Gastroenterology

ISSN 1007-9327 CN 14-1219/R

Gastroenterology

®

Volume 13 Number 40 October 28, 2007

World Journal of Gastroenterology

World Journal of

www.wjgnet.com Oct 28

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

Number 40

Baishideng

Volume 13

National Journal Award 2005

2007

ISSN 1007-9327 CN 14-1219/R Local Post Offices Code No. 82-261

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.


World J Gastroenterol 2007 October 28; 13(40): 5295-5420 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

Baishideng http://www.wjgnet.com E-mail: [email protected] 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 EDITOR-IN-CHIEF Lian-Sheng Ma, Taiyuan 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 MEMBERS OF THE EDITORIAL BOARD Albania Bashkim Resuli, Tirana

Argentina Julio Horacio Carri, Córdoba Adriana M Torres, Rosario Australia Minoti Vivek Apte, Liverpool Richard B Banati, Lidcombe Michael R Beard, Adelaide Patrick Bertolino, Sydney Filip Braet, Sydney Andrew D Clouston, Sydney Darrell HG Crawford, Brisbane Guy D Eslick, Sydney Michael Anthony 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 Daniel Markovich, Brisbane Phillip S Oates, Perth Stephen M Riordan, Sydney IC Roberts-Thomson, Adelaide Arthur Shulkes, Melbourne Ross C Smith, Sydney Kevin John Spring, Brisbane Nathan Subramaniam, Brisbane Herbert Tilg, Innsbruck Martin John Veysey, Gosford DL Worthley, Bedford Austria Valentin Fuhrmann, Vienna Alfred Gangl, Vienna Christoph Gasche, Vienna Kurt Lenz, Linz M Peck-Radosavljevic, Vienna RE Stauber, Auenbruggerplatz Michael Trauner, Graz Harald Vogelsang, Vienna Guenter Weiss, Innsbruck Belarus Yury K Marakhouski, Minsk

www.wjgnet.com

Belgium Rudi Beyaert, Gent Bart Rik De Geest, Leuven Inge Irma Depoortere, Leuven Olivier Detry, Liège BY 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 Canada Fernando Alvarez, Québec David Armstrong, Ontario Olivier Barbier, Québec Nancy Baxter, Toronto Matthew Bjerknes, Toronto Frank J Burczynski, Winnipeg Michael F Byrne, Vancouver Wang-Xue Chen, Ottawa Hugh J Freeman, Vancouver Chantal Guillemette, Québec Samuel S Lee, Calgary Gary A Levy, Toronto Andrew Lawrence Mason, Alberta John K Marshall, Ontario Donna-Marie McCafferty, Calgary Thomas I Michalak, St. John's Gerald Y Minuk, Manitoba Paul Moayyedi, Hamilton Eldon Shaffer, Calgary Morris Sherman, Toronto Alan BR Thomson, Edmonton EF Verdu, Ontario

I

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 Cuba Damian Casadesus Rodriguez, Havana Czech Milan Jirsa, Praha Denmark Peter Bytzer, Copenhagen Hans Gregersen, Aalborg Jens H Henriksen, Hvidovre Claus Peter Hovendal, Odense Fin Stolze Larsen, Copenhagen SØren MØller, Hvidovre Egypt Abdel-Rahman El-Zayadi, Giza Amr Mohamed Helmy, Cairo Sanaa Moharram Kamal, Cairo Ayman Yosry, Cairo Finland Irma Elisabet Jarvela, Helsinki Katri Maria Kaukinen, Tampere Minna Nyström, Helsinki Pentti Sipponen, Espoo France Bettaieb Ali, Dijon Corlu Anne, Rennes Denis Ardid, Clermont-Ferrand Charles Paul Balabaud, Bordeaux Soumeya Bekri, Rouen Jacques Belghiti, Clichy

Ⅱ

Pierre Brissot, Rennes Patrice Philippe Cacoub, Paris Franck Carbonnel, Besancon Laurent Castera, Pessac Bruno Clément, Rennes Jacques Cosnes, Paris Thomas Decaens, Cedex Francoise Lunel Fabiani, Angers Gérard Feldmann, Paris Jean Fioramonti, Toulouse Catherine Guettier, Villejuif Chantal Housset, Paris Juan Lucio Iovanna, Marseille Rene Lambert, Lyon Philippe Mathurin, Lille 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 Ahnini Tazi, Clichy Emmanuel Tiret, Paris Baumert F Thomas, Strasbourg MC Vozenin-brotons, Villejuif Jean-Pierre Henri Zarski, Grenoble Jessica Zucman-Rossi, Paris Germany HD 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 Katja Breitkopf, Mannheim Dunja Bruder, Braunschweig Markus W Büchler, Heidelberg Christa Buechler, Regensburg Reinhard Buettner, Bonn Elke Cario, Essen CF Dietrich, Bad Mergentheim Rainer Josef Duchmann, Berlin Paul Enck, Tuebingen Fred Fändrich, Kiel Ulrich Robert Fölsch, Kiel Helmut Friess, Heidelberg Peter R Galle, Mainz Nikolaus Gassler, Aachen Andreas Geier, Aachen Dieter Glebe, Giessen Burkhard Göke, Munich Florian Graepler, Tuebingen Axel M Gressner, Aachen Veit Gülberg, Munich Rainer Haas, Munich Eckhart Georg Hahn, Erlangen Stephan Hellmig, Kiel Martin Hennenberg, Bonn Johannes Herkel, Hamburg Klaus Herrlinger, Stuttgart Eberhard Hildt, Berlin Joerg C Hoffmann, Berlin Ferdinand Hofstaedter, Regensburg Werner Hohenberger, Erlangen RG 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 www.wjgnet.com

Michael Peter Manns, Hannover 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 Steffen Rickes, Magdeburg Gerhard Rogler, Regensburg Tilman Sauerbruch, Bonn Dieter Saur, Munich Hans Scherubl, Berlin Joerg Schirra, Munich Roland M Schmid, München Volker Schmitz, Bonn AG Schreyer, Regensburg Tobias Schroeder, Essen Hans Seifert, Oldenburg Manfred V Singer, Mannheim Gisela Sparmann, Rostock Jurgen M Stein, Frankfurt Ulrike Susanne Stein, Berlin Manfred Stolte, Bayreuth Christian P Strassburg, Hannover WR Stremmel, Heidelberg Harald F Teutsch, Ulm Robert Thimme, Freiburg HL Tillmann, Leipzig Tung-Yu Tsui, Regensburg Axel Ulsenheimer, Munich Patrick Veit, Essen Claudia Veltkamp, Heidelberg Siegfried Wagner, Deggendorf Henning Walczak, Heidelberg Fritz von Weizsacker, Berlin Jens Werner, Heidelberg Bertram Wiedenmann, Berlin Reiner Wiest, Regensburg Stefan Wirth, Wuppertal Stefan JP Zeuzem, Homburg Greece Elias A Kouroumalis, Heraklion Ioannis E Koutroubakis, Heraklion Spiros Sgouros, Athens Hungary Peter Laszlo Lakatos, Budapest Zsuzsa Szondy, Debrecen Iceland H Gudjonsson, Reykjavik India KA Balasubramanian, Vellore Sujit K Bhattacharya, Kolkata Yogesh K Chawla, Chandigarh Radha K Dhiman, Chandigarh Sri Prakash Misra, Allahabad ND Reddy, Hyderabad Iran Seyed-Moayed Alavian, Tehran Reza Malekzadeh, Tehran Seyed Alireza Taghavi, Shiraz Ireland Billy Bourke, Dublin Ronan A Cahill, Cork Anthony P Moran, Galway Israel Simon Bar-Meir, Hashomer Abraham Rami Eliakim, Haifa

Yaron Ilan, Jerusalem Avidan U Neumann, Ramat-Gan Yaron Niv, Pardesia Ran Oren, Tel Aviv Italy Giovanni Addolorato, Roma Luigi E Adinolfi, Naples Domenico Alvaro, Rome V Annese, San Giovanni Rotond Adolfo Francesco Attili, Roma Giovanni Barbara, Bologna Gabrio Bassotti, Perugia Pier Maria 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 Roberto 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 ED Mangoni, Napoli 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 F Perri, San Giovanni Rotondo Raffaele Pezzilli, Bologna A Pilotto, San Giovanni Rotondo Mario Pirisi, Novara Paolo Del Poggio, Treviglio Gabriele Bianchi Porro, Milano Piero Portincasa, Bari Bernardino Rampone, Siena Claudio Romano, Messina Marco Romano, Napoli Gerardo Rosati, Potenza Enrico Roda, Bologna Domenico Sansonno, Bari Vincenzo Savarino, Genova Mario Del Tacca, Pisa Giovanni Tarantino, Naples Roberto Testa, Genoa Pier Alberto Testoni, Milan

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 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 www.wjgnet.com

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 Yuji 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 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 Shinichi Wada, Tochigi Hiroyuki Watanabe, Kanazawa Toshio Watanabe, Osaka Yuji Watanabe, Ehime Chun-Yang Wen, Nagasaki Koji Yamaguchi, Fukuoka Takayuki Yamamoto, Yokkaichi Takashi Yao, Fukuoka

Ⅲ

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 Daoud Boujaoude, Beirut Lithuania Limas Kupcinskas, Kaunas Macedonia Vladimir Cirko Serafimoski, Skopje Malaysia Andrew Seng Boon Chua, Ipoh Khean-Lee Goh, Kuala Lumpur Jayaram Menon, Sabah Mexico Garcia-Compean Diego, Monterrey E R Marin-Lopez, Jesús García Saúl Villa-Treviño, México JK Yamamoto-Furusho, México Monaco Patrick Rampal, Monaco Netherlands Ulrich Beuers, Amsterdam Gerd Bouma, Amsterdam Lee Bouwman, Leiden J Bart A Crusius, Amsterdam Janine K Kruit, Groningen Ernst Johan Kuipers, Rotterdam Ton Lisman, Utrecht Yi Liu, Amsterdam Servaas Morré, Amsterdam Chris JJ Mulder, Amsterdam Michael Müller, Wageningen Amado Salvador Peña, Amsterdam Robert J Porte, Groningen Ingrid B Renes, Rotterdam Andreas Smout, Utrecht RW Stockbrugger, Maastricht Luc JW van der Laan, Rotterdam Karel van Erpecum, Utrecht GP VanBerge-Henegouwen,Utrecht New Zealand Ian David Wallace, Auckland Nigeria Samuel Babafemi Olaleye, Ibadan Norway Trond Berg, Oslo Tom Hemming Karlsen, Oslo Helge Lyder Waldum, Trondheim Pakistan Muhammad S Khokhar, Lahore Poland Tomasz Brzozowski, Cracow Robert Flisiak, Bialystok Hanna Gregorek, Warsaw DM Lebensztejn, Bialystok Wojciech G Polak, Wroclaw Marek Hartleb, Katowice

IV

Portugal MP Cecília, Lisbon Miguel Carneiro De Moura, Lisbon Russia Vladimir T Ivashkin, Moscow Leonid Lazebnik, Moscow Vasiliy I Reshetnyak, Moscow Saudi Arabia Ibrahim Abdulkarim Al Mofleh, Riyadh Serbia DM Jovanovic, Sremska Kamenica Singapore Bow Ho, Kent Ridge Khek-Yu Ho, Singapore Francis Seow-Choen, Singapore Slovakia Anton Vavrecka, Bratislava Slovenia Sasa Markovic, Ljubljana South Africa Michael C Kew, Parktown South Korea Byung Ihn Choi, Seoul Ho Soon Choi, Seoul M Yeo, Suwon Sun Pyo Hong, Gyeonggi-do Jae J Kim, Seoul Jin-Hong Kim, Suwon Myung-Hwan Kim, Seoul Chang Hong Lee, Seoul Jong Kyun Lee, Seoul Eun-Yi Moon, Seoul Jae-Gahb Park, Seoul Dong Wan Seo, Seoul Dong jin Suh, Seoul 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 Bosch Genover, Barcelona Jaime Guardia, Barcelona Angel Lanas, Zaragoza María Isabel Torres López, Jaén José M Mato, Derio Juan F Medina, Pamplona MA Muñoz-Navas, Pamplona Julian Panes, Barcelona Miguel Minguez 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 Stefan Björnsson, Gothenburg Curt Einarsson, Huddinge www.wjgnet.com

Ulf Hindorf, Lund Hanns-Ulrich Marschall, Stockholm Lars Christer Olbe, Molndal Matti Sallberg, Stockholm Magnus Simrén, Göteborg Xiao-Feng Sun, Linköping Ervin Tóth, Malmö Weimin Ye, Stockholm Switzerland Chrish Beglinger, Basel Pierre A Clavien, Zurich Jean-Francois Dufour, Bern Franco Fortunato, Zürich Jean Louis Frossard, Geneva Gerd A Kullak-Ublick, Zurich Pierre Michetti, Lausanne Francesco Negro, Genève Bruno Stieger, Zurich Radu Tutuian, Zurich Stephan Robert Vavricka, Zurich Arthur Zimmermann, Berne Turkey Yusuf Bayraktar, Ankara Figen Gurakan, Ankara Aydin Karabacakoglu, Konya Serdar Karakose, Konya Hizir Kurtel, Istanbu Osman Cavit Ozdogan, Istanbul Özlem Yilmaz, Izmir Cihan Yurdaydin, Ankara United Arab Emirates Sherif M Karam, Al-Ain United Kingdom David Adams, Birmingham NK Ahluwalia, Stockport CG Antoniades, London Anthony TR Axon, Leeds Qasim Aziz, Manchester Nicholas M Barnes, Birmingham Jim D Bell, London Mairi Brittan, London Alastair David Burt, Newcastle Simon Scott Campbell, Manchester Simon R Carding, Leeds Paul Jonathan Ciclitira, London Eithne Costello, Liverpool Tatjana Crnogorac-Jurcevic, London Amar Paul Dhillon, London Emad M El-Omar, Aberdeen Annette Fristscher-Ravens, London Elizabeth Furrie, Dundee Daniel Richard Gaya, Edinburgh Subrata Ghosh, London William Greenhalf, Liverpool Indra Neil Guha, Southampton Peter Clive Hayes, Edinburgh Gwo-Tzer Ho, Edinburgh Anthony R Hobson, Salford Stefan G Hübscher, Birmingham Robin Hughes, London Pali Hungin, Stockton David Paul Hurlstone, Sheffield Rajiv Jalan, London Janusz AZ Jankowski, Oxford Brian T Johnston, Belfast David EJ Jones, Newcastle Michael A Kamm, Harrow Peter Karayiannis, London Laurens Kruidenier, Harlow Patricia F Lalor, Birmingham Hong-Xiang Liu, Cambridge K E L McColl, Glasgow Stuart AC McDonald, London

Dermot Patrick 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 Paul Thompson, Newcastle David Tosh, Bath Frank Ivor Tovey, London Chris Tselepis, Birmingham Diego Vergani, London Geoffrey Warhurst, Salford Peter James Whorwell, Manchester Roger Williams, London Karen Leslie Wright, Bath Min Zhao, Foresterhill United States Gary A Abrams, Birmingham Golo Ahlenstiel, Bethesda BS Anand, Houston Frank A Anania, Atlanta M Ananthanarayanan, New York Gavin Edward Arteel, Louisville Jasmohan Singh Bajaj, Milwaukee Jamie S Barkin, Miami Beach Kim Elaine Barrett, San Diego Marc Basson, Detroit Wallace F Berman, Durham Timothy R Billiar, Pittsburgh Edmund J Bini, New York Jennifer D Black, Buffalo Herbert L Bonkovsky, Farmington Andrea D Branch, New York Robert S Bresalier, Houston Alan L Buchman, Chicago Alan Cahill, Philadelphia John M Carethers, San Diego David L Carr-Locke, Boston 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 Mark G Clemens, Charlotte Vincent Coghlan, Beaverton David Cronin II, New Haven John Cuppoletti, Cincinnati Mark James Czaja, New York Peter V Danenberg, Los Angeles Kiron Moy Das, New Brunswick Sharon DeMorrow, Temple Deborah L Diamond, Seattle Peter Draganov, Florida Bijan Eghtesad, Cleveland Hala El-Zimaity, Houston Michelle Embree-Ku, Providence Ronnie Fass, Tucson Mark A Feitelson, Philadelphia Ariel E Feldstein, Cleveland Alessandro Fichera, Chicago 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 Julia Butler Greer, Pittsburgh James Henry 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 McLean Heitkemper, Seattle Alan W Hemming, Gainesville Samuel B Ho, San Diego Colin William 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 James Kahrilas, Chicago AN Kalloo, Baltimore Neil Kaplowitz, 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 Daryl Tan Yeung Lau, Galvesto 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 Ming Li, New Orleans Zhiping Li, Baltimore LM Lichtenberger, Houston GR Lichtenstein, Philadelphia Otto Schiueh-Tzang Lin, Seattle Martin Lipkin, New York Edward V Loftus, Rocheste Robin G Lorenz, Birmingham Michael Ronan Lucey, Madison JD Luketich, Pittsburgh Henry Thomson Lynch, Omaha Patrick M Lynch, Houston Peter J Mannon, Bethesda Charles Milton Mansbach, Memphis John Frank Di Mari, Texas John M Mariadason, Bronx WM 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 G Michalopoulos, Pittsburgh James Michael Millis, Chicago Albert D Min, New York Pramod Kumar Mistry, New Haven www.wjgnet.com

Smruti Ranjan Mohanty, Chicago Satdarshan Singh Monga, Pittsburgh Timothy H Moran, Baltimore Steven F Moss, Providence Masaki Nagaya, Boston Laura Eleanor Nagy, Cleveland Hiroshi Nakagawa, Philadelphia Douglas B Nelson, Minneaplis 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 Jay Pasricha, Gaveston Zhiheng Pei, New York Michael A Pezzone, Pittsburgh CS Pitchumoni, New Brunswiuc Jay Pravda, Gainesville M Raimondo, Jacksonville GS Raju, Galveston Murray B Resnick, Providence Adrian Reuben, Charleston Douglas K Rex, Indianapolis Victor E Reyes, Galveston Richard A Rippe, Chapel Hill Marcos Rojkind, Washington Philip Rosenthal, San Francisco Hemant Kumar Roy, Evanston Shawn David Safford, Norfolk Bruce E Sands, Boston NJ Shaheen, Chapel Hill Harvey L Sharp, Minneapolis Stuart Sherman, Indianapolis Shivendra Shukla, Columbia Alphonse E Sirica, Virginia Shanthi V Sitaraman, Atlanta Shanthi Srinivasan, Atlanta Michael Steer, Boston Gary D Stoner, Columbus Liping Su, Chicago Christina Surawicz, Seattle Gyongyi Szabo, Worcester Yvette Taché, Los Angeles Seng-Lai Tan, Seattle Andrzej Tarnawski, Long Beach Andrzej S Tarnawski, Orange K-M Tchou-Wong, New York Neil D Theise, New York PJ Thuluvath, Baltimore Swan Nio Thung, New York Natalie J Torok, Sacramento RA Travagli, Baton Rouge G Triadafilopoulos, Stanford James F Trotter, Denver Chung-Jyi Tsai, Lexington Andrew Ukleja, Florida Hugo E Vargas, Scottsdale Scott A Waldman, Philadelphia Jian-Ying Wang, Baltimore Steven David Wexner, Weston Keith Tucker Wilson, Baltimore Jacqueline L Wolf, Boston Jackie Wood, Ohio George Y Wu, Farmington Jian Wu, Sacramento Samuel Wyllie, Houston Wen Xie, Pittsburgh Yoshio Yamaoka, Houston Vincent W Yang, Atlanta Francis Y Yao, 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 Uruguay Henry Cohen, Montevideo

Ⅴ

World Journal of

Gastroenterology



Weekly Established in October 1995 National Journal Award 2005


Baishideng

Contents REVIEW

5295 Colonic gas explosion during therapeutic colonoscopy with electrocautery

LIVER CANCER

5299

Ladas SD, Karamanolis G, Ben-Soussan E

Effects and mechanisms of silibinin on human hepatoma cell lines Lah JJ, Cui W, Hu KQ

5306 Survivin expression in early hepatocellular carcinoma and post-treatment with anti-cancer drug under hypoxic culture condition Mamori S, Asakura T, Ohkawa K, Tajiri H

COLORECTAL CANCER

5312 RNA interference-mediated gene silencing of vascular endothelial growth factor in colon cancer cells Li TJ, Song JN, Kang K, Tong SS, Hu ZL, He TC, Zhang BQ, Zhang CQ

VIRAL HEPATITIS

5317

Antioxidant therapy for chronic hepatitis C after failure of interferon: Results of phase Ⅱ randomized, double-blind placebo controlled clinical trial Gabbay E, Zigmond E, Pappo O, Hemed N, Rowe M, Zabrecky G, Cohen R, Ilan Y

5324

Establishment and primary application of a mouse model with hepatitis B virus replication Liu FJ, Liu L, He F, Wang S, Zhou TY, Liu C, Deng LY, Tang H

CLINICAL RESEARCH

5331

Right liver lobe/albumin ratio: Contribution to non-invasive assessment of portal hypertension Alempijevic T, Bulat V, Djuranovic S, Kovacevic N, Jesic R, Tomic D, Krstic S, Krstic M

RAPID COMMUNICATION 5336 Liver biopsy in a district general hospital: Changes over two decades Syn WK, Bruckner-Holt C, Farmer A, Howdle S, Bateman J

5343

Effects of a 24-week course of interferon-a therapy after curative treatment of hepatitis C virus-associated hepatocellular carcinoma Jeong SC, Aikata H, Katamura Y, Azakami T, Kawaoka T, Saneto H, Uka K, Mori N, Takaki S, Kodama H, Waki K, Imamura M, Shirakawa H, Kawakami Y, Takahashi S, Chayama K

5351

Pancreatic fistula after pancreaticoduodenectomy: A comparison between the two pancreaticojejunostomy methods for approximating the pancreatic parenchyma to the jejunal seromuscular layer: Interrupted vs continuous stitches Lee SE, Yang SH, Jang JY, Kim SW

5357

Type 2 dıabetes mellıtus and CA 19-9 levels Uygur-Bayramiçli O, Dabak R, Orbay E, Dolapçıoğlu C, Sargın M, Kılıçoğlu G, Güleryüzlü Y, Mayadağlı A

5360 Holistic Acupuncture approach to idiopathic refractory nausea, abdominal pain and bloating Ouyang A, Xu L www.wjgnet.com


Contents

Volume 13 Number 40 October 28, 2007 5367 Epidemiology of upper gastrointestinal cancers in Iran: A Sub site analysis of 761 cases Taghavi N, Nasrollahzadeh D, Merat S, Yazdanbod A, Hormazdi M, Sotoudeh M, Semnani S, Eslami F, Marjani HA, Fahimi S, Khademi H, Malekzadeh R

5371 Colorectal neoplasm: Magnetic resonance colonography with fat enema initial clinical experience Zhang S, Peng JW, Shi QY, Tang F, Zhong MG

5376 Treatment of gastric outlet and duodenal obstructions with uncovered expandable metal stents Huang Q, Dai DK, Qian XJ, Zhai RY

5380

Effects of H pylori therapy on erythrocytic and iron parameters in iron deficiency anemia patients with H pylori -positive chronic gastristis Chen LH, Luo HS

5384 Heme oxygenase-1 induction by hemin protects liver cells from ischemia/reperfusion injury in cirrhotic rats Xue H, Guo H, Li YC, Hao ZM

CASE REPORTS

5391 Sequential stenotic strictures of the small bowel leading to obstruction Van Buren II G, Teichgraeber DC, Ghorbani RP, Souchon EA

5394 Occasional finding of mesenteric lipodystrophy during laparoscopy: A difficult diagnosis Vettoretto N, Diana DR, Poiatti R, Matteucci A, Chioda C, Giovanetti M

5397

Complications and treatment of migrated biliary endoprostheses: A review of the literature Namdar T, Raffel AM, Topp SA, Namdar L, Alldinger I, Schmitt M, Knoefel WT, Eisenberger CF

5400 Sigmoid colon endometriosis treated with laparoscopy-assisted sigmoidectomy: Significance of preoperative diagnosis Yoshida M, Watanabe Y, Horiuchi A, Yamamoto Y, Sugishita H, Kawachi K

5403 Reactive lymphoid hyperplasia of the liver: A case report and review of literature Machida T, Takahashi T, Itoh T, Hirayama M, Morita T, Horita S

5408 Clindamycin-induced acute cholestatic hepatitis Aygün C, Kocaman O, Gürbüz Y, Şentürk Ö, Hülagü S

5411 Sudden hearing loss associated with peginterferon and ribavirin combination therapy during hepatitis C treatment Elloumi H, Houissa F, Hadj NB, Gargouri D, Romani M, Kharrat J, Ghorbel A

5413 Positron emission tomography/computer tomography in guidance of extrahepatic hepatocellular carcinoma metastasis management Sun L, Guan YS, Pan WM, Chen GB, Luo ZM, Wu H

ACKNOWLEDGMENTS

5416 Acknowledgments to Reviewers of World Journal of Gastroenterology

APPENDIX

5417 M eetings 5418

Instructions to authors www.wjgnet.com


Contents FLYLEAF

Volume 13 Number 40 October 28, 2007 I-V

Editorial Board

INSIDE FRONT COVER

Online Submissions

INSIDE BACK COVER

Online Submissions

Responsible E-Editor for this issue: Wen-Hua Ma C-Editor for this issue: Thierry Piche, MD, PhD Responsible S-Editor for this issue: Ye Liu

World Journal of Gastroenterology ( World J Gastroenterol , WJG ), a leading international journal in gastroenterology and hepatology, has an established reputation for publishing first class research on esophageal cancer, gastric cancer, liver cancer, viral hepatitis, colorectal cancer, and H pylori infection, providing a forum for both clinicians and scientists, and has been 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. WJG is a weekly journal published by WJG. The publication date is on 7th, 14th, 21st, and 28th every month. The WJG is supported by The National Natural Science Foundation of China, No. 30224801 and No.30424812, which was founded with a name of China National Journal of New Gastroenterology on October 1, 1995, and renamed as WJG on January 25, 1998. You-Yong Lu, Beijing Masao Omata, Tokyo Harry HX Xia, Hanover

NAME OF JOURNAL World Journal of Gastroenterology

EDITOR-IN -CHIEF Lian-Sheng Ma, Taiyuan

RESPONSIBLE INSTITUTION Department of Science and Technology of Shanxi Province

SUBSCRIPTION RMB 50 Yuan for each issue, RMB 2400 Yuan for one year

SPONSOR Taiyuan Research and Treatment Center for Digestive Diseases, Taiyuan 77, Shuangta Xijie, Taiyuan 030001, Shanxi Province, China

CSSN ISSN 1007-9327 CN 14-1219/R

LANGUAGE EDITORS Director: Jing-Yun Ma, Beijing Deputy Director: Xian-Lin Wang, Beijing

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

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

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 OVERSEAS DISTRIBUTOR Beijing Bureau for Distribution of Newspapers and Journals (Code No. 82-261) China International Book Trading Corporation PO Box 399, Beijing, China (Code No. M4481) PUBLICATION DATE October 28, 2007

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

SCIENCE EDITORS Deputy Director: Ye Liu, Beijing Jian-Zhong Zhang, Beijing

NEWS EDITOR Lixin Zhu, Berkeley COPY EDITORS Gianfranco D Alpini, Temple Sujit Kumar Bhattacharya, Kolkata Filip Braet, Sydney

www.wjgnet.com

Kirsteen N Browning, Baton Rouge Radha K Dhiman, Chandigarh John Frank Di Mari, Texas Shannon S Glaser, Temple 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 © 2007 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.

World J Gastroenterol 2007 October 28; 13(40): 5295-5298 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

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

REVIEW

Colonic gas explosion during therapeutic colonoscopy with electrocautery Spiros D Ladas, George Karamanolis, Emmanuel Ben-Soussan Spiros D Ladas, George Karamanolis, Hepatogastroenterology Unit, 2nd Department of Internal Medicine-Propaedeutic, Medical School, Athens University, “Attikon” University General Hospital, Athens, Greece Emmanuel Ben-Soussan, Clinique de l’Alma 166, rue de l’ université 750007, Paris, France Correspondence to: Spiros D Ladas MD, PhD, Hepatogastroenterology Unit, 2nd Propedeutic Internal Medicine Department, “ATTIKON” General Hospital, Rimini 1, Athens 12462, Greece. [email protected] Telephone: +30-210-5832087 Fax: +30-210-5326422 Revised: June 26, 2007 Revised: August 10, 2007

Abstract Therapeutic colonoscopy with electrocautery is widely used around the world. Adequate colonic cleansing is considered a crucial factor for the safety of this procedure. Colonic gas explosion, although rare, is one of the most frightening iatrogenic complications during colonoscopy with electrocautery. This complication is the result of an accumulation of colonic gases to explosive concentrations, but may be prevented by meticulous bowel preparation. The purpose of this review is to discuss the indications and the types of bowel preparations for therapeutic colonoscopy, and to contribute recommendations for the adequate bowel preparation for colonoscopy with electrocautery. © 2007 WJG . All rights reserved.

Key words: Colonic gas explosion; Electrocautery; Therapeutic polypectomy; Argon plasma coagulation; Polypectomy Ladas SD, Karamanolis G, Ben-Soussan E. Colonic gas explosion during therapeutic colonoscopy with electrocautery. World J Gastroenterol 2007; 13(40): 5295-5298

http://www.wjgnet.com/1007-9327/13/5295.asp

INTRODUCTION Endoscopic polypectomy and argon plasma coagulation for hemostasis of colonic vascular lesions are considered relatively safe procedures. Among complications that have been reported, gas explosion is rare, but its nature could be dramatic, as perforation could complicate colonic explosion and urgent surgery is needed.

Three factors are necessary to trigger an explosion of colonic gases: presence of combustible gases (hydrogen, methane) produced by the fermentation of non-absorbable carbohydrates in the colon by the colonic bacteria, presence of combustive gas (oxygen), and application of a heat source (electrocautery or argon plasma coagulation)[1-3]. Five major components of gases have been identified in colon: nitrogen (23%-80%), oxygen (0.1%-2.3%), hydrogen (0.06%-47%), methane (0%-26%), and carbon dioxide (5.1%-29%). Only hydrogen and methane are combustible[4]. They are produced in the colonic lumen from fermentation of non absorbable (e.g. lactulose, mannitol) or incompletely absorbed (lactose, fructose, sorbitol) carbohydrates by the colonic flora[4-6]. Concentrations of hydrogen more than 4% and/or methane more than 5% are considered potentially explosive[7]. Almost half of the patients (42.8%) with unprepared colon have potentially explosive concentrations of hydrogen and methane[8]. Nevertheless, an explosion with these two gases can occur only if the oxygen concentration is over 5%[9]. Following bowel preparation with a combination of clear liquids, cathartics, and enemas, mean concentration of hydrogen (0.024% ± 0.007%) and methane (0.0023% ± 0.001%) were below of their minimal explosive concentration[4]. Thus, safety of therapeutic colonoscopy could be in part related to the quality of preparation before the procedure. An accumulation of colonic gas to potentially explosive concentrations due to poor colon preparation is considered an initiating factor in the complication of colonic gas explosion. Therefore, quality of bowel preparation as well as type of preparation and dietary restrictions are all essential for an uneventful therapeutic colonoscopy.

INDICATIONS FOR COLONOSCOPY WITH ELECTROCAUTERY The main indication for application of electrosurgical energy is snare colonoscopic polypectomy with blended or pure coagulation current[10]. Argon plasma has been successfully used for hemostasis of vascular ectasias, for ablation of intestinal polyps or residual adenomatous tissue after colonic polypectomy, and for the endoscopic therapy of postradiation colitis[11]. Electrosurgical generators Electrosurgical generators are used to supply electrical energy to endoscopic accessories. When electrical energy is introduced to tissue, it produces excitation of molecules, www.wjgnet.com

5296

ISSN 1007-9327

CN 14-1219/R

World J Gastroenterol

which results in generation of heat[12]. Electrosurgical generators may supply two types of circuits, monopolar and bipolar. Electrosurgical cutting is achieved by a high voltage (> 200 V) continuous current[10]. Argon plasma coagulation Argon plasma coagulation is a noncontact electrocoagulation device that uses high-frequency monopolar current conducted to target tissues through ionized argon gas[10]. Argon plasma delivered through a flexible probe passed through the accessory channel and allows treatment of a large area quickly[13]. In general, low power and low argon flow rates are used for hemostasis with settings of 40-50 W and 0.8 to 1.2 L/mn, whereas higher settings (70-90 W and over 1.2 L/mn) are used for tissue ablation[14].

BOWEL PREPARATION Bowel preparation with purgatives The selection of purgative used for colon preparation is an important factor that makes the bowel safe for therapeutic procedures. Earlier than 1980’s, mannitol was considered as the reference agent for colonic preparation. Explosions during therapeutic colonoscopy have been reported after mannitol preparation and its use is now avoided as cleansing colonic solution[1-3,5,15-17]. Use of oral manittol increased hydrogen and methane excretion [3,5,9,18-19]. Aspiration of colonic gas at the time of colonoscopy showed that mean intracolonic hydrogen concentration was significantly higher after mannitol than after castor oil. Moreover, potentially explosive concentrations were present in 60% of patients given mannitol compared to 0%-20% of patients given castor oil[3,19]. Fermentation of mannitol by E.coli is thought to be responsible for the production of potentially explosive gas mixture after oral mannitol preparation[20-21]. Significantly higher counts of gas-producing E.coli were recovered from patients prepared with mannitol alone compared with mannitol preceded by oral antimicrobials[20]. Therefore, the use of antibiotics prior to therapeutic colonoscopy could be a measure that would lower the load of intracolonic bacteria. An alternative approach to reduce the risk of explosion of colonic gases if mannitol preparation is used before electrocautery is insufflation of an inert gas such as carbon dioxide instead of air[3,22]. Since 1990, a major progress occurred with new agents, such as polyethylene glycol electrolyte lavage solution (PEG-ELS) and oral sodium phosphate (NaP) solutions. Several studies agree that these agents provide a climate safe for electrocautery during colonoscopy by decreasing the concentrations of combustible gases[14,23-26]. The highest hydrogen and methane concentrations after a PEGELS preparation are well below the combustible level[26]. A recent report described a case of colonic gas explosion in a patient that underwent bowel preparation with a polyethylene glycol solution containing sorbitol[27]. Sorbitol is an important carbohydrate that is daily included in humans diet. Studies have shown that the frequency of sorbitol malabsorption may be as high as 60% in healthy subjects[3,28]. Thus, fermentation of this malabsorded carbohydrate by colonic bacteria could result in raised combustible gas concentrations in the colon and explain the explosion that occurred in the above mentioned case report[27]. www.wjgnet.com

October 28, 2007

Volume 13

Number 40

Partial bowel preparation by enemas For lesions that need electrocautery and are located up to the level of sigmoid colon, a flexible sigmoidoscopy with enema preparation is the procedure of choice by the majority of gastroenterologists. However, studies have reported cases of gas explosion in patients prepared by enemas[29-31]. In these cases, the used enema did not contain any fermentable agents and the extension of colonic preparation was thought to be the initiating factor in this complications. Due to the partial colonic preparation, presence of residual stools above the lesions could enhance gas production and explain gas explosion. In a prospective study, sixty patients were evaluated to compare the presence of the combustible gases hydrogen and methane during colonoscopy after a PEG-ELS preparation and flexible sigmoidoscopy after phosphosoda enemas alone[32]. During colonoscopy, the concentrations of hydrogen and methane remained below combustible levels in all patients, whereas 10% of patients had combustible levels of either hydrogen or methane during flexible sigmoidoscopy. Patients had combustible levels even after air insufflation during sigmoidoscopy and the possibility of explosion was clinically significant. Another important observation of this elegant study was that even segments of colon with excess retained stools did not have combustible levels of these two gases. It seems that insufflation of air during colonoscopy equalizes the distribution of combustible gases, overcoming the compartmentation of the colon. In a more recent study using argon plasma coagulation for hemorrhagic radiation proctitis, incidence of gas explosion was higher after local colon preparation (3/19 sessions) compared with oral preparation (0/53 sessions)[31]. All three explosions after enema preparation occurred in patients with persistent solid stool above the coagulated lesions. Thus, the presence of stools could constitute the main risk for the colonic explosion.

REVIEW OF PUBLISHED STUDIES A systematic review of the medical research published in English language from 1952 to October 2006 was performed, by using MEDLINE, SCOPUS, SCIRUS, and EMBASE to obtain studies published on colonic gas explosion. The search terms included were combinations of “colonic explosion” or “gas explosion” with “surgery”, “electrocautery”, “polypectomy”, and “argon plasma coagulation”. A total of 20 cases of colonic gas explosion were identified (Figure 1). Eleven cases of gas explosion during surgery[2,16,33-39] and 9 cases during colonoscopic procedures have been published. Argon plasma coagulation provided the initiating heat source in five of the nine colonoscopic cases[29-31], whereas the remaining four cases were associated with endoscopic polypectomy[1,15,17,27]. Recently, we also experienced a case of colonic explosion during an argon plasma coagulation procedure for postradiation colitis. Although seven successive, uneventful sessions of argon plasma coagulation were performed with enema preparation, gas explosion without colonic perforation occurred upon finishing the last procedure. Nine of the 20 published cases (45%) of gas explosion

Ladas SD et al . Colonic gas explosion 2

20 colonic gas explosion

11 surgery

5 APC

5297

4 polypectomy

3 4

3 perforations

2 perforations

4 perforations

5 6

1 death

Figure 1 Flow chart of published cases with colonic gas explosion.

7 8

were complicated with colon perforation. Perforation was observed in all of the four polypectomy cases[1,15,17,27], in two cases using argon plasma coagulation[29,31], and in three cases during electrosurgery[2,33-34]. One of the four perforations during polypectomy was fatal[1]. Bowel preparation by ingestion of a mannitol solution was used in 14 cases and of a cleansing solution containg sorbitol in one case[27]. Preparation by enemas containg no fermentable agent was used in all five cases treated with argon plasma coagulation for post-radiation colitis[29-31].

CONCLUSION Colonic gas explosion is a rare, but potentially serious complication during colonoscopy with electrocautery. Accumulation of colonic combustible gases at potentially explosive concentrations due to poor colon preparation is the cause of gas explosion. Cleansing purgatives (PEG, NaP) that make the bowel safe for electrocautery by decreasing the concentrations of the combustible gases are adequate for colon preparation. Argon plasma coagulation carries an increased risk of explosion during sigmoidoscopy following enemas, and it should only be performed after full bowel preparation. In conclusion, we recommend the following to avoid colonic gas explosion during colonoscopy with electrocautery: (1) Cleansing solution containing mannitol [3,8,19] or other malabsorbed carbohydrates (e.g. sorbitol)[6] should be avoided in the preparation of the colon since intracolonic concentrations of H2 or/and CH4 could be at combustible levels. (2) During flexible sigmoidoscopy after standard enema preparation, concentrations of H2 or/and CH4 could be at explosive levels[32]. (3) Argon plasma coagulation for post-radiation colitis should be performed only after complete bowel preparation with PEG or NaP, to avoid the risk of explosion[31]. (4) Polypectomy with electrocautery should only be performed after full bowel preparation with PEG or NaP to prevent colonic explosion[1,15,17,27].

REFERENCES 1

Bigard MA, Gaucher P, Lassalle C. Fatal colonic explosion during colonoscopic polypectomy. Gastroenterology 1979; 77: 1307-1310

9 10

11

12 13 14 15 16 17 18 19 20

21 22 23

24

25 26

Raillat A, de Saint-Julien J, Abgrall J. Colonic explosion during an endoscopic electrocoagulation after preparation with mannitol. Gastroenterol Clin Biol 1982; 6: 301-302 Avgerinos A, Kalantzis N, Rekoumis G, Pallikaris G, Arapakis G, Kanaghinis T. Bowel preparation and the risk of explosion during colonoscopic polypectomy. Gut 1984; 25: 361-364 Levitt MD, Bond JH Jr. Volume, composition, and source of intestinal gas. Gastroenterology 1970; 59: 921-929 Bond JH, Levitt MD. Colonic gas explosion--is a fire extinguisher necessary? Gastroenterology 1979; 77: 1349-1350 Ladas SD, Grammenos I, Tassios PS, Raptis SA. Coincidental malabsorption of lactose, fructose, and sorbitol ingested at low doses is not common in normal adults. Dig Dis Sci 2000; 45: 2357-2362 Levy EI. Explosions during lower bowel electrosurgery. Am J Surg 1954; 88: 754-758 Ragins H, Shinya H, Wolff WI. The explosive potential of colonic gas during colonoscopic electrosurgical polypectomy. Surg Gynecol Obstet 1974; 138: 554-556 Taylor EW, Bentley S, Youngs D, Keighley MR. Bowel preparation and the safety of colonoscopic polypectomy. Gastroenterology 1981; 81: 1-4 Slivka A, Bosco JJ, Barkun AN, Isenberg GA, Nguyen CC, Petersen BT, Silverman WB, Taitelbaum G, Ginsberg GG. Electrosurgical generators: MAY 2003. Gastrointest Endosc 2003; 58: 656-660 Ginsberg GG, Barkun AN, Bosco JJ, Burdick JS, Isenberg GA, Nakao NL, Petersen BT, Silverman WB, Slivka A, Kelsey PB. The argon plasma coagulator: February 2002. Gastrointest Endosc 2002; 55: 807-810 Tucker RD. Principles of electrosurgery. In: Sivak MV, editor. Gastroenterologic endoscopy. 2nd ed. Philadelphia: WB Saunders, 2000: 125 Farin G, Grund KE. Technology of argon plasma coagulation with particular regard to endoscopic applications. Endosc Surg Allied Technol 1994; 2: 71-77 Waye J. How I use the argon plasma coagulation. Clin Perspect Gastronetrol 1999; 2: 249-252 Bisson B. Methane gas explosion during colonoscopy. Gastroenterol Nurs 1997; 20: 136-137 Shinagawa N, Mizuno H, Shibata Y, Yura J, Katsumi K, Ito M, Takeuchi T. Gas explosion during diathermy colotomy. Br J Surg 1985; 72: 306 Bond JH, Levy M, Levitt MD. Explosion of hydrogen gas in the colon during proctosigmoidoscopy. Gastrointest Endosc 1976; 23: 41-42 Beck DE, Fazio VW, Jagelman DG. Comparison of the oral lavage methods for preoperative colonic cleansing. Dis Colon Rectum 1986; 29: 699-703 La Brooy SJ, Avgerinos A, Fendick CL, Williams CB, Misiewicz JJ. Potentially explosive colonic concentrations of hydrogen after bowel preparation with mannitol. Lancet 1981; 1: 634-636 Keighley MR, Taylor EW, Hares MM, Arabi Y, Youngs D, Bentley S, Burdon DW. Influence of oral mannitol bowel preparation on colonic microflora and the risk of explosion during endoscopic diathermy. Br J Surg 1981; 68: 554-556 K e i g h l e y M R, L e e J R , A m b r o s e N S . I n d i c a t ions and techniques for bowel preparation in colorectal cancer. Int Adv Surg Oncol 1983; 6: 257-270 Rogers BH. Carbon dioxide for colonoscopy. Gastroenterology 1980; 78: 1659-1660 Ambrose NS, Johnson M, Burdon DW, Keighley MR. A physiological appraisal of polyethylene glycol and a balanced electrolyte solution as bowel preparation. Br J Surg 1983; 70: 428-430 DiPalma JA, Brady CE 3rd, Stewart DL, Karlin DA, McKinney MK, Clement DJ, Coleman TW, Pierson WP. Comparison of colon cleansing methods in preparation for colonoscopy. Gastroenterology 1984; 86: 856-860 DiPalma JA, Brady CE 3rd. Colon cleansing for diagnostic and surgical procedures: polyethylene glycol-electrolyte lavage solution. Am J Gastroenterol 1989; 84: 1008-1016 Strocchi A, Bond JH, Ellis C, Levitt MD. Colonic concentrations www.wjgnet.com

5298

ISSN 1007-9327

CN 14-1219/R


of hydrogen and methane following colonoscopic preparation with an oral lavage solution. Gastrointest Endosc 1990; 36: 580-582 27 Josemanders DF, Spillenaar Bilgen EJ, van Sorge AA, Wahab PJ, de Vries RA. Colonic explosion during endoscopic polypectomy: avoidable complication or bad luck? Endoscopy 2006; 38: 943-944 28 Nelis GF, Vermeeren MA, Jansen W. Role of fructosesorbitol malabsorption in the irritable bowel syndrome. Gastroenterology 1990; 99: 1016-1020 29 Zinsser E, Will U, Gottschalk P, Bosseckert H. Bowel gas explosion during argon plasma coagulation. Endoscopy 1999; 31: S26 30 Pichon N, Maisonnette F, Cessot F, Sodji M, Sautereau D. Colonic perforations after gas explosion induced by argon plasma coagulation. Endoscopy 2004; 36: 573 31 Ben-Soussan E, Antonietti M, Savoye G, Herve S, Ducrotte P, Lerebours E. Argon plasma coagulation in the treatment of hemorrhagic radiation proctitis is efficient but requires a perfect colonic cleansing to be safe. Eur J Gastroenterol Hepatol

October 28, 2007

Volume 13

Number 40

2004; 16: 1315-1318 Monahan DW, Peluso FE, Goldner F. Combustible colonic gas levels during flexible sigmoidoscopy and colonoscopy. Gastrointest Endosc 1992; 38: 40-43 33 Zanoni CE, Bergamini C, Bertoncini M, Bertoncini L, Garbini A. Whole-gut lavage for surgery. A case of intraoperative colonic explosion after administration of mannitol. Dis Colon Rectum 1982; 25: 580-581 34 Freund PR, Radke HM. Intraoperative explosion: methane gas and diet. Anesthesiology 1981; 55: 700-701 35 De Wilt JH, Borel Rinkes IH, Brouwer KJ. Gas explosion during colonic surgery. J R Coll Surg Edinb 1996; 41: 419 36 Altomare DF, Memeo V. Colonic explosion during diathermy colotomy. Report of a case. Dis Colon Rectum 1993; 36: 291-292 37 Sadanaga M, Kano T, Morioka T. A case of colonic gas explosion caused by electrocautery. J Anesth 1992; 6: 117-119 38. Carter HG. Explosion in the colon during electrodesiccation of polyps. Am J Surg 1952; 84: 514-517 39 Becker GL. The prevention or gas explosions in the large bowel during electrosurgery. Surg Gynecol Obstet 1953; 97: 463-467 32

S- Editor Liu Y L- Editor Kremer M E- Editor Li JL

www.wjgnet.com



LIVER CANCER

Effects and mechanisms of silibinin on human hepatoma cell lines John J Lah, Wei Cui, Ke-Qin Hu John J Lah, Wei Cui, Ke-Qin Hu, Division of Gastroenterology, University of California, Irvine Medical Center, 101 The City Drive, Building 53, Suite 113, Orange, CA 92868, United States Supported by UCI institutional research grants from GI Division and Chao Family Comprehensive Cancer Center (K.-Q.H.) Correspondence to: Ke-Qin Hu, Division of Gastroenterology, University of California, Irvine Medical Center, 101 The City Drive, Building 53, Suite 113, Orange, CA 92868, United States. [email protected] Telephone: +1-714-4566745 Fax: +1-714-4567753 Received: April 7, 2007 Revised: July 25, 2007

Abstract AIM: To investigate in vitro effects and mechanisms of silibinin on hepatocellular carcinoma (HCC) cell growth. METHODS: Human HCC cell lines were treated with different doses of silibinin. The effects of silibinin on HCC cell growth and proliferation, apoptosis, cell cycle progression, histone acetylation, and other related signal transductions were systematically examined. RESULTS: We demonstrated that silibinin significantly reduced the growth of HuH7, HepG2, Hep3B, and PLC/PRF/5 human hepatoma cells. Silibinin-reduced HuH7 cell growth was associated with significantly upregulated p21/CDK4 and p27/CDK4 complexes, downregulated Rb-phosphorylation and E2F1/DP1 complex. Silibinin promoted apoptosis of HuH7 cells that was associated with down-regulated survivin and upregulated activated caspase-3 and -9. Silibinin's antiangiogenic effects were indicated by down-regulated metalloproteinase-2 (MMP2) and CD34. We found that silibinin-reduced growth of HuH7 cells was associated with increased activity of phosphatase and tensin homolog deleted on chromosome ten (PTEN) and decreased p-Akt production, indicating the role of PTEN/ PI3K/Akt pathway in silibinin-mediated anti-HCC effects. We also demonstrated that silibinin increased acetylation of histone H3 and H4 (AC-H3 and AC-H4), indicating a possible role of altered histone acetylation in silibininreduced HCC cell proliferation. CONCLUSION: Our results defined silibinin's in vitro anti-HCC effects and possible mechanisms, and

provided a rationale to further test silibinin for HCC chemoprevention. © 2007 WJG . All rights reserved.

Key words: Hepatocellular carcinoma; HuH7 cells; Silibinin; Chemoprevention; Cell cycle; Cell cycle progression; Apoptosis; Acetylation of histone Lah JJ, Cui W, Hu KQ. Effects and mechanisms of silibinin on human hepatoma cel l lines. World J Gastroenterol 2007; 13(40): 5299-5305 http://www.wjgnet.com/1007-9327/13/5299.asp

INTRODUCTION Hepatocellular carcinoma (HCC) is one of the most common malignancies related to a high mortality globally[1,2]. Recent studies have noted a significant rise in the incidence of HCC in the United States in the past 2 decades[2]. Less than 1% of HCC patients underwent a radical surgical resection in the US between 1974 and 1996[3]. HCC's limited treatment remedies and the poor prognosis emphasize the importance in developing an effective chemoprevention for this disease. Milk thistle (Silybum marianum) has been widely utilized as a folk remedy for liver diseases. It is a popular dietary supplement widely used in the United States and Europe[4]. Silibinin is a polyphenolic flavonoid and the major biologically active compound of milk thistle[4-6]. It is well known that milk thistle is safe and well-tolerated, and it protects the liver from drug or alcohol-related injury[7,8]. Studies demonstrated silibinin's inhibitory effects on multiple cancer cell lines, including prostate[9-12], colon[13,14], skin[15-17], bladder[18,19] and lung cancers[20]. Recently, we and Varghese et al reported silibinin's anti-HCC effects[21,22], but further studies are needed to define silibinin's inhibitory effects and mechanisms on human HCC cell growth. Searching for non-invasive biomarkers is another important filed of HCC chemoprevention. Plasma alphafetoprotein (AFP) has been used as a clinical marker for diagnosing and monitoring recurrent HCC [23-25] . However, AFP's value in monitoring effect of HCC chemoprevention has not been tested before. Phosphatase and tensin homolog deleted on chromosome ten (PTEN), phosphatidylinositol 3'-kinase (PI3K) and Akt (PTEN/PI3K/Akt) pathway has been associated with carcinogenesis [26] . Activated PI 3 K-Akt signaling promotes carcinogenesis[27,28]. PTEN is a negative regulator of PI3K-Akt signaling[29] and one of the most frequently inactivated genes in malignancies[30, 31]. Akt is a downstream protein kinase of PI3K (PTEN) and is a signal transduction www.wjgnet.com

5300

ISSN 1007-9327

CN 14-1219/R


protein that has been identified as one of the key elements in protecting cells from apoptosis. If unregulated, Akt promotes uncontrolled cell replication[32,33]. It was reported that silibinin affects Akt expression in prostate cancer cells[16], but it remains unknown whether silibinin affects HCC growth through a PTEN/PI 3K/Akt pathway in human liver cancer cells. Histone acetylation modifies nucleosome structure that leads to DNA relaxation, reduces the affinity of histone complexes with DNA, and enhances the access of transcriptional factor to DNA[34]. Accumulating evidence has indicated that alteration of histone acetylation plays an important role in carcinogenesis[35,36], but it remains unknown whether it is associated with silibinin's anti-HCC effects. In the present study, we demonstrated that silibinin significantly inhibited the growth of HuH7, HepG2, Hep3B, and PLC/PRF/5 human HCC cells that was associated with decreased Ki-67 expression, and cell cycle progression by arresting G1-S transition, and promoted apoptosis. These effects of silibinin were associated with increased PTEN activity and decreased p-Akt production, indicating the role of PTEN/PI 3 K/Akt pathway in silibinin-mediated anti-HCC effects. We also demonstrated that silibinin increased AC-H3 and AC-H4 expression, indicating that altered histone acetylation is involved in silibinin-reduced HCC cell proliferation.

MATERIALS AND METHODS Reagents The cell culture media were the same, as previously reported [37,38] . Anti-activated caspase-3 antibody was purchased from Sigma Chemical Co. (St. Louis, MO). The antibodies against human Ki-67, AFP, p-Rb, E2F1, DP1, CD1, CDK4, p21 and p27, activated caspase-9, bcl-2, survivin, CD34, metalloproteinase (MMP)-2, MMP-9, phosphorylated-AktThr308, PTEN, AC-histone3 and AChistone4, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PTEN activity assay kit was from Biomol Research Laboratories, Inc (Plymouth Meeting, PA). An EIA kit for cell death detection was from Roche Applies Science (Indianapolis, IN). Cell culture Human HCC cell lines, HuH7, HepG2, PLC/PRF/5, and Hep3B cells[37,38], were used in the present study. All the cells were cultured, as previously reported[37,38]. The experiments were performed when cells reached about 80% confluence and cultured in FBS-free media for 24 h to synchronize the cell growth[37,38]. Cell proliferation assay Cell proliferation was determined using MTT assay, as previously reported[37,38]. Briefly, the effects of silibinin on HCC cell growth were then determined after 24 h of incubation by optical density absorbance at 490 nm according to the manufacturer's instruction[37,38]. Apoptosis assays Apoptosis was determined in duplicate using an EIA kit for cell death detection, as previously reported[37,38]. www.wjgnet.com

October 28, 2007

Volume 13

Number 40

Immunoprecipitation (IP) and immunoblot (IB) assays After 24 h of treatment with silibinin at 25% inhibitory concentration (IC 25) or IC 50 dose, the cell pellets were lysed and the supernatants were used to detect Ki-67, AFP, p-Rb, E2F1, CD1, CDK4, p21waf1/cip1, p27kip1, bcl-2, survivin, activated caspase-3 and caspase-9, CD34, MMP-2, MMP-9, phosphorylated-Akt Thr308, PTEN, and AC-H3 and AC-H4. The IP assays were same, as previously reported[37,38]. β-actin was used as an internal control. The relative amount of each protein was quantified by digitally scanning its hybridizing bands, as previously reported[37,38]. PTEN activity assay PTEN protein was immunoprecipitated with 10 μL of rabbit anti-human antibody at 4℃ overnight, followed by addition of 25 μ L of anti-rabbit IgG-conjugated agarose beads for 2 h at 4℃, washing and centrifugation. The phosphatase reaction was performed in 50 µL of assay buffer containing 200 µmol/L water-soluble diC8PIP 3 and the immunoprecipitated PTEN protein. The release of phosphate from the substrate was measured in a colorimetric assay using the Biomol Green Reagent (Plymouth Meeting, PA)[39]. The OD absorbance at 650 nm was recorded in an ELISA plate reader[37,38]. Statistical analysis The descriptive statistics was provided with mean ± SD. A repeated-measure ANOVA test was used to assess dose dependent effects of silibinin on HuH7, HepG2, Hep3B, and PLC/PRF/5 cells. An independent sample t-test was used to assess the effects (i.e. mean differences) of silibinin treatment on apoptosis, and IB results. A P value < 0.05 was considered statistically significant.

RESULTS Potent dose-dependent anti-proliferative effects of silibinin on human HCC cells Effects of silibinin were initially assessed in HuH7 cells by MTT assay. As shown in Figure 1A, silibinin resulted in a dose-dependent inhibition of HuH7 cell growth. Compared to the control, there was a dose-dependent inhibitory which became significant at the dose greater than 180 µ mol/L (P < 0.05). As shown in Figure 1B, silibinin also significantly inhibited the growth of HepG2, Hep3B, and PLC/PRF/5 human HCC cell lines, indicating a wide spectrum of silibinin's inhibitory effects on human HCC cell growth, as previously reported[21,22]. Because the HuH7 cell line is one of the most commonly used human HCC lines [37,38], it was then used to further determine silibinin's anti-HCC effects and mechanisms. For further characterization of dose-related mechanistic effect of silibinin, approximate IC25 (i.e. 120 µmol/L) and IC50 (i.e. 240 µmol/L) concentration were subsequently used for the remainder of the study. Ki-67 is a commonly used biomarker for cell proliferation[40]. Consistent with the data derived from MTT assay, silibinin treatment resulted in a significantly dosedependent decrease in Ki-67 expression, as shown in Figures 2B (P < 0.05). These data further demonstrated

Lah JJ et al. Silibinin’s effects on hepatoma cells

5301

A

Control

120 100 80

a

a

60

a

Silibinin 120 μmol/L

A

β-actin (Internal control)

B

IB: Ki-67

a

40 20

15 ± 1.1

Co nt ro

l

0

B

10

20

40

2.0

a

9.1 ± 0.8

C

IP: Cyclin D1; IB: CDK4 28 ± 1.3

Control 120 240

1.6

a

12.3 ± 0.8

80 100 120 140 160 180 200 220 240 Silibinin (Micromolar)

1.8 OD value (490 nm)

60


a

b

9.3 ± 1.1

5.4 ± 0.2

D

1.4

IB: p-Rb

1.2 12.2 ± 1.5

1.0

b

8.3 ± 0.9

b

2.5 ± 0.4

0.8 0.6 0.4 0.2 0.0

a a

a

a

a

a

E

IP: E2F1; IB: DP1 11.5 ± 1.6

PLC

HepG2

b

7.6 ± 0.9

b

6.6 ± 0.4

Hep3B

Dose (Micromolar)

Figure 1 Silibinin’s effects on growth of human hepatoma cells. A: MTT assay showed silibinin’s dose dependent anti-proliferative effects on HuH7 cells. A significant decrease in proliferation compared to control was noted from silibinin ≥ 180 μmol/L. The IC25 is determined to be 120 μmol/L and the IC50 is determined to be 240 μmol/L; B: Silibinin’s effects on other human HCC cell growth. PLC/PRF/5, HepG2, and Hep3B HCC cells were treated with silibinin at IC25 and IC50 doses for HuH7 cells. Silibinin significantly reduced growth of all three HCC cells in different rates. aP < 0.05 vs control.

F

IB: p21 9.4 ± 1.5

b

11.5 ± 1.2

17.8 ± 2.1

G

IB: p27 7.1 ± 1.2

b

9.2 ± 1.2

11.3 ± 1.5

H

silibinin's significant dose-dependent anti-proliferative effects on human HCC cells. Effects of silibinin on cell cycle progression Uncontrolled progression of the cell cycle promotes growth of cancer cells[41]. A major activity of the CD1/CDK4 complex is to initiate phosphorylation of retinoblastoma (Rb) that then fails to maintain it's binding to E2F1, and thus releases the transcription factor to promote cell cycle progression[42]. Previous studies on other cancer cell lines showed a significant inhibitory effect of silibinin on the cell cycle progression[11-13]. In the present study, we found that silibinin resulted in a significant dose-dependent inhibition of CD1/CDK4 complex that was associated with reduced Rb phosphorylation and, E2F1/DP1 complex in HuH7 cells (P < 0.01), as shown in Figure 2C-E. By binding to the cyclin/CDK complexes, cyclin dependent kinase inhibitors (CDKIs), such as p21 and p27, halt uncontrolled cell proliferation[43]. As noted in previous studies on other cancer cell lines[12-14], we demonstrated that silibinin not only significantly increased p21 and p27 expression (P < 0.01), but also increased formation of p21/CDK4 and p27/CDK4 complexes (Figure 2F-I) in a dose-dependent fashion. Thus, our results demonstrate

IP: p21; IB: CDK4

6.3 ± 0.4

a

9.1 ± 2.3

14.9 ± 2.4

IP: p27; IB: CDK4

I 14.7 ± 2.1

b

24.7 ± 2.4

b

30.5 ± 2.8

Figure 2 Silibinin’s effects on proliferation of HCC cells. After HuH7 cells were treated with silibinin at IC 25 and IC 50 doses, immunoprecipitation (IP) and immunoblots (IB) were performed in triplicate for each specimen. A mean densitometer reading was expressed in the respective box and used for statistical analysis. A: β-actin for internal control; B: Ki-67; C: CD1/CDK4 complex; D: p-Rb; E: E2F1-DP1 complex; F: p21Waf1/Cip1; G: p27Kip1; H: p21Waf1/Cip/CDK4 complex; I: p27Kip1/CDK4 complex. aP < 0.05, bP < 0.01, vs control.

silibinin inhibits the growth of human hepatoma cells through inhibiting CDK activity. Effects of silibinin on AFP production and secretion from HuH7 cells As shown in Figure 3B, compared to untreated HuH7 cells, silibinin at the dose of 120 µmol/L resulted in a significant decrease in AFP production in HuH7 cells (P < 0.05) that was associated with a reduced AFP level in the culture medium (Figure 3C, P < 0.05). www.wjgnet.com

5302

ISSN 1007-9327

Control

CN 14-1219/R


A


October 28, 2007

Volume 13

2.0

Number 40

b

1.8 β-actin (Internal control)

A

1.6

AFP in HuH7 cells IP: AFP; IB: AFP

B 35.6 ± 3.2

a

15.9 ± 2.2

AFP in HuH7 culture medium IP: AFP; IB: AFP

C

Od values (405 nm)

1.4 1.2

b

1.0 0.8 0.6 0.4

12.8 ± 1.6

a

5.8 ± 1.6

0.2

Figure 3 Silibinin’s effects on AFP production and secretion from HuH7 cells. A: β-actin for internal control; B: Silibinin at IC25 dose decreased AFP production in HuH7 cells; C: Silibinin at IC25 dose decreased AFP secretion from HuH7 cells. a P < 0.05 vs control.

0.0

Control

Control

Effects of Silibinin on Apoptosis Apoptosis is another important regulatory step in controlling cancer cell proliferation[4]. Studies indicated that silibinin induces apoptosis in several malignant cell lines[13,14,18], but such effects have not been tested in human hepatoma cells. We demonstrated silibinin dose-dependently increases of apoptosis in HuH7 cells, as shown in Figure 4A (P < 0.01). To understand the mechanisms of silibinin-induced apoptosis, we examined the expression of Bcl-2, survivin, and activated caspase-3 and 9. Our results showed that silibinin-induced apoptosis did not alter bcl-2 expression (data not shown), but resulted in a dose-dependent inhibition of survivin expression (Figure 4C, P < 0.01) that was associated with increased levels of activated caspases-3 and -9 (Figures 4D and 4E, P < 0.01). Possible effects of silibinin on angiogenesis In previous studies, silibinin has been reported to inhibit angiogenesis in non-HCC cancer cell lines[13]. To evaluate whether silibinin affects angiogenesis in human HCC cells, we measured the expression of CD34, a transmembrane glycoprotein on vascular cells associated with angiogenesis[44], and MMP-2 and MMP-9, which are markers associated with angiogenesis as well as metastatic invasion[45]. As shown in Figure 5B, silibinin at IC50, but not IC25 dose decreased the expression of CD34 (P < 0.01). At the higher dose, Silibinin also resulted in decrease of MMP-2 (Figure 5C, P < 0.01), but not MMP-9 (data not shown) in HuH7 cells. Effects of silibinin on PTEN/PI3K/Akt pathway It has been reported that the PTEN/PI3K/Akt pathway is involved in cancer growth[16,46], As shown in Figure 6B, silibinin-reduced HuH7 cell proliferation was associated with a dose-dependent decrease in p-Akt (P < 0.01) in these cells. PTEN is an upstream negative regulator of Akt. It was reported that altered PTEN expression or activity is associated with the pathogenesis of HCC[47-49]. In the present study, we found that silibinin at IC50 dose did not significantly change PTEN expression (data not shown), but significantly increased PTEN activity (Figure 6E). These results suggested that the PTEN/PI3K/Akt pathway is involved in silibinin-reduced growth of human HCC cells. www.wjgnet.com

120 μmol/L Silibinin dose


240 μmol/L


B


C

IB: Survivin a

33.4 ± 4.1

12.5 ± 2.1

b

4.7 ± 1.2

D

IB: Cleaved caspase-3 4.4 ± 0.3

a

5.7 ± 1.2

b

15.4 ± 2.0

E

IB: Cleaved caspase-9 8.3 ± 2.1

b

13.3 ± 1.8

b

22.2 ± 2.5

Figure 4 Silibinin’s effects on apoptosis in HuH7 cells. A: Silibinin at IC25 and IC50 doses significantly promoted apoptosis of HuH7 cells; B: β-actin for internal control. Silibinin at IC25 and IC50 doses decreased survivin expression (C), but increased activated caspase-3 (D), and activated caspase-9 (E). aP < 0.05, b P < 0.01, vs control.

Effects of silibinin on AC-H3 and AC-H4 expression We then examined the association of AC-H3 and AC-H4 expression with silibinin-reduced HCC cell growth. Our results demonstrated that silibinin-reduced HuH7 cell growth was associated with increased AC-H3 and AC-H4 expression (Figures 6 C and D, P < 0.05). These results suggest that increased AC-H3 and AC-H4 expression may play an important role in silibinin-reduced HCC growth.

DISCUSSION Searching for an effective chemoprevention of HCC has been an active field of research. Silibinin is a polyphenolic flavonoid and the major biologically active compound of milk thistle. It is well known that milk thistle is safe and well tolerated, and it protects the liver from drug or alcohol-related injury[7,8]. Recent demonstration of silibinin's anti-HCC


Control


5303


Control



A


A


B

IB: CD34

B

IB: p-Akt

24.9 ± 2.7

27.3 ± 2.7

b

16.2 ± 1.5

35.9 ± 3.7 IB: MMP2

C 34.1 ± 3.5

32.8 ± 4.2

25.8 ± 2.7

b

6.4 ± 1.3

IB: AC-H3 25.3 ± 3.5

a

39.7 ± 2.6

a

48.2 ± 4.1

D

IB: AC-H4 63.5 ± 5.5

E

a

79.7 ± 2.6

a

98.2 ± 4.1

a

0.40 0.38

Od values (650 nm)

effects[21,22] provided us with a rationale to further define the related effects and mechanisms of HCC chemoprevention. In the present study, we examined the effects and mechanisms of silibinin on growth of human HCC cells. Using MTT assay[37,38], we demonstrated that silibinin treatment resulted in a potent inhibition of four different human HCC cell lines, indicating its broad spectrum of anti-HCC effects. We also revealed silibinin's linear dosedependent inhibition of HuH7 cell growth. Silibinin at IC 25 and IC 50 doses for HuH7 cells also resulted in reduced growth of HepG2, Hep3B, and PLC/PRF/5 cells, confirming the previous reports[21,22], These results promote us to further test silibinin for HCC chemoprevention. Both PCNA and Ki-67 are biomarkers for cell proliferation[40]. Singh et al reported silibinin significantly decreases PCNA and Ki-67 expression in nude mice bearing xenografts of human prostate cancer[11]. Consistent with this, we have demonstrated that silibinin significantly reduced Ki-67 expression in HuH7 cells in a dose-dependent fashion. These suggest that silibinin reduces growth of human HCC cells by down regulating their proliferation. AFP is associated with HCC differentiation and has been widely used for diagnosing HCC and assessing treatment effects or recurrence of HCC in humans[24,25]. Our results showed that silibinin treatment resulted in significant decrease in AFP production and secretion that was well correlated with growth inhibition of HuH7 cells. These findings suggest silibinin may promote HCC cell differentiation, and AFP may serve as a non-invasive biomarker to determine silibinin's in vivo anti-HCC effects. An uncontrolled G1-S progression results in continued proliferation with potential malignant transformation and carcinogenesis. Increased CDK4/CD1 complex enhances Rb phosphorylation that results in release of E2F1 from p-Rb/E2F1 complex and promotes E2F1/DP1 complex formation and stimulates cell cycle progression[42]. Tyagi et al reported that silibinin causes a significant decrease in p-Rb in human prostate cancer cells[10]. Our results indicate that silibinin-inhibited CDK4/CD1 complex formation is one of the important steps that inhibit Rb phosphorylation, followed by reduction of E2F1/DP1 complex formation. CDKIs are important regulators of the activity of the CD1/CDK4 complex. By binding to the cyclin/CDK

b

C

b

Figure 5 Silibinin’s effects on angiogenesis in HuH7 cells. A: β-actin for internal control. Silibinin decreased CD34 (B), and MMP-2 (C). bP < 0.01 vs control.

25.2 ± 2.7

0.36 0.34 0.32 0.30 0.28

Control


Figure 6 Silibinin’s Effects on PTEN, p-Akt, AC-H3 and AC-H4 in HuH7 Cells (A). β-actin for internal control. Silibinin significantly decreased p-Akt (B); increased AC-H3 (C); AC-H4 (D). Silibinin at 240 µmol/L significantly increased PTEN activity in HuH7 cells (E). aP < 0.05, bP < 0.01, vs control.

complexes, two very important CDKIs, p21 and p27, inhibit their activities. Varghese, et al reported that silibinin increases levels of p27 in human hepatoma cells[22]. Silibinin was also reported increasing expression of p21 in several non-HCC cancer cells[14]. In the present study, we demonstrated that silibinin resulted in a significantly dose-dependent increase in both p21 and p27, which was well correlated with their respective binding to CDK4, the bioactive forms of these CDKIs. Taken together, our data demonstrated that silibinin reduces cell cycle progression in human hepatoma cells by arresting G1-S transition that involves a comprehensive signaling of cell cycle modulators. Previous data on other cancer cell lines have demonstrated that silibinin has effects on the apoptotic control[10,14,21]. We demonstrated that silibinin causes a significant increase in apoptosis of HuH7 cells which was associated with decreased survivin expression and increased activated caspase-3 and -9. Because survivin can bind with caspases[19,50,51], our results suggest that silininin-induced apoptosis of HuH7 cells is mediated by decreased survivin that results in increased caspase-3 and 9 activation. Angiogenesis is an important aspect of cancer invasion and survival. CD34 is a valuable marker to demonstrate this issue[44]. A previous study demonstrated that silibinin decreases angiogenesis in colon cancer cells[15]. In the present study, we revealed that silibinin at IC50 decreased CD34 www.wjgnet.com

5304

ISSN 1007-9327

CN 14-1219/R


protein expression. MMP-2 and MMP-9 have been used as markers for angiogenesis and malignant invasion[45]. It was reported that silibinin resulted in a significant decrease in MMP-2, but not MMP-9 levels in human lung cancer cells[20]. In the present study, we found that silibinin also resulted in a dose-dependent and significant decrease in MMP-2, but not MMP-9 expression. Although our data suggest that silibinin may reduce angiogenesis in human HCC cells, further in vivo studies with quantification of microvessel density[52] will be needed to validate these findings. There is growing evidence of PTEN/PI3K/Akt pathway in hepatocarcinogenesis[27,32,47-49]. PTEN is a tumor suppressor gene and the deletion or inactivation of this gene has been described in a variety of cancer cell lines[30,33,53]. As a result, the tumor suppressive properties of PTEN relates in part to its ability to down-regulate the Akt pathway and thus inhibit cell proliferation[32,33]. Paramio et al showed that PTEN decreases p-Rb and resulted in down-regulation of CD1 [54]. Furthermore, Weng et al demonstrated through the use of breast cancer cells that PTEN also up-regulates p27 and down-regulates CD1[55]. However, it remains to be determined whether PTEN/ PI3K/Akt pathway is involved in silibinin-reduced growth of cancers. In the present study, we found that silibinin significantly increased PTEN activity in association with decreased p-Akt in HuH7 cells. Since silibinin treatment also resulted in significant decrease of p-Rb and proliferation in these cells, it is evident that silibinin alters PTEN activity to assist in cellular growth control through downstream regulation of Akt and also possibly in promoting the up-regulation of p27 and the downregulation of both p-Rb and CD1 as suggested by previous studies [54]. It was also reported that overexpression of PTEN reduces survivin expression [56]. We found that silibinin-mediated increase in PTEN activity and decrease in p-Akt was associated with decreased survivin expression and enhanced apoptosis in HuH7 cells. These data support the notion that PTEN/PI3K/Akt pathway may mediate cancer cell apoptosis by modulating surviving expression, and silibinin may play an important role in this interaction. Additional studies will be needed to further detail the role of PTEN/PI3K/Akt signaling in silibinin-reduced growth of human HCC cells. Histone acetylation alters chromatin conformation by m a k i n g p r o m o t e r r e g i o n s m o r e a c c e s s i b l e t o transcription factors and permissive to transcriptional activation[34]. Studies have reported that histone acetylation is involved in cell proliferation, differentiation, and cell cycle regulation[34]. Decrease in acetylation status in the cell is associated with carcinogenesis [35,36]. Our results demonstrated that silibinin-reduced HuH7 cell growth was significantly associated with increased AC-H3 and AC-H4 expression, suggesting that increased histone acetylation may mediate silibinin-reduced HCC growth. Our findings not only indicate silibinin's novel anti-cancer mechanisms, but also provide additional targets for searching new agents for HCC chemoprevention.

2 3 4 5 6 7 8 9

10

11

12 13 14

15

16

17

18

19

REFERENCES 1

Chen CJ, Yu MW, Liaw YF. Epidemiological characteristics and risk factors of hepatocellular carcinoma. J Gastroenterol

www.wjgnet.com

20

October 28, 2007

Volume 13

Number 40

Hepatol 1997; 12: S294-S308 El-Serag HB, Mason AC. Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med 1999; 340: 745-750 El-Serag HB, Mason AC, Key C. Trends in survival of patients with hepatocellular carcinoma between 1977 and 1996 in the United States. Hepatology 2001; 33: 62-65 Kroll DJ, Shaw HS, Oberlies NH. Milk thistle nomenclature: why it matters in cancer research and pharmacokinetic studies. Integr Cancer Ther 2007; 6: 110-119 Gazak R, Walterova D, Kren V. Silybin and silymarin--new and emerging applications in medicine. Curr Med Chem 2007; 14: 315-338 Singh RP, Agarwal R. A cancer chemopreventive agent silibinin, targets mitogenic and survival signaling in prostate cancer. Mutat Res 2004; 555: 21-32 Jacobs BP, Dennehy C, Ramirez G, Sapp J, Lawrence VA. Milk thistle for the treatment of liver disease: a systematic review and meta-analysis. Am J Med 2002; 113: 506-515 Lieber CS, Leo MA, Cao Q, Ren C, DeCarli LM. Silymarin retards the progression of alcohol-induced hepatic fibrosis in baboons. J Clin Gastroenterol 2003; 37: 336-339 Singh RP, Sharma G, Dhanalakshmi S, Agarwal C, Agarwal R. Suppression of advanced human prostate tumor growth in athymic mice by silibinin feeding is associated with reduced cell proliferation, increased apoptosis, and inhibition of angiogenesis. Cancer Epidemiol Biomarkers Prev 2003; 12: 933-939 Tyagi A, Agarwal C, Agarwal R. Inhibition of retinoblastoma protein (Rb) phosphorylation at serine sites and an increase in Rb-E2F complex formation by silibinin in androgen-dependent human prostate carcinoma LNCaP cells: role in prostate cancer prevention. Mol Cancer Ther 2002; 1: 525-532 Singh RP, Dhanalakshmi S, Tyagi AK, Chan DC, Agarwal C, Agarwal R. Dietary feeding of silibinin inhibits advance human prostate carcinoma growth in athymic nude mice and increases plasma insulin-like growth factor-binding protein-3 levels. Cancer Res 2002; 62: 3063-3069 Tyagi A, Bhatia N, Condon MS, Bosland MC, Agarwal C, Agarwal R. Antiproliferative and apoptotic effects of silibinin in rat prostate cancer cells. Prostate 2002; 53: 211-217 Yang SH, Lin JK, Chen WS, Chiu JH. Anti-angiogenic effect of silymarin on colon cancer LoVo cell line. J Surg Res 2003; 113: 133-138 Agarwal C, Singh RP, Dhanalakshmi S, Tyagi AK, Tecklenburg M, Sclafani RA, Agarwal R. Silibinin upregulates the expression of cyclin-dependent kinase inhibitors and causes cell cycle arrest and apoptosis in human colon carcinoma HT-29 cells. Oncogene 2003; 22: 8271-8282 Mohan S, Dhanalakshmi S, Mallikarjuna GU, Singh RP, Agarwal R. Silibinin modulates UVB-induced apoptosis via mitochondrial proteins, caspases activation, and mitogenactivated protein kinase signaling in human epidermoid carcinoma A431 cells. Biochem Biophys Res Commun 2004; 320: 183-189 Mallikarjuna G, Dhanalakshmi S, Singh RP, Agarwal C, Agarwal R. Silibinin protects against photocarcinogenesis via modulation of cell cycle regulators, mitogen-activated protein kinases, and Akt signaling. Cancer Res 2004; 64: 6349-6356 Singh RP, Tyagi AK, Zhao J, Agarwal R. Silymarin inhibits growth and causes regression of established skin tumors in SENCAR mice via modulation of mitogen-activated protein kinases and induction of apoptosis. Carcinogenesis 2002; 23: 499-510 Tyagi A, Agarwal C, Harrison G, Glode LM, Agarwal R. Silibinin causes cell cycle arrest and apoptosis in human bladder transitional cell carcinoma cells by regulating CDKICDK-cyclin cascade, and caspase 3 and PARP cleavages. Carcinogenesis 2004; 25: 1711-1720 Tyagi AK, Agarwal C, Singh RP, Shroyer KR, Glode LM, Agarwal R. Silibinin down-regulates survivin protein and mRNA expression and causes caspases activation and apoptosis in human bladder transitional-cell papilloma RT4 cells. Biochem Biophys Res Commun 2003; 312: 1178-1184 Chu SC, Chiou HL, Chen PN, Yang SF, Hsieh YS. Silibinin


21 22 23 24

25

26 27 28 29

30

31

32

33 34 35 36

37

38 39

inhibits the invasion of human lung cancer cells via decreased productions of urokinase-plasminogen activator and matrix metalloproteinase-2. Mol Carcinog 2004; 40: 143-149 Lah J, Cui W, Hu KQ. Inhibitory effects and mechanisms of silibinin on growth of human hepatoma cell lines. Hepatology 2005; 42: Suppl 309A (Abstract) Varghese L, Agarwal C, Tyagi A, Singh RP, Agarwal R. Silibinin efficacy against human hepatocellular carcinoma. Clin Cancer Res 2005; 11: 8441-8448 Johnson PJ. The role of serum alpha-fetoprotein estimation in the diagnosis and management of hepatocellular carcinoma. Clin Liver Dis 2001; 5: 145-159 Shirabe K, Takenaka K, Gion T, Shimada M, Fujiwara Y, Sugimachi K. Significance of alpha-fetoprotein levels for detection of early recurrence of hepatocellular carcinoma after hepatic resection. J Surg Oncol 1997; 64: 143-146 Peng SY, Chen WJ, Lai PL, Jeng YM, Sheu JC, Hsu HC. High alpha-fetoprotein level correlates with high stage, early recurrence and poor prognosis of hepatocellular carcinoma: significance of hepatitis virus infection, age, p53 and betacatenin mutations. Int J Cancer 2004; 112: 44-50 Osaki M, Oshimura M, Ito H. PI3K-Akt pathway: its functions and alterations in human cancer. Apoptosis 2004; 9: 667-676 Lawlor MA, Alessi DR. PKB/Akt: a key mediator of cell proliferation, survival and insulin responses? J Cell Sci 2001; 114: 2903-2910 Yao R, Cooper GM. Requirement for phosphatidylinositol-3 kinase in the prevention of apoptosis by nerve growth factor. Science 1995; 267: 2003-2006 Li J, Yen C, Liaw D, Podsypanina K, Bose S, Wang SI, Puc J, Miliaresis C, Rodgers L, McCombie R, Bigner SH, Giovanella BC, Ittmann M, Tycko B, Hibshoosh H, Wigler MH, Parsons R. PTEN, a putative protein tyrosine phosphatase gene mutated in human brain, breast, and prostate cancer. Science 1997; 275: 1943-1947 Steck PA, Pershouse MA, Jasser SA, Yung WK, Lin H, Ligon AH, Langford LA, Baumgard ML, Hattier T, Davis T, Frye C, Hu R, Swedlund B, Teng DH, Tavtigian SV. Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers. Nat Genet 1997; 15: 356-362 Wan XW, Jiang M, Cao HF, He YQ, Liu SQ, Qiu XH, Wu MC, Wang HY. The alteration of PTEN tumor suppressor expression and its association with the histopathological features of human primary hepatocellular carcinoma. J Cancer Res Clin Oncol 2003; 129: 100-106 Liu LZ, Zhou XD, Qian G, Shi X, Fang J, Jiang BH. AKT1 amplification regulates cisplatin resistance in human lung cancer cells through the mammalian target of rapamycin/ p70S6K1 pathway. Cancer Res 2007; 67: 6325-6332 Sansal I, Sellers WR. The biology and clinical relevance of the PTEN tumor suppressor pathway. J Clin Oncol 2004; 22: 2954-2963 Grunstein M. Histone acetylation in chromatin structure and transcription. Nature 1997; 389: 349-352 Cress WD, Seto E. Histone deacetylases, transcriptional control, and cancer. J Cell Physiol 2000; 184: 1-16 Song J, Noh JH, Lee JH, Eun JW, Ahn YM, Kim SY, Lee SH, Park WS, Yoo NJ, Lee JY, Nam SW. Increased expression of histone deacetylase 2 is found in human gastric cancer. APMIS 2005; 113: 264-268 Hu KQ, Yu CH, Mineyama Y, McCracken JD, Hillebrand DJ, Hasan M. Inhibited proliferation of cyclooxygenase-2 expressing human hepatoma cells by NS-398, a selective COX-2 inhibitor. Int J Oncol 2003; 22: 757-763 Cui W, Yu CH, Hu KQ. In vitro and in vivo effects and mechanisms of celecoxib-induced growth inhibition of human hepatocellular carcinoma cells. Clin Cancer Res 2005; 11: 8213-8221 Meuillet EJ, Mahadevan D, Berggren M, Coon A, Powis G.

