**5. Epigenetic abnormalities of EBV-associated gastric carcinoma: DNA methylation**

EBV-associated gastric cancer has been reported as the most extensive CpG island methylation at both human and viral genomes, being more extensive than any other tumor type from the TCGA network [14, 21, 57, 58]. CpG island methylation is an epigenetic mechanism of gene expression regulation, affecting all cellular pathways [59]. It consists of methyl groups attached to the 5′ position of cytosines, which are followed by a guanine nucleotide (CpG site) [60] (**Figure 6**). More than 50% of human genes contain CpG site in the promoter region and are known as CpG islands [62]. Methylation of CpG sites within a promoter region may inhibit the binding of transcription factors in their consensus sequence of tumor suppressor genes, thus precluding the transcription of the gene, and resulting in gene silencing [63]. Methylation of tumor suppressor genes is usually seen at early stages of gastric cancer and increases during the stomach carcinogenesis [64]. These observations suggest that the silencing of these genes by DNA methylation mechanisms plays an important role in the gastric carcinogenesis. In addition, aberrant methylation is unusually detected in EBVaGC nonneoplastic surrounding mucosa, which might indicate a critical role of EBV in tumorigenesis mechanisms [65]. Although the extensive CpG island methylation is present in EBVaGC, a distinctive pattern of methylation has been suggested, as promoter methylation of tumor suppressor gene CDKN2A, but not promoter methylation of MLH1,

**4. EBV strains and EBV-associated gastric carcinoma**

The results are shown in a log-scale. Taken from [13] with permission.

are shown in a log-scale. Taken from [4] with permission.

14 Gastric Cancer

Previous restriction fragment length polymorphism (RFLP) studies and the recently completed genome sequencing of 31 viruses head to the first global approach of the diversity of EBV. Seven of these sequences were obtained from healthy donors or benign lesions and twenty-five from strains present in tumors including nine EBVaGC [40–44]. By this approach, the genome diversity of EBV can be classified into five types (A–F). A substitution of 1.8 kb in the C-terminal part of the EBNA-2 gene defines types A and B [45]. Subtype A is the most common strain in the West and in Asia, while subtype B is the most common one in Africa [45, 46]. Polymorphisms at

**Figure 5.** An estimated odds ratio (95% CI) for diffuse-type histology of EBV-associated gastric carcinoma in the Americas. Meta-analyses were performed by a random effects model with the inverse variance method, using the DerSimonian-Laird estimator, a logit transformation and the Clopper-Pearson confidence interval for individual studies.

**Figure 4.** An estimated odds ratio (95% CI) for male predominance of EBV-associated gastric cancer in the Americas. Meta-analyses were performed by a random effects model with the inverse variance method, using the DerSimonian-Laird estimator, a logit transformation and the Clopper-Pearson confidence interval for individual studies. The results

**Figure 6.** DNA methylation. (A) CpG methylation mechanism is mediated by DNA methyltransferases and consists in the addition of a methyl group to the carbon in the 5th position of cytosines that precedes guanine nucleotides. (B) CpG islands are DNA sequences rich in CpG sites (>50% CpG sites within a 200bp sequence). Methylation of CpG islands inside a promoter region may control gene expression.

characteristic of the microsatellite instability (MSI) GC subtype [21, 27, 66–71]. Individual methylation of p14ARF and p16INK4a, alternative reading frames of CDKN2A locus, has also been described, and many studies have proved a significant association between their methylation and EBV positivity [72, 73]. Ushiku et al. [74] observed a uniform methylation of all CpG sites on promoter regions of both genes in EBVaGC, whereas it was variable in EBV-negative tumors. In addition, methylation frequency of p16INK4a appears to be about three times higher in EBVaGC than in EBV-negative tumors [65, 75]. CDH1, p15 and p73 tumor suppressor genes are also frequently methylated in EBVaGC, representing one of the most common abnormalities described in this tumor [65, 74].

Despite EBV-induced host gene methylation in EBVaGC is well established, the exact underlying mechanisms are not entirely understood. It has been proposed that when the host cell detects the viral genome, it defends itself by starting a host-driven extensive methylation of the foreign genome, which may trigger the subsequent host genome methylation [75–77]. However, based on the specific methylation patterns observed, a possible participation of EBV in maintenance and de novo methylation has been proposed [72, 74]. Several studies have shown that EBV can modulate the expression of DNA methyltransferases (DNMT), which catalyze the transfer of methyl groups to DNA. Specifically, LMP-2A, EBV latent gene, has been shown to upregulate DNMT1 and DNMT3b expression in gastric cancer cell lines, which further induced methylation of several tumor suppressor genes, such as PTEN [78, 79]. Therefore, LMP-2A may play an essential role in the epigenetic abnormalities in host cells and in the development and maintenance of EBV-associated cancer.
