter JM, Christensen MR, Kaplan LM, Nishioka NS. Effectiveness of current technology in the diagnosis and management of lower gastrointestinal hemorrhage. Gastrointest Endosc 1995; 41: 93-98 Geller A, Mayoral W, Balm R, Geller N, Gostout C. Colonoscopy in acute lower gastrointestinal bleeding [abstract]. Gastrointest Endosc 1997; 45: AB107 Kok KY, Kum CK, Goh PM. Colonoscopic evaluation of severe hematochezia in an Oriental population. Endoscopy 1998; 30:

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675-680 Prakash C, Zuckerman GR, Aliperti G, Walden DT, Royal HD, Willis J-R. Prospective analysis of work-up of acute lower gastrointestinal bleeding. Can an optimal algorithm be designed? Gastrointest Endosc 1998; 47: AB102 Chaudhry V, Hyser MJ, Gracias VH, Gau FC. Colonoscopy: the initial test for acute lower gastrointestinal bleeding. Am Surg 1998; 64: 723-728 Ohyama T, Sakurai Y, Ito M, Daito K, Sezai S, Sato Y. Analysis of urgent colonoscopy for lower gastrointestinal tract bleeding. Digestion 2000; 61: 189-192 Schmulewitz N, Fisher DA, Rockey DC. Early colonoscopy for acute lower GI bleeding predicts shorter hospital stay: a retrospective study of experience in a single center. Gastrointest Endosc 2003; 58: 841-846 Strate LL, Syngal S. Timing of colonoscopy: impact on length of hospital stay in patients with acute lower intestinal bleeding. Am J Gastroenterol 2003; 98: 317-322 Smoot RL, Gostout CJ, Rajan E, Pardi DS, Schleck CD, Harmsen WS, Zinsmeister AR, Nolte T, Melton LJ. Is early colonoscopy after admission for acute diverticular bleeding needed? Am J Gastroenterol 2003; 98: 1996-1999 Zuckerman GR, Prakash C. Acute lower intestinal bleeding: part I: clinical presentation and diagnosis. Gastrointest Endosc 1998; 48: 606-617 Jensen DM, Machicado GA, Jutabha R, Kovacs TO. Urgent colonoscopy for the diagnosis and treatment of severe diverticular hemorrhage. N Engl J Med 2000; 342: 78-82 Elta GH. Urgent colonoscopy for acute lower-GI bleeding. Gastrointest Endosc 2004; 59: 402-408 Farrell JJ, Graeme-Cook F, Kelsey PB. Treatment of bleeding colonic diverticula by endoscopic band ligation: an in-vivo and ex-vivo pilot study. Endoscopy 2003; 35: 823-829 Witte JT. Band ligation for colonic bleeding: modification of multiband ligating devices for use with a colonoscope.

1983 Gastrointest Endosc 2000; 52: 762-765 Bloomfeld RS, Rockey DC, Shetzline MA. Endoscopic therapy of acute diverticular hemorrhage. Am J Gastroenterol 2001; 96: 2367-2372 21 Johnston J, Sones J. Endoscopic heater probe coagulation of the bleeding colonic diverticulum. Gastrointest Endosc 1986; 84: AB168 22 Bertoni G, Conigliaro R, Ricci E, Mortilla MG, Bedogni G, Fornaciari G. Endoscopic injection hemostasis of colonic diverticular bleeding: a case report. Endoscopy 1990; 22: 154-155 23 Kim YI, Marcon NE. Injection therapy for colonic diverticular bleeding. A case study. J Clin Gastroenterol 1993; 17: 46-48 24 Savides TJ, Jensen DM. Colonoscopic hemostasis for recurrent diverticular hemorrhage associated with a visible vessel: a report of three cases. Gastrointest Endosc 1994; 40: 70-73 25 Foutch PG, Zimmerman K. Diverticular bleeding and the pigmented protuberance (sentinel clot): clinical implications, histopathological correlation, and results of endoscopic intervention. Am J Gastroenterol 1996; 91: 2589-2593 26 Ramirez FC, Johnson DA, Zierer ST, Walker GJ, Sanowski RA. Successful endoscopic hemostasis of bleeding colonic diverticula with epinephrine injection. Gastrointest Endosc 1996; 43: 167-170 27 Hokama A, Uehara T, Nakayoshi T, Uezu Y, Tokuyama K, Kinjo F, Saito A. Utility of endoscopic hemoclipping for colonic diverticular bleeding. Am J Gastroenterol 1997; 92: 543-546 28 Prakash C, Chokshi H, Walden DT, Aliperti G. Endoscopic hemostasis in acute diverticular bleeding. Endoscopy 1999; 31: 460-463 29 Cuillerier E, Landi B, Berger A, Barbier JP, Marteau P. Endoscopic treatment of lower intestinal haemorrhage of diverticular origin. Gastroenterol Clin Biol 2003; 27: 558-559 30 Simpson PW, Nguyen MH, Lim JK, Soetikno RM. Use of endoclips in the treatment of massive colonic diverticular bleeding. Gastrointest Endosc 2004; 59: 433-437 20

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World J Gastroenterol 2008 April 7; 14(13): 1984-1989 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

TOPIC HIGHLIGHT Peter Draganov, Dr, Series Editor

Endoscopic mucosal resection in the upper gastrointestinal tract Anis Ahmadi, Peter Draganov Anis Ahmadi, Peter Draganov, Division Gastroenterology Hepatology and Nutrition, University of Florida, Gainesville, Florida 32610, United States Author contributions: Ahmadi A and Draganov P contributed equally to this work. Correspondence to: Peter Draganov, Dr, Division Gastroenterology Hepatology and Nutrition, University of Florida, Gainesville, 1600 SW Archer Road PO Box 100214, Florida 32610, United States. [email protected] Telephone: +1-352-3922877  Fax: +1-352-3923618 Received: December 15, 2007 Revised: January 31, 2008

Abstract Endoscopic mucosal resection (EMR) is a technique used to locally excise lesions confined to the mucosa. Its main role is the treatment of advanced dysplasia and early gastrointestinal cancers. EMR was originally described as a therapy for early gastric cancer. Recently its use has expanded as a therapeutic option for ampullary masses, colorectal cancer, and large colorectal polyps. In the Western world, the predominant indication for EMR in the upper gastrointestinal tract is the staging and treatment of advance dysplasia and early neoplasia in Barrett’s esophagus. This review will describe the basis, indications, techniques, and complications of EMR, and its role in the management of Barrett’s esophagus. © 2008 WJG . All rights reserved.

Key words: Endoscopic mucosal resection; Mucosal resection; Barrett’s esophagus; Barrett’s dysplasia; Therapeutic endoscopy Peer reviewer: Ryuichi Iwakiri, Dr, Department of Medicine and Gastrointestinal Endoscopy, Saga Medical School, 5-1-1 Nabeshima, Saga 849-8501, Japan

Ahmadi A, Draganov P. Endoscopic mucosal resection in the upper gastrointestinal tract. World J Gastroenterol 2008; 14(13): 1984-1989 Available from URL: http://www.wjgnet. com/1007-9327/14/1984.asp DOI: http://dx.doi.org/10.3748/ wjg.14.1984

INTRODUCTION Endoscopic mucosal resection (EMR) is a technique used www.wjgnet.com

to locally excise lesions confined to the mucosa. Its main role in therapeutic endoscopy is the treatment of advanced dysplasia and early gastrointestinal (GI) cancers[1-4]. Tada et al published the first case report of EMR in 1984 as a treatment option for early-stage gastric carcinoma[5]. Since then, EMR has been used diagnostically and therapeutically in both the upper and lower GI tracts, and has significantly lower morbidity and mortality compared to surgical management[6]. EMR is most commonly used to locally treat early gastric and esophageal dysplasia/cancer. However, there are several reports describing EMR as a therapeutic option for ampullary masses, colorectal cancer, and large colorectal polyps [7-10]. In the Western world, the predominant indication for EMR in the upper GI tract is the staging and treatment of early neoplasia in Barrett’s esophagus. This review will describe the basis, indications, techniques, and complications of EMR, and its role in the management of Barrett’s esophagus.

BASIS OF EMR The GI wall is comprised of 5 layers: mucosa, deep mucosa, submucosa, muscularis propria, and serosa or adventitia. The two major components are the mucosal and the muscular layer. The submucosa consists of loose connective tissue that attaches the above layers together. The mucosal and muscular layers can be separated from each other by injection of fluid into the submucosal layer. This allows for safe resection of mucosal lesions without causing damage of the deeper muscle layer[11]. EMR has become standard practice because of its following distinct advantages: (1) advanced dysplasia and most early neoplastic lesions are free of lymph-node metastases, and can be treated with curative intent simply by local resection[12]; (2) EMR provides tissue specimen for histology and staging[13]; (3) EMR is minimally invasive and carries lower morbidity and mortality compared to traditional surgical approaches[14]; and (4) surgery can be performed after EMR if advanced neoplasia or incomplete resection is detected on histologic evaluation of the EMR specimen. EMR also has some disadvantages: (1) EMR is labor intense, time consuming, and requires advanced endoscopic skills; (2) larger lesions can only be resected in piecemeal fashion which precludes evaluation for completeness of the resection at the lateral margins; (3)

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there is uncertainty regarding the long term outcome of patients treated with EMR due to the lack of randomized trials directly comparing EMR with surgery; and (4) EMR is poorly reimbursed in the US.

TECHNIQUES OF EMR Once a mucosal lesion is identified, it is helpful to perform chromoendoscopy ± endoscopic ultrasound to further define the size and borders of the lesion, and determine its depth of invasion[15-17]. Both chromoendoscopy and narrow band imaging can help improve detection of dysplastic lesions, and further delineate the borders of the lesion. Additionally, computer tomography (CT) imaging can help determine the size of the lesion and rule out distant metastatic disease prior to proceeding with EUS. Although it can be challenging to determine intramucosal from submucosal neoplasias, the risk of pre-existing lymph node metastases must be discussed with the patient prior to pursuing EMR. Ultimately, histopathologic evaluation of the EMR specimen is the most important predictor of lymph node metastasis. Surgical management of early esophageal/gastric malignancies should be based on the histological analysis of the EMR specimen (i.e. depth of invasion) as well as each patient’s surgical morbidity and mortality risks. Several EMR techniques have been described in the literature. Multiple EMR techniques are available: (1) stripoff biopsy; (2) “inject, lift, and cut” method; (3) the “cup and suction” or EMR-cap technique; and lastly (4) EMR with band ligation[18]. Prior to pursuing any of the above methods, it is recommended that the periphery of the lesion be marked with either a needle knife, electrocautery, or argon plasma coagulator (APC). This allows for distinct identification of the borders of the lesion that is being excised. The absence of all markings assists in determining if the resection is complete. Depending on the EMR technique used, lift injection may be required. No standardization of the type of injection solution exists. Various injectates that have been used include normal saline, normal saline plus epinephrine solution, 50% dextrose in normal saline, 10% glycerine/5% fructose in normal saline, hyaluronic acid, and a mixture of methylene blue and normal saline[18,19]. Injection of these various solutions can help lift the mucosa from the submucosa, and theoretically decrease the risk of perforation and reduce the risk of hemorrhage[20]. An additional advantage of injecting prior to EMR is identification of lesions that do not successfully lift, which generally suggests involvement of the submucosal layer, and thus are not candidates for resection[15]. Multiple electrosurgical currents are used during EMR, including blend, cut, and coagulation settings, depending primarily on operator preference. The electrosurgical setting most commonly used in the esophagus at Leeds is the ERBE “endo-cut mode” with a power setting of 45 watts[21]. In a recent editorial by Seewald et al, a pure coagulation current with the Erbotom at a setting 3- and 60-W output was used for electrosurgical resection[22]. (1) The strip biopsy is the least complex EMR technique, but is often limited to polypoid or nodular

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lesions only [23]. It is similar to standard polypectomy. Injection into the submucosa is not done. A diathermy snare is tightened around a lesion, which is subsequently removed with the application of an electrical cutting current. The strip biopsy technique can be applied to flat lesions by using either a barbed snare or ultra stiff snare[24]. (2) The “inject, lift, and cut” method is similar to the strip-off method. A two channel upper endoscope is needed. Prior to snaring the lesion, a submucosal injection is used to effectively lift the mucosa from the submucosa, thereby potentially reducing the risk of perforating the muscular layer. The lesion is then lifted by forceps and situated into a snare (via the second channel of a dualchannel endoscope), such that the lesion is resected at the base by applying electrocautery[20]. (3) The “cup and suction” or EMR-cap technique is the most frequently used method of EMR in the esophagus[25]. This technique was first described by Inohue et al in 1993[26]. This method requires a transparent plastic cap be attached to the distal tip of a single-channel endoscope. This is followed by injection of approximately 20 mL of lifting solution into the submucosa. A designated “duck bill” small-diameter snare is then placed within the rim of the transparent cap. Following this, the lesion is sucked into the cap as the snare is closed at the base of the lesion. Once suction has been released and it has been determined that the entire lesion is contained within the snare, the lesion is removed using electrocautery[27]. The advantages of the EMR-cap technique are that a standard single-channel endoscope can be used, and only one endoscopy assistant is required. In addition, it appears that there is a lower risk of bleeding compared to the stripoff method[21,25]. The disadvantage is that it may be difficult to ensure that the entire lesion has been aspirated into the cap, and occasionally, visualization of the lesion can be obscured by the cap itself. Furthermore, the snare tends to loose its shape after a single use and thus a new snare is usually required for each piece of tissue removed. This can quickly add to the cost of the procedure, particularly if a large surface area needs to be removed (e.g. long segment Barrett’s esophagus). (4) EMR with ligation is similar to the EMR-cap technique, in that suction of the lesion is required. However, unlike the EMR-cap technique, the lesion is suctioned into a ligation cylinder without prior submucosal injection. A rubber band is then deployed at the base to create a pseudopolyp. The pseudopolyp is subsequently removed at its base by tightening a snare just below the level of the rubber band[28]. The standard band ligator can be used, but a designated ligator that fits the single channel therapeutic upper scope is available (Duette, Cook Medical, Winston-Salem, NC). The main disadvantage of this technique with the standard ligator is that the endoscope must be withdrawn to remove the ligation cylinder before reinsertion for resection[29]. The Duette system, on the other hand, allows the passage of a snare via the therapeutic channel of the scope and multiple resections can be carried out sequentially without the need of removing the scope. The EMR-cap and EMR with ligation techniques have been prospectively compared to each other in a singlewww.wjgnet.com

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center study performed by May A et al. In this study, 50 EMR-cap resections were compared to 50 EMR with ligation resections of early stage esophageal cancer. No significant difference in the maximal diameter of the resection specimen area was noted between the two groups. In addition, only one minor episode of bleeding was seen in each group, with no severe complications in either group. Therefore, it was concluded that both techniques are similar in efficacy and safety[30].

clipping, nasogastric tube suction, and broad-spectrum antibiotics[43]. Patients are at an increased risk of developing strictures if circumferential resections of the esophagus or gastric pylorus are performed[29,44,45]. These strictures are often responsive to dilation. To date, animal studies have not identified that prophylactic balloon dilation, esophageal stenting, or deep mural steroid injections prevent the formation of strictures[44].

ENDOSCOPIC SUBMUCOSAL DISSECTION (ESD)

INDICATIONS FOR EMR IN THE UPPER GI TRACT

ESD, one of the more recently described techniques, has been developed to perform single en-block resections of large mucosal lesions[31]. This technique involves the use of an electrocautery knife to dissect out mucosal lesions. Several knives have been developed for ESD, including triangle-tip knives, hook knives, insulation-tip knives, and flex knives[32-34]. In ESD with use of a triangle-tip knife, the borders of a mucosal lesion are marked by electrocautery and then injection of an epinephrine-saline solution into the submucosa is performed. This is followed by marginal cutting circumferentially, via electrocautery, around the previous markings. At this point, a high-viscosity solution (such as hyaluronic acid) is injected to provide a longer period of mucosal lifting to allow for actual submucosal dissection. Dissection is carried out by electro cauterization using the tip of the triangle-tip knife to free the mucosal lesion from the submucosa. Once completed, the freed mucosa is removed[27]. At present, the various knives used for ESD are not available in the US.

Gastric malignancy Per the Japanese literature, the indications for EMR in resection of superficial gastric cancers applies to well- or moderately differentiated adenocarcinoma and/or papillary carcinoma. Gastric cancers that penetrate the submucosa are at increased risk of lymph node metastases. Gastric cancer confined to the mucosa has a 0%-5% risk of lymph node metastases, compared to 10%-20% risk if the cancer involves the submucosa[46-49]. Thus, gastric lesions must meet the following criteria to be candidates for EMR: confined to the mucosa, < 2 cm for elevated lesions, < 1 cm for flat or depressed lesions, cannot be associated with an ulcer or ulcer scar, and cannot have venous or lymphatic involvement[50].

EMR COMPLICATIONS As with any endoscopic procedure, complication rates are operator dependent and diminish with increased experience. The Japanese Society of Gastrointestinal Endoscopy calculated a complication rate of 0.5% based on all upper GI EMR’s performed between 1993 to 1997[35]. A lower overall complication rate of 0.17% was reported by Kaneko et al in 1995. The mortality rate calculated by Kaneko et al was 0.0001%[36]. The risks of EMR include bleeding, pain, perforation, and stricture formation. Bleeding can occur at the time of the procedure, or be delayed for up to 12 h. Bleeding rarely occurs beyond 24 h after the procedure. Venous oozing is more common following esophageal EMR, whereas brisk bleeding is more common after gastric EMR of large and fundic lesions. Bleeding is most commonly treated endoscopically via electrocauterization, APC, or placement of metallic clips[37-40]. Dull pain following EMR generally results from denudation of the mucosa and subsequent exposure to gastric acid. This pain can often be controlled by protonpump inhibitors. Sudden sharp pain, especially during or at the completion of the procedure, should raise suspicion of a perforation[41]. The risk of perforation following EMR of gastric lesions is 0.06%-5%[42]. Upper GI tract perforation can be managed conservatively with the combination of www.wjgnet.com

Esophageal squamous cell carcinoma Currently, EMR is generally indicated for superficial wellor moderately differentiated squamous cell carcinoma without venous or lymphatic involvement that is limited to the lamina propria. This is based on a 0% risk of metastasis when the neoplasia is limited to the epithelium, compared to the 12% and 26% risk of metastasis when the neoplasia involves the muscularis mucosa and submucosa, respectively[51]. There is no consensus on the maximum size of the lesion that can be resected. EMR is not recommended for circumferential lesions secondary to the risk of subsequent stricture formation[15]. High-grade dysplasia and early adenocarcinoma in Barrett’s esophagus (BE) BE is associated with a 30-fold increased risk of esophageal adenocarcinoma, which remains one of most rapidly rising cancers in the Western world[52-54]. Although EMR has a clear role in squamous cell carcinoma, its role in BE is not clearly defined, although studies have determined that EMR is effective in removing visible lesions in BE[24,30,55]. In fact, current evidence suggests that EMR of high-grade dysplasia (HGD) and early cancer (EC) has similar success rates as surgical treatment[56-60]. According to Ell et al, the indications for EMR in the setting of Barrett’s neoplasia include the following: lesions limited to the mucosa that are macroscopically flat, tumor size between 20-30 mm, and good to moderate differentiation on histology [56]. Additionally, research suggests that EMR has better diagnostic reproducibility compared to mucosal biopsies alone, implying a possible role in routine surveillance[61]. Certainly, the cost-effectiveness and availability of EMR

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would need to be considered prior to pursuing EMR as a primary tool in the surveillance of BE. In one of the largest studies evaluating the efficacy of EMR for treatment of HGD and EC, 97% complete remission was achieved in resection of “low-risk” lesions and 59% complete remission in resection of “high-risk” lesions. “Low-risk” lesions were defined as macroscopic lesions measuring up to 20 mm and limited to the mucosa. However, at an average 12 mo follow-up period, the combined recurrence and metachronous cancer rate was 14% [56]. In an Italian study, EMR was found to be an effective method of treating HGD and intramucosal cancer in 34 patients that did not have submucosal involvement, with all patients remaining in remission at a median followup of 34.9 mo. In addition, EMR changed the pretreatment diagnosis in 25.6% of the studied patients[62]. Recurrence of neoplastic disease after EMR is a potential limitation[56,59]. Therefore, circumferential resection, in which the targeted dysplastic lesion and the surrounding Barrett’s mucosa are removed, has also been studied[63]. In two separate studies from 2003 and 2006, no recurrent or metachronous lesions were reported. This is in stark contrast to an 11% recurrence rate with circumferential resections at a mean follow-up of 18 mo in a study by Giovannini et al[24,29,55]. In addition to EMR, multiple ablative techniques have been evaluated in the management of HGD and EC in BE. These include photodynamic therapy (PDT), argon beam coagulation therapy, lasers, radiofrequency ablation, and yttrium-aluminum-garnet laser therapy[64-68]. In fact, a recently-published multicenter, randomized controlled trial has shown that photodynamic therapy with Photofrin is superior than omeprazole alone in eliminating HGD at 5 years follow-up (77% vs 39%, respectively)[69]. To improve eradication of neoplastic tissue and decrease recurrence rates, the combined use of EMR with ablative techniques have been described[59,70]. Theoretically, recurrence rates are expected to be much lower with combined modalities, because both visible and non-visible lesions would be eradicated. Combined modality has been evaluated by the Wiesbaden group in two separate studies. In the first study, 28 patients underwent EMR, 13 underwent PDT, 3 underwent APC, and 6 patients received a combination of these therapies for the treatment of HGD or EC. Metachronous or recurrent lesions were seen in 23% of the patients at a mean follow-up period of 34 ± 10 mo. Amongst the patients treated with EMR alone, 6/28 patients (21.4%) developed metachronous or recurrent lesions, compared to 1/6 patients (16.6%) treated with combined modalities[59]. In a follow-up study of a total of 115 patients (EMR = 70, PDT = 32, APC = 3, EMR + PDT = 10) undergoing endoscopic treatment for HGD or EC, there was a 31% rate of metachronous or recurrent lesions over an average follow-up time of 34 ± 10 mo. Individually, the metachronous or recurrence rate was 30% (21/70) in the EMR group vs 37.5% in the EMR + PDT group[70]. It is important to note that the number of patients treated with the combined modality is markedly less than that treated with EMR alone.

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Conclusion Since its introduction as a potential treatment option of GI mucosal cancers in 1984, the indications for EMR are continuing to expand. Today, EMR has become an integral part of the therapeutic endoscopy ar mamentarium. Although there are no specific guidelines for EMR as a treatment option for HGD or early cancer in Barrett’s esophagus, the literature indicates that EMR is similar to surgery in efficacy, but has less morbidity and mortality. As newer techniques of EMR, including circumferential mucosectomy, are developed, the potential of reducing recurrence and metachronous rates are inviting.

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Kaneko E, Hanada H, Kasugai T, Sakita T. The survey of gastrointestinal endoscopic complications in Japan (1993-1997, in Japanese). Gastroenterol Endosc 2000; 42: 308-313 36 Kaneko E, Harada H, Kasugai T, Sakita T. The results of a multi-center analysis from 1988-1992 (in Japanese). Gastroenterol Endosc 1995; 37: 642-652 37 Fujishiro M, Ono H, Gotoda T, Yamaguchi H, Kondo H, Saito D. Usefulness of Maalox for detection of the precise bleeding points and confirmation of hemostasis on gastrointestinal hemorrhage. Endoscopy 2001; 33: 196 38 Szaloki T, Toth V, Tiszlavicz L, Czako L. Flat gastric polyps: results of forceps biopsy, endoscopic mucosal resection, and long-term follow-up. Scand J Gastroenterol 2006; 41: 1105-1109 39 Fujishiro M, Yahagi N, Nakamura M, Kakushima N, Kodashima S, Ono S, Kobayashi K, Hashimoto T, Yamamichi N, Tateishi A, Shimizu Y, Oka M, Ichinose M, Omata M. Safety of argon plasma coagulation for hemostasis during endoscopic mucosal resection. Surg Laparosc Endosc Percutan Tech 2006; 16: 137-140 40 Peters FP, Kara MA, Rosmolen WD, Aalders MC, Ten Kate FJ, Bultje BC, Krishnadath KK, Fockens P, van Lanschot JJ, van Deventer SJ, Bergman JJ. Endoscopic treatment of highgrade dysplasia and early stage cancer in Barrett's esophagus. Gastrointest Endosc 2005; 61: 506-514 41 Soetikno RM, Gotoda T, Nakanishi Y, Soehendra N. Endoscopic mucosal resection. Gastrointest Endosc 2003; 57: 567-579 42 Tada M. One piece resection and piecemeal resection of early gastric cancer by strip biopsy. Tokyo: Igaku-Shoin, 1998: 68-87 43 Takeshita K, Tani M, Inoue H, Saeki I, Hayashi S, Honda T, Kando F, Saito N, Endo M. Endoscopic treatment of early oesophageal or gastric cancer. Gut 1997; 40: 123-127 44 Rajan E, Gostout C, Feitoza A, Herman L, Knipschield M, Burgart L, Chung S, Cotton P, Hawes R, Kalloo A, Kantsevoy S, Pasricha P. Widespread endoscopic mucosal resection of the esophagus with strategies for stricture prevention: a preclinical study. Endoscopy 2005; 37: 1111-1115 45 Katada C, Muto M, Manabe T, Boku N, Ohtsu A, Yoshida S. Esophageal stenosis after endoscopic mucosal resection of superficial esophageal lesions. Gastrointest Endosc 2003; 57: 165-169 46 Moreaux J, Bougaran J. Early gastric cancer. A 25-year surgical experience. Ann Surg 1993; 217: 347-355 47 Perri F, Iuliano R, Valente G, Angelillo IF, Arrigoni A, Campra D, Recchia S, Andriulli A. Minute and small early gastric cancers in a Western population: a clinicopathologic study. Gastrointest Endosc 1995; 41: 475-480 48 Baba H, Maehara Y, Okuyama T, Orita H, Anai H, Akazawa K, Sugimachi K. Lymph node metastasis and macroscopic features in early gastric cancer. Hepatogastroenterology 1994; 41: 380-383 49 Endo M, Habu H. Clinical studies of early gastric cancer. Hepatogastroenterology 1990; 37: 408-410 50 Tsujitani S, Oka S, Saito H, Kondo A, Ikeguchi M, Maeta M, Kaibara N. Less invasive surgery for early gastric cancer based on the low probability of lymph node metastasis. Surgery 1999; 125: 148-154 51 Kodama M, Kakegawa T. Treatment of superficial cancer of the esophagus: a summary of responses to a questionnaire on superficial cancer of the esophagus in Japan. Surgery 1998; 123: 432-439 52 Brown LM, Devesa SS. Epidemiologic trends in esophageal and gastric cancer in the United States. Surg Oncol Clin N Am 2002; 11: 235-256 53 Devesa SS, Blot WJ, Fraumeni JF Jr. Changing patterns in the incidence of esophageal and gastric carcinoma in the United States. Cancer 1998; 83: 2049-2053 54 Falk GW. Barrett's esophagus. Gastroenterology 2002; 122: 1569-1591 55 Giovannini M, Bories E, Pesenti C, Moutardier V, Monges G, Danisi C, Lelong B, Delpero JR. Circumferential endoscopic mucosal resection in Barrett's esophagus with high-grade intraepithelial neoplasia or mucosal cancer. Preliminary 35

Ahmadi A et al. Upper GI endoscopic mucosal resection results in 21 patients. Endoscopy 2004; 36: 782-787 56 Ell C, May A, Gossner L, Pech O, Gunter E, Mayer G, Henrich R, Vieth M, Muller H, Seitz G, Stolte M. Endoscopic mucosal resection of early cancer and high-grade dysplasia in Barrett's esophagus. Gastroenterology 2000; 118: 670-677 57 Ciocirlan M, Lapalus MG, Hervieu V, Souquet JC, Napoleon B, Scoazec JY, Lefort C, Saurin JC, Ponchon T. Endoscopic mucosal resection for squamous premalignant and early malignant lesions of the esophagus. Endoscopy 2007; 39: 24-29 58 Esaki M, Matsumoto T, Hirakawa K, Nakamura S, Umeno J, Koga H, Yao T, Iida M. Risk factors for local recurrence of superficial esophageal cancer after treatment by endoscopic mucosal resection. Endoscopy 2007; 39: 41-45 59 May A, Gossner L, Pech O, Fritz A, Gunter E, Mayer G, Muller H, Seitz G, Vieth M, Stolte M, Ell C. Local endoscopic therapy for intraepithelial high-grade neoplasia and early adenocarcinoma in Barrett's oesophagus: acute-phase and intermediate results of a new treatment approach. Eur J Gastroenterol Hepatol 2002; 14: 1085-1091 60 Ell C, May A, Pech O, Gossner L, Guenter E, Behrens A, Nachbar L, Huijsmans J, Vieth M, Stolte M. Curative endoscopic resection of early esophageal adenocarcinomas (Barrett's cancer). Gastrointest Endosc 2007; 65: 3-10 61 Mino-Kenudson M, Hull MJ, Brown I, Muzikansky A, Srivastava A, Glickman J, Park DY, Zuckerberg L, Misdraji J, Odze RD, Lauwers GY. EMR for Barrett's esophagus-related superficial neoplasms offers better diagnostic reproducibility than mucosal biopsy. Gastrointest Endosc 2007; 66: 660-666; quiz 767, 769 62 Conio M, Repici A, Cestari R, Blanchi S, Lapertosa G, Missale G, Della Casa D, Villanacci V, Calandri PG, Filiberti R. Endoscopic mucosal resection for high-grade dysplasia and intramucosal carcinoma in Barrett's esophagus: an Italian

1989 experience. World J Gastroenterol 2005; 11: 6650-6655 63 Seewald S, Ang TL, Soehendra N. Endoscopic mucosal resection of Barrett's oesophagus containing dysplasia or intramucosal cancer. Postgrad Med J 2007; 83: 367-372 64 Barham CP, Jones RL, Biddlestone LR, Hardwick RH, Shepherd NA, Barr H. Photothermal laser ablation of Barrett's oesophagus: endoscopic and histological evidence of squamous re-epithelialisation. Gut 1997; 41: 281-284 65 Dumoulin FL, Terjung B, Neubrand M, Scheurlen C, Fischer HP, Sauerbruch T. Treatment of Barrett's esophagus by endoscopic argon plasma coagulation. Endoscopy 1997; 29: 751-753 66 Attwood SE, Lewis CJ, Caplin S, Hemming K, Armstrong G. Argon beam plasma coagulation as therapy for high-grade dysplasia in Barrett's esophagus. Clin Gastroenterol Hepatol 2003; 1: 258-263 67 Weston AP, Sharma P. Neodymium:yttrium-aluminum garnet contact laser ablation of Barrett's high grade dysplasia and early adenocarcinoma. Am J Gastroenterol 2002; 97: 2998-3006 68 Panjehpour M, Overholt BF, Haydek JM, Lee SG. Results of photodynamic therapy for ablation of dysplasia and early cancer in Barrett's esophagus and effect of oral steroids on stricture formation. Am J Gastroenterol 2000; 95: 2177-2184 69 Overholt BF, Wang KK, Burdick JS, Lightdale CJ, Kimmey M, Nava HR, Sivak MV Jr, Nishioka N, Barr H, Marcon N, Pedrosa M, Bronner MP, Grace M, Depot M. Five-year efficacy and safety of photodynamic therapy with Photofrin in Barrett's high-grade dysplasia. Gastrointest Endosc 2007; 66: 460-468 70 May A, Gossner L, Pech O, Muller H, Vieth M, Stolte M, Ell C. Intraepithelial high-grade neoplasia and early adenocarcinoma in short-segment Barrett's esophagus (SSBE): curative treatment using local endoscopic treatment techniques. Endoscopy 2002; 34: 604-610 S- Editor Li DL L- Editor Rippe RA E- Editor Liu Y

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World J Gastroenterol 2008 April 7; 14(13): 1990-1996 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

GASTRIC CANCER

Relationship between cell adhesion molecules expression and the biological behavior of gastric carcinoma Yong-Quan Chu, Zai-Yuan Ye, Hou-Quan Tao, Yuan-Yu Wang, Zhong-Sheng Zhao

Yong-Quan Chu, Wenzhou Medical College, Wenzhou 325035, Zhejiang Province, China Zai-Yuan Ye, Hou-Quan Tao, Yuan-Yu Wang, Department of Gastroenterology, Zhejiang Provincial People’s Hospital, Hangzhou 310014, Zhejiang Province, China Zhong-Sheng Zhao, Department of Pathology, Zhejiang Provincial People’s Hospital, Hangzhou 310014, Zhejiang Province, China Author contributions: Chu YQ and Zhao ZS contributed equally to this work; Chu YQ, Zhao ZS, Tao HQ, Wang YY, Ye ZY, designed research; Chu YQ, Wang YY, Ma HF, Li SG and Tao HQ performed research; Zhao ZS contributed new reagents/analytic tools; Chu YQ, Wang YY analyzed data; and Chu YQ, Ma HF, and Wang YY wrote the paper. Supported by The Grant of Zhejiang Province Natural Science Foundation, No. M303843 Correspondence to: Dr. Zhong-Sheng Zhao, Department of Pathology, Zhejiang Provincial People’s Hospital, Hangzhou 310014, Zhejiang Province, China. [email protected] Telephone: +86-571-85893289 Fax: +86-571-85131448 Received: August 29, 2007 Revised: January 3, 2008

analysis demonstrated that the mean survival time and 5-year survival rate were lower in the cases with low expressions of syndecan-1 and E-cadherin and high expression of integrin β3 (P < 0.01, in all cases). COX multivariate analysis showed that the expression level of syndecan-1 could be an independent prognostic index of gastric carcinoma (P < 0.01), whereas E-cadherin and integrin β3 could not be independent indexes (P > 0.05, P > 0.05 respectively). CONCLUSION: The low expression of syndecan-1 and E-cadherin and the high expression of integrin β3 are significantly correlated with the invasion and metastasis of gastric carcinoma, and they are highly correlated with each other. Therefore they may serve as important prognostic markers of gastric carcinoma. © 2008 WJG . All rights reserved.

Key words: Cell adhesion molecules; Gastric Carcinoma; Invasion; Metastasis; Prognosis

Abstract AIM: To evaluate the relationship between the expression of cell adhesion molecules (CAMs) and the biological behavior of gastric carcinoma. METHODS: Expression of syndecan-1, E-cadherin and integrin β3 were evaluated by immunohistochemical study in a total of 118 gastric carcinomas and 20 nontumor gastric mucosas. RESULTS: T h e e x p r e s s i o n s o f s y n d e c a n - 1 a n d E-cadherin were significantly lower in gastric carcinoma compared to non-tumor gastric mucosa, and the low expression rates were positively correlated to the tumor invasion depth, vessel invasion, lymph node metastasis and distant metastasis (P < 0.01 in all cases). However, the expression of integrin β3 was significantly higher in gastric carcinoma compared to non-tumor gastric mucosa, and the high expression rates were positively correlated to the tumor invasion depth, vessel invasion, lymph node metastasis and distant metastasis (P < 0.01 in all cases). In addition, the three protein expressions were correlated to the tumor growth pattern (P < 0.01, P < 0.01, and P < 0.05 respectively), but not correlated to tumor differentiation (P > 0.05, P > 0.05 and P > 0.05 respectively). Positive correlation was observed between the expressions of syndecan-1 and E-cadherin, but they which were negatively correlated to the expression of integrin β3 (P < 0.01 in all cases). Univariate www.wjgnet.com

Peer reviewer: Toru Ishikawa, MD, Department of Gastroenterology, Saiseikai Niigata Second Hospital, Teraji 280-7, Niigata, Niigata 950-1104, Japan

Chu YQ, Ye ZY, Tao HQ, Wang YY, Zhao ZS. Relationship between cell adhesion molecules expression and the biological behavior of gastric carcinoma. World J Gastroenterol 2008; 14(13): 1990-1996 Available from: URL: http://www.wjgnet.com/1007-9327/14/1990. asp DOI: http://dx.doi.org/10.3748/wjg.14.1990

INTRODUCTION Gastric carcinoma is the most common malignant tumor of the digestive system. Two of the most important causes for the high mortality are invasion and metastasis. However, the mechanism of invasion and metastasis of gastric carcinoma is still not definitely clear at present[1]. Cell adhesion is one of the important steps in metastasis. Syndecan-1, E-cadherin and integrin β3 make up cell adhesion molecules (CAMs) and participate in the adhesion between the cell and the extracellular matrix[2]. Syndecan-1 is a set of transmembrane heparitin sulfate glycoproteins (HSPGS), which is present at the surface of most epithelia cells and take part in the adhesion between the cell and the extracellular matrix. The expression of syndecan-1 was augmented during epithelial regeneration and rearrangement in the stomach and other tissues[3,4]. E-cadherin is a calcium dependent intercellular

Chu YQ et al . CAMs expression in gastric carcinoma 

adhesion molecule and it is present in normal cells to maintain the normal structure of tissues which has special biological characters and is highly related to invasion and metastasis of cancer cells[3]. Studies show that the reduction or absence of syndecan-1 and E-cadherin expressions could induce the growth, invasion and metastasis of tumors[5]. Integrin is a heterodimer consisting of a group of α and β polypeptide chains which can be divided into three groups based on the difference of a common β chain (β1, β2 and β3). By identifying the extracellular matrix such as laminin, fibronectin and immunoglobulin superfamily on the cell membrane (I-CAM for example) etc., mediating adhesive reaction of cell-extracellular matrix and cell-cell and receiving and conducting cascade signals, Integrin regulates the survival, apoptosis, movement, proliferation and inflammatory reaction of cells[6]. To our knowledge, the co-expressions of syndecan-1, E-cadherin and integrin β3 in gastric carcinoma and their clinical significances have not been reported before. We thus studied the expressions of CAMs in gastric carcinoma patients to find out the relationship among three of them using the immunochemical S-P method and to explore the correlation in their expression and pathological indexes of gastric carcinoma and survival time.

MATERIALS AND METHODS Specimen One hundred and eighteen specimens were collected from gastric carcinoma patients (91 men, 27 women, average age 56.3 years, range 25-79 years). They received the radical operations in our hospital from October 1986 to November 2002. Follow-up period was up to 5 years and the survival period was calculated from the day of surgery to the end of the follow-up or to the day of death. The censored value was zero. Of 118 gastric carcinomas, 70 were highly or moderately differentiated, while 48 were poorly or undifferentiated. According to the tumor, lymph node, and metastasis (TNM) standard stadiation, 47 were in T1-T2 stages and 71 were in T3-T4 stages. Eighty nine cases had vascular invasion and 29 without. Eighty three had lymph node metastasis and 35 without; distant metastases of carcinoma were found in 55 cases, while no distant metastasis in 63 cases. Twenty gastric mucosa specimens were collected 5 cm away from the cutting edges of radical operation as normal controls, which were detected as nontumor mucosa. Main reagents Mouse anti-human syndecan-1, E-cadherin and integrin β3 were purchased from ZYMED Company. SP kit was purchased from Maixin Biotect Company, Fuzhou, China. Streptavidin peroxidase staining was performed according to the kit instruction. Normal gastric mucosa was used as a positive control and PBS was used to replace the first antibody as a negative control. Immunohistochemistry Immunohistochemistry was made according to the streptavidin peroxidase (SP) methods. The staining step followed the routine process. In order to examine the

1991

specificity of immunostaining, both positive and negative controls were run at the same time in each experiment. The normal gastric mucous was used as the negative control. Results evaluation Based on the proportion of positively stained cells in the sections, the criteria of syndecan-1 and E-cadherin were set as follows[7]: (1) if more than 90% of the tumor cells exhibited intense membranous staining similar to that of normal cells, the result was considered as positive (++); (2) if the staining intensity was demonstrably reduced relative to that of normal cells and/or the staining pattern was heterogeneous (10%-90% positive), the result was deemed to be weakly positive (+); (3) if its expression was completely lost or positive in less than 10% of cells, the result was defined as negative (-). In statistical analysis, ± were classified as the low expression group and ++ were set as the high expression group. In addition, the criteria for integrin β3 were classified into four grades[8]. Briefly, -, no staining in fewer than 10% of tumor cells; +, weak staining in only 10%-50% of tumor cells; ++, moderate staining in 50%-75% of tumor cells; and +++, strong staining of more than 75% of tumor cells. The sections for integrin β3 were judged as a high expression group when more than 50% of cancer cells (++ or +++) were stained; others were judged as a low expression group. Statistical analysis SPSS 11.5 software package was used in data processing. Non-parameter Spearman rank correlation analysis was used to determine the relationship between the expressions of syndecan-1, E-cadherin and integrin β3 and the pathological indexes of the progression of gastric carcinoma. The survival rate was estimated by the Kaplan-Meier method and analyzed by the long-rank test. Fisher’s exact test was used to differentiate the rates of different groups. Univariate analysis and Cox-multivariate analysis were used to analyze the effect of the pathologic parameters (differentiation level, invasion depth, vessel invasion and lymph node metastasis), the expression of syndecan-1, E-cadherin and integrin β3 on the total survival.

RESULTS The expressions of syndecan-1, E-cadherin and integrin β 3 in gastric carcinoma and non-tumor gastric mucosa Of a total of 118 gastric carcinomas the following was found: syndecan-1 low expression 57.6% (68/118) (Figure 1A) and high expression 42.4% (50/118); E-cadherin low expression 57% (68/118) (Figure 2A) and high expression 42.4% (50/118); and integrin β3 low expression 50% (59/118) and high expression 50% (59/118) (Figure 3A). However, of 20 non-tumor gastric mucosa, syndecan-1 high expression (Figure 1B) 90% (18/20) and low expression 10% (2/20); E-cadherin high expression 85% (7/20) (Figure 2B) and low expression 15% (3/20); and integrin β3 high expression 15% (3/20) (Figure 3B) and low expression 85% (17/20). Compared to normal tissues, the gastric carcinoma tissues showed lower syndecan-1 expression (χ2 = 15. 5, P < 0.01), lower E-cadherin expression rate and density with www.wjgnet.com

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the increase of invasive depth of cancer cells (χ2 = 12.4, P < 0.01) and higher integrin β3 expression (χ2 = 8.5, P < 0.01). There are significant differences in the expressions of these three proteins in gastric carcinoma and non-tumor gastric mucosa. Relationship between the expression levels of syndecan-1, E-cadherin and integrin β 3 and the pathological indexes of progression of gastric carcinoma The low expressions of syndecan-1 and E-cadherin were positively correlated with the gastric carcinoma growth mode (χ2 = 12.47, P < 0.01; χ2 = 15.27, P < 0.01), invasion depth (χ2 = 32.95, P < 0.01; χ2 = 28.73, P < 0.01), vessel invasion (χ2 = 46.22, P 0.05). The high expression of integrin β3 protein was positively correlated with gastric carcinoma growth modes (χ2 = 5.83, P < 0.05), invasion depth (χ 2 = 29.74, P < 0.01), vessel invasion (χ2 = 33.33, P < 0.01), lymph node metastasis (χ2 = 29.61, P < 0.01) and distant metastasis (χ2 = 41.72, P < 0.01). The gastric carcinoma growing in invasive style had high expressions of integrin β3 protein, which was not correlated to the differentiation level of gastric carcinoma (χ2 = 0.14, P > 0.05) (Table 1). Relationship among the expressions of syndecan-1, E-cadherin and integrin β 3 in gastric carcinoma There was significant positive correlation between the expression levels of syndecan-1 and E-cadherin. Both of them had negative correlation with integrin β3 expression (Table 2).

Chu YQ et al . CAMs expression in gastric carcinoma 

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Table 1 Correlation between expressions of syndecan-1, E-cadherin and integrin b3 and pathological parameters in 118 gastric carcinoma patients (n )

Variable

Syndecan-1 expression

Integrin b3 expression

E-cadherin expression 2

2

2

Low

High

χ

P

Low

High

χ

P

Low

High

P

Invasion 67 Expansion 51 Histologic differentiations

48 20

19 31

12.47

0.000

49 19

18 32

15.27

0.000

27 32

40 19

5.836 0.016

Well/moderate Poor Depth of invasion

70 48

37 31

33 17

1.60

0.205

37 31

33 17

1.60

0.205

36 23

34 25

0.140 0.708

T1-T2 T3-T4 Vascular invasion

47 71

12 56

35 15

32.95

0.000

13 55

34 16

28.73

0.000

38 21

9 50

29.74

0.000

Negative Positive Lymphatic metastasis

29 89

1 67

28 22

46.22

0.000

2 66

27 23

40.52

0.000

28 31

1 58

33.33

0.000

Negative Positive Distant metastasis

35 83

4 64

31 19

43.49

0.000

5 63

30 20

38.28

0.000

31 28

4 55

29.61

0.000

63 55

15 53

48 2

63.30

0.000

17 51

46 4

51.98

0.000

49 10

14 45

41.72

0.000

χ

Growth mode of tumors

Negative Positive

Table 2 Relationship among the expressions of syndecan-1, E-cadherin and integrin b3 in gastric carcinoma Groups Syndecan-1 + ++ E-cadherin + ++ Integrin b3 + ++ +++

Syndecan-1 + ++

r

P -

43

38 11 3

25 50 0.837 38 4 1

11 12 2 6 1 8 10

48 1 1 0

4 12 0

r

P Groups

1 2 47

0

3 0 47

52 16 50 -0.792

2 1 18 22

E-cadherin + ++

0

-0.666 0 9 2 19 22

1 0 8 7

Table 3 Relationship between the expressions of syndecan-1, E-cadherin and integrin b3 and the prognosis of gastric carcinoma patients

46 1 2 1

Factors that affect the survival rate of gastric carcinoma Univariate analysis showed that the patients with high syndecan-1 expression had a 5-year survival rate of 91.66%, while it was 12.8% in those with low syndecan-1 expression. There was significant difference between the rates (χ2 = 53.13, P < 0.01). The patients with high E-cadherin expression level had a 5-year-survival rate of 93.59%, which was significantly different from that of the patients with low E-cadherin expression with the rate of 12.8%. There was significant difference between the rates (χ2 = 43.36, P < 0.01). The patients with high integrin β3 protein expression had a 5-year-survival rate of 13.85% and the patients with low integrin β3 expression had the rate of 72.75%. There was significant difference between the rates (χ2 = 35.11, P < 0.01). Kaplan-Meier analysis indicated that the patients with low syndecan-1/E-cadherin protein expression level and high integrin β3 expression level had poor prognosis (Table 3, Figure 4A-C). COX-multivariate analysis showed that syndecan-1 expression could be used as a prognostic marker of gastric

Syndecan-1 expression Low expression High expression E-cadherin expression Low expression High expression Integrin b3 expression Low expression High expression

2

n

Mean survival time (mo)

5-yr survival rate (%)

68 50

32.10 ± 4.16 123.80 ± 5.56

12.80 91.66

53.13 0

68 50

33.69 ± 4.33 119.78 ± 5.87

12.80 93.59

43.36 0

59 59

103.30 ± 7.73 33.12 ± 4.64

72.75 13.85

35.11 0

χ

P

cancer patients (P < 0.01). However, E-cadherin and integrin β3 could not be used as independent prognosis markers (P > 0.05 and P > 0.05 respectively). (Syndecan-1: B = 3.447, SE = 0.988, Wald = 12.183, P < 0.01; E-cadherin: B = 0.019, SE = 0.686, Wald = 0.001, P > 0.05; integrin β3: B = 0.098, SE = 0.364, Wald = 27.711, P > 0.05).

DISCUSSION Gastric carcinoma is highly malignant and usually results in a poor prognosis. Although the achievement in early diagnosis and treatment of gastric cancer has improved the patients’ outcome, it is still one of the leading causes of mortality in countries such as China and Japan. Currently, about 39% of gastric cancer cases occur in the Chinese population, ranking the leading cause of cancermortality in China, particularly in rural areas[9]. The overall 5-year survival rate for patients who undergo curative surgical resection for gastric carcinoma ranges from 47% to 60.4%[10]. The typical characteristics of malignant tumor are invasion and metastasis, which are the main cause for their lethality. Tumor progression is considered to be www.wjgnet.com

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Cumulative survival rate (%)

1.0

Syndecan-1 (++)

0.8 0.6 0.4 0.2

Syndecan-1 (-/+)

0

20

40

Cumulative survival rate (%)

1.0

100

120

140

Survival functions E-cadherin (++)

0.6 0.4 E-cadherin (-/+)

0.2

0

20

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1.0 Cumulative survival rate (%)

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Survival functions Integrin β3 (-/+)

0.8 0.6 0.4

Integrin β3 (++/+++) 0.2 0.0 0

20

40

60 80 100 Survival t /mo

120

140

Figure 4 Correlation between syndecan-1, E-cadherin and integrin β3 expressions and the survival time. A: Survival curves by the Kaplan-Meier method. Long-rank test revealing a significant difference between negative and positive expression of Syndecan-1 (P < 0.05); B: Survival curves by Kaplan-Meier method. Long-rank test revealing a significant difference between negative and positive expression of E-cadherin (P < 0.05); C: Survival curves by the Kaplan-Meier method. There was a significant difference in Long-rank test between positive and negative expression of integrin β3 (P < 0.05).

dynamic, complex, and a multi-step process, where the essential steps are the breakdown of cell-cell adhesion and degradation of basement membrane (BM)[11]. The cellular and molecular steps required for metastases of neoplastic cells are starting to be elucidated. The metastatic cascade starts with a downregulation of the epithelial bonds which enables tumor cells to leave epithelial structures, to invade the stroma, to enter the blood stream or the lymphatics, to extravasate, and to colonize the target organs. Interestingly, invasive growth and metastases often recapitulate embryonic development and are greatly influenced by www.wjgnet.com

April 7, 2008

Volume 14

Number 13

the stromal microenvironment[12]. Thus, carcinoma cells interact bidirectionally with neighboring fibroblasts, endothelial cells, and lymphocytes through growth factors, chemokines, cell adhesion molecules, and extracellular matrix proteases. Loss of cell adhesion may contribute to loss of contact inhibition of growth, which is an early step in the neoplastic process. It has been shown that various cell adhesion molecules expressed on carcinoma cells play crucial roles. However, the mechanisms of invasion and metastasis are still under investigation. Until now there is no satisfactory tumor marker for predicting its evolution. Syndecan-1, E-cadherin and integrin β3 make up CAMs together[2] to participate in adhesion between cell and extracellular matrix. Experimental studies show that changes in cell-cell and cell-matrix adhesion are central to the conversion from premalignant lesions to early invasive carcinoma[13]. Syndecan-1 (CD138) is a member of the transmembrane heparin sulfate proteoglycan (HSPG) family, taking part in and improving adhesion between cell and extracellular matrix[14], improving cell proliferation, maintaining the differentiation phenotype of cells and inhibiting the growth of tumor cells[15]. Wiksten et al[16] reported that abatement or loss of syndecan-1 expression is highly correlated to the focal size, lymphatic metastasis, invasion depth, TNM stage and prognosis of gastric carcinoma. It is reported that the expression of syndecan-1 increases gradually from large intestine adenoma to carcinoma, then to invasive carcinoma[17,18]. E-cadherin is a calcium dependent transmembrane glycoprotein and has the functions of mediating the adhesion of homogeneous cells among epithelia and of maintaining the integrity and polarity of tissue structures [19] . The abatement or loss of E-cadherin expression may induce the decrease of adhesion among cells and thus make cancer cells disunite, grow invasively toward peripheral tissues and leave original focal to form metastasis once the necessary conditions are met[20,21]. Documents indicate that in gastric carcinomas, the reduction in E-cadherin expression activation of E-cadherin gene varies from 17% to 92% [22,23] . This phenomenon has been confirmed in many kinds of tumors such as lung cancer, breast cancer, large intestine cancer, liver cancer and gastric cancer[24-27]. Integrins belong to the family of transmembrane glycoprotein hetero-dimer. They mediate adhesion of neighboring cells and participate in the growth and repair of cells and vascular proliferation etc as important receptors of extracellular matrix protein[28]. Molecular biological studies on melanoma, colon and rectal cancer and other carcinomas in the past few years showed that the dissociation from or penetration through BM of tumor cells, caused by the adhesion of α3β1, α5β1, β3 and other Integrins on tumor cell surface to extracellular matrix, is the initial step for the invasive growth and remote metastasis of malignant tumor. Moreover the high expression of integrin β3 in malignant melanoma and malignant ovarian tumor cells is positively correlated to invasion and metastasis of cancer cells[29]. Documented data shows that these three proteins, syndecan-1, E-cadherin and integrin β3, cooperate with

Chu YQ et al . CAMs expression in gastric carcinoma 

each other for expressions and functions; moreover their expression levels are correlated with the progression of gastric carcinoma. Experimental studies show that syndecan-1 and E-cadherin are all present in epithelia and both can form immunoprecipitation with transcription regulatory factor β-cadherin and this indicates that they are materially and functionally correlated with each other[30]. The expression of syndecan-1 and E-cadherin in gastric carcinoma is low and their expression levels are positively correlated [31] . The abatement or loss of E-cadherin expression is involved in lymph node micro-metastasis of gastric carcinoma[32]. Sun et al[33] proposed the following point of view based on different studies: the decrease of in vitro syndecan-1 expression inhibits E-cadherin expression, and/or lowers E-cadherin expression at the same time as the beginning of epithelium-stroma transformation and induces effective and timely epithelium-stroma transformation[33]. Signal conduction mediated by syndecan-1 needs the cooperation of integrin β3[34]. Mammary glandular epithelia short of syndecan-1 show rearrangement of integrin β3 and markedly low expression of E-cadherin at the same time[35]. Wound healing theory indicates that E-cadherin activates the migration of integrin β3-transfected cells and constrains them to separate from wound margin. The study of Ohta et al shows that over expression of the homeobox gene HOXD3 promotes the non-expression of E-cadherin and increased expression of integrin β3, which plays an important role in the quick migration and isolation of tumor cells[29]. Zhang et al[11] reported that E-cadherin loss in epithelial tumor progression was not only related to severing cell-cell adhesion but also associated with increased integrins expression, which induced cell-matrix adhesion of these cells[13]. The results of this study indicate that the expressions of syndecan-1 and E-cadherin in gastric tumor tissues are low and their levels are significantly correlated. This suggests that syndecan-1 and E-cadherin play a positively cooperative role in the genesis and development of gastric carcinoma. They are negatively correlated to the level of integrin β3 expression and this suggests that Integrins have different effects on the progression of gastric carcinoma. The functional consequence of enhanced cell-matrix adhesion is the initial attachment and retention of these cells at the epithelial-stromal interface, thus providing the appropriate microenvironmental conditions for incipient tumor cell invasion[13]. Basing on the biological characters of syndecan-1, E-cadherin and Integrins, on relevant documents and on the results of this study, the correlation of CAMs with the progression of gastric carcinoma can be summarized as follows: (1) at the early stage of gastric carcinoma, tumor cells have low E-cadherin and syndecan-1 expression, adhesion between cells is weak and tumor cells are isolated from primary tumor, which is the initial step of invasion and metastasis of gastric carcinoma; (2) the decrease of the expressions of E-cadherin and syndecan-1 reduces adhesion between tumor cells and BM or extracellular matrix mediated by them and this is propitious to local growth and dispersion of tumor. At the same time, various hydrolytic enzymes will be released, after the adhesion of

1995

cancer cells to BM or extracellular matrix, to degrade the BM or extracellular matrix which tumor cells adhere to. Thus cancer cells may enter into the blood circulation. Experimental studies show that Integrins are the main receptors for cells adhesion to extracellular matrix and syndecan-1 increases the combination of them; (3) the increase of Integrin expression, after that cancer cells enter the blood circulation, is in favor of adhesion of tumor cells to endothelia to induce invasive growth and remote metastasis of gastric carcinoma. In these processes, adhesion of tumor cells to endothelia and the BM under endothelia is the key process for the invasion and metastasis. The pathological indexes and survival analysis results confirm that gastric cancer tissues with low syndecan-1 and E-cadherin protein expressions and high integrin β3 protein expression have deeper invasion depth, higher occurrences of vascular invasion, lymph node metastasis and remote metastasis, shorter average survival time and lower 5-year survival rate, which is consistent with the study results of Wiksten et al[16], Trikh et al[36] and other researchers. In conclusion, low expressions of syndecan-1 and E-cadherin protein and high expression of integrin β3 protein are significantly correlated to invasion of gastric carcinoma. As intracellular adhesion molecular complexes, these three proteins are highly correlated with each other. Therefore, the results of co-examination of them can be important indexes for prognosis of gastric carcinoma.

COMMENTS Background

Cell adhesion is one of the important steps in invasion and metastasis Syndecan-1, E-cadherin and integrin β3 make up intercellular adhesion molecules (CAMs) and participate in the adhesion between cell and extracellular matrix. One or two of them in gastric carcinoma has been reported before; however, in order to better understand the coordinated regulation of cell-cell and cell-matrix interactions during malignant transformation, we study the coexpression of E-cadherin, syndecan-1 and integrin β3 by immunohistochemical study in gastric carcinomas.

Research frontiers

Recent investigations have suggested that frozen tissue-based molecular classifications effectively predict prognosis of gastric cancer, prognostic classification on formalin-fixed tissue is needed. Therefore additional markers are required in the prognosis of patients with gastric cancer.

Innovations and breakthroughs

In this article, we identified that syndecan-1, E-cadherin and integrins were highly correlated with each other as intracellular adhesion molecular complexes. We suggest that the results of co-examination can be important indexes for prognosis of gastric carcinoma.

Applications

The results from the study confirm the correlation between expressions of CAMs and gastric cancer. It suggests that the coexpression of them can be used to identify the prognosis of gastric carcinoma.

Terminology

CAMs are proteins located on the cell surface involved in the binding with other cells or with the extracellular matrix in the process called cell adhesion.

Peer review

This report analyzed that advanced gastric cancer including with/without distant

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metastases. Prognostic factor of each stage of gastric cancer about CAMs should be analyzed and author should speculate other factors too.

REFERENCES 1

Sakakura C, Hagiwara A, Nakanishi M, Shimomura K, Takagi T, Yasuoka R, Fujita Y, Abe T, Ichikawa Y, Takahashi S, Ishikawa T, Nishizuka I, Morita T, Shimada H, Okazaki Y, Hayashizaki Y, Yamagishi H. Differential gene expression profiles of gastric cancer cells established from primary tumour and malignant ascites. Br J Cancer 2002; 87: 1153-1161 Day RM, Hao X, Ilyas M, Daszak P, Talbot IC, Forbes A. 2 Changes in the expression of syndecan-1 in the colorectal adenoma-carcinoma sequence. Virchows Arch 1999; 434: 121-125 3 Siitonen SM, Kononen JT, Helin HJ, Rantala IS, Holli KA, Isola JJ. Reduced E-cadherin expression is associated with invasiveness and unfavorable prognosis in breast cancer. Am J Clin Pathol 1996; 105: 394-402 4 Tanabe H, Yokota K, Kohgo Y. Localization of syndecan-1 in human gastric mucosa associated with ulceration. J Pathol 1999; 187: 338-344 Kurokawa H, Zhang M, Matsumoto S, Yamashita Y, Tanaka T, 5 Takamori K, Igawa K, Yoshida M, Fukuyama H, Takahashi T, Sakoda S. Reduced syndecan-1 expression is correlated with the histological grade of malignancy at the deep invasive front in oral squamous cell carcinoma. J Oral Pathol Med 2006; 35: 301-306 Yao M, Zhou XD, Zha XL, Shi DR, Fu J, He JY, Lu HF, Tang 6 ZY. Expression of the integrin alpha5 subunit and its mediated cell adhesion in hepatocellular carcinoma. J Cancer Res Clin Oncol 1997; 123: 435-440 Liu YC, Shen CY, Wu HS, Hsieh TY, Chan DC, Chen CJ, Yu 7 JC, Yu CP, Harn HJ, Chen PJ, Hsieh CB, Chen TW, Hsu HM. Mechanisms inactivating the gene for E-cadherin in sporadic gastric carcinomas. World J Gastroenterol 2006; 12: 2168-2173 Yin F, Qiao T, Shi Y, Xiao B, Chen B, Miao J, Fan D. In situ 8 hybridization of tight junction molecule occludin mRNA in gastric cancer. Zhonghua Zhongliu Zazhi 2002; 24: 557-560 9 Parkin DM. Global cancer statistics in the year 2000. Lancet Oncol 2001; 2: 533-543 10 Samson PS, Escovidal LA, Yrastorza SG, Veneracion RG, Nerves MY. Re-study of gastric cancer: analysis of outcome. World J Surg 2002; 26: 428-433 11 Zhang JF, Zhang YP, Hao FY, Zhang CX, Li YJ, Ji XR. DNA ploidy analysis and expression of MMP-9, TIMP-2, and E-cadherin in gastric carcinoma. World J Gastroenterol 2005; 11: 5592-5600 12 Hirohashi S, Kanai Y. Cell adhesion system and human cancer morphogenesis. Cancer Sci 2003; 94: 575-581 13 Zhang W, Alt-Holland A, Margulis A, Shamis Y, Fusenig NE, Rodeck U, Garlick JA. E-cadherin loss promotes the initiation of squamous cell carcinoma invasion through modulation of integrin-mediated adhesion. J Cell Sci 2006; 119: 283-291 14 Bernfield M, Gotte M, Park PW, Reizes O, Fitzgerald ML, Lincecum J, Zako M. Functions of cell surface heparan sulfate proteoglycans. Annu Rev Biochem 1999; 68: 729-777 15 Kato M, Saunders S, Nguyen H, Bernfield M. Loss of cell surface syndecan-1 causes epithelia to transform into anchorage-independent mesenchyme-like cells. Mol Biol Cell 1995; 6: 559-576 16 Wiksten JP, Lundin J, Nordling S, Lundin M, Kokkola A, von Boguslawski K, Haglund C. Epithelial and stromal syndecan-1 expression as predictor of outcome in patients with gastric cancer. Int J Cancer 2001; 95: 1-6 17 Fujiya M, Watari J, Ashida T, Honda M, Tanabe H, Fujiki T, Saitoh Y, Kohgo Y. Reduced expression of syndecan-1 affects metastatic potential and clinical outcome in patients with colorectal cancer. Jpn J Cancer Res 2001; 92: 1074-1081 18 Hayashida K, Johnston DR, Goldberger O, Park PW. Syndecan-1 expression in epithelial cells is induced by transforming growth

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factor beta through a PKA-dependent pathway. J Biol Chem 2006; 281: 24365-24374 Shiozaki H, Oka H, Inoue M, Tamura S, Monden M. E-cadherin mediated adhesion system in cancer cells. Cancer 1996; 77: 1605-1613 Hirohashi S. Inactivation of the E-cadherin-mediated cell adhesion system in human cancers. Am J Pathol 1998; 153: 333-339 Shiozaki H, Tahara H, Oka H, Miyata M, Kobayashi K, Tamura S, Iihara K, Doki Y, Hirano S, Takeichi M. Expression of immunoreactive E-cadherin adhesion molecules in human cancers. Am J Pathol 1991; 139: 17-23 Shimoyama Y, Hirohashi S. Expression of E- and P-cadherin in gastric carcinomas. Cancer Res 1991; 51: 2185-2192 Oka H, Shiozaki H, Kobayashi K, Tahara H, Tamura S, Miyata M, Doki Y, Iihara K, Matsuyoshi N, Hirano S. Immunohistochemical evaluation of E-cadherin adhesion molecule expression in human gastric cancer. Virchows Arch A Pathol Anat Histopathol 1992; 421: 149-156 Elzagheid A, Algars A, Bendardaf R, Lamlum H, Ristamaki R, Collan Y, Syrjanen K, Pyrhonen S. E-cadherin expression pattern in primary colorectal carcinomas and their metastases reflects disease outcome. World J Gastroenterol 2006; 12: 4304-4309 Wu ZY, Zhan WH, Li JH, He YL, Wang JP, Lan P, Peng JS, Cai SR. Expression of E-cadherin in gastric carcinoma and its correlation with lymph node micrometastasis. World J Gastroenterol 2005; 11: 3139-3143 Kase S, Sugio K, Yamazaki K, Okamoto T, Yano T, Sugimachi K. Expression of E-cadherin and beta-catenin in human non-small cell lung cancer and the clinical significance. Clin Cancer Res 2000; 6: 4789-4796 Kowalski PJ, Rubin MA, Kleer CG. E-cadherin expression in primary carcinomas of the breast and its distant metastases. Breast Cancer Res 2003; 5: R217-R222 Springer TA, Wang JH. The three-dimensional structure of integrins and their ligands, and conformational regulation of cell adhesion. Adv Protein Chem 2004; 68: 29-63 Maubant S, Cruet-Hennequart S, Dutoit S, Denoux Y, Crouet H, Henry-Amar M, Gauduchon P. Expression of alpha V-associated integrin beta subunits in epithelial ovarian cancer and its relation to prognosis in patients treated with platinumbased regimens. J Mol Histol 2005; 36: 119-129 Zimmermann P, Tomatis D, Rosas M, Grootjans J, Leenaerts I, Degeest G, Reekmans G, Coomans C, David G. Characterization of syntenin, a syndecan-binding PDZ protein, as a component of cell adhesion sites and microfilaments. Mol Biol Cell 2001; 12: 339-350 Huang MF, Zhu YQ, Chen ZF, Xiao J, Huang X, Xiong YY, Yang GF. Syndecan-1 and E-cadherin expression in differentiated type of early gastric cancer. World J Gastroenterol 2005; 11: 2975-2980 Koriyama C, Akiba S, Itoh T, Sueyoshi K, Minakami Y, Corvalan A, Yonezawa S, Eizuru Y. E-cadherin and beta-catenin expression in Epstein-Barr virus-associated gastric carcinoma and their prognostic significance. World J Gastroenterol 2007; 13: 3925-3931 Sun D, Mcalmon KR, Davies JA, Bernfield M, Hay ED. Simultaneous loss of expression of syndecan-1 and E-cadherin in the embryonic palate during epithelial-mesenchymal transformation. Int J Dev Biol 1998; 42: 733-736 Beauvais DM, Rapraeger AC. Syndecan-1-mediated cell spreading requires signaling by alphavbeta3 integrins in human breast carcinoma cells. Exp Cell Res 2003; 286: 219-232 Ohta H, Hamada J, Tada M, Aoyama T, Furuuchi K, Takahashi Y, Totsuka Y, Moriuchi T. HOXD3-overexpression increases integrin alpha v beta 3 expression and deprives E-cadherin while it enhances cell motility in A549 cells. Clin Exp Metastasis 2006; 23: 381-390 Seftor RE, Seftor EA, Hendrix MJ. Molecular role(s) for integrins in human melanoma invasion. Cancer Metastasis Rev 1999; 18: 359-375 S- Editor Ma L L- Editor Alpini GD

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World J Gastroenterol 2008 April 7; 14(13): 1997-2002 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

LIVER CANCER

Long-term outcome of percutaneous ethanol injection therapy for minimum-sized hepatocellular carcinoma Miyuki Taniguchi, Soo Ryang Kim, Susumu Imoto, Hirotsugu Ikawa, Kenji Ando, Keiji Mita, Shuichi Fuki, Noriko Sasase, Toshiyuki Matsuoka, Masatoshi Kudo, Yoshitake Hayashi Miyuki Taniguchi, Soo Ryang Kim, Susumu Imoto, Hirotsugu Ikawa, Kenji Ando, Keiji Mita, Shuichi Fuki, Noriko Sasase, Department of Gastroenterology, Kobe Asahi Hospital, Kobe, Japan Toshiyuki Matsuoka, Department of Radiology, Osaka City University Medical School, Osaka, Japan Masatoshi Kudo, Department of Gastroenterology, Kinki University School of Medicine, Osakasayama, Japan Yoshitake Hayashi, Division of Molecular Medicine & Medical Genetics, International Center for Medical Research and Treatment (ICMRT), Kobe University Graduate School of Medicine, Kobe, Japan Correspondence to: Soo Ryang Kim, MD, Department of Gastroenterology, Kobe Asahi Hospital, 3-5-25 Bouoji-cho, Nagata-ku, Kobe 653-0801, Japan. [email protected] Telephone: +81-78-6125151 Fax: +81-78-6125152 Received: October 18, 2007 Revised: January 9, 2008

Abstract AIM: To evaluate long-term follow-up of minimum-sized hepatocellular carcinoma (HCC) treated with percutaneous ethanol injection (PEI). METHODS: PEI was applied to 42 lesions in 31 patients (23 male and eight female) with HCC < 15 mm in diameter, over the past 15 years. RESULTS: Overall survival rate was 74.1% at 3 years, 49.9% at 5 years, 27.2% at 7 years and 14.5% at 10 years. These results are superior to, or at least the same as those for hepatic resection and radiofrequency ablation. Survival was affected only by liver function, but not by sex, age, etiology of Hepatitis B virus or Hepatitis C virus, α-fetoprotein levels, arterial and portal blood flow, histological characteristics, and tumor multiplicity or size. Patients in Child-Pugh class A and B had 5-, 7- and 10-years survival rates of 76.0%, 42.2% and 15.8%, and 17.1%, 8.6% and 0%, respectively (P = 0.025). CONCLUSION: Treatment with PEI is best indicated for patients with HCC < 15 mm in Child-Pugh class A. © 2008 WJG . All rights reserved.