5305

40

41 42 43 44 45

46

47

48 49

50 51 52

53 54

55

56

Thioredoxin-1 binds to the C2 domain of PTEN inhibiting PTEN's lipid phosphatase activity and membrane binding: a mechanism for the functional loss of PTEN's tumor suppressor activity. Arch Biochem Biophys 2004; 429: 123-133 Hall PA, Levison DA, Woods AL, Yu CC, Kellock DB, Watkins JA, Barnes DM, Gillett CE, Camplejohn R, Dover R. Proliferating cell nuclear antigen (PCNA) immunolocalization in paraffin sections: an index of cell proliferation with evidence of deregulated expression in some neoplasms. J Pathol 1990; 162: 285-294 Deshpande A, Sicinski P, Hinds PW. Cyclins and cdks in development and cancer: a perspective. Oncogene 2005; 24: 2909-2915 Seville LL, Shah N, Westwell AD, Chan WC. Modulation of pRB/E2F functions in the regulation of cell cycle and in cancer. Curr Cancer Drug Targets 2005; 5: 159-170 Megargee EI, Cook PE. Negative response bias and the MMPI overcontrolled-hostility scale: a response to Deiker. J Consult Clin Psychol 1975; 43: 725-729 Xu J, You C, Zhang S, Huang S, Cai B, Wu Z, Li H. Angiogenesis and cell proliferation in human craniopharyngioma xenografts in nude mice. J Neurosurg 2006; 105: 306-310 Tutton MG, George ML, Eccles SA, Burton S, Swift RI, Abulafi AM. Use of plasma MMP-2 and MMP-9 levels as a surrogate for tumour expression in colorectal cancer patients. Int J Cancer 2003; 107: 541-550 Mitsuuchi Y, Johnson SW, Selvakumaran M, Williams SJ, Hamilton TC, Testa JR. The phosphatidylinositol 3-kinase/ AKT signal transduction pathway plays a critical role in the expression of p21WAF1/CIP1/SDI1 induced by cisplatin and paclitaxel. Cancer Res 2000; 60: 5390-6394 Rahman MA, Kyriazanos ID, Ono T, Yamanoi A, Kohno H, Tsuchiya M, Nagasue N. Impact of PTEN expression on the outcome of hepatitis C virus-positive cirrhotic hepatocellular carcinoma patients: possible relationship with COX II and inducible nitric oxide synthase. Int J Cancer 2002; 100: 152-157 Dong-Dong L, Xi-Ran Z, Xiang-Rong C. Expression and significance of new tumor suppressor gene PTEN in primary liver cancer. J Cell Mol Med 2003; 7: 67-71 Hu TH, Huang CC, Lin PR, Chang HW, Ger LP, Lin YW, Changchien CS, Lee CM, Tai MH. Expression and prognostic role of tumor suppressor gene PTEN/MMAC1/TEP1 in hepatocellular carcinoma. Cancer 2003; 97: 1929-1940 Ladas EJ, Kelly KM. Milk thistle: is there a role for its use as an adjunct therapy in patients with cancer? J Altern Complement Med 2003; 9: 411-416 Sah NK, Khan Z, Khan GJ, Bisen PS. Structural, functional and therapeutic biology of survivin. Cancer Lett 2006; 244: 164-171 Kinoshita S, Hirai R, Yamano T, Yuasa I, Tsukuda K, Shimizu N. Angiogenesis inhibitor TNP-470 can suppress hepatocellular carcinoma growth without retarding liver regeneration after partial hepatectomy. Surg Today 2004; 34: 40-46 Besson A, Robbins SM, Yong VW. PTEN/MMAC1/TEP1 in signal transduction and tumorigenesis. Eur J Biochem 1999; 263: 605-611 Paramio JM, Navarro M, Segrelles C, Gomez-Casero E, Jorcano JL. PTEN tumour suppressor is linked to the cell cycle control through the retinoblastoma protein. Oncogene 1999; 18: 7462-7468 Weng LP, Brown JL, Eng C. PTEN coordinates G(1) arrest by down-regulating cyclin D1 via its protein phosphatase activity and up-regulating p27 via its lipid phosphatase activity in a breast cancer model. Hum Mol Genet 2001; 10: 599-604 Wu ZX, Song TB, Li DM, Zhang XT, Wu XL. Overexpression of PTEN suppresses growth and induces apoptosis by inhibiting the expression of survivin in bladder cancer cells. Tumour Biol 2007; 28: 9-15 S- Editor Liu Y L- Editor Alpini GD

E- Editor Li HY

www.wjgnet.com



LIVER CANCER

Survivin expression in early hepatocellular carcinoma and post-treatment with anti-cancer drug under hypoxic culture condition Satoshi Mamori, Tadashi Asakura, Kiyoshi Ohkawa, Hisao Tajiri Satoshi Mamori, Hisao Tajiri, Division of Gastroenterology and Hepatology, Department of Internal Medicine, The Jikei University School of Medicine, Tokyo 105-8461, Japan Tadashi Asakura, Kiyoshi Ohkawa, Department of Biochemistry 1, The Jikei University School of Medicine, Tokyo 105-8461, Japan 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: July 07, 2007 Revised: August 17, 2007

Abstract AIM: To investigate the expression of survivin during the early stages of hepatocellular carcinoma (HCC). METHODS: Immunohistochemical expression of survivin in liver tumor and non-tumor tissue specimens taken from 17 patients was compared. In addition, to determine the survivin expression in response to anticancer drugs in early stage HCC, the survivin expression was determined after the treatment of the HCC cells with anti-cancer drugs under hypoxic culture conditions. RESULTS: Survivin proteins were expressed in 64.7% of cells in early HCC specimens. A correlation between the survivin expression rate in the peritumoral hepatocytes and the rate of expression in the HCC specimens (low-rate group vs high-rate group) was observed. The survivin protein concentration in HCC cells was increased by the combination of hypoxia and anti-cancer drugs. CONCLUSION: This study suggests that survivin could be used as a therapeutic target in early HCC. © 2007 WJG . All rights reserved.

Key words: Survivin; Hepatocellular carcinoma; Hypoxia Mamori S, Asakura T, Ohkawa K, Tajiri H. Survivin expression in early hepatocellular carcinoma and post-treatment with anti-cancer drug under hypoxic culture condition. World J Gastroenterol 2007; 13(40): 5306-5311


www.wjgnet.com

INTRODUCTION Hepatocellular carcinoma (HCC) is a major health problem worldwide. There are more than 500 000 new cases diagnosed each year, with an age-adjusted incidence of 5.5-14.9 per 100 000 people[1]. In some areas of Asia and the Middle East, HCC ranks as the most frequent cancer-related cause of death[2]. The incidence of HCC is also increasing in Europe and the United States[3]. A more effective therapy thus needs to be developed from early stages. Survivin is a member of a family of inhibitors of apoptosis protein (IAP), which has been implicated in both the control of cell division and the inhibition of apoptosis. Specifically, its anti-apoptotic function is associated with the ability to directly or indirectly inhibit caspases. By inhibiting apoptosis and promoting mitosis, survivin facilitates cancer cell survival and growth [4-8]. Survivin is selectively expressed in the most common human neoplasms and appears to be involved in tumor cell resistance to some anticancer agents and ionizing radiation[9]. Several preclinical studies have demonstrated that the down-regulation of survivin expression/function by the use of anti-sense oligonucleotide, dominant negative mutants, ribozymes, small interfering RNAs and cyclin-dependent kinase inhibitors increased the rate of apoptosis, reduced tumor growth potential and sensitized tumor cells to various chemotherapeutic drugs and γ-irradiation in in vitro and in vivo models of various types of human tumors[9]. Moreover, YM155 is the first agent designed to inhibit survivin. Some early phase clinical studies demonstrated that this novel anticancer agent was well tolerated and shrank tumors in some patients with non-Hodgkin lymphoma and hormone-refractory prostate cancer that recurred after conventional chemotherapy. In addition, interim reports indicate that there are few side effects. These results suggest the possible efficacy of the survivin inhibitor on HCC. It may be effective for patients with early stages of HCC. Sur vivin is expressed in HCC[10]. However, the expression during the early stages of HCC has not been characterized pathologically. In addition, previous results have shown that survivin gene transcription is increased in hypoxic tumor cells[11]. The well-differentiated HCC has portal blood flow and is not hypervascular[12]. In order to compare the expression of

Mamori S et al . Survivin expression in early HCC and treatment under hypoxia

survivin and the efficacy of anti-cancer drugs, HCC cells were cultured in a hypoxic environment.

MATERIALS AND METHODS Patients The study population included 17 patients (11 men and 6 women; median age 68 years, range 56-81 years) who underwent a tumor biopsy between January 2004 and December 2005 in the Jikei University Daisan Hospital, Tokyo, Japan (Table 1). All patients underwent biopsies to confirm the diagnosis of HCC. These tissue specimens were examined retrospectively. This study was approved by the Jikei University Ethics Committee Institutional Review Broad. Pathologic specimens Tumor specimens were obtained by tumor biopsies with a 21-G fine-needle aspiration kit. Non-tumorous liver tissue specimens were concurrently obtained by an 18-20-G needle liver biopsy. Formalin-fixed, paraffin-embedded specimens of liver tumor and non-tumor tissues were processed for conventional histological assessment by hematoxylin and eosin (H&E) staining. The tumors were histologically graded as well or moderately differentiated. Immunohistochemical analysis For the immunohistochemical analysis, for malinfixed, paraffin-embedded specimens were used after deparaffinization. A rabbit anti-human survivin polyclonal antibody (Diagnostic BioSystems, USA) was used at dilution of 1:2000 as the primary antibody, which was detected with ENVISION + Rabbit/HRP (Dako, Japan). The specimens were heated in a microwave oven containing antigen retrieval solution (10 mmol/L citrate buffer, pH 6.4) at 121℃ for 15 min for the retrieval of the antigens and then cooled to room temperature. 3, 3-Diaminobenzidine and hematoxylin were used for color development and counterstaining, respectively. Cells with brown-colored nuclei were regarded as positive. The mean percentage of survivin-positive HCC cells was determined in three areas at 100 × magnification with the nuclear labeling index (labeled nuclei/500 nuclei). The same method was performed for hepatocytes in nontumorous biopsy specimens. HCC cell line cultured in the combination of hypoxia and anti-cancer drugs environment Human hepatocellular carcinoma cell line FLC-7 was cultured with RPMI-1640 (Invitrogen, Carlsbad, CA) medium supplemented with 100 mL/L heat-inactivated fetal bovine serum (FBS) under conventional conditions at 37℃ in a humidified atmosphere containing 50 mL/L CO2[13] until the cells were 70%-80% confluent. Cells were then used for culture under hypoxic conditions employing the AnaeroPack for cell (Mitsubishi Gas Chemical Co., Tokyo, Japan) packaging device. The cells were sealed tightly and incubated at 37℃ for either 6 or 96 h. In addition, for the anti-cancer drug therapy, the cells were cultured continuously with 0.1 µmol/L farmorubicin (EPI)

5307

Table 1 Characteristics of the patients undergoing tumor biopsies (n = 17) Features Age, yr Sex (Male/Female) AFP (ng/mL) HBsAg/HCVAb Tumor size (mm) Cirrhosis (positive/negative) Differentiation (well/moderate)

Values 68 (56-81) 11/6 21 (3-444) 3/14 15 (8-23) 5/12 11/6

Data are expressed as the medians with ranges in parentheses unless indicated otherwise. HBsAg: Anti-hepatitis B surface antigen; HCVAb: Antihepatitis C antibody; Well: Well-differentiated HCC; Moderate: Moderatedifferentiated HCC; Normal ranges: AFP (alpha-feto protein) > 20 ng/mL.

Table 2 PCR primer sequences Name Survivin β-actin

Forward sequence (5'-3') GCCCAGTGTTTCTTCT GCTT AGCCATGTACGTAGC CATCC

Reverse sequence (5'-3') GCACTTTCTTCGCAGT TTCC AAGTGGTGGTGTCGAC TCTC

containing growth medium for 6 or 96 h. The cytotoxicity (IC50) with EPI of FLC-7 cells determined the medication concentration (Normoxia cultured for 96 h, data not shown). Reverse transcription-polymerase chain reaction (RT-PCR) Total RNA was extracted from the cells using the RNeasy kit (Qiagen, Hilden, Germany). The mRNA was reverse transcribed into cDNA using the Prime script (TAKARA BIO INC, Shiga, Japan). The specific cDNA target sequences for survivin were amplified by a PCR reaction mixture consisting of 1 μL cDNA template, 10 μmol/L each primer (Primer sequences are listed in Table 2), PCR Master Mix (Go taq, Promega, Madison, WI, USA). The PCR conditions were: initial pre-denaturation at 95℃ for 5 min; 30 amplification cycles of denaturation at 95℃ for 30 s, primer annealing at 55℃ for 30 s, and elongation at 72℃ for 60 s; and a final extension at 72℃ for 5 min. PCR products were analyzed on a 20 g/L agarose/TBE gel electrophoresis and compared to the expression of β-actin as a housekeeping gene. Western blotting The harvested cells cultured in either a normal or hypoxic environment for 96 h with or without 0.1 μ mol/L of EPI were washed with ice-cold PBS and lysed in icecold 5 mL/L Triton X-100 containing 10 mmol/L EDTA. The cell lysate was centrifuged at 15 000 g for 5 min and the supernatant was used for Western blotting. Thirty micrograms of protein was separated on 150 g/L polyacr ylamide g els and transfer red onto 0.2- μ m nitrocellulose membranes by wet blotting (20 mA for 60 min). Membranes were blocked with blocking buffer (1 × TBS, 1 g/L Tween-20, 1 g/L casein gelatin) for 0.5 h at 37℃ and stained with the specific antibody for survivin www.wjgnet.com

5308

ISSN 1007-9327

CN 14-1219/R


October 28, 2007

Volume 13

Survivin expression

A

High group 100

Survivin rate of hepatocytes in non-tumor biopsies Low

45 40 35 30 25 20 15 10 5 0

High

%

80 Positive rate (%)

Number 40

60 40

Group

20

P < 0.01

Low group 0 Tumor biopsy (Avarage: 64.7%) Non-tumor biopsy (Avarage: 36.3%)

B

Figure 2 Nuclear survivin expression rates in HCC biopsies and non-tumor biopsies samples. In tumor biopsies, > 500 survivin-expressing HCC cells were counted in three areas at 100 х magnification using the nuclear labeling index. In non-tumor biopsies, > 500 survivin-expressing hepatocyte cell were counted in three areas at 100 х magnification using the nuclear labeling index.

Protein determination Protein concentration was assayed by a Bio-Rad protein assay kit (Bio-Rad Lab., Tokyo, Japan) using BSA as the standard. C

Statistical analysis Statistical analyses were performed using the WilcoxonMann-Whitney two-sample rank-sum test. A P value less than 0.05 was considered statistically significant.

RESULTS

D

Figure 1 Immunopathological staining of survivin in HCC and peritumoral biopsy tissues. A: Non-tumor biopsy (х 100); B: Tumor biopsy (х 100); C: Nontumor biopsy (х 400); D: Tumor biopsy (х 400).

(1:1000; Novus Biologicals, Littleton, USA). The complex of antigen with the primary antibody was completely labeled with the secondary antibody, anti-rabbit IgG alkaline phosphatase conjugate (1:2000; Sigma-Aldrich Japan, Tokyo, Japan). The survivin band was visualized with 5-bromo-4-chlor-3-indoly-phosphate/nitro blue terazulium (Sigma-Aldrich Japan, Tokyo, Japan). www.wjgnet.com

Rates of survivin expression in early HCC and in nontumorous liver tissues In HCC tissues, the rate of survivin expression was determined by counting survivin-positive cancer cells (Figure 1). The average survivin expression rate was 64.7% (median). The samples with a survivin expression rate over 50% were regarded as high-rate group, while the three samples with a survivin expression rate under 50% were regarded as low-rate group (Figure 2). In early-stage HCC with a tumor size > 10 mm (n = 3), the expression rate ranged for 67.7%-83.7%. The expression rate of survivin in HCC had no significant correlation to the level of differentiation of HCC. In non-tumorous liver tissues, survivin expression rates were counted in all hepatocytes. The average of survivin expression was 36.3% (median, Figure 2). A correlation between the survivin expression rate in peritumoral cells and the rate of expression in the HCC specimens (low-rate group vs high-rate group) was observed. A significant difference in survivin expression in the hepatocyte was observed between the high-rate group and low-rate group (P < 0.01, Figure 2). Survivin expression of HCC cells in hypoxic conditions and post-treatment with anti-cancer drugs T he hypoxic environment increased the sur vivin mRNA expression in both short-term and long-term cultures (Figure 3). Under both normoxia and hypoxia, the survivin mRNA concentrations increased in the presence of anti-cancer drugs in the short-term culture.


5309

Survivin β-action 6 h cultured EPI (-) EPI (+) N N H H

96 h cultured EPI (-) EPI (+) N N H H

EPI: 0.1 μm EPI/mL N: Normoxia cultured H: Hypoxia cultured

Figure 3 Expression of survivin mRNA in a hypoxic and anti-cancer drugcontaining medium. The survivin mRNA expression increased under hypoxia, by anti-cancer drug treatment, and in presence of the both conditions in the 6-h culture. The survivin mRNA expression increased under hypoxia and in the combined conditions of hypoxia and anti-cancer drug in the 96-h culture.

Nevertheless, the survivin mRNA concentrations only increased in the combination of hypoxic culture and anti-cancer drugs in the long-term culture (Figure 3). No survivin protein expression was observed in the hypoxia culture (Figure 4). In contrast, the survivin protein concentration increased with the anti-cancer drug concentrations. Moreover, the survivin protein concentrations increased when cultured in a combination of hypoxia and anti-cancer drugs (Figure 4).

DISCUSSION The suppression of apoptosis is thought to contribute to carcinogenesis due to several mechanisms, including unusually prolonging the cellular lifespan, facilitating the accumulation of gene mutations and permitting growth factor-independent cell survival[14]. In addition, since the host's immune system normally eliminates cancer cells by induction of apoptosis, inhibition of this process is critical for cancer cells survival. Several proteins, including the bcl-2 family and the IAP family, are involved in the inhibition of apoptotic signaling[15-16]. Survivin, a novel member of the IAP family, inhibits the activation of caspase-3 and -7, which are downstream effectors of apoptosis, in cells exposed to apoptotic stimuli [17-20] . Previous studies have shown that survivin is expressed at a high level in 60%-100% of the most common human tumor types, including colon, pancreas, breast, lung, liver, brain, lymphoma, melanoma and prostate cancers [21-24]. The elevated expression of survivin is associated with poor patient survival[25-28]. In the present study, positive nuclear survivin expression was observed in all tumor biopsy samples. It is possible that this result was based on dyeing conditions and the nuclear labeling index. The differential nuclear and cytoplasmic localization of survivin has been shown to be due to differences in the amino-acid sequence of its carboxy-terminal domain[29]. In HCC, the predominant function of survivin is its cell cycle nuclear distribution, and not the cytoplasmic caspase-3-dependent anti-apoptotic effect[30]. So in HCC, the prognostic significance of survivin immunostain relates to the cel l cycl e i n nu cl ei , a n d n o t to i ts cytoplasmic anti-apoptotic effect[31]. These reports suggest that most cytoplasm in the early HCC samples might be stained moderately under the conditions employed in this study. In the present study, nuclear survivin is expressed in 64.7% of cells (median) from early-stage HCC specimens.

A

B

C

D

16.5 kDa Marker

Figure 4 Western blotting showing the expression of survivin protein in the combined conditions of hypoxia and anti-cancer drugs in the 96-h culture. Survivin expression increased under anti-cancer drug-containing medium. Moreover, survivin further increased after the administration of a combination of hypoxia and anti-cancer drug. A: Normoxia; B: Hypoxia; C: Normoxia + 0.1 μmol/L EPI; D: Hypoxia + 0.1 μmol/L EPI.

Moreover, in all early HCCs of tumor size > 10 mm (n = 3), survivin expression was always above the median average. These data indicate that survivin could be an effective target of gene therapy for HCC, even at an early stage. Moreover, a previous study reported that in surgically removed tissues, the expression of survivin had no correlation with the patient's age, gender, tumor size and differentiation level of HCC [11]. This is consistent with the rate of nuclear expression in the small biopsy samples from early-stage HCC observed in the present study. However, a recent report showed that alpha-feto protein (AFP) blocked the X-linked inhibitor of apoptosis protein-mediated inhibition of endogenous active caspases in the cytosolic lysates of tumor cells[32]. Further immunohistological analyses of other proteins of the IAP family must be compared to the clinical parameters in early-stage HCC. In this study, the survivin expression in HCC samples could be divided into two groups: a high-rate group (rate > 50%) and low-rate group (rate < 50%). Ikeguchi et al[33,34] detected survivin mRNA over-expression in 21 of 51 (41%) of HCC biopsies, and suggested that this could be useful as a prognostic factor for patients with HCC. From the early stages, the level of nuclear survivin expression may correlate with the prognosis of HCC. HBV X and HCV core proteins activate NF- k B and/or STAT-3, which regulate the gene expression for cell survival factors such as the anti-apoptosis proteins, including survivin[35,36]. A resulting up-regulation of antiapoptosis factors during HCV or HBV infection may contribute to hepatocarcinogenesis [35,36] . A previous report demonstrated that HBx promotes the upregulation of survivin expression in hepatoma and normal liver cells, regardless of apoptosis. These findings suggest that survivin and HBx may play important roles in the carcinogenesis of HCC [37]. Other studies have shown that HCV NS5A protein can stimulate survivin protein expression, and this may result from induced survivin gene transcription[38]. In the present study, the average nuclear survivin expression was 36.3% in hepatocyte of non-tumor specimens. This result may indicate that the hepatitis virus is associated with survivin expression in peritumoral cells. The expression of survivin has been detected in a variety of pre-neoplastic and/or benign lesions, including polyps of the colon, breast adenomas, Bowen's disease and hypertrophic actinic keratosis [39], suggesting that expression of survivin may occur during early malignant transformation or following a disturbance in the balance between cell proliferation and death[9]. The same process may also occur in viral hepatitis. www.wjgnet.com

5310

ISSN 1007-9327

CN 14-1219/R


A previous study reported that inhibition of apoptosis by survivin plays a pivotal role in the metastasis of HCC, and it has some correlation with tumorigenesis. The expression of survivin in the primary lesion can be an indicator of metastasis and the prognosis of HCC[10]. In this study, a correlation between the survivin expression rate in the non-tumor cells and that in the HCC specimens of the high-rate group (rate > 50%) and low-rate group (rate < 50%) was observed. A significant difference in the survivin expression in the peritumoral hepatocyte was obser ved between the high-rate group and the low-rate group (P < 0.01). The survivin expression of peritumoral cells may, therefore, also be a prognostic factor for patients with HCC. Interestingly, we observed that when the amount of survivin expression was low in the adjacent non-tumor tissues, the corresponding tumor tissues also showed low expression. So in the future, when taking the target therapy of survivin into consideration, a curative effect may be possible if the amount of survivin expression with non-tumorous tissue is evaluated with liver biopsies. Saitoh et al [40] demonstrated that the portal blood flow is lost before the increase in arterial flow develops in well-differentiated HCC. When the well-differentiated HCC has portal blood flow and is not hypervascular, it shows slow growth[12]. Yamaguchi et al[41] suggested that this phenomenon is related to hypoxia, because the welldifferentiated HCC would be in a transitional stage from the portal blood supply to the arterial blood supply, but the reduction in portal flow appears prior to the increase in arterial flow. Therefore, the current research indicates that the impairment of the normal liver blood system probably causes local hypoxic regions at an early stage of hepatocarcinogensis and eventually induces angiogenesis[12]. Recent studies have shown that human solid tumors, even those less than 1 cm in diameter, may have substantial hypoxic fractions[42-43]. Hence tumor growth is restricted by limited oxygen and nutrients when they are too distant from nearby vessels[12]. Therefore, to observe the expression of survivin in early-stage HCC, HCC cells were cultured in a hypoxic environment. Previous studies have shown that survivin gene transcription is increased in hypoxic tumor cells[11]. In the current study, the hypoxic environment increased the survivin mRNA expression in both the short-term and long-term cultures. Moreover, the appearance of survivin protein is thought to control the survivin mRNA levels in the presence of anti-cancer drugs. On the contrary, in the present study, the survivin protein concentrations increased when both hypoxia and anti-cancer drugs were combined. These data suggest that survivin inhibition could therefore potentially be as effective as interventional therapy for the treatment of early HCC. In conclusion, sur vivin is expressed at a rate of 64.7% (median) in early HCC. Moreover, survivin protein concentration of HCC cells increases when cultured with anti-cancer drugs under hypoxic conditions. These data suggest that survivin inhibition for early HCC could therefore be potentially useful as an effective interventional radiological treatment modality. www.wjgnet.com

October 28, 2007

Volume 13

Number 40

COMMENTS Background

Several preclinical studies have demonstrated that the down-regulation of survivin expression/function increases the rate of apoptosis, reduces the tumor-growth potential and sensitizes tumor cells to various chemotherapeutic drugs and γ-irradiation in both in vitro and in vivo models of various types of human tumors.

Research frontiers

Previous reports have shown survivin to be expressed in post-operative HCC tissues. However, it has not yet been fully elucidated regarding whether survivin can be used as a therapeutic target in early HCC.

Innovations and breakthrough

We studied biopsy tissue specimens to confirm the diagnosis of HCC. In all early HCCs of tumor size > 10 mm, survivin expression was always above the median average (64.7%). Moreover, the survivin protein concentrations increased when cultured in a combination of hypoxia and anti-cancer drugs.

Applications

These data suggest that survivin inhibition for early HCC could therefore be potentially useful as an effective interventional radiological treatment modality, such as transcatheter arterial chemoembolization (TACE), etc.

Terminology

In this study, a hypoxia model of cultured HCC cells was employed using an AnaeroPack for cell culture. The Anaeropack is a gas concentration-controlling reagent yielding a hypoxic atmosphere. The principal ingredient of this reagent is sodium ascorbate, which absorbs oxygen and generates carbon dioxide by oxidative degradation. The culture dishes were placed into an airtight jar with the Anaeropack and then the lid was closed. The jar was then incubated at 37℃ for 2 h. The concentration of oxygen decreased to less than 1% within 1 h and the carbon dioxide concentration was maintained at about 5% as reported previously.

Peer review

This paper investigated survivin expression in early-stage, small HCC and the results are interesting. The methods and results were clearly written, and the authors gave thoughtful discussions on this topic and their findings. This is an interesting paper that is generally well written.

REFERENCES 1 2 3 4 5

6 7 8 9 10

Parkin DM, Bray F, Ferlay J, Pisani P. Estimating the world cancer burden: Globocan 2000. Int J Cancer 2001; 94: 153-156 Bruix J, Sala M, Llovet JM. Chemoembolization for hepatocellular carcinoma. Gastroenterology 2004; 127: S179-S188 El-Serag HB, Mason AC. Rising incidence of hepatocellular carcinoma in the United States. N Engl J Med 1999; 340: 745-750 Tarnawski A, Pai R, Chiou SK, Chai J, Chu EC. Rebamipide inhibits gastric cancer growth by targeting survivin and Aurora-B. Biochem Biophys Res Commun 2005; 334: 207-212 Wakana Y, Kasuya K, Katayanagi S, Tsuchida A, Aoki T, Koyanagi Y, Ishii H, Ebihara Y. Effect of survivin on cell proliferation and apoptosis in gastric cancer. Oncol Rep 2002; 9: 1213-1218 Kania J, Konturek SJ, Marlicz K, Hahn EG, Konturek PC. Expression of survivin and caspase-3 in gastric cancer. Dig Dis Sci 2003; 48: 266-271 Zhu XD, Lin GJ, Qian LP, Chen ZQ. Expression of survivin in human gastric carcinoma and gastric carcinoma model of rats. World J Gastroenterol 2003; 9: 1435-1438 Ikeguchi M, Liu J, Kaibara N. Expression of survivin mRNA and protein in gastric cancer cell line (MKN-45) during cisplatin treatment. Apoptosis 2002; 7: 23-29 Zaffaroni N, Pennati M, Daidone MG. Survivin as a target for new anticancer interventions. J Cell Mol Med 2005; 9: 360-372 Zhu H, Chen XP, Zhang WG, Luo SF, Zhang BX. Expression and significance of new inhibitor of apoptosis protein survivin


11

12 13

14 15 16 17

18

19 20 21 22 23

24

25

26

in hepatocellular carcinoma. World J Gastroenterol 2005; 11: 3855-3859 Yang L, Cao Z, Li F, Post DE, Van Meir EG, Zhong H, Wood WC. Tumor-specific gene expression using the survivin promoter is further increased by hypoxia. Gene Ther 2004; 11: 1215-1223 Kim KR, Moon HE, Kim KW. Hypoxia-induced angiogenesis in human hepatocellular carcinoma. J Mol Med 2002; 80: 703-714 Asakura T, Ohkawa K, Takahashi N, Takada K, Inoue T, Yokoyama S. Glutathione-doxorubicin conjugate expresses potent cytotoxicity by suppression of glutathione S-transferase activity: comparison between doxorubicin-sensitive and -resistant rat hepatoma cells. Br J Cancer 1997; 76: 1333-1337 Thompson CB. Apoptosis in the pathogenesis and treatment of disease. Science 1995; 267: 1456-1462 Saikumar P, Dong Z, Mikhailov V, Denton M, Weinberg JM, Venkatachalam MA. Apoptosis: definition, mechanisms, and relevance to disease. Am J Med 1999; 107: 489-506 Jaattela M. Escaping cell death: survival proteins in cancer. Exp Cell Res 1999; 248: 30-43 Tamm I, Wang Y, Sausville E, Scudiero DA, Vigna N, Oltersdorf T, Reed JC. IAP-family protein survivin inhibits caspase activity and apoptosis induced by Fas (CD95), Bax, caspases, and anticancer drugs. Cancer Res 1998; 58: 5315-5320 Shin S, Sung BJ, Cho YS, Kim HJ, Ha NC, Hwang JI, Chung CW, Jung YK, Oh BH. An anti-apoptotic protein human survivin is a direct inhibitor of caspase-3 and -7. Biochemistry 2001; 40: 1117-1123 Asanuma K, Moriai R, Yajima T, Yagihashi A, Yamada M, Kobayashi D, Watanabe N. Survivin as a radioresistance factor in pancreatic cancer. Jpn J Cancer Res 2000; 91: 1204-1209 Asanuma K, Kobayashi D, Furuya D, Tsuji N, Yagihashi A, Watanabe N. A role for survivin in radioresistance of pancreatic cancer cells. Jpn J Cancer Res 2002; 93: 1057-1062 Altieri DC. Validating survivin as a cancer therapeutic target. Nat Rev Cancer 2003; 3: 46-54 Li F. Survivin study: what is the next wave? J Cell Physiol 2003; 197: 8-29 Tanaka K, Iwamoto S, Gon G, Nohara T, Iwamoto M, Tanigawa N. Expression of survivin and its relationship to loss of apoptosis in breast carcinomas. Clin Cancer Res 2000; 6: 127-134 Satoh K, Kaneko K, Hirota M, Masamune A, Satoh A, Shimosegawa T. Expression of survivin is correlated with cancer cell apoptosis and is involved in the development of human pancreatic duct cell tumors. Cancer 2001; 92: 271-278 Kato J, Kuwabara Y, Mitani M, Shinoda N, Sato A, Toyama T, Mitsui A, Nishiwaki T, Moriyama S, Kudo J, Fujii Y. Expression of survivin in esophageal cancer: correlation with the prognosis and response to chemotherapy. Int J Cancer 2001; 95: 92-95 Nakamura M, Tsuji N, Asanuma K, Kobayashi D, Yagihashi A, Hirata K, Torigoe T, Sato N, Watanabe N. Survivin as a predictor of cis-diamminedichloroplatinum sensitivity in

27 28 29

30

31

32

33

34 35 36 37

38 39 40

41 42 43

5311

gastric cancer patients. Cancer Sci 2004; 95: 44-51 Kawasaki H, Altieri DC, Lu CD, Toyoda M, Tenjo T, Tanigawa N. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res 1998; 58: 5071-5074 Sarela AI, Macadam RC, Farmery SM, Markham AF, Guillou PJ. Expression of the antiapoptosis gene, survivin, predicts death from recurrent colorectal carcinoma. Gut 2000; 46: 645-650 Grabowski P, Kuhnel T, Muhr-Wilkenshoff F, Heine B, Stein H, Hopfner M, Germer CT, Scherubl H. Prognostic value of nuclear survivin expression in oesophageal squamous cell carcinoma. Br J Cancer 2003; 88: 115-119 Ito T, Shiraki K, Sugimoto K, Yamanaka T, Fujikawa K, Ito M, Takase K, Moriyama M, Kawano H, Hayashida M, Nakano T, Suzuki A. Survivin promotes cell proliferation in human hepatocellular carcinoma. Hepatology 2000; 31: 1080-1085 Fields AC, Cotsonis G, Sexton D, Santoianni R, Cohen C. Survivin expression in hepatocellular carcinoma: correlation with proliferation, prognostic parameters, and outcome. Mod Pathol 2004; 17: 1378-1385 Dudich E, Semenkova L, Dudich I, Denesyuk A, Tatulov E, Korpela T. Alpha-fetoprotein antagonizes X-linked inhibitor of apoptosis protein anticaspase activity and disrupts XIAPcaspase interaction. FEBS J 2006; 273: 3837-3849 Ikeguchi M, Ueda T, Sakatani T, Hirooka Y, Kaibara N. Expression of survivin messenger RNA correlates with poor prognosis in patients with hepatocellular carcinoma. Diagn Mol Pathol 2002; 11: 33-40 Ikeguchi M, Hirooka Y, Kaibara N. Quantitative analysis of apoptosis-related gene expression in hepatocellular carcinoma. Cancer 2002; 95: 1938-1945 Waris G, Siddiqui A. Regulatory mechanisms of viral hepatitis B and C. J Biosci 2003; 28: 311-321 Kountouras J, Zavos C, Chatzopoulos D. Apoptosis in hepatitis C. J Viral Hepat 2003; 10: 335-342 Zhang X, Dong N, Yin L, Cai N, Ma H, You J, Zhang H, Wang H, He R, Ye L. Hepatitis B virus X protein upregulates survivin expression in hepatoma tissues. J Med Virol 2005; 77: 374-381 Zhou Y, Gong GZ. Hepatitis C virus NS5A protein upregulates survivin gene expression. Zhonghua Ganzangbing Zazhi 2006; 14: 414-417 Altieri DC. Survivin, versatile modulation of cell division and apoptosis in cancer. Oncogene 2003; 22: 8581-8589 Saitoh S, Ikeda K, Koida I, Tsubota A, Arase Y, Chayama K, Kumada H. Serial hemodynamic measurements in welldifferentiated hepatocellular carcinomas. Hepatology 1995; 21: 1530-1534 Yamaguchi R, Yano H, Iemura A, Ogasawara S, Haramaki M, Kojiro M. Expression of vascular endothelial growth factor in human hepatocellular carcinoma. Hepatology 1998; 28: 68-77 Dang CV, Semenza GL. Oncogenic alterations of metabolism. Trends Biochem Sci 1999; 24: 68-72 Brown JM, Giaccia AJ. The unique physiology of solid tumors: opportunities (and problems) for cancer therapy. Cancer Res 1998; 58: 1408-1416 S- Editor Liu Y L- Editor Kumar M E- Editor Yin DH

www.wjgnet.com



COLORECTAL CANCER

RNA interference-mediated gene silencing of vascular endothelial growth factor in colon cancer cells Tie-Jun Li, Jian-Ning Song, Kai Kang, Shu-Sheng Tong, Zan-Lan Hu, Tong-Chuan He, Bing-Qiang Zhang, Cai-Quan Zhang Tie-Jun Li, Kai Kang, Bing-Qiang Zhang, Cai-Quan Zhang, The First Affiliated Hospital, Chongqing University of Medical Sciences, Chongqing 400016, China Jian-Ning Song, Shu-sheng Tong, Zan-Lan Hu, The Ninth Hospital of Chongqing, Chongqing 400700, China Tong-Chuan He, Molecular Oncology Laboratory, University of Chicago Medical Center, IL 60637, United States Supported in part by research grants from the National Natural Science Foundation of China, No. 30300298, and the National Natural Science Foundation of China's Joint Research Fund for Overseas Chinese Young Scholars, No. 30228026 Correspondence to: Professor Cai-Quan Zhang, Department in General Surgery, the First Affiliated Hospital, Chongqing University of Medical Science, Chongqing 400016, China. [email protected] Telephone: +86-13983764504 Received: March 28, 2007 Revised: August 10, 2007

Abstract AIM: To inhibit the expression of vascular endothelial growth factor (VEGF) in colon cancer cell line by RNA interference (RNAi). METHODS: Followed the service of E-RNAi, we designed and constructed two kinds of shRNA expression vectors aiming at the VEGF gene, then transfected them into TM colon cancer HT29 cells by lipofectamine 2000. The level of VEGF mRNA was investigated by RT-PCR and Northern blotting. The protein expression of VEGF was observed by immunofluoresence staining and Western blotting. RESULTS: We got two kinds of VEGF specific shRNA expression vectors which could efficiently inhibit the expression of VEGF in HT29 cells. RT-PCR, Northern blotting, immunofluoresence staining and Western blotting showed that inhibition rate for VEGF expression was up to 42%, 89%, 73% and 82%, respectively. CONCLUSION: The expression of VEGF can be inhibited by RNA interference in HT29 cells. Key words: RNA interference; Vascular endothelial growth factor; Colon cancer; Northern blotting; Western blotting Li TJ, Song JN, Kang K, Tong SS, Hu ZL, He TC, Zhang BQ, Zhang CQ. RNA interference mediated gene silencing of vascular endothelial growth factor in colon cancer cells. www.wjgnet.com

World J Gastroenterol 2007; 13(40): 5312-5316 http://www.wjgnet.com/1007-9327/13/5312.asp

INTRODUCTION Angiogenesis is a common process that is essential for tumor growth beyond 2 mm[1]. Although numerous growth factors are involved, vascular endothelial growth factor (VEGF), particularly VEGF-A, has been shown to play an important role in tumor angiogenesis[2]. VEGF, a 45 kDa heparin-binding growth factor, is induced by hypoxiainducible factor-1a. Binding of VEGF-A to tyrosine kinase receptors, especially VEGFR-2, mediates many key components of angiogenesis, including endothelial cell proliferation, invasion, migration, survival, as well as vessel permeability. VEGF is secreted by most rumors, including tumors of the lung, gastrointestinal tract, kidney, thyroid, bladder, ovary, and cervix, and the level of VEGF is correlated with tumor progression, invasion[3]. RNAi is the sequence-specific, posttranscriptional gene silencing method initiated by double-stranded RNAs, which are homologous to the suppressed gene. Doublestranded RNAs are processed by Dicer, a cellular RNase Ⅲ, to generate duplexes of about 21nt with 3’-overhang small interfering RNA (siRNA), which mediate sequencespecific mRNA degradation. RNAi technology is not only an extremely powerful instrument for functional genomic analysis but also a potentially useful method to develop highly specific gene silencing therapeutics[4-9]. In this study, we constructed vector-based expression systems in which sense and antisense strands of short VEGF sequences were transcribed into the hairpin structure under control of the U6 promoter. A number of studies are available on VEGF in the treatment of tumors, such as colon cancer and liver cancer[10-15]. However, due to the “off-target” of RNAi, experiments have to be done to verify the more effective sequence of VEGF before it is used in clinical practice. This study was to find a better VEGF specific RNAi for colon cancer.

MATERIALS AND METHODS Construction of RNAi vectors RNAi vectors pShRNA-V1 and pShRNA-V2 were constr ucted as previously described [16-19] . In brief,

Li TJ et al . RNAi-mediated gene silencing of VEGF 1

2

3

4

5

6

7

8

9

10

bp 19329 7743 4254 1882 1489 925 421

Figure 1 Restriction map of recombinant plasmid pShRNA. Lanes 1-2: pShRNA undigested; Lane 3: undigested null vector pTZU6+1; Lanes 4-5: pShRNA digested by salⅠ; Lane 6: pTZU6+1 digested by salⅠ; Lanes 7-8: pShRNA digested by Hind Ⅲ and EcoRⅠ(2800 + 395 bp); Lane 9: pTZU6+1 digested by Hind Ⅲ and EcoRⅠ(2800 + 352 bp); Lane 10: λT14 DNA Marker.

21-nucleotide-long inverted repeats (separated by a 4-nucleotide linker, ttcg) were inserted downstream of the U6 promoter. The transcribed RNA thus comprised a 21-base pair of double-stranded RNAs. Five thymidines were inserted downstream the antisense strand to provide a stop signal for the RNA polymerase. The sense strand of hairpin was homologous to a 21-nucleotide region in the target mRNA. The target sequences were selected following the advice of E-RNAi services (http://e-rnai. dkfz.de/). The sequence of V1 and V2 is 5’-TGAAGTT CATGGATGTCTATC-3’ and 5’-ACATCACCATGCAG ATTATGC-3’, respectively. An irrelevant RNAi control plasmid was constructed for green fluorescent protein (GFP) gene, pShRNA-GFP. The sequence (5’-AGCTG ACCCTGAAGTTCATCT-3’) was designed to target the nucleotides 126-144 of the GFP coding region. Cell culture and transfection Human colon cancer cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS), 100 μg/mL streptomycin, and 100-units/mL ampicillin. The cells were plated in 24- or 6- well plates at 50%-70% confluence 24 h prior to transfection. Transfection of cells was carried out with LipofectamineTM 2000 reagent (Invitrogen, Carlsbad, CA). Real-time polymerse chain reaction for VEGF Total RNA was isolated from cultured cells and realtime polymerase chain reaction (RT-PCR) was performed using the RNeasy and one step RT-PCR kit from Qiagen Corp. RT-PCR of hGAPDH, a housekeeping gene ser ved as a control. The sequences used for primers are 5’ ctacctccaccatgccaagt-3’ (sense) and 5’aaatgctttctccgctctga-3’ (antisense) for VEGF (411 bp), 5’-GGCTCTCCAGAACATCAT-3’(sense) and 5’CACCTGGTGCTCAGTGTA-3’ (antisense) for hGAPDH (240 bp). For RT-PCR, two pairs of primers were added into a reaction tube, the program consisted of an initial reverse transcription at 50℃ for 30 min, denaturation at 95℃ for 10 min, followed by 24 cycles of amplification (denaturation at 95℃ for 30 s, annealing at 55℃ for 1 min, and extension at 68℃ for 1 min) and a final extension at 68℃ for 10 min. The products were then separated by electrophoresis on 1.5% agarose gel, the bands were visualized using UV light and analyzed by Genetools software.

5313

Northern blotting analysis Total RNA was extracted from transfected cells on d 3 post-transfection, and purified using the RNeasy Mini Kit (Qiagen). Twenty micrograms of total RNA was separated on 1.2% agarose-formaldehyde gels and transferred onto a positively charged nylon membrane (Amersham). The presence of VEGF mRNA was probed with 32P-labeled VEGF DNA, which was generated with a random-primed labeling kit (Amersham). Immunofluorescence staining Cells were har vested on d 2 post-transfection for analysis, washed once with PBS and fixed with 4% paraformaldehyde in PBS for 20 min at 4℃. After blocked with goat serum, the cells were incubated with monoclonal mouse anti-VEGF for 2 h at 37℃. After three washes, the cells were incubated with Cy3-conjugated rabbit antimouse secondary antibodies for 1 h at 37℃ and washed three times with PBS. The stained cells were mounted and analyzed under fluorescence microscope. Western blotting Cells were harvested on d 3 post-transfection, washed twice with 10 mL of PBS, lysed with SDS buffer, boiled for 5 min, separated by 10% SDS-PAGE gel electrophoresis, transferred onto a nitrocellulose membrane, incubated with VEGF antibodies at a dilution of 1/400 and HRPconjugated rabbit anti-mouse antibody at a dilution of 1/4000. The HRP substrate was observed on the NC membrane. After three washes, the NC membrane was incubated with actin antibody and HRP-conjugated second antibody. The HRP substrate was observed again.

RESULTS Identification of recombinant plasmid pShRNA and sequencing Recombinant plasmid pShRNAs could not be digested by Sal I due to the loss of Sal I site. However, the blank plasmid pTZU6+1 could be lined by sal I. When digested by Hind Ⅲ and EcoR I, pShRNAs could be separated into two parts (2800 and 395 bp), and pTZU6+1 into 2800 bp and 352 bp. The correct recombinant plasmids were shown in gel electrophoresis and verified by DNA sequencing (Figure 1). Gel electrophoresis of VEGF The size of VEGF was 411 bp, and consisted of the Marker in gel electrophoresis. After cloned into T vector, its sequence was verified by DNA sequencing (Figure 2). VEGF mRNA inhibition in HT29 cells by RT-PCR The inhibition rate of pShRNA-V1 and pShRNA-V2 was 42% and 40% respectively in HT29 cells compared with the control plasmid pShRNA-GFP (Figure 3A and B). VEGF mRNA inhibition in HT29 cells by Northern blotting The inhibition rate of pShRNA-V1 and pShRNA-V2 was 87% and 89% respectively in HT29 cells compared with the control plasmid pShRNA-GFP (Figure 4A and B). www.wjgnet.com

5314 bp

1

ISSN 1007-9327

CN 14-1219/R


Figure 2 Gel electrophoresis of VEGF. Lane 1: 100 bp Marker; Lane 2: RT-PCR for VEGF (411 bp).

2

1500

October 28, 2007

A

1

Volume 13 2

Number 40

3 VEGF

1000

VEGF

500

100

A

1

2

3

4

bp 1500 28S

1000 800 600 500 400

VEGF

B 1.2

GAPDH

Level of VEGF nRNA

300 200 100

B Level of VEGF nRNA

1.2 1.0

1.00

0.6

1.00

0.8 0.6 0.4 0.2 0.0

VEGF expression inhibited by shRNA in HT29 cells

0.8

1.0

VEGF mRNA inhibited in HT29 cells

pShRNA-GFP

0.13

0.11

pShRNA-V2

pShRNA-V1

Different kinds of shRNA vectors 0.60

0.58

pShRNA-V2

pShRNA-V1

0.4

Figure 4 VEGF mRNA inhibited in HT29 cells by Northern blotting analysis (A) and the inhibition rate of pShRNA-V1 and pShRNA-V2 (B). Lane 1: pShRNA-GFP; Lane 2: pShRNA-V1; Lane3: pShRNA-V2.

0.2 0.0

pShRNA-GFP

Different kinds of shRNA vectors

Figure 3 VEGF gene expression inhibited by shRNAs (A) and the inhibition rate of pShRNA-V1 and pShRNA-V2 (B) in HT29 cells. Lane 1: pShRNA-GFP; Lane 2: pShRNA-V2; Lane 3: pShRNA-V1; Lane 4: 100 bp Marker.

VEGF protein inhibition in HT29 cells by immunofluoresence staining The inhibition rate of pShRNA-V1 and pShRNA-V2 was 63% and 73% respectively in HT29 cells compared with the control plasmid pShRNA-GFP and pTZU6+1 (Figure 5A and B). VEGF was stained red and located in plasma of cells. VEGF protein inhibition in HT29 cells by Western blotting The inhibition rate of pShRNA-V1 and pShRNA-V2 was 69% and 82% respectively in HT29 cells compared with the control plasmid pShRNA-GFP and pTZU6+1 (Figure 6A and B).

DISCUSSION Angiogenesis is a process of generating new capillaries from pre-existing blood vessels, which involves multiple gene products expressed by various cell types. This uncontrolled process of new blood vessel growth from the www.wjgnet.com

preexisting circulation network is an important pathogenic cause of tumor growth[20-24]. Although several proteins such as hepatocyte growth factor, tumor necrosis factor-α, and fibroblast growth factor 2 (FGF2) have been identified as stimulators of angiogenesis in various settings, the most important angiogenic growth factor is VEGF, which is over-expressed in many human cancers. VEGF expression in tumors can be induced by more than one mechanism. Hypoxia, which is found in most tumors, has long been known to be a potent inducer of VEGF[25-28]. In this study, shRNAs targeting VEGF efficiently reduced the transcript levels of VEGF mRNAs, and ultimately resulted in the reduction in VEGF protein levels. Furthermore, this inhibition was shown to be highly selective and sequence-specific, since control siRNAs had almost no inhibitory effect on the expression and transcription of VEGF. There has been a considerable interest in treating a wide range of diseases with RNAi therapeutics[29,30]. In this study, in addition to the above-reported target sites in the VEGF genome, the specific 21-bp siRNAs targeting VEGF could efficiently and specifically inhibit VEGF expression, suggesting that it is a good method to inhibit the expression of VEGF. The results of this study demonstrated that the constructed shRNA could efficiently reduce the level of VEGF transcripts and expression, suggest that shRNA-

Li TJ et al . RNAi-mediated gene silencing of VEGF

A

5315

A

B

1

2

3

4 VEGF

Actin

C

D

B

VEGF expression inhibited in HT29 cells

Level of VEGF

1.2 1.00

0.74

0.8 0.6 0.4

0.31 0.18

0.2

E Total fluoresence/Total cells

1.0

1.20 1.00

0.0

Immunofluoresence staining for VEGF in HT29 cells 1.00

pTZU6+1

1.05

pShRNA-V1

pShRNA-V2

Different kinds of shRNA vectors

0.80 0.60 0.40

pShRNA-GFP

0.33

0.27

Figure 6 Western blotting for VEGF (A) and the inhibition rate of pShRNA-V1 and pShRNA-V2 (B) in HT29 cells. Lane 1: pTZU6+1; Lane 2: pShRNA-G; Lane 3: pShRNA-V1; Lane 4: pShRNA-V2.

0.20 0.00

pTZU6+1

pTZU6-GFP

pTZU6-V1

pTZU6-V2

Different kinds of shRNA

Figure 5 Immunofluoresence staining for pTZU6+1 (A), pTZU6-GFP (B), pTZU6-V1 (C), pTZU6-V2 (D), and the inhibition rate of pShRNA-V1 and pShRNA-V2 (E).

expressing vectors can be used as RNAi-based anti-VEGF therapeutics[31]. Future studies should be centered on the evaluation of the anti-VEGF efficacy of RNAi vectors in animal models, as well as on the preclinical elucidation using the RNAi technology.

ACKNOWLEDGMENTS The authors thank Dr. David Engelke of University of Michigan for generously providing the pTZU6+1 plasmid. This work was supported in part by research grants from the National Natural Science Foundation of China (No. 30300298) to Bing-Qiang Zhang, and the National Natural Science Foundation of China’s Joint Research Fund for Overseas Chinese Young Scholars to Tong-Chuan He (No. 30228026).

cancer, liver cancer. There has been a considerable interest in treating a wide range of diseases with RNAi.

Innovations and breakthroughs

The results of our study suggest that shRNA-expressing vectors can be used as RNAi-based anti-VEGF therapeutics. Future studies should be centered on the evaluation of the anti-VEGF efficacy of RNAi vectors in animal models, as well as on the preclinical elucidation using the RNAi technology.

Applications

shRNA-expressing vectors can be used as RNAi-based anti-VEGF therapeutics

Terminology

RNAi is the sequence-specific, posttranscriptional gene silencing method initiated by double-stranded RNAs, which are homologous to the suppressed gene. Double-strand RNAs are processed by Dicer, a cellular RNase Ⅲ, to generate duplexes of about 21nt with 3’-overhang small interfering RNA (siRNA), which mediate sequence-specific mRNA degradation.

Peer review

This manuscript describes the methodology for RNAi of VEGF in HT29 cells by lipofectamine. The process was evaluated quite well. No application of the technique has reported prior to this study.

REFERENCES 1

COMMENTS Background

Vascular endothelial growth factor (VEGF) has been shown to play an important role in tumor angiogenesis. RNAi is the sequence-specific, posttranscriptional gene silencing method initiated by double-stranded RNAs. A number of studies are available on VEGF used in the treatment of tumors, such as colon cancer, liver cancer. However, due to the “off-target” of RNAi, experiments have to be done to verify the more effective sequence of VEGF before it is used in clinical practice. This study was to find a better VEGF specific RNAi for colon cancer.

Research frontiers

Other researches have applied VEGF in the treatment of tumors, such as colon

2 3 4 5 6

Potgens AJ, Westphal HR, de Waal RM, Ruiter DJ. The role of vascular permeability factor and basic fibroblast growth factor in tumor angiogenesis. Biol Chem Hoppe Seyler 1995; 376: 57-70 Kraizer Y, Mawasi N, Seagal J, Paizi M, Assy N, Spira G. Vascular endothelial growth factor and angiopoietin in liver regeneration. Biochem Biophys Res Commun 2001; 287: 209-215 Lu PY, Xie FY, Woodle MC. Modulation of angiogenesis with siRNA inhibitors for novel therapeutics. Trends Mol Med 2005; 11: 104-113 Bernstein E, Caudy AA, Hammond SM, Hannon GJ. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 2001; 409: 363-366 Agami R. RNAi and related mechanisms and their potential use for therapy. Curr Opin Chem Biol 2002; 6: 829-834 Brummelkamp TR, Bernards R, Agami R. A system for stable www.wjgnet.com

5316

ISSN 1007-9327

CN 14-1219/R


expression of short interfering RNAs in mammalian cells. Science 2002; 296: 550-553 Wilda M, Fuchs U, Wossmann W, Borkhardt A. Killing 7 of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi). Oncogene 2002; 21: 5716-5724 Cioca DP, Aoki Y, Kiyosawa K. RNA interference is a 8 functional pathway with therapeutic potential in human myeloid leukemia cell lines. Cancer Gene Ther 2003; 10: 125-133 Aoki Y, Cioca DP, Oidaira H, Kamiya J, Kiyosawa K. RNA 9 interference may be more potent than antisense RNA in human cancer cell lines. Clin Exp Pharmacol Physiol 2003; 30: 96-102 10 Mulkeen AL, Silva T, Yoo PS, Schmitz JC, Uchio E, Chu E, Cha C. Short interfering RNA-mediated gene silencing of vascular endothelial growth factor: effects on cellular proliferation in colon cancer cells. Arch Surg 2006; 141: 367-374; discussion 374 11 Wannenes F, Ciafre SA, Niola F, Frajese G, Farace MG. Vectorbased RNA interference against vascular endothelial growth factor-A significantly limits vascularization and growth of prostate cancer in vivo. Cancer Gene Ther 2005; 12: 926-934 12 Murata M, Takanami T, Shimizu S, Kubota Y, Horiuchi S, Habano W, Ma JX, Sato S. Inhibition of ocular angiogenesis by diced small interfering RNAs (siRNAs) specific to vascular endothelial growth factor (VEGF). Curr Eye Res 2006; 31: 171-180 13 Fan Y, Xin XY, Chen BL, Ma X. Knockdown of RAB25 expression by RNAi inhibits growth of human epithelial ovarian cancer cells in vitro and in vivo. Pathology 2006; 38: 561-567 14 Hammond SM, Boettcher S, Caudy AA, Kobayashi R, Hannon GJ. Argonaute2, a link between genetic and biochemical analyses of RNAi. Science 2001; 293: 1146-1150 15 Zamore PD, Tuschl T, Sharp PA, Bartel DP. RNAi: doublestranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 2000; 101: 25-33 16 Paul CP, Good PD, Winer I, Engelke DR. Effective expression of small interfering RNA in human cells. Nat Biotechnol 2002; 20: 505-508 17 Sui G, Soohoo C, Affar el B, Gay F, Shi Y, Forrester WC, Shi Y. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells. Proc Natl Acad Sci USA 2002; 99: 5515-5520 18 Hannon GJ. RNA interference. Nature 2002; 418: 244-251

19

20 21 22 23 24 25 26

27

28

29

30 31

October 28, 2007

Volume 13

Uchida H, Tanaka T, Sasaki K, Kato K, Dehari H, Ito Y, Kobune M, Miyagishi M, Taira K, Tahara H, Hamada H. Adenovirus-mediated transfer of siRNA against survivin induced apoptosis and attenuated tumor cell growth in vitro and in vivo. Mol Ther 2004; 10: 162-171 Kerbel R, Folkman J. Clinical translation of angiogenesis inhibitors. Nat Rev Cancer 2002; 2: 727-739 McManus MT, Sharp PA. Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 2002; 3: 737-747 Valdes VJ, Sampieri A, Sepulveda J, Vaca L. Using doublestranded RNA to prevent in vitro and in vivo viral infections by recombinant baculovirus. J Biol Chem 2003; 278: 19317-19324 Waterhouse PM, Wang MB, Finnegan EJ. Role of short RNAs in gene silencing. Trends Plant Sci 2001; 6: 297-301 Shlomai A, Shaul Y. RNA interference--small RNAs effectively fight viral hepatitis. Liver Int 2004; 24: 526-531 Shweiki D, Itin A, Soffer D, Keshet E. Vascular endothelial growth factor induced by hypoxia may mediate hypoxiainitiated angiogenesis. Nature 1992; 359: 843-845 Ravi R, Mookerjee B, Bhujwalla ZM, Sutter CH, Artemov D, Zeng Q, Dillehay LE, Madan A, Semenza GL, Bedi A. Regulation of tumor angiogenesis by p53-induced degradation of hypoxia-inducible factor 1alpha. Genes Dev 2000; 14: 34-44 Bohula EA, Salisbury AJ, Sohail M, Playford MP, Riedemann J, Southern EM, Macaulay VM. The efficacy of small interfering RNAs targeted to the type 1 insulin-like growth factor receptor (IGF1R) is influenced by secondary structure in the IGF1R transcript. J Biol Chem 2003; 278: 15991-15997 Du J, Pan Y, Shi Y, Guo C, Jin X, Sun L, Liu N, Qiao T, Fan D. Overexpression and significance of prion protein in gastric cancer and multidrug-resistant gastric carcinoma cell line SGC7901/ADR. Int J Cancer 2005; 113: 213-220 Dias S, Shmelkov SV, Lam G, Rafii S. VEGF(165) promotes survival of leukemic cells by Hsp90-mediated induction of Bcl-2 expression and apoptosis inhibition. Blood 2002; 99: 2532-2540 Beierle EA, Strande LF, Chen MK. VEGF upregulates Bcl-2 expression and is associated with decreased apoptosis in neuroblastoma cells. J Pediatr Surg 2002; 37: 467-471 Gerber HP, Dixit V, Ferrara N. Vascular endothelial growth factor induces expression of the antiapoptotic proteins Bcl-2 and A1 in vascular endothelial cells. J Biol Chem 1998; 273: 13313-13316 S- Editor Liu Y L- Editor Wang XL

www.wjgnet.com

Number 40

E- Editor Li HY



VIRAL HEPATITIS

Antioxidant therapy for chronic hepatitis C after failure of interferon: Results of phase Ⅱ randomized, double-blind placebo controlled clinical trial Ezra Gabbay, Ehud Zigmond, Orit Pappo, Nila Hemed, Mina Rowe, George Zabrecky, Robert Cohen, Yaron Ilan Ezra Gabbay, Ehud Zigmond, Nila Hemed, Mina Rowe, Yaron Ilan, Orit Pappo, Liver Unit, Department of Medicine; Department of Pathology, Hebrew University, Hadassah Medical Center, Jerusalem IL-91120, Israel George Zabrecky, Robert Cohen, Marcus Foundation, Atlanta, United States Supported by The Bernie Markus Foundation Correspondence to: Yaron Ilan, MD, Liver Unit, Department of Medicine, Hebrew University, Hadassah Medical Center, POB 12000, Jerusalem IL-91120, Israel. [email protected] Telephone: +972-2-6778231 Fax: +972-2-6431021 Received: December 14, 2006 Revised: August 17, 2007

Abstract AIM: To assess the safety and efficacy of antioxidant therapy for patients with chronic hepatitis C virus (HCV) infection. METHODS: One hundred chronic HCV infection patients failed in interferon treatment were enrolled and randomly assigned to receive combined intravenous and oral antioxidants or placebo, or oral treatment alone. Primary end points were liver enzymes, HCV-RNA levels and histology. RESULTS: Combined oral and intravenous antioxidant therapy was associated with a significant decline in ALT levels in 52% of patients who received antioxidant therapy vs 20% of patients who received placebo (P = 0.05). Histology activity index (HAI) score at the end of treatment was reduced in 48% of patients who received antioxidant therapy vs 26% of patients who received placebo (P = 0.21). HCV-RNA levels decreased by 1-log or more in 28% of patients who received antioxidant therapy vs 12% who received placebo (P = NS). In part Ⅱ of the trial, oral administration of antioxidants was not associated with significant alterations in any of the end points. CONCLUSION: Antioxidant therapy has a mild beneficial effect on the inflammatory response of chronic HCV infection patients who are non-responders to interferon. Combined antiviral and antioxidant therapy may be beneficial for these patients. © 2007 WJG . All rights reserved.

Key words: Hepatitis C virus; Antioxidant therapy; Histology activity index; Glycyrrhiza; L-glutathione Gabbay E, Zigmond E, Pappo O, Hemed N, Rowe M, Zabrecky G, Cohen R, Ilan Y. Antioxidant therapy for chronic hepatitis C after failure of interferon: Results of phase Ⅱ randomized, double-blind placebo controlled clinical trial. World J Gastroenterol 2007; 13(40): 5317-5323


INTRODUCTION The pathogenesis of hepatitis C virus (HCV) infection involves a complex interaction between viral factors and host immune responses. A major component of the latter involves oxidative stress[1]. Oxidative stress has been attributed to both host inflammatory processes and induction by viral proteins, with the two mechanisms possibly acting in synergy. HCV non-structural proteins have been shown to induce activation of STAT-3 via oxidative stress and Ca 2+ signaling [2]. This induction is influenced by the activation of cellular kinases, including p38 mitogen-activated protein kinase, JNK, JAK-2 and Src, and inhibited in vitro in the presence of antioxidant 3. It was reported that HCV core protein increases radical oxygen species (ROS) as well as lipid peroxidation products and antioxidant gene expression [3]. Increased intrahepatic lipid peroxidation products have been observed in HCV transgenic mice, following exposure to carbon tetrachloride[4]. These processes may contribute to fibrosis and carcinogenesis in chronic HCV[5]. Analysis of HCV-sub genomic RNA replicon cell lines has shown a drastic reduction in cellular glutathione peroxidase, increasing cellular susceptibility to oxidative stress[6]. Based on these data, a rationale for antioxidant treatment of chronic hepatitis C was suggested. Currently, the mainstay of treatment of chronic hepatitis C is a combination of pegylated interferons and ribavirin[7]. However, the overall response rate of its pharmacological efficacy and adverse effects is approximately 55% in genotype I patients[8]. The use of complimentary and alternative medicine is common in patients with chronic liver disease. In a recent study, 41% of patients reported use of these modalities,

www.wjgnet.com

5318

ISSN 1007-9327

CN 14-1219/R


with 20% reporting use of herbal medicine, including sylamarin (12%), licorice root (glycyrrhizin) and St. John’s wart[9]. In a review of medicinal herbs for HCV infection, the authors concluded that some of these agents may have an effect on liver enzymes but there is no firm evidence that supports their efficacy on chronic HCV[10]. In a recently published phase-I, open label clinical trial, 50 patients with chronic HCV infection were treated orally with a combination of seven antioxidative oral preparations (glycyrrhizin, schisandra, silymarin, ascorbic acid, lipoic acid, L-glutathione, and alpha-tocopherol) on a daily basis for 20 wk, along with four different intravenous preparations (glycyrrhizin, ascorbic acid, L-glutathione, B-complex) twice weekly for the first 10 wk[11]. Normalization of liver enzymes occurred in 44% of patients who had elevated pretreatment ALT levels. A decrease in viral load by one log or more was observed in 25% of the patients. Histological improvement, with twopoint reduction in the HAI score, was noted in 36.1% of the patients. The SF-36 quality of life score improved in 26 of 45 patients (58%) throughout the trial. No major adverse reactions were noted. These findings suggest that antioxidant therapy may have a beneficial effect on necroinflammatory variables in these patients. The aim of the present trial was to determine the effect of a mixture of antioxidants on the inflammatory response of chronic HCV infection patients who were non-responders to interferon, in a double-blind placebo controlled trial. Two different treatment regimens were studied and compared. The data suggest that combined intravenous and oral antioxidant therapy mildly alleviates the inflammatory response in these patients.

MATERIALS AND METHODS Subjects A total of 100 chronic HCV infection patients were enrolled in a double-blind, placebo controlled single-center trial. The study focused on 2 treatment options, with 50 patients in each section. Part I looked at administration of intravenous and oral antioxidant preparations versus placebo. Part Ⅱ tested administration of oral preparations only versus placebo. Patients were randomly assigned to treatment or placebo groups, 25 patients in each group. Antioxidants were administered as described below. All experiments were carried out in accordance with the guidelines of the Hebrew University-Hadassah Institutional Committee for Human Clinical Trials. The Israel Ministry of Health Committee for Human Trials approved all experiments. Inclusion criteria: Eligible participants were male and female chronic HCV infection patients at the age of 18-75 years. All patients were required to display positive HCV RNA on two tests for at least 6 mo prior to enrollment. Diagnosis of chronic HCV infection was based on positive HCV RNA levels with liver biopsy (within 1 year of initiation of study) with or without elevated liver enzymes. Informed consent was obtained from patients prior to participation in the trial. Patients screened for the trial included those in whom treatment with interferon had previously failed, or was contra-indicated. Both partial www.wjgnet.com

October 28, 2007

Volume 13

Number 40

responders and non-responders were enrolled. Abstinence from any alternative medications or vitamin supplements for 6 mo prior to initiation of therapy was stipulated. Exclusion criteria: Patients excluded from the trial included those with decompensated liver disease as evidenced by Child’s B or C status; those with creatinine levels above 150 μ mol/L, hemoglobin levels below 100 mg/L, white blood cell count below 3 × 109/L, or platelet count below 1 × 1011/L; those with a history of varices, ascites, or encephalopathy. Also ineligible were patients who exhibited irreversible neurologic deficit or active co-infection with HBV, HAV, HDV, or HIV. Those treated with interferon or pegylated interferon or ribavirin less than 6 mo prior to enrollment or with a history of having received any systemic anti-neoplastic or immune modulatory treatment 6 mo prior to the first dose of study medication were excluded. Evidence of other causes of liver disease or other severe debilitating diseases precluded the patient from participating in this study. Patients who used alcohol or anti-viral medication during the trial, pregnancy or breast-feeding, and had a history of hepatic, renal, or other major organ transplantation were also exclude from the study. In part I, 50 patients (30 males and 20 females) with a mean age of 56.1 (range, 22-75) years were enrolled in the trial. Sixty-four percent of patients (n = 32) had elevated liver enzymes on screening, and 36% (n = 18) had normal liver enzymes despite viremia and active inflammation on biopsy. All other markers for additional causes of liver disease were negative. HCV genotype 1, genotype 1a, and genotype 1b were found in 33, 5, and 28 patients, respectively. HCV genotype 2, genotype 3, and genotype 4 were found in 8, 6 and 3 patients, respectively. In part Ⅱ, 50 patients (34 males and 16 females) were randomly assigned to treatment (n = 25) and placebo (n = 25) groups. Their mean age was 57.4 (range 24-75) years. Thirty-five patients had genotype 1 (31 had genotype 1b and 4 had genotype 1a). Two patients had genotype 2a2c, two had genotype 3a, and one had genotype 4e. Drug administration Patients in the treatment group received a combination of seven different anti-oxidants orally at the appropriate dose (glycyrrhiza capsules, 500 mg, bid; schizandrae capsules, 500 mg, tid; ascorbate capsules, 2000 mg, tid; L-glutathione capsules, 150 mg, bid; silymarin capsules, 250 mg, tid; lipoic Acid capsules, 150 mg, bid; d-alpha tocopherol, 800 IU/d) prepared by Vital Nutrients Middletown (CT, USA), once daily for 24 wk. In addition, all patients in part I of the trial received intravenously a combination of four different preparations (120 mg glycyrrhiza, 10 g ascorbate, 750 mg L-glutathione, 1 mL B-comple) at the appropriate dose, twice a week for the first 10 wk of the study. Medications were prepared by GY&N Nutriment Pharmacology, Unique Pharmaceuticals Company, Palmdale, CA, USA. Dosages were determined based on previous trials in humans. Products for intravenous injection were added to 400 mL of normal saline for infusion. Patients were followed up for an additional 24 wk following completion of the treatment as described below. Patients in the placebo group received identical pills and normal saline intravenously.

Gabbay E et al . Treatment of chronic HCV with antioxidants

ALT (IU)

A

in patients with chronic HCV infection; (2) evaluation of the effect of glycyrrhizin, antioxidants, and vitamins on inflammatory indices and HCV viral load. The two primary variables were: (1) safety and tolerability of intravenous and oral administration of glycyrrhizin, antioxidants, and vitamins in patients with chronic HCV infection; (2) amelioration of inflammation and existing liver damage, and reduction in viral load.

80

60

Statistical analysis Data were analyzed using Fisher’s exact test. P < 0.05 was considered statistically significant.

Treatment Placebo 40

AST (IU)

B

0

10

24

t /wk

32

48

40

RESULTS

100

80

60 Treatment Placebo 40

5319

0

10

24

t /wk

32

40

48

Figure 1 Effect of antioxidant treatment on ALT (A) and AST (B) serum levels in partⅠwith treatment group in solid lines and placebo in dashed lines.

Follow-up parameters Patients were followed up for clinical, biochemical, virological, and histological parameters. Patients underwent a bi-weekly physical examination throughout the treatment period, and every 4 wk during the follow-up period. Serum bilirubin and liver enzymes, kidney function, prothrombin time, and complete blood counts were checked bi-weekly during the treatment period and every 4 wk during followup. HCV RNA was detected every 4 wk throughout the study by standard assays (Roche Diagnostics Systems Inc., Branchburg, NJ). The lower limit of detection for this assay was 100 copies/L. All patients underwent a liver biopsy within 2 years prior to treatment. A repeat biopsy was performed 2 to 6 wk following completion of the antioxidant therapy. Intra-hepatic inflammatory score was evaluated by two blinded pathologists using the standard histological activity index (HAI). Evaluation of the effect of treatment on quality of life was performed using the eighth paradigm (self assessment of general health) of the SF-36 quality of life questionnaire. The questionnaire was completed 2 wk before and within 4 wk after the study. Primary and secondary variables The objectives of the study were: (1) evaluation of the safety and tolerability of intravenous and oral administration of glycyrrhizin, antioxidants, and vitamins

Safety and adverse effects In part I, no major adverse effects related to treatment were noted. Side effects possibly related to the mineral corticoid effect of glycyrrhizin treatment included hypokalemia in 6 patients of the treatment group (24%), in 5 patients of the placebo group (20%), and mild exacerbation of hypertension in 1 patient of the treatment group. All cases were mild and treated symptomatically. Two patients in the treatment group developed pedal edema and a sense of bloating. One patient in the treatment group was hospitalized twice due to fever during the trial period which was considered unrelated to treatment. In part Ⅱ, One patient in the treatment group had exacerbation of hypertension at wk 6 of treatment, requiring treatment with enalapril and lercanidipine. Subsequent blood pressure during treatment was normal. Six patients in the treatment group had hypokalemia of 2.9 to 3.3 and one patient in the placebo group had hypokalemia of 3.4. One patient in the treatment group had a high systolic blood pressure (up to 190 mmHg) on the day of his second biopsy, and resolved after administration of oral oxazepam. One patient in the treatment group had a weight loss of 5.5 kg during treatment, but regained three kg by wk 32. One patient in the placebo group reported weakness, upper abdominal pain and arthralgia. Effect of antioxidative treatment on liver enzymes In part I, at the end of 24 wk of treatment a decline in ALT and AST levels was observed in the treatment group compared to the placebo group. The ALT level at this point was 64.8 IU in the treatment group and 75.04 IU in the placebo group (P = 0.42, Figure 1A), the AST level at this point was 61.72 IU in the treatment group and 78 IU in the placebo group (P = 0.1, Figure 1B). This effect diminished during the follow-up period. At wk 48, the ALT level was 63 IU in the treatment group and 58.27 IU in the placebo group (P = 0.68), the AST level was 73.13 IU in the treatment group and 69.33 IU in the placebo group (P = 0.75). At the end of the treatment period, the ALT level improved in 54% of treated patients and in 20% of patients receiving the placebo (P = 0.05, Figure 2A), the AST level declined in 50% of treated patients and 20% of patients receving placebo (P = 0.02, Figure 2B). At www.wjgnet.com

5320

A

ISSN 1007-9327

70

CN 14-1219/R


A

Treatment

October 28, 2007

100

Placebo

90 ALT (IU)

50 40

Number 40

Treatment

Placebo 60

Volume 13

80 70

%

60 30 50 20

40

0

12

24

10 0

Normal

Deteriorated

Improved

32

40

48

Treatment

100

Placebo 90

60

Treatment

AST (IU)

B

B Unchanged

t /wk

Placebo 50

80 70 60

%

40

50

30

40 20 10 0

0

12

24

t /wk

32

40

48

Figure 3 Effect of antioxidant treatment on ALT (A) and AST (B) serum levels in partⅡwith treatment group in solid lines and placebo in dashed lines. Unchanged

Normal

Deteriorated

Improved

Figure 2 Proportions of patients with improvement in ALT (A) and AST (B).

the end of the follow-up period (at wk 48), the ALT level improved in 46% of patients in the treatment group and in 45% of patients in the placebo group, the AST level improved in 35% of patients in the treatment group and 34% of patients in the placebo group compared to their baseline. In part Ⅱ, a minor non-significant decline in liver enzyme levels was noted after 24 wk of treatment. The ALT level at this point was 63.5 IU in the treatment group and 81.81 IU in the placebo group (P = 0.35, Figure 3A), the AST level was 65.71 IU in the treatment group and 81.82 IU in the placebo group (P = 0.24, Figure 3B). This effect disappeared during the follow-up period. At wk 48, the ALT level was 61.75 IU in the placebo group and 83.94 IU in the treatment group (P = 0.24, Figure 3A), the AST level was 69.5 IU in the placebo group and 92.66 IU in the treatment group (P = 0.39, Figure 3B). Effect of antioxidant treatment on biopsy scores In part I, the HAI reduced from 8.87 before to 8.13 after treatment in the treatment group and from 7.85 to 7.4 in the placebo group (P = 0.42). Compared to the pretreatment score, the total biopsy HAI score at the end of treatment reduced in 48% of patients in the treatment group and 27% of patients in the placebo group, and

www.wjgnet.com

unchanged in 26% of patients in the treatment group and 49% of patients in the placebo group (P = 0.21). In part Ⅱ, the HAI scores increased from 7.619 to 8.36 in the treatment group, and from 8.53 to 9 in the placebo group (P = 0.42). Effect of antioxidant treatment on HCV RNA serum levels In par t I, antioxidant treatment had no effect on diminishing viral load, and the average log of HCV RNA at wk 24 was 5.08 for the treatment group and 5.04 for the placebo group (Figure 4A). In part Ⅱ, similarly, no effect of treatment on viral load was observed. The average log of HCV RNA at wk 24 was 5.36 for the treatment group and 5.28 for the placebo group (Figure 4B). Effect of antioxidant treatment on quality of life In part I, data were available from thirty-eight patients. The average baseline quality of life in the treatment group was 52.88 out of a maximum of 100, and the post-treatment value was 52.12 (P = NS). The value in the placebo group was 48.5 before and 47.25 after treatment (P = NS). In part Ⅱ, data were available from thirty-seven patients. The average baseline quality of life in the treatment group was 53.1 out of a maximum of 100, and the post-treatment value was 48.8 (P = 0.50). The value in the placebo group was 43.44 before and 50.31 after treatment (P = 0.38).

