Dual immunohistochemistry for PD-L1 and PD-1 expression was performed using Cell Signaling Technologies #13684 (clone E1L3N) and Abcam AB52587 (Nat105) in the Tissue Core Facility at Moffitt Cancer Center and evaluated on 37 cases total and 27/36 cases with evaluable responses to immunotherapy (Supplementary Fig. S2 ). PD-L1 expression was quantified using manual morphometric analysis in blinded fashion by a dermatopathologist (KYT). Given the large variability of tissue sample sizes, the following approach was used. PD-L1 expression was largely confined to the tumor periphery and quantitated on the basis of estimating the proportion of tumor cells expressing membranous or membranous and cytoplasmic PD-L1. The tumor periphery was defined here as the 50 micron-thick outer edge of every physically distinct tumor nodule. Within this region, the proportion of tumor cells exhibiting expression was quantitated as <1% (negative) or ≥1% (positive). All evaluated tumors had some expression of PD-L1.
Because of the relative paucity of PD-1 expression overall, the staining was scored solely on the basis of presence (1 (link)) or absence (0) of expression on peritumoral (within 0.02 mm = 20 microns of tumor periphery) lymphocytes (35 (link)).
Because of the relative paucity of PD-1 expression overall, the staining was scored solely on the basis of presence (1 (link)) or absence (0) of expression on peritumoral (within 0.02 mm = 20 microns of tumor periphery) lymphocytes (35 (link)).
