Epstein-Barr virus (EBV) is associated with Burkitt lymphoma, nasopharyngeal carcinoma, opportunistic lymphomas in immunocompromised hosts, and a fraction of gastric cancers. Aberrant promoter methylation accompanies human gastric carcinogenesis, though the contribution of EBV to such somatic methylation changes has not been fully clarified. We analyzed promoter methylation in gastric cancer cases with Illumina's Infinium BeadArray and used hierarchical clustering analysis to classify gastric

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... ncers into 3 subgroups: EBV À /low methylation, EBV À /high methylation, and EBV þ /high methylation. The 3 epigenotypes were characterized by 3 groups of genes: genes methylated specifically in the EBV þ tumors (e.g., CXXC4, TIMP2, and PLXND1), genes methylated both in EBV þ and EBV À /high tumors (e.g., COL9A2, EYA1, and ZNF365), and genes methylated in all of the gastric cancers (e.g., AMPH, SORCS3, and AJAP1). Polycomb repressive complex (PRC) target genes in embryonic stem cells were significantly enriched among EBV À /high-methylation genes and commonly methylated gastric cancer genes (P ¼ 2 Â 10 -15 and 2 Â 10 -34 , respectively), but not among EBV þ tumor-specific methylation genes (P ¼ 0.2), suggesting a different cause for EBV þ -associated de novo methylation. When recombinant EBV was introduced into the EBV À /low-methylation epigenotype gastric cancer cell, MKN7, 3 independently established subclones displayed increases in DNA methylation. The promoters targeted by methylation were mostly shared among the 3 subclones, and the new methylation changes caused gene repression. In summary, DNA methylation profiling classified gastric cancer into 3 epigenotypes, and EBV þ gastric cancers showed distinct methylation patterns likely attributable to EBV infection. Cancer Res; 71(23); 7187-97. Ó2011 AACR. þ -markers were 53 genes methylated in EBV þ -epigenotype only. High-markers were 79 genes methylated in EBV þ -and High-epigenotypes, but not in low-epigenotype or NGM. Common-markers were 72 genes methylated commonly in EBV þ -, high-, and low-epigenotypes. Right, PRC target genes in ES cells (black bar; ref. 38). B, the ratio of PRC target genes. PRC target genes were significantly enriched in high-markers (P ¼ 2 Â 10 -15 ) and common-markers (P ¼ 2 Â 10 -34 ), but not in EBV þ -markers (P ¼ 0.2), compared with the ratio in the whole 13,897 genes. C, validation of 9 markers by pyrosequencing. Top, Infinium result. Bottom, pyrosequencing result. Although Infinium probe determines methylation status of single CpG site, pyrosequencing data not only validated the methylation status of the probe site (red number in each marker), but also analyzed multiple surrounding CpG sites quantitatively (see also Supplementary Fig. S1 ).