Key words: Percutaneous ethanol injection; Interventional ablation; Ultrasound; Hepatocellular carcinoma; Prognosis Peer reviewer: Reinhard Buettner, Professor, Institute of

Pathology, University Hospital Bonn, Sigmund-Freud-Str. 25, D-53127 Bonn, Germany Taniguchi M, Kim SR, Imoto S, Ikawa H, Ando K, Mita K, Fuki S, Sasase N, Matsuoka T, Kudo M, Hayashi Y. Long-term outcome of percutaneous ethanol injection therapy for minimumsized hepatocellular carcinoma. World J Gastroenterol 2008; 14(13): 1997-2002 Available from: URL: http://www.wjgnet. com/1007-9327/14/1997.asp DOI: http://dx.doi.org/10.3748/ wjg.14.1997

INTRODUCTION Hepatocellular carcinoma (HCC) is one of the major malignancies worldwide [1-4] . With recent advances in diagnostic imaging, particularly in ultrasound (US), an increasing number of small or early-stage HCCs have been detected. In patients with early-stage HCC, percutaneous ethanol injection (PEI) has been a second choice when surgical techniques have been precluded, although PEI has been used as a first-line treatment option in some centers in Italy and Japan. Over the past few years, several methods for thermal tumor destruction through localized heating or freezing, including radiofrequency ablation (RFA), laser ablation, microwave ablation, and cryoablation, have been developed and clinically tested. Among these, RFA has recently emerged as a real competitor to PEI. At this point, there are no unequivocal data to back up percutaneous ablation as a replacement for resection as first-line treatment for patients with early-stage HCC. Therefore, whether percutaneous ablation replaces resection as first-line option for very early HCC will be resolved by launching a large international randomized controlled trial. In this study, early-stage HCC (< 15 mm diameter) is referred to as minimum-sized HCC[5]. The present study is a long-term follow-up of minimum-sized HCC treated with PEI.

MATERIALS AND METHODS Subjects We analyzed the clinical features of consecutive patients treated with PEI in the 1990s at Kobe Asahi Hospital. The diagnosis of HCC was made by imaging, including US and computed topography (CT) and confirmed by www.wjgnet.com

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tumor-targeted biopsies. Thirty-one patients with liver cirrhosis and HCC were treated with PEI as the first-line anticancer treatment. The characteristics of the patients are summarized in Table 1. The criteria for treatment with PEI were as follows: (1) Uninodular HCC ≤ 15 mm in diameter, or multinodular HCC lesions ≤ 15 mm in diameter (in one or both hepatic lobes); (2) absence of portal vein thrombosis and extra-hepatic metastases; (3) age < 75 years; (4) liver cirrhosis of Child-Pugh class A or B; and (5) prothrombin time ratio (normal/patient) > 40% and platelet count > 40 000/μL. The number of tumorous nodules and portal vein patency were established by US and CT. Maximum tumor diameter was measured by US. The absence of extrahepatic metastases was ascertained by chest X-ray, and abdominal CT and US. L ive r c i r r h o s i s wa s d i a g n o s e d h i s t o l o g i c a l l y, radiologically or clinically. Serum hepatitis B surface antigen (HBsAg) was positive in two patients, and antihepatitis C virus antibody (anti-HCV) was positive in 29. Arterial blood flow was confirmed by CO2 US-angiography or CT during arteriography (CTA); portal blood flow was confirmed by CT during arterial portography (CTAP) (Table 1). Surgery was contraindicated in most patients because of liver dysfunction, presence of lesions in locations that made hepatic resection inappropriate, advanced age, coexistence of another disease, or a combination of these factors. However, during the past few years, some patients, including possible candidates for surgery, were treated with PEI. Informed consent was obtained from all patients after the nature of the procedure had been fully explained. The diameter of the tumors ranged between 8 and 15 (mean 11.9 ± 2.4) mm. The diagnosis of HCC was established by histological biopsy with the needle guided by sonography in 31 patients. Treatment schedule and follow-up protocol PEI [6] was administered to each patient (3-6 sessions; once or twice weekly) by one or two injections of 95% sterile ethyl alcohol (1.6-73.9 mL, mean 17.6 ± 16.7 mL) delivered to each lesion with a multiple-side-hole 21-gauge needle (Et-hanoject, TSK, Tokyo, Japan), depending on the size of the lesion and the distribution of the injected ethanol within the tumor. One month after the end of PEI treatment, the α-fetoprotein (AFP) level was measured, CT was repeated, and multiple percutaneous biopsies of the treated lesions were carried out to evaluate treatment outcome. Lesions appearing as hypoattenuated, non-enhanced areas on CT scans were diagnosed as necrotic, whereas enhanced areas were suspected of being persistent tumors. The biopsies under US guidance were carried out by placing the needle in the enhanced areas of the tumor. Histological samples were obtained from all of the patients. The treatment was terminated and the patients entered the follow-up protocol in the absence of residual tumors, confirmed by CT and biopsies, and of suspected persistent tumors, confirmed by AFP levels. Such patients were given additional PEIs targeted in areas where viable tumors had previously been observed, and were examined again by CT www.wjgnet.com

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Table 1 Characteristics of 31 patients with liver cirrhosis and small HCC Parameter Sex (male:female) Age (yr) (mean ± SD) Cause of cirrhosis HCV Non-HCV Liver dysfunction Child-Pugh A Child-Pugh B AFP level (mg/L) ≤ 20 > 20 Tumor multiplicity Uninodular Multinodular Tumor diameter (mm) (mean ± SD) ≤ 10 11-15 Histological characteristics Well-differentiated Well to moderately differentiated Arterial blood flow Positive Negative Portal blood flow Positive Negative

23:8 63.8 ± 8.9 29 2 17 14 15 16 21 10 11.9 ± 2.4 12 19 18 13 9 13 12 9

and biopsies after 1 mo. The follow-up protocol included clinical assessment, measurement of hepatic functional serum indexes and AFP levels, and US examinations conducted at 3-mo intervals. The duration of the followup was calculated from the beginning of PEI and lasted for 2-167 (mean ± SD, 57.5 ± 37.7) mo. Statistical analysis Student’s t test and χ2 test were used to identify differences in patient characteristics in the various subgroups with prognostic factors. The Kaplan-Meier method was used to analyze the factors associated with post-PEI survival of patients with HCC and distant intrahepatic recurrence of HCC, and the difference was determined by log rank test. Stepwise regression analysis was used to identify factors that affected the survival rate of post-PEI patients. P < 0.05 was considered to indicate a statistically significant difference.

RESULTS All patients completed the planned treatment course. No major treatment-related complication had occurred by the end of the study. Overall survival rate was 74.1% at 3 years, 49.9% at 5 years, 27.2% at 7 years and 14.5% at 10 years. The longest survival period was 13 years 11 mo, and three patients lived longer than 10 years after PEI treatment. Up to December 2003, 23 post-PEI patients died: Four from cancer (17.4%), 15 from hepatic failure (65.2%) and four from other causes (17.4%). According to the Child-Pugh classification, two (20%) died from cancer and seven (70%) from hepatic failure (class A), and

Taniguchi M et al. Percutaneous ethanol injection for small HCC

Table 2 Cause of death of post-PEI patients with small HCC, according to Child-Pugh classification and number of treatment sessions Child-Pugh classification Number of treatment sessions A (n = 10) B (n = 13) 1st Cancer growth Hepatic failure Cancer (+) Cancer (–) Other

2nd

3rd

4th

5th

2

2

0

3

0

0

0

7 0 1

6 2 3

3

2

2

1

5

3

0

0

0

0

Table 3 Analysis of the prognostic value of patient- and tumorrelated factors Probability of survival (%) Factor Sex Male Female Age (yr) ≤ 65 > 65 Cause of cirrhosis HCV Non-HCV Liver dysfunction Child-Pugh A Child-Pugh B AFP level (mg/L) ≤ 20 > 20 Tumor multiplicity Uninodular Multinodular Tumor diameter (mm) ≤ 10 11-15 Histological characteristics Well-differentiated Well to moderately differentiated Arterial blood flow Positive Negative Portal blood flow Positive Negative

n

3-yr

5-yr

7-yr

10-yr P value

23 8

73.7 62.5

59.5 33.3

29.7 0

11.1 0.0

18 13

77.4 61.5

59.5 35.9

29.8 23.9

9.9 0.0

29 2

72.2 -

46.2 -

23.1 -

15.4 -

17 14

76.0 64.3

76.0 17.1

42.2 8.6

15.8 0.0

15 16

79.4 62.5

56.9 37.5

42.6 18.7

21.3 0.0

21 10

75.9 60.0

50.6 37.5

36.1 12.5

13.5 12.5

12 19

74.1 68.4

64.8 40.9

32.4 24.6

21.6 12.2

0.595

0.475

0.025

0.139

0.751

0.336



1999

Table 4 Factors affecting survival of patients with HCC treated by PEI, determined by stepwise regression analysis Factors Child-Pugh class A B

95% CI

P value

1.50-23.15

0.011

Hazard ratio 1 5.89

Table 5 Characteristics of patients according to Child-Pugh class Parameter

Class A (n = 17)

Class B (n = 14)

P value

Sex (male:female) Age (yr) (mean ± SD) Cause of cirrhosis HCV Non- HCV AFP level ≤ 20 mg/L > 20 mg/L Tumor multiplicity Uninodular Multinodular Tumor diameter (mm) (mean ± SD) ≤ 10 11-15 Histological characteristics Well-differentiated Well to moderately differentiated Arterial blood flow Positive Negative Portal blood flow Positive Negative

16:1 61.2 ± 8.3

7:7 66.9 ± 8.9

0.017 NS

15 2

14 0

NS

9 8

6 8

NS

11 6

10 4

NS

7 10

5 9

NS

9 8

9 5

NS

5 5

4 8

NS

7 4

5 5

NS

NS: Not significant. 0.119

18 13

83.3 61.5

58.0 38.5

33.2 19.2

22.1 0.0

9 13

64.8 61.5

38.9 30.8

38.9 10.2

38.9 0.0

12 9

58.3 64.8

33.3 38.9

16.7 38.9

0.0 0.0

0.269

0.458

two (15.3%) from cancer and eight (61.5%) from hepatic failure (class B) (Table 2). Prognostic factors The influence of patient- and tumor-related factors on survival is shown in Table 3. Survival was affected by liver function, but not by sex, age, etiology of cirrhosis (HCV or non-HCV), AFP level, arterial blood flow, portal blood flow, histological characteristics, and tumor multiplicity or size. The final step of stepwise variable showed function was significantly associated with survival rate. Child-Pugh class A patients showed a higher survival rate than ChildPugh class B (P = 0.011, Table 4). The 5-, 7- and 10-years

survival rates of 76.0%, 42.2% and 15.8%, respectively, for patients in Child-Pugh class A were significantly higher than those for patients in Child-Pugh class B (17.1%, 8.6% and 0%, respectively) (P = 0.025, Figure 1). Patient characteristics by Child-Pugh classification showed that prognosis of class A and class B patients was affected by sex and not by any other patient- and tumor-related factors (Table 5). The two groups were similar with respect to the other patient- and tumor-related factors. Lesions in intrahepatic areas other than the site treated by PEI were observed in 25 patients (81.8%), and cumulative recurrence rates at 1, 3, 5, 7 and 10 years after PEI were 47.0%, 73.4%, 87.7%, 87.7% and 87.7%, respectively (Figure 2). The frequency of initial recurrence according to segment was 32.0% in segments other than the initially treated segment (other segments), 32.0% in the same segment as the initially treated segments, and 28.0% in both the same and other segments. The tumor diameter of recurrent lesions was < 20 mm in 84.0% of cases, 21-30 mm in 8.0%, and no tumor was > 30 mm at detection (Table 6). The recurrent lesions that occurred in 25 post-PEI patients after initial treatment were managed as follows: PEI in 15

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Child-Pugh A Child-Pugh B

 0

20

40

 60

 80

100

120

140

160

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Table 6 Recurrence of HCC initially treated by PEI (n = 25) Location of recurrence Same segment Same and other segments Other segments Others1 Tumor diameter at detection (mm) ≤ 10 11-15 16-20 21-30 ≥ 31 Others

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Figure 1 Survivial of patients with HCC treated by PEI according to Child-Pugh classification.

8 (32.0%) 7 (28.0%) 8 (32.0%) 2 (8.0%) 8 (32.0%) 9 (36.0%) 4 (16.0%) 2 (8.0%) 0 2 (8.0%)

Recurrence (%)

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100 90 80 70 60 50 40 30 20 10 0

In two cases, tumor thrombus was seen in portal vein and HCC metastasizing to distant site (sacrum and lumbar vertebra), respectively.

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Figure 2 Recurrence of HCC treated by PEI at remote site from initial treated site.

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n =8 3rd

n = 12 2nd

n = 19 1st

n = 25 0  10 20

30

40 50 60 Percentage (%)

70

80

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100

Figure 3 Treatment for repeated recurrences in patients with HCC initially treated by PEI.

(60.0%); transcatheter chemoembolization (TACE) in two (8.0%); TACE + PEI in four (16.0%); transcatheter arterial infusion chemotherapy in one (4.0%); and other methods in two (8.0%). One patient (4.0%) was untreatable because of severe liver dysfunction or poor general condition. Second, third, fourth and fifth recurrent lesions were treated with PEI in eight (42.1%), five (41.6%), two (25.0%) and three (42.8%) patients, respectively (Figure 3).

DISCUSSION In a multicenter trial conducted in Italy[7], the 3-, 5- and 7-years survival rates after PEI in patients with a single www.wjgnet.com

HCC ≤ 30 mm in diameter have been reported as 78%, 54% and 28%, respectively; those in patients with a single HCC 30.1-50 mm as 61%, 32% and 16%, respectively; and those in patients with multiple lesions as 51%, 21% and 0%, respectively. In a series of 270 patients in Japan with fewer than three small lesions (≤ 30 mm in diameter) of HCC, overall 3- and 5-years survival rates after PEI were 81.6% and 60.3%, respectively, but the rates were higher, 87.3% and 78.3% in Child-Pugh class A patients with a solitary tumor [8] ≤ 20 mm in diameter . The overall 3-, 5-, 7- and 10-years survival rates after PEI in the present study were 74.1%, 49.9%, 27.2% and 14.5%, respectively, in our population with minimum-sized HCC. Patients of Child-Pugh class A had 5-, 7- and 10-years survival rates of 76.0%, 42.2% and 15.8%, respectively. The outcome in this study was superior, or at least equal to that reported in other studies. The superiority or equality of our results, including those for multinodular HCC, compared with other studies can be explained by tumor size alone. Generally speaking, the therapeutic effect of PEI is largely dependent on tumor size [9]. Strictly speaking, the difference between tumors 15 and 16-20 mm in diameter is very important. Pathological events identified in 106 small resected HCCs < 20 mm in diameter have demonstrated local metastases (located ≤ 10 mm from the nodule), and microscopic portal invasion among the most frequently occurring tumors (the so-called distinctly nodular types). The frequency of portal invasion has been reported as significantly higher in HCC 16-20 mm in diameter (40%) than in HCC 11-15 mm in diameter (25%, P < 0.01)[9,10]. In the current study, survival was not influenced by sex, age, HBsAg positivity, anti-HCV positivity, AFP levels, arterial blood flow, portal blood flow, histological characteristics, tumor multiplicity, or tumor size. However, a statistically significant difference was observed in long-term survival probability attributed only to liver dysfunction. PEI-treated patients of Child-Pugh class A had longer survival than those of class B, which was comparable with the Italian and Japanese studies[7,8,11]. Generally speaking, in treating HCC, prognosis depends not only on the grade of cancer spread (tumor stage)[12], but also on the grade of residual liver function (liver disease stage). Kudo et al[13] have proposed a prognostic staging system for HCC called

Taniguchi M et al. Percutaneous ethanol injection for small HCC

the Japan Integrated Staging Score (JIS score), and have suggested that the prognosis of stageⅠHCC (solitary, < 20 mm in diameter, no vascular invasion) depends on liver function. Here, the long-term results of PEI were equivalent to those achieved with patients treated by hepatic resection. According to the Liver Cancer Study Group of Japan[14], the 5- and 7-years survival rates among 3674 patients with single, clinical stageⅠHCC lesions < 20 mm in diameter, treated by hepatic resection, reached 65.4% and 47.1%, respectively. In our study, the respective rates of 77.3% and 43.0% were obtained by restricting the final analysis to a selected group of 20 patients with single or multiple HCC nodules ≤ 15 mm in diameter and with Child-Pugh class A cirrhosis. Hence, although no prospective randomized trials comparing PEI versus surgery have been conducted, the long-term results of the two treatments seem to be quite similar. Recently, new thermal therapeutic techniques for HCC have been developed, including RFA, laser microwaves and cryotherapy. Among these, RFA has attracted much international interest and is now widely used in clinical practice. In a comparison of PEI and RFA in 86 patients with 112 HCCs[15], a complete response was reached in 90.3% by RFA and 80% by PEI, with an average of 1.2 sessions for RFA and 4.8 sessions for PEI. However, more complications arose by RFA: one severe (hemothorax that required drainage) and four minor (intraperitoneal bleeding, hemobilia, pleural effusion and cholecytitis), compared with none by PEI. Recently, three randomized studies which compared RFA versus PEI for first-line treatment of early-stage HCC have been published[16-18]. European groups have failed to show a statistically significant difference in overall survival between patients who received RFA and PEI[16]. On the other hand, survival advantages have been identified in studies in Japan and Taiwan[17,18]. In Japan, Shiina et al[17] have described 232 patients, 118 treated by RFA and 112 by PEI. Four-year survival rate was 74% (95% CI: 65-84) for RFA and 57% (95% CI: 45-71) for PEI. RFA had a 46% smaller risk of death [adjusted relative risk, 0.54 (95% CI: 0.33-0.89), P = 0.02], a 43% smaller risk of overall recurrence [adjusted relative risk 0.57 (95% CI: 0.41-0.80), P = 0.0009], and an 88% smaller risk of local tumor progression [relative risk, 0.12 (95% CI: 0.03-0.55), P = 0.006] than PEI. Similarly, benefits in survival were also suggested in a subgroup analysis of a trial in Taiwan[18]. Most trials comparing RFA and PEI for treatment of small HCC have yielded better survival, local efficacy, local recurrence and duration of treatment in favor of RFA; the only advantage in favor of PEI being a slightly lower rate of complications[16]. In the present study, the frequency of HCC recurrence was considered to be high, with new lesions appearing in 25 of the 31 patients. However, after PEI, almost all recurrence was caused by the emergence of new nodular lesions in hepatic segments other than at the locations of the treated tumors, and were therefore probably unrelated to the original tumor. Repeat PEI alone was feasible for recurrence in 60.0%, 42.1%, 41.6%, 25.0% and 42.8% of these cases at detection of the first to fifth recurrence, respectively. As the number of lesions also



2001

increases whenever a tumor recurs, local treatment such as PEI is of limited value, and PEI must be replaced by another form of treatment such as TACE or transcatheter infusion chemotherapy. All the studies mentioned above confirm the high efficacy of PEI in the treatment of minimum-sized HCC. In conclusion, to achieve the best possible prognosis in its treatment, early detection of HCC < 15 mm in diameter by imaging and histological diagnoses, and early treatment by PEI are essential.

comments COMMENTS Background

In treatment of hepatocellular carcinoma (HCC), only 20%-30% of patients are candidates for surgery. Thus, various non-surgical therapies, such as percutaneous ethanol injection (PEI), microwave coagulation and radiofrequency ablation (RFA) have been widely used for small HCC. Although PEI was used as first-line treatment in some Japanese and Italian centers in the 1980s and 1990s when surgical techniques were precluded, RFA has recently emerged as a real competitor to PEI. At this time, there are no unequivocal data to back up PEI as a replacement for resection as a first-line treatment for patients with early-stage HCC.

Research frontiers

PEI is a standard therapy. However, there has been a drastic shift from PEI to RFA since the introduction of the latter into clinical practice, because efficacy seems more reproducible in RFA than in PEI and microwave coagulation. Most trials comparing RFA and PEI for the treatment of small HCC have yielded not only local efficacy but also survival in favor of RFA. In addition, RFA requires shorter hospitalization than PEI, which improves quality of life. However, severe complications arise with RFA, such as hemothorax, intraperitoneal bleeding, liver abscess, liver infarction and diaphragmatic hernia, compared with none with PEI.

Innovations and breakthroughs

PEI can be safely performed. In fact, severe complications such as intraperitoneal bleeding, liver abscess, liver infarction and diaphragmatic hernia did not occur in our study. After PEI however, almost all recurrence was caused by the emergence of new nodular lesions in hepatic segments other than at the locations of the treated tumors. In those cases, PEI was used not only for the initial treatment of small HCC, but also for recurrent lesions at untreated sites after treatment. The post-PEI survival rates in our patients with Child-Pugh class A cirrhosis were at least equal to those in the post-surgery group. Tumor size (< 15 mm in diameter) and liver function (Child-Pugh class A cirrhosis) were significant survival predictors, and such patients were the best candidates for percutaneous ablation. PEI is considered not to compete with but to be complementary to RFA in the treatment of small HCC, because of its excellent safety and efficacy.

Applications

RFA is superior to PEI in the treatment of small HCC from the viewpoint of treatment response and long-term survival. PEI however, seems feasible, efficacious and is very safe. RFA is difficult with tumors located near the gall bladder, bile ducts and diaphragm. Therefore, the usefulness and importance of PEI for HCC, especially for small-sized (< 15 mm in diameter) HCC, should be emphasized. Early detection of HCC < 15 mm by imaging and histological diagnosis and early treatment by PEI are essential.

Terminology

Small HCC: < 15 mm in diameter. Local ablation therapy: non-surgical imagingguided therapy (using US and/or CT) such as PEI, microwave coagulation and RFA. PEI: absolute ethanol is injected directly into lesions through 21-22-G needles, which are inserted under US guidance. It can destroy a considerably large volume of tissue in one session. RFA: electrodes are inserted into the tumor under imaging guidance. Radiofrequency energy is emitted from the exposed portion of the electrode, which is converted into heat and causes necrosis of the tumor. Child-Pugh class A liver cirrhosis: Cirrhosis with relatively good liver function (bilirubin < 2 mg/dL, albumin > 3.5 g/dL, and prothrombin time > 80%), without ascites and encephalopathy.

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Peer review

This study reported a small cohort of 31 patients with HCC < 15 mm in diameter treated by PEI. Overall survival of the patients was equal or possibly slightly superior to that with treatment by hepatic resection or radioablation. Although a small number of patients was analyzed, it represents an interesting and potentially important clinical finding.

REFERENCES 1 2 3

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Edmondson HA, Steiner PE. Primary carcinoma of the liver: a study of 100 cases among 48,900 necropsies. Cancer 1954; 7: 462-503 Okuda K, Peters RL, Simson IW. Gross anatomic features of hepatocellular carcinoma from three disparate geographic areas. Proposal of new classification. Cancer 1984; 54: 2165-2173 Trevisani F, Caraceni P, Bernardi M, D'Intino PE, Arienti V, Amorati P, Stefanini GF, Grazi G, Mazziotti A, Fornale L. Gross pathologic types of hepatocellular carcinoma in Italian patients. Relationship with demographic, environmental, and clinical factors. Cancer 1993; 72: 1557-1563 Okuda K. Early recognition of hepatocellular carcinoma. Hepatology 1986; 6: 729-738 Kim SR, Kang KB, Soh CG, Kim JH, Hayashi Y, Hanioka K, Itoh H. Clinicopathological study of minimum-sized hepatocellular carcinoma: an approach to the definition of early hepatocellular carcinoma. J Gastroenterol Hepatol 1995; 10: 498-508 Bartolozzi C, Lencioni R. Ethanol injection for the treatment of hepatic tumours. Eur Radiol 1996; 6: 682-696 Lencioni R, Pinto F, Armillotta N, Bassi AM, Moretti M, Di Giulio M, Marchi S, Uliana M, Della Capanna S, Lencioni M, Bartolozzi C. Long-term results of percutaneous ethanol injection therapy for hepatocellular carcinoma in cirrhosis: a European experience. Eur Radiol 1997; 7: 514-519 Ebara M, Okabe S, Kita K, Sugiura N, Fukuda H, Yoshikawa M, Kondo F, Saisho H. Percutaneous ethanol injection for small hepatocellular carcinoma: therapeutic efficacy based on 20-year observation. J Hepatol 2005; 43: 458-464 Vilana R, Bruix J, Bru C, Ayuso C, Sole M, Rodes J. Tumor size determines the efficacy of percutaneous ethanol injection

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for the treatment of small hepatocellular carcinoma. Hepatology 1992; 16: 353-357 10 Kojiro M. The evolution of pathologic features of hepatocellular carcinoma. In: Tabor E, ed. Viruses and liver cancer. Amsterdam: Elsevier, 2002: 113-122 11 Lencioni R, Caramella D, Bartolozzi C. Hepatocellular carcinoma: use of color Doppler US to evaluate response to treatment with percutaneous ethanol injection. Radiology 1995; 194: 113-118 12 Marsh JW, Dvorchik I, Bonham CA, Iwatsuki S. Is the pathologic TNM staging system for patients with hepatoma predictive of outcome? Cancer 2000; 88: 538-543 13 Kudo M, Chung H, Haji S, Osaki Y, Oka H, Seki T, Kasugai H, Sasaki Y, Matsunaga T. Validation of a new prognostic staging system for hepatocellular carcinoma: the JIS score compared with the CLIP score. Hepatology 2004; 40: 1396-1405 14 Arii S, Yamaoka Y, Futagawa S, Inoue K, Kobayashi K, Kojiro M, Makuuchi M, Nakamura Y, Okita K, Yamada R. Results of surgical and nonsurgical treatment for small-sized hepatocellular carcinomas: a retrospective and nationwide survey in Japan. The Liver Cancer Study Group of Japan. Hepatology 2000; 32: 1224-1229 15 Livraghi T, Goldberg SN, Lazzaroni S, Meloni F, Solbiati L, Gazelle GS. Small hepatocellular carcinoma: treatment with radio-frequency ablation versus ethanol injection. Radiology 1999; 210: 655-661 16 Lencioni RA, Allgaier HP, Cioni D, Olschewski M, Deibert P, Crocetti L, Frings H, Laubenberger J, Zuber I, Blum HE, Bartolozzi C. Small hepatocellular carcinoma in cirrhosis: randomized comparison of radio-frequency thermal ablation versus percutaneous ethanol injection. Radiology 2003; 228: 235-240 17 Shiina S, Teratani T, Obi S, Sato S, Tateishi R, Fujishima T, Ishikawa T, Koike Y, Yoshida H, Kawabe T, Omata M. A randomized controlled trial of radiofrequency ablation with ethanol injection for small hepatocellular carcinoma. Gastroenterology 2005; 129: 122-130 18 Lin SM, Lin CJ, Lin CC, Hsu CW, Chen YC. Radiofrequency ablation improves prognosis compared with ethanol injection for hepatocellular carcinoma < or = 4 cm. Gastroenterology 2004; 127: 1714-1723 S- Editor Liu JN L- Editor Kerr C

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World J Gastroenterol 2008 April 7; 14(13): 2003-2009 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

LIVER CANCER

Anti-cancer and anti-angiogenic effects of curcumin and tetrahydrocurcumin on implanted hepatocellular carcinoma in nude mice Pornprom Yoysungnoen, Ponthip Wirachwong, Chatchawan Changtam, Apichart Suksamrarn, Suthiluk Patumraj Pornprom Yoysungnoen, Department of Physiology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand Ponthip Wirachwong, The Government Pharmaceutical Organization, Rama VI, Rajtevi, Bangkok 10300, Thailand Chatchawan Changtam, Apichart Suksamrarn, Department of Chemistry, Faculty of Science, Ramkhamhaeng University, Bangkok 10240, Thailand Suthiluk Patumraj, Department of Physiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10300, Thailand Author contributions: Yoysungnoen P and Patumraj S designed research; Yoysungnoen P performed research; Wirachwong P contributed HepG2 cell lines; Changtam C and Suksamrarn A prepared curcumin and its analog; Yoysungnoen P and Patumraj S analyzed data; and Yoysungnoen P and Patumraj S wrote the paper. Supported by The Thailand Research Fund, No. MRG4980032 and partially supported by The National Center for Genetic Engineering and Biotechnology, Thailand Correspondence to: Pornprom Yoysungnoen, Department of Physiology, Faculty of Medical Science, Naresuan University, Phitsanulok 65000, Thailand. [email protected] Telephone: +66-81-7009474  Fax: +66-55-261197 Received: December 10, 2007 Revised: February 21, 2008

Abstract AIM: To determine the effect of tetrahydrocurcumin (THC) on tumor angiogenesis compared with curcumin (CUR) by using both in vitro and in vivo models of human hepatocellular carcinoma cell line (HepG2). METHODS: T h e 3 - ( 4, 5 - d i m e t hy l t h i a zo l - 2 -y l ) - 2 , 5-diphenyl-tetrazolium bromide (MTT) assay was used for testing the anti-proliferating activities of CUR and THC. In 6 male BALB/c nude mice, 2 × 10 human HepG2 cells were inoculated onto a dorsal skin-fold chamber. One day after HepG2 inoculation, the experimental groups were fed oral daily with CUR or THC (300 mg/kg or 3000 mg/kg). On d 7, 14 and 21, the tumor microvasculature was observed using fluorescence videomicroscopy and capillary vascularity (CV) was measured. RESULTS: Pathological angiogenic features including microvascular dilatation, tortuosity, and hyper-permeability were observed. CUR and THC could attenuate these pathologic features. In HepG2-groups, the CV were significantly increased on d 7 (52.43%), 14 (69.17%), and 21 (74.08%), as compared to controls (33.04%,

P < 0.001). Treatment with CUR and THC resulted in significant decrease in the CV (P < 0.005 and P < 0.001,

respectively). In particular, the anti-angiogenic effects of CUR and THC were dose-dependent manner. However, the beneficial effect of THC treatment than CUR was observed, in particular, from the 21 d CV (44.96% and 52.86%, P < 0.05). CONCLUSION: THC expressed its anti-angiogenesis without any cytotoxic activities to HepG2 cells even at the highest doses. It is suggested that anti-angiogenic properties of CUR and THC represent a common potential mechanism for their anti-cancer actions. © 2008 WJG . All rights reserved.

Key words: Tumor angiogenesis; HepG2; Curcumin; Tetrahydrocurcumin; Intravital fluorescence videomicroscopy Peer reviewer: Kazuhiro Hanazaki, MD, Professor and Chairman, Department of Surgery, Kochi Medical School, Kochi University, Kohasu, Okohcho, Nankoku, Kochi 783-8505, Japan

Yoysungnoen P, Wirachwong P, Changtam C, Suksamrarn A, Patumraj S. Anti-cancer and anti-angiogenic effects of curcumin and tetrahydrocurcumin on implanted hepatocellular carcinoma in nude mice. World J Gastroenterol 2008; 14(13): 2003-2009 Available from: URL: http://www.wjgnet.com/1007-9327/14/2003. asp DOI: http://dx.doi.org/10.3748/wjg.14.2003

INTRODUCTION Hepatocellular carcinoma (HCC) is a highly malignant tumor characterized by active neovascularization[1]. Since HCC recruit new blood vessels to support tumor growth, an anti-angiogenic agent is one of the goal drugs to treatment of HCC. Cancer cells have a very high rate of mutation in contrast to endothelial cells, which are the main components of blood vessels and have a lower rate of mutation. Because of this genetic stability, anti-cancer treatmenting tumor-induced angiogenesis is expected to be less vulnerable to such drug tolerance. Moreover, it may work on a broad spectrum of solid tumors because all these tumors need to induce angiogenesis for their processes. It is of great interest to apply the idea of antiangiogenesis treatment to the prevention of cancer. If www.wjgnet.com

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food factors that can inhibit angiogenesis were to be found, such factors could be used to stop small cancers from progression. Curcumin (CUR) is considered to be among such candidates. Curcumin (diferuloylmethane) is a phenolic compound from the plant Curcuma longa. A variety of pharmacological effects of curcumin have been reported, including anti-inflammatory[2], anti-oxidant[3,4], and anti-carcinogenic activities[5-7]. Recently, it has been shown that the anti-cancer property of curcumin is mediated in part by its anti-angiogenic activity[8-11]. As the active metabolite of curcumin obtained in gastrointestinal tract, tetrahydrocurcumin (THC) is a reduced analog of curcumin with phenolic and β-diketo moieties as well as curcumin (Figure 1). Sugiyama et al[12] demonstrated that THC exhibited similar physiological and pharmacological properties, in particular, THC has possessed strong antioxidant action than curcuminoids including curcumin, demethoxycurcumin, and bisdemethoxycurcumin [13] . Although the role of THC in anti-cancer activity has been implicated[13], its possible mechanism(s) and efficacy related to curcumin anti-cancer responsibility are still controversial. For instance, THC has been reported to be a less effective chemopreventive agent in mouse skin than curcumin[14]. In contrast, 0.5% THC mixed diet showed a stronger inhibitory effect on 1,2-dimethylhydrazine induced mouse colon carcinogenesis than curcumin[15]. Therefore, the present study was aimed to determine the effect of THC on tumor angiogenesis in comparison with curcumin by using both in vitro and in vivo models of human hepatocellular carcinoma cell line (HepG2).

MATERIALS AND METHODS Preparation of curcumin and THC The curcuminoid mixture obtained from the rhizomes of Curcuma longa was subjected to silica gel column chromatography, using hexane-dichloromethane, dichloromethane and dichloromethane-methanol as eluents to afford curcumin (CUR) as the major constituent. Recrystallization was accomplished by dissolving the evaporated eluate with a small quantity of dichloromethane and ethanol was then added. CUR crystallized out as yellow needles, melting point (m.p.) 181-183℃. THC was synthesized from CUR by catalytic hydrogenation reaction, with palladium on charcoal as a catalyst. The product was purified by silica gel column chromatography followed by recrystallization with dichloromethane-hexane to give 75% yield of THC as colorless needles, m.p. 93-94℃. The spectroscopic (IR, 1 H-NMR and mass spectra) data of the synthesized THC were consistent with the reported values[16]. In vitro study of anti-proliferation assay The effects of CUR and THC on the growth and survival of human hepatocellular carcinoma cell lines were measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay. Briefly, HepG2 cells (7.5 × 10 4 per well) were plated in 0.2 mL medium containing 10% FBS in triplicate in 96 well plate after 24 h medium was removed and then treated with 0.2 mL medium containing the indicated concentrations of CUR or THC at 37℃

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Figure 1 Chemical structures of CUR and THC. CUR and THC have similar b-diketo and phenolic moieties.

for 24 h. At the end of incubation, 0.050 mL of MTT solution (5 mg/mL) was added to each well. After 20 min incubated at 37℃, 0.030 mL of isopropanol was added to dissolve the formazan crystals. The absorbance of the MTT formazan was determined at 570 nm in an enzyme-linked immunosorbent assay (ELISA) reader. Cell growth index was defined as a percentage of the absorbance of treated cells to untreated cells. Animal preparation The experiments were perfor med in BALB/c-nude mice (b.w. 20-25 g; n = 90). The animal experiment was conducted according to the guideline of experimental animals by The National Research Council of Thailand (1999). The mice were bred and maintained in a specific pathogen germ-free environment. The mice were divided into four groups: (1) normal (control) mice with vehicle treatment (Con, n = 15), (2) HepG2-induced tumor mice (HepG2, n = 15), (3) HepG2induced tumor mice with CUR treatment (HepG2-CUR, n = 30) and (4) HepG2-induced tumor mice with THC treatment (HepG2-THC, n = 30). In order to implant HepG2 a dorsal skin-fold chamber (7 mm diameter)[16] was used. After the anesthetization by sodium pentobarbital (50 mg/100 g BW, i.p.), 30 µL of 2 × 10-6 HepG2 cells were inoculated in the middle area of dorsal skin-fold chamber and then covered with 7 mm glass slip. All surgical procedures were performed under aseptic conditions. The animals were then housed one animal per cage with free access to sterile water and standard laboratory chow. In the CUR and THC treated groups (HepG2-CUR and HepG2-THC groups), the mice were daily oral treated by 2 mL of 300 and 3000 mg/kg BW CUR and THC dissolved in 0.1% dimethyl sulfoxide (DMSO; Sigma, USA). These treatments were started twenty-four hours after the inoculation. In the control group (Con and HepG2), the mice received vehicle (0.1% DMSO) instead. Intravital fluorescence videomicroscopy study The experiments were performed d 7, 14 and 21 after vehicle, CUR or THC treatments. T he mice were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg BW). A catheter was inserted into

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Figure 2 An example of 5 windows selected for measurement of capillary vascularity (CV) from the video-image at a low magnification (× 10, Bar = 100 mm).

Measurement of capillary vascularity Based on the recorded video image, we measured capillary vascularity (CV) defined as follows: CV = (number of pixels within the capillaries) × 100 (%)/total number of pixels within the selected window area. In each mouse, we observed and recorded at 5 positions on the surface of tumors by moving the microscopic stage. Figure 2 shows an example of 5 windows selected on one low-magnified image. Each window (video frame of 100 × 100 pixels) was selected so as to cover any no large vessel. By determining both minimum and maximum intensities of pixels, we counted the total number of pixels over all capillaries in each window, using digital image processing software (Global Lab Ⅱ) and expressing the CV as percents of capillary area to total area. Averaging the CV’s (CVi, i = 1-5) measured over 5 video-frames (positions), we calculated the mean CV in one mouse: Mean CV = (1/5) Σ CV(1-5). Statistical analysis Results were shown as mean ± SE. One-way ANOVA was used to evaluate the difference of means. The statistical differences were considered at the probability level (P value) of less than 0.05.

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a jugular vein for application of fluorescence tracers. Then, the dorsal skin-fold chamber was removed and skin area around the chamber was fixed with modeling wax on a plate. The microcirculation within a studied area was observed under an intravital fluorescence microscope using a 10 × objective. During the experiment, a videocamera (Sony, Japan) was used to project the image onto a monitor (Sony, Japan) and to record the interested areas within the tumorbearing chambers by using a video-recorder (Sony, Japan). The videotape of each experiment was then analyzed offline using digital image processing software (Global Lab Ⅱ). For visualization of the microvascular lumen, a bolus of 0.1 mL of 5% fluorescein isothiocyanate-labeled dextran (FITC-dextran) was injected into the jugular vein 5 min prior to the recording. The recorded videoimages were analyzed and calculated for capillary vascularity, using digital image processing software (Global Lab Ⅱ) and then expressed in percentage as described previously [9]. The capillary vascularity (CV) level was used as an index of angiogenesis.

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Figure 3 IC50 and cell growth index by MTT assay. A: Effects of CUR on cell proliferation of HepG2 cell; B: Effects of THC on cell proliferation of HepG2 cell. Values given represent the mean ± SE of three independent experiments carried out in triplicate. The IC50 value was required to decrease viability from 100% to 50%.

RESULTS Anti-proliferation effects of curcumin and its analog on HepG2 cells The anti-proliferation activity of CUR and its analog, THC, were examined in HepG2 cell lines by MTT assay. It was found that CUR is a more potent anti-proliferative agent than THC. The IC50 of CUR and THC were 85.98 and 233.12 µmol/L, respectively (Figure 3). Tumor angiogenesis in HepG2-implanted nude mice Intravital fluorescence microscopic observation demonstrated a number of neocapillaries in HepG2 groups. Figure 4 shows fluorescence videoimages of the microvasculature for control and HepG2 groups on d 7, 14 and 21 after tumor cells implantation. In addition, pathological angiogenic features including abrupt changes in the diameter, tortuosity, and hyper-permeability were also observed in HepG2 groups. Effects of curcumin and its analog on tumor angiogenesis in HepG2-implanted nude mice Figure 5 demonstrated the intravital fluorescent microscopic observation of tumor angiogenesis affected by CUR and THC treatment. The result showed that the appearance of www.wjgnet.com

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Figure 4 A: Fluorescence videoimage of the microvasculature for control; B: Fluorescence videoimage of the microvasculature for 7 d HepG2 groups; C: Fluorescence videoimage of the microvasculature for 14 d HepG2 groups 14; D: Fluorescence videoimage of the microvasculature for 21 d HepG2 groups (× 10, Bar = 100 mm).

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neocapillaries induced by HepG2 was markedly reduced on 14 and 21 d after treatment of CUR and THC (3000 mg/kg BW). In addition, the abnormalities of neocapillary network pattern were attenuated after both treatments. Capillary vascularity of tumor tissue in HepG2-implanted nude mice For analysis of microvascular parameters, CV in the surface

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Figure 5 A: Fluorescence videoimage of capillary vascularity on 14 d after the implantation of tumor cells with treatment of CUR (3000 mg/kg); B: Fluorescence videoimage of capillary vascularity on 14 d after the implantation of tumor cells with treatment of THC (3000 mg/kg); C: Fluorescence videoimage of capillary vascularity on 21 d after the implantation of tumor cells with treatment of CUR (3000 mg/kg); D: Fluorescence videoimage of capillary vascularity on 21 d after the implantation of tumor cells with treatment of THC (3000 mg/kg, × 10, Bar = 100 mm).

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area of tumor was calculated at different periods after tumor cell inoculation. Figure 6 shows CV of 7, 14 and 21 d after vehicle, CUR or THC treatment in control and HepG2 groups. In HepG2-group, the percentage of CV was significantly increased on d 7 (52.43%), 14 (69.17%), and 21 (74.08%), as compared to age-matched controls (33.04%, P < 0.001). Treatment with CUR and THC showed significant decrease in the percentage of CV (P < 0.005

Yoysungnoen P et al. CUR and THC effects on anti-angiogenesis Control

HepG2

HepG2 + CUR 300

HepG2 + THC 300

 HepG2 + CUR 3000

HepG2 + THC 3000

100

CV (%)

80 60

a

a a

b

c c

e

e e,f,g

c,d

40 20 0

7

14 Days after treatment

21

Figure 6 Capillary vascularity (mean ± SE) of 7 d, 14 d, and 21 d after vehicle (0.1% DMSO), CUR or THC (300 and 3000 mg/kg BW) treatment in control and HepG2 groups. aP < 0.001, vs control group with vehicle; bP < 0.005, vs 14 d HepG2 group with vehicle; cP < 0.001, vs 14 d HepG2 group with vehicle; dP < 0.01, vs 14 d HepG2-THC 300 group; eP < 0.001, vs 21 d HepG2 group with vehicle; fP < 0.001, vs 21 d HepG2-THC 300 group; gP < 0.05, vs 21 d HepG2-CUR 3000 group.

and P < 0.001, respectively). In particular, the antiangiogenic effects of CUR and THC were dose-dependent manner. However, the beneficial effect of (3000 mg/kg) THC treatment than CUR was suggested, in particular, from the 21 d percent of neocapillaries density (44.96% and 52.86%, P < 0.05).

DISCUSSION By using MTT assay, the anti-proliferation properties of CUR and THC were examined in He pG2 cell lines. It was found that CUR has more potent antiproliferation properties than THC. The IC 50 of CUR and THC were 85.98 and 233.12 µmol/L, respectively. CUR has been shown to inhibit cell proliferation in a wide variety of human cancer cell lines in vitro[18] and in various xenotransplant and orthotopic models of human cancer in rodent [18,19] . CUR suppresses the activation of several transcription factors that are implicated in carcinogenesis [18] , including nuclear factor kappa B (NF-κB)[20], activator protein 1 (AP-1)[21], and at least two of the signal transducer and activator of transcription proteins (STAT3, STAT5), and modulates the expression of early growth response protein 1 (Erg-1), peroxisome proliferators-associated receptor gamma (PPAR-γ)[22]. It also suppresses the expression of cyclin D1[23] and induces apoptosis of tumor cells[24,25]. According to the inhibitory effects of CUR on these cell signaling pathways, CUR may mediate its anti-proliferation by inhibiting either expression or activation of proteins that required for cell survival or cell proliferation. Furthermore, it might imply that THC could be able to suppress these key factors of signaling pathways at lesser efficacies than CUR. By using intravital fluorescence videomicroscopy, the results showed that: (1) there was a significant increase in the numbers of CV with the heterogeneous network in HepG2 groups as compared to controls as which consistency with our previous reports [9,10]. (2) In this study, it was confirmed that more neocapillary density was observed as a time-dependent manner during tumor

2007

progression (CV of 21 d > CV of 14 d > CV of 7 d). (3) THC is a stronger anti-angiogenic agent than CUR. Therefore, in the present study, CUR and THC could exert both direct and indirect actions by inhibiting tumor cell proliferation and by inhibiting tumor angiogenesis, respectively. Although the precise mechanisms that lead to tumor angiogenesis are not fully understood, several studies have shown that tumor angiogenesis which is the common process necessary for every tumor types requires the expressions of cyclooxygenase-2 (COX-2), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-9 (MMP-9). Similar to our findings, CUR has been shown to suppress the proliferation of human vascular endothelial cells in vitro[26] and to abrogate angiogenic response in vivo[10]. It has also been shown that CUR inhibited Akt activation and down-regulated the expression of 5-lipooxygenase[20]. The significant finding initiated from the current study is that THC has efficacy in anti-angiogenic activity than CUR. Although THC is shown to be less anti-proliferative activity than CUR, several studies agreed to demonstrate that THC is a more potent anti-oxidant than CUR[12,27], and the mechanism could be implied by its β-diketo moiety[12]. A number of evidence also suggested that tumor-mediated inflammatory response could generate an intensive local accumulation of reactive oxygen species (ROS). ROS may play a role as the mediator for the consequence of tumor induced the expression of tumor biomarkers involved tumor angiogenesis. The activation for VEGF and angiopoietin-1 induced EC migration and/or proliferation through an increase in ROS mainly proposed by a number of researchers[28,29]. It was found that ethanol stimulated actin cytoskeletal reorganization, cell motility and tube formation in a ROS-dependent manner in ECs [30]. Furthermore, Leptin, a circulating adipocytokine, upregulated VEGF mRNA and stimulates cell proliferation through an increase in ROS in ECs[31]. Based on the idea of ROS, several other antioxidants such as green tea catechins, vitamin E, and natural polyphenols from red wine have been documented as the inhibitors of tumor angiogenic responses[32]. According to the more potent anti-oxidant activity of THC, the more potent anti-angiogenic activity of THC than CUR could be used to describe the different anti-angiogenic results. Finally, it was concluded that both CUR and THC have shown to produce both anti-proliferation and antiangiogenesis at different manner. More potent tumor antiangiogenesis was observed for THC, and it might be due to its higher anti-oxidant activity than CUR. It is implied that THC might be a promising candidate for tumor antiangiogenesis in the near future.

comments COMMENTS Background

Anti-angiogenesis, the postulated mechanism of anti-cancer activities of tetrahydrocurcumin (THC) and curcumin (CUR), was examined using hepatocellular carcinoma cells (HepG2)-implanted nude mice. CUR has been found to be an angiogenic inhibitor. However, THC, a potent anti-oxidative agent is responsible for the reported effect is still to be determined. The present study was aimed to determine the effect of THC on tumor angiogenesis in comparison with CUR by using both in vitro and in vivo models of HepG2.

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Research frontiers

THC contains both a phenolic moiety and a β-diketone moiety in the same structure. THC exhibits many of the same physiologic and pharmacological activities as CUR and in some systems may exert greater anti-oxidant activity than CUR. Therefore, the role of THC in preventive and therapeutic of cancer is gain interest. Anti-angiogenic therapy is one of the most promising strategies for cancer treatment. In this study, anti-angiogenic activity was investigated by evaluating the density of neovascularization induced by Hepatocellular carcinoma cell (HepG2) in nude mice, using intravital fluorescence videomicroscope. The results showed that THC exerts significant anti-angiogenic activity. Therefore, THC might be a promising candidate for tumor anti-angiogenesis in the near future.

12 13 14

Innovations and breakthroughs

The present study showed the anti-cancer and anti-angiogenic activities of CUR and THC. CUR and THC have shown to produce both anti-proliferation and antiangiogenesis at different manner. More potent tumor anti-angiogenesis was observed for THC, and it might be due to its higher anti-oxidant activity than CUR.

15

Applications

The findings from this study support the idea that anti-oxidative substances can be a therapeutic target for treating cancer.

16

Peer review

In this study, THC, a novel type of anti-oxidant showed anti-angiogenic activities without any cytotoxic effect. Importantly, our results have provided originally an in vivo evidence for anti-angiogenic activity of THC, in particular by using hepatocellular-carcinoma inoculated skin-chamber model.

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Armaiz-Pena GN, Kamat AA, Spannuth WA, Gershenson DM, Lutgendorf SK, Aggarwal BB, Sood AK. Curcumin inhibits tumor growth and angiogenesis in ovarian carcinoma by targeting the nuclear factor-kappaB pathway. Clin Cancer Res 2007; 13: 3423-3430 Sugiyama Y, Kawakishi S, Osawa T. Involvement of the beta-diketone moiety in the antioxidative mechanism of tetrahydrocurcumin. Biochem Pharmacol 1996; 52: 519-525 Pari L, Murugan P. Protective role of tetrahydrocurcumin against erythromycin estolate-induced hepatotoxicity. Pharmacol Res 2004; 49: 481-486 Huang MT, Ma W, Lu YP, Chang RL, Fisher C, Manchand PS, Newmark HL, Conney AH. Effects of curcumin, demethoxycurcumin, bisdemethoxycurcumin and tetrahydrocurcumin on 12-O-tetradecanoylphorbol-13-acetateinduced tumor promotion. Carcinogenesis 1995; 16: 2493-2497 Kim JM, Araki S, Kim DJ, Park CB, Takasuka N, BabaToriyama H, Ota T, Nir Z, Khachik F, Shimidzu N, Tanaka Y, Osawa T, Uraji T, Murakoshi M, Nishino H, Tsuda H. Chemopreventive effects of carotenoids and curcumins on mouse colon carcinogenesis after 1,2-dimethylhydrazine initiation. Carcinogenesis 1998; 19: 81-85 Lee SL, Huang WJ, Lin WW, Lee SS, Chen CH. Preparation and anti-inflammatory activities of diarylheptanoid and diarylheptylamine analogs. Bioorg Med Chem 2005; 13: 6175-6181 Lehr HA, Leunig M, Menger MD, Nolte D, Messmer K. Dorsal skinfold chamber technique for intravital microscopy in nude mice. Am J Pathol 1993; 143: 1055-1062 Aggarwal BB, Kumar A, Bharti AC. Anticancer potential of curcumin: preclinical and clinical studies. Anticancer Res 2003; 23: 363-398 Aggarwal BB, Shishodia S, Takada Y, Banerjee S, Newman RA, Bueso-Ramos CE, Price JE. Curcumin suppresses the paclitaxelinduced nuclear factor-kappaB pathway in breast cancer cells and inhibits lung metastasis of human breast cancer in nude mice. Clin Cancer Res 2005; 11: 7490-7488 Aggarwal S, Ichikawa H, Takada Y, Sandur SK, Shishodia S, Aggarwal BB. Curcumin (diferuloylmethane) down-regulates expression of cell proliferation and antiapoptotic and metastatic gene products through suppression of IkappaBalpha kinase and Akt activation. Mol Pharmacol 2006; 69: 195-206 Tomita M, Kawakami H, Uchihara JN, Okudaira T, Masuda M, Takasu N, Matsuda T, Ohta T, Tanaka Y, Mori N. Curcumin suppresses constitutive activation of AP-1 by downregulation of JunD protein in HTLV-1-infected T-cell lines. Leuk Res 2006; 30: 313-321 Chen A, Xu J. Activation of PPAR{gamma} by curcumin inhibits Moser cell growth and mediates suppression of gene expression of cyclin D1 and EGFR. Am J Physiol Gastrointest Liver Physiol 2005; 288: G447-G456 Mukhopadhyay A, Banerjee S, Stafford LJ, Xia C, Liu M, Aggarwal BB. Curcumin-induced suppression of cell proliferation correlates with down-regulation of cyclin D1 expression and CDK4-mediated retinoblastoma protein phosphorylation. Oncogene 2002; 21: 8852-8861 Choudhuri T, Pal S, Das T, Sa G. Curcumin selectively induces apoptosis in deregulated cyclin D1-expressed cells at G2 phase of cell cycle in a p53-dependent manner. J Biol Chem 2005; 280: 20059-20068 Sandur SK, Ichikawa H, Pandey MK, Kunnumakkara AB, Sung B, Sethi G, Aggarwal BB. Role of pro-oxidants and antioxidants in the anti-inflammatory and apoptotic effects of curcumin (diferuloylmethane). Free Radic Biol Med 2007; 43: 568-580 Singh AK, Sidhu GS, Deepa T, Maheshwari RK. Curcumin inhibits the proliferation and cell cycle progression of human umbilical vein endothelial cell. Cancer Lett 1996; 107: 109-115 Osawa T, Sugiyama Y, Inayoshi M, Kawakishi S. Antioxidative activity of tetrahydrocurcuminoids. Biosci Biotechnol Biochem 1995; 59: 1609-1612 Harfouche R, Malak NA, Brandes RP, Karsan A, Irani K, Hussain SN. Roles of reactive oxygen species in angiopoietin-1/ tie-2 receptor signaling. FASEB J 2005; 19: 1728-1730

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Ikeda S, Ushio-Fukai M, Zuo L, Tojo T, Dikalov S, Patrushev NA, Alexander RW. Novel role of ARF6 in vascular endothelial growth factor-induced signaling and angiogenesis. Circ Res 2005; 96: 467-475 Qian Y, Luo J, Leonard SS, Harris GK, Millecchia L, Flynn DC, Shi X. Hydrogen peroxide formation and actin filament reorganization by Cdc42 are essential for ethanol-induced in vitro angiogenesis. J Biol Chem 2003; 278: 16189-16197

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World J Gastroenterol 2008 April 7; 14(13): 2010-2022 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

VIRAL HEPATITIS

Serial changes in expression of functionally clustered genes in progression of liver fibrosis in hepatitis C patients Yoshiyuki Takahara, Mitsuo Takahashi, Qing-Wei Zhang, Hirotaka Wagatsuma, Maiko Mori, Akihiro Tamori, Susumu Shiomi, Shuhei Nishiguchi Yoshiyuki Takahara, Mitsuo Takahashi, Qing-Wei Zhang, Hirotaka Wagatsuma, Maiko Mori, Exploratory & Applied Pharmaceutical Research Department, Pharmaceutical Company, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan Akihiro Tamori, Susumu Shiomi, Department of Nuclear Medicine, Graduate School of Medicine, Osaka City University, Osaka, Japan Shuhei Nishiguchi, Division of Hepatobiliary and Pancreatic Diseases, Department of Internal Medicine, Hyogo College of Medicine, Nishinomiya, Japan Correspondence to: Yoshiyuki Takahara, Exploratory & Applied Pharmaceutical Research Department, Pharmaceutical Company, Ajinomoto Co., Inc., 1-1 Suzuki-cho, Kawasaki-ku, Kawasaki 210-8681, Japan. [email protected] Telephone: +81-44-2105822 Received: April 23, 2007 Revised: July 18, 2007

Abstract AIM: To investigate the relationship of changes in expression of marker genes in functional categories or molecular networks comprising one functional category or multiple categories in progression of hepatic fibrosis in hepatitis C (HCV) patients. METHODS: Marker genes were initially identified using DNA microarray data from a rat liver fibrosis model. The expression level of each fibrosis associated marker gene was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) in clinical biopsy specimens from HCV-positive patients (n = 61). Analysis of changes in expression patterns and interactions of marker genes in functional categories was used to assess the biological mechanism of fibrosis. RESULTS: The profile data showed several biological changes associated with progression of hepatic fibrosis. Clustered genes in functional categories showed sequential changes in expression. Several sets of clustered genes, including those related to the extracellular matrix (ECM), inflammation, lipid metabolism, steroid metabolism, and some transcription factors important for hepatic biology showed expression changes in the immediate early phase (F1/F2) of fibrosis. Genes associated with aromatic amino acid (AA) metabolism, sulfur-containing AA metabolism and insulin/ Wnt signaling showed expression changes in the middle phase (F2/F3), and some genes related to glucose www.wjgnet.com

metabolism showed altered expression in the late phase of fibrosis (F3/F4). Therefore, molecular networks showing serial changes in gene expression are present in liver fibrosis progression in hepatitis C patients. CONCLUSION: Analysis of gene expression profiles from a perspective of functional categories or molecular networks provides an understanding of disease and suggests new diagnostic methods. Selected marker genes have potential utility for biological identification of advanced fibrosis. © 2008 WJG . All rights reserved.

Key words: Hepatitis C; Liver fibrosis; Marker gene; Gene expression; RT-PCR; Molecular network; Metabolism; Transcription factor; Diagnosis Peer reviewer: Richard A Rippe, Dr, Department of Medicine, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7038, United States

Takahara Y, Takahashi M, Zhang QW, Wagatsuma H, Mori M, Tamori A, Shiomi S, Nishiguchi S. Serial changes in expression of functionally clustered genes in progression of liver fibrosis in hepatitis C patients. World J Gastroenterol 2008; 14(13): 2010-2022 Available from: URL: http://www.wjgnet.com/1007-9327/14/2010. asp DOI: http://dx.doi.org/10.3748/wjg.14.2010

INTRODUCTION Liver fibrosis is caused by liver disorders such as hepatitis C, hepatitis B, alcoholic hepatitis and non-alcoholic hepatitis. Fibrosis progresses gradually and finally disrupts liver structure and function over several decades, leading to fatal diseases such as cirrhosis and hepatocellular carcinoma (HCC). Classification of fibrosis progression is usually based on histological criteria using the METAVIR scoring system[1], which includes five stages: F0 (no fibrosis), F1, F2, F3, and F4 (cirrhosis). Such a classification is essential in decisions regarding treatment of liver fibrosis. Prominent subjective symptoms do not occur from F1 to F3, but patients begin to be aware of symptoms after F4. However, the F4 stage of cirrhosis is almost incurable and diagnosis of fibrosis at an earlier stage is desirable. The biology after F4 (cirrhosis) has been well studied, due to the interest in diagnosis and therapy for hepatocellular carcinoma (HCC),

Takahara Y et al . Marker genes for liver fibrosis in hepatitis C patients

2011

but progression to HCC may begin in the early stage of fibrosis in hepatitis C[2]. Prevention of HCC and inhibition of fibrosis in the early phase is important, but the detailed hepatic biological changes corresponding to the F stage are unclear. To understand the background of the early stage of fibrosis, we previously identified genes that can be used as markers of biological changes in progression of hepatic fibrosis and diagnosis of fibrotic progression; these data were obtained from DNA microarray data from an experimental DMN (dimethylnitrosamine)-treated rat model of hepatic fibrosis [3] . This work led to marker genes that were arranged in functional categories related to fibrosis, based on genes associated with hepatic cell types such as Kupffer cells, hepatic stellate cells, and hepatocytes. These marker genes give information on cell-specific and time-dependent behavior of each hepatic cell in fibrogenesis. In the current work, the behavior of marker genes associated with a particular F stage was analyzed using RT-PCR in clinical biopsy specimens from hepatitis C patients. This profile data revealed several biological changes in progression of hepatic fibrosis. Since many functionally clustered genes showed similar changes in expression, we propose serial expression changes in molecular networks associated with liver fibrosis progression in hepatitis C patients. Many functionally clustered genes showed large changes in expression in the early stage of fibrosis, suggesting the importance of therapy at this early stage. Alteration of gene expression also suggested qualitative changes in biological status in the transition from F3 to F4, which is a suspected risk factor for development of HCC. We conclude that analysis of gene expression profiles from a perspective of molecular networks provides improved understanding of disease and indicates potential methods of diagnosis.

an ISOGEN kit (Nippongene) and reverse transcribed using a High Capacity cDNA Archive Kit (ABI, Foster City, CA), in each case according to the manufacturer’s instructions. The total RNA in the final reaction mixture was 10 ng/ µ L. Real-time PCR was performed on an Applied Biosystems 7500 Real-Time PCR System (ABI) data collection system, and analyses were performed using the accompanying software. RT-PCR was performed using 0.8 µL cDNA in each well, with a final concentration of 1X the probes of the TaqMan® Gene Expression Assay and 1X the Taqman Universal PCR Master Mix (ABI). The final reaction volume was 20 µL. Each sample was analyzed in duplicate. The thermal cycler conditions were 2 min at 50℃ and 10 min at 95℃, followed by 40 cycles of 15 s at 95℃ and 1 min at 60℃. Data were analyzed using the comparative CT method, in which the expression level of a target gene is normalized relative to an endogenous reference. GAPDH was used as the endogenous reference in all experiments. The target CT and endogenous control CT were calculated for each sample, and the target gene expression level was then calculated using the for mula 2 (34-CT) . The average of duplicate measurements was obtained, and the relative expression of each gene in a sample was calculated by setting the expression of GAPDH equal to 1000. PCR fluorogenic probes for all the target genes and the endogenous reference were purchased as TaqMan® Gene Expression Assays (ABI).

MATERIALS AND METHODS

Pathway analysis of PCR data The behavior and relationships of marker genes in pathways associated with lipid metabolism were analyzed with bioSpace Explorer, a system for analysis of expression profile data. This system was produced collaboratively by Pharmafrontier Co. Ltd. and World Fusion Co. Ltd. to examine molecular interactions in expression profile data, using both manual and computational text mining.

Patients in the clinical study All patients were recruited from the Osaka City University Hospital (Osaka, Japan). Sixty-one patients with seropositive results in diagnosis using the third-generation hepatitis C virus enzyme-linked immunosorbent assay (Lumipulse Ⅱ Ortho HCV, Ortho-Clinical Diagnostics, Tokyo, Japan) and positive serum HCV-RNA were included in the study. Informed consent was obtained from all patients. Liver biopsies were performed on all patients enrolled in the study, and the histological features of the liver specimens were analyzed and graded using the METAVIR scoring system[1]. The liver fibrotic stage (F stage) and inflammatory activity (A grade) were determined histologically: at least four subjects were found to be in each F stage classification. Determination for chymase 1 exceptionally has been done with three subjects in F4 stage due to the lack of appropriate samples. Part of the biopsy sample from each patient was immediately immersed in RNAlater (QIAGEN, The Netherlands) to inhibit RNAase and then kept at 4℃ overnight before being transferred to another tube and frozen at -80℃. RT-PCR analysis Total RNA was extracted from liver biopsy samples using

Statistical analysis A Kruskal-Wallis test was applied to select marker genes with statistically significant changes (P < 0.05) in expression level during the fibrosis progression. This calculation was performed using SPSS (SPSS Inc., IL, USA). The gene expression data were subjected to hierarchical clustering analysis using Genowiz™ software (Ocimum Biosolutions).

