Spatial T lymphocyte infiltration into the primary melanoma tumor associates with survival

Tumor infiltrating lymphocyte grade is a prognostic indicator for patients with cutaneous invasive primary melanoma and assigned one of three-levels (brisk, nonbrisk, or absent). We sought to develop objective, quantitative measures of T lymphocyte subtypes spatially within the tumor microenvironment. We applied multiplex whole-slide immunohistochemistry/immunofluorescence to 80 melanomas, using S100 to identify tumor cells (S100+) and CD3 and CD8 to identify noncytotoxic T lymphocytes (CD3+CD8-), cytotoxic CD8+ T lymphocytes (CD3+CD8+), and total T lymphocytes (CD3+CD8- plus CD3+CD8+). We used image analysis to spatially locate the T lymphocyte subtypes within the whole tumor, the tumor center (175 µm into the tumor), and the tumor margin (within 100 µm outside the tumor). T lymphocyte subtypes were quantified for density as CD3+CD8- and CD3+CD8+ cells colocalized with intact nuclei/µm 2 . Melanoma-specific survival, adjusted for American Joint Committee on Cancer eighth edition stage, improved when a higher density of each T lymphocyte subtype (CD3+CD8-; CD3+CD8+) infiltrated 175 µm into the tumor center or total T lymphocytes infiltrated into the whole tumor (hazard ratios: 0.72-0.77; all P  < 0.05). Improved melanoma-specific survival is significantly associated with high density of CD3+CD8- T lymphocytes in the margin after stage adjustment. Our results indicate spatial location of T lymphocyte subset infiltration within the tumor microenvironment is important for predicting melanoma-specific survival.