We obtained tumor tissues from different origins, including the lung, liver, lymph nodes, soft tissue and bones. According to manufacturer’s recommendations, IHC was conducted using 4–5 μm formalin-fixed and paraffin-embedded (FFPE) sections. The PD-L1 clone 28-8 pharmDx kit and the Dako Automated Link 48 platform were used to measure the expression of PD-L1. CD4 (clone B468A, diluted at 1:200, Santa Cruz, Texas, USA) and CD8 (clone 144B, diluted at 1:100, Abcam, Cambridge, UK) expression in T cells, and CD68 (clone PG-M1, diluted at 1:600, Abcam, Cambridge, UK) expression on macrophages were also assessed. Two pathologists independently scored the stained tissues, and the clinical parameters were kept confidential from these two pathologists.
The tumor proportion score (TPS) was used to assess PD-L1 expression. The percentage of tumor cells stained with partial or complete membranes was the definition of TPS. The positive expression of PD-L1 was considered as TPS ≥ 1%. Among all nucleated cells in the tumor mesenchyme, the proportion of positive cells for CD4, CD8, and CD68 expression was assessed. Positivity on lymphocytes was set at 5, 5, and 20% for CD4, CD8, and CD68, respectively. Based on PD-L1 expression and CD8+ T cell infiltration, the tumor microenvironment was divided into three subgroups (both negative, both positive, or single-positive).
The tumor proportion score (TPS) was used to assess PD-L1 expression. The percentage of tumor cells stained with partial or complete membranes was the definition of TPS. The positive expression of PD-L1 was considered as TPS ≥ 1%. Among all nucleated cells in the tumor mesenchyme, the proportion of positive cells for CD4, CD8, and CD68 expression was assessed. Positivity on lymphocytes was set at 5, 5, and 20% for CD4, CD8, and CD68, respectively. Based on PD-L1 expression and CD8+ T cell infiltration, the tumor microenvironment was divided into three subgroups (both negative, both positive, or single-positive).