World Journal of Gastroenterology

Transcription

ISSN 1007-9327 (print)
ISSN 2219-2840 (online)
World Journal of
Gastroenterology
Volume 17 Number 16
April 28, 2011
World Journal of
Gastroenterology
World J Gastroenterol 2011 April 28; 17(16): 2063-2160
www.wjgnet.com
Volume 17
Number 16
Apr 28
Published by Baishideng Publishing Group Co., Limited,
Room 1701, 17/F, Henan Building,
No. 90 Jaffe Road, Wanchai, Hong Kong, China
Fax: +852-3115-8812
Telephone: +852-5804-2046
E-mail: [email protected]
http://www.wjgnet.com
2011
I S S N 1 0 0 7 - 9 3 2 7
1 6
ISSN 1007-9327 CN 14-1219/R Local Post Offices Code No. 82-261
9 7 7 1 0 0 7 9 3 2 0 45
www.wjgnet.com
Editorial Board
2010-2013
The World Journal of Gastroenterology Editorial Board consists of 1144 members, representing a team of worldwide
experts in gastroenterology and hepatology. They are from 60 countries, including Albania (1), Argentina (8),
Australia (29), Austria (14), Belgium (12), Brazil (10), Brunei Darussalam (1), Bulgaria (2), Canada (20), Chile (3),
China (69), Colombia (1), Croatia (2), Cuba (1), Czech (4), Denmark (8), Ecuador (1), Egypt (2), Estonia (2), Finland
(8), France (24), Germany (75), Greece (14), Hungary (10), India (26), Iran (6), Ireland (7), Israel (12), Italy (101),
Japan (112), Jordan (1), Kuwait (1), Lebanon (3), Lithuania (2), Malaysia (1), Mexico (10), Moldova (1), Netherlands
(29), New Zealand (2), Norway (11), Pakistan (2), Poland (11), Portugal (4), Romania (3), Russia (1), Saudi Arabia
(3), Serbia (3), Singapore (10), South Africa (2), South Korea (32), Spain (38), Sweden (18), Switzerland (11),
Thailand (1), Trinidad and Tobago (1), Turkey (24), United Arab Emirates (2), United Kingdom (82), United States
(249), and Uruguay (1).
HONORARY EDITORS-IN-CHIEF
James L Boyer, New Haven
Ke-Ji Chen, Beijing
Martin H Floch, New Haven
Emmet B Keeffe, Palo Alto
Geng-Tao Liu, Beijing
Lein-Ray Mo, Tainan
Eamonn M Quigley, Cork
Rafiq A Sheikh, Sacramento
Nicholas J Talley, Rochester
Ming-Lung Yu, Kaohsiung
Natalia A Osna, Omaha
Wei Tang, Tokyo
Alan BR Thomson, Edmonton
Harry HX Xia, Hanover
Jesus K Yamamoto-Furusho, Mexico
Yoshio Yamaoka, Houston
PRESIDENT AND EDITOR-INCHIEF
Lian-Sheng Ma, Beijing
GUEST EDITORIAL BOARD
MEMBERS
Chien-Jen Chen, Taipei
Yang-Yuan Chen, Changhua
Jen-Hwey Chiu, Taipei
Seng-Kee Chuah, Kaohsiung
Wan-Long Chuang, Kaohsiun
Ming-Chih Hou, Taipei
Kevin Cheng-Wen Hsiao, Taipei
Po-Shiuan Hsieh, Taipei
Tsung-Hui Hu, Kaohsiung
Wen-Hsin Huang, Taichung
Chao-Hung Hung, Kaohsiung
I-Rue Lai, Taipei
Teng-Yu Lee, Taichung
Ching Chung Lin, Taipei
Hui-Kang Liu, Taipei
Hon-Yi Shi, Kaohsiung
Chih-Chi Wang, Kaohsiung
Jin-Town Wang, Taipei
Cheng-Shyong Wu, Chia-Yi
Jaw-Ching Wu, Taipei
Jiunn-Jong Wu, Tainan
Ming-Shiang Wu, Taipei
ACADEMIC EDITOR-IN-CHIEF
Tauseef Ali, Oklahoma City
Mauro Bortolotti, Bologna
Tarkan Karakan, Ankara
Weekitt Kittisupamongkol, Bangkok
Anastasios Koulaouzidis, Edinburgh
Bo-Rong Pan, Xi’an
Sylvia LF Pender, Southampton
Max S Petrov, Auckland
George Y Wu, Farmington
STRATEGY ASSOCIATE
EDITORS-IN-CHIEF
Peter Draganov, Florida
Hugh J Freeman, Vancouver
Maria C Gutiérrez-Ruiz, Mexico
Kazuhiro Hanazaki, Kochi
Akio Inui, Kagoshima
Kalpesh Jani, Baroda
Javier S Martin, Punta del Este
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ASSOCIATE EDITORS-IN-CHIEF
You-Yong Lu, Beijing
John M Luk, Singapore
Hiroshi Shimada, Yokohama
Ta-Sen Yeh, Taoyuan
Hsu-Heng Yen, Changhua
Ming-Whei Yu, Taipei
MEMBERS OF THE EDITORIAL
BOARD
Albania
Bashkim Resuli, Tirana
Argentina
Julio H Carri, Córdoba
Eduardo de Santibañes, Buenos Aires
Bernardo Frider, Buenos Aires
Carlos J Pirola, Buenos Aires
Bernabe Matias Quesada, Buenos Aires
Silvia Sookoian, Buenos Aires
Adriana M Torres, Rosario
Maria Ines Vaccaro, Buenos Aires
Australia
Leon Anton Adams, Nedlands
Richard Anderson, Victoria
Minoti V Apte, New South Wales
Andrew V Biankin, Sydney
Filip Braet, Sydney
Christopher Christophi, Melbourne
Philip G Dinning, Koagarah
Guy D Eslick, Sydney
Michael A Fink, Melbourne
January 7, 2011
Robert JL Fraser, Daw Park
Jacob George, Westmead
Mark D Gorrell, Sydney
Alexander G Heriot, Melbourne
Michael Horowitz, Adelaide
John E Kellow, Sydney
William Kemp, Melbourne
Finlay A Macrae, Victoria
Daniel Markovich, Brisbane
Vance Matthews, Melbourne
Phillip S Oates, Perth
Shan Rajendra, Tasmania
Rajvinder Singh, Elizabeth Vale
Ross C Smith, Sydney
Kevin J Spring, Brisbane
Nathan Subramaniam, Brisbane
Phil Sutton, Melbourne
Cuong D Tran, North Adelaide
Debbie Trinder, Fremantle
David Ian Watson, Bedford Park
Austria
Herwig R Cerwenka, Graz
Ashraf Dahaba, Graz
Peter Ferenci, Vienna
Valentin Fuhrmann, Vienna
Alfred Gangl, Vienna
Alexander M Hirschl, Wien
Kurt Lenz, Linz
Dietmar Öfner, Salzburg
Markus Peck-Radosavljevic, Vienna
Markus Raderer, Vienna
Stefan Riss, Vienna
Georg Roth, Vienna
Michael Trauner, Graz
Thomas Wild, Kapellerfeld
Belgium
Rudi Beyaert, Gent
Benedicte Y De Winter, Antwerp
Inge I Depoortere, Leuven
Olivier Detry, Liège
Philip Meuleman, Ghent
Marc Peeters, De Pintelaan
Freddy Penninckx, Leuven
Jean-Yves L Reginster, Liège
Mark De Ridder, Brussels
Etienne M Sokal, Brussels
Kristin Verbeke, Leuven
Eddie Wisse, Keerbergen
Brazil
José LF Caboclo, São José do Rio Preto
Roberto J Carvalho-Filho, São Paulo
Jaime Natan Eisig, São Paulo
Andre Castro Lyra, Salvador
Marcelo Lima Ribeiro, Braganca Paulista
Joao Batista Teixeira Rocha, Santa Maria
Heitor Rosa, Goiania
Damiao C Moraes Santos, Rio de Janeiro
Ana Cristina Simões e Silva, Belo Horizonte
Eduardo Garcia Vilela, Belo Horizonte
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Brunei Darussalam
Vui Heng Chong, Bandar Seri Begawan
Bulgaria
Zahariy Krastev, Sofia
Mihaela Petrova, Sofia
Canada
Alain Bitton, Montreal
Michael F Byrne, Vancouver
Kris Chadee, Calgary
Wangxue Chen, Ottawa
Ram Prakash Galwa, Ottawa
Philip H Gordon, Montreal
Waliul Khan, Ontario
Qiang Liu, Saskatoon
John K Marshall, Ontario
Andrew L Mason, Alberta
Kostas Pantopoulos, Quebec
Nathalie Perreault, Sherbrooke
Baljinder Singh Salh, Vancouver
Eldon Shaffer, Calgary
Martin Storr, Calgary
Pingchang Yang, Hamilton
Eric M Yoshida, Vancouver
Claudia Zwingmann, Montreal
Chile
Marcelo A Beltran, La Serena
Xabier De Aretxabala, Santiago
Silvana Zanlungo, Santiago
China
Hui-Jie Bian, Xi’an
San-Jun Cai, Shanghai
Guang-Wen Cao, Shanghai
Xiao-Ping Chen, Wuhan
Chi-Hin Cho, Hong Kong
Zong-Jie Cui, Beijing
Jing-Yuan Fang, Shanghai
De-Liang Fu, Shanghai
Ze-Guang Han, Shanghai
Chun-Yi Hao, Beijing
Ming-Liang He, Hong Kong
Ching-Lung Lai, Hong Kong
Simon Law, Hong Kong
Yuk-Tong Lee, Hong Kong
En-Min Li, Shantou
Fei Li, Beijing
Yu-Yuan Li, Guangzhou
Zhao-Shen Li, Shanghai
Xing-Hua Lu, Beijing
Yi-Min Mao, Shanghai
Qin Su, Beijing
Paul Kwong-Hang Tam, Hong Kong
Yuk Him Tam, Hong Kong
Ren-Xiang Tan, Nanjing
Wei-Dong Tong, Chongqing
Eric WC Tse, Hong Kong
II
Fu-Sheng Wang, Beijing
Xiang-Dong Wang, Shanghai
Nathalie Wong, Hong Kong
Justin CY Wu, Hong Kong
Wen-Rong Xu, Zhenjiang
An-Gang Yang, Xi’an
Wei-Cheng You, Beijing
Chun-Qing Zhang, Jinan
Jian-Zhong Zhang, Beijing
Xiao-Peng Zhang, Beijing
Xuan Zhang, Beijing
Colombia
Germán Campuzano-Maya, Medellín
Croatia
Tamara Cacev, Zagreb
Marko Duvnjak, Zagreb
Cuba
Damian C Rodriguez, Havana
Czech
Jan Bures, Hradec Kralove
Milan Jirsa, Praha
Marcela Kopacova, Hradec Kralove
Pavel Trunečka, Prague
Denmark
Leif Percival Andersen, Copenhagen
Asbjørn M Drewes, Aalborg
Morten Frisch, Copenhagen
Jan Mollenhauer, Odense
Morten Hylander Møller, Holte
Søren Rafaelsen, Vejle
Jorgen Rask-Madsen, Skodsborg
Peer Wille-Jørgensen, Copenhagen
Ecuador
Fernando E Sempértegui, Quito
Egypt
Zeinab Nabil Ahmed, Cairo
Hussein M Atta, El-Minia
Estonia
Riina Salupere, Tartu
Tamara Vorobjova, Tartu
Finland
Saila Kauhanen, Turku
January 7, 2011
Thomas Kietzmann, Oulu
Kaija-Leena Kolho, Helsinki
Jukka-Pekka Mecklin, Jyvaskyla
Minna Nyström, Helsinki
Pauli Antero Puolakkainen, Turku
Juhani Sand, Tampere
Lea Veijola, Helsinki
France
Claire Bonithon-Kopp, Dijon
Lionel Bueno, Toulouse
Sabine Colnot, Paris
Catherine Daniel, Lille Cedex
Alexis Desmoulière, Limoges
Thabut Dominique, Paris
Francoise L Fabiani, Angers
Jean-Luc Faucheron, Grenoble
Jean Paul Galmiche, Nantes cedex
Boris Guiu, Dijon
Paul Hofman, Nice
Laurent Huwart, Paris
Juan Iovanna, Marseille
Abdel-Majid Khatib, Paris
Philippe Lehours, Bordeaux
Flavio Maina,Marseille
Patrick Marcellin, Paris
Rene Gerolami Santandera, Marseille
Annie Schmid-Alliana, Nice cedex
Alain L Servin, Châtenay-Malabry
Stephane Supiot, Nantes
Baumert F Thomas, Strasbourg
Jean-Jacques Tuech, Rouen
Frank Zerbib, Bordeaux Cedex
Germany
Erwin Biecker, Siegburg
Hubert Blum, Freiburg
Thomas Bock, Tuebingen
Dean Bogoevski, Hamburg
Elfriede Bollschweiler, Köln
Jürgen Borlak, Hannover
Christa Buechler, Regensburg
Jürgen Büning, Lübeck
Elke Cario, Essen
Bruno Christ, Halle/Saale
Christoph F Dietrich, Bad Mergentheim
Ulrich R Fölsch, Kiel
Nikolaus Gassler, Aachen
Markus Gerhard, Munich
Dieter Glebe, Giessen
Ralph Graeser, Freiburg
Axel M Gressner, Aachen
Nils Habbe, Marburg
Thilo Hackert, Heidelberg
Wolfgang Hagmann, Heidelberg
Dirk Haller, Freising
Philip D Hard, Giessen
Claus Hellerbrand, Regensburg
Klaus R Herrlinger, Stuttgart
Eberhard Hildt, Berlin
Andrea Hille, Goettingen
Joerg C Hoffmann, Berlin
Philipe N Khalil, Munich
Andrej Khandoga, Munich
Jorg Kleeff, Munich
Ingmar Königsrainer, Tübingen
Peter Konturek, Erlangen
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Stefan Kubicka, Hannover
Joachim Labenz, Siegen
Michael Linnebacher, Rostock
Jutta Elisabeth Lüttges, Riegelsberg
Peter Malfertheiner, Magdeburg
Oliver Mann, Hamburg
Peter N Meier, Hannover
Sabine Mihm, Göttingen
Klaus Mönkemüller, Bottrop
Jonas Mudter, Erlangen
Sebastian Mueller, Heidelberg
Robert Obermaier, Freiburg
Matthias Ocker, Erlangen
Stephan Johannes Ott, Kiel
Gustav Paumgartner, Munich
Christoph Reichel, Bad Brückenau
Markus Reiser, Bochum
Steffen Rickes, Magdeburg
Elke Roeb, Giessen
Christian Rust, Munich
Hans Scherubl, Berlin
Martin K Schilling, Homburg
Joerg F Schlaak, Essen
Rene Schmidt, Freiburg
Andreas G Schreyer, Regensburg
Karsten Schulmann, Bochum
Henning Schulze-Bergkamen, Mainz
Manfred V Singer, Mannheim
Jens Standop, Bonn
Jurgen M Stein, Frankfurt
Ulrike S Stein, Berlin
Wolfgang R Stremmel, Heidelberg
Harald F Teutsch, Ulm
Hans L Tillmann, Leipzig
Christian Trautwein, Aachen
Joerg Trojan, Frankfurt
Arndt Vogel, Hannover
Siegfried Wagner, Deggendorf
Frank Ulrich Weiss, Greifswald
Fritz von Weizsäcker, Berlin
Thomas Wex, Magdeburg
Stefan Wirth, Wuppertal
Marty Zdichavsky, Tübingen
Yvette Mándi, Szeged
Zoltan Rakonczay, Szeged
Ferenc Sipos, Budapest
Zsuzsa Szondy, Debrecen
Gabor Veres, Budapest
India
Philip Abraham, Mumbai
Vineet Ahuja, New Delhi
Giriraj Ratan Chandak, Hyderabad
Devinder Kumar Dhawan, Chandigarh
Radha K Dhiman, Chandigarh
Pankaj Garg, Panchkula
Pramod Kumar Garg, New Delhi
Debidas Ghosh, Midnpore
Uday C Ghoshal, Lucknow
Bhupendra Kumar Jain, Delhi
Ashok Kumar, Lucknow
Bikash Medhi, Chandigarh
Sri P Misra, Allahabad
Gopal Nath, Varanasi
Samiran Nundy, New Delhi
Jagannath Palepu, Mumbai
Vandana Panda, Mumbai
Benjamin Perakath, Tamil Nadu
Ramesh Roop Rai, Jaipur
Nageshwar D Reddy, Hyderabad
Barjesh Chander Sharma, New Delhi
Virendra Singh, Chandigarh
Rupjyoti Talukdar, Guwahati
Rakesh Kumar Tandon, New Delhi
Jai Dev Wig, Chandigarh
Iran
Mohammad Abdollahi, Tehran
Peyman Adibi, Isfahan
Seyed-Moayed Alavian, Tehran
Seyed Mohsen Dehghani, Shiraz
Reza Malekzadeh, Tehran
Alireza Mani, Tehran
Greece
Helen Christopoulou-Aletra, Thessaloniki
T Choli-Papadopoulou, Thessaloniki
Tsianos Epameinondas, Ioannina
Ioannis Kanellos, Thessaloniki
Elias A Kouroumalis, Heraklion
Ioannis E Koutroubakis, Heraklion
Michael Koutsilieris, Athens
Andreas Larentzakis, Athens
Emanuel K Manesis, Athens
Spilios Manolakopoulos, Athens
Konstantinos Mimidis, Alexandroupolis
George Papatheodoridis, Athens
Spiros Sgouros, Athens
Evangelos Tsiambas, Ag Paraskevi Attiki
Hungary
György M Buzás, Budapest
László Czakó, Szeged
Gyula Farkas, Szeged
Peter Hegyi, Szeged
Peter L Lakatos, Budapest
III
Ireland
Billy Bourke, Dublin
Ted Dinan, Cork
Catherine Greene, Dublin
Ross McManus, Dublin
Anthony P Moran, Galway
Marion Rowland, Dublin
Israel
Simon Bar-Meir, Hashomer
Alexander Becker, Afula
Abraham R Eliakim, Haifa
Sigal Fishman, Tel Aviv
Boris Kirshtein, Beer Sheva
Eli Magen, Ashdod
Menachem Moshkowitz, Tel-Aviv
Assy Nimer, Safed
Shmuel Odes, Beer Sheva
Mark Pines, Bet Dagan
Ron Shaoul, Haifa
Ami D Sperber, Beer-Sheva
January 7, 2011
Italy
Donato F Altomare, Bari
Piero Amodio, Padova
Angelo Andriulli, San Giovanni Rotondo
Paolo Angeli, Padova
Bruno Annibale, Rome
Paolo Aurello, Rome
Salvatore Auricchio, Naples
Antonio Basoli, Rome
Claudio Bassi, Verona
Gabrio Bassotti, Perugia
Mauro Bernardi, Bologna
Alberto Biondi, Rome
Luigi Bonavina, Milano
Guglielmo Borgia, Naples
Roberto Berni Canani, Naples
Maria Gabriella Caruso, Bari
Fausto Catena, Bologna
Giuseppe Chiarioni, Valeggio
Michele Cicala, Rome
Dario Conte, Milano
Francesco Costa, Pisa
Antonio Craxì, Palermo
Salvatore Cucchiara, Rome
Giuseppe Currò, Messina
Mario M D’Elios, Florence
Mirko D’Onofrio, Verona
Silvio Danese, Milano
Roberto de Franchis, Milano
Paola De Nardi, Milan
Giovanni D De Palma, Naples
Giuliana Decorti, Trieste
Gianlorenzo Dionigi, Varese
Massimo Falconi, Verona
Silvia Fargion, Milan
Giammarco Fava, Ancona
Francesco Feo, Sassari
Alessandra Ferlini, Ferrara
Alessandro Ferrero, Torino
Mirella Fraquelli, Milan
Luca Frulloni, Verona
Giovanni B Gaeta, Napoli
Antonio Gasbarrini, Rome
Edoardo G Giannini, Genoa
Alessandro Granito, Bologna
Fabio Grizzi, Milan
Salvatore Gruttadauria, Palermo
Pietro Invernizzi, Milan
Achille Iolascon, Naples
Angelo A Izzo, Naples
Ezio Laconi, Cagliari
Giovanni Latella, L’Aquila
Massimo Levrero, Rome
Francesco Luzza, Catanzaro
Lucia Malaguarnera, Catania
Francesco Manguso, Napoli
Pier Mannuccio Mannucci, Milan
Giancarlo Mansueto, Verona
Giulio Marchesini, Bologna
Mara Massimi, Coppito
Giovanni Milito, Rome
Giuseppe Montalto, Palermo
Giovanni Monteleone, Rome
Luca Morelli, Trento
Giovanni Musso, Torino
Mario Nano, Torino
Gerardo Nardone, Napoli
Riccardo Nascimbeni, Brescia
Valerio Nobili, Rome
Fabio Pace, Milan
Nadia Peparini, Rome
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Marcello Persico, Naples
Mario Pescatori, Rome
Raffaele Pezzilli, Bologna
Alberto Piperno, Monza
Anna C Piscaglia, Rome
Piero Portincasa, Bari
Michele Reni, Milan
Vittorio Ricci, Pavia
Oliviero Riggio, Rome
Mario Rizzetto, Torino
Ballarin Roberto, Modena
Gerardo Rosati, Potenza
Franco Roviello, Siena
Cesare Ruffolo, Treviso
Massimo Rugge, Padova
Marco Scarpa, Padova
C armelo Scarpignato, Parma
Giuseppe Sica, Rome
Marco Silano, Rome
Pierpaolo Sileri, Rome
Vincenzo Stanghellini, Bologna
Fiorucci Stefano, Perugia
Giovanni Tarantino, Naples
Alberto Tommasini, Trieste
Guido Torzilli, Rozzano Milan
Cesare Tosetti, Porretta Terme
Antonello Trecca, Rome
Vincenzo Villanacci, Brescia
Lucia Ricci Vitiani, Rome
Marco Vivarelli, Bologna
Japan
Kyoichi Adachi, Izumo
Yasushi Adachi, Sapporo
Takafumi Ando, Nagoya
Akira Andoh, Otsu
Masahiro Arai, Tokyo
Hitoshi Asakura, Tokyo
Kazuo Chijiiwa, Miyazaki
Yuichiro Eguchi, Saga
Itaru Endo, Yokohama
Munechika Enjoji, Fukuoka
Yasuhiro Fujino, Akashi
Mitsuhiro Fujishiro, Tokyo
Kouhei Fukushima, Sendai
Masanori Hatakeyama, Tokyo
Keiji Hirata, Kitakyushu
Toru Hiyama, Higashihiroshima
Masahiro Iizuka, Akita
Susumu Ikehara, Osaka
Kenichi Ikejima, Bunkyo-ku
Yutaka Inagaki, Kanagawa
Hiromi Ishibashi, Nagasaki
Shunji Ishihara, Izumo
Toru Ishikawa, Niigata
Toshiyuki Ishiwata, Tokyo
Hajime Isomoto, Nagasaki
Yoshiaki Iwasaki, Okayama
Satoru Kakizaki, Gunma
Terumi Kamisawa, Tokyo
Mototsugu Kato, Sapporo
Naoya Kato, Tokyo
Takumi Kawaguchi, Kurume
Yohei Kida, Kainan
Shogo Kikuchi, Aichi
Tsuneo Kitamura, Chiba
Takashi Kobayashi, Tokyo
Yasuhiro Koga, Isehara
Takashi Kojima, Sapporo
Norihiro Kokudo, Tokyo
Masatoshi Kudo, Osaka
Shin Maeda, Tokyo
IV
Satoshi Mamori, Hyogo
Atsushi Masamune, Sendai
Yasushi Matsuzaki, Tsukuba
Kenji Miki, Tokyo
Toshihiro Mitaka, Sapporo
Hiroto Miwa, Hyogo
Kotaro Miyake, Tokushima
Manabu Morimoto, Yokohama
Yoshiharu Motoo, Kanazawa
Yoshiaki Murakami, Hiroshima
Yoshiki Murakami, Kyoto
Kunihiko Murase, Tusima
Akihito Nagahara, Tokyo
Yuji Naito, Kyoto
Atsushi Nakajima, Yokohama
Hisato Nakajima, Tokyo
Hiroki Nakamura, Yamaguchi
Shotaro Nakamura, Fukuoka
Akimasa Nakao, Nagogya
Shuhei Nishiguchi, Hyogo
Mikio Nishioka, Niihama
Keiji Ogura, Tokyo
Susumu Ohmada, Maebashi
Hirohide Ohnishi, Akita
Kenji Okajima, Nagoya
Kazuichi Okazaki, Osaka
Morikazu Onji, Ehime
Satoshi Osawa, Hamamatsu
Hidetsugu Saito, Tokyo
Yutaka Saito, Tokyo
Naoaki Sakata, Sendai
Yasushi Sano, Chiba
Tokihiko Sawada, Tochigi
Tomohiko Shimatan, Hiroshima
Yukihiro Shimizu, Kyoto
Shinji Shimoda, Fukuoka
Yoshio Shirai, Niigata
Masayuki Sho, Nara
Shoichiro Sumi, Kyoto
Hidekazu Suzuki, Tokyo
Masahiro Tajika, Nagoya
Yoshihisa Takahashi, Tokyo
Toshinari Takamura, Kanazawa
Hiroaki Takeuchi, Kochi
Yoshitaka Takuma, Okayama
Akihiro Tamori, Osaka
Atsushi Tanaka, Tokyo
Shinji Tanaka, Hiroshima
Satoshi Tanno, Hokkaido
Shinji Togo, Yokohama
Hitoshi Tsuda, Tokyo
Hiroyuki Uehara, Osaka
Masahito Uemura, Kashihara
Yoshiyuki Ueno, Sendai
Mitsuyoshi Urashima, Tokyo
Takuya Watanabe, Niigata
Satoshi Yamagiwa, Niigata
Taketo Yamaguchi, Chiba
Mitsunori Yamakawa, Yamagata
Takayuki Yamamoto, Yokkaichi
Yutaka Yata, Maebashi
Hiroshi Yoshida, Tokyo
Norimasa Yoshida, Kyoto
Yuichi Yoshida, Osaka
Kentaro Yoshika, Toyoake
Hitoshi Yoshiji, Nara
Katsutoshi Yoshizato, Higashihiroshima
Tomoharu Yoshizumi, Fukuoka
Jordan
Ismail Matalka, Irbid
January 7, 2011
Robert Christiaan Verdonk, Groningen
Erwin G Zoetendal, Wageningen
Kuwait
Serbia
Islam Khan, Safat
New Zealand
Andrew S Day, Christchurch
Tamara M Alempijevic, Belgrade
Dusan M Jovanovic, Sremska Kamenica
Zoran Krivokapic, Belgrade
Lebanon
Bassam N Abboud, Beirut
Ala I Sharara, Beirut
Rita Slim, Beirut
Lithuania
Giedrius Barauskas, Kaunas
Limas Kupcinskas, Kaunas
Malaysia
Andrew Seng Boon Chua, Ipoh
Mexico
Richard A Awad, Mexico
Aldo Torre Delgadillo, Mexico
Diego Garcia-Compean, Monterrey
Paulino M Hernández Magro, Celaya
Miguel Angel Mercado, Distrito Federal
Arturo Panduro, Jalisco
Omar Vergara-Fernandez, Tlalpan
Saúl Villa-Trevio, Mexico
Moldova
Igor Mishin, Kishinev
Netherlands
Ulrich Beuers, Amsterdam
Lee Bouwman, Leiden
Albert J Bredenoord, Nieuwegein
Lodewijk AA Brosens, Utrecht
J Bart A Crusius, Amsterdam
Wouter de Herder, Rotterdam
Pieter JF de Jonge, Rotterdam
Robert J de Knegt, Rotterdam
Wendy W Johanna de Leng, Utrecht
Annemarie de Vries, Rotterdam
James CH Hardwick, Leiden
Frank Hoentjen, Haarlem
Misha Luyer, Sittard
Jeroen Maljaars, Maastricht
Gerrit A Meijer, Amsterdam
Servaas Morré, Amsterdam
Chris JJ Mulder, Amsterdam
John Plukker, Groningen
Albert Frederik Pull ter Gunne, Tilburg
Paul E Sijens, Groningen
BW Marcel Spanier, Arnhem
Shiri Sverdlov, Maastricht
Maarten Tushuizen, Amsterdam
Jantine van Baal, Heidelberglaan
Astrid van der Velde, The Hague
Karel van Erpecum, Utrecht
Loes van Keimpema, Nijmegen
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Norway
Olav Dalgard, Oslo
Trond Peder Flaten, Trondheim
Reidar Fossmark, Trondheim
Rasmus Goll, Tromso
Ole Høie, Arendal
Asle W Medhus, Oslo
Espen Melum, Oslo
Trine Olsen, Tromso
Eyvind J Paulssen, Tromso
Jon Arne Søreide, Stavanger
Kjetil Soreide, Stavanger
Singapore
Madhav Bhatia, Singapore
Kong Weng Eu, Singapore
Brian Kim Poh Goh, Singapore
Khek-Yu Ho, Singapore
Kok Sun Ho, Singapore
Fock Kwong Ming, Singapore
London Lucien Ooi, Singapore
Nagarajan Perumal, Singapore
Francis Seow-Choen, Singapore
South Africa
Pakistan
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Michael A Zimmerman, Colorado
January 7, 2011
S
Contents
EDITORIAL
Weekly Volume 17 Number 16 April 28, 2011
2063
Targeting the cell cycle in esophageal adenocarcinoma: An adjunct to
anticancer treatment
Dibb M, Ang YS
2070
Optimizing management in autoimmune hepatitis with liver failure at initial
presentation
Potts JR, Verma S
TOPIC HIGHLIGHT
2076
A practical approach to the diagnosis of autoimmune pancreatitis
Frulloni L, Amodio A, Katsotourchi AM, Vantini I
2080
Endoscopic ultrasonography findings in autoimmune pancreatitis
Buscarini E, De Lisi S, Arcidiacono PG, Petrone MC, Fuini A, Conigliaro R, Manfredi G,
Manta R, Reggio D, De Angelis C
ORIGINAL ARTICLE
2086
Effects of α-mangostin on apoptosis induction of human colon cancer
Watanapokasin R, Jarinthanan F, Nakamura Y, Sawasjirakij N, Jaratrungtawee A,
Suksamrarn S
2096
Chemometrics of differentially expressed proteins from colorectal cancer
patients
Yeoh LC, Dharmaraj S, Gooi BH, Singh M, Gam LH
BRIEF ARTICLE
2104
Dietary treatment of colic caused by excess gas in infants: Biochemical
evidence
Infante D, Segarra O, Luyer BL
2109
Levels of matrix metalloproteinase-1 and tissue inhibitors of
metalloproteinase-1 in gastric cancer
Kemik O, Kemik AS, Sümer A, Dulger AC, Adas M, Begenik H, Hasirci I, Yilmaz O,
Purisa S, Kisli E, Tuzun S, Kotan C
WJG|www.wjgnet.com
April 28, 2011|Volume 17|Issue 16|
Contents
BRIEF ARTICLE
Volume 17 Number 16 April 28, 2011
2113
Sunitinib for Taiwanese patients with gastrointestinal stromal tumor after
imatinib treatment failure or intolerance
Chen YY, Yeh CN, Cheng CT, Chen TW, Rau KM, Jan YY, Chen MF
2120
MELD score can predict early mortality in patients with rebleeding after band
ligation for variceal bleeding
Chen WT, Lin CY, Sheen IS, Huang CW, Lin TN, Lin CJ, Jeng WJ, Huang CH, Ho YP,
Chiu CT
2126
Study on chronic pancreatitis and pancreatic cancer using MRS and pancreatic
juice samples
Wang J, Ma C, Liao Z, Tian B, Lu JP
2131
Ku80 gene G-1401T promoter polymorphism and risk of gastric cancer
Li JQ, Chen J, Liu NN, Yang L, Zeng Y, Wang B, Wang XR
2137
Effects of penehyclidine hydrochloride on rat intestinal barrier function during
cardiopulmonary bypass
Sun YJ, Cao HJ, Jin Q, Diao YG, Zhang TZ
2143
p53 gene therapy in combination with transcatheter arterial
chemoembolization for HCC: One-year follow-up
Guan YS, Liu Y, He Q, Li X, Yang L, Hu Y, La Z
CASE REPORT
2150
Celiac disease and microscopic colitis: A report of 4 cases
Barta Z, Zold E, Nagy A, Zeher M, Csipo I
2155
Pure red cell aplasia caused by pegylated interferon-α-2a plus ribavirin in the
treatment of chronic hepatitis C
Chang CS, Yan SL, Lin HY, Yu FL, Tsai CY
LETTERS TO THE EDITOR 2159
Enucleation for gastrointestinal stromal tumors at the esophagogastric
junction: Is this an adequate solution?
Peparini N, Carbotta G, Chirletti P
WJG|www.wjgnet.com
II
Contents
Volume 17 Number 16 April 28, 2011
ACKNOWLEDGMENTS
I
Acknowledgments to reviewers of World Journal of Gastroenterology
APPENDIX
I
Meetings
I-VI
Instructions to authors
ABOUT COVER
Watanapokasin R, Jarinthanan F, Nakamura Y, Sawasjirakij N, Jaratrungtawee
A, Suksamrarn S. Effects of α-mangostin on apoptosis induction of human colon
cancer.
World J Gastroenterol 2011; 17(16): 2086-2095
http://www.wjgnet.com/1007-9327/full/v17/i16/2086.htm
AIM AND SCOPE
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The major task of WJG is to report rapidly the most recent results in basic and clinical
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gastrointestinal bleeding, infection and tumors; gastric and duodenal disorders; intestinal
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III
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doi:10.3748/wjg.v17.i16.2063
ISSN 1007-9327 (print) ISSN 2219-2840 (online)
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EDITORIAL
Targeting the cell cycle in esophageal adenocarcinoma: An
adjunct to anticancer treatment
Martyn Dibb, Yeng S Ang
Key words: Esophageal adenocarcinoma; Cell cycle;
Cyclin-dependent kinase; Aurora kinases; Polo-like
kinase
Martyn Dibb, Department of Gastroenterology, Royal Albert Edward Infirmary, Wigan Lane, Wigan WN1 2NN, United Kingdom
Yeng S Ang, School of Translational Medicine, Faculty of
Medical and Human Sciences, The University of Manchester,
Manchester M13 9PL, United Kingdom
Author contributions: Literature review and manuscript by
Dibb M, concepts and corrections by Ang YS.
Supported by UK National Institute of Health Research/Cancer Research Network and Research and Development Department of Wrightington Wigan and Leigh NHS Foundation Trust
(to Ang YS); Wrightington Wigan and Leigh NHS Foundation
Trust Cancer Therapy Fund (to Dibb M)
Correspondence to: Yeng S Ang, MD, FRCP, FRCPI, FEBG,
Consultant Gastroenterologist/Honorary Senior Lecturer,
Royal Albert Edward Infirmary, Wigan Lane, Wigan WN1 2NN,
United Kingdom. [email protected]
Telephone: +44-1942-773119 Fax: +44-1942-822340
Received: December 2, 2010 Revised: January 11, 2011
Accepted: January 18, 2011
Published online: April 28, 2011
Peer reviewer: Luis Grande, Professor, Department of Surgery,
Hospital del Mar, Passeig Marítim 25-29, Barcelona 08003,
Spain
Dibb M, Ang YS. Targeting the cell cycle in esophageal
adenocarcinoma: An adjunct to anticancer treatment. World J
Gastroenterol 2011; 17(16): 2063-2069 Available from: URL:
http://www.wjgnet.com/1007-9327/full/v17/i16/2063.htm DOI:
http://dx.doi.org/10.3748/wjg.v17.i16.2063
INTRODUCTION
Esophageal cancer is a major cause of cancer death worldwide[1]. It was the fourth most common cause of death
from cancer in men in the United Kingdom between
2004 and 2006[2]. Although in the developed world the
incidence and mortality of cancer in general has decreased
with advances in diagnosis and treatment, the incidence
and mortality of esophageal carcinoma have increased[1].
Esophageal cancer carries a poor prognosis with a
5-year survival rate of < 10%[3]. This probably reflects
the fact that the majority of esophageal cancers present late with symptoms after invasion of the muscularis
propria and lymph node metastasis have occurred[4]. Extensive disease means that few patients are suitable for
definitive surgical therapy[4,5]. Poor outcomes from conventional therapies including surgery and radiochemotherapy have led to increasing interest in understanding
the molecular mechanisms that underpin the development of esophageal cancer. This may assist in developing new diagnostic techniques and identifying potential
therapeutic targets.
The mechanism by which cells reproduce has fascinated biologists since Virchow’s 1855 observation that
cells could only arise from pre-existing cells. By the
Abstract
Esophageal adenocarcinoma is a major cause of cancer
death in men in the developed world. Continuing poor
outcomes with conventional therapies that predominantly target apoptosis pathways have lead to increasing interest in treatments that target the cell cycle. A
large international effort has led to the development
of a large number of inhibitors, which target cell cycle
kinases, including cyclin-dependent kinases, Aurora kinases and polo-like kinase. Initial phase Ⅰ/Ⅱ trials in
solid tumors have often demonstrated only modest clinical benefits of monotherapy. This may relate in part to
a failure to identify the patient populations that will gain
the most clinical benefit. Newer compounds lacking the
side effect profile of first-generation compounds may
show utility as adjunctive treatments targeted to an individual’s predicted response to treatment.
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2063
Dibb M et al . Esophageal adenocarcinoma and cell cycle
early 20th century, pathologists had recorded extensive
descriptions of the cytological events of cell division,
including division of the nucleus and partitioning of
the cytoplasm to the formation of two daughter cells[6].
It has become increasingly clear since those early descriptions of the normal cell cycle that disorders in this
process can lead to disease. It was not however until the
1970s, that molecular biology allowed a deeper understanding of the cell cycle and its role in health, disease
and cancer development. The past three decades, in
particular, have seen major advances in our understanding of the genetic and molecular mechanisms by which
cells reproduce and how this process is regulated and
controlled. It has also been aptly described that cell cycle
deregulation, in the form of growth self-sufficiency
and insensitivity to growth inhibitory signals, have become fundamental hallmarks of cancer development[7-9].
Targeting these pathways in cancer development for
diagnostic and therapeutic use has become increasingly
important. We assess in this review the potential for targeting the cell cycle to treat esophageal adenocarcinoma.
PLK1
AURORA A
••
G2/M checkpoint
•
CDK1
Legend
DNA synthesis
Kinase
Active replication
Gap phase
Cyclin/CD complex
Main action
Checkpoint
•
G1/S
checkpoint
•
Figure 1 Cell cycle.
Bl-2536
kinase 1
like
Polo
BUB-1
AZD1152
MP
S-1
ra
ro
Au
Danusertib
It is clear that cellular reproduction is carefully controlled and regulated to prevent uncontrolled proliferation of cells[10]. A number of alterations in cell physiology are required to lead to carcinogenesis[7]. First, a cell
must become able to move from its dormant inactive
state (known as quiescence) to enter the cell cycle without stimulation from external growth factors. Second,
the cell must lose response to growth-inhibitory signals.
Cells must evade senescence and programmed cell death
to gain limitless replicative potential. Finally, it must be
able to develop and maintain an adequate blood supply
(angiogenesis), which allows the cancer cell to invade
and metastasize throughout the organism[11].
Many genes responsible for the carcinogenesis have
been identified. Broadly, they fall into two categories:
oncogenes and tumor suppressor genes. Oncogenes are
created by mutations in genes that cause them to become constitutively active, whereas in tumor suppressor
genes, mutations reduce or inactive the gene product[12].
Oncogenes and tumor suppressor genes increase tumor
cell number by stimulation of cell division or prevention
of cell death.
CDK1
Cylin
B
Au
ro
ra
A
B
Drug
Active replication
Gap phase
Cell cycle target
Inhibition
Flavopiridol
C
CDK4/6 ylin D
Legend
HALLMARKS OF CANCER
Figure 2 Compounds targeting the cell cycle.
Events in the cell cycle happen in a temporally organized sequence, with later events depending on successful completion of earlier events[14].
Control of the cell cycle is driven by the cyclin-dependent kinases (CDKs), a family of serine/threonine kinases. Cells cannot enter S phase, without CDK activation. In
order to become catalytically active, CDKs need to bind
to a cyclin subunit that acts as an activator. CDKs can also
be modulated by inhibitors such as CDK inhibitor 1A
(p21CIP1), CDK inhibitor 1B (p27KIP1) or CDK inhibitor
2B (p15INK4B)[10]. It has previously been thought that mammalian cells require the sequential activation of a number
of the CDKs to complete the cell cycle successfully[15].
Recent evidence from mouse models has suggested that
CDK1 alone is sufficient to complete the cell cycle, although other CDKs are required for normal development
and cell type specialization[16]. Cell cycle defects can contribute to esophageal cancer development in a number of
different ways (Figure 2).
Mitosis itself contains a series of phases that lead to
chromosome separation and cell division. Mitosis is a
vital step in the cell cycle, which involves carefully regulated interactions between multiple proteins. Abnormalities throughout the cell cycle can lead to genomic instability through unrestrained proliferation or defects in the
transmission of genetic information to daughter cells. A
number of established chemotherapy agents, including
CELL CYCLE
Embyronic cells can undergo DNA replication and
nuclear division at rapid rates. A full cycle of embyronic
cell division can last just 30 min[13]. Division of adult stem
cells requires more complex control (Figure 1). Gaps or
pauses are inserted between the phases of nuclear division
(M phase) and DNA synthesis (S phase). These gaps are
known as G1 (between M and S phases) and G2 (between
S and M phases).
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C
CDK4/6 ylin D
Cylin
B
Cell division
2064
at T210[25]. Phosphorylated PLK1 then activates CDC25
phosphatases that remove inhibitory phosphates from the
ATP-binding site located at Thr14 and Tyr15 in human
CDK1. This causes the activation of the CDK1/cyclin
B complex and drives the cell into mitosis[26]. PLK1 also
increases phosphorylation-dependent cyclin B import to
the nucleus[27]. PLK1 phosphorylates WEE1 and MYT1,
which leads to ubiquitination and degradation of WEE1
and inhibition of MYT1[28,29]. PLK1 is then inactivated
and degraded during anaphase by ubiquitin-dependent
degradation mediated by the anaphase promoting complex[30]. Cell cultures show severely impaired growth when
PLK1 is either overexpressed or functionally depleted[31,32].
the vinca alkaloids and the taxanes work by targeting the
mitotic phase of the cell cycle.
CELL CYCLE CHECKPOINTS
Cells need mechanisms that prevent progression of the
cell cycle if there is significant genomic damage, until
the damage is repaired or the cell undergoes apoptosis.
These have become known as cell cycle checkpoints.
There are two major checkpoints: the G1/S checkpoint
and the G2/M checkpoint. Checkpoint kinases ATM
and ATR mediate these checkpoints, through effector
kinases such as CHK1 and CHK2, by preventing activation of CDKs and progression through the cell cycle[17].
Double-stranded DNA breaks activate preferentially
ATM, whereas UV light activates ATR kinase. Defects
in this DNA damage response can contribute to cancer
formation by allowing tumor cell survival despite genome instability and enhanced mutation rates[18,19]. The
DNA damage response is commonly activated in early
neoplastic lesions[20,21].
CELL CYCLE AS A TARGET FOR CANCER
THERAPEUTICS
Many oncogenes and tumor suppressors have downstream
effects on cellular functions involving cell cycle entry and
exit. Healthy or normal cells have the ability to stop at predetermined checkpoints in the cell cycle in the presence of
damage or unfavorable conditions. Cancer cells develop
mechanisms that eliminate these checkpoints, which leads
to uncontrolled proliferation. One example of this is the
INK4 family member p16. This occurs as a result of epigenetic silencing by DNA hypermethylation at the p16
promoter, which leads to reduced transcription and loss of
gene expression. p16 is a CDK inhibitor and loss of p16
function leads to unrestrained cellular proliferation. This has
been demonstrated to occur with a number of different tumors[33]. Abnormalities of p16 function have been described
in Barrett’s esophagus and esophageal adenocarcinoma[34].
DNA hypermethylation of the p16 promoter has also been
shown to be a strong predictor of the progression to highgrade dysplasia and esophageal adenocarcinoma[35].
G1/S checkpoint
The G1/S checkpoint occurs towards the end of the G1
phase, prior to entry into G2. During G1, the cell remains
responsive to external mitogenic and anti-mitogenic
stimuli. These can either cause the cell to become quiescent (entering the GO phase) or allow re-entry to the cell
cycle. This decision is controlled by the pocket protein
RB. Immediately after mitosis, RB is dephosphorylated by
protein phosphatase type 1. Whilst in this dephosphorylated state, RB binds to a group of transcription factors
called E2Fs and inhibits their activity. During G1, RB is
hypophosphorylated by the complex of CDK4 and cyclin
D. CDK2 and cyclin E complexes then act to hyperphosphorylate RB, which causes dissociation from E2Fs. Free
E2Fs trigger increased transcription of CDK2 and cyclin
E, which creates a positive feedback loop that drives the
cell into DNA synthesis (S phase). CDC25 phosphatases
act to regulate CDK and cyclin complexes by removing
inhibitory phosphate groups thereby promoting cell cycle
progression[13]. In genomic damage, CHK2 activates the
p53 pathway, which stimulates production of p21CIP1 as
well as phosphorylation of CDC25A. This prevents activation of the CDK/cyclin complexes[13].
CDK inhibitors
Abnormal expression of CDKs and their partner cyclins
has been noted in esophageal cancer[36-39]. Polymorphisms
of CCND1, which encodes cyclin D1 has been shown to
be associated with an increased risk of esophageal adenocarcinoma[40]. CCND1 amplification and nuclear staining
of cyclin D1 have been shown to correlate negatively with
survival[41,42]. Abnormal activity of the CDK/cyclin complexes in esophageal adenocarcinoma has been shown to
be a marker of acquired chemoradioresistance[42,43]. The
observation that inhibition of CDKs leads to cell cycle
arrest and apoptosis has lead to the development of CDK
inhibitors as antitumor drugs. There are a number of
drugs that target these pathways. The pioneer compound
for this group is flavopiridol, a semi-synthetic inhibitory
flavonoid of CDKs. Flavopiridol prevents the phosphorylation and activation of CDK1, CDK2, CDK4 and
CDK6, which leads to reduced expression of cyclin D1,
cell cycle arrest, and induction of apoptosis[44].
In vitro, it has been demonstrated that even at nanomolar doses, flavopiridol can enhance the antitumor activity
G2/M Checkpoint
The G2/M checkpoint acts as a final check to prevent
mitosis occurring if the genome is damaged. A complex
of cyclin B and CDK1 regulates this transition. Throughout G2, the inhibitory kinases CHK1, WEE1 and MYT1
phosphorylate CDK1, which prevents its activation and
progression to mitosis. Polo-like kinase 1 (PLK1) protein
levels begin to accumulate during S phase and G2/M
phases, having been relatively low during G1[22,23]. PLK1
transcription is most abundant in cells that are in G2/
M phase[24]. In the absence of DNA damage, PLK1 is
phosphorylated by Aurora A at its phosphorylation site
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2065
of cytotoxic drugs by increasing apoptosis[45]. Phase Ⅰ and
Ⅱ studies have been undertaken with various combinations of chemotherapeutic agents with variable results.
Most promising is the combination with irinotecan and
cisplatin. A phase I trial of relapsed gastric and esophageal
cancer patients showed that eight out 14 patients achieved
a partial response[46]. Further clinical studies are awaited.
fied as a phosphopeptide-binding motif[60]. The polo box
motif is only observed in the PLK family and contains
a characteristic sequence. Drugs that target the PBD are
specific to the human family of PLKs.
PLK1 is overexpressed in a broad range of primary
gastrointestinal tumors, including gastric, colorectal and
pancreatic carcinoma[61-63]. In contrast, one study has
noted downregulation of PLK1 within tumor cells[64].
There is now increasing evidence that PLK1 expression levels have prognostic significance within different
cancers, including esophageal cancer[63]. Two separate reports of PLK1 overexpression in esophageal carcinoma
primarily relate to squamous cell carcinoma (SCC) in
the far east[63,65]. Given the high impact of environmental factors (e.g. aflatoxin) on SCC development in these
populations, it is unclear whether the findings can be directly applied to western populations. There are no data
on PLK1 expression in adenocarcinoma patients. Some
reports of other cancers have suggested that PLK1 expression is a reliable marker of metastasis[66]. PLK1 has
also been used in the context of larger arrays of genes
as a prognostic marker to predict metastasis in breast
cancers[67]. Current cancer staging systems and histological assessments often fail to predict individual outcomes
reliably but correlation of PLK1 protein and mRNA
expression levels with clinical stage has the potential to
improve clinical decision making in a number of different tumors[68].
The unique PLD of PLK1 also makes it a good candidate for the development of alternative cancer therapies. Initial efforts have focused on specific phosphorothioate antisense oligonucleotides that are able to block
protein translation[69]. Use of siRNAs, which cause depletion of PLK1, has also been considered. Whilst there
are drawbacks of siRNAs, including off-target effects
and nuclease sensitivity, these hold promise in cancers
such as bladder cancer in which they can act locally[70].
There are now a number of small molecule inhibitors
of PLK1, which act either in an ATP-competitive or
non-ATP-competitive manner[68]. The multiple actions
of PLK1 throughout the cell cycle mean that these new
agents need to be carefully assessed for specificity and
side effects. In particular, it is possible that anti-PLK1
agents have similar toxicity to other microtubule inhibitors. PLK1 inhibitors are now in early clinical testing
(phase Ⅰ and Ⅱ). Early clinical experience suggests that
neutropenia and thrombocytopenia are dose-related effects, although neuropathy has not been seen[71].
Aurora kinases inhibitors
The Aurora kinase family is an important family of serine/threonine kinases that are evolutionarily conserved
and act as mitotic regulators throughout the cell cycle.
There are three mammalian aurora kinases, Aurora A,
Aurora B, and Aurora C, which have differing roles
throughout mitosis[47]. Aurora A is required for centrosome maturation and spindle formation, in addition to its
role at the G2/M checkpoint described above. Aurora B
is required for chromosome segregation and cytokinesis.
Small molecule inhibitors of Aurora B lead to premature
mitotic exit without successful chromosome separation.
Continued inhibition of Aurora B results in large multiploid cells that eventually undergo apoptosis[48]. This
potentially has the advantage that Aurora B inhibitors
could be combined with other agents that act during
other phases of the cell cycle. Aurora C is abundant in
the testes. Its global functions are unclear, however, it has
recently been shown to have some overlap with the functions of Aurora B during mitosis[49]. Aurora kinases have
been shown to be overexpressed in a number of different
tumors. Aurora A has been shown to be overexpressed in
Barrett’s esophagus and esophageal adenocarcinoma[50,51].
Cell line models suggest that Aurora A overexpression
protects developing esophageal adenocarcinoma cells
against drug-induced apoptosis[51]. In other forms of
cancer, Aurora A expression has been shown to correlate
with chromosomal instability[52]. A number of Aurora kinase inhibitors are undergoing phase Ⅰ and Ⅱ evaluation.
Danusertib, a pan-Aurora kinase inhibitor has undergone
phase I testing in patients with advanced solid tumors.
Forty-six percent of patients treated with danuserib had
stable disease following treatment and a number of prolonged objective responses were noted[53,54]. The major
dose limiting effect of these drugs is neutropenia.
PLK1 inhibitors
PLKs form a group of prominent mitotic kinases. They
were first described in mutants that failed to undergo a
normal mitosis in Drosophilia melanogaster (polo)[55,56]. They
are highly conserved from yeast to humans. There are four
members of the polo family in mammals (PLK1-4)[57,58].
They are involved in multiple functions throughout the
cell cycle in mitosis and meiosis. PLK1 is the best characterized of the four known PLKs[58].
PLK1 is a candidate for development as a therapeutic target because it contains two functionally relevant
sites: a C-terminal regulatory region containing two polo
box domains (PBDs) and an N-terminal catalytic kinase
domain[59]. The highly conserved PBD has been identi-
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MPS1 inhibitors
Cell cycle translational research has focused on the development of inhibitors of the major kinases discussed
above. There are additional mitotic kinases that may have
relevance for inhibiting tumor growth. Inhibitors of
MPS1, a kinetochore-associated kinase that is involved in
the spindle assembly checkpoint, have been shown to arrest tumor cell proliferation in vitro[72,73]. This appears to
be mediated at least in part by impaired Aurora B func-
2066
Table 1 Compounds targeting the cell cycle under active development
Inhibitor
Main target
Sponsor
Clinical trials
BI2536
Danusertib
(Formerly PHA-739358)
MLN8237
BI6267
P276-00
PLK1 (partial inhibition of PLK2/3)
Pan-aurora kinase inhibitor
Boehringer Ingelheim
Pfizer Italia
Phase Ⅱ pancreatic cancer
Phase Ⅱ advanced solid tumors
Aurora a inhibitor
PLK1 inhibitor
Small molecule cyclin inhibitor
Millennium
Boehringer Ingelheim
Piramal Life Sciences
NMS-1286937
P1446A-05
SCH727965
Seliciclib (Roscovitine)
PLK1 selective inhibitor
CDK selective inhibitor
CDK inhibitor
CDK inhibitor
Nerviano Medical Sciences
Piramal Life Sciences
Schering–Plough
Cyclacel Pharmaceuticals
Phase Ⅰ/Ⅱ advanced solid tumours
Phase Ⅱ ovarian cancer/phase Ⅰ advanced solid tumors
Phase Ⅰ advanced malignancy/phase Ⅱ head and neck
malignancy
Phase Ⅰ advanced solid tumours
Phase Ⅰ advanced malignancy
CDK: Cyclin-dependent kinase; PLK1: Polo-like kinase 1.
tion at centromeres, which leads to impaired alignment
of chromosomes[74]. Detailed information on MPS1 in
esophageal cancer is lacking, however, MPS1 inhibition
has been demonstrated as a chemotherapy sensitization
strategy in vitro[75].
ease. Given the large number of cells involved it is likely
that some tumor cells will abrogate the inhibited pathways and escape from chemotherapy-induced apoptosis.
Targeted cell cycle therapy in esophageal cancer presents
an alternate strategy as cell cycle inhibitors affect multiple essential pathways involved in replication and DNA
damage repair. They may provide a useful adjunct in patients with late presenting esophageal tumors who have
failed standard chemotherapy regimens.
CONCLUSION
Established esophageal carcinoma chemotherapy regimes
are relatively blunt tools that predominantly target apoptosis pathways and are often associated with significant
side effects. This has led to a large international effort to
develop targeted therapy.
Current therapies that target the cell cycle have largely
disappointed with relatively modest effects seen in phase Ⅰ
/Ⅱ trials (Table 1). This may be in part related to failure
to identify the patient populations that will gain the most
clinical benefit. Few treatments are targeted towards specific pathways or personalized to the individual tumor proteome or genomic signature.
Efforts are now being made to assess gene expression
profiles from histological specimens from solid tumors such
as breast cancer in an attempt to predict response to chemotherapy[76]. Initial steps in this direction have been taken
by the UK Oesophageal Cancer Clinical and Molecular
Stratification (OCCAMS) Study Group, which has demonstrated a four-gene signature associated with poor prognosis
in esophageal adenocarcinoma, as well as a larger group
of genes associated with lymph node metastasis[77]. Efforts
have also been made to identify Barrett’s esophagus patients
who are likely to progress to adenocarcinoma, however, little
work has been undertaken on response to chemotherapy in
the esophagus[78]. Careful studies are needed in esophageal
adenocarcinoma to define patient populations that are likely
to respond well to treatment with both established and novel
chemotherapy regimes. Optimizing individual chemotherapy
regimens for patients will assume greater significance as
health economies demand most clinical benefit from limited
resources. In this setting of personalized targeted therapy,
new cell cycle treatments may hold promise as carefully selected adjuncts to existing chemotherapy regimes.
Patients with esophageal adenocarcinoma unfortunately often still present late with a large burden of dis-
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S- Editor Tian L L- Editor Kerr C
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E- Editor Ma WH
[email protected]
doi:10.3748/wjg.v17.i16.2070
EDITORIAL
Optimizing management in autoimmune hepatitis with liver
failure at initial presentation
Jonathan R Potts, Sumita Verma
understatement that multicenter prospective studies are
urgently needed to address this important clinical issue.
Jonathan R Potts, Sumita Verma, Department of Medicine,
Brighton and Sussex Medical School, Brighton, BN1 9PX, United
Kingdom
Author contributions: Verma S conceived the idea; Potts JR
performed the literature search; Verma S wrote the initial draft;
Potts JR and Verma S contributed equally to writing the final
draft of the manuscript.
Correspondence to: Dr. Sumita Verma, Senior Lecturer
Medicine, Honorary Consultant Hepatologist, Department of
medcine Brighton and Sussex Medical School, Falmer, Department of medcine Brighton, BN1 9PX,
United Kingdom. [email protected]
Telephone: +44-1273-877890 Fax: +44-1273-877576
Received: October 12, 2010 Revised: November 4, 2010
Accepted: November 11, 2010
Key words: Autoimmune hepatitis; Liver failure; Liver
transplantation; Corticosteroids
Peer reviewer: Atsushi Tanaka, MD, PhD, Associate Professor,
Department of Medicine, Teikyo University School of Medicine,
2-11-1, Kaga, Itabashi-ku, Tokyo 173-8605, Japan
Potts JR, Verma S. Optimizing management in autoimmune
hepatitis with liver failure at initial presentation. World J Gastroenterol 2011; 17(16): 2070-2075 Available from: URL:
Abstract
Autoimmune hepatitis (AIH) is a disease of unknown
etiology, its hallmark being ongoing hepatic inflammation. By its very nature, it is a chronic condition, although increasingly, we are becoming aware of patients
with acute presentations, some of whom may have
liver failure. There are very limited published data on
patients with AIH with liver failure at initial diagnosis,
which consist mostly of small retrospective studies. As
a consequence, the clinical features and optimal management of this cohort remain poorly defined. A subset
of patients with AIH who present with liver failure do
respond to corticosteroids, but for the vast majority, an
urgent liver transplantation may offer the only hope of
long-term survival. At present, there is uncertainty on
how best to stratify such a cohort into responders and
non- responders to corticosteroids as soon as possible
after hospitalization, thus optimizing their management.
This editorial attempts to answer some of the unresolved issues relating to management of patients with
AIH with liver failure at initial presentation. However,
it must be emphasized that, at present, this editorial is
based mostly on small retrospective studies, and it is an
WJG|www.wjgnet.com
INTRODUCTION
Autoimmune hepatitis (AIH) is a disease that is characterized by chronic hepatic inflammation, presence of autoantibodies [antinuclear antibody (ANA), anti-smooth muscle
antibody (SMA), and liver kidney microsomal (LKM) antibody], female preponderance and elevated serum gammaglobulins, especially IgG[1]. Earlier studies have established
the beneficial effects of corticosteroids in AIH and up
to 80% of patients can now achieve remission with immunosuppressants[2,3]. At accession, 10%-20% of patients
with AIH can be negative for the conventional autoantibodies[4], although their outcomes, especially response to
immunosuppression, are no different from those that are
autoantibody-positive[5].
AIH can have protean manifestations, with the majority
of patients presenting with subclinical or chronic disease.
However, in > 25%, the disease may present acutely with
jaundice, a subset of whom may have fulminant or subacute liver failure (LF)[6-8]. Fulminant hepatic failure (FHF)
is a devastating clinical condition that occurs in patients
2070
April 28|Volume 17|Issue 16|
Potts JR et al . Autoimmune hepatitis and liver failure
with no prior history of liver disease, and is characterized
by development of hepatic encephalopathy and coagulopathy within 8 wk after onset of jaundice[9]. In contrast, those
with subacute LF present with encephalopathy at 8-26
wk after onset of symptoms[10]. In a survey in the United
States carried out between 1998 and 2008, the major etiologies of FHF in 1147 patients were acetaminophen overdose (46%), followed by indeterminate causes (14%), druginduced (11%), hepatitis B virus (7%), other causes (7%),
AIH (5%), ischemic hepatitis (4%), hepatitis A virus (3%)
and Wilson’s disease (2%)[11]. Similar data were reported
from Europe where 2%-5% of patients with FHF have
AIH as the underlying etiology[12,13]. Unfortunately, neither
the International Autoimmune Hepatitis Group (IAIHG)
criteria[14] nor the simplified diagnostic criteria for diagnosis
of AIH[15] have been extensively validated in patients with
LF; largely because of the small number of cases encountered. Thus, diagnosis of AIH and LF remains clinical
and is supported by positive autoantibodies, negative viral
serology, absence of alcohol excess and culprit drugs, and
compatible liver biopsy. This has been corroborated by an
earlier study in which 28 patients with FHF were clinically
diagnosed with AIH, but after application of the IAIHG
criteria and simplified scoring systems only 50% and 46%,
respectively, fulfilled the criteria, with the concordance of
the two scoring systems being only 46%[16].
Immunoparesis is commonly seen in critically ill patients with LF in whom both autoantibodies and/or elevated IgG concentrations may be absent[17]. In addition,
because of the severity of the hepatic insult (massive/submassive necrosis), histological evaluation may be difficult
or impossible[16]. Although challenging, AIH can still be
diagnosed in such a scenario by excluding other liver diseases, and by testing for other autoantibodies [perinuclear
antineutrophil cytoplasmic antibodies (pANCA), and antibodies to soluble liver antigen (SLA)][18,19]. Furthermore,
if the patient is HLA B8, DR3 or DR4 positive, has a concurrent immunological disorder, and responds to corticosteroid therapy, this further lends credence to the diagnosis
of AIH[4]. Nonetheless, the decision to initiate corticosteroids in patients who do not fulfill conventional diagnostic
criteria for AIH must be made on an individual basis, and
remains the prerogative of the treating hepatologist.
management of patients with AIH that present with LF.
We therefore searched the medical literature (PubMed)
to collect published data on AIH with initial presentation
with LF. Only studies providing data on type and duration
of immunosuppressive therapy and outcomes were included. Case reports/small case series, and studies in which
authors reported acute AIH in the absence of LF were
excluded. We identified five studies that met our inclusion
criteria and these included a total of 85 patients with AIH
and LF[7,22-25] (Table 1). In three of the five studies[7,23,24],
patients were diagnosed with AIH according to IAIHG
criteria, although information regarding probable or definite AIH was only available in two[7,24]. In the remaining
two studies[22,25], the diagnosis of AIH was based on the
presence of autoantibodies, elevated IgG levels, exclusion
of Wilson’s disease, negative viral serology, absence of culprit drugs, and compatible liver histology (1). The patients
were very heterogeneous as regards ethnicity, presence/absence of cirrhosis, and inclusion of acute and subacute LF.
It is well known that these factors have a prognostic value
in patients with AIH and in those with LF[7,26-28]. In addition, all the studies were retrospective, and one has only
been published in an abstract form[22]. Nonetheless, these
five studies do provide valuable information about the
natural history of AIH with LF at initial presentation.
In these five studies, the prevalence of LF at initial
presentation in patients with AIH varied from 8.7% to
19.8%[7,23]. In all but one patient this was the first presentation of their disease. The majority (> 75%) were women
in the third to the sixth decade with type 1 AIH. Almost
all patients had either encephalopathy at admission and/or
had significant coagulopathy (Table 1). IgG levels were
available in two studies[24,25], and 74% had levels in excess
of 1800 mg/dL.
OUTCOMES IN PATIENTS WITH AIH AND
LF
Table 2 shows treatment data and outcomes in these five
above studies. Of the total of 85 patients, 69 (89.2%) received immunosuppression, mostly corticosteroids (Table
2). For the majority of the patients, there was no rationale
provided for initiation or withholding corticosteroids, and
the decision appeared to have been made on an ad hoc basis.
The remission rates with immunosuppression varied from
8.3% to 50% (average: 33.3%, 23/69) (Table 2). Overall,
43.5% (37/85) either underwent or were listed for LT and
32.9% (28/85) died. These outcomes are certainly poorer
than those reported in patients with chronic AIH (remission
with corticosteroids ~80%[2,3], need for LT 1.4%-8.4% and
mortality 1.8%-4.9%[27, 29]), and makes for dismal reading.
The variability in remission rates with corticosteroid
therapy in these five studies is most certainly a reflection
of the heterogeneous patient population. Unsurprisingly,
the lowest remission rates were seen in the study of Ichai
et al[25], which had the sickest patients, as reflected by their
high admission MELD scores. However, those patients
with AIH and LF that did respond to corticosteroid
AIH AND LF
There is a paucity of published data on patients with AIH
with LF at initial diagnosis; consisting mostly of anecdotal
case reports or small case series[20,21]. Thus the clinical characteristics, response to immunosuppression, and outcomes
with/without liver transplantation (LT) of this cohort remain poorly described. Much of the controversy hinges on
a critical management issue, namely should such patients
be given a trial of corticosteroids, be priority listed for LT,
or both. If corticosteroids are indeed initiated, how and at
what time point do we define failure of medical treatment?
This editorial attempts to address some of these controversies with the aim to develop strategies that could optimize
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2071
Table 1 Clinical characteristics of patients with autoimmune hepatitis with liver failure at initial presentation
[22]1
Villamil et al
(n = 28)
Study design
Age (yr)2
Definition of LF
Symptoms duration2
Female
Ethnicity or country of origin
Definite/probable AIH (IAIHG4
criteria)
Retrospective
41
NA
Retrospective
40 ± 15.9
NA
NA
NA
South American
NA
6 (21.4)
22 (78.5%)
3988
LC/LKM6 positive
ANA/SMA7 positive
Bilirubin2 (mg/dL)
AST or ALT2
INR2 or PT
HE9 at onset
Cirrhosis
MELD2
Sub-massive or massive necrosis
(SMN, MN)
Immunosuppressant regimen used
Poor prognostic criteria
[23]
Kessler et al
(n = 10)
NA
30%
28 (100%)
None
NA
19/23 (82.6%)
17 needed LT and/or
died
Prednisone 60 mg/d
1: PT < 20%; 2: Grade 4
HE; 3: SMN at diagnosis; 4: 20% increase in
PT at day 3 of steroids
Septic events
NA
[24]
Miyake et al
(n = 11)
[25]
Ichai et al
(n = 16)
[7]
Verma et al
(n = 20)
3.2 wk
8 (80%)
80% White
NA5
Retrospective
53 (16-75)
PT < 40% and HE
≥ grade 2
24 (16-52) d
11 (100%)
Japanese
3(36%)/8 (64%)
Retrospective
36 ± 13.1
HE within 12 wk of
jaundice
NA
14/16 (87.5%)
French
NA
Retrospective
41.3 ± 14.2
Any grade HE and/or
INR > 2
2.1 ± 2.5 mo3
15 (75%)
70% black
9(45%)/11(55%)
1 (10%)
7 (70%)
16.97 ± 9.83
NA
20.6 (5.9-31)
3 (18.7%)
11 (68.7%)
425 (278-850)8
NA
20 (100%)
19.3 ± 10.3
1179 ± 1127.17
49.3 ± 66.9
8 (80%)
2/10 (20%)
NA
5/10 (50%)
220 (59-1094)
29% (6%-38%)
11 (100%)
NA
NA
NA
678 (60-2867)
5.36 (1.7-12.2)
10 (62.5%)
None
37 (24-47)
16/16 (100%)
15 needed LT and/or
died
1147.1 ± 711.4
2.7 ± 1.4
19 (95%)
8/20 (40%)
28 ± 7.41
12/19 (63.1%),
10 needed LT and or
died
Corticosteroids
(Dose NA) and
other10
NA
NA
Prednisolone
Prednisone 1 mg/kg
per day and other10
40-60 mg/d and
steroid pulse
1: High bilirubin at
NA
onset; 2: Worsening
bilirubin during
days 8-15 of steroid
therapy
NA
7 (43.7%), of whom 6
had received steroids
Corticosteroids11
20-1250 mg/d
1: Absence of cirrhosis; 2:
MELD > 28; 3: Worsening
trend in bilirubin and
INR after 3.7 ± 0.6 d of
steroid therapy
2 (10%), of whom 1
received steroids
1
Published only in abstract form; 2Data presented as mean ± SD or median (range); 3Duration from first symptom (and not necessarily jaundice/hepatic encephalopathy) to hospitalization; 4IAIHG: International Autoimmune Hepatitis Group; 5Met IAIHG criteria, data on probable or definite disease unavailable;
6
LKM/LC: Liver kidney microsomal antibody/liver cytosol antibody; 7ANA/SMA: antinuclear antibody/anti-smooth muscle antibody; 8Values in µmol/L;
9
HE: Hepatic encephalopathy; 10Additional immunosuppression was used in nine patients in the study of Kessler et al (azathioprine, tacrolimus, mycophenolate mofetil, 6-mercaptopurine, cyclosporine) and in one patient in the study of Ichai et al (azathioprine and cyclosporine); 11Included prednisone, hydrocortisone and methylprednisone, (converted to equivalent doses of prednisone); LT:Liver transplantation; PT: Prothrombin time; AIH: Autoimmune hepatitis.
corticosteroids[7]. It is more likely that non-responders to
corticosteroids had aggressive disease at the time of diagnosis with a critical degree of liver cell death already having
occurred prior to the introduction of medical treatment[24].
This hypothesis is supported by the study of Ichai et al[25],
in which all patients had massive/sub-massive liver necrosis (median MELD score at admission: 37), with only 8.3%
responding to corticosteroids and > 80% needing LT.
therapy survived, obviating the need for a subsequent LT.
Unfortunately, among the non-responders to corticosteroids in these five studies (n = 46), death was the inevitable
outcome in the absence of LT (Table 2). The duration of
steroid therapy prior to death was highly variable (3-95 d).
Clearly, in some, the illness was so fulminant that death
occurred rapidly after hospitalization, thereby precluding
LT, and in others, there were active contraindications to
transplantation, such as sepsis (Table 2). Nevertheless, in
these five studies, there were a subset of patients with AIH
and LF in whom death may have been preventable had LT
been more aggressively pursued. It is conceivable that initiation of steroids provided a false sense of security, thereby
delaying transplant evaluation.
One could argue that the low remission rates to corticosteroids in this cohort were partly related to delay in
initiating therapy. However, where available, the data do
not support this conclusion, as corticosteroids were initiated promptly, especially in the sicker patients. In our study,
subsequent non-responders to corticosteroids were commenced on therapy within 2.6 ± 1.8 d of admission, compared to 6.4 ± 5.5 d in those who eventually responded to
WJG|www.wjgnet.com
OPTIMIZING MANAGEMENT IN PATIENTS WITH AIH AND LF
Assessing patients with LF for LT is a complex process.
The most widely used criteria for prioritizing patients for
LT are the King’s College criteria[30]. However, neither the
King’s College criteria[29] nor the more recently developed
MELD score[31] have been validated in patients with AIH
and LF. This is most likely due to the fact that the prevalence of AIH in patients with LF being evaluated for LT
is low (0%-5%)[12,13,32]. As is evident from the published
data[7,22-25], there certainly are a subset of patients with AIH
and LF who will respond to corticosteroids. Inappropri-
2072
Table 2 Outcomes of patients with autoimmune hepatitis and initial presentation with liver failure
[22]
[23]
[24]
Study
Villamil et al
(n = 28)
Kessler et al
(n = 10)
Miyake et al
(n = 11)
Treated with IS1
Responders to steroids
Non responders
LT
Listed for LT
Died without LT
Not treated with IS1
Spontaneous survival
LT
Listed for LT
Died
Overall underwent LT or listed for LT
Overall mortality
25
9 (36%) (alive)
16
11 (2 Died)
5
3
1
2
12/28 (42.8%)
9/28 (32.1%)
10
4 (40%) (alive)
6
3
1
2
4/10 (40%)
2/10 (20%)
8
2 (25%) (alive)
6
1
5
33
3
1/11 (9%)
5/11 (45.4%)
[25]
Ichai et al
(n = 16)
12
1 (8.3%) (alive)
11
10 (1 Died)
14
4
3
1
13/16 (81.2%)
3/16 (18.7%)
[7]
Verma et al
(n = 20)
14
7 (50%) (alive)
7
1 (Died)
1 (Died)
52
6
5 (1 Died)
1
7/20 (35%)
9/20 (45%)
IS: Immunosuppression; 2Four died while being evaluated for liver transplantation, in 1 sepsis precluded liver transplantation evaluation; 3Treated with
plasmapheresis and or stronger neo-minophagen; 4Not evaluated for LT due to sepsis; LT: Liver transplantation. Additional outcome data obtained by personal communication with authors.
1
ate transplantation in such patients would mean subjecting
them to unnecessary surgery (and its attendant complications) and lifelong immunosuppression. In addition, it
would deprive another more suitable recipient from receiving the graft[33]. On the other hand, denying LT to a patient with AIH and LF who is unlikely to respond to corticosteroids means condemning them to a certain death,
which is unacceptable, especially since post-transplant
survival for AIH is excellent [estimated 5-year survival
probability after first LT is 0.73 (95% CI: 0.67-0.77)][34].
The contentious issue thus is how best to stratify patients with AIH and LF into likely responders and nonresponders to corticosteroids as soon as possible after
hospitalization; hence optimizing their management. In
our study[7], all responders to corticosteroid therapy had a
MELD score ≤ 28 at admission. This is also supported by
Ichai et al[25], who showed that the only patient to respond
to corticosteroids had a MELD score of 24, and none with
an initial MELD score > 28 responded to corticosteroids.
Furthermore, in our study, responders to corticosteroids
were more likely to have either an improvement or stabilization in bilirubin and INR within 3.7 ± 0.6 d of initiation of
corticosteroid therapy, whereas non-responders tended to
have a trend for higher bilirubin and INR[7]. Villamil et al[22]
also observed that a 20% increase in prothrombin time (PT)
at day 3 of corticosteroid therapy to be a predictor of poor
outcome, along with PT < 20% , grade 4 encephalopathy,
and LKM antibody/liver cytosol (LC) antibody positivity
at diagnosis. Histological evidence of sub-massive/massive
necrosis is also invariably associated with need for LT and/
or death (Table 1). Surprisingly, in our study, the presence
of cirrhosis was more likely was associated with response
to corticosteroids[7]. Although the impact of cirrhosis on
the natural history of AIH remains controversial[27,28,35,36], it
is likely that this group has long-standing indolent disease
that progresses to cirrhosis, with LF representing an acute
relapse of AIH[37]. This is in contrast with the study of Ichai
et al[25], in which absence of significant hepatic fibrosis in all
the patients indicated a de novo fulminant disease process.
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CORTICOSTEROIDS AND INFECTIONS
Whether steroids increase the risk of septic complications
in patients with severe liver disease is subject to an ongoing debate. The issue becomes even more contentious in
the presence of LF because in itself that has been associated with an increased risk of bacterial and fungal infections[25,38,39]. In fact, earlier studies have shown that up to
35% of patients with LF can develop bacteremia in the
pre-transplant period[39]. This increased propensity for sepsis is further aggravated in the post-transplant setting due
to use of immunosuppression. Therefore, not surprisingly,
sepsis with or without multiorgan failure, accounts for
almost one-third of all deaths in patients undergoing LT
for LF, and is the most common cause of mortality in this
cohort[40]. In the study of Ichai et al[25] (which had the sickest cohort of patients with a median MELD score of 37 at
admission), 42.3% developed a septic event, and this prevalence is not higher than that reported previously[39]. It is
however noteworthy that in Ichai et al’s study septic events
were more likely to occur in those initiated (6/12) versus
those not initiated (1/4) on corticosteroids[25]. It is unclear
whether patients received prophylactic antibiotics in this
study. Reich et al[41] also have reported an increased trend
for wound infection in corticosteroid-treated patients with
AIH undergoing LT (30.7% vs 5.2%). In a recent publication that analyzed data from the European Transplant
Registry, in comparison with transplantation for primary
biliary cirrhosis and alcoholic cirrhosis, the probability of
infectious complications limiting patient survival was significantly increased after transplantation for AIH. This was
especially relevant to patients aged > 50 years and within
the first 3 mo of transplantation[34]. Unfortunately, data on
disease severity and use of pre-transplant immunosuppression and prophylactic antibiotics were not available in that
study. On the other hand, others have reported corticosteroids not to be associated with increased risk of infections
in patients with severe AIH[42]. These discordant results
most likely reflect the heterogeneous patient groups (in-
2073
cluding the whole spectrum from chronic disease to FHF),
use of varying immunosuppressive regimens, and inconsistent use of prophylactic antibiotics. Nonetheless, Ichai
et al[25] caution against injudicious use of corticosteroids in
patients with AIH and LF, and on the contrary, emphasize
the need for expedited LT evaluation in such a cohort.
Furthermore, it lends credence to the argument for the use
of prophylactic antibiotics and antifungal agents, because
such a strategy has been shown to reduce the risk of infections in the pre-transplant setting[43].
mune hepatitis. Hepatology 2002; 36: 479-497
Murray-Lyon IM, Stern RB, Williams R. Controlled trial
of prednisone and azathioprine in active chronic hepatitis.
Lancet 1973; 1: 735-737
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liver disease: a controlled study of treatments and early
prognosis. Gastroenterology 1972; 63: 820-833
McFarlane IG. Autoimmune hepatitis: diagnostic criteria,
4
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Czaja AJ, Hay JE, Rakela J. Clinical features and prognostic
5
implications of severe corticosteroid-treated cryptogenic
chronic active hepatitis. Mayo Clin Proc 1990; 65: 23-30
Ferrari R, Pappas G, Agostinelli D, Muratori P, Muratori L,
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Lenzi M, Verucchi G, Cassani F, Chiodo F, Bianchi FB. Type
1 autoimmune hepatitis: patterns of clinical presentation and
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outcomes. Hepatology 2009; 49: 1396-1397
Trey C, Davidson LS. The management of fulminant hepatic
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10 Gimson AE, O’Grady J, Ede RJ, Portmann B, Williams R.
Late onset hepatic failure: clinical, serological and histological features. Hepatology 1986; 6: 288-294
11 Lee WM, Squires RH Jr, Nyberg SL, Doo E, Hoofnagle JH.
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nordic countries. Liver Transpl 2002; 8: 1055-1062
14 Alvarez F, Berg PA, Bianchi FB, Bianchi L, Burroughs AK,
Cancado EL, Chapman RW, Cooksley WG, Czaja AJ, Desmet VJ, Donaldson PT, Eddleston AL, Fainboim L, Heathcote J, Homberg JC, Hoofnagle JH, Kakumu S, Krawitt
EL, Mackay IR, MacSween RN, Maddrey WC, Manns MP,
McFarlane IG, Meyer zum Büschenfelde KH, Zeniya M.
International Autoimmune Hepatitis Group Report: review
of criteria for diagnosis of autoimmune hepatitis. J Hepatol
1999; 31: 929-938
15 Hennes EM, Zeniya M, Czaja AJ, Parés A, Dalekos GN,
Krawitt EL, Bittencourt PL, Porta G, Boberg KM, Hofer H,
Bianchi FB, Shibata M, Schramm C, Eisenmann de Torres
B, Galle PR, McFarlane I, Dienes HP, Lohse AW. Simplified
criteria for the diagnosis of autoimmune hepatitis. Hepatology 2008; 48: 169-176
16 Yeoman AD, Westbrook RH, Al-Chalabi T, Carey I, Heaton
ND, Portmann BC, Heneghan MA. Diagnostic value and
utility of the simplified International Autoimmune Hepatitis
Group (IAIHG) criteria in acute and chronic liver disease.
Hepatology 2009; 50: 538-545
17 Gregorio GV, Portmann B, Reid F, Donaldson PT, Doherty
DG, McCartney M, Mowat AP, Vergani D, Mieli-Vergani G.
Autoimmune hepatitis in childhood: a 20-year experience.
Hepatology 1997; 25: 541-547
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neutrophil cytoplasmic antibodies in autoimmune liver diseases. Hepatology 1993; 17: 411-447
2
THE FUTURE
Prospective multicenter studies are clearly needed to address this complex and important clinical issue. In future,
testing for additional autoantibodies and HLA typing
might also help risk-stratify patients. For example, presence
of antibodies to SLA have been associated with DRB1
*0301, and such patients have aggressive disease and are
more likely to require LT and/or die[44,45].
CONCLUSION
The diagnosis and management of patients with AIH with
AF at initial diagnosis can be challenging. Although there
are only limited published data available, mostly in the
form of small retrospective studies, up to 8.7%-19.8% of
patients with AIH may have this form of presentation. On
the whole, about one-third can respond to corticosteroids
and have a good outcome, although for the vast majority, LT may offer the only hope of long-term survival. A
MELD score at admission of ≤ 28, more severe hepatic
fibrosis, absence of sub-massive/massive necrosis, and
early (within 4 d) improvement or stabilization in bilirubin and INR, identify those who are likely to respond to
corticosteroid therapy, and thus survive without the need
for LT. If clinical and biochemical improvement does not
occur within the first few days, then continuation of corticosteroids may be a futile exercise, as it would be unlikely
to change the clinical outcome, and on the contrary, may
result in adverse events, especially sepsis. Nonetheless, if a
decision is made to continue therapy with corticosteroids
it is imperative that LT be actively pursued concomitantly.
Furthermore, it may not be unreasonable to consider
prophylactic antimicrobial and antifungal agents in such
high-risk patients. It must however be emphasized that,
at present, these recommendations are based on small
retrospective studies. This underlines the urgent need for
prospective multicenter studies to address this important
clinical issue.
ACKNOWLEDGEMENTS
SV is grateful to Dr. Villamil, Dr. Kessler and Dr. Miyake for providing additional data upon request.
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failure secondary to autoimmune hepatitis. P R Health Sci J
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an unusual form of presentation. J Hepatol 1997; 27: 578-582
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Ciardullo M, et al. Fulminant autoimmune hepatitis type 1:
Clinical presentation, outcome and prognostic factors. (Abstract 480) AJT 2005; 5 (Suppl 11): 278
23 Kessler WR, Cummings OW, Eckert G, Chalasani N, Lumeng L, Kwo PY. Fulminant hepatic failure as the initial
presentation of acute autoimmune hepatitis. Clin Gastroenterol Hepatol 2004; 2: 625-631
24 Miyake Y, Iwasaki Y, Terada R, Onishi T, Okamoto R,
Sakai N, Sakaguchi K, Shiratori Y. Clinical characteristics of
fulminant-type autoimmune hepatitis: an analysis of eleven
cases. Aliment Pharmacol Ther 2006; 23: 1347-1353
25 Ichai P, Duclos-Vallée JC, Guettier C, Hamida SB, Antonini
T, Delvart V, Saliba F, Azoulay D, Castaing D, Samuel D.
Usefulness of corticosteroids for the treatment of severe
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26 O’Grady JG, Schalm SW, Williams R. Acute liver failure:
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27 Verma S, Gunuwan B, Mendler M, Govindrajan S, Redeker
A. Factors predicting relapse and poor outcome in type I autoimmune hepatitis: role of cirrhosis development, patterns
of transaminases during remission and plasma cell activity
in the liver biopsy. Am J Gastroenterol 2004; 99: 1510-1516
28 Roberts SK, Therneau TM, Czaja AJ. Prognosis of histological cirrhosis in type 1 autoimmune hepatitis. Gastroenterology 1996; 110: 848-857
29 Montano-Loza AJ, Carpenter HA, Czaja AJ. Features associated with treatment failure in type 1 autoimmune hepatitis
and predictive value of the model of end-stage liver disease.
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30 O’Grady JG, Alexander GJ, Hayllar KM, Williams R. Early
indicators of prognosis in fulminant hepatic failure. Gastroenterology 1989; 97: 439-445
31 Kamath PS, Wiesner RH, Malinchoc M, Kremers W, Therneau TM, Kosberg CL, D’Amico G, Dickson ER, Kim WR.
A model to predict survival in patients with end-stage liver
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32 Gow PJ, Jones RM, Dobson JL, Angus PW. Etiology and outcome of fulminant hepatic failure managed at an Australian
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33 Bernal W, Auzinger G, Dhawan A, Wendon J. Acute liver
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S- Editor Tian L L- Editor Kerr C
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2075
E- Editor Ma WH
[email protected]
doi:10.3748/wjg.v17.i16.2076
TOPIC HIGHLIGHT
Luca Frulloni, MD, PhD, Professor, Series Editor
A practical approach to the diagnosis of autoimmune
pancreatitis
Luca Frulloni, Antonio Amodio, Anna Maria Katsotourchi, Italo Vantini
Peer reviewers: Yoshiharu Motoo, MD, PhD, FACP, FACG,
Luca Frulloni, Antonio Amodio, Anna Maria Katsotourchi,
Italo Vantini, Department of Medicine, University of Verona,
37134 Verona, Italy
Luca Frulloni, Professor, Cattedra di Gastroenterologia, Policlinico GB Rossi, P.le LA Scuro, 10, 37134 Verona, Italy
Author contributions: Frulloni L and Amodio A wrote the paper;
Katsotourchi AM searched the literature for papers on autoimmune
pancreatitis up to December 2009 and for papers on the frequency
of benign lesions in resected pancreatic masses; Vantini I revised
the paper.
Correspondence to: Luca Frulloni, Professor, Cattedra di Gastroenterologia, Policlinico GB Rossi, P.le LA Scuro, 10, 37134
Verona, Italy. [email protected]
Telephone: +39-45-8124191 Fax: +39-45-8027495
Received: September 21, 2010 Revised: January 29, 2011
Accepted: February 5, 2011
Professor and Chairman, Department of Medical Oncology,
Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan; Richard Hu, MD, MSc, Division of
Gastroenterology, Department of Medicine, Olive view-UCLA
Medical Center, 14445 Olive View Drive, Los Angeles, CA
91342, United States
Frulloni L, Amodio A, Katsotourchi AM, Vantini I. A practical
approach to the diagnosis of autoimmune pancreatitis. World J
INTRODUCTION
Autoimmune pancreatitis (AIP) is now a well defined entity among the inflammatory diseases of the pancreas[1-3].
The number of studies in literature has constantly increased since the first one published in 1995 by Yoshida
et al[4] (Figure 1). Despite the fact that in the first paper
from Japan the disease was described as diffusely involving the pancreatic gland[5-8], later publications pointed
out that the pancreas may also be focally involved by the
autoimmune process[3,9-12]. Therefore, some authors have
classified AIP as focal or diffuse[3]. Focal AIP is characterized by a segmental involvement of the parenchyma
with the possibility of a low-density mass being present
at imaging.
Clinically, the focal form, particularly in the presence
of a low-density pancreatic mass, requires a more careful patient evaluation, since it may be easily confused with
pancreatic cancer. Several series indicate that in 5%-21%
of resected pancreatic masses suspected of being cancer,
the final diagnosis excluded malignancy (Table 1)[13-20]. Since
AIP responds dramatically to steroid treatment[1], a correct
diagnosis of the disease is important to avoid surgery. On
the other hand, in the presence of a resectable pancreatic
mass, the probability of cancer is very high (> 90%). A
Abstract
Autoimmune pancreatitis is a disease characterized by
specific pathological features, different from those of
other forms of pancreatitis, that responds dramatically
to steroid therapy. The pancreatic parenchyma may
be diffusely or focally involved with the possibility of a
low-density mass being present at imaging, mimicking
pancreatic cancer. Clinically, the most relevant problems
lie in the diagnosis of autoimmune pancreatitis and in
distinguishing autoimmune pancreatitis from pancreatic
cancer. Since in the presence of a pancreatic mass the
probability of tumour is much higher than that of pancreatitis, the physician should be aware that in focal autoimmune pancreatitis the first step before using steroids is
to exclude pancreatic adenocarcinoma. In this review, we
briefly analyse the strategies to be followed for a correct
diagnosis of autoimmune pancreatitis.
Key words: Autoimmune diseases; Pancreatitis; Therapy; Diagnosis
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2076
Frulloni L et al . A practical approach to the diagnosis of autoimmune pancreatitis
misdiagnosis of AIP implies 2-3 week’s steroid treatment
and a one month delay in surgery, with the consequent risk
of not operating because of the progression of the malignancy with the onset of metastasis or of vascular involvement. A correct and quick diagnosis of AIP is therefore an
important goal in clinical practice, particularly in focal AIP.
AIP diagnosis may be attained through well established
diagnostic criteria. There is agreement on the use of four
main criteria based on histological findings, radiological features, other organ involvement and clinical and instrumental
response to steroid therapy. HISORt criteria introduced by
Chari et al[21] in 2006 and based on surgical specimens of operated AIP patients can be considered standard criteria for
the diagnosis of AIP. Serum IgG4[22-24] and positive IgG4+
plasma cells in pancreatic surgical specimens or pancreatic
biopsies may also support the diagnosis of AIP[25-29].
There is agreement on the use of these diagnostic criteria (pathology, imaging, presence of other organ involvement, response to steroids), but not on the strategy to be
followed in making the diagnosis.
120
60
40
2009
2008
2007
2006
2005
2004
2003
2002
2001
2000
1999
1998
1997
1996
20
0
Figure 1 Increased number of published papers on autoimmune pancreatitis obtained by searching in Pubmed up to 2009 (search terms: Autoimmune pancreatitis, limit: Field title).
Table 1 Frequency of benign lesions in patients who undergo
pancreatico-duodenectomy in the presence of a pancreatic
mass suspected of being pancreatic adenocarcinoma
THE STRATEGIES IN THE DIAGNOSIS OF
AIP
Authors
Yr
No. of pts
1994
1996
1997
2003
2003
2006
2010
2010
-
603
510
220
442
1287
162
494
461
4179
Frequency of benign lesions
n
Smith et al[13]
Barens et al[14]
van Gulik et al[15]
Abraham et al[16]
Weber et al[17]
Kennedy et al[18]
De La Fuente et al[19]
Hurtuk et al[20]
All studies
Three main strategies, from Japan, the USA and Italy, have
been suggested. The clinical approach to the disease by
these strategies is different.
In the USA distinguishing the different pathological
subtypes of AIP[21,30,31] is considered prominent for the diagnosis. In the USA and in Europe, AIP may be classified
as type 1 (or Lympho-Plasmacytic Sclerosing PancreatitisLPSP) and type 2 (or Idiopathic Duct-centric Chronic
Pancreatitis- IDCP)[31-34]. Since the clinical evolution of
these forms seems to be different, some authors have
suggested obtaining the diagnosis of AIP subtypes from
EUS-guided biopsy[31,35].
The main pathological and serological features in type
1 AIP are[31,36]: (1) Prevalence of storiform fibrosis, with
obstructive phlebitis; (2) high levels of serum IgG4; (3)
presence of IgG4+ plasma cells in the involved pancreatic
tissue; and (4) absence of granulocytic epithelial lesions
(GEL), that are the expression of an aggression against
epithelial ductal cells, with rupture and destruction of ductal structures. The pathological characteristics in type 2 AIP
are on the contrary[31,36]: (1) prevalence of inflammation; (2)
presence of GEL; and (3) absence of serum IgG4 and of
IgG4+ plasmavcells in the inflamed pancreatic tissue.
The clinical aspects and the evolution are different in
type 1 and 2 AIP[31,36,37]. In type 1 AIP (LPSP), there is a
prevalence of males, patients are older, other organs may
be involved (more commonly salivary glands, biliary tract,
kidney, lung, retroperitoneum) and the relapse of the disease is more frequent after steroid treatment. In type 2
(IDCP), male/female ratio is about 1, patients are younger,
the colon only may be involved (ulcerative colitis) and
relapse after steroids is infrequent. Both forms respond
quickly to steroid treatment[31,36-38].
The diagnostic approach is therefore aimed at diagnosing
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80
1995
No. of published papers
100
29
108
14
47
159
21
37
35
450
%
5
21
6
10.4
12
12.9
7.4
8
10.8
AIP subtypes, mainly through pancreatic core biopsy, and
this appears to have a good sensitivity and specificity[28,29,39].
In Japan, only type 1 AIP (LPSP) is considered an autoimmune disorder and an IgG4-mediated systemic disorder
associated with pancreatic lesions[40]. Only in a few cases
has type 2 AIP (IDCP) been described in Japan and it is
not considered an autoimmune disease, despite its quick response to steroids just as type 1 AIP. Instrumentally, in the
majority of cases the disease diffusely involves the pancreas.
Several diagnostic algorithms have been suggested in Japan
and Korea[8,41-44]. A comprehensive diagnosis should be
based on pancreatic imaging (including ERCP), serological
tests (IgG4, total IgG, non organ specific autoantibodies,
antibodies to carbonic anhydrase type Ⅰ and Ⅱ, antibodies
to lactoferrin) and pathological findings. The presence of
extrapancreatic lesions may suggest the possibility of AIP.
THE ITALIAN STRATEGY: A CLINICAL
APPROACH TO THE DISEASE
The Italian proposal for the diagnosis of AIP, which is different from that suggested in Japan and the USA, is based
on the instrumental distinction between focal and diffuse
forms of the disease[2,3].
2077
A wide range of symptoms are reported by patients at
the clinical onset of the disease. Jaundice, abdominal pain,
usually mild, symptoms secondary to pancreatic exocrine
and endocrine insufficiency (weight loss, diabetes), and
persistent elevation of serum levels of pancreatic enzymes
may be observed in AIP patients. In a few cases AIP is
discovered incidentally by US or other imaging techniques
performed without an indication for a pancreatic disorder.
On the basis of imaging, these patients can be divided
in those with focal involvement of the pancreas and those
with diffuse enlargement of the pancreatic gland[12]. In the
case of focal AIP, particularly in the presence of a lowdensity pancreatic mass, the clinical challenge is to exclude
pancreatic cancer and correctly diagnose AIP. Therefore,
focal and diffuse types AIP should be strictly separated,
since the problem of differential diagnosis with pancreatic
cancer involves only focal AIP.
Diffuse AIP may be confused with acute pancreatitis.
The clinical picture of diffuse AIP, however, differs from
those observed in acute pancreatitis. In AIP, pain, if present, is mild, no risk factors for pancreatitis (biliary lithiasis,
alcohol) are present, a persistent increase in serum pancreatic enzymes may be observed, jaundice is caused by
enlargement of the pancreas without the presence of a
mass, with a stricture on the intrapancreatic tract of the
common bile duct. Since pancreatic necrosis has never
been described in AIP, the differential diagnosis should
be with oedematous pancreatitis. This can be achieved
through imaging, since the radiologic features of AIP are
different from those observed in acute oedematous pancreatitis. Hypodensity of the pancreas in arterial phase and
the absence of a peripancreatic strand appear to differentiate AIP from acute oedematous pancreatitis, where the
pancreatic gland shows normal perfusion and the peripancreatic strand is a common radiological picture (personal
unpublished data). We do not suggest pancreatic biopsy
in diffuse AIP. The diagnosis may be definitely made after
treatment with steroids, which produces complete disappearance of the pancreatic changes.
In the diffuse form associated with jaundice secondary
to a common bile duct stricture, a diagnosis of cholangiocarcinoma should be considered and, if necessary, ruled
out before steroid therapy through ERCP with biliary biopsies and/or intraductal biliary ultrasonography.
In the focal form, particularly in the presence of a lowdensity pancreatic mass at imaging, the first diagnostic goal
is to exclude pancreatic cancer, even if the presence of
clinical (young age, other organ involvement), radiological
(perfusion of the pancreatic mass suggestive of inflammation, no or mild dilation of the main pancreatic duct) and
serological (high level of IgG4, presence of autoantibodies, low serum levels of Ca 19-9) findings are suggestive of
AIP. Therefore, pancreatic biopsy is mandatory, preferably
EUS-guided, first of all to exclude neoplasia and possibly
to confirm the diagnosis of AIP.
If pancreatic biopsy confirms the diagnosis of AIP, a
3 wk steroid treatment is indicated. The diagnosis of AIP
is final in the presence of a significant clinical and radiological response. Since significant improvement/resolution
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of jaundice is an indication of response to steroid therapy,
biliary stenting is not recommended, unless serum bilirubin levels are very high.
If pancreatic biopsy is only suggestive of AIP or non
diagnostic, a careful evaluation of HISORt criteria is necessary to decide whether the patient should be treated with
steroids or undergo resective surgery. The decision is actually a challenge and should be made in experienced centres
only, because it requires expert clinicians, radiologists,
pathologists and surgeons. After a complete or significant
response to steroid therapy, a definitive diagnosis of AIP
may be made.
CONCLUSION
The diagnosis of AIP still remains difficult. The diagnostic
algorithm is different in the diffuse and focal forms of the
disease, particularly in the presence of a low-density pancreatic mass at imaging. Biopsy or fine needle aspiration cytology is mandatory in the presence of a low-density pancreatic mass. In some cases, only a full or significant response
to steroids allows a final diagnosis of AIP to be made.
Agreement among experienced clinicians, radiologists, pathologists and surgeons is needed to adopt the response to
steroid therapy as a diagnostic criterion in patients where
the diagnosis cannot be made through pancreatic biopsy.
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28 Detlefsen S, Mohr Drewes A, Vyberg M, Klöppel G. Diagnosis of autoimmune pancreatitis by core needle biopsy:
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30 Levy MJ. Endoscopic ultrasound-guided trucut biopsy of
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31 Sah RP, Chari ST, Pannala R, Sugumar A, Clain JE, Levy MJ,
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35 Sah RP, Pannala R, Chari ST, Sugumar A, Clain JE, Levy MJ,
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36 Sugumar A, Klöppel G, Chari ST. Autoimmune pancreatitis:
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37 Maire F, Le Baleur Y, Rebours V, Vullierme MP, Couvelard A,
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38 Sugumar A, Chari ST. Diagnosis and treatment of autoimmune pancreatitis. Curr Opin Gastroenterol 2010; 26: 513-518
39 Hirano K, Fukushima N, Tada M, Isayama H, Mizuno S,
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40 Okazaki K, Kawa S, Kamisawa T, Shimosegawa T, Tanaka M.
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41 Choi EK, Kim MH, Kim JC, Han J, Seo DW, Lee SS, Lee SK.
The Japanese diagnostic criteria for autoimmune chronic pancreatitis: is it completely satisfactory? Pancreas 2006; 33: 13-19
42 Kamisawa T, Chung JB, Irie H, Nishin T, Ueki T, Takase M,
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43 Kamisawa T, Okazaki K, Kawa S. Diagnostic criteria for autoimmune pancreatitis in Japan. World J Gastroenterol 2008; 14:
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A, Kubo K, Notohara K, Hasebe O, Fujinaga Y, Ohara H, Tanaka S, Nishino T, Nishimori I, Nishiyama T, Suda K, Shiratori K, Shimosegawa T, Tanaka M. Japanese clinical guidelines
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S- Editor Tian L L- Editor O’Neill M E- Editor Ma WH
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[email protected]
doi:10.3748/wjg.v17.i16.2080
TOPIC HIGHLIGHT
Luca Frulloni, MD, PhD, Professor, Series Editor
Endoscopic ultrasonography findings in autoimmune
pancreatitis
Elisabetta Buscarini, Stefania De Lisi, Paolo Giorgio Arcidiacono, Maria Chiara Petrone, Arnaldo Fuini,
Rita Conigliaro, Guido Manfredi, Raffaele Manta, Dario Reggio, Claudio De Angelis
Key words: Pancreatitis; Autoimmune; Endoscopic ultrasound; IgG4 cholangitis
Elisabetta Buscarini, Guido Manfredi, Department of Gastroenterology, Maggiore Hospital, Crema 26013, Italy
Stefania De Lisi, Department of Gastroenterology, Di.Bi.M.I.S.,
University of Palermo, Palermo 90121, Italy
Paolo Giorgio Arcidiacono, Maria Chiara Petrone, Department
of Gastroenterology, I.R.C.C.S San Raffaele, Università Vita e
Salute San Raffaele, Milan 20122, Italy
Arnaldo Fuini, Department of Gastroenterology and Digestive
Endoscopy, Ospedale Civile Maggiore, Verona 37142, Italy
Rita Conigliaro, Guido Manfredi, Raffaele Manta, Department
of Gastroenterology and Digestive Endoscopy, New Civil Hospital S. Agostino Estense, Modena 41126, Italy
Dario Reggio, Chirurgia Trapianti di Fegato, San Giovanni Battista Hospital, Torino 10156, Italy
Claudio De Angelis, Department of Gastroenterology, San
Giovanni Battista Hospital, Torino 10156, Italy
Author contributions: All authors contributed to conception and
design, drafting the article or revising it, and to final approval.
Correspondence to: Elisabetta Buscarini, MD, Department of
Gastroenterology, Maggiore Hospital, Largo Dossena 2, Crema
26013, Italy. [email protected]
Telephone: +39-373-280278 Fax: +39-373-280654
Received: September 21, 2010 Revised: November 16, 2010
Peer reviewer: Dr. Jeremy FL Cobbold, PhD, Clinical Lecturer
in Hepatology, Department of Hepatology and Gastroenterology,
Liver Unit, Imperial College London, St Mary’s Hospital, 10th
Floor, QEQM building, Praed Street, London, W2 1NY, United
Kingdom
Buscarini E, De Lisi S, Arcidiacono PG, Petrone MC, Fuini A,
Conigliaro R, Manfredi G, Manta R, Reggio D, De Angelis C.
Endoscopic ultrasonography findings in autoimmune pancreatitis.
World J Gastroenterol 2011; 17(16): 2080-2085 Available from:
URL: http://www.wjgnet.com/1007-9327/full/v17/i16/2080.htm
DOI: http://dx.doi.org/10.3748/wjg.v17.i16.2080
INTRODUCTION
Endoscopic ultrasonography (EUS) is superior to standard imaging techniques in detecting pancreatic cancer
or masses and in the assessment of early parenchymal
changes in chronic pancreatitis[1,2]; however, its role in the
diagnosis of autoimmune pancreatitis (AIP) has yet to be
standardized, even though its high accuracy together with
its safety make it a promising tool in the management of
this disease.
To date, no consensus about the diagnosis of AIP has
been reached[3]. Several criteria have recently been proposed, reflecting the different clinical entities that AIP
can adopt worldwide[4-9]. The diffuse form of AIP has
typically been included in the first set of criteria[4], and in
2008 only, a focal pancreatic enlargement evident upon
imaging was classified as a form of AIP.
Obstructive jaundice is the most common presentation of AIP and, together with biochemical and imaging
features, can mimic neoplastic conditions[10,11]. Because
Abstract
Endoscopic ultrasonography is an established diagnostic
tool for pancreatic masses and chronic pancreatitis. In
recent years there has been a growing interest in the
worldwide medical community in autoimmune pancreatitis (AIP), a form of chronic pancreatitis caused by
an autoimmune process. This paper reviews the current available literature about the endoscopic ultrasonographic findings of AIP and the role of this imaging
technique in the management of this protean disease.
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2080
Buscarini E et al . EUS and autoimmune pancreatitis
Table 1 Endoscopic ultrasonography features of autoimmune pancreatitis
Pancreas
EUS
Lymph nodes
Caliber: dilated
Volume: enlarged
(also substantially)
Diffuse AIP
Focal AIP
Gland volume:
increased
Gland volume: focal
enlargement/s
Echotexture: echopoor,
with echogenic
interlobular septa
Gland border:
thickened
Wirsung: narrowed
Echotexture: echopoor, Wall: diffuse and
with echogenic
uniform thickening,
interlobular septa
“sandwich-pattern”1
Wirsung wall:
thickened1
1
Extrahepatic bile duct Gallbladder
Wall: diffuse and
uniform thickening,
“sandwich-pattern”1
Peripancreatic vessels
Loss of interface between
pancreas and portal or
mesenteric veins1
Echotexture:
echopoor
Sites: liver hylum,
peripancreatic, celiac
Wirsung: narrowed
within the lesion,
dilated upstream to the
lesion
Wirsung wall:
thickened1
IDUS
Wall: diffuse and
uniform thickening,
“sandwich-pattern”;
differential
diagnosis with
cholangiocarcinoma1
1
Indicates the features which are detected only or substantially better by endoscopic ultrasonography (EUS) and not seen in conventional cross-sectional
imaging. AIP: Autoimmune pancreatitis; IDUS: Intraductal ultrasonography.
AIP is a benign disease, a definitive diagnosis without the
need for surgery is desirable.
AIP can present with extrapancreatic lesions, the most
frequent being IgG4-related sclerosing cholangitis (IgG4SC), followed by hilar lymphadenopathy[12].
EUS can display both of these conditions in addition
to parenchymal, ductal and vascular lesions. Moreover,
this technique offers the advantage over other diagnostic tools of allowing clinicians to perform biopsies to
achieve a definitive diagnosis[13-16].
In this article, we describe the EUS findings of AIP
(Table 1) by reviewing the currently available literature in
this field.
to be inadequate to evaluate parenchymal and ductal
changes in AIP[19]. According to the scoring system, a series of 25 patients with AIP were classified as normal or
displaying mild disease[16].
In the focal form of AIP a solitary (Figure 2), irregular hypoechoic mass, generally located in the head of the
pancreas, is observed[13-15]. In addition, upstream dilatation of the main pancreatic duct could be observed[13,17].
In this setting, the overlap with EUS findings of pancreatic cancer is remarkable, and EUS-elastography (Figure 1)
can provide further information about pancreatic lesions.
In a case-control study of five patients with AIP, EUSelastography showed a typical and homogeneous stiffness pattern of the focal lesions and of the surrounding
parenchyma that is different from that observed in ductal
adenocarcinoma[20].
PANCREATIC FINDINGS
The predominant finding in the diffuse form of AIP is a
diffuse pancreatic enlargement with altered echotexture
(Figure 1)[14,15]. A recent retrospective study proposed to
differentiate early from advanced stage AIP according to
EUS findings[15]. In 19 patients with AIP, the presence
of parenchymal lobularity and a hyperechoic pancreatic
duct margin were significantly correlated with early stage
AIP[17]. Other EUS findings that are indicative of AIP,
such as reduced echogenicity, hyperechoic foci and hyperechoic strands (Figure 1), are found in both AIP stages[17].
Should these results be confirmed in prospective studies,
EUS would acquire an essential role in the identification
of early stage AIP, which is characterized by a prompt
response to steroid therapy. Stones and cysts similar to
those described in chronic alcoholic pancreatitis can occur in the late stage of AIP[18].
The Sahai criteria for chronic pancreatitis were found
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COMMON BILE DUCT FINDINGS
The common bile duct is the most frequent extrapancreatic organ involved in AIP and was found to affect 58%
of patients in a Japanese survey[12]. Biliary strictures can
mimic both sclerosing cholangitis and biliary cancer. EUS
allows visualization of the entire common bile duct and
enables identification of the cause of a biliary stricture.
In patients with either diffuse or focal AIP, EUS can
show dilatation of the common bile duct and thickening
of its wall better than other diagnostic techniques[3,13-16,21].
The typical EUS feature of the common bile duct is a
homogeneous, regular thickening of the bile duct wall,
called “sandwich-pattern”, which is characterized by an
echopoor intermediate layer and hyperechoic outer and
inner layers, has been described as a EUS feature of the
2081
A
B
SV
C
Soft
D
Hard
D1
c
Figure 1 Diffuse form of autoimmune pancreatitis. A: Endoscopic ultrasonography (EUS) linear scanning shows a diffuse pancreatic enlargement (arrowheads)
with echopoor echotexture, and with loss of interface with splenic vein (arrows); B: Parenchymal lobularity and hyperechoic strands (arrows) are visible in the enlarged
gland; C: Pancreatic duct calliper is 1.8 mm; D: EUS-elastography demonstrates the diffuse pancreatic stiffness (arrowheads).
A
B
C
D
c
w
Figure 2 Focal form of autoimmune pancreatitis. A: Endoscopic ultrasonography (EUS) shows a focal lesion (arrows) of pancreatic head which is echopoor with
hyperechoic strands; B: A EUS-guided fine needle aspiration is performed (arrow) for tissue characterization; C: Another case of focal autoimmune pancreatitis (AIP)
with echopoor lesion of pancreatic head (between callipers) and marked echopoor thickening of the choledochal wall (arrow); D: In this case of focal AIP EUS shows a
echopoor lesion (arrows) of pancreatic head, with upstream dilatation of both common bile duct (c) and pancreatic duct (w); notice the thickened choledochal wall.
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2082
A
B
c
C
D
g
c
c
E
c
Figure 3 Biliary and peripancreatic findings in autoimmune pancreatitis. Autoimmune pancreatitis presenting with jaundice: A: Endoscopic ultrasonography (EUS)
shows a dilated common bile duct (c) upstream to a distal funnel-shaped stenosis; EUS demonstrates the diffuse thickening of the biliary wall (between arrows) with
“sandwich-pattern”, either of common bile duct or of cystic duct (arrowheads). This thickening is equally visible both in the dilated region of the common bile duct; B:
In the distal strictured tract (arrows); C: After contrast administration (Sonovue, Bracco) the biliary wall shows an early and persistent enhancement (arrowheads); D:
EUS shows the same thickening of the gallbladder (g) wall (arrowheads); E: Enlarged lymph nodes to the hepatic hylum (arrows).
common bile duct; the cause of the biliary stricture is the
thickened wall itself rather than extrinsic pancreatic compression (Figure 3)[3,13].
