The mongolian gerbil gastric epithelial cell line immortalized by SV-40 large T antigen

already have comparablelevels of Cox-2 expression to GC and can therefore be regardedas precancerous lesions. ~ i p

i:

4005 Long Term Infection With Helicobacter Pylori Induces Intestinal Methaplasia, Carcinoid And Cancer In The Stomachs Of Mongolian Gerbils. Effect Of Eradication. Yoshihiro Keto, Misako Ebata, Atsuko Ono, Kikuko Amagase, Susumu Okabe, Kyoto Pharmaceutical University, Kyoto Japan

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Watanabeet al. 1999 reported that H. pylori induced gastric carcinoid and cancer 62 wk after infection in Mongolian gerbils (M. gerbils). In this study, we examinedwhether eradication of H. pylorican preventthe developmentof gastric mucosal injuries including gastric cancer. [Methods] H. py/ofi (TN2GF4, 2 × 108 CFU/head)was orally given once to male M. gerbils (6 wk). At 4 or 8 mo after H. pylori inoculation, eradication was performed by omeprazole (60 mg/kg, 4 wk) + clarithromycin (100 mg/kg, 2 wk). After such treatment, two groups of animals were killed at around 5 and 9 mo to confirm the achievementof eradication and to determine MPO activity in the gastric mucosa. The other animals had been maintained until 18 mo after H. pylori inoculation. Subsequently,the animals were killed and the histological study was performed with H&E and AB-PAS staining. The titer of anti-/-/, pylori IgG antibody was determined. [Results] In the control animals, gastric ulcers were observed 5 or 9 mo after H. pylofi inoculation, the ulcer size being about 3 or 10 mm2, respectively. Viable H. pylori (determined by culture method) and higher MPO activity were also detected. In the eradicationgroup, gastric ulcers and viable H. pyloriwere not determined,yet MPO activity was markedlyreduced.At 18 mo after H. pylofiinoculation, carcinoid (3/1O) and adenocarcinoma(2/ 1O) were observed,in the submucosal layerand underthe muscle layerto serosa, respectively. Atrophic gastritis (9/10), gastric ulcer (7/10) and intestinal mataplasia(9/10) in the gastric mucosa were also observed. Such changeswere not observed in the animals eradicatedwith drugs 4 mo after infection. However,the atrophy-like changes (6/10) and intestinal metaplasia (5/10) were remainedin animals eradicatedat 8 mo after infection. [Conclusions] We demonstrated that while H. pylori infection alone induced intestinal metaplasiaand gastric cancer, fully eradication of H. pylori prevented from gastric carcinogenesis in M. gerbils.

4003 SP Expressing Melnplasia (SPEM) and Early Gastric Cancer Anna Margret Halldorsdottir, Dept of Pathology and Medicine, Univ Hosp of Iceland, Reykjavik Iceland; Jon G. Jonasson, Dept of Pathology, Univ Hosp of Iceland, Reykjavik Iceland; Margret Oddsdottir, Jonas Magnusson, Dept of Surg, Univ Hosp of Iceland, Reykjavik Iceland; Jeffrey R. Lee, James R. Goldenring, Augusta VAMC and Institute of Molecular Med and Genetics, Augusta, GA BACKGROUNDAlthough the association of H. pylori with gastric adenocarcinoma is now well established,the mechanism by which chronic H. pylori infection contributes to gastric adenocarcinomais poorly understood. Recently, a novel metaplastic cell lineage in the body mucosawas described,which has morphologicalsimilarities to Brunnersglandsand expresses the trefoil peptidespasmolytic polypeptide(SP). DesignatedSPEM(SP expressingmetaplasia), this lineagewas strongly associatedwith both chronic H. pytori infection and gastric adenocarcinoma. This study investigates the association between early gastric adenocarcinomaand SPEM. METHODSTwenty-nine patientswith early gastric cancer diagnosed in the years 1983 to 2000 who had archival tissues from gastric resections, were found in databasesprovided by the Surgical and Pathology Departments of the Icelandic University Hospital. For each case, six sections were selected (two containing cancer,the others without), recur and stained with H&E, PAS-DIA, and gaetrin plus SP immunostaining. These histological sections were examined and the results analysed. The presence of SPEM and IM adjacent to and distant from the cancerwas comparedand SP immunostaining within dysplastic/cancerouscells was identified. RESULTSSixteen early gastric cancers were located in the antrum, three at the junction of the body and antrum and ten in the body. In all the thirteen cases where the tumor was located in the body or at the body/antrum junction, SPEM was observed in the vicinity of the cancer. In 76 % of all the twenty-nine cases, SPEM was present in the body mucosa distant from the cancer and was usually associated with atrophic gastritis. On the other hand, IM was found adjacent to the tumor in 76 % of cases and in body sections in 52 % of resections. SP immunostaining was noted within cancer cells in 62 % of tumors, and within dysplastic cells in 76 % of resectionswhere dysplasiawas present.The SP positivity was almost always located in the deepest portions of the dysplastic/cancerousglands and (sub)apically in the cells. CONCLUSIONThese results support the hypothesis that SPEM is associatedwith gastric adenocarcinoma.High levelsof SP staining in dysplastic and neoplastic cells associated with early gastric cancers suggests that SPEM may be a precursor to the evolution of dysplasia and adenocarcinomain gastric mucosa infected with H. pylori.

