PD-L1 and EBV Status Assessment in Gastric Cancer

IHC was performed on 4 -µm-thick tissue sections using an automated IHC Stainer (Ventana, Tucson, AZ, USA). The assessment of PD-L1 protein expression in GC is a qualitative IHC assay that uses anti-PD-L1 antibodies (Dako, 22C3) to detect PD-L1 protein in formalin-fixed, paraffin-embedded tissues from gastric adenocarcinomas. A minimum of 100 tumor cells must be present in the PD-L1 stained slide for the specimen to be considered adequate for PD-L1 evaluation. Expression of PD-L1 was reported as CPS, defined as the total number of PD-L1 positive cells (lymphocytes, macrophages, and tumor cells) divided by the total number of viable tumor cells^{10 (link)}. The CPS ≥ 1 and ≥ 5 were chosen to define PD-L1 positive. A monoclonal antibody against Latent Membrane Protein (LMP)-1 (CS1-4; Dako, Glostrup, Denmark) was used to detect EBV-specific protein to identify EBV status for GC with CPS ≥ 1. IHC for LMP-1 was done according to the method previously described^{11 (link)}. Brown granular cytoplasmic and membrane staining was interpreted as positive for EBV LMP-1, whereas bluish staining of the cytoplasm and membrane was interpreted as negative for EBV LMP-1. A positive control included a tissue known to have EBV infection, whereas, for negative controls, the test antibody was omitted and replaced by phosphate-buffered saline.