Gabbay E et al . Treatment of chronic HCV with antioxidants

A

5.8

Treatment Placebo

5.7

5.5 5.4 5.3

3

10 Copies/mL (log)

5.6

5.2 5.1 5.0

B

0

12

24

5.8

Treatment

5.7

Placebo

t /wk

32

40

48

32

40

48

5.6

AST (IU)

5.5 5.4 5.3 5.2 5.1 5.0 4.9 4.8

0

10

24

t /wk

Figure 4 Effect of antioxidant therapy on viral RNA serum levels in partⅠ (A) and partⅡ(B).

DISCUSSION A combination of intravenous and oral antioxidants could mildly alleviate the intra-hepatic inflammatory response in chronic HCV infection patients who nonresponders to interferon therapy. This was manifested as a significant decrease in ALT and AST serum levels, along with improvement in the biopsy inflammatory scores. No effect was noted when oral antioxidants were used alone. The rationale for treatment of chronic HCV infection patients with antioxidants is based on the role of oxidative stress in the pathogenesis of disease progression in animal models and humans. Results of uncontrolled trials suggest that this treatment may have a beneficial effect on inflammatory indices such as liver enzymes and biopsy HAI scores, as well as quality of life [11] . Intravenous glycyrrhizin was previously tested in patients with chronic HCV infection, and lowered ALT levels (26% vs 6% with placebo) within 4 wk were noted. The effect disappears after cessation of therapy[12]. A retrospective study examining the effects of stronger neo-minophagen C (SNMC), which contains glycyrrhizin as an active component, revealed that treatment with this agent reduces the long term relative risk of developing hepatocellular carcinoma by a factor of 2.49[13]. When SNMC is used in combination with interferon, a higher rate of improvement in liver enzymes and viral clearance can be observed with no statistical significance[14]. It was reported that a combination therapy of interferon (IFN) with glycyrrhizin

5321

induces normalization of serum ALT levels in 64.3% of non-responders with serum HVC RNA disappeared in 38.5%[13]. The efficacy of glycyrrhizin in European studies was less than that reported on Japanese subjects, which can be explained by the genetic polymorphism in drug metabolism[15]. Silybum marianum (milk thistle) is a herbal medicine commonly used in treatment of liver disease. Its active component, silybinin, has been shown to have anti-fibrotic and antioxidant properties, and hepatoprotective effects in animal models of acetaminophen-induced liver damage[16]. It was reported that silymarin protects liver cells from other toxins (including ethanol, carbon tetra-chloride, and D-galactosamine) and ischemic injury, radiation, iron toxicity, and viral hepatitis[17,18]. A combination of three potent anti-oxidants [alphalipoic acid (thioctic acid), silymarin, and selenium] induces marked clinical, laboratory, and histological improvements in chronic HCV infection patients[19,20]. However, a Cochrane systematic review of trials of medicinal herbs in HCV, reported that silybin is significantly reduced in serum AST and GGT levels in only one trial, with no firm evidence for the use of herbal medicines in this condition[21]. Schizandrae chinensis, a potent anti-oxidant, lowers ALT levels in patients with chronic viral hepatitis[22]. Administration of glutathione to patients with chronic hepatitis significantly decreases GSH-Px activity of catalase (CAT), and increases superoxide dismutase (SOD) activity [23,24] . N-acetyl-cysteine, sodium selenite and vitamin E have also been studied as supplementation to interferon therapy for patients with chronic HCV infection[25]. Vitamin E supplementation increases the chance for a complete response[25]. A randomized double-blind trial of thioctic acid (alpha-lipoic acid) in chronic hepatitis patients showed that 55% patients have significant improvements in mean ALT levels, and 77% patients have histological improvements on liver biopsy [26,27]. The results of the present study showed a modest reduction in liver enzymes at the end of 24 wk of treatment in patients receiving the combined intravenous and oral protocol. The proportion of patients on the combination therapy with improvements, was statistically significant, the difference in absolute values did reach significance. In both parts of the trial, the beneficial effect on enzymes did not sustain throughout the follow-up period, which is consistent with some of the previous published data[13]. The lack of significant improvement on liver biopsy seems to reflect the relatively transient nature of the anti-inflammatory effect of antioxidants in this setting. No significant effect on viral load was noted in either part of the trial. This finding was not surprising given the lack of known direct anti-viral effects of antioxidants. A study reported that induction of gastrointestinal glutathione oxidase (which counteracts oxidative stress) by all trans retinoic acids, down-regulates HCV replicon in cell lines [28]. Adverse events that are attributable to therapy such as exacerbation of hypertension, pedal edema, and hypokalemia, are mostly related to pseudoaldosteronism associated with the 11-hydroxy-steroid dehydrogenase inhibitory activity of glycyrrhiza and L-glutathione. The excess endogenous cortisol produced by both agents, reacts with the renal www.wjgnet.com

5322

ISSN 1007-9327

CN 14-1219/R


mineral corticoid receptor, promoting an aldosterone-like action. Similar adverse events have also been described[11]. These findings suggest that it is important to have a close follow-up of patients’ blood pressure and serum potassium levels throughout antioxidant therapy. In the present study, the use of antioxidants had no significant effect on quality of life (QOL) as measured in the general health paradigm of the SF-36 questionnaire which is in contrast with uncontrolled trials, suggesting that antioxidants may exert a beneficial effect on quality of life in chronic HCV infection patients[11]. The combined intravenous and oral antioxidants had a better effect than oral combination alone, further supporting the potential of the intravenous antioxidants, specifically of glycyrrhiza, as a possible basis for the beneficial anti-inflammatory effect noted. Although not directly tested in the present study, the use of a combination of multiple antioxidants administered both intravenously and orally, may have enhanced the anti-oxidative effect, thus contributing to the observed effect. The data of the present study support the role of oxidative stress in the pathogenesis of HCV-induced inflammation. The modest effect noted should be viewed in light of the fact that patients enrolled in the trial were non-responders to anti-viral therapy. Therefore, they represent a relatively “hard to treat” subset of chronic HCV infection patients. Treatment in the present study was lasted for a relatively short period of time, less than that of the current treatment with anti-viral drugs. If the main effect of antioxidants is anti-inflammatory, it would be expected that a combination of antioxidants with current anti-viral treatment may be of benefit for these patients. Indeed, previous reports suggest an added value for antioxidants in interferon-treated chronic HCV infection patients. It has been demonstrated that vitamin E-treated patients have a 2.4 times higher chance of obtaining a complete response and a more significant reduction in viral load than patients not treated with vitamin E[29]. In conclusion, a combination of intravenous and oral antioxidants can reduce intra-hepatic inflammatory response in chronic HCV infection patients who are nonresponders to interferon. It is plausible to examine whether such a treatment in combination with anti-viral therapy may be useful.

COMMENTS

Related publications

Melhem A, Stern M, Shibolet O, Israeli E, Ackerman Z, Pappo O, Hemed N, Rowe www.wjgnet.com

Number 40


Antioxidant therapy has a mild beneficial effect on the inflammatory response in chronic HCV infection patients who are non-responders to interferon. A combination of antiviral and antioxidant therapy may be beneficial for these patients.

Applications

It is plausible to examine whether such a treatment in combination with anti-viral therapy may be useful.

Peer review

This is the first double blind trial using a combination of several anti oxidants in patients with chronic hepatitis C who failed in interferon therapy. The study was designed well and the results were summarized and discussed appropriately.

REFERENCES 1 2

3 4

5 6

7 8 9 10 11

12

Research frontiers

The aim of the present trial was to determine the effect of a mixture of antioxidants on the inflammatory response of chronic HCV infection patients who were non-responders to interferon, in a double blind placebo controlled trial. Two different treatment regimens were studied and compared. The data suggest that the combined use of intravenous and oral antioxidants mildly alleviates the inflammatory response in these patients.

Volume 13

M, Ohana H, Zabrecky G, Cohen R, Ilan Y. Treatment of chronic hepatitis C virus infection via antioxidants: Results of a phaseⅠclinical trial. J Clin Gastroenterol 2005; 39: 737-342.

Background

The pathogenesis of hepatitis C virus (HCV) infection involves a complex interaction between viral factors and host immune responses. A major component of the latter involves oxidative stress[1].

October 28, 2007

13

14 15

Koike K, Miyoshi H. Oxidative stress and hepatitis C viral infection. Hepatol Res 2006; 34: 65-73 Gong G, Waris G, Tanveer R, Siddiqui A. Human hepatitis C virus NS5A protein alters intracellular calcium levels, induces oxidative stress, and activates STAT-3 and NF-kappa B. Proc Natl Acad Sci USA 2001; 98: 9599-9604 Waris G, Turkson J, Hassanein T, Siddiqui A. Hepatitis C virus (HCV) constitutively activates STAT-3 via oxidative stress: role of STAT-3 in HCV replication. J Virol 2005; 79: 1569-1580 Okuda M, Li K, Beard MR, Showalter LA, Scholle F, Lemon SM, Weinman SA. Mitochondrial injury, oxidative stress, and antioxidant gene expression are induced by hepatitis C virus core protein. Gastroenterology 2002; 122: 366-375 Koike K. Molecular basis of hepatitis C virus-associated hepatocarcinogenesis: lessons from animal model studies. Clin Gastroenterol Hepatol 2005; 3: S132-S135 Morbitzer M, Herget T. Expression of gastrointestinal glutathione peroxidase is inversely correlated to the presence of hepatitis C virus subgenomic RNA in human liver cells. J Biol Chem 2005; 280: 8831-8841 Pawlotsky JM. Current and future concepts in hepatitis C therapy. Semin Liver Dis 2005; 25: 72-83 Hughes CA, Shafran SD. Chronic hepatitis C virus management: 2000-2005 update. Ann Pharmacother 2006; 40: 74-82 Seeff LB, Lindsay KL, Bacon BR, Kresina TF, Hoofnagle JH. Complementary and alternative medicine in chronic liver disease. Hepatology 2001; 34: 595-603 Liu JP, Manheimer E, Tsutani K, Gluud C. Medicinal herbs for hepatitis C virus infection. Cochrane Database Syst Rev 2001; CD003183 Melhem A, Stern M, Shibolet O, Israeli E, Ackerman Z, Pappo O, Hemed N, Rowe M, Ohana H, Zabrecky G, Cohen R, Ilan Y. Treatment of chronic hepatitis C virus infection via antioxidants: results of a phase I clinical trial. J Clin Gastroenterol 2005; 39: 737-742 van Rossum TG, Vulto AG, Hop WC, Brouwer JT, Niesters HG, Schalm SW. Intravenous glycyrrhizin for the treatment of chronic hepatitis C: a double-blind, randomized, placebocontrolled phase I/II trial. J Gastroenterol Hepatol 1999; 14: 1093-1099 Arase Y, Ikeda K, Murashima N, Chayama K, Tsubota A, Koida I, Suzuki Y, Saitoh S, Kobayashi M, Kumada H. The long term efficacy of glycyrrhizin in chronic hepatitis C patients. Cancer 1997; 79: 1494-1500 Abe Y, Ueda T, Kato T, Kohli Y. Effectiveness of interferon, glycyrrhizin combination therapy in patients with chronic hepatitis C. Nippon Rinsho 1994; 52: 1817-1822 van Rossum TG, Vulto AG, Hop WC, Schalm SW. Pharmacokinetics of intravenous glycyrrhizin after single and

Gabbay E et al . Treatment of chronic HCV with antioxidants multiple doses in patients with chronic hepatitis C infection. Clin Ther 1999; 21: 2080-2090 16 Muriel P, Garciapina T, Perez-Alvarez V, Mourelle M. Silymarin protects against paracetamol-induced lipid peroxidation and liver damage. J Appl Toxicol 1992; 12: 439-442 17 Ferenci P, Dragosics B, Dittrich H, Frank H, Benda L, Lochs H, Meryn S, Base W, Schneider B. Randomized controlled trial of silymarin treatment in patients with cirrhosis of the liver. J Hepatol 1989; 9: 105-113 18 Boigk G, Stroedter L, Herbst H, Waldschmidt J, Riecken EO, Schuppan D. Silymarin retards collagen accumulation in early and advanced biliary fibrosis secondary to complete bile duct obliteration in rats. Hepatology 1997; 26: 643-649 19 Salmi HA, Sarna S. Effect of silymarin on chemical, functional, and morphological alterations of the liver. A double-blind controlled study. Scand J Gastroenterol 1982; 17: 517-521 20 Buzzelli G, Moscarella S, Giusti A, Duchini A, Marena C, Lampertico M. A pilot study on the liver protective effect of silybin-phosphatidylcholine complex (IdB1016) in chronic active hepatitis. Int J Clin Pharmacol Ther Toxicol 1993; 31: 456-460 21 Liu J, Manheimer E, Tsutani K, Gluud C. Medicinal herbs for hepatitis C virus infection: a Cochrane hepatobiliary systematic review of randomized trials. Am J Gastroenterol 2003; 98: 538-544 22 Liu GT. Pharmacological actions and clinical use of fructus schizandrae. Chin Med J (Engl) 1989; 102: 740-749 23 Loguercio C, Caporaso N, Tuccillo C, Morisco F, Del Vecchio Blanco G, Del Vecchio Blanco C. Alpha-glutathione

5323 transferases in HCV-related chronic hepatitis: a new predictive index of response to interferon therapy? J Hepatol 1998; 28: 390-395 24 Sun F, Hayami S, Ogiri Y, Haruna S, Tanaka K, Yamada Y, Tokumaru S, Kojo S. Evaluation of oxidative stress based on lipid hydroperoxide, vitamin C and vitamin E during apoptosis and necrosis caused by thioacetamide in rat liver. Biochim Biophys Acta 2000; 1500: 181-185 25 Look MP, Gerard A, Rao GS, Sudhop T, Fischer HP, Sauerbruch T, Spengler U. Interferon/antioxidant combination therapy for chronic hepatitis C--a controlled pilot trial. Antiviral Res 1999; 43: 113-122 26 Bustamante J, Lodge JK, Marcocci L, Tritschler HJ, Packer L, Rihn BH. Alpha-lipoic acid in liver metabolism and disease. Free Radic Biol Med 1998; 24: 1023-1039 27 Houglum K, Venkataramani A, Lyche K, Chojkier M. A pilot study of the effects of d-alpha-tocopherol on hepatic stellate cell activation in chronic hepatitis C. Gastroenterology 1997; 113: 1069-1073 28 Morbitzer M, Herget T. Expression of gastrointestinal glutathione peroxidase is inversely correlated to the presence of hepatitis C virus subgenomic RNA in human liver cells. J Biol Chem 2005; 280: 8831-8841 29 Meydani SN, Meydani M, Blumberg JB, Leka LS, Siber G, Loszewski R, Thompson C, Pedrosa MC, Diamond RD, Stollar BD. Vitamin E supplementation and in vivo immune response in healthy elderly subjects. A randomized controlled trial. JAMA 1997; 277: 1380-1386 S- Editor Ma N L- Editor Wang XL

E- Editor Li HY

www.wjgnet.com



VIRAL HEPATITIS

Establishment and primary application of a mouse model with hepatitis B virus replication Feng-Jun Liu, Li Liu, Fang He, Su Wang, Tao-You Zhou, Cong Liu, Lin-Yu Deng, Hong Tang Feng-Jun Liu, Li Liu, Fang He, Su Wang, Tao-You Zhou, Cong Liu, Hong Tang, Center of Infectious Diseases, Division of Molecular Biology of Infectious Diseases, National Key Laboratory of Biotherapy (Sichuan University), West China Hospital of Sichuan University, Chengdu 610041, Sichuan Province, China Lin-Yu Deng, National Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, Sichuan Province, China Supported by the National Science Fund for Distinguished Young Scholars from the National Natural Science Foundation of China, No. 30325036, and a grant from the National Natural Science Foundation of China, No. 30571640 Correspondence to: Professor Hong Tang, Center of Infectious Diseases, Division of Molecular Biology of infectious Diseases, National Key Laboratory of Biotherapy (Sichuan University), West China Hospital of Sichuan University, Chengdu 610041, Sichuan Province, China. [email protected] Telephone: +86-28-85422650 Fax: +86-28-85423052 Received: March 29, 2007 Revised: May 24, 2007

Abstract AIM: To establish a rapid and convenient animal model with hepatitis B virus (HBV) replication. METHODS: A naked DNA solution of HBV-replicationcompetent plasmid was transferred to BALB/C mice via the tail vein, using a hydrodynamic in vivo transfection procedure. After injection, these mice were sacrificed on d 1, 3, 4, 5, 7 and 10. HBV DNA replication intermediates in the liver were analyzed by Southern blot hybridization. The expression of hepatitis B core antigen (HBcAg) and hepatitis B surface antigen (HBsAg) in the liver was checked by immunohistochemistry. Serum HBsAg and hepatitis B e antigen (HBeAg) was detected by enzymelinked immunosorbent assay (ELISA). Inhibition of HBV replication was compared in HBV replication model mice treated intraperitoneally with polyinosinic-polytidylin acid (polyIC) or phosphate-buffered saline (PBS). RESULTS: After hydrodynamic in vivo transfection, HBV DNA replication intermediates in the mouse liver were detectable on d 1 and abundant on d 3 and 4, the levels were slightly decreased and remained relatively stable between d 5 and 7, and were almost undetectable on d 10. The expression patterns of HBcAg and HBsAg were similar to that of HBV replication intermediate DNA, except that they reached a peak on d 1 after injection. No obvious differences in HBV DNA replication

www.wjgnet.com

intermediates were observed in the left, right and middle lobes of the liver. After treatment with polyIC, the level of HBV intermediate DNA in the liver was lower than that in the control mice injected with PBS. CONCLUSION: A rapid and convenient mouse model with a high level of HBV replication was developed and used to investigate the inhibitory effect of polyIC on HBV replication, which provides a useful tool for future functional studies of the HBV genome. © 2007 WJG . All rights reserved.

Key words: Animal model; Gene expression; Hepatitis B Virus; Hydrodynamic transfection; Polyinosinic-polytidylin acid; Virus replication Liu FJ, Liu L, He F, Wang S, Zhou TY, Liu C, Deng LY, Tang H. Establishment and primary application of a mouse model with hepatitis B virus replication. World J Gastroenterol 2007; 13(40): 5324-5330


INTRODUCTION Hepatitis B virus (HBV) infection is a major health problem with more than 350 million chronic carriers worldwide, who are at high risk for developing liver cirrhosis and hepatocellular carcinoma [1,2] . Because the pathogenic mechanisms of HBV remain unclear, molecular-biological studies are required. Until recently, most in vivo studies on the mechanism by which HBV causes liver diseases, and the mechanism of anti-HBV drugs, have been performed by using HBV transgenic mice[3]. However, the acquisition of transgenic mice is very time-consuming and laborious, because expensive microinjection equipment and complicated technology is needed. We have reported previously that high-level expression of HBV surface antigen (HBsAg) in mouse hepatocytes is achieved after injection of an HBsAg expression plasmid via the tail vein using a hydrodynamic method [4]. In this study, we developed a mouse model for HBV replication by using a hydrodynamic procedure that provides a simple, convenient and useful tool for molecular-biological studies on HBV, and for selection of antiviral drugs to treat HBV infection.

Liu FJ et al . Establishment and primary application of a mouse model with HBV replication

MATERIALS AND METHODS Preparation of plasmid DNA The HBV replication-competent plasmid used in this study was pHBV4.1, which contains 1.3 copies of the HBV genome (subtype ayw). This construct has been proven to be able to initiate replication of HBV efficiently after being transfected into human hepatoma HepG2 cell lines[5,6]. The plasmid DNA was prepared by using a plasmid-purifying Kit (QIAGEN TIP-500) according to the manufacturer's instructions Injection of naked plasmid DNA Male BALB/C mice at specific-pathogen-free (SPF) level, weighing 18-20 g, were provided by the Huaxi Laboratory Animal Center of Sichuan University. Mice were injected via the tail vein with plasmid DNA at different doses in 1.8 mL saline within 5-8 s (hydrodynamic in vivo transfection)[7]. The animals were sacrificed at different time points after injection, and the serum and liver were collected. The serum was stored at -20℃. The liver tissue was separated into two parts: one was fixed in 40 g/L neutral-buffered formalin, and the other was snap frozen in liquid nitrogen and stored at -70℃ for subsequent analysis of HBV DNA replication intermediates. Polyinosinic-polytidylin acid (polyIC) treatment Twenty-four hours after hydrodynamic injection with pHBV4.1, the HBV replication-competent mice were matched by body weight, age and serum levels of hepatitis B e antigen (HBeAg), and were divided into two groups of five mice each. The mice in the experimental group were injected intraperitoneally with 200 μg polyIC (P-0913; Sigma) in 200 μL phosphate-buffered saline (PBS), three times at 24-h intervals, while the control group mice received only 200 μL PBS. The mice were sacrificed 4-6 h after the final injection of polyIC. Liver tissue was frozen in liquid nitrogen and stored at -70℃ for DNA extraction and analysis. Detection of HBV DNA replication intermediates Frozen liver tissue was mechanically pulverized in liquid nitrogen and HBV DNA replicative inter mediates were isolated as described previously, with some modifications[8]. One hundred and twenty micrograms of liver tissue powder was lysed in 0.6 mL 100 mmol/L Tris hydrochloride (pH 8.0) and 2 mL/L NP40. The lysate was centrifuged for 1 min at 14000 rpm in a microcentrifuge. The supernatant was adjusted to 6.75 mmol/L magnesium acetate plus 200 μg DNaseI/mL, and incubated for 1 h at 37℃. The supernatant was readjusted to 10 mmol/L ethylenediaminetetraacetic acid EDTA, 8 g/L sodium lauryl sulfate, 100 mmol/L NaCl and 1.6 mg pronase/mL, and was incubated for an additional 1 h at 37℃. The supernatant was extracted twice with phenol/chloroform, precipitated with 0.7 volume of isopropanol, and resuspended in 30 μL 10 mmol/L Tris hydrochloride (pH 8.0) and 1 mmol/L EDTA. DNA Southern blot hybridization analysis was performed with 30 μL viral replication intermediates, as previously described[7]. HBV DNA probe was labeled by

5325

using DIG Luminescent Detection Kit (Roche Applied Science). A digoxigenin-labeled, full-length HBV DNA probe was used for Southern filter hybridization analysis. Detection of liver HBsAg and HBcAg by immunohistochemistry The number and location of cells in the liver that expressed HBV core antigen (HBcAg) or HBsAg were assessed by immunohistochemical staining. Liver tissue fixed in for malin was embedded with paraffin and sectioned (3 μm thick). The paraffin-embedded sections in PBS were treated for 15 min at 37℃ with 30 g/L hydrogen peroxide, and washed with PBS. After the sections were blocked with normal goat serum for 20 min at 37℃, rabbit anti-HbsAg (Biodesign) or rabbit antiHBc (NEOMARKERS) primary antibody was applied at 1:100 or 1:150 dilution for 60 min at 37℃, or overnight at 4℃, respectively. After washing with PBS, the secondary antiser um consisting of Polymer-HRP Anti-Rabbit (Zhongshan, Beijing) was applied at 1:350 dilution for 50 min at 37℃, according to the manufacturer's instructions. The antibody-coated sections were washed with PBS, stained with 3', 3'-diaminobenzidine tetrahydrochloride (DAB), and counterstained with hematoxylin, before being mounted. Serological analysis for HBV antigen HBeAg and HBsAg analysis was performed with 50 μL mouse serum by using enzyme-linked immunosorbent assay (ELISA). The HBeAg and HBsAg Detection Kits (Shanghai Shiye Kehua Company, China) were used respectively.

RESULTS Assessment of the appropriate amount of pHBV4.1 plasmid DNA for hydrodynamic injection To determine an appropriate amount of pHBV4.1 DNA for establishing the mouse model for HBV replication, mice were divided into different groups (two in each group) and injected with various amount of plasmid DNA (5, 10 and 25 μg per mouse). HBV DNA replicative intermediates were examined on d 4 after injection. The results of three independent experiments showed that a significant level of HBV DNA replicative intermediates was detected in mouse liver when the amount of plasmid DNA injected was as low as 5 μg per mouse. The level of HBV replicative intermediates increased when the amount of plasmid DNA was increased to 10 μg per mouse. A further increase in the amount of plasmid DNA to 25 μg per mouse did not result in a significant increase in HBV replicative intermediates in mouse liver. Therefore, we used 10 μg per mouse as the appropriate dose of pHBV4.1 to develop an HBVreplication-competent mouse model (Figure 1). Establishment of a mouse model with HBV replication Thirty mice were injected with pHBV4.1 plasmid DNA at 10 μg per mouse using a hydrodynamic procedure. These mice were sacrificed on d 1, 3, 4, 5, 7 and 10 after injection (n = 5 at each time point). The level of HBV replication and expression were analyzed. www.wjgnet.com

5326

ISSN 1007-9327

5 μg

CN 14-1219/R

10 μg


25 μg

Day

October 28, 2007 1

3

4

Volume 13 5

7

Number 40 10 HBV RC DNA

HBV RC DNA

HBV SS DNA

HBV SS DNA

Figure 1 DNA-dose-dependent HBV replication. Various amount of pHBV4.1 in 1.8 mL saline were injected into BALB/c mice within 5-8 s. Mice were sacrificed on d 4 after injection, and HBV DNA intermediates were detected by Southern blotting.

A

B

Figure 2 Time-dependent viral replication after hydrodynamic in vivo transfection with pHBV4.1. Mice were injected hydrodynamically with 10 μg pHBV4.1 and were sacrificed at different time points after injection. HBV DNA intermediates were detected by Southern blotting.

C

D

Figure 3 Detection of HBcAg expression in the liver by immunohistochemical staining (DAB, × 40). Mice were injected hydrodynamically with 10 μg pHBV4.1 and were sacrificed at different time points after injection and HBcAg in mouse liver was detected. A: 1 d after transfection; B: 4 d after transfection; C: 7 d after transfection; D: 10 d after transfection.

A

B

C

D

Figure 4 Detection of HBsAg expression in liver by immunohistochemical staining (DAB, × 40). Mice were injected hydrodynamically with 10 μg pHBV4.1 and were sacrificed at different time points after injection and HBsAg in mouse liver was detected. A: 1 d after transfection; B: 4 d after transfection; C: 7 d after transfection; D: 10 d after transfection.

HBV replication intermediates As shown in Figure 2, HBV DNA replication intermediates were detected on d 1 [but without the relaxed circular DNA (RC DNA) form], and were abundant on d 3 and 4. By d 5, the number of HBV replication intermediates decreased, and were eventually undetectable by d 10. These results suggested that HBV could replicate in the liver of the mice, and that the level of HBV replication reached a peak on d 3 and 4 after hydrodynamic transfection. Expression of HBcAg and HBsAg in mouse liver Expression of HBcAg and HBsAg in the liver collected on d 1, 4, 7 and 10 after injection was detected by immunowww.wjgnet.com

histochemistry. As shown in Figures 3 and 4, HBcAg- and HBsAg-positive hepatocytes were randomly distributed throughout the liver lobules, with a tendency for localization in the centrilobular area. HBcAg was present in the nuclei and cytoplasm of the hepatocytes, with most expression in the nuclei. HBsAg staining was only seen in the cytoplasm. The expression of both antigens exhibited a similar time-dependent expression pattern that reached a maximum level on d 1, and remained steady until d 4 after injection, followed by a sharp decline. There were only a few HBcAg- and HBsAg-positive cells in whole liver sections by d 7 and 10. Serum from mice sacrificed on d 1, 3, 4, 5, 7 and 10

Liu FJ et al . Establishment and primary application of a mouse model with HBV replication 45

Liver lobe

L

R

M

5327 L

R

M HBV RC DNA

40

Serum HBsAg (OD value)

35 HBV SS DNA

30 25 A

20

B

Figure 6 Viral replication in different parts of the mouse liver after hydrodynamic transfection. The results from two mice (A and B) are shown. L: left lobe; R: right lobe; M, middle lobe.

15 10 5 0

PBS 1

3 4 5 7 Days after hydrodynamic injection

10

Poly I.C

d HBV RC DNA

16 14

Serum HBeAg (OD value)

HBV SS DNA 12

Figure 7 Effect of polyIC on HBV replication in mice. After matched by body weight, age and serum HBeAg, mice were treated with PBS or polyIC. HBV replication levels in the liver of the two groups of mice were compared.

10 8 6

the left, right and middle lobules in each mouse was similar (Figure 6).

4 2 0

1

3 4 5 7 Days after hydrodynamic injection

10

d

Figure 5 Time-dependent HBV antigen expression in serum after hydrodynamic transfection. Mice were injected with 10 μg pHBV4.1. At different time points, HBsAg and HBeAg in the serum were measured by ELISA.

after injection was measured by ELISA for the level of HBeAg and HBsAg (OD450). Samples were considered positive at OD450 ≥ 2.1. The mean level of HBeAg or HBsAg expression at each point was derived from five mice. Figure 5 shows that the expression of HBeAg and HBsAg in the serum was detected on d 1, 3, 4 and 5, reached a peak on d 1 after injection, and decreased thereafter. All samples from mice on d 7 and 10 after injection were negative for HBeAg and HBsAg. HBV replication levels in different parts of mouse liver To investigate whether the levels of HBV replication in different parts of the liver are similar, mice were injected with 10 μg pHBV4.1 plasmid DNA and sacrificed 3 d later. The liver of each mouse was divided into three parts: left, right and middle lobules. The level of HBV DNA replication intermediates in each part was analyzed and compared. We found that the level of HBV replication in

Effect of polyIC injection on HBV replication To investigate whether the mouse model established in this study can be used for the study of HBV, we used it to evaluate the effect of polyIC on HBV replication. As shown in Figure 7, compared with the control animals that were injected with PBS only, HBV DNA replicative intermediates dramatically decreased in the liver from mice injected with polyIC. This result suggested that repetitive polyIC injection decreased the level of viral replication in the liver.

DISCUSSION HBV is a member of the hepadnavirus family, which contains a group of closely related hepatotropic DNA viruses that infect their respective animal hosts, such as HBV, woodchuck hepatitis virus (WHV)[9] and duck hepatitis B virus (DHBV)[10]. HBV naturally infects human and certain non-human primates such as chimpanzees. However, experiments with chimpanzees are limited by cost, availability and ethical considerations. Also, the ability of HBV to infect tupia and the significance of tupia as an HBV animal model remain controversial. Therefore, much current understanding about HBV is derived from studies of WHV and DHBV infection in their natural host[11]. In addition to their use for the selection of anti-HBV drugs[12,13], they are also used to study the mechanism of HBV replication by reverse transcription[14] www.wjgnet.com

5328

ISSN 1007-9327

CN 14-1219/R


and pathog enesis of HBV-related he patocellular carcinoma[15-17]. However, these two hepadnaviruses have some differences to HBV in terms of genome structure and transcriptional and replicative regulation; therefore, they cannot serve as a substitute for research into HBV[18]. Recently, much attention has been paid to HBV transgenic mice, especially HBV-replication-competent transgenic mice with 1.3 copies of the genome of HBV. It is one of the most widely-used animal models in studies on the mechanism of HBV replication[19-21], the pathogenesis of HBV-related liver diseases[22-24], and the selection of antiHBV drugs[25-27]. However, the production of transgenic mice is very laborious and time-consuming. Therefore, it is necessary to develop a convenient, rapid and useful animal model for HBV-related studies. Hydrodynamic in vivo transfection is an efficient, rapid and convenient gene-delivery method, invented in 1999 [7] . We have reported previously that with the hydrodynamic injection of plasmid DNA encoding HBsAg via the tail vein into mice, efficient expression of HBsAg was detected mainly in the mouse liver, which persisted for about 10 d. In this study, by taking advantage of this hydrodynamic in vivo transfection method, an HBV-replication mouse model was developed by using the HBV-replication-competent plasmid pHBV4.1. The replication of HBV was confirmed by the presence of HBV-replication-intermediate DNA, HBcAg and HBsAg in the mouse liver, and the sera of mice were positive for HBeAg and HBsAg. For different plasmid DNA, the doses needed for hydrodynamic injection are presumably different; therefore, we first determined the appropriate amount of pHBV4.1 for hydrodynamic transfection. The results of three independent experiments demonstrated that the level of HBV replication was significant and reached a steadystate level when the amount of plasmid DNA injected was as low as 10 μg per mouse. To keep the replication at a high level and also save the plasmid, we used 10 μg per mouse as the appropriate amount of pHBV4.1 to establish an HBV-replication-competent mouse model. HBV is an enveloped vir us containing a 3.2-kb partially double-stranded DNA genome within its nu c l e o c a p s i d s [ 2 8 , 2 9 ] . U p o n H B V i n f e c t i o n o f t h e hepatocyte, the viral genome is converted to covalently closed circular DNA (cccDNA) in the nucleus. The cccDNA serves as the template for transcription by the host RNA polymerase Ⅱ, which generates the 3.5-, 2.4-, 2.1- and 0.7-kb viral transcriptions that encode the HBV core (HBcAg and HBeAg) and polymerase polypeptide, the large surface antigen polypeptide, the middle and major surface antigen polypeptides (HBsAg)[30-32] and the HBx polypeptide[33]. Besides encoding for the HBV core polypeptide and HBV DNA polymerase that compose the viral capsid, the greater-than-genome-length 3.5-kb pregenomic RNA (pgRNA ) is encapsulated and serves as the template for reverse transcription to synthesize HBV replication intermediates [34-36]. The time-course of the results of our experiments provided the characteristics of HBV replication in the mouse model: within 24 h after transfection, the input DNA apparently reached the nuclei of the hepatocytes, in which it was transcribed and translated to produce HBV-related antigens, and these www.wjgnet.com

October 28, 2007

Volume 13

Number 40

antigens were soon secreted into the serum. The time at which HBV antigen expression peaked was on d 1 after transfection. The expression level of viral antigen in mouse liver was stable, but the titer of HBeAg and HBsAg in the serum decreased from d 1 to 4 accordingly. The reason for this difference is unclear at present. Viral replication was not robust on d 1, although the presence of small amounts of single-stranded DNA, the earliest replicative DNA intermediates, indicated that viral replication had begun. By d 3 and 4, viral replication reached its peak level and decreased substantially until d 10, which is the time that HBV replication intermediates could not be detected. All these findings suggest that it probably requires about 2 d for 3.5-kb mRNA to produce HBV replication intermediates by reverse transcription abundantly. The basic mechanism of the hydrodynamic transfection procedure is that when a large volume of DNA solution is injected rapidly via the tail vein, a high hydrostatic pressure develops in the inferior vena cava and causes heart failure, which forces a rapid flow of DNA solution to the liver tissue and DNA molecules into liver cells[7,37-39]. In this way, digestion of the input DNA by DNAase in the blood is greatly reduced. To assess whether the levels of gene expression in different liver lobes are different, we compared the level of HBV replication in left, right and middle liver lobes from the same mouse. The results suggested that, for a given mouse, the level of HBV-DNA-replicative intermediates was similar in all parts of the liver. This also suggests that the experimental results were not influenced by the use of different liver tissue parts. As shown in Figures 1 and 2, however, there was some variation in HBV replication between different mice, even under similar experimental conditions. This suggests that if this animal model is to be used for selecting anti-HBV drugs or for other research in which HBV replication levels need to be compared, it is necessary to match the HBV-DNA-replication levels by checking serum HBeAg before treatment, and that repeated experiments are needed to guarantee the reliability and accuracy of the test findings. As a strong inducer of alpha/beta interferon, polyIC has been widely used in HBV transgenic mice and exerts an inhibitory effect on HBV replication[40-42]. Therefore, we applied polyIC treatment to the HBV replication mouse model established in this study to investigate whether this model can be used for studies of HBV. We found that polyIC could inhibit HBV replication, which suggests that the mouse model developed in our study can be used for future research on HBV. To understand the mechanisms of HBV transcription, replication and pathogenesis, it is usually necessary to mutate the HBV genome, and then measure the transcription, replication and expression levels of viral mutants; in this way, the function of various HBV genome sequences can be understood. If HBV transgenic mice are used, it is necessary for every mutated HBV genome to be transferred into mouse fertilized eggs, and then transgenic mice can be cultivated, but that is very timeconsuming, costly and laborious. In contrast, by using the HBV-replication-competent mouse model developed in this study, no surgical techniques or special equipment

Liu FJ et al . Establishment and primary application of a mouse model with HBV replication

are needed, which suggests that this model is very simple, convenient, rapid and economical. As a result, this model affords new opportunities for studying HBV; in particular, it is useful for investigating HBV genomic function, especially its role and mechanism in HBV replication through mutation of the HBV genome. In conclusion, we developed a rapid and convenient mouse model with a high level of HBV replication, and applied it to detecting the inhibitory effect of polyIC on HBV. This provides a very useful tool for functional studies on HBV replication and selection of anti-HBV drugs.

5

ACKNOWLEDGMENTS

10

We are grateful to Alan McLachlan (The Scripps Research Institute, La Jolla, CA, USA) for plasmid pHBV4.1.

COMMENTS Backgrond

Hepatitis B Virus(HBV) infection is a worldwide health problem. Because the pathogenic mechanisms of HBV remain unclear, molecular-biological studies for HBV are required. Untill recently, most in vivo studies about HBV have been performed by using HBV transgenic mice. However, the acquisition of transgenic mice is very time-consuming and laborious, Therefore, it is necessary to develop a convenient , rapid and useful animal model.

Research frontiers

HBV mouse model developed by using the hydrodynamic in vivo transfection method is convenient and useful for HBV-related studies. If the mouse genetic background or plasmid backbonne are changed, many different HBV mouse models can be developed for different purposes.By using immunodeficient mice or using AAV as backbone of transferred plasmid, chronic HBV carrier mouse models were established.

6 7 8

9

11

12

13

14

15 16


In this study, we established a mouse model with HBV.replication with naked HBV plasmid through a hydrodynamic precedure and found that for a certain mouse, the levels of HBVDNA replication are similar in left, right, and middle parts of liver tissue. Non-isotope labeled probe and chemiluminescent detetion system was used to detect hepatitis B virus replication levels in vivo by southern hybridization.

Applications

This mouse model can be used for functional study of HBV genome, for studies about HBV replication mechanism and for selection of anti-HBV drugs.

Peer review

This paper describes the establishment of a mouse model with hepatitis B virus replication, with the aim to provide a tool for selection of antiviral drugs to inhibit HBV replication. The work is well developed, and shows promising results.

REFERENCES 1 2 3 4

Lupberger J, Hildt E. Hepatitis B virus-induced oncogenesis. World J Gastroenterol 2007; 13: 74-81 Beasley RP, Hwang LY, Lin CC, Chien CS. Hepatocellular carcinoma and hepatitis B virus. A prospective study of 22 707 men in Taiwan. Lancet 1981; 2: 1129-1133 Akbar SK, Onji M. Hepatitis B virus (HBV)-transgenic mice as an investigative tool to study immunopathology during HBV infection. Int J Exp Pathol 1998; 79: 279-291 Liu FJ, Liu C, Deng LY, Zhou TY, Liu L, Tang H. Hydrodynamics-based in vivo transfection results in high level expression of HBsAg in mouse liver. Huaxi Yixue 2005; 20: 697-699

17 18 19

20 21 22

23

24

25

5329

Tang H, McLachlan A. Transcriptional regulation of hepatitis B virus by nuclear hormone receptors is a critical determinant of viral tropism. Proc Natl Acad Sci USA 2001; 98: 1841-1846 Tang H, McLachlan A. A pregenomic RNA sequence adjacent to DR1 and complementary to epsilon influences hepatitis B virus replication efficiency. Virology 2002; 303: 199-210 Liu F, Song Y, Liu D. Hydrodynamics-based transfection in animals by systemic administration of plasmid DNA. Gene Ther 1999; 6: 1258-1266 Tang H, Delgermaa L, Huang F, Oishi N, Liu L, He F, Zhao L, Murakami S. The transcriptional transactivation function of HBx protein is important for its augmentation role in hepatitis B virus replication. J Virol 2005; 79: 5548-5556 Summers J, Smolec JM, Snyder R. A virus similar to human hepatitis B virus associated with hepatitis and hepatoma in woodchucks. Proc Natl Acad Sci USA 1978; 75: 4533-4537 Mason WS, Seal G, Summers J. Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus. J Virol 1980; 36: 829-836 Botta A, Lu M, Zhen X, Kemper T, Roggendorf M. Naturally occurring woodchuck hepatitis virus (WHV) deletion mutants in chronically WHV-infected woodchucks. Virology 2000; 277: 226-234 Menne S, Cote PJ, Korba BE, Butler SD, George AL, Tochkov IA, Delaney WE 4th, Xiong S, Gerin JL, Tennant BC. Antiviral effect of oral administration of tenofovir disoproxil fumarate in woodchucks with chronic woodchuck hepatitis virus infection. Antimicrob Agents Chemother 2005; 49: 2720-2728 Howe AY, Robins MJ, Wilson JS, Tyrrell DL. Selective inhibition of the reverse transcription of duck hepatitis B virus by binding of 2',3'-dideoxyguanosine 5'-triphosphate to the viral polymerase. Hepatology 1996; 23: 87-96 Hu J, Toft DO, Seeger C. Hepadnavirus assembly and reverse transcription require a multi-component chaperone complex which is incorporated into nucleocapsids. EMBO J 1997; 16: 59-68 Imazeki F, Yaginuma K, Omata M, Okuda K, Kobayashi M, Koike K. Integrated structures of duck hepatitis B virus DNA in hepatocellular carcinoma. J Virol 1988; 62: 861-865 Lisi D, Kondili LA, Ramieri MT, Giuseppetti R, Bruni R, Della Rocca C, De Santis A, Rapicetta M. Ultrasonography in the study of hepatocellular carcinoma in woodchucks chronically infected with WHV. Lab Anim 2003; 37: 233-240 Menne S, Cote PJ. The woodchuck as an animal model for pathogenesis and therapy of chronic hepatitis B virus infection. World J Gastroenterol 2007; 13: 104-124 Tang H, McLachlan A. Avian and Mammalian hepadnaviruses have distinct transcription factor requirements for viral replication. J Virol 2002; 76: 7468-7472 Guidotti LG, Matzke B, Pasquinelli C, Shoenberger JM, Rogler CE, Chisari FV. The hepatitis B virus (HBV) precore protein inhibits HBV replication in transgenic mice. J Virol 1996; 70: 7056-7061 Cavanaugh VJ, Guidotti LG, Chisari FV. Interleukin-12 inhibits hepatitis B virus replication in transgenic mice. J Virol 1997; 71: 3236-3243 Kimura K, Kakimi K, Wieland S, Guidotti LG, Chisari FV. Interleukin-18 inhibits hepatitis B virus replication in the livers of transgenic mice. J Virol 2002; 76: 10702-10707 Larkin J, Clayton M, Sun B, Perchonock CE, Morgan JL, Siracusa LD, Michaels FH, Feitelson MA. Hepatitis B virus transgenic mouse model of chronic liver disease. Nat Med 1999; 5: 907-912 Hsieh YH, Su IJ, Wang HC, Chang WW, Lei HY, Lai MD, Chang WT, Huang W. Pre-S mutant surface antigens in chronic hepatitis B virus infection induce oxidative stress and DNA damage. Carcinogenesis 2004; 25: 2023-2032 Benn J, Schneider RJ. Hepatitis B virus HBx protein activates Ras-GTP complex formation and establishes a Ras, Raf, MAP kinase signaling cascade. Proc Natl Acad Sci USA 1994; 91: 10350-10354 Madden CR, Finegold MJ, Slagle BL. Altered DNA mutation

www.wjgnet.com

5330

26

27 28 29

30

31

32

33 34

ISSN 1007-9327

CN 14-1219/R


spectrum in aflatoxin b1-treated transgenic mice that express the hepatitis B virus x protein. J Virol 2002; 76: 11770-11774 Wieland SF, Vega RG, Muller R, Evans CF, Hilbush B, Guidotti LG, Sutcliffe JG, Schultz PG, Chisari FV. Searching for interferon-induced genes that inhibit hepatitis B virus replication in transgenic mouse hepatocytes. J Virol 2003; 77: 1227-1236 Robek MD, Boyd BS, Wieland SF, Chisari FV. Signal transduction pathways that inhibit hepatitis B virus replication. Proc Natl Acad Sci USA 2004; 101: 1743-1747 Ganem D, Varmus HE. The molecular biology of the hepatitis B viruses. Annu Rev Biochem 1987; 56: 651-693 Zarski JP, Thelu MA, Rachail M, Seigneurin JM. Molecular biology of hepatitis B virus. I: Structure, genetic organization, replication, transcription. Gastroenterol Clin Biol 1991; 15: 489-496 Yaginuma K, Shirakata Y, Kobayashi M, Koike K. Hepatitis B virus (HBV) particles are produced in a cell culture system by transient expression of transfected HBV DNA. Proc Natl Acad Sci USA 1987; 84: 2678-2682 Standring DN, Rutter WJ, Varmus HE, Ganem D. Transcription of the hepatitis B surface antigen gene in cultured murine cells initiates within the presurface region. J Virol 1984; 50: 563-571 Simonsen CC, Levinson AD. Analysis of processing and polyadenylation signals of the hepatitis B virus surface antigen gene by using simian virus 40-hepatitis B virus chimeric plasmids. Mol Cell Biol 1983; 3: 2250-2258 Kaneko S, Miller RH. X-region-specific transcript in mammalian hepatitis B virus-infected liver. J Virol 1988; 62: 3979-3984 Will H, Reiser W, Weimer T, Pfaff E, Buscher M, Sprengel R, Cattaneo R, Schaller H. Replication strategy of human

Volume 13

Number 40

hepatitis B virus. J Virol 1987; 61: 904-911 35 W a n g G H , S e e g e r C . N o v e l m e c h a n i s m f o r r e v e r s e transcription in hepatitis B viruses. J Virol 1993; 67: 6507-6512 36 Nassal M, Rieger A. A bulged region of the hepatitis B virus RNA encapsidation signal contains the replication origin for discontinuous first-strand DNA synthesis. J Virol 1996; 70: 2764-2773 37 Z h a n g G , S o n g Y K , L i u D . L o n g - t e r m e x p r e s s i o n o f human alpha1-antitrypsin gene in mouse liver achieved by intravenous administration of plasmid DNA using a hydrodynamics-based procedure. Gene Ther 2000; 7: 1344-1349 38 K o b a y a s h i N , N i s h i k a w a M , H i r a t a K , T a k a k u r a Y . Hydrodynamics-based procedure involves transient hyperpermeability in the hepatic cellular membrane: implication of a nonspecific process in efficient intracellular gene delivery. J Gene Med 2004; 6: 584-592 39 Zhang G, Gao X, Song YK, Vollmer R, Stolz DB, Gasiorowski JZ, Dean DA, Liu D. Hydroporation as the mechanism of hydrodynamic delivery. Gene Ther 2004; 11: 675-682 40 Wieland SF, Guidotti LG, Chisari FV. Intrahepatic induction of alpha/beta interferon eliminates viral RNA-containing capsids in hepatitis B virus transgenic mice. J Virol 2000; 74: 4165-4173 41 Anderson AL, Banks KE, Pontoglio M, Yaniv M, McLachlan A. Alpha/beta interferon differentially modulates the clearance of cytoplasmic encapsidated replication intermediates and nuclear covalently closed circular hepatitis B virus (HBV) DNA from the livers of hepatocyte nuclear factor 1alpha-null HBV transgenic mice. J Virol 2005; 79: 11045-11052 42 U p r i c h a r d S L , W i e l a n d S F , A l t h a g e A , C h i s a r i F V . Transcriptional and posttranscriptional control of hepatitis B virus gene expression. Proc Natl Acad Sci USA 2003; 100: 1310-1315

www.wjgnet.com

October 28, 2007

S- Editor Liu Y L- Editor Kerr C

E- Editor Li HY



CLINICAL RESEARCH

Right liver lobe/albumin ratio: Contribution to non-invasive assessment of portal hypertension Tamara Alempijevic, Vladislava Bulat, Srdjan Djuranovic, Nada Kovacevic, Rada Jesic, Dragan Tomic, Slobodan Krstic, Miodrag Krstic Tamara Alempijevic, Vladislava Bulat, Srdjan Djuranovic, Nada Kovacevic, Rada Jesic, Dragan Tomic, Slobodan Krstic, Miodrag Krstic, Clinic for Gastroenterology and Hepatology, Institute for Digestive Diseases, Clinical Centre of Serbia, 6 Dr Koste Todorovica St., 11000 Belgrade, Serbia C o r r e s p o n d e n c e t o : Ta m a r a A l e m p i j e v i c , C l i n i c a l Centre of Serbia, Institute for Digestive Diseases, Clinic for Gastroenterology and Hepatology, 6 Dr Koste Todorovica St. 11000 Belgrade, Serbia. [email protected] Telephone: +381-11-3635417 Fax: +381-11-3615432 Received: June 16, 2007 Revised: August 8, 2007

Key words: Liver cirrhosis; Esophageal varices; Portal hypertension; Ultrasonography

Abstract

INTRODUCTION

AIM: To study the value of biochemical and ultrasonographic parameters in prediction of presence and size of esophageal varices.

Esophageal varices development is among the major complications of liver cirrhosis, with an estimated prevalence of approximately 50%. The risk of bleeding from varices is 25%-35% with majority of the initial bleeding occurring within 1 year from varices detection[1]. The mortality from each episode of variceal bleeding is 17%-57%[2-4]. Bleeding episodes can be predicted by the presence of red signs (“red cherry spots”) on the varices, and by the variceal size[5,6]. The incidence of bleeding can be reduced with nonselective beta-blockers[7,8]. It is also suggested that prophylactic endoscopic variceal ligation can decrease the incidence of first variceal bleeding and mortality in patients with liver cirrhosis who have large varices [9,10]. Therefore, annual endoscopic screening is highly recommended for patients with small esophageal varices while the procedure should be conducted once every two years for patients suffering from liver cirrhosis without diagnosed varices [11,12]. Nevertheless, repeated endoscopic examinations are unpleasant for patients, and have cost impact on health care insurance, while only half of cirrhotic patients have esophageal varices, and up to 30% have large varices. Therefore, the sensitivity and specificity of numerous non-invasive parameters have been investigated for assessment of presence and size of esophageal varices, and risk prediction for bleeding. We aim to identify non-invasive parameters based on ultrasonographic measurement and laboratory test that have a potential in assessment of presence and size of esophageal varices in patients with liver cirrhosis.

METHODS: The study includes selected cirrhotic patients who underwent a complete biochemical workup, upper digestive endoscopic and ultrasonographic examinations. Albumin/right liver lobe diameter and platelet count/spleen diameter ratios were calculated. The correlation between calculated ratio and the presence and degree of esophageal varices was evaluated. RESULTS: Ninety-four subjects (62 males, 32 females), with a mean age of 52.32 ± 13.60 years, were studied. Child-Pugh class A accounted for 42.6%, class B 37.2%, whereas class C 20.2%. Esophageal varices (OE) were not demonstrated by upper digestive endoscopy in 24.5%, while OE gradeⅠwas found in 22.3% patients, grade Ⅱ in 33.0%, grade Ⅲ in 16.0%, and grade Ⅳ in 4.3%. The mean value of right liver lobe diameter/ albumin ratio was 5.51 ± 1.82 (range from 2.76 to 11.44), while the mean platelet count/spleen diameter ratio was 1017.75 ± 729.36 (range from 117.39 to 3362.50), respectively. Statistically significant correlation was proved by Spearman's test between OE grade and calculated ratios. The P values were 0.481 and -0.686, respectively. CONCLUSION: The right liver lobe diameter/albumin and platelet count/spleen diameter ratios are noninvasive parameters providing accurate information pertinent to determination of presence of esophageal varices, and their grading in patients with liver cirrhosis. © 2007 WJG . All rights reserved.

Alempijevic T, Bulat V, Djuranovic S, Kovacevic N, Jesic R, Tomic D, Krstic S, Krstic M. Right liver lobe/albumin ratio: Contribution to non-invasive assessment of portal hypertension. World J Gastroenterol 2007; 13(40): 5331-5335


MATERIALS AND METHODS This study includes 94 patients treated for liver cirrhosis in Clinic for Gastroenterology and Hepatology, Clinical Centre of Serbia in 2006. The diagnosis of cirrhosis www.wjgnet.com

5332

ISSN 1007-9327

CN 14-1219/R


Table 1 Clinical characteristics of patients Sex (M/F) Age (yr; mean ± SD) Age range (yr) Aetiology of liver disease (alcoholic/infective/autoimmune/other) Child-Pugh class (A/B/C) Mean size of the right liver lobe (mean ± SD) Mean size of the spleen (mean ± SD) Mean albumin concentration (g/L; mean ± SD) Mean thrombocyte number (mean ± SD) Oesophageal varices grade (0/Ⅰ/Ⅱ/Ⅲ/Ⅳ)

62/32 52.32 ± 13.60 17-79 43/19/17/15 40/35/19 157.71 ± 23.37 148.05 ± 33.73 30.79 ± 7.97 134 632.98 ± 75 937.83 23/21/31/15/4

Table 2 Calculated ratios and varice size Varices Grade 0 GradeⅠ Grade Ⅱ Grade Ⅲ Grade Ⅳ

Right lobe/albumin ratio mean ± SD Range 4.19 ± 1.36 2.76-8.19 5.43 ± 1.58 3.25-9.44 5.84 ± 1.25 3.40-8.48 6.57 ± 2.42 4.07-10.00 6.68 ± 2.52 4.07-11.44

Platelet count/spleen ratio mean ± SD Range 1822.53 ± 804.06 608.70-3362.50 1072.86 ± 493.49 366.67-2021.05 696.46 ± 439.43 150.00-2283.33 549.14 ± 289.90 127.27-1275.00 348.19 ± 231.86 117.39-633.33

was based on clinical features, laboratory test, imaging diagnostics, and liver histology whenever possible. The following information was collected for each patient: age, gender, etiology of cirrhosis, biochemical par ameters (aspar tate amino tr ansfer ase, a lan ine aminotransferase, total bilir ubin, ser um albumin, prothrombin activity, and platelet count), presence and degree of esophageal varices and degree of liver function impairment by Child-Pugh classes. Cirrhosis etiology was determined as viral if hepatitis B surface antigen or hepatitis C serum markers were positive. If viral markers were negative, and the history obtained indicated alcohol consumption of at least 50 g per day in the past five years, liver cirrhosis was considered as alcoholic. Positive immunological markers were characteristic for immunological liver disease. The other studied cases had liver cirrhosis of different etiology (Wilson’s disease, α1 antitrypsin deficiency, hemochromatosis, etc.). All studied subjects underwent ultrasonographic examination of the upper abdomen. The right lobe diameter in the medioclavicular line, as well as the spleen bipolar diameter were measured for three times and the mean value was recorded. One investigator performed all measurements (TA) in order to reduce the inter- and intraobserver errors in assessing the diameters. Using the laboratory and ultrasonographic values, we calculated two ratios: right lobe diameter/serum albumin concentration and platelet count/spleen diameter. An experienced endoscopist (SD) who used the grade [13] Ⅰ-Ⅳ classification did the endoscopies. Varices in the level of mucosa were recognized as grade I, those smaller than 5 mm fulfilling less than 1/3 of the esophageal lumen were recognized as g rade Ⅱ, g rade Ⅲ were varices larger than 5 mm fulfilling more than 1/3 of the esophageal, while grade Ⅳ varices occupied more than 2/3 of esophageal lumen. Patients with previous variceal www.wjgnet.com

October 28, 2007

Volume 13

Number 40

bleeding, porto-systemic shunts and those taking betablockers medications and patients with coexistent illness or infection that could influence the liver and spleen size, were excluded. Child-Pugh score was calculated using five variables (ascites, ence phalopathy, bilir ubin, albumin, and prothrombin time). Values 1, 2 or 3 were assigned to each of these variables due to increasing abnormality, and the score calculated as sum of the five variables for each patient. A Child-Pugh score less than 7 was considered as class A, from 7 to 9 as class B, while any score greater than 9 was as class C[14]. Laboratory test and ultrasonographic and endoscopic examinations were performed within one week. The Ethic Committee of our institution approved the study and all patients gave an informed consent prior to inclusion into this investigation. All collected data were analyzed and correlated. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS, version 10.0). Basic descriptive statistics included means, standard deviations, ranges and percentages. For correlation analysis, we used Spearman’s test. Differences were considered statistically significant if the two-tailed P value was less than 0.05. Sensitivity and specificity, as well as the best cut-off value for the diagnosis of varices were calculated using ROC curves.

RESULTS Clinical characteristics of the patients are presented in Table 1. The mean value of the ratio of the platelet count/ spleen size was 1017.75 ± 729.36 (range 117.39-3362.50). The mean value of the ratio of the right liver lobe size/ serum albumin concentration was 5.51 ± 1.82 (range 2.76-11.44). These ratios were correlated with the presence and size of esophageal varices (Table 2, Figure 1A and B). Spearman’s test for nonparametric correlation showed that, the platelet count/spleen size ratio significantly correlated with the presence (P = 0.585, P < 0.01) and size (P = 0.686, P < 0.01) of esophageal varices, so does the right liver lobe/serum albumin concentration ratio (P = 0.488, P < 0.01 and P = 0.481, P < 0.01 respectively). We also studied the correlation with Child-Pugh score of liver function impairment (Table 3, Figure 2A and B). These ratios had a highly significant correlation (P < 0.01) with Child Pugh score. For the platelet count/spleen size ratio, P = -0.585, while for the right liver lobe/serum albumin concentration ratio, P = 0.491. We counted the best cut-off value of the right liver lobe/serum albumin concentration ratio for diagnosis of varices. For the value of 4.425, the sensitivity was 83.1% and the specificity was 73.9% (Figure 3).

DISCUSSION Incidence of esophageal varices in patients with liver cir rhosis is approximately 90%, but only 9%-35% have been described[15-17]. Variceal bleeding in cirrhotic

Alempijevic T et al . Non-invasive assessment of portal hypertension

A

A

2000

1500

1000

B

500

5333

8 7 6 5 4 3 2 1 0

Ⅰ

0

Ⅱ

Ⅲ

Ⅳ

8 7

0

Ⅰ

0

Ⅱ

Ⅲ

6

Ⅳ

5

B

4

1600 1400 1200 1000 800 600 400 200 0

3 2 1 0 Child A

Child A

Child B

Child C

Child B

Child C

Figure 2 A: Right lobe/albumin ratio in correlation to varice size; B: Right lobe/ albumin ratio in correlation to Child-Pugh score.

Figure 1 A: Platelet count/spleen ratio in correlation to varice size; B: Platelet count/spleen ratio in correlation to Child-Pugh score.

1.0

Table 3 Calculated ratios and Child score for hepatic insufficiency ChildPugh score A B C

Right lobe/albumin ratio Platelet count/spleen ratio mean ± SD Range mean ± SD Range 4.59 ± 1.38 2.76-8.19 1355.34 ± 796.62 365.52-3362.50 5.92 ± 1.60 3.25-9.44 946.67 ± 612.48 127.27-2975.00 6.68 ± 2.12 4.07-11.44 437.97 ± 215.99 117.39-900.00

patients is associated mainly with fatal outcome. Portal hypertension is an essential element of survival in cirrhotic patients[3]. Therefore, regular control and evaluation of esophageal varices with timely introduction of nonselective beta-blockers and variceal ligation play an important role in prevention of bleeding. Endoscopy is an invasive and costly diagnostic procedure. Therefore, introduction of non-invasive parameters for assessment of presence and size of esophageal varices is a major goal of numerous studies. Platelet count and spleen size were the most frequently explored non-invasive parameters in recent studies. Zaman[15] reported that patients with platelet counts of less than 88 000/mm3 have five times greater likelihood of having large esophageal or gastric varices as compared with the patients with higher platelet counts. Ng[16] identified correlation between presence of ascites, thrombocytopenia, hyperbilirubinemia and larger varices in the Chinese population. Similarly, Chalasani [11] concluded that large esophageal varices are predictable in thrombocytopenic patients who have enlarged spleen, while platelet count of less than 88 000/mm 3 indicates a higher risk for esophageal bleeding. The same study also demonstrated that endoscopy is incrementally cost-effective. Madthora reported that platelet count less than 68 000/mm3 have a

Sensitivity

0.8 0.6 0.4 0.2 0.0

0.0

0.2

0.4 0.5 1-Specificity

0.8

1.0

Figure 3 ROC curve for diagnosis of esophageal varices using right lobe/albumin ratio.

larger discriminatory value[12]. Splenomegaly and hypersplenism are prevalent in patients with liver cirrhosis and portal hypertension[11]. Thrombocytopenia is a common and highly specific manifestation of hypersplenism, but with low sensitivity for presence of portal hypertension[18]. It is suggested that the main mechanism of thrombocytopenia is splenic sequestration and pooling[19], and other mechanisms are also involved. Madthora and co-authors reported that 32% of patients had platelet count less than 68 000/mm3 without detectable splenomeg aly, which might be explained by insufficient synthesis of thrombopoietin. It is also indicated that platelet count and thrombopoietin level are returning to referent values following liver transplantation[20]. Other potential explanations for this phenomenon are presence of antithrombocytic antibodies and thrombocyte associated immunoglobulin, which can be found in the sera of patients with liver diseases[21]. Some investigators reported that the use of noninvasive indicators is invalid for avoiding the endoscopic screening in patients with primary biliary cirrhosis and www.wjgnet.com

5334

ISSN 1007-9327

CN 14-1219/R


sclerosing cholangitis. The probability of esophageal varices presence is lower in these patients if they have platelet count less than 200 000/mm 3, possibly due to the fact that thrombopoietin production is preserved in primary biliary cirrhosis and sclerosing cholangitis[22]. It is well documented that the spleen size is correlated with severity of esophageal varices [23-25]. Watanabe [25] calculated the splenic ratio (length × width × height of the spleen size on computed tomography) and showed that patients having a ratio greater than 963 cm 3 have esophageal varices, and that a high ratio may predict esophageal bleeding in patients with liver cirrhosis. It is concluded that ultrasonographic measurement of splenic cranio-caudal diameter results in lower inter- and intraobserver variability unlike Doppler sonographic assessment of hepatic flow[26,27]. The value of Doppler sonographic assessment of portal hypertension is controversial. It is indicated that congestive ratio (counted from the cross section area and mean velocity of portal flow)[26,28,29], as well as resistance ratio of the hepatic artery[30] and splenic artery[24] are correlated with portal hypertension. Portal hemodynamic parameters (mean and maximum portal vein velocity) seam to be weak in assessment of portal hypertension [31]. In addition, it is important to keep in mind that performing Doppler sonographic examination requires sophisticated skills and equipment. The pathophysiologic mechanisms are combined based on the integration of two non-invasive parameters, i.e., platelet count and spleen size into one ratio. Calculation of this ratio is very easy for routine clinical practice. Gianinni and co-authors [32,33] reported the results of a retrospective and prospective study and concluded that this ratio is sensitive for prediction of presence and size of esophageal varices. The same study group suggests as especially important, the use of non-invasive parameters in the diagnostic algorithm for identifying patients without esophageal varices. A cut-off value of 909 has been proposed for platelet count/spleen size ratio[34]. Patient having the ratio greater than cut-off value should not receive nonselective beta-blockers prophylactic therapy because they are less likely to develop esophageal varices. These patients should less frequently undergo endoscopy. Ta k i n g i n t o a c c o u n t t h e r e s u l t s o f p r e v i o u s studies in the field, we also combined laboratory and ultrasonographic parameters and counted an original ratio. For the first time we reported the value of the right liver lobe/serum albumin concentration in assessment of portal hypertension[35]. We used serum albumin concentration as a parameter of liver function in combination with right liver lobe size. Previously published studies reported that platelet count/spleen size ratios are good predictors of esophageal varices. Despite a good correlation between these ratios and esophageal varices grade, it is unlikely that these ratios could be used to exclude patients from initial endoscopic screening. Nevertheless, these ratios may ser ve for selection of patients who need more frequent endoscopies. These ratios will help identify patients at higher risks for development of esophageal varices. It will provide insight into the relationship between clinical, biochemical, www.wjgnet.com

October 28, 2007

Volume 13

Number 40

hematological and imaging abnormalities and development of clinically significant esophageal varices. The right liver lobe diameter/albumin, as well as platelet count/spleen diameter ratios are non-invasive parameters that can provide accurate information pertinent to determination of the presence and grade of esophageal varices in patients with liver cirrhosis. Further validation of the results will be achieved through long-term follow-up of the patients and a larger number of studied subjects.

COMMENTS Background

Esophageal varices are frequent in patients with liver cirrhosis. There is an increasing mortality rate of cirrhotic patients due to high risk of bleeding. The correlation of varices size and risk of bleeding is well-known, yet non-invasive techniques for variceal size estimation have not been intensively studied.

Research frontiers

Valuable laboratory tests and clinical examination may help predict the risk of esophageal varices bleeding. This study shows that excellent correlation exists between platelet count/spleen size and size of esophageal varices, so does the right liver lobe/serum albumin concentration ratio. In addition, the authors suggest that the use of non-invasive and less expensive methods may be of benefit for patients and consumers.


Accumulated literature shows that it is necessary to investigate non-invasive parameters in liver cirrhosis for prediction of the size of esophageal varices. Development of these methods may decrease the exposure of cirrhotic patients to repeated endoscopic procedures.

Applications

Thet can be used for non-invasive estimation of esophageal varice size in cirrhotic patients.

Peer review

The manuscript reports the results from an investigation of non-invasive parameters in estimation of variceal size and risk for bleeding. The authors conclude that combination of laboratory and ultrasonographic parameters such as right liver lobe/serum albumin concentration may be useful in assessment of portal hypertension. The appropriate figures and tables are supporting the results.

REFERENCES 1

2 3 4 5 6

The North Italian Endoscopic Club for the Study and Treatment of Esophageal varices. Prediction of the first variceal hemorrhage in patients with cirrhosis of the liver and esophageal varices. A prospective multicenter study. The North Italian Endoscopic Club for the Study and Treatment of Esophageal Varices. N Engl J Med 1988; 319: 983-989 Graham DY, Smith JL. The course of patients after variceal hemorrhage. Gastroenterology 1981; 80: 800-809 D'Amico G, Luca A. Natural history. Clinical-haemodynamic correlations. Prediction of the risk of bleeding. Baillieres Clin Gastroenterol 1997; 11: 243-256 Jensen DM. Endoscopic screening for varices in cirrhosis: findings, implications, and outcomes. Gastroenterology 2002; 122: 1620-1630 Bhasin DK, Malhi NJ. Variceal bleeding and portal hypertension: much to learn, much to explore. Endoscopy 2002; 34: 119-128 Merkel C, Zoli M, Siringo S, van Buuren H, Magalotti D, Angeli P, Sacerdoti D, Bolondi L, Gatta A. Prognostic indicators of risk for first variceal bleeding in cirrhosis: a multicenter study in 711 patients to validate and improve the North Italian Endoscopic Club (NIEC) index. Am J Gastroenterol 2000; 95: 2915-2920

Alempijevic T et al . Non-invasive assessment of portal hypertension 7

D'Amico G, Pagliaro L, Bosch J. The treatment of portal hypertension: a meta-analytic review. Hepatology 1995; 22: 332-354 Tripathi D, Hayes PC. Review article: a drug therapy for the 8 prevention of variceal haemorrhage. Aliment Pharmacol Ther 2001; 15: 291-310 Khuroo MS, Khuroo NS, Farahat KL, Khuroo YS, Sofi AA, 9 Dahab ST. Meta-analysis: endoscopic variceal ligation for primary prophylaxis of oesophageal variceal bleeding. Aliment Pharmacol Ther 2005; 21: 347-361 10 Psilopoulos D, Galanis P, Goulas S, Papanikolaou IS, Elefsiniotis I, Liatsos C, Sparos L, Mavrogiannis C. Endoscopic variceal ligation vs. propranolol for prevention of first variceal bleeding: a randomized controlled trial. Eur J Gastroenterol Hepatol 2005; 17: 1111-1117 11 Chalasani N, Imperiale TF, Ismail A, Sood G, Carey M, Wilcox CM, Madichetty H, Kwo PY, Boyer TD. Predictors of large esophageal varices in patients with cirrhosis. Am J Gastroenterol 1999; 94: 3285-3291 12 Madhotra R, Mulcahy HE, Willner I, Reuben A. Prediction of esophageal varices in patients with cirrhosis. J Clin Gastroenterol 2002; 34: 81-85 13 Wehrmann T. Öseophagus Diagnostisch und therapeutisch. In: Lembcke B and Wehrmann T, editors. Die gastroenterologische Endoskopie. Reinbeck: Einhorn-Presse Verlag, 1999: 56-83 14 Pugh RN, Murray-Lyon IM, Dawson JL, Pietroni MC, Williams R. Transection of the oesophagus for bleeding oesophageal varices. Br J Surg 1973; 60: 646-649 15 Zaman A, Hapke R, Flora K, Rosen HR, Benner K. Factors predicting the presence of esophageal or gastric varices in patients with advanced liver disease. Am J Gastroenterol 1999; 94: 3292-3296 16 Ng FH, Wong SY, Loo CK, Lam KM, Lai CW, Cheng CS. Prediction of oesophagogastric varices in patients with liver cirrhosis. J Gastroenterol Hepatol 1999; 14: 785-790 17 Pagliaro L, D'Amico G, Pasta F. Portal hypertension in cirrhosis: natural history. In Bosch J, Groszmann RJ, editors. Portal hypertension: pathophysiology and treatment. London: Blackwell Scientific, 1994: 72-92 18 Thomopoulos KC, Labropoulou-Karatza C, Mimidis KP, Katsakoulis EC, Iconomou G, Nikolopoulou VN. Non-invasive predictors of the presence of large oesophageal varices in patients with cirrhosis. Dig Liver Dis 2003; 35: 473-478 19 Mc Cormick PA. The spleen, hypersplenism, and other relationships between the liver and spleen. In: Bircher J, Benhamou J-P, McIntyre N, Rizzetto M, Rodés J, editors. Oxford textbook of clinical hepatology. Oxford: Oxford University Press 1999; 787-795 20 Goulis J, Chau TN, Jordan S, Mehta AB, Watkinson A, Rolles K, Burroughs AK. Thrombopoietin concentrations are low in patients with cirrhosis and thrombocytopenia and are restored after orthotopic liver transplantation. Gut 1999; 44: 754-758 21 Winkfield B, Aube C, Burtin P, Cales P. Inter-observer and intra-observer variability in hepatology. Eur J Gastroenterol Hepatol 2003; 15: 959-966 22 Bressler B, Pinto R, El-Ashry D, Heathcote EJ. Which patients with primary biliary cirrhosis or primary sclerosing cholangitis

23 24

25

26

27

28 29

30 31

32

33

34

35

5335

should undergo endoscopic screening for oesophageal varices detection? Gut 2005; 54: 407-410 Pilette C, Oberti F, Aube C, Rousselet MC, Bedossa P, Gallois Y, Rifflet H, Cales P. Non-invasive diagnosis of esophageal varices in chronic liver diseases. J Hepatol 1999; 31: 867-873 Piscaglia F, Donati G, Cecilioni L, Celli N, Stagni B, Pini P, Gaiani S, Gherlinzoni F, Bolondi L. Influence of the spleen on portal haemodynamics: a non-invasive study with Doppler ultrasound in chronic liver disease and haematological disorders. Scand J Gastroenterol 2002; 37: 1220-1227 Watanabe S, Hosomi N, Kitade Y, Kurokohchi K, Arima K, Kawabata H, Uchida Y, Nishioka M. Assessment of the presence and severity of esophagogastric varices by splenic index in patients with liver cirrhosis. J Comput Assist Tomogr 2000; 24: 788-794 Aube C, Winkfield B, Oberti F, Vuillemin E, Rousselet MC, Caron C, Cales P. New Doppler ultrasound signs improve the non-invasive diagnosis of cirrhosis or severe liver fibrosis. Eur J Gastroenterol Hepatol 2004; 16: 743-751 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 Bolondi L, Li Bassi S, Gaiani S, Barbara L. Doppler flowmetry in portal hypertension. J Gastroenterol Hepatol 1990; 5: 459-67 Martins RD, Szejnfeld J, Lima FG, Ferrari AP. Endoscopic, ultrasonographic, and US-Doppler parameters as indicators of variceal bleeding in patients with schistosomiasis. Dig Dis Sci 2000; 45: 1013-1018 Schneider AW, Kalk JF, Klein CP. Hepatic arterial pulsatility index in cirrhosis: correlation with portal pressure. J Hepatol 1999; 30: 876-881 Schepis F, Camma C, Niceforo D, Magnano A, Pallio S, Cinquegrani M, D'amico G, Pasta L, Craxi A, Saitta A, Raimondo G. Which patients with cirrhosis should undergo endoscopic screening for esophageal varices detection? Hepatology 2001; 33: 333-338 Giannini E, Botta F, Borro P, Risso D, Romagnoli P, Fasoli A, Mele MR, Testa E, Mansi C, Savarino V, Testa R. Platelet count/spleen diameter ratio: proposal and validation of a non-invasive parameter to predict the presence of oesophageal varices in patients with liver cirrhosis. Gut 2003; 52: 1200-1205 Giannini EG, Botta F, Borro P, Dulbecco P, Testa E, Mansi C, Savarino V, Testa R. Application of the platelet count/spleen diameter ratio to rule out the presence of oesophageal varices in patients with cirrhosis: a validation study based on followup. Dig Liver Dis 2005; 37: 779-785 Giannini EG, Zaman A, Kreil A, Floreani A, Dulbecco P, Testa E, Sohaey R, Verhey P, Peck-Radosavljevic M, Mansi C, Savarino V, Testa R. Platelet count/spleen diameter ratio for the noninvasive diagnosis of esophageal varices: results of a multicenter, prospective, validation study. Am J Gastroenterol 2006; 101: 2511-2519 Alempijevic T, Kovacevic N. Right liver lobe diameter: albumin ratio: a new non-invasive parameter for prediction of oesophageal varices in patients with liver cirrhosis (preliminary report). Gut 2007; 56: 1166-1167; authror reply 1167 S- Editor Liu Y L- Editor Ma JY E- Editor Yin DH

www.wjgnet.com



RAPID COMMUNICATION

Liver biopsy in a district general hospital: Changes over two decades Wing-Kin Syn, Caroline Bruckner-Holt, Adam Farmer, Sarah Howdle, Jeffrey Bateman Wing-Kin Syn, Liver and Hepatobiliary Unit, Queen Elizabeth Hospital, Birmingham, B15 2TH, United Kingdom Caroline Bruckner-Holt, Jeffrey Bateman, Department of Gastroenterology, Princess Royal Hospital, Telford, United Kingdom Adam Farmer, Department of Gastroenterology, City Hospital, Birmingham, United Kingdom Sarah Howdle, Department of Radiology, Princess Royal Hospital, Telford, United Kingdom Correspondence to: Dr. Wing-Kin Syn, Liver and Hepatobiliary Unit, Queen Elizabeth Hospital, Birmingham, B15 2TH, United Kingdom. [email protected] Telephone: +44-121-4721311-3414 Fax: +44-121-6272449 Received: June 20, 2007 Revised: August 17, 2007

Abstract AIM: To study liver biopsy practice over two decades in a district general hospital in the United Kingdom. METHODS: We identified all patients who had at least one liver biopsy between 1986 and 2006 from the databases of the radiology and gastroenterology departments. Subjects with incomplete clinical data were excluded from the study. RESULTS: A total of 103 liver biopsies were performed. Clinical data was available for 88 patients, with 95 biopsies. Between 1986 and 1996, 18 (95%) out of the 19 liver biopsies performed were blind and 6 (33%) were for primary biliary cirrhosis. Between 1996 and 2006, 14 (18%) out of 76 biopsies were blind; and the indications were abnormal liver tests (33%), hepatitis C (12%) and targeted-biopsies (11%). Liver biopsies were unhelpful in 5 (5%) subjects. Pain was the most common complication of liver biopsy (5%). No biopsy-related mortality was reported. There was a trend towards more technical failures and complications with the blind biopsy technique. CONCLUSION: Liver biopsies performed in small district hospitals are safe and useful for diagnostic and staging purposes. Abnormal liver tests, non-alcoholic fatty liver disease and targeted biopsies are increasingly common indications. Ultrasound-guided liver biopsies are now the preferred method and are associated with fewer complications. © 2007 WJG . All rights reserved.

Key words: Complication; District general; Indication; www.wjgnet.com

Liver biopsy; Non-alcoholic fatty liver Syn WK, Bruckner-Holt C, Farmer A, Howdle S, Bateman J. Liver biopsy in a district general hospital: Changes over two decades. World J Gastroenterol 2007; 13(40): 5336-5342


INTRODUCTION Since the first reported percutaneous liver biopsy in 1923, the liver biopsy has become widely used in the investigation of liver disease and is currently the gold standard for confirming the diagnosis and for staging of liver disease[1]. However, it remains an invasive procedure, with a mortality risk ranging between 0.01% and 0.17%[2]. Studies have shown that less than one-third of the biopsies altered the treatment[3]; hence biopsies should only be performed in patients who would benefit from this procedure. Over the last few decades, there has been a significant improvement in diagnostic and imaging techniques, as well as in the drug therapy of liver disease[1]. These have led to changes in our management of liver diseases. At the same time, changes in socio-economic status and life-style, have resulted in increased prevalence of obesity[4,5] and rising incidence of non-insulin diabetes mellitus[6,7]. Nonalcoholic steatohepatitis (NASH), the hepatic manifestation of the metabolic syndrome or insulin-resistant state [8], is now one of the most common indications for liver transplantation in the USA[9]. Consequently, the indications for liver biopsies in the UK will be expected to evolve over time. We reviewed all liver biopsies performed in a single district general hospital (DGH) in the UK over two decades. We examined the indications, findings and complications of liver biopsy and explored the changes in our practice over the last two decades.

MATERIALS AND METHODS Patients All patients who had a liver biopsy were identified from the databases of the radiology and gastroenterology departments. Relevant clinical and laboratory information was collected retrospectively. Patients whose clinical information was incomplete were excluded from the analysis.

Syn WK et al . Liver biopsy over two decades

5337

Table 1 Patient demographics and clinical features

Age (yr) Sex (F/M)

Median values (range) 57 (25-86) 51:37

Normal values

66.5 (11-1103) 13 (5-430) 221 (20-1394) 145 (44-994) 239 (93-813)

10-31 1-17 0-50 45-145 150-500

Liver biochemistry ALT (U/L) Bilirubin (μmol/L) GGT (U/L) ALP (U/L) Platelets (× 109/L) Indications for biopsy: Acute hepatitis Chronic hepatitis/Chronic elevated liver tests/Staging of disease Abnormal imaging Disease follow-up Routes of biopsy: Blind USS guided TJB Laparoscopic

5 75 8 3 32 (31) 60 (11) 2 1

ALT: Alanine aminotransferase; GGT: Gamma gluteryltransferase; ALP: Alkaline phosphatase; USS: Ultrasonography; TJB: Transjugular liver biopsy. 1 Failed.

