EBV-infection in cardiac and non-cardiac gastric adenocarcinomas is associated with promoter methylation of p16, p14 and APC, but not hMLH1.
ABSTRACT Epstein-Barr virus (EBV)-associated gastric carcinomas (GC) constitute a distinct clinicopathological entity of gastric cancer. In order to determine underlying distinct aberrant promoter methylation we tested cardiac and non-cardiac GC with regard to the presence of EBV.
One hundred GC were tested by RNA-in situ hybridization for the presence of EBV by EBV-encoded small RNA (EBER). Aberrant promoter methylation was investigated by methylation-specific real-time PCR for p16, p14, APC and hMLH1. P16 protein expression was assessed by immunohistochemistry.
In our selected study cohort, EBER-transcripts were detected in 19.6% (18/92) of GC. EBV-positive GC revealed significantly more often gene hypermethylation of p16, p14 and APC (p<0.0001, p<0.0001 and p=0.02, respectively) than EBV-negative GC. The majority of GC with p16 hypermethylation showed a p16 protein loss (22/28). In contrast, no correlation between the presence of EBV and hMLH1 hypermethylation was found (p=0.7). EBV-positive GC showed a trend towards non-cardiac location (p=0.06) and lower stages (I/II) according to the WHO (p=0.05).
Hypermethylation of tumor suppressor genes is significantly more frequent in EBV-associated GC compared to EBV-negative GC. Our data add new insights to the role of EBV in gastric carcinogenesis and underline that EBV-associated GC comprise a distinct molecular-pathologic as well as a distinct clinicopathological entity of GC.
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ABSTRACT: EBV has been detected in 2-16% of gastric carcinomas. However, there is little information available about the gene expression profile of EBV-positive gastric carcinomas. EBV infection was examined using EBV-encoded small RNAs (EBERs) in situ hybridization, and 63 (5.6%) of 1127 consecutive gastric carcinomas were found to be EBV-positive. The expressions of 27 tumor-associated proteins were evaluated immunohistochemically in 63 EBV-positive gastric carcinomas and 287 EBV-negative carcinomas using the tissue array method. In addition, the genotype of EBV was investigated by PCR amplification of LMP1 (latent membrane protein 1), Epstein-Barr nuclear antigen 2 (EBNA2), and EBNA3B genes. EBV-positive gastric carcinomas are characterized by the presence of lymphoid stroma, proximal location, and predominance in males. In comparison with EBV-negative carcinomas, EBV-positive carcinomas showed frequent loss of expression of p16, smad4, FHIT, and KAI-1 (kangai 1; P < 0.05), but retained the expression of APC (adenomatous polyposis coli), DCC (deleted in colorectal cancer), and some DNA repair proteins (P < 0.05). There was negative association between EBV infection and the expression of MUC1, MUC2, MUC5AC, p53, CEA, C-erbB2, and smad7. Using hierarchical cluster analysis, we divided EBV-positive gastric carcinomas into two clusters. Those patients with cluster 1 (42 cases) carcinomas had a better prognosis than those with cluster 2 (12 cases; P = 0.0002) or those with EBV-negative carcinomas (280 cases; P = 0.0251). Fifty-one (92.7%) of 55 EBV-positive carcinomas demonstrated the 30-bp deletion in LMP1 gene, and 53 (96.4%) of 55 cases were type 1 for EBNA2 and EBNA3B genes. EBV-positive gastric carcinomas have a distinct protein expression profile as well as distinct clinicopathological features, as compared with EBV-negative carcinomas. The subclassification of EBV-positive carcinomas, by hierarchical cluster analysis, is significantly associated with patient survival.Clinical Cancer Research 03/2004; 10(5):1698-705. · 7.84 Impact Factor