4006 Analysis of p53 Mutations and Helicobacter pylori Infection in Human and Animal Models Kazunari Murakami, Toshio Fujioka, Masaaki Kodama,Shoji Honda, Tadayoshi Okimoto, Touta Oda, Akira Nishizono, Ryugo Sato, Jiro Kagawa,Masaru Nasu, Oita Medical Univ, Oita Japan Background: It is consideredthat p53 gene mutations play a critical role in the development of gastric carcinome. We examined the relationship between Helicobacter pylori (H.pylori) infection and p53 gene mutation of gastric mucosa in human and animal models. Methods: To detect original p53 DNA sequence of Japanese monkey and Mongolian gerbil, p53 gene of these animals were amlified using nested PCR method with the primers for human p53 gene. Direct DNA sequencing of exons 5, 6, 7, and 8 of the p53 genes were performed by dyedeoxyterminator method for gastric mucosa of human, Japanesemonkey and Mongolian gerbil. Expressionof p53 was examinedimmunohistochemically in Japanesemonkey model. Results: Mutations in p53 genewere identified in 52.4 % of human H. pylod positive mucosa and in 100% of monkey H. pylori positive mucosa. However, no mutations of p53 gene were found in gastric mucosa of Mongolian gerbil infected with H.pylori. There was no mutation in H. pylori negativegastritis mucosa of human, monkey and Mongolian gerbil. In Japanese monkey, nuclear staining p53 was seen in the grandular cells of the mucosa infected with H.pylori, especially in the neck region of the glands. Conclusions:Thesefindings demonstrate that the H. pylori infection can induce p53 point mutations in human and Japanesemonkey and appear to be involved in the pathway leading to dysplasia or carcinoma. However,there was no p53 mutation in Mongolian gerbil model at present in our Direct DNA sequencing method, further studies about this model are needed.

4OO4 Expression of the DNA Repair Enzyme Apurinic/Apyrimidinic Endonuclensein Gastric Mucoss and the Effect of Helicobacterpylori Infection. Simon M. Everett, Tetsuro Yoshimura, Peter D. Howdle, Jean E. Crabtree, St James's Univ Hosp, Leeds United Kingdom