Liver biopsy protocol In the absence of any contraindications, liver biopsies were performed when clinically indicated, as determined by the supervising clinician. The clinician performing the biopsy obtained an informed consent. A full blood count (FBC) and clotting profile were obtained one day prior to the biopsy to ensure that the platelet counts were > 80 000/mm3 and the international normalized ratio (INR) was < 1.3[1,2,10]. Appropriate platelet and clotting factors were given as necessary. An abdominal ultrasound of the liver was performed within six months of biopsy to ensure that there were no anatomical variants, biliary dilatation or focal or cystic lesions that may require a targeted biopsy[1,11]. The presence of moderate to severe ascites was considered a contraindication to percutaneous biopsy[1]. If considered safe and feasible, patients with massive ascites underwent paracentesis until completely dry, prior to the liver biopsy. All liver biopsies were performed either as a ‘blind’ procedure or under ultrasound (USS)-guidance, depending on the personal preference of the clinician or the availability of a radiologist. Patients were discharged after six hours if the procedure was uncomplicated, and if the patient was clinically stable and pain free. A transjugular liver biopsy (TJB) was perfor med in patients with contraindications to a percutaneous biopsy. Liver biochemistry, serology and immunology All patients had liver biochemistry (serum bilirubin, alanine aminotransferase (ALT), alkaline phosphatase (ALP), gamma gluteryltransferase (GGT)) taken at the time of the initial clinical assessment. The normal ranges of these tests were: INR, 1; platelets 150-500 × 109/L; bilirubin, 1-17 µmol/L; ALT, 10-31 U/L; ALP, 45-145 U/L; GGT

0-50 U/L. Serum aspartate transaminase (AST) is not part of the routine liver panel at our centre. A liver screen comprising of immunological tests (anti-nuclear antibodies, anti-mitochondrial antibodies, anti-smooth muscle antibodies) for autoimmune diseases, serological tests for hepatitis B and C viruses (hepatitis B surface antigen, antihepatitis C antibodies) and serum levels for metabolic disease (caeruloplasmin, alpha-1 antitrypsin, ferritin) was performed in all patients with abnormal liver biochemistry or abnormal USS. Patients with elevated ferritin levels had additional blood samples taken for haemochromatosis gene (C282Y/H63D) mutation analysis. Usefulness of liver biopsy In the present study, the usefulness of a liver biopsy was determined qualitatively by a) the success in establishing the diagnosis and/or b) any change (s) in the management based on the results of the liver biopsy

RESULTS Patient population A total of 103 liver biopsies were obtained between 1986 and 2006. Clinical data was available on 88 patients who had 95 biopsies. The number of successful liver biopsies was 91; 88 had one biopsy, 7 (4 failed 1st attempt; 3 followup biopsies) had two biopsies, while none of the subjects had > 2 biopsies. The median age of the patients was 57 (range, 25-86) years; 51 patients were female. 5 patients had acute hepatitis and 8 patients had focal lesions in the liver on abdominal imaging. The remaining patients underwent staging biopsies or diagnostic biopsies for chronic hepatitis or chronic elevations in liver function tests. Liver function tests, platelets and clotting profile The median INR was 1 (range 0.9-1.6). All patients had platelet levels > 80 000/mm3 (median 239; range 93-813). The median values of the liver tests were as follows: ALT 66.5 (range 11-1103) U/L, ALP 145 (range 44-994) U/L, GGT 221 (range 20-1394) U/L and bilirubin 13 (range 5-430) µmol/L (Table 1). Liver biopsy Between 1986 and 1996: Number of patients and approach used: Eighteen patients underwent 19 biopsies. One biopsy was USS-guided and 18 were ‘blind’ biopsies (94.7%). One patient needed a repeat biopsy because of inadequate sample (blind) at the first attempt. The median size of liver biopsies was 1.5 cm (range: 0.5-2.5) (Table 2). Liver biopsy findings: One-third of biopsies were performed for primary biliary cirrhosis (PBC). Two patients had elevated serum ferritin levels but only one was homozygous for the C282Y haemochromatosis mutation. Liver biopsy of the second patient showed features of porphyria cutanea tarda that was confirmed on faecal and plasma porphyrin tests. Between 1996 and 2006: Number of patients and approach used: A total of 70 patients underwent 76 liver www.wjgnet.com

5338

ISSN 1007-9327

CN 14-1219/R


October 28, 2007

Volume 13

Number 40

Table 2 Liver biopsies between 1986 and 1995 Number of successful liver biopsies 6 3 1 3 3 1

1

Diagnosis pre-biopsy PBC AIH LKM hepatitis ALD

History Abnormal liver tests Y Y N Y N Y

Viral serology N N N

Autoimmune / Immunoglobulins Y Y Y LKM + AMA N

Imaging

Others

Final Diagnosis

Normal Normal Spleen +

n/a n/a n/a

Normal/Spleen +

n/a

PBC AIH: 3 (staging) Possible overlap AIH/PBC ALD: 3 (cirrhosis in 1) NAFLD (simple steatosis): 3 Porphyria cutanea tarda (plasma porphyrin 618, faecal porphyrin +) Primary haemochromatosis

Y

Y

N

Abnormal Liver tests ALD/Iron overload

N

Y

N

N

Echogenic liver

Elevated ferritin

Y

Y

N

N

Normal

Elevated ferritin

Iron overload

N

Y

N

N

Normal

Elevated ferritin and homozygous C282Y

Total number of biopsies performed: 19; Failed biopsies: 1; Successful biopsies: 18. Y: Present; N: Negative or not present; n/a: Not applicable; PBC: Primary biliary cirrhosis; AIH: Autoimmune hepatitis; ALD: Alcoholic liver disease; NAFLD: Non-alcoholic fatty liver disease; LKM: Liver, kidney, microsomal antibody; AMA: Anti-mitochondrial antibody.

biopsies; 59 biopsies were USS-guided and 14 (18.4%) were ‘blind’ procedures. Two patients had contraindications to a percutaneous biopsy (one had large volume ascites with raised INR and one had elevated INR), and underwent TJB. Laparoscopic liver biopsy was performed in one patient during laparoscopy performed for abdominal pain. Three patients needed a second biopsy after a failed initial attempt (2 blind, 1 USS-guided) while a further 3 had follow-up biopsies. The median size of liver biopsies was 1.7 (range, 0.1-3) cm (Table 3). Liver biopsy findings: One-third of the biopsies were performed for undiagnosed elevation in the liver function tests and 12% were for staging of hepatitis C infection. Out of 59 USS-guided biopsies, 8 were performed in patients with focal lesions seen on abdominal imaging. Seven out of eight patients were found to have metastatic lesions while one patient had macrovesicular steatosis. Changes in indications and/or findings over two decades Between 1996 and 2006, 26% of patients were diagnosed with non-alcoholic fatty liver disease (NAFLD) (including non-alcoholic steatohepatitis), compared with 16.7% between 1986 and 1995. In the last ten years, targeted biopsies were also more common (11%; Table 3) but only one patient underwent biopsy for PBC. Usefulness of liver biopsy Diagnosis: In patients with elevated liver function tests but non-specific liver screen, liver biopsy confirmed the diagnoses in 25 out of 27 patients. It provided histological confirmation of metastases in 7 patients and was useful in confirming the overlap syndrome (autoimmune hepatitis/ PBC) in 3 patients. Non-specific features were seen in 5 patients (5%). Staging disease and treatment: Biopsy findings were used to stage autoimmune hepatitis, haemochromatosis, hepatitis B, hepatitis C, alcoholic liver disease and NAFLD. www.wjgnet.com

Those with moderate to severe fibrosis and cirrhosis at liver histology were placed on six-monthly hepatocellular carcinoma USS screening programme and had an upper gastrointestinal endoscopy to assess the presence and size of varices. Six patients with NASH were followed on a regimen of exercise and dietary changes, with tight glycaemic and blood pressure control. Nine patients with hepatitis C infections were treated with pegylated interferon and ribavarin. Complications: Five patients experienced severe pain, requiring hospital admission and treatment with opiates. One patient developed a sub-capsular haematoma (blind biopsy); one patient developed pneumothorax (blind biopsy) that was managed conservatively; and another patient had an intra-abdominal bleed during TJB, requiring admission to the intensive care unit. There was no biopsyrelated mortality.

DISCUSSION In the present study, we observed an increasing number of liver biopsies being performed for elevated liver function tests. NAFLD was the most common diagnosis (70%) in these patients. When all the biopsies were taken in account, NAFLD accounted for nearly 30% of biopsies in our study population. These findings are consistent with the results of previous studies[12-14], and likely reflect that an increasing proportion of our population suffers from obesity[15-18] and non-insulin dependent diabetes mellitus[19,20]. Although the diagnosis of NAFLD can often be made clinically in patients with a combination of elevated liver tests, negative liver screen, ‘bright’ liver on abdominal USS and the presence of metabolic risk factors such as hypertension, non-insulin dependent diabetes mellitus, hyperlipidaemia and obesity[16,21,22], the distinction between simple steatosis and steatohepatitis can only be made by liver biopsy[23,24]. This is important because simple steatosis is considered a benign condition, while steatohepatitis can progress to


5339

Table 3 Liver biopsies between 1996 and 2006 Number of successful liver biopsies 24

Diagnosis pre-biopsy

History

Abnormal liver tests

Viral serology

Autoimmune/ Immunoglobulins

Imaging

Others

Final Diagnosis

Abnormal liver tests

Non-specific Abdominal pains in 2

Y

N

SMA in 1 IgG in 1

Normal/ Echogenic

n/a

Simple steatosis: 12 NASH: 4 Autoimmune (including overlap): 2 Drug-related: 2 Microharmatomas: 1 (laparoscopy) Non-specific: 2 Venous congestion: 1

6

NAFLD

Metabolic features present

Y

N

IgA elevated in 2

Echogenic 3

n/a

NASH: 1 Simple steatosis: 4 Normal: 1

3

Alcohol

Strong alcohol history

Y

N

Elevated IgG in 1 Autoimmune normal

Normal 2 Ascites 1

n/a

ALD: 1 (staging) ALD: 1 (diagnosis; transjugular) AIH: 1

6

Iron overload

N

Y in 2

N

N

Echogenic 3

Elevated Ferritin in all

C282Y/C282Y: 4 C282Y/H63D: 1 NAFLD (simple steatosis): 1

2

Alcohol/Iron overload?

Strong alcohol history

Y

N

Elevated IgA

Echogenic 2

Elevated Ferritin in 2

C282Y/H63D + alcohol features: 2

8

Liver lesions on imaging

N

Y in 2

N

N

Multiple lesions noted on US/CT

n/a

Lung primary: 3 GIST: 1 GI tract: 2 Pancreas: 1 NASH: 1

9

Hepatitis C

Y

Y

PCR positive

N

Normal

n/a

Hepatitis C (staging) Genotype 1:6 Genotype 3:3

1

Hepatitis B

Y

Normal

Y

N

Normal

n/a

Hepatitis B (staging)

1

PBC

N

Y

N

Elevated IgM AMA: 1: 640

Normal

n/a

PBC

8

AIH

N

Y

N

AIH: ANA + SMA in 3 Isolated SMA in 1 LKM + AMA in 1 Non-specific: Isolated SMA in 2 ANA only in 1

Normal (7); Splenomegaly in 1

n/a

AIH: 3 (staging) (transjugular in 1) AIH: 1 (diagnosis) Possible AIH/PBC overlap: 1 Non-specific: 3

2

Drug induced

Y

Y

N

Polyclonal increase in Ig

Normal

n/a

Drug-cholestasis: 1 NAFLD(simple steatosis): 1

3

Miscellaneous

Y

Y in PUO

N

N

Normal

n/a

PUO: 1 Methotrexate: 2 (staging)

Total number of biopsies: 76; Failed biopsies: 3; Number of successful biopsies: 73. Y: Present; N: negative or not present; n/a: Not applicable. PBC: Primary biliary cirrhosis; PUO: Pyrexia of unknown origin; AIH: Autoimmune hepatitis; ALD: Alcoholic liver disease; NAFLD: Non-alcoholic fatty liver disease. NASH: Non-alcoholic steatohepatitis; LKM: Liver, kidney, microsomal antibody; SMA: Smooth muscle antibody. ANA: Anti-nuclear antibody; AMA: Antimitochondrial antibody; IgG: Immunoglobulin G; IgA: Immunoglobulin A; IgM: Immunoglobulin M; US: Ultrasound; CT: Computed tomography.

cirrhosis[25]. Aggressive control of metabolic risk factors has been shown to improve the liver function tests and liver histology[26-31]. Although it is not feasible to biopsy all patients with possible NAFLD, this should be considered

in patients who are older (over 40 years), obese or have non-insulin dependent diabetes[18,32]. With improvements in chemo-radiotherapy regimens, many patients with metastatic diseases are offered neowww.wjgnet.com

5340

ISSN 1007-9327

CN 14-1219/R


Table 4 Liver biopsies for acute hepatitis Acute hepatitis 1

Pre-biopsy diagnosis Cause unknown

2 3 4 5

Cause unknown Pyrexia of unknown origin Cause unknown Cause unknown

Final diagnosis Likely drug-induced; prednisolone started Mild steatohepatitis Tuberculosis excluded No specific features Microharmatomas with cholestatic hepatitis

adjuvant and adjuvant therapy. Hepatic resection for colorectal metastases limited to the liver, has become the standard of care [33,34] . Imaging guided biopsy of liver lesions is being performed increasingly to confirm metastases and to determine the primary tumor. By contrast, a liver biopsy is no longer necessary to make a diagnosis of PBC. A persistently elevated E2AMA strongly suggests PBC, even in asymptomatic patients[35]. Moreover, Cox-regression analyses have shown that the main prognostic marker in PBC is serum bilirubin[36]; thus indicating that the presence of fibrosis or cirrhosis in PBC is of limited prognostic use. Liver biopsies are currently recommended in patients with chronic hepatitis C before treatment to stage and grade the disease[37,38]. Six patients with genotype 1 disease and three with genotype 3 disease received combination treatment with pegylated interferon and ribavarin. Since over 75% of patients infected with genotype 3 hepatitis C have a sustained virologic response after six months of combination treatment with pegylated interferon and ribavarin[39]; a liver biopsy may be unnecessary in some of these patients. In the future, non-invasive fibrosis markers are likely to play a bigger role in staging liver disease. We found that in patients with isolated autoantibodies or complex clinical and laboratory features, a liver biopsy was able to confirm the disease processes. For example, in a patient with excessive alcohol consumption who also had elevated immunoglobulin levels, liver biopsy indicated a diagnosis of autoimmune hepatitis instead of alcoholic hepatitis. Liver biopsies from five patients showed non-specific features (5%). It has been suggested that for evaluation of diffuse liver disease, a core of at least 1.5cm is required[40] as it provides at least 6 to 8 portal tracts for adequate histological assessment. Although the median biopsy size in the study was 1.5 cm, many samples were inadequate, with the smallest recorded size being 0.1 cm. Three of these patients had biopsy fragments measuring less than 1cm in size, which may account for the nonspecific findings. Liver biopsies are generally not required in patients with acute hepatitis[1]. We however, found it helpful in three patients (Table 4). The most common complication of liver biopsy is abdominal discomfort. Hospitalization is needed in up to 3% of patients for pain and hypotension[41,42], but major complications are rare[43]. At our centre, five patients (5.6% of patients, 5.2% of biopsies) were admitted for severe pain while another patient developed clinically significant intraperitoneal bleed after a TJB and required intensive care treatment. Consistent with published reports[44,45], www.wjgnet.com

October 28, 2007

Volume 13

Number 40

we found a trend towards greater technical failures and complications with the ‘blind’ biopsy technique. Three out of four patients who needed a second liver biopsy had their initial biopsies performed ‘blind’; two other patients, one with a pneumothorax and the second with sub-capsular haematoma also had ‘blind’ biopsies. In the last decade, nearly 78% of all biopsies in our unit were USS-guided. This increase is likely to be related to the increasing availability and safety of USS-guided liver biopsy[46,47]. Although our study population was small, it is reflective of the current DGH practice in the UK. Post-liver transplant, protocol liver biopsies are performed at the tertiary transplant units, hence the absence such data in the present study. In conclusion, our findings confirm that liver biopsies in the DGH are safe and useful in the evaluation and staging of liver diseases. USS-guided rather than ‘blind’ liver biopsies are likely to be the preferred technique by patients and clinicians. NAFLD rather than PBC or viral hepatitis, will increasingly constitute the majority share of the liver biopsy workload.

COMMENTS Background

Liver biopsy is a widely used tool in the investigation of liver diseases. However, it is an invasive procedure and studies have shown that less than one-third of the biopsies actually alter the management of patients. A liver biopsy should therefore only be performed in those patients who would benefit from such a procedure. Over the last two decades, changes in the socio-economic status and life-style have led to alterations in disease profiles. There is an increasing burden of obesity, type Ⅱ diabetes mellitus and non-alcoholic steatohepatitis. Non-alcoholic steatohepatitis is now one of the most common causes of chronic liver disease and indications for liver transplantation in the USA. Concurrently, there have been rapid improvements in diagnostic and treatment options for patients with liver disease. With such changes, we expect that indications for liver biopsy will also evolve over time.

Research frontiers

Between 1986 and 1996, liver biopsies were performed mainly in patients with primary biliary cirrhosis as part of the staging process. With the development of reliable immunological markers designed to confirm the diagnosis, and prognostic markers to predict outcome of primary biliary cirrhosis, liver biopsy is no longer necessary. Between 1996 and 2006, the majority of biopsies (> 40%) were performed for raised liver function tests and hepatitis C staging. Non-alcoholic fatty liver disease was also found to be more common. In the present study, only 5% of biopsies showed non-specific features and were therefore unhelpful in patient management. The rest of the biopsies confirmed the diagnoses of the primary liver disease or the presence of metastasis. Patients with end-stage liver disease were placed on regular surveillance and those with treatable diseases were managed according to the treatment protocols. The complication rates after a liver biopsy in a district general hospital were found to be similar to the published rates. Between 5% and 6% of patients were admitted for severe pain. There was a trend towards increasing technical failures and complications with blind liver biopsy. In the last decade, ultrasound-guided biopsies were obtained more frequently than blind biopsies.


This study confirms that a liver biopsy is safe and useful in evaluating and staging of liver disease, even in district general hospitals. The risks from a biopsy remains small, but ultrasound-guided biopsies should be the preferred technique. In the future, non-alcoholic fatty liver disease will account for the vast share of the biopsy workload.

Applications

Useful for practicing clinicians in smaller hospitals as it confirms the safety and


5341

utility of a liver biopsy. Ultrasound-guidance may be safer. Non-alcoholic fatty liver disease is increasingly common.

20

Peer review

21

This is a very nice review that describes the historical changes in liver biopsy methodology over the past 20 years. The data presented is clear and concise and has been described clearly in the text.

22 23

REFERENCES Grant A, Neuberger J. Guidelines on the use of liver biopsy in clinical practice. British Society of Gastroenterology. Gut 1999; 45 Suppl 4: IV1-IV11 Sherlock S, Dooley J. Chapter 3: Biopsy of the Liver. In: 2 Sherlock S, Dooley J. Diseases of the liver and biliary system. 11th ed. Oxford, UK: Blackwell Science Ltd, 2002: 37-44 3 Sorbi D, McGill DB, Thistle JL, Therneau TM, Henry J, Lindor KD. An assessment of the role of liver biopsies in asymptomatic patients with chronic liver test abnormalities. Am J Gastroenterol 2000; 95: 3206-3210 Office for National Statistics. Obesity among adults: by sex 4 and NS-SeC, 2001: Social Trends 34. Available from: URL: http://www.statistics.gov.uk Livingstone MB. Childhood obesity in Europe: a growing 5 concern. Public Health Nutr 2001; 4: 109-116 6 Lipscombe LL, Hux JE. Trends in diabetes prevalence, incidence, and mortality in Ontario, Canada 1995-2005: a population-based study. Lancet 2007; 369: 750-756 Ehtisham S, Barrett TG. The emergence of type 2 diabetes in 7 childhood. Ann Clin Biochem 2004; 41: 10-16 8 Marchesini G, Bugianesi E, Forlani G, Cerrelli F, Lenzi M, Manini R, Natale S, Vanni E, Villanova N, Melchionda N, Rizzetto M. Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome. Hepatology 2003; 37: 917-923 Clark JM, Diehl AM. Nonalcoholic fatty liver disease: an 9 underrecognized cause of cryptogenic cirrhosis. JAMA 2003; 289: 3000-3004 10 Gilmore IT, Burroughs A, Murray-Lyon IM, Williams R, Jenkins D, Hopkins A. Indications, methods, and outcomes of percutaneous liver biopsy in England and Wales: an audit by the British Society of Gastroenterology and the Royal College of Physicians of London. Gut 1995; 36: 437-441 11 Dixon AK, Nunez DJ, Bradley JR, Seymour CA. Failure of percutaneous liver biopsy: anatomical variation. Lancet 1987; 2: 437-439 12 Mathiesen UL, Franzen LE, Fryden A, Foberg U, Bodemar G. The clinical significance of slightly to moderately increased liver transaminase values in asymptomatic patients. Scand J Gastroenterol 1999; 34: 85-91 13 D a n i e l S , B e n - M e n a c h e m T , V a s u d e v a n G , M a C K , Blumenkehl M. Prospective evaluation of unexplained chronic liver transaminase abnormalities in asymptomatic and symptomatic patients. Am J Gastroenterol 1999; 94: 3010-3014 14 Skelly MM, James PD, Ryder SD. Findings on liver biopsy to investigate abnormal liver function tests in the absence of diagnostic serology. J Hepatol 2001; 35: 195-199 15 Ludwig J, Viggiano TR, McGill DB, Oh BJ. Nonalcoholic steatohepatitis: Mayo Clinic experiences with a hitherto unnamed disease. Mayo Clin Proc 1980; 55: 434-438 16 Powell EE, Cooksley WG, Hanson R, Searle J, Halliday JW, Powell LW. The natural history of nonalcoholic steatohepatitis: a follow-up study of forty-two patients for up to 21 years. Hepatology 1990; 11: 74-80 17 Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCullough AJ. Nonalcoholic fatty liver disease: a spectrum of clinical and pathological severity. Gastroenterology 1999; 116: 1413-1419 18 Angulo P, Keach JC, Batts KP, Lindor KD. Independent predictors of liver fibrosis in patients with nonalcoholic steatohepatitis. Hepatology 1999; 30: 1356-1362 19 Creutzfeldt W, Frerichs H, Sickinger K. Liver diseases and diabetes mellitus. Prog Liver Dis 1970; 3: 371-407

24

1

25

26 27

28 29 30

31

32

33 34 35 36 37

38

39

de Marco R, Locatelli F, Zoppini G, Verlato G, Bonora E, Muggeo M. Cause-specific mortality in type 2 diabetes. The Verona Diabetes Study. Diabetes Care 1999; 22: 756-761 Chitturi S, Farrell GC. Etiopathogenesis of nonalcoholic steatohepatitis. Semin Liver Dis 2001; 21: 27-41 Bacon BR, Farahvash MJ, Janney CG, Neuschwander-Tetri BA. Nonalcoholic steatohepatitis: an expanded clinical entity. Gastroenterology 1994; 107: 1103-1109 Bianchi L. Liver biopsy in elevated liver functions tests? An old question revisited. J Hepatol 2001; 35: 290-294 Saadeh S, Younossi ZM, Remer EM, Gramlich T, Ong JP, Hurley M, Mullen KD, Cooper JN, Sheridan MJ. The utility of radiological imaging in nonalcoholic fatty liver disease. Gastroenterology 2002; 123: 745-750 Caldwell SH, Hylton AI. The clinical outcome of NAFLD including cryptogenic cirrhosis. In: Farrell GC, George J, de la M. Hall P, McCullough AJ. Fatty Liver Disease: NASH and Related Disorders. Oxford, UK: Blackwell Publishing Ltd, 2005: 168-180 Akyüz F, Demir K, Ozdil S et al. The Effects of Rosiglitazone, Metformin, and Diet with Exercise in Nonalcoholic Fatty Liver Disease. Dig Dis Sci 2007; Epub ahead of print Yamamoto M, Iwasa M, Iwata K, Kaito M, Sugimoto R, Urawa N, Mifuji R, Konishi M, Kobayashi Y, Adachi Y. Restriction of dietary calories, fat and iron improves non-alcoholic fatty liver disease. J Gastroenterol Hepatol 2007; 22: 498-503 Portincasa P, Grattagliano I, Palmieri VO, Palasciano G. Current pharmacological treatment of nonalcoholic fatty liver. Curr Med Chem 2006; 13: 2889-2900 Lavoie JM, Gauthier MS. Regulation of fat metabolism in the liver: link to non-alcoholic hepatic steatosis and impact of physical exercise. Cell Mol Life Sci 2006; 63: 1393-1409 Ekstedt M, Franzen LE, Mathiesen UL, Holmqvist M, Bodemar G, Kechagias S. Statins in non-alcoholic fatty liver disease and chronically elevated liver enzymes: a histopathological followup study. J Hepatol 2007; 47: 135-141 Belfort R, Harrison SA, Brown K, Darland C, Finch J, Hardies J, Balas B, Gastaldelli A, Tio F, Pulcini J, Berria R, Ma JZ, Dwivedi S, Havranek R, Fincke C, DeFronzo R, Bannayan GA, Schenker S, Cusi K. A placebo-controlled trial of pioglitazone in subjects with nonalcoholic steatohepatitis. N Engl J Med 2006; 355: 2297-2307 Ratziu V, Giral P, Charlotte F, Bruckert E, Thibault V, Theodorou I, Khalil L, Turpin G, Opolon P, Poynard T. Liver fibrosis in overweight patients. Gastroenterology 2000; 118: 1117-1123 Welsh FK, Tilney HS, Tekkis PP, John TG, Rees M. Safe liver resection following chemotherapy for colorectal metastases is a matter of timing. Br J Cancer 2007; 96: 1037-1042 Cummings LC, Payes JD, Cooper GS. Survival after hepatic resection in metastatic colorectal cancer: a population-based study. Cancer 2007; 109: 718-726 Metcalf JV, Mitchison HC, Palmer JM, Jones DE, Bassendine MF, James OF. Natural history of early primary biliary cirrhosis. Lancet 1996; 348: 1399-1402 Neuberger J. Primary biliary cirrhosis. In: O’Grady JG, Lake JR, Howdle PD, editors. Comprehensive Clinical Hepatology. Mosby, London: Harcourt Publishers Limited, 2000: 17.1-17.13 National Institute for Health and Clinical Excellence. Peginterferon alfa and ribavirin for the treatment of mild chronic hepatitis C. Technology appraisal 2006 (106). Available from: URL: http://www.nice.org.uk/TA106 National Institute for Health and Clinical Excellence. Interferon alfa (pegylated and non-pegylated) and ribavirin for the treatment of chronic hepatitis C. Technology appraisal 2004 (75). Available from: URL: http://www.nice.org.uk/ TA75 Brandao C, Barone A, Carrilho F, Silva A, Patelli M, Caramori C, Focaccia R, Pereira L, Pedroso M, Tatsch F, Pessoa M. The results of a randomized trial looking at 24 weeks vs 48 weeks of treatment with peginterferon alpha-2a (40 kDa) and ribavirin combination therapy in patients with chronic hepatitis C genotype 1. J Viral Hepat 2006; 13: 552-559 www.wjgnet.com

5342

ISSN 1007-9327

CN 14-1219/R


Bravo AA, Sheth SG, Chopra S. Liver biopsy. N Engl J Med 2001; 344: 495-500 41 Garcia-Tsao G, Boyer JL. Outpatient liver biopsy: how safe is it? Ann Intern Med 1993; 118: 150-153 42 Janes CH, Lindor KD. Outcome of patients hospitalized for complications after outpatient liver biopsy. Ann Intern Med 1993; 118: 96-98 43 Piccinino F, Sagnelli E, Pasquale G, Giusti G. Complications following percutaneous liver biopsy. A multicentre retrospective study on 68,276 biopsies. J Hepatol 1986; 2: 165-173 44 Farrell RJ, Smiddy PF, Pilkington RM, Tobin AA, Mooney EE, Temperley IJ, McDonald GS, Bowmer HA, Wilson GF, Kelleher D. Guided versus blind liver biopsy for chronic 40

45

46 47

October 28, 2007

Volume 13

Number 40

hepatitis C: clinical benefits and costs. J Hepatol 1999; 30: 580-587 Nobili V, Comparcola D, Sartorelli MR, Natali G, Monti L, Falappa P, Marcellini M. Blind and ultrasound-guided percutaneous liver biopsy in children. Pediatr Radiol 2003; 33: 772-775 Vautier G, Scott B, Jenkins D. Liver biopsy: blind or guided? BMJ 1994; 309: 1455-1456 Caturelli E, Giacobbe A, Facciorusso D, Bisceglia M, Villani MR, Siena DA, Fusilli S, Squillante MM, Andriulli A. Percutaneous biopsy in diffuse liver disease: increasing diagnostic yield and decreasing complication rate by routine ultrasound assessment of puncture site. Am J Gastroenterol 1996; 91: 1318-1321 S- Editor Zhu LH L- Editor Anand BS E- Editor Yin DH

www.wjgnet.com



RAPID COMMUNICATION

Effects of a 24-week course of interferon-a therapy after curative treatment of hepatitis C virus-associated hepatocellular carcinoma Soo Cheol Jeong, Hiroshi Aikata, Yoshio Katamura, Takahiro Azakami, Tomokazu Kawaoka, Hiromi Saneto, Kiminori Uka, Nami Mori, Shintaro Takaki, Hideaki Kodama, Koji Waki, Michio Imamura, Hiroo Shirakawa, Yoshiiku Kawakami, Shoichi Takahashi, Kazuaki Chayama Soo Cheol Jeong, Hiroshi Aikata, Yoshio Katamura, Takahiro Azakami, Tomokazu Kawaoka, Hiromi Saneto, Kiminori Uka, Nami Mori, Shintaro Takaki, Hideaki Kodama, Koji Waki, Michio Imamura, Hiroo Shirakawa, Yoshiiku Kawakami, Shoichi Takahashi, Kazuaki Chayama, Department of Medicine and Molecular Science, Division of Frontier Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan Correspondence to: Hiroshi Aikata, MD, Department of Medicine and Molecular Science, Division of Frontier Medical Science, Programs for Biomedical Research, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3, Kasumi, Minami-ku, Hiroshima 734-8551, Japan. [email protected] Telephone: +81-82-2575192 Fax: +81-82-2575194 Received: June 6, 2007 Revised: August 8, 2007

Abstract AIM: To assess whether a 24-wk course of interferon (IFN) could prevent hepatocellular carcinoma (HCC) recurrence and worsening of liver function in patients with hepatitis C virus (HCV)-infected patients after receiving curative treatment for primary HCC. METHODS: Outcomes in 42 patients with HCV infection treated with IFN-a, after curative treatment for primary HCC (IFN group), were compared with 42 matched curatively treated historical controls not given IFN (nonIFN group). RESULTS: Although the rate of initial recurrence did not differ significantly between IFN group and non-IFN group (0%, 44%, 61%, and 67% vs 4.8%, 53%, 81%, and 87% at 1, 3, 5, and 7 years, P = 0.153, respectively), IFN group showed a lower rate than the non-IFN group for second recurrence (0%, 10.4%, 28%, and 35% vs 0%, 30%, 59%, and 66% at 1, 3, 5 and 7 years, P = 0.022, respectively). Among the IFN group, patients with sustained virologic response (SVR) were less likely to have a second HCC recurrence than IFN patients without an SVR, or non-IFN patients. Multivariate analysis identified the lack of SVR as the only independent risk factor for a second recurrence, while SVR and Child-Pugh class A independently favored overall survival. CONCLUSION: Most intrahepatic recurrences of HCV-

related HCC occurred during persistent viral infection. Eradication of HCV is essential for the prevention of HCC recurrence and improvement of survival. © 2007 WJG . All rights reserved.

Key words: Hepatitis C virus, Hepatocellular carcinoma, Recurrence, Survival, Sustained virological response Jeong SC, Aikata H, Katamura Y, Azakami T, Kawaoka T, Saneto H, Uka K, Mori N, Takaki S, Kodama H, Waki K, Imamura M, Shirakawa H, Kawakami Y, Takahashi S, Chayama K. Effects of a 24-week course of interferon-a therapy after curative treatment of hepatitis C virusassociated hepatocellular carcinoma. World J Gastroenterol 2007; 13(40): 5343-5350


INTRODUCTION Hepatocellular carcinoma (HCC) is one of the most common malignant neoplasms worldwide. Chronic infection with hepatitis C virus (HCV) has been causally associated with HCC [1-3]. Recent advances in imaging and treatment have brought about some improvement in prognosis of patients with HCV-related HCC, but outcomes are still unsatisfactory. The 5-year survival rate is only 50%-70%, even after curative treatment such as hepatic resection or local ablation [4]. Reasons for this unfavorable prognosis are considered to include high intrahepatic tumor recurrence rates and sustained hepatic damage, both resulting from HCV infection[5]. Even after curative hepatic resection for HCV-related HCC, the rate of intrahepatic tumor recurrence within 1 year is 20%-40%, rising to about 80% by 5 years[4,6-8]. Intrahepatic recur rence of HCC may result from intrahepatic metastasis originating from the primary HCC, or from ongoing multicentric carcinogenesis related to chronic HCV infection. Underlying HCV-related hepatic damage may also compromise hepatic functional reserve, and worsen clinical outcome. Thus prevention of HCC recurrence and preservation of liver function are both high priorities for improving prognosis of patients with HCVwww.wjgnet.com

5344

ISSN 1007-9327

CN 14-1219/R


related HCC. Interferon (IFN) therapy for patients with HCV i n f e c t i o n i s e f f e c t ive i n r e d u c i n g s e r u m a l a n i n e transaminase (ALT) activity and in eradicating HCV[9,10], and thus IFN could have value in minimizing hepatic necrosis, inflammation and fibrosis, as well as reducing the incidence of HCC. Several recent studies have reported that IFN therapy, even after curative treatment for HCVrelated HCC, could prevent HCC recurrence and improve sur vival [11-17] . Unfortunately, since these studies are characterized by differing IFN regimens, definitions of IFN responses, and background characteristics of patients, results have varied and no standard IFN regimen has been established for after curative treatment of HCV-related HCC. As well, the mechanisms by which IFN suppresses HCC recurrence, including possible direct anti-tumor and anti-inflammatory effects, remain uncertain. In the present study, recurrence and survival outcomes in matched historical controls were compared with those in patients receiving a 24-wk course of IFN-a therapy after receiving curative treatment for HCC.

MATERIALS AND METHODS Patients We retrospectively reviewed 495 consecutive patients treated for primary HCC associated with HCV infection at Hiroshima University Hospital from March 1992 to March 2004. Of these, 384 with HCC initially underwent therapeutic intervention with curative intent. Curative treatment was defined as complete tumor eradication, with no residual tumor visible by computed tomography, or resection of all evident tumor tissue. Medical treatment included percutaneous radiofrequency ablation (RFA), ethanol injection, and microwave coagulation therapy (MCT). Surgical treatment included hepatic resection and ablation during laparotomy. Among these 384 patients, we administered IFN therapy to 42 who met the following eligibility criteria: age under 70 years; up to three tumors with none exceeding 30 mm in diameter, or a solitary tumor less than 50 mm in diameter; tumor-node-metastasis (TNM) stageⅠ, Ⅱ, or Ⅲ; detectable serum HCV RNA; seronegativity for hepatitis B surface antigen; chronic hepatitis or compensated cirrhosis with a Child-Pugh class of A or B; platelet count above 70 000/μL; absence of local recurrence during the follow-up period; and absence of ectopic intrahepatic recurrence within 24 wk after treatment for primary HCC. We used the TNM classification system of the Liver Cancer Study Group of Japan as the staging system for HCC[18]. Underlying liver conditions such as hepatitis or cirrhosis were confirmed by laboratory, pathologic and radiologic examinations. We classified liver function in chronic hepatitis as Child class A because chronic hepatitis is a known pre-cirrhotic condition. There were only a few chronic hepatitis cases: three in the IFN group and four in the non-IFN group. As historical control subjects, we selected 42 patients with no IFN therapy after treatment for primary HCC (non-IFN group). These 42 patients, who met the eligibility

www.wjgnet.com

October 28, 2007

Volume 13

Number 40

Table 1 Patient characteristics

Median age in years (range) Gender (male/female) Alb (g/dL) PLT (× 10 000/μL) ICG R-15 (%) CH or Child A/B Size of main tumor (mm) AFP (ng/mL) No. of HCC (single/two or three) Stage (Ⅰ/Ⅱ or Ⅲ) Treatment of HCC (medical/surgical)

IFN group Non-IFN group (n = 42) (n = 42) 621 (45-69) 631 (40-69) 36/6 29/13 3.91 3.91 1 12 11.51 171 181 35/7 35/7 201 (10-50) 151 (10-50) 1 26 31.41 30/12 36/6 14/28 23/19 18/24 20/22

P NS NS NS NS NS NS NS NS NS NS NS

IFN: interferon; Alb: albumin; PLT: platelet; ICG-R15: indocyanine green retention at 15 min; CH: chronic hepatitis; AFP: alpha-fetoprotein; HCC: hepatocellular carcinoma. 1Median.

criteria noted above, were matched by age, gender, tumor size, TNM stage of HCC, serum albumin, platelet counts, and Child-Pugh class with patients who received IFN therapy (IFN group). Thus, a total of 84 patients (42 in the IFN group and 42 in the non-IFN group) were enrolled. All agreed to participate in the research protocol, which was approved by the hospital research ethics board. Table 1 shows the baseline characteristics of the two groups, indicating no significant differences for age, gender, liver function, tumor characteristics, or therapeutic methods used against HCC. IFN therapy In the IFN group, patients received 6 MIU of natural IFN- a (human lymphoblastoid IFN, Sumiferon; Dainippon Sumitomo Pharmaceuticals, Osaka, Japan) intramuscularly every day for 2 wk, followed by three times weekly for 22 wk. IFN therapy began within 24 wk after the initial treatment for HCC. All patients were evaluated every week in an outpatient setting during IFN treatment. Qualitative detection of HCV-RNA was performed by a standardized qualitative reverse transcription-polymerase chain reaction (RT-PCR) assay at every 4 wk during and after IFN treatment. Among the patients who received IFN therapy, 28 were of HCV genotype 1 and 14 were of HCV genotype 2. These 42 patients had various pretreatment viral loads. Twenty patients (genotype 1, n = 11; genotype 2, n = 9) had high viral loads (≥ 100 kIU/mL by PCR), and 22 (genotype 1, n = 17; genotype 2, n = 5) had low viral loads (≤ 100 kIU/mL by PCR). The 42 patients were divided into two subgroups according to virologic response, i.e. patients with vs without a sustained virologic response (SVR). SVR was defined as the sustained absence of serum HCV RNA for more than 24 wk after completion of IFN treatment. Absence of SVR included both persistent viremia (no response) and transient viral disappearance (transient response) during or after IFN therapy. Biochemical response was defined as ALT activity declining to a value within the normal reference range in the presence of viremia.

Jeong SC et al . Interferon after curative HCC treatment

Follow-up After curative treatment for primary HCC, all patients studied underwent liver function tests, serum tumor marker assays, such as those for α-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist Ⅱ (PIVKAⅡ) every month, abdominal ultrasonography every 3 mo, and dynamic computed tomography (CT) every 6 mo. If recurrence of HCC was suspected, additional examinations including CT during arteriography or tumor biopsy were performed. Recurrence of HCC was defined as any new nodules indicated by CT as hyperattenuation during hepatic arteriography or by hypoattenuation in CT performed during arterioportography. Hypovascular HCC was confirmed histopathologically after fine-needle aspiration biopsy. Patients with recurrent HCC were treated medically or surgically, with curative intent if possible. In IFN patients, including those with or without SVR, and in the non-IFN group, we compared both the rate of HCC recurrence and the survival rate. We also sought to identify significant prognostic indicators for survival and recurrence after curative treatment of primary HCC. Statistical analysis Chi-squared and Fisher exact tests were used for categorical variables, while Student’s t test and the MannWhitney U test were used for continuous and ordinal variables, as appropriate. The Kaplan-Meier method was used to assess cumulative survival and recurrence rates, calculated from the date of diagnosis to the date of disease recurrence or death. Surviving patients and those who died of causes unrelated to the liver were defined as censored cases, while patients who died of causes related to the liver were defined as non-censored cases. The log-rank test was used to compare survival and recurrence curves. Univariate and multivariate predictors of survival or recurrence time were determined using the Cox proportional hazard model. Hazard ratios and their 95% confidence intervals (95% CI) were computed. P < 0.05 was considered to indicate statistical significance. The JMP version 5.1 statistical software package (SAS Institute, Cary, NC, USA) was used for analysis of data.

RESULTS Virologic and biochemical responses to IFN therapy and side effects The 42 patients receiving IFN therapy included 29 in the SVR group and 13 in the group without SVR (10 transient virological responders, 3 with no virological response). In the group without SVR, 7 biochemical responders who had a normalized ALT included 5 with transient virological responses and 2 with no virological response. Although there was no significant difference in the population of patients with HCV genotype 1 between the SVR and non-SVR group, patients in the former had significantly lower pre-IFN viral loads than patients in the latter group. In the SVR group, 24 patients received full-dose IFN therapy without dose reduction, while five patients received a reduced dose of IFN until completion

5345

of treatment. In the group without SVR, one patient with no response discontinued IFN treatment at 16 wk because of a recurrence of HCC, while three patients with a transient response discontinued treatment because of generalized fatigue. The remainder of the group without SVR received the full course of IFN therapy. Thus, most patients were able to complete the 24-wk course. Recurrence of HCC In the IFN group, first recurrences of HCC developed in 20 patients after the initial treatment for HCC during a median follow-up period of 32 mo. Of these recurrences, 10 were in patients with SVR (10/29) and 10 in patients without SVR (10/13), including 7 transient virological responders and 3 with no virological response. For the 7 biochemical responders without SVR, HCC recurred in 6 patients, including 5 transient virological responders and 1 with no virological response. Of these 20 patients with recurrence, 18 were treated with local ablation therapy or surgical resection without leaving any residual tumor. The remaining 2 patients developed uncontrolled multiple HCC and were excluded from the subsequent study concerning the next recurrence. One died of HCC, while the other was treated repeatedly with hepatic arterial infusion, and has survived. Three patients in the SVR group and 7 in the group without SVR (5 transient virological responders and 2 with no virological response) had a second recurrence of HCC. Of these 10 patients with a second recurrence, 3 (2 transient virological responders and one with no virological response) developed uncontrolled HCC, while others were treated curatively with hepatic resection or local ablation therapy. In the non-IFN group, a first recurrence of HCC occurred in 30 patients during a median follow-up period of 31 mo. HCC recurred in 11 of the 17 who had a normal ALT level. Among the 30 patients with recurrent HCC, 25 were treated with local ablation therapy or surgical treatment, with no residual tumor. The remaining 5 patients who did not undergo curative therapy were treated repeatedly with transarterial chemoembolization. A second recurrence developed in 15 of the 25 patients who had curative treatment for a first recurrence. Among these 15 patients, 10 were treated curatively (9 with local ablation and 1 with hepatic resection). The remaining 5 patients had uncontrolled multiple HCC as their second recurrence. Overall cumulative rates for first and second recurrence of HCC were compared between the groups. The 1-, 3-, 5and 7-year rates for first recurrence in the IFN and nonIFN group were 0% vs 4.8%, 44% vs 53%, 61% vs 81%, and 67% vs 87%, respectively (Figure 1A, P = 0.153; no significant difference between groups). However, the 1-, 3-, 5-, and 7-year rates for second recurrence in the IFN and non-IFN group were 0% vs 0%, 10.4% vs 30%, 28% vs 59%, and 35% vs 66%, respectively (Figure 1B, P = 0.022). Thus, the second-recurrence rate was significantly lower in the IFN group than in the non-IFN group. Next, the recurrence rates of HCC were compared between the SVR group, the non-SVR group and the nonIFN group. The rate of first recurrence was significantly lower in the SVR group than in the non-SVR and non-IFN group (Figure 2A). The rate of second recurrence in the

www.wjgnet.com

5346

ISSN 1007-9327

CN 14-1219/R


IFN group (n = 42) Non-IFN group (n = 42)

Recurrence rate (%)

A

100 90 80 70 60 50 40 30 20 10 0

P = 0.153

0

1

2

3

4

5

6

7

8

9 10 11 12

Years after initial treatment of HCC

Recurrence rate (%)

B

100 90

P = 0.022

80 70 60 50 40 30 20 10 0

0

1

2

3

4

5

6

7

8

9 10 11 12


Figure 1 Cumulative recurrence rates after curative treatment of HCC. A: Rates of first recurrence compared between IFN and non-IFN groups, showed no significant difference (P = 0.153); B: Rates of second recurrence compared between IFN and non-IFN groups. The second recurrence rate for the IFN group was lower than that for the non-IFN group (P = 0.022).

SVR group was also lower than that in the non-SVR and non-IFN groups; this decrease was significantly greater than that for the rate of first recurrence (Figure 2B). No significant difference was seen in cumulative rates for first or second recurrence between the non-SVR and non-IFN groups. We also confirmed that biochemical responders in the non-SVR and non-IFN groups showed similar Kaplan-Meier curves for cumulative recurrence (data not shown). Recurrence curves were similar between the non-SVR group, including biochemical responders, and the non-IFN group, therefore, we defined these two groups as “non-SVR status” for statistical analysis. Factors found to be significantly associated with first recurrence by univariate analysis were tumor size (≥ 20 mm) and non-SVR status (P = 0.019, P = 0.0067, respectively). Multivariate analysis showed that no independent risk factor was associated with the first recurrence of HCC (data not shown), although non-SVR status tended to be associated with first recurrence (P = 0.0657). As shown in Table 2, univariate analysis indicated that non-SVR status, low platelet count (< 100 000) and high indocyanine green retention (≥ 20%) were significantly associated with second recurrence. Multivariate analysis identified only SVR status as a significant independent inhibiting factor for second recurrence of HCC. Survival of patients During the observation period, 13 of the total patients www.wjgnet.com

October 28, 2007

Volume 13

Number 40

studied died of liver disease. Nine died of HCC and 4 of liver failure. When we compared cumulative survival rates between the IFN and the non-IFN groups (Figure 3A), the respective rates were 100% vs 95% at 3 years, 100% vs 72% at 5 years, and 86% vs 63% at 7 years. The cumulative survival rate was significantly higher in the IFN group than in the non-IFN group (P = 0.039). Median survival time following the first treatment of HCC was 52.3 mo (range, 12-158) in the IFN group and 51.8 mo (range, 11-126) in the non-IFN group. In the IFN group, 2 patients died of advanced HCC, 1 with an SVR and the other without. No patients in the IFN group died of hepatic failure. In the non-IFN group, 7 patients died of HCC and four of hepatic failure. Figure 3B shows cumulative survival curves for the SVR, non-SVR and non-IFN groups. The rate of survival in the SVR group was significantly better than that in the non-IFN group (P = 0.029), while no significant difference was evident between the non-SVR and non-IFN group (P = 0.248). Pretreatment factors found to be significantly associated with survival by univariate analysis subsequently were evaluated by Cox regression analysis to determine independent factors. Multivariate analysis showed that SVR status and Child-Pugh class A were independent factors favorably associated with long survival (Table 3). Liver function Compared with the non-IFN group, patients who received IFN therapy were less likely to have worsening of hepatic dysfunction. For the SVR, non-SVR and non-IFN groups, we compared the average score for Child-Pugh classification at initial treatment of HCC with that at the time of data analysis. Median observation time was 59.8 mo in the SVR group, 45 mo in the non-SVR group, and 51.8 mo in the non-IFN group. There were no significant differences in the Child-Pugh classification score among these three groups at the time of initial treatment of HCC; however, at the time of data analysis, scores in the nonIFN group were significantly worse than in the SVR group (P = 0.003). No significant difference was seen between the non-SVR and non-IFN groups (Figure 4).

DISCUSSION The present study compared historical control subjects with no IFN treatment with other subjects who were treated with IFN. Background characteristics showed no significant difference between the groups. IFN and non-IFN group did not differ significantly in their rate of first recurrence, but did differ significantly in their rate of second recurrence. According to IFN response, the recurrence rate in the SVR group was significantly lower than that in the non-SVR and non-IFN group, while recurrence rates in the non-SVR and non-IFN group did not differ significantly. Thus, SVR (i.e. HCV eradication) was the most important, and only, inhibiting factor for decreasing risk of HCC recurrence, associated with a 24-wk course of IFN-a therapy following HCC treatment. Although several recent studies have reported the


5347

Table 2 Factors associated with second recurrence Variables

Univariate analysis 95% CI 0.246-0.728 0.373-0.814 0.450-0.965

Hazard ratio 0.454 0.553 0.667

SVR PLT > 100 000/μ ICG R-15 (< 20%)

P 0.0005 0.003 0.032

Multivariate Hazard ratio 0.457 0.694 0.685

analysis 95% CI 0.243-0.757 0.445-1.069 0.447-1.035

P 0.0015 0.0973 0.0721

Cox’s proportional hazards model was used.

SVR group (n = 29) Group without SVR (n = 13) Non-IFN group (n = 42)

90 80

Recurrence rate (%)

70

P = 0.039

60 50 40 30 20 10 0

0

1

2

3 4 5 6 7 8 9 Years after initial treatment of HCC

10

11

12

B 100 90 80 70 60 50 40 30 20 10 0

IFN group (n = 42) Non-IFN group (n = 42)

100

Ssurvival rate (%)

100 90 80 70 60 50 40 30 20 10 0

A

0

1

2 3 4 5 6 7 8 9 10 11 12 Years after initial treatment of HCC SVR group (n = 29) Group without SVR (n = 13) Non-IFN group (n = 42)

B 100 90

P = 0.538 P = 0.029

80

0

1

2

3

4

5

6

7

8

9

10

11

12


Ssurvival rate (%)

Recurrence rate (%)

A

70 50 40 30 20 10 0

Figure 2 Cumulative recurrence rates according to SVR to IFN therapy after curative treatment of HCC. A: Rates of first recurrence compared among SVR, non-SVR and non-IFN groups. The rate of first recurrence of HCC in the SVR group was significantly lower than in the non-SVR and non-IFN groups (P = 0.002, P = 0.016, respectively). No significant difference in first recurrence rate was seen between the non-SVR and non-IFN groups (P = 0.381); B: Rates of second recurrence compared among the three groups. Second recurrence of HCC was suppressed in the SVR group compared with the non-SVR and non-IFN groups (P = 0.0037, P = 0.0019, respectively), and to a more pronounced degree than for the first recurrence rate. No significant difference in second recurrence rate was seen between the non-SVR and non-IFN groups (P = 0.90).

efficacy of chemoprevention with IFN after treatment of HCV-related HCC, the basis of this benefit has not been deter mined, since IFN has a variety of biologic effects, including antiviral, antiproliferative, immunomodulator y [19-22] and anti-fibrog enic [23,24] activities; growth inhibition through changes in signal transduction[19,25,26]; and activation of natural killer cells[27] and T cells [28,29] . Through these various effects, IFN therapy is thought to suppress tumor recurrence directly and/or indirectly. Sakaguchi et al[15] have reported that low-dose, longterm, intermittent IFN-a therapy can, by a direct anticancer effect, inhibit intrahepatic metastasis but not

P = 0.248

60

0

1 2 3 4 5 6 7 8 9 10 11 12 Years after initial treatment of HCC

Figure 3 Cumulative survival rates after curative treatment of HCC. A: Comparison of cumulative survival rates in the IFN and non-IFN groups. The cumulative survival rate was significantly higher in the IFN group than in the nonIFN group (P = 0.039); B: Comparison of cumulative survival rates in the SVR, non-SVR and non-IFN groups. Although no significant overall difference was found between the SVR and non-SVR groups (P = 0.538), the SVR group had a particularly high survival rate compared with the non-IFN group (P = 0.029).

multicentric occurrences. Lai et al[29] have reported that IFN- a therapy is effective in advanced HCC. Several experimental studies have shown that IFN inhibits the growth of a human hepatoma cell line [11,15]. In partial disagreement, however, Nishiguchi et al[12,14], Suou et al[16] and Shiratori et al[17] have reported that the rate of HCC recurrence was not different between IFN and nonIFN group during the first few years, but later became significantly lower in the IFN group. They suggested that IFN reduced HCC recurrence in the later period of observation by suppressing multicentric occurrence, as an indirect anti-tumor effect that was related to sustained hepatic inflammation. Although the present study did not have a randomized controlled design, and details of the www.wjgnet.com

5348

ISSN 1007-9327

CN 14-1219/R


October 28, 2007

Volume 13

Number 40

Table 3 Factors associated with survival Variables SVR Child-Pugh class A ICG R-15 (< 20%)

Hazard ratio 0.409 0.521 0.551

Univariate analysis 95% CI 0.096-0.922 0.299-0.922 0.286-0.968

P 0.028 0.027 0.038

Multivariate analysis Hazard ratio 95% CI 0.329 0.076-0.761 0.463 0.238-0.875 0.724 0.351-1.429

P 0.006 0.019 0.350

Cox’s proportional hazards model was used.

Child-Pugh score (mean)

9

SVR group (n = 29) Group without SVR (n = 13) Non-IFN group (n = 42)

8 7

P = 0.33

P = 0.003

P = 0.2

6 5 Initial

Final

Figure 4 Influence of IFN therapy after curative treatment of HCC on ChildPugh scores. IFN-treated patients were less likely to show deterioration of hepatic function. In particular, liver function scores in the SVR group were significantly better preserved than in the non-IFN group (P = 0.003). Median observation time was 59.8 mo in the SVR group, 45 mo in the non-SVR group, and 51.8 mo in the non-IFN group.

IFN protocol differed from those of others, the long-term results appear to be similar among studies. Recurrence during the first few years might involve undetectable intrahepatic metastasis, or a potential malignant tumor already existing at the time of treatment of the primary HCC; afterward, HCC might recur as multicentric new liver tumor, accompanied by sustained hepatic necrosis and inflammation. Although a direct anti-cancer effect of IFN might to some extent have directly inhibited HCC recurrence, our IFN doses were insufficient to suppress intrahepatic metastatic tumors because there was only a 24-wk treatment. Therefore, in our study, we believe that IFN therapy suppressed HCC recurrence less by a direct anti-tumor effect than by an indirect effect through inhibition of the chronic inflammation associated with HCV infection in the later period of observation. Several studies have reported that recurrence was suppressed not only in virologic responders to IFN, but also in biochemical responders, even though HCV was not eradicated[12-14]. However, the recurrence rates in our study did not differ significantly between biochemical responders and the non-IFN group. HCV eradication appeared to stand alone as an IFN effect capable of inhibiting recurrence, with eradication having a stronger influence against second recurrence than the first. The differences between the results of the various studies might be due to several reasons. In most previous studies, IFN therapy was given for more than 48 wk, compared with our 24 wk. Differences may also have been present in underlying hepatic inflammatory conditions such as chronic hepatitis and cirrhosis. Although such differences introduce some uncertainty to the conclusions, several recent studies suggest that HCV core protein might directly participate in hepatocarcinogenesis[28,29], which supports the importance www.wjgnet.com

of virus eradication. Although some other recent studies have reported that IFN therapy following HCC treatment also improves liver function and survival of patients with HCV-related HCC, which of the specific IFN actions is important for these benefits remains unknown. We found that overall survival rate and preservation of liver function were significantly better in the SVR group than in the other groups, even including biochemical responders, with all subgroups without SVR resembling non-IFN patients. Favorable independent factors associated with survival by multivariate analysis were SVR and Child–Pugh class A. Thus, with a 24-wk course of IFN-a therapy, HCV eradication appears necessary for prolonging survival, suppressing HCC recurrence, and preserving liver function. As stated above, effective management of HCV infection is needed, as well as direct treatment of the primary HCC. Although our study had limitations, such as the use of historical controls and a small number of patients, we could demonstrate a clear requirement for HCV eradication to improve survival after a short-course IFN-a therapy. Ribavirin combination or pegylated IFN therapy are considered more effective in HCV eradication than conventional IFN monotherapy[32-34]. Several studies have indicated that pegylated IFN therapy is superior to conventional IFN when administered for 48 wk[34-41]. Pegylated IFN therapy, with or without ribavirin, may improve prognosis in selected patients with no sustained initial response to conventional IFN. For patients who cannot undergo standard-dose IFN therapy because of limited hepatic reserve or thrombocytopenia, lowdose IFN therapy for a longer course might be effective. Nonetheless, further studies with larger controlled groups and long-term follow-up need to be performed to establish what constitutes optimal management of HCV infection after HCC treatment.

COMMENTS Background

Risk of multicentric recurrence of hepatocellular carcinoma (HCC) and liver function deterioration remains high in hepatitis C virus (HCV)-infected patients even after receiving curative treatment for primary HCC. Most intrahepatic recurrences occurred during persistent viral infection. Although several recent studies have reported the efficacy of chemoprevention with interferon (IFN) therapy after treatment of HCV-related HCC, there was no standard IFN regimen. We investigated whether 24-week course of IFN-a therapy following curative treatment for primary HCC associated with HCV infection could suppress HCC recurrence and improve prognosis.

Research frontiers

To obtain sustained virological response (SVR) was important for suppression of HCC recurrence and for long-term survival in a 24-week course of IFN-a therapy.


5349


12

Applications

13

Peer review

14

Our study demonstrated that only SVR status by a 24-wk IFN-a therapy was the most important factor for decreasing risk of HCC recurrence in the later period of observation including second recurrence.

This study demonstrated that compared with non-IFN and non-SVR group, SVR group decreased the rate of recurrence, preserved liver function, and prolonged survival time in a 24-wk course of IFN-a therapy.

This is a matched historical case controlled study concerning about the effect of 24-week short course IFN-a therapy after receiving curative treatment for primary HCC. The paper is well written and the results show that the most important factor associated with the improvement of prognosis is the SVR status.

15

REFERENCES 1

2

3

4

5

6

7

8

9

10

11

Tsukuma H, Hiyama T, Tanaka S, Nakao M, Yabuuchi T, Kitamura T, Nakanishi K, Fujimoto I, Inoue A, Yamazaki H. Risk factors for hepatocellular carcinoma among patients with chronic liver disease. N Engl J Med 1993; 328: 1797-1801 Takano S, Yokosuka O, Imazeki F, Tagawa M, Omata M. Incidence of hepatocellular carcinoma in chronic hepatitis B and C: a prospective study of 251 patients. Hepatology 1995; 21: 650-655 Shiratori Y, Shiina S, Imamura M, Kato N, Kanai F, Okudaira T, Teratani T, Tohgo G, Toda N, Ohashi M. Characteristic difference of hepatocellular carcinoma between hepatitis Band C- viral infection in Japan. Hepatology 1995; 22: 1027-1033 Ikeda K, Saitoh S, Tsubota A, Arase Y, Chayama K, Kumada H, Watanabe G, Tsurumaru M. Risk factors for tumor recurrence and prognosis after curative resection of hepatocellular carcinoma. Cancer 1993; 71: 19-25 Kubo S, Nishiguchi S, Shuto T, Tanaka H, Tsukamoto T, Hirohashi K, Ikebe T, Wakasa K, Kuroki T, Kinoshita H. Effects of continuous hepatitis with persistent hepatitis C viremia on outcome after resection of hepatocellular carcinoma. Jpn J Cancer Res 1999; 90: 162-170 Kumada T, Nakano S, Takeda I, Sugiyama K, Osada T, Kiriyama S, Sone Y, Toyoda H, Shimada S, Takahashi M, Sassa T. Patterns of recurrence after initial treatment in patients with small hepatocellular carcinoma. Hepatology 1997; 25: 87-92 Shimada M, Takenaka K, Gion T, Fujiwara Y, Kajiyama K, Maeda T, Shirabe K, Nishizaki T, Yanaga K, Sugimachi K. Prognosis of recurrent hepatocellular carcinoma: a 10-year surgical experience in Japan. Gastroenterology 1996; 111: 720-726 Nagasue N, Uchida M, Makino Y, Takemoto Y, Yamanoi A, Hayashi T, Chang YC, Kohno H, Nakamura T, Yukaya H. Incidence and factors associated with intrahepatic recurrence following resection of hepatocellular carcinoma. Gastroenterology 1993; 105: 488-494 Davis GL, Balart LA, Schiff ER, Lindsay K, Bodenheimer HC Jr, Perrillo RP, Carey W, Jacobson IM, Payne J, Dienstag JL. Treatment of chronic hepatitis C with recombinant interferon alfa. A multicenter randomized, controlled trial. Hepatitis Interventional Therapy Group. N Engl J Med 1989; 321: 1501-1506 Di Bisceglie AM, Martin P, Kassianides C, Lisker-Melman M, Murray L, Waggoner J, Goodman Z, Banks SM, Hoofnagle JH. Recombinant interferon alfa therapy for chronic hepatitis C. A randomized, double-blind, placebo-controlled trial. N Engl J Med 1989; 321: 1506-1510 Ikeda K, Arase Y, Saitoh S, Kobayashi M, Suzuki Y, Suzuki F, Tsubota A, Chayama K, Murashima N, Kumada H. Interferon beta prevents recurrence of hepatocellular carcinoma after complete resection or ablation of the primary tumor-A prospective randomized study of hepatitis C virus-related liver cancer. Hepatology 2000; 32: 228-232

16

17

18 19

20

21

22

23

24

25 26

27

28

Kubo S, Nishiguchi S, Hirohashi K, Tanaka H, Shuto T, Yamazaki O, Shiomi S, Tamori A, Oka H, Igawa S, Kuroki T, Kinoshita H. Effects of long-term postoperative interferonalpha therapy on intrahepatic recurrence after resection of hepatitis C virus-related hepatocellular carcinoma. A randomized, controlled trial. Ann Intern Med 2001; 134: 963-967 Kubo S, Nishiguchi S, Hirohashi K, Tanaka H, Shuto T, Kinoshita H. Randomized clinical trial of long-term outcome after resection of hepatitis C virus-related hepatocellular carcinoma by postoperative interferon therapy. Br J Surg 2002; 89: 418-422 Nishiguchi S, Tamori A, Kubo S. Effect of long-term postoperative interferon therapy on intrahepatic recurrence and survival rate after resection of hepatitis C virus-related hepatocellular carcinoma. Intervirology 2005; 48: 71-75 Sakaguchi Y, Kudo M, Fukunaga T, Minami Y, Chung H, Kawasaki T. Low-dose, long-term, intermittent interferonalpha-2b therapy after radical treatment by radiofrequency ablation delays clinical recurrence in patients with hepatitis C virus-related hepatocellular carcinoma. Intervirology 2005; 48: 64-70 Suou T, Mitsuda A, Koda M, Matsuda H, Maruyama S, Tanaka H, Kishimoto Y, Kohno M, Hirooka Y, Kawasaki H. Interferon alpha inhibits intrahepatic recurrence in hepatocellular carcinoma with chronic hepatitis C: a pilot study. Hepatol Res 2001; 20: 301-311 Shiratori Y, Shiina S, Teratani T, Imamura M, Obi S, Sato S, Koike Y, Yoshida H, Omata M. Interferon therapy after tumor ablation improves prognosis in patients with hepatocellular carcinoma associated with hepatitis C virus. Ann Intern Med 2003; 138: 299-306 The general rules for the clinical and pathological study of primary liver cancer. Liver Cancer Study Group of Japan. Jpn J Surg 1989; 19: 98-129 Harada H, Kitagawa M, Tanaka N, Yamamoto H, Harada K, Ishihara M, Taniguchi T. Anti-oncogenic and oncogenic potentials of interferon regulatory factors-1 and -2. Science 1993; 259: 971-974 Tanaka N, Ishihara M, Kitagawa M, Harada H, Kimura T, Matsuyama T, Lamphier MS, Aizawa S, Mak TW, Taniguchi T. Cellular commitment to oncogene-induced transformation or apoptosis is dependent on the transcription factor IRF-1. Cell 1994; 77: 829-839 Tanaka N, Ishihara M, Lamphier MS, Nozawa H, Matsuyama T, Mak TW, Aizawa S, Tokino T, Oren M, Taniguchi T. Cooperation of the tumour suppressors IRF-1 and p53 in response to DNA damage. Nature 1996; 382: 816-818 Czaja MJ, Weiner FR, Eghbali M, Giambrone MA, Eghbali M, Zern MA. Differential effects of gamma-interferon on collagen and fibronectin gene expression. J Biol Chem 1987; 262: 13348-13351 Capra F, Casaril M, Gabrielli GB, Tognella P, Rizzi A, Dolci L, Colombari R, Mezzelani P, Corrocher R, De Sandre G. alphaInterferon in the treatment of chronic viral hepatitis: effects on fibrogenesis serum markers. J Hepatol 1993; 18: 112-118 Ishibashi K, Kashiwagi T, Ito A, Tanaka Y, Nagasawa M, Toyama T, Ozaki S, Naito M, Azuma M. Changes in serum fibrogenesis markers during interferon therapy for chronic hepatitis type C. Hepatology 1996; 24: 27-31 Hannigan GE, Williams BR. Signal transduction by interferonalpha through arachidonic acid metabolism. Science 1991; 251: 204-207 Swaminathan N, Lai CM, Beilharz MW, Boyer SJ, Klinken SP. Biological activities of recombinant murine interferons alpha 1 and alpha 4: large difference in antiproliferative effect. Antiviral Res 1992; 19: 149-159 Chen LK, Tourvieille B, Burns GF, Bach FH, MathieuMahul D, Sasportes M, Bensussan A. Interferon: a cytotoxic T lymphocyte differentiation signal. Eur J Immunol 1986; 16: 767-770 Tabata Y, Uno K, Yamaoka T, Ikada Y, Muramatsu S. Effects of recombinant alpha-interferon-gelatin conjugate on in vivo

www.wjgnet.com

5350

29

30 31

32

33

34

35

ISSN 1007-9327

CN 14-1219/R


murine tumor cell growth. Cancer Res 1991; 51: 5532-5538 Lai CL, Lau JY, Wu PC, Ngan H, Chung HT, Mitchell SJ, Corbett TJ, Chow AW, Lin HJ. Recombinant interferonalpha in inoperable hepatocellular carcinoma: a randomized controlled trial. Hepatology 1993; 17: 389-394 Koike K. Molecular basis of hepatitis C virus-associated hepatocarcinogenesis: lessons from animal model studies. Clin Gastroenterol Hepatol 2005; 3: S132-S135 Sato Y, Kato J, Takimoto R, Takada K, Kawano Y, Miyanishi K, Kobune M, Sato Y, Takayama T, Matunaga T, Niitsu Y. Hepatitis C virus core protein promotes proliferation of human hepatoma cells through enhancement of transforming growth factor alpha expression via activation of nuclear factorkappaB. Gut 2006; 55: 1801-1808 Poynard T, Marcellin P, Lee SS, Niederau C, Minuk GS, Ideo G, Bain V, Heathcote J, Zeuzem S, Trepo C, Albrecht J. Randomised trial of interferon alpha2b plus ribavirin for 48 weeks or for 24 weeks versus interferon alpha2b plus placebo for 48 weeks for treatment of chronic infection with hepatitis C virus. International Hepatitis Interventional Therapy Group (IHIT). Lancet 1998; 352: 1426-1432 McHutchison JG, Gordon SC, Schiff ER, Shiffman ML, Lee WM, Rustgi VK, Goodman ZD, Ling MH, Cort S, Albrecht JK. Interferon alfa-2b alone or in combination with ribavirin as initial treatment for chronic hepatitis C. Hepatitis Interventional Therapy Group. N Engl J Med 1998; 339: 1485-1492 Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Gonçales FL Jr, Häussinger D, Diago M, Carosi G, Dhumeaux D, Craxi A, Lin A, Hoffman J, Yu J. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002; 347: 975-982 Bailon P, Palleroni A, Schaffer CA, Spence CL, Fung WJ,

October 28, 2007

Volume 13

Porter JE, Ehrlich GK, Pan W, Xu ZX, Modi MW, Farid A, Berthold W, Graves M. Rational design of a potent, longlasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon alpha-2a for the treatment of hepatitis C. Bioconjug Chem 2001; 12: 195-202 36 Harris JM, Martin NE, Modi M. Pegylation: a novel process for modifying pharmacokinetics. Clin Pharmacokinet 2001; 40: 539-551 37 Glue P, Fang JW, Rouzier-Panis R, Raffanel C, Sabo R, Gupta SK, Salfi M, Jacobs S. Pegylated interferon-alpha2b: pharmacokinetics, pharmacodynamics, safety, and preliminary efficacy data. Hepatitis C Intervention Therapy Group. Clin Pharmacol Ther 2000; 68: 556-567 38 Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, Goodman ZD, Koury K, Ling M, Albrecht JK. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 2001; 358: 958-965 39 Lindsay KL, Trepo C, Heintges T, Shiffman ML, Gordon SC, Hoefs JC, Schiff ER, Goodman ZD, Laughlin M, Yao R, Albrecht JK. A randomized, double-blind trial comparing pegylated interferon alfa-2b to interferon alfa-2b as initial treatment for chronic hepatitis C. Hepatology 2001; 34: 395-403 40 Zeuzem S, Feinman SV, Rasenack J, Heathcote EJ, Lai MY, Gane E, O'Grady J, Reichen J, Diago M, Lin A, Hoffman J, Brunda MJ. Peginterferon alfa-2a in patients with chronic hepatitis C. N Engl J Med 2000; 343: 1666-1672 41 Reddy KR, Wright TL, Pockros PJ, Shiffman M, Everson G, Reindollar R, Fried MW, Purdum PP 3rd, Jensen D, Smith C, Lee WM, Boyer TD, Lin A, Pedder S, DePamphilis J. Efficacy and safety of pegylated (40-kd) interferon alpha-2a compared with interferon alpha-2a in noncirrhotic patients with chronic hepatitis C. Hepatology 2001; 33: 433-438 S- Editor Liu Y L- Editor Kerr C

www.wjgnet.com

Number 40

E- Editor Yin DH



RAPID COMMUNICATION

Pancreatic fistula after pancreaticoduodenectomy: A comparison between the two pancreaticojejunostomy methods for approximating the pancreatic parenchyma to the jejunal seromuscular layer: Interrupted vs continuous stitches Seung Eun Lee, Sung Hoon Yang, Jin-Young Jang, Sun-Whe Kim Seung Eun Lee, Sung Hoon Yang, Jin-Young Jang, Sun-Whe Kim, Department of Surgery, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea Supported by grant from the National R&D Program for Cancer Control, Ministry of Health & Welfare, Republic of Korea, No.0520320 Correspondence to: Sun-Whe Kim, MD, PhD, Department of Surgery, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744, Korea. [email protected] Telephone: +82-2-20722315 Fax: +82-2-7452282 Received: June 30, 2007 Revised: August 9, 2007

Abstract AIM: The purpose of this study is to find a better operative technique by comparing interrupted stitches with continuous stitches for the outer layer of the pancreaticojejunostomy, i.e. the stitches between the stump parenchyma of the pancreas and the jejunal seromuscular layer, and other risk factors for the incidence of pancreatic leakage. METHODS: During the period January 1997 to October 2004, 133 patients have undergone the end-to-side and duct-to-mucosa pancreaticojejunostomy reconstruction after pancreaticoduodenectomy with interrupted suture for outer layer of the pancreaticojejunostomy and 170 patients with a continuous suture at our institution by one surgeon. RESULTS: There were no significant differences between the two groups in the diagnosis, texture of the pancreas, use of octreotide and pathologic stage. Pancreatic fistula occurred in 14 patients (11%) among the interrupted suture cases and in 10 (6%) among the continuous suture cases (P = 0.102). Major pancreatic leakage developed in three interrupted suture patients (2%) and zero continuous suture patients (P = 0.026). In multivariate analysis, soft pancreatic consistency (odds ratio, 5.5; 95% confidence interval 2.3-13.1) and common bile duct cancer (odds ratio, 3.7; 95% CI 1.6-8.5) were predictive of pancreatic leakage.

CONCLUSION: Pancreatic texture and pathology are the most important factors in determining the fate of pancreaticojejunal anastomosis and our continuous suture method was performed with significantly decreased occurrence of major pancreatic fistula. In conclusion, the continuous suture method is more feasible and safer in performing duct-to-mucosa pancreaticojejunostomy. © 2007 WJG . All rights reserved.

Key words: Pancreaticoduodenectomy; Pancreaticojejunostomy; Pancreatic fistula Lee SE, Yang SH, Jang JY, Kim SW. Pancreatic fistula after pancreaticoduodenectomy: A comparison between the two pancreaticojejunostomy methods for approximating the pancreatic parenchyma to the jejunal seromuscular layer: Interrupted vs continuous stitches. World J Gastroenterol

2007; 13(40): 5351-5356


INTRODUCTION Despite improvement of the operative technique, materials and instr uments, pancreatic fistula after pancreaticoduodenctomy is the most common and serious complication. Recent large series have reported that the failure rate of the pancreaticoenteric anastomosis is 9%-18% [1-6] , a complication rate not far improved from Dr. Whipple's report of a 19.5% fistula rate more than 50 years ago[7]. A number of methods for reducing the incidence of pancreatic fistula have been proposed and tested. Many of these involve technical aspects of the anastomosis, including the site of reconstruction (pancreaticogastrostomy versus pancreaticojejunostomy) [8,9] , the anastomotic technique (duct-to-mucosa anastomosis versus stump invagination) [10,11] , the use of biologic adhesive[12,13] and the use of intraoperative transanastomotic stents [14] . In addition, in order to determine how to prevent pancreatic fistula, the risk

www.wjgnet.com

5352

ISSN 1007-9327

CN 14-1219/R


October 28, 2007

Volume 13

factors for pancreatic fistula have been extensively studied. They include the patient's comorbid illness[8], age[15], texture of the pancreas[4], pancreatic duct size[16], intraoperative blood loss[15] and the surgeon's experience[15]. T h e p r e s e n t s t u d y t e s t e d t h e hy p o t h e s i s t h a t using continuous stitches for the outer layer of the panceaticojejunostomy (i.e. the stitches between the stump parenchyma of the pancreas and the jejunal seromuscular layer) is a better operative technique than using interrupted stitches in terms of safety and efficiency.

A

B

MATERIALS AND METHODS

C

D

During the period from January 1997 to October 2004, 133 patients underwent duct-to-mucosa pancreaticojejunostomy reconstruction after pancreaticoduodenectomy with the interrupted suture method for the outer layer of the pancreaticojejunostomy and 170 patients underwent the procedure with the continuous suture method at our institution. From 1997 to 2000, the interrupted suture method was performed, and from 2001 to 2004, the continuous suture method was performed. The operations were performed by one surgeon who had experienced more than 500 cases of pancreaticoduodenectomy before this study. We retrospectively reviewed the medical records o f t h e p a t i e n t s w h o u n d e r we n t d u c t - t o - mu c o s a pancreaticojejunostomy after pancreaticoduodenectomy noting parameters such as the existence of pancreatic fistula, age, sex, preoperative symptoms, preoperative laboratory tests results, amount of intraoperative bleeding, and postoperative octreotide usage. Postoperative octreotide was given subcutaneously (dose 100 mg every 8 hours) for the patients considered high risk for pancreatic fistula based on gland texture and duct size. Pancreaticoduodenectomy was perfor med with conventional pancreaticoduodenectomy or pyloruspreser ving pancreaticoduodenectomy (PPPD). Anastomosis for the remnant pancreas was performed between the pancreas and jejunum by a two layer pancreaticojejunostomy. The outer layer consisted of the remnant pancreatic parenchyma and the seromuscular layer of jejunum and interrupted suture or continuous suture between these two was performed with 5-0 polypropylene (Prolene * , Ethicon, Somerville, NJ). The inner layer consisted of the pancreatic duct and mucosa of the jejunum, and interrupted suture for duct-to-mucosa was performed with 5-0 polydioxanone (PDSTM Ⅱ, Ethicon, Somerville, NJ). A silastic polyethylene tube was inserted into the pancreatic duct as a stent for all patients, and external drainage was done (Figure 1). Two or three drains were routinely placed anterior and posterior to the pancreatico-jejunal anastomosis and exteriorized through the lateral abdominal wall. A pancreatic fistula was defined as the drainage of more than 30 mL of fluid with an amylase level higher than 600 U/dL on or after postoperative week 1[17]. Also, three grades of fistula severity (A, B, C) were classified according to the International Study Group for Pancreatic Fistulas (ISGPF) clinical criteria[18] as follows: Grade A www.wjgnet.com

Number 40

E

Figure 1 Continuous suture method for the outer layer of pancreaticojejunostomy. A: The posterior outer layer consisted of the remnant pancreatic parenchyma and the seromuscular layer of jejunum and continuous suture between these two was performed with 5-0 polypropylene (Prolene*, Ethicon, Somerville, NJ); B: The posterior inner layer consisted of the pancreatic duct and mucosa of the jejunum, and interrupted suture for duct-to-mucosa was performed with 5-0 polydioxanone (PDSTMⅡ, Ethicon, Somerville, NJ); C: A silastic polyethylene tube was inserted into the pancreatic duct and external drainage was done; D: For anterior inner layer consisted of the pancreatic duct and mucosa of the jejunum, interrupted suture was performed; E: Continuous suture for anterior outer layer was performed.

fistulas are transient, asymptomatic fistulas, with only elevated drain amylase levels and treatments or deviation in clinical management are not required. Grade B fistulas are symptomatic, clinically apparent fistulas that require diagnostic evaluation and therapeutic management. Grade C fistulas are severe, clinically significant fistulas that require major deviations in clinical management and aggressive therapeutic interventions are unquestionably warranted. Major pancreatic leakage was defined as the drainage of more than 200 mL of fluid or the development of an intra-abdominal abscess. Pancreatic fistulas, according to the operative methods and clinicopathologic factors causing pancreatic fistula, were analyzed.

Lee SE et al . Pancreatic fistula after pancreaticoduodenectomy

Table 1 Comparison of clinical characteristics between the interrupted suture group and the continuous suture group n (%) Interrupted suture group (n = 133)

Continuous suture group (n = 170)

5353

Table 2 Comparison of postoperative complications and mortality between the interrupted suture group and the continuous suture group n (%)

P value

Age (yr) Male:Female Pancreas texture Hard Firm Soft

58.2 ± 12.4 1.8:1

60.4 ± 10.9 1.5:1

0.072 0.484 0.893

35 (26) 86 (65) 12 (9)

52 (31) 76 (45) 42 (24)

Use of prophylactic octreotide

83 (62)

122 (72)

0.072

Jaundice Diabetes mellitus Diagnosis Ampulla of vater cancer Common bile duct cancer Pancreatic cancer Duodenal cancer Etc.

59 (44) 21 (16)

69 (41) 33 (19)

0.643 0.544 0.433

35 (26)

45 (26)

38 (29)

46 (27)

34 (25) 6 (5) 20 (15)

57 (34) 6 (4) 16 (9)

Etc.: Ampulla of Vater adenoma, choledochal cyst, chronic pancreatitis, duodenal GIST, gallbladder cancer, intraductal papillary mucinous tumor, islet cell tumor of pancreas, peripancreatic neurilemmoma, pseudocyst, serous cystic adenoma.

Data comparisons between the two groups were made using the χ2 test for qualitative parameters, a Student's t-test for quantitative parameters and logistic regression n for determining the effect of multiple risk factors on pancreatic leakage. A value of P < 0.05 was accepted as significant.