A further application of EUS is intraductal ultrasonography (IDUS), which can be performed during endoscopic retrograde cholangiography for the characterization of biliary stenosis. Naitoh et al[22] recently evaluated
IDUS findings in 23 patients with IgG4-SC. They found
that a circular, symmetric wall thickness, smooth inner
and outer margins and a homogeneous intermediate layer
in the stricture were significantly more common in AIP
than in cholangiocarcinoma. The wall thickness in IgG4SC in regions of non-stricture on the cholangiogram was
significantly greater than that in cholangiocarcinoma and
therefore a bile duct wall thickness exceeding 0.8 mm
in regions of non-stricture on the cholangiogram was
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highly suggestive of IgG4-SC. Contrast enhancement of
conventional EUS and IDUS showed an inflammatory
pattern of the bile duct wall, with a long-lasting enhancement starting in the early phase instead of the poor enhancement found in bile duct cancer (Figure 3)[21].
PERIPANCREATIC FINDINGS
Hilar lymphadenopathy is one of the most frequently described extrapancreatic lesions[12]. Other sites of enlarged
lymph nodes are the peripancreatic and celiac regions.
EUS nodal features that accurately predict nodal metastasis have been previously identified in patients with esophageal cancer[23]; they include size (> 1 cm in diameter on
the short axis), hypoechoic appearance, round shape, and
smooth border. However, these conventional EUS cri-
2083
teria have proven inaccurate for staging non-esophageal
cancers, including those that are biliopancreatic[24,25]. EUS
can detect single or multiple enlarged lymph nodes in
patients with AIP (Figure 3), reflecting the underlying
inflammatory process, which can involve extra-pancreatic
organs[14,15].
Hoki et al[16] reported a significant difference in detection of lymphadenopathy by EUS imaging over CT (72%
vs 8%) in patients with AIP. Moreover, in the same series,
a trend toward a higher prevalence of lymphadenopathy
in AIP compared to pancreatic cancer was reported.
In the absence of specific nodal features indicating
malignancy, the differential diagnosis with biliopancreatic
neoplasms is arduous but can be achieved by evaluating
the broad spectrum of clinical and imaging data of AIP
patients.
EUS criteria for vascular invasion of pancreatic cancer have been established[26,27].
In a series of 14 patients with AIP, EUS suspected
invasion of the portal or mesenteric veins in 21% of
patients compared to 14% on CT. No pancreatic cancer
developed during the follow-up of these patients. Such
EUS features, easily mistaken for malignancy, are due to
the inflammatory process of AIP, which can involve medium and large-sized vessels (Figure 1)[15].
Peripancreatic fluid collections are less common and
not specific for AIP.
tively). Both procedures were found to be safe, with no
complications[33].
However, the diagnostic accuracy of EUS-FNA for
pancreatic cancer has been reported to range between
60% and 90%[34-36], and the shortcomings of EUS-TCB
due to technical difficulties of the sampling of lesions in
the pancreatic head should also be considered.
Hence, when AIP is suspected, a sequential sampling
strategy has been proposed based on using EUS-FNA
first, which is followed by EUS-TCB when cytologic examination is inconclusive[33].
In cases of inconclusive cytology, an additional aid
for AIP diagnosis could come from molecular analysis
of EUS-FNA samples, which has shown high accuracy
in the differential diagnosis between AIP and pancreatic
cancer[37].
CONCLUSION
AIP represents 20%-25% of benign diagnoses undergoing resection for presumed malignancy[38,39]. Thus, a definitive diagnosis based on safe and reliable methods should
be obtained. In this setting, EUS could play an important
role in diagnosis, identifying typical features of AIP and
distinguishing it from biliopancreatic neoplasms. The
higher sensitivity over standard imaging for pancreatic,
biliary and nodal lesions should make it a cornerstone
in the process of diagnosing AIP. If validated in large
prospective series, newly available techniques such as
EUS-elastography and CE-EUS could add useful information about focal lesions without resorting to invasive
procedures. Finally, EUS-FNA or EUS-TCB can provide
pathological specimens, as required by some diagnostic
criteria.
INTERVENTIONAL EUS IN AIP
In recent years, the possibility of guiding tissue sampling
with either fine needle aspiration (EUS-FNA) (Figure 2)
or Tru-cut biopsy (EUS-TCB) has increased the diagnostic potential of EUS by the acquisition of cytological and
histological specimens from gastrointestinal lesions[28-30].
In the setting of AIP, EUS-FNA can be employed to
yield specimens of pancreatic lesions, the common bile
duct wall or lymph nodes[13,15]. Although a cytologic pattern specific for AIP has not been identified, high cellularity of stromal fragments with lymphoplasmacytic infiltrate
has emerged as a discriminating feature in a retrospective
series of 16 patients with an AIP diagnosis confirmed by
histology of the respective specimens. Indeed, 56% of
AIP patients presented such a feature vs 19% of patients
with pancreatic carcinoma, and none of the chronic pancreatitis controls exhibited this feature[31]. Immunohistochemical staining can show IgG4-positive plasma cells
which are a useful marker for the tissue diagnosis of AIP.
EUS-FNA can fail in diagnosis because of the small
size of the specimens that do not have preserved tissue
architecture. Moreover, sampling error due to the patchy
distribution of AIP can occur. EUS-TCB can overcome
these limitations by acquiring large samples fit for histological examination[30-32].
A recent study compared EUS-FNA and EUS-TCB
performed in 14 patients for the diagnosis of AIP. EUSTCB showed higher sensitivity (100%) and specificity
(100%) compared to EUS-FNA (36% and 33%, respec-
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H, Okumura F, Takahashi S, Joh T. Endoscopic transpapillary intraductal ultrasonography and biopsy in the diagnosis
of IgG4-related sclerosing cholangitis. J Gastroenterol 2009;
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Endosonographic features predictive of lymph node metastasis. Gastrointest Endosc 1994; 40: 442-446
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GJ. EUS-guided FNA of regional lymph nodes in patients
with unresectable hilar cholangiocarcinoma. Gastrointest Endosc 2008; 67: 438-443
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Lorenz R, Allescher HD, Kassem AM, Gerhardt P, Siewert
JR, Höfler H, Classen M. Endoscopic ultrasound criteria for
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pancreas: a blind reevaluation of videotapes. Gastrointest Endosc 2000; 52: 469-477
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L. Endoscopic ultrasonographic criteria of vascular invasion
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Harewood GC, Pearson RK, Rajan E, Topazian MD, Yusuf
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E, Jondal ML, Wiersema LM. Initial experience with EUSguided trucut needle biopsies of perigastric organs. Gastrointest Endosc 2002; 56: 275-278
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Fernandez-del Castillo C, Warshaw AL, Lauwers GY. Endoscopic ultrasound guided fine needle aspiration biopsy of
autoimmune pancreatitis: diagnostic criteria and pitfalls. Am
J Surg Pathol 2005; 29: 1464-1471
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Pederzoli P, Leins A, Longnecker D, Klöppel G. Histopathological features of diagnostic and clinical relevance in autoimmune pancreatitis: a study on 53 resection specimens and
9 biopsy specimens. Virchows Arch 2004; 445: 552-563
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S- Editor Tian L L- Editor O’Neill M E- Editor Zheng XM
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2085
[email protected]
doi:10.3748/wjg.v17.i16.2086
ORIGINAL ARTICLE
Effects of α-mangostin on apoptosis induction of human
colon cancer
Ramida Watanapokasin, Faongchat Jarinthanan, Yukio Nakamura, Nitisak Sawasjirakij,
Amornmart Jaratrungtawee, Sunit Suksamrarn
Ramida Watanapokasin, Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok 10110,
Thailand
Faongchat Jarinthanan, Faculty of Medical Technology, Rangsit University, Pratumthani 12130, Thailand
Yukio Nakamura, Clinical Research Center, Murayama Medical
Center, Gakuen 2-37-1, Tokyo 208-0011, Japan
Nitisak Sawasjirakij, Department of Research, North Bangkok
University, Bangkok 10220, Thailand
Amornmart Jaratrungtawee, Sunit Suksamrarn, Department
of Chemistry, Faculty of Science, Srinakharinwirot University,
Bangkok 10110, Thailand
Author contributions: Watanapokasin R conceived, initiated
the project and designed the research, provided project guidance
and supervision and wrote the paper; Jarinthanan F performed the
experiments; Nakamura Y and Sawasjirakij N revised the manuscript and analyzed the data; Jaratrungtawee A and Suksamrarn S
participated in purifying of the α-mangostin.
Supported by The Thailand Research Fund, Grant No. RMU
4980043
Correspondence to: Ramida Watanapokasin, PhD, Associate Professor and Head, Department of Biochemistry, Faculty
of Medicine, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, Thailand. [email protected]
Telephone: +66-2-6495369
Fax: +66-2-6495834
Received: November 11, 2010 Revised: January 10, 2011
mitochondrial membrane potential. The molecular
mechanisms of α-mangostin mediated apoptosis were
further investigated by Western blotting analysis including activation of caspase cascade, cytochrome c
release, Bax, Bid, p53 and Bcl-2 modifying factor.
Abstract
AIM: To investigate the effect of α-mangostin on the
growth and apoptosis induction of human colon cancer
cells.
Key words: α-mangostin; Apoptosis; Caspases; Colon
cancer; Mitochondria
RESULTS: The highest inhibitory effect of α-mangostin
on cell proliferation of COLO 205, MIP-101 and SW 620
were 9.74 ± 0.85 μg/mL, 11.35 ± 1.12 μg/mL and 19.6
± 1.53 μg/mL, respectively. Further study showed that
α-mangostin induced apoptotic cell death in COLO 205
cells as indicated by membrane blebbing, chromatin
condensation, DNA fragmentation, cell cycle analysis,
sub-G1 peak (P < 0.05) and phosphatidylserine exposure. The executioner caspase, caspase-3, the initiator
caspase, caspase-8, and caspase-9 were expressed
upon treatment with α-mangostin. Further studies of
apoptotic proteins were determined by Western blotting
analysis showing increased mitochondrial cytochrome
c release, Bax, p53 and Bmf as well as reduced mitochondrial membrane potential (P < 0.05). In addition,
up-regulation of tBid and Fas were evident upon treatment with α-mangostin (P < 0.01).
CONCLUSION: α-Mangostin may be effective as an
anti-cancer agent that induced apoptotic cell death
in COLO 205 via a link between extrinsic and intrinsic
pathways.
Peer reviewer: Masahiro Iizuka, MD, PhD, Director, Akita
METHODS: The three colorectal adenocarcinoma cell
lines tested (COLO 205, MIP-101 and SW 620) were
treated with α-mangostin to determine the effect on
cell proliferation by MTT assay, cell morphology, chromatin condensation, cell cycle analysis, DNA fragmentation, phosphatidylserine exposure and changing of
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Health Care Center, Akita Red Cross Hospital, 3-4-23, Nakadori,
Akita, 010-0001, Japan
Watanapokasin R, Jarinthanan F, Nakamura Y, Sawasjirakij N,
Jaratrungtawee A, Suksamrarn S. Effects of α-mangostin on
apoptosis induction of human colon cancer. World J Gastroenterol
2086
Watanapokasin R et al . Colon cancer apoptosis with mangostin
2011; 17(16): 2086-2095 Available from: URL: http://www.
wjgnet.com/1007-9327/full/v17/i16/2086.htm DOI: http://dx.doi.
org/10.3748/wjg.v17.i16.2086
The objective of the present study was to purify the
-mangostin
from the fruit hull of Garcinia mangostana L.
α
and explore its effect on apoptosis induction and mechanisms involved in COLO 205 cells.
INTRODUCTION
MATERIALS AND METHODS
α -mangostin preparation
Mangosteen fruit (G. mangostana) was collected from
Kombang District, Chantaburi Province, Thailand in
April, 2007. A voucher specimen (Ms Porntip Wongnapa
No. 002) was deposited at the Faculty of Science, Ramkhamhaeng University. The dried and pulverized fruit hull
of G. mangostana (0.5 kg) was thoroughly extracted with
ethyl acetate (EtOAc) at 50℃. The combined extract after filtration was concentrated under reduced pressure to
yield the extract as a yellowish solid (285 g). A portion of
the extract was subjected to repeated column chromatography over silica gel using a gradient of hexane/acetone
which yielded the pure major compound, α-mangostin,
including other minor xanthones. Purity of α-mangostin
exceeded 98% as determined by LC analysis and its spectroscopic data (NMR and MS) was consistent with the
reported values[12].
Searching for new biologically active compounds, novel
chemotherapeutic agents derived from active phytochemicals, could be used to improve the anti-carcinogenicity of
standard drug treatment. A variety of tropical plants have
useful biological activities and some offer potential therapeutic applications. Mangosteen (Garcinia mangostana L.)
in the Clusiaceae family has been used in Southeast Asia
as traditional medicine for treatment of wounds, skin
infection, diarrhea and chronic ulcer[1]. Phytochemical
studies showed that the fruit hull of mangosteen is rich
in a variety of oxygenated and prenylated xanthones[2,3]
which possess different biological properties, such as
anti-mycobacterial[4], anti-fungal[5], anti-oxidant[6-8], cytotoxicity[9-12] and anti-inflammatory activities[13]. However,
the underlying molecular mechanisms of α-mangostin in
COLO 205 cells are not yet reported.
Apoptosis plays a vital role in controlling cell number
in many physiological and developmental stages, tissue
homeostasis, and regulation of immune system[14], while
insufficient apoptosis is an integral part of cancer development[15]. Mammalian cells have two major apoptotic
pathways. One pathway (extrinsic pathway) is triggered
when ligands [Fas/CD95, tumor necrosis factor (TNF)-α]
bind to receptors on cell surface leading to the activation
of caspase-8 and -3[16], respectively. The other involves
mitochondrial (intrinsic) pathway induced by anti-cancer
drugs, prostaglandin, etc. resulting in disruption of mitochondrial membrane and release of various pro-apoptotic
factors[17-19]. The pro-apoptotic and anti-apoptotic members of B-cell CLL/lymphoma 2 (Bcl-2) family regulate
the release of cytochrome c, a mitochondrial protein that
can activate caspases.
Fas/CD95 belongs to the TNF superfamily and is
the prototype of death receptor that initiates an apoptotic cascade[20]. FasL, the tumor necrosis factor-related
cytokine, is a ligand of Fas and synthesized as a type
[21]
Ⅱ membrane protein . Upon FasL binding, activated
death receptors engage the Fas associated death domain
(FADD)[22], which in turn recruits caspase-8 and forming
the death inducing signaling complex (DISC). The DISC
then activates caspase-8 through a proximity-inducing
dimerization mechanism. In type Ⅰ cells, caspase-8 directly activates caspase-3, -6 and -7, leading to cell death.
In contrast, in type Ⅱ cells the small amount of active
caspase-8 generated at the DISC is not sufficient to induce cell death, therefore the mitochondria-dependent
apoptosis pathway is needed[23]. As such, the pro-apoptotic signal has to be amplified via cleavage of the BH3-only
protein Bid, Bax/Bak-assisted release of cytochrome c
from the mitochondria, an activation of caspase-9 and
subsequently caspase-3[24].
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Cell lines and culture conditions
Three human colorectal cancer cell lines were used: COLO
205 (colorectal adenocarcinoma), MIP-101 (colorectal carcinoma) and SW620 (colorectal adenocarcinoma). COLO
205 and SW620 were obtained from the American Type
Culture Collection (Manassas, VA). MIP-101 was a generous gift from Peter Thomas, Boston University School
of Medicine, Boston, MA. COLO 205 and MIP-101
were maintained in the RPMI 1640 medium (Invitrogen),
supplemented with 10% fetal calf serum (Invitrogen).
SW620 were cultured in Dulbecco’s modified Eagle’s
medium (Invitrogen), supplemented with 10% fetal calf
serum. All cell lines were maintained in culture at 37°C in
an atmosphere of 5% CO2.
Cell proliferation and cell viability assays
The cytotoxic activity of α-mangostin was determined
by cell proliferation analysis using MTT assays as previously described[25]. Briefly, cells were cultured in 96-well
plates at a density of 1 × 104/well in complete medium.
Then the cells were treated with varying concentrations
of α-mangostin and incubated at 37℃ for 24 h. The final
DMSO concentration in each well was 0.05%, at which
concentration no appreciable effect on cell proliferation
was seen. Then, 100 μL of 5.0 mg/mL MTT in culture media was added to each well and incubated at 37℃ for 2 h.
The metabolic product of MTT, formazan, in each well
was dissolved in DMSO, and the absorbance was determined at 595 nm. Effect of α-mangostin on the viability
of COLO 205 cells was analyzed by using a trypan blue
exclusion method. Briefly, cells were cultured in 96-well
plates at cell density of 1 × 104/well at 37℃ for 24 h.
α-mangostin was then added to culture wells at 0, 10, 20,
2087
Cell cycle analysis using flow cytometer
COLO 205 cells were cultured in 6-well culture plates
at 4 × 10 6 cells/well and treated with 0, 10, 20 and
30 μg/mL of α-mangostin for 3 h. The cells were then harvested, washed with PBS and resuspended in 200 μL PBS
and fixed in 800 μL of ice-cold 70% ethanol at -20℃,
overnight. The cells were stained with 1 mL of 50 μg/mL
propidium iodide solution (containing 0.1% Triton X-100,
and 0.1% sodium citrate) for 30 min at 37℃. The samples
were then analyzed by a flow cytometer (FACScan, Becton Dickinson). Excitation was done at 488 nm, and emission filter at 600 nm. Histograms generated by FACS were
analyzed by Cell Quest™ software (Becton Dickinson) to
determine the percentage of cells in each phase.
30 and 40 μg/mL or vehicle (in DMSO), incubated at
37℃ then collected periodically (0, 3, 6, 9 and 12 h). The
number of viable cells was determined with hemocytometers under a light microscope. Cell viability was expressed
as a percentage of the number of viable cells to that of
the control, to which no α-mangostin was applied.
Apoptosis assay
Characterization of the anticancer activity of α-mangostin
in COLO 205 cells was further conducted. Based on the
preliminary experiments, 20 μg/mL of α-mangostin was
used to study cell and nuclear morphology, cytochrome c
release, mitochondrial transmembrane potential, expression of pro-apoptotic proteins, Fas and truncated-Bid (tBid). However, a selective range of test concentrations,
ranging from 10 to 30 μg/mL was used to study DNA
fragmentation and for cell cycle analysis and annexin
V-FITC assays.
Annexin V-FITC assay
Percentage of α-mangostin-treated cells undergoing apoptosis was determined using an annexin V-fluorescein
isothiocyanate (FITC) apoptosis detection kit (BD)
Bioscience, San Jose, CA). COLO 205 cells at 1 × 105
cells/mL were treated with 0, 10, 20 and 30 μg/mL of
α-mangostin for 3 h and resuspended in 100 μL of annexin V binding buffer (10 mmol/L HEPES, 150 mmol/L
NaCl, 5 mmol/L KCl, 1 mmol/L MgCl2, 1.8 mmol/L
CaCl2), then incubated with 5 μL of 1 μg/mL FITCconjugated annexin V and 1 μg/mL propidium iodide
for 15 min at room temperature prior to analysis on a
FACScan, Becton Dickinson.
Microscopic analysis of cell and nuclear morphology
COLO 205 cells were cultured in 24-well culture plates
at the initial number of 2 × 104/well in the presence of
20 μg/mL α-mangostin for 3, 6, 9 and 12 h. As controls,
cells were cultured in the same fashion in the absence of
α-mangostin. The cells were examined under a phasecontrast inverted microscope (model CKX31/CKX41,
Olympus) for cell morphology.
The nuclear morphology was analyzed by treatment
of COLO 205 cells with 20 μg/mL α-mangostin for 3, 6,
9 and 12 h. Control cells were grown in the same manner
in the absence of α-mangostin. Cells were trypsinized
and fixed with methanol. Then, cell nuclei were stained
by treatment with 1 μg/mL Hoechst 33342 (Sigma) at
37℃ for 15 min in the dark. Stained cells were examined
under a fluorescence inverted microscope (model BX50,
Olympus).
Analysis of mitochondrial transmembrane potential
COLO 205 cells at 1 × 106 cells/mL were treated with
20 μg/mL of α-mangostin for 3 h, and then incubated
with 10 μ g/mL JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’tetraethylbenzimidazolcarbocyanine iodide) at 37℃ for
10 min in darkness. Stained cells were washed with PBS,
followed by FACS analysis. The mitochondrial function
was assessed as JC-1 green (uncoupled mitochondria) or
red (contact mitochondria).
Analysis of DNA fragmentation
COLO 205 cells were treated with varying concentrations,
10, 20 and 30 μg/mL, of α-mangostin for 12 h and then
lysed in 500 μL of lysis solution, consisting of 5 mmol/L
Tris-Cl (pH 8.0), 0.5% Triton X-100, and 20 mmol/L
ethylenediaminetetraacetic acid (EDTA). The cells were
then treated with RNase A (0.5 mg/mL) for 1 h at 37℃.
DNA fractions were prepared using phenol-chloroformisoamyl alcohol (25:24:1) and electrophoresed on 1.8%
agarose gels. Approximately 20 μg of DNA was loaded
in each well and the agarose gels were run at 50 V for 2 h
in Tris-borate/EDTA electrophoresis buffer. DNA was
stained with ethidium bromide and visualized under a UV
light trans-illuminator and photographed.
Western blotting analysis of cytochrome c
COLO 205 cells were cultured in 6-well culture plates at
4 × 106 cells/well and then incubated in the absence or
presence of 20 μg/mL α-mangostin for 3, 6, and 9 h. Cell
lysates was prepared as previously described[25]. Briefly, cell
suspensions were sonicated for 10 s and the cell lysates was
centrifuged at 4℃ at 10 000 × g for 30 min. The supernatants (cytosol fractions) were subjected to SDS-PAGE
using 12% polyacrylamide gels, and transferred onto Immobilon P membrane and subjected to immuno-detection
of cytochrome c using a mouse monoclonal antibody
against human cytochrome c (7H8, mouse monoclonal
Ig G 2b, Santa Cruz, Biotechnology) with goat anti-mouse
IgG conjugates to horse radish peroxidase (Cell Signaling Technology) and detected using an ECL Plus Western
Blotting Detection System (Amersham Biosciences).
Flow cytometer analysis
The cell cycle analysis, annexin V binding and mitochondrial transmembrane potential were investigated by flow
cytometric analysis (FACScan, Becton Dickinson). The
proper filters and optimal setting of the instrument were
chosen, the histograms generated by FACS were analyzed
by Cell Quest™ software (Becton Dickinson).
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Western blotting analysis of caspases, Bid, p53, Bax,
Bmf and Fas
COLO 205 cells were treated with 20 μg/mL α-mangostin
2088
125
100
% Cell viability
A
0 mg/mL
10 mg/mL
20 mg/mL
30 mg/mL
40 mg/mL
75
a-mangostin (20 mg/mL)
0h
50
25
0
B
Control
0
3
6
9
12
50 mm
50 mm
50 mm
50 mm
50 mm
50 mm
50 mm
50 mm
50 mm
50 mm
15
t /h
Figure 1 Effect of α-mangostin on the viability of COLO 205 cells. Different
concentrations of α-mangostin (0-40 μg/mL) at different incubation times were
studied. Cell viability is expressed as a percentage of the number of viable cells
to that of the control, to which no α-mangostin was applied. Each data point
shown is the mean ± SD from three independent experiments.
3h
for 3 h, lysed in lysis buffer. Cell lysates were subjected
to SDS-PAGE using 12% Tris/HCl ready gels (BioRad),
The transfered proteins were incubated with appropriate antibodies at 4℃ overnight: rabbit polyclonal anticaspase-3 (8G10); mouse polyclonal anti-caspase-8 (1C12);
mouse monoclonal anti-caspase-9 (C9); rabbit polyclonal
anti-Bid; mouse monoclonal anti-p53; rabbit polyclonal
anti-Bax, anti-Bmf (Cell Signaling Technology) and
mouse monoclonal anti-Fas (CD 95) (CH 11, MBL international). After the removal of unbound primary antibodies, the blots were incubated with a secondary antibody
(goat anti-rabbit IgG and goat anti-mouse IgG, each of
which was conjugated with horse radish peroxidase; (Cell
Signaling Technology) as described for Western blotting
analysis of cytochrome c above.
6h
9h
Statistical analysis
Data were expressed as mean ± SD. Statistical comparisons were performed by using one-way analysis of variance. A P value less than 0.05 was considered statistically
significance.
12 h
RESULTS
Cell growth inhibition by α -mangostin
The IC50 value of the three human colonic cancer cell lines,
after 24 h incubation with serial dilutions of α-mangostin,
COLO 205, MIP-101 and SW 620 were 9.74 ± 0.85 μg/mL,
11.35 ± 1.12 μg/mL and 19.6 ± 1.53 μg/mL, respectively.
Among the three cell lines tested, the highest inhibitory
effect of α-mangostin on cell proliferation was detected
with COLO 205. Thus, COLO 205 cells were used as the
primary target in subsequent experiments.
The viability of COLO 205 cells decreased by the
treatment with α-mangostin in both concentration- and
time-dependent fashions (Figure 1). Treatment of COLO
205 cells with α-mangostin at 20 μg/mL or higher for
12 h reduced the number of viable cells to approximately
5%-10% of the control cells, to which no α-mangostin
was applied.
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Figure 2 Effect of α-mangostin on the cell morphology and nuclear condensation of COLO 205 cells. A: Untreated control cells were examined for cell
morphology and Hoescht 33342 stained cells were examined for nuclei morphology; B: Cells were cultured for 0, 3, 6, 9 and 12 h in the presence of 20 μg/mL
α-mangostin.
Morphological and nuclei changes
Cells treated with 20 μg/mL α-mangostin for 3, 6, 9 and
12 h showed evident morphological changes including
rounding and blebbing as well as the presence of apoptotic bodies (Figure 2A, 3, 6, 9 and 12 h). Such morphological changes were not seen with control cells (without
the α-mangostin treatment) (Figure 2A, 0 h). For nuclei
staining with Hoechst 33342, chromatin condensation and
destructive fragmentation of the nucleus with intact cell
membrane was seen with COLO 205 cells which had been
2089
0 mg/mL
1%
5
10
4
10
FL-2H
FL-2H
4
3
10
2
10
2
5
3
4
10
10
FL-1H
10
5
2
5
10
3
4
10
10
FL-1H
10
5
30 mg/mL
10%
3
10
2
10%
Negative
2
10
3
4
10
10
FL-1H
10
5
a
a
5
a
10
0
3
10
2
10
a
10
4
FL-2H
FL-2H
4%
Negative
10
4
10
10
15
3
10
2
20 mg/mL
8%
10
10
0.5%
Negative
10
10
10 mg/mL
2%
Apoptotic cell (%)
5
10
13%
Negative
2
10
3
4
10
10
FL-1H
10
0
10
20
a-mangostin (mg/mL)
30
5
Figure 3 Fluorescent-activated cell sorter analysis of COLO 205 cells stained with Annexin V-FITC. Cells were treated with 10, 20 and 30 μg/mL α-mangostin
for 3 h. α-mangostin induced early apoptosis in a concentration dependent manner. The values are expressed as mean ± SD; aP < 0.05.
treated with 20 μg/mL of α-mangostin for 3, 6, 9 and
12 h (Figure 2B, 3, 6, 9 and 12 h), indicated early apoptosis while the nuclei of control cells without α-mangostin
treatment showed normal morphology (Figure 2B, 0 h).
Accordingly, cleaved activated forms of caspase-3 (19
and 20 kDa), caspase-8 (41 and 43 kDa) and caspase-9
(35 and 37 kDa) (P < 0.01) became apparent upon treatment of α-mangostin at 20 μg/mL or higher. Activated
caspase-3 was apparent upon treatment with 20 μg/mL
α-mangostin but not at higher concentrations (30 and
40 μg/mL α-mangostin) due to cell death at higher concentrations, as the effector caspase-3 is the late event of
the pathways. On the other hand, caspase-8 and -9 were
also detected implying the consecutive activation of caspases of the intrinsic pathway. In addition, induction of
apoptosis by α-mangostin was accompanied by increased
phospho-p53, pro-apoptotic Bax and Bmf (P < 0.01)
(Figure 7). The release of cytochrome c from mitochondria
to cytosol was evident upon treatment of COLO 205 cells
with 20 μg/mL α-mangostin for 3 h and 6 h (Figure 8).
The non-cytosolic fraction (pellet) of the treated cells
showed no cytochrome c expression (data not shown). At
9 h of α-mangostin treatment, cytochrome c was reduced
due to increasing cell death. No appreciable amount of
cytochrome c was detected in the cytosol fraction of control COLO 205 cells, to which no α-mangostin was applied (Figure 8). This implied that α-mangostin mediated
apoptosis is accompanied by mitochondrial dysfunction,
which could be further strengthened by mitochondrial
membrane depolarization detected by a carbocyanine
fluorescence dye, JC-1, upon treatment with α-mangostin.
The results indicated the increased percentage of cells
with depolarized mitochondrial membrane potential (red
to green) approximately 82% after α-mangostin treatment
for 3 h (Figure 9). These results further confirmed that
α-mangostin is an efficient inducer of apoptosis that both
extrinsic and intrinsic pathway may be involved. Thus, we
further conducted the Western blotting analysis of Bid,
t-Bid, the linker between extrinsic and intrinsic pathway,
and Fas receptor and found that they were up-regulated
upon α-mangostin treatment (P < 0.01) (Figure 7).
α -mangostin mediated apoptotic cell death
The early apoptosis was detected upon treatment of COLO
205 cells with 0, 10, 20 and 30 μg/mL of α-mangostin for
3 h using Annexin V-FITC assay. The results indicated
significant increases in apoptotic populations in COLO
205 cells approximately 0.50% ± 0.02%, 4.00% ± 0.25%,
10.00% ± 0.50% and 13.00% ± 0.30%, (P < 0.05) respectively (Figure 3). Exposure of COLO 205 cells to
increasing concentrations (0, 10, 20 and 30 μg/mL) of
α-mangostin for 3 h resulted in increased percentage of
cells arrested in sub G-1 phase (apoptotic cell death) of
1.03% ± 0.15%, 7.00% ± 0.20%, 51.00% ± 1.53% and
80.00% ± 2.08% (P < 0.05), respectively (Figure 4). It is
evident that the formation of apoptotic cells at sub-G1
phase was directly proportional to the increased concentration of α-mangostin.
Fragmentation of chromosomal DNA
Fragmentation of genomic DNA was apparent by the
presence of DNA ladders when COLO 205 cells were
treated with 10, 20 and 30 μg/mL α-mangostin for 12 h
(Figure 5). The characteristic ladder pattern of discontinuous DNA fragments was observed only in the treated
cells, whereas the untreated control showed no DNA
fragmentation.
Activation of caspases upon α -mangostin treatment
The activation of caspases-3, -8 and -9 was detected
(Figure 6). The amount of the pro-enzyme form of caspase-3 (pro-caspase-3, 35 kDa), pro-caspase-8 (57 kDa)
and pro-caspase-9) (47 kDa) decreased with increasing
concentration of α-mangostin (10, 20, 30 and 40 μg/mL).
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0 mg/mL
Counts
a-mangostin (3 h)
800
700
600
500
400
300
200
100
0
a-mangostin (12 h)
M
Sub-G1
G0/G1
S
Counts
10 mg/mL
Counts
20 mg/mL
Counts
30 mg/mL
100
150
200
Sub-G1
250
20 kb
Sub-G1: 7%
G0/G1
S
6 kb
G0/G1: 55%
G2/M
S: 23%
G2/M: 15%
50
100
150
200
Sub-G1
Figure 5 Analysis of DNA integrity in COLO 205 cells upon treatment
with α-mangostin. COLO 205 cells were treated with varying concentrations
of α-mangostin (10, 20 and 30 μg/mL) for 12 h. Cells were lysed, followed by
phenol-chloroform extraction. DNA fractions were then electrophoresed on 1.8%
agarose gels. DNA was stained with ethidium bromide and visualized under a
UV light trans-illuminator.
250
(× 1000)
Sub-G1: 52%
G0/G1
S
G0/G1: 36%
G2/M
apy[26]. In the present study, we showed that α-mangostin
treatment of human colon COLO 205 induced cytotoxic
effects in a dose and time dependent fashion. We then
investigated the apoptotic effects of α -mangostin in
COLO 205 cells to advance our knowledge of its biological functions and also health advantages. The membrane
shrinkage, chromatin condensation and fragmentation
were detected. As cancer growth is associated with the
loss of cell cycle checkpoints, which regulate the DNA
integrity and ensure that the genes are co-ordinately expressed[27]. The sub-G1 fraction and phosphatidylserine
translocation is an indication of apoptosis cell death
that naturally occurs in cells and is beneficial for cancer
therapy[28]. Therefore, to characterize apoptotic cells upon
treatment of COLO 205 cells with α-mangostin, a biparametric cytofluorimetric analysis was performed using
PI and annexin V-FITC, which stained DNA and phosphatidylserine residues, respectively. In the early apoptotic
process, a phosphatidylserine residue became exposed on
the cell surface by flipping from the inner to outer leaflet
of the cytoplasmic membrane[29,30]. Our results demonstrated the increased sub-G1 population and numbers
of early apoptotic cells upon treatment of COLO 205
cells with 20 μg/mL α-mangostin for 3 h as compared to
untreated control. The Bcl-2 family of proteins regulates
apoptosis and it has been shown that the gene products of Bcl-2 and Bax play important roles in apoptotic
cell death[14]. The Bcl-2 family comprises of both proapoptotic and anti-apoptotic proteins that elicit opposite
effects on mitochondria. Anti-apoptotic members include
Bcl-2, Bcl-xL, Bcl-W, Mcl-1, whereas pro-apoptotic members are Bid, Bax, Bakm, Bmf and others. Several pathways involve p53-mediated apoptosis, and one of these
is the Bcl-2 and Bax proteins. The Bax protein is a p53
target and known to promote cytochrome c release from
mitochondria which in turn activates caspase-3. Regulation of Bax/Bcl-2 and caspases activity becomes impor-
S: 9%
G2/M: 3%
50
100
150
200
Sub-G1
250
(× 1000)
Sub-G1: 80%
G0/G1
S
G0/G1: 14%
G2/M
S: 5%
G2/M: 1%
50
100
150
200
250
(× 1000)
100
a
Apoptotic cells (%)
kb
kb
kb
kb
30 kb
(× 1000)
PI-A
75
a
50
25
0
(mg/mL)
40 kb
50
PI-A
800
700
600
500
400
300
200
100
0
200
120
100
50
S: 29%
G2/M: 15%
PI-A
800
700
600
500
400
300
200
100
0
10 20 30
G0/G1: 55%
G2/M
PI-A
800
700
600
500
400
300
200
100
0
0
Sub-G1: 1%
a
0
a
10
20
a-mangostin (mg/mL)
30
Figure 4 Flow analysis of cell cycle. Representative plots of PI staining
of COLO 205 cells that were treated with 0 (control), 10, 20 and 30 μg/mL
a
α-mangostin for 3 h. The values are expressed as mean ± SD; P < 0.05.
DISCUSSION
The evident goal of medical research is to be able to manipulate the machinery of cell death. Regulation of apoptosis might also lead to new possibilities for cancer ther-
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2091
a-mangostin (3 h)
0
10
20
30
Caspase-3/actin
40
1.00
(mg/mL)
Pro-caspase-9
P37
P35
b
b
0.25
40
20
20
10
0
Actin (42 kDa)
pr
of
or
pr
of
or
m
m
0.00
30
P43
P41
b
0.50
m
cle
av
ed
-fo
rm
Pro-caspase-8
0.75
pr
of
or
P20
P19
Relative quantity
Pro-caspase-3
a-mangostin (mg/mL)
Caspase-8/actin
1.00
Caspase-9/actin
1.00
b
0.75
0.50
b
b
b
Relative quantity
Relative quantity
b
0.25
0.75
0.50
b
0.25
b
cle
m
av
ed
-fo
rm
30
pr
of
30
or
cle
m
av
ed
-fo
rm
40
pr
of
40
or
cle
m
av
ed
-fo
rm
pr
of
or
20
20
10
pr
of
or
m
m
pr
of
or
40
0
-fo
rm
cle
av
ed
-fo
rm
m
Cl
ea
ve
d
pr
of
or
30
pr
of
or
m
20
pr
of
or
10
pr
of
or
0
m
0.00
m
0.00
a-mangostin (mg/mL)
a-mangostin (mg/mL)
Figure 6 Effects of α-mangostin on the activation of caspase-3, -8 and -9 in COLO 205 cells. Cells were treated with 10, 20, 30 and 40 μg/mL α-mangostin for 3 h.
Cell lysates were separated by SDS-PAGE using 12% polyacrylamide gels. Proteins were subjected to immuno-detection of caspases-3, -8, and -9 using appropriate
anti-caspase antibodies at 4℃. The expression of cleaved-caspase-3, -8 and -9 were detected. The density of each band was determined, equal protein loading was
verified by β-actin staining. The values are expressed as mean ± SD; bP < 0.01.
Time (3 h)
(mg/mL)
Cleaved-Bid
42 kDa
Actin
id
15 kDa
0
t-B
Bid
d
22 kDa
b
5
Bi
FAS clone CH11
b
b
FA
S
43 kDa
36 kDa
10
3
Phospho-p53 (Ser15)
pp5
53 kDa
p5
3
p53
b
15
f
53 kDa
20
Bm
Bmf
x
18 kDa
25
Ba
Bax
ro
l
20 kDa
Co
nt
20
Relative quantity of proteins
(proteins/actin)
0
Figure 7 Effects of α-mangostin on the activation of Bax, Bmf, p53, Fas and Bid. Cells were treated with 20 μg/mL α-mangostin for 3 h. Cell lysates were
separated by SDS-PAGE using 12% polyacrylamide gels. Proteins were subjected to immuno-detection of Bax, Bmf, p53, p-p53, Fas, Bid and t-Bid using appropriate
antibodies. The density of each band was determined, equal protein loading was verified by β-actin staining. The values are expressed as mean ± SD; bP < 0.01.
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2092
a-mangostin (20 mg/mL)
0
3
6
9
h
Cytochrome c (15 kDa)
Actin (42 kDa)
Relative quantity of cytochrome c
1.5
a
a
1.0
a
0.5
0.0
0
3
6
9
t /h
Figure 8 Release of cytochrome c from the mitochondria in COLO 205 cells. Upon treatment with 20 μg/mL α-mangostin for 3, 6 and 9 h, cytosol fractions were
prepared from these cells and separated by SDS-PAGE using 12% polyacrylamide gels. Proteins were subjected to immuno-detection of cytochrome c using a mouse
monoclonal antibody against human cytochrome c. The density of each band was determined, equal protein loading was verified by β-actin staining. The values are
expressed as mean ± SD; aP < 0.05.
0 mg/mL a-mangostin
97%
0 mg/mL a-mangostin
3%
360
Freq of events
300
260
200
160
100
60
0
2
10
10
3
4
10
10
50 mm
5
FITC-A
20 mg/mL a-mangostin
8%
20 mg/mL a-mangostin
82%
300
Freq of events
260
200
160
100
60
0
2
10
10
3
4
10
10
50 mm
5
FITC-A
% JC-1 positive cell
100
a
75
50
25
a
0
Control
20 μg/mL α-mangostin
Figure 9 Measurements of mitochondrial membrane depolarization in COLO 205 cells. Cells were treated with 0 (control) and 20 μg/mL α-mangostin for 3h and
then incubated with JC-1 (10 μg/mL in PBS) at 37℃ for 10 min. Stained cells were subjected to FACS analysis. The mitochondrial function was assessed as JC-1
green. The values are expressed as mean ± SD; aP < 0.05.
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2093
up-regulated the expression of Bax in COLO 205 cells at
protein level, suggesting that Bcl-2 family protein regulate
α-mangostin mediated apoptotic cell death.
Taken together our results evaluating the molecular
mechanism that α -mangostin induced apoptosis cell
death in COLO 205 cells may occur via caspase-8 dependent cleavage of Bid to tBid providing a link between extrinsic and intrinsic pathways (Figure 10). This could be
a promising chemotherapeutic agent and may also serve
as a model to develop and design new derivatives which
may be more potent.
FasL
Cell membrane
O
HO
O
O
Fas
OH
OH
Procaspase 8
p53
Caspase 8
Mitochondria
Bid
t-Bid p53 Bmf
Bax
DYm loss
Cytochrome c
Procaspase 9
Caspase 9
COMMENTS
COMMENTS
Background
Procaspase 3
Apoptosis
Cancer causes significant morbidity and mortality and is a major public health
problem worldwide. Globally, colorectal cancer is one of the most common
types of cancer in both men and women. Phytochemical studies showed that a
variety of tropical plants have useful biological activities and some offer potential therapeutic applications. However, the molecular mechanisms underlying
α-mangostin induced apoptosis in human colorectal adenocarcinoma have not
yet been fully understood.
Caspase 3
Figure 10 A proposed diagram for α-mangostin-induced apoptosis in COLO
205 cells. Upon α-mangostin treatment, extrinsic pathway was activated, procaspase-8 was cleaved to caspase-8 which then further activated the cleavage of
Bid to t-Bid. The t-Bid then translocates to mitochondria resulting in the activation of mitochondrial apoptotic pathway.
Research frontiers
Apoptosis plays a vital role in controlling cell number in many physiological
and developmental stages, tissue homeostasis, and regulation of the immune
system, while insufficient apoptosis is an integral part of cancer development.
The main function of apoptosis is to dispose of a cell without causing damage
or stress to neighbouring cells. Thus, the anti-cancer drug that induces apoptotic cell death would be more suitable for use in patients and should be further
developed. Therefore, the effect of α-mangostin on the growth and apoptosis
induction of human colon cancer cells was investigated in this study.
tant targets for cancer intervention. Caspase-3 being the
major executioner caspase[31], thus we examined whether
activated caspase-3, -8 and -9 is involved in apoptotic
induction and found evident expression of activated caspase-3, -8 and -9.
Under the influence of α-mangostin treatment in
COLO 205 cells, a cell death pathway both via death
receptor pathway and mitochondrial pathway may be
involved, as caspase-8 and -9 were expressed. We further demonstrated the loss of mitochondrial membrane
potential, release of cytochrome c into the cytosol and
DNA fragmentation. Our results are in agreement with
a study showing that apoptosis was associated with a
loss of mitochondrial membrane potential, which may
correspond to the opening of an outer membrane pore,
leading to cytochrome c release from mitochondria into
the cytosol. The released cytochrome c later triggered the
cleavage and activation of caspases and onset of apoptosis[32]. The expression of Fas and caspase-8 implies the
death receptor pathway, while regulation of mitochondrial membrane permeability by upregulation of Bax and
p-Bad triggering the release of cytochrome c from mitochondria to cytosol. Our further investigation showed
the expression of Fas, Bid, t-Bid, p-53, phospho-p53 and
the proteins in Bcl-2 family (Bax and Bmf). Expression
of t-Bid is the important linkage between death receptor
and mitochondrial pathway. When Bid is cleaved into t-Bid
by the activated caspase-8 and translocated towards the
mitochondria to activate Bax and Bak, then changing the
mitochondrial membrane permeability and the release of
cytochrome c[33] which form a complex with apoptotic
enzyme activators and caspase-9. It activates and starts
a caspase cascade reaction, which further activates the
downstream caspase-3 and other caspase family members
for apoptosis induction. We also noticed that α-mangostin
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Innovations and breakthroughs
The results showed that α-mangostin induced apoptotic cell death in COLO
205 cells indicating by membrane blebbing, chromatin condensation, DNA
fragmentation, cell cycle analysis, sub-G1 peak, and phosphatidylserine exposure. The expression of caspase-3, caspase-8 and caspase-9, cytochrome
c release, Bax, p53 and Bcl-2 modifying factor (Bmf) as well as reduced
mitochondrial membrane potential were demonstrated. In addition, upon treatment with α-mangostin, up-regulation of tBid and Fas were evident. Therefore,
α-mangostin may be effective as an anti-cancer agent that induces apoptotic
cell death in COLO 205 via a link between extrinsic and intrinsic pathways.
Applications
α-mangostin could be used as a future promising anti-cancer agent for the
treatment of colorectal adenocarcinoma cells.
Terminology
The fruit hull of mangosteen (Garcinia mangostana L.) in the Clusiaceae family is rich in a variety of oxygenated and prenylated xanthones which possess
different biological properties, such as anti-mycobacterial, anti-fungal, antioxidant, cytotoxicity and anti-inflammatory activities.
Peer review
In this study the authors examined the effect of α-mangostin on the growth and
apoptosis induction of human colon cancer cells. They showed that α-mangostin
induced apoptotic cell death in COLO 205 cells, activated caspase-3, -8, and -9,
increased the mitochondrial cytochrome c release, Bax, p53 and Bmf, reduced
mitochondrial membrane potential, and up-regulated tBid and Fas. From these
results, the authors suggested that α-mangostin may be effective as an anticancer agent via a link between extrinsic and intrinsic pathways. It is interesting
that the authors clearly showed the mechanism through which α-mangostin
induced apoptosis in colon cancer cells.
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2095
[email protected]
doi:10.3748/wjg.v17.i16.2096
ORIGINAL ARTICLE
Chemometrics of differentially expressed proteins from
colorectal cancer patients
Lay-Chin Yeoh, Saravanan Dharmaraj, Boon-Hui Gooi, Manjit Singh, Lay-Harn Gam
by applying an eigenvalue > 1 was successfully used to
reduce the number of principal components (PCs) into
12 and seven PCs for Tris and TLB extracts, respectively,
and subsequently six PCs, respectively from both the
extracts were used for LDA. The LDA classification for
Tris extract showed 82.7% of original samples were correctly classified, whereas 82.7% were correctly classified
for the cross-validated samples. The LDA for TLB extract
showed that 78.8% of original samples and 71.2% of
the cross-validated samples were correctly classified.
Lay-Chin Yeoh, Lay-Harn Gam, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, 11800, Malaysia
Saravanan Dharmaraj, Centre for Drug Research, Universiti
Sains Malaysia, Penang, 11800, Malaysia
Boon-Hui Gooi, Manjit Singh, Department of Surgery, Penang
General Hospital, Penang, 10990, Malaysia
Author contributions: Gam LH conceived the design of the
study and edited the manuscript; Yeoh LC carried out the experimental work and manuscript writing; Dharmaraj S carried out the
statistical analyses; Gooi BH and Singh M provided the colorectal cancer specimens and patient information.
Supported by Research Universiti Grant, Grant No. 1001/PFAR
MASI/815007
Correspondence to: Lay-Harn Gam, PhD, School of Pharmaceutical Sciences, Universiti Sains Malaysia, Penang, 11800,
Malaysia. [email protected]
Telephone: +60-4-6533888 Fax: +60-4-6570017
Received: August 13, 2010 Revised: September 18, 2010
Accepted: September 25, 2010
CONCLUSION: The classification of CRC tissues by
PCA and LDA provided a promising distinction between
normal and cancer types. These methods can possibly be used for identification of potential biomarkers
among the differentially expressed proteins identified.
Key words: Colorectal cancer; Proteomics; Marker protein; Principal component analysis; Linear discriminant
analysis
Abstract
AIM: To evaluate the usefulness of differentially expressed proteins from colorectal cancer (CRC) tissues
for differentiating cancer and normal tissues.
Peer reviewer: Ki-Baik Hahm, MD, PhD, Professor, Gachon
METHODS: A Proteomic approach was used to identify the differentially expressed proteins between CRC
and normal tissues. The proteins were extracted using
Tris buffer and thiourea lysis buffer (TLB) for extraction
of aqueous soluble and membrane-associated proteins,
respectively. Chemometrics, namely principal component analysis (PCA) and linear discriminant analysis
(LDA), were used to assess the usefulness of these
proteins for identifying the cancerous state of tissues.
Yeoh LC, Dharmaraj S, Gooi BH, Singh M, Gam LH. Chemometrics of differentially expressed proteins from colorectal cancer patients. World J Gastroenterol 2011; 17(16): 2096-2103 Available
from: URL: http://www.wjgnet.com/1007-9327/full/v17/i16/
2096.htm DOI: http://dx.doi.org/10.3748/wjg.v17.i16.2096
Graduate School of Medicine, Department of Gastroenterology,
Lee Gil Ya Cancer and Diabetes Institute, Lab of Translational
Medicine, 7-45 Songdo-dong, Yeonsu-gu, Incheon, 406-840,
South Korea
RESULTS: Differentially expressed proteins identified
were 37 aqueous soluble proteins in Tris extracts and 24
membrane-associated proteins in TLB extracts. Based
on the protein spots intensity on 2D-gel images, PCA
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INTRODUCTION
Proteomic research has made great achievements in biomarker discovery, especially when incorporated with high-
2096
Yeoh LC et al . Chemometrics of colorectal cancer biomarkers
throughput analytical tools and technology, for example
2D-PAGE and LC-MS/MS[1]. Two-dimensional gel electrophoresis is a fundamental tool for protein analysis to
detect alterations in protein expression between control
and disease states of cells, which can lead to the discovery of various biomarkers that contribute to pathogenesis
or carcinogenesis[2]. Biomarkers can be used to discriminate variables for subsequent classification of normal and
diseased groups[3]. The complexity of variables generated
by mass spectra, microarray and immunohistochemistry
often requires advanced statistical techniques or chemometrics to evaluate their clinical value.
Multivariate analyses including the dimension reduction method known as principal component analysis
(PCA), and classification methods such as linear discriminant analysis (LDA) are often employed in proteomic
studies. PCA reduces the number of variables for further
data analysis and interpretation while identifying the variables that retain most of the data variance[4]. A principal
component (PC) is defined as a new variable to explain
the maximum amount of variance in the original data
and corresponds to a linear combination of the original
variables. PCs are presented orthogonally to each other,
which provides a more effective representation of the
data than the original variables[2]. LDA is a multivariate
technique to classify observations into groups or categories. LDA forms new variables from the original data and
identifies the variables that provide the best discrimination between the groups[5].
Djidja et al[6] have used a novel approach that combines
matrix-assisted laser desorption ionization-ion mobility
separation-mass spectrometry (MALDI-IMS-MS) and
PCA-discriminant analysis (PCA-DA) to generate tumor
classification models based on pancreatic cancer protein
patterns. Furthermore, Kamath et al[7] have used PCAbased k-nearest neighbor analysis to classify normal and
cancerous autofluorescence spectra of colonic mucosal
tissues. Zwielly et al[8] have investigated the use of Fourier
transform infrared microscopy for colon cancer diagnosis.
Their model uses PCA to define spectral changes among
normal and cancerous human biopsied colon tissues.
Ragazzi et al[9] have reported the use of multivariate techniques on plasma proteins to diagnose colorectal cancer
(CRC). The plasma protein profile generated by MALDIMS is analyzed by PCA and LDA to discriminate ionic
species from normal subjects and CRC patients.
In this study, we carried out the comparison of 2-D
images of cancerous and normal colorectal tissues. The
differentially expressed proteins from Tris and thiourea
lysis buffer (TLB) extractions were respectively tested on
a PCA-LDA model to find out the possibility of using
protein expression to classify the disease and non-disease
tissues of CRC.
CRC patients were collected after surgery at the Penang
General Hospital, Penang, Malaysia. The study was approved by the Human Ethical Committee of Universiti
Sains Malaysia. Informed written consent was received
from all patients before the study was conducted. Prior to
surgery, the patients did not receive preoperative neoadjuvant chemotherapy and radiotherapy. The tissues were
confirmed as cancerous and normal, respectively, by the
hospital’s pathologist. The cancerous tissues were classified using the TNM system. Surgically removed samples
were stored at -80℃ until use.
The differential expression of the proteins was tested by
the paired Student’s t test that is included in PDQuest,
to determine their statistical significance (P < 0.05). For
Protein analysis
The method of protein analysis was as described in Yeoh
et al[10]. Frozen tissue (250 mg) was rinsed in distilled water
to remove cell debris and excess blood. The tissues were
homogenized in ice-cold Tris buffer (0.5 g tissue/mL buffer) [40 mmol/L Tris and 1 × Protease Inhibitor Cocktail
(Sigma, St Louis, MO, USA)] and centrifuged at 12 000 rpm
for 15 min at 18℃. The supernatant was recovered and
labeled as Tris extract. The pellet was subjected to further
extraction using TLB (1 g tissue/1 mL buffer) [8 mol/L
urea, 2 mol/L thiourea, 4% (w/v) CHAPS, 0.4% (w/v)
carrier ampholytes and 50 mmol/L dithiothreitol] and
centrifuged at 12 000 rpm for 15 min at 18℃. The supernatant was recovered and labeled as TLB extract. The
extracts were subjected to 2D gel separation on 11 cm
ReadyStrip™ IPG strip (linear pH 4-7, Bio-Rad, USA)
followed by separation on 10% (w/v) PAGE at a constant
voltage of 200 V. The gels were stained with Coomassie
Blue. The images obtained were analyzed by PDQuest
version 7.3 (Bio-Rad). Comparison of the protein expression levels was carried out between cancerous and
normal tissues. Differentially expressed proteins were
defined as proteins with a spot intensity that was 1.5-fold
higher or lower in cancerous tissues when compared to
that in the corresponding normal tissues. A differentially
expressed protein was defined as upregulated when it was
found at greater intensity in cancerous tissue than in the
corresponding normal tissue. The downregulated proteins were detected at greater intensity in normal tissues
than in the corresponding CRC cancerous tissues.
Protein identification
The differentially expressed proteins were excised from
the gel and subjected to in-gel digestion using trypsin and
the tryptic peptides were analyzed by LC/MS/MS using
an electrospray ionization ion trap mass analyzer (Agilent
Technologies, Santa Clara, CA, USA). The MS/MS data
were subjected to the MASCOT protein database search
engine for protein identification. The identities of a few
proteins (dependent on the availability of antibodies)
were further confirmed using western blotting.
Tissue specimen collection
Matching pairs of normal colonic mucosa and cancerous
colonic tissue (located 10 cm from each other) from 26
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2097
Table 1 Clinicopathological features of 26 colorectal cancer patients involved in study
Patient No.
