Epstein-Barr Virus-associated gastric cancer: a histopathologic study with comprehensive molecular profiling

: A subset of gastric cancers (GCs) is linked to Epstein-Barr virus (EBV) infection. This study aims to characterize the histopathological and molecular features of EBV-associated GCs (EBVaGCs), focusing on predictive biomarkers and genomic and transcriptomic analysis. A total of 35 primary EBVaGCs were considered. The presence of EBV was confirmed with in-situ hybridization. Immunohistochemical analyses for HER2, PD-L1, Claudin 18.2 (CLDN18), and mismatch repair (MMR) proteins were performed. Genomic and transcriptomic profiles were assessed using AmoyDX Master panel which can identify, at DNA level, SNV, InDels, and CNV on 571 hot genes, microsatellite status, TMB, and HRD, while at RNA level, in addition to rearrangements/fusions in 45 genes, also quantifies the expression of 2396 cancer-related transcripts. The following histotypes were identified: carcinoma with lymphoid stroma (CLS; 69%), tubular (20%), and mixed (11%). Most cases were associated with atrophic gastritis (71%) and only 11% with dysplasia. The vast majority (94%) of EBVaGCs expressed EBER in all tumor cells. MMR deficiency and HER2 overexpression were each observed in 6% of cases, while all tumors had a PD-L1 Combined Positive Score ≥10. Sixty-six percent of cases showed moderate/strong CLDN18 expression in ≥75% of cancer cells. The most frequently altered genes were PIK3CA (41%) and ARID1A (17%). Transcriptomic analysis revealed substantial differential gene expression between EBVaGCs and EBV-negative controls, with upregulation of genes involved in antigen presentation, natural killer cell-mediated cytotoxicity, and cytokine-cytokine receptor interaction in EBVaGCs. Within EBVaGC, CLS showed higher expression of immune-related transcripts and higher PD-L1 expression than other histotypes. This study establishes EBVaGC as a distinct molecular class, with a distinctive profile of genomic alterations and expression of predictive biomarkers and also with a unique immune microenvironment with enhanced cytotoxic activity. The findings highlight EBV's role in early tumor development and EBVaG-CLS as a distinct subgroup within EBVaGC, characterized by unique morphologic features and a pronounced immune activation profile.