RESULTS There were no significant differences in mean age, sex, prophylactic use of octreotide, pancreas texture, stage, and indication for pancreaticoduodenectomy between the two groups (Table 1). Although there was no significant difference in the mean total operation time between the interrupted suture and continuous suture groups, there was a statistically significant difference in the mean operation time of the outer layer anastomosis of pancreaticojejunostomy. The mean operation time of the interrupted suture method for the outer layer anastomosis of pancreaticojejunostomy was 35.4 ± 4.8 minutes, and the mean operation time of the continuous suture method was 29.1 ± 3.9 min (P < 0.001). Pancreatic fistula occurred in 14 patients (11%) of the 133 patients who underwent interrupted suture for the outer layer and in 10 patients (6%) of the 170 patients who underwent continuous suture (P = 0.102). There were 5 grade A fistulas, 6 grade B fistulas, and 3 grade C fistulas in the interrupted suture group. There were 4 grade A fistulas, 5 grade B fistulas, and no grade C fistula in the continuous suture group. There was no significant difference between the two groups (P = 0.085). Major pancreatic leakage occurred in three patients (2%) in the interrupted suture group and zero patients in the continuous suture group (P = 0.026). For one patient of the interrupted suture group

Number of patients Interrupted suture Continuous suture P value group (n = 133) group (n = 170) Pancreatic fistula ISGPF grade Grade A Grade B Grade C Major pancreatic fistula Disruption Daily drainage > 200 cc Intra-abdominal abscess Pseudoaneurysm Reoperation for pancreatic fistula Hospital mortality

14 (10.5)

10 (5.9)

5 (3.8) 6 (4.5) 3 (2.3) 3 (2.3) 1 (0.8) 0 2 (1.5) 0 1 (0.8)

4 (2.4) 6 (3.5) 0 0 0 0 0 2 0

0

0

0.102 0.085

0.026

0.128 0.199

who experienced pancreaticojejunal anastomotic rupture, externalization of the pancreatic duct was performed at the 10th postoperative day. For the other two patients of the interrupted suture group with major pancreatic leakage, percutaneous drainage was added. Two patients of the continuous suture group with distal common bile duct (CBD) cancer developed pseudoaneurysm with preceding pancreatic fistula. They were successfully managed by radiologic intervention. There was no postoperative hospital mortality in the two groups (Table 2). Of the total of 303 patients, 24 patients (8%) developed postoperative pancreatic fistula. There were no significant differences in age, sex, preoperative bilirubin and albumin levels, operation methods, amount of intraoperative bleeding, total operation time, postoperative prophylactic octreotide usage, and stage between the pancreatic fistula group and the non-pancreatic fistula group. There was a significant difference in pathologic features between the pancreatic fistula group and the nonpancreatic fistula group (P = 0.039). When the pathologic features were divided into CBD cancer and non-CBD cancer, there was a significant difference (P = 0.004). The consistency of the remnant pancreas correlates strongly with subsequent postoperative fistula rates. In the nonpancreatic fistula group, 43 (15%) were classified as soft and 85 as hard. In the pancreatic fistula group, 11 (46%) were classified as soft and 2 as hard (P < 0.001) (Table 3). In multivariate analysis, soft pancreatic consistency (odds ratio, 5.5; 95% confidence interval 2.3-13.1) and CBD cancer (odds ratio, 3.7; 95% confidence interval 1.6-8.5) were predictive of pancreatic leakage.

DISCUSSION Recently, pancreaticoduodenectomy has become popularized as the standard treatment for various benign and malignant diseases of the periampullary region, including the pancreas head. Although the mortality rate has markedly decreased, the incidence of pancreatic fistula, the most catastrophic complication, remains high www.wjgnet.com

5354

ISSN 1007-9327

CN 14-1219/R


Table 3 Perioperative risk factors for pancreatic fistula

Age (yr) Male:Female Preoperative disease Diabetes mellitus Laboratory findings Hypoalbuminemia (3 < g/dL) Hyperbilirubinemia (> 2 mg/dL) Pathologic feature Ampulla of Vater cancer Common bile duct cancer Pancreatic cancer Duodenal cancer Others Type of resection PPPD Whipple’s op. Outer layer suture method Interrupted Continuous Pancreas consistency Hard Firm Soft Total operative time (min) Estimated blood loss (mL) Use of prophylactic Octreotide Lymph node metastasis Yes No Positive resection margin Yes No

Pancreatic fistula (+) (n = 24) 62.3 ± 10.2 2:1

P value 0.565 0.582

4 (7%, 4/54)

0.959

3 (9%, 3/33) 14 (7%, 14/195)

0.729 0.665 0.039

4 (5%, 4/80) 13 (16%, 13/84) 4 (4%, 4/91) 0 (0%, 0/12) 3 (8%, 3/36) 0.097 22 (10%, 22/218) 2 (2%, 2/85) 0.102 14 (11%, 14/133) 10 (6%, 10/170) < 0.001 2 (2%, 2/87) 11 (7%, 11/162) 11 (20%, 11/54) 383 ± 52 564 ± 220 14 (7%, 14/205)

0.515 0.831 0.317 0.351

17 (9%, 17/197) 7 (7%, 7/106) 0.105 1 (4%, 1/26) 23 (8%, 23/277)

after pancreaticoduodenectomy. After the first successful pancreaticoduodenectomy was performed by the German surgeon Kausch in 1912[19], the risk factors for pancreatic anastomotic leakage and methods aimed at prevention of pancreatic anastomotic leakage have been extensively studied. Several factors related to pancreatic anastomotic leakage have been described in the literature. They can be conveniently divided into disease factors (pancreatic texture[4], pancreatic pathology[8], pancreatic duct size[16], pancreatic juice output [8] ), procedure-related factors (intraoperative blood loss[15], operative techniques[15]) and patient factors (age[15], sex[8], comorbid illness[8], jaundice[8]). Still, it is very difficult to predict the relationship of the risk factors and pancreatic fistula, and many studies have revealed heterogeneous results. In our study, two risk factors, common bile duct cancer and soft pancreatic texture were correlated with increased incidence of pancreatic fistula. The relative proportion of bile duct and ampulla of Vater cancers is much larger in Korea than in Western countries. The reason is not clear. The high incidence of hepatolithiasis, choledocholithiasis, choledochal cyst, and clonorchiasis in Korea is a possible explanation, at least in a proportion of cases. Unlike in the Johns Hopkins series[8], the leakage rate was lower in cases of ampulla of Vater cancer (5%, 4/80) than in others (9%, 20/223). Patients with ampulla of Vater cancer are diagnosed early, and their general condition is www.wjgnet.com

October 28, 2007

Volume 13

Number 40

relatively good. More importantly, most of the patients have a dilated pancreatic duct and a firm pancreas, which facilitates the anastomosis and its healing. On the other hand, most of the patients with common bile duct cancer have a non-dilated pancreatic duct and a soft pancreas. The increased fistula rates with soft pancreatic texture may be interpreted by three explanations. First, most soft pancreases have no pancreatic duct dilatation, which makes secure duct-to-mucosa anastomosis difficult[20,21]. Second, a soft pancreas is more easily injured directly or via ischemia by stitches placed between the pancreas parenchyma and the seromuscular layer of the jejunum [20]. Third, a soft pancreas has a good exocrine function, secreting more pancreatic juices rich in proteolytic enzymes[20-22]. Prophylactic use of octreotide is expected theoretically to reduce the incidence rate of the pancreatic fistula through decreasing pancreatic juice secretion. However, randomized trials from Europe and United states showed opposite results[23-26]. In our study, there was no significant difference in the incidence rate of pancreatic fistula between postoperative prophylactic octreotide usage group and no usage group (P = 0.347). To prove the effect of octreotide in reducing the amount of postoperative pancreatic fistula, more organized randomized controlled studies are needed. Various techniques for managing the pancreatic remnant have been studied with the aim of reducing the anastomotic leakage rate, including varying the site of the jejunum for pancreaticojejunostomy (end vs side), varying the type of anastomosis (duct-to-mucosa vs invagination) [10,11], comparing the difference between pancreaticogastrostomy and pancreaticojejunostomy [8,9], use of fibrin glue [12,13] and pancreatic duct stenting [14]. Unfortunately, randomized trials on these technical measures are scarce[11]. As a result, there is no universal agreement on which operative technique is safer and less prone to pancreatic leakage[11]. Because our study is not a prospective randomized controlled study, there is potential for a beta error. However, it could be thought as a periodic randomized s t u d y. T h e o p e r a t i o n s w e r e p e r f o r m e d b y o n e surgeon who had experienced more than 500 cases of pancreaticoduodenectomy before this study, and the improved results are unlikely associated with a learning curve by doing more procedures. Although there was no significant difference between the interrupted suture and continuous suture methods for preventing pancreatic fistula in our study, the incidence of major pancreatic fistula decreased significantly in the continuous suture group (P = 0.026). Theoretically, the continuous suture method has many advantages over the interrupted suture method[27-32]. First of all, a more even distribution of tension is possible in the continuous suture between the pancreatic parenchyma and jejunum[27]. Due to the coiled spring effect, the continuous suture method provides a reduction in the likelihood of focal tissue ischemia[27], an increase in tensile strength[27], and a reduction of the risk of pancreaticojejunal rupture. A continuous suture provides an enhanced air and watertight seal[28,29]. As our study shows, a continuous suture reduces anastomosis time[30,31]. A continuous suture is technically

Lee SE et al . Pancreatic fistula after pancreaticoduodenectomy

easier[30,31] and costs less[31,32]. For continuous sutures, we use one polypropylene (Prolene *, Ethicon, Somerville, NJ) thread, and the thread costs 5.7 dollars. However, for interrupted sutures, we use 16 polypropylene sutures and it costs 86 dollars more than continuous suture. The study of two methods for approximating the pancreatic parenchyma to the jejunal seromuscular layer in duct-to-mucosa pancreaticojejunostomy revealed that the incidence of major pancreatic fistula decreased significantly and operation time is reduced significantly in the continuous suture group. In conclusion, the continuous suture method is more feasible and safe to use in performing pancreaticojejunostomy.

5355

2

3 4

5

COMMENTS Background

6

Leakage of the pancreaticojejunal anastomosis has been a major complication after pancreaticoduodenectomy. Over the past decades, various measures directed towards prevention of pancreatic leakage have been studied.

Research frontiers

The purpose of this study is to find better operative technique as comparing the interrupted stitches with the continuous stitches for the outer layer of the panceaticojejunostomy, ie. the stitches between the stump parenchyma of the pancreas and the jejunal seromuscular layer, and other risk factors for the incidence of pancreatic leakage.

7 8

9


Soft pancreatic consistency (odds ratio, 5.5; 95% confidence interval 2.3-13.1) and CBD cancer (odds ratio, 3.7; 95% confidence interval 1.6-8.5) were predictive of pancreatic leakage and our continuous suture method performed with significantly decreasing major pancreatic fistula.

Applications

The study of two methods for approximating the pancreatic parenchyma to the jejunal seromuscular layer in duct-to-mucosa pancreaticojejunostomy revealed that the incidence of major pancreatic fistula decreased significantly and operation time is reduced significantly in the continuous suture group. In conclusion, the continuous suture method is more feasible and safe to use in performing pancreaticojejunostomy.

10 11 12

13

Terminology

A pancreatic fistula was defined as the drainage of more than 30 mL of fluid with an amylase level higher than 600 U/dL on or after postoperative week 1. Also, three grades of fistula severity (A, B, C) were classified according to ISGPF clinical criteria as follows: Grade A fistulas are transient, asymptomatic fistulas, with only elevated drain amylase levels and treatments or deviations in clinical management are not required. Grade B fistulas are symptomatic, clinically apparent fistulas that require diagnostic evaluation and therapeutic management. Grade C fistulas are severe, clinically significant fistulas that require major deviations in clinical management and aggressive therapeutic interventions are unquestionably warranted. Major pancreatic leakage was defined as the drainage of more than 200 mL of fluid or the development of an intra-abdominal abscess.

Peer review

As this was a retrospective, non-randomized study in which the continuous suture was used after experience with 133 patients in whom an interrupted suture technique was used, the "better" results with the continuous suturing may simply be a learning curve.

14

15 16

17

18

REFERENCES 1

Balcom JH 4th, Rattner DW, Warshaw AL, Chang Y, Fernandez-del Castillo C. Ten-year experience with 733 pancreatic resections: changing indications, older patients,

19 20

and decreasing length of hospitalization. Arch Surg 2001; 136: 391-398 Bassi C, Falconi M, Salvia R, Mascetta G, Molinari E, Pederzoli P. Management of complications after pancreaticoduodenectomy in a high volume centre: results on 150 consecutive patients. Dig Surg 2001; 18: 453-457; discussion 458 Marcus SG, Cohen H, Ranson JH. Optimal management of the pancreatic remnant after pancreaticoduodenectomy. Ann Surg 1995; 221: 635-645; discussion 645-648 van Berge Henegouwen MI, De Wit LT, Van Gulik TM, Obertop H, Gouma DJ. Incidence, risk factors, and treatment of pancreatic leakage after pancreaticoduodenectomy: drainage versus resection of the pancreatic remnant. J Am Coll Surg 1997; 185: 18-24 Yeh TS, Jan YY, Jeng LB, Hwang TL, Wang CS, Chen SC, Chao TC, Chen MF. Pancreaticojejunal anastomotic leak after pancreaticoduodenectomy--multivariate analysis of perioperative risk factors. J Surg Res 1997; 67: 119-125 Yeo CJ, Cameron JL, Sohn TA, Lillemoe KD, Pitt HA, Talamini MA, Hruban RH, Ord SE, Sauter PK, Coleman J, Zahurak ML, Grochow LB, Abrams RA. Six hundred fifty consecutive pancr eaticoduodenectomies in the 1990s: pathology, complications, and outcomes. Ann Surg 1997; 226: 248-257; discussion 257-260 Whipple AO. The rationale of radical surgery for cancer of the pancreas and ampullary region. Ann Surg 1941; 114: 612-615 Yeo CJ, Cameron JL, Maher MM, Sauter PK, Zahurak ML, Talamini MA, Lillemoe KD, Pitt HA. A prospective randomized trial of pancreaticogastrostomy versus pancreaticojejunostomy after pancreaticoduodenectomy. Ann Surg 1995; 222: 580-588; discussion 588-592 Aranha GV, Hodul P, Golts E, Oh D, Pickleman J, Creech S. A comparison of pancreaticogastrostomy and pancreaticojejunostomy following pancreaticoduodenectomy. J Gastrointest Surg 2003; 7: 672-682 Greene BS, Loubeau JM, Peoples JB, Elliott DW. Are pancreatoenteric anastomoses improved by duct-to-mucosa sutures? Am J Surg 1991; 161: 45-49; discussion 49-50 Poon RT, Lo SH, Fong D, Fan ST, Wong J. Prevention of pancreatic anastomotic leakage after pancreaticoduodenectomy. Am J Surg 2002; 183: 42-52 D’Andrea AA, Costantino V, Sperti S, Pedrazzoli S. Human fibrin sealant in pancreatic surgery: it is useful in preventing fistulas? A prospective randomized study. Italy J Gastroenterol 1994; 26: 283-286 Suc B, Msika S, Fingerhut A, Fourtanier G, Hay JM, Holmieres F, Sastre B, Fagniez PL. Temporary fibrin glue occlusion of the main pancreatic duct in the prevention of intra-abdominal complications after pancreatic resection: prospective randomized trial. Ann Surg 2003; 237: 57-65 Ohwada S, Tanahashi Y, Ogawa T, Kawate S, Hamada K, Tago KI, Yamada T, Morishita Y. In situ vs ex situ pancreatic duct stents of duct-to-mucosa pancreaticojejunostomy after pancreaticoduodenectomy with billroth I-type reconstruction. Arch Surg 2002; 137: 1289-1293 Lerut JP, Gianello PR, Otte JB, Kestens PJ. Pancreaticoduodenal resection. Surgical experience and evaluation risk factors in 103 patients. Ann Surg 1984; 199: 432-437 Miedema BW, Sarr MG, van Heerden JA, Nagorney DM, McIlrath DC, Ilstrup D. Complications following pancreaticoduodenectomy. Current management. Arch Surg 1992; 127: 945-949; discussion 949-950 Kim SW, Youk EG, Park YH. Comparison of pancreatogastrostomy and pancreatojejunostomy after pancreatoduodenectomy performed by one surgeon. World J Surg 1997; 21: 640-643 Bassi C, Dervenis C, Butturini G, Fingerhut A, Yeo C, Izbicki J, Neoptolemos J, Sarr M, Traverso W, Buchler M. Postoperative pancreatic fistula: an international study group (ISGPF) definition. Surgery 2005; 138: 8-13 Kausch W. Das Carcinoma der Papilla duodeni und serine radikale Entfernung. Beitrag Z Klin Chir. 1912; 78: 439-486 Suzuki Y, Fujino Y, Tanioka Y, Hiraoka K, Takada

www.wjgnet.com

5356

21 22 23

24

25

26

ISSN 1007-9327

CN 14-1219/R


M, Ajiki T, Takeyama Y, Ku Y, Kuroda Y. Selection of pancreaticojejunostomy techniques according to pancreatic texture and duct size. Arch Surg 2002; 137: 1044-1047; discussion 1048 Lin JW, Cameron JL, Yeo CJ, Riall TS, Lillemoe KD. Risk factors and outcomes in postpancreaticoduodenectomy pancreaticocutaneous fistula. J Gastrointest Surg 2004; 8: 951-959 Sato N, Yamaguchi K, Chijiiwa K, Tanaka M. Risk analysis of pancreatic fistula after pancreatic head resection. Arch Surg 1998; 133: 1094-1098 Montorsi M, Zago M, Mosca F, Capussotti L, Zotti E, Ribotta G, Fegiz G, Fissi S, Roviaro G, Peracchia A. Efficacy of octreotide in the prevention of pancreatic fistula after elective pancreatic resections: a prospective, controlled, randomized clinical trial. Surgery 1995; 117: 26-31 Friess H, Beger HG, Sulkowski U, Becker H, Hofbauer B, Dennler HJ, Buchler MW. Randomized controlled multicentre study of the prevention of complications by octreotide in patients undergoing surgery for chronic pancreatitis. Br J Surg 1995; 82: 1270-1273 Lowy AM, Lee JE, Pisters PW, Davidson BS, Fenoglio CJ, Stanford P, Jinnah R, Evans DB. Prospective, randomized trial of octreotide to prevent pancreatic fistula after pancreaticoduodenectomy for malignant disease. Ann Surg 1997; 226: 632-641 Yeo CJ, Cameron JL, Lillemoe KD, Sauter PK, Coleman J, Sohn

27 28

29

30 31

32

October 28, 2007

Volume 13

Number 40

TA, Campbell KA, Choti MA. Does prophylactic octreotide decrease the rates of pancreatic fistula and other complications after pancreaticoduodenectomy? Results of a prospective randomized placebo-controlled trial. Ann Surg 2000; 232: 419-429 Behrend M, Kluge E, Schuttler W, Klempnauer J. A comparison of interrupted and continuous sutures for tracheal anastomoses in sheep. Eur J Surg 2002; 168: 101-106 Friedman E, Perez-Atayde AR, Silvera M, Jonas RA. Growth of tracheal anastomoses in lambs. Comparison of PDS and Vicryl suture material and interrupted and continuous techniques. J Thorac Cardiovasc Surg 1990; 100: 188-193 Max E, Sweeney WB, Bailey HR, Oommen SC, Butts DR, Smith KW, Zamora LF, Skakun GB. Results of 1,000 singlelayer continuous polypropylene intestinal anastomoses. Am J Surg 1991; 162: 461-467 Chen YX, Chen LE, Seaber AV, Urbaniak JR. Comparison of continuous and interrupted suture techniques in microvascular anastomosis. J Hand Surg [Am] 2001; 26: 530-539 Bardini R, Bonavina L, Asolati M, Ruol A, Castoro C, Tiso E. Single-layered cervical esophageal anastomoses: a prospective study of two suturing techniques. Ann Thorac Surg 1994; 58: 1087-1089; discussion 1089-1090 Burch JM, Franciose RJ, Moore EE, Biffl WL, Offner PJ. Singlelayer continuous versus two-layer interrupted intestinal anastomosis: a prospective randomized trial. Ann Surg 2000; 231: 832-837 S- Editor Liu Y L- Editor Kremer M E- Editor Li HY

www.wjgnet.com



RAPID COMMUNICATION

Type 2 dıabetes mellıtus and CA 19-9 levels Oya Uygur-Bayramiçli, Resat Dabak, Ekrem Orbay, Can Dolapçıoğlu, Mehmet Sargın, Gamze Kılıçoğlu, Yüksel Güleryüzlü, Alpaslan Mayadağlı Oya Uygur-Bayramiçli, Can Dolapçıoğlu, Department of Gastroenterology, Kartal State Hospital, Istanbul 81110, Turkey Resat Dabak, Ekrem Orbay, Mehmet Sargın, Department of Family Medicine, Kartal State Hospital, Istanbul 81110, Turkey Gamze Kılıçoğlu, Department of Radiology, Haydarpasa Numune State Hospital, Istanbul 81110, Turkey Yüksel Güleryüzlü, Department of Internal Medicine, Kartal State Hospital, Istanbul 81110, Turkey Alpaslan Mayadağlı, Department of Radiotherapy, Kartal State Hospital, Istanbul 81110, Turkey Correspondence to: Resat Dabak, MD, Department of Family Medicine, Kartal State Hospital, Prof Dr. Ali Nihat Tarlan Cad. No. 55/8 Bostancı, Istanbul 81110, Turkey. [email protected] Telephone: +90-216-5414318 Fax: +90-216-5414318 Received: June 17, 2007 Revised: July 31, 2007

Abstract AIM: To prospectively investigate serum CA 19-9 levels in type 2 diabetic patients in comparison with age- and gender-matched control subjects. METHODS: We recorded duration of diabetes and examined fasting glucose levels, HbA1c levels and serum CA 19-9 levels in 76 type 2 diabetic patients and 76 controls. Abdominal CT was performed in order to eliminate abdominal malignancy in the diabetic and control groups. RESULTS: The average CA 19-9 level was 46.0 ± 22.4 U/mL for diabetic patients whereas it was 9.97 ± 7.1 U/mL for the control group (P < 0.001 ). Regression analysis showed a positive correlation between diabetes and CA 19-9 independent from age, gender, glucose level and HbA1c level (t = 8.8, P < 001 ). Two of the diabetic patients were excluded from the study because of abdominal malignancy shown by CT at the initial evaluation. For all patients, abdominal CT showed no pancreatic abnormalities. CONCLUSION: CA 19-9 is a tumor-associated antigen, which is elevated in pancreatic, upper gastrointestinal tract, ovarian hepatocellular, and colorectal cancers, as well as in inflammatory conditions of the hepatobiliary system, biliary obstruction and in thyroid diseases. Diabetes has been claimed to be a risk factor for pancreatic cancer, which is increasing its incidence and has one of the lowest survival rates of all cancers. CA 19-9 is used in the diagnosis of pancreatic cancer but is also a marker of pancreatic tissue damage that might

be caused by diabetes. We propose that a higher cutoff value of CA 19-9 should be used in diabetics to differentiate benign and malignant pancreatic disease, and subtle elevations of CA 19-9 in diabetics should be considered as the indication of exocrine pancreatic dysfunction. © 2007 WJG . All rights reserved.

Key words: CA 19-9; Diabetes mellitus; Chronic pancreatitis Uygur-Bayramiçli O, Dabak R, Orbay E, Dolapçıoğlu C, Sargın M, Kılıçoğlu G, Güleryüzlü Y, Mayadağlı A. Type 2 dıabetes mellıtus and CA 19-9 levels. World J Gastroenterol 2007; 13(40): 5357-5359


INTRODUCTION CA 19-9 is a tumor-associated antigen that was originally defined by a monoclonal antibody produced by a hybridoma prepared from murine spleen cells immunized with a human colorectal cancer cell line. CA 19-9 is elevated in pancreatic cancers, in cancers of the upper gastrointestinal tract, in ovarian cancer, hepatocellular cancer, colorectal cancer, as well as in inflammatory conditions of the hepatobiliary system and in thyroid diseases[1]. It may also be elevated in malignant and benign cases of biliary obstruction[2]. Diabetes has been claimed to be a risk factor for pancreatic cancer, which is increasing its incidence and has one of the lowest survival rates of all cancers[3]. The association between diabetes and pancreatic cancer remains controversial. A meta-analysis of 20 studies found a two-fold increased risk of pancreatic cancer among diabetic patients of 5 years duration, suggesting that diabetes is a risk factor for the tumor[4]. Other studies have concluded that cancer precedeed and caused diabetes[5]. There are even some studies suggesting that diabetes protects against pancreatic cancer[6]. CA 19-9 is used in the diagnosis of pancreatic cancer but is also a marker of pancreatic tissue damage that might be caused by diabetes. Therefore it is necessary to define the normal range of CA 19-9 in type 2 diabetic patients in order to eliminate additional interventional approaches. In this study, we prospectively evaluated serum CA 19-9 levels in type 2 diabetic patients in comparison with age- and gender-matched control subjects. www.wjgnet.com

ISSN 1007-9327

CN 14-1219/R


MATERIALS AND METHODS 76 consecutive, type 2 diabetic patients and 76 age- and gender-matched healthy controls comprised the study groups. We recorded duration of diabetes and examined fasting glucose levels, HbA1c and serum CA 19-9 levels. CA 19-9 was measured with a chemiilluminecence immunoassay (Roche Diagnostics E 170 analyser). The normal range of CA 19-9 was defined as less than 27 U/mL. Abdominal CT was performed in order to eliminate abdominal malignities for the diabetic and control groups.

RESULTS Characteristic features of the control and study groups are shown in Table 1. Two of the diabetic patients were excluded from the study because of abdominal malignancy found with CT at the initial evaluation (one had an adrenal mass and the other prostatic carcinoma). Average CA 19-9 levels were 46.0 ± 22.4 U/mL in diabetic patients whereas it was found to be 9.97 ± 7.1 U/mL in the control group (P < 0.001). Regression analysis showed a positive correlation between diabetes and CA 19-9, independent from age, gender, glucose level and HbA1c level (t = 8.8, P < 001) (Figure 1). For all patients, abdominal CT showed no pancreatic abnormalities.

DISCUSSION Diabetes mellitus is a chronic inflammatory disease of the pancreas. The mechanisms of glucose intolerance include insulin resistance and destruction of islet beta cells. Most of these conditions damage exocrine tissue and islet cells and both of these systems are anatomically and functionally related. There are no capsules or basement membranes around islets and there are cell-to-cell contacts between exocrine and endocrine cells. There are direct connections between the capillaries of islets and acini that underlie the regulatory connections between islet hormones and exocrine pancreatic secretion, or in other words the relation between parenchymal disease and beta-cell dysfunction[3]. Insulitis is a progressive phenomenon in diabetes mellitus. The United Kingdom Prospective Diabetes Study evaluated beta cell function in diabetes using the homeostasis model assessment and found that beta cell function was already reduced by 50% at diagnosis and that there was subsequent deterioration regardless of therapy[7]. Diabetes can even be described as the last step of chronic pancreatitis with the new developing concepts of pathogenesis[8]. On the other hand, patients with chronic pancreatitis often suffer from endocrine pancreatic dysfunction, which leads to a secondary form of diabetes. This form of diabetes accounts for < 1% of all diabetic cases[8]. Eighty percent of patients with chronic pancreatitis develop an overt diabetes mellitus in the long run and diabetes mellitus is an independent risk factor for mortality in patients with chronic pancreatitis[9,10]. Chronic pancreatitis is a risk factor for pancreatic cancer[11] and the same is also true for diabetes[3]. Therefore diabetic patients have to be followed up for pancreatic cancer. CA 19-9 is a tumor marker used for the diagnosis of www.wjgnet.com

October 28, 2007

Volume 13

Number 40

Table 1 Sex distribution and mean age of patients Sex Male 19 17

Type Ⅱ DM Control

Female 55 59

Mean age (yr) 53.3 52.5

50.00

40.00 CA 19-9 level (U/mL)

5358

30.00 20.00 10.00 0.00 Type Ⅱ DM

Control

Figure 1 Mean CA 19-9 levels in Type Ⅱ DM and Control groups.

pancreatic cancer with a sensitivity of 70%-90% and a specificity of 68%-91%[12]. The main limitation of CA 19-9 is that it can also be elevated in patients with nonmalignant obstructive jaundice, thyroid disease and ovarian diseases resulting in impaired specificity of the marker. The solution to this problem was found with the use of higher cut-off values and a combination of tumor markers in differential diagnosis of gastrointestinal cancers. Lewis blood group status is also important for the interpretation of CA 19-9 because 10% of the Caucasian population who are Lewis genotype negative are unable to express CA 19-9[13]. Diabetes is also associated with an increased risk of hepatocellular carcinoma[14]. Taking the close relation between exocrine and endocrine pancreatic function into consideration, the use of CA 19-9 in diabetics is controversial. The relationship between diabetes mellitus and CA 19-9 has been studied by Benhamou et al[15] and they investigated the relationship between CA 19-9 and metabolic control of diabetes in 51 adult patients. They concluded that CA 19-9 in diabetic patients is raised in acute metabolic situations, which correlated very well with blood glucose concentration. In contrast to Benhamou et al, 64 diabetic patients examined by Banfi et al[16] showed no correlation between CA 19-9 and biochemical markers of metabolic compensation in diabetes. In our study we also found that CA 19-9 levels were higher in diabetic patients in comparison to controls (P < 0.001 ). The elevation of CA 19-9 in diabetics was less than twice of the normal range. Regression analysis showed a positive correlation between diabetes and CA 19-9 independent from age, gender, glucose level and HbA1c level (t = 8.8, P < 001). We could not identify any pancreatic or gastrointestinal cancer in the diabetic patients with a high level of CA 19-9, which is consistent with the concept of exocrine pancreatic dysfunction in diabetes. We propose that a higher cut-off value for CA 19-9

Uygur-Bayramiçli O et al . CA 19-9 and diabetes

should be used for diabetics to differentiate benign and malignant pancreatic disease, and subtle elevations of CA 19-9 in diabetics should be considered as an indication of exocrine pancreatic dysfunction.

COMMENTS Background

Ca 19-9 is a widely used antigen in the diagnosis of pancreatic and gastrointestinal cancers but its elevation might also be due to some nonspecific causes. Chronic inflammation such as diabetes mellitus can lead to pancreatic tissue damage and to a nonmalignant cause of high Ca 19-9 levels. Therefore the “normal “ interval of Ca19-9 should be in defined in type 2 diabetic patients.

Research frontiers

Insulitis is a progressive phenomenon in type 2 diabetes and United Kingdom Prospective Diabetes Study confirmed this entity.Chronic pancreatitis is a risk factor for diabetes and also for pancreatic cancer. The studies by Benhamou and Banfi found controversial data regarding the correlation between Ca 19-9 and diabetes.


Elevation of Ca 19-9 in type 2 diabetics has been associated with bad metabolic control in previous studies and no morphological studies was done. We performed abdominal CT in all the patients in order to exclude any pancreatic malignancy and found out that Ca 19-9 was elevated in all the diabetic patients.

Applications

We have to define a new cut-off value for Ca 19-9 level in type 2 diabetics in order to eliminate unnecessary investigations for pancreatic cancer in this subgroup.This article can lead to further study the effect of insulitis and chronic pancreatitis on Ca 19-9 expression in experimental and morphological studies.

Terminology

Ca 19-9 is a tumor-associated antigen which is originally defined by a monoclonal antibody that has been produced by a hybridoma prepared from murine spleen cells immunized with a human colorectal cancer cell line.

Peer review

This study has interesting scientific and innovative contents, and good readability.

REFERENCES 1

Locker GY, Hamilton S, Harris J, Jessup JM, Kemeny N, Macdonald JS, Somerfield MR, Hayes DF, Bast RC Jr. ASCO 2006 update of recommendations for the use of tumor markers in gastrointestinal cancer. J Clin Oncol 2006; 24: 5313-5327

5359 2

Murray MD, Burton FR, Di Bisceglie AM. Markedly elevated serum CA 19-9 levels in association with a benign biliary stricture due to primary sclerosing cholangitis. J Clin Gastroenterol 2007; 41: 115-117 Mohan V,Premalatha G and Pitchumoni CS. Pancreatic 3 diseases and diabetes. In: Textbook of diabetes. Eds: Pickup JC and Williams G. Third edition. USA: Blackwell Publishing, 2003: 28: 1-15 Rosewicz S, Wiedenmann B. Pancreatic carcinoma. Lancet 4 1997; 349: 485-489 Everhart J, Wright D. Diabetes mellitus as a risk factor for 5 pancreatic cancer. A meta-analysis. JAMA 1995; 273: 1605-1609 Gullo L. Diabetes and the risk of pancreatic cancer. Ann Oncol 6 1999; 10 Suppl 4: 79-81 U.K. Prospective Diabetes Study 16. Overview of 6 years' 7 therapy of type II diabetes: a progressive disease. U.K. Prospective Diabetes Study Group. Diabetes 1995; 44: 1249-1258 8 Göke FJM, Göke B. Optimal control of diabetes mellitus in pancreatitis. Pancreatitis: Advances in pathobiology, diagnosis and treatment. In: Ammann RW, Büchler MW, Asler G, DiMagno EP, Sarner M, editors. Dordrecht: Springer, 2005: 226-232 Malka D, Hammel P, Sauvanet A, Rufat P, O'Toole D, Bardet P, 9 Belghiti J, Bernades P, Ruszniewski P, Levy P. Risk factors for diabetes mellitus in chronic pancreatitis. Gastroenterology 2000; 119: 1324-1332 10 Levy P, Milan C, Pignon JP, Baetz A, Bernades P. Mortality factors associated with chronic pancreatitis. Unidimensional and multidimensional analysis of a medical-surgical series of 240 patients. Gastroenterology 1989; 96: 1165-1172 11 Lowenfels AB, Maisonneuve P. Chronic pancreatitis: precursor of carcinoma? Pancreatitis: Advances in pathobiology, diagnosis and treatment. In: Ammann RW, Büchler MW, Asler G, DiMagno EP, Sarner M, editors. Dordrecht; The Netherlands: Springer, 2005: 232-239 12 Goonetilleke KS, Siriwardena AK. Systematic review of carbohydrate antigen (CA 19-9) as a biochemical marker in the diagnosis of pancreatic cancer. Eur J Surg Oncol 2007; 33: 266-270 13 Aoki Y, Yanagisawa Y, Ohfusa H, Kawa S, Oguchi H, Furuta S. Elevation of serum CA 19-9 in parallel with HbA1c in a diabetic female with the Lewis(a+b-) blood group. Diabetes Res Clin Pract 1991; 13: 77-81 14 El-Serag HB, Hampel H, Javadi F. The association between diabetes and hepatocellular carcinoma: a systematic review of epidemiologic evidence. Clin Gastroenterol Hepatol 2006; 4: 369-380 15 Benhamou PY, Vuillez JP, Halimi S, Meffre G, Bachelot I. Influence of metabolic disturbances of diabetes mellitus on serum CA 19-9 tumor marker. Diabete Metab 1991; 17: 39-43 16 Banfi G, Ardemagni A, Bravi S, Pacchioni M, Bonini P. Are diabetic metabolic compensation and CA19.9 really correlated? Int J Biol Markers 1996; 11: 207-210 S- Editor Liu Y L- Editor Lutze M E- Editor Li JL

www.wjgnet.com



RAPID COMMUNICATION

Holistic Acupuncture approach to idiopathic refractory nausea, abdominal pain and bloating Ann Ouyang, Lihua Xu Ann Ouyang, Lihua Xu, Division of Gastroenterology and Hepatology, Department of Medicine, The Milton S. Hershey Medical Center, College of Medicine, Pennsylvania State University, Hershey, PA 17033, United States Correspondence to: Ann Ouyang, MD, H045, Division of Gastroenterology and Hepatology, The Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033, United States. [email protected] Telephone: +1-717-5318741 Fax: +1-717-5316770 Received: May 29, 2007 Revised: August 8, 2007

Abstract AIM: To evaluate the effectiveness of a holistic acupuncture approach on nausea, pain, bloating and electrogastrogram (EGG) parameters in patients with intractable symptoms. METHODS: Twelve patients with no or mild nausea (those without nausea had bloating or pain) and 10 with a history of moderate to severe nausea were referred for acupuncture. All underwent an EGG and were treated at acupuncture points PC6, SP4 and DU20. Visual analog scales (VAS) assessing severity of nausea, pain and bloating were obtained before and after acupuncture treatment. Nineteen patients received three and three patients received two treatments. RESULTS: VAS scores for nausea reflected the clinical assessment and differed significantly between mild and moderate/severe nausea groups. Acupuncture significantly improved severity of nausea in both groups with improved pre-treatment nausea between the first and third treatments in the moderate/severe nausea group. Pain scores improved with acupuncture in the mild nausea group only and bloating improved only with the first treatment in this group. Patients with bloating with VAS scores greater than 35 pre-treatment improved with acupuncture and over all VAS scores for pain improved with treatment. Acupuncture increased the power in the 2.7 to 3.5 cpm range in the EGG. CONCLUSION: In this uncontrolled clinical study, a holistic acupuncture approach significantly improved nausea in patients with refractory symptoms and increased the power in the 2.7-3.5 cpm component of the electrogastrogram. Bloating and pain VAS scores improved acutely with treatment. This study suggests that acupuncture may be effective in this refractory group of patients and further study using appropriate controls is warranted. www.wjgnet.com

© 2007 WJG . All rights reserved.

Key words: Acupuncture; Functional bowel disorder; nausea; Pain; Holistic medicine Ouyang A, Xu L. Holistic Acupuncture approach to idiopathic refractory nausea, abdominal pain and bloating. World J Gastroenterol 2007; 13(40): 5360-5366


INTRODUCTION Nausea is a debilitating symptom that severely impacts on quality of life. Surveys of patients with functional dyspepsia (chronic or recurrent abdominal pain without an anatomic or inflammatory cause) and patients undergoing chemotherapy, identify nausea as one of the symptoms with the greatest negative impact on quality of life[1,2]. It is a complicated symptom to study and treat because it is often a co-morbid symptom and multiple factors impact on its etiology and severity. Nausea is a prominent symptom in approximately 20% of patients recruited to dyspepsia studies in whom pain is the major complaint[1]. Patients who present with chronic or recurrent nausea in whom no biochemical, anatomic or inflammatory abnormalities can be found, are diagnosed as having functional nausea[3]. Efforts to determine a single pathophysiologic process in patients with functional nausea have been unsuccessful. Some patients have gastroparesis, or delayed emptying of solid foods from the stomach, either related to other medical conditions such as diabetes mellitus or without any obvious underlying systemic disorder. Others have normal gastric emptying despite severe nausea. Even when a condition such as gastroparesis is identified, it is unclear whether the symptom of nausea can be directly ascribed to that condition[4]. Symptoms of nausea can vary in severity over time despite persistent delayed gastric emptying, suggesting that gastroparesis alone does not explain the severity of nausea. Medications for treatment of nausea are limited to those that increase gastric or duodenal contractile activity, the prokinetic agents, and those affecting the chemoreceptor trigger zone[3]. Many prokinetic and centrally acting medications have significant side effects. Psychological factors are often important. Even following chemotherapy, a condition where nausea is known to be a direct effect of medications, the severity of nausea is impacted by psychological stress from family

Ouyang A et al . Holistic acupuncture treatment for nausea

relations[5]. Psychotropic agents may be helpful in the treatment of nausea, but many have side effects that may be counterproductive to gastric function, such as the anticholinergic action of tricyclic antidepressants. The complexity of the processes contributing to nausea does not lend itself to the usual “modern” medical approach to disease, which includes treating each abnormal condition with separate medications. In contrast, the ancient Chinese paradigm of medicine involves a holistic approach that uses a paradigm of deviation from balance in energy or “Qi” and accepts the concept that many conditions that would appear to involve disparate organ systems may be intertwined resulting in “imbalance”. Improvement in symptoms requires the treatment of multiple conditions to restore balance[6]. Acupuncture has been used since antiquity to treat patients with nausea 7 and has been subjected to clinical study. It has been used with moderate success in patients with chemotherapy induced nausea and post-operative nausea[7,8,9]. A search of the literature revealed no reports of its use in treating functional nausea. The aims of this study were to examine the effect of acupuncture, using a holistic approach, in patients referred for nausea and/or abdominal pain, refractory to routine medical treatment. We examined the effect on the symptoms of nausea, bloating, and pain and on gastric motor activity, as assessed by electrogastrography. The holistic approach included acupuncture therapy at three points, PC6, SP4 and DU20. These are described in detail in the methods section.

MATERIALS AND METHODS Subjects and treatment Charts were reviewed of 22 patients (3 male, 19 female. Mean 44.1 ± 2.7 years, range 17-66) who were treated with acupuncture between June 2003 and December 2005. Patients were referred to Hershey Medical Center for care and treatment with prokinetic agents and usual therapy had failed. All patients were interested in pursuing alternative medical therapy. All patients had undergone the same treatment plan. Charts were reviewed for the history of the severity and duration of nausea, abdominal pain and bloating and prior gastric emptying studies. Fourteen of the patients had a documented diagnosis of depression on their chart. The other patients had no documentation of psychiatric disorders but no documentation stated specifically that there were no psychological issues. From the chart narrative, patients were determined to have either no nausea or mild nausea (those with no nausea complained primarily of abdominal bloating and pain) or moderate to severe nausea. The severity of each symptom was also assessed before and after each acupuncture treatment using a visual analog scale (VAS) with 0 for no symptoms and 100 for the worst symptom during the first three treatments. The assessment by the physician in clinic was not known to the subject who completed the VAS score and was based on patients’ history of the complaint. EGGs were also performed on all patients[10]. During the treatment sessions, the acupuncture points P6, SP4 bilaterally and DU20 were needled for 30 min, while the EGG was recorded. A

5361

VAS was completed for each of the symptoms of nausea, pain and bloating before and after each acupuncture treatment. Patients returned for at least one or two additional treatments. Nineteen patients had three treatment and three patients underwent two treatments. The average duration between treatments was 15 ± 1.9 d (mean ± SE), range 7-33 d. The study was approved by the Institutional Review Board of the Hershey Medical Center (November 8, 2006). Electrogastrogram (EGG) recording and analysis All patients underwent a clinical EGG following our standard protocol[10]. Patients fasted overnight. On the day of treatment, each patient consumed a standard 200 Kcal snack of 8oz of apple juice and two slices of toast. Each subject then fasted for an additional two hours after which time EGG recordings were obtained. On the first treatment day, a water load test was performed after 15 min of baseline EGG. EGG recording was then continued during the 30 min of acupuncture treatment. Signals were passed through 0.016 Hz high-pass filter and a 0.25 Hz low-pass filter. The EGG signal was reviewed to remove areas of artifact and the resulting EGG signal was digitized for computer analysis and subjected to fast Fourier transform to extract the frequency information present in the EGG signal. A running spectral analysis of the component wave form was followed over time. The percent power in the frequency ranges of 1.0-2.5 cpm, 2.5-3.7 cpm and 3.7 to 10 cpm were recorded. Water load test A water load test was performed at the first treatment session. After a 15 min baseline EGG was recorded, the patients ingested water “until full” within five minutes. The water was non-carbonated spring water at room temperature (23℃). The volume (mL) of water drunk was recorded[10]. Visual analog scale (VAS) for symptoms A visual analog scale (VAS) with a score from 0-100 points was utilized to assess the intensity of each of the following visceral sensations: nausea, pain, bloating, with 0 being none and 100 being unbearable. Each subject completed a VAS score prior to the study and before and after each acupuncture treatment. Acupuncture points Acupuncture points P6, SP4 and DU20 were selected for this treatment regimen[7]. The point PC6 (Neiguan) is on the pericardium meridian of hand-jueyin. It is located 2 cm above the transverse crease of the wrist, between the tendons of muscularis palmaris longus and the flexor radialis. The traditional indications for needling of the PC6 point are stomach ache, nausea, vomiting, hiccough, mental disorders, epilepsy, insomnia, febrile disease, irritability, and malaria. The point SP4 (Gong sun) lies on the Spleen meridian of the foot-taiyin. It is located in the depression distal and inferior to the base of the first metatarsal bone. The traditional indications for treatment of the SP4 point relate to the gastrointestinal (GI) or central nervous system and are abdominal pain and distension, diarrhea, dysentery, www.wjgnet.com

5362

ISSN 1007-9327

CN 14-1219/R

World J Gastroenterol 100

Table 1 Demographics and clinical characteristics of patients in the mild or severe nausea groups

143 ± 7 1/12 0.25 ± 0.04

150.8 ± 10.4

NS

P = 0.0002

Number 40 Baseline nausea Baseline pain Baseline bloating

80

P NS NS

Volume 13

90

70 VAS score

Gender Age (yr ± SE) Weight (lbs ± SE) Diabetes Severity of nausea1 Severity of pain1 Severity of bloating1 Duration of symptoms in months (mean ± SE)

Mild nausea (n = 12) Severe nausea (n = 10) symptom score 0 or 1 symptom score 2 or 3 10F:1M 9F:1M 47.5 ± 2.9 40 ± 4.6

October 28, 2007

60 50 40 30

1/10 2.6 ± 0.15

P < 0.0001

20

2 ± 0.3

1.1 ± 0.37

NS

10

2.3 ± 0.37

1.2 ± 0.5

NS

24.9 ± 5

15.3 ± 3.4

NS2

Range 7-60

Range 4-36

0

Maild nausea

Severe nausea

Figure 1 VAS score for symptoms of nausea, abdominal pain and bloating at baseline for patients in either the mild or severe nausea groups. A significant difference was seen in the VAS score for nausea between the groups (P = 0.0002).

1

Symptom key: 0-none, 1-mild, 2-moderate, 3-severe (based on narrative and complaint in patient chart). 2Using Mann-Whitney test for non-parametric data.

borborygmus, nausea, vomiting, hiccough, and insomnia. The point DU20 (Baihui or GV 20) lies on the Governor vessel. It is located in the midline of the head, at the intersection of the line connecting the apices of both ears and a line that lies in the midline in the saggital orientation on the head. The traditional indications for using the DU20 point are vertigo, headache, tinnitus, nasal obstruction, aphasia by apoplexy, coma, mental disorders, prolapse of the rectum and the uterus. It was felt that this combination of points would treat specific nausea symptoms and gastrointestinal complaints as well as depression, which was felt to be a common problem for many patients with refractory nausea. Statistical analysis The charts were reviewed for age, weight, gender, and assessment of the severity of the symptoms of nausea, vomiting, bloating and pain at the clinic visit. As the degree of nausea was variable patients were analyzed in two groups based on the severity of nausea determined by history at the initial clinic assessment-either in a “none to mild” nausea group or a “moderate to severe” nausea group. Separation of patients into the two groups was based on clinical assessment of the chart and was determined prior to any knowledge of the outcome of treatment. The effect of acupuncture before and after treatment in the different groups and its effect on the parameters of VAS scores for nausea, bloating and pain and power of EGG in the different frequencies was analyzed using ANOVA. If a significance level of P < 0.05 was determined, direct comparisons between specific parameters was performed using a student t test (two-tailed) for parametric data or Mann-Whitney test for non-parametric data. Data are presented as mean ± SE.

RESULTS Patients were primarily analyzed as two groups, based on www.wjgnet.com

an assessment of the severity of nausea as determined by obtaining a history from patients at their initial clinic visit. Those with no or mild nausea were grouped in the mild nausea group (n = 12), those with moderate or severe nausea were grouped in the severe nausea group (n = 10). The demographics and clinical features are shown in Table 1. In the group who complained of mild to no nausea, nine patients did not complain of nausea, but complained more of pain and/or bloating. The frequency of nausea (25% vs 100%) and the severity of nausea was significantly different between the groups (P < 0.05). There was no difference in either the prevalence of abdominal pain or bloating or severity of these symptoms between the two groups. The duration of nausea when present was between seven months and five years in the mild nausea group and between four months and three years in the severe nausea group (not significant [NS]). Only one out of each group was diabetic. At the first visit for acupuncture treatment, the VAS scores for nausea were significantly greater in the severe nausea group than the mild nausea group (68.2 ± 4.4 vs 33.3 ± 5.8, P < 0.0002, Figure 1) and these subjects tolerated the water load test more poorly than did the mild nausea group (429 ± 33.9 vs 642 ± 22 mL, P < 0.0001). There was no significant difference in VAS scores for bloating or pain at baseline between the two groups. Bloating was the predominant symptom in the mild nausea group while both nausea and bloating were equally prominent in the severe nausea group. Effect of acupuncture on symptoms Acupuncture significantly improved the VAS score for nausea in both the mild nausea and severe nausea groups (Figure 2A). There was improvement in the pre-acupuncture nausea score between the first and third treatment in the severe nausea group (P < 0.03) suggesting a persistent effect between treatments. In the mild nausea group, there was no significant difference in the baseline score between the first and third treatment. Acupuncture was less effective for the symptoms of pain or bloating (Figure 2B and 2C). Pain scores were significantly improved during acupuncture in the mild


A

Nausea scores before and after each acupuncture treatment

a

1

2 3 Mild nausea

1

10

2 3 Severe nausea

0

1

2

3

Mod/severe bloating (VAS > 35)

Pain scores before and after each acupunture treatment a a

60

30 20

1

1

2 3 Mild nausea

2

3

Severe nausea

Bloating scores before and after each acupuncture treatment 70

1 2 3 Mild bloating (VAS ≤ 35)

70

VAS score

VAS score

a

14

17

4

5

5

80

40

80

17

b

90

a

10

a

50 40 30 20

0

9

a

10 13

9

8

a 13

10

1 2 Mild pain (VAS ≤ 35)

3

1 2 3 Mod/severe pain (VAS > 35)

Figure 3 Effect of acupuncture on the symptoms of bloating and pain. Response of patients with a baseline pre-treatment VAS of > 35 (moderate/severe) or < 35 (mild) are shown. The numbers of patients in each group are shown for each treatment session. A significant improvement was seen with acupuncture at the first treatment only for bloating although there was a significant decrease in pretreatment bloating between the first and third treatment. Acupuncture improved pain with most treatments but the improvement was not sustained.

60 VAS score

40

20

50

C

n Number of patients

50

Pain scores before and after each acupuncture treatment

0

P < 0.05 b P < 0.0005

30

10

60

b

a

60 VAS score

VAS score

b

20

B

After treatment

70

a

50

0

Before treatment

80

P < 0.05 b P < 0.005

b

30

a

90

a

60 40

1 First treatment 2 Second treatment 3 Third treatment

b

70

Bloating scores before and after each acupunture treatment

Before acupuncture Rx After acupuncture Rx

P < 0.03

80

5363

50 40 30 20 10 0

1

2 Mild nausea

3

1

2 3 Severe nausea

Figure 2 Effect of acupuncture on the symptoms of nausea (A), abdominal pain (B) and bloating (C) in the patients with mild or severe nausea. The response to each acupuncture treatment is shown.

nausea group. No significant improvement was seen in the severe nausea group although the trend was towards improvement. Although bloating improved during the first acupuncture treatment in the mild nausea group, no significant improvement was seen in the severe nausea group. Additional categorization of patients examined the response of bloating and pain to acupuncture by the pretreatment VAS score, as the examining physician did not score the symptoms of pain and bloating clinically. The VAS for bloating improved with the first acupuncture treatment (n = 22) from 56.2 ± 7.7 to 30.4 ± 5.4 (P < 0.01). The overall pain score for all patients decreased from 35.9 ± 7.8 to 17.8 ± 0.8 (P = 0.08) with the first treatment. We also compared responses to acupuncture in patients with mild vs moderate/severe symptoms of bloating or pain.

We chose an arbitrary cut off of an initial VAS score of 35 out of 100 to differentiate patients with mild bloating or pain from those with moderate or severe bloating or pain (Figure 3). The VAS for bloating improved with initial treatment in those with a baseline VAS score of greater than 35. While there was no acute improvement with subsequent treatments, there was a significant improvement in the pre-treatment VAS for bloating over the three treatments (Figure 3). Acupuncture treatment resulted in improvement of the VAS score for pain in both those with severe/moderate pain and mild pain after the acute acupuncture treatment (Figure 3). However, there was no significant improvement in pre-treatment pain between the first and third treatments. Effect of acupuncture on EGG Acupuncture resulted in a significant improvement in the percent of power in the normal frequency. At baseline, there was no difference in overall EGG diagnosis between the severe and mild nausea groups. When comparing the power in the different frequency components before and after acupuncture, there was a significant improvement in the power in the 2.5-3.7 cpm between the pre-acupuncture www.wjgnet.com

5364

ISSN 1007-9327

CN 14-1219/R


and post-acupuncture EGG following the first and second treatments in the severe nausea patients. The percent power in this range improved from 23.4 ± 7.5 to 30.8 ± 10 following the first treatment (P < 0.05) and from 19.35 ± 10 to 31.2 ± 10.8 with the second treatment (P < 0.03). The third treatment increased the percent power in this frequency range from 28.1 ± 11.3 to 37.3 ± 15 (NS). In the mild nausea group, the percent power in this normal frequency range also improved comparing pre- and postacupuncture treatment but did not reach statistical significance. There was no correlation between the percent power in the 2.5-3.7 cpm range and the symptoms of bloating or pain.

DISCUSSION We report the results of our experience with the use of acupuncture in patients with functional dyspepsia and nausea. A significant improvement in the level of nausea was seen following acupuncture in patients with none to mild nausea (mild nausea group) and those who complained of moderate to severe nausea (severe nausea group). A significant response was seen in both, suggesting that acupuncture given in the holistic method was effective for both mild to severe nausea. Only one patient in each group was diabetic and most had failed other treatment modalities. A review of Pubmed using the search term “acupuncture and nausea” revealed no prior reports of the use of acupuncture for functional dyspepsia or nausea, other than one study of 15 patients with diabetes and symptoms suggestive of gastric dysmotility[11]. There was a significant improvement in the VAS score for pain in the mild nausea group and the first treatment only improved bloating in the mild nausea group. This difference in response between complaints of nausea and the other complaints of pain and bloating suggest that the response seen was not purely placebo. This is also the first reported study in which acupuncture was given in a holistic approach for nausea. Multiple studies report the use of acupuncture in the treatment of chemotherapy induced nausea, postoperative nausea and nausea in pregnancy. Acupuncture is effective in most studies without controls, but its effectiveness is less noticeable if compared to a placebo or sham treatment[7-9]. Controlled studies of acupuncture in the treatment of chemotherapy induced nausea and vomiting suggest a moderate effect with less acute vomiting episodes and an improvement in the mean worst nausea severity in patients treated by acupressure and concurrent anti-emetics[8]. Following elective gynecological surgery, a greater percentage of patients treated with acupressure of PC6 point (also known as P6 point) reported no nausea or vomiting compared to sham treated patients[9]. Several studies suggest effectiveness of acupuncture/acupressure in treating nausea of pregnancy. A randomized controlled trial of acupressure at P6 vs placebo showed improvement of symptoms of nausea over time in both groups with nausea symptoms being less severe in the acupressure treated group[12]. Others report an improvement in duration but not intensity of nausea in pregnancy compared to placebo treatment[13]. Although well-conducted placebo controlled studies failed to show a difference compared to www.wjgnet.com

October 28, 2007

Volume 13

Number 40

sham treatment, acupuncture may prove to be as effective as medications[13,14]. Comparison between studies is difficult because the criteria for recruitment of subjects and presence of potentially confounding factors, such as depression, are often not discussed. Most of the trials involve acupuncture at one or two acupuncture points only, mainly P6, and are therefore different from the usual clinical holistic approach when using acupuncture to treat a patient. A review of several studies suggests that these studies may be underpowered and a true difference between placebo and sham may be missed[15]. Our treatment included acupuncture at three points, two related to the GI tract and one at a point recommended for psychological complaints. These points were chosen based on clinical experience and training and our observation that many of the subjects with nausea have psychological distress. We did not formally test for any psychiatric diagnosis, as this was a clinical treatment approach. The results suggest that acupuncture at the points chosen for this study may be effective in treating nausea and less for pain or bloating when patients are grouped by degree of nausea. When the subjects are grouped into moderate/severe or mild bloating or pain by their first pre-treatment VAS score, an improvement in VAS score was seen in bloating and pain. It is unclear if this can be extrapolated to treatment of functional bloating or pain as the degree of symptom was assessed by the pre-treatment VAS score and it is unknown if this correlates with the degree of chronic symptom complaint. There was an increase in the power of gastric myoelectric activity in the normal range, 2.5-3.7 cpm, as assessed by EGG. Whether this is a primary effect of acupuncture or secondary to a central nervous system response cannot be determined in this study. These data support further study using a placebo-controlled approach. The mechanism of the action of acupuncture is unknown. There are few studies in patients examining the effect in nausea in humans. Hu et al demonstrated that acupressure at the PC6 point decreased symptoms of nausea and decreased the gastric dysrhythmia induced by vection in an optokinetic drum[16]. An increase in the percent of normal frequency in the EGG has been reported in diabetic patients with symptoms suggesting an action of acupuncture on gastric motor function[17]. In these subjects, the ST36 points and electrical stimulation of needles was used. In humans, acupuncture and manipulation at the PC6 point in healthy volunteers selectively activated regions of the superior frontal gyrus, anterior cingulated gyrus and dorsomedial nucleus of the thalamus as well as areas of the cerebellum[17]. The subjects did experience more unpleasantness with active acupuncture than with sham treatment which may explain the cerebral cortical findings. The cerebellar findings may be more specific to the action of acupuncture at PC6, as activation of this brain area is probably unrelated to the pain of needle placement. In animals, acupuncture of the PC6 and ST36 points results in an increased rate of gastric emptying and an increase in the percent of time that the dominant frequency is within the normal range[18]. Based on the effect of acupuncture on the heart rate variability, it was concluded that this effect was associated with increased vagal activity. A


central effect of electroacupuncture has also been suggested by Tatewaki et al who demonstrated that electroacupuncture at PC6 reduced the number of retching and vomiting episodes following vasopressin injection in dogs, an anti-emetic effect which was abolished by pretreatment with naloxone[19]. Whether this proves to be similar for humans is unknown. The effect of acupuncture is complex in animals, with studies showing that in the rat, electrical stimulation of the ST36 point can induce either excitatory or inhibitory effects on gastric contraction[20]. Multiple variables are possible to explain differences in animals: the site of stimulation and the intensity and type of stimulation. These effects in animals support a true physiologic response to stimulation of acupuncture points. In humans, additional variables are present. These include co-morbid conditions, particularly other pain or psychological conditions. The effect may be influenced by belief in the treatment that may contribute to a placebo effect, or may be associated with central nervous system biological responses that may impact on response to acupuncture via a non-placebo mechanism. For example, a study by Pariente et al demonstrated that real acupuncture stimulated activity in the brain, as assessed by fMRI, to a greater extent than placebo and that belief in acupuncture affected the response at other areas of the brain[21]. Our clinical study was not designed to test the effect of placebo or belief in the response. However, the differential effect of acupuncture on the symptoms of nausea compared to pain and bloating in the same subjects suggests that the effect on nausea may not be entirely placebo. These results would support the value of a rigorously designed study to examine the effects of acupuncture on the symptoms of patients with functional nausea.

ACKNOWLEDGMENTS The efforts of Dr. Xu were supported in part by a grant from the American College of Gastroenterology.

5365

Applications

Data from this study would support the need for a controlled study to examine the effect of treatment and to control for factors such as concurrent psychological conditions and belief in acupuncture as a treatment.

Terminology

Functional nausea-symptom of nausea in the absence of anatomic or biochemical abnormalities, such as pregnancy, ulcer disease, obstruction etc.Dyspepsiachronic upper abdominal pain. The symptom may be associated with ulcer, but functional dyspepsia is a condition with similar symptoms but in the absence of identifiable pathology in the stomach.

Peer review

It is an interesting paper integrating Traditional Chinese Medicine (acupuncture) with Western Medicine. The author has included a lot of thought provoking factors in this paper, such as the causes of nausea, the Chinese medical theory of imbalance, the conducted placebo controlled studies failed to show a difference compared to sham treatment, activation of brain region related to needle stimulation, and that acupuncture affected the response, and so on.

REFERENCES 1 2

3 4

5 6 7 8

COMMENTS Nausea is a common and debilitating symptom complaint. In many patients the underlying pathophysiology is unknown and likely represents a combination of gastric pathophysiology and psychological issues. There are few medications available, many of which have significant side effects. The traditional approach of acupuncture allows several likely underlying conditions to be treated at the same time.

9

10

Research frontiers

While acupuncture has been studied in treatment of nausea of chemotherapy, postoperatively and during pregnancy, no studies have been published on its use in functional nausea. Most published studies also use acupuncture and one site, which is a different approach to the general clinical acupuncture approach. Based on experience and the finding that 14/22 subjects had documented history of depression, a holistic approach which treated multiple clinical conditions was used. The same acupuncture points were used in all patients.

11 12 13


The study showed a significant improvement in degree of nausea in subjects who presented with moderate/severe nausea. The degree of nausea improved over the three treatment visits suggesting a lasting effect. Both bloating and pain were improved by acute treatment with acupuncture, but it is unclear whether the pretreatment VAS score accurately reflects the severity of chronic complaints by the patients. The main innovation in this study was the holistic approach to treat both gastrointestinal and psychological conditions concurrently.

14 15 16

Talley NJ, Locke GR 3rd, Lahr BD, Zinsmeister AR, Tougas G, Ligozio G, Rojavin MA, Tack J. Functional dyspepsia, delayed gastric emptying, and impaired quality of life. Gut 2006; 55: 933-939 Stromgren AS, Sjogren P, Goldschmidt D, Petersen MA, Pedersen L, Groenvold M. Symptom priority and course of symptomatology in specialized palliative care. J Pain Symptom Manage 2006; 31: 199-206 Tack J, Talley NJ, Camilleri M, Holtmann G, Hu P, Malagelada JR, Stanghellini V. Functional gastroduodenal disorders. Gastroenterology 2006; 130: 1466-1479 Nowak TV, Johnson CP, Kalbfleisch JH, Roza AM, Wood CM, Weisbruch JP, Soergel KH. Highly variable gastric emptying in patients with insulin dependent diabetes mellitus. Gut 1995; 37: 23-29 Kim Y, Morrow GR. Changes in family relationships affect the development of chemotherapy-related nausea symptoms. Support Care Cancer 2003; 11: 171-177 Chen XN. Chinese Acupuncture and Moxibustion. Beijing: Foreign Languages Press, 1999: 200-201, 416-420, 430-431 Kaptchuk TJ. Acupuncture: theory, efficacy, and practice. Ann Intern Med 2002; 136: 374-383 Ezzo J, Vickers A, Richardson MA, Allen C, Dibble SL, Issell B, Lao L, Pearl M, Ramirez G, Roscoe JA, Shen J, Shivnan J, Streitberger K, Treish I, Zhang G. Acupuncture-point stimulation for chemotherapy-induced nausea and vomiting. J Clin Oncol 2005; 23: 7188-7198 Alkaissi A, Evertsson K, Johnsson VA, Ofenbartl L, Kalman S. P6 acupressure may relieve nausea and vomiting after gynecological surgery: an effectiveness study in 410 women. Can J Anaesth 2002; 49: 1034-1039 Ko ch KL, Hong SP, Xu L. Rep roducib ilit y of gastric myoelectrical activity and the water load test in patients with dysmotility-like dyspepsia symptoms and in control subjects. J Clin Gastroenterol 2000; 31: 125-129 Chang CS, Ko CW, Wu CY, Chen GH. Effect of electrical stimulation on acupuncture points in diabetic patients with gastric dysrhythmia: a pilot study. Digestion 2001; 64: 184-190 Belluomini J, Litt RC, Lee KA, Katz M. Acupressure for nausea and vomiting of pregnancy: a randomized, blinded study. Obstet Gynecol 1994; 84: 245-248 Norheim AJ, Pedersen EJ, Fonnebo V, Berge L. Acupressure treatment of morning sickness in pregnancy. A randomised, double-blind, placebo-controlled study. Scand J Prim Health Care 2001; 19: 43-47 Jewell D, Young G. Interventions for nausea and vomiting in early pregnancy. Cochrane Database Syst Rev 2003: CD000145 Vickers AJ. Statistical reanalysis of four recent randomized trials of acupuncture for pain using analysis of covariance. Clin J Pain 2004; 20: 319-323 Hu S, Stritzel R, Chandler A, Stern RM. P6 acupressure

www.wjgnet.com

5366

ISSN 1007-9327

CN 14-1219/R


reduces symptoms of vection-induced motion sickness. Aviat Space Environ Med 1995; 66: 631-634 17 Yoo SS, Teh EK, Blinder RA, Jolesz FA. Modulation of cerebellar activities by acupuncture stimulation: evidence from fMRI study. Neuroimage 2004; 22: 932-940 18 O u y a n g H , Y i n J , W a n g Z , P a s r i c h a P J , C h e n J D . Electroacupuncture accelerates gastric emptying in association with changes in vagal activity. Am J Physiol Gastrointest Liver Physiol 2002; 282: G390-G396 19 Tatewaki M, Strickland C, Fukuda H, Tsuchida D, Hoshino

20

21

October 28, 2007

Volume 13

Number 40

E, Pappas TN, Takahashi T. Effects of acupuncture on vasopressin-induced emesis in conscious dogs. Am J Physiol Regul Integr Comp Physiol 2005; 288: R401-R408 Tatewaki M, Harris M, Uemura K, Ueno T, Hoshino E, Shiotani A, Pappas TN, Takahashi T. Dual effects of acupuncture on gastric motility in conscious rats. Am J Physiol Regul Integr Comp Physiol 2003; 285: R862-R872 Pariente J, White P, Frackowiak RS, Lewith G. Expectancy and belief modulate the neuronal substrates of pain treated by acupuncture. Neuroimage 2005; 25: 1161-1167 S- Editor Liu Y L- Editor Roberts SE E- Editor Li JL

www.wjgnet.com



RAPID COMMUNICATION

Epidemiology of upper gastrointestinal cancers in Iran: A sub site analysis of 761 cases Noushin Taghavi, Dariush Nasrollahzadeh, Shahin Merat, Abbas Yazdanbod, Mahshid Hormazdi, Masoud Sotoudeh, Shahriar Semnani, Farhad Eslami, Haji-Amin Marjani, Saman Fahimi, Hooman Khademi, Reza Malekzadeh Noushin Taghavi, Dariush Nasrollahzadeh, Shahin Merat, Masoud Sotoudeh, Farhad Eslami, Haji-Amin Marjani, Saman Fahimi, Hooman Khademi, Reza Malekzadeh, Digestive Disease Research Center, Shariati Hospital, Medical Sciences, University of Tehran, Tehran, Iran Abbas Yazdanbod, Ardabil University of Medical Sciences, Tehran, Iran Mahshid Hormazdi, Mehr General Hospital, Iran University of Medical Sciences, Tehran, Iran Shahriar Semnani, Golestan University of Medical Sciences, Tehran, Iran Correspondence to: Shahin Merat, MD, Digestive Disease Research Center, Shariati Hospital, N Kargar St, Tehran 14144, Iran. [email protected] Telephone: +98-21-88012992 Fax: +98-21-22253635 Received: June 15, 2007 Revised: August 17, 2007


Key words: Upper gastrointestinal tract; Gastrointestinal neoplasms; Iran Taghavi N, Nasrollahzadeh D, Merat S, Yazdanbod A, Hormazdi M, Sotoudeh M, Semnani S, Eslami F, Marjani HA, Fahimi S, Khademi H, Malekzadeh R. Epidemiology of upper gastrointestinal cancers in Iran: A sub site analysis of 761 cases. World J Gastroenterol 2007; 13(40): 5367-5370


INTRODUCTION Abstract AIM: To define the sub site distribution of upper gastrointestinal cancers in three provinces of Iran. METHODS: T h e s t u d y wa s c a r r i e d o u t i n t h re e provinces in Iran: Ardabil, Golestan, and Tehran. In Arbabil and Golestan, the data was collected from the sole referral center for gastrointestinal cancers and the local cancer registry. For Tehran province, data from two major private hospitals were used. All gastric and esophageal cancer patients diagnosed during the period from September 2000 and April 2002 were included in the study. R E S U LT S : A t o t a l o f 7 6 1 p a t i e n t s w i t h u p p e r gastrointestinal cancers were identified, 314 from Ardabil, 261 from Golestan, and 186 from Tehran. In Tehran, the relative rate of cancer increased from the upper esophagus to the distal stomach. In Golestan, the reverse pattern was observed. In Ardabil, the mid portion (distal esophagus and proximal stomach) was involved most frequently. CONCLUSION: There were considerable variations in the sub site of upper gastrointestinal cancers in the three provinces studied. We cannot provide any explanation for this variation. Further research aimed at explaining the discrepancies in sub site distribution of upper gastrointestinal cancers may help identify important risk factors.

Gastric cancer is the third most common malignancy worldwide [1] . According to a recent report by Iran’s ministry of health, cancer is the third most common cause of mortality, constituting 14% of all deaths in Iran. Upper gastrointestinal (GI) cancers cause 55% of all cancer-related deaths in Iran, with gastric cancer being the most common. Gastric cancer accounts for nearly 50% of all GI cancers[2]. The incidence of squamous cell carcinoma of the esophagus and cancer of the stomach are very high as compared to their incidence in western countries[3,4]. A major problem in classifying upper GI cancers is the lack of a universally accepted and clearly reproducible anatomic landmark separating the gastric cardia from the distal esophagus. Even when the landmarks are defined, cancer frequently destroys the anatomy to the extent that the landmarks become unrecognizable. Therefore, misclassification of gastric cancer occurs frequently[5,6]. Recently, several studies have indicated an increase in the incidence of adenocarcinoma of the distal esophagus compared to cancer of the gastric cardia in western countries. These studies postulate that a significant proportion of adenocarcinomas of the distal esophagus may have been misclassified as cancer of gastric cardia, leading to the apparent increase in cardia cancer[7,8]. In view of recent recognition of the importance of the sub site involvement in upper GI cancers, we aimed to examine the sub sites of upper GI cancers in Tehran and two provinces in north Iran.

www.wjgnet.com

5368

ISSN 1007-9327

CN 14-1219/R


MATERIALS AND METHODS Subjects The study was carried out in three provinces: Golestan, Ardabil, and Tehran. Golestan, located in the south-east of the Caspian Sea, is distinguished from other Caspian littoral provinces by an ethnic composition that has 40% Turkmen ethnicity. This province is located on longitude 51'-56' E, and latitude 36'-38' N, with an altitude of 50-500 meters above sea level. Ardabil is situated near the south-western border of the Caspian Sea on 48'-49' E, 38'-39' N, and altitude of 3010 m. In this mountainous land, 98% of the population is of Azeri ethnicity. Tehran is the most densely populated province of Iran and is located between the other two provinces (51'-52' E, 35'-36' N, altitude 1100-1700 m). Tehran has a diverse ethnicity which is representative of the entire Iranian population. Western lifestyle is prevalent in Tehran. By contrast; traditional lifestyle is still dominant in Golestan and Ardabil. Methods The data was collected from three different sources. The first source was Atrak clinic located in Gonbad, the main Turkmen-resident city of Golestan. Atrak clinic is the sole referral center for gastroesophageal cancers in the region and it also hosts an active cancer surveillance program. We estimate that over 95% of cancers in the region are registered in this center. The second source was Aras clinic established in Ardabil for the management of gastroesophageal cancers. This clinic is also the sole referral center for such cancers in the Ardabil province and is a host to another active cancer surveillance program registering over 95% of cancers in the region. The third source was two private general hospitals in Tehran. These two hospitals together are the primary referral centers for nearly 2 million upper socioeconomic class residents of Tehran. All patients with gastric and esophageal cancer diagnosed during the period from September 2000 to April 2002, were included in the study. An essential inclusion criterion was that the subjects were born in the specified provinces and had been residing there for at least the last 10 years. Endoscopic and pathologic tests were performed by the same team, according to the study protocol in the regions[9]. Uniform endoscopic, surgical, and histologic criteria were used to define the tumor sub site. The sub-site of esophageal tumors was recorded as upper, middle, or lower based on the endoscopy or surgery report. If the location of the main tumor bulk was not obvious, the cancer was recorded as “esophageal”. Non-cardia gastric cancers where defined as those where the center of the tumor was located over 2 cm distal to the gastro-esophageal junction. The gastro-esophageal junction was defined as the most proximal site of the gastric folds. Non-cardia gastric cancers were further divided into antrum, body, and fundus, according to the location of the main bulk of tumor. If the main bulk was www.wjgnet.com

October 28, 2007

Volume 13

Number 40

Table 1 The demographic characteristics of patients in the study

Patients No. Age, yr (SD) Male/Female Urban/Rural residents

Ardabil 314 63 (10.8) 224/90 97/217

Golestan 261 64.4 (11.8) 161/100 85/176

Tehran 186 60.1 (12.7) 123/63 186/0

Total 761 62.8 (11.7) 508/253 368/393

not obvious, the cancer was recorded as “gastric”. Adenocarcinomas of the gastro-esophageal junction area were classified according to the WHO/IARC guidelines as a tumor which crosses the gastro-esophageal junction regardless of the main site of the tumor bulk. Adenocarcinoma of esophagus was located entirely above the gastro-esophageal junction, and adenocarcinoma of gastric cardia was located below the gastro-esophageal junction and was centered within 2 cm from the junction. Statistical analysis Statistical analysis was performed using SPSS for windows, version 11.5. Analysis of variance was applied for comparison between normal numeric variables and chisquare for categorical variables.

RESULTS A total of 761 patients with a confirmed diagnosis of gastric and esophageal cancer were identified. Of the patients included, 41.3% were from Ardabil, 34.3% from Golestan, and 24.4% from Tehran. The demographic characteristics are shown in Table 1. The mean age of the subjects was 63 years (SD 12, range 18-96). There was no difference with respect to the age of the subjects in the three provinces (ANOVA, F = 2.7, P = 0.64). The male to female ratio was 2.0; there was no difference in the sex ratios between the three provinces (χ2 = 9.16, P = 0.10). None of the subjects from Tehran resided in rural regions. This observation is in agreement with the fact that 87% of Tehran’s population resides in urban areas. In Ardabil and Golestan provinces this ratio was 43.1%, and 54.5% respectively. In 54 (7%) cases, the location of the tumor sub site was determined by review of the surgical report. Endoscopic data was used in the remaining subjects. Localization to a specific sub site was not possible in 5.2% of gastric cancers and 2% of esophageal cancers. The details of the anatomic distribution of cancers are shown in Table 2.

DISCUSSION We observed distinct patterns of sub site involvement of upper GI cancers in the three geographic regions of Ardabil, Gonbad and Tehran. Tehran province had a higher rate of antral cancer compared to esophageal or junctional cancers. By contrast, in Ardabil and Golestan provinces, junctional and esophageal cancers were more common respectively, compared to antral cancers.

Taghavi N et al . Sub sites of upper GI cancers in Iran

Table 2 The distribution of upper gastrointestinal cancers in three provinces of Iran n (%)

Stomach Antrum Body Fundus Cardia Unspecified

Ardabil 178 (56.7) 49 (15.6) 47 (15.0) 0 (0) 82 (26.1) 0 (0)

Golestan 93 (35.6) 18 (6.9) 32 (12.3) 1 (0.4) 41 (15.7) 1 (0.4)

Tehran 137 (73.7) 64 (34.4) 29 (15.6) 3 (1.6) 21 (11.3) 20 (10.8)

Total 408 (53.6) 131 (17.2) 108 (14.2) 4 (0.5) 144 (18.9) 21 (2.8)

Junction Esophagus Lower SCC ADC Mid Upper Unspecified

24 (7.6) 112 (35.7) 41 (13.1) 26 15 57 (18.2) 14 (4.5) 0 (0)

13 (5.0) 155 (59.4) 42 (16.1) 41 1 91 (34.9) 19 (7.3) 3 (1.1)

13 (7.0) 36 (19.4) 21 (11.3) 18 3 9 (4.8) 3 (1.6) 3 (1.6)

50 (6.6) 303 (39.8) 104 (13.7) 85 19 157 (20.6) 36 (4.7) 6 (0.8)

Total

314

261

186

761

SCC: Squamous cell carcinoma; ADC: Adenocarcinoma.

The disparity in the relative frequencies of upper GI cancers in different regions could be explained by variations in the environmental and genetic factors. There are several reports that dietary, behavioral, life style and environmental factors may play a causative role in high-risk populations[10]. HP infection is a well recognized factor involved in the pathogenesis of gastric cancer[11,12]. All the three provinces studied have HP prevalence rates of nearly 90% [13,14]. Therefore, in the present study, HP infection is unlikely to have accounted for the observed disparity in the sub-site prevalence of cancer. Gastro-esophageal reflux (GER) is another risk factor associated with lower esophageal and cardia cancers[15]. The prevalence of GER, defined as having reflux symptoms at least once a week, has been found to be 21.2% in Tehran[16] and 30% in Golestan[17,18]. The endoscopic prevalence of erosive GER in Ardabil was 34.7%[19]. The progressive increase in the prevalence rate of GER from Tehran to Golestan to Ardabil correlates with the increase in the rate of cancer of gastric cardia in these provinces, from 11.3% in Tehran to 15.7% in Golestan and 26.1% in Ardabil (Table 2). It is possible that the prevalence of GER may explain the differences in cancer rate, especially that of cardia cancer. However, other explanations are required to determine the basis of the other cancer variations in the present study. In the west, adenocarcinoma of the distal stomach (non-cardia cancer) has shown a decreasing incidence over the last 50 years [20]. By contrast, adenocarcinoma of the cardia of the stomach and the adjacent gastroesophageal junction has increased over the past 25 years. This increase is occurring at rate that exceeds that of any other cancer[21-24]. The opposite trends in the incidence may indicate distinct etiologies for the cancer at these two sub sites of the stomach. Moreover, cancers at these sub sites differ with respect to the underlying gastric phenotype. Non-cardia gastric cancers occur in patients with HP-induced atrophic gastritis and accompanying hypochlorhydria. These patients usually have elevated

5369

serum gastrin and low serum pepsinogen levels[25,26]. On the other hand, cancer of the cardia and gastro-esophageal junction occur in subjects with normal acid secretion and is generally not associated with HP infection[26,27]. Although the distinctive patterns observed in gastric cardia and non-cardia cancers have received much attention in explaining the underlying etiology of cancer, junctional cancers have not excited much interest. One type of cancer pattern is observed in western countries where the incidence of junctional cancer is increasing while that of non-junctional esophageal cancer (SCC) is decreasing. The other pattern is observed in high incidence regions such as Linxian in China and North Iran. In these areas, an increase in junctional cancer is accompanied by high rates of esophageal SCC (not only of the lower esophagus), which is in sharp contrast to the pattern in the west where esophageal SCC is uncommon [28,29]. As with the case of cardia vs noncardia gastric cancers, it is possible that distinct etiologies are involved in the pathogenesis of junctional cancers such that junctional cancers associated with high rates of esophageal SCC may have different etiologies than those not associated with high rates of high esophageal SCC. In summary, the present study represents the largest report from Iran identifying sub site involvement of upper GI cancers. We observed distinct pattern of cancers in the three provinces of Tehran, Ardabil, and Golestan. There is no clear explanation for this disparity, although we postulate that different etiologic factors are likely to be involved.