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Introduction: The multi-function enzyme apudnic/apydmidinic endonuclease(APE) regulates a number of transcription factors including NF-kB, AP-1 and p53 and is essential in DNA base excision repair. Patterns of expression are altered in cervical and ovarian cancers and APE is upregulatedby oxidative stress. This has recently beendemonstratedin gastric cancer cell lines, suggesting that upregulation in vivo may limit oxidative DNA damage induced by H. pylori. Our aim, therefore, was to determine the effect of H. pylori infection on expression of APE in gastric biopsies. Methods: At endoscopy, antral and corpus biopsies were taken from 36 patients (mean age 51 years, range 19-70) who had not been taking proton pump inhibitors or NSAID's. R. pylori infection was determined by histology and urease testing. APE mRNA and G3PDH mRNA (control) were analysed by semi-quantitative RT-PCR and computer image analysis. Presence of APE protein was determined immunohistochemically in formalin fixed gastric biopsies using a mouse monoclonal antibody against APE. Results: APE/G3PDH ratio was similar in antral biopsies from H.pylori positive (1.02, SEM 0.24) and negative (0.98, SEM 0.23) patients (p = 0.9, 95% CI of difference -.064 to 0.73). APE mRNA expression was observed in all but 3 antral biopsies and in all corpus biopsies. Expression of APE/G3PDHwas higher in the corpus (0.76, SEM 0.13) than antrum (0.49, SEM 0.10) but this did not reach statistical significance (p = 0.09, 95%CI of difference -0.60 to 0.05, n = 15). Immunohistochemistry of infected and uninfected biopsies demonstrated APE expression in virtually all foveolar and glandular epithelial cells, with relative sparing of the superficial mucosa. Staining was predominantly nuclear although cytoplasmic staining was also seen, mainly in the glandular epithelium. Conclusions:Although H. pylori increasesoxidative stress in the gastric mucosa, the DNA repair enzyme APE is not upregulated in infected samples. APE mRNA and protein are expressedconstitutively in gastric epithelial cells. This study was funded by a grant from Yorkshire Cancer Research.

The Mongolian Gerbil Gastric Epithelial Cell Line Immortalized by SV-40 Large T Antigen Kenta Yoshiura, Tadahito Shimada, Takahiro Mitsuhashi, Kumi Takahashi, Hideyuki Hiraishi, Akira Terano, Dokkyo medical Sch, Mibu Japan Background: Helicobacter pylori has been assumed as a gastric carcinogen in human. To investigatethe molecular mechanism of gastric carcinogenesisby H. py/ori, an in vitro system to investigate the interaction between the bacteria and the host gastric epithelial cells is crucial. So far, H. pylofi infection without any carcinogen has been shown to cause the gastric adenocarcinomaonly in the Mongolian gerbil as an animal model. Aim: To make an immortalized gastric epithelial cell line from the Mongolian gerbil by introducing SV-40 large T antigen (LTA). Methods: Stomach of the Mongolian gerbil (five weeks) was excised and treated with proteinases,followed by incubation in the buffer solution containing 3mM EDTA at room temperature. Isolatedcells were harvestedby centrifuge and cultured in MEM medium with 10% fetal bovine serum (FBS) on a collagen-coatedculture plate in 5% C02 incubator at 37°C. The primary culture cells were transfected with the pSVori- plasmid harboring SV40 large T antigen (kindly gifted by Dr. S. Yasumoto, Kanagawa,Japan) by the lipofection method. Cell proliferation was assessed by the WST-1 assay. Results: Primary cultures of the Mongolian gerbil gastric epithelial cells formed a monolayeron a culture plate.The original cells did not survive more than four weeks.When the pSVori was transfected with the primary cultures on the third day, surviving cells appearedafter four weeks. After a subculture, they continued to grow and underwent repeated re-plating. After four months, the cells, named MGS (Mongolian gerbil Gastric epithelial cells with SV-40), were vigorously growing at the passage 22. The MGS cells were positive for PAS staining, indicating an epithelial origin. Immunofluorostaining for SV-40 LTA was positive in the nucleus of the MGS cells at a comparable level to GSM06, a gastric epithelial cell line originated from the temperaturesensitive SV-40 LTA transgenic mouse. The MGS cells formed a spheroid-shapedcolony in the soft agar culture. Examinationof a vertical section of the colony revealedthat MGS cells formed an epithelial monolayer at the surface of the spheroid. Epidermal growth factor (10 ng/ml) significantly stimulated the cell proliferation in the absence of FBS. Conclusion: We

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established the Mongolian gerbil gastric epithelial cell line (MGS) by introduction of the SV40 LTA. The MGS cell line is expected to provide an in vitro model for gastric carcinogenesis by H. pylori infection.

4008 Conservative Treatment of Primary Gastric Low-Grade B-Cell Lymphomaof MncalaAssociated LymphoidTissue: Predictive Factors of Response and Outcome. Michael Levy, Service de Gastroenterologie Hosp Henri Mondor, Creteil France; Christiane Copie-Bergman, Pathology et EA 2348 Hosp Henri Mondor, Creteil France; Catherine Traulle, Service de Medicine Interne Hosp Nord, Amiens France; Anne Lavergne-Slove, Service d'Anatomo-pathologie Hosp Lariboisiere, Paris France; Nicole Brousse, Service d'Anatomo-pathologie Hosp Necker Enfants-Malades, Paris France; Jean-Francois Rejou, Service d'Anatomo-pathologie Hosp Saint-Antoine, Paris France; Antoine De Mascarel, Service d'Anatomo-pathologie CHU de Bordeaux, Bordeaux France; Francois Hemery, Biostatistiques et Informatique Hosp Henri Mondor, Creteil France; Philippe Geuiard, Pathology et EA 2348 Hosp Henri Mondor, Creteil France; Jean-Charles Delctiier, Service de Gastroenterologie Hosp Henri Mondor, Creteil France;, Groupe d'Etudes du Lymphome de I'Adulte (GEt.A) Background. Primary gastric low-grade B-cell lymphoma (PGL) of mucosa-associated lymphoid tissue (MALT) may regress with conservative treatment such as anti-Helicobacter pylori (H pylon) therapy or monochemotherapy. The aims of the present study were to analyse the predictive factors of response to anti-H pyloritreatment, to assess the effects of an adjuvant therapy in responding patients and to evaluate an altemative therapy in non responding patients. Methods. From 1995 to 2000, 48 H py/ori infected patients with Iocalised PGL of MALT were treated with anti-H pyloritreatment. Endoscopic and endoscopic ultrasonography (EUS) features and the large cells rate were analysed. Eradication of H py/od and tumoral response were assessed at 2 and 6 months respectively. From 1996, patients in remission at 6 months were randomised to receive either chlorambucil per os for 6 months or no treatment. Patients who did not respond to H pylori eradication received chlorambucil per os for 1 year. Results. Among the 48 treated patients, 33 (69%) were in complete (n = 28) or in partial (n = 5) remission, and 15 (31%) in treatment failure at 6 months. H py/or/was eradicated in 47 patients. The response was not correlated to the endoscopic features nor to the large cells rate. in contrast it was related to ultrasonngraphic features: remission was achieved in 75% of cases when gastric wall was normal or thicker than 5 mm without lymph nodes versus only 33% when endoscopic ultrasonography showed enhanced gastric wall thicker than 5 mm with presence of lymph nodes (p = 0.033). All responding patients remained in remission (median 34 months) whatever the treatment they received (no treatment or chlorambucil). Remission could be achievedwith chlorambucil in 58% of the non responding patients to anti-H pyloritreatment. Conclusions. The major negative predictive factor of the tumoral response to anti-H pylori treatment in patients with PGL of MALT was the presence of lymph nodes in patients with tumoral infiltration greater than 5 mm on EUS. In responding patients, remission remained stable suggesting that an adjuvant chemotherapy was not useful. In patients who failed to respond to Hpylorieradication, monochemotharapywith chlorambucil proved to be efficient but new therapeutic modalities should be evaluated to improve the control of the tumoral process.

