Characterization of PD-L1 Expression and Associated T-cell Infiltrates in Metastatic Melanoma Samples from Variable Anatomic Sites

Abstract

Purpose: Programmed death ligand-1 (PD-L1) tumor expression represents a mechanism of immune escape for melanoma cells. Drugs blocking PD-L1 or its receptor have shown unprecedented activity in melanoma, and our purpose was to characterize tumor PD-L1 expression and associated T-cell infiltration in metastatic melanomas.

Experimental design: We used a tissue microarray (TMA) consisting of two cores from 95 metastatic melanomas characterized for clinical stage, outcome, and anatomic site of disease. We assessed PD-L1 expression and tumor-infiltrating lymphocyte (TIL) content (total T cells and CD4/CD8 subsets) by quantitative immunofluorescence.

Results: High PD-L1 expression was associated with improved survival (P = 0.02) and higher T-cell content (P = 0.0005). Higher T-cell content (total and CD8 cells) was independently associated with improved overall survival; PD-L1 expression was not independently prognostic. High TIL content in extracerebral metastases was associated with increased time to developing brain metastases (P = 0.03). Cerebral and dermal metastases had slightly lower PD-L1 expression than other sites, not statistically significant. Cerebral metastases had less T cells (P = 0.01).

Conclusions: T-cell-infiltrated melanomas, particularly those with high CD8 T-cell content, are more likely to be associated with PD-L1 expression in tumor cells, an improved prognosis, and increased time to development of brain metastases. Studies of T-cell content and subsets should be incorporated into trials of PD-1/PD-L1 inhibitors to determine their predictive value. Furthermore, additional studies of anatomic sites with less PD-L1 expression and T-cell infiltrate are needed to determine if discordant responses to PD-1/PD-L1 inhibitors are seen at those sites.

Figure 1

Quantitative immunofluorescent staining of PD-L1 on tumor cells and quantification of CD3-, CD4- and CD8 TILs. A. Staining is shown in histospots of formalin fixed, paraffin embedded MEL624 cells transfected to overexpress PD-L1 or non-transfected and human placental tissue. DAPI (4,6-diamidine-2-phenylindole) was utilized to identify nuclei and Cy5 to visualize the target. B. Example of a tumor spot showing high PD-L1 expression and high CD3-, CD4- and CD8 lymphocytic content. PD-L1 intensity on tumor cells was calculated on a scale of 1-250. The percentage of either CD3-positive T-cell area or of the CD4- or CD8-positive T-cell subsets within the total tumor area (including stroma) was used to assess the degree of tumor lymphocytic infiltration (TIL density) on a cohort of metastatic melanoma specimens.

Figure 2

Kaplan-Meier curves showing the association between PD-L1 expression or TIL density and overall survival. The median PD-L1 intensity score in our cohort was utilized to dichotomize scores into low/high categories while the median CD3 TIL area was used as a threshold for defining high/low TIL density. High PD-L1 expression and high density of either CD3 positive TILs or the CD8 positive subset were significantly associated with longer overall survival.

Figure 3

Association between PD-L1 expression or TIL density and tumor characteristics. ANOVA was used to compare PD-L1 expression (continuous intensity scores) or TIL density among specimens of different origin. Brain and skin metastases had the lowest PD-L1 expression and the least TIL infiltration and this association was statistically significant for CD3 TILs. Twenty six specimens were from lymph nodes, 11 from skin, 25 from soft tissue, 40 from the brain and 13 from other (extracerebral) visceral sites.

Figure 4

Kaplan-Meier curves showing the association between PD-L1 expression or TIL density and time to development of brain metastases from the time of diagnosis of metastatic melanoma. The median PD-L1 intensity score was utilized to dichotomize scores into low/high while the median CD3 TIL areas was used as thresholds for defining high/low TIL density. High density of CD3 positive TILs was significantly associated with a longer time to development of brain metastasis.