COMMENTS Background

Gastric cancer is the world’s third most common malignancy. Defining the exact origin of gastric cancers is sometimes difficult especially when the gastroesophageal junction is involved. This difficulty often leads to misclassification of cancers, especially those arising from the gastric cardia and the lower esophagus.

Research frontiers

The incidence of cancer of the gastric cardia and the lower esophagus has shown a rapid change in several areas of the world. This rapid change offers a unique opportunity to study the etiology of these cancers. Many scientists are working on the epidemiologic characteristics of these changes in order to identify the etiologic factors. Thus, the exact determination of the sub site of origin of cancers and their epidemiology is very important.


The decreased prevalence of H pylori infection in some areas is believed to have resulted in a decrease in the incidence of gastric cancers. On the other hand, the increase in prevalence of gastro-esophageal reflux disease is believed to have lead to an increase in lower esophageal cancer.

Applications

We observed different patterns of sub site involvement in different parts of Iran. This difference could indicate different etiologies. Further epidemiologic studies aimed at identifying the probable etiologic factors are required.

Terminology

Gastric cardia is the most proximal 2 cm of stomach located distal to the gastroesophageal junction.

Peer review

In this prospective study, the authors studied the sub site distribution of upper www.wjgnet.com

5370

ISSN 1007-9327

CN 14-1219/R


gastrointestinal cancers in three provinces of Iran and they found that there are considerable variations in the sub site of upper gastrointestinal cancers in the three provinces studied.

15 16

REFERENCES 1

Parkin DM, Pisani P, Ferlay J. Estimates of the worldwide incidence of 25 major cancers in 1990. Int J Cancer 1999; 80: 827-841 Naghavi M. Death in eighteen provinces of Iran. In: Annual 2 report of Iran Ministry of Health and Medical Education. Tehran: Ministry of Health and Medical Education, 2001: 127-135 Derakhshan MH, Yazdanbod A, Sadjadi AR, Shokoohi B, 3 McColl KE, Malekzadeh R. High incidence of adenocarcinoma arising from the right side of the gastric cardia in NW Iran. Gut 2004; 53: 1262-1266 4 Sadjadi A, Malekzadeh R, Derakhshan MH, Sepehr A, Nouraie M, Sotoudeh M, Yazdanbod A, Shokoohi B, Mashayekhi A, Arshi S, Majidpour A, Babaei M, Mosavi A, Mohagheghi MA, Alimohammadian M. Cancer occurrence in Ardabil: results of a population-based cancer registry from Iran. Int J Cancer 2003; 107: 113-118 Ekstrom AM, Signorello LB, Hansson LE, Bergstrom 5 R, Lindgren A, Nyren O. Evaluating gastric cancer misclassification: a potential explanation for the rise in cardia cancer incidence. J Natl Cancer Inst 1999; 91: 786-790 Chandrasoma P, Wickramasinghe K, Ma Y, DeMeester T. 6 Adenocarcinomas of the distal esophagus and "gastric cardia" are predominantly esophageal carcinomas. Am J Surg Pathol 2007; 31: 569-575 Corley DA, Kubo A. Influence of site classification on cancer 7 incidence rates: an analysis of gastric cardia carcinomas. J Natl Cancer Inst 2004; 96: 1383-1387 Chandrasoma P, Wickramasinghe K, Ma Y, DeMeester T. 8 Adenocarcinomas of the distal esophagus and "gastric cardia" are predominantly esophageal carcinomas. Am J Surg Pathol 2007; 31: 569-575 Islami F, Kamangar F, Aghcheli K, Fahimi S, Semnani S, 9 Taghavi N, Marjani HA, Merat S, Nasseri-Moghaddam S, Pourshams A, Nouraie M, Khatibian M, Abedi B, Brazandeh MH, Ghaziani R, Sotoudeh M, Dawsey SM, Abnet CC, Taylor PR, Malekzadeh R. Epidemiologic features of upper gastrointestinal tract cancers in Northeastern Iran. Br J Cancer 2004; 90: 1402-1406 10 Kelley JR, Duggan JM. Gastric cancer epidemiology and risk factors. J Clin Epidemiol 2003; 56: 1-9 11 Uemura N , Ok a mo t o S , Y a ma mo t o S , Ma t su mu r a N , Yamaguchi S, Yamakido M, Taniyama K, Sasaki N, Schlemper RJ. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med 2001; 345: 784-789 12 Dawsey SM, Mark SD, Taylor PR, Limburg PJ. Gastric cancer and H pylori. Gut 2002; 51: 457-458 13 Malekzadeh R, Sotoudeh M, Derakhshan MH, Mikaeli J, Yazdanbod A, Merat S, Yoonessi A, Tavangar M, Abedi BA, Sotoudehmanesh R, Pourshams A, Asgari AA, Doulatshahi S, Alizadeh BZ, Arshi S, Madjidpoor A, Mir Moomen S, Fleischer DE. Prevalence of gastric precancerous lesions in Ardabil, a high incidence province for gastric adenocarcinoma in the northwest of Iran. J Clin Pathol 2004; 57: 37-42 14 Massarrat S, Saberi-Firoozi M, Soleimani A, Himmelmann GW, Hitzges M, Keshavarz H. Peptic ulcer disease, irritable bowel syndrome and constipation in two populations in Iran. Eur J Gastroenterol Hepatol 1995; 7: 427-433

17

18 19

20 21 22

23 24

25

26

27 28

29

October 28, 2007

Volume 13

Number 40

Lagergren J, Bergstrom R, Lindgren A, Nyren O. Symptomatic gastroesophageal reflux as a risk factor for esophageal adenocarcinoma. N Engl J Med 1999; 340: 825-831 Nouraie M, Radmard AR, Zaer-Rezaii H, Razjouyan H, Nasseri-Moghaddam S, Malekzadeh R. Hygiene could affect GERD prevalence independently: a population-based study in Tehran. Am J Gastroenterol 2007; 102: 1353-1360 Pourshams A, Saadatian-Elahi M, Nouraie M, Malekshah AF, Rakhshani N, Salahi R, Yoonessi A, Semnani S, Islami F, Sotoudeh M, Fahimi S, Sadjadi AR, Nasrollahzadeh D, Aghcheli K, Kamangar F, Abnet CC, Saidi F, Sewram V, Strickland PT, Dawsey SM, Brennan P, Boffetta P, Malekzadeh R. Golestan cohort study of oesophageal cancer: feasibility and first results. Br J Cancer 2005; 92: 176-181 Pourshams A, Rahmani AR, Hatami K. Gastroesophageal Reflux Disease in Iran. Govaresh 2005; 10: 48-53 Malekzadeh R, Sotoudeh M, Derakhshan MH, Mikaeli J, Yazdanbod A, Merat S, Yoonessi A, Tavangar M, Abedi BA, Sotoudehmanesh R, Pourshams A, Asgari AA, Doulatshahi S, Alizadeh BZ, Arshi S, Madjidpoor A, Mir Moomen S, Fleischer DE. Prevalence of gastric precancerous lesions in Ardabil, a high incidence province for gastric adenocarcinoma in the northwest of Iran. J Clin Pathol 2004; 57: 37-42 Kelley JR, Duggan JM. Gastric cancer epidemiology and risk factors. J Clin Epidemiol 2003; 56: 1-9 Blot WJ, Devesa SS, Kneller RW, Fraumeni JF Jr. Rising incidence of adenocarcinoma of the esophagus and gastric cardia. JAMA 1991; 265: 1287-1289 Botterweck AA, Schouten LJ, Volovics A, Dorant E, van Den Brandt PA. Trends in incidence of adenocarcinoma of the oesophagus and gastric cardia in ten European countries. Int J Epidemiol 2000; 29: 645-654 Powell J, McConkey CC. Increasing incidence of adenocarcinoma of the gastric cardia and adjacent sites. Br J Cancer 1990; 62: 440-443 Crane SJ, Richard Locke G 3rd, Harmsen WS, Diehl NN, Zinsmeister AR, Joseph Melton L 3rd, Romero Y, Talley NJ. The changing incidence of oesophageal and gastric adenocarcinoma by anatomic sub-site. Aliment Pharmacol Ther 2007; 25: 447-453 Hansen S, Vollset SE, Melby K, Aase S, Jellun E, Fyfe V, Ferguson J, Mccoll KEL. Gastric mucosal atrophy is a strong predictor of non-cardia gastric cancer but not of cardia cancer. Gastroenterology 1998; 114: A606 Ue m ur a N , O k a mo t o S , Y a ma mo t o S , Ma t sumura N, Yamaguchi S, Yamakido M, Taniyama K, Sasaki N, Schlemper RJ. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med 2001; 345: 784-789 MacDonald WC. Clinical and pathologic features of adenocarcinoma of the gastric cardia. Cancer 1972; 29: 724-732 Islami F, Kamangar F, Aghcheli K, Fahimi S, Semnani S, Taghavi N, Marjani HA, Merat S, Nasseri-Moghaddam S, Pourshams A, Nouraie M, Khatibian M, Abedi B, Brazandeh MH, Ghaziani R, Sotoudeh M, Dawsey SM, Abnet CC, Taylor PR, Malekzadeh R. Epidemiologic features of upper gastrointestinal tract cancers in Northeastern Iran. Br J Cancer 2004; 90: 1402-1406 Malekzadeh R, Sotoudeh M, Derakhshan MH, Mikaeli J, Yazdanbod A, Merat S, Yoonessi A, Tavangar M, Abedi BA, Sotoudehmanesh R, Pourshams A, Asgari AA, Doulatshahi S, Alizadeh BZ, Arshi S, Madjidpoor A, Mir Moomen S, Fleischer DE. Prevalence of gastric precancerous lesions in Ardabil, a high incidence province for gastric adenocarcinoma in the northwest of Iran. J Clin Pathol 2004; 57: 37-42 S- Editor Zhu LH L- Editor Anand BS E- Editor Yin DH

www.wjgnet.com



RAPID COMMUNICATION

Colorectal neoplasm: Magnetic resonance colonography with fat enema-initial clinical experience Shuai Zhang, Jun-Wei Peng, Qiang-Ying Shi, Feng Tang, Min-Guo Zhong Shuai Zhang, Jun-Wei Peng, Feng Tang, Min-Guo Zhong, Department of Radiology, Fudan University Cancer Hospital; Depatment of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China Qiang-Ying Shi, Department of Surgery, Fudan University Cancer Hospital; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China Supported by the Developing Research Programs of Science and Technology Commission Foundation of Shanghai, No. 34958038 Correspondence to: Dr. Jun-Wei Peng, Department of Radiology, Fudan University Cancer Hospital; Depatment of Oncology, Shanghai Medical College, Fudan University, 270 Dong’an, Shanghai 200032, China. [email protected] Telephone: +86-21-64433384 Fax: +86-21-64433384 Received: June 4, 2007 Revised: July 20, 2007

echo with inversion recovery sequence is feasible in detecting colorectal neoplasm larger than 10 mm. © 2007 WJG . All rights reserved.

Key words: Magnetic resonance colonography; Contrastto-noise ratio; Virtual endoscopy; Colorectal neoplasm; Fat contrast medium Zhang S, Peng JW, Shi QY, Tang F, Zhong MG. Colorectal neoplasm: Magnetic resonance colonography with fat enema-initial clinical experience. World J Gastroenterol 2007; 13(40): 5371-5375


Abstract AIM: To assess Magnetic resonance colonography with fat enema as a method for detection of colorectal neoplasm. METHODS: Consecutive twenty-two patients underwent MR colonography with fat enema before colonoscopy. T1-weighted three-dimensional fast spoiled gradientecho with inversion recovery sequence was acquired with the patient in the supine position before and 75 s after Gadopentetate Dimelumine administration. Where by, pre and post MR coronal images were obtained with a single breath hold for about 20 s to cover the entire colon. The quality of MR colonographs and patients’ tolerance to fat contrast medium was investigated. Colorectal neoplasms identified by MR colonography were compared with those identified on colonoscopy and sensitivity of detecting the lesions was calculated accordingly. RESULTS: MR colonography with fat enema was well tolerated without sedation and analgesia. 120 out of 132 (90.9%) colonic segments were well distended and only 1 (0.8%) colonic segment was poor distension. After contrast enhancement scan, mean contrast-to-noise ratio (CNR) value between the normal colonic wall and lumen was 18.5 ± 2.9 while mean CNR value between colorectal neoplasm and lumen was 20.2 ± 3.1. By Magnetic resonance colonography, 26 of 35 neoplasms (sensitivity 74.3%) were detected. However, sensitivity of MRC was 95.5% (21 of 22) for neoplasm larger than 10 mm and 55.6% (5 of 9) for 5-10 mm neoplasm. CONCLUSION: MR colonography with fat enema and T1-weighted three-dimensional fast spoiled gradient-

INTRODUCTION Conventional colonoscopy, with its ability to visualize the mucosa directly, is the standard procedure for evaluating the colon[1]. But poor patients’ acceptance and high cost limit the utility of the technique for colorectal screening[2]. This catalyzes the investigation for non-invasive methods to visualize the colon. A great number of studies about computed tomography (CT) colonography have been reported in the United States and Europe [3-6]. But CT colonography has its intrinsic limitation of exposing patients to ionizing radiation and magnetic resonance colonography (MRC) has theoretic capacity for originating soft-tissue contrast 10-1000 times greater than that on CT[7,8]. So the future of colorectal screening has shifted to MRC. To date, most of the MRC research needs administration of a rectal enema containing paramagnetic contrast medium and the acquisition of three-dimensional (3D) gradient-echo pulse sequence[9-12]. This approach directly demonstrates the lumen but not the bowel wall and requires an effective breath hold of up to 30 s. In addition, enema containing paramagnetic contrast medium needs to be compounded by staff themselves and has high cost. These factors may limit its widespread application in the symptomatic and screening population. Recently, several MRC studies used air, carbon dioxide and water as intraluminal contrast medium which provides negative contrast within bowel lumen and have a high accuracy in diagnosing colorectal neoplasm[13-16]. But there are few patients in the MRC studies with air enema and the data required from MRC with water enema could not provide the magnetic resonance virtual endoscopy (MRVE) mode. www.wjgnet.com

5372

ISSN 1007-9327

CN 14-1219/R


In this paper, we evaluated the feasibility of detection of colorectal neoplasm with fat enema in MRC using T1weighted 3D fast spoiled gradient-echo with inversion recovery sequence.

MATERIALS AND METHODS Materials The study was approved by the Ethics Committee of the Fudan University Cancer Hospital. All patients who participated in the study were requested to review and sign a written informed consent. No patient has a history of severe arrhythmia and glaucoma. MRC was performed in consecutive 22 patients (9 men, 13 women) aged 46-86 (mean age 58.6) years. Owing to rectal bleeding, positive fecal occult blood test or altered bowel habits, these patients had been referred for conventional colonoscopy. Exclusion criteria included contraindications to magnetic resonance imaging, such as the presence of a pacemaker, metallic implants in the central nervous system, as well as claustrophobia. Methods Following a standard bowel preparation (oral ingestion of 0.5 L of 20% mannite and 1 L of 5% glucose sodium chloride solution), MRC was performed with a 1.5T MR system (Signa twinspeed with excite; GE medical system, Milwaukee, Wis). No sedative or analgesic agents were administered. An eight-channel body coil was used for signal transmission and reception to permit coverage of the entire colon. To minimize bowel peristalsis, 20 mg of raceanisodamine hydrochloride (No.1 Biochem & Pharm, Shanghai, China) were injected intramuscularly. Following placement of a rectal enema tube (202-26, Shuangling Medical Device, China), fat contrast medium (Kangque; Atai medical system, Inner Mongolia, China) were gently insufflated into the colon at up to 1 m of hydrostatic pressure with the patient lying in the prone position. Fat insufflation was stopped when the patient began to feel uncomfortable. Reinsufflation was not a routine procedure. The administered volume was 1200-1500 mL. MRC was perfor med using T1-weighted threedimensional fast spoiled gradient-echo with inversion recovery sequence. Coronal images were obtained in the supine position with a single breath hold that lasted about 20 s to cover the entire colon. Imaging parameters included: repetition time 4 ms, echo time 2 ms, flip angle 14°, field of view 38-42 cm, matrix 256 × 256 and section thickness 5 mm. Zero interpolation was applied in coronal plane, rendering an effective section thickness of 2.5 mm and a matrix of 512 × 512. About 68 sections were required. Subsequently, using an automatic injector (Optistar, Mallinckrodt; Tyco, Missouri, USA), gadopentetate dimelumine (Magnevist; Schering, Berlin, Germany) was intravenously administered by the basilic vein. Injection parameters included a dosage of 0.2 mmol/kg and a flow rate of 2.0 mL/s, followed by rapid injection of 10 mL of normal saline at the same rate. After a delay of 75 s, 3D acquisition was repeated with identical imaging parameters with the patient in the supine position. The 3D data sets were transferred to a post processing www.wjgnet.com

October 28, 2007

Volume 13

Number 40

workstation (Advantage 4.0; GE Medical System) and evaluated by two radiologists who were blinded to the colonoscopic data. All the data sets were assessed in the multiplanar reformation and MRVE mode. For the purpose of analysis, the entire colon was divided into six segments: cecum, ascending colon, transverse colon, descending colon, sigmoid colon, and rectum. The colonic distension was classified by scores as follows: score 1 = well distension; score 2 = moderate distension, if it was distended enough to visualize the mucosa and fat interface; and score 3 = poor distension, if it was collapsed. For quantitative assessment, region of interest (ROI) was placed in the lumen, all the colonic neoplasm and the adjacent healthy colonic walls. Image noise, defined as the standard deviation of signal intensities (SI) measured in a ROI placed outside the body, were determined[14]. On the basis of these measurements, contrast-to-noise ratio (CNR) values were calculated: CNR = [SI (colonic wall/colonic lesion)-SI (lumen)]/noise. Localization and size of all detected colonic neoplasm was recorded. Colorectal neoplasm was classified based on size: (a) < 5 mm; (b) 5-10 mm; (c) > 10 mm in diameter. Extracolonic lesions, including hepatic metastasis, were also recorded as additional findings. Colonoscopic findings were considered as the standard of reference.

RESULTS All patients completed the MRC without any complication, but 4 patients did not complete colonoscopy for obvious stenosis of colon. Of the 132 colonic segments examined, distensions were documented as follows: 120 (90.9%) had a score of 1, 11 (8.3%) had a score of 2, and 1 (0.8%) had a score of 3. Distinct motion artefacts affecting diagnosis were not found. Mean CNR values were documented as follows: between the normal colonic wall and lumen, pre contrast enhancement scan 8.4 ± 2.1, post contrast enhancement scan 18.5 ± 2.9; between colorectal neoplasm and lumen, post contrast enhancement scan 20.2 ± 3.1. MRC revealed 26 neoplasms including an inflammatory neoplasm (Figure 1), 16 carcinomas (Figure 2) and 9 polyps in 22 patients. The size of five polyps (Figure 3) was between 5 mm and 10 mm and the size of the other 21 neoplasm (Figure 4) was larger than 10 mm in diameter. In patients, there were no false positive MR findings. However, four polyps less than 5 mm, four 5-10 mm polyps and one 20 mm polyp were not found by MRC but by colonoscopy. The 20 mm polyp was a flat polyp. On MRC, neoplasm larger than 10 mm had a higher sensitivity and neoplasm less than 10 mm had a lower sensitivity. The sensitivity for neoplasm of a diameter of < 5 mm, 5-10 mm, and > 10 mm was 0% (0/4), 55.5% (5/9), and 95.5% (21/22). The overall sensitivity was 74.3% (26/35). MRC permitted assessment of extracolonic organs. Hepatic metastases were discovered in two patients. Hepatic and/or renal cysts were seen in three patients.

DISCUSSION MRC is a promising, less intrusive imaging technique that can image the entire colon. The potential advantages of

Zhang S et al . MRC with fat enema on colorectal neoplasm

A

A

B

B

C

C

Figure 1 74-year-old man with history of altered bowel habits. A: The thickened and increased-contrast-uptake bowel wall of sigmoid colon is seen (white arrow) in the coronal gadolinium enhanced T1-weighted image. In liver, an obvious high-intensity neoplasm is discovered (white curved arrow). Subsequent MR scan confirms the diagnosis of a hepatic angioma; B: MR virtual endoscopic rendering confirms stenosis of sigmoid colon (white arrows); C: Post-operation pathology confirms the condition of an inflammatory neoplasm. Specimen shows inflammatory mucosa change (white arrows).

A

5373

B

Figure 2 79-year-old woman with history of right upper quadrant pain. A: Coronal gadolinium enhanced T1-weighted image shows an obviously enhanced 6 cm carcinoma (white arrowheads) in the transverse colon; B: Virtual endoscopic rendering confirms the carcinoma (white arrowheads).

MRC over CT colonography include powerful multi-planar data acquisition, superior contrast resolution and absence of ionizing radiation[8]. To date, most MRC techniques require a water enema containing paramagnetic contrast material gadolinium at 10 mmol/L concentration[9,11,12]. Despite Luboldt et al[9] reporting promising results, the gadolinium enema may limit extensive use of MRC. Given 10 mmol/L as optimal concentration of gadolinium, 1.5 L enema would need 30 mL of 0.5 mol/L gadopentetate dimeglumine. The

Figure 3 54-year-old man with positive results of FOBT. A: Coronal gadolinium enhanced T1-weighted image shows a 7 mm polyp (white arrow) in the sigmoid colon and a cyst (white arrowhead) in the left kidney; B: Virtual endoscopic rendering shows a well-defined filling defect (white arrow) in the sigmoid colon; C: Endoscopy confirms the polyp (white arrows).

Figure 4 66-year-old man with history of right lower quadrant pain. Coronal gadolinium enhanced T1-weighted image shows a 1.1 cm polyp (white arrow) covered by residual water in the ascending colon and multiple hepatic metastases (white curved arrows).

cost of dilute gadolinium enema would be considered as big setback to MRC. In addition, a 3D gradient echo sequence method that is designed to generate maximal signal from interior of the lumen and depress all the other tissue including the luminal wall was performed. Colorectal neoplasms were identified solely on filling defects in the bright colonic lumen. Hence the differentiation between polyps or carcinomas and residual fecal materials or air bubbles proved to be difficult and sometimes even impossible. So the MRC technique needs to be further amplified by the acquisition of 2D gradient echo sequence datasets. And to compensate for the residual air in the lumen, the MRC requires data acquisition both in the supine and the prone patient positions, which results in the complicated examination and longer examination time. Recently, MRC has been used with air or carbon www.wjgnet.com

5374

ISSN 1007-9327

CN 14-1219/R


dioxide insufflation and Susceptibility artefacts which thought to be a big obstacle had been minimized by using a half-Fourier single-shot fast spin-echo (SE) sequence or a short echo time (TE) multi-slice half-Fourier acquisition single-shot turbo spin-echo (HASTE ) sequence [14,17,18]. Although air is cheap and abundant, HASTE sequence acquires two or more breath hold acquisitions to cover the entire colon, and can not afford virtual endoscopic observation. In addition, the MRC with air or carbon dioxide insufflation analysis had shortage of clinical cases. The fat contrast medium that we used in MRC mainly contains salad oil, acacia, menthol and distilled water. It is not absorbed by gastrointestinal tract and has no obvious side-effects. The estimated price of 500 mL of fat contrast medium is $1.25. It is cheaper than paramagnetic contrasts such as gadolinium and less sensitive to susceptibility artefacts than air. It could provide negative contrast within the bowel lumen with a fat-depression sequence. On T1WI, the intensity of fat contrast medium was slightly lower compared to pericolonic fat; however it was too low to visualize air-fat interface when the fat-depression sequence was used. But the intensity of residual water in colon on T1-weighted three-dimensional fast spoiled gradient-echo with inversion recovery sequence was higher than that of fat, which led to distinction of fat-water interface. At the same time, residual water blurred colonic wall and neoplasm, even resulting in neoplasm being missed in diagnosis. But after gadolinium administration, the intensity of colonic wall and neoplasm increased considerably, and the intensity difference between colonic wall, neoplasm and water became distinct. Thus the diagnosis of neoplasm obscured by residual water could be done without a need to change patients’ position. Residual fecal materials can not take up paramagnetic contrast, so it is easy to differentiate between residual fecal materials and neoplasm. This form of direct visualization of the colonic wall and colorectal pathologies stemming from it can reduce the possibility of false positive findings that might mimic neoplasm in MRC with gadolinium solution. T1-weighted three-dimensional fast spoiled gradientecho with inversion recovery sequence is a 3D SPGR acquisition that automatically uses a Partial K spatial filling technique and a segmented special technique. Thus T1-weighted three-dimensional fast spoiled gradientecho with inversion recovery sequence allows shortening of acquisition time, enlargement of spectrum of scan, optimization of fat saturation, and boosting spatial resolution. However, it is essential that the patients maintain the breath-hold position throughout the data acquisition. Minor movements can lead to significant motion artefacts. After being trained, all patients can hold their breath for approximately 25 s. The mean acquisition time of 20 s for covering entire colon can be tolerated by the patients. And thus T1-weighted three-dimensional fast spoiled gradient-echo with inversion recovery sequence is able to accomplish MRC. In terms of lesion detection, the sensitivity of barium enema in detecting polyps of 10 mm or larger has been reported to be varied between 39% and 56%[19]. For CT colonography, the sensitivities of detecting > 9 mm, 6-9 www.wjgnet.com

October 28, 2007

Volume 13

Number 40

mm and < 6 mm polyps were 85%, 70% and 48% and specificities of detecting > 9 mm, 6-9 mm and < 6 mm polyps were 97%, 93% and 92%, respectively[20]. Using a water enema containing paramagnetic contrast material as a contrast medium, MRC of colorectal neoplasm larger than 10 mm has been reported to have a sensitivity of 93%, specificity of 99%, positive predictive value of 92%, and negative predictive value of 98%[9]. While using water as a contrast medium, sensitivity was 93% and specificity was 100% for colonic neoplasm exceeding 5 mm [15] . However, colorectal neoplasm less than 5 mm are often missed in diagnosis[9,15,16,21]. In contrast, MRC using air has shortcomings of low sensitivity, limited coverage and signal drop-off at the borders of the field of view as well as needs further large-scale studies[14,22]. Although the number of patients in our study was small, our results suggest that MRC with fat enema could correctly detect neoplasm lager than 10 mm and neoplasm less than 5 mm would most likely were missed. This technique can be helpful in patients with colonic carcinoma. It can demonstrate the entire colon and exclude synchronous colorectal neoplasm, as well as detect extracolonic involvement, such as hepatic metastases. But there are three limitations of this study. The first is the relatively large slice thickness, 5 mm slice thickness. It may be a reason for missing colorectal neoplasm less than 5 mm in diameter. With technical advancements, it is more likely that colorectal neoplasm less than 5 mm would be identified on MRC with fat enema. The second is that patients required a standard bowel preparation, which results in the limitation of patient acceptance for the technique. Recent studies have shown that fecal tagging technique which does not need a cleansed colon can improve patient acceptance, evaluate the majority of colonic segments inaccessible with conventional colonoscopy and depict polyps exceeding 5 mm in size[23-25]. The third is the small number of the patients and almost all patients with colorectal carcinoma that have large neoplasm. It could be a reason for high sensitivity. In summary, the present data indicates that MRC with fat enema is a promising alternative to colonoscopy for the detection of colorectal neoplasm larger than 10 mm. Fat contrast medium distention seems to be well tolerated by patients. In the future, advancements in technique together with large-scale clinical cases analysis might improve the sensitivity of the method and hence reliability of its results.

COMMENTS Background

MR colonograhpy (MRC) is an effective diagnostic tool for colorectal lesions. Currently two techniques were introduced for MRC based on the signal in the colorectal lumen: “bright lumen” and “dark lumen”. Usually, bowel cleansing is essential for two techniques. Bright lumen MRC can be performed by fast T1 weighted 3D gradient-echo acquisitions within the conforms of a single breath hold and requires a rectal enema containing paramagnetic contrast. Dark lumen MRC is based on contrast generated between an enhancing colorectal wall and a homogeneously dark colonic lumen. The technique requires intravenous administration of paramagnetic contrast and administration of a water or air enema.

Zhang S et al . MRC with fat enema on colorectal neoplasm

5375

Research frontiers

Recently, several studies laid emphasis on dark lumen MRC. Compared with bright lumen MRC, dark lumen MRC has considerable advantages including reduced examination and post-processing time, good differentiation between polyps and stool, direct analysis of the bowel wall and comprehensive assessment of parenchymal abdominal organs within the field of view. However, bowel cleansing is not well accepted by more than half of patients. To assure high patient acceptance of MRC, fecal tagging has been introduced. It is based on the principle of altering the signal intensity of fecal material by adding contrast compounds to regular meals. For fecal tagging, a paramagnetic MR contrast medium and a highly concentrated barium sulphate containing contrast are administrated for bright lumen MRC and dark lumen MRC, respectively.


MRC with fat enema is a technique that could generate a dark colonic lumen with a fat-depression sequence. So it has almost all of the advantages of dark lumen MRC. The data sets required could render an endoluminal virtual view through post-processing. In addition, fat contrast medium is cheaper than paramagnetic contrasts such as gadolinium and less sensitive to susceptibility artefacts than air.

Applications

MRC with fat enema is a promising alternative to colonoscopy for the detection of colorectal neoplasms larger than 10 mm. Moreover, it could depict the infiltration of extra-colonic fat and evaluate other abdominal organs.

9 10

11

12

13 14 15

Terminology

MRC is based on 3D datasets required from cross sectional images. Compared with a conventional colonography, MRC is not limited to endoscopic viewing. Multiplanar reformation analysis can not only depict the colonic lumen and colonic wall, but also assess parenchymal abdominal organs.

16

Peer review

17

The authors assessed Magnetic resonance colonography with fat enema as a method for detection of colorectal neoplasms. Colorectal neoplasms identified by MR colonography were compared with those identified on colonoscopy and sensitivity of detecting the lesions was calculated accordingly. They concluded that MR colonography with fat enema and T1-weighted three-dimensional fast spoiled gradient-echo with inversion recovery sequence is feasible in detecting colorectal neoplasms larger than 10 mm.

18

19

REFERENCES 1 2

3 4

5

6 7 8

Dachman AH, Yoshida H. Virtual colonoscopy: past, present, and future. Radiol Clin North Am 2003; 41: 377-393 Angtuaco TL, Banaad-Omiotek GD, Howden CW. Differing attitudes toward virtual and conventional colonoscopy for colorectal cancer screening: surveys among primary care physicians and potential patients. Am J Gastroenterol 2001; 96: 887-893 Chung DJ, Huh KC, Choi WJ, Kim JK. CT colonography using 16-MDCT in the evaluation of colorectal cancer. AJR Am J Roentgenol 2005; 184: 98-103 Johnson KT, Carston MJ, Wentz RJ, Manduca A, Anderson SM, Johnson CD. Development of a cathartic-free colorectal cancer screening test using virtual colonoscopy: a feasibility study. AJR Am J Roentgenol 2007; 188: W29-W36 Pickhardt PJ, Taylor AJ, Kim DH, Reichelderfer M, Gopal DV, Pfau PR. Screening for colorectal neoplasia with CT colonography: initial experience from the 1st year of coverage by third-party payers. Radiology 2006; 241: 417-425 Doshi T, Rusinak D, Halvorsen RA, Rockey DC, Suzuki K, Dachman AH. CT colonography: false-negative interpretations. Radiology 2007; 244: 165-173 Martin DR, Yang M, Thomasson D, Acheson C. MR colonography: development of optimized method with ex vivo and in vivo systems. Radiology 2002; 225: 597-602 Saar B, Rosch T, Rummeny EJ. Colorectal cancer screening:

20 21

22

23 24

25

a challenge for magnetic resonance colonography. Top Magn Reson Imaging 2002; 13: 427-434 Luboldt W, Bauerfeind P, Wildermuth S, Marincek B, Fried M, Debatin JF. Colonic masses: detection with MR colonography. Radiology 2000; 216: 383-388 Pappalardo G, Polettini E, Frattaroli FM, Casciani E, D'Orta C, D'Amato M, Gualdi GF. Magnetic resonance colonography versus conventional colonoscopy for the detection of colonic endoluminal lesions. Gastroenterology 2000; 119: 300-304 Haykir R, Karakose S, Karabacakoglu A, Sahin M, Kayacetin E. Three-dimensional MR and axial CT colonography versus conventional colonoscopy for detection of colon pathologies. World J Gastroenterol 2006; 12: 2345-2350 Saar B, Meining A, Beer A, Settles M, Helmberger H, Frimberger E, Rummeny EJ, Rosch T. Prospective study on bright lumen magnetic resonance colonography in comparison with conventional colonoscopy. Br J Radiol 2007; 80: 235-241 Lam WW, Leung WK, Wu JK, So NM, Sung JJ. Screening of colonic tumors by air-inflated magnetic resonance (MR) colonography. J Magn Reson Imaging 2004; 19: 447-452 Lomas DJ, Sood RR, Graves MJ, Miller R, Hall NR, Dixon AK. Colon carcinoma: MR imaging with CO2 enema--pilot study. Radiology 2001; 219: 558-562 Ajaj W, Pelster G, Treichel U, Vogt FM, Debatin JF, Ruehm SG, Lauenstein TC. Dark lumen magnetic resonance colonography: comparison with conventional colonoscopy for the detection of colorectal pathology. Gut 2003; 52: 1738-1743 Hartmann D, Bassler B, Schilling D, Adamek HE, Jakobs R, Pfeifer B, Eickhoff A, Zindel C, Riemann JF, Layer G. Colorectal polyps: detection with dark-lumen MR colonography versus conventional colonoscopy. Radiology 2006; 238: 143-149 Semelka RC, Kelekis NL, Thomasson D, Brown MA, Laub GA. HASTE MR imaging: description of technique and preliminary results in the abdomen. J Magn Reson Imaging 1996; 6: 698-699 Morrin MM, Hochman MG, Farrell RJ, Marquesuzaa H, Rosenberg S, Edelman RR. MR colonography using colonic distention with air as the contrast material: work in progress. AJR Am J Roentgenol 2001; 176: 144-146 Johnson CD, MacCarty RL, Welch TJ, Wilson LA, Harmsen WS, Ilstrup DM, Ahlquist DA. Comparison of the relative sensitivity of CT colonography and double-contrast barium enema for screen detection of colorectal polyps. Clin Gastroenterol Hepatol 2004; 2: 314-321 Mulhall BP, Veerappan GR, Jackson JL. Meta-analysis: computed tomographic colonography. Ann Intern Med 2005; 142: 635-650 Ajaj W, Ruehm SG, Gerken G, Goyen M. Strengths and weaknesses of dark-lumen MR colonography: clinical relevance of polyps smaller than 5 mm in diameter at the moment of their detection. J Magn Reson Imaging 2006; 24: 1088-1094 Bielen DJ, Bosmans HT, De Wever LL, Maes F, Tejpar S, Vanbeckevoort D, Marchal GJ. Clinical validation of highresolution fast spin-echo MR colonography after colon distention with air. J Magn Reson Imaging 2005; 22: 400-405 Lauenstein TC, Goehde SC, Ruehm SG, Holtmann G, Debatin JF. MR colonography with barium-based fecal tagging: initial clinical experience. Radiology 2002; 223: 248-254 Ajaj W, Lauenstein TC, Pelster G, Holtmann G, Ruehm SG, Debatin JF, Goehde SC. MR colonography in patients with incomplete conventional colonoscopy. Radiology 2005; 234: 452-459 Kuehle CA, Langhorst J, Ladd SC, Zoepf T, Nuefer M, Grabellus F, Barkhausen J, Gerken G, Lauenstein TC. Magnetic resonance colonography without bowel cleansing: a prospective cross sectional study in a screening population. Gut 2007; 56: 1079-1085 S- Editor Zhu LH L- Editor Li M E- Editor Yin DH

www.wjgnet.com



RAPID COMMUNICATION

Treatment of gastric outlet and duodenal obstructions with uncovered expandable metal stents Qiang Huang, Ding-Ke Dai, Xiao-Jun Qian, Ren-You Zhai Qiang Huang, Ding-Ke Dai, Xiao-Jun Qian, Ren-You Zhai, Department of Radiology, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100021, China Correspondence to: Ren-You Zhai, MD, Director of the Department of Radiology, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100021, China. [email protected] Telephone: +86-10-85231882 Fax: +86-10-65935214 Received: June 15, 2007 Revised: August 3, 2007


Key words: Gastric outlet obstruction; Duodenal obstruction; Expandable metal stent Huang Q, Dai DK, Qian XJ, Zhai RY. Treatment of gastric outlet and duodenum obstructions in the uncovered expandable metal stents. World J Gastroenterol 2007; 13(40): 5376-5379


Abstract AIM: To investigate and evaluate the technical feasibility and clinical effectiveness of fluoroscopically guided peroral uncovered expandable metal stent placement to treat gastric outlet and duodenal obstructions. METHODS: Fifteen consecutive patients underwent TM peroral placement of Wallstent Enteral Endoprosthesis to treat gastric outlet and duodenal obstructions (14 malignant, 1 benign). All procedures were completed under fluoroscopic guidance without endoscopic assistance. Follow-up was completed until the patients died or were lost, and the clinical outcomes were analyzed. RESULTS: The technique success rate was 100%, and the oral intake was maintained in 12 of 14 patients varying from 7 d to 270 d. Two patients remained unable to resume oral intake, although their stents were proven to be patent with the barium study. One patient with acute necrotizing pancreatitis underwent enteral stenting to treat intestinal obstruction, and nausea and vomiting disappeared. Ten patients died during the followup period, and their mean oral intake time was 50 d. No procedure-related complications occurred. Stent migration to the gastric antrum occurred in one patient 1 year after the procedure, a tumor grew at the proximal end of the stent in another patient 38 d post-stent insertion. CONCLUSION: Fluoroscopically guided peroral metal stent implantation is a safe and effective method to treat malignant gastrointestinal obstructions, and complications can be ignored based on our short-term study. Indications for this procedure should be discreetly considered because a few patients may not benefit from gastrointestinal insertion, but some benign gastrointestinal obstructions can be treated using this procedure.

www.wjgnet.com

INTRODUCTION Gastroduodenal obstruction is usually a preterminal event in patients with advanced malignancies of the stomach, pancreas, and duodenum. Surgery is associated with a high complication rate, and relatively high morbidity and mortality rates, due to poor nutrition and general status or progressing tumor infiltration in these patients [1-8]. Fluoroscopically guided peroral placement of selfexpandable metal stents to treat gastric and duodenal obstructions have been demonstrated to be an effective alternative to surgical bypass with lower morbidity and mortality rates, shorter hospitalization, and a lower cost of the overall treatment [1,3,5,6,8-10] . Various stents including vascular stents have been used to treat gastric and duodenal obstructions before the design of special duodenal stents[1]. Here, we report our experience in the treatment of gastric and duodenal obstructions using uncovered Wallstent Enteral Endoprosthesis in recent years. There have been few reports on enteral stenting for benign gastrointestinal obstructions[2,11,12]. Here, we discuss our methods used for intestinal stenting for one patient with acute necrotizing pancreatitis. We also discuss our opinions on the placement technique, indications, complications, and follow-up results.

MATERIALS AND METHODS Subjects A total of 15 consecutive patients underwent fluoroscopically guided peroral placement of Wallstent Enteral Endoprosthesis to treat gastroduodenal obstructions from June 2004 to December 2006, in our department. There were 11 men and 4 women with an average age of 62.4 years (age range, 32-87 years). The underlying causes of

Huang Q et al . Gastric out and duodenum obstructions

5377

Table 1 Patient information Case

Sex/age (yr)

Underlying disease

1 2 3 4 5 6

F/68 M/72 F/39 F/65 M/86 M/87

Ampullary cancer Gastric cancer Pancreatic cancer Pancreatic cancer Pancreatic cancer Pancreatic cancer

7 8

M/80 F/72

Ampullary cancer Colon cancer

9 10

M/66 M/33

Gallbladder cancer Acute necrotizing pancreatitis

11 12 13 14 15

M/67 M/68 M/48 M/32 M/53

Duodenal cancer Pancreatic cancer Pancreatic cancer Colon cancer Esophageal cancer

Stent size (diameter × length, mm) Duration of oral intake after stenting Follow-up time 22 × 90 22 × 60 20 × 90 22 × 90 20 × 90 20 × 60 22 × 60 20 × 60 22 × 90 22 × 60 22 × 90 20 × 60 22 × 60 22 × 90 20 × 90 20 × 90 20 × 90 20 × 60 20 × 60

obstructions were pancreatic carcinoma (n = 6), ampullary carcinoma (n = 2), colon carcinoma (n = 2), gastric cancer (n = 1), duodenal cancer (n = 1), gallbladder cancer (n = 1), esophageal carcinoma (n = 1), and acute necrotizing pancreatitis (n = 1) (Table 1). The diagnosis was established by means of CT (n = 7), endoscopy (n = 2) or after surgery (n = 6). Gastrointestinal obstructions were confirmed by a barium study (n = 11), endoscopy (n = 2), and CT (n = 2). The sites of obstructions were the gastric antrum (n = 1), pylorus (n = 1), gastrointestinal anastomotic stoma (n = 1), duodenum (n = 12, including duodenal metastasis from colon carcinoma in two patients). Fourteen patients with malignant obstructions were considered inoperative because of the risk of recurrence after surgery, extensive tumor growth, metastatic cancer or the presence of peritoneal seeding or distant metastasis. They all presented with severe nausea and recurrent vomiting. The patient with acute necrotizing pancreatitis developed severe intestinal obstruction following surgery. Although jejunostomy had been performed, nausea and vomiting persisted in this patient, and duodenal obstruction was finally detected. There was no amelioration in conditions after 4 months, so we decided to insert a metal stent in order to relieve the obstructive syndrome. Methods The Wallstent Enteral Endoprosthesis (Boston Scientific Medi-Tech, USA) was used in all patients. The sizes of the stents were as follows: 20 mm (diameter) × 60 mm (length), 22 mm × 60 mm, 20 mm × 90 mm, and 22 mm × 90 mm. Informed consent was obtained from all patients before all procedures. Stent placement procedures were performed according to a standard technique as described in the literature [1,13,14]. More than 2 d before the stent placement, a barium study was performed to evaluate the site, length, and severity of the obstruction. A nasogastric tube was placed overnight to empty the stomach, and was removed just before the stent insertion. All procedures were performed using a digital angiographic unit with the patient in the supine position. Pharyngeal

At least 10 d 15 d At least 4 mo 38 d 80 d

10 d 24 d 4 mo 74 d 85 d

At least 20 d

20 d

9 mo

Outcome Dead Dead Dead

10 mo

Dead

22 d

26 d

Dead

7d

50 d

Dead

1y 60 d 7d 0d At least 7 d 0d

70 d 10 d 6d 7d 40 d

Dead Dead Dead Dead

anesthesia was usually performed using lidocaine aerosol spray (n = 13), but basal anesthesia (n = 1) or intravenous general anesthesia (n = 1) was also offered depending on the patients’ or their relatives’ demands. A catheter and guiding wire were inserted perorally into the esophagus under fluoroscopic guidance, and were advanced into the stomach. A small amount of contrast medium was injected through the catheter to identify the gastric outlet or the site of obstruction. The guiding wire and catheter were carefully manipulated to cross the obstruction using a standard catheter technique. After the catheter and guiding wire were located distal to the obstruction and the length of the obstruction was measured, the wire was exchanged for a 260-cm super-stiff guide wire to provide better support for the stent insertion. The Wallstent Enteral Endoprosthesis was then performed under fluoroscopic monitoring to ensure the perfect position of the stent. A stent 4 cm longer than the obstruction length was routinely chosen to completely overlap the affected region. Balloon dilation was performed when the stent assembly could not pass the stenosis. We also performed balloon dilation after stent deployment in 2 of our patients, which was a different procedure from the literature[1]. The patients were monitored hourly for at least 8 hours after the procedure. If they did not complain of abdominal pain and the hemoglobin level was normal, they were allowed to resume oral intake of liquids. Their diet progressed to soft or solid foods when they could tolerate liquids well. A barium study or endoscopy was performed to verify the position and patency of the stent in cases of bad tolerance of foods or recurrence of nausea and vomiting. Follow-up was continued by phone calls after patient discharge, and a further barium study or endoscopy was suggested only when symptoms recurred.

RESULTS Peroral stent deployment was successfully completed under fluoroscopic guidance in our 15 patients, indicating www.wjgnet.com

5378

ISSN 1007-9327

CN 14-1219/R


a technique success rate of 100%. No procedure-related complication occurred. Two stents were needed in 2 patients, and three stents were needed in one due to the lengths of the obstructions. Therefore, a total of 19 stents were used in our 15 patients. The follow-up period was 6 d to 365 d (mean = 80.5 d). Stent migration to the gastric antrum occurred in one patient 1 year after the procedure, and tumor ingrowth at the proximal end of the stent was detected in another patient 38 d after stent insertion. Oral intake was maintained in 12 of 14 patients (85.7%) varying from 7 d to 270 d. Two patients were still unable to resume oral intake, although their stents were proven to be patent with the barium study. The patient with acute necrotizing pancreatitis (benign g astrointestinal obstr uction) underwent enteral stenting in order to treat the intestinal obstruction, and nausea and vomiting disappeared after the procedure. Ten patients died during the follow-up period, and their mean oral intake time was 50 d (0 d to 270 d). The time between their death and the stent insertion varied from 6 d to 300 d (mean = 68.5 d). Percutaneous transhepatic biliary drainage (PTBD) and/or bile duct stent embedding were perfor med before enteral stenting in 13 patients, 4 of whom further underwent biliary interventional procedures as jaundice recurred after the procedure. The times between the relapse of jaundice and the enteral stent procedure were 7 d, 14 d, 15 d, and 130 d for these 4 patients, respectively. No evidence showed that jaundice recurred because of enteral stenting. Three of the 14 patients (malignant gastrointestinal obstructions) underwent transcatheter arterial chemotherapy (TAC), and their nutritional status improved after the procedure. Their survival time seemed to be longer than that of the others. However, we did not analyze the influence of TAC considering the small number of patients.

DISCUSSION A malignant gastrointestinal obstruction is usually a preter minal event in patients with pancreatobiliary, stomach or duodenum malignancies. Such patients often cannot undergo curative resections because of the inoperability or unresectability of their tumors. Palliative surgery is associated with a high complication rate, and relatively high morbidity and mortality rates, due to poor nutritional and general status or progressing tumor infiltration in such patients[1-8]. Fluoroscopically guided peroral placement of self-expandable metal stents to treat gastric and duodenal obstructions have been demonstrated to be an effective alternative to surgical bypass with lower morbidity and mortality rates, shorter hospitalization, and a lower cost of the overall treatment[1,3,5,6,8-10]. Various stents including vascular stents have been used for the treatment of gastric and duodenal obstructions before the availability of specially designed duodenal stents[13]. However, only expandable metal stents have been approved by the Food and Drug Administration for the treatment of gastrointestinal obstructions due to cancer risks[5,15]. We chose the Wallstent Enteral Endoprosthesis (Boston Scientific Medi-Tech, USA) because of its flexibility and safety. Covered stents have been used to prevent tumor www.wjgnet.com

October 28, 2007

Volume 13

Number 40

ingrowth or overgrowth[4,6,10,12,16-19]. Because we thought the expected survival time of these patients was limited, and tumor ingrowth or overgrowth did not need to be considered [19], we chose to use uncovered metal stents to obviate migration. In fact, only one case of tumor ingrowth was identified in our 15 patients 38 d after stent insertion, with an ingrowth rate of 6.67%. Additionally, the implantation of peroral uncovered stents is technically easier than covered stents[1,6,8,16-18]. According to the literature, f luoroscopic stent placement for duodenal obstructions is a technically more difficult procedure than for gastric outlet obstructions, not only because of a loop formation of the stent delivery system in the distended stomach, but also because of the curved configuration of the duodenal C loop[1,16,17,20]. Endoscopic assistance and gastrostomy have been used after guide wire navigation to advance past the duodenal obstructions[1,3,16]. In our series, the site of obstruction was located in the duodenum in 12 patients, but no endoscopic assistance or gastrostomy was required in any of them because of the flexibility of the endoprosthetic system and the patience of the interventional radiologists. PTBD before enteral stenting is considered to be necessary by some authors, but there are different opinions [1,18]. Thirteen patients in our series developed jaundice and underwent PTBD or biliary stenting before gastrointestinal obstructions occurred, and enteral stenting was scheduled. Three of them underwent a second biliary intervention after the enteral stenting, but there was no evidence showing that enteral stenting caused recurrence of jaundice. TAC was performed in three patients who showed a relatively longer survival time than those who could not tolerate TAC. Considering the small number of patients, we did not analyze the influence of TAC on survival time. Further studies are required to elucidate this relationship. Nausea and vomiting disappeared or mitigated, and oral intake was maintained in 12 of 14 (85.7%) patients with malignant gastrointestinal obstructions ranging from 7 d to 270 d (mean = 54.7 d). Two patients were still unable to resume oral intake while nausea and vomiting continued after the procedure, although their stents were proven to be patent according to the barium study. A clinical success rate of 85.7% is acceptable because many factors may contribute to the symptoms in malignant gastrointestinal obstructions [21]. Further studies on the indications of enteral stenting for malignant gastric and duodenal obstructions are warranted. The patient with acute necrotizing pancreatitis (benign gastrointestinal obstruction) underwent enteral stenting to treat the intestinal obstruction, and nausea and vomiting disappeared after the procedure. His symptoms of nausea and vomiting continued for over 4 mo even after jejunostomy had been performed. Therefore, we decided to perform enteral stenting after discussion with his surgeons. The convention has been that stent placement is rarely indicated for benign gastrointestinal strictures[12,14], and further studies are needed to elucidate such issues. No procedure-related complication occurred, and only two complications were observed during the follow-up period. Stent migration to the gastric antrum happened in one

Huang Q et al . Gastric out and duodenum obstructions

patient 1 year after the procedure, and a gastric ulcer with hemorrhage was found around the proximal end of the stent by endoscopy. The patient refused further treatment, and left. Tumor ingrowth at the proximal end of the stent was detected by the barium study in another patient 38 d after stent insertion. Due to progressing tumor infiltration and a hepatic abscess leading to deterioration, a second stent implantation could not be performed, and the patient died 36 d later. In conclusion, fluoroscopic peroral placement of selfexpandable metal stents is a safe and effective treatment for malignant gastric outlet and duodenal obstructions. The technique success rate is high, and complications can be ignored due to the short survival time of the patients in our study. However, indications should be carefully considered before the procedure because a minority of patients may not benefit from gastrointestinal insertion, while some benign gastrointestinal obstructions can also be treated with this procedure.

5379 3

4

5 6

7

8

9

COMMENTS Background

Gastroduodenal obstruction is usually a preterminal event in patients with advanced malignant diseases. It can lead to severe malnutrition and even death in the patients. Many studies have reported that enteral stents can be used to achieve gastroduodenal patency, and the effects have been satisfactory.

10

11

Research frontiers

Stents have been widely used in interventional procedures. A research hotspot has been to modify the craft of stents to obtain more flexible and durable materials. Various covered stents have been studied to improve effectiveness and reduce adverse reactions.


In previous applications of stents to treat gastroduodenal obstructions, it was found that tumor ingrowth or overgrowth could cause restenosis in uncovered stents, while migration might happen in covered stents. Endoscopic assistance or gastrostomy has been used after guide wire navigation to advance past the duodenal obstructions. Here, we proved that fluoroscopic peroral placement of uncovered metal stents can be safe and effective for most cases.

Applications

The study results suggest that fluoroscopic peroral placement of uncovered metal stents is safe and effective to treat gastroduodenal obstructions. However, indications should be discreetly considered before the procedure.

12

13 14 15 16

17

Peer review

This is a clinical study in which authors investigate and evaluate the technique feasibility and clinical effectiveness of fluoroscopically guided peroral uncovered expandable metal stent placement to treat gastric outlet and duodenal obstructions.

18

19

REFERENCES 1

2

Yoon CJ, Song HY, Shin JH, Bae JI, Jung GS, Kichikawa K, Lopera JE, Castaneda-Zuniga W. Malignant duodenal obstructions: palliative treatment using self-expandable nitinol stents. J Vasc Interv Radiol 2006; 17: 319-326 Binkert CA, Jost R, Steiner A, Zollikofer CL. Benign and malignant stenoses of the stomach and duodenum: treatment with self-expanding metallic endoprostheses. Radiology 1996; 199: 335-338

20

21

Bessoud B, de Baere T, Denys A, Kuoch V, Ducreux M, Precetti S, Roche A, Menu Y. Malignant gastroduodenal obstruction: palliation with self-expanding metallic stents. J Vasc Interv Radiol 2005; 16: 247-253 Park HS, Do YS, Suh SW, Choo SW, Lim HK, Kim SH, Shim YM, Park KC, Choo IW. Upper gastrointestinal tract malignant obstruction: initial results of palliation with a flexible covered stent. Radiology 1999; 210: 865-870 Lopera JE, Brazzini A, Gonzales A, Castaneda-Zuniga WR. Gastroduodenal stent placement: current status. Radiographics 2004; 24: 1561-1573 Jung GS, Song HY, Kang SG, Huh JD, Park SJ, Koo JY, Cho YD. Malignant gastroduodenal obstructions: treatment by means of a covered expandable metallic stent-initial experience. Radiology 2000; 216: 758-763 Wai CT, Ho KY, Yeoh KG, Lim SG. Palliation of malignant gastric outlet obstruction caused by gastric cancer with selfexpandable metal stents. Surg Laparosc Endosc Percutan Tech 2001; 11: 161-164 Lopera JE, Alvarez O, Castano R, Castaneda-Zuniga W. Initial experience with Song's covered duodenal stent in the treatment of malignant gastroduodenal obstruction. J Vasc Interv Radiol 2001; 12: 1297-1303 de Baere T, Harry G, Ducreux M, Elias D, Briquet R, Kuoch V, Roche A. Self-expanding metallic stents as palliative treatment of malignant gastroduodenal stenosis. AJR Am J Roentgenol 1997; 169: 1079-1083 Jeong JY, Han JK, Kim AY, Lee KH, Lee JY, Kang JW, Kim TJ, Shin SH, Choi BI. Fluoroscopically guided placement of a covered self-expandable metallic stent for malignant antroduodenal obstructions: preliminary results in 18 patients. AJR Am J Roentgenol 2002; 178: 847-852 Morris CD, Lynch N, Mehta S, Deans GT. Management of ischaemic stricture at a gastro-jejunostomy by self-expanding metallic stent insertion. Eur J Gastroenterol Hepatol 2002; 14: 331-332 Bae JI, Shin JH, Song HY, Lee GH. Treatment of a b e n i g n a n a s t o m o t i c d u o d e n o j e j u n a l s t r i c t u re with a polytetrafluoroethylene-covered retrievable expandable nitinol stent. J Vasc Interv Radiol 2004; 15: 769-772 Strecker EP, Boos I, Husfeldt KJ. Malignant duodenal stenosis: palliation with peroral implantation of a self-expanding nitinol stent. Radiology 1995; 196: 349-351 Morgan R, Adam A. Use of metallic stents and balloons in the esophagus and gastrointestinal tract. J Vasc Interv Radiol 2001; 12: 283-297 Baron TH. Expandable metal stents for the treatment of cancerous obstruction of the gastrointestinal tract. N Engl J Med 2001; 344: 1681-1687 Park KB, Do YS, Kang WK, Choo SW, Han YH, Suh SW, Lee SJ, Park KS, Choo IW. Malignant obstruction of gastric outlet and duodenum: palliation with flexible covered metallic stents. Radiology 2001; 219: 679-683 Jung GS, Song HY, Seo TS, Park SJ, Koo JY, Huh JD, Cho YD. Malignant gastric outlet obstructions: treatment by means of coaxial placement of uncovered and covered expandable nitinol stents. J Vasc Interv Radiol 2002; 13: 275-283 Song HY, Shin JH, Yoon CJ, Lee GH, Kim TW, Lee SK, Yook JH, Kim BS. A dual expandable nitinol stent: experience in 102 patients with malignant gastroduodenal strictures. J Vasc Interv Radiol 2004; 15: 1443-1449 Mauro MA, Koehler RE, Baron TH. Advances in gastrointestinal intervention: the treatment of gastroduodenal and colorectal obstructions with metallic stents. Radiology 2000; 215: 659-669 Song HY, Shin JH, Lim JO, Kim TH, Lee GH, Lee SK. Use of a newly designed multifunctional coil catheter for stent placement in the upper gastrointestinal tract. J Vasc Interv Radiol 2004; 15: 369-373 Cherny NI. Taking care of the terminally ill cancer patient: management of gastrointestinal symptoms in patients with advanced cancer. Ann Oncol 2004; 15 Suppl 4: iv205-iv213 S- Editor Ma N L- Editor Ma N E- Editor Li HY

www.wjgnet.com



RAPID COMMUNICATION

Effects of H pylori therapy on erythrocytic and iron parameters in iron deficiency anemia patients with H pylori -positive chronic gastristis Lun-Hua Chen, He-Sheng Luo Lun-Hua Chen, Department of Hematology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China He-Sheng Luo, Department of Gastroenterology, Renmin Hospital of Wuhan University, Wuhan 430060, Hubei Province, China Correspondence to: He-Sheng Luo, Department of Gastroenterology, Renmin Hospital of Wuhan University, no. 238 Jiefang Road, Wuhan 430060, Hubei Province, China. [email protected] Telephone: +86-27-88041911213 Fax: +86-27-88042292 Received: April 10, 2007 Revised: August 3, 2007

efficacy of ferrous succinate therapy in IDA patients with

H pylori -positive chronic gastritis. © 2007 WJG . All rights reserved.

Key words: H pylori ; Iron deficiency anemia; Oral iron treatment; Chronic gastritis Chen LH, Luo HS. Effects of H pylori therapy on erythrocytic and iron parameters in iron deficiency anemia patients with H pylori -positive chronic gastritis. World J Gastroenterol

2007; 13(40): 5380-5383

Abstract


AIM: To elucidate the influences of H pylori infection on oral iron treatment for iron deficiency anemia (IDA). METHODS: A total of 86 patients were divided into two groups: group A, receiving ferrous succinate combined with triple therapy for H pylori eradication, and group B (control), treated with ferrous succinate only. During treatment of IDA, dynamic changes in hemoglobin (Hb) level, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), serum iron (SI), and serum ferritin (SF) were compared between the groups. RESULTS: Hb was slightly higher in group A at d 14 after the start of triple therapy for H pylori eradication (P > 0.05). After the therapy, the increase of Hb in group A became significantly faster than that in group B (P < 0.05). At d 56, the mean Hb in group A returned to the normal level, however, in group B, it was lower than that in group A (P < 0.05) although it had also increased compared with that before oral iron treatment. The MCV and MCH in group A recovered to the normal level, and were much higher than those in group B (P < 0.05) at d 21. In Group B, the MCV and MCH remained at lower than normal levels until d 42 after the start of therapy. And then, they reached a plateau in both groups and the differences disappeared (P > 0.05). The SF in group A was higher than that in group B (P < 0.05) 28 d after the treatment and its improvement was quicker in group A (P < 0.05) , and the difference between the two groups was even more significant (P < 0.01) at d 56. The SI in group A was higher than that in group B (P < 0.05) at d 14 and this persisted until d 56 when the follow-up of this research was finished. CONCLUSION: Treatment of H pylori can enhance the www.wjgnet.com

INTRODUCTION Spontaneous iron excretion in adults is minimal and iron deficiency anemia (IDA) is generally attributed to abnormal blood loss[1]. IDA is often caused by gastrointestinal bleeding due to peptic ulcer and chronic gastritis, or in women of reproductive age by increased menstrual flow and the greater requirements of pregnancy. Recently, several seroepidemiological studies have suggested a link between IDA and H pylori infection[2,3]. A high percentage of H pylori-positive IDA patients exhibit atrophic changes in the gastric body, and the remainders have chronic superficial gastritis extending to the fundic mucosa. H pylori infection has even been implicated as a cause of IDA refractory to oral iron treatment[4,5]. Some patients with refractory IDA have no gastrointestinal symptoms but H pylori gastritis, as the only cause of their anemia[6]. Most dietary iron is in the non-hemic ferric form, and an acidic intragastric pH is needed to reduce it to the ferrous form for absorption. This reaction is promoted by gastric acidity and ascorbic acid (AA), which is thus considered the most potent regulator of iron absorption[7]. Patients with H pylori gastritis showed an increase in intragastric pH with a median of > 3, a pH that is known to be critical in the process of iron absorption. Moreover, AA is actively secreted from plasma to the gastric juice[8], but the concentration of AA in the gastric juice of patients with H pylori gastritis and IDA is clearly reduced in comparison with both healthy and nonanemic H pylori-positive controls[9-11]. The availability of convenient, non-invasive methods for identifying H pylori gastritis has greatly facilitated the recognition of infected patients, resulting in progressive

Chen LH et al . H pylori therapy in IDA patients 160

5381

A B

120 Blood Hb (g/L)

awareness of its influences and possible role in the causation of IDA[12]. Therefore, we first examined the prevalence of H pylori infection and serum markers of iron deficiency. We then evaluated the effects of subsequent H pylori eradication on the response to oral iron therapy, which could provide valuable information for further clinical applications.

a

a

a

a

a

a

80

40

MATERIALS AND METHODS Patients From January 2002 to December 2005, 86 IDA adult patients with H pylori-positive chronic gastritis were enrolled. The participants comprised 36 women and 50 men; median age 53 years, range 18-76. According to Sydney System, the patients were diagnosed as having atrophy, intestinal metaplasia, or antral versus body gastritis, in 34, 27, and 25 cases, respectively. According to the criteria[13], IDA was defined as hemoglobin (Hb) concentration < 120 g/L in the men and < 110 g/L in the women, serum ferritin (SF) < 12 μg/L, mean corpuscular hemoglobin (MCH) < 27 pg, and mean corpuscular volume (MCV) < 80 fL. Obvious causes of blood loss, such as active gastrointestinal hemorrhage, menometorrhagia or heavy menstrual loss, and any other non-gastrointestinal disease likely to cause IDA, were exclusion criteria for this study. Also excluded from the study protocol were patients with previous gastric surgery, and those who received anti-H pylori treatment or anti-secretory drugs before. Patients who were lactating or pregnant and those with malnutrition or cancer were also excluded. Methods The 14C-urease breath test was performed as described previously[14]. Briefly, the patients fasted overnight and were then given urea labeled with 37 kBq of 14C dissolved in water. Breath samples were collected before and 15 min after ingestion, when enzymolysis of labeled urea occurred and 14CO2 had been released into the peripheral circulation. The 14CO2 in these samples was trapped in 1 mmol/L of hyamine hydroxide in ethanol and transferred into scintillation liquid. The 14C content was measured in Bq mode using a liquid β-scintillation counter (Headway, China). Samples with < 3.33 Bq were considered negative for H pylori, while samples with > 3.33 Bq were considered positive. The peripheral hemogram including MCH and MCV was measured with a Sysmex automated analyzer (Kobe, Japan). Biochemical assays for serum iron (SI) and Hb were performed as routine. SF was determined with a chemiluminescent immunometric analyzer and kit from Bayer Co. (Germany). The 86 patients were randomly divided into two groups of equal size. Group A comprised 43 cases and received oral iron treatment combined with triple therapy for H pylori eradication, while the 43 cases in group B received oral iron treatment only as controls. Oral iron treatment comprised ferrous succinate (with an Fe 2+ content of 34%-36%) 200 mg supplemented with ascorbic acid 100 mg three times daily. Treatment was given until the SF recovered to normal. Triple therapy for H pylori eradication included deutero-bismuth citrate 240 mg × 2/d, amoxicillin 500 mg × 2/d, and metronidazole 400 mg × 2/d for 2 wk; in all cases, H pylori was eradicated.

0

0

7

14

21

28

35

42

49

56

t /d

Figure 1 Blood Hb during iron treatment (mean ± SD, n = 43; aP < 0.05 vs group B).

Follow-up The mean Hb, MCH, and MCV in the peripheral blood of the two groups were recorded before and at d 0, 7, 14, 21, 28, 35, 42, 49, and 56 after iron treatment. SI and SF were compared before and at d 0, 14, 28, 42, and 56. Statistical analysis Each assay was made at least three times and data were expressed as the mean ± SD. The differences between groups were determined using t test for paired analysis. P < 0.05 was considered statistically significant.

RESULTS Influence of H pylori on Hb Hb was slightly higher in group A at d 14 after the start of triple therapy for H pylori eradication, but the difference was not significant (P > 0.05). After the complete course of triple therapy, the increase of Hb in group A became significantly faster than that in group B (P < 0.05). At d 56, the mean Hb in group A returned to the normal level referred to the criteria. In group B, the mean Hb had also increased compared with that before oral iron treatment, but it was still lower than that in group A (P < 0.05, Figure 1). Influence of H pylori on MCV and MCH Similar to the trend seen for Hb, the increase of MCV and MCH in group A was not significantly greater than that in group B (P > 0.05). At d 21, the MCV and MCH in group A became normal, and were much higher than those in group B (P < 0.05). In group B, the MCV and MCH remained at lower than normal levels until 42 d after the start of therapy. At this time, the MCV and MCH reached a plateau in both groups and the differences disappeared (P > 0.05, Figures 2 and 3). Influence of H pylori on iron status There were no significant differences in parameters of iron status between the two groups at the start of treatment (P > 0.05), but after 28 d the SF in group A was higher than that in group B (P < 0.05). Thereafter, the improvement of SF was much quicker in group A, and at d 56 the difference between the two groups was even more significant (P < 0.01). At d 14, the SI in group A was higher than in group B (P < 0.05) and this persisted until 56 d after the start of oral iron treatment, when the follow-up of this research was finished (Table 1). www.wjgnet.com

5382

ISSN 1007-9327

120

A B

a

a

a

28

35


80

MCH (pg)

MCV (fL)

100

CN 14-1219/R

60 40 20 0

0

7

14

21

42

49

56

40 35 30 25 20 15 10 5 0

October 28, 2007

Volume 13

A B

a

0

a

7

14

21

28

t /d

Number 40

a

35

42

49

56

t /d

Figure 2 MCV during iron treatment (mean ± SD, n = 43; aP < 0.05 vs group B).

Figure 3 MCH during iron treatment (mean ± SD, n = 43; aP < 0.05 vs group B).

Table 1 Alterations of SI (μmol/L) and SF (μg/L) during iron treatment (mean ± SD, n = 43) Groups A B

d0

d 14

d 28

d 42

d 56

SI

SF

SI

SF

SI

SF

SI

SF

SI

SF

3.2 ± 1.1 3.3 ± 1.2

7.2 ± 1.6 7.5 ± 1.8

11.3 ± 3.2a 8.9 ± 2.9

9.4 ± 2.6 8.8 ± 2.4

13.2 ± 3.4a 9.2 ± 2.2

11.3 ± 2.6a 9.6 ± 2.7

18.2 ± 3.6a 13.2 ± 2.9

18.2 ± 5.3a 13.6 ± 3.2

23.2±4.7a 18.2±3.7

38.5 ± 9.2a 21.6 ± 5.6

a

P < 0.05 vs group B.

DISCUSSION H pylori infection has been reported to have various manifestations in adolescents and adults, including IDA. A double-blind, placebo-controlled trial performed in adolescents with IDA and H pylori infection suggested that SF was significantly lower in the H pylori-infected group and that H pylori eradication led to resolution of iron deficiency[15,16]. A large serosurvey of H pylori in adults also found that SF was significantly lower in adults positive for H pylori IgG than that in non-infected controls[17,18]. Based on our medical records of adult patients with chronic gastritis, we found that IDA occurred more frequently in H pylori-positive cases. The coexistence of H pylori gastritis in the 86 IDA adult patients enrolled in this study was determined by endoscopy and the urease breath test. To avoid influences from other clinical factors, the subjects were selected according to strict criteria. It has recently been shown that extension of gastritis to the corporal mucosa occurs in a higher percentage of patients with H pylori infection and IDA compared with non-anemic H pylori-infected controls[19]. The blood loss in chronic gastritis, and bleeding from duodenal or gastric ulcers related to H pylori infection, plays an important role in the development of iron deficiency in adults. In response to H pylori chronic gastric inflammation, the epithelial cells in the mucosa are damaged, leading to detachment and apoptosis. In the absence of bleeding lesions, the possible mechanisms by which H pylori is involved in the development of IDA remain unclear. Preliminary studies suggest that the growth and proliferation of H pylori requires iron from the host and that some H pylori strains have a specific ability to interfere with iron metabolism by binding iron to their outer membrane proteins[20]. However, other studies have demonstrated that neither virulence factors such as Cag-A4 nor mutations in the bacterial genes involved in iron uptake are associated with IDA. Given the high prevalence of H pylori infection, this hypothesis cannot explain why not all patients with H pylori gastritis develop IDA[21]. www.wjgnet.com

Besides the occult gastrointestinal bleeding and competition for dietary iron, H pylori infection can affect the gastric body and initiate the development of atrophic body gastritis that can in turn cause decreased gastric acid secretion and increased intragastric pH[5,22]. H pylori infection adversely influences the composition of the gastric juice; for example, in terms of its acidity and ascorbate content, both of which are critical for normal iron absorption[23]. These findings suggest that the physiological mechanisms that are necessary for the absorption of alimentary iron in the duodenal mucosa are impaired in patients with H pylori gastritis and IDA. Thus, we planned to determine the relationship between H pylori infection status and indices of IDA such as the peripheral hemogram and SI. These indices were compared between group A, which received triple therapy combined with oral iron treatment, and group B, which received oral iron treatment only. Before the completion of H pylori eradication, there were no significant differences in parameters reflecting iron status between the two groups, though the indices were slightly higher in group A. After the 2 wk triple therapy was finished, our observations indicate that the response to oral iron treatment was significantly greater in group A than in group B. Since supplementation with ascorbic acid has been commonly shown to reduce the pH of gastric juice thereby increasing iron absorption, its influence was not evaluated in this research. It has been clearly demonstrated in previous studies that H pylori eradication can reverse the negative effect of H pylori infection on iron absorption and lead to improvement of IDA in case series and in clinical trials in both children and adults[4,15,16]. Successful H pylori eradication resulted first in a significant post-treatment decrease of serum gastrin and H pylori IgG antibody titers, and then an increase in the peripheral hemogram and SI. This normalization of iron metabolism in H pylori-positive patients increased the MCV and MCH to a plateau similar to the normal level. The H pylori gastritis is increasingly considered a pos-

Chen LH et al . H pylori therapy in IDA patients

sible cause of IDA refractory to oral iron treatment, and eradication of H pylori may be followed by an improved response to oral iron in previously refractory IDA patients[4,5]. In this study, H pylori infection slowed the trend of recovery from IDA recovery, but the indices in group B still reached normal levels. In our opinion, H pylori eradication is necessary for the resolution of IDA with a lower risk of recurrence. Patients with H pylori infection and IDA should receive oral iron and triple therapy simultaneously.

6

7 8

COMMENTS Background

Iron deficiency anemia (IDA) is often caused by gastrointestinal bleeding due to peptic ulcer and chronic gastritis. H pylori infection, which has been proved to play the main role in peptic ulcer and chronic gastritis, has been implicated as a cause of IDA refractory to oral iron treatment. The role of H pylori and its eradication in IDA in patients with H pylori-positive gastritis has been unclear.

Research frontiers

In some cases, refractory IDA is not sensitive to oral iron treatment, especially in patients with H pylori-positive gastritis. It is important to elucidate the relation of IDA to H pylori infection and the effect of H pylori eradication on the treatment of IDA.

9

10

11 12


Related publications are rare.


This study showed that the H pylori infection could slow the trend of recovery from IDA and that H pylori eradication is necessary for the resolution of IDA with a lower risk of recurrence. The efficacy of simultaneous oral iron and triple therapy was evaluated.

13 14 15

Applications

For the treatment of IDA in patients with H pylori-positive chronic gastritis, it is effective and necessary to eradicate H pylori infection, which can lead to satisfactory recovery of this category of IDA.

Terminology

H pylori infection: H pylori infection is the main cause of peptic ulcer and chronic gastritis. IDA: iron deficiency anemia is generally attributed to abnormal blood loss, which is often caused by gastrointestinal bleeding due to peptic ulcer and chronic gastritis, or in women of reproductive age by increased menstrual flow and the greater requirements of pregnancy.

Peer review

This is a good review on an interesting topic with an appropriate number of patients. The major point greatly enhances the conclusions.

16 17

18

19

REFERENCES 1 2

3 4 5

Ross EM. Evaluation and treatment of iron deficiency in adults. Nutr Clin Care 2002; 5: 220-224 Ciacci C, Sabbatini F, Cavallaro R, Castiglione F, Di Bella S, Iovino P, Palumbo A, Tortora R, Amoruso D, Mazzacca G. Helicobacter pylori impairs iron absorption in infected individuals. Dig Liver Dis 2004; 36: 455-460 Ashorn M. Acid and iron-disturbances related to Helicobacter pylori infection. J Pediatr Gastroenterol Nutr 2004; 38: 137-139 Konno M, Muraoka S, Takahashi M, Imai T. Iron-deficiency anemia associated with Helicobacter pylori gastritis. J Pediatr Gastroenterol Nutr 2000; 31: 52-56 Hacihanefioglu A, Edebali F, Celebi A, Karakaya T, Senturk O, Hulagu S. Improvement of complete blood count in

20 21

22 23

5383

patients with iron deficiency anemia and Helicobacter pylori infection after the eradication of Helicobacter pylori. Hepatogastroenterology 2004; 51: 313-315 Annibale B, Capurso G, Chistolini A, D'Ambra G, DiGiulio E, Monarca B, DelleFave G. Gastrointestinal causes of refractory iron deficiency anemia in patients without gastrointestinal symptoms. Am J Med 2001; 111: 439-445 Conrad ME, Umbreit JN. Iron absorption and transport-an update. Am J Hematol 2000; 64: 287-298 Schulz HU, Schurer M, Krupp S, Dammann HG, Timm J, Gessner U. Effects of acetylsalicylic acid on ascorbic acid concentrations in plasma, gastric mucosa, gastric juice and urine--a double-blind study in healthy subjects. Int J Clin Pharmacol Ther 2004; 42: 481-487 Capurso G, Ricci R, Panzuto F, Baccini F, Passi S, Di Giulio E, Delle Fave G, Annibale B. Intragastric ascorbic but not uric acid is depleted in relation with the increased pH in patients with atrophic body gastritis and H. pylori gastritis. Helicobacter 2003; 8: 300-306 Park JH, Kim SY, Kim DW, Lee WG, Rhee KH, Youn HS. Correlation between Helicobacter pylori infection and vitamin C levels in whole blood, plasma, and gastric juice, and the pH of gastric juice in Korean children. J Pediatr Gastroenterol Nutr 2003; 37: 53-62 Furuta T, Baba S, Takashima M, Futami H, Arai H, Kajimura M, Hanai H, Kaneko E. Effect of Helicobacter pylori infection on gastric juice pH. Scand J Gastroenterol 1998; 33: 357-363 Kato S, Nakayama K, Minoura T, Konno M, Tajiri H, Matsuhisa T, Iinuma K. Comparison between the 13C-urea breath test and stool antigen test for the diagnosis of childhood Helicobacter pylori infection. J Gastroenterol 2004; 39: 1045-1050 Zhang ZN, Shen T. Standard diagnosis and curative effect of hematologic disease. Beijing: Science Press, 1998: 10-19 Mirbagheri SA, Sohrabpour AA, Hasibi M, Moghimi B, Mohamadnejad M. 14C-urea breath test in patients undergoing anti-tuberculosis therapy. World J Gastroenterol 2005; 11: 1712-1714 Choe YH, Kim SK, Son BK, Lee DH, Hong YC, Pai SH. Randomized placebo-controlled trial of Helicobacter pylori eradication for iron-deficiency anemia in preadolescent children and adolescents. Helicobacter 1999; 4: 135-139 Choe YH, Kwon YS, Jung MK, Kang SK, Hwang TS, Hong YC. Helicobacter pylori-associated iron-deficiency anemia in adolescent female athletes. J Pediatr 2001; 139: 100-104 Milman N, Rosenstock S, Andersen L, Jorgensen T, Bonnevie O. Serum ferritin, hemoglobin, and Helicobacter pylori infection: a seroepidemiologic survey comprising 2794 Danish adults. Gastroenterology 1998; 115: 268-274 Parkinson AJ, Gold BD, Bulkow L, Wainwright RB, Swaminathan B, Khanna B, Petersen KM, Fitzgerald MA. High prevalence of Helicobacter pylori in the Alaska native population and association with low serum ferritin levels in young adults. Clin Diagn Lab Immunol 2000; 7: 885-888 Capurso G, Lahner E, Marcheggiano A, Caruana P, Carnuccio A, Bordi C, Delle Fave G, Annibale B. Involvement of the corporal mucosa and related changes in gastric acid secretion characterize patients with iron deficiency anaemia associated with Helicobacter pylori infection. Aliment Pharmacol Ther 2001; 15: 1753-1761 Perez-Perez GI, Israel DA. Role of iron in Helicobacter pylori: its influence in outer membrane protein expression and in pathogenicity. Eur J Gastroenterol Hepatol 2000; 12: 1263-1265 Annibale B, Negrini R, Caruana P, Lahner E, Grossi C, Bordi C, Delle Fave G. Two-thirds of atrophic body gastritis patients have evidence of Helicobacter pylori infection. Helicobacter 2001; 6: 225-233 Bini EJ. Helicobacter pylori and iron deficiency anemia: guilty as charged? Am J Med 2001; 111: 495-497 Koike T, Ohara S, Sekine H, Iijima K, Abe Y, Kato K, Toyota T, Shimosegawa T. Helicobacter pylori infection prevents erosive reflux oesophagitis by decreasing gastric acid secretion. Gut 2001; 49: 330-334 S- Editor Ma N L- Editor Ma JY E- Editor Li JL

www.wjgnet.com



RAPID COMMUNICATION

Heme oxygenase-1 induction by hemin protects liver cells from ischemia/reperfusion injury in cirrhotic rats Hui Xue, Hua Guo, Ying-Chao Li, Zhi-Ming Hao Hui Xue, Hua Guo, Ying-Chao Li, Zhi-Ming Hao, Department of Gastroenterology, The First Affiliated Hospital, Xi’an Jiaotong University Medical College, Xi’an 710061, Shaanxi Province, China Correspondence to: Dr. Hui Xue, Department of Gastroenterology, The First Affiliated Hospital, Xi’an Jiaotong University Medical College, Xi’an 710061, Shaanxi Province, China. [email protected] Telephone: +86-29-85324001 Fax: +86-29-85263190 Received: March 28, 2007 Revised: August 12, 2007

Abstract AIM: To investigate the potential protective effect of HO-1 on cirrhotic liver cells in rats. METHODS: Male Wistar rats included in the current study were randomly divided into 5 groups as follows: normal (N) group; liver cirrhotic (LC) group; sham (S) group; I/R group and I/R + hemin group. The model for inducing liver cirrhosis in rats was established according to a previously published protocol. Following this the segmental hepatic ischemia reperfusion operation was carried out. The rats were treated with 30 μmol/kg hemin (HO-1 inducer, ferric portoporphyrin IX chloride) i.p. or 0.9% NaCl (control) 24 h and 12 h before hepatic ischemia for 30 min or sham laparotomy. Blood was collected for serum enzymatic measurement 6 and 12 h after reperfusion or sham laparotomy. HO-1, NF-κB and caspase-3 expressions were assessed by immunohistochemical analysis. RESULTS: The expressions of proteins are inversely correlated to the gray values. HO-1 expression in the I/R + hemin group was increased significantly than I/R group at 6 h and 12 h after hepatic I/R (6 h: 112.0 ± 8.3 vs 125.1 ± 5.7, P < 0.01; 12 h: 120.8 ± 11.0 vs 132.4 ± 6.2, P < 0.01). Hemin improved serum manganese superoxide dismutase (MnSOD) (6 h: 131.3 ± 17.6 vs 107.0 ± 13.9, P < 0.01; 12 h: 141.4 ± 12.5 vs 118.3 ± 10.2, P < 0.01), lessened liver cell injury, decreased caspase-3(6 h: 166.7 ± 8.1 vs 145.5 ± 14.6, P < 0.01; 12 h: 172.8 ± 3.8 vs 148.0 ± 6.5, P < 0.01) and NF-κB expression (6 h: 150.2 ± 8.6 vs 139.7 ± 6.0, P < 0.01; 12 h: 151.1 ± 5.9 vs 148.1 ± 5.3, P > 0.05) and serum alanine aminotransferase (ALT) (6 h: 413.3 ± 104.1 vs 626.8 ± 208.2, P < 0.01; 12 h: 322.2 ± 98.8 vs 425.8 ± 115.4, P < 0.05), aspartate aminotransferase (AST) (6 h: 665.2 ± 70.1 vs 864.3 ± 70.4, P < 0.01; 12 h: 531.1 ± 98.6 vs 664.4 ± 115.6, P < 0.01), malondialdehyde www.wjgnet.com

(MDA) levels (6 h: 11.1 ± 2.17 vs 13.5 ± 2.01, P < 0.01; 12 h: 9.36 ± 1.10 vs 10.8 ± 1.62, P < 0.05) in the I/R + hemin group when compared with the I/R group. CONCLUSION: These results suggest that HO-1 plays an important role in protecting liver cells from hepatic I/R injury in cirrhotic rats by decreasing oxidative stress, apoptosis and inflammation. © 2007 WJG . All rights reserved. Key words: Heme oxygenase-1; Ischemia reperfusion; Caspase-3; Nuclear factor-κB; Liver cirrhosis Xue H, Guo H, Li YC, Hao ZM. Heme oxygenase-1 induction by hemin protects liver cells from ischemia/reperfusion injury in cirrhotic rats. World J Gastroenterol 2007; 13(40): 5384-5390


INTRODUCTION Gastrointestinal bleeding, liver surgery and liver transplantation may lead to hepatic ischemia/reperfusion (I/R) injury. Previous studies have shown that pro-inflammatory, apoptosis, oxidative stress and microcirculation dysfunction are strongly correlated with hepatic I/R[1,2]. Many patients presenting with hepatic ischemia have liver cirrhosis disease. Some studies have shown that cells from a cirrhotic liver are more fragile than the cells from a normal liver. Similarly, other studies have found that the anti-oxidative capacity during stress decreases in patients with hepatic decompensated cirrhosis[3,4], and further, the tolerance time of ischemia is shorter in cirrhotic rats than in normal rats[5]. Heme oxygenases (HOs), the rate-limiting enzymes in heme catabolism, catalyzes the oxidative degradation of heme into carbon monoxide (CO), free iron, and biliverdin. Three isoforms of the enzyme have been identified: The inducible HO-1, also known as heat shock protein 32, the constitutive HO-2, and a not fully defined HO-3. It has been found that HO-1 serves as a protective gene by virtue of anti-inflammatory, anti-apoptotic and antioxidative actions and improves microcirculation in many cell types[6,7]. Amersi et al[8] reported that up-regulation of HO-1 protects genetically fat Zucker rat livers from I/R injury.

