Multiplex quantitative analysis of cancer-associated fibroblasts and immunotherapy updates outcome in metastatic melanoma

Background: The cancer-associated fibroblast (CAF) population is implicated in immune dysregulation. Here, we test the hypothesis that CAF profiles in pretreatment tumor specimens are associated with response to immune checkpoint blockade of programmed cell death 1 (PD-1). Methods: Pretreatment whole tissue sections from 117 melanoma patients treated with anti-PD-1 therapy were assessed by multiplex immunofluorescence to detect CAFs defined by Thy1, smooth muscle actin (SMA), and fibroblast activation protein (FAP). Two independent image analysis technologies were used: inForm software (PerkinElmer) to quantify cell counts, and AQUA (TM) to measure protein by quantitative immunofluorescence (QIF). CAF parameters by both methodologies were assessed for association with previously measured immune markers (CD3, CD4, CD8, CD20, CD68, PD-L1), best overall response, progression-free survival (PFS), and overall survival (OS). Results: CAF parameters, by cell counts or QIF, did not correlate with immune markers nor with best overall response. However, both Thy1 and FAP cell counts had significant positive associations with PFS (all P < 0.05) and OS (all P < 0.003). SMA cell counts showed negative associations with outcome in anti-PD-1 treated patients. Similar associations were not observed in a control cohort of historical melanoma patients predating immunotherapy. Instead, FAP was a negative prognostic biomarker (P = 0.01) in the absence of immunotherapy. Multivariable analyses revealed significant PFS and OS associations with the CAF parameters were independent of baseline variables. Conclusions: Pretreatment CAF profiles are associated with melanoma immunotherapy outcome. Multiplex CAF analysis has potential as an objective companion diagnostic in immuno-oncology.