Open AccessViral loads correlate with upregulation of PD-L1 and worse patient prognosis in Epstein–Barr Virus-associated gastric carcinoma
Author(s) -
Atsuhito Nakayama,
Hiroyuki Abé,
Akiko Kunita,
Rui Saito,
Teru Kanda,
Hiroharu Yamashita,
Yasuyuki Seto,
Shumpei Ishikawa,
Masashi Fukayama
Publication year - 2019
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0211358
Subject(s) - epstein–barr virus , virus , biology , viral load , cancer , genome , cancer research , virology , carcinoma , in situ hybridization , real time polymerase chain reaction , microbiology and biotechnology , gene , gene expression , genetics
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.