Xue H et al . Hemin protects liver cells from I/R injury in cirrhotic rats by upregulating HO-1

Some researchers have demonstrated that HO-1 has potent protecting effects against I/R injury in various organ systems[9,10]. Kaizu et al[11] reported that prior induction of the HO-1 protein may lead to anti-inflammatory and antiapoptotic effects on warm renal I/R injury. HO-1 has been shown to protect liver cells from I/R injury[12,13]. However, there are few reports regarding the protective effect of HO-1 on liver cells affected by the pathological condition of cirrhosis. Investigations into the role of HO-1 in protecting liver cells from hepatic I/R injury in cirrhotic rats and the mechanism by which it achieves this protective effect have great significance. Thus, the objective of the present study was to investigate whether upregulating HO-1 would result in reduced damage to liver cells as a result of hepatic I/R in a liver cirrhotic rat model. Secondly, we investigated the possible mechanism/s by which HO-1 exerts its protective effect.

MATERIALS AND METHODS Rat cirrhosis and grouping Adult male Wistar rats (weighting 200-250 g) were obtained from the experimental Animal Center of Xi’an Jiaotong University. Ethical approval for this study was obtained from the Ethical Committee on Animal Experiments at the Medical College of Xi’an Jiaotong University. The rats had free access to food and water, were kept in an air-conditioned room at 23℃, with a 12 h/12 h light/dark cycle, and were handled humanely. Liver cirrhosis was induced by subcutaneous injection of 400 mL/L CCl4-olive oil solution twice a week at an initial dose of 5 mL/kg. Subsequent doses were adjusted to body mass changes at a dose of 3 mL/kg for 11 wk as described previously[14,15]. The rats in the normal group received the same dose of pure olive oil only. The I/R operation was carried out in wk 12. All 91 animals were randomly divided into 5 groups as follows: Normal (N) group; liver cirrhotic (LC) group; sham (S) group (cirrhotic group operated); I/R group and I/R + hemin group. The last three groups were divided into two subgroups for sampling at different time-points. The I/R + hemin group was administered 30 μmol/kg hemin[16] (HO-1 inducer, Sigma chemical Co., USA) 24 h and 12 h before hepatic I/R. Hemin solution was prepared under subdued lighting by dissolving the compound in 1 mL of 0.2 mol/L NaOH, adjusting the pH to 7.4 with 1 mol/L HCl, and diluting the solution with 9 g/L NaCl[11]. The stock concentration was 5 g/L. The solution was kept in the dark and used within 1 h. The concentration dose of hemin used in the present study was selected according to previous reports. Hepatic I/R model Rats were anaesthetized using ketamine hydrochloride (800 mg/kg) intraperitoneally. The laparotomy was performed through a midline abdominal incision. Partial (70%) hepatic ischemia of the median and the left lobes was induced by placing non-crushing microvascular clamps around the appropriate branches of the portal vein and hepatic artery for 30 min. The portal branches to the right

5385

and caudate lobes were left open to prevent mesenteric venous congestion[17]. Reperfusion was initiated by removal of the clamps. Hepatic I/R injury was induced by 30 min of ischemia followed by 6 h and 12 h of reperfusion. After 30 min of ischemia, the clamps were removed allowing the liver to reperfuse, and the wound was closed with 3-0 silk. Sham operated rats underwent isolation of the portal vein and hepatic artery without occlusions. At the indicated time after the start of reperfusion, rats were anesthetized and sacrificed. Blood was collected from the portal vein and liver samples were taken from the left lobe. The blood samples were centrifuged to obtain the serum for the biochemical analyses, and the left lobe liver tissue samples (0.8 cm × 0.4 cm × 0.4 cm) were fixed in 40 g/L paraformaldehyde for immunohistochemical analysis. Immunohistochemical analysis of HO-1, NF-κB and caspase-3 expression Serial 5 μmol/L sections were prepared after the samples had been dehydrated in graded ethanol solutions, cleared in chloroform and embedded in paraffin. Immunohistochemical staining was performed to detect expression of NF-κB (diluted 1:300; Boster, China), caspase-3 (diluted 1:100; Boster, China), and HO-1 (diluted 1:100; Boster, China) using the MaxVisionTM kit-5004 (Maxin Biotechnology Co., Fujian, China). Sections were boiled in 0.01 mol/L citrate buffer (pH 6.0) to retrieve the antigens. Endogenous peroxidases were inactivated by immersing the sections in 30 mL/L hydrogen peroxide for 20 min. The sections were then incubated overnight at 4℃ with the relevant primary rabbit anti-rat polyclonal antibodies. After washing, the sections were overlaid with peroxidase-conjugated goat anti-rabbit secondary antibodies (MaxVisionTM kit-5004, Maxin Biotechnology Co., Fujian, China) for 15 min. The chromogenic reaction was developed with diaminobenzidine. Some sections were counterstained with hematoxylin. The immunohistochemical signal was analyzed using an image acquiring and analysis system (QWin500CW, Leica, Germany). At least four random fields of each section were examined at a magnification of × 400, and analyzed using a computer image analysis system (Image-Pro Plus, Media Cybernetics, Silver Spring, MD). The expressions of proteins are inversely correlated to the gray values. Serum enzyme analysis For the assessment of hepatic injury, serum levels of aspartate transaminase (AST) and alanine transaminase (ALT) activities were measured using an automatic biochemical analyzer (Vitros 250, Johnson & Johnson Co., USA). Serum Malondialdehyde (MDA) and superoxide dismutase (SOD) activity were measured with a commercially available kit (Nanjing Jiancheng Bioengineering Institute, China, NJBI). Total SOD and Cu/Zn-SOD activities were measured. Mn-SOD activity was calculated as the difference between total SOD and Cu/Zn-SOD activity. Statistical analysis All results were expressed as mean ± SD. The statistical analysis was performed using a one-way analysis of variance. The results were considered statistically significant if P < 0.05. www.wjgnet.com

5386

ISSN 1007-9327

CN 14-1219/R


October 28, 2007

B

A

Volume 13

Number 40

C

200 180 160 140 120 100 80 60 40 20 0

0h 6h 12 h

d d

f

e

b

N group

S group

LC group

d

N group

LC group

S group

c

I/R group

f

f

I/R + hemin group

0h 6h 12 h

1000 800

d d

f

f

600 400 b

200 0

I/R group I/R + hemin group

0h 6h 12 h

b

Serum AST level (U/L)

1000 900 800 700 600 500 400 300 200 100 0

Serum MDA level (nmol/mL)

Serum MnSOD level (U/mL)

Serum ALT activity (U/L)

Figure 1 A: No evidence of collagen deposition was observed in the liver of the normal group (HE, × 400); B: The collagens deposited in portal areas of liver cirrhosis group (HE, × 400); C: Severe fibrosis with regenerating nodules was observed in liver cirrhosis group (Masson, × 100).

18 16 14 12 10 8 6 4 2 0

N group

LC group

S group

I/R group I/R + hemin group

0h 6h 12 h

d d

a

N group

LC group

S group

I/R group

f

e

I/R + hemin group

Figure 2 Serum ALT, AST activity, MnSOD and MDA levels of each group. N group: normal group; LC group: liver cirrhosis group; S group: sham group; I/R group: the rats underwent hepatic ischemia/reperfusion operation; I/R + hemin group: the rats were administered with 30 μmol/kg hemin before hepatic ischemia/reperfusion operation. 6 h: 6 h after reperfusion. 12 h: 12 h after reperfusion. After hepatic ischemia/reperfusion, the ALT, AST and MDA levels were increased and the levels were all decreased after hemin administration. The level of MnSOD was low in I/R group, and after giving hemin, its level in I/R + hemin group was higher than I/R group. Values are mean ± SD. b P < 0.01 vs N group, aP < 0.05 vs N group, dP < 0.01 vs S group, cP < 0.05 vs S group, fP < 0.01 vs I/R group, eP < 0.05 vs I/R group.

Table 1 Gray values of caspase-3, NF-kB and HO-1 of rats (mean ± SD) Group Normal Liver cirrhosis Sham I/R I/R+hemin

b

6h 12 h 6h 12 h 6h 12 h

n

NF-kB

Caspase-3

HO-1

6 10 10 10 10 9 10 9

172.0 ± 1.7 164.1 ± 3.5b 159.4 ± 4.7d 159.6 ± 4.2d 139.7 ± 6.0 148.1 ± 5.3 150.2 ± 8.6d 151.1 ± 5.9c

188.3 ± 3.5 176.8 ± 4.9b 177.2 ± 2.7d 178.5 ± 3.9d 145.5 ± 14.6 148.0 ± 6.5 166.7 ± 8.1d 172.8 ± 3.8d

158.9 ± 2.6 148.1 ± 3.7b 145.4 ± 2.9d 146.8 ± 3.8d 125.1 ± 5.7 132.4 ± 6.2 112.0 ± 8.3d 120.8 ± 11.0d

P < 0.01 vs N group; cP > 0.05 vs I/R group; dP < 0.01 vs I/R group.

RESULTS Changes in the CCl4 induced liver cirrhotic rats To confirm the occurrence of liver cirrhosis, the liver tissues were examined following hematoxylin and eosin www.wjgnet.com

staining (HE staining) and Masson’s trichrome staining (Figure 1). Biochemical analyses of the serum and immunohistochemical analysis of the liver samples were also performed. The levels of serum ALT and AST activities are markers for hepatic injury, MDA and MnSOD reflect the anti-oxidative ability. Following the administration of CCl4 for 11 wk, the activities of ALT, AST and MDA in the LC group were increased when compared with those observed in the N group. The MnSOD in the LC group was decreased when compared with the N group (Figure 2). Hemin induced HO-1 high expression in liver tissue Animals in the I/R + Hemin group were given hemin prior to the induction of I/R. We examined the changes in HO-1 expression in liver tissues after I/R using the gray value method. HO-1 expression in the I/R + hemin group was increased significantly, especially 6 h after reperfusion (Table 1 and Figure 3). Hepatic I/R resulted in an increase in serum ALT, AST and MDA levels in the I/R group

Xue H et al . Hemin protects liver cells from I/R injury in cirrhotic rats by upregulating HO-1

A1

A2

A3

B1

B2

B3

C1

C2

C3

D1

D2

D3

5387

Figure 3 Immunohistochemistry of NF-κB, caspase-3 and HO-1 at 6 h and 12 h after hepatic ischemia/reperfusion in cirrhotic rats. A: I/R group, 6 h after reperfusion; B: I/R group, 12 h after reperfusion; C: I/R + hemin group, 6 h after reperfusion; D: I/R + hemin group, 12 h after reperfusion; 1: NF-κB: Most positive NF-κB expression was in the nuclear of liver cells. After giving hemin to induce HO-1, the positive expression of NF-κB in I/R + hemin group was decreased compared with I/R group at 6 h after reperfusion (P < 0.01). There is no difference between I/R group and I/R + hemin group at 12 h after reperfusion (P > 0.05) (Figure A1-D1); 2: Caspase-3: The positive caspase-3 expression was in the cytoplasm of hepatic cells. After hemin administration, the caspase-3 expression was decreased at 6 h and 12 h after reperfusion when compared with I/R group (P < 0.01) (Figure A2-D2); 3: HO-1: The positive HO-1 expression was in the plasma of hepatic cells. After hepatic ischemia/reperfusion, the expression of HO-1 was increased in I/R group, and through giving hemin, the expression of HO-1 was higher than I/R group at 6 h and 12 h after reperfusion (P < 0.01) (Figure A3-D3).

compared with the S group, especially at 6 h after reperfusion. In contrast, the I/R + hemin group had significantly lower serum ALT, AST and MDA levels at both timepoints relative to the I/R group (Figure 2). MnSOD levels were markedly decreased in the I/R group at 6 h and 12 h after hepatic I/R compared with that of the S group. However, hemin administration increased MnSOD levels in the I/R+hemin group at both time-points (Figure 2).

Caspase-3 and NF-κB immunohistochemical staining To examine the protective effects of elevated HO-1 expression on apoptosis and inflammation in the liver following hepatic I/R, caspase-3 and NF-κB expression in the liver were assessed by immunohistochemical staining. Most positive NF-κB expression was observed in the nucleus of liver cells, while caspase-3 expression was localized in the cytoplasm of hepatocytes. After hepatic I/R, the expreswww.wjgnet.com

5388

ISSN 1007-9327

CN 14-1219/R


sion of NF-κB and caspase-3 was significantly stronger with a lower gray value in the I/R group compared with the S group (P < 0.01). After giving hemin to upregulate HO-1, caspase-3 expression in the I/R + hemin group was decreased 6 h and 12 h after reperfusion and the gray values were increased (P < 0.01). Following upregulation of HO-1, the expression of NF-κB in the I/R + hemin group was lower 6 h after reperfusion than in the I/R group (P < 0.01), but there was no difference 12 h after reperfusion between the two groups (P > 0.05, Figure 3).

DISCUSSION In this study, we have shown that HO-1 upregulation following hemin administration protects cirrhotic rats from hepatic I/R injury. Hepatic injury induced by I/R has been proposed as a key clinical problem associated with liver upper gastrointestinal bleeding, liver surgery or liver transplantation. There are two distinct phases of liver injury after I/R. The initial phase (< 2 h after reperfusion) is characterized by oxidant stress, where production and release of reactive oxygen species (ROS) appears to directly result in hepatocellular injury. The late phase of liver injury, from 6 h to 48 h after hepatic reperfusion, is an inflammatory disorder mediated by recruited neutrophils[18-20]. Our results regarding the mechanism leading to I/R injury will undoubtedly provide important clues for developing an approach to decreasing liver cell damage caused by I/R. Protection against hepatic I/R injury is, clinically, one of the most critical problems in liver cirrhotic patients. Recently, it was demonstrated that HO-1 upregulation ameliorates hepatic I/R injury[12,13]. In addition, Coito et al[21] reported that HO-1 gene transfer inhibits inducible nitric oxide synthase expression and protects genetically fat Zucker rat livers from I/R injury. In the present study, our data support the hypothesis that HO-1 provides a protective effect against I/R injury in cirrhotic rats via antioxidative, anti-apoptotic and anti-inflammatory actions. Our results also highlight that administration of hemin may be an important new therapy for the treatment of hepatic I/R. Although these mechanisms are complicated and largely unknown, our findings suggest the possibility that upregulation of HO-1 may reduce the potential risk in hepatocellular I/R complicated with liver cirrhosis. HO-1 is one of the most critical of the cytoprotective mechanisms activated during cellular stress, exerting antioxidative and anti-inflammatory functions, modulating the cell cycle, and maintaining the microcirculation. The upregulation of HO-1 is thought to be a protective response from cellular stress following ischemia, inflammation and radiation. Given the multi-factorial cytoprotective properties of HO-1, it is used as a novel strategy to prevent I/R injury[22]. In line with this, we have found that hemin administration significantly protects the cells in the hepatic I/R as compared with control treatment. The role of SOD in hepatic I/R has received more attention. There are three isoforms of SOD. The Cu/ZnSOD is located in the cytosol, the MnSOD is primarily a mitochondrial enzyme, and extracellular SOD is usually found on the outside of the plasma membrane interacting with matrix component. Marczin et al[23] reported that, by www.wjgnet.com

October 28, 2007

Volume 13

Number 40

utilizing adenovial gene transfer, overexpression of MnSOD may reduce the extent of in vivo regional I/ R injury in the rat heart. Similarly, in the current study we found that MnSOD levels were increased, and the injury due to I/R was lessened, after giving hemin to induce HO-1 expression. Based on this, we suggest that HO-1 has an antioxidant function during hepatic I/R. MDA is the main product of lipid peroxidation, and is reactive with thiobarbituric acid (TBA). MDA concentration is therefore generally presented as the total level of lipid peroxidation products[24]. As the end product of lipid peroxidation MDA can produce ozone, which reacts rapidly with cellular structures, generates hydrogen peroxide and other active oxygen species and causes peroxidation and denaturation of membranes[25]. Our findings suggest that HO-1, induced by hemin administration, decreases the serum MDA level after hepatic I/R. Therefore, we speculate that high HO-1 expression induced by hemin may decrease the lipid peroxidation and play an anti-oxidative role in hepatic I/R. Apoptosis plays a vital role in lethal hepatic I/R injury as indicated by many studies in various animal models[2,26]. There are two major pathways of apoptosis, and caspase-3 is the final executor of apoptosis[27]. Although the TUNEL assay is a very sensitive and widely used method[28,29], data is also available suggesting that caspase-3 immunohistochemistry is an easy, sensitive, and reliable method for detecting and qualifying apoptosis in tissue sections[30]. In the present study, to determine the effect of HO-1 on apoptosis, we carried out an immunohistochemical analysis of caspase-3. The expression of caspase-3 was elevated following hepatic I/R. However, caspase-3 expression was lower in the I/R + hemin group than in the I/R group. These data suggest that the increase in HO-1 following treatment with hemin results in a decrease in apoptosis during hepatic I/R in liver cirrhotic rats. Our findings in this regard are consistent with other studies[31]. NF-κB is activated during I/R of the liver, and plays an important and complex role in the gene expression of proinflammatory cytokines (TNF-α and IL-1), chemokines and adhesion molecules (ICAM-1 and VCAM-1), which will lead to the tissue injury[32]. In the present study, the level of NF-κB at 6 h after reperfusion was decreased in the I/R + hemin group compared with the I/R group. However, there was no statistical difference 12 h after reperfusion between the I/R group and the I/R + hemin group. We believe that high levels of HO-1 expression may decrease NF-κB expression to lessen the inflammatory reaction at 6 h after reperfusion. Hepatic I/R injury may cause more severe tissue damage in cirrhotic liver than in normal livers. Our data show that HO-1 overexpression, induced by hemin, plays a crucial role in protecting liver cells from hepatic I/R injury in cirrhotic rats. Our results also raise the possibility of a new treatment for reducing hepatic I/R injury in the pathological condition of cirrhosis.

COMMENTS Background

Gastrointestinal bleeding, liver surgery and liver transplantation may lead to

Xue H et al . Hemin protects liver cells from I/R injury in cirrhotic rats by upregulating HO-1 hepatic ischemia/reperfusion (I/R) injury. Previous studies have shown that pro-inflammatory, apoptosis, oxidative stress and microcirculation dysfunction are strongly correlated with hepatic I/R. Many patients presenting with hepatic ischemia have liver cirrhosis disease. Some studies have shown that cells from a cirrhotic liver are more fragile than the cells from a normal liver. Similarly, other studies have found that the anti-oxidative capacity during stress decreases in patients with hepatic decompensated cirrhosis, and further, the tolerance time of ischemia is shorter in cirrhotic rats than in normal rats.

4

Research frontiers

6

Three isoforms of the Heme oxygenases (HOs) have been identified: The inducible HO-1, also known as heat shock protein 32, the constitutive HO-2, and a not fully defined HO-3. It has been found that HO-1 serves as a protective gene by virtue of anti-inflammatory, anti-apoptotic and anti-oxidative actions and improves microcirculation in many cell types.


Researchers have demonstrated that HO-1 has potent protecting effects against I/R injury in various organ systems. It has also been reported that upregulation of HO-1 protects genetically fat Zucker rat livers from I/R injury and that prior induction of the HO-1 protein may lead to anti-inflammatory and anti-apoptotic effects on warm renal I/R injury. HO-1 has been shown to protect liver cells from I/R injury. Investigations into the role of HO-1 in protecting liver cells from hepatic I/R injury in cirrhotic rats and the mechanism by which it achieves this protective effect have great significance.

5

7

8

9 10


There are few reports regarding the protective effect of HO-1 on liver cells affected by the pathological condition of cirrhosis. The objective of the present study was to investigate whether upregulation of HO-1 would lessen the liver cell damage caused by hepatic I/R in a liver cirrhotic rat model and, secondly, to study the possible mechanisms for its protective effect. Our data support the hypothesis that HO-1 has a protective effect from I/R injury in cirrhotic rats. Our data further suggest that this effect is mediated by the anti-oxidative, anti-apoptotic and antiinflammatory actions of HO-1, and highlight that administration of hemin may be an important new therapy method in hepatic I/R. Although many of these mechanisms are still unknown, our findings suggest the possibility that upregulation of HO-1 may reduce the potential risk in hepatocellular I/R complicated with liver cirrhosis.

Applications

Hepatic I/R injury may cause more severe tissue damage in a cirrhotic liver than in a normal liver. Our data have shown that HO-1 over-expression, induced by hemin, plays a crucial role in protecting liver cells from hepatic I/R injury in cirrhotic rats, and raises the possibility of a new treatment in hepatic I/R injury in the pathological condition of cirrhosis.

Terminology

Heme oxygenases (HOs): Heme oxygenases (HOs) are the rate-limiting enzymes in heme catabolism. HOs catalyse the oxidative degradation of heme into carbon monoxide (CO), free iron, and biliverdin. Three isoforms of the enzyme have been identified: HO-1, HO-2 and HO-3. Superoxide dismutase (SOD): Superoxide dismutase (SOD) is a ubiquitous enzyme with an essential function in protecting cells against oxidative stress by catalytic removal of superoxide radicals and conversion to H2O2 by the dismutation reaction.

Peer review

In this manuscript, Hui Xu et al have analyzed the beneficial effects of hemindependent induction of hepatic heme-oxygenase-1 expression in cirrhotic livers after ischemia/reperfusion which is an interesting paper.

11

12

13

14

15 16 17

18 19 20

REFERENCES 1 2 3

Serracino-Inglott F, Habib NA, Mathie RT. Hepatic ischemiareperfusion injury. Am J Surg 2001; 181: 160-166 Kang KJ. Mechanism of hepatic ischemia/reperfusion injury and protection against reperfusion injury. Transplant Proc 2002; 34: 2659-2661 Koruk M, Aksoy H, Akcay F, Onuk MD. Antioxidant capacity and nitric oxide in patients with hepatic cirrhosis. Ann Clin Lab Sci 2002; 32: 252-256

21

22

5389

Rhoden EL, Pereira-Lima L, Kalil AN, Lucas ML, Mauri M, Menti E, Rhoden CR, Pereira-Lima J, Zettler CG, Bello-Klein A. Effects of ischemia and reperfusion on oxidative stress in hepatic cirrhosis induced by carbon tetrachloride in rats. Kobe J Med Sci 2000; 46: 171-180 Figueras J, Farran L, Benasco C, Ribas Y, Ramos E, Borobia FG, Fradera R, Castellvi J, Lama C, Jaurrieta E. Vascular occlusion in hepatic resections in cirrhotic rat livers: an experimental study in rats. Liver Transpl Surg 1997; 3: 617-623 Camara NO, Soares MP. Heme oxygenase-1 (HO-1), a protective gene that prevents chronic graft dysfunction. Free Radic Biol Med 2005; 38: 426-435 Wagener FA, Volk HD, Willis D, Abraham NG, Soares MP, Adema GJ, Figdor CG. Different faces of the heme-heme oxygenase system in inflammation. Pharmacol Rev 2003; 55: 551-571 Amersi F, Buelow R, Kato H, Ke B, Coito AJ, Shen XD, Zhao D, Zaky J, Melinek J, Lassman CR, Kolls JK, Alam J, Ritter T, Volk HD, Farmer DG, Ghobrial RM, Busuttil RW, Kupiec-Weglinski JW. Upregulation of heme oxygenase-1 protects genetically fat Zucker rat livers from ischemia/reperfusion injury. J Clin Invest 1999; 104: 1631-1639 Fondevila C, Busuttil RW, Kupiec-Weglinski JW. Hepatic ischemia/reperfusion injury--a fresh look. Exp Mol Pathol 2003; 74: 86-93 Yang S, Shih HJ, Chow YC, Tsai PS, Wang TY, Wang PS, Huang CJ. The protective role of heme oxygenase-1 induction on testicular tissues after testicular torsion and detorsion. J Urol 2007; 177: 1928-1933 Kaizu T, Tamaki T, Tanaka M, Uchida Y, Tsuchihashi S, Kawamura A, Kakita A. Preconditioning with tinprotoporphyrin IX attenuates ischemia/reperfusion injury in the rat kidney. Kidney Int 2003; 63: 1393-1403 Wang XH, Wang K, Zhang F, Li XC, Qian XF, Cheng F, Li GQ, Fan Y. Alleviating ischemia-reperfusion injury in aged rat liver by induction of heme oxygenase-1. Transplant Proc 2004; 36: 2917-2923 Katori M, Anselmo DM, Busuttil RW, Kupiec-Weglinski JW. A novel strategy against ischemia and reperfusion injury: cytoprotection with heme oxygenase system. Transpl Immunol 2002; 9: 227-233 Gandhi CR, Nemoto EM, Watkins SC, Subbotin VM. An endothelin receptor antagonist TAK-044 ameliorates carbon tetrachloride-induced acute liver injury and portal hypertension in rats. Liver 1998; 18: 39-48 Xu JW, Gong J, Chang XM, Luo JY, Dong L, Hao ZM, Jia A, Xu GP. Estrogen reduces CCL4- induced liver fibrosis in rats. World J Gastroenterol 2002; 8: 883-887 Datta PK, Duann P, Lianos EA. Long-term effect of heme oxygenase (HO)-1 induction in glomerular immune injury. J Lab Clin Med 2006; 147: 150-155 Ku Y, Kusunoki N, Shiotani M, Maeda I, Iwasaki T, Tominaga M, Kitagawa T, Fukumoto T, Suzuki Y, Kuroda Y. Stimulation of haematogenous liver metastases by ischaemia-reperfusion in rats. Eur J Surg 1999; 165: 801-807 Fan C, Zwacka RM, Engelhardt JF. Therapeutic approaches for ischemia/reperfusion injury in the liver. J Mol Med 1999; 77: 577-592 Teoh NC, Farrell GC. Hepatic ischemia reperfusion injury: pathogenic mechanisms and basis for hepatoprotection. J Gastroenterol Hepatol 2003; 18: 891-902 Arii S, Teramoto K, Kawamura T. Current progress in the understanding of and therapeutic strategies for ischemia and reperfusion injury of the liver. J Hepatobiliary Pancreat Surg 2003; 10: 189-194 Coito AJ, Buelow R, Shen XD, Amersi F, Moore C, Volk HD, Busuttil RW, Kupiec-Weglinski JW. Heme oxygenase-1 gene transfer inhibits inducible nitric oxide synthase expression and protects genetically fat Zucker rat livers from ischemiareperfusion injury. Transplantation 2002; 74: 96-102 Kupiec-Weglinski JW, Busuttil RW. Ischemia and reperfusion injury in liver transplantation. Transplant Proc 2005; 37: 1653-1656 www.wjgnet.com

5390

ISSN 1007-9327

23

CN 14-1219/R


Marczin N, El-Habashi N, Hoare GS, Bundy RE, Yacoub M. Antioxidants in myocardial ischemia-reperfusion injury: therapeutic potential and basic mechanisms. Arch Biochem Biophys 2003; 420: 222-236 24 Drewa G, Krzyzynska-Malinowska E, Wozniak A, ProtasDrozd F, Mila-Kierzenkowska C, Rozwodowska M, Kowaliszyn B, Czajkowski R. Activity of superoxide dismutase and catalase and the level of lipid peroxidation products reactive with TBA in patients with psoriasis. Med Sci Monit 2002; 8: BR338-BR343 25 Ajamieh HH, Menendez S, Martinez-Sanchez G, CandelarioJalil E, Re L, Giuliani A, Fernandez OS. Effects of ozone oxidative preconditioning on nitric oxide generation and cellular redox balance in a rat model of hepatic ischaemiareperfusion. Liver Int 2004; 24: 55-62 26 Zhao ZQ. Oxidative stress-elicited myocardial apoptosis during reperfusion. Curr Opin Pharmacol 2004; 4: 159-165 27 Kam PC, Ferch NI. Apoptosis: mechanisms and clinical implications. Anaesthesia 2000; 55: 1081-1093

October 28, 2007

Volume 13

Number 40

28

Elsasser A, Suzuki K, Schaper J. Unresolved issues regarding the role of apoptosis in the pathogenesis of ischemic injury and heart failure. J Mol Cell Cardiol 2000; 32: 711-724 29 Eefting F, Rensing B, Wigman J, Pannekoek WJ, Liu WM, Cramer MJ, Lips DJ, Doevendans PA. Role of apoptosis in reperfusion injury. Cardiovasc Res 2004; 61: 414-426 30 Duan WR, Garner DS, Williams SD, Funckes-Shippy CL, Spath IS, Blomme EA. Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts. J Pathol 2003; 199: 221-228 31 Choi BM, Pae HO, Jeong YR, Oh GS, Jun CD, Kim BR, Kim YM, Chung HT. Overexpression of heme oxygenase (HO)-1 renders Jurkat T cells resistant to fas-mediated apoptosis: involvement of iron released by HO-1. Free Radic Biol Med 2004; 36: 858-871 32 Ali S, Mann DA. Signal transduction via the NF-kappaB pathway: a targeted treatment modality for infection, inflammation and repair. Cell Biochem Funct 2004; 22: 67-79 S- Editor Ma N L- Editor Li M E-Editor Li JL

www.wjgnet.com



CASE REPORT

Sequential stenotic strictures of the small bowel leading to obstruction George Van Buren II, Davis C Teichgraeber, Rhonda P Ghorbani, Eduardo A Souchon George Van Buren II, Eduardo A Souchon, Department of General Surgery, The University of Texas Health Science Center at Houston, Houston, TX 77030, United States Davis C Teichgraeber, Department of Radiology, The University of Texas Health Science Center at Houston, Houston, TX 77030, United States Rhonda P Ghorbani, Department of Pathology, The University of Texas Health Science Center at Houston, Houston, TX 77030, United States Correspondence to: George Van Buren II, MD, The University of Texas Health Science Center Houston, Department of Surgery, 6431 Fannin Street, MSB 4.169, Houston, Texas 77030, United States. [email protected] Telephone: +1-713-5007216 Fax: +1-713-5007213 Received: May 15, 2007 Revised: July 29, 2007

Abstract Small bowel obstructions (SBOs) are primarily caused by adhesions, hernias, neoplasms, or inflammatory strictures. Intraluminal strictures are an uncommon cause of SBO. This report describes our findings in a unique case of sequential, stenotic intraluminal strictures of the small intestine, discusses the differential diagnosis of intraluminal intestinal strictures, and reviews the literature regarding intraluminal pathology. © 2007 WJG . All rights reserved.

Key words: Small bowel obstruction; Stenosis; Stricture Van Buren II G, Teichgraeber DC, Ghorbani RP, Souchon EA. Sequential stenotic strictures of the small bowel leading to obstruction. World J Gastroenterol 2007; 13(40): 5391-5393


INTRODUCTION Small bowel obstructions (SBOs) are most commonly caused by adhesions, hernias, neoplasms, or inflammatory strictures[1], with most caused by extraluminal adhesions due to postoperative inflammatory changes[2]. Intraluminal strictures are less common and are primarily caused by congenital defects, inflammation, ischemia, neoplasms, or radiation. We present an unusual case of sequential, stenotic strictures of the small intestine with no clearly defined etiology.

CASE REPORT A 43-year-old Hispanic female presented to a surgical ser vice for evaluation of SBO. Four days prior to presentation she developed nausea and mid-epigastric, cramping abdominal pain. She had not been able to tolerate liquids or solids for 2 d. She had a 22-year history of chronic abdominal pain. An outpatient work-up involving an abdominal computed tomography (CT) scan the previous year revealed dilated loops of small intestine, with areas of diffuse thickening of the intestinal wall, but no evidence of obstruction. An esophagogastroduodenoscopy revealed gastritis but no major pathology. Stool cultures sent for Clostridium difficile toxin, ova, and parasites (Iodine and trichrome staining for worms (i.e. Ascaris, Necator, Enterobius, Taenia), amoeba (i.e. Entamoeba), flagellates (i.e. Giardia), cryptosporidium, and others) were negative. Her surgical history included bilateral tubal lig ation, open appendectomy, and laparoscopic cholecystectomy. She had no personal or family history of trauma, cancer, or inflammatory bowel disease. The patient was awake and alert with stable vital signs. Her abdomen was soft and mildly distended, with mid-epigastric tenderness upon palpation. She had no peritoneal signs. Her laboratory test values were all within normal limits. A CT scan of the abdomen revealed a proximal SBO with dilated loops of small intestine and a transition point in the mid jejunum. Distal to the transition point, the intestine was thickened for several centimeters (Figure 1). Distal to the thickened portion, the intestine was normal in appearance. The terminal ileum and colon were normal, and no retroperitoneal lymphadenopathy was present. On exploratory laparotomy, we found a thickened, dilated proximal small intestine with a clear transition point to the decompressed distal jejunum. The intestine was pink and viable. No extraluminal bands, adhesions or hernias were present. The duodenum and ligament of trietz appeared normal. Distal to the duodenum, the jejunum became thickened and dilated. We found deposits of mesenteric fat on the serosal edge of the intestine every 5-10 cm. Associated with these areas of mesenteric fat were palpable intraluminal stenoses. The rest of the intestine was normal in appearance. There were no intestinal or mesenteric masses. Strong pulses were palpated in all of the mesenteric vessels. We resected the thickened portion of jejunum and mesentery, and performed a jejunojejuno anastomosis. www.wjgnet.com

5392

ISSN 1007-9327

F

CN 14-1219/R


October 28, 2007

Volume 13

Number 40

GASTRO × 3 & 150 CC OMNIPA QUE LOC: -286.93 THK: S FFS

L R

Figure 1 CT scan of the abdomen. A CT scan of the abdomen revealed an SBO with dilation of small-intestinal loops, and a transition point in the mid-jejunum. A distended small was observed anteriorly of the air fluid level, and distal to this, a dilated loop and cross section was filled with feces, the so-called small bowel feces sign, which is indicative of SBO. Distal to the obstruction, there was a clear transition point in the mid-jejunum.

A

B

C

Figure 2 Gross specimens. Seventy-six centimeter segment of small intestine. On gross examination, fat surrounded the serosal surface of the intestine at irregular intervals, at the mesenteric border (A). Associated with these deposits of fat were 14 areas of mucosal stricture. In the normal intestine, the luminal diameter ranged from 4.7 to 9.5 cm. In the areas of luminal constriction, the diameter was 0.7 to 1.0 cm (B, C).

A 76-cm segment of small intestine was resected. On gross examination, fat was seen surrounding the serosal surface of the intestine at irregular intervals at the mesenteric border (Figure 2A). Associated with these deposits of fat were 14 areas of mucosal stricture causing luminal constriction (Figure 2B and C). The lumen between the constrictions was dilated and showed mucosal thinning. In the mesentery, four benign reactive perienteric lymph nodes were present. On microscopic examination, the intestinal strictures contained mucosa and submucosa. Muscularis propria was not present within the webs (Figure 3). There was no evidence of inflammatory bowel disease, malignancy, or pathologic inflammation of the bowel or mesentery. Postoperatively, bowel function was slow to return to www.wjgnet.com

Figure 3 Microscopic image. On microscopic examination, the intestinal webs contained mucosa and submucosa. Muscularis propria was not present within the webs. There was no evidence of inflammatory bowel disease, malignancy, or pathologic inflammation of the bowel or mesentery.

normal. The patient had minimal bowel sounds and did not pass flatus until postoperative day (POD) 8. After POD 8, the patient progressed quickly; first, she tolerated a clear liquid diet and then a regular diet. On POD 10, the patient had an upper gastrointestinal barium study with a small intestine follow through, which showed normal motility, prompt emptying through the anastomosis, and barium reached the cecum within an hour. She was discharged on POD 10 and had no postoperative complications.

DISCUSSION SBOs are most commonly caused by adhesions, hernias, neoplasms, or inflammatory strictures [1] , with most caused by extraluminal adhesions due to postoperative inflammatory changes [2] . Although our patient had a history of abdominal surgery, she had no evidence of adhesions or extraluminal patholog y. Her SBO was secondary to intraluminal strictures. Intraluminal strictures are primarily caused by congenital defects, inflammation, ischemia, neoplasms or radiation. Congenital atresia is a common cause of neonatal intestinal obstruction. Jejunoileal atresia primarily results from intrauterine vascular events and tends to present early in life[3]. Late presentation of jejunal atresia has been reported; however, this is associated with other abnormalities[4]. It is unlikely that our patient had such a condition that had remained undiagnosed since birth. Inf lammator y bowel diseases, such as Crohn’ s disease, may also cause intestinal strictures. Crohn’s disease presents with full thickness inflammation of the intestinal wall. Obstruction occurs through either acute inflammation leading to occlusion, or through chronic inflammation leading to strictures. Our patient had no clinical or pathologic evidence of acute or chronic inflammatory bowel disease. Chronic inflammatory bowel disease strictures also tend to be longitudinal and localized to the ileum[5,6]; whereas our patient’s strictures were distinct, concentric jejunal bands. Intestinal ischemia may also cause intestinal strictures and usually occurs in the presence of trauma, drug use, or vascular disease. The patient had no history of vascular disease or abdominal

Van Buren II G et al . Stenotic strictures of the small bowel

trauma, and reported no history of postprandial abdominal pain or fecal blood, which might indicate chronic intestinal ischemia. Cases of intestinal ischemia usually reveal some degree of mucosal ulceration and necrosis, with associated intestinal fibrosis and vascular disease[7]. A Medline search revealed no previous cases of sequential, stenotic strictures of the small intestine. Intraoperatively, the intestine was viable and the mesenteric vessels had strong pulses. On pathologic examination, the patient had no evidence of mucosal irritation, intestinal fibrosis, or vascular pathology. Malignancies may be another cause of intraluminal strictures. Benign tumors are the most common (i.e. leiomyomas); however, malignant lesions are more likely to have a symptomatic presentation. Melanoma is the most common metastatic malignancy of the small intestine and adenocarcinoma is the most common primary malignancy [8]. Additionally, radiation therapy used to treat intraabdominal masses may cause intestinal webs or scarring[9]. However, the patient had no history of malignancy or radiation therapy, and the pathology revealed no evidence of malignancy. Chronic nonsteroidal anti-inflammatory (NSAID) usage may also cause intraluminal pathology [10]. With newer imaging modalities such as capsule endoscopy, the small intestine is increasingly being recognized as a site of NSAID-induced toxicity. Mucosal damage is primarily mediated by a cycloxygenase-independent mechanism, but may also be caused by cycloxygenasedependent mechanisms [10] . NSAID usage may cause either longitudinal or concentric strictures; however, our patient had no history of chronic NSAID usage, and there was no pathologic evidence of mucosal ulceration or inflammation. Other possible sources of intestinal stricture are parasites (e.g. Ascaris) or bacteria (e.g. Mycobacterium tuberculosis); however, the patient had no evidence of bacterial or parasitic infection. In conclusion, in this report, we describe our finding

5393

of sequential, stenotic intraluminal strictures of the small intestine, which led to SBO. The unusual sequential, concentric nature of the strictures, along with the lack of evidence of inflammatory bowel disease, vascular disease, or malignancy is believed to be unique. This lack of a clearly defined etiology of the patient’s strictures made this an intriguing case.

REFERENCES 1

Markogiannakis H, Messaris E, Dardamanis D, Pararas N, Tzertzemelis D, Giannopoulos P, Larentzakis A, Lagoudianakis E, Manouras A, Bramis I. Acute mechanical bowel obstruction: clinical presentation, etiology, management and outcome. World J Gastroenterol 2007; 13: 432-437 Nagle A, Ujiki M, Denham W, Murayama K. Laparoscopic 2 adhesiolysis for small bowel obstruction. Am J Surg 2004; 187: 464-470 Dalla Vecchia LK, Grosfeld JL, West KW, Rescorla FJ, Scherer 3 LR, Engum SA. Intestinal atresia and stenosis: a 25-year experience with 277 cases. Arch Surg 1998; 133: 490-496; discussion 496-497 Peetsold MG, Ekkelkamp S, Heij HA. Late presentation of a 4 duodenal web in a patient with situs inversus and apple peel jejunal atresia. Pediatr Surg Int 2004; 20: 301-303 5 Hurst RD, Molinari M, Chung TP, Rubin M, Michelassi F. Prospective study of the features, indications, and surgical treatment in 513 consecutive patients affected by Crohn's disease. Surgery 1997; 122: 661-667; discussion 667-668 6 Taschieri AM, Cristaldi M, Elli M, Danelli PG, Molteni B, Rovati M, Bianchi Porro G. Description of new "bowelsparing" techniques for long strictures of Crohn's disease. Am J Surg 1997; 173: 509-512 7 Lee-Elliott C, Landells W, Keane A. Using CT to reveal traumatic ischemic stricture of the terminal ileum. AJR Am J Roentgenol 2002; 178: 403-404 8 Gill SS, Heuman DM, Mihas AA. Small intestinal neoplasms. J Clin Gastroenterol 2001; 33: 267-282 9 H a t o u m O A , B i n i o n D G , P h i l l i p s S A , O ' L o ug hlin C, Komorowski RA, Gutterman DD, Otterson MF. Radiation induced small bowel "web" formation is associated with acquired microvascular dysfunction. Gut 2005; 54: 1797-800 10 Fortun PJ, Hawkey CJ. Nonsteroidal antiinflammatory drugs and the small intestine. Curr Opin Gastroenterol 2005; 21: 169-175 S- Editor Liu Y L- Editor Kerr C

E- Editor Yin DH

www.wjgnet.com



CASE REPORT

Occasional finding of mesenteric lipodystrophy during laparoscopy: A difficult diagnosis Nereo Vettoretto, Domenico Roberto Diana, Roberto Poiatti, Armando Matteucci, Caterina Chioda, Maurizio Giovanetti Nereo Vettoretto, Domenico Roberto Diana, Roberto Poiatti, Maurizio Giovanetti, Department of General and Vascular Surgery, M Mellini Hospital, Chiari (BS) 25032, Italy Armando Matteucci, Surgical Clinic, University of Brescia, Brescia, Italy Caterina Chioda, Department of Pathology, M Mellini Hospital, Chiari (BS) 25032, Italy Correspondence to: Nereo Vettoretto, MD, General and Vascular Surgery, Az. Osp. M. Mellini, V.le Mazzini 4, Chiari (BS) 25032, Italy. [email protected] Telephone: +39-30-7102799 Fax: +39-30-7102478 Received: March 28, 2007 Revised: August 17, 2007

Abstract Mesenteric lipodystrophy is a rare pathological condition affecting the mesentery. Its initial presentation is typically asymptomatic. Pathological characteristics are unspecific, and generally attributed to inflammation, unless the diagnosis is suspected. Laparoscopy done for other reasons has been, as in this case, unsuccessful in providing evidence for the correct diagnosis, thus requiring laparotomy due to lack of diagnostic tissue. After 6 mo no further medical therapy is required, as the patient remains asymptomatic. Discussion of this case and a brief review of the literature are presented in the following paragraphs. © 2007 WJG . All rights reserved.

Key words: Mesenteritis; Lypodystrophy; Laparoscopy; Panniculitis; Sclerosing mesenteritis Vettoretto N, Diana DR, Poiatti R, Matteucci A, Chioda C, Giovanetti M. Occasional finding of mesenteric lipodystrophy d u r i n g l a p a r o s c o p y : A d i ff i c u l t d i a g n o s i s . World J Gastroenterol 2007; 13(40): 5394-5396


INTRODUCTION Mesenteric lipodystrophy is an uncommon diagnosis with few, if any, symptoms at presentation. Only 200 cases have been reported in the world literature[1]. These cases mirror the presentation and symptoms of this case. Therefore, the diagnosis requires the astute clinical www.wjgnet.com

suspicion of the surgeon, radiologist, and pathologist, as it may resemble other diseases which require different therapeutic approaches. Diagnosis is usually achieved through laparotomy performed for a mesenteric mass with lipomatous characteristics. The first report is attributed to Jura[2], an Italian surgeon, who described a condition of “retractile sclerosing mesenteritis” in 1924. The term was reprised by Durst[3], who reviews 68 cases previously described in the literature and defines it as a benign disease. Today most articles describe the pathologic condition as “sclerosing mesenteritis”, and divide it into three major phases, each regarding a progressively worsening state. The pathologic findings begin with fat necrosis and end with regenerative fibrosis. Within this progress of disease state, fat necrosis predominates in the first step, therefore called mesenteric lipodystrophy. This is then followed by mesenteric panniculitis, characterized by intense inflammatory reaction, and lastly by retractile mesenteritis, when fibrosis becomes the main feature.

CASE REPORT An obese (body mass index = 41), 56-year old Caucasian man underwent laparoscopy for the repair of an umbilical hernia (> 3 cm). On exploration chylous ascites was noted in the pouch of Douglas and taken for examination. Because of the patient’s obesity no mass or other abnormalities were noted, with the exception of ceruleus stains on the surface of the mesentery, at first attributed to an unknown previous pancreatitis. A laparoscopic repair was performed, using a polytetrafluoroethylene (PTFE) mesh. The recovery was uneventful with dismissal on post operative day five. The patient did have chylous drainage in the first two days which then ceased by dismissal. Cytological and chemical examination of the fluid was negative for malignant cells, even though mononuclear inflammatory cells with enlarged cytoplasm were noted. Abdominal ultrasound and blood chemistr y were normal, comprising amylase, lypase, lipidic asset, carcinoembryonal antigen (CEA) and Ca 19-9. After a month, a follow-up computed tomography (CT) scan (Figure 1) revealed hyperdensity of mesenteric fat, with regular margins, and radiological characteristics of a mesenteric lipoma (8 cm wide). Exploratory laparotomy was then performed to exclude definitely any malignancy. The mass was biopsied, however a complete removal was impossible due to its encasement of the superior mesenteric vessels

Vettoretto N et al . Mesenteric lipodystrophy, a difficult laparoscopic diagnosis

Figure 1 CT scan visualizing a mesenteric lipoma-like mass.

Figure 2 Intraoperative view of pseudonodular thickening of the mesentery.

(Figure 2). The ileum showed chronic ischemic condition with scars on the serosal surface. Macro-biopsies were performed which excluded neoplastic origin and diagnosed fatty necrosis, with scarce fibrotic component, adipose cells with foamy cytoplasm, infiltration of lipid-laden macrophages and foreign body giant cells (Figure 3). The patient was asymptomatic in the next six months, without any medical therapy, and did not complain of any digestive discomfort. The follow-up CT after three months was unchanged, except for a minimal reduction in the diameter of the mesenteric enlargement.

DISCUSSION A nosological classification has been recently proposed[4] for this rare pathology with unknown origin, defining under the word of “sclerosing mesenteritis”, a disease previously described as mesenteric panniculitis, retractile mesenteritis, isolated lipodystrophy, mesenteric lipogranuloma, mesenteric manifestation of WeberChristian disease, etc. Only three large series have been collected in Literature, one by Durst[3], one by Kipfer[5] and one more recently by Emory[4] of 68, 53 and 42 cases, respectively. Still this disease is largely unknown to many surgeons and pathologists. It usually affects males (2-3:1), in the fifth or sixth decade of life (age range 20-80 years), and might take on different clinical aspects. In almost half of the cases (as in our patient) the mass is incidentally

5395

Figure 3 Mesenteric adipose tissue showing lipid necrosis and foamy macrophages (HE, x 10).

discovered during abdominal surg er y (i.e. during cholecystectomy). The remaining patients might complain of various degrees of intestinal obstruction, from postprandial discomfort to acute occlusion. Other uncommon clinical findings may be fever, protein, losing enteropathy or abdominal mass. The biochemistry is mostly unhelpful in diagnosis. CT scan appearance varies from increased attenuation (“misty mesentery”) to solid soft-tissue mass, which might envelope the mesenteric vessels preserving a fatty area around (“fat ring sign”)[6,7]. Other tumors of the mesentery may show a similar radiologic appearance, like lipoma, lymphoma, carcinoid tumor, carcinomatosis or tubercolosis, mesothelioma, edema or hematoma (from cirrhosis, trauma, hypoalbuminemia, heart failure or vasculitis)[8]. Percutaneous needle aspiration or biopsy can give a hint to diagnosis, but generally laparotomy and surgical biopsy is required. More recently laparoscopic biopsy has been described [9,10] , but a pre-operation suspicion is mandatory, given, for example, by a CT scan. In our case, obesity (hiding the diffuse thickness of the mesentery) and the peritoneal aspect (mimicking that of a pancreatitis) misled laparoscopic diagnosis. A CT scan, performed after dismissal, led to laparotomy, in order to obtain a radical excision of the described mass. Intraoperative findings might be a diffuse thickening of the mesentery, a single tumor, multiple tumors or also a various mix of the nodular and hypertrophic components. Also the mesocolon may be involved, as well as, rarely, mesoappendix, peripancreatic area, omentum and pelvis. Its gross appearance explains how easy it is to mimick many other intra-abdominal neoplastic diseases. Histology displays different grades of involvement. Lipodystrophy is characterized by mesenteric infiltration by lipid-filled macrophages in the narrow septa inside the adipose tissue, as inflammation increases (panniculitis) lymphocytic infiltrate and lipid cystic necrosis can be noted. The last stage is distinguished by a diffuse presence of necrosis and fibrosis that contribute to tissue retraction as seen in retractile mesenteritis. Also calcifications and giant multinulceolated cells have been seen in some cases. In the case presented, macrophage infiltration was the main feature, even if a component of fatty necrosis and fibrosis was evidenced. Haematoxylin and eosin stain

www.wjgnet.com

5396

ISSN 1007-9327

CN 14-1219/R


is generally enough to provide a correct diagnosis, but also immunochemistry might prove useful in some cases (between antibodies’ panel nuclear beta-catenin has proved interesting)[11]. The differential diagnosis comprises foreign body reaction (more fibrosis and fewer lipid laden macrophages) and Weber-Christian disease (predominantly localized in the subcutaneous tissue and with enhanced lymphocytic infiltrate) [12]. Lipomatosis, lymphoma and retroperitoneal fibrosis have quite different microscopic features, and no fatty necrosis[13]. Although the aetiology remains obscure, association with other diseases has been described. AIDS [14], non-Hodgkin lymphoma [15], tuberculitis [1], cholesterol ester storage disease [16] have been found together with sclerosing mesenteritis. Drug therapy is not standardized, and should be based on the stage of the disease. In the first stage, when fat necrosis is predominant, authors agree not to treat the disease as it can regress spontaneously. Chronic inflammation requires therapy based on corticosteroids and various types of immunosuppressants. Good results are reported with cyclophosphamide, colchicine, azathioprine and also with oral progesteron[17]. As intense fibrosis appears, bowel obstruction may occur. Intestinal resections, bypasses or neostomy might be required[18,19]. In summary, diagnosis of this unspecific, benign inflammatory disease is a challenge, both for the radiologist, surgeon and pathologist. Some authors[20] suggest that this condition might be more common than reported. In fact, as Poniachik et al has supposed[21], the suspect of sclerosing mesenteritis has to be arisen in order to prevent such conditions to be catalogued as “unspecific inflammatory process”. Since we also met the difficulty in diagnosis by laparoscopic means, especially for large masses in obese patients, we acknowledge that laparotomy is required to achieve complete removal or at least a macro-biopsy of the mass, as pathologic examination on frozen section might be incomplete, and radiology is unhelpful.

3 4 5 6 7

8

9 10 11

12 13 14

15

16 17

ACKNOWLEDGMENTS The authors thank David W Larson, MD, from Mayo Clinic (Rochester, MN, USA) for his help and suggestions.

18

REFERENCES

19

1 2

Ege G, Akman H, Cakiroglu G. Mesenteric panniculitis associated with abdominal tuberculous lymphadenitis: a case report and review of the literature. Br J Radiol 2002; 75: 378-380 Jura V. Mesenterite retrattile-caso clinico: risultati sperimentali, rilievi patogenetici, considerazoni cliniche. Policlinico (sez. Chir) 1927; 34: 535-556, 566-599

20 21

October 28, 2007

Volume 13

Number 40

Durst AL, Freund H, Rosenmann E, Birnbaum D. Mesenteric panniculitis: review of the leterature and presentation of cases. Surgery 1977; 81: 203-211 Emory TS, Monihan JM, Carr NJ, Sobin LH. Sclerosing mesenteritis, mesenteric panniculitis and mesenteric lipodystrophy: a single entity? Am J Surg Pathol 1997; 21: 392-398 Kipfer RE, Moertel CG, Dahlin DC. Mesenteric lipodystrophy. Ann Intern Med 1974; 80: 582-588 Horton KM, Lawler LP, Fishman EK. CT findings in sclerosing mesenteritis (panniculitis): spectrum of disease. Radiographics 2003; 23: 1561-1567 Patel N, Saleeb SF, Teplick SK. General case of the day. Mesenteric panniculitis with extensive inflammatory involvement of the peritoneum and intraperitoneal structures. Radiographics 1999; 19: 1083-1085 Seo BK, Ha HK, Kim AY, Kim TK, Kim MJ, Byun JH, Kim PN, Lee MG, Yang SK, Yu ES, Kim JH. Segmental misty mesentery: analysis of CT features and primary causes. Radiology 2003; 226: 86-94 Weiser J, Salky B, Slepian A, Dikman S. Laparoscopic diagnosis of retractile mesenteritis. Gastrointest Endosc 1992; 38: 615-617 Rajendran B, Duerksen DR. Retractile mesenteritis presenting as protein-losing gastroenteropathy. Can J Gastroenterol 2006; 20: 787-789 Montgomery E, Torbenson MS, Kaushal M, Fisher C, Abraham SC. Beta-catenin immunohistochemistry separates mesenteric fibromatosis from gastrointestinal stromal tumor and sclerosing mesenteritis. Am J Surg Pathol 2002; 26: 1296-1301 Wang H, Recant W, Montag AG, Hart J. Pathologic quiz case. A large mesentric mass in 40-year-old man. Arch Pathol Lab Med 2001; 125: 443-444 Bashir MS, Abbott CR. Mesenteric lipodystrophy. J Clin Pathol 1993; 46: 872-874 Aboulafia DM. Inflammatory pseudotumor causing small bowel obstruction and mimicking lymphoma in a patient with AIDS: clinical improvement after initiation of thalidomide treatment. Clin Infect Dis 2000; 30: 826-831 Petrovic D, Jovanovic D, Guduric B, Filipovic B, Mutibaric A. Primary non-Hodgkin lymphoma of the jejunum associated with mesenteric lipodystrophy: a case report. Arch Oncol 2004; 12: 67-70 Cherayil GD, Scaria KS, Hensley GT, Elliott WH. Abnormal l i p i d co mp o si t i o n o f f a t t i s su e i n h u ma n mesenteric panniculitis. Lipids 1981; 16: 199-202 Colomer Rubio E, Blanes Gallego A, Carbonell Biot C, Villar Grimalt A, Tomas Ivorra H, Llamusi Lorente A. Mesenteric panniculitis with retroperitoneal involvement resolved after treatment with intravenous cyclophosphamide pulses. An Med Interna 2003; 20: 31-33 Shah DM, Patel SB, Shah SR, Goswami KG. Mesenteric panniculitis a case report and review of literature. Ind J Radiol Imag 2005; 15: 191-192 Parra-Davila E, McKenney MG, Sleeman D, Hartmann R, Rao RK, McKenney K, Compton RP. Mesenteric panniculitis: case report and literature review. Am Surg 1998; 64: 768-771 Khachaturian T, Hughes J. Mesenteric panniculitis. West J Med 1988; 148: 700-701 Jaime Poniachik T, Gladys Smok S. Mesenterits retractil. Comunicacion de cuatro casos. Rev Med Chile 2000; 128(11): 1250-1254 S- Editor Zhu LH L- Editor Wang XL E- Editor Li HY

www.wjgnet.com



CASE REPORT

Complications and treatment of migrated biliary endoprostheses: A review of the literature Thomas Namdar, Andreas Martin Raffel, Stefan Andreas Topp, Lisa Namdar, Ingo Alldinger, Marcus Schmitt, Wolfram Trudo Knoefel, Claus Ferdinand Eisenberger Thomas Namdar, Andreas Martin Raffel, Stefan Andreas Topp, Lisa Namdar, Ingo Alldinger, Wolfram Trudo Knoefel, Claus Ferdinand Eisenberger, Department of General, Visceral and Pediatric Surgery, Heinrich-Heine University, Düsseldorf 40225, Germany Marcus Schmitt, Department of Gastroenterology, Hepatology and Infectiology, Heinrich-Heine University, Düsseldorf 40225, Germany Correspondence to: Thomas Namdar, MD, Department of General, Visceral and Pediatric Surgery, Heinrich-Heine University, Moorenstrasse 5, Düsseldorf 40225, Germany. [email protected] Telephone: +49-211-8117351 Fax: +49-211-8117359 Received: March 9, 2007 Revised: July 23, 2007

Abstract Endoscopic biliary stent insertion is a well-established procedure. It is especially successful in treating postoperative biliary leaks, and may prevent surgical intervention. A routine change of endoprostheses after 3 mo is a common practice but this can be prolonged to 6 mo. We reported a colonic perforation due to biliary stent dislocation and migration to the rectosigmoid colon, and reviewed the literature. © 2007 WJG . All rights reserved.

Key words: Biliary endoprostheses; Migrated biliary stent; Colonic perforation; Biliary stent complications Namdar T, Raffel AM, Topp SA, Namdar L, Alldinger I, Schmitt M, Knoefel WT, Eisenberger CF. Complications and treatment of migrated biliary endoprostheses: A review of the literature. World J Gastroenterol 2007; 13(40): 5397-5399


INTRODUCTION Endoscopic biliary stent insertion is a well-established treatment for hepatic, biliary or pancreatic disorders (e.g. chronic pancreatitis or pancreatic carcinoma). It is especially successful in treating postoperative biliary leaks, and may prevent surgical intervention[1,2]. Stenting of the biliary duct can be performed with a variety of prosthese that can

differ by size, design and material[3,4]. The available biliary endoprostheses can be classified by material into two categories: plastic (i.e. polyethylene, polyurethane and “Teflon”) and metallic stents. Plastic endoprostheses are less expensive but have a higher risk of clogging and dislocation[5]. On the other hand, they are easier to remove or to change. A routine change of an endoprosthesis after 3 mo is common practice, but this can be prolonged to 6 mo[6]. To avoid stent migration, the biliary stent should be placed across the sphincter of Oddi[7]. Distal stent migration is an infrequent late complication, but occurs in up to 6% of cases[3,8]. The majority of biliary endoprostheses pass through the intestine without any problems. Infrequently, however, stents get stuck in the bowel, leading to complications. Endoscopic retrieval is often possible and surgical intervention is rarely necessary[9,10]. The most common site of a migrated biliary stent is the duodenum[11-17], whereas complications in the rest of the small intestine [18,19] or colon[20-30] are rare.

CASE REPORT A 65-year-old female patient presented with 4 d of persistent diffuse colicky abdominal pain and localized peritonitis in the supra-symphysial area. Normal leukocytes, elevated C-reactive protein (14.5 mg/dL; normal value < 0.5) and simultaneous cystitis (6500 bacteria/µL; normal value < 600) were found. Three months previously, the patient had undergone a laparoscopic cholecystectomy 4 wk after acute cholangitis with choledocholithiasis. The intraoperatively placed easy-flow drainage showed early postoperative biliary secretion. Endoscopic retrograde cholangiography was performed and showed a small peripheral leakage from the liver. A residual bile duct stone that was causing partial obstruction was removed. A 12 French 10 cm plastic stent was placed in the common bile duct without sphincterotomy. Biliary drainage stopped concurrently. The patient was discharged on the ninth postoperative day. A biliary stent extraction was advised at 4-6 wk later, but this was not carried out because of the patient’s noncompliance. A computed tomography (CT) scan was performed because of extensive pain. A biliary stent dislocation and migration to the rectosigmoid colon was detected. The CT scan showed rectal perforation, and the stent was found in www.wjgnet.com

5398

ISSN 1007-9327

CN 14-1219/R


October 28, 2007

Volume 13

Number 40

Table 1 Colonic perforation due to biliary stent migration. Review of the literature to January 2007 Reference

Year

Stent

Material

Anderson et al[28] Wilhelm et al[27] Diller et al[19] Elliott et al[21]

2006 2003 2003 2003

Uk Uk Teflon Uk

Benign Benign Benign Benign

5 mo 18 mo 1 mo 4 mo

Figueras et al[29] Klein et al[26]

2001 2001

Polyethylene Teflon

Benign Benign

3 mo 3 yr

Colocutaneous fistula Surgery Sigmoid diverticular perforation Surgery

Storkson et al[30] Lenzo et al[22] Baty et al[20] Schaafsma et al[23] D´Costa et al[24]

2000 1998 1996 1996 1994

Uk Straight 7 French, 10 cm 10 French, 10 cm 10 French, 10 cm Straight Straight 7 French, 5 cm, Straight 7 French, 7, 5 cm Uk Straight Uk

Type of bile duct lesion

Time to migration

Complication

Plastic Polyethylene Polyethylene Uk Uk

Benign Benign Malign Benign Malign

2 wk 1 mo 1 mo 6 mo Uk

Sigmoid perforation Sigmoid diverticula perforation Sigmoid diverticula perforation Sigmoid perforation Sigmoid perforation

Sigmoid diverticula perforation Colovesicular fistula Stuck in sigmiod diverticula Sigmoid perforation

Therapy Endoscopy Surgery Endoscopy Surgery

Surgery Surgery Surgery Surgery Surgery

Uk: unknown.

F i g u r e 1 C T- s c a n o f colonic perforation caused by migrated biliary endoprosthesis. Arrow marks dislocated stent.

the rectal mesentery (Figure 1). A rectal resection was performed. After an uncomplicated postoperative recuperation, the patient was discharged on the seventh postoperative day.

DISCUSSION The incidence of postoperative biliary fistula is elevated in settings of acute or chronic inflammation[32-34]. Treatment options are surgical biliary leak repair, percutaneous biliary drainage, and endoscopic biliary drainage. Endoscopic placement of a biliary endoprosthesis is a well-established procedure for treatment of biliary outflow obstruction. The most frequent complication associated with bile duct stents is early occlusion caused by clogging, with resultant cholangitis, or by tumor over-growth. Stent dislocation and proximal or distal migration are uncommon, with an overall incidence of up to 6% [3]. Several complications of stent migration have been reported and can be classified into categories of penetration, intestinal perforation, and obstruction. Other organs are usually unaffected[9,10]. Most ingested foreign bodies pass through the intestine without major problems. Neither size nor shape of the stent predicts the likelihood of successful conservative management. In all patients, a close follow-up after biliary stent placement is mandatory. Patients with diverticular disease, hernia or intra-abdominal adhesions, are

www.wjgnet.com

especially at risk. Intestinal-wall weakness and increased resistance during bowel movement can produce localized complications[26,39]. Lesions of the intestinal wall due to biliary-stent migration have been reported in the duodenum, and are related to retroperitoneal fixation of this intestinal segment. Penetration requires adherence between the perforated and the penetrated organ, and does not induce intra-abdominal contamination, but eventually causes fistulae (i.e. interenteric or biliocolic[36], colovaginal[25] or colovesicular[27]). Downstream migration is more frequent in benign than in malignant biliary duct strictures[9,35]. Benign stenoses are not as tight because of regression of inflammatory reactions after placement of the stent. By contrast, tumor growth can anchor a stent in malignant stenoses. In the case of intestinal perforation, surgical stent removal and drainage of consecutive intra-abdominal or retroperitoneal abscesses is mandatory. A pelvic abscess[28], and colovesicular[27] or colocutaneous[29] fistula are typical consequences (Table 1). A case of necrotizing fasciitis due to colonic perforation has also been reported[31]. A review of the literature published to January 2007 has revealed 11 cases of colonic perforation due to biliary stent migration, with the majority being straight plastic endoprostheses[10,17,26,27,29,38,40]. In conclusion, endoscopic insertion of biliary stents is a useful and powerful procedure for short-term decompression of the biliary system. In the case of long-term therapy, stent-migration-associated complications have to be considered as a differential diagnosis that may lead to life-threatening situations. A correct diagnosis is sometimes difficult to make because of the absence of typical symptoms. A migrated biliary stent, symptomatic or not, should be removed immediately[19]. In cases of intestinal perforation, immediate surgical therapy is required. In cases of benign lesions of the bile duct, the stent should be either removed early to decrease the risk of secondary complications, or replaced regularly to prevent stent obstruction, infection or migration. Caution should be exercised when straight plastic stents are inserted, and these should be closely followed-up.

Namdar T et al . Biliary stent migration

REFERENCES 1

2 3 4

5

6 7

8

9 10 11 12

13 14 15 16 17

18

Costamagna G, Pandolfi M, Mutignani M, Spada C, Perri V. Long-term results of endoscopic management of postoperative bile duct strictures with increasing numbers of stents. Gastrointest Endosc 2001; 54: 162-168 Agarwal N, Sh a r m a B C , G a r g S , K u m a r R , S a r i n S K . Endoscopic management of postoperative bile leaks. Hepatobiliary Pancreat Dis Int 2006; 5: 273-277 Johanson JF, Schmalz MJ, Geenen JE. Incidence and risk factors for biliary and pancreatic stent migration. Gastrointest Endosc 1992; 38: 341-346 Moesch C, Sautereau D, Cessot F, Berry P, Mounier M, Gainant A, Pillegand B. Physicochemical and bacteriological analysis of the contents of occluded biliary endoprostheses. Hepatology 1991; 14: 1142-1146 Levy MJ, Baron TH, Gostout CJ, Petersen BT, Farnell MB. Palliation of malignant extrahepatic biliary obstruction with plastic versus expandable metal stents: An evidence-based approach. Clin Gastroenterol Hepatol 2004; 2: 273-285 Frakes JT, Johanson JF, Stake JJ. Optimal timing for stent replacement in malignant biliary tract obstruction. Gastrointest Endosc 1993; 39: 164-167 Pedersen FM, Lassen AT, Schaffalitzky de Muckadell OB. Randomized trial of stent placed above and across the sphincter of Oddi in malignant bile duct obstruction. Gastrointest Endosc 1998; 48: 574-579 Mueller PR, Ferrucci JT Jr, Teplick SK, vanSonnenberg E, Haskin PH, Butch RJ, Papanicolaou N. Biliary stent endoprosthesis: analysis of complications in 113 patients. Radiology 1985; 156: 637-639 Jendresen MB, Svendsen LB. Proximal displacement of biliary stent with distal perforation and impaction in the pancreas. Endoscopy 2001; 33: 195 Liebich-Bartholain L, Kleinau U, Elsbernd H, Buchsel R. Biliary pneumonitis after proximal stent migration. Gastrointest Endosc 2001; 54: 382-384 Miller G, Yim D, Macari M, Harris M, Shamamian P. Retroperitoneal perforation of the duodenum from biliary stent erosion. Curr Surg 2005; 62: 512-515 Bui BT, Oliva VL, Ghattas G, Daloze P, Bourdon F, Carignan L. Percutaneous removal of a biliary stent after acute spontaneous duodenal perforation. Cardiovasc Intervent Radiol 1995; 18: 200-202 Elder J, Stevenson G. Delayed perforation of a duodenal diverticulum by a biliary endoprosthesis. Can Assoc Radiol J 1993; 44: 45-48 Gould J, Train JS, Dan SJ, Mitty HA. Duodenal perforation as a delayed complication of placement of a biliary endoprosthesis. Radiology 1988; 167: 467-469 Humar A, Barron PT, Sekar AS, Lum A. Pancreatitis and duodenal perforation as complications of an endoscopically placed biliary stent. Gastrointest Endosc 1994; 40: 365-366 Melita G, Curro G, Iapichino G, Princiotta S, Cucinotta E. Duodenal perforation secondary to biliary stent dislocation: a case report and review of the literature. Chir Ital 2005; 57: 385-388 Basile A, Macri' A, Lamberto S, Caloggero S, Versaci A, Famulari C. Duodenoscrotal fistula secondary to retroperitoneal migration of an endoscopically placed plastic biliary stent. Gastrointest Endosc 2003; 57: 136-138 Esterl RM Jr, St Laurent M, Bay MK, Speeg KV, Halff GA. Endoscopic biliary stent migration with small bowel perforation in a liver transplant recipient. J Clin Gastroenterol 1997; 24: 106-110

5399 19 20 21 22 23 24 25 26 27 28 29 30 31 32

33 34 35 36

37 38 39 40

Diller R, Senninger N, Kautz G, Tubergen D. Stent migration necessitating surgical intervention. Surg Endosc 2003; 17: 1803-1807 Baty V, Denis B, Bigard MA, Gaucher P. Sigmoid diverticular perforation relating to the migration of a polyethylene endoprosthesis. Endoscopy 1996; 28: 781 Elliott M, Boland S. Sigmoid colon perforation following a migrated biliary stent. ANZ J Surg 2003; 73: 669-670 Lenzo NP, Garas G. Biliary stent migration with colonic diverticular perforation. Gastrointest Endosc 1998; 47: 543-544 Schaafsma RJ, Spoelstra P, Pakan J, Huibregtse K. Sigmoid perforation: a rare complication of a migrated biliary endoprosthesis. Endoscopy 1996; 28: 469-470 D'Costa H, Toy E, Dennis MJ, Brown C. Case report: intestinal perforation--an unusual complication of endoscopic biliary stenting. Br J Radiol 1994; 67: 1270-1271 Blake AM, Monga N, Dunn EM. Biliary stent causing colovaginal fistula: case report. JSLS 2004; 8: 73-75 Klein U, Weiss F, Wittkugel O. Migration of a biliary Tannenbaum stent with perforation of sigmoid diverticulum. Rofo 2001; 173: 1057 Wilhelm A, Langer C, Zoeller G, Nustede R, Becker H. Complex colovesicular fistula: A severe complication caused by biliary stent migration. Gastrointest Endosc 2003; 57: 124-126 Anderson EM, Phillips-Hughes J, Chapman R. Sigmoid colonic perforation and pelvic abscess complicating biliary stent migration. Abdom Imaging 2007; 32: 317-319 Figueiras RG, Echart MO, Figueiras AG, Gonzalez GP. Colocutaneous fistula relating to the migration of a biliary stent. Eur J Gastroenterol Hepatol 2001; 13: 1251-1253 Storkson RH, Edwin B, Reiertsen O, Faerden AE, Sortland O, Rosseland AR. Gut perforation caused by biliary endoprosthesis. Endoscopy 2000; 32: 87-89 Marsman JW, Hoedemaker HP. Necrotizing fasciitis: fatal complication of migrated biliary stent. Australas Radiol 1996; 40: 80-83 Binmoeller KF, Katon RM, Shneidman R. Endoscopic management of postoperative biliary leaks: review of 77 cases and report of two cases with biloma formation. Am J Gastroenterol 1991; 86: 227-231 Peters JH, Gibbons GD, Innes JT, Nichols KE, Front ME, Roby SR, Ellison EC. Complications of laparoscopic cholecystectomy. Surgery 1991; 110: 769-777; discussion 777-778 Airan MC. Complications of laparoscopic cholecystectomy: a national survey of 4,292 hospitals and an analysis of 77,604 cases. Am J Surg 1993; 165: 9-14 Culp WC, McCowan TC, Lieberman RP, Goertzen TC, LeVeen RF, Heffron TG. Biliary strictures in liver transplant recipients: treatment with metal stents. Radiology 1996; 199: 339-346 Fiori E, Mazzoni G, Galati G, Lutzu SE, Cesare A, Bononi M, Bolognese A, Tocchi A. Unusual breakage of a plastic biliary endoprosthesis causing an enterocutaneous fistula. Surg Endosc 2002; 16: 870 Levey JM. Intestinal perforation in a parastomal hernia by a migrated plastic biliary stent. Surg Endosc 2002; 16: 1636-1637 Mistry BM, Memon MA, Silverman R, Burton FR, Varma CR, Solomon H, Garvin PJ. Small bowel perforation from a migrated biliary stent. Surg Endosc 2001; 15: 1043 Ruffolo TA, Lehman GA, Sherman S, Aycock R, Hayes A. Biliary stent migration with colonic diverticular impaction. Gastrointest Endosc 1992; 38: 81-83 Mofidi R, Ahmed K, Mofidi A, Joyce WP, Khan Z. Perforation of ileum: an unusual complication of distal biliary stent migration. Endoscopy 2000; 32: S67 S- Editor Liu Y L- Editor Kerr C

E- Editor Li JL

www.wjgnet.com



CASE REPORT

Sigmoid colon endometriosis treated with laparoscopy-assisted sigmoidectomy: Significance of preoperative diagnosis Motohira Yoshida, Yuji Watanabe, Atsushi Horiuchi, Yuji Yamamoto, Hiroki Sugishita, Kanji Kawachi Motohira Yoshida, Yuji Watanabe, Atsushi Horiuchi, Yuji Yamamoto, Hiroki Sugishita, Kanji Kawachi, Department of Organ Regenerative Surgery, Ehime University Graduate School of Medicine, Shitsukawa, Toon, Ehime 791-0295, Japan Correspondence to: Motohira Yoshida, Ehime University, Shitsukawa, Toon, Ehime 791-0295, Japan. [email protected] Telephone: +81-89-9605331 Fax: +81-89-9605335 Revised: August 14, 2007 Received: July 10, 2007

Abstract We present a female patient with sigmoid colon endometriosis who was diagnosed correctly preoperatively and underwent minimally invasive surgery. She was admitted to our hospital with rectal bleeding and constipation. We performed several workups. Colonoscopy and endoscopic ultrasonography showed sigmoid colon stenosis caused by submucosal tumor, and magnetic resonance imaging revealed a sigmoid colon tumor displaying signal hypointensity on both T1- and T2-weighted imaging. However, colonoscopic ultrasonography-assisted needle aspiration biopsy could not specify tumor characteristics. From these examinations, the lesion was diagnosed as sigmoid colon endometriosis and laparoscopy-assisted sigmoidectomy was performed. Pathological diagnosis from the resected specimen was identical to preoperative diagnosis, i.e., colonic endometriosis. Since differential diagnosis of intestinal endometriosis seems difficult, a cautious preoperative diagnosis is required to select treatments including minimally invasive surgery. © 2007 WJG . All rights reserved.

Key words: Intestinal endometriosis; Preoperative diagnosis; Laparoscopy-assisted surgery Yoshida M, Watanabe Y, Horiuchi A, Yamamoto Y, Sugishita H, Kawachi K. Sigmoid colon endometriosis treated with laparoscopy-assisted sigmoidectomy: Significance of preoperative diagnosis. World J Gastroenterol 2007;

13(40): 5400-5402


www.wjgnet.com

INTRODUCTION Endometriosis is a common benign disease among women of reproductive age, and affects the intestinal tract in 15%-37% of all patients with pelvic endometriosis[1]. The disease is thus comparatively common, but preoperative diagnosis is reportedly difficult[2-4]. Herein, we report a case of sigmoid colon endometriosis that was diagnosed preoperatively using magnetic resonance imaging (MRI) and then treated by minimally invasive surgery.