1
2
3
4
5
6
7
8
9
10
11
Age (yr)
Race
Sex
pTNM
Stage
Degree of differentiation
Tumor location
62
79
74
37
58
59
69
63
84
58
Malay
Malay
Malay
Malay
Malay
Malay
Malay
Malay
Malay
Chinese
Chinese
Male
Male
Male
Male
Male
Female
Female
Male
Female
Female
Male
pT3N1Mx
pT2NoM0
pT3N0M0
pT3N2Mx
pT3N0M0
pT3N0Mx
pT4N2Mx
pT3N0Mx
pT3N0Mx
pT4N0M0
pT3N0Mx
ⅢB
MD
MD
MD
MD
MD
MD
MD
MD
MD
MD
MD
Sigmoid colon
Descending colon
Ascending colon
Rectum
Transverse colon
Recto-sigmoid
Ileocecal
Sigmoid colon
Recto-sigmoid
Rectum
Recto-sigmoid
Ⅰ
ⅡA
ⅢC
ⅡA
ⅡA
ⅢC
ⅡA
ⅡA
ⅡB
ⅡA
MD: Moderately differentiated adenocarcinoma.
30 000
25 000
Protein intensity
Normal
Cancer
20 000
15 000
10 000
5000
0
Normal
Cancer
Figure 1 Comparison of protein spot intensity between normal and colorectal cancer tissues for glutathione S-transferase P.
PCA and LDA, the protein spot intensities were exported
out from PDQuest and imported into SPSS version 15.0
(Chicago, IL, USA) to perform multivariate analyses. Protein spot intensities were used as variables.
pressed proteins in all patients for Tris and TLB extracts,
respectively. An example of the differentially expressed
protein, as represented by different intensities of protein
spots between normal and cancerous tissues for glutathione S-transferase P (GST-P), is shown in Figure 1; the bar
chart was plotted according to the intensity of the respective protein spots. GST-P was detected as upregulated in
cancerous tissues.
RESULTS
The tissues specimens from each patient were collected
in pairs of cancerous and normal tissues. Table 1 shows
the details of the tissues used in the analysis. The tissues
were subjected to a sequential extraction method to extract aqueous soluble proteins and membrane-associated
proteins in two different fractions using Tris and TLB, respectively. Tables 2 and 3 show the 37 and 24 differentially expressed proteins identified in Tris and TLB extracts,
respectively. The average fold change indicates the degree
of differentiation in expression levels of the protein in
cancerous tissues compared to normal tissues in all the
patients tested, where a positive sign indicates a greater
expression level in cancerous tissues, whereas a negative
sign indicates a greater expression level in normal tissues. The MOWSE score refers to the score values given
by the MASCOT search. Tables 4 and 5 show the mean
intensity of spots and SD, and percentage coefficient of
variation (%CV) of spot intensity of differentially ex-
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Data analysis
The significance of the expression levels of the differentially expressed proteins in both Tris and TLB extracts
was analyzed by Student’s t test. After univariate analysis
was performed, the normalized intensities of 37 differentially expressed protein spots in Tris extracts were
subjected to PCA. The PCA reduced the original data to
12 PCs based on an eigenvalue of > 1, and these 12 PCs
contributed 76.43% of the total data variance of the Tris
extract data. Figure 2 shows the 3D PC plot with the xy- and z-axes representing the first, second and third PC
number. The variables that had the highest loadings were
those that contributed most to the differentiation of the
disease state. Figure 3 shows the scree plot of Tris extracts. Six PCs were chosen and these components contributed 53.97% of the total variance of the Tris extract
2098
Table 2 List of proteins found in 2D gel of Tris extracts
Spot
No.
Protein name
Swissprot
1
No.
MOWSE
2
score
MW (Da)
pI
Sequence
coverage (%)
GRAVY
Average fold
3
change
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Proteasome subunit β type 6
14-3-3 protein ζ
Tropomyosin α-3C-like protein
Rho GDP-dissociation inhibitor 1
14-3-3 protein ζ
Tubulin β-2C chain
Cathepsin B
Rho GDP-dissociation inhibitor 2
SEC13 homolog
Hsc70-interacting protein
Apolipoprotein A-I
Proteasome subunit α type 3
Actin, cytoplasmic 2
60 kDa heat shock protein
Peroxiredoxin-2
Guanine nucleotide binding protein subunit β 2
F-actin-capping protein subunit β
GST-P
Haptoglobin-related protein
Cathepsin Z
F-actin-capping protein subunit β
Actin-related protein 3
Abhydrolase domain-containing protein 14B
Nucleoside diphosphate kinase A
L-lactate dehydrogenase B chain
Fibrinogen β chain
Leukocyte elastase inhibitor
PDI A3
Gelsolin
Heat shock 27 kDa protein
DJ-1 protein
Fibrinogen β chain
Selenium-binding protein 1
Leukotriene A-4 hydrolase
Proteasome subunit α type 6
P28072
P63104
A6NL28
P52565
P63104
P68371
P07858
P52566
P55735
P50502
P02647
P25788
P63261
P10809
P32119
P62879
P47756
P09211
P00739
Q9UBR2
P47756
P61158
Q96IU4
P15531
P07195
P02675
P30740
P30101
P06396
P04792
Q99497
P02675
Q13228
Q13228
Q13228
P09960
P60900
134
336
127
167
282
524
74
48
78
164
143
201
105
151
283
112
259
730
49
100
245
148
200
87
228
151
170
674
238
256
122
75
502
592
979
215
71
25573
35567
27407
23120
27919
50304
22981
22901
36040
28464
30777
15958
26169
61348
21935
37954
34187
23442
39529
27787
21280
47704
25429
19873
36928
56624
42857
57202
86103
22840
20079
56624
52971
52938
52938
69792
20988
4.80
6.97
4.71
5.03
4.73
4.83
5.20
5.10
5.22
8.92
5.56
6.82
5.65
5.70
5.67
5.60
6.02
5.44
6.42
5.48
7.93
5.61
6.82
5.42
5.71
8.54
5.90
5.98
5.90
5.98
6.33
8.54
5.93
5.93
5.93
5.80
8.57
16
40
31
29
16
40
18
18
9
21
26
41
14
14
42
11
37
60
3
15
34
27
26
36
14
22
11
35
20
47
54
22
21
30
37
22
39
0.034
-0.744
-0.992
-0.700
-0.621
-0.362
-0.433
-0.799
-0.372
-0.653
-0.717
0.008
-0.156
-0.074
-0.210
-0.183
-0.574
-0.131
-0.308
-0.545
-0.540
-0.271
-0.023
-0.075
0.056
-0.758
-0.249
-0.506
-0.415
-0.567
0.004
-0.758
-0.254
-0.254
-0.254
-0.259
-0.247
-2.967
11.659
44.183
-7.607
4.127
-52.184
33.149
-10.625
6.873
20.959
-4.478
4.249
28.601
131.219
1.250
-14.442
33.554
4.834
56.209
-60.766
13.278
15.881
0.765
73.120
3.513
41.329
10.458
7.579
-11.917
-1.508
4.981
-72.722
-26.544
27.403
-1.887
29.759
0.768
1
Protein accession number at SwissProt at http://www.expasy.org/uniprot; 2MOWSE score from MASCOT protein database search at http://www.
matrixscience.com, where score > 41 is statistically significant (P < 0.05); 3Average ratio of the spot intensity in normal mucosa over tumor tissue (negative
variation or decrease) or tumor tissue over normal tissue (positive variation or increase). GST-P: Glutathione S-transferase P; PDI: Protein disulfide isomerase.
4.0
Type
3.0
Cancer
Normal
6
5
Eigenvalue
2.0
PC2
1.0
0.0
4
3
2
-1.0
1
-2.0
3.0
1.0
PC1
2.0
0.0
-1.0
0
-2.0
-3.0
Scree plot
7
.0
.0 -1
.0 0
0 1
.
2
PC3
3.0
3.0
2.0 -
1
-
9
13 17 21 25 29
Principal component
33
37
Figure 3 Scree plot showing principal components and their eigenvalues
in Tris extracts.
Figure 2 Principal component plot of Tris proteins.
out of 26 original cancer tissues were correctly classified.
In cross-validated samples, 22 out of 26 normal tissues
and 21 out of 26 cancer tissues were correctly classified.
data. Table 6 shows the LDA results for Tris extract proteins, where 22 out of 26 original normal tissues, and 21
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5
2099
Table 3 List of proteins found in 2D gel in thiourea lysis buffer extracts
Spot
No.
Protein name
SwissProt
1
No.
MOWSE
2
score
MW (Da)
pI
Sequence
coverage (%)
GRAVY
Average fold
3
change
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Tropomyosin α-4 chain
Putative tropomyosin α-3-chain-like protein
GC1q-R, mitochondrial
Calreticulin
Prohibitin
Heat shock 70 kDa protein
Tubulin β-2C chain
PDI
ATP synthase subunit β, mitochondrial
ATP synthase D chain
Chloride intracellular channel protein 1
Tubulin α-1 chain
Apolipoprotein A-I
Actin, cytoplasmic 2
Actin, aortic smooth muscle
Stomatin-like protein 2
60 kDa heat shock protein, mitochondrial
Triosephosphate isomerase
Annexin A5
Cytochrome b-c1 complex subunit 1, mitochondrial
Annexin A3
Annexin A4
α-enolase
Lamin-A/C
P67936
A6NL28
Q07021
P27797
P35232
P11021
P68371
P07237
P06576
O75947
O00299
Q71U36
P02647
P63261
P62736
Q9UJZ1
P10809
P60174
P08758
P31930
P12429
P09525
P06733
P02545
139
53
123
73
421
775
299
266
1096
117
299
61
129
52
261
151
451
167
195
96
140
165
143
198
28506
27407
31768
47092
29890
72488
48142
57510
56559
18406
27123
50800
28078
42009
42154
38644
61386
26828
35994
53342
36396
35983
47385
65192
4.67
4.71
4.74
4.30
5.57
5.07
4.70
4.82
5.26
5.22
5.09
4.94
5.27
5.31
5.23
6.88
5.70
6.51
4.94
5.94
5.63
5.85
6.99
6.40
33
25
20
11
41
42
25
42
43
32
30
6
37
4
21
28
28
24
39
18
22
33
12
25
-1.033
-0.992
-0.461
-1.191
0.024
-0.487
-0.347
-0.450
0.018
-0.569
-0.293
-0.229
-0.840
-0.205
-0.233
-0.161
-0.074
-0.126
-0.330
-0.141
-0.430
-0.447
-0.226
-0.947
-51.151
4.922
-3.333
1.394
0.032
-32.940
-9.060
-1.515
-15.661
-5.129
20.288
-30.291
78.135
-26.716
46.181
-29.709
14.023
16.757
-2.019
13.151
31.244
11.890
85.960
-3.378
1
Protein accession number as SwissProt at http://www.expasy.org/uniprot; 2MOWSE score from MASCOT protein database search at http://www.
matrixscience.com, where score > 41 is statistically significant (P < 0.05); 3Average ratio of the spot intensity in normal mucosa over tumor tissue (negative
variation or decrease) or tumor tissue over normal tissue (positive variation or increase). PDI: Protein disulfide isomerase.
4.0
Type
3.0
Cancer
Normal
Scree plot
5
4
Eigenvalue
PC2
2.0
1.0
0.0
3
2
-1.0
1
-2.0
0
0
3.
-
1
.0
-2
.0
-1
0
0.
1
PC
0
1.
0
2.
0
3.
3.0
2.0
1.0
0.0
PC3
-1.0
-2.0
5
7
9
12 15
18
Principal component
21
24
Figure 5 Scree plot showing principal components and their eigenvalues
in thiourea lysis buffer extracts.
-3.0
67.61% of the total data variance of TLB extracts. Table 7
shows the LDA results of TLB extracts, where 22 out of
26 original normal tissues, and 19 out of 26 original cancerous tissues were correctly classified. In cross-validated
samples, 21 out of 26 normal tissues and 16 out of 26
cancerous tissues were correctly classified. The average
percentages of correct classification for original and crossvalidation samples were 78.8% and 71.2%, respectively.
Figure 4 Principal component plot of thiourea lysis buffer proteins.
Both original and cross-validation samples had an average
82.7% correct classification.
Figure 4 shows the 3D view of the PCs plot for the
TLB extract. PCA reduced the original data of the TLB
extract to seven PCs based on an eigenvalue one of >
1, and the seven PCs accounted for 72.46% of the total
data variance. The 3D view indicates that tissues can be
grouped according to CRC disease state. Figure 5 shows
the scree plot of the TLB extracts. Six PCs were chosen based on the slope of scree plot, which contributed
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3
DISCUSSION
The expression levels of the differentially expressed protein between colorectal cancerous and normal tissues were
2100
Table 4 mean ± SD and percentage coefficient of variation
of spot intensities of Tris proteins
Table 5 mean ± SD and percentage coefficient of variation
of spot intensities of thiourea lysis buffer proteins
Protein spot
No.
Protein spot
No.
Intensity of spots
(mean ± SD)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
10 918.80 ± 8005.09
8516.42 ± 7898.33
3986.45 ± 3471.51
36 146.18 ± 24 859.84
13 329.50 ± 7123.20
4091.51 ± 4636.51
6512.40 ± 6048.73
13 401.28 ± 8031.43
24 196.99 ± 14 907.64
4861.29 ± 4327.71
4128.52 ± 3764.18
3522.46 ± 2821.84
9624.81 ± 8295.52
5407.19 ± 5270.17
4683.89 ± 6994.94
2633.26 ± 2593.91
10 104.77 ± 10 369.91
16 086.82 ± 19 928.39
6791.99 ± 5063.21
7596.19 ± 4759.49
2685.37 ± 3298.54
5022.01 ± 3735.74
5957.62 ± 7526.42
2323.67 ± 2269.62
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
Intensity of spots
(mean ± SD)
% CV of spot intensity
2565.84 ± 2247.86
3865.47 ± 3766.11
2424.01 ± 1847.71
4957.17 ± 2923.49
3901.55 ± 3900.52
2105.64 ± 2444.14
2572.91 ± 1765.28
2959.95 ± 2177.86
2478.29 ± 1697.98
1253.48 ± 1472.88
3373.93 ± 2451.35
3247.26 ± 2519.26
9413.58 ± 10 685.11
2735.49 ± 2665.85
8354.35 ± 4824.59
7370.39 ± 7935.67
14 200.72 ± 16 194.91
6254.81 ± 5105.54
14 364.73 ± 10 849.77
10 753.33 ± 14 509.06
5171.49 ± 3304.12
3230.12 ± 1905.24
2114.69 ± 1164.19
2331.41 ± 2122.56
9254.07 ± 4830.01
9118.41 ± 9336.23
3750.45 ± 3869.35
8098.16 ± 5450.79
3984.55 ± 2658.12
4236.70 ± 4229.74
3932.80 ± 2507.88
1681.49 ± 2019.10
6600.04 ± 4860.85
3121.51 ± 2694.58
8587.77 ± 5871.40
939.46 ± 1682.25
3780.67 ± 1967.05
87.60
97.42
76.23
58.97
99.97
116.08
68.61
73.58
68.51
117.50
72.66
77.58
113.51
97.45
57.75
100.34
114.04
81.63
75.53
134.93
63.89
58.98
55.05
91.04
52.19
102.39
103.17
67.31
66.71
99.84
63.77
120.08
73.65
86.32
68.37
179.07
52.03
73.31
92.74
87.08
68.78
53.44
113.32
92.88
59.93
61.61
89.02
91.18
80.11
86.19
97.47
149.34
98.51
102.62
123.88
74.55
62.66
122.84
74.39
124.65
97.67
CV: Coefficient of variation.
Table 6 Percentage of correct classification of normal and
colorectal cancer tissues in Tris extracts using linear discriminant analysis
Type
Original count
Cancer (26)
Normal (26)
Cross-validated count
Cancer (26)
Normal (26)
CV: Coefficient of variation.
analyzed using PCA based on a multivariate analysis approach, to assess their usefulness in classifying colorectal
tissues as cancerous or normal. The differentially expressed
proteins identified showed good consistency in their expression levels in cancerous and normal tissues. The proteins were extracted in two fractions according to their
polarities. In the PCA-LDA model, the selected proteins
from the first few PCs were able to discriminate colorectal tissues with and without CRC.
A scree plot was derived by plotting the eigenvalues
against the PC number. The shape of the plot was used
to evaluate the number of PCs to be retained. In general,
the point at which the scree plot straightens out indicates
the number of PCs to be extracted[11]. Cross-validation
is a method to estimate the accuracy of a predicted classification model if performed using new future data sets
(samples); this is because a classification model is considered incomplete until the prediction error is estimated[12].
One method of cross validation is leave-one-out crossvalidation, where one sample from the data set of N
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% CV of spot intensity
Predicted group membership
% correct
Cancer
Normal
classification
21
4
5
22
82.7
21
4
5
22
82.7
Table 7 Percentage of correct classification of normal and
colorectal cancer tissues in thiourea lysis buffer extracts using
linear discriminant analysis
Type
Original count
Cancer (26)
Normal (26)
Cross-validated count
Cancer (26)
Normal (26)
Predicted group membership
% correct
classification
Cancer
Normal
19
4
7
22
78.8
16
5
10
21
71.2
samples is removed, the discriminant rule is recalibrated,
and a classification model is built based on the remaining
N - 1 data. The one sample that is left out is classified in
this model and the process repeated N times[12].
2101
PCA and LDA results from Tris extract indicated that
six out of 37 proteins were reliable to determine the tissues with CRC. The proteins comprised five upregulated
proteins, namely GST-P, tropomyosin α-3C-like protein,
F-actin capping protein subunit β, selenium binding protein 1 and DJ-1 protein, and one downregulated protein,
namely, proteasome subunit β type 6. DJ-1 protein and
GST-P contributed the most to the first PC based on
the weight of their loadings. This was followed by the
tropomyosin α-3C-like protein and proteasome subunit
β type 6 that contributed to the second PC, while F-actin
capping protein subunit β and selenium binding protein
1 contributed to the third PC. The initial PCA reduced
the original data and therefore enabled LDA to be carried
out because LDA is sensitive to the number of variables.
In LDA, the six PCs chosen were shown to be capable of
predicting whether the tissues were with or without CRC.
Two-way validation by using original and cross-validation
analyses was applied to validate the state of the tissues,
where the cancerous and normal tissues were classified
correctly at 82.7% for both original and cross-validation
samples.
Two proteins that contributed most to PC1 in Tris extract were DJ-1 and GST. DJ-1 is a putative oncoprotein
that is able to transform cells with H-Ras[13]. Overexpression of DJ-1 activates protein kinase B, which subsequently increases cell survival. Furthermore, increased DJ-1
expression also activates Nrf2 (nuclear factor erythroid
2-related factor), which in turn increases expression of
antioxidant enzymes that confer a survival advantage to
tumor cells[14]. Upregulation of DJ-1 protein in esophageal squamous cell carcinoma is correlated with lymph
node metastasis[15]. Although there is no reported role of
DJ-1 in CRC, its upregulation in CRC is undeniable, and
we have shown that its expression can be used to discriminate between CRC cancerous and normal tissues.
GST catalyzes the conjugation of reduced glutathione
to electrophiles[16]. GST functions to remove peroxides
from endogenous compounds such as lipids and DNA[17].
Overexpression of GST-P1 in CRC may be involved in
cell proliferation, differentiation and apoptosis[18]. GST-P1
is overexpressed in liver cancer cells[19].
In TLB extract, six of the 24 differentially expressed
proteins identified were found to be useful in discriminating CRC cancerous from normal tissues. These proteins were protein disulfide isomerase (PDI), complement component 1 Q subcomponent-binding protein
(GC1q-R), chloride intracellular channel protein 1, triosephosphate isomerase, annexin A5 and actin cytoplasmic
2. All the proteins were downregulated in TLB extracts,
except chloride intracellular channel protein 1 and triosephosphate isomerase. PDI and GC1q-R contributed
the most to the first PC based on the weight of their
loadings. This was followed by the chloride intracellular
channel protein 1 and triosephosphate isomerase that
contributed the most to the second PC, while annexin A5
and actin cytoplasmic 2 contributed most to the third PC.
In LDA, the six PCs that explained 67.61% of the total
variance were able to distinguish CRC cancerous from
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normal tissues. The leave-one-out cross-validation obtained 71.2% correct classification of normal and cancerous tissues. The value for original grouped samples was
higher with 78.8% correct classification.
Two proteins that contributed most to PC1 in TLB
extracts were PDI and GC1q-R. PDI catalyzes the formation and breakage of disulfide bonds between two
cysteine residues[20]. PDI regulates cell transformation
and intracellular and extracellular redox activities via its
reductase activity[21]. PDI regulates STAT3 signaling and
proliferation, which is thought to induce malignancy[22].
PDI is upregulated in CRC cell lines and its upregulation
is correlated with cancer cell differentiation[23,24].
GC1q-R is a cell surface glycoprotein, which binds to
the globular heads of C1q molecules[25]. C1q molecules
bind to a variety of cells such as B cells, monocytes, macrophages, endothelial and smooth muscle cells[26]. C1q
elicits responses such as phagocytosis in monocytes and
activation of tumor cytotoxicity of macrophages[27,28].
GC1q-R is overexpressed in colon cancer cells and may
be involved in tumor metastasis. However, PDI and
GC1q-R were downregulated when using average fold
change to determine their expression levels.
Proteins are the expression components that regulate
cell activity. Differential expression of proteins is expected upon transformation of normal cells to cancerous
cells. These differentially expressed proteins are useful
in diagnosis and prognosis of the disease. In the present study, the specimens used in the analysis comprised
tissues from female and male patients who were diagnosed with various stages, grades and locations of CRC.
Regardless of the sex of the patients and pathological
specification of the tissues, we showed that the differentially expressed protein identified from 2D protein
profiles of cancerous and normal tissues could be used
to separate and classify normal and cancerous tissues
by combining PCA and LDA. The data reduction technique of PCA was sufficient to provide a classification
of tissues according to CRC disease state. These statistical models simplify the data management through the
reduced dimensionality of protein spots from the 2D gel
images. Therefore, multivariate analysis of differentially
expressed proteins identified from cancerous and normal
tissues may be used as a tool for diagnosis and prognosis
of CRC disease state.
ACKNOWLEDGMENTS
We want to express our appreciation to Universiti Sains
Malaysia for provided the grant under RU funding to
conduct this project. We would also like to thank National Institute of Pharmaceutical and Neutraceutical for
their generosity in allowing us to conduct the LC/MS/
MS experiments.
COMMENTS
COMMENTS
Background
Colorectal cancer (CRC) is one of the leading causes of death worldwide. Dif-
2102
ferentially expressed proteins between cancerous and normal colonic tissues
were identified using 2D gel separation followed by LC/MS/MS analysis. The
protein spot intensities of the 2D gel images were analyzed using principal component analysis (PCA) and linear discriminant analysis (LDA) for their possible
use in classification of disease state.
11
12
Research frontiers
Multivariate analyses, including the dimension reduction method known as PCA
and classification methods such as LDA, are used in cancer proteomic studies
to identify the protein variables that provide the best discrimination between the
cancerous and normal tissues.
13
The authors used sequential protein extraction to extract aqueous soluble and
membrane-associated proteins from colorectal tissues. Differentially expressed
proteins were analyzed using a combination of PCA and LDA to determine
their usability in differentiating normal and cancerous colonic tissues. Using
this method, the authors successfully classified the tissues according to their
respective types. DJ-1 protein and glutathione S transferase P1 of the aqueous
soluble proteins, protein disulfide isomerase and complement component 1 Q
subcomponent-binding protein of the membrane-associated proteins gave the
best classification of the tissues.
14
15
16
Applications
The identified biomarkers may be used for the diagnosis and prognosis of CRC.
Terminology
17
Chemometrics is defined as the information aspects of complex biological and
chemical systems. Chemometrics utilize mathematical, statistical or formal
logic-based methods to extract chemical information, which in this case, is for
biomarker discovery.
18
Peer review
This study investigated the use of PCA and LDA of differential protein expression between normal and cancerous tissues for classification of disease state.
The method gave good classification of cancerous and normal colonic tissues.
19
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S- Editor Sun H L- Editor Kerr C
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E- Editor Zheng XM
[email protected]
doi:10.3748/wjg.v17.i16.2104
BRIEF ARTICLE
Dietary treatment of colic caused by excess gas in infants:
Biochemical evidence
Dámaso Infante, Oscar Segarra, Bernard Le Luyer
Dámaso Infante, Oscar Segarra, Unit of Gastroenterology,
Hepatology and Nutrition, Children’s Hospital Vall d’Hebron,
Autonomous University, Barcelona 08035, Spain
Bernard Le Luyer, Medical Director UP International I.C.C
Route de Pré-Bois, CH-1215, Geneva, Switzerland
Author contributions: Infante D designed, performed the research, analyzed the data and wrote the paper; Segarra O performed the research; Luyer BL revised the data and wrote the
paper.
Supported by United Pharmaceuticals SAS, 55 Avenue Hoche,
75008 Paris, France
Correspondence to: Dámaso Infante, MD, Chief, Unit of
Gastroenterology, Hepatology and Nutrition, Children’s Hospital Vall d’Hebron, Autonomous University, Pg Vall d´Hebro nº
119-129, Barcelona 08035, Spain. [email protected]
Telephone: +34-93-4893000 Fax: +34-93-4174892
Received: June 22, 2010
Revised: September 9, 2010
Accepted: September 16, 2010
breath test decreased significantly (10 ± 2.5 ppm, P <
0.01).
Abstract
Infante D, Segarra O, Luyer BL. Dietary treatment of colic
caused by excess gas in infants: Biochemical evidence. World J
CONCLUSION: This study showed an association between clinical improvement and evidence of decreased
levels of hydrogen when the infants were fed with a
specially designed, low-lactose formula.
Key words: Infants; Colic; Lactose; Hydrogen breath
test
Peer reviewers: Guang-Yin Xu, MD, PhD, Assistant Professor,
Division of Gastroenterology, Department of Internal Medicine,
University of Texas Medical Branch, Galveston, TX 77555-0655,
United States; Loes van Keimpema, MSc, PhD, Deparment
of Gastroenterology and Hepatology, Radboud University Nijmegen Medical Center, PO Box 9101, 6500 HB, Nijmegen,
The Netherlands
AIM: To evaluate the impact of feeding colicky infants
with an adapted formula on the hydrogen breath test
and clinical symptoms.
METHODS: Hydrogen expiration was measured by SC
MicroLyzer gas chromatography at inclusion and 15 d
after treatment with an adapted low-lactose formula in
20 colicky infants.
INTRODUCTION
Infant colic continues to be one of the most disconcerting issues in pediatric medicine. Wessel in 1954 established the famous “rule of three” criteria: “a symptomatic
disorder characterized by paroxysms of fussing, agitation
or crying, lasting more than 3 h a day and occurring more
than 3 d a week for at least 3 wk”[1]. These criteria are
now outdated and as there is no clear definition for the
condition, studies on its causes, prevalence and treatment
inevitably include a heterogeneous group of infants with
different problems[2-4]. The definition of “excessive infant crying syndrome”[5] is preferred, although the word
RESULTS: All babies were symptomatic: 85% with
excess gas, 75% with abnormal feeding pattern, and
85% with excessive crying. The hydrogen breath test
at inclusion was abnormal: 35 ± 3.1 ppm. After 15 d
feeding with an adapted low-lactose formula, crying and
flatulence decreased in 85% of patients (P < 0.001).
For infants in whom no decrease of gas was reported,
crying was still reduced (P < 0.01). Moreover, the feeding pattern was improved in 50% of infants when it was
initially considered as abnormal. Finally, the hydrogen
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2104
Infante D et al . Dietary treatment of functional colic
“colic” is still used and can be defined as an acronym
standing for “Cause Obscure Lengthy Infant Crying”. It
is characterized by paroxysms of excessive and inconsolable crying. The infant might present with a tense abdomen, flex the leg to the abdomen, and appear flushed.
Symptoms typically start around the second week of life,
peak around 3-6 wk and resolve by 3 mo[4,6]. This term
now includes digestive disorders such as constipation,
gastroesophageal reflux, allergy to cow milk proteins, and
excess intestinal gas due to malabsorption of lactose, and
its prevalence has been established[6,7].
These disorders, although not serious from a medical
point of view, can be very distressing for the baby and
his/her family, and can be associated with symptoms of
depression in the mother in the first months after birth[8].
For most of these disorders, some dietary solutions have
been developed in compliance with the international
expert group coordinated by ESPGHAN (European
Society for Paediatric Gastroenterology, Hepatology and
Nutrition)[9].
The objective of this study was to provide clinical
and biochemical evidence of the efficacy of an adapted
formula in colic caused by excessive gas, due to physiological hypolactasia, which led to excessive infant crying
syndrome.
Table 1 Composition of Novalac AC®
Average composition for 100 mL
Energy (kcal)
Proteins (g)
Carbohydrates (g)
Lactose (g)
Maltodextrin (g)
Fat (g)
C18:2 (mg)
C18:3 (mg)
Calcium (mg)
Phosphorus (mg)
The persons legally responsible for the children were
invited to give their informed consent for participation
in the study. The study was approved by the local ethics
committee.
Method of hydrogen breath test
Breath samples were collected before the start of feeding,
as well as at 90, 120 and 180 min after the beginning of
the meal. The feeding schedule was not modified. Samples
were taken using face masks with a two-way valve and a
pot system. Breath samples were collected in duplicate.
The samples were injected in an SC MicroLyzer gas chromatograph (Quinton Instrument Company, USA) for
simultaneous detection of hydrogen, CO2 and methane[10].
This model had an internal gas chromatographic column
through which the sample was flushed. Material in the
column retarded components which might have interfered
with the measurement, and hydrogen thus appeared by
itself at the detector and was accurately measured. The
gas was inserted in a SvRite-10 cartridge before being
analyzed. Prior calibration was performed with a standard
gas that contained 102 ppm hydrogen, 23 ppm methane,
and 5% CO2. The results were expressed as parts per million (ppm). The result was considered to be positive when
there was an increase > 20 ppm; the normal level for
methane being < 10 ppm applying a correction factor for
CO2. The hydrogen breath data were analyzed as a nested
factorial design by analysis of variance. For each infant,
the maximum hydrogen value was defined as being the
highest mean of the hydrogen breath test, because the actual time from the beginning of the feeding did not have a
consistent impact on the value.
We included formula-fed infants who were referred to their
pediatrician and/or the Unit of Gastroenterology, Hepatology and Nutrition, Children’s Hospital, Vall d´Hebron, Barcelona, Spain because of excessive crying reported by their
parents. Infants with vomiting/regurgitation, constipation or
cutaneous rash were excluded. When the parents reported
“rumbling tummies” excessive flatus, and frothy stools, we
considered it as suggestive of carbohydrate malabsorption.
Other symptoms were taken into account such as feeding
difficulties (e.g. crying during meals) and excessive crying
time per 24 h. The hydrogen breath test was performed in
case of suspected excess intestinal gas, or infant crying for
> 3 h/d to diagnose possible reduced lactose absorption.
Twenty consecutive infants with positive hydrogen
breath test were included in this study. All infants were fed
with an adapted formula, from various brands having a
lactose content of 7 g/100 mL equivalent to 10.4 g of lactose/100 kcal (caloric density of formulas 67 kcal/100 mL).
All of them were eutrophic, Caucasian, healthy full
term infants whose growth and development had been
normal since birth. Infants were 3.7 wk old on average
(range: 1.7-6 wk). They were switched to an adapted formula, Novalac AC (United Pharmaceuticals SA, Paris,
France) during the intervention period (Table 1).
Duration of crying, intestinal bloating and behavior
during feeding (such as interruption of the meal due to
crying) were evaluated through a questionnaire at baseline
and during a second consultation 15 d later. A second hydrogen breath test was also performed during the second
visit in these 20 infants.
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65.7
1.4
7.5
3.0
4.5
3.3
610.0
59.8
50.7
31.2
Hydrogen values were expressed as mean ± SE. The values of expired hydrogen were compared using Student’s
t test. A χ2 test was used for categorical variables when
both groups were compared. P < 0.05 was considered
significant. The statistical analysis was performed using
SPSS version 18.0 (SPSS Inc., Chicago, IL, USA).
RESULTS
Table 2 shows the clinical evolution (data reported by
2105
Miller et al[12] have shown that the expired hydrogen in 65
infants with colic was significantly higher than in control
subjects (29 ppm vs 11 ppm, P < 0.001). Sixty-two percent
of children with colic had expired hydrogen > 20 ppm,
but 38% of control subjects also had abnormal levels of
expired hydrogen. Similar results have been shown by
Moore et al[14], with 80% of colicky infants having positive
expired hydrogen vs 36% in normal infants. The assignment of infants to the colicky or control groups according to the mothers’ perception of the duration of crying
might not distinguish clearly colicky or non-colicky infants. This might explain the dissociation between clinical
and hydrogen values reported by some studies[12-14]. Moreover, among infants with positive expired hydrogen, individual response to abdominal distension might vary, and
this is why some infants cry more than others. Therefore,
the issue of personal susceptibility to stimuli and a lengthy
crying response varies considerably between infants.
Our baseline data are close to those reported by Miller
et al[12]. In this pilot study, we assessed the efficacy of the
studied formula only in children younger than 6 wk who
had a positive expired hydrogen test and clearly defined
symptoms.
Mature breast milk contains 7 g lactose per 100 mL
(10.2 g/100 kcal), as do several standard infants formulas.
In the first few weeks, infants present a physiological or
functional lactase deficiency that limits the amount of
lactose that they can digest[15]. Twenty-seven point five
percent of neonates present a positive hydrogen breath
test (i.e. > 20 ppm) after lactose ingestion, regardless of
sex or gestational age, with only weight playing a significant role: hydrogen expired is more important in infants
with a birth weight < 2.5 kg than in those > 2.5 kg[16].
One study[17] has found that infants who weigh < 1.8 kg,
fed with a low-lactose formula (< 5% lactose) ingested
more calories, finished their bottles sooner, presented less
milk residue in the stomach, and required feeding more
often and with less interruptions than those fed with a
formula with normal lactose content (7 g/100 mL). The
study of Douwes et al[18] has indicated that abnormal expired hydrogen is more frequent in breast-fed infants or
those fed with a 7.5% lactose formula, than in infants fed
with a 1% lactose formula.
Furthermore, the concentration of hydrogen in the
breath of healthy infants increases from birth to reach its
highest level in the second month of life, and declines to
low concentrations by 3-4 mo of age. This pattern is parallel to the evolution of crying in infants with COLIC[4].
Children selected in our study were almost as old as those
in the clinical studies of Barr et al[4] or Moore et al[14] (3.7 wk
vs 28.4 d and 2.6 wk, respectively) and younger than those
studied by Miller et al[12] (median age: 8 wk).
Fermentation of the disaccharide generates osmotic
active substances such as lactic acid, short chain fatty acids, and hydrogen and/or methane. Methane production
in lactose malabsorbers is normal, and without significance[10].
Colic can result directly from the hyper-peristaltic
Table 2 Clinical evaluation of associated symptoms in infants
with crying secondary to excess gas n (%)
Excess gas
Abnormal feeding
Duration of crying
< 1 h/d
1-3 h/d
> 3 h/d
2
P
Inclusion
After 15 d
χ
17 (85)
15 (75)
5 (25)
6 (30)
14.56
8.22
< 0.001
< 0.01
13 (65)
7 (35)
85%
3 (15)
0 (0)
10.41
8.48
< 0.001
< 0.01
40
Hydrogen
Methane
SE 3.1
Gas (ppm)
30
20
SE 2.5
10
SE 0.2
0
SE 0.1
D0
D15
Figure 1 Evolution of breath hydrogen and methane before (D0) and after
15 d (D15) of consumption of low-lactose formula. Values are expressed as
mean ± SE. Only the change in expired hydrogen was significant (P < 0.01).
relatives) of infants who were included because of crying secondary to excess gas. The duration of crying was
reduced in all infants regardless of the initial duration,
and 85% cried for < 1 h/d. Moreover, of the 85% of
infants reported with excessive gas at inclusion, only 25%
still had excessive gas at the end of the study period (P <
0.001). Four out of the five infants who were described
by their parents as having excessive gas were crying for <
1 h/d. The proportion of infants for whom feeding was
described as abnormal decreased from 75% at inclusion
to 30% after 2 wk feeding with Novalac AC (P < 0.01).
The level of hydrogen expired (biochemical evidence) decreased from 35 ± 3.1 ppm at inclusion to 10 ± 2.5 ppm
(P < 0.01) after 2 wk feeding with Novalac AC. The
methane excretion was unchanged (Figure 1).
DISCUSSION
Many authors tend to include digestive disorders in the
etiology of “excessive infant crying syndrome” (COLIC),
and recommend dietary treatment of this functional
disorder[6,7]. Therefore, this study focused on transient
lactose intolerance in infants with colic[11,12] and we do
not discuss other etiologies such as food hypersensitivity,
which has been reviewed by Hill et al[13].
As a result of the failure to break down all the lactose
ingested, part of it enters the large bowel where it becomes a substrate for lactobacilli and bifidobacteria. This
reaction (fermentation) produces hydrogen and other substances. Therefore, subsequent increases in breath hydrogen are accepted as an indirect sign of hypolactasia[6,11,12,14].
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2106
stimulus of the fluid load imposed by the osmotic action
of unabsorbed lactose in the small intestine, and gas or
pharmacologically active metabolites might be responsible for the symptomatic signs. In many susceptible infants, this excess of gas is responsible for triggering colic.
Several studies have investigated lengthy crying and
have related it to excess intestinal gas[14,19,20]. The rapid
production of hydrogen in the lower bowel distends the
colon, which causes different symptoms. This model of
colic implies that symptoms could be relieved by reducing
the lactose content of the infant feed. Four clinical trials
that have used artificial lactase have been published[19,21-23].
When lactase is added to a formula 30 min before it is fed,
30% of the lactose is not hydrolyzed. In a double-blind,
placebo-controlled crossover study, 10 colicky infants were
fed breast milk and cows’ milk formulas, untreated and
treated with lactase[19]. This study showed no evidence that
low-lactose milk reduced the severity and amount of crying[19]. In another double-blind, placebo-controlled crossover trial in 12 infants, no effects on duration of crying
and fussing were demonstrated[21]. In a third study with the
same methodology, the lactase-treated formula reduced
crying time by 1.14 h/d[22]. Yet another double-blind
placebo-controlled crossover study was performed on 53
infants with colic who were treated with placebo or lactase
added to their formula 4 h before they were fed. Data on
46 infants were available for crying time analysis and hydrogen breath tests were available in 34. Only 32 infants
complied with treatment. In these infants, crying time and
median expired hydrogen were significantly lower in the
active group than in the placebo group[23]. The results of
our study were in agreement with Kanabar’s study that
also included an expired hydrogen measurement.
Differences in hydrogen breath excretion between
colicky and control infants might also be associated with
factors other than the amount or rate of delivery of lactose to the colon. The microbiota, the colonic bacterial
metabolic pathways, the partial pressure of hydrogen in
the colon, the buffering capacity of the colon, gut perfusion, and incomplete monosaccharide absorption might
all play a part. Therefore, the volume of gas released by a
fecal sample reflects the end result of a complex interaction of several factors.
This pathophysiological mechanism explains the
clinical and biochemical response of these infants to an
adapted, low-lactose diet[22,23]. However, when calcium
absorption is enhanced by the presence of lactose[24], the
formula has a lactose content (3 g/100 mL) that provides
a daily amount close to absorption capacity in young infants of 4.5 g/kg per day)[25].
Even in the absence of a placebo control group, we
believe that the clinical improvement observed in our
study was related to dietary management. This clinical
improvement appeared earlier (after 15 d feeding with
the test formula only) than the usual resolution of colic.
Moreover this improvement was endorsed by a decrease
in expired hydrogen.
In conclusion this non-randomized, non-placebo-con-
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trolled pilot study demonstrates that the use of an adapted
infant formula with a low lactose concentration leads to
clinical improvement and a decrease in expired hydrogen in
colicky infants. Thus, infants with colic might benefit from
a switch from standard formula to this specific adapted
formula. Larger randomized clinical trials on the efficacy
of this formula are needed.
COMMENTS
COMMENTS
Background
Infant colic is still one of the most disconcerting issues in pediatric medicine.
This term now includes digestive disorders such as constipation, gastroesophageal reflux, allergy to cow milk proteins, and excess intestinal gas due to lactose malabsorption.
Research frontiers
In infants with colic, the possibility of functional lactase deficiency has led to
clinical trials with lactase supplementation of infant formulas. These studies
have given conflicting results.
This article reports the clinical and biological efficiency of an adapted low lactose formula in infants with colic and positive hydrogen expiration.
Applications
In colicky infants with excess abdominal gas, a diet with an adapted low lactose
formula can be tried for several days. If the results are positive, the diet can be
continued for several months.
Peer review
The experiments were well designed and the paper is well written. However,
there is one major concern about the data analysis.
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S- Editor Shi ZF
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2108
L- Editor Kerr C
E- Editor Zheng XM
[email protected]
doi:10.3748/wjg.v17.i16.2109
BRIEF ARTICLE
Levels of matrix metalloproteinase-1 and tissue inhibitors
of metalloproteinase-1 in gastric cancer
Ozgur Kemik, Ahu Sarbay Kemik, Aziz Sümer, Ahmet Cumhur Dulger, Mine Adas, Huseyin Begenik,
Ismail Hasirci, Ozkan Yilmaz, Sevim Purisa, Erol Kisli, Sefa Tuzun, Cetin Kotan
using an enzyme-linked immunosorbent assay.
Ozgur Kemik, Aziz Sümer, Ismail Hasirci, Ozkan Yilmaz, Erol
Kisli, Cetin Kotan, Department of General Surgery, Yuzuncu Yıl
University Medical Faculty, Van, 6500, Turkey
Ahu Sarbay Kemik, Department of Biochemistry, Cerrahpasa
Medical Faculty, University of Istanbul, Istanbul, 3400, Turkey
Ahmet Cumhur Dulger, Department of Gastroenterology, Medical Faculty, University of Yüzüncü Yıl, Van, 6500, Turkey
Mine Adas, Department of Endocrinology, Okmeydani Education and Research Hospital, Istanbul, 3400, Turkey
Huseyin Begenik, Department of Internal Medicine, Medical
Faculty, University of Yüzüncü Yıl, Van, 6500, Turkey
Sevim Purisa, Department of Biostatistics, Istanbul Medical
Faculty, University of Istanbul, Istanbul, 3400, Turkey
Sefa Tuzun, II. General Surgery, Haseki Education and Research
Hospital, Istanbul, 3400, Turkey
Author contributions: Kemik O, Kemik AS and Sümer A designed the study and wrote the paper; Kemik AS performed the
biochemical evaluation, and collected and analyzed the data;
Purisa S performed the statistical analyzis; Adas M, Begenik H,
Yilmaz O, Hasirci I, Dulger AC, Kisli E, Tuzun S and Kotan C
contributed to the discussion.
Correspondence to: Ozgur Kemik, MD, Assistant Professor,
Department of General Surgery, Yuzuncu Yıl University Medical
Faculty, Van, 6500, Turkey. [email protected]
Telephone: +90-432-2251024 Fax: +90-432-2164705
Received: August 10, 2010
Revised: January 18, 2011
RESULTS: Higher serum MMP-1 and TIMP-1 levels
were observed in patients than in controls (P < 0.001).
Serum MMP-1 and TIMP-1 levels were positively associated with morphological appearance, tumor size, depth
of wall invasion, lymph node metastasis, liver metastasis, perineural invasion, and pathological stage. They
were not significantly associated with age, gender, tumor location, or histological type.
CONCLUSION: Increased MMP-1 and TIMP-1 were
associated with gastric cancer. Although these markers are not good markers for diagnosis, these markers
show in advanced gastric cancer.
Key words: Gastric cancer; Matrix metalloproteinase-1;
Tissue matrix metalloproteinase-1
Peer reviewer: Peter JK Kuppen, PhD, Associate Professor, Department of Surgery, Leiden University Medical Center, 2300 RC
Leiden, The Netherlands
Kemik O, Kemik AS, Sümer A, Dulger AC, Adas M, Begenik H,
Hasirci I, Yilmaz O, Purisa S, Kisli E, Tuzun S, Kotan C. Levels
of matrix metalloproteinase-1 and tissue inhibitors of metalloproteinase-1 in gastric cancer. World J Gastroenterol 2011;
17(16): 2109-2112 Available from: URL: http://www.wjgnet.
com/1007-9327/full/v17/i16/2109.htm DOI: http://dx.doi.
org/10.3748/wjg.v17.i16.2109
Abstract
AIM: To evaluate the levels of preoperative serum matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of
metalloproteinase-1 (TIMP-1) in gastric cancer.
INTRODUCTION
METHODS: One hundred gastric cancer patients who
underwent gastrectomy were enrolled in this study. The
serum concentrations of MMP-1 and TIMP-1 in these
patients and in fifty healthy controls were determined
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Matrix metalloproteinases (MMPs) are a family of zincdependent neutral endopeptidases that play a significant
role in the degradation of all matrix partitions, which
2109
Kemik O et al . MMP-1 and TIMP-1 in gastric cancer
are crucial for malignant tumor growth, invasion, and
metastasis[1,2]. MMPs are inhibited by tissue inhibitors of
metalloproteinase (TIMPs), which are secreted proteins.
TIMPs bind to enzymatically active MMPs at a 1:1 molar
stoichiometry, thus inhibiting proteolysis[3]. The role of
TIMPs in the imbalance of the extracellular matrix is significant and may inhibit or stimulate tumorigenesis[4].
MMP-1 is also known as collagenase (EC 3.4.23.7)[5].
Saffarian et al[6] showed that activated MMP-1 acts by
processing on the collagen fibril. The biological implications of MMP-1 acting as a molecular retainer, tied to the
cell surface, prompted recent mechanisms for its status in
tissue remodeling and cell-matrix interaction to be proposed. MMP-1 in the stromal tumor microenvironment
can change the behavior of cancer cells to promote cell
migration and invasion[7].
TIMP-1 is a 28.5 kDa glycoprotein that has been studied in many human malignancies, including gastric cancer[8]. TIMP-1 mRNA expression is increased in gastric,
esophageal, and pancreatic cancer[9-11]. TIMP-1 is present
in human peripheral blood and body fluids[12]. MMP-1
and TIMP-1 levels have been studied in plasma or serum
of patients with cumulative malignancies[13,14].
Our study was carried out to analyze serum MMP-1
and TIMP-1 levels in gastric cancer patients and to investigate their clinicopathological correlations.
Table 1 Serum matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 levels in patients and controls
Variables
Age (yr)
Gender female (%)
MMP-1 (ng/mL)
TIMP-1 (ng/mL)
56 (48-65)
37
256 (109-342)
220 (198-267)
58 (47-64)
40
785 (457-900)
725 (417-1134)
P
< 0.0001
< 0.0001
MMP-1: Matrix metalloproteinase-1; TIMP-1: Tissue inhibitor of metalloproteinase-1.
A
1600
1400
MMP-1 (ng/mL)
1200
Patients
Controls
1000
800
600
400
200
0
B
1800
1600
TIMP-1 (ng/mL)
1400
A total of 100 patients who underwent gastrectomy with
gastric cancer between December 2007 and April 2010
were enrolled. Their median age was 58.5 years (range,
34-78 years), and the ratio of men/women was 47/53.
There were 50 healthy volunteer controls without family
history of cancer, whose average age was 56 years (range,
48-65 years) (22 men, 28 women). Peripheral venous
blood of patients and controls was taken before gastrectomy and stored at 4℃. Blood from controls was taken
on the day of a physical examination. The blood samples
were centrifuged 1000 rpm, in 15 min, at 20℃ to separate
the serum, which was stored at -70℃ until analysis. The
mean storage time of all samples was 2 mo (45-80 d).
Resected tumor specimens were studied pathologically according to the criteria of the UICC’s pTNM classification[15]. Information recorded included age, gender,
tumor location, tumor size, wall invasion, resection margin, histological type, lymph node metastasis, vascular
invasion, lymphatic invasion, and perineural invasion.
The histological features were classified into two types:
(1) intestinal or differentiated type, consisting of papillary and/or tubular adenocarcinomas; and (2) diffuse or
undifferentiated type, consisting of poorly differentiated,
signet-ring cells, and/or mucinous adenocarcinomas.
Enzyme-linked immunosorbent assay (ELISA) for serum MMP-1 and TIMP-1 was performed using an ELISA
kit (R&D System, USA) following the manufacturer’s
instructions.
As appropriate, the Mann-Whitney U test or Fisher’s
exact test was used for group comparisons. Correlations
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Controls (n = 50) Patients (n = 100)
Patients
Controls
1200
1000
800
600
400
200
0
Figure 1 Serum matrix metalloproteinase-1 (A) and tissue inhibitor of metalloproteinase-1 (B) levels of controls and patients. MMP-1: Matrix metalloproteinase-1; TIMP-1: Tissue inhibitor of metalloproteinase-1.
between parameters were tested by Spearman’s correlation coefficient. A P < 0.05 was considered statistically
significant.
RESULTS
Serum MMP-1 and TIMP-1 levels in gastric cancer patients
and controls are shown in Table 1 and Figure 1A and B.
The serum levels of MMP-1 and TIMP-1 in gastric cancer patients were significantly higher than in the control
group (P < 0.0001). Clinicopathological variables are
shown in Table 2. Serum MMP-1 and TIMP-1 levels were
positively associated with the depth of wall invasion (P <
0.01), lymph node metastasis (P < 0.001), and lymphatic
invasion (P < 0.001). The serum levels of MMP-1 and
TIMP-1 were closely associated with distant metastasis (P
< 0.001). In particular, higher MMP-1 and TIMP-1 levels
were significantly associated with positive lymphovascular invasion (P < 0.001), tumor size ≥ 4 cm (P < 0.001),
positive lymph node metastasis (P < 0.001), T stage
2110
obvious that matrix metalloproteinases also alter the biological functions of ECM molecules by definite proteolysis. MMP-1 and TIMP-1 are thought out to be involved
in dissemination of cancer cells by dissolving the ECM,
but they are also important in creating an environment
that supports the initiation and growth of primary and
metastatic tumors. These effects may be associated with
proteolytic release of growth factors and/or modification
of cellular environments[16].
The most important finding in our study was the association between high MMP-1 and TIMP-1 levels in
gastric cancer patients. In addition, high MMP-1 and
TIMP-1 levels were significantly associated with certain
clinicopathological variables. High MMP-1 expression
has been associated with hematogenous metastasis[17,18],
rising depth of invasion, and metastasis in colorectal cancer[18,19]. Our study also suggested that MMP-1 levels are
associated with depth of invasion and metastasis.
Patients with colorectal cancer, ovary, lung, and liver
diseases have increased TIMP-1 levels compared to control
groups[14,20-22]. Wang et al[23] suggested that serum TIMP-1
levels were higher in gastric cancer patients than control groups and were associated with clinicopathological
variables. However, they suggested that serum TIMP-1
levels were associated with depth of wall invasion, distant
metastasis, peritoneal seeding, lymphatic invasion, lymph
node metastasis, and perineural invasion. However, we did
not find that serum TIMP-1 levels were associated with
peritoneal seeding and perineural invasion.
MMP-1 is associated with the primal pace of invasion and angiogenesis in gastric cancer, which may make
it a useful marker for prognosis. TIMP-1 is more simply
released into the blood[24]; therefore, the sensitivity of the
assay is higher than that for MMP-1.
High blood levels of MMP-1 and TIMP-1 are associated with poor prognosis of malignancies. Thus, they
might useful as markers for malignant potential (i.e. tumor growth and/or differentiation) for cancer. Notably,
serum TIMP-1 levels have been established as an independent factor in gastric cancer[23].
Some metalloproteinases have been shown to degrade
over time when measured in stored blood samples. However, we do not think that such protein decay is a significant factor when proteins are stored for 2 mo. This assumption is supported by the work of Papazoglou et al[25],
Kardeşler et al[26] and Karapanagiotidis et al[27].
MMP-1 and TMP-1 can be considered as ‘traditional’
and conventional serum biomarkers; many studies have
measured both of these proteins as serum biomarkers[28].
This study demonstrated that high serum MMP-1
and TIMP-1 levels in gastric cancer patients are significantly associated with disease progression. Their levels
are important markers of tumor progression or advanced
tumor stages.
Table 2 Clinicopathological variables of serum matrix metalloproteinase-1 and tissue inhibitor of metalloproteinase-1 in
patients
Variables
Lymphovascular invasion
Negative
Positive
Tumor size (cm)
<4
≥4
Lymph node metastasis
Negative
Positive
T stage
T0-2
T3-4
TNM stage
Ⅰ
Ⅱ
Ⅲ
Ⅳ
MMP-1
TIMP-1
P
543 (500-678)
801 (768-845)
489 (450-573)
642 (567-703)
< 0.001
478 (460-501)
675 (509-725)
429 (425-479)
671 (532-690)
< 0.001
563 (503-650)
742 (657-799)
642 (598-709)
756 (570-876)
< 0.001
521 (498-599)
674 (578-783)
598 (564-783)
749 (570-794)
< 0.001
469 (458-502)
534 (467-563)
714 (546-857)
765 (699-900)
476 (423-512)
521 (478-589)
753 (512-699)
975 (812-1134)
< 0.001
< 0.001
MMP-1: Matrix metalloproteinase-1; TIMP-1: Tissue inhibitor of metalloproteinase-1.
(T3-T4) (P < 0.001), or TNM stage (Ⅲ and Ⅳ) (P < 0.001).
MMP-1 and TIMP-1 levels were not significantly associated with negative lymphovascular invasion, tumor size <
4 cm, negative lymph node metastasis, T stage (T0-T2),
and TNM stage (Ⅰ and Ⅱ). Overall, they were associated
with pathological stage (P < 0.001). Serum MMP-1 and
TIMP-1 levels were not associated with age (P = 0.237),
gender (P = 0.281), tumor location (P < 0.142), histological type (P = 0.103), vascular invasion (P = 0.247), or peritoneal seeding (P = 0.271).
Higher serum MMP-1 and TIMP-1 levels were correlated with gastric cancer (P < 0.001, r = 0.77). Figure 1A
shows that MMP-1 levels in patients with gastric cancer
were significantly higher than in control groups. Figure 1B
shows that TIMP-1 levels in patients with gastric cancer
were significantly higher than in control groups.
DISCUSSION
In our study, we investigated MMP-1 and TIMP-1 levels
in gastric cancer patients and compared them with a control group. We also investigated their associations with
clinicopathological features.
