Purpose: EBV-associated gastric carcinoma shows global CpG island methylation of the promoter region of various cancer-related genes. To further clarify the significance of CpG island methylator phenotype (CIMP) status in gastric carcinoma, we investigated methylation profile and clinicopathologic features including overall survival in four subgroups defined by EBV infection and CIMP status: EBV-associated gastric carcinoma and EBV-negative/CIMP-high (H), EBV-intermediate (I), and EBV-negative (N) gastric carcinoma. Experimental Design: Methylation-specific PCR was applied to 106 gastric carcinoma cases. CIMP-N, CIMP-I, and CIMP-H status was determined by the number (0, 1-3, and 4-5, respectively) of methylated marker genes (LOX, HRASLS, FLNc, HAND1, and TM), that were newly identified as highly methylated in gastric cancer cell lines. The methylation status of 10 other cancer-related genes (p14, p15, p16, p73, TIMP-3, E-cadherin, DAPK, GSTP1, hMLH1, and MGMT) was also evaluated. Results: Nearly all (14 of 15) of EBV-associated gastric carcinoma exhibited CIMP-H, constituting a homogenous group (14%). EBV-negative gastric carcinoma consisted of CIMP-H (24%), CIMP-I (38%), and CIMP-N (24%). EBV-associated gastric carcinoma showed significantly higher frequencies of methylation of cancer-related genes (mean number ± SD = 6.9 ± 1.5) even if compared with EBV-negative/CIMP-H gastric carcinoma (3.5 ± 1.8). Among EBV-negative gastric carcinoma subgroups, CIMP-H gastric carcinoma showed comparatively higher frequency of methylation than CIMP-I or CIMP-N, especially of p16 and hMLH1. CIMP-N gastric carcinoma predominantly consisted of advanced carcinoma with significantly higher frequency of lymph node metastasis. The prognosis of the patients of CIMP-N was significantly worse compared with other groups overall by univariate analysis (P = 0.0313). Conclusion: The methylation profile of five representative genes is useful to stratify gastric carcinomas into biologically different subgroups. EBV-associated gastric carcinoma showed global CpG island methylation, comprising a pathogenetically distinct subgroup in CIMP-H gastric carcinoma.

To clarify the significance of p73 in Epstein-Barr virus (EBV)-associated gastric carcinoma (GC), the immunohistochemical expression and CpG-island methylation of p73 were evaluated in cancer tissues and adjacent nonneoplastic tissues of GC with and without EBV infection. Loss of p73 expression by immunohistochemistry was specific to EBV-associated GC (11/13) compared to EBV-negative GC (3/38), which was independent of abnormal p53 expression. With methylation-specific polymerase chain reaction (MSP), the aberrant methylation of p73 exon 1 was similarly specific to EBV-associated GC (12/13), and also rare in EBV-negative GC (2/38). Bisulfite sequencing for p73 exon 1 and its 5 0 region confirmed the MSP results, showing uniform and high-density methylation in EBV-associated GC. Comparative MSP analysis of p14, p16 and p73 methylation, using 20 cases each of formalin-fixed and paraffin-embedded tissues of early GC with and without EBV infection, confirmed 2 types of methylation: global methylation with increased rates (p14 and p16) and specific methylation of p73 in EBV-associated GC. In nonneoplastic mucosa, p14, p16 and p73 methylation occurred in both EBV-associated (8/33, 6/34 and 3/38, respectively) and EBV-negative GC (6/23, 4/35, and 1/35). p73 methylation was observed in the mucosa without H. pylori infection in all 4 samples. Loss of p73 expression through aberrant methylation of the p73 promoter occurs specifically in EBV-associated GC, together with the global methylation of p14 and p16. A specific type of gastritis, prone to a higher grade of atrophy and p73 methylation, may facilitate the development of EBV-associated GC. ' 2006 Wiley-Liss, Inc.Key words: gastric carcinoma; Epstein-Barr virus; CpG island methylation; p73 Epstein-Barr virus (EBV) is a human oncogenic virus that has been identified in a wide variety of malignancies. Representative examples are nasopharyngeal carcinoma (80,043 new cases/year), about half of all Hodgkin's lymphoma (62,329 new cases/year), and posttransplant lymphoproliferative disease (<2% of recipients of solid organ transplants). 1,2 EBV-associated gastric carcinoma (GC) comprises about 10% of all GC throughout the world. 3 Since the worldwide incidence of GC is reportedly 934,000 new cases/year, EBV-associated GC is estimated to occur in nearly 93,400 new cases/year. 1 A causal role of EBV in GC has been suggested on the basis of the clonal nature of EBV in neoplastic cells, the presence of EBV in almost all cancer cells and the absence of EBV in noncancerous mucosa. 3 EBV-associated GC not only has several distinct clinicopathological features, such as male predominance, localization of gastric corpus, and the accompaniment of lymphocytic infiltration, but it exhibits global and nonrandom DNA methylation of the promoter regions of various cancer-associated genes. [4][5][6][7] This promoter methylation correlated well with p16 and E-cadherin expression abnormalities in EBV-associated GC, whereas such correlation was indistinct in EBV-negative GC. 5,7 Regarding methyl...

Neoplastic epithelial cells of EBV-associated GC did not express MUC2 or CD10, and most of them were categorized as null or gastric types. EBV infection may occur in the epithelial cells of null or gastric phenotypes, which may be devoid of transdifferentiation potential toward intestinal phenotypes.