Helicobacter pylori and EBV in gastric carcinomas: methylation status and microsatellite instability

Abstract

Aim: To verify the methylation status of CDH1, DAPK, COX2, hMLH1 and CDKN2A genes and to evaluate their association with Helicobacter pylori (H. pylori)-cagA(+) and Epstein Barr virus (EBV) infections in gastric adenocarcinomas.

Methods: Methylation-specific PCR (MSP) assay was performed in 89 primary gastric carcinomas (intestinal and diffuse types). Microsatellite instability (MSI) analysis was performed using the BAT26 primer set and PCR products were analyzed with the ABI PRISM 3100 Genetic Analyzer using Genescan 3.7 software (Applied Biosystems). Detection of H. pylori and genotyping were performed by PCR, using specific primers for ureaseC and cagA genes. The presence of EBV was assessed by in situ hybridization. Statistical analyses were performed using the chi(2) or Fisher's exact test.

Results: The most frequent hypermethylated gene was COX-2 (63.5%) followed by DAPK (55.7%), CDH1 (51%), CDKN2A (36%) and hMLH1 (30.3%). Intestinal and diffuse adenocarcinomas showed different methylation profiles and there was an association between methylation of E-CDH1 and H. pylori-cagA(+) in the intestinal adenocarcinoma type. MSI was correlated with hMLH1 methylation. There was an inverse correlation between DAPK hypermethylation and MSI.

Conclusion: We found a strong association between CDH1 methylation and H. pylori-cagA(+) in intestinal-type gastric cancer, association of MSI and better prognosis and an heterogeneous COX-2 overexpression.

Figure 1

Example of results from methylation-specific PCR analysis performed in tumoral samples from patients with gastric cancer. The studied gene is given on the right of the each panel. Lane U: Amplified product with primers recognizing unmethylated sequences; Lane M: Amplified product with primers recognizing methylated sequences; L: Ladder 50 bp.

Figure 2

Frequencies of methylation at CDH1, DAPK, hMLH1, COX2 and CDKN2A genes in gastric carcinomas. A: Frequencies of methylation at CDH1, DAPK, hMLH1, COX2 and CDKN2A distributed according to tumor stages in diffuse adenocarcinoma; B: Frequencies of methylation at CDH1, DAPK, hMLH1, COX2 and CDKN2A distributed according to tumor stages in intestinal adenocarcinoma; C: Frequencies of methylation at CDH1, DAPK, hMLH1, COX2 and CDKN2A distributed with regard to age groups.