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ABSTRACT: To investigate the interrelationship of Epstein-Barr virus (EBV) and EBV- encoded proteins with Helicobacter pylori (H pylori) infection and the expression of c-met and c-myc oncogene proteins in gastric carcinoma, and to explore their role in gastric carcinogenesis. One hundred and eighty-five gastric carcinoma tissues were detected by polymerase chain reaction (PCR)-Southern blot for EBV genome and in situ hybridization (ISH) for EBV-encoded small RNA 1 (EBER1). Gastric carcinoma with positive EBER1 signals was confirmed EBV-associated gastric carcinoma (EBVaGC). The status of H pylori infection in 185 gastric carcinomas was assessed by rapid urease test and PCR. The samples with positive PCR and urease test were defined as H pylori infection. The expression of c-met and c-myc oncogene proteins in tissues of EBVaGC and matched EBV-negative gastric carcinoma (EBVnGC) were examined by immunohistochemistry. RT-PCR and Southern hybridization were used to detect the expression of nuclear antigens (EBNAs) 1 and 2, latent membrane protein (LMP) 1, early genes BARF1 and BHRF1 in EBVaGC cases. The positive rate of H pylori and EBV in 185 gastric carcinomas was 59.45% (110/185) and 7.03% (13/185) respectively. No difference was found in sex, age, pathological differentiation, clinical stages and lymph node metastasis between H pylori-positive and H pylori-negative gastric carcinomas. However, the positive rate of H pylori infection in the antrum gastric carcinomas was higher than that of cardia and body gastric carcinomas. In our series, age, pathological differentiation, clinical stages, lymph node metastasis and location of cancer were not different between EBVnGC and EBVaGC, while the positive rate of EBV in male patients was significantly higher than that of female patients. The positivity of H pylori in EBV-associated and EBV-negative gastric carcinomas was 46.15% (6/13) and 81.40%(104/172) respectively. There was no significant correlation between EBV and H pylori infection. The c-met overexpression was significantly higher in the EBVaGC group than in the EBVnGC group. However, c-met and c-myc expression did not show significant difference between the two groups. Transcripts of EBNA1 were detected in all 13 EBVaGCs, while both EBNA2 and LMP1 mRNA were not detected. Six of the 13 cases exhibited BARF1 transcripts and 2 exhibited BHRF1 transcripts. The positivity of H pylori in EBVnGCs is higher than that of EBVaGCs, but no significant correlation is found between EBV infection and H pylori infection. H pylori-positive gastric carcinoma is predominant in antrum location, while EBVaGC has a tendency of predominance in cardia/body location. EBV infection is associated with c-met abnormal expression but not with c-myc protein in EBVaGC. c-met overexpression is not induced by LMP1. BARF1 and BHRF1 may play important roles in the tumorigenesis of EBVaGC through different pathways.World Journal of Gastroenterology 04/2006; 12(12):1842-8. · 2.55 Impact Factor
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ABSTRACT: Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC) is a unique type of gastric carcinoma (GC), which is considered to develop in a different pathway from EBV-negative GC. To evaluate a possible role of p16, an inhibitor of G1/S transition of the cell cycle, in the carcinogenesis of EBVaGC, p16-immunohistochemistry and methylation-specific PCR analysis (MSP) were applied to surgically resected gastric carcinomas. When the percentage of p16-positive cells in more than 1000 carcinoma cells was expressed as p16 labeling index (p16-LI), it ranged from 2.5 to 88.1 (mean 42.9+/-24.4) in 70 gastric carcinomas. EBVaGC showed significantly lower values (n=15, 26.1+/ -22.1) than EBV-negative GC (n=55, 47.5+/-23.2) (P=0.0036). Fresh frozen tissues of 55 gastric carcinomas (16 EBVaGC and 39 EBV-negative GC) were further subjected to MSP, to evaluate abnormal methylation of the promoter region of the p16 gene. The frequency of methylation was significantly higher in EBVaGC (14/16) than in EBV-negative GC (9/39) (<0.0001). The methylation-positive carcinomas showed significantly lower p16-LI (35.9+/-21.6) than the unmethylated ones (55.2+/-22.7) (P=0.0014). Thus, a marked decrease of p16 expression, caused by the aberrant methylation of the p16 gene promoter, is closely associated with the development of EBVaGC.Japanese journal of cancer research: Gann 12/2002; 93(11):1195-200.