RESULTS Expression behavior of marker genes All marker genes determined in this study are listed in Table 1. For each gene, since the quantitative limitation of biopsy specimens resulted in a difference in sample number for each probe, the type of samples, indicating the number of biopsy specimens, is listed in Table 2. Marker genes were selected as representative members of functional categories or molecular networks based on their expression changes in an experimental hepatic fibrosis model[3]. Marker genes with statistically significant changes in expression are listed in Table 1. Genes that showed statistically insignificant changes in expression are listed with the gene name only in Table 1. Marker genes that showed statistically significant changes by t test during a www.wjgnet.com

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World J Gastroenterol

April 7, 2008

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Table 1 Expression profiles of marker genes with statistically significant changes during fibrosis progression Functional category Description ECM (or other HSC marker)

Inflammation (or apoptosis)

Growth factor

Insulin/ Wnt signal

Others signal Transcription factors

Transporter

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Gene name

Expression at F1 Group

Expression ratio Serial Type F2/F1 F3/F2 F4/F3

Decorin DCN 217.6 2 1.2 1.1 1.1 1 1 Matrix metalloproteinase 2 MMP2 11.7 2 1.5 1.2 0.9 2 1 Hyaluronan-mediated motility receptor HMMR 1.2 2 2.6 0.6 1.1 3 1 Lysyl oxidase LOX 1.0 2 1.4 1.2 0.9 4 1 Lysyl oxidase-like 1 LOXL1 0.8 2 1.8 1.4 1.0 5 1 Tropomyosin 1 TPM1 35.0 2 1.8 1.5 1.1 6 1 Prion PRNP 19.4 3 1.2 0.7 1.0 7 1 Collagen, type I, alpha 1 COL1A1 16.2 2 1.3 2.2 0.6 8 1 Collagen type Ⅲ alpha 1 COL3A1 143.9 2 1.3 1.5 0.8 9 1 Collagen type alpha 1 COL4A1 16.8 2 1.3 1.9 0.6 10 1 Humican LUM 22.8 2 1.2 2.0 0.9 11 1 Sialoprotein SPP1 13.2 2 1.2 2.2 1.4 12 1 Glypican 3 GPC3 23.5 2 1.8 2.5 1.0 13 1 Proline 4-hydroxylase, alpha polypeptide I P4HA1 34.0 1 1.1 0.4 1.2 14 1 Insignificant change: MGP, BGN, TAGLN, LGALS1, EDG2, EDG5, TNNT2 Lysozyme LYZ 185.3 2 2.1 0.8 1.0 15 2 TGF beta TGFB1 51.9 3 1.5 0.8 0.9 16 1 TGF beta 3 TGFB3 3.3 3 1.4 0.9 0.9 17 3 TNF TNF 2.6 3 1.7 0.6 1.2 18 3 Natural killer cell proteinase 1 GZMB 1.7 3 1.6 0.5 1.1 19 1 IL1 beta IL1B 2.6 3 1.5 0.7 1.1 20 3 Hemopoietic cell kinase HCK 22.4 3 1.3 0.6 0.9 21 4 Interleukin 6 receptor IL6R 142.9 1 0.9 0.7 1.0 22 3 BCL2-related ovarian killer BOK 205.9 1 0.9 0.7 1.1 23 5 Caspase 2 CASP2 1.3 1 1.2 0.7 0.8 24 5 Chymase 1, mast cell CMA1 0.2 2 0.7 1.8 2.2 25 6 Insignificant change: LTBP1, LBP, TNFRSF1B, DEFB1, IL1RN, S100A8, BRIC3, CARD12, CASP1, CASP4, CASP8, PAWR, CD19, CD3Z, MS4A1, CD37, TRA@ Growth hormone receptor GHR 102.3 1 0.8 0.9 0.9 26 4 IGF1 IGF1 56.5 1 0.9 0.7 1.1 27 1 Insignificant change: PTN, FST, PRLR Cyclin D1 CCND1 190.6 2 1.7 1.0 1.0 28 5 Forkhead box M1 FOXM1 0.6 3 3.7 0.8 1.1 29 4 Gap junction protein, alpha 1, 43 kDa (connexin 43) GJA1 4.0 3 2.4 0.5 1.0 30 5 V-akt murine thymoma viral oncogene homolog 1 AKT1 77.6 1 0.9 0.9 1.0 31 4 V-akt murine thymoma viral oncogene homolog 2 AKT2 59.6 1 0.9 0.7 0.8 32 7 Catenin (cadherin-associated protein), beta 1, 88 kDa CTNNB1 159.0 1 1.1 0.7 1.0 33 5 Catenin, beta interacting protein 1 CTNNBIP1 24.0 1 1.1 0.7 0.8 34 5 Glycogen synthase kinase 3 beta GSK3B 44.1 1 1.1 0.8 0.8 35 5 Dishevelled, dsh homolog 1 (Drosophila) DVL1 12.2 1 1.1 0.7 0.9 36 7 Membrane-bound transcription factor peptidase, site 1 MBTPS1 72.4 1 0.9 0.7 1.0 37 7 Membrane-bound transcription factor peptidase, site 2 MBTPS2 13.2 1 0.9 0.7 1.0 38 7 Tribbles homolog 3 (Drosophila) TRIB3 18.4 1 1.1 0.9 0.4 39 7 Insignificant change: GSK3A, INSIG1, INSIG2, PRKCB1, PRKCD Regucalcin (senescence marker protein-30) RGN 697.8 1 0.9 0.8 0.8 56 4 Insignificant change: DAB2, PMP22, S100A10, LCN2 CCAAT/enhancer binding protein (C/EBP), alpha CEBPA 352.2 1 0.7 1.2 0.7 40 4 Retinoid X receptor, alpha RXRA 409.6 1 0.7 1.0 0.8 41 4 Hepatocyte nuclear factor 4, alpha HNF4A 708.7 1 0.7 1.1 0.9 42 4 Transcription factor 1 (HNF1) TCF1 26.7 1 0.8 0.9 0.8 43 4 Nuclear receptor subfamily 0, group B, member 2 NR0B2 166.7 1 0.7 0.9 0.8 44 4 Peroxisome proliferative activated receptor, alpha PPARA 62.9 1 0.7 0.8 0.9 45 4 Inhibitor of DNA binding 1 (splice variation) ID1 463.4 3 1.6 0.5 1.4 46 1 AE binding protein 1 AEBP1 24.6 2 1.4 1.2 1.0 47 4 Nuclear receptor subfamily 1, group H, member 2 NR1H2 (LXRB) 9.0 1 1.2 0.7 1.0 48 7 Nuclear receptor subfamily 1, group H, member 3 NR1H3 (LXRA) 27.8 1 1.0 0.7 0.8 49 7 Nuclear receptor subfamily 1, group H, member 4 NR1H4 (FXR) 107.0 1 1.0 0.7 0.9 50 7 c/EBPbeta CEBPB 274.3 1 0.9 0.7 0.9 51 2 Upstream transcription factor 2, c-fos interacting USF2 323.7 1 0.9 0.7 0.9 52 4 Estrogen-related receptor alpha ESRRA 79.6 1 1.2 0.6 0.8 53 7 C-met MET 73.5 1 1.0 0.7 1.1 54 3 Upstream transcription factor 1 USF1 30.8 1 1.0 0.9 0.8 55 4 Insignificant change: ONECUT1, JUNB, NR3C1, PPARG, PPARGC1B, PPARGC1A, SREBF2, FHL2 Solute carrier family 6, member 6 SLC6A6 3.7 3 2.5 0.6 1.0 57 5 Solute carrier family 7, member 1 SLC7A1 0.9 3 2.3 0.7 1.3 58 5 Solute carrier family 38, member 2 Alanine-transporter) SLC38A2 68.6 3 1.3 0.6 1.0 59 5 Solute carrier family 25 member 15 SLC25A15 85.7 1 1.1 0.7 0.9 60 5 Solute carrier family 7, member 7 SLC7A7 7.1 1 1.1 0.6 1.0 61 5 Solute carrier family 17 (sodium phosphate), member 1 SLC17A1 23.6 1 0.9 0.7 1.0 62 4

Takahara Y et al . Marker genes for liver fibrosis in hepatitis C patients

Redox Blood coagulation

Lipid metabolism

Steroid (or drug) metabolism

Bile acid metabolism

Prostanoid

Aromatic amino acid metabolism

Sulfur-containing amino acid metabolism

Insignificant change: SLC38A3, ABCB1, SLC15A4 Catalase CAT 1977.7 Paraoxonase 1 PON1 191.1 Coagulation factor X F10 187.5 Angiotensinogen AGT 1958.4 Fibrinogen, A alpha polypeptide FGA 8337.5 Plasminogen PLG 6156.1 Pai I SERPINE1 11.6 Acyl-Coenzyme A oxidase 2, branched chain ACOX2 122.2 L-3-hydroxyacyl-Coenzyme A dehydrogenase, short chain HADHSC 139.8 Acyl-CoA synthetase long-chain family member 1 ACSL1 2800.8 Acyl-Coenzyme A oxidase 1, palmitoyl ACOX1 357.7 Carnitine O-octanoyltransferase CROT 10.3 2,4-dienoyl CoA reductase 2, peroxisomal DECR2 135.2 Acetyl-Coenzyme A acyltransferase 2 ACAA2 1167.7 Acetyl-Coenzyme A acetyltransferase 1 ACAT1 714.2 Acyl-CoA synthetase long-chain family member 5 ACSL5 57.3 Dodecenoyl-Coenzyme A delta isomerase DCI 327.2 Enoyl Coenzyme A hydratase, short chain, 1, mitochondrial ECHS1 324.3 Hydroxyacyl-Coenzyme A dehydrogenase, type Ⅱ HADH2 422.5 Hydroxyacyl-Coenzyme A dehydrogenase, beta subunit HADHB 398.8 Lipase, hepatic LIPC 561.5 Hydroxyacyl-Coenzyme A dehydrogenase, alpha subunit HADHA 103.2 Palmitoyl-protein thioesterase 1 PPT1 82.1 Fatty acid synthase FASN 105.3 Peroxisomal D3, D2-enoyl-CoA isomerase PECI 292.8 Acyl-CoA synthetase long-chain family member 4 ACSL4 5.8 Insignificant change: BHHADH, ACAA1, CPT1A, ACADM, ACACA, CPT2 Aldo-keto reductase family 1, member D1 AKR1D1 248.3 HMT1 hnRNP methyltransferase-like 2 HRMT1L2 6.6 Hydroxysteroid (11-beta) dehydrogenase 1 HSD11B1 1064.6 Hydroxysteroid (17-beta) dehydrogenase 4 HSD17B4 66.7 Steroid-5-alpha-reductase, alpha polypeptide 1 SRD5A1 74.3 UDP glycosyltransferase 2 family, polypeptide B7 UGT2B7 530.3 Sulfotransferase family 1E, estrogen-preferring, member 1 SULT1E1 52.8 Aldo-keto reductase family 1, member C4 AKR1C4 137.1 Hydroxysteroid (17-beta) dehydrogenase 2 HSD17B2 496.9 Sulfotransferase family, cytosolic, 2A, member 1 SULT2A1 689.0 Hydroxysteroid (17-beta) dehydrogenase 8 HSD17B8 72.2 Steroid sulfatase (microsomal), arylsulfatase C, isozyme S STS 10.9 Emopamil binding protein (sterol isomerase) EBP 197.0 Farnesyl-diphosphate farnesyltransferase 1 FDFT1 252.6 Insignificant change: HSD17B2, HSD3B1, LCMT1, SULT2A1, HMGCR, DHCR7, CES2 Sterol O-acyltransferase 1 SOAT1 9.1 Alcohol dehydrogenase 1C (class I), gamma polypeptide ADH1C 312.7 Alcohol dehydrogenase, iron containing, 1 ADHFE1 112.7 Cytochrome P450, family 7, subfamily A, polypeptide 1 CYP7A1 104.1 Arachidonate 5-lipoxygenase-activating protein ALOX5AP 7.9 Leukotriene B4 receptor 2 LTB4R2 2.5 Insignificant change: LTA4H, CYSLTR1, CYSLTR2, LTC4S, PPT1 Dopa decarboxylase DDC 51.4 Monoamine oxidase B MAOB 426.6 Kynurenine 3-monooxygenase KMO 51.7 Kynureninase KYNU 63.3 Tyrosine aminotransferase TAT 658.2 GTP cyclohydrolase 1 GCH1 49.6 Insignificant change: HPD MAT2 MAT2B 111.7 Cystathionase (cystathionine gamma-lyase) CTH 112.0 Cystathionine-beta-synthase CBS 350.4 Betaine-homocysteine methyltransferase BHMT 1032.1 Methionine adenosyltransferase I, alpha MAT1A 547.6 Cysteine dioxygenase, type I CDO1 84.4 Glutamate-cysteine ligase, catalytic subunit GCLC 175.9 Glutathione S-transferase A1 GSTA1 2812.3 Alanyl (membrane) aminopeptidase ANPEP 501.9 Bile acid Coenzyme A: amino acid N-acyltransferase BAAT 284.4 Glutathione synthetase GSS 70.1 Lactate dehydrogenase A LDHA 716.3 Mercaptopyruvate sulfurtransferase MPST 684.3 Serine dehydratase SDS 440.4 Methionine adenosyltransferase Ⅱ, alpha MAT2A 122.9 Insignificant change: GGT1, GSR, MTR, DNMMT1, CSAD, GCLM, LDHB

2013

1 1 1 1 1 1 3 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 3 1 2

0.8 0.8 0.8 0.8 0.6 0.8 0.9 0.8 0.8 0.8 0.7 0.7 0.8 0.8 0.7 0.8 0.9 0.7 0.7 0.8 0.5 0.9 1.0 0.8 0.8 1.1

0.9 0.7 1.0 1.0 1.0 0.8 2.9 0.9 0.9 0.9 0.9 0.8 0.9 0.8 1.1 0.9 0.8 1.0 0.9 0.9 1.5 0.9 0.8 1.8 0.8 1.4

1.0 0.9 0.9 1.0 0.9 1.0 0.5 0.9 0.9 0.9 1.0 1.1 0.9 1.1 0.8 1.0 0.9 0.9 0.9 1.1 0.9 1.0 0.8 0.6 0.8 2.3

63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88

4 4 4 4 4 4 1 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4

1 1 1 1 1 1 1 1 1 1 1 1 1 1

0.6 0.8 0.7 0.8 0.7 0.8 0.5 0.9 1.0 0.9 0.7 1.0 1.0 0.7

0.8 1.0 0.8 0.9 0.8 0.8 0.5 0.6 0.8 0.8 0.7 1.0 0.7 0.9

0.6 0.8 0.8 0.9 0.9 0.9 1.4 1.2 1.1 0.9 1.0 0.6 0.7 0.7

89 90 91 92 93 94 95 96 97 98 99 100 101 102

4 4 4 4 4 4 7 4 4 4 7 4 5 5

1 1 1 1 2 1

1.0 0.7 0.7 0.7 1.7 1.1

0.8 0.6 0.7 0.6 0.7 0.7

0.9 1.1 0.8 2.9 1.0 1.0

103 104 105 106 107 108

5 5 5 5 3 3

1 1 1 1 1 1

1.0 0.8 1.0 0.9 0.9 0.9

0.8 0.8 0.7 0.7 0.5 0.7

0.8 1.0 1.0 0.8 1.3 0.9

109 110 111 112 113 114

4 4 4 4 4 4

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

1.1 1.0 1.1 1.0 1.0 0.9 1.1 1.0 0.9 1.0 1.1 0.8 0.9 0.9 1.0

0.7 0.7 0.7 0.7 0.5 0.7 0.6 0.7 0.8 0.6 0.8 0.7 0.7 0.4 0.9

1.1 1.1 0.9 1.0 1.1 1.0 1.1 1.0 0.9 1.0 0.8 1.0 0.9 1.0 0.8

115 116 117 118 119 120 121 122 124 125 126 127 128 129 130

3 3 3 3 2 1 1 1 5 5 5 5 5 5 5

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ISSN 1007-9327

Energy source amino acid metabolism

Glucose metabolism

Urea cycle

CN 14-1219/R

World J Gastroenterol

Phosphoenolpyruvate carboxykinase 2 (mitochondrial) PCK2 Alanine-glyoxylate aminotransferase AGXT Alanine-glyoxylate aminotransferase 2 AGXT2 Aldehyde dehydrogenase 2 family (mitochondrial) ALDH2 Aldehyde dehydrogenase 9 family, member A1 ALDH9A1 Pyruvate kinase, liver and RBC PKLR Aldehyde dehydrogenase 3 family, member A2 ALDH3A2 Phosphoenolpyruvate carboxykinase 1 (soluble) PCK1 Dihydrolipoamide dehydrogenase DLD Glutaminase 2 (liver, mitochondrial) GLS2 Glutamate-ammonia ligase GLUL Glutamic-oxaloacetic transaminase 1, soluble GOT1 Glutamic-pyruvate transaminase GPT Pyruvate carboxylase PC Phosphoglucomutase 1 PGM1 Pyruvate dehydrogenase kinase, isoenzyme 2 PDK2 Pyruvate dehydrogenase kinase, isoenzyme 4 PDK4 Fumarylacetoacetate hydrolase FAH Malic enzyme 1, NADP (+) -dependent, cytosolic ME1 Insignificant change: ALDOA, ASNS, GOT2, MGC33309, PDHB, PDK1 Phosphorylase, glycogen; liver PYGL Aldolase B, fructose-bisphosphate ALDOB Hexokinase 3 (white cell) HK3 Glycogen synthase 2 (liver) GYS2 Sterol regulatory element binding transcription factor 1 SREBF1 Glycerol-3-phosphate dehydrogenase 1 (soluble) GPD1 Ketohexokinase (fructokinase) KHK Glucokinase (hexokinase 4) regulator GCKR Aldolase C, fructose-bisphosphate ALDOC Insignificant change: GCK, PFKFB1, G6PC, HK2 Carbamoyl-phosphate synthetase 1, mitochondrial CPS1 Ornithine aminotransferase (gyrate atrophy) OAT Insignificant change: OTC

April 7, 2008

Volume 14

Number 13

791.8 3352.3 122.8 2027.7 160.9 211.3 237.4 3166.8 72.3 133.8 6.2 388.2 298.8 27.8 218.8 24.0 85.6 85.8 4.9

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2

0.8 0.7 0.6 0.7 0.8 0.6 0.9 1.0 0.9 0.8 1.0 0.9 0.8 0.7 0.8 0.9 0.7 0.9 1.0

0.9 0.9 0.8 0.9 0.8 1.1 0.8 0.5 0.8 0.7 0.6 0.7 0.6 0.7 0.8 0.8 0.5 0.9 1.0

1.0 1.0 1.0 0.9 0.9 0.7 1.1 1.2 0.9 1.0 1.2 0.8 1.1 0.9 1.0 0.9 1.4 0.8 1.3

131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149

4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 7 7 4 4

37.2 13141.8 3.3 276.6 10.6 130.8 411.8 204.8 36.8

1 1 1 1 2 1 1 1 1

0.8 0.7 1.2 0.9 1.1 0.8 0.7 0.7 0.7

1.0 0.8 0.6 0.6 1.2 1.0 1.0 1.0 0.9

1.0 0.9 0.9 0.9 0.9 0.8 0.8 0.8 0.6

150 151 152 153 154 155 156 157 158

4 4 4 7 4 4 4 4 4

1230.8 94.8

1 1

0.7 0.5

0.9 0.6

0.9 1.6

159 160

7 7

Liver biopsy samples were analyzed by RT-PCR using a TaqMan® Gene Expression Assay probe, as described in the Materials and Methods. The genes were selected from DNA microarray data from a rat fibrosis model presented in a previous paper 3. Genes in the ECM category (or other HSC markers) were selected as marker genes for HSC. Genes in the inflammation category were selected as markers of inflammatory cells in liver. Other genes were selected as markers of hepatocytes. Expression profiles of marker genes that showed statistically significant changes during fibrosis by a Kruskal-Wallis test are listed. Genes that showed statistically insignificant changes in expression are listed with the gene name only in Table 1. Columns from left to right indicate functional category, gene description, gene name, expression intensity at F1 stage, group classification (expression of marker genes in group 1 decreased almost linearly along with the F stages, expression of marker genes in group 2 increased almost linearly along with the F stages, and expression of marker genes in group 3 had a peak in the middle of the F stages), expression ratio between neighbouring F stages (a decreased ratio is shown in light gray and an increased ratio in deep gray), serial gene number and types of samples, which indicates the number of biopsy specimens, as shown in the Table 2. Actual alteration behavior of some characteristic genes are shown in Figure 1.

Table 2 Types of samples indicating the number of biopsy specimens Type of sample 1 2 3 4 5 6 7

F1

Number of samples F2 F3

F4

25 23 22 12 7 6 5

13 13 12 6 4 4 4

9 8 8 4 4 3 5

14 14 14 9 5 4 5

Quantitative limitation of biopsy specimens resulted in a difference in sample number for each probe, according to the interest in the particular gene.

transition to an adjacent F stage are similar to the selected genes in Table 1. An additional four marker genes selected in the t test analysis were added to the genes in Table 1. Over half of the selected marker genes from the DNA microarray data from the animal model showed statistically significant changes in the human samples, showing the effectiveness of the animal model for selection of genes www.wjgnet.com

of significance in humans. Genes showing changes in expression were roughly divided into three groups: genes in group 1 showed an almost linear decrease in expression along with an increase in F stage; genes in group 2 showed an almost linear increase with an increase in F stage; and genes in group 3 showed a peak in the middle of the F stage scale. Almost all genes in one category belonged to one or two groups, suggesting that genes in one category showed similar changes in expression during progression of fibrosis. The expression ratio between F stages is also shown in Table 1, with the peak ratio shown in bold. The peak for almost all genes in a given category occurred at the transition to the same F stage, again suggesting that genes in one category underwent changes in expression under similar mechanistic control. Expression changes in gene clusters in the early phase (F1 to F2) of fibrosis The functional categories showing a peak change in expression ratio in the early phase of fibrosis were inflammation, ECM, blood coagulation, lipid metabolism, half of the

Takahara Y et al . Marker genes for liver fibrosis in hepatitis C patients

A

B

Inflammation (or apoptosis)

ECM or other HSC marker 13

4.0 Signal ratio

Signal ratio

2.0

5.0

15

1.5

17 16

1.0

20

18

12

3.0

6

2.0

19

1

2

3

1.0

4

1

2

4.0

Signal ratio

3.0

5 11

8

4

F stage

C

10

9 2

21 0.5

2015

1

3

4

F stage

D

Insulin/Wnt signal

Blood coagulation

1.0

2.0

Signal ratio

0.9 29

28 1.0

2

3

66

0.7

0.5

4

67 1

2

3

F stage

1.0 Signal ratio

F

Lipid metabolism

1.1

85

0.9 0.8

0.6 0.5

78 81

1

2

82

84

75 79 71 77 80

0.7

76

73

74 72

70

3

Sulfur-containing animo acid metabolism 1.1 0.9

0.3

H 4.0

Insulin/Wnt signal 36

54 51 43 52

40 42 45

41

1

Signal ratio

Signal ratio

0.5

2

50 44 49 4

3

3.0 33 35

2.0

1.0

38

3.0

Signal ratio

2.0

37

31 34 32

1

F stage

I

4

2

53

0.7

3

129 1

F stage

48

0.9

121 116 123 117118 124 122120 128125127 119

0.5

4

Transcription factor

1.1

115 0.7

F stage

G 1.3

4

F stage

Signal ratio

E

65

68

0.6

30 1

0.8

Figure 1 Clusters of genes in functional categories. The characteristic behavior of gene clusters for each functional category is shown separately in Figure 1A-I. The numbers in each figure refer to the serial number of genes in Table 1. The expression ratio relative to the F1 stage is plotted in each graph. A: Inflammation gene cluster with a peak at F2; B: ECM gene cluster showing increased expression along with fibrosis progression; C: Insulin/Wnt signaling gene cluster with increased expression in the early phase of fibrosis; D: Blood coagulation factor gene cluster showing decreased expression along with fibrosis progression; E: Lipid metabolism gene cluster showing decreased expression along with fibrosis progression; F: Sulfurcontaining amino acid metabolism gene cluster showing a synchronous decrease in expression from F2 to F3; G: Two transcription factor gene clusters showing sequential decreases in expression; H: Insulin/Wnt signaling gene cluster showing decreased expression from F2 to F3; I: A gene cluster showing a peak or bottom at F3.

2

3

4

F stage

8 11

10 9 69

1.0

86 106

0.0

1

2

3

160 4

F stage

genes in steroid metabolism, half of those in energy source amino AA metabolism, and half of transcription factors.

Expression of marker genes in the inflammation and ECM categories started to increase in the early phase. Expression www.wjgnet.com

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World J Gastroenterol

Table 3 Classification of gene clusters based on serial alteration of gene expression along with the fibrosis progression F1-F2

F2-F3

Inflammation Wound-healing (ECM) Blood coagulation Transcription factors Transcription factors (cluster 1) (cluster 2) Insulin/Wnt signal Lipid metabolism Steroid metabolism Steroid metabolism (cluster 1) (cluster 1) Bile acid metabolism Energy source AA Energy source AA metabolism (cluster 1) metabolism (cluster 1) Aromatic AA metabolism Sulfur-containing AA metabolism Glucose metabolism (cluster 1)

F3-F4

Glucose metabolism (cluster 2)

Gene clusters are indicated under the name of gene category. The pattern of serial alteration for gene cluster was determined based on the maximum changing point of the expression ratio shown in Table 1.

of marker genes related to inflammation, such as LYZ, GZMB, IL1B, TNF and TGFB1, occurred in clusters and reached a peak at fibrotic stage F2, as shown in Figure 1A, suggesting that inflammatory events are particularly active at the F2 stage. However, histological classification of inflammatory activity shows a tendency for an increase in inflammation that is correlated with an increase in F stage; therefore, the conclusion regarding inflammatory events based on expression of marker genes appears to differ from that based on histological classification. Gene expression in the ECM category also increased until F3 or F4, as shown in Figure 1B; expression of such genes might indicate an inflammatory response for wound healing. Increased expression of some clustered genes related to the cell cycle, CCND1, FOXM1 and GJA1 (Connexin 43), was also found at an early stage, as shown in Figure 1C, and might reflect a response to hepatic cell injury. Almost all other genes were linearly down-regulated. Several genes related to blood coagulation, i.e. F10, AGT, FGA and PLG, were down-regulated as a cluster in the early phase, as shown in Figure 1D; this down-regulation may be linked to prolongation of the blood coagulation time in cirrhosis. An early response of many clustered genes associated with lipid metabolism was also found, as shown in Figure 1E. Expression of these genes decreased consecutively during fibrosis and the early response of genes affecting lipid metabolism is of interest. Expression changes in gene clusters in the middle phase (F2 to F3) of fibrosis The peak change in the expression ratio of marker genes in metabolism of sulfur-containing AA and aromatic AA was delayed, compared to genes associated with other kinds of metabolism. Marker genes for sulfur-containing AA metabolism decreased remarkably as a cluster in the phase from F2 to F3, as shown in Figure 1F. Decreased expression of marker genes for steroid metabolism, energy www.wjgnet.com

April 7, 2008

Volume 14

Number 13

source AA, and transcription factors were separable into two groups: early-response and middle-response genes. All the down-regulated transcription factors, including TCF1 (HNF-1), HNF4A (HNF-4), CEBPA (C/EBP alpha), CEBPB (C/EBP beta), PPARA (PPAR alpha), RXRA (RXR alpha), NR1H3 (LXRA), NR1H2 (LXRB), NR1H4 (FXR), USF-1, USF-2, and NR0B2 are important in hepatic metabolism and other regulatory mechanisms. Alteration of expression of these genes might be related to abnormal expression of metabolic enzymes. Two clusters of transcription factors were clearly distinguishable based on their expression pattern, as shown in Figure 1G: the first cluster, including HNF-4, C/EBPA, RXR, TCF1 (HNF1), PPARA, and NR0B2, which showed altered expression in the early phase, might influence expression of the second cluster, including CEBPB, NR1H2(LXRB), NR1H3(LXRA), NR1H4(FXR), ESRRA and USF2, which showed altered expression in the middle phase of fibrosis. A cluster of genes associated with insulin/Wnt signaling were down-regulated, as shown in Figure 1H, suggesting a common regulatory mechanism. These expression changes are likely to be related to changes in expression of transcription factors and genes in metabolic networks. The down-regulated genes included GSK3B and CTNNB1 (catenin beta 1), which participates in Wg/Wnt signaling for regulation of cell proliferation and differentiation[4]; GJA1 (connexin 43), which forms gap junctions and is regulated by Wg/Wnt signaling [5]; and FOXM1, which is associated with cell proliferation[6] and liver regeneration[7]. All of these genes had peak expression at F2 in a clustered manner, as shown in Figure 1C and G. Enhancement of cell proliferation for wound healing might be linked with a peak of inflammation at F2, and expression of genes such as CCND1, GJA1 and FOXM1 in the downstream part of the insulin/Wnt pathway were altered ahead of genes involved in insulin/Wnt signaling. The relationship between these genes requires further study. Expression changes in gene clusters in the late phase (F3 to F4) of fibrosis Few genes showed altered expression in the late phase of fibrosis, but a cluster of genes in the glucose metabolism category showed decreased expression. It was also of interest that expression of several genes reversed direction or abruptly altered in the late phase, as shown in Figure 1I. These results suggest different biological changes from the start of the late stage in the transition from F3 to F4. Serial expression changes for the functional categories are summarized in Table 3 and these data indicate associations between clustered genes in one category and inter-category relationships. Down-regulated individual marker genes (group 1) Regeneration of hepatic cells is suppressed during fibrosis and such suppression is thought to then cause further fibrosis. IGF1, GHR and IL6R (inflammation) support the regeneration of hepatocytes and down-regulation of the expression of these genes may be linked directly to formation of fibrosis. Down-regulation of PON1, which associates with HDL (high-density lipoprotein)

Takahara Y et al . Marker genes for liver fibrosis in hepatitis C patients

and regulates the cellular redox state, and PPT, which is known as a lysosomal hydrolase of long chain fatty acyl CoA and has a role in maintenance of synaptic function, may be related to mitochondrial damage, as we and others have suggested[3,8,9]. RGN, a calcium-binding protein that plays a pivotal role in maintaining cell homeostasis and function, was also down-regulated. Down-regulation of these genes may impair liver function. TRIB3 inhibits Akt/ PKB activation by insulin[10], and this gene was suddenly down-regulated from the F3 to F4 phase, suggesting new conditions in the insulin signaling pathway in the transition from F3 to F4. Up-regulated individual marker genes (group 2) Most inflammatory marker genes showed peak expression in the middle phase of fibrosis, as shown in Figure 1A, but CMA1 (chymase 1), which is produced by mast cells, underwent a linear increase in expression with progression of fibrosis. This is of note, since chymase has been reported to be involved in chronic hepatic fibrosis in autoimmune hepatitis and primary biliary cirrhosis[11], and mast cells may have a special role in fibrogenesis. The role of mast cells in chronic inflammation, however, deserves further study, because of the shortage of determined sample and low expression of CMA1. Individual marker genes with peak expression in the middle phase of fibrosis (group 3) The only hepatic stellate cell (HSC)-specific marker gene to show peak expression at the F2 stage was PRNP, which is reported to be a marker for the early phase of HSC activation. The amino acid transporters SLC38A2 (ATA2), SLC6A6 (TAUT) and SLC7A1 (CAT-1) showed peak expression at F2, which may also suggest enhancement of cell proliferation at this stage of fibrosis. Increased expression of SLC38A2, which preferentially transports alanine, has been reported in regeneration of hepatocytes[12], since hepatocytes require alanine as an energy source[13]. Based on our results, down-regulation of SLC38A2 in the late phase of fibrosis suggests that utilization of alanine as an energy source decreases at this stage of fibrosis. Upregulation of SLC6A6, a taurine transporter, in the early phase of fibrosis can be understood as protective behavior against injury of hepatocytes [14]. FASN, a marker gene for fatty acid metabolism, showed peak expression at the F3 stage of fibrosis. We have observed suppression of biosynthesis and degradation of fatty acids in the liver in a CCl4-induced cirrhotic rat model, and clinical results show temporal enhancement of fatty acid biosynthesis before subsequent suppression in an advanced phase of fibrosis; this may be a compensative action related to suppression of other forms of energy metabolism in fibrosis, as we have previously suggested[3]. Candidate marker genes for diagnosis of fibrosis and discrimination of fibrotic stages Diagnosis in the early stage of fibrosis may be important for monitoring progression of fibrosis and hepatic biological changes. Candidate marker genes at each step of fibrosis were selected based on an expression change

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ratio > 1.5 and an intensity > 10. Up-regulation of LYZ and down-regulation of FGA, OAT and AGXT2 were noteworthy in the transition from F1 to F2, and upregulation of genes in the ECM category and downregulation of genes in metabolism of energy source AA, aromatic AA, steroids and sulfur-containing AA occurred in the transition from F2 to F3. Diagnosis of the late stage of fibrosis (the transition from F3 to F4) is important because of the risk of tumorigenesis. Some genes showed a reversal of expression in the F3 to F4 transition, suggesting that biological changes in the stage from F3 to F4 are qualitatively different from those at earlier stages. Remarkable down-regulation of TRIB3, which inhibits insulin signaling and NFκB signaling, was noteworthy in the F3 to F4 transition. The reversal in expression of FASN in this phase may indicate changes in lipid metabolism and that of OAT indicates changes in ornithine metabolism or the urea cycle. The early increase in collagen expression began to decline in the F3 to F4 stage and the similar decline of SERPINE1 (Pai1) expression may reflect increased fibrolysis. Consecutive analysis of marker molecules in plasma will be important for monitoring progression of fibrosis at each step, and the data in this paper provides useful information for the selection of serum markers and interpretation of changes in the levels of these markers. Coordinated regulation of functional categories All genes in Figure 1 were subjected to hierarchical clustering analysis. Statistical clustering of the expression ratio between neighboring F stages for the genes in Table 1 was combined with functional categories, as shown in Figure 2. Functional categories clearly corresponded to the statistical clusters, suggesting coordinated regulation of genes in one functional category. Overlap of functional categories in statistical clusters suggested that these categories might be regulated by correlated mechanisms. In contrast, separation of members of a category into multiple positions of a statistical cluster suggests that the functional category may be divided into subgroups with respect to regulation.

DISCUSSION Biological interpretation of changes in gene expression Changes in expression of hepatic cell-specific marker genes reflects biological changes in the progression of hepatic fibrosis. Shimizu et al[15] reported co-localization of chymase with fibrotic tissue, and we have reported increased expression of marker genes for mast cells, including chymase, in progression of fibrosis in a DNMinduced rat fibrosis model. The results reported here also show correlation of the expression of chymase with the stage of fibrosis. Therefore, these results suggest that a certain cell population producing chymase has an important role in the pathogenesis of fibrosis. Many marker genes related to inflammation showed peak expression at F2. Inflammation has been reported to induce activation of HSCs[16], and we have shown a peak in activated inflammatory cells in a DMN-induced fibrosis model. These results suggest that a temporal www.wjgnet.com

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FOXM1 3 GPC3 2 SPP1 2 SERPINE1 4 FASN LUM 2 COL4A1 2 COL4A1 2 CYP7A1 TPM1 2 LOXL1 2 DCN 2 COL3A1 2 LOX 2 MMP2 2 GJA1 3 LYZ 1 CCND1 3 TGFB3 1 TGFB1 1 IL1B 1 GZMB 1 TNF 1 OAT SDS 3 MAT1A 3 ESRRA 7 HCK 1 NRH2 7 CTNNB1 8 GCLC 6 MAT2B 6 BAAT 6 GSTA1 6 BHMT 6 CTH 6 AHCY 6 MET 7 PPT1 5 GSK3B 8 CTNNBIP18 CBS 6 DVL1 8 NR1H4 7 NR1H3 7 AKT2 8 USF2 7 MPST 6 CEBPB 7 CEBPA 7 ACAT1 5 HNF4A 7 FGA 4 ECHS1 5 RXRA 7 ACOX1 5 CROT 5 HADHB 5 ACAA2 5 LDHA 6 MBTPS2 8 CDO1 6 MBTPS1 8 PLG 4 ACSL5 5 HADHA 5 AKT1 8 AGT 4 F10 4 PPATA 7 HADH2 5 NROB2 7 ANPEP 6 DCI 5 TCF1 7 ACSL1 5 HADHSC 5 DECR2 5 ACOX2 5

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Figure 2 Comparison between statistical clustering analysis and functional cluster. All genes in Figure 1 were subjected to hierarchical clustering analysis using Genowiz™. Statistical clustering of the expression ratio between neighboring F stages for the genes in Table 1 was combined with functional categories. The color coding in the center column shows the expression ratio between neighboring F stages for the genes in Table 1: Ratio increases from green to red. The left dendrogram shows the result of hierarchical clustering by Genowiz™ software. The numbers on the right of gene names indicate the subgraph numer in Figure 1, which represents functional categories, and the same numbers are tied with color bars.

strong inflammatory reaction is required for initiation of auto-stimulatory activation of HSCs. In the DMNinduced fibrosis model, inflammation was suppressed after it reached a peak, but weaker inflammation might still be sufficient to maintain activation of HSCs. Expression of HSC-specific marker genes such as COL1A1, LUM, TAGLN, MGP, SERPINE1 (PAI-1) and LOX did not increase or decrease continuously from F3 to F4, as shown in Figure 1B and 1I, whereas expression of DCN, BGN and GPC3 increased comparatively from F3 to F4, as shown in Figure 1B. This suggests some changes in the behavior of HSCs between F3 and F4, and it has been hypothesized that the perpetuation phase [17] may change to a new phase in fibrosis of stage F4. Therefore, profiles of HSC-specific marker genes may be useful for www.wjgnet.com

indication of the phase of fibrosis. A cluster of down-regulated genes associated with sulfur-containing AA metabolism is shown in Figure 1F. Changes in expression of the clustered genes occurred simultaneously between the F2 and F3 stages; this transition may reflect a biological change causing fibrotic progression, and the change in expression of the clustered genes seems to suggest an increased demand for glutathione[18]. Abnormalities in metabolism of sulfur-containing AA in thioacetamide-induced cirrhosis in rat liver have been found in a proteomics analysis[19], and similar abnormalities were also reported based on hepatic gene expression changes in patients with alcoholic hepatitis[20]. There is only limited information on altered expression of transcription factors in fibrotic liver, but down-regulation

Takahara Y et al . Marker genes for liver fibrosis in hepatitis C patients

of HNF-4 in human cirrhosis[21] and of PPARs in hepatitis C virus genotype 3[22] have been reported. C/EBP alpha and C/EBP beta regulate proliferation of hepatocytes[23] and glucose and lipid homeostasis[24-27], and HNF-1 and HNF-4 broadly regulate hepatic functions such as carbohydrate metabolism[28], lipid metabolism[29], bile acid metabolism and HDL-cholesterol metabolism[30]; expression of HNF-1 is also regulated by HNF-4[31]. USF1 and USF2 have been reported as glucose signals[32,33], and the USF1 and USF2 homodimers and the USF1-USF2 heterodimer regulate expression of liver-specific genes such as apolipoprotein A2 and pyruvate kinase. HNF-4 and USF2a bind to the enhancer sequence cooperatively[34]. HNF-4 also regulates PPAR alpha[35], which in turn regulates glucose[36], lipid[37] and cholesterol metabolism[38]. RXR alpha regulates lipid, bile acid and cholesterol homeostasis[39], and LXR alpha, LXR beta and FXR are associated with lipid[40,41], bile acid and cholesterol homeostasis [40,42]. RXR and FXR form heterodimers with other transcription factors, including other members of the same family or with PPAR alpha[40,43,44]. NR0B2 (SHP) regulates cholesterol metabolism[45], glucose metabolism[45] and bile acid synthesis[46], and interacts with LXR[47]. AKT1 and AKT2 are important kinases in the pathway of insulin regulation of glucose homeostasis[48] and in fatty acid synthesis[49]. Hence, the altered expression of these transcription factors may relate to altered expression of metabolic enzymes in glucose and lipid metabolism in the fibrotic liver of hepatitis C patients. The continuous increase in expression of cyclin D1 (CCND1) correlated with F stage, as shown in Figure 1C, and appears to be important for hepatic tumorigenesis. The association of Wnt signaling[50,51] and cyclin D[52] with tumorigenesis is well known. Catenin beta 1 (CTNNB1) regulates cyclin D expression [53] and is itself regulated through phosphorylation by GSK3B[54] or other kinases[55]. Down-regulation of expression of CTNNB1 occurred in advanced fibrotic stages, as shown in Figure 1H, and beta interacting protein 1 (CTNNBIP1), which is a negative regulator of CTNNB1, was also down-regulated, as also shown in Figure 1H. From this perspective, it was interesting that alterations in expression of genes associated with insulin signaling, including GSK3B, CTNNB1, CTNNBIP1 and downstream genes such as CCND1 and GJA1 (connexin 43), were clustered, as shown in Figures 1C and H. Suppression of insulin signaling has been reported in cirrhosis[56]; however, the response of downstream genes in this pathway was inconsistent with suppression of the insulin signal. Therefore, further studies will be necessary to clarify whether this inconsistency arises from differences between expression levels and the activity of the protein products of these genes, or if another signal[57] is involved in the Wnt and insulin signaling pathways. The bioSpace Explorer is a system for analysis of DNA microarray data that may allow an understanding of the molecular relationships underlying the above results. The network of genes from previous reports revealed integral relationships among insulin/Wnt signal, CEBPA, PPARA, RXRA, glucose metabolism, lipid synthesis and lipid metabolism. An initial version of bioSpace Explorer was constructed based on molecular networks related to lipid metabolism, with entries showing relationships between

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molecules via a line between the molecules. This bird’s eye view of the pathway including lipid metabolism with the input PCR data is illustrated in Figure 3; molecules related to inflammation were up-regulated and many genes related to lipid metabolism were down-regulated. Hypothetical causes of biological changes in progression of fibrosis Biological changes in fibrosis can be summarized as follows. Initially, Kupffer cells or other inflammatory cells are activated in the transition from F1 to F2. This event immediately influences production of ECM and cell cycle genes for wound-healing[58]. Blood coagulation is quickly suppressed in moving from F1 to F2, as shown by down-regulation of coagulation factor genes and upregulation of the inhibitor, PAI-1. Several transcription factor genes are also immediately influenced, probably due to inflammation, as shown by the down-regulation of CEBPA, HNF4A, TCF1 and NR0B2. Expression of many genes associated with lipid metabolism also changed quickly in the transition from F1 to F2. Down-regulation of these genes may be controlled by down-regulation of the transcription factors, especially RXRA, PPARA, LXRs and FXR. Some genes related to steroid metabolism also responded quickly for control of inflammation. Expression of genes associated with sulfur-containing AA metabolism and aromatic AA metabolism changed simultaneously in the transition from F2 to F3. The first type of metabolism relates to the redox state[59] and the second is associated with production of active metabolites such as catecholamines and serotonin. Such important biological states are controlled to maintain homeostasis through several mechanisms[60] and this may explain the delayed change in expression of these genes. The expression of many genes related to intracellular signaling, including insulin/Wnt signaling, also changed simultaneously in the transition from F2 to F3. This delayed change may also reflect compensative action for hepatic cellular defects on metabolism for energy supply and/or hepatic cellular proliferation. The main molecules in fibrosis, such as collagens, increase in expression from F2 to F3 and cause development of fibrosis through activation of HSCs through a stimulatory cycle involving inflammatory cells, HSCs and hepatocytes, as described previously[3]. Some quantitative biological changes started in the transition from F3 to F4. Down-regulation of genes associated with sugar metabolism and fatty acid synthesis at this stage might induce persistent defects in energy storage and supply to the liver. The liver transits into an inescapable negative cycle between this defect in the hepatic energy state and mitochondrial damage in cirrhosis. This negative cycle will be discussed in a future paper describing DNA microarray analysis of an animal model of cirrhosis. Coordinated regulation of functional categories Statistical cluster analysis showed coordinated regulation of functional categories in liver fibrosis. These regulatory mechanisms can be examined prospectively using bioSpace Explorer, and these results will be discussed in a future paper. In the current work, the clinical gene expression profiles assembled using RT-PCR, using genes www.wjgnet.com

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Figure 3 Molecular network associated with lipid metabolism. Gene expression changes in pathways related to lipid metabolism, illustrated with bioSpace Explorer (a system for analysis of DNA microarray data for lipid metabolism; Pharma Frontier Co. Ltd / World Fusion Co. Ltd.; see texts for details). The bird’s-eye view of the lipid metabolism is displayed. The up-regulated and down-regulated gene expression ratios at F3 vs F1 in Table 1 are displayed in red and green, respectively, with the color gradation proportional to the ratio. Genes in Table 1 that did not show a statistically significant change in expression are indicated with a blue circle with gray background. An entry with a gray background only indicates no input data. Most of the entity names in Figure 2 are the same as the gene name in Table 1, but the names “C20orf97”, “SCEH”, “Acaa2”, and “ACS5” in Figure 2 refer to “TRIB3”, “ECHS1”, “ACAA2”, and “ACSL5”, respectively, in Table 1. These differences are due to the software used in bioSpace Explorer.

originally selected based on DNA microarray data from an experimental animal model, provided an improved understanding of disease and suggested new methods of diagnosis. Therefore, statistical analysis and functional clustering or network analysis of the transcriptome, alone or in combination, can provide an overview of a dynamic biological system.

ACKNOWLEDGMENTS We thank Ms. Tomoko Kobayashi of Pharmafrontier Co. Ltd. and Mr. Ken Osaki of World Fusion Co. Ltd for statistical analysis and operation of the bioSpace Explorer for analysis of DNA microarray data for lipid metabolism.

COMMENTS Background

Information from clinical specimens is very important, but there is a limitation in sample preparation. With DNA microarray, it is difficult to determine gene expression profile from a small amount of sample such as a clinical needle biopsy sample. RT-PCR with TaqMan probe can make it possible with high quality. We have to get maximum information with a minimum number of TaqMan probes

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because the amount of samples is limited and probes are expensive. Selection of probes is a key factor. We extensively determined gene expression profiles from animal models of liver fibrosis with DNA microarray (WJG 2006; 12: 6473). The gene marker sets were arranged to show the change of gene expression of a molecular network or functionally clustered gene sets. In this paper, the selected gene marker sets effectively showed the dynamic behavior of global gene network change during liver fibrosis.

Research frontiers

Dynamic behavior of genome-wide genes expression is now measured with DNA microarray. This technology must be applied to clinical samples. Such information can greatly advance the study of disease pathogenesis, diagnosis and therapy. One problem is the interpretation of huge expression profile data from DNA microarray. Advanced technology of computational text-mining has recently shown the genomewide molecular networks or functional molecular clusters. This genome-wide network is going to be applied to the analysis of DNA microarray data. When expression profile data are arranged as a change in their networks of functional clusters, these huge data are expected to suggest effectively the biological meaning in terms of broad aspects of research interest. Therefore, we are developing an analysis system genome-wide molecular network which was made possible by a combination system based on both computational and manual text-mining. This system was partially applied on the interpretation of gene expression profile in this paper. Prevention therapy for individual patients at an early stage is required because genomic polymorphism is going to reveal the personal risk of diseases. The biological background of progression to disease onset must be understood for development of prevention therapy. Prediction of liver cancer risk is going to be tried in the early

Takahara Y et al . Marker genes for liver fibrosis in hepatitis C patients stage of liver fibrosis before cirrhosis. The accumulation process of hepatic stress which leads to onset of liver cancer has to be elucidated. For example, there is a question why BCAA (branched chain amino acids), which improves hepatic metabolism, reduces onset of liver cancer.

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Innovations and breakthroughs

We have already prepared the gene marker sets which can show the change of molecular networks or functionally clustered genes by DNA microarray experiment on liver fibrosis of animal models. The gene marker sets were linked to biological events in each hepatic cell such as hepatocytes, immune cells and stellate cells. Application of gene marker sets and RT-PCR with TaqMan probe technology on clinical specimens successfully showed the serial change of gene expression in molecular network or functionally clustered gene sets in the progression of liver fibrosis. It was proved that network analysis is a powerful tool for biological research. Our sequential approach (animal model/DNA microarray → selection of appropriate gene marker sets in molecular networks → clinical samples/RT-PCR → analysis functionally clustered gene markers in molecular networks → analysis of the relation between molecular networks) can effectively advance clinical research. Serial change of clustered gene expression during liver fibrosis progression, which was made clear in this paper, will reveal the molecular mechanism of many symptoms before and after the onset of cirrhosis and liver cancer.

Application

Gene marker sets and RT-PCR on clinical specimens as well as analytical methods with genome-wide gene networks can be applied to get the information of dynamic biological progression on various diseases. Serial change of clustered gene expression during liver fibrosis progression can provide a method of diagnosis and therapy in liver fibrosis. For example, the question why BCAA, which improves hepatic metabolism, reduces onset of liver cancer, will be solved.

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Terminology

Text-mining: all published information about molecular interaction and their function are collected and arranged to make new valuable information such as gene-wide molecular networks; TaqMan-PCR probe: Applied Biosystems offers more than 700 000 TaqMan® Gene Expression Assays, the most comprehensive set of predesigned Real-Time PCR assays available. All TaqMan® Gene Expression Assays have been designed using validated bioinformatics pipeline of Applied Biosystems, and run with the same PCR protocol, eliminating the need for primer design or PCR optimization.

Peer review

This paper revealed that metabolic deficiency occurs before the onset of cirrhosis. It had already been found in animal models with hepatic toxic substances in the preceding paper. Metabolic deficiency in hepatitis which was caused by a virus was found to be the same as animal models in this paper. Gene marker sets, selected from analysis of animal models, and analysis methods using molecular networks can lead to success in finding the serial change of functionally clustered genes expression during liver fibrosis progression. A set of appropriate gene markers in each network was a key to analysis. The sequential approach (animal model/DNA microarray → appropriate gene marker sets in molecular networks → clinical samples/RT-PCR → analysis functionally clustered gene markers in molecular network → analysis of the relation between molecular networks) is useful to elucidate the molecular mechanism of disease.

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for maintenance of the differentiated state during liver growth. J Clin Invest 1995; 96: 1351-1365 Arizmendi C, Liu S, Croniger C, Poli V, Friedman JE. The transcription factor CCAAT/enhancer-binding protein beta regulates gluconeogenesis and phosphoenolpyruvate carboxykinase (GTP) gene transcription during diabetes. J Biol Chem 1999; 274: 13033-13040 Gautier-Stein A, Mithieux G, Rajas F. A distal region involving hepatocyte nuclear factor 4alpha and CAAT/ enhancer binding protein markedly potentiates the protein kinase A stimulation of the glucose-6-phosphatase promoter. Mol Endocrinol 2005; 19: 163-174 Qiao L, MacLean PS, You H, Schaack J, Shao J. knocking down liver ccaat/enhancer-binding protein alpha by adenovirustransduced silent interfering ribonucleic acid improves hepatic gluconeogenesis and lipid homeostasis in db/db mice. Endocrinology 2006; 147: 3060-3069 Wang ND, Finegold MJ, Bradley A, Ou CN, Abdelsayed SV, Wilde MD, Taylor LR, Wilson DR, Darlington GJ. Impaired energy homeostasis in C/EBP alpha knockout mice. Science 1995; 269: 1108-1112 Pontoglio M. Hepatocyte nuclear factor 1, a transcription factor at the crossroads of glucose homeostasis. J Am Soc Nephrol 2000; 11 Suppl 16: S140-S143 Louet JF, Hayhurst G, Gonzalez FJ, Girard J, Decaux JF. The coactivator PGC-1 is involved in the regulation of the liver carnitine palmitoyltransferase I gene expression by cAMP in combination with HNF4 alpha and cAMP-response elementbinding protein (CREB). J Biol Chem 2002; 277: 37991-38000 Shih DQ, Bussen M, Sehayek E, Ananthanarayanan M, Shneider BL, Suchy FJ, Shefer S, Bollileni JS, Gonzalez FJ, Breslow JL, Stoffel M. Hepatocyte nuclear factor-1alpha is an essential regulator of bile acid and plasma cholesterol metabolism. Nat Genet 2001; 27: 375-382 Miura N, Tanaka K. Analysis of the rat hepatocyte nuclear factor (HNF) 1 gene promoter: synergistic activation by HNF4 and HNF1 proteins. Nucleic Acids Res 1993; 21: 3731-3736 Corre S, Galibert MD. Upstream stimulating factors: highly versatile stress-responsive transcription factors. Pigment Cell Res 2005; 18: 337-348 Kahn A. From the glycogenic function of the liver to gene regulation by glucose. C R Seances Soc Biol Fil 1998; 192: 813-827 Moriizumi S, Gourdon L, Lefrancois-Martinez AM, Kahn A, Raymondjean M. Effect of different basic helix-loop-helix leucine zipper factors on the glucose response unit of the L-type pyruvate kinase gene. Gene Expr 1998; 7: 103-113 Pineda Torra I, Jamshidi Y, Flavell DM, Fruchart JC, Staels B. Characterization of the human PPARalpha promoter: identification of a functional nuclear receptor response element. Mol Endocrinol 2002; 16: 1013-1028 Xu J, Chang V, Joseph SB, Trujillo C, Bassilian S, Saad MF, Lee WN, Kurland IJ. Peroxisomal proliferator-activated receptor alpha deficiency diminishes insulin-responsiveness of gluconeogenic/glycolytic/pentose gene expression and substrate cycle flux. Endocrinology 2004; 145: 1087-1095 Lee SS, Chan WY, Lo CK, Wan DC, Tsang DS, Cheung WT. Requirement of PPARalpha in maintaining phospholipid and triacylglycerol homeostasis during energy deprivation. J Lipid Res 2004; 45: 2025-2037 Chakravarthy MV, Pan Z, Zhu Y, Tordjman K, Schneider JG, Coleman T, Turk J, Semenkovich CF. "New" hepatic fat activates PPARalpha to maintain glucose, lipid, and cholesterol homeostasis. Cell Metab 2005; 1: 309-322 Wan YJ, Cai Y, Lungo W, Fu P, Locker J, French S, Sucov HM. Peroxisome proliferator-activated receptor alpha-mediated pathways are altered in hepatocyte-specific retinoid X receptor alpha-deficient mice. J Biol Chem 2000; 275: 28285-28290 Edwards PA, Kennedy MA, Mak PA. LXRs; oxysterolactivated nuclear receptors that regulate genes controlling lipid homeostasis. Vascul Pharmacol 2002; 38: 249-256 Sinal CJ, Tohkin M, Miyata M, Ward JM, Lambert G, Gonzalez FJ. Targeted disruption of the nuclear receptor FXR/BAR

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impairs bile acid and lipid homeostasis. Cell 2000; 102: 731-744 Lambert G, Amar MJ, Guo G, Brewer HB Jr, Gonzalez FJ, Sinal CJ. The farnesoid X-receptor is an essential regulator of cholesterol homeostasis. J Biol Chem 2003; 278: 2563-2570 Vidal H. PPAR receptors: recent data. Ann Endocrinol (Paris) 2005; 66: 1S5-1S9 Yoshikawa T, Shimano H, Amemiya-Kudo M, Yahagi N, Hasty AH, Matsuzaka T, Okazaki H, Tamura Y, Iizuka Y, Ohashi K, Osuga J, Harada K, Gotoda T, Kimura S, Ishibashi S, Yamada N. Identification of liver X receptor-retinoid X receptor as an activator of the sterol regulatory element-binding protein 1c gene promoter. Mol Cell Biol 2001; 21: 2991-3000 Kim HJ, Kim JY, Kim JY, Park SK, Seo JH, Kim JB, Lee IK, Kim KS, Choi HS. Differential regulation of human and mouse orphan nuclear receptor small heterodimer partner promoter by sterol regulatory element binding protein-1. J Biol Chem 2004; 279: 28122-28131 Ito S, Fujimori T, Furuya A, Satoh J, Nabeshima Y, Nabeshima Y. Impaired negative feedback suppression of bile acid synthesis in mice lacking betaKlotho. J Clin Invest 2005; 115: 2202-2208 Brendel C, Schoonjans K, Botrugno OA, Treuter E, Auwerx J. The small heterodimer partner interacts with the liver X receptor alpha and represses its transcriptional activity. Mol Endocrinol 2002; 16: 2065-2076 Cho H, Thorvaldsen JL, Chu Q, Feng F, Birnbaum MJ. Akt1/ PKBalpha is required for normal growth but dispensable for maintenance of glucose homeostasis in mice. J Biol Chem 2001; 276: 38349-38352 Ono H, Shimano H, Katagiri H, Yahagi N, Sakoda H, Onishi Y, Anai M, Ogihara T, Fujishiro M, Viana AY, Fukushima Y, Abe M, Shojima N, Kikuchi M, Yamada N, Oka Y, Asano T. Hepatic Akt activation induces marked hypoglycemia, hepatomegaly, and hypertriglyceridemia with sterol regulatory element binding protein involvement. Diabetes 2003; 52: 2905-2913 Colnot S, Decaens T, Niwa-Kawakita M, Godard C, Hamard G, Kahn A, Giovannini M, Perret C. Liver-targeted disruption of Apc in mice activates beta-catenin signaling and leads to hepatocellular carcinomas. Proc Natl Acad Sci USA 2004; 101: 17216-17221 Murata M, Miyoshi Y, Ohsawa M, Shibata K, Ohta T, Imai Y, Nishikawa M, Iwao K, Tateishi H, Shimano T, Kobayashi T, Nakamura Y. Accumulation of beta-catenin in the cytoplasm and the nuclei during the early hepatic tumorigenesis. Hepatol Res 2001; 21: 126-135 Yamaoka H, Ohtsu K, Sueda T, Yokoyama T, Hiyama E. Diagnostic and prognostic impact of beta-catenin alterations in pediatric liver tumors. Oncol Rep 2006; 15: 551-556 Kolligs FT, Bommer G, Goke B. Wnt/beta-catenin/tcf signaling: a critical pathway in gastrointestinal tumorigenesis. Digestion 2002; 66: 131-144 Zeng X, Tamai K, Doble B, Li S, Huang H, Habas R, Okamura H, Woodgett J, He X. A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation. Nature 2005; 438: 873-877 Taurin S, Sandbo N, Qin Y, Browning D, Dulin NO. Phosphorylation of beta-catenin by cyclic AMP-dependent protein kinase. J Biol Chem 2006; 281: 9971-9976 Picardi A, D'Avola D, Gentilucci UV, Galati G, Fiori E, Spataro S, Afeltra A. Diabetes in chronic liver disease: from old concepts to new evidence. Diabetes Metab Res Rev 2006; 22: 274-283 Gotoh J, Obata M, Yoshie M, Kasai S, Ogawa K. Cyclin D1 over-expression correlates with beta-catenin activation, but not with H-ras mutations, and phosphorylation of Akt, GSK3 beta and ERK1/2 in mouse hepatic carcinogenesis. Carcinogenesis 2003; 24: 435-442 Kershenobich Stalnikowitz D, Weissbrod AB. Liver fibrosis and inflammation. A review. Ann Hepatol 2003; 2: 159-163 Nagahara N, Katayama A. Post-translational regulation of mercaptopyruvate sulfurtransferase via a low redox potential cysteine-sulfenate in the maintenance of redox homeostasis. J Biol Chem 2005; 280: 34569-34576 Fitzpatrick PF. The aromatic amino acid hydroxylases. Adv Enzymol Relat Areas Mol Biol 2000; 74: 235-294 S- Editor Liu Y L- Editor Roberts SE E- Editor Liu Y

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World J Gastroenterol 2008 April 7; 14(13): 2023-2028 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

BASIC RESEARCH

Pancreatic stellate cells promote proliferation and invasiveness of human pancreatic cancer cells via galectin-3 Hai-Biao Jiang, Ming Xu, Xing-Peng Wang Hai-Biao Jiang, Ming Xu, Xing-Peng Wang, Department of Gastroenterology, Shanghai First People’s Hospital Affiliated to Shanghai Jiaotong University, 85 Wujin Road, Shanghai 200080, China Author contributions: Jiang HB designed the study, performed most of the research work and wrote the paper; Xu M performed part of the research work; Wang XP instructed Jiang HB and Xu M on their research work and reviewed the paper. Correspondence to: Xing-Peng Wang, PhD, Department of Gastroenterology, Shanghai First People’s Hospital Affiliated to Shanghai Jiaotong University, 85 Wujin Road, Shanghai 200080, China. [email protected] Telephone: +86-21-63240090-3142 Fax: +86-21-63240825 Received: October 22, 2007 Revised: January 15, 2008

Peer reviewer: Yoshiharu Motoo, MD, PhD, FACP, FACG,

Professor and Chairman, Department of Medical Oncology, Kanazawa Medical University,1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan Jiang HB, Xu M, Wang XP. Pancreatic stellate cells promote proliferation and invasiveness of human pancreatic cancer cells via galectin-3. World J Gastroenterol 2008; 14(13): 2023-2028 Available from: URL: http://www.wjgnet.com/1007-9327/14/2023. asp DOI: http://dx.doi.org/10.3748/wjg.14.2023

INTRODUCTION Abstract AIM: To investigate the role of pancreatic stellate cells (PSCs) and galectin-3 (GAL-3) in the proliferation and infiltration of pancreatic cancer cell line SW1990. METHODS: Human pancreatic cancer cell line SW1990 and PSCs were cultured in vitro . Supernatant fluid of cultured PSCs and SW1990 cells was collected. Expression of GAL-3 in SW1990 cells and PSCs was detected by ELISA, RT-PCR and Western blotting. Proliferation of cultured PSCs and SW1990 cells was measured by 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and flow cytometry. Infiltration of SW1990 cells was detected by a cell infiltration kit. RESULTS: SW1990 cells expressed GAL-3 and this was up-regulated by the supernatant fluid of cultured PSCs. PSCs did not express GAL-3. SW1990 cells stimulated proliferation of PSCs via GAL-3. GAL-3 antibody inhibited SW1990 cell proliferation, while the supernatant fluid of PSCs stimulated proliferation of SW1990 cells through interaction with GAL-3 protein. The supernatant fluid of PSCs enhanced the invasiveness of SW1990 cells through interaction with GAL-3. CONCLUSION: GAL-3 and PSCs were involved in the proliferation and infiltration process of pancreatic cancer cells. © 2008 WJG . All rights reserved.

Key words: Cell proliferation; Galectin-3; Infiltration; Desmoplastic reaction; Pancreatic cancer cell; Pancreatic stellate cell

Tumor desmoplasia, a process in which fibrous tissue (e.g., collagen, fibronectin and laminin) infiltrates and envelops neoplasia, is one of the representative histopathological findings in ductual adenocarcinoma of the pancreas. The desmoplastic reaction may contribute to the rapid progression, early metastasis, and a limited response to chemotherapy and radiotherapy of pancreatic carcinoma[1-5]. Some studies have confirmed that pancreatic cancer cells activate pancreatic stellate cells (PSCs) via transforming growth factor (TGF)-β and other cytokines[6,7]. Although pancreatic carcinoma cells are able to produce the fibrotic extracellular matrix (ECM) that surrounds carcinoma, most studies have indicated that the fibrotic ECM is mainly produced and secreted by PSCs[8]. Galectin-3 (GAL-3), is a member of the β-galactosidebinding protein family which recognizes the N-acetyllactosamine structure of various glycoconjugates[9,10]. Studies on hepatic stellate cells (HSCs) have shown that GAL-3 stimulates HSC DNA synthesis in a dose-dependent manner, but no report on the effect of GAL-3 on PSCs has been published[11]. Some studies have been carried out to evaluate the role of GAL-3 in carcinoma proliferation, infiltration and metastasis[12-18], but few of these were on pancreatic cancer. Some immunohistochemical studies have reported that GAL-3 is expressed in pancreatic cancer[19,20]. It has been confirmed that laminin, one important component of the fibrotic ECM that surrounds pancreatic cancer, can be recognized by GAL-3, so GAL-3 may play an important role in the progression of pancreatic cancer.

MATERIALS AND METHODS Cell isolation and culture SW1990 cells were cultured in 100 mL culture bottles containing Dulbecco’s modified Eagle’s medium (DMEM, www.wjgnet.com

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Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) 100 U/mL penicillin and 100 U/mL streptomycin at 37℃ in a 5% CO2/air humidified atmosphere. PSCs were provided by our laboratory and were cultured in DMEM/F-12 supplemented with 10% FBS and antibiotics under the same conditions. Collecting supernatants of cultured cells SW1990 cells and PSCs cultured in 100 mL bottles were washed in PBS and then incubated with serum-free DMEM (5 mL/bottle) for 24 h. Supernatants of cells were collected and filtered under sterilized conditions to remove cell debris, and were stored at -80℃. The concentration of GAL-3 protein in these supernatants was detected by ELISA (Bender Systems). RT-PCR SW1990 cells were washed in PBS and incubated with serum-free DMEM for 24 h. The cells were divided into two groups, one was still incubated with serum-free medium, and the other was exposed to serum-free DMEM supplemented with 40% (v/v) supernatants of PSCs (SPSCs). Cells were harvested and counted 24 h later. Total RNA of 106 cells in each group was extracted according to the manufacturer’s instructions (TRIzol, Gibco-BRL, Rockville, MD, USA). The concentration and purity of RNA was determined by measuring the absorbance at 260 and 280 nm. After that, 1 μg total RNA in each group was reversed-transcribed to cDNA and amplified by RT-PCR (Gibco) according to the manufacturer’s instructions. For detection of GAL-3 mRNA, the following oligonucleotide primers were used: 5'-ATGATGCGTTATCTGGGT CT-3' and 3'-TATTGGACGGAAACGGAC-5'. The amplification reaction involved denaturation at 94℃ for 5 min followed by 35 cycles of denaturation at 94℃ for 1 min, annealing for 1 min at 58℃, and extension for 1 min 30 s at 97℃. Expression of GAL-3 by PSCs was also detected by RT-PCR. Western blotting Two groups of SW1990 cells were prepared as mentioned for RT-PCR. SW1990 cells were centrifuged at 600 g for 10 min. The cell pellet was washed twice with icecold PBS, resuspended in 150 mL lysis buffer (1% Triton X-100 in 5 mmol/L Tris-HCl, pH 8.0, 15 mmol/L NaCl, 2 mmol/L PMSF). The fragmented cells were scraped and removed into a sterilized Eppendorf tube and conserved on ice for 20 min, then centrifuged at 12 000 r/min for 20 min, and unresolved debris was discarded. Proteins were transferred onto PVDF membranes using the wet transfer technique, and the membranes were incubated overnight with monoclonal mouse anti-human-GAL-3 antibodies (R&D) diluted 1:200 in TBS. After 1 h incubation with horseradish peroxidase-labeled secondary antibodies (goat anti-mouse IgM diluted 1:10 000 in TBS), GAL-3 was visualized with the ECL Western Blot Detection Kit (Gibco). Cell proliferation test Cell growth experiments were performed using the 3-(4, 5-methylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and were reconfirmed by cell cycle analysis, www.wjgnet.com

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which was performed by flow cytometry. In the MTT assay, cells were seeded with medium that contained 10% FBS at a density of 6000 cells/well in 96-well plates, grown overnight, washed in PBS, and incubated with serum-free medium for 24 h. Cells were exposed to different concentration of SPSC (5%, 10%, 20% or 40%), supernatants of SW1990 cells (SSW; 5%, 10%, 20% or 40%), GAL-3 monoclonal antibody (GAL-3 MA; 10, 50 or 250 ng/mL, or 1.25 μg/mL), or recombinant human GAL-3 protein (5, 25, 125 or 625 ng/mL). Twenty-four hours later, MTT was added (50 μg/well) for 4 h. Formazan products were solubilized with DMSO, and the optical density was measured at 490 nm. For flow cytometry, cells cultured in 100-mL culture bottles were washed in PBS and incubated with serum-free medium for 24 h. Cells were then exposed to 40% SPSC (for SW1990 cells), 40% SSW (for PSCs), 1 μg/mL GAL-3 MA or 100 ng/mL recombinant GAL-3 protein for 24 h. Cells were harvested and resuspended in fixation fluid at a density of 106/mL, then 1800 μL trypsin solution was added to the fixation fluid, followed by 1500 μL RNase solution. Several minutes later, 1500 μL propidium iodide solution was added, and 15 min later, cells were filtered, and the cell cycle was detected by FACSCaliber (Becton Dickinson). Cell invasion analysis Invasion assays were carried out following the manufacturer’s instructions of cell invasion assay kit (Chemicon International Inc., catalog: ECM550). For the invasion assay, we used a modified Boyden chamber. The chamber had two compartments divided by a polycarbonate filter (8 μm pore size), coated with a reconstituted basement membrane (ECMatrix solution). 3 × 105 SW1990 cells were added to each upper compartment, and chemoattractant fluid was added to the lower compartment (Group A: control group, serum-free medium in the upper compartment and medium containing 2% FBS in the lower compartment; Group B, serum-free medium containing 1 mg/mL GAL-3 MA in the upper compartment and medium containing 2% FBS in the lower compartment; Group C, serum-free medium in the upper compartment and medium containing 2% FBS and 40% SPSC in the lower compartment; Group D, serum-free medium containing 1 mg/mL GAL-3 MA in the upper compartment and medium containing 2% FBS and 40% SPSC in the lower compartment). After 48 h incubation, non-invading cells were removed by cottontipped swabs and the filters were stained in the staining solution for 20 min and rinsed several times in water and air dried. Three filters were used per group. The number of invading cells was counted in 10 random high-powered fields per filter under a Zeiss microscope. Statistical analysis The data were expressed as mean ± SD and compared by ANOVA and the bivariate correlate test. P < 0.05 was considered statistically significant.

