and tumors were correlated to dinicopathologlcal data including survival. Results: Hierarchi- cal cluster analysis revealed throe different genomic gastric cancer profiles (cluster 1, 2, and 3), with cluster 2 showing a pattern intermediate between cluster 1 and 3. These profiles were significantly correlated with lymph node status (p = 0.02). Moreover, gastric cancer cases from cluster 3 (n = 14) showed a significantly better prognosis than those from cluster 1 (n = 9) and 2 (n = 6), (p = 0.02). Conclusions: Genomic profiling of gastric adenocarcinomas based on microarray analysis of chromosomal copy number changes pre- dicts lymph node status and allows for discriminating a subgroup of patients with high risk of lymph node metastasis. This latter observation could clinically be highly relevant for selecting candidate patients lor extended lymph node resection.

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Differential Expression of Caveolin-1 in Gastric Cancer Cells and Adjacent Stromal Tissue Mark Juhasz, Jie Chen, Ju[iane Hoffmann, Christoph Roecken, Zsoh Tulassay, Peter Malfertbeiner, Matthias Ebert

Introduction: Caveolin-1 acts as the driving force of caveolae formation, and is responsible for organizing the suhcellular distnbution of signalling molecules. Permrbances in caveolin- 1 gene expression were therefore hypothesized to play an important part in carcinogenesis. The role of caveolin-I in gastric cancer has not been previously investigated. MateriaLs and Methods: Quantitative caveolin-1 mRNA expression were studied in 19 gastric cancer specimens with real-time PCR. The P132L mutation, previously described in breast cancer cells, was evaluated by RFLP analysis. Hypermethylation of the promoter region of the caveolin- 1 gene was examined by incubating gastric cancer cells with 5-aza-2'-deoxycytidine. Caveolin- 1 protein expression was evaluated in 41 gasmc cancer specimens using immuno- bistochemistry and in 3 randomly selected cases by western blot analysis. Results: Caveolin- 1 mRNA levels were reduced in 14/19 gasmc cancer tissues (74%) as compared to the tumour-free tissue. No mutation of the caveolin-1 gene and no hypermethylation of the promoter region could be detected Caveolin-1 expression was found only in three cases (7%) in the gastric cancer cells Interestingly, expression was more frequent and more intense in the stromal compartment of well-differentiated tumours compared to undifferentiated and metastasing gastric cancers. Conclusion: Caveolin-1 expression is frequently lost in gastric cancer cells and is only retained in the stmmal compartment of wen-differentiated cancers. In agreement with results on caveolin-1 expression in other turnouts, cavedin-1 might act as a tumour-suppressor gene in gastric cancer as well.

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New Mutations in APC and B-Catenin Gene in Wnt Signaling Pathway in Iranian Gastric Adenocarcinoma Mohammad Reza Zali, Harold Asadzadeh, Nazanin Pazouki, Kamran Ghaflarzadegan, Seyed Javad Mirhassani Moghaddam, Shahin Ansari, Mohammad Reza Abbaszadeghan