CASE REPORT A 43-year-old woman was admitted to our hospital with chronic constipation and decreased stool caliber for 3 mo before admission. Rectal bleeding was also reported, but was not associated with her menstrual cycle. She did not have any other symptoms, including abdominal pain. She was 14-years-old at first menstruation and her cycle was irregular at every 27-50 d, lasting 7-12 d. The patient was nulliparous and had not had an abortion. Physical examination revealed no specific findings, including abdominal pain or palpable masses in the abdomen. Laboratory examination showed an elevation of cancer antigen (CA) 125 level to 90.5 IU/mL, but other values were within normal limits. Gynecological examination revealed a normal vagina, uterus and uterine cervix. Transvaginal ultrasonography revealed multiple myomas with diameters of 1-2 cm in the uterine wall, but no abnormality in either ovary. Barium enema showed stenosis of the sigmoid colon caused by tumor, but the mucous membrane was smooth (Figure 1). Colonoscopy also revealed sigmoidal stenosis with a smooth mucosal surface. Endoscopic ultrasonography was performed at the same time and revealed an iso- to highechoic lesion in the 3rd echoic layer (submucosal layer) (Figure 2). In addition, endoscopic ultrasonography-guided fine needle aspiration biopsy (EUS-FNAB) was attempted, but sufficient specimens could not be obtained due to severe stenosis of the sigmoid colon. MRI was performed for evaluation and differential

Yoshida M et al . Sigmoid endometriosis

5401

Figure 1 Barium enema shows sigmoidal stenosis with smooth inner lumen.

Figure 4 Gross examination of the resected specimen.

Figure 2 EUS shows iso to high echoic submucosal tumor in the 3rd echoic layer.

Figure 5 Tumor comprising endometrial-like grands and smooth muscle cells (× 200).

Figure 3 MRI (T2 weighted image) shows a sigmoidal tumor lesion with low signal intensity.

diagnosis of the submucosal tumor, revealing a tumor 3 cm in diameter in the sigmoid colon with signal hypointensity on both T1- and T2-weighted imaging (Figure 3; T2-weighted image only). Several small myomas were also seen in the uterus. The sigmoid tumor was diagnosed as intestinal endometriosis from these examinations, for the following reasons: (1) the patient was a woman of reproductive age; (2) tumor was located in the sigmoid colon; (3) a submucosal tumor containing muscle and/or fibrous components was detected on MRI. As stenosis of the colon was severe and the patient wanted to have children

in the near future, we selected sigmoid resection by laparoscopy-assisted sigmoidectomy with a minimal skin incision to prevent postoperative abdominal adhesions. Surgery was perfor med after infor med consent was obtained from the patient. Three trocars, including a 5-mm laparoscope, were introduced and a 4-cm minilaparotomy was performed in the left lower abdomen to retrieve the resected specimen and anastomose the colon. The operation was successful and she was discharged uneventfully 7 d after surgery. Gross examination of the resected specimen revealed a 30 mm × 20 mm × 15 mm hard submucosal mass, and the serous membrane of this site was indented to the tumor (Figure 4). No abnormality in the mucosal surface was identified. Histological examination revealed a mixture of smooth muscle cells and endometrial glands, without atypical cells and goblet cells, so the diagnosis of sigmoid endometriosis was confirmed (Figure 5).

DISCUSSION Endometriosis is histologically defined as the presence of endometrial-like tissue outside the uterus[5]. This condition is thought to affect the intestinal tract in 15%-37% of patients with pelvic endometriosis[1]. The disease is not particularly rare among women of childbearing age, but many authors have reported that correct preoperative www.wjgnet.com

5402

ISSN 1007-9327

CN 14-1219/R


diagnosis is difficult[2-4]. This is because the symptoms such as abdominal pain, hemostool and decreased stool caliber are not specific for this disease, and hemostool or rectal bleeding are thought to result from mucosal injury during passage of stools through the narrowed intestinal lumen[2]. Some patients reportedly display symptoms associated with the menstrual cycle[2,4], but these patients represent only about 40% of all patients with endometriosis[6]. In addition, some reports have described aggravation of inflammatory bowel disease and irritable bowel syndrome during menstruation [7]. Symptoms alone are thus not helpful for differential diagnosis, and a similar situation is seen for laboratory examination of the blood. Mol et al[8] reported a systematic review of the diagnosis of endometriosis, and concluded that serum CA125 level may be elevated in endometriosis, but this measurement had no value as a diagnostic tool compared to laparoscopy. Imaging examination is thus essential for the preoperative diagnosis of intestinal endometriosis, but some reports have described preoperative confusion between this disease and cancer according to colonoscopy and CT with barium enema, particularly in patients with lesions involving the mucosal surface[3,4]. In such patients, MRI is helpful for differential diagnosis. In a typical endometrial lesion, MRI showed signal hyperintensity on T1-weighted imaging and signal hypointensity on T2-weighted imaging [9]. However, smooth muscle components are reportedly recognized frequently in endometrial lesions[10]. In such lesions, as seen in the present case, MRI indicates signal hypointensity on both T1- and T2-weighted imaging, and differential diagnosis from other diseases such as cancer and gastrointestinal stromal tumor is thus difficult. In fact, Chapron et al[11] reported that MRI specificity for deeply infiltrating endometriosis was 97.9%, but sensitivity was only 76.5%. Endoscopic ultrasonography is also a useful and noninvasive examination for the diagnosis of intestinal endometriosis. Sensitivity and specificity are reportedly about 97% for the diagnosis of rectal involvement in patients with known pelvic endometriosis[12]. In addition, EUS-FNAB provides accurate tissue and may be the only procedure for correct preoperative diagnosis of intestinal endometriosis, but the overall specificity, sensitivity and accuracy of EUS-FNA for neoplasms of the gastrointestinal tract are reportedly 88%, 89% and 89%, respectively[13]. Biscaldi et al[14] reported the usefulness of multislice CT combined with distention of the colon by rectal enteroclysis for intestinal endometriosis. The sensitivity was 98.7% and specificity was 100% in identifying women with intestinal endometriosis. This method is thought to be very helpful for diagnosing intestinal endometriosis, but requires bowel preparation, such as the need for a low-residue diet for 3 d, drinking of 4-6 doses of a granular powder dissolved in 500 mL of water per dose and intravenous administration of iodinated contrast medium. This technique is thus inappropriate for patients with obstructive symptoms or

October 28, 2007

Volume 13

Number 40

allergy to iodinated contrast medium. Among these examinations, we consider MRI and EUS (and/or EUS-FNAB) are the most useful examinations for intestinal endometriosis. However, it is important to perform valuable examinations for diagnosis of intestinal endometriosis, including radiological, histological and etiological examinations, as the condition basically involves a benign lesion requiring minimally invasive treatment. This disease should also be considered in the differential diagnosis for women of childbearing age presenting with gastrointestinal tract symptoms.

REFERENCES 1

Yantiss RK, Clement PB, Young RH. Endometriosis of the intestinal tract: a study of 44 cases of a disease that may cause diverse challenges in clinical and pathologic evaluation. Am J Surg Pathol 2001; 25: 445-454 Bartkowiak R, Zieniewicz K, Kaminski P, Krawczyk M, 2 Marianowski L, Szymanska K. Diagnosis and treatment of sigmoidal endometriosis--a case report. Med Sci Monit 2000; 6: 787-790 Chu PW, Su HY, Ko CS. Endometriosis of the colon and rectum 3 mimicking colon cancer. Int J Gynaecol Obstet 2004; 87: 167-168 Dimoulios P, Koutroubakis IE, Tzardi M, Antoniou P, 4 Matalliotakis IM, Kouroumalis EA. A case of sigmoid endometriosis difficult to differentiate from colon cancer. BMC Gastroenterol 2003; 3: 18 Kennedy S, Bergqvist A, Chapron C, D'Hooghe T, Dunselman 5 G, Greb R, Hummelshoj L, Prentice A, Saridogan E. ESHRE guideline for the diagnosis and treatment of endometriosis. Hum Reprod 2005; 20: 2698-2704 Jubanyik KJ, Comite F. Extrapelvic endometriosis. Obstet 6 Gynecol Clin North Am 1997; 24: 411-440 7 Kane SV, Sable K, Hanauer SB. The menstrual cycle and its effect on inflammatory bowel disease and irritable bowel syndrome: a prevalence study. Am J Gastroenterol 1998; 93: 1867-1872 Mol BW, Bayram N, Lijmer JG, Wiegerinck MA, Bongers MY, 8 van der Veen F, Bossuyt PM. The performance of CA-125 measurement in the detection of endometriosis: a metaanalysis. Fertil Steril 1998; 70: 1101-1108 9 Puglielli E, Di Cesare E, Masciocchi C. Rectal endometriosis: MRI study with rectal coil. Eur Radiol 2004; 14: 2362-2363 10 Anaf V, Simon P, Fayt I, Noel J. Smooth muscles are frequent components of endometriotic lesions. Hum Reprod 2000; 15: 767-771 11 Chapron C, Vieira M, Chopin N, Balleyguier C, Barakat H, Dumontier I, Roseau G, Fauconnier A, Foulot H, Dousset B. Accuracy of rectal endoscopic ultrasonography and magnetic resonance imaging in the diagnosis of rectal involvement for patients presenting with deeply infiltrating endometriosis. Ultrasound Obstet Gynecol 2004; 24: 175-179 12 Doniec JM, Kahlke V, Peetz F, Schniewind B, Mundhenke C, Lohnert MS, Kremer B. Rectal endometriosis: high sensitivity and specificity of endorectal ultrasound with an impact for the operative management. Dis Colon Rectum 2003; 46: 1667-1673 13 Vander Noot MR 3rd, Eloubeidi MA, Chen VK, Eltoum I, Jhala D, Jhala N, Syed S, Chhieng DC. Diagnosis of gastrointestinal tract lesions by endoscopic ultrasound-guided fine-needle aspiration biopsy. Cancer 2004; 102: 157-163 14 Biscaldi E, Ferrero S, Fulcheri E, Ragni N, Remorgida V, Rollandi GA. Multislice CT enteroclysis in the diagnosis of bowel endometriosis. Eur Radiol 2007; 17: 211-219 S-Editor Liu Y L-Editor Ma JY E- Editor Li HY

www.wjgnet.com



CASE REPORT

Reactive lymphoid hyperplasia of the liver: A case report and review of literature Takuro Machida, Toshiyuki Takahashi, Tomoo Itoh, Michiaki Hirayama, Takayuki Morita, Shoichi Horita Takuro Machida, Michiaki Hirayama, Shoichi Horita, Department of Internal Medicine, Hokkaido Gastroenterology Hospital, Sapporo, Hokkaido 065-0041, Japan Toshiyuki Takahashi, Department of Pathology, Hokkaido Gastroenterology Hospital, Sapporo, Hokkaido 065-0041, Japan Tomoo Itoh, Department of Pathology, Hokkaido University Hospital, Sapporo, Hokkaido, 060-8648, Japan Ta k a y u k i M o r i t a , D e p a r t m e n t o f S u rg e r y, H o k k a i d o Gastroenterology Hospital, Sapporo, Hokkaido 065-0041, Japan Correspondence to: Toshiyuki Takahashi, MD, PhD, Department of Pathology, Hokkaido Gastroenterology Hospital, Honcho 1-1-2-10, Higashi-ku, Sapporo, Hokkaido 065-0041, Japan. [email protected] Telephone: +81-11-7841811-540 Fax: +81-11-7841838 Received: June 28, 2007 Revised: August 6, 2007

Abstract A case of a 53-year-old female patient with reactive lymphoid hyperplasia (RLH), clinically designated as pseudolymphoma of the liver is described in this article. The patient was admitted to our hospital for further evaluation of hepatic tumors incidentally discovered at another hospital. Various diagnostic methods, including ultrasonography (US), computerized tomography (CT), magnetic resonance imaging (MRI) and hepatic angiography displayed three small lesions in the liver with outstanding findings consistent with hepatocellular carcinoma (HCC). Surgical resection was performed and the three lesions were microscopically diagnosed as RLH of the liver. The lesions comprised a massive infiltration of lymphoid cells with follicles and hyalinized interfollicular spaces. Immunohistochemical examination revealed that infiltrating lymphocytes had no prominent nuclear atypia and polyclonality. RLH of the liver is a very rare condition and only twelve cases have been reported in the English literature. Majority of the reported cases were middle-aged women and about half of them had some immunologic abnormalities such as autoimmune thyroiditis, Sjogren’s syndrome, primary immunodeficiency, primary biliary cirrhosis. Since they are often clinically misdiagnosed as HCC, surgery is the choice of treatment for these patients. Although their pathology resembles malignant lymphoma, the clinical course is completely benign. The authors propose that RLH of the liver can be discriminated from HCC by its clinical features. © 2007 WJG . All rights reserved.

Key words: Reactive lymphoid hyperplasia; Pseudo-

lymphoma; Hepatocellular carcinoma; Autoimmune thyroiditis; Immunohistochemistry Machida T, Takahashi T, Itoh T, Hirayama M, Morita T, Horita S. Reactive lymphoid hyperplasia of the liver: A case report and review of literature. World J Gastroenterol 2007; 13(40): 5403-5407


INTRODUCTION Reactive lymphoid hyperplasia (RLH) is a benign nodular lesion, histopathologically characterized by marked proliferation of non-neoplastic, polyclonal lymphocytes forming follicles with an active germinal center. The lesion is encountered in various organs such as the orbit[1], lung[2], skin[3,4] and gastrointestinal tract[5-7]. However, disease of the liver is quite rare, with only 12 cases reported in the English literature. We have recently encountered a patient with 3 RLH lesions occurred in the liver. In this paper, the clinicopathological and radiographic characteristics of this unique disorder are discussed. Problems in differential diagnosis from malignant diseases, including malignant lymphoma and hepatocellular carcinoma, are also discussed.

CASE REPORT A 53-year-old Japanese woman with a history of autoimmune thyroiditis was admitted to our hospital for further evaluation of hepatic lesions incidentally discovered on abdominal ultrasonography. The patient was asymptomatic on admission and her condition was generally good. Physical examination revealed no abnormalities. Relevant laboratory tests disclosed slightly elevated lactate dehydrogenase activity (547 IU/L; normal range: 120-245 IU/L), thymol turbidity (7.2 U/L; normal range: 0.0-4.0 U/L) and zinc sulfate turbidity (13.0 U/L; normal range: 4.0-12.0 U/L), although other hepatic function tests including serum asparate aminotransferase, serum alanine aminotransaminase and total bilirubin were within normal range. Levels of serum thyroid-stimulating hormone (10.22 µIU/mL; normal range: 0.35-3.73 µU/mL) and antinuclear antibody (80 ×; normal range: < 40 ×) were elevated due to autoimmune thyroiditis. Both hepatitis B surface antigen and antihepatitis C virus antibody were negative. Tumor markers including alpha-fetoprotein and protein induced by vitamin K antagonist-Ⅱ were negative. www.wjgnet.com

5404

ISSN 1007-9327

A

A

CN 14-1219/R


October 28, 2007

Volume 13

Number 40

Figure 1 CT angiography showing the conspicuously enhanced lesion in segment 7 through the early phase (arrows) (A) and intensified tumor rim enhancement through the delayed phase along with radial enhancement (arrowheads) (B).

B

B

C

Figure 2 MRI displaying a hypointense signal on T1-weighted imaging (arrow) (A), a hyperintense signal on T2-weighted imaging of the lesion (arrow) (B), and enhanced tumor rim in the delayed phase of MRI (arrow) (C).

Figure 3 Macroscopy of tumor in segment 7 demonstrating the lesion as a wellcircumscribed, white-yellowish nodule on the cut surface.

Precise US examination disclosed 2 hypoechoic nodules in the posterior segment and 1 nodule in the medial segment of the liver (13, 11 and 8 mm in diameter) respectively. Dynamic CT performed in the planes of lesions demonstrated a slight hyperdensity in the arterial phase followed by hypodense areas with pronounced enhancement along the tumor rims in the portal phase. The lesion in segment 7 was conspicuously enhanced through the early phase on CT angiography (Figure 1A). The tumor rim was continuatively intensified through the delayed phase, and radial enhancement toward surrounding liver parenchyma was also recognized (Figure 1B). The lesion was identified as a portal flow defect on CT with arterial portography. On MRI, tumor gave a hypointense signal on T1-weighted imaging (Figure 2A) and a hyperintense signal on T2-weighted imaging (Figure 2B). The tumor rim www.wjgnet.com

was underscored as an annular enhancement in the delayed phase, similar to dynamic CT (Figure 2C). Other lesions within segments 4 and 6 showed equivalent hemodynamics to the tumor in segment 7. Hepatic angiography of the 3 lesions also demonstrated distinct tumor staining in the arterial phase, following dense staining along tumor rims in the parenchymal-venous phase. As all images suggested hepatocellular carcinoma, posterior segmentectomy and partial resection of the medial segment of the liver were performed. The resected liver displayed smooth surfaces and normal appearance. On the cut section, lesions were wellcircumscribed, but not encapsulated white-yellowish nodules with irregular shapes (Figure 3). The 3 nodules exhibited similar microscopic findings, comprising a massive infiltration of lymphoid cells with follicles and hyalinized interfollicular spaces at low magnification (Figure 4A). Lymph follicles varied in size and shape with germinal centers composed of small or large lymphoid cells and tingible body macrophages. Interfollicular areas mostly comprised mature lymphocytes with no prominent nuclear atypia and a small amount of infiltrated plasma cells and histiocytes (Figure 4B). Strands of amorphous, somewhatdense hyalinized material accompanied with capillaries were observed in interfollicular areas (Figure 4C). In some parts, fibrous materials aggregated as nodular structures of various sizes. Bile ductule proliferation was seen along the lesion edges. Portal areas apart from nodules showed irregular expansion with infiltration of small mature lymphocytes (Figure 4D). Immunohistochemical studies on

Machida T et al . Reactive lymphoid hyperplasia of the liver

A

B

C

D

A

B

5405 Figure 4 Microscopy revealing lesions comprising massive lymphoid cell infiltration with prominent follicles and hyalinized interfollicular spaces at low magnification (HE, X 2.5) (A), interfollicular areas mainly comprising mature lymphocytes with no nuclear atypia (HE, X 50) (B), strands of amorphous hyalinized material accompanying capillaries in interfollicular areas (HE, X 50) (C), and portal areas apart from the nodules with irregular expansion of infiltration of small mature lymphocytes (HE, X 25) (D).

that the lesion was composed of polyclonal lymphocytes and plasma cells. A genetic investigation of clonality in the immunoglobulin heavy chains (IgH) using a previously reported polymerase chain reaction method[8] with DNA from paraffin-embedded tissue was also performed. No clonal IgH gene rearrangement was detected with satisfactory negative and positive controls. The 3 lesions were thus pathologically diagnosed as RLH of the liver. Postoperative course was relatively good and the patient experienced no signs of recurrence for 22 mo.

DISCUSSION Figure 5 Immunohistochemistry discovering follicles mainly comprising CD20positive lymphocytes (X 50) (A), and interfollicularly and circumferentially distributed CD3-positive cells compared to follicles (X 50) (B).

paraffin-embedded tissue sections for CD3, CD5, CD10, CD20, bcl-2 and both κ and λ immunoglobulin light chains were undertaken using the avidin-biotin-peroxidase complex technique. Follicles mainly comprised CD20-positive lymphocytes, while CD3-positive cells were distributed interfollicularly and circumferentially around follicles (Figure 5). Distributions were similar to that seen in reactive hyperplasia of lymph nodes. Lymphoid cells in follicles showed ordinary immunoreactivity, while cells in the mantle zone were positive for CD5 and cells in the germinal center were positive for CD10. Bcl-2 reacted only to lymphocytes in marginal or circumferential areas of follicles, but not in central regions. Scattered plasma cells were positive for both κ and λ light chains at an equal frequency. These immunohistochemical results indicate that lymph follicles were of reactive origin, but not of neoplastic, and

Although RLH can be found in various organs, including the gastrointestinal tract[5-7], orbit[1], lung[2] and skin[3,4], its occurrence in the liver is rare. To the best of our knowledge, only 14 lesions in 12 other cases of hepatic RLH of the liver have been reported in the English literature since the first case report by Snover et al[9] in 1981, with this case representing the thirteenth (Table 1). RLH occurs predominantly in middle-aged women, with a mean age of 54.1 years (range 15-72 years, Table 1). Although the pathogenesis remains unknown, an association between development of hepatic RLH and systemic or local immunological abnormalities has been suggested in earlier reports[10,11]. In fact, 6 of 13 patients, including the present, have displayed some immunological complications, such as autoimmune thyroiditis [10], Sjogren’s syndrome [11], primary immunodeficiency (PI)[9], and primary biliary cirrhosis (PBC)[12] (Table 1). Two cases have been reported in association with hepatitis B (HB) virus or hepatitis C (HC) virus infection[13,14] (Table 1). The patient with hepatitis B was treated with interferon (IFN)-α[13]. Whether hepatitis viral infection is involved in hepatic RLH remains unclear. However, www.wjgnet.com

5406

ISSN 1007-9327

CN 14-1219/R


October 28, 2007

Volume 13

Number 40

Table 1 Reported cases of RLH of the liver Case Reference # Age (yr) 1 2 3 4 5 6 7 8 9 10 11 12 13

9 13 10 11 12 12 12 19 16 17 18 14 Our case

15 42 47 49 52 56 56 59 66 67 69 72 53

Sex Complications F F F F F F M F F F F M F

PI HB Autoimmune thyroiditis Sjogren’s syndrome PBC PBC Diverticulitis Diabetes Diabetes None Renal cell carcinoma Gastric carcinoma HC Autoimmune thyroiditis

Needle biopsy Preoperative diagnosis Done None None Done None None None None Done None Done None None

the findings in these patients indicate that continuous stimulation from cytokines such as IFN-α or hepatitis viruses may cause RLH of the liver. Saltzstein et al[15] has defined RLH as propagation of lymph-follicles constructed with lymphoid cells without cytological atypia, accompanied with a conspicuous, reactive germinal center. Katayanagi et al[16] and Tanizawa et al[17] have also proposed RLH as a localized lesion welldemarcated from the surrounding tissues and characterized by the presence of hyperplastic lymphoid follicles with polymorphic and polyclonal cell populations composed of small mature lymphocytes, mature plasma cells, macrophages and stromal fibrosis. The nodular lesion described herein seems to satisfy these histological criteria. When RLH is diagnosed, malignant lymphoma must be excluded from the differential diagnosis. In the present case, immunohistochemical examination was performed to rule out malignant lymphoma. Germinal centers were considered non-neoplastic, based on negative results for bcl-2. In addition, lymphoid cells, including plasma cells, proliferating in the interfollicular area displayed polyclonal immunophenotypes. The results of a genetic investigation pertaining to IgH clonality also supported the histological findings. A diagnosis of RLH of the liver thus seems justified. The lesion has been often misdiagnosed as a malignant tumor, since features are shared with hepatocellular carcinoma on various imaging modalities, namely detection as a hypoechoic mass on US, low density on CT with or without enhancement, low intensity on T1-weighted imaging and high intensity on T2-weighted imaging with magnetic resonance imaging, and tumor staining on hepatic angiography. Distinguishing RLH from hepatocellular carcinoma (HCC) is thus extremely difficult. Hepatectomy has therefore been performed for many patients[10-14,16-19] (Table 1). In the present case, the tumor rim displayed a sustained intensity accompanied with radial enhancement in addition to intratumoral hyperintensity on various imaging modalities. This prominent finding may represent increased capillaries within the nodule and massive infiltration of lymphocytes into portal areas around the lesion. Needle biopsy was indicated for only 4 patients in past reports[9,11,16,18] (Table 1), but it is not helpful for a confident diagnosis of this disease. In conclusion, accumulation of reported cases with this www.wjgnet.com

Tumor number Tumor size (mm)

Cirrhosis HCC HCC Suspicious HCC PBC PBC Not reported Malignant tumor Suspicious lymphoma HCC Metastatic tumor HCC HCC

3 1 1 1 1 1 1 1 1 1 1 3

15, 14, and smaller 17 20 4 15 7 9 15 20 17 17 15, 12, and 10

Treatment Resection Resection Resection Transplantation Transplantation Resection Resection Resection Resection Resection Resection Resection

benign lesion, including our patient, reveals some knowledge concerning the characteristics of this rare condition, such as female predominance, small tumor size, coexistent immunological disorder, and imaging features resembling HCC. The authors would like to emphasize that RLH should be included in the differential diagnosis of small hepatic lesions, particularly in middle-aged women with suspected HCC.

REFERENCES 1

2 3 4

5 6 7

8

9

10

11

Knowles DM, Jakobiec FA, McNally L, Burke JS. Lymphoid hyperplasia and malignant lymphoma occurring in the ocular adnexa (orbit, conjunctiva, and eyelids): a prospective multiparametric analysis of 108 cases during 1977 to 1987. Hum Pathol 1990; 21: 959-973 Abbondanzo SL, Rush W, Bijwaard KE, Koss MN. Nodular lymphoid hyperplasia of the lung: a clinicopathologic study of 14 cases. Am J Surg Pathol 2000; 24: 587-597 Caro WA, Helwig HB. Cutaneous lymphoid hyperplasia. Cancer 1969; 24: 487-502 Baldassano MF, Bailey EM, Ferry JA, Harris NL, Duncan LM. Cutaneous lymphoid hyperplasia and cutaneous marginal zone lymphoma: comparison of morphologic and immunophenotypic features. Am J Surg Pathol 1999; 23: 88-96 Tokunaga O, Watanabe T, Morimatsu M. Pseudolymphoma of the stomach. A clinicopathologic study of 15 cases. Cancer 1987; 59: 1320-1327 Abbondanzo SL, Sobin LH. Gastric "pseudolymphoma": a retrospective morphologic and immunophenotypic study of 97 cases. Cancer 1997; 79: 1656-1663 Kojima M, Itoh H, Motegi A, Sakata N, Masawa N. Localized lymphoid hyperplasia of the rectum resembling polypoid mucosa-associated lymphoid tissue lymphoma: a report of three cases. Pathol Res Pract 2005; 201: 757-761 Orba Y, Tanaka S, Nishihara H, Kawamura N, Itoh T, Shimizu M, Sawa H, Nagashima K. Application of laser capture microdissection to cytologic specimens for the detection of immunoglobulin heavy chain gene rearrangement in patients with malignant lymphoma. Cancer 2003; 99: 198-204 Snover DC, Filipovich AH, Dehner LP, Krivit W. 'Pseudolymphoma'. A case associated with primary immunodeficiency disease and polyglandular failure syndrome. Arch Pathol Lab Med 1981; 105: 46-49 Nagano K, Fukuda Y, Nakano I, Katano Y, Toyoda H, Nonami T, Nagasaka T, Hayakawa T. Reactive lymphoid hyperplasia of liver coexisting with chronic thyroiditis: radiographical characteristics of the disorder. J Gastroenterol Hepatol 1999; 14: 163-167 Okubo H, Maekawa H, Ogawa K, Wada R, Sekigawa I, Iida N, Maekawa T, Hashimoto H, Sato N. Pseudolymphoma of the

Machida T et al . Reactive lymphoid hyperplasia of the liver liver associated with Sjogren's syndrome. Scand J Rheumatol 2001; 30: 117-119 12 Sharifi S, Murphy M, Loda M, Pinkus GS, Khettry U. Nodular lymphoid lesion of the liver: an immune-mediated disorder mimicking low-grade malignant lymphoma. Am J Surg Pathol 1999; 23: 302-308 13 Ohtsu T, Sasaki Y, Tanizaki H, Kawano N, Ryu M, Satake M, Hasebe T, Mukai K, Fujikura M, Tamai M. Development of pseudolymphoma of liver following interferon-alpha therapy for chronic hepatitis B. Intern Med 1994; 33: 18-22 14 Kim SR, Hayashi Y, Kang KB, Soe CG, Kim JH, Yang MK, Itoh H. A case of pseudolymphoma of the liver with chronic hepatitis C. J Hepatol 1997; 26: 209-214 15 Saltzstein SL. Pulmonary malignant lymphomas and pseu-

5407 dolymphomas: Classification, therapy, and prognosis. Cancer 1963; 16: 928-955 16 Katayanagi K, Terada T, Nakanuma Y, Ueno T. A case of pseudolymphoma of the liver. Pathol Int 1994; 44: 704-711 17 Tanizawa T, Eishi Y, Kamiyama R, Nakahara M, Abo Y, Sumita T, Kawano N. Reactive lymphoid hyperplasia of the liver characterized by an angiofollicular pattern mimicking Castleman's disease. Pathol Int 1996; 46: 782-786 18 Pantanowitz L, Saldinger PF, Kadin ME. Pathologic quiz case: Hepatic mass in a patient with renal cell carcinoma. Arch Pathol Lab Med 2001; 125: 577-578 19 Isobe H, Sakamoto S, Sakai H, Masumoto A, Sonoda T, Adachi E, Nawata H. Reactive lymphoid hyperplasia of the liver. J Clin Gastroenterol 1993; 16: 240-244 S- Editor Liu Y L- Editor Wang XL

E- Editor Li JL

www.wjgnet.com



CASE REPORT

Clindamycin-induced acute cholestatic hepatitis Cem Aygün, Orhan Kocaman, Yeşim Gürbüz, Ömer Şentürk, Sadettin Hülagü Cem Aygün, Orhan Kocaman, Yeşim Gürbüz, Ömer Şentürk, Sadettin Hülagü, Department of Gastroenterology and Pathology, Kocaeli University Medical Faculty, Kocaeli 41380, Turkey Correspondence to: Cem Aygün, MD, Department of Gastroenterology, Kocaeli University Medical Faculty, Kocaeli 41380, Turkey. [email protected] Telephone: +90-532-7130491 Fax: +90-262-3038003 Revised: July 28, 2007 Received: June 12,2007

Abstract We report a case of acute hepatotoxicity in a 42-yearold woman after administration of clindamycin for a dental infection. After 6 d of treatment, she had fatigue, nausea, vomiting, anorexia, pruritus and jaundice. Her laboratory analysis showed alanine aminotransferase (ALT), 1795 IU/L (normal range 0-40); aspartate aminotransferase (AST), 1337 IU/L (normal range 5-34); alkaline phosphatase (ALP), 339 IU/L (normal range 40-150); γ -glutamyl transpeptidase (GGT), 148 IU/L (normal range 9-64 IU/L); total bilirubin, 4.1 mg/dL; direct bilirubin, 2.9 mg/dL and prothrombin time (PT), 13.5 s, with international normalized ratio (INR), 1.04. She was hospitalized, with immediate drug discontinuation. Her liver biopsy specimen showed mixed-type (both hepatocellular and cholestatic) hepatic injury, compatible with a diagnosis of drug-induced hepatitis. An objective causality assessment using the Naranjo probability scale suggested that clindamycin was the probable cause of the acute hepatitis. In susceptible individuals, clindamycin use may lead to acute mixedtype liver toxicity. Complete recovery may be possible if the drug is discontinued before severe liver injury is established. © 2007 WJG . All rights reserved.

Key words: Clindamycin; Hepatic injury; Hepatitis Aygün C, Kocaman O, Gürbüz Y, Şentürk Ö, Hülagü S. Clindamycin-induced acute cholestatic hepatitis. World J

Gastroenterol 2007; 13(40): 5408-5410


INTRODUCTION Liver injury may be caused by various drugs and chemicals. The severity of injury may vary from minor non-specific changes in hepatic structure and function to fulminant www.wjgnet.com

hepatic failure, cirrhosis and even hepatocellular cancer. The term drug-induced liver disease should be confined to cases in which the nature of the liver injury has been characterized histologically, because biochemical parameters used to detect liver injury may also be elevated as an adaptive response to drugs. The lincosamide antibiotics seem to be most useful against the bacteria classified as Gram-positive cocci. In addition, they are helpful against protozoa, such as Toxoplasma and Mycoplasma, as well as many anaerobic b a c t e r i a [ 1 ] . C l i n d a my c i n ( 7 - ch l o r o l i n c o my c i n ) , a semisynthetic derivate of lincomycin, has been extensively used in the therapy of obstetric, gynecologic and oral infections for over 20 years. It is more active than erythromycin or clarithromycin against anaerobic bacteria. In humans, absorption of clindamycin is rapid and virtually complete (90%) following oral administration, and is widely distributed throughout the body[2]. We describe herein a case of acute hepatitis following clindamycin use for dental infection.

CASE REPORT A 42-year-old woman was admitted to hospital for pruritus, jaundice and asthenia. Her medical history included recurrent gingival infections treated with clindamycin on several previous occasions. Six days after the initiation of the last clindamycin (150 mg PO qid) treatment, the patient had fatigue, nausea, vomiting, anorexia, pruritis and jaundice. Emergency laboratory analysis showed the following results: Alanine aminotransferase (ALT), 1795 IU/L (normal range 0-40); aspartate aminotransferase (AST), 1337 IU/L (normal range 5-34); alkaline phosphatase (ALP), 339 IU/L (normal range 40-150); γ -glutamyl transpeptidase (GGT), 148 IU/L (normal range 9-64); total bilirubin, 4.1 mg/dL; direct bilirubin, 2.9 mg/dL and prothrombin time (PT), 13.5 s, with international normalized ratio (INR), 1.04. Blood tests for glucose, blood urea nitrogen (BUN), serum creatinine, total protein, albumin, uric acid, total cholesterol and triglycerides were within normal limits. There was no peripheral eosinophilia. The patient was hospitalized and clindamycin treatment was discontinued. Additional test results for hepatitis A, B and C viruses, cytomegalovirus, and Epstein-Barr virus were negative. Ferritin and cerulleoplasmin levels were within normal limits. Autoantibodies (antinuclear, antimitochondrial, anti-smooth-muscle, anti-liver-kidney microsomal enzymes and anti-soluble liver antigen) were also negative. Hepatobiliary imaging with ultrasonography was normal. She denied taking any other medications,

Aygün C et al . Clindamycin-induced hepatitis

5409

Figure 1 Extensive hepatocellular injury showing centrolobular and portal cholestatic hepatitis with inflammatory infiltration (HE, × 40).

Figure 2 Intense inflammatory reaction in liver without evidence of fibrosis (Mason's Trichrome, × 40).

or using alcohol or any herbal or folk remedies at any time. There were no known contributing environmental issues. One week after hospitalization, her liver function tests were as follows: ALT, 1579 IU/L; AST, 1031 IU/L; ALP, 345 IU/L; GGT, 112 IU/L; total bilir ubin, 3.34 mg/dL; and direct bilirubin, 2.4 mg/dL. Her medical history and laboratory results suggested severe acute hepatitis of mixed-type, with hepatocellular and cholestatic hepatic injury, and liver biopsy was performed. The histopathological samples revealed centrolobular and portal cholestatic hepatitis, without fibrosis or necrosis, highly suggestive of drug-related hepatotoxicity (Figures 1 and 2). Within 3 wk of hospitalization, the patient had significant subjective improvement, and showed improvement of liver function tests (ALT, 754 IU/L; AST, 429 IU/L; ALP, 175 IU/L; GGT, 82 IU/L; total bilirubin, 1.8 mg/dL; and direct bilirubin, 1.26 mg/dL). After 8 wk hospitalization, her liver function test results were within normal limits (ALT, 28 IU/L; AST, 23 IU/L; and total bilir ubin, 0.74 mg/dL). The patient was discharged in good condition. The probability that the symptoms of hepatotoxicity that occurred after clindamycin treatment were an adverse drug reaction (ADR) was assessed using the Naranjo ADR probability scale. The total Naranjo score for the patient was 7, which is in the "probable" range[3].

and (5) recovery after clindamycin withdrawal. Cholestasis with hepatitis is a frequent type of hepatic drug reaction and it is characterized by conspicuous cholestasis with hepatocellular injury. Histological lesions may show lobular and portal tract inflammation, often with neutrophils, eosinophils or mononuclear cells[4]. The clinical spectrum of cholestatic hepatitis was indicated in our case by pruritis and jaundice. It was also reflected by the markedly elevated liver tests, with increased serum bilirubin, GGT and ALP. Cases of mixed cholestasis and hepatitis are considered highly suggestive of a drug reaction[5]. To exclude dilation of large bile ducts produced by biliary obstruction, and hepatic or pancreatic masses, hepatobiliary imaging is essential. Ultrasound is the most preferred method to rule out possible bile duct obstruction. Endoscopic retrograde cholangiopancrea tography, percutaneous transhepatic cholangiography or computed tomography may be necessary in difficult cases. In the absence of the symptoms mentioned earlier, drug-induced cholestasis is more likely, and a liver biopsy is often advisable. Certain histological findings, such as lobular and portal tract inflammation, suggest a hepatic drug reaction, whereas others such as edema of the portal tracts suggest biliary obstruction. When the temporal relationship to drug ingestion indicates a high probability of a drug reaction, particularly when the agent is known to be potentially hepatotoxic, it is appropriate to discontinue the incriminated drug and observe whether improvement occurs. Recovery may be seen rapidly if the drug is discontinued before severe liver injury is established[6]. Hepatocytes, because of their capability to metabolize drugs, form minute amounts of drug-protein adducts, for which the immune system normally shows tolerance. Hypersensitivity reactions occur when this tolerance is impaired. Additional signals, such as a concomitant inflammatory reaction, may eventually be needed to break this tolerance. The allergic hepatitis induced by drugs is generally a type Ⅳ hypersensitivity reaction involving CD4+, CD8+ cytotoxic lymphocytes, as well as natural killer cells. Antibodies directed to the drug are much less common. Antibodies against cellular components may, however, occur when the sensitization process evolves towards an autoimmune reaction[7,8]. In our patient, autoantibodies were negative and several features, such as the absence of

DISCUSSION Six days after the start of treatment with clindamycin, our 42-year-old female patient experienced signs of hepatotoxicity with jaundice and asthenia. Laboratory testing showed marked elevations in ser um ALT, AST, ALP, GGT and total bilirubin concentrations. Her symptoms, as well as the liver tests, resolved on discontinuation of the drug treatment. The subtype of the hepatotoxicity observed in the patient was considered as mixed hepatocellular and cholestatic. The diagnosis of severe clindamycin-induced hepatotoxicity in our case was suggested by: (1) symptoms such as fatigue, nausea, vomiting, anorexia, pruritis and jaundice 6 d after clindamycin initiation; (2) the chronology between clindamycin introduction and liver test abnormalities; (3) histological findings that were highly suggestive of a toxic mechanism; (4) absence of any other clear etiology;

www.wjgnet.com

5410

ISSN 1007-9327

CN 14-1219/R


predictable dose-dependent toxicity of clindamycin and pruritis (hypersensitivity manifestations), were consistent with a metabolic type of idiosyncratic toxicity. Clindamycin is a widely used antibiotic that is available in many countries. Previously, another case of hepatotoxicity associated with use of clindamycin has been reported, in which the clinicopathological spectrum encompassed cholestatic liver disease with portal inflammation, bile duct injury and bile duct paucity (ductopenia). In the same patient, a second biopsy after clinical recovery showed resolution of cholestasis but persistence of duct paucity, whereupon the authors concluded that early onset hepatic injury with cholestasis and duct paucity may occur after clindamycin use, and long term duct paucity may persist on repeat biopsy[9]. Bile duct paucity as a biopsy finding was not significant in our patient. In summary, with this report, we presented a case of hepatotoxicity after 6 d of oral clindamycin treatment for dental infection. The toxicity appeared to be associated with drug use. In conclusion, although rare, mixed-type (hepatocellular and cholestatic) hepatic injury might be associated with clindamycin use in some cases. Patients may present with fatigue, nausea, vomiting, anorexia, pruritis and jaundice. Complete recovery may be possible if the drug is discontinued before critical liver injury is established.

Volume 13

Number 40

REFERENCES 1

2 3

4 5

6 7 8 9

Mazur D, Schug BS, Evers G, Larsimont V, Fieger-Buschges H, Gimbel W, Keilbach-Bermann A, Blume HH. Bioavailability and selected pharmacokinetic parameters of clindamycin hydrochloride after administration of a new 600 mg tablet formulation. Int J Clin Pharmacol Ther 1999; 37: 386-392 DeHaan RM, Metzler CM, Schellenberg D, VandenBosch WD, Masson EL. Pharmacokinetic studies of clindamycin hydrochloride in humans. Int J Clin Pharmacol 1972; 6: 105-119 Naranjo CA, Busto U, Sellers EM, Sandor P, Ruiz I, Roberts EA, Janecek E, Domecq C, Greenblatt DJ. A method for estimating the probability of adverse drug reactions. Clin Pharmacol Ther 1981; 30: 239-245 Lee WM. Drug-induced hepatotoxicity. N Engl J Med 2003; 349: 474-485 Farrel GC. Liver disease caused by drugs, anesthetics and toxins. In: Mark Feldman, Lawrence S Friedman, Marvin H, editors. Sleisenger and Fordtran’s gastrointestinal and liver disease: pathophysiology, diagnosis, management. 7th ed. Philadelphia: Saunders, 2002: 1403-1437 Chitturi S, Farrell GC. Drug-Induced Liver Disease. Curr Treat Options Gastroenterol 2000; 3: 457-462 Pichler WJ. Delayed drug hypersensitivity reactions. Ann Intern Med 2003; 139: 683-693 Depta JP, Pichler WJ. Cross-reactivity with drugs at the T cell level. Curr Opin Allergy Clin Immunol 2003; 3: 261-267 Altraif I, Lilly L, Wanless IR, Heathcote J. Cholestatic liver disease with ductopenia (vanishing bile duct syndrome) after administration of clindamycin and trimethoprimsulfamethoxazole. Am J Gastroenterol 1994; 89: 1230-1234

www.wjgnet.com

October 28, 2007

S- Editor Liu Y L- Editor Kerr C

E- Editor Li HY



CASE REPORT

Sudden hearing loss associated with peginterferon and ribavirin combination therapy during hepatitis C treatment Héla Elloumi, Fatma Houissa, Najet Bel Hadj, Dalila Gargouri, Malika Romani, Jamel Kharrat, AbdelJabbar Ghorbel Héla Elloumi, Fatma Houissa, Najet Bel Hadj, Dalila Gargouri, Malika Romani, Jamel Kharrat, AbdelJabbar Ghorbel, Department of Gastroenterology and Hepatology, Habib Thameur Hospital, Tunis, Tunisia Correspondence to: Héla Elloumi, Department of Gastroenterology and Hepatology, Habib Thameur Hospital, Tunis, Tunisia. [email protected] Telephone: +216-98-208498 Fax: +216-71-493167 Received: June 7, 2007 Revised: July 23, 2007

Abstract Adverse effects associated with peginterferon and ribavirin during hepatitis C treatment are well known. Sudden hearing loss has rarely been reported. Possible m e c h a n i s m s i nvo l ve d i n c l u d e d i r e c t o t o t ox i c i ty o f i n t e r fe ro n , a ut oimmuni ty, and he mat ologic al changes. Hearing loss is frequently fully resolved after discontinuation of antiviral therapy. We report a 47-yearold man with chronic hepatitis C, genotype 2 ac who developed sudden hearing loss 22 wk after starting therapy with peginterferon alpha 2a at a dose of 180 μg per week and ribavirin 800 mg per day. Since symptoms did not worsen, antiviral therapy was continued for 2 wk, according to the patient’s wish. Hearing loss resolved within 2 wk after the end of treatment. Serum liver alanine aminotransferase remained normal during and after the end of antiviral therapy. HCV RNA was undetectable at the end of therapy and remained negative 24 wk later. Thus, patients should be aware that hearing loss may occur with peginterferon therapy, but the decision whether to continue or to stop the treatment is based on the clinical judgment of the physician and the wishes of the patient. © 2007 WJG . All rights reserved.

Key words: Adverse effects; Hearing loss; Hepatitis C; Interferon; Ribavirin Elloumi H, Houissa F, Hadj NB, Gargouri D, Romani M, Kharrat J, Ghorbel A. Sudden hearing loss associated with peginterferon and ribavirin combination therapy during hepatitis C treatment. World J Gastroenterol 2007; 13(40): 5411-5412


INTRODUCTION Chronic hepatitis C affects 250-300 million people worldwide, and in Tunisia, the prevalence is estimated to be 0.4%-1.6% of the general population[1-3]. The higher prevalence is in the north which corresponds to the south side of the Mediterranean basin. The most frequent genotype in Tunisia is 1b[4]. Treatment of hepatitis C virus infection with the combination of peginterferon alpha (2a or 2b) and ribavirin therapy is now recommended[5,6]. Both peginterferon and ribavirin are well known to be associated with significant adverse effects[7]. Hearing loss has been reported as a consequence of therapy with pegylated and non-pegylated interferon[8-10]. However, we still pay little attention to auditory acuity. Moreover, contrary to standard interferon, hearing loss seems to not be fully resolved after discontinuation of peginterferon treatment[8,10]. We report a case of sudden hearing loss in a patient with HCV infection treated with a combination of peginterferon and ribavirin. The interesting aspect of our case is that peginterferon therapy was continued during the last month and hearing loss was fully resolved after the end of treatment.

CASE REPORT A 47-year-old Tunisian man with chronic hepatitis C, with 106 UI/L HCV RNA and genotype 2a/2c, attended the Gastroenterology Department for follow-up. He did not have any pathological history and he had not taken any drugs prior to his visit. His initial physical and neurological examinations were normal. Serum levels of glucose, urea, creatinin, lipids, electrolytes, alkaline phosphatase, bilirubin, gammaglutamyl transferase, blood cell count, ferritin and cryoglobulin were within normal limits. Alanine aminotransferase (ALT) was 62 UI/L (upper limit of normal < 45), aspartate aminotransferase (AST) was 39 UI/L (upper limit of normal < 45 UI/L). Antinuclear, smooth muscle, anti-liver-kidney microsome 1 and anti-mitochondrial antibodies were negative. He was treated with peginterferon alpha 2a (180 μ g/wk) and ribavirin (800 mg/d) for a predetermined period of 24 wk. Twenty-two weeks after beginning therapy, he developed otalgia and sudden onset of left-sided hearing loss. He was seen in consultation by an otolaryngologist who reported that ear examination was normal. Since

www.wjgnet.com

5412

ISSN 1007-9327

CN 14-1219/R


symptoms did not worsen, and in accord with the patient’s wishes, antiviral therapy was continued for 2 wk. Hearing loss resolved within 2 wk after the end of treatment. Serum liver ALT remained normal during and after the end of antiviral therapy, and no sign of thrombocytopenia was detected during blood cell count monitoring. HCV RNA was undetectable at the end of therapy and remained negative 24 wk later.

DISCUSSION This is another report on sudden hearing loss after treatment with peginterferon alpha, which adds to the observations made in patients treated with peginterferon and standard interferon for chronic hepatitis C[8-11]. Such hearing loss is mostly unilateral and sensorineural[12]. In one prospective study of 73 patients receiving interferon, hearing impair ment (tinnitus and/or hearing loss) occurred in 32 patients (43.8%), among whom audiometry documented hearing loss in 27 cases (36.9%)[13]. Several factors may cause sensorineural hearing loss in patients treated with interferon. First, the drug itself may be directly toxic to the auditory nerve hairy cells[9]. Also, an indirect effect of interferon due to its immunoregulatory and antiviral activity can explain this sensorineural pathology. Cadoni has detected antiendothelial-cell antibodies in an HCV patient with sudden hearing loss during interferon therapy[11]. The finding of these antibodies suggests microvascular damage during therapy for vasculitis. In addition, interferon-induced thrombocytopenia may cause a microvascular accident in the inner ear. Some authors have reported that hearing loss fully resolves 5 d to 1 mo after discontinuation of antiviral therapy with interferon [9,14] ; whereas others have demonstrated that hearing impairment does not completely recover after cessation of peginterferon, and worsens with continued treatment[8,10]. In the case reported here, hearing loss fully resolved after the end of treatment with peginterferon. Auditory disability frequently develops in the later stages of treatment[15]; in our case, it developed 2 wk before the end of the treatment (i.e., after 22 wk). The average cumulative dose until development of auditory disability is about 176.9 mV[13]. In a recent study, hearing loss was detected in nine of 27 patients after 7 d, and the degree of hearing loss increased until 21 d of treatment[14]. Therefore, it is recommended that patients on interferon therapy should be monitored during treatment for changes in hearing perception. It remains possible that those who develop hearing loss can continue with inter-

October 28, 2007

Volume 13

feron therapy. The decision whether to continue or to stop the treatment when signs of ototoxicity appear is based on the clinical judgement of the treating physician and the wishes of the patient.

REFERENCES 1

2 3

4 5 6 7

8

9

10

11

12 13 14

15

World Health Organization. Weekly Epidemiological Record. No. 49, 10 December 1999; Hepatitis C Fact Sheet No. 164, October 2000. Available from: URL: http:// www.who.int/ inf-fs/en/fact164.html Habib Ben Khalifa. Epidemiologie Du Virus C En Tunisie: Ou En Sommes Nous? Available from: URL: http://www.stmi. org.tn Triki H, Said N, Ben Salah A, Arrouji A, Ben Ahmed F, Bouguerra A, Hmida S, Dhahri R, Dellagi K. Seroepidemiology of hepatitis B, C and delta viruses in Tunisia. Trans R Soc Trop Med Hyg 1997; 91: 11-14 Djebbi A, Triki H, Bahri O, Cheikh I, Sadraoui A, Ben Ammar A, Dellagi K. Genotypes of hepatitis C virus circulating in Tunisia. Epidemiol Infect 2003; 130: 501-505 National Institutes of Health Consensus Development Conference Statement: Management of hepatitis C: 2002-June 10-12, 2002. Hepatology 2002; 36: S3-S20 Dhumeaux D, Marcellin P, Lerebours E. Treatment of hepatitis C. The 2002 French consensus. Gut 2003; 52: 1784-1787 Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, Goodman ZD, Koury K, Ling M, Albrecht JK. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatment of chronic hepatitis C: a randomised trial. Lancet 2001; 358: 958-965 Wong VK, Cheong-Lee C, Ford JA, Yoshida EM. Acute sensorineural hearing loss associated with peginterferon and ribavirin combination therapy during hepatitis C treatment: outcome after resumption of therapy. World J Gastroenterol 2005; 11: 5392-5393 Tunca A, Erbayrak M, Aytac S, Turkay C. Axonal neuropathy and hearing loss associated with alpha interferon treatment in chronic hepatitis B: a case report. Turk J Gastroenterol 2004; 15: 97-99 Formann E, Stauber R, Denk DM, Jessner W, Zollner G, Munda-Steindl P, Gangl A, Ferenci P. Sudden hearing loss in patients with chronic hepatitis C treated with pegylated interferon/ribavirin. Am J Gastroenterol 2004; 99: 873-877 Cadoni G, Marinelli L, De Santis A, Romito A, Manna R, Ottaviani F. Sudden hearing loss in a patient hepatitis C virus (HCV) positive on therapy with alpha-interferon: a possible autoimmune-microvascular pathogenesis. J Laryngol Otol 1998; 112: 962-963 Dusheiko G. Side effects of alpha interferon in chronic hepatitis C. Hepatology 1997; 26: 112S-121S Kanda Y, Shigeno K, Matsuo H, Yano M, Yamada N, Kumagami H. Interferon-induced sudden hearing loss. Audiology 1995; 34: 98-102 Gorur K, Kandemir O, Unal M, Ozcan C. The effect of recombinant interferon alpha treatment on hearing thresholds in patients with chronic viral hepatitis B. Auris Nasus Larynx 2003; 30: 41-44 Kanda Y, Shigeno K, Kinoshita N, Nakao K, Yano M, Matsuo H. Sudden hearing loss associated with interferon. Lancet 1994; 343: 1134-1135 S- Editor Liu Y L- Editor Kerr C

www.wjgnet.com

Number 40

E- Editor Ma WH



CASE REPORT

Positron emission tomography/computer tomography in guidance of extrahepatic hepatocellular carcinoma metastasis management Long Sun, Yong-Song Guan, Wei-Ming Pan, Gui-Bing Chen, Zuo-Ming Luo, Hua Wu Long Sun, Wei-Ming Pan, Gui-Bing Chen, Zuo-Ming Luo, Hua Wu, Minnan PET Center, The First Hospital of Xiamen, Fujian Medical University, Xiamen 316003, Fujian Province, China Yong-Song Guan, West China Hospital of Sichuan University; State Key Laboratory of Biotherapy, West China Medical School, Sichuan University, Gaopeng Street, Keyuan Road 4, Chengdu 610041, Sichuan Province, China Correspondence to: Hua Wu, Minnan PET Center, The First Hospital of Xiamen, Fujian Medical University, Xiamen 316003, Fujian Province, China. [email protected] Telephone: +86-592-2139521 Fax: +86-592-2139521 Revised: July 9, 2007 Received: June 29, 2007

Abstract Hepatocellular carcinoma (HCC) is one of the most common primary cancers in the world. Surgery is the gold standard for treatment of patients with HCC. Recurrence and metastasis are the major obstacles to further improve the prognosis of HCC. Most recurrences are intrahepatic. However, 30% of the recurrences are extrahepatic. The role of resection in intrahepatic recurrences is widely accepted. The role of resection in extrahepatic HCC recurrence and metastasis is not well established. 18F fluorodeoxyglucose (18F-FDG) positron emission tomography/computer tomography (PET/CT) is useful in detecting distant metastasis from a variety of malignancies and shows superior accuracy to conventional imaging modalities in identification of intrahepatic and extrahepatic metastasis. We present one patient with one new isolated omental lymph node metastasis, who had a history of huge HCC resected six years ago. The metastatic focus was identified with 18 F-FDG PET/CT and resected. The follow-up revealed good prognosis with a long-term survival potential after resection of the omental lymphatic metastasis. © 2007 WJG . All rights reserved.

Key words: Fluorodeoxyglucose; Positron emission tomography/computer tomography; Huge hepatocellular carcinoma; Omental node metastasis; Extrahepatic metastasis Sun L, Guan YS, Pan WM, Chen GB, Luo ZM, Wu H. Positron emission tomography/computer tomography in guidance of extrahepatic hepatocellular carcinoma metastasis management. World J Gastroenterol 2007; 13(40): 5413-5415


INTRODUCTION Recurrence and metastasis are the major obstacles to further improve the prognosis of hepatocellular carcinoma (HCC). Tumor recurrence is common after hepatic resection of huge HCC greater than 10 cm in diameter. The overall 1- and 3-year survival rates are 66% and 44%, respectively. The overall 5-year survival ranges from 11% to 76%, with a median around 30% after hepatectomy[1]. Extrahepatic recurrence occurs in 30% of the patients. Resection of isolated extrahepatic HCC metastasis has been advocated to obtain a possibility of long-term survival[2,3]. Accurate re-staging plays the most important role in making a decision on extrahepatic metastasis resection. Nowadays, PET/CT is more and more widely available, and its application with 18F-FDG in oncology has become one of the standard imaging modalities in diagnosing, staging and re-staging HCC[4]. We present here a patient with huge HCC resected six years ago and a new omental lymph node metastasis recently identified by 18 F-FDG PET/CT. Long-term survival was achieved in this patient after resection of the omental lymph node metastasis.

CASE REPORT A 60-year old woman with a medical history of HBV infection and no cirrhosis was referred to our hospital for a liver tumor found by coincidence. The AFP level was 13.4 ng/mL. HCC was suspected (Figure 1) and a left hemihepatectomy was performed in December 2000. Histology showed a HCC (Edmondson-Steiner’s gradeⅠ) with a diameter of 13.0 cm with central necrosis. No microscopic vascular invasion was found. After partial liver resection, AFP values returned to normal. The patient accepted trancatheter arterial chemo-embolization (TACE) treatment 1 mo after hepatectomy and no special change was found during angiography. After 78 mo of follow-up, her AFP increased to 597.8 ng/mL. Conventional chest and abdominal CT did not find any evidence of intrahepatic recurrence and extrahepatic metastasis. To find out the reason for AFP increasing, the patient was referred to our center for a whole body PET/CT examination. 18F-FDG PET/CT detected a high metabolic lymph node in the omental position (Figure 2). Due to the maximum stanwww.wjgnet.com

5414

ISSN 1007-9327

CN 14-1219/R


October 28, 2007

Volume 13

Number 40

A

Figure 1 Contrast CT demonstrating a huge lesion at the left lobe of liver (black arrows, portal phase).

B

dard uptake value (SUVmax), lymph node metastasis was considered. The high metabolic lymph node was excised. Histopathological examination of the resected nodule revealed a metastatic hepatocellular carcinoma. After excision of the tumor, her AFP level decreased to normal. Ten months after resection of the extrahepatic metastasis and 88 mo after partial liver resection, the patient was in good condition with no recurrence at the time when we wrote this paper.

DISCUSSION HCC over 10 cm in diameter is found in a number of patients undergoing hepatic resection. A large HCC has its specific clinicopathological features. Multiple tumor nodules, venous invasion and impaired liver function are factors associated with its recurrence[5]. Hepatic resection of HCC with a diameter ≥ 10 cm at stage Ⅱ or Ⅲ is safe and effective for selected patients without liver cirrhosis. The overall incidence of postoperative complications is 39%. The 3-, 5- and 9-year overall survival rates after hepatic resection are 32%, 27%, and 17%, respectively[6]. Appropriate selection of candidates for partial hepatectomy based on the above prognostic factors may play an important role in decreasing the high mortality rate and poor long-term survival of such patients[7]. Approximately 50% of patients with resected HCC may have tumor recurrence. The overall 1-, 3- and 5-year disease-free survival rates are reported to be 43%, 26% and 20%, respectively[8]. Thus, it is of importance to predict its relapse and treat it with other modalities[9]. The role of resection in case of intrahepatic recurrences is widely accepted. If the distribution and localization of extrahepatic metastasis are fully confirmed, these lesions may be resected. In our case, conventional CT did not find any intrahepatic and extrahepatic metastasis. However, the whole body PET/CT detected isolated extrahepatic metastasis which we treated with surgery. 18F-FDG PET/CT is not only a useful tool for preoperative staging, but also a follow-up means better than conventional diagnostic modalities, especially for staging and re-staging after hepatectomy. In case of an AFP producing HCC, unexplained rise of serum AFP after treatment is an early indicator of tuwww.wjgnet.com

C

D

Figure 2 Whole body PET showing an isolated highly metabolic focus in the upper abdomen (white arrow) (A), non-enhanced CT (C) detecting a median density lesion in the omenta (white arrow), 18F-FDG PET (B) and fused imaging of PET/CT (D) revealing a highly metabolic lesion at the same position.

mor recurrence or extrahepatic metastasis. However, the sensitivity of conventional imaging techniques is limited in detecting recurrent diseases in such patients[10]. 18F-FDG

Sun L et al . PET/CT application in extrahepatic hepatocellular carcinoma metastasis

PET/CT has proved to be an effective whole-body imaging technique that detects metabolic changes preceding structural findings. It was reported that tumor restaging with PET/CT can detect and localize disease recurrence among patients with no or mild symptoms and an elevated tumor marker level[11]. PET/CT also can provide information about whether the detected disease is resectable. 18F-FDG PET/CT provides fused images that demonstrate the complementary role of functional and anatomic assessments in the diagnosis of cancer recurrence through the precise localization of suspected 18FFDG uptake foci and their characterization as malignant or benign[12]. TACE is a procedure involving injection of chemotherapeutic agents into the hepatic artery supplying the tumor, followed by embolization with lipiodol or gelfoam pieces. It appears that postoperative adjuvant hepatic arterial chemotherapy is more likely beneficial for preventing recurrence than preoperative TACE. To date, convincing evidence supporting the effectiveness of adjuvant therapy in preventing HCC recurrence is lacking. Validated information in the literature is limited in a paucity of randomized studies. It was reported that meaningful meta-analysis is impossible because of different therapeutic regimens used and the lack of stratification of patients according to the risk of recurrence[13]. Better understanding of biological characteristics of HCC can lead to better outcomes of adjuvant surgical treatment[14]. For example, if patients at a high risk of HCC recurrence could be identified before the resection, they would benefit from additional treatment modalities, such as neoadjuvant therapy, adjuvant therapy and biotherapy. It is not necessary for patients at a low risk of tumor recurrence to receive the above therapies[15]. Molecular imaging in oncology is the noninvasive imaging of the key molecules and molecule-based events characterized by the genotype and phenotype of human cancer. Tumors with increased 18F-FDG uptake are more metabolically active and biologically aggressive. PET/CT with 18F-FDG would play a key role in evaluating recurrence risk of HCC before hepatectomy[16]. Further research is warranted because of the limited number of reports and the absence of randomized trials. Aggressive management with combined resection of isolated extrahepatic recurrence and re-resection or local therapy for intrahepatic recurrence may offer long-term survival in selected HCC patients. Our case demonstrates that 18F-FDG PET/CT provides the most significant additional information related to the accurate detection of extrahepatic spread of tumors and additional important information in patients with presumed resectable extrahepatic metastases. This test should be used for patients at a high risk of extrahepatic disease and should be evaluated prospectively for all patients under consideration for intrahepatic recurrence and extrahepatic metastasis resection.

5415

REFERENCES 1

Lee SG, Hwang S, Jung JP, Lee YJ, Kim KH, Ahn CS. Outcome of patients with huge hepatocellular carcinoma after primary resection and treatment of recurrent lesions. Br J Surg 2007; 94: 320-326 Verhoef C, Holman FA, Hussain SM, de Man RA, de Wilt 2 JH, IJzermans JN. Resection of extrahepatic hepatocellular carcinoma metastasis can result in long-term survival. Acta Chir Belg 2005; 105: 533-536 Tomimaru Y, Sasaki Y, Yamada T, Eguchi H, Takami K, 3 Ohigashi H, Higashiyama M, Ishikawa O, Kodama K, Imaoka S. The significance of surgical resection for pulmonary metastasis from hepatocellular carcinoma. Am J Surg 2006; 192: 46-51 Anderson GS, Brinkmann F, Soulen MC, Alavi A, Zhuang 4 H. FDG positron emission tomography in the surveillance of hepatic tumors treated with radiofrequency ablation. Clin Nucl Med 2003; 28: 192-197 5 Chen XP, Qiu FZ, Wu ZD, Zhang BX. Hepatectomy for huge hepatocellular carcinoma in 634 cases. World J Gastroenterol 2006; 12: 4652-4655 Yuan YF, Li BK, Li JQ, Zhang YQ, Guo RP, Lin XJ, Li GH. 6 Long-term prognosis after hepatic resection for huge primary liver cancer. Aizheng 2004; 23: 821-824 Chen XP, Qiu FZ, Wu ZD, Zhang BX. Chinese experience with 7 hepatectomy for huge hepatocellular carcinoma. Br J Surg 2004; 91: 322-326 Lee SG, Hwang S, Jung JP, Lee YJ, Kim KH, Ahn CS. Outcome 8 of patients with huge hepatocellular carcinoma after primary resection and treatment of recurrent lesions. Br J Surg 2007; 94: 320-326 Arriagada R, Bergman B, Dunant A, Le Chevalier T, Pignon 9 JP, Vansteenkiste J. Cisplatin-based adjuvant chemotherapy in patients with completely resected non-small-cell lung cancer. N Engl J Med 2004; 350: 351-360 10 Zhao M, Wu PH, Zeng YX, Zhang FJ, Huang JH, Fan WJ, Gu YK, Zhang L, Tan ZB, Lin YE. Evaluating efficacy of transcatheter arterial chemo-embolization combined with radiofrequency ablation on patients with hepatocellular carcinoma by 18FDG-PET/CT. Aizheng 2005; 24: 1118-1123 11 Liu FY, Chen JS, Changchien CR, Yeh CY, Liu SH, Ho KC, Yen TC. Utility of 2-fluoro-2-deoxy-D-glucose positron emission tomography in managing patients of colorectal cancer with unexplained carcinoembryonic antigen elevation at different levels. Dis Colon Rectum 2005; 48: 1900-1912 12 P a r k Y A , L e e K Y , K i m N K , B a i k S H , S o h n S K , C h o CW. Prognostic effect of perioperative change of serum carcinoembryonic antigen level: a useful tool for detection of systemic recurrence in rectal cancer. Ann Surg Oncol 2006; 13: 645-650 13 Tung-Ping Poon R, Fan ST, Wong J. Risk factors, prevention, and management of postoperative recurrence after resection of hepatocellular carcinoma. Ann Surg 2000; 232: 10-24 14 Larson SM. Positron emission tomography-based molecular imaging in human cancer: exploring the link between hypoxia and accelerated glucose metabolism. Clin Cancer Res 2004; 10: 2203-2204 15 Larson SM, Schwartz LH. 18F-FDG PET as a candidate for "qualified biomarker": functional assessment of treatment response in oncology. J Nucl Med 2006; 47: 901-903 16 Hatano E, Ikai I, Higashi T, Teramukai S, Torizuka T, Saga T, Fujii H, Shimahara Y. Preoperative positron emission tomography with fluorine-18-fluorodeoxyglucose is predictive of prognosis in patients with hepatocellular carcinoma after resection. World J Surg 2006; 30: 1736-1741 S- Editor Liu Y L- Editor Wang XL

E- Editor Li JL

www.wjgnet.com


World J Gastroenterol 2007 October 28; 13(40): 5416 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.

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 were published and those were rejected in this issue) during the last editing period of time. Olivier Barbier CHUQ-CHUL Research Center, 2705 Laurier Boulevard, Québec G1V 4G2, Canada Thomas Bock, PhD, Professor Department of Molecular Pathology, Institute of Pathology, University Hospital of Tuebingen, D-72076 Tuebingen, Germany Andres Cardenas Institut de Malalties Digestives i Metaboliques Hospital Clinic, Villaroel 170, Esc 7-4, Barcelona 08036, Spain 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 Mitsuhiro Fujishiro, Dr Department of Gastroenterology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan Andreas Geier, Associate Professor Department of Internal Medicine III, University Hospital Aachen (UKA), Aachen University (RWTH), Pauwelsstrasse 30, D-52074 Aache, Germany Naohiko Harada, PhD Department of Gastroenterology, Fukuoka Higashi Medical Center, Chidori 1-1-1, Koga, Fukuoka 811-3195, Japan Anthony R Hobson, Dr Section of Gastrointestinal Sciences, University of Manchester, Eccles Old Road, Hope Hospital, Clinical Sciences Building, Salford M6 8HD, United Kingdom 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

Emanuele Durante Mangoni, MD Dottorando di Ricerca, Cattedra di Medicina Interna-II Università di Napoli, Dirigente Medico, UOC Medicina Infettivologica e dei Trapianti - Ospedale Monaldi, Napoli 80135, Italy Donna-Marie McCafferty Gastrointestinal Research Group, Department of Physiology and Biophysics, Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1, Canada Laura E Matarese, MS RD, LDN, FADA, CNSD, Thomas E Starzl Transplantation Institute, UPMC Montefiore, 7 South, 3459 Fifth Avenue, Pittsburgh, PA 15213, United States Chris Jacob Johan Mulder, Professor Department of Gastroenterology, VU University Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands Hisato Nakajima, MD Department of Gastroenterology and Hepatology, The Jikei University School of Medicine, 3-25-8, Nishi-Shinbashi, Minato-ku, Tokyo 105-8461, Japan Francesco Negro, MD Divisions of Gastroenterology and Hepatology and of Clinical Pathology, Hôpital Cantonal Universitaire, 24 rue Micheli-du-Crest, CH-1211 Genève 14, Switzerland Raffaele Pezzilli, MD Department of Internal Medicine and Gastroenterology, Sant’Orsola-Malpighi Hospital, Via Massarenti, 9, Bologna 40138, Italy Vasiliy I Reshetnyak, MD, PhD, Professor Scientist Secretary of the Scientific Research Institute of General Reanimatology, 25-2, Petrovka str., 107031, Moscow, Russia Damian Casadesus Rodriguez, MD, PhD Calixto Garcia University Hospital, J and University, Vedado, Havana City, Cuba Ross C Smith, Professor Department of Surgery, University of Sydney, Royal North Shore Hospital, St Leonards, New South Wales 2065, Australia Qin Su, Professor Department of Pathology, Cancer Hospital and Cancer Institute, Chinese Academy of Medical Sciences and Peking Medical College, PO Box 2258, Beijing 100021, China Seng-Lai Tan, Principal Scientist Eli Lilly and Company, Indianapolis 46285, United States

Yoshiaki Iwasaki, Dr Department of Gastroenterology and Hepatology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1, Shikata-cho, Okayama 700-8558, Japan

Saúl Villa-Trevio, MD, PhD Departamento de Biología Celular, Centro de Investigación y de Estudios Avanzados del IPN (Cinvestav), Ave. IPN No. 2508. Col. San Pedro, Zacatenco, C.P. 07360, México, DF, Mexico

Neil Kaplowitz, MD Research Center for Liver Disease, Keck School of Medicine, University of Southern California 2011 Zonal Avenue, HMR101, Los Angeles, California 90033, United States

Ian David Wallace, MD Shakespeare Specialist Group, 181 Shakesperare Rd, Milford, Auckland 1309, New Zealand

Richard A Kozarek, MD Department of Gastroenterology, Virginia Mason Medical Center, 1100 Ninth Avenue, PO Box 900, Seattle 98111-0900, United States

Francis Y Yao, MD, Professor Division of Gastroenterology, Department of. Medicine, and. b. Division of Transplantation, Department of Surgery, University of California,. San Francisco, CA, United States

Reza Malekzadeh, Professor, Director Digestive Disease Research Center, Tehran University of Medical Sciences, Shariati Hospital, Kargar Shomali Avenue, 19119 Tehran, Iran

Hiroshi Yoshida, MD First Department of Surgery, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan

World J Gastroenterol 2007 October 28; 13(40): 5417 World Journal of Gastroenterology ISSN 1007-9327 © 2007 WJG. All rights reserved.


Meetings MAJOR MEETINGS COMING UP Meeting Falk Research Workshop: Morphogenesis and Cancerogenesis of the Liver 25-26 January 2007 Goettingen [email protected] Meeting Canadian Digestive Diseases Week (CDDW) 16-20 February 2007 Banff-AB [email protected] www.cag-acg.org/cddw/cddw2007. htm Meeting Falk Symposium 158: Intestinal Inflammation and Colorectal Cancer 23-24 March 2007 Sevilla [email protected] Meeting BSG Annual Meeting 26-29 March 2007 Glasgow www.bsg.org.uk/

NEXT 6 MONTHS Meeting 42nd Annual Meeting of the European Association for the Study of the Liver 11-15 April 2007 Barcelona [email protected] www.easl.ch/liver-meeting/ Meeting Falk Symposium 159: IBD 2007 - Achievements in Research and Clinical Practice 4-5 May 2007 Istanbul [email protected] Meeting European Society for Paediatric Gastroenterology, Hepatology and Nutrition Congress 2007 9-12 May 2007 Barcelona [email protected] Digestive Disease Week 19-24 May 2007 Washington Convention Center, Washington DC Meeting Gastrointestinal Endoscopy Best Practices: Today and Tomorrow, ASGE Annual Postgraduate Course at DDW 23-24 May 2007 Washington-DC [email protected] Meeting ESGAR 2007 18th Annual Meeting and Postgraduate Course 12-15 June 2007 Lisbon [email protected] Meeting Falk Symposium 160: Pathogenesis and Clinical Practice in

Gastroenterology 15-16 June 2007 Portoroz [email protected] Meeting ILTS 13th Annual International Congress 20-23 June 2007 Rio De Janeiro www.ilts.org Meeting 9th World Congress on Gastrointestinal Cancer 27-30 June 2007 Barcelona [email protected]

EVENTS AND MEETINGS IN 2007 Meeting Falk Research Workshop: Morphogenesis and Cancerogenesis of the Liver 25-26 January 2007 Goettingen [email protected] Meeting Canadian Digestive Diseases Week (CDDW) 16-20 February 2007 Banff-AB [email protected] www.cag-acg.org/cddw/cddw2007. htm Meeting Falk Symposium 158: Intestinal Inflammation and Colorectal Cancer 23-24 March 2007 Sevilla [email protected] Meeting BSG Annual Meeting 26-29 March 2007 Glasgow www.bsg.org.uk/ Meeting 42nd Annual Meeting of the European Association for the Study of the Liver 11-15 April 2007 Barcelona [email protected] www.easl.ch/liver-meeting/ Meeting Falk Symposium 159: IBD 2007 - Achievements in Research and Clinical Practice 4-5 May 2007 Istanbul [email protected] Meeting European Society for Paediatric Gastroenterology, Hepatology and Nutrition Congress 2007 9-12 May 2007 Barcelona [email protected] Meeting Gastrointestinal Endoscopy Best Practices: Today and Tomorrow, ASGE Annual Postgraduate Course at DDW 23-24 May 2007 Washington-DC [email protected] Meeting ESGAR 2007 18th Annual Meeting and Postgraduate Course 12-15 June 2007 Lisbon [email protected]

Meeting Falk Symposium 160: Pathogenesis and Clinical Practice in Gastroenterology 15-16 June 2007 Portoroz [email protected] Meeting ILTS 13th Annual International Congress 20-23 June 2007 Rio De Janeiro www.ilts.org Meeting 9th World Congress on Gastrointestinal Cancer 27-30 June 2007 Barcelona [email protected] Meeting 15th International Congress of the European Association for Endoscopic Surgery 4-7 July 2007 Athens [email protected] congresses.eaes-eur.org/ Meeting 39th Meeting of the European Pancreatic Club 4-7 July 2007 Newcastle www.e-p-c2007.com Meeting XXth International Workshop on Heliobacter and related bacteria in cronic degistive inflammation 20-22 September 2007 Istanbul www.heliobacter.org Meeting Falk Workshop: Mechanisms of Intestinal Inflammation 10 October 2007 Dresden [email protected] Meeting Falk Symposium 161: Future Perspectives in Gastroenterology 11-12 October 2007 Dresden [email protected] Meeting Falk Symposium 162: Liver Cirrhosis - From Pathophysiology to Disease Management 13-14 October 2007 Dresden [email protected] American College of Gastroenterology Annual Scientific Meeting 12-17 October 2007 Pennsylvania Convention Center Philadelphia, PA Meeting APDW 2007 - Asian Pacific Digestive Disease Week 2007 15-18 October 2007 Kobe [email protected] www.apdw2007.org 15th United European Gastroenterology Week, UEGW 27-31 October 2007 Le Palais des Congrès de Paris, Paris, France Meeting The Liver Meeting ® 2007 57th Annual Meeting of the American Association for the Study of Liver Diseases

2-6 November 2007 Boston-MA www.aasld.org Gastro 2009, World Congress of Gastroenterology and Endoscopy London, United Kingdom 2009



Instructions to authors GENERAL INFORMATION World Journal of Gastroenterology (WJG, World J Gastroenterol ISSN 1007-9327 CN 14-1219/R) is a weekly journal of more than 48 000 circulation, published on the 7th, 14th, 21st and 28th of every month. Original Research, Clinical Trials, Reviews, Comments, and Case Reports in esophageal cancer, gastric cancer, colon cancer, liver cancer, viral liver diseases, etc., from all over the world are welcome on the condition that they have not been published previously and have not been submitted simultaneously elsewhere.

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 WJG

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 is responsible for the opinions expressed by contributors. Manuscripts formally accepted for publication become the permanent property of WJG, 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 put onto our website and copy-edit accepted manuscripts. Authors should also follow the guidelines for the care and use of laboratory animals of their institution or national animal welfare committee. Authors should retain one copy of the text, tables, photographs and illustrations, as rejected manuscripts will not be returned to the author(s) and the editors will not be responsible for the loss or damage to photographs and illustrations in mailing process.

Online submission

Online submission is strongly advised. Manuscripts should be submitted through the Online Submission System at: http://www.wjgnet.com/index.jsp. Authors are highly recommended to consult the ONLINE INSTRUCTIONS TO AUTHORS (http://www.wjgnet.com/wjg/help/instructions.jsp) before attempting to submit online. Authors encountering problems with the Online Submission System may send an email you describing the problem to wjg@ wjgnet.com for assistance. If you submit your manuscript online, do not make a postal contribution. A repeated online submission for the same manuscript is strictly prohibited.

Postal submission

Send 3 duplicate hard copies of the full-text manuscript typed double-spaced on A4 (297 mm × 210 mm) white paper together with any original photographs or illustrations and a 3.5 inch computer diskette or CD-ROM containing an electronic copy of the manuscript including all the figures, graphs and tables in native Microsoft Word format or *.rtf format to:

line spacing and in word size 12 with ample margins. The letter font is Tahoma. For authors from China, one copy of the Chinese translation of the manuscript is also required (excluding references). 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 accomplished, 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 (removing 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.

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 equipments, and the experimental procedures should be included. RESULTS: The observatory and experimental results, including data, effects, outcome, etc. should be included. Authors should present P value where necessary, and the significant data should accompany. CONCLUSION: Accurate view and the value of the results should be included. The format of structured abstracts is at: http://www.wjgnet.com/wjg/ help/11.doc

Key words

Please list 5-10 key words that could reflect content of the study mainly from Index Medicus.

Text

For most article types, the main text should be structured into the following sections: INTRODUCTION, MATERIALS AND METHODS, RESULTS and DISCUSSION, and should include in appropriate Figures and Tables. Data should be presented in the body text or in Figures and Tables, but not in both.

Illustrations

Figures should be numbered as 1, 2, 3 and so on, and mentioned clearly in the main text. Provide a brief title for each figure on a separate page. No detailed legend should be involved under the figures. This part should be added into the text where the figures are applicable. Digital images: black and white photographs should be scanned and saved in TIFF format at a resolution of 300 dpi; color images should be saved as CMYK (print files) but not as RGB (screen-viewing files). Place each photograph in a separate file. Print images: supply images of size no smaller than 126 mm × 85 mm printed on smooth surface paper; label the image by writing the Figure number and orientation using an arrow. Photomicrographs: indicate the original magnification and stain in the legend. Digital Drawings: supply files in EPS if created by freehand and illustrator, or TIFF from photoshops. EPS files must be accompanied by a version in native file format for editing purposes. Existing line drawings should be scanned at a resolution of 1200 dpi and as close as possible to the size where they will appear when printed. Please use uniform legends for the same subjects. For example: Figure 1 Pathological changes of atrophic gastritis after treatment. A: ...; B: ...; C: ...; D: ...; E: ...; F: ...; G: ...

Tables

Editorial Office

Three-line tables should be numbered as 1, 2, 3 and so on, and mentioned clearly in the main text. Provide a brief title for each table. No detailed legend should be included under the tables. This part should be added into the text where the tables are applicable. The information should complement but not duplicate that contained in the text. Use one horizontal line under the title, a second under the column heads, and a third below the Table, above any footnotes. Vertical and italic lines should be omitted.


Editorial Department: Apartment 1066, Yishou Garden, 58 North Langxinzhuang Road, PO Box 2345, Beijing 100023, China E-mail: [email protected] http: //www.wjgnet.com Telephone: +86-10-85381892 Fax: +86-10-85381893

Notes in tables and illustrations

MANUSCRIPT PREPARATION All contributions should be written in English. All articles must be submitted using a word-processing software. All submissions must be typed in 1.5

Data that are not statistically significant should not be noted. aP

Gastroenterology - CiteSeerX

Gastroenterology - CiteSeerX

Suggest Documents