RESULTS GAL-3 in SSWs and SPSCs GAL-3 protein in SSWs and SPSCs was measured by

Jiang HB et al . Pancreatic stellate cells promote progress of pancreatic cancer 2.0 SSW

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Table 1 PSC cycle detected by flow cytometry (%)

A 450 nm

1.5 Group

1.0 SPSC

0.5 0.0

0

0.156

0.312 0.625 1.25 2.5 5 Concentration of GAL-3 protein (ng/mL)

10

Figure 1 Concentration of GAL-3 in SPSC and SSW (ELISA).

M

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2

3

4

Medium

1 (Control) Serum-free medium 2 Serum-free medium containing 40% SSW 3 Serum-free medium containing 40% SSW and 1 μg/mL GAL-3 antibody 4 Serum-free medium containing 100 ng/mL GAL-3 protein

G0 + G1 S phase G2/M S + G2/M fraction fraction fraction fraction 89.81 89.63

1.94 3.23

8.25 7.14

10.19 10.37

89.42

2.70

7.88

10.58

87.04

3.68

9.28

12.96

Group 1: PSCs exposed to serum-free medium (control group); Group 2: PSCs exposed to serum-free medium containing 40% SSW; Group 3: PSCs exposed to serum-free medium containing 40% SSW and 1 μg/mL GAL-3 antibody; Group 4: PSCs exposed to serum-free medium containing 100 ng/mL GAL-3 protein.

Figure 2 GAL-3 mRNA of SW1990 cells detected by RT-PCR. M: Marker; 1: GAPDH of control group; 2: GAPDH after stimulation with 40% SPSC; 3: GAL-3 of control group; 4: GAL-3 after stimulation with 40% SPSC.

1

2

Figure 3 GAL-3 protein of SW1990 cells detected by Western blotting. 1: GAL-3 of control group; 2: GAL-3 after stimulation with 40% SPSC.

ELISA kit (Figure 1). The concentration of GAL-3 in SSWs was above the upper limit of the ELISA kit detection range (10 ng/mL), which meant that SW1990 cells could secrete relatively large amounts of GAL-3 into the extracellular fluid. There was little GAL-3 in SPSCs. GAL-3 expression at the mRNA and protein level Both RT-PCR (Figure 2) and Western blotting (Figure 3) confirmed that SW1990 cells expressed GAL-3, and the SPSCs up-regulated expression of GAL-3. PSCs showed no expression of GAL-3 according to RT-PCR and Western blotting. PSC proliferation According to MTT assay (Figure 4), GAL-3 antibody had no effect on the proliferation of PSCs. SSW stimulated PSC proliferation and this was partly inhibited by GAL-3 MA, which suggested that the stimulatory effect of SSW on PSC proliferation was partly mediated via GAL-3. This was confirmed by the effect of recombinant GAL-3 protein on PSC proliferation. According to the GAL-3 ELISA kit, the concentration of GAL-3 in SSW was > 10 ng/mL, and in the MTT assay, 5 ng/mL GAL-3 was sufficient to significantly stimulate PSC proliferation, which meant that GAL-3 secreted by pancreatic cells played a role in PSC proliferation in pancreatic cancer. PSC proliferation detected by flow cytometry is show

in Table 1. The S-phase fraction of the control group was the lowest, that of group 4 was the highest, and group 2 had a higher S-phase fraction than group 3. This result was consistent with that of the MTT assay. PSCs are normal cells that grow very slowly, and have high demand on serum during culturing, so the S-phase fraction of all four groups was not very high. Proliferation of SW1990 cells According to MTT assay (Figure 5), GAL-3 MA inhibited proliferation of SW1990 cells, and this was positively related to antibody concentration, which sug gested that GAL-3 protein was involved in the proliferation process of SW1990 cells. SW1990 cells may increase their proliferation by paracrine or autocrine GAL-3 protein. SPSC stimulated proliferation of SW1990 cells. After GAL-3 MA was added to serum-free DMEM containing 40% SPSC, absorbance declined with the increase in antibody concentration, but it was still higher than that in groups that only used GAL-3 MA. There was a significant correlation between the effect of GAL-3 MA and SPSC on absorbance, which suggested that the stimulatory effect of SPSC on proliferation of SW1990 cells was partly related to GAL-3. SW1990 cell proliferation detected by flow cytometry is show in Table 2. SW1990 cells are quickly proliferating cancer cells, and the S-phase fraction may not properly reflect their proliferation. Therefore, we use the total S phase plus G2/M phase fraction to measure their proliferation. Compared with the control group, GAL-3 MA distinctly inhibited their proliferation, SPSC obviously stimulated proliferation, and GAL-3 MA partly inhibited the SPSC-induced stimulation. This result was consistent with the results of the MTT assay. Invasion of SW1990 cells The results of cell invasion are shown in Figures 6 and 7. GAL-3 MA had no effect on invasion of SW1990 cells. SPSC stimulated invasion of SW1990 cells and this was partly inhibited by GAL-3 MA, which suggested that the www.wjgnet.com

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A 490 nm

A 490 nm

0.20

0.25

0.20

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Figure 4 Proliferation of PSCs detected by MTT assay. (A) Absorbance at 490 nm after exposure to different concentrations of GAL-3 MA; (B) Absorbance at 490 nm after exposure to different concentrations of recombinant human GAL-3 protein; (C) Absorbance at 490 nm after exposure to different concentrations of SSW; (D) Absorbance at 490 nm after exposure to 40% SSW plus different concentrations of GAL-3 MA (aP < 0.05).

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GAL-3 protein (ng/mL)

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Figure 5 Proliferation of SW1990 cells detected by MTT assay. (A) Absorbance at 490 nm after exposure to different concentrations of GAL-3 MA; (B) Absorbance at 490 nm after exposure to different concentrations of SPSC; (C) Absorbance at 490 nm after exposure to 40% SPSC plus different concentrations of GAL-3 MA (aP < 0.05).

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Group

Medium

1 (control) Serum-free DMEM 2 Serum-free DMEM containing 1 μg/mL GAL-3 MA 3 Serum-free DMEM containing 40% SPSC 4 Serum-free DMEM containing 40% SPSC and 1 μg/mL GAL-3 MA

G0 + G1 S phase G2/M S + G2/M fraction fraction fraction fraction 71.80% 79.28%

16.76% 13.87%

11.45% 6.85%

28.20% 20.72%

62.51%

20.34%

17.15%

37.49%

67.91%

20.56%

11.53%

32.09%

Group 1: Sw1990 cells exposed to serum-free DMEM (control group); Group 2: SW1990 cells exposed to serum-free DMEM containing 1 μg/mL GAL-3 MA; Group 3: PSCs exposed to serum-free DMEM containing 40% SPSC; Group 4: PSCs exposed to serum-free DMEM containing 40% SPSC and 1 μg/mL GAL-3 MA.

stimulatory effect of SPSC on SW1990 cell invasion was partly mediated via GAL-3 expressed on the cells. www.wjgnet.com

Number of infiltrated cells

Table 2 Proliferation of SW1990 cells detected by flow cytometry

a

100 c

80 60 40 20 0

Control

GAL-3 MA

40% SPSC 40% PSC + GAL-3 MA

Figure 6 Number of infiltrating cells in each group. aP < 0.05 vs control; cP < 0.05 vs 40% SPSC.

DISCUSSION Our study confirmed that human pancreatic cancer cell line SW1990 expressed and secreted GAL-3 protein, and that GAL-3 MA inhibited proliferation of SW1990 cells, which suggests that cancer cells such as SW1990 can stimulate their proliferation by autocrine or paracrine GAL-3.

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However, more studies should be done to clarify this and to establish the signaling pathway through which GAL-3 stimulates proliferation of cancer cells. Ductal adenocarcinoma of the pancreas is composed of infiltrating cancer cells surrounded by a predominant dense fibroblastic stroma. Previous studies have shown that the fibrotic ECM is mainly produced and secreted by PSCs, and pancreatic cancer cells activate PSCs via TGF-β and other cytokines such as basic fibroblast growth factor to increase production of ECM[8]. Our study, including MTT assay and flow cytometry, confirmed that pancreatic cancer cells stimulated proliferation of PSCs via GAL-3, but further studies should be performed to elucidate the stimulation mechanism of GAL-3 on the proliferation of PSCs. Our study also confirmed that PSCs, through interaction with GAL-3, stimulated pancreatic cancer cells to proliferate, but the exact mechanism needs to be further investigated. Our study confirmed that pancreatic cancer cells and PSCs stimulated proliferation of each other. This may be one of the important reasons for the rapid progression of pancreatic cancer, and preventing this counter-stimulation may be very important in slowing the progression of the disease. GAL-3 is involved in the counter-stimulation between pancreatic cancer cells and PSCs, and this may help us to establish new methods for treating pancreatic cancer. It has been shown by many studies that GAL-3 plays an important role in the interaction between cells and ECM, and this interaction is very important for carcinoma infiltration and metastasis. Activated PSCs can produce and secrete a lot of ECM, including collagen, fibronectin and laminin. Our infiltration study confirmed that the SPSC stimulated the infiltration of pancreatic cancer cells through interaction with GAL-3, which suggests that PSCs

2027 Figure 7 Infiltrating SW1990 cells. (A) Control group. (B) GAL-3 MA group. (C) SPSC group. (D) SPSC plus GAL-3 MA group. Compared with the control group, GAL-3 antibody had no significant effect on invasion of SW1990 cells. SPSC stimulated the invasion process of SW1990 cells, and this stimulation was partly inhibited by GAL-3 antibody.

play an important role in the early infiltration of pancreatic cancer. Since GAL-3 stimulated the proliferation of pancreatic cancer cells and PSCs, and was involved in neoplastic and ECM interaction, we suggest that it plays an important role in the infiltration and metastasis of pancreatic cancer.

COMMENTS Background

Bachem et al[8] discovered pancreatic stellate cells (PSCs) in 1998. After that, several studies showed that there are activated PSCs around pancreatic cancer tissues, pancreatic cancer cells can activate PSCs, and activated PSCs can produce a lot of extracellular matrix (ECM), which lead to the desmoplasia reaction in pancreatic cancer. Galectin-3 (GAL-3) is a member of the β-galactoside-binding protein family which recognizes the N-acetyllactosamine structure of various glycoconjugates. Many studies have shown that GAL-3 plays a role in carcinoma proliferation, infiltration and metastasis. One study has also shown that pancreatic cancer may express GAL-3, but the role of GAL-3 in pancreatic cancer has not been investigated.

Research frontiers

Previous studies have shown that pancreatic cancer cells can activate PSCs via cytokines such as TFG-β and platelet-derived growth factor, which are expressed by cancer cells, but few have discussed the role of activated PSCs in the progression of pancreatic caner. Bachem et al[8] have shown that GAL-1 can activate PSCs, and several others have shown that Gal-3 activates HSCs, but up till now, no study on the effect of GAL-3 on PSCs has been published.

Innovations and breakthroughs

This study showed that pancreatic cancer cells activated PSCs via GAL-3 expressed by cancer cells besides TGF-β and PDGF. It showed that activated PSCs promoted progress of pancreatic cancer by stimulating the proliferation and invasion of cancer cells. This study showed that there were complicated interactions between pancreatic cancer cells and PSCs. It also suggests that ECM and polysaccharides might play a role in the progress of pancreatic cancer, since GAL-3 is a lectin that recognizes the N-acetyllactosamine structure of various glycoconjugates.

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Applications

This study provides a basis for future studies on the mechanisms of how GAL-3 activates PSCs, and how PSCs promote the proliferation and invasion of pancreatic cancer. It also provides a basis for future studies on treating pancreatic cancer through inhibition of GAL-3.

8

Terminology

SW1990 is a commonly used human pancreatic cancer cell line in pancreatic cancer research, which was first derived from spleen metastasis of a grade 2 ductal pancreatic cancer in the early 1980s. PSCs are vitamin A-storage cells that resemble HSCs in the healthy pancreas and comprise approximately 4% of all pancreatic cells; they show a periacinar distribution. GAL-3 is an endogenous β-galactoside-binding protein that is expressed widely in normal and neoplastic cells.

Peer review

This was a well-executed study. It explored the role of GAL-3 in the proliferation and invasion of pancreatic cancer cells in vitro, and showed that GAL-3 may play a role in the progression of pancreatic cancer.

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REFERENCES 1

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Koenig A, Mueller C, Hasel C, Adler G, Menke A. Collagen type I induces disruption of E-cadherin-mediated cell-cell contacts and promotes proliferation of pancreatic carcinoma cells. Cancer Res 2006; 66: 4662-4671 Edderkaoui M, Hong P, Vaquero EC, Lee JK, Fischer L, Friess H, Buchler MW, Lerch MM, Pandol SJ, Gukovskaya AS. Extracellular matrix stimulates reactive oxygen species production and increases pancreatic cancer cell survival through 5-lipoxygenase and NADPH oxidase. Am J Physiol Gastrointest Liver Physiol 2005; 289: G1137-G1147 Miyamoto H, Murakami T, Tsuchida K, Sugino H, Miyake H, Tashiro S. Tumor-stroma interaction of human pancreatic cancer: acquired resistance to anticancer drugs and proliferation regulation is dependent on extracellular matrix proteins. Pancreas 2004; 28: 38-44 Vaquero EC, Edderkaoui M, Nam KJ, Gukovsky I, Pandol SJ, Gukovskaya AS. Extracellular matrix proteins protect pancreatic cancer cells from death via mitochondrial and nonmitochondrial pathways. Gastroenterology 2003; 125: 1188-1202 Shintani Y, Hollingsworth MA, Wheelock MJ, Johnson KR. Collagen I promotes metastasis in pancreatic cancer by activating c-Jun NH(2)-terminal kinase 1 and up-regulating N-cadherin expression. Cancer Res 2006; 66: 11745-11753 Lohr M, Schmidt C, Ringel J, Kluth M, Muller P, Nizze H, Jesnowski R. Transforming growth factor-beta1 induces desmoplasia in an experimental model of human pancreatic carcinoma. Cancer Res 2001; 61: 550-555 Yoshida S, Yokota T, Ujiki M, Ding XZ, Pelham C, Adrian

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TE, Talamonti MS, Bell RH Jr, Denham W. Pancreatic cancer stimulates pancreatic stellate cell proliferation and TIMP-1 production through the MAP kinase pathway. Biochem Biophys Res Commun 2004; 323: 1241-1245 Bachem MG, Schunemann M, Ramadani M, Siech M, Beger H, Buck A, Zhou S, Schmid-Kotsas A, Adler G. Pancreatic carcinoma cells induce fibrosis by stimulating proliferation and matrix synthesis of stellate cells. Gastroenterology 2005; 128: 907-921 Barondes SH, Castronovo V, Cooper DN, Cummings RD, Drickamer K, Feizi T, Gitt MA, Hirabayashi J, Hughes C, Kasai K. Galectins: a family of animal beta-galactoside-binding lectins. Cell 1994; 76: 597-598 Barondes SH, Cooper DN, Gitt MA, Leffler H. Galectins. Structure and function of a large family of animal lectins. J Biol Chem 1994; 269: 20807-20810 Maeda N, Kawada N, Seki S, Ikeda K, Okuyama H, Hirabayashi J, Kasai KI, Yoshizato K. Involvement of Galectin-1 and Galectin-3 in Proliferation and Migration of Rat Hepatic Stellate Cells in Culture. Comp Hepatol 2004; 3 Suppl 1: S10 Allen HJ, Sucato D, Woynarowska B, Gottstine S, Sharma A, Bernacki RJ. Role of galaptin in ovarian carcinoma adhesion to extracellular matrix in vitro. J Cell Biochem 1990; 43: 43-57 Oda Y, Leffler H, Sakakura Y, Kasai K, Barondes SH. Human breast carcinoma cDNA encoding a galactoside-binding lectin homologous to mouse Mac-2 antigen. Gene 1991; 99: 279-283 Miyazaki J, Hokari R, Kato S, Tsuzuki Y, Kawaguchi A, Nagao S, Itoh K, Miura S. Increased expression of galectin-3 in primary gastric cancer and the metastatic lymph nodes. Oncol Rep 2002; 9: 1307-1312 Hittelet A, Legendre H, Nagy N, Bronckart Y, Pector JC, Salmon I, Yeaton P, Gabius HJ, Kiss R, Camby I. Upregulation of galectins-1 and -3 in human colon cancer and their role in regulating cell migration. Int J Cancer 2003; 103: 370-379 Song YK, Billiar TR, Lee YJ. Role of galectin-3 in breast cancer metastasis: involvement of nitric oxide. Am J Pathol 2002; 160: 1069-1075 Inufusa H, Nakamura M, Adachi T, Aga M, Kurimoto M, Nakatani Y, Wakano T, Miyake M, Okuno K, Shiozaki H, Yasutomi M. Role of galectin-3 in adenocarcinoma liver metastasis. Int J Oncol 2001; 19: 913-919 Moon BK, Lee YJ, Battle P, Jessup JM, Raz A, Kim HR. Galectin-3 protects human breast carcinoma cells against nitric oxide-induced apoptosis: implication of galectin-3 function during metastasis. Am J Pathol 2001; 159: 1055-1060 Schaffert C, Pour PM, Chaney WG. Localization of galectin-3 in normal and diseased pancreatic tissue. Int J Pancreatol 1998; 23: 1-9 Berberat PO, Friess H, Wang L, Zhu Z, Bley T, Frigeri L, Zimmermann A, Buchler MW. Comparative analysis of galectins in primary tumors and tumor metastasis in human pancreatic cancer. J Histochem Cytochem 2001; 49: 539-549 S- Editor Li DL L- Editor Kerr C

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World J Gastroenterol 2008 April 7; 14(13): 2029-2036 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

CLINICAL RESEARCH

Probiotic intervention has strain-specific anti-inflammatory effects in healthy adults Riina A Kekkonen, Netta Lummela, Heli Karjalainen, Sinikka Latvala, Soile Tynkkynen, Salme Järvenpää, Hannu Kautiainen, Ilkka Julkunen, Heikki Vapaatalo, Riitta Korpela Riina A Kekkonen, Riitta Korpela, University of Helsinki, Institute of Biomedicine, Pharmacology, PO Box 63, 00014 Helsinki, Finland and Valio Ltd, Research Centre, Meijeritie 4, Helsinki 00370, Finland Netta Lummela, Heli Karjalainen, Soile Tynkkynen, Valio Ltd, Research Centre, Meijeritie 4, Helsinki 00370, Finland Sinikka Latvala, Ilkka Julkunen, National Public Health Institute, Department of Viral Diseases and Immunology, Mannerheimintie 166, Helsinki 00300, Finland Salme Järvenpää, Hannu Kautiainen, Medcare Foundation, Äänekoski, Finland Heikki Vapaatalo, University of Helsinki, Institute of Biomedicine, Pharmacology, PO Box 63, Helsinki 00014, Finland Author contributions: Kekkonen RA designed and coordinated the study, did the PBMC analysis participated in the interpretation of the results and wrote the paper; Lummela N participated in planning the study, was responsible for the study visits and created the database; Latvala S helped in the PBMC analysis; Karjalainen H and Tynkkynen S were responsible for the microbiological analysis; Kautiainen H and Järvenpää S participated in planning the study, carried out the statistical analysis, participated in the interpretation of the results and revised the manuscript; Julkunen I offered expertise in immunology and revised the manuscript; Vapaatalo H participated in planning the study and in the interpretation of the results, supervised the study and revised the manuscript; Korpela R initiated the study, participated in planning the study and the interpretation of the results, was responsible for the management of the study, and revised the manuscript. Supported by The Research Council for Health of the Academy of Finland, and Valio Research Centre Correspondence to: Riitta Korpela, PhD, Assistant Professor, Institute of Biomedicine, Pharmacology, PO Box 63, 00014 University of Helsinki, Helsinki 00014, Finland. [email protected] Telephone: +35-810-3813026 Fax: +35-810-3813019 Received: December 11, 2007 Revised: February 21, 2008

freudenreichii ssp. shermanii JS (PJS) or a placebo drink

for 3 wk. Venous blood and saliva samples were taken at baseline and on d 1, 7 and 21. Fecal samples were collected at baseline and at the end of intervention.

RESULTS: The serum hsCRP expressed as the median AUC0-21 (minus baseline) was 0.018 mg/L in the placebo group, -0.240 mg/L in the LGG group, 0.090 mg/L in the Bb12 group and -0.085 mg/L in the PJS group (P = 0.014). In vitro production of TNF-α from in vitro cultured peripheral blood mononuclear cells (PBMC) was significantly lower in subjects receiving LGG vs placebo. IL-2 production from PBMC in the Bb12 group was significantly lower compared with the other groups. CONCLUSION: In conclusion, probiotic bacteria have strain-specific anti-inflammatory effects in healthy adults. © 2008 WJG . All rights reserved.

Key words: Probiotic; Highly sensitive C-reactive protein; Cytokine; Inflammation; Immune response; Mononuclear cells Peer reviewer: Francesco Costa, Dr, Dipartimento di Medicina Interna-U.O. di Gastroenterologia Universitá di Pisa-Via Roma, 67-56122-Pisa, Italy

Kekkonen RA, Lummela N, Karjalainen H, Latvala S, Tynkkynen S, Järvenpää S, Kautiainen H, Julkunen I, Vapaatalo H, Korpela R. Probiotic intervention has strain-specific anti-inflammatory effects in healthy adults. World J Gastroenterol 2008; 14(13): 2029-2036 Available from: URL: http://www.wjgnet.com/1007-9327/14/2029. asp DOI: http://dx.doi.org/10.3748/wjg.14.2029

Abstract AIM: To evaluate the effects of three potentially antiinflammatory probiotic bacteria from three different genera on immune variables in healthy adults in a clinical setting based on previous in vitro characterization of cytokine responses. METHODS: A total of 62 volunteers participated in this randomized, double-blind and placebo-controlled parallel group intervention study. The volunteers were randomized to receive a milk-based drink containing either Lactobacillus rhamnosus GG (LGG), Bifidobacterium animalis ssp. lactis Bb12 (Bb12), or Propionibacterium

INTRODUCTION Probiotics are defined as living microorganisms that have beneficial effects on human health[1]. The immunomodulatory effects of probiotics have mostly been studied in certain disease conditions, such as allergies[2] and inflammatory diseases[3,4], though the general, healthy population mostly consumes probiotics. The immunomodulatory effects of probiotics in healthy populations have not been fully established and only a few randomized, double blind, placebo-controlled studies have addressed this question[5-9]. Also, there are few studies where the effects of different

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probiotic bacteria have been compared within the same clinical setting. Isolauri et al[10] and Viljanen et al[11] have compared the effects of two different probiotics or a probiotic mixture with placebo in allergic infants. Schiffrin et al[12] and Gill et al[13] evaluated the effects of two different probiotics in healthy adults, but these studies did not have a placebo group. Efforts trying to compare the in vitro results of one probiotic to its results in an in vivo setting are even more scarce and are at the moment limited to comparisons between in vitro and experimental animal studies[14-16]. In our previous studies, we have characterized the capacity of potentially probiotic bacteria to induce cytokine production in human leukocyte cell culture and found that probiotic bacteria direct immune responses to either the Th1 type or the anti-inflammatory direction in a manner specific to the bacterial genera[17]. Based on these findings we selected probiotic bacteria from three different genera for the present study and compared their effects on immune variables in healthy adults in a 3-wk intervention trial.

MATERIALS AND METHODS Subjects T he subjects were healthy adults recr uited by an advertisement in the Helsinki area. The inclusion criteria were to be healthy (no chronic illnesses), to exercise regularly (at least three times per week), and to not be participating in any other clinical trials. The exclusion criteria was comprised of milk allergies (due to the nature of the study products), use of antibiotics during the two months before the study, acute gastrointestinal disorders during the two months before the study, gastrointestinal diseases and related medications, pregnancy, and lactation. Before entering the study, the subjects gave their written informed consent. The study protocol was approved by the Ethics Committee of the Hospital District of Helsinki and Uusimaa. A total of 68 subjects were recruited for the study. Six subjects withdrew from the study during the run-in period and were not included in the analysis. The mean age for the subjects was 44 years (range 23-58) and their mean BMI was 24 kg/m2 (range 18-30). Of these 62 subjects (45 females, 17 males), one subject withdrew from the study due to a back injury after two study visits and one subject due to an antibiotic treatment after four study visits. These two subjects were included in the statistical analysis. Study design and intervention The study was a randomized, double-blind and placebocontrolled parallel group intervention study. Prior to the intervention period, there was a 3-wk run-in period during which no probiotic-containing products were allowed. Thereafter the subjects received either Lactobacillus rhamnosus GG (n = 13), Bifidobacterium animalis ssp. lactis Bb12 (n = 16), Propionibacterium freudenreichii ssp. shermanii JS (n = 17) or placebo (n = 16) drink for 3 wk. After the intervention period, subjects were followed up for 3 wk without any study drink. A list of probiotic-containing products was given to the subjects, and they were asked

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not to consume any other probiotic-containing products at any time during the study. Study products The subjects were advised to consume a 250 mL milk-based fruit drink daily for 3 wk containing either: L. rhamnosus GG (ATCC 53103) (LGG) bacteria, on average 6.2 × 107 cfu/mL (daily dose of 1.6 × 1010 cfu); B. animalis ssp. lactis Bb12 (Bb12) bacteria, 1.4 × 108 cfu/mL (daily dose of 3.5 × 1010 cfu); P. freudenreichii ssp. shermanii JS (DSM 7067) (PJS) bacteria, 1.3 × 108 cfu/mL (daily dose of 3.3 × 1010 cfu); or a placebo drink without any probiotic bacteria. The subjects consumed the study drinks throughout the 3-wk intervention period after the baseline blood sampling. The amount of probiotic bacteria in the study drinks was measured right after packaging and after 3 wk. The appearance and taste of the study drinks were the same. Blood samples Venous blood samples from the antecubital vein were taken at baseline, on 1, 7 and 21 d, and after the 3-wk follow-up period after an overnight fast. The samples were taken into standard serum tubes and EDTA tubes, centrifuged, and the plasma/serum was collected and stored at -20℃ for further analyses. Three EDTA tubes were used in the purification of PBMC. Blood cells and immunoglobulins: Blood cells (leukocytes, monocytes, and lymphocytes) from all time points were determined using an electronic counter (Coulter MAXM Hematology Analyzer, Beckman Coulter, Fullerton, CA, USA). Immunoglobulins (IgA, IgG and IgM) from all time points were measured by immunoturbidimetric method with Tina-quant Roche/Hitachi System reagent using a Roche Hitachi 912 analyzer (Roche Diagnostics GmbH, Mannheim, Germany). Highly sensitive C-reactive protein: Serum levels of C-reactive protein (CRP) were measured at all time points by a highly sensitive particle-enhanced immunoturbidimetric CRP (hsCRP) assay using a Tina-quant C-reactive protein (latex) high sensitive reagent and a Roche Hitachi 912 analyzer (Roche Diagnostics GmbH) with a detection limit of 0.04 mg/L. Cytokine levels from serum: Baseline and 21 d cytokine levels (TNF- α , IL-6, IFN- γ and IL-10) in serum were deter mined using Quantikine HS, Human TNF- α / TNFSF1A (Catalog Number HSTA00D), IL-6 (HS600B), IFN- γ (DIF50) and IL-10 (HS100B) immunoassays purchased from R&D Systems (Minneapolis MN, USA). These assays were carried out according to the manufacturer’s instructions. The detection limit was 0.5 pg/mL for TNF-α, 0.16 pg/mL for IL-6, 15.6 pg/mL for IFN-γ and 0.78 pg/mL for IL-10. For TNF-α, 94% of the samples were over the detection limit, and for IL-6, 89%. For statistical analyses, a detection limit divided by two was given as a value for those samples under the detection limit. None of the IFN-γ samples and only 39%

Kekkonen RA et al. Probiotics and inflammatory markers

of the IL-10 samples was over the detection limit and were therefore not further analyzed. PBMC cell culture Purification: Human PBMC were purified by density gradient centrifugation over a Ficoll-Paque gradient (Amersham-Pharmacia Biotech, Uppsala, Sweden), as described previously[18], from freshly collected EDTA blood on the study days (baseline, d 1, 7 and 21 wk and 3 wk after intervention). After washing, the cells were resuspended in RPMI 1640 medium (Sigma, USA) containing 10% heatinactivated fetal calf serum (FCS) (Integro, Zaandam, Holland) and supplemented with 2 mmol/L L-glutamine (Sigma), 100 U/mL penicillin and 100 μg/mL streptomycin (Gibco BRL, Paisley, Scotland). In stimulation experiments, purified leukocytes (2 × 106 cells/mL) were incubated with stimulants in a final volume of one ml in 24-well plates (Nunc, Roskilde, Denmark) for 24 h in 5% CO2 at 37℃. Stimulations: During the stimulation experiments, the PBMC were maintained in RPMI-1640 medium containing 10% FCS. PBMC were left unstimulated or were stimulated with one of three different stimulants, simulating Grampositive bacteria, a Gram-negative bacteria or a virus. Live Group A streptococci S. pyogenes serotype T1M1 obtained from the National Public Health Institute, Helsinki, Finland, grown as previously described[19], was used as a Gram-positive bacteria at 1:1 host-cell:bacteria ratio; lipopolysaccharide (LPS) from E. coli serotype 0111:B4 (L-3024, Sigma) was used as a model for Gram-negative bacteria at a concentration of 100 ng/mL; and Influenza A H3N2 virus (A/Beijing/353/89) was used to infect cells at a multiplicity of infection of 5. Cell culture supernatants were collected individually at the 24 h time point and stored at -20℃ before analysis. Cytokine levels from cell culture supernatants of stimulated PBMC: Cytokine levels (TNF-α, IFN-γ, IL-1β, Il-2, IL-4, IL-5, IL-6, IL-8, IL-10 and IL-12p70) in cell culture supernatants from each time point (baseline, 1 d, 7 d, 21 d and 3 wk after intervention) were determined using the FlowCytomix human Th1/Th2 10 plex kit Ⅱ (BMS716FFCE) from Bender MedSystems (Vienna, Austria) according to manufacturer’s instructions. The detection limit was 4.5 pg/mL for IL-1β, 8.9 pg/mL for IL-2, 6.4 pg/mL for IL-4, 5.3 pg/mL for IL-5, 4.7 pg/mL for IL-6, 6.4 pg/mL for IL-8, 6.9 pg/mL for IL-10, 7.9 pg/mL for TNF- α , 9.7 pg/mL for IL-12p70 and 7.0 pg/mL for IFN-γ. Only those cytokines from which over 80% of the samples were above the detection limit were statistically analyzed. Therefore, all unstimulated samples, IL-4 and IL-5 in all stimulated samples, and IFN-γ in LPS stimulated samples were not included in further analyses. For statistical analyses, samples under the detection limit were replaced by the values obtained by dividing the detection limit by two. Saliva samples and secretory IgA An unstimulated saliva sample was taken at every visit (at baseline, d 1, 7 and 21 and 3 wk after the intervention)





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after the blood sampling. The saliva samples were placed in Eppendorf tubes, chilled, and stored at -20℃ until secretory IgA was analyzed. SIgA from saliva was determined with an ELISA assay (catalog number K8870) purchased from Gentaur (Brussels, Belgium) according to the manufacturers’ instructions. Fecal samples and microbiological analyses The fecal samples were collected at home at baseline and at the end of the 3-wk intervention period. Immediately after the collection the subjects were asked to deep-freeze (-20℃) the samples at home. They were subsequently transported to the study center on the morning of the study day and the samples were immediately put on dry ice and stored at -70℃ until analysis. The amounts of the probiotic strains L. rhamnosus GG, B. animalis ssp. lactis Bb12 and P. freudenreichii ssp. shermanii JS in the fecal samples were analyzed with a previously described real-time quantitative PCR method[20]. Study diary Subjects were asked to fill in a structured study diary throughout the study. The study diary included questions about the use of the study product, the presence of any symptoms of respiratory infection, gastrointestinal symptoms or any other symptoms, the amount of exercise, and the use of any medication. No respiratory tract infections or major symptoms were recorded by the subjects during the study. The amount of weekly exercise carried out by the study subjects remained the same throughout the study. Outcome measures and statistical analysis The intention-to-treat population (all randomized patients who took at least one dose of the study product) was included in the analysis. The last-observation-carriedforward (LOCF) approach was used for missing data and for subjects who withdrew early. The main outcome measures were the serum hsCRP levels and the cytokines produced by PBMCs. The responses for these outcomes were calculated as the area under the curve from the 0, 1 d, 7 d and 21 d, subtracted by the baseline value (AUC0-21 minus baseline). Data is presented as mean with standard deviation (SD) or as median with interquartile range (IQR). The differences between the groups were tested using the Kruskal-Wallis test or median regression analysis with Holm’s adjustment for pair wise comparisons. A P-value below 0.05 was regarded as statistically significant, but no adjustment was made for multiple testing.

RESULTS Highly sensitive CRP In order to study the effect of probiotic bacteria on inflammatory markers, we determined serum CRP levels at different time points during the intervention. The median AUC 0-21 minus baseline (IQR) for hsCRP was 0.018 (-0.209-0.244) mg/L in the placebo group, -0.240 (-0.424-0.017) mg/L in the LGG group, 0.090 (-0.199-0.191) mg/L in the Bb12 group and -0.085 (-0.303-0.032) mg/L

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Figure 1 The median AUC0-21 (minus baseline) with IQR for serum highly sensitive CRP (hsCRP) levels (A), for Streptococcus pyogenes (GAS) -stimulated TNF-a production from peripheral blood mononuclear cells (B) and for Influenza A virus-stimulated IL-2 production from peripheral blood mononuclear cells (C) during the 3-wk intervention period in healthy adults (n = 62). LGG: Lactobacillus rhamnosus GG; Bb12: Bifidobacterium animalis ssp. lactis Bb12; PJS: Propionibacterium freudenreichii ssp. shermanii JS.

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Table 1 Counts of cells of innate and adaptive immunity (10 /L) and levels of immunoglobulins (g/L) in serum and secretory IgA (g/mL) in saliva in healthy adults (n = 62) at baseline presented as median (IQR)

Leukocytes Monocytes Neutrophils Basophils Eosinophils Lymphocytes IgM IgG IgA sIgA

Placebo (n = 16)

LGG (n = 13)

Bb12 (n = 16)

4.90 (3.90-7.05) 5.00 (4.25-6.75) 2.05 (1.67-3.72) 0.05 (0.00-0.10) 4.00 (3.00-5.75) 38.0 (33.5-48.7) 1.28 (0.97-1.65) 10.7 (9.5-12.2) 2.65 (2.45-3.21) 0.23 (0.15- 0.34)

5.20 (4.90-6.40) 5.00 (5.00-6.00) 3.10 (2.50-3.30) 0.10 (0.00-0.10) 3.00 (2.00-6.00) 35.0 (31.5-37.5) 0.87 (0.69-1.32) 10.3 (9.3-11.7) 2.42 (2.04-3.44) 0.27 (0.14-0.42)

5.25 (4.60-6.00) 6.00 (5.00-6.00) 2.95 (2.12-3.47) 0.05 (0.00-0.10) 3.00 (2.25-4.75) 34.0 (30.0-40.5) 1.10 (0.73-1.66) 10.6 (9.1-12.1) 2.20 (1.73-2.94) 0.40 (0.27-0.88)

PJS (n = 17) 4.90 (4.35 -5.70) 6.00 (4.50-6.50) 3.10 (2.15-3.45) 0.00 (0.00-0.10) 2.00 (1.50-3.00) 31.0 (26.0-40.5) 1.44 (0.80-1.77) 10.4 (8.4-11.7) 2.44 (1.53-2.85) 0.28 (0.17-0.49)

P value1 0.55 0.84 0.39 0.73 0.077 0.24 0.31 0.72 0.22 0.065

1

Kruskall-Wallis test with Monte Carlo P values. IQR: Interquartile range.

in the PJS group (P = 0.014); a statistically significant difference was observed between LGG and Bb12 group by pair wise comparisons. In the LGG and PJS groups, hsCRP appeared to be at a lower level during the 3-wk intervention period compared with the Bb12 and placebo groups (Figure 1A). Serum cytokines The baseline values for pro-inflammatory cytokine TNF-α in serum were 1.2 pg/mL in the placebo group, 1.0 pg/mL in the LGG, 1.0 pg/mL in the Bb12 and 0.8 pg/mL in the PJS. The change (median with IQR) from baseline to the end of 3-wk intervention for TNF-α in these study groups was 0.1 (-0.1-0.3) pg/mL, 0.1 (-0.02-0.2) pg/mL, 0.3 (-0.04-0.4) pg/mL and 0.0 (-0.1-0.3) pg/mL, respectively (P = 0.44). The baseline values for pro-inflammatory cytokine IL-6 were 0.3 pg/mL in the placebo group, 0.6 pg/mL in the LGG, 0.3 pg/mL in the Bb12 and 0.4 pg/mL in the PJS. The change (median with IQR) from baseline to the end of 3-wk intervention for IL-6 in these study groups was -0.5 (-0.6-0.0) pg/mL, -0.2 (-0.3-0.2) pg/mL, 0.1 (-0.3-0.3) www.wjgnet.com

pg/mL and -0.04 (-0.3-0.1) pg/mL, respectively (P = 0.26). There were no statistically significant differences between the study groups with respect to serum cytokine levels. Blood cells and immunoglobulins Baseline values for leukocytes, monocytes, neutrophils, basophils, lymphocytes and immunoglobulins are presented in Table 1. There were no differences in these variables between the groups during the intervention. Cytokines produced by PBMC We also determined whether the use of probiotic bacteria has an effect on the overall responsiveness of PBMC to various microbial stimuli in in vitro cultured cells. The microbe-induced cytokine production by PBMC is presented in Table 2. S. pyogenes-stimulated production of pro-inflammatory cytokine TNF-α was significantly different between the groups (P = 0.025); a statistically significant difference was observed between LGG and placebo groups by pair wise comparisons (Figure 1B). Influenza A virus-stimulated production of Th1 cytokine IL-2 was significantly different between the groups (P
65℃. Laparoscopic time-lag EI-RFA (L-EI-RFA) L-EI-RFA was perfor med for the HCC protr uding from the surface of the liver. First, the laparoscope was inserted into the abdominal cavity beginning in the leftupper portion of the navel. RFA electrode and PEI needle were inserted at a second abdominal site according to the location of the tumor. A sonde was inserted into the abdominal cavity to lift up the liver when the HCC was protruding from the reverse surface of the liver. Under laparoscopic observation, a RFA electrode was directly inserted into the tumor, and then a 21-gauge PEI needle was inserted and ethanol was injected[18]. Thoracoscopic time-lag EI-RFA (T-EI-RFA) T-EI-RFA was performed for HCC close to the right diaphragm. Patients were put under general anesthesia with one-lung (left lung) ventilation in a left-decubitus position. Three ports for the thoracoscope, End-fire laparoscopic probe (ALOKA UST-52109), and water-pouring tool were inserted into the pleural cavity through the intercostal space. After putting the collapsed right lung aside by a laparoscope, the End-fire laparoscopic probe was guided to the surface of the exposed diaphragm, and the tumor close to the diaphragm was identified by US (ALOKA ProSound SSD-3500). After visualizing the tumor by US, an RFA electrode was inserted into the tumor through the channel along the End-fire laparoscopic probe. A 21-gauge

Kurokohchi K et al. Ethanol injection and radiofrequency ablation under general anesthesia

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Table 2 Comparison of the volume of coagulated necrosis and energy requirement between the groups treated with expandable and straight electrode

T-S (cm)

ETOH (mL) L (cm)

S (cm)

H (cm)

3

V (cm )

3

M (cm )

3

3

T-ENE (J) T-ENE/V (J/cm )  T-ENE/M (J/cm )

Group L (n = 40)

2.0 ± 0.8

3.0 ± 1.9

3.5 ± 0.6

2.8 ± 0.4

3.0 ± 0.5

15.9 ± 5.2

10.1 ± 8.2

21 565 ± 9631

1636 ± 791

2406 ± 1819

Group G (n = 15)

2.1 ± 0.8

6.1 ± 3.4

3.6 ± 1.1

3.2 ± 0.6

3.2 ± 0.8

22.5 ± 14.9

15.9 ± 12.1

37 207 ± 20 583

2409 ± 2045

3594 ± 2727

0.56

0.0007

0.64

0.10

0.31

0.045

0.06

0.0095

0.10

0.11

P value

Twenty-four HCC were treated with time-lag PEI-RFA with straight type electrode, while 15 HCC were treated with time-lag PEI-RFA with expandable type electrode. After treatment, the longest and the shortest diameters, and the height of the coagulated necrosis were estimated by helical dynamic CT and the approximation volume of total and marginal coagulated necrosis were calculated. Each datum expresses mean ± SD. Each abbreviation in the table is expressing as follows: T-S: Tumor size; EtOH: Amount of ethanol; L: Longest diameter; S: Shortest diameter; H: Height; V: Volume of coagulated necrosis; M: Volume of marginal coagulation; D: Duration of ablation; T-ENE: Total energy requirement; T-ENE/V: Energy requirement per unit volume for whole coagulation; T-ENE/ M: Energy requirement per unit volume for inducing marginal coagulation.

PEI needle was inserted and pure ethanol or lipiodol containing ethanol was injected[19]. Open surgery-assisted time-lag EI-RFA (O-EI-RFA) O-EI-RFA was performed for HCC which were difficult to treat with other EI-RFA approaches or when splenectomy was simultaneously performed to improve cirrhotic liver dysfunction accompanying severe esophageal varices or a decrease of platelet count. After exposing the liver and confirming the surface location of liver tumors by US, the RFA electrode was directly inserted into the liver tumors and time-lag EI-RFA was performed. Evaluation of therapeutic efficacy Five to seven days after treatment, plain or contrast enhanced CT was performed to evaluate the response to L-EI-RFA, T-EI-RFA, and O-EI-RFA. Tumor necrosis was considered to be complete when no foci of early enhancement were seen around the original regions. Statistical analysis Statistical analysis was performed using Statview Ⅱ (Version 5.0), statistical significance between the group L and group G was analyzed by a Chi-square test for independence and significant difference was accepted at P < 0.05.

RESULTS Comparison of the volume of coagulated area and the applied energy requirement for total and unit volume coagulation in patients treated with EI-RFA under local anesthesia (group L) and general anesthesia (group G) Group L (40 cases) received time-lag EI-RFA under local anesthesia, while group G (22 cases) received time-lag EIRFA under general anesthesia. The patients underwent RFA therapy by means of the Cool-tip RF system, RTC system, and RITA system. No major adverse effects were observed in either group. Among treated cases, 40 cases in group L and 15 cases in group G (L-EI-RFA; 6 cases, T-EI-RFA; 8 cases, O-EI-RFA; 1 case) were treated with EI-RFA by means of the Cool-tip RF system. Between these patients treated by the Cool-tip RF system, the effect of EI-RFA was compared using several parameters drawn from the treatments. Comparison of the amount of injected ethanol, the volume of the induced coagulated necrosis, total applied energy for total and per unit volume coagulated necrosis

in both groups are summarized in Table 2. The tumor size was approximately 3 cm in diameter in both groups, and no significant difference was detected between groups. Although the longest and the shortest diameter and the height of the coagulated necrosis did not show a significant difference between the groups, the mean values of all of these parameters in group G were higher than those in group L. Thus, the volume of total coagulated necrosis in group G was significantly larger than that in group L. Furthermore, the volume of marginal coagulated necrosis in group G was also larger than that in group L. According to the analysis of parameters which affect the volume of induced coagulated necrosis in EI-RFA, both the quantity of ethanol and applied energy for ablation in group G were significantly larger than those in group L. Because both, the volume of coagulated necrosis and total applied, energy, were increased in group G compared with group L, the applied energy per unit volume for whole and marginal coagulated necrosis were comparable. These results suggest that a higher volume of coagulated necrosis was induced in group G compared with group L because higher amounts of ethanol and energy for ablation could be applied. Rate of local recurrence within a year in group G and L Among the 22 patients treated with EI-RFA under general anesthesia (group G), local recurrence was observed in only one case (4.5%) within a year after the treatment. By contrast, local recurrence was detected within a year in four cases (10%) among the 40 cases treated with EI-RFA under local anesthesia (group L). Although the difference of the rate of local recurrence between group G and group L did not reach statistical significance, the rate was extremely low in group G. In one case (patient No. 21) with local recurrence, the volume of marginal coagulated necrosis around the original tumor was lower than in cases without recurrence. Comparison of the effects of L-EI-RFA, T-EI-RFA, and O-EI-RFA The results for treatment with L-EI-RFA, T-EI-RFA, and O-EI-RFA are summarized in the Table 3. L-EI-RFA was performed on the HCC located in the left lobe of the liver (segment 2-4) and T-EI-RFA was performed on the HCC of segment 8 of the liver close to the diaphragm (except for one HCC located in the segment 6). O-EI-RFA was performed on the liver HCC of segment 3, 7, and 8. The www.wjgnet.com

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Table 3 Results of L-EI-RFA, T-EI-RFA and O-EI-RFA Location

T-S (cm)

Ins

EtOH (mL)

A-A (L x S x H) (cm)

L-EI-RFA No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8

S3 S2 S4 S2 S3 S4 S2 S2

1.5 3.0 1.5 3.0 2.5 1.0 1.5 1.5

Cool-tip Cool-tip Cool-tip Cool-tip Cool-tip Cool-tip RITA RITA

15 8 2 7 8 7 2 2

3.2 × 2.7 × 2.6 4.2 × 4.2 × 4.2 2.2 × 2.2 × 2.2 6.7 × 5.0 × 3.7 3.7 × 3.7 × 3.4 2.2 × 2.2 × 2.3 4.0 × 4.0 × 4.0 3.5 × 2.5 × 3.0

T-EI-RFA No. 9 No. 10 No. 11 No. 12 No. 13 No. 14 No. 15 No. 16 No. 17

S6 S8 S8 S8 S8 S8 S8 S8 S8

2.5 1.0 2.0 2.5 2.0 2.0 2.0 3.5 2.0

Cool-tip RTC Cool-tip Cool-tip Cool-tip Cool-tip Cool-tip Cool-tip Cool-tip

7 3 12 5 4 5 5 3 4

3.7 × .2 × 3.2 4.1 × 2.5 × 3.2 4.2 × 4.2 × 3.7 3.2 × 3.2 × 3.2 3.0 × 3.0 × 2.5 3.5 × 3.5 × 3.5 4.5 × 3.0 × 3.5 2.0 × 2.0 × 2.0 2.5 × 2.5 × 4.0

O-EI-RFA No. 18 No. 19 No. 20 No. 21 No. 22

S3 S8 S7 S3 S3

1.5 2.5 1.0 1.5 1.5

RITA Cool-tip RTC RTC RTC

2 7 2 2 2

3.3 × 2.5 × 3.0 4.2 × 3.2 × 3.7 3.0 × 2.5 × 2.5 2.0 × 2.0 × 1.5 4.0 × 4.0 × 4.0



3

V (cm )

3

M (cm )

11.8 38.7 5.60 64.8 24.4 5.83 33.0 19.0

10.0 24.0 3.81 50.7 16.2 5.30 31.0 17.0

19.8 17.2 34.1 17.1 11.8 22.4 24.7 4.20 13.1

11.7 16.6 30.0 8.97 7.59 18.3 20.5 3.70 8.90

13.0 26.0 9.81 3.14 25.1

8.90 17.9 9.29 1.37 23.4

Eight HCC were treated with L-EI-RFA, Nine HCC were treated with T-EI-RFA and five HCC were treated with O-EI-RFA. Location of the tumor, tumor size (T-S), instruments for ablation (Ins), amounts of injected ethanol (EtOH), ablated area (A-A), [longest diameter (L) × shortest diameter (S) × height (H)], volume of coagulated necrosis (V) and volume of marginal coagulated necrosis (M) are shown.

approximate estimated volume of the original tumor, volume of whole and the marginal coagulated necrosis were calculated from the CT image after the treatments. In most treated cases, a larger volume of coagulated necrosis and marginal coagulated necrosis was induced compared with the volume of original tumor. In most patients treated with a large amount of ethanol (over 7 mL), larger volume of whole and marginal coagulated necrosis were induced in patients such as No. 2, 4, 5, 9, 11, and 19. The volume of whole and marginal coagulated necrosis was comparable in the patients treated with L-EI-RFA, T-EI-RFA, and O-EIRFA. Relationship between the amount of ethanol, applied energy, and the volume of coagulated necrosis In group G, the induced coagulated necrosis increased with the amount of ethanol and applied energy. We previously showed that the amount of ethanol was positively correlated with the volume of coagulated necrosis in patients treated with P-EI-RFA using an RFA instrument equipped with a straight electrode (Cool-tip RF system)[19]. In the present study, the relationship between the amount of ethanol or applied energy and the volume of induced coagulated necrosis were evaluated in patients treated with EI-RFA under general anesthesia. Furthermore, the relationship between the product of the amount of ethanol and the applied energy vs the volume of coagulated necrosis was also analyzed. The results showed that both the amounts of injected ethanol and applied energy were significantly and positively correlated with the volume of www.wjgnet.com

coagulated necrosis (Figure 1A and B). The product of the amount of ethanol and the applied energy was also positively correlated with the volume of coagulated necrosis (Figure 1D). These results clearly indicated that both the amount of ethanol and applied energy were critical factors that regulate the volume of coagulated necrosis in the EIRFA under general anesthesia. Representative case treated with L-EI-RFA By analyzing the effects of time-lag EI-RFA under general anesthesia, one point became evident: Total applied energy and the applied energy per unit volume of whole and marginal coagulated necrosis were significantly larger in patients treated under general anesthesia than in those treated under local anesthesia. This lead to the induction of a larger volume of coagulated necrosis. A representative case treated with T-EI-RFA was shown in our previous manuscript[22]. A case treated with L-EI-RFA is presented. One case with HCC successfully treated with L-EI-RFA is shown in Figure 2. An HCC (2 cm in diameter) was located in the S2 region of the liver protruding from the reverse surface and with an enhanced early vascular phase (Figure 2A) via dynamic CT. The laparoscope was inserted from the left-upper portion of the abdomen into the abdominal cavity and the liver was lifted by a sonde. An RFA electrode and PEI needle were percutaneously inserted into the center of the tumor, monitoring the depth of the inserted electrode by the linear type US. The electrode and needle were firstly inserted into the tumor from the upper surface of the liver (Figure 2B), and then inserted into the

40

30

30

60

3

20

20

40

20

10

0

3

40

2041

80

V (cm )

50

V (cm )

50

3

V (cm )

Kurokohchi K et al. Ethanol injection and radiofrequency ablation under general anesthesia

10

 0

 2

 4

 6  8

 10

 12

0

0 10 000 20 000 30 000 40 000 50 000 60 000 70 000

14

Ethanol (mL)



0

Energy (Joule)

100 000 200 000 300 000 400 000 500 000 Ethanol × energy

Figure 1 Relationship between the volume of coagulated necrosis induced and the amounts of ethanol injected or total applied energy or the product of the amounts of ethanol and the total applied energy. All the amounts of ethanol, total applied energy, or the products of the amount of ethanol and total applied energy showed a significant positive correlation with the volume of coagulated necrosis. (Ethanol vs volume, r = 0.54, P = 0.018; energy vs volume, r = 0.61, P = 0.0057; ethanol × energy vs volume, r = 0.61, P = 0.0078).

A

B

C

D

E

F

Figure 2 A case of HCC located on the reverse surface of the liver (S2 of the liver) treated with L-EI-RFA. A HCC (2 cm in diameter) showed an enhancement in early vascular phase of helical dynamic CT (A); RFA electrode and PEI needle were firstly inserted from the surface of the liver (B) and secondly inserted from the reverse surface of the liver (C); After injecting the ethanol containing 15% lipiodol, RFA was performed. Dynamic CT after the treatment showed lipiodol deposit associated with the original tumor and low density area was observed around the tumor (D); Laparoscopic observation of the tumor from the surface (E) and from the reverse surface (F) of the liver after the treatment.

tumor from the reverse surface of the liver (Figure 2C). We previously reported the usefulness of injecting the mixture of ethanol and lipiodol to visualize the original tumor by dynamic CT[23]. Therefore, a mixture of ethanol and lipiodol (15% lipiodol in ethanol) was injected into the tumor. Five minutes after injection of ethanol containing lipiodol, RFA was started at 30 W, and the power output was stepwise increased to 80 W by a Cool-tip RF system. During ablation, the tumor was constantly lifted by a sonde to prevent the transmission of heat to the mesentery. Abdominal dynamic CT taken after the operation clearly

showed a lipiodol deposit associated with the tumor and the ablated region reached beyond the tumor (Figure 2D). A safety margin was shown to be sufficiently obtained by L-EI-RFA. The laparoscopic findings for the tumor after ablation are shown in Figure 2E and F.

DISCUSSION HCC is one of the most serious and common malignancies worldwide [1,4]. As a treatment for HCC, RFA now plays a central role for local control of HCC, because RFA www.wjgnet.com

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can induce wider coagulated necrosis in a few sessions compared with PEI which is frequently used for relatively small-encapsulated HCC. However, the region of coagulated necrosis induced by RFA is still limited and only considered applicable to tumors within a 3 cm diameter. Furthermore, it is also pointed out that relatively frequent local recurrences of tumor occur after RFA treatment. Therefore, the RFA technique could be further developed to improve the therapeutic effects of this treatment. To enhance the therapeutic effect of RFA, several treatment modalities have been applied in addition to local treatment[24-29]. As one of the optional combination therapies, we have developed a novel combination therapy of P-EIRFA and showed that this combination therapy accurately enlarged the area of induced coagulated necrosis. Total applied energy and the applied energy per unit volume of whole and marginal coagulated necrosis was significantly lower in the P-EI-RFA than RFA alone. Furthermore, we found that the time-lag performance of RFA after ethanol injection (time-lag P-EI-RFA) resulted in a lower energy requirement per total and unit volume of coagulated necrosis than without time-lag performance of RFA after ethanol injection. In this regard, we suggest that timelag P-EI-RFA can induce wider coagulated necrosis with a smaller energy requirement. Although P-EI-RFA was shown to enlarge the area of coagulated necrosis, there are HCC cases that are difficult to treat with the percutaneous RFA due to the location of the HCC. For these situations, we applied the combined therapy of ethanol injection and RFA (EI-RFA) with laparoscopic, thoracoscopic and open-surgery assisted. Among 22 patients treated with EIRFA under general, the number of local recurrences was very small [1 case (4.5%)] and its frequency was kept in extremely low level. Analysis of the amount of injected ethanol, applied energy and the volume of coagulated necrosis showed that these parameters in the group treated with EI-RFA under general anesthesia were significantly larger than those in the group treated under local anesthesia. One of the most relevant differences between the EI-RFA under general anesthesia and local anesthesia is presence or absence of pain felt by the treated patients. We have reported in a series of analyses that P-EI-RFA under local anesthesia enabled a comparable coagulated necrosis with smaller energy requirement relative to RFA alone. P-EI-RFA was likely to be less invasive than RFA alone[16]. However, in the present study, the rate of local recurrence was reduced in the patients treated under general anesthesia compared with the patients treated under local anesthesia. Taken collectively, these results suggest that higher amounts of ethanol and energy administrated under pain-free conditions may result in a decreased rate of local tumor recurrences after RFA treatment. Indeed, although we still believe that PEI-RFA is less invasive for the treatment of HCC, we are sometimes obliged to cease the RFA treatment due to the pain felt by the patient during the percutaneous RFA treatment. Therefore, the results in the present study suggest that it is important to apply enough ethanol and energy for RFA treatment to decrease the local recurrence after percutaneous treatment as well as treatment under general anesthesia. For this purpose, it is beneficial to use anesthesia intravenously to decrease the www.wjgnet.com

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pain felt by the patients during the percutaneous treatment as well. Patients under pain-free conditions during treatment may have a decreased rate of local tumor recurrence. Recently, it was reported that there were no differences in tumor control and complications under general anesthesia and analog-sedation in RFA treatment of pulmonary tumors[30]. This result is not in accordance with our results obtained during treatment of HCC. In the treatment of HCC located near the surface of the liver, patients often complain about pain originating from the membrane of the liver. In our patients, we usually use pentazocine and non-steroid anti-inflammatory drugs (NSAIDs) (if necessary diazepam is also used on a case by case basis) for the percutaneous RFA treatment. Therefore, it may be better to consider stronger pain relief during the treatment of percutaneous RFA treatment. In conclusion, we compared the clinical effects, amounts of ethanol, and applied energy in P-EI-RFA between patients under general anesthesia and local anesthesia. The volume of induced coagulated necrosis, amounts of ethanol, and applied energy were significantly larger in the group treated under general anesthesia than that under local anesthesia. The rate of local tumor recurrence in the former group was kept at an extremely low level.

COMMENTS Background

Radiofrequency ablation (RFA) plays a central role for the treatment of hepatocellular carcinoma (HCC) because this newly developed technology appears very effective to induce wider coagulated necrosis. However, several disadvantages have been pointed out for RFA and improvement of RFA technique will be desirable.

Research frontiers

RFA treatments are performed percutaneously under local anesthesia in many cases. Local tumor recurrence varies according to the location of tumor in the liver, size of tumors, and level of RFA technique. A few reports compared the effects of RFA treatment under local and general anesthesia.

Innovations and breakthroughs

This report showed that the total applied energy and the applied energy per unit volume of whole and marginal coagulated necrosis were significantly larger in the group treated under general anesthesia (group G) resulting in a larger volume of coagulated necrosis.

Applications

Patients under pain-free condition during treatment may have a decreased rate of local tumor recurrence. It thus may be better to consider stronger pain relief during the treatment of percutaneous RFA treatment.

Peer review

Dr. Kurokohchi and colleagues reported the advantage and features of the combination therapy of ethanol injection and radiofrequency ablation (EI-RFA) under general anesthesia for HCC. This manuscript arouses interest for readers and provides an important clue to effectively treat patients with HCC.

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World J Gastroenterol 2008 April 7; 14(13): 2044-2048 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

RAPID COMMUNICATION

Serum type Ⅳ collagen level is predictive for esophageal varices in patients with severe alcoholic disease Satoshi Mamori, Yasuyuki Searashi, Masato Matsushima, Kenichi Hashimoto, Shinichiro Uetake, Hiroshi Matsudaira, Shuji Ito, Hisato Nakajima, Hisao Tajiri Satoshi Mamori, Yasuyuki Searashi, Shinichiro Uetake, Hiroshi Matsudaira, Shuji Ito, Hisato Nakajima, Hisao Tajiri, Division of Gastroenterology and Hepatology, Department of Internal Medicine, the Jikei University School of Medicine, Tokyo 105-8461, Japan Masato Matsushima, Division of Clinical Research and development, the Jikei University School of Medicine, Tokyo 105-8461, Japan Kenichi Hashimoto, Tokyo Medical Center of Alcohol Related Disabilities, Tokyo 175-0091, Japan Author contributions: Mamori S and Searashi Y contributed equally to this work; Mamori S designed research; Hashimoto K, Searashi Y, and Uetake S performed research; Mamori S, Matsushima M, Matsudaira H, and Ito S analyzed data; Mamori S wrote the manuscript. Correspondence to: Satoshi Mamori, MD, Division of Gastroenterology and Hepatology, Department of Internal Medicine, the Jikei University School of Medicine, 3-25-8 Nishi-shinbashi, Minato-ku, Tokyo 105-8461, Japan. [email protected] Telephone: +81-3-34331111-3201 Fax: +81-3-34350569 Received: November 30, 2007 Revised: January 28, 2008

detection of esophageal varices in SAD. © 2008 WJG . All rights reserved.

Key words: Type Ⅳ collagen; Esophageal varice; Alcoholic disease; Abdominal ultrasonography; Alcoholism Peer reviewers: Kazuma Fujimoto, Professor, Department

of Internal Medicine, Saga Medical School, Nabeshima, Saga, Saga 849-8501, Japan; Volker F Eckardt, Chief, MD, Professor, Department of Gastroenterology, Deutsche Klinik für Diagnostik, Aukammallee 33, Wiesbaden 65191, Germany; Hitoshi Togashi, Associate Professor, Department of Gastroenterology, Course of Internal Medicine and Therapeutics, Yamagata University School of Medicine, 2-2-2 Iida-Nishi, Yamagata 990-9585, Japan Mamori S, Searashi Y, Matsushima M, Hashimoto K, Uetake S, Matsudaira H, Ito S, Nakajima H, Tajiri H. Serum type Ⅳ collagen level is predictive for esophageal varices in patients with severe alcoholic disease. World J Gastroenterol 2008; 14(13): 2044-2048 Available from: URL: http://www.wjgnet.com/1007-9327/14/2044. asp DOI: http://dx.doi.org/10.3748/wjg.14.2044

Abstract AIM: To determine factors predictive for esophageal varices in severe alcoholic disease (SAD). METHODS: Abdominal ultrasonography (US) was performed on 444 patients suffering from alcoholism. Forty-four patients found to have splenomegaly and/ or withering of the right liver lobe were defined as those with SAD. SAD patients were examined by upper gastrointestinal (UGI) endoscopy for the presence of esophageal varices. The existence of esophageal varices was then related to clinical variables. RESULTS: Twenty-five patients (56.8%) had esophageal varices. A univariate analysis revealed a significant difference in age and type Ⅳ collagen levels between patients with and without esophageal varices. A logistic regression analysis identified type Ⅳ collagen as the only independent variable predictive for esophageal varices (P = 0.017). The area under the curve (AUC) for type Ⅳ collagen as determined by the receiver operating characteristic (ROC) for predicting esophageal varices was 0.78. CONCLUSION: This study suggests that the level of type Ⅳ collagen has a high diagnostic accuracy for the

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INTRODUCTION Regular daily drinking is more likely to result in liver damage than intermittent drinking. The longer this pattern is maintained, the more likely it is that alcoholic hepatitis, and subsequently cirrhosis, will develop[1]. In patients with cirrhosis, the incidence of esophageal varices increases by nearly 5% per year, and the rate of progression from small to large varices is approximately 5%-10% per year[2,3]. Bleeding from esophageal varices is common among patients with cirrhosis. Bleeding from varices may occur in 15%-68% of patients with varices[4]. Variceal hemorrhaging is associated with a high mortality and with high hospital costs[5]. Both, beta-blockers and endoscopic procedures, have been established as effective preventive modalities for variceal hemorrhage[5,6]. Therefore, the early detection of esophageal varices is critical for the effective prevention of variceal hemorrhage [7] . Adding an accurate serum marker for hepatic fibrosis to the model may improve the diagnostic accuracy in predicting esophageal varices without performing liver biopsy. Moreover, developing an accurate non-invasive diagnostic model might also decrease the costs for the prevention of hemorrhaging from varices[7]. In daily medical practice, it is common to encounter

Mamori S et al. Type Ⅳ collagen predicts esophageal varice in alcoholic disease

patients with liver damage from chronic alcohol consumption. Moreover, when the alcoholic patient is examined, it is often evident that alcoholic liver damage is progressing. Once alcoholic cirrhosis is established, esophageal varices develop in the majority of patients, as found during prolonged follow-up[8]. Nevertheless, alcoholic patients tend to be indifferent regarding self health, and are not likely to undergo periodic consultations. We therefore examined predictive factors for esophageal varices in severe alcoholic disease.

