2. PD-L1 IHC and interpretation

Two PD-L1 IHC diagnostic assays were performed on each specimen according to the manufacturer’s instructions: 22C3 pharmDx (mouse monoclonal primary anti–PD-L1 antibody, prediluted, clone 22C3, Dako, Carpinteria, CA) on the Autostainer Link 48 with EnVision DAB Detection System (Agilent Technologies, Santa Clara, CA), and Ventana SP263 (rabbit monoclonal primary anti–PD-L1 antibody, prediluted, Ventana Medical Systems, Tucson, AZ) on the Benchmark XT staining systems and Ultra with OptiView Universal DAB Detection Kit (Ventana Medical Systems) [8,13].

Interpretation of the 22C3 pharmDx and SP263 assays was performed from stained slides by two of the authors (Y.P. and Y.K.) who received appropriate training. PD-L1 expression in the tumor cell membrane and membrane and/or cytoplasm of tumor-associated mononuclear inflammatory cells such as lymphocytes and macrophages was scored. The CPS was defined as the total number of tumor cells and immune cells (including lymphocytes and macrophages) stained with PD-L1 divided by the number of all viable tumor cells, then multiplied by 100 [9]. Each countable array core section contained at least 100 viable GC cells.