Four-micrometer-thick sequential histologic sections were prepared from representative formalin-fixed, paraffin-embedded tumor blocks. Immunohistochemical (IHC) analysis was performed using an automated immunostainer (Nexes; Ventana, Tucson, AZ, USA). The following primary antibodies were used according to the manufacturer’s instructions and according to a previously described protocol: a mouse monoclonal anti-PD-L1 antibody (1: 50, clone 28–8: ab205921; Abcam, Cambridge, MA, USA) and CD8 (1:50, clone C8/144B, Dako; Agilent Technologies, Inc., Santa Clara, CA, USA).10 (link),16 (link)
The expression levels of each marker protein were examined and evaluated according to a previously reported original protocol. PD-L1 expression was categorized as positive when staining of the tumor-cell membrane (at any intensity) was present. PD-L1 high expression was observed at prespecified expression levels of 5% of all cells in a section that included at least 100 evaluable tumor cells.10 (link) To evaluate the CD8+TIL count, 10 digital high-power field images of the tumor area were selected, and the absolute number of CD8+TILs in these images was determined.16 (link)
The expression levels of each marker protein were examined and evaluated according to a previously reported original protocol. PD-L1 expression was categorized as positive when staining of the tumor-cell membrane (at any intensity) was present. PD-L1 high expression was observed at prespecified expression levels of 5% of all cells in a section that included at least 100 evaluable tumor cells.10 (link) To evaluate the CD8+TIL count, 10 digital high-power field images of the tumor area were selected, and the absolute number of CD8+TILs in these images was determined.16 (link)