MATERIALS AND METHODS Patients The 444 consecutive patients considered for this study were hospitalized at the Tokyo Medical Center of Alcohol Related Disabilities in Tokyo, Japan, between April and September 2005, July 2006, and June 2007 for alcoholism. A complete physical examination was performed by a senior physician. The recorded variables included age, gender, height, body weight, mean alcohol consumption, duration of alcohol abuse, jaundice, ascites, and hepatic encephalopathy. After an overnight fast, serum samples were obtained from all patients for test purposes, including a complete blood cell count, blood platelets, bilirubin, aspartate transaminase (AST), alanine aminotransferase (ALT), gamma glutamyl transpeptidase (GTP), albumin, prothrombin index (ratio between patient and control Quick time expressed in percentage), and type Ⅳ collagen (Mitsubishi Chemical Medience Corporation, Tokyo, Japan). Ultrasonography (US) was performed by experienced gastroenterologists during the stay in hospital. The spleen was visualized with the patient in the right lateral decubitus position. Measurements were then taken in the sagittal (S) and transverse (T) planes, with the maximum dimension being recorded in each plane[9]. Splenomegaly was defined as a spleen index (S × T × 0.9) > 30. Alcoholic patients with splenomegaly and/or withering of the right liver lobe were defined as severe alcoholic disease patients (SAD) and included into the study. Patients with the following criteria were excluded: (1) the presence of suspected hepatocellular carcinoma on US; (2) the presence of extrahepatic infectious or inflammatory disease; (3) treatment by any drug known to affect liver fibrosis; (4) seropositivity for the hepatitis B surface antigen, hepatitis C virus, and/ or human autoimmune antibodies. Finally, we identified 44 SAD patients (Table 1). For each patient, upper g astrointestinal (UGI) endoscopy was performed by an endoscopist. The purpose of endoscopy was to evaluate the presence of esophageal varices (Figure 1). The endoscopist evaluated esophageal varices with the Esophagogastric Varices Grading System of the Japan Society for Portal Hypertension [10]. The endoscopist performed UGI endoscopy without knowledge of serum data. Statistical analysis The results were expressed as the mean ± SD. Differences between the groups were examined for statistical signifi-



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Table 1 Characteristics of the study population (n = 44) Esophageal varice

Yes (n = 25)

No (n = 19)

P value

Age (yr) Sex (male/female) Total alcohol intake (kg) MCV (fL) Plt (/μL) PT (%) AST (IU/L) ALT (IU/L) GTP (IU/L) T -Bil (mg/dL) Alb (g/dL) Collagen type Ⅳ (ng/mL) Ascites (yes/no) Encephalopathy (yes/no)

49.6 ± 7.0 20/5 1075.9 ± 646.9 97.7 ± 12.4 12.6 ± 6.1 62.6 ± 16.2 80.9 ± 72.6 44.4 ± 2.6 453.6 ± 594 2.7 ± 3.2 3.7 ± 0.6 712.3 ± 355.6 6/19 1/24

55.5 ± 9.2 15/4 1018.4 ± 684.9 95.9 ± 12.0 13.9 ± 9.8 69.3 ± 18.8 96.1 ± 85.1 45.2 ± 28.5 410.2 ± 374.3 2.4 ± 1.9 3.9 ± 0.5 404.3 ± 198 2/17 2/17

< 0.05 NS NS NS NS NS NS NS NS NS NS < 0.001 NS NS

NS: Not significant; Normal ranges: MCV (mean corpuscular volume), 85-102/fL; Plt (platelet count), 14-34 × 10 3/μL; PT (prothrombin index), 70%-100%; AST(aspartate aminotransferase), 10-40 IU/L; ALT(alanine aminotransferase), 5-45 IU/L; GTP (gamma glutamyl transpeptidase), male < 80 IU/L, female < 30 IU/L; T-Bil (total bilirubin), 0.2-1.1 mg/dL; Alb (albumin), 3.8-5.3 g/dL; collagen type Ⅳ, < 150 ng/mL.

444 patients for alcoholism Physical examination Serum samples Ultrasonography (US) 44 severe alcoholic disease (SAD) patients Upper gastrointestinal (UGI) endoscopy 25 with EV

19 no EV

Figure 1 Flow chart of patients in this study. EV: Esophageal varice.

cance using the Mann-Whitney U test and a c2 test where appropriate. Independent predictive factors associated with esophageal varices were assessed by a multivariate analysis using a logistic regression model. The sensitivity and specificity of collag en type Ⅳ for predicting esophageal varices was determined using receiver operating characteristic (ROC) curves. A P value of less than 0.05 was considered to be statistically significant. All analyses were performed using the STATA 10.0 software program (STATA Corporation, College Station, Texas, USA).

RESULTS Twenty-five patients (56.8%) had esophageal varices, and 19 (43.2%) had no varice (Figure 1). A univariate analysis revealed a significant difference between patients with and without esophageal varices with regard to age and type Ⅳ collagen levels (Table 1 and Figure 2). These two variables that were significantly linked to the presence of esophageal varices in the univariate analysis, and a factor previously reported[11], age, PT, and type Ⅳ collagen, were assessed by multivariate analysis. A logistic regression

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25

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> 300



1000

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80

0 60

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Figure 3 The positive predictive value for esophageal varices of collagen type Ⅳ. Y-axis: Positive percentages of esophageal varices; X-axis: The total predictive value of esophageal varices; > 300 (600, 900): The positive predictive value of esophageal varices with a collagen type Ⅳ value > 300 (600, 900) ng/mL.

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200 0.75

30 (+)

(-)

(+)

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(-) Varix

Figure 2 Two independent factors correlated with the appearance of esophageal varices. Collagen: Collagen type Ⅳ (ng/mL); (+): Positive patients; (-): Negative patients.

Sensitivity

0

0.50

0.25

0.00

Table 2 An independent factor for the presence of esophageal varices (odds ratio) Variable Collagen type Ⅳ

Odds ratio

95% CI

P value

2.02

1.13-3.60

0.017

0

0.25

0.5

0.75

1

1-Specificity

Figure 4 Receiver operating characteristics (ROC) curve of collagen type Ⅳ for the diagnosis of esophageal varices [area under curve = 0.7802, se (area) = 0.0704].

Data: Collagen type Ⅳ per 150 ng/mL.

analysis identified type Ⅳ collagen as the only independent variable predictive for esophageal varices (P = 0.017) (Table 2). Whenever the type Ⅳ collagen level raised every 150 ng/mL, the odds ratio of esophageal varices doubled. Figure 3 shows the positive predictive values at each cut off point of type Ⅳ collagen. The positive predictive value of esophageal varices with a type Ⅳ collagen value > 900 ng/mL (n = 6) was 100%. Finally, the area under the curve (AUC) of type Ⅳ collagen as determined by ROC for predicting the presence of esophageal varices was 0.78 (Figure 4).

DISCUSSION A rupture of esophageal varices is the most frequent complication of portal hypertension, occurring in one third of all cirrhotic patients, and is associated with a high mortality[12,13]. The mortality rate from variceal bleeding is about 20% when patients are treated optimally in a hospital[14]. However, an appreciable proportion of patients with variceal bleeding die before reaching the hospital[15]. Numerous studies have shown that the prevention of UGI bleeding and early detection of esophageal varices reduces mortality, morbidity, and health care costs[16]. Nevertheless, www.wjgnet.com

Suzuki et al demanded further studies to determine which strategies are the most beneficial to patients and society in terms of preventing and treating esophageal varices, in a recent article[7]. We thus examined potential esophageal varice prediction factors for SAD on a medical checkup level. In this study, a non-invasive marker for hepatic fibrosis (type Ⅳ collagen) had a high diagnostic accuracy for the detection of esophageal varices. The combination of abdominal ultrasound scan and this marker correctly identified, at a high rate, patients with esophageal varices. These examinations can be conducted on a medical checkup level, so we considered this approach to be of considerable diagnostic value. A previous repor t showed the type Ⅳ collagen concentration was the most accurate in cor rectly identifying patients with severe histologic alcoholic hepatitis. At a cut-off of 150 ng/mL, type Ⅳ collagen was 89% sensitive and 77% specific[17]. First, patient sorting was conducted via the abdominal ultrasound test in this study. The alcoholic patients with splenomegaly and/or withering of the right liver lobe participated in this study. In these 44 patients, the value of type Ⅳ collagen was > 150 ng/mL in all specimens. As a result, when suspecting SAD, we thought it very meaningful to add the abdominal

Mamori S et al. Type Ⅳ collagen predicts esophageal varice in alcoholic disease

ultrasound test to the characteristics under evaluation. In a previous repor t, Geoffroy and colleagues demonstrated that the independent factors of prothrombin index, alkaline phosphatase activity, and hyaluronate level predicted the presence of esophageal varices [11]. Nevertheless, their proposed model included two agedependent serum markers, hyaluronate and alkaline phosphatase, both of which rise in serum with aging[18]. We therefore added only the prothrombin index as an examination item. Moreover, another report demonstrated a rise in amino-terminal procollagen Ⅲ peptide (PⅢNP) following alcohol withdrawal that is likely to be caused by intact PⅢNP[19]. We thus decided not to include this fibrosis marker as an examination item at the time of hospitalization. Finally, we elected to add GTP and collagen type Ⅳ as examination items. The serum concentration of laminin and type Ⅳ collagen have been reported to be increased in patients with alcoholic hepatitis and to correlate with the degree of inflammation [20-26]. In our study, logistic regression identified type Ⅳ collagen as the only independent variable predictive for esophageal varices. While based on the findings of these studies, laminin may be predictive for esophageal varices. However, the use of such testing is not covered by the national health insurance program in Japan, so we decided to exclude laminin from this study. We therefore hope that further study of esophageal varices in other countries will help to elucidate and confirm the predictive potential of laminin. Antler et al found that in younger patients, the most common bleeding sites are those associated with alcoholism (esophageal varices, Mallory-Weiss tears, and hemorrhagic gastritis), accounting for 40%-60% of lesions in patients less than 55 years-of-age[27]. Another report demonstrated that younger patients had a trend toward more variceal bleeds (P = 0.39)[28]. In our research, there was a positive correlation of esophageal varices with younger age (P > 0.05). We think that younger alcoholic patients with high fibrosis markers should be evaluated by GI endoscopy. In conclusion, this study suggests that the level of type Ⅳ collagen has a high diagnostic accuracy for the detection of esophageal varices in SAD. These results show that the non-invasive screening of patients who are at risk for variceal bleeding is possible, and that this approach may assist in the prevention of this most serious complication.

COMMENTS COMMENTS Background

Bleeding from varices may occur in 15%-68% of patients with varices. Variceal hemorrhaging is associated with a high mortality rate, as well as high hospital costs.

Research frontiers

The early detection of esophageal varices is critical for the effective prevention of variceal hemorrhaging. Adding an accurate non-invasive diagnostic model may improve the diagnostic accuracy in predicting esophageal varices without performing liver biopsy.

Innovations and breakthroughs

First, patient sorting for alcoholism was conducted via the abdominal ultrasound



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test in this study. The existence of esophageal varices with severe alcoholic disease was compared according to a number of clinical background variables. A univariate analysis revealed a significant difference in age and type Ⅳ collagen levels between the patients with and without esophageal varices. A logistic regression analysis identified type Ⅳ collagen as the only independent variable predictive for esophageal varices (P = 0.017). AUC of type Ⅳ collagen as determined by ROC for predicting expressed esophageal varices was 0.78.

Applications

The combination of an abdominal ultrasound scan and type Ⅳ collagen correctly identified, at a high rate, alcoholism patients with esophageal varices.

Terminology

The type Ⅳ collagen and laminin are the major components of basement membranes. Early accumulation of type Ⅳ collagen and laminin, thus leading to the formation of basement membrane-like material in the space of Disse (capillarization), is considered a typical characteristic of alcoholic liver disease. The amount of collagen in the space of Disse has been shown to correlate significantly with the presence of alcoholic hepatitis and portal blood pressure.

Peer review

This short paper summarized well the relevance of type Ⅳ collagen as a predictive factor for varices in alcoholic liver cirrhosis.

REFERENCES 1 2

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Sutton R, Shields R. Alcohol and oesophageal varices. Alcohol Alcohol 1995; 30: 581-589 Merli M, Nicolini G, Angeloni S, Rinaldi V, De Santis A, Merkel C, Attili AF, Riggio O. Incidence and natural history of small esophageal varices in cirrhotic patients. J Hepatol 2003; 38: 266-272 D'Amico G, Morabito A. Noninvasive markers of esophageal varices: another round, not the last. Hepatology 2004; 39: 30-34 Groszmann RJ, de Franchis R. Portal hypertension. In: Schiff ER, Sorrell MF, Maddrey WC. Schiff’s Disease of the Liver. Philadelphia, New York: Lippincott-Raven, 1999: 387-442 Sharara AI, Rockey DC. Gastroesophageal variceal hemorrhage. N Engl J Med 2001; 345: 669-681 Grace ND, Groszmann RJ, Garcia-Tsao G, Burroughs AK, Pagliaro L, Makuch RW, Bosch J, Stiegmann GV, Henderson JM, de Franchis R, Wagner JL, Conn HO, Rodes J. Portal hypertension and variceal bleeding: an AASLD single topic symposium. Hepatology 1998; 28: 868-880 Suzuki A, Mendes F, Lindor K. Diagnostic model of esophageal varices in alcoholic liver disease. Eur J Gastroenterol Hepatol 2005; 17: 307-309 Burroughs AK, McCormick PA. Natural history and prognosis of variceal bleeding. Baillieres Clin Gastroenterol 1992; 6: 437-450 Hosey RG, Mattacola CG, Kriss V, Armsey T, Quarles JD, Jagger J. Ultrasound assessment of spleen size in collegiate athletes. Br J Sports Med 2006; 40: 251-254; discussion 251-254 Yoshida H, Mamada Y, Taniai N, Tajiri T. New methods for the management of gastric varices. World J Gastroenterol 2006; 12: 5926-5931 Vanbiervliet G, Barjoan-Marine E, Anty R, Piche T, Hastier P, Rakotoarisoa C, Benzaken S, Rampal P, Tran A. Serum fibrosis markers can detect large oesophageal varices with a high accuracy. Eur J Gastroenterol Hepatol 2005; 17: 333-338 Graham DY, Smith JL. The course of patients after variceal hemorrhage. Gastroenterology 1981; 80: 800-809 D'Amico G, Pagliaro L, Bosch J. The treatment of portal hypertension: a meta-analytic review. Hepatology 1995; 22: 332-354 D'Amico G, De Franchis R. Upper digestive bleeding in cirrhosis. Post-therapeutic outcome and prognostic indicators. Hepatology 2003; 38: 599-612 Nidegger D, Ragot S, Berthelemy P, Masliah C, Pilette C, Martin T, Bianchi A, Paupard T, Silvain C, Beauchant M. Cirrhosis and bleeding: the need for very early management. J Hepatol 2003; 39: 509-514

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Jensen DM. Endoscopic screening for varices in cirrhosis: findings, implications, and outcomes. Gastroenterology 2002; 122: 1620-1630 Castera L, Hartmann DJ, Chapel F, Guettier C, Mall F, Lons T, Richardet JP, Grimbert S, Morassi O, Beaugrand M, Trinchet JC. Serum laminin and type IV collagen are accurate markers of histologically severe alcoholic hepatitis in patients with cirrhosis. J Hepatol 2000; 32: 412-418 Guechot J, Poupon RE, Poupon R. Serum hyaluronan as a marker of liver fibrosis. J Hepatol 1995; 22: 103-106 Campbell S, Timms PM, Maxwell PR, Doherty EM, Rahman MZ, Lean ME, Danesh BJ. Effect of alcohol withdrawal on liver transaminase levels and markers of liver fibrosis. J Gastroenterol Hepatol 2001; 16: 1254-1259 Annoni G, Colombo M, Cantaluppi MC, Khlat B, Lampertico P, Rojkind M. Serum type III procollagen peptide and laminin (Lam-P1) detect alcoholic hepatitis in chronic alcohol abusers. Hepatology 1989; 9: 693-697 Lotterer E, Gressner AM, Kropf J, Grobe E, von Knebel D, Bircher J. Higher levels of serum aminoterminal type III procollagen peptide, and laminin in alcoholic than in nonalcoholic cirrhosis of equal severity. J Hepatol 1992; 14: 71-77

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Niemela O, Risteli L, Sotaniemi EA, Risteli J. Type IV collagen and laminin-related antigens in human serum in alcoholic liver disease. Eur J Clin Invest 1985; 15: 132-137 Niemela O, Risteli J, Blake JE, Risteli L, Compton KV, Orrego H. Markers of fibrogenesis and basement membrane formation in alcoholic liver disease. Relation to severity, presence of hepatitis, and alcohol intake. Gastroenterology 1990; 98: 1612-1619 Niemela O, Risteli J, Blake JE, Risteli L, Compton KV, Orrego H. Connective tissue metabolism and alcohol intake in alcoholic liver disease. Alcohol Alcohol Suppl 1991; 1: 351-355 Nouchi T, Worner TM, Sato S, Lieber CS. Serum procollagen type III N-terminal peptides and laminin P1 peptide in alcoholic liver disease. Alcohol Clin Exp Res 1987; 11: 287-291 Robert P, Champigneulle B, Kreher I, Gueant JL, Foliguet B, Dollet JM, Bigard MA, Gaucher P. Evaluation of fibrosis in the disse space in noncirrhotic alcoholic liver disease. Alcohol Clin Exp Res 1989; 13: 176-180 Antler AS, Pitchumoni CS, Thomas E, Orangio G, Scanlan BC. Gastrointestinal bleeding in the elderly. Morbidity, mortality and cause. Am J Surg 1981; 142: 271-273 Segal WN, Cello JP. Hemorrhage in the upper gastrointestinal tract in the older patient. Am J Gastroenterol 1997; 92: 42-46 S- Editor Li DL L- Editor Mihm S E- Editor Ma WH

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World J Gastroenterol 2008 April 7; 14(13): 2049-2054 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

RAPID COMMUNICATION

Early effects of Lansoprazole orally disintegrating tablets on intragastric pH in CYP2C19 extensive metabolizers Hatsushi Yamagishi, Tomoyuki Koike, Shuichi Ohara, Toru Horii, Ryousuke Kikuchi, Shigeyuki Kobayashi, Yasuhiko Abe, Katsunori Iijima, Akira Imatani, Kaori Suzuki, Takanori Hishinuma, Junichi Goto, Tooru Shimosegawa Hatsushi Yamagishi, Tomoyuki Koike, Shuichi Ohara, Toru Horii, Ryousuke Kikuchi, Shigeyuki Kobayashi, Yasuhiko Abe, Katsunori Iijima, Akira Imatani, Tooru Shimosegawa, Division of Gastroenterology, Tohoku University Graduate School of Medicine, 1-1 Seiryou-Machi, Aobaku, Sendai 9808574, Japan Kaori Suzuki, Junichi Goto, Division of Clinical Pharmacy, Tohoku University Graduate School of Pharmaceutical Sciences, 1-1 Seiryou-Machi, Aobaku, Sendai 9808574, Japan Takanori Hishinuma, Division of Pharmacotherapy, Tohoku University Graduate School of Pharmaceutical Sciences; Department of Pharmaceutical Sciences, Tohoku University Hospital, 1-1 Seiryou-Machi, Aobaku, Sendai 9808574, Japan Author contributions: Yamagishi H, Koike T and Ohara S designed the research; Yamagishi H, Koike T, Ohara S, Horii T, Kikuchi R, Kobayashi S, Abe Y, Iijima K, Imatani A, Suzuki K, Hishinuma T, Goto J and Shimosegawa T performed the research; Yamagishi H, Koike T, Ohara S, Suzuki K and Hishinuma T analyzed the data; Yamagishi H, Koike T and Ohara S wrote the paper. Correspondence to: Hatsushi Yamagishi, MD, Division of Gastroenterology Tohoku University graduate school of medicine, 1-1 Seiryou-Machi, Aobaku, Sendai 9808574, Japan. [email protected] Telephone: +81-22-7177171 Fax: +81-22-7177177 Received: October 15, 2007 Revised: November 12, 2007

Abstract AIM: To compare rabeprazole (RPZ; 10 mg) with Lansoprazole orally disintegrating tablets (LPZ; 30 mg OD) in terms of antisecretory activity and blood drug concentration after a single dose. METHODS: Eight H pylori -negative cytochrome P450 (CYP) 2C19 extensive metabolizers were assigned to receive a single oral dose of RPZ 10 mg or LPZ 30 mg OD. Twelve hour intragastric pH monitoring was performed on the day of treatment. Blood samples were also collected after the administration of each drug. RESULTS: LPZ 30 mg OD induced a significantly earlier rise in blood drug concentration than RPZ 10 mg; consequently, LPZ 30 mg OD induced a significantly earlier rise in median pH in the third and fourth hours of the study. CONCLUSION: In H pylori -negative CYP2C19 extensive metabolizers, LPZ 30 mg OD induced a significantly faster inhibition of gastric acid secretion than RPZ 10 mg.

© 2008 WJG . All rights reserved.

Key words: LPZ 30 mg orally disintegrating tablets; Intragastric pH; Blood drug concentration; Cytochrome P450 2C19 extensive metabolizers; H pylori -negative Peer reviewer: Tomohiko Shimatani, Assistant Professor, Department of General Medicine, Hiroshima University Hospital, 1-2-3 Kasumi, Minami-ku, Hiroshima 7348551, Japan

Yamagishi H, Koike T, Ohara S, Horii T, Kikuchi R, Kobayashi S, Abe Y, Iijima K, Imatani A, Suzuki K, Hishinuma T, Goto J, Shimosegawa T. Early effects of Lansoprazole orally disintegrating tablets on intragastric pH in CYP2C19 extensive metabolizers. World J Gastroenterol 2008; 14(13): 2049-2054 Available from: URL: http://www.wjgnet.com/1007-9327/14/2049. asp DOI: http://dx.doi.org/10.3748/wjg.14.2049

INTRODUCTION Gastroesophageal reflux disease (GERD) is a common disease in the West [1-3] , with increasing prevalence in Japan[4-7]. A recent study in Japan by Ohara et al has shown that a total of 42.2% of Japanese experienced heartburn[6], which is a similar proportion to the estimated 42.4% reported in Western studies [1]. Moreover, endoscopic studies have shown the overall prevalence of reflux esophagitis (RE) among the adult population of Japanese outpatients is 14%-16%[4-7]. The mechanism of GERD is closely associated with gastric acid; thereby, gastric acid suppression is the most common therapeutic approach, and stronger and prompter gastric acid suppression is required[8]. At present, drug therapy for reflux esophagitis is common because of its effectiveness; acid suppressing dr ugs such as H 2 receptor antag onist (H 2 RA) and proton pump inhibitors (PPIs) are commonly used. As PPIs have been shown to be more effective against RE than H 2RA [9-11] , PPIs such as lansoprazole (LPZ) and rabeprazole (RPZ) are now widely used as first-line acid inhibitors. Continuous maintenance with PPIs is considered to be the mainstay of GERD treatment. However, there are some reports not showing that all GERD patients need continuous acid inhibition. Bour et al reported that on-demand therapy with PPI provides an alternative to continuous therapy in patients with mild to moderate gastro-esophageal reflux[12]. Several reports have www.wjgnet.com

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demonstrated that on-demand therapy with PPI provides an alternative to continuous therapy in patients with mild to moderate gastro-esophageal reflux[13]. In Japan, many studies on RE have been done and although each report differs slightly, an obvious trend is apparent, showing most patients suffered from mild RE[5-7], which is milder than that experienced in Western countries. As a result, some Japanese patients take PPIs when required according to their symptoms in the clinical setting. It is reported that rapid acid suppression is important for effective pain relief at the onset of treatment in GERD patients [14]. Thus, the aim of this study was to examine the correlation between pH value and blood drug concentration in patients treated with RPZ 10 mg or LPZ OD 30 mg at the early post-administration phase (1-12 h). RPZ and LPZ are generally administered at doses of 10 mg or 30 mg, respectively, in the clinical setting in Japan. Thus, in the present study, the RPZ and LPZ doses were set at 10 mg and 30 mg, respectively. F u r t h e r m o r e, i n Ja p a n m a n y o u t p a t i e n t s a r e administered multiple drugs. Hence, in order to improve dr ug compliance, RPZ and LPZ are also generally administered after a meal with other drugs. Therefore, we administered both RPZ and LPZ after a meal. The acid-inhibitory effects of PPIs are significantly dependent on the cytochrome P450 (CYP) 2C19 genotype status, as well as on their intrinsic pharmacokinetic and pharmacodynamic characteristic and dosing schemes[15-19]. According to these reports, the metabolism of PPIs is affected by the CYP2C19 polymorphism; the plasma PPI levels and intragastric pH values in extensive metabolizers are significantly lower than those in poor metabolizers[20-22]. Therefore, in this study, the subjects were all CYP2C19 extensive metabolizers.

MATERIALS AND METHODS Subjects The subjects were 8 healthy male volunteers, aged between 24 and 48 years (median, 23 years) and weighing 52-78 kg (median, 54 kg). No patient had a history of gastrointestinal or hepatobiliary disease or of eradication therapy for H pylori, and none took regular medications. All volunteers gave written informed consent. The study protocol was approved by the ethical committee of the Tohoku University Graduate School of Medicine. Detection of H pylori infection H pylori infection was determined by the 13C-urea breath test[23]. A total of 8 H pylori-negative subjects were invited and approved to participate in this study. CYP2C19 genotyping After obtaining informed consent, a venous blood sample was collected from all patients. DNA was extracted from the nucleus of venous white blood cells. The genetic mutation was analyzed by either the polymerase chain reactionrestriction fragment length polymorphism (PCR-RFLP) method [24] or the TaqMan polymerase chain reaction

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amplification method (Applied Biosystems Japan, Chiba, Japan) [25]. Based on point mutations in exons 4 and 5 of the CYP2C19 gene, individuals can be classified into homo-extensive metabolizers (homo-EMs), hetero-extensive metabolizers (hetero-EMs) and poor metabolizers (PMs)[20-22]. Homo-EMs have the wild type alleles (wt/wt) without any mutations in exons 5 and 4; PMs have mutated alleles (m1/m2) with mutations in both exons 5 and 4 (m1/m2, m1/m1 or m2, m2); and hetero-EMs have a mutated allele in either exon 5 or 4 (wt/m1 or wt/m2). Study protocol All subjects (homo EM = 4, hetero EM = 4) participated in an open-label crossover study with RPZ 10 mg tablets or LPZ 30 mg OD. They were randomly assigned to receive a single oral dose of RPZ 10 mg tablet or LPZ 30 mg OD 30 min after eating a standardized meal. There was a washout period of at least 14 d between the two study periods. Twenty-four-hour intragastric pH monitoring was performed on the day of treatment. To monitor gastric pH, a pH electrode was inserted transnasally, and positioned fluoroscopically in the gastric corpus, approximately 5 cm-10 cm below the esophago-gastric junction. Gastric pH was measured at 10 s intervals by means of a portable pH meter attached to a glass pH electrode (Chemical Instrument, Tokyo, Japan). The pH electrode was calibrated before each recording, using standard buffers of pH 1.68, 4.01 and 6.86. The pH data were analyzed with the use of established software (Chemical Instrument). At fixed times (Breakfast 8.30 AM, lunch at noon, snack at 15:00 and dinner at 7:00 PM), standardized meals were consumed (total 1359 kcal; protein, 24 g lipid, 18.5 g glucose 267 g. The individual calorie contents of breakfast, lunch, snacks and supper were 356, 324, 355 and 324 kcal respectively). No additional food was allowed, and 100 mL of tap water was allowed only when the subjects felt thirsty. All subjects were instructed to remain upright until 21:00. Normal daily activities were not restricted. Sample collection and assay of LPZ, RPZ concentration in plasma In order to study the correlation between intragastric pH and blood drug concentration, blood samples were collected in heparinized tubes before and 0.5, 1, 1.5, 2, 3.5, 6, 9 and 12 h after the administration of each drug. After collection, the blood samples were immediately centrifuged at 3000 r/min for 10 min. For the determination of RPZ levels in plasma, 100 μL of 1% diethylamine solution was added to 1 mL of plasma; this was not required for the determination of plasma concentrations of LPZ. All samples were stored at -20℃ until assayed. Plasma levels of LPZ and RPZ were measured by high-performance liquid chromatography/tandem mass spectrometry[26,27]. This method required only 20 μL of serum and is a simple procedure. Analytes and the internal standard (lansoprazole deuterium derivatives) were separated using a mobile phase of acetonitrile/1 mmol/L ammonium formate (140/60, v/v) on a C18 analytical column and analyzed in the selected reaction-monitoring (SRM) mode. The lower limit of quantification was 500 fg/20 μL.

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Table 1 Characteristics of the subjects in this study Subject 1 2 3 4 5 6 7 8

CYP2C19

Age

Height (cm)

Body weight (kg)

BMI

Hetero Hetero Hetero Hetero Homo Homo Homo Homo

33 50 31 22 31 26 23 25

175 175 168 165 168 173 171 184

70 73 65 60 65 68 70 75

22.8 23.8 23.0 22.0 23.0 22.7 23.9 22.1

Statistical analysis Intragastric pH were expressed as median values (ranges). Differences in these parameters among each regimen were determined by the Wilcoxon signed rank test. P values less than 0.05 were considered significant.

RESULTS Eight volunteers (all men; mean age 30.3 years, range years) completed the study. There were no adverse events during the study, which was completed according to the protocol by all 8 subjects. Four subjects were homo-EMs, and the other 4 subjects were hetero-EMs (Table 1). The 12-h trendgram and the profiles of correlation between intragastric pH and blood drug concentration are shown in Figure 1. The lower limit for quantification of blood drug concentration was 500 fg/20 μL in this study. However, the blood drug concentration of RPZ was not detectable until 2 h after drug administration, while LPZ was detectable 0.5 h after drug administration. LPZ 30 mg OD induced a significantly earlier rise in blood drug concentration than RPZ 10 mg tablets. As a result of this prompter rise in blood LPZ concentration, there was a prompter onset of median pH. The 12-h (median pH per hour) trendgrams of intragastric pH values obtained without medication are shown in Figure 1. The intragastric pH values increased significantly with both drugs. LPZ 30 mg OD increased the pH value after the second hour of the study, while RPZ 10 mg tablets increased the pH value after the fourth hour of the study, compared with those pH values of individuals without medication. LPZ 30 mg OD induced a significantly earlier rise in median pH in the third and fourth hours of the study than RPZ 10 mg tablets (Figure 1).

DISCUSSION PPIs, such as omeprazole, LPZ and RPZ, are widely used for the treatment of acid-related diseases. The frequency of GERD has increased recently, because of increased average fat intake[28], increased rates of obese patients[29,30], and declining rates of H pylori infection [31,32]. GERD is a common disease in the West [1-3] and appears to be increasing in prevalence in Japan[4-7]. Recent endoscopic studies have shown overall prevalence of reflux esophagitis among the adult population in Japan is 14%-16% [5-7]. Each report differs slightly, but an obvious trend is that

most patients suffer from mild RE [5-7]. Moreover, it is reported that the incidence of atrophic gastritis in the general population is higher in Japan than in Western countries[33-35], and that gastric acid secretion levels in the general population are lower in Japan than in Western countries [33-36] . Thereby, some patients in Japan want administration of the drug on demand. Patients with GERD mainly suffer of intermittent symptoms rather than continuous symptoms[37]. On-demand therapy with PPIs is reported to provide an alternative to continuous therapy in patients with mild to moderate gastro-esophageal reflux disease suffering from frequent symptomatic relapse[12]. It is important to use medicines that immediately ameliorate the clinical symptoms. Therefore, it is useful to administer antisecretory drugs which have a faster and stronger onset of pH rise in the stomach among patients with acid-related disorders. Thus, we studied the effect in the early post-administration phase (1-12 h) of a single dosing of each PPI. In this study, LPZ 30 mg OD induced a significantly earlier rise in blood drug concentration than RPZ 10 mg tablets. As a result of this prompter rise in blood LPZ concentration, LPZ 30 mg OD induced a significantly earlier rise in intragastric pH values compared with the values in individuals without medication after the second hour of the study, while RPZ 10 mg tablets induced an earlier rise in the fourth hour of the study. Moreover, LPZ 30 mg OD also induced prompter and stronger inhibition of gastric acid secretion than RPZ 10 mg tablets in the early post-administration phase, in the third and fourth hours following a single oral dose of treatment. These results differ slightly from those of previous reports[17-19,38,39] in which RPZ induced an earlier rise in intragastric pH than other PPIs. However, most previous studies examined the effects after administration of RPZ or other PPIs on days 3-7[17-19,38,39], not at the early postadministration phase (1-12 h) of a single dose of treatment, whereas the present study examined the effect in this phase. There are interindividual variations in the metabolism of PPI, resulting in differences in the acid-suppressing effect of each PPI[17-19,38,39]. Each report differs slightly, but CYP2C19 genotype status is shown to influence gastric acid suppression by LPZ and most other PPIs. The metabolism of LPZ, OPZ and other PPIs is affected by the CYP2C19 polymorphism, and the plasma PPI level and intragastric pH values of EMs are significantly lower than those of PMs[20-22]. On the other hand, several studies have demonstrated that after a dose of RPZ, intragastric pH is not affected by the CYP2C19 polymorphism on 3 d-7 d of treatment[17-19,38,39]. However, Horai et al[15] reported the pharmacodynamic effects and pharmacokinetics of RPZ depend on the CYP2C19 genotype status on the first day after a single dose. As the present study is on the effect on the first day after a single dose of PPIs, both RPZ and LPZ are considered to be influenced by CYP2C19 genotype status. Previous reports have indicated that H pylori infection of the gastric mucosa potentiates the effects of proton pump inhibitors[40,41]. Therefore, in the present study, the study subjects were all H pylori-negative CYP2C19 EMs. www.wjgnet.com

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LPZ pH

LPZ plasma concentration

Basal pH

RPZ pH

RPZ plasma concentration

n = 8 ng/mL 800.0

Hourly median intragastric pH

7

700.0

6

600.0

5

a

4 3

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c

a

c

c

c

c

c c

c

c

2

c

c

c c c c

500.0 c

c

c

c c

300.0 200.0

1

100.0

9:00 10:00 11:00 12:30

B

400.0

L

15:00

S

18:0019:00

21:00

0.0 Time

D

Drug administration

Figure 1 The 12-h (median pH per hour) trendgrams for all subjects (n = 8) and correlations with the blood concentration of each drug. The solid line (●) shows hourly intragastric median pH of individuals administered LPZ 30 mg OD, and the broken line (■) shows that of individuals administered RPZ. The solid line (○) shows the blood drug concentration level in individuals administered LPZ OD 30 mg, and the broken line (□) shows those in individuals administered RPZ 10 mg tablets. The solid line (×) shows the intragastric pH values of all subjects obtained without medication. LPZ 30 mg OD induces an earlier rise in both blood concentration level and median pH than RPZ 10 mg tablets. Arrows: Drug administration. B: Breakfast, L: Lunch, S: Snacks, D: Dinner. Blood sample were collected 1, 1.5, 2, 3.5, 6, 9 and 12 h after the administration of each drug. The significance of differences in these intragastric pHs among each regimen was determined by the Wilcoxon signed rank test. aP < 0.05 vs RPZ; cP < 0.05 vs baseline data.

Thus, we clearly state that LPZ 30 mg OD induces a significantly earlier rise in intragastric pH and stronger inhibition of gastric acid secretion than RPZ 10 mg tablets in the early post-administration phase (1-12 h) of a single dose of treatment. Pipkin et al reported that rapid acid suppression is important for effective pain relief at the onset of treatment in GERD patients[14]. Thereby, our results perhaps show that administration of LPZ 30 mg OD as an on-demand therapy is useful for mild GERD patients, because of its faster onset of pH rising action. So why does LPZ 30 mg OD induce a prompter rise in intragastric pH than RPZ 10 mg tablets? This may be partly because of the difference between the dosage forms of RPZ and LPZ. In this study, we compared an entericcoated tablet formulation of RPZ 10 mg tablets with an enteric-coated microgranule formulation of LPZ OD 30 mg in terms of antisecretory activity and the onset of action of a single dose. PPIs are degenerated by gastric acid; therefore, for immediate passage through the stomach, some PPIs are formulated as granules or microgranules. This is necessary to ensure their intact passage through the stomach to allow for absorption in the intestine. The discharge speed, namely passage over time through the stomach, depends upon the particle diameter[42-45]. According to physiological reports [42-46], complexes of high amplitude action potentials occur in the stomach and duodenum. The interdigestive complex in the dog is looked upon as a “housekeeper”, which sweeps the www.wjgnet.com

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bowel clear of contents at the end of the digestive phase. Using a test food labeled with radionuclide, Davis SS et al[46] reported that food emptied into the duodenum immediately, consisting of particles smaller than 2 mm without a “housekeeper”. Moreover, particles larger than 2 mm emptied into the duodenum after the “housekeeper,” which occurs after all meals have emptied from the stomach. As a result, particles smaller than 2 mm empty from the stomach faster than particles larger than 2 mm. LPZ OD particles are smaller than 2 mm; thereby they may passage through the stomach into the duodenum and small intestine faster than RPZ, which is larger than 2 mm. They are absorbed in the small intestine and reach the gastric parietal cells via systemic circulation, where they bind to the proton pump, thereby resulting in potent acid inhibition[16]. In fact, the plasma concentration level of LPZ 30 mg OD induced a prompter effect than RPZ 10 mg tablets, and consequently, LPZ 30 mg OD induced a prompter rise in intragastric pH than RPZ 10 mg tablets. These findings suggest that LPZ 30 mg OD is suitable for administration as an on-demand PPI, because of the prompter rise in plasma concentration level and the faster rise in intragastric pH. In conclusion, LPZ 30 mg OD induced a significantly earlier rise in plasma concentration level in the early postadministration phase of a single oral dose than RPZ 10 mg tablets. As a result, LPZ 30 mg OD induced a significantly earlier rise in median pH in the early post-administration phase of a single oral dose than RPZ 10 mg tablets.

COMMENTS Background

The prevalence of gastroesophageal reflux disease (GERD) symptoms is now increasing in Japan. GERD has a high rate of relapse. The rising use of proton pump inhibitor (PPI) therapy on demand has raised issues regarding efficacy.

Research frontiers

To compare RPZ 10 mg to LPZ 30 mg OD in terms of their antisecretory activity and blood drug concentration in the ultra-early phase after a single dose.

Innovations and breakthroughs

Most previous studies have examined pH monitoring after administration of PPI on days 3-7, not at the early post-administration phase.

Applications

We clearly state that LPZ 30 mg OD induced a significantly earlier rise in intragastric pH and stronger inhibition of gastric acid secretion than RPZ 10 mg tablets during the early post-administration phase (1-12 h) of a single dose of treatment. It is reported that rapid acid suppression is important for effective pain relief at the onset of treatment in GERD patients. Thereby, our results show administration of LPZ 30 mg OD as an on-demand therapy might be useful for mild GERD patients because of its faster onset of pH rising action.

Peer review

In this manuscript, the authors ascertained the effectiveness of LPZ 30 mg OD compared with RPZ 10 mg in the elevation of intragastric pH in the ultra-early state after a single oral administration. The study was well performed and the conclusion was clinically useful.

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2053 on intragastric pH. Aliment Pharmacol Ther 2001; 15: 1929-1937 Saitoh T, Fukushima Y, Otsuka H, Hirakawa J, Mori H, Asano T, Ishikawa T, Katsube T, Ogawa K, Ohkawa S. Effects of rabeprazole, lansoprazole and omeprazole on intragastric pH in CYP2C19 extensive metabolizers. Aliment Pharmacol Ther 2002; 16: 1811-1817 19 Shimatani T, Inoue M, Kuroiwa T, Xu J, Mieno H, Nakamura M, Tazuma S. Acid-suppressive effects of rabeprazole, omeprazole, and lansoprazole at reduced and standard doses: a crossover comparative study in homozygous extensive metabolizers of cytochrome P450 2C19. Clin Pharmacol Ther 2006; 79: 144-152 20 Chang M, Tybring G, Dahl ML, Gotharson E, Sagar M, Seensalu R, Bertilsson L. Interphenotype differences in disposition and effect on gastrin levels of omeprazole-suitability of omeprazole as a probe for CYP2C19. Br J Clin Pharmacol 1995; 39: 511-518 21 Furuta T, Shirai N, Sugimoto M, Nakamura A, Okudaira K, Kajimura M, Hishida A. Effect of concomitant dosing of famotidine with lansoprazole on gastric acid secretion in relation to CYP2C19 genotype status. Aliment Pharmacol Ther 2005; 22: 67-74 22 Pearce RE, Rodrigues AD, Goldstein JA, Parkinson A. Identification of the human P450 enzymes involved in lansoprazole metabolism. J Pharmacol Exp Ther 1996; 277: 805-816 23 Ohara S, Kato M, Asaka M, Toyota T. Studies of 13C-urea breath test for diagnosis of Helicobacter pylori infection in Japan. J Gastroenterol 1998; 33: 6-13 24 De Morais SM, Wilkinson GR, Blaisdell J, Meyer UA, Nakamura K, Goldstein JA. Identification of a new genetic defect responsible for the polymorphism of (S)-mephenytoin metabolism in Japanese. Mol Pharmacol 1994; 46: 594-598 25 Heid CA, Stevens J, Livak KJ, Williams PM. Real time quantitative PCR. Genome Res 1996; 6: 986-994 26 Oliveira CH, Barrientos-Astigarraga RE, Abib E, Mendes GD, da Silva DR, de Nucci G. Lansoprazole quantification in human plasma by liquid chromatography-electrospray tandem mass spectrometry. J Chromatogr B Analyt Technol Biomed Life Sci 2003; 783: 453-459 27 Huang J, Xu Y, Gao S, Rui L, Guo Q. Development of a liquid chromatography/tandem mass spectrometry assay for the quantification of rabeprazole in human plasma. Rapid Commun Mass Spectrom 2005; 19: 2321-2324 28 Yoshiike N, Matsumura Y, Yamaguchi M, Seino F, Kawano M, Inoue K, Furuhata T, Otani Y. Trends of average intake of macronutrients in 47 prefectures of Japan from 1975 to 1994--possible factors that may bias the trend data. J Epidemiol 1998; 8: 160-167 29 Sakamoto M. The situation of the epidemiology and management of obesity in Japan. Int J Vitam Nutr Res 2006; 76: 253-256 30 Wu JC, Mui LM, Cheung CM, Chan Y, Sung JJ. Obesity is associated with increased transient lower esophageal sphincter relaxation. Gastroenterology 2007; 132: 883-889 31 Fujisawa T, Kumagai T, Akamatsu T, Kiyosawa K, Matsunaga Y. Changes in seroepidemiological pattern of Helicobacter pylori and hepatitis A virus over the last 20 years in Japan. Am J Gastroenterol 1999; 94: 2094-2099 32 Haruma K, Hamada H, Mihara M, Kamada T, Yoshihara M, Sumii K, Kajiyama G, Kawanishi M. Negative association between Helicobacter pylori infection and reflux esophagitis in older patients: case-control study in Japan. Helicobacter 2000; 5: 24-29 33 Kawaguchi H, Haruma K, Komoto K, Yoshihara M, Sumii K, Kajiyama G. Helicobacter pylori infection is the major risk factor for atrophic gastritis. Am J Gastroenterol 1996; 91: 959-962 34 Mihara M, Haruma K, Kamada T, Komoto K, Yoshihara M, Sumii K, Kajiyama G. The role of endoscopic findings for the diagnosis of Helicobacter pylori infection: evaluation in a country with high prevalence of atrophic gastritis. Helicobacter 1999; 4: 40-48 35 Haruma K, Kamada T, Kawaguchi H, Okamoto S, Yoshihara 18

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M, Sumii K, Inoue M, Kishimoto S, Kajiyama G, Miyoshi A. Effect of age and Helicobacter pylori infection on gastric acid secretion. J Gastroenterol Hepatol 2000; 15: 277-283 36 Feldman M, Cryer B, McArthur KE, Huet BA, Lee E. Effects of aging and gastritis on gastric acid and pepsin secretion in humans: a prospective study. Gastroenterology 1996; 110: 1043-1052 37 Vallot T, Bruley des Varannes S, Grimaud JC, Ruszniewski P, Richard A, Gentin F, Slama A. Epidemiology of gastroesophageal-reflux in general practice. . Predictive factors for health care utilization in the course of a year. Gastroenterol Clin Biol 1999; 23: 1139-1144 38 Adachi K, Katsube T, Kawamura A, Takashima T, Yuki M, Amano K, Ishihara S, Fukuda R, Watanabe M, Kinoshita Y. CYP2C19 genotype status and intragastric pH during dosing with lansoprazole or rabeprazole. Aliment Pharmacol Ther 2000; 14: 1259-1266 39 Williams MP, Sercombe J, Hamilton MI, Pounder RE. A placebo-controlled trial to assess the effects of 8 days of dosing with rabeprazole versus omeprazole on 24-h intragastric acidity and plasma gastrin concentrations in young healthy male subjects. Aliment Pharmacol Ther 1998; 12: 1079-1089

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Martinek J, Blum AL, Stolte M, Hartmann M, Verdu EF, Luhmann R, Dorta G, Wiesel P. Effects of pumaprazole (BY841), a novel reversible proton pump antagonist, and of omeprazole, on intragastric acidity before and after cure of Helicobacter pylori infection. Aliment Pharmacol Ther 1999; 13: 27-34 41 Labenz J, Tillenburg B, Peitz U, Idstrom JP, Verdu EF, Stolte M, Borsch G, Blum AL. Helicobacter pylori augments the pHincreasing effect of omeprazole in patients with duodenal ulcer. Gastroenterology 1996; 110: 725-732 42 Code CF. The interdigestive housekeeper of the gastrointestinal tract. Perspect Biol Med 1979; 22: S49-S55 43 Aeberhard P. Gastrointestinal myoelectric complex. Z Gastroenterol 1977; 15: 202-208 44 Malagelada JR, Longstreth GF, Summerskill WH, Go VL. Measurement of gastric functions during digestion of ordinary solid meals in man. Gastroenterology 1976; 70: 203-210 45 Meyer JH, Ohashi H, Jehn D, Thomson JB. Size of liver particles emptied from the human stomach. Gastroenterology 1981; 80: 1489-1496 46 Davis SS, Hardy JG, Fara JW. Transit of pharmaceutical dosage forms through the small intestine. Gut 1986; 27: 886-892 S- Editor Zhong XY

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RAPID COMMUNICATION

p16 promoter hypermethylation: A useful serum marker for early detection of gastric cancer Mohammad Reza Abbaszadegan, Omeed Moaven, Hamid Reza Sima, Kamran Ghafarzadegan, Azadeh A'rabi, Mohammad Naser Forghani, Hamid Reza Raziee, Ali Mashhadinejad, Mostafa Jafarzadeh, Ehsan Esmaili-Shandiz, Ezzat Dadkhah M o h a m m a d Re z a A b b a s z a d e g a n , O m e e d M o a v e n , Azadeh A'rabi, Ezzat Dadkhah, Division of Human Genetics, Immunology Research Center, Avicenna Research Institute, Mashhad University of Medical Sciences, Mashhad 9196773117, Iran Hamid Reza Sima, Department of Internal Medicine, Imam Reza Hospital, Mashhad University of Medical Sciences, Mashhad 9137913316, Iran Kamran Ghafarzadegan, Department of Pathology, Omid Hospital, Mashhad University of Medical Sciences, Mashhad 9176613775, Iran Mohammad Naser Forghani, Department of Surgery, Omid Hospital, Mashhad University of Medical Sciences, Mashhad 9176613775, Iran Hamid Reza Raziee, Department of Oncology, Omid Hospital and Cancer Research Center, Mashhad 9176613775, Iran Ali Mashhadinejad, Mostafa Jafarzadeh, Ehsan EsmailiShandiz, Gastroenterology Research Center, Mashhad University of Medical Sciences, Mashhad 9137913316, Iran Author contributions: Abbaszadegan MR and Moaven O contributed equally to this work; Abbaszadegan MR, Moaven O, Sima HR, Ghafarzadegan K and Raziee HR designed the research; Abbaszadegan MR, A’rabi A, Moaven O, Ghafarzadegan K, Mashhadinejad A, Forghani MN, Esmaili-Shandiz E, and Dadkhah E performed the research; Moaven O and Jafarzade M analyzed the data; and Abbaszadegan MR, Moaven O and Sima HR wrote the paper. Supported by A grant offered by Mashhad University of Medical Sciences, No. 84129 Correspondence to: Mohammad Reza Abbaszadegan, MT (ASCP), PhD, Associate Professor, Director, Division of Human Genetics, Avicenna Research Institute, Mashhad University of Medical Sciences, Mashhad 9196773117, Iran. [email protected] Telephone: +98-511-7112343 Fax: +98-511-7112343 Received: December 21, 2007  Revised: February 10, 2008

Abstract AIM: To determine p16 promoter hypermethylation in gastric tumoral tissue and serum samples, its impact on p16-protein expression, and correlation with clinical and histological features. METHODS: Samples were obtained from 52 histologically confirmed cases of gastric adenocarcinoma. Gastric tissue and serum of 50 age- and sex-matched individuals with normal gastroscopy and biopsy were obtained as control samples. Methylation-specific polymerase chain reaction (MSP) was used to evaluate

methylation status of p16 promoter. p16-protein expression was analyzed by immunohistochemical staining on paraffin-embedded sections. RESULTS: Methylation was detected in 44.2% (23/52) of tumoral tissues. 60.9% of them were also methylated in serum, i.e., 26.9% of all patients (14/52). Methylation was not detected in tissue and sera of control samples. p16-protein expression was decreased in 61.5% of cases (32/52), and was significantly associated with promoter hypermethylation (P < 0.001). Methylation was significantly more frequent in higher pathological grades (P < 0.05). Methylation was not associated with other clinicopathological features and environmental factors including H pylori infection and smoking. CONCLUSION: p16 promoter hypermethylation is an important event in gastric carcinogenesis. It is the principle mechanism of p16 gene silencing. It is related to malignant tumor behavior. Detection of DNA methylation in serum may be a biomarker for early detection of gastric cancer. © 2008 WJG . All rights reserved.

Key words: Gastric cancer; p16 ; Hypermethylation; Methylation specific PCR Peer reviewer: Shingo Tsuji, Professor, Department of Internal

Medicine and Therapeutics, Osaka University Graduate School of Medicine (A8), 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan Abbaszadegan MR, Moaven O, Sima HR, Ghafarzadegan K, A'rabi A, Forghani MN, Raziee HR, Mashhadinejad A, Jafarzadeh M, Esmaili-Shandiz E, Dadkhah E. p16 promoter hypermethylation: A useful serum marker for early detection of gastric cancer. World J Gastroenterol 2008; 14(13): 2055-2060 Available from: URL: http://www.wjgnet.com/1007-9327/14/2055.asp DOI: http:// dx.doi.org/10.3748/wjg.14.2055

INTRODUCTION Gastric cancer is one of the most widespread cancers and the second leading cause of cancer-related death worldwide[1]. It is estimated that gastric cancer is the most common cancer in Iran (Age Standardized Rate = 26.1 per 105), and the incidence rate is higher than the world www.wjgnet.com

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average[2]. Several genetic and epigenetic alterations have been suggested to play important roles in the carcinogenesis pathway, affecting oncogenes, tumor suppressor genes, apoptosis-regulating or mismatch repair genes[3]. Repression of genes by CpG island methylation in the promoter region, which is normally unmethylated, is the most frequent epigenetic alteration, in which the DNA structure is affected while the genetic code remains intact[4]. Thus, an increasing number of genes, methylated at the promoter region, are targeted as possible tumor markers for different purposes such as early detection, classification and tumor prognosis, therapeutic strategies and patient follow up[5-9]. Detection of circulating tumoral DNA was first reported about three decades ago[10]. Free DNA is thought to originate from apoptotic and necrotic tumoral cells[11]. More recently, detection of promoter hypermethylation in serum of patients has been reported in some malignancies such as colorectal or esophageal cancer[12,13]. Correlation of clinicopathological features with methylation patterns, which help to predict patient outcome, has been indicated in several studies[14-18]. p16 is a cell-cycle regulator that induces G1-phase arrest by inhibition of cyclin D-dependent protein kinase 4 (CDK4) and 6 (CDK6), thus interfering with phosphorylation of the retinoblastoma protein (pRb) and further inhibition of transcription of proteins that promote passage of the cell through the restriction point of the G1 stage[19]. p16 inactivation breaks down the regulatory mechanism of the cell cycle. As a tumor suppressor gene, being silenced by any mechanism will promote carcinogenesis. This study was conducted to assess the methylation of p16 promoter in gastric tumoral tissue and serum samples and its impact on gene expression, and correlation with clinical and histological features for the first time in the Iranian population.

MATERIALS AND METHODS Sample collection and DNA preparation Tumoral tissue and corresponding serum samples were obtained from 52 consecutive histologically confirmed gastric adenocarcinoma patients. Patients undergoing any therapeutic intervention were excluded. Tumoral tissues were obtained by gastrectomy or endoscopy in unresectable metastatic cases, formalin-fixed and paraffinembedded. Tumors were histologically verified as gastric adenocarcinoma and subtyped into intestinal, diffuse, or mixed type, as suggested by Lauren[20]. Grading was also determined and staging was performed using tumor, node and metastasis (TNM) classification, sixth edition. Fifty age- and sex-matched individuals, with normal gastroscopy and biopsy, were included as a control group. Paraffinembedded tissue and corresponding serum samples were taken as well. All patients and control individuals gave informed consent according to institutional guidelines, and the study was approved by the research ethics committee of Mashhad University of Medical Sciences. Paraffin-embedded tissues were retrieved by using xylene and alcohol, digested by proteinase K, extracted with phenol/chloroform/isoamyl alcohol, and precipitated in ethanol. Serum samples were isolated by DNA extraction www.wjgnet.com

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Table 1 Primer sets used in MSP Primer sets Sense primer: 5'-3'

Antisense primer: 5'-3'

p16-W

CGGGCCGCGGCCGTGG

140

GACCCCGAACCGCGAC CGTAA CAACCCCAAACCACAA CCATAA

150

p16-M p16-U

CAGAGGGTGGGG CGGACCGC TTATTAGAGGGTG GGGCGGATCGC TTATTAGAGGGTG GGGTGGATTGT

Size (bp)

151

W: Unmodified or wild-type primers; M: Methylated-specific primers; U: Unmethylated-specific primers.

kit (QIAamp DNA mini kit, QIAGEN, Canada). H pylori infection was detected and double checked in serum with an ELISA (Trinity H pylori kit, Ireland). None of the patients had a history of H pylori eradication. DNA modification (bisulfite treatment) DNA modification with sodium bisulfite causes unmethylated cytosine bases to convert to uracil, while methylated cytosine is resistant and remains unchanged. After treatment, methylated alleles have a different sequence as compared with unmethylated alleles, which can be used to design allele-specific PCR primers, and methylation-specific PCR (MSP) takes advantage of this fact[21]. Two micrograms of genomic DNA was first denatured by heating (97℃ for 10 min followed by chilling on ice at 0℃ for 5 min), and incubated for 20 min at 48℃ after adding 3 mol/L NaOH (2 µL). Bisulfite solution (2.5 mol/L sodium metabisulfite and 125 mmol/L hydroquinone) was added and incubated for 12 h at 48℃ in the dark, for modification. Modified DNA was purified by using Wizard DNA purification resin (DNA Cleanup Kit; Promega, Madison, WI, USA) according to the manufacturer’s instructions. Modified DNA was treated with 3 mol/L NaOH (5 µL) in 37℃ for 10 min and precipitated with ammonium acetate 5 mol/L (75 µL), 2.5 volumes 100% ethanol and 2 µL glycogen (20 mg/ mL; Fermentase; UAB, Lithuania) and dissolved in 20 µL 5 mmol/L Tris (pH 8.0). MSP Specific primer sets for unmethylated (p16U) and methylated (p16M) DNAs, described by Herman et al[21], were utilized (Table 1). The PCR mixture contained 1 × buffer (Finzymes OY, Finland) with 2 mmol/L MgCl2, 500 nmol/L each primer, 0.2 mmol/L dNTPs, 1 U Hot Start Taq polymerase (Finzymes OY, Finland). The PCR amplification of the modified DNA samples consisted of one cycle of 95℃ for 10 min, 40 cycles of 94℃ for 45 s, 60℃ for 45 s, and 72℃ for 1 min; then one cycle of 72℃ for 10 min. DNA from L132 (embryonic lung cell line) cells was used as a positive control for unmethylated DNA. In order to make a positive control for methylated DNA, normal lymphocyte DNA was treated with M.Sss1 CpG methyltransferase (New England BioLabs, USA) before bisulfite treatment. Six microliters of amplified PCR products were loaded onto 2.5% agarose gels and nondenaturing 8% polyacrylamide gels, stained with ethidium bromide, and directly visualized under UV illumination.

Abbaszadegan MR et al . p16 hypermethylation in gastric cancer IVD

A

M

U

L132 M

Case 4 U

M

U

Case 10 Case 24 M

U

M

U

Case 43 M

 

Table 3 Clinicopathological features of p16 promoter hypermethylation

U

Variable

B

Figure 1 Analysis of p16 promoter hypermethylation in tissue and corresponding serum of patients. (A) MSP analysis. IVD served as a positive control for hypermethylated DNA and L132 as a positive control for unmethylated DNA. Patients 4, 24 and 43 were hypermethylated, which revealed 150 bp bands with hypermethylated primers. Patient 10 was not methylated. (B) MSP analysis in corresponding sera of samples depicted in A. Patients 4 and 24 were hypermethylated in serum as well. Patients 43 and 10 were not methylated.

Table 2 Results of methylation in tumoral tissue and corresponding serum

Serum (+) Serum (-)

2057

Tissue (+)

Tissue (-)

14 9

0 29

(+): Methylated; (–): Unmethylated.

Immunohistochemical staining Immunohistochemical staining was performed using the CINtec p16INK4A Histology Kit, clone E6H4 (Dako, Carpinteria, CA, USA) and the DakoCytomation Autostainer Instrument, according to the manufacturer’s instructions. Briefly, 4-µm-thick formalin-fixed, paraffinembedded sections were dewaxed, rehydrated and boiled in Target Retrieval Solution of Dako in a microwave oven for 40 min. After endogenous peroxidase blocking, the slides were incubated with primary p16 INK4A antibody (clone E6H4) at 1:25 dilutions for 30 min. The antigen-antibody reaction was visualized by 3,3'-diaminobenzidine (DAB) chromogen for 10 min, followed by acidified hematoxyline counterstaining for 1 min. p16-positive cervical squamous cell carcinoma was used as an external positive control, and non-neoplastic stromal cells served as internal positive controls for p16 in every tumor section. Statistical analysis Statistical analysis was performed using SPSS software (ver. 11.5). The correlation between two variables was evaluated using Pearson’s χ 2 and Fisher’s exact test. Statistical significance was defined as P < 0.05.

RESULTS The studied population consisted of 38 men and 14 women. The median age was 64.5 and the average was 63.7 years (range, 38-81). Hypermethylation of p16 promoter was detected in 44.2% of tumoral gastric tissue (23/52), while normal gastric samples were all unmethylated. MSP analysis of the p16 promoter in gastric cancer is shown in Figure 1. Corresponding serum samples were also examined. Among the patients with methylated gastric

Total Gender Male Female Age (yr) < 64 > 64

n (%)

Methylated Unmethylated

P value

23

29

38 (73.1) 14 (26.9)

17 6

21 8

0.904

26 (50.0) 26 (50.0)

8 15

18 11

0.051

Pathological grade 1 20 (39.2) 2 11 (21.6) 3 20 (39.2)

5 7 10

15 4 10

< 0.05

Pathological type Intestinal 27 (62.7)

13

14

(1 vs 2&3) 0.754 (Intestinal vs Diffuse) 0.161 (Intestinal vs Mix) 0.140 (Diffuse vs Mix)

Diffuse

14 (27.5)

5

9

Mixed

5 (9.8)

4

1

Anatomical site Cardia 22 (44.0) Body 15 (30.0) Pylorus 13 (26.0)

7 9 6

15 6 7

Distant metastasis Absent 39 (75.0) Present 13 (25.0)

20 9

19 4

0.259

Smoking Yes No

9 (17.6) 42 (82.4)

4 19

5 23

1.000

H pylori infection Positive 30 (60.0) Negative 20 (40.0)

11 11

19 9

0.201

0.124 (Cardia vs Noncardia)

tissue, 60.9% were also methylated in their corresponding serum, i.e., 26.9% of all cases (14/52). All normal individuals in the control group were unmethylated in their sera (Table 2). Clinicopathological features of p16 promoter hypermethylation are tabulated in Table 3. There was no association between gender and promoter hypermethylation. Methylation had higher frequency in older patients (P = 0.051). Methylation was significantly lower in welldifferentiated tumors as compared with higher pathological grades (P < 0.05). There was no significant difference in methylation status between intestinal and diffuse types. However, it was observed that 4/5 mixed type tumors were methylated, and all five were negative for p16 protein expression. Methylation was more frequent in non-cardiac type of tumor as compared with cardiac type (68.2% vs 31.8%), without statistical significance. p16 promoter methylation had no correlation with distant metastasis. No association was observed between methylated circulating DNA and lymph node metastasis. Among environmental factors, we assessed H pylori infection and smoking. Sixty percent of patients (30/50) were infected with H pylori and 17.6% (9/51) were smokers. p16 promoter methylation had no correlation with smoking and H pylori infection. Immunohistochemical staining of p16 Nuclear p16 immunostaining was positive for protein www.wjgnet.com

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p16-positive p16-negative

expression. Positive tumors varied in intensity of nuclear staining, with the proportion of cells ranging from 10% to 95%. In a few positive tumors, we observed marked heterogeneity in different areas with respect to p16 reactivity. Negative staining was observed in 61.5% of patients (32/52). Immunohistochemical staining is depicted in Figure 2. We assessed the correlation between immunohistochemical staining and methylation status (Table 4). Among the p16-negative tumors, 62.5% were methylated, which showed strong correlation between negative immunostaining and promoter region hypermethylation (P < 0.001). There was significant correlation with pathological grade and mixed subtype, as well as methylation pattern, but no association was observed with other clinicopathological and demographic features and environmental factors.

DISCUSSION Several genetic and epigenetic alterations play an important role in gastric carcinogenesis. Tumor suppressor and other tumor-related genes are the main targets. Aberrant alterations of p16, as a tumor suppressor gene, are important events in several tumors, including gastric cancer, and hypermethylation of CpG islands in the promoter region is responsible for a great proportion of tumors[22-25]. In this study, we demonstrated that 44.2% of tissues from gastric adenocarcinoma were methylated. The result was consistent with previous studies by Shim et al[26] and Ding et al[27] who reported 42% and 45% methylation of p16 promoter in gastric cancer. Different mechanisms are suggested for decreased p16 protein expression, including homozygote deletion, point mutation and promoter methylation[28]. The first two mechanisms occur in < 10% of tumors[29,30]. We report that 61.5% of gastric adenocarcinomas were negative for p16 protein expression. Among p16-negative tumors, 62.5% were methylated in their promoter region, which was strongly correlated with decreased protein expression. We conclude that the principle mechanism for decreased p16protein expression in gastric cancer is hypermethylation of the promoter region. There were three patients with normal protein expression despite promoter hypermethylation. All of these exhibited tumor heterogeneity, although www.wjgnet.com

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Table 4 Results of methylation analysis in p16-positive and negative cases Immunohistochemistry

Figure 2 Immunohistochemical staining with monoclonal anti-p16 protein. (A) Nuclear reactivity showed expression of p16 protein (case 48). (B) p16-negative tumor (case 15) failed to stain due to decreased expression of p16 protein.

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Methylated

Unmethylated

3 20

17 12

partial methylation in one or both alleles might have been another possible reason. There were also twelve p16negative patients with normal methylation status. Alternative mechanisms such as point mutation or homozygote deletion might be responsible for decreased protein expression. Promoter hypermethylation was detected from sera of 60.9% of patients with tumor methylation, which accounted for 26.9% of all cases. This was similar to the study performed by Koike et al[31] who reported 27% methylated sera in gastric cancer patients. There are some interesting features that introduce p16 hypermethylation as a good serum marker for early detection of gastric cancer. As compared with other methods, serum markers are easier to use, less expensive and less invasive. Among serum markers, hypermethylation has some distinct features. It is a frequent event in cancer, while rare or absent in normal tissue, which leads to high specificity for the purpose of tumor diagnosis. Small specific regions of genome are affected, which make it easily detectable, in contrast with mutations. MSP is a sensitive technique that detects free circulating DNA even in small amounts[32]. Among many genes altered by methylation in gastric cancer, p16 promoter methylation is an early event in carcinogenesis[33]. We conclude that p16 is a good serum marker for early detection of gastric cancer. In this study we showed that hypermethylation is less frequent in well-differentiated tumors. We conclude that p16 promoter hypermethylation is associated with tumor malignant behavior. There were no significant differences between intestinal and diffuse types in our population, but mixed subtype was strongly associated with silenced expression. However, there were only five cases with mixed type among our patients, and additional studies with a larger number of cases are needed to confirm the finding. In the population studied, 86.5% were at stage 3 and 4, which showed that gastric cancer was diagnosed in advanced stages, which necessitates early detection. We did not statistically compare patients at different stages, because of unbalanced distribution and the consequent bias. Methylation status in cardiac tumors was also compared with that of non-cardiac tumors. We know that cardiac tumors have different behavior from non-cardiac tumors. Methylation tends to occur more in distal tumors (more than two-fold higher), without statistical significance. We did not find any association between H pylori and methylation in our population, despite a few previous studies that suggested H pylori induces methylation[34,35]. Smoking, the other studied environmental factor, had no correlation with promoter hypermethylation. However, our patients consisted of only 17.6% (9/51) smokers, which was not sufficient to draw any strong conclusion. Promoter hypermethylation was not affected by gender, although it tended to occur more often in older patients. Precise

Abbaszadegan MR et al . p16 hypermethylation in gastric cancer

matching and absence of promoter hypermethylation in the control individuals decreased the possibility of methylation induction by aging, unrelated to cancer. No association was observed between circulating methylated DNA and lymph node metastasis. This indicates that the origin of circulating methylated DNA is not from lymph node metastasis. Hypermethylation status in serum was not associated with other clinicopathological features either. In conclusion, p16 promoter hypermethylation is an important event in gastric carcinogenesis. It is the principle mechanism of p16 gene silencing. It is associated with tumor malignant behavior. Detection of p16 hypermethylation in serum may be a useful biomarker for early detection of gastric cancer. Assessment of other serum markers may increase the sensitivity of screening in future studies. Moreover, promoter hypermethylation will regress after treatment. Further studies will determine whether promoter hypermethylation is a good surveillance marker for patient follow-up after treatment.