Introduction: Gastric cancer is one of the most prevalent cancers in the world particularly in Iran. Unlike colorectal cancer, genetic alterations during the tumorigenesis of gastric cancer are not characterized First studies of gastric adeuocarcinomas determined loss of heterozygosity (LOH) in the chromosomal regions of 5q. Two putative tumor suppressor genes exist at 5q21-22 known as Adenomatous Pdyposis Cofi (APC) and Muted in Colorectal Cancer (MCC) genes. APC protein phosphoD'lates B-catenin, which is central event in Wnt signaling and oncogenic pathway; therefore, determining any genetic alterations of APC and B-catenin affecting mmauon and progression of gastric cancer in Iranian population is very important. Methods: During a ten months period from December 2000 to September 2001, all documented gastric cancer cases reported from pathology centers from eight provinces in Iran were registered in the study and the pedigree of all patients was illustrated. Mutational analysis o( APC, B-catenin genes and analysis 5q LOH (regions of APC & MCC genes) was performed in 40 gastric adenocarcinoma patients. Four Mutation Cluster Regions (MCR) of APC gene was screened for mutations using PCR-SSCP The mutation hotspot region of B- catenin gene (exon 3) was analyzed by PCR-SSCP and PCR-RFLP. LOH of APC and MCC reglmrs was analyzed by PCR-based methods. Results: Following the sequencing of SSCP band shifts, in addition to reported mutations in these genes, two new mutations in APC gene (MCR-D cds 1520; CAT (O)/CAG (H)) and (MCR-C cds; 1513 ATG (M)/ATT (ocher) and 2 new mutations in B-catenin gene (exon3 cds: 45 AGT (S)/ACT (T)) and (exon3 cds; 45 AGT (S)/ATT (ocher) were detected, Conclusion: In conclusion, the genetic alterations found in this study can be implicated in deregulation of Wnt signaling pathway and initiation of gastric cancer in Iranian population because a total of 62.5% of paraffin-embedded gastric adenocarcinoma specimens contained at least one of the following genetic alterations: B- catenin gene mutation (25 %), 5q LOH (22.5%) and APC (15%) gene mutation.

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The Differences in Microsatellite Instability between Diffuse-type and Intestinal- type Gastric Cancer from a High-risk Population Wh Choe, Jl Kim, Sy Lee, Jh Lee, Y.-H. Kim, Hj Son, P-L. Rhee, Jc Rhee, Cs Ki, Hb Jeon, Jw Kim, Sy Song

Background: Microsatellite alterations are common feature of gastric cancers in the Korean population, but, there have been few reports of associations between histological classification and microsatdlite instability (MSI). So, we examined MSI of gastric adenocarcinomas to determine whether the histological subtypes according to Laureu's classification are correlated with MSI status.Methods: Paraffin embedded tissue samples from 28 gastric cancer patmnts (14 diffuse type, 14 intestinal type) were obtained. The specimens of diffuse type and intestinal type were matched up according to the patient's sex, age. MS1 was analyzed by using PCR amplitication and automated DNA sequencing. Loss of hMLH1, hMSH2 protein expression was studied by immunohistochemistry Mononucleotide markers (BAT-25, BAT- 26 ) and dinudeotide markers (D2S123, D5S346, D17S250 ) were used in the analyses of MSI Microsatellite genotypes were categonzed as high incidence of MSI (MS1-H, more than

30% positive marker) and low incidence of MS1 (MSI-L, less than 30% positive marker). Results: Of the 28 cases, MSI-H was found in 14cases (50%). MSI-H was more frequently observed in adenocarcinomas of the intestinal-type than the diffuse-type (11/14, 3/14 respec- tively ; p<0.05). Ten (71%) and 3 (21%) of 14 MSI-H tumors showed loss of hMLH1, hMSH2 protein expression, respectively. Two(14%) and 1(11%) of 14 MSI-L tumors showed loss of hMLH1, hMSH2 protein expression, respectively. Immunostaining results on loss of hMLH1 protein expression revealed significant correlation with MS1 statns.(P<0.05) Conclusion: These findings suggest that intestinal-type and diffuse-type of gastric adenocarci- noma may have different molecular pathway for carcinogenesis. And gastric cancer with MSI-H shows a defective expression of hMLH1 protein.

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Microsatellite Alterations in Serum DNA in Patients with Ulcerative Colitis Peter P, auh, 5teffen Rickes, Michael Fleischhacker

