Multiplex Quantitative Analysis of Tumor-Infiltrating Lymphocytes and Immunotherapy Outcome in Metastatic Melanoma

Purpose: Because durable response to programmed cell death 1 (PD-1) inhibition is limited to a subset of melanoma patients, new predictive biomarkers could have clinical utility. We hypothesize that pretreatment tumorinfiltrating lymphocyte (TIL) profiles could be associated with response. Experimental Design: Pretreatment whole tissue sections from 94 melanoma patients treated with anti-PD-1 therapy were profiled by multiplex immunofluorescence to perform TIL quantification (CD4, CD8, CD20) and assess TIL activation (CD3, GZMB, Ki67). Two independent image analysis technologies were used: inForm (PerkinElmer) to determine cell counts, and AQUA to measure protein by quantitative immunofluorescence (QIF). TIL parameters by both methodologies were correlated with objective response or disease control rate (ORR/DCR) by RECIST 1.1 and survival outcome. Results: Pretreatment lymphocytic infiltration, by cell counts or QIF, was significantly higher in complete or partial response than in stable or progressive disease, particularly for CD8 (P < 0.0001). Neither TIL activation nor dormancy was associated with outcome. CD8 associations with progressionfree survival (HR > 3) were independently significant in multivariable analyses and accounted for similar CD3 associations in anti-PD-1-treated patients. CD8 was not associated with melanoma prognosis in the absence of immunotherapy. Predictive performance of CD8 cell count (and QIF) had an area under the ROC curve above 0.75 (ORR/DCR), which reached 0.83 for ipilimumab plus nivolumab. Conclusions: Pretreatment lymphocytic infiltration is associated with anti-PD-1 response in metastatic melanoma. Quantitative TIL analysis has potential for application in digital precision immuno-oncology as an indicative companion diagnostic.