ACKNOWLEDGMENTS We would like to thank Dr. Velayati, Dr. Gholamin and Mrs. Asadi for their technical assistance and Miss hoseinnezhad for her help in collecting gastric cancer specimens.

COMMENTS

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3 4 5

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Background

Gastric cancer is the second leading cause of cancer-related death worldwide. This is because most cases are diagnosed at an advanced stage, and there is no standard method for early detection. Several genetic and epigenetic alterations play important roles in gastric carcinogenesis. Methylation, the most important epigenetic alteration, influences gene repression without affecting genetic coding. Several recent studies have been conducted to assess the clinical implications of epigenetics in cancer. Free circulating, tumor-derived DNA is a good target for early detection as a serum marker. p16 is a tumor suppressor gene, which acts as a cell-cycle regulator. It is silenced in early stages of gastric carcinogenesis.

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Research frontiers

P16 promoter hypermethylation related to clinicopathological features and early detection of gastric cancer.

Innovations and breakthroughs

A thorough analysis of p16 promoter hypermethylation, correlations with demographic and clinicopathological features and environmental factors, and implications as an early diagnostic marker were performed for the first time in the Iranian population.

Applications

Although p16 promoter hypermethylation is very specific for early detection of gastric cancer, further assessment of other genes will be useful to raise the sensitivity and provide a panel of serum markers for the purpose.

Peer review

This was a very interesting study. It examined p16 promoter hypermethylation in gastric adenocarcinoma and free DNA. Methylation was detected in 44% of tumors, and in 26.9% of serum. p16 promoter hypermethylation in serum was closely correlated with that in gastric tissue. Methylation was significantly associated with pathological grade.

REFERENCES 1

Munoz N, Franceschi S. Epidemiology of gastric cancer and perspectives for prevention. Salud Publica Mex 1997; 39: 318-330

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9821-9826 Lee WH, Isaacs WB, Bova GS, Nelson WG. CG island methylation changes near the GSTP1 gene in prostatic carcinoma cells detected using the polymerase chain reaction: a new prostate cancer biomarker. Cancer Epidemiol Biomarkers Prev 1997; 6: 443-450 Belinsky SA, Nikula KJ, Palmisano WA, Michels R, Saccomanno G, Gabrielson E, Baylin SB, Herman JG. Aberrant methylation of p16(INK4a) is an early event in lung cancer and a potential biomarker for early diagnosis. Proc Natl Acad Sci USA 1998; 95: 11891-11896 Sato F, Meltzer SJ. CpG island hypermethylation in progression of esophageal and gastric cancer. Cancer 2006; 106: 483-493 Zhao YF, Zhang YG, Tian XX, Juan Du, Jie Zheng. Aberrant methylation of multiple genes in gastric carcinomas. Int J Surg Pathol 2007; 15: 242-251 Shim YH, Kang GH, Ro JY. Correlation of p16 hypermethylation with p16 protein loss in sporadic gastric carcinomas. Lab Invest 2000; 80: 689-695 Ding Y, Le XP, Zhang QX, Du P. Methylation and mutation analysis of p16 gene in gastric cancer. World J Gastroenterol 2003; 9: 423-426 Kamb A, Gruis NA, Weaver-Feldhaus J, Liu Q, Harshman K, Tavtigian SV, Stockert E, Day RS 3rd, Johnson BE, Skolnick MH. A cell cycle regulator potentially involved in genesis of many

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tumor types. Science 1994; 264: 436-440 Wu MS, Shun CT, Sheu JC, Wang HP, Wang JT, Lee WJ, Chen CJ, Wang TH, Lin JT. Overexpression of mutant p53 and c-erbB-2 proteins and mutations of the p15 and p16 genes in human gastric carcinoma: with respect to histological subtypes and stages. J Gastroenterol Hepatol 1998; 13: 305-310 Lee YY, Kang SH, Seo JY, Jung CW, Lee KU, Choe KJ, Kim BK, Kim NK, Koeffler HP, Bang YJ. Alterations of p16INK4A and p15INK4B genes in gastric carcinomas. Cancer 1997; 80: 1889-1896 Koike H, Ichikawa D, Ikoma H, Tani N, Ikoma D, Otsuji E, Okamoto K, Ueda Y, Kitamura K, Yamagishi H. Comparison of serum aberrant methylation and conventional tumor markers in gastric cancer patients. Hepatogastroenterology 2005; 52: 1293-1296 Miyamoto K, Ushijima T. Diagnostic and therapeutic applications of epigenetics. Jpn J Clin Oncol 2005; 35: 293-301 Kang GH, Lee S, Kim JS, Jung HY. Profile of aberrant CpG island methylation along the multistep pathway of gastric carcinogenesis. Lab Invest 2003; 83: 635-641 Perri F, Cotugno R, Piepoli A, Merla A, Quitadamo M, Gentile A, Pilotto A, Annese V, Andriulli A. Aberrant DNA methylation in non-neoplastic gastric mucosa of H. Pylori infected patients and effect of eradication. Am J Gastroenterol 2007; 102: 1361-1371 Ushijima T. Epigenetic field for cancerization. J Biochem Mol Biol 2007; 40: 142-150 S- Editor Li DL L- Editor Kerr C

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Online Submissions: wjg.wjgnet.com www.wjgnet.com [email protected]

World J Gastroenterol 2008 April 7; 14(13): 2061-2064 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

RAPID COMMUNICATION

Prospective evaluation of small bowel preparation with bisacodyl and sodium phosphate for capsule endoscopy Andreas Franke, Frank Hummel, Phillip Knebel, Christoph Antoni, Ulrich Böcker, Manfred V Singer, Matthias Löhr

Andreas Franke, Frank Hummel, Phillip Knebel, Christoph Antoni, Ulrich Böcker, Manfred V Singer, Matthias Löhr, Department of Medicine Ⅱ (Gastroenterology, Hepatology and Infectious Diseases), University Hospital of Heidelberg at Mannheim, Mannheim D-68167, Germany F ra n k H u m m e l , Department of Medicine, Rotes Kreuz Krankenhaus Kassel, Kassel D-34121, Germany Author contributions: Franke A, Hummel F, Knebel P and Löhr M designed research and analyzed data; Löhr M, Antoni C, Singer MV and Böcker U evaluated the quality of preparation; Franke A and Löhr M wrote the paper. Correspondence to: Andreas Franke, MD, Department of Med icine Ⅱ (Gastroenterology, Hepatology and Infectious Diseases), University Hospital of Heidelberg at Mannheim, Theodor-KutzerUfer 1-3, Mannheim D-68167, Germany. [email protected] Telephone: +49-621-3833359 Fax: +49-621-3833805    Revised: February 25, 2008 Received: January 3, 2008

Abstract AIM: To determine the effect of Prepacol®, a combination of sodium phosphate and bisacodyl, on transit and quality of capsule endoscopy (CE). METHODS: Fivety two consecutive patients were included in this prospective study. CE was performed following a 12 h fasting period. Twenty six patients were ® randomized for additional preparation with Prepacol . The quality of CE was assessed separately for the proximal and the distal small bowel by 3 experienced endoscopists on the basis of a graduation which was initially developed with 20 previous CE. RESULTS: Preparation with Prepacol® accelerated small bowel transit time (262 ± 55 min vs 287 ± 97 min), but had no effect on the quality of CE. Visibility was significantly reduced in the distal compared to the proximal small bowel. CONCLUSION: The significantly reduced visibility of CE in the distal small bowel allocates the need for a good ® preparation. Since Prepacol has no beneficial effect on CE the modality of preparation and the ideal time of application remains unclear. Further standardized examinations are necessary to identify sufficient preparation procedures and to determine the impact of the volume of the preparation solution. © 2008 WJG . All rights reserved.

Key words: Small bowel; Capsule endoscopy; Preparation;

Laxative; Visibility; Transit time

Peer reviewer: Chris JJ Mulder, Professor, Department of Gastroenterology, VU University Medical Center, PO Box 7057, Amsterdam1007 MB, The Netherlands Franke A, Hummel F, Knebel P, Antoni C, Böcker U, Singer MV, Löhr M. Prospective evaluation of small bowel preparation with bisacodyl and sodium phosphate for capsule endoscopy. World J Gastroenterol 2008; 14(13): 2061-2064 Available from: URL: http://www.wjgnet.com/1007-9327/14/2061.asp DOI: http://dx.doi. org/10.3748/wjg.14.2061

INTRODUCTION Capsule endoscopy (CE) is a well accepted tool for evaluation of small bowel pathologies [1-4]. However, it has some limitations due to restricted recording time and reduced visibility by air and residual material especially in the distal small bowel. Therefore, prokinetic drugs, laxatives and defoaming agents have been tested to improve the quality of the examination. Prokinetic drugs were used to avoid incomplete small bowel examinations due to long gastric retention and slow bowel transit of the capsule. It was shown that domperidone shortened gastric emptying time of the capsule [5]. The results on metoclopramide were inconsistent: Keuchel et al [5] found no effect, whereas Selby[6] demonstrated an increased gastric emptying time. Erythromycin accelerated gastric emptying[7,8], however, this treatment had no effect on the visibility in one study[7] and led even to an impaired visibility in another study[8]. In order to clean the small bowel from residual material different laxatives were tested. Sodium phosphate improved the view in some studies[9-11]. Preparation with polyethylenglycol produced controversial results: In two studies the visibility was improved[12,13], whereas in others it was unchanged[14-16]. Preparation with simethicone, a defoaming agent, resulted in fewer air bubbles and better visibility in one study[17]. However, since the data is scanty and partially inconsistent, to date no standardized protocol has been recommended for bowel preparation for CE. Prepacol® (Guerbet GmbH, Sulzbach, Ger many) is a combination of a saline (sodium phosphate) and a stimulant laxative (bisacodyl). It consists of 30 mL of a sodium phosphate solution (containing 6.9 sodium www.wjgnet.com

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monohydrogenphosphatedodecahydrate and 16.4 mg sodiumdihydrogenphosphatedihydrate) and 4 tablets (5 mg bisacodyl each). Prepacol® is available in several European countries and mainly applied for preparation before gastrointestinal operations, radiological and endoscopic bowel examinations. The sodium phosphate solution is poorly absorbable. Water absorption from the gut is therefore impeded by the osmotic gradient. Besides its effects on colonic motor and secretory function, bisacodyl changes the net absorption of sodium and water in the small bowel into a net secretion[18] and accelerates small intestinal transit[18,19]. It was shown that bisacodyl elicits propulsive contractions of the terminal ileum[20,21]. The combination of the osmotic purgative effect of sodium phosphate with the secretory and prokinetic effect of bisacodyl makes Prepacol ® at least theoretically an ideal candidate for small bowel preparation for CE. However, its effect on the quality and gastrointestinal transit of CE has not been studied yet.

April 7, 2008

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GIVEN (R)

C

GIVEN (R)

MATERIALS AND METHODS Fifty two consecutive patients receiving capsule endoscopy were included. The patients were prospectively randomized into two groups. Group A fasted at least 8 h before the examination; group B received additionally Prepacol ®. A written informed consent was obtained from all patients. The research protocol was approved by the ethics committee of the University of Heidelberg. All patients fasted from 7 p.m. the day before CE, patients in group B received additionally at 7 p.m. 30 mL of the sodium phosphate-solution diluted with 70 mL of tap water. Subsequently, they drank 250 mL of water. At 10 p.m. the patients received 4 tablets Prepacol® (20 mg Bisacodyl totally) again with 250 mL of water. All patients were allowed to drink water until 2 h before the examination. The capsule was swallowed at 10 a.m. with 250 mL of plain water. Capsule endoscopy films were evaluated by three independent, endoscopically experienced investigators who were blinded concerning the kind of preparation. In a runin-phase the three investigators corporately generated the appraisal factors and their graduation on the basis of 20 retrospective CE examinations. The following parameters were assessed: total quality of the film, visibility of small bowel mucosa, velocity of the capsule and occurrence of foam, air and residual food. Every parameter was graduated from 1 to 4, accordingly, excellent, good, limited and poor quality. Graduation for occurrence of foam is shown in Figure 1. To evaluate the effect of the preparation with Prepacol® two one-hour-lasting periods were evaluated. The first period started one hour after the capsule left the stomach, the second period ended when the capsule reached the ileocecal valve. Investigators examined the films at a rate of 20 pictures per second. Quantitative data are expressed as mean ± SEM and were analyzed by student’s t-test for significant differences. Categorical data were evaluated by Chi 2 and Fisher exact test. P < 0.05 was chosen as the level of statistical significance.

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B

GIVEN (R)

D

GIVEN (R)

Figure 1 Graduation of visibility concerning occurrence of foam. A: Excellent; B: Good; C: Limited; D: Poor visibility.

Table 1 Patient’s data and indications for CE: Group A (fasting) and group B (additionally Prepacol®)

Gender (w/m) Age (yr) Weight (kg) Length (cm) Indication GI-bleeding Inflammatory bowel disease Miscellaneous

Group A

Group B

P value

13/13 54 ± 17 71 ± 15 170 ± 10

10/16 56 ± 20 79 ± 17 171 ± 8

NS NS NS NS

17 (65%) 4 (15%) 5 (19%)

19 (73%) 3 (12%) 4 (15%)

NS NS NS

Data are mean ± SEM; NS: Not significant.

RESULTS Both groups were not different concerning age, weight, length and gender of the patients. Obscure gastrointestinal (GI) bleeding was the main indication for CE in both groups (Table 1). Other indications were suspicion for or follow-up in IBD, celiac disease, small bowel polyps or malignancy and no difference was observed between the groups (Table 1). There were no differences in gastric emptying time of the capsule between the two groups (Table 2). Small bowel transit time was slightly but significantly shorter in the Prepacol®-group (262 ± 55 min vs 287 ± 97 min, P = 0.05) (Table 2). Recording time was not different between both groups. Figure 2 demonstrates median assessment of investigators concerning visibility of small bowel mucosa, occurrence of foam, air and residual food, as well as velocity of the capsule and total quality of the film separately for the proximal and distal small bowel. Compared to exclusive fasting additional preparation with Prepacol ® did not improve any parameter.

Franke A et al . Preparation for CE

 

P value

38 ± 23 287 ± 97 441 ± 36

44 ± 47 262 ± 55 424 ± 49

NS 0.05 NS

Data are mean ± SEM; NS: Not significant.

4

Fasting Prepacol

Median rank

  60 a   40

a

  20    0

Fasting Prepacol Proximal small bowel

Fasting Prepacol Distal small bowel

Figure 3 Percentage of good or excellent quality as assessed by three independent investigators, separately shown for proximal and distal small bowel, data are median of 52 patients, n = 26 for each group, aP < 0.05 vs proximal small bowel.

3

2

1

0

  80 Good/excellent quality (%)

Gastric retention (min) Small bowel transit (min) Total recording time (min)

Group B

2063

100

Table 2 GI transit times: Group A (fasting) and group B ® (additionally Prepacol ) Group A

 

Visibility Food Foam

Air Velocity Total Visibility Food Foam quality

Proximal small bowel

Air Velocity Total quality

Distal small bowel

Figure 2 Quality of CE as assessed by three independent investigators, separately shown for proximal and distal small bowel, data are median of 52 patients, n = 26 for each group.

The quality was significantly more frequently judged as excellent or good in the proximal compared to the distal small bowel (Figure 3). Concordance in the assessment between each of the investigators was good (82%, 78% and 87%, respectively).

DISCUSSION An adequately cleaned bowel is an important precondition for any gastrointestinal endoscopic procedure. Turbid fluid due to intestinal secretion and food residues in the small bowel may limit visibility and therewith the information obtained by capsule endoscopy. Although in some studies preparation with prokinetic agents or laxatives improved quality of CE [5-16] , no standard procedure has been reached since the data is scanty and partially inconsistent. The selection of an appropriate preparation is further aggravated by the fact that the evaluation of the quality of capsule endoscopy is subjective. Therefore we chose three independent investigators to estimate the influence of Prepacol® on the quality of CE. In the present study preparation with Prepacol® had no advantages concerning visibility of the mucosa, occurrence of foam, frequency and extent of air filled segments, food residues, velocity of the capsule and total quality as compared to exclusive overnight fasting. Quality was significantly inferior in distal small bowel segments compared to proximal segments. This demonstrates that sufficient preparation would be of great help to obtain best possible conditions throughout the whole small bowel. Prepacol® is not effective as a preparation for capsule endoscopy. This may be due to the pharmacological effect,

the dosage and the time of application in relation to CE. Bisacodyl is mainly activated by bacterial metabolism in the colon. Although both, sodium phosphate and bisacodyl increase luminal fluid in the small bowel, their main effect is documented in colon preparation[22,23]. The dose of the sodium phosphates could be too low. Niv and colleagues could show that 90 mL of sodium phosphate in combination with 2 liters of water improved CE quality[9,10]. Those studies showing a positive effect of PEG on CE visibility also used volumes of at least 2 liters[12,15]. Therefore, not only the pharmacological effect may be responsible, but also the volume itself. Patients in our study were allowed to drink until 2 h before the examination, but no minimum volume was recommended. The volume was not documented, it was therefore not possible to examine if the individual amount of fluid intake had any effect on CE quality. Double balloon enteroscopy (DBE) is another recent tool for examination of the small bowel[24-26]. Requirement for more manpower and a slightly increased complication rate are some disadvantages of DBE compared to CE[27,28]. However, DBE has a working channel which enables the examiner for example to take biopsies or intervene in bleedings[29]. In contrast to CE no other preparation than fasting is essential when DBE is performed orally, since the small bowel content can be cleaned during the examination by means of the suction channel[27,28]. CE is an expensive and also time consuming examination. An effective preparation is essential to minimize false results at the best possible rate. Therefore, additional studies are necessary to identify sufficient preparation procedures and to determine the impact of the volume of the preparation solution. This has to be performed in consideration of the patient’s compliance, which might be reduced by the taste of the preparation solution[30].

COMMENTS Background

Capsule endoscopy (CE), a well accepted tool for evaluation of small bowel pathologies, has some limitations due to reduced visibility by air and residual material especially in the distal small bowel.

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Research frontiers

Several forms of preparation (e.g. prokinetic drugs, laxatives and defoaming agents) have been tried to improve the quality of the examination. However, since the data is scanty and partially inconsistent, to date no standardized protocol has been recommended for bowel preparation for CE.

Innovations and breakthroughs ®

The effect of Prepacol (Guerbet GmbH, Sulzbach, Germany), a combination of a saline (sodium phosphate) and a stimulant laxative (bisacodyl), on the quality and ® transit time of CE was tested. It has been shown that preparation with Prepacol accelerated small bowel transit time, but had no effect on the quality of CE.

13 14 15 16

Applications

17

Peer review

18

®

Since Prepacol has no beneficial effect on CE the modality of preparation and the ideal time of application remains unclear.

Interesting effort to do proper research on SB-preparation.

REFERENCES Iddan G, Meron G, Glukhovsky A, Swain P. Wireless capsule endoscopy. Nature 2000; 405: 417 Costamagna G, Shah SK, Riccioni ME, Foschia F, Mutignani 2 M, Perri V, Vecchioli A, Brizi MG, Picciocchi A, Marano P. A prospective trial comparing small bowel radiographs and video capsule endoscopy for suspected small bowel disease. Gastroenterology 2002; 123: 999-1005 3 Ell C, Remke S, May A, Helou L, Henrich R, Mayer G. The first prospective controlled trial comparing wireless capsule endoscopy with push enteroscopy in chronic gastrointestinal bleeding. Endoscopy 2002; 34: 685-689 4 Fireman Z, Mahajna E, Broide E, Shapiro M, Fich L, Sternberg A, Kopelman Y, Scapa E. Diagnosing small bowel Crohn's disease with wireless capsule endoscopy. Gut 2003; 52: 390-392 5 K e u c h e l M , V o r d e r h o l z e r W , S c h e n k G , C s o m o s G , Hagenmuller F, Lochs H. Domperidone shortens gastric transit time of video capsule endoscope. Endoscopy 2003; 35 (Suppl II) A185 Selby W. Complete small-bowel transit in patients undergoing 6 capsule endoscopy: determining factors and improvement with metoclopramide. Gastrointest Endosc 2005; 61: 80-85 7 Leung WK, Chan FK, Fung SS, Wong MY, Sung JJ. Effect of oral erythromycin on gastric and small bowel transit time of capsule endoscopy. World J Gastroenterol 2005; 11: 4865-4868 Fireman Z, Paz D, Kopelman Y. Capsule endoscopy: 8 improving transit time and image view. World J Gastroenterol 2005; 11: 5863-5866 Niv Y, Niv G, Wiser K, Demarco DC. Capsule endoscopy 9 comparison of two strategies of bowel preparation. Aliment Pharmacol Ther 2005; 22: 957-962 10 Niv Y, Niv G. Capsule endoscopy: role of bowel preparation in successful visualization. Scand J Gastroenterol 2004; 39: 1005-1009 11 Lapalus M, Saurin J, Mion F, Ponchon T. Prospective randomized single-blind trial on oral sodium phosphate efficacy for small intestine preparation before capsule endoscopy. Endoscopy 2003; 35 (Suppl II) A183 12 Viazis N, Sgouros S, Papaxoinis K, Vlachogiannakos J, Bergele C, Sklavos P, Panani A, Avgerinos A. Bowel preparation increases the diagnostic yield of capsule endoscopy: a prospective, randomized, controlled study. Gastrointest Endosc

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2004; 60: 534-538 Fireman Z, Kopelman Y, Fish L, Sternberg A, Scapa E, Mahaina E. Effect of oral purgatives on gastric and small bowel transit time in capsule endoscopy. Isr Med Assoc J 2004; 6: 521-523 Ben-Soussan E, Savoye G, Antonietti M, Ramirez S, Ducrotte P, Lerebours E. Is a 2-liter PEG preparation useful before capsule endoscopy? J Clin Gastroenterol 2005; 39: 381-384 Dai N, Gubler C, Hengstler P, Meyenberger C, Bauerfeind P. Improved capsule endoscopy after bowel preparation. Gastrointest Endosc 2005; 61: 28-31 Lee HS, Um SH, Lee SW, Choi JH,. Kim CD, Ryu HS, Hyun JH, Uhm CS. Comparison of two bowel prep-aration for capsule endoscopy: NPO only versus PEG. Endoscopy 2003; 35 (Suppl II) A117 Albert J, Gobel CM, Lesske J, Lotterer E, Nietsch H, Fleig WE. Simethicone for small bowel preparation for capsule endoscopy: a systematic, single-blinded, controlled study. Gastrointest Endosc 2004; 59: 487-491 Ewe K. Effect of bisacodyl on intestinal electrolyte and water net transport and transit. Perfusion studies in men. Digestion 1987; 37: 247-253 Ewe K, Ueberschaer B, Press AG, Kurreck C, Klump M. Effect of lactose, lactulose and bisacodyl on gastrointestinal transit studied by metal detector. Aliment Pharmacol Ther 1995; 9: 69-73 Saunders DR, Sillery J, Rachmilewitz D, Rubin CE, Tytgat GN. Effect of bisacodyl on the structure and function of rodent and human intestine. Gastroenterology 1977; 72: 849-856 Pescatori M. Myoelectric and motor activity of the terminal ileum after pelvic pouch for ulcerative colitis. Dis Colon Rectum 1985; 28: 246-253 Adams WJ, Meagher AP, Lubowski DZ, King DW. Bisacodyl reduces the volume of polyethylene glycol solution required for bowel preparation. Dis Colon Rectum 1994; 37: 229-233; discussion 233-234 Ker TS. Comparison of reduced volume versus four-liter electrolyte lavage solutions for colon cleansing. Am Surg 2006; 72: 909-911 May A, Nachbar L, Wardak A, Yamamoto H, Ell C. Doubleballoon enteroscopy: preliminary experience in patients with obscure gastrointestinal bleeding or chronic abdominal pain. Endoscopy 2003; 35: 985-991 May A, Nachbar L, Ell C. Double-balloon enteroscopy (pushand-pull enteroscopy) of the small bowel: feasibility and diagnostic and therapeutic yield in patients with suspected small bowel disease. Gastrointest Endosc 2005; 62: 62-70 Ell C, May A, Nachbar L, Cellier C, Landi B, di Caro S, Gasbarrini A. Push-and-pull enteroscopy in the small bowel using the double-balloon technique: results of a prospective European multicenter study. Endoscopy 2005; 37: 613-616 Fujimori S, Seo T, Gudis K, Tanaka S, Mitsui K, Kobayashi T, Ehara A, Yonezawa M, Tatsuguchi A, Sakamoto C. Diagnosis and treatment of obscure gastrointestinal bleeding using combined capsule endoscopy and double balloon endoscopy: 1-year follow-up study. Endoscopy 2007; 39: 1053-1058 Kaffes AJ, Siah C, Koo JH. Clinical outcomes after doubleballoon enteroscopy in patients with obscure GI bleeding and a positive capsule endoscopy. Gastrointest Endosc 2007; 66: 304-309 May A, Nachbar L, Pohl J, Ell C. Endoscopic interventions in the small bowel using double balloon enteroscopy: feasibility and limitations. Am J Gastroenterol 2007; 102: 527-535 Szojda MM, Mulder CJ, Felt-Bersma RJ. Differences in taste between two polyethylene glycol preparations. J Gastrointestin Liver Dis 2007; 16: 379-381 S- Editor Li DL L- Editor Alpini GD

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World J Gastroenterol 2008 April 7; 14(13): 2065-2071 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

RAPID COMMUNICATION

Effect of Prometheus liver assist system on systemic hemodynamics in patients with cirrhosis: A randomized controlled study Thomas Dethloff, Flemming Tofteng, Hans-Jorgen Frederiksen, Michael Hojskov, Bent Adel Hansen, Fin Stolze Larsen

Thomas Dethloff, Flemming Tofteng, Bent Adel Hansen, Fin Stolze Larsen, Department of Hepatology, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark Hans-Jorgen Frederiksen, Michael Hojskov, Department of Anaesthesia, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark Supported by The NOVO Nordic Foundation, Savværksejer Jeppe & Ovita Mindelegat, Fabricant Vilhelm Pedersen & Wifes Mindelegat, A.P. Moller Scientific Foundation, the Danish Medical Association Research Fund and the Laerdal Foundation for Acute Medicine, and by an unrestricted grant from the Fresenius Medical Care GmBH Correspondence to: Fin Stolze Larsen, Department of Hepatology, Section A-2121, Copenhagen University Hospital Rigshospitalet, Blegdamsvej 9, DK-2100 Copenhagen O, Denmark. [email protected] Telephone: +45-35452358 Fax: +45-35452913 Received: September 21, 2007 Revised: December 14, 2007

Abstract AIM: To evaluate treatment safety and hemodynamic changes during a single 6-h treatment with the Prometheus™ liver assist system in a randomized, controlled study. METHODS: Twenty-four patients were randomized to either the study group or to one of two control groups: Fractionated Plasma Separation Adsorption and Dialysis, Prometheus™ system (Study group; n = 8); Molecular Adsorbent Recirculation System (MARS)™ (Control group 1, n = 8); or hemodialysis (Control group 2; n = 8). All patients included in the study had decompensated cirrhosis at the time of the inclusion into the study. Circulatory changes were monitored with a Swan-Ganz catheter and bilirubin and creatinine were monitored as measures of protein-bound and water-soluble toxins. RESULTS: Systemic hemodynamics did not differ between treatment and control groups apart from an increase in arterial pressure in the MARS group (P = 0.008). No adverse effects were observed in any of the groups. Creatinine levels significantly decreased in the MARS group (P = 0.03) and hemodialysis group (P = 0.04). Platelet count deceased in the Prometheus group (P = 0.04).

CONCLUSION: Extra-corporal liver support with Prometheus is proven to be safe in patients with endstage liver disease but does not exert the beneficial effects on arterial pressure as seen in the MARS group. © 2008 WJG . All rights reserved.

Key words: Extra-corporal liver therapy; Prometheus; Molecular Adsorbent Recirculation System; Systemic hemodynamics Peer reviewers: Vasiliy I Reshetnyak, MD, PhD, Professor,

Scientist Secretary of the Scientific Research Institute of General Reanimatology, 25-2, Petrovka str., 107031, Moscow, Russia; Curt Einarsson, Professor, Department of Medicine, Karolinska institute, Karolinska University Hospital Huddinge, Department of Gastroenterology and Hepatology, K 63, Huddinge SE-141 86, Sweden Dethloff T, Tofteng F, Frederiksen HJ, Hojskov M, Hansen BA, Larsen FS. Effect of Prometheus liver assist system on systemic hemodynamics in patients with cirrhosis: A randomized controlled trial. World J Gastroenterol 2008; 14(13): 2065-2071 Available from: URL: http://www.wjgnet.com/1007-9327/14/2065.asp DOI: http://dx.doi.org/10.3748/wjg.14.2065

INTRODUCTION The fact that not all patients with end-stage liver disease are suitable for liver transplantation, and the shortage of grafts enhance the need for supportive liver therapy either to secure time to stabilize hepatic functions or to enable bridging to liver transplantation. Indeed, extra-corporal liver support has turned out to be a valuable supplement to standard medical therapy (SMT)[1]. Especially, albumin dialysis improves not only the general condition, but also both cardiovascular and renal function [2-7] as well as the degree of hepatic encephalopathy [1,8,9] . The Fractionated Plasma Separation, Adsorption and Dialysis (Prometheus™) system and the Molecular Adsorbent Recirculation System (MARS™) both represent such treatment modalities. End-stage liver disease is often accompanied by a hyper-dynamic systemic circulation[10-13]. This circulatory

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change is caused by a variety of vasoactive factors, such as cytokines, prostacyclins, and nitric oxide[14-18]. Infection or bleeding, which are frequent complications in cirrhotic patients, increase the nitric oxide production and result in aggravation of the hyperdynamic circulation[19]. Studies comparing the hemodynamic alterations in stable patients with cirrhosis during extra-corporal intervention remain scarce though such knowledge might evidently improve patient safety and perhaps lessen reluctance towards the use of extra-corporal liver support at an early time in the treatment. This randomized, controlled study was designed to clarify the hemodynamic effects of intervention with Prometheus™, using MARS™ and hemodialysis as control groups, in patients with end-stage liver disease.

MATERIALS AND METHODS Subjects The clinical and paraclinical characteristics of the patients included in the study are listed in Table 1. The study was approved by the Ethics Committee of Copenhagen (jr. nr. KF 01-186/04) and the study protocol conformed to the ethical guidelines of the 1975 Declaration of Helsinki. Twenty-six patients (10 females and 16 males) were screened and 24 (9 females and 15 males) were enrolled in the study after obtaining written and oral consent from the patient or next-of-kin. All patients included in the study were under evaluation for liver transplantation and had a right-sided heart catheterization by a Swan-Ganz catheter. Former studies from our department have evaluated the effects of a 6-h albumin dialysis on patients with acuteon-chronic liver failure as well as patients with acute liver failure[20,21]. Consequently, the present study was designed accordingly with a 6-h treatment to match and complete the former studies. Patients were divided into three groups each receiving a 6-h extra-corporal treatment: Prometheus treatment with MARS and hemodialysis as control groups. The randomization was performed using sealed, opaque envelopes containing a computerized sequence code. Envelopes were drawn by a third person not involved in the study. Inclusion criteria were pre-existing liver disease with decompensated cirrhosis (verified by histological examination and/or CT/MRI scanning), ascites and a history of hepatic encephalopathy or repeated variceal bleeding. Exclusion criteria were uncontrolled systemic or intracranial bleeding, uncontrolled systemic infection, extrahepatic cholestasis, necrotic pancreatitis, cardiovascular failure necessitating > 0.05 µg/kg per minute of norepinephrine, and a history of albumin dialysis within the last 7 d before entering the study. Treatment All patients were treated in our liver ward but they were admitted to our liver intensive care unit for the duration of the extra-corporal treatment. Red blood cells, platelets and fresh frozen plasma were transfused according to the attending physician’s orders. Pre-treatment albumin levels were not corrected (Table 1). Measurements of pulmonary www.wjgnet.com

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artery core temperature, cardiac output (CO), pulmonary artery mean pressure (PAPM), pulmonary artery wedged pressure (PAWP) and central venous pressure (CVP) were obtained through thermo dilution technique using a four-lumen balloon-tipped catheter (Swan-Ganz; Baxter, Copenhagen, Denmark). Using ultrasound supervision, all patients were equipped with a double-lumen catheter in the femoral vein (24 cm, med. COMP, Harlysville PA, USA) as well as a Swan-Ganz catheter in the right internal jugular vein. Extra-corporal treatment: It was performed using the 4008 hemodialysis machine (Fresenius Medical Care Denmark A/S, Albertslund, Denmark) capable of performing both conventional hemodialysis as well as Prometheus treatments. The 4008 machine was also combined with a MARS monitor to perform MARS treatments. The thermostat of the machine was set to 36.5℃ to avoid cooling of the patients. Patients in all three groups were treated with identical blood and dialysate flow rates (225 mL/min and 500 mL/min, respectively). Anticoagulation: Citrate/calcium-anticoagulation was used for all patients using the 4008 hemodialysis machine’s built-in citrate/calcium algorithm. Calcium is automatically infused with a rate of 3.33 mmol citrate per litre of perfused blood. The citrate dose is calculated from the ionized calcium content in the patient’s venous blood. Venous blood samples were taken every 30 min and immediately analyzed in heparinized syringes (Radiometer, Copenhagen, Denmark). Blood tests before and after the treatment included creatinine, platelet count and bilirubin. The Prometheus system removes albumin bound toxins in the patient’s blood by combining fractionated plasma separation and adsorption (FPSA) with conventional dialysis. The patient’s blood first passes trough a plasma separator with a pore size of 250 kDa. The filtered plasma fraction then passes over two adsorption columns, a neutral resin and an anion exchange resin adsorber, before it is filtered back to the systemic circulation. The blood then passes through a high-flux dialyzer (F60S, Fresenius, Denmark). The flow rate in the plasma circuit was set to 300 mL/min according to the manufacturer’s recommendations. MARS is an extra-corporal high-flux hemofiltration that removes albumin-bound toxins from the blood over a specialized hybrid (albumin-impermeable) membrane into an albumin-enriched dialysate (500 mL of 200 g/L albumin). MARS combines a standard dialysis machine with a closed-loop albumin circuit, which is re-circulated by the MARS monitor (Gambro, Lyon, France). The albumin dialysate is passed through a conventional dialyzer and afterwards a charcoal and an anion-exchanger column. The flow rate in the closed albumin circuit was set to the maximum rate of 250 mL/min. Hemodialysis was performed using a high-flux dialyzer (F60S, Fresenius, Denmark). Measurements All measurements determined heart rate (HR), systolic/ diastolic and mean arterial blood pressure (MAP), stroke

Dethloff T et al . Hemodynamics during Prometheus treatment   

 

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Table 1 Pre-treatment clinical status of patients with liver failure No Randomization Sex Age Cause (yr)

MELD Child-Pugh Coma INR Bilirubin Albumin Creatinine Hg Platelet count 4 score score grade (μmol/L) (g/L) (μmol/L) (g/L) (× 10 /L)

6-mo outcome

1

Hemodialysis

M

51

Alcoholic cirrhosis

2 3 4 5 6 7

Prometheus Prometheus MARS MARS Hemodialysis Hemodialysis

M M M F F F

66 60 59 44 54 57

Hemochromatosis Alcoholic cirrhosis Alcoholic cirrhosis Alcoholic cirrhosis Alcoholic cirrhosis PBC

24 22 25 24 32

C/13 B/9 C/14 C/13 C/14

Ⅱ Ⅰ Ⅰ Ⅰ Ⅰ

2.5 1.5 2.7 2.2 2.7

7.1 2.8 8.0 11.9 41.0

23.6 39.8 25.6 17.8 19.3

0.7 2.1 0.6 0.8 0.7

6.2 5.2 5.4 6.0 6.5

105 126 22 148 66

Died OLT/survived Died Survived Died

8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24

Prometheus Prometheus Prometheus Prometheus MARS MARS MARS MARS Hemodialysis Hemodialysis Hemodialysis Prometheus Prometheus MARS MARS Hemodialysis Hemodialysis

M F F F F F M M M M M F M M M M M

39 25 59 49 43 45 59 63 54 67 61 63 57 65 57 59 52

Porphyria Alcoholic cirrhosis Autoimmune hepatitis Alcoholic cirrhosis Alcoholic cirrhosis Cholest. stor. dis. Autoimmune hepatitis Alcoholic cirrhosis Hepatitis C Alcoholic cirrhosis Alcoholic cirrhosis Unknown Alcoholic cirrhosis Alcoholic cirrhosis Hepatitis C Alcoholic cirrhosis Alcoholic cirrhosis

22 31 35 25 29 22 26 11 20 12 12 10 48 18 38 23 23

C/10 C/12 C/12 C/12 C/12 C/11 C/10 B/8 C/10 B/9 B/8 B/8 C/13 B/7 C/12 C/14 C/10

0 0 Ⅰ Ⅰ Ⅰ Ⅰ 0 0 Ⅰ Ⅰ Ⅰ Ⅰ Ⅱ 0 Ⅰ Ⅰ Ⅰ

1.4 2.3 1.8 2.4 1.8 2.2 1.7 1.2 1.9 1.4 1.2 1.3 6.0 1.3 2.2 2.4 1.5

22.8 26.9 15.6 9.6 35.8 5.0 35.1 1.9 1.1 1.6 1.2 1.1 36.6 1.6 36.8 5.5 4.0

31.8 20.0 34.0 29.2 25.0 29.3 31.3 31.7 35.2 30.4 35.1 40.8 28.5 39.0 32.1 24.6 41.8

0.4 1.3 3.3 0.7 1.3 1.1 0.9 0.8 1.8 0.7 1.4 0.8 2.3 2.0 2.5 0.4 0.5

8.0 6.2 6.2 7.1 6.3 5.2 8.8 7.0 7.5 6.6 8.3 8.0 6.0 7.2 5.8 5.6 6.8

70 109 71 63 123 57 31 65 87 91 140 146 74 99 37 46 221

OLT/survived OLT/survived Died Survived Survived OLT/survived Survived Survived Survived OLT/survived Died Survived Died Survived Died Died Survived

PBC: Primary biliary cirrhosis; Cholest. stor. dis: Cholesterol esther storage disease.

volume (SV), CO, CVP, PAMP, and PAWP. Baseline measurements were performed 30 min before starting the extra-corporal treatment. Throughout the 6-h treatment, hemodynamic measurements were performed and registered approximately every hour. Blood samples before and after the treatment included platelet count, international normalized ratio (INR), bilirubin, creatinine and alanine transaminase (ALT). During the treatment, we monitored the venous levels of ionized calcium as well as potassium and sodium, hemoglobin, glucose and magnesium. If necessary, substitution by continuous infusion was performed. Calculations were performed as follows: Cardiac index (CI) (L/min•m2) = CO divided by the body surface area. SV (mL/beat) = CO/HR; SVRI (DS/m2•cm5) = 80 × (MAP - CVP)/CI; PVRI (DS/m2•cm5) = 80 × (PAPM PAWP)/CI. Unless stated differently, data were expressed as mean ± SEM. Statistical analysis Comparison within a group was performed using the paired t-test or Wilcoxon’s rank sum test. For comparison between groups, the one-way-ANOVA or KruskalWallis rank sum test were applied. P values < 0.05 were considered statistically significant.

RESULTS Of the 24 patients included, 22 patients completed the 6-h treatment without complications. Patient number 1 and 7, both randomized to hemodialysis treatment, dropped out of the study due to repeated clotting of the dialysis filters. During the treatment, we recorded no serious adverse

events, i.e. no drop in MAP and no hemolysis or bleeding requiring therapeutic intervention. Patient number 15 from the MARS group experienced bleeding after removal of the dialysis catheter 10 h after termination of the treatment. None of the conscious patients complained of any discomfort that could be related to the extracorporal treatment. Nine patients were discharged 2 to 19 d after the treatment; 5 patients underwent orthotopic liver transplantation (OLT) and were alive 6 mo after the transplantation, whereas 11 patients died within 4 to 143 d after participating in the study (Table 1). There was no statistical correlation between the study group and the 6-mo outcome (P = 0.397). All hemodynamic measurements are listed in Table 2. A one-way analysis of variance between the three groups comparing baseline values of age, MELD score, INR, bilirubin, albumin, creatinine, hemoglobin, and platelets showed no significant differences. Pre-treatment MAP values were within the normal range (mean 76 mmHg; range 57-98 mmHg), while the other hemodynamic parameters were in accordance with the normal findings in end-stage liver disease: CO was elevated (mean 8.9 L/min; range 4.4-13.9 L/min), CI high (mean 4.6 L/min• m2; range 2.3-7.1 L/min•m2), and SVRI low (mean 1231 Ds*s/cm5•m2; range 139-3616). Only small hemodynamic changes from pre- to posttreatment in all three groups were noted. However, the MARS group showed, during the treatment, a significant increase in the systolic and diastolic blood pressure of 10.5% and 15.2%, respectively. Heart rate, CI and SV remained constant. Paraclinical values of bilirubin, creatinine, and platelet count measured before and after the treatment were as www.wjgnet.com

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Table 2 Hemodynamic variables (mean ± SEM) Hemodialysis Variables CO HR MAP PAPM PAWP CVPM SV CI SVRI PVRI

Pre-treatment L/min beats/min mmHg mmHg mmHg mmHg mL L/min•m2 DS*m2•cm5 DS*m2•cm5

8.26 ± 1.07 74.5 ± 5.89 75.17 ± 6.99 14.17 ± 2.98 7.17 ± 1.56 6.83 ± 2.8 107.78 ± 11.86 4.12 ± 0.5 1454.17 ± 478.92 138.5 ± 32.22

Post-treatment 8.37 ± 1.01 78.5 ± 8.45 76.5 ± 5.13 12.83 ± 2.23 5.83 ± 1.74 3.83 ± 1.38 112.18 ± 10.95 4.26 ± 0.49 1541.17 ± 353.82 119.67 ± 29.05

MARS

Prometheus

Pre-treatment

Post-treatment

Pre-treatment

Post-treatment

8.66 ± 0.87 79.5 ± 6.06 68.88 ± 3.82 18.5 ± 2.25 11.38 ± 1.61 9.5 ± 2.15 106.95 ± 8.78 4.71 ± 0.56 1223.38 ± 344.76 121.5 ± 30.65

8.74 ± 0.88 81.13 ± 6.53 78.13 ± 4.87 19.88 ± 2.09 13 ± 2.1 8.63 ± 1.69 107.56 ± 7.28 4.71 ± 0.51 1372.25 ± 271.5 100.05 ± 28.13

9.52 ± 1.26 82.25 ± 6.06 74 ± 3.52 24.88 ± 5.71 13.63 ± 2.24 13.25 ± 1.81 116.11 ± 13.54 4.9 ± 0.56 1073.5 ± 126.69 236 ± 141.71

9.36 ± 1.4 81.63 ± 6.88 74.38 ± 5 23.25 ± 5.32 14.63 ± 1.86 12 ± 2.05 110.91 ± 13.49 4.9 ± 0.67 1177.38 ± 194.2 210.63 ± 133.63

CO: Cardiac output; HR: Heart rate; MAP: Mean arterial blood pressure; PAPM: Pulmonary artery pressure mean; PAWP: Pulmonary artery wedged pressure; CVPM: Central venous pressure middle; SV: Stroke volume; CI: Cardiac index; SVRI: Systemic vascular resistance index; PVRI: Pulmonary vascular resistance index.

2.0

160

P = 0.07

1.8

100

1.2 1.0

P = 0.04

0.8 0.6 0.4

P = 0.09

120

1.4 Platelet count

Plasma creatinine

1.6

P = 0.04 P = 0.10

  80   60   40   20

0.2

   0

0.0

t re ost pre pre s pos ost Sp S p heus sis sp u R i y R l e s A A a y h t l t M M di me dia me mo Pro mo Pro He He

Figure 1 Plasma creatinine pre- and post-treatment using hemodialysis, MARS and Prometheus. All values are given as mean ± SEM.

25

P = 0.13 P = 0.08

20

P = 0.42 Plasma bilirubin

140

P = 0.03

15

10

5

0

e st st pre pre post S pr po po is us us RS he lys lysis MAR e t A a i h e M t d ia m od me mo Pro He Hem Pro

Figure 2 Plasma bilirubin pre- and post-treatment using hemodialysis, MARS and Prometheus. All values are given as mean ± SEM.

follows: (1) Prometheus group: pre-treatment values: bilirubin (mean 15.3 × 10-2 g/L; SEM 4.4 × 10-2 g/L), creatinine (mean 1.44 × 10-2 g/L; SEM 0.35 × 10-2 g/L) and platelet count (mean 96 × 109/L; SEM 11 × 109/L);

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t t t re pre pre pos pos pos is p us RS eus RS lys e sis A h A a y h t i l t M M d me dia me mo Pro mo Pro He He

Figure 3 Platelet count pre- and post-treatment using hemodialysis, MARS and Prometheus. All values are given as mean ± SEM.

post-treatment values: bilirubin (mean 12.6 × 10-2 g/L; SEM 3.6 × 10-2 g/L), creatinine (mean 1.09 × 10-2 g/L; SEM 0.39 × 10-2 g/L) and platelet count (mean 76 × 109/L; SEM 11 × 109/L); (2) MARS group: pre-treatment values: bilirubin (mean 17.0 × 10-2 g/L; SEM 5.6 × 10-2 g/L), creatinine (mean 1.2 × 10-2 g/L; SEM 0.24 × 10-2 g/L) and platelet count (mean 73 × 109/L; SEM 16 × 109/L); post-treatment values: bilirubin (mean 13.4 × 10-2 g/L; SEM 3.8 × 10-2 g/L), creatinine (mean 0.86 × 10-2 g/L; SEM 0.17 × 10-2 g/L) (P = 0.03), and platelet count (mean 63 × 109/L; SEM 14 × 109/L). (3) Hemodialysis group: pre-treatment values: bilirubin (mean 9.1 × 10-2 g/L; SEM 6.4 × 10-2 g/L), creatinine (mean 0.64 × 10-2 g/L; SEM 0.16 × 10-2 g/L) and platelet count (mean 109 × 109/L; SEM 25 × 109/L); post-treatment values: bilirubin (mean 9.9 × 10-2 g/L; SEM 6.8 × 10-2 g/L), creatinine (mean 0.49 × 10-2 g/L; SEM 0.06 × 10-2 g/L) and platelet count (mean 94 × 109/L; SEM 28 × 109/L). The results of the paired t-test comparing pre- and posttreatment values in each group are shown in Figures 1-3. Anticoagulation was performed using citrate/calcium infusion. In all patients, we followed the 4008 machine’s built-in algorithm, which calculates the citrate infusion rate

Dethloff T et al . Hemodynamics during Prometheus treatment   

from the patient’s ionized calcium. Both during and after the treatment, none of the patients required calcium or citrate supplements and no correction of the pH was necessary.

DISCUSSION In this study, we compared the systemic circulatory changes during a single treatment of Prometheus with MARS and hemodialysis as control groups in patients with end-stage liver disease. The hemodynamic differences between the three groups showed insignificant differences besides a rise in MAP in the MARS treated group. None of the patients experienced any serious adverse events. Though we have some experience both about the clinical and the hemodynamic effects of the available extracorporal treatments, i.e. MARS and Prometheus, there is a growing recognition that patients may benefit from the initiation of extra-corporal liver support before treatment for multi-organ failure in the ICU setting is needed[22]. We have been using both MARS and Prometheus for several years in our liver failure unit. Former studies from our unit have determined the hemodynamic changes during a single 6-h MARS treatment both in patients with acute-on-chronic liver failure (AoCLF) as well as hyper acute liver failure[20,21]. None of the patients in this study had an acute exacerbation at the time of randomization. Thus our patients represent a group that differs from AoCLF patients but can be characterized as having a chronic liver failure with decompensation. The Child-Pugh score for our patients supports this view with 6 group B and 18 group C patients. Arterial hypotension is a well-described adverse effect induced by hemodialysis. In the light of this fact, it is interesting that hemodynamic studies have indicated that the MARS system exerts a beneficial influence on CO, SV, SVRI[20-23] and MAP[20-26]. The present study cannot demonstrate such beneficial changes, neither in the Prometheus nor in the hemodialysis group; however, there was a significant increase in MAP in the MARS group. The overall lack of significant hemodynamic changes (especially on CO, SV, and SVRI as would have been expected in the MARS group) could be attributed to the fact that the patients in the present study were treated early before deterioration of their chronic liver disease: the plasma bilirubin in our patient group was 42% of the value seen in the study by Schmidt et al[20] and 48% of the mean value of all three groups in the study by Laleman et al[2]. Both the Prometheus and MARS systems are capable of removing both albumin-bound as well as water-soluble substances. As shown in Figure 2, both albumin dialysis systems, as expected, decreased the plasma bilirubin level but the removal did not reach statistical significance. This was most likely due to heterogeneity regarding pretreatment bilirubin levels among our group of patients and a low pre-treatment bilirubin level. In addition, the absolute amount of bilirubin eliminated during a treatment depends on blood concentration: higher pre-treatment values will yield higher clearances. The toxin concentration in the blood is generally thought to cause the hyperdynamic circulation seen in liver failure. If we consider the bilirubin level merely as an

 

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indicator for the general level of albumin-bound toxins in the blood, it could also explain why potential positive hemodynamic effects could not be demonstrated. In our study, the pre-treatment bilirubin level was low and the toxin level would accordingly also be low. Therefore, no significant removal of bilirubin/toxins could be achieved and possible beneficial hemodynamic changes would thus become difficult to demonstrate. No significant removal of creatinine could be demonstrated in the Prometheus group (Figure 1). This is most likely due to a low pre-treatment concentration and a high standard deviation as mentioned above. As expected, the survival of the patients was not statistically correlated to the randomized study group. Regarding the safety of the treatment, none of the patients experienced arterial hypotension requiring cessation of the treatment. As a standard routine, the initial blood flow on the 4008 machine was set to 120 mL/min for the first 10 min and was then increased to 225 over the next 5-10 min. As mentioned, one episode of post-treatment bleeding occurred. The event was related to the removal of the dialysis catheter and occurred despite vessel compression for 15 min and a 30-min rest after dialysis-catheter removal, all according to standard guidelines. Both the study group and the two control groups exhibited a drop in platelet count, yet, it was only statistically significant in the Prometheus group (Figure 3). Low platelet count is well known in cirrhotic patients. In addition, thrombelastography (TEG) often discloses dysfunction of the platelets. Consequently, monitoring the platelet count closely before, during, and after an extracorporal treatment, especially when using Prometheus treatment, seems advisable as well as substitution, if necessary. Two patients in the hemodialysis group dropped out of the study due to repeated clotting. Patient number 7 showed obvious signs of hyper-coagulation: apart from clotting three hemodialysis filters, the dialysis catheter also clotted during a blood pump stop of 3-5 min. The clotting problems in the hemodialysis group opposed our former clinical experience, which had pointed at patients treated with the Prometheus system in combination with citrate anticoagulation as being most prone to clotting problems. The paraclinical data from patient number 1 and 7 did not account for the repeated clotting. Apart from the reported data, the activated prothrombin time values were within normal range for both patients (data not shown). In conclusion, the decision to use extra-corporal liver support in the treatment of patients with end-stage liver disease hinges on many factors, with safety considerations as a major concern. The choice of treatment will depend on the risk of adverse events, and possible positive or negative hemodynamic influences between the available treatment modalities. Our study adds to the clarification of these considerations in showing that the Prometheus system does not aggravate the systemic hemodynamics; however, in our study, Prometheus does not exert an equally beneficial influence on MAP as seen during MARS treatment. We conclude that an intervention using extracorporal liver support with albumin-dialysis should not be withheld merely because of safety considerations. www.wjgnet.com

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COMMENTS Background

Patients with end-stage liver disease often need supportive liver therapy either to secure time to stabilize hepatic functions or to enable bridging to liver transplantation. Albumin dialysis improves not only the general condition, but also both cardiovascular and renal function as well as the degree of hepatic encephalopathy. Studies comparing the hemodynamic alterations in stable patients with cirrhosis during extra-corporal intervention remain scarce though such knowledge might evidently improve patient safety and perhaps lessen reluctance towards the use of extra-corporal liver support at an early time in the treatment. This randomized, controlled study was designed to clarify the hemodynamic profile of intervention with Prometheus™, using MARS™ and hemodialysis as control groups, in patients with end-stage liver disease.

5

6

7 8

Research frontiers

This paper is related to the scientific efforts to develop clinically valuable artificial liver assist devices to support patients with fulminant liver failure.

9

Innovations and breakthroughs

http://www.ncbi.nlm.nih.gov/sites/entrez. See under “liver assist devices“ or Sen S, Williams R, Jalan R. Emerging indications ofr albumin dialysis. Am J Gastroenterol 2005; 100: 468-475.

Applications

The decision to use extra-corporal liver support in the treatment of patients with end-stage liver disease hinges on many factors, with safety considerations as a major concern. The choice of treatment will depend on the risk of adverse events, and possible positive or negative hemodynamic influences between the available treatment modalities. Our study adds to the clarification of these considerations in showing that the Prometheus system does not aggravate the systemic hemodynamics; however, in our study, Prometheus does not exert an equally beneficial influence on arterial pressure as seen during MARS treatment. We conclude that an intervention using extra-corporal liver support with albumindialysis should not be withheld merely because of safety considerations.

10

11

12

13

Terminology

Acute-on-chronic liver failure is defined as an acute deterioration in liver function in a patient with cirrhosis that results in dysfunction of other organs, such as the brain or the kidneys.

Peer review

This is an interesting and well performed study. The abstract gives a clear delineation of the research background, aim, materials and methods, results and conclusion. The design of the study is rational and reliable. The work is of the practical importance.

REFERENCES 1

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Hassanein TI, Tofteng F, Brown RS Jr, McGuire B, Lynch P, Mehta R, Larsen FS, Gornbein J, Stange J, Blei AT. Randomized controlled study of extracorporeal albumin dialysis for hepatic encephalopathy in advanced cirrhosis. Hepatology 2007; 46: 1853-1862 Laleman W, Wilmer A, Evenepoel P, Elst IV, Zeegers M, Zaman Z, Verslype C, Fevery J, Nevens F. Effect of the molecular adsorbent recirculating system and Prometheus devices on systemic haemodynamics and vasoactive agents in patients with acute-on-chronic alcoholic liver failure. Crit Care 2006; 10: R108 Stefoni S, Coli L, Bolondi L, Donati G, Ruggeri G, Feliciangeli G, Piscaglia F, Silvagni E, Sirri M, Donati G, Baraldi O, Soverini ML, Cianciolo G, Boni P, Patrono D, Ramazzotti E, Motta R, Roda A, Simoni P, Magliulo M, Borgnino LC, Ricci D, Mezzopane D, Cappuccilli ML. Molecular adsorbent recirculating system (MARS) application in liver failure: clinical and hemodepurative results in 22 patients. Int J Artif Organs 2006; 29: 207-218 Jalan R, Sen S, Steiner C, Kapoor D, Alisa A, Williams R. Extracorporeal liver support with molecular adsorbents

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recirculating system in patients with severe acute alcoholic hepatitis. J Hepatol 2003; 38: 24-31 Lai WK, Haydon G, Mutimer D, Murphy N. The effect of molecular adsorbent recirculating system on pathophysiological parameters in patients with acute liver failure. Intensive Care Med 2005; 31: 1544-1549 Sorkine P, Ben Abraham R, Szold O, Biderman P, Kidron A, Merchav H, Brill S, Oren R. Role of the molecular adsorbent recycling system (MARS) in the treatment of patients with acute exacerbation of chronic liver failure. Crit Care Med 2001; 29: 1332-1336 Sen S, Williams R, Jalan R. Emerging indications for albumin dialysis. Am J Gastroenterol 2005; 100: 468-475 Camus C, Lavoue S, Gacouin A, Le Tulzo Y, Lorho R, Boudjema K, Jacquelinet C, Thomas R. Molecular adsorbent recirculating system dialysis in patients with acute liver failure who are assessed for liver transplantation. Intensive Care Med 2006; 32: 1817-1825 Sen S, Davies NA, Mookerjee RP, Cheshire LM, Hodges SJ, Williams R, Jalan R. Pathophysiological effects of albumin dialysis in acute-on-chronic liver failure: a randomized controlled study. Liver Transpl 2004; 10: 1109-1119 Catalina MV, Barrio J, Anaya F, Salcedo M, Rincon D, Clemente G, Banares R. Hepatic and systemic haemodynamic changes after MARS in patients with acute on chronic liver failure. Liver Int 2003; 23 Suppl 3: 39-43 Schmidt LE, Svendsen LB, Sorensen VR, Hansen BA, Larsen FS. Cerebral blood flow velocity increases during a single treatment with the molecular adsorbents recirculating system in patients with acute on chronic liver failure. Liver Transpl 2001; 7: 709-712 Clemmesen JO, Larsen FS, Ejlersen E, Schiodt FV, Ott P, Hansen BA. Haemodynamic changes after high-volume plasmapheresis in patients with chronic and acute liver failure. Eur J Gastroenterol Hepatol 1997; 9: 55-60 Larsen FS, Ejlersen E, Hansen BA, Mogensen T, Tygstrup N, Secher NH. Systemic vascular resistance during high-volume plasmapheresis in patients with fulminant hepatic failure: relationship with oxygen consumption. Eur J Gastroenterol Hepatol 1995; 7: 887-892 Liu H, Gaskari SA, Lee SS. Cardiac and vascular changes in cirrhosis: pathogenic mechanisms. World J Gastroenterol 2006; 12: 837-842 Wang JJ, Gao GW, Gao RZ, Liu CA, Ding X, Yao ZX. Effects of tumor necrosis factor, endothelin and nitric oxide on hyperdynamic circulation of rats with acute and chronic portal hypertension. World J Gastroenterol 2004; 10: 689-693 Guarner C, Soriano G. Prostaglandin and portal hypertension. Prostaglandins Leukot Essent Fatty Acids 1993; 48: 203-206 Jalan R, Hayes PC. Hepatic encephalopathy and ascites. Lancet 1997; 350: 1309-1315 Wong F, Bernardi M, Balk R, Christman B, Moreau R, GarciaTsao G, Patch D, Soriano G, Hoefs J, Navasa M. Sepsis in cirrhosis: report on the 7th meeting of the International Ascites Club. Gut 2005; 54: 718-725 Mohammed NA, Abd El-Aleem S, Appleton I, Maklouf MM, Said M, McMahon RF. Expression of nitric oxide synthase isoforms in human liver cirrhosis. J Pathol 2003; 200: 647-655 Schmidt LE, Sorensen VR, Svendsen LB, Hansen BA, Larsen FS. Hemodynamic changes during a single treatment with the molecular adsorbents recirculating system in patients with acute-on-chronic liver failure. Liver Transpl 2001; 7: 1034-1039 Schmidt LE, Wang LP, Hansen BA, Larsen FS. Systemic hemodynamic effects of treatment with the molecular adsorbents recirculating system in patients with hyperacute liver failure: a prospective controlled trial. Liver Transpl 2003; 9: 290-297 Chiu A, Fan ST. MARS in the treatment of liver failure: controversies and evidence. Int J Artif Organs 2006; 29: 660-667 Lai WK, Haydon G, Mutimer D, Murphy N. The effect of molecular adsorbent recirculating system on pathophysiological parameters in patients with acute liver failure. Intensive Care Med 2005; 31: 1544-1549 Sorkine P, Ben Abraham R, Szold O, Biderman P, Kidron A,

Dethloff T et al . Hemodynamics during Prometheus treatment   

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Merchav H, Brill S, Oren R. Role of the molecular adsorbent recycling system (MARS) in the treatment of patients with acute exacerbation of chronic liver failure. Crit Care Med 2001; 29: 1332-1336 Jalan R, Sen S, Steiner C, Kapoor D, Alisa A, Williams R. Extracorporeal liver support with molecular adsorbents recirculating system in patients with severe acute alcoholic hepatitis. J Hepatol 2003; 38: 24-31

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Stefoni S, Coli L, Bolondi L, Donati G, Ruggeri G, Feliciangeli G, Piscaglia F, Silvagni E, Sirri M, Donati G, Baraldi O, Soverini ML, Cianciolo G, Boni P, Patrono D, Ramazzotti E, Motta R, Roda A, Simoni P, Magliulo M, Borgnino LC, Ricci D, Mezzopane D, Cappuccilli ML. Molecular adsorbent recirculating system (MARS) application in liver failure: clinical and hemodepurative results in 22 patients. Int J Artif Organs 2006; 29: 207-218 S- Editor Li DL L- Editor Kumar M E- Editor Yin DH

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World J Gastroenterol 2008 April 7; 14(13): 2072-2079 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

RAPID COMMUNICATION

Ultrasonography in differentiation between chronic viral hepatitis and compensated early stage cirrhosis Panagiotis Iliopoulos, Marianna Vlychou, Chrisoula Karatza, Spyros D Yarmenitis, Maria Repanti, Ioannis Tsamis, Kostantinos Tepetes Panagiotis Iliopoulos, Ioannis Tsamis, Department of Radiology, “Agios Andreas” General Hospital, Patras, Greece Marianna Vlychou, Department of Radiology, University of Thessaly, Greece Chrisoula Karatza, Department of Hepatology-Gastroenterology, University of Patras, Greece Spyros D Yarmenitis, Department of Radiology, University of Crete, Greece Maria Repanti, Department of Pathology, “Agios Andreas” General Hospital, Patras, Greece Kostantinos Tepetes, Department of Surgery, University of Thessaly, Greece Correspondence to: Panagiotis Iliopoulos, Department of Radiology, “Agios Andreas” General Hospital, Diagora 24A, Patras 26441, Greece. [email protected] Telephone: +30-2610-461977 Fax: +30-2610-461977  Revised: October 31, 2007 Received: July 17, 2007

Abstract AIM: To assess the value of gray scale (GS) and colour Doppler ultrasonography (CDU) in differentiating the progression of chronic viral hepatitis (CVH) and compensated liver cirrhosis (CIR). METHODS: Seventy-two patients and 32 normal individuals who were used as controls were studied. Forty-four patients suffered from CVH and 28 from CIR. All patients were underwent to liver biopsy. Multiple qualitative and quantitative variables were studied in liver, portal vein (PV), hepatic artery (HA) and spleen with GS and CDU. On the basis of the obtained CDU data, several known indexes were calculated. In addition, alternative indices [PV diameter (D)/time average mean velocity (VTAM), HA/PV VTAM ratio] were calculated and studied. RESULTS: ROC analysis showed that PV congestion index, PV D/VTAM and HA/PV VTAM indices had the best sensitivity and specificity in discriminating CVH from CIR. Stepwise discriminant analysis showed that 88.9% of the originally grouped cases could be correctly classified by the three qualitative and four quantitative variables selected as statistically significant predictors. Among the CVH patients who underwent to biopsy, statistically significant changes were found in those at fibrosis stage 5 compared to fibrosis stages 1-4. CONCLUSION: Simple GS and CDU parameters discriwww.wjgnet.com

minate CVH from CIR. The alternative Doppler indexes can accurately differentiate chronic virus hepatitis from cirrhosis. These indexes can be used in monitoring chronic virus hepatitis and avoiding unnecessary biopsies. © 2008 WJG . All rights reserved.

Key words: Liver cirrhosis; Virus hepatitis; Portal hypertension; Doppler ultrasonography Peer reviewer: Sri Prakash Misra, Professor, Gastroenterology, Moti Lal Nehru Medical College, Allahabad 211001, India

Iliopoulos P, Vlychou M, Karatza C, Yarmenitis SD, Repanti M, Tsamis I, Tepetes K. Ultrasonography in differentiation between chronic viral hepatitis and compensated early stage cirrhosis. World J Gastroenterol 2008; 14(13): 2072-2079 Available from: URL: http://www.wjgnet.com/1007-9327/14/2072.asp DOI: http://dx.doi.org/10.3748/wjg.14.2072

INTRODUCTION Chronic viral hepatitis, mainly caused by hepatitis virus B or C, results in liver parenchyma damage and inflammation and may lead to fibrosis, cirrhosis and/or hepatocellular carcinoma[1-3]. Cirrhosis often occurs as an indolent disease and a lot of patients remain asymptomatic[4,5] until the occurrence of decompensation, and are characterised by portal hypertension, variceal bleeding, ascites and hepatic encephalopathy. Liver biopsy is the gold standard for diagnosis and determination of the fibrosis and necroinflammatory changes in chronic viral hepatitis and cirrhosis. However, the use of biopsy in clinical practice has some limitations related to sample errors with an estimated false negative percentage of 24% morbidity and mortality in series of blind biopsies[6] and complications[7]. The non-invasive assessment of chronic liver disease has been attempted by various research groups using either clinical signs[8,9], gray scale[10-15] and colour Doppler ultrasound (CDU) signs and indexes[16-32], or biochemical parameters in the blood[9]. The use of CDU in diagnosis and staging of chronic viral liver disease has been based on the hypothesis that alteration in liver haemodynamics due to chronic inflammatory changes may indirectly reflect histological alterations. Therefore, positive correlation studies have usually referred to the velocity ratio of hepatic

Iliopoulos P et al . Ultrasonograpgy in CVH and CIR

artery to portal vein or the resistive index in hepatic artery. However, the role of CDU remains controversial regarding the reproducibility[33-36] and the statistical significance[37,38] of the measurements in hepatic fibrosis and cirrhosis. The aim of the present study was to determine the alterations in liver haemodynamics by using the Doppler indexes and measurements of spleen size that may lead to differentiation between chronic viral liver disease and compensated cirrhosis in a group of patients with a welldelineated histological profile. Furthermore, an effort was made to isolate predictive factors for discrimination between patients with chronic viral hepatitis and cirrhosis.

