Prognostic perspectives of PD-L1 combined with tumor-infiltrating lymphocytes, Epstein-Barr virus, and microsatellite instability in gastric carcinomas

BACKGROUND: The prognostic potential of PD-L1 is currently unclear in gastric carcinomas, although the immune checkpoint PD-1/PD-L1 inhibitors have produced promising results in clinical trials. METHODS: We explored the prognostic implications of programmed death ligand 1 (PD-L1) in 514 consecutive surgically-resected gastric carcinomas. Overall survival and recurrence-free survival were evaluated. Immunohistochemistry for PD-L1, CD8, FOXP3, and PD-1, and molecular grouping by in situ hybridization for Epstein-Barr virus (EBV)-encoded small RNAs and multiplex PCR for microsatellite instability (MSI) markers were performed. Additionally, to explore the function inherent to PD-L1, PD-L1-specific siRNA transfection, cell proliferation, invasion, migration and apoptosis assays were conducted in five gastric carcinoma cell lines. RESULTS: PD-L1(+) tumor and immune cells were observed in 101 (20%) and 244 patients (47%), respectively. “Tumoral PD-L1(+)/immune cell PD-L1(-)/CD8(+/low) tumor-infiltrating lymphocytes (TILs),” and more advanced-stage tumors were associated with unfavorable clinical outcomes in the entire cohort through multivariate analysis. Furthermore, tumoral PD-L1(+)/FOXP3(+/low) TILs were associated with worse clinical outcomes in EBV-positive and MSI-high carcinomas. Tumoral PD-L1(+) alone was an adverse prognostic factor in EBV-positive carcinomas, but not in MSI-high carcinomas, whereas PD-L1(+) immune cells or FOXP3(+/high) TILs alone were correlated with a favorable prognosis. PD-L1 knockdown in gastric carcinoma cells suppressed cell proliferation, invasion and migration, and increased apoptosis, which were all statistically significant in two EBV(+) cell lines, but not all in three EBV(−) cell lines. CONCLUSIONS: The prognostic impact of PD-L1 may depend on the tumor microenvironment, and statuses of EBV and MSI, although PD-L1 innately promotes cancer cell survival in cell-based assays. The combination of “tumoral PD-L1/immune cell PD-L1/CD8(+) TILs” may serve as an independent prognostic factor. Tumoral PD-L1(+)/immune cell PD-L1(−)/CD8(+/low) TILs showing a worse prognosis may be beneficial for combinatorial therapies of anti-PD-L1/PD-1 and anti-cytotoxic T-lymphocyte associated antigen 4 (CTLA4) that would promote effector T cells, thus attack the tumor.