4010 Long-Term Outcome of Patients with Localized Gastric Marginal Zone B-Cell Lymphoma of MALT Following Exclusively Helicoabcter pylori Eradication Therapy. Wolfgang Fischhach, Dept of Internal Medicine, Aschaffenburg Germany; Maria Elisabeth Kolve-Gnebeler, Mediznische Poliklinik, Wuerzburg Germany; Brigitte Dragosics, Gesundhaitszentmm Sd, Vienna Germany; Martin Stolte, Oept of Pathology, Bayreuth Germany; Axel Grainer, Hans Konrad Mller-Hermelink, Dept of Pathology, Wuerzburg Germany Background: Helicobacter pylori (Hp) is nowadays accepted as a decisive pathogenetic factor of low-grade gastric lymphoma of MALT-type. Eradication therapy has become the well accepted initial treatment of stage El. However, data on the long-term outcome of these patients are rare and the curative potential of this approach is still under debate. We, here, report a large serie of 95 patients with a follow-up of at least 12 months. Methods: From March 1993 on, 95 patients (35 female, 60 male, mean age 55.8 vears, range 27-85) were diagnosed to have gastric marginal zone B-cell lymphoma of MALT-type (according to the REAL/WHO-classification) by centralized histologic review. Complete diagnostic work-up including abdominal and cervical ultrasound, CT scan of the chest and abdomen, bone marrow biopsy and endoscopic ultrasound revealed stage El according to the Ann Arbor staging system modified by Musshoff in all cases. Triple therapy using omeprazole, clarithromycin and metronidazole or amoxicilline (OMC, OAC) was performed. Follow-up examinations were carried out at 3- and 6-monthly intervals during the first two and five years, respectively, and yearly thereafter. Complete remission (CR) was defined as the total disappearanceof lymphoma, and partial remission (PR) was assumed in case of tumor reduction of at least 50%. Those with normalization of the macroscopic findings but histologically persisting residual lymphoma infiltrates were classified as minimal residual disease (MRD). Results: The median follow-up was 49.5 months (range 12-89). Hp eradication was successful in 93/95 patients (98%) as determined by rapid urease test and histology. Based on an ITF analysis the long term outcome was as follows: see table There were four relapses (2 high-grade, 2 low-grade lymphoma) within a time period of 6-20 months after initial CR, one with an Hp-reinfection. Conclusion: These data show that i) Hp-eradication is an effective treatment modality in localized gastric marginal zone B-cell lymphoma of MALT; ii) the majority of patients treated this way have a favourable long-term outcome obviously offering a real chance of cure. CR 62 (65%)

MRD

PR

15 (16%)

9 (10%}

Stable disease 7 (7%)

Progress unknown 1 (1%)

1 (1%)

total 95 (100%)

4111 Gactdn, C y c t o o x y g ~ in Gastric MALT-Lymphoma Before and After Eradication of HelioMIxter pylori (HP) Stanisiaw J. Konturek, Dept of Physiology, Univ Medical Sch, Cracow Poland; Peter C. Kocturek, Dept of Medicine, Univ of Eflangen-Nuremberg, Eriangen Germany; Teresa Star-zynska,Krzysztof Madicz, Dept of Gastroenterology, Pomeranian Medical Acad, $zczecin Poland; Piotr Pierzchalski, Dept of Physiology, Univ Medical Sch, Cracow Poland; Eckhart G. Hahn, Dept of Medicine, Univ of Erlangen-Nuremberg, Eriangen Germany Background: HP infection is considered as an important risk factor of gastric cancer but the mechanism of this HP-induced carcinogenesis has not been explained. Low grade, mucosal associated lymphoid tissue (MALT)-Iymphoma is an unique among gastric malignancies where causal involvement of this infection has been proposed based on the regression of the tumor following the HP eradication. Methods: In this report ten well-defined primary, low-grade gastric MALT-lymphomas infected with HP have been examined before and 6 months after one week of successful HP-eradication (clarithromycin + amoxicillin + omaprazole) as proved by negative ~3C-UBT.Gastric biopsy samples from tumor and intact antrum and corpus mucosa were obtained during endoscopy before and after HP-eraclicationfor assessment of expression of gustrin and gastric receptor (CCK~R)aswell as cycloo~genase (COX)-1 and COX-2 using RT-PCR and Western blot. The gastric lumen and serum gastrin and mucosal and tumor tissue Oastrin content and PGE2biosynthesis were determined by RIA before and after HP eradication. Results: Eradication of HP resulted in complete endoscopic and histological remission of MALT-iymphoma in 9 out of 10 patients as assessed 6 months after this eradication. Before eradication, the mRNA expression for gastrin and CCKo-Ras welt as for COX-1 and COX-2 were observed in tumor tissue and infected mucosa. Gestrin content in tumor tissue was about 5 times higher than that in antrum mucosa. Corpus mucusa expressed only mRNA for CCKB-Rand antrum mucusa only mRNA for gastrin. Six months upon the HP-eradication when MALT-lymphoma regressed both endoscopically and histologically, the expression of gastrin and COX-2 disappeared from the former area of MALT-lymphoma. Gastrin mRNA remained detectable only in antrum mucosa, CCKe-R mRNA only in corpus mucosa and COX-1 mRNA both in antrum and corpus mucosa. Gastric luminal and antrum gastrin levels and gastric mucosa and tumor PGE2,which were greatly elevated before HPeradication, became normalized after this eradication. Conclusions: This study demonstrates that low-grade gastric MALT-lymphoma is linked to HP infection which promotes mRNA expression of gastrin and COX-2 and results in excessive release of gastdn and PGE2that may contribute to the development of MALT-lymphoma.

4009 Conservative Treatment Of Primary Gastric B-Cell LymphomaOf MALT-Type Arising In The Setting Of HIV Infection And Posttrensplantation Christiane Copie Bergman, Tibor Ponnelle, Michael Levy, Yves Levy, Hosp Henri Mondor, Creteil France; Cecile Goujard, Pierre Bedossa, Hosp Bicetre, Bicetre France; Olivier Toupance, Hosp de la maison Blanche, Reims France; Md Diebold, CHRU Reims, Reims France; Gilles Salles, Francoise Berger, Hosp Lyon Sud, Lyon France; Jean Charles Delchier, Philippe Gaulard, Hosp Henri Mondor, Creteil France Marginal zone B-cell lymphomas of MALT-type are the most frequent extranndal lymphomas but occurrence of these lymphomas in the setting of immunosuppression is unusual. We describe 8 cases in patients with HIV infection or organ transplant. Methods: 8 patients (6 males, mean age 51 years, range 29-66 ), HIV infected (n = 3) or transplant recipients (renal = 4, cardiac = 1), with primary gastric MALT-type lymphomas were followed. Hpy/oriwas present in seven patients. Mean time between diagnosis of lymphoma and organ transplant was 8 years (range 6-13 ). Seven patients were stage IE, 1 patient was stage liE, according to Ann Arbor system modified by Musshof. Seven patients received anti-H pyloritreatment consisting of a combination of omeprazole, amoxicillin, and clarithromycin (OAC). One of these patients subsequently received chlorambucil. One patient without H pylori infection at presentation was treated surgically and subsequent chemotherapy (CHOP).Results: All patients had morphologic features of low-grade marginal zone B-cell lymphoma of MALT-type at presentation. All cases were negative for EBV by in situ hybridization. Six patients treated conservatively with OAC were in complete remission with no further treatment after a mean time of 9 months (range 5-12). One patient who received OAC plus chlorambucil was in complete remission at 12 months. The HIV patient treated surgically and with CHOP had a minor large cell component documented histologically on the gastrectomy specimen and relapsed at 20 months. Further treatment with chlorambucil resulted in complete remission at 30 months. Remission persisted in all patients (median follow-up 20 months, range 5-46 months). One patient died of heart failure at 46 months. Conclusion: Primary gastric B-cell lymphomas of MALT-type may occur in immunosuppressed patients. They differ from usual immunosuppression-associated lymphoid malignancies by their lack of association with EBV and their favourable outcome. These results suggest that these patients should be treated conservatively as immunocompetent patients.

4012 Relationship Between Gastric Localization Of Hepatitis C Vires And Gastric MucecaAssociated Lymphoid Tissue Related To Helicobecter Pylori Infection Giovanni Cammarota, Rosaella Cianci, Luigi Maria Larocca, Franco Pandotfi, Antonio Gasbarfini, Maurizio Pompili, Giulia Pignataro, Cristiana Di Campli, Lucio Cuoco, Monica Vustola, Manuela Cammarota, Giovanni Gasbarrini, Catholic Univ, Rome Italy Background It has beensuggestedthat hepatitis C virus (HCV), a hepatotropic and lymphotropic virus, plays a role in the pathogenesis of B-cell non-Hbdgkin lymphoma. In particular, gastric lymphoma has been reported in patients with HCV infection. HCV has been localized in several tissues besides the liver. Therefore, the definition of the possible gastric localization of the

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