Matrix metalloproteinases are involved in many normal biological processes (e.g. embryonic development,
blastocyst implantation, organ morphogenesis, nerve
growth, ovulation, cervical dilatation, postpartum uterine
involution, endometrial cycling, hair follicle cycling, bone
remodeling, wound healing, angiogenesis, and apoptosis)
and pathological processes (e.g. arthritis, cancer, cardiovascular disease, nephritis, neurological disease, breakdown of the blood brain barrier, periodontal disease, skin
ulceration, corneal ulceration, liver fibrosis, emphysema,
and fibrotic lung disease). Although the main function
of matrix metalloproteinases is elevation of ECM during
tissue resorption and progression of many diseases, it is
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COMMENTS
COMMENTS
Background
The incidence of gastric cancer is rising worldwide. Collagenases may play a role
2111
in degradation of the cell matrix, possibly leading to growth of malignant tumors,
lymph node metastasis, increased depth of invasion and other metastases.
13
Matrix metalloproteinase-1 (MMP-1) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) change the environment of cancer cells to promote cell
migration and invasion. Changes caused by these endopeptidases have a role
in the progression of the gastric cancer.
14
Research frontiers
15
High blood levels of MMP-1 and TIMP-1 are associated with poor prognosis
of malignancies, making them potentially useful biomarkers for the malignant
potential (i.e. tumor growth and/or differentiation) of cancer. These effects may
be associated with proteolytic release of growth factors and/or modification of
tumor cells.
16
17
Applications
The date generated in this paper might be used to explain the development of
gastric cancer, to prevent metastasis, and to aid early diagnosis.
18
MMP-1 and TIMP-1 zinc-dependent neutral endopeptidases. The role of MMP-1
and TIMP-1 in the imbalance of the extracellular matrix is significant and may
inhibit or stimulate tumorigenesis. These effects have been demonstrated, and
these molecules may represent useful markers of tumorigenesis.
19
Terminology
Peer review
It is a nice study, with interesting results.
20
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S- Editor Tian L L- Editor Stewart GJ
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2112
E- Editor Zheng XM
[email protected]
doi:10.3748/wjg.v17.i16.2113
BRIEF ARTICLE
Sunitinib for Taiwanese patients with gastrointestinal
stromal tumor after imatinib treatment failure or intolerance
Yen-Yang Chen, Chun-Nan Yeh, Chi-Tung Cheng, Tsung-Wen Chen, Kun-Ming Rau, Yi-Yin Jan, Miin-Fu Chen
(PR), and 9 stationary disease (SD); 15/23]. In 12 patients harboring mutations of the kit gene at exon 11,
the clinical benefit rate (CR, PR, and SD) was 75.0%
and 6 patients with tumors containing kit exon 9 mutations had a clinical benefit of 50.0% (not significant,
P = 0.344). The progression free survival (PFS) and
overall survival (OS) did not differ between patients
whose GISTs had wild type, KIT exon 9, or KIT exon 11
mutations. Hand-foot syndrome was the most common
cause of grade Ⅲ adverse effect (26.1%), followed by
anemia (17.4%), and neutropenia (13.0%). During the
median 7.5-mo follow-up after sunitinib use, the median PFS and OS of these 23 GIST patients after sunitinib
treatment were 8.4 and 14.1 mo, respectively.
Yen-Yang Chen, Kun-Ming Rau, Division of Hematology-Oncology, Department of Internal Medicine, Chang Gung Memorial
Hospital, Kaohsiung Medical Center, Taoyuan 333, Taiwan, China
Chun-Nan Yeh, Chi-Tung Cheng, Tsung-Wen Chen, Yi-Yin
Jan, Miin-Fu Chen, GIST team, Department of Surgery, Chang
Gung Memorial Hospital and University, Taoyuan 333, Taiwan,
China
Author contributions: Chen YY helped collect the data and
wrote the manuscript; Yeh CN was in charge of this project and
revised the manuscript; Cheng CT, Chen TW, Rau KM, Jan YY
and Chen MF helped to review this paper.
Supported by Chang Gung Medical Research Program 380711
Grant to Dr. Yeh CN
Correspondence to: Chun-Nan Yeh, MD, GIST team, Department of Surgery, Chang Gung Memorial Hospital and University,
5, Fu-Hsing Street, Kwei-Shan, Taoyuan 333, Taiwan,
China. [email protected]
Telephone: +886-3-3281200 Fax: +886-3-3285818
Received: November 8, 2010 Revised: January 3, 2011
CONCLUSION: Sunitinib appears to be an effective
treatment for Taiwanese with IM-resistant/intolerant
GISTs and induced a sustained clinical benefit in more
than 50% of Taiwanese advanced GIST patients.
Key words: Suintinib; Gastrointestinal stromal tumors;
Imaitinib; Failure or intolerance
Abstract
AIM: To report preliminary results of the efficacy and
safety of sunitinib in the management of Taiwanese
gastrointestinal stromal tumors (GIST) patients facing
imatinib mesylate (IM) intolerance or failure.
Peer reviewer: I-Rue Lai, Assistant professor, Department of
Anatomy and Cell Biology, Medical College, National Taiwan
University, 7, Chun-San S. Rd, Taipei 106, Taiwan, China
Chen YY, Yeh CN, Cheng CT, Chen TW, Rau KM, Jan YY, Chen
MF. Sunitinib for Taiwanese patients with gastrointestinal stromal tumor after imatinib treatment failure or intolerance. World
J Gastroenterol 2011; 17(16): 2113-2119 Available from: URL:
METHODS: Between 2001 and May 2010, 199 Taiwanese patients with metastatic GIST were treated at Chang
Gung Memorial Hospital. Among them, 23 (11.6%) patients receiving sunitinib were investigated.
RESULTS: Sixteen male and 7 female patients with a
median age of 59 years (range: 24-83 years) received
sunitinib. Twenty-two GIST patients changed to sunitinib because of IM failure and 1 because of intolerance. The median duration of sunitinib administration
was 6.0 mo (range: 2-29 mo). The clinical benefit was
65.2% [2 complete response (CR), 4 partial response
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INTRODUCTION
Gastrointestinal stromal tumors (GISTs) primarily arise
from mesenchymal tissue in the gastrointestinal (GI) tract
2113
Chen YY et al . Sunitinib for GIST after imatinib failure
and abdomen. Although GISTs are rare, representing
only an estimated 0.1% to 3% of all GI tract tumors[1]
GISTs account for the most common mesenchymal
malignancy of the GI tract with unknown incidence[2].
GISTs appear to be related to the interstitial cells of
Cajal[3] and express the cell surface transmembrane receptor KIT, which has tyrosine kinase activity. Gain-offunction mutations of KIT are frequent in GISTs and
result in constitutive activation of KIT signaling and
lead to uncontrolled cell proliferation and resistance to
apoptosis[4,5]. The KIT tyrosine kinase inhibitor imatinib
mesylate (IM) has shown a promising clinical result for an
advanced GIST patient[6], and several trials have shown a
promising effect of this target therapy[6,7]. Our previous
study showed that IM significantly affected survival in
GIST patients[8,9].
Surgical resection remains the mainstay therapy for
GIST, but recurrence is common. The 5-year survival
rates for GIST after complete resection range from 40%
to 65%[6,10-13]. Unresectable or metastatic GIST is a fatal
disease that resists conventional chemotherapy. IM selectively inhibits certain protein tyrosine kinases: intracellular
ABL kinase, chimeric BCR-ABL fusion oncoprotein of
chronic myeloid leukemia, the transmembrane receptor
KIT, and platelet-derived growth factor receptors (PDGFR)[14-17]. IM induced a sustained objective response in
more than 50% of patients with advanced GISTs in the
West and in Taiwan[8,9]. However, progression of GIST
eventually develops and emerges as a challenge.
Sunitinib is an oral multi-targeted tyrosine kinase inhibitor with activity against KIT and PDGFRs, as well as
vascular endothelial growth factor receptors (VEGFRs),
glial cell line-derived neurotrophic factor receptor (rearranged during transfection; RET), colony-stimulating factor 1 receptor (CSF-1R), and FMS- like tyrosine kinase-3
receptor (FLT3)[18-23]. Sunitinib received multi-national
approval for the treatment of GIST after failure of IM
because of resistance or intolerance based on the results
of an international, randomized, double-blind, placebocontrolled phase Ⅲ trial[24]. The clinical safety and efficacy of both IM and sunitinib in GIST have primarily
been established in Western patients living in the USA or
Europe and have not been thoroughly studied in Asian
patients. Fifty-six centers in 11 countries participated in
the phase Ⅲ trial of sunitinib in GIST, but only 15 of the
312 patients were of Asian descent (10 and 5 in the sunitinib and placebo groups, respectively)[25]. Therefore, we
report our preliminary results to clarify the efficacy and
safety of sunitinib in management of Taiwanese GIST
patients facing IM intolerance or failure.
spectively reviewed. Failure of prior IM therapy, as demonstrated by disease progression [based on Response
Evaluation Criteria in Solid Tumors (RECIST)][26] or discontinuation of IM due to toxicity was the inclusion criteria in this study. Additional eligibility criteria included
an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1 and adequate cardiac, hepatic,
renal, coagulation, and hematologic function. Key exclusion criteria included lack of recovery from the acute
toxic effects of previous anticancer therapy or imatinib
treatment, discontinuation of imatinib therapy within 2
wk or of any other approved or investigational drug for
GIST within 4 wk before starting sunitinib treatment,
clinically significant cardiovascular events or disease in
the previous 12 mo, diabetes mellitus with clinical evidence of peripheral vascular disease or diabetic ulcers,
or a diagnosis of any second malignancy within the
previous 5 years. Patients could have previously received
chemotherapeutic regimens (the last chemotherapy treatment must have been at least 4 wk before study entry)
and undergone radiotherapy, or surgery, or both. The
study was approved by the local institutional review
board of Chang Gung Memorial Hospital, and written
informed consent for drug administration and the analysis of tumor-associated genetic alteration was obtained
independently from each patient.
Study design and follow-up study
A retrospective study was conducted to evaluate the effect
of sunitinib in inducing objective response in Taiwanese
GIST patients. Patients were administered 50 mg (4 wk
on and 2 wk off; for clinical trial) or 37.5 mg continuously of sunitinib in 12.5 mg capsules taken orally daily
with food. Patients had regular physical examinations and
evaluations of performance status, body weight, complete
blood count, and serum chemistry. The administration of
each dose and any adverse events were recorded for each
patient. Standard computed tomography (CT) was performed on each patient every 3 mo for the first 3 years
and every 6 mo for the following 2 years to assess patient
response. Measurement of efficacy was based on objective tumor assessments made using RECIST with a minor
modification to allow use of standard radiographic protocols for spiral CT. Time to response (TTR) was defined
as the interval for better drug response during sunitinib
treatment. Time to progression (TTP) was defined as the
interval for worse drug response during sunitinib treatment. Progression free survival (PFS) was defined as no
progression after sunitinib use. Overall survival (OS) was
defined as survival after administration of sunitinib and
death was the endpoint of the study. Response rate, PFS,
OS, TTR, duration of response, and TTP were recorded.
Safety and tolerability were assessed by analysis of adverse
events, physical examinations, vital signs, ECOG performance status, and laboratory abnormality assessments (for
example, complete blood count with differential count,
serum electrolyte measurements, and electrocardiogram).
Cardiac function was assessed at screening, at day 28 of
Between 2001 and May 2010, 199 patients who had histologically confirmed, recurrent, unresectable, or metastatic GIST that expressed CD117 or CD34 and were
treated at the Department of Medical Oncology, and
Surgery, Chang Gung Memorial Hospital were retro-
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2114
all treatment cycles, and treatment end with 12-lead electrocardiogram and multigated acquisition scans. Toxic
effects were recorded in accordance with the National
Cancer Institute Common Toxicity Criteria[27].
Table 1 Demographic and genetic data of 23 Taiwanese
gastrointestinal stromal tumor patients with imatinib failure
or intolerance treated with sunitinib n (%)
Sunitinib (n = 23)
Analysis of KIT and PDGFRA mutations
Sections were prepared from formalin-fixed, paraffinembedded pretreatment specimens trimmed to enrich
tumor cells. Polymerase chain reaction amplification of
genomic DNA for KIT and PDGFRA was performed
and amplification was analyzed for mutations as previously described[28].
Age (median/range, yr)
Gender (male:female)
Location
Stomach
Duodenum
Jejunum
Ileum
Mesentery
Rectum
Tumor recurrence
Liver
Peritoneum
Local recurrence
Genetic spectrum
Exon 11
Deletion mutation
Deletion and insertion mutation
Missense mutation
Exon 9 (insertion mutation)
Exon 13
No mutation (wild type)
PDGFRA (exon 18)
Median duration of sunitinib use (mo)
All data are presented as percentages of patients or means
with standard deviation. Pearson χ2 test and Fisher exact
test were used for nominal variables. Survival rate was calculated and plots constructed by the Kaplan-Meier method
and compared between groups with a log-rank test. All
statistical analyses were performed using the SPSS computer software package (Version 10.0, Chicago, IL, USA).
A P-value < 0.05 was considered statistically significant.
RESULTS
8 (26.6)
1 (12.5)
5 (23.4)
5 (14.1)
1 (18.8)
3 (4.7)
15
6
2
21 (84.4)
12
6
1
1
1
6
PDGFRA: Platelet derived growth factor α.
Clinical features
Table 1 summarizes the demographic features of 23
GIST patients receiving sunitinib. There were 16 male
and 7 female patients with a median age of 59 years (range
from 24 to 83 years). The stomach was the most common site for GISTs treated with sunitinib (8/23; 35%),
followed by the jejunum (5/23; 22%), the ileum (5/23;
22%), and the rectum (3/23; 13%) (Table 1).
Table 2 Antitumor response of 23 Taiwanese with advanced
gastrointestinal stromal tumor treated with sunitinib
n (%)
CR
PR
SD
PD
Treatment and outcomes before and after use of
sunitinib
In Taiwan, sunitinib has been approved for treatment
of metastatic GIST patients facing IM intolerance or
failure since February 2009. Before 2009, sunitinib was
administered to selected patients with unresectable or
metastatic (advanced) GISTs facing IM failure or intolerance because they were enrolled in clinical trials. Sunitinib
(12.5-50 mg/d) was given to 23 patients and all 23 patients were followed after administration of sunitinib at
regular intervals until death or until the time of this manuscript writing. The median follow-up time after sunitinib
was 7.5 mo, range: 1.2-58.0 mo. Overall, 2 patients (8.7%)
had a complete response (CR), 4 (17.4%) had a partial response (PR), 9 had stationary disease (SD) (39.1 %), and
8 had progressive disease (PD) (34.8%). A clinical benefit
was observed in 65.2% of GIST patients. Among the
23 patients, the median TTR for 2 patients with CR was
3.73 mo and was 3.67 mo for 4 PR patients. The median
TTP was 2.37 mo and the median survival is still unknown in the 8 PD patients (Table 2). During the median
7.5 mo follow-up after sunitinib use, the median PFS and
OS of these 23 GIST patients after sunitinib treatment
was 8.4 and 14.1 mo, respectively (Figures 1 and 2).
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59.0/24-83
16:7
2 (8.7)
4 (17.4)
9 (39.1)
8 (34.8)
Sunitinib duration
(median, mo)
TTR/TTP
(median, mo)
OS (median,
mo)
9.85
12.3
11.9
3.63
3.73/NA
3.67/12.71
1.87/13.53
2.37
NA
NA
14.03
NA
CR: Complete response; PR: Partial response; SD: Stationary disease; PD:
Progression of disease; TTR: Time to response; TTP: Time to progression;
OS: Overall survival.
Spectrum of mutations in 23 advanced GIST patients
Tumor specimens suitable for genetic analysis were available from 21 (84.4%) of the 23 GIST patients with IM
failure or intolerance. Overall, 18 (85.7%) of the 21 examined GISTs had activated mutations of KIT exon 9
and 11. Six of 21 (28.6%) GISTs had exon 9 mutation,
12 (57.1%) had exon 11 mutation, and 1 (4.8%) had no
mutation of KIT. One PDGFRA exon 18 mutation was
found. One patient had a concurrent deletion mutation in
exon 11 and a missense mutation in exon 13; however, the
exon 13 mutation was followed by the deletion mutation
in exon 11. This patient developed acquired resistance and
expired from disease progression. All 6 GISTs had KIT
exon 9 mutation and displayed in-frame duplication of nucleotides, resulting in insertion of alanine (A) and tyrosine
(Y) at codons 502 and 503. The KIT exon 11 mutations in
the 12 GIST patients included insertion and deletion mutations, deletion mutations, and missense mutations.
2115
1.0
Use of sunitinib (n = 23)
0.9
0.9
0.8
0.8
0.7
Cumulative survival rate
1.0
Median/mean: 8.4/8.37 mo
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Exon 9 (n = 6)
Exon 11 (n = 12)
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
2
4
6
8
10 12
t /mo
14
16
18
0.0
20
2
4
6
8
10
12
14
16
18
20
t /mo
Figure 1 Progression free survival of 23 Taiwanese with metastatic gastrointestinal stromal tumor treated with sunitinib after imatinib failure or
intolerance.
1.0
0
Figure 3 Progression free survival of 18 Taiwanese with metastatic gastrointestinal stromal tumor treated with sunitinib after imatinib failure or
intolerance (exon 9 vs exon 11). Median/mean (mo): 3.7/5.6 (exon 9), 8.8/10.2
(exon 11); 95% CI: 0-7.9/2.5-8.6 (exon 9), 2.24-14.4/6.5-14 (exon 11). Log-rank
test, P = 0.221.
Use of sunitinib (n = 23)
1.0
0.8
0.7
0.5
0.4
0.3
0.2
0.1
0.0
0
6
12
18
24
30
Exon 9 (n = 6)
Exon 11 (n = 12)
Exon 13 (n = 1)
No mutation
(n = 1)
PDGFRA (n = 1)
0.5
0.4
0.3
0.2
P
PD
0
2
0
0
1
2
0
0
2
5
0
1
3
3
1
1
0
1
0
0
0.610
CR + PR PD
+ SD
1
3
9
3
3
12
18
24
30
patients with tumors containing a KIT exon 9 mutation
were 3.7 and 13.5 mo, respectively. The twelve GIST patients who had KIT exon 11 mutations had similar PFS
and OS to that of 6 patients with tumors containing a
KIT exon 9 mutation (Figures 3 and 4).
1
Adverse events in 23 advanced GIST patients receiving
sunitinib
Hand-foot syndrome was the most common cause of
grade Ⅲ adverse effects (26.1%), followed by anemia
(17.4%), and neutropenia (13.0%). None of 11 patients
had hypothyroidism after use of sunitinib (Table 4).
1
Exon 9 vs exon 11. CR: Complete response; PR: Partial response; SD:
Stationary disease; PD: Progression of disease; PDGFRA: Platelet derived
growth factor α.
Treatment and outcomes after use of sunitinib in terms
of mutation status
In 12 patients with GISTs harboring KIT exon 11 mutations, the clinical benefit rate was 75% (2 CR, 2PR, and
5 PR) and 3 of 6 patients with tumors containing a KIT
exon 9 mutation had a clinical benefit of 50% (1 PR and
2 SD) (not significant, P = 0.344) (Table 3). The median
PFS and OS for the 12 GIST patients who had KIT exon
11 mutations after sunitinib use was 8.8 mo and still not
reached, respectively. The median PFS and OS for the 6
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6
Figure 4 Overall survival of 18 Taiwanese with metastatic gastrointestinal
stromal tumor treated with sunitinib after imatinib failure or intolerance
(exon 9 vs exon 11). Median/mean (mo): 13.5/13.7 (exon 9), Not achieved/22.6
(exon 11); 95% CI: 0.9-26.1/9.8-17.7 (exon 9), NA/16.1-29.1 (exon 11). Logrank test, P = 0.473.
P
0.344
0
t /mo
Table 3 Correlation between antitumor response and mutation status of 21 Taiwanese with advanced gastrointestinal
stromal tumor treated with sunitinib
SD
0.7
0.6
0.0
Figure 2 Overall survival of 23 Taiwanese with metastatic gastrointestinal
stromal tumor treated with sunitinib after imatinib failure or intolerance.
PR
0.8
0.1
t /mo
CR
Exon 9 (n = 6)
Exon 11 (n = 12)
0.9
Median/mean: 14.1/19.3 mo
0.6
0.9
DISCUSSION
We had shown that IM significantly prolongs the postrecurrence and OS of Taiwanese patients with advanced
GISTs[8,9]. However, approximately 50% of GIST patients eventually develop progression in 24 mo after IM
treatment and emerge as a challenge[7]. This study confirmed the positive effect of sunitinib on improving PFS
2116
significantly impacted by both primary and secondary
mutations in the predominant pathogenic kinases, which
has implications on optimal treatment of patients with
GIST. Heinrich reported that both the clinical benefit
and the objective response rates with sunitinib were
higher in patients with primary KIT exon 9 mutations
than with exon 11 mutations. Similarly, PFS and OS were
significantly longer in patients with primary KIT exon
9 mutations or a wild-type genotype than in those with
KIT exon 11 mutations[29]. A possible explanation is
that the potency of sunitinib against wild-type and exon
9 mutant KIT was superior to that of imatinib in vitro,
whereas both drugs exhibited similar potency against
KIT exon 11 mutant kinases. These results suggest that
the greater clinical benefit seen in sunitinib-treated patients with exon 9 mutant or wild-type imatinib-resistant
GISTs may be related to the greater potency of sunitinib
against these kinases[29]. In contrast to Heinrich’s study,
the clinical benefit, PFS, and OS did not differ between
the groups of patients whose GISTs had KIT exon 9
or exon 11 mutation. Although the KIT oncoproteins
encoded by exon 9 and exon 11 mutants were unequally
sensitive to sunitinib in vitro[29], the limited case number
and racial difference might partly explain the similar clinical response rate of sunitinib in terms of KIT exon mutations in Taiwanese GIST patients.
Sunitinib was reasonably well tolerated in our study
and the most common treatment-related adverse events
were fatigue, diarrhea, skin discoloration, and nausea.
Treatment-related adverse events of any severity grade
were reported in 83% of sunitinib-treated patients, and
serious treatment-related adverse events were reported
in 20% of patients[24]. In contrast to western GIST patients, hand-foot syndrome was the most common cause
of grade Ⅲ adverse events in our study. The reason for
this discrepant incidence of hand-foot syndrome is still
unknown and needs to be fully clarified. Racial differences in drug metabolism or pharmacokinetics are possible reasons for this observation[33]. However, Lee et al[34]
reported a higher frequency of hand-foot syndrome in
Asian patients at Asian sites compared to Asian patients
at non-Asian sites and in non-Asian patients in more than
4000 renal cell carcinoma patients receiving sunitinib. A
lower frequency of some GI-related adverse events (AEs)
in Asian patients at non-Asian sites compared to frequencies in Asian patients at Asian sites and in non-Asian
patients has been observed. Recent evidence suggest that
heterogeneity in toxicity and efficacy among patients receiving anti-VEGF therapy can be partially explained by
genomic variability, including single-nucleotide polymorphisms, providing a possible explanation for the differences in AE frequencies between Asians and non-Asians
in this analysis[34].
Sunitinib-induced hypothyroidism was reported as a
side effect in 12% of GIST patients. No hypothyroidism
was noted in our series and primary hypothyroidism is
not a common complication of therapeutic drugs. Drugs
known to affect thyroid function are lithium, thioamides,
Table 4 Adverse events and selected laboratory abnormalities
Sunitinib (n = 23)
Variable
Adverse event
Anorexia
Diarrhea
Constipation
Fatigue
Nausea
Mucositis/stomatitis
Vomiting
Hypertension
Hand-foot syndrome
Rash
Skin discoloration
Fever
Laboratory abnormalities
Leukopenia
Neutropenia
Febrile neutropenia
Anemia
Elevated creatinine
Thrombocytopenia
AST
ALT
Total bilirubin
GFR
Hypothyroidism
Grade 1
Grade 2
Grade 3
Grade 4
5
6
1
2
0
4
1
4
1
1
5
2
1
8
2
2
0
0
0
4
2
4
0
0
0
0
0
0
0
0
0
1
6
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
4
2
0
8
4
6
7
4
3
3
0
6
4
0
6
4
7
1
0
1
2
0
1
3
1
4
0
0
0
0
1
0
0
0
0
0
0
0
0
0
0
0
0
0
AST: Aspartate aminotransferase; ALT: Alanine aminotransferase; GFR:
Glomerular filtration rate.
and OS of advanced GIST patients facing IM failure
or intolerance. This study reported a median PFS and
OS for 23 advanced GIST patients of 8.4 and 14.1 mo,
respectively, after sunitinib administration for a median
period of 6.0 mo.
Sunitinib induced a sustained clinical benefit in more
than 50% of Taiwanese patients with advanced GISTs
(15/23; 65.2%)[29] in our study, which was better than
Henrich’s report. A CR induced by tyrosine kinase inhibitors on GIST patients has been sporadically reported. The
US S0033 phase Ⅲ study revealed that the CR rate was
3% for 751 metastatic or unresectable GIST patients receiving 400 or 800 mg IM daily[30]. In the EORTC 62005
phase Ⅲ study, the CR rate was 4.76% for 923 metastatic
or unresectable patients receiving 400 or 800 mg imatinib
daily[31]. In contrast to the 2 previous studies, the CR rate
in this study was 8.7% (2/23) and the median TTR for 2
patients that had a CR was 3.73 mo. The high incidence
of CR in this study, even for patients using the second
line tyrosine kinase, is because 1 of these 2 patients underwent surgery to achieve complete tumor removal. The
limited experience on CR after sunitinib treatment for
advanced or metastatic GIST patients facing IM failure or
intolerance may still not justify the use of surgery as an
adjunct method for target therapy in selected patients.
Regarding the relationship between response rate
and kinase mutation, KIT exon 11 and exon 9 mutations
predict a favorable response to IM[32]. Henrich reported
that the clinical activity of sunitinib after IM failure is
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2117
amiodarone, and cytokines such as interferon and interleukin-2. The molecular mechanisms of sunitinib-induced
hypothyroidism are currently unknown but one possible
mechanism by which sunitinib direct affects the thyroid is
through the inhibition of VEGFR and/or PDGFR. Recent studies in a mouse model have shown that VEGFR
inhibition can induce capillary regression in various organs,
including the thyroid. Moreover, the vasculature of the
thyroid showed the greatest regression of all organs[35,36].
In conclusion, sunitinib appears to be a safe and effective treatment for Taiwanese patients with imatinibresistant/intolerant GIST. Sunitinib induced a sustained
clinical benefit in more than 50% of Taiwanese advanced
GIST patients, even those facing imatinib failure or intolerance, with a median 8.4 mo PFS. ORR, PFS, and OS
did not differ between patients whose GISTs had wild
type KIT, KIT exon 9 mutation, or KIT exon 11 mutation. However, hand-foot syndrome accounted for the
most common cause of grade Ⅲ adverse event.
2
3
4
5
6
7
ACKNOWLEDGMENTS
We would like to thank Novartis (Taiwan) Co., Ltd. for
financial support of genetic analysis.
8
COMMENTS
COMMENTS
9
Background
The clinical safety and efficacy of both imatinib mesylate (IM) and sunitinib in
gastrointestinal stromal tumors (GIST) have primarily been established in Western patients living in the USA or Europe and have not been thoroughly studied
in Asian patients. Fifty-six centers in 11 countries participated in the phase Ⅲ
trial of sunitinib in GIST, but only 15 of the 312 patients were of Asian descent (10
and 5 in the sunitinib and placebo groups, respectively).
10
Research frontiers
11
To clarify the efficacy and safety of sunitinib in management of Taiwanese GIST
patients facing IM intolerance or failure. The response of this second line target
therapy also correlates with genetic status of the tumor.
12
Sunitinib appears to be a safe and effective treatment for Taiwanese patients
with imatinib-resistant/intolerant GIST. Sunitinib induced a sustained clinical
benefit in more than 50% of Taiwanese advanced GIST patients, even those
facing imatinib failure or intolerance, with a median 8.4 mo progression free
survival (PFS). ORR, PFS, and overall survival did not differ between patients
whose GISTs had wild type KIT, KIT exon 9 mutation, or KIT exon 11 mutation.
However, hand-foot syndrome accounted for the most common cause of grade
Ⅲ adverse event.
13
14
Applications
15
The preliminary report helps to clarify the efficacy and safety of sunitinib in
management of Taiwanese GIST patients facing IM intolerance or failure.
Peer review
This is a review of therapeutic effects of sunitinib on 22 Taiwanese patients with
metastatic GISTs after IM failure. Their data showed that sunitinib was helpful
in 15 of the 23 patients, and the clinical benefits of sunitinib did not differ in patients with either primary KIT exon 9 or exon 11 mutation. Although the finding
of this study is not new for sunitinib has been shown effective for IM failure, it is
an interesting report showing authors’ experience in using sunitinib in Taiwanese patients.
16
17
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[email protected]
doi:10.3748/wjg.v17.i16.2120
BRIEF ARTICLE
MELD score can predict early mortality in patients with
rebleeding after band ligation for variceal bleeding
Wei-Ting Chen, Chun-Yen Lin, I-shyan Sheen, Chang-Wen Huang, Tsung-Nan Lin, Chun-Jung Lin, Wen-Juei Jeng,
Chien-Hao Huang, Yu-Pin Ho, Cheng-Tang Chiu
shock, and higher model for end-stage liver disease
(MELD) score at the time of rebleeding were independent
predictors for 6-wk mortality. A cut-off value of 21.5 for
the MELD score was found with an area under ROC curve
of 0.862 (P < 0.001). The sensitivity, specificity, positive
predictive value, and negative predictive value were
77.6%, 81%, 71.7%, and 85.3%, respectively. As for the
6-mo survival rate, patients with a MELD score ≥ 21.5
had a significantly lower survival rate than patients with a
MELD score < 21.5 (P < 0.001).
Wei-Ting Chen, Chun-Yen Lin, I-Shyan Sheen, Chang-Wen
Huang, Tsung-Nan Lin, Chun-Jung Lin, Wen-Juei Jeng,
Chien-Hao Huang, Yu-Pin Ho, Cheng-Tang Chiu, Department
of Hepato-Gastroenterology, Linkou Medical Center, Chang
Gung Memorial Hospital and Chang Gung University College of
Medicine, Taoyuan 333, Taipei, Taiwan, China
Author Contributions: Ho YP and Chiu CT designed the
research; Chen WT, Jeng WJ and Huang CH performed data
collection; Chen WT, Lin CY and Ho YP analyzed and interpreted
the data; Chen WT, Ho YP and Lin CJ wrote the manuscript; Lin
CY and Sheen IS contributed critical revision of the manuscript
for important intellectual content; Huang CW contributed
statistical analysis; Lin CJ provided material support; the final
version of the manuscript was approved by all authors.
Correspondence to: Yu-Pin Ho, MD, Assistant Professor,
Department of Hepato-Gastroenterology, Linkou Medical Center,
Chang Gung Memorial Hospital and Chang Gung University
College of Medicine, 5 Fu-Shin Street, Kweishan, Taoyuan 333,
Taipei, Taiwan, China. [email protected]
Telephone: +886-3-3281200 Fax: +886-3-3272236
Received: October 7, 2010
Revised: December 1, 2010
Accepted: December 8, 2010
CONCLUSION: This study demonstrated that the MELD
score is an easy and powerful predictor for 6-wk mortality
and outcomes of patients with early rebleeding after EVL
for EVH.
Key words: Model for end-stage liver disease score; Es
ophageal variceal hemorrhage; Rebleeding; Cirrhosis;
Mortality
Peer reviewer: Igor Mishin, MD, PhD, First Department of
Surgery “N.Anestiadi” and Laboratory of Hepato-PancreatoBiliary Surgery, Medical University "N.Testemitsanu", National
Center of Emergency Medicine, Str. T Chorba 1, 2001 Kishinev,
Republic of Moldova
Abstract
AIM: To investigate the outcomes, as well as risk factors
for 6-wk mortality, in patients with early rebleeding after
endoscopic variceal band ligation (EVL) for esophageal
variceal hemorrhage (EVH).
Chen WT, Lin CY, Sheen IS, Huang CW, Lin TN, Lin CJ,
Jeng WJ, Huang CH, Ho YP, Chiu CT. MELD score can pre
dict early mortality in patients with rebleeding after band
ligation for variceal bleeding. World J Gastroenterol 2011;
org/10.3748/wjg.v17.i16.2120
METHODS: Among 817 EVL procedures performed for
EVH between January 2007 and December 2008, 128
patients with early rebleeding, defined as rebleeding
within 6 wk after EVL, were enrolled for analysis.
RESULT: The rate of early rebleeding after EVL for acute
EVH was 15.6% (128/817). The 5-d, 6-wk, 3-mo, and
6-mo mortality rates were 7.8%, 38.3%, 55.5%, and
58.6%, respectively, in these early rebleeding patients.
The use of beta-blockers, occurrence of hypovolemic
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INTRODUCTION
Esophageal variceal hemorrhage (EVH) is a serious com
2120
Chen WT et al . Predictors for early mortality with rebleeding
plication of liver cirrhosis and causes 70% of all upper
gastrointestinal bleeding episodes in patients with portal
hypertension[1]. According to the Baveno Consensus Wo
rkshop in portal hypertension, endoscopic variceal band
ligation (EVL) therapy is recommended for acute EVH,
although endoscopic sclerotherapy may be used if ligation
is technically difficult[2]. According to the natural course of
EVH, the risk of a recurrent episode of EVH increases
after the first EVH but becomes similar to non-bleeding
esophageal varices (EV) after 6 wk[3]. Therefore, rebleeding
within 6 wk after the first EVH is coined as early rebleeding.
Secondary prophylaxis could reduce the early rebleeding rate
to 20%[1]. Several factors have been identified as predictors
of mortality after EVH, including early rebleeding, bacterial
infection[4], hepatic venous pressure gradient (HVPG) > 20
mmHg measured shortly after admission[5], active bleeding
at initial endoscopy, severity of initial bleeding, hematocrit
level, AST levels, presence of portal vein thrombosis or
of hepatocellular carcinoma (HCC), alcoholic liver disease,
serum bilirubin and albumin levels, Child-Turcotte-Pugh
(CTP) score[1], and Model for End-stage Liver Disease
(MELD) score[6-8]. Among these predictors, early rebleeding
is the most important one[3,9]. However, little information is
known about the risk factors for mortality in the group of
patients with early rebleeding. Thus, the goal of this retro
spective study was to investigate the predictive factors for
mortality in patients with early rebleeding.
bined typical dynamic imaging appearance and elevated
α-fetoprotein (AFP). According to the tumor size, patients
with HCC were divided into early (one nodule ≤ 5 cm or
maximum three nodules, each < 3 cm) or advanced (one
nodule > 5 cm or > 3 nodules). The diagnosis of infec
tion was made by positive results of blood, sputum, urine,
and ascites bacterial culture or elevated ascites fluid and
absolute neutrophil count (ANC) ≥ 250 cells/μL. In addi
tion, 6-wk mortality was defined as death occurring within
6 wk after rebleeding. The first EVL procedure applied
to esophageal variceal bleeding during our study period
was considered as the index EVL and index bleeding. The
following definitions were used on the basis of the rec
ommendations of the Baveno Consensus Workshop: (1)
esophageal variceal bleeding: (a) visible oozing or spurting
of blood from a esophageal varix, (b) white nipple sign or
blood clot adherent to a varix, (c) presence of medium or
large esophageal varices with no other potential bleeding
lesion; (2) EVL ulcer bleeding: bleeding from esophageal
ulcers after endoscopic EVL with one of the following:
(a) active bleeding from the ulcer site, (b) adherent clot at
the ulcer site, or (c) absence of other potential bleeding le
sions; (3) bleeding duration: the acute bleeding episode was
considered finished at the beginning of the first 24-h in
terval with no hematemesis, stable hemoglobin concentra
tion without blood transfusions, and stable hemodynamic
condition; (4) early rebleeding: recurrence of clinically sig
nificant hemorrhage (hematemesis/melena, aspiration of
greater than 100 mL of fresh blood from nasogastric tube
or > 3 g/dL decrease of Hb if no transfusion is given)
within 6 wk after index bleeding episode was considered
finished; (5) rebleeding 5-d failure: uncontrolled bleeding,
death, or recurrent hemorrhage within 5 d since rebleed
ing; and (6) portal hypertensive gastropathy (PHG) bleed
ing: a macroscopic finding of a characteristic mosaic-like
pattern of gastric mucosa with red-point lesions, cherry
red spots, and/or black-brown spots (severe PHG) and the
absence of other potential bleeding lesions.
A total of 817 consecutive EVL procedures for esopha
geal variceal bleeding were recorded and evaluated in a
3500-bed tertiary referral medical center between January
2007 and December 2008. All of the patients with early
rebleeding, defined as rebleeding between one day and
6 wk after ligation, were enrolled. The patients without
endoscopic confirmation of rebleeding focus were ex
cluded. The appropriately convened Institutional Review
Board approved this study. Finally, 128 cirrhotic patients
(15.6%) with early rebleeding were enrolled in our study.
Among these patients, 49 patients who died within 6-wk
after rebleeding were classified as the mortality group. The
remaining 79 patients who survived more than 6 wk were
classified as the survival group. The clinical characteristics
and laboratory data of the patients in these 2 groups were
collected for comparison. Vasoactive drug therapy (terlip
ressin, somatostatin, or octreotide) was routinely adminis
tered before diagnostic endoscopic examination and was
continued for at least 3 d according to national insurance
guidelines of Taiwan for variceal hemorrhage. Prophylactic
antibiotic treatment with intravenous ceftriaxone and nonselective beta-blockers were prescribed for some, depend
ing on the patients’ clinical condition, contraindication,
adverse effect with tolerability, and physician’s preference.
Diagnosis of liver cirrhosis was based on a previous liver
biopsy or compatible clinical, laboratory, and imaging
findings. Hepatocellular carcinoma (HCC) was diagnosed
by liver biopsy, fine needle aspiration cytology, or com
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Statistical analysis was performed after proper tabulation
of data. Continuous variables were expressed as mean with
range, and categorical variables were expressed as count
with percentage. Groups were compared using Student’s
software t-test for continuous variables and χ2 test for cate
gorical variables. Multivariate analysis was performed using
logistic regression, and a receiver operating characteristic
(ROC) curve was generated to assess the predictive accu
racy of the variables. All of these values were considered
statistically significant if the P-value was < 0.05. Cumula
tive survival estimates were calculated by using the KaplanMeier method. All statistical analyses were performed with
SPSS statistical for Windows (Version 16; SPSS. Inc., Chi
cago, IL, USA).
RESULTS
The relevant characteristics of these 128 rebleeding pa
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Table 1 Characteristics of patients with rebleeding after endoscopic variceal band ligation for esophageal varices bleeding
(mean ± SD) n (%)
Variable
Overall (n = 128)
Sex (male/female)
Age (yr)
Etiology
Virus
Alcohol
Virus + alcohol
Others
Index EGD finding
Active EV bleeding
Blood in lumen
Clean
Duration between rebleeding and Index EVL
1-5 d
6-42 d
Index EVL
CTP score
CTP classification
A
B
C
MELD
Rebleeding
CTP score
CTP classification
A
B
C
MELD
107 (83.6)/21 (16.4)
53.6 ± 13.9
Table 2 Rebleeding focus, therapy and outcome of patients
with rebleeding after endoscopic variceal band ligation for
esophageal varices bleeding n (%)
Overall (n = 128)
Rebleeding focus
EV bleeding
Post-EVL ulcer bleeding
GV bleeding
Peptic ulcer bleeding
PHG
Therapy for rebleeding
Endoscopic and pharmacologic therapy
Pharmacologic therapy only
Surgery
Rebleeding 5-d failure
Uncontrolled bleeding and died within 5 d
Died of non-bleeding cause
Recurrent bleeding within 5 d
5-d mortality
6-wk mortality
6-wk mortality causes
Sepsis-induced multi-organ failure
GI bleeding
Liver failure
3-mo mortality
6-mo mortality
61 (47.7)
31 (24.2)
28 (21.9)
8 (6.2)
39 (30.5)
49 (38.3)
40 (31.2)
14.0 (10.8)
33 (25.8)
95 (74.2)
9.8 ± 2.3
12 (9.40)
44 (34.4)
72 (56.2)
19.8 ± 8.9
92 (71.9)
35 (27.3)
1 (0.8)
24 (18.8)
8 (6.3)
2 (1.6)
14 (10.9)
10 (7.8)
49 (38.3)
26 (53.1)
18 (36.7)
5 (10.2)
71 (55.5)
75 (58.6)
10.1 ± 2.6
EV: Esophageal varices; EVL: Endoscopic variceal band ligation; GV: Gastric varices; PHG: Portal hypertensive gastropathy; GI: Gastrointestinal.
14 (10.9)
36 (28.1)
78 (60.9)
21.4 ± 9.8
tients (18.8%) were associated with 5-d failure at rebleed
ing of which 8 patients had uncontrolled bleeding and
died within 5 d, 2 patients died of other causes within 5 d,
and 14 patients had recurrent bleeding within 5 d. The re
bleeding mortality rates at 5 d, 6 wk, 3 mo, and 6 mo were
7.8%, 38.3%, 55.5%, and 58.6%, respectively. The causes
of death within 6 wk after rebleeding were sepsis-induced
multiple organ failure (53.1%), upper gastrointestinal tract
hemorrhage (36.7%), and liver failure (10.2%).
We then further divided the patients into two groups
according to their mortality or survival during the 6-wk
period after rebleeding. The clinical characteristics of pa
tients in the 6-wk mortality group and the survival group
are displayed and compared in Table 3. There were no sig
nificant differences between these two groups with regard
to gender, age, etiology of cirrhosis, HCC, PVT, duration
between rebleeding and index EVL, rebleeding focus and
treatment methods, antibiotic use, serum platelet count,
and sodium and potassium level at rebleeding. However,
higher CTP score (11.9 vs 8.9), higher MELD score (28.9
vs 16.8), and hypovolemic shock during rebleeding (P <
0.001); higher serum total bilirubin, creatinine, and white
cell count levels; lower serum albumin and hemoglobin
levels; longer prothrombin time (INR) and active bleeding
on endoscopy; and higher hepatic encephalopathy grade
were markedly seen in the 6-wk mortality group. Betablocker use after rebleeding was also significantly associ
ated with 6-wk mortality.
Furthermore, by multivariate logistic regression analysis,
hypovolemic shock (OR = 9.25, 95% CI: 1.68-50.93, P =
0.011), beta-blocker use after rebleeding (OR = 0.18, 95%
EVL: Endoscopic variceal band ligation; EV: Esophageal varices; CTP: Childturcotte-pugh; MELD: Model for end-stage liver disease; EGD: Esophagogastroduodenoscopy.
tients are reported in Table 1. The mean age of the pa
tients was 54 years old (range 14-82); 83.6% were male and
16.4% were female. The etiologies of liver cirrhosis were
virus (47.7%), alcohol (24.2%), or combined virus and al
cohol (21.9%). The endoscopic findings at index bleeding
were active EV bleeding (30.5%), blood in the esophageal
or gastric lumen (38.3%), and clean esophago-gastro-duo
denal lumen (31.2%). The average interval between index
EVL and rebleeding was 14 ± 10.8 d. The average CTP
scores at the time of index bleeding and rebleeding were 9.8
± 2.3 and 10.1 ± 2.6, and the average MELD scores were
19.8 ± 8.9 and 21.4 ± 9.8, respectively.
The surveillance of rebleeding sites was carried out by
upper GI endoscopy within 1 day for all enrolled patients;
the rebleeding site, therapeutic methods, and outcomes
are show in Table 2. The surveillance revealed 75 patients
with residual EV bleeding (58.6%), 24 patients with EVLrelated esophageal ulcer bleeding (18.8%), 7 patients with
gastric variceal bleeding (5.5%), 11 patients with peptic
ulcer bleeding (8.6%), and 11 patients with PHG-related
bleeding (8.6%). The management methods for rebleeding
included combined endoscopic and pharmacologic therapy
(71.9%) and pharmacologic therapy only for EVL ulcers
or PHG with mild oozing (27.3%). Only one patient was
treated with surgical intervention (0.8%). In total, 24 pa
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75 (58.6)
24 (18.8)
7 (5.5)
11 (8.6)
11 (8.6)
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Table 3 Variables associated with 6-wk mortality in patients with rebleeding after endoscopic variceal band ligation for esophageal
varices bleeding (mean ± SD) n (%)
Variable
Sex (male/female)
Age (yr)
Etiology
Virus
Alcohol
Virus + alcohol
Others
HCC
No or small
Advanced
PVT
Main trunk
Branch
Duration between rebleeding and index EVL
1-5 d
6-42 d
Rebleeding laboratory findings
CTP score
CTP classification
A
B
C
MELD score
HE (grade)
Cr (μmol/L)
Na (mmol/L)
K (mmol/L)
Bil (μmol/L)
Alb (g/L)
Hb (g/L)
WBC (109/L)
Platelets (109/L)
INR (PT)
Rebleeding 5-d failure
Died within 5 d
Recurrent bleeding within 5 d
Hypovolemic shock
Rebleeding focus
EV bleeding
Post-EVL ulcer bleeding
GV bleeding
Peptic ulcer bleeding
PHG
Rebleeding EGD finding
Active bleeding
Blood in lumen
Clean
EGD Tx
Endoscopic therapy + medical
Medical therapy only
Surgery
Rebleeding infection
Rebleeding antibiotic use
Rebleeding Inderal use
6-wk mortality (n = 49, 38.3%)
6-wk survival (n = 79, 61.7%)
P -value
40 (81.6)/9 (18.8)
53.3 ± 13.3
67 (84.8)/12 (17.6)
53.7 ± 14.3
0.637
0.866
0.683
23 (46.9)
11 (22.4)
13 (26.5)
2 (4.1)
38 (48.1)
20 (25.3)
15 (19.0)
6 (7.6)
29 (59.2)
20 (40.8)
54 (68.4)
25 (31.6)
12 (24.5)
5 (10.2)
12.8 ± 9.0
13 (26.5)
36 (73.5)
18 (22.8)
9 (11.4)
14.7 ± 11.7
20 (25.3)
59 (74.7)
11.9 ± 1.9
8.9 ± 2.3
0.000
1 (2.0)
3 (6.1)
45 (91.8)
28.9 ± 9.0
1.3 ± 1.5
203.3 ± 159.1
138.7 ± 9.6
4.2 ± 1.0
263.3 ± 236.0
25 ± 5
83 ± 18
12.5 ± 6.4
102.4 ± 69.2
2.2 ± 0.9
18 (36.7)
10
8
21 (42.9)
13 (16.5)
33 (41.8)
33 (41.8)
16.8 ± 7.0
0.3 ± 0.8
114.9 ± 97.2
134.6 ± 14.3
3.9 ± 0.9
90.6 ± 135.1
29 ± 6
90 ± 17
8.2 ± 4.7
101.0 ± 72.3
1.5 (0.4)
6 (7.6)
0
6
3 (3.8)
0.017
0.000
0.000
0.000
0.000
0.000
0.084
0.171
0.000
0.000
0.041
0.000
0.915
0.000
0.000
0.000
0.150
0.000
0.500
28 (57.1)
9 (18.4)
3 (6.1)
6 (12.2)
3 (6.1)
47 (59.5)
15 (19.0)
4 (5.1)
5 (6.3)
8 (10.1)
20 (40.8)
25 (51.0)
4 (8.2)
30 (38.0)
27 (34.2)
22 (27.8)
36 (73.5)
13 (26.5)
0 (0.0)
42 (85.7)
35 (71.4)
8 (16.3)
56 (70.9)
22 (27.8)
1 (1.3)
36 (45.6)
46 (58.2)
37 (46.8)
0.291
0.786
0.528
0.879
0.019
0716
0.000
0.132
0.000
The P-values were calculated by using Student’s t-test for continuous variables and c2 test for categorical variables. EV: Esophageal varices; EVL: Endoscopic
variceal band ligation; HCC: Hepatocellular carcinoma; PVT: Portal vein thrombosis; CTP: Child-turcotte-pugh; MELD: Model for end-stage liver disease; INR
(PT): International normalised ratio of a patient's prothrombin time to a normal control sample; PHG: portal hypertensive gastropathy; EGD: Esophagogastroduodenoscopy; GV: Gastric varices.
CI: 0.05-0.63, P = 0.007), and higher MELD score (OR
= 1.17, 95% CI: 1.10-1.25, P < 0.001) at rebleeding were
found to be independent factors for 6-wk mortality in these
patients and this is reported in Table 4. The ROC curve
WJG|www.wjgnet.com
was used for predicting 6-wk mortality in cirrhotic patients
with early rebleeding, and the area under ROC curve (AU
ROC) of the MELD score for predicting 6-wk mortality
was 0.862 (95% CI: 0.80-0.93, P < 0.001). An optimized
2123
Table 4 Logistic regression models for variables associated with
6-wk mortality in patients with rebleeding after endoscopic
variceal band ligation for esophageal varices bleeding
UV
Estimate P -value
Rebleeding laboratory
CTP score
0.61
MELD score
0.17
HE (grade)
0.76
Cr
0.61
Bil
0.09
Alb
-1.18
Hb
-0.23
WBC
0.15
INR (PT)
1.85
Rebleeding
1.96
5-d failure
2.94
Hypovolemic
shock
Rebleeding
-1.47
EGD finding
Rebleeding
1.97
infection
Rebleeding
-1.51
Inderal use
< 0.001
< 0.001
< 0.001
0.001
< 0.001
0.001
0.045
< 0.001
< 0.001
< 0.001
< 0.001
Estimate
0.16
OR
1.17
MV
95% CI
1.10-1.25
P -value
6-wk mortality
< 0.001
MELD score ≥ 21.5
Hypovolemic shock (+)
Beta-blocker use after rebleeding
Sensitivity Specificity
77.55
42.86
83.67
81.01
96.20
46.84
PPV
NPV
71.7
87.5
49.4
85.33
26.92
82.22
PPV: Positive predictive value; NPV: Negative predictive value; MELD:
Model for end-stage liver disease.
1.0
Rebleeding MELD < 21.5
Rebleeding MELD ≥ 21.5
0.8
2.23
9.25 1.68-50.93
0.011
Cum survival
Variables
Table 5 Sensitivity, specificity, positive predictive value, and
negative predictive value for predicting 6-wk mortality in patients with rebleeding after endoscopic variceal band ligation
for esophageal varices bleeding n (%)
0.011
< 0.001
0.6
0.4
0.2
0.001
-1.7
0.18
0.05-0.63
0.007
0.0
0
UV: Univariate analysis; MV: Multivariate analysis; CTP: Child-turcottepugh; MELD: Model for end-stage liver disease; HE: Hepatic encephalopathy; INR (PT): International normalised ratio of a patient's prothrombin
time to a normal control sample; EGD: Esophagogastroduodenoscopy.
60
90
120
150
180
Time since rebleeding (d)
Figure 1 Kaplan-Meier survival curves in patients classified according
to model for end-stage liver disease score < 21.5 or ≥ 21.5 (P < 0.001).
MELD: Model for end-stage liver disease.
cut-off value of the MELD score is 21.5. As shown in
Table 5, the MELD score had a good sensitivity of 78%,
specificity of 81%, positive predictive value (PPV) of 72%,
and negative predictive value (NPV) of 85% for predicting
6-wk mortality. By the above analyses, a MELD score of ≥
21.5 was subsequently chosen as the value for identifying
patients with a high risk of death at 6 wk after rebleeding.
The Kaplan-Meier survival curves in patients classified
according to a MELD score of < 21.5 and ≥ 21.5 revealed
a significant difference as shown in Figure 1. The mortality
rate was 14.7% in patients with MELD <21.5 and 71.7%
in patients with MELD ≥ 21.5 at 6 wk (P < 0.001); 36% in
patients with MELD < 21.5 and 83% in patients MELD ≥
21.5 at 3 mo (P < 0.001); and 40% in patients with MELD
< 21.5 and 84.9% in patients MELD ≥ 21.5 at 6 mo (P <
0.001), respectively.
71.7% for patients with MELD scores more than 21.5.
The mortality rate within 6 wk in our study was 38.3%,
which is higher than the mortality rate of patients after acute
variceal bleeding[1,6]. This is probably because the patients
with rebleeding after EVH were more advanced in disease
severity than patients with initial acute EVH. This difference
in severity could be reflected in the mean MELD score; the
mean score was 21.4 in the rebleeding patients of our study
group, higher than the group of acute variceal bleeding with
a MELD score of 12 as previously reported[6].
The presence of HCC could influence both early re
bleeding and mortality in patients with EVH, as reported
previously[1,10]. However, in the present study, advanced
HCC and portal vein thrombosis were not predictors of
6-wk mortality, which is consistent with previous reports
that advanced HCC is not an independent risk factor but
MELD score is a good predictor for early mortality after
EVH[11].
Another independent factor associated with 6-wk mor
tality in our study was rebleeding related to hypovolemic
shock. This observation was similar to previous studies that
found that the severity of the hemorrhage was predictive
of 6-wk mortality in acute EVH of all cirrhotic patients[1,6].
The third independent factor associated with 6-wk mortal
ity was the use of beta-blockers, which reflected the general
consensus that the use of beta-blockers for the secondary
prevention of EVH could reduce mortality[12]. Overall, 49
patients (38.3%) died within 6 wk after early rebleeding;
DISCUSSION
Our study has revealed that potential rebleeding sources
in cirrhosis cases after index EVL for EVH were esopha
geal varices (58.6%), esophageal ulcer (18.8%), peptic
ulcer (8.6%), PHG (8.6%), and gastric varices (5.5%). The
results were consistent with a previous study[1] reporting
that residual esophageal varices were a major source of
rebleeding. In addition, beta-blocker usage after rebleeding,
hypovolemic shock, and higher MELD score at the time of
rebleeding were independent predictors of 6-wk mortal
ity. In addition, we found that the 6-wk mortality rate was
14.7% for patients with MELD scores less than 21.5 and
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30
2124
to predict death within three months of surgery in patients who had undergone a transjugular intrahepatic protosystemic shut procedure, and was
subsequently found to be useful in determining prognosis and prioritizing
for receipt of liver transplant instead of the older Child-Pugh score. MELD
Score = [0.957 × In(Serum Cr) + 0.378 × In(Serum Bilirubin) + 1.120 ×
In(INR) + 0.643] × 10.
among of them, 10 died within 5 d, and 39 died from day
6 to day 42. In our study, the causes of death were sepsisrelated multiple organ failure (53.1%), GI bleeding-related
complications (36.7%), and liver failure-related complica
tions (10.2%); our results were similar to a recent study
reporting that early mortality after cessation of initial EV
bleeding is significantly associated with bacterial infection
and rebleeding[13]. This finding provides evidence to sup
port the AASLD guidelines for the treatment of acute
variceal bleeding regarding the early use of pharmacologi
cal agents and emergent endoscopic procedure within 12 h[14].
