Abstract
Epstein–Barr virus (EBV)-associated gastric carcinoma (EBVaGC), one of four major gastric cancer types, consists of clonal growth of EBV-infected epithelial cells. However, the significance of viral loads in each tumor cell has not been evaluated. EBV-DNA is stably maintained in episomal form in the nucleus of each cancer cell. To estimate EBV copy number per genome (EBV-CN), qPCR of viral EBNA1 and host GAPDH, standardized by Namalwa DNA (one copy/genome), was applied to the formalin-fixed paraffin embedded (FFPE) surgically resected EBVaGC specimens (n = 43) and EBVaGC cell lines (SNU-719 and NCC-24). In surgical specimens, the cancer cell ratio (CCR) was determined with image analysis, and EBV-CN was obtained by adjusting qPCR value with CCR. Fluorescent in situ hybridization (FISH) was also applied to the FFPE sections using the whole EBV-genome as a probe. In surgical specimens, EBV-CN obtained by qPCR/CCR was between 1.2 and 185 copies with a median of 9.9. EBV-CN of SNU-719 and NCC-24 was 42.0 and 1.1, respectively. A linear correlation was observed with qPCR/CCR data up to 20 copies/genome (40 signals/nucleus), the limit of FISH analysis. In addition, substantial variation in the number of EBV foci was observed. Based on qPCR/CCR, high EBV-CN (>10 copies) correlated with PD-L1 expression in cancer cells (P = 0.015), but not with other pathological indicators. Furthermore, EBVaGC with high EBV-CN showed worse disease-specific survival (P = 0.041). Our findings suggest that cancer cell viral loads may contribute to expression of the immune checkpoint molecule and promotion of cancer progression in EBVaGC.
Highlights
Epstein–Barr virus (EBV)-associated gastric cancer (EBVaGC), one of the four major types of gastric cancer, consists of clonal growth of EBV-infected epithelial cells
EBV-associated gastric carcinoma (EBVaGC) consists of clonal growth of stomach epithelial cells that are infected with EBV
Many studies have investigated the role of viral latent gene products in EBVaGC, but few studies have focused on the significance of viral load in cancer cells
Summary
Epstein–Barr virus (EBV)-associated gastric cancer (EBVaGC), one of the four major types of gastric cancer, consists of clonal growth of EBV-infected epithelial cells. When in situ hybridization (ISH) targeting EBV-encoded small RNA (EBER) is applied to the tissue sections of EBVaGC, all of the cancer cells show positive signals in the nuclei with extremely rare. Viral copy number in EBV-associated gastric cancer. EBVaGC comprises 5%–10% of gastric cancer cases and has several characteristics distinct from EBV-negative gastric cancers, such as massive lymphocytic infiltration, frequent genetic mutations in PIK3CA and ARID1A, and global hypermethylation of CpG islands [1, 2]. The viral copy number in EBV-related tumor tissue has not yet been investigated. The precise EBV copy number in each cancer cell has not yet been examined, because EBVaGC consists of both EBV-infected cancer cells and EBV-negative stromal cells, including many inflammatory cells
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