Background: Patients with long standing ulcerative colitis have a high risk for the development of colorectal cancer. In order to detect dysplastic or malignant alterations as early as possible, these patients are frequently investigated by colonoscopy. The aim of this study was i) to establish a panel of genetic alterations which might be useful markers for diagnostic purposes, staging, or follow up, and it) to examine whether these alterations are also detectable in free circulating nucleic acid. For this purpose we used microsatellites, which are short interspersed, highly repetitive and polymorphic sequences, mostly located in non-coding sequences. Methods: We used a set of 11 microsatellties located on different chromosomes for the detection of aherations in free circulating serum DNA in comparison to DNA from white blood ceUs (which is considered wild type) in 50 patients with ulcerative colitis. PCR primers were radiolabeled using T4 kinase, the PCR products from amplified serum and white blood cell DNA were separated by running through a denaturing polyacrylamide gel and analyzed by autoradiography. Results: Eight out of 50 patients (16%) showed one or more microsatellite aheration/s in their serum DNA Four of them had an alteration in one marker, 2 patients showed alterations in 2 markers and 2 more patients had changes in 4 and 5 markers, respectively. There were no alterations in BAI-25, BAT-26, BAT-40 and D13S175 markers in any patient, whereas the D12S90 marker showed ahemtions in 50% of all patients who tested positive. The only patient who showed dysplastic areas in his biopsy samples, aLso had the highest number of microsatellite ahemtions (5 changes) in free circulating serum DNA. Conclusion: From the panel of 11 microsatellite markers used for these studies, some of them seem to be well suited for the detection of ulcerative colitis - associated alterations in free circulating serum DNA. Ongoing studies will show whether the same alterations are also detectable in biopsy samples of patients with microsatdlite alterations in their serum DNA and whether this panel of genetic markers might be useful for an early detection of ulcerative colitis - associated colorectal tumor. The fact, that no alterations were found in BAT-40, BAT-25 and BAT-26 markers, which are used to differenti- ate microsatelhte instable from microsatellite stable colorectal tumors, is another indication for different pathways for the development of spontaneous and ulcerative colitis - associ- ated tumors.

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Immunohistochemical Expression of Bcl-2, Bcl-xL, Bax, p53 Proteins in Gastric Adenoma and Adenocarcinoma Dongsoo Lee, Sokwon Han, Jongtae Back, Byunmin Ahn, Eunhee Lee, lnsik Chung, Dooho Park

Object; The aim of this study was to investigate the immunohistochemical expression of the bel-2, bcl-xL, bax, and p53 proteins accordmg to the pathological parameters such as grade of dysplasia, histological type, depth of invasion, lymph node metastasis, and TNM stage in the gastric adenoma and gastric adenocarcinoma. Methods: Immunohistochemical staining using monoclonal bcl-2, bd-xL, bax, p53, was performed on paraffin embedded specimens from 41 gasmc adenomas and from 100 gastric adenocarcinomas. Ki-67 labelling index and apoptosis index were also examined. Results: The results were as follows : 1. The expression rate of the bd-2 was higher in adenomas(34.2%, P<O.O0001), especially in high grade dysplasia(52.4%, P=O.O020), than adenocarcinomas(2%). 2. The expression rate of the bd-xL was higher in adenocarcinomas(55%, P=0.00158) than adenomas(22%). /he degree of the bcl-xL expression was higher in the intestinal type than in the diffuse type(P= 0.0161). 3. The expression rate of the bax was higher in adenocarcinomas(58%, P=O.0003) than adenomas(14.6%). In the adenocarcinoma, the expression of the bax was significantly related with the depth of invasion, lymph node metastasis, and TNM stage(P = 0.002, P = 0.001, P = 0.009). The higher intensity of the bax expression was signifi- cantly related with the intestinal type, advanced cancer, lymph node metastasis, and advanced TNM stage(P = 0.02554, P = 0.00409, P = 0.00196, P = 0.0142). 4. The expression rate of the p53 was higher in adenocarcinomas(64%, P=O.O0001) than adenomas(14.6%). The intensity of the p53 expression was higher in the adenocarcinomas with lymph node metasta- sis(P=0.O1485). 5. Ki-67 labelling index was higher in adenocarcinomas than adeno- mas(P = 0.0479) and apoptotic index was higher in adenomas than adenocarcino- mas(P= 0.00026). Conclusion: Bcl-2 protein would be related with the development of gastric adenoma, especially with high grade dysplasia. Bcl-xL and p53 proteins would be involved in the development of relatively early stage of gastric adenocarcinoma but not in tumor progression. Bax protein would be involved in the development of gastric adenocarci- noma and related with depth of invasion, lymph node metastasis, and TNM stage.

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