MATERIALS AND METHODS Patient population Seventy-two patients were enrolled in this controlled prospective study and divided into chronic viral hepatitis (CVH) group and cirrhotic (CIR) group. The CVH group included 44 patients (mean age 53 ± 12 years, 29 males and 18 females) with chronic viral hepatitis. Of them, 20 were positive for hepatitis B surface antigen and 24 were positive for hepatitis C serum markers. All patients underwent needle biopsy at the time of study. The CIR group consisted of 28 patients suffering from compensated early stage cirrhosis (Child-Pugh A score, mean age 63 ± 9 years, 16 males and 13 females) due to viral hepatitis B or C. All cirrhotic patients had previous needle biopsy that confirmed their disease and endoscopic investigation of the upper GI tract. All patients included in the study gave their written informed consent and had no known liver tumour or decompensated liver disease. The study was approved by the ethics committee of our institution. Thirty-two healthy individuals (mean age 50 ± 15 years, 18 males and 14 females) served as controls. They were chosen from healthy volunteers with normal blood profile without evidence of liver disease. Volunteers with complex anatomy related to the hepatic artery were excluded from the study. The alcohol consumption was no more than 28 units a week (one unit = 8 g) for each individual in the control group. None of them had a history of cardiac or liver disease and risk factors for viral hepatitis, or was receiving therapy known to alter liver haemodynamics. CDU technique and indexes All sonographic scans were perfor med by a single experienced radiologist (first author), who was unaware of the clinical and laboratory data. All asymptomatic patients and healthy adults were fasted overnight before the examination. Patients did not take drugs that could affect their portal or systemic haemodynamics twenty-four hours prior to examination. All scans were performed with the individuals lying supine using the same sonography system (ATL, HDI 3500) with a curvilinear 2.5-5 MHz transducer. The machine was supported with the proper software for direct and automatic calculation of the haemodynamic parameters based on the spectral Doppler waveform. The examination was started with the observation in gray-

  

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scale scanning of the liver size (normal or enlarged if the midclavicular longitudinal diameter of the organ was greater than 12.6 cm[39], taking the value 1 for enlarged and 0 for normal), contour (nodular yes/no, taking the value 1 for yes and 0 for no) and parenchyma (homogeneity diffuse yes/no, and echocenic yes/no, taking the value 1 for yes and 0 for no). Subsequently, the examination proceeded to study CDU and a transverse section was obtained at the epigastrium to locate the proper hepatic artery in its longitudinal axis. The same method was used at the midlevel of the portal vein trunk to calculate venous indices, since no aberrant anatomy was present in the subjects participating in this study. To decrease the effect of respiration on the portal blood flow, all measurements were obtained during a short time breath-holding to avoid deep respiration. An occasional problem of overlying bowel gas was handled either by extending the scanning time or by setting a new appointment on the following day. For quantitative flow measurements, the position of the scanner was optimised until a Doppler angle of less than 60° was achieved. Haemodynamic parameters were calculated over four cardiac cycles. The sample volume size was always equal to the lumen diameter of blood vessels. The following portal vein (PV) variables were measured: diameter (D) in cm, cross-sectional area (AR) in cm2, time-averaged maximum velocity (VMAX) in cm/s, time-averaged mean velocity (VTAM) in cm/s, blood flow volume (BF) in mL/min and the congestion index which was calculated as the ratio between cross-sectional area and time-averaged mean velocity (CI = AR/V TAM) in cm*s[11]. The time-averaged mean portal venous velocity was determined electronically using the software package provided with the ultrasound machine. Hepatic artery (HA) measurements included: diameter (D) in cm, cross-sectional area (AR) in cm2, time-averaged mean velocity (VTAM) in cm/s and blood flow volume (BF) in mL/min. Resistance index (RI) of HA (percentage) was the ratio of 100 × the difference of peak systolic velocity minus end diastolic velocity to peak systolic velocity, automatically given by machine’s software. In addition, Doppler perfusion index (DPI)[22] was calculated according to the formula: DPI = BFHA/(BFHA + BFPV). We also evaluated two alternative indexes for liver haemodynamics: the ratio of portal vein diameter to VTAM (PV r1 = D/VTAM) in cm and the artery to portal vein ratio (A/P), which was calculated by the following formula: Time-averaged HA mean velocity (VTAM)/time-averaged PV mean velocity (VTAM). Two consecutive measurements of the anatomic and Doppler parameters were made in each blood vessel and the average value was taken for statistical analysis. The spleen size was estimated by measuring the maximum craniocaudal and transverse diameters[40,41]. Liver biopsies Liver biopsies were fixed in formalin and embedded in paraffin. Individual histological sections were prepared and stained using standard procedures. All patients were classified on the basis of the histologic activity index according to Ishak et al[42] in six fibrosis stages (F1-F6) with

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the sixth (F6) to be cirrhosis stage and necroinflammatory score varying from 0 to 18 in each stage. Statistical analysis The quantitative variables (predictors) were compared by t-test and ANOVA between CVH and CIR patients and between controls. Quantitative variables between CVH patients at fibrosis stages lower than 5 and between patients with CVH at the fifth fibrosis stage were compared using Wilcoxon rank sum test since few observations were carried out in the second group and therefore parametric assumption of the t-test was violated. P < 0.05 was considered statistically significant. Analysis of variance between the two consecutive Doppler measurements showed a very high reproducibility. The predictive value for each of the predictors was evaluated by the area under the receiver operating characteristic (ROC) curves. Accuracy was calculated for the best cut-off value (BCV) of the current data set, defined as the highest sum of sensitivity and specificity. Stepwise discriminant analysis was performed to predict group membership from the set of predictors by the classification functions. All statistical analyses were performed using the SPSS program (version 13).

RESULTS According to the histological findings (Table 1), 9 CVH patients were at F1 stage with necroinflammatory score (NI) ranging from 2 to 4, 9 patients at F2 stage with NI ranging from 3 to 6, 9 patients at F3 stage with NI ranging from 3 to 8, 7 patients at F4 stage with NI ranging 5 to 8 and 10 patients at F5 stage with NI ranging 3 to 9. All the 28 cirrhotic patients were at F6 fibrosis stage. No change was observed endoscopically in 8 cirrhotic patients, while first degree varices were found in 16 patients, portal gastropathy in 3 patients, first degree varices and portal gastropathy in 1 patient, respectively. Ultrasonography CVH group vs control group: There was a significant decrease in the portal vein mean value related to time average maximum velocity (V TAM) and diameter (D) to VTAM ratio between CVH and control groups (P = 0.03 and 0.037, respectively). In addition, there was a significant increase in the spleen volume between the two groups (P = 0.011). The other haemodynamic parameters and indexes did no show any statistical significance (Table 2). CIR group vs CVH group: There was a significant decrease related to the mean value of portal vein blood flow velocities (V MAX and V TAM) and blood flow (BF) between the CIR and CVH groups (P < 0.00007, P < 0.00002, P < 0.005 respectively, Table 1). In addition, there was a significant increase in BF of the hepatic artery and the spleen volume (P < 0.013, P > 0.002, Table 2). According to the qualitative data, liver in early stage cirrhotic patients had nodular surface, diffuse parenchymal echogenicity and was larger than that in CVH patients (Table 1). Descriptive statistics and comparative data on qualitative,

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Table 1 Liver qualitative parameters in controls, CVH and CIR groups Chronic viral hepatitis group (CVH) Variables Size Contour Echogenic Homogeneity

Enlarged Nodular Raised Diffuse

Cirrhosis group (CIR)

n

n

10 6 11 11

12 13 1 18

quantitative anatomic and haemodynamic variables as well as the calculated indexes in portal vein, hepatic artery and spleen are presented in Table 2. The P values for the quantitative variables between the two groups are listed in Table 2. In comparison with the CVH patients, the mean values of portal vein congestion index, diameter to time average mean velocity ratio, Doppler perfusion index (DPI) as well as hepatic artery RI and HA/PV time average mean velocity ratio were all significantly increased in the earlystage cirrhotic patients (P < 0.01, Table 2). CVH patients at F5 stage vs other fibrosis stages (F1-F4): Wilcoxon’s test between CVH patients at F5 stage to F1-F4 showed a significant increase in portal vein congestion index (CI) (P = 0.041). In addition, there was a marginally significant increase in diameter, cross sectional area (P = 0.051) and D/VTAM ratio (P = 0.055) in CVH patients (Table 3). Portal Vein VMAX and VTAM values were marginally decreased (P = 0.056 and 0.08 respectively) while all other variables were not significantly different (Table 3). CIR group and F5 CVH group vs other CVH groups (F1-F4): A significant increase in portal vein’s diameter (P < 0.05) and cross section area (P < 0.05) was observed, while PV VMAX and PV VTAM were significantly decreased (P < 0.001) in cirrhotic patients and CVH patients (incomplete cirrhosis) at F1-F4. In addition, a significant increase was observed in PV CI and PV D/V TAM ratio (P < 0.01) as well as in DPI (P < 0.05) and HA/PV VTAM ratio between the two groups of patients (P < 0.001) (Table 2). Spleen volume was also increased (P < 0.05, Table 3). ROC and stepwise discriminant analysis of CIR group vs CVH group: Portal vein D/VTAM ratio, CI and HA/PV VTAM ratio had the same sensitivity of 85.71% and specificities of 59.09%-68.18% respectively, between CIR and CVH patients. Doppler perfusion index (best cutoff value of 0.29) had a very good specificity (90.91%) but a low sensitivity (42.86%) between the two groups. The area under the cur ve (AUC), comparing CVH group with CIR group, for each variable as measured by receiver operating characteristic curve (ROC) analysis is presented in Table 4. PV CI, D/VTAM ratio and HA/PV VTAM ratio under AUC curves are shown in Figure 1 A-C. The best cut-off value (BCV) for each statistically significant quantitative variable defined as the highest sum of sensitivity and specificity is summarized in Table 5.

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Table 2 Quantitative and haemodynamic parameters in controls, CVH and CIR groupsgroups Quantitative variables Portal vein D (cm) AR (cm2) VMAX (cm/s) VTAM (cm/s) BF Hepatic artery AR (cm2) VTAM (cm/s) BF (mL/min) Indexes PV D/VTAM [cm/(cm*s)] PV CI (cm*s) HA RI Total BF (mL/min) DPI HA/PV VTAM Spleen volume (cm3)

t-test a vs b

CIR (c)

ANOVA a/b/c

b vs c

Controls (a)

CVH (b)

mean ± SD 1.14 ± 0.12 1.02 ± 0.20 41.56 ± 9.30 22.88 ± 5.69 1369.76 ± 349.41

mean ± SD 1.14 ± 0.17 1.03 ± 0.32 36.27 ± 9.40 20.64 ± 5.92 1238.06 ± 392.03

mean ± SD 1.17 ± 0.19 1.11 ± 0.37 27.60 ± 6.75 14.80 ± 3.82 980.06 ± 319.58

0.88 0.81 0.03 0.14 0.17

0.16 ± 0.05 29.62 ± 10.55 281.07 ± 125.79

0.16 ± 0.05 29.39 ± 12.70 288.79 ± 163.30

0.17 ± 0.06 34.06 ± 12.34 341.27 ± 144.16

0.75 0.92 0.84

0.772 0.128 0.013

0.867 0.244 0.067

0.05 ± 0.02 0.05 ± 0.02 0.73 ± 0.08 1657.21 ± 379.72 0.17 ± 0.07 1.34 ± 0.50 364.63 ± 114.01

0.06 ± 0.02 0.06 ± 0.03 0.70 ± 0.07 1526.85 ± 477.80 0.19 ± 0.07 1.49 ± 0.66 587.09 ± 408.30

0.09 ± 0.05 0.09 ± 0.07 0.74 ± 0.04 1321.33 ± 345.78 0.26 ± 0.11 2.50 ± 1.25 937.13 ± 525.98

0.166 0.251 0.252 0.254 0.41 0.34 0.011

0.002 0.01 0.169 0.053 0.001 3.00E-05 0.002

2.00E-04 0.003 0.257 0.016 3.00E-04 1.00E-06 6.00E-06

P value 0.481 0.37 7.00E-05 2.00E-05 0.005

0.739 0.509 4.00E-07 6.00E-07 6.00E-04

PV: Portal vein; HA: Hepatic artery; n: Number of patients; D: Diameter; AR: Area; VMAX: Time averaged maximum velocity; VTAM: Time averaged mean velocity; RI: Resistance index; CI: Congestion index; BF: Blood flow volume; DPI: Doppler perfusion index; CVH: Chronic virus hepatitis; CIR: Cirrhosis.

Table 3 Quantitative variables in CVH patients at other fibrosis stages and those at fibrosis stage 5

Variables Portal vein D (cm) AR (cm2) VMAX (cm/s) VTAM (cm/s) FV (mL/min) Hepatic Artery AR (cm2) VTAM (cm/s) FV (mL/min) Indexes PV CI (cm*s) PV D/VTAM [cm/(cm*s)] HA RI TOTAL BF (mL/min) DPI HA/PV VTAM Spleen volume (cm3)

1th-4th fibrotic stages (a)  mean ± SD 1.1 ± 0.16 0.97 ± 0.28 37.5 ± 9.86 21.4 ± 6.06 1227.3 ± 423.02

Wilcoxon’s test a vs b

t -test a vs c

5th fibrotic stage (b)

5th stage and cirrhotics (c)

mean ± SD 1.25 ± 0.19 1.25 ± 0.37 32.15 ± 6.44 18.02 ± 4.79 1274.55 ± 276.1

mean ± SD 1.19 ± 0.19 1.14 ± 0.37 28.8 ± 6.9 15.65 ± 4.27 1057.55 ± 332.2

P 0.051 0.051 0.056 0.08 0.68

P< 0.05 0.05 0.001 0.001 0.06

0.16 ± 0.057 29.9 ± 13.1 291.7 ± 171.32

0.17 ± 0.04 27.61 ± 11.69 278.79 ± 140.17

0.17 ± 0.06 32.37 ± 12.35 324.83 ± 143.9

0.85 0.60 0.81

0.75 0.41 0.37

  0.05 ± 0.023 0.056 ± 0.02 0.7 ± 0.07   1519.05 ± 517.6 0.19 ± 0.075 1.46 ± 0.66 574.06 ± 402.11

0.077 ± 0.035 0.075 ± 0.026 0.697 ± 0.034 1553.33 ± 327.8 0.177 ± 0.074 1.6 ± 0.67 631.4 ± 448.12

0.045 ± 0.02 0.085 ± 0.046 0.73 ± 0.045 1382.38 ± 352.3 0.24 ± 0.11 2.26 ± 1.19 856.7 ± 519

0.04 0.055 0.63 0.8 0.6 0.56 0.72

0.01 0.01 0.2 0.2 0.05 0.001 0.05

PV: Portal vein; HA: Hartery, n: Number of patients; D: Diameter; AR: Area; VMAX: Time averaged maximum velocity; VTAM: Time averaged mean velocity; RI: resistance index; CI: Congestion index; BF: Blood flow volume; DPI: Doppler perfusion index.

The statistically significant variables selected by the stepwise discriminant analysis are ENLARGED, ECHOGENIC, DIFFUSE, PV AR, HA RI, HA/PV VTAM, and SPLEEN. The classification scores for the CVH and CIR groups are * = multiply W1 = -89.090 - 1.657*ENLARGED + 12.769* ECHOGENIC + 4.202*DIFFUSE - 0.764*PV AR + 235.564*HA RI + 2.961*HA/PV VTAM + 0.007*SPLEEN, and W2 = -105.029 + 0.755*ENLARGED + 10.406* ECHOGENIC + 7.683*DIFFUSE - 5.849*PV AR + 251.99*HA RI + 5.473*HA/PV VTAM+ 0.010*SPLEEN. Based on these scores, we classified any new patient in the CVH group if w1 > w2 and in the CIR group if

w 2 > w 1. According to the classification formula, 41 CVH patients (93.18%) were correctly classified in CVH group while 23 cirrhotic patients (82.14%) were correctly classified in CIR group.

DISCUSSION In our study, two major findings are of interest to note regarding haemodynamic parameters and indexes. The first finding is related to a statistically significant increase in PVCI between CVH patients at fibrosis stage 5 and those at fibrosis stages 1-4. At the same time, a marginally significant increase was recorded in PV diameter, cross www.wjgnet.com

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Table 4 Area under the curve (AUC) for each variable measured by receiver operating characteristic curve (ROC) analysis Variables Liver Enlarged Echogenic1 Diffuse Nodular Portal vein D AR (cm2) V MAX1 (cm/s) VTAM1 (cm/s) FV1 (mL/min) Hepatic artery AR (cm2) VTAM (cm/s) FV (mL/min) Indexes PV CI (cm*s) PV D/VTAM [cm/(cm/s)] HA RI Total BF (mL/min) DPI HA/PV VTAM Spleen volume (cm3)

ROC Area Std. Err.

P value

95% CI

0.60 0.61 0.70 0.66

0.06 0.04 0.06 0.05

0.49 0.53 0.59 0.56

0.71 0.68 0.81 0.77

0.54 0.56 0.77 0.79 0.68

0.07 0.07 0.06 0.05 0.06

0.40 0.42 0.66 0.68 0.56

0.68 0.56 0.70 0.38 0.88 < 0.0002 0.89 < 0.0001 0.81 < 0.01

0.51 0.64 0.63

0.07 0.07 0.07

0.37 0.51 0.49

0.65 0.77 0.76

0.86 0.053 0.07

0.74 0.74 0.66 0.37 0.70 0.80 0.71

0.06 0.06 0.06 0.07 0.06 0.05 0.07

0.62 0.63 0.53 0.24 0.58 0.70 0.58

0.86 0.86 0.78 0.50 0.83 0.90 0.84

< 0.001 < 0.001 < 0.05 0.058 < 0.005 < 0.0001 < 0.005

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PV Cl

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Area under ROC curve = 0.74

0.00 0.00

B

0.25

Table 5 The best cut-of value (BCV) defined as the highest sum of sensitivity and specificity as statistically significant predictors BCV 30.10 16.00 1106.64 0.06 0.07 0.72 0.29 1.45 553.00

Sensitivity (%)

Specificity (%)

77.27 75.00 59.09 85.71 85.71 71.43 42.86 85.71 75.00

71.43 71.43 75.00 65.91 68.18 54.55 90.91 61.36 70.45

PV: Portal vein; HA: Hepatic artery; D: Diameter; AR: Area; V MAX: Time averaged maximum velocity; V TAM: Time averaged mean velocity; RI: Resistance index; CI: Congestion index; BF: Blood flow volume; DPI: Doppler perfusion index. 1Denotes CIR vs CVH.

section area and D/V TAM ratio, while V MAX and V TAM velocities were marginally reduced. Blood flow volume in the hepatic artery and the portal vein as well as DPI index remained unchanged at all CVH fibrosis stages. Decrease in portal vein blood flow velocities related to fibrotic stage in CVH patients was firstly described by Koda et al[23], who also found that portal vein blood flow volume is not significantly affected in the same patients. Tziafalia et al[32] reported that portal vein blood velocities are decreased in CVH patients. In our study, PV VMAX velocity was also decreased in CVH patients. Gaiani et al[25] also found that portal vein VTAM velocity www.wjgnet.com

0.50 1-specificity

0.75

1.00

1.00 0.75 0.50 PV D/VTAM ratio 0.25 0.00

C

Area under ROC curve = 0.7439

0.00

0.25

0.50 1-specificity

0.75

1.00

1.00 0.75 0.50 HA PV VTAM ratio Area under ROC curve = 0.80

0.25

PV VMAX1 (cm/s) PV VTAM1 (cm/s) PV BF1 (mL/min) PV CI (cm*s) PV D/VTAM [cm/(cm*s)] HA RI DPI HA/PV VTAM Spleen volume (cm3)

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PV: Portal vein; HA: Hepatic artery; D: Diameter; AR: Area, V MAX: Time averaged maximum velocity; V TAM: Time averaged mean velocity; RI: Resistance index; CI: Congestion index; BF: Blood flow volume; DPI: Doppler perfusion index; CIR: Compensated liver cirrhosis; CVH: Chronic viral hepatitis. 1Denotes CIR vs CVH.

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0.00 0.00

0.25

0.50 1-specificity

0.75

1.00

Figure 1 Portal vein congestion index (A), portal vein diameter/time averaged mean velocity (VTAM) ratio (B), and hepatic arterial/portal vein time averaged mean velocity (VTAM) ratio (C).

is the only haemodynamic variable that is independently associated with the histopathological diagnosis in CVH patients. Bernatik et al[37] reported that VMAX and VTAM velocities are reduced at end-stage fibrosis, while DPI does not change significantly and progression of liver fibrosis is associated with a continuous increase in HA resistive index (RI), suggesting that Doppler parameters are not useful in assessing the stage of liver fibrosis. In our study, although the mean HA RI value was significantly increased (P = 0.013) in CIR group compared to CVH group, it was not affected in CVH patients at different fibrosis stages. In our study, the portal vein diameter (D) was significantly enlarged at end-stage fibrosis patients, which is consistent with the reported data[31]. Walsh et al[29] found that hepatic artery blood flow (BF) and DPI are increased in CVH C patients at different fibrosis stages, while portal vein CI values remain unchanged. Our data do not support these findings regarding HA BF volume and DPI. However, we observed a significant change in PV CI. The second major finding is that there were differences in anatomic, haemodynamic parameters and indexes

Iliopoulos P et al . Ultrasonograpgy in CVH and CIR

between CVH and CIR groups, suggesting that when early cirrhosis and portal hypertension are settled, portal vein blood flow velocity is reduced. This phenomenon is accompanied with enlargement of portal vein D and AR at end-stage fibrosis. On the other hand, hepatic artery blood flow volume increases in an effort to maintain liver blood flow volume. Portosystemic shunts and varices that may subsequently occur decrease portal hypertension. Portal vein D and AR remain for some time unchanged while blood flow velocities are further reduced. These phenomena dramatically increase most haemodynamic indexes such as portal vein CI and DPI as well as the calculated alternative ratios of PV D/VTAM, HA/PV VTAM. The same observations regarding portal vein CI and DPI have been reported by other investigators [17,20-26], which are consistent with our findings. In the present study, ROC analysis showed that portal vein CI and PVD/ VTAM for the best cut-off values of 0.06 and 0.07 also had a very good sensitivity of 85.71% and a sensitivity of 65.91%-68.18% (Figures 1A and B, AUC = 0.74). In our study, spleen volume was increased in CVH and cirrhotic patients compared with the controls, which is consistent with the findings in other studies[19,20,22,24-26,28-31,38]. It is well known that a relative interobser ver variability may limit the value for gray scale and Doppler ultrasonography[33-36]. In this study, we tried to simplify Doppler indexes and haemodynamic parameters such as congestion index[17] which is the ratio of area to time average mean blood velocity in portal vein. In the most recent studies, portal vein’s area was assumed to be circular and is automatically calculated by the machine’s software from the equation area = π*r2, because r2 = (diameter/2)2. Therefore, we replaced the area with diameter of the vessel. The HA/PV VTAM ratio at proper hepatic artery has never been described. Stepwise discriminant analysis showed that the main predictors for discriminating CVH from CIR patients included three liver qualitative variables: ENLARGED, ECHOGENIC, DIFFUSE and four quantitative variables: PV AR, HA RI, HA/PV VTAM ratio and SPLEEN volume. By calculating the formula, 88.9% of the patients were correctly classified either in CVH group or in CIR group. The interesting finding is that the HA/PV VTAM alternative ratio was included in the classification function, while other indexes such as PVCI and DPI were not included. ROC analysis confirmed the high predictive value of HA/PV VTAM index for discriminating CVH form CIR patients (best-cut-off value = 1.45, sensitivity = 85.71% and specificity = 61.36%, = 0.80, Figure 1C). In the past, the “arterioportal index”[43] was described as the ratio between VMAX velocities in HA and PV right and left branches. We consider that the calculation of HA/ PV VTAM ratio would be easier to perform. We also suggest that it can be used in routine practice since these velocities are automatically measured in most studies evaluating liver haemodynamics. The accuracy of US in assessing diffuse liver disease has been evaluated in previous studies[25,26]. The reported sensitivity of gray scale and US is 57%-95% in distinguishing normal from abnormal livers[44-47]. However, attempts to identify specific pathological processes, such as fatty

  

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infiltration and fibrosis, have produced conflicting results[31,45], probably related to the different US criteria employed in the studies, such as distribution of the parenchymal echoes and attenuation of the ultrasonic beam. In our study, liver parenchymal changes were simply described and classified by yes or no (1 or 0). For routine practice and simplicity reasons, no further analysis was attempted in echo structure of the liver. Percutaneous needle biopsy and histological examination of the samples are considered the gold standard for the severity of fibrosis and cirrhosis. However, several studies[25,28] have questioned this because liver biopsies lead to false negative diagnoses of cirrhosis due to sampling errors in an estimated average of 24% pooled blind liver biopsy series [6]. Schalm [28] has reviewed the diagnostic methodology of liver cirrhosis and found that percutaneus liver biopsy has a sensitivity of below 85% in detection of liver cirrhosis. It was reported that percutaneous liver biopsy sampling errors are significantly decreased when automated spring loaded true-cut needles are used [47]. The standard of practice we used in liver biopsies is in agreement with the recently published data[47] and unsuccessful biopsies and complications were kept at their minimum. We routinely performed a thorough sonographic investigation of the liver to assess the liver parenchyma and exclude the presence of lesions, such as a cyst. Subsequently, we performed the biopsy after marking the skin with automated cutting needles that according to our experience provide superior liver biopsy specimens in subjects with advanced fibrosis and cirrhosis; more than one samples were always taken. On the other hand, cirrhosis is a common disease, which is frequently undiagnosed[4,5]. The risk of biopsy (morbidity 3%, mortality 0.03%)[7] may limit its use in screening for this disease. Finally, cirrhosis is reversible[48], making the use of alternative non-invasive diagnostic tools essential. Gaiani et al [25] suggested that ultrasonography may be used to identify cirrhosis with a diagnostic accuracy of 80% for cirrhosis even in the absence of a typical histopathological pattern. In our study, stepwise discriminant analysis showed that it diagnostic accuracy in discriminating cirrhosis from chronic viral hepatitis patients was 82.14%. In conclusion, gray scale and Doppler ultrasonography can accurately and non-invasively assess liver haemodynamics and discriminate CVH patients at end fibrosis stage from those at other CVH stages as well as CVH patients from early stage cirrhotic patients with compensated function. The method can easily be performed with routine upper abdominal ultrasonography, and is inexpensive and safe.

COMMENTS Background

In recent years, many papers have been published regarding the efforts to correctly classify chronic virus hepatitis (CVH) and distinguish this entity from liver cirrhosis by bloodless means. Blood tests and ultrasonography, or other imaging modalities (CT, MRI) are used to achieve this goal. Doppler ultrasonography of portal vein and hepatic artery has gained its ground in the estimation of portal haemodynamics. Simple recording of portal vein’s blood flow velocities is common in daily practice in many institutions. Haemodynamic indexes, although old as a www.wjgnet.com

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conception, have not been thoroughly investigated, probably due to the difficulty of producing them from the Doppler measurements in day practice.

7

Research frontiers

8

Our study investigated the value of the most popular liver haemodynamic indexes (congestion index, Doppler perfusion index, arterioportal index). We also simplified these indexes by introducing new alternative to them. Finally our data were compared to those published in literature.

9

Innovations and breakthroughs

The major finding of our study is that end CVH at the 5th fibrotic stage can be distinguished from those at other stages. In daily practice, incomplete and complete early stage cirrhosis may be predicted by simple haemodynamic indexes. Stepwise discriminant analysis produced a formula (including qualitative and quantitative variables) that differentiates liver cirrhosis from CVH in 80% of the originally classified cases. ROC analysis could find the best cut off values for discriminating CVH from cirrhosis. The new alternative indexes, particularly “arterioportal index” have been proved to be of great value in discriminating CVH from cirrhotic patients. The “arterioportal index” is first used in porta hepatis (portal vein’s trunk and proper hepatic artery). Another major point is that the best cut-off values are similar to those from other investigators contributing to the establishment of internationally accepted values for Doppler haemodynamic indexes.

10 11

12

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Applications

Our data suggest that the “congestion index” and alternative indexes are of value and can be added in daily practice for monitoring CVH and cirrhotic patients. Because the arterioportal index of porta hepatis is first used, more studies are needed to establish more precise best cut off values.

Terminology

“Arterioportal index”: The ratio of time average mean blood velocity in proper hepatic artery to time average blood velocity in portal vein’s trunk.

Peer review

In this study, the authors assessed the value of gray scale (GS) and colour Doppler ultrasonography (CDU) in differentiating the progression of CVH and compensated liver cirrhosis (CIR). Significant differences in haemodynamic parameters and indexes were found between CVH patients at fibrosis stage 5 and those at other fibrosis stages, suggesting that simple GS and CDU parameters can discriminate CVH from CIR.

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Aube C, Oberti F, Korali N, Namour MA, Loisel D, Tanguy JY, Valsesia E, Pilette C, Rousselet MC, Bedossa P, Rifflet H, Maiga MY, Penneau-Fontbonne D, Caron C, Cales P. Ultrasonographic diagnosis of hepatic fibrosis or cirrhosis. J Hepatol 1999; 30: 472-478 Cardi M, Muttillo IA, Amadori L, Petroni R, Mingazzini P, Barillari P, Lisi D, Bolognese A. Superiority of laparoscopy compared to ultrasonography in diagnosis of widespread liver diseases. Dig Dis Sci 1997; 42: 546-548 Schalm SW. The diagnosis of cirrhosis: clinical relevance and methodology. J Hepatol 1997; 27: 1118-1119 Walsh KM, Leen E, MacSween RN, Morris AJ. Hepatic blood flow changes in chronic hepatitis C measured by duplex Doppler color sonography: relationship to histological features. Dig Dis Sci 1998; 43: 2584-2590 O'Donohue J, Ng C, Catnach S, Farrant P, Williams R. Diagnostic value of Doppler assessment of the hepatic and portal vessels and ultrasound of the spleen in liver disease. Eur J Gastroenterol Hepatol 2004; 16: 147-155 Shen L, Li JQ, Zeng MD, Lu LG, Fan ST, Bao H. Correlation between ultrasonographic and pathologic diagnosis of liver fibrosis due to chronic virus hepatitis. World J Gastroenterol 2006; 12: 1292-1295 Tziafalia C, Vlychou M, Tepetes K, Kelekis N, Fezoulidis IV. Echo-Doppler measurements of portal vein and hepatic artery in asymptomatic patients with hepatitis B virus and healthy adults. J Gastrointestin Liver Dis 2006; 15: 343-346 Iwao T, Toyonaga A, Shigemori H, Oho K, Sumino M, Sato M, Tanikawa K. Echo-Doppler measurements of portal vein and superior mesenteric artery blood flow in humans: inter- and intra-observer short-term reproducibility. J Gastroenterol Hepatol 1996; 11: 40-46 Sabba C, Merkel C, Zoli M, Ferraioli G, Gaiani S, Sacerdoti D, Bolondi L. Interobserver and interequipment variability of echoDoppler examination of the portal vein: effect of a cooperative training program. Hepatology 1995; 21: 428-433 Sabba C, Weltin GG, Cicchetti DV, Ferraioli G, Taylor KJ, Nakamura T, Moriyasu F, Groszmann RJ. Observer variability in echo-Doppler measurements of portal flow in cirrhotic patients and normal volunteers. Gastroenterology 1990; 98: 1603-1611 Oppo K, Leen E, Angerson WJ, Cooke TG, McArdle CS.

  

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World J Gastroenterol 2008 April 7; 14(13): 2080-2084 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

RAPID COMMUNICATION

Endoscopic band ligation and endoscopic hemoclip placement for patients with Mallory-Weiss syndrome and active bleeding Young-Seok Cho, Hiun-Suk Chae, Hyung-Keun Kim, Jin-Soo Kim, Byung-Wook Kim, Sung-Soo Kim, Sok-Won Han, Kyu-Yong Choi

Young-Seok Cho, Hiun-Suk Chae, Hyung-Keun Kim, JinSoo Kim, Sung-Soo Kim, Department of Internal Medicine, Uijeongbu St. Mary’s Hospital, The Catholic University of Korea, Ujeongbu 480-717, Korea Byung-Wook Kim, Kyu-Yong Choi, Department of Internal Medicine, Our Lady Mercy Hospital, The Catholic University of Korea, Bupyeong 403720, Korea Sok-Won Han, Department of Internal Medicine, Holy Family Hospital, The Catholic University of Korea, Bucheon 420717, Korea Author contributions: Chae HS, Han SW and Choi KY designed study; Kim BW and Kim SS preformed study; Kim HK and Kim JS analyzed data; Cho YS mainly wrote the paper. Correspondence to: Hiun-Suk Chae, MD, Division of Gastroenterology, Department of Internal Medicine, Uijeongbu St. Mary’s Hospital, 65-1 Geumo-dong, Uijeongbu 480-717, Korea. [email protected] Telephone: +82-31-8203019 Fax: +82-31-8472719 Received: October 20, 2007  Revised: January 13, 2008

Abstract AIM: To compare the hemostatic efficacy and safety of two mechanical endoscopic methods: endoscopic band ligation (EBL) and endoscopic hemoclip placement (EHP) in patients with actively bleeding Mallory-Weiss syndrome (MWS). METHODS: A prospective randomized study to compare the efficacy and safety of EHP with EBL was performed from January 2002 to August 2005. Forty-one patients with active bleeding from MWS were treated with EHP (n = 21) or EBL (n = 20). RESULTS: There were no significant differences between groups with respect to clinical and endoscopic characteristics. The mean number of hemoclips applied was 3.2 ± 1.5 and the mean number of bands applied was 1.2 ± 0.4. Primary hemostasis was achieved in all patients. Recurrent bleeding was observed in one patient from the EHP group and two from the EBL group. Patients with recurrent bleeding were treated by the same modality as at randomization and secondary hemostasis was achieved in all. There were no significant differences between the two groups in total transfusion amount or duration of hospital stay. No complications or bleeding-related death resulted. CONCLUSION: EHP and EBL are equally effective and www.wjgnet.com

safe for the management of active bleeding in patients with Mallory-Weiss syndrome, even in those with shock or comorbid diseases. © 2008 WJG . All rights reserved.

Key words: Mallory-Weiss syndrome; Hemostasis; Endoscopic band ligation; Endoscopic clipping Peer reviewer: Kyoichi Adachi, MD, Department of Gastroenterology and Hepatology, Shimane University, School of Medicine Shimane, 89-1 Enya-cho, Izumo-shi Shimane 693-8501, Japan

Cho YS, Chae HS, Kim HK, Kim JS, Kim BW, Kim SS, Han SW, Choi KY. Endoscopic band ligation and endoscopic hemoclip placement for patients with Mallory-Weiss syndrome and active bleeding. World J Gastroenterol 2008; 14(13): 2080-2084 Available from: URL: http://www.wjgnet.com/1007-9327/14/2080.asp DOI: http://dx.doi.org/10.3748/wjg.14.2080

INTRODUCTION Mallory-Weiss syndrome (MWS), vomiting-induced mucosal lacerations in the region of the gastroesophageal junction, is one cause of nonvariceal upper gastrointestinal (UGI) bleeding and its incidence is considered to be 5% to 15%[1]. In most cases, MWS-related bleeding requires no intervention other than hemodynamic support[2]. However, some patients may require intensive care[3], especially those with risk factors such as evidence of active bleeding (for example, fresh blood hematemesis and hemodynamic instability), presence of stigmata of recurrent bleeding (such as visible vessel and adherent clots), and comorbid diseases or bleeding diathesis. Surgery or other therapeutic approaches such as balloon tamponade of the esophagus, transcatheter arterial embolization, and systemic or selective arterial infusion of vasopressin have been used to control active bleeding in patients with MWS[4-7]. In recent decades, endoscopic treatment has been the treatment of choice [8] . Various endoscopic techniques, mainly consisting of endoscopic coagulation or injection, have been used in the management of patients with MWS at high risk for recurrent bleeding[9-12]. However, injection hemostasis may be incomplete for patients with a large and/or long plexuses of vessels and coagulation has the risk of producing transmural injury and perforation due to relatively thin esophageal wall [13]. Mechanical

Cho YS et al . Hemostatic methods of MWS

endoscopic methods have recently become one of the therapeutic options for treating patients with actively bleeding MWS [13-20]. Among the mechanical methods, endoscopic band ligation (EBL) and endoscopic hemoclip placement (EHP) both has merits and problems related to the hemostatic mechanism and technical procedure itself. However, there have been few studies comparing the hemostatic efficacy of different mechanical endoscopic methods in the treatment of actively bleeding MWS. Therefore, this prospective randomized study was carried out to compare the hemostatic efficacy and safety of EBL with EHP in the treatment of actively bleeding MWS.

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A

B

Figure 1 Endoscopic view of a Mallory-Weiss tear at the cardia of the stomach. A: An actively bleeding vessel ; B: After band placement.

MATERIALS AND METHODS Subjects Among patients undergoing upper endoscopy at the gastrointestinal endoscopy center of Uijeongbu St Mary’s Hospital from January 2002 to August 2005 because of acute UGI bleeding, all consecutive patients with endoscopically verified MWS (defined as a mucosal tear or laceration near the esophagogastric junction with active bleeding, either spurting or oozing) were considered for inclusion in the study. Written informed consent for endoscopy and participation in the study was obtained from patients or near relatives before the procedure. This research was carried out in accordance with the Helsinki declaration. After receiving basic life support, all patients underwent endoscopy within 12 h of the onset of UGI bleeding. Patients were excluded if they were unable or unwilling to give informed consent for endoscopic therapy, if active bleeding was not present at endoscopy, or if more than one source of bleeding was identified. Patients with old adherent clots and clean-based longitudinal mucosal tears near the esophagogastric junction as the only possible origin of bleeding were treated conservatively. All patients’ clinical characteristics, including age, gender, presenting symptoms, alcohol use, blood pressure, hemoglobin level, coagulopathy, and comorbid diseases, were recorded. Shock was defined as a systolic blood pressure of less than 90 mmHg and a pulse rate greater than 100 beats/min, accompanied by pallor or cold sweating. Therapy Forty-one patients were randomized to undergo EBL (n = 20) or EHP (n = 21) by concealed allocation determined according to a table of random numbers. All diagnostic and therapeutic endoscopic procedures were performed by members of the research team of four staff gastroenterologists. They did not participate in the postprocedure care of the patients, which was conducted by other physicians. Endoscopic procedures were performed with videoendoscopes (GIF-Q230, GIFXQ230, Olympus Optical Co. Ltd, Tokyo, Japan). In the EBL group, an endoscopic ligating device (Pneumoactive EVL device; Sumitomo Bakelite Company, Tokyo, Japan) was used with no overtube. After endoscopic identification of the lesion, the endoscope was withdrawn and was reinserted after attachment of the band ligator. After the hood had been placed over the bleeding site, endoscopic

A

B

Figure 2 Endoscopic view of a Mallory-Weiss tear at the esophagogastric junction. A: Oozing vessel; B: After hemoclip application to the bleeding vessel.

suction was activated to draw the bleeding site into the banding cylinder. After suction and band release, a polypoid mass of mucosa 1 cm in diameter was formed and active bleeding stopped immediately (Figure 1). In the EHP group, hemoclips (MDS50, Olympus) were placed with a clip application device (HX-3L, HX-5 LR-1; Olympus). In terms of technique, hemoclips were applied directly on spurting or oozing vessels along with surrounding tissues (Figure 2). Once hemostasis was achieved, the bleeding site was observed for at least 1 min. Primary hemostasis was defined as no endoscopic evidence of bleeding during this time after band ligation or after hemoclipping, during the first endoscopic session. Patients who continued to bleed, despite receiving EBL or EHP, were given endoscopic injections of epinephrine. If patients continued to bleed despite endoscopic treatment, we planned to undergo transcatheter arterial embolization or emergency surgery, depending on the status of the patient and/or the volume of blood transfused. After endoscopic treatment, all patients were observed closely. During admission, treatment included partial parenteral nutrition and intravenous administration of histamine H 2 receptor antagonists at standard doses. Evidence of recurrent bleeding included the following signs of ongoing bleeding: (1) new hematemesis; (2) fresh blood aspirated via a nasogastric tube; (3) continuous melena with instability of vital signs (systolic blood pressure ≤ 90 mm Hg, heart rate ≥ 100 beats/min, or orthostatic changes in systolic blood pressure of ≥ 20 mmHg or heart rate of www.wjgnet.com

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Table 1 Clinical characteristics and outcome of patients in endoscopic band ligation (EBL) and endoscopic hemoclip placement (EHP) groups EHP (n = 21)

EBL (n = 20)

P value

Age (yr) Gender (male/female) Alcohol use Hematemesis/melena Shock Hemoglobin level (g/L) Comorbid disease Number of tears Bleeding stigmata

49.5 ± 13.2 21/0 13 (62%) 21/1 3 (14%) 125 ± 33 9 (43%) 1.7 ± 0.9

47.1 ± 11.9 17/3 13 (65%) 20/2 4 (20%) 112 ± 25 9 (45%) 1.4 ± 0.6

0.58 0.10 1.00 0.61 0.70 0.14 1.00 0.29 0.45

Spurting vessel Oozing vessel Tear location

3 (14%) 18 (86%)

5 (25%) 15 (75%)

Distal esophagus Esophagogastric junction Cardia Length of tear (cm) Primary outcomes

6 (29%) 9 (43%) 6 (27%) 2.2 ± 0.6

4 (25%) 14 (75%) 2 (10%) 2.1 ± 0.5

0.34

Primary hemostasis First episode of recurrent bleeding Permanent primary hemostasis Secondary hemostasis Other outcomes

21 (100%) 1 (6%) 20 (94%) 1 (100%)

20 (100%) 2 (10%) 18 (90%) 2 (100%)

1.00 0.61 0.61 1.00

Blood transfusion (mean units) Total procedure time (mean min) Additional epinephrine injection Bleeding-related deaths Hospital stay (mean days)

2.0 ± 2.5 16.7 ± 3.2 0 0 6.7 ± 5.1

3.3 ± 2.9 17.1 ± 2.6 0 0 7.3 ± 3.3

0.14 0.68 1.00 1.00 0.20

0.63

≥ 20 beats/min), or a decrease in hemoglobin level

of more than 20 g/L within 24 h of obtaining primary hemostasis. When recurrent bleeding was suspected, endoscopy was performed immediately and the same initial therapeutic modality used at randomization was used. After patients were discharged, the clinical outcome was evaluated by physicians who were blinded to the type of endoscopic treatment and who followed the patients for 30 d including initial hospitalization. Permanent hemostasis was defined as the absence of recurrent bleeding during this period. To evaluate the efficacy of the two hemostatic procedures, data for each patient were collected during hospitalization and included information on demographics, medical history, presenting symptoms, initial hemodynamic status, laboratory values, rate of primary hemostasis, permanent primary hemostasis rate, number of therapeutic endoscopic sessions, need for emergency operation or transcatheter arterial embolization, bleeding-related deaths, transfusion requirements, and duration of hospitalization. Statistical analysis Quantitative data are summarized as the mean ± SD. The Mann-Whitney nonparametric U test was used to compare the mean values of continuous variables and Fisher’s exact test was used for the comparison of discrete variables. P < 0.05 was accepted as indicating statistical significance. The analyses were performed using SPSS version 11.0 for Windows (SPSS Inc., Chicago, IL, USA). www.wjgnet.com

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RESULTS During the study period, 137 patients with MWS tears underwent emergency endoscopy, but only 41 patients (30%) met the entry criteria and were randomized. Clinical and endoscopic characteristics for the patients at entry are outlined in Table 1. No differences were noted between the two groups with respect to age, gender, presenting symptoms, alcohol use, shock, hemoglobin level, coagulopathy, comorbid diseases, number of bleeding points, length of tear, stigmata of bleeding, or locations of the lacerations. Comorbid diseases in the EBL group included liver cirrhosis, coronary artery disease, stroke, and chronic renal failure; in the EHP group, comorbid diseases included liver cirrhosis, pancreatitis, valvular heart disease, and rheumatoid arthritis. However, no differences were noted between the two groups with respect to frequency of having a spurting vessel or oozing. The most common location of the tear was the esophagogastric junction in all patients (23/41, 56%; Table 1). Primary hemostasis was obtained in all patients in each group. In the EBL group, treatment was completed in a single session (mean number of bands 1.2 ± 0.4). A single elastic band was applied in 17 patients; two bands were placed in three patients (Table 1). In the EHP group, the mean number of hemoclips applied was 3.2 ± 1.5. Two patients (10%) in the EBL group and one patient (6%) in the EHP group had recurrent bleeding; this was controlled in all three with endoscopic treatment and no patient required surgery. No differences were noted in primary outcomes including rates of primary hemostasis, recurrent bleeding, and permanent primary hemostasis. No differences were noted in the other secondary outcomes, including the number of endoscopic sessions, total procedure time, the need for additional epinephrine injection, the need for emergency operation or transcatheter arterial embolization, bleeding-related deaths, transfusion requirements, or the duration of hospitalization. No significant complications or adverse events attributable to endoscopic treatment were noted in either group, and no recurrent bleeding was noted in either group during outpatient follow-up.

DISCUSSION MWS is a relatively common cause of nonvariceal UGI bleeding, in which an abrupt rise in abdominal pressure caused by retching or vomiting induces mucosal tears near the esophagogastric junction[1,2]. Because most patients stop bleeding spontaneously, emergency treatment is reserved for those showing active bleeding [3]. The rate of recurrent bleeding in patients with MWS is also lower than that for other nonvariceal bleeding[8]. However, when bleeding is active and severe, patients require surgical treatment or nonsurgical therapeutic approaches[4,5]. Several endoscopic methods have been used to treat actively bleeding MWS, including injection of different agents, electrocoagulation, application of hemoclips, and band ligation[8,18]. Thermal coagulation or injection therapies have been used successfully to control active bleeding from MWS [10-12,21] . Laine [21] reported that

Cho YS et al . Hemostatic methods of MWS

multipolar electrocoagulation significantly improved hemostasis and reduced surgery in patients with active bleeding from MWS and has been associated with few complications. However, repeated coagulation has the risk of producing transmural injury and perforation because the esophagus lacks serosa and is very thin at the tear site[22]. Injection therapy with various agents is an effective, simple, and inexpensive first-line approach[11,12]. Llach et al[11]. Reported that endoscopic injection therapy using epinephrine and polidocanol improved outcomes (rate of recurrent bleeding, hospital stay, and transfusion requirement) compared with supportive measures alone in a prospective, randomized controlled trial. However, injection therapy can produce cardiovascular complications such as ventricular tachycardia and should be avoided in patients with a history of coronary artery disease because of the potential for systemic absorption[23]. EBL, commonly used in variceal bleeding, has also been used to treat nonvariceal bleeding [8]. EBL is technically easier to perform than other methods, with the lesions well viewed under direct pressure and suction from the transparent ligation cap[20]. The use of EBL for treatment of patients with bleeding MWS has been described in several studies[13-15,18,20,24]. Our study also demonstrated high successful rates of primary and permanent hemostasis in such cases. EHP is acceptable for treating bleeding lesions in nonfibrotic tissues and has advantages over other hemostatic methods because it rarely causes perforation[25]. It is effective in the management of bleeding from a Mallory-Weiss tear [18,19,26]. Compared with cautery or sclerotherapy, it may be a safer option in the management of bleeding from MWS because of the lack of additional tissue damage with endoclips[8,19]. In the event of a deeper extension of the tear with an esophageal perforation, the placement of endoclips can fix both problems simultaneously[27]. EHP has been shown to be an effective alternative treatment in critically ill patients with severe gastrointestinal bleeding[28,29]. Park et al[30] reported that EHP and EBL showed similar efficacy and safety in the management of bleeding gastric Dieulafoy’s lesions. However, there have been no reports comparing the efficacy and safety of EHP with those of EBL for the treatment of active bleeding from MWS. In our study, EBL and EHP both achieved low rates of recurrent bleeding and high rates of primary hemostasis. The recurrent bleeding was successfully treated using the same method as at randomization. Shock and comorbid diseases were present in four (20%) and nine (45%) patients in the EBL group, respectively; and three (14%) and nine (43%) patients in the EHP group. Despite these adverse factors, primary hemostasis was achieved in all patients. No patient in either group required additional epinephrine injection or advanced invasive therapy such as transcatheter arterial embolization or emergency surgery, and there was no procedure-related complication in either group. Therefore, the results of our study indicate that both EBL and EHP are effective and safe for treatment of patients with actively bleeding MWS, including those with shock, comorbid diseases, and/or coagulopathy. In conclusion, EBL and EHP are equally safe and

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effective for the control of active bleeding in patients with hemodynamically unstable MWS and/or combined major diseases. Moreover, after treatment, the frequency of recurrent bleeding is low and the rate of permanent hemostasis is high.

COMMENTS Background

Mallory-Weiss syndrome (MWS) is a relatively common cause of nonvariceal upper gastrointestinal bleeding. When bleeding is active and severe, patients require treatment. Several endoscopic methods have been used to treat actively bleeding MWS. We aimed to compare the hemostatic efficacy and safety of endoscopic band ligation (EBL) with endoscopic hemoclip placement (EHP) in the treatment of actively bleeding MWS.

Research frontiers

Endoscopic injection of epinephrine is effective treatment for MWS but has higher recurrent bleeding rate. Mechanical endoscopic methods is one of treatment options that are effective and safe for actively bleeding MWS.

Innovations and breakthroughs

EBL and EHP both have merits and problems related to the hemostatic mechanism and technical procedure itself. Although many investigators have reported the usefulness of mechanical endoscopic methods, there have been few studies comparing the hemostatic efficacy of different mechanical endoscopic methods in the treatment of actively bleeding MWS. We showed that both EHP and EBL are effective for MWS. These two methods are also used effectively in patients with hemodynamically unstable MWS or combined diseases.

Applications

This finding provides evidence that EBL or EHP in the management of actively bleeding MWS can be selected depending on physician’s expertise.

Peer review

This is a report designed to compare the efficacy of two different mechanical endoscopic methods in the management of actively bleeding MWS. It is concluded that EBL and EHP are equally safe and effective. After treatment with both, the frequency of recurrent bleeding is low and the rate of permanent hemostasis is high.

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in bleeding Mallory-Weiss syndrome: a randomized controlled trial. Gastrointest Endosc 2001; 54: 679-681 Peng YC, Tung CF, Chow WK, Chang CS, Chen GH, Hu WH, Yang DY. Efficacy of endoscopic isotonic saline-epinephrine injection for the management of active Mallory-Weiss tears. J Clin Gastroenterol 2001; 32: 119-122 Terada R, Ito S, Akama F, Kidogawa H, Kashima K, Yamayoshi T, Ooe H. Mallory-Weiss syndrome with severe bleeding: treatment by endoscopic ligation. Am J Emerg Med 2000; 18: 812-815 Myung SJ, Kim HR, Moon YS. Severe Mallory-Weiss tear after endoscopy treated by endoscopic band ligation. Gastrointest Endosc 2000; 52: 99-101 Gunay K, Cabioglu N, Barbaros U, Taviloglu K, Ertekin C. Endoscopic ligation for patients with active bleeding MalloryWeiss tears. Surg Endosc 2001; 15: 1305-1307 Lin LF, Siauw CP, Ho KS, Tung JC. Endoscopic hemoclip treatment of gastrointestinal bleeding. Chang Gung Med J 2001; 24: 307-312 Will U, Seidel T, Bosseckert H. Endoscopic hemoclip treatment for bleeding artificially induced Mallory-Weiss tears. Endoscopy 2002; 34: 748 Chung IK, Kim EJ, Hwang KY, Kim IH, Kim HS, Park SH, Lee MH, Kim SJ. Evaluation of endoscopic hemostasis in upper gastrointestinal bleeding related to Mallory-Weiss syndrome. Endoscopy 2002; 34: 474-479 Huang SP, Wang HP, Lee YC, Lin CC, Yang CS, Wu MS, Lin JT. Endoscopic hemoclip placement and epinephrine injection for Mallory-Weiss syndrome with active bleeding. Gastrointest Endosc 2002; 55: 842-846 Park CH, Min SW, Sohn YH, Lee WS, Joo YE, Kim HS, Choi SK, Rew JS, Kim SJ. A prospective, randomized trial of endoscopic band ligation vs. epinephrine injection for actively bleeding Mallory-Weiss syndrome. Gastrointest Endosc 2004; 60: 22-27 Laine L. Multipolar electrocoagulation in the treatment of

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active upper gastrointestinal tract hemorrhage. A prospective controlled trial. N Engl J Med 1987; 316: 1613-1617 Lum DF, McQuaid K, Lee JG. Endoscopic hemostasis of nonvariceal, non-peptic ulcer hemorrhage. Gastrointest Endosc Clin N Am 1997; 7: 657-670 Stevens PD, Lebwohl O. Hypertensive emergency and ventricular tachycardia after endoscopic epinephrine injection of a Mallory-Weiss tear. Gastrointest Endosc 1994; 40: 77-78 Higuchi N, Akahoshi K, Sumida Y, Kubokawa M, Motomura Y, Kimura M, Matsumoto M, Nakamura K, Nawata H. Endoscopic band ligation therapy for upper gastrointestinal bleeding related to Mallory-Weiss syndrome. Surg Endosc 2006; 20: 1431-1434 Hachisu T. Evaluation of endoscopic hemostasis using an improved clipping apparatus. Surg Endosc 1988; 2: 13-17 Yamaguchi Y, Yamato T, Katsumi N, Morozumi K, Abe T, Ishida H, Takahashi S. Endoscopic hemoclipping for upper GI bleeding due to Mallory-Weiss syndrome. Gastrointest Endosc 2001; 53: 427-430 Hurlstone DP. Successful endoscopic haemoclipping in Mallory-Weiss syndrome with concurrent closure of oesophageal perforation: further prospective evaluation of the technique is required. Scand J Gastroenterol 2002; 37: 866 Ohta S, Yukioka T, Ohta S, Miyagatani Y, Matsuda H, Shimazaki S. Hemostasis with endoscopic hemoclipping for severe gastrointestinal bleeding in critically ill patients. Am J Gastroenterol 1996; 91: 701-704 Ohta S, Goto H, Yukioka T, Mishima S, Shimazaki S. Efficacy of endoscopic hemoclipping for GI bleeding in relation to severity of shock. Hepatogastroenterology 2003; 50: 721-724 Park CH, Joo YE, Kim HS, Choi SK, Rew JS, Kim SJ. A prospective, randomized trial of endoscopic band ligation versus endoscopic hemoclip placement for bleeding gastric Dieulafoy's lesions. Endoscopy 2004; 36: 677-681 S- Editor Zhong XY

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World J Gastroenterol 2008 April 7; 14(13): 2085-2088 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

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Effects of honey as a scolicidal agent on the hepatobiliary system Bulent Kilicoglu, Kemal Kismet, Sibel Serin Kilicoglu, Serap Erel, Omur Gencay, Kadriye Sorkun, Esra Erdemli, Okan Akhan, Mehmet Ali Akkus, Iskender Sayek Bulent Kilicoglu, Kemal Kismet, Serap Erel, Mehmet Ali Akkus, Department of General Surgery, Ankara Training and Research Hospital, Ankara 06340, Turkey Sibel Serin Kilicoglu, Department of Histology-Embryology, Ufuk University, Ankara 06340, Turkey Omur Gencay, Kadriye Sorkun, Department of Biology, Faculty of Science, Hacettepe University, Ankara 06340, Turkey Esra Erdemli, Department of Histology-Embryology, Ankara University, Ankara 06340, Turkey Okan Akhan, Department of Radiology, Hacettepe University, Ankara 06340, Turkey Iskender Sayek, Department of General Surgery, Hacettepe University, Ankara 06340, Turkey Author contributions: Kilicoglu B and Kismet K contributed equally to this work, Kilicoglu SS and Erdemli E performed histological examination, Gencay O and Sorkun K analyzed honey, Akhan O performed radiological examination, Akkus MA and Erel S analyzed data; and Sayek I, Kismet K and Kilicoglu B wrote the paper. Correspondence to: Bulent Kilicoglu, Ankara Training and Research Hospital Ulucanlar, Ankara 06340, Turkey. [email protected] Telephone: +90-312-5953449 Fax: +90-312-3633697 Received: December 26, 2007 Revised: February 27, 2008

Abstract AIM: To examine the effects of 10% diluted honey, which has been shown to be scolicidal, on the liver and biliary system and determine whether it could be used as a scolicidal agent in the presence of biliary-cystic communication. METHODS: Thirty Wistar-Albino rats were divided into two groups. Honey with 10% dilution in the study group and 0.9% saline (NaCl) in the control group were injected into the common bile ducts of rats through a 3-mm duodenotomy. The animals were sacrificed 6 mo after the procedure. Histopathological, biochemical, and radiological examinations were performed for evaluation of side effects. RESULTS: At the end of the sixth month, liver function tests were found to be normal in both groups. The tissue samples of liver and ductus choledochus of the honey group showed no histomorphologic difference from the control group. No stricture on the biliary tree was detected on the retrograde cholangiograms. CONCLUSION: According to these results, we concluded

that 10% diluted honey could be used as scolicidal agent safely in the presence of biliary-cystic communication. © 2008 WJG . All rights reserved.

Key words: Scolocidal agent; Honey; Hepato biliary system Peer reviewer: Sharon DeMorrow, Division of Research and Education, Scott and White Hospital and The Texas A&M University System, Health Science Center College of Medicine, Temple, Texas 76504, United States

Kilicoglu B, Kismet K, Kilicoglu SS, Erel S, Gencay O, Sorkun K, Erdemli E, Akhan O, Akkus MA, Sayek I. Effects of honey as a scolicidal agent on the hepatobiliary system. World J Gastroenterol 2008; 14(13): 2085-2088 Available from: URL: http://www.wjgnet.com/1007-9327/14/2085.asp DOI: http:// dx.doi.org/10.3748/wjg.14.2085

INTRODUCTION Hydatid disease is still an important endemic problem in Turkey and in many other parts of the world. Dissemination of protoscolex-rich fluid during surgery is a major cause of recurrence. Instillation of scolicidal agent into a hepatic hydatid cyst is the most commonly employed measure to prevent this serious complication[1]. Biliary system strictures or “caustic sclerosing cholangitis” can develop from the caustic effect of the scolicidal solution diffused from the cyst into the biliary system, during the surgical intervention of hydatid cysts of the liver[2]. Surgical treatment has been used around the world for years as the primary choice of treatment. Although percutaneous treatment of hydatid cysts was considered to be contraindicated due to potential risks of anaphylactic shock and dissemination of the hydatid fluid into the abdomen, this method has been used successfully for treatment of hydatid cysts since 1980s[3]. Up to date, many scolicidal agents have been used for inactivation of the cyst content, but there is no ideal agent that is both effective and safe[4]. The surgical treatment of hydatid disease of the liver includes evacuation of the cyst with scolocidal irrigation and either excision or drainage of the cyst[5]. The objectives of surgical treatment are inactivating scolices, preventing spillage of cyst contents, eliminating all viable elements of the cyst, and managing the residual cavity of the cyst. www.wjgnet.com

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Inactivation of scolices with various scolicidal agents has been tried with varying success[6]. Honey is the foodstuff made by honeybees from the nectar of flowers or secretions from other parts of the plants, which they gather, transform together with their own specific materials, and store in honeycomb. Honey is considered as healthy and wholesome food with curative properties. It has antimicrobial effects against many bacteria and this property may be due to its osmolarity, acidity, flavonoids, aromatic acid substances, and hydrogen peroxide[7]. In a previous study, 10% diluted honey has been shown to be highly effective on protoscolices[8]. The aim of the present study was to investigate whether diluted honey would cause caustic sclerosing cholangitis when injected directly into the common bile duct of rats.

MATERIALS AND METHODS Animals Thirty Wistar-Albino female rats, weighing 225 ± 25 g, were included in this study. Animals were deprived of food 12 h before anesthesia, but had free access to water 2 h before anesthesia. No enteral or parenteral antibiotics were administered at any time. Rats were housed under constant temperature (21℃ ± 2℃) individually in wire cages with 12 h light-dark cycle. Rats that died during the study were excluded. The procedures in this experimental study were performed in accordance with the National Guidelines for the Use and Care of Laboratory Animals and approved by the Animal Ethics Committee of Ankara Research and Training Hospital. Surgical procedure The rats were randomly divided into two equal groups of 15 rats each. Rats were anesthetized by an intramuscular injection of ketamine HCl (Ketalar, Parke-Davis, Eczacibasi, Istanbul, Turkey; 40 mg/kg body weight) and xylazine (Rompun, Bayer, Leverkusen, Germany; 5 mg/kg body weight). All animals were allowed to breath spontaneously during the experiments. After the abdomen was shaved and cleaned with povidone iodine, a midline laparotomy was carried out, and the intestines were covered with sterile gauze pads soaked with isotonic saline at 36℃-38℃. A 3-mm duodenotomy was performed. Test solutions, 0.15 mL, either sterile isotonic saline solution (control group) or 10% dilutions of honey (study group) (Anzer honey, Rize, Turkey), were injected without pressure into the common bile duct with 27 gauge syringe. Immediately after the injection, the common bile duct was clamped with an atraumatic vascular clamp (bulldog). The catheter was then withdrawn. The clamp was removed 5 min later, and the duodenotomy was closed with a 6-0 polypropylene (Prolene) suture. There was no operative mortality. The study animals were kept for 6 mo, during which time they were fed with rat chow ad libitum and tap water and kept at room temperature (18℃-20℃) in separate cages. Blood samples were obtained 1 wk after the surgical procedure and at the end of the experimental study (6 mo

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after the procedure) for liver function tests including bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma glutamyl transferase (GGT). Six months after the procedure, retrograde cholangiography was performed under ketamine hydrochloride anesthesia. Midline laparotomy was carried out, and a 3-mm duodenotomy was performed. Radiopaque solution, 0.15 mL, was injected without pressure into the common bile duct using a 27 gauge syringe. Immediately after the injection, the common bile duct was clamped with an atraumatic vascular clamp and antero-posterior cholangiograms were obtained. Following cholangiography, blood samples for determination of liver function tests were obtained. Liver, common bile duct and duodenum were excised en-bloc for histopathological examination. Biochemical and histopathological examinations The biochemical analyses were made by an autoanalyzer (Olympus AU 640, Japan) using commercial kits. The liver specimens of the right and left lobes and common bile duct were taken and immediately fixed in 10% neutral buffered formalin solution for one week. Tissues were washed in flowing water and dehydrated with rising concentrations of ethanol (50%, 75%, 96% and 100%). After dehydration, specimens were put into xylene to obtain transparency and were then infiltrated with and embedded in paraffin. Histological sections of the specimens in thickness of 6 μm from all the groups were stained with hematoxylin and eosin. The whole tissue blocks were sectioned and histopathological examinations were performed on systematically randomly sampled preparations by a blinded researcher. The specimens were photographed by Nikon eclipse E 600. Liver specimens were evaluated to assess the morphology of the hepatocytes, portal areas, sinusoidal lesions, cellular infiltration in the lobule or portal spaces and parenchymal lesions. Histopathological examination of the common bile duct was performed to assess the histomorphology of the epithelium, connective tissue, inflammation, fibroblastic proliferation and necrosis. Statistical analysis Differences between the groups were analyzed with MannWhitney U test. Statistical analysis was performed with the Statistical Package for the Social Sciences (SPSS) version 13.0 for Windows (SPSS Inc., Chicago, USA). P values less than 0.05 were considered to be significant.

RESULTS Five rats from each group died within 5 d after the procedure. Two of ten died in the early postoperative period possibly due to anesthesia, and the others died because of trauma to the common bile duct and leakage into the peritoneum. The remaining 20 rats were alive until the end of the study without any problem. Biochemical and radiological results Liver function tests were slightly elevated 1 wk after the procedure in both groups, and this might be due to the

Kilicoglu B et al . Honey and the hepatobiliary system

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Figure 2 Common bile duct (ductus choledochus) sections stained with hematoxyline and eosin. A: Control group; B: Treatment group. L: Lumen; sb: Sacculi of Beale; c: Capillary; s: Stroma; arrow head: Simple columnar epithelium. The right column of the panel is the larger view of the left column.

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Figure 1 Liver sections stained with hematoxyline and eosin. A, B: Control group showing the regular architecture; C, D: Treatment group. vc: Central vein; pv: Portal vein; ha: Hepatic artery; bd: Bile ductules. Arrow, hepatic sinusoids; *, cell infiltration. The right column of the panel is the larger view of the left column.

in regular architecture in both groups. All three layers, mucosa, muscularis and serosa of the common bile duct were viewed normal. There was no inflammation and/or fibroblastic proliferation in the stroma of the control and honey groups. The lining cells of the sacculi of Beale were mucous gland cells that were clearly viewed in the honey group. The tissue samples of liver and common bile duct of the honey group showed no histomorphologic difference from the control group (Figure 2).

DISCUSSION cannulation and injection of common bile duct and there was no difference between the groups. At the end of the first and sixth month, liver function tests were found to be normal in both groups. No stricture in the biliary tree was found on the retrograde cholangiograms. Histopathological results The histopathological evaluation of the liver sections did not reveal any difference between the control and honey groups. Polygonal shaped hepatocytes radiating outward from a central vein in the center and arranging into hepatic cords separated by adjacent sinusoids were demonstrated. Both the hepatic portal vein and hepatic artery branch and also the bile ductules in the corners of hepatic lobules were in normal architecture histologically. There was neither inflammatory cellular infiltration nor bile pigment accumulation in either group (Figure 1). When we examined the tissue samples of the common bile duct, no adverse effect of the honey was demonstrated. The epithelium of the common bile duct was penetrated into the stroma to form pits known as the sacculi of Beale. The dense connective tissue and smooth muscle were

The ideal treatment for hepatic hydatid disease should completely eliminate the parasite and prevent recurrence of the disease with minimum morbidity and mortality. There are three available therapeutic modalities for hepatic hydatid disease: systemic chemotherapy, surgery, and percutaneous treatment[9]. Meticulous packing of the operative field is necessary irrespective of the surgical technique employed with sponges soaked in scolicidal agents to inactivate the scolices which may leak from the cyst during surgical manipulation. In conventional or minimally invasive hydatid disease surgery, inactivation of the cyst content is essential, justifying the routine use of scolicidal solutions. In the presence of cystobiliary communications, the passage of these solutions may cause hepatic stasis, edema and necrosis in the hepatic tissue as well as histopathological changes in the biliary tree [2]. Various experimental studies investigated the effects of 95% alcohol, 10% povidone iodine, 0.9%, 5.0%, 10.0%, 20.0% NaCl, 3% H2O2, 5% formalin, 0.5% AgNO3, cetrimide on the liver and the biliary tree. Severe hepatobiliary complications have been reported for formalin, alcohol and 10%-20% NaCl[10,11]. Sclerosing cholangitis may be due to immunological,

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infectious, vascular, or chemical factors. In patients with hydatid disease of the liver, various factors, including injection of scolicidal agent into the cyst cavity, a communication between the cyst and biliary tree, and a particular sensitivity to the scolicidal agent seems to be necessary to promote caustic sclerosing cholangitis[5]. Histopathological changes in sclerosing cholangitis is spotty necrosis in the liver parenchyma, widening of sinusoids, regenerative changes in hepatocytes, Kupffer cell hyperplasia, pigment accumulation, periductal fibrosis, inflammation, fibroblastic proliferation, and necrosis in the extrahepatic biliary ducts [5,12]. In our study, no histopathological difference was detected in honey group when compared with the control group. None of the above mentioned pathological changes was present in the liver and common bile duct specimens of honey group. The retrograde cholangiograms were all within normal limits without any evidence for biliary stricture. Liver function tests at the end of the first and sixth month were also within the normal ranges. In a previous study, we found that honey was a potent scolicidal agent in vitro[8]. Honey concentrations of 10% or greater killed all protoscolices. The scolicidal effects of honey began at the end of the third minute. Intraperitoneal application of honey resulted in adverse effects with this concentration. According to these results, we concluded that honey might be used as a potent scolicidal agent after the evaluation of side effects on hepatobiliary system and the in vivo activity. The ideal scolicidal agent should have rapid and complete scolicidal effects with minimal local and systemic side effects[13]. No systemic side effects, such as anaphylactic reaction or hyperglycemia, and no local side effects in peritoneal surface developed with intraperitoneal administration[8]. Since an important and life-threatening side effect of scolicidal agents is sclerosing cholangitis, we planned to evaluate the effects of honey on hepatobiliary system with the present study in which we did not find any side effects on hepatobiliary system evaluated by using biochemical, histological, and radiologic parameters. In conclusion, although sclerosing cholangitis is a major complication that restrict the use of many scolicidal agents in the presence of a biliary-cystic communication in hydatid liver disease, our experience from the present study shows that honey can be used safely in this situation.