Additionally, for reducing sepsis-related multi-organ failure,
prophylactic use of antibiotics for all patients with cirrhosis
and GI hemorrhage should be encouraged[2,15]. Patients with
a CTP classification of A respond well to current therapies
with minimal risk of death and represented only 2% of the
patients in the 6-wk mortality group in our study. Whether
current treatment recommendations should be applied to
all patients should be further investigated[16].
In conclusion, this study examined the focuses of re
bleeding and treatment outcomes in cirrhotic patients with
early rebleeding after EVL for acute EVH. Specifically, the
study revealed that hypovolemic shock and MELD scores
≥ 21.5 at the time of rebleeding are predictors for 6-wk
mortality in patients with early rebleeding after EVL for
acute EVH. Also, beta-blocker use after rebleeding was
associated with lower 6-wk mortality.
Peer review
The manuscript is a well designed retrospective study with the aim to investigate
the predictive factors for mortality in patients with early rebleeding.
REFERENCES
1
2
3
4
5
6
COMMENTS
COMMENTS
7
Background
The management of variceal bleeding remains a clinical challenge with high
mortality. At the present time, available treatments have reduced the 6-wk rebleeding rate to 20%. Early rebleeding is a strong predictor of death from variceal bleeding. Endoscopic therapy increases control of bleeding and decreases
the risk of rebleeding and mortality. Despite the fact that endoscopic variceal
ligation (EVL) is recommended for acute esophageal variceal bleeding in recent
practice guidelines, there has been relatively little research investigating the
situation of early rebleeding after EVL for esophageal variceal bleeding.
8
9
10
Research frontiers
Currently, treatment recommendations are applied to all patients with variceal
bleeding. At present, only 40% of deaths are directly related to bleeding, while the
majority are caused by infection-related multiple organ failure that is paralleled
with the severity of liver cirrhosis. Patients with Child-Pugh classification A have
good response to current therapy, with a minimal risk of mortality. However, treatment strategies might be different with different Child-Pugh classification.
11
12
This study provides evidence that there are independent predictors for 6-wk
mortality and rebleeding origin in cirrhotic patients with early rebleeding after
therapeutic endoscopic band ligation of initial esophageal varices bleeding.
13
Applications
This article shows significantly less beta blocker use in the mortality group and
recognizes this as an independent poor predictor. To prevent rebleeding with
associated mortality, secondary prophylaxis with beta blockers should start
as soon as possible from the day after stopping usage of vasoactive drugs.
Furthermore, the authors demonstrate that Model for End-Stage Liver Disease
(MELD) score is an easy and accurate predictor of 6-wk mortality of patients
with early rebleeding after EVL for esophageal variceal bleeding. Accurate predictive rules are provided for early recognition of high risk patients.
14
15
Terminology
The Model for End-Stage Liver Disease, or MELD, is a scoring system for
assessing the severity of chronic liver disease. It was initially developed
16
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Abraldes JG, Villanueva C, Bañares R, Aracil C, Catalina MV,
Garci A-Pagán JC, Bosch J. Hepatic venous pressure gradient
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TM, Kosberg CL, D'Amico G, Dickson ER, Kim WR. A model
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(MELD). Hepatology 2007; 45: 797-805
Bosch J, Abraldes JG, Berzigotti A, Garcia-Pagan JC. Portal hypertension and gastrointestinal bleeding. Semin Liver Dis 2008;
28: 3-25
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JH. Endoscopic injection sclerotherapy vs. endoscopic variceal
ligation in arresting acute variceal bleeding for patients with advanced hepatocellular carcinoma. J Hepatol 1994; 21: 1048-1052
Amitrano L, Guardascione MA, Bennato R, Manguso F, Balzano A. MELD score and hepatocellular carcinoma identify patients at different risk of short-term mortality among cirrhotics
bleeding from esophageal varices. J Hepatol 2005; 42: 820-825
D'Amico G, Pagliaro L, Bosch J. Pharmacological treatment of
portal hypertension: an evidence-based approach. Semin Liver
Dis 1999; 19: 475-505
Lee SW, Lee TY, Chang CS. Independent factors associated
with recurrent bleeding in cirrhotic patients with esophageal
variceal hemorrhage. Dig Dis Sci 2009; 54: 1128-1134
Garcia-Tsao G, Sanyal AJ, Grace ND, Carey W. Prevention
and management of gastroesophageal varices and variceal
hemorrhage in cirrhosis. Hepatology 2007; 46: 922-938
Fernández J, Ruiz del Arbol L, Gómez C, Durandez R, Serradilla R, Guarner C, Planas R, Arroyo V, Navasa M. Norfloxacin
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S- Editor Tian L L- Editor Logan S E- Editor Ma WH
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2125
[email protected]
doi:10.3748/wjg.v17.i16.2126
BRIEF ARTICLE
Study on chronic pancreatitis and pancreatic cancer using
MRS and pancreatic juice samples
Jian Wang, Chao Ma, Zhuan Liao, Bing Tian, Jian-Ping Lu
The parameters were as follows: spectral width, 15
KHz; time domain, 64 K; number of scans, 512; and
acquisition time, 2.128 s.
Jian Wang, Chao Ma, Bing Tian, Jian-Ping Lu, Department of
Radiology, Changhai Hospital, The Second Military Medical Uni
versity, Shanghai 200433, China
Zhuan Liao, Department of Gastroenterology, Changhai Hospital,
The Second Military Medical University, Shanghai 200433, China
Author contributions: Wang J and Ma C contributed equally to
this work and performed the majority of experiments and data
analysis; Liao Z provided the vital reagents and analytical tools;
Tian B collected of pancreatic juice samples; Lu JP designed the
study and wrote the manuscript.
Supported by Grants from the National Natural Science Founda
tion of China, No. 30870709 and the Program of Shanghai Subje
ct Chief Scientist, No. 08XD14002(A)
Correspondence to: Jian-Ping Lu, MD, Department of Radiolo
gy, Changhai Hospital, The Second Military Medical University,
168 Changhai Road, Shanghai 200433, China. [email protected]
Telephone: +86-21-81873637 Fax: +86-21-81873637
RESULTS: The main component of pancreatic juice inc
luded leucine, iso-leucine, valine, lactate, alanine, acetate,
aspartate, lysine, glycine, threonine, tyrosine, histidine,
tryptophan, and phenylalanine. On performing 1D 1H
and 2D total correlation spectroscopy, we found a triplet
peak at the chemical shift of 1.19 ppm, which only ap
peared in the spectra of pancreatic juice obtained from
patients with alcoholic chronic pancreatitis. This triplet
peak was considered the resonance of the methyl of
ethoxy group, which may be associated with the metab
olism of alcohol in the pancreas.
CONCLUSION: The triplet peak, at the chemical shift of
1.19 ppm is likely to be the characteristic metabolite of
alcoholic chronic pancreatitis.
Abstract
Key words: Pancreatic juice; Pancreatic cancer; Chronic
pancreatitis; Magnetic resonance spectroscopy; Magnetic
resonance imaging
AIM: To investigate the markers of pancreatic diseases
and provide basic data and experimental methods for
the diagnosis of pancreatic diseases.
Peer reviewer: Vinay Kumar Kapoor, Professor, Department
of Surgical Gastroenterology, Sanjay Gandhi Post-Graduate
Institute of Medical Sciences, Lucknow 226014, India
METHODS: There were 15 patients in the present
study, among whom 10 had pancreatic cancer and 5,
chronic pancreatitis. In all patients, pancreatic cancer
or chronic pancreatitis was located on the head of the
pancreas. Pathology data of all patients was confirmed
by biopsy and surgery. Among the 10 patients with pa
ncreatic cancer, 3 people had a medical history of longterm alcohol consumption. Of 5 patients with chronic
pancreatitis, 4 men suffered from alcoholic chronic
pancreatitis. Pancreatic juice samples were obtained
from patients by endoscopic retrograde cholangiopancreatography. Magnetic resonance spectroscopyn
was performed on an 11.7-T scanner (Bruker DRX-500)
using Call-Purcell-Meiboom-Gill pulse sequences.
WJG|www.wjgnet.com
Wang J, Ma C, Liao Z, Tian B, Lu JP. Study on chronic pancreatitis
and pancreatic cancer using MRS and pancreatic juice samples.
World J Gastroenterol 2011; 17(16): 2126-2130 Available from:
URL: http://www.wjgnet.com/1007-9327/full/v17/i16/2126.htm
DOI: http://dx.doi.org/10.3748/wjg.v17.i16.2126
INTRODUCTION
Pancreatic cancer accounts for about 2% of all cancer cases,
but it has the worst prognosis of all cancers with a 5-year
2126
Wang J et al . MRS for pancreatic diseases
mum lesion diameter was 26.1 mm (range, 11-51 mm),
and all lesions were located in the head of the pancreas.
We chose 5 patients (4 men and 1 woman; mean age, 58.3
years) with chronic pancreatitis, confirmed by in vivo biopsy, and lesions located at the head of the pancreas. The
medical history of every patient was recorded in detail. All
study protocols were approved by our Institutional Review Board, and informed consent was obtained from all
patients before they were enrolled in this study.
Pancreatic juice samples were obtained from patients
by endoscopic retrograde cholangio-pancreatography
(ERCP) in frozen tubes and immediately placed in liquid
nitrogen before storing the tubes in a -80℃ refrigerator
for MRS experiments. All patients were diagnosed by biopsy analyses of pathology data. Both the tumor marker
CA19-9 and the cancer gene maker p53 were detected in
patients with pancreatic cancer, whereas these markers
were not detected in patients with chronic pancreatitis.
survival rate of less than 3%[1,2]. Because of the deep-seated
location of the pancreas and no apparent symptoms at the
initial stages of pancreatic cancer, it is difficult to diagnose
this disease in the early stages. Chronic pancreatitis is a kind
of localized or diffuse inflammation, and is caused by many
factors. One of the medical dilemmas is to distinguish
pancreatic cancer from chronic pancreatitis with a mass in
the head of the pancreas; both these diseases have similar
clinical behavior and imaging features[3]. A puncture biopsy
is usually preferred over an operation when diagnosing the
disease, but this is an injurious procedure and may lead to
some complications[4].
Magnetic resonance imaging (MRI) is the most common procedure used in the diagnosis of pancreatic cancer.
An MRI helps obtain images of the pancreas and its surrounding structures[5]. The deep-seated location of the
pancreas and similar clinical manifestations of chronic
pancreatitis and pancreatic cancer are the main barriers in
differentiating between these two diseases even with advanced MRI techniques. Magnetic resonance spectroscopy
(MRS) has high sensitivity and resolution, allows in vitro
testing of metabolites, and has been widely used in the
field of metabolomics[6-12]. MRS will be the most potent
tool to help differentiate between pancreatic cancer and
chronic pancreatitis, and it will be the most effective tool
for the early diagnosis of a pancreatic tumor.
Usually, in the process of cancerization, gene and
metabolite abnormalities appear before tissue structure
transformation. Detection of abnormalities in metabolites
facilitates early diagnoses of tumors. Clinically, the serum
marker CA19-9[13] and gene tumor markers such as the
K-ras gene[14,15], p53 anti-oncogene, and p53 protein[16-19] are
widely used as markers of pancreatic cancer; however, these
markers are not sensitive, show low specificity, and are used
as auxiliary tools[20]. Beger et al[10] had successfully used MRS
and mass spectrum (MS) to analyze blood constituents of
patients with pancreatic cancer and of healthy volunteers;
they were able to make a good distinction between pancreatic cancer and the control group by performing lipid profiling of the blood. Pancreatic juice is the exocrine of the
pancreas, and is closely related with pancreatic tissues. We
wanted to investigate whether it is possible to obtain some
information to help differentiate between chronic pancreatitis and pancreatic cancer by analyzing pancreatic juice
samples. In the present study, we used MRS technology to
analyze the pancreatic juice of patients with pancreatic cancer or chronic pancreatitis with a mass in the head of the
pancreas and tried to explore the markers of these diseases.
We provided basic data and experimental methods for the
study of pancreatopathy.
MRS experiments
MRS experiments were performed on a Bruker DRX-500
spectrometer (1H frequency, 500.13 MHz; Bruker Biospin, Rheinstetten, Germany). Pancreatic juice samples
were diluted with phosphate buffer in D2O and placed in
sample tubes (diameter, 5 mm), which are used in MRS
experiments. Spectra were acquired at 300.0 K using CallPurcell-Meiboom-Gill (CPMG) pulse sequence[21,22] along
with water presaturation during the relaxation delay of
2 s. The CPMG pulse sequence was applied as a T2 filter
to suppress signals from molecules with short T2 values
(such as macromolecules and lipids), using a total echo time
(TE) time of 320 ms. The main parameters for the 1D 1HMRS spectra were as follows: spectral width (SW), 15 KHz;
time domain (TD), 64 K; number of scans (NS), 512; and
acquisition time (AQ), 2.128 s. Spectral assignments were
confirmed by 2D 1H-1H TOCSY[23] and J-resolved (JRES)
along with the values obtained from the literature[24]. The
main parameters used for TOCSY were as follows: TD
(F1-dimensional), 512; TD, (F2-dimensional), 1 K; SW (F1
and F2-dimensional), 5 KHz; and NS, 32. The main parameters used for JRES were as follows: TD (F1-dimensional),
256; TD (F2-dimensional), 8 K; SW (F1-dimensional), 78
Hz; SW (F2-dimensional), 8 KHz; and NS, 32. In both the
2D MRS experiments, the delay time was 2 s. The stability
of the pancreatic juice samples was evaluated by repeating a 1D MRS experiment after overall acquisition. No
biochemical degradation of any of the pancreatic juice
samples was observed.
RESULTS
On combining 2D MRS experimental results (Figure 1)
obtained in the present study with results from related literature[25-27] we identified the main resonances in 1H MRS
spectra of pancreatic juice. TOCSY is a useful 2D MRS
technology, it can be used to distinguish the frequencies
in the total spin system and improve the sensitivity of detecting small J couplings[28]. On the basis of TOCSY data,
the resonances in 1D 1H MRS spectra (Figure 2) of some
The initial subject population comprised 35 patients with
pancreatic cancer (24 men and 11 women; mean age, 67.2
years; age range, 47-85 years) recruited between January
2006 and June 2009. We selected 10 subjects (7 men and 3
women; mean age, 67.7 years; age range, 57-74 years) with
surgically confirmed pancreatic cancer. The mean maxi-
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2127
Phe
A
Tyr
Tyr
F1
Tyr
His
7.0
Trp
Trp
His
His
7.2
δH
Trp
Phe
7.4
7.8
7.6
7.2
7.0
Val + Leu + Ileu
His
7.8
7.4
δH
7.6
7.8
7.6
7.4
7.2
7.0
Ala
F2
Ala + Lys Gly
Lys
Tyr
Val Thr
Asp
Thr Lac
Asp
δH
B
Lac
Ace
F1
-0.05
1.2
F2
δH
2.0
1.5
1.0
Among the 10 patients with pancreatic cancer, diagnosed by postoperative analyses of pathology data, 3 people had a medical history of long-term alcohol consumption. Of 5 patients with chronic pancreatitis, 4 men had a
medical history of long-term alcohol consumption, and
they were typical patients with alcoholic chronic pancreatitis. The female patient suffered from auto-immune chronic
pancreatitis by analyses of the pathology data.
Figure 1 Assignments of partial resonances in 1H spectra of pancreatic
juice. A: 2D Total correlation spectroscopy (TOCSY) of human pancreatic juice; B:
JRES spectra of human pancreatic juice. Tyr: Tyrosine; His: Histidine; Trp: Tryptophan; Phe: Phenylalanine.
amino acids were identified. The components, including
leucine (Leu), iso-leucine (Ileu), valine (Val), lactate (Lac),
alanine (Ala), acetate (Ace), aspartate (Asp), lysine (Lys),
glycine (Gly), threonine (Thr), tyrosine (Tyr), histidine
(His), tryptophan (Trp) and phenylalanine (Phe), and the
locations were also identified.
From the analysis of 1D 1H MRS spectra of all panc
reatic juice samples, it was easy to find a triplet peak at the
chemical shift of 1.19 ppm (Figure 3), which only appeared
in some spectra. Four of the pancreatic juice samples of
patients with chronic pancreatitis showed a triplet peak at
the chemical shift of 1.19 ppm on 1H MRS, whereas the
spectra of the pancreatic juice of patients with pancreatic
cancer did not show a peak at the chemical shift of 1.19 ppm.
When subjected to 2D TOCSY, the spectra of pancreatic
juice samples of patients with chronic pancreatitis only
showed one correlation peak at the chemical shift of 1.19
ppm and 3.36 ppm (Figure 4). By chemical shift and J
coupling constant, we found that the peak at the chemical
shift of 1.19 ppm was the 1H peak of the methyl of ethoxy
group (CH3CH2O-). The 1D 1H spectra of the 4 panc
reatic juice samples of patients with chronic pancreatitis
showed a triplet peak at the chemical shift of 1.19 ppm;
further, these patients had a history of drinking, which was
found from the analysis of pathology data. The 1D 1H
spectra of pancreatic juice obtained from the female patient
with chronic pancreatitis did not show a triplet peak at the
chemical shift of 1.19 ppm.
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2.5
Figure 2 The assignment of proton magnetic resonance spectroscopy spectra of pancreatic juice with chronic pancreatitis. Ace: Acetate; Ala: Alanine; Asp:
Aspartate; Gly: Glycine; His: Histidine; Ileu: Iso-leucine; Lac: Lactate; Leu: Leucine;
Lys: Lysine; Phe: Phenylalanine; Thr: Threonine; Tyr: Tyrosine; Trp: Tryptophan;
Val: Valine.
0.10
3.6
3.0
δH
0.05
3.7
3.5
δH
0
4.0
DISCUSSION
In recent years, there has been much debate on the relati
on between chronic pancreatitis and pancreatic cancer.
While some scholars[29] believe that both diseases have a
close connection, others[30] disagree. The results of our
study show that there is no apparent difference between
the components of pancreatic juice obtained from patien
ts with chronic pancreatitis and pancreatic cancer, except
that 1D 1H spectra of pancreatic juice obtained from the
former group of patients who suffered from alcoholic
chronic pancreatitis shows a triplet peak of the methyl of
ethoxy group (CH3CH2O-) at the chemical shift of 1.19 ppm.
This finding may be used to differentiate pancreatic cancer from alcoholic chronic pancreatitis with a mass in the
head of the pancreas.
In the present study of the 10 patients with pancreatic cancer, 3 had a medical history of long-term alcohol
consumption, but the 1D 1H spectra of their pancreatic
juice did not show the triplet peak of the ethoxy group
(CH3CH2O-); further, the pathology data of these patients
did not show symptoms related to chronic pancreatitis.
Hence, we can conclude that alcohol is not the main factor
that causes pancreatic cancer. It is controversial whether
2128
A
A
Val
Thr
Lys
C
Lys
F1
Lys
Ileu
Leu
D
1.5
2.0
2.5
E
Phe
F
3.0
Val
G
Ala
Lac
Tyr
H
3.5
3.0
2.5
2.0
4.0
1.5
3.5
3.0
2.5
2.0
1.5
3.5
4.0
F2
δH
δH
B
Figure 3 1H magnetic resonance spectroscopy spectra of human pancreatic juice with chronic pancreatitis and pancreatic cancer. A-E: 1D 1H MRS
spectra of pancreatic juice of patients with chronic pancreatitis; F-H: 1D 1H magnetic resonance spectroscopy spectra of pancreatic juice of patients with pancreatic
cancer, other 7 similar spectra of pancreatic juice of patients with pancreatic cancer
are not shown.
F1
long-term alcohol consumption can cause pancreatic cancer. Riediger et al[31] found that the association between alcohol and pancreatic cancer was not apparent during epidemiological investigation, which is in accord with our results.
We applied MRS to study pancreatic juice obtained
from patients with chronic pancreatitis and pancreatic
cancer, and separated the various components of different amino acids in human pancreatic juice by 1D and 2D
1
H spectra. Recently, many analyses on the components
of pancreatic juice have focused on the aspect of proteomics. Our study in the field of metabolomics is auxiliary to proteomics, and goes a step further in the study of
pancreatopathy.
It is difficult to differentiate between pancreatic cancer
and chronic pancreatitis with a mass in the head of the
pancreas solely by MRI, because both these diseases have
similar clinical behaviors and imaging features. There is
no noninvasive method that can be successfully used to
diagnose these diseases. Biopsy is frequently used to diagnose pancreatic cancer or chronic pancreatitis with a mass
in the head of the pancreas but has some disadvantages,
e.g. false negative results, many complications (bleeding,
seepage of bile and pancreatic juice), and risk of tumor
metastasis. ERCP causes some injury, but it is better than
biopsy, because it causes fewer complications. ERCP,
when combined with MRS technology, helps obtain more
information on metabolites, which cannot be obtained
from biopsy or MRI, and is likely to be used to distinguish
pancreatic cancer from alcoholic chronic pancreatitis with
a mass in the head of pancreas on the basis of 1H MRS
spectra of pancreatic juice.
There are some limitations to this study. Lack of control groups and the content of metabolites in the pancreatic juice being small meant we could not perform a quantitative analysis, and only obtained some qualitative results.
The excreta of patients with pancreatic cancer or chronic
pancreatitis may not only have different components but
also differ in quantity.
3.5
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1.5
2.0
2.5
δH
4.0
δH
B
3.0
4.0
4.0
3.5
3.0
2.5
2.0
1.5
F2
δH
Figure 4 2D total correlation spectroscopy spectra of pancreatic juice
with chronic pancreatitis and pancreatic cancer. A: Total correlation spectroscopy (TOCSY) spectra of pancreatic juice from patients with alcoholic
chronic pancreatitis; B: TOCSY spectra of pancreatic juice from patients with
pancreatic cancer. Ala: Alanine; Ileu: Iso-leucine; Lac: Lactate; Leu: Leucine;
Lys: Lysine; Phe: Phenylalanine; Thr: Threonine; Tyr: Tyrosine; Val: Valine.
In conclusions, MRS is a powerful tool that can be
applied to the study of pancreatic juice obtained from
patients by ERCP, and does not cause injury. The triplet
peak, which is at the chemical shift of 1.19 ppm in 1D
1
H-MRS data of pancreatic juice obtained from the patients with alcoholic chronic pancreatitis, was identified as
resonance of the methyl of ethoxy group (CH3CH2O-).
The ethoxy group may be associated with alcohol metabolism in the pancreas, and is likely to be used to distinguish
pancreatic cancer from alcoholic chronic pancreatitis with
a mass in the head of the pancreas. In view of small numbers, further confirmation of the results in a larger number of patients is required.
COMMENTS
COMMENTS
Background
Pancreatic cancer is a malignant neoplasm of the pancreas, and it has a high
death rate. One of the medical dilemmas is to distinguish pancreatic cancer
from chronic pancreatitis with a mass in the head of the pancreas; both diseases have similar clinical behavior and imaging features. Exploring the markers to
distinguish the diseases is very important to the therapies of patients in clinic.
Research frontiers
CA19-9, K-ras gene, p53 anti-oncogene, and p53 protein are widely used as
markers of pancreatic cancer, but they are not sensitive and show low specificity. Searching characteristic markers of the diseases is still the goal of tireless
pursuit. Magnetic resonance spectroscopy (MRS) has high sensitivity and
2129
resolution, allows in vitro testing of metabolites, and has been widely used in
the field of metabolomics. MRS will be the most potent tool to help differentiate
between pancreatic cancer and chronic pancreatitis.
12
It is difficult to differentiate between pancreatic cancer and chronic pancreatitis with
a mass in the head of the pancreas solely by Magnetic Resonance Imaging (MRI).
Biopsy is frequently used to diagnose the diseases but has many complications and
risk of tumor metastasis. Endoscopic retrograde cholangio-pancreatography (ERCP)
combined with MRS helps obtain more information on metabolites, which cannot
be obtained from biopsy or MRI. We separated the various components of different
amino acids in human pancreatic juice. The triplet peak which is at the chemical
shift of 1.19 ppm in 1D 1H MRS spectra was identified as resonance of the methyl
of ethoxy group (CH3CH2O-), and it may be the characteristic metabolites of the
patients with alcoholic chronic pancreatitis.
13
14
15
Applications
16
This study provides basic data and experimental methods for the diagnosis of
pancreatic diseases. The ethoxy group is likely to be used to distinguish pancreatic
cancer from alcoholic chronic pancreatitis with a mass in the head of the pancreas.
Terminology
17
1
Chemical shift is a basic concept in MRS, the pick appearing in the H MRS spectra with different chemical shift means the nuclear of proton spins with different
frequency. The same rotation frequency of nuclear of protons only shows one peak
in the 1H MRS spectra. The triplet peak at the chemical shift of 1.19 ppm is caused
by the interaction of nearby nuclear of protons. In fact, it is split by one peak.
18
Peer review
19
REFERENCES
20
In my opinion, the article is acceptable for publication.
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S- Editor Sun H L- Editor O’Neill M E- Editor Ma WH
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[email protected]
doi:10.3748/wjg.v17.i16.2131
BRIEF ARTICLE
Ku80 gene G-1401T promoter polymorphism and risk of
gastric cancer
Jia-Qi Li, Jie Chen, Nan-Nan Liu, Li Yang, Ying Zeng, Bin Wang, Xue-Rong Wang
TT were 65.6%, 22.8% and 11.6% in gastric cancer
cases, respectively, and 75.8%, 17.6% and 6.6% in
controls, respectively. There were significant differences between gastric cancer and control groups in the
distribution of their genotypes (P = 0.03) and allelic
frequencies (P = 0.002) in the Ku80 promoter G-1401T
polymorphism.
Jia-Qi Li, Jie Chen, Nan-Nan Liu, Ying Zeng, Bin Wang,
Xue-Rong Wang, Key Laboratory of Reproductive Medicine,
Department of Pharmacology, Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
Li Yang, Department of General Surgery, First Affiliated Hospital
of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
Author contributions: Yang L, Zeng Y, Wang B and Wang XR
designed the research and enrolled the patients; Li JQ, Chen J and
Liu NN performed the research; Li JQ analyzed the data; Li JQ
wrote the paper.
Supported by Grants from the National Natural Science Foundation of China, No. 30672486; the Natural Science Foundation
of Jiangsu Province, No. BK2006525; “333 Project” and “Qinglan
Project” Funds for the Young Academic Leader of Jiangsu Province to Wang B
Correspondence to: Xue-Rong Wang, PhD, Key Laboratory of Reproductive Medicine, Department of Pharmacology,
Nanjing Medical University, Nanjing 210029, Jiangsu Province,
Telephone: +86-25-86862884 Fax: +86-25-86862884
CONCLUSION: The T allele of Ku80 G-1401T may be
associated with the development of gastric cancer.
Key words: Ku80 ; Gastric cancer; Polymorphism; Promoter; Carcinogenesis
Peer reviewer: Pete Muscarella, MD, Division of Gastrointestinal Surgery, The Ohio State University, N711 Doan Hall, 410 W.
10th Ave., Columbus, OH 43210, United States
Li JQ, Chen J, Liu NN, Yang L, Zeng Y, Wang B, Wang XR.
Ku80 gene G-1401T promoter polymorphism and risk of gastric cancer. World J Gastroenterol 2011; 17(16): 2131-2136
Available from: URL: http://www.wjgnet.com/1007-9327/full/
v17/i16/2131.htm DOI: http://dx.doi.org/10.3748/wjg.v17.i16.
2131
Abstract
AIM: To evaluate the possible relationship between the
Ku80 gene polymorphism and the risk of gastric cancer
INTRODUCTION
in China.
Gastric cancer is one of the most frequent malignancies
in many countries, accounting for 8.7% of all cancers
and 10.4% of all cancer deaths in the year of 2000[1].
In China, gastric cancer remains the leading cause of
cancer-related mortality among men and women[1,2]. It is
estimated that about 39% of gastric cancer cases occur in
Chinese population[1,2]. The environmental factors, diet,
tobacco, alcohol and Helicobacter pylori infection are wellknown causes of gastric cancer in China[3-5]. However,
only a fraction of individuals exposed to these factors
METHODS: In this hospital-based case-control study
of gastric cancer in Jiangsu Province, China, we investigated the association of the Ku80 G-1401T (rs828907)
polymorphism with gastric cancer risk. A total of 241
patients with gastric cancer and 273 age- and sexmatched control subjects were genotyped and analyzed
by polymerase chain reaction-restriction fragment length
polymorphism.
RESULTS: The frequencies of genotypes GG, GT and
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Li JQ et al . Ku80 and gastric cancer
develop gastric cancer, suggesting that individual susceptibility to gastric cancer should be different. Currently, the
genomic etiology of gastric cancer is of great interest but
largely unknown.
DNA damage drives the formation and development
of malignant tumors that ameliorate this damage, and
its sequelae can be categorized as either gatekeeper or
caretaker tumor suppressors, depending on their mode
of action[6]. Nonhomologous end joining (NHEJ) repairs
DNA double-strand breaks (DSBs) by joining ends without using a homologous template strand and has been
described as a caretaker[7,8]. Many studies have shown
that NHEJ is the predominant repair system in humans,
which included the DNA ligase IV and its associated
protein XRCC4, and the three components of the DNAdependent protein kinase (DNA-PK) complex, Ku70,
Ku80, and the catalytic subunit PKcs[9]. The Ku80 gene,
also known as XRCC5, is an important and specific member of NHEJ. Ku70 and Ku80 form a heterodimer called
Ku that is well known for its role in NHEJ pathway[10].
Ku acts as a regulator of transcription by interacting
with the recombination signal binding protein Jκ and the
nuclear factor (NF)-κB p50 homodimer to up-regulate
p50 expression, which may regulate the proliferation of
gastric cancer cells[11]. Gastric cancer cells with a low level
of constitutive NF-κB had a lower expression level of
Ku70 and Ku80, which was reflected in the lower nuclear
levels of Ku proteins, than the wild-type cells and the
cells transfected with control vector[12,13]. In addition,
several studies reported that gastric cancer patients with
a lower Ku80 expression level had a slightly prolonged
survival after neoadjuvant chemotherapy[14-16].
Genetic polymorphisms in Ku80 genes influence DNA
repair capacity and change predisposition of several cancers, including colorectal[17], bladder[18] and oral cancers[19].
In addition, in these hospital-based case-control studies
of other cancers, it was reported that the frequency of
GT/TT type of the Ku80 gene at promoter G-1401T
(rs828907) was significantly higher in cases than in controls[17-19]. Thus, we assumed that the specific polymorphism of Ku80 gene may also contribute to gastric cancer.
To test the hypothesis that the promoter G-1401T polymorphism is associated with the risk of gastric cancer,
we used polymerase chain reaction-restriction fragment
length polymorphism (PCR-RELP) to genotype this polymorphism in a hospital-based case-control study of 241
patients with gastric cancer and 273 age- and sex-matched
cancer-free controls. The results of this research will lead
to a better understanding of the role of SNPs in the Ku80
genes in gastric cancer carcinogenesis. Such knowledge
may eventually lead to the development of better preventive measures for gastric cancer.
gastric cancer patients were confirmed histologically. Genetically unrelated cancer-free individuals were recruited
as controls who were selected by matching for age and
gender during the same period. All subjects were Han
Chinese from the eastern region of China and randomly
selected from the Department of General Surgery of the
First Affiliated Hospital of Nanjing Medical University
between 2005 and 2009. All patients and control subjects
voluntarily participated in the study, completed a selfadministered questionnaire and donated 5 mL of blood
samples. The questionnaire included questions on sex,
age, residence, diabetes, hypertension and smoking status.
Smoking was defined as ≥ 10 cigarettes per day. This research protocol was approved by the Institutional Review
Board of Nanjing Medical University.
Genotyping analysis
Genomic DNA was isolated from peripheral blood lymphocytes using standard phenol-chloroform extraction,
as previously described[20,21]. PCR-RELP assay was used
to type the Ku80 G-1401T (rs828907) polymorphisms. In
brief, the primers of the Ku80 G-1401T polymorphism
were 5'-TAGCTGACAACCTCACAGAT-3' (forward)
and 5'-ATTCAGAGGTGCTCATAGAG-3' (reverse)[19],
which generated a 252-bp fragment. The PCR reaction
was performed in a total volume of 20 μL containing
2 μL 10 × PCR buffer, 1.25 mmol/L MgCl2, 0.1 mmol/L
dNTPs, 0.25 μmol/L each primer, 200 ng of genomic
DNA and 1 U of Taq DNA polymerase (MBI Fermentas). The PCR was performed at 94℃ for 5 min and followed by 35 cycles of 30 s at 94℃, 30 s at 55℃ and 30 s
at 72℃, with a final elongation at 72℃ for 10 min. The
restriction enzyme BfaI (New England BioLabs) was used
to distinguish the PCR product, and the genotypes were
discriminated on 3% agarose gel and visualized by staining
with 0.5 μg/mL ethidium bromide. The wild-type G-allele
produced a single 252-bp fragment, and the polymorphic
T-allele produced 2 fragments of 81-bp and 171-bp. Approximately, 10%-15% of the samples were randomly
selected for repeated assays, and the results were 100%
concordant.
Continuous variables are presented as mean ± SD and
compared by unpaired Student’s t test. Continuous variables departing from the normal distribution were presented as median and interquartile range and analyzed
by Mann-Whitney U-test. Discrete variables were represented as frequencies and percentages and evaluated by
the Pearson’s χ2 test. Pearson’s χ2 test was also used to
compare the distribution of the Ku80 genotypes between
cases and controls. The association between the Ku80
G-1401T polymorphism and the risk of gastric cancer
was estimated by odds ratio (OR) and 95% CI using
multivariate logistic regression. P < 0.05 was considered
statistically significant. All statistical analyses were performed using SPSS version 13.0 for Windows (SPSS Inc.,
Chicago, IL, USA).
Study population
The case-control study consisted of 241 patients with
gastric cancer and 273 cancer-free control subjects. The
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Table 3 Allele distribution of Ku80 G-1401T polymorphism
in cases and controls n (%)
Table 1 Baseline characteristics of cases and controls n (%)
Characteristics
Cases (n = 241)
Controls (n = 273)
181 (75.1)
57.9 ± 12.9
61 (25.3)
111 (46.1)
21 (8.7)
15 (6.2)
193 (70.7)
56.9 ± 14.1
39 (14.3)
142 (52.0)
28 (10.3)
22 (8.1)
Sex (male)
Age (yr)
Smoking
Residence (rural)
Hypertension
Diabetes
P
Allele
0.43
0.52
0.014
0.32
0.58
0.51
G
T
GG
GT
TT
Cases
Control
158 (65.6)
55 (22.8)
28 (11.6)
207 (75.8)
48 (17.6)
18 (6.6)
371 (77.0)
111 (23.0)
462 (84.6)
84 (15.4)
residence, diabetes and hypertension) with 95% CI for
mutant genotypes was all described. In statistical analyses stratified by the median age of controls (58 years),
the increased risk associated with the GT/TT genotypes
tended to be more evident in the younger subjects aged <
58 years (adjusted OR = 1.97, 95% CI: 1.05-2.90). However, we did not note a statistically significant inverse association with gastric cancer risk in older subjects aged ≥
58 years (adjusted OR = 1.31, 95% CI: 0.88-1.96).
The adjusted OR for the GT/GT genotypes was 1.81
(95% CI: 1.28-2.52) in male subjects and 1.33 (95% CI:
0.81-2.24) in female subjects. We did not note a statistically significant inverse association with gastric cancer
risk in both non-smokers (adjusted OR = 1.48; 95% CI:
1.08-2.02) and smokers (adjusted OR = 2.52; 95% CI:
1.25-5.18). In urban subjects, there was significant evidence of an increased risk of gastric cancer in the variant genotypes (adjusted OR = 1.88; 95% CI: 1.26-2.76),
while the association was not statistically significant in
rural subjects (adjusted OR = 1.48; 95% CI: 0.99-2.15).
2
χ = 7.26, df = 2, P = 0.03.
RESULTS
Baseline characteristics
The frequency distributions of selected characteristics
of the cases and controls are presented in Table 1. There
was no significant difference between the cases and controls in sex (male: 75.1% vs 70.7%, P = 0.43) and age (57.9
± 12.9 years vs 56.9 ± 14.1 years, P = 0.52), indicating
that the matching for the subjects was successful. More
smokers were found among gastric cancer cases compared with controls (25.3% vs 14.3%, P = 0.014). No
significant differences were noted in residing in the rural
area (46.1% vs 52.0%, P = 0.32), hypertension (8.7% vs
10.3%, P = 0.58) and diabetes (6.2% vs 8.1%, P = 0.51).
DISCUSSION
In this hospital-based, case-control study, we assessed the
potential association between the Ku80 G-1401T polymorphism and the presence of gastric cancer in Chinese
population. To our best knowledge, this is the first study
linking the Ku80 G-1401T polymorphism with gastric
cancer risk. Our data showed that the Ku80 -1401 G to T
variant was associated with the increased risk of gastric
cancer.
Gastric cancer is a genetic disease developing from a
multifactorial, multigenetic and multistage process[22,23]. It
was widely accepted that both genetic and environmental
factors may be involved in the etiology of gastric cancer[24]. During the multistage carcinogenesis, Ku80 may be
involved in multiple important cellular processes. To date,
several studies have reported abnormal expression of
Ku80 protein in various cancers[13,25-28]. Over-expression of
Ku80 increased the capability of cancer acquired resistance
to radiation and chemical drugs[29-31], while suppression of
Ku80 expression decreased cellular proliferation, colony
formation and inhibited tumorigenicity in a xenograft
model[32]. As an important component of NHEJ, Ku80
and Ku70 form a heterodimer, which acts as a regulatory
subunit of the DNA-dependent protein kinase complex
DNA-PK by increasing the affinity of the catalytic subunit PRKDC to DNA[17]. The Ku80 gene plays an important and specific role in removing DSBs. Chang et al[18]
Genotype distributions and allele frequencies
Table 2 shows the distribution of the genotypic for the
Ku80 G-1401T (rs828907) between gastric cancer patients and controls. The genotypic frequencies in both
gastric cancer and control groups were in agreement
with those predicted by Hardy-Weinberg equilibrium (P
= NS). The distribution of the Ku80 G-1401T genotypes
(GG, GT and TT) was markedly different between cases
(65.6%, 22.8%, and 11.6%) and controls (75.8%, 17.6%,
and 6.6%, P = 0.03). A significantly different distribution of the Ku80 G-1401T genotype was demonstrated
among the cases and controls. As shown in Table 3, the
frequency of T allele was significantly higher in gastric
cancer patients than in control subjects (23.0% vs 15.4%,
P = 0.002).
Stratified analyses for the variant Ku80 genotype in
cases and controls
The multivariate logistic regression analysis was further
used to evaluate the association between the G-1401T
polymorphism and gastric cancer stratified by risk factors
including age, sex, smoking and residence under control
(Table 4). Adjusted OR (for age, sex, smoking status,
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Controls
2
χ = 9.73, df = 1, P = 0.002.
Table 2 Genotype of Ku80 G-1401T polymorphism in cases
and controls n (%)
Genotype
Cases
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Table 4 Stratification analyses of the association between Ku80 polymorphism and risk of gastric cancer n (%)
Cases (n = 241)
Variable
Age (yr) (median)
< 58
≥ 58
Sex
Male
Female
Smoking status
Smokers
Non-smokers
Residence
Urban
Rural
Controls (n = 273)
Adjusted OR (95% CI)
1
P
GG
GT + TT
GG
GT + TT
76 (62.3)
82 (68.9)
46 (37.7)
37 (31.1)
109 (76.8)
98 (74.8)
33 (23.2)
33 (25.2)
1.97 (1.05-2.90)
1.31 (0.88-1.96)
0.01
0.3
118 (65.2)
40 (66.7)
63 (34.8)
20 (33.3)
149 (77.2)
58 (72.5)
44 (22.8)
22 (27.5)
1.81 (1.28-2.52)
1.33 (0.81-2.24)
0.01
0.46
39 (63.9)
119 (66.1)
22 (36.1)
61 (33.9)
32 (82.1)
175 (74.8)
7 (17.9)
59 (25.2)
2.52 (1.25-5.18)
1.48 (1.08-2.02)
0.051
0.054
85 (65.4)
73 (65.8)
45 (34.6)
38 (34.2)
102 (77.7)
105 (73.9)
29 (22.3)
37 (26.1)
1.88 (1.26-2.76)
1.48 (0.99-2.15)
0.025
0.16
1
Adjusted for age, sex, smoking status, hypertension, diabetes and residence.
found evidence that the Ku80 G-1401T variant was associated with increased risk of bladder cancer in a central Taiwanese population. A recent study, involving 362 patients
with colorectal cancer and 362 age- and gender-matched
healthy controls, showed that the T allele Ku80 G-1401T
conferred a significantly (P = 0.0069) increased risk of
colorectal cancer[17]. These observations were consistent
with the findings previously described by other investigators from Asian populations[19].
To further investigate the association between the
Ku80 promoter G-1401T polymorphism and the risk of
gastric cancer, we conducted this hospital-based casecontrol study in a Chinese population which incorporated
the information on exposure to smoking, residence and
other potential confounding factors (age and sex) that
were frequency matched between cases and controls and
further adjusted in the analysis. In our study, a significant
difference of the Ku80 G-1401T genotype distribution
was found between gastric cancer cases and controls. The
frequency of T allele was significantly higher in gastric
cancer patients than in control subjects.
The precise mechanisms underlying the relationship
between Ku80 polymorphism and stomach carcinogenesis remain unclear. Although the Ku80 promoter G-1401T
genetic variation does not directly lead to amino acid
coding change, presumably, it is plausible that this SNP
influences the expression level or stability of the Ku80
protein by the alternative spicing, intervention, modification, determination or involvement. It is similar to another important member of NHEJ, XRCC4. A few reports
provided evidence that its SNPs located on the promoter
region are significant in various cancers[33,34].
Our data also showed that the association between
increased gastric cancer risk and the mutant genotypes
(GT + TT) was more evident in younger subjects aged <
58 years than in older subjects. We also found an interaction between genotype and sex. The adjusted OR was
1.81 (95% CI: 1.28-2.52) for GT/TT genotype compared
with GG genotype among male subjects. But the OR
(adjusted OR = 1.33; 95% CI: 0.81-2.24) was not statistically significant among female subjects. Our findings
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were inconsistent with previous observations by Yang
et al[17] and Chang et al[18]. The reason for the different observations remains unclear.
In addition, we did not note a statistically significant
inverse association with gastric cancer risk in both nonsmokers (adjusted OR = 1.48; 95% CI: 1.08-2.02) and
smokers (adjusted OR = 2.52; 95% CI: 1.25-5.18). But
Yang et al[17] reported that the GT and TT genotypes, in
association with smoking, conferred an increased risk
(adjusted OR = 2.537; 95% CI: 1.398-4.601) for colorectal cancer. Similarly, Chang et al[18] and Hsu et al[19] found
a significantly decreased risk of bladder cancer (adjusted
OR = 2.053; 95% CI: 1.232-3.419) and oral cancer in
smokers with GT or TT genotypes[18,19]. The results are
inconsistent with our findings. The reason for the different observations remains unclear. Several studies have
reported that smoking is associated with free radicalinduced DNA damage and strand breaks[26], and tobacco
smoke contains some potential carcinogens including
polycyclic aromatic hydrocarbons, tobacco nitro-amines,
aromatic amines and BPDE, which form DNA bulky adducts and DNA strand breaks[27,35].
The stratified analyses by residence revealed that the
association was significant in variant genotypes in urban
subjects (OR = 1.88; 95% CI: 1.26-2.76) but not in rural
subjects (adjusted OR = 1.48; 95% CI: 0.99-2.15). The
different results may be explained, at least in part, between rural and urban subjects. Environmental factors,
including air, soil, diet, occupation and lifestyle, may be
responsible for the different observations between rural
and urban subjects. It was plausible, considering the better environment in rural areas[36].
The potential limitations of the present study should
be stressed. Firstly, in this hospital-based case-control
study, we selected controls from individuals with a variety
of nonmalignant diseases. These may cause the possibility of selection bias and confound the results. Nevertheless, the frequencies of Ku80 G-1401T polymorphism
variant alleles were similar to those reported in the NCBI
Website in the Asian population studies. T allele frequencies of Ku80 promoter G-1401T are 15.4% in our
2134
control group and 17.4% for Asian population in NCBI.
The genotype distribution of controls in our study met
Hardy-Weinberg equilibrium conditions. Secondly, the
sample size of the present study was relatively small,
which may limit the statistical power. Finally, our study
was conducted in Chinese population. Caution should
be exercised when extrapolating the data to other ethnic
groups.
In conclusion, we found a significant difference in the
Ku80 G-1401T polymorphism distribution between the
patients with gastric cancer and the control group. The T
allele of the Ku80 G-1401T was found more frequently in
patients with gastric cancer and it may be associated with
an increased risk of gastric cancer, suggesting that the
polymorphism of Ku80 G-1401T, involved in the gastric
tract carcinogenesis, may be a useful marker for primary
prevention and anticancer intervention. Further studies
are needed to determine the exact nature of this relationship.
4
5
6
7
8
9
10
11
12
COMMENTS
COMMENTS
Background
The Ku80 gene is an important and specific member of NHEJ. Genetic polymorphisms in Ku80 genes (G-1401T) influence DNA repair capacity and change the
predisposition of several cancers, including colorectal, bladder and oral cancer.
Whether genetic variants are involved in the risk of gastric cancer in a Chinese
population is unknown.
13
14
Research frontiers
In this study, the frequency of the Ku80 G-1401T GT/TT genotypes was significantly higher in the gastric cancer patients than in control subjects. This is
the first analysis of the association between genetic predisposition and gastric
cancer risk in Chinese population.
15
Applications
16
The Ku80 G-1401T polymorphisms may modulate the development of gastric
cancer in a Chinese population.
The Ku80 G-1401T GT/TT genotypes can be used as biomarkers for selecting
patients from the individuals at high risk for gastric cancer in China. Identifying
such susceptibility polymorphisms may lead to the development of tests that allow more focused follow-ups of high-risk groups.
17
Terminology
The Ku80 gene, also known as XRCC5, is an important and specific member of
NHEJ. As an important component of NHEJ, Ku80 and Ku70 form a heterodimer, which acts as a regulatory subunit of the DNA-dependent protein kinase
complex DNA-PK by increasing the affinity of the catalytic subunit PRKDC to
DNA.
18
Peer review
19
The quality of the work and the methodology are sound. The conclusions are
appropriate, although it seems unlikely that these findings represent a major
breakthrough.
20
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S- Editor Wang YR
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2136
L- Editor Ma JY E- Editor Zheng XM
[email protected]
doi:10.3748/wjg.v17.i16.2137
BRIEF ARTICLE
Effects of penehyclidine hydrochloride on rat intestinal
barrier function during cardiopulmonary bypass
Ying-Jie Sun, Hui-Juan Cao, Qiang Jin, Yu-Gang Diao, Tie-Zheng Zhang
intestinal bacterial translocation (BT). Serum levels
of DAO, D-lactate, endotoxin and the incidence of
intestinal BT were significantly increased in the surgical groups, compared with the sham-operated groups
(0.543 ± 0.061, 5.697 ± 0.272, 14.75 ± 2.46, and 0/40
vs 1.038 ± 0.252, 9.377 ± 0.769, 60.37 ± 5.63, and
30/40, respectively, all P < 0.05). PHC alleviated the
biochemical and histopathological changes in a dosedependent manner. Serum levels of DAO, D-lactate,
and endotoxin and the incidence of intestinal BT in the
high-dose PHC group were significantly lower than in
the low-dose PHC group (0.637 ± 0.064, 6.972 ± 0.349,
29.64 ± 5.49, and 14/40 vs 0.998 ± 0.062, 7.835 ±
0.330, 38.56 ± 4.28, and 6/40, respectively, all P <
0.05).
Ying-Jie Sun, Hui-Juan Cao, Qiang Jin, Yu-Gang Diao, TieZheng Zhang, Department of Anesthesiology, General Hospital of
Shenyang Commend, Shenyang 110840, Liaoning Province, China
Author contributions: Sun YJ and Jin Q contributed equally to
this work; Sun YJ, Jin Q and Zhang TZ designed the research;
Sun YJ, Cao HJ and Jin Q performed the research; Diao YG contributed new reagents/analytic tools; Sun YJ, Cao HJ and Jin Q
analyzed the data; Sun YJ, Cao HJ and Jin Q wrote the paper.
Supported by A grant from the Doctor Priming Foundation of
Liaoning Province, No. 20091099
Correspondence to: Qiang Jin, PhD, Associate Chief Physician, Department of Anesthesiology, General Hospital of
Shenyang Commend, Shenyang 110840, Liaoning Province,
Telephone: +86-24-28851366 Fax: +86-24-28851368
Received: October 21, 2010
CONCLUSION: PHC protects the structure and function of the intestinal mucosa from injury after CPB in
rats.
Abstract
AIM: To test the ability of penehyclidine hydrochloride
(PHC) to attenuate intestinal injury in a rat cardiopulmonary bypass (CPB) model.
Key words: Penehyclidine hydrochloride; Intestinal mucosa injury; Cardiopulmonary bypass
Peer reviewer: Dr. Richard A Rippe, Department of Medicine,
METHODS: Male Sprague-Dawley rats were randomly
divided into six groups (eight each): sham-operated control; sham-operated low-dose PHC control (0.6 mg/kg);
sham-operated high-dose PHC control (2.0 mg/kg); CPB
vehicle control; CPB low-dose PHC (0.6 mg/kg); and
CPB high-dose PHC (2.0 mg/kg). Blood samples were
collected from the femoral artery 2 h after CPB for determination of plasma diamine oxidase (DAO), D-lactate
and endotoxin levels. Spleen, liver, mesenteric lymph
nodes and lung were removed for biochemical analyses.
Intestinal tissue ultrastructure was examined by electron microscopy.
The University of North Carolina at Chapel Hill, Chapel Hill, NC
27599-7038, United States
Sun YJ, Cao HJ, Jin Q, Diao YG, Zhang TZ. Effects of penehyclidine hydrochloride on rat intestinal barrier function during cardiopulmonary bypass. World J Gastroenterol 2011;
org/10.3748/wjg.v17.i16.2137
INTRODUCTION
RESULTS: In the sham-operated groups, high- and
low-dose-PHC had no significant impact on the levels
of DAO, D-lactate and endotoxin, or the incidence of
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Cardiopulmonary bypass (CPB) is essential during some
cardiovascular surgical procedures; however, it can cause
2137
Sun YJ et al . Intestinal protection by penehyclidine
peripheral hypoperfusion as a result of non-pulsatile
flow, low blood pressure, hemodilution, and other nonphysiological conditions. Furthermore, an increase in intestinal permeability and bacterial translocation (BT) has
been demonstrated, not only in animal models, but also
in patients during CPB[1-3]. Perioperative gastrointestinal
integrity is therefore now recognized as an important factor determining the outcome of cardiac surgical procedures[4]. In general, changes in mucosal permeability and
morphology during CPB reflect the degree of damage to
intestinal mucosal barrier function.
Previous in vitro studies have demonstrated that tropane
alkaloids can stabilize the cell membrane and prevent oxidative stress. The new anticholinergic drug, penehyclidine
hydrochloride (PHC), has been evaluated for its protective effects on the cardiovascular system[5-7]. Its selective
blocking of M1, M3 and N receptors means that PHC
has few M2 receptor-associated cardiovascular side effects. It has been shown to reduce endotoxin-stimulated
acute lung injury and to attenuate liver damage during
CPB in a rat model[8-10].
Based on the potential roles of PHC as an antioxidant
and a cell membrane stabilizer, we hypothesized that its
administration might reverse CPB-associated intestinal
damage. Zhan et al[11] have suggested that PHC concentrations of 0.18-3.60 mg/kg were found to be safe, and
we therefore tested this hypothesis in a rat CPB model,
using high- (2.0 mg/kg) and low-dose (0.6 mg/kg) PHC.
while maintaining spontaneous ventilation during the entire operative procedure. All subsequent procedures were
performed under aseptic conditions.
After surgical-level anesthesia was achieved, the left
femoral artery was cannulated using a 22-gauge Teflon
heparinized catheter. Arterial pressure was monitored
and blood samples were collected for gas analysis, using
a blood gas analyzer (GEM Premier 3000; Mallinckrodt,
Lexington, MA, USA). Following administration of heparin (250 U/kg), an 18-gauge catheter was inserted into
the right jugular vein and advanced to the right atrium. A
22-gauge catheter was cannulated into the tail artery, to
serve as an arterial infusion line for the CPB circuit. The
mini-CPB circuit comprised a venous reservoir, a specially
designed membrane oxygenator, a roller pump, and sterile tubing with inner diameters of 4 mm for the venous
line and 1.6 mm for the arterial line. The CPB circuit was
primed with a total volume of 15 mL of synthetic colloid
solution. The perfusion flow rate was gradually adjusted
to sustain a mean arterial pressure of 60-80 mmHg. The
gas flow (95% O2, 5% CO2) was initiated at around 5075 mL/kg per minute and adjusted to maintain blood gas
analysis parameters within the physiological range. When
the flow rate reached 80-100 mL/kg per minute, it was
maintained for 60 min. At the end of CPB, the flow rate
was reduced stepwise to achieve hemodynamic stabilization. Throughout the experiment, central body temperature was monitored with a rectal probe and kept at 37.5 ±
1.0℃ using a heat lamp placed above the animal and CPB
equipment. The mean arterial pressure was maintained at
60-80 mmHg.
Animals and treatments
Forty-eight male Sprague-Dawley rats (weighing 300-450 g,
18-22 wk old) were randomly assigned to one of six
groups (eight each): sham-operated control; sham-operated control + low-dose PHC (0.6 mg/kg) (sham L-PHC);
sham-operated control + high-dose PHC (2 mg/kg)
(sham H-PHC); CPB + vehicle (control); CPB + lowdose PHC (0.6 mg/kg) (L-PHC); and CPB + high-dose
PHC (2.0 mg/kg) (H-PHC). PHC (Lisite Pharmacology
Co. Chendou, China, No. 080301) was dissolved in absolute ethanol and diluted in saline (final concentration of
ethanol < 1.0%), and added to the priming solution for
CPB. All animals received humane care in compliance
with the Principles of Laboratory Animal Care. The experimental protocol was approved by the local animal use
and care committee at the General Hospital of Shenyang
Commend, China.
