Unmasking Epstein-Barr Role in Gastric Carcinogenesis: A Gene Expression Approach to Virus-Positive Tumors

Human gammaherpesvirus 4 or Epstein-Barr virus (EBV) is an oncogenic virus linked to malignancies like gastric adenocarcinoma. Notably, EBV infection induces genetic and epigenetic modifications that play a crucial role in oncogenesis and tumor progression, underscoring the importance of analyzing viral gene expression in the context of gastric cancer (GC) to elucidate its unique characteristics. This study aimed to perform a molecular characterization of EBV gene expression using next-generation sequencing (NGS). The analysis included human gene expression patterns in EBV-positive and EBV-negative samples and the expression of viral genes in EBV-positive samples. The study received approval from the Ethics and Research Committee of João de Barros Barreto University Hospital under reference number 47580121.9.0000.5634. It utilized 76 tumor tissue samples from patients with gastric cancer who had undergone surgical resection, and both fresh and paraffin-embedded samples were gathered for total RNA sequencing (RNA-seq) and in situ hybridization (ISH). The RNA-seq was conducted in a pair-end manner on the NextSeq® platform (Illumina®, US). The NextSeq® 500 MID Output V2 kit - 150 cycles (Illumina®) were utilized following the manufacturer's instructions. ISH targeting RNA-1 of EBER1 (Y5200, DAKO, Carpinteria) was performed using the automated Dako system.  Molecular characterization was conducted using the Kraken2 software. Subsequently, to elucidate the mechanisms through which EBV may influence gastric cancer, we analyzed the patterns of human gene expression in EBV-positive and EBV-negative samples. Of the 76 samples, 8 were classified as EBV-positive according to the applied methodology. Our analysis identified approximately 834 differentially expressed genes, 92 of which exhibited an AUC > 0.85. These genes are implicated in tumor progression, cellular metabolism, and both innate and adaptive immune responses. Additionally, viral genes expressed in the positive samples were evaluated, and we found manifestations of both lytic phase and latent phase genes. Finally, our study presents an efficient strategy for molecular classification of EBV-positive gastric cancer based on NGS and shows the effects of EBV on human gene expression.