COMMENTS Background

To examine the effects of 10% diluted honey, which has been shown to be scolicidal, on the liver and biliary system whether it could be used as a scolicidal agent in the presence of biliary-cystic communication.

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Research frontiers

The present study investigated whether diluted honey would cause caustic sclerosing cholangitis when injected directly into the common bile duct of rats.

Innovations and breakthroughs

Sclerosing cholangitis is a major complication that restricts the use of many scolicidal agents in the presence of a biliary-cystic communication in hydatid liver disease, our experience from the present study shows that honey can be used safely in this situation.

Applications

Evaluates the effects of a 10% honey solution on the liver as a possible scolicidal agent in the treatment of hydatid cysts.

Peer review

The rationale behind this study is that the authors have previously shown that a 10% honey solution has scolicidal properties in vitro and that current scolicidal agents often cause sclerosing cholangitis. It is an interesting paper.

REFERENCES 1

Tozar E, Topcu O, Karayalcin K, Akbay SI, Hengirmen S. The effects of cetrimide-chlorhexidine combination on the hepatopancreatico-biliary system. World J Surg 2005; 29: 754-758 2 Belghiti J, Benhamou JP, Houry S, Grenier P, Huguier M, Fekete F. Caustic sclerosing cholangitis. A complication of the surgical treatment of hydatid disease of the liver. Arch Surg 1986; 121: 1162-1165 Akhan O, Ozmen MN. Percutaneous treatment of liver 3 hydatid cysts. Eur J Radiol 1999; 32: 76-85 4 McManus DP, Zhang W, Li J, Bartley PB. Echinococcosis. Lancet 2003; 362: 1295-1304 Sahin M, Eryilmaz R, Bulbuloglu E. The effect of scolicidal 5 agents on liver and biliary tree (experimental study). J Invest Surg 2004; 17: 323-326 6 Sayek I, Onat D. Diagnosis and treatment of uncomplicated hydatid cyst of the liver. World J Surg 2001; 25: 21-27 7 Orsolic N, Basic I. Antimetastatic effect of honey. Mellifera 2004; 4: 38-43 Kilicoglu B, Kismet K, Koru O, Tanyuksel M, Oruc MT, Sorkun 8 K, Akkus MA. The scolicidal effects of honey. Adv Ther 2006; 23: 1077-1083 Sayek I, Tirnaksiz MB, Dogan R. Cystic hydatid disease: 9 current trends in diagnosis and management. Surg Today 2004; 34: 987-996 10 Yetim I, Erzurumlu K, Hokelek M, Baris S, Dervisoglu A, Polat C, Belet U, Buyukkarabacak Y, Guvenli A. Results of alcohol and albendazole injections in hepatic hydatidosis: experimental study. J Gastroenterol Hepatol 2005; 20: 1442-1447 11 Topcu O, Aydin C, Arici S, Duman M, Sen M, Koyuncu A. The effects of various scolicidal agents on the hepatopancreatic biliary system. Chir Gastroenterol 2006; 22: 185-190 12 Houry S, Languille O, Huguier M, Benhamou JP, Belghiti J, Msika S. Sclerosing cholangitis induced by formaldehyde solution injected into the biliary tree of rats. Arch Surg 1990; 125: 1059-1061 13 Altindis M, Arikan Y, Cetinkaya Z, Polat C, Yilmaz S, Akbulut G, Dilek ON, Gokce O. Octenidine hydrochloride in hydatid disease. J Invest Surg 2004; 17: 41-44 S- Editor Zhong XY

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World J Gastroenterol 2008 April 7; 14(13): 2089-2093 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

RAPID COMMUNICATION

Predictive factors for early aspiration in liver abscess Rustam Khan, Saeed Hamid, Shahab Abid, Wasim Jafri, Zaigham Abbas, Mohammed Islam, Hasnain Shah, Shaalan Beg Rustam Khan, Saeed Hamid, Shahab Abid, Wasim Jafri, Zaigham Abbas, Mohammed Islam, Hasnain Shan, Shaalan Beg, Section of Gastroenterology Department of Medicine, The Aga Khan University Hospital, Karachi, Pakistan Author contributions: Khan R collected and entered the data on SPSS software, did literature searches and wrote manuscript drafts; Hamid S coordinated data interpretation, drafted and wrote the manuscript; Abid S conceived the idea, designed the study, helped in drafting and writing the manuscript; Jafri W helped in data interpretation, general support, drafting and manuscript writing; Abbas Z helped in data interpretation and manuscript writing; Islam M Performed the statistical analysis; Shan H helped in drafting and manuscript writing; Beg S helped in data collection and manuscript writing. Correspondence to: Dr. Saeed Hamid, MBBS, FRCP, FRCPI, FACP, FACG, Department of Medicine, The Aga Khan University, Stadium Road, Karachi 74800, Pakistan. [email protected] Telephone: +92-21-4930051-4672 Fax: +92-21-4934294 Received: September 27, 2007 Revised: December 8, 2007

Key words: Liver abscess; Aspiration and liver abscess; Needle aspiration and liver abscess; Amebic liver abscess; Pyogenic liver abscess; Liver abscess and management Peer reviewer: Pietro Invernizzi, Dr, Division of Internal Medicine, Department of Medicine, Surgery, Dentistry, San Paolo School of Medicine, University of Milan, Via Di Rudinfi 8, 20142 Milan, Italy

Khan R, Hamid S, Abid S, Jafri W, Abbas Z, Islam M, Shah H, Beg S. Predictive factors for early aspiration in liver abscess. World J Gastroenterol 2008; 14(13): 2089-2093 Available from: URL: http://www.wjgnet.com/1007-9327/14/2089.asp DOI: http://dx.doi.org/10.3748/wjg.14.2089

INTRODUCTION Abstract AIM: To determine the predictive factors for early aspiration in liver abscess. METHODS: A retrospective analysis of all patients with liver abscess from 1995 to 2004 was performed. Abscess was diagnosed as amebic in 661 (68%) patients, pyogenic in 200 (21%), indeterminate in 73 (8%) and mixed in 32 (3%). Multiple logistic regression analysis was performed to determine predictive factors for aspiration of liver abscess. RESULTS: A total of 966 patients, 738 (76%) male, mean age 43 ± 17 years, were evaluated: 540 patients r e s p o n d e d t o m e d i c a l t h e ra p y w h i l e a d j u n c t i ve percutaneous aspiration was performed in 426 patients. Predictive factors for aspiration of liver abscess were: age ≥ 55 years, size of abscess ≥ 5 cm, involvement of both lobes of the liver and duration of symptoms ≥ 7 d. Hospital stay in the aspiration group was relatively longer than in the non aspiration group. Twelve patients died in the aspiration group and this mortality was not statistically significant when compared to the non aspiration group. CONCLUSION: Patients with advanced age, abscess size > 5 cm, both lobes of the liver involvement and duration of symptoms > 7 d were likely to undergo aspiration of the liver abscess, regardless of etiology. © 2008 WJG . All rights reserved.

Liver abscess, particularly due to amebiasis, is an important clinical problem in tropical regions of the world and accounts for a high number of hospital admissions[1-5]. It is usually an easily treatable condition with good clinical outcomes. There is however potential for morbidity and even mortality if proper and timely treatment is not provided[6-9]. The standard treatment of liver abscess is the use of appropriate antibiotics and supportive care. Needle aspiration can be used as an additional mode of therapy and has been promoted by some authors for routine use in the treatment of uncomplicated liver abscess. It is suggested that needle aspiration can improve response to antibiotic treatment, reduce hospital stay and the total cost of treatment[10-12]. Although ultrasound guided needle aspiration is fairly safe, it is nonetheless an invasive procedure requiring the passage of a wide bore needle into a highly vascular organ, and can be associated with the risk of bleeding. Needle aspirations, especially at the time of intervention has therefore remained a debatable issue and it seems important to determine its possible role in the treatment of liver abscess[13-16]. We have used a large database of patients admitted to hospital with liver abscess in order to determine the factors associated with the likelihood of liver abscess aspiration in the treatment of patients with uncomplicated liver abscess.

MATERIALS AND METHODS Medical records of all patients admitted to our hospital with liver abscess over a ten-year period (Jan. 1995 to Dec. www.wjgnet.com

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2004) were identified using the International classification of diseases 9th revision with clinical modification (ICD9-CM-USA) and reviewed retrospectively. Patients with complicated liver abscess, generally due to rupture of abscess, were excluded from this analysis, as the indications for needle aspiration in these patients are rather different (Figure 1). Diagnosis of liver abscess was based upon clinical history and abdominal ultrasound or CT scan findings. The following data was collected in all the patients who were diagnosed with uncomplicated liver abscess: demographic information, chief complaint, duration of fever or abdominal pain, associated illnesses, malignancy and history of biliary surgery or other procedures. Results of laboratory investigations and imaging studies done at the time of admission were recorded as were the clinical course of disease, modalities of treatments used and outcome of the patients. Patients with uncomplicated (non-ruptured) liver abscess were underwent to the following investigations: Complete blood counts, imaging by ultrasound, Indirect Hem-agglutination Assay (IHA) for amebiasis, blood culture and pus culture if the abscess was aspirated. IHA was done with serology reagent “Cellognost Amebiasis” supplied by (Dade Behring Marburg GmbH Germany) and a titer of ≥ 1:128 was taken as diagnostic for amebic liver abscess, as per the manufacturer’s recommendations. Based upon the results of these investigations, patients with liver abscess were categorized into four groups according to the following criteria: (1) Amebic liver abscess (ALA): IHA titer ≥ 1:128 with negative blood or pus culture. (2) Pyogenic liver abscess (PLA): IHA titer < 1:32 with or without positive blood and/or pus culture. (3) Mixed liver abscess (MLA): IHA titer ≥ 1:128 with positive blood and/or pus culture and (4) Indeterminate liver abscess (ILA): IHA titer between 1:32 and 1:128 with negative blood or pus culture. According to our usual practice, patients were started on standard treatment of liver abscess and if no clinical response was observed within three days, therapeutic percutaneous needle aspiration was carried out at the discretion of the treating physician. Needle aspiration was done under local anesthesia and ultrasound guidance without catheter drainage and the procedure was repeated in 3-4 d if optimal response was not obtained. Antibiotics were continued for 10-14 d for ALA, 4-6 wk for PLA and mixed infection and 2-6 weeks for indeterminate abscess. Statistical analysis A descriptive analysis was done for demographic, clinical and radiographic features and results were presented as mean ± SD for quantitative variables and number (percentage) for qualitative variables. In univariate analyses, differences in proportions for the group of patient underwent to needle aspiration and no aspiration was done by using the Chi-square test or Fisher exact test where appropriate. One-way analysis of variance and independent samples t-test were used to assess the difference of means for contrasts of continuous variables. Multiple logistic regression analysis was done and factors associated with likelihood of abscess aspiration were identified.

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Total patients with liver abscess (1020)

Complicated cases excluded (54) Uncomplicated liver abscess cases (966)

Pyogenic liver abscess (200) Indeterminate liver abscess (73) Amebic liver abscess (661)

Mixed liver abscess (32)

Uncomplicated liver abscess cases (966)

Aspiration done (426)

Medical therapy only (540)

Figure 1 Flow diagram of the patients with liver abscess and treatment received.

RESULTS A total of 1020 patients with liver abscess were admitted during the study period. Fifty four patients with complicated liver abscess were excluded from the study and 966 patients with uncomplicated liver abscess were evaluated (Figure 1). The mean age was 43 ± 17 years and 738 (76%) were males. The abscess was diagnosed as amebic (ALA) in 661 (68%), pyogenic (PLA) in 200 (21%), indeterminate (ILA) in 73 (8%) and mixed (MLA) in 32 (3%) patients. Clinical features of the patients in different types of liver abscess are presented in Table 1. Five hundred and forty patients responded to medical therapy alone; adjunctive percutaneous aspiration was performed in 426 patients. Demographic, clinical and laboratory features of the two groups are compared in Tables 2-4. There were significant differences between aspiration and non aspiration groups for many covariates. In the aspiration group, more patients were older than 55 years (OR = 1.008; 95% CI = 1.0-1.01), duration of symptoms was more than 7 d (OR=1.60; 95% CI = 1.21-2.11), they were more likely to be jaundiced (OR = 1.55; 95% CI = 1.18-2.17), have tender hepatomegaly (OR = 0.68; 95% CI = 0.48-0.97) and hospital stay of more than 5 d (OR = 2.99; 95% CI = 1.75-2.99), as compared to the non-aspiration group (Table 2). In the laboratory features the meaningful predictors of aspiration were elevated total bilirubin (OR = 1.09; 95% CI = 1.04-1.15), ALT (OR = 1.002; 95% CI = 1.0-1.004), alkaline phosphatase (OR = 1.002; 95% CI = 1.001-1.003), total leukocyte count (OR = 1.01; 95% CI = 1.004-1.03) and platelet count (OR = 1.001; 95% CI = 1.0-1.002), whereas relatively low serum albumin (OR = 0.59; 95% CI = 0.44-0.80) was found in the aspiration group as compared to the non aspiration group (Table 3). The aspiration group, when compared with the nonaspiration group, was also found to have more patients with abscess sizes larger than 5 cm (OR = 1.59; 95% CI = 1.21-2.09), multiple abscesses (OR = 1.66; 95% CI = 1.23-2.24), involvement of both lobes of the liver (OR =

Khan R et al . Liver abscess and aspiration

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Table 1 Clinical features of the patients with different types of liver abscess n (%) Characteristics

Table 3 Laboratory features of the patients with liver abscess Characteristics (mean ± SD)

Amebic Pyogenic Mixed Indeterminate P abscess abscess abscess abscess value (n = 661) (n = 200) (n = 32) (n = 73)

Gender Male 568 (86) Female 93 (14) Age < 55 yr 471 (71) 190 (29) ≥ 55 yr Duration of symptoms1 453 (69) ≥7d 5 cm

0.352

Aspiration Non Odds 95% CI P group aspiration ratio value (n = 426) group (n = 540) 454 (84.1) 86 (15.9)

2.24 ± 0.50 1.37 ± 1.20

P

value

Table 4 Radiological features of patients with liver abscess (%)

Table 2 Clinical features of patients with liver abscess

365 (85.7) 61 (14.3)

81.29 ± 98.87 18.45 ± 141.3

95% CI

Variable compared to reference category (OR = 1), aP < 0.05; ALT: Alanine aminotransferase.

Fever or abdominal pain.

Gender Male Female Age < 55 yr ≥ 55 yr Duration of symptoms1 (n%) 5 cm Location of abscess One lobe Both lobes Duration of symptoms C COX-2 polymorphism. Clin Cancer Res 2005; 11: 4090-4096 11 Chell S, Patsos HA, Qualtrough D, H-Zadeh AM, Hicks DJ, Kaidi A, Witherden IR, Williams AC, Paraskeva C. Prospects in NSAID-derived chemoprevention of colorectal cancer. Biochem Soc Trans 2005; 33: 667-671 12 Chen ZG, En BT, Zhang ZQ. Simultaneous determination of eight organic acids in Fructus mume by RP-HPLC. Zhongguo Zhongyao Zazhi 2006; 31: 1783-1786 13 Yin ZQ, Ye WC, Zhao SX. Studies on the chemical constituents of Bombyx batryticatus. Zhongguo Zhongyao Zazhi 2004; 29: 52-54 14 E l k h a r w i l y A , G o t t l i e b K . T h e p a n c r e a s i n f a m i l i a l adenomatous polyposis. JOP 2008; 9: 9-18 15 Kumar SK, Ram S, Jorgensen MG, Shuler CF, Sedghizadeh PP. Multicentric peripheral ossifying fibroma. J Oral Sci 2006; 48: 239-243 16 Chimenos-Kustner E, Pascual M, Blanco I, Finestres F. Hereditary familial polyposis and Gardner's syndrome: contribution of the odonto-stomatology examination in its diagnosis and a case description. Med Oral Patol Oral Cir Bucal 2005; 10: 402-409 S- Editor Li DL L- Editor Wang XL

E- Editor Yin DH

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BOOK REVIEW

Rome Ⅲ: The functional gastrointestinal disorders, third edition, 2006 Randa Mostafa Randa Mostafa, Department of Basic Medical Sciences, College of Medicine, Sharjah University, Sharjah, United Arab Emirates Author contributions: Randa Mostafa summarized the content of the 3rd edition of Rome Ⅲ, assessed its quality and noted its contribution to the field; Randa Mostafa analyzed and compared Rome Ⅲ with the previous edition of Rome Ⅱ. Correspondence to: Professor Randa Mostafa, Department of Basic Medical Sciences, College of Medicine, Sharjah University, PO Box: 27272, Sharjah, United Arab Emirates. [email protected] Telephone: +971-6-5057204  Fax: +971-6-5585879 Received: November 16, 2007 Revised: December 14, 2007

Abstract Functional gastrointestinal disorders (FGIDs) represent a common and important class of disorders within gastroenterology. RomeⅠ, the first edition was published in 1994, with symptom-based diagnostic criteria for FGIDs. These criteria began to change the diagnostic approach to FGIDs, and no longer considered "diagnoses of exclusion" but rather "diagnoses of inclusion". Rome Ⅱ, the second edition published in 2000, resulted from the continual process of analyzing new scientific and clinical evidence in the study of FGIDs. Rome Ⅱ, diagnostic criteria for irritable bowel syndrome (IBS), was extended with a focus on the frequency of symptoms occurring twelve weeks (not necessarily consecutive weeks) within twelve months. ROME Ⅲ, the third edition, conservative one, was published in September 2006, with changes made only where there is good evidence to do so. Some of the differences between Rome Ⅱ and Rome Ⅲ criteria are highlighted in this issue.

a common and important class of disorders within gastroenterology. They are a group of disorders in clinical medicine that have often posed immense problems for patients to experience, for clinicians to diagnose and treat, and for researchers to study. The “road to Rome” began in Rome, Italy, in 1988 during the 12th International Congress of Gastroenterology, during which a working team was set up to create guidelines for the management and study of irritable bowel syndrome (IBS). After a publication outlining the classification system in 1990, several committees convened in Rome, Italy, throughout 1994 and began a process of review and analysis of the medical literature to improve the methodology for studying, diagnosing and treating 21 FGIDs. The ultimate goal was to improve the lives of patients and their families. The process has matured through three generations, producing a series of publications (RomeⅠ, Ⅱ and Ⅲ), with an increased evidence-based approach to the recommendations.

ROME Ⅰ AND ROME Ⅱ

Mostafa R. Rome Ⅲ: The functional gastrointestinal disorders, third edition, 2006. World J Gastroenterol 2008; 14(13): 2124-2125 Available from: URL: http://www.wjgnet.com/1007-9327/14/2124. asp DOI: http://dx.doi.org/10.3748/wjg.14.2124

RomeⅠ, the first edition published in1994, is a compilation of documents previously published in Gastroenterology International over a period of 5 years by 30 international investigators who categorized the FGIDs from the esophagus to the anus. The most striking result of this process is the creation of the RomeⅠsymptom-based diagnostic criteria for FGIDs. These criteria began to change the diagnostic approach to FGIDs, and no longer considered “diagnoses of exclusion” but rather “diagnoses of inclusion”. The Rome criteria have enabled positive diagnoses without the need for extensive and unnecessary diagnostic studies to “rule out organic diseases”. Rome Ⅱ, the second edition, published in 2000, resulted from the continual process of analyzing new scientific and clinical evidence in the study of FGIDs. Rome Ⅱ , diagnostic criteria for irritable bowel syndrome (IBS), is extended with a focus on the frequency of symptoms occurring twelve weeks (not necessarily consecutive weeks) within twelve months. For the first time, pediatric FGIDs were categorized, and chapters highlighting physiology of motility, sensation, brain-gut interactions, and psychosocial aspects were included.

INTRODUCTION

ROME Ⅲ

Functional gastrointestinal disorders (FGIDs) represent

ROME Ⅲ , the third edition, published in September

© 2008 WJG . All rights reserved.

Key words: Rome Ⅲ; Functional gastrointestinal disorders; Diagnosis; Classification Peer reviewer: Olivier Barbier, CHUQ-CHUL Research Center, 2705 Laurier Boulevard, Québec G1V 4G2, Canada

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Mostafa RM. Rome Ⅲ: The functional gastrointestinal disorders

2006, is a 1048-page document written by a collaborative effort of 82 international experts. The book consists of seventeen chapters that contain the most recent information on the epidemiology, pathophysiology, diagnosis, and treatment of FGIDs. Diagnostic criteria for some of the FGIDs have been revised. “Red flag” symptoms and signs that warrant further diagnostic evaluation have been included. Suggestions for when to make a mental health referral have also been given with new chapters on pharmacology and pharmacokinetics, sociocultural perspectives related to gender, age, and cultural impact. One chapter is also devoted to the development and validation of the Rome Ⅲ: Diagnostic Questionnaire. New appendices contain validated Rome Ⅲ: adult and pediatric questionnaires and a table comparing Rome Ⅱ and Rome Ⅲ diagnostic criteria.

CHANGES IN ROME Ⅲ The Rome Ⅲ process is a conservative one, with changes made only where there is good evidence to do so. The following is a summary of the changes in criteria and other recom­mendations along with their justification. Time frame change for FGIDs The time frame for a diagnosis now originates at 6 mo prior to clinical presentation and diagnosis and must be currently active (i.e., meet criteria) for 3 mo. This time frame is less restrictive than that in Rome Ⅱ (12 wk of symp­toms over 12 mo) and is easier to understand in a questionnaire or for research and clinical practice. Changes in classification categories The Rome Ⅱ : category of Childhood Functional GI Dis­orders, is now classified as Childhood Functional GI Disorders: Neonate/Toddler (Category G) and Childhood Functional GI Disorders: Child/Adolescent (Category H). This reflects the differ­ent clinical conditions existing between the two categories relating to the growth and develop­ment of the child, thus removing Functional abdominal pain syndrome (FAPS) from functional bowel disorders (Category C) into its own category (Category D). This is based on the growing evi­dence that FAPS relates more to CNS amplification of normal regulatory visceral signals rather than functional abnormalities per se within the GI tract. The committee members selected for this new category included psychologists, psychiatrists and gastroenterologists involved in brain gut interactions. Criteria changes In Rome Ⅲ, functional dyspepsia is de-­emphasized as an entity for research, due to its symptom het­erogeneity. Instead, the gastro­duodenal committee has recom­ mended using an umbrella term “dyspepsia symptom com­plex” which is subclassified into two conditions that may overlap: (1) Postprandial Distress Syndrome, and (2) Epigastric Pain Syndrome. Although similar to the

  

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dysmotility like and ulcer like dyspepsia in Rome Ⅱ, there now are several items for the criteria derived from factor analytic studies and physiological sup­port instead of being based on the single symptom of epigastric discomfort or pain. Further studies are needed to vali­date this change. Functional biliary tract disorders have been a challenging group of disorders to diagnose and treat. These disorders are of low prevalence in comparison to other FGIDs, but they tend to be investigated with invasive and risky studies, such as endoscopic retrograde pancreatography (ERCP) or sphincter of Oddi manometry, and treated with unnecessary endoscopic sphincterotomy and surger y. Rome Ⅲ recommends more restrictive evaluation of these disorders. Rome Ⅲ is the single most comprehensive and authoritative resource on the subject of FGIDs. It is readable, well organized, clearly labeled, and extensively referenced. The 82 experts who participated in the Rome Ⅲ process have created an outstanding work from which clinicians, clinical investigators, basic scientists, mental health providers, the pharmaceutical industry and, most of all, patients with FGIDs will greatly benefit.

CONCLUSION The information obtained in Rome Ⅲ is comprehensive although certainly not complete. It is likely that the next few years will bring considerable advances in our understanding and treatment of these disorders, and when that occurs, revision of such information should be planned before moving to Rome Ⅳ. Brain imaging, using positron emission tomography, functional magnetic resonance imaging, or other modalities[1-3] (Drossman 2005, Hobson 2004, and Hobson 2005) provides an opportunity to assess brain function in response to visceral stimulation[4,5] (Kern 2002, Yaguez 2005) among healthy subjects and patients with FGIDs. Developing standardization for brain imaging assessment and making recommendations relating to symptom severity for research and clinical care should be emphasized in the next edition.

REFERENCES 1 2 3

4 5

Drossman DA. Brain imaging and its implications for studying centrally targeted treatments in irritable bowel syndrome: a primer for gastroenterologists. Gut 2005; 54: 569-573 Hobson AR, Aziz Q. Brain imaging and functional gastrointestinal disorders: has it helped our understanding? Gut 2004; 53: 1198-1206 Hobson AR, Furlong PL, Worthen SF, Hillebrand A, Barnes GR, Singh KD, Aziz Q. Real-time imaging of human cortical activity evoked by painful esophageal stimulation. Gastroenterology 2005; 128: 610-619 Kern MK, Shaker R. Cerebral cortical registration of subliminal visceral stimulation. Gastroenterology 2002; 122: 290-298 Yaguez L, Coen S, Gregory LJ, Amaro E Jr, Altman C, Brammer MJ, Bullmore ET, Williams SC, Aziz Q. Brain response to visceral aversive conditioning: a functional magnetic resonance imaging study. Gastroenterology 2005; 128: 1819-1829 S- Editor Li DL L- Editor Wang XL E- Editor Lu W

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World J Gastroenterol 2008 April 7; 14(13): 2126-2127 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

LETTERS TO THE EDITOR

Role of ABCC2 common variants in intrahepatic cholestasis of pregnancy Silvia Sookoian, Gustavo Castaño, Carlos J Pirola Silvia Sookoian, Carlos J Pirola, Department of Molecular Genetics and Biology of Complex Diseases, Institute of Medical Research A. Lanari, University of Buenos Aires-CONICET, Buenos Aires 1427, Argentina Silvia Sookoian, Gustavo Castaño, Research Council of GCBA, Buenos Aires 1427, Argentina Author contributions: Sookoian S, Castaño G, and Pirola CJ designed and performed the research; Sookoian S and Pirola CJ wrote the paper; Pirola CJ the analyzed data. Correspondence to: Silvia Sookoian, MD, PhD, Instituto de Investigaciones Médicas A. Lanari-Conicet, Combatiente de Malvinas 3150, Buenos Aires 1427, Argentina. [email protected] Telephone: +54-11-45148701 Fax: +54-11-45238947 Received: January 9, 2008 Revised: February 19, 2008

Abstract The pathogenesis of intrahepatic cholestasis of pregnancy (ICP), a disorder that adversely affects maternal wellbeing and fetal outcome, is unclear. However, multiple factors probably interact along with a genetic predisposition. We would like to add some comments on a paper recently published concerning the role of ABCB11 and ABCC2 polymorphisms in both ICP and contraceptive-induced cholestasis, especially in the light of our recently published findings about a positive association between ICP and ABCC2 common variants. © 2008 WJG . All rights reserved.

Key words: Intrahepatic cholestasis of pregnancy;

ABCC2 ; MRP2; Gene variants

Peer reviewers: Eldon Shaffer, Professor of Medicine, Division

of Gastroenterology, Department of Medicine, Health Science Centre, University of Calgary, 3330 Hospital Dr N.W., Calgary, AB, T2N4N1, Canada; Limas Kupcinskas, Gastroenterology of Kaunas University of Medicine, Mickeviciaus 9, Kaunas LT 44307, Lithuania Sookoian S, Castaño G, Pirola CJ. Role of ABCC2 common variants in intrahepatic cholestasis of pregnancy. World J Gastroenterol 2008; 14(13): 2126-2127 Available from: URL: http:// www.wjgnet.com/1007-9327/14/2126.asp DOI: http://dx.doi. org/10.3748/wjg.14.2126

To the editor We read with great interest the article of Meier et al. www.wjgnet.com

about the role of ABCB11 and ABCC2 polymorphisms in both intrahepatic cholestasis of pregnancy (ICP) and contraceptive-induced cholestasis [1]. The authors observed an association between the ABCB11 1331T > C polymorphism and the above mentioned cholestatic disorders. Additionally, the authors claimed of a lack of association with 3 single nucleotide polymorphisms (SNPs) in ABCC2 gene, concluding that common ABCC2 polymorphisms are not associated with the development of ICP. We would like to make several comments on these findings, particularly concerning to the lack of association with ABCC2 gene variants. First of all, a note of caution should be added to the reported findings, as the markers assumed by the authors as common variants (rs2273697, rs17222723 and rs8187710), except for rs2273697, include one allele at a very low frequency, as the rs17222723-A allele frequency is 0.067 and the rs8187710-A allele frequency is 0.059, at least for data from the HapMap project for Caucasians and from a rough estimation of own author’s data[1]. Consequently, in the group of patients included by the authors only 5 out 33 patients showed either the rs172227234 or rs8187710 heterozygous AT or AG genotype, respectively, and none of them showed the homozygous AA genotype. The same happened in the pregnant control group. Thus, in this frame, the statistical power of the study even taking into account the additive genetic model is very low (less than 20%) owing to the lower MAF of these variants. Second, we wish to note that we recently published[2] a candidate gene association study showing the contribution of six ABCC2 gene variants to the risk of ICP. The study involved promoter, coding and non-coding regions of ABCC2 (4 tag SNPs representing 46 polymorphic sites located in 70 kb of the gene, in addition to the rs17222723 and rs8187710), and showed that, at least, one of the ABCC2 variants (rs3740066) at the exon 28 was significantly associated with ICP with its estimated risk of disease for homozygous AA subjects being 4-fold higher than that for homozygous GG subjects (OR, 4.44; 95% CI, 1.83-10.78; P < 0.001). Although more studies are necessary to establish whether rs3740066 is the causal variant or one linked to it, our results suggest that ICP may be associated with the common variants of ABCC2. Interestingly, we also included in the analysis rs172227234 and rs8187710, and we did not obser ve significant differences in genotype frequencies of the 2 SNPs in ICP and controls.

Sookoian S et al. ABCC2 and cholestasis of pregnancy



In conclusion, we consider that the pathogenic involvement of ABCC2 (MRP2) in ICP is still an open issue, particularly in the frame of a small number of studies about the role of the ABCC2 gene variants in cholestatic disorders, with the exception of the Dubin Johnson phenotype. Although the major physiological function of ABCC2 is to transport conjugated metabolites into the bile canaliculus, previous data demonstrated that a major metabolite of human estrogen metabolism, estradiol17- β -D-glucuronide (E 217 β G), has been shown to be transported by both MRP2 and MRP3[3]. These findings support that ABCC2 represents an alternative candidate protein involved in the pathogenesis of hor monal cholestasis. Additionally, it has been shown that MRP2 is regulated by three distinct nuclear receptor signaling pathways that converge on a common response element in the 5'-flanking region of this gene[4]. Hence, the intimate mechanism, by which gene variants including ABCC2 may influence ICP susceptibility, is not fully explored and a complex network involving nuclear receptors and other transcription factors may be the cause of liver injury in cholestatic disorders[5].



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REFERENCES 1

2

3

4

5

Meier Y, Zodan T, Lang C, Zimmermann R, Kullak-Ublick GA, Meier PJ, Stieger B, Pauli-Magnus C. Increased susceptibility for intrahepatic cholestasis of pregnancy and contraceptiveinduced cholestasis in carriers of the 1331T > C polymorphism in the bile salt export pump. World J Gastroenterol 2008; 14: 38-45 Sookoian S, Castano G, Burgueno A, Gianotti TF, Pirola CJ. Association of the multidrug-resistance-associated protein gene (ABCC2) variants with intrahepatic cholestasis of pregnancy. J Hepatol 2008; 48: 125-132 Ito K, Oleschuk CJ, Westlake C, Vasa MZ, Deeley RG, Cole SP. Mutation of Trp1254 in the multispecific organic anion transporter, multidrug resistance protein 2 (MRP2) (ABCC2), alters substrate specificity and results in loss of methotrexate transport activity. J Biol Chem 2001; 276: 38108-38114 Kast HR, Goodwin B, Tarr PT, Jones SA, Anisfeld AM, Stoltz CM, Tontonoz P, Kliewer S, Willson TM, Edwards PA. Regulation of multidrug resistance-associated protein 2 (ABCC2) by the nuclear receptors pregnane X receptor, farnesoid X-activated receptor, and constitutive androstane receptor. J Biol Chem 2002; 277: 2908-2915 Zollner G, Marschall HU, Wagner M, Trauner M. Role of nuclear receptors in the adaptive response to bile acids and cholestasis: pathogenetic and therapeutic considerations. Mol Pharm 2006; 3: 231-251 S- Editor Zhong XY

L- Editor Wang XL

E- Editor Ma WH

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ACKNOWLEDGMENTS

Acknowledgments to Reviewers of World Journal of Gastroenterology Many reviewers have contributed their expertise and time to the peer review, a critical process to ensure the quality of World Journal of Gastroenterology. The editors and authors of the articles submitted to the journal are grateful to the following reviewers for evaluating the articles (including those published in this issue and those rejected for this issue) during the last editing time period. Bruno Annibale, Professor, Digestive and Liver Disease Unit, University “La Sapienza” II School of Medicine, Via di Grottarossa 1035, Roma 00189, Italy Juan G Abraldes, MD, Hepatic Hemodynamic Laboratory, Liver Unit. Hospital Clinic. University of Barcelona, Hepatic Hemodynamic Lab. Liver Unit, Hospital Clinic, Villarroel 170, Barcelona 08036, Spain Philip Abraham, Dr. Professor, Consultant Gastroenterologist & Hepatologist, P. D. Hinduja National Hospital & Medical Research Centre, Veer Savarkar Marg, Mahim, Mumbai 400 016, India Luigi Bonavina, Professor, Department of Surgery, Policlinico San Donato, University of Milano, via Morandi 30, Milano 20097, Italy Wallace F Berman, MD, Professor, Box 3009 DUMC, Durham, NC 27710, United States Adrian G Cummins, Dr, Department of Gastroenterology and Hepatology, (DX 465384), 28 Woodville Road, Woodville South, 5011, South Australia, Australia Andrew Seng Boon Chua, MD, Department of Gastroenterology, Gastro Centre Ipoh, 1, lorong Rani, 31, lebuhraya Tmn Ipoh, Ipoh Garden South, IPOH 30350, Malaysia Giuseppe Chiarioni, Dr, Gastroenterological Rehabilitation Division of the University of Verona, Valeggio sul Mincio Hospital, Azienda Ospedale di Valeggio s/M, Valeggio s/M 37067, Italy Bijan Eghtesad, Dr, Associate Professor, Department of General Surgery, Cleveland Clinic Foundation, 9500 Euclid Avenue, Cleveland OH 44195, United States Volker F Eckardt, Chief, MD, Professor, Department of Gastroenterology, Deutsche Klinik für Diagnostik, Aukammallee 33, 65191 Wiesbaden, Germany Ulrich R Fölsch, Professor, 1st Department of Medicine, Christian-Albrechts-University of Kiel, Schittenhelmstrasse 12, Kiel 24105, Germany Daniel R Gaya, Dr, Gastrointestinal Unit, Molecular Medicine Centre, School of Molecular and Clinical Medicine, University of Edinburgh, Western General Hospital, Crewe Road, Edinburgh EH4 2XU, United Kingdom Chee-Kin Hui, MD, University of Hong Kong, Queen Mary Hospital, Department of Medicine, 102 Pokfulam Road, Hong Kong SAR, China Kazuhiro Hanazaki, MD, Professor and Chairman, Department of Surgery, Kochi Medical School, Kochi University, Kohasu, Okohcho, Nankoku, Kochi 783-8505, Japan Dusan M Jovanovic, Professor, Institute of Oncology, Institutski Put 4, Sremska Kamenica 21204, Serbia

Janusz AZ Jankowski, MD, PhD, Professor, University of Oxford Dept Clinical Pharmacology, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, United Kingdom Milan Jirsa, Dr, Laboratory of Experimental Medicine - building Z1,Institute for Clinical and Experimental Medicine, Videnska 1958/9, Praha 4, 140 00, Czech Tom H Karlsen, MD, Institute of Immunology, Rikshospitalet University Hospital, N-0027 Oslo, Norway Walter Edwin Longo, Professor, Department of Surgery, Yale University School of Medicine, 205 Cedar Street, New Haven 06510, United States Finlay A Macrae, MD, Professor, Royal Melbourne Hospital, Po Box 2010, Victoria 3050, Australia Morito Monden, Professor, Department of Surgery and Clinical Oncology, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita 565-0871, Japan Thomas I Michalak, MD, PhD, Molecular Virology and Hepatology Research, Faculty of Medicine, Health Sciences Center, Memorial University, 300 Prince Philip Drive, St. Jonh’s, NL A1B 3V6, Canada Susumu Ohwada, Associate Professor, Department of Surgery, Gunma University Graduate School of Medicine, 3-39-15 Shoma-Machi, Maebashi 371-8511, Japan Ian C Roberts-Thomson, Professor, Department of Gastroenterology and Hepatology, The Queen Elizabeth Hospital, 28 Woodville Road, Woodville South 5011, Australia Vasiliy I Reshetnyak, MD, PhD, Professor, Scientist Secretary of the Scientific Research Institute of General Reanimatology, 25-2, Petrovka str., 107031, Moscow, Russia Mitsuo Shimada, Professor, Department of Digestive and Pediatric Surgery, Tokushima University, Kuramoto 3-18-15, Tokushima 770-8503, Japan Shanthi V Sitaraman, MD, Division of Digestive Diseases, Room 201-F, 615, Michael Street, Whitehead Research Building, Emory University, Atlanta, Georgia 30322, United States Vincenzo Stanghellini, MD, Professor of Medicine, Department of Internal Medicine and Gastroenterology, Policlinico S.OrsolaMalpighi, University of Bologna, VIA MASSARENTI 9, Bologna I-40138, Italy Natalie J Torok, Dr, UC Davis Medical Center, Patient Support Services Building, 4150 V Street, Suite 3500, Sacramento, CA 95817, United States Stefan Wirth, Professor, Dr, Children's Hospital, Heusnerstt. 40, Wuppertal 42349, Germany Harry HX Xia, PhD, MD, Novartis Pharmaceuticals Corporation, One Health Plaza, East Hanover, NJ 07936-1080, United States Jia-Yu Xu, Professor, Shanghai Second Medical University, Rui Jin Hospital, 197 Rui Jin Er Road, Shanghai 200025, China Takayuki Yamamoto, MD, Inflammatory Bowel Disease Center, Yokkaichi Social Insurance Hospital, 10-8 Hazuyamacho, Yokkaichi 510-0016, Japan Shu Zheng, Professor, Scientific Director of Cancer Institute, Zhejiang University, Secondary Affiliated Hospital, Zhejiang University, 88# Jiefang Road, Hangzhou 310009, Zhejiang Province, China

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Meetings Events Calendar 2008-2009 FALK SYMPOSIA 2008 January 24-25, Frankfurt, Germany Falk Workshop: Perspectives in Liver Transplantation International Gastroenterological Congresses 2008 February 14-16, Paris, France EASL-AASLD-APASL-ALEH-IASL Conference Hepatitis B and C virus resistance to antiviral therapies www.easl.ch/hepatitis-conference February 14-17, Berlin, Germany 8th International Conference on New Trends in Immunosuppression and Immunotherapy www.kenes.com/immuno February 28, Lyon, France 3rd Congress of ECCO - the European Crohn’s and Colitis Organisation Inflammatory Bowel Diseases 2008 www.ecco-ibd.eu March 10-13, Birmingham, UK British Society of Gastroenterology Annual Meeting E-mail: [email protected] March 14-15, HangZhou, China Falk Symposium 163: Chronic Inflammation of Liver and Gut March 23-26, Seoul, Korea Asian Pacific Association for the Study of the Liver 18th Conference of APASL: New Horizons in Hepatology www.apaslseoul2008.org March 29-April 1, Shanghai, China Shanghai - Hong Kong International Liver Congress www.livercongress.org April 05-09, Monte-Carlo (Grimaldi Forum), Monaco OESO 9th World Congress, The Gastro-esophageal Reflux Disease: from Reflux to Mucosal Inflammation - Management of Adeno- carcinomas Email: [email protected] April 18-22, Buenos Aires, Argentina 9th World Congress of the International Hepato-Pancreato Biliary Association Association for the Study of the Liver www.ca-ihpba.com.ar April 23-27, Milan, Italy 43rd Annual Meeting of the European

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Association for the Study of the Liver www.easl.ch May 2-3, Budapest, Hungary Falk Symposium 164: Intestinal Disorders

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May 17-20, Denver, Colorado, USA Digestive Disease Week 2009

May 18-21, San Diego, California, USA Digestive Disease Week 2008 June 4-7, Helsinki, Finland The 39th Nordic Meeting of Gastroenterology www.congrex.com/ngc2008 June 6-8, Prague, Czech Republic 3rd Annual European Meeting: Perspectives in Inflammatory Bowel Diseases Email: [email protected] June 13-14, Amsterdam, Netherlands Falk Symposium 165: XX International Bile Acid Meeting. B ile Acid Biology and Therapeutic Actions June 25-28, Barcelona, Spain 10th World Congress on Gastrointestinal Cancer Imedex and ESMO Email: [email protected] June 25-28, Lodz, Poland Joint Meeting of the European Pancreatic Club (EPC) and the International Association of Pancreatology (IAP) E-mail: [email protected] www.e-p-c.org www.pancreatology.org June 26-28, Bratislava, Slovakia 5th Central European Gastroenterology Meeting www.ceurgem2008.cz September 10-13, Budapest, Hungary 11th World Congress of the International Society for Diseases of the Esophagus Email: [email protected] September 13-16, New Delhi, India Asia Pacific Digestive Week E-mail: [email protected] III FALK GASTRO-CONFERENCE September 17, Mainz, Germany Falk Workshop: Strategies of Cancer Prevention in Gastroenterology September 18-19, Mainz, Germany Falk Symposium 166: GI Endoscopy - Standards & Innovations September 18-20, Prague, Czech Republic Prague Hepatology Meeting 2008 www.czech-hepatology.cz/phm2008 September 20-21, Mainz, Germany Falk Symposium 167: Liver Under Constant Attack - From

Congresses 2009 March 23-26, Glasgow, Scotland Meeting of the British Society of Gastroenterology (BSG) E-mail: [email protected]

November 21-25, London, UK Gastro 2009 UEGW/World Congress of Gastroenterology www.gastro2009.org October 8-11, Istanbul, Turkey 18th World Congress of the International Association of Surgeons, Gastroenterologists and Oncologists E-mail: [email protected] October 18-22, Vienna, Austria 16th United European Gastroenterology Week www.negf.org www.acv.at October 22-25, Brisbane, Australia Anstralian Gastroenterology Week 2008 Email: [email protected] October 31-November 4, Moscone West Convention Center, San Francisco, CA 59th AASLD Annual Meeting and Postgraduate Course The Liver Meeting Information: www.aasld.org November 6-9, Lucerne, Switzerland Neurogastroenterology & Motility Joint International Meeting 2008 Email: [email protected] www.ngm2008.com November 12, Santiago de Chile, Chile Falk Workshop: Digestive Diseases: State of the Art and Daily Practice December 7-9, Seoul, Korea 6th International Meeting Hepatocellular Carcinoma: Eastern and Western Experiences E-mail: [email protected] INFORMATION FOR ALL FALK FOUNDATION e.V. Email: [email protected] www.falkfoundation.de Advanced Courses - European Institute of Telesurgery EITS - 2008 Strasbourg, France January 18-19, March 28-29, June 6-7, October 3-4 N.O.T.E.S April 3-5, November 27-29 Laparoscopic Digestive Surgery June 27-28, November 7-8 Laparoscopic Colorectal Surgery July 3-5 Interventional GI Endoscopy Techniques Contact address for all courses: [email protected] International Gastroenterological

Global Collaboration for Gastroenterology For the first time in the history of gastroenterology, an international conference will take place which joins together the forces of four pre-eminent organisations: Gastro 2009, UEGW/WCOG London. The United European Gastroenterology Federation (UEGF) and the World Gastroenterology Organisation (WGO), together with the World Organisation of Digestive Endoscopy (OMED) and the British Society of Gastroenterology (BSG), are jointly organising a landmark meeting in London from November 21-25, 2009. This collaboration will ensure the perfect balance of basic science and clinical practice, will cover all disciplines in gastroenterology (endoscopy, digestive oncology, nutrition, digestive surgery, hepatology, gastroenterology) and ensure a truly global context; all presented in the exciting setting of the city of London. Attendance is expected to reach record heights as participants are provided with a compact “all-in-one” programme merging the best of several GI meetings. Faculty and participants from all corners of the earth will merge to provide a truly global environment conducive to the exchange of ideas and the forming of friendships and collaborations.

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World J Gastroenterol 2008 April 7; 14(13): 2130-2132 World Journal of Gastroenterology ISSN 1007-9327 © 2008 WJG. All rights reserved.

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Instructions to authors GENERAL INFORMATION World Journal of Gastroenterology (World J Gastroenterol ISSN 1007-9327 CN 14-1219/R) is a weekly open access peer-reviewed journal supported by an editorial board consisting of 1215 experts in gastroenterology and hepatology from 60 countries. The aim of the journal is to deliver the most clinically relevant original and commentary articles to readers, and to make the full text publicly available to all clinicians, scientists, patients and biomedical students on an unrestricted platform, so that they can access and learn about the most recent key advances in the field. In addition to the open access nature, another key characteristic of WJG is its reading guidance for each article which includes background, research frontier, related reports, breakthroughs, applications, terminology, and comments of peer reviewers for the general readers. WJG publishes articles on esophageal, gastrointestinal, hepatobiliary and pancreatic tumors, and other esophageal, gastrointestinal, hepatic-biliary and pancreatic diseases in relation to epidermiology, immunology, microbiology, motility & nerve-gut interaction, endocrinology, nutrition & obesity, endoscopy, imaging and advanced hi-technology. The main goal of WJG is to publish high quality commentary articles contributed by leading experts in gastroenterology and hepatology and original articles that combine the clinical practice and advanced basic research, to provide an interactive platform for clinicians and researchers in internal medicine, surgery, infectious diseases, traditional Chinese medicine, oncology, integrated Chinese and Western medicine, imaging, endoscopy, interventional therapy, pathology and other basic medical specialities, and thus eventually improving the clinical practice and healthcare for patients. Indexed and abstracted in Current Contents®/Clinical Medicine, Science Citation Index Expanded (also known as SciSearch®) and Journal Citation Reports/ Science Edition, Index Medicus, MEDLINE and PubMed, Chemical Abstracts, EMBASE/Excerpta Medica, Abstracts Journals, Nature Clinical Practice Gastroenterology and Hepatology, CAB Abstracts and Global Health. ISI JCR 2003-2000 IF: 3.318, 2.532, 1.445 and 0.993. Published by The WJG Press

SUBMISSION OF MANUSCRIPTS Manuscripts should be typed double-spaced on A4 (297 mm × 210 mm) white paper with outer margins of 2.5 cm. Number all pages consecutively, and start each of the following sections on a new page: Title Page, Abstract, Introduction, Materials and Methods, Results, Discussion, Acknowledgements, References, Tables, Figures, and Figure Legends. Neither the editors nor the publisher are responsible for the opinions expressed by contributors. Manuscripts formally accepted for publication become the permanent property of The WJG Press, and may not be reproduced by any means, in whole or in part, without the written permission of both the authors and the publisher. We reserve the right to copy-edit and put onto our website accepted manuscripts. Authors should follow the relevant guidelines for the care and use of laboratory animals of their institution or national animal welfare committee. For the sake of transparency in regard to the performance and reporting of clinical trials, we endorse the policy of the International Committee of Medical Journal Editors to refuse to publish papers on clinical trial results if the trial was not recorded in a publicly-accessible registry at its outset. The only register now available, to our knowledge, is http://www. clinicaltrials.gov sponsored by the United States National Library of Medicine and we encourage all potential contributors to register with it. However, in the case that other registers become available you will be duly notified. A letter of recommendation from each author’s organization should be provided with the contributed article to ensure the privacy and secrecy of research is protected. Authors should retain one copy of the text, tables, photographs and illustrations because rejected manuscripts will not be returned to the author(s) and the editors will not be responsible for loss or

Online submissions Manuscripts should be submitted through the Online Submission System at: http://wjg.wjgnet.com. Authors are highly recommended to consult the ONLINE INSTRUCTIONS TO AUTHORS (http:// www.wjgnet.com/wjg/help/instructions.jsp) before attempting to submit online. For assistance, authors encountering problems with the Online Submission System may send an email describing the problem to [email protected], or by telephone: +86-10-85381892. If you submit your manuscript online, do not make a postal contribution. Repeated online submission for the same manuscript is strictly prohibited.

MANUSCRIPT PREPARATION All contributions should be written in English. All articles must be submitted using word-processing software. All submissions must be typed in 1.5 line spacing and 12 pt. font with ample margins. The preferred font is Book Antiqua. Style should conform to our house format. Required information for each of the manuscript sections is as follows: Title page Full manuscript title, running title, all author(s) name(s), affiliations, institution(s) and/or department(s) where the work was carried out; author contributions; disclosure of any financial support for the research; and the name, full address, telephone and fax numbers and email address of the corresponding author should be included. Titles should be concise and informative (remove all unnecessary words), emphasize what is new, and avoid abbreviations. A short running title of less than 40 letters should be provided. List the author(s)’ name(s) as follows: initial and/or first name, middle name or initial(s), and full family name. Author controbutions: The format of this section should be like this: Author contributions: Wang CL and Liang L contributed equally to this work; Wang CL, Liang L, Fu JF, Zou CC, Hong F and Wu XM designed research; Wang CL, Zou CC, Hong F and Wu XM performed research; Xue JZ and Lu JR contributed new reagents/ analytic tools; Wang CL, Liang L and Fu JF analyzed data; and Wang CL, Liang L and Fu JF wrote the paper. Peer reviewers: All articles received are subject to peer review. Normally, three experts are invited for each article. Decision for acceptance is made only when at least two experts recommend an article for publication. Reviewers for accepted manuscripts are acknowledged in each manuscript, and reviewers of articles which were not accepted will be acknowledged at the end of each issue. To ensure the quality of the articles published in WJG, reviewers of accepted manuscripts will be announced by publishing the name, title/ position and institution of the reviewer in the footnote accompanying the printed article. For example, reviewers: Professor Jing-Yuan Fang, Shanghai Institute of Digestive Disease, Shanghai, Affiliated Renji Hospital, Medical Faculty, Shanghai Jiaotong University, Shanghai, China; Professor Xin-Wei Han, Department of Radiology, The First Affiliated Hospital, Zhengzhou University, Zhengzhou, Henan Province, China; and Professor Anren Kuang, Department of Nuclear Medicine, Huaxi Hospital, Sichuan University, Chengdu, Sichuan Province, China. Abstract An informative, structured abstract of no more than 350 words should accompany each manuscript. Abstracts for original contributions should be structured into the following sections: AIM: Only the purpose should be included. METHODS: The materials, techniques, instruments and equipment, and the experimental procedures should be included. RESULTS: The observed and experimental results, including data, effects, outcome, etc. should be included. Authors should present P value where necessary, and also include any significant data. CONCLUSION: Accurate view and the value of the results should be included. The format for structured abstracts can be found at: http:// www.wjgnet.com/wjg/help/11.doc.

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Key words Please list 5-10 key words, selected mainly from Index Medicus, which reflect the content of the study. Text For articles of these sections, original articles, rapid communication and case reports, the main text should be structured into the following sections: INTRODUCTION, MATERIALS AND METHODS, RESULTS and DISCUSSION, and should include appropriate Figures and Tables. Data should be presented in the body text or in Figures and Tables, but not in both. The main text format of these sections, editorial, topic highlight, case report, letters to the editors, should be found at: http://www.wjgnet.com/wjg/help/instructions.jsp. Illustrations Figures should be numbered as 1, 2, 3, etc., and mentioned clearly in the main text. Provide a brief title for each figure on a separate page. Detailed legends should not be provided under the figures. This part should be added into the text where the figures are applicable. Figures should be either Photoshop or Illustrator files (in tiff, eps, jpeg formats) at high-resolution. Examples can be found at: http://www.wjgnet.com/1007-9327/13/4520. pdf; http://www.wjgnet.com/1007-9327/13/4554.pdf; http:// www.wjgnet.com/1007-9327/13/4891.pdf; http://www. wjgnet.com/1007-9327/13/4986.pdf; http://www.wjgnet. com/1007-9327/13/4498.pdf. Keeping all elements compiled is necessary in line-art image. Scale bars should be used rather than magnification factors, with the length of the bar defined in the legend rather than on the bar itself. File names should identify the figure and panel. Avoid layering type directly over shaded or textured areas. Please use uniform legends for the same subjects. For example: Figure 1 Pathological changes in atrophic gastritis after treatment. A: ...; B: ...; C: ...; D: ...; E: ...; F: ...; G: …etc. It is our principle to publish high resolution-figures for the printed and E-versions. Tables Three-line tables should be numbered 1, 2, 3, etc., and mentioned clearly in the main text. Provide a brief title for each table. Detailed legends should not be included under tables, but rather added into the text where applicable. The information should complement but not duplicate the text. Use one horizontal line under the title, a second under column heads, and a third below the Table, above any footnotes. Vertical and italic lines should be omitted. Notes in tables and illustrations Data that are not statistically significant should not be noted. aP < 0.05, b P < 0.01 should be noted (P > 0.05 should not be noted). If there are other series of P values, cP < 0.05 and dP < 0.01 are used. A third series of P values can be expressed as eP < 0.05 and fP < 0.01. Other notes in tables or under illustrations should be expressed as 1F, 2F, 3F; or sometimes as other symbols with a superscript (Arabic numerals) in the upper left corner. In a multi-curve illustration, each curve should be labeled with ●, ○, ■, □, ▲, △, etc., in a certain sequence. Acknowledgments Brief acknowledgments of persons who have made genuine contributions to the manuscripts and who endorse the data and conclusions should be included. Authors are responsible for obtaining written permission to use any copyrighted text and/or illustrations.

REFERENCES Coding system The author should number the references in Arabic numerals according to the citation order in the text. Put reference numbers in square brackets in superscript at the end of citation content or after the cited author’s name. For citation content which is part of the narration, the coding number and square brackets should be typeset normally. For example, “Crohn’s disease (CD) is associated with increased intestinal permeability[1,2]”. If references are cited directly in the text, they should be put together within the text, for example, “From references[19,22-24], we know that...” When the authors write the references, please ensure that the order in text is the same as in the references section, and also ensure the spelling accuracy of the first author’s name. Do not list the same citation twice.

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PMID requirement PMID roots in the abstract serial number indexed by PubMed (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed). The author should supply the PMID for journal citation. For those references that have not been indexed by PubMed, a printed copy of the first page of the full reference should be submitted. The accuracy of the information for journal citations is very important. Using the reference testing system, the authors and editor should check the authors name, title, journal title, publication date, volume number, start page, and end page. We will interlink all references with PubMed in an ASP file so that the readers can immediately access the abstract of the citations online. DOI requirement A CrossRef DOI® (Digital Object Identifier) name is a unique string created to identify a piece of scholarly content in the online environment. The author should supply the DOIs for journal citation(doi:10.3748/wjg.13.6458). This link (http://www.crossref.org/ SimpleTextQuery/) allows you to retrieve Digital Object Identifiers (DOIs) for journal articles, books, and chapters by simply cutting and pasting the reference list into the box. You may use the form with any reference style, although the tool works most reliably if references are formatted in a standard style such as shown in this example: Assimakopoulos SF, Scopa CD, Vagianos CE. Pathophysiology of increased intestinal permeability in obstructive jaundice. World J Gastroenterol 2007; 13(48): 6458-6464 The accuracy of the information of journal citations is very important. We will interlink all references with DOI in ASP file so that readers can access the abstracts of cited articles online immediately. Style for journal references Authors: the name of the first author should be typed in bold-faced letters. The family name of all authors should be typed with the initial letter capitalized, followed by their abbreviated first and middle initials. (For example, Lian-Sheng Ma is abbreviated as Ma LS, BoRong Pan as Pan BR). The title of the cited article and italicized journal title (journal title should be in its abbreviated form as shown in PubMed), publication date, volume number (in black), start page, and end page [PMID: 11819634 DOI: 10.3748/wjg.13.5396]. Style for book references Authors: the name of the first author should be typed in bold-faced letters. The surname of all authors should be typed with the initial letter capitalized, followed by their abbreviated middle and first initials. (For example, Lian-Sheng Ma is abbreviated as Ma LS, Bo-Rong Pan as Pan BR) Book title. Publication number. Publication place: Publication press, Year: start page and end page. Format Journals English journal article (list all authors and include the PMID where applicable) 1 Jung EM, Clevert DA, Schreyer AG, Schmitt S, Rennert J, Kubale R, Feuerbach S, Jung F. Evaluation of quantitative contrast harmonic imaging to assess malignancy of liver tumors: A prospective controlled two-center study. World J Gastroenterol 2007; 13: 6356-6364 [PMID: 18081224 DOI: 10.3748/wjg.13.6356] Chinese journal article (list all authors and include the PMID where applicable) 2 Lin GZ, Wang XZ, Wang P, Lin J, Yang FD. Immunologic effect of Jianpi Yishen decoction in treatment of Pixu-diarrhoea. Shijie Huaren Xiaohua Zazhi 1999; 7: 285-287 In press 3 Tian D, Araki H, Stahl E, Bergelson J, Kreitman M. Signature of balancing selection in Arabidopsis. Proc Natl Acad Sci USA 2006; In press Organization as author 4 Diabetes Prevention Program Research Group. Hypertension, insulin, and proinsulin in participants with impaired glucose tolerance. Hypertension 2002; 40: 679-686 [PMID: 12411462] Both personal authors and an organization as author 5 Vallancien G, Emberton M, Harving N, van Moorselaar RJ; AlfOne Study Group. Sexual dysfunction in 1, 274 European men suffering from lower urinary tract symptoms. J Urol 2003; 169: 2257-2261 [PMID: 12771764] No author given 6 21st century heart solution may have a sting in the tail. BMJ

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2002; 325: 184 [PMID: 12142303] Volume with supplement 7 Geraud G, Spierings EL, Keywood C. Tolerability and safety of frovatriptan with short- and long-term use for treatment of migraine and in comparison with sumatriptan. Headache 2002; 42 Suppl 2: S93-99 [PMID: 12028325] Issue with no volume 8 Banit DM, Kaufer H, Hartford JM. Intraoperative frozen section analysis in revision total joint arthroplasty. Clin Orthop Relat Res 2002; (401): 230-238 [PMID: 12151900] No volume or issue 9 Outreach: Bringing HIV-positive individuals into care. HRSA Careaction 2002; 1-6 [PMID: 12154804] Books Personal author(s) 10 Sherlock S, Dooley J. Diseases of the liver and billiary system. 9th ed. Oxford: Blackwell Sci Pub, 1993: 258-296 Chapter in a book (list all authors) 11 Lam SK. Academic investigator’s perspectives of medical treatment for peptic ulcer. In: Swabb EA, Azabo S. Ulcer disease: investigation and basis for therapy. New York: Marcel Dekker, 1991: 431-450 Author(s) and editor(s) 12 Breedlove GK, Schorfheide AM. Adolescent pregnancy. 2nd ed. Wieczorek RR, editor. White Plains (NY): March of Dimes Education Services, 2001: 20-34 Conference proceedings 13 Harnden P, Joffe JK, Jones WG, editors. Germ cell tumours V. Proceedings of the 5th Germ cell tumours Conference; 2001 Sep 13-15; Leeds, UK. New York: Springer, 2002: 30-56 Conference paper 14 Christensen S, Oppacher F. An analysis of Koza's computational effort statistic for genetic programming. In: Foster JA, Lutton E, Miller J, Ryan C, Tettamanzi AG, editors. Genetic programming. EuroGP 2002: Proceedings of the 5th European Conference on Genetic Programming; 2002 Apr 3-5; Kinsdale, Ireland. Berlin: Springer, 2002: 182-191 Electronic journal (list all authors) Morse SS. Factors in the emergence of infectious diseases. Emerg Infect Dis serial online, 1995-01-03, cited 1996-06-05; 1(1): 24 screens. Available from: URL: http//www.cdc.gov/ncidod/ EID/eid.htm Patent (list all authors) 16 Pagedas AC, inventor; Ancel Surgical R&D Inc., assignee. Flexible endoscopic grasping and cutting device and positioning tool assembly. United States patent US 20020103498. 2002 Aug 1 Inappropriate references Authors should always cite references that are relevant to their article, and avoid any inappropriate references. Inappropriate references include those linked with a hyphen when the difference between the two numbers is greater than five. For example, [1-6], [2-14] and [1, 3, 4-10, 22] are all considered inappropriate references. Authors should not cite their own unrelated published articles. Statistical data Write as mean ± SD or mean ± SE. Statistical expression Express t test as t (in italics), F test as F (in italics), chi square test as χ2 (in Greek), related coefficient as r (in italics), degree of freedom as υ (in Greek), sample number as n (in italics), and probability as P (in italics). Units Use SI units. For example: body mass, m (B) = 78 kg; blood pressure, p (B) = 16.2/12.3 kPa; incubation time, t (incubation) = 96 h, blood glucose concentration, c (glucose) 6.4 ± 2.1 mmol/L; blood CEA mass concentration, p (CEA) = 8.6 24.5 μg/L; CO2 volume fraction, 50 mL/L CO2, not 5% CO2; likewise for 40 g/L formaldehyde, not 10% formalin; and mass fraction, 8 ng/g, etc. Arabic numerals such as 23, 243, 641 should be read 23 243 641. The format for how to accurately write common units and quantums can be found at: http://www.wjgnet.com/wjg/help/15.doc. Abbreviations Standard abbreviations should be defined in the abstract and on first mention in the text. In general, terms should not be abbreviated unless

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they are used repeatedly and the abbreviation is helpful to the reader. Permissible abbreviations are listed in Units, Symbols and Abbreviations: A Guide for Biological and Medical Editors and Authors (Ed. Baron DN, 1988) published by The Royal Society of Medicine, London. Certain commonly used abbreviations, such as DNA, RNA, HIV, LD50, PCR, HBV, ECG, WBC, RBC, CT, ESR, CSF, IgG, ELISA, PBS, ATP, EDTA, mAb, can be used directly without further explanation. Italics Quantities: t time or temperature, c concentration, A area, l length, m mass, V volume. Genotypes: gyrA, arg 1, c myc, c fos, etc. Restriction enzymes: EcoRI, HindI, BamHI, Kbo I, Kpn I, etc. Biology: H pylori, E coli, etc.

SUBMISSION OF THE REVISED MANUSCRIPTS AFTER ACCEPTED Please revise your article according to the revision policies of WJG. The revised version including manuscript and high-resolution image figures (if any) should be copied on a floppy or compact disk. The author should send the revised manuscript, along with printed highresolution color or black and white photos, copyright transfer letter, and responses to the reviewers by courier (such as EMS/DHL).

Editorial Office World Journal of Gastroenterology Editorial Department: Room 903 Ocean International Center, Building D No. 62 Dongsihuan Zhonglu Chaoyang District, Beijing 100025, China E-mail: [email protected] http://www.wjgnet.com Telephone: +86-10-59080039 Fax: +86-10-85381893 Language evaluation The language of a manuscript will be graded before it is sent for revision. (1) Grade A: priority publishing; (2) Grade B: minor language polishing; (3) Grade C: a great deal of language polishing needed; (4) Grade D: rejected. Revised articles should reach Grade A or B. Copyright assignment form Please download a Copyright assignment form from http://www. wjgnet.com/wjg/help/9.doc. Responses to reviewers Please revise your article according to the comments/suggestions provided by the reviewers. The format for responses to the reviewers’ comments can be found at: http://www.wjgnet.com/wjg/help/10.doc. Proof of financial support For paper supported by a foundation, authors should provide a copy of the document and serial number of the foundation. Links to documents related to the manuscript WJG will be initiating a platform to promote dynamic interactions between the editors, peer reviewers, readers and authors. After a manuscript is published online, links to the PDF version of the submitted manuscript, the peer-reviewers’ report and the revised manuscript will be put on-line. Readers can make comments on the peer reviewer’s report, authors’ responses to peer reviewers, and the revised manuscript. We hope that authors will benefit from this feedback and be able to revise the manuscript accordingly in a timely manner. Science news releases Authors of accepted manuscripts are suggested to write a science news item to promote their articles. The news will be released rapidly at EurekAlert/AAAS (http://www.eurekalert.org). The title for news items should be less than 90 characters; the summary should be less than 75 words; and main body less than 500 words. Science news items should be lawful, ethical, and strictly based on your original content with an attractive title and interesting pictures. Publication fee Authors of accepted articles must pay a publication fee. EDITORIAL, TOPIC HIGHLIGHTS, BOOK REVIEWS and LETTERS TO THE EDITOR are published free of charge.