Specimen collection
Rats from each group were sacrificed by decapitation, and
arterial blood (2.0 mL) and terminal ileums were sampled
at 2 h after CPB. Plasma was prepared by centrifugation
at 3000 g for 5-10 min at 4℃ and stored at -70℃ for determination of serum diamine oxidase (DAO), D-lactate,
and endotoxin. Intestinal (ileum) tissue samples were obtained for electron microscopy.
Intestinal permeability
The permeability of the intestinal mucosa was assayed
by measuring D-lactate and DAO levels in plasma. Plasma D-lactate levels were measured by enzymatic spectrophotometric assay using a centrifugal analyzer at 30℃,
as described previously[13]. Plasma DAO activities were
also determined by enzymatic spectrophotometry, as described previously[14]. D-lactate, D-lactate dehydrogenase,
NAD+, O-dianisidine, cadaverine dihydrochloride and
DAO were purchased from Sigma Chemical Company
(Milan, Italy).
Surgical procedure
None of the sham-operated control groups underwent
CPB. After PHC injection, the respiratory rate (RR),
heart rate (HR), blood pressure (BP) and electrocardiography (ECG) were continually monitored. The rat
CPB model was established as previously described, with
some modifications[12]. In brief, rats were anesthetized
by intraperitoneal administration of 10% chloral hydrate
(0.3 mL/100 g body weight) to provide stable anesthesia,
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Plasma endotoxin determination
The endotoxin content of the plasma sample was assayed
using the Limulus amebocyte lysate test using the Endochrome K test kit (CoaChrom, Vienna, Austria). In brief,
heparinized plasma was diluted 1:10 in pyrogen-free water
2138
Table 1 Physiological data (mean ± SD, n = 8)
MAP (mmHg)
HR (beats/min)
PH
BE (mmol/L)
HCT (%)
Groups
Pre-CPB
Controls
CPB + vehicle
CPB + L-PHC
CPB + H-PHC
Controls
CPB + vehicle
CPB + L-PHC
CPB + H-PH
Controls
CPB + vehicle
CPB + L-PHC
CPB + H-PHC
Controls
CPB + vehicle
CPB + L-PHC
CPB + H-PHC
Controls
CPB + vehicle
CPB + L-PHC
CPB + H-PHC
86.33 ± 16.82
84.50 ± 7.05
85.45 ± 9.36
87.54 ± 10.43
325 ± 34
315 ± 30
305 ± 26
310 ± 20
7.40 ± 0.02
7.41 ± 0.03
7.38 ± 0.04
7.42 ± 0.01
-1.96 ± 0.45
-1.86 ± 0.35
-1.75 ± 0.26
-1.85 ± 0.36
41.10 ± 1.85
41.80 ± 3.73
42.20 ± 2.45
42.45 ± 2.50
CPB 30 min
CPB 60 min
84.26 ± 14.55
62.14 ± 15.23a,c
66.58 ± 11.26a,c
65.73 ± 9.85a,c
320 ± 25
286 ± 28
315 ± 34
296 ± 25
7.41 ± 0.03
7.38 ± 0.05
7.43 ± 0.02
7.42 ± 0.03
-2.36 ± 0.75
-1.36 ± 0.26
-1.55 ± 0.96
-2.05 ± 0.90
40.20 ± 1.53
26.45 ± 4.24a,c
27.65 ± 3.65a,c
28.76 ± 5.38a,c
85.45 ± 10.36
67.08 ± 19.12a,c
69.78 ± 14.05a,c
70.85 ± 12.36a,c
315 ± 20
305 ± 42
302 ± 38
301 ± 35
7.39 ± 0.02
7.35 ± 0.06
7.41 ± 0.03
7.38 ± 0.04
-1.54 ± 0.85
-1.60 ± 0.84
-1.95 ± 0.63
-1.74 ± 0.42
40.60 ± 1.46
21.54 ± 3.71a,c
23.05 ± 5.30a,c
22.46 ± 4.25a,c
Post-CPB 2 h
83.63 ± 11.24
72.18 ± 17.39
76.15 ± 13.65
77.84 ± 12.36
324 ± 15
310 ± 37
304 ± 27
305 ± 30
7.40 ± 0.02
7.39 ± 0.02
7.36 ± 0.04
7.43 ± 0.02
-1.95 ± 0.54
-2.30 ± 1.50
-2.04 ± 0.72
-2.45 ± 0.14
39.80 ± 1.35
27.82 ± 3.66a,c
29.53 ± 5.45a,c
28.75 ± 4.56a,c
Controls summarize the results from sham-operated rats treated with saline or penehyclidine hydrochloride. aP < 0.05 vs baseline; cP < 0.05 vs controls. CPB:
Cardiopulmonary bypass; MAP: Mean arterial pressure; HCT: Hematocrit; BE: Buffer excess; HR: Heart rate.
and kept heated at 75℃ for 10 min to remove non-specific inhibitors. A quantitative chromogenic kinetic method
was used, as specified by the manufacturer, using a Thermo microplate reader (Tecan Spectra, Salzburg, Austria).
The method had a detection limit of 0.75 pg/mL at a 1:10
plasma dilution.
with 1% osmium tetroxide for 2 h, washed again, and
then embedded in Araldite 6005. Tissue sections were
cut with a Leica EM FCS (Vienna, Austria) ultramicrotome. Tissue sections (1 μm) were initially stained with
toluidine blue-Azur Ⅱ to select the region of interest for
subsequent procedures. Thin sections (60-70 nm) were
stained with uranyl acetate and lead citrate and examined
and photographed using an H-7200 transmission electron
microscope (80 kV; Oberkochen, Germany). Electron
microscopy pictures were evaluated twice by two independent histologists with at least 10 years of experience,
who were blinded to our study.
Bacteriological cultures
A midline incision was made using a sterile technique.
Mesenteric lymph nodes (MLNs), portal vein, and samples from the liver, spleen, and lung were harvested and
weighed prior to determination of bacterial growth. The
samples were homogenized in test tubes containing 3 mL
Brain Heart Infusion Broth (Difco, Detroit, IL, USA).
The supernatant (0.2 mL) was cultured for growth of
aerobic, microaerophilic and anaerobic bacteria. All media for aerobic cultures were incubated at 37℃ for at least
3-5 d, while organ samples for anaerobic bacteria were
cultured at 37℃ for 7 d. Enteric Gram-negative bacteria
were identified using the API 20 system (BioMérieux SA,
Marcy-l’Etoile, France) and Lactobacillus acidophilus by API
50 CH (Analytab Products Inc., Plainview, NY, USA). All
other aerobic, microaerophilic and anaerobic microbes
isolated were identified by standard procedures. The
numbers of living bacteria were calculated and expressed
as the numbers of living organisms per gram of organ
tissue.
All experimental data were expressed as mean ± SD and
analyzed using a SPSS for Windows v. 13.0 (Chicago,
IL, USA). One-way ANOVA was used for comparisons
among various treatment groups. Post-hoc comparisons
were analyzed using least significant difference test or
Dunnett’s T3 test. P < 0.05 was considered to be statistically significant.
RESULTS
PHC reverses the increase in intestinal mucosal
permeability after CPB
There were no obvious changes in the RR, HR, BP or
ECG at any time points (0, 30, 60, or 120 min) after anesthesia in the sham-operated groups (Table 1). Hemodynamic changes in the vehicle control, L-PHC and H-PHC
groups are shown in Table 1. As shown in Table 2, DAO
and D-lactate levels increased significantly in vehicletreated CPB rats, compared with the sham group (P <
0.05), which effect was largely reversed by treatment with
Transmission electron microscopy
For transmission electron microscopy, ileum tissues were
removed immediately from anesthetized rats 2 h after
CPB, and then fixed with 2% paraformaldehyde and 2.5%
glutaraldehyde in PBS (pH 7.3) for 2 h at room temperature (25℃). The tissues were washed with PBS, fixed
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2139
Table 2 Plasma diamine oxidase, D-lactate and endotoxin
levels (mean ± SD, n = 8)
DAO (U/L)
Controls
CPB + vehicle
CPB + L-PHC
CPB + H-PHC
D-lactate
(mg/L)
0.543 ± 0.061
1.038 ± 0.252a
0.998 ± 0.062a,e
0.637 ± 0.064a,c
A
B
C
D
Endotoxin
(pg/mL)
5.697 ± 0.272
9.377 ± 0.769a
7.835 ± 0.330a,e
6.972 ± 0.349a,c
14.75 ± 2.46
60.37 ± 5.63a
38.56 ± 4.28a,e
29.64 ± 5.49a,c
Controls summarize the results from sham-operated rats treated with
saline or penehyclidine hydrochloride (PHC). Data are shown as medians,
n = 8 rats for each group. CPB: Cardiopulmonary bypass; H-PHC: Highdose PHC; L-PHC: Low-dose PHC; DAO: Diamine oxidase. The level of
significance was set at P < 0.05. aP < 0.05 vs baseline; cP < 0.05 vs CPB group;
e
P < 0.05 vs H-PHC group.
Table 3 Numbers of animals pretreated with vehicle or penehyclidine hydrochloride with positive bacteriological cultures
from blood (portal vein), mesenteric lymph nodes, liver,
spleen and lungs after induction of cardiopulmonary bypass
Portal vein MLN
Controls
CPB + vehicle
CPB + L-PHC
CPB + H-PHC
0
7
3
2
0
8
4
3
Liver
0
6
3
1
Lungs Spleen
0
4
2
0
0
5
2
0
Total
0/40
30/40a,c
14/30a,c
6/40a
Figure 1 Effect of cardiopulmonary bypass on the ileal epithelial cells. A:
Control group (× 5800), regularly-aligned microvilli in the intestinal epithelium,
intact mitochondria and rough endoplasmic reticulum (RER), and distinct junctional complexes were observed; B: Cardiopulmonary bypass group (× 7200),
epithelial damage was demonstrated by swollen mitochondria and loss of cristae, and tight junctions were disrupted; C: Mitochondrial swelling with damage
to mitochondrial cristae and vacuolar degeneration were present. The nuclear
structure was incomplete; D: Microvilli in the intestinal epithelium were regularly
aligned, and the structure of the tight junctions became tight. However, mild
swelling of mitochondria and RER were observed.
Controls summarize the results from sham-operated rats treated with
saline or penehyclidine hydrochloride (PHC). MLN: Mesenteric lymph
node; CPB: Cardiopulmonary bypass; H-PHC: High-dose PHC; L-PHC:
Low-dose PHC. aP < 0.05 vs controls; cP < 0.05 vs L-PHC group.
PHC in a dose-dependent manner (P < 0.05). H-PHC
had significantly greater effects on the values of DAO
and D-lactate than L-PHC (Table 2, P < 0.05).
(Figure 1A). In the vehicle control group, the microvilli
were reduced in number, and showed irregular lengths
and arrangements. The mitochondria were swollen with
cracked and vacuolated cristae. Some RER structures
were destroyed, and the intercellular spaces between
epithelial cells were widened. The structure of the tight
junctions became shortened (Figure 1B). Mitochondrial
swelling with damage to mitochondrial cristae and vacuolar degeneration was present in the L-PHC group. The
nuclear structure was incomplete (Figure 1C). In the
H-PHC group, microvilli in the intestinal epithelium were
regularly aligned, and the tight junction structure became
tight. However, mild swelling of the mitochondria and
RER were seen (Figure 1D). These results indicated dosedependent effects of PHC on the reduction of cellular
damage after CPB.
PHC prevents CPB-induced BT
Bacteriological cultures from all sham-operated animals
were negative. The incidence of Escherichia coli-positive
cultures was significantly increased in the CPB-vehicle
group, while pretreatment with PHC seemed to prevent
systemic dissemination. The incidence of intestinal BT to
the MLNs, spleen, liver, lung and blood was significantly
higher in the CPB-vehicle group, compared with that in
the sham groups (Table 3, P < 0.05), which was largely
reversed by treatment with PHC in a dose-dependent
manner (Table 3, P < 0.05). The plasma endotoxin level
was increased in the CPB-vehicle group compared with
the sham groups (Table 2, P < 0.05) and PHC decreased
the plasma endotoxin levels in a dose-dependent manner
(Table 2, P < 0.05).
DISCUSSION
PHC prevents CPB-induced damage to the intestinal
mucosa ultrastructure
Transmission electronic microscopy demonstrated normal intestinal ultrastructure in the sham-operated group,
including regularly aligned microvilli in the intestinal
epithelium, integral mitochondria and rough endoplasmic reticulum (RER) and distinct junction complexes
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The present study focused on intestinal barrier injury after CPB and the potential of PHC as a therapeutic agent.
We demonstrated that application of PHC during CPB
preserved intestinal barrier function in a dose-dependent
manner, and histological evidence is provided to support
these biochemical results.
DAO reduces the concentration of polyamines re-
2140
quired for cell proliferation. DAO is localized to the small
intestine and placenta, which are both organs with rapid
cell turnover rates. In humans, DAO activity is especially
high in the upper portion of the small intestinal villi, and
has therefore been used as an index of small intestinal
mucosal mass and integrity. Serum DAO levels have been
found to increase markedly when the small intestine is
strangulated, and elevations are thus believed to reflect
small intestine mucosal ischemia[15]. Tsunooka et al[4,16]
have demonstrated simultaneous increases in serum DAO
activity and peptidoglycan concentrations during clinical
CPB, suggesting the occurrence of small intestinal mucosal ischemia and BT.
D-Lactate is habitually tested for in the intensive care
unit. Mammals only have one type of enzyme: L-lactate
dehydrogenase. L-Lactate is a marker of cell hypoxemia,
and its levels correlate with survival in patients with septic
shock[17,18]. Microorganisms however, particularly bacteria,
are equipped with D-lactate dehydrogenase and produce
D-lactate during fermentation, and D-lactate therefore
acts as a marker of bacterial infection. D-Lactate has also
recently been proposed as a sensitive, specific and early
marker of translocation in gut ischemia[19].
In the current study, all the rats survived the CPB
procedures. DAO and D-lactate activities remained low in
the sham-operated animals (based on previously reported
normal DAO value of 0.46 ± 0.087 U/L, and D-lactate
value of 5.245 ± 0.653 mg/L[13,14]). However, significant
increases in DAO, D-lactate and endotoxin levels were
found in the vehicle-treated CPB group. The incidence of
intestinal BT to the MLNs, spleen, liver, lung and blood
was also significantly higher in the vehicle-treated CPB
group, compared with that in the sham groups. These
results indicate the occurrence of severe intestinal barrier injury after CPB. Transmission electron microscopic
examination of intestinal tissues further confirmed the
intestinal barrier injury in this rat CPB model.
There is increasing interest in developing PHC as a
novel therapeutic agent. PHC is a new anticholinergic drug
derived from hyoscyamine, which has few M2 receptorassociated cardiovascular side effects, because of its selective blocking of M1, M3 and N receptors. Recent clinical
results have demonstrated that PHC has curative effects
in soman poisoning and pulmonary dysfunction associated with chronic obstructive pulmonary disease[6,9,20]. In
addition to improving microcirculation, PHC can inhibit
lipid peroxidation, attenuate the release of lysosomes, and
depress microvascular permeability[9,20]. Moreover, it can
significantly decrease brain nuclear factor (NF)-κB expression in cerebral ischemia/reperfusion (I/R) injury. Furthermore, PHC can improve acute lung injury stimulated
by endotoxin and attenuate liver damage during CPB in a
rat model[11].
In the present study, PHC lowered DAO, D-lactate
and endotoxin levels in PHC-treated rats in a dose-dependent manner, suggesting its potential clinical application.
The incidences of intestinal BT to MLNs, spleen, liver,
lung and blood were lower in PHC-treated CPB rats than
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in untreated CPB ones, suggesting that the use of PHC
resulted in an overall decrease in bacteria. In intestinal mucosal injury, PHC can efficiently inhibit NF-κB expression
in intestinal mucosal I/R injury. More importantly, PHC
can improve the microcirculation, inhibit lipid peroxidation, attenuate the release of lysosomes, and decrease
microvascular permeability, leading to the inhibition of
inflammation[21].
However, our research was subject to some limitations. PHC treatment was only performed prior to CPB;
although this pretreatment was effective, this study did
not establish the efficacy of PHC given after CPB. Future
studies are required to assess the effects of postoperative
treatment with PHC. Additional research is also needed
to establish the optimal time of PHC administration.
In conclusion, the present study demonstrated that
PHC protected rat intestine from morphological and
functional mucosal injury after CPB. These results suggest that PHC could be clinically useful for the treatment
of intestinal injury induced by CPB.
COMMENTS
COMMENTS
Background
An increase in intestinal permeability and bacterial translocation has been
demonstrated not only in animal models, but also in patients during cardiopulmonary bypass (CPB). Previous in vitro studies have demonstrated that tropane
alkaloids could stabilize the cell membrane and prevent oxidative stress. The
new anticholinergic drug penehyclidine hydrochloride (PHC) has been evaluated for its protective effects on the cardiovascular system.
Research frontiers
PHC can improve microcirculation, inhibit lipid peroxidation, attenuate the
release of lysosomes, and decrease microvascular permeability, leading to
the inhibition of inflammation. The effects of PHC on intestinal barrier function
during CPB have not been unequivocally addressed. We hypothesized that the
administration of PHC could reverse CPB-associated intestinal damage.
The selectivity of PHC in blocking M1, M3 and N receptors means that it has
few M2 receptor-associated cardiovascular side effects. It can reduce endotoxin-stimulated acute lung injury and attenuate liver damage during CPB in a rat
model. The present study demonstrated that the application of PHC during CPB
preserved intestinal barrier function in a dose-dependent manner, and provided
histological findings to support these biochemical results.
Applications
The present study demonstrated the ability of PHC to protect rat intestine from
morphological and functional mucosal injury after CPB. These results suggest
that PHC could be clinically useful in the treatment of intestinal injury induced
by CPB.
Terminology
PHC is a new anticholinergic drug with antioxidant and cell membrane-stabilizing activities.
Peer review
The authors have demonstrated a protective role for PHC in preventing intestinal breakdown during CPB. The conclusion was reached that the administration of PHC could prevent intestinal damage and its sequelae following CPB
surgery. This manuscript describes an interesting and well-performed study with
convincing results.
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S, Watanabe Y, Kawachi K. Bacterial translocation secondary
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S- Editor Sun H L- Editor Kerr C
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2142
E- Editor Zheng XM
[email protected]
doi:10.3748/wjg.v17.i16.2143
BRIEF ARTICLE
p53 gene therapy in combination with transcatheter arterial
chemoembolization for HCC: One-year follow-up
Yong-Song Guan, Yuan Liu, Qing He, Xiao Li, Lin Yang, Ying Hu, Zi La
control group (P < 0.01). The combination treatment
was well tolerated with such adverse events as fever
(51.5%, P = 0.006) and pain of muscles and joints
(13.2%, P = 0.003), which were significantly higher
than the chemotherapy. Except for these minor adverse effects, no severe vector-related complications
were identified. With respect to the efficacy, patients in
p53 treatment group had less gastrointerestinal symptoms (P = 0.062), better improvement in tumor-related
pain (P = 0.003), less downgrade of leukocyte counts (P
= 0.003) and more upgrade of Karnofsky performance
score (P = 0.029) than those in control group. The
total effective rate (CR + PR) for p53 treatment group
and control group was 58.3% and 26.5%, respectively,
with distributions of different effect in two groups (P
= 0.042). The survival rates were 89.71%, 76.13%,
and 43.30% for p53 treatment group, and 68.15%,
36.98%, and 24.02% for control group, respectively,
3, 6 and 12 mo after treatment, suggesting that the
survival rates are significantly higher for p53 treatment
group than for control group (P = 0.0002).
Yong-Song Guan, Yuan Liu, Qing He, Xiao Li, Lin Yang,
Ying Hu, Zi La, Department of Oncology, West China Hospital,
Sichuan University, Chengdu 610041, Sichuan Province, China
Author contributions: Guan YS supervised the entire process of
design and execution of the study and writing of the manuscript,
and corrected the paper; Liu Y drafted the paper and organized
the figures and patient data; He Q, Li X, Yang L, Hu Y and La Z
completed the follow-up of patients and data collection.
Correspondence to: Yong-Song Guan, Professor, Department of Oncology West China Hospital, Sichuan University, 37
Guoxuexiang, Chengdu 610041, Sichuan Province,
Telephone: +86-28-85421008 Fax: +86-28-85538359
Revised: November 13, 2010
Abstract
AIM: To evaluate the efficacy and safety of combination therapy with recombinant adenovirus p53 injection
(rAdp53) and transcatheter hepatic arterial chemoembolization (TACE) for advanced hepatocellular carcinoma (HCC).
CONCLUSION: The rAd-p53 gene therapy in combination with TACE is a safe and effective treatment modality for advanced HCC.
METHODS: A total of 82 patients with advanced HCC
treated only with TACE served as control group. Another 68 patients with HCC treated with TACE in combination with recombinant adenovirus-p53 injection served
as p53 treatment group. Patients were followed up for
12 mo. Safety and therapeutic effects were evaluated
according to the improvement in clinical symptoms,
leukocyte count, Karnofsky and RECIST criteria. Survival rate was calculated with Kaplan-Meier method.
Key words: Adenovirus p53 ; Clinical trial; Hepatocellular carcinoma; Transcatheter hepatic arterial chemoembolization; p53 gene therapy
Peer reviewer: Dr. Jun Qin, Baylor College of Medicine, One
Baylor Plaza, Houston 77030, United States
RESULTS: The total effective rate was 58.3% for p53
treatment group, and 26.5% for control group (P <
0.05). The incidence of gastrointestinal symptoms was
lower in p53 treatment group than in control group
(P < 0.05). The 3-, 6- and 12-mo survival rates were
significantly higher for p53 treatment group than for
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Guan YS, Liu Y, He Q, Li X, Yang L, Hu Y, La Z. p53 gene
therapy in combination with transcatheter arterial chemoembolization for HCC: One-year follow-up. World J Gastroenterol
wjgnet.com/1007-9327/full/v17/i16/2143.htm DOI: http://
dx.doi.org/10.3748/wjg.v17.i16.2143
2143
Guan YS et al . p53 combing TACE for HCC
INTRODUCTION
Table 1 Characteristics of enrolled patients with hepatocellular carcinoma
Gene therapy is a potentially new treatment modality for
cancer patients and an engineered recombinant replicationdefective adenovirus can express the tumor suppressor
gene p53 (rAd-p53) with encouraging clinical responses[1-3].
rAd-p53 has been recently approved by the State Food and
Drug Administration of China as the very first gene therapy product for head and neck squamous cell carcinoma
(HNSCC)[4].
Hepatocellular carcinoma (HCC) is one of the major
cancers in China with a poor prognosis due to its occult
onset, rapid infiltrating growth and complicating liver
cirrhosis. No effective treatment modality is available
for it at present. Although transcatheter hepatic arterial
chemoembolization (TACE) is currently one of the most
popular treatment modalities for unresectable advanced
HCC, the long-term survival rate of such patients remains low with a reported 5-year survival rate of 17%[5].
In this study, the safety and efficacy of rAd-p53 therapy
in combination with TACE were examined in patients
with advanced HCC.
Characteristics
Age
43.5 (20 - 72)
Sex (M/F)
43/25
Child class A
41
Child class B
27
UICC TNM classification
0
Stage Ⅰ and Ⅱ
Stage Ⅲ
31 (46.5%)
Stage Ⅳ
37 (54.4%)
Size of main tumors
≥ 5 cm
53 (77.9%)
< 5 cm
15 (22.1%)
Control group
(n = 82)
Statistic
analysis
45.7 (18 - 75)
40/42
43
39
NS
NS
NS
NS
0
60 (73.2%)
22 (26.8%)
NS
NS
NS
61 (74.3%)
21 (25.7%)
NS
NS
NS: No statistical difference.
Ⅲ and 59 patients as stage Ⅳ according to the Interna-
tional Union against Cancer TNM classification[7].
Patients who gave their informed consent to receive
Ad-p53 gene therapy served as gene treatment group,
while those not willing to receive gene therapy served as
control group. Patients in gene treatment group underwent
rAd-p53 gene therapy and TACE while those in control
group received only TACE. Although this was a retrospective nonrandomized study, no statistical difference was
observed in baseline between the two groups. The characteristics of the two groups are illustrated in Table 1.
rAd-p53
rAd-p53 is a recombinant human serotype 5 adenovirus
in which the E1 region is replaced by a human wild-type
p53 expression cassette. The p53 gene is driven by a Rous
sarcoma virus promoter with a bovine growth hormone
poly (A) tail. The recombinant adenovirus is produced in
human embryonic kidney 293 cells and manufactured by
Shenzhen SiBionoGenTech Co. Ltd (Shenzhen, China)
and marketed under the trade name of Gendince®. Before
p53 gene therapy, a vial of rAd-p53 is taken out from a refrigerator in which the temperature is about -20℃. When
thawed, the solution, diluted with 1 mL NS, is sucked into
a 5-mL syringe for intra-tumor injection.
Procedure of rAd-p53 intra-tumor injection
The patients in gene treatment group were placed in a supine, prone or lateral position on the CT scanning bed and
asked to hold their breath after an inhalation. The slice for
puncture was carefully determined, the puncture site on
the surface of body as well as the needle-traveling depth
and angle within the body were determined. The bed was
moved to the slice and a marker for puncture was made
on the body surface according to the laser beam emitted
from the gantry. The bed was then moved out and the
puncture site was sterilized. After local anesthesia, a 19-G
needle was inserted into the puncture site according to the
determined angle and depth as the operator asked the patient to hold his or her breath after an inhalation. Finally,
another scan was performed to make sure that the tip of
the needle was within the tumor, and the rAd-p53 gene
was injected into the tumor in a multi-point fashion. Usually, this procedure is repeated according to the patient’s
clinical condition and the interval between two procedures
is about 1 wk. At each injection, 1-4 rAd-p53 injections
are administered at a viral dose of 1-4 × 1012 VP (viral
particles) according to the diameter of the lesion, and the
intra-tumor injection usually lasts 1-2 min.
Patients and trial design
One hundred and fifty patients (83 men and 67 women)
with advanced HCC were enrolled in this study from
March to July 2004. Patients with Child C disease[6], tumor thrombus in the main portal trunk, or extrahepatic
metastasis were excluded. These exclusion criteria were
implemented to ensure at least a 3-mo life span in the
enrolled patients so as to have enough time to follow
up. All patients did not receive local ethanol injection,
microwave coagulation, systemic chemotherapy or radiotherapy before and after TACE or gene therapy. All tumors were diagnosed according to pathologic examination, distinctive findings on computed tomography (CT),
conventional angiography, magnetic resonance imaging
(MRI), or serum tumor markers [alpha-fetoprotein (AFP)
or ferritin]. The patients were divided into gene treatment group (n = 68) with a mean age of 43 years (range
20-72 years) and control group (n = 82) with a mean age
of 45 years (range 18-75 years). No patient was classified
as stage Ⅰ or Ⅱ while 91 patients were classified as stage
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Gene group
(n = 68)
TACE
TACE was performed through the femoral artery using
the Seldinger technique with local anesthesia. Arteriography of the celiac trunk and superior mesenteric artery
was performed to visualize the arterial vascularization of
liver and evaluate portal vein patency. An angiographic
2144
catheter was inserted into the right or left hepatic artery
where the target tumor was located. TACE agents, involving embolic agent (Lipiodol) and anticancer drugs,
were injected through the right or left hepatic artery. In
both groups, the dose of Lipiodol, ranging 3-20 mL, was
determined according to the tumor location, tumor size,
number of tumors, and functional hepatic reserve. Anticancer drugs used were 5-Fluorouracil (800-1000 mg)
and vinorelbine (30-40 mg). TACE was repeated according to the patient’s clinical condition at a 1-mo interval.
Table 2 Clinical synptoms after treatments
Group
Gastrointestinal Palliation of
Pain of
symptoms
mass-associated muscles or
pain
joint
Gene group
35 (51.5)a
Control group 24 (29.3)
20 (29.4)b
28 (34.1)
30 (44.1)c
21 (25.6)
9 (13.2)d
1 (1.2)
2
a
2
b
2
c
2
χ = 7.679, P = 0.006; χ = 4.001, P = 0.062; χ = 5.674, P = 0.017; χ = 8.626,
P = 0.003.
d
Follow-up protocol
Clinical symptoms, leukocyte counts and Karnofsky index evaluation were recorded before and after treatment.
After treatment, CT scan or MRI was performed every
three months with or without contrast enhancement to
evaluate the features of Lipiodol deposit and the therapeutic effect according to the response evaluation criteria for solid tumors[8]. If elevated tumor markers (AFP
and ferritin), diminished Lipiodol, or enlarged lesions or
new nodules were observed, the patients were readmitted for angiography and treatment. The starting point of
survival analysis was regulated as the day of initial treatment. The Kaplan-Meier method was used to analyze
the survival rates in the two groups.
Table 3 Changes in leukocytes before and after treatment
Group
Gene group
Control group
9
Change degree (×10 /L)
< 4.0
< 3.0
< 2.0
12 (25.0)
8 (13.3)
4 (8.3)
20 (33.3)
2 (4.2)
11 (18.3)
n (%)
18 (37.5)
39 (65.0)
Rank sum tests (Wilcoxon text), T = -3.018, P = 0.003 < 0.05.
frequent adverse event occurred in patients receiving
rAd-p53 gene therapy in combination with TACE was
the flu-like symptom associated with fever. Of the 68
patients in gene treatment group, 35 (51.5%) had a fever
at 38-39.5℃, usually occurred 3-10 h after p53 intratumor injection and decreased after physic cooling, and 9
(13.2%) had pain of muscles or joints which often faded
away (Table 2). No other severe gene therapy-associated
complications were encountered in this study.
Statistical analysis was performed to assess the baseline,
leukocyte counts, Karnofsky index, clinical symptoms and
survival curve between the two groups using the SPSS
11.0. P < 0.05 was considered statistically significant.
Efficacy
The clinical symptoms were carefully recorded after treatment (Table 2). The patients in gene treatment group had
less gastrointerestinal symptoms such as nausea, vomiting,
abdominal pain or belling than those in control group.
The palliative rate of mass-associated pain one week after
treatment was 44.1% (30/68) for patients in gene treatment group, higher than that for those in control group.
Before and one week after treatment, the number of
leukocytes was calculated (Table 3). Statistical analysis
showed that the number of leukocytes was smaller in
gene treatment group than in control group (P = 0.003).
Karnofsky index was changed in gene treatment group
one month after treatment (Table 4). Generally speaking,
the patients in gene treatment group had a higher Karnofsky index than those in control group (P = 0.029).
The therapeutic effect was evaluated following the
response evaluation criteria for solid tumors after treatment. CR, PR, NC and PD in the two groups are listed
in Table 5. The total effective rate (CR + PR) was 58.3%
and 26.5% for the gene treatment group and control
group, respectively (P < 0.05). Chi-square test showed
that the distributions of therapeutic effect were statistically different (P = 0.042, Figures 1 and 2)
The patients were followed up for 12 mo. The number of withdrawal patients in gene treatment group and
control group was 4 and 7, respectively. The survival
rate was 89.71% (standard error 0.036), 76.13% (standard error 0.052), and 43.30% (standard error 0.061),
RESULTS
Two hundred and fifty-one p53 intra-tumor injections
were performed for 83 lesions in 68 patients of gene treatment group. Of the 68 patients, 9 received one injection,
13 received two injections, 15 received three injections,
20 received four injections, 7 received five injections, 3
received six injections and 1 received seven injections. One
hundred and ninety-two 2 (mean 2.82 procedures) and 167
(mean 2.03 procedures) procedures of TACE were performed in gene treatment and control groups, respectively.
Arterial portal vein shunt (AVS), arterial hepatic vein shunt
(APS) or/and portal vein involvement, signs that meant a
high invasion and a poor prognosis were found in 27.9%
(19/68) patients of gene treatment group and 36.6%
(30/82) patients of control group, respectively, during the
TACE. Although the patients with tumor thrombus in the
main portal trunk were excluded, some of them developed
vascular invasion because of tumor progression after they
were enrolled in this study. No difference was observed
in the incidence of malignancy signs such as AVS, APS or
portal vein involvement between the two groups.
Safety
The clinical symptoms were carefully recorded after
treatment (Table 2). Overall, rAd-p53 gene therapy in
combination with TRCE was well tolerated. The most
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Fever
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A
B
C
Figure 1 Contrast computed tomography showing a nodule (3.5 cm in diameter) in the right upper liver lobe manifested as homogenous enhancement (A);
computed tomography scan (b) demonstrating the course of fine needle biopsy under computed tomography guidance with the diagnosis of hepatocellular
carcinoma confirmed (B); computed tomography follow-up (c) revealing lipiodol deposit in the mass and spleen infarction after spleen embolization (C) in a
52-year-old man with multiple hepatic nodules, liver cirrhosis, splenomegaly and elevated alpha-fetoprotein.
A
B
Figure 2 Contrast computed tomography scan showing a 15 cm × 11.5 cm hepatocellular carcinoma in the right liver lobe manifested as a heterogenous lower
density, partial enhancement and well-differentiated contour (A) and computed tomography follow-up displaying the significant regression of a 8.5 cm x 6 cm
lesion with compact lipiodol deposit in a 72-year-old man after 3 p53 gene injections and 4 courses of transcatheter hepatic arterial chemoembolization.
Table 4 Changes in Karnofsky index before and after treatment
Table 5 Therapeutic effect evaluated following response
evaluation criteria for solid tumors 2 mo after treatment
Group
Group
n
CR
PR
NC
PD Effective rate (CR + PR)
Gene group
Control group
68
82
0
0
46
42
15
27
7
13
Upgrade
> 20
points
Gene group
Control group
14
12
Upgrade
No
Downgrade
> 10
changes
> 10
points
points
28
24
18
18
8
28
Total
upgrade
[n (%)]
42 (61.8)
36 (43.9)
2
χ = 4.137, P = 0.042 < 0.05. CR: Complete response; PR: Partial response; NC:
No change; PD: Progressed disease.
2
χ = 4.752, P = 0.029.
interventional therapies[9-12]. However, its therapeutic effect is also limited due to the lack of appropriate and reliable embolic agents, and the infiltrative or hypovascular
nature, too large or small in size[13-15]. Another limitation
of TACE is the need for repeated treatment, thus resulting in deterioration of liver function[16]. So, lots of efforts
have been made to explore other new therapies in order
to achieve the better efficacy of multiple treatments. PEI
or RFA gene therapy in combination with TACE may
improve the survival rate of HCC patients and decrease
the risk of liver failure[17-19]. In this study, p53 gene therapy
in combination with TACE could overcome the downside
of TACE and improve the prognosis of HCC patients.
The p53 tumor suppressor gene is a gene guardian and
loss of p53 is responsible for the lack of apoptotic signals
respectively, for the patients in gene treatment group 3, 6,
and 12 mo after treatment. The survival rate was 68.15%
(standard error 0.051), 36.98% (standard error 0.054), and
24.02% (standard error 0.049), respectively, for those in
control group 3, 6, and 12 mo after treatment. Log-rank
test showed that the survival rate for the two groups was
significantly different (P = 0.0002, Figure 3).
DISCUSSION
Hepatocellular carcinoma (HCC) is a highly malignant
tumor with a very high morbidity and mortality. Since
TACE was introduced as a palliative treatment of unresectable HCC, it has become one of the most common
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67.60%
51.20%
2146
were observed. Although these adverse events have been
observed in clinical practice, they can be well tolerated by
most patients with no severe physical and mental harm.
The patients receiving p53 gene therapy had less severe
post embolization syndrome than others after TACE.
Gastrointestinal symptoms, such as nausea, vomiting and
abdominal pain or belling, were less frequently observed
in gene treatment group than in control group. The decreased number of leukocytes in gene treatment group
was a pleasing phenomenon. However, its mechanism remains to be studied. The Karnofsky index was significantly
higher, suggesting that the life quality of patients is largely
improved in gene treatment group. It could be concluded
that the rAd-p53 gene therapy could reduce the side effects
of chemical drugs and Lipiodol embolization. Also, it was
noticed that many patients in gene treatment group had a
compact Lipiodol deposit manifested as a high homogenous density occupying the majority of tumor mass (Figures
1 and 2). Compact deposit means tumor necrosis. Further
study is needed to observe whether p53 gene therapy is related to the better deposit of Lipiodol in lesions.
Theoretically, in-vitro p53 protein can bring about specific anti-tumor cells into effect in such ways as induction of
apoptosis or necrosis, incentive of body immune response,
regulation of cell cycle, etc. Two months after treatment,
the distributions of therapeutic effect in the two groups
were statistically different and the effective rate (CR + PR)
was higher for p53 gene treatment group than for control
group, suggesting that p53 gene therapy can enhance the
efficacy of TACE, radiotherapy and chemotherapy.
Kaplan-Meier analysis showed that the survival rate
was higher for gene treatment group than for control
group. Because no other control study is available, the
outcome of p53 gene therapy for such a large number of
patients was not compared with that in other studies. The
1-year survival rate was lower in our study than in another study (67% vs 81%)[31], which may be attributed to the
different baselines, in which our enrolled patients might
have a larger lesion and a poorer liver function reserve.
Although it seems that the higher survival rate in gene
treatment group may be attributed to the longer mean
TACE time in patients of gene treatment group than in
those of control group (2.82 vs 2.03), it was the clinical improvement after p53 gene therapy that made the patients in
gene treatment group have more chance to receive repeated TACE. On the other hand, no difference was found in
the incidence of malignancy DSA signs between the two
groups. However, these signs appeared later with a lower
incidence in gene treatment group than in control group,
which is an interesting phenomena, and further study with
a larger sample size is needed to confirm it.
Usually, the rAd-p53 gene begins to express p53
protein 3 h after intra-tumor injection, reaches its peak
on day 3, and then gradually decreases according to the
specification of Gendicine®. On day 5 after injection, the
expression decreases to 30%. Because most of the chemotherapeutic drugs can affect DNA or RNA duplication or expression, cell cycle or nucleic acid metabolism
would likewise affect the expression of p53 gene in
Treatment measures
1.2
Control group
Cum survival
1.0
Control group-censored
Combo group
0.8
Combo group-censored
0.6
0.4
0.2
-2
0
2
4
6
8
10
12
Survival time-month
Figure 3 Survival curves for patients following treatment.
in tumor cells and thus for their uncontrolled proliferation and recurrence[20]. Many human tumors carry mutations in the p53 gene[21,22] and mutant or absent p53 gene
is associated with the resistance to radiotherapy and
apoptosis-inducing chemotherapy[23]. It has been shown
that p53 gene therapy in combination with radiotherapy
or chemotherapy can control local tumor, suggesting
that it is superior to either radiotherapy or chemotherapy
alone[24,25]. It was reported that the incidence of p53 mutation is 61% in HCC[22]. Chen et al[26] also reported that
mutations in the p53 gene are frequently detectable in
recurrent HCC and the interval between surgical resection
and recurrence of HCC is significantly longer in patients
with the wild-type p53 gene than in those with mutant
p53 gene mutations, strongly suggesting that the mutant
p53 gene plays a role in pathogenesis of HCC. Jeng et al[27]
demonstrated that the biological behavior of the mutant p53 gene is strongly related to the invasiveness of
HCC and may also influence the postoperative course of
HCC. Many scholars suggest that immunopositivity of
the mutant p53 gene plays a role in predicting the prognosis of patients with HCC after resection[27-29].
The rAd-p53 gene has been approved in China under
the trade name of Gendicine for the treatment of head
and neck squamous cell carcinoma (HNSCC). In one of
the trials[3], 75% tumors experienced complete regression
following 8 wk of therapy involving 1 injection per week,
which was significantly higher than that in control group,
and combined chemotherapy and radiotherapy improved
the treatment efficacy of over 3-fold. Although its recommended indications are limited in HNSCC according to the specification, good treatment efficacy can be
achieved in HCC patients when rAd-p53 is used[30]. In the
current study, Gendicine was used in treatment of HCC
to evaluate its effect in order to provide some evidence
for its off-table use in treatment of HCC.
As for the safety of rAd-p53 used in treatment of advanced HCC, just fever at 38-39.5℃ was observed in our
study, which was returned to normal after symptomatic
treatment. In addition, some patients suffered from pain
of muscles or joints and its cause is still controversial.
However, no severe complications caused by Gendicine
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2147
tumor tissue. In this study, TACE was started 3-4 d after
p53 injection when the p53 protein was highly expressed
in tumor tissue, indicating that these anti-tumor drugs
do not interfere with the expression of p53. However,
the optimal interval remains to be further studied.
In conclusion, rAd-p53 gene therapy in combination
with TACE is well tolerated and its anti-tumor efficacy is
superior to that of TACE alone in terms of the survival
rate and improved symptoms of HCC patients. Further
clinical study with a large sample size is warranted to
optimize the administration procedure and assess the
impact of anti-p53 antibody on its therapeutic effect.
kajo M. Transcatheter arterial chemoembolization therapy
using iodized oil for patients with unresectable hepatocellular carcinoma: evaluation of three kinds of regimens and
analysis of prognostic factors. Cancer 2000; 88: 1574-1581
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Comparison of CT findings with resected specimens after
chemoembolization with iodized oil for hepatocellular carcinoma. AJR Am J Roentgenol 2000; 175: 699-704
Sobin LH, Wittekind CH, editors. UICC TNM classification
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Sons, 1997
Therasse P, Arbuck SG, Eisenhauer EA, Wanders J, Kaplan
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RS, Rubinstein L, Verweij J, Van Glabbeke M, van Oosterom
AT, Christian MC, Gwyther SG. New guidelines to evaluate
the response to treatment in solid tumors. European Organization for Research and Treatment of Cancer, National
Cancer Institute of the United States, National Cancer Institute of Canada. J Natl Cancer Inst 2000; 92: 205-216
Llovet JM, Real MI, Montaña X, Planas R, Coll S, Aponte J,
9
Ayuso C, Sala M, Muchart J, Solà R, Rodés J, Bruix J. Arterial embolisation or chemoembolisation versus symptomatic treatment in patients with unresectable hepatocellular
carcinoma: a randomised controlled trial. Lancet 2002; 359:
1734-1739
10 Li L, Wu PH, Li JQ, Zhang WZ, Lin HG, Zhang YQ. Segmental transcatheter arterial embolization for primary hepatocellular carcinoma. World J Gastroenterol 1998; 4: 511-512
11 Mizoe A, Yamaguchi J, Azuma T, Fujioka H, Furui J, Kanematsu T. Transcatheter arterial embolization for advanced
hepatocellular carcinoma resulting in a curative resection: report of two cases. Hepatogastroenterology 2000; 47:
1706-1710
12 Llovet JM, Bruix J. Systematic review of randomized trials
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13 Qian J, Truebenbach J, Graepler F, Pereira P, Huppert P, Eul
T, Wiemann G, Claussen C. Application of poly-lactide-coglycolide-microspheres in the transarterial chemoembolization in an animal model of hepatocellular carcinoma. World
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14 Fan J, Ten GJ, He SC, Guo JH, Yang DP, Wang GY. Arterial
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Gastroenterol 1998; 4: 33-37
15 Lin DY, Lin SM, Liaw YF. Non-surgical treatment of hepatocellular carcinoma. J Gastroenterol Hepatol 1997; 12: S319-S328
16 Ahrar K, Gupta S. Hepatic artery embolization for hepatocellular carcinoma: technique, patient selection, and outcomes. Surg Oncol Clin N Am 2003; 12: 105-126
17 Qian J, Feng GS, Vogl T. Combined interventional therapies
of hepatocellular carcinoma. World J Gastroenterol 2003; 9:
1885-1891
18 Chen XM, Luo PF, Lin HH, Zhou ZJ, Shao PJ, Fu L, Li WK.
[Long-term result of combination of transcatheter arterial
chemoembolization and percutaneous ethanol injection for
treatment of hepatocellular carcinoma]. Ai Zheng 2004; 23:
829-832
19 Guo WJ, Yu EX, Liu LM, Li J, Chen Z, Lin JH, Meng ZQ,
Feng Y. Comparison between chemoembolization combined
with radiotherapy and chemoembolization alone for large
hepatocellular carcinoma. World J Gastroenterol 2003; 9:
1697-1701
20 Kouraklis G. Progress in cancer gene therapy. Acta Oncol
1999; 38: 675-683
21 Friedmann T. Gene therapy of cancer through restoration of
tumor-suppressor functions? Cancer 1992; 70: 1810-1817
22 Hsia CC, Nakashima Y, Thorgeirsson SS, Harris CC, Minemura M, Momosaki S, Wang NJ, Tabor E. Correlation of immunohistochemical staining and mutations of p53 in human
hepatocellular carcinoma. Oncol Rep 2000; 7: 353-356
23 Lowe SW, Bodis S, McClatchey A, Remington L, Ruley HE,
COMMENTS
COMMENTS
Background
Hepatocellular carcinoma (HCC) is one of the major cancers in China with a
poor prognosis due to its occult onset, rapid infiltrating growth and complicating
liver cirrhosis. Although transcatheter arterial chemoembolization (TACE) has
been used in treatment of HCC for years, its effect is often unsatisfactory.
Research frontiers
Among the actively studied novel treatment modalities for HCC, the majority
of experts hold that comprehensive or combination ones are most promising.
In addition, gene therapy with p53 (rAd-p53) is a potentially new treatment
modality for cancer.
TACE in combination of rAd-p53 injection has a synergistic effect on HCC and
its strategy is gene addition. Tumor with mutant of the rAd-p53 gene is a better
candidate for p53 therapy. However, this treatment is also effective in those with
inactivated wild-type p53, a common condition in tumors. Injection of rAd-p53 can
lead to apoptosis of tumor cells and TACE can result in necrosis of tumor tissue.
Applications
The results of this study demonstrate that TACE in combination with rAd-p53
with is well tolerated and its anti-tumor efficacy is superior to that of TACE alone
with respect to the survival rate and improved symptoms. Further study with a
large sample size would provide an alternative treatment modality for HCC.
Terminology
p53 gene is a tumor suppressor gene which can prevent the formation of tumors. Mutations in p53 are found in most tumor types and contribute to complex
molecular events leading to tumor formation. Recombinant adenovirus is one of
the viral vectors which are commonly used to deliver genetic materials into cells.
Gene therapy for diseases is to insert, alterate, or remove such materials in cells.
Peer review
This is a well-designed study in which the authors analyzed the clinical effect
of rAd-p53 injection and TACE on advanced HCC. The data show that the
combination therapy is a safe and effective treatment modality for advanced
HCC, and can significantly improve the survival rate of HCC patients.
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S- Editor Sun H L- Editor Wang XL
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E- Editor Ma WH
[email protected]
doi:10.3748/wjg.v17.i16.2150
CASE REPORT
Celiac disease and microscopic colitis: A report of 4 cases
Zsolt Barta, Eva Zold, Arpad Nagy, Margit Zeher, Istvan Csipo
milial co-existence and prevalence of MC in patients with
a prior diagnosis of CD are unclear. Clinicians managing
celiac disease should be aware of these associations and
understand when to consider colon investigation.
Zsolt Barta, Eva Zold, Arpad Nagy, Margit Zeher, Istvan
Csipo, 3rd Department of Medicine, Institute for Internal Medicine, Medical and Health Science Center, University of Debrecen, 4032 Moricz Zs. krt. 22, Debrecen, Hungary
Author contributions: All authors gave substantial contributions
to acquisition, analysis and interpretation of data and participated
in writing the paper; Barta Z gave final approval of the version to
be published.
Correspondence to: Zsolt Barta, MD, PhD, 3rd Department of Medicine, Institute for Internal Medicine, Medical and
Health Science Center, University of Debrecen, 4032 Moricz
Zs. krt. 22, Debrecen, Hungary. [email protected]
Telephone: +36-52-255218 Fax: +36-52-255218
Received: October 27, 2010 Revised: December 30, 2010
Key words: Collagen colitis; Lymphocytic colitis; Celiac
disease; Fibrosing alveolitis; Anti-saccharomyces cerevi
siae antibody
Peer reviewer: Dr. Alberto Tommasini, MD, Professor, Labo-
ratory of Immunopathology, Institute for Maternal and Child
Health, IRCCS Burlo Garofolo, Via dell’Istria 65/1, Trieste
34137, Italy
Barta Z, Zold E, Nagy A, Zeher M, Csipo I. Celiac disease and
microscopic colitis: A report of 4 cases. World J Gastroenterol
wjgnet.com/1007-9327/full/v17/i16/2150.htm DOI: http://
dx.doi.org/10.3748/wjg.v17.i16.2150
Abstract
Celiac disease (CD) is an autoimmune disorder of the sm
all intestine that occurs in genetically predisposed people
at all ages. However, it can be associated also to other im
munopathological disorders, and may be associated with
abnormal histology in segments of the gut other than the
small bowel including colonic inflammation. While guide
lines for endoscopic investigation of the jejunum are well
defined, no indication is defined for colonic investigation.
We describe four cases of concurrent CD and microscopic
colitis (MC) diagnosed at our department over a 10-year
period and analyzed the main features and outcomes of
CD in this setting. The symptoms of these patients were
improved initially by a gluten-free diet before the onset
of MC symptoms. Two of the patients were siblings and
had an atypical form of CD. The other two patients with
CD and MC also presented with fibrosing alveolitis and
were anti-Saccharomyces cerevisiae antibody positive.
The co-existence of immune-mediated small bowel and
colonic inflammatory and pulmonary diseases are not
well-known, and no systematic approach has been used
to identify the lifelong patterns of these immune-based
diseases. Patients can develop, or present with CD at any
stage in life, which can co-exist with other gastrointesti
nal diseases of (auto-) immune origin. In addition, the fa
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INTRODUCTION
Celiac disease (CD) is an immune-mediated disorder,
an autoimmune enteropathy, triggered by the ingestion
of gluten in genetically susceptible persons. The dis
ease primarily affects the gastrointestinal tract and is
characterized by chronic inflammation of the small bowel
mucosa that may result in atrophy of intestinal villi, ma
labsorption, and a variety of clinical manifestations. Of
genetic factors, the strongest recognized association is
with HLA-DQ2 and/or -DQ8: 95%-100% of the patients
carry these molecules. Dietary glutens interact with
these HLA molecules to activate an abnormal mucosal
immune response and induce tissue damage. Most affected
individuals experience remission after gluten is excluded
from their diet.
The diagnosis of CD is established by serologic testing, biopsy evidence of villous atrophy, and improvement
of symptoms on a gluten-free diet. Avoidance of gluten
2150
Barta Z et al . CD and MC
exposure is crucial for CD patients to reduce the risk of
complications so the follow-up serological assessment of
treatment effectiveness should be added to be sure of a
good compliance.
There are atypical forms of CD. For example, silent
CD is found in individuals who are asymptomatic but have
a positive serologic test and villous atrophy on biopsy, and
latent CD is defined by a positive serology but no villous
atrophy on biopsy. These individuals are asymptomatic, but
later may develop symptoms and/or histological changes[1].
The late concordance in the appearance of CD in monozygotic twins also suggests that the disorder may remain in
the latent stage for a long time[2,3]. Small bowel villous atrop
hy with crypt hyperplasia and recovery of the lesion on a
gluten-free diet suggest that villous atrophy comprises only
the end stage of the clinical course of the disease and that
CD clearly develops gradually from mucosal inflammation
to crypt hyperplasia and finally to overt villous atrophy.
A typical feature of CD, in addition to mucosal changes,
is gluten-dependent serum IgA class autoantibodies against
transglutaminase 2 (TG2). These serum autoantibodies,
endomysial and TG2, are powerful tools in disclosing CD
with overt villous atrophy. Furthermore, positive serum
celiac autoantibodies can predict impending CD in many
patients evincing normal small bowel mucosal villous architecture. Hence, patients having “false-positive” celiac
autoantibodies in serum are in fact at risk of developing
overt CD. Some patients with positive serum endomysial
or tissue TG2 antibodies may still seroconvert negatively
during follow-up. However, it is well recognized that serum celiac autoantibodies in some cases fluctuate before a
patient eventually develops overt CD after a longer followup period. The reason for this still remains obscure.
Transglutaminases are a family of 8 currently known
calcium-dependent enzymes that catalyze the cross-linking
or deamidation of proteins and are involved in important
biological processes such as wound healing, tissue repair, fibrogenesis, apoptosis, inflammation, and cell-cycle control.
Therefore, they play an important role in the pathomechanisms of autoimmune, inflammatory, and degenerative
diseases, many of which affect the gastrointestinal system.
Transglutaminase 2 is prominent, since it is central to the
pathogenesis of CD, and modulates inflammation and fibrosis in inflammatory bowel and chronic liver diseases[4].
Respiratory disease and subclinical pulmonary abnormalities are the recognized complications of both CD and
inflammatory bowel disease (IBD) but the mechanisms of
lung disease in CD differ from that in IBD and support the
hypothesis of a common mucosal defect in lung and small
intestine in CD that allow increased permeability[5].
Lymphocytic colitis (LC), together with collagenous
colitis (CC), is included under the umbrella term “microscopic colitis” (MC), in which chronic gastrointestinal
symptoms, including diarrhea, abdominal pain, fecal urgency, incontinence, and nausea, are not associated with endoscopic or radiological alterations. It is not known whether
LC and CC are two different diseases or distinct manifestations of the same clinical condition. Data on pathophysiology conflict and different hypotheses refer to genetic pre-
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disposition, immune dysregulation, autoimmunity, bile acid
malabsorption, infection, and drug effect. Familial occurrence of MC has been identified in some families[6-13]. The
central role of an altered immune system in MC pathogenesis is supported by the association with several conditions
in which an immune dysregulation is involved, such as CD,
rheumatoid arthritis, and hypo- and hyperthyroidism. Up to
now, it has not been clear whether CC (or LC) is a distinct
entity or only an epiphenomenon of another disease that
leads to thickening of the collagen layer. However, whether
MC (both CC and LC) is an autoimmune disease has not
been conclusively established[14]. Diagnosis of MC can be
established only by colonic biopsies and subsequent histopathological examination, when an increase in inflammatory infiltration and/or a thickening of the collagen layer
are found. A number of papers have documented an association between CD and MC[15-18]. However, the prevalence
of MC in patients with a prior diagnosis of CD is unclear,
but it does feature prominently in several series of patients
with CD who have persisting symptoms despite gluten
exclusion. When continuing gluten ingestion, inadvertent
or covert, has been excluded, colon investigation should be
considered as part of the investigation of these patients.
The link may be genetic, at least in part. Both types of MC
are known to resolve spontaneously in a majority of cases.
Data are limited regarding pharmacological therapies, but
budesonide appears best documented as showing an efficacy against CC and MC[19].
We report here 4 cases with sequential development of
CD and MC and discuss the possible connection of these
co-existences.
CASE REPORT
Case 1
A 42-year-old female with a previous history of both
cognitive and neurovegetative symptoms of depression,
including depressed mood, anhedonia, feelings of wor
thlessness, low energy, troubled sleep, and poor concen
tration, was evaluated in the local medical center for com
plaints of watery diarrhea. She had longstanding lactose
intolerance for which she was taking a lactose-free diet. As
her mother had manifestations of CD, enteroscopy was
performed. However, the first endoscopic and histological
evaluation showed no duodenal mucosal alterations
(Marsh 0). Six months later, endoscopic findings were
persistent and duodenal biopsies were taken which were
not diagnostic for CD, and biochemical laboratory tests
were within normal ranges. She was then referred to our
clinic and additional laboratory tests showed increased
antibody titers against gliadin, endomysium, and tTG. Her
psychiatric disease was controlled after treatment and then
remained stable. She was given a gluten-free diet, which
resolved her diarrhea and allowed her to regain her lost
body weight. Five years later, the patient presented with
watery diarrhea occurring 8-10 times daily and mild body
weight loss. At the beginning, this condition was associated
with urgency, nocturnal stools, abdominal cramping,
nausea, mild body weight loss, and fatigue, and persisted
2151
despite strict adherence to the gluten- and lactose-free diet.
With the loss of patience of the diet and of her symptoms
she broke her diet and clinical signs remained. Small bo
wel biopsies at upper endoscopy demonstrated nothing
(Marsh 0) but the gluten panel was unambiguously positive.
Stool cultures and Clostridium difficile toxin assay were
negative. After consultation with dietitians, the patient was
maintained on a gluten- and lactose-free diet for six months
but with mild improvement. Colonoscopy was performed
later, and biopsies from her colon demonstrated LC. Her
symptoms responded partially to mesalazine treatment over
the subsequent two months, at which point her medical
therapy was changed to budesonide (9 mg/d). After she
was put on budesonide with a strict diet, both abdominal
complaints and psychiatric problems resolved (Figure 1).
Mother
Celiac
disease
Case 2
Latent CD
and
MC-CC
Figure 1 Family of cases 1 and 2.
to our hospital from an outside hospital because of contin
ued signs and symptoms of CD that persisted despite selfreported adherence to a gluten-free diet. The patient rep
orted abdominal pain, bowel distension, and body weight
loss over the past few years. Diagnosis of CD was made 4
years ago, based on the small bowel biopsy results showing
evidence of villous blunting with increased chronic infl
ammatory cells, positive laboratory tests, and typical
gastrointestinal signs and symptoms with negative stool
cultures and Clostridium difficile toxin assay. Repeated
laboratory tests showed elevated antibodies against gliadin,
endomysium, and tTG, and small bowel biopsy proved
villous atrophy. The patient met with a nutritionist and
implemented recommended dietary changes to eliminate
gluten. Her symptoms temporarily improved with her
bowel function returned to normal, but after a short time
her symptoms recurred. Results of further tests excluded
conditions known to complicate or coexist with CD,
including bacterial overgrowth and lactose intolerance.
Because of chronic watery stools, a colonoscopy was do
ne with random biopsies from the colon for histological
investigations. Based on the typical picture of prominent
intraepithelial lymphocytes but no thickened collagenous
layer, the pathologist diagnosed her with LC. She was
started on strict gluten-free diet and budesonide with
success. Five years later, she was free of complaints of
CD and LC.
Case 2
A 45-year-old woman with suspected irritable bowel syn
drome was admitted to the hospital. Her bowel movements
increased from one to six or eight a day with watery stools.
She did not note any mucus or blood in the stool and
could not identify any alleviating or aggravating factors.
She consumed a normal diet, including meat, wheat,
and dairy. Over-the-counter anti-diarrheal medications
did not relieve her symptoms. She had no fevers, chills,
or night sweats, but body weight loss. Her medical hist
ory included major depression for 10 years, which was
controlled after treatment and remained stable at admis
sion. Results of basic laboratory tests, including thyroidstimulating hormone (TSH), complete blood count, blood
chemistries, renal function, and liver function, were nor
mal. Colonoscopy showed normal mucosa as far as the
cecum. Colonic biopsy revealed a mildly expanded lamina
propria and intraepithelial lymphocytosis with significantly
thickening of the subepithelial collagen table. This set of
features was consistent with CC, a variant of MC. Her
symptoms were eventually controlled after a 6-mo course
of oral budesonide (9 mg) and ongoing intermittent use
of loperamide (Imodium). Six years later, similar problems
with body weight loss caused her to be hospitalized at our
clinic. A detailed previous history unraveled the familial
connection with Case 1 and her mother with known CD.
Psychiatric disease was controlled, so control and further
GI investigations were organized. The histopathology
report of colonic biopsy showed aspecific inflammation
without MC. Further laboratory investigation revealed
that the entire celiac antibody panel was positive. Results
of duodenal biopsy did not reveal typical lymphocyte
infiltration, crypt hyperplasia, and villous atrophy but
normal mucosal architecture, without significant intraepi
thelial lymphocytic infiltration (Marsh 0). The diagnosis
was latent CD, as the patient had abnormal antibody blood
tests for CD but normal small intestines. After she was put
on a strict gluten-free diet, both abdominal complaints and
psychiatric problems resolved (Figure 1).
Case 4
A man at age 31, with a previous history of bronchial ast
hma, was investigated for abdominal pain, chronic watery
diarrhea, and body weight loss with negative stool samples
(both the cultures and Clostridium difficile toxin assay).
Findings from an upper gastrointestinal endoscopy were
normal, but distal duodenal biopsies showed subtotal
villous atrophy, inflammatory infiltration of the lamina
propria, and an increase in intraepithelial lymphocytes.
Based on the histology and positive laboratory tests, CD
was diagnosed and the patient was started on a glutenfree diet. Abdominal pain ceased but he did not gain body
weight and diarrhea remained a problem. Compliance
with a gluten-free diet was confirmed by the assessment
of dietitians. Repeated biopsies of the duodenal mucosa
showed mild improvement in villous atrophy but serology
Case 3
A 56-year-old woman with a previous history of chronic
(non-specific) colitis and fibrosing alveolitis was referred
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Case 1
Latent CD
and
MC-LC
2152
Table 1 Summary of the cases
Case 1
Case 2
Case 3
Case 4
Sex/birth (yr)
Female/1956
Female/1955
Female/1945
Male/1964
Small bowel histology Normal small-bowel Normal small-bowel
Partial villous atrophy (Marsh 3A)
Total villous atrophy (Marsh 3C)
mucosal structure
mucosal structure
according to the modified Marsh criteria according to the modified Marsh criteria
HLA-DQ2
Present
Present
Present
Present
lgA TTG
+
+
+
+
lgG/lgA EMA
+/+
-/+
-/+
+/+
lgG/lgA Gliadin
+/+
-/+
-/+
+/+
lgG/lgA ASCA
-/-/+/+
+/+
Celiac disease
Latent
Latent
Manifest
Manifest
Colon histology
Lymphocytic colitis
Collagenous colitis
Lymphocytic colitis
Collagenous colitis
Therapy
GFD and budesonide GFD and budesonide
GFD and budesonide
GFD and budesonide
Other disease
Fibrosing alveolitis
Fibrosing alveolitis
Laboratory tests and histology of the small bowel before gluten-free diet, colon histology after gluten-free diet, therapy, and concomitant lung disease. TTG:
Tissue transglutaminase; EMA: Endomysial antibody; ASCA: Anti-saccharomyces cerevisiae antibody; GFD: Gluten-free diet; HLA: Human leukocyte antigen.
was negative. Four years later, a dietitian again confirmed
adherence to a strict gluten-free diet and colonic biopsies
showed no alteration. A barium follow-through showed
mild jejunal and rather featureless ileal mucosa but no
obstructive lesion of the small bowel, nothing abnormal
was seen on an ultrasound scan of the abdomen. Because
of bloody stools and in view of his worsening symptoms
despite the gluten-free diet, repeated colonoscopy with
random biopsies was done for histological investigations
from ileal and colonic samples. Both proved a submucosal
thickened collagen layer, thus the diagnosis of collagenous
entero-colitis (with CD) was made. He was started on
mesalazine and budesonide but without e’clat. The next
step was methylprednisolone, initially 32 mg/d, and then
the dose was decreased to 4 mg/d. This therapy was
continued with corticosteroids for three months. Over the
next year, his clinical condition improved, with resolution
of his diarrhea and a body weight gain of 3 kg. Three
years later, his symptoms recurred. Results of further tests
excluded conditions known to complicate or coexist with
CD, including bacterial overgrowth and lactose intolerance.
Repeated biopsies excluded collagenous entero-colitis, thus
fibrosing alveolitis was diagnosed by the pulmonologist
based on the lung function, laboratory and radio-imaging
tests, chest X-ray, and high-resolution CT scanning
(HRCT). Because the abdominal symptoms of the patient
were refractory to treatment, he was treated again with
budesonide and his clinical condition improved.
The diagnoses of CD, MC, and fibrosing alveolitis in all
cases were made according to the formally accepted criteria. Two independent pathologists certified the diagnosis of
MC by verifying the subsequent sections and completing
the check with additional investigations (intraepithelial lymphocytes, tenascin labeling of the collagen layer, mast cells,
and other lamina propria cell components). Fibrosing alveolitis was proved by HRCT and upon the ATS/ERS clinical criteria. The laboratory tests were performed. In brief,
the HLA-DQ alleles were determined from whole blood
samples by PCR with sequence-specific primers, traditional
IgG and IgA AGA were detected by ELISA (α-gliatest IgG
and IgA; Eurospital, Trieste, Italy), anti-tTG was measured
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by ELISA using recombinant human tissue transglutaminase as an antigen (EutTG, Eurospital), IgG and IgA EmA
were investigated by indirect immunofluorescence using
human umbilical cord cryostat sections prepared in our
laboratory as a substrate, and both serum IgG and IgA
levels in anti-Saccharomyces cerevisiae antibody (ASCA)
were evaluated (separately) according to the manufacturer’s
protocol (ASCA IgG, ASCA IgA, QUANTA Lite, INOVA
Diagnostics) (Table 1).
DISCUSSION
Is CD much ado about nothing? This report presents four
cases of CD with MC. The symptoms of these patients
were improved initially by a gluten-free diet before the onset
of MC symptoms. Their history indicates and underlines
that patients can develop, or present with CD at any stage
in life and that CD can co-exist with other gastrointestinal
diseases of an (auto-) immune origin. Patients with CD
can fail to respond to the initial introduction of a glutenfree diet or have a recurrence of symptoms after initial
improvement, despite maintaining gluten exclusion. The
most feared causes of either scenario are complicating
malignancy, notably enteropathy-associated T-cell lymp
homa, or refractory sprue. Other causes of persistent sy
mptoms with increased prevalence in CD include lactose
intolerance, exocrine pancreatic insufficiency, bacterial
overgrowth, and microscopic (lymphocytic or collagenous)
colitis. Thus, in patients whose symptoms fail to respond
or who later relapse, despite the exclusion of gluten from
their diet, the possibility of additional pathology should be
considered and colonoscopy should, therefore, be part of
the follow-up in patients who present with chronic watery
diarrhea, even if initial tests indicate only CD.
Relations between CD and ulcerative ileojejunitis,
polymyositis, and fibrosing alveolitis have been previously
described[20,21], and it is of interest that an auto-immune
pathophysiology has been implicated in each of these conditions. An association has been suggested between CD
and diffuse interstitial lung disease of the hypersensitivity
pneumonitis type in several reports from Europe[22]. A case
2153
of lymphocytic bronchoalveolitis and CD with improvement following a gluten free diet was also reported[23].
Our patients with manifestations of CD and MC
presented with fibrosing alveolitis and were ASCA (antiyeast antibodies to yeast antigens that are found in bread
and other cereal derived products) positive (both IgG and
IgA types). Previously, ASCA positivity was shown to be
evident in up to 40%-60% of CD patients and 13%-15%
of MC patients, but its implication is disputed[24]. A possible connection between alveolitis and ASCA is also
not known. Only one case of a Japanese patient was
published: lung biopsy specimens showed alveolitis and
serum-precipitating antibody gave a positive reaction for
an extract from S. cerevisiae[25].
In conclusion, the co-existence of immune-mediated
small bowel and colonic inflammatory diseases (i.e. CD
and IBD) and pulmonary diseases is not well-known
and no systematic approach has been used to identify
the life-long patterns of these immune-based diseases[26].
Such information may be useful for both disease prevention and treatment approaches. Clinicians managing CD
should be aware of these associations and when to consider colon investigation.
11
12
13
14
15
16
17
18
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2
3
4
5
6
7
8
9
10
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Dewar DH, Ciclitira PJ. Clinical features and diagnosis of celiac disease. Gastroenterology 2005; 128: S19-S24
Kaukinen K, Mäki M, Partanen J, Sievänen H, Collin P. Celiac disease without villous atrophy: revision of criteria called
for. Dig Dis Sci 2001; 46: 879-887
Kaukinen K, Collin P, Mäki M. Latent coeliac disease or coeliac disease beyond villous atrophy? Gut 2007; 56: 1339-1340
Elli L, Bergamini CM, Bardella MT, Schuppan D. Transglutaminases in inflammation and fibrosis of the gastrointestinal
tract and the liver. Dig Liver Dis 2009; 41: 541-550
Robertson DA, Taylor N, Sidhu H, Britten A, Smith CL,
Holdstock G. Pulmonary permeability in coeliac disease and
inflammatory bowel disease. Digestion 1989; 42: 98-103
Vernier G, Cocq P, Baron P, Paquet PY, Colombel JF. [Familial
occurrence of collagenous colitis]. Gastroenterol Clin Biol 2005;
29: 474-476
Kong SC, Keogh S, Carter MJ, Lobo AJ, Sanders DS. Familial
occurrence of microscopic colitis: an opportunity to study the
relationship between microscopic colitis and coeliac disease?
Scand J Gastroenterol 2002; 37: 1344-1345
Thomson A, Kaye G. Further report of familial occurrence of
collagenous colitis. Scand J Gastroenterol 2002; 37: 1116
Freeman HJ. Familial occurrence of lymphocytic colitis. Can J
Järnerot G, Hertervig E, Grännö C, Thorhallsson E, Eriksson
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S, Tysk C, Hansson I, Björknäs H, Bohr J, Olesen M, Willén R,
Kagevi I, Danielsson A. Familial occurrence of microscopic
colitis: a report on five families. Scand J Gastroenterol 2001; 36:
959-962
Abdo AA, Zetler PJ, Halparin LS. Familial microscopic colitis. Can J Gastroenterol 2001; 15: 341-343
Chutkan R, Sternthal M, Janowitz HD. A family with collagenous colitis, ulcerative colitis, and Crohn’s disease. Am J
Gastroenterol 2000; 95: 3640-3641
van Tilburg AJ, Lam HG, Seldenrijk CA, Stel HV, Blok P,
Dekker W, Meuwissen SG. Familial occurrence of collagenous colitis. A report of two families. J Clin Gastroenterol 1990;
12: 279-285
Abdo AA, Urbanski SJ, Beck PL. Lymphocytic and collagenous colitis: the emerging entity of microscopic colitis. An
update on pathophysiology, diagnosis and management. Can
J Gastroenterol 2003; 17: 425-432
Green PH, Yang J, Cheng J, Lee AR, Harper JW, Bhagat G.
An association between microscopic colitis and celiac disease.
Clin Gastroenterol Hepatol 2009; 7: 1210-1216
Matteoni CA, Goldblum JR, Wang N, Brzezinski A, Achkar E,
Soffer EE. Celiac disease is highly prevalent in lymphocytic
colitis. J Clin Gastroenterol 2001; 32: 225-227
Fine KD, Do K, Schulte K, Ogunji F, Guerra R, Osowski L,
McCormack J. High prevalence of celiac sprue-like HLA-DQ
genes and enteropathy in patients with the microscopic colitis syndrome. Am J Gastroenterol 2000; 95: 1974-1982
DuBois RN, Lazenby AJ, Yardley JH, Hendrix TR, Bayless
TM, Giardiello FM. Lymphocytic enterocolitis in patients
with ‘refractory sprue’. JAMA 1989; 262: 935-937
Rubio-Tapia A, Talley NJ, Gurudu SR, Wu TT, Murray JA.
Gluten-free diet and steroid treatment are effective therapy
for most patients with collagenous sprue. Clin Gastroenterol
Hepatol 2010; 8: 344-349.e3
Coupe MO, Barnard ML, Stamp G, Hodgson HJ. Ulcerative
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Ben M’rad S, Dogui MH, Merai S, Djenayah F. [Respiratory
manifestation of celiac disease]. Presse Med 1998; 27: 1384
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R, Pavord ID. A case of cough, lymphocytic bronchoalveolitis
and coeliac disease with improvement following a gluten free
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S- Editor Sun H
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2154
L- Editor Wang XL
E- Editor Ma WH
[email protected]
doi:10.3748/wjg.v17.i16.2155
CASE REPORT
Pure red cell aplasia caused by pegylated interferon-α-2a
plus ribavirin in the treatment of chronic hepatitis C
Cheng-Shyong Chang, Sheng-Lei Yan, Hsuan-Yu Lin, Fu-Lien Yu, Chien-Yu Tsai
Key words: Chronic hepatitis C; Pegylated interferon-α2a; Pure red cell aplasia; Ribavirin
Cheng-Shyong Chang, Hsuan-Yu Lin, Fu-Lien Yu, Chien-Yu
Tsai, Division of Hematology and Oncology, Department of Internal Medicine, Changhua Christian Hospital, Changhua City
500, Taiwan, China
Sheng-Lei Yan, Division of Gastroenterology and Hepatology,
Department of Internal Medicine, Chang-Bing Show Chwan
Memorial Hospital, Changuha County 505, Taiwan, China
Sheng-Lei Yan, Department and Graduate Program of Bioindustrial Technology, Dayeh University, Changhua County
51591, Taiwan, China
Fu-Lien Yu, Graduate Institute of Nursing, Chung Shan Medical University, Taichung City 40201, Taiwan, China
Author contributions: Chang CS substantial contributions to
conception and design; Yan SL draft the article and final approval of the version; Lin HY revise it critically for important
intellectual content; Yu FL and Tsai CY help acquisition and
interpretation of data.
Correspondence to: Sheng-Lei Yan, MD, Division of Gastroenterology and Hepatology, Department of Internal Medicine,
Chang-Bing Show Chwan Memorial Hospital, No 6, Lu-Gong
Rd., Lugang Township, Changhua County 505, Taiwan,
Telephone: +886-4-7813888 Fax: +886-4-7812401
Peer reviewer: William Dickey, Professor, Altnagelvin Hospital,
Londonderry, BT47 6SB, Northern Ireland, United Kingdom
Chang CS, Yan SL, Lin HY, Yu FL, Tsai CY. Pure red cell aplasia caused by pegylated interferon-α-2a plus ribavirin in the
treatment of chronic hepatitis C. World J Gastroenterol 2011;
org/10.3748/wjg.v17.i16.2155
INTRODUCTION
Pure red cell aplasia (PRCA) is a rare hematological disorder which is characterized by severe anemia, reticulocytopenia and almost complete absence of erythroid precursors
in bone marrow[1]. Patients typically present with symptoms
of severe anemia in the absence of hemorrhagic phenomena. Common causes of PRCA include human parvovirus
B19 infection, lymphoproliferative disorder, humoral or cellular immunity, production of erythropoietin-neutralizing
antibody, and drugs such as ribavirin and standard interferon[1-5]; however, to our knowledge, there is only one case
report in the English literature of PRCA after pegylated interferon combination therapy for chronic hepatitis C[6]. We
report a second case of PRCA during combination therapy
for chronic hepatitis C.
Abstract
Pure red cell aplasia (PRCA) is a rare hematological disorder which is characterized by severe anemia, reticulocytopenia and almost complete absence of erythroid precursors in bone marrow. The pathophysiology of PRCA
may be congenital or acquired. To our knowledge, there
is only one case report in the English literature of PRCA
after pegylated interferon combination therapy for chronic hepatitis C. We report a second case of PRCA after
pegylated interferon combination treatment for chronic
hepatitis C. The diagnosis of PRCA was confirmed by the
typical findings of bone marrow biopsy. The possible etiologies of our case are also discussed in this paper.
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CASE REPORT
A 69-year-old male was referred to our hospital for treatment of chronic hepatitis C. His past medical history was
remarkable for megaloblastic anemia due to vitamin B12
deficiency. He received regular vitamin B12 injection therapy with a stable hemoglobin level of around 11 g/dL. At
2155
Chang CS et al . PRCA caused by chronic HCV treatment
Methylprednisolone
Termination of combination treatment
Hemoglobin
2
12
Reticulocyte count
Hemoglobin (g/dL)
10
RBC transfusions
1.6
1.4
8
1.2
1
6
0.8
4
0.6
Reticulocyte conmt (%)
1.8
0.4
2
0.2
0
0
0 4 7 9 12 16 20 24 28 32
Weeks after combination treatment
Figure 1 Hemoglobin level and reticulocyte count over time. RBC: Red blood cells.
Figure 2 Photomicrograph of bone marrow biopsy showing overall hypocellularity of 10% (HE, x 40).
Figure 3 Photomicrograph of higher magnification showing normal maturation of myeloid precursors and absence of erythroid precursors in this
field (HE, x 100).
initial presentation, elevated aminotransferase levels and
positive antibody to hepatitis C virus were detected. HCV
RNA level was 85 000 IU/mL. The genotype was 2. The
pretreatment hemoglobin level was 11.2 g/dL and the
reticulocyte count was 1.8% (normal range 0.5-1.5%). Serum level of vitamin B12 was 288 pg/mL (normal range
272-1078 pg/mL). Serum level of folic acid was 11.0 ng/mL
(normal range 5-26 ng/mL). Treatment with peginterferon alfa-2a 180 ug weekly and ribavirin 800 mg daily
was started. After four weeks of treatment, the ribavirin
dose was reduced to 600 mg due to a prominent decrease
in the hemoglobin level to 8.4 g/dL. HCV RNA level at
the 4th wk was undetected. Three weeks after dose reduction of ribavirin, the hemoglobin level continued to drop
to 6.9 g/dL. The combination therapy was discontinued
after 7 wk of treatment due to the patient’s intolerance of
anemia (Figure 1). Serum levels of indirect bilirubin, lactate dehydrogenase and haptoglobin level remained normal during combination treatment. Parvovirus serologies
revealed positive immunoglobin (Ig) G but negative IgM
antibodies, which were consistent with past exposure.
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He received follow up at a previous institution after
termination of combination treatment. However, his anemia persisted and became transfusion-dependent. Blood
transfusion with packed red blood cells was administered
every month (Figure 1). Three months after discontinuation of combination treatment, the hemoglobin dropped
to 5.5 g/dL and the reticulocyte count was 0.1% (normal range 0.5-1.5%). Levels of vitamin B12 and folic
acid were within the normal range. Bone marrow biopsy
revealed severe hypocellularity of 10% (Figure 2) with
normal maturation of myeloid precursor cells (Figure 3).
Erythroid precursor cells were markedly decreased with
a ratio of myeloid to erythroid precursors of 10. The
diagnosis of PRCA was made based on these histopathological findings. Oral methylprednisolone 15 mg daily was
administered. After four weeks of oral methylprednisolone therapy, his hemoglobin level increased to 9.2 g/dL
and the reticulocyte count increased to 1.7% (Figure 1).
He needed no further blood transfusions in the following
months.
2156
alter significantly before and after combination treatment,
thus excluding vitamin B12 deficiency as the cause of severe anemia after combination treatment. Therefore, it is
reasonable to assume that pegylated interferon might have
played a role in the pathogenesis of PRCA in our case.
Several studies have also suggested that interferon may
play a role in the development of acquired PRCA[4,5,23].
However, further investigations may be needed to determine whether PRCA is caused by pegylated interferon
alone or by combination treatment.
In conclusion, this case highlights the importance of
considering PRCA when severe anemia associated with
reticulocytopenia develops during the combination treatment of patients with chronic hepatitis C.
DISCUSSION
Combination treatment of pegylated interferon α and
ribavirin has become the standard therapy for patients
with chronic hepatitis C infection[7]. Patients with chronic
hepatitis C receiving combination treatment develop
anemia because of ribavirin-induced hemolysis[8] and
interferon-induced bone marrow suppression [9]. The
ribavirin-induced anemia is dose-dependent and reversible[10]. Conversely, interferon directly suppresses the bone
marrow synthesis of granulocytes, erythrocytes and megakaryocytes[9]. PRCA is rarely encountered in chronic hepatitis C patients receiving combination treatment. To our
knowledge, there is only one previous case report in the
English literature of PRCA after combination treatment
in patients with chronic hepatitis C[6].
The pathophysiology of PRCA is heterogenous,
which may be congenital or acquired[1]. Diamond-Blackfan anemia is a congenital form of PRCA with genetic
defects affecting erythropoietic lineage. Acquired causes
of PRCA include human parvovirus B19 infection, lymphoproliferative disorder, humoral or cellular immunity,
production of erythropoietin-neutralizing antibody, and
drugs such as ribavirin and standard interferon[1-5]. Other
reported causes of PRCA include hepatitis A infection[11],
malignant thymoma[12], and systemic lupus erythematosus[13]. The diagnosis of PRCA is based on bone marrow
findings such as severe hypocellularity, a markedly elevated
myeloid: erythroid ratio, and severe decreased erythroid
precursors[6,14]. With regard to the treatment of PRCA,
corticosteroid therapy is considered the treatment of first
choice, although relapse is not uncommon[15]. In the study
of Clark et al[16], 80% of patients relapsed as the dosage of
steroid was tapered during the first year after remission. In
contrast, cyclosporine A is suggested when the long-term
feasibility of maintenance is considered[15].
As to the etiology of PRCA in our case, several possibilities should be considered. Firstly, although HCV
infection itself has been associated with PRCA[17], the
HCV RNA in our case was undetected at the 4th wk of
combination treatment. Furthermore, parvovirus serologies revealed positive IgG but negative IgM antibodies,
which excluded parvovirus infection as the cause of
PRCA in our case. Secondly, although ribavirin-induced
PRCA has been reported previously[3], the anemia in our
case might not have been caused solely by ribavirin since
the anemia persisted for 3 mon after discontinuation of
ribavirin. Drug-induced PRCA generally resolves within
1-2 wk after removal of the causative agent[18]. Thirdly,
one might consider the possible association of underlying pernicious anemia with PRCA in this case. Pernicious
anemia is generally considered an autoimmune disease
resulting in deficiency of intrinsic factor and subsequent
vitamin B12 deficiency[19]. Coexistence of pernicious anemia and PRCA in the same patient has been reported in
earlier literature[20-22], suggesting that an immunological
process is involved in the pathogenesis of PRCA in these
cases. In our case, serum levels of vitamin B12 did not
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REFERENCES
1
Fisch P, Handgretinger R, Schaefer HE. Pure red cell aplasia. Br J Haematol 2000; 111: 1010-1022
2 Schecter JM, Mears JG, Alobeid B, Gaglio PJ. Anti-erythropoietin antibody-mediated pure red cell aplasia in a living
donor liver transplant recipient treated for hepatitis C virus.
Liver Transpl 2007; 13: 1589-1592
3 Tanaka N, Ishida F, Tanaka E. Ribavirin-induced pure redcell aplasia during treatment of chronic hepatitis C. N Engl J
Med 2004; 350: 1264-1265
4 Tomita N, Motomura S, Ishigatsubo Y. Interferon-alpha-induced pure red cell aplasia following chronic myelogenous
leukemia. Anticancer Drugs 2001; 12: 7-8
5 Hirri HM, Green PJ. Pure red cell aplasia in a patient with
chronic granulocytic leukaemia treated with interferonalpha. Clin Lab Haematol 2000; 22: 53-54
6 Miura Y, Kami M, Yotsuya R, Toda N, Komatsu T. Pure
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with chronic hepatitis C virus infection: role of membrane
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M, Tanaka N, Osuga T, Sato Y, Abe T. Severe acute hepatitis
A associated with acute pure red cell aplasia. J Gastroenterol
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thymoma, myasthenia gravis, polyclonal large granular
lymphocytosis and clonal thymic T cell expansion. J Clin
Pathol 1994; 47: 676-679
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14 Arcasoy MO, Rockey DC, Heneghan MA. Pure red cell
aplasia following pegylated interferon alpha treatment. Am
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18 Mamiya S, Itoh T, Miura AB. Acquired pure red cell aplasia
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15
S- Editor Tian L L- Editor Webster JR
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E- Editor Ma WH
[email protected]
doi:10.3748/wjg.v17.i16.2159
LETTERS TO THE EDITOR
Enucleation for gastrointestinal stromal tumors at the
esophagogastric junction: Is this an adequate solution?
Nadia Peparini, Giovanni Carbotta, Piero Chirletti
Nadia Peparini, Giovanni Carbotta, Piero Chirletti, Department of General Surgery “Francesco Durante”, La Sapienza University, viale del Policlinico 155, 00161 Rome, Italy
Author contributions: Peparini N and Chirletti P contributed equally to this manuscript, conceived and drafted the
manuscript, critically revised the manuscript and gave its final
approval; Carbotta G contributed to the manuscript draft and
gave its final approval.
Correspondence to: Nadia Peparini, MD, PhD, Department
of General Surgery “Francesco Durante” viale del Policlinico
155, 00161 Rome, Italy. [email protected]
Telephone: +39-339-2203940 Fax: +39-6-49970385
Received: October 16, 2010 Revised: December 17, 2010
Accepted: December 24,2010
org/10.3748/wjg.v17.i16.2159
TO THE EDITOR
We read with great interest the article by Coccolini and
colleagues on the treatment of gastrointestinal stromal
tumors (GISTs) at the esophagogastric junction[1]. They
stated the problems related to the choice of extended
esophageal and gastroesophageal resection (i.e. a better guarantee of R0 resection but a higher prevalence of
morbidity and mortality) or enucleation (i.e. a higher risk
of microscopically positive margins but a better postoperative outcome).
The impact of microscopically negative margins on
long-term survival remains controversial and there is no
evidence that extensive resections are related to a better
survival rate. The authors suggested that, for GISTs at the
esophagastric junction, enucleation and adjuvant therapies
can be useful alternatives to avoid the high prevalence of
morbidity and mortality associated with esophageal and esophagogastric resections. However, the 2009 edition of the
TNM Classification of Malignant Tumors states that, in the
absence of nodal metastasis, esophageal GISTs ≤ 2 cm (T1,
i.e. tumors that may be treated with enucleation more frequently) are classified as stage Ⅰ in the case of a low mitotic
rate but as stage ⅢA in the case of a high mitotic rate. This
case is different from T1 gastric GISTs that are classified as
stage Ⅰ or stage Ⅱ in the presence of a low or high mitotic
rate, respectively[2]. In the case of a high mitotic rate, the
prognostic impact of a T1 esophageal GIST is worse than
that of a gastric GIST with an identical size. Prospective,
multicenter evaluation of the different treatment strategies
for esophagogastric GISTs is sorely needed. However, enucleation may not be an adequate surgery for esophagogastric GISTs with a high-mitotic rate in which the guarantee
of negative resection margins and adjuvant therapies can be
the only chance of survival.
Abstract
The authors discussed the proposal by Coccolini and
colleagues to treat gastrointestinal stromal tumors
(GISTs) at the esophagogastric junction with enucleation and, if indicated, adjuvant therapy, reducing the
risks related to esophageal and gastroesophageal resection. They concluded that, because the prognostic
impact of a T1 high-mitotic rate on esophageal GIST
is worse than that of a T1 high-mitotic rate on gastric
GIST, enucleation may not be an adequate surgery
for esophagogastric GISTs with a high mitotic rate in
which the guarantee of negative resection margins and
adjuvant therapies can be the only chance of survival.
Key words: Gastrointestinal stromal tumor; Esophagogastric
junction; Surgery; Resection; Enucleation
Peer reviewer: Alexander Becker, MD, Department of Surgery,
Haemek Medical Center, Afula 18000, Israel
Peparini N, Carbotta G, Chirletti P. Enucleation for gastro
intestinal stromal tumors at the esophagogastric junction:
Is this an adequate solution? World J Gastroenterol 2011;
WJG|www.wjgnet.com
2159
Peparini N et al . GISTs at esophagogastric junction
REFERENCES
1
2
Coccolini F, Catena F, Ansaloni L, Lazzareschi D, Pinna AD.
Esophagogastric junction gastrointestinal stromal tumor: resec-
tion vs enucleation. World J Gastroenterol 2010; 16: 4374-4376
Sobin LH, Gospodarowicz MK, Wittekind Ch, editors.
TNM Classification of Malignant Tumors. Seventh edition
2009. Wiley-Blackwell, 2010
S- Editor Tian L L- Editor Wang XL
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2160
E- Editor Ma WH
World J Gastroenterol 2011 April 28; 17(16): I
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ACKNOWLEDGMENTS
Acknowledgments to reviewers of World Journal of
Gastroenterology
Many reviewers have contributed their expertise and
time to the peer review, a critical process to ensure the
quality of World Journal of Gastroenterology. The editors
and authors of the articles submitted to the journal are
grateful to the following reviewers for evaluating the
articles (including those published in this issue and
those rejected for this issue) during the last editing
time period.
Giulio Marchesini, Professor, Department of Internal Medicine and
Gastroenterology, “Alma Mater Studiorum” University of Bologna, Polic
linico S. Orsola, Via Massarenti 9, Bologna 40138, Italy
Luis Bujanda, PhD,Professor, Departament of Gastroenterology,
CIBEREHD, University of Country Basque, Donostia Hospital, Paseo Dr.
Beguiristain s/n, 20014 San Sebastián, Spain
Frank G Schaap, PhD, Tytgat Institute for Liver and Intestinal Research,
Academic Medical Center, Meibergdreef 69-71, 1105 BK Amsterdam, The
Netherlands
Olivier Detry, Dr., Department of Abdominal Surgery and Transplantation,
University of Liège, CHU Sart Tilman B35, B-4000 Liège, Belgium
Richie Soong, Associate Professor, Centre for Life Sciences #02-15,
National University of Singapore, 28 Medical Drive, Singapore 117456,
Singapore
Julio Mayol, MD, PhD, Department of Digestive surgery, Hospital Clinico
San Carlos, MARTIN-LAGOS S/n, Madrid, 28040, Spain
C Bart Rountree, MD, Assistant Professor of Pediatrics and Phar
macology, Penn State College of Medicine, 500 University Drive, H085,
Hershey, PA 17033, United States
Marek Hartleb, Professor, Department of Gastroenterology, Silesian
Medical School, ul. Medyków 14, Katowice 40-752, Poland
Gisela Sparmann, MD, Division of Gastroenterology, Department of
Internal Medicine, University of Rostock, Ernst-Heydemann-Str. 6, Rostock
D-18057, Germany
Kok Sun Ho, Dr., Department of Colorectal Surgery, Singapore General
Hospital, Outram Road, Singapore 169608, Singapore
Yoshitaka Takuma, MD, PhD, Department of Gastroenterology, Kurashiki
Central Hospital, 1-1-1 Miwa, Kurashiki, Okayama, 710-8602 Japan
Richard Hu, MD, MSc, Division of Gastroenterology, Department of
Medicine, Olive view-UCLA Medical Center, 14445 Olive View Drive, Los
Angeles, CA 91342, United States
Yasuhito Tanaka, MD, PhD, Professor, Department of Virology and
Liver unit, Nagoya City University Graduate School of Medical Sciences,
Kawasumi, Mizuho, Nagoya 467-8601, Japan
Hartmut Jaeschke, Professor, Liver Research Institute, University of
Arizona, College of Medicine, 1501 N Campbell Ave, Room 6309, Tucson,
Arizona 85724, United States
Kam-Meng Tchou-Wong, Assistant Professor, Departments of Enviro
nmental Medicine and Medicine, NYU School ofMedicine, 57 Old Forge
Road, Tuxedo, New York, NY 10987, United States
Ioannis Kanellos, Professor, 4th Surgical Department, Aristotle University
of Thessaloniki, Antheon 1, Panorama, Thessaloniki, 55236, Greece
Luca VC Valenti, MD, Internal Medicine, Università degli Studi di Milano,
Fondazione IRCCS Ospedale Maggiore Policlinico, Padiglione Granelli, via F
Sforza 35, Milano, 20122, Italy
Islam Khan, PhD, Professor, Departmenet of Biochemistry, Faculty of
Medicine, Kuwait University, PO box 24923, Safat 13110, Kuwait
Marco Vivarelli, MD, Assistant Professor, Department of Surgery and
Transplantation, University of Bologna, S.Orsola Hospital, Bologna 40123, Italy
Jae J Kim, MD, PhD, Associate Professor, Department of Medicine,
Samsung Medical Center, Sungkyunkwan University School of Medicine, 50,
Irwon-dong, Gangnam-gu, Seoul 135-710, South Korea
Dinesh Vyas, Dr., Department of Minimally and Endosopic Surgery, St John
Mercy Hospital, 851 E Fifth Street, Washington, MO 63090, United States
Won Ho Kim, MD, Professor, Department of Internal Medicine, Yonsei
Uiversity College of Medicine, 134 Shinchon-dong Seodaemun-ku, Seoul
120-752, South Korea
Yutaka Yata, MD, PhD, Director, Department of Gastroenterology,
Saiseikai Maebashi Hospital, 564-1 Kamishinden-machi, Maebashi-city,
Gunma 371-0821, Japan
Ashok Kumar, MD, Dr., Department of Surgical Gastroenterology, Sanjay
Gandhi Post Graduate Institute of Medical Sciences, Lucknow, 226014, India
Huiping Zhou, PhD, Assistant Professor, Department of Microbiology
and Immunology, School of Medicine, Virginia Commonwealth University,
1217 East Marshall Street, MSB#533, Richmond, VA 23298, United States
Rene Lambert, Professor, International Agency for Research on Cancer,
150 Cours Albert Thomas, Lyon 69372 cedex 8, France
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MEETINGS
Events Calendar 2011
January 14-15, 2011
AGA Clinical Congress of
Gastroenterology and Hepatology:
Best Practices in 2011 Miami, FL
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Gastrointestinal Cancers Symposium
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United States
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9. Gastro Forum München, Munich,
Germany
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February 13-27, 2011
Gastroenterology: New Zealand
CME Cruise Conference, Sydney,
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APASL 2011-The 21st Conference of
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February 22, 2011-March 04, 2011
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Inflammatory Bowel Diseases
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of the Saudi Society of Pediatric
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Nutrition, Riyadh, Saudi Arabia
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Annual Meeting 2011, Birmingham,
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Surgery, Budapest, Hungary
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(C-Hep), Palau de Congressos de
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Research, Istanbul, Turkey
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Radiology Annual Meeting and
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March 26-27, 2011
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4th Congress of the Gastroenterology
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June 11-12, 2011
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Forum 2011, Hong Kong, China
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Falk Symposium 177, Endoscopy
Live Berlin 2011 Intestinal Disease
Meeting, Stauffenbergstr. 26, 10785
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SPIGC, II ESYS, Napoli, Italy
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on Probiotics and PrebioticsIPC2011, Kosice, Slovakia
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June 22-25, 2011
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September 2-3, 2011 Falk Symposium
178, Diverticular Disease, A Fresh
Approach to a Neglected Disease,
Gürzenich Cologne,
Martinstr. 29-37, 50667 Cologne,
Germany
September 10-11, 2011
New Advances in Inflammatory
Bowel Disease, La Jolla, CA 92093,
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ICE 2011-International Congress of
Endoscopy, Los Angeles Convention
Center, 1201 South Figueroa Street
Los Angeles, CA 90015,
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IBD Management: Dogmas to be
Challenged, Sheraton Brussels
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Cardiology & Gastroenterology |
Tahiti 10 night CME Cruise,
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October 28-November 2, 2011
ACG Annual Scientific Meeting &
Postgraduate Course,
Washington, DC 20001,
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November 11-12, 2011
Falk Symposium 180, IBD 2011:
Progress and Future for Lifelong
Management, ANA Interconti Hotel,
1-12-33 Akasaka, Minato-ku,
Tokyo 107-0052, Japan
December 1-4, 2011
2011 Advances in Inflammatory
Bowel Diseases/Crohn's & Colitis
Foundation's Clinical & Research
Conference, Hollywood, FL 34234,
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ISSN 1007-9327, online ISSN 2219-2840, DOI: 10.3748) is a
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Title: Title should be less than 12 words.
Abstract
There are unstructured abstracts (no more than 256 words)
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An informative, structured abstracts of no more than 480
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140 words); RESULTS (no more than 294 words): You should
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data to illustrate how they were obtained, e.g. 6.92 ± 3.86 vs 3.61
± 1.67, P < 0.001; CONCLUSION (no more than 26 words).
Running title: A short running title of less than 6 words should
be provided.
Authorship: Authorship credit should be in accordance with the
standard proposed by ICMJE, based on (1) substantial contributions to conception and design, acquisition of data, or analysis and
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for important intellectual content; and (3) final approval of the
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Institution: Author names should be given first, then the com
plete name of institution, city, province and postcode. For ex
ample, Xu-Chen Zhang, Li-Xin Mei, Department of Pathology,
Chengde Medical College, Chengde 067000, Hebei Province,
China. One author may be represented from two institutions,
for example, George Sgourakis, Department of General, Visceral, and Transplantation Surgery, Essen 45122, Germany; George
Sgourakis, 2nd Surgical Department, Korgialenio-Benakio Red
Cross Hospital, Athens 15451, Greece.
Key words
Please list 5-10 key words, selected mainly from Index Medicus,
which reflect the content of the study.
Text
For articles of these sections, original articles and brief articles, the main text should be structured into the following sections: INTRODUCTION, MATERIALS AND METHODS,
RESULTS and DISCUSSION, and should include appropriate Figures and Tables. Data should be presented in the main
text or in Figures and Tables, but not in both. The main text
format of these sections, editorial, topic highlight, case report,
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com/1007-9327/g_info_20100315215714.htm.
Author contributions: The format of this section should be:
Author contributions: Wang CL and Liang L contributed equally
to this work; Wang CL, Liang L, Fu JF, Zou CC, Hong F and Wu
XM designed the research; Wang CL, Zou CC, Hong F and Wu
XM performed the research; Xue JZ and Lu JR contributed new
reagents/analytic tools; Wang CL, Liang L and Fu JF analyzed the
data; and Wang CL, Liang L and Fu JF wrote the paper.
Supportive foundations: The complete name and number of
supportive foundations should be provided, e.g. Supported by
National Natural Science Foundation of China, No. 30224801
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http://www.wjgnet.com/1007-9327/13/4891.pdf; http://
www.wjgnet.com/1007-9327/13/4986.pdf; http://www.
wjgnet.com/1007-9327/13/4498.pdf. Keeping all elements
compiled is necessary in line-art image. Scale bars should be
Correspondence to: Only one corresponding address should be
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affiliation, the complete name of institution, city, postcode, province, country, and email. All the letters in the email should be in
lower case. A space interval should be inserted between country
name and email address. For example, Montgomery Bissell, MD,
Professor of Medicine, Chief, Liver Center, Gastroenterology
Division, University of California, Box 0538, San Francisco, CA
94143, United States. [email protected]
Telephone and fax: Telephone and fax should consist of +,
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Pathological changes in atrophic gastritis after treatment. A:...;
B:...; C:...; D:...; E:...; F:...; G: …etc. It is our principle to publish
high resolution-figures for the printed and E-versions.
first and middle initials. (For example, Lian-Sheng Ma is abbreviated as Ma LS, Bo-Rong Pan as Pan BR). The title of the
cited article and italicized journal title (journal title should be
in its abbreviated form as shown in PubMed), publication date,
volume number (in black), start page, and end page [PMID:
11819634 DOI: 10.3748/wjg.13.5396].
Style for book references
Authors: the name of the first author should be typed in boldfaced letters. The surname of all authors should be typed with the
initial letter capitalized, followed by their abbreviated middle and
first initials. (For example, Lian-Sheng Ma is abbreviated as Ma LS,
Bo-Rong Pan as Pan BR) Book title. Publication number. Publication place: Publication press, Year: start page and end page.
Tables
Three-line tables should be numbered 1, 2, 3, etc., and mentioned clearly in the main text. Provide a brief title for each
table. Detailed legends should not be included under tables,
but rather added into the text where applicable. The information should complement, but not duplicate the text. Use one
horizontal line under the title, a second under column heads,
and a third below the Table, above any footnotes. Vertical and
italic lines should be omitted.
Format
Journals
English journal article (list all authors and include the PMID where applicable)
1 Jung EM, Clevert DA, Schreyer AG, Schmitt S, Rennert J,
Kubale R, Feuerbach S, Jung F. Evaluation of quantitative
contrast harmonic imaging to assess malignancy of liver
tumors: A prospective controlled two-center study. World J
Gastroenterol 2007; 13: 6356-6364 [PMID: 18081224 DOI:
10.3748/wjg.13.6356]
Chinese journal article (list all authors and include the PMID where applicable)
2 Lin GZ, Wang XZ, Wang P, Lin J, Yang FD. Immunologic effect of Jianpi Yishen decoction in treatment of Pixudiarrhoea. Shijie Huaren Xiaohua Zazhi 1999; 7: 285-287
In press
3 Tian D, Araki H, Stahl E, Bergelson J, Kreitman M.
Signature of balancing selection in Arabidopsis. Proc Natl
Acad Sci USA 2006; In press
Organization as author
4 Diabetes Prevention Program Research Group. Hyper
tension, insulin, and proinsulin in participants with impaired
glucose tolerance. Hypertension 2002; 40: 679-686 [PMID:
12411462 PMCID:2516377 DOI:10.1161/01.HYP.00000
35706.28494.09]
Both personal authors and an organization as author
5 Vallancien G, Emberton M, Harving N, van Moorselaar RJ; Alf-One Study Group. Sexual dysfunction in 1,
274 European men suffering from lower urinary tract
symptoms. J Urol 2003; 169: 2257-2261 [PMID: 12771764
DOI:10.1097/01.ju.0000067940.76090.73]
No author given
6 21st century heart solution may have a sting in the tail. BMJ
2002; 325: 184 [PMID: 12142303 DOI:10.1136/bmj.325.
7357.184]
Volume with supplement
7 Geraud G, Spierings EL, Keywood C. Tolerability and
safety of frovatriptan with short- and long-term use for
treatment of migraine and in comparison with sumatriptan. Headache 2002; 42 Suppl 2: S93-99 [PMID: 12028325
DOI:10.1046/j.1526-4610.42.s2.7.x]
Issue with no volume
8 Banit DM, Kaufer H, Hartford JM. Intraoperative frozen
section analysis in revision total joint arthroplasty. Clin
Orthop Relat Res 2002; (401): 230-238 [PMID: 12151900
DOI:10.1097/00003086-200208000-00026]
No volume or issue
9 Outreach: Bringing HIV-positive individuals into care.
HRSA Careaction 2002; 1-6 [PMID: 12154804]
Notes in tables and illustrations
Data that are not statistically significant should not be noted.
a
P < 0.05, bP < 0.01 should be noted (P > 0.05 should not be
noted). If there are other series of P values, cP < 0.05 and dP
< 0.01 are used. A third series of P values can be expressed as
e
P < 0.05 and fP < 0.01. Other notes in tables or under illustrations should be expressed as 1F, 2F, 3F; or sometimes as other
symbols with a superscript (Arabic numerals) in the upper left
corner. In a multi-curve illustration, each curve should be labeled with ●, ○, ■, □, ▲, △, etc., in a certain sequence.
Acknowledgments
Brief acknowledgments of persons who have made genuine
contributions to the manuscript and who endorse the data and
conclusions should be included. Authors are responsible for
obtaining written permission to use any copyrighted text and/or
illustrations.
REFERENCES
Coding system
The author should number the references in Arabic numerals according to the citation order in the text. Put reference numbers
in square brackets in superscript at the end of citation content or
after the cited author’s name. For citation content which is part of
the narration, the coding number and square brackets should be
typeset normally. For example, “Crohn’s disease (CD) is associated
with increased intestinal permeability[1,2]”. If references are cited
directly in the text, they should be put together within the text, for
example, “From references[19,22-24], we know that...”.
When the authors write the references, please ensure that
the order in text is the same as in the references section, and also
ensure the spelling accuracy of the first author’s name. Do not list
the same citation twice.
PMID and DOI
Pleased provide PubMed citation numbers to the reference list,
e.g. PMID and DOI, which can be found at http://www.ncbi.
nlm.nih.gov/sites/entrez?db=pubmed and http://www.crossref.org/SimpleTextQuery/, respectively. The numbers will be
used in E-version of this journal.
Style for journal references
Authors: the name of the first author should be typed in boldfaced letters. The family name of all authors should be typed
with the initial letter capitalized, followed by their abbreviated
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Books
Personal author(s)
10 Sherlock S, Dooley J. Diseases of the liver and billiary
system. 9th ed. Oxford: Blackwell Sci Pub, 1993: 258-296
Chapter in a book (list all authors)
11 Lam SK. Academic investigator’s perspectives of medical
treatment for peptic ulcer. In: Swabb EA, Azabo S. Ulcer
disease: investigation and basis for therapy. New York:
Marcel Dekker, 1991: 431-450
Author(s) and editor(s)
12 Breedlove GK, Schorfheide AM. Adolescent pregnancy.
2nd ed. Wieczorek RR, editor. White Plains (NY): March
of Dimes Education Services, 2001: 20-34
Conference proceedings
13 Harnden P, Joffe JK, Jones WG, editors. Germ cell tumours V. Proceedings of the 5th Germ cell tumours Conference; 2001 Sep 13-15; Leeds, UK. New York: Springer,
2002: 30-56
Conference paper
14 Christensen S, Oppacher F. An analysis of Koza’s compu
tational effort statistic for genetic programming. In: Foster
JA, Lutton E, Miller J, Ryan C, Tettamanzi AG, editors. Genetic programming. EuroGP 2002: Proceedings of the 5th
European Conference on Genetic Programming; 2002 Apr
3-5; Kinsdale, Ireland. Berlin: Springer, 2002: 182-191
Electronic journal (list all authors)
15 Morse SS. Factors in the emergence of infectious diseases. Emerg Infect Dis serial online, 1995-01-03, cited
1996-06-05; 1(1): 24 screens. Available from: URL: http://
www.cdc.gov/ncidod/eid/index.htm
Patent (list all authors)
16 Pagedas AC, inventor; Ancel Surgical R&D Inc., assignee. Flexible endoscopic grasping and cutting device
and positioning tool assembly. United States patent US
20020103498. 2002 Aug 1
by The Royal Society of Medicine, London. Certain commonly
used abbreviations, such as DNA, RNA, HIV, LD50, PCR,
HBV, ECG, WBC, RBC, CT, ESR, CSF, IgG, ELISA, PBS, ATP,
EDTA, mAb, can be used directly without further explanation.
Italics
Quantities: t time or temperature, c concentration, A area, l length,
m mass, V volume.
Genotypes: gyrA, arg 1, c myc, c fos, etc.
Restriction enzymes: EcoRI, HindI, BamHI, Kbo I, Kpn I, etc.
Biology: H. pylori, E coli, etc.
Examples for paper writing
Editorial: http://www.wjgnet.com/1007-9327/g_info_20100315
220036.htm
Frontier: http://www.wjgnet.com/1007-9327/g_info_20100315
220305.htm
Topic highlight: http://www.wjgnet.com/1007-9327/g_info_20
100315220601.htm
Observation: http://www.wjgnet.com/1007-9327/g_info_201003
12232427.htm
Guidelines for basic research: http://www.wjgnet.com/1007-93
27/g_info_20100315220730.htm
Guidelines for clinical practice: http://www.wjgnet.com/10079327/g_info_20100315221301.htm
Review: http://www.wjgnet.com/1007-9327/g_info_20100315
221554.htm
Original articles: http://www.wjgnet.com/1007-9327/g_info_20
100315221814.htm
Brief articles: http://www.wjgnet.com/1007-9327/g_info_2010
0312231400.htm
Statistical data
Write as mean ± SD or mean ± SE.
Case report: http://www.wjgnet.com/1007-9327/g_info_2010
0315221946.htm
Statistical expression
Express t test as t (in italics), F test as F (in italics), chi square test
as χ2 (in Greek), related coefficient as r (in italics), degree of freedom as υ (in Greek), sample number as n (in italics), and probability as P (in italics).
Letters to the editor: http://www.wjgnet.com/1007-9327/g_info_
20100315222254.htm
Book reviews: http://www.wjgnet.com/1007-9327/g_info_2010
0312231947.htm
Units
Use SI units. For example: body mass, m (B) = 78 kg; blood pressure, p (B) = 16.2/12.3 kPa; incubation time, t (incubation) = 96 h,
blood glucose concentration, c (glucose) 6.4 ± 2.1 mmol/L; blood
CEA mass concentration, p (CEA) = 8.6 24.5 mg/L; CO2 volume
fraction, 50 mL/L CO2, not 5% CO2; likewise for 40 g/L formaldehyde, not 10% formalin; and mass fraction, 8 ng/g, etc. Arabic
numerals such as 23, 243, 641 should be read 23 243 641.
The format for how to accurately write common units and
quantums can be found at: http://www.wjgnet.com/1007-9327/
g_info_20100315223018.htm.
Guidelines: http://www.wjgnet.com/1007-9327/g_info_2010
0312232134.htm
RESUBMISSION OF THE REVISED
MANUSCRIPTS
Please revise your article according to the revision policies of
WJG. The revised version includes manuscript and high-resolution image figures. The author should re-submit the revised
manuscript online, along with printed high-resolution color or
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to the reviewers, and science news are sent to us via email.
Abbreviations
Standard abbreviations should be defined in the abstract and
on first mention in the text. In general, terms should not be abbreviated unless they are used repeatedly and the abbreviation
is helpful to the reader. Permissible abbreviations are listed in
Units, Symbols and Abbreviations: A Guide for Biological and
Medical Editors and Authors (Ed. Baron DN, 1988) published
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After a manuscript is published online, links to the PDF version
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The language of a manuscript will be graded before it is sent for
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Authors of accepted manuscripts are suggested to write a science
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WJG is an international, peer-reviewed, Open-Access, online journal. Articles published by this journal are distributed under the
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Authors of accepted articles must pay a publication fee. The
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