Comprehensive RNA Sequencing Analysis Reveals Heterogeneous Expression of Epstein-Barr Virus Genes in Gastric Cancer

Epstein-Barr virus-associated gastric cancer (EBVaGC) is a distinct histological and molecular subtype of gastric cancer. Histologically, it is characterized by marked lymphocytic infiltration; molecularly, it exhibits a hypermethylated phenotype. The diagnosis of surgical pathology relies on the detection of BEV-encoded RNA in situ hybridization (EBER-ISH) in tumor cells. To diagnose the histological type related to EBV is crucial, as EBVaGC is a potential target for treatment with programed cell death-1/programed cell death ligand-1 inhibitors. This study comprehensively analyzed viral and tumor gene expression in EBVaGC using RNA sequencing (RNA-seq). Six cases of EBVaGC, including one with heterogeneous EBER-ISH expression, were investigated. RNA was extracted from formalin-fixed, paraffin-embedded tissue sections and analyzed using RNA-seq. EBER-ISH-positive and negative areas from tumors with heterogeneous EBER-ISH expression were separately collected using modified manual microdissection techniques. EBV gene expression in EBVaGCs varied among the tumors. Viral gene expression was observed even in EBER-ISH-negative lesions in tumors showing heterogeneous EBER-ISH expression. Gene expression and set enrichment analyses revealed that EBVaGC was highly immunogenic, whereas EBER-ISH-negative lesions were not. However, EBVaGC tumor gene expression patterns, including EBER-ISH-negative lesions, were classified into the same cluster. Viral RNA expression in EBVaGC was heterogenous and even EBER expression fluctuates, suggesting that EBV infection in EBVaGC may not be detected by EBER-ISH alone.