Cs1 4

IHC for PD-L1 was performed on formalin-fixed paraffin-embedded tissue sections using antibodies directed against the N-terminal (E1J2J, Cell Signaling Technology, Beverly, MA, USA) and C-terminal (SP142, Spring Bioscience, Fremont, CA, USA) domains of PD-L1. IHC for Ki-67 was performed using a mouse monoclonal antibody (MIB-1, Dako, Tokyo, Japan). The antigen–antibody complexes were visualized with Histofine Simple Stain MAX PO (Nichirei Bioscience, Tokyo, Japan). Some sections were double-stained with PD-L1 (E1J2J) and CD3, using a mouse monoclonal antibody (F7.2.38, Dako) with PolyView IHC reagent (mouse-AP, Enzo Life Sciences, Farmingdale, NY, USA) (Supplemental Figure S2). A tumor sample was considered positive for PD-L1 expression, when >5% of tumor cells were stained, where PD-L1 expression in tumor cells was discriminated from that in immune cells on the basis of cell morphology and/or cytoplasmic CD3 expression. LMP1 expression was detected using a mouse monoclonal antibody (CS1-4, Dako) with the EnVision system (Dako). EBER-ISH was performed on the Leica Bond-III Automatic Stainer (Leica Microsystems, Wetzlar, Germany) using Bond Ready-to-Use ISH EBER Probe and Bond Ready-to-Use Anti-Fluorescein Antibody (Leica Biosystems, Wetzlar, Germany).

The expression of LMP1 was determined in FFPE NPC sections by immunohistochemical staining. After de-waxing, the sections were subjected to antigen retrieval and staining in the automated slide processing system BenchMark XT (Ventana Medical systems Inc., Tucson, AZ) with the OptiView Amplification kit (Ventana Medical Systems Inc.). The primary antibody used in this study was anti-LMP1 mouse monoclonal antibody (CS.1-4, Dako). The LMP1 expression was assessed by assigning a proportion score and an intensity score36 (link). The proportion score was according to the percentage of tumour cells with positive membrane and cytoplasmic staining (0–100). The intensity score was assigned for the average intensity of positive tumour cells (0, none; 1, weak; 2, intermediate; 3, strong). The LMP1 staining score was the product of proportion and intensity scores, ranging from 0 to 300. The LMP1 expression was categorized into absence/low (score 0–100) and high (score 101–300).

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.