Dissecting the Tumor Microenvironment to Identify Biomarkers of Outcome to Anti–PD-1 Therapy in Clear-Cell Renal Cell Carcinoma: Analyses of the HCRN GU16-260 Trial

PURPOSE: We investigated components of the immune tumor microenvironment as determinants of clinical outcome to anti-PD-1 therapy in patients with metastatic clear-cell renal cell carcinoma (ccRCC) treated with first-line nivolumab in the phase II, nonrandomized HCRN GU16-260 trial. EXPERIMENTAL DESIGN: Pretreatment primary ccRCCs from 72 patients were analyzed via multiplex immunofluorescence and image analysis to assess nonterminally exhausted CD8+ (CD8+PD-1+TIM-3-LAG-3-) tumor-infiltrating lymphocytes (TIL), PD-1+ regulatory T cells (Treg), total and peritumoral tertiary lymphoid structures (TLS), and CD163+ tumor-associated macrophages (TAM). Clinical endpoints included objective response rate (ORR) and progression-free survival (PFS). RESULTS: The densities of CD8+PD-1+TIM-3-LAG-3- TILs, total and peritumoral TLSs, and CD163+ TAMs, as continuous variables, were associated with improved ORR [odds ratio: 1.54 (1.10-2.16) for TILs; 1.18 (1.02-1.37) for total TLSs; 1.10 (1.02-1.18) for peritumoral TLSs; and 2.21 (1.33-3.69) