Isolated lymphoid follicles in colon - World Journal of Gastroenterology

<strong>World</strong> <strong>Journal</strong> <strong>of</strong><br />

<strong>Gastroenterology</strong><br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): 1655-1790<br />

www.wjgnet.com<br />

ISSN 1007-9327 (pr<strong>in</strong>t)<br />

ISSN 2219-2840 (onl<strong>in</strong>e)

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Editorial Board<br />

2010-2013<br />

The <strong>World</strong> <strong>Journal</strong> <strong>of</strong> <strong>Gastroenterology</strong> Editorial Board consists <strong>of</strong> 1144 members, represent<strong>in</strong>g a team <strong>of</strong> worldwide<br />

experts <strong>in</strong> gastroenterology and hepatology. They are from 60 countries, <strong>in</strong>clud<strong>in</strong>g Albania (1), Argent<strong>in</strong>a (8),<br />

Australia (29), Austria (14), Belgium (12), Brazil (10), Brunei Darussalam (1), Bulgaria (2), Canada (20), Chile (3),<br />

Ch<strong>in</strong>a (69), Colombia (1), Croatia (2), Cuba (1), Czech (4), Denmark (8), Ecuador (1), Egypt (2), Estonia (2), F<strong>in</strong>land<br />

(8), France (24), Germany (75), Greece (14), Hungary (10), India (26), Iran (6), Ireland (7), Israel (12), Italy (101),<br />

Japan (112), Jordan (1), Kuwait (1), Lebanon (3), Lithuania (2), Malaysia (1), Mexico (10), Moldova (1), Netherlands<br />

(29), New Zealand (2), Norway (11), Pakistan (2), Poland (11), Portugal (4), Romania (3), Russia (1), Saudi Arabia<br />

(3), Serbia (3), S<strong>in</strong>gapore (10), South Africa (2), South Korea (32), Spa<strong>in</strong> (38), Sweden (18), Switzerland (11),<br />

Thailand (1), Tr<strong>in</strong>idad and Tobago (1), Turkey (24), United Arab Emirates (2), United K<strong>in</strong>gdom (82), United States<br />

(249), and Uruguay (1).<br />

HONORARY EDITORS-IN-CHIEF<br />

James L Boyer, New Haven<br />

Ke-Ji Chen, Beij<strong>in</strong>g<br />

Mart<strong>in</strong> H Floch, New Haven<br />

Emmet B Keeffe, Palo Alto<br />

Geng-Tao Liu, Beij<strong>in</strong>g<br />

Le<strong>in</strong>-Ray Mo, Ta<strong>in</strong>an<br />

Eamonn M Quigley, Cork<br />

Rafiq A Sheikh, Sacramento<br />

Nicholas J Talley, Rochester<br />

M<strong>in</strong>g-Lung Yu, Kaohsiung<br />

PRESIDENT AND EDITOR-IN-<br />

CHIEF<br />

Lian-Sheng Ma, Beij<strong>in</strong>g<br />

ACADEMIC EDITOR-IN-CHIEF<br />

Tauseef Ali, Oklahoma City<br />

Mauro Bortolotti, Bologna<br />

Tarkan Karakan, Ankara<br />

Weekitt Kittisupamongkol, Bangkok<br />

Anastasios Koulaouzidis, Ed<strong>in</strong>burgh<br />

Bo-Rong Pan, Xi’an<br />

Sylvia LF Pender, Southampton<br />

Max S Petrov, Auckland<br />

George Y Wu, Farm<strong>in</strong>gton<br />

STRATEGY ASSOCIATE<br />

EDITORS-IN-CHIEF<br />

Peter Draganov, Florida<br />

Hugh J Freeman, Vancouver<br />

Maria C Gutiérrez-Ruiz, Mexico<br />

Kazuhiro Hanazaki, Kochi<br />

Akio Inui, Kagoshima<br />

Kalpesh Jani, Baroda<br />

Javier S Mart<strong>in</strong>, Punta del Este<br />

WJG|www.wjgnet.com<br />

Natalia A Osna, Omaha<br />

Wei Tang, Tokyo<br />

Alan BR Thomson, Edmonton<br />

Harry HX Xia, Hanover<br />

Jesus K Yamamoto-Furusho, Mexico<br />

Yoshio Yamaoka, Houston<br />

ASSOCIATE EDITORS-IN-CHIEF<br />

You-Yong Lu, Beij<strong>in</strong>g<br />

John M Luk, S<strong>in</strong>gapore<br />

Hiroshi Shimada, Yokohama<br />

GUEST EDITORIAL BOARD<br />

MEMBERS<br />

Chien-Jen Chen, Taipei<br />

Yang-Yuan Chen, Changhua<br />

Jen-Hwey Chiu, Taipei<br />

Seng-Kee Chuah, Kaohsiung<br />

Wan-Long Chuang, Kaohsiun<br />

M<strong>in</strong>g-Chih Hou, Taipei<br />

Kev<strong>in</strong> Cheng-Wen Hsiao, Taipei<br />

Po-Shiuan Hsieh, Taipei<br />

Tsung-Hui Hu, Kaohsiung<br />

Wen-Hs<strong>in</strong> Huang, Taichung<br />

Chao-Hung Hung, Kaohsiung<br />

I-Rue Lai, Taipei<br />

Teng-Yu Lee, Taichung<br />

Ch<strong>in</strong>g Chung L<strong>in</strong>, Taipei<br />

Hui-Kang Liu, Taipei<br />

Hon-Yi Shi, Kaohsiung<br />

Chih-Chi Wang, Kaohsiung<br />

J<strong>in</strong>-Town Wang, Taipei<br />

Cheng-Shyong Wu, Chia-Yi<br />

Jaw-Ch<strong>in</strong>g Wu, Taipei<br />

Jiunn-Jong Wu, Ta<strong>in</strong>an<br />

M<strong>in</strong>g-Shiang Wu, Taipei<br />

I<br />

Ta-Sen Yeh, Taoyuan<br />

Hsu-Heng Yen, Changhua<br />

M<strong>in</strong>g-Whei Yu, Taipei<br />

MEMBERS OF THE EDITORIAL<br />

BOARD<br />

Albania<br />

Bashkim Resuli, Tirana<br />

Argent<strong>in</strong>a<br />

Julio H Carri, Córdoba<br />

Eduardo de Santibañes, Buenos Aires<br />

Bernardo Frider, Buenos Aires<br />

Carlos J Pirola, Buenos Aires<br />

Bernabe Matias Quesada, Buenos Aires<br />

Silvia Sookoian, Buenos Aires<br />

Adriana M Torres, Rosario<br />

Maria Ines Vaccaro, Buenos Aires<br />

Australia<br />

Leon Anton Adams, Nedlands<br />

Richard Anderson, Victoria<br />

M<strong>in</strong>oti V Apte, New South Wales<br />

Andrew V Biank<strong>in</strong>, Sydney<br />

Filip Braet, Sydney<br />

Christopher Christophi, Melbourne<br />

Philip G D<strong>in</strong>n<strong>in</strong>g, Koagarah<br />

Guy D Eslick, Sydney<br />

Michael A F<strong>in</strong>k, Melbourne<br />

January 7, 2011

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Robert JL Fraser, Daw Park<br />

Jacob George, Westmead<br />

Mark D Gorrell, Sydney<br />

Alexander G Heriot, Melbourne<br />

Michael Horowitz, Adelaide<br />

John E Kellow, Sydney<br />

William Kemp, Melbourne<br />

F<strong>in</strong>lay A Macrae, Victoria<br />

Daniel Markovich, Brisbane<br />

Vance Matthews, Melbourne<br />

Phillip S Oates, Perth<br />

Shan Rajendra, Tasmania<br />

Rajv<strong>in</strong>der S<strong>in</strong>gh, Elizabeth Vale<br />

Ross C Smith, Sydney<br />

Kev<strong>in</strong> J Spr<strong>in</strong>g, Brisbane<br />

Nathan Subramaniam, Brisbane<br />

Phil Sutton, Melbourne<br />

Cuong D Tran, North Adelaide<br />

Debbie Tr<strong>in</strong>der, Fremantle<br />

David Ian Watson, Bedford Park<br />

Austria<br />

Herwig R Cerwenka, Graz<br />

Ashraf Dahaba, Graz<br />

Peter Ferenci, Vienna<br />

Valent<strong>in</strong> Fuhrmann, Vienna<br />

Alfred Gangl, Vienna<br />

Alexander M Hirschl, Wien<br />

Kurt Lenz, L<strong>in</strong>z<br />

Dietmar Öfner, Salzburg<br />

Markus Peck-Radosavljevic, Vienna<br />

Markus Raderer, Vienna<br />

Stefan Riss, Vienna<br />

Georg Roth, Vienna<br />

Michael Trauner, Graz<br />

Thomas Wild, Kapellerfeld<br />

Belgium<br />

Rudi Beyaert, Gent<br />

Benedicte Y De W<strong>in</strong>ter, Antwerp<br />

Inge I Depoortere, Leuven<br />

Olivier Detry, Liège<br />

Philip Meuleman, Ghent<br />

Marc Peeters, De P<strong>in</strong>telaan<br />

Freddy Penn<strong>in</strong>ckx, Leuven<br />

Jean-Yves L Reg<strong>in</strong>ster, Liège<br />

Mark De Ridder, Brussels<br />

Etienne M Sokal, Brussels<br />

Krist<strong>in</strong> Verbeke, Leuven<br />

Eddie Wisse, Keerbergen<br />

Brazil<br />

José LF Caboclo, São José do Rio Preto<br />

Roberto J Carvalho-Filho, São Paulo<br />

Jaime Natan Eisig, São Paulo<br />

Andre Castro Lyra, Salvador<br />

Marcelo Lima Ribeiro, Braganca Paulista<br />

Joao Batista Teixeira Rocha, Santa Maria<br />

Heitor Rosa, Goiania<br />

Damiao C Moraes Santos, Rio de Janeiro<br />

Ana Crist<strong>in</strong>a Simões e Silva, Belo Horizonte<br />

Eduardo Garcia Vilela, Belo Horizonte<br />

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Brunei Darussalam<br />

Vui Heng Chong, Bandar Seri Begawan<br />

Bulgaria<br />

Zahariy Krastev, S<strong>of</strong>ia<br />

Mihaela Petrova, S<strong>of</strong>ia<br />

Canada<br />

Ala<strong>in</strong> Bitton, Montreal<br />

Michael F Byrne, Vancouver<br />

Kris Chadee, Calgary<br />

Wangxue Chen, Ottawa<br />

Ram Prakash Galwa, Ottawa<br />

Philip H Gordon, Montreal<br />

Waliul Khan, Ontario<br />

Qiang Liu, Saskatoon<br />

John K Marshall, Ontario<br />

Andrew L Mason, Alberta<br />

Kostas Pantopoulos, Quebec<br />

Nathalie Perreault, Sherbrooke<br />

Balj<strong>in</strong>der S<strong>in</strong>gh Salh, Vancouver<br />

Eldon Shaffer, Calgary<br />

Mart<strong>in</strong> Storr, Calgary<br />

P<strong>in</strong>gchang Yang, Hamilton<br />

Eric M Yoshida, Vancouver<br />

Claudia Zw<strong>in</strong>gmann, Montreal<br />

Chile<br />

Marcelo A Beltran, La Serena<br />

Xabier De Aretxabala, Santiago<br />

Silvana Zanlungo, Santiago<br />

Ch<strong>in</strong>a<br />

Hui-Jie Bian, Xi’an<br />

San-Jun Cai, Shanghai<br />

Guang-Wen Cao, Shanghai<br />

Xiao-P<strong>in</strong>g Chen, Wuhan<br />

Chi-H<strong>in</strong> Cho, Hong Kong<br />

Zong-Jie Cui, Beij<strong>in</strong>g<br />

J<strong>in</strong>g-Yuan Fang, Shanghai<br />

De-Liang Fu, Shanghai<br />

Ze-Guang Han, Shanghai<br />

Chun-Yi Hao, Beij<strong>in</strong>g<br />

M<strong>in</strong>g-Liang He, Hong Kong<br />

Ch<strong>in</strong>g-Lung Lai, Hong Kong<br />

Simon Law, Hong Kong<br />

Yuk-Tong Lee, Hong Kong<br />

En-M<strong>in</strong> Li, Shantou<br />

Fei Li, Beij<strong>in</strong>g<br />

Yu-Yuan Li, Guangzhou<br />

Zhao-Shen Li, Shanghai<br />

X<strong>in</strong>g-Hua Lu, Beij<strong>in</strong>g<br />

Yi-M<strong>in</strong> Mao, Shanghai<br />

Q<strong>in</strong> Su, Beij<strong>in</strong>g<br />

Paul Kwong-Hang Tam, Hong Kong<br />

Yuk Him Tam, Hong Kong<br />

Ren-Xiang Tan, Nanj<strong>in</strong>g<br />

Wei-Dong Tong, Chongq<strong>in</strong>g<br />

Eric WC Tse, Hong Kong<br />

II<br />

Fu-Sheng Wang, Beij<strong>in</strong>g<br />

Xiang-Dong Wang, Shanghai<br />

Nathalie Wong, Hong Kong<br />

Just<strong>in</strong> CY Wu, Hong Kong<br />

Wen-Rong Xu, Zhenjiang<br />

An-Gang Yang, Xi’an<br />

Wei-Cheng You, Beij<strong>in</strong>g<br />

Chun-Q<strong>in</strong>g Zhang, J<strong>in</strong>an<br />

Jian-Zhong Zhang, Beij<strong>in</strong>g<br />

Xiao-Peng Zhang, Beij<strong>in</strong>g<br />

Xuan Zhang, Beij<strong>in</strong>g<br />

Colombia<br />

Germán Campuzano-Maya, Medellín<br />

Croatia<br />

Tamara Cacev, Zagreb<br />

Marko Duvnjak, Zagreb<br />

Cuba<br />

Damian C Rodriguez, Havana<br />

Czech<br />

Jan Bures, Hradec Kralove<br />

Milan Jirsa, Praha<br />

Marcela Kopacova, Hradec Kralove<br />

Pavel Trunečka, Prague<br />

Denmark<br />

Leif Percival Andersen, Copenhagen<br />

Asbjørn M Drewes, Aalborg<br />

Morten Frisch, Copenhagen<br />

Jan Mollenhauer, Odense<br />

Morten Hylander Møller, Holte<br />

Søren Rafaelsen, Vejle<br />

Jorgen Rask-Madsen, Skodsborg<br />

Peer Wille-Jørgensen, Copenhagen<br />

Ecuador<br />

Fernando E Sempértegui, Quito<br />

Egypt<br />

Ze<strong>in</strong>ab Nabil Ahmed, Cairo<br />

Husse<strong>in</strong> M Atta, El-M<strong>in</strong>ia<br />

Estonia<br />

Ri<strong>in</strong>a Salupere, Tartu<br />

Tamara Vorobjova, Tartu<br />

F<strong>in</strong>land<br />

Saila Kauhanen, Turku<br />

January 7, 2011

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Thomas Kietzmann, Oulu<br />

Kaija-Leena Kolho, Hels<strong>in</strong>ki<br />

Jukka-Pekka Meckl<strong>in</strong>, Jyvaskyla<br />

M<strong>in</strong>na Nyström, Hels<strong>in</strong>ki<br />

Pauli Antero Puolakka<strong>in</strong>en, Turku<br />

Juhani Sand, Tampere<br />

Lea Veijola, Hels<strong>in</strong>ki<br />

France<br />

Claire Bonithon-Kopp, Dijon<br />

Lionel Bueno, Toulouse<br />

Sab<strong>in</strong>e Colnot, Paris<br />

Cather<strong>in</strong>e Daniel, Lille Cedex<br />

Alexis Desmoulière, Limoges<br />

Thabut Dom<strong>in</strong>ique, Paris<br />

Francoise L Fabiani, Angers<br />

Jean-Luc Faucheron, Grenoble<br />

Jean Paul Galmiche, Nantes cedex<br />

Boris Guiu, Dijon<br />

Paul H<strong>of</strong>man, Nice<br />

Laurent Huwart, Paris<br />

Juan Iovanna, Marseille<br />

Abdel-Majid Khatib, Paris<br />

Philippe Lehours, Bordeaux<br />

Flavio Ma<strong>in</strong>a, Marseille<br />

Patrick Marcell<strong>in</strong>, Paris<br />

Rene Gerolami Santandera, Marseille<br />

Annie Schmid-Alliana, Nice cedex<br />

Ala<strong>in</strong> L Serv<strong>in</strong>, Châtenay-Malabry<br />

Stephane Supiot, Nantes<br />

Baumert F Thomas, Strasbourg<br />

Jean-Jacques Tuech, Rouen<br />

Frank Zerbib, Bordeaux Cedex<br />

Germany<br />

Erw<strong>in</strong> Biecker, Siegburg<br />

Hubert Blum, Freiburg<br />

Thomas Bock, Tueb<strong>in</strong>gen<br />

Dean Bogoevski, Hamburg<br />

Elfriede Bollschweiler, Köln<br />

Jürgen Borlak, Hannover<br />

Christa Buechler, Regensburg<br />

Jürgen Bün<strong>in</strong>g, Lübeck<br />

Elke Cario, Essen<br />

Bruno Christ, Halle/Saale<br />

Christoph F Dietrich, Bad Mergentheim<br />

Ulrich R Fölsch, Kiel<br />

Nikolaus Gassler, Aachen<br />

Markus Gerhard, Munich<br />

Dieter Glebe, Giessen<br />

Ralph Graeser, Freiburg<br />

Axel M Gressner, Aachen<br />

Nils Habbe, Marburg<br />

Thilo Hackert, Heidelberg<br />

Wolfgang Hagmann, Heidelberg<br />

Dirk Haller, Freis<strong>in</strong>g<br />

Philip D Hard, Giessen<br />

Claus Hellerbrand, Regensburg<br />

Klaus R Herrl<strong>in</strong>ger, Stuttgart<br />

Eberhard Hildt, Berl<strong>in</strong><br />

Andrea Hille, Goett<strong>in</strong>gen<br />

Joerg C H<strong>of</strong>fmann, Berl<strong>in</strong><br />

Philipe N Khalil, Munich<br />

Andrej Khandoga, Munich<br />

Jorg Kleeff, Munich<br />

Ingmar Königsra<strong>in</strong>er, Tüb<strong>in</strong>gen<br />

Peter Konturek, Erlangen<br />

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Stefan Kubicka, Hannover<br />

Joachim Labenz, Siegen<br />

Michael L<strong>in</strong>nebacher, Rostock<br />

Jutta Elisabeth Lüttges, Riegelsberg<br />

Peter Malferthe<strong>in</strong>er, Magdeburg<br />

Oliver Mann, Hamburg<br />

Peter N Meier, Hannover<br />

Sab<strong>in</strong>e Mihm, Gött<strong>in</strong>gen<br />

Klaus Mönkemüller, Bottrop<br />

Jonas Mudter, Erlangen<br />

Sebastian Mueller, Heidelberg<br />

Robert Obermaier, Freiburg<br />

Matthias Ocker, Erlangen<br />

Stephan Johannes Ott, Kiel<br />

Gustav Paumgartner, Munich<br />

Christoph Reichel, Bad Brückenau<br />

Markus Reiser, Bochum<br />

Steffen Rickes, Magdeburg<br />

Elke Roeb, Giessen<br />

Christian Rust, Munich<br />

Hans Scherubl, Berl<strong>in</strong><br />

Mart<strong>in</strong> K Schill<strong>in</strong>g, Homburg<br />

Joerg F Schlaak, Essen<br />

Rene Schmidt, Freiburg<br />

Andreas G Schreyer, Regensburg<br />

Karsten Schulmann, Bochum<br />

Henn<strong>in</strong>g Schulze-Bergkamen, Ma<strong>in</strong>z<br />

Manfred V S<strong>in</strong>ger, Mannheim<br />

Jens Standop, Bonn<br />

Jurgen M Ste<strong>in</strong>, Frankfurt<br />

Ulrike S Ste<strong>in</strong>, Berl<strong>in</strong><br />

Wolfgang R Stremmel, Heidelberg<br />

Harald F Teutsch, Ulm<br />

Hans L Tillmann, Leipzig<br />

Christian Trautwe<strong>in</strong>, Aachen<br />

Joerg Trojan, Frankfurt<br />

Arndt Vogel, Hannover<br />

Siegfried Wagner, Deggendorf<br />

Frank Ulrich Weiss, Greifswald<br />

Fritz von Weizsäcker, Berl<strong>in</strong><br />

Thomas Wex, Magdeburg<br />

Stefan Wirth, Wuppertal<br />

Marty Zdichavsky, Tüb<strong>in</strong>gen<br />

Greece<br />

Helen Christopoulou-Aletra, Thessaloniki<br />

T Choli-Papadopoulou, Thessaloniki<br />

Tsianos Epame<strong>in</strong>ondas, Ioann<strong>in</strong>a<br />

Ioannis Kanellos, Thessaloniki<br />

Elias A Kouroumalis, Heraklion<br />

Ioannis E Koutroubakis, Heraklion<br />

Michael Koutsilieris, Athens<br />

Andreas Larentzakis, Athens<br />

Emanuel K Manesis, Athens<br />

Spilios Manolakopoulos, Athens<br />

Konstant<strong>in</strong>os Mimidis, Alexandroupolis<br />

George Papatheodoridis, Athens<br />

Spiros Sgouros, Athens<br />

Evangelos Tsiambas, Ag Paraskevi Attiki<br />

Hungary<br />

György M Buzás, Budapest<br />

László Czakó, Szeged<br />

Gyula Farkas, Szeged<br />

Peter Hegyi, Szeged<br />

Peter L Lakatos, Budapest<br />

III<br />

Yvette Mándi, Szeged<br />

Zoltan Rakonczay, Szeged<br />

Ferenc Sipos, Budapest<br />

Zsuzsa Szondy, Debrecen<br />

Gabor Veres, Budapest<br />

India<br />

Philip Abraham, Mumbai<br />

V<strong>in</strong>eet Ahuja, New Delhi<br />

Giriraj Ratan Chandak, Hyderabad<br />

Dev<strong>in</strong>der Kumar Dhawan, Chandigarh<br />

Radha K Dhiman, Chandigarh<br />

Pankaj Garg, Panchkula<br />

Pramod Kumar Garg, New Delhi<br />

Debidas Ghosh, Midnpore<br />

Uday C Ghoshal, Lucknow<br />

Bhupendra Kumar Ja<strong>in</strong>, Delhi<br />

Ashok Kumar, Lucknow<br />

Bikash Medhi, Chandigarh<br />

Sri P Misra, Allahabad<br />

Gopal Nath, Varanasi<br />

Samiran Nundy, New Delhi<br />

Jagannath Palepu, Mumbai<br />

Vandana Panda, Mumbai<br />

Benjam<strong>in</strong> Perakath, Tamil Nadu<br />

Ramesh Roop Rai, Jaipur<br />

Nageshwar D Reddy, Hyderabad<br />

Barjesh Chander Sharma, New Delhi<br />

Virendra S<strong>in</strong>gh, Chandigarh<br />

Rupjyoti Talukdar, Guwahati<br />

Rakesh Kumar Tandon, New Delhi<br />

Jai Dev Wig, Chandigarh<br />

Iran<br />

Mohammad Abdollahi, Tehran<br />

Peyman Adibi, Isfahan<br />

Seyed-Moayed Alavian, Tehran<br />

Seyed Mohsen Dehghani, Shiraz<br />

Reza Malekzadeh, Tehran<br />

Alireza Mani, Tehran<br />

Ireland<br />

Billy Bourke, Dubl<strong>in</strong><br />

Ted D<strong>in</strong>an, Cork<br />

Cather<strong>in</strong>e Greene, Dubl<strong>in</strong><br />

Ross McManus, Dubl<strong>in</strong><br />

Anthony P Moran, Galway<br />

Marion Rowland, Dubl<strong>in</strong><br />

Israel<br />

Simon Bar-Meir, Hashomer<br />

Alexander Becker, Afula<br />

Abraham R Eliakim, Haifa<br />

Sigal Fishman, Tel Aviv<br />

Boris Kirshte<strong>in</strong>, Beer Sheva<br />

Eli Magen, Ashdod<br />

Menachem Moshkowitz, Tel-Aviv<br />

Assy Nimer, Safed<br />

Shmuel Odes, Beer Sheva<br />

Mark P<strong>in</strong>es, Bet Dagan<br />

Ron Shaoul, Haifa<br />

Ami D Sperber, Beer-Sheva<br />

January 7, 2011

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Italy<br />

Donato F Altomare, Bari<br />

Piero Amodio, Padova<br />

Angelo Andriulli, San Giovanni Rotondo<br />

Paolo Angeli, Padova<br />

Bruno Annibale, Rome<br />

Paolo Aurello, Rome<br />

Salvatore Auricchio, Naples<br />

Antonio Basoli, Rome<br />

Claudio Bassi, Verona<br />

Gabrio Bassotti, Perugia<br />

Mauro Bernardi, Bologna<br />

Alberto Biondi, Rome<br />

Luigi Bonav<strong>in</strong>a, Milano<br />

Guglielmo Borgia, Naples<br />

Roberto Berni Canani, Naples<br />

Maria Gabriella Caruso, Bari<br />

Fausto Catena, Bologna<br />

Giuseppe Chiarioni, Valeggio<br />

Michele Cicala, Rome<br />

Dario Conte, Milano<br />

Francesco Costa, Pisa<br />

Antonio Craxì, Palermo<br />

Salvatore Cucchiara, Rome<br />

Giuseppe Currò, Mess<strong>in</strong>a<br />

Mario M D’Elios, Florence<br />

Mirko D’On<strong>of</strong>rio, Verona<br />

Silvio Danese, Milano<br />

Roberto de Franchis, Milano<br />

Paola De Nardi, Milan<br />

Giovanni D De Palma, Naples<br />

Giuliana Decorti, Trieste<br />

Gianlorenzo Dionigi, Varese<br />

Massimo Falconi, Verona<br />

Silvia Fargion, Milan<br />

Giammarco Fava, Ancona<br />

Francesco Feo, Sassari<br />

Alessandra Ferl<strong>in</strong>i, Ferrara<br />

Alessandro Ferrero, Tor<strong>in</strong>o<br />

Mirella Fraquelli, Milan<br />

Luca Frulloni, Verona<br />

Giovanni B Gaeta, Napoli<br />

Antonio Gasbarr<strong>in</strong>i, Rome<br />

Edoardo G Giann<strong>in</strong>i, Genoa<br />

Alessandro Granito, Bologna<br />

Fabio Grizzi, Milan<br />

Salvatore Gruttadauria, Palermo<br />

Pietro Invernizzi, Milan<br />

Achille Iolascon, Naples<br />

Angelo A Izzo, Naples<br />

Ezio Laconi, Cagliari<br />

Giovanni Latella, L’Aquila<br />

Massimo Levrero, Rome<br />

Francesco Luzza, Catanzaro<br />

Lucia Malaguarnera, Catania<br />

Francesco Manguso, Napoli<br />

Pier Mannuccio Mannucci, Milan<br />

Giancarlo Mansueto, Verona<br />

Giulio Marches<strong>in</strong>i, Bologna<br />

Mara Massimi, Coppito<br />

Giovanni Milito, Rome<br />

Giuseppe Montalto, Palermo<br />

Giovanni Monteleone, Rome<br />

Luca Morelli, Trento<br />

Giovanni Musso, Tor<strong>in</strong>o<br />

Mario Nano, Tor<strong>in</strong>o<br />

Gerardo Nardone, Napoli<br />

Riccardo Nascimbeni, Brescia<br />

Valerio Nobili, Rome<br />

Fabio Pace, Milan<br />

Nadia Pepar<strong>in</strong>i, Rome<br />

WJG|www.wjgnet.com<br />

Marcello Persico, Naples<br />

Mario Pescatori, Rome<br />

Raffaele Pezzilli, Bologna<br />

Alberto Piperno, Monza<br />

Anna C Piscaglia, Rome<br />

Piero Port<strong>in</strong>casa, Bari<br />

Michele Reni, Milan<br />

Vittorio Ricci, Pavia<br />

Oliviero Riggio, Rome<br />

Mario Rizzetto, Tor<strong>in</strong>o<br />

Ballar<strong>in</strong> Roberto, Modena<br />

Gerardo Rosati, Potenza<br />

Franco Roviello, Siena<br />

Cesare Ruffolo, Treviso<br />

Massimo Rugge, Padova<br />

Marco Scarpa, Padova<br />

C armelo Scarpignato, Parma<br />

Giuseppe Sica, Rome<br />

Marco Silano, Rome<br />

Pierpaolo Sileri, Rome<br />

V<strong>in</strong>cenzo Stanghell<strong>in</strong>i, Bologna<br />

Fiorucci Stefano, Perugia<br />

Giovanni Tarant<strong>in</strong>o, Naples<br />

Alberto Tommas<strong>in</strong>i, Trieste<br />

Guido Torzilli, Rozzano Milan<br />

Cesare Tosetti, Porretta Terme<br />

Antonello Trecca, Rome<br />

V<strong>in</strong>cenzo Villanacci, Brescia<br />

Lucia Ricci Vitiani, Rome<br />

Marco Vivarelli, Bologna<br />

Japan<br />

Kyoichi Adachi, Izumo<br />

Yasushi Adachi, Sapporo<br />

Takafumi Ando, Nagoya<br />

Akira Andoh, Otsu<br />

Masahiro Arai, Tokyo<br />

Hitoshi Asakura, Tokyo<br />

Kazuo Chijiiwa, Miyazaki<br />

Yuichiro Eguchi, Saga<br />

Itaru Endo, Yokohama<br />

Munechika Enjoji, Fukuoka<br />

Yasuhiro Fuj<strong>in</strong>o, Akashi<br />

Mitsuhiro Fujishiro, Tokyo<br />

Kouhei Fukushima, Sendai<br />

Masanori Hatakeyama, Tokyo<br />

Keiji Hirata, Kitakyushu<br />

Toru Hiyama, Higashihiroshima<br />

Masahiro Iizuka, Akita<br />

Susumu Ikehara, Osaka<br />

Kenichi Ikejima, Bunkyo-ku<br />

Yutaka Inagaki, Kanagawa<br />

Hiromi Ishibashi, Nagasaki<br />

Shunji Ishihara, Izumo<br />

Toru Ishikawa, Niigata<br />

Toshiyuki Ishiwata, Tokyo<br />

Hajime Isomoto, Nagasaki<br />

Yoshiaki Iwasaki, Okayama<br />

Satoru Kakizaki, Gunma<br />

Terumi Kamisawa, Tokyo<br />

Mototsugu Kato, Sapporo<br />

Naoya Kato, Tokyo<br />

Takumi Kawaguchi, Kurume<br />

Yohei Kida, Ka<strong>in</strong>an<br />

Shogo Kikuchi, Aichi<br />

Tsuneo Kitamura, Chiba<br />

Takashi Kobayashi, Tokyo<br />

Yasuhiro Koga, Isehara<br />

Takashi Kojima, Sapporo<br />

Norihiro Kokudo, Tokyo<br />

Masatoshi Kudo, Osaka<br />

Sh<strong>in</strong> Maeda, Tokyo<br />

IV<br />

Satoshi Mamori, Hyogo<br />

Atsushi Masamune, Sendai<br />

Yasushi Matsuzaki, Tsukuba<br />

Kenji Miki, Tokyo<br />

Toshihiro Mitaka, Sapporo<br />

Hiroto Miwa, Hyogo<br />

Kotaro Miyake, Tokushima<br />

Manabu Morimoto, Yokohama<br />

Yoshiharu Motoo, Kanazawa<br />

Yoshiaki Murakami, Hiroshima<br />

Yoshiki Murakami, Kyoto<br />

Kunihiko Murase, Tusima<br />

Akihito Nagahara, Tokyo<br />

Yuji Naito, Kyoto<br />

Atsushi Nakajima, Yokohama<br />

Hisato Nakajima, Tokyo<br />

Hiroki Nakamura, Yamaguchi<br />

Shotaro Nakamura, Fukuoka<br />

Akimasa Nakao, Nagogya<br />

Shuhei Nishiguchi, Hyogo<br />

Mikio Nishioka, Niihama<br />

Keiji Ogura, Tokyo<br />

Susumu Ohmada, Maebashi<br />

Hirohide Ohnishi, Akita<br />

Kenji Okajima, Nagoya<br />

Kazuichi Okazaki, Osaka<br />

Morikazu Onji, Ehime<br />

Satoshi Osawa, Hamamatsu<br />

Hidetsugu Saito, Tokyo<br />

Yutaka Saito, Tokyo<br />

Naoaki Sakata, Sendai<br />

Yasushi Sano, Chiba<br />

Tokihiko Sawada, Tochigi<br />

Tomohiko Shimatan, Hiroshima<br />

Yukihiro Shimizu, Kyoto<br />

Sh<strong>in</strong>ji Shimoda, Fukuoka<br />

Yoshio Shirai, Niigata<br />

Masayuki Sho, Nara<br />

Shoichiro Sumi, Kyoto<br />

Hidekazu Suzuki, Tokyo<br />

Masahiro Tajika, Nagoya<br />

Yoshihisa Takahashi, Tokyo<br />

Tosh<strong>in</strong>ari Takamura, Kanazawa<br />

Hiroaki Takeuchi, Kochi<br />

Yoshitaka Takuma, Okayama<br />

Akihiro Tamori, Osaka<br />

Atsushi Tanaka, Tokyo<br />

Sh<strong>in</strong>ji Tanaka, Hiroshima<br />

Satoshi Tanno, Hokkaido<br />

Sh<strong>in</strong>ji Togo, Yokohama<br />

Hitoshi Tsuda, Tokyo<br />

Hiroyuki Uehara, Osaka<br />

Masahito Uemura, Kashihara<br />

Yoshiyuki Ueno, Sendai<br />

Mitsuyoshi Urashima, Tokyo<br />

Takuya Watanabe, Niigata<br />

Satoshi Yamagiwa, Niigata<br />

Taketo Yamaguchi, Chiba<br />

Mitsunori Yamakawa, Yamagata<br />

Takayuki Yamamoto, Yokkaichi<br />

Yutaka Yata, Maebashi<br />

Hiroshi Yoshida, Tokyo<br />

Norimasa Yoshida, Kyoto<br />

Yuichi Yoshida, Osaka<br />

Kentaro Yoshika, Toyoake<br />

Hitoshi Yoshiji, Nara<br />

Katsutoshi Yoshizato, Higashihiroshima<br />

Tomoharu Yoshizumi, Fukuoka<br />

Jordan<br />

Ismail Matalka, Irbid<br />

January 7, 2011

---

Islam Khan, Safat<br />

Kuwait<br />

Lebanon<br />

Bassam N Abboud, Beirut<br />

Ala I Sharara, Beirut<br />

Rita Slim, Beirut<br />

Lithuania<br />

Giedrius Barauskas, Kaunas<br />

Limas Kupc<strong>in</strong>skas, Kaunas<br />

Malaysia<br />

Andrew Seng Boon Chua, Ipoh<br />

Mexico<br />

Richard A Awad, Mexico<br />

Aldo Torre Delgadillo, Mexico<br />

Diego Garcia-Compean, Monterrey<br />

Paul<strong>in</strong>o M Hernández Magro, Celaya<br />

Miguel Angel Mercado, Distrito Federal<br />

Arturo Panduro, Jalisco<br />

Omar Vergara-Fernandez, Tlalpan<br />

Saúl Villa-Trevio, Mexico<br />

Moldova<br />

Igor Mish<strong>in</strong>, Kish<strong>in</strong>ev<br />

Netherlands<br />

Ulrich Beuers, Amsterdam<br />

Lee Bouwman, Leiden<br />

Albert J Bredenoord, Nieuwege<strong>in</strong><br />

Lodewijk AA Brosens, Utrecht<br />

J Bart A Crusius, Amsterdam<br />

Wouter de Herder, Rotterdam<br />

Pieter JF de Jonge, Rotterdam<br />

Robert J de Knegt, Rotterdam<br />

Wendy W Johanna de Leng, Utrecht<br />

Annemarie de Vries, Rotterdam<br />

James CH Hardwick, Leiden<br />

Frank Hoentjen, Haarlem<br />

Misha Luyer, Sittard<br />

Jeroen Maljaars, Maastricht<br />

Gerrit A Meijer, Amsterdam<br />

Servaas Morré, Amsterdam<br />

Chris JJ Mulder, Amsterdam<br />

John Plukker, Gron<strong>in</strong>gen<br />

Albert Frederik Pull ter Gunne, Tilburg<br />

Paul E Sijens, Gron<strong>in</strong>gen<br />

BW Marcel Spanier, Arnhem<br />

Shiri Sverdlov, Maastricht<br />

Maarten Tushuizen, Amsterdam<br />

Jant<strong>in</strong>e van Baal, Heidelberglaan<br />

Astrid van der Velde, The Hague<br />

Karel van Erpecum, Utrecht<br />

Loes van Keimpema, Nijmegen<br />

WJG|www.wjgnet.com<br />

Robert Christiaan Verdonk, Gron<strong>in</strong>gen<br />

Erw<strong>in</strong> G Zoetendal, Wagen<strong>in</strong>gen<br />

New Zealand<br />

Andrew S Day, Christchurch<br />

Norway<br />

Olav Dalgard, Oslo<br />

Trond Peder Flaten, Trondheim<br />

Reidar Fossmark, Trondheim<br />

Rasmus Goll, Tromso<br />

Ole Høie, Arendal<br />

Asle W Medhus, Oslo<br />

Espen Melum, Oslo<br />

Tr<strong>in</strong>e Olsen, Tromso<br />

Eyv<strong>in</strong>d J Paulssen, Tromso<br />

Jon Arne Søreide, Stavanger<br />

Kjetil Soreide, Stavanger<br />

Pakistan<br />

Shahab Abid, Karachi<br />

Syed MW Jafri, Karachi<br />

Poland<br />

Marek Bebenek, Wroclaw<br />

Tomasz Brzozowski, Cracow<br />

Hal<strong>in</strong>a Cichoż-Lach, Lubl<strong>in</strong><br />

Andrzej Dabrowski, Bialystok<br />

Hanna Gregorek, Warsaw<br />

Marek Hartleb, Katowice<br />

Beata Jolanta Jablońska, Katowice<br />

Stanislaw J Konturek, Krakow<br />

Jan Kulig, Krakow<br />

Dariusz M Lebensztejn, Bialystok<br />

Julian Swierczynski, Gdansk<br />

Portugal<br />

Raquel Almeida, Porto<br />

Ana Isabel Lopes, Lisboa Codex<br />

Ricardo Marcos, Porto<br />

Guida Portela-Gomes, Estoril<br />

Romania<br />

Dan L Dumitrascu, Cluj<br />

Adrian Saftoiu, Craiova<br />

Andrada Seicean, Cluj-Napoca<br />

Russia<br />

Vasiliy I Reshetnyak, Moscow<br />

Saudi Arabia<br />

Ibrahim A Al M<strong>of</strong>leh, Riyadh<br />

Abdul-Wahed Meshikhes, Qatif<br />

Faisal Sanai, Riyadh<br />

V<br />

Serbia<br />

Tamara M Alempijevic, Belgrade<br />

Dusan M Jovanovic, Sremska Kamenica<br />

Zoran Krivokapic, Belgrade<br />

S<strong>in</strong>gapore<br />

Madhav Bhatia, S<strong>in</strong>gapore<br />

Kong Weng Eu, S<strong>in</strong>gapore<br />

Brian Kim Poh Goh, S<strong>in</strong>gapore<br />

Khek-Yu Ho, S<strong>in</strong>gapore<br />

Kok Sun Ho, S<strong>in</strong>gapore<br />

Fock Kwong M<strong>in</strong>g, S<strong>in</strong>gapore<br />

London Lucien Ooi, S<strong>in</strong>gapore<br />

Nagarajan Perumal, S<strong>in</strong>gapore<br />

Francis Seow-Choen, S<strong>in</strong>gapore<br />

South Africa<br />

Rosemary Joyce Burnett, Pretoria<br />

Michael Kew, Cape Town<br />

South Korea<br />

Sang Hoon Ahn, Seoul<br />

Sung-Gil Chi, Seoul<br />

Myung-Gyu Choi, Seoul<br />

Hoon Jai Chun, Seoul<br />

Yeun-Jun Chung, Seoul<br />

Young-Hwa Chung, Seoul<br />

Kim Donghee, Seoul<br />

Ki-Baik Hahm, Incheon<br />

Sun Pyo Hong, Geonggi-do<br />

Seong Gyu Hwang, Seongnam<br />

Hong Joo Kim, Seoul<br />

Jae J Kim, Seoul<br />

J<strong>in</strong>-Hong Kim, Suwon<br />

Nayoung Kim, Seongnam-si<br />

Sang Geon Kim, Seoul<br />

Seon Hahn Kim, Seoul<br />

Sung Kim, Seoul<br />

Won Ho Kim, Seoul<br />

Jeong M<strong>in</strong> Lee, Seoul<br />

Kyu Taek Lee, Seoul<br />

Sang Kil Lee, Seoul<br />

Sang Yeoup Lee, Gyeongsangnam-do<br />

Yong Chan Lee, Seoul<br />

Eun-Yi Moon, Seoul<br />

Hyoung-Chul Oh, Seoul<br />

Seung Woon Paik, Seoul<br />

Joong-Won Park, Goyang<br />

Ji Kon Ryu, Seoul<br />

Si Young Song, Seoul<br />

Marie Yeo, Suwon<br />

Byung Chul Yoo, Seoul<br />

Dae-Yeul Yu, Daejeon<br />

Spa<strong>in</strong><br />

Maria-Angeles Aller, Madrid<br />

Raul J Andrade, Málaga<br />

Luis Aparisi, Valencia<br />

Gloria González Asegu<strong>in</strong>olaza, Navarra<br />

Matias A Avila, Pamplona<br />

January 7, 2011

---

Fernando Azpiroz, Barcelona<br />

Ramon Bataller, Barcelona<br />

Belén Beltrán, Valencia<br />

Adolfo Benages, Valencia<br />

Josep M Bordas, Barcelona<br />

Lisardo Boscá, Madrid<br />

Luis Bujanda, San Sebastián<br />

Juli Busquets, Barcelona<br />

Matilde Bustos, Pamplona<br />

José Julián calvo Andrés, Salamanca<br />

Andres Cardenas, Barcelona<br />

Antoni Castells, Barcelona<br />

Fernando J Corrales, Pamplona<br />

J E Domínguez-Muñoz, Santiago de Compostela<br />

Juan Carlos Laguna Egea, Barcelona<br />

Isabel Fabregat, Barcelona<br />

Antoni Farré, Barcelona<br />

Vicente Felipo, Valencia<br />

Laureano Fernández-Cruz, Barcelona<br />

Luis Grande, Barcelona<br />

Angel Lanas, Zaragoza<br />

Juan-Ramón Larrubia, Guadalajara<br />

María IT López, Jaén<br />

Juan Macías, Seville<br />

Javier Mart<strong>in</strong>, Granada<br />

José Manuel Mart<strong>in</strong>-Villa, Madrid<br />

Julio Mayol, Madrid<br />

Mireia Miquel, Sabadell<br />

Albert Parés, Barcelona<br />

Jesús M Prieto, Pamplona<br />

Pedro L Majano Rodriguez, Madrid<br />

Joan Roselló-Catafau, Barcelona<br />

Eva Vaquero, Barcelona<br />

Sweden<br />

Lars Erik Agréus, Stockholm<br />

Mats Andersson, Stockholm<br />

Roland Andersson, Lund<br />

Mauro D’Amato, Hudd<strong>in</strong>ge<br />

Evangelos Kalaitzakis, Gothenburg<br />

Greger L<strong>in</strong>dberg, Stockholm<br />

Annika L<strong>in</strong>dblom, Stockholm<br />

Sara L<strong>in</strong>dén, Göteborg<br />

Hanns-Ulrich Marschall, Stockholm<br />

Pär Erik Myrelid, L<strong>in</strong>köp<strong>in</strong>g<br />

Åke Nilsson, Lund<br />

Helena Nordenstedt, Stockholm<br />

Kjell Öberg, Uppsala<br />

Lars A Pahlman, Uppsala<br />

Stefan G Pierzynowski, Lund<br />

Sara Regnér, Malmö<br />

Bobby T<strong>in</strong>gstedt, Lund<br />

Zongli Zheng, Stockholm<br />

Switzerland<br />

Pascal Bucher, Geneva<br />

Michelangelo Foti, Geneva<br />

Jean L Frossard, Geneva<br />

Andreas Geier, Zürich<br />

Pascal Gervaz, Geneva<br />

Gerd A Kullak-Ublick, Zürich<br />

Fabrizio Montecucco, Geneva<br />

Paul M Schneider, Zürich<br />

Felix Stickel, Berne<br />

Bruno Stieger, Zürich<br />

Inti Zlobec, Basel<br />

WJG|www.wjgnet.com<br />

Tr<strong>in</strong>idad and Tobago<br />

Shivananda Nayak, Mount Hope<br />

Turkey<br />

S<strong>in</strong>an Akay, Tekirdag<br />

Met<strong>in</strong> Basaranoglu, Istanbul<br />

Yusuf Bayraktar, Ankara<br />

A Mithat Bozdayi, Ankara<br />

Hayrullah Derici, Balıkesir<br />

Eren Ersoy, Ankara<br />

Mukaddes Esrefoglu, Malatya<br />

Can Goen, Kutahya<br />

Sel<strong>in</strong> Kapan, Istanbul<br />

Ayd<strong>in</strong> Karabacakoglu, Konya<br />

Cuneyt Kayaalp, Malatya<br />

Kemal Kismet, Ankara<br />

Seyfett<strong>in</strong> Köklü, Ankara<br />

Mehmet Refik Mas, Etlik-Ankara<br />

Osman C Ozdogan, Istanbul<br />

Bülent Salman, Ankara<br />

Orhan Sezg<strong>in</strong>, Mers<strong>in</strong><br />

Ilker Tasci, Ankara<br />

Müge Tecder-Ünal, Ankara<br />

Ahmet Tek<strong>in</strong>, Mers<strong>in</strong><br />

Mesut Tez, Ankara<br />

Ekmel Tezel, Ankara<br />

Özlem Yilmaz, Izmir<br />

United Arab Emirates<br />

Fikri M Abu-Zidan, Al-A<strong>in</strong><br />

Sherif M Karam, Al-A<strong>in</strong><br />

United K<strong>in</strong>gdom<br />

Simon Afford, Birm<strong>in</strong>gham<br />

Navneet K Ahluwalia, Stockport<br />

Mohamed H Ahmed, Southampton<br />

Basil Ammori, Salford<br />

Lesley A Anderson, Belfast<br />

Ch<strong>in</strong> Wee Ang, Liverpool<br />

Yeng S Ang, Wigan<br />

Anthony TR Axon, Leeds<br />

Kathleen B Bamford, London<br />

Jim D Bell, London<br />

John Beynon, Swansea<br />

Chris Briggs, Sheffield<br />

Ge<strong>of</strong>frey Burnstock, London<br />

Alastair D Burt, Newcastle<br />

Jeff Butterworth, Shrewsbury<br />

Jeremy FL Cobbold, London<br />

Jean E Crabtree, Leeds<br />

Tatjana Crnogorac-Jurcevic, London<br />

William Dickey, Londonderry<br />

Sunil Dolwani, Cardiff<br />

Emad M El-Omar, Aberdeen<br />

A M El-Tawil, Birm<strong>in</strong>gham<br />

Charles B Ferguson, Belfast<br />

Andrew Fowell, Southampton<br />

Piers Gatenby, London<br />

Daniel R Gaya, Ed<strong>in</strong>burgh<br />

Anil George, London<br />

Rob Glynne-Jones, Northwood<br />

Jason CB Goh, Birm<strong>in</strong>gham<br />

Gianpiero Gravante, Leicester<br />

VI<br />

Brian Green, Belfast<br />

William Greenhalf, Liverpool<br />

Indra N Guha, Nott<strong>in</strong>gham<br />

Stefan G Hübscher, Birm<strong>in</strong>gham<br />

Rob<strong>in</strong> Hughes, London<br />

Pali Hung<strong>in</strong>, Stockton<br />

Nawfal Husse<strong>in</strong>, Nott<strong>in</strong>gham<br />

Clement W Imrie, Glasgow<br />

Janusz AZ Jankowski, Oxford<br />

Sharad Karandikar, Birm<strong>in</strong>gham<br />

Peter Karayiannis, London<br />

Shahid A Khan, London<br />

Patricia F Lalor, Birm<strong>in</strong>gham<br />

John S Leeds, Sheffield<br />

Ian L<strong>in</strong>dsey, Oxford<br />

Hong-Xiang Liu, Cambridge<br />

Dileep N Lobo, Nott<strong>in</strong>gham<br />

Graham MacKay, Glasgow<br />

Mark Edward McAl<strong>in</strong>don, Sheffield<br />

Anne McCune, Bristol<br />

Donald Campbell McMillan, Glasgow<br />

Giorg<strong>in</strong>a Mieli-Vergani, London<br />

Jamie Murphy, London<br />

Guy Fairbairn Nash, Poole<br />

James Neuberger, Birm<strong>in</strong>gham<br />

Patrick O’Dwyer, Glasgow<br />

Christos Paraskeva, Bristol<br />

Richard Parker, North Staffordshire<br />

Thamara Perera, Birm<strong>in</strong>gham<br />

Kondragunta Rajendra Prasad, Leeds<br />

D Mark Pritchard, Liverpool<br />

Alberto Quaglia, London<br />

Akhilesh B Reddy, Cambridge<br />

Kev<strong>in</strong> Robertson, Glasgow<br />

Sanchoy Sarkar, Liverpool<br />

John B Sch<strong>of</strong>ield, Kent<br />

Marco Senzolo, Padova<br />

Venkatesh Shanmugam, Derby<br />

Paul Sharp, London<br />

Chew Thean Soon, Manchester<br />

Arav<strong>in</strong>d Suppiah, East Yorkshire<br />

Noriko Suzuki, Middlesex<br />

Simon D Taylor-Rob<strong>in</strong>son, London<br />

Frank I Tovey, London<br />

A McCulloch Veitch, Wolverhampton<br />

Vamsi R Velchuru, Lowest<strong>of</strong>t<br />

Sumita Verma, Brighton<br />

Cather<strong>in</strong>e Walter, Cheltenham<br />

Julian RF Walters, London<br />

Roger Williams, London<br />

United States<br />

Kareem M Abu-Elmagd, Pittsburgh<br />

Sami R Achem, Florida<br />

Golo Ahlenstiel, Bethesda<br />

Bhup<strong>in</strong>der S Anand, Houston<br />

M Ananthanarayanan, New York<br />

Balamurugan N Appakalal, M<strong>in</strong>neapolis<br />

Dimitrios V Avger<strong>in</strong>os, New York<br />

Shashi Bala, Worcester<br />

Anthony J Bauer, Pittsburgh<br />

Kev<strong>in</strong> E Behrns, Ga<strong>in</strong>esville<br />

Roberto Bergamaschi, New York<br />

Henry J B<strong>in</strong>der, New Haven<br />

Edmund J B<strong>in</strong>i, New York<br />

Wojciech Blonski, Philadelphia<br />

Mark Bloomston, Columbus<br />

Edward L Bradley III, Sarasota<br />

Carla W Brady, Durham<br />

January 7, 2011

---

David A Brenner, San Diego<br />

Adeel A Butt, Pittsburgh<br />

Shi-Y<strong>in</strong>g Cai, New Haven<br />

Just<strong>in</strong> MM Cates, Nashville<br />

Eugene P Ceppa, Durham<br />

Jianyuan Chai, Long Beach<br />

Ronald S Chamberla<strong>in</strong>, Liv<strong>in</strong>gston<br />

Fei Chen, Morgantown<br />

Xian-M<strong>in</strong>g Chen, Omaha<br />

Ramsey Chi-man Cheung, Palo Alto<br />

Denesh Chitkara, East Brunswick<br />

Clifford S Cho, Madison<br />

Parimal Chowdhury, Arkansas<br />

John David Christe<strong>in</strong>, Birm<strong>in</strong>gham<br />

Thomas Clancy, Boston<br />

Ana J Coito, Los Angeles<br />

Ricardo Alberto Cruciani, New York<br />

Joseph J Cullen, Iowa City<br />

Mark J Czaja, New York<br />

Mariana D Dabeva, Bronx<br />

Jessica A Davila, Houston<br />

Conor P Delaney, Cleveland<br />

Laurie DeLeve, Los Angeles<br />

Anthony J Demetris, Pittsburgh<br />

Sharon DeMorrow, Temple<br />

Bijan Eghtesad, Cleveland<br />

Yoram Elitsur, Hunt<strong>in</strong>gton<br />

Mohamad A Eloubeidi, Alabama<br />

Wael El-Rifai, Nashville<br />

Sukru H Emre, New Haven<br />

Giamila Fantuzzi, Chicago<br />

Ashkan Farhadi, Irv<strong>in</strong>e<br />

Ronnie Fass, Tucson<br />

Martín E Fernández-Zapico, Rochester<br />

Alessandro Fichera, Chicago<br />

Josef E Fischer, Boston<br />

Piero Marco Fisichella, Maywood<br />

Fritz Francois, New York<br />

Glenn T Furuta, Aurora<br />

T Clark Gambl<strong>in</strong>, Pittsburgh<br />

Henn<strong>in</strong>g Gerke, Iowa City<br />

Jean-Francois Geschw<strong>in</strong>d, Baltimore<br />

R Mark Ghobrial, Texas<br />

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Chang Kim, West Lafayette<br />

Dean Y Kim, Detroit<br />

Miran Kim, Providence<br />

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Santhi Swaroop Vege, Rochester<br />

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Steven D Wexner, Weston<br />

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Guang-Y<strong>in</strong> Xu, Galveston<br />

Fang Yan, Nashville<br />

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Liq<strong>in</strong>g Yu, W<strong>in</strong>ston-Salem<br />

Run Yu, Los Angeles<br />

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Michael E Zenilman, New York<br />

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L<strong>in</strong> Zhang, Pittsburgh<br />

Mart<strong>in</strong> D Ziel<strong>in</strong>ski, Rochester<br />

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January 7, 2011

---

S<br />

Contents<br />

EDITORIAL<br />

TOPIC HIGHLIGHT<br />

GUIDELINES FOR<br />

CLINICAL PRACTICE<br />

REVIEW<br />

ORIGINAL ARTICLE<br />

WJG|www.wjgnet.com<br />

1655 Natural orifice translumenal endoscopic surgery: Progress <strong>in</strong> humans s<strong>in</strong>ce<br />

white paper<br />

Santos BF, Hungness ES<br />

1666 <strong>Isolated</strong> <strong>lymphoid</strong> <strong>follicles</strong> <strong>in</strong> <strong>colon</strong>: Switch po<strong>in</strong>ts between <strong>in</strong>flammation and<br />

colorectal cancer?<br />

Sipos F, Műzes G<br />

1674 Patterns <strong>of</strong> local recurrence <strong>in</strong> rectal cancer after a multidiscipl<strong>in</strong>ary approach<br />

Enríquez-Navascués JM, Borda N, Lizerazu A, Placer C, Elosegui JL, Ciria JP, Lacasta A,<br />

Bujanda L<br />

1685 Therapeutic options for <strong>in</strong>termediate-advanced hepatocellular carc<strong>in</strong>oma<br />

Zhang ZM, Guo JX, Zhang ZC, Jiang N, Zhang ZY, Pan LJ<br />

1690 How to assess the severity <strong>of</strong> atrophic gastritis<br />

Dai YC, Tang ZP, Zhang YL<br />

1694 Legalon-SIL downregulates HCV core and NS5A <strong>in</strong> human hepatocytes<br />

express<strong>in</strong>g full-length HCV<br />

Mehrab-Mohseni M, Sendi H, Steuerwald N, Ghosh S, Schrum LW, Bonkovsky HL<br />

1701 Endoscopic submucosal dissection for premalignant lesions and non<strong>in</strong>vasive<br />

early gastro<strong>in</strong>test<strong>in</strong>al cancers<br />

Hulagu S, Senturk O, Aygun C, Kocaman O, Celebi A, Konduk T, Koc D, Sir<strong>in</strong> G,<br />

Korkmaz U, Duman AE, Bozkurt N, D<strong>in</strong>dar G, Attila T, Gurbuz Y, Tarc<strong>in</strong> O, Kalayci C<br />

1710 Discovery and validation <strong>of</strong> prognostic markers <strong>in</strong> gastric cancer by<br />

genome-wide expression pr<strong>of</strong>il<strong>in</strong>g<br />

Zhang YZ, Zhang LH, Gao Y, Li CH, Jia SQ, Liu N, Cheng F, Niu DY, Cho WCS, Ji JF,<br />

Zeng CQ<br />

1718 Differential expression <strong>of</strong> Bcl-2 and Bax dur<strong>in</strong>g gastric ischemia-reperfusion<br />

<strong>of</strong> rats<br />

Weekly Volume 17 Number 13 April 7, 2011<br />

Qiao WL, Wang GM, Shi Y, Wu JX, Qi YJ, Zhang JF, Sun H, Yan CD<br />

April 7, 2011|Volume 17| ssue 13|

---

Contents<br />

BRIEF ARTICLE<br />

CASE REPORT<br />

WJG|www.wjgnet.com<br />

<strong>World</strong> <strong>Journal</strong> <strong>of</strong> <strong>Gastroenterology</strong><br />

Volume 17 Number 13 April 7, 2011<br />

1725 Intrahepatic natural killer T cell populations are <strong>in</strong>creased <strong>in</strong> human hepatic<br />

steatosis<br />

Adler M, Taylor S, Okebugwu K, Yee H, Field<strong>in</strong>g C, Field<strong>in</strong>g G, Poles M<br />

1732 Gastrotomy closure with a new tissue anchor<strong>in</strong>g device: A porc<strong>in</strong>e survival<br />

study<br />

Guarner-Argente C, Córdova H, Martínez-Pallí G, Navarro-Ripoll R, Rodríguez-d'Jesús A,<br />

Rodríguez de Miguel C, Beltrán M, Fernández-Esparrach G<br />

1739 MR-arterioportography: A new technical approach for detection <strong>of</strong> liver<br />

lesions<br />

Rennert J, Jung EM, Schreyer AG, H<strong>of</strong>fstetter P, Heiss P, Feuerbach S, Zorger N<br />

1746 Carbachol promotes gastro<strong>in</strong>test<strong>in</strong>al function dur<strong>in</strong>g oral resuscitation <strong>of</strong> burn<br />

shock<br />

Hu S, Che JW, Tian YJ, Sheng ZY<br />

1753 Gastroesophageal reflux <strong>in</strong> cirrhotic patients without esophageal varices<br />

Zhang J, Cui PL, Lv D, Yao SW, Xu YQ, Yang ZX<br />

1759 Association between polymorphism rs6983267 and gastric cancer risk <strong>in</strong><br />

Ch<strong>in</strong>ese population<br />

Guo Y, Fang J, Liu Y, Sheng HH, Zhang XY, Chai HN, J<strong>in</strong> W, Zhang KH, Yang CQ, Gao HJ<br />

1766 EUS for choos<strong>in</strong>g best endoscopic treatment <strong>of</strong> mesenchymal tumors <strong>of</strong><br />

upper gastro<strong>in</strong>test<strong>in</strong>al tract<br />

Zhou XX, Ji F, Xu L, Li L, Chen YP, Lu JJ, Wang CW, Huang W<br />

1772 β-caten<strong>in</strong> accumulation <strong>in</strong> nuclei <strong>of</strong> hepatocellular carc<strong>in</strong>oma cells<br />

up-regulates glutathione-s-transferase M3 mRNA<br />

Li YS, Liu M, Nakata Y, Tang HB<br />

1779 Nutrition support <strong>in</strong> surgical patients with colorectal cancer<br />

Chen Y, Liu BL, Shang B, Chen AS, Liu SQ, Sun W, Y<strong>in</strong> HZ, Y<strong>in</strong> JQ, Su Q<br />

1787 Application <strong>of</strong> a wire-guided side-view<strong>in</strong>g duodenoscope <strong>in</strong> total<br />

esophagectomy with <strong>colon</strong>ic <strong>in</strong>terposition<br />

Yii CY, Chou JW, Peng YC, Chow WK<br />

April 7, 2011|Volume 17| ssue 13|

---

Contents<br />

APPENDIX<br />

FLYLEAF<br />

EDITORS FOR<br />

THIS ISSUE<br />

NAME OF JOURNAL<br />

<strong>World</strong> <strong>Journal</strong> <strong>of</strong> <strong>Gastroenterology</strong><br />

LAUNCH DATE<br />

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RESPONSIBLE INSTITUTION<br />

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SPONSOR<br />

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Prov<strong>in</strong>ce, Ch<strong>in</strong>a<br />

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<strong>World</strong> <strong>Journal</strong> <strong>of</strong> <strong>Gastroenterology</strong><br />

Volume 17 Number 13 April 7, 2011<br />

ACKNOWLEDGMENTS I Acknowledgments to reviewers <strong>of</strong> <strong>World</strong> <strong>Journal</strong> <strong>of</strong> <strong>Gastroenterology</strong><br />

ABOUT COVER<br />

AIM AND SCOPE<br />

WJG|www.wjgnet.com<br />

I Meet<strong>in</strong>gs<br />

I-VI Instructions to authors<br />

Santos BF, Hungness ES. Natural orifice translumenal endoscopic surgery: Progress<br />

<strong>in</strong> humans s<strong>in</strong>ce white paper.<br />

<strong>World</strong> J Gastroenterol 2011; 17(13): 1655-1665<br />

http://www.wjgnet.com/1007-9327/full/v17/i13/1655.htm<br />

<strong>World</strong> <strong>Journal</strong> <strong>of</strong> <strong>Gastroenterology</strong> (<strong>World</strong> J Gastroenterol, WJG, pr<strong>in</strong>t ISSN 1007-9327, DOI:<br />

10.3748) is a weekly, open-access, peer-reviewed journal supported by an editorial board <strong>of</strong><br />

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research on esophageal, gastro<strong>in</strong>test<strong>in</strong>al, liver, pancreas and biliary tract diseases, Helicobacter<br />

pylori, endoscopy and gastro<strong>in</strong>test<strong>in</strong>al surgery, <strong>in</strong>clud<strong>in</strong>g: gastroesophageal reflux disease,<br />

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Ke-Ji Chen, Beij<strong>in</strong>g<br />

Mart<strong>in</strong> H Floch, New Haven<br />

Geng-Tao Liu, Beij<strong>in</strong>g<br />

Emmet B Keeffe, Palo Alto<br />

Le<strong>in</strong>-Ray Mo, Ta<strong>in</strong>an<br />

Eamonn M Quigley, Cork<br />

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PRESIDENT AND EDITOR-IN-CHIEF<br />

Lian-Sheng Ma, Beij<strong>in</strong>g<br />

ACADEMIC EDITOR-IN-CHIEF<br />

Tauseef Ali, Oklahoma<br />

Mauro Bortolotti, Bologna<br />

Tarkan Karakan, Ankara<br />

Weekitt Kittisupamongkol, Bangkok<br />

Anastasios Koulaouzidis, Ed<strong>in</strong>burgh<br />

Gerd A Kullak-Ublick, Zürich<br />

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George Y Wu, Farm<strong>in</strong>gton<br />

STRATEGY ASSOCIATE EDITORS-IN-CHIEF<br />

Peter Draganov, Florida<br />

Hugh J Freeman, Vancouver<br />

Maria Concepción Gutiérrez-Ruiz, México<br />

Kazuhiro Hanazaki, Kochi<br />

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Kalpesh Jani, Baroda<br />

Javier S Mart<strong>in</strong>, Punta del Este<br />

Natalia A Osna, Omaha<br />

Wei Tang, Tokyo<br />

Alan BR Thomson, Edmonton<br />

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ASSOCIATE EDITORS-IN-CHIEF<br />

You-Yong Lu, Beij<strong>in</strong>g<br />

John M Luk, Pokfulam<br />

Hiroshi Shimada, Yokohama<br />

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<strong>World</strong> <strong>Journal</strong> <strong>of</strong> <strong>Gastroenterology</strong><br />

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Full <strong>in</strong>structions are available onl<strong>in</strong>e at http://www.<br />

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Onl<strong>in</strong>e Submissions: http://www.wjgnet.com/1007-9327<strong>of</strong>fice<br />

wjg@wjgnet.com<br />

doi:10.3748/wjg.v17.i13.1655<br />

EDITORIAL<br />

Natural orifice translumenal endoscopic surgery: Progress<br />

<strong>in</strong> humans s<strong>in</strong>ce white paper<br />

Byron F Santos, Eric S Hungness<br />

Byron F Santos, Department <strong>of</strong> Surgery, Fe<strong>in</strong>berg School <strong>of</strong><br />

Medic<strong>in</strong>e, Northwestern University, Chicago, IL 60611,<br />

United States<br />

Eric S Hungness, Department <strong>of</strong> Surgery, Fe<strong>in</strong>berg School <strong>of</strong><br />

Medic<strong>in</strong>e, Northwestern University, Chicago, IL 60611,<br />

United States<br />

Author contributions: Santos BF and Hungness ES contributed<br />

equally to this paper.<br />

Correspondence to: Eric S Hungness, MD, Assistant Pr<strong>of</strong>essor,<br />

Department <strong>of</strong> Surgery, Fe<strong>in</strong>berg School <strong>of</strong> Medic<strong>in</strong>e,<br />

Northwestern University, Suite 650, 676 North St. Clair Street,<br />

Chicago, IL 60611, United States. ehungnes@nmh.org<br />

Telephone: +1-312-6952534 Fax: +1-312-6951462<br />

Received: October 11, 2010 Revised: February 22, 2011<br />

Accepted: March 1, 2011<br />

Published onl<strong>in</strong>e: April 7, 2011<br />

Abstract<br />

S<strong>in</strong>ce the first description <strong>of</strong> the concept <strong>of</strong> natural orifice<br />

translumenal endoscopic surgery (NOTES), a substantial<br />

number <strong>of</strong> cl<strong>in</strong>ical NOTES reports have appeared <strong>in</strong> the<br />

literature. This editorial reviews the available human<br />

data address<strong>in</strong>g research questions orig<strong>in</strong>ally proposed<br />

by the white paper, <strong>in</strong>clud<strong>in</strong>g determ<strong>in</strong><strong>in</strong>g the optimal<br />

method <strong>of</strong> access for NOTES, develop<strong>in</strong>g safe methods<br />

<strong>of</strong> lumenal closure, sutur<strong>in</strong>g and anastomotic devices,<br />

advanced multitask<strong>in</strong>g platforms, address<strong>in</strong>g the risk<br />

<strong>of</strong> <strong>in</strong>fection, manag<strong>in</strong>g complications, address<strong>in</strong>g challenges<br />

with visualization, and tra<strong>in</strong><strong>in</strong>g for NOTES procedures.<br />

An analysis <strong>of</strong> the literature reveals that so far<br />

transvag<strong>in</strong>al access and closure appear to be the most<br />

feasible techniques for NOTES, with a limited, but grow<strong>in</strong>g<br />

transgastric, transrectal, and transesophageal NOTES<br />

experience <strong>in</strong> humans. The theoretically <strong>in</strong>creased risk <strong>of</strong><br />

<strong>in</strong>fection as a result <strong>of</strong> NOTES procedures has not been<br />

substantiated <strong>in</strong> transvag<strong>in</strong>al and transgastric procedures<br />

so far. Development <strong>of</strong> sutur<strong>in</strong>g and anastomotic devices<br />

and advanced platforms for NOTES has progressed slowly,<br />

with limited cl<strong>in</strong>ical data on their use so far. Data on<br />

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1655<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): 1655-1665<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

the optimal management and <strong>in</strong>cidence <strong>of</strong> <strong>in</strong>traoperative<br />

complications rema<strong>in</strong> sparse, although possible factors<br />

contribut<strong>in</strong>g to complications are discussed. F<strong>in</strong>ally, this<br />

editorial discusses the likely direction <strong>of</strong> future NOTES<br />

development and its possible role <strong>in</strong> cl<strong>in</strong>ical practice.<br />

© 2011 Baishideng. All rights reserved.<br />

Key words: Natural orifice translumenal endoscopic surgery;<br />

Outcomes; Complications; Endoscopic; Surgery<br />

Peer reviewers: Mitsuhiro Asakuma, MD, PhD, Assistant Pr<strong>of</strong>essor,<br />

Department <strong>of</strong> General and Gastroenterological Surgery,<br />

Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka<br />

569-8686, Japan; Mohammad Al-Haddad, MD, Assistant<br />

Pr<strong>of</strong>essor <strong>of</strong> Cl<strong>in</strong>ical Medic<strong>in</strong>e, Director, Endoscopic Ultrasound<br />

Fellowship Program, Indiana University School <strong>of</strong> Medic<strong>in</strong>e,<br />

550 N. University Blvd, Suite 4100, Indianapolis, IN 46 202,<br />

United States; Jeffrey Michael Marks, MD, Associate Pr<strong>of</strong>essor,<br />

Department <strong>of</strong> Surgery, Director <strong>of</strong> Surgical Endoscopy,<br />

University Hospitals, 11 100 Euclid Avenue, Cleveland, OH<br />

44106, United States<br />

Santos BF, Hungness ES. Natural orifice translumenal endoscopic<br />

surgery: Progress <strong>in</strong> humans s<strong>in</strong>ce white paper. <strong>World</strong> J<br />

Gastroenterol 2011; 17(13): 1655-1665 Available from: URL:<br />

http://www.wjgnet.com/1007-9327/full/v17/i13/1655.htm DOI:<br />

http://dx.doi.org/10.3748/wjg.v17.i13.1655<br />

INTRODUCTION<br />

The concept <strong>of</strong> natural orifice translumenal endoscopic<br />

surgery (NOTES ® ) has generated <strong>in</strong>tense <strong>in</strong>terest <strong>in</strong> the<br />

surgical and gastroenterology communities. Access<strong>in</strong>g<br />

the peritoneal or thoracic spaces through <strong>in</strong>ternal, transvisceral<br />

<strong>in</strong>cisions <strong>in</strong>stead <strong>of</strong> transabdom<strong>in</strong>al <strong>in</strong>cisions has<br />

the potential benefits <strong>of</strong> decreas<strong>in</strong>g postoperative pa<strong>in</strong>,<br />

wound complications, improv<strong>in</strong>g cosmesis, decreas<strong>in</strong>g the<br />

physiologic and immune response to surgery, decreas<strong>in</strong>g<br />

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Santos BF et al . Progress <strong>in</strong> humans<br />

anesthesia requirements, accelerat<strong>in</strong>g patient recovery and<br />

return to normal function, and improv<strong>in</strong>g access to organs<br />

that are currently difficult to reach with conventional<br />

open or laparoscopic approaches (e.g. esophagus, rectum).<br />

Given the <strong>in</strong>tense <strong>in</strong>terest <strong>in</strong> NOTES and its potential<br />

to revolutionize current surgical therapy, several work<strong>in</strong>g<br />

groups throughout the world have been formed to help<br />

guide NOTES research and cl<strong>in</strong>ical development. These<br />

groups <strong>in</strong>clude EURO-NOTES, EATS (European Association<br />

for Translum<strong>in</strong>al Surgery), D-NOTES, ASIA-<br />

NOTES, NOSLA (Natural Orifice Surgery Lat<strong>in</strong> America),<br />

Japan-NOTES, India NOTES, NOTES Research<br />

Group Brazil, and NOSCAR, which published a white paper<br />

<strong>in</strong> 2006 outl<strong>in</strong><strong>in</strong>g the perceived barriers to the cl<strong>in</strong>ical<br />

adoption <strong>of</strong> NOTES [1] . These barriers <strong>in</strong>cluded determ<strong>in</strong><strong>in</strong>g<br />

the optimal orifice to access the peritoneal cavity, develop<strong>in</strong>g<br />

a reliable means to close a viscotomy, m<strong>in</strong>imiz<strong>in</strong>g<br />

the risk <strong>of</strong> <strong>in</strong>fection as a result <strong>of</strong> access through a nonsterile<br />

orifice, develop<strong>in</strong>g an endoscopic sutur<strong>in</strong>g device,<br />

address<strong>in</strong>g difficulties with spatial orientation <strong>in</strong>herent to<br />

a NOTES technique, develop<strong>in</strong>g multi-task<strong>in</strong>g platforms<br />

to perform NOTES procedures, manag<strong>in</strong>g <strong>in</strong>traoperative<br />

complications, and develop<strong>in</strong>g NOTES tra<strong>in</strong><strong>in</strong>g to allow<br />

safe, widespread adoption <strong>of</strong> the techniques. Although<br />

there have been numerous studies address<strong>in</strong>g some <strong>of</strong><br />

these questions <strong>in</strong> animal and cadaver models, reports <strong>of</strong><br />

cl<strong>in</strong>ical NOTES procedures <strong>in</strong> humans, and human data<br />

address<strong>in</strong>g these questions have only started to appear<br />

s<strong>in</strong>ce 2007. This editorial will discuss the progress made<br />

on these questions by review<strong>in</strong>g the currently available human<br />

outcomes data and cl<strong>in</strong>ical NOTES publications <strong>in</strong><br />

the literature.<br />

ACCESS TO THE PERITONEAL CAVITY<br />

A comprehensive review <strong>of</strong> the human NOTES literature<br />

was conducted us<strong>in</strong>g PubMed to search the MEDLINE<br />

database with the search terms <strong>of</strong> “human natural orifice<br />

surgery, human transvag<strong>in</strong>al, human transrectal, human<br />

transgastric, or human NOTES surgery,” for articles<br />

published between January 1, 2 004 and September 1,<br />

2 010. Manuscripts describ<strong>in</strong>g cl<strong>in</strong>ical human NOTES<br />

procedures <strong>in</strong>clude the use <strong>of</strong> transgastric, transvag<strong>in</strong>al,<br />

transrectal, and transesophageal approaches. Currently,<br />

the most frequently used orifice for NOTES is the vag<strong>in</strong>a,<br />

with cholecystectomy account<strong>in</strong>g for the highest number<br />

<strong>of</strong> cases <strong>in</strong> the published literature [2] . Transvag<strong>in</strong>al access<br />

has the longest history <strong>of</strong> use for <strong>in</strong>traperitoneal procedures,<br />

prior to the recent description <strong>of</strong> NOTES. In 1949,<br />

Bueno described a series <strong>of</strong> transvag<strong>in</strong>al appendectomies<br />

performed with open <strong>in</strong>struments (without an endoscope)<br />

at the time <strong>of</strong> hysterectomy [3] . S<strong>in</strong>ce then, transvag<strong>in</strong>al<br />

access for <strong>in</strong>traperitoneal procedures <strong>in</strong> the form <strong>of</strong> culdoscopy<br />

has developed as an accepted, safe procedure <strong>in</strong><br />

the gynecology community [4-7] . Transvag<strong>in</strong>al access can<br />

be established us<strong>in</strong>g a posterior colpotomy created under<br />

direct vision with open <strong>in</strong>struments, or with the use <strong>of</strong><br />

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direct trocar <strong>in</strong>sertion under laparoscopic guidance. Establishment<br />

<strong>of</strong> transvag<strong>in</strong>al access does not require the use <strong>of</strong><br />

a flexible endoscope or transanal endoscopic microsurgery<br />

(TEM) platform, unlike transgastric, transrectal, and transesophageal<br />

approaches that have been described to date.<br />

Likewise, closure <strong>of</strong> transvag<strong>in</strong>al access sites is performed<br />

with direct sutur<strong>in</strong>g us<strong>in</strong>g open <strong>in</strong>struments.<br />

While transvag<strong>in</strong>al access is the most frequently used<br />

NOTES approach to date and can be safely performed,<br />

the potential for complications should not be overlooked.<br />

The close proximity <strong>of</strong> the rectum posteriorly, the ureters<br />

laterally, and the tendency for the small <strong>in</strong>test<strong>in</strong>e to occupy<br />

the pelvis should be kept <strong>in</strong> m<strong>in</strong>d while perform<strong>in</strong>g<br />

transvag<strong>in</strong>al NOTES. Reported complications <strong>of</strong> NOTES<br />

transvag<strong>in</strong>al access <strong>in</strong>clude rectal and <strong>colon</strong>ic <strong>in</strong>juries, small<br />

bowel <strong>in</strong>juries, ureterovag<strong>in</strong>al fistula formation, vulvar lacerations,<br />

and bladder <strong>in</strong>juries [8-14] . Given the possibility <strong>of</strong><br />

these complications, assistance from a gynecologist experienced<br />

<strong>in</strong> transvag<strong>in</strong>al access should be considered, at least<br />

<strong>in</strong>itially, <strong>in</strong> the performance <strong>of</strong> transvag<strong>in</strong>al NOTES. In<br />

addition, simultaneous visualization <strong>of</strong> colpotomy creation<br />

with a transumbilical laparoscope, along with the use <strong>of</strong><br />

a uter<strong>in</strong>e manipulator to anteriorly retract the uterus may<br />

m<strong>in</strong>imize the likelihood <strong>of</strong> rectal, bladder, or bowel <strong>in</strong>juries<br />

dur<strong>in</strong>g the creation <strong>of</strong> transvag<strong>in</strong>al access. Most cases<br />

reported so far have utilized a “hybrid” NOTES approach,<br />

with at least one laparoscopic port used for <strong>in</strong>itial visualization,<br />

retraction, and assistance with the dissection. Until<br />

<strong>in</strong>struments for NOTES improve, a “hybrid” NOTES<br />

approach may be preferable to a “pure” NOTES approach<br />

(without any percutaneous or laparoscopic assistance) <strong>in</strong><br />

order to <strong>in</strong>crease the safety <strong>of</strong> the procedures.<br />

Transgastric access is the second-most frequently<br />

reported access route after transvag<strong>in</strong>al access <strong>in</strong> the literature.<br />

Experience with transgastric NOTES <strong>in</strong>cludes at<br />

least 70 transgastric peritoneoscopy procedures reported<br />

by Nau et al [15,16] and Nikfarjam et al [17] , as well as several<br />

series which have reported at least 42 cholecystectomies,<br />

15 appendectomies, PEG rescue, and 6 cases <strong>of</strong> transgastric,<br />

stapled cystogastrostomy [11,15,16,18-24] . Transgastric<br />

access <strong>in</strong> all <strong>of</strong> these cases was obta<strong>in</strong>ed <strong>in</strong> the anterior<br />

stomach (antrum or body) us<strong>in</strong>g needle knife cautery and<br />

balloon dilation through a flexible endoscope, except <strong>in</strong><br />

cases <strong>of</strong> PEG-rescue and cystogastrostomy. Most cases<br />

were performed with placement <strong>of</strong> a laparoscopic port<br />

prior to gastrotomy creation to allow laparoscopic guidance<br />

and <strong>in</strong>sufflation, while some were performed without<br />

any previous laparoscopic ports or <strong>in</strong>sufflation. It is<br />

<strong>in</strong>terest<strong>in</strong>g to note that although no bowel <strong>in</strong>juries were<br />

recorded <strong>in</strong> transgastric peritoneoscopy cases performed<br />

without prior laparoscopic port placement, the authors<br />

noted there were <strong>in</strong>stances <strong>of</strong> cautery burns to the anterior<br />

peritoneum or the under surface <strong>of</strong> the liver that<br />

were discovered after subsequent abdom<strong>in</strong>al <strong>in</strong>spection<br />

with a laparoscope [15] . As such, it is not surpris<strong>in</strong>g that<br />

the majority <strong>of</strong> transgastric cases have been performed<br />

<strong>in</strong> a hybrid fashion, with laparoscopic visualization <strong>of</strong><br />

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the access po<strong>in</strong>t <strong>in</strong> order to prevent <strong>in</strong>juries to surround<strong>in</strong>g<br />

organs or the gastroepiploic vessels, which may be<br />

difficult or impossible to see from <strong>in</strong>side the stomach [2] .<br />

Transesophageal access has been used to perform<br />

esophageal myotomies <strong>in</strong> a series <strong>of</strong> 17 patients with<br />

achalasia, reported by Inoue et al [25] . This procedure,<br />

termed Per-Oral Esophageal Myotomy (POEM), <strong>in</strong>cises<br />

the <strong>in</strong>ner circular muscle layer <strong>of</strong> the distal esophagus and<br />

lower esophageal sph<strong>in</strong>cter, while completely avoid<strong>in</strong>g the<br />

hiatal dissection and disruption <strong>of</strong> the phrenoesophageal<br />

ligament that occurs dur<strong>in</strong>g laparoscopic Heller myotomy.<br />

Transesophageal access beg<strong>in</strong>s at the anterior, midesophagus<br />

with the creation <strong>of</strong> a submucosal bleb us<strong>in</strong>g<br />

a sclerotherapy needle. The submucosal bleb is then <strong>in</strong>cised<br />

us<strong>in</strong>g electrocautery and the endoscope is advanced<br />

through the <strong>in</strong>cision to create a submucosal tunnel distally<br />

past the gastro-esophageal (GE) junction onto the cardia<br />

<strong>of</strong> the stomach. The <strong>in</strong>ner circular muscle distal to the<br />

mucosal <strong>in</strong>cision is then <strong>in</strong>cised. Currently transesophageal<br />

access has been used to perform only procedures<br />

on the esophageal wall. In one case, full separation <strong>of</strong><br />

the outer longitud<strong>in</strong>al esophageal muscle layer occurred,<br />

expos<strong>in</strong>g the mediast<strong>in</strong>um. Per the authors, however, this<br />

patient did not have any adverse consequences as a result;<br />

this suggests that as long as closure <strong>of</strong> the proximal mucosal<br />

<strong>in</strong>cision is ensured, transesophageal mediast<strong>in</strong>al or<br />

thoracic access through a submucosal tunnel may be cl<strong>in</strong>ically<br />

feasible <strong>in</strong> the future. However, no cl<strong>in</strong>ical studies<br />

have been performed to date <strong>in</strong>vestigat<strong>in</strong>g the safety <strong>of</strong><br />

transesophageal mediast<strong>in</strong>al or thoracic access.<br />

In contrast to other forms <strong>of</strong> NOTES access, transrectal<br />

access has been the least reported <strong>in</strong> the literature.<br />

The only two published cases are <strong>of</strong> a proctosigmoidectomy<br />

for cancer [26] , and a transanal pull-through for<br />

Hirschsprung’s disease [27] . The proctosigmoidectomy was<br />

performed us<strong>in</strong>g a TEM platform, with a circumferential<br />

rectal dissection proceed<strong>in</strong>g cephalad from the distal rectum,<br />

assisted by laparoscopy. The transanal pull-through<br />

was performed <strong>in</strong> an <strong>in</strong>fant, without the use <strong>of</strong> the TEM;<br />

<strong>in</strong>stead the authors reported us<strong>in</strong>g trocars <strong>in</strong>serted directly<br />

through the rectal wall to allow passage <strong>of</strong> a rigid laparoscope<br />

and rigid <strong>in</strong>struments. Although no complications<br />

were reported to have occurred <strong>in</strong> either case, further data<br />

is needed <strong>in</strong> order to accurately determ<strong>in</strong>e the risks <strong>of</strong> this<br />

approach.<br />

Although so far various access po<strong>in</strong>ts for a variety <strong>of</strong><br />

NOTES procedures have been attempted, the specific<br />

<strong>in</strong>dications that are best suited for each orifice will need to<br />

be def<strong>in</strong>ed. For example, the ideal <strong>in</strong>dications for transoral<br />

access may end up be<strong>in</strong>g limited to therapeutic esophageal<br />

or gastric procedures, or diagnostic procedures <strong>in</strong> the <strong>in</strong>traperitoneal<br />

cavity. Transoral access may be poorly suited<br />

to advanced therapeutic <strong>in</strong>traperitoneal procedures given<br />

the requirement for complex, flexible <strong>in</strong>strumentation, as<br />

well as the small native diameter <strong>of</strong> the esophagus which<br />

makes extraction <strong>of</strong> large, bulky specimens potentially<br />

hazardous. Similarly, transrectal access may be best suited<br />

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Santos BF et al . Progress <strong>in</strong> humans<br />

to colorectal applications, and transvag<strong>in</strong>al access may end<br />

up be<strong>in</strong>g ideally suited for gynecologic <strong>in</strong>dications. However,<br />

if these two approaches prove to be the most forgiv<strong>in</strong>g<br />

<strong>in</strong> terms <strong>of</strong> ease <strong>of</strong> access, ability to reach the upper<br />

abdomen, complications, and the ability to <strong>in</strong>troduce both<br />

flexible and rigid <strong>in</strong>struments through the orifice, it is possible<br />

that these approaches may become “workhorse” approaches<br />

for <strong>in</strong>traperitoneal NOTES procedures or specimen<br />

removal <strong>in</strong> female and male patients, respectively.<br />

VISCERAL CLOSURE<br />

Transvag<strong>in</strong>al closure is currently the most feasible closure<br />

method for NOTES, as the <strong>in</strong>cision is closed by direct sutur<strong>in</strong>g.<br />

Aside from potential <strong>in</strong>juries to surround<strong>in</strong>g structures<br />

as previously mentioned, there have been no reports<br />

<strong>of</strong> vag<strong>in</strong>al dehiscence or herniation through the vag<strong>in</strong>al<br />

<strong>in</strong>cision. Also, the consequences <strong>of</strong> a vag<strong>in</strong>al wound dehiscence<br />

would likely not be as potentially dangerous as a<br />

gastric leak or a rectal leak, which would <strong>in</strong>troduce highly<br />

caustic or <strong>in</strong>fectious lum<strong>in</strong>al contents <strong>in</strong>to the abdomen.<br />

In contrast to transvag<strong>in</strong>al closure, transgastric closure<br />

currently requires the use <strong>of</strong> flexible endoscopic clips or<br />

tissue anchors, with or without laparoscopic sutures to buttress<br />

the closure. Although several groups have reported<br />

successful performance <strong>of</strong> transgastric closures without<br />

leaks, data on the true safety <strong>of</strong> current transgastric closure<br />

techniques are sparse at best. In 2010, Zorron et al [14]<br />

reported results from a prospective, multi-center NOTES<br />

registry, <strong>in</strong>clud<strong>in</strong>g data from 43 transgastric operations (29<br />

cholecystectomies and 14 appendectomies), <strong>in</strong> which the<br />

stomach was closed us<strong>in</strong>g laparoscopic sutur<strong>in</strong>g. No gastric<br />

leaks were reported <strong>in</strong> this study. Similarly, reports <strong>of</strong><br />

transgastric closure by other groups us<strong>in</strong>g endoscopic clips<br />

or anchors, with or without laparoscopic sutures, account<strong>in</strong>g<br />

for a total <strong>of</strong> approximately 30 patients, did not <strong>in</strong>clude<br />

any postoperative gastric leaks [18-21,23,24] . However, there<br />

has been at least one reported complication <strong>of</strong> gastric closure:<br />

a pneumothorax which occurred due to the aberrant<br />

placement <strong>of</strong> a tissue anchor through the diaphragm [24] .<br />

Innovative solutions for transgastric closure that have<br />

been reported <strong>in</strong> humans <strong>in</strong>clude the creation <strong>of</strong> a gastric<br />

valve mechanism made with tissue anchors, through<br />

which a gastrotomy is created [21] . The gastrotomy is then<br />

closed with additional tissue anchors once the procedure<br />

is f<strong>in</strong>ished. Although this technique has been successfully<br />

used <strong>in</strong> 5 patients so far, the majority <strong>of</strong> transgastric cases<br />

reported <strong>in</strong> the literature cont<strong>in</strong>ue to rely on laparoscopic<br />

sutur<strong>in</strong>g alone or <strong>in</strong> comb<strong>in</strong>ation with endoscopic <strong>in</strong>struments.<br />

Completely endoscopic means for clos<strong>in</strong>g gastrostomies<br />

will need to be developed and evaluated <strong>in</strong> human<br />

studies for transgastric NOTES to become feasible without<br />

laparoscopic assistance. Numerous prototype closure<br />

devices and techniques have been developed and tested<br />

<strong>in</strong> pre-cl<strong>in</strong>ical models. However, a detailed discussion <strong>of</strong><br />

these devices and their results <strong>in</strong> animals are beyond the<br />

scope <strong>of</strong> this editorial.<br />

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dures, along with some form <strong>of</strong> lum<strong>in</strong>al dis<strong>in</strong>fection for<br />

transvag<strong>in</strong>al, transrectal or transesophageal procedures<br />

will be the ultimate strategy adopted cl<strong>in</strong>ically.<br />

DEVELOPMENT OF ENDOSCOPIC<br />

SUTURING OR ANASTOMOTIC DEVICES<br />

The development <strong>of</strong> endoscopic sutur<strong>in</strong>g and anastomotic<br />

devices was deemed by the white paper to be necessary<br />

<strong>in</strong> order for NOTES to ultimately be applied to the wide<br />

spectrum <strong>of</strong> current surgical therapy [1] . However, the development<br />

<strong>of</strong> these devices and their use <strong>in</strong> cl<strong>in</strong>ical trials<br />

has proceeded slowly s<strong>in</strong>ce 2005. Currently, two types <strong>of</strong><br />

endoscopic sutur<strong>in</strong>g devices have been approved: Over-<br />

Stitch (Apollo Endosurgery, Inc., Aust<strong>in</strong>, TX, USA)<br />

and the Tissue Apposition System (TAS, Ethicon Endosurgery,<br />

C<strong>in</strong>c<strong>in</strong>nati, OH, USA). However, only use <strong>of</strong> the<br />

TAS has so far been reported cl<strong>in</strong>ically to approximate<br />

partial <strong>colon</strong>ic wall defects at the time <strong>of</strong> laparoscopicassisted<br />

polypectomy [32] . The TAS system works by sequentially<br />

deploy<strong>in</strong>g a threaded T-tag through the bowel<br />

wall on each side <strong>of</strong> a defect us<strong>in</strong>g an endoscopic hollow<br />

bore needle; once two threaded T-tags have been placed<br />

on either side <strong>of</strong> a defect, the two threads are c<strong>in</strong>ched<br />

together and trimmed by a one-way lock<strong>in</strong>g mechanism<br />

<strong>in</strong> order to approximate both sides <strong>of</strong> the lum<strong>in</strong>al defect.<br />

A similar endoscopic T-tag closure technique us<strong>in</strong>g <strong>in</strong>struments<br />

from Cook Medical (Bloom<strong>in</strong>gton, IN, USA)<br />

was employed by Park et al. to close gastrotomy defects<br />

dur<strong>in</strong>g transgastric NOTES [24] . One <strong>of</strong> the difficulties<br />

with exist<strong>in</strong>g endoscopic T-tag systems, however, is the <strong>in</strong>ability<br />

to directly visualize deployment <strong>of</strong> the T-tag from<br />

the extralum<strong>in</strong>al side <strong>of</strong> the defect without a laparoscope.<br />

This impaired visualization may contribute to the risk <strong>of</strong><br />

<strong>in</strong>advertent <strong>in</strong>jury to surround<strong>in</strong>g organs, or deployment<br />

through a vessel. As mentioned previously, <strong>in</strong>advertent<br />

deployment <strong>of</strong> a T-tag through the diaphragm dur<strong>in</strong>g a<br />

gastric closure was reported to have resulted <strong>in</strong> a pneumothorax<br />

discovered post-operatively [24] . The OverStitch, <strong>in</strong><br />

contrast to T-tag based systems, employs a lateral needlepass<strong>in</strong>g<br />

mechanism more similar to conventional sutur<strong>in</strong>g<br />

techniques. However, the OverStitch still requires assistance<br />

from an endoscopic grasper, and may be limited<br />

by the visual and mechanical constra<strong>in</strong>ts <strong>of</strong> conventional<br />

flexible endoscopes. Human use data will be needed to<br />

adequately evaluate the potential <strong>of</strong> the OverStitch for<br />

use <strong>in</strong> lum<strong>in</strong>al closures.<br />

The development <strong>of</strong> endoscopic anastomotic devices<br />

for NOTES has proceeded even more slowly than the<br />

development <strong>of</strong> sutur<strong>in</strong>g devices. The only reports <strong>of</strong><br />

NOTES procedures with anastomoses have utilized handsewn<br />

coloanal anastomoses dur<strong>in</strong>g colorectal resections,<br />

or a flexible, powered surgical stapler (SurgAssist SLC<br />

55, Power Medical Interventions, Langhorn, PA, USA)<br />

dur<strong>in</strong>g cystogastrostomy [33] . In the cystogastrostomy cases,<br />

the stapler was passed down the esophagus through an<br />

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overtube, alongside a flexible gastroscope. Although this<br />

stapler was used successfully to create a cystogastrostomy,<br />

the authors reported significant difficulty <strong>in</strong> pass<strong>in</strong>g the<br />

rigid part <strong>of</strong> the stapler through the esophagus, even<br />

through a previously placed overtube. In addition, the<br />

authors reported significant difficulty direct<strong>in</strong>g the stapler<br />

<strong>in</strong>to the appropriate angle once <strong>in</strong>side the stomach. Unfortunately,<br />

s<strong>in</strong>ce the publication <strong>of</strong> the study, the stapler<br />

has been removed from the market due to the acquisition<br />

<strong>of</strong> Power Medical, Inc. by Covidien (Mansfield, MA) and<br />

is not currently available for use. Development <strong>of</strong> flexible,<br />

articulat<strong>in</strong>g, low-pr<strong>of</strong>ile staplers is needed to make creation<br />

<strong>of</strong> anastomoses or lum<strong>in</strong>al closures dur<strong>in</strong>g NOTES<br />

more feasible. Additional features which may make application<br />

<strong>of</strong> stapl<strong>in</strong>g technology to NOTES more feasible<br />

<strong>in</strong>clude the addition <strong>of</strong> visualization and steer<strong>in</strong>g capabilities.<br />

These features might allow staplers to be more precisely<br />

directed <strong>in</strong>to difficult to reach areas and fired with<br />

more confidence.<br />

SPATIAL ORIENTATION<br />

Santos BF et al . Progress <strong>in</strong> humans<br />

The difficulty with correct spatial orientation dur<strong>in</strong>g NO-<br />

TES and its consequences <strong>in</strong> h<strong>in</strong>der<strong>in</strong>g the performance<br />

<strong>of</strong> advanced procedures was foreseen <strong>in</strong> the white paper.<br />

These difficulties are <strong>in</strong>herent to the use <strong>of</strong> current flexible<br />

endoscopes to perform NOTES, and have the potential to<br />

create not only a difficult operation, but may also <strong>in</strong>crease<br />

the risk <strong>of</strong> complications dur<strong>in</strong>g NOTES. Perretta et al [34]<br />

reported a case <strong>of</strong> mis<strong>in</strong>terpretation <strong>of</strong> biliary anatomy<br />

dur<strong>in</strong>g transgastric NOTES which was fortunately recognized,<br />

prevent<strong>in</strong>g the occurrence <strong>of</strong> a common bile duct<br />

<strong>in</strong>jury. The authors <strong>in</strong> this case converted to a laparoscopic<br />

view temporarily to clarify the unclear biliary anatomy. As<br />

emphasized by the authors, current NOTES techniques<br />

may alter the usual surgical anatomy that is seen due to the<br />

difficulty <strong>in</strong> achiev<strong>in</strong>g adequate retraction without laparoscopic<br />

<strong>in</strong>struments, and the spatial confusion created by<br />

retr<strong>of</strong>lexion when us<strong>in</strong>g a flexible endoscope. A solution to<br />

the problem <strong>of</strong> difficult spatial orientation dur<strong>in</strong>g NOTES<br />

may be the use <strong>of</strong> rigid endoscopes whenever possible.<br />

However, the use <strong>of</strong> rigid endoscopes is potentially feasible<br />

only through transvag<strong>in</strong>al or transrectal approaches, or<br />

through the umbilicus <strong>in</strong> the case <strong>of</strong> transgastric surgery.<br />

Short <strong>of</strong> us<strong>in</strong>g a rigid endoscope rout<strong>in</strong>ely, surgeons perform<strong>in</strong>g<br />

NOTES with flexible endoscopes should have a<br />

low threshold to convert to a laparoscopic view, even temporarily,<br />

to resolve any confusion <strong>in</strong> regards to the surgical<br />

anatomy. Although image-guided systems have been described<br />

as hav<strong>in</strong>g potential applications for NOTES, none<br />

<strong>of</strong> these systems have been applied <strong>in</strong> a cl<strong>in</strong>ical sett<strong>in</strong>g so<br />

far [35] . Future solutions to the problem <strong>of</strong> spatial orientation<br />

may also <strong>in</strong>volve the use <strong>of</strong> small, wireless cameras<br />

that are able to provide a wider, overhead view <strong>of</strong> the surgical<br />

field, and can be moved to the appropriate location as<br />

needed. Use <strong>of</strong> this type <strong>of</strong> camera has been described for<br />

human s<strong>in</strong>gle-<strong>in</strong>cision laparoscopy (SIL) cases [36] . However,<br />

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Santos BF et al . Progress <strong>in</strong> humans<br />

Figure 1 The Transanal Endoscopic Operations device from Karl-Storz<br />

allows the <strong>in</strong>sertion <strong>of</strong> rigid or flexible <strong>in</strong>struments through the anus and<br />

is currently used for perform<strong>in</strong>g transanal endoscopic microsurgery excisions<br />

<strong>of</strong> rectal tumors. It also has the potential to serve as a stable transrectal<br />

natural orifice translumenal endoscopic surgery (NOTES ® ) platform. Image used<br />

with permission (©Karl Storz).<br />

Figure 2 The TransPort multi-channel access device from USGI has<br />

been used as a transgastric natural orifice translumenal endoscopic surgery<br />

platform. It has a steer<strong>in</strong>g mechanism similar to a flexible endoscope, along<br />

with multiple, large-diameter channels to accommodate a small-diameter flexible<br />

endoscope and other large caliber flexible endoscopic <strong>in</strong>struments (g-Prox ® tissue<br />

anchor device is shown). Image used with permission (©USGI Medical).<br />

it should be noted that these cameras are not currently<br />

FDA-approved.<br />

DEVELOPMENT OF A MULTITASKING<br />

PLATFORM<br />

The creation <strong>of</strong> a multitask<strong>in</strong>g platform to allow the performance<br />

<strong>of</strong> multiple NOTES procedures with the same<br />

platform cont<strong>in</strong>ues to be an issue <strong>of</strong> the highest priority<br />

<strong>in</strong> the development <strong>of</strong> NOTES as a viable technique.<br />

Although several types <strong>of</strong> advanced operations (nephrectomy<br />

[12,37] , partial gastrectomy [38,39] , sigmoidectomy [40,41] ,<br />

and splenectomy [42] ) have been reported us<strong>in</strong>g a NOTES<br />

technique, many <strong>of</strong> these procedures have relied heavily<br />

on laparoscopic <strong>in</strong>struments for visualization and dissection.<br />

In order for “pure” NOTES (without laparoscopic or<br />

percutaneous assistance) to become feasible, a multi-task<strong>in</strong>g<br />

platform that balances flexibility and maneuverability with<br />

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Figure 3 The Anubis ® platform from Karl-Storz is an advanced flexible natural<br />

orifice translumenal endoscopic surgery platform (<strong>in</strong> development), with a<br />

tip that opens to expose work<strong>in</strong>g <strong>in</strong>struments capable <strong>of</strong> multiple degrees <strong>of</strong><br />

freedom controlled by the surgeon. Image used with permission (©Karl Storz).<br />

the ability to provide powerful retraction and <strong>in</strong>strument<br />

mobility, as well as an <strong>in</strong>tuitive <strong>in</strong>terface for the surgeon will<br />

need to be developed. Examples <strong>of</strong> the novel application<br />

<strong>of</strong> multi-lumen operat<strong>in</strong>g platforms for NOTES <strong>in</strong>clude<br />

use <strong>of</strong> a TEM platform (Figure 1) to perform proctosigmoidectomy,<br />

and the use <strong>of</strong> the TransPort (USGI Medical,<br />

San Clemente, CA, USA) multi-channel access port for<br />

transgastric NOTES (Figure 2). Use <strong>of</strong> a TEM platform<br />

for transrectal surgery allows the simultaneous use <strong>of</strong> rigid<br />

<strong>in</strong>struments with a flexible or rigid endoscope to perform<br />

<strong>in</strong>tra-abdom<strong>in</strong>al surgery. This comb<strong>in</strong>ation <strong>of</strong> operat<strong>in</strong>g <strong>in</strong>struments<br />

permits strong retraction, while allow<strong>in</strong>g flexible<br />

visualization and dissection capabilities through the flexible<br />

endoscope. Current limitations <strong>of</strong> such a system, however,<br />

<strong>in</strong>clude the difficulty <strong>of</strong> reach<strong>in</strong>g beyond the sacral promontory<br />

with rigid <strong>in</strong>struments, and the limitations <strong>of</strong> dissection<br />

performed with current flexible endoscopes. The<br />

TransPort device is a flexible, multi-channel device which<br />

allows passage <strong>of</strong> a flexible endoscope through one channel<br />

as well as additional flexible <strong>in</strong>struments through the<br />

other channels. This device is flexible enough to be passed<br />

transgastrically and has the ability to retr<strong>of</strong>lex and assume a<br />

rigid configuration <strong>in</strong>dependent <strong>of</strong> the endoscope. While it<br />

has been used for transgastric cholecystectomy and appendectomy<br />

[19,21,23] , use <strong>of</strong> the flexible <strong>in</strong>struments through its<br />

channels is similar to the use <strong>of</strong> accessories through a conventional<br />

flexible endoscope <strong>in</strong> that the <strong>in</strong>struments have<br />

limited degrees <strong>of</strong> freedom and lack the ability to make lateral<br />

or vertical movements <strong>in</strong>dependent <strong>of</strong> the endoscope<br />

<strong>in</strong> an <strong>in</strong>tuitive fashion. The limitations <strong>of</strong> both <strong>of</strong> these<br />

platforms make them less than ideal multi-task<strong>in</strong>g platforms<br />

for NOTES. However, the development <strong>of</strong> a system<br />

comb<strong>in</strong><strong>in</strong>g aspects <strong>of</strong> both platforms, along with robotic<br />

control may greatly facilitate the performance <strong>of</strong> NOTES<br />

procedures and may be the crucial enabl<strong>in</strong>g technology<br />

that would allow NOTES development to proceed exponentially,<br />

similar to the way the development <strong>of</strong> the chargecoupled<br />

device (CCD) camera revolutionized laparoscopy.<br />

Examples <strong>of</strong> experimental platforms with some <strong>of</strong> these<br />

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Figure 4 EndoSamurai is a prototype, advanced platform <strong>in</strong> development<br />

by Olympus. To operate the system, a surgeon uses an <strong>in</strong>tuitive, bi-manual<br />

<strong>in</strong>terface to control <strong>in</strong>struments with multiple degrees <strong>of</strong> freedom (<strong>in</strong>set shows<br />

close-up <strong>of</strong> endoscope tip with work<strong>in</strong>g <strong>in</strong>struments). Image used with permission<br />

(©Olympus Medical Systems Corp.).<br />

capabilities that may be seen <strong>in</strong> future cl<strong>in</strong>ical reports <strong>in</strong>clude<br />

Anubis (Karl-Storz, Tuttl<strong>in</strong>gen, Germany, Figure 3),<br />

EndoSamurai (Olympus, Tokyo, Japan, Figure 4), and the<br />

Direct-Drive Endoscopic System (Boston Scientific, Natick,<br />

MA, USA, Figure 5). Economic concerns cont<strong>in</strong>ue to be an<br />

issue with regard to the development <strong>of</strong> these platforms,<br />

however. The emergence <strong>of</strong> s<strong>in</strong>gle-<strong>in</strong>cision laparosopy<br />

(SIL) has caused a tremendous amount <strong>of</strong> resources to be<br />

redirected away from NOTES, towards the development<br />

<strong>of</strong> SIL technology. Although some <strong>of</strong> this technology may<br />

end up be<strong>in</strong>g adapted for NOTES, the development <strong>of</strong><br />

SIL will likely delay the development <strong>of</strong> NOTES-specific<br />

technology such as advanced multitask<strong>in</strong>g platforms. Both<br />

<strong>in</strong>dustry and <strong>in</strong>novators <strong>in</strong> m<strong>in</strong>imally-<strong>in</strong>vasive surgery need<br />

to not lose site <strong>of</strong> the potential promise <strong>of</strong> NOTES, while<br />

SIL occupies the spotlight.<br />

TRAINING<br />

There are currently not enough data from human studies<br />

to make quantitative recommendations <strong>in</strong> regards to<br />

the ideal amount <strong>of</strong> previous endoscopic or laparoscopic<br />

cl<strong>in</strong>ical or laboratory NOTES tra<strong>in</strong><strong>in</strong>g prior to the performance<br />

<strong>of</strong> cl<strong>in</strong>ical NOTES procedures. Nevertheless,<br />

a conservative approach described <strong>in</strong> the white paper<br />

recommends that NOTES procedures be performed by<br />

multi-discipl<strong>in</strong>ary teams after a period <strong>of</strong> laboratory tra<strong>in</strong><strong>in</strong>g<br />

<strong>in</strong> a properly equipped facility <strong>in</strong> order to maximize<br />

patient safety and ensure cont<strong>in</strong>u<strong>in</strong>g regulatory acceptance<br />

<strong>of</strong> early NOTES development.<br />

Future NOTES practitioners will likely need some<br />

form <strong>of</strong> fundamental surgical tra<strong>in</strong><strong>in</strong>g, along with platform-specific<br />

and procedure-specific tra<strong>in</strong><strong>in</strong>g once the<br />

field has undergone significant development. The current<br />

paradigm <strong>of</strong> perform<strong>in</strong>g NOTES primarily with flexible<br />

endoscopes is reach<strong>in</strong>g the limits <strong>of</strong> practicality and<br />

safety, and will arguably become quickly obsolete with the<br />

availability <strong>of</strong> advanced multitask<strong>in</strong>g platforms. Thus, rec-<br />

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Santos BF et al . Progress <strong>in</strong> humans<br />

Figure 5 Direct-Drive Endoscopic System from Boston Scientific is a prototype,<br />

advanced multi-channel platform currently <strong>in</strong> development, featur<strong>in</strong>g<br />

<strong>in</strong>struments with multiple degrees <strong>of</strong> freedom controlled through a bi-manual<br />

user <strong>in</strong>terface. Inset figure shows close-up <strong>of</strong> device tip with a small diameter<br />

flexible endoscope <strong>in</strong> place. Image used with permission (©Boston Scientific).<br />

ommendations made <strong>in</strong> regards to tra<strong>in</strong><strong>in</strong>g surgeons for<br />

NOTES us<strong>in</strong>g currently available <strong>in</strong>struments and techniques<br />

may quickly become obsolete.<br />

COMPLICATIONS OF NOTES<br />

Complications are an <strong>in</strong>evitable part <strong>of</strong> surgical practice,<br />

especially dur<strong>in</strong>g the application <strong>of</strong> new techniques such<br />

as NOTES. Intraoperative complications <strong>in</strong>herent to all<br />

procedures, such as bleed<strong>in</strong>g, will need to be managed<br />

appropriately to ensure patient safety. Along with the development<br />

<strong>of</strong> better endoscopic <strong>in</strong>struments to manage<br />

hemorrhage, surgical decision-mak<strong>in</strong>g will need to evolve<br />

based on laboratory and cl<strong>in</strong>ical data. Although the method<br />

<strong>of</strong> hemorrhage control will always depend on the situation<br />

and surgeon judgment, it will be useful to determ<strong>in</strong>e<br />

what is realistically manageable us<strong>in</strong>g a pure versus hybrid<br />

NOTES technique, and when it would be most beneficial<br />

to convert to a full laparoscopic procedure. Unfortunately,<br />

data from human studies to answer this question are currently<br />

limited. The reported <strong>in</strong>cidence <strong>of</strong> bleed<strong>in</strong>g <strong>in</strong> a<br />

prospective NOTES registry <strong>of</strong> 488 transvag<strong>in</strong>al cholecystectomy<br />

patients was 0% for <strong>in</strong>traoperative bleed<strong>in</strong>g,<br />

and 0.6% for postoperative bleed<strong>in</strong>g, comparable with<br />

the <strong>in</strong>cidence <strong>of</strong> bleed<strong>in</strong>g dur<strong>in</strong>g laparoscopic cholecystectomy<br />

[13] . However, it should be kept <strong>in</strong> m<strong>in</strong>d that the<br />

dom<strong>in</strong>ant technique <strong>in</strong> these cases did not <strong>in</strong>clude the use<br />

<strong>of</strong> any flexible endoscopes or accessories, and relied heavily<br />

on dissection through a transumbilical laparoscopic<br />

port. These results may thus only apply to NOTES performed<br />

exclusively with rigid <strong>in</strong>struments, as opposed to<br />

NOTES performed us<strong>in</strong>g a comb<strong>in</strong>ation <strong>of</strong> flexible and<br />

rigid <strong>in</strong>struments. A more accurate picture <strong>of</strong> NOTES<br />

outcomes from operations performed primarily with flexible<br />

endoscopes (with or without laparoscopic assistance)<br />

may be derived from the registry by Zorron et al [14] , which<br />

reported the <strong>in</strong>cidence <strong>of</strong> <strong>in</strong>traoperative bleed<strong>in</strong>g to be approximately<br />

2% for transvag<strong>in</strong>al cholecystectomy (all from<br />

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the cystic artery), 8% for transvag<strong>in</strong>al appendectomy (all<br />

from the appendiceal artery), and a comb<strong>in</strong>ed <strong>in</strong>cidence<br />

<strong>of</strong> 4.7% for all transgastric procedures (7% for the appendiceal<br />

artery and 3.4% from the gastroepiploic artery) [14] .<br />

Although these rates <strong>of</strong> bleed<strong>in</strong>g may seem high, it should<br />

be kept <strong>in</strong> m<strong>in</strong>d that all <strong>of</strong> these bleed<strong>in</strong>g complications<br />

occurred <strong>in</strong>traoperatively and were managed laparoscopically<br />

or endoscopically with the exception <strong>of</strong> 1 <strong>in</strong>stance <strong>of</strong><br />

gastroepiploic bleed<strong>in</strong>g dur<strong>in</strong>g transgastric access which<br />

required conversion to an open procedure. In addition,<br />

no cases <strong>of</strong> delayed postoperative bleed<strong>in</strong>g were reported.<br />

Future research on the optimal method to control hemorrhage<br />

dur<strong>in</strong>g NOTES will likely need to be performed <strong>in</strong><br />

animal models (for ethical reasons), and should <strong>in</strong>volve<br />

both the development <strong>of</strong> new <strong>in</strong>struments and algorithms<br />

to help guide <strong>in</strong>traoperative decision-mak<strong>in</strong>g.<br />

In addition to bleed<strong>in</strong>g, the authors <strong>of</strong> the white paper<br />

foresaw the possibility that physiologic complications and<br />

compression syndromes might be more frequently seen<br />

dur<strong>in</strong>g NOTES procedures, compared to exist<strong>in</strong>g laparoscopic<br />

procedures. So far the <strong>in</strong>cidence <strong>of</strong> these complications<br />

<strong>in</strong> the literature has been low (0.8% <strong>of</strong> 362 NOTES<br />

procedures), however, as reported by Zorron et al [14] <strong>in</strong><br />

a large, multi-<strong>in</strong>stitutional registry. These complications<br />

consisted <strong>of</strong> two episodes <strong>of</strong> <strong>in</strong>traoperative abdom<strong>in</strong>al<br />

hypertension which resolved with desufflation <strong>of</strong> gas and<br />

fluid therapy, as well as one episode <strong>of</strong> facial and cervical<br />

subcutaneous emphysema follow<strong>in</strong>g transvag<strong>in</strong>al, retroperitoneal<br />

cyst excision from the kidney [14,43] . This complication<br />

was reported to have been managed with oxygen<br />

therapy and observation <strong>in</strong> the <strong>in</strong>tensive care unit, without<br />

requir<strong>in</strong>g re-<strong>in</strong>tubation. Although it was reported that<br />

most groups <strong>in</strong> the registry used laparoscopic <strong>in</strong>sufflators<br />

through a transabdom<strong>in</strong>al port or Veress needle, the<br />

case with subcutaneous emphysema used a laparoscopic<br />

carbon dioxide (CO2) <strong>in</strong>sufflator connected to one <strong>of</strong> the<br />

flexible endoscopic channels with pressure ma<strong>in</strong>ta<strong>in</strong>ed<br />

between 12 to 16 mmHg. Although the overall <strong>in</strong>cidence<br />

<strong>of</strong> physiologic and compression syndrome complications<br />

was low <strong>in</strong> this registry report, surgeons perform<strong>in</strong>g<br />

NOTES should be aware that these complications may<br />

still occur and the risk <strong>of</strong> their occurrence may depend on<br />

the <strong>in</strong>sufflation gas or <strong>in</strong>sufflators used, and the anatomic<br />

compartment where dissection is performed.<br />

Although the use <strong>of</strong> pressure-controlled CO2 <strong>in</strong>sufflation<br />

is likely to cont<strong>in</strong>ue be<strong>in</strong>g a key component <strong>of</strong><br />

NOTES procedures, lower <strong>in</strong>sufflation pressures compared<br />

to conventional laparoscopy may be feasible, further<br />

reduc<strong>in</strong>g the risk <strong>of</strong> compression syndromes and subcutaneous<br />

emphysema.<br />

More serious complications dur<strong>in</strong>g NOTES cases have<br />

been reported that would otherwise be rare <strong>in</strong> the correspond<strong>in</strong>g<br />

laparoscopic operations. These are worth not<strong>in</strong>g<br />

to caution those who might be tempted to prematurely<br />

or over-enthusiastically adopt this still nascent approach<br />

to <strong>in</strong>tra-abdom<strong>in</strong>al surgery, and also to prioritize areas for<br />

potential improvement through better patient selection or<br />

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technical modifications to NOTES procedures. Reported<br />

complications <strong>of</strong> this k<strong>in</strong>d dur<strong>in</strong>g transvag<strong>in</strong>al cholecystectomy<br />

<strong>in</strong>clude 4 bladder <strong>in</strong>juries (0.8%), 2 rectal <strong>in</strong>juries<br />

(0.4%), and 1 small bowel <strong>in</strong>jury (0.2%). All bladder <strong>in</strong>juries<br />

were reported to have occurred <strong>in</strong> older, obese women.<br />

However, it was not clear from the report whether these <strong>in</strong>juries<br />

occurred dur<strong>in</strong>g the establishment <strong>of</strong> transvag<strong>in</strong>al access<br />

us<strong>in</strong>g a transvag<strong>in</strong>al trocar <strong>in</strong>serted under laparoscopic<br />

guidance or whether they occurred dur<strong>in</strong>g the latter parts<br />

<strong>of</strong> the procedures. The occurrence <strong>of</strong> these complications<br />

emphasizes the extreme care that should be taken when<br />

establish<strong>in</strong>g transvag<strong>in</strong>al access, clos<strong>in</strong>g the defect, and<br />

with the use <strong>of</strong> rigid transvag<strong>in</strong>al laparoscopic <strong>in</strong>struments.<br />

Similarly, the registry report by Zorron et al noted 2 esophageal<br />

hematomas, 1 esophageal laceration, and 1 esophageal<br />

perforation dur<strong>in</strong>g 29 transgastric cholecystectomies, account<strong>in</strong>g<br />

for a comb<strong>in</strong>ed rate <strong>of</strong> 13.7% esophageal complications.<br />

This is an unacceptably high rate <strong>of</strong> complications<br />

compared to conventional laparoscopic cholecystectomy,<br />

which is normally performed with m<strong>in</strong>imal morbidity and<br />

mortality. Investigators from the International Multicenter<br />

Trial on Cl<strong>in</strong>ical Natural Orifice Surgery (IMTN) <strong>in</strong>vestigators<br />

addressed this high rate <strong>of</strong> esophageal complications<br />

and recommended the use <strong>of</strong> esophageal overtubes to protect<br />

the esophagus dur<strong>in</strong>g the procedures, especially dur<strong>in</strong>g<br />

specimen extraction. In agreement with this recommendation,<br />

a study conducted by our group found that preoperative<br />

ultrasound measurements <strong>of</strong> gallbladder stones can<br />

be used to help predict which gallbladders are able to be<br />

extracted through an esophageal overtube [44] . Gallbladders<br />

found to be full <strong>of</strong> multiple small stones, <strong>in</strong> which the size<br />

<strong>of</strong> the largest stone cannot be determ<strong>in</strong>ed, as well as those<br />

<strong>in</strong> which the largest gallstone is greater than or equal to<br />

10 mm, are unlikely to pass through an overtube. Patients<br />

with these ultrasound f<strong>in</strong>d<strong>in</strong>gs may be better managed with<br />

conventional laparoscopic cholecystectomy. Criteria such<br />

as these may help improve patient selection for transgastric<br />

cholecystectomy, for example.<br />

Ultimately, once more human data on the risks and<br />

benefits <strong>of</strong> NOTES procedures become available surgeons<br />

will have to decide whether the benefits <strong>of</strong> NOTES<br />

are worth the risks. It should be kept <strong>in</strong> m<strong>in</strong>d that just<br />

because a procedure has an <strong>in</strong>herently higher rate <strong>of</strong> a<br />

specific complication doesn’t mean it is not worthwhile.<br />

Laparoscopic cholecystectomy, for example, has been<br />

shown have <strong>in</strong>creased rates <strong>of</strong> common bile duct <strong>in</strong>jury<br />

compared to open cholecystectomy [45,46] . However, this risk<br />

is acceptable given that the other benefits <strong>of</strong> laparoscopic<br />

cholecystectomy (decreased postoperative pa<strong>in</strong>, decreased<br />

wound complications, improved cosmesis, and a faster rate<br />

<strong>of</strong> recovery for patients) outweigh its potential for harm.<br />

The same type <strong>of</strong> analysis weigh<strong>in</strong>g the risks and benefits<br />

<strong>of</strong> NOTES will need to be applied to determ<strong>in</strong>e its ultimate<br />

role <strong>in</strong> surgical practice.<br />

NOTES MOVING FORWARD<br />

In the five years s<strong>in</strong>ce the publication <strong>of</strong> the NOTES<br />

1662 April 7, 2011|Volume 17|Issue 13|

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Santos BF et al . Progress <strong>in</strong> humans<br />

drolaparoscopy. J Obstet Gynaecol Res 2007; 33: 705-709<br />

7 Ts<strong>in</strong> DA, Bumaschny E, Helman M, Colombero LT. Culdolaparoscopic<br />

oophorectomy with vag<strong>in</strong>al hysterectomy: an<br />

optional m<strong>in</strong>imal-access surgical technique. J Laparoendosc<br />

Adv Surg Tech A 2002; 12: 269-271<br />

8 Michalik M, Orlowski M, Bobowicz M, Frask A, Trybull<br />

A. The First Report on Hybrid NOTES Adjustable Gastric<br />

Band<strong>in</strong>g <strong>in</strong> Human. Obes Surg 2010; Epub ahead <strong>of</strong> pr<strong>in</strong>t<br />

9 Decarli LA, Zorron R, Branco A, Lima FC, Tang M, Pioneer<br />

SR, Sansever<strong>in</strong>o JI, Menguer R, Bigol<strong>in</strong> AV, Gagner M. New<br />

hybrid approach for NOTES transvag<strong>in</strong>al cholecystectomy:<br />

prelim<strong>in</strong>ary cl<strong>in</strong>ical experience. Surg Innov 2009; 16: 181-186<br />

10 Federle<strong>in</strong> M, Borchert D, Müller V, Atas Y, Fritze F, Burghardt<br />

J, Ell<strong>in</strong>g D, Gellert K. Transvag<strong>in</strong>al video-assisted<br />

cholecystectomy <strong>in</strong> cl<strong>in</strong>ical practice. Surg Endosc 2010; 24:<br />

2444-2452<br />

11 Sal<strong>in</strong>as G, Saavedra L, Agurto H, Quispe R, Ramírez E,<br />

Grande J, Tamayo J, Sánchez V, Málaga D, Marks JM. Early<br />

experience <strong>in</strong> human hybrid transgastric and transvag<strong>in</strong>al<br />

endoscopic cholecystectomy. Surg Endosc 2010; 24: 1092-1098<br />

12 Sotelo R, de Andrade R, Fernández G, Ramirez D, Di Grazia<br />

E Carmona O, Moreira O, Berger A, Aron M, Desai MM,<br />

Gill IS. NOTES hybrid transvag<strong>in</strong>al radical nephrectomy for<br />

tumor: stepwise progression toward a first successful cl<strong>in</strong>ical<br />

case. Eur Urol 2010; 57: 138-144<br />

13 Lehmann KS, Ritz JP, Wibmer A, Gellert K, Zornig C,<br />

Burghardt J, Büs<strong>in</strong>g M, Runkel N, Kohlhaw K, Albrecht<br />

R, Kirchner TG, Arlt G, Mall JW, Butters M, Bulian DR,<br />

Bretschneider J, Holmer C, Buhr HJ. The German registry<br />

for natural orifice translumenal endoscopic surgery: report<br />

<strong>of</strong> the first 551 patients. Ann Surg 2010; 252: 263-270<br />

14 Zorron R, Palanivelu C, Galvão Neto MP, Ramos A, Sal<strong>in</strong>as G,<br />

Burghardt J, DeCarli L, Henrique Sousa L, Forgione A, Pugliese<br />

R, Branco AJ, Balashanmugan TS, Boza C, Corcione F, D’<br />

Avila Avila F, Arturo Gómez N, Galvão Ribeiro PA, Mart<strong>in</strong>s<br />

S, Filgueiras M, Gellert K, Wood Branco A, Kondo W, Inacio<br />

Sansever<strong>in</strong>o J, de Sousa JA, Saavedra L, Ramírez E, Campos<br />

J, Sivakumar K, Rajan PS, Jategaonkar PA, Ranagrajan M,<br />

Parthasarathi R, Senthilnathan P, Prasad M, Cuccurullo D,<br />

Müller V. International multicenter trial on cl<strong>in</strong>ical natural<br />

orifice surgery--NOTES IMTN study: prelim<strong>in</strong>ary results <strong>of</strong><br />

362 patients. Surg Innov 2010; 17: 142-158<br />

15 Nau P, Anderson J, Happel L, Yuh B, Narula VK, Needleman<br />

B, Ellison EC, Melv<strong>in</strong> WS, Hazey JW. Safe alternative<br />

transgastric peritoneal access <strong>in</strong> humans: NOTES. Surgery<br />

2011; 149: 147-152<br />

16 Nau P, Anderson J, Yuh B, Muscarella P Jr, Christopher Ellison<br />

E, Happel L, Narula VK, Melv<strong>in</strong> WS, Hazey JW. Diagnostic<br />

transgastric endoscopic peritoneoscopy: extension <strong>of</strong><br />

the <strong>in</strong>itial human trial for stag<strong>in</strong>g <strong>of</strong> pancreatic head masses.<br />

Surg Endosc 2010; 24: 1440-1446<br />

17 Nikfarjam M, McGee MF, Trunzo JA, Onders RP, Pearl JP,<br />

Poulose BK, Chak A, Ponsky JL, Marks JM. Transgastric<br />

natural-orifice translum<strong>in</strong>al endoscopic surgery peritoneoscopy<br />

<strong>in</strong> humans: a pilot study <strong>in</strong> efficacy and gastrotomy<br />

site selection by us<strong>in</strong>g a hybrid technique. Gastro<strong>in</strong>test Endosc<br />

2010; 72: 279-283<br />

18 Asakuma M, Perretta S, Allemann P, Cahill R, Con SA, Solano<br />

C, Pasupathy S, Mutter D, Dallemagne B, Marescaux J.<br />

Challenges and lessons learned from NOTES cholecystectomy<br />

<strong>in</strong>itial experience: a stepwise approach from the laboratory<br />

to cl<strong>in</strong>ical application. J Hepatobiliary Pancreat Surg<br />

2009; 16: 249-254<br />

19 Auyang ED, Hungness ES, Vaziri K, Mart<strong>in</strong> JA, Soper NJ.<br />

Human NOTES cholecystectomy: transgastric hybrid technique.<br />

J Gastro<strong>in</strong>test Surg 2009; 13: 1149-1150<br />

20 Rao GV, Reddy DN, Banerjee R. NOTES: human experience.<br />

Gastro<strong>in</strong>test Endosc Cl<strong>in</strong> N Am 2008; 18: 361-370; x<br />

21 Ujiki MB, Mart<strong>in</strong>ec DV, Diwan TS, Denk PM, Dunst CM,<br />

WJG|www.wjgnet.com<br />

Swanström LL. Video: natural orifice translumenal endoscopic<br />

surgery (NOTES): creation <strong>of</strong> a gastric valve for safe<br />

and effective transgastric surgery <strong>in</strong> humans. Surg Endosc<br />

2010; 24: 220<br />

22 Marks JM, Ponsky JL, Pearl JP, McGee MF. PEG “Rescue”:<br />

a practical NOTES technique. Surg Endosc 2007; 21: 816-819<br />

23 Horgan S, Cullen JP, Talam<strong>in</strong>i MA, M<strong>in</strong>tz Y, Ferreres A,<br />

Jacobsen GR, Sandler B, Bosia J, Savides T, Easter DW, Savu<br />

MK, Ramamoorthy SL, Whitcomb E, Agarwal S, Lukacz E,<br />

Dom<strong>in</strong>guez G, Ferra<strong>in</strong>a P. Natural orifice surgery: <strong>in</strong>itial<br />

cl<strong>in</strong>ical experience. Surg Endosc 2009; 23: 1512-1518<br />

24 Park PO, Bergström M. Transgastric peritoneoscopy and<br />

appendectomy: thoughts on our first experience <strong>in</strong> humans.<br />

Endoscopy 2010; 42: 81-84<br />

25 Inoue H, M<strong>in</strong>ami H, Kobayashi Y, Sato Y, Kaga M, Suzuki M,<br />

Satodate H, Odaka N, Itoh H, Kudo S. Peroral endoscopic<br />

myotomy (POEM) for esophageal achalasia. Endoscopy 2010;<br />

42: 265-271<br />

26 Sylla P, Rattner DW, Delgado S, Lacy AM. NOTES transanal<br />

rectal cancer resection us<strong>in</strong>g transanal endoscopic microsurgery<br />

and laparoscopic assistance. Surg Endosc 2010; 24:<br />

1205-1210<br />

27 Velhote MC, Velhote CE. A NOTES modification <strong>of</strong> the<br />

transanal pull-through. J Laparoendosc Adv Surg Tech A 2009;<br />

19: 255-257<br />

28 Gavagan JA, Whiteford MH, Swanstrom LL. Full-thickness<br />

<strong>in</strong>traperitoneal excision by transanal endoscopic microsurgery<br />

does not <strong>in</strong>crease short-term complications. Am J Surg<br />

2004; 187: 630-634<br />

29 A prospective analysis <strong>of</strong> 1518 laparoscopic cholecystectomies.<br />

The Southern Surgeons Club. N Engl J Med 1991; 324:<br />

1073-1078<br />

30 Narula VK, Happel LC, Volt K, Bergman S, Roland JC, Dettorre<br />

R, Renton DB, Reavis KM, Needleman BJ, Mikami DJ,<br />

Ellison EC, Melv<strong>in</strong> WS, Hazey JW. Transgastric endoscopic<br />

peritoneoscopy does not require decontam<strong>in</strong>ation <strong>of</strong> the<br />

stomach <strong>in</strong> humans. Surg Endosc 2009; 23: 1331-1336<br />

31 Narula VK, Hazey JW, Renton DB, Reavis KM, Paul CM,<br />

H<strong>in</strong>shaw KE, Needleman BJ, Mikami DJ, Ellison EC, Melv<strong>in</strong><br />

WS. Transgastric <strong>in</strong>strumentation and bacterial contam<strong>in</strong>ation<br />

<strong>of</strong> the peritoneal cavity. Surg Endosc 2008; 22: 605-611<br />

32 Delaney CP, Champagne BJ, Marks JM, Sanuk L, Ermlich B,<br />

Chak A. Tissue apposition system: new technology to m<strong>in</strong>imize<br />

surgery for endoscopically unresectable <strong>colon</strong>ic polyps.<br />

Surg Endosc 2010; 24: 3113-3118<br />

33 Pallapothu R, Earle DB, Desilets DJ, Romanelli JR. NOTES(®)<br />

stapled cystgastrostomy: a novel approach for surgical management<br />

<strong>of</strong> pancreatic pseudocysts. Surg Endosc 2011; 25:<br />

883-839<br />

34 Perretta S, Dallemagne B, Donatelli G, Mutter D, Marescaux J.<br />

Multimedia article. The fear <strong>of</strong> transgastric cholecystectomy:<br />

mis<strong>in</strong>terpretation <strong>of</strong> the biliary anatomy. Surg Endosc 2011;<br />

25: 648<br />

35 Coughl<strong>in</strong> G, Samavedi S, Palmer KJ, Patel VR. Role <strong>of</strong> imageguidance<br />

systems dur<strong>in</strong>g NOTES. J Endourol 2009; 23: 803-812<br />

36 Cadeddu J, Fernandez R, Desai M, Bergs R, Tracy C, Tang<br />

SJ, Rao P, Desai M, Scott D. Novel magnetically guided <strong>in</strong>tra-abdom<strong>in</strong>al<br />

camera to facilitate laparoendoscopic s<strong>in</strong>glesite<br />

surgery: <strong>in</strong>itial human experience. Surg Endosc 2009; 23:<br />

1894-1899<br />

37 Kaouk JH, Haber GP, Goel RK, Crouzet S, Brethauer S,<br />

Firoozi F, Goldman HB, White WM. Pure natural orifice<br />

translumenal endoscopic surgery (NOTES) transvag<strong>in</strong>al nephrectomy.<br />

Eur Urol 2010; 57: 723-726<br />

38 Fischer LJ, Jacobsen G, Wong B, Thompson K, Bosia J, Talam<strong>in</strong>i<br />

M, Horgan S. NOTES laparoscopic-assisted transvag<strong>in</strong>al<br />

sleeve gastrectomy <strong>in</strong> humans-description <strong>of</strong> prelim<strong>in</strong>ary<br />

experience <strong>in</strong> the United States. Surg Obes Relat Dis 2009; 5:<br />

633-636<br />

1664 April 7, 2011|Volume 17|Issue 13|

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39 Nakajima K, Nishida T, Takahashi T, Souma Y, Hara J,<br />

Yamada T, Yoshio T, Tsutsui T, Yokoi T, Mori M, Doki Y.<br />

Partial gastrectomy us<strong>in</strong>g natural orifice translumenal endoscopic<br />

surgery (NOTES) for gastric submucosal tumors: early<br />

experience <strong>in</strong> humans. Surg Endosc 2009; Epub ahead <strong>of</strong> pr<strong>in</strong>t<br />

40 Lacy AM, Delgado S, Rojas OA, Almenara R, Blasi A, Llach<br />

J. MA-NOS radical sigmoidectomy: report <strong>of</strong> a transvag<strong>in</strong>al<br />

resection <strong>in</strong> the human. Surg Endosc 2008; 22: 1717-1723<br />

41 Sanchez JE, Rasheid SH, Krieger BR, Fratt<strong>in</strong>i JC, Marcet JE.<br />

Laparoscopic-assisted transvag<strong>in</strong>al approach for sigmoidectomy<br />

and rectocolpopexy. JSLS 2009; 13: 217-220<br />

42 Targarona EM, Gomez C, Rovira R, Pernas JC, Balague<br />

C, Guarner-Argente C, Sa<strong>in</strong>z S, Trias M. NOTES-assisted<br />

transvag<strong>in</strong>al splenectomy: the next step <strong>in</strong> the m<strong>in</strong>imally <strong>in</strong>vasive<br />

approach to the spleen. Surg Innov 2009; 16: 218-222<br />

43 Zorron R, Goncalves L, Leal D, Kanaan E, Cabral I, Saraiva P.<br />

WJG|www.wjgnet.com<br />

Santos BF et al . Progress <strong>in</strong> humans<br />

Transvag<strong>in</strong>al hybrid natural orifice translum<strong>in</strong>al endoscopic<br />

surgery retroperitoneoscopy--the first human case report. J<br />

Endourol 2010; 24: 233-237<br />

44 Santos BF, Auyang ED, Hungness ES, Desai KR, Chan ES,<br />

van Beek DB, Wang EC, Soper NJ. Preoperative ultrasound<br />

measurements predict the feasibility <strong>of</strong> gallbladder extraction<br />

dur<strong>in</strong>g transgastric natural orifice translumenal endoscopic<br />

surgery cholecystectomy. Surg Endosc 2010; Epub<br />

ahead <strong>of</strong> pr<strong>in</strong>t<br />

45 Russell JC, Walsh SJ, Mattie AS, Lynch JT. Bile duct <strong>in</strong>juries,<br />

1989-1993. A statewide experience. Connecticut Laparoscopic<br />

Cholecystectomy Registry. Arch Surg 1996; 131: 382-388<br />

46 Targarona EM, Marco C, Balagué C, Rodriguez J, Cugat E,<br />

Hoyuela C, Veloso E, Trias M. How, when, and why bile<br />

duct <strong>in</strong>jury occurs. A comparison between open and laparoscopic<br />

cholecystectomy. Surg Endosc 1998; 12: 322-326<br />

S- Editor Sun H L- Editor O’Neill M E- Editor Ma WH<br />

1665 April 7, 2011|Volume 17|Issue 13|

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Onl<strong>in</strong>e Submissions: http://www.wjgnet.com/1007-9327<strong>of</strong>fice<br />

wjg@wjgnet.com<br />

doi:10.3748/wjg.v17.i13.1666<br />

EDITORIAL<br />

<strong>Isolated</strong> <strong>lymphoid</strong> <strong>follicles</strong> <strong>in</strong> <strong>colon</strong>: Switch po<strong>in</strong>ts between<br />

<strong>in</strong>flammation and colorectal cancer?<br />

Ferenc Sipos, Györgyi Műzes<br />

Ferenc Sipos, Györgyi Műzes, 2nd Department <strong>of</strong> Internal<br />

Medic<strong>in</strong>e, Semmelweis University, 1088 Budapest, Hungary<br />

Author contributions: Sipos F and Műzes G contributed equally<br />

to the writ<strong>in</strong>g and revis<strong>in</strong>g <strong>of</strong> this paper.<br />

Correspondence to: Ferenc Sipos, MD, PhD, Cell Analysis<br />

Laboratory, 2nd Department <strong>of</strong> Internal Medic<strong>in</strong>e, Semmelweis<br />

University, 1088 Budapest, Hungary. dr.siposf@gmail.com<br />

Telephone: +36-20-4780752 Fax: +36-1-2660816<br />

Received: January 5, 2011 Revised: February 12, 2011<br />

Accepted: February 19, 2011<br />

Published onl<strong>in</strong>e: April 7, 2011<br />

Abstract<br />

Gut-associated <strong>lymphoid</strong> tissue is supposed to play a<br />

central role <strong>in</strong> both the organization <strong>of</strong> <strong>colon</strong>ic repair<br />

mechanisms and colorectal carc<strong>in</strong>ogenesis. In <strong>in</strong>flammatory<br />

conditions, the number, diameter and density <strong>of</strong><br />

isolated <strong>lymphoid</strong> <strong>follicles</strong> (ILFs) <strong>in</strong>creases. They are not<br />

only <strong>in</strong>volved <strong>in</strong> immune surveillance, but their presence<br />

is also <strong>in</strong>dispensable <strong>in</strong> normal mucosal regeneration <strong>of</strong><br />

the <strong>colon</strong>. In carc<strong>in</strong>ogenesis, ILFs may play a dual role.<br />

On the one hand they may support tumor growth and<br />

the metastatic process by vascular endothelial growth<br />

factor receptor signal<strong>in</strong>g and produc<strong>in</strong>g a specific cytok<strong>in</strong>e<br />

and cellular milieu, but on the other hand their<br />

presence is sometimes associated with a better prognosis.<br />

The relation <strong>of</strong> ILFs to bone marrow derived stem<br />

cells, follicular dendritic cells, subepithelial my<strong>of</strong>ibroblasts<br />

or crypt formation, which are all <strong>in</strong>volved <strong>in</strong> mucosal<br />

repair and carc<strong>in</strong>ogenesis, has not been directly<br />

studied. Data about the putative organizer role <strong>of</strong> ILFs<br />

is scattered <strong>in</strong> scientific literature.<br />

© 2011 Baishideng. All rights reserved.<br />

Key words: <strong>Isolated</strong> <strong>lymphoid</strong> follicle; Colon; Mucosal<br />

repair; Colorectal cancer; Epithelial stem cell; My<strong>of</strong>ibroblast;<br />

Follicular dendritic cell; Mesenchymal-epithelial<br />

transition; Epithelial-mesenchymal transition<br />

WJG|www.wjgnet.com<br />

1666<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): 1666-1673<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

Peer reviewer: Akira Andoh, MD, Department <strong>of</strong> Internal<br />

Medic<strong>in</strong>e, Shiga University <strong>of</strong> Medical Science, Seta Tuk<strong>in</strong>owa,<br />

Otsu 520-2192, Japan<br />

Sipos F, Műzes G. <strong>Isolated</strong> <strong>lymphoid</strong> <strong>follicles</strong> <strong>in</strong> <strong>colon</strong>: Switch<br />

po<strong>in</strong>ts between <strong>in</strong>flammation and colorectal cancer? <strong>World</strong> J<br />

Gastroenterol 2011; 17(13): 1666-1673 Available from: URL:<br />

http://www.wjgnet.com/1007-9327/full/v17/i13/1666.htm DOI:<br />

http://dx.doi.org/10.3748/wjg.v17.i13.1666<br />

INTRODUCTION<br />

The imbalance <strong>of</strong> <strong>colon</strong>ic epithelial proliferation and<br />

apoptosis may lead to both ulcer- and carc<strong>in</strong>oma development<br />

<strong>of</strong> the mucosa. The f<strong>in</strong>al direction <strong>of</strong> this imbalance<br />

depends on complex pathogenetic pathways <strong>in</strong><br />

which isolated <strong>lymphoid</strong> <strong>follicles</strong> (ILFs) seem to have a<br />

specific role.<br />

Some steps <strong>of</strong> <strong>colon</strong>ic epithelial regeneration are<br />

known, but the connection among them is not fully understood.<br />

The cont<strong>in</strong>uous reformation <strong>of</strong> the epithelial<br />

layer is important <strong>in</strong> avoid<strong>in</strong>g the aggregation <strong>of</strong> pernicious<br />

mutations <strong>in</strong>duced by <strong>in</strong>tralum<strong>in</strong>al factors. In <strong>in</strong>flammation,<br />

the lack <strong>of</strong> regenerative factors and the disturbance<br />

<strong>of</strong> the regulation <strong>of</strong> regenerative mechanisms<br />

favour ulcer development. It has also been observed that<br />

<strong>in</strong> <strong>colon</strong>ic <strong>in</strong>flammation there is a tight connection between<br />

the degree <strong>of</strong> epithelial damage and the number,<br />

diameter and cellular compounds <strong>of</strong> subepithelial <strong>lymphoid</strong><br />

<strong>follicles</strong> [1,2] . The more severe the epithelial destruction<br />

that develops, the higher the number <strong>of</strong> ILFs that<br />

can be found <strong>in</strong> adjacent mucosa.<br />

It has recently been reported that <strong>lymphoid</strong> <strong>follicles</strong><br />

are also present <strong>in</strong> carc<strong>in</strong>omas <strong>of</strong> the lung [3] , endometrium<br />

[4] , liver [5] , and <strong>colon</strong> [1] . They are supposed to have<br />

immune-mediated anti-tumoral effects, as their elevated<br />

number is <strong>in</strong> positive correlation with a better prognosis<br />

and a longer survival [3] . However, the density <strong>of</strong> <strong>lymphoid</strong><br />

April 7, 2011|Volume 17|Issue 13|

---

follicle-associated flat dysplastic aberrant crypt foci was<br />

significantly higher compared to the rest <strong>of</strong> the mucosa<br />

<strong>in</strong> azoxymethane-treated rats [6] . Several reports have <strong>in</strong>vestigated<br />

the association between <strong>lymphoid</strong> aggregates<br />

and <strong>colon</strong>ic tumors <strong>in</strong> rodents [7,8] . The results <strong>in</strong>dicate that<br />

<strong>colon</strong>ic crypts overly<strong>in</strong>g ILFs show a significantly higher<br />

proliferative activity, which may also <strong>in</strong>fluence genetically<br />

defected epithelial cells. Hence, the risk <strong>of</strong> carc<strong>in</strong>oma is<br />

<strong>in</strong>creased <strong>in</strong> the <strong>colon</strong>ic mucosa <strong>of</strong> ILFs compared to<br />

mucosa without ILFs. It has also been shown that the<br />

<strong>in</strong>cidence <strong>of</strong> ILFs <strong>in</strong> early human colorectal cancers significantly<br />

differs by gender, location, macroscopic type<br />

and histology, but moreover, their localization significantly<br />

differs by their macroscopic type [9] .<br />

However, the exact role <strong>of</strong> ILFs <strong>in</strong> <strong>colon</strong>ic epithelial repair<br />

and colorectal carc<strong>in</strong>ogenesis is not yet known. Some<br />

data show [10] that the lack <strong>of</strong> <strong>lymphoid</strong> <strong>follicles</strong> results <strong>in</strong><br />

abnormal crypt formation <strong>in</strong> the case <strong>of</strong> epithelial destruction.<br />

On the other hand, Apc gene mutation causes<br />

impairment <strong>of</strong> developmental and apparent differentiation<br />

blockade <strong>in</strong> proliferative tissues, <strong>in</strong>clud<strong>in</strong>g those <strong>of</strong> the <strong>lymphoid</strong><br />

<strong>follicles</strong> [11] . Whether ILFs act as a regenerative pool<br />

conta<strong>in</strong><strong>in</strong>g putative stem cells <strong>in</strong> case <strong>of</strong> mucosal damage,<br />

or they are responsible only for the optimal cytok<strong>in</strong>e milieu<br />

for the differentiation <strong>of</strong> immigrat<strong>in</strong>g stem cells or <strong>in</strong>vasive<br />

carc<strong>in</strong>oma cells [12] need to be further exam<strong>in</strong>ed.<br />

THE ORGANIZATION OF THE GUT-<br />

ASSOCIATED LYMPHOID TISSUE<br />

The gut-associated <strong>lymphoid</strong> tissue (GALT) is a component<br />

<strong>of</strong> the mucosa-associated <strong>lymphoid</strong> tissue, <strong>in</strong> which<br />

approximately 70 percent <strong>of</strong> the body’s immune cells are<br />

found [13,14] . GALT differentiates between pathogens and<br />

commensal bacteria.<br />

The majority <strong>of</strong> GALT is composed <strong>of</strong> isolated and<br />

aggregated <strong>lymphoid</strong> <strong>follicles</strong> dispersed throughout the<br />

small and large <strong>in</strong>test<strong>in</strong>es [15] . These <strong>lymphoid</strong> <strong>follicles</strong>,<br />

<strong>in</strong>clud<strong>in</strong>g Peyer’s patches (PPs) <strong>of</strong> the small <strong>in</strong>test<strong>in</strong>e and<br />

ILFs <strong>of</strong> the large <strong>in</strong>test<strong>in</strong>e, are composed <strong>of</strong> a specialised<br />

follicle associated epithelium (FAE), which overlies a subepithelial<br />

dome conta<strong>in</strong><strong>in</strong>g numerous macrophages, dendritic<br />

cells, T, B lymphocytes, and special antigen sampl<strong>in</strong>g<br />

micr<strong>of</strong>old/M/cells [15-17] . The FAE has a crucial role <strong>in</strong> the<br />

<strong>in</strong>itiation <strong>of</strong> the mucosal and systemic immune response [18] .<br />

ILFs have, <strong>in</strong> general, an average diameter <strong>of</strong> 0.1-0.7 mm<br />

and number <strong>of</strong> around 30 000 <strong>in</strong> humans [19] .<br />

ILFs are <strong>in</strong>nervated sites <strong>of</strong> GALT. Functionally,<br />

antigen-triggered mast cell and eos<strong>in</strong>ophil activation affects<br />

both the secretory and motor functions <strong>of</strong> the <strong>in</strong>test<strong>in</strong>es [20] ,<br />

and these defensive reactions can be modulated by the enteric<br />

nervous system [21] . It has been recently recognised that<br />

there is a dense neuronal network at the level <strong>of</strong> the suprafollicular<br />

dome region, but not with<strong>in</strong> the germ<strong>in</strong>al centers<br />

<strong>in</strong> <strong>lymphoid</strong> <strong>follicles</strong> [22] . Neuronal alterations <strong>of</strong> PPs and<br />

ILFs, such as nerve-eos<strong>in</strong>ophil associations or <strong>in</strong>creas<strong>in</strong>g<br />

neuronal cell adhesion molecule expression, may have consequences<br />

on the uptake <strong>of</strong> particular pathogens [16,23] .<br />

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Sipos F et al . Colonic isolated <strong>lymphoid</strong> <strong>follicles</strong><br />

VASCULARIZATION OF ILFS<br />

ILFs have rich blood and lymphatic vascularization [14,19] .<br />

Vasculogenesis may play a dual role <strong>in</strong> mucosal organization,<br />

<strong>in</strong> that it is not only necessary for nutritional and<br />

metabolic processes, but the hom<strong>in</strong>g <strong>of</strong> the repopulat<strong>in</strong>g<br />

bone marrow derived stem cells to the site <strong>of</strong> tissue damage<br />

may happen via blood vessels. In the case <strong>of</strong> cancer<br />

development, the vascular system is essentially <strong>in</strong>volved <strong>in</strong><br />

tumor growth, <strong>in</strong>vasion and metastasis formation.<br />

Revascularization is a key po<strong>in</strong>t <strong>of</strong> <strong>colon</strong>ic mucosal<br />

repair. Dur<strong>in</strong>g <strong>in</strong>flammatory stages, due to cytok<strong>in</strong>e action<br />

and <strong>in</strong>tercellular adhesion, molecules signall<strong>in</strong>g some<br />

<strong>of</strong> the vessels differentiate <strong>in</strong>to high endothelial venules<br />

(HEVs) [24,25] . In the case <strong>of</strong> lymphocytes and neutrophils,<br />

it is supposed that they firstly reach the <strong>in</strong>flammatory<br />

sites via a transcellular pathway through the HEVs [26] , but<br />

an <strong>in</strong>tercellular pathway is also known [27] . Upon epithelial<br />

<strong>in</strong>jury the circulat<strong>in</strong>g bone marrow derived cells (BMDCs)<br />

migrate to the stromal layer <strong>of</strong> the damaged <strong>colon</strong>ic wall,<br />

presumably via HEVs at an <strong>in</strong>creased number regulated by<br />

overexpressed <strong>in</strong>flammatory chemok<strong>in</strong>es [28] .<br />

Based on the result <strong>of</strong> Witmer et al [29] , it has been<br />

suggested that <strong>in</strong> <strong>lymphoid</strong> tissues, <strong>in</strong>clud<strong>in</strong>g GALT, the<br />

signal<strong>in</strong>g system <strong>of</strong> the vascular endothelial growth factor<br />

(VEGF) and its receptor play a permanent role <strong>in</strong> the<br />

vasculogenesis <strong>of</strong> ILFs. Whereas the <strong>in</strong>hibition <strong>of</strong> VEGF<br />

has shown promis<strong>in</strong>g results <strong>in</strong> sporadic <strong>colon</strong> cancer, it<br />

has been recently published that VEGF receptor signal<strong>in</strong>g<br />

acts as a direct growth factor for tumor cells <strong>in</strong> colitisassociated<br />

cancer, provid<strong>in</strong>g a molecular l<strong>in</strong>k between<br />

<strong>in</strong>flammation and the development <strong>of</strong> <strong>colon</strong> cancer [30] .<br />

BONE MARROW DERIVED STEM CELLS<br />

OF ILFS<br />

Based on the former results [31-33] , emerg<strong>in</strong>g evidence suggests<br />

that bone marrow derived stem cells contribute to<br />

tissue regeneration partly by promot<strong>in</strong>g neovascularization<br />

or arteriogenesis. After human hematopoietic cell transplantation<br />

epithelial tissue chimerism appears [34-36] .<br />

The bone marrow orig<strong>in</strong> <strong>of</strong> epithelial cells may be<br />

supposed by observations <strong>in</strong> which epithelial cell markers<br />

and leukocyte markers showed that double positive cells<br />

were found <strong>in</strong> <strong>in</strong>flamed mucosa adjacent to <strong>lymphoid</strong><br />

aggregates [2,37-39] . The presence <strong>of</strong> cytokerat<strong>in</strong>, epithelial<br />

growth factor receptor, hepatocyte-derived growth factor<br />

receptor or CDX2 co-expression <strong>in</strong> CD45+ cells <strong>of</strong><br />

ILFs may support the mesenchymal orig<strong>in</strong> <strong>of</strong> epithelial<br />

stem cells. Based on these results, it seems that ILFs are<br />

<strong>in</strong>volved <strong>in</strong> the hom<strong>in</strong>g and differentiation <strong>of</strong> BMDCs <strong>in</strong><br />

the case <strong>of</strong> <strong>colon</strong>ic mucosal damage (Figure 1).<br />

The cause <strong>of</strong> metastasis rema<strong>in</strong>s elusive despite a vast<br />

amount <strong>of</strong> <strong>in</strong>formation on cancer cells. Accord<strong>in</strong>g to recent<br />

research, cancer cell fusion with macrophages or immigrat<strong>in</strong>g<br />

BMDCs provides an explanation [40,41] . BMDCs fused<br />

with tumor cells were present not just <strong>in</strong> animal tumor xenografts<br />

where they were associated with metastases, but <strong>in</strong><br />

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Sipos F et al . Colonic isolated <strong>lymphoid</strong> <strong>follicles</strong><br />

A 10 μm<br />

FISH<br />

human carc<strong>in</strong>omas, <strong>in</strong>clud<strong>in</strong>g <strong>colon</strong> cancer. BMDC-tumor<br />

cell fusion expla<strong>in</strong>s the epidermal-mesenchymal transition<br />

<strong>in</strong> cancer s<strong>in</strong>ce BMDCs express mesodermal traits and<br />

epithelial-mesenchymal transition regulators (i.e.: Twist,<br />

SPARC). If BMDC-tumor cell fusion underlies <strong>in</strong>vasion<br />

and metastasis <strong>in</strong> human cancer, new therapeutic strategies<br />

would be mandated.<br />

DENDRITIC CELLS IN ILFS<br />

Follicular dendritic cells (FDCs) <strong>in</strong> <strong>lymphoid</strong> <strong>follicles</strong> reta<strong>in</strong><br />

native antigens <strong>in</strong> the form <strong>of</strong> immune complexes on<br />

their membrane for months, and present these antigens to<br />

B cells dur<strong>in</strong>g the secondary response [42,43] . The orig<strong>in</strong> and<br />

cell l<strong>in</strong>eage <strong>of</strong> FDCs are controversial. Whereas their immune<br />

functions and expression <strong>of</strong> hemopoietic cell-associated<br />

antigens suggest that they belong to the hemopoietic<br />

l<strong>in</strong>eage [44] , their sp<strong>in</strong>dle-shaped morphology “<strong>in</strong> vitro”, lack<br />

<strong>of</strong> CD45, and presence <strong>of</strong> antigens expressed by fibroblasts<br />

[45] <strong>in</strong>dicate that FDCs may be mesenchymal cells.<br />

Based on studies with mouse radiation chimeras, Humphrey<br />

et al [46] concluded that FDCs were not derived from<br />

the bone marrow, but came from a local mesenchymal precursor.<br />

However, Kapasi et al [44] , us<strong>in</strong>g mice homozygous<br />

for the SCID mutation, which lack T, B lymphocytes, and<br />

FDCs, demonstrated that after reconstitution with bone<br />

marrow from donor mice, the FDCs <strong>of</strong> the reconstituted<br />

mice expressed the donor phenotype. These authors concluded<br />

that FDC precursors came from bone marrow.<br />

Accord<strong>in</strong>g to the results <strong>of</strong> Muñoz-Fernández et al [47] ,<br />

FDCs seem to be a specialized form <strong>of</strong> my<strong>of</strong>ibroblasts and<br />

derive from bone marrow stromal cell progenitors. The authors<br />

were able to isolate and culture 18 follicular dendritic<br />

cell l<strong>in</strong>es from human tonsils. These cells were CD45negative<br />

and expressed antigens associated with FDCs<br />

(CD21, CD23, CD35, CD40, CD73, BAFF, ICAM-1, and<br />

VCAM-1) and antigens specific for FDC (DRC-1, CNA.42,<br />

and HJ2). These cell l<strong>in</strong>es were also able to b<strong>in</strong>d B cells and<br />

secrete CXCL13, and they had functional activities characteristic<br />

<strong>of</strong> FDCs. Nevertheless, the additional expression<br />

<strong>of</strong> STRO-1, together with CD10, CD13, CD29, CD34,<br />

CD63, CD73, CD90, ICAM-1, VCAM-1, HLA-DR, al-<br />

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B<br />

10 μm<br />

FISH + F-IHC<br />

Figure 1 Intraepithelial male donor bone marrow orig<strong>in</strong> CD45-/Y-FISH+ cell (white arrow) and CD45+/Y-FISH+ <strong>in</strong>traepithelial lymphocyte (red arrow) <strong>in</strong> the<br />

<strong>colon</strong>ic biopsy specimen <strong>of</strong> a female acceptor. A: Chromosomal detection (green: Y-chromosome, red: X-chromosome; fluorescence <strong>in</strong> situ hybridization); B: CD45<br />

and cytokerat<strong>in</strong> (green: cytokerat<strong>in</strong>, red: CD45; fluorescence immunohistochemistry; 130 × magnification).<br />

kal<strong>in</strong>e phosphatase, and α-smooth muscle act<strong>in</strong> (α-SMA)<br />

<strong>in</strong>dicated that FDCs are closely related to bone marrow<br />

stromal cell progenitors. The expression <strong>of</strong> α-SMA also relates<br />

FDCs with my<strong>of</strong>ibroblasts. Like my<strong>of</strong>ibroblasts, FDC<br />

l<strong>in</strong>es expressed stress fibers conta<strong>in</strong><strong>in</strong>g α-SMA and were<br />

able to contract collagen gels under the effect <strong>of</strong> TGFβ1<br />

and platelet-derived growth factor.<br />

In various <strong>in</strong>flammation models, tissue-derived dendritic<br />

cells have been shown to migrate from the <strong>in</strong>flammatory<br />

site via lymphatics to secondary <strong>lymphoid</strong> organs where<br />

they <strong>in</strong>teract with lymphocytes [48] . Based on their dual<br />

phenotype, follicular dendritic cells may represent a transformation<br />

switch po<strong>in</strong>t among immigrat<strong>in</strong>g bone marrow<br />

derived stem cells <strong>in</strong> ILFs and the surround<strong>in</strong>g subepithelial<br />

my<strong>of</strong>ibroblasts.<br />

The orig<strong>in</strong> <strong>of</strong> dendritic cells (DCs) <strong>in</strong> tumors rema<strong>in</strong>s<br />

obscure. Recent studies <strong>in</strong>dicate that conventional DCs <strong>in</strong><br />

<strong>lymphoid</strong> tissues arise from a dist<strong>in</strong>ct population <strong>of</strong> committed<br />

conventional DC precursors (pre-cDCs) that orig<strong>in</strong>ate<br />

<strong>in</strong> bone marrow and migrate via blood. Diao et al [49]<br />

showed that pre-cDCs are precursors for conventional<br />

DCs <strong>in</strong> tumors, and they migrate from blood <strong>in</strong>to the tumor<br />

where they generate conventional DCs. The chemok<strong>in</strong>e<br />

CCL3, which is markedly upregulated <strong>in</strong> tumors (<strong>in</strong>clud<strong>in</strong>g<br />

<strong>colon</strong> cancer) and <strong>in</strong> tumor-<strong>in</strong>filtrat<strong>in</strong>g stromal and<br />

immune cells, promotes pre-cDC recruitment. Both precDCs<br />

and their conventional DC progeny actively proliferate<br />

with<strong>in</strong> the tumor, and have the ability to mature and<br />

stimulate Ag-specific lymphocytes. This f<strong>in</strong>d<strong>in</strong>g suggests<br />

that <strong>in</strong> several cases the migration <strong>of</strong> pre-cDCs to tumors<br />

may represent a normal response to <strong>in</strong>flammation. Further<br />

studies are needed to del<strong>in</strong>eate the role <strong>of</strong> pre-cDCs<br />

<strong>in</strong> other <strong>in</strong>flammatory processes and to compare them<br />

with monocytes, which are currently considered the ma<strong>in</strong><br />

source <strong>of</strong> <strong>in</strong>flammatory DCs <strong>in</strong> peripheral tissues [50,51] .<br />

MYOFIBROBLASTS SORROUNDING ILF<br />

ADJACENT EPITHELIUM<br />

Subepithelial my<strong>of</strong>ibroblasts (SEMFs) exist as a syncytium<br />

that extends throughout the <strong>colon</strong>ic lam<strong>in</strong>a propria, merg-<br />

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<strong>in</strong>g with the pericytes surround<strong>in</strong>g the blood vessels [52,53] .<br />

SEMFs are <strong>in</strong>volved <strong>in</strong> two epithelial repair processes [54,55] .<br />

One process is called restitution [56] . This is an important<br />

response to m<strong>in</strong>or to moderate <strong>in</strong>jury. The other process<br />

is observed when the wound is deep, and the subepithelial<br />

tissues and the basement membrane need to be reconstituted<br />

[55] .<br />

Accord<strong>in</strong>g to recent studies [54,57,58] , my<strong>of</strong>ibroblasts are<br />

thought to derive from two major sources, bone marrow<br />

or locally activated fibroblasts, <strong>in</strong> response to transform<strong>in</strong>g<br />

growth factor-β1. In the case <strong>of</strong> serious tissue <strong>in</strong>jury<br />

(i.e. active ulcerative colitis) the regeneration capacity <strong>of</strong><br />

local stem cells is not enough to complete tissue repair. In<br />

this case, bone marrow derived mesenchymal stem cells<br />

migrate <strong>in</strong>to the gastro<strong>in</strong>test<strong>in</strong>al wall where they may contribute<br />

to the repair progress [59,60] as differentiated mesenchymal<br />

cells (e.g. my<strong>of</strong>ibroblasts) [61] .<br />

Despite the <strong>in</strong>creas<strong>in</strong>g number <strong>of</strong> publications illustrat<strong>in</strong>g<br />

the role <strong>of</strong> tumor-associated stromal cells <strong>in</strong> cancer<br />

progression, there still exists a significant ambiguity with<br />

respect to the identification <strong>of</strong> cancer-associated fibroblasts,<br />

my<strong>of</strong>ibroblasts and peritumoral fibroblasts <strong>in</strong> the<br />

cancer tissue. SEMFs appear early <strong>in</strong> the cancer’s development.<br />

The mutual <strong>in</strong>teraction (through direct cell-cell<br />

contacts and paracr<strong>in</strong>e signals) between cancer cells and<br />

SEMFs is essential for <strong>in</strong>vasive growth and is translated<br />

<strong>in</strong>to a poor cl<strong>in</strong>ical prognosis [62] .<br />

TOLL-LIKE RECEPTOR EXPRESSION IN<br />

ILFS<br />

Beside immune functions, PPs and ILFs are supposed<br />

to be <strong>in</strong>volved <strong>in</strong> mucosal repair via Toll-like receptors<br />

(TLRs). In ILFs, TLRs are expressed on the cells <strong>of</strong> the<br />

monocyte/macrophage system, on some k<strong>in</strong>ds <strong>of</strong> T cells,<br />

as well as on <strong>in</strong>test<strong>in</strong>al epithelial, endothelial and stromal<br />

cells [63] . Us<strong>in</strong>g the dextran sodium sulfate (DSS) model <strong>of</strong><br />

colitis, mice lack<strong>in</strong>g TLR2, TLR4 or MyD88 all developed<br />

more severe colitis than wild type mice when exposed to<br />

orally adm<strong>in</strong>istered DSS [64] . These f<strong>in</strong>d<strong>in</strong>gs suggest that<br />

signal<strong>in</strong>g from commensal bacteria throughout TLRs resulted<br />

<strong>in</strong> protection from DSS colitis through enhanced<br />

epithelial cell proliferation, and worked as a compensatory<br />

factor aga<strong>in</strong>st epithelial damage [64] .<br />

TLRs can also b<strong>in</strong>d endogenous ligands <strong>in</strong>clud<strong>in</strong>g<br />

necrotic cells, heat shock prote<strong>in</strong>s, and extracellular matrix<br />

components [65-67] . Necrotic cells may activate NF-κB<br />

through TLR2, lead<strong>in</strong>g to the expression <strong>of</strong> tissue repairassociated<br />

genes [65] . It is supposed that necrosis <strong>in</strong>duced<br />

<strong>in</strong>flammation <strong>in</strong> tissue damage may provide danger signals<br />

function<strong>in</strong>g as <strong>in</strong>ducers <strong>of</strong> tissue repair responses through<br />

TLRs. The TLR ligands released from necrotic cells have<br />

not been identified, although heat shock prote<strong>in</strong>s produced<br />

by damaged cells are known to be TLR ligands [66] .<br />

Components <strong>of</strong> the extracellular matrix, such as hyaluronan,<br />

can be an endogenous ligand for TLR4 [67] . Increased<br />

hyaluronan production has been demonstrated <strong>in</strong> both<br />

DSS colitis <strong>in</strong> mice and <strong>in</strong> human Crohn’s disease [68] . It<br />

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Sipos F et al . Colonic isolated <strong>lymphoid</strong> <strong>follicles</strong><br />

is possible that TLR activation may occur <strong>in</strong> the absence<br />

<strong>of</strong> microbial products [68] . In the case <strong>of</strong> <strong>in</strong>flammatory<br />

mucosal damage, ILFs may <strong>in</strong>duce repair mechanisms via<br />

endogenous TLR activation.<br />

TLR4 was also shown to be expressed on human <strong>colon</strong><br />

carc<strong>in</strong>oma cells and functionally active. It may play<br />

important roles <strong>in</strong> promot<strong>in</strong>g immune escape <strong>of</strong> human<br />

<strong>colon</strong> carc<strong>in</strong>oma cells by <strong>in</strong>duc<strong>in</strong>g immunosuppressive factors<br />

and apoptosis resistance, and it may also promote the<br />

proliferation and migration <strong>of</strong> cancer cells [69,70] .<br />

The analysis <strong>of</strong> isolated tumor cells from primary <strong>colon</strong><br />

cancers showed co-expression <strong>of</strong> TLR7 and TLR8 with<br />

CD133 and gave evidence for a subpopulation <strong>of</strong> <strong>colon</strong><br />

cancer-<strong>in</strong>itiat<strong>in</strong>g cells [71] . Persistent TLR-specific activation<br />

<strong>of</strong> NF-κB <strong>in</strong> colorectal cancer, particularly <strong>in</strong> tumor-<strong>in</strong>itiat<strong>in</strong>g<br />

cells, may susta<strong>in</strong> further tumor growth and progression<br />

through perpetuated signal<strong>in</strong>g known from <strong>in</strong>flammatory<br />

and tissue repair mechanisms with consecutive self-renewal<br />

<strong>in</strong> pluripotent tumor cells. Activation through self-ligands<br />

or viral RNA fragments from tumor-associated <strong>lymphoid</strong><br />

aggregates may putatively ma<strong>in</strong>ta<strong>in</strong> this <strong>in</strong>flammatory process,<br />

suggest<strong>in</strong>g a key role <strong>in</strong> cancer progression.<br />

THE EFFECT OF THE PRESENCE OF ILFS<br />

ON MUCOSAL REPAIR<br />

The epithelia <strong>of</strong> <strong>in</strong>test<strong>in</strong>al crypts associated with ILFs and<br />

PPs have an <strong>in</strong>creased proliferation rate [10,14] . Saxena et al [10]<br />

showed that PPs <strong>in</strong> rats have a facilitative effect on the<br />

heal<strong>in</strong>g <strong>of</strong> <strong>in</strong>test<strong>in</strong>al wounds by promot<strong>in</strong>g both epithelial<br />

cell migration on the defect and epithelial cell proliferation<br />

<strong>in</strong> the crypts adjacent to the wound and by decreas<strong>in</strong>g the<br />

rate <strong>of</strong> wound contraction.<br />

In rats, a difference <strong>in</strong> epithelial apoptosis between the<br />

FAE <strong>of</strong> PPs and <strong>in</strong>test<strong>in</strong>al villi was described [72] . Onishi<br />

et al [72] showed that the progression <strong>of</strong> the apoptotic process<br />

<strong>in</strong> the epithelial cells <strong>of</strong> FAE occurs later than <strong>in</strong> the<br />

<strong>in</strong>test<strong>in</strong>al villi, so the possibility <strong>of</strong> epithelial differentiation<br />

might rema<strong>in</strong> <strong>in</strong> FAE, unlike <strong>in</strong> <strong>in</strong>test<strong>in</strong>al villi. PPs<br />

are supposed to have a regulatory effect on the epithelial<br />

proliferation as well [73] .<br />

The Wnt signal<strong>in</strong>g pathway is critical for regulat<strong>in</strong>g a<br />

number <strong>of</strong> basic cell functions, such as cell proliferation,<br />

cell fate, polarity, differentiation, and migration, lead<strong>in</strong>g to<br />

morphogenesis and organogenesis [74,75] . There is strong genetic<br />

evidence that Wnt signal<strong>in</strong>g play critical roles <strong>in</strong> the<br />

regulation <strong>of</strong> epithelial stem cells <strong>in</strong> the <strong>in</strong>test<strong>in</strong>al tract [76] .<br />

The Wnt target gene Lgr5 has been recently identified as<br />

a novel stem cell marker <strong>of</strong> the <strong>in</strong>test<strong>in</strong>al epithelium and<br />

the hair follicle [77] . In the <strong>in</strong>test<strong>in</strong>e, Lgr5 is exclusively expressed<br />

<strong>in</strong> cycl<strong>in</strong>g crypt base columnar cells [77] . Many Wnt<br />

family prote<strong>in</strong>s are expressed <strong>in</strong> hematopoietic tissues, and<br />

can also be secreted by <strong>lymphoid</strong> cells [78,79] . The Wnt-Lgr5<br />

pathway may be a potential switch between the ILFs and<br />

<strong>colon</strong>ic epithelial renewal. Lymphoid cells <strong>of</strong> ILFs may<br />

produce Wnts which are essential components <strong>of</strong> a milieu<br />

<strong>in</strong> which bone marrow derived stem cells immigrated to<br />

ILFs to engage <strong>in</strong> epithelial differentiation (Figure 2).<br />

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Sipos F et al . Colonic isolated <strong>lymphoid</strong> <strong>follicles</strong><br />

*<br />

Figure 2 3D reconstruction <strong>of</strong> a human <strong>colon</strong>ic surgical sample (MIRAX<br />

Viewer, 3D, 3DHISTECH Ltd., Budapest). A large subepithelial isolated <strong>lymphoid</strong><br />

follicle (white star) can be seen. Colonic crypts (white arrow) with no connection<br />

to the lum<strong>in</strong>al surface “outgrow” from the isolated <strong>lymphoid</strong> follicle.<br />

THE EFFECT OF THE PRESENCE OF ILFS<br />

ON COLORECTAL CARCINOGENESIS<br />

Results from experimental <strong>colon</strong> cancer studies <strong>in</strong>dicated<br />

that ILFs might promote the development <strong>of</strong> adenocarc<strong>in</strong>omas<br />

[7,8] . However, studies <strong>in</strong> experimental animals have also<br />

shown that the <strong>in</strong>test<strong>in</strong>al <strong>lymphoid</strong> system plays an important<br />

role <strong>in</strong> immunologic defense mechanisms; that is, antigenic<br />

stimuli result <strong>in</strong> germ<strong>in</strong>al center formation, antibody<br />

production, and f<strong>in</strong>ally enlargement <strong>of</strong> the <strong>follicles</strong> [80] . In<br />

humans, the presence <strong>of</strong> tumor-<strong>in</strong>filtrat<strong>in</strong>g lymphocytes is<br />

associated with an improved prognosis <strong>in</strong> colorectal cancers,<br />

as does the presence <strong>of</strong> high level DNA microsatellite <strong>in</strong>stability<br />

[81] . These results suggest that ILFs <strong>in</strong> early colorectal<br />

neoplasms play an important role <strong>in</strong> defense rather than <strong>in</strong><br />

promotion.<br />

In a recent study, Fu et al [9] found that the <strong>in</strong>cidence<br />

<strong>of</strong> ILFs <strong>in</strong> early human colorectal neoplasms significantly<br />

differs by gender, location, macroscopic type, and histology,<br />

but moreover, their localization significantly differs<br />

by macroscopic type.<br />

In squamous cell carc<strong>in</strong>omas <strong>of</strong> the esophagus, cycl<strong>in</strong><br />

A expression <strong>in</strong> the germ<strong>in</strong>al center cells <strong>of</strong> ILFs beneath<br />

the superficial tumorous lesions was shown to be an immunological<br />

signal toward the proliferation and progression<br />

<strong>of</strong> the tumors [82] . Gutfeld et al [83] found that the cells<br />

<strong>of</strong> <strong>colon</strong>ic ILFs, <strong>in</strong>flammatory cells, ganglion cells, and<br />

endothelial cells express serum amyloid A, an acute phase<br />

reactant, whose level <strong>in</strong> the blood is elevated <strong>in</strong> response<br />

to trauma, <strong>in</strong>fection, <strong>in</strong>flammation, and neoplasia, on<br />

both mRNA and prote<strong>in</strong> levels. The serum amyloid A<br />

mRNA expression <strong>in</strong> epithelial cells was found to gradually<br />

<strong>in</strong>crease as it progressed through different stages <strong>of</strong><br />

dysplasia to overt carc<strong>in</strong>oma. While expression <strong>of</strong> the<br />

serum amyloid A1 and -4 genes <strong>in</strong> <strong>colon</strong> carc<strong>in</strong>omas was<br />

confirmed by RT-PCR analysis, this expression was barely<br />

detectable <strong>in</strong> normal <strong>colon</strong> tissues. Their f<strong>in</strong>d<strong>in</strong>gs <strong>in</strong>dicate<br />

local and differential expression <strong>of</strong> serum amyloid A <strong>in</strong><br />

human <strong>colon</strong> cancer and tumor-associated ILFs, and suggest<br />

its role <strong>in</strong> colorectal carc<strong>in</strong>ogenesis.<br />

WJG|www.wjgnet.com<br />

MESENCHYMAL-EPITHELIAL AND EPI-<br />

THELIAL-MESENCHYMAL TRANSITION<br />

IN ILFS<br />

Epithelial-mesenchymal transition (EMT) is a physiological<br />

mechanism present dur<strong>in</strong>g development, and is also<br />

encountered <strong>in</strong> several pathological situations such as renal<br />

<strong>in</strong>terstitial fibrosis, endometrial adhesion, and cancer metastasis<br />

[84] . A reverse phenomenon, mesenchymal-epithelial<br />

transition (MET) also takes place dur<strong>in</strong>g normal development<br />

<strong>in</strong> processes such as somitogenesis, kidney development<br />

and coelomic cavity formation [85] . In adult organisms,<br />

it has been proposed that restrictive mechanisms repress<br />

EMT and MET [86] . Dur<strong>in</strong>g tumor development, these<br />

mechanisms appear to fail, allow<strong>in</strong>g EMT described <strong>in</strong> metastasis<br />

generation [87] .<br />

In <strong>in</strong>flammation, MET can also be altered because<br />

mesenchymal stem cells are mobilized to these sites <strong>of</strong><br />

<strong>in</strong>jury and consequently subjected to the <strong>in</strong>flammatory response<br />

[88] . BMDCs could differentiate <strong>in</strong>to mature-appear<strong>in</strong>g<br />

epithelial cells <strong>in</strong> response to tissue damage [89] . It was<br />

recently published that versican, a large chondroit<strong>in</strong> sulfate<br />

proteoglycan, mediates MET [90] . The results <strong>of</strong> Hirose<br />

et al [91] <strong>in</strong>dicate that versican can b<strong>in</strong>d specific chemok<strong>in</strong>es<br />

through its chondroit<strong>in</strong> sulfate cha<strong>in</strong>s and that the b<strong>in</strong>d<strong>in</strong>g<br />

tends to down-regulate the chemok<strong>in</strong>e function. This<br />

raises the possibility that versican may act as a regenerative<br />

factor <strong>in</strong> <strong>colon</strong>ic mucosa, and may be an important switch<br />

po<strong>in</strong>t between ILFs and MET. The presence <strong>of</strong> CDX2<br />

and cytokerat<strong>in</strong> positive subepithelial cells <strong>in</strong> the marg<strong>in</strong>al<br />

zone <strong>of</strong> ILFs also suggests that MET may take place <strong>in</strong><br />

these immune formations [2] .<br />

Stroma-tissue, <strong>in</strong>clud<strong>in</strong>g <strong>lymphoid</strong> aggregates and ILFs<br />

surround<strong>in</strong>g the cancer cells, plays an important role <strong>in</strong> the<br />

tumor behavior. Mesker et al [92] analyzed the expression <strong>of</strong><br />

markers <strong>in</strong>volved <strong>in</strong> pathways related to stroma production<br />

and EMT (β-caten<strong>in</strong>, TGF-β-R2, SMAD4) <strong>in</strong> highrisk<br />

colorectal cancer patients, and found that patients with<br />

stroma-high and SMAD4 loss are <strong>of</strong> high risk. The anti-<br />

EMT effect <strong>of</strong> SMAD4 was also proven <strong>in</strong> <strong>colon</strong> carc<strong>in</strong>oma<br />

cells [93] .<br />

CONCLUSION<br />

Based on the summarized results <strong>of</strong> literature, it seems<br />

that ILFs act like a switch between <strong>colon</strong>ic mucosal regeneration<br />

and colorectal carc<strong>in</strong>ogenesis.<br />

Subepithelial revascularization after mucosal damage<br />

takes place partly under the direction <strong>of</strong> ILFs with the<br />

prom<strong>in</strong>ent help <strong>of</strong> vascular endothelial growth factor and<br />

its receptors. Immigrat<strong>in</strong>g stem cells from bone marrow<br />

may leave circulation via high endothelial venules <strong>in</strong> ILFs<br />

and their surround<strong>in</strong>gs. Their differentiation throughout<br />

mesenchymal-to-epithelial transition may also happen <strong>in</strong><br />

ILFs, and follicular dendritic cells, as well as the subepithelial<br />

my<strong>of</strong>ibroblasts, seem to be crucial parts <strong>of</strong> <strong>colon</strong>ic<br />

crypt formation and epithelial renewal.<br />

Vasculogenesis <strong>in</strong> ILFs supports not just tumor growth<br />

1670 April 7, 2011|Volume 17|Issue 13|

---

and the metastatic process, but the VEGF receptor signal<strong>in</strong>g<br />

acts like a direct growth factor for tumor cells. The fusion<br />

<strong>of</strong> BMDCs immigrat<strong>in</strong>g to ILFs with tumor cells may<br />

expla<strong>in</strong> EMT <strong>in</strong> colorectal cancers. The presence <strong>of</strong> ILFs,<br />

dendritic cells and subepithelial my<strong>of</strong>ibroblasts may also<br />

result <strong>in</strong> a specific milieu for tumor formation, growth and<br />

<strong>in</strong>vasion.<br />

Better understand<strong>in</strong>g <strong>of</strong> the role <strong>of</strong> ILFs <strong>in</strong> mucosal<br />

repair may lead to the development <strong>of</strong> new therapeutic<br />

agents for <strong>in</strong>flammatory <strong>colon</strong> diseases that not only decrease<br />

the activity <strong>of</strong> <strong>in</strong>flammation, but also accelerate<br />

epithelial barrier recovery, hence dramatically decreas<strong>in</strong>g<br />

cl<strong>in</strong>ical symptoms. Moreover, by reveal<strong>in</strong>g the exact connections<br />

between ILFs and colorectal carc<strong>in</strong>ogenesis, the<br />

basis <strong>of</strong> <strong>in</strong>dividualized anti-cancer immunotherapies may<br />

be established.<br />

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Medzhitov R. Recognition <strong>of</strong> commensal micr<strong>of</strong>lora by tolllike<br />

receptors is required for <strong>in</strong>test<strong>in</strong>al homeostasis. Cell 2004;<br />

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65 Li M, Carpio DF, Zheng Y, Bruzzo P, S<strong>in</strong>gh V, Ouaaz F,<br />

Medzhitov RM, Beg AA. An essential role <strong>of</strong> the NF-kappa<br />

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and tissue-repair gene expression by necrotic cells. J Immunol<br />

2001; 166: 7128-7135<br />

66 Beg AA. Endogenous ligands <strong>of</strong> Toll-like receptors: implications<br />

for regulat<strong>in</strong>g <strong>in</strong>flammatory and immune responses.<br />

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67 Taylor KR, Trowbridge JM, Rudisill JA, Termeer CC, Simon<br />

JC, Gallo RL. Hyaluronan fragments stimulate endothelial<br />

recognition <strong>of</strong> <strong>in</strong>jury through TLR4. J Biol Chem 2004; 279:<br />

17079-17084<br />

68 Majors AK, Aust<strong>in</strong> RC, de la Motte CA, Pyeritz RE, Hascall<br />

VC, Kessler SP, Sen G, Strong SA. Endoplasmic reticulum<br />

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69 Tang XY, Zhu YQ, Wei B, Wang H. Expression and functional<br />

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70 Zhou B, Zhou H, L<strong>in</strong>g S, Guo D, Yan Y, Zhou F, Wu Y.<br />

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proliferation and migration via phosphorylation <strong>of</strong> ERK1/2.<br />

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71 Gasser M, Grimm M, Kim M, Rosenwald A, Germer CT,<br />

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progression. J Cl<strong>in</strong> Oncol 2010; 28: 15s<br />

72 Onishi S, Miyata H, Inamoto T, Qi WM, Yamamoto K,<br />

Yokoyama T, Warita K, Hoshi N, Kitagawa H. Immunohistochemical<br />

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apoptosis <strong>in</strong> follicle-associated epithelium <strong>of</strong> rat Peyer’s<br />

patch. J Vet Med Sci 2007; 69: 1123-1129<br />

73 Renes IB, Verburg M, Buls<strong>in</strong>g NP, Ferd<strong>in</strong>andusse S, Büller<br />

HA, Dekker J, E<strong>in</strong>erhand AW. Protection <strong>of</strong> the Peyer’s<br />

patch-associated crypt and villus epithelium aga<strong>in</strong>st methotrexate-<strong>in</strong>duced<br />

damage is based on its dist<strong>in</strong>ct regulation<br />

<strong>of</strong> proliferation. J Pathol 2002; 198: 60-68<br />

74 Cadigan KM, Nusse R. Wnt signal<strong>in</strong>g: a common theme <strong>in</strong><br />

animal development. Genes Dev 1997; 11: 3286-3305<br />

75 DasGupta R, Fuchs E. Multiple roles for activated LEF/TCF<br />

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76 Kor<strong>in</strong>ek V, Barker N, Moerer P, van Donselaar E, Huls G,<br />

Peters PJ, Clevers H. Depletion <strong>of</strong> epithelial stem-cell compartments<br />

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Genet 1998; 19: 379-383<br />

77 Haegebarth A, Clevers H. Wnt signal<strong>in</strong>g, lgr5, and stem<br />

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78 Ouji Y, Yoshikawa M, Shiroi A, Ishizaka S. Wnt-10b secreted<br />

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cells. Biochem Biophys Res Commun 2006; 342: 1063-1069<br />

79 Malhotra S, K<strong>in</strong>cade PW. Wnt-related molecules and signal<strong>in</strong>g<br />

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80 O’Leary AD, Sweeney EC. Lymphoglandular complexes <strong>of</strong><br />

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81 Michael-Rob<strong>in</strong>son JM, Biemer-Hüttmann A, Purdie DM,<br />

Walsh MD, Simms LA, Biden KG, Young JP, Leggett BA, Jass<br />

JR, Radford-Smith GL. Tumour <strong>in</strong>filtrat<strong>in</strong>g lymphocytes and<br />

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accord<strong>in</strong>g to microsatellite <strong>in</strong>stability status. Gut 2001; 48:<br />

360-366<br />

82 Nozoe T, Korenaga D, Futatsugi M, Saeki H, Ohga T, Sugi-<br />

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Sipos F et al . Colonic isolated <strong>lymphoid</strong> <strong>follicles</strong><br />

machi K. Cycl<strong>in</strong> A expression <strong>in</strong> superficial squamous cell<br />

carc<strong>in</strong>oma <strong>of</strong> the esophagus and coexist<strong>in</strong>g <strong>in</strong>filtrated lymphocyte<br />

follicle. Cancer Lett 2002; 188: 221-229<br />

83 Gutfeld O, Prus D, Ackerman Z, Dishon S, L<strong>in</strong>ke RP, Lev<strong>in</strong><br />

M, Urieli-Shoval S. Expression <strong>of</strong> serum amyloid A, <strong>in</strong><br />

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implication for a role <strong>in</strong> <strong>colon</strong>ic tumorigenesis. J Histochem<br />

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84 Thiery JP. Epithelial-mesenchymal transitions <strong>in</strong> development<br />

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85 Rubio D, Garcia S, De la Cueva T, Paz MF, Lloyd AC, Bernad A,<br />

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86 Pr<strong>in</strong>dull G, Zipori D. Environmental guidance <strong>of</strong> normal<br />

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87 Kang Y, Massagué J. Epithelial-mesenchymal transitions:<br />

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88 Spees JL, Olson SD, Ylostalo J, Lynch PJ, Smith J, Perry A,<br />

Peister A, Wang MY, Prockop DJ. Differentiation, cell fusion,<br />

and nuclear fusion dur<strong>in</strong>g ex vivo repair <strong>of</strong> epithelium<br />

by human adult stem cells from bone marrow stroma. Proc<br />

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89 Van Arnam JS, Herzog E, Grove J, Bruscia E, Ziegler E, Swenson<br />

S, Krause DS. Engraftment <strong>of</strong> bone marrow-derived<br />

epithelial cells. Stem Cell Rev 2005; 1: 21-27<br />

90 Sheng W, Wang G, La Pierre DP, Wen J, Deng Z, Wong CK,<br />

Lee DY, Yang BB. Versican mediates mesenchymal-epithelial<br />

transition. Mol Biol Cell 2006; 17: 2009-2020<br />

91 Hirose J, Kawashima H, Yoshie O, Tashiro K, Miyasaka M.<br />

Versican <strong>in</strong>teracts with chemok<strong>in</strong>es and modulates cellular<br />

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92 Mesker WE, Liefers GJ, Junggeburt JM, van Pelt GW, Alberici<br />

P, Kuppen PJ, Miranda NF, van Leeuwen KA, Morreau<br />

H, Szuhai K, Tollenaar RA, Tanke HJ. Presence <strong>of</strong> a<br />

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93 Pohl M, Radacz Y, Pawlik N, Schoeneck A, Baldus SE,<br />

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2603-2613<br />

S- Editor Tian L L- Editor Rutherford A E- Editor Ma WH<br />

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INTRODUCTION<br />

Over the past two decades, there have been improvements<br />

<strong>in</strong> the management <strong>of</strong> rectal cancer <strong>in</strong> terms <strong>of</strong> postoperative<br />

death (fall<strong>in</strong>g from 10% to 2%), locoregional failure<br />

(dropp<strong>in</strong>g from 30%-40% to less than 15%), conservative<br />

surgery rates (<strong>in</strong>creas<strong>in</strong>g from 20% to 60%) and survival,<br />

with advances made <strong>in</strong> the understand<strong>in</strong>g <strong>of</strong> the biology<br />

<strong>of</strong> this type <strong>of</strong> tumor as well as stag<strong>in</strong>g and the use <strong>of</strong><br />

comb<strong>in</strong>ed therapies [1] . The anatomical and technical basis<br />

<strong>of</strong> tumor recurrence with<strong>in</strong> the pelvis has been extensively<br />

<strong>in</strong>vestigated by surgeons and pathologists, and this has led<br />

to major improvements <strong>in</strong> surgical therapy. Nonetheless,<br />

although surgery rema<strong>in</strong>s the ma<strong>in</strong>stay <strong>of</strong> treatment aimed<br />

at achiev<strong>in</strong>g locoregional control, nowadays the therapeutic<br />

approach to rectal cancer is em<strong>in</strong>ently multidiscipl<strong>in</strong>ary.<br />

The key to successful surgery is complete excision <strong>of</strong><br />

the tumor proximally, distally and around its circumference<br />

with sufficient marg<strong>in</strong> <strong>of</strong> normal tissue (R0 resection). In<br />

total mesorectal excision (TME) surgery the rectum and<br />

its perirectal lymphatic and fatty tissue (mesorectum) is<br />

completely mobilized by sharp dissection, as an <strong>in</strong>tact package<br />

surrounded by an undamaged peri-mesorectal layer <strong>of</strong><br />

proper rectal (visceral) fascia, avoid<strong>in</strong>g spillage and growth<br />

<strong>of</strong> residual tumor cells <strong>in</strong>to the pelvis and subsequent<br />

development <strong>of</strong> a local recurrence (LR). Educational<br />

programs aimed at tra<strong>in</strong><strong>in</strong>g surgeons <strong>in</strong> this pelvic dissection<br />

technique have demonstrated reproducible results <strong>in</strong><br />

achiev<strong>in</strong>g a reduction <strong>of</strong> the LR <strong>of</strong> rectal cancer rate by<br />

40% [2] and even greater when associated with radiotherapy<br />

(RT) [3] or neoadjuvant chemoradiotherapy (CRT) [4] .<br />

Nevertheless, LR <strong>of</strong> rectal cancer rema<strong>in</strong>s a significant<br />

cl<strong>in</strong>ical problem, associated with severe morbidity,<br />

low rates <strong>of</strong> success <strong>of</strong> salvage procedures, and eventual<br />

death <strong>in</strong> the majority <strong>of</strong> patients [5] .<br />

It is important to review the patterns <strong>of</strong> treatment<br />

failure result<strong>in</strong>g after rectal cancer management. Improvements<br />

<strong>in</strong> surgical and adjuvant therapies may affect not only<br />

the likelihood <strong>of</strong> tumor recurrence <strong>in</strong> the pelvis but also<br />

the pattern <strong>of</strong> pelvic recurrence itself (i.e. pelvic subsites).<br />

Knowledge <strong>of</strong> the pattern and natural history <strong>of</strong> LR, the<br />

associated risk factors for their development and the mechanism<br />

by which they occur may serve as the foundation for<br />

efforts to improve the results <strong>of</strong> multidiscipl<strong>in</strong>ary care (i.e.<br />

RT field design, suitability <strong>of</strong> lymphadenectomies, strategy<br />

<strong>in</strong> the follow-up monitor<strong>in</strong>g, etc.).<br />

The aim <strong>of</strong> this review is to characterize and analyze<br />

the pattern <strong>of</strong> LR today follow<strong>in</strong>g different curative approaches<br />

for rectal cancer, with special emphasis on the<br />

correlation between subsites <strong>of</strong> pelvic recurrences and<br />

treatment modalities.<br />

LIMITATIONS OF THIS REVIEW<br />

Many <strong>of</strong> the studies that report patterns <strong>of</strong> pelvic recurrence<br />

have multiple limitations. Some are outdated or do<br />

not give exact anatomical <strong>in</strong>formation <strong>of</strong> the location <strong>of</strong><br />

recurrent tumors <strong>in</strong> the pelvis. In particular, the diagnostic<br />

procedures, methods <strong>of</strong> documentation, acknowledge-<br />

WJG|www.wjgnet.com<br />

Enríquez-Navascués JM et al . Recurrences after curative treatment <strong>of</strong> rectal cancer<br />

ment or confirmation <strong>of</strong> diagnosis, presence or absence<br />

<strong>of</strong> histology, the use <strong>of</strong> <strong>in</strong>terval pa<strong>in</strong>, anatomic def<strong>in</strong>itions<br />

<strong>of</strong> the rectum, first site <strong>of</strong> recurrence and cumulative<br />

recurrence data, as well as the def<strong>in</strong>ition <strong>of</strong> the LR itself<br />

and other details all affect the analysis <strong>of</strong> <strong>in</strong>cidence rates,<br />

tim<strong>in</strong>g, and patterns [6] .<br />

Old literature concern<strong>in</strong>g the pattern <strong>of</strong> local failure<br />

<strong>in</strong> rectal cancer was based on planned or symptomatic reoperations<br />

data or autopsy series. Planned “second look”<br />

procedures and symptomatic surgery were performed <strong>in</strong><br />

the pre-TME surgery era and LR data obta<strong>in</strong>ed may be<br />

outdated. Furthermore, autopsies reveal only the end pattern<br />

<strong>of</strong> failure.<br />

On the other hand, most recent reports are based on<br />

cl<strong>in</strong>ical or imag<strong>in</strong>g data which can be also mislead<strong>in</strong>g as<br />

the methods <strong>of</strong> diagnos<strong>in</strong>g and confirm<strong>in</strong>g LR and length<br />

<strong>of</strong> follow-up are not described consistently. The actual<br />

rate <strong>of</strong> pelvic recurrence may be somewhat higher than<br />

estimated by these reports, as some studies report only<br />

first sites <strong>of</strong> failure, and pelvic relapse later <strong>in</strong> the course<br />

<strong>of</strong> disease is not always assessed <strong>in</strong> patients under palliative<br />

chemotherapy for distant metastasis.<br />

Trials <strong>of</strong> preoperative RT or CRT <strong>in</strong> resectable rectal<br />

cancer are characterized by multiple methodological problems<br />

because treatments are comb<strong>in</strong>ed (RT and surgery)<br />

to address a heterogeneous condition (various populations<br />

and stages <strong>of</strong> rectal carc<strong>in</strong>oma) and to achieve a variety <strong>of</strong><br />

goals (downstag<strong>in</strong>g and improv<strong>in</strong>g resectability, as well as<br />

decreas<strong>in</strong>g local and possibly distant recurrences and improv<strong>in</strong>g<br />

survival).<br />

DEFINITION AND CLASSIFICATION OF<br />

LOCAL RECURRENCE<br />

Although by def<strong>in</strong>ition the term LR is only applicable when<br />

the <strong>in</strong>itial or primary surgery is expected to be curative (no<br />

rema<strong>in</strong><strong>in</strong>g macroscopic evidence <strong>of</strong> disease locally, that is<br />

R0 and R1 accord<strong>in</strong>g to the UICC (International Union<br />

Aga<strong>in</strong>st Cancer), it must be seen as the further development<br />

<strong>of</strong> tumor cell remnants: there is a close biological<br />

similarity between a primary tumor and an LR, <strong>in</strong> contrast<br />

to the situation with correspond<strong>in</strong>g organ metastases [7] .<br />

Recurrent rectal cancer may be isolated (local or metastatic)<br />

or comb<strong>in</strong>ed (local and metastasis). Indeed LR can<br />

be def<strong>in</strong>ed as any tumor located with<strong>in</strong> the pelvis, either<br />

alone or <strong>in</strong> conjunction with metastases [6] . Several authors<br />

have classified locoregional pelvic recurrence <strong>in</strong> order to<br />

facilitate treatment and compare outcomes. Specifically,<br />

the dist<strong>in</strong>ction between localized and diffuse pelvic recurrence<br />

is pivotal <strong>in</strong> def<strong>in</strong><strong>in</strong>g subsequent management and<br />

prognosis.<br />

The Mayo Cl<strong>in</strong>ic [8] described recurrence <strong>in</strong> terms <strong>of</strong><br />

degree <strong>of</strong> fixation both <strong>in</strong> term <strong>of</strong> site (anterior, sacral,<br />

right or left) and number <strong>of</strong> po<strong>in</strong>ts <strong>of</strong> fixation (F0-F3).<br />

Wanebo et al [9] proposed a classification based on the<br />

UICC TNM system: TR1 and TR2 correspond<strong>in</strong>g to<br />

<strong>in</strong>tralum<strong>in</strong>al LR, either follow<strong>in</strong>g local excision or at the<br />

anastomosis; TR3 correspond<strong>in</strong>g to LR at or around the<br />

level <strong>of</strong> the anastomosis with limited extramural spread<br />

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the preoperative RT group (4.7%) than the postoperative<br />

CRT group (11.5%). However, <strong>in</strong> those patients with a<br />

positive CRM, the LR rates were not statistically different<br />

(16% preoperative vs 23% postoperative).<br />

PATTERN OF PELVIC FAILURE AFTER<br />

LONG COURSE RT PLUS CHEMOTHERAPY<br />

Postoperative adjuvant strategies to improve outcomes follow<strong>in</strong>g<br />

rectal resection have ma<strong>in</strong>ly been explored <strong>in</strong> the<br />

United States. Indeed, <strong>in</strong> 1990, after positive trials conducted<br />

by the Gastro<strong>in</strong>test<strong>in</strong>al Tumor Study Group [45] and the<br />

Mayo Cl<strong>in</strong>ic/North Central Cancer Treatment Group [46] ,<br />

the NCI issued a statement declar<strong>in</strong>g comb<strong>in</strong>ed postoperative<br />

therapy the new standard <strong>of</strong> care <strong>in</strong> this sett<strong>in</strong>g [47] .<br />

Researchers, however, were question<strong>in</strong>g whether preoperative<br />

comb<strong>in</strong>ed therapy would be even more beneficial.<br />

In the 1990s, data from the Memorial Sloan-Ketter<strong>in</strong>g Cancer<br />

Center and the MD Anderson Cancer Center accumulated<br />

[48] <strong>in</strong> support <strong>of</strong> that benefit. Moreover, results from<br />

three randomized trials (the Uppsala trial [49] , NSABP R03 [50]<br />

and above all the German CAO/ARO/AIO trial [25] ), demonstrated<br />

the clear superiority <strong>of</strong> preoperative RT regimens<br />

over postoperative therapy <strong>in</strong> terms <strong>of</strong> local control with<br />

better compliance to treatment and lower toxicity.<br />

The next step was to test the hypothesis that chemotherapy<br />

plus preoperative RT significantly improved local<br />

control, tumor downsiz<strong>in</strong>g and downstag<strong>in</strong>g compared<br />

with RT alone. Two randomized trials compared preoperative<br />

RT vs preoperative CRT, the study by the Fédération<br />

Francophone de Cancérologie Digestive (FFCD 9203) [51]<br />

and the EORTC 22921 trial [52] , and similar results were<br />

reported. In the latter, the five-year results showed that<br />

chemotherapy <strong>in</strong>creased the rate <strong>of</strong> pCR (14% vs 5.3%),<br />

translated <strong>in</strong>to a 3% benefit <strong>in</strong> terms <strong>of</strong> sph<strong>in</strong>cter preservation<br />

and significantly reduced LR rate from 17% without<br />

chemotherapy down to 8% with CRT. Thus chemotherapy,<br />

regardless <strong>of</strong> whether it is adm<strong>in</strong>istered before or after surgery,<br />

confers a significant benefit with respect to local control.<br />

The ma<strong>in</strong> criticism that can be made <strong>of</strong> those trials is<br />

that TME resections were not uniformly implemented.<br />

On the other hand, the favorable effect <strong>of</strong> delay<strong>in</strong>g surgery<br />

after CRT on downstag<strong>in</strong>g (and possibly also sph<strong>in</strong>cter<br />

preservation) was shown <strong>in</strong> the Lyon R90-01 trial [53] .<br />

There is, however, limited data on patterns <strong>of</strong> relapse<br />

<strong>in</strong> rectal cancer patients treated with TME surgery and<br />

CRT. Such <strong>in</strong>formation might help determ<strong>in</strong>e whether<br />

modifications <strong>in</strong> RT dose or field design are warranted (i.e.<br />

a local recurrence after RT may be <strong>in</strong>side or outside the<br />

RT field; recurrence outside the field requires an <strong>in</strong>crease<br />

<strong>in</strong> the size <strong>of</strong> the field, and recurrence <strong>in</strong>side the field implies<br />

the need for an <strong>in</strong>crease <strong>in</strong> the total dose). We have<br />

identified reports on only four series <strong>of</strong> patients that conta<strong>in</strong><br />

detailed analysis <strong>of</strong> the pattern <strong>of</strong> pelvic recurrence<br />

after CRT and TME surgery.<br />

From the MD Anderson Cancer center, Yu et al [54]<br />

presented a thorough study attempt<strong>in</strong>g to identify subsites<br />

<strong>of</strong> pelvic LR <strong>in</strong> an effort to correlate sites <strong>of</strong> relapse on<br />

WJG|www.wjgnet.com<br />

Enríquez-Navascués JM et al . Recurrences after curative treatment <strong>of</strong> rectal cancer<br />

CT images with RT simulation films <strong>in</strong> 46 rectal cancer<br />

patients. Of all the LR, approximately two-thirds were <strong>in</strong>field<br />

(with<strong>in</strong> the radiation field) recurrences and only onethird<br />

were marg<strong>in</strong>al (<strong>in</strong>side but with<strong>in</strong> 1 cm <strong>of</strong> the border<br />

<strong>of</strong> the field) or out-<strong>of</strong>-field (more than 1 cm from the<br />

border) recurrences. Of the <strong>in</strong>-field recurrences, nearly<br />

80% occurred <strong>in</strong> the low pelvic and presacral regions.<br />

Multivariate analysis showed that the risk <strong>of</strong> <strong>in</strong>-field LR<br />

was significantly associated with pathological N stage,<br />

while it was notably not with positive CRM or downstag<strong>in</strong>g.<br />

The authors suggested various strategies to improve<br />

locoregional control <strong>in</strong> low pelvic and presacral regions.<br />

Hötch et al [12] published a large-scale multicenter study<br />

based <strong>in</strong> Germany to evaluate pelvic sites <strong>of</strong> recurrence<br />

with special attention to radiation ports. Nearly 80% <strong>of</strong><br />

LR occurred with<strong>in</strong> the treated volume, <strong>in</strong> the central<br />

pelvis, and the pelvic sidewall structures were <strong>in</strong>volved<br />

<strong>in</strong> fewer than 5% <strong>of</strong> tumor relapses. They found no significant<br />

differences <strong>in</strong> the <strong>in</strong>cidence <strong>of</strong> pelvic sidewall<br />

<strong>in</strong>volvement between APR and LAR cases, however there<br />

was a significant difference <strong>in</strong> the spread <strong>of</strong> recurrent tumors<br />

<strong>in</strong> the <strong>in</strong>ferior part <strong>of</strong> the pelvis.<br />

However, a quite different picture has been reported<br />

from Korea, where Kim et al [56] exam<strong>in</strong>ed the patterns <strong>of</strong><br />

locoregional recurrences <strong>in</strong> 366 patients with locally advanced<br />

rectal cancer who underwent preoperative CRT and<br />

curative TME surgery, and assessed the effect <strong>of</strong> cl<strong>in</strong>ical<br />

parameters on lateral pelvic recurrence. Eight percent <strong>of</strong><br />

the patients had LR, <strong>of</strong> which around 20% and 80% occurred<br />

<strong>in</strong> central and lateral pelvic areas respectively. Multivariable<br />

analysis showed that lateral pelvic recurrence was<br />

significantly associated with ypN classification (lymph node<br />

status after preoperative CRT) and lateral lymph node size.<br />

The authors suggested that lateral lymph node metastasis<br />

is a risk factor for LR and could be a potentially curable<br />

regional disease rather than a sign <strong>of</strong> systemic disease. Accord<strong>in</strong>gly,<br />

they suggested that patients with lateral lymph<br />

node size <strong>of</strong> > 10 mm and ypN0 or lateral lymph node size<br />

<strong>of</strong> 5 mm and ypN+ are a potential subgroup <strong>of</strong> patients<br />

who might benefit from lateral lymph node dissection.<br />

In tumors <strong>of</strong> the middle and lower rectum, lateral<br />

lymph nodes rema<strong>in</strong> a potential cause <strong>of</strong> locoregional<br />

recurrence after conventional TME because they are not<br />

removed. EL has been championed ma<strong>in</strong>ly by Asian surgeons,<br />

who are <strong>in</strong>ternationally renowned for their skills <strong>in</strong><br />

radical surgery.<br />

PATTERN OF RECURRENCES AFTER<br />

EXTENDED LYMPHADENECTOMY AND<br />

TME, WITH OR WITHOUT ADJUVANT<br />

TREATMENT<br />

Around 40% <strong>of</strong> patients treated for rectal cancer present<br />

with lymph node metastases, which occur along the mesorectal<br />

nodal cha<strong>in</strong>, along the <strong>in</strong>ferior mesenteric artery<br />

lymph nodes or <strong>in</strong> the lateral pelvic lymph nodes (along<br />

the obturator, <strong>in</strong>ternal iliac or medial aspect <strong>of</strong> the external<br />

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Table 2 Relative <strong>in</strong>cidence <strong>of</strong> sub-site locations <strong>of</strong> pelvic recurrences<br />

Authors (yr) Treatment Axial or central (%) Lateral (%) Other (%)<br />

patients with advanced rectal cancer could not be safely<br />

answered by this meta-analysis.<br />

PATTERNS OF RECURRENCE AFTER IN-<br />

TRA-OPERATIVE RADIOTHERAPY PLUS<br />

EXTENDED SURGERY<br />

Several authors reported the impact <strong>of</strong> <strong>in</strong>tra-operative radiotherapy<br />

(IORT) with or without preoperative external<br />

beam irradiation and surgical resection <strong>in</strong> patients with<br />

locally advanced or recurrent cancer. The overall available<br />

data on IORT showed a favorable impact on local control<br />

and <strong>in</strong> overall survival for patients resected for cure (R0,<br />

R1); However, there is a need for randomized studies <strong>of</strong><br />

the effect <strong>of</strong> IORT. From a Dutch national referral center,<br />

the pattern <strong>of</strong> LR <strong>in</strong> 247 patients with locally advanced<br />

rectal carc<strong>in</strong>oma after IORT <strong>in</strong>clud<strong>in</strong>g multimodal treatment<br />

(preoperative CRT and extended surgery) has been<br />

analyzed <strong>in</strong> detail [55] . The 5-year LR rate was 13.2% (7.5%<br />

after R0 resections). The most prom<strong>in</strong>ent sites <strong>of</strong> LR<br />

were the presacral (44%) followed by the anterior (21%)<br />

subsites and lateral spread accounted for less than 10%<br />

<strong>of</strong> recurrences. Around 50% <strong>of</strong> the LRs appeared <strong>in</strong> the<br />

IORT field, particularly high rates <strong>of</strong> <strong>in</strong>field recurrences<br />

be<strong>in</strong>g observed after dorsal IORT (75%). The authors hypothesize<br />

that migration <strong>of</strong> rema<strong>in</strong><strong>in</strong>g tumor cells to the<br />

presacral space would expla<strong>in</strong> the occurrence <strong>of</strong> this LR.<br />

PATTERNS OF RECURRENCE FOLLOW-<br />

ING COMPLETE CLINICAL RESPONSE<br />

AFTER CRT<br />

The def<strong>in</strong>itive role <strong>of</strong> an <strong>in</strong>itially non-surgical approach<br />

to treatment follow<strong>in</strong>g complete cl<strong>in</strong>ical response (cCR)<br />

after CRT has not yet been determ<strong>in</strong>ed and no def<strong>in</strong>itive<br />

conclusions can be drawn before long-term results concern<strong>in</strong>g<br />

LR and distant failure are available.<br />

WJG|www.wjgnet.com<br />

Enríquez-Navascués JM et al . Recurrences after curative treatment <strong>of</strong> rectal cancer<br />

Anastomotic<br />

(perianastomotic)<br />

Anterior<br />

(genitour<strong>in</strong>ary)<br />

Posterior<br />

(presacral)<br />

However, Habr-Gama et al [66] have reported the pattern<br />

<strong>of</strong> recurrence and survival <strong>of</strong> 99 patients with distal rectal<br />

cancer (0-7 cm) and cCR follow<strong>in</strong>g adjuvant CRT, susta<strong>in</strong>ed<br />

for at least 12 mo, managed by <strong>in</strong>itial non-operative<br />

treatment. They observed 13 recurrences: five endorectal<br />

(limited to the rectal wall), seven systemic, one comb<strong>in</strong>ed<br />

(endorectal and distant), and no pelvic recurrence outside<br />

the rectal wall was detected. There were no significant cl<strong>in</strong>ical<br />

differences either between patients with and without<br />

recurrence, or these same patients accord<strong>in</strong>g to the location<br />

<strong>of</strong> the recurrence. Surpris<strong>in</strong>gly, systemic recurrence occurred<br />

sooner than LR. The authors suggest that a change<br />

<strong>in</strong> the approach to follow-up monitor<strong>in</strong>g may be necessary.<br />

CLOSING REMARKS<br />

Per<strong>in</strong>eal<br />

Gunderson et al [31]<br />

Pre-TME surgery - 1 40 19 31 10<br />

Pilipshen et al [32] (1968-1976) Pre-TME surgery 40 10 40 2<br />

10<br />

Hruby et al [33] (1979-1996) Pre-TME surgery 21 10.7 47 11 11<br />

Dutch Trial [41] (1996-1999) TME surgery 24 18 32 5 18 2.5<br />

Dutch Trial [41] (1996-1999) sRT+ TME 13 16 41 - 25 2.7<br />

Syk et al [40] (1995-1999) sRT +TME 37 3 30 10 18<br />

Yu et al [54] (1989-2001) CRT+ CRT - 44 28 - 10 18.0<br />

Hötch et al [12] (1998-2001) CRT+TME 60 4<br />

29 10<br />

Kim et al [56] (2001-2005) CRT+TME 20 5<br />

80<br />

Kusters et al [58] (1993-2002) Unilateral EL 25 - 16 16 40 6<br />

Kusters et al [58] (1993-2002) Bilateral EL 20 10 16 16 40<br />

APR: Abdom<strong>in</strong>oper<strong>in</strong>eal resection; Anast: Anastomotic; per<strong>in</strong>: Per<strong>in</strong>eal; Ant: Anterior. 1 0% were APR; 2 Post + per<strong>in</strong>eum; 3 Anast + ant; 4 Per<strong>in</strong> + ant +<br />

anast; 5 Axial o central; 6 20% ipsilateral and 20% contralateral. TME: Total mesorectal excision; sRT: Short-term preoperative radiotherapy; EL: Extended<br />

lymphadenectomy; CRT: Chemoradiotherapy.<br />

The reported patterns and rates <strong>of</strong> local recurrence, after<br />

the aforementioned range <strong>of</strong> treatment approaches to<br />

rectal cancer, used <strong>in</strong> isolation and as comb<strong>in</strong>ed therapies<br />

are summarized <strong>in</strong> Table 1. Overall, comb<strong>in</strong><strong>in</strong>g therapies<br />

reduces LR rates, delays the appearance <strong>of</strong> LR and means<br />

that when there is recurrence it is less <strong>of</strong>ten isolated than<br />

after surgery alone.<br />

Table 2 lists the relative frequency <strong>of</strong> LRs <strong>in</strong> various<br />

pelvic subsites. In recent years, a subtle change has been<br />

observed <strong>in</strong> the distribution <strong>of</strong> LRs <strong>in</strong> terms <strong>of</strong> location<br />

with<strong>in</strong> the pelvis, imply<strong>in</strong>g the <strong>in</strong>volvement <strong>of</strong> a different<br />

mechanism <strong>in</strong> their development. In general terms, <strong>in</strong> the<br />

pre-TME years most recurrences were central, perianastomotic<br />

and anterior and s<strong>in</strong>ce the adoption <strong>of</strong> comb<strong>in</strong>ed<br />

therapies lateral and posterior (presacral) forms dom<strong>in</strong>ate.<br />

However, the LR distribution is not only related to therapy<br />

modality but also to the height <strong>of</strong> the tumor. LRs <strong>in</strong> the<br />

upper rectum are relatively rare, but when they do occur<br />

are usually perianastomotic, orig<strong>in</strong>at<strong>in</strong>g <strong>in</strong> the residual mesorectal<br />

fatty tissue (as they are treated with partial mesorectal<br />

excision, transect<strong>in</strong>g the rectum at about 5 cm below<br />

the tumor), and are comparatively more common when<br />

only surgical treatment is used. This suggests that while<br />

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Enríquez-Navascués JM et al . Recurrences after curative treatment <strong>of</strong> rectal cancer<br />

preoperative RT helps to prevent LRs at all sites, it is especially<br />

effective <strong>in</strong> prevent<strong>in</strong>g anastomotic recurrences [67] .<br />

<strong>Isolated</strong> anastomotic recurrences are also seen <strong>in</strong> select<br />

cases <strong>of</strong> very low rectal cancer treated us<strong>in</strong>g <strong>in</strong>tersph<strong>in</strong>cteric<br />

resection (ISR) [68] . Surgical technique and attention<br />

to distal marg<strong>in</strong> can also play a role <strong>in</strong> prevent<strong>in</strong>g this type<br />

<strong>of</strong> LR. The LR rate after ISR is higher <strong>in</strong> poorly selected<br />

cases <strong>of</strong> pT3 with no previous RT, due to accidental tumor<br />

spillage <strong>in</strong>to the <strong>in</strong>tersph<strong>in</strong>cteric space or positive CRM [69] .<br />

A lower LR rate has been reported with stapled coloanal<br />

anastomosis than for ISR even <strong>in</strong> T1-T2 patients [70] . As expected,<br />

after transanal endoscopic microsurgery, <strong>in</strong>tramural<br />

recurrence is the most common type <strong>of</strong> LR [71] .<br />

Most local recurrences <strong>of</strong> mid-rectal cancers treated<br />

with RT or CRT + TME are related to the advanced<br />

nature <strong>of</strong> the disease. Tumor height <strong>of</strong> 5 cm or more is<br />

associated with a higher <strong>in</strong>cidence <strong>of</strong> presacral and lateral<br />

LR [72] . If the CRM is found to be positive after CRT, the<br />

hazard ratio for LR after surgery is significantly higher<br />

than if the CMR is <strong>in</strong>volved when no preoperative CRT<br />

has been adm<strong>in</strong>istered (6.3 vs 2.0), possibly because <strong>of</strong><br />

selection <strong>of</strong> a population <strong>of</strong> tumor cells that are resistant<br />

to therapy [73] . LR may also sometimes occur even <strong>in</strong> the<br />

absence <strong>of</strong> an <strong>in</strong>volved CRM possibly ow<strong>in</strong>g to lymphatic<br />

spread from the distal rectum to lymph nodes <strong>in</strong> the pelvic<br />

side wall [57] . Unilateral EL (lateral lymph nodes on one<br />

side <strong>of</strong> the pelvis are left <strong>in</strong>tact) result <strong>in</strong> more LR than bilateral<br />

ELs, and it has been suggested that the mechanism<br />

for the formation <strong>of</strong> posterior-lateral recurrences may be<br />

the migration <strong>of</strong> tumor cells through the lateral lymphatic<br />

vessels to the presacral space under gravity [55] . This would<br />

expla<strong>in</strong> why presacral local recurrence is more common <strong>in</strong><br />

advanced disease than <strong>in</strong> limited disease.<br />

The highest rates <strong>of</strong> positive CRM are found with lower<br />

rectum tumors. Indeed, TME surgery is not a universal<br />

solution for all rectal carc<strong>in</strong>omas: <strong>in</strong> low rectal cancer<br />

TME may be <strong>in</strong>sufficient to obta<strong>in</strong> the desired circumferential<br />

clearance because <strong>of</strong> this lack <strong>of</strong> mesorectum at the<br />

level <strong>of</strong> the pelvic floor. On the other hand, APR surgery<br />

ma<strong>in</strong>ly results <strong>in</strong> per<strong>in</strong>eal and presacral LR, which may be<br />

prevented by a wider resection [37] .<br />

The pelvic pattern <strong>of</strong> recurrence itself (i.e. pelvic subsites)<br />

also has important prognostic value and is related to<br />

the potential success <strong>of</strong> repeat curative <strong>in</strong>tent surgery [74,75] .<br />

The operability for cure and prognosis for anastomotic and<br />

anterior recurrence are generally better than for presacral<br />

and lateral recurrences [75] . Moreover, the upper sacral/lateral<br />

<strong>in</strong>vasive type <strong>of</strong> LR is <strong>of</strong>ten associated with synchronous<br />

metastatic disease [74] . The type <strong>of</strong> pelvic <strong>in</strong>vasion is<br />

also closely associated with survival after re-resection [74-76] .<br />

F<strong>in</strong>ally, it is worth not<strong>in</strong>g that overall survival after LR diagnosis<br />

is lower with RT and CRT+TME approaches, than<br />

after TME surgery alone [74] .<br />

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71 Bach SP, Hill J, Monson JR, Simson JN, Lane L, Merrie A,<br />

Warren B, Mortensen NJ. A predictive model for local recurrence<br />

after transanal endoscopic microsurgery for rectal<br />

cancer. Br J Surg 2009; 96: 280-290<br />

72 Nagtegaal ID, Quirke P. What is the role for the circumferential<br />

marg<strong>in</strong> <strong>in</strong> the modern treatment <strong>of</strong> rectal cancer? J<br />

Cl<strong>in</strong> Oncol 2008; 26: 303-312<br />

73 den Dulk M, Collette L, van de Velde CJ, Marijnen CA, Calais<br />

G, M<strong>in</strong>eur L, Ma<strong>in</strong>gon P, Radosevic-Jelic L, Daban A, Bosset<br />

JF. Quality <strong>of</strong> surgery <strong>in</strong> T3-4 rectal cancer: <strong>in</strong>volvement <strong>of</strong> circumferential<br />

resection marg<strong>in</strong> not <strong>in</strong>fluenced by preoperative<br />

treatment. Results from EORTC trial 22921. Eur J Cancer 2007;<br />

43: 1821-1828<br />

74 Kusters M, Dresen RC, Martijn H, Nieuwenhuijzen GA, van<br />

de Velde CJ, van den Berg HA, Beets-Tan RG, Rutten HJ. Radicality<br />

<strong>of</strong> resection and survival after multimodality treatment<br />

is <strong>in</strong>fluenced by subsite <strong>of</strong> locally recurrent rectal cancer. Int J<br />

Radiat Oncol Biol Phys 2009; 75: 1444-1449<br />

75 Kanemitsu Y, Hirai T, Komori K, Kato T. Prediction <strong>of</strong> residual<br />

disease or distant metastasis after resection <strong>of</strong> locally<br />

recurrent rectal cancer. Dis Colon Rectum 2010; 53: 779-789<br />

76 Yamada K, Ishizawa T, Niwa K, Chuman Y, Akiba S, Aikou T.<br />

Patterns <strong>of</strong> pelvic <strong>in</strong>vasion are prognostic <strong>in</strong> the treatment <strong>of</strong><br />

locally recurrent rectal cancer. Br J Surg 2001; 88: 988-993<br />

S- Editor Tian L L- Editor Cant MR E- Editor Ma WH<br />

1684 April 7, 2011|Volume 17|Issue 13|

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Onl<strong>in</strong>e Submissions: http://www.wjgnet.com/1007-9327<strong>of</strong>fice<br />

wjg@wjgnet.com<br />

doi:10.3748/wjg.v17.i13.1685<br />

Therapeutic options for <strong>in</strong>termediate-advanced<br />

hepatocellular carc<strong>in</strong>oma<br />

Zong-M<strong>in</strong>g Zhang, J<strong>in</strong>-X<strong>in</strong>g Guo, Zi-Chao Zhang, Nan Jiang, Zhen-Ya Zhang, Li-Jie Pan<br />

Zong-M<strong>in</strong>g Zhang, J<strong>in</strong>-X<strong>in</strong>g Guo, Zi-Chao Zhang, Nan Jiang,<br />

Zhen-Ya Zhang, Li-Jie Pan, Department <strong>of</strong> General Surgery,<br />

Digestive Medical Center, The First Affiliated Hospital, School<br />

<strong>of</strong> Medic<strong>in</strong>e, Ts<strong>in</strong>ghua University, Beij<strong>in</strong>g 100016, Ch<strong>in</strong>a<br />

Author contributions: Zhang ZM designed and wrote the paper;<br />

and all other authors revised and approved the f<strong>in</strong>al version<br />

<strong>of</strong> the paper.<br />

Supported by the National Natural Science Foundation <strong>of</strong> Ch<strong>in</strong>a,<br />

No. 81071996<br />

Correspondence to: Zong-M<strong>in</strong>g Zhang, MD, PhD, Pr<strong>of</strong>essor,<br />

Department <strong>of</strong> General Surgery, Digestive Medical Center, The<br />

First Affiliated Hospital, School <strong>of</strong> Medic<strong>in</strong>e, Ts<strong>in</strong>ghua University,<br />

Beij<strong>in</strong>g 100016,<br />

Ch<strong>in</strong>a. zhangzongm<strong>in</strong>g@mail.ts<strong>in</strong>ghua.edu.cn<br />

Telephone: +86-10-64372362 Fax: +86-10-64361322<br />

Received: December 6, 2010 Revised: January 18, 2011<br />

Accepted: January 25, 2011<br />

Published onl<strong>in</strong>e: April 7, 2011<br />

Abstract<br />

Hepatocellular carc<strong>in</strong>oma (HCC) is one <strong>of</strong> the most common<br />

malignancies, rank<strong>in</strong>g the sixth <strong>in</strong> the world, with<br />

55% <strong>of</strong> cases occurr<strong>in</strong>g <strong>in</strong> Ch<strong>in</strong>a. Usually, patients with<br />

HCC did not present until the late stage <strong>of</strong> the disease,<br />

thus limit<strong>in</strong>g their therapeutic options. Although surgical<br />

resection is a potentially curative modality for HCC,<br />

most patients with <strong>in</strong>termediate-advanced HCC are not<br />

suitable candidates. The current therapeutic modalities<br />

for <strong>in</strong>termediate-advanced HCC <strong>in</strong>clude: (1) surgical procedures,<br />

such as radical resection, palliative resection,<br />

<strong>in</strong>traoperative radi<strong>of</strong>requency ablation or cryosurgical<br />

ablation, <strong>in</strong>traoperative hepatic artery and portal ve<strong>in</strong><br />

chemotherapeutic pump placement, two-stage hepatectomy<br />

and liver transplantation; (2) <strong>in</strong>terventional treatment,<br />

such as transcatheter arterial chemoembolization,<br />

portal ve<strong>in</strong> embolization and image-guided locoregional<br />

therapies; and (3) molecularly targeted therapies. So<br />

far, how to choose the therapeutic modalities rema<strong>in</strong>s<br />

controversial. Surgeons are faced with the challenge <strong>of</strong><br />

provid<strong>in</strong>g the most appropriate treatment for patients<br />

with <strong>in</strong>termediate-advanced HCC. This review focuses<br />

WJG|www.wjgnet.com<br />

1685<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): 1685-1689<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

GUIDELINES FOR CLINICAL PRACTICE<br />

on the optional therapeutic modalities for <strong>in</strong>termediateadvanced<br />

HCC.<br />

© 2011 Baishideng. All rights reserved.<br />

Key words: Hepatocellular carc<strong>in</strong>oma; Intermediateadvanced;<br />

Surgical procedure; Interventional treatment;<br />

Molecularly targeted therapy<br />

Peer reviewers: Satoshi Mamori, MD, PhD, Department <strong>of</strong><br />

<strong>Gastroenterology</strong> and Hepatology, Sh<strong>in</strong>ko Hospital, 1-4-47<br />

Wakihama-cho, Chuo-ku, Kobe, Hyogo 651-0072, Japan; Jian Wu,<br />

Associate Pr<strong>of</strong>essor <strong>of</strong> Medic<strong>in</strong>e, Internal Medic<strong>in</strong>e/Transplant<br />

Research Program, University <strong>of</strong> California, Davis Medical Center,<br />

4635 2nd Ave. Suite 1001, Sacramento, CA 95817, United States<br />

Zhang ZM, Guo JX, Zhang ZC, Jiang N, Zhang ZY, Pan LJ.<br />

Therapeutic options for <strong>in</strong>termediate-advanced hepatocellular<br />

carc<strong>in</strong>oma. <strong>World</strong> J Gastroenterol 2011; 17(13): 1685-1689<br />

Available from: URL: http://www.wjgnet.com/1007-9327/full/<br />

v17/i13/1685.htm DOI: http://dx.doi.org/10.3748/wjg.v17.i13.<br />

1685<br />

INTRODUCTION<br />

Hepatocellular carc<strong>in</strong>oma (HCC) is the sixth most common<br />

cancer <strong>in</strong> the world and the third most common<br />

cause <strong>of</strong> cancer-related death [1] . Patients at the early stage<br />

are those who present with an asymptomatic s<strong>in</strong>gle HCC<br />

with the nodule < 5 cm <strong>in</strong> diameter or ≤ 3 <strong>in</strong> number.<br />

Patients exceed<strong>in</strong>g these limits, but free <strong>of</strong> cancer-related<br />

symptoms and vascular <strong>in</strong>vasion or extrahepatic spread,<br />

are considered at the <strong>in</strong>termediate stage. The patients with<br />

the cancer-related symptoms and vascular <strong>in</strong>vasion or extrahepatic<br />

spread are deemed at the advanced stage. HCC<br />

is frequently diagnosed at the late stage and has a high<br />

mortality rate. Surgical resection is a potentially curative<br />

therapy for HCC, however, only 10%-30% <strong>of</strong> patients<br />

with HCC are eligible for curative hepatectomy. Comprehensive<br />

therapy for HCC has become the focus <strong>of</strong> <strong>in</strong>terest<br />

<strong>in</strong> recent years [2-6] . The current therapeutic modalities<br />

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Zhang ZM et al . Treatment <strong>of</strong> <strong>in</strong>termediate-advanced HCC<br />

for <strong>in</strong>termediate-advanced HCC are collected and evaluated<br />

as follows.<br />

SURGICAL PROCEDURES<br />

Radical resection is still the first choice for treatment <strong>of</strong><br />

HCC [7,8] , even at the <strong>in</strong>termediate or advanced stage [9,10] .<br />

If radical resection is impractical, palliative resection comb<strong>in</strong>ed<br />

with comprehensive therapy can significantly prolong<br />

patients’ survival time [11,12] . Intraoperative comprehensive<br />

therapy <strong>in</strong>cludes radi<strong>of</strong>requency ablation, cryosurgical<br />

ablation, and hepatic artery and portal ve<strong>in</strong> chemotherapeutic<br />

pump placement. Two-stage hepatectomy can improve<br />

the survival rate <strong>in</strong> selected patients with advanced<br />

HCC [13,14] . Liver transplantation has been shown to achieve<br />

excellent survival rate <strong>in</strong> appropriate HCC patients [15,16] .<br />

Radical resection<br />

Radical resection for <strong>in</strong>termediate-advanced HCC is <strong>in</strong>dicated<br />

as follows: (1) s<strong>in</strong>gle HCC with large or huge tumor<br />

nodule, swell<strong>in</strong>g outward, clear border or pseudocapsule,<br />

and less than 30% hepatic tissue destroyed measured by<br />

computed tomography (CT) or magnetic resonance imag<strong>in</strong>g<br />

(MRI) scan, or more than 50% compensatory hepatic<br />

hypertrophy; (2) multiple HCC with 3 or fewer nodules<br />

localized <strong>in</strong> one lobe or segment <strong>of</strong> the liver [17,18] . It should<br />

be po<strong>in</strong>ted out that tumor nodules limited to the liver are<br />

not the absolute operative <strong>in</strong>dication. The outcome <strong>of</strong> radical<br />

resection could be affected by multicentric occurrence<br />

<strong>of</strong> HCC, tumor nodule adjacent to major blood vessel or<br />

bile duct, and the hepatic <strong>in</strong>sufficiency <strong>in</strong>duced by coexist<strong>in</strong>g<br />

cirrhosis [19] .<br />

With the deeper recognition <strong>of</strong> the pathology <strong>of</strong><br />

HCC, the rational criteria <strong>of</strong> negative surgical marg<strong>in</strong> are<br />

<strong>in</strong>itially determ<strong>in</strong>ed as follows: (1) > 2 cm marg<strong>in</strong> free<br />

from tumors < 5 cm <strong>in</strong> diameter; (2) > 1 cm marg<strong>in</strong> free<br />

from tumors 5-10 cm; and (3) > 0.5 cm marg<strong>in</strong> free from<br />

tumors > 10 cm. More than 90% hepatectomies fulfill<strong>in</strong>g<br />

the above-mentioned criteria can achieve negative surgical<br />

marg<strong>in</strong> [20] . Thereby, healthy hepatic tissue should be reserved<br />

as much as possible dur<strong>in</strong>g radical resection so as to<br />

enhance the operative security, to facilitate the postoperative<br />

recovery and to help with further treatment.<br />

Palliative resection<br />

The <strong>in</strong>dications <strong>of</strong> palliative resection for <strong>in</strong>termediateadvanced<br />

HCC are: (1) multiple HCC with 3-5 tumor<br />

nodules, exceed<strong>in</strong>g half <strong>of</strong> the liver; (2) multiple HCC<br />

with nodules localized <strong>in</strong> 2-3 adjacent segments or half<br />

<strong>of</strong> the liver, more than 50% compensatory hypertrophy<br />

<strong>in</strong> the tumor-free liver demonstrated by image exam<strong>in</strong>ations;<br />

(3) central HCC with more than 50% compensatory<br />

hypertrophy <strong>in</strong> the tumor-free liver; (4) hilar lymph node<br />

metastasis should be cleared up dur<strong>in</strong>g hepatectomy; and (5)<br />

<strong>in</strong>vaded organs around the liver, such as <strong>colon</strong>, stomach,<br />

diaphragm, right adrenal gland, etc., and s<strong>in</strong>gle metastatic<br />

neoplasm far from the liver (e.g. lung metastasis) should be<br />

resected [17] .<br />

WJG|www.wjgnet.com<br />

Intraoperative radi<strong>of</strong>requency ablation<br />

Radi<strong>of</strong>requency ablation (RFA) is a technique <strong>in</strong> which<br />

an electromagnetic energy deposition is used to thermally<br />

ablate the hepatic tumor tissue [21] . Dur<strong>in</strong>g RFA treatment,<br />

heat energy generated by high-frequency alternat<strong>in</strong>g currents<br />

targeted at the liv<strong>in</strong>g tissues causes prote<strong>in</strong> denaturation<br />

at a temperature <strong>of</strong> 60-110℃ through ionic vibration,<br />

result<strong>in</strong>g <strong>in</strong> coagulative necrosis <strong>of</strong> the target lesion.<br />

In addition, RFA treatment stimulates the immune system<br />

and provides an easy way to achieve <strong>in</strong> vivo vacc<strong>in</strong>ation<br />

aga<strong>in</strong>st tumoral antigens [22] .<br />

RFA is generally <strong>in</strong>dicated for HCC patients who are<br />

not candidates for either liver resection or transplantation<br />

[23] . HCC patients are required to have ≤ 5 nodules,<br />

each < 3 cm <strong>in</strong> diameter, no evidence <strong>of</strong> vascular <strong>in</strong>vasion<br />

or extrahepatic spread, 0 score performance status <strong>of</strong> the<br />

Eastern Cooperative Oncology Group (ECOG), and liver<br />

cirrhosis <strong>in</strong> Child-Pugh class A or B. The more versatile<br />

radi<strong>of</strong>requency probes allow ablation <strong>of</strong> nodule > 5 cm.<br />

When complete resection by major hepatectomy is dangerous<br />

because <strong>of</strong> difficult nodule location, selective use<br />

<strong>of</strong> <strong>in</strong>traoperative RFA will be helpful [24] . The <strong>in</strong>tegration<br />

<strong>of</strong> <strong>in</strong>traoperative RFA <strong>in</strong>to resection surgery contributes<br />

to complete removal <strong>of</strong> nodules with adequate marg<strong>in</strong>,<br />

dim<strong>in</strong>ishes the extent <strong>of</strong> parenchymal resection, and improves<br />

the resectability rate for patients with advanced<br />

HCC [24] .<br />

Pretreatment imag<strong>in</strong>g must carefully def<strong>in</strong>e the location<br />

<strong>of</strong> tumor nodule with respect to the surround<strong>in</strong>g structures<br />

for RFA <strong>in</strong> HCC: nodules located on the surface <strong>of</strong><br />

the liver can be considered; nodules adjacent to the hepatic<br />

vessels may be considered because flow<strong>in</strong>g blood usually<br />

protects the vascular wall from thermal <strong>in</strong>jury; nodules adjacent<br />

to the hepatic hilum represents a relative contra<strong>in</strong>dication<br />

due to the risk <strong>of</strong> thermal <strong>in</strong>jury <strong>of</strong> the biliary tract;<br />

and nodules adjacent to any part <strong>of</strong> the gastro<strong>in</strong>test<strong>in</strong>al<br />

tract must be avoided [25] .<br />

Intraoperative cryosurgical ablation<br />

Although RFA has been the most widely utilized ablation<br />

modality for HCC, cryosurgical ablation has several advantages<br />

(most significantly, the ability to produce larger<br />

and more precise zones <strong>of</strong> ablation) over RAF [26] .<br />

Cryosurgical ablation for HCC patient relies on nonspecific<br />

tissue necrosis due to freez<strong>in</strong>g as well as microvascular<br />

thrombosis. Argon-helium cryosurgical ablation is able to<br />

<strong>in</strong>duce the necrosis <strong>of</strong> tumor cells through the formation<br />

<strong>of</strong> extracellular and <strong>in</strong>tracellular ice crystals and then cell<br />

dehydration due to rapidly freez<strong>in</strong>g (< -140℃) as well as<br />

rapidly thaw<strong>in</strong>g (20-40℃) the tumor tissues with argon/helium<br />

gas. Therefore, argon-helium cryosurgical ablation has<br />

become one <strong>of</strong> the major therapeutic approaches for unresectable<br />

<strong>in</strong>termediate-advanced HCC.<br />

The <strong>in</strong>dications <strong>of</strong> cryosurgical ablation for HCC patient<br />

are: (1) nodules < 5 cm <strong>in</strong> diameter, ≤ 3 <strong>in</strong> number;<br />

(2) nodule > 5 cm with irregular marg<strong>in</strong>, may be given<br />

<strong>in</strong>traoperative cryosurgical ablation with or without excision<br />

<strong>of</strong> nodule. Intraoperative cryosurgical ablation <strong>of</strong>fers<br />

1686 April 7, 2011|Volume 17|Issue 13|

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an effective and safe option for management <strong>of</strong> advanced<br />

HCC [27] . HCC patients with diffuse <strong>in</strong>filtrative disease or<br />

large bilobar nodules (> 50% <strong>of</strong> liver volume) are not candidates<br />

for cryosurgical ablation because complete ablation<br />

<strong>of</strong> the nodules might <strong>in</strong>duce hepatic failure.<br />

Intraoperative hepatic artery and portal ve<strong>in</strong><br />

chemotherapeutic pump placement<br />

The liver has a dual blood supply from the hepatic artery<br />

and the portal venous system. For HCC patients who are<br />

not suitable for hepatectomy confirmed by <strong>in</strong>traoperative<br />

exploration, two chemotherapeutic pumps could be<br />

implanted subcutaneously <strong>in</strong>to the upper abdom<strong>in</strong>al wall<br />

near the <strong>in</strong>cision, with the tip <strong>of</strong> pump catheter separately<br />

<strong>in</strong>serted <strong>in</strong>to the hepatic artery and portal ve<strong>in</strong> dur<strong>in</strong>g the<br />

operation, followed by postoperative chemotherapy. The<br />

advantage <strong>of</strong> <strong>in</strong>traoperatively implanted chemotherapeutic<br />

pump is the ability to accurately and selectively place <strong>in</strong>to<br />

the ma<strong>in</strong> trunk or branch <strong>of</strong> hepatic artery and portal ve<strong>in</strong>.<br />

For resectable <strong>in</strong>termediate-advanced HCC, the postoperative<br />

hepatic artery and portal ve<strong>in</strong> dual perfusion chemotherapy<br />

via chemotherapeutic pumps could prevent tumor<br />

recurrence [28] .<br />

Two-stage hepatectomy<br />

Two-stage hepatectomy has been developed as a surgical<br />

strategy for extremely difficult patients with <strong>in</strong>termediateadvanced<br />

HCC [7] . This strategy is applied when it is impossible<br />

to resect the tumor <strong>in</strong> a s<strong>in</strong>gle procedure. The ma<strong>in</strong><br />

pr<strong>in</strong>ciples <strong>of</strong> this strategy are: huge HCC with the remnant<br />

liver volume cannot ma<strong>in</strong>ta<strong>in</strong> hepatic function after hepatectomy;<br />

central or hilar HCC adjacent to or <strong>in</strong>vaded major<br />

blood vessel; and serious cirrhosis with possible hepatic<br />

decompensation after hepatectomy.<br />

For unresectable HCC, preoperative <strong>in</strong>tervention with<br />

transcatheter arterial chemoembolization (TACE) [29] , portal<br />

ve<strong>in</strong> embolization (PVE) [30,31] , or percutaneous RFA could<br />

control tumor progression and <strong>in</strong>vasion, downstage tumor<br />

status, <strong>in</strong>crease remnant liver volume, and decrease tumor<br />

recurrence rate, thus mak<strong>in</strong>g the two-stage hepatectomy<br />

possible. The <strong>in</strong>dication <strong>of</strong> two-stage hepatectomy is that<br />

tumor diameter reduced to 50% <strong>of</strong> the <strong>in</strong>itial size, and<br />

nontumorous liver tissue had significant compensatory<br />

hyperplasia. Sequential TACE and PVE could broaden the<br />

surgical <strong>in</strong>dication and the safety <strong>of</strong> major hepatic resection<br />

for advanced HCC patient with damaged liver [32,33] .<br />

Non-anatomic local excision <strong>of</strong> liver cancer or hepatic<br />

segmentectomy should be used <strong>in</strong> the two-stage hepatectomy<br />

so as to maximally preserve the normal liver tissue.<br />

For the patients with HCC <strong>in</strong>vad<strong>in</strong>g the hepatic hilum and<br />

<strong>in</strong>ferior vena cava, total hepatic vascular exclusion (HVE)<br />

should be prepared to avoid massive hemorrhage dur<strong>in</strong>g<br />

hepatectomy.<br />

Liver transplantation<br />

Liver transplantation is an ideal treatment option, as it<br />

simultaneously cures HCC. However, up to date, there<br />

are no uniform criteria <strong>of</strong> liver transplantation for HCC<br />

WJG|www.wjgnet.com<br />

Zhang ZM et al . Treatment <strong>of</strong> <strong>in</strong>termediate-advanced HCC<br />

patients <strong>in</strong> Ch<strong>in</strong>a. The United Network for Organ Shar<strong>in</strong>g<br />

(UNOS) criteria for liver transplantation are usually adopted<br />

<strong>in</strong> the world: s<strong>in</strong>gle tumor ≤ 5 cm; 2-3 tumors, each<br />

≤ 3 cm; no macrovascular <strong>in</strong>vasion; and no extrahepatic<br />

spread to surround<strong>in</strong>g lymph nodes, lungs, abdom<strong>in</strong>al<br />

organs, or bones [34] . However, if the UNOS criteria are<br />

strictly adopted <strong>in</strong> Ch<strong>in</strong>a, it means that most HCC patients<br />

will lose the opportunity <strong>of</strong> liver transplantation, because<br />

more than 100 000 patients die <strong>of</strong> advanced HCC each<br />

year. For this reason, the <strong>in</strong>dication <strong>of</strong> liver transplantation<br />

for advanced HCC should be relatively loose <strong>in</strong> Ch<strong>in</strong>a.<br />

For the patients with unresectable huge or multiple HCC,<br />

if no vascular <strong>in</strong>vasion and no extrahepatic spread, liver<br />

transplantation is the treatment <strong>of</strong> choice. Consider<strong>in</strong>g the<br />

limited organ supply, high cost, and considerable risk, we<br />

suggest that only those HCC patients with a high probability<br />

<strong>of</strong> survival benefit should be selected to receive liver<br />

transplantation. The shortage <strong>of</strong> donor livers is the major<br />

constra<strong>in</strong>t <strong>of</strong> liver transplantation.<br />

INTERVENTIONAL TREATMENT<br />

Although surgical resection has been the first choice for<br />

treatment <strong>of</strong> HCC, a simple surgical exploration could<br />

accelerate the process <strong>of</strong> disease and even cause death<br />

due to the postoperative complication <strong>of</strong> patients with<br />

unresectable HCC. With advances <strong>of</strong> medical imag<strong>in</strong>g and<br />

improvement <strong>of</strong> <strong>in</strong>terventional technology, <strong>in</strong>terventional<br />

treatment has become an effective approach to <strong>in</strong>operable<br />

HCC [35-37] . The common approaches <strong>of</strong> <strong>in</strong>terventional<br />

treatments for <strong>in</strong>operable HCC <strong>in</strong>clude transcatheter arterial<br />

chemoembolization, portal ve<strong>in</strong> embolization, and<br />

image-guided locoregional therapies.<br />

Transcatheter arterial chemoembolization<br />

For the treatment <strong>of</strong> <strong>in</strong>operable HCC demonstrated by<br />

preoperative image exam<strong>in</strong>ation, the priority is transcatheter<br />

arterial chemoembolization (TACE). The theoretical basis<br />

<strong>of</strong> TACE is the special vascular supply <strong>of</strong> liver and HCC.<br />

Liver derives dual blood supply from portal ve<strong>in</strong> and hepatic<br />

artery, the former accounts for 2/3 to 3/4 while the<br />

latter for only 1/4 to 1/3. HCC derives 90% blood supply<br />

from hepatic artery and only 10% from portal ve<strong>in</strong>. Thus,<br />

TACE provides a higher local concentration <strong>of</strong> chemotherapeutic<br />

drugs <strong>in</strong>to tumor compared with <strong>in</strong>travenous<br />

perfusion chemotherapy, and meanwhile, it blocks blood<br />

supply <strong>of</strong> HCC, but only exerts little <strong>in</strong>fluence on blood<br />

supply <strong>of</strong> the liver. The consequence is that the major<br />

portion <strong>of</strong> cancer nodule becomes necrotic, while hepatic<br />

function rema<strong>in</strong>s unchanged or little impaired.<br />

Better patient selection and selective segmental chemoembolization<br />

may improve the benefit-risk ratio <strong>of</strong><br />

TACE [38] . TACE is <strong>in</strong>dicated <strong>in</strong> <strong>in</strong>termediate-advanced<br />

HCC even <strong>in</strong> the sett<strong>in</strong>g <strong>of</strong> portal ve<strong>in</strong> <strong>in</strong>volvement (exclud<strong>in</strong>g<br />

ma<strong>in</strong> portal ve<strong>in</strong>) [39] . The presence <strong>of</strong> ma<strong>in</strong> portal<br />

ve<strong>in</strong> thrombosis, extrahepatic metastasis, Child-Pugh class<br />

C liver function, and severe hepatic arterio-portal shunts<br />

is considered as contra<strong>in</strong>dications for TACE.<br />

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Zhang ZM et al . Treatment <strong>of</strong> <strong>in</strong>termediate-advanced HCC<br />

Portal ve<strong>in</strong> embolization<br />

Percutaneous transhepatic portal ve<strong>in</strong> embolization (PVE)<br />

is a useful procedure for the preoperative <strong>in</strong>tervention <strong>of</strong><br />

advanced HCC patients selected for hepatectomy. PVE<br />

could <strong>in</strong>crease the volume and function <strong>of</strong> the future remnant<br />

liver through the acceleration <strong>of</strong> hepatocyte proliferation,<br />

and embolize possible hepatic arterio-portal shunts, so<br />

as to prevent postoperative liver <strong>in</strong>sufficiency.<br />

For the treatment <strong>of</strong> <strong>in</strong>termediate-advanced HCC, the<br />

comb<strong>in</strong>ation <strong>of</strong> TACE and PVE not only blocks most<br />

blood supply <strong>of</strong> ma<strong>in</strong> tumor and satellite lesions, but also<br />

<strong>in</strong>creases the local concentration <strong>of</strong> chemotherapeutic<br />

drugs <strong>in</strong>to tumor, so as to more effectively control the<br />

tumor growth and decrease tumor recurrence. Contra<strong>in</strong>dications<br />

to PVE <strong>in</strong>clude distant metastases, uncontrolled<br />

coagulopathy, active cholangitis, portal hypertension, and<br />

renal failure [40] .<br />

Image-guided locoregional therapies<br />

Ultrasound or CT guided locoregional therapies have a<br />

therapeutic effect <strong>in</strong> advanced HCC patients by means<br />

<strong>of</strong> thermoablative therapy (radi<strong>of</strong>requency ablation, microwave<br />

coagulation, laser ablation), cryotherapy (argonhelium<br />

knife, liquid nitrogen), or chemical therapy (ethanol<br />

<strong>in</strong>jection, acetic acid <strong>in</strong>jection) to destroy tumor tissues. To<br />

date, the commonly used therapies <strong>in</strong>clude percutaneous<br />

RFA, microwave coagulation, cryoablation therapy, and<br />

ethanol <strong>in</strong>jection, especially with percutaneous RFA as<br />

the first choice to <strong>in</strong>operable HCC. The roles <strong>of</strong> different<br />

locoregional therapies may change with further development<br />

<strong>of</strong> technology and availability <strong>of</strong> data from future<br />

prospective randomized trials [38,41-43] .<br />

MOLECULARLY TARGETED THERAPIES<br />

Recently, molecularly targeted therapies, <strong>in</strong>clud<strong>in</strong>g<br />

sorafenib, sunit<strong>in</strong>ib, brivanib, cetuximab, erlot<strong>in</strong>ib plus bevacizumab,<br />

and lapat<strong>in</strong>ib, have emerged as promis<strong>in</strong>g therapeutic<br />

approaches for advanced HCC [44,45] . Sorafenib, as an<br />

orally-active multik<strong>in</strong>ase <strong>in</strong>hibitor target<strong>in</strong>g both tumor cells<br />

and the tumor vasculature, and the first agent to improve<br />

the overall survival status for patients with advanced HCC,<br />

has been approved for systemic therapy <strong>in</strong> patients with<br />

advanced HCC <strong>in</strong> Eastern and Western countries [3,46-48] .<br />

Many other molecularly targeted agents <strong>of</strong> block<strong>in</strong>g epidermal<br />

growth factor receptor (EGFR), vascular endothelial<br />

growth factor receptor (VEGFR), platelet-derived growth<br />

factor receptor (PDGFR), and mammalian target <strong>of</strong> rapamyc<strong>in</strong><br />

(mTOR) are at different stages <strong>of</strong> cl<strong>in</strong>ical development<br />

for the treatment <strong>of</strong> advanced HCC [49-51] .<br />

CONCLUSION<br />

For the treatment <strong>of</strong> <strong>in</strong>termediate-advanced HCC, various<br />

surgical procedures may produce the def<strong>in</strong>ite therapeutic<br />

effects. The <strong>in</strong>terventional treatment can also improve the<br />

prognosis to a great extent, but so far there is still lack <strong>of</strong><br />

a special effective approach. In recent years, the model<br />

<strong>of</strong> comprehensive therapies ma<strong>in</strong>ly based on surgical<br />

WJG|www.wjgnet.com<br />

resection has been adopted to further enhance the curative<br />

effect, prolong the survival time, and improve the life<br />

quality <strong>of</strong> the patients. Accord<strong>in</strong>g to the <strong>in</strong>dications and<br />

advantages <strong>of</strong> each therapeutic method, comb<strong>in</strong>ed with<br />

the patient’s cl<strong>in</strong>ical stage, the selection <strong>of</strong> therapeutic<br />

approaches to maximize the efficacy and m<strong>in</strong>imize the<br />

adverse effect is very important for design<strong>in</strong>g a more rational<br />

therapeutic plan for <strong>in</strong>termediate-advanced HCC.<br />

REFERENCES<br />

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2 He AR, Soe K, El Zouhairi M. Current problems with systemic<br />

treatment <strong>of</strong> advanced hepatocellular cancer. Curr<br />

Probl Cancer 2010; 34: 131-149<br />

3 Han KH, Park JY. Systemic treatment <strong>in</strong> advanced/metastatic<br />

hepatocellular carc<strong>in</strong>oma <strong>in</strong> the era <strong>of</strong> targeted therapy.<br />

J Gastroenterol Hepatol 2010; 25: 1023-1025<br />

4 Porta C, Pagl<strong>in</strong>o C. Medical treatment <strong>of</strong> unresectable hepatocellular<br />

carc<strong>in</strong>oma: Go<strong>in</strong>g beyond sorafenib. <strong>World</strong> J Hepatol<br />

2010; 2: 103-113<br />

5 Rampone B, Schiavone B, Mart<strong>in</strong>o A, Viviano C, Confuorto<br />

G. Current management strategy <strong>of</strong> hepatocellular carc<strong>in</strong>oma.<br />

<strong>World</strong> J Gastroenterol 2009; 15: 3210-3216<br />

6 Yau T, Chan P, Epste<strong>in</strong> R, Poon RT. Evolution <strong>of</strong> systemic<br />

therapy <strong>of</strong> advanced hepatocellular carc<strong>in</strong>oma. <strong>World</strong> J Gastroenterol<br />

2008; 14: 6437-6441<br />

7 Wu MC. Status and prospect <strong>of</strong> comprehensive treatment <strong>of</strong><br />

liver cancer. Zhonghua Gandan Waike Zazhi 2006; 12: 1-4<br />

8 Liu JH, Chen PW, Asch SM, Busuttil RW, Ko CY. Surgery<br />

for hepatocellular carc<strong>in</strong>oma: does it improve survival? Ann<br />

Surg Oncol 2004; 11: 298-303<br />

9 Wakabayashi H, Ushiyama T, Ishimura K, Izuishi K, Karasawa<br />

Y, Masaki T, Watanabe S, Kuriyama S, Maeta H. Significance<br />

<strong>of</strong> reduction surgery <strong>in</strong> multidiscipl<strong>in</strong>ary treatment<br />

<strong>of</strong> advanced hepatocellular carc<strong>in</strong>oma with multiple <strong>in</strong>trahepatic<br />

lesions. J Surg Oncol 2003; 82: 98-103<br />

10 Yu YQ, Xu DB, Zhou XD, Lu JZ, Tang ZY, Mack P. Experience<br />

with liver resection after hepatic arterial chemoembolization<br />

for hepatocellular carc<strong>in</strong>oma. Cancer 1993; 71: 62-65<br />

11 L<strong>in</strong> DX, Zhang QY, Li X, Ye QW, L<strong>in</strong> F, Li LL. An aggressive<br />

approach leads to improved survival <strong>in</strong> hepatocellular carc<strong>in</strong>oma<br />

patients with portal ve<strong>in</strong> tumor thrombus. J Cancer Res<br />

Cl<strong>in</strong> Oncol 2011; 137: 139-149<br />

12 Morris-Stiff G, Gomez D, de Liguori Car<strong>in</strong>o N, Prasad KR.<br />

Surgical management <strong>of</strong> hepatocellular carc<strong>in</strong>oma: is the<br />

jury still out? Surg Oncol 2009; 18: 298-321<br />

13 Zhang Z, Liu Q, He J, Yang J, Yang G, Wu M. The effect <strong>of</strong><br />

preoperative transcatheter hepatic arterial chemoembolization<br />

on disease-free survival after hepatectomy for hepatocellular<br />

carc<strong>in</strong>oma. Cancer 2000; 89: 2606-2612<br />

14 Choi GH, Kim DH, Kang CM, Kim KS, Choi JS, Lee WJ,<br />

Kim BR. Is preoperative transarterial chemoembolization<br />

needed for a resectable hepatocellular carc<strong>in</strong>oma? <strong>World</strong> J<br />

Surg 2007; 31: 2370-2377<br />

15 Bruix J, Sherman M. Management <strong>of</strong> hepatocellular carc<strong>in</strong>oma.<br />

Hepatology 2005; 42: 1208-1236<br />

16 Bismuth H, Chiche L, Adam R, Casta<strong>in</strong>g D, Diamond T,<br />

Dennison A. Liver resection versus transplantation for hepatocellular<br />

carc<strong>in</strong>oma <strong>in</strong> cirrhotic patients. Ann Surg 1993;<br />

218: 145-151<br />

17 Chen XP, Zhang ZW. Attach great importance to standardization<br />

<strong>of</strong> surgical treatment <strong>of</strong> hepatoma. Zhonghua Xiaohua<br />

Waike Zazhi 2007; 6: 5-7<br />

18 Ruzzenente A, Capra F, Pachera S, Iacono C, Piccirillo G,<br />

Lunardi M, Pistoso S, Valdegamberi A, D‘On<strong>of</strong>rio M, Guglielmi<br />

A. Is liver resection justified <strong>in</strong> advanced hepatocellular<br />

carc<strong>in</strong>oma? Results <strong>of</strong> an observational study <strong>in</strong> 464<br />

1688 April 7, 2011|Volume 17|Issue 13|

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Onl<strong>in</strong>e Submissions: http://www.wjgnet.com/1007-9327<strong>of</strong>fice<br />

wjg@wjgnet.com<br />

doi:10.3748/wjg.v17.i13.1690<br />

How to assess the severity <strong>of</strong> atrophic gastritis<br />

Yan-Cheng Dai, Zhi-Peng Tang, Ya-Li Zhang<br />

Yan-Cheng Dai, Zhi-Peng Tang, Ya-Li Zhang, Department <strong>of</strong><br />

<strong>Gastroenterology</strong>, Longhua Hospital Affiliated to Shanghai University<br />

<strong>of</strong> Traditional Ch<strong>in</strong>ese Medic<strong>in</strong>e, Shanghai 200032, Ch<strong>in</strong>a<br />

Yan-Cheng Dai, Zhi-Peng Tang, Ya-Li Zhang,The Institute <strong>of</strong><br />

Digestive Disease Affiliated to Shanghai University <strong>of</strong> Traditional<br />

Ch<strong>in</strong>ese Medic<strong>in</strong>e, Shanghai 200032, Ch<strong>in</strong>a<br />

Author contributions: Dai YC, Tang ZP and Zhang YL contributed<br />

equally to this paper.<br />

Supported by the Shanghai Lead<strong>in</strong>g Academic Discipl<strong>in</strong>e Project,<br />

No. J50305<br />

Correspondence to: Zhi-Peng Tang, MD, Department <strong>of</strong> <strong>Gastroenterology</strong>,<br />

Longhua Hospital Affiliated to Shanghai University<br />

<strong>of</strong> Traditional Ch<strong>in</strong>ese Medic<strong>in</strong>e, 725 South Wanp<strong>in</strong> Road,<br />

Shanghai 200032, Ch<strong>in</strong>a. zhipengtang@sohu.com<br />

Telephone: +86­21­64385700 Fax: +86­21­64392310<br />

Received: September 16, 2010 Revised: December 1, 2010<br />

Accepted: December 8, 2010<br />

Published onl<strong>in</strong>e: April 7, 2011<br />

Abstract<br />

Atrophic gastritis, is the ma<strong>in</strong> consequence <strong>of</strong> long-stand<strong>in</strong>g<br />

Helicobacter pylori <strong>in</strong>fection, and is l<strong>in</strong>ked to the<br />

development <strong>of</strong> gastric cancer. The severity <strong>of</strong> atrophic<br />

gastritis is related to the lifetime risk <strong>of</strong> gastric cancer<br />

development, especially <strong>in</strong> terms <strong>of</strong> its degree and extent<br />

<strong>of</strong> mucosal damage. Therefore, it is important for<br />

cl<strong>in</strong>icians to assess the severity <strong>of</strong> atrophic gastritis,<br />

<strong>in</strong>terfere with the disease progress, and reverse gastric<br />

mucosal atrophy. In the article, we demonstrated some<br />

methods (conventional endoscopy, modern endoscopic<br />

technology and non<strong>in</strong>vasive methods) that may help assess<br />

the severity <strong>of</strong> atrophic gastritis and select the reasonable<br />

treatment protocols.<br />

© 2011 Baishideng. All rights reserved.<br />

Key words: Atrophic gastritis; Endoscopy; Peps<strong>in</strong>ogen<br />

Peer reviewer: Gerardo Nardone, MD, Pr<strong>of</strong>essor, Department<br />

<strong>of</strong> Cl<strong>in</strong>ical and Internal Medic<strong>in</strong>e, <strong>Gastroenterology</strong> Unit, Via<br />

Pans<strong>in</strong>i 5 Napoli80131, Italy<br />

Dai YC, Tang ZP, Zhang YL. How to assess the severity <strong>of</strong> atr­<br />

WJG|www.wjgnet.com<br />

1690<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): 1690-1693<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

REVIEW<br />

ophic gastritis. <strong>World</strong> J Gastroenterol 2011; 17(13): 1690­1693<br />

Available from: URL: http://www.wjgnet.com/1007­9327/full/<br />

v17/i13/1690.htm DOI: http://dx.doi.org/10.3748/wjg.v17.i13.<br />

1690<br />

INTRODUCTION<br />

Atrophic gastritis (AG) is a histopathological entity that<br />

is characterized by chronic <strong>in</strong>flammation <strong>of</strong> the gastric<br />

mucosa with loss <strong>of</strong> gastric glandular cells and replacement<br />

by <strong>in</strong>test<strong>in</strong>al-type epithelium, pyloric-type glands, and<br />

fibrous tissue. Atrophy <strong>of</strong> the gastric mucosa is the<br />

endpo<strong>in</strong>t <strong>of</strong> chronic processes, such as chronic gastritis<br />

associated with Helicobacter pylori (H. pylori) <strong>in</strong>fection, other<br />

unidentified environmental factors, and autoimmunity<br />

directed aga<strong>in</strong>st gastric glandular cells [1] . It has been<br />

established that people with AG have a high risk for gastric<br />

cancer [2,3] , and it has been reported that about 10% <strong>of</strong> the<br />

patients with moderate-severe AG will develop gastric<br />

malignancies dur<strong>in</strong>g a mean follow-up <strong>of</strong> 7.8 years [4] . Thus,<br />

the assessment <strong>of</strong> the severity <strong>of</strong> AG may be an important<br />

challenge for the management <strong>of</strong> these patients because its<br />

features (i.e. extension <strong>of</strong> atrophy and <strong>in</strong>test<strong>in</strong>al metaplasia,<br />

and hypochlorhydria) may be considered as potential<br />

surrogate markers for the <strong>in</strong>creased risk for gastric cancer.<br />

Here, we demonstrate some methods used to assess the<br />

severity <strong>of</strong> AG.<br />

DEFINITION AND CLASSIFICATION OF AG<br />

Gastric mucosal atrophy is def<strong>in</strong>ed as the loss <strong>of</strong> appropriate<br />

glands, which occurs when glands damaged by<br />

<strong>in</strong>flammation are replaced either by connective tissue<br />

(scarr<strong>in</strong>g) or by glandular structures <strong>in</strong>appropriate for<br />

location (metaplasia). Most <strong>of</strong>ten, as <strong>in</strong> the antral mucosa,<br />

the metaplastic transformation assumes the phenotype<br />

<strong>of</strong> the glands l<strong>in</strong>ed by <strong>in</strong>test<strong>in</strong>al-type epithelium (IM), but<br />

<strong>in</strong> the oxyntic mucosa, it may also take the form <strong>of</strong> muc<strong>in</strong>-secret<strong>in</strong>g<br />

antral glands (pseudopyloric metaplasia) [5] .<br />

Traditionally, AG can be divided <strong>in</strong>to gastric body atrophy<br />

April 7, 2011|Volume 17|Issue 13|

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and s<strong>in</strong>uses ventriculi atrophy: the former is mostly associated<br />

with autoimmune diseases, and the latter is <strong>of</strong>ten<br />

associated with H. pylori <strong>in</strong>fection [6,7] . However, <strong>in</strong> general<br />

practice, the diagnosis <strong>of</strong> atrophy and IM is troublesome<br />

due to an unsatisfactory <strong>in</strong>terobserver agreement among<br />

pathologists, therefore <strong>in</strong> 2000, an <strong>in</strong>ternational group<br />

<strong>of</strong> pathologists from Atrophy Club reviewed once aga<strong>in</strong><br />

the spectrum <strong>of</strong> gastric atrophy and IM, and proposed<br />

a simplified def<strong>in</strong>ition <strong>of</strong> atrophy, which <strong>in</strong>cludes a metaplastic<br />

and a non-metaplastic category, thus mak<strong>in</strong>g metaplasia<br />

an absolute concept to demonstrate the severity <strong>of</strong><br />

the disease [5] .<br />

CONVENTIONAL ENDOSCOPY AND AG<br />

In 2003, the Ch<strong>in</strong>ese Society <strong>of</strong> Digestive Endoscopy<br />

established endoscopic criteria for chronic gastritis <strong>in</strong><br />

Dalian meet<strong>in</strong>g. The scar lesions were characterized by the<br />

follow<strong>in</strong>g attributes: mucosal atrophy, granular mucosa,<br />

flattened folds, gray <strong>in</strong>test<strong>in</strong>al-type epithelium and blood<br />

vessel permeability. AG was classified <strong>in</strong>to three patterns<br />

<strong>of</strong> ridges: (1) f<strong>in</strong>e granular mucosa, permeability <strong>of</strong> some<br />

blood vessels and a s<strong>in</strong>gle nodule <strong>of</strong> gray <strong>in</strong>test<strong>in</strong>al-type<br />

epithelium; (2) medium granular mucosa, permeability<br />

<strong>of</strong> blood vessels, multiple nodules <strong>of</strong> gray <strong>in</strong>test<strong>in</strong>al-type<br />

epithelium; and (3) coarse granular mucosa, blood vessels<br />

can be seen up to the surface, diffuse nodules <strong>of</strong> gray<br />

<strong>in</strong>test<strong>in</strong>al-type epithelium [8] .<br />

MAGNIFYING ENDOSCOPY AND AG<br />

Magnify<strong>in</strong>g endoscopy has been developed to visualize<br />

the microstructure <strong>of</strong> gastro<strong>in</strong>test<strong>in</strong>al surface mucosa and<br />

mucosal vascularity, which provides a magnified image <strong>of</strong><br />

up to 200 times [9] . The pit patterns observed on the mucosal<br />

surface are considered to reflect the arrangement<br />

and structure <strong>of</strong> surface epithelia, morphology, number,<br />

distribution and function <strong>of</strong> glands, mucosal edema and<br />

<strong>in</strong>flammation, and vascular morphology, arrangement,<br />

number and distribution. The basic units <strong>of</strong> the microstructures<br />

on the surface <strong>of</strong> gastric mucosa are countless<br />

gastric pits that form gastric areas separated by m<strong>in</strong>or gastric<br />

grooves (also called <strong>in</strong>terval grooves). As the open<strong>in</strong>gs <strong>of</strong><br />

glands, gastric pits are the first to undergo structural change<br />

due to gastric mucosal lesions. Yagi et al [10] thought that the<br />

presentation <strong>of</strong> gastric mucosal atrophy was that gastric pit<br />

became white, expanded <strong>in</strong> size, and was surrounded by<br />

areas <strong>of</strong> erythema. In the study <strong>of</strong> Sakaki et al, magnify<strong>in</strong>g<br />

endoscopy patterns <strong>of</strong> gastric erosion pits were classified<br />

<strong>in</strong>to six types: A (round spot pits), B (short rod pits), C<br />

(sparsely and thickly l<strong>in</strong>ear), D (patchy), E (villous) and F<br />

(unclear or disappearance <strong>of</strong> pits or abnormal hyperplasia<br />

blood capillary) [11] . Yuan et al [12] used magnify<strong>in</strong>g endoscopy<br />

<strong>in</strong> comb<strong>in</strong>ation with methylene blue sta<strong>in</strong><strong>in</strong>g to exam<strong>in</strong>ate<br />

the microstructures <strong>of</strong> gastric mucosa <strong>in</strong> 180 patients with<br />

gastric erosion. Their results showed that types A and B<br />

were found <strong>in</strong> normal gastric mucosa, while types C-F were<br />

found <strong>in</strong> gastric mucosa with active <strong>in</strong>flammation, atrophic<br />

WJG|www.wjgnet.com<br />

Dai YC et al . Assessment <strong>of</strong> atrophic gastritis<br />

<strong>in</strong>flammation, <strong>in</strong>test<strong>in</strong>al metaplasia and dysplasia <strong>of</strong> vary<strong>in</strong>g<br />

degrees. Type E mucosa (81.8%) suggested <strong>in</strong>test<strong>in</strong>al<br />

metaplasia, type F <strong>in</strong>dicated existence <strong>of</strong> dysplasia (86.3%),<br />

and type F with abnormal hyperplasia blood capillary<br />

suggested dysplasia (89.9%).<br />

MAGNIFYING NARROW-BAND IMAGING<br />

AND AG<br />

Narrow-band imag<strong>in</strong>g (NBI) is an endoscopic imag<strong>in</strong>g<br />

technique for the enhanced visualization <strong>of</strong> mucosal<br />

microscopic structure and capillaries <strong>of</strong> the superficial<br />

mucosal layer. Images are obta<strong>in</strong>ed us<strong>in</strong>g narrower bands<br />

<strong>of</strong> red, blue and green filters, which are different from<br />

conventional red-green-blue filters [13] . Comb<strong>in</strong><strong>in</strong>g the<br />

NBI system and magnify<strong>in</strong>g endoscopy allows for simple<br />

and clear visualization <strong>of</strong> microscopic structures <strong>of</strong> the<br />

superficial mucosa and its capillary patterns [14] . In the<br />

study <strong>of</strong> Tahara et al [15] , gastric mucosal patterns seen with<br />

magnify<strong>in</strong>g NBI <strong>in</strong> un<strong>in</strong>volved gastric corpus were divided<br />

<strong>in</strong>to the follow<strong>in</strong>g categories: normal small, round pits<br />

with regular subepithelial capillary networks; type 1, slightly<br />

enlarged, round pits with unclear or irregular subepithelial<br />

capillary networks; type 2, obviously enlarged, oval or<br />

prolonged pits with <strong>in</strong>creased density <strong>of</strong> irregular vessels;<br />

and type 3, well-demarcated oval or tubulovillous pits<br />

with clearly visible coiled or wavy vessels. They found that<br />

the mucosal patterns were associated with the degree <strong>of</strong><br />

endoscopic gastric atrophy. As mucosal patterns advanced<br />

from normal to types 1, 2 and 3, the degree <strong>of</strong> endoscopic<br />

gastric mucosal atrophy <strong>in</strong>creased simultaneously. The<br />

sensitivity and specificity for types 1, 2 and 3 for detection<br />

<strong>of</strong> H. pylori <strong>in</strong>fection and type 3 for detection <strong>of</strong> <strong>in</strong>test<strong>in</strong>al<br />

metaplasia were 95.2%, 82.2%, 73.3%, and 95.6%,<br />

respectively. Uedo et al [16] found <strong>in</strong> their study that the<br />

appearance <strong>of</strong> a light blue crest on the epithelial surface<br />

was correlated with histological evidence <strong>of</strong> <strong>in</strong>test<strong>in</strong>al<br />

metaplasia with a sensitivity <strong>of</strong> 89% (95% CI: 83-96),<br />

specificity <strong>of</strong> 93% (95% CI: 88-97), positive predictive<br />

value <strong>of</strong> 91% (95% CI: 85-96), negative predictive value<br />

<strong>of</strong> 92% (95% CI: 87-97), and accuracy <strong>of</strong> 91% (95% CI:<br />

88-95).<br />

AUTO-FLUORESCENCE IMAGING<br />

VIDEOENDOSCOPY AND AG<br />

Auto-fluorescence imag<strong>in</strong>g (AFI) produces real-time<br />

pseudocolor images based on natural tissue aut<strong>of</strong>luorescence<br />

emitted by light excitation from endogenous<br />

fluorophores such as collagen, nicot<strong>in</strong>amide, aden<strong>in</strong>e<br />

d<strong>in</strong>ucleotide, flav<strong>in</strong> and porphyr<strong>in</strong>s. AFI enables the<br />

detection <strong>of</strong> mucosal features not visible with conventional<br />

endoscopy, therefore, it might improve the identification<br />

and characterization <strong>of</strong> the premalignant status <strong>in</strong> gastric<br />

mucosa [17,18] .<br />

The fluorescence is almost purple, weaker <strong>in</strong> the normal<br />

gastric gland mucosa than that <strong>in</strong> the pyloric gland mucosa.<br />

1691 April 7, 2011|Volume 17|Issue 13|

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<strong>of</strong> atrophic body gastritis <strong>in</strong> patients with parietal cell antibodies.<br />

J Cl<strong>in</strong> Endocr<strong>in</strong>ol Metab 2007; 92: 4346-4351<br />

7 Veijola LI, Oksanen AM, Sipponen PI, Rautel<strong>in</strong> HI. Association<br />

<strong>of</strong> autoimmune type atrophic corpus gastritis with Helicobacter<br />

pylori <strong>in</strong>fection. <strong>World</strong> J Gastroenterol 2010; 16: 83-88<br />

8 Ch<strong>in</strong>ese Medical Association <strong>of</strong> Digestive Endoscopy. Endoscopic<br />

classification and trial standards on treatment <strong>of</strong> chronic<br />

gastritis. Zhonghua Xiaohua Neij<strong>in</strong>g Zazhi 2004; 21: 77-78<br />

9 Lee SH, Ryu CB, Jang JY, Cho JY. [Magnify<strong>in</strong>g endoscopy<br />

<strong>in</strong> upper gastro<strong>in</strong>test<strong>in</strong>al tract]. Korean J Gastroenterol 2006;<br />

48: 145-155<br />

10 Yagi K, Nakamura A, Sek<strong>in</strong>e A. Comparison between magnify<strong>in</strong>g<br />

endoscopy and histological, culture and urease test<br />

f<strong>in</strong>d<strong>in</strong>gs from the gastric mucosa <strong>of</strong> the corpus. Endoscopy<br />

2002; 34: 376-381<br />

11 Yang JM, Chen L, Fan YL, Li XH, Yu X, Fang DC. Endoscopic<br />

patterns <strong>of</strong> gastric mucosa and its cl<strong>in</strong>icopathological<br />

significance. <strong>World</strong> J Gastroenterol 2003; 9: 2552-2556<br />

12 Yuan HF, Tang SB, Huang S, Xi JY. Diagnostic value <strong>of</strong><br />

magnify<strong>in</strong>g chromoendoscopy <strong>in</strong> gastric premalignant lesions.<br />

Shijie Huaren Xiaohua Zazhi 2008; 16: 2052-2055<br />

13 Gono K, Obi T, Yamaguchi M, Ohyama N, Machida H,<br />

Sano Y, Yoshida S, Hamamoto Y, Endo T. Appearance <strong>of</strong><br />

enhanced tissue features <strong>in</strong> narrow-band endoscopic imag<strong>in</strong>g.<br />

J Biomed Opt 2004; 9: 568-577<br />

14 Emura F, Saito Y, Ikematsu H. Narrow-band imag<strong>in</strong>g optical<br />

chromo<strong>colon</strong>oscopy: advantages and limitations. <strong>World</strong> J<br />

Gastroenterol 2008; 14: 4867-4872<br />

15 Tahara T, Shibata T, Nakamura M, Yoshioka D, Okubo M,<br />

Arisawa T, Hirata I. Gastric mucosal pattern by us<strong>in</strong>g magnify<strong>in</strong>g<br />

narrow-band imag<strong>in</strong>g endoscopy clearly dist<strong>in</strong>guishes<br />

histological and serological severity <strong>of</strong> chronic gastritis. Gastro<strong>in</strong>test<br />

Endosc 2009; 70: 246-253<br />

16 Uedo N, Ishihara R, Iishi H, Yamamoto S, Yamamoto S,<br />

Yamada T, Imanaka K, Takeuchi Y, Higash<strong>in</strong>o K, Ishiguro<br />

S, Tatsuta M. A new method <strong>of</strong> diagnos<strong>in</strong>g gastric <strong>in</strong>test<strong>in</strong>al<br />

metaplasia: narrow-band imag<strong>in</strong>g with magnify<strong>in</strong>g endoscopy.<br />

Endoscopy 2006; 38: 819-824<br />

17 Har<strong>in</strong>gsma J, Tytgat GN, Yano H, Iishi H, Tatsuta M, Ogihara<br />

T, Watanabe H, Sato N, Marcon N, Wilson BC, Cl<strong>in</strong>e<br />

RW. Aut<strong>of</strong>luorescence endoscopy: feasibility <strong>of</strong> detection <strong>of</strong><br />

GI neoplasms unapparent to white light endoscopy with an<br />

evolv<strong>in</strong>g technology. Gastro<strong>in</strong>test Endosc 2001; 53: 642-650<br />

18 Kara MA, Bergman JJ. Aut<strong>of</strong>luorescence imag<strong>in</strong>g and narrow-band<br />

imag<strong>in</strong>g for the detection <strong>of</strong> early neoplasia <strong>in</strong> patients<br />

with Barrett's esophagus. Endoscopy 2006; 38: 627-631<br />

19 Kato M, Uedo N, Ishihara R, Kizu T, Chatani R, Inoue T,<br />

Masuda E, Tatsumi K, Takeuchi Y, Higash<strong>in</strong>o K, Iishi H,<br />

Tomita Y, Tatsuta M. Analysis <strong>of</strong> the color patterns <strong>of</strong> early<br />

gastric cancer us<strong>in</strong>g an aut<strong>of</strong>luorescence imag<strong>in</strong>g video endoscopy<br />

system. Gastric Cancer 2009; 12: 219-224<br />

20 Inoue T, Uedo N, Ishihara R, Kawaguchi T, Kawada N,<br />

WJG|www.wjgnet.com<br />

Dai YC et al . Assessment <strong>of</strong> atrophic gastritis<br />

Chatani R, Kizu T, Tamai C, Takeuchi Y, Higash<strong>in</strong>o K, Iishi<br />

H, Tatsuta M, Tomita Y, Tóth E. Aut<strong>of</strong>luorescence imag<strong>in</strong>g<br />

videoendoscopy <strong>in</strong> the diagnosis <strong>of</strong> chronic atrophic fundal<br />

gastritis. J Gastroenterol 2010; 45: 45-51<br />

21 Saml<strong>of</strong>f IM, Taggart RT. Peps<strong>in</strong>ogens, peps<strong>in</strong>s, and peptic<br />

ulcer. Cl<strong>in</strong> Invest Med 1987; 10: 215-221<br />

22 Saml<strong>of</strong>f IM, Varis K, Ihamaki T, Siurala M, Rotter JI. Relationships<br />

among serum peps<strong>in</strong>ogen I, serum peps<strong>in</strong>ogen II,<br />

and gastric mucosal histology. A study <strong>in</strong> relatives <strong>of</strong> patients<br />

with pernicious anemia. <strong>Gastroenterology</strong> 1982; 83: 204-209<br />

23 Ren JS, Kamangar F, Qiao YL, Taylor PR, Liang H, Dawsey<br />

SM, Liu B, Fan JH, Abnet CC. Serum peps<strong>in</strong>ogens and risk<br />

<strong>of</strong> gastric and oesophageal cancers <strong>in</strong> the General Population<br />

Nutrition Intervention Trial cohort. Gut 2009; 58: 636-642<br />

24 Storskrubb T, Aro P, Ronka<strong>in</strong>en J, Sipponen P, Nyhl<strong>in</strong> H,<br />

Talley NJ, Engstrand L, Stolte M, Vieth M, Walker M, Agréus<br />

L. Serum biomarkers provide an accurate method for diagnosis<br />

<strong>of</strong> atrophic gastritis <strong>in</strong> a general population: The Kalixanda<br />

study. Scand J Gastroenterol 2008; 43: 1448-1455<br />

25 Miki K, Morita M, Sasajima M, Hosh<strong>in</strong>a R, Kanda E, Urita<br />

Y. Usefulness <strong>of</strong> gastric cancer screen<strong>in</strong>g us<strong>in</strong>g the serum<br />

peps<strong>in</strong>ogen test method. Am J Gastroenterol 2003; 98: 735-739<br />

26 Hattori Y, Tashiro H, Kawamoto T, Kodama Y. Sensitivity<br />

and specificity <strong>of</strong> mass screen<strong>in</strong>g for gastric cancer us<strong>in</strong>g the<br />

measurment <strong>of</strong> serum peps<strong>in</strong>ogens. Jpn J Cancer Res 1995; 86:<br />

1210-1215<br />

27 Oishi Y, Kiyohara Y, Kubo M, Tanaka K, Tanizaki Y, N<strong>in</strong>omiya<br />

T, Doi Y, Shikata K, Yonemoto K, Shirota T, Matsumoto T,<br />

Iida M. The serum peps<strong>in</strong>ogen test as a predictor <strong>of</strong> gastric<br />

cancer: the Hisayama study. Am J Epidemiol 2006; 163: 629-637<br />

28 Sipponen P, Mäki T, Ranta P, L<strong>in</strong>nala A, Kääriä<strong>in</strong>en I,<br />

Helske T, Suovaniemi O, Härkönen M. Application <strong>of</strong> blood<br />

levels <strong>of</strong> gastr<strong>in</strong>-17, peps<strong>in</strong>ogen I and H.pylori antibody for<br />

nonendoscopic diagnosis <strong>of</strong> atrophic gastritis. Available<br />

from: URL: http://www.biohit.com/pdf/ddwsip.pdf<br />

29 Leja M, Kupc<strong>in</strong>skas L, Funka K, Sudraba A, Jonaitis L, Ivanauskas<br />

A, Janciauskas D, Kiudelis G, Chiu HM, L<strong>in</strong> JT. The<br />

validity <strong>of</strong> a biomarker method for <strong>in</strong>direct detection <strong>of</strong> gastric<br />

mucosal atrophy versus standard histopathology. Dig<br />

Dis Sci 2009; 54: 2377-2384<br />

30 Schistosomes, liver flukes and Helicobacter pylori. IARC<br />

Work<strong>in</strong>g Group on the Evaluation <strong>of</strong> Carc<strong>in</strong>ogenic Risks to<br />

Humans. Lyon, 7-14 June 1994. IARC Monogr Eval Carc<strong>in</strong>og<br />

Risks Hum 1994; 61: 1-241<br />

31 Fox JG, Wang TC. Helicobacter pylori-not a good bug after<br />

all! N Engl J Med 2001; 345: 829-832<br />

32 Correa P. A human model <strong>of</strong> gastric carc<strong>in</strong>ogenesis. Cancer<br />

Res 1988; 48: 3554-3560<br />

33 Weck MN, Stegmaier C, Rothenbacher D, Brenner H. Epidemiology<br />

<strong>of</strong> chronic atrophic gastritis: population-based study<br />

among 9444 older adults from Germany. Aliment Pharmacol<br />

Ther 2007; 26: 879-887<br />

S- Editor Sun H L- Editor Ma JY E- Editor Ma WH<br />

1693 April 7, 2011|Volume 17|Issue 13|

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lead to fibrosis, cirrhosis, end-stage liver disease and hepatocellular<br />

carc<strong>in</strong>oma [1] . The ultimate goal <strong>of</strong> antiviral treatment<br />

for hepatitis C is the susta<strong>in</strong>ed elim<strong>in</strong>ation <strong>of</strong> HCV.<br />

Currently, the standard <strong>of</strong> care for <strong>in</strong>dividuals with CHC is<br />

pegylated <strong>in</strong>terferon (IFN) α-2a or IFN α-2b plus ribavir<strong>in</strong>.<br />

However, this protocol is far from ideal. Even under the<br />

best conditions <strong>of</strong> sponsored cl<strong>in</strong>ical trials, susta<strong>in</strong>ed virologic<br />

responses have been achieved <strong>in</strong> only 40%-50% <strong>of</strong><br />

those with HCV genotype 1 <strong>in</strong>fection [2,3] . Furthermore, serious<br />

side effects are associated with this therapy. The paucity<br />

<strong>of</strong> effective and affordable treatments for HCV-<strong>in</strong>fected<br />

patients has led scientists to seek alternative therapies. At<br />

present, novel therapeutic agents with various mechanisms<br />

<strong>of</strong> action are under development or <strong>in</strong> cl<strong>in</strong>ical trials.<br />

Although the precise mechanisms underly<strong>in</strong>g hepatocellular<br />

<strong>in</strong>jury associated with HCV has yet to be determ<strong>in</strong>ed,<br />

there is compell<strong>in</strong>g evidence that HCV produces<br />

<strong>in</strong>creased oxidative stress <strong>in</strong> human liver cells that is l<strong>in</strong>ked<br />

to the production <strong>of</strong> reactive oxygen species (ROS), and<br />

consequent <strong>in</strong>creases <strong>in</strong> cellular lipid peroxidation and<br />

other oxidative damage. Oxidative stress appears to be an<br />

important aspect <strong>of</strong> HCV-<strong>in</strong>duced hepatocellular <strong>in</strong>jury [4] .<br />

Microsomal heme oxygenase-1 (HMOX-1) is an <strong>in</strong>ducible<br />

cytoprotective enzyme that catalyzes the <strong>in</strong>itial and<br />

rate-limit<strong>in</strong>g reaction <strong>in</strong> heme catabolism to release free<br />

iron and equimolar amounts <strong>of</strong> carbon monoxide and<br />

biliverd<strong>in</strong> [5,6] . A variety <strong>of</strong> DNA-b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong>s <strong>in</strong>teract<br />

with regions that conta<strong>in</strong> multiple antioxidant response elements<br />

(ARE). Among these are nuclear factor erythroid<br />

2-related factor 2 (Nrf2) and Bach1, a leuc<strong>in</strong>e b-zipper<br />

transcription prote<strong>in</strong>, which form heterodimers with the<br />

small Maf prote<strong>in</strong>s [7,8] . Nrf2 is known to be associated<br />

with activation <strong>of</strong> HMOX-1 and numerous other antioxidant<br />

genes <strong>in</strong> response to multiple agents [9] , while Bach1 is<br />

a negative regulator <strong>of</strong> HMOX-1 [10] .<br />

Milk thistle (Silybum marianum) has been used s<strong>in</strong>ce<br />

ancient times as a liver tonic. Silymar<strong>in</strong> (SI), a purified extract<br />

<strong>of</strong> polyphenolic flavonoids isolated from milk thistle,<br />

is composed ma<strong>in</strong>ly <strong>of</strong> silychrist<strong>in</strong>, silydian<strong>in</strong>, silyb<strong>in</strong> A,<br />

silyb<strong>in</strong> B, isosilyb<strong>in</strong> A and isosilyb<strong>in</strong> B. After oral adm<strong>in</strong>istration,<br />

the SI flavonolignans are rapidly metabolized [11] .<br />

Silyb<strong>in</strong> (SBN) constitutes approximately 50% <strong>of</strong> SI and<br />

is the most biologically active component [12] . A number<br />

<strong>of</strong> studies have shown that SI has potent antioxidant<br />

and immunomodulatory effects <strong>in</strong> addition to numerous<br />

metabolic actions that may contribute to its purported<br />

hepatoprotective actions [13-15] .<br />

We recently showed that SI downregulates HCV RNA<br />

(core region) and prote<strong>in</strong> <strong>in</strong> CNS3 cells that stably express<br />

HCV RNA core to the am<strong>in</strong>o term<strong>in</strong>al <strong>of</strong> NS3 prote<strong>in</strong>s<br />

[16] . Another recent study <strong>in</strong> vitro showed that SI exerts<br />

antiviral and anti<strong>in</strong>flammatory effects <strong>in</strong> hepatoma cell<br />

l<strong>in</strong>es express<strong>in</strong>g the HCV full length genome <strong>of</strong> genotype<br />

2a [17] . On the other hand, a randomized, double-bl<strong>in</strong>d,<br />

placebo-controlled study adm<strong>in</strong>ister<strong>in</strong>g oral SI to CHC<br />

patients failed to show a significant effect on either serum<br />

am<strong>in</strong>otransferase levels or quality-<strong>of</strong>-life measures [18] .<br />

Legalon-SIL (LS) is a form <strong>of</strong> SBN which is a watersoluble<br />

formulation <strong>of</strong> the dihydro-succ<strong>in</strong>ate sodium salt<br />

<strong>of</strong> SBN A and SBN B <strong>in</strong> equal proportion. Recent results<br />

WJG|www.wjgnet.com<br />

Mehrab-Mohseni M et al . Legalon-SIL downregulates HCV<br />

from a pilot study <strong>in</strong> patients with chronic HCV us<strong>in</strong>g LS<br />

<strong>in</strong>dicate that some SI flavonolignans may have antiviral<br />

activity [19] . In this study we assessed the effects <strong>of</strong> LS on<br />

HCV RNA and prote<strong>in</strong> levels <strong>in</strong> cell l<strong>in</strong>es express<strong>in</strong>g the<br />

full length genome <strong>of</strong> HCV genotype 1b. We also determ<strong>in</strong>ed<br />

the effects <strong>of</strong> LS on HMOX-1, Bach1, and Nrf2<br />

expression <strong>in</strong> these cells.<br />

MATERIALS AND METHODS<br />

Chemicals and antibodies<br />

LS was obta<strong>in</strong>ed from Rottapharm-Madaus (Italy). Dimethyl<br />

sulphoxide (DMSO) was purchased from Thermo<br />

Fisher Scientific Inc (Rock ford, IL, USA). A 100 mmol/L<br />

LS stock solution (molecular weight = 726) was prepared<br />

<strong>in</strong> DMSO and filtered through a 0.2 μmol/L nylon filter.<br />

LS was prepared fresh just prior to use <strong>in</strong> each experiment.<br />

Mouse monoclonal antibody aga<strong>in</strong>st HCV core<br />

prote<strong>in</strong> was purchased from Abcam (Cambridge, MA,<br />

USA). Mouse monoclonal antibody aga<strong>in</strong>st HCV NS5A<br />

was purchased from Virogen Corporation (Watertown,<br />

MA, USA). Rabbit polyclonal antibody aga<strong>in</strong>st HMOX-1<br />

was purchased from Stress Gene (Ann Arbor, MI, USA).<br />

Goat monoclonal antibody aga<strong>in</strong>st Bach1, rabbit polyclonal<br />

antibody aga<strong>in</strong>st Nrf2, and mouse anti-glyceraldehyde-3-phosphate<br />

dehydrogenase (GAPDH) monoclonal<br />

antibody were purchased from Santa Cruz Biotechnology<br />

Inc. (Santa Cruz, CA, USA). Enhanced chemilum<strong>in</strong>escence<br />

(ECL)-Plus Western blott<strong>in</strong>g detection reagent was<br />

obta<strong>in</strong>ed from Amersham Biosciences (Piscataway, NJ,<br />

USA).<br />

Cell cultures and treatments<br />

Huh-7.5 and CON1 subgenomic genotype 1b HCV cell<br />

l<strong>in</strong>es were from Apath LLC (St. Louis, MO, USA). Huh-7.5<br />

is a highly permissive, IFN-cured Huh-7 human hepatocellular<br />

carc<strong>in</strong>oma cell l<strong>in</strong>e derivative. The CON1 cell l<strong>in</strong>e is<br />

a Huh-7.5 cell population conta<strong>in</strong><strong>in</strong>g the full-length HCV<br />

genotype 1b replicon.<br />

Huh-7.5 and CON1 cells were ma<strong>in</strong>ta<strong>in</strong>ed <strong>in</strong> Dulbecco’s<br />

Modified Eagle’s Medium (DMEM) supplemented with<br />

10% fetal bov<strong>in</strong>e serum, 100 U/mL penicill<strong>in</strong>, 100 μg/mL<br />

streptomyc<strong>in</strong>, and selection antibiotic for CON1 cells<br />

(750 μg/mL G418).<br />

Colorimetric MTT assay<br />

Cellular proliferation <strong>of</strong> treated CON1 cells was assessed<br />

by measur<strong>in</strong>g the conversion <strong>of</strong> MTT [3-(4, 5-dimethylthiazol-2-yl)-2,<br />

5-diphenyl tetrazolium bromide] to MTT<br />

formazan (Sigma-Aldrich). The absorbance was measured<br />

on a Synergy HT microtiter plate reader (Biotek Instruments,<br />

W<strong>in</strong>ooski, VT, USA), at a wavelength <strong>of</strong> 570 nm<br />

with background subtraction at 690 nm. Decreases <strong>in</strong><br />

absorption were taken as an <strong>in</strong>dex <strong>of</strong> decreased cellular<br />

proliferation.<br />

Propidium iodide assay<br />

The viability <strong>of</strong> CON1 cells treated with LS was also confirmed<br />

by the standard propidium iodide [(PI); Invitrogen,<br />

1695 April 7, 2011|Volume 17|Issue 13|

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Mehrab-Mohseni M et al . Legalon-SIL downregulates HCV<br />

USA)] assay. The experiment was performed accord<strong>in</strong>g to<br />

the manufacturer’s recommended protocol. CON1 cells<br />

were plated <strong>in</strong> 12-well plates 24 h before treatment and <strong>in</strong>cubated<br />

at 37℃. LS was dissolved <strong>in</strong> DMSO and added to<br />

the cell culture medium. The effects <strong>of</strong> LS on cell viability<br />

were studied at various concentrations (0, 50, 100, 200,<br />

300, 400 and 500) μmol/L and at different time po<strong>in</strong>ts<br />

(4, 8, and 24 h). Percent cell viability was determ<strong>in</strong>ed by<br />

count<strong>in</strong>g cell density <strong>in</strong> drug-treated cells and <strong>in</strong> DMSOtreated<br />

cells as control <strong>in</strong> the same <strong>in</strong>cubation period [percentage<br />

<strong>of</strong> cellular viability = (total cell count-PI positive<br />

cell count)/total cell count*100]. All experiments were<br />

repeated 3 times.<br />

RNA isolation and quantitative reverse transcriptase<br />

polymerase cha<strong>in</strong> reaction<br />

RNA from treated cells was isolated by TRIZOL reagent<br />

(Invitrogen). The RNA concentration and purity were<br />

determ<strong>in</strong>ed by measur<strong>in</strong>g absorbance at 260/280 nm. Reverse<br />

transcription was performed on 1 μg <strong>of</strong> total RNA<br />

to generate cDNA us<strong>in</strong>g the iScript cDNA Synthesis Kit<br />

(Bio-Rad Laboratories, Hercules, CA, USA). Quantitative<br />

real time reverse transcriptase (RT)-polymerase cha<strong>in</strong><br />

reaction (PCR) was performed us<strong>in</strong>g a CFX-96 Real-Time<br />

PCR Detection System (Bio-Rad Laboratories) and iQTM<br />

SYBR Green Supermix (Bio-Rad Laboratories). Sequence<br />

specific primers for HMOX-1, Bach1, and GAPDH were<br />

designed as described [20] . Nucleotide sequences <strong>of</strong> other<br />

primers are as follows: NS5A: Forward primer, 5'-CG-<br />

GACGTAGCAGTGCTCACTTC-3' and reverse primer,<br />

5-'TGATGAGCTGGCCAAGGAGG-3'; Nrf2: Forward<br />

primer, 5'-CCTTTCTCGCCTAGGCATCA-3', reverse<br />

primer, 5'-CCCTTCAGCTCTCCCTACCG-3'. Fold<br />

change values were calculated by comparative cycle threshold<br />

(Ct) value analysis after normaliz<strong>in</strong>g for the quantity <strong>of</strong><br />

GAPDH mRNA <strong>in</strong> samples.<br />

Prote<strong>in</strong> preparation and Western blott<strong>in</strong>g<br />

Cells were grown to near confluence and washed with<br />

phosphate-buffered sal<strong>in</strong>e (PBS), lysed <strong>in</strong> a buffer conta<strong>in</strong><strong>in</strong>g<br />

1% Triton X-100 with PBS and Halt Protease Inhibitor<br />

Cocktail (Pierce Chemicals, Rockford, IL, USA). Prote<strong>in</strong><br />

concentrations were measured us<strong>in</strong>g the bic<strong>in</strong>chon<strong>in</strong>ic acid<br />

method. Total prote<strong>in</strong>s (10 μg) were separated on 4%-12%<br />

gradient sodium dodecyl sulphate-polyacrylamide gel (Invitrogen<br />

Laboratories) and electrophoretically transferred<br />

onto an Immun-Blot PVDF (Invitrogen Laboratories).<br />

The membranes were blocked for 1 h <strong>in</strong> PBS conta<strong>in</strong><strong>in</strong>g 5%<br />

nonfat dry milk, and then <strong>in</strong>cubated for 1 h with the primary<br />

antibody at room temperature. The dilutions <strong>of</strong> the<br />

primary antibodies were as follows: 1:1000 for anti-HCV<br />

core antibody; 1:1000 for anti-HCV NS5A antibody; 1:500<br />

for anti-HMOX-1, and 1:1000 for anti-Bach1, anti-Nrf2,<br />

and anti-GAPDH antibody. After 4 washes with 0.1%<br />

Tween 20 <strong>in</strong> PBS (PBS-T), the membranes were <strong>in</strong>cubated<br />

for 1 h with a secondary antibody (anti-rabbit, anti-goat or<br />

anti-mouse immunoglobul<strong>in</strong> G; dilution 1:10 000). F<strong>in</strong>ally,<br />

the membranes were washed 4 times with PBS-T, and the<br />

WJG|www.wjgnet.com<br />

Cell viability (%)<br />

120<br />

100<br />

80<br />

60<br />

40<br />

20<br />

4 h<br />

8 h<br />

24 h<br />

a a<br />

0<br />

0 100 200 300 400 500<br />

Legalon-SIL (μmol/L)<br />

Figure 1 Effects <strong>of</strong> legalon-SIL on CON1 cell viability. Cellular viability <strong>of</strong><br />

CON1 cells was measured us<strong>in</strong>g the propidium iodide assay after 4, 8, and<br />

24 h <strong>of</strong> exposure to legalon-SIL (LS) at vary<strong>in</strong>g concentrations rang<strong>in</strong>g from<br />

0-500 μm/L. The mean ± SE from 3 <strong>in</strong>dependent experiments. a P < 0.05 vs<br />

DMSO control.<br />

bound antibodies were visualized with the ECL-Plus chemilum<strong>in</strong>escence<br />

system. A computer based imag<strong>in</strong>g system,<br />

LAS 3000 (Fuji Film, USA) was used to measure the<br />

relative optical density <strong>of</strong> each specific band obta<strong>in</strong>ed after<br />

Western blott<strong>in</strong>g.<br />

Statistical analysis<br />

Data are expressed as mean ± SE <strong>of</strong> the mean. Statistical<br />

differences between groups were analyzed by analysis <strong>of</strong><br />

variance followed by Dunnett’s test. P < 0.05 was considered<br />

significant.<br />

RESULTS<br />

Cytotoxicity <strong>of</strong> Legalon-SIL <strong>in</strong> CON1<br />

Appropriate doses <strong>of</strong> LS <strong>in</strong> the cell l<strong>in</strong>es were determ<strong>in</strong>ed.<br />

Effects <strong>of</strong> different doses <strong>of</strong> LS on cell viability <strong>in</strong> CON1<br />

cells were assessed by PI sta<strong>in</strong><strong>in</strong>g (Figure 1). LS concentrations<br />

<strong>of</strong> 50-300 μmol/L had no significant effect on cell<br />

viability, whereas concentrations equal to or more than<br />

400 μmol/L caused significant cytotoxicity <strong>in</strong> the cells (P<br />

< 0.05). Similar results were demonstrated with the MTT<br />

proliferation assay (data not shown).<br />

Legalon-SIL downregulates HCV RNA as well as HCV<br />

core and NS5A prote<strong>in</strong>s <strong>in</strong> CON1 cells<br />

LS downregulated HCV RNA (core region) <strong>in</strong> a dose-dependent<br />

and also a time-dependent manner <strong>in</strong> CON1 cells.<br />

The HCV RNA (core region) level was decreased 21% follow<strong>in</strong>g<br />

6 h treatment with LS 200 μmol/L compared with<br />

the DMSO control (P < 0.05, Figure 2A). HCV RNA (core<br />

region) levels were further decreased after 24 h treatment<br />

by both LS 50 μmol/L (55% decrease, P < 0.05) and 200<br />

μmol/L (88% decrease, P < 0.01) when compared with vehicle<br />

(DMSO) control (Figure 2A). The HCV RNA (NS5A<br />

region) level was also decreased 43% follow<strong>in</strong>g 6 h treatment<br />

with LS 200 μmol/L compared with DMSO control<br />

(P < 0.01 Figure 2B), and was also further decreased after<br />

1696 April 7, 2011|Volume 17|Issue 13|

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A<br />

HCV RNA (normalized)<br />

120<br />

100<br />

80<br />

60<br />

40<br />

20<br />

24 h treatment by both LS 50 μmol/L (62% decrease, P<br />

< 0.01) and 200 μmol/L (87% decrease, P < 0.01) (Figure<br />

2B). LS 200 μmol/L also downregulated HCV core (by<br />

57%) and NS5A prote<strong>in</strong> (by 49%) after 24 h <strong>of</strong> treatment<br />

although this was statistically significant only for HCV<br />

core prote<strong>in</strong>, P < 0.05). This effect was more pronounced<br />

follow<strong>in</strong>g 48 h <strong>of</strong> treatment: LS 200 μmol/L decreased<br />

HCV NS5A prote<strong>in</strong> expression by 65% (P < 0.05), while<br />

LS significantly decreased HCV core prote<strong>in</strong> expression <strong>in</strong><br />

a dose-dependent manner (52% reduction at 50 μmol/L<br />

and 85% reduction at 200 μmol/L P < 0.01) (Figure 3).<br />

Legalon-SIL downregulates HMOX-1 and Bach1 mRNA<br />

levels <strong>in</strong> CON1 cells while it upregulates Nrf2 prote<strong>in</strong><br />

expression<br />

HMOX-1 and Bach1 mRNA levels were significantly decreased<br />

follow<strong>in</strong>g 24 h treatment by both LS 50 μmol/L<br />

and 200 μmol/L when compared with the DMSO control<br />

(HMOX-1 decreased by 40%, P < 0.01; Bach1 decreased<br />

by 35%, P < 0.01; Figure 4). LS treatment decreased Bach1<br />

prote<strong>in</strong> level, although not significantly, while it significantly<br />

<strong>in</strong>creased Nrf2 prote<strong>in</strong> expression (P < 0.05) (Figure 5).<br />

HMOX-1 expression is <strong>in</strong>creased <strong>in</strong> CON1 cells <strong>in</strong><br />

comparison with its expression <strong>in</strong> the parental Huh7.5<br />

cell l<strong>in</strong>e<br />

For <strong>in</strong>vestigation <strong>of</strong> possible effects <strong>of</strong> stable transfection<br />

<strong>of</strong> the HCV genome <strong>in</strong> CON1 replicon system, we compared<br />

HMOX-1, Bach1, and Nrf2 mRNA levels between<br />

CON1 cells and Huh7.5 cells, the ‘parental’ cell l<strong>in</strong>e. In untreated<br />

CON1 cells, the HMOX-1 mRNA level was 3-fold<br />

higher than <strong>in</strong> untreated Huh7.5 cells (P < 0.01), while<br />

there was no significant difference <strong>in</strong> the level <strong>of</strong> Bach1 or<br />

Nrf2 mRNA between CON1 and Huh7.5 cells (Figure 6).<br />

DISCUSSION<br />

a<br />

DMSO<br />

LS 50<br />

LS 200<br />

Core region<br />

0<br />

0 4 8 12 16 20 24 28<br />

t /h<br />

The current treatment for CHC is a comb<strong>in</strong>ation <strong>of</strong> pe-<br />

WJG|www.wjgnet.com<br />

b<br />

a<br />

Mehrab-Mohseni M et al . Legalon-SIL downregulates HCV<br />

Figure 2 Time course <strong>of</strong> effects <strong>of</strong> legalon-SIL on hepatitis C virus RNA <strong>in</strong> CON1 cells. CON1 cells were grown to near confluence and the medium was changed to 5%<br />

fetal bov<strong>in</strong>e serum (FBS) plus Dulbecco’s modified Eagle’s medium, then treated with vehicle only (DMSO) or 50 or 200 μmol/L legalon-SIL (LS). The cells were harvested<br />

after 2, 6, and 24 h after treatment. The levels <strong>of</strong> hepatitis C virus (HCV) RNA [core region (A) and NS5A region (B)] were quantified us<strong>in</strong>g qRT-PCR as described <strong>in</strong> “Materials<br />

and Methods”. The amounts <strong>of</strong> HCV RNA were normalized to glyceraldehyde-3-phosphate dehydrogenase. A: LS 50 μmol/L downregulated HCV RNA (core region) after<br />

24 h. LS 200 μmol/L downregulated HCV RNA (core region) after 6 and 24 h; B: LS 50 μmol/L downregulated HCV RNA (NS5A region) after 24 h. LS 200 μM downregulated<br />

HCV RNA (core region) after 6 and 24 h. Data for RNA levels are mean ± SE (n = 3 <strong>in</strong>dependent experiments). a P < 0.05, b P < 0.01 vs DMSO control.<br />

B<br />

HCV RNA (normalized)<br />

120<br />

100<br />

80<br />

60<br />

40<br />

20<br />

b<br />

DMSO<br />

LS 50<br />

LS 200<br />

NS5A region<br />

0<br />

0 4 8 12 16 20 24 28<br />

t /h<br />

gylated IFN-α and ribavir<strong>in</strong> which is effective only <strong>in</strong><br />

40%-50% <strong>of</strong> treated patients <strong>in</strong>fected with genotype-1,<br />

by far the most frequent HCV genotype worldwide. This<br />

therapeutic regimen is expensive, prolonged (usually at<br />

least 48 wk) and causes serious side effects. Therefore <strong>in</strong>vestigations<br />

cont<strong>in</strong>ue to search for alternative treatments<br />

for hepatitis C. SI is an herbal remedy that has been used<br />

to treat acute and chronic liver diseases for millennia [12-22] .<br />

Despite this broad use, the exact molecular mechanism by<br />

which SI confers hepatoprotection is yet to be elucidated.<br />

Ferenci et al [19] recently showed that LS, as used <strong>in</strong> our<br />

studies, significantly reduced serum HCV RNA level <strong>in</strong> patients<br />

who had not responded to comb<strong>in</strong>ation therapy with<br />

full dose pegylated IFN and ribavir<strong>in</strong>. They showed that<br />

SBN was effective only when adm<strong>in</strong>istered <strong>in</strong>travenously,<br />

as LS, and that the antiviral effect was dose-dependent.<br />

They reported that HCV RNA was undetectable <strong>in</strong> 7 (out<br />

<strong>of</strong> 14) patients receiv<strong>in</strong>g 15 and 20 mg/kg SBN as LS.<br />

In a recent study <strong>in</strong> vitro [23] , SBN A, SBN B, a mixture <strong>of</strong><br />

SBN A and SBN B, and LS were shown to <strong>in</strong>hibit HCV<br />

RNA-dependent RNA polymerase (RdRP) function and<br />

<strong>in</strong>hibited HCV genotype 1b sub-genomic replicon replication<br />

and HCV genotype 2a stra<strong>in</strong> JFH1 replication <strong>in</strong> cell<br />

culture. Our results extend these f<strong>in</strong>d<strong>in</strong>gs show<strong>in</strong>g that LS<br />

<strong>in</strong>hibits HCV replication <strong>in</strong> human hepatoma cells express<strong>in</strong>g<br />

full-length HCV genotype 1b. In this study, we also<br />

showed that LS doses equal to 400 μmol/L and higher are<br />

cytotoxic for human hepatoma cells similar to other recent<br />

results [23] . The exact pharmacok<strong>in</strong>etics <strong>of</strong> SI and LS rema<strong>in</strong><br />

to be determ<strong>in</strong>ed. However, it is suggested that oral<br />

doses <strong>of</strong> SI up to 2.1 g/d are safe and well-tolerated [24] . In<br />

the present study, we showed that LS started to downregulate<br />

HCV RNA <strong>in</strong> a dose-dependent manner after 2 h, but<br />

the significant effect was observed after 6 h <strong>of</strong> treatment.<br />

Although LS 200 μmol/L was found to downregulate<br />

HCV RNA and prote<strong>in</strong>s significantly <strong>in</strong> CON1 cells, LS<br />

50 μmol/L was also found to be effective <strong>in</strong> downregulation<br />

<strong>of</strong> HCV RNA.<br />

1697 April 7, 2011|Volume 17|Issue 13|<br />

b<br />

b

---

Mehrab-Mohseni M et al . Legalon-SIL downregulates HCV<br />

Peer review<br />

The paper will appeal to cl<strong>in</strong>icians as well as researchers <strong>in</strong>volved <strong>in</strong> the field <strong>of</strong><br />

elucidat<strong>in</strong>g the mechanisms <strong>of</strong> silymar<strong>in</strong> <strong>in</strong> HCV positive patients.<br />

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agent <strong>in</strong> experimental cholestasis: new possibilities for an<br />

ancient medication. Curr Med Chem 2006; 13: 1055-1074<br />

13 Dehmlow C, Murawski N, de Groot H. Scaveng<strong>in</strong>g <strong>of</strong> reactive<br />

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by silib<strong>in</strong><strong>in</strong> <strong>in</strong> human cells. Life Sci 1996; 58: 1591-1600<br />

14 Lee JS, Kim SG, Kim HK, Lee TH, Jeong YI, Lee CM, Yoon<br />

MS, Na YJ, Suh DS, Park NC, Choi IH, Kim GY, Choi YH,<br />

Chung HY, Park YM. Silib<strong>in</strong><strong>in</strong> polarizes Th1/Th2 immune<br />

responses through the <strong>in</strong>hibition <strong>of</strong> immunostimulatory<br />

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15 Manna SK, Mukhopadhyay A, Van NT, Aggarwal BB.<br />

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Silymar<strong>in</strong> suppresses TNF-<strong>in</strong>duced activation <strong>of</strong> NF-kappa<br />

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16 Bonifaz V, Shan Y, Lambrecht RW, Donohue SE, Moschenross<br />

D, Bonkovsky HL. Effects <strong>of</strong> silymar<strong>in</strong> on hepatitis C virus<br />

and haem oxygenase-1 gene expression <strong>in</strong> human hepatoma<br />

cells. Liver Int 2009; 29: 366-373<br />

17 Polyak SJ, Morishima C, Shuhart MC, Wang CC, Liu Y, Lee<br />

DY. Inhibition <strong>of</strong> T-cell <strong>in</strong>flammatory cytok<strong>in</strong>es, hepatocyte<br />

NF-kappaB signal<strong>in</strong>g, and HCV <strong>in</strong>fection by standardized<br />

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18 Gordon A, Hobbs DA, Bowden DS, Bailey MJ, Mitchell J,<br />

Francis AJ, Roberts SK. Effects <strong>of</strong> Silybum marianum on<br />

serum hepatitis C virus RNA, alan<strong>in</strong>e am<strong>in</strong>otransferase<br />

levels and well-be<strong>in</strong>g <strong>in</strong> patients with chronic hepatitis C. J<br />

Gastroenterol Hepatol 2006; 21: 275-280<br />

19 Ferenci P, Scherzer TM, Kerschner H, Rutter K, Be<strong>in</strong>hardt S,<br />

H<strong>of</strong>er H, Schöniger-Hekele M, Holzmann H, Ste<strong>in</strong>dl-Munda<br />

P. Silib<strong>in</strong><strong>in</strong> is a potent antiviral agent <strong>in</strong> patients with chronic<br />

hepatitis C not respond<strong>in</strong>g to pegylated <strong>in</strong>terferon/ribavir<strong>in</strong><br />

therapy. <strong>Gastroenterology</strong> 2008; 135: 1561-1567<br />

20 Ghaziani T, Shan Y, Lambrecht RW, Donohue SE, Pietschmann<br />

T, Bartenschlager R, Bonkovsky HL. HCV prote<strong>in</strong>s<br />

<strong>in</strong>crease expression <strong>of</strong> heme oxygenase-1 (HO-1) and<br />

decrease expression <strong>of</strong> Bach1 <strong>in</strong> human hepatoma cells. J<br />

Hepatol 2006; 45: 5-12<br />

21 Seeff LB, Curto TM, Szabo G, Everson GT, Bonkovsky HL,<br />

Dienstag JL, Shiffman ML, L<strong>in</strong>dsay KL, Lok AS, Di Bisceglie<br />

AM, Lee WM, Ghany MG. Herbal product use by persons<br />

enrolled <strong>in</strong> the hepatitis C Antiviral Long-Term Treatment<br />

Aga<strong>in</strong>st Cirrhosis (HALT-C) Trial. Hepatology 2008; 47: 605-612<br />

22 Zhang L, Wang G, Hou W, Li P, Dul<strong>in</strong> A, Bonkovsky HL.<br />

Contemporary cl<strong>in</strong>ical research <strong>of</strong> traditional Ch<strong>in</strong>ese<br />

medic<strong>in</strong>es for chronic hepatitis B <strong>in</strong> Ch<strong>in</strong>a: an analytical<br />

review. Hepatology 2010; 51: 690-698<br />

23 Ahmed-Belkacem A, Ahnou N, Barbotte L, Wychowski C,<br />

Pallier C, Brillet R, Pohl RT, Pawlotsky JM. Silib<strong>in</strong><strong>in</strong> and<br />

related compounds are direct <strong>in</strong>hibitors <strong>of</strong> hepatitis C virus<br />

RNA-dependent RNA polymerase. <strong>Gastroenterology</strong> 2010;<br />

138: 1112-1122<br />

24 Hawke RL, Schrieber SJ, Soule TA, Wen Z, Smith PC,<br />

Reddy KR, Wahed AS, Belle SH, Afdhal NH, Navarro VJ,<br />

Berman J, Liu QY, Doo E, Fried MW. Silymar<strong>in</strong> ascend<strong>in</strong>g<br />

multiple oral dos<strong>in</strong>g phase I study <strong>in</strong> noncirrhotic patients<br />

with chronic hepatitis C. J Cl<strong>in</strong> Pharmacol 2010; 50: 434-449<br />

25 Polyak SJ, Morishima C, Lohmann V, Pal S, Lee DY, Liu Y,<br />

Graf TN, Oberlies NH. Identification <strong>of</strong> hepatoprotective<br />

flavonolignans from silymar<strong>in</strong>. Proc Natl Acad Sci USA 2010;<br />

107: 5995-5999<br />

26 Reichard JF, Motz GT, Puga A. Heme oxygenase-1 <strong>in</strong>duction<br />

by NRF2 requires <strong>in</strong>activation <strong>of</strong> the transcriptional repressor<br />

BACH1. Nucleic Acids Res 2007; 35: 7074-7086<br />

27 Gozzel<strong>in</strong>o R, Jeney V, Soares MP. Mechanisms <strong>of</strong> cell<br />

protection by heme oxygenase-1. Annu Rev Pharmacol Toxicol<br />

2010; 50: 323-354<br />

28 Gong G, Waris G, Tanveer R, Siddiqui A. Human hepatitis C<br />

virus NS5A prote<strong>in</strong> alters <strong>in</strong>tracellular calcium levels, <strong>in</strong>duces<br />

oxidative stress, and activates STAT-3 and NF-kappa B. Proc<br />

Natl Acad Sci USA 2001; 98: 9599-9604<br />

29 Sendi H, Mehrab-Mohseni M, Steuerwald N, Bonkovsky HL.<br />

Heme oxygenase 1 [HMOX1] expression is up-regulated by<br />

HCV <strong>in</strong> human hepatoma cells and is normalized through<br />

IFN treatment. Hepatology 2009; 50: S936A<br />

S- Editor Tian L L- Editor Cant MR E- Editor Ma WH<br />

1700 April 7, 2011|Volume 17|Issue 13|

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Onl<strong>in</strong>e Submissions: http://www.wjgnet.com/1007-9327<strong>of</strong>fice<br />

wjg@wjgnet.com<br />

doi:10.3748/wjg.v17.i13.1701<br />

ORIGINAL ARTICLE<br />

Endoscopic submucosal dissection for premalignant lesions<br />

and non<strong>in</strong>vasive early gastro<strong>in</strong>test<strong>in</strong>al cancers<br />

Sadett<strong>in</strong> Hulagu, Omer Senturk, Cem Aygun, Orhan Kocaman, Altay Celebi, Tolga Konduk, Deniz Koc,<br />

Goktug Sir<strong>in</strong>, Ugur Korkmaz, Ali Erkan Duman, Neslihan Bozkurt, Gokhan D<strong>in</strong>dar, Tan Attila, Yesim Gurbuz,<br />

Orhan Tarc<strong>in</strong>, Cem Kalayci<br />

Sadett<strong>in</strong> Hulagu, Omer Senturk, Cem Aygun, Orhan Kocaman,<br />

Altay Celebi, Tolga Konduk, Deniz Koc, Goktug<br />

Sir<strong>in</strong>, Ugur Korkmaz, Ali Erkan Duman, Neslihan Bozkurt,<br />

Gokhan D<strong>in</strong>dar, Division <strong>of</strong> <strong>Gastroenterology</strong>, Kocaeli University<br />

Medical School, Kocaeli 41000, Turkey<br />

Tan Attila, Division <strong>of</strong> <strong>Gastroenterology</strong>, American Hospital,<br />

Istanbul 34365, Turkey<br />

Yesim Gurbuz, Department <strong>of</strong> Pathology, Kocaeli University<br />

Medical School, Kocaeli 41000, Turkey<br />

Orhan Tarc<strong>in</strong>, Division <strong>of</strong> <strong>Gastroenterology</strong>, Der<strong>in</strong>ce State Hospital,<br />

Kocaeli 41900, Turkey<br />

Cem Kalayci, Division <strong>of</strong> <strong>Gastroenterology</strong>, Marmara University<br />

School <strong>of</strong> Medic<strong>in</strong>e, Altunizade, Istanbul 34662, Turkey<br />

Author contributions: Hulagu S performed all ESD procedures;<br />

Senturk O assisted dur<strong>in</strong>g the ESD procedure and literature<br />

review; Aygun C assisted dur<strong>in</strong>g the ESD procedure,<br />

data collection and statistical analysis; Kocaman O, Celebi A,<br />

Konduk T, Koc D, Sir<strong>in</strong> G, Korkmaz U, Duman AE, Bozkurt<br />

N and D<strong>in</strong>dar G assisted dur<strong>in</strong>g the ESD procedure and data<br />

collection; Attila T, Tarc<strong>in</strong> O and Kalayci C performed the endosonographic<br />

evaluation <strong>of</strong> patients; Gurbuz Y performed the<br />

histologic exam<strong>in</strong>ation <strong>of</strong> ESD specimens.<br />

Correspondence to: Dr. Sadett<strong>in</strong> Hulagu, Division <strong>of</strong> <strong>Gastroenterology</strong>,<br />

Kocaeli University Medical School, Kocaeli<br />

41000, Turkey. shulagu@hotmail.com<br />

Telephone: +90-532-3252996 Fax: +90-262-3038003<br />

Received: October 21, 2010 Revised: November 20, 2010<br />

Accepted: November 27, 2010<br />

Published onl<strong>in</strong>e: April 7, 2011<br />

Abstract<br />

AIM: To <strong>in</strong>vestigate the <strong>in</strong>dication, feasibility, safety,<br />

and cl<strong>in</strong>ical utility <strong>of</strong> endoscopic submucosal dissection<br />

(ESD) <strong>in</strong> the management <strong>of</strong> various gastro<strong>in</strong>test<strong>in</strong>al<br />

pathologies.<br />

METHODS: The medical records <strong>of</strong> 60 consecutive patients<br />

(34 female, 26 male) who underwent ESD at the<br />

gastroenterology department <strong>of</strong> Kocaeli University from<br />

2006-2010 were exam<strong>in</strong>ed. Patients selected for ESD<br />

WJG|www.wjgnet.com<br />

1701<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): 1701-1709<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

had premalignant lesions or non-<strong>in</strong>vasive early cancers<br />

<strong>of</strong> the gastro<strong>in</strong>test<strong>in</strong>al tract and had endoscopic and<br />

histological diagnoses. Early cancers were considered<br />

to be conf<strong>in</strong>ed to the submucosa, with no lymph node<br />

<strong>in</strong>volvement by means <strong>of</strong> computed tomography and<br />

endosonography.<br />

RESULTS: Sixty ESD procedures were performed. The<br />

<strong>in</strong>dications were epithelial lesions (n = 39) (33/39 adenoma<br />

with high grade dysplasia, 6/39 adenoma with<br />

low grade dysplasia), neuroendocr<strong>in</strong>e tumor (n = 7),<br />

cancer (n = 7) (5/7 early colorectal cancer, 2/7 early<br />

gastric cancer), granular cell tumor (n = 3), gastro<strong>in</strong>test<strong>in</strong>al<br />

stromal tumor (n = 2), and leiomyoma (n = 2). En<br />

bloc and piecemeal resection rates were 91.6% (55/60)<br />

and 8.3% (5/60), respectively. Complete and <strong>in</strong>complete<br />

resection rates were 96.6% (58/60) and 3.3%<br />

(2/60), respectively. Complications were major bleed<strong>in</strong>g<br />

[n = 3 (5%)] and perforations [n = 5 (8.3%)] (4<br />

<strong>colon</strong>, 1 stomach). Two patients with <strong>colon</strong>ic perforations<br />

and two patients with submucosal lymphatic and<br />

microvasculature <strong>in</strong>vasion (1 gastric carc<strong>in</strong>oid tumor,<br />

1 <strong>colon</strong>ic adenocarc<strong>in</strong>oma) were referred to surgery.<br />

Dur<strong>in</strong>g a mean follow-up <strong>of</strong> 12 mo, 1 patient with adenoma<br />

with high grade dysplasia underwent a second<br />

ESD procedure to resect a local recurrence.<br />

CONCLUSION: ESD is a feasible and safe method for<br />

treatment <strong>of</strong> premalignant lesions and early malignant<br />

gastro<strong>in</strong>test<strong>in</strong>al epithelial and subepithelial lesions. Successful<br />

en bloc and complete resection <strong>of</strong> lesions yield<br />

high cure rates with low recurrence.<br />

Key words: Endoscopic submucosal dissection; Premalignant<br />

gastro<strong>in</strong>test<strong>in</strong>al lesion; Non<strong>in</strong>vasive early gastro<strong>in</strong>test<strong>in</strong>al<br />

cancer; Neuroendocr<strong>in</strong>e tumor; Gastro<strong>in</strong>test<strong>in</strong>al<br />

stromal tumor<br />

Peer reviewers: Marcela Kopacova, Associate Pr<strong>of</strong>essor, MD,<br />

PhD, 2nd Department <strong>of</strong> Internal Medic<strong>in</strong>e, Charles University<br />

April 7, 2011|Volume 17|Issue 13|

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electrocautery (30 W s<strong>of</strong>t coagulation) to determ<strong>in</strong>e the<br />

resection border (except <strong>in</strong> the <strong>colon</strong>). Then submucosal<br />

<strong>in</strong>jection was performed to lift the lesion. For the <strong>in</strong>jection,<br />

a special mixture (1 unit <strong>of</strong> 2% sodium hyaluronic acid,<br />

3 units <strong>of</strong> sal<strong>in</strong>e, 0.5 mL <strong>of</strong> ep<strong>in</strong>ephr<strong>in</strong>e (1/10 000) and<br />

0.5% <strong>of</strong> <strong>in</strong>digo carm<strong>in</strong>e) was used. After sufficient lift<strong>in</strong>g,<br />

a flush knife (DK2618JN 20; Fuj<strong>in</strong>on), <strong>in</strong>sulated-tip knife<br />

(KD-610L; Olympus) or needle knife (KD-11Q-1) was<br />

used to create a circumferential <strong>in</strong>cision around the lesion<br />

extend<strong>in</strong>g <strong>in</strong>to the submucosa. After circumferential <strong>in</strong>cision,<br />

a submucosal dissection was performed to remove<br />

the lesion <strong>in</strong> an en bloc fashion.<br />

A high frequency generator with an automatically controlled<br />

system (Endo-cut mode; ERBE ICC 200, Elektromediz<strong>in</strong><br />

GmbH, Germany) was used for dissection and<br />

coagulation. A specialized cap (EMR ST Hood DH 15CR,<br />

Fuj<strong>in</strong>on) was placed on the tip <strong>of</strong> the endoscope to make<br />

the dissection easier by <strong>in</strong>creas<strong>in</strong>g stability. Initially marked<br />

lesions were dissected with a diathermic knife (Olympus<br />

or Fuj<strong>in</strong>on) circumferentially us<strong>in</strong>g endo-cut mode (2-3/80<br />

W). An <strong>in</strong>sulated-tip (IT) knife (KD-610L; Olympus)<br />

was used to dissect the borders <strong>of</strong> the lesions with a high<br />

perforation risk (wide based lesions and <strong>colon</strong>ic lesions<br />

between haustral folds) us<strong>in</strong>g endo-cut mode (3/120 W).<br />

Submucosal dissection was performed with spray coagulation<br />

(45 W). Small vessels were coagulated with spray coagulation.<br />

Larger vessels or arteries with high bleed<strong>in</strong>g risk<br />

were coagulated with hemostatic forceps (Fuj<strong>in</strong>on).<br />

Circumferential <strong>in</strong>cision was completed <strong>in</strong> all cases. In<br />

<strong>colon</strong>ic lesions, semi-circumferential <strong>in</strong>cision was performed<br />

<strong>in</strong>itially. After submucosal dissection, circumferential<br />

<strong>in</strong>cision was completed. A few cases were f<strong>in</strong>ished<br />

with snare resection, but only after 80% <strong>of</strong> ESDs were<br />

completed. Standard or therapeutic gastroscopes (Fuj<strong>in</strong>on<br />

EG-530 D) were used for lesions located <strong>in</strong> the rectum<br />

and sigmoid <strong>colon</strong>. Lesions close to the anus were<br />

treated <strong>in</strong> the retr<strong>of</strong>lexion position. Colonoscopes were<br />

used for lesions proximal to the splenic flexure.<br />

The first gastric lesions treated by ESD were located<br />

at the antrum <strong>in</strong> our series, as most gastric lesions were.<br />

Later on, cardiac lesions were treated by ESD <strong>in</strong> the retr<strong>of</strong>lexion<br />

position.<br />

All <strong>of</strong> the ESD procedures were performed under<br />

deep sedation. A comb<strong>in</strong>ation <strong>of</strong> prop<strong>of</strong>ol and fentanyl<br />

was provided by an anesthesiologist. Patients were cont<strong>in</strong>uously<br />

monitored with an electrocardiogram, and blood<br />

pressure and oxygen saturation were monitored. The<br />

position <strong>of</strong> the patient could be easily changed whenever<br />

required with the help <strong>of</strong> medical attendants under the<br />

control <strong>of</strong> the anesthesiologist.<br />

Def<strong>in</strong>itions and follow-up strategy<br />

All the specimens were exam<strong>in</strong>ed by a s<strong>in</strong>gle pathologist<br />

who is specialized <strong>in</strong> gastro<strong>in</strong>test<strong>in</strong>al pathology. En bloc<br />

resection was def<strong>in</strong>ed as the removal <strong>of</strong> a lesion <strong>in</strong> a s<strong>in</strong>gle<br />

piece. Piecemeal resection was def<strong>in</strong>ed as the removal<br />

<strong>of</strong> a lesion <strong>in</strong> more than one piece.<br />

A recurrent disease was def<strong>in</strong>ed as the reappearance<br />

<strong>of</strong> neoplastic tissue at the site <strong>of</strong> <strong>in</strong>itial ESD at the 6th mo<br />

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Hulagu S et al . Endoscopic submucosal dissection<br />

follow-up endoscopy. In the case <strong>of</strong> a perforation, hemoclips<br />

were used. Bleed<strong>in</strong>g that could be managed with<br />

endoscopic <strong>in</strong>tervention was considered as m<strong>in</strong>or bleed<strong>in</strong>g.<br />

Bleed<strong>in</strong>g with hemodynamic <strong>in</strong>stability and blood transfusion<br />

requirement with or without the need for surgical<br />

<strong>in</strong>tervention was considered as major bleed<strong>in</strong>g.<br />

A lesion was considered to be completely removed<br />

(R0 resection), when the vertical and lateral surgical marg<strong>in</strong>s<br />

were 2 mm away from the lesion. When neoplastic<br />

cells were present at surgical marg<strong>in</strong>s, this was considered<br />

as an <strong>in</strong>complete resection (R1). Patients found to have<br />

undifferentiated or signet cell adenocarc<strong>in</strong>oma and submucosal/lymphovascular<br />

<strong>in</strong>vasion on histopathological<br />

evaluation were referred to surgery. Patients were hospitalized<br />

for observation after the procedure and underwent<br />

a control endoscopy with<strong>in</strong> 2 d <strong>of</strong> the ESD procedure.<br />

Patients underwent follow-up endoscopies at 3 and 6 mo.<br />

After a normal endoscopy at the 6th mo, annual followup<br />

was <strong>of</strong>fered.<br />

Statistical analysis<br />

A median <strong>of</strong> cont<strong>in</strong>uous variables was used to present<br />

data. The Kruskal-Wallis test was used to compare median<br />

procedure time <strong>of</strong> ESD groups. When Kruskal-Wallis test<br />

results were statistically significant (P < 0.05), a Mann-<br />

Whitney test us<strong>in</strong>g Bonferroni correction was used to<br />

compare median procedure time between ESD groups. P<br />

< 0.01 was accepted as statistically significant. Statistical<br />

Packages for Social Sciences version 16.0 for W<strong>in</strong>dows<br />

(SPSS, Chicago, IL, USA) was used for statistical analysis.<br />

RESULTS<br />

Over the 46-mo period, 60 ESD procedures were performed<br />

by a s<strong>in</strong>gle operator (S.H.). There were 34 female<br />

(56.6%) and 26 male (43.3%) patients. The mean age (via<br />

standard deviation) <strong>of</strong> patients was 54.6 (± 14.1) years.<br />

The majority <strong>of</strong> ESD procedures (65%) were performed<br />

for <strong>in</strong>traepithelial lesions (n = 39) (33/39 adenoma<br />

with high grade dysplasia, 6/39 adenoma with low grade<br />

dysplasia). The <strong>in</strong>dication <strong>of</strong> the rema<strong>in</strong><strong>in</strong>g ESD procedures<br />

were as follow<strong>in</strong>g: neuroendocr<strong>in</strong>e tumor (NET) (n<br />

= 7), cancer (n = 7) [5/7 early colorectal cancer (ECC), 2/7<br />

early gastric cancer (EGC)], granular cell tumor (n = 3), gastro<strong>in</strong>test<strong>in</strong>al<br />

stromal tumor (GIST) (n = 2), and leiomyoma (n<br />

= 2). Microscopic types <strong>of</strong> the lesions <strong>in</strong> the different locations<br />

accord<strong>in</strong>g to Paris classification is given <strong>in</strong> Table 1. En<br />

bloc and piecemeal resection rates were 91.6% (55/60) and<br />

8.3% (5/60), complete and <strong>in</strong>complete resection rates were<br />

96.6% (58/60) and 3.3% (2/60), respectively.<br />

An adenoma (piecemeal resection is done on purpose,<br />

Figure 1), early gastric cancer located <strong>in</strong> the antrum,<br />

and three flat adenomas with lateral <strong>in</strong>vasion <strong>in</strong> the <strong>colon</strong><br />

were resected <strong>in</strong> piecemeal fashion. A patient with<br />

a NET that was <strong>in</strong>completely resected was referred to<br />

surgery due to vascular <strong>in</strong>vasion noted on histopathologic<br />

evaluation. A patient with an <strong>in</strong>completely resected early<br />

<strong>colon</strong> cancer (ECC) due to lateral spread<strong>in</strong>g was referred<br />

to surgery. Invasion <strong>in</strong>to the muscularis propria was seen<br />

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A B C D<br />

E<br />

ed <strong>in</strong> the 7 patients that had follow-up data. The mean<br />

follow-up period for these 7 patients was 15 mo.<br />

Gastric lesions<br />

Twenty four gastric lesions, consist<strong>in</strong>g <strong>of</strong> 16 adenomas (12<br />

adenomas with HGD, 4 adenomas with LGD), 4 carci-<br />

F<br />

I K L<br />

Figure 2 Endoscopic submucosal dissection procedure for adenoma with high grade dysplasia at cardia. A: Adenoma at cardia; B: View <strong>of</strong> the lesion<br />

from esophageal aspect; C: Endosonographic image <strong>of</strong> the lesion; D, E: Mark<strong>in</strong>g the borders <strong>of</strong> the lesion with needle knife and lift<strong>in</strong>g it; F, G: Cutt<strong>in</strong>g the lesion<br />

circumferentially with endo-cut above Z l<strong>in</strong>e, <strong>in</strong> retr<strong>of</strong>lexion; H: Dissection <strong>of</strong> the submucosal area; I: Appearance <strong>of</strong> the mucosa after the lesion be<strong>in</strong>g extracted; K:<br />

Microscopic view <strong>of</strong> the lesion; L: Histology: mucosa, muscularis mucosa and superficial submucosa <strong>of</strong> stomach (HE × 20). Adenoma structure <strong>in</strong>clud<strong>in</strong>g adenomatous<br />

epithelium formed by irregular glands at mucosa; M: Endoscopic view six months after the procedure.<br />

Table 2 En bloc, piecemeal, complete, <strong>in</strong>complete resection rates, median follow up and recurrence rates<br />

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G<br />

En bloc res. rate Piecemeal res. rate Complete res. rate Incomplete res. rate Median follow-up (mo) Local recurrence<br />

Esophagus 7/7 (100%) 0 7/7 (100%) 0 15 0<br />

Stomach 22/24 (91.7%) 2/24 (8.3%) 23/24 (95.8%) 1/24 (4.1%) 11.8 1<br />

Small <strong>in</strong>test<strong>in</strong>e and <strong>colon</strong> 26/29 (89.7%) 3/29 (10.3%) 28/29 (96.5%) 1/29 (3.4%) 12.5 0<br />

Total 55/60 (91.6%) 5/60 (8.3%) 58/60 (96.6%) 2/60 (3.3%) 12.51 1/58 (1.7%) 1<br />

1 Two patients who underwent surgery were excluded. Res: Resection.<br />

Table 3 Esophageal endoscopic submucosal dissection cases<br />

Location Number Histology (n )<br />

Proximal esophagus 1 Granular cell tumor (1)<br />

Middle esophagus 2 Granular cell tumor (2)<br />

Distal esophagus 4 GIST (1)<br />

HGD-A (2)<br />

Leiomyoma (1)<br />

Specimen size (median) 2.34 cm² (1.5-3 cm 2 )<br />

Procedure time (median) 50.5 m<strong>in</strong> (21.8 m<strong>in</strong>/cm²)<br />

GIST: Gastro<strong>in</strong>test<strong>in</strong>al stromal tumor; HGD-A: Adenoma with high grade<br />

dysplasia.<br />

Hulagu S et al . Endoscopic submucosal dissection<br />

noid tumors, 2 early gastric cancers, 1 GIST (submucosal),<br />

and 1 leiomyoma (submucosal) were treated with ESD<br />

(Table 4) (Figures 1 and 2). One lesion was located <strong>in</strong> the<br />

cardia (adenoma with HGD), 3 lesions were located <strong>in</strong> the<br />

gastric corpus (3 carc<strong>in</strong>oid tumors), and 20 lesions were<br />

located <strong>in</strong> the antrum (11 adenomas with HGD, 4 adenomas<br />

with LGD, 2 EGC, 1 GIST, 1 carc<strong>in</strong>oid tumor, 1<br />

leiomyoma). A patient with a gastric adenoma with HGD<br />

had recurrence at the site <strong>of</strong> prior resection. This patient<br />

had a second ESD, 12 mo after the first ESD.<br />

Colonic and small <strong>in</strong>test<strong>in</strong>al lesions<br />

Twenty-n<strong>in</strong>e lesions <strong>in</strong>clud<strong>in</strong>g 21 adenomas (2 tubulovillous<br />

adenoma with HGD <strong>in</strong> duodenal bulb, 1 tubulovillous<br />

adenoma with HGD <strong>in</strong> cecum, 2 tubulovillous<br />

adenoma with HGD <strong>in</strong> transverse <strong>colon</strong>, 3 tubulovillous<br />

adenoma with HGD <strong>in</strong> sigmoid <strong>colon</strong>, 1 tubular adenoma<br />

<strong>in</strong> sigmoid <strong>colon</strong>, 9 tubulovillous adenoma with HGD <strong>in</strong><br />

rectum, and 3 tubular adenoma <strong>in</strong> rectum), 3 carc<strong>in</strong>oid<br />

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A B C<br />

D<br />

G H<br />

<strong>in</strong> three patients (5%). One <strong>of</strong> these patients had a lesion<br />

located <strong>in</strong> the <strong>colon</strong> and two <strong>of</strong> them had lesions located<br />

<strong>in</strong> the stomach. Contribut<strong>in</strong>g factors to major bleed<strong>in</strong>g<br />

may possibly be the nature as well as the location <strong>of</strong> the lesion,<br />

possibly due to the rich vascularization <strong>of</strong> the lesion.<br />

Both <strong>of</strong> the bleed<strong>in</strong>g lesions <strong>in</strong> the stomach were NETs<br />

and located <strong>in</strong> the lesser curvature. Bleed<strong>in</strong>g from gastric<br />

lesions was delayed and required blood transfusion. The<br />

<strong>colon</strong>ic case that had a major bleed was an early <strong>colon</strong>ic<br />

cancer. M<strong>in</strong>or bleed<strong>in</strong>g occurred <strong>in</strong> 13 cases (7 lesions<br />

located <strong>in</strong> the <strong>colon</strong> and 6 lesions located <strong>in</strong> the stomach)<br />

(21.7%). Table 6 shows complications <strong>in</strong> detail.<br />

DISCUSSION<br />

Although endoscopic resection-based therapeutic modalities<br />

(EMR and ESD) are considered to be the treatment<br />

<strong>of</strong> choice for premalignant and early gastro<strong>in</strong>test<strong>in</strong>al neoplasias<br />

<strong>in</strong> Japan, they are not widely practiced by Western<br />

endoscopists [10] . This is the first study from Turkey and<br />

among the few studies from outside <strong>of</strong> Japan and Asia on<br />

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E F<br />

Hulagu S et al . Endoscopic submucosal dissection<br />

Figure 4 Endoscopic submucosal dissection procedure for pseudo-depressed type lateral spread<strong>in</strong>g tumor with high grade dysplasia at rectum. A:<br />

Pseudo-depressed type lateral spread<strong>in</strong>g tumor at rectum; B: Cutt<strong>in</strong>g the lesion circumferentially with endo-cut; C, D: Submucosal dissection with semipermeable cap;<br />

E: Endoscopic view just before complet<strong>in</strong>g submucosal dissection; F: Appearance <strong>of</strong> the mucosa after the lesion be<strong>in</strong>g extracted; G: Microscopic view <strong>of</strong> the lesion; H:<br />

Histology; tubulovillous adenoma <strong>in</strong>clud<strong>in</strong>g fields <strong>of</strong> focal pattern loss and dysplasia (HE × 20); I: Endoscopic view ten weeks after the procedure.<br />

I<br />

the application <strong>of</strong> ESD <strong>in</strong> the management <strong>of</strong> premalignant<br />

and non<strong>in</strong>vasive early gastro<strong>in</strong>test<strong>in</strong>al cancers from<br />

various anatomic locations <strong>in</strong> the gastro<strong>in</strong>test<strong>in</strong>al tract.<br />

It is difficult or impossible to remove large lesions<br />

with EMR technique <strong>in</strong> one fragment. Removal <strong>of</strong> a<br />

lesion <strong>in</strong> one piece is very important to accurately diagnose<br />

the tumor depth as well as decreas<strong>in</strong>g the risk <strong>of</strong><br />

local recurrence. A recent study has illustrated that this<br />

problem can be solved with the use <strong>of</strong> ESD for larger<br />

lesions [11] . In early esophageal cancers with a diameter <strong>of</strong><br />

less than 20 mm, en bloc and curative resection rate <strong>of</strong><br />

ESD (97%) was found to be significantly higher than that<br />

<strong>of</strong> EMR us<strong>in</strong>g a transparent cap (71%) and 2-channel<br />

EMR (46%) [11] . However, no significant difference was<br />

found between ESD and EMR us<strong>in</strong>g a transparent cap <strong>in</strong><br />

en bloc and curative resection rate <strong>of</strong> lesions less than 15<br />

mm <strong>in</strong> diameter. Therefore, ESD would be a better therapeutic<br />

modality than EMR for esophageal lesions with a<br />

diameter <strong>of</strong> greater than 15 mm. Given the size (median<br />

size <strong>of</strong> esophageal/gastric/<strong>colon</strong>ic lesions 2.34 cm²/3.25<br />

cm²/8.61 cm 2 respectively), various anatomic location <strong>of</strong><br />

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Hulagu S et al . Endoscopic submucosal dissection<br />

Table 6 Complications with regard to location and diagnosis<br />

n Diagnosis M<strong>in</strong>or<br />

bleed<strong>in</strong>g<br />

lesions and subepithelial nature <strong>of</strong> some lesions, ESD<br />

would be the treatment <strong>of</strong> choice for our cases.<br />

There are few studies from Europe that were published<br />

<strong>in</strong> full manuscript format. D<strong>in</strong>is-Ribeiro et al [12] .<br />

evaluated feasibility and effectiveness <strong>of</strong> ESD <strong>in</strong> 19 gastric<br />

superficial lesions with HGD, LGD and non<strong>in</strong>vasive epithelial<br />

neoplasias. Probst et al [13] . evaluated ESD <strong>in</strong> 71 flat<br />

adenomas, early cancers and submucosal tumors located<br />

<strong>in</strong> various locations <strong>of</strong> the gastro<strong>in</strong>test<strong>in</strong>al tract (51 gastric,<br />

17 rectal, 2 esophageal and 1 duodenal). In the study <strong>of</strong><br />

D<strong>in</strong>is-Ribeiro et al, complete and en bloc resection rates<br />

were 89% and 79%, respectively. Major bleed<strong>in</strong>g occurred<br />

<strong>in</strong> 1 case (5%). There were no perforations. Recurrence<br />

<strong>of</strong> a lesion (5%) was noted with<strong>in</strong> a mean follow-up <strong>of</strong><br />

10 mo. In order to evaluate ESD learn<strong>in</strong>g curve, Probst<br />

et al [13] . compared various aspects <strong>of</strong> ESD procedures performed<br />

<strong>in</strong> the first and second halves <strong>of</strong> the study.<br />

A statistically significant <strong>in</strong>crease <strong>in</strong> specimen size and<br />

decrease <strong>in</strong> procedural duration were noted between the<br />

two groups. En bloc resection rates and R0 en bloc resection<br />

rates <strong>in</strong> the first half <strong>of</strong> the study (77.1% and 65.7%,<br />

respectively) <strong>in</strong>creased when compared with the second<br />

half <strong>of</strong> the study (86.1% and 72.2%, respectively), however<br />

this difference did not reach statistical significance.<br />

No recurrence occurred after R0 en bloc resection; however<br />

38% recurrence occurred after piecemeal resection.<br />

Complications <strong>in</strong> the study <strong>of</strong> Probst <strong>in</strong>cluded 2 perforations<br />

(gastric submucosal tumors) that required surgery<br />

(2.7%), 2 other perforations (large flat rectal lesions)<br />

(2.7%), 8 m<strong>in</strong>or bleed<strong>in</strong>gs (10.9%) and 3 pyloric stenosis<br />

(4.1%) that were endoscopically managed.<br />

ESD complication rates among the published studies<br />

have been variable depend<strong>in</strong>g on the size <strong>of</strong> the study<br />

as well as the experience <strong>of</strong> the operator. In our study,<br />

a patient (1.7%) was found to have a recurrence <strong>of</strong> an<br />

adenoma with HGD at the site <strong>of</strong> prior ESD on 12-mo<br />

follow-up endoscopy. The recurrent lesion was treated<br />

with a repeat ESD. The patient was free <strong>of</strong> disease at the<br />

6-mo follow-up. No recurrences were noted with esophageal<br />

and <strong>colon</strong>ic lesions. Compared to other studies, lower<br />

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Major Perforation<br />

bleed<strong>in</strong>g<br />

Esophagus 7 3 granular cell tumor<br />

1 GIST<br />

2 Premalignant lesions<br />

1 Leiomyoma<br />

Stomach 24 4 NETs 4 2 1<br />

2 EGC 2<br />

18 Premalignant lesions<br />

Small<br />

<strong>in</strong>test<strong>in</strong>e<br />

4 2 Premalignant lesions<br />

2 NETs<br />

Colon 25 5 ECCs 3 1 1<br />

19 Premalignant lesions 4 3<br />

1 NETs<br />

Total 60 13<br />

(21.7%)<br />

3<br />

(5%)<br />

5<br />

(8.3%)<br />

GIST: Gastro<strong>in</strong>test<strong>in</strong>al stromal tumor; NET: Neuroendocr<strong>in</strong>e tumor; EGC:<br />

Early gastric cancer; ECC: Early colorectal cancer.<br />

recurrence rate <strong>in</strong> our study may be related to the relatively<br />

shorter follow up (median = 12.5 mo) <strong>of</strong> the patients<br />

after ESD. Our bleed<strong>in</strong>g rate is consistent with other<br />

studies. Our perforation rate is higher than the study <strong>of</strong><br />

Probst et al [13] . However, all <strong>of</strong> our perforations occurred<br />

at the first half <strong>of</strong> the study, which is a reflection <strong>of</strong> the<br />

impact <strong>of</strong> the operator’s experience with the success <strong>of</strong><br />

ESD procedures. Eighty percent <strong>of</strong> perforations occurred<br />

with <strong>colon</strong>ic cases, which may be related to the relatively<br />

th<strong>in</strong>ner <strong>colon</strong>ic wall thickness and larger size <strong>of</strong> <strong>colon</strong>ic<br />

lesions. Most perforations took place <strong>in</strong> <strong>in</strong>itial cases. In<br />

those cases needle knives were used. We believe that the<br />

use <strong>of</strong> these knives also contributed to this relatively high<br />

number <strong>of</strong> perforations. After provid<strong>in</strong>g IT-knives we<br />

did not encounter any perforations. As stated above we<br />

could not refuse the patients and it is true that we had to<br />

perform colorectal cases with <strong>in</strong>sufficient experience <strong>in</strong><br />

gastric ESD, result<strong>in</strong>g <strong>in</strong> relatively high perforation rates.<br />

In a review article from Japan, the en bloc resection<br />

rate <strong>of</strong> early gastric cancers was reported to be<br />

79%-100%, with local recurrence, bleed<strong>in</strong>g and perforation<br />

rates <strong>of</strong> 0%-1%, 1.7%-38%, 0%-5%, respectively [14] .<br />

Another study from Japan evaluat<strong>in</strong>g ESD <strong>in</strong> colorectal<br />

epithelial neoplasms revealed the rate <strong>of</strong> en bloc resection<br />

and en bloc resection with tumor free marg<strong>in</strong>s to be<br />

91.5% and 70.5% [15] . In this study, perforation and local<br />

recurrence rates were found to be 5% and 1.7%, respectively.<br />

The sample size <strong>of</strong> studies com<strong>in</strong>g from outside<br />

<strong>of</strong> Japan and Asia is quite modest; therefore it is premature<br />

to compare Western experience with the Asian one.<br />

Ideally one should beg<strong>in</strong> with gastric cases located <strong>in</strong><br />

the antrum. After gett<strong>in</strong>g sufficient experience <strong>in</strong> gastric<br />

cases they can proceed with esophageal and colorectal cases,<br />

which are more risky. Our practice seems <strong>in</strong>compatible<br />

with this idea. But ESD is only performed <strong>in</strong> our <strong>in</strong>stitute<br />

<strong>in</strong> Turkey and patients are referred to our hospital from<br />

the entire country. So we did not have the chance to refuse<br />

the patients and performed esophageal and colorectal<br />

cases before hav<strong>in</strong>g sufficient experience <strong>in</strong> gastric cases.<br />

We believe that, besides EMR and endoscopic piecemeal<br />

mucosal resection, ESD will be a good alternative <strong>in</strong><br />

the treatment <strong>of</strong> non-epithelial esophageal lesions. We observed<br />

that neuroendocr<strong>in</strong>e tumors which have rich vascularization<br />

are more likely to bleed, so more attention should<br />

be paid when operat<strong>in</strong>g on them. Dur<strong>in</strong>g the ESD procedures<br />

<strong>in</strong> the <strong>colon</strong> and esophagus, a needle knife should be<br />

avoided <strong>in</strong> endo-cutt<strong>in</strong>g because <strong>of</strong> the perforation risk.<br />

Perforation risk is even higher <strong>in</strong> laterally spread<strong>in</strong>g <strong>colon</strong>ic<br />

lesions so IT-knife is a better choice for those lesions.<br />

In summary, ESD, which orig<strong>in</strong>ates <strong>in</strong> Japan, has been<br />

ga<strong>in</strong><strong>in</strong>g popularity <strong>in</strong> other parts <strong>of</strong> the world as well.<br />

Comparable outcomes <strong>of</strong> ESD to surgery play an important<br />

role <strong>in</strong> the rapid propagation <strong>of</strong> this therapeutic endoscopic<br />

modality. Although no procedure related mortality<br />

has been reported, there is considerable morbidity with this<br />

technique. There is a significant learn<strong>in</strong>g curve to achieve<br />

pr<strong>of</strong>iciency <strong>in</strong> order to acquire skills to perform ESD safely<br />

and effectively. Therefore, importance <strong>of</strong> tra<strong>in</strong><strong>in</strong>g can not<br />

be overemphasized. Further studies from outside <strong>of</strong> Japan<br />

and Asia are needed to better determ<strong>in</strong>e the global role <strong>of</strong><br />

1708 April 7, 2011|Volume 17|Issue 13|

---

ESD <strong>in</strong> the management <strong>of</strong> premalignant and early malignant<br />

epithelial, as well as subepithelial lesions.<br />

COMMENTS<br />

Background<br />

Advances <strong>in</strong> endoscopic diagnosis techniques allowed premalignant lesions and<br />

non-<strong>in</strong>vasive cancers <strong>of</strong> the gastro<strong>in</strong>test<strong>in</strong>al system to be detected early and be<br />

treated effectively and safely by endoscopic methods. Among these methods,<br />

endoscopic submucosal dissection (ESD) is be<strong>in</strong>g used more and more commonly<br />

and has pleas<strong>in</strong>g results.<br />

Research frontiers<br />

ESD is a safe and effective modality for the treatment <strong>of</strong> premalignant lesions and<br />

early non-<strong>in</strong>vasive cancers <strong>of</strong> the gastro<strong>in</strong>test<strong>in</strong>al system and when compared<br />

to surgery it has the advantage <strong>of</strong> preserv<strong>in</strong>g the gastro<strong>in</strong>test<strong>in</strong>al system and its<br />

functions. This is the first study on ESD reported from Turkey.<br />

Innovations and breakthroughs<br />

Complications related to ESD are similar to other studies <strong>in</strong> the literature except<br />

for the complication rate. Recurrence rates are lower compared to other studies.<br />

In this study we observed that neuroendocr<strong>in</strong>e tumors had higher bleed<strong>in</strong>g<br />

rates due to hypervascularization and the use <strong>of</strong> a needle knife <strong>in</strong> <strong>colon</strong>ic and<br />

esophageal lesions <strong>in</strong>creased the risk <strong>of</strong> perforation.<br />

Applications<br />

When performed by experienced endoscopists ESD has pleas<strong>in</strong>g results and<br />

can be safely performed for the treatment <strong>of</strong> premalignant lesions and early<br />

cancers <strong>of</strong> gastro<strong>in</strong>test<strong>in</strong>al system.<br />

Term<strong>in</strong>ology<br />

ESD is be<strong>in</strong>g used for the treatment <strong>of</strong> premalignant and lesions (with no lymph<br />

node <strong>in</strong>volvement) <strong>of</strong> the gastro<strong>in</strong>test<strong>in</strong>al tractus conf<strong>in</strong>ed to mucosa and submucosa.<br />

After lift<strong>in</strong>g the lesion by <strong>in</strong>ject<strong>in</strong>g the specially prepared solution (Na - Hyaluronate<br />

+ Adrenal<strong>in</strong> + Sal<strong>in</strong>e + Indigo carm<strong>in</strong>e), the basement <strong>of</strong> the lesion, along<br />

with the surround<strong>in</strong>g area, are cut with special knives and the lesion is extracted.<br />

Peer review<br />

This retrospective study sets out to evaluate the feasibility, safety and cl<strong>in</strong>ical<br />

outcomes <strong>of</strong> ESD for premalignant lesions and early gastro<strong>in</strong>test<strong>in</strong>al cancers.<br />

It establishes that the rates <strong>of</strong> en bloc and complete resection <strong>of</strong> these lesions<br />

were good, and comparable those <strong>of</strong> previous studies.<br />

REFERENCES<br />

1 Uedo N, Iishi H, Tatsuta M, Ishihara R, Higash<strong>in</strong>o K, Takeuchi<br />

Y, Imanaka K, Yamada T, Yamamoto S, Yamamoto S,<br />

Tsukuma H, Ishiguro S. Longterm outcomes after endoscopic<br />

mucosal resection for early gastric cancer. Gastric Cancer 2006;<br />

9: 88-92<br />

WJG|www.wjgnet.com<br />

Hulagu S et al . Endoscopic submucosal dissection<br />

2 Muto M, Miyamoto S, Hosokawa A, Doi T, Ohtsu A, Yoshida<br />

S, Endo Y, Hosokawa K, Saito D, Shim CS, Gossner<br />

L. Endoscopic mucosal resection <strong>in</strong> the stomach us<strong>in</strong>g the<br />

<strong>in</strong>sulated-tip needle-knife. Endoscopy 2005; 37: 178-182<br />

3 Kim JJ, Lee JH, Jung HY, Lee GH, Cho JY, Ryu CB, Chun<br />

HJ, Park JJ, Lee WS, Kim HS, Chung MG, Moon JS, Choi SR,<br />

Song GA, Jeong HY, Jee SR, Seol SY, Yoon YB. EMR for early<br />

gastric cancer <strong>in</strong> Korea: a multicenter retrospective study.<br />

Gastro<strong>in</strong>test Endosc 2007; 66: 693-700<br />

4 Hyatt BJ, Paull PE, Wassef W. Gastric oncology: an update.<br />

Curr Op<strong>in</strong> Gastroenterol 2009; 25: 570-578<br />

5 Yamamoto H, Kita H. Endoscopic therapy <strong>of</strong> early gastric<br />

cancer. Best Pract Res Cl<strong>in</strong> Gastroenterol 2005; 19: 909-926<br />

6 Soetikno RM, Gotoda T, Nakanishi Y, Soehendra N. Endoscopic<br />

mucosal resection. Gastro<strong>in</strong>test Endosc 2003; 57: 567-579<br />

7 Gotoda T. Endoscopic resection for premalignant and malignant<br />

lesions <strong>of</strong> the gastro<strong>in</strong>test<strong>in</strong>al tract from the esophagus<br />

to the <strong>colon</strong>. Gastro<strong>in</strong>test Endosc Cl<strong>in</strong> N Am 2008; 18: 435-450,<br />

VIII<br />

8 Update on the paris classification <strong>of</strong> superficial neoplastic<br />

lesions <strong>in</strong> the digestive tract. Endoscopy 2005; 37: 570-578<br />

9 Kudo S, Hirota S, Nakajima T, Hosobe S, Kusaka H, Kobayashi<br />

T, Himori M, Yagyuu A. Colorectal tumours and pit<br />

pattern. J Cl<strong>in</strong> Pathol 1994; 47: 880-885<br />

10 Bergman JJ. How to justify endoscopic submucosal dissection<br />

<strong>in</strong> the Western world. Endoscopy 2009; 41: 988-990<br />

11 Ishihara R, Iishi H, Uedo N, Takeuchi Y, Yamamoto S, Yamada<br />

T, Masuda E, Higash<strong>in</strong>o K, Kato M, Narahara H, Tatsuta M.<br />

Comparison <strong>of</strong> EMR and endoscopic submucosal dissection<br />

for en bloc resection <strong>of</strong> early esophageal cancers <strong>in</strong> Japan.<br />

Gastro<strong>in</strong>test Endosc 2008; 68: 1066-1072<br />

12 D<strong>in</strong>is-Ribeiro M, Pimentel-Nunes P, Afonso M, Costa N,<br />

Lopes C, Moreira-Dias L. A European case series <strong>of</strong> endoscopic<br />

submucosal dissection for gastric superficial lesions.<br />

Gastro<strong>in</strong>test Endosc 2009; 69: 350-355<br />

13 Probst A, Golger D, Arnholdt H, Messmann H. Endoscopic<br />

submucosal dissection <strong>of</strong> early cancers, flat adenomas, and<br />

submucosal tumors <strong>in</strong> the gastro<strong>in</strong>test<strong>in</strong>al tract. Cl<strong>in</strong> Gastroenterol<br />

Hepatol 2009; 7: 149-155<br />

14 Gotoda T, Yamamoto H, Soetikno RM. Endoscopic submucosal<br />

dissection <strong>of</strong> early gastric cancer. J Gastroenterol 2006;<br />

41: 929-942<br />

15 Fujishiro M, Yahagi N, Kakushima N, Kodashima S, Muraki<br />

Y, Ono S, Yamamichi N, Tateishi A, Oka M, Ogura K, Kawabe<br />

T, Ich<strong>in</strong>ose M, Omata M. Outcomes <strong>of</strong> endoscopic submucosal<br />

dissection for colorectal epithelial neoplasms <strong>in</strong> 200<br />

consecutive cases. Cl<strong>in</strong> Gastroenterol Hepatol 2007; 5: 678-683;<br />

quiz 645<br />

S- Editor Tian L L- Editor Rutherford A E- Editor Ma WH<br />

1709 April 7, 2011|Volume 17|Issue 13|

---

Onl<strong>in</strong>e Submissions: http://www.wjgnet.com/1007-9327<strong>of</strong>fice<br />

wjg@wjgnet.com<br />

doi:10.3748/wjg.v17.i13.1710<br />

ORIGINAL ARTICLE<br />

Discovery and validation <strong>of</strong> prognostic markers <strong>in</strong> gastric<br />

cancer by genome-wide expression pr<strong>of</strong>il<strong>in</strong>g<br />

Yue-Zheng Zhang, Lian-Hai Zhang, Yang Gao, Chao-Hua Li, Shu-Q<strong>in</strong> Jia, Ni Liu, Feng Cheng, De-Yun Niu,<br />

William CS Cho, Jia-Fu Ji, Chang-Q<strong>in</strong>g Zeng<br />

Yue-Zheng Zhang, Yang Gao, Chao-Hua Li, Feng Cheng,<br />

De-Yun Niu, Chang-Q<strong>in</strong>g Zeng, Beij<strong>in</strong>g Institute <strong>of</strong> Genomics,<br />

Ch<strong>in</strong>ese Academy <strong>of</strong> Sciences, Beij<strong>in</strong>g 100029, Ch<strong>in</strong>a<br />

Yue-Zheng Zhang, Yang Gao, Chao-Hua Li, Feng Cheng,<br />

De-Yun Niu, CAS Key Laboratory <strong>of</strong> Genome Sciences and<br />

Information, Ch<strong>in</strong>ese Academy <strong>of</strong> Sciences, Beij<strong>in</strong>g Institute <strong>of</strong><br />

Genomics, Beij<strong>in</strong>g 100029, Ch<strong>in</strong>a<br />

Yue-Zheng Zhang, De-Yun Niu, Graduate School <strong>of</strong> Ch<strong>in</strong>ese<br />

Academy <strong>of</strong> Sciences, Beij<strong>in</strong>g 100049, Ch<strong>in</strong>a<br />

Lian-Hai Zhang, Shu-Q<strong>in</strong> Jia, Ni Liu, Jia-Fu Ji, Key Laboratory<br />

<strong>of</strong> Carc<strong>in</strong>ogenesis and Translational Research (M<strong>in</strong>istry <strong>of</strong><br />

Education), Department <strong>of</strong> Surgery, Beij<strong>in</strong>g Cancer Hospital<br />

and Institute, Pek<strong>in</strong>g University School <strong>of</strong> Oncology, Beij<strong>in</strong>g<br />

100142, Ch<strong>in</strong>a<br />

William CS Cho, Department <strong>of</strong> Cl<strong>in</strong>ical Oncology, Queen<br />

Elizabeth Hospital, Hong Kong, Ch<strong>in</strong>a<br />

Chang-Q<strong>in</strong>g Zeng, Laboratory <strong>of</strong> Cancer Genomics and Personalized<br />

Medic<strong>in</strong>e, Beij<strong>in</strong>g Institute <strong>of</strong> Genomics, Ch<strong>in</strong>ese<br />

Academy <strong>of</strong> Sciences, Beij<strong>in</strong>g 100029, Ch<strong>in</strong>a<br />

Author contributions: Zhang YZ and Zhang LH contributed<br />

equally to this work; Ji JF and Zeng CQ are correspond<strong>in</strong>g authors;<br />

Ji JF, Zhang LH, Gao Y and Zeng CQ contributed to the<br />

conception <strong>of</strong> this study; Gao Y, Zhang YZ, Zhang LH, and Ji JF<br />

designed the research strategy and experiments; Ji JF and Zhang<br />

LH collected samples and cl<strong>in</strong>ical data; Li CH, Zhang LH, Jia SQ,<br />

Zhang YZ, Liu N and Niu DY did sample preparation and chip<br />

array; Zhang YZ, Zhang LH, Gao Y and Cheng F did the data<br />

analysis and <strong>in</strong>terpretation; Zhang YZ and Zhang LH prepared the<br />

manuscript; Zeng CQ, Ji JF and Cho WCS revised the manuscript.<br />

Supported by the National 863 Program (SQ2009AA02­<br />

XK1482570 and 2006AA02A402), Beij<strong>in</strong>g Municipal Committee<br />

<strong>of</strong> Science and Technology (D0905001040631) and Beij<strong>in</strong>g Capital<br />

Development Foundation <strong>of</strong> Health Bureau (2007­2051)<br />

Correspondence to: Chang-Q<strong>in</strong>g Zeng, Pr<strong>of</strong>essor, Beij<strong>in</strong>g<br />

Institute <strong>of</strong> Genomics, Ch<strong>in</strong>ese Academy <strong>of</strong> Sciences, Key Laboratory<br />

<strong>of</strong> Genome Sciences and Information, Ch<strong>in</strong>ese Academy<br />

<strong>of</strong> Sciences, Beij<strong>in</strong>g Institute <strong>of</strong> Genomics, Ch<strong>in</strong>ese Academy <strong>of</strong><br />

Sciences, Beij<strong>in</strong>g 100029, Ch<strong>in</strong>a. czeng@big.ac.cn<br />

Telephone: +86­10­82275720 Fax: +86­10­82275694<br />

Received: September 27, 2010 Revised: November 3, 2010<br />

Accepted: November 10, 2010<br />

Published onl<strong>in</strong>e: April 7, 2011<br />

WJG|www.wjgnet.com<br />

1710<br />

Abstract<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): 1710-1717<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

AIM: To develop a prognostic gene set that can predict<br />

patient overall survival status based on the whole genome<br />

expression analysis.<br />

METHODS: Us<strong>in</strong>g Illum<strong>in</strong>a HumanWG-6 BeadChip followed<br />

by semi-supervised analysis, we analyzed the<br />

expression <strong>of</strong> 47 296 transcripts <strong>in</strong> two batches <strong>of</strong> gastric<br />

cancer patients who underwent surgical resection.<br />

Thirty-n<strong>in</strong>e samples <strong>in</strong> the first batch were used as the<br />

tra<strong>in</strong><strong>in</strong>g set to discover candidate markers correlated to<br />

overall survival, and thirty-three samples <strong>in</strong> the second<br />

batch were used for validation.<br />

RESULTS: A panel <strong>of</strong> ten genes were identified as prognostic<br />

marker <strong>in</strong> the first batch samples and classified patients<br />

<strong>in</strong>to a low- and a high-risk group with significantly<br />

different survival times (P = 0.000047). This prognostic<br />

marker was then verified <strong>in</strong> an <strong>in</strong>dependent validation<br />

sample batch (P = 0.0009). By compar<strong>in</strong>g with the traditional<br />

Tumor-node-metastasis (TNM) stag<strong>in</strong>g system, this<br />

ten-gene prognostic marker showed consistent prognosis<br />

results. It was the only <strong>in</strong>dependent prognostic value by<br />

multivariate Cox regression analysis (P = 0.007). Interest<strong>in</strong>gly,<br />

six <strong>of</strong> these ten genes are ribosomal prote<strong>in</strong>s,<br />

suggest<strong>in</strong>g a possible association between the deregulation<br />

<strong>of</strong> ribosome related gene expression and the poor<br />

prognosis.<br />

CONCLUSION: A ten-gene marker correlated with overall<br />

prognosis, <strong>in</strong>clud<strong>in</strong>g 6 ribosomal prote<strong>in</strong>s, was identified<br />

and verified, which may complement the predictive<br />

value <strong>of</strong> TNM stag<strong>in</strong>g system.<br />

© 2011 Baishideng. All rights reserved.<br />

Key words: Gastric cancer; Gene expression pr<strong>of</strong>il<strong>in</strong>g;<br />

Survival markers; Prognosis; Ribosomal prote<strong>in</strong>s<br />

April 7, 2011|Volume 17|Issue 13|

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Peer reviewer: Jian­Zhong Zhang, Pr<strong>of</strong>essor, Department <strong>of</strong><br />

Pathology and Laboratory Medic<strong>in</strong>e, Beij<strong>in</strong>g 306 Hospital, 9<br />

North Anxiang Road, PO Box 9720, Beij<strong>in</strong>g 100101, Ch<strong>in</strong>a<br />

Zhang YZ, Zhang LH, Gao Y, Li CH, Jia SQ, Liu N, Cheng F,<br />

Niu DY, Cho WCS, Ji JF, Zeng CQ. Discovery and validation <strong>of</strong><br />

prognostic markers <strong>in</strong> gastric cancer by genome­wide expression<br />

pr<strong>of</strong>il<strong>in</strong>g. <strong>World</strong> J Gastroenterol 2011; 17(13): 1710­1717 Available<br />

from: URL: http://www.wjgnet.com/1007­9327/full/v17/<br />

i13/1710.htm DOI: http://dx.doi.org/10.3748/wjg.v17.i13.1710<br />

INTRODUCTION<br />

Gastric cancer is the second lead<strong>in</strong>g cause <strong>of</strong> cancer related<br />

death worldwide [1] . As a complex and heterogeneous<br />

disease, it comprises multiple tumor entities associated<br />

with dist<strong>in</strong>ctive histological patterns and biological features,<br />

as well as cl<strong>in</strong>ical behaviours [2] . The 5­year survival rate <strong>of</strong><br />

patients with advanced disease is only 20%­30% [3] . The<br />

current treatment plan and prognosis prediction for gastric<br />

cancer ma<strong>in</strong>ly depend on the cl<strong>in</strong>icopathologic stag<strong>in</strong>g <strong>of</strong><br />

the disease, and TNM stag<strong>in</strong>g system is still the golden<br />

standard for survival prediction among gastric cancer<br />

patients. However, prognosis varies among patients with a<br />

similar tumor stage, therefore disease stag<strong>in</strong>g alone can not<br />

accurately predict the outcome for <strong>in</strong>dividual patients.<br />

Although great efforts have been made <strong>in</strong> the identification<br />

<strong>of</strong> prognostic markers from gene expression<br />

pr<strong>of</strong>il<strong>in</strong>g to improve prognosis prediction for many<br />

cancers especially breast cancer [4] , limited research has<br />

been conducted <strong>in</strong> the field <strong>of</strong> gastric cancer. To date,<br />

most studies on the selection <strong>of</strong> prognosis markers were<br />

conducted by cDNA array or quantitative RT­PCR, <strong>in</strong><br />

which only a few thousand genes were analyzed [5­9] . In an<br />

attempt to predict peritoneal relapse after gastrectomy for<br />

gastric cancer, the whole genome microarray consist<strong>in</strong>g<br />

<strong>of</strong> 30K transcripts was employed <strong>in</strong> a very recent gene<br />

expression analysis [10] . Such a robust approach may provide<br />

not only more signals <strong>in</strong> marker selection, but also more<br />

comprehensive <strong>in</strong>formation <strong>in</strong> understand<strong>in</strong>g molecular<br />

mechanisms <strong>of</strong> tumor­related processes. In this study, we<br />

explored the gene expression by microarray conta<strong>in</strong><strong>in</strong>g<br />

over 47K probes <strong>in</strong> two batches <strong>of</strong> surgical samples from<br />

79 Ch<strong>in</strong>ese gastric cancer patients. A ten­gene marker<br />

for overall survival was identified and verified <strong>in</strong> an<br />

<strong>in</strong>dependent batch <strong>of</strong> samples.<br />

MATERIALS AND METHODS<br />

Patients and samples<br />

Seventy­n<strong>in</strong>e tissue samples from patients who were surgically<br />

treated for primary gastric carc<strong>in</strong>oma were procured<br />

at the Beij<strong>in</strong>g Cancer Hospital (Pek<strong>in</strong>g University, School<br />

<strong>of</strong> Oncology) from 1999 to 2003. No patient received<br />

chemotherapy or radiotherapy before surgery. All patients<br />

were treated with curative surgical resection, which was, <strong>in</strong><br />

some cases, followed by second­l<strong>in</strong>e treatment at the time<br />

WJG|www.wjgnet.com<br />

Zhang YZ et al . Prognostic markers <strong>in</strong> gastric cancer<br />

<strong>of</strong> recurrence. Macroscopic and microscopic evaluations<br />

were conducted by pathologist accord<strong>in</strong>g to the general<br />

rules for gastric cancer. Follow­up was performed every<br />

three month for the first two years, and every three to six<br />

months thereafter. Stage <strong>of</strong> gastric cancer was classified<br />

accord<strong>in</strong>g to 2002 tumor-node-metastasis (TNM) classification<br />

system recommended by the American Jo<strong>in</strong>t Committee<br />

on Cancer.<br />

Overall survival was calculated from the date <strong>of</strong> primary<br />

surgery to the date <strong>of</strong> last follow­up or to the date<br />

<strong>of</strong> death due to cancer relapse or metastasis. All tumor<br />

samples were obta<strong>in</strong>ed at the surgery, followed by fresh<br />

freez<strong>in</strong>g <strong>in</strong> liquid nitrogen and stored at ­80°C. Informed<br />

consent was obta<strong>in</strong>ed from each patient for the collection<br />

and storage <strong>of</strong> tissue samples <strong>in</strong> a tissue bank for future<br />

research. This <strong>in</strong>vestigation was performed after approval<br />

by Ethics Committee <strong>of</strong> Pek<strong>in</strong>g University.<br />

RNA preparation and microarray analysis<br />

Total RNA was purified from cl<strong>in</strong>ical samples us<strong>in</strong>g<br />

TRIzol reagent (GibcoBRL, Grand Island, New York,<br />

USA). And mRNA was l<strong>in</strong>early amplified by <strong>in</strong> vitro<br />

transcription us<strong>in</strong>g T7 RNA polymerase (MEGAscript T7<br />

kit, Ambion, Inc, USA). The quality and <strong>in</strong>tegrity <strong>of</strong> total<br />

and amplified mRNA (cRNA) was monitored by both<br />

spectrophotometry (OD UV 260/280 ratio > 1.8) and<br />

agarose gel electrophoresis.<br />

Gene­expression pr<strong>of</strong>il<strong>in</strong>g was performed us<strong>in</strong>g Illum<strong>in</strong>a<br />

HumanWG­6 BeadChip, which conta<strong>in</strong>s 47 296 transcripts.<br />

BeadChips were scanned with a BeadStation 500<br />

GX and data are available at Gene Expression Omnibus<br />

(GSE21983). http://www.ncbi.nlm.nih.gov/geo).<br />

Statistical analysis<br />

Average normalization <strong>in</strong> BeadStudio s<strong>of</strong>tware was<br />

conducted for probe level average normalization and<br />

background correction. A detection P value was used <strong>in</strong><br />

BeadChip to calculate probability to see a certa<strong>in</strong> signal<br />

level without specific probe-target hybridization. All genes<br />

and probes with P value > 0.01 were filtered and removed<br />

from the analysis. Among 47 296 transcripts, 18 819 were<br />

expressed with P values < 0.01.<br />

The supervised pr<strong>in</strong>cipal components method was used<br />

for survival pr<strong>of</strong>il<strong>in</strong>g [11] . In the tra<strong>in</strong><strong>in</strong>g set, we calculated<br />

the modified univariate Cox proportional­hazard scores<br />

for all genes (n = 18 819), which were measured to identify<br />

genes with their expression correlated to the duration <strong>of</strong><br />

survival. We selected a set <strong>of</strong> genes whose absolute Cox<br />

score exceeded a threshold us<strong>in</strong>g cross­validation. For each<br />

iteration <strong>of</strong> the complete cross­validation, 10% <strong>of</strong> the cases<br />

were omitted, and pr<strong>in</strong>cipal components derived from the<br />

rema<strong>in</strong><strong>in</strong>g 90% <strong>of</strong> the cases were <strong>in</strong>cluded <strong>in</strong> a Cox model<br />

to predict the survival <strong>in</strong> 10% <strong>of</strong> the cases. By repeat<strong>in</strong>g the<br />

iteration process for 10 times, we found that a threshold <strong>of</strong><br />

2.6 yielded the highest average partial log­likelihood ratio.<br />

Pr<strong>in</strong>cipal component analysis (PCA) was then performed<br />

us<strong>in</strong>g 10 transcripts whose absolute Cox score equalled or<br />

exceeded the threshold for all cases <strong>in</strong> the tra<strong>in</strong><strong>in</strong>g data set.<br />

1711 April 7, 2011|Volume 17|Issue 13|

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Zhang YZ et al . Prognostic markers <strong>in</strong> gastric cancer<br />

Table 1 Cl<strong>in</strong>icopathological characteristics <strong>of</strong> all patients<br />

Variables Cases Tra<strong>in</strong><strong>in</strong>g dataset<br />

(n = 39)<br />

1 The multiple comparisons <strong>of</strong> different subclasses; 2 Data was <strong>in</strong>complete.<br />

Kaplan­Meier survival curves were then plotted to predict<br />

overall survival. All analysis and plott<strong>in</strong>g were conducted<br />

us<strong>in</strong>g R package superpc (http://www­stat.stanford.edu/<br />

~tibs/superpc).<br />

Based on the transcript level <strong>in</strong> the 10 transcripts and<br />

the weight assigned to each transcript from the tra<strong>in</strong><strong>in</strong>g set,<br />

a discrete risk score (the supervised pr<strong>in</strong>cipal components<br />

risk score) was then calculated for each patient <strong>in</strong> the validation<br />

dataset.<br />

Multivariate analysis was conducted to evaluate the prediction<br />

accuracy <strong>of</strong> our survival pr<strong>of</strong>ile <strong>in</strong> comparison with<br />

the standard cl<strong>in</strong>icopathological covariates by Cox proportional<br />

hazards regression us<strong>in</strong>g SPSS s<strong>of</strong>tware.<br />

Functional gene set enrichment analysis was performed<br />

to f<strong>in</strong>d the pathways associated with prolonged and poor<br />

survivals. A total <strong>of</strong> 249 sets <strong>of</strong> canonical pathways (Gene<br />

Set Enrichment Analysis­Molecular Signatures Database)<br />

were analysed to <strong>in</strong>dicate their correlations with overall<br />

survival to a greater degree than expected by chance [12] .<br />

RESULTS<br />

Patient characteristics<br />

Totally, 79 gastric cancer patients treated with surgical resection<br />

were recruited <strong>in</strong> this study. Samples were randomly<br />

separated <strong>in</strong>to two batches with no significant differences<br />

between the two sets with respect to age, sex and other<br />

cl<strong>in</strong>icopathological features. Microarray was conducted <strong>in</strong><br />

all samples, and the data <strong>of</strong> batch one served as the tra<strong>in</strong><strong>in</strong>g<br />

dataset for marker discovery and data <strong>of</strong> batch two as the<br />

validation dataset. In batch one, microarray Quality Control<br />

WJG|www.wjgnet.com<br />

Validation dataset<br />

(n = 33)<br />

Sex<br />

Male 53 28 25 0.79<br />

Female<br />

Age (yr)<br />

19 11 8<br />

mean ± SE 72 60.9 ± 1.5 61.6 ± 1.3 0.74<br />

Depth <strong>of</strong> wall <strong>in</strong>vasion<br />

T2 4 3 1 0.12 1<br />

T3 56 32 24<br />

T4<br />

Differentiation<br />

12 4 8<br />

Well 7 5 2 0.18 1<br />

Moderate 31 14 17<br />

Poor 27 18 9<br />

Undifferentiated 2<br />

7<br />

Lymph node metastasis<br />

Negative 16 10 6 0.57<br />

Positive<br />

Distance metastasis<br />

56 29 27<br />

M0 66 38 28 0.09<br />

M1<br />

TNM stages<br />

6 1 5<br />

I + II 17 12 5 0.30 1<br />

III 33 18 15<br />

IV 22 9 13<br />

P<br />

Tra<strong>in</strong><strong>in</strong>g set (n = 39)<br />

Cox scores <strong>of</strong> each gene<br />

Threshold determ<strong>in</strong>ation<br />

by cross-validation<br />

Survival gene expression<br />

pr<strong>of</strong>ile exceed<strong>in</strong>g threshold<br />

Pr<strong>in</strong>cipal component<br />

analysis <strong>of</strong> the pr<strong>of</strong>ile<br />

To predict survival us<strong>in</strong>g<br />

PCs <strong>in</strong> the tra<strong>in</strong><strong>in</strong>g set<br />

Validation set (n = 33)<br />

Risk scores <strong>of</strong> candidate survival markers<br />

derived from the tra<strong>in</strong><strong>in</strong>g set<br />

Evaluation <strong>of</strong> predictive<br />

value by Cox model<br />

Figure 1 Overview <strong>of</strong> the strategy used for the development and validation<br />

<strong>of</strong> prognostic markers.<br />

(QC) removed 7 samples due to failure <strong>in</strong> hybridization or<br />

failure to meet the analysis criteria, result<strong>in</strong>g <strong>in</strong> a total <strong>of</strong><br />

39 samples <strong>in</strong>cluded <strong>in</strong> the tra<strong>in</strong><strong>in</strong>g set. All <strong>of</strong> 33 samples<br />

<strong>in</strong> the second batch passed QC and were used <strong>in</strong> validation<br />

phase. The characteristics <strong>of</strong> the 72 patients are summarized<br />

<strong>in</strong> Table 1. The median overall survival time <strong>of</strong> all<br />

samples was 31 mo, rang<strong>in</strong>g from 4.2 to 73.6 mo, and the<br />

5­year overall survival was 33%.<br />

Gene expression pr<strong>of</strong>ile associated with the overall<br />

survival<br />

The “semi­supervised” learn<strong>in</strong>g approach was used to<br />

identify the gene expression pr<strong>of</strong>ile related to the overall<br />

survival <strong>in</strong> the tra<strong>in</strong><strong>in</strong>g dataset [12] (Figure 1). A total <strong>of</strong> 18<br />

819 expression signals passed QC. First, we calculated<br />

the Cox scores <strong>of</strong> all 18 819 genes based on the survival<br />

times versus the expression levels obta<strong>in</strong>ed <strong>in</strong> 39 tra<strong>in</strong><strong>in</strong>g<br />

observations. To choose the genes with the best prediction<br />

power, the threshold <strong>of</strong> Cox scores was calculated by<br />

10-fold cross-validation. The expression pr<strong>of</strong>ile <strong>of</strong> 10 transcripts<br />

whose Cox score equalled or exceeded the threshold<br />

was obta<strong>in</strong>ed (Table 2). Next, we performed PCA on the<br />

entire tra<strong>in</strong><strong>in</strong>g set. For each case, a risk score that represents<br />

the sum <strong>of</strong> the weighted expression levels <strong>of</strong> the 10 prognostic<br />

transcripts was computed by supervised component<br />

analysis <strong>in</strong> a regression model. As shown <strong>in</strong> the Kaplan­<br />

Meier survival curves <strong>in</strong> Figure 2A and B, the patients were<br />

categorized <strong>in</strong>to two groups based on their scores above<br />

or below the median risk <strong>of</strong> death. The low­risk group (n<br />

= 20) had a median survival <strong>of</strong> 42.1 mo, whereas the highrisk<br />

group (n = 19) had a median survival <strong>of</strong> only 26.5 mo.<br />

1712 April 7, 2011|Volume 17|Issue 13|

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markers <strong>of</strong> diagnosis or prognosis <strong>in</strong> various types <strong>of</strong> cancers<br />

[13,17,18] . TMSB10, a migration­<strong>in</strong>duc<strong>in</strong>g gene, was shown<br />

to relate to cancer metastasis [19] . Additionally, a few genes<br />

(RPS19, RPLP2, GLTSCR2) are known factors <strong>in</strong>volved <strong>in</strong><br />

cell cycle control and apoptosis [20­22] . None <strong>of</strong> our 10 markers<br />

was reported <strong>in</strong> other sets <strong>of</strong> candidate genes for gastric<br />

cancer prognosis [5­8] . This is not a surprise s<strong>in</strong>ce these 10<br />

genes were selected based on the whole genome expression<br />

pr<strong>of</strong>il<strong>in</strong>g followed by supervised PCA, whereas much<br />

less genes were <strong>in</strong>cluded <strong>in</strong> earlier studies with the analysis<br />

strategy <strong>of</strong> supervised classification. Patients’ genetic background<br />

may also contribute to such diversity.<br />

Unexpectedly but also <strong>in</strong>terest<strong>in</strong>gly, 6 out <strong>of</strong> 10 candidate<br />

markers identified are ribosomal prote<strong>in</strong>s (RPs). There<br />

may be a few explanations for this phenomenon. First, RPs<br />

have been shown to be the targets <strong>of</strong> several tumor suppressors<br />

and proto­oncogenes which affect the formation<br />

<strong>of</strong> the mature ribosomes or regulate the activity <strong>of</strong> prote<strong>in</strong>s<br />

[23] . Moreover, the deregulated expression <strong>of</strong> RPs was<br />

reported to associate with the carc<strong>in</strong>ogenesis and metastasis<br />

<strong>of</strong> various cancers [14] . Therefore, besides their unknown<br />

mechanisms possibly related to p53 and MYC [24,25] . RPs appear<br />

to have various cellular roles <strong>in</strong>dependent <strong>of</strong> prote<strong>in</strong><br />

biosynthesis, <strong>in</strong>clud<strong>in</strong>g their functions <strong>in</strong> DNA replication<br />

and DNA repair, transcription, RNA splic<strong>in</strong>g and modification,<br />

cell proliferation, apoptosis, and cellular transformation.<br />

[26] Among 6 RPs <strong>of</strong> our candidate prognosis markers,<br />

RPLP2, RPL19, RPS8 and RPS12 were all found to be<br />

<strong>in</strong>volved <strong>in</strong> the carc<strong>in</strong>ogenesis and progression <strong>of</strong> various<br />

cancers [13,17,27] . RPS12 was also seen to have significant higher<br />

expression <strong>in</strong> gastric tumors <strong>in</strong> comparison with normal<br />

tissues <strong>in</strong> Ch<strong>in</strong>ese [28] .<br />

In a number <strong>of</strong> diagnosis and prognosis sets identified<br />

<strong>in</strong> expression pr<strong>of</strong>il<strong>in</strong>g from various cancer researches, the<br />

gene pr<strong>of</strong>ile <strong>in</strong> most panels came from various pathways<br />

with different cellular functions. The result <strong>of</strong> our ten­gene<br />

prognostic marker conta<strong>in</strong><strong>in</strong>g 6 RPs raised another <strong>in</strong>terest<strong>in</strong>g<br />

issue on the molecular composition <strong>of</strong> biomarkers, i.e.<br />

which type is more powerful and more accurate <strong>in</strong> prediction,<br />

a set consist<strong>in</strong>g <strong>of</strong> s<strong>in</strong>gle gene tags from multiple <strong>in</strong>dividual<br />

pathways, or a group <strong>of</strong> genes from a few and related<br />

pathways. This issue needs more tests and evaluations<br />

for conv<strong>in</strong>cible answers. At this po<strong>in</strong>t, however, a few facts<br />

shall be brought <strong>in</strong>to attention. First, our prognostic marker<br />

resulted from systematic analysis <strong>of</strong> whole genome expression<br />

pr<strong>of</strong>il<strong>in</strong>g, and our strategy <strong>of</strong> supervised PCA largely<br />

reduced subjective attribution <strong>in</strong> analysis. Thus, a group <strong>of</strong><br />

p<strong>in</strong>po<strong>in</strong>ted signals will be more representative <strong>in</strong> biological<br />

mean<strong>in</strong>g, thus provid<strong>in</strong>g more accurate prediction. Second,<br />

obviously <strong>in</strong> comparison with <strong>in</strong>dividual s<strong>in</strong>gle signatures<br />

from multi-pathways, a group <strong>of</strong> signals would significantly<br />

overcome the <strong>in</strong>dividual bias, <strong>in</strong> which the pathway components<br />

<strong>in</strong> tumors vary widely [29] . F<strong>in</strong>ally, it has been shown<br />

that even for genetic alterations <strong>of</strong> a large number <strong>of</strong> genes<br />

<strong>in</strong> cancer, these variations may function through a relatively<br />

small number <strong>of</strong> pathways and processes [29] . In our prognosis<br />

marker, although the details <strong>of</strong> the <strong>in</strong>terrelationship<br />

WJG|www.wjgnet.com<br />

Zhang YZ et al . Prognostic markers <strong>in</strong> gastric cancer<br />

among those 6 RPs are still unknown, they have the same<br />

elevation <strong>in</strong> high­risk group, <strong>in</strong>dicat<strong>in</strong>g the concordance <strong>of</strong><br />

their functions <strong>in</strong> gastric cancer.<br />

To reduce the heterogeneity among patients and samples<br />

which may br<strong>in</strong>g bias to the analysis <strong>in</strong> this study,<br />

samples were randomly separated <strong>in</strong>to tra<strong>in</strong><strong>in</strong>g and validation<br />

batches. And no significant difference with respect to<br />

age, sex and other cl<strong>in</strong>icopathological factors was found<br />

between the two batches (Table 1). And, by compar<strong>in</strong>g<br />

cl<strong>in</strong>icopathological factors between the high­risk group and<br />

low­risk group predicted by ten­gene markers, there was no<br />

significant difference between the two groups except for<br />

TNM stag<strong>in</strong>g (Table 3). Then we compared this ten­gene<br />

prognostic marker with TNM stag<strong>in</strong>g system. Both tengene<br />

prognostic marker and TNM classification can predict<br />

survival with statistical significances <strong>in</strong> discovery and validation<br />

sample batches (Figure 2C and 3C), <strong>in</strong>dicat<strong>in</strong>g that our<br />

prognosis set can effectively complement traditional cl<strong>in</strong>icopathological<br />

stag<strong>in</strong>g (Figure 2B and C, Figure 3B and C).<br />

The applicability <strong>of</strong> a marker with only 10 genes also suggests<br />

its potential to be developed as the prognosis marker<br />

panel for pre­operative molecular stag<strong>in</strong>g from endoscopic<br />

biopsy. Further validation with large scale samples are warranted<br />

for cl<strong>in</strong>ical application.<br />

In conclusion, based on the whole genome expression<br />

pr<strong>of</strong>il<strong>in</strong>g, we found and validated a ten­gene prognostic<br />

marker for overall survival prognosis <strong>of</strong> gastric cancer patients,<br />

which may be used with the TNM stag<strong>in</strong>g system<br />

as a parallel and complementary approach. However, the<br />

predom<strong>in</strong>ance <strong>of</strong> ribosome prote<strong>in</strong> genes <strong>in</strong> our molecular<br />

prognostic marker warrants further research on their roles<br />

<strong>in</strong> cancer progression.<br />

ACKNOWLEDGMENTS<br />

We would like to thank Dr. Yong Tao and Dr. Wei­Wei Zhai<br />

for their excellent suggestions and assistance <strong>in</strong> improv<strong>in</strong>g<br />

our manuscript.<br />

COMMENTS<br />

Background<br />

Gastric cancer is the second lead<strong>in</strong>g cause <strong>of</strong> cancer related death <strong>in</strong> Ch<strong>in</strong>a<br />

and worldwide. The 5-year survival rate <strong>of</strong> patients with advanced disease is<br />

very poor. Currently, treatment plan and prognosis prediction for gastric cancer<br />

ma<strong>in</strong>ly depend on the cl<strong>in</strong>icopathological stag<strong>in</strong>g. However, prognosis varies<br />

among patients with the same cl<strong>in</strong>icalpathological stage. An <strong>in</strong>dividualized expression<br />

test for selected markers <strong>in</strong> biopsy and surgical samples will complement<br />

the current stag<strong>in</strong>g system, especially for prognosis prediction.<br />

Research frontiers<br />

The gene expression pr<strong>of</strong>il<strong>in</strong>g has enabled researchers to quantify the biological<br />

states and consequently to uncover the subtle phenotypes <strong>in</strong> cancer. Such<br />

analyses have provided unique opportunities to develop various pr<strong>of</strong>iles that<br />

can dist<strong>in</strong>guish, identify, and classify discrete subsets <strong>of</strong> disease, predict the<br />

disease outcome, and even predict the response to therapy.<br />

Innovations and breakthroughs<br />

In this study, based on the whole genome expression pr<strong>of</strong>il<strong>in</strong>g, the authors<br />

identified and validated a ten-gene set that can be further developed as cl<strong>in</strong>ical<br />

prognosis markers to predict overall survival <strong>of</strong> gastric cancer patients. This<br />

marker set showed consistent prognosis results with the traditional Tumor-<br />

1715 April 7, 2011|Volume 17|Issue 13|

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Zhang YZ et al . Prognostic markers <strong>in</strong> gastric cancer<br />

node-metastasis (TNM) stag<strong>in</strong>g system. The f<strong>in</strong>d<strong>in</strong>gs <strong>in</strong> this study also provided<br />

new clues about the possible association between the deregulation <strong>of</strong> ribosome<br />

related gene expression and survival status <strong>of</strong> the patients after surgery.<br />

Applications<br />

Based on the whole genome expression pr<strong>of</strong>il<strong>in</strong>g, a ten-gene prognostic marker<br />

set for overall survival prognosis <strong>of</strong> gastric cancer patients may be applied <strong>in</strong><br />

comb<strong>in</strong>ation with the TNM stag<strong>in</strong>g system as a parallel and complementary<br />

approach. However, the predom<strong>in</strong>ance <strong>of</strong> ribosome prote<strong>in</strong> genes <strong>in</strong> these molecular<br />

prognostic markers awaits for further research on their roles <strong>in</strong> cancer<br />

progression.<br />

Term<strong>in</strong>ology<br />

TNM: The TNM system is one <strong>of</strong> the most widely used stag<strong>in</strong>g systems <strong>in</strong> tumor<br />

classification. The system is based on the extent <strong>of</strong> the tumor (T), the extent <strong>of</strong><br />

spread to the lymph nodes (N), and the presence <strong>of</strong> distant metastasis (M). A<br />

number is added to each letter to <strong>in</strong>dicate the size or extent <strong>of</strong> the primary tumor<br />

and the extent <strong>of</strong> cancer spread. Pr<strong>in</strong>cipal component analysis (PCA): A mathematical<br />

tool used to reduce the number <strong>of</strong> variables while reta<strong>in</strong><strong>in</strong>g the orig<strong>in</strong>al<br />

variability <strong>of</strong> the data. The first pr<strong>in</strong>cipal component accounts for as much <strong>of</strong> the<br />

variability <strong>in</strong> the data as possible, and each succeed<strong>in</strong>g component accounts for<br />

as much <strong>of</strong> the rema<strong>in</strong><strong>in</strong>g variability as possible. Gene set enrichment analysis<br />

(GSEA): A computational method that determ<strong>in</strong>es whether a prior identified set <strong>of</strong><br />

genes shows statistically significant, concordant differences between two biological<br />

states. It is a method which focuses on the analysis at the level <strong>of</strong> functional<br />

related gene sets <strong>in</strong>stead <strong>of</strong> a s<strong>in</strong>gle gene. It helps biologists to <strong>in</strong>terpret the DNA<br />

microarray data by their previous biological knowledge <strong>of</strong> the genes <strong>in</strong> a gene set.<br />

GSEA has been shown to efficiently identify gene sets conta<strong>in</strong><strong>in</strong>g known diseaserelated<br />

genes <strong>in</strong> the real experiments.<br />

Peer review<br />

A ten-gene prognostic marker, <strong>in</strong>clud<strong>in</strong>g 6 ribosomal prote<strong>in</strong>s, for overall survival<br />

prognosis <strong>of</strong> gastric cancer were identified and validated based on whole<br />

genome expression pr<strong>of</strong>il<strong>in</strong>g. By compar<strong>in</strong>g with the traditional TNM stag<strong>in</strong>g<br />

system, this ten-gene prognostic marker showed consistent prognosis results,<br />

which may complement the predictive value <strong>of</strong> current TNM stag<strong>in</strong>g system.<br />

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ribosomal prote<strong>in</strong> haplo<strong>in</strong>sufficiency. Nature 2008; 456: 971-975<br />

26 Wool IG. Extraribosomal functions <strong>of</strong> ribosomal prote<strong>in</strong>s.<br />

Trends Biochem Sci 1996; 21: 164-165<br />

27 Gardner-Thorpe J, Ito H, Ashley SW, Whang EE. Ribosomal<br />

prote<strong>in</strong> P2: a potential molecular target for antisense therapy<br />

1716 April 7, 2011|Volume 17|Issue 13|

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<strong>of</strong> human malignancies. Anticancer Res 2003; 23: 4549-4560<br />

28 Sun XJ, Hao DM, Zheng ZH, Fu H, Xu HM, Wang MX, Sun<br />

KL. [Screen<strong>in</strong>g and analysis <strong>of</strong> associated genes <strong>in</strong> the carc<strong>in</strong>ogenesis<br />

and progression <strong>of</strong> gastric cancer]. Zhonghua Yixue<br />

Yichuanxue Zazhi 2005; 22: 31-34<br />

29 Jones S, Zhang X, Parsons DW, L<strong>in</strong> JC, Leary RJ, Angenendt<br />

P, Mankoo P, Carter H, Kamiyama H, Jimeno A, Hong SM, Fu<br />

WJG|www.wjgnet.com<br />

Zhang YZ et al . Prognostic markers <strong>in</strong> gastric cancer<br />

B, L<strong>in</strong> MT, Calhoun ES, Kamiyama M, Walter K, Nikolskaya T,<br />

Nikolsky Y, Hartigan J, Smith DR, Hidalgo M, Leach SD, Kle<strong>in</strong><br />

AP, Jaffee EM, Gogg<strong>in</strong>s M, Maitra A, Iacobuzio-Donahue C,<br />

Eshleman JR, Kern SE, Hruban RH, Karch<strong>in</strong> R, Papadopoulos<br />

N, Parmigiani G, Vogelste<strong>in</strong> B, Velculescu VE, K<strong>in</strong>zler KW.<br />

Core signal<strong>in</strong>g pathways <strong>in</strong> human pancreatic cancers revealed<br />

by global genomic analyses. Science 2008; 321: 1801-1806<br />

S- Editor Sun H L- Editor Ma JY E- Editor Ma WH<br />

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Onl<strong>in</strong>e Submissions: http://www.wjgnet.com/1007-9327<strong>of</strong>fice<br />

wjg@wjgnet.com<br />

doi:10.3748/wjg.v17.i13.1718<br />

ORIGINAL ARTICLE<br />

Differential expression <strong>of</strong> Bcl-2 and Bax dur<strong>in</strong>g gastric<br />

ischemia-reperfusion <strong>of</strong> rats<br />

Wei-Li Qiao, Guang-M<strong>in</strong>g Wang, Yue Shi, J<strong>in</strong>-Xia Wu, You-Jian Qi, Jian-Fu Zhang, Hong Sun, Chang-Dong Yan<br />

Wei-Li Qiao, Guang-M<strong>in</strong>g Wang, Yue Shi, J<strong>in</strong>-Xia Wu,<br />

You-Jian Qi, Jian-Fu Zhang, Hong Sun, Chang-Dong Yan,<br />

Department <strong>of</strong> Physiology, Xuzhou Medical College, 84 West<br />

Huaihai Road, Xuzhou 221002, Jiangsu Prov<strong>in</strong>ce, Ch<strong>in</strong>a<br />

Author contributions: Qiao WL, Wang GM, Shi Y, Yan CD,<br />

Zhang JF, Wu JX, Qi YJ and Sun H designed the research; Qiao<br />

WL, Wang GM and Shi Y performed the research; Qiao WL,<br />

Wu JX and Qi YJ contributed new reagents/analytic tools; Qiao<br />

WL, Yan CD, Zhang JF and Sun H analyzed the data and were<br />

<strong>in</strong>volved <strong>in</strong> edit<strong>in</strong>g the manuscript; Qiao WL and Yan CD wrote<br />

the paper.<br />

Supported by grants from the National Natural Science Foundation<br />

<strong>of</strong> Ch<strong>in</strong>a, No. 30570671, the Natural Science Foundation<br />

<strong>of</strong> Jiangsu Prov<strong>in</strong>ce, No. BK2009088, the Natural Science<br />

Fund for Colleges and Universities <strong>in</strong> Jiangsu Prov<strong>in</strong>ce, No.<br />

10KJB310015 and the Xuzhou Social Development Fund, No.<br />

XM08C062<br />

Correspondence to: Chang-Dong Yan, MD, Pr<strong>of</strong>essor, Director,<br />

Department <strong>of</strong> Physiology, Xuzhou Medical College, 84<br />

West Huaihai Road, Xuzhou 221002, Jiangsu Prov<strong>in</strong>ce,<br />

Ch<strong>in</strong>a. yancd55@hotmail.com<br />

Telephone:+86-516-83262105 Fax: +86-516-83262014<br />

Received: October 31, 2010 Revised: December 21, 2010<br />

Accepted: December 28, 2010<br />

Published onl<strong>in</strong>e: April 7, 2011<br />

Abstract<br />

AIM: To <strong>in</strong>vestigate expression <strong>of</strong> Bcl-2 and Bax <strong>in</strong> gastric<br />

ischemia-reperfusion (GI-R) and <strong>in</strong>volvement <strong>of</strong> extracellular<br />

signal-regulated k<strong>in</strong>ase (ERK) 1/2 activation.<br />

METHODS: The GI-R model was established by ligature<br />

<strong>of</strong> the celiac artery for 30 m<strong>in</strong> and reperfusion <strong>in</strong> Sprague-<br />

Dawley rats. Rats were assigned to groups <strong>in</strong> accordance<br />

with their evaluation period: control, 0, 0.5, 1, 3, 6, 24,<br />

48, and 72 h. Expression and distribution <strong>of</strong> Bcl-2 and Bax<br />

prote<strong>in</strong>s were analyzed by immunohistochemistry and<br />

western blott<strong>in</strong>g <strong>in</strong> gastric tissue samples after sacrifice.<br />

RESULTS: Compared with controls, the percentage <strong>of</strong><br />

positive cells and prote<strong>in</strong> levels <strong>of</strong> Bcl-2 decreased <strong>in</strong><br />

WJG|www.wjgnet.com<br />

1718<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): 1718-1724<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

the early phases <strong>of</strong> reperfusion, reached its m<strong>in</strong>imum<br />

at 1 h (P < 0.05); it then <strong>in</strong>creased, reach<strong>in</strong>g its peak<br />

at 24 h <strong>of</strong> reperfusion (P < 0.05). The pattern <strong>of</strong> Bax<br />

expression was opposite to that <strong>of</strong> Bcl-2. Bax expression<br />

<strong>in</strong>creased after reperfusion, with its peak at 1 h <strong>of</strong><br />

reperfusion (P < 0.05), and then it decreased gradually<br />

to a m<strong>in</strong>imum at 24 h after reperfusion (P < 0.05).<br />

On the other hand, <strong>in</strong>hibition <strong>of</strong> activation <strong>of</strong> ERK1/2<br />

<strong>in</strong>duced by PD98059, a specific upstream MEK <strong>in</strong>hibitor,<br />

had significant effects on Bcl-2 and Bax <strong>in</strong> GI-R.<br />

Compared with GI-R treatment only at 3 h <strong>of</strong> reperfusion,<br />

PD98059 reduced the number <strong>of</strong> Bcl-2 positive<br />

cells (0.58% <strong>of</strong> R3h group, P < 0.05) and Bcl-2 prote<strong>in</strong><br />

level (74% <strong>of</strong> R3h group, P < 0.05) but <strong>in</strong>creased the<br />

number <strong>of</strong> Bax-positive cells (1.33-fold vs R3h group, P<br />

< 0.05) and Bax prote<strong>in</strong> level (1.35-fold <strong>of</strong> R3h group,<br />

P < 0.05).<br />

CONCLUSION: These results <strong>in</strong>dicated that the Bcl-2<br />

and Bax played a pivotal role <strong>in</strong> the gastric mucosal I-R<br />

<strong>in</strong>jury and repair by activation <strong>of</strong> ERK1/2.<br />

© 2011 Baishideng. All rights reserved.<br />

Key words: Stomach; Ischemia-reperfusion; Bcl-2; Bax;<br />

Extracellular signal-regulated k<strong>in</strong>ase 1/2<br />

Peer reviewer: Dr. José Liberato Ferreira Caboclo, Pr<strong>of</strong>essor,<br />

Rua Antônio de Godoy, 4120, São José do Rio Preto, Brazil<br />

Qiao WL, Wang GM, Shi Y, Wu JX, Qi YJ, Zhang JF, Sun<br />

H, Yan CD. Differential expression <strong>of</strong> Bcl-2 and Bax dur<strong>in</strong>g<br />

gastric ischemia-reperfusion <strong>of</strong> rats. <strong>World</strong> J Gastroenterol<br />

2011; 17(13): 1718-1724 Available from: URL: http://www.<br />

wjgnet.com/1007-9327/full/v17/i13/1718.htm DOI: http://dx.doi.<br />

org/10.3748/wjg.v17.i13.1718<br />

INTRODUCTION<br />

Current research <strong>in</strong>to gastric ischemia-reperfusion (GI-R)<br />

April 7, 2011|Volume 17|Issue 13|

---

has focused on its pathogenic and underly<strong>in</strong>g molecular<br />

mechanism [1-8] . GI-R <strong>in</strong>jury and repair is related to the<br />

changes <strong>in</strong> gastric mucosal cellular apoptosis and proliferation<br />

<strong>in</strong>duced by GI-R <strong>in</strong> rats. In our previous experiments,<br />

we have explored the time course <strong>of</strong> gastric mucosal apoptosis<br />

and proliferation <strong>in</strong>duced by GI-R, and the role <strong>of</strong><br />

the extracellular signal-regulated k<strong>in</strong>ase 1 and 2 (ERK1/2)<br />

signal<strong>in</strong>g pathway <strong>in</strong> GI-R-<strong>in</strong>duced gastric mucosal <strong>in</strong>jury<br />

and repair [7] . We have found that serious gastric mucosal<br />

damage occurs rapidly at the early stage <strong>of</strong> reperfusion<br />

and is closely related to the suppression <strong>of</strong> ERK1/2 activation.<br />

The activity <strong>of</strong> ERK1/2 <strong>in</strong>creases as the time <strong>of</strong><br />

reperfusion is extended, and the activated ERK1/2 might<br />

<strong>in</strong>hibit apoptosis and promote proliferation <strong>in</strong> gastric mucosal<br />

cells. However, the precise mechanisms by which activated<br />

ERK1/2 accomplishes gastric mucosal apoptosis<br />

and proliferation are unknown.<br />

The balance between apoptosis and cellular proliferation<br />

is a key <strong>in</strong> gastric <strong>in</strong>jury and repair, and is regulated by<br />

several genes, <strong>in</strong>clud<strong>in</strong>g p53 and members <strong>of</strong> the Bcl-2 family<br />

such as Bax and Bcl-2 [9-11] . The Bax gene is a proliferative<br />

suppressor gene that encodes Bax prote<strong>in</strong> that promotes<br />

apoptosis. On the other hand, bcl gene encodes Bcl-2 prote<strong>in</strong><br />

that blocks wild type p53-mediated apoptosis, and heterodimers<br />

with Bax, antagoniz<strong>in</strong>g the function <strong>of</strong> Bax [12] .<br />

Therefore, it is conceivable that <strong>in</strong>creased cellular apoptosis<br />

and proliferation, because <strong>of</strong> altered expression <strong>of</strong> the<br />

regulat<strong>in</strong>g prote<strong>in</strong>s such as Bax and Bcl-2, may be associated<br />

with gastric <strong>in</strong>jury and repair <strong>in</strong>duced by GI-R. Although<br />

Bcl-2 and Bax are expressed <strong>in</strong> gastric mucosa, their presence<br />

<strong>in</strong> normal gastric mucosa is controversial. Liu et al [13]<br />

have reported that Bcl-1 mRNA and prote<strong>in</strong> are expressed<br />

<strong>in</strong> the gastric gland zone at a middle level and Bax prote<strong>in</strong><br />

is expressed <strong>in</strong> the epithelial cells <strong>of</strong> normal gastric mucosa.<br />

Xia et al [14] have found that, <strong>in</strong> <strong>in</strong>tact gastric tissue, Bcl-2<br />

and Bax are localized predom<strong>in</strong>antly <strong>in</strong> the glandular base<br />

region <strong>in</strong> chief cells <strong>in</strong> normal rat gastric mucosa. However,<br />

a conflict<strong>in</strong>g study has found that no expression <strong>of</strong> Bcl-2<br />

prote<strong>in</strong> is detected <strong>in</strong> the glandular epithelium <strong>of</strong> normal<br />

gastric mucosa [15] . On the other hand, Bcl-2 and Bax show<br />

significant changes <strong>in</strong> many conditions <strong>in</strong>clud<strong>in</strong>g gastric<br />

cancer, gastritis, and GI-R [15-18] . El Eter et al [15] have reported<br />

that cytoplasmic expression <strong>of</strong> Bcl-2 prote<strong>in</strong> is observed <strong>in</strong><br />

the superficial portion <strong>of</strong> gastric mucosa sections obta<strong>in</strong>ed<br />

from rats subjected to GI-R <strong>in</strong>jury.<br />

The available data on expression <strong>of</strong> Bcl-2 and Bax<br />

prote<strong>in</strong>s <strong>in</strong> the stomach, and their relation to apoptosis <strong>of</strong><br />

gastric mucosal cells, seem equivocal, thus, a further study<br />

<strong>of</strong> Bcl-2 and Bax expression <strong>in</strong> the stomach is clearly<br />

important. In the present study, we used an immunohistochemical<br />

assay and western blott<strong>in</strong>g to determ<strong>in</strong>e the<br />

changed courses <strong>of</strong> Bcl-2 and Bax at different reperfusion<br />

durations after GI-R, and whether ERK1/2 activation was<br />

<strong>in</strong>volved <strong>in</strong> this process.<br />

MATERIALS AND METHODS<br />

Animals<br />

Groups <strong>of</strong> six adult Sprague-Dawley rats, regardless <strong>of</strong><br />

WJG|www.wjgnet.com<br />

Qiao WL et al . Bcl-2 and Bax <strong>in</strong> ischemia-reperfusion<br />

sex, weigh<strong>in</strong>g 220-270 g, were provided by the Experimental<br />

Animal Centre <strong>of</strong> Xuzhou Medical College. All experiments<br />

were performed <strong>in</strong> accordance with the National<br />

Institutes <strong>of</strong> Health Guidel<strong>in</strong>es for the Care and Use <strong>of</strong><br />

Laboratory Animals. Rats were housed under controlled<br />

temperature (22-24℃) and photoperiod (12 h light/12 h<br />

dark), and allowed food and water ad libitum. Rats were<br />

fasted for 24 h before the experiment, but were allowed<br />

free access to tap water. Animals were randomly assigned<br />

to groups: GI-R (different reperfusion time po<strong>in</strong>ts after 30<br />

m<strong>in</strong> <strong>of</strong> ischemia); PD98059 + R3h (PD98059 + reperfusion<br />

for 3 h after 30 m<strong>in</strong> <strong>of</strong> ischemia); and vehicle control<br />

(PD98059 replaced with vehicle but otherwise the same<br />

as PD98059 + R3h). PD98059 was given 20 m<strong>in</strong> before<br />

operation [150 μg/kg, adm<strong>in</strong>istered <strong>in</strong>traperitoneally (i.p.),<br />

dissolved <strong>in</strong> dimethyl sulfoxide]. A sham group <strong>in</strong> which<br />

only the same surgical procedure without clamp<strong>in</strong>g the<br />

celiac artery was performed served as a control.<br />

Reagents<br />

PowerVision TM two-step immunohistochemistry detection<br />

kit were purchased from Zhongshan Biotech Co.<br />

(Beij<strong>in</strong>g, Ch<strong>in</strong>a), anti-Bcl-2 and anti-Bax polyclonal antibodies<br />

were purchased from Santa Cruz Biotechnology<br />

(Santa Cruz, CA, USA), alkal<strong>in</strong>e-phosphorylase-tagged<br />

goat anti-rat IgG antibody, PD98059 was from Promega<br />

(Madison, WI, USA), and sodium pentobarbital was purchased<br />

from Sigma (St. Louis, MO, USA).<br />

Preparation <strong>of</strong> GI-R model<br />

GI-R models were <strong>in</strong>duced accord<strong>in</strong>g to the method <strong>of</strong><br />

Qiao et al [7] . The randomly grouped rats were all anesthetized<br />

with sodium pentobarbital (40 mg/kg, i.p.). Their<br />

abdomens were <strong>in</strong>cised along the midl<strong>in</strong>e, and the celiac<br />

artery and its adjacent tissues were carefully isolated. The<br />

celiac artery was clamped with a small non-traumatic vascular<br />

clamp for 30 m<strong>in</strong> to <strong>in</strong>duce gastric ischemia and then<br />

released for 0, 0.5, 1, 3, 6, 24, 48 and 72 h to allow reperfusion.<br />

Follow<strong>in</strong>g reperfusion, the rats were sacrificed and<br />

the stomachs were removed immediately. The stomachs<br />

were <strong>in</strong>cised along the greater curvature and flushed with<br />

ice-cold PBS (0.1 mol/L). One half <strong>of</strong> the gastric mucosa<br />

was frozen at -80℃ for western blott<strong>in</strong>g, and the other<br />

was fixed <strong>in</strong> Bou<strong>in</strong>’s fixative for immunohistochemical<br />

sta<strong>in</strong><strong>in</strong>g.<br />

Immunohistochemical sta<strong>in</strong><strong>in</strong>g<br />

The fixed stomach was embedded <strong>in</strong> paraff<strong>in</strong>, sliced <strong>in</strong>to<br />

4-μm-thick sections, and mounted on glass slides. The<br />

immunohistochemistry was performed with a PowerVision<br />

two-step immunohistochemistry detection kit. The<br />

sections were sta<strong>in</strong>ed with 3,3’-diam<strong>in</strong>obenzid<strong>in</strong>e (DAB),<br />

then countersta<strong>in</strong>ed us<strong>in</strong>g hematoxyl<strong>in</strong>. The sections were<br />

exam<strong>in</strong>ed with a microscope (Model IX71; Olympus, Tokyo,<br />

Japan). Gastric mucosal cells with brown granules visible<br />

<strong>in</strong> the cytoplasm or nucleus were considered positive.<br />

The number <strong>of</strong> positive cells per section was counted <strong>in</strong><br />

10 random lower-power (× 10) fields, and the percentage<br />

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Qiao WL et al . Bcl-2 and Bax <strong>in</strong> ischemia-reperfusion<br />

<strong>of</strong> positive cells (positive cells/total cells × 100%) was<br />

calculated. Three non-consecutive sections were selected<br />

from each specimen and those <strong>in</strong>dexes were averaged.<br />

Western blott<strong>in</strong>g<br />

The frozen gastric mucosa was homogenized with a Teflon<br />

glass homogenizer <strong>in</strong> 1:10 (w/v) ice-cold homogenization<br />

buffer consist<strong>in</strong>g <strong>of</strong> 50 mmol/L 3-(N-morphol<strong>in</strong>o)<br />

propanesulfonic acid (MOPS, pH 7.4), 50 mmol/L NaF,<br />

20 mmol/L sodium pyrophosphate (NaPPi), 20 mmol/L<br />

b-glycerophosphate, 1 mmol/L EDTA, 1 mmol/L EGTA,<br />

1 mmol/L phenylmethylsulphonyl fluoride, 10 mg/mL<br />

leupept<strong>in</strong>, 10 mg/mL aprot<strong>in</strong><strong>in</strong> and 10 mg/mL pepstat<strong>in</strong> A.<br />

The homogenate was centrifuged at 800 g for 15 m<strong>in</strong> at 4℃,<br />

and the supernatant was reta<strong>in</strong>ed as cytoplasmic parts. Prote<strong>in</strong><br />

concentrations were determ<strong>in</strong>ed by Coomassie brilliant<br />

blue prote<strong>in</strong> assay. The prote<strong>in</strong>s was heated at 100℃<br />

for 5 m<strong>in</strong> with load<strong>in</strong>g buffer conta<strong>in</strong><strong>in</strong>g 0.125 mol/L<br />

Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 10% mercaptoethanol<br />

and 0.002% bromophenol blue, then separated<br />

by 10% SDS-PAGE. The prote<strong>in</strong>s were isolated by 12.5%<br />

SDS-PAGE and transferred to a nitrocellulose membrane.<br />

The blots were <strong>in</strong>cubated with 4% bov<strong>in</strong>e serum album<strong>in</strong><br />

<strong>in</strong> TBST (10 mmol/L Tris, pH 7.5; 150 mmol/L NaCl,<br />

0.05% Tween-20) at 4℃ for 6 h and probed with primary<br />

antibodies (anti-Bcl-2 polyclonal antibody 1:500, anti-Bax<br />

polyclonal antibody 1:400) at 4℃ overnight. Membranes<br />

were r<strong>in</strong>sed and <strong>in</strong>cubated with secondary antibody for<br />

2 h and were detected with an NBT/BCIP assay kit. After<br />

immunoblott<strong>in</strong>g, the bands were scanned and analyzed by<br />

Image J s<strong>of</strong>tware. The optical density (OD) <strong>of</strong> the band <strong>in</strong><br />

each lane was expressed as the fold change versus the OD<br />

<strong>of</strong> the sham control.<br />

Statistical analysis<br />

All results are presented as mean ± SD. Comparisons between<br />

two groups were made with Student’s t test; multiplegroup<br />

analyses were made by one-way ANOVA. Statistical<br />

analyses were performed with SPSS for W<strong>in</strong>dows version<br />

11.5. P < 0.05 was considered statistically significant.<br />

RESULTS<br />

Quantitative changes <strong>in</strong> Bcl-2 and Bax positive cells <strong>of</strong><br />

the gastric mucosa for different reperfusion durations<br />

after ischemia<br />

Immunohistochemical sta<strong>in</strong><strong>in</strong>g clearly showed that Bcl-2<br />

and Bax were expressed and limited to the cytosol <strong>of</strong><br />

the cells <strong>in</strong> the gastric mucosa (Figure 1). Bcl-2 express<strong>in</strong>g<br />

cells were found predom<strong>in</strong>antly <strong>in</strong> the lower part <strong>of</strong><br />

the gastric gland, and were present only <strong>in</strong> gland cells <strong>of</strong><br />

the stomach fundus (Figure 1A-D). Bax positive cellular<br />

distribution was similar to that <strong>of</strong> Bcl-2, with prom<strong>in</strong>ent<br />

expression <strong>in</strong> the base, but sta<strong>in</strong><strong>in</strong>g for Bax was also<br />

noticeable <strong>in</strong> the cells <strong>of</strong> the pit. Bax appeared to be absent<br />

from the middle part <strong>of</strong> the gastric gland, and weak<br />

expression <strong>of</strong> Bax was detected <strong>in</strong> most cells <strong>of</strong> the gastric<br />

mucosa (Figure 1E-H). The control sample (shamoperated)<br />

obta<strong>in</strong>ed prior to the ischemic period showed a<br />

WJG|www.wjgnet.com<br />

normal appearance <strong>of</strong> Bcl-2 (Figure 1A and B) and Bax<br />

(Figure 1E and F), and there were significant differences<br />

between the GI-R (Figure 1C, D, G and H) and control<br />

groups (Figure 1A and E) <strong>in</strong> the quantities <strong>of</strong> Bcl-2 and<br />

Bax immunoreactive cells.<br />

The percentage <strong>of</strong> Bcl-2 and Bax positive cells <strong>in</strong> various<br />

groups is shown <strong>in</strong> Figure 2. Bcl-2 and Bax positive<br />

cells decreased <strong>in</strong> the early phase <strong>of</strong> reperfusion, with a<br />

nadir (10.02% ± 1.21%) at 1 h <strong>of</strong> reperfusion, then <strong>in</strong>creased<br />

significantly after reperfusion for 3 h, with a peak<br />

(29.76% ± 3.32%) at 24 h <strong>of</strong> reperfusion, and returned to<br />

near the base level (20.47% ± 2.97%) at 72 h <strong>of</strong> reperfusion.<br />

The opposite pattern was observed for Bax positive<br />

cells. The percentage <strong>of</strong> Bax positive cells <strong>in</strong>creased <strong>in</strong> the<br />

<strong>in</strong>itial stages <strong>of</strong> reperfusion, reached the highest Bax positive<br />

cell count (49.34% ± 3.83%) at 1 h <strong>of</strong> reperfusion,<br />

then decreased gradually, with its nadir (13.36% ± 3.05%)<br />

at 24 h <strong>of</strong> reperfusion, and recovered to base level (24.94%<br />

± 2.83%) at 72 h <strong>of</strong> reperfusion.<br />

Prote<strong>in</strong> expression <strong>of</strong> Bcl-2 and Bax <strong>in</strong> gastric mucosa<br />

at different reperfusion durations after ischemia<br />

Figure 3 show the Bcl-2 and Bax prote<strong>in</strong> levels <strong>in</strong> the<br />

gastric mucosa <strong>in</strong> different groups <strong>of</strong> the study. After<br />

reperfusion, expression <strong>of</strong> Bcl-2 prote<strong>in</strong> was significantly<br />

lower than that <strong>of</strong> the controls, and the lowest level was<br />

observed at 1 h <strong>of</strong> reperfusion (0.59% <strong>of</strong> sham group, P<br />

< 0.05). A peak <strong>of</strong> Bcl-2 prote<strong>in</strong> expression was displayed<br />

<strong>in</strong> the 24 h reperfusion group (1.36 fold vs sham group, P<br />

< 0.05). The Bax prote<strong>in</strong> level <strong>in</strong>creased <strong>in</strong> the early stage<br />

<strong>of</strong> reperfusion, reached its peak (1.62-fold vs sham group,<br />

P < 0.05) at 1 h <strong>of</strong> reperfusion, and then decreased gradually<br />

to its lowest levels (0.57% <strong>of</strong> sham group, P < 0.05)<br />

at 24 h <strong>of</strong> reperfusion. At 72 h <strong>of</strong> reperfusion, Bcl-2 and<br />

Bax prote<strong>in</strong> levels were the same as that <strong>of</strong> the control<br />

group.<br />

Effects <strong>of</strong> PD98059 on expression <strong>of</strong> Bcl-2 and Bax<br />

PD98059 is a specific upstream <strong>in</strong>hibitor <strong>of</strong> ERK1/2.<br />

By immunohistochemical assay, we found PD98059 had<br />

significant effects on Bcl-2 and Bax expression <strong>in</strong> GI-R.<br />

Compared with the control group (R3h group), the<br />

PD98059 + R3h group showed a fall <strong>in</strong> the number <strong>of</strong><br />

Bcl-2 positive cells (0.58% <strong>of</strong> R3h group, P < 0.05) but an<br />

<strong>in</strong>crease <strong>in</strong> Bax positive cells (1.31-fold vs R3h group, P <<br />

0.05) (Figure 4). To ascerta<strong>in</strong> the effect <strong>of</strong> PD98059 on<br />

expression levels <strong>of</strong> Bcl-2 and Bax prote<strong>in</strong>, western blott<strong>in</strong>g<br />

was performed. In the PD98059 + R3h group, gastric<br />

mucosal Bcl-2 prote<strong>in</strong> level was 74% <strong>of</strong> that <strong>in</strong> the R3h<br />

group (P < 0.05), whereas Bax prote<strong>in</strong> level was 1.35-fold<br />

more than that <strong>in</strong> the R3h group (P < 0.05) (Figure 5).<br />

DISCUSSION<br />

Previous studies have shown that many stress conditions,<br />

such as hemorrhagic shock, burns, sepsis, major surgery,<br />

ischemia and trauma can lead to GI-R <strong>in</strong>jury. In recent<br />

years, studies on GI-R <strong>in</strong>jury have revealed that reactive<br />

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Positive cells (%)<br />

A B C<br />

D E<br />

G<br />

60<br />

50<br />

40<br />

30<br />

20<br />

10<br />

0<br />

Bcl-2<br />

Bax<br />

a<br />

a<br />

a<br />

Sham 0 0.5 1 3 6 24 48 72<br />

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a<br />

a<br />

a<br />

a<br />

a<br />

a<br />

Reperfusion time (h)<br />

Figure 2 Quantitative changes <strong>in</strong> Bcl-2 and Bax positive cells <strong>in</strong> rat gastric<br />

mucosa for different reperfusion times after GI-R. Reperfusion was<br />

ma<strong>in</strong>ta<strong>in</strong>ed for 0, 0.5, 1, 3, 6, 24, 48 and 72 h after 30 m<strong>in</strong> <strong>of</strong> ischemia. Sham:<br />

sham-operated. Values are percentage <strong>of</strong> positive cells (positive cells/total cells)<br />

counted <strong>in</strong> 10 microscopic fields. Each column represents mean ± SD, n = 6. a P <<br />

0.05 vs sham.<br />

H<br />

Figure 1 Histological exhibition <strong>of</strong> Bcl-2 and Bax positive cells <strong>in</strong> the gastric mucosa at different reperfusion times after ischemia, by immunohistochemical<br />

sta<strong>in</strong><strong>in</strong>g <strong>in</strong> rats. The Bcl-2 and Bax positive cells were respectively probed with anti-Bcl-2 and anti-Bax polyclonal antibodies <strong>in</strong> rat gastric mucosa. Nuclear countersta<strong>in</strong><strong>in</strong>g<br />

was performed with hematoxyl<strong>in</strong>. The examples <strong>of</strong> immunoreactive cells are those with dark brown sta<strong>in</strong><strong>in</strong>g <strong>in</strong> their cytosol (arrows). A and B: Bcl-2, control; C: Bcl-2,<br />

GI-R at 1 h after reperfusion; D: Bcl-2, GI-R at 24 h after reperfusion; E and F: Bax, control; G: Bax, GI-R at 1 h after reperfusion; H: Bax, GI-R at 24 h after reperfusion.<br />

Images were obta<strong>in</strong>ed at × 100 (A, C, D, E, G and H, Bar 100 μm) and × 400 (B and F, Bar 400 μm).<br />

a<br />

a<br />

a<br />

Qiao WL et al . Bcl-2 and Bax <strong>in</strong> ischemia-reperfusion<br />

F<br />

oxygen species, endothel<strong>in</strong>, microvascular dysfunction,<br />

polymorphonuclear leukocyte <strong>in</strong>filtration, nitric oxide release,<br />

gastric acid secretion and decreased prostagland<strong>in</strong><br />

concentrations may play a role <strong>in</strong> the pathogenesis <strong>of</strong> gastric<br />

mucosal <strong>in</strong>jury <strong>in</strong>duced by GI-R [1,5,19-25] . Although the<br />

gastric mucosa is vulnerable to damage by various factors,<br />

it can quickly repair the damage [26] . Mucosal <strong>in</strong>tegrity is<br />

ma<strong>in</strong>ta<strong>in</strong>ed by a balance between proliferation and apoptosis<br />

<strong>of</strong> the gastric mucosal cells. To understand better<br />

the causes <strong>of</strong> gastric lesions, it is important to study the<br />

imbalance between proliferation and apoptosis [27,28] .<br />

In previous experiments [7] , we have shown the changed<br />

courses <strong>of</strong> gastric mucosal <strong>in</strong>jury and repair <strong>in</strong>duced by<br />

GI-R, and the role <strong>of</strong> ERK1/2 <strong>in</strong> this process. Our results<br />

<strong>in</strong>dicated clearly that the gastric mucosal <strong>in</strong>jury <strong>in</strong>duced<br />

by GI-R was ma<strong>in</strong>ly the result <strong>of</strong> reperfusion. The serious<br />

gastric mucosal lesions occurred <strong>in</strong> the <strong>in</strong>itial stages<br />

<strong>of</strong> reperfusion and the aggravat<strong>in</strong>g processes <strong>of</strong> mucosal<br />

lesions were at 1 h after reperfusion, which were ma<strong>in</strong>-<br />

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A<br />

B<br />

O.D. (fold vs R3h)<br />

Qiao WL et al . Bcl-2 and Bax <strong>in</strong> ischemia-reperfusion<br />

Bcl-2<br />

Bax<br />

b-act<strong>in</strong><br />

2.0<br />

1.8<br />

1.6<br />

1.4<br />

1.2<br />

1.0<br />

0.8<br />

0.6<br />

0.4<br />

0.2<br />

0.0<br />

Sham 0 0.5 1 3 6 24 48 72 h<br />

Bcl-2<br />

Bax<br />

a<br />

a<br />

Figure 3 Expression <strong>of</strong> Bcl-2 and Bax prote<strong>in</strong>s <strong>in</strong> cytoplasm extracts from<br />

rat gastric mucosa for different reperfusion durations after gastric ischemia-reperfusion.<br />

Extracts were obta<strong>in</strong>ed from sham-operated rats or from<br />

gastric ischemia-reperfusion rats with different reperfusion durations (0, 0.5, 1,<br />

3, 6, 24, 48, or 72 h) after 30 m<strong>in</strong> <strong>of</strong> ischemia, and were analyzed respectively<br />

by western blott<strong>in</strong>g with anti-Bcl-2 and anti-Bax antibodies. A: Representative<br />

blots correspond<strong>in</strong>g to expression levels <strong>of</strong> Bcl-2 and Bax prote<strong>in</strong>s; B: Semiquantitative<br />

analysis <strong>of</strong> the levels <strong>of</strong> Bcl-2 and Bax. Sham: Sham-operated.<br />

Values are means ± SD, n = 6. a P < 0.05 vs sham.<br />

ta<strong>in</strong>ed about 3 h after reperfusion. The gastric mucosal<br />

repairs started after 3 h <strong>of</strong> reperfusion, and the complete<br />

recovery took almost 3 d. Based on these facts, it <strong>in</strong>dicated<br />

that gastric mucosa has an amaz<strong>in</strong>g self-repair<strong>in</strong>g<br />

ability. ERK1/2 are important members <strong>of</strong> the mitogenactivated<br />

prote<strong>in</strong> k<strong>in</strong>ase family. The activation <strong>of</strong> ERK1/2<br />

participates <strong>in</strong> the regulation <strong>of</strong> cellular <strong>in</strong>jury and repair<br />

<strong>in</strong> many tissues. Our researches have also shown that the<br />

p-ERK1/2 prote<strong>in</strong> level decreased at 0.5 h after reperfusion<br />

began, and then gradually <strong>in</strong>creased, reach<strong>in</strong>g its peak<br />

after 3 h <strong>of</strong> reperfusion. Inhibition <strong>of</strong> the activation <strong>of</strong><br />

ERK1/2 aggravated the gastric mucosal <strong>in</strong>jury, with apoptosis<br />

<strong>in</strong>creased and proliferation reduced <strong>in</strong> the gastric<br />

mucosal cells at the same duration <strong>of</strong> reperfusion. Therefore,<br />

activated ERK1/2 <strong>in</strong>hibited apoptosis and promoted<br />

proliferation <strong>in</strong> gastric mucosal cells.<br />

Apoptosis and proliferation are fundamental mechanisms<br />

for cell death and survival and differentiation <strong>in</strong> the<br />

gastric mucosa. The status <strong>of</strong> the Bcl-2 family prote<strong>in</strong>s<br />

determ<strong>in</strong>es whether a cell will live or die through the regulation<br />

<strong>of</strong> cytochrome c release from the mitochondria [29,30] .<br />

Bcl-2 prote<strong>in</strong> ma<strong>in</strong>ly <strong>in</strong>hibits apoptosis and facilitates cellular<br />

survival and differentiation, whereas overexpression<br />

<strong>of</strong> Bax prote<strong>in</strong> <strong>in</strong>duces apoptosis and <strong>in</strong>hibits the effect<br />

<strong>of</strong> Bcl-2 [31-33] . Our data showed that Bcl-2 expression<br />

decreased significantly after the start <strong>of</strong> the reperfusion,<br />

reach<strong>in</strong>g its nadir at 1 h, before <strong>in</strong>creas<strong>in</strong>g gradually to a<br />

peak after 24 h <strong>of</strong> reperfusion. The pattern <strong>of</strong> change <strong>in</strong><br />

Bax expression was opposite to that <strong>of</strong> Bcl-2 expression.<br />

Bax expression <strong>in</strong>creased at first, reach<strong>in</strong>g its maximum<br />

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a<br />

a<br />

a<br />

a a<br />

sham 0 0.5 1 3 6 24 48 72<br />

Reperfusion time (h)<br />

a<br />

a<br />

a<br />

a<br />

Positive cells (%)<br />

60<br />

50<br />

40<br />

30<br />

20<br />

10<br />

0<br />

R3h Vehi + R3h PD + R3h<br />

Figure 4 Effects <strong>of</strong> PD98059 (ERK1/2 <strong>in</strong>hibitor) on quantitative changes <strong>of</strong><br />

Bcl-2 and Bax positive cells <strong>in</strong> rat gastric mucosa after gastric ischemiareperfusion.<br />

R3h: Reperfusion for 3 h after 30 m<strong>in</strong> <strong>of</strong> ischemia; Vehi + R3h:<br />

Vehicle + R3h; PD + R3h: PD98059 + R3h; Sham: Sham-operated. Values are<br />

percentage <strong>of</strong> positive cells (positive cells/total cells) counted <strong>in</strong> 10 microscopic<br />

fields. Each column represents mean ± SD, n = 6. a P < 0.05 vs R3h.<br />

A<br />

B<br />

O.D. (fold vs R3h)<br />

Bcl-2<br />

Bax<br />

b-act<strong>in</strong><br />

1.6<br />

1.4<br />

1.2<br />

1.0<br />

0.8<br />

0.6<br />

0.4<br />

0.2<br />

0.0<br />

Bcl-2<br />

Bax<br />

R3h Vehi + R3h PD + R3h<br />

Bcl-2<br />

Bax<br />

R3h Vehi + R3h PD + R3h<br />

Figure 5 Effects <strong>of</strong> PD98059 (ERK1/2 <strong>in</strong>hibitor) on expression <strong>of</strong> Bcl-2 and<br />

Bax prote<strong>in</strong>s <strong>in</strong> cytoplasm extracts from rat gastric mucosa after gastric<br />

ischemia-reperfusion. R3h: Reperfusion for 3 h after 30 m<strong>in</strong> <strong>of</strong> ischemia; Vehi<br />

+ R3h: Vehicle + R3h; PD + R3h: PD98059 + R3h. Extracts were obta<strong>in</strong>ed for<br />

analysis by western blott<strong>in</strong>g with anti-Bcl-2 and anti-Bax antibodies. A: Representative<br />

blots correspond<strong>in</strong>g to expression levels <strong>of</strong> Bcl-2 and Bax prote<strong>in</strong>s; B:<br />

Semi-quantitative analysis <strong>of</strong> the levels <strong>of</strong> Bcl-2 and Bax. Values are means ±<br />

SD, n = 6. a P < 0.05 vs R3h.<br />

after 1 h <strong>of</strong> reperfusion, and then decreased. Bcl-2 and<br />

Bax recovered gradually to base level at 72 h <strong>of</strong> reperfusion.<br />

PD98059, a specific upstream <strong>in</strong>hibitor <strong>of</strong> ERK1/2,<br />

downregulated expression <strong>of</strong> Bcl-2 and upregulated expression<br />

<strong>of</strong> Bax <strong>in</strong> GI-R. These results suggest that the<br />

course <strong>of</strong> expression <strong>of</strong> Bcl-2 and Bax were closely correlated<br />

with p-ERK1/2. Activation <strong>of</strong> ERK1/2 causes<br />

upregulation <strong>of</strong> Bcl-2 and downregulation <strong>of</strong> Bax.<br />

In conclusion, Bcl-2 and Bax played a pivotal role <strong>in</strong><br />

GI-R <strong>in</strong>jury and repair by activation <strong>of</strong> ERK1/2. Bcl-2<br />

was <strong>in</strong>volved <strong>in</strong> recovery <strong>of</strong> GI-R-mediated gastric mucosa<br />

<strong>in</strong>jury by promot<strong>in</strong>g cellular proliferation, and Bax<br />

1722 April 7, 2011|Volume 17|Issue 13|<br />

a<br />

a<br />

a<br />

a

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was <strong>in</strong>volved <strong>in</strong> gastric mucosal <strong>in</strong>jury <strong>in</strong>duced by GI-R<br />

by promot<strong>in</strong>g apoptosis.<br />

COMMENTS<br />

Background<br />

It is well known that many hemorrhagic and stress conditions lead to gastric<br />

ischemia-reperfusion (GI-R) <strong>in</strong>jury. Gastric ulceration is very prevalent <strong>in</strong><br />

humans and is usually preceded by burns, sepsis, major surgery, ischemia,<br />

trauma and other heterogeneous forms <strong>of</strong> stress. Erosions <strong>in</strong> the gastric mucosa<br />

can be demonstrated <strong>in</strong> as many as 75%-100% <strong>of</strong> patients with<strong>in</strong> 24 h <strong>of</strong><br />

admission to the <strong>in</strong>tensive care unit (ICU). Cl<strong>in</strong>ically apparent gastro<strong>in</strong>test<strong>in</strong>al<br />

bleed<strong>in</strong>g can occur <strong>in</strong> as many as 25% <strong>of</strong> ICU patients.<br />

Research frontiers<br />

In recent years, studies on GI-R <strong>in</strong>jury have focused on its pathogenic and<br />

underly<strong>in</strong>g molecular mechanism. In recent years, studies on GI-R <strong>in</strong>jury have<br />

revealed that reactive oxygen species, endothel<strong>in</strong>, microvascular dysfunction,<br />

polymorphonuclear leukocyte <strong>in</strong>filtration, nitric oxide release, gastric acid secretion<br />

and decreased prostagland<strong>in</strong> concentrations dur<strong>in</strong>g reperfusion may play<br />

a role <strong>in</strong> the pathogenesis <strong>of</strong> gastric mucosal <strong>in</strong>jury <strong>in</strong>duced by GI-R. Mucosal<br />

<strong>in</strong>tegrity is ma<strong>in</strong>ta<strong>in</strong>ed by the equilibrium between proliferation and apoptosis <strong>of</strong><br />

the gastric mucosal cells. To understand better the pathogenesis <strong>of</strong> gastric lesions,<br />

it is <strong>of</strong> great importance to study the imbalance between proliferation and<br />

apoptosis.<br />

Innovations and breakthroughs<br />

This is believed to be the first study to <strong>in</strong>vestigate changes <strong>in</strong> expression <strong>of</strong><br />

Bcl-2 and Bax at different times <strong>of</strong> reperfusion after gastric ischemia, and<br />

whether extracellular signal-regulated k<strong>in</strong>ase 1/2 activation was <strong>in</strong>volved <strong>in</strong> this<br />

process.<br />

Applications<br />

Not only does our study provide <strong>in</strong>sights <strong>in</strong>to the mechanism <strong>of</strong> gastric mucosal<br />

tissue <strong>in</strong>jury and repair; it also provides <strong>in</strong>formation that could potentially guide<br />

development <strong>of</strong> a new therapeutic strategy.<br />

Peer review<br />

The quality <strong>of</strong> the paper is excellent and deserves a fast publication, consider<strong>in</strong>g<br />

the contribution importance.<br />

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Qiao WL et al . Bcl-2 and Bax <strong>in</strong> ischemia-reperfusion<br />

27 Wada K, Nakajima A, Takahashi H, Yoneda M, Fujisawa N,<br />

Ohsawa E, Kadowaki T, Kubota N, Terauchi Y, Matsuhashi<br />

N, Saubermann LJ, Nakajima N, Blumberg RS. Protective<br />

effect <strong>of</strong> endogenous PPARgamma aga<strong>in</strong>st acute gastric<br />

mucosal lesions associated with ischemia-reperfusion. Am J<br />

Physiol Gastro<strong>in</strong>test Liver Physiol 2004; 287: G452-G458<br />

28 Villegas I, Martín AR, Toma W, de la Lastra CA. Rosiglitazone,<br />

an agonist <strong>of</strong> peroxisome proliferator-activated receptor<br />

gamma, protects aga<strong>in</strong>st gastric ischemia-reperfusion<br />

damage <strong>in</strong> rats: role <strong>of</strong> oxygen free radicals generation. Eur J<br />

Pharmacol 2004; 505: 195-203<br />

29 Adams JM, Cory S. The Bcl-2 prote<strong>in</strong> family: arbiters <strong>of</strong> cell<br />

survival. Science 1998; 281: 1322-1326<br />

WJG|www.wjgnet.com<br />

30 Maga G, Hubscher U. Proliferat<strong>in</strong>g cell nuclear antigen (PCNA):<br />

a dancer with many partners. J Cell Sci 2003; 116: 3051-3060<br />

31 Konturek PC, Brzozowski T, Konturek SJ, Pajdo R, Konturek<br />

JE, Kwiecień S, Taut A, Hahn EG. Apoptosis <strong>in</strong> gastric<br />

mucosa with stress-<strong>in</strong>duced gastric ulcers. J Physiol Pharmacol<br />

1999; 50: 211-225<br />

32 L<strong>in</strong>dsten T, Zong WX, Thompson CB. Def<strong>in</strong><strong>in</strong>g the role <strong>of</strong><br />

the Bcl-2 family <strong>of</strong> prote<strong>in</strong>s <strong>in</strong> the nervous system. Neuroscientist<br />

2005; 11: 10-15<br />

33 Maeda S, Yoshida H, Mitsuno Y, Hirata Y, Ogura K, Shiratori<br />

Y, Omata M. Analysis <strong>of</strong> apoptotic and antiapoptotic<br />

signall<strong>in</strong>g pathways <strong>in</strong>duced by Helicobacter pylori. Gut<br />

2002; 50: 771-778<br />

S- Editor Tian L L- Editor Kerr C E- Editor Ma WH<br />

1724 April 7, 2011|Volume 17|Issue 13|

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Onl<strong>in</strong>e Submissions: http://www.wjgnet.com/1007-9327<strong>of</strong>fice<br />

wjg@wjgnet.com<br />

doi:10.3748/wjg.v17.i13.1725<br />

BRIEF ARTICLE<br />

Intrahepatic natural killer T cell populations are <strong>in</strong>creased<br />

<strong>in</strong> human hepatic steatosis<br />

Michael Adler, Sarah Taylor, Kamalu Okebugwu, Herman Yee, Christ<strong>in</strong>e Field<strong>in</strong>g, George Field<strong>in</strong>g, Michael Poles<br />

Michael Adler, Sarah Taylor, Kamalu Okebugwu, Herman<br />

Yee, Christ<strong>in</strong>e Field<strong>in</strong>g, George Field<strong>in</strong>g, Michael Poles,<br />

New York University School <strong>of</strong> Medic<strong>in</strong>e New York, NY 10016,<br />

United States<br />

Author contributions: Poles M was responsible for study concept<br />

design direction, supervision, formulat<strong>in</strong>g the hypothesis<br />

and obta<strong>in</strong><strong>in</strong>g fund<strong>in</strong>g; Field<strong>in</strong>g G and Field<strong>in</strong>g C were responsible<br />

for obta<strong>in</strong><strong>in</strong>g the liver samples dur<strong>in</strong>g bariatric surgery;<br />

Adler M enrolled patients, performed flow cytometry and drafted<br />

the manuscript; Taylor S enrolled patients and performed<br />

flow cytometry; Okebugwu K performed flow cytometry; Yee H<br />

reviewed the liver biopsies for steatosis.<br />

Supported by The Irma T Hirschl/Monique Weill-Caulier Charitable<br />

Trust and The Michael Saperste<strong>in</strong> Medical Scholars Research<br />

Fund<br />

Correspondence to: Michael A Poles, MD, PhD, Division<br />

<strong>of</strong> <strong>Gastroenterology</strong>, Manhattan VA Medical Center, New York<br />

University School <strong>of</strong> Medic<strong>in</strong>e, 423 East 23rd Street, Room<br />

11097-S, New York, NY 10010,<br />

United States. michael.poles@nyumc.org<br />

Telephone: +1-212-6867500 Fax: +1-212-9513481<br />

Received: June 1, 2010 Revised: August 6, 2010<br />

Accepted: August 13, 2010<br />

Published onl<strong>in</strong>e: April 7, 2011<br />

Abstract<br />

AIM: To determ<strong>in</strong>e if natural killer T cell (NKT) populations<br />

are affected <strong>in</strong> nonalcoholic fatty liver disease<br />

(NAFLD).<br />

METHODS: Patients undergo<strong>in</strong>g bariatric surgery underwent<br />

liver biopsy and blood sampl<strong>in</strong>g dur<strong>in</strong>g surgery.<br />

The biopsy was assessed for steatosis and immunocyte<br />

<strong>in</strong>filtration. Intrahepatic lymphocytes (IHLs)<br />

were isolated from the rema<strong>in</strong>der <strong>of</strong> the liver biopsy,<br />

and peripheral blood mononuclear cells (PBMCs) were<br />

isolated from the blood. Expression <strong>of</strong> surface prote<strong>in</strong>s<br />

on both IHLs and PBMCs were quantified us<strong>in</strong>g flow<br />

cytometry.<br />

RESULTS: Twenty­seven subjects participated <strong>in</strong> this<br />

WJG|www.wjgnet.com<br />

1725<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): 1725-1731<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

study. Subjects with moderate or severe steatosis had<br />

a higher percentage <strong>of</strong> <strong>in</strong>trahepatic CD3+/CD56+ NKT<br />

cells (38.6%) than did patients with mild steatosis<br />

(24.1%, P = 0.05) or those without steatosis (21.5%, P<br />

= 0.03). Patients with moderate to severe steatosis also<br />

had a higher percentage <strong>of</strong> NKT cells <strong>in</strong> the blood (12.3%)<br />

as compared to patients with mild steatosis (2.5% P =<br />

0.02) and those without steatosis (5.1%, P = 0.05).<br />

CONCLUSION: NKT cells are significantly <strong>in</strong>creased <strong>in</strong><br />

the liver and blood <strong>of</strong> patients with moderate to severe<br />

steatosis and support the role <strong>of</strong> NKT cells <strong>in</strong> NAFLD.<br />

© 2011 Baishideng. All rights reserved.<br />

Key words: Nonalcoholic fatty liver disease; Natural killer<br />

T cells; Natural killer T­like cells; Lymphocytes; Hepatic<br />

steatosis<br />

Peer reviewer: Dongchang Zhao, Pr<strong>of</strong>essor <strong>of</strong> Beckman Research<br />

Institute, Department <strong>of</strong> Surgery, 1500 Duarte Road, Duarte,<br />

CA 91010, United States<br />

Adler M, Taylor S, Okebugwu K, Yee H, Field<strong>in</strong>g C, Field<strong>in</strong>g<br />

G, Poles M. Intrahepatic natural killer T cell populations are <strong>in</strong>creased<br />

<strong>in</strong> human hepatic steatosis. <strong>World</strong> J Gastroenterol 2011;<br />

17(13): 1725-1731 Available from: URL: http://www.wjgnet.<br />

com/1007-9327/full/v17/i13/1725.htm DOI: http://dx.doi.<br />

org/10.3748/wjg.v17.i13.1725<br />

INTRODUCTION<br />

With the epidemic <strong>of</strong> obesity burgeon<strong>in</strong>g across much <strong>of</strong><br />

the world, nonalcoholic fatty liver disease (NAFLD) has<br />

become an <strong>in</strong>creas<strong>in</strong>gly press<strong>in</strong>g problem. The prevalence<br />

<strong>of</strong> NAFLD <strong>in</strong> western countries is as high as 17%-33% [1] ,<br />

and the more severe form <strong>of</strong> NAFLD, non-alcoholic steatohepatitis<br />

(NASH), will progress to cirrhosis <strong>in</strong> 20% <strong>of</strong><br />

patients [1] . Due to its <strong>in</strong>creas<strong>in</strong>g prevalence, NAFLD has become<br />

the third lead<strong>in</strong>g <strong>in</strong>dication for liver transplantation [2] .<br />

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Table 2 Percentage <strong>of</strong> CD3+ lymphocyte populations <strong>in</strong> patients with normal livers, mild hepatic steatosis, and moderate to severe<br />

steatosis n (%)<br />

Lymphocyte population N steatosis M steatosis MS steatosis P value<br />

(N vs M)<br />

by ELISA, compared to peripheral lymphocytes, though<br />

no differences were noted between patient cohorts (data<br />

not shown). In the majority <strong>of</strong> samples, IL-4 secretion<br />

rema<strong>in</strong>ed undetectable.<br />

Expression <strong>of</strong> CD161+ on NKT cells is <strong>in</strong>creased <strong>in</strong> patients<br />

with moderate to severe steatosis<br />

CD161 (NKR-P1A) is a receptor that is primarily associ-<br />

WJG|www.wjgnet.com<br />

ated with NK cells, but is also expressed on NKT cells,<br />

and may <strong>in</strong>dicate an effector and memory subset <strong>of</strong> such<br />

cells [19] . We therefore assessed the expression <strong>of</strong> CD161<br />

on the CD3+/CD56+ populations <strong>in</strong> the liver and blood<br />

(Figure 1). Aga<strong>in</strong>, <strong>in</strong> each cohort, there were a higher percentage<br />

<strong>of</strong> CD3+/CD56+ cells that expressed CD161<br />

<strong>in</strong> the liver, compared to the blood (Table 2). Further, the<br />

percentage <strong>of</strong> CD161-express<strong>in</strong>g CD3+/CD56+ cells<br />

<strong>in</strong> the liver (35.8% ± 9.1%) and blood (9.6% ± 4.9%) <strong>of</strong><br />

subjects with moderate-to-severe hepatic steatosis were<br />

significantly <strong>in</strong>creased compared to those without steatosis<br />

(liver: 15.5% ± 12.6%, P = 0.01, blood: 2.5% ± 3.8%, P =<br />

0.03) and those with mild hepatic steatosis (liver: 18.9% ±<br />

12.5%, P = 0.02, blood: 1.2% ± 1.1%, P = 0.02).<br />

Moderate-to-severe steatosis alters the percentages <strong>of</strong><br />

non NKT cell lymphocyte population<br />

In addition to <strong>in</strong>creases <strong>in</strong> the percentages <strong>of</strong> NKT cells,<br />

other m<strong>in</strong>or lymphocyte subsets were significantly affected<br />

<strong>in</strong> patients with moderate-to-severe hepatic steatosis. Intrahepatic<br />

percentages <strong>of</strong> double negative T cells (CD3+,<br />

CD4-, CD8-) were <strong>in</strong>creased <strong>in</strong> the liver <strong>of</strong> subjects with<br />

moderate-severe steatosis (26.6% ± 17.0%), compared to<br />

those without steatosis [12.6% ± 10.4%, P = 0.05 (Table 2)].<br />

The CD3+/CD8+ lymphocytes were the only lymphocyte<br />

population found to significantly decrease <strong>in</strong> patients<br />

with moderate-to-severe steatosis. In these patients,<br />

the percentage <strong>of</strong> CD3+/CD8+ lymphocytes (27.3%<br />

± 9.6%) decreased significantly as compared to patients<br />

with mild steatosis (49.%3 ± 10.7%, P < 0.001) or without<br />

steatosis (55.6% ± 14.3%, P < 0.001). CD3+/CD8+<br />

lymphocytes also decreased <strong>in</strong> the peripheral blood <strong>in</strong><br />

patients with moderate-to-severe steatosis as compared to<br />

normal livers and approached significance (17.4% ± 8.5%<br />

vs 26.2% ± 7.0%, P = 0.06).<br />

DISCUSSION<br />

P value<br />

(N vs MS)<br />

P value<br />

(M vs MS)<br />

CD3+/CD4-/CD8- PBMC 8.13 3.62 22.88 0.22 0.1 0.04 a<br />

Correlation<br />

coefficient<br />

CD3+/CD4-/CD8- IHL 12.61 9.12 26.58 0.4 0.1 0.05 a<br />

0.76<br />

CD3+/CD56+ PBMC 5.09 2.45 12.32 0.22 0.049 a<br />

0.016 a<br />

0.71<br />

CD3+/CD56+ IHL 21.49 24.13 38.62 0.7 0.03 a<br />

0.048 a<br />

0.93<br />

CD3+/CD56+/CD161+ PBMC 2.45 1.15 9.64 0.3 0.027 a<br />

0.017 a<br />

0.79<br />

CD3+/CD56+/CD161+ IHL 15.50 18.90 35.81 0.6 0.006 a<br />

0.017 a<br />

0.93<br />

CD3+/Vα24+ PBMC 0.60 0.53 0.57 0.48 0.23 0.14 -0.43<br />

CD3+/Vα24+ IHL 0.43 0.42 0.76 0.9 0.37 0.36 0.85<br />

CD3+/CD8+ IHL 55.59 49.30 26.58 0.51 0.0003 a<br />

0.006 a<br />

-0.95<br />

Each percentage is the proportion <strong>of</strong> a specific CD3+ lymphocyte population out <strong>of</strong> all CD3+ lymphocytes. a P < 0.05. PBMC: Peripheral blood mononuclear<br />

cell; IHL: Intrahepatic lymphocyte. N: Normal; M: Mild; MS: Mod/sev.<br />

A<br />

CD56 FITC<br />

B<br />

CD56 FITC<br />

Adler M et al . Natural killer T cells <strong>in</strong> human hepatic steatosis<br />

10 5<br />

10 4<br />

10 3<br />

10 2<br />

0<br />

10 5<br />

10 4<br />

10 3<br />

10 2<br />

0<br />

8.3%<br />

46.0%<br />

6.6%<br />

39.1%<br />

0 10 2 10 3 10 4 10 5<br />

CD161 PE<br />

11.9% 20.1%<br />

36.0%<br />

32.1%<br />

0 10 2 10 3 10 4 10 5<br />

CD161 PE<br />

Figure 1 Flow cytometry <strong>of</strong> CD3+/CD56+/CD161+ <strong>in</strong>trahepatic lymphocytes<br />

<strong>in</strong> a patient with a normal liver versus a patient with moderate<br />

steatosis. Cells were <strong>in</strong>itially selected via CD3+ gat<strong>in</strong>g prior to analysis for<br />

expression <strong>of</strong> CD56 and CD161. There are a greater percentage <strong>of</strong> natural<br />

killer T cells (CD56+/CD161+) <strong>in</strong> patients with moderate steatosis (20.1%) as<br />

compared to patients with normal livers (6.6%). A: Normal liver; B: Moderate<br />

steatosis.<br />

NAFLD and NASH are <strong>in</strong>creas<strong>in</strong>g <strong>in</strong> importance throughout<br />

the world. While our immune system plays an important<br />

role <strong>in</strong> the pathogenesis <strong>of</strong> this disease, our under-<br />

1728 April 7, 2011|Volume 17|Issue 13|<br />

0.73

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stand<strong>in</strong>g <strong>of</strong> the specifics <strong>of</strong> the immunopathogenesis <strong>of</strong><br />

NAFLD is limited. Much <strong>of</strong> our <strong>in</strong>formation regard<strong>in</strong>g<br />

NAFLD has come from mur<strong>in</strong>e models, and NKT cells<br />

have been shown to be a key mediator <strong>of</strong> mur<strong>in</strong>e fatty<br />

liver disease [12] . However, there are very few studies <strong>of</strong> <strong>in</strong>trahepatic<br />

NKT cells <strong>in</strong> humans. In this study, we sought<br />

to <strong>in</strong>vestigate the changes <strong>in</strong> lymphocyte populations, with<br />

a focus on NKT cells, <strong>in</strong> obese patients with histologically<br />

confirmed steatosis or steatohepatitis. We found that<br />

NKT cells, def<strong>in</strong>ed as CD3+/CD56+ lymphocytes, are<br />

significantly <strong>in</strong>creased <strong>in</strong> patients with moderate to severe<br />

steatosis as compared to patients with no steatosis or mild<br />

steatosis. These f<strong>in</strong>d<strong>in</strong>gs differ from the numerous studies<br />

performed <strong>in</strong> mice and suggest a different role <strong>of</strong> NKT<br />

cells <strong>in</strong> fatty liver disease <strong>in</strong> humans.<br />

There have been 4 previous studies <strong>in</strong>vestigat<strong>in</strong>g NKT<br />

cells and fatty liver disease <strong>in</strong> humans, each us<strong>in</strong>g different<br />

techniques and yield<strong>in</strong>g different results. In a study<br />

by Xu and colleagues, the <strong>in</strong>vestigators found a decrease<br />

<strong>in</strong> peripheral vα24+ NKT cells as compared to healthy<br />

matched non-obese controls [14] . In that study, the diagnosis<br />

<strong>of</strong> NAFLD was made on a cl<strong>in</strong>ical basis, as opposed to<br />

our utilization <strong>of</strong> histology, which is a more specific means<br />

<strong>of</strong> diagnosis, and IHLs were not exam<strong>in</strong>ed. In a study<br />

by Kremer and colleagues, the <strong>in</strong>vestigators also found a<br />

decrease <strong>in</strong> NKT cells <strong>in</strong> patients with moderate to severe<br />

steatosis [15] . However, they def<strong>in</strong>ed NKT cells by expression<br />

<strong>of</strong> CD3+/CD57+, and used immunohistochemistry<br />

sta<strong>in</strong><strong>in</strong>g <strong>in</strong>stead <strong>of</strong> flow cytometry for quantification, both<br />

<strong>of</strong> which can account for the differences <strong>in</strong> their results<br />

and ours. F<strong>in</strong>ally, Tajiri and colleagues evaluated liver biopsy<br />

specimens <strong>of</strong> patients with NAFLD and performed<br />

flow cytometry on 20 <strong>of</strong> the specimens. In these 20 specimens,<br />

they found that <strong>in</strong> patients with more severe steatosis<br />

there was an <strong>in</strong>crease <strong>in</strong> CD3+/CD56+ NKT cells [16] ,<br />

and is <strong>in</strong> agreement with the results reported here. F<strong>in</strong>ally<br />

Syn et al [17] studied 6 liver biopsies, 2 <strong>of</strong> which had confirmed<br />

NASH cirrhosis, and found an <strong>in</strong>crease percentage<br />

<strong>of</strong> NKT cells <strong>in</strong> the livers with NASH cirrhosis compared<br />

to healthy controls and patients with other forms <strong>of</strong><br />

hepatitis. With 27 patients enrolled <strong>in</strong> this study, this is the<br />

largest sample size to date to evaluate lymphocyte populations<br />

<strong>in</strong> patients with NAFLD. Further, we also quantified<br />

the presence <strong>of</strong> <strong>in</strong>variant NKT cells, expression <strong>of</strong><br />

CD161 and other m<strong>in</strong>or T cell populations <strong>in</strong> our biologic<br />

samples, as well as exam<strong>in</strong><strong>in</strong>g cytok<strong>in</strong>e production.<br />

NKT cells may play a number <strong>of</strong> immunoregulatory<br />

roles <strong>in</strong> the liver and are considered by some to be a bridge<br />

between the <strong>in</strong>nate and adaptive immune systems [20] .<br />

NKT cells participate <strong>in</strong> pro-<strong>in</strong>flammatory, Th1, and anti<strong>in</strong>flammatory<br />

Th2 mediated pathways via the secretion <strong>of</strong><br />

IFN-γ and IL-4, respectively. In mur<strong>in</strong>e models, it has been<br />

proposed that depletion <strong>of</strong> NKT cells shifts the hepatic<br />

immune environment toward a Th1 milieu, lead<strong>in</strong>g to immunocyte<br />

<strong>in</strong>filtration and development <strong>of</strong> steatohepatitis [9] .<br />

Lept<strong>in</strong> deficient mice develop steatosis and NASH, but<br />

they do not develop cirrhosis [20] . Alternatively, NKT cells,<br />

when shift<strong>in</strong>g the immune environment toward a Th2<br />

WJG|www.wjgnet.com<br />

Adler M et al . Natural killer T cells <strong>in</strong> human hepatic steatosis<br />

milieu may be responsible for collagen deposition <strong>in</strong> the<br />

liver. Stimulation and proliferation <strong>of</strong> NKT cells <strong>in</strong> lept<strong>in</strong><br />

deficient mice, through adrenergic stimulation, results<br />

<strong>in</strong> hepatic collagen deposition and fibrosis secondary to<br />

IL-4 and IL-13 secretion and activation <strong>of</strong> Th2 mediated<br />

pathways [12,20] . In our study, we found an <strong>in</strong>creased percentage<br />

<strong>of</strong> <strong>in</strong>trahepatic CD3+/CD56+ NKT cells <strong>in</strong> patients<br />

with moderate to severe steatosis and a low <strong>in</strong>cidence <strong>of</strong><br />

steatohepatitis, which could support a protective role <strong>of</strong><br />

NKT cells aga<strong>in</strong>st steatohepatitis. In addition we found<br />

a decrease <strong>in</strong> CD3+/CD8+ <strong>in</strong>trahepatic lymphocytes<br />

which may implicate NKT cells <strong>in</strong> shift<strong>in</strong>g the hepatic immunoregulatory<br />

environment towards more Th2 mediated<br />

mechanisms. We were unable to identify a difference <strong>in</strong><br />

the secretion <strong>of</strong> IFN-γ or IL-4 by NKT cells <strong>in</strong> patients<br />

with various degrees <strong>of</strong> steatosis, although <strong>in</strong>terferon,<br />

but not IL-4 production was elaborated when NKT cells<br />

were stimulated <strong>in</strong> the liver samples studied. Future studies<br />

should focus on <strong>in</strong>vestigat<strong>in</strong>g the functional role <strong>of</strong> NKT<br />

cells <strong>in</strong> human fatty liver disease.<br />

The multiple def<strong>in</strong>itions <strong>of</strong> NKT cells can lead to<br />

much confusion when discuss<strong>in</strong>g their role <strong>in</strong> the liver.<br />

We classified NKT cells <strong>in</strong> two different ways, both by<br />

expression <strong>of</strong> CD3+/CD56+, as well as by expression<br />

<strong>of</strong> vα24+. Human NKT cells were <strong>in</strong>itially described <strong>in</strong><br />

liver donor patients by Doherty et al [21] as CD3+/CD56+<br />

cells and were shown to be capable <strong>of</strong> lys<strong>in</strong>g NK sensitive<br />

cells. CD3+/CD56+ lymphocytes have been analyzed<br />

for mRNA expression <strong>of</strong> vα24 and approximately 5% <strong>of</strong><br />

human hepatic CD3+/CD56+ lymphocytes expressed<br />

vα24 mRNA, which encodes the TCR that recognizes<br />

CD1d ligands [21] . Thus, NKT cells are also def<strong>in</strong>ed functionally<br />

as vα24+ lymphocytes or via isolation <strong>of</strong> CD3+<br />

lymphocytes us<strong>in</strong>g CD1d ligands, and are classified as<br />

<strong>in</strong>variant NKT cells. The CD3+/CD56+ lymphocytes,<br />

which are also called NKT-like cells, are populations that<br />

<strong>in</strong>corporate many different type <strong>of</strong> lymphocytes such as<br />

<strong>in</strong>variant T-cells and CD161+ lymphocytes which can potentially<br />

create confusion [22,23] . Thus, although we f<strong>in</strong>d that<br />

this broader more diverse population (CD3+/CD56+<br />

NKT cells) is significantly <strong>in</strong>creased with greater degrees<br />

<strong>of</strong> steatosis, the more specific subgroup <strong>of</strong> <strong>in</strong>variant<br />

vα24 NKT cells were unchanged. It is possible that other<br />

functional subgroups <strong>of</strong> CD3+/CD56+ lymphocytes<br />

such as CD161+ lymphocytes play a larger role <strong>in</strong> human<br />

NAFLD and NASH. This is <strong>in</strong> contrast to the mur<strong>in</strong>e<br />

model where there are higher percentages <strong>of</strong> <strong>in</strong>variant<br />

NKT cells normally found <strong>in</strong> the liver [24] . These f<strong>in</strong>d<strong>in</strong>gs<br />

highlight the importance <strong>of</strong> <strong>in</strong>vestigat<strong>in</strong>g the role <strong>of</strong> <strong>in</strong>variant<br />

NKT and NKT-like lymphocytes <strong>in</strong> human disease,<br />

rather than just us<strong>in</strong>g mur<strong>in</strong>e models.<br />

The results <strong>of</strong> the study are limited by the small sample<br />

size, and impaired our ability to further characterize the<br />

role <strong>of</strong> NKT cells <strong>in</strong> NAFLD. Nevertheless the <strong>in</strong>crease <strong>in</strong><br />

NKT cells <strong>in</strong> moderate to severe steatosis was significant<br />

and correlates with other studies. Absolute lymphocyte<br />

numbers were not reported here because the values were<br />

affected by the varied size <strong>of</strong> the liver biopsy samples<br />

1729 April 7, 2011|Volume 17|Issue 13|

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Adler M et al . Natural killer T cells <strong>in</strong> human hepatic steatosis<br />

taken <strong>in</strong> each patient. Thus, NKT cell percentages <strong>of</strong> total<br />

lymphocyte were reported for more precise comparison<br />

between subjects. Immunohistochemistry has not yet been<br />

performed on the liver biopsies, however we hope to conduct<br />

future studies to further elucidate the role <strong>of</strong> NKT<br />

cells <strong>in</strong> NAFLD.<br />

In this study, we exam<strong>in</strong>ed the change <strong>in</strong> lymphocyte<br />

populations <strong>in</strong> obese patients with NAFLD, with a focus<br />

on <strong>in</strong>trahepatic NKT cells. We found an <strong>in</strong>crease <strong>in</strong> NKT<br />

cells, def<strong>in</strong>ed as CD3+/CD56+ and as well as CD161+<br />

lymphocytes, <strong>in</strong> obese patients with moderate and severe<br />

steatosis. These results differ from previous mur<strong>in</strong>e models<br />

and some human studies. In addition we reported other<br />

changes <strong>in</strong> lymphocyte populations, such as depletion<br />

<strong>in</strong> CD3+/CD8+ lymphocytes and an <strong>in</strong>crease <strong>in</strong> CD3+/<br />

CD4-/CD8- cells, which have not yet been reported <strong>in</strong><br />

NAFLD. The results <strong>of</strong> this study highlight the importance<br />

<strong>of</strong> <strong>in</strong>vestigat<strong>in</strong>g NKT cells and other lymphocyte<br />

populations <strong>in</strong> humans with NAFLD s<strong>in</strong>ce the pathophysiology<br />

<strong>of</strong> human NAFLD likely differs from that <strong>in</strong><br />

mur<strong>in</strong>e models. Future studies to <strong>in</strong>vestigate the role <strong>of</strong><br />

NKT cells <strong>in</strong> NAFLD <strong>in</strong> humans are warranted <strong>in</strong> order<br />

to elucidate the mechanisms beh<strong>in</strong>d the pervasive disease<br />

<strong>of</strong> NAFLD.<br />

COMMENTS<br />

Background<br />

Non alcoholic fatty liver disease (NAFLD) is a common disease where fat <strong>in</strong>filtrates<br />

the liver, which can lead to <strong>in</strong>flammation and cirrhosis. natural killer T<br />

(NKT) cells have been implicated <strong>in</strong> the pathogenesis <strong>of</strong> NAFLD. In obese mice,<br />

NKT cells are depleted <strong>in</strong> the liver and are associated with a greater degree <strong>of</strong><br />

steatosis. When the NKT cells are upregulated <strong>in</strong> mice, the degree <strong>of</strong> fatty <strong>in</strong>filtration<br />

dim<strong>in</strong>ishes. There is limited data about NKT cells <strong>in</strong> human NAFLD, and<br />

this study adds to our understand<strong>in</strong>g <strong>of</strong> NKT cells <strong>in</strong> human NAFLD.<br />

Research frontiers<br />

The data on <strong>in</strong>trahepatic NKT cells and its role <strong>in</strong> human steatosis has been<br />

mixed. There have been 3 studies <strong>in</strong>vestigat<strong>in</strong>g NKT cells and NAFLD. One<br />

found that NKT cells are depleted with <strong>in</strong>creased steatosis whereas the others<br />

found an <strong>in</strong>crease <strong>in</strong> NKT cells with steatosis. This study conta<strong>in</strong>s the largest<br />

sample to date which <strong>in</strong>vestigates NKT cells <strong>in</strong> human NAFLD.<br />

Innovations and breakthroughs<br />

The authors found that NKT cells, def<strong>in</strong>ed as CD3+/CD56+ lymphocytes are<br />

<strong>in</strong>creased <strong>in</strong> human livers with moderate and severe steatosis. In addition, they<br />

reported other changes <strong>in</strong> lymphocyte populations with steatosis, such as depletion<br />

<strong>in</strong> CD3+/CD8+ lymphocytes and an <strong>in</strong>crease <strong>in</strong> CD3+/CD4-/CD8- cells, which<br />

have not yet been reported <strong>in</strong> NAFLD.<br />

Applications<br />

These f<strong>in</strong>d<strong>in</strong>gs further support the role <strong>of</strong> NKT cells <strong>in</strong> NAFLD and highlight an<br />

important difference between NKT cells <strong>in</strong> the mur<strong>in</strong>e model <strong>of</strong> fatty liver disease<br />

and human NAFLD.<br />

Term<strong>in</strong>ology<br />

NKT cells are a highly conserved subset <strong>of</strong> lymphocytes with properties <strong>of</strong> both<br />

T cells (CD3+ expression) and NK cells (CD56+ and CD161+ expression). The<br />

liver conta<strong>in</strong>s a high percentage <strong>of</strong> these unique lymphocytes, which have been<br />

implicated <strong>in</strong> the pathogenesis <strong>of</strong> non alcoholic fatty liver disease.<br />

Peer review<br />

The authors showed that patients with moderate or severe steatosis had a higher<br />

percentage <strong>of</strong> <strong>in</strong>trahepatic CD3+/CD56+ NKT cells than that with mild steatosis<br />

or without steatosis. Further, the percentage <strong>of</strong> CD3+/CD56+CD161+ cells <strong>in</strong><br />

the liver <strong>of</strong> subjects with moderate-to-severe hepatic steatosis were significantly<br />

<strong>in</strong>creased compared to those with mild hepatic steatosis or without steatosis.<br />

This is an <strong>in</strong>terest<strong>in</strong>g f<strong>in</strong>d<strong>in</strong>g and may provide more <strong>in</strong>formation about the NKT<br />

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cells <strong>in</strong> human NAFLD because the data on NKT cells <strong>in</strong> human NAFLD is limited<br />

at present. However, there are several areas <strong>of</strong> the manuscript that the authors<br />

should expand upon that would enhance the presentation.<br />

REFERENCES<br />

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Gastroenterol 2008; 14: 2474-2486<br />

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Bodmer-Narkevitch V, Plettenburg O, Kronenberg M, Tsuji<br />

M, Ho DD, Wong CH. Bacterial glycolipids and analogs as<br />

antigens for CD1d-restricted NKT cells. Proc Natl Acad Sci<br />

USA 2005; 102: 1351-1356<br />

5 Godfrey DI, Hammond KJ, Poulton LD, Smyth MJ, Baxter<br />

AG. NKT cells: facts, functions and fallacies. Immunol Today<br />

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6 K<strong>in</strong>jo Y, Wu D, Kim G, X<strong>in</strong>g GW, Poles MA, Ho DD, Tsuji<br />

M, Kawahara K, Wong CH, Kronenberg M. Recognition <strong>of</strong><br />

bacterial glycosph<strong>in</strong>golipids by natural killer T cells. Nature<br />

2005; 434: 520-525<br />

7 Berz<strong>of</strong>sky JA, Terabe M. NKT cells <strong>in</strong> tumor immunity: oppos<strong>in</strong>g<br />

subsets def<strong>in</strong>e a new immunoregulatory axis. J Immunol<br />

2008; 180: 3627-3635<br />

8 Li Z, L<strong>in</strong> H, Yang S, Diehl AM. Mur<strong>in</strong>e lept<strong>in</strong> deficiency<br />

alters Kupffer cell production <strong>of</strong> cytok<strong>in</strong>es that regulate the<br />

<strong>in</strong>nate immune system. <strong>Gastroenterology</strong> 2002; 123: 1304-1310<br />

9 McClure N, Henry O, Harley JM. Pure XY gonadal dysgenesis<br />

present<strong>in</strong>g as secondary amenorrhea. A case report. J<br />

Reprod Med 1992; 37: 291-292<br />

10 Shiratori Y, Kawase T, Komatsu Y, Hikiba Y, Okano K, Kamii<br />

K, Omata M. Endotox<strong>in</strong> <strong>in</strong>duced cellular communication<br />

<strong>in</strong> the liver: mur<strong>in</strong>e models for clarification <strong>of</strong> the role <strong>of</strong> LPSresponsive<br />

macrophages <strong>in</strong> the pathogenesis <strong>of</strong> liver diseases.<br />

J Gastroenterol Hepatol 1995; 10 Suppl 1: S97-S100<br />

11 El<strong>in</strong>av E, Pappo O, Sklair-Levy M, Margalit M, Shibolet O,<br />

Gomori M, Alper R, Thalenfeld B, Engelhardt D, Rabbani E,<br />

Ilan Y. Adoptive transfer <strong>of</strong> regulatory NKT lymphocytes<br />

ameliorates non-alcoholic steatohepatitis and glucose <strong>in</strong>tolerance<br />

<strong>in</strong> ob/ob mice and is associated with <strong>in</strong>trahepatic CD8<br />

trapp<strong>in</strong>g. J Pathol 2006; 209: 121-128<br />

12 Li Z, Oben JA, Yang S, L<strong>in</strong> H, Stafford EA, Soloski MJ,<br />

Thomas SA, Diehl AM. Norep<strong>in</strong>ephr<strong>in</strong>e regulates hepatic<br />

<strong>in</strong>nate immune system <strong>in</strong> lept<strong>in</strong>-deficient mice with nonalcoholic<br />

steatohepatitis. Hepatology 2004; 40: 434-441<br />

13 Margalit M, Shalev Z, Pappo O, Sklair-Levy M, Alper R,<br />

Gomori M, Engelhardt D, Rabbani E, Ilan Y. Glucocerebroside<br />

ameliorates the metabolic syndrome <strong>in</strong> OB/OB mice. J<br />

Pharmacol Exp Ther 2006; 319: 105-110<br />

14 Xu CF, Yu CH, Li YM, Xu L, Du J, Shen Z. Association <strong>of</strong> the<br />

frequency <strong>of</strong> peripheral natural killer T cells with nonalcoholic<br />

fatty liver disease. <strong>World</strong> J Gastroenterol 2007; 13: 4504-4508<br />

15 Kremer M, Thomas E, Milton RJ, Perry AW, van Rooijen<br />

N, Wheeler MD, Zacks S, Fried M, Rippe RA, H<strong>in</strong>es IN.<br />

Kupffer cell and <strong>in</strong>terleuk<strong>in</strong>-12-dependent loss <strong>of</strong> natural<br />

killer T cells <strong>in</strong> hepatosteatosis. Hepatology 2010; 51: 130-141<br />

16 Tajiri K, Shimizu Y, Tsuneyama K, Sugiyama T. Role <strong>of</strong><br />

liver-<strong>in</strong>filtrat<strong>in</strong>g CD3+CD56+ natural killer T cells <strong>in</strong> the<br />

pathogenesis <strong>of</strong> nonalcoholic fatty liver disease. Eur J Gastroenterol<br />

Hepatol 2009; 21: 673-680<br />

17 Syn WK, Oo YH, Pereira TA, Karaca GF, Jung Y, Omenetti<br />

A, Witek RP, Choi SS, Guy CD, Fear<strong>in</strong>g CM, Teaberry V,<br />

Pereira FE, Adams DH, Diehl AM. Accumulation <strong>of</strong> natural<br />

killer T cells <strong>in</strong> progressive nonalcoholic fatty liver disease.<br />

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Hepatology 2010; 51: 1998-2007<br />

18 Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-<br />

Tetri BA, Bacon BR. Nonalcoholic steatohepatitis: a proposal<br />

for grad<strong>in</strong>g and stag<strong>in</strong>g the histological lesions. Am J Gastroenterol<br />

1999; 94: 2467-2474<br />

19 Diehl AM. Nonalcoholic steatosis and steatohepatitis IV.<br />

Nonalcoholic fatty liver disease abnormalities <strong>in</strong> macrophage<br />

function and cytok<strong>in</strong>es. Am J Physiol Gastro<strong>in</strong>test Liver<br />

Physiol 2002; 282: G1-G5<br />

20 Takahashi T, Dejbakhsh-Jones S, Strober S. Expression <strong>of</strong><br />

CD161 (NKR-P1A) def<strong>in</strong>es subsets <strong>of</strong> human CD4 and CD8 T<br />

cells with different functional activities. J Immunol 2006; 176:<br />

211-216<br />

WJG|www.wjgnet.com<br />

Adler M et al . Natural killer T cells <strong>in</strong> human hepatic steatosis<br />

21 Doherty DG, Norris S, Madrigal-Estebas L, McEntee G,<br />

Traynor O, Hegarty JE, O'Farrelly C. The human liver<br />

conta<strong>in</strong>s multiple populations <strong>of</strong> NK cells, T cells, and<br />

CD3+CD56+ natural T cells with dist<strong>in</strong>ct cytotoxic activities<br />

and Th1, Th2, and Th0 cytok<strong>in</strong>e secretion patterns. J Immunol<br />

1999; 163: 2314-2321<br />

22 Choi S, Diehl AM. Role <strong>of</strong> <strong>in</strong>flammation <strong>in</strong> nonalcoholic<br />

steatohepatitis. Curr Op<strong>in</strong> Gastroenterol 2005; 21: 702-707<br />

23 Peralbo E, Alonso C, Solana R. Invariant NKT and NKT-like<br />

lymphocytes: two different T cell subsets that are differentially<br />

affected by age<strong>in</strong>g. Exp Gerontol 2007; 42: 703-708<br />

24 Exley MA, Koziel MJ. To be or not to be NKT: natural killer<br />

T cells <strong>in</strong> the liver. Hepatology 2004; 40: 1033-1040<br />

S- Editor Tian L L- Editor O’Neill M E- Editor Ma WH<br />

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few years [1-5] . This novel technique permits access to the<br />

peritoneal organs through the mouth, rectosigmoid or<br />

vag<strong>in</strong>a with diagnostic and therapeutic purposes. Numerous<br />

hybrid NOTES procedures (comb<strong>in</strong><strong>in</strong>g NOTES with<br />

laparoscopy) have been described <strong>in</strong> the last five years [6-9] ,<br />

but it was not until 2007 that the first pure NOTES<br />

procedures <strong>in</strong> humans were reported [10-13] . Although the<br />

translum<strong>in</strong>al approach holds great potential, secure access<br />

site closure rema<strong>in</strong>s a critical issue [14] . In recent cases and<br />

series, endoscopic closure is substituted by use <strong>of</strong> rigid <strong>in</strong>struments,<br />

us<strong>in</strong>g the transvag<strong>in</strong>al access <strong>in</strong> almost all cases.<br />

However, this approach excludes the male population.<br />

Consider<strong>in</strong>g the safety <strong>of</strong> laparoscopy, studies are mandatory<br />

to evaluate secure and reproducible closure methods<br />

<strong>in</strong> NOTES procedures [15] . Several closure techniques have<br />

been tested [16] , <strong>in</strong>clud<strong>in</strong>g clips [1,17-19] , septal occluders [20] ,<br />

T-tags [21-23] , more complex sutur<strong>in</strong>g devices [24-26] , and l<strong>in</strong>ear<br />

endoscopic staplers [27] . T-tags have been tested recently to<br />

treat gastrogastric fistulas <strong>in</strong> humans [28] . However, most <strong>of</strong><br />

these devices are time consum<strong>in</strong>g and <strong>of</strong>ten difficult to implement<br />

endoscopically and the current data do not allow<br />

def<strong>in</strong>itive conclusions regard<strong>in</strong>g the different options [16,29] .<br />

The aim <strong>of</strong> this study was to assess the feasibility,<br />

reproducibility and efficacy <strong>of</strong> a new tissue anchor<strong>in</strong>g device<br />

as a gastric suture system <strong>in</strong> a porc<strong>in</strong>e survival model.<br />

MATERIALS AND METHODS<br />

Animals<br />

A total <strong>of</strong> ten female Yorkshire pigs weigh<strong>in</strong>g 30-35 kg<br />

were <strong>in</strong>cluded <strong>in</strong> the study. Animals underwent a 3-d<br />

quarant<strong>in</strong>e and acclimation period. Dur<strong>in</strong>g this period <strong>of</strong><br />

time, veter<strong>in</strong>ary personnel evaluated each animal to ensure<br />

basel<strong>in</strong>e health. Animals were fed the same diet and had<br />

unlimited access to water. The study was conducted at the<br />

University <strong>of</strong> Barcelona Medical School’s animal facilities.<br />

The protocol was approved by the University <strong>of</strong> Barcelona’s<br />

Animal Ethics Committee.<br />

Preoperative care and anesthesia<br />

Animals fasted from solids 24 h prior to the procedure.<br />

All procedures were performed with pigs under general<br />

anesthesia with endotracheal <strong>in</strong>tubation and mechanical<br />

ventilation.<br />

Procedure<br />

A non sterile endoscope (GIF 160, Olympus Medical<br />

Systems, Europe, Hamburg, Germany) was first <strong>in</strong>serted<br />

through the pig’s mouth and the esophagus and stomach<br />

were <strong>in</strong>spected. Afterwards, gastric lavage was performed<br />

with water until the stomach was free <strong>of</strong> solid particles. An<br />

iodated solution followed by an antibiotic suspension (ceftriaxone<br />

1 g/300 mL sal<strong>in</strong>e solution) was <strong>in</strong>stilled and the<br />

antibiotic solution was left <strong>in</strong> the stomach for 10 m<strong>in</strong>. From<br />

this po<strong>in</strong>t on, all the <strong>in</strong>struments used were sterile or high<br />

level dis<strong>in</strong>fected. With a regular endoscope, an overtube was<br />

<strong>in</strong>serted and a double channel gastroscope (GIF 2T160,<br />

Olympus Medical Systems, Europe, Hamburg, Germany)<br />

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Guarner-Argente C et al . Gastrotomy closure <strong>in</strong> a porc<strong>in</strong>e survival model<br />

Figure 1 The <strong>in</strong>cision is enlarged with a balloon dilator. Through the balloon<br />

we can see peritoneal structures.<br />

was used until the end <strong>of</strong> the peritoneoscopy. By external<br />

palpation, the anterior gastric wall was selected to perform<br />

the gastric access. A 5 mm <strong>in</strong>cision was made with a needleknife<br />

(KD-V451M, Olympus Europe, Hamburg, Germany)<br />

and it was subsequently dilated with an 18 mm balloon (CRE<br />

wire-guided balloon, Boston Scientific Microvasive, Natick,<br />

MA) (Figure 1). Then, the scope was passed through the<br />

gastric wall for a 30 m<strong>in</strong> peritoneoscopy.<br />

A Veress needle was placed at the lower left quadrant<br />

<strong>of</strong> the abdomen to control <strong>in</strong>traperitoneal pressure. To<br />

avoid respiratory compromise and impaired venous return,<br />

<strong>in</strong>traperitoneal pressures were monitored and ma<strong>in</strong>ta<strong>in</strong>ed<br />

below 15 mm H2O. Pneumoperitoneum was ma<strong>in</strong>ta<strong>in</strong>ed<br />

with CO 2 <strong>in</strong>sufflation through the scope.<br />

Tissue-anchor<strong>in</strong>g device<br />

The tissue-anchor<strong>in</strong>g device prototype is called brace-bar<br />

(Olympus Medical Systems, Europe, Hamburg, Germany)<br />

and is an evolution <strong>of</strong> a former prototype [21] . It consists<br />

<strong>of</strong> a s<strong>in</strong>gle 18-gauge flexible needle catheter (Figure 2A),<br />

and a bifurcated nylon thread (“Y” shaped) with 3 small<br />

tags (2 regular tags fixed at both bifurcated distal ends and<br />

the other tag stopper at the s<strong>in</strong>gle proximal end, which<br />

will be used for tighten<strong>in</strong>g) (Figure 2B). The tag stopper is<br />

movable and can be slid forward for c<strong>in</strong>ch<strong>in</strong>g <strong>of</strong> the other<br />

tissue-anchor<strong>in</strong>g tags. The proximal end <strong>of</strong> the thread is<br />

fixed to the needle with a small metallic guide (Figure 2C).<br />

Before deployment, the device has to be extracorporally<br />

loaded <strong>in</strong>side the needle catheter. The two distal tags are<br />

consecutively <strong>in</strong>serted <strong>in</strong>to the needle (Figure 2D) and,<br />

f<strong>in</strong>ally, the needle is pulled back <strong>in</strong>to the sheath <strong>in</strong>sert<strong>in</strong>g<br />

also the stopper tag (Figure 2E). Once <strong>in</strong>side the gastric<br />

cavity, the needle is pushed forward and the device is<br />

ready for use. The pusher button (Figure 2F) allows release<br />

<strong>of</strong> one tag at each side <strong>of</strong> the <strong>in</strong>cision and the suture<br />

is tightened by press<strong>in</strong>g the tag Stopper with the needle<br />

sheath. F<strong>in</strong>ally, the suture is released by extract<strong>in</strong>g the metallic<br />

guide that fixed it to the needle.<br />

The tightness <strong>of</strong> the closure was confirmed by means<br />

<strong>of</strong> air <strong>in</strong>sufflation and the ability to ma<strong>in</strong>ta<strong>in</strong> gastric distension<br />

with stability <strong>in</strong> peritoneal pressure measured with<br />

the Veress needle.<br />

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A<br />

Guarner-Argente C et al . Gastrotomy closure <strong>in</strong> a porc<strong>in</strong>e survival model<br />

D<br />

Figure 2 Description <strong>of</strong> the tissue anchor<strong>in</strong>g device. A: S<strong>in</strong>gle 18-gauge flexible needle catheter; B: Bifurcated nylon thread (“Y” shaped) with 3 small tags (2<br />

regular tags fixed at both bifurcated distal ends and the other tag stopper at the s<strong>in</strong>gle proximal end, which is used for tighten<strong>in</strong>g); C: The proximal end <strong>of</strong> the thread is<br />

fixed to the needle with a small metallic guide; D: The two distal tags are consecutively <strong>in</strong>serted <strong>in</strong>to the needle; E: The needle is pulled back <strong>in</strong>to the sheath <strong>in</strong>sert<strong>in</strong>g<br />

also the stopper tag; F: The pusher button allows releas<strong>in</strong>g one tag at each side <strong>of</strong> the <strong>in</strong>cision.<br />

Figure 3 The <strong>in</strong>cision looks completely sealed after the <strong>in</strong>sertion <strong>of</strong> one<br />

brace-bar and the stomach is able to ma<strong>in</strong>ta<strong>in</strong> air distension.<br />

A s<strong>in</strong>gle channel scope (Olympus GIG 160) was only<br />

used for the suture. Endoclips were added when the sutures<br />

were not placed at the middle <strong>of</strong> the <strong>in</strong>cision and<br />

one <strong>of</strong> the sides seemed not completely sealed.<br />

Postoperative care and necropsy<br />

Water was immediately allowed and food was allowed<br />

after 24 h. All animals received <strong>in</strong>travenous ceftriaxone<br />

1 g daily for 3 d and they were monitored daily for signs<br />

<strong>of</strong> peritonitis and sepsis dur<strong>in</strong>g the next 14 d. Weight was<br />

controlled prior to surgery and necropsy. Dur<strong>in</strong>g necropsy,<br />

the peritoneal cavity and the gastric access site were exam<strong>in</strong>ed<br />

for signs <strong>of</strong> peritonitis (exudates, abscesses) or other<br />

complications.<br />

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B<br />

E<br />

Statistical analysis<br />

Data were expressed as mean ± SD or range. Results were<br />

analyzed us<strong>in</strong>g the χ 2 test with Yates correction and Fisher<br />

exact test for qualitative variables and Mann-Whitney test<br />

for quantitative parameters. A P value < 0.05 was considered<br />

statistically significant. Statistical analysis was performed<br />

with SPSS Statistical Package (version 17.0, SPSS<br />

Inc., Chicago, IL).<br />

RESULTS<br />

C<br />

F<br />

All transgastric accesses were achieved with no difficulties<br />

and a mean time <strong>of</strong> 5.7 ± 3.6 m<strong>in</strong> (range 2-14). No major<br />

complications occurred. M<strong>in</strong>or complications <strong>in</strong>cluded an<br />

accidental <strong>in</strong>jury to the anterior abdom<strong>in</strong>al wall and 4 m<strong>in</strong>or<br />

bleed<strong>in</strong>gs dur<strong>in</strong>g the use <strong>of</strong> the needle-knife. A 30 m<strong>in</strong><br />

peritoneoscopy was possible <strong>in</strong> all animals.<br />

The brace-bar was used <strong>in</strong> all cases and closure was<br />

easily achieved <strong>in</strong> 18.1 ± 19.2 m<strong>in</strong>. This time was reduced<br />

to 11.4 ± 5.9 m<strong>in</strong> when consider<strong>in</strong>g only the last 5 cases<br />

and was less than 9 m<strong>in</strong> <strong>in</strong> the last 3 cases, whereas it<br />

was 24.8 ± 13.9 m<strong>in</strong> <strong>in</strong> the first 5 (P = 0.1). Details <strong>of</strong> all<br />

procedures are described <strong>in</strong> Table 1. A total <strong>of</strong> 21 sets <strong>of</strong><br />

sutures (mean 2.1 ± 1, range 1-4) were used to achieve closure<br />

but only 15 (mean 1.5 ± 0.5, range 1-2) could be completely<br />

tightened. Therefore, <strong>in</strong> 5 cases, the <strong>in</strong>cision was<br />

closed with only 1 suture (Figure 3), but <strong>in</strong> two <strong>of</strong> them 1<br />

and 3 clips respectively, were added.<br />

In total, 6 sutures (29%) were <strong>in</strong>effectively positioned.<br />

In 5 attempts one <strong>of</strong> the tags did not stay attached to the<br />

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Table A 1 Summary <strong>of</strong> procedure details B C<br />

Case Number <strong>of</strong><br />

brace-bars used<br />

mucosa either immediately after the tag release or when<br />

tighten<strong>in</strong>g the suture. The rema<strong>in</strong><strong>in</strong>g failure was caused by<br />

a thread rupture after steady tighten<strong>in</strong>g.<br />

Immediately after gastrotomy closure, all brace-bars<br />

seemed well positioned and gastric distension with air was<br />

possible <strong>in</strong> all cases without changes <strong>in</strong> <strong>in</strong>traperitoneal pressure,<br />

suggest<strong>in</strong>g the closure was correct (mean peritoneal<br />

pressure before and after the closure: 14.3 ± 3.3 mmHg,<br />

range 3-15).<br />

The mean procedure time, <strong>in</strong>clud<strong>in</strong>g gastric access<br />

creation, peritoneoscopy and gastrotomy suture, was<br />

63.7 ± 18.2 m<strong>in</strong>.<br />

All the pigs completed the 14 d follow-up period. They<br />

had a weight ga<strong>in</strong> <strong>of</strong> 3.3 ± 3.2 kg. At necropsy, the gastric<br />

access site was completely closed <strong>in</strong> all cases and all bracebars<br />

were present (Table 2). The tags were usually attached<br />

WJG|www.wjgnet.com<br />

Number <strong>of</strong> brace-bars<br />

correctly placed<br />

Cause <strong>of</strong><br />

Misplaced brace-bar<br />

at the gastric serosa (n = 15) or <strong>in</strong>side the gastric wall (n =<br />

14). In the first case, 1 tag was misplaced <strong>in</strong>side the meso<strong>colon</strong>.<br />

In this case, an omental patch had been added<br />

to the suture pull<strong>in</strong>g the omentum <strong>in</strong>side the stomach<br />

through the <strong>in</strong>cision. M<strong>in</strong>imal adhesions were observed <strong>in</strong><br />

3 pigs and signs <strong>of</strong> mild peritonitis and adhesions <strong>in</strong> one.<br />

DISCUSSION<br />

Adjunctive method Closure time<br />

(m<strong>in</strong>)<br />

Endoscopic view<br />

1 2 2 Omental patch 8 Correct<br />

2 1 1 3 endoclips 42 Correct<br />

3 4 2 1 tag detached<br />

1 brace-bar not tightened<br />

None 36 Correct<br />

4 2 2 None 19 Correct<br />

5 2 1 1 tag detached 1 endoclip 19 Correct<br />

6 3 2 1 tag detached None 15 Correct<br />

7 3 1 2 tags detached None 20 Correct<br />

8 2 2 None 9 Correct<br />

9 1 1 None 7 Correct<br />

10 1 1 None 6 Correct<br />

Table 2 Summary <strong>of</strong> necropsy f<strong>in</strong>d<strong>in</strong>gs<br />

Case Weight ga<strong>in</strong> (kg) Abdom<strong>in</strong>al cavity Incision Location <strong>of</strong> tags<br />

1 6.85 M<strong>in</strong>imal adhesions Totally closed 1 tag at meso<strong>colon</strong><br />

3 tags at gastric serosa<br />

2 tag stoppers at gastric mucosa<br />

2 6.90 Normal Totally closed 2 tags at gastric serosa.<br />

1 tag stopper at gastric mucosa<br />

3 5.00 M<strong>in</strong>imal adhesions Totally closed 4 tags at gastric serosa<br />

2 tag stoppers at gastric mucosa<br />

4 3.70 Normal Totally closed 4 tags <strong>in</strong>side gastric wall<br />

2 tag stoppers at gastric mucosa<br />

5 1.90 Normal Totally closed 1 tag at gastric serosa<br />

1 tag <strong>in</strong>side gastric wall<br />

1 tag stopper at gastric mucosa<br />

6 3.74 Small clot Totally closed 3 tags at gastric serosa<br />

1 tag <strong>in</strong>side gastric wall<br />

1 tag stopper at gastric mucosa<br />

7 0.90 Normal Totally closed 1 tag at gastric serosa<br />

1 tag at gastric wall<br />

1 tag stopper at gastric mucosa<br />

8 0.00 Normal Totally closed 1 tag at gastric serosa<br />

3 tags at gastric wall<br />

2 tag stoppers at gastric mucosa<br />

9 -2.78 Fibr<strong>in</strong> exudates<br />

M<strong>in</strong>imal adhesions<br />

Guarner-Argente C et al . Gastrotomy closure <strong>in</strong> a porc<strong>in</strong>e survival model<br />

Totally closed 2 tags at gastric wall<br />

1 tag stopper at gastric mucosa<br />

10 6.42 M<strong>in</strong>imal adhesions Totally closed 2 tags at gastric wall<br />

1 tag stopper at gastric mucosa<br />

NOTES holds great appeal as a less <strong>in</strong>vasive alternative<br />

to laparoscopic surgery. As NOTES heads toward human<br />

trials, it is essential that the creation and closure <strong>of</strong> translum<strong>in</strong>al<br />

<strong>in</strong>cisions be performed <strong>in</strong> a safe, rapid, and reproducible<br />

manner [14-16,28] .<br />

In this study, we assessed the feasibility <strong>of</strong> a new generation<br />

tissue anchor<strong>in</strong>g device with relatively good results.<br />

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Guarner-Argente C et al . Gastrotomy closure <strong>in</strong> a porc<strong>in</strong>e survival model<br />

Moreover, it turned out to be easy and <strong>in</strong>tuitive to use<br />

and the time for placement was short and progressively<br />

reduced. It was not necessary to use complementary clips<br />

when we ga<strong>in</strong>ed experience with the system. One <strong>of</strong> the<br />

advantages <strong>of</strong> this device (and the ma<strong>in</strong> difference with<br />

the former prototype used by Sumiyama et al [22] ) is that it<br />

can be used with a s<strong>in</strong>gle channel endoscope. The same<br />

needle catheter is used for releas<strong>in</strong>g the tags and tighten<strong>in</strong>g<br />

them later without need for a different forceps grasper,<br />

and this makes the procedure shorter. Furthermore,<br />

the depth <strong>of</strong> the needle <strong>in</strong>sertion is limited to 20 mm and<br />

this might decrease the risk <strong>of</strong> complications.<br />

However, we still found some problems with the<br />

device: the needle had to be loaded extracorporally after<br />

each set <strong>of</strong> tissue anchors was applied and this prevented<br />

sequential stitch<strong>in</strong>g. Moreover, we observed a dysfunction<br />

<strong>of</strong> the needle after several attempts which could expla<strong>in</strong><br />

the high rate <strong>of</strong> sutures be<strong>in</strong>g <strong>in</strong>effectively positioned<br />

because the tags could not be released deeply enough. We<br />

th<strong>in</strong>k that pre-charged and non-reusable devices might improve<br />

the procedure time and security. On the other hand,<br />

we did not drop any tags <strong>in</strong> the peritoneal cavity and, s<strong>in</strong>ce<br />

each pair <strong>of</strong> tags is attached to a thread, we th<strong>in</strong>k that the<br />

possibility <strong>of</strong> dropp<strong>in</strong>g one <strong>in</strong> the peritoneum is extremely<br />

low.<br />

The possibility <strong>of</strong> an <strong>in</strong>advertent <strong>in</strong>jury <strong>of</strong> organs and<br />

structures outside <strong>of</strong> the gut wall has been described as a<br />

possible limitation <strong>of</strong> T-tag based systems [30,31] . Sumiyama<br />

et al [22] produced 12 gastric perforations <strong>in</strong> 6 pigs that<br />

were closed with 48 tissue anchor sets and three <strong>of</strong> the 24<br />

used <strong>in</strong> the anterior gastric wall (12.5%) penetrated surround<strong>in</strong>g<br />

organs (2 penetrated the liver and 1 the anterior<br />

abdom<strong>in</strong>al wall). However, as mentioned above, with the<br />

new brace-bar prototype the depth <strong>of</strong> the needle <strong>in</strong>sertion<br />

is limited to 20 mm and this fact was crucial <strong>in</strong> the low<br />

<strong>in</strong>cidence <strong>of</strong> surround<strong>in</strong>g structure <strong>in</strong>juries <strong>in</strong> our series<br />

(1 tag out <strong>of</strong> 21 sets, 4.8%). This was the first case and we<br />

tried to perform an omental patch pull<strong>in</strong>g the omentum<br />

through the <strong>in</strong>cision. Dur<strong>in</strong>g this maneuver, the meso<strong>colon</strong><br />

was probably unsafely moved towards the gastric<br />

wall caus<strong>in</strong>g this complication. In the rema<strong>in</strong><strong>in</strong>g cases <strong>in</strong><br />

which the omental patch was not attempted no lesions<br />

occurred at the adjacent structures. The importance <strong>of</strong><br />

the depth <strong>of</strong> the needle to avoid complications has been<br />

demonstrated very recently by Park et al [32] These authors<br />

performed needle punctures <strong>of</strong> 1-1.5 mm us<strong>in</strong>g a different<br />

anchor-based endoscopic system (the TAS o tissue apposition<br />

system) and they did not have any adjacent organ<br />

penetration with a 100% <strong>of</strong> closure effectiveness.<br />

Although the ex-vivo study <strong>of</strong> Voermans et al [33] suggested<br />

that t-tag based methods do not permit the serosa<br />

to serosa approach and leak<strong>in</strong>g pressure is lower than with<br />

other devices, the surviv<strong>in</strong>g pigs showed a good postoperative<br />

course. This fact could be expla<strong>in</strong>ed because<br />

physiological <strong>in</strong>tralum<strong>in</strong>al pressures are much lower than<br />

pressures obta<strong>in</strong>ed <strong>in</strong> acute burst<strong>in</strong>g tests and, therefore,<br />

might not be a necessary objective test for viscerotomy<br />

closure [34] . From a cl<strong>in</strong>ical standpo<strong>in</strong>t, the critical test for a<br />

WJG|www.wjgnet.com<br />

gastric closure is animal survival without cl<strong>in</strong>ical signs <strong>of</strong><br />

leakage or complications.<br />

Previous studies have demonstrated that peritoneal<br />

contam<strong>in</strong>ation occurs when us<strong>in</strong>g transgastric access. A<br />

conservative <strong>in</strong>terpretation <strong>of</strong> these f<strong>in</strong>d<strong>in</strong>gs is that the<br />

current “aseptic” technique may require further ref<strong>in</strong>ement,<br />

as suggested by Rolanda et al [18] and Ryou et al [35] .<br />

In fact, we had some difficulties <strong>in</strong> completely clean<strong>in</strong>g<br />

some <strong>of</strong> the stomachs and it was very common to notice<br />

residual liquid near the <strong>in</strong>cision. Only one pig did not have<br />

a satisfactory recovery and the necropsy showed the presence<br />

<strong>of</strong> fibr<strong>in</strong> exudates <strong>in</strong> the upper abdomen. Although<br />

the <strong>in</strong>cision site was seen completely sealed at necropsy,<br />

we cannot totally exclude the possibility that an <strong>in</strong>itial<br />

suture failure occurred but it could be also related with a<br />

potential contam<strong>in</strong>ation <strong>of</strong> the abdom<strong>in</strong>al cavity by the<br />

stomach content. Because <strong>in</strong> this case we used only one<br />

brace-bar, we now consider it prudent to use two devices<br />

to ensure a safer closure.<br />

The present study has some limitations: first, the number<br />

<strong>of</strong> cases is low and we did not <strong>in</strong>clude a control group.<br />

Second, the 14 d survival period might be short to evaluate<br />

late complications. F<strong>in</strong>ally, the use <strong>of</strong> complementary<br />

clips <strong>in</strong> two cases might modify the results <strong>of</strong> the study.<br />

In conclusion, the use <strong>of</strong> a brace-bar <strong>in</strong> a gastric porc<strong>in</strong>e<br />

model is easy, fast, and reproducible after a short<br />

learn<strong>in</strong>g curve and permits the use <strong>of</strong> a s<strong>in</strong>gle channel<br />

endoscope. We believe this tissue anchor<strong>in</strong>g system holds<br />

tremendous potential as a sutur<strong>in</strong>g method for both iatrogenic<br />

and <strong>in</strong>tentional perforations <strong>of</strong> the gastric wall. Unfortunately,<br />

it is still far from a safe application <strong>in</strong> humans.<br />

Further studies and more technological improvements are<br />

still mandatory before expand<strong>in</strong>g its use to humans.<br />

ACKNOWLEDGMENTS<br />

Dr. Fernández-Esparrach thanks the Generalitat de Catalunya<br />

(AGAUR, BE-100022) and the Societat Catalana de<br />

Digestologia for support<strong>in</strong>g her tra<strong>in</strong><strong>in</strong>g and research <strong>in</strong><br />

NOTES. The authors would like to thank Olympus Europe<br />

(Hamburg, Germany) for provid<strong>in</strong>g with endoscopic<br />

closure devices for free for this study.<br />

COMMENTS<br />

Background<br />

Natural orifice translum<strong>in</strong>al endoscopic surgery (NOTES) has changed the approach<br />

to the peritoneum <strong>in</strong> the last few years. Secure closure <strong>of</strong> the gastrotomy<br />

access is one <strong>of</strong> the most important issues for the development <strong>of</strong> NOTES.<br />

However, most new sutur<strong>in</strong>g devices are time consum<strong>in</strong>g and <strong>of</strong>ten difficult to<br />

implement endoscopically.<br />

Research frontiers<br />

T-tag based systems have already been used with variable results. The possibility<br />

<strong>of</strong> an <strong>in</strong>advertent <strong>in</strong>jury <strong>of</strong> organs and structures outside <strong>of</strong> the gut wall<br />

has been described as a possible limitation <strong>of</strong> these devices. In this study, the<br />

authors demonstrate the usefulness and safety <strong>of</strong> a new tissue-anchor<strong>in</strong>gdevice<br />

prototype.<br />

Innovations and breakthroughs<br />

One <strong>of</strong> the advantages <strong>of</strong> this device (and the ma<strong>in</strong> difference with the former<br />

prototype) is that it can be used with a s<strong>in</strong>gle channel endoscope. On the other<br />

hand, the same needle catheter is used for releas<strong>in</strong>g the tags and tighten<strong>in</strong>g<br />

1736 April 7, 2011|Volume 17|Issue 13|

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them later without need for a different forceps grasper, and this makes the procedure<br />

shorter. Furthermore, the depth <strong>of</strong> the needle <strong>in</strong>sertion is limited to 20<br />

mm and this might decrease the risk <strong>of</strong> complications.<br />

Applications<br />

Because the use <strong>of</strong> the brace-bar is easy, fast, and reproducible after a short<br />

learn<strong>in</strong>g curve, we believe this tissue anchor<strong>in</strong>g system holds tremendous potential<br />

as a sutur<strong>in</strong>g method for both iatrogenic and <strong>in</strong>tentional perforations <strong>of</strong><br />

the gastric wall.<br />

Term<strong>in</strong>ology<br />

Natural orifice translum<strong>in</strong>al endoscopic surgery permits access to the peritoneal<br />

organs without the need <strong>of</strong> sk<strong>in</strong> <strong>in</strong>cisions. Tissue-anchor<strong>in</strong>g devices are endoscopic<br />

sutur<strong>in</strong>g devices based on a nylon thread and a small tag at the distal<br />

end that are deployed with<strong>in</strong> the gastric wall. When two or more <strong>of</strong> them are<br />

tight together, the marg<strong>in</strong>s <strong>of</strong> the <strong>in</strong>cision approach and the <strong>in</strong>cision is sealed.<br />

Peer review<br />

This is well written and succ<strong>in</strong>ct with appropriate <strong>in</strong>terpretation and caution <strong>in</strong><br />

the discussion.<br />

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31 Kantsevoy SV. Endoscopic full-thickness resection: new<br />

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natural orifice translum<strong>in</strong>al endoscopic surgery access gastrotomy<br />

compared with open surgical closure <strong>in</strong> a porc<strong>in</strong>e<br />

model. A randomized, multicenter controlled trial. Endoscopy<br />

2010; 42: 311-317<br />

33 Voermans RP, Worm AM, van Berge Henegouwen MI,<br />

Breedveld P, Bemelman WA, Fockens P. In vitro comparison<br />

and evaluation <strong>of</strong> seven gastric closure modalities for<br />

WJG|www.wjgnet.com<br />

natural orifice translum<strong>in</strong>al endoscopic surgery (NOTES).<br />

Endoscopy 2008; 40: 595-601<br />

34 Trunzo JA, Schomisch SJ, Mishra T, Andrews J, Ponsky JL,<br />

Marks JM. The validity <strong>of</strong> bust<strong>in</strong>g pressure as an objective<br />

measure <strong>in</strong> Natural Orifice Translumenal Endsocopic Surgery<br />

(NOTES) closure test<strong>in</strong>g. Gastro<strong>in</strong>test Endosc 2009; 69:<br />

AB169<br />

35 Ryou M, Fong DG, Pai RD, Tavakkolizadeh A, Rattner<br />

DW, Thompson CC. Dual-port distal pancreatectomy us<strong>in</strong>g<br />

a prototype endoscope and endoscopic stapler: a natural<br />

orifice translum<strong>in</strong>al endoscopic surgery (NOTES) survival<br />

study <strong>in</strong> a porc<strong>in</strong>e model. Endoscopy 2007; 39: 881-887<br />

S- Editor Tian L L- Editor O’Neill M E- Editor Ma WH<br />

1738 April 7, 2011|Volume 17|Issue 13|

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Onl<strong>in</strong>e Submissions: http://www.wjgnet.com/1007-9327<strong>of</strong>fice<br />

wjg@wjgnet.com<br />

doi:10.3748/wjg.v17.i13.1739<br />

MR-arterioportography: A new technical approach for<br />

detection <strong>of</strong> liver lesions<br />

BRIEF ARTICLE<br />

Jan<strong>in</strong>e Rennert, Ernst-Michael Jung, Andreas G Schreyer, Patrick H<strong>of</strong>fstetter, Peter Heiss, Stefan Feuerbach,<br />

Niels Zorger<br />

Jan<strong>in</strong>e Rennert, Ernst-Michael Jung, Andreas G Schreyer,<br />

Patrick H<strong>of</strong>fstetter, Peter Heiss, Stefan Feuerbach, Niels<br />

Zorger, Department <strong>of</strong> Radiology, Regensburg University School<br />

<strong>of</strong> Medic<strong>in</strong>e, D-93053 Regensburg, Germany<br />

Author contributions: Rennert J, Schreyer AG and Zorger N<br />

evaluated the majority <strong>of</strong> the data acquired; Rennert J also wrote<br />

the manuscript; Jung EM, H<strong>of</strong>fstetter P, Feuerbach S and Heiss<br />

P were <strong>in</strong>volved <strong>in</strong> adapt<strong>in</strong>g the study design, patient contact and<br />

correction <strong>of</strong> the manuscript.<br />

Correspondence to: Jan<strong>in</strong>e Rennert, MD, Department <strong>of</strong> Radiology,<br />

Regensburg University School <strong>of</strong> Medic<strong>in</strong>e, Franz-Josef-<br />

Strauss-Allee 11, D-93053 Regensburg,<br />

Germany. jarennert@yahoo.de<br />

Telephone: +49-941-9447401 Fax: +49-941-9447402<br />

Received: September 14, 2010 Revised: January 17, 2011<br />

Accepted: January 22,2011<br />

Published onl<strong>in</strong>e: April 7, 2011<br />

Abstract<br />

AIM: To evaluate the benefit and effectiveness <strong>of</strong> MRarterioportography<br />

(MR-AP) to achieve the highest<br />

sensitivity for detection and evaluation <strong>of</strong> hepatocellular<br />

carc<strong>in</strong>oma (HCC).<br />

METHODS: Twenty liver cirrhosis patients with suspected<br />

HCC were <strong>in</strong>cluded before transarterial chemoembolization.<br />

In all patients double-enhanced Magnetic<br />

resonance imag<strong>in</strong>g (MRI) was performed. A bolus <strong>of</strong><br />

10 mL Magnevist ® was <strong>in</strong>jected through a selectively<br />

placed catheter <strong>in</strong> the superior mesenteric artery and<br />

MRI <strong>of</strong> the liver was performed <strong>in</strong> arterioportographic<br />

phase. Two <strong>in</strong>dependent readers evaluated number, size<br />

and localization <strong>of</strong> detected lesions. Diagnostic quality<br />

was determ<strong>in</strong>ed us<strong>in</strong>g a 4-po<strong>in</strong>t scale. Differences were<br />

analyzed for significance us<strong>in</strong>g a t -test. Interobserver<br />

variability was calculated.<br />

RESULTS: In all 20 patients (100%), MR-AP was feasible.<br />

Diagnostic quality was, <strong>in</strong> all cases, between 1 and<br />

WJG|www.wjgnet.com<br />

1739<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): 1739-1745<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

2 for both modalities and readers. MR-AP detected significantly<br />

more lesions than double-enhanced MRI (102.5<br />

vs 61, respectively, P < 0.0024). The <strong>in</strong>ter-observer<br />

variability was 0.881 for MRI and 0.903 for MR-AP.<br />

CONCLUSION: Our study confirmed that the MR-AP as<br />

an additional modality for detection <strong>of</strong> HCC is beneficial,<br />

as significantly more lesions were detected compared to<br />

MRI with liver-specific contrast.<br />

© 2011 Baishideng. All rights reserved.<br />

Key words: MR-arterioportography; Magnetic resonance<br />

imag<strong>in</strong>g; Hepatocellular carc<strong>in</strong>oma; Liver lesions<br />

Peer reviewers: Anuj Mishra, MD, Pr<strong>of</strong>essor, Department <strong>of</strong><br />

Radiology, National Organ Transplant Program, Central Hospital,<br />

Tripoli, PO Box 7913, Libya; Sri P Misra, Pr<strong>of</strong>essor, <strong>Gastroenterology</strong>,<br />

Moti Lal Nehru Medical College, Allahabad 211001, India<br />

Rennert J, Jung EM, Schreyer AG, H<strong>of</strong>fstetter P, Heiss P, Feuerbach<br />

S, Zorger N. MR-arterioportography: A new technical<br />

approach for detection <strong>of</strong> liver lesions. <strong>World</strong> J Gastroenterol<br />

2011; 17(13): 1739-1745 Available from: URL: http://www.wjgnet.com/1007-9327/full/v17/i13/1739.htm<br />

DOI: http://dx.doi.<br />

org/10.3748/wjg.v17.i13.1739<br />

INTRODUCTION<br />

Hepatocellular carc<strong>in</strong>oma (HCC) is the most common<br />

primary malignant tumor <strong>of</strong> the liver and <strong>of</strong>ten develops<br />

<strong>in</strong> patients with underly<strong>in</strong>g liver cirrhosis due to excessive<br />

alcohol <strong>in</strong>take, chronic hepatitis or primary biliary<br />

cirrhosis.<br />

In the treatment <strong>of</strong> hepatocellular carc<strong>in</strong>oma, surgical<br />

resection is considered the only potentially curative therapy.<br />

However, technical improvements <strong>in</strong> hepatic surgery<br />

have extended the <strong>in</strong>dications for surgery remarkably, and<br />

also regional therapeutic procedures such as transcatheter<br />

April 7, 2011|Volume 17|Issue 13|

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Rennert J et al . MR-AP-a new method for detection <strong>of</strong> liver lesions<br />

arterial chemoembolization (TACE) [1-3] and radi<strong>of</strong>requency<br />

ablation (RFA) [1,4,5] have proved to be very successful. A<br />

prolonged time <strong>of</strong> survival follow<strong>in</strong>g diagnosis is noted.<br />

Therefore, the pre-operative or pre-<strong>in</strong>terventional workup<br />

<strong>of</strong> patients with suspected liver malignancy is even more<br />

important, especially concern<strong>in</strong>g the evaluation and characterization<br />

<strong>of</strong> focal or diffuse lesions <strong>in</strong> the cirrhotic<br />

liver. Magnetic resonance imag<strong>in</strong>g (MRI) has been used<br />

to improve identification <strong>of</strong> focal hepatic masses <strong>in</strong> a cirrhotic<br />

liver.<br />

Dynamic MRI after a bolus <strong>in</strong>jection <strong>of</strong> gadopentetate<br />

dimeglum<strong>in</strong>e has been accepted as a valuable method<br />

for the detection and characterization <strong>of</strong> liver tumors [6-9] .<br />

Studies have shown that superparamagnetic iron oxideenhanced<br />

magnetic resonance imag<strong>in</strong>g (SPIO-MRI) <strong>in</strong>creases<br />

sensitivity [4,10] .<br />

In order to determ<strong>in</strong>e the treatment <strong>of</strong> choice for HCC,<br />

studies have shown that exam<strong>in</strong>ations by both computed<br />

tomography angiography (CTA) and computed tomography<br />

arterioportography (CT-AP) are <strong>in</strong>dispensable<br />

because <strong>of</strong> the high sensitivity <strong>of</strong> CT-AP <strong>in</strong> detect<strong>in</strong>g<br />

hepatic lesions and the capability <strong>of</strong> CTA to characterize<br />

them [11,12] . However, <strong>in</strong> contrast to its high sensitivity <strong>in</strong><br />

detect<strong>in</strong>g lesions, the specificity <strong>of</strong> CT-AP for characteriz<strong>in</strong>g<br />

<strong>in</strong>trahepatic lesions is low. Tumor-mimick<strong>in</strong>g benign<br />

perfusion abnormalities and benign lesions (e.g. hemangiomas,<br />

arterio-venous shunts) have led to a reported <strong>in</strong>cidence<br />

<strong>of</strong> false-positive lesions between 9% and 63% <strong>in</strong><br />

primary and secondary liver lesions [13,14] .<br />

Despite advances <strong>in</strong> CT or MRI, ultrasound (US) with<br />

or without application <strong>of</strong> contrast agents also plays a key<br />

role <strong>in</strong> the diagnostic algorithm <strong>of</strong> HCC due to its low<br />

cost, availability and non-<strong>in</strong>vasiveness.<br />

Until now, there have hardly been any studies compar<strong>in</strong>g<br />

the effectiveness <strong>of</strong> MR-arterioportography (MR-AP)<br />

and contrast-enhanced MRI for diagnosis <strong>of</strong> malignant<br />

liver lesions. Thus, the purpose <strong>of</strong> this study was to comb<strong>in</strong>e<br />

the advantages <strong>of</strong> modern contrast-enhanced MRI<br />

with the technique <strong>of</strong> arterioportography to achieve the<br />

highest sensitivity for diagnosis <strong>of</strong> malignant liver lesions<br />

<strong>in</strong> patients suffer<strong>in</strong>g from HCC.<br />

MATERIALS AND METHODS<br />

Patients<br />

Approval for this study was obta<strong>in</strong>ed from the <strong>in</strong>stitutional<br />

review board <strong>in</strong> conformity with the Declaration<br />

<strong>of</strong> Hels<strong>in</strong>ki. Before the procedures were conducted,<br />

written <strong>in</strong>formed consent was obta<strong>in</strong>ed from each patient<br />

for MRI, MR-AP and angiography after the nature<br />

<strong>of</strong> the procedure was fully expla<strong>in</strong>ed.<br />

Dur<strong>in</strong>g the period from February 2005 to September<br />

2007, 20 patients [18 men, 2 women, age range from 47 to<br />

76 years (mean age, 62 years)] with symptoms suggestive<br />

<strong>of</strong> primary malignant hepatic tumors were referred to our<br />

department. As HCC is commonly associated with liver<br />

cirrhosis, all patients had deteriorated liver but a tolerable<br />

renal function, and the cardiovascular status was stable.<br />

Twelve <strong>of</strong> our 20 patients suffered from alcohol toxic<br />

WJG|www.wjgnet.com<br />

liver cirrhosis. In 4 out <strong>of</strong> 20 patients the underly<strong>in</strong>g disease<br />

was chronic hepatitis (2 patients with chronic hepatitis<br />

B, 2 patients with chronic hepatitis C). In 4 out <strong>of</strong> 20<br />

patients, the fundamental disease could not be elicited.<br />

Concern<strong>in</strong>g the severity <strong>of</strong> cirrhosis, 8 out <strong>of</strong> 20 patients<br />

were classified as Child Pugh score A, 7 out <strong>of</strong> 20 patients<br />

as Child Pugh score B and 5 out <strong>of</strong> 20 patients as Child<br />

Pugh score C.<br />

The existence <strong>of</strong> malignant hepatic tumors was confirmed<br />

us<strong>in</strong>g multislice MRI and CT. MR-AP was performed<br />

to evaluate the tumor extent <strong>in</strong> order to suggest<br />

<strong>in</strong>terventional therapy, surgery or chemotherapy.<br />

Diagnosis <strong>of</strong> HCC was histologically confirmed <strong>in</strong> 15<br />

out <strong>of</strong> 19 patients. In one patient (patient No. 16), existence<br />

<strong>of</strong> a malignant hepatic lesion was excluded histologically<br />

follow<strong>in</strong>g liver transplantation.<br />

In 13 out <strong>of</strong> 20 patients the a-fetoprote<strong>in</strong> (AFP)<br />

level was elevated, rang<strong>in</strong>g from 16.4-2513 ng/mL (mean<br />

488.1 ng/mL). In 7 patients (<strong>in</strong>clud<strong>in</strong>g patient No. 16) AFP<br />

levels were with<strong>in</strong> a normal range.<br />

Imag<strong>in</strong>g procedures<br />

Angiography: Before TACE and for MR-arterioportography,<br />

the femoral common artery was punctured<br />

under local anesthesia us<strong>in</strong>g the Seld<strong>in</strong>ger technique and<br />

a 5-French angiographic catheter (Cobra, Cook Medical,<br />

USA) was positioned <strong>in</strong> the proximal superior mesenteric<br />

artery. A diagnostic angiography was performed to visualize<br />

the portal ve<strong>in</strong> and to exclude shunts which could<br />

<strong>in</strong>volve contrast<strong>in</strong>g via the portal ve<strong>in</strong>.<br />

MRI: MRI was performed on a 1.5-T whole-body scanner<br />

(Magnetom Sonata, Siemens Medical Solutions, Germany)<br />

equipped with a high-performance gradient (Quantum)<br />

system (maximum gradient strength, 30 mT/m; slew<br />

rate, 125 T/ms). A comb<strong>in</strong>ation <strong>of</strong> the standard body<br />

phased-array coil with sp<strong>in</strong>e array coils was used for signal<br />

reception.<br />

MR-AP standard protocol (Table 1): For MR-AP, 10 mL<br />

gadopentetate dimeglum<strong>in</strong>e was <strong>in</strong>jected through the catheter<br />

placed <strong>in</strong> the superior mesenteric artery at a rate <strong>of</strong><br />

2 mL/s with a power <strong>in</strong>jector (Medrad Spectris MR Injector,<br />

USA).<br />

MRI standard protocol (Table 2): For MRI, 0.2 mmol/<br />

kg body weight gadopentetate dimeglum<strong>in</strong>e was <strong>in</strong>jected <strong>in</strong>travenously<br />

at a rate <strong>of</strong> 2 mL/s with a power <strong>in</strong>jector (Medrad<br />

Spectris MR Injector). T1-weighted VIBE transversal<br />

Dynamic scans were acquired 20, 40, and 120 s after application<br />

<strong>of</strong> gadopentetate dimeglum<strong>in</strong>e. T2-star-weighted<br />

Flash 2D scans and T2-weighted TSE FS scans were obta<strong>in</strong>ed<br />

after application <strong>of</strong> 1.4 mL Ferucarbotran (Resovist ® ,<br />

Bayer Scher<strong>in</strong>g Pharma AG, Germany).<br />

Image analysis<br />

In the retrospective review<strong>in</strong>g procedure, all images <strong>of</strong> each<br />

technique were <strong>in</strong>terpreted and evaluated <strong>in</strong>dependently<br />

by two observers with great experience <strong>in</strong> abdom<strong>in</strong>al MRI.<br />

1740 April 7, 2011|Volume 17|Issue 13|

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Table 1 MR-arterioportography standard protocol<br />

Scans Plane TE TR Flip angle<br />

Unenhanced<br />

T2-weighted TRUFI Coronal 1.9 3.8 71°<br />

T2-weighted TRUFI Transversal 1.88 3.76 71°<br />

T1-weighted VIBE<br />

Enhanced<br />

Transversal 2.02 4.78 10°<br />

T1-weighted FLASH FS CE Transversal 4.76 123 70°<br />

T1-weighted VIBE dynamic 1<br />

Transversal 2.02 4.78 10°<br />

1 Dynamic scans were started immediately follow<strong>in</strong>g application <strong>of</strong> 10 mL<br />

Gd-DTPA. MRI: Magnetic resonance imag<strong>in</strong>g.<br />

Table 2 Magnetic resonance imag<strong>in</strong>g standard protocol<br />

Scans Plane TE TR Flip angle<br />

Unenhanced<br />

T1-weighted VIBE Transversal 1.59 4.37 10°<br />

T2-star-weighted FLASH 2D Transversal 10.00 169.00 90°<br />

T2-weighted TSE FS Transversal 105.00 2740.00 170°<br />

T1-weighted FLASH opp Transversal 2.71 100.00 70°<br />

T1-weighted FLASH <strong>in</strong> Transversal 4.76 87.00 60°<br />

T2-weighted HASTE Transversal 85.00 1000.00 150°<br />

T2-weighted TRUFI Coronal 1.83 3.65 71°<br />

T1-weighted VIBE<br />

Enhanced<br />

Transversal 1.55 4.81 10°<br />

T1-weighted VIBE dynamic 1 Transversal 1.55 4.81 10°<br />

T1-weighted FLASH FS Transversal 4.76 123.00 70°<br />

T1-weighted FLASH FS Coronal 4.76 94.00 70°<br />

T2-star-weighted FLASH 2 2 Transversal 10.00 169.00 90°<br />

T2-weighted TSH FS 2<br />

Transversal 105.00 2740.00 170°<br />

1 Dynamic scans were started immediately follow<strong>in</strong>g application <strong>of</strong> Gd-<br />

DTPA (0.2 mmol/kg); 2 Follow<strong>in</strong>g application <strong>of</strong> 1.4 mL Resovist.<br />

No cl<strong>in</strong>ical <strong>in</strong>formation or patient diagnosis was given to<br />

the observers. The images from each technique were <strong>in</strong>terpreted<br />

<strong>in</strong> separate sessions <strong>in</strong> a randomized sequence. In<br />

the first session, the two observers reviewed a set <strong>of</strong> images<br />

that <strong>in</strong>cluded both unenhanced and gadopentetate dimeglum<strong>in</strong>e-enhanced,<br />

as well as Resovist-enhanced, images.<br />

In the second session, each observer reviewed a set <strong>of</strong><br />

images (MR-AP set) that <strong>in</strong>cluded gadopentetate dimeglum<strong>in</strong>e-enhanced<br />

images after <strong>in</strong>jection via the superior<br />

mesenteric artery.<br />

For characterization <strong>of</strong> liver lesions, all images <strong>of</strong><br />

each exam<strong>in</strong>ation were reviewed together us<strong>in</strong>g all the<br />

sequences available. Each observer recorded the number<br />

<strong>of</strong> suspected lesions noted, their size, and the segmental<br />

location. Furthermore, the image quality <strong>of</strong> MR and<br />

MR-arterioportography was documented on a four po<strong>in</strong>t<br />

scale: 1-excellent, 2-m<strong>in</strong>or diagnostic limitations, 3-major<br />

diagnostic limitations, 4-non-diagnostic.<br />

Statistical analysis<br />

Statistical s<strong>of</strong>tware (SPSS, version 14, Chicago, USA) was<br />

used for statistical analysis. We evaluated the differences<br />

with regard to number <strong>of</strong> lesions found us<strong>in</strong>g MR-arterioportography<br />

and MRI. Furthermore, the <strong>in</strong>ter-observer<br />

differences <strong>in</strong> evaluation <strong>of</strong> MR-arterioportography<br />

and double-enhanced MRI were analyzed. Paired-samples<br />

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Rennert J et al . MR-AP-a new method for detection <strong>of</strong> liver lesions<br />

Table 3 Number <strong>of</strong> lesions detected <strong>in</strong> MR-arterioportography<br />

and magnetic resonance imag<strong>in</strong>g<br />

Patient Age (yr) MR-AP<br />

number <strong>of</strong> lesions<br />

∑102/103<br />

t tests and χ 2 test were used to compare. In pairedsamples<br />

t tests, P < 0.05 <strong>in</strong>dicated a statistically significant<br />

difference.<br />

RESULTS<br />

Double-enhanced MRI<br />

number <strong>of</strong> lesions<br />

∑60/56<br />

Reader 1 Reader 2 Reader 1 Reader 2<br />

1 62 9 9 5 4<br />

2 47 7 8 2 2<br />

3 70 4 5 2 2<br />

4 74 7 8 4 3<br />

5 51 3 3 3 3<br />

6 76 1 2 1 1<br />

7 70 6 6 9 8<br />

8 1<br />

63 - - (7) (7)<br />

9 70 4 4 2 2<br />

10 63 9 8 2 2<br />

11 51 9 8 6 5<br />

12 63 10 11 3 2<br />

13 57 6 6 2 2<br />

14 67 5 4 4 5<br />

15 51 4 3 3 2<br />

16 53 0 0 1 1<br />

17 51 2 2 2 2<br />

18 54 5 6 3 3<br />

19 57 9 8 4 4<br />

20 73 2 2 3 3<br />

1 Patient No. 8 was excluded from the evaluation for diffuse <strong>in</strong>filtration <strong>of</strong><br />

virtually all liver segments. P = 0.0024 (relation <strong>of</strong> the number <strong>of</strong> lesions<br />

detected <strong>in</strong> MR-AP and MRI). MR-AP: MR-arterioportography; MRI: Magnetic<br />

resonance imag<strong>in</strong>g.<br />

Twenty patients with liver cirrhosis underwent comb<strong>in</strong>ed<br />

MR-arterioportography and SPIO-MRI exam<strong>in</strong>ations.<br />

No adverse reactions were experienced by any <strong>of</strong> the patients<br />

who received Gd-DTPA and SPIO.<br />

In all 20 patients (100%), MR-arterioportography was<br />

feasible. The image quality <strong>in</strong> both modalities (MR-AP<br />

and MRI) was excellent (1-2 <strong>in</strong> MR-arterioportography,<br />

SD: 0.4865; 1-2 <strong>in</strong> MR, SD: 0.4292). In patient No. 8, a<br />

MR-AP evaluation was not suitable due to a diffuse <strong>in</strong>filtration<br />

<strong>of</strong> virtually all liver segments, thus the patient was<br />

excluded. Altogether, 102.5 hepatic lesions were detected<br />

us<strong>in</strong>g MR-AP, whereas only 61 lesions could be detected<br />

us<strong>in</strong>g MRI (Table 3). This difference is considered to be<br />

statistically significant (P < 0.0024).<br />

The kappa analyses <strong>of</strong> two observers regard<strong>in</strong>g the<br />

number <strong>of</strong> lesions detected by both modalities showed substantial<br />

to excellent agreement (κ = 0.903, 95% CI: 0.844 to<br />

0.962 for MR-AP; and κ = 0.881, 95% CI: 0.795 to 0.966<br />

for MRI). In particular, κ values with MR-AP imag<strong>in</strong>g <strong>in</strong>dicated<br />

excellent agreement.<br />

The lesions found <strong>in</strong> all patients ranged <strong>in</strong> size from<br />

7-120 mm (mean 28.24 mm) for MRI and from 4-120 mm<br />

(mean 24.62 mm) for MR-AP. This difference is not con-<br />

1741 April 7, 2011|Volume 17|Issue 13|

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Rennert J et al . MR-AP-a new method for detection <strong>of</strong> liver lesions<br />

Term<strong>in</strong>ology<br />

MR-AP is an MRI procedure where the contrast agent is <strong>in</strong>jected through a<br />

catheter placed <strong>in</strong> the superior mesenteric artery.<br />

Peer review<br />

It is well-written and is novel work.<br />

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7 Ito K, Honjo K, Fujita T, Matsui M, Awaya H, Matsumoto T,<br />

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9 Seki T, Tamai T, Nakagawa T, Imamura M, Nishimura A,<br />

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Cancer 2000; 89: 1245-1251<br />

10 Senéterre E, Taourel P, Bouvier Y, Pradel J, Van Beers B,<br />

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metastases: ferumoxides-enhanced MR imag<strong>in</strong>g versus<br />

unenhanced MR imag<strong>in</strong>g and CT dur<strong>in</strong>g arterial portography.<br />

Radiology 1996; 200: 785-792<br />

11 Kim SR, Ando K, Mita K, Fuki S, Ikawa H, Kanbara Y, Imoto<br />

S, Matsuoka T, Hayashi Y, Kudo M. Superiority <strong>of</strong> CT arterioportal<br />

angiography to contrast-enhanced CT and MRI <strong>in</strong><br />

the diagnosis <strong>of</strong> hepatocellular carc<strong>in</strong>oma <strong>in</strong> nodules smaller<br />

than 2 cm. Oncology 2007; 72 Suppl 1: 58-66<br />

12 Kim SR, Imoto S, Ikawa H, Ando K, Mita K, Fuki S, Sakamoto<br />

M, Kanbara Y, Matsuoka T, Kudo M, Hayashi Y. Wellto<br />

moderately-differentiated HCC manifest<strong>in</strong>g hyperattenuation<br />

on both CT dur<strong>in</strong>g arteriography and arterial portography.<br />

<strong>World</strong> J Gastroenterol 2007; 13: 5775-5778<br />

13 Kondo H, Kanematsu M, Hoshi H, Murakami T, Kim T,<br />

Hori M, Matsuo M, Nakamura H. Preoperative detection <strong>of</strong><br />

malignant hepatic tumors: comparison <strong>of</strong> comb<strong>in</strong>ed methods<br />

<strong>of</strong> MR imag<strong>in</strong>g with comb<strong>in</strong>ed methods <strong>of</strong> CT. AJR Am<br />

J Roentgenol 2000; 174: 947-954<br />

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14 Soyer P, Bluemke DA, Hruban RH, Sitzmann JV, Fishman<br />

EK. Hepatic metastases from colorectal cancer: detection<br />

and false-positive f<strong>in</strong>d<strong>in</strong>gs with helical CT dur<strong>in</strong>g arterial<br />

portography. Radiology 1994; 193: 71-74<br />

15 Kanematsu M, Hoshi H, Murakami T, Inaba Y, Kim T,<br />

Yamada T, Kato M, Yokoyama R, Nakamura H. Detection <strong>of</strong><br />

hepatocellular carc<strong>in</strong>oma <strong>in</strong> patients with cirrhosis: MR imag<strong>in</strong>g<br />

versus angiographically assisted helical CT. AJR Am J<br />

Roentgenol 1997; 169: 1507-1515<br />

16 Noguchi Y, Murakami T, Kim T, Hori M, Osuga K, Kawata<br />

S, Kumano S, Okada A, Sugiura T, Nakamura H. Detection<br />

<strong>of</strong> hepatocellular carc<strong>in</strong>oma: comparison <strong>of</strong> dynamic MR<br />

imag<strong>in</strong>g with dynamic double arterial phase helical CT. AJR<br />

Am J Roentgenol 2003; 180: 455-460<br />

17 Semelka RC, Cance WG, Marcos HB, Mauro MA. Liver<br />

metastases: comparison <strong>of</strong> current MR techniques and spiral<br />

CT dur<strong>in</strong>g arterial portography for detection <strong>in</strong> 20 surgically<br />

staged cases. Radiology 1999; 213: 86-91<br />

18 Tang Y, Yamashita Y, Arakawa A, Namimoto T, Mitsuzaki<br />

K, Abe Y, Katahira K, Takahashi M. Detection <strong>of</strong> hepatocellular<br />

carc<strong>in</strong>oma aris<strong>in</strong>g <strong>in</strong> cirrhotic livers: comparison <strong>of</strong><br />

gadol<strong>in</strong>ium- and ferumoxides-enhanced MR imag<strong>in</strong>g. AJR<br />

Am J Roentgenol 1999; 172: 1547-1554<br />

19 Yamamoto H, Yamashita Y, Yoshimatsu S, Baba Y, Hatanaka<br />

Y, Murakami R, Nishiharu T, Takahashi M, Higashida<br />

Y, Moribe N. Hepatocellular carc<strong>in</strong>oma <strong>in</strong> cirrhotic livers:<br />

detection with unenhanced and iron oxide-enhanced MR<br />

imag<strong>in</strong>g. Radiology 1995; 195: 106-112<br />

20 Bell<strong>in</strong> MF, Zaim S, Auberton E, Sarfati G, Duron JJ, Khayat<br />

D, Grellet J. Liver metastases: safety and efficacy <strong>of</strong> detection<br />

with superparamagnetic iron oxide <strong>in</strong> MR imag<strong>in</strong>g.<br />

Radiology 1994; 193: 657-663<br />

21 Fretz CJ, Elizondo G, Weissleder R, Hahn PF, Stark DD,<br />

Ferrucci JT Jr. Superparamagnetic iron oxide-enhanced MR<br />

imag<strong>in</strong>g: pulse sequence optimization for detection <strong>of</strong> liver<br />

cancer. Radiology 1989; 172: 393-397<br />

22 Hagspiel KD, Neidl KF, Eichenberger AC, Weder W, Mar<strong>in</strong>cek<br />

B. Detection <strong>of</strong> liver metastases: comparison <strong>of</strong> superparamagnetic<br />

iron oxide-enhanced and unenhanced MR imag<strong>in</strong>g<br />

at 1.5 T with dynamic CT, <strong>in</strong>traoperative US, and percutaneous<br />

US. Radiology 1995; 196: 471-478<br />

23 Ward J, Naik KS, Guthrie JA, Wilson D, Rob<strong>in</strong>son PJ. Hepatic<br />

lesion detection: comparison <strong>of</strong> MR imag<strong>in</strong>g after the adm<strong>in</strong>istration<br />

<strong>of</strong> superparamagnetic iron oxide with dual-phase<br />

CT by us<strong>in</strong>g alternative-free response receiver operat<strong>in</strong>g<br />

characteristic analysis. Radiology 1999; 210: 459-466<br />

24 Choi D, Kim S, Lim J, Lee W, Jang H, Lee S, Lim H. Preop<br />

erative detection <strong>of</strong> hepatocellular carc<strong>in</strong>oma: ferumoxidesenhanced<br />

mr imag<strong>in</strong>g versus comb<strong>in</strong>ed helical CT dur<strong>in</strong>g<br />

arterial portography and CT hepatic arteriography. AJR Am J<br />

Roentgenol 2001; 176: 475-482<br />

25 Murakami T, Oi H, Hori M, Kim T, Takahashi S, Tomoda K,<br />

Narumi Y, Nakamura H. Helical CT dur<strong>in</strong>g arterial portography<br />

and hepatic arteriography for detect<strong>in</strong>g hypervascular hepatocellular<br />

carc<strong>in</strong>oma. AJR Am J Roentgenol 1997; 169: 131-135<br />

26 Dai Y, Chen MH, Fan ZH, Yan K, Y<strong>in</strong> SS, Zhang XP. Diagnosis<br />

<strong>of</strong> small hepatic nodules detected by surveillance<br />

ultrasound <strong>in</strong> patients with cirrhosis: Comparison between<br />

contrast-enhanced ultrasound and contrast-enhanced helical<br />

computed tomography. Hepatol Res 2008; 38: 281-290<br />

27 Hosch WP, Schmidt SM, Plaza S, Dechow C, Schmidt J, Ley<br />

S, Kauffmann GW, Hansmann J. Comparison <strong>of</strong> CT dur<strong>in</strong>g<br />

arterial portography and MR dur<strong>in</strong>g arterial portography <strong>in</strong><br />

the detection <strong>of</strong> liver metastases. AJR Am J Roentgenol 2006;<br />

186: 1502-1511<br />

28 Yu JS, Kim KW, Lee JT, Yoo HS. MR imag<strong>in</strong>g dur<strong>in</strong>g arterial<br />

portography for assessment <strong>of</strong> hepatocellular carc<strong>in</strong>oma:<br />

comparison with CT dur<strong>in</strong>g arterial portography. AJR Am J<br />

Roentgenol 1998; 170: 1501-1506<br />

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29 Takahara T, Imai Y, Yamashita T, Yasuda S, Nasu S, Van<br />

Cauteren M. Diffusion weighted whole body imag<strong>in</strong>g with<br />

background body signal suppression (DWIBS): technical<br />

improvement us<strong>in</strong>g free breath<strong>in</strong>g, STIR and high resolution<br />

3D display. Radiat Med 2004; 22: 275-282<br />

30 Kim T, Murakami T, Takahashi S, Hori M, Tsuda K, Nakamura<br />

H. Diffusion-weighted s<strong>in</strong>gle-shot echoplanar MR imag<strong>in</strong>g<br />

for liver disease. AJR Am J Roentgenol 1999; 173: 393-398<br />

31 Nasu K, Kuroki Y, Nawano S, Kuroki S, Tsukamoto T,<br />

Yamamoto S, Motoori K, Ueda T. Hepatic metastases: diffu-<br />

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Rennert J et al . MR-AP-a new method for detection <strong>of</strong> liver lesions<br />

sion-weighted sensitivity-encod<strong>in</strong>g versus SPIO-enhanced<br />

MR imag<strong>in</strong>g. Radiology 2006; 239: 122-130<br />

32 Parikh T, Drew SJ, Lee VS, Wong S, Hecht EM, Babb JS,<br />

Taouli B. Focal liver lesion detection and characterization<br />

with diffusion-weighted MR imag<strong>in</strong>g: comparison with standard<br />

breath-hold T2-weighted imag<strong>in</strong>g. Radiology 2008; 246:<br />

812-822<br />

33 Koyama T, Tamai K, Togashi K. Current status <strong>of</strong> body MR<br />

imag<strong>in</strong>g: fast MR imag<strong>in</strong>g and diffusion-weighted imag<strong>in</strong>g.<br />

Int J Cl<strong>in</strong> Oncol 2006; 11: 278-85<br />

S- Editor Sun H L- Editor Logan S E- Editor Ma WH<br />

1745 April 7, 2011|Volume 17|Issue 13|

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Onl<strong>in</strong>e Submissions: http://www.wjgnet.com/1007-9327<strong>of</strong>fice<br />

wjg@wjgnet.com<br />

doi:10.3748/wjg.v17.i13.1746<br />

BRIEF ARTICLE<br />

Carbachol promotes gastro<strong>in</strong>test<strong>in</strong>al function dur<strong>in</strong>g oral<br />

resuscitation <strong>of</strong> burn shock<br />

Sen Hu, J<strong>in</strong>-Wei Che, Yi-Jun Tian, Zhi-Yong Sheng<br />

Sen Hu, J<strong>in</strong>-Wei Che, Yi-Jun Tian, Zhi-Yong Sheng, Research<br />

Laboratory <strong>of</strong> Shock and Multiple Organ Dysfunction,<br />

Burns Institute, First Hospital Affiliated to the PLA General<br />

Hospital, Beij<strong>in</strong>g 100048, Ch<strong>in</strong>a<br />

Author contributions: Hu S, Che JW and Sheng ZY designed<br />

the research; Hu S, Che JW and Tian YJ performed the research;<br />

Hu S and Che JW analyzed the data; Hu S and Sheng ZY wrote<br />

the paper.<br />

Supported by The Special Foundation <strong>of</strong> the 11th five-year<br />

Plan for Military Medical Projects, No. 06Z055<br />

Correspondence to: Sen Hu, MD, PhD, Pr<strong>of</strong>essor, Chief, Research<br />

Laboratory <strong>of</strong> Shock and Multiple Organ Dysfunction,<br />

Burns Institute, First Hospital Affiliated to the PLA General<br />

Hospital, No.51 Fu Cheng Road, Beij<strong>in</strong>g 100048,<br />

Ch<strong>in</strong>a. hs82080@yahoo.com.cn<br />

Telephone: +86-10-66867397 Fax: +86-10-68989139<br />

Received: November 9,2010 Revised: January 11,2011<br />

Accepted: January 18,2011<br />

Published onl<strong>in</strong>e: April 7, 2011<br />

Abstract<br />

AIM: To <strong>in</strong>vestigate the effect <strong>of</strong> carbachol on gastro<strong>in</strong>test<strong>in</strong>al<br />

function <strong>in</strong> a dog model <strong>of</strong> oral resuscitation<br />

for burn shock.<br />

METHODS: Twenty Beagle dogs with <strong>in</strong>tubation <strong>of</strong><br />

the carotid artery, jugular ve<strong>in</strong> and jejunum for 24 h<br />

were subjected to 35% total body surface area fullthickness<br />

burns, and were divided <strong>in</strong>to three groups:<br />

no fluid resuscitation (NR, n = 10), <strong>in</strong> which animals did<br />

not receive fluid by any means <strong>in</strong> the first 24 h postburn;<br />

oral fluid resuscitation (OR, n = 8), <strong>in</strong> which dogs<br />

were gavaged with glucose-electrolyte solution (GES)<br />

with volume and rate consistent with the Parkland<br />

formula; and oral fluid with carbachol group (OR/CAR,<br />

n = 8), <strong>in</strong> which dogs were gavaged with GES conta<strong>in</strong><strong>in</strong>g<br />

carbachol (20 μg/kg), with the same volume and rate as<br />

the OR group. Twenty-four hours after burns, all animals<br />

were given <strong>in</strong>travenous fluid replacement, and 72 h after<br />

<strong>in</strong>jury, they received nutritional support. Hemodynamic<br />

WJG|www.wjgnet.com<br />

1746<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): 1746-1752<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

and gastro<strong>in</strong>test<strong>in</strong>al parameters were measured serially<br />

with animals <strong>in</strong> conscious and cooperative state.<br />

RESULTS: The mean arterial pressure, cardiac<br />

output and plasma volume dropped markedly, and<br />

gastro<strong>in</strong>test<strong>in</strong>al tissue perfusion was reduced obviously<br />

after the burn <strong>in</strong>jury <strong>in</strong> all the three groups.<br />

Hemodynamic parameters and gastro<strong>in</strong>test<strong>in</strong>al<br />

tissue perfusion <strong>in</strong> the OR and OR/CAR groups<br />

were promoted to pre-<strong>in</strong>jury level at 48 and 72 h,<br />

respectively, while hemodynamic parameters <strong>in</strong> the<br />

NR group did not return to pre-<strong>in</strong>jury level till 72 h,<br />

and gastro<strong>in</strong>test<strong>in</strong>al tissue perfusion rema<strong>in</strong>ed lower<br />

than pre-<strong>in</strong>jury level until 120 h post-burn. CO2 <strong>of</strong><br />

the gastric mucosa and <strong>in</strong>test<strong>in</strong>al mucosa blood<br />

flow <strong>of</strong> OR/CAR groups were 56.4 ± 4.7 mmHg and<br />

157.7 ± 17.7 blood perfusion units (BPU) at 24 h postburn,<br />

respectively, which were significantly superior<br />

to those <strong>in</strong> the OR group (65.8 ± 5.8 mmHg and<br />

127.7 ± 11.9 BPU, respectively, all P < 0.05). Gastric<br />

empty<strong>in</strong>g and <strong>in</strong>test<strong>in</strong>al absorption rates <strong>of</strong> GES were<br />

significantly reduced to the lowest level (52.8% and<br />

23.7% <strong>of</strong> pre-<strong>in</strong>jury levels) <strong>in</strong> the OR group at about<br />

2 and 4 h post-burn, and did not return to 80% <strong>of</strong><br />

pre-<strong>in</strong>jury level until 24 h. In the first 24 h postburn,<br />

the rate <strong>of</strong> gastric empty<strong>in</strong>g and <strong>in</strong>test<strong>in</strong>al water<br />

absorption were elevated by a mean 15.7% and 11.5%,<br />

respectively, <strong>in</strong> the OR/CAR group compared with the<br />

OR group. At 5 days, the mortality <strong>in</strong> the NR group was<br />

30% (3/10), 12.5% <strong>in</strong> the OR group (1/8), and none <strong>in</strong><br />

the OR/CAR group.<br />

CONCLUSION: Carbachol had a beneficial effect on<br />

oral resuscitation <strong>of</strong> burn shock by promot<strong>in</strong>g gastric<br />

empty<strong>in</strong>g and <strong>in</strong>test<strong>in</strong>al absorption <strong>in</strong> our can<strong>in</strong>e<br />

model.<br />

© 2011 Baishideng. All rights reserved.<br />

Key words: Burn shock; Fluid therapy; Oral rehydration;<br />

Carbachol; Animal model; Gastric empty<strong>in</strong>g; Intest<strong>in</strong>al<br />

absorption<br />

April 7, 2011|Volume 17|Issue 13|

---

Peer reviewer: Damian Casadesus Rodriguez, MD, PhD,<br />

Calixto Garcia University Hospital, J and University, Vedado,<br />

Havana City, Cuba<br />

Hu S, Che JW, Tian YJ, Sheng ZY. Carbachol promotes gastro<strong>in</strong>test<strong>in</strong>al<br />

function dur<strong>in</strong>g oral resuscitation <strong>of</strong> burn shock. <strong>World</strong><br />

J Gastroenterol 2011; 17(13): 1746-1752 Available from: URL:<br />

http://www.wjgnet.com/1007-9327/full/v17/i13/1746.htm DOI:<br />

http://dx.doi.org/10.3748/wjg.v17.i13.1746<br />

INTRODUCTION<br />

Rapid <strong>in</strong>travenous <strong>in</strong>fusion <strong>of</strong> large quantity <strong>of</strong> fluids<br />

conta<strong>in</strong><strong>in</strong>g electrolytes and colloidal solutions rema<strong>in</strong> the<br />

key measure to resuscitate hypovolemic shock as a result<br />

<strong>of</strong> a massive burn <strong>in</strong>jury. This life-sav<strong>in</strong>g measure has<br />

unanimously been accepted worldwide. It has also been<br />

recognized that a delay <strong>in</strong> such replenishment could sometimes<br />

be fatal due to complications subsequent to delayed<br />

resuscitation <strong>of</strong> hypovolemic shock. Unfortunately, <strong>in</strong> certa<strong>in</strong><br />

cases, such as mass casualties <strong>in</strong> an <strong>in</strong>cendiary bomb<br />

attack <strong>in</strong> battlefields, or a forest or prairie fire <strong>in</strong> regions<br />

with an austere environment with poor medical support<br />

and transportation facilities due to geographical barriers,<br />

not only there would be a shortage <strong>of</strong> medics to <strong>in</strong>troduce<br />

an <strong>in</strong>travenous needle, but also the weight and bulk <strong>of</strong> the<br />

necessary <strong>in</strong>travenous fluids would make the treatment<br />

unrealistic. In such cases, it is our supposition that oral adm<strong>in</strong>istration<br />

<strong>of</strong> fluids might be more practical, and it might<br />

be able to ma<strong>in</strong>ta<strong>in</strong> the life <strong>of</strong> the victims till <strong>in</strong>travenous<br />

fluid replacement was available.<br />

Oral fluid resuscitation has been reported with success<br />

<strong>in</strong> early cl<strong>in</strong>ical studies <strong>of</strong> burn care [1] . Thomas et al [2] have<br />

described oral fluid replacement <strong>in</strong> burn patients, and<br />

have concluded that oral resuscitation may have a slower<br />

<strong>in</strong>itial onset <strong>of</strong> hemodynamic effectiveness, but after 3-4 h,<br />

it can be similarly effective. It is unanimously recognized<br />

that the ma<strong>in</strong> limit<strong>in</strong>g factors for effective oral resuscitation<br />

<strong>of</strong> burn shock are dim<strong>in</strong>ution <strong>of</strong> gastric empty<strong>in</strong>g<br />

capacity and <strong>in</strong>test<strong>in</strong>al absorption <strong>of</strong> fluid and electrolytes<br />

due to underm<strong>in</strong>ed gastro<strong>in</strong>test<strong>in</strong>al perfusion. It seems to<br />

be necessary to overcome these two hurdles before oral<br />

hydration can be successful for treatment <strong>of</strong> burn shock.<br />

As enough data <strong>of</strong> oral resuscitation <strong>of</strong> burn shock<br />

could not be accumulated <strong>in</strong> cl<strong>in</strong>ical practice <strong>in</strong> ord<strong>in</strong>ary<br />

situations, most <strong>in</strong>vestigations have been done with animals<br />

[3,4] . We found that the previous animal experiments<br />

have been limited by their short experimental duration<br />

<strong>of</strong> < 24 h post-burn, and performed under general anesthesia,<br />

which might <strong>in</strong>terfere with the observation <strong>of</strong><br />

gastric empty<strong>in</strong>g and <strong>in</strong>test<strong>in</strong>al absorption <strong>of</strong> fluid and<br />

electrolytes. With these <strong>in</strong> m<strong>in</strong>d, a can<strong>in</strong>e model <strong>of</strong> burn<br />

shock was devised <strong>in</strong> which oral resuscitation was given<br />

<strong>in</strong> the first 24 h post-burn, followed by delayed <strong>in</strong>travenous<br />

fluid replacement, and the whole course <strong>of</strong> the<br />

experiment lasted for 120 h. Also, <strong>in</strong> this experiment, the<br />

effect <strong>of</strong> general anesthesia was elim<strong>in</strong>ated, and carbachol,<br />

which is a chol<strong>in</strong>ergic receptor agonist, was used <strong>in</strong><br />

WJG|www.wjgnet.com<br />

Hu S et al . Carbachol, gastro<strong>in</strong>test<strong>in</strong>al function and burns<br />

an attempt to shorten gastric empty<strong>in</strong>g time and restore<br />

<strong>in</strong>test<strong>in</strong>al peristalsis.<br />

MATERIALS AND METHODS<br />

All the experimental protocols were reviewed and approved<br />

by the Committee <strong>of</strong> Scientific Research <strong>of</strong> First Affiliated<br />

Hospital <strong>of</strong> General Hospital <strong>of</strong> PLA (Beij<strong>in</strong>g, Ch<strong>in</strong>a).<br />

Surgical preparation<br />

Pure bred Beagle dogs (purchased from Experimental<br />

Animal Center <strong>of</strong> Academy <strong>of</strong> Military Medical Sciences<br />

<strong>of</strong> PLA, Beij<strong>in</strong>g, Ch<strong>in</strong>a, License <strong>of</strong> qualification SCX<br />

2005-0005), aged 16-20 mo, body weight 11-13 kg, were<br />

used. They were acclimatized <strong>in</strong> the animal house <strong>of</strong> our<br />

Research Laboratory for 2 wk before use. They were fasted<br />

for 24 h, and water was withheld for 4 h before the surgical<br />

preparation. Under anesthesia with 8 mg/kg ketam<strong>in</strong>e (Gu-<br />

Tian Pharmacy, Fu Jian Prov<strong>in</strong>ce, Ch<strong>in</strong>a), the right carotid<br />

artery and jugular ve<strong>in</strong> were <strong>in</strong>dividually cannulated for<br />

hemodynamic monitor<strong>in</strong>g, collect<strong>in</strong>g blood samples, and adm<strong>in</strong>istration<br />

<strong>of</strong> drugs. Both cannula were led out through a<br />

subcutaneous passageway and fixed to the sk<strong>in</strong>. A midl<strong>in</strong>e <strong>in</strong>cision<br />

was made to open the peritoneal cavity to expose the<br />

proximal part <strong>of</strong> the jejunum, and small <strong>in</strong>cisions were made<br />

on the jejunum 10, 20, and 50 cm distal to the Treitz ligament.<br />

A Silastic tube, 3 mm <strong>in</strong> diameter and 25 cm <strong>in</strong> length,<br />

was <strong>in</strong>troduced <strong>in</strong>to the jejunal lumen through each <strong>of</strong> the<br />

above open<strong>in</strong>gs. They were fixed with purse-str<strong>in</strong>g sutures,<br />

led out through a subcutaneous tunnel and fixed to the sk<strong>in</strong>.<br />

A cystostomy was done for collect<strong>in</strong>g ur<strong>in</strong>e.<br />

Burn protocol<br />

Twenty-four hours after the above surgical procedures, an<br />

<strong>in</strong>travenous <strong>in</strong>jection <strong>of</strong> 0.5 mL/kg prop<strong>of</strong>ol was given to<br />

all the animals to produce brief anesthesia for 10-15 m<strong>in</strong>,<br />

which was long enough to elim<strong>in</strong>ate pa<strong>in</strong> dur<strong>in</strong>g burn<br />

<strong>in</strong>jury. Napalm (3%) was applied to the shaved neck and<br />

back <strong>of</strong> the dogs, and it was ignited for 30 s to produce a<br />

full-thickness burn that <strong>in</strong>volved about 35% <strong>of</strong> the total<br />

body surface area (TBSA). The depth <strong>of</strong> the burn <strong>in</strong>jury<br />

was verified by pathological exam<strong>in</strong>ation.<br />

Experimental groups<br />

The <strong>in</strong>jured dogs were grouped <strong>in</strong>to no fluid replacement<br />

(NR, n = 10), oral fluid replacement (OR, n = 8), and oral<br />

replacement <strong>of</strong> fluid with addition <strong>of</strong> carbachol (OR/<br />

CAR, n = 8). For NR, the animals received no fluid replacement<br />

or any other treatment. Dogs <strong>in</strong> the OR group received<br />

<strong>in</strong>tragastric, pre-warmed glucose sal<strong>in</strong>e solution (each<br />

1 L conta<strong>in</strong><strong>in</strong>g 59.83 mmol/L NaCl, 29.76 mmol/L NaH-<br />

CO3, 20.18 mmol/L KCl, and 114.94 mmol/L glucose <strong>in</strong><br />

distilled water), and the rate <strong>of</strong> gastric <strong>in</strong>fusion was consistent<br />

with that <strong>of</strong> the Parkland formula [5] (4 mL/kg for each<br />

% TBSA burn, half <strong>of</strong> the total amount given <strong>in</strong> the first<br />

8 h). In the OR/CAR group, 20 μg/kg carbachol (Sigma,<br />

St Louis, MO, USA) was added to the glucose-sal<strong>in</strong>e solution.<br />

Twenty-four hours after the burn <strong>in</strong>jury, all the<br />

animals <strong>in</strong> the three groups were given <strong>in</strong>travenous fluid<br />

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Table 1 Effects <strong>of</strong> carbachol on hemodynamic parameters <strong>in</strong> oral resuscitation <strong>of</strong> burn shock (mean ± SD)<br />

Post-burn (h) MAP (mmHg) CO (L/m<strong>in</strong>) PV (mL/kg)<br />

NR OR OR/CAR NR OR OR/CAR NR OR OR/CAR<br />

0 135 ± 9.3 131 ± 18.7 128 ± 12.6 2.46 ± 0.13 2.43 ± 0.17 2.39 ± 0.19 49.6 ± 5.6 46.8 ± 4.2 48.3 ± 4.0<br />

2 100 ± 7.2 a<br />

94 ± 15.3 a<br />

89 ± 10.0 a<br />

1.28 ± 0.11 a<br />

1.34 ± 0.1 a<br />

1.30 ± 0.11 a<br />

38.2 ± 3.8 a<br />

40.3 ± 3.0 a<br />

41.3 ± 3.1 a<br />

4 108 ± 8.3 a<br />

136 ± 18.9 b<br />

130 ± 15.1 b<br />

1.12 ± 0.10 a<br />

a, b<br />

1.54 ± 0.11<br />

a, b<br />

1.48 ± 0.12 32.8 ± 2.3 a<br />

39.0 ± 3.8 a<br />

40.8 ± 3.9 a<br />

8 120 ± 14.3 125 ± 15.9 127 ± 14.3 1.20 ± 0.17 a<br />

a, b<br />

1.65 ± 0.12<br />

a, b, c<br />

1.86 ± 0.14 30.9 ± 3.4 a<br />

a, b<br />

36.2 ± 3.4<br />

a, b, c<br />

42.2 ± 3.4<br />

24 124 ± 8.9 126 ± 8.8 129 ± 8.9 1.07 ± 0.17 a<br />

a, b<br />

1.94 ± 0.18 2.16 ± 0.15 a, b, c 30.6 ± 4.4 a<br />

a, b<br />

40.4 ± 3.0<br />

b, c<br />

45.8 ± 3.6<br />

48 129 ± 8.7 135 ± 20.8 123 ± 15.8 1.88 ± 0.15 a<br />

2.39 ± 0.23 b<br />

2.49 ± 0.16 b<br />

34.5 ± 2.4 a<br />

43.0 ± 3.8 b<br />

45.6 ± 3.6 b<br />

72 115 ± 13.7 a<br />

137 ± 11.0 b<br />

135 ± 13.1 b<br />

2.10 ± 0.13 a<br />

2.34 ± 0.12 b<br />

2.41 ± 0.20 b<br />

35.8 ± 2.9 a<br />

43.8 ± 3.4 b<br />

45.8 ± 4.0 b<br />

120 112 ± 11.4 a<br />

134 ± 14.6 b<br />

131 ± 14.2 b<br />

2.15 ± 0.15 a<br />

2.39 ± 0.15 b<br />

2.41 ± 0.20 b<br />

37.6 ± 3.5 a<br />

44.7 ± 3.2 b<br />

46.9 ± 3.4 b<br />

Compared with that at 0 h, a P < 0.05; compared with no fluid resuscitation (NR), b P < 0.05; compared with oral fluid resuscitation (OR), c P < 0.05. MAP:<br />

mean arterial pressure; CO: Cardiac output; PV: Plasma volume; CAR: Carbachol.<br />

Table 2 Effect <strong>of</strong> carbachol on gastro<strong>in</strong>test<strong>in</strong>al perfusion <strong>in</strong> oral resuscitation <strong>of</strong> burn shock (mean ± SD)<br />

Post-burn (h) PgCO2 (mmHg) IMBF (BPU)<br />

NR OR OR/CAR NR OR OR/CAR<br />

0 32.2 ± 3.7 33.2 ± 6.1 33.8 ± 6.8 203.8 ± 17.6 198.3 ± 11.9 207.3 ± 13.9<br />

2 73.1 ± 7.7 a<br />

a, b<br />

60.8 ± 8.2<br />

a, b<br />

57.4 ± 8.0 74.2 ± 10.8 a<br />

a, b<br />

101.2 ± 12.2<br />

a, b<br />

112.6 ± 10.2<br />

4 83.1 ± 6.5 a<br />

a, b<br />

74.0 ± 6.5<br />

a, b<br />

70.0 ± 6.2 71.5 ± 15.3 a<br />

a, b<br />

108.8 ± 12.2<br />

a, b, c<br />

138.8 ± 14.1<br />

8 86.4 ± 8.6 a<br />

a, b<br />

69.2 ± 6.8<br />

a, b<br />

68.5 ± 5.8 77.8 ± 10.0 a<br />

a, b<br />

114.7 ± 12.0<br />

a, b, c<br />

134.7 ± 13.9<br />

24 82.5 ± 7.6 a<br />

a, b<br />

65.8 ± 5.8<br />

a, b, c<br />

56.4 ± 4.7 79.2 ± 17.3 a<br />

a, b<br />

127.7 ± 11.9<br />

a, b, c<br />

157.7 ± 17.7<br />

48 61.5 ± 8.2 a<br />

56.0 ± 8.4 a<br />

57.0 ± 6.4 a<br />

146.8 ± 13.8 a<br />

159.3 ± 19.1 a<br />

a, b<br />

179.3 ± 19.1<br />

72 56.8 ± 6.6 a<br />

39.4 ± 8.9 b<br />

35.4 ± 5.6 b<br />

168.5 ± 9.7 a<br />

180.7 ± 18.5 198.7 ± 16.5 b<br />

120 45.8 ± 6.2 a<br />

31.2 ± 5.0 b<br />

34.2 ± 4.0 b<br />

178.8 ± 16.5 a<br />

203.5 ± 23.2 b<br />

200.5 ± 18.2 b<br />

Compared with that at 0 h, a P < 0.05; compared with no fluid resuscitation (NR), b P < 0.05; compared with oral fluid resuscitation (OR), c P < 0.05. IMBF:<br />

Intest<strong>in</strong>al mucosal blood flow; CAR: Carbachol.<br />

Gastric empty<strong>in</strong>g (%)<br />

110<br />

100<br />

90<br />

80<br />

70<br />

60<br />

50<br />

40<br />

30<br />

a<br />

a<br />

OR OR/CAR<br />

a,c<br />

a<br />

a,c a,c<br />

20<br />

0 1 2 3 4 6 8 24<br />

Postburn (h)<br />

Figure 1 Carbachol promoted gastric empty<strong>in</strong>g rate <strong>in</strong> oral fluid resuscitation/Carbachol<br />

group compared with those <strong>of</strong> oral fluid resuscitation<br />

group at 2, 3, 4 and 6 h after burn <strong>in</strong>jury. a P < 0.05 vs 0 h, c P < 0.05 vs oral<br />

fluid resuscitation (OR) group (one-way ANOVA). Error bars represent mean ±<br />

SD. CAR: Carbachol.<br />

though it was higher than that <strong>in</strong> the OR/CAR group, but<br />

there was no significant difference (P > 0.05). IMBF was<br />

always higher <strong>in</strong> the OR and OR/CAR groups compared<br />

with the NR group (all P < 0.05), but the value was lower<br />

<strong>in</strong> the OR group compared with the OR/CAR group at 4,<br />

8 and 24 h post-burn (all P < 0.05).<br />

Gastric empty<strong>in</strong>g rate<br />

Figure 1 shows that, <strong>in</strong> the OR and OR/CAR groups,<br />

gastric empty<strong>in</strong>g rate was lowered, especially <strong>in</strong> the for-<br />

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a<br />

a,c<br />

a<br />

a a<br />

a<br />

a<br />

Hu S et al . Carbachol, gastro<strong>in</strong>test<strong>in</strong>al function and burns<br />

mer group, with 52.8% <strong>of</strong> the normal empty<strong>in</strong>g rate at<br />

2 h, and 70.5% at 24 h post-burn. In the OR/CAR group,<br />

it was also lowered, but reached 65.2% at 2 h, and 73.0% at<br />

24 h post-burn, and the values were all significantly higher<br />

than those <strong>in</strong> the OR group at 2, 3, 4 and 6 h post-burn (all<br />

P < 0.05). Thus, it was estimated that approximately 15.7%<br />

more gastric content was expelled from the stomach <strong>in</strong> 24<br />

h <strong>in</strong> the OR/CAR group as compared with the OR group.<br />

Intest<strong>in</strong>al absorption rate<br />

As shown <strong>in</strong> Figures 2 and 3, the rate <strong>of</strong> absorption <strong>of</strong><br />

orally adm<strong>in</strong>istered water and Na + was lowest at 3 h postburn<br />

<strong>in</strong> the OR group, and the values were 23.7% and<br />

50.3% <strong>of</strong> pre-<strong>in</strong>jury levels. These absorption rates were<br />

gradually <strong>in</strong>creased to 44.4% and 65.1%, respectively, at<br />

24 h post-burn. In the OR/CAR group, all these values<br />

were higher than those <strong>in</strong> the OR group at 3, 4 and 6 h<br />

post-burn (all P < 0.05). In the first 24 h post-burn, the<br />

rate <strong>of</strong> <strong>in</strong>test<strong>in</strong>al water absorption was elevated by 11.5%<br />

<strong>in</strong> the OR/CAR group. It was estimated that with<strong>in</strong> 24 h<br />

after <strong>in</strong>jury, the water absorption rate at 24 h <strong>in</strong> the OR<br />

and OR/CAR groups was 110.9 ± 17.1 mL/h.m and<br />

127.8 ± 17.3 mL/h.m, respectively, and they were actually<br />

higher than the pre-requisite <strong>of</strong> the Parkland formula<br />

82.1 ± 11.2 mL/h.m (P < 0.05).<br />

DISCUSSION<br />

Mass casualties from burn <strong>in</strong>jury may occur <strong>in</strong> a region<br />

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Water absorption rate (mL/h.m)<br />

Hu S et al . Carbachol, gastro<strong>in</strong>test<strong>in</strong>al function and burns<br />

250.00<br />

200.00<br />

150.00<br />

100.00<br />

50.00<br />

0.00<br />

OR OR/CAR Infusion rate<br />

a,c<br />

0 1 2 3 4 6 8 24<br />

Postburn (h)<br />

Figure 2 Carbachol significantly improved rate <strong>of</strong> water absorption <strong>of</strong> <strong>in</strong>test<strong>in</strong>e<br />

<strong>in</strong> oral fluid resuscitation/Carbachol group compared with those <strong>of</strong><br />

oral fluid resuscitation group at 3, 4 and 6 h after burn <strong>in</strong>jury. a P < 0.05 vs 0<br />

h, c P < 0.05 vs oral fluid resuscitation (OR) group (one-way ANOVA). Error bars<br />

represent mean ± SD. CAR: Carbachol.<br />

where the major problems fac<strong>in</strong>g medical personnel are<br />

the availability and probability <strong>of</strong> fluid replacement with<br />

sterile <strong>in</strong>travenous fluids. If we resuscitate a s<strong>in</strong>gle burn<br />

victim weigh<strong>in</strong>g > 60 kg with a burn <strong>of</strong> 30% TBSA it<br />

would require > 5-7 kg <strong>of</strong> fluid for a medic to carry. In<br />

a very harsh environment with lack <strong>of</strong> decent medical<br />

support, resuscitation <strong>of</strong> burn shock is severely handicapped,<br />

and the life <strong>of</strong> those with extensive burn <strong>in</strong>jury<br />

is jeopardized. One strategy for reduc<strong>in</strong>g such a hazard<br />

is to try to supplement liquid with sufficient electrolytes<br />

by mouth until <strong>in</strong>travenous <strong>in</strong>fusion fluids are available [9] .<br />

Early <strong>in</strong> 1970, Monafo [10] reported a cl<strong>in</strong>ical trial <strong>of</strong> oral<br />

adm<strong>in</strong>istration <strong>of</strong> hypertonic lactated sal<strong>in</strong>e solution to<br />

patients with burn <strong>in</strong>jury <strong>of</strong> various extents, and he found<br />

that at least partial oral resuscitation <strong>of</strong> severe burns could<br />

be successful. However, oral resuscitation <strong>of</strong> burn shock<br />

has not been popular, because, ord<strong>in</strong>arily, <strong>in</strong>travenous<br />

resuscitation is almost always available, especially <strong>in</strong> cities,<br />

and even <strong>in</strong> many rural areas where medical facilities have<br />

been established. Nevertheless, <strong>in</strong> certa<strong>in</strong> scenarios, such<br />

as fire disasters <strong>in</strong> cities after a strong earthquake, forest<br />

fires <strong>in</strong> mounta<strong>in</strong>ous terra<strong>in</strong>, or <strong>in</strong> combat zones after<br />

<strong>in</strong>cendiary devices have exploded, when transportation is<br />

seriously lack<strong>in</strong>g or hampered due to geographical barriers,<br />

and medical support is lack<strong>in</strong>g, so that sterile <strong>in</strong>travenous<br />

fluids and tra<strong>in</strong>ed personnel are not available, oral<br />

<strong>in</strong>take <strong>of</strong> fluids should be considered, <strong>in</strong> the hope that the<br />

victims can be tided over burn shock.<br />

Sufficient data about oral resuscitation <strong>of</strong> burn shock<br />

can not be accumulated <strong>in</strong> cl<strong>in</strong>ical practice under normal<br />

circumstances, therefore, most <strong>in</strong>vestigations have been<br />

done with animals. In these experiments, animals were<br />

always under general anesthesia to prevent restlessness <strong>of</strong><br />

the animals [1,3,4] . Thus, the results <strong>of</strong> oral hydration might<br />

not reflect the true states <strong>of</strong> fluid absorption because<br />

the anesthesia <strong>in</strong>evitably <strong>in</strong>hibits gastro<strong>in</strong>test<strong>in</strong>al motility<br />

and other functions. The present study was planned to<br />

measure all the physiological parameters <strong>in</strong> a conscious<br />

state. All the catheters for measur<strong>in</strong>g physiological <strong>in</strong>dexes<br />

were implanted 1 d before burn <strong>in</strong>jury. Burn <strong>in</strong>jury was<br />

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a<br />

a<br />

a<br />

a,c<br />

a<br />

a,c<br />

a<br />

a<br />

a<br />

a a<br />

Na + absorption rate (mL/h.m)<br />

7<br />

6<br />

5<br />

4<br />

3<br />

2<br />

1<br />

a<br />

OR OR/CAR<br />

a<br />

*<br />

a<br />

a,c<br />

0 1 2 3 4 6 8 24<br />

Postburn (h)<br />

produced under brief anesthesia to elim<strong>in</strong>ate mental stra<strong>in</strong><br />

and pa<strong>in</strong>. Thereafter, all the measurements were made<br />

with the animals <strong>in</strong> a fully conscious state. Pure bred Beagle<br />

dogs were used because they were tame, docile and cooperative.<br />

No restriction <strong>of</strong> the body was necessary dur<strong>in</strong>g<br />

the whole course <strong>of</strong> the experiment, which rendered all<br />

the measurements complete without any restriction <strong>of</strong> the<br />

animals or general anesthesia. Thus, all the disturbances<br />

to gastro<strong>in</strong>test<strong>in</strong>al mobility or absorption were greatly alleviated.<br />

Therefore, there was no external <strong>in</strong>terference to<br />

<strong>in</strong>fluence the measurements dur<strong>in</strong>g the whole course <strong>of</strong><br />

the experiment, which lasted for 120 h, which <strong>in</strong>creased<br />

the reliability <strong>of</strong> the measurements.<br />

The degree <strong>of</strong> perfusion <strong>of</strong> the gastro<strong>in</strong>test<strong>in</strong>al tract is<br />

at present considered as an important <strong>in</strong>dex <strong>of</strong> circulatory<br />

shock and tissue oxygen delivery. The determ<strong>in</strong>ation <strong>of</strong><br />

PCO2 <strong>of</strong> the gastric mucosa has been used to estimate the<br />

pH <strong>of</strong> the mucosa, and the change <strong>in</strong> pH <strong>of</strong> the mucosa<br />

reflects the condition <strong>of</strong> blood perfusion and oxygen delivery<br />

to the gastric tissue [11] . The procedure is untraumatic,<br />

and the catheter can also be used to give necessary fluids<br />

as well as a convenient tool for measur<strong>in</strong>g gastric empty<strong>in</strong>g<br />

rate. To determ<strong>in</strong>e blood perfusion <strong>of</strong> the <strong>in</strong>test<strong>in</strong>al mucosa<br />

by way <strong>of</strong> preformed fistulae has already been a standard<br />

method <strong>in</strong> monitor<strong>in</strong>g the circulatory state <strong>of</strong> transplanted<br />

<strong>in</strong>test<strong>in</strong>e [12] . In our experiment, a flexible fiberoptic<br />

detector <strong>of</strong> a laser Doppler flow monitor was passed<br />

through the preformed fistulae. Each read<strong>in</strong>g took about<br />

1 m<strong>in</strong> only, so that it did not give any discomfort to the<br />

animal, and sequential measurements were assured. The<br />

experimental results showed that, <strong>in</strong> <strong>in</strong>jured dogs without<br />

the benefit <strong>of</strong> fluid gavage, blood perfusion was rapidly<br />

dim<strong>in</strong>ished <strong>in</strong> the <strong>in</strong>test<strong>in</strong>al mucosa. It was also shown<br />

that this change was consistent with a decrease gastro<strong>in</strong>test<strong>in</strong>al<br />

mucosal perfusion, and its recovery lagged beh<strong>in</strong>d<br />

the improvement <strong>in</strong> hemodynamic parameters. Up to<br />

120 h after <strong>in</strong>jury, it was still lower than that before <strong>in</strong>jury.<br />

These phenomena corroborated that which was found <strong>in</strong><br />

human patients with hypodynamic shock.<br />

When a solution with electrolytes and glucose is <strong>in</strong>tro-<br />

1750 April 7, 2011|Volume 17|Issue 13|<br />

a<br />

a,c<br />

a,c a<br />

Figure 3 Carbachol significantly improved rate <strong>of</strong> Na+ absorption <strong>of</strong> <strong>in</strong>test<strong>in</strong>e<br />

<strong>in</strong> oral fluid resuscitation/Carbachol group compared with those <strong>of</strong> oral<br />

fluid resuscitation group at 3, 4 and 6 h after burn <strong>in</strong>jury. a P < 0.05 vs 0 h, c P<br />

< 0.05 vs oral fluid resuscitation (OR) group (one-way ANOVA). Error bars represent<br />

mean ± SD. CAR: Carbachol.<br />

a<br />

a<br />

a<br />

a<br />

a

---

duced <strong>in</strong>to the stomach, the empty<strong>in</strong>g rate <strong>of</strong> the liquid<br />

from the stomach depends on the pressure gradient between<br />

the stomach and duodenum. It also is <strong>in</strong>fluenced by<br />

the state <strong>of</strong> blood supply to the stomach and regulatory<br />

activity <strong>of</strong> the vagus nerve (chol<strong>in</strong>ergic nerve) and humoral<br />

agents (motil<strong>in</strong>) [13] . Absorption <strong>of</strong> water through the<br />

<strong>in</strong>test<strong>in</strong>al mucosa depends on translocation <strong>of</strong> Na + ions,<br />

while the latter process could only be realized with the<br />

presence and activity <strong>of</strong> Na + /K + -ATPase, which is localized<br />

<strong>in</strong> the basal layer <strong>of</strong> the <strong>in</strong>test<strong>in</strong>al mucosal epithelium.<br />

Therefore, it is obvious that the rate <strong>of</strong> absorption <strong>of</strong><br />

water by the <strong>in</strong>test<strong>in</strong>al mucosa is under the <strong>in</strong>fluence <strong>of</strong><br />

the <strong>in</strong>test<strong>in</strong>al blood flow, activity <strong>of</strong> Na + /K + -ATPase, and<br />

aquapor<strong>in</strong> (AQP)-1 expression, along with consumption<br />

<strong>of</strong> ATP. Thus, it is evident that with ischemia and hypoxia<br />

<strong>of</strong> the <strong>in</strong>test<strong>in</strong>al mucosa dur<strong>in</strong>g hypovolemic shock, water<br />

absorption by the <strong>in</strong>test<strong>in</strong>al mucosa is hampered.<br />

Heavy leakage <strong>of</strong> fluid from the circulation results <strong>in</strong><br />

a sharp decrease <strong>in</strong> ATP. This shortage <strong>in</strong> ATP is further<br />

amplified by an <strong>in</strong>creased demand created by feed<strong>in</strong>g and<br />

absorption <strong>of</strong> water and electrolytes. Under such conditions,<br />

the presence <strong>of</strong> glucose, which can be metabolized<br />

<strong>in</strong>to lactic acid, alan<strong>in</strong>e and CO2 to produce ATP under<br />

anaerobic conditions, is essential. This additional ATP is<br />

helpful for the absorption potential <strong>of</strong> the <strong>in</strong>test<strong>in</strong>al mucosa.<br />

Dur<strong>in</strong>g the process <strong>of</strong> absorption, the transport <strong>of</strong><br />

glucose is coupled with the transport <strong>of</strong> water molecules<br />

and Na + ions, and two Na + ions and 223 water molecules<br />

are absorbed. Therefore, addition <strong>of</strong> glucose to an electrolyte<br />

solution is an ideal liquid for oral replacement dur<strong>in</strong>g<br />

hypovolemic shock [14,15] .<br />

Thomas et al [2] have described an experiment to study<br />

gastric empty<strong>in</strong>g with oral replacement <strong>of</strong> fluid <strong>in</strong> hypovolemic<br />

shock. They gavaged glucose-electrolyte solution<br />

(GES) <strong>in</strong>to the stomach <strong>of</strong> pigs with 40% TBSA<br />

burn <strong>in</strong>jury, accord<strong>in</strong>g to the Parkland formula. They<br />

showed that the gastric empty<strong>in</strong>g volume <strong>in</strong>creased with<br />

an <strong>in</strong>crease <strong>in</strong> the volume <strong>of</strong> gavaged fluid. However, the<br />

volume <strong>of</strong> fluid passed <strong>in</strong>to the duodenum was one half<br />

<strong>of</strong> the volume required by the Parkland formula, and hemodynamic<br />

parameters did not recover to the pre-<strong>in</strong>jury<br />

level. Michell et al [4] have performed a study <strong>of</strong> duodenal<br />

<strong>in</strong>fusion <strong>of</strong> glucose-electrolyte solution <strong>in</strong> pigs with 40%<br />

TBSA burns, and demonstrated that a total <strong>of</strong> 93% <strong>of</strong><br />

<strong>in</strong>fused solution was absorbed dur<strong>in</strong>g the course <strong>of</strong> the<br />

4 h experiment. However, these experiments were performed<br />

<strong>in</strong> animals under general anesthesia, and there<br />

was unavoidable impairment <strong>of</strong> gastro<strong>in</strong>test<strong>in</strong>al peristalsis<br />

and absorption ability. Therefore, the results could<br />

not be considered as reflect<strong>in</strong>g the true condition <strong>of</strong> the<br />

gastro<strong>in</strong>test<strong>in</strong>al tract.<br />

In our present study, we determ<strong>in</strong>ed the gastric empty<strong>in</strong>g<br />

and <strong>in</strong>test<strong>in</strong>al absorption rate <strong>of</strong> GES without the<br />

<strong>in</strong>terfer<strong>in</strong>g effects <strong>of</strong> general anesthesia. We found that<br />

gastric empty<strong>in</strong>g and <strong>in</strong>test<strong>in</strong>al absorption were significantly<br />

reduced to the lowest level (52.8% and 23.7% <strong>of</strong><br />

pre-<strong>in</strong>jury levels) <strong>in</strong> the OR group at about 2 and 4 h<br />

post-burn, respectively, and did not return to 80% <strong>of</strong> pre<strong>in</strong>jury<br />

level until 24 h. It was estimated that with<strong>in</strong> 24 h<br />

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Hu S et al . Carbachol, gastro<strong>in</strong>test<strong>in</strong>al function and burns<br />

after <strong>in</strong>jury, the water absorption rate <strong>in</strong> the OR group<br />

was 110.9 ± 17.1 mL/h.m, and it was actually higher<br />

than the pre-requisite <strong>of</strong> the Parkland formula (82.1 ±<br />

11.2 mL/h.m). This <strong>in</strong>test<strong>in</strong>al absorptive rate was similar<br />

to that <strong>of</strong> Michael et al [4] . The above results suggest that, <strong>in</strong><br />

large animals (e.g. pigs or dogs), with < 40% TBSA burns,<br />

almost all GES <strong>in</strong>fused accord<strong>in</strong>g to the Parkland formula<br />

could be fully absorbed, although <strong>in</strong>test<strong>in</strong>al absorption<br />

was <strong>in</strong>hibited due to gut hypoperfusion. Therefore, we<br />

considered that gastric empty<strong>in</strong>g is the ma<strong>in</strong> limitation to<br />

effective gastro<strong>in</strong>test<strong>in</strong>al resuscitation.<br />

The results <strong>of</strong> us<strong>in</strong>g carbachol <strong>in</strong> our experiment<br />

clearly demonstrated that this drug could improve tissue<br />

blood perfusion <strong>of</strong> the gastro<strong>in</strong>test<strong>in</strong>al tract. Thus, it was<br />

helpful <strong>in</strong> expedit<strong>in</strong>g gastric empty<strong>in</strong>g <strong>of</strong> its contents and<br />

improv<strong>in</strong>g <strong>in</strong>test<strong>in</strong>al absorption <strong>of</strong> water and electrolytes.<br />

Carbachol is a chol<strong>in</strong>ergic receptor agonist. It stimulates<br />

peristalsis <strong>of</strong> the gastro<strong>in</strong>test<strong>in</strong>al tract by activat<strong>in</strong>g M<br />

chol<strong>in</strong>ergic receptors, and also activates a7 subunits <strong>of</strong><br />

chol<strong>in</strong>ergic nicot<strong>in</strong>ic receptors on macrophage and endothelial<br />

cells which leads to cellular deactivation and <strong>in</strong>hibition<br />

<strong>of</strong> cytok<strong>in</strong>e release, thus attenuat<strong>in</strong>g the systemic or<br />

regional <strong>in</strong>flammatory response [16-18] . In addition, it is an<br />

antioxidant and <strong>in</strong>hibitor <strong>of</strong> apoptosis [19,20] . In our study,<br />

the addition <strong>of</strong> carbachol to the resuscitation fluid did<br />

improve blood perfusion <strong>of</strong> the gastro<strong>in</strong>test<strong>in</strong>al tissues,<br />

expedite gastric empty<strong>in</strong>g, and improve <strong>in</strong>test<strong>in</strong>al absorption<br />

rate <strong>of</strong> water and electrolytes. These beneficial effects<br />

<strong>of</strong> carbachol may be attributable to the follow<strong>in</strong>g mechanisms:<br />

(1) <strong>in</strong>hibition <strong>of</strong> release <strong>of</strong> pro<strong>in</strong>flammatory cytok<strong>in</strong>es<br />

alleviates the <strong>in</strong>flammatory reaction <strong>of</strong> the gastro<strong>in</strong>test<strong>in</strong>al<br />

tissue, thus result<strong>in</strong>g <strong>in</strong> reduced loss <strong>of</strong> AQP1 [21,22] ;<br />

(2) improvement <strong>in</strong> <strong>in</strong>test<strong>in</strong>al peristalsis and blood perfusion<br />

due to stimulation <strong>of</strong> M receptors facilitates <strong>in</strong>test<strong>in</strong>al<br />

absorption; and (3) promotion <strong>of</strong> activity <strong>of</strong> Na – /K Na + -<br />

ATPase, which is essential for absorption <strong>of</strong> water and<br />

especially Na + by <strong>in</strong>test<strong>in</strong>al mucosa [23] . Our experiment<br />

showed that the gastric empty<strong>in</strong>g time was promoted by<br />

15.7%, and the <strong>in</strong>test<strong>in</strong>al absorption rate was <strong>in</strong>creased by<br />

11.5%.<br />

In conclusion, our experiment has successfully reproduced<br />

a can<strong>in</strong>e model <strong>of</strong> serious burn <strong>in</strong>jury. In this<br />

model, we are able to study the effects <strong>of</strong> oral replenishment<br />

<strong>of</strong> GES for resuscitation <strong>of</strong> burn shock. The<br />

hemodynamic parameters, gastric empty<strong>in</strong>g rate, and<br />

<strong>in</strong>test<strong>in</strong>al absorption <strong>of</strong> GES can be relatively accurately<br />

determ<strong>in</strong>ed. The results <strong>of</strong> the experiment also show<br />

that burn shock can be ameliorated to a certa<strong>in</strong> extent by<br />

adm<strong>in</strong>istration <strong>of</strong> GES, with the addition <strong>of</strong> carbachol<br />

to enhance gastric empty<strong>in</strong>g and <strong>in</strong>test<strong>in</strong>al absorption <strong>of</strong><br />

fluids. Oral resuscitation for burn shock might be a surrogate<br />

measure where mass casualties from burn <strong>in</strong>jury<br />

occur <strong>in</strong> an area where medical support is m<strong>in</strong>imal and<br />

transportation is difficult. Carbachol may be beneficial<br />

because it is a chol<strong>in</strong>ergic receptor agonist, and it possesses<br />

the function <strong>of</strong> enhanc<strong>in</strong>g gastric empty<strong>in</strong>g and<br />

<strong>in</strong>test<strong>in</strong>al absorption <strong>of</strong> fluid when given by mouth.<br />

Therefore, <strong>in</strong> circumstances when <strong>in</strong>travenous replacement<br />

<strong>of</strong> fluids for resuscitation <strong>of</strong> burn shock is not fea-<br />

1751 April 7, 2011|Volume 17|Issue 13|

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Hu S et al . Carbachol, gastro<strong>in</strong>test<strong>in</strong>al function and burns<br />

sible, oral feed<strong>in</strong>g <strong>of</strong> GES, with addition <strong>of</strong> carbachol,<br />

may be possible.<br />

COMMENTS<br />

Background<br />

Rapid <strong>in</strong>travenous <strong>in</strong>fusion rema<strong>in</strong>s unanimously the key measure to resuscitate<br />

hypovolemic shock as a result <strong>of</strong> massive burn <strong>in</strong>jury. Unfortunately, <strong>in</strong><br />

armed conflicts or massive disasters (such as forest fire, earthquake, or terrorist<br />

attack) mass casualties occur <strong>in</strong> an austere environment with poor medical support<br />

and transportation facilities. In such cases, it is our supposition that oral or<br />

gastro<strong>in</strong>test<strong>in</strong>al adm<strong>in</strong>istration <strong>of</strong> fluids might be more practical, and could have<br />

a positive effect <strong>in</strong> ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g the life <strong>of</strong> the victims until <strong>in</strong>travenous replacement<br />

<strong>of</strong> fluids is available.<br />

Research frontiers<br />

Gastro<strong>in</strong>test<strong>in</strong>al tract ischemia and hypoxia due to massive surgical stresses<br />

such as severe trauma, extensive burns and major surgery result<strong>in</strong>g <strong>in</strong> hypovolemic<br />

shock can lead to dysfunction <strong>of</strong> gastric empty<strong>in</strong>g and <strong>in</strong>test<strong>in</strong>al absorption,<br />

followed by poor transportation and absorption <strong>of</strong> oral electrolytes and<br />

nutrients <strong>in</strong> the gastro<strong>in</strong>test<strong>in</strong>al tract. Improvement <strong>of</strong> mucosal blood perfusion,<br />

and enhancement <strong>of</strong> gastro<strong>in</strong>test<strong>in</strong>al tolerance to oral rehydration fluid and<br />

enteral nutrition are not only the foci <strong>of</strong> research <strong>in</strong> the surgical and critical care<br />

fields, but they are key factors for facilitation <strong>of</strong> oral resuscitation <strong>of</strong> hypovolemic<br />

shock.<br />

Innovations and breakthroughs<br />

A large animal model <strong>of</strong> severe burn <strong>in</strong>jury was reproduced to <strong>in</strong>vestigate the<br />

feasibility <strong>of</strong> oral resuscitation <strong>of</strong> burn shock, without the disturb<strong>in</strong>g effects <strong>of</strong><br />

general anesthesia. The effects <strong>of</strong> carbachol, which is a chol<strong>in</strong>ergic receptor<br />

agonist, on blood circulation, gastro<strong>in</strong>test<strong>in</strong>al perfusion, gastric empty<strong>in</strong>g and<br />

<strong>in</strong>test<strong>in</strong>al absorption <strong>of</strong> fluid and electrolytes were <strong>in</strong>vestigated. The results<br />

<strong>in</strong>dicated that oral resuscitation with the help <strong>of</strong> such a drug might be an ideal<br />

way <strong>in</strong> lieu <strong>of</strong> <strong>in</strong>travenous resuscitation for burn shock, especially <strong>in</strong> battlefields<br />

or other sites <strong>of</strong> mass casualties.<br />

Applications<br />

Orally adm<strong>in</strong>istered fluid can be considered to be a simple and effective means<br />

<strong>of</strong> replacement <strong>of</strong> body fluid that is feasible for resuscitation <strong>of</strong> hypovolemic<br />

shock, especially when there is an extreme shortage <strong>of</strong> means <strong>of</strong> medical support<br />

<strong>in</strong> an austere environment such as battlefields and disasters.<br />

Peer review<br />

The manuscript is very well written and the conclusions applicable.<br />

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acts on M3 muscar<strong>in</strong>ic receptors and <strong>in</strong>duces the<br />

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lung epithelial cells. J Biol Chem 2001; 276: 18657-18664<br />

23 Bao C, Hu S, Zhou G, Tian Y, Wu Y, Sheng Z. Effect <strong>of</strong> carbachol<br />

on <strong>in</strong>test<strong>in</strong>al mucosal blood flow, activity <strong>of</strong> Na+-K+-<br />

ATPase, expression <strong>of</strong> aquapor<strong>in</strong>-1, and <strong>in</strong>test<strong>in</strong>al absorption<br />

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S- Editor Tian L L- Editor Kerr C E- Editor Ma WH<br />

1752 April 7, 2011|Volume 17|Issue 13|

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Onl<strong>in</strong>e Submissions: http://www.wjgnet.com/1007-9327<strong>of</strong>fice<br />

wjg@wjgnet.com<br />

doi:10.3748/wjg.v17.i13.1753<br />

Gastroesophageal reflux <strong>in</strong> cirrhotic patients without<br />

esophageal varices<br />

Jun Zhang, Pei-L<strong>in</strong> Cui, Dong Lv, Shi-Wei Yao, You-Q<strong>in</strong>g Xu, Zhao-Xu Yang<br />

Jun Zhang, Pei-L<strong>in</strong> Cui, Dong Lv, Shi-Wei Yao, You-Q<strong>in</strong>g Xu,<br />

Zhao-Xu Yang, Department <strong>of</strong> <strong>Gastroenterology</strong>, Beij<strong>in</strong>g Tiantan<br />

Hospital, Capital Medical University, Beij<strong>in</strong>g 100050, Ch<strong>in</strong>a<br />

Author contributions: Zhang J and Cui PL contributed equally to<br />

this work; Zhang J and Cui PL designed this research and wrote the<br />

paper; Lv D and Yao SW collected cl<strong>in</strong>ical data; Xu YQ and Yang<br />

ZX worked as academic advisors <strong>of</strong> this research.<br />

Correspondence to: Pei-L<strong>in</strong> Cui, Associated Chief, Department<br />

<strong>of</strong> <strong>Gastroenterology</strong>, Beij<strong>in</strong>g Tiantan Hospital, Capital<br />

Medical University, Beij<strong>in</strong>g 100050, Ch<strong>in</strong>a. cuipl@yahoo.cn<br />

Telephone: +86-10-67096644 Fax:+86-10-67096644<br />

Received: January 4, 2011 Revised: February 21, 2011<br />

Accepted: February 28, 2011<br />

Published onl<strong>in</strong>e: April 7, 2011<br />

Abstract<br />

AIM: To evaluate the esophageal motility and abnormal<br />

acid and bile reflux <strong>in</strong>cidence <strong>in</strong> cirrhotic patients without<br />

esophageal varices (EV).<br />

METHODS: Seventy-eight patients with liver cirrhosis<br />

without EV confirmed by upper gastroesophageal endoscopy<br />

and 30 healthy control volunteers were prospectively<br />

enrolled <strong>in</strong> this study. All the patients were<br />

evaluated us<strong>in</strong>g a modified protocol <strong>in</strong>clud<strong>in</strong>g Child-Pugh<br />

score, upper gastro<strong>in</strong>test<strong>in</strong>al endoscopy, esophageal<br />

manometry, simultaneous ambulatory 24-h esophageal<br />

pH and bilirub<strong>in</strong> monitor<strong>in</strong>g. All the patients and volunteers<br />

accepted the manometric study.<br />

RESULTS: In the liver cirrhosis group, lower esophageal<br />

sph<strong>in</strong>cter pressure (LESP, 15.32 ± 2.91 mmHg), peristaltic<br />

amplitude (PA, 61.41 ± 10.52 mmHg), peristaltic<br />

duration (PD, 5.32 ± 1.22 s), and peristaltic velocity<br />

(PV, 5.22 ± 1.11 cm/s) were all significantly abnormal <strong>in</strong><br />

comparison with those <strong>in</strong> the control group (P < 0.05),<br />

and LESP was negatively correlated with Child-Pugh<br />

score. The <strong>in</strong>cidence <strong>of</strong> reflux esophagitis (RE) and<br />

pathologic reflux was 37.18% and 55.13%, respectively<br />

WJG|www.wjgnet.com<br />

1753<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): 1753-1758<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

BRIEF ARTICLE<br />

(vs control, P < 0.05). And the <strong>in</strong>cidence <strong>of</strong> isolated<br />

abnormal acid reflux, bile reflux and mixed reflux was<br />

12.82%, 14.10% and 28.21% <strong>in</strong> patients with liver cirrhosis<br />

without EV.<br />

CONCLUSION: Cirrhotic patients without EV presented<br />

esophageal motor disorders and mixed acid and bile<br />

reflux was the ma<strong>in</strong> pattern; the cirrhosis itself was an<br />

important causative factor.<br />

© 2011 Baishideng. All rights reserved.<br />

Key words: Gastroesophageal reflux disease; Liver cirrhosis;<br />

Esophageal varices; Esophageal manometry; pH;<br />

Bilirub<strong>in</strong>; Monitor<strong>in</strong>g<br />

Peer reviewer: Helena Nordenstedt, MD, PhD, Upper Gastro<strong>in</strong>test<strong>in</strong>al<br />

Research, Department <strong>of</strong> Molecular Medic<strong>in</strong>e and<br />

Surgery, Karol<strong>in</strong>ska Institute, Stockholm 17176, Sweden<br />

Zhang J, Cui PL, Lv D, Yao SW, Xu YQ, Yang ZX. Gastroesophageal<br />

reflux <strong>in</strong> cirrhotic patients without esophageal varices. <strong>World</strong> J<br />

Gastroenterol 2011; 17(13): 1753-1758 Available from: URL:<br />

http://www.wjgnet.com/1007-9327/full/v17/i13/1753.htm DOI:<br />

http://dx.doi.org/10.3748/wjg.v17.i13.1753<br />

INTRODUCTION<br />

Gastroesophageal reflux disease (GERD) is one <strong>of</strong> the<br />

most common diseases <strong>in</strong> modern civilization, which<br />

greatly affects people’s health and quality <strong>of</strong> life [1] . GERD<br />

is def<strong>in</strong>ed as reflux <strong>of</strong> gastroduodenal content to the<br />

esophagus, and <strong>in</strong>cludes reflux esophagitis (RE), nonerosive<br />

reflux disease (NERD) and Barrett’s esophagus (BE).<br />

GERD orig<strong>in</strong>ates from a disturbance <strong>in</strong> the structure and<br />

function <strong>of</strong> the lower esophageal sph<strong>in</strong>cter (LES) barrier,<br />

and dysfunctional esophageal motility coupled with a weak<br />

LES can cause uncoord<strong>in</strong>ated propulsion, regurgitation <strong>of</strong><br />

gastric and/or duodenal contents <strong>in</strong>to the esophagus [2] .<br />

Gastroesophageal reflux consists <strong>of</strong> a broad mixture<br />

April 7, 2011|Volume 17|Issue 13|

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Table 1 Results <strong>of</strong> esophageal manometry <strong>in</strong> liver cirrhosis patients and controls (mean ± SD)<br />

Group LESP (mmHg) PA (mmHg) PD (s) PV (cm/s)<br />

Liver cirrhosis (n = 78) 15.32 ± 2.91 a<br />

61.41 ± 10.52 a<br />

5.32 ± 1.22 a<br />

5.22 ± 1.11 a<br />

Child A (n = 28) 16.18 ± 2.81 70.52 ± 8.93 a<br />

3.91 ± 1.03 a<br />

4.56 ± 1.22 a<br />

Child B (n = 27) 15.41 ± 3.13 c<br />

67.4 ± 9.3 c<br />

5.11 ± 1.21 c<br />

5.10 ± 1.02 c<br />

Child C (n = 23) 14.52 ± 2.91 e<br />

56.13 ± 10.06 e<br />

6.02 ± 1.23 e<br />

5.91 ± 1.01 e<br />

Control (n =30) 16.21 ± 5.33 74.41 ± 17.53 2.70 ± 0.81 3.71 ± 1.82<br />

a Compared with control, P < 0.05; c Compared with Child A, P < 0.05; e Compared with Child B, P < 0.05. LESP: Lower esoph-<br />

ageal sph<strong>in</strong>cter pressure; PA: Peristaltic amplitude; PD: Peristaltic duration; PV: Peristaltic velocity.<br />

exam<strong>in</strong>ation, H2 blockers at least 48-72 h and prok<strong>in</strong>etics<br />

agents 24 h. An esophageal pH <strong>of</strong> less than 4 for at least<br />

15 s was considered to be a reflux episode. Pathological<br />

acid reflux was considered if the percentage <strong>of</strong> the time<br />

with the <strong>in</strong>traesophageal pH less than 4 was greater than<br />

4%, the number <strong>of</strong> reflux episodes was larger than 50 or<br />

the DeMeester value was higher than 14.72 [17] .<br />

The fiber optic spectrophotometer Bilitec 2000 was<br />

used to quantify DGER. The system consisted <strong>of</strong> a<br />

m<strong>in</strong>iaturized probe measur<strong>in</strong>g 1.5 mm <strong>in</strong> diameter that<br />

carried light signals <strong>in</strong>to the esophagus and backed via a<br />

plastic fiberoptic bundle. Before each study, the probe was<br />

calibrated <strong>in</strong> water, and the probe tip was checked for obstruction<br />

after completion <strong>of</strong> the study.<br />

Patients were also encouraged to ma<strong>in</strong>ta<strong>in</strong> normal activities,<br />

sleep schedule, and to follow a particular low-fat<br />

diet conta<strong>in</strong><strong>in</strong>g light food elements, and not to take c<strong>of</strong>fee,<br />

tea and fruit juice, <strong>in</strong> order to prevent any <strong>in</strong>terference<br />

with the spectrophotometric record<strong>in</strong>g. Skimmed<br />

milk and non-sparkl<strong>in</strong>g water were allowed. An episode<br />

<strong>of</strong> DGER was def<strong>in</strong>ed as an <strong>in</strong>crease <strong>in</strong> esophageal bilirub<strong>in</strong><br />

absorbance 0.14 for more than 10 s [18,19] .<br />

Blood sample detection<br />

Blood samples were drawn for a complete analysis <strong>of</strong><br />

blood cell count and levels <strong>of</strong> prothromb<strong>in</strong>, album<strong>in</strong>, alan<strong>in</strong>e<br />

transam<strong>in</strong>ase (ALT), aspartate transam<strong>in</strong>ase (AST),<br />

alkal<strong>in</strong>e phosphatase, gamma glutamyl transferase, bilirub<strong>in</strong>,<br />

cholesterol, creat<strong>in</strong><strong>in</strong>e.<br />

Statistical analysis<br />

Statistical analysis was performed us<strong>in</strong>g the statistical program<br />

SPSS 13.0 for W<strong>in</strong>dows (SPSS Inc., Chicago, USA).<br />

All data were presented as mean ± SD, and P values lower<br />

than 0.05 were considered statistically significant.<br />

RESULTS<br />

Patient characteristics<br />

Seventy-eight patients met the <strong>in</strong>clusion criteria, 40 males<br />

(51.28%) and 38 females (48.72%), with a mean age <strong>of</strong><br />

56.41 ± 9.72 years (range, 18-75 years). Twenty-eight patients<br />

were classified as Child A, 27 as Child B and 23 as<br />

Child C patients. Typical symptoms <strong>of</strong> gastroesophageal<br />

reflux disease were present <strong>in</strong> 25 (32.05%) patients. The<br />

RDQ scores were significantly higher <strong>in</strong> liver cirrhosis<br />

group (11.32 ± 3.14) than <strong>in</strong> control group (6.25 ± 3.31)<br />

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Zhang J et al . GERD and liver cirrhosis without EV<br />

(P < 0.01). There were no statistical differences <strong>of</strong> RDQ<br />

scores among the liver cirrhosis subgroups, and no relationship<br />

between Child-Pugh score and abnormal reflux<br />

(P > 0.05).<br />

Esophageal manometry<br />

In the liver cirrhosis group, LESP (15.32 ± 2.91 mm Hg),<br />

PA (61.41 ± 10.52 mmHg), PD (5.32 ± 1.22 s), and PV<br />

(5.22 ± 1.11 cm/s) were all significantly abnormal <strong>in</strong> comparison<br />

with those <strong>in</strong> the control group (P < 0.05) (Table<br />

1). The results showed a gradual decrease <strong>of</strong> LESP and<br />

PA, also an extension <strong>of</strong> PD and PV <strong>in</strong> the liver cirrhosis<br />

group from Child A to Child C. LESP was negatively correlated<br />

with Child-Pugh score (P < 0.01, r = -0.625).<br />

24-h esophageal pH monitor<strong>in</strong>g<br />

The results demonstrated a stepwise <strong>in</strong>crease <strong>of</strong> pathologic<br />

esophageal pH-metry <strong>in</strong> liver cirrhosis patients, and an<br />

<strong>in</strong>crease <strong>of</strong> acid reflux episodes and percentage <strong>of</strong> a pH <<br />

4 <strong>in</strong> the upright, sup<strong>in</strong>e and total phases <strong>of</strong> measurement (P<br />

< 0.05) (Table 2).<br />

24-h esophageal bilirub<strong>in</strong> monitor<strong>in</strong>g<br />

The results showed a significant stepwise <strong>in</strong>crease <strong>of</strong><br />

pathologic esophageal bilirub<strong>in</strong>-metry <strong>in</strong> liver cirrhosis<br />

patients, along with significant <strong>in</strong>creases <strong>of</strong> bile reflux<br />

episodes and percentage <strong>of</strong> absorbance > 0.14 <strong>in</strong> the<br />

upright, sup<strong>in</strong>e, and total phases <strong>of</strong> measurement (P <<br />

0.05) (Table 3).<br />

Incidence <strong>of</strong> RE and abnormal reflux<br />

The <strong>in</strong>cidence <strong>of</strong> RE and pathologic reflux was 37.18%<br />

and 55.13% <strong>in</strong> patients with liver cirrhosis, respectively,<br />

which were all higher than those <strong>in</strong> the control group (P<br />

< 0.05) (Table 4 and Figure 1). The <strong>in</strong>cidence <strong>of</strong> isolated<br />

abnormal acid reflux, bile reflux and mixed reflux was<br />

12.82%, 14.10% and 28.21% <strong>in</strong> patients with liver cirrhosis,<br />

respectively (Table 5). And the <strong>in</strong>cidence <strong>of</strong> BE was<br />

5.13% (4/78) <strong>in</strong> patients with liver cirrhosis, and none was<br />

found <strong>in</strong> the control group.<br />

DISCUSSION<br />

As a complication <strong>of</strong> chronic liver disease, GERD <strong>in</strong> cirrhotic<br />

patients with EV accounted for about 20%, which<br />

ma<strong>in</strong>ly belongs to a dysk<strong>in</strong>etic type [20] . Previous studies<br />

found that esophageal varices played an important role<br />

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Zhang J et al . GERD and liver cirrhosis without EV<br />

Table 2 Results <strong>of</strong> ambulatory 24-h esophageal pH monitor<strong>in</strong>g <strong>in</strong> liver cirrhosis patients and controls (mean ± SD)<br />

Group Number <strong>of</strong> acid reflux episodes Number <strong>of</strong> acid reflux episodes last<strong>in</strong>g ≥ 5 m<strong>in</strong> Mean time pH < 4 (%)<br />

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Total Upright Sup<strong>in</strong>e<br />

Liver cirrhosis (n = 78) 61.17 ± 33.35 a<br />

15.25 ± 5.73 a<br />

10.34 ± 4.45 a<br />

5.22 ± 2.71 a<br />

9.56 ± 3.42 a<br />

Child A (n = 28) 51.24 ± 20.54 a<br />

10.66 ± 7.28 a<br />

8.11 ± 2.32 a<br />

4.48 ± 1.76 a<br />

7.32 ± 5.44 a<br />

Child B (n = 27) 60.35 ± 18.66 c<br />

12.35 ± 9.83 c<br />

10.51 ± 1.62 c<br />

5.64 ± 1.31 c<br />

9.14 ± 4.37 c<br />

Child C (n = 23) 73.52 ± 28.63 e<br />

17.34 ± 12.46 e<br />

12.34 ± 2.15 e<br />

6.79 ± 1.51 e<br />

11.56 ± 5.43 e<br />

Child D (n = 30) 39.62 ± 29.32 4.81 ± 2.04 2.35 ± 1.53 3.58 ± 1.34 8.69 ± 3.45<br />

a Compared with control, P < 0.05; c Compared with Child A, P < 0.05; e Compared with Child B, P < 0.05.<br />

Table 3 Results <strong>of</strong> ambulatory 24-h esophageal bilirub<strong>in</strong> monitor<strong>in</strong>g <strong>in</strong> liver cirrhosis patients and controls (mean ± SD)<br />

Group Number <strong>of</strong> bile reflux episodes Number <strong>of</strong> bile reflux episodes last<strong>in</strong>g ≥ 5 m<strong>in</strong> Mean time Abs > 0.14 (%)<br />

Total Upright Sup<strong>in</strong>e<br />

Liver cirrhosis (n = 78) 36.53 ± 9.31 a<br />

4.09 ± 1.15 a<br />

6.73 ± 1.15 a<br />

3.32 ± 1.05 a<br />

4.37 ± 1.44 a<br />

Child A (n = 28) 27.32 ± 10.31 a<br />

3.85 ± 1.34 a<br />

5.12 ± 1.45 a<br />

3.15 ± 0.92 a<br />

4.12 ± 0.97 a<br />

Child B (n = 27) 39.46 ± 18.31 c<br />

4.11 ± 1.65 c<br />

6.54 ± 1.21 c<br />

3.37 ± 1.13 c<br />

5.04 ± 1.11 c<br />

Child C (n = 23) 48.54 ± 26.41 e<br />

4.23 ± 2.14 e<br />

7.32 ± 1.34 e<br />

4.28 ± 1.22 e<br />

5.52 ± 1.12 e<br />

Control (n = 30) 12.76 ± 6.97 2.15 ± 1.36 1.98 ± 0.86 1.03 ± 0.23 0.83 ± 0.62<br />

a Compared with control, P < 0.05; c Compared with Child A, P < 0.05; e Compared with Child B, P < 0.05. Abs: Aborbance.<br />

0.00<br />

0.08<br />

0.16<br />

0.24<br />

0.32<br />

0.40<br />

0.48<br />

0.56<br />

0.64<br />

0.72<br />

0.80<br />

0.88<br />

0.96<br />

1.04<br />

0.0<br />

0.8<br />

1.6<br />

2.4<br />

3.2<br />

4.0<br />

4.8<br />

5.6<br />

6.4<br />

7.2<br />

8.0<br />

8.8<br />

9.6<br />

10.4<br />

11.2<br />

8:22 12:55:22 14:55:22 16:55:22 18:55:22 20:55:22 22:55:22 00:55:22 02:55:22 04:55:22 06:55:22 08:55:22<br />

Figure 1 Mixed abnormal acid and bilirub<strong>in</strong> reflux curves <strong>in</strong> a typical Child C patient.<br />

Table 4 Relationship between liver function classification <strong>of</strong><br />

cirrhotic patients and gastroesophageal reflux disease n (%)<br />

Group RE Abnormal reflux<br />

Child A (n = 28) 8 (28.57) 12 (42.86)<br />

Child B (n = 27) 11 (40.74) 15 (55.56)<br />

Child C (n = 23) 10 (43.48) 16 (69.57)<br />

Total (n = 78) 29 (37.18) 43 (55.13)<br />

RE: Reflux esophagitis.<br />

<strong>in</strong> the development <strong>of</strong> esophageal motor disorders and<br />

abnormal gastroesophageal reflux <strong>in</strong> these patients, who<br />

presented obvious esophageal motor and motility disorders<br />

[21,22] . The most prevalent disorder was the <strong>in</strong>efficient<br />

esophageal motility, along with abnormal PA, PD and<br />

PV [12,21-23] . Some studies found that motor disorders existed<br />

<strong>in</strong> the esophageal body <strong>in</strong> these cirrhotic patients with EV,<br />

as compared with the cirrhotic patients without varices<br />

and control group [24] . Thus, it seemed that EV itself, <strong>in</strong>de-<br />

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Table 5 Abnormal reflux <strong>in</strong> liver cirrhosis patients<br />

pendent <strong>of</strong> the cirrhosis, delayed esophageal clearance and<br />

<strong>in</strong>creased the contact time between acid and mucosa.<br />

In this study, LESP, PA, PD and PV <strong>in</strong> cirrhotic patients<br />

without esophageal varices were significantly abnormal as<br />

compared with those <strong>in</strong> the control group. LESP was markedly<br />

lower <strong>in</strong> patients with severe liver function damage,<br />

and negatively correlated with Child-Pugh score (P < 0.01,<br />

r = -0.625). The results showed that cirrhosis itself was another<br />

important factor for the esophageal motor disorder.<br />

The <strong>in</strong>cidence <strong>of</strong> esophageal acid reflux among cirrhotic<br />

patients with EV has also been studied <strong>in</strong> the last<br />

decades us<strong>in</strong>g pH-metry record<strong>in</strong>g. It has been postulated<br />

that acid reflux may contribute to esophagitis and variceal<br />

bleed<strong>in</strong>g <strong>in</strong> cirrhotic patients, and it occurs at a high frequency<br />

(64%) <strong>in</strong> patients with liver cirrhosis and portal<br />

hypertension, irrespective <strong>of</strong> the etiology <strong>of</strong> cirrhosis and<br />

the grade <strong>of</strong> esophageal varices [8] . The results <strong>in</strong>dicated<br />

that there was a correlation between typical gastroesophageal<br />

reflux disease and abnormal reflux, but no relationship<br />

between ascites, variceal size, congestive gastropathy<br />

and Child-pugh score and abnormal reflux.<br />

The high <strong>in</strong>cidence <strong>of</strong> RE <strong>in</strong> patients with severe<br />

chronic liver disease was also demonstrated, and asymptomatic<br />

RE was more common <strong>in</strong> cirrhotic and liver<br />

failure patients [25,26] . In the present study, abnormal reflux<br />

and RE were demonstrated <strong>in</strong> 55.13% and 37.18% <strong>of</strong><br />

the cirrhotic patients without esophageal varices, and the<br />

more severe liver function damage, the more abnormal<br />

parameters <strong>of</strong> acid and bilirub<strong>in</strong> reflux. In the mean time,<br />

typical symptoms <strong>of</strong> gastroesophageal reflux disease were<br />

presented <strong>in</strong> only 32.05% <strong>of</strong> the cirrhotic patients <strong>in</strong> this<br />

study, and abnormal reflux was found <strong>in</strong> 62% <strong>of</strong> the patients<br />

<strong>in</strong> the night possibly due to the lowered esophageal<br />

defenses dur<strong>in</strong>g this period, with reduction <strong>of</strong> saliva production,<br />

swallow<strong>in</strong>g and esophageal clearance.<br />

GERD may occur <strong>in</strong> acid, bile or a mixed form, and<br />

DGER is considered as an <strong>in</strong>dependent risk factor for<br />

complicated GERD. However, few studies have reported<br />

the <strong>in</strong>cidence <strong>of</strong> DGER among cirrhotic patients. Patients<br />

with Barrett’s esophagus had significantly higher levels <strong>of</strong><br />

DGER than patients with uncomplicated GERD, and bile<br />

reflux either alone or mixed with acid reflux contributed<br />

obviously to the severity <strong>of</strong> erosive and non-erosive reflux<br />

disease [6] . Moreover, DGER <strong>in</strong> acid medium was more <strong>in</strong>jurious<br />

to the esophagus than DGER <strong>in</strong> alkal<strong>in</strong>e pH [7] . We<br />

studied for the first time the <strong>in</strong>cidence <strong>of</strong> BE and DGER<br />

<strong>in</strong> cirrhotic patients without esophageal varices. We found<br />

that the mixed acid and bile reflux was the predom<strong>in</strong>ant<br />

pattern <strong>of</strong> reflux <strong>in</strong> GERD patients, and the reflux <strong>in</strong>cidence<br />

was also higher <strong>in</strong> Child B or C group than <strong>in</strong> Child<br />

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Zhang J et al . GERD and liver cirrhosis without EV<br />

Group <strong>Isolated</strong> abnormal acid reflux <strong>Isolated</strong> abnormal bile reflux Mixed abnormal reflux No abnormal reflux<br />

Child A (n = 28) 4 3 5 16<br />

Child B (n = 27) 3 4 8 12<br />

Child C (n = 23) 3 4 9 7<br />

Total (n = 78) 10 (12.82%) 11 (14.10%) 22 (28.21%) 35 (44.87%)<br />

A group. A stepwise <strong>in</strong>crease <strong>of</strong> mixed reflux was demonstrated<br />

along with the severity <strong>of</strong> liver function damage.<br />

Four BE patients (2 with mixed abnormal reflux, 2 with<br />

DGER) were found <strong>in</strong> Child C group.<br />

The causes and the mechanism <strong>of</strong> liver cirrhosis <strong>in</strong> patients<br />

with abnormal GERD have not been fully elucidated.<br />

In this study, we demonstrated an obvious esophageal<br />

motility disorder and abnormal gastroesophageal reflux <strong>in</strong><br />

cirrhotic patients without esophageal varices, and abnormalities<br />

<strong>of</strong> esophageal motility and reflux parameter were<br />

correlated with the severity <strong>of</strong> liver function damage. It<br />

seemed that not only mechanical effect (EV), but also neural<br />

and humoral factor are related to the high <strong>in</strong>cidence <strong>of</strong><br />

GERD <strong>in</strong> patients with liver cirrhosis. The progress <strong>of</strong> liver<br />

dysfunction decreased the <strong>in</strong>cidence <strong>of</strong> LESP, worsened the<br />

esophageal motility and the reflux <strong>in</strong> the cirrhotic patients.<br />

In some studies, the levels <strong>of</strong> plasma vasoactive peptides<br />

and neurotens<strong>in</strong> were markedly higher <strong>in</strong> patients with liver<br />

cirrhosis than <strong>in</strong> the normal population, which were also<br />

known to lower the pressure <strong>of</strong> the LES, facilitat<strong>in</strong>g the reflux<br />

<strong>of</strong> the stomach content [21,27] .<br />

The importance <strong>of</strong> nitrous oxide (NO) <strong>in</strong> the exacerbation<br />

<strong>of</strong> portal hypertension <strong>in</strong> liver cirrhosis was also reported<br />

[28,29] . This substance can be found <strong>in</strong> large amounts<br />

<strong>in</strong> the systemic circulation <strong>of</strong> cirrhotic patients, and NO<br />

concentration <strong>in</strong>creased significantly <strong>in</strong> patients with liver<br />

disease, which was closely related to the transient LES<br />

relaxation, suggest<strong>in</strong>g that NO played an important role<br />

<strong>in</strong> the process <strong>of</strong> GERD. Whether the excessive NO <strong>in</strong><br />

cirrhotic patients could exacerbate these manifestations,<br />

needs to be further confirmed.<br />

We found that gastric half-empty<strong>in</strong>g <strong>of</strong> liquid food<br />

was delayed <strong>in</strong> patients with liver cirrhosis, and the function<br />

<strong>of</strong> gastric empty<strong>in</strong>g was also <strong>in</strong>fluenced by the damaged<br />

liver function [30] . Ascites <strong>in</strong>duced an <strong>in</strong>crease <strong>in</strong> <strong>in</strong>traabdom<strong>in</strong>al<br />

pressure, compress<strong>in</strong>g the stomach and the<br />

stomach content reflux [31] .<br />

In summary, the majority <strong>of</strong> cirrhotic patients without<br />

EV presented esophageal motor disorders; mixed<br />

acid and bile reflux was the ma<strong>in</strong> pattern <strong>of</strong> reflux <strong>in</strong><br />

GERD patients; and the presence <strong>of</strong> cirrhosis itself was an<br />

important causative factor for the onset <strong>of</strong> gastroesophageal<br />

reflux. Further researches on the functional and humoral<br />

factors and mechanism <strong>of</strong> GERD <strong>in</strong> liver diseases will ga<strong>in</strong><br />

a broad attention and <strong>in</strong>terest <strong>in</strong> this field.<br />

COMMENTS<br />

Background<br />

Gastroesophageal reflux disease (GERD) is one <strong>of</strong> the most common diseases<br />

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Zhang J et al . GERD and liver cirrhosis without EV<br />

<strong>in</strong> modern civilization, and it has been reported that gastroesophageal reflux<br />

disease occurs at a high frequency <strong>in</strong> patients with liver cirrhosis.<br />

Research frontiers<br />

The relationship between esophageal motor disorders and acid gastroesophageal<br />

reflux <strong>in</strong> cirrhotic patients with esophageal varices has been reported. This<br />

study was designed to evaluate the esophageal motility and abnormal acid and<br />

bile reflux <strong>in</strong>cidence <strong>in</strong> cirrhotic patients without esophageal varices.<br />

Innovations and breakthroughs<br />

This study showed that the presence <strong>of</strong> cirrhosis itself was an important<br />

causative factor for the onset <strong>of</strong> gastroesophageal reflux <strong>in</strong> patients with liver<br />

cirrhosis without varices. It is the first research on the <strong>in</strong>cidence <strong>of</strong> Barrett’s<br />

esophagus and DGER <strong>in</strong> cirrhotic patients without esophageal varices.<br />

Applications<br />

This study helped better understand the mechanism <strong>of</strong> GERD <strong>in</strong> patients with<br />

liver cirrhosis, and contributed to the diagnosis and treatment <strong>of</strong> liver cirrhosis<br />

and its complications <strong>in</strong> cl<strong>in</strong>ical practice.<br />

Peer review<br />

This is an <strong>in</strong>terest<strong>in</strong>g study on GERD <strong>in</strong> patients with liver cirrhosis, but without<br />

esophageal varices. S<strong>in</strong>ce it has before been thought that esophageal varices<br />

somehow have someth<strong>in</strong>g to do with the <strong>in</strong>creased frequency <strong>of</strong> GERD <strong>in</strong> patients<br />

with liver disease, the authors have made an <strong>in</strong>terest<strong>in</strong>g contribution to<br />

the literature by show<strong>in</strong>g that reflux symptoms and pathologic esophageal motility<br />

changes are more common <strong>in</strong> patients with cirrhosis but without varices.<br />

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for the system <strong>of</strong> GERD]. Fukuoka Igaku Zasshi 2007; 98: 373-378<br />

21 Grassi M, Albiani B, De Matteis A, Fontana M, Lucchetta<br />

MC, Raffa S. [Prevalence <strong>of</strong> dyspepsia <strong>in</strong> liver cirrhosis: a<br />

cl<strong>in</strong>ical and epidemiological <strong>in</strong>vestigation]. M<strong>in</strong>erva Med<br />

2001; 92: 7-12<br />

22 Suzuki K, Suzuki K, Koizumi K, Takada H, Nishiki R,<br />

Ichimura H, Oka S, Kuwayama H. Effect <strong>of</strong> symptomatic<br />

gastroesophageal reflux disease on quality <strong>of</strong> life <strong>of</strong> patients<br />

with chronic liver disease. Hepatol Res 2008; 38: 335-339<br />

23 Guo Q, Chen Y, Long Y. [Study on esophageal motility and<br />

its effect by EVL <strong>in</strong> patients with esophageal varices]. Zhonghua<br />

Ganzangb<strong>in</strong>g Zazhi 1999; 7: 201-202<br />

24 Passaretti S, Mazzotti G, de Franchis R, Cipolla M, Testoni<br />

PA, Tittobello A. Esophageal motility <strong>in</strong> cirrhotics with and<br />

without esophageal varices. Scand J Gastroenterol 1989; 24:<br />

334-338<br />

25 Schechter RB, Lemme EM, Coelho HS. Gastroesophageal<br />

reflux <strong>in</strong> cirrhotic patients with esophageal varices without<br />

endoscopic treatment. Arq Gastroenterol 2007; 44: 145-150<br />

26 Li B, Zhang B, Ma JW, Li P, Li L, Song YM, D<strong>in</strong>g HG. High<br />

prevalence <strong>of</strong> reflux esophagitis among upper endoscopies<br />

<strong>in</strong> Ch<strong>in</strong>ese patients with chronic liver diseases. BMC Gastroenterol<br />

2010; 10: 54<br />

27 Richter JE. Role <strong>of</strong> the gastric refluxate <strong>in</strong> gastroesophageal<br />

reflux disease: acid, weak acid and bile. Am J Med Sci 2009;<br />

338: 89-95<br />

28 Okamoto E, Amano Y, Fukuhara H, Furuta K, Miyake T,<br />

Sato S, Ishihara S, K<strong>in</strong>oshita Y. Does gastroesophageal reflux<br />

have an <strong>in</strong>fluence on bleed<strong>in</strong>g from esophageal varices?<br />

J Gastroenterol 2008; 43: 803-808<br />

29 Papadopoulos N, Soultati A, Goritsas C, Lazaropoulou C,<br />

Achimastos A, Adamopoulos A, Dourakis SP. Nitric oxide,<br />

ammonia, and CRP levels <strong>in</strong> cirrhotic patients with hepatic<br />

encephalopathy: is there a connection? J Cl<strong>in</strong> Gastroenterol<br />

2010; 44: 713-719<br />

30 Souza RC, Lima JH. Helicobacter pylori and gastroesophageal<br />

reflux disease: a review <strong>of</strong> this <strong>in</strong>trigu<strong>in</strong>g relationship.<br />

Dis Esophagus 2009; 22: 256-263<br />

31 Navarro-Rodriguez T, Hashimoto CL, Carrilho FJ, Strauss<br />

E, Laudanna AA, Moraes-Filho JP. Reduction <strong>of</strong> abdom<strong>in</strong>al<br />

pressure <strong>in</strong> patients with ascites reduces gastroesophageal<br />

reflux. Dis Esophagus 2003; 16: 77-82<br />

S- Editor Tian L L- Editor Ma JY E- Editor Ma WH<br />

1758 April 7, 2011|Volume 17|Issue 13|

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Onl<strong>in</strong>e Submissions: http://www.wjgnet.com/1007-9327<strong>of</strong>fice<br />

wjg@wjgnet.com<br />

doi:10.3748/wjg.v17.i13.1759<br />

BRIEF ARTICLE<br />

Association between polymorphism rs6983267 and gastric<br />

cancer risk <strong>in</strong> Ch<strong>in</strong>ese population<br />

Yi Guo, J<strong>in</strong>g Fang, Yan Liu, Hai-Hui Sheng, Xiao-Yan Zhang, Hai-Na Chai, Wei J<strong>in</strong>, Ke-Hao Zhang,<br />

Chang-Q<strong>in</strong>g Yang, Heng-Jun Gao<br />

Yi Guo, Hai-Na Chai, Chang-Q<strong>in</strong>g Yang, Heng-Jun Gao,<br />

Department <strong>of</strong> <strong>Gastroenterology</strong>, Tongji Hospital Affiliated to<br />

Tongji University, Shanghai 200065, Ch<strong>in</strong>a<br />

Yi Guo, Hai-Na Chai, Heng-Jun Gao, National Eng<strong>in</strong>eer<strong>in</strong>g<br />

Center for Biochip at Shanghai, Shanghai 201203, Ch<strong>in</strong>a<br />

J<strong>in</strong>g Fang, Xiao-Yan Zhang, Ke-Hao Zhang, Hai-Hui Sheng,<br />

National Eng<strong>in</strong>eer<strong>in</strong>g Center for Biochip at Shanghai, Shanghai<br />

201203, Ch<strong>in</strong>a<br />

Yan Liu, Wei J<strong>in</strong>, Department <strong>of</strong> Cardiology, Ruij<strong>in</strong> Hospital,<br />

Shanghai Jiao Tong University School <strong>of</strong> Medic<strong>in</strong>e, Shanghai<br />

200025, Ch<strong>in</strong>a<br />

Author contributions: Guo Y, Fang J, Liu Y and Sheng HH contributed<br />

equally to this work; Gao HJ and Sheng HH designed the<br />

research; Guo Y, Fang J and Chai HN performed all the experiments;<br />

Liu Y, Zhang XY, J<strong>in</strong> W and Zhang KH recruited patients<br />

and collected <strong>in</strong>formation; Guo Y, Fang J and Liu Y analyzed<br />

data and wrote the manuscript; Sheng HH, Yang CQ and Gao HJ<br />

reviewed the manuscript.<br />

Supported by Shanghai “Phosphor” Science Foundation, Ch<strong>in</strong>a,<br />

No. 09QB1403100, the National High Technology Research<br />

and Development Program <strong>of</strong> Ch<strong>in</strong>a, No. 2006AA020704 and<br />

2006AA02A407, the Funds for Key Programs <strong>of</strong> M<strong>in</strong>istry <strong>of</strong><br />

Health <strong>of</strong> Ch<strong>in</strong>a, No. 2008ZX10002-017<br />

Correspondence to: Heng-Jun Gao, Pr<strong>of</strong>essor, National<br />

Eng<strong>in</strong>eer<strong>in</strong>g Center for Biochip at Shanghai, No. 151, Lib<strong>in</strong>g<br />

Road, Shanghai 201203, Ch<strong>in</strong>a. hengjun_gao@shbiochip.com<br />

Telephone: +86-21-51320288 Fax: +86-21-51320287<br />

Received: December 5, 2010 Revised: December 23, 2010<br />

Accepted: December 30, 2010<br />

Published onl<strong>in</strong>e: April 7, 2011<br />

Abstract<br />

AIM: To explore the association between s<strong>in</strong>gle nucleotide<br />

polymorphisms (SNPs) at 8q24 and gastric cancer risk.<br />

METHODS: A case-control <strong>in</strong>vestigation <strong>in</strong>clud<strong>in</strong>g<br />

212 gastric cancer patients and 377 healthy controls<br />

was conducted. The genotypes <strong>of</strong> SNPs (rs6983267,<br />

rs7008482 and rs10808555) were exam<strong>in</strong>ed and established<br />

through polymerase cha<strong>in</strong> reaction-restriction<br />

WJG|www.wjgnet.com<br />

1759<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): 1759-1765<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

fragment length polymorphism (PCR-RFLP). Multivariate<br />

logistic regression models were used to evaluate the association<br />

between SNPs and gastric cancer.<br />

RESULTS: The genotype frequencies <strong>of</strong> rs6983267<br />

<strong>in</strong> gastric cancer patients were obviously different<br />

from those <strong>in</strong> the control (P = 0.005). GT genotype <strong>of</strong><br />

rs6983267 was associated with an <strong>in</strong>creased risk <strong>of</strong> gastric<br />

cancer compared with GG genotype (adjusted odds<br />

ratio = 2.01, 95% confidence <strong>in</strong>terval: 1.28-3.14). Further<br />

stratified analysis <strong>in</strong>dicated that rs6983267 GT genotype<br />

facilitated the risk <strong>of</strong> gastric cancer <strong>of</strong> non-cardiac<br />

and <strong>in</strong>test<strong>in</strong>al type (OR: 2.638, 95% CI: 1.464-4.753;<br />

OR: 1.916, 95% CI: 1.166-3.150, respectively).<br />

CONCLUSION: This study demonstrates for the first<br />

time that rs6983267 is <strong>in</strong>volved <strong>in</strong> susceptibility to<br />

gastric cancer, although further large-sample <strong>in</strong>vestigations<br />

are still needed.<br />

© 2011 Baishideng. All rights reserved.<br />

Key words: Gastric cancer; Genetic susceptibility; S<strong>in</strong>gle<br />

nucleotide polymorphism; MYC; 8q24<br />

Peer reviewer: Dr. Guang-Wen Cao, MD, PhD, Pr<strong>of</strong>essor and<br />

Chairman, Department <strong>of</strong> Epidemiology, The Second Military<br />

Medical University, 800 Xiangy<strong>in</strong> Road, Shanghai 200433, Ch<strong>in</strong>a<br />

Guo Y, Fang J, Liu Y, Sheng HH, Zhang XY, Chai HN, J<strong>in</strong> W,<br />

Zhang KH, Yang CQ, Gao HJ. Association between polymorphism<br />

rs6983267 and gastric cancer risk <strong>in</strong> Ch<strong>in</strong>ese population. <strong>World</strong><br />

J Gastroenterol 2011; 17(13): 1759-1765 Available from: URL:<br />

http://www.wjgnet.com/1007-9327/full/v17/i13/1759.htm DOI:<br />

http://dx.doi.org/10.3748/wjg.v17.i13.1759<br />

INTRODUCTION<br />

Gastric cancer is the second most common cause <strong>of</strong> death<br />

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Guo Y et al . 8q24 variants and gastric cancer risk<br />

from cancer <strong>in</strong> the world [1] and the <strong>in</strong>cidence rate was 16.2<br />

per 100 000 [2] . Despite <strong>of</strong> a marked decrease <strong>in</strong> gastric<br />

cancer mortality rate <strong>in</strong> many countries, there is a higher<br />

prevalence <strong>of</strong> gastric cancer <strong>in</strong> the Ch<strong>in</strong>ese population than<br />

<strong>in</strong> other races. No doubt, either a high absolute number or<br />

a high mortality <strong>of</strong> gastric cancer has become a key public<br />

health issue <strong>in</strong> Ch<strong>in</strong>a.<br />

Although numerous biological and epidemiological<br />

studies have shown risk factors for gastric cancer, the<br />

available knowledge is still <strong>in</strong>sufficient to reveal the exact<br />

mechanism <strong>of</strong> gastric cancer. Current researches have<br />

shown that both genetic and environmental factors play<br />

an important role <strong>in</strong> gastric carc<strong>in</strong>ogenesis [3,4] and genetic<br />

susceptibility accounts for 35% <strong>of</strong> disease etiology [5] . Recently,<br />

the association between variants at 8q24 and breast,<br />

prostate and colorectal cancers has been discovered and<br />

confirmed by several research groups [6-15] , which suggested<br />

a complex contribution <strong>of</strong> polymorphisms at 8q24 to the<br />

formation <strong>of</strong> multiple adenomas. However, whether these<br />

common variants <strong>in</strong> 8q24 are also associated with the risk<br />

<strong>of</strong> gastric cancer has so far not been published.<br />

In the present study, we conducted a case-control association<br />

study to evaluate the effect <strong>of</strong> rs6983267, rs7008482<br />

and rs10808555 <strong>in</strong> 8q24 <strong>in</strong> the risk <strong>of</strong> gastric cancer <strong>in</strong> the<br />

Ch<strong>in</strong>ese population.<br />

MATERIALS AND METHODS<br />

Subjects<br />

A total number <strong>of</strong> 216 cases and 400 controls were enrolled<br />

from January 2009 to January 2010 <strong>in</strong> Tongji Hospital,<br />

Shanghai. Among the 1360 subjects who were <strong>in</strong>vited<br />

to take part <strong>in</strong> this study, only 45% <strong>in</strong>dividuals agreed to<br />

participate and donated 3 mL venous blood sample. All the<br />

gastric cancer cases had been checked by the gastroscopy<br />

and diagnosed by the specialized physician. The exclusion<br />

criteria <strong>of</strong> cases <strong>in</strong>cluded: (1) Hav<strong>in</strong>g a history <strong>of</strong> any other<br />

cancers or any metastasized cancer (carc<strong>in</strong>omas were not<br />

orig<strong>in</strong>ally from stomach); and (2) Hav<strong>in</strong>g undergone radiotherapy<br />

or chemotherapy. Controls were randomly selected<br />

among the first-visit outpatients who were confirmed to<br />

have no cancer or a prior history <strong>of</strong> neoplasm. Available<br />

basel<strong>in</strong>e characteristics, <strong>in</strong>clud<strong>in</strong>g age, gender, race, tumor<br />

location, histological type, were recorded. All the subjects<br />

were genetically unrelated ethnic Han Ch<strong>in</strong>ese. This study<br />

was approved by the <strong>in</strong>stitutional review board <strong>of</strong> Tongji<br />

University School <strong>of</strong> Medic<strong>in</strong>e. Written <strong>in</strong>formed consent<br />

was obta<strong>in</strong>ed from all participants.<br />

Genotyp<strong>in</strong>g<br />

Accord<strong>in</strong>g to the manufacturer’s protocol, we used Flexi<br />

Gene DNA Kit (Qiagen, Hilden, Germany) to extract<br />

genomic DNA from peripheral blood leukocytes <strong>of</strong> the<br />

subjects and stored extracted DNA at -20℃. Unique<br />

primer sequences were designed <strong>in</strong> the website <strong>of</strong> primer3<br />

(http://frodo.wi.mit.edu/primer3/<strong>in</strong>put.htm) and primer<br />

sequences for rs6983267, rs7008482 and rs10808555 were<br />

as follows: 5'-ATGAAGGCGTCGTCCAAATGA-3'<br />

WJG|www.wjgnet.com<br />

(forward) and 5'-TTGGCTGGCACTGTCTGTATA-3'<br />

(reverse); 5'-CCAAGCAGAGAGGAACCAACT-3'<br />

(forward) and 5'-GCCACCCTTTATTCTCCAACC-3'<br />

(reverse); 5'-ATATGGTCCCTGCCCTCAAG-3' (forward)<br />

and 5'-CACTGTGCTAAAGGAATCAGCAA-3'<br />

(reverse), respectively. Polymorphisms genotyp<strong>in</strong>g was<br />

performed by polymerase cha<strong>in</strong> reaction-restriction fragment<br />

length polymorphism (PCR-RFLP). Amplification<br />

reactions were carried out <strong>in</strong> a total volume <strong>of</strong> 15 μL<br />

conta<strong>in</strong><strong>in</strong>g 0.3 mmol/L <strong>of</strong> each deoxynucleoside triphosphate,<br />

10 mmol/L Tris-HCl, 50 mmol/L KCl, 2 mmol/L<br />

MgCl2, 20% Q solution (Qiagen, Hilden, Germany),<br />

0.16 μmol/L <strong>of</strong> each primer, 10 ng genomic DNA, and<br />

1 U Taq (TaKaRa, Otsu, Shiga, Japan). Cycl<strong>in</strong>g conditions<br />

were: 94℃ for 3 m<strong>in</strong>, followed by 10 cycles <strong>of</strong> 94℃ for 30 s,<br />

64℃ for 30 s with a 0.5℃ decrement <strong>of</strong> the anneal<strong>in</strong>g<br />

temperature per cycle and 72℃ for 30-45 s, followed by<br />

30 cycles <strong>of</strong> 94℃ for 30 s, 59℃ for 30 s and 72℃ for 30 s,<br />

followed by 72℃ for 8 m<strong>in</strong>. PCR products were digested<br />

overnight at 37℃ with a predicted restriction enzyme, Tsp45I<br />

(Fermentas, Vilnius, Lithuania) for rs6983267, CviQI<br />

(Fermentas, Vilnius, Lithuania) for rs7008482, Eco130I<br />

(Fermentas, Vilnius, Lithuania) for rs10808555 and were<br />

analyzed on 3% agarose with ethidium bromide sta<strong>in</strong><strong>in</strong>g.<br />

Three sorts <strong>of</strong> PCR products were digested <strong>in</strong>to 3 different<br />

types <strong>of</strong> fragments. For rs6983267, the G allele resulted<br />

<strong>in</strong> two fragments <strong>of</strong> 198-bp and 344-bp, and the C allele<br />

produced one fragment <strong>of</strong> 498-bp. For rs7008482, the<br />

G allele resulted <strong>in</strong> two fragments <strong>of</strong> 270-bp and 131-bp,<br />

while the T allele produced one fragment <strong>of</strong> 401-bp. For<br />

rs10808555, the G allele digested <strong>in</strong>to two fragments <strong>of</strong><br />

193-bp and 110-bp, and the A allele generated one fragment<br />

<strong>of</strong> 303-bp.<br />

All the samples were assayed bl<strong>in</strong>dly without know<strong>in</strong>g<br />

the case or control status. After genotyp<strong>in</strong>g was performed,<br />

two research assistants read the gel pictures <strong>in</strong>dependently.<br />

When they failed to reach a consensus on the tested genotypes<br />

(< 1%), they would repeat the genotyp<strong>in</strong>g aga<strong>in</strong> so<br />

as to achieve a f<strong>in</strong>al consensus. To ensure the genotyp<strong>in</strong>g<br />

accuracy, randomly selected PCR products were reevaluated<br />

by DNA sequenc<strong>in</strong>g [16] . In addition, 5% <strong>of</strong> all samples<br />

were randomly selected and genotyped <strong>in</strong> duplicate, and the<br />

results were 100% concordant.<br />

Statistical analysis<br />

Hardy-We<strong>in</strong>berg equilibrium was tested us<strong>in</strong>g the twosided<br />

χ 2 . χ 2 test was used to compare genotype frequency<br />

and demographic distributions between cases and controls.<br />

Multivariate unconditional logistic regression was<br />

used to calculate odds ratios (OR) and 95% confidence<br />

<strong>in</strong>tervals (CIs) for the association between genotypes and<br />

gastric cancer, adjust<strong>in</strong>g for age and gender. The co-dom<strong>in</strong>ant<br />

and the dom<strong>in</strong>ant models were used for the analysis.<br />

In the co-dom<strong>in</strong>ant model, each SNP was separated <strong>in</strong>to<br />

three categories, 1 for each genotype, with one genotype<br />

chosen as the reference group. For the dom<strong>in</strong>ant model,<br />

each SNP was modeled as a dichotomous variable with<br />

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Table 1 Characteristics <strong>of</strong> cases and control<br />

Variables Gastric cancer n (%) Control n (%) P value 2<br />

Overall 212 377<br />

Sex 0.063<br />

Male 152 (71.7) 242 (64.2)<br />

Female 60 (28.3) 135 (35.8)<br />

Age 0.670<br />

Mean ± SD (yr) 62.47 ± 11.6 62.89 ± 11.3<br />

Histological types<br />

Intest<strong>in</strong>al 155 (73.1)<br />

Diffuse 44 (20.8)<br />

Mixed 13 (6.1)<br />

Tumor location 1<br />

Cardia 74 (38.5)<br />

Noncardia 118 (61.5)<br />

1 The number <strong>of</strong> subjects <strong>in</strong> cases for tumor location (n = 192) was less than<br />

the total number (n = 212) because some <strong>in</strong>formation was not obta<strong>in</strong>ed;<br />

2 Two-sided χ 2 test for the frequency distribution <strong>of</strong> variants between<br />

gastric cancer cases and controls.<br />

1 genotype chosen as the reference group, and the other<br />

two genotypes comb<strong>in</strong>ed <strong>in</strong>to one category. All tests were<br />

two-sided and P values < 0.05 were considered statistically<br />

significant. All statistical analyses were performed us<strong>in</strong>g<br />

SPSS 16.0 s<strong>of</strong>tware package (SPSS, Chicago, IL)<br />

RESULTS<br />

Demography<br />

Among 216 gastric cancer cases and 400 controls, 4 cases<br />

and 23 controls were dropped out due to poor-quality<br />

genomic DNA. Of the 212 cases <strong>of</strong> gastric cancer, 155<br />

were <strong>in</strong>test<strong>in</strong>al type, 44 diffuse type and 13 mixed-type.<br />

The mean age <strong>of</strong> cases was 62 years and the mean age <strong>of</strong><br />

controls was 63 years. The characteristics <strong>of</strong> the cases and<br />

controls are summarized <strong>in</strong> Table 1. There was no significant<br />

difference between the groups with respect to the age<br />

and gender distributions.<br />

SNPs and gastric cancer risk<br />

Among the controls, the genotype distributions <strong>of</strong><br />

rs6983267 and rs7008482 were <strong>in</strong> Hardy-We<strong>in</strong>berg equilibrium<br />

(P > 0.1 and P > 0.9, respectively), but rs10808555<br />

did not fit Hardy-We<strong>in</strong>berg equilibrium (P < 0.05). The<br />

genotype frequencies <strong>of</strong> rs6983267 were obviously different<br />

between gastric cancer patients and control (χ 2 = 10.8,<br />

P = 0.005). Analysis under both co-dom<strong>in</strong>ant model and<br />

dom<strong>in</strong>ant model showed that only rs6983267 was significantly<br />

associated with gastric cancer risk, after adjustment<br />

for age and gender (Table 2). In the co-dom<strong>in</strong>ant model,<br />

rs6983267 GT genotype was associated with approximately<br />

2 times higher odds <strong>of</strong> gastric cancer risk (OR: 2.01,<br />

95% CI: 1.28-3.15) compared with the GG genotype. In<br />

the dom<strong>in</strong>ant model, comb<strong>in</strong>ed genotypes (GT + TT) <strong>of</strong><br />

rs6983267 were significantly associated with <strong>in</strong>creased risk<br />

<strong>of</strong> gastric cancer <strong>in</strong> comparison with GG genotype (OR:<br />

1.82, 95% CI: 1.18-2.81). However, the genotype frequencies<br />

<strong>of</strong> rs7008482 were similar between gastric cancer<br />

WJG|www.wjgnet.com<br />

Table 2 Association between variation <strong>in</strong> s<strong>in</strong>gle nucleotide<br />

polymorphisms rs6983267 and rs708482 and risk <strong>of</strong> gastric<br />

cancer<br />

Genotype Control n (%) Cases n (%) P value<br />

rs6983267<br />

Guo Y et al . 8q24 variants and gastric cancer risk<br />

GT/TT 268 (72.8) 166 (83.0) 0.007<br />

TT 72 (19.6) 32 (16.0) 0.369<br />

GT 196 (53.3) 134 (67.0) 0.002<br />

GG 100 (27.2) 34 (17.0)<br />

rs7008482<br />

GT/GG 224 (61.0) 142 (67.0) 0.138<br />

GG 52 (14.2) 38 (17.9) 0.108<br />

GT 172 (46.9) 104 (49.1) 0.250<br />

TT 143 (39.0) 70 (33.0)<br />

patients and controls (P > 0.05).<br />

Furthermore, we evaluated the contributions <strong>of</strong> SNPs<br />

to subgroups accord<strong>in</strong>g to age, gender, different histological<br />

types and tumor locations (Table 3). In the subgroup<br />

aged ≤ 60 years, rs6983267 GT genotype markedly <strong>in</strong>creased<br />

the risk <strong>of</strong> gastric cancer referr<strong>in</strong>g to GG genotype<br />

(OR: 3.21, 95% CI: 1.52-6.68), but <strong>in</strong> the subgroup aged ><br />

60 years, no significant difference was found (P = 0.137).<br />

In addition, rs6983267 GT genotype was significantly associated<br />

with augmentation <strong>of</strong> gastric cancer risk <strong>in</strong> both<br />

male and female. As to the histological types and tumor<br />

sites, rs6983267 GT heterozygote had a significantly <strong>in</strong>creased<br />

risk for non-cardiac gastric cancer (OR: 2.64, 95%<br />

CI: 1.46-4.75) and <strong>in</strong>test<strong>in</strong>al-type gastric cancer (OR: 1.92,<br />

95% CI: 1.17-3.15) <strong>in</strong> contrast with GG genotype. Further<br />

analysis <strong>in</strong> Table 4 demonstrated that rs6983267 GT genotype<br />

<strong>in</strong>creased the risk <strong>of</strong> an <strong>in</strong>test<strong>in</strong>al-type gastric adenocarc<strong>in</strong>oma<br />

from non-cardiac region. For rs7008482, only<br />

GG genotype was associated with significantly <strong>in</strong>creas<strong>in</strong>g<br />

risk <strong>of</strong> gastric cancer compared with TT genotype <strong>in</strong> male<br />

subgroup (OR: 1.88, 95% CI: 1.01-3.47) (Table 3).<br />

DISCUSSION<br />

This is the first study to discover the association between<br />

rs6983267 at 8q24 and the susceptibility <strong>of</strong> gastric cancer,<br />

although other previous studies had reported that<br />

rs6983267 was associated with the risk <strong>of</strong> colorectal cancer<br />

and prostate cancer [13,17] . Our observation and analysis <strong>in</strong>dicated<br />

that compared with GG genotype <strong>of</strong> rs6983267, GT<br />

genotype and comb<strong>in</strong>ed genotypes (GT + TT) were both<br />

markedly associated with the <strong>in</strong>creas<strong>in</strong>g risk for gastric<br />

cancer. And further stratified <strong>in</strong>vestigation confirmed that<br />

rs6983267 GT genotype facilitated the risk <strong>of</strong> non-cardiac<br />

and <strong>in</strong>test<strong>in</strong>al-type gastric cancer. Therefore, rs6983267 is<br />

a novel gastric cancer associated polymorphism <strong>in</strong> 8q24 <strong>in</strong><br />

Ch<strong>in</strong>ese Han population.<br />

Rs6983267 resides at 8q24, proximal to a processed<br />

pseudogene, POU5F1P1, which is a retrotransposed copy<br />

<strong>of</strong> the POU-doma<strong>in</strong> transcription factor Oct4 [18] . At least<br />

one mouse Oct4 pseudogene has been shown to mediate<br />

stem cell regulatory function [19] , suggest<strong>in</strong>g that Oct4<br />

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Guo Y et al . 8q24 variants and gastric cancer risk<br />

Table 3 Association between rs6983267 and rs7008482 polymorphism and cl<strong>in</strong>icopathological features <strong>of</strong> gastric cancer<br />

Age (yr)<br />

pseudogene may exert <strong>in</strong>fluence <strong>in</strong> regulat<strong>in</strong>g stem cell<br />

proliferation [7] . Oct4 also plays a critical role <strong>in</strong> ma<strong>in</strong>ta<strong>in</strong><strong>in</strong>g<br />

stem cell pluripotency [20] , self-renewal, and l<strong>in</strong>eage<br />

commitment [21] . Oct4 has been found to promote tumor<br />

growth <strong>in</strong> a dose-dependent manner [22] and epithelial dysplasia<br />

by <strong>in</strong>terfer<strong>in</strong>g with progenitor cell differentiation [23] .<br />

Although the expression <strong>of</strong> many Oct4 pseudogenes <strong>in</strong><br />

poorly differentiated tumors [24] has been observed, the<br />

related molecular mechanism <strong>in</strong> cancer is unknown.<br />

On the other hand, rs6983267 is located <strong>in</strong> the region<br />

which is 335 kb away from the nearest gene, MYC.<br />

MYC is able to <strong>in</strong>crease the growth and proliferation <strong>of</strong><br />

normal gastric cells [25] , and may enhance the canceration<br />

<strong>of</strong> gastric epithelial cells by regulat<strong>in</strong>g a variety <strong>of</strong> genes<br />

related to proliferation, differentiation [26] , and apoptosis [27] .<br />

WJG|www.wjgnet.com<br />

rs6983267 rs7008482<br />

GG GT TT TT GT GG<br />

HC/GC HC/GC OR P value HC/GC OR P value HC/GC HC/GC OR P value HC/GC OR<br />

(95% CI)<br />

(95% CI)<br />

(95% CI)<br />

(95% CI)<br />

≤ 60 46/11 76/59 3.21<br />

(1.52-6.77)<br />

> 60<br />

Sex<br />

54/23 120/75 1.54<br />

(0.87-2.74)<br />

Male 60/26 125/94 1.77<br />

(1.04-3.02)<br />

Female 40/8 71/40 3.68<br />

(1.49-9.06)<br />

Histological types<br />

Intest<strong>in</strong>al 100/26 196/98 1.92<br />

(1.17-3.15)<br />

Diffuse 100/7 196/25 1.93<br />

(0.80-4.66)<br />

Mixed<br />

Location<br />

100/1 196/11 5.56<br />

(0.71-44.52)<br />

Cardia 100/16 196/40 1.26<br />

(0.67-2.38)<br />

Noncardia 100/16 196/82 2.64<br />

(1.46-4.75)<br />

0.002 29/13 1.86<br />

(0.73-4.72)<br />

0.137 43/19 1.05<br />

(0.50-2.16)<br />

0.036 48/23 1.10<br />

(0.56-2.17)<br />

0.005 24/9 2.22<br />

(0.73-6.74)<br />

0.010 72/23 1.20<br />

(0.63-2.28)<br />

0.144 72/8 1.74<br />

(0.60-5.09)<br />

0.103 72/1 1.37<br />

(0.08-21.06)<br />

0.469 72/14 1.18<br />

(0.54-2.58)<br />

0.001 72/12 1.06<br />

(0.47-2.38)<br />

0.191 66/30 71/46 1.42<br />

(0.80-2.51)<br />

0.901 77/40 101/58 1.15<br />

(0.69-1.90)<br />

0.783 98/46 107/77 1.52<br />

(0.96-2.41)<br />

0.160 45/24 65/27 0.83<br />

(0.42-1.65)<br />

0.571 143/52 172/73 1.17<br />

(0.77-1.78)<br />

0.309 143/15 172/23 1.36<br />

(0.68-2.71)<br />

0.827 143/3 172/8 2.29<br />

(0.63-9.43)<br />

0.681 143/25 172/35 1.18<br />

(0.67-2.07)<br />

0.888 14/39 172/59 1.28<br />

(0.81-2.04)<br />

0.230 15/11 1.61<br />

(0.66-3.92)<br />

0.592 37/27 1.44<br />

(0.77-2.70)<br />

0.072 32/29 1.88<br />

(1.01-3.47)<br />

0.599 20/9 0.98<br />

(0.38-2.54)<br />

0.047 52/30 1.56<br />

(0.89-2.72)<br />

0.390 52/6 1.26<br />

(0.46-3.47)<br />

0.228 52/2 1.95<br />

(0.34-13.47)<br />

0.574 52/14 1.52<br />

(0.73-3.18)<br />

0.295 52/20 1.47<br />

(0.78-2.77)<br />

Multivariate unconditional logistic regression was used to calculate odds ratios (OR) and 95% confidence <strong>in</strong>tervals (CIs) for the association between<br />

genotypes and gastric cancer, adjust<strong>in</strong>g for age and gender. HC: Health control; GC: Gastric cancer.<br />

Table 4 Stratified analysis <strong>of</strong> rs6983267 genotypes and gastric cancer<br />

Control n (%) Intest<strong>in</strong>al case Diffuse case<br />

P value<br />

0.295<br />

0.258<br />

0.045<br />

0.967<br />

0.118<br />

0.651<br />

0.474<br />

0.263<br />

0.228<br />

n (%) OR (95% CI) n (%) OR (95% CI)<br />

Cardia<br />

TT 72 (19.6) 12 (22.2) 1.243 (0.53-2.90) 1 (8.3) 0.44 (0.04-4.31)<br />

GT 196 (53.3) 29 (53.7) 1.131 (0.56-2.28) 8 (66.7) 1.36 (0.35-5.28)<br />

GG 100 (27.1) 13 (24.1) 3 (25)<br />

Noncardia<br />

TT 72 (19.6) 8 (9.6) 0.99 (0.38-2.59) 4 (16.7) 1.91 (0.44-8.25)<br />

GT 196 (53.3) 64 (77.1) 2.95 (1.49-5.86) 16 (66.6) 2.56 (0.80-8.17)<br />

GG 100 (27.1) 11 (13.3) 4 (16.7)<br />

MYC overexpression has been described <strong>in</strong> over 40% <strong>of</strong><br />

gastric cancer (<strong>in</strong> both <strong>in</strong>test<strong>in</strong>al- and diffuse-type gastric<br />

adenocarc<strong>in</strong>oma) [28] . Overexpression <strong>of</strong> MYC gene can<br />

<strong>in</strong>fluence some biological characteristics <strong>of</strong> normal gastric<br />

cells, directly regulate the genes <strong>in</strong>volved <strong>in</strong> cell cycle regulation<br />

[29] , such as cycl<strong>in</strong> A, cycl<strong>in</strong> B and cdk4 [30] , and accelerate<br />

cancerous growth ultimately. The promotion <strong>of</strong> the<br />

growth and proliferation <strong>of</strong> these cells helps tumor cells<br />

ma<strong>in</strong>ta<strong>in</strong> malignant phenotype. Moreover, the therapeutic<br />

medic<strong>in</strong>e <strong>in</strong>hibits gastric cancer cell growth by suppress<strong>in</strong>g<br />

MYC gene expressions, which consistently confirms the<br />

crucial function <strong>of</strong> MYC <strong>in</strong> gastric cancer cell growth [31] .<br />

The region harbor<strong>in</strong>g rs6983267 is a transcriptional enhancer<br />

and differentially b<strong>in</strong>ds transcription factor 7-like 2<br />

(TCF7L2) due to rs6983267, lead<strong>in</strong>g to a different physi-<br />

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cal <strong>in</strong>teraction with MYC [32] . Given that the cancer riskassociated<br />

SNP enhances the expression <strong>of</strong> MYC through<br />

<strong>in</strong>creased distal enhancer activity [32,33] , it is reasonable to<br />

speculate that rs6983267 may alter expression <strong>of</strong> MYC<br />

through modify<strong>in</strong>g regulatory sequences <strong>in</strong> this region.<br />

Despite the research progress, further studies are needed<br />

about the concrete molecular mechanisms <strong>of</strong> the jo<strong>in</strong>t effect<br />

between MYC and rs6983267 polymorphism.<br />

Previous studies have demonstrated that rs6983267 is<br />

possibly related to some k<strong>in</strong>ds <strong>of</strong> malignant tumor. In the<br />

present study we found that rs6983267 is a novel gastric<br />

cancer related polymorphism. Stratified analysis <strong>in</strong>dicated<br />

that the associations between rs6983267 GT genotype<br />

and gastric cancer tended to vary with tumor sites and<br />

histological types. Rs6983267 GT genotype was associated<br />

with both <strong>in</strong>test<strong>in</strong>al and non-cardiac type <strong>of</strong> gastric cancer<br />

but not associated with the diffuse and cardiac type, and<br />

<strong>in</strong>creased the risk <strong>of</strong> <strong>in</strong>test<strong>in</strong>al type among the non-cardiac<br />

gastric cancer, which suggested that rs6983267 GT genotype<br />

is more important <strong>in</strong> modulat<strong>in</strong>g the <strong>in</strong>test<strong>in</strong>al and<br />

non-cardiac type <strong>of</strong> gastric cancer. However, TT genotype<br />

<strong>in</strong> rs6983267 tended to be a protective factor <strong>in</strong> <strong>in</strong>test<strong>in</strong>al<br />

type among the non-cardial gastric cancer although this was<br />

not significant <strong>in</strong> the association analysis. This phenomenon<br />

could be expla<strong>in</strong>ed, because dist<strong>in</strong>ct cl<strong>in</strong>ical, epidemiological<br />

and molecular features have been noted among<br />

tumors aris<strong>in</strong>g from cardia or non-cardia, and among<br />

<strong>in</strong>test<strong>in</strong>al or diffuse histological subtypes [34] . For <strong>in</strong>stance,<br />

the loss <strong>of</strong> p16 and smad4 prote<strong>in</strong> expression and the<br />

positive EPste<strong>in</strong>-Barr virus (EBV) status are more frequent<br />

<strong>in</strong> cardiac carc<strong>in</strong>omas than that <strong>in</strong> non-cardiac carc<strong>in</strong>omas<br />

reported by Kim et al [35] . Lu et al [36] reported that <strong>in</strong>test<strong>in</strong>altype<br />

gastric cancer predom<strong>in</strong>ates <strong>in</strong> high-risk geographic<br />

areas, especially <strong>in</strong> Japan, Korea and Ch<strong>in</strong>a, whereas the<br />

diffuse-type gastric cancer has a uniform geographic distribution.<br />

The observed differences between gastric cancers<br />

<strong>in</strong> tumor location and histological types suggest that they<br />

are dist<strong>in</strong>ct diseases with different etiologies [37] . Thus, various<br />

genetic factors, <strong>in</strong>clud<strong>in</strong>g rs6983267, may be <strong>in</strong>volved<br />

<strong>in</strong> different subtypes <strong>of</strong> gastric cancer (cardiac or noncardiac;<br />

<strong>in</strong>test<strong>in</strong>al or diffuse). Another plausible explanation<br />

for this situation may be the genetic heterogeneity which<br />

may limit the ability to detect an association between TT<br />

genotype and gastric cancer. Other variants, which have a<br />

strong association with risk <strong>of</strong> gastric cancer, <strong>in</strong>clud<strong>in</strong>g as<br />

yet undiscovered susceptibility genes, may affect the outcome<br />

<strong>of</strong> this research. Moreover, the limited sample size<br />

may be not sufficient to generate this association. Overall,<br />

the genetic susceptibility and environmental factors have<br />

been proposed to play an important role <strong>in</strong> the etiology <strong>of</strong><br />

gastric cancer, and different subtypes <strong>of</strong> gastric cancer may<br />

have diverse biological mechanisms.<br />

Apart from the discovery <strong>in</strong> rs6983267, this study failed<br />

to demonstrate the association between rs7008482 and the<br />

risk <strong>of</strong> gastric cancer, although rs7008482 was reported to<br />

be associated with prostate and colorectal cancer [10,11] . Nevertheless,<br />

positive association between rs7008482 GG genotype<br />

and risk <strong>of</strong> gastric cancer <strong>in</strong> male subgroup has been<br />

WJG|www.wjgnet.com<br />

Guo Y et al . 8q24 variants and gastric cancer risk<br />

shown. Rs7008482 lies with<strong>in</strong> an <strong>in</strong>tronic region <strong>of</strong> the<br />

NSMCE2 (also called MMS21) gene, and MMS21 prote<strong>in</strong><br />

is a SUMO ligase which is required for DNA replication,<br />

recomb<strong>in</strong>ation and repair [11] . Consider<strong>in</strong>g the function <strong>of</strong><br />

NSMCE2 gene and MMS21 prote<strong>in</strong>, we wonder whether<br />

the limited sample size hampered the detection <strong>of</strong> association<br />

between rs7008482 and gastric cancer risk. Therefore,<br />

further studies are still needed to confirm it.<br />

In conclusion, our data demonstrated for the first time<br />

that rs6983267 may predispose to the susceptibility <strong>of</strong><br />

gastric cancer, especially the <strong>in</strong>test<strong>in</strong>al and non-cardiac<br />

type. However, as the sample size <strong>of</strong> the present study is<br />

relatively small, additional tests <strong>of</strong> variant at 8q24 for its<br />

association with gastric cancer <strong>in</strong> a larger population, and<br />

functional studies <strong>of</strong> MYC and other nearby genes will<br />

be required to fully understand the mechanisms <strong>of</strong> the<br />

cancer-specific risk at 8q24.<br />

COMMENTS<br />

Background<br />

Gastric cancer (GC) is one <strong>of</strong> the most common cancers, and the second most<br />

frequent cause <strong>of</strong> cancer-related deaths <strong>in</strong> the world. Epidemiological studies<br />

have shown that genetic factors play a crucial role <strong>in</strong> gastric carc<strong>in</strong>ogenesis.<br />

Recently, common polymorphisms located at chromosome 8q24 have been<br />

identified to <strong>in</strong>crease the tumor risk. The authors <strong>in</strong>vestigated the associations<br />

between rs6983267 polymorphisms and GC risk.<br />

Research frontiers<br />

Chromosome 8q24 is an established risk locus for many common epithelial<br />

cancers. Polymorphism rs6983267 is a susceptibility marker for prostate and<br />

<strong>colon</strong> cancers, and perhaps also ovarian and other cancers. The relationship<br />

between rs6983267 polymorphism and GC needs to be addressed.<br />

Innovations and breakthroughs<br />

To our knowledge, this is the first study <strong>of</strong> GC risk variant at 8q24 <strong>in</strong> a Ch<strong>in</strong>ese<br />

population. Polymorphism rs6983267 was found to be associated with an <strong>in</strong>creased<br />

risk <strong>of</strong> GC, which had been not reported before. The result <strong>of</strong> stratified<br />

analysis accord<strong>in</strong>g to histological types confirms the contribution <strong>of</strong> rs6983267<br />

<strong>in</strong> non-cardiac and <strong>in</strong>test<strong>in</strong>al type <strong>of</strong> gastric carc<strong>in</strong>ogenesis.<br />

Applications<br />

These f<strong>in</strong>d<strong>in</strong>gs might be <strong>of</strong> value <strong>in</strong> the explanation <strong>of</strong> gastric carc<strong>in</strong>ogenesis.<br />

They could to be used for further <strong>in</strong>vestigations about the association between<br />

genetic predisposition and the risk <strong>of</strong> GC at 8q24.<br />

Term<strong>in</strong>ology<br />

MYC is an oncogene, the prote<strong>in</strong> encoded by this gene is a multifunctional,<br />

nuclear phosphoprote<strong>in</strong> that plays a role <strong>in</strong> cell cycle progression, apoptosis<br />

and proliferations. S<strong>in</strong>gle nucleotide polymorphism is a DNA sequence variation<br />

occurr<strong>in</strong>g when a s<strong>in</strong>gle nucleotide-A, T, C, or G-<strong>in</strong> the genome (or other shared<br />

sequence) differs between members <strong>of</strong> a species or paired chromosomes <strong>in</strong> an<br />

<strong>in</strong>dividual.<br />

Peer review<br />

The authors <strong>in</strong>vestigated the association <strong>of</strong> the 3 s<strong>in</strong>gle nucleotide polymorphisms<br />

(SNPs) (rs6983267, rs7008482 and rs10808555) with the risk <strong>of</strong> gastric<br />

cancer by a case-control study, and found that GT genotype <strong>of</strong> rs6983267 was<br />

associated with an <strong>in</strong>creased risk <strong>of</strong> gastric cancer compared with GG genotype<br />

(AOR = 2.01, 95% CI: 1.28-3.14). After stratification, rs6983267 GT genotype<br />

was associated the risk <strong>of</strong> non-cardiac and <strong>in</strong>test<strong>in</strong>al type <strong>of</strong> gastric cancer. This<br />

study provides some new SNP for the evaluation <strong>of</strong> gastric cancer risk.<br />

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17 Wasserman NF, Aneas I, Nobrega MA. An 8q24 gene desert<br />

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18 Schöler HR, Ruppert S, Suzuki N, Chowdhury K, Gruss<br />

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19 L<strong>in</strong> H, Shabbir A, Molnar M, Lee T. Stem cell regulatory<br />

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20 Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-<br />

Nebenius D, Chambers I, Schöler H, Smith A. Formation <strong>of</strong><br />

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on the POU transcription factor Oct4. Cell 1998; 95: 379-391<br />

21 Campbell PA, Perez-Iratxeta C, Andrade-Navarro MA, Rudnicki<br />

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23 Hochedl<strong>in</strong>ger K, Yamada Y, Beard C, Jaenisch R. Ectopic<br />

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24 Ben-Porath I, Thomson MW, Carey VJ, Ge R, Bell GW,<br />

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tumors. Nat Genet 2008; 40: 499-507<br />

25 Zhang L, Hou Y, Ashktorab H, Gao L, Xu Y, Wu K, Zhai J,<br />

Zhang L. The impact <strong>of</strong> C-MYC gene expression on gastric<br />

cancer cell. Mol Cell Biochem 2010; 344: 125-135<br />

26 Dang CV, Resar LM, Emison E, Kim S, Li Q, Prescott JE,<br />

Wonsey D, Zeller K. Function <strong>of</strong> the c-Myc oncogenic transcription<br />

factor. Exp Cell Res 1999; 253: 63-77<br />

27 Nilsson JA, Cleveland JL. Myc pathways provok<strong>in</strong>g cell<br />

suicide and cancer. Oncogene 2003; 22: 9007-9021<br />

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29 Rob<strong>in</strong>son K, Asawachaicharn N, Galloway DA, Grandori C.<br />

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stress. PLoS One 2009; 4: e5951<br />

30 Orian A, van Steensel B, Delrow J, Bussemaker HJ, Li L,<br />

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by the Drosophila Myc, Max, Mad/Mnt transcription factor<br />

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Guo Y et al . 8q24 variants and gastric cancer risk<br />

network. Genes Dev 2003; 17: 1101-1114<br />

31 Luo J, Li YN, Wang F, Zhang WM, Geng X. S-adenosylmethion<strong>in</strong>e<br />

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32 Pomerantz MM, Ahmadiyeh N, Jia L, Herman P, Verzi MP,<br />

Doddapaneni H, Beckwith CA, Chan JA, Hills A, Davis M,<br />

Yao K, Kehoe SM, Lenz HJ, Haiman CA, Yan C, Henderson<br />

BE, Frenkel B, Barret<strong>in</strong>a J, Bass A, Tabernero J, Baselga J, Regan<br />

MM, Manak JR, Shivdasani R, Coetzee GA, Freedman<br />

ML. The 8q24 cancer risk variant rs6983267 shows longrange<br />

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33 Haiman CA, Le Marchand L, Yamamato J, Stram DO, Sheng<br />

X, Kolonel LN, Wu AH, Reich D, Henderson BE. A common<br />

genetic risk factor for colorectal and prostate cancer. Nat<br />

Genet 2007; 39: 954-956<br />

34 Wu X, Zeng Z, Chen B, Yu J, Xue L, Hao Y, Chen M, Sung JJ,<br />

Hu P. Association between polymorphisms <strong>in</strong> <strong>in</strong>terleuk<strong>in</strong>-<br />

17A and <strong>in</strong>terleuk<strong>in</strong>-17F genes and risks <strong>of</strong> gastric cancer.<br />

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35 Kim MA, Lee HS, Yang HK, Kim WH. Cl<strong>in</strong>icopathologic<br />

and prote<strong>in</strong> expression differences between cardia carc<strong>in</strong>oma<br />

and noncardia carc<strong>in</strong>oma <strong>of</strong> the stomach. Cancer 2005; 103:<br />

1439-1446<br />

36 Lu Y, Chen J, D<strong>in</strong>g Y, J<strong>in</strong> G, Wu J, Huang H, Deng B, Hua<br />

Z, Zhou Y, Shu Y, Liu P, Hu Z, Shen J, Xu Y, Shen H. Genetic<br />

variation <strong>of</strong> PSCA gene is associated with the risk <strong>of</strong><br />

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population. Int J Cancer 2010; 127: 2183-2189<br />

37 Crew KD, Neugut AI. Epidemiology <strong>of</strong> gastric cancer. <strong>World</strong><br />

J Gastroenterol 2006; 12: 354-362<br />

S- Editor Tian L L- Editor Ma JY E- Editor Ma WH<br />

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Onl<strong>in</strong>e Submissions: http://www.wjgnet.com/1007-9327<strong>of</strong>fice<br />

wjg@wjgnet.com<br />

doi:10.3748/wjg.v17.i13.1766<br />

EUS for choos<strong>in</strong>g best endoscopic treatment <strong>of</strong><br />

mesenchymal tumors <strong>of</strong> upper gastro<strong>in</strong>test<strong>in</strong>al tract<br />

X<strong>in</strong>-X<strong>in</strong> Zhou, Feng Ji, Liang Xu, L<strong>in</strong> Li, Yi-Peng Chen, J<strong>in</strong>g-J<strong>in</strong>g Lu, Chun-Wei Wang, Wei Huang<br />

X<strong>in</strong>-X<strong>in</strong> Zhou, Feng Ji, Liang Xu, L<strong>in</strong> Li, Yi-Peng Chen,<br />

Department <strong>of</strong> <strong>Gastroenterology</strong>, the First Affiliated Hospital,<br />

College <strong>of</strong> Medic<strong>in</strong>e, Zhejiang University, Hangzhou 310003,<br />

Zhejiang Prov<strong>in</strong>ce, Ch<strong>in</strong>a<br />

J<strong>in</strong>g-J<strong>in</strong>g Lu, Department <strong>of</strong> <strong>Gastroenterology</strong>, Beilun Zongrui<br />

Hospital, N<strong>in</strong>gbo 315806, Zhejiang Prov<strong>in</strong>ce, Ch<strong>in</strong>a<br />

Chun-Wei Wang, Department <strong>of</strong> <strong>Gastroenterology</strong>, the Traditional<br />

Ch<strong>in</strong>ese Medic<strong>in</strong>e Hospital <strong>of</strong> N<strong>in</strong>ghai, N<strong>in</strong>ghai 315600,<br />

Zhejiang Prov<strong>in</strong>ce, Ch<strong>in</strong>a<br />

Wei Huang, Department <strong>of</strong> <strong>Gastroenterology</strong>, J<strong>in</strong>hua Wenrong<br />

Hospital, J<strong>in</strong>hua 321017, Zhejiang Prov<strong>in</strong>ce, Ch<strong>in</strong>a<br />

Author contributions: Ji F and Zhou XX designed the research<br />

and wrote the paper; Ji F, Zhou XX, Xu L, Li L, Chen YP, Lu<br />

JJ, Wang CW and Huang W performed the research; Zhou XX<br />

and Xu L analyzed the data.<br />

Correspondence to: Feng Ji, MD, PhD, Department <strong>of</strong> <strong>Gastroenterology</strong>,<br />

the First Affiliated Hospital, College <strong>of</strong> Medic<strong>in</strong>e,<br />

Zhejiang University, Hangzhou 310003, Zhejiang Prov<strong>in</strong>ce,<br />

Ch<strong>in</strong>a. jifeng1126@s<strong>in</strong>a.com<br />

Telephone: +86-571-87236568 Fax: +86-571-87236611<br />

Received: October 19, 2010 Revised: November 17, 2010<br />

Accepted: November 24, 2010<br />

Published onl<strong>in</strong>e: April 7, 2011<br />

Abstract<br />

AIM: To evaluate the value <strong>of</strong> endoscopic ultrasonography<br />

(EUS) <strong>in</strong> the choice <strong>of</strong> endoscopic therapy strategies<br />

for mesenchymal tumors <strong>of</strong> the upper gastro<strong>in</strong>test<strong>in</strong>al<br />

tract.<br />

METHODS: From July 2004 to September 2010, 1050<br />

patients with upper gastro<strong>in</strong>test<strong>in</strong>al mesenchymal tumors<br />

(GIMTs) were diagnosed us<strong>in</strong>g EUS. Among them,<br />

201 patients underwent different endoscopic therapies<br />

based on the deriv<strong>in</strong>g layers, growth patterns and lesion<br />

sizes.<br />

RESULTS: Us<strong>in</strong>g EUS, we found 543 leiomyomas and<br />

507 stromal tumors. One hundred and thirty-three leiomyomas<br />

and 24 stromal tumors were treated by snare<br />

electrosection, 6 leiomyomas and 20 stromal tumors<br />

WJG|www.wjgnet.com<br />

1766<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): 1766-1771<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

BRIEF ARTICLE<br />

were treated by endoloop, 10 stromal tumors were<br />

treated by endoscopic mucosal resection and 8 stromal<br />

tumors were treated by endoscopic submucosal dissection.<br />

Complete resection <strong>of</strong> the lesion was achieved <strong>in</strong><br />

all cases. Of the mesenchymal tumors, 90.38% diagnosed<br />

by EUS were also identified by pathohistology.<br />

All wounds were closed up nicely and no recurrence<br />

was found <strong>in</strong> the follow-up after 2 mo.<br />

CONCLUSION: EUS is an effective means <strong>of</strong> diagnosis<br />

for upper GIMTs and is an important tool <strong>in</strong> choos<strong>in</strong>g<br />

the endoscopic therapy for GIMTs, by which the lesions<br />

can be treated safely and effectively.<br />

© 2011 Baishideng. All rights reserved.<br />

Key words: Leiomyoma; Stromal tumor; Endoscopic<br />

ultrasonography; Endoscopic therapy<br />

Peer reviewer: Massimo Raimondo, Division <strong>of</strong> <strong>Gastroenterology</strong><br />

and Hepatology, Mayo Cl<strong>in</strong>ic, 4500 San Pablo Road,<br />

Jacksonville, FL 32224, United States<br />

Zhou XX, Ji F, Xu L, Li L, Chen YP, Lu JJ, Wang CW, Huang<br />

W. EUS for choos<strong>in</strong>g best endoscopic treatment <strong>of</strong> mesenchymal<br />

tumors <strong>of</strong> upper gastro<strong>in</strong>test<strong>in</strong>al tract. <strong>World</strong> J Gastroenterol<br />

2011; 17(13): 1766-1771 Available from: URL: http://www.wjgnet.com/1007-9327/full/v17/i13/1766.htm<br />

DOI: http://dx.doi.<br />

org/10.3748/wjg.v17.i13.1766<br />

INTRODUCTION<br />

Gastro<strong>in</strong>test<strong>in</strong>al mesenchymal tumors (GIMTs) orig<strong>in</strong>ate<br />

from mesenchymal cells other than epithelial cells or lymphocytes.<br />

They are further classified as stromal tumors,<br />

leiomyomas, leiomyosarcomas, neural tumors, fibroblast<br />

tumors or liparomphalus. Cl<strong>in</strong>ically, mesenchymal tumors<br />

are usually <strong>in</strong>cidentally discovered as subepithelial bulges<br />

dur<strong>in</strong>g rout<strong>in</strong>e endoscopic exam<strong>in</strong>ations for unrelated<br />

conditions. The classification and management <strong>of</strong> these<br />

April 7, 2011|Volume 17|Issue 13|

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lesions can be challeng<strong>in</strong>g. In recent years, with the wide<br />

use <strong>of</strong> endoscopic ultrasound (EUS) to clarify the nature<br />

and orig<strong>in</strong> <strong>of</strong> the subepithelial tumor, great progress has<br />

been made <strong>in</strong> diagnosis and treatment <strong>of</strong> GIMTs [1,2] . Importantly,<br />

under the guidance <strong>of</strong> the EUS, GIMTs can be<br />

removed by appropriate endoscopic treatment without<br />

severe complications [2-4] .<br />

From July 2004 to September 2010, we analyzed 1050<br />

patients with GIMTs diagnosed by EUS <strong>in</strong> our hospital.<br />

Of these patients, 201 underwent different endoscopic<br />

therapies based on the EUS results. Our aim <strong>in</strong> this retrospective<br />

study was to evaluate the value <strong>of</strong> EUS <strong>in</strong> the<br />

choice <strong>of</strong> endoscopic therapy strategies for mesenchymal<br />

tumors <strong>of</strong> the upper gastro<strong>in</strong>test<strong>in</strong>al tract.<br />

MATERIALS AND METHODS<br />

Patients<br />

The medical records <strong>of</strong> 1050 patients with upper GIMTs<br />

diagnosed by EUS exam<strong>in</strong>ation <strong>in</strong> the First Affiliated<br />

Hospital <strong>of</strong> Zhejiang University were retrospectively<br />

reviewed. All these patients with submucosal protrud<strong>in</strong>g<br />

lesions <strong>in</strong> the upper gastro<strong>in</strong>test<strong>in</strong>al tract by rout<strong>in</strong>e<br />

endoscopy were exam<strong>in</strong>ed by EUS. There were 499 men<br />

and 551 women, with a mean age <strong>of</strong> 52.6 years (range,<br />

19-86 years). Of these patients, 201 patients underwent<br />

endoscopic therapy <strong>in</strong> the First Affiliated Hospital <strong>of</strong><br />

Zhejiang University, Beilun Zongrui Hospital, the Traditional<br />

Ch<strong>in</strong>ese Medical Hospital <strong>of</strong> N<strong>in</strong>ghai and J<strong>in</strong>hua<br />

Wenrong Hospital, respectively.<br />

Methods<br />

A two-channel endoscope (GIF-2T240, Olympus, Tokyo,<br />

Japan) and a 12 MHz probe (GF-UM 2R, Olympus,<br />

Tokyo, Japan) were used for the ultrasonographic study.<br />

Scann<strong>in</strong>g <strong>of</strong> the tumor was performed after fill<strong>in</strong>g the<br />

upper gastro<strong>in</strong>test<strong>in</strong>al tract with 100-500 mL <strong>of</strong> deaerated<br />

water. Diagnosis was made accord<strong>in</strong>g to the layer<br />

<strong>of</strong> orig<strong>in</strong>, size, nature, <strong>in</strong>ternal echo pattern, outer marg<strong>in</strong><br />

and grow pattern <strong>of</strong> the lesion. Follow<strong>in</strong>g the EUS<br />

procedure, if the lesion was identified as an <strong>in</strong>tramural<br />

lesion ≤ 2.5 cm, endoscopic treatment was performed.<br />

A lesion > 2.5 cm <strong>in</strong> size and suspected to be malignant<br />

was suggested for surgery. A large proportion <strong>of</strong> patients<br />

were followed up with EUS, because their poor<br />

conditions were unsuited for the therapy or the lesion<br />

was too small.<br />

An Olympus GIF-XQ240/260 gastroscope (Tokyo,<br />

Japan) was used for the resection when it was <strong>in</strong>dicated.<br />

Informed consent was given by each patient before the<br />

endoscopic therapy. Four different resection techniques<br />

were used: snare electrosection, endoloop, endoscopic<br />

mucosal resection (EMR) and endoscopic submucosal<br />

dissection (ESD).<br />

EMR procedure: Ep<strong>in</strong>ephr<strong>in</strong>e (0.001%) was <strong>in</strong>jected<br />

<strong>in</strong>to the submucosal layer to lift the lesion, and then a<br />

conventional electrosurgical snare (FD-IU, Olympus,<br />

Tokyo, Japan) and an electrosurgical unit (VIO 200D,<br />

WJG|www.wjgnet.com<br />

Zhou XX et al . EUS and endoscopic treatment <strong>of</strong> GIMT<br />

ERBE, Tub<strong>in</strong>gen, Germany) were used for removal <strong>of</strong><br />

the overly<strong>in</strong>g mucosa and resection <strong>of</strong> the tumor.<br />

ESD procedure: the surround<strong>in</strong>g area <strong>of</strong> the lesion<br />

was marked with argon plasma coagulation (APC 300A,<br />

ERBE, Tub<strong>in</strong>gen, Germany). Normal sal<strong>in</strong>e solution<br />

with 0.002% <strong>in</strong>digo carm<strong>in</strong>e and 0.001% ep<strong>in</strong>ephr<strong>in</strong>e was<br />

<strong>in</strong>jected <strong>in</strong>to the submucosal layer to lift the lesion. An<br />

<strong>in</strong>itial <strong>in</strong>cision was made outside the mark<strong>in</strong>g dots with<br />

a hook-knife (KD-620LR, Olympus, Tokyo, Japan). The<br />

submucosal resection under the lesion was done with<br />

<strong>in</strong>sulation-tipped (IT) electrosurgical knife (KD-610L,<br />

Olympus, Tokyo, Japan). F<strong>in</strong>ally, a snare was used to remove<br />

the surround<strong>in</strong>g tissues. Bleed<strong>in</strong>g and visible vessels<br />

<strong>in</strong> the resection area were closed us<strong>in</strong>g hemoclips (HX-<br />

201YR-135, Olympus, Tokyo, Japan).<br />

Postoperative EUS exam<strong>in</strong>ation was made to check<br />

whether the lesions were completely removed except<br />

those with endoloop ligation. The specimens were sent<br />

for pathologic study, some <strong>of</strong> which were assayed by<br />

immunohistochemistry. All 201 patients were exam<strong>in</strong>ed<br />

two months later with EUS.<br />

RESULTS<br />

Us<strong>in</strong>g EUS, we identified 1050 patients with GIMTs,<br />

543 <strong>of</strong> them had leiomyomas. Five hundred and twenty<br />

lesions were <strong>in</strong> the esophagus, 22 <strong>in</strong> the stomach, and 1<br />

<strong>in</strong> the duodenum. The mean maximal tumor diameter<br />

was 3.3 cm. Five hundred and seven cases were stromal<br />

tumors. Forty-n<strong>in</strong>e lesions were <strong>in</strong> the esophagus, 428<br />

were <strong>in</strong> the stomach, and 30 were <strong>in</strong> the duodenum. The<br />

mean maximal tumor diameter was 6.6 cm. EUS features<br />

<strong>of</strong> leiomyomas and stromal tumors were characteristic<br />

with regular borders, a hypoechoic mass with homogeneous<br />

or heterogeneous echo patterns (Figures 1-4).<br />

The echogenicity <strong>of</strong> leiomyomas was slightly lower than<br />

the normal proper muscle layer, while that <strong>of</strong> stromal<br />

tumors was slightly higher. Malignant stromal tumors<br />

<strong>of</strong>ten appeared as a heterogeneous mass with irregular<br />

borders.<br />

One hundred and thirty-n<strong>in</strong>e leiomyomas and 62 stromal<br />

tumors underwent endoscopic therapy after EUS exam<strong>in</strong>ation.<br />

No obvious malignant signs were seen <strong>in</strong> these<br />

lesions. The location, orig<strong>in</strong> level and removal methods<br />

<strong>of</strong> the lesions are shown <strong>in</strong> Table 1. The mean maximal<br />

tumor diameter was 2.5 cm. For a tumor protrud<strong>in</strong>g <strong>in</strong>to<br />

the cavity, if it orig<strong>in</strong>ated from the muscularis mucosa or<br />

from submucosa ≤ 1 cm, snare electrosection was directly<br />

used. If the lesion orig<strong>in</strong>at<strong>in</strong>g from submucosa was flat<br />

and > 1 cm, electrosection would be expected to fail, and<br />

other treatments, such as endoloop, EMR or ESD, were<br />

used. For a lesion orig<strong>in</strong>at<strong>in</strong>g from muscularis propria<br />

but not grow<strong>in</strong>g outward, endoloop or ESD was used.<br />

Among these patients, 133 leiomyomas and 24 stromal<br />

tumors were treated by snare electrosection, 6 leiomyomas<br />

and 20 stromal tumors were treated by endoloop, 10 stromal<br />

tumors were treated by EMR and 8 stromal tumors<br />

were treated by ESD (Figures 1-4). Complete resection <strong>of</strong><br />

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A<br />

Figure 1 An esophageal leiomyoma treated by snare electrosection. A: An elevated lesion <strong>in</strong> the lower esophagus; B: A homogeneous, hypoechoic mass (1.0<br />

× 0.7 cm 2 ) with a regular border orig<strong>in</strong>ated from muscularis mucosa, which was diagnosed as a leiomyoma by endoscopic ultrasonography; C: The tumor was snared<br />

at the base, and then it was resected by snare electrosection; D: Postoperative wounds.<br />

WJG|www.wjgnet.com<br />

B C D<br />

A B<br />

C D<br />

Figure 2 A gastric stromal tumor treated by endoscopic mucosal resection. A: An elevated lesion <strong>in</strong> the cardia; B: A homogeneous, hypoechoic mass (1.4 ×<br />

0.8 cm 2 ) with a regular border orig<strong>in</strong>at<strong>in</strong>g from submucosa, which was diagnosed as a stromal tumor by endoscopic ultrasonography; C: Ep<strong>in</strong>ephr<strong>in</strong>e (0.001%) was<br />

<strong>in</strong>jected <strong>in</strong>to the submucosa to lift the lesion; D: Postoperative wounds.<br />

A B<br />

C<br />

Figure 3 A gastric stromal tumor treated by endoloop. A: An elevated lesion <strong>in</strong> gastric fundus; B: A homogeneous, hypoechoic mass (2.0 × 1.5 cm 2 ) with a regular<br />

border orig<strong>in</strong>at<strong>in</strong>g from muscularis propria, which was diagnosed as a stromal tumor by endoscopic ultrasonography; C: Endoscopic ligation with an endoloop; D:<br />

Endoscopic view <strong>of</strong> an ulcer scar without tumor recurrence at the ligation site 2 mo later.<br />

A<br />

Zhou XX et al . EUS and endoscopic treatment <strong>of</strong> GIMT<br />

B C<br />

D<br />

Figure 4 A gastric stromal tumor treated by endoscopic submucosal dissection. A: An elevated lesion <strong>in</strong> the gastric antrum; B: A homogeneous, hypoechoic<br />

mass (2.0 × 1.2 cm 2 ) with a regular border orig<strong>in</strong>at<strong>in</strong>g from muscularis propria, which was diagnosed as a stromal tumor by endoscopic ultrasonography; C: The<br />

surround<strong>in</strong>g area <strong>of</strong> the lesion was marked with argon plasma coagulation. After normal sal<strong>in</strong>e solution with 0.002% <strong>in</strong>digo carm<strong>in</strong>e and 0.001% ep<strong>in</strong>ephr<strong>in</strong>e was<br />

<strong>in</strong>jected <strong>in</strong>to the submucosal layer to lift the lesion, an <strong>in</strong>itial <strong>in</strong>cision was made outside the mark<strong>in</strong>g dots with hook-knife. Submucosal dissection under the lesion<br />

was performed with an IT knife; D: The tumor was dissected and the postoperative wounds were closed us<strong>in</strong>g hemoclip.<br />

D<br />

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Table 1 Location, orig<strong>in</strong> and treatment <strong>of</strong> 201 gastro<strong>in</strong>test<strong>in</strong>al mesenchymal tumors<br />

Diagnosis by EUS Location Layer <strong>of</strong> orig<strong>in</strong> Treatment<br />

the lesion was achieved <strong>in</strong> all cases. No residual lesion<br />

was detected by postoperative EUS exam<strong>in</strong>ation except<br />

for those by endoloop ligation. None <strong>of</strong> the patients<br />

suffered from severe hemorrhage or resection-related<br />

perforation. Postoperative histological results showed<br />

that 141 <strong>of</strong> 156 patients were <strong>in</strong> agreement with the<br />

preoperative diagnosis <strong>of</strong> EUS. All the specimens tested<br />

had complete envelope and negative resection marg<strong>in</strong> <strong>in</strong><br />

pathology. Wounds were closed up nicely <strong>in</strong> all patients<br />

when rechecked after two months. No residual lesion<br />

was detected by EUS exam<strong>in</strong>ation and pathology demonstrated<br />

negative results at the same time.<br />

DISCUSSION<br />

Esophagus Stomach Muscularis mucosa Submucosa Muscularis propria Snare electrosection Endoloop EMR ESD<br />

Leiomyoma 134 5 121 15 3 133 6 0 0<br />

Stromal tumor 22 40 18 19 25 24 20 10 8<br />

Total 156 45 139 34 28 157 26 10 8<br />

EUS: Endoscopic ultrasonography; EMR: Endoscopic mucosal resection; ESD: Endoscopic submucosal dissection.<br />

Leiomyomas and stromal tumors are the most common<br />

GIMTs <strong>of</strong> the upper gastro<strong>in</strong>test<strong>in</strong>al tract. Many lesions<br />

are subepithelial, and they are <strong>of</strong>ten difficult to diagnose<br />

by general endoscopy. Some also need to be identified<br />

with extr<strong>in</strong>sic compression. EUS can reliably characterize<br />

the nature, size, and layer <strong>of</strong> orig<strong>in</strong> <strong>of</strong> lesions, and<br />

accurately differentiate <strong>in</strong>tramural from extramural, lead<strong>in</strong>g<br />

to a diagnosis [5] . Features <strong>of</strong> leiomyomas and stromal<br />

tumors seen with EUS <strong>of</strong>ten <strong>in</strong>clude: a round shape, and<br />

a homogeneous, hypoechoic mass with regular borders [6] .<br />

A marg<strong>in</strong>al halo, hyperechogenic spots and higher echogenicity<br />

as compared with the normal muscle layer is<br />

seen more frequently <strong>in</strong> stromal tumors than <strong>in</strong> the leiomyomas<br />

[7] . Malignant stromal tumors are characterized<br />

by large size (> 5 cm), irregular borders, and echogenic<br />

foci [8,9] .<br />

In this study, we identified 1050 patients with GIMTs<br />

us<strong>in</strong>g EUS. There were 543 leiomyomas and 507 stromal<br />

tumors. The majority <strong>of</strong> leiomyomas were located<br />

<strong>in</strong> the esophagus while most stromal tumors were located<br />

<strong>in</strong> the stomach, which is <strong>in</strong> accordance with other<br />

studies [6-10] . For these mesenchymal tumors, 90.38%<br />

diagnosed by EUS were also identified by pathohistology.<br />

Among these, 5 retention cysts, 4 stromal tumors<br />

with leiomyoma differentiation, and 1 hyperplastic polyp<br />

were diagnosed as leiomyoma, and 3 leiomyoma and 2<br />

hyperplastic polyps were diagnosed as stromal tumors<br />

by EUS. Submucosal retention cysts are small and <strong>of</strong>ten<br />

filled with thick fluid, and thus the ultrasonophic image<br />

is <strong>of</strong> a hypoechoic mass that may be confused with<br />

mesenchymal tumors. Stromal tumors with leiomyoma<br />

differentiation are also difficult to discrim<strong>in</strong>ate by rout<strong>in</strong>e<br />

pathology and should be identified by immuno-<br />

WJG|www.wjgnet.com<br />

Zhou XX et al . EUS and endoscopic treatment <strong>of</strong> GIMT<br />

histochemistry. Samples from EUS-guided f<strong>in</strong>e-needle<br />

aspiration biopsies can be sent for cytological, pathological<br />

and immunohistochemical assays which may enable<br />

cl<strong>in</strong>icians to make more accurate diagnoses than us<strong>in</strong>g<br />

EUS exam<strong>in</strong>ation alone [6-11] .<br />

In the past, conventional endoscopy could not accurately<br />

determ<strong>in</strong>e the location and categorization <strong>of</strong> subepithelial<br />

lesions. Therefore, GIMTs were usually treated by surgery.<br />

The <strong>in</strong>troduction <strong>of</strong> EUS has solved these problems and it<br />

has played an important role <strong>in</strong> the choice <strong>of</strong> endoscopic<br />

therapy for mesenchymal tumors. Based on EUS images,<br />

we treated 201 GIMTs with different endoscopic therapies,<br />

<strong>in</strong>clud<strong>in</strong>g snare electrosection, endoloop, EMR and ESD.<br />

Complete resection <strong>of</strong> the lesions was achieved <strong>in</strong> all cases.<br />

None <strong>of</strong> the patients suffered from severe hemorrhage or<br />

resection-related perforation. All wounds were closed up<br />

nicely and no recurrence was found <strong>in</strong> the follow-up after<br />

2 mo.<br />

Electrosection is the most common endoscopic treatment,<br />

and its value for the treatment <strong>of</strong> gastro<strong>in</strong>test<strong>in</strong>al<br />

submucosal tumors has been recognized [12,13]<br />

. It is ma<strong>in</strong>ly<br />

used for protuberant lesions (especially the pedunculated<br />

ones). In this study, 157 GIMTs aris<strong>in</strong>g from non-muscularis<br />

propria, with a diameter <strong>of</strong> ≤ 1 cm, were treated by<br />

snare electrosection after EUS exam<strong>in</strong>ation. It is reported<br />

that serious complications rarely occurred when electrosection<br />

is used to cut non-muscularis propria tumors with<br />

a diameter ≤ 3 cm [3]<br />

. Tumors orig<strong>in</strong>at<strong>in</strong>g from muscularis<br />

propria are associated with an <strong>in</strong>creased risk <strong>of</strong> perforation<br />

and hemorrhage complications dur<strong>in</strong>g endoscopic<br />

treatment, and snare electrosection was not used <strong>in</strong> these<br />

cases.<br />

Compared with ord<strong>in</strong>ary snare removal, EMR is more<br />

suitable for the treatment <strong>of</strong> flat lesions generally conf<strong>in</strong>ed<br />

to < 2 cm [14] . In this study, 10 flat lesions were treated by<br />

EMR. We <strong>in</strong>jected 0.001% ep<strong>in</strong>ephr<strong>in</strong>e <strong>in</strong>to the submucosal<br />

layer to lift the lesion and made it easy to snare. Furthermore,<br />

this may provide a buffer to protect the <strong>in</strong>herent<br />

muscle function, which could reduce the bleed<strong>in</strong>g and<br />

perforation risk dur<strong>in</strong>g the process <strong>of</strong> muscle removal.<br />

Exam<strong>in</strong>ation by EUS before surgery to determ<strong>in</strong>e the size<br />

and depth <strong>of</strong> lesions could help determ<strong>in</strong>e the <strong>in</strong>jection<br />

site and the resection scope.<br />

Endoloop ligation <strong>of</strong> tumors at the base, block<strong>in</strong>g<br />

blood supply and caus<strong>in</strong>g tumor necrosis, could significantly<br />

reduce the risk <strong>of</strong> hemorrhage and perforation<br />

[15,16] . But, the procedure is not suitable for large<br />

lesions. Incomplete ligation might leave residual tumors,<br />

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Zhou XX et al . EUS and endoscopic treatment <strong>of</strong> GIMT<br />

while ligation could <strong>in</strong>crease the risk <strong>of</strong> hemorrhage and<br />

perforation. Therefore, the range and depth <strong>of</strong> ligation<br />

should be strictly controlled accord<strong>in</strong>g to the results <strong>of</strong><br />

EUS dur<strong>in</strong>g surgery. In the past, the majority <strong>of</strong> tumors<br />

studied have been only the muscularis mucosa and submucosa<br />

[3] . Recently, it was reported that endoloop could<br />

remove tumors aris<strong>in</strong>g from muscularis propria safely<br />

and effectively [15,17] . In this study, we also used endoloop<br />

removal <strong>of</strong> lesions aris<strong>in</strong>g from muscularis propria, without<br />

hemorrhage or perforation. The tumor from the<br />

muscularis propria can grow <strong>in</strong>side or outside the cavity,<br />

therefore, preoperative EUS for def<strong>in</strong><strong>in</strong>g the tumor<br />

growth pattern is very important to determ<strong>in</strong>e whether<br />

the lesion can be safely and completely removed.<br />

ESD should be performed us<strong>in</strong>g a high-frequency<br />

electric knife to dissect the subepithelial tumor, which<br />

is more suitable for treatment <strong>of</strong> large and flat lesions.<br />

Tumors derived from the muscularis mucosa and submucosa<br />

can be completely dissected [18,19] . It is difficult<br />

to dissect lesions from the muscularis propria because<br />

<strong>of</strong> the <strong>in</strong>creased risk <strong>of</strong> hemorrhage and perforation. In<br />

Lee et al ’s [20] study, among 12 cases <strong>of</strong> gastro<strong>in</strong>test<strong>in</strong>al<br />

submucosal muscle tumors aris<strong>in</strong>g from muscularis propria<br />

treated by ESD, 9 tumors were completely dissected.<br />

The size <strong>of</strong> these tumors ranged from 0.6 to 4 cm (average,<br />

2 cm). In this study, 8 stromal tumors aris<strong>in</strong>g from<br />

submucosa or muscularis propria were treated safely by<br />

ESD. All <strong>of</strong> them were dissected once and clipp<strong>in</strong>g was<br />

used to close deep wounds to reduce hemorrhage and<br />

perforation risk.<br />

Our cl<strong>in</strong>ical practice demonstrates that endoscopic<br />

treatment can be applied to GIMTs aris<strong>in</strong>g from muscularis<br />

mucosa, submucosa and muscularis propria. Based<br />

on the results <strong>of</strong> the EUS procedure, lesions > 2.5 cm <strong>in</strong><br />

size and suspected to be malignant should be considered<br />

for surgery. Moreover, if the tumor grew outside the<br />

cavity, endoscopic treatment should be aborted as well.<br />

We also suggested a follow-up with EUS for the few patients<br />

who are not <strong>in</strong>dicated for the endoscopic therapy<br />

or whose tumor is too small.<br />

In conclusion, EUS can help determ<strong>in</strong>e the orig<strong>in</strong>,<br />

size, shape, nature and growth pattern <strong>of</strong> lesions, with a<br />

high diagnostic accuracy for upper GIMTs. Preoperative<br />

EUS exam<strong>in</strong>ation is important for choos<strong>in</strong>g the type <strong>of</strong><br />

endoscopic therapy for mesenchymal tumors, by which<br />

the lesions can be treated safely and effectively.<br />

COMMENTS<br />

Background<br />

Cl<strong>in</strong>ically, gastro<strong>in</strong>test<strong>in</strong>al mesenchymal tumors (GIMTs) are usually <strong>in</strong>cidentally<br />

discovered as subepithelial bulges dur<strong>in</strong>g rout<strong>in</strong>e endoscopy for unrelated conditions.<br />

The classification and management <strong>of</strong> these lesions can be challeng<strong>in</strong>g.<br />

Research frontiers<br />

With the wide use <strong>of</strong> endoscopic ultrasonography (EUS) to clarify the nature<br />

and orig<strong>in</strong> <strong>of</strong> the subepithelial tumor, great progress has been made <strong>in</strong> diagnosis<br />

and treatment <strong>of</strong> GIMTs. However, the value <strong>of</strong> EUS <strong>in</strong> the choice <strong>of</strong> endoscopic<br />

treatment strategies for GIMTs has not been well established.<br />

Innovations and breakthroughs<br />

This study <strong>in</strong>dicated that EUS could help determ<strong>in</strong>e the orig<strong>in</strong>, size, shape,<br />

WJG|www.wjgnet.com<br />

nature and growth pattern <strong>of</strong> lesions, with a high diagnostic accuracy for upper<br />

GIMTs. Under the guidance <strong>of</strong> the EUS, GIMTs could be removed by appropriate<br />

endoscopic treatment, such as snare electrosection, endoloop, endoscopic<br />

mucosal resection (EMR) and endoscopic submucosal dissection (ESD) without<br />

severe complications.<br />

Applications<br />

The results <strong>of</strong> this study demonstrate that EUS is an effective means <strong>of</strong> diagnosis<br />

for upper GIMTs. Preoperative EUS exam<strong>in</strong>ation is important for choos<strong>in</strong>g<br />

the type <strong>of</strong> endoscopic therapy for mesenchymal tumors. The study will guide<br />

the cl<strong>in</strong>ical application <strong>of</strong> EUS <strong>in</strong> the endoscopic therapy for upper GIMTs.<br />

Term<strong>in</strong>ology<br />

GIMTs are tumors which orig<strong>in</strong>ate from mesenchymal cells other than epithelial<br />

cells or lymphocytes. They are further classified as stromal tumors, leiomyomas,<br />

leiomyosarcomas, neural tumors, fibroblast tumors or liparomphalus. EMR is a<br />

m<strong>in</strong>imally <strong>in</strong>vasive technique for resection <strong>of</strong> a lesion that requires the separation<br />

<strong>of</strong> the submucosa by <strong>in</strong>ject<strong>in</strong>g a fluid agent. ESD is a new endoscopic<br />

method us<strong>in</strong>g special knife for complete en bloc resection <strong>of</strong> early gastro<strong>in</strong>test<strong>in</strong>al<br />

neoplasms.<br />

Peer review<br />

This is a well written paper which describes the experience <strong>of</strong> the authors <strong>in</strong><br />

the EUS diagnosis and subsequent endoscopic treatment <strong>of</strong> gastro<strong>in</strong>test<strong>in</strong>al<br />

mesenchymal tumors. The pictures well support the authors’ f<strong>in</strong>d<strong>in</strong>gs and conclusions.<br />

REFERENCES<br />

1 Oh YS, Early DS, Azar RR. Cl<strong>in</strong>ical applications <strong>of</strong> endoscopic<br />

ultrasound to oncology. Oncology 2005; 68: 526-537<br />

2 Huang WH, Feng CL, Lai HC, Yu CJ, Chou JW, Peng CY,<br />

Yang MD, Chiang IP. Endoscopic ligation and resection for<br />

the treatment <strong>of</strong> small EUS-suspected gastric GI stromal tumors.<br />

Gastro<strong>in</strong>test Endosc 2010; 71: 1076-1081<br />

3 Martínez-Ares D, Lorenzo MJ, Souto-Ruzo J, Pérez JC, López<br />

JY, Belando RA, Vilas JD, Colell JM, Iglesias JL. Endoscopic<br />

resection <strong>of</strong> gastro<strong>in</strong>test<strong>in</strong>al submucosal tumors assisted by<br />

endoscopic ultrasonography. Surg Endosc 2005; 19: 854-858<br />

4 Sun S, Ge N, Wang S, Liu X, Lü Q. EUS-assisted band ligation<br />

<strong>of</strong> small duodenal stromal tumors and follow-up by<br />

EUS. Gastro<strong>in</strong>test Endosc 2009; 69:492-496<br />

5 Shim CS, Jung IS. Endoscopic removal <strong>of</strong> submucosal<br />

tumors: preprocedure diagnosis, technical options, and results.<br />

Endoscopy 2005; 37: 646-654<br />

6 Ji F, Wang ZW, Wang LJ, N<strong>in</strong>g JW, Xu GQ. Cl<strong>in</strong>icopathological<br />

characteristics <strong>of</strong> gastro<strong>in</strong>test<strong>in</strong>al mesenchymal tumors<br />

and diagnostic value <strong>of</strong> endoscopic ultrasonography. J<br />

Gastroenterol Hepatol 2008; 23: e318-e324<br />

7 Kim GH, Park do Y, Kim S, Kim DH, Kim DH, Choi CW,<br />

Heo J, Song GA. Is it possible to differentiate gastric GISTs<br />

from gastric leiomyomas by EUS? <strong>World</strong> J Gastroenterol 2009;<br />

15: 3376-3381<br />

8 Okubo K, Yamao K, Nakamura T, Tajika M, Sawaki A, Hara<br />

K, Kawai H, Yamamura Y, Mochizuki Y, Koshikawa T, Inada<br />

K. Endoscopic ultrasound-guided f<strong>in</strong>e-needle aspiration biopsy<br />

for the diagnosis <strong>of</strong> gastro<strong>in</strong>test<strong>in</strong>al stromal tumors <strong>in</strong><br />

the stomach. J Gastroenterol 2004; 39: 747-753<br />

9 Shah P, Gao F, Edmundowicz SA, Azar RR, Early DS. Predict<strong>in</strong>g<br />

malignant potential <strong>of</strong> gastro<strong>in</strong>test<strong>in</strong>al stromal tumors<br />

us<strong>in</strong>g endoscopic ultrasound. Dig Dis Sci 2009; 54: 1265-1269<br />

10 Kang YN, Jung HR, Hwang I. Cl<strong>in</strong>icopathological and immunohistochemical<br />

features <strong>of</strong> gasto<strong>in</strong>test<strong>in</strong>al stromal tumors.<br />

Cancer Res Treat 2010; 42: 135-143<br />

11 Scarpa M, Bert<strong>in</strong> M, Ruffolo C, Polese L, D’Amico DF, Angriman<br />

I. A systematic review on the cl<strong>in</strong>ical diagnosis <strong>of</strong> gastro<strong>in</strong>test<strong>in</strong>al<br />

stromal tumors. J Surg Oncol 2008; 98: 384-392<br />

12 Wehrmann T, Martchenko K, Nakamura M, Riphaus A,<br />

Stergiou N. Endoscopic resection <strong>of</strong> submucosal esophageal<br />

tumors: a prospective case series. Endoscopy 2004; 36:<br />

802-807<br />

13 Stergiou N, Riphaus A, Lange P, Menke D, Köckerl<strong>in</strong>g F,<br />

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Wehrmann T. Endoscopic snare resection <strong>of</strong> large <strong>colon</strong>ic polyps:<br />

how far can we go? Int J Colorectal Dis 2003; 18: 131-135<br />

14 Li H, Lu P, Lu Y, Liu C, Xu H, Wang S, Chen J. Predictive factors<br />

<strong>of</strong> lymph node metastasis <strong>in</strong> undifferentiated early gastric<br />

cancers and application <strong>of</strong> endoscopic mucosal resection.<br />

Surg Oncol 2010; 19: 221-226<br />

15 Sun S, J<strong>in</strong> Y, Chang G, Wang C, Li X, Wang Z. Endoscopic<br />

band ligation without electrosurgery: a new technique for<br />

excision <strong>of</strong> small upper-GI leiomyoma. Gastro<strong>in</strong>test Endosc<br />

2004; 60: 218-222<br />

16 Lee SH, Park JH, Park do H, Chung IK, Kim HS, Park SH,<br />

Kim SJ, Cho HD. Endoloop ligation <strong>of</strong> large pedunculated<br />

submucosal tumors (with videos). Gastro<strong>in</strong>test Endosc 2008;<br />

67: 556-560<br />

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Zhou XX et al . EUS and endoscopic treatment <strong>of</strong> GIMT<br />

17 Sun S, Ge N, Wang C, Wang M, Lü Q. Endoscopic band<br />

ligation <strong>of</strong> small gastric stromal tumors and follow-up by<br />

endoscopic ultrasonography. Surg Endosc 2007; 21: 574-578<br />

18 Kakushima N, Fujishiro M. Endoscopic submucosal dissection<br />

for gastro<strong>in</strong>test<strong>in</strong>al neoplasms. <strong>World</strong> J Gastroenterol<br />

2008; 14: 2962-2967<br />

19 Ono S, Fujishiro M, Niimi K, Goto O, Kodashima S, Yamamichi<br />

N, Omata M. Long-term outcomes <strong>of</strong> endoscopic submucosal<br />

dissection for superficial esophageal squamous cell<br />

neoplasms. Gastro<strong>in</strong>test Endosc 2009; 70: 860-866<br />

20 Lee IL, L<strong>in</strong> PY, Tung SY, Shen CH, Wei KL, Wu CS. Endoscopic<br />

submucosal dissection for the treatment <strong>of</strong> <strong>in</strong>tralum<strong>in</strong>al<br />

gastric subepithelial tumors orig<strong>in</strong>at<strong>in</strong>g from the<br />

muscularis propria layer. Endoscopy 2006; 38: 1024-1028<br />

S- Editor Tian L L- Editor Ma JY E- Editor Ma WH<br />

1771 April 7, 2011|Volume 17|Issue 13|

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INTRODUCTION<br />

Hepatocellular carc<strong>in</strong>oma (HCC) is a primary cancer <strong>of</strong><br />

the liver chronically <strong>in</strong>jured by <strong>in</strong>fection, metabolic disease<br />

or various drugs [1] . As generally observed <strong>in</strong> other carc<strong>in</strong>omas,<br />

HCC is attributed to accumulated genetic alterations,<br />

<strong>in</strong>clud<strong>in</strong>g (1) Activation <strong>of</strong> oncogenes N-ras, H-ras, and<br />

K-ras, c-erbA, c-met, RB and c-myc [2-5] ; (2) Transcriptional<br />

activation <strong>of</strong> c-jun and nuclear factor kB by hepatitis B<br />

virus factors [6] ; (3) Repression or mutation <strong>of</strong> the p53<br />

anti-oncogene [7] ; and (4) Accumulation <strong>of</strong> β-caten<strong>in</strong> [8] .<br />

Although the genetic events responsible for either HCC<br />

<strong>in</strong>itiation or progression are not clear, they <strong>in</strong>volve at least<br />

three carc<strong>in</strong>ogenesis pathways: the p53, RB and Wnt/<br />

β-caten<strong>in</strong> signal<strong>in</strong>g pathways [1-10] .<br />

β-caten<strong>in</strong> is an essential downstream effector <strong>of</strong> the canonical<br />

Wnt signal<strong>in</strong>g pathway [11,12] . Approximately 20% <strong>of</strong><br />

HCC cells display β-caten<strong>in</strong> aberrant activation [9,13] . In the<br />

normal steady state, β-caten<strong>in</strong> is cont<strong>in</strong>uously phosphorylated<br />

at ser<strong>in</strong>e and threon<strong>in</strong>e residues by glycogen synthase<br />

k<strong>in</strong>ase 3β (GSK-3β) <strong>in</strong> a complex with adenomatous polyposis<br />

coli (APC)-ax<strong>in</strong>/conduct<strong>in</strong> and is quickly degraded<br />

through the ubiquit<strong>in</strong>/proteasome pathway. In mice, liverspecific<br />

deletion <strong>of</strong> APC <strong>in</strong>duces β-caten<strong>in</strong> stabilization<br />

and <strong>in</strong>creases the number <strong>of</strong> HCC cells. Although the activation<br />

<strong>of</strong> β-caten<strong>in</strong> is likely an <strong>in</strong>itiat<strong>in</strong>g or contributory<br />

factor for HCC, more fundamental <strong>in</strong>formation is required<br />

for a better understand<strong>in</strong>g <strong>of</strong> the detailed genetic mechanism<br />

underly<strong>in</strong>g HCC associated with β-caten<strong>in</strong>.<br />

To uncover the detailed genetic mechanisms underly<strong>in</strong>g<br />

HCC <strong>in</strong> the present study, several genes <strong>in</strong> mouse cancerous<br />

liver tissue samples were identified to disclose more <strong>of</strong><br />

the genes that play a very important role <strong>in</strong> the regulation<br />

<strong>of</strong> cell proliferation and the development <strong>of</strong> HCC. We<br />

identified several cDNA fragments that were differentially<br />

expressed <strong>in</strong> radiation-<strong>in</strong>duced mouse HCC and compared<br />

with those <strong>in</strong> matched nontumorous liver tissue. Samples<br />

us<strong>in</strong>g a differential display technique [14] . We determ<strong>in</strong>ed<br />

whether the nearby upstream doma<strong>in</strong> <strong>of</strong> those genes<br />

conta<strong>in</strong> the β-caten<strong>in</strong>/Tcf-Lef consensus b<strong>in</strong>d<strong>in</strong>g site<br />

sequences. The <strong>in</strong>fluence <strong>of</strong> β-caten<strong>in</strong> accumulation <strong>in</strong><br />

nuclei <strong>of</strong> HCC cells on activation <strong>of</strong> prote<strong>in</strong> encoded by<br />

the gene conta<strong>in</strong><strong>in</strong>g β-caten<strong>in</strong>/Tcf-Lef consensus b<strong>in</strong>d<strong>in</strong>g<br />

site sequences was further <strong>in</strong>vestigated.<br />

MATERIALS AND METHODS<br />

Sample preparation<br />

Surgically resected HCC and adjacent nontumorous tissue<br />

samples were taken from the livers <strong>of</strong> 18-mo-old mice irradiated<br />

by 3.5 Gy 60Co γ-ray for 1 wk immediately after<br />

they were born. B6C3F1 mouse HCC and matched nontumorous<br />

liver tissue samples were obta<strong>in</strong>ed immediately<br />

under the same conditions for measurement <strong>of</strong> total GST<br />

activity [15] , isolation <strong>of</strong> total RNA [16] , and immunohistochemical<br />

expression <strong>of</strong> β-caten<strong>in</strong> and hematoxyl<strong>in</strong>-eos<strong>in</strong><br />

(HE) sta<strong>in</strong><strong>in</strong>g. Histological analysis <strong>of</strong> HCC tissue samples<br />

from mice was carried out accord<strong>in</strong>g to the general rules<br />

for cl<strong>in</strong>ical and pathological study <strong>of</strong> primary liver cancer.<br />

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Li YS et al . GSTM3 mRNA up-regulation by β-caten<strong>in</strong> accumulation<br />

Table 1 Primer sets used <strong>in</strong> mRNA differential display analysis<br />

Anchor primers Arbitrary primers<br />

T12MG (10 μmol/L) AP-10 (2 μmol/L), 5’-TAGCAAGTGC-3’<br />

T12MA (10 μmol/L) AP-11 (2 μmol/L), 5’-CAGACCGTTC-3’<br />

T12MT (10 μmol/L) AP-12 (2 μmol/L), 5’-TGCTGACCTG-3’<br />

T12MC (10 μmol/L) AP-14 (2 μmol/L), 5’-AATGGGCTGA-3’<br />

M represents a degenerated mixture <strong>of</strong> dA, dG and dC. The 16 different<br />

primer sets used for PCR amplification were randomly comb<strong>in</strong>ed from the<br />

four arbitrary primers and the four anchor primers.<br />

On the other hand, HepG2 cells obta<strong>in</strong>ed from Ch<strong>in</strong>a<br />

Center for Type Culture Collection were ma<strong>in</strong>ta<strong>in</strong>ed <strong>in</strong><br />

DMEM supplemented with 10% fetal calf serum (Sigma,<br />

Louis, MO), 200 mmol/L L-glutam<strong>in</strong>e and 1% penicill<strong>in</strong>/streptomyc<strong>in</strong><br />

(Invitrogen, Carlsbad, CA) at 37℃ <strong>in</strong><br />

a water-saturated atmosphere conta<strong>in</strong><strong>in</strong>g 5% CO2. Cell<br />

cultures were allowed to reach 90% confluence. The cells<br />

were then treated with or without 1 μmol/L <strong>of</strong> 4, 6-disubstituted<br />

pyrrolopyrimid<strong>in</strong>e (TWS119, Cayman Chemical,<br />

Ann Arbor, MI) and <strong>in</strong>cubated at 37℃ for 24 h. F<strong>in</strong>ally,<br />

total RNA or cytoplasmic prote<strong>in</strong>s, <strong>in</strong>clud<strong>in</strong>g total GST<br />

prote<strong>in</strong>s, were extracted from surgically resected frozen<br />

tissue samples or HepG2 cells by homogenization with a<br />

Vibra cell sonicator (Sonics and Materials, Inc., Danbury,<br />

CT) under a regularity condition.<br />

mRNA differential display analysis and DNA sequenc<strong>in</strong>g<br />

mRNA differential display analysis was performed as previously<br />

described [14] with a RNAmap kit A (Genhunter,<br />

Nashville, TN) (Table 1). Total RNA (0.4 μg) extracted<br />

from radiation-<strong>in</strong>duced mouse HCC and matched nontumorous<br />

liver tissue samples was reverse-transcribed with<br />

different comb<strong>in</strong>ations <strong>of</strong> arbitrary and anchor primers<br />

(Table 1) for <strong>in</strong>itial cDNA synthesis. The thermal cycler<br />

parameters were as follows: 1 cycle at 94℃ for 4 m<strong>in</strong>, followed<br />

by 40 cycles at 94℃ for 30 s, at 40℃ for 2 m<strong>in</strong> and<br />

at 72℃ for 30 s. Amplified subpopulations were distributed<br />

on a 6% DNA sequenc<strong>in</strong>g gel. The bands <strong>of</strong> <strong>in</strong>terest<br />

were cut out from the polyacrylamide gel, and cDNA fragments<br />

were re-amplified us<strong>in</strong>g the same pair <strong>of</strong> primers<br />

and the same cycle parameters as described above. The<br />

re-amplified cDNA fragments were purified from 2% agarose<br />

gels and subcloned <strong>in</strong>to pCRII-TOPO vectors us<strong>in</strong>g a<br />

TOPO TA clon<strong>in</strong>g kit (Invitrogen, Carlsbad, CA). Clones<br />

were selected by the same size <strong>of</strong> bands cut from the<br />

polyacrylamide gel as described above, followed by <strong>in</strong>verse<br />

hybridization and DNA sequenc<strong>in</strong>g. DNA sequences were<br />

compared with those <strong>in</strong> GenBank by the Blast Service<br />

provided by NIH (Bethesda, MD).<br />

Northern blot analysis<br />

Total RNA extraction (10 μg) was denatured and electrophoresed<br />

<strong>in</strong> a 1.2% agarose gel conta<strong>in</strong><strong>in</strong>g 0.66 mol/L<br />

formaldehyde and then transferred onto a Hybond-nylon<br />

membrane (Amersham Biosciences, Buck<strong>in</strong>gham, England).<br />

The membranes were UV cross-l<strong>in</strong>ked, pre-hybrid-<br />

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0.40<br />

y = 0.0044x + 0.309<br />

R 2 A B<br />

= 0.999 TWS119<br />

OD (A340)<br />

0.38<br />

0.36<br />

0.34<br />

0.32<br />

TWS119<br />

0.30<br />

y = 0.004x + 0.2922<br />

R<br />

0.28<br />

2 = 0.999 control<br />

Control<br />

0 4 8 12 16<br />

Time<br />

Figure 4 Total glutathione-s-transferase activity (A) and glutathione-s-transferase M3 mRNA expression (B) <strong>in</strong> HepG2 cells. a P < 0.05, b P < 0.01 vs TWS119<br />

at 24 h by one-way analysis <strong>of</strong> variance followed by Bonferroni’s test.<br />

Tcf-Lef family <strong>of</strong> DNA b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong>s, and regulate the<br />

transcription <strong>of</strong> target genes (for example, c-myc, gastr<strong>in</strong>,<br />

cycl<strong>in</strong> D1 and PPAR are identified as target genes <strong>of</strong> the<br />

β-caten<strong>in</strong>/Tcf-Lef complex) [30-33] . In the present study, the<br />

GSTM3 mRNA level was higher <strong>in</strong> B6C3F1 mouse HCC<br />

cells with β-caten<strong>in</strong> accumulation than <strong>in</strong> those without<br />

β-caten<strong>in</strong> accumulation. To our knowledge, no similar<br />

observation has been reported. The total GST activity was<br />

much higher <strong>in</strong> B6C3F1 mouse HCC cells with β-caten<strong>in</strong><br />

accumulation than <strong>in</strong> normal tissue samples without<br />

β-caten<strong>in</strong> accumulation (Table 2). The averaged GST<br />

activity ratio was significantly higher <strong>in</strong> HCC cells with<br />

β-caten<strong>in</strong> accumulation than <strong>in</strong> those without β-caten<strong>in</strong><br />

accumulation (0.747 ± 0.360 vs 0.071 ± 0.213, P < 0.001),<br />

suggest<strong>in</strong>g that the GST activity ratio is significantly different<br />

(Table 2). Furthermore, by search<strong>in</strong>g the GenBank,<br />

we found that the upstream region <strong>of</strong> the GSTM3 gene<br />

conta<strong>in</strong>ed three β-caten<strong>in</strong>/Tcf-Lef consensus b<strong>in</strong>d<strong>in</strong>g site<br />

sequences <strong>in</strong> mouse GST polymorphisms. The canonical<br />

Wnt pathway <strong>in</strong> cultured HepG2 cells was further mimicked<br />

us<strong>in</strong>g TWS119, a GSK-3β <strong>in</strong>hibitor, which caused<br />

abnormal β-caten<strong>in</strong> accumulation. As a result, TWS119<strong>in</strong>duced<br />

β-caten<strong>in</strong> accumulation enhanced the GST activity<br />

and the GSTM3 mRNA expression <strong>in</strong> HepG2 cells,<br />

suggest<strong>in</strong>g that β-caten<strong>in</strong> accumulation <strong>in</strong> nuclei <strong>of</strong> HCC<br />

cells can <strong>in</strong>crease the activity <strong>of</strong> mouse GSTM3, one <strong>of</strong><br />

the enzymes responsible for the metabolism <strong>of</strong> a variety<br />

<strong>of</strong> xenobiotics and carc<strong>in</strong>ogens, and that mouse GSTM3<br />

may be a novel downstream target gene <strong>of</strong> the β-caten<strong>in</strong>/<br />

Tcf-Lef complex <strong>in</strong> mouse HCC.<br />

It was reported that GLNS can catalyze the synthesis<br />

<strong>of</strong> glutam<strong>in</strong>e [34] , a major energy source <strong>of</strong> cells (an important<br />

ATP source), and is a precursor for the synthesis <strong>of</strong><br />

nucleotides and numerous am<strong>in</strong>o acids, and up-regulated<br />

<strong>in</strong> a subset <strong>of</strong> human HCC [35] . In this study, at 3 wk after<br />

tumor implantation, the glutam<strong>in</strong>e synthetase activity <strong>in</strong><br />

rats <strong>in</strong>creased by 34%, which is consistent with the reported<br />

f<strong>in</strong>d<strong>in</strong>gs [36] . However, further study is needed to observe<br />

the possible pharmacological action (s) <strong>of</strong> β-caten<strong>in</strong> <strong>in</strong> the<br />

up-regulated expression <strong>of</strong> GLNS mRNA.<br />

In conclusion, GSTM3 and GLNS genes are differen-<br />

WJG|www.wjgnet.com<br />

Li YS et al . GSTM3 mRNA up-regulation by β-caten<strong>in</strong> accumulation<br />

GSTM3/β-act<strong>in</strong> expression<br />

(% <strong>of</strong> 12 h)<br />

150<br />

100<br />

50<br />

0<br />

b<br />

tially expressed <strong>in</strong> mouse HCC cells. The expression level<br />

<strong>of</strong> GSTM3 mRNA and total GST activity are higher <strong>in</strong><br />

B6C3F1 mouse HCC cells with β-caten<strong>in</strong> accumulation<br />

than <strong>in</strong> those without β-caten<strong>in</strong> accumulation, <strong>in</strong>dicat<strong>in</strong>g<br />

that GSTM3 may be a novel target gene for the β-caten<strong>in</strong>/<br />

Tcf-Lef complex <strong>in</strong> mouse HCC.<br />

COMMENTS<br />

Background<br />

Hepatocellular carc<strong>in</strong>oma (HCC) is a primary cancer <strong>of</strong> the liver. However, the<br />

genetic events responsible for HCC <strong>in</strong>itiation and progression are not clear.<br />

S<strong>in</strong>ce approximately 20% <strong>of</strong> HCC display β-caten<strong>in</strong> aberrant activation, Wnt/<br />

β-caten<strong>in</strong> signal<strong>in</strong>g pathways may be <strong>in</strong>volved <strong>in</strong> HCC occurrence.<br />

Research frontiers<br />

Recent data show that β-caten<strong>in</strong> may be an <strong>in</strong>itiat<strong>in</strong>g or contributory factor for HCC.<br />

In this study, the authors demonstrated that glutathione-s-transferase M3 (GSTM3)<br />

might be a novel target gene <strong>of</strong> the β-caten<strong>in</strong>/Tcf-Lef complex <strong>in</strong> mouse HCC.<br />

Innovations and breakthroughs<br />

The authors identified two up-regulated genes, glutam<strong>in</strong>e synthetase (GLNS)<br />

and GSTM3, <strong>in</strong> nuclei <strong>of</strong> radiation-<strong>in</strong>duced mouse HCC cells with β-caten<strong>in</strong><br />

accumulation. Three β-caten<strong>in</strong>/Tcf-Lef consensus b<strong>in</strong>d<strong>in</strong>g site sequences were<br />

observed <strong>in</strong> mouse glutathione-s-transferase (GST) polymorphisms. GST activity<br />

and GSTM3 mRNA levels were <strong>in</strong>duced <strong>in</strong> cultured HepG2 cells by TWS119 (an<br />

<strong>in</strong>hibitor <strong>of</strong> GSK-3β). To our knowledge, no similar observation has been reported.<br />

Applications<br />

By demonstrat<strong>in</strong>g that GSTM3 may be a novel target gene <strong>of</strong> the β-caten<strong>in</strong>/Tcf-<br />

Lef complex <strong>in</strong> mouse HCC, this study may represent a future strategy for<br />

therapeutic <strong>in</strong>tervention <strong>in</strong> patients with HCC.<br />

Term<strong>in</strong>ology<br />

β-caten<strong>in</strong> plays an important role <strong>in</strong> cell-cell adhesion and <strong>in</strong> Wnt signal<strong>in</strong>g<br />

pathway, can enter nuclei by b<strong>in</strong>d<strong>in</strong>g to the Tcf-Lef family <strong>of</strong> DNA b<strong>in</strong>d<strong>in</strong>g<br />

prote<strong>in</strong>s and regulate the transcription <strong>of</strong> target genes.<br />

Peer review<br />

This paper reports the results <strong>of</strong> <strong>in</strong>vestigations on some differentially overexpressed<br />

genes associated with β-caten<strong>in</strong> accumulation <strong>in</strong> nuclei <strong>of</strong> HCC<br />

cells, show<strong>in</strong>g that GSTM3 may be a novel target gene <strong>of</strong> the β-caten<strong>in</strong>/Tcf-Lef<br />

complex <strong>in</strong> mouse HCC us<strong>in</strong>g mRNA differential display, Northern blot analysis,<br />

immunosta<strong>in</strong><strong>in</strong>g and RT-PCR techniques, respectively. It is worthy <strong>of</strong> publication.<br />

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S- Editor Sun H L- Editor Wang XL E- Editor Ma WH<br />

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Onl<strong>in</strong>e Submissions: http://www.wjgnet.com/1007-9327<strong>of</strong>fice<br />

wjg@wjgnet.com<br />

doi:10.3748/wjg.v17.i13.1779<br />

BRIEF ARTICLE<br />

Nutrition support <strong>in</strong> surgical patients with colorectal cancer<br />

Yang Chen, Bao-L<strong>in</strong> Liu, B<strong>in</strong> Shang, Ai-Shan Chen, Shi-Q<strong>in</strong>g Liu, Wei Sun, Hong-Zhuan Y<strong>in</strong>, Jian-Qiao Y<strong>in</strong>, Qi Su<br />

Yang Chen, Bao-L<strong>in</strong> Liu, B<strong>in</strong> Shang, Ai-Shan Chen, Shi-Q<strong>in</strong>g<br />

Liu, Wei Sun, Hong-Zhuan Y<strong>in</strong>, Jian-Qiao Y<strong>in</strong>, Qi Su, Department<br />

<strong>of</strong> Surgery, Shengj<strong>in</strong>g Hospital, Ch<strong>in</strong>a Medical University,<br />

Shenyang 110004, Liaon<strong>in</strong>g Prov<strong>in</strong>ce, Ch<strong>in</strong>a<br />

Author contributions: Liu BL and Chen Y contributed equally<br />

to this work; Liu BL, Su Q and Chen Y designed the research;<br />

Chen Y, Shang B, Chen AS, Liu SQ, Sun W, Y<strong>in</strong> HZ and Y<strong>in</strong> JQ<br />

performed the research; Sun W, Y<strong>in</strong> HZ and Y<strong>in</strong> JQ analyzed the<br />

data; Chen Y, Shang B, Chen AS and Liu SQ wrote the manuscript;<br />

Liu BL and Chen Y revised the paper.<br />

Supported by the Postgraduate Scientific Research Fund <strong>of</strong><br />

Shengj<strong>in</strong>g Hospital, Ch<strong>in</strong>a Medical University<br />

Correspondence to: Bao-L<strong>in</strong> Liu, MD, PhD, Pr<strong>of</strong>essor, Department<br />

<strong>of</strong> Surgery, Shengj<strong>in</strong>g Hospital, Ch<strong>in</strong>a Medical University,<br />

No. 36 Sanhao Street, Hep<strong>in</strong>g District, Shenyang 110004,<br />

Liaon<strong>in</strong>g Prov<strong>in</strong>ce, Ch<strong>in</strong>a. liubl55@hotmail.com<br />

Telephone: +86­24­96615­1­31912 Fax: +86­24­96615­1­13631<br />

Received: November 8, 2010 Revised: February 15, 2011<br />

Accepted: February 22, 2011<br />

Published onl<strong>in</strong>e: April 7, 2011<br />

Abstract<br />

AIM: To review the application <strong>of</strong> nutrition support <strong>in</strong><br />

patients after surgery for colorectal cancer, and to propose<br />

appropriate nutrition strategies.<br />

METHODS: A total <strong>of</strong> 202 consecutive surgical patients<br />

admitted to our hospital with a diagnosis <strong>of</strong> <strong>colon</strong> cancer<br />

or rectal cancer from January 2010 to July 2010, meet<strong>in</strong>g<br />

the requirements <strong>of</strong> Nutrition Risk Screen<strong>in</strong>g 2002,<br />

were enrolled <strong>in</strong> our study. Laboratory tests were performed<br />

to analyze the nutrition status <strong>of</strong> each patient,<br />

and the cl<strong>in</strong>ical outcome variables, <strong>in</strong>clud<strong>in</strong>g postoperative<br />

complications, hospital stay, cost <strong>of</strong> hospitalization<br />

and postoperative outcome, were analyzed.<br />

RESULTS: The “non-risk” patients who did not receive<br />

postoperative nutrition support had a higher rate <strong>of</strong><br />

postoperative complications than patients who received<br />

postoperative nutrition support (2.40 ± 1.51 vs 1.23 ±<br />

0.60, P = 0.000), and had a longer postoperative hospital<br />

stay (23.00 ± 15.84 d vs 15.27 ± 5.89 d, P = 0.009).<br />

There was higher cost <strong>of</strong> hospitalization for patients who<br />

received preoperative total parenteral nutrition (TPN)<br />

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1779<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): 1779-1786<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

than for patients who did not receive preoperative TPN<br />

(62 713.50 ± 5070.66 RMB Yuan vs 43178.00 ± 3596.68<br />

RMB Yuan, P = 0.014). Apply<strong>in</strong>g postoperative enteral nutrition<br />

significantly shortened postoperative fast<strong>in</strong>g time<br />

(5.16 ± 1.21 d vs 6.40 ± 1.84 d, P = 0.001) and postoperative<br />

hospital stay (11.92 ± 4.34 d vs 15.77 ± 6.03 d,<br />

P = 0.002). The patients who received postoperative TPN<br />

for no less than 7 d had <strong>in</strong>creased serum glucose levels<br />

(7.59 ± 3.57 mmol/L vs 6.48 ± 1.32 mmol/L, P = 0.006)<br />

and cost <strong>of</strong> hospitalization (47 724.14 ± 16 945.17 Yuan<br />

vs 38 598.73 ± 8349.79 Yuan, P = 0.000). The patients<br />

who received postoperative omega-3 fatty acids had a<br />

higher rate <strong>of</strong> postoperative complications than the patients<br />

who did not (1.33 ± 0.64 vs 1.13 ± 0.49, P = 0.041).<br />

High level <strong>of</strong> serum glucose was associated with a high<br />

risk <strong>of</strong> postoperative complications <strong>of</strong> <strong>in</strong>fection.<br />

CONCLUSION: Appropriate and moderate nutritional<br />

<strong>in</strong>tervention can improve the postoperative outcome <strong>of</strong><br />

colorectal cancer patients.<br />

© 2011 Baishideng. All rights reserved.<br />

Key words: Nutritional support; Nutrition assessment;<br />

Colorectal cancer; Surgery; Prognosis<br />

Peer reviewers: Laura E Matarese, PhD, RD, LDN, FADA,<br />

CNSD, Assistant Pr<strong>of</strong>essor <strong>of</strong> Surgery, University <strong>of</strong> Pittsburgh<br />

Medical Center, Director <strong>of</strong> Nutrition, Intest<strong>in</strong>al Rehabilitation<br />

and Transplantation Center, Thomas E. Starzl Transplantation<br />

Institute, UPMC Montefiore, 7 South, 3459 Fifth Avenue, Pittsburgh,<br />

PA 15213, United States; Dr. Jose Perea, Department <strong>of</strong><br />

Surgery, 12 De Octubre University Hospital, Rosas De Aravaca,<br />

82a, Madrid 28023, Spa<strong>in</strong><br />

Chen Y, Liu BL, Shang B, Chen AS, Liu SQ, Sun W, Y<strong>in</strong> HZ, Y<strong>in</strong><br />

JQ, Su Q. Nutrition support <strong>in</strong> surgical patients with colorectal<br />

cancer. <strong>World</strong> J Gastroenterol 2011; 17(13): 1779­1786 Available<br />

from: URL: http://www.wjgnet.com/1007­9327/full/v17/<br />

i13/1779.htm DOI: http://dx.doi.org/10.3748/wjg.v17.i13.1779<br />

INTRODUCTION<br />

Colorectal cancer is the fourth most common cancer<br />

<strong>in</strong> men and the third most common cancer <strong>in</strong> women<br />

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Chen Y et al . Nutrition support <strong>in</strong> colorectal cancer<br />

worldwide [1] . It is also a significant cause <strong>of</strong> morbidity<br />

and mortality throughout the world [2] . Malnutrition is<br />

common <strong>in</strong> patients present<strong>in</strong>g for surgical management<br />

<strong>of</strong> colorectal cancer, and multiple factors, such as tumor<br />

location, tumor type, tumor stage, and preoperative<br />

radiation or chemotherapy, may predispose the patients to<br />

malnutrition [3] . Postoperative outcomes, <strong>in</strong>clud<strong>in</strong>g <strong>in</strong>cidence<br />

<strong>of</strong> complications, morbidity and survival, are usually better<br />

<strong>in</strong> the patients who are <strong>in</strong> a good nutritional condition [4] .<br />

Comprehensive cl<strong>in</strong>ical application <strong>of</strong> nutrition support <strong>in</strong><br />

colorectal cancer patients appears to be necessary.<br />

Unfortunately, malnutrition has rema<strong>in</strong>ed a troublesome<br />

problem because <strong>of</strong> lack <strong>of</strong> nutrition support rout<strong>in</strong>es and<br />

a discrepancy between cl<strong>in</strong>ical practice and guidel<strong>in</strong>es regard<strong>in</strong>g<br />

nutrition support [5] .<br />

Currently, <strong>in</strong>ternational guidel<strong>in</strong>es on nutrition support<br />

have been established, such as the European Society for<br />

Cl<strong>in</strong>ical Nutrition and Metabolism (ESPEN) guidel<strong>in</strong>es and<br />

the American Society for Parenteral and Enteral Nutrition<br />

(ASPEN) guidel<strong>in</strong>es. Both are the authoritative guidel<strong>in</strong>es<br />

at present, and should be followed and used <strong>in</strong> cl<strong>in</strong>ical practice<br />

as appropriate to the specific medical condition. However,<br />

s<strong>in</strong>ce fewer than one sixth <strong>of</strong> the recommendations <strong>in</strong><br />

the current guidel<strong>in</strong>es are Grade A, and more than 50% are<br />

Grade C [6] , more and better controlled trials are needed <strong>in</strong><br />

the specific fields.<br />

We carried out a retrospective study to evaluate the<br />

nutritional risk <strong>of</strong> colorectal cancer patients who underwent<br />

elective surgery, and assessed the nutrition support process<br />

by analyz<strong>in</strong>g the postoperative cl<strong>in</strong>ical outcomes and<br />

compar<strong>in</strong>g with the <strong>in</strong>ternational recommendations or<br />

guidel<strong>in</strong>es. In particular, we <strong>in</strong>vestigated the current status<br />

<strong>of</strong> nutrition support for patients undergo<strong>in</strong>g surgery for<br />

colorectal cancer, and determ<strong>in</strong>ed the requirements <strong>of</strong><br />

feasible and appropriate nutrition support strategies for<br />

such patients.<br />

MATERIALS AND METHODS<br />

Case selection<br />

We reviewed a total <strong>of</strong> 220 consecutive patients admitted<br />

to our hospital with a diagnosis <strong>of</strong> <strong>colon</strong> cancer or rectal<br />

cancer from January 2010 to July 2010, and excluded 18<br />

patients, <strong>in</strong>clud<strong>in</strong>g one with hydroperitoneum accord<strong>in</strong>g to<br />

the exclusion criteria <strong>of</strong> the Nutrition Risk Screen<strong>in</strong>g (NRS)<br />

2002 [7] , and 17 who had received non­surgical treatment.<br />

The rema<strong>in</strong><strong>in</strong>g 202 patients were enrolled <strong>in</strong> this study.<br />

Methods<br />

In order to evaluate the cl<strong>in</strong>ical effect <strong>of</strong> different nutritional<br />

strategies <strong>in</strong> colorectal cancer patients with different<br />

nutritional status, we stratified the patients <strong>in</strong>to<br />

five groups.<br />

In Group A, to evaluate the effect <strong>of</strong> NRS score, we<br />

excluded the patients who received preoperative nutritional<br />

support, and divided the rema<strong>in</strong><strong>in</strong>g 199 patients <strong>in</strong>to a<br />

“non­risk” group (n = 148) whose NRS score was 0­2, and<br />

an “at­risk” group (n = 51) whose NRS score was ≥ 3.<br />

We further divided the two groups <strong>in</strong>to two subgroups, a<br />

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nutrition support group (NS) who received postoperative<br />

nutrition support and a non­nutrition support group (NNS)<br />

who did not receive postoperative nutrition support (Table<br />

1). Diagnosis and tumor stage were used to illustrate preoperative<br />

health status. The tumor stage was determ<strong>in</strong>ed<br />

accord<strong>in</strong>g to the 7th edition <strong>of</strong> the AJCC cancer stag<strong>in</strong>g<br />

manual (American Jo<strong>in</strong>t Committee on Cancer) [8] . Complications,<br />

postoperative hospital stay, cost <strong>of</strong> hospitalization<br />

and postoperative outcomes were used to assess the<br />

cl<strong>in</strong>ical effect <strong>of</strong> postoperative nutritional <strong>in</strong>tervention. In<br />

addition, we graded complications as none = 1, <strong>in</strong>fection<br />

= 2, fistula = 3, others = 4, and postoperative outcome<br />

as recovery = 1, no recovery = 2, death = 3, for statistical<br />

evaluation <strong>of</strong> the results.<br />

Group B consisted <strong>of</strong> five patients whose NRS score<br />

was > 4, the cl<strong>in</strong>ical effect <strong>of</strong> preoperative TPN <strong>in</strong> patients<br />

with severe malnutrition was evaluated. They were<br />

divided <strong>in</strong>to two groups: group 1 (n = 2) who received<br />

preoperative TPN and group 2 (n = 3) who did not (Table<br />

2). Diagnosis, tumor stage, and preoperative album<strong>in</strong>,<br />

potassium, and sodium levels reflected the preoperative<br />

nutrition status. Postoperative enteral nutrition (EN),<br />

postoperative TPN and postoperative TPN duration were<br />

<strong>in</strong>dicative <strong>of</strong> the postoperative nutritional <strong>in</strong>tervention.<br />

Postoperative serum glucose level was fluctuated accord<strong>in</strong>g<br />

to the proportion <strong>of</strong> <strong>in</strong>sul<strong>in</strong> <strong>in</strong> TPN. Postoperative day<br />

1 (POD1) album<strong>in</strong>, potassium and sodium, and POD5<br />

album<strong>in</strong>, potassium and sodium levels reflected the postoperative<br />

nutritional status. Complications, postoperative<br />

hospital stay, cost <strong>of</strong> hospitalization, and postoperative<br />

outcomes were used to assess cl<strong>in</strong>ical outcome.<br />

In Group C, the application <strong>of</strong> postoperative EN <strong>in</strong><br />

colorectal cancer patients was assessed. Patients who received<br />

preoperative nutrition support were excluded, and<br />

the rema<strong>in</strong><strong>in</strong>g 199 patients were divided <strong>in</strong>to two groups:<br />

group 1 (n = 25) who received postoperative EN and<br />

group 2 (n = 174) who did not (Table 3). Diagnosis, tumor<br />

stage and NRS 2002 score reflected the preoperative nutrition<br />

status. As an <strong>in</strong>terferential factor <strong>in</strong> this group, postoperative<br />

TPN was used <strong>in</strong> the statistical analysis to identify<br />

the effect <strong>of</strong> postoperative EN. Postoperative fast<strong>in</strong>g<br />

time, occurrence <strong>of</strong> complications, postoperative hospital<br />

stay, cost <strong>of</strong> hospitalization, and postoperative outcome<br />

<strong>in</strong>dicated cl<strong>in</strong>ical outcome.<br />

In Group D, to determ<strong>in</strong>e the effect <strong>of</strong> postoperative<br />

TPN duration, we excluded the patients who received<br />

preoperative nutrition support and postoperative EN,<br />

and <strong>in</strong>cluded the rema<strong>in</strong><strong>in</strong>g 174 patients who received<br />

postoperative TPN only, divid<strong>in</strong>g them <strong>in</strong>to two groups:<br />

group 1 (n = 66) with postoperative TPN duration <strong>of</strong><br />

no less than 7 d, and group 2 (n = 108) with a duration<br />

<strong>of</strong> less than 7 d (Table 4). Diagnosis, tumor stage, NRS<br />

2002 score, preoperative album<strong>in</strong>, potassium and sodium<br />

levels gave an <strong>in</strong>dication <strong>of</strong> preoperative nutrition status.<br />

POD1 album<strong>in</strong>, potassium and sodium, and POD5<br />

album<strong>in</strong>, potassium and sodium reflected postoperative<br />

nutritional status. Complications, postoperative hospital<br />

stay, cost <strong>of</strong> hospitalization and postoperative outcome<br />

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Table 1 Nutrition risk screen<strong>in</strong>g<br />

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Non-risk P At-risk P<br />

NS NNS NS NNS<br />

Patient number 143 5 49 2<br />

Diagnosis (<strong>colon</strong> / rectal cancer) 50/93 1/4 24/25 2/0<br />

Gender (male/female) 63/80 2/3 0.893 30/19 2/0 0.088<br />

Tumor stage 1<br />

0.066 0.358<br />

Complications 2<br />

None (= 1)<br />

Infection (= 2)<br />

Fistula (= 3)<br />

Others (= 4)<br />

1.23 ± 0.60 2.40 ± 1.51 0.000 1.20 ± 0.45 1.50 ± 0.70 0.348<br />

Postoperative hospital stay (d) 15.27 ± 5.89 23.00 ± 15.84 0.009 14.55 ± 4.11 14.50 ± 2.12 0.986<br />

Cost <strong>of</strong> hospitalization (RMB Yuan) 43 469.88 ± 9961.67 35 825.00 ± 16 271.94 0.301 41 802.97 ± 13 300. 99 33 845.80 ± 8374.80 0.187<br />

Postoperative outcome 2<br />

1.02 ± 0.16 1.00 ± 0.00 0.707 1.10 ± 0.30 1.50 ± 0.70 0.090<br />

1 The tumor stage <strong>of</strong> the patients was judged accord<strong>in</strong>g to the 7th edition <strong>of</strong> American Jo<strong>in</strong>t Committee on Cancer stag<strong>in</strong>g manual [8] ; 2 Complications are def<strong>in</strong>ed<br />

as none = 1, <strong>in</strong>fection = 2, fistula = 3, others = 4, and postoperative outcome as recovery = 1, no recovery = 2, death = 3, for easier statistical presentation<br />

<strong>of</strong> the results. NS: Nutrition support; NNS: Non-nutrition.<br />

Table 2 Preoperative total parenteral nutrition <strong>in</strong> malnourished<br />

patients<br />

Group 1 Group 2 P<br />

Patient number 2 3<br />

Diagnosis<br />

(<strong>colon</strong>/rectal cancer)<br />

2/0 1/2<br />

Gender (male/female) 2/0 1/2 0.219<br />

Tumor stage 1 2<br />

Preoperative album<strong>in</strong> (g/L) 36.30 ± 5.65 32.83 ± 9.00 0.669<br />

Preoperative potassium (mmol/L) 4.53 ± 0.36 3.75 ± 0.92 0.352<br />

Preoperative sodium (mmol/L) 138.45 ± 2.05 142.13 ± 3.40 0.274<br />

Postoperative EN 2 0<br />

Postoperative TPN 2 2<br />

Postoperative TPN duration (d) 7.00 ± 1.41 3.66 ± 3.2 0.276<br />

POD1 a serum glucose (mmol/L) 6.91 ± 1.11 9.05 ± 3.65 0.498<br />

POD1 album<strong>in</strong> (g/L) 27.80 ± 3.11 26.13 ± 3.19 0.605<br />

POD1 potassium (mmol/L) 4.49 ± 0.36 4.45 ± 0.78 0.953<br />

POD1 sodium (mmol/L) 135.8 ± 3.11 137.66 ± 2.51 0.508<br />

POD5 a serum glucose (mmol/L) 6.29 ± 0.24 6.39 ± 2.88 0.968<br />

POD5 album<strong>in</strong> (g/L) 28.65 ± 5.16 32.33 ± 4.67 0.466<br />

POD5 potassium (mmol/L) 4.67 ± 0.11 4.42 ± 0.12 0.115<br />

POD5 sodium (mmol/L) 135.95 ± 1.34 135.93 ± 2.72 0.994<br />

Complications 0.445<br />

Postoperative hospital stay(d) 10.50 ± 4.94 13.00 ± 2.64 0.500<br />

Cost <strong>of</strong> hospitalization (RMB) 62713.50 ± 43178.00 ± 0.014<br />

5070.66 3596.68<br />

Postoperative outcome 0.495<br />

a<br />

POD1: Postoperative day 1; POD5: Postoperative day 5; EN: Enteral nutrition;<br />

TPN: Total parenteral nutrition.<br />

were used to assess the cl<strong>in</strong>ical outcome. In addition,<br />

the comparison <strong>of</strong> preoperative serum glucose, POD1<br />

serum glucose, and POD5 serum glucose reflected the<br />

contribution <strong>of</strong> postoperative TPN duration to postoperative<br />

serum glucose.<br />

Group E excluded the patients who received preoperative<br />

nutrition support or postoperative EN, and <strong>in</strong>cluded<br />

the rema<strong>in</strong><strong>in</strong>g 167 patients who received postoperative<br />

TPN. This group was subdivided <strong>in</strong>to two groups: group<br />

1 (n = 102), those who received postoperative application<br />

<strong>of</strong> omega­3 fatty acids, and group 2 (n = 65), those who<br />

Chen Y et al . Nutrition support <strong>in</strong> colorectal cancer<br />

Table 3 Postoperative enteral nutrition and cl<strong>in</strong>ical outcome<br />

Group 1 Group 2 P<br />

Patient number 25 174<br />

Diagnosis (<strong>colon</strong> /rectal<br />

cancer)<br />

8/17 66/108 0.568<br />

Gender (male/female) 9/16 91/83 0.129<br />

Tumor stage 0.777<br />

NRS 2002 score 1.88 ± 0.88 1.96 ± 1.01 0.689<br />

Postoperative fast<strong>in</strong>g<br />

time (d)<br />

5.16 ± 1.21 6.40 ± 1.84 0.001<br />

Postoperative TPN 27 167 0.996<br />

Complications<br />

None (= 1)<br />

Infection (= 2)<br />

Fistula (= 3)<br />

Others (= 4)<br />

1.04 ± 0.20 1.29 ± 0.66 0.060<br />

Postoperative hospital<br />

stay (d)<br />

11.92 ± 4.34 15.77 ± 6.03 0.002<br />

Cost <strong>of</strong> hospitalization<br />

(RMB)<br />

44210.88 ± 7635.85 42060.09 ± 13066.15 0.752<br />

Postoperative outcome<br />

Recovery (= 1)<br />

Unrecovery (= 2)<br />

Dead (= 3)<br />

1.00 ± 0.00 1.06 ± 0.27 0.214<br />

Group 1: Patients who received enteral nutrition postoperatively; Group 2:<br />

Patients who did not receive enteral nutrition postoperatively. TPN: Total<br />

parenteral nutrition; NRS: Nutrition risk screen<strong>in</strong>g.<br />

did not, as shown <strong>in</strong> Table 5. Diagnosis, tumor stage, and<br />

NRS 2002 score were <strong>in</strong>dicative <strong>of</strong> preoperative nutrition<br />

status. The total lymphocyte count reflected the immune<br />

status. Complications, postoperative hospital stay, cost <strong>of</strong><br />

hospitalization, and postoperative outcome were used to<br />

assess the cl<strong>in</strong>ical outcome.<br />

We also analyzed the relationship between postoperative<br />

day 5 serum glucose levels and postoperative complications<br />

<strong>of</strong> <strong>in</strong>fection (Figure 1).<br />

Statistical analysis<br />

Analyses were performed us<strong>in</strong>g SPSS statistical s<strong>of</strong>tware<br />

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Chen Y et al . Nutrition support <strong>in</strong> colorectal cancer<br />

Table 4 Postoperative total parenteral nutrition duration<br />

Group 1 Group 2 P<br />

Patient number 66 108<br />

Diagnosis (<strong>colon</strong>/rectal cancer) 31/35 35/73<br />

Gender (male/female) 32/34 59/49 0.434<br />

Tumor stage 0.493<br />

NRS 2002 score 1.92 ± 0.94 1.99 ± 1.05 0.676<br />

Preoperative serum<br />

glucose (mmol/L)<br />

6.10 ± 1.86 5.75 ± 1.17 0.134<br />

Preoperative album<strong>in</strong> (g/L) 38.47 ± 4.44 39.51 ± 6.37 0.249<br />

Preoperative potassium (mmol/L) 3.98 ± 0.38 6.57 ± 1.84 0.270<br />

Preoperative sodium (mmol/L) 140.98 ± 3.23 139.58 ± 14.1 0.435<br />

POD1 serum glucose (mmol/L) 8.59 ± 3.39 7.37 ± 2.06 0.100<br />

POD1 album<strong>in</strong> (g/L) 32.24 ± 3.65 35.49 ± 4.11 0.725<br />

POD1 potassium (mmol/L) 4.05 ± 0.44 3.99 ± 0.45 0.424<br />

POD1 sodium (mmol/L) 136.35 ± 3.59 135.72 ± 14.50 0.763<br />

POD5 serum glucose (mmol/L) 7.59 ± 3.57 6.48 ± 1.32 0.006<br />

POD5 album<strong>in</strong> (g/L) 31.79 ± 3.53 39.91 ± 3.66 0.063<br />

POD5 potassium (mmol/L) 4.21 ± 0.50 4.16 ± 0.53 0.553<br />

POD5 sodium (mmol/L) 136.5 ± 18.60 137.84 ± 2.81 0.348<br />

Complications 0.533<br />

Postoperative hospital stay (d) 15.59 ± 5.32 15.87 ± 6.45 0.761<br />

Cost <strong>of</strong> hospitalization (RMB) 47724.14 ± 38598.73 ± 0.000<br />

16945.17 8349.79<br />

Postoperative outcome 0.166<br />

Group 1: The duration <strong>of</strong> postoperative total parenteral nutrition (TPN) was<br />

not less than 7 d; Group 2: The duration <strong>of</strong> postoperative TPN was less<br />

than 7 d. NRS: Nutrition Risk Screen<strong>in</strong>g.<br />

(SPSS for W<strong>in</strong>dows Ver. 11.5). Results <strong>of</strong> different groups<br />

were compared us<strong>in</strong>g descriptive statistics (mean ± SD). P<br />

≤ 0.05 was considered statistically significant.<br />

RESULTS<br />

Nutrition risk screen<strong>in</strong>g is a necessary and effective tool<br />

to identify the nutritional status <strong>of</strong> colorectal cancer patients,<br />

and to aid <strong>in</strong> provid<strong>in</strong>g the appropriate nutrition<br />

<strong>in</strong>tervention. As Table 1 shows, the “non­risk” patients<br />

who did not receive postoperative nutrition support had<br />

a higher rate <strong>of</strong> postoperative complications than those<br />

who received postoperative nutrition support (2.40 ±<br />

1.51 vs 1.23 ± 0.60, P = 0.000), and also had a longer postoperative<br />

hospital stay (23.00 ± 15.84 vs 15.27 ± 5.89,<br />

P = 0.009), which <strong>in</strong>dicated that postoperative nutrition<br />

support may be necessary for “non­risk” patients.<br />

Postoperative nutrition support or not did not show a<br />

significant difference <strong>in</strong> the outcome <strong>of</strong> “at-risk” patients,<br />

though postoperative nutrition support tended to improve<br />

the postoperative outcome (1.10 ± 0.30 vs 1.50 ± 0.70, P<br />

= 0.090), thus moderate nutrition support is allowable for<br />

“at­risk” patients.<br />

Table 2 shows that the cost <strong>of</strong> hospitalization for malnourished<br />

patients who received preoperative TPN was<br />

significantly higher than <strong>in</strong> patients who did not (62 713.50<br />

± 5070.66 RMB Yuan vs 43 178.00 ± 3596.68 RMB Yuan,<br />

P = 0.014) with no significant difference <strong>in</strong> the outcome.<br />

Postoperative EN markedly improved postoperative<br />

recovery course, <strong>in</strong>clud<strong>in</strong>g a reduction <strong>in</strong> postoperative<br />

fast<strong>in</strong>g time (5.16 ± 1.21 d vs 6.40 ± 1.84 d, P = 0.001)<br />

and postoperative hospital stay (11.92 ± 4.34 d vs 15.77<br />

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Table 5 Postoperative adm<strong>in</strong>istration <strong>of</strong> omega-3 fatty acids<br />

Group 1 Group 2 P<br />

Patient number 102 65<br />

Diagnosis (<strong>colon</strong> /rectal cancer) 42/60 40/25<br />

Gender (male/female) 49/53 45/38 0.089<br />

Tumor stage 0.317<br />

NRS 2002 score 1.94 ± 0.87 2.00 ± 1.15 0.710<br />

Preoperative total lymphocyte count 1.80 ± 0.63 1.93 ± 0.59 0.186<br />

POD1 total lymphocyte count 0.99 ± 0.34 1.11 ± 0.40 0.067<br />

POD5 total lymphocyte count 1.26 ± 0.59 1.29 ± 0.35 0.660<br />

Complications<br />

None (= 1)<br />

Infection (= 2)<br />

Fistula (= 3)<br />

Others (= 4)<br />

1.33 ± 0.64 1.13 ± 0.49 0.041<br />

Postoperative hospital stay (d) 16.04 ± 5.81 14.81 ± 4.29 0.159<br />

Cost <strong>of</strong> hospitalization (RMB) 43936.75 ± 39938.89 ± 0.055<br />

14260.31 10741.40<br />

Postoperative outcome<br />

Recovery (= 1)<br />

Unrecovery (= 2)<br />

Dead (= 3)<br />

1.09 ± 0.33 1.06 ± 0.27 0.055<br />

Group 1: Patients who received omega-3 fatty acids; Group 2: Patients<br />

who did not receive omega-3 fatty acids.<br />

POD5 serum glucose level<br />

(mmol/L)<br />

35<br />

30<br />

25<br />

20<br />

15<br />

10<br />

5<br />

0<br />

1 25 49 73 97 121 145 169 193<br />

Serial number <strong>of</strong> each patient<br />

POD5 serum<br />

glucose<br />

Postoperative<br />

complications<br />

<strong>of</strong> <strong>in</strong>fection<br />

Figure 1 Relationship between postoperative serum glucose level and<br />

complications <strong>of</strong> <strong>in</strong>fection. Abscissa: the serial number <strong>of</strong> each patient, arranged<br />

accord<strong>in</strong>g to the Postoperative day 5 serum glucose level; Ord<strong>in</strong>ate: numerical<br />

value; The red l<strong>in</strong>e shows the <strong>in</strong>cidence <strong>of</strong> postoperative complications<br />

<strong>of</strong> <strong>in</strong>fection.<br />

± 6.03 d, P = 0.002), as shown <strong>in</strong> Table 3.<br />

Longer postoperative TPN was not associated with<br />

better cl<strong>in</strong>ical outcome (Table 4). The patients who received<br />

postoperative TPN for no less than 7 d had <strong>in</strong>creased<br />

POD5 serum glucose (7.59 ± 3.57 mmol/L vs 6.48<br />

± 1.32 mmol/L, P = 0.006) and cost <strong>of</strong> hospitalization (47<br />

724.14 ± 16 945.17 Yuan vs 38598.73 ± 8349.79 Yuan, P =<br />

0.000), compared to those with less than 7 d postoperative<br />

TPN, suggest<strong>in</strong>g that less than 7 d nutrition support for<br />

postoperative colorectal cancer patients is adequate.<br />

More postoperative complications occurred <strong>in</strong> the<br />

patients with postoperative adm<strong>in</strong>istration <strong>of</strong> omega­3<br />

fatty acid (1.33 ± 0.64 vs 1.13 ± 0.49, P = 0.041) than <strong>in</strong><br />

patients who did not receive the fatty acid (Table 5).<br />

Postoperative complications were positively correlated<br />

with the postoperative serum glucose level, a high postoperative<br />

serum glucose level be<strong>in</strong>g associated with a higher<br />

risk <strong>of</strong> complications <strong>of</strong> <strong>in</strong>fection (Figure 1).<br />

1782 April 7, 2011|Volume 17|Issue 13|

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DISCUSSION<br />

Malnutrition is common <strong>in</strong> patients with colorectal cancer,<br />

and <strong>in</strong> our study, 52 (25.7%) <strong>of</strong> the 202 cases had<br />

a NRS score <strong>of</strong> more than 3, and had a high nutrition<br />

risk [7] . Poor nutrition status impacts on the recovery<br />

<strong>of</strong> physical performance status <strong>in</strong> cancer patients after<br />

treatment [9] . It was reported that about 20% <strong>of</strong> cancer<br />

patients died <strong>of</strong> malnutrition or related complications<br />

rather than the malignant disease itself [10] . Malnutrition<br />

is <strong>of</strong>ten neglected <strong>in</strong> our daily cl<strong>in</strong>ical practice, and can<br />

also <strong>in</strong>duce many cl<strong>in</strong>ical problems, <strong>in</strong>clud<strong>in</strong>g impaired<br />

wound­heal<strong>in</strong>g, immunocompromization, dim<strong>in</strong>ished<br />

cardiac and respiratory function, and a host <strong>of</strong> other complications<br />

that can lead to longer hospitalization and a<br />

higher mortality rate [11] . Although provision <strong>of</strong> nutrition<br />

support to cancer patients may cause tumors to grow<br />

more quickly, nutrition support is recommended when<br />

the nutrition status is so compromised that patients are<br />

at a high risk <strong>of</strong> complications, or cannot comply with<br />

the oncologic therapy as reported <strong>in</strong> the cl<strong>in</strong>ical practice<br />

ESPEN guidel<strong>in</strong>es [12] . Thus perioperative nutrition support<br />

is beneficial for moderately or severely malnourished<br />

gastro<strong>in</strong>test<strong>in</strong>al cancer patients [13] . The implementation<br />

<strong>of</strong> nutrition support guidel<strong>in</strong>es has facilitated many<br />

appropriate nutritional support procedures for colorectal<br />

cancer patients [4,6,14­16] .<br />

Preoperative nutrition risk screen<strong>in</strong>g can identify nutritional<br />

risks<br />

Patients with cancer are at a risk <strong>of</strong> malnutrition, and nutrition<br />

screen<strong>in</strong>g should be performed to identify those who<br />

require nutrition support [17] . When a patient is admitted to<br />

our ward, knowledge <strong>of</strong> the nutritional status, which is a<br />

cl<strong>in</strong>ical predictor <strong>of</strong> postoperative mortality and morbidity<br />

<strong>in</strong> surgery for colorectal cancer [18­20] , is essential, not only<br />

for screen<strong>in</strong>g malnourished or non­malnourished patients,<br />

but also for multimodal oncological treatment [21] . There are<br />

various k<strong>in</strong>ds <strong>of</strong> screen<strong>in</strong>g methods, <strong>in</strong>clud<strong>in</strong>g NRS 2002,<br />

which is a rapid screen<strong>in</strong>g tool recommended by ESPEN [7] ,<br />

and has been proven to be an appropriate scor<strong>in</strong>g system<br />

for predict<strong>in</strong>g unfavorable cl<strong>in</strong>ical outcomes [22] . ASPEN<br />

suggested us<strong>in</strong>g a subjective global assessment (SGA) as<br />

a screen<strong>in</strong>g tool [23] , and was shown to be a reliable assessment<br />

tool which could predict hospital stay and medical<br />

expenditure <strong>of</strong> surgical gastro<strong>in</strong>test<strong>in</strong>al cancer patients [24,25] .<br />

We believe that the assessment <strong>of</strong> nutritional status requires<br />

a multidimensional approach, which <strong>in</strong>cludes different<br />

cl<strong>in</strong>ical <strong>in</strong>dices and various nutritional parameters, so it<br />

is better to use both SGA and NRS 2002 to predict the<br />

cl<strong>in</strong>ical outcome [26] . Our study <strong>in</strong>dicated that, for “nonrisk”<br />

colorectal patients, postoperative nutrition support<br />

is necessary to avoid postoperative complications and shorten<br />

postoperative hospital stay. Although postoperative<br />

nutrition support to “at­risk” colorectal patients showed no<br />

significant advantage, <strong>in</strong> our op<strong>in</strong>ion, moderate nutrition<br />

support is allowable, as no harm or economic burden was<br />

<strong>in</strong>curred. Further prospective studies are necessary to confirm<br />

this.<br />

WJG|www.wjgnet.com<br />

Chen Y et al . Nutrition support <strong>in</strong> colorectal cancer<br />

Preoperative TPN is not always necessary<br />

The goal <strong>of</strong> preoperative nutrition support is to m<strong>in</strong>imize<br />

negative prote<strong>in</strong> balance by avoid<strong>in</strong>g starvation, to ma<strong>in</strong>ta<strong>in</strong><br />

muscle, immune and cognitive functions, and to enhance<br />

postoperative recovery, as the ESPEN guidel<strong>in</strong>es <strong>in</strong>dicated<br />

[27] . Preoperative parenteral nutrition is <strong>in</strong>dicated <strong>in</strong> severely<br />

undernourished patients <strong>in</strong> whom enteral nutrition<br />

cannot be adequately adm<strong>in</strong>istered either orally or enterally.<br />

Conversely, its use <strong>in</strong> well-nourished patients has no benefit<br />

but <strong>in</strong>creased morbidity. In our study, preoperative nutrition<br />

support <strong>in</strong> severely malnourished colorectal cancer patients<br />

only <strong>in</strong>creased the economic burden, with little beneficial<br />

effect. This is <strong>in</strong> agreement with the ASPEN guidel<strong>in</strong>es,<br />

which recently recommended that nutrition support should<br />

not be used rout<strong>in</strong>ely <strong>in</strong> patients undergo<strong>in</strong>g major cancer<br />

surgeries [17] . Because <strong>of</strong> the limited sample size, further prospective<br />

studies with a larger sample size should be carried<br />

out.<br />

Gunerhan’s study [28] recently showed that preoperative<br />

immunonutrition resulted <strong>in</strong> a significant <strong>in</strong>crease <strong>in</strong> serum<br />

prealbum<strong>in</strong> levels, but it did not significantly alter the T lymphocyte<br />

subpopulation count, the rate <strong>of</strong> postoperative<br />

complications and the hospitalization duration, thus preoperative<br />

immunonutrition should not be provided rout<strong>in</strong>ely.<br />

None <strong>of</strong> our patients received preoperative immunonutrition.<br />

Postoperative EN can shorten the fast<strong>in</strong>g time and hospital<br />

stay<br />

Previously, many colorectal doctors believed that nutrients<br />

<strong>in</strong> the gut disrupted anastomoses, so they preferred delay<strong>in</strong>g<br />

the EN postoperatively, and adm<strong>in</strong>istered TPN <strong>in</strong>stead<br />

to avoid anastomotic leak, which requires substantial use<br />

<strong>of</strong> hospital resources [29] . However, Seidner [11] emphasized<br />

that there were no significant differences <strong>in</strong> morbidity<br />

and mortality between patients who received EN or TPN,<br />

and recommended the guidel<strong>in</strong>e: if the gut works, use it.<br />

The available evidence lends support to the use <strong>of</strong> enteral<br />

over parenteral feed<strong>in</strong>g <strong>in</strong> <strong>in</strong>patients with function<strong>in</strong>g gastro<strong>in</strong>test<strong>in</strong>al<br />

tracts [30] . The application <strong>of</strong> EN can reverse<br />

the loss <strong>of</strong> gut mucosal <strong>in</strong>tegrity result<strong>in</strong>g from surgical<br />

trauma [31] , and early nutrition support (EEN) is associated<br />

with a decreased <strong>in</strong>fection risk, a decreased mortality, a<br />

reduced hospital stay, an <strong>in</strong>crease <strong>in</strong> collagen deposition<br />

at anastomosis and wound strength, and a clear trend <strong>of</strong><br />

a reduction <strong>in</strong> anastomotic breakdown [32,33] . In addition,<br />

EEN can reduce the use <strong>of</strong> nasogastric tubes, which<br />

may delay the return <strong>of</strong> bowel function and <strong>in</strong>crease<br />

pulmonary complications [34,35] . Osland [3] even suggested<br />

adopt<strong>in</strong>g EEN as a standard <strong>of</strong> care <strong>in</strong> cancer patients<br />

undergo<strong>in</strong>g gastro<strong>in</strong>test<strong>in</strong>al resections. As Table 3 shows,<br />

postoperative EN <strong>in</strong> colorectal cancer patients can significantly<br />

shorten the postoperative fast<strong>in</strong>g time and postoperative<br />

hospital stay, and there is a tendency to reduce<br />

postoperative complications (P = 0.060). Although it<br />

rema<strong>in</strong>s to be determ<strong>in</strong>ed how much should be provided<br />

<strong>in</strong>itially, underfeed<strong>in</strong>g with a small amount <strong>of</strong> nutrients,<br />

which “bathe” the gut mucosa, makes EEN necessary or<br />

desirable.<br />

1783 April 7, 2011|Volume 17|Issue 13|

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Chen Y et al . Nutrition support <strong>in</strong> colorectal cancer<br />

The risk <strong>of</strong> overfeed<strong>in</strong>g should not be neglected, as it<br />

can overwhelm the digestive and absorptive capacity <strong>of</strong><br />

the gastro<strong>in</strong>test<strong>in</strong>al tract, and lead to occurrence <strong>of</strong> some<br />

cl<strong>in</strong>ical complications, such as gastric distention, nausea,<br />

and diarrhea [32] .<br />

Postoperative TPN can <strong>of</strong>fer a smooth postoperative<br />

recovery<br />

Parenteral nutrition (PN) has been widely used <strong>in</strong> cl<strong>in</strong>ical<br />

practice, and a safe PN system must be developed which<br />

m<strong>in</strong>imizes procedural <strong>in</strong>cidents and maximizes the ability<br />

to meet <strong>in</strong>dividual patient requirements [36] . Thus, it is<br />

desirable to provide, devise, or make available customized<br />

PN formulations for <strong>in</strong>dividuals who have complex<br />

requirements secondary to disease or underly<strong>in</strong>g illness,<br />

or when otherwise warranted by rout<strong>in</strong>e monitor<strong>in</strong>g <strong>of</strong><br />

electrolytes, organ function, growth, and development.<br />

Not only fat and carbohydrates, but also a full range<br />

<strong>of</strong> vitam<strong>in</strong>s and trace elements should be important<br />

components <strong>of</strong> the TPN bag, and optimal nitrogenspar<strong>in</strong>g<br />

can be achieved when all components <strong>of</strong> the PN<br />

mix are adm<strong>in</strong>istered simultaneously over 24 h. However,<br />

when early oral food <strong>in</strong>take or EN is comb<strong>in</strong>ed with PN,<br />

<strong>in</strong>travenous supplementation with vitam<strong>in</strong>s appears to be<br />

unnecessary [27] .<br />

Should colorectal cancer patients be adm<strong>in</strong>istered postoperative<br />

TPN? Planas [4] recommended that such patients<br />

hav<strong>in</strong>g elective surgery should not be given postoperative<br />

PN rout<strong>in</strong>ely. Seidner [11] implied that adm<strong>in</strong>ister<strong>in</strong>g PN <strong>in</strong><br />

disregard <strong>of</strong> the patient’s nutritional status could do more<br />

harm than good, and suggested that postoperative TPN<br />

should be reserved for patients who have a prolonged postoperative<br />

ileus, generally more than 7­10 d, and for those<br />

who are severely malnourished and whose feed<strong>in</strong>g cannot<br />

be started with<strong>in</strong> 3­5 d. Accord<strong>in</strong>g to the ESPEN guidel<strong>in</strong>es<br />

[27] , postoperative PN is recommended <strong>in</strong> patients who<br />

cannot meet their caloric requirements with<strong>in</strong> 7­10 d both<br />

orally or enterally, and <strong>in</strong> patients who require postoperative<br />

artificial nutrition, enteral feed<strong>in</strong>g or a comb<strong>in</strong>ation <strong>of</strong><br />

enteral and supplementary parenteral feed<strong>in</strong>g.<br />

In our study, most colorectal cancer patients could<br />

resume feed<strong>in</strong>g 5­8 d postoperatively (Table 3), so postoperative<br />

TPN may be beneficial dur<strong>in</strong>g the period <strong>of</strong> postoperative<br />

fast<strong>in</strong>g. Should we give the patients TPN for 7 d<br />

or more, or is less than 7 d adequate? The results <strong>in</strong> Table<br />

4 <strong>in</strong>dicate that a longer duration <strong>of</strong> TPN <strong>in</strong>curs high hospitalization<br />

costs and <strong>in</strong>duces hyperglycemia, which is associated<br />

with a higher rate <strong>of</strong> postoperative complications<br />

(Figure 1), thus less than 7 d’ postoperative TPN appears<br />

to be appropriate.<br />

PN can be delivered through short­term, non­tunneled<br />

central venous catheters, and the appropriate choice, <strong>in</strong>sertion,<br />

and monitor<strong>in</strong>g <strong>of</strong> the venous access are <strong>of</strong> paramount<br />

importance to avoid a catheter­related bloodstream<br />

<strong>in</strong>fection, an important and still very common complication<br />

<strong>of</strong> PN [37] . Such <strong>in</strong>fections can be reduced by adopt<strong>in</strong>g costeffective,<br />

evidence­based <strong>in</strong>terventions, <strong>in</strong>clud<strong>in</strong>g specific<br />

tra<strong>in</strong><strong>in</strong>g <strong>of</strong> staff, an adequate handwash<strong>in</strong>g, the correct type<br />

WJG|www.wjgnet.com<br />

<strong>of</strong> device and site <strong>of</strong> <strong>in</strong>sertion, the use <strong>of</strong> maximal barrier<br />

protection dur<strong>in</strong>g <strong>in</strong>sertion, and removal <strong>of</strong> central l<strong>in</strong>es as<br />

soon as they are no longer necessary.<br />

Postoperative application <strong>of</strong> omega­3 fatty acids<br />

There is controversy as to whether visceral prote<strong>in</strong>s should<br />

be used to assess nutrient status <strong>in</strong> hospitalized patients.<br />

Seidner [11] suggested that visceral prote<strong>in</strong>s can be used <strong>in</strong><br />

the hospital sett<strong>in</strong>g, because they can identify patients at<br />

risk <strong>of</strong> a poor outcome who may benefit from nutrition support.<br />

In addition, the total lymphocyte count can be used to<br />

assess a patient’s immune function, which has been shown<br />

to correlate with the degree <strong>of</strong> visceral prote<strong>in</strong> depletion<br />

and cl<strong>in</strong>ical outcome. Therefore, total lymphocyte counts<br />

were used <strong>in</strong> our study to assess the effect <strong>of</strong> the omega­3<br />

fatty acids.<br />

Postoperative supplementation <strong>of</strong> omega­3 fatty acids<br />

by TPN has been reported to have a favorable effect <strong>in</strong> the<br />

outcomes <strong>of</strong> colorectal cancer patients undergo<strong>in</strong>g radical<br />

resection, by lower<strong>in</strong>g the magnitude <strong>of</strong> the <strong>in</strong>flammatory<br />

response and modulat<strong>in</strong>g the immune response [38,39] . In<br />

contrast, the application <strong>of</strong> omega­3 fatty acids showed<br />

no significant benefit <strong>in</strong> our study, and <strong>in</strong>deed there was a<br />

trend <strong>of</strong> an <strong>in</strong>creased risk <strong>of</strong> postoperative complications,<br />

an <strong>in</strong>creased economic burden, and a poorer postoperative<br />

outcome. Further prospective research is necessary with a<br />

larger sample to assess the functional benefit or otherwise<br />

<strong>of</strong> omega­3 fatty acids <strong>in</strong> the postoperative sett<strong>in</strong>g.<br />

Currently, many barriers, <strong>in</strong>clud<strong>in</strong>g low priority <strong>of</strong> nutritional<br />

support, no rout<strong>in</strong>e or established procedures <strong>in</strong><br />

many medical centers, <strong>in</strong>sufficient knowledge <strong>of</strong> nutritional<br />

support, lack <strong>of</strong> qualified and optional nutritional menus<br />

for the patients, and lack <strong>of</strong> leadership support from the<br />

medical team, make the nutritional therapy difficult to carry<br />

out <strong>in</strong> many hospitals [40] . A greater effort should be made <strong>in</strong><br />

the nutritional assessment <strong>of</strong> patients.<br />

In conclusion, nutrition support is an important therapy<br />

for colorectal cancer patients, and appropriate and moderate<br />

nutritional <strong>in</strong>tervention can significantly improve the<br />

postoperative recovery course, relieve the patient’s suffer<strong>in</strong>g,<br />

and reduce the medical cost <strong>of</strong> the patients. Cl<strong>in</strong>icians<br />

must be aware <strong>of</strong> nutrition support pr<strong>in</strong>ciples and methods<br />

<strong>in</strong> order to adm<strong>in</strong>ister appropriate nutrition support and<br />

avoid bl<strong>in</strong>d nutrition adm<strong>in</strong>istration.<br />

ACKNOWLEDGMENTS<br />

The authors thank Sangeeta Sharma for the help with manuscript<br />

preparation.<br />

COMMENTS<br />

Backgrounds<br />

Nutrition support has been widely used <strong>in</strong> the area <strong>of</strong> surgery, where the benefit on<br />

patients’ prognosis is evident. Colorectal cancer is the fourth most common cancer<br />

<strong>in</strong> men and the third most common cancer <strong>in</strong> women worldwide, and is also a<br />

significant cause <strong>of</strong> morbidity and mortality throughout the world, thus an appropriate<br />

and feasible nutrition support strategy is necessary and beneficial for patients’<br />

prognosis.<br />

1784 April 7, 2011|Volume 17|Issue 13|

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Research frontiers<br />

Nutritional support is widely used <strong>in</strong> postoperative colorectal cancer patients, but<br />

the role <strong>of</strong> nutrients has not been clearly def<strong>in</strong>ed. This study <strong>in</strong>vestigated the effect<br />

<strong>of</strong> nutrition support on the outcomes <strong>of</strong> patients with different nutritional status.<br />

Innovations and breakthroughs<br />

The authors found that appropriate and moderate nutritional <strong>in</strong>tervention can significantly<br />

improve the postoperative outcome <strong>of</strong> the patients with colorectal cancer.<br />

Applications<br />

The study provides a reference for daily cl<strong>in</strong>ical practice and future research. A prospective,<br />

multicenter, randomized, controlled trial with a larger sample is necessary<br />

to validate the statistical results and dim<strong>in</strong>ish bias.<br />

Peer review<br />

Although this is a retrospective review, I believe it will be <strong>of</strong> <strong>in</strong>terest to the readers.<br />

And, it does add someth<strong>in</strong>g to the literature.<br />

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Chen Y et al . Nutrition support <strong>in</strong> colorectal cancer<br />

nutrition support therapy dur<strong>in</strong>g adult anticancer treatment<br />

and <strong>in</strong> hematopoietic cell transplantation. JPEN J Parenter Enteral<br />

Nutr 2009; 33: 472-500<br />

18 Schwegler I, von Holzen A, Gutzwiller JP, Schlumpf R, Mühlebach<br />

S, Stanga Z. Nutritional risk is a cl<strong>in</strong>ical predictor <strong>of</strong><br />

postoperative mortality and morbidity <strong>in</strong> surgery for colorectal<br />

cancer. Br J Surg 2010; 97: 92-97<br />

19 Salv<strong>in</strong>o RM, Dechicco RS, Seidner DL. Perioperative nutrition<br />

support: who and how. Cleve Cl<strong>in</strong> J Med 2004; 71: 345-351<br />

20 Karlsson S, Andersson L, Berglund B. Early assessment <strong>of</strong><br />

nutritional status <strong>in</strong> patients scheduled for colorectal cancer<br />

surgery. Gastroenterol Nurs 2009; 32: 265-270<br />

21 Senesse P, Assenat E, Schneider S, Chargari C, Magné N,<br />

Azria D, Hébuterne X. Nutritional support dur<strong>in</strong>g oncologic<br />

treatment <strong>of</strong> patients with gastro<strong>in</strong>test<strong>in</strong>al cancer: who could<br />

benefit? Cancer Treat Rev 2008; 34: 568-575<br />

22 Raslan M, Gonzalez MC, Dias MC, Nascimento M, Castro M,<br />

Marques P, Segatto S, Torr<strong>in</strong>has RS, Cecconello I, Waitzberg<br />

DL. Comparison <strong>of</strong> nutritional risk screen<strong>in</strong>g tools for predict<strong>in</strong>g<br />

cl<strong>in</strong>ical outcomes <strong>in</strong> hospitalized patients. Nutrition<br />

2010; 26: 721-726<br />

23 Detsky AS, McLaughl<strong>in</strong> JR, Baker JP, Johnston N, Whittaker<br />

S, Mendelson RA, Jeejeebhoy KN. What is subjective global<br />

assessment <strong>of</strong> nutritional status? JPEN J Parenter Enteral Nutr<br />

1987; 11: 8-13<br />

24 Wu BW, Y<strong>in</strong> T, Cao WX, Gu ZD, Wang XJ, Yan M, Liu BY.<br />

Cl<strong>in</strong>ical application <strong>of</strong> subjective global assessment <strong>in</strong> Ch<strong>in</strong>ese<br />

patients with gastro<strong>in</strong>test<strong>in</strong>al cancer. <strong>World</strong> J Gastroenterol<br />

2009; 15: 3542-3549<br />

25 Filipović BF, Gajić M, Mil<strong>in</strong>ić N, Milovanović B, Filipović<br />

BR, Cvetković M, Sibalić N. Comparison <strong>of</strong> two nutritional<br />

assessment methods <strong>in</strong> gastroenterology patients. <strong>World</strong> J<br />

Gastroenterol 2010; 16: 1999-2004<br />

26 Raslan M, Gonzalez MC, Torr<strong>in</strong>has RS, Ravacci GR, Pereira<br />

JC, Waitzberg DL. Complementarity <strong>of</strong> Subjective Global Assessment<br />

(SGA) and Nutritional Risk Screen<strong>in</strong>g 2002 (NRS<br />

2002) for predict<strong>in</strong>g poor cl<strong>in</strong>ical outcomes <strong>in</strong> hospitalized<br />

patients. Cl<strong>in</strong> Nutr 2011; 30: 49-53<br />

27 Braga M, Ljungqvist O, Soeters P, Fearon K, Weimann A,<br />

Bozzetti F. ESPEN Guidel<strong>in</strong>es on Parenteral Nutrition: surgery.<br />

Cl<strong>in</strong> Nutr 2009; 28: 378-386<br />

28 Gunerhan Y, Koksal N, Sah<strong>in</strong> UY, Uzun MA, Ekşioglu-<br />

Demiralp E. Effect <strong>of</strong> preoperative immunonutrition and<br />

other nutrition models on cellular immune parameters. <strong>World</strong><br />

J Gastroenterol 2009; 15: 467-472<br />

29 Frye J, Bokey EL, Chapuis PH, S<strong>in</strong>clair G, Dent OF. Anastomotic<br />

leakage after resection <strong>of</strong> colorectal cancer generates<br />

prodigious use <strong>of</strong> hospital resources. Colorectal Dis 2009; 11:<br />

917-920<br />

30 Zaloga GP. Parenteral nutrition <strong>in</strong> adult <strong>in</strong>patients with function<strong>in</strong>g<br />

gastro<strong>in</strong>test<strong>in</strong>al tracts: assessment <strong>of</strong> outcomes. Lancet<br />

2006; 367: 1101-1111<br />

31 Jiang XH, Li N, Li JS. Intest<strong>in</strong>al permeability <strong>in</strong> patients after<br />

surgical trauma and effect <strong>of</strong> enteral nutrition versus parenteral<br />

nutrition. <strong>World</strong> J Gastroenterol 2003; 9: 1878-1880<br />

32 Ochoa JB, Caba D. Advances <strong>in</strong> surgical nutrition. Surg Cl<strong>in</strong><br />

North Am 2006; 86: 1483-1493<br />

33 Fearon KC, Ljungqvist O, Von Meyenfeldt M, Revhaug A,<br />

Dejong CH, Lassen K, Nygren J, Hausel J, Soop M, Andersen<br />

J, Kehlet H. Enhanced recovery after surgery: a consensus<br />

review <strong>of</strong> cl<strong>in</strong>ical care for patients undergo<strong>in</strong>g <strong>colon</strong>ic resection.<br />

Cl<strong>in</strong> Nutr 2005; 24: 466-4`77<br />

34 Nelson R, Tse B, Edwards S. Systematic review <strong>of</strong> prophylactic<br />

nasogastric decompression after abdom<strong>in</strong>al operations. Br<br />

J Surg 2005; 92: 673-680<br />

35 Zhou T, Wu XT, Zhou YJ, Huang X, Fan W, Li YC. Early<br />

remov<strong>in</strong>g gastro<strong>in</strong>test<strong>in</strong>al decompression and early oral feed<strong>in</strong>g<br />

improve patients’ rehabilitation after colorectostomy.<br />

<strong>World</strong> J Gastroenterol 2006; 12: 2459-2463<br />

36 Kochevar M, Guenter P, Holcombe B, Malone A, Mirtallo J.<br />

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Chen Y et al . Nutrition support <strong>in</strong> colorectal cancer<br />

ASPEN statement on parenteral nutrition standardization.<br />

JPEN J Parenter Enteral Nutr 2007; 31: 441-448<br />

37 Pittiruti M, Hamilton H, Biffi R, MacFie J, Pertkiewicz M. ES-<br />

PEN Guidel<strong>in</strong>es on Parenteral Nutrition: central venous catheters<br />

(access, care, diagnosis and therapy <strong>of</strong> complications).<br />

Cl<strong>in</strong> Nutr 2009; 28: 365-377<br />

38 Liang B, Wang S, Ye YJ, Yang XD, Wang YL, Qu J, Xie QW, Y<strong>in</strong><br />

MJ. Impact <strong>of</strong> postoperative omega-3 fatty acid-supplemented<br />

parenteral nutrition on cl<strong>in</strong>ical outcomes and immunomodulations<br />

<strong>in</strong> colorectal cancer patients. <strong>World</strong> J Gastroen-<br />

WJG|www.wjgnet.com<br />

terol 2008; 14: 2434-2439<br />

39 Jiang ZM, Wilmore DW, Wang XR, Wei JM, Zhang ZT, Gu<br />

ZY, Wang S, Han SM, Jiang H, Yu K. Randomized cl<strong>in</strong>ical<br />

trial <strong>of</strong> <strong>in</strong>travenous soybean oil alone versus soybean oil plus<br />

fish oil emulsion after gastro<strong>in</strong>test<strong>in</strong>al cancer surgery. Br J<br />

Surg 2010; 97: 804-809<br />

40 Rasmussen HH, Kondrup J, Staun M, Ladefoged K, L<strong>in</strong>dorff<br />

K, Jørgensen LM, Jakobsen J, Kristensen H, Wengler A. A<br />

method for implementation <strong>of</strong> nutritional therapy <strong>in</strong> hospitals.<br />

Cl<strong>in</strong> Nutr 2006; 25: 515-523<br />

S- Editor Sun H L- Editor Ma JY E- Editor Ma WH<br />

1786 April 7, 2011|Volume 17|Issue 13|

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Onl<strong>in</strong>e Submissions: http://www.wjgnet.com/1007-9327<strong>of</strong>fice<br />

wjg@wjgnet.com<br />

doi:10.3748/wjg.v17.i13.1787<br />

CASE REPORT<br />

Application <strong>of</strong> a wire-guided side-view<strong>in</strong>g duodenoscope <strong>in</strong><br />

total esophagectomy with <strong>colon</strong>ic <strong>in</strong>terposition<br />

Ch<strong>in</strong>-Yuan Yii, Jen-Wei Chou, Yen-Chun Peng, Wai-Keung Chow<br />

Ch<strong>in</strong>-Yuan Yii, Jen-Wei Chou, Wai-Keung Chow, Division <strong>of</strong><br />

<strong>Gastroenterology</strong> and Hepatology, Department <strong>of</strong> Internal Medic<strong>in</strong>e,<br />

Ch<strong>in</strong>a Medical University Hospital, Taichung 40447, Taiwan,<br />

Ch<strong>in</strong>a<br />

Yen-Chun Peng, Division <strong>of</strong> <strong>Gastroenterology</strong> and Hepatology,<br />

Department <strong>of</strong> Internal Medic<strong>in</strong>e, Taichung Veterans General<br />

Hospital, Taichung 40447, Taiwan, Ch<strong>in</strong>a<br />

Author contributions: Yii CY and Chow WK contributed equally<br />

to this work; Chow WK performed the therapeutic ERCP; Chou<br />

JW and Peng YC critically revised the manuscript; Yii CY and<br />

Chow WK wrote the paper.<br />

Correspondence to: Dr. Wai-Keung Chow, MD, Division <strong>of</strong><br />

<strong>Gastroenterology</strong> and Hepatology, Department <strong>of</strong> Internal Medic<strong>in</strong>e,<br />

Ch<strong>in</strong>a Medical University Hospital, No. 2, Yuh-Der Road,<br />

North District, Taichung 40447,<br />

Taiwan, Ch<strong>in</strong>a. wkchow2010@gmail.com<br />

Telephone: +886-4-22052121 Fax: +886-4-22023119<br />

Received: September 10, 2010 Revised: November 3, 2010<br />

Accepted: November 10, 2010<br />

Published onl<strong>in</strong>e: April 7, 2011<br />

Abstract<br />

Therapeutic endoscopic retrograde cholangiopancreatography<br />

(ERCP) is the ma<strong>in</strong>stay treatment for bile<br />

duct disease. The procedure is difficult per se, especially<br />

when a side-view<strong>in</strong>g duodenoscope is used, and when<br />

the patient has altered anatomical features, such as<br />

<strong>colon</strong>ic <strong>in</strong>terposition. Currently, there is no consensus on<br />

the standard approach for therapeutic ERCP <strong>in</strong> patients<br />

with total esophagectomy and <strong>colon</strong>ic <strong>in</strong>terposition. We<br />

describe a novel treatment design that <strong>in</strong>volves the use<br />

<strong>of</strong> a side-view<strong>in</strong>g duodenoscope to perform therapeutic<br />

ERCP <strong>in</strong> patients with total esophagectomy and <strong>colon</strong>ic<br />

<strong>in</strong>terposition. A gastroscope was <strong>in</strong>itially <strong>in</strong>troduced<br />

<strong>in</strong>to the <strong>in</strong>terposed <strong>colon</strong> and a radio-opaque standard<br />

guidewire was advanced to a distance beyond the papilla<br />

<strong>of</strong> Vater, before the gastroscope was withdrawn. A sideview<strong>in</strong>g<br />

duodenoscope was then <strong>in</strong>troduced along the<br />

guidewire under fluoroscopic guidance. After cannulation<br />

<strong>in</strong>to the papilla <strong>of</strong> Vater, endoscopic retrograde chol-<br />

WJG|www.wjgnet.com<br />

1787<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): 1787-1790<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

angiography (ERC) revealed a fill<strong>in</strong>g defect (maximum<br />

diameter: 15 cm) at the distal portion <strong>of</strong> the common bile<br />

duct (CBD). This defect was determ<strong>in</strong>ed to be a stone,<br />

which was successfully retrieved by a Dormia basket after<br />

complete sph<strong>in</strong>cterotomy. With this treatment design, it<br />

is possible to perform therapeutic ERCP <strong>in</strong> patients with<br />

<strong>colon</strong>ic <strong>in</strong>terposition, thereby preclud<strong>in</strong>g the need for<br />

percutaneous dra<strong>in</strong>age or surgery.<br />

© 2011 Baishideng. All rights reserved.<br />

Key words: Wire-guided; Duodenoscope; Endoscopic<br />

retrograde cholangiopancreatography; Esophagectomy;<br />

Interposition <strong>of</strong> <strong>colon</strong><br />

Peer reviewer: Klaus Mönkemüller, MD, PhD, FASGE, Division<br />

<strong>of</strong> Internal Medic<strong>in</strong>e and <strong>Gastroenterology</strong>, Marien Hospital,<br />

Josef-Albers-Str. 70, 46236 Bottrop, Germany.<br />

Yii CY, Chou JW, Peng YC, Chow WK. Application <strong>of</strong> a<br />

wire-guided side-view<strong>in</strong>g duodenoscope <strong>in</strong> total esophagectomy<br />

with <strong>colon</strong>ic <strong>in</strong>terposition. <strong>World</strong> J Gastroenterol 2011;<br />

17(13): 1787-1790 Available from: URL: http://www.wjgnet.<br />

com/1007-9327/full/v17/i13/1787.htm DOI: http://dx.doi.<br />

org/10.3748/wjg.v17.i13.1787<br />

INTRODUCTION<br />

The application <strong>of</strong> a side-view<strong>in</strong>g duodenoscope <strong>in</strong> total<br />

esophagectomy with <strong>colon</strong>ic <strong>in</strong>terposition is technically<br />

difficult, because <strong>of</strong> the altered structure <strong>of</strong> the <strong>colon</strong> and<br />

the redundancy <strong>of</strong> the endoscopic route. We report a wire-guided<br />

treatment designed to overcome this pitfall by<br />

<strong>in</strong>troduc<strong>in</strong>g a side-view<strong>in</strong>g duodenoscope along a radioopaque<br />

standard guidewire to facilitate therapeutic ERCP<br />

<strong>in</strong> patients undergo<strong>in</strong>g esophagectomy with <strong>colon</strong>ic <strong>in</strong>terposition.<br />

The use <strong>of</strong> this treatment method ensured the safety<br />

<strong>of</strong> wire-guided therapeutic ERCP <strong>in</strong> patients undergo<strong>in</strong>g<br />

total esophagectomy with <strong>colon</strong>ic <strong>in</strong>terposition.<br />

April 7, 2011|Volume 17|Issue 13|

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Yii CY et al . Wire-guided duodenoscope <strong>in</strong> <strong>colon</strong>ic <strong>in</strong>terposition<br />

CASE REPORT<br />

An 87-year-old man was referred to our hospital, a tertiary<br />

referral medical center, for the management <strong>of</strong> episodic fever,<br />

chills, and right upper quadrant abdom<strong>in</strong>al pa<strong>in</strong>, which<br />

had been occurr<strong>in</strong>g <strong>in</strong>termittently for two months. He had<br />

undergone total esophagectomy with <strong>colon</strong>ic <strong>in</strong>terposition<br />

17 years ago for the treatment <strong>of</strong> <strong>in</strong>tractable esophageal<br />

ulcers with massive bleed<strong>in</strong>g (Figure 1). He denied hav<strong>in</strong>g<br />

passed tea-colored ur<strong>in</strong>e or clay-colored stool. Abdom<strong>in</strong>al<br />

ultrasonography revealed dilatation <strong>of</strong> the common hepatic<br />

duct (CHD) and common bile duct (CBD; diameter:<br />

1.45 cm). Magnetic resonance cholangiopancreatography<br />

(MRCP) showed the presence <strong>of</strong> a stone impacted at the<br />

distal portion <strong>of</strong> the CBD (Figure 2). The patient was<br />

<strong>in</strong>travenously adm<strong>in</strong>istered midazolam (3 mg), pethid<strong>in</strong>e<br />

(50 mg), and butylscopolam<strong>in</strong>e (20 mg), and ERCP was<br />

performed with the patient <strong>in</strong> the left lateral position. A<br />

forward-view<strong>in</strong>g gastroscope (GIF-Q260, Olympus) was<br />

<strong>in</strong>itially <strong>in</strong>troduced; it was advanced through the <strong>in</strong>terposed<br />

<strong>colon</strong>ic segment, gastric remnant, and duodenum to reach<br />

the papilla <strong>of</strong> Vater. A radio-opaque standard guidewire<br />

(THSF-35-480, Wilson-Cook) was <strong>in</strong>serted deep <strong>in</strong>to the<br />

small <strong>in</strong>test<strong>in</strong>e, up to a distance beyond the papilla <strong>of</strong> Vater,<br />

via the accessory channel (Figure 3). The gastroscope was<br />

then withdrawn over-the-wire. Under fluoroscopic guidance,<br />

and with the patient <strong>in</strong> the left-lateral position, a sideview<strong>in</strong>g<br />

duodenoscope (TJF-240, Olympus) was <strong>in</strong>troduced<br />

carefully along the guidewire until it reached the papilla <strong>of</strong><br />

Vater. After cannulation with an ERCP catheter (StarTip<br />

cannula, PR-106Q-1, Olympus) as usual, cholangiography<br />

showed a fill<strong>in</strong>g defect (diameter, 1.5 cm) <strong>in</strong> the distal portion<br />

<strong>of</strong> the CBD; the lesion was determ<strong>in</strong>ed to be a CBD<br />

stone (Figure 4). Complete sph<strong>in</strong>cterotomy with a traction<br />

sph<strong>in</strong>cterotome was performed (Figure 5). The pigmented<br />

stone was successfully retrieved us<strong>in</strong>g a Dormia basket<br />

(Figure 6). Subsequent balloon-occlusion cholangiography<br />

showed complete clearance <strong>of</strong> the CBD. The patient was<br />

followed up <strong>in</strong> the outpatient department and rema<strong>in</strong>s well.<br />

DISCUSSION<br />

The <strong>colon</strong> has been used as an esophageal substitute s<strong>in</strong>ce<br />

1911. It has been proven to be superior to other substitutes,<br />

such as the stomach and small <strong>in</strong>test<strong>in</strong>e, because <strong>of</strong> it is length,<br />

acid resistance, and richness <strong>of</strong> vascular supply. It affords<br />

good overall satisfaction and allows ma<strong>in</strong>tenance <strong>of</strong> a<br />

wider surgical resection marg<strong>in</strong> <strong>in</strong> patients with cancers <strong>of</strong><br />

the gastroesophageal junction. The disadvantages <strong>of</strong> its application<br />

<strong>in</strong>clude prolonged operation time, extensive preoperative<br />

preparation, and the late redundancy <strong>of</strong> <strong>colon</strong>ic<br />

grafts [1,2] .<br />

Therapeutic ERCP with the application <strong>of</strong> a side-view<strong>in</strong>g<br />

duodenoscope is widely used <strong>in</strong> the management <strong>of</strong><br />

pancreatic or hepatobiliary diseases, such as biliary stones [3] .<br />

Technically, it is difficult to advance a side-view<strong>in</strong>g duodenoscope<br />

through the <strong>colon</strong> because the duodenoscope<br />

affords visualization <strong>of</strong> only areas to the sides <strong>of</strong> the scope,<br />

and because <strong>of</strong> the presence <strong>of</strong> <strong>colon</strong>ic <strong>in</strong>terhaustral folds,<br />

WJG|www.wjgnet.com<br />

C<br />

Figure 1 Esophagography show<strong>in</strong>g the <strong>in</strong>terposition <strong>of</strong> the <strong>colon</strong> (C) and<br />

the gastric remnant (S).<br />

Figure 2 Magnetic resonance cholangiopancreatography show<strong>in</strong>g a stone<br />

<strong>in</strong> the distal common bile duct (arrow). The arrowhead shows the second<br />

portion <strong>of</strong> the duodenum.<br />

Figure 3 The radio-opaque standard guidewire (arrowhead) was <strong>in</strong>serted<br />

through the work<strong>in</strong>g channel <strong>of</strong> the gastroscope.<br />

the angulation <strong>of</strong> the <strong>colon</strong>, and the redundancy <strong>of</strong> the<br />

<strong>colon</strong>ic graft [4] . To date, several techniques have been described<br />

for us<strong>in</strong>g the side-view<strong>in</strong>g duodenoscope to visualize<br />

the <strong>colon</strong>. Dafnis reported the successful application <strong>of</strong><br />

a unique technique for approach<strong>in</strong>g an <strong>in</strong>accessible <strong>colon</strong>ic<br />

polyp at the splenic flexure us<strong>in</strong>g an overtube to advance<br />

the side-view<strong>in</strong>g duodenoscope [5] . Another report <strong>of</strong> a case<br />

series on the management <strong>of</strong> <strong>in</strong>accessible <strong>colon</strong>ic polyps,<br />

advocated the technique <strong>of</strong> slightly bend<strong>in</strong>g the tip <strong>of</strong> the<br />

side-view<strong>in</strong>g duodenoscope, thereby provid<strong>in</strong>g a slopedforward<br />

view for perform<strong>in</strong>g polypectomy [6] . We believe<br />

that the use <strong>of</strong> a wire-guided side-view<strong>in</strong>g duodenoscope<br />

1788 April 7, 2011|Volume 17|Issue 13|<br />

C<br />

S

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Figure 4 With the patient <strong>in</strong> the left-lateral position, endoscopic retrograde<br />

cholangiopancreatography showed a fill<strong>in</strong>g defect <strong>in</strong> the distal part <strong>of</strong><br />

the common bile duct (arrow). The arrowhead shows the pancreatic duct.<br />

Figure 5 Complete sph<strong>in</strong>cterotomy.<br />

might represent a safe technique for approach<strong>in</strong>g <strong>in</strong>accessible<br />

<strong>colon</strong>ic polyps.<br />

In the present case, our most important concern was<br />

the smooth advancement <strong>of</strong> the duodenoscope through the<br />

<strong>colon</strong>ic graft. To address this concern, we <strong>in</strong>serted a radioopaque<br />

guidewire to serve as a roadmap. Fry et al [7] reported<br />

an over-the-wire method by us<strong>in</strong>g a Super-Stiff Amplatz<br />

guidewire, which was actually designed for cardiac catheterization,<br />

to <strong>in</strong>tubate the duodenum with a side-view<strong>in</strong>g<br />

duodenoscope <strong>in</strong> a patient with large paraesophageal hernia.<br />

The reason we chose the standard guidewire, <strong>in</strong>stead <strong>of</strong> a<br />

Super-Stiff Amplatz guidewire, was because it is entirely<br />

radio-opaque. It facilitated the localization and visualization<br />

<strong>of</strong> the tip <strong>of</strong> the duodenoscope under close fluoroscopic<br />

guidance. Despite this, the duodenoscope did, at one po<strong>in</strong>t,<br />

move away from the appropriate path <strong>in</strong> the gastro<strong>in</strong>test<strong>in</strong>al<br />

tract, dur<strong>in</strong>g the procedure. When the graft lumen could not<br />

be visualized on the endoscopic screen, we pushed the duodenoscope<br />

forward once its axis was the same as that <strong>of</strong> the<br />

wire, as determ<strong>in</strong>ed by fluoroscopy; the scope was advanced<br />

<strong>in</strong> this manner until the graft lumen could be seen (Figure 7).<br />

The duodenoscope was advanced through the graft, and the<br />

CBD stone was eventually retrieved.<br />

Manipulation <strong>of</strong> the guidewire is an art. One <strong>of</strong> its pr<strong>in</strong>ciples<br />

is to avoid loop<strong>in</strong>g, especially <strong>in</strong> a spacious cavity,<br />

such as the stomach. In our experience, we have observed<br />

WJG|www.wjgnet.com<br />

Yii CY et al . Wire-guided duodenoscope <strong>in</strong> <strong>colon</strong>ic <strong>in</strong>terposition<br />

Figure 6 The pigment stone retrieved by a Dormia basket.<br />

Figure 7 The duodenoscope (arrowhead) was pushed along the guidewire<br />

(arrow) at the same axis under fluoroscopic guidance .<br />

that the loop<strong>in</strong>g <strong>of</strong> the guidewire may cause the failure<br />

<strong>of</strong> esophageal or duodenal metallic stent implantation <strong>in</strong><br />

patients with malignant obstruction. The loop<strong>in</strong>g <strong>of</strong> the<br />

guidewire could render it difficult to <strong>in</strong>troduce the scope<br />

further. To avoid this loop<strong>in</strong>g, we advanced the tip <strong>of</strong> the<br />

guidewire to a distance beyond the papilla <strong>of</strong> Vater, <strong>in</strong>stead<br />

<strong>of</strong> stopp<strong>in</strong>g with<strong>in</strong> the stomach.<br />

Some experienced endoscopists prefer to backload the<br />

guidewire through the work<strong>in</strong>g channel <strong>of</strong> the duodenoscope.<br />

However, we th<strong>in</strong>k that this is not feasible because<br />

the side-view<strong>in</strong>g characteristic, with its acute angle <strong>of</strong> elevation.<br />

Backload<strong>in</strong>g would render it difficult to <strong>in</strong>sert the<br />

duodenoscope and would <strong>in</strong>crease the number <strong>of</strong> loops<br />

formed. Furthermore, the double-balloon enteroscope<br />

could not be applied <strong>in</strong> our case because it is a forwardview<strong>in</strong>g<br />

scope and lacks the angle <strong>of</strong> elevation required to<br />

support the use <strong>of</strong> ERCP accessories.<br />

Another technique that could have been considered <strong>in</strong><br />

the present case would be the direct <strong>in</strong>troduction <strong>of</strong> the<br />

side-view<strong>in</strong>g duodenoscope without the <strong>in</strong>itial use <strong>of</strong> the<br />

forward-view<strong>in</strong>g gastroscope; however, this would have<br />

made it difficult to clearly visualize the lumen, especially<br />

as this patient had undergone <strong>colon</strong>ic <strong>in</strong>terposition. Such<br />

an approach would be accompanied by a high risk <strong>of</strong> perforation.<br />

The successful application <strong>of</strong> our technique for<br />

perform<strong>in</strong>g therapeutic ERCP is pro<strong>of</strong> <strong>of</strong> the feasibility<br />

<strong>of</strong> this technique. To the best <strong>of</strong> our knowledge, this is the<br />

1789 April 7, 2011|Volume 17|Issue 13|

---

Yii CY et al . Wire-guided duodenoscope <strong>in</strong> <strong>colon</strong>ic <strong>in</strong>terposition<br />

first report on the use <strong>of</strong> this novel technique for treat<strong>in</strong>g<br />

a CBD stone <strong>in</strong> a patient with esophagectomy and <strong>colon</strong>ic<br />

<strong>in</strong>terposition.<br />

In conclusion, <strong>in</strong> cases with rare cl<strong>in</strong>ical presentations,<br />

it is necessary to carefully and accurately estimate possible<br />

h<strong>in</strong>drances and develop appropriate solutions to successfully<br />

overcome them.<br />

REFERENCES<br />

1 Yildirim S, Köksal H, Celayir F, Erdem L, Oner M, Baykan A.<br />

Colonic <strong>in</strong>terposition vs. gastric pull-up after total esophagectomy.<br />

J Gastro<strong>in</strong>test Surg 2004; 8: 675-678<br />

2 DeMeester SR. Colon <strong>in</strong>terposition follow<strong>in</strong>g esophagectomy.<br />

Dis Esophagus 2001; 14: 169-172<br />

WJG|www.wjgnet.com<br />

3 Cohen S, Bacon BR, Berl<strong>in</strong> JA, Fleischer D, Hecht GA, Loehrer<br />

PJ Sr, McNair AE Jr, Mulholland M, Norton NJ, Rabeneck<br />

L, Ransoh<strong>of</strong>f DF, Sonnenberg A, Vannier MW. National Institutes<br />

<strong>of</strong> Health State-<strong>of</strong>-the-Science Conference Statement:<br />

ERCP for diagnosis and therapy, January 14-16, 2002. Gastro<strong>in</strong>test<br />

Endosc 2002; 56: 803-809<br />

4 Grisolano SW, Petersen BT. Use <strong>of</strong> a duodenoscope to identify<br />

and treat a <strong>colon</strong>ic vascular malformation. Gastro<strong>in</strong>test<br />

Endosc 2004; 59: 323-325<br />

5 Dafnis G. A novel technique for endoscopic snare polypectomy<br />

us<strong>in</strong>g a duodenoscope <strong>in</strong> comb<strong>in</strong>ation with a <strong>colon</strong>oscope<br />

for the <strong>in</strong>accessible <strong>colon</strong>ic polyp. Endoscopy 2006; 38: 279-281<br />

6 Frimberger E, von Delius S, Rösch T, Schmid RM. Colonoscopy<br />

and polypectomy with a side-view<strong>in</strong>g endoscope. Endoscopy<br />

2007; 39: 462-465<br />

7 Fry LC, Howell CA, Mönkemüller KE. Use <strong>of</strong> a super-stiff<br />

Amplatz guidewire to <strong>in</strong>tubate the duodenum with a duodenoscope.<br />

Gastro<strong>in</strong>test Endosc 2002; 56: 773-774<br />

S- Editor Sun H L- Editor Stewart GJ E- Editor Ma WH<br />

1790 April 7, 2011|Volume 17|Issue 13|

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Onl<strong>in</strong>e Submissions: http://www.wjgnet.com/1007-9327<strong>of</strong>fice<br />

wjg@wjgnet.com<br />

www.wjgnet.com<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): I<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

ACKNOWLEDGMENTS<br />

Acknowledgments to reviewers <strong>of</strong> <strong>World</strong> <strong>Journal</strong> <strong>of</strong><br />

<strong>Gastroenterology</strong><br />

Many reviewers have contributed their expertise and<br />

time to the peer review, a critical process to ensure the<br />

quality <strong>of</strong> <strong>World</strong> <strong>Journal</strong> <strong>of</strong> <strong>Gastroenterology</strong>. The editors<br />

and authors <strong>of</strong> the articles submitted to the journal are<br />

grateful to the follow<strong>in</strong>g reviewers for evaluat<strong>in</strong>g the<br />

articles (<strong>in</strong>clud<strong>in</strong>g those published <strong>in</strong> this issue and<br />

those rejected for this issue) dur<strong>in</strong>g the last edit<strong>in</strong>g<br />

time period.<br />

Shiu-M<strong>in</strong>g Kuo, MD, University at Buffalo, 15 Farber Hall, 3435 Ma<strong>in</strong><br />

Street, Buffalo, NY 14214, United States<br />

Ezio Laconi, MD, PhD, Pr<strong>of</strong>essor <strong>of</strong> General Pathology, Department<br />

<strong>of</strong> Sciences and Biomedical Technologies, Unit <strong>of</strong> Experimental Pathology,<br />

University <strong>of</strong> Cagliari, Via Porcell, 4 - IV Piano, 09125 Cagliari, Italy<br />

María IT López, Pr<strong>of</strong>essor, Experimental Biology, University <strong>of</strong> Jaen,<br />

araje de las Lagunillas s/n, Jaén 23071, Spa<strong>in</strong><br />

Francesco Luzza, Pr<strong>of</strong>essor, MD, Department <strong>of</strong> Cl<strong>in</strong>ical and Experimental<br />

Medic<strong>in</strong>e, University <strong>of</strong> Catanzaro “Magna Graecia”, Campus<br />

Universitario di Germaneto, Viale Europa, Catanzaro 88100, Italy<br />

Nahum Méndez-Sánchez, MD, PhD, Departments <strong>of</strong> Biomedical<br />

Research, <strong>Gastroenterology</strong> and Liver Unit, Medica Sur Cl<strong>in</strong>ic and Foundation,<br />

Puente de Piedra 150, Col. Toriello Guerra, Tlalpan 14050, México,<br />

City, México<br />

Sebastian Mueller, MD, PhD, Pr<strong>of</strong>essor <strong>of</strong> Medic<strong>in</strong>e, Department <strong>of</strong><br />

Internal Medic<strong>in</strong>e, Salem Medical Center, and Center for Alcohol Research,<br />

University <strong>of</strong> Heidelberg, Zeppel<strong>in</strong>straße 11 - 33, Heidelberg, 69121,<br />

Germany<br />

Patrick O'Dwyer, MB, BCh, BAO, FRCS (1),MCh, FRCS (Glasg),<br />

University Department <strong>of</strong> Surgery,Western Infirmary, Glasgow, G11 6NT,<br />

United K<strong>in</strong>gdom<br />

Piero Port<strong>in</strong>casa, Pr<strong>of</strong>essor, Internal Medic<strong>in</strong>e - DIMIMP, University <strong>of</strong><br />

Bari Medical School, Hospital Policl<strong>in</strong>ico Piazza G. Cesare 11, Bari 70124,<br />

Italy<br />

Shan Rajendra, Associate Pr<strong>of</strong>essor, Department <strong>of</strong> Medic<strong>in</strong>e,<br />

Launceston General Hospital, Launceston, Tasmania 7250, Australia<br />

Robert V Rege, MD, Department <strong>of</strong> Surgery, University <strong>of</strong> Texas<br />

Southwestern Medical Center, 5323 Harry H<strong>in</strong>es Boulevard, Dallas, Texas,<br />

TX 75390-9031, United States<br />

Richard A Rippe, Dr., Department <strong>of</strong> Medic<strong>in</strong>e, The University <strong>of</strong><br />

WJG|www.wjgnet.com<br />

North Carol<strong>in</strong>a at Chapel Hill, Chapel Hill, NC 27599-7038, United States<br />

Heitor Rosa, Pr<strong>of</strong>essor, Department <strong>of</strong> <strong>Gastroenterology</strong> and<br />

Hepatology, Federal University School <strong>of</strong> Medic<strong>in</strong>e, Rua 126 n.21, Goiania<br />

- GO 74093-080, Brazil<br />

Ala<strong>in</strong> L Serv<strong>in</strong>, PhD, Faculty <strong>of</strong> Pharmacy, French National Institute<br />

<strong>of</strong> Health and Medical Research, Unit 756, Rue J.-B. Clément, F-922296<br />

Châtenay-Malabry, France<br />

Ana Crist<strong>in</strong>a Simões e Silva, MD, PhD, Pr<strong>of</strong>essor, Faculdade de<br />

Medic<strong>in</strong>a UFMG, Departamento de Pediatria, sala 267, Avenida Pr<strong>of</strong>essor<br />

Alfredo Balena, 190, Bairro Santa Efigênia, Belo Horizonte, M<strong>in</strong>as Gerais<br />

30130-100, Brazil<br />

Scott Steele, MD, FACS, FASCRS, Chief, Colon and Rectal Surgery,<br />

Dept <strong>of</strong> Surgery,Madigan Army Medical Center, Fort Lewis, WA 98431,<br />

United States<br />

Kazuaki Takabe, MD, PhD, Assistant Pr<strong>of</strong>essor <strong>of</strong> Surgery and Assistant<br />

Pr<strong>of</strong>essor <strong>of</strong> Biochemistry and Molecular Biology, Surgical Oncology, VCU<br />

Massey Cancer Center, Virg<strong>in</strong>ia Commonwealth University/Medical College<br />

<strong>of</strong> Virg<strong>in</strong>ia, PO Box 980011, Richmond VA 23298-0011, United States<br />

Paul Kwong-Hang Tam, MBBS (HK), ChM (Liverpool), FRCS<br />

(England, Ed<strong>in</strong>burgh, Glasgow, Ireland), FACS, FHKAM, FRCPCH<br />

(UK), Pro-Vice-Chancellor and Vice-President (Research), Chair<br />

<strong>of</strong> Paediatric Surgery, Department <strong>of</strong> Surgery, University <strong>of</strong> Hong Kong<br />

Medical Center, The University <strong>of</strong> Hong Kong, Queen Mary Hospital,<br />

Pokfulam Road, Hong Kong, Ch<strong>in</strong>a<br />

Hitoshi Tsuda, MD, PhD, Diagnostic Pathology Section,Cl<strong>in</strong>ical<br />

Laboratory Division, National Cancer Center Hospital, 5-1-1 Tsukiji, Chuoku,<br />

Tokyo 104-0045, Japan<br />

Eduardo Garcia Vilela, Pr<strong>of</strong>essor, PhD, Department <strong>of</strong> Internal<br />

Medic<strong>in</strong>e, Faculty <strong>of</strong> Medic<strong>in</strong>e, Federal University <strong>of</strong> M<strong>in</strong>as Gerais, Avenida<br />

Pr<strong>of</strong>essor Alfredo Balena, 190/ 2 andar, Belo Horizonte 30130-100, Brazil<br />

Hsiu-Po Wang, Pr<strong>of</strong>essor, Department <strong>of</strong> Emergency Medic<strong>in</strong>e,<br />

National Taiwan University Hospital, No. 7, Chung-Shan South Road,<br />

Taipei, Taiwan, Ch<strong>in</strong>a<br />

Toshiaki Watanabe, MD, PhD, Deptartment <strong>of</strong> Surgery, Teikyo<br />

University School <strong>of</strong> Medic<strong>in</strong>e, 2-11-1, Kaga, Itabashi-ku, Tokyo, 173-8605,<br />

Japan<br />

P<strong>in</strong>gchang Yang, Dr., MD, PhD, Department <strong>of</strong> Pathology and<br />

Molecular Medic<strong>in</strong>e, McMaster University, BBI-T3330, 50 Charlton Ave<br />

East, Hamilton, L8N 4A6, Canada<br />

I April 7, 2011|Volume 17|Issue 13|

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www.wjgnet.com<br />

Meet<strong>in</strong>gs<br />

Events Calendar 2011<br />

January 14-15, 2011<br />

AGA Cl<strong>in</strong>ical Congress <strong>of</strong><br />

<strong>Gastroenterology</strong> and Hepatology:<br />

Best Practices <strong>in</strong> 2011 Miami, FL<br />

33101, United States<br />

January 20-22, 2011<br />

Gastro<strong>in</strong>test<strong>in</strong>al Cancers Symposium<br />

2011, San Francisco, CA 94143,<br />

United States<br />

January 27-28, 2011<br />

Falk Workshop, Liver and<br />

Immunology, Medical University,<br />

Franz-Josef-Strauss-Allee 11, 93053<br />

Regensburg, Germany<br />

January 28-29, 2011<br />

9. Gastro Forum München, Munich,<br />

Germany<br />

February 4-5, 2011<br />

13th Duesseldorf International<br />

Endoscopy Symposium,<br />

Duesseldorf, Germany<br />

February 13-27, 2011<br />

<strong>Gastroenterology</strong>: New Zealand<br />

CME Cruise Conference, Sydney,<br />

NSW, Australia<br />

February 17-20, 2011<br />

APASL 2011-The 21st Conference <strong>of</strong><br />

the Asian Pacific Association for the<br />

Study <strong>of</strong> the Liver<br />

Bangkok, Thailand<br />

February 22, 2011-March 04, 2011<br />

Canadian Digestive Diseases Week<br />

2011, Vancouver, BC, Canada<br />

February 24-26, 2011<br />

Inflammatory Bowel Diseases<br />

2011-6th Congress <strong>of</strong> the European<br />

Crohn's and Colitis Organisation,<br />

Dubl<strong>in</strong>, Ireland<br />

February 24-26, 2011<br />

2nd International Congress on<br />

Abdom<strong>in</strong>al Obesity, Buenos Aires,<br />

Brazil<br />

February 24-26, 2011<br />

International Colorectal Disease<br />

Symposium 2011, Hong Kong, Ch<strong>in</strong>a<br />

February 26-March 1, 2011<br />

Canadian Digestive Diseases Week,<br />

WJG|www.wjgnet.com<br />

West<strong>in</strong> Bayshore, Vancouver, British<br />

Columbia, Canada<br />

February 28-March 1, 2011<br />

Childhood & Adolescent Obesity:<br />

A whole-system strategic approach,<br />

Abu Dhabi, United Arab Emirates<br />

March 3-5, 2011<br />

42nd Annual Topics <strong>in</strong> Internal<br />

Medic<strong>in</strong>e, Ga<strong>in</strong>esville, FL 32614,<br />

United States<br />

March 7-11, 2011<br />

Infectious Diseases: Adult Issues<br />

<strong>in</strong> the Outpatient and Inpatient<br />

Sett<strong>in</strong>gs, Sarasota, FL 34234,<br />

United States<br />

March 14-17, 2011<br />

British Society <strong>of</strong> <strong>Gastroenterology</strong><br />

Annual Meet<strong>in</strong>g 2011, Birm<strong>in</strong>gham,<br />

England, United K<strong>in</strong>gdom<br />

March 17-19, 2011<br />

41. Kongress der Deutschen<br />

Gesellschaft für Endoskopie und<br />

Bildgebende Verfahren e.V., Munich,<br />

Germany<br />

March 17-20, 2011<br />

Mayo Cl<strong>in</strong>ic <strong>Gastroenterology</strong> &<br />

Hepatology 2011, Jacksonville, FL<br />

34234, United States<br />

March 18, 2011<br />

UC Davis Health Informatics:<br />

Change Management and Health<br />

Informatics, The Keys to Health<br />

Reform, Sacramento, CA 94143,<br />

United States<br />

March 25-27, 2011<br />

MedicReS IC 2011 Good Medical<br />

Research, Istanbul, Turkey<br />

March 26-27, 2011<br />

26th Annual New Treatments <strong>in</strong><br />

Chronic Liver Disease, San Diego,<br />

CA 94143, United States<br />

April 6-7, 2011<br />

IBS-A Global Perspective, Pfister<br />

Hotel, 424 East Wiscons<strong>in</strong> Avenue,<br />

Milwaukee, WI 53202, United States<br />

April 7-9, 2011<br />

International and Interdiscipl<strong>in</strong>ary<br />

Conference Excellence <strong>in</strong> Female<br />

Surgery, Florence, Italy<br />

April 15-16, 2011<br />

Falk Symposium 177, Endoscopy<br />

Live Berl<strong>in</strong> 2011 Intest<strong>in</strong>al Disease<br />

Meet<strong>in</strong>g, Stauffenbergstr. 26, 10785<br />

Berl<strong>in</strong>, Germany<br />

April 18-22, 2011<br />

Pediatric Emergency Medic<strong>in</strong>e:<br />

Detection, Diagnosis and Develop<strong>in</strong>g<br />

Treatment Plans, Sarasota, FL 34234,<br />

United States<br />

April 20-23, 2011<br />

9th International Gastric Cancer<br />

Congress, COEX, <strong>World</strong> Trade<br />

Center, Samseong-dong, Gangnamgu,<br />

Seoul 135-731, South Korea<br />

April 25-27, 2011<br />

The Second International Conference<br />

<strong>of</strong> the Saudi Society <strong>of</strong> Pediatric<br />

<strong>Gastroenterology</strong>, Hepatology &<br />

Nutrition, Riyadh, Saudi Arabia<br />

April 25-29, 2011<br />

Neurology Updates for Primary<br />

Care, Sarasota, FL 34230-6947,<br />

United States<br />

April 28-30, 2011<br />

4th Central European Congress <strong>of</strong><br />

Surgery, Budapest, Hungary<br />

May 7-10, 2011<br />

Digestive Disease Week, Chicago, IL<br />

60446, United States<br />

May 12-13, 2011<br />

2nd National Conference Cl<strong>in</strong>ical<br />

Advances <strong>in</strong> Cystic Fibrosis, London,<br />

England, United K<strong>in</strong>gdom<br />

May 19-22, 2011<br />

1st <strong>World</strong> Congress on Controversies<br />

<strong>in</strong> the Management <strong>of</strong> Viral Hepatitis<br />

(C-Hep), Palau de Congressos de<br />

Catalunya, Av. Diagonal, 661-671<br />

Barcelona 08028, Spa<strong>in</strong><br />

May 21-24, 2011<br />

22nd European Society <strong>of</strong><br />

Gastro<strong>in</strong>test<strong>in</strong>al and Abdom<strong>in</strong>al<br />

Radiology Annual Meet<strong>in</strong>g and<br />

Postgraduate Course, Venise, Italy<br />

May 25-28, 2011<br />

4th Congress <strong>of</strong> the <strong>Gastroenterology</strong><br />

Association <strong>of</strong> Bosnia and<br />

Herzegov<strong>in</strong>a with <strong>in</strong>ternational<br />

participation, Hotel Holiday Inn,<br />

Sarajevo, Bosnia and Herzegov<strong>in</strong>a<br />

June 11-12, 2011<br />

The International Digestive Disease<br />

Forum 2011, Hong Kong, Ch<strong>in</strong>a<br />

June 13-16, 2011<br />

Surgery and Disillusion XXIV<br />

SPIGC, II ESYS, Napoli, Italy<br />

June 14-16, 2011<br />

International Scientific Conference<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): I<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

on Probiotics and Prebiotics-<br />

IPC2011, Kosice, Slovakia<br />

June 22-25, 2011<br />

ESMO Conference: 13th <strong>World</strong><br />

Congress on Gastro<strong>in</strong>test<strong>in</strong>al Cancer,<br />

Barcelona, Spa<strong>in</strong><br />

June 29-2, 2011<br />

XI Congreso Interamericano<br />

de Pediatria "Monterrey 2011",<br />

Monterrey, Mexico<br />

September 2-3, 2011 Falk Symposium<br />

178, Diverticular Disease, A Fresh<br />

Approach to a Neglected Disease,<br />

Gürzenich Cologne, Mart<strong>in</strong>str. 29-37,<br />

50667 Cologne, Germany<br />

September 10-11, 2011<br />

New Advances <strong>in</strong> Inflammatory<br />

Bowel Disease, La Jolla, CA 92093,<br />

United States<br />

September 10-14, 2011<br />

ICE 2011-International Congress <strong>of</strong><br />

Endoscopy, Los Angeles Convention<br />

Center, 1201 South Figueroa Street<br />

Los Angeles, CA 90015,<br />

United States<br />

September 30-October 1, 2011<br />

Falk Symposium 179, Revisit<strong>in</strong>g<br />

IBD Management: Dogmas to be<br />

Challenged, Sheraton Brussels<br />

Hotel, Place Rogier 3, 1210 Brussels,<br />

Belgium<br />

October 19-29, 2011<br />

Cardiology & <strong>Gastroenterology</strong> |<br />

Tahiti 10 night CME Cruise, Papeete,<br />

French Polynesia<br />

October 22-26, 2011<br />

19th United European<br />

<strong>Gastroenterology</strong> Week, Stockholm,<br />

Sweden<br />

October 28-November 2, 2011<br />

ACG Annual Scientific Meet<strong>in</strong>g &<br />

Postgraduate Course, Wash<strong>in</strong>gton,<br />

DC 20001, United States<br />

November 11-12, 2011<br />

Falk Symposium 180, IBD 2011:<br />

Progress and Future for Lifelong<br />

Management, ANA Interconti Hotel,<br />

1-12-33 Akasaka, M<strong>in</strong>ato-ku, Tokyo<br />

107-0052, Japan<br />

December 1-4, 2011<br />

2011 Advances <strong>in</strong> Inflammatory<br />

Bowel Diseases/Crohn's & Colitis<br />

Foundation's Cl<strong>in</strong>ical & Research<br />

Conference, Hollywood, FL 34234,<br />

United States<br />

I April 7, 2011|Volume 17|Issue 13|

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GENERAL INFORMATION<br />

<strong>World</strong> <strong>Journal</strong> <strong>of</strong> <strong>Gastroenterology</strong> (<strong>World</strong> J Gastroenterol, WJG, pr<strong>in</strong>t<br />

ISSN 1007-9327, onl<strong>in</strong>e ISSN 2219-2840, DOI: 10.3748) is a<br />

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validate the <strong>in</strong>novativeness, scientific and practical values <strong>of</strong> their<br />

own research achievements, thus ensur<strong>in</strong>g that their articles have<br />

novel arguments or viewpo<strong>in</strong>ts, solid evidence and correct conclu-<br />

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sion; and (4) Maximization <strong>of</strong> the benefits <strong>of</strong> employees: It is an<br />

iron law that a first-class journal is unable to exist without first-class<br />

editors, and only first-class editors can create a first-class academic<br />

journal. We <strong>in</strong>sist on strengthen<strong>in</strong>g our team cultivation and construction<br />

so that every employee, <strong>in</strong> an open, fair and transparent<br />

environment, could contribute their wisdom to edit and publish<br />

high-quality articles, thereby realiz<strong>in</strong>g the maximization <strong>of</strong> the<br />

personal benefits <strong>of</strong> editorial board members, authors and readers,<br />

and yield<strong>in</strong>g the greatest social and economic benefits.<br />

Aims and scope<br />

The major task <strong>of</strong> WJG is to report rapidly the most recent results<br />

<strong>in</strong> basic and cl<strong>in</strong>ical research on esophageal, gastro<strong>in</strong>test<strong>in</strong>al,<br />

liver, pancreas and biliary tract diseases, Helicobacter pylori, endoscopy<br />

and gastro<strong>in</strong>test<strong>in</strong>al surgery, <strong>in</strong>clud<strong>in</strong>g: gastroesophageal<br />

reflux disease, gastro<strong>in</strong>test<strong>in</strong>al bleed<strong>in</strong>g, <strong>in</strong>fection and tumors;<br />

gastric and duodenal disorders; <strong>in</strong>test<strong>in</strong>al <strong>in</strong>flammation, micr<strong>of</strong>lora<br />

and immunity; celiac disease, dyspepsia and nutrition; viral<br />

hepatitis, portal hypertension, liver fibrosis, liver cirrhosis, liver<br />

transplantation, and metabolic liver disease; molecular and cell<br />

biology; geriatric and pediatric gastroenterology; diagnosis and<br />

screen<strong>in</strong>g, imag<strong>in</strong>g and advanced technology.<br />

Columns<br />

The columns <strong>in</strong> the issues <strong>of</strong> WJG will <strong>in</strong>clude: (1) Editorial: To<br />

<strong>in</strong>troduce and comment on major advances and developments <strong>in</strong><br />

the field; (2) Frontier: To review representative achievements, comment<br />

on the state <strong>of</strong> current research, and propose directions for<br />

future research; (3) Topic Highlight: This column consists <strong>of</strong> three<br />

formats, <strong>in</strong>clud<strong>in</strong>g (A) 10 <strong>in</strong>vited review articles on a hot topic, (B)<br />

a commentary on common issues <strong>of</strong> this hot topic, and (C) a commentary<br />

on the 10 <strong>in</strong>dividual articles; (4) Observation: To update<br />

the development <strong>of</strong> old and new questions, highlight unsolved<br />

problems, and provide strategies on how to solve the questions;<br />

(5) Guidel<strong>in</strong>es for Basic Research: To provide guidel<strong>in</strong>es for basic<br />

research; (6) Guidel<strong>in</strong>es for Cl<strong>in</strong>ical Practice: To provide guidel<strong>in</strong>es<br />

for cl<strong>in</strong>ical diagnosis and treatment; (7) Review: To review systemically<br />

progress and unresolved problems <strong>in</strong> the field, comment<br />

on the state <strong>of</strong> current research, and make suggestions for future<br />

work; (8) Orig<strong>in</strong>al Article: To report <strong>in</strong>novative and orig<strong>in</strong>al f<strong>in</strong>d<strong>in</strong>gs<br />

<strong>in</strong> gastroenterology; (9) Brief Article: To briefly report the<br />

novel and <strong>in</strong>novative f<strong>in</strong>d<strong>in</strong>gs <strong>in</strong> gastroenterology and hepatology;<br />

(10) Case Report: To report a rare or typical case; (11) Letters to the<br />

Editor: To discuss and make reply to the contributions published<br />

<strong>in</strong> WJG, or to <strong>in</strong>troduce and comment on a controversial issue <strong>of</strong><br />

general <strong>in</strong>terest; (12) Book Reviews: To <strong>in</strong>troduce and comment on<br />

quality monographs <strong>of</strong> gastroenterology and hepatology; and (13)<br />

Guidel<strong>in</strong>es: To <strong>in</strong>troduce consensuses and guidel<strong>in</strong>es reached by<br />

<strong>in</strong>ternational and national academic authorities worldwide on basic<br />

research and cl<strong>in</strong>ical practice gastroenterology and hepatology.<br />

Name <strong>of</strong> journal<br />

<strong>World</strong> <strong>Journal</strong> <strong>of</strong> <strong>Gastroenterology</strong><br />

ISSN and EISSN<br />

ISSN 1007-9327 (pr<strong>in</strong>t)<br />

ISSN 2219-2840 (onl<strong>in</strong>e)<br />

<strong>World</strong> J Gastroenterol 2011 April 7; 17(13): I-VI<br />

ISSN 1007-9327 (pr<strong>in</strong>t) ISSN 2219-2840 (onl<strong>in</strong>e)<br />

© 2011 Baishideng. All rights reserved.<br />

I April 7, 2011|Volume 17|Issue 13|

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Instructions to authors<br />

Indexed and Abstracted <strong>in</strong><br />

Current Contents ® /Cl<strong>in</strong>ical Medic<strong>in</strong>e, Science Citation Index<br />

Expanded (also known as SciSearch ® ), <strong>Journal</strong> Citation Reports ® ,<br />

Index Medicus, MEDLINE, PubMed, PubMed Central, Digital<br />

Object Identifer, and Directory <strong>of</strong> Open Access <strong>Journal</strong>s. ISI,<br />

Thomson Reuters, 2009 Impact Factor: 2.092 (33/65 <strong>Gastroenterology</strong><br />

and Hepatology).<br />

Published by<br />

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All articles published <strong>in</strong> this journal represent the viewpo<strong>in</strong>ts<br />

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<strong>of</strong> covariance, etc. The review<strong>in</strong>g po<strong>in</strong>ts <strong>in</strong>clude: (1) Statistical<br />

methods should be described when they are used to verify the results;<br />

(2) Whether the statistical techniques are suitable or correct;<br />

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UK Medic<strong>in</strong>es Research Council Guidel<strong>in</strong>es for Good Cl<strong>in</strong>ical<br />

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the authors must expla<strong>in</strong> the rationale for their approach<br />

and demonstrate that the <strong>in</strong>stitutional review body explicitly approved<br />

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Before submitt<strong>in</strong>g, authors should make their study approved<br />

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If experimental animals were used, the materials and methods<br />

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Authors should reta<strong>in</strong> one copy <strong>of</strong> the text, tables, photographs<br />

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II April 7, 2011|Volume 17|Issue 13|

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l<strong>in</strong>e. For assistance, authors encounter<strong>in</strong>g problems with the Onl<strong>in</strong>e<br />

Submission System may send an email describ<strong>in</strong>g the problem<br />

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MANUSCRIPT PREPARATION<br />

All contributions should be written <strong>in</strong> English. All articles must be<br />

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for each <strong>of</strong> the manuscript sections is as follows:<br />

Title page<br />

Title: Title should be less than 12 words.<br />

Runn<strong>in</strong>g title: A short runn<strong>in</strong>g title <strong>of</strong> less than 6 words should<br />

be provided.<br />

Authorship: Authorship credit should be <strong>in</strong> accordance with the<br />

standard proposed by ICMJE, based on (1) substantial contributions<br />

to conception and design, acquisition <strong>of</strong> data, or analysis and<br />

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Institution: Author names should be given first, then the complete<br />

name <strong>of</strong> <strong>in</strong>stitution, city, prov<strong>in</strong>ce and postcode. For example,<br />

Xu-Chen Zhang, Li-X<strong>in</strong> Mei, Department <strong>of</strong> Pathology,<br />

Chengde Medical College, Chengde 067000, Hebei Prov<strong>in</strong>ce,<br />

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for example, George Sgourakis, Department <strong>of</strong> General, Visceral,<br />

and Transplantation Surgery, Essen 45122, Germany; George<br />

Sgourakis, 2nd Surgical Department, Korgialenio-Benakio Red<br />

Cross Hospital, Athens 15451, Greece.<br />

Author contributions: The format <strong>of</strong> this section should be:<br />

Author contributions: Wang CL and Liang L contributed equally<br />

to this work; Wang CL, Liang L, Fu JF, Zou CC, Hong F and Wu<br />

XM designed the research; Wang CL, Zou CC, Hong F and Wu<br />

XM performed the research; Xue JZ and Lu JR contributed new<br />

reagents/analytic tools; Wang CL, Liang L and Fu JF analyzed the<br />

data; and Wang CL, Liang L and Fu JF wrote the paper.<br />

Supportive foundations: The complete name and number <strong>of</strong><br />

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Pr<strong>of</strong>essor <strong>of</strong> Medic<strong>in</strong>e, Chief, Liver Center, <strong>Gastroenterology</strong><br />

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Normally, three experts are <strong>in</strong>vited for each article. Decision for<br />

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Pr<strong>of</strong>essor J<strong>in</strong>g-Yuan Fang, Shanghai Institute <strong>of</strong> Digestive<br />

Disease, Shanghai, Affiliated Renji Hospital, Medical Faculty,<br />

Shanghai Jiaotong University, Shanghai, Ch<strong>in</strong>a; Pr<strong>of</strong>essor X<strong>in</strong>-<br />

Wei Han, Department <strong>of</strong> Radiology, The First Affiliated Hospital,<br />

Zhengzhou University, Zhengzhou, Henan Prov<strong>in</strong>ce, Ch<strong>in</strong>a; and<br />

Pr<strong>of</strong>essor Anren Kuang, Department <strong>of</strong> Nuclear Medic<strong>in</strong>e, Huaxi<br />

Hospital, Sichuan University, Chengdu, Sichuan Prov<strong>in</strong>ce, Ch<strong>in</strong>a.<br />

Abstract<br />

There are unstructured abstracts (no more than 256 words)<br />

and structured abstracts (no more than 480). The specific requirements<br />

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An <strong>in</strong>formative, structured abstracts <strong>of</strong> no more than 480<br />

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study/…”; MATERIALS AND METHODS (no more than<br />

140 words); RESULTS (no more than 294 words): You should<br />

present P values where appropriate and must provide relevant<br />

data to illustrate how they were obta<strong>in</strong>ed, e.g. 6.92 ± 3.86 vs 3.61<br />

± 1.67, P < 0.001; CONCLUSION (no more than 26 words).<br />

Key words<br />

Please list 5-10 key words, selected ma<strong>in</strong>ly from Index Medicus,<br />

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Text<br />

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INTRODUCTION, MATERIALS AND METHODS,<br />

RESULTS and DISCUSSION, and should <strong>in</strong>clude appropriate<br />

Figures and Tables. Data should be presented <strong>in</strong> the ma<strong>in</strong><br />

text or <strong>in</strong> Figures and Tables, but not <strong>in</strong> both. The ma<strong>in</strong> text<br />

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III April 7, 2011|Volume 17|Issue 13|

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Instructions to authors<br />

uniform legends for the same subjects. For example: Figure 1<br />

Pathological changes <strong>in</strong> atrophic gastritis after treatment. A:...;<br />

B:...; C:...; D:...; E:...; F:...; G: …etc. It is our pr<strong>in</strong>ciple to publish<br />

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Tables<br />

Three-l<strong>in</strong>e tables should be numbered 1, 2, 3, etc., and mentioned<br />

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but rather added <strong>in</strong>to the text where applicable. The <strong>in</strong>formation<br />

should complement, but not duplicate the text. Use one<br />

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and a third below the Table, above any footnotes. Vertical and<br />

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Notes <strong>in</strong> tables and illustrations<br />

Data that are not statistically significant should not be noted.<br />

a P < 0.05, b P < 0.01 should be noted (P > 0.05 should not be<br />

noted). If there are other series <strong>of</strong> P values, c P < 0.05 and d P<br />

< 0.01 are used. A third series <strong>of</strong> P values can be expressed as<br />

e P < 0.05 and f P < 0.01. Other notes <strong>in</strong> tables or under illustrations<br />

should be expressed as 1 F, 2 F, 3 F; or sometimes as other<br />

symbols with a superscript (Arabic numerals) <strong>in</strong> the upper left<br />

corner. In a multi-curve illustration, each curve should be labeled<br />

with ●, ○, ■, □, ▲, △, etc., <strong>in</strong> a certa<strong>in</strong> sequence.<br />

Acknowledgments<br />

Brief acknowledgments <strong>of</strong> persons who have made genu<strong>in</strong>e<br />

contributions to the manuscript and who endorse the data and<br />

conclusions should be <strong>in</strong>cluded. Authors are responsible for<br />

obta<strong>in</strong><strong>in</strong>g written permission to use any copyrighted text and/or<br />

illustrations.<br />

REFERENCES<br />

Cod<strong>in</strong>g system<br />

The author should number the references <strong>in</strong> Arabic numerals accord<strong>in</strong>g<br />

to the citation order <strong>in</strong> the text. Put reference numbers<br />

<strong>in</strong> square brackets <strong>in</strong> superscript at the end <strong>of</strong> citation content or<br />

after the cited author’s name. For citation content which is part <strong>of</strong><br />

the narration, the cod<strong>in</strong>g number and square brackets should be<br />

typeset normally. For example, “Crohn’s disease (CD) is associated<br />

with <strong>in</strong>creased <strong>in</strong>test<strong>in</strong>al permeability [1,2] ”. If references are cited<br />

directly <strong>in</strong> the text, they should be put together with<strong>in</strong> the text, for<br />

example, “From references [19,22-24] , we know that...”.<br />

When the authors write the references, please ensure that<br />

the order <strong>in</strong> text is the same as <strong>in</strong> the references section, and also<br />

ensure the spell<strong>in</strong>g accuracy <strong>of</strong> the first author’s name. Do not list<br />

the same citation twice.<br />

PMID and DOI<br />

Pleased provide PubMed citation numbers to the reference list,<br />

e.g. PMID and DOI, which can be found at http://www.ncbi.<br />

nlm.nih.gov/sites/entrez?db=pubmed and http://www.crossref.org/SimpleTextQuery/,<br />

respectively. The numbers will be<br />

used <strong>in</strong> E-version <strong>of</strong> this journal.<br />

Style for journal references<br />

Authors: the name <strong>of</strong> the first author should be typed <strong>in</strong> boldfaced<br />

letters. The family name <strong>of</strong> all authors should be typed<br />

with the <strong>in</strong>itial letter capitalized, followed by their abbreviated<br />

first and middle <strong>in</strong>itials. (For example, Lian-Sheng Ma is abbreviated<br />

as Ma LS, Bo-Rong Pan as Pan BR). The title <strong>of</strong> the<br />

cited article and italicized journal title (journal title should be<br />

<strong>in</strong> its abbreviated form as shown <strong>in</strong> PubMed), publication date,<br />

WJG|www.wjgnet.com<br />

volume number (<strong>in</strong> black), start page, and end page [PMID:<br />

11819634 DOI: 10.3748/wjg.13.5396].<br />

Style for book references<br />

Authors: the name <strong>of</strong> the first author should be typed <strong>in</strong> boldfaced<br />

letters. The surname <strong>of</strong> all authors should be typed with the<br />

<strong>in</strong>itial letter capitalized, followed by their abbreviated middle and<br />

first <strong>in</strong>itials. (For example, Lian-Sheng Ma is abbreviated as Ma LS,<br />

Bo-Rong Pan as Pan BR) Book title. Publication number. Publication<br />

place: Publication press, Year: start page and end page.<br />

Format<br />

<strong>Journal</strong>s<br />

English journal article (list all authors and <strong>in</strong>clude the PMID where applicable)<br />

1 Jung EM, Clevert DA, Schreyer AG, Schmitt S, Rennert J,<br />

Kubale R, Feuerbach S, Jung F. Evaluation <strong>of</strong> quantitative<br />

contrast harmonic imag<strong>in</strong>g to assess malignancy <strong>of</strong> liver<br />

tumors: A prospective controlled two-center study. <strong>World</strong> J<br />

Gastroenterol 2007; 13: 6356-6364 [PMID: 18081224 DOI:<br />

10.3748/wjg.13.6356]<br />

Ch<strong>in</strong>ese journal article (list all authors and <strong>in</strong>clude the PMID where applicable)<br />

2 L<strong>in</strong> GZ, Wang XZ, Wang P, L<strong>in</strong> J, Yang FD. Immunologic<br />

effect <strong>of</strong> Jianpi Yishen decoction <strong>in</strong> treatment <strong>of</strong> Pixudiarrhoea.<br />

Shijie Huaren Xiaohua Zazhi 1999; 7: 285-287<br />

In press<br />

3 Tian D, Araki H, Stahl E, Bergelson J, Kreitman M.<br />

Signature <strong>of</strong> balanc<strong>in</strong>g selection <strong>in</strong> Arabidopsis. Proc Natl<br />

Acad Sci USA 2006; In press<br />

Organization as author<br />

4 Diabetes Prevention Program Research Group. Hypertension,<br />

<strong>in</strong>sul<strong>in</strong>, and pro<strong>in</strong>sul<strong>in</strong> <strong>in</strong> participants with impaired<br />

glucose tolerance. Hypertension 2002; 40: 679-686 [PMID:<br />

12411462 PMCID:2516377 DOI:10.1161/01.HYP.00000<br />

35706.28494.09]<br />

Both personal authors and an organization as author<br />

5 Vallancien G, Emberton M, Harv<strong>in</strong>g N, van Moorselaar<br />

RJ; Alf-One Study Group. Sexual dysfunction <strong>in</strong> 1,<br />

274 European men suffer<strong>in</strong>g from lower ur<strong>in</strong>ary tract<br />

symptoms. J Urol 2003; 169: 2257-2261 [PMID: 12771764<br />

DOI:10.1097/01.ju.0000067940.76090.73]<br />

No author given<br />

6 21st century heart solution may have a st<strong>in</strong>g <strong>in</strong> the tail. BMJ<br />

2002; 325: 184 [PMID: 12142303 DOI:10.1136/bmj.325.<br />

7357.184]<br />

Volume with supplement<br />

7 Geraud G, Spier<strong>in</strong>gs EL, Keywood C. Tolerability and<br />

safety <strong>of</strong> frovatriptan with short- and long-term use for<br />

treatment <strong>of</strong> migra<strong>in</strong>e and <strong>in</strong> comparison with sumatriptan.<br />

Headache 2002; 42 Suppl 2: S93-99 [PMID: 12028325<br />

DOI:10.1046/j.1526-4610.42.s2.7.x]<br />

Issue with no volume<br />

8 Banit DM, Kaufer H, Hartford JM. Intraoperative frozen<br />

section analysis <strong>in</strong> revision total jo<strong>in</strong>t arthroplasty. Cl<strong>in</strong><br />

Orthop Relat Res 2002; (401): 230-238 [PMID: 12151900<br />

DOI:10.1097/00003086-200208000-00026]<br />

No volume or issue<br />

9 Outreach: Br<strong>in</strong>g<strong>in</strong>g HIV-positive <strong>in</strong>dividuals <strong>in</strong>to care.<br />

HRSA Careaction 2002; 1-6 [PMID: 12154804]<br />

Books<br />

Personal author(s)<br />

10 Sherlock S, Dooley J. Diseases <strong>of</strong> the liver and billiary<br />

system. 9th ed. Oxford: Blackwell Sci Pub, 1993: 258-296<br />

IV April 7, 2011|Volume 17|Issue 13|

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Chapter <strong>in</strong> a book (list all authors)<br />

11 Lam SK. Academic <strong>in</strong>vestigator’s perspectives <strong>of</strong> medical<br />

treatment for peptic ulcer. In: Swabb EA, Azabo S. Ulcer<br />

disease: <strong>in</strong>vestigation and basis for therapy. New York:<br />

Marcel Dekker, 1991: 431-450<br />

Author(s) and editor(s)<br />

12 Breedlove GK, Schorfheide AM. Adolescent pregnancy.<br />

2nd ed. Wieczorek RR, editor. White Pla<strong>in</strong>s (NY): March<br />

<strong>of</strong> Dimes Education Services, 2001: 20-34<br />

Conference proceed<strong>in</strong>gs<br />

13 Harnden P, J<strong>of</strong>fe JK, Jones WG, editors. Germ cell tumours<br />

V. Proceed<strong>in</strong>gs <strong>of</strong> the 5th Germ cell tumours Conference;<br />

2001 Sep 13-15; Leeds, UK. New York: Spr<strong>in</strong>ger,<br />

2002: 30-56<br />

Conference paper<br />

14 Christensen S, Oppacher F. An analysis <strong>of</strong> Koza’s computational<br />

effort statistic for genetic programm<strong>in</strong>g. In: Foster<br />

JA, Lutton E, Miller J, Ryan C, Tettamanzi AG, editors. Genetic<br />

programm<strong>in</strong>g. EuroGP 2002: Proceed<strong>in</strong>gs <strong>of</strong> the 5th<br />

European Conference on Genetic Programm<strong>in</strong>g; 2002 Apr<br />

3-5; K<strong>in</strong>sdale, Ireland. Berl<strong>in</strong>: Spr<strong>in</strong>ger, 2002: 182-191<br />

Electronic journal (list all authors)<br />

15 Morse SS. Factors <strong>in</strong> the emergence <strong>of</strong> <strong>in</strong>fectious diseases.<br />

Emerg Infect Dis serial onl<strong>in</strong>e, 1995-01-03, cited<br />

1996-06-05; 1(1): 24 screens. Available from: URL: http://<br />

www.cdc.gov/ncidod/eid/<strong>in</strong>dex.htm<br />

Patent (list all authors)<br />

16 Pagedas AC, <strong>in</strong>ventor; Ancel Surgical R&D Inc., assignee.<br />

Flexible endoscopic grasp<strong>in</strong>g and cutt<strong>in</strong>g device<br />

and position<strong>in</strong>g tool assembly. United States patent US<br />

20020103498. 2002 Aug 1<br />

Statistical data<br />

Write as mean ± SD or mean ± SE.<br />

Statistical expression<br />

Express t test as t (<strong>in</strong> italics), F test as F (<strong>in</strong> italics), chi square test<br />

as χ 2 (<strong>in</strong> Greek), related coefficient as r (<strong>in</strong> italics), degree <strong>of</strong> freedom<br />

as υ (<strong>in</strong> Greek), sample number as n (<strong>in</strong> italics), and probability<br />

as P (<strong>in</strong> italics).<br />

Units<br />

Use SI units. For example: body mass, m (B) = 78 kg; blood pressure,<br />

p (B) = 16.2/12.3 kPa; <strong>in</strong>cubation time, t (<strong>in</strong>cubation) = 96 h,<br />

blood glucose concentration, c (glucose) 6.4 ± 2.1 mmol/L; blood<br />

CEA mass concentration, p (CEA) = 8.6 24.5 mg/L; CO 2 volume<br />

fraction, 50 mL/L CO 2, not 5% CO 2; likewise for 40 g/L formaldehyde,<br />

not 10% formal<strong>in</strong>; and mass fraction, 8 ng/g, etc. Arabic<br />

numerals such as 23, 243, 641 should be read 23 243 641.<br />

The format for how to accurately write common units and<br />

quantums can be found at: http://www.wjgnet.com/1007-9327/<br />

g_<strong>in</strong>fo_20100315223018.htm.<br />

Abbreviations<br />

Standard abbreviations should be def<strong>in</strong>ed <strong>in</strong> the abstract and<br />

on first mention <strong>in</strong> the text. In general, terms should not be abbreviated<br />

unless they are used repeatedly and the abbreviation<br />

is helpful to the reader. Permissible abbreviations are listed <strong>in</strong><br />

Units, Symbols and Abbreviations: A Guide for Biological and<br />

Medical Editors and Authors (Ed. Baron DN, 1988) published<br />

by The Royal Society <strong>of</strong> Medic<strong>in</strong>e, London. Certa<strong>in</strong> commonly<br />

used abbreviations, such as DNA, RNA, HIV, LD50, PCR,<br />

HBV, ECG, WBC, RBC, CT, ESR, CSF, IgG, ELISA, PBS, ATP,<br />

EDTA, mAb, can be used directly without further explanation.<br />

WJG|www.wjgnet.com<br />

Italics<br />

Quantities: t time or temperature, c concentration, A area, l length,<br />

m mass, V volume.<br />

Genotypes: gyrA, arg 1, c myc, c fos, etc.<br />

Restriction enzymes: EcoRI, H<strong>in</strong>dI, BamHI, Kbo I, Kpn I, etc.<br />

Biology: H. pylori, E coli, etc.<br />

Examples for paper writ<strong>in</strong>g<br />

Editorial: http://www.wjgnet.com/1007-9327/g_<strong>in</strong>fo_20100315<br />

220036.htm<br />

Frontier: http://www.wjgnet.com/1007-9327/g_<strong>in</strong>fo_20100315<br />

220305.htm<br />

Topic highlight: http://www.wjgnet.com/1007-9327/g_<strong>in</strong>fo_20<br />

100315220601.htm<br />

Observation: http://www.wjgnet.com/1007-9327/g_<strong>in</strong>fo_201003<br />

12232427.htm<br />

Guidel<strong>in</strong>es for basic research: http://www.wjgnet.com/1007-93<br />

27/g_<strong>in</strong>fo_20100315220730.htm<br />

Guidel<strong>in</strong>es for cl<strong>in</strong>ical practice: http://www.wjgnet.com/1007-<br />

9327/g_<strong>in</strong>fo_20100315221301.htm<br />

Review: http://www.wjgnet.com/1007-9327/g_<strong>in</strong>fo_20100315<br />

221554.htm<br />

Orig<strong>in</strong>al articles: http://www.wjgnet.com/1007-9327/g_<strong>in</strong>fo_20<br />

100315221814.htm<br />

Brief articles: http://www.wjgnet.com/1007-9327/g_<strong>in</strong>fo_2010<br />

0312231400.htm<br />

Case report: http://www.wjgnet.com/1007-9327/g_<strong>in</strong>fo_2010<br />

0315221946.htm<br />

Letters to the editor: http://www.wjgnet.com/1007-9327/g_<strong>in</strong>fo_<br />

20100315222254.htm<br />

Book reviews: http://www.wjgnet.com/1007-9327/g_<strong>in</strong>fo_2010<br />

0312231947.htm<br />

Guidel<strong>in</strong>es: http://www.wjgnet.com/1007-9327/g_<strong>in</strong>fo_2010<br />

0312232134.htm<br />

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V April 7, 2011|Volume 17|Issue 13|

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Instructions to authors<br />

Language evaluation<br />

The language <strong>of</strong> a manuscript will be graded before it is sent for<br />

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