Evaluation of potential immunotherapy biomarkers in female genital tract malignant melanomas

Abstract

Background: Female genital tract malignant melanomas may response to immune checkpoint inhibitors. However, immunohistochemical assessment of the biomarker PD-L1 is difficult due to interobserver variation and intra-tumoral and temporal heterogeneity. Other predictive biomarkers are needed. The FDA has recently approved the use of pembrolizumab for mismatch repair (MMR) deficient solid tumors but the MMR status in genital melanomas has not been explored Design: Immunohistochemical evaluation of CD8+ tumor infiltrating lymphocytes (TIL) density and MMR proteins status was done on 55 genital tract melanomas. Anti-CD8 antibody (clone C8/144B. Dako) was used. Assessment of TIL density was made in an area with highest lymphoid cells density identified on low-magnification. An absolute count of CD8+ cells per high power field (x40 objective, 0.55 mm field diameter) was made. Survival analysis was performed by using a Cox proportional hazard model. Using receiver operating characteristic analysis, the optimal cut-off value for TIL count was determined by choosing the maximum value of Youden’s index. The relationship between TIL count and PD-L1 expression (previously performed on the same patients by using Roche Ventana SP263 antibody) was done with Pearson’s Product-moment correlation analysis. MMR protein (Roche Ventana) antibodies used were anti-MLH1 (clone M1), anti-MSH2 (clone G219-1129), anti-MSH6 (clone SP93) and anti-PMS2 (clone A16-4). A tumor was considered to have abnormal MMR expression when nuclear staining was absent. Results: There were 20 vulvar, 32 vaginal and 3 cervical melanomas. The median counts of CD8+ TILs in vulvar, vaginal and cervical melanomas were 158, 65.5 and 150 per high power field, respectively. CD8+ TILs density was significantly higher among vulvar melanomas compared with non-vulvar melanomas (p<0.05) and showed a positive correlation with the PD-L1 score with a coefficient of 0.355 (p = 0.01). Univariable analysis showed that a CD8+ TIL count <93 per high power field was significantly associated with an adverse outcome. Loss of MLH1 and PMS2 staining was observed in 4 vulvar melanomas (21.1%) and 3 vaginal melanomas (9.38%). All 3 cervical melanomas had intact staining for all 4 proteins. Conclusion: CD8+ TIL count showed a positive correlation with PD-L1 immunohistochemistry and interpretation of staining was easier. The combined evaluation of CD8+ TIL and MMR protein status in genital melanomas may be useful predictive biomarkers for immunotherapy.