Integrated Bulk and Single-Cell Transcriptomic Analyses Identify a Five-Gene Signature Associated With Prognosis and the Tumor Microenvironment in Epstein-Barr Virus-Associated Gastric Cancer.

BACKGROUND: Epstein-Barr virus-associated gastric cancer (EBVaGC) is a distinct molecular subtype of gastric cancer, but reliable biomarkers linking EBV-related biology, prognosis, and microenvironmental remodeling remain limited. METHODS: Differentially expressed genes associated with EBVaGC were identified from TCGA and GEO datasets. Weighted gene co-expression network analysis and machine learning were used to screen core genes. Functional enrichment, diagnostic and prognostic modeling, immune infiltration analysis, single-cell transcriptomic analysis of a public scRNA-seq dataset, cell-cell communication analysis, drug sensitivity prediction, external cohort validation, and RT-qPCR validation in gastric cancer cell lines were subsequently performed. RESULTS: Five core genes (ASPA, CHODL, GNG7, P2RY14, and PI16) were identified. The combined classifier showed high apparent diagnostic performance in the discovery datasets (AUC = 1.000 in the EBV cohort and 0.981 in TCGA-STAD) and retained value in GSE27342 (AUC = 0.771). RT-qPCR confirmed differential expression of these genes between a gastric epithelial cell line and a gastric cancer cell line, providing general tumor-versus-normal experimental support rather than EBV-specific validation. A five-gene risk signature stratified overall survival, with 1-, 2-, and 3-year AUCs of 0.634, 0.645, and 0.622, which improved to 0.716, 0.768, and 0.693 after integration with age and stage. Single-cell analysis localized the strongest signature signal to fibroblasts and B cells and revealed enhanced MIF-, PTN-, and TNFSF13B-related communication in tumors. High-risk tumors showed higher predicted IC50 values for paclitaxel and 5-fluorouracil. CONCLUSION: We identified a five-gene signature with strong diagnostic value and biologically meaningful prognostic relevance in EBVaGC, highlighting fibroblast- and B-cell-associated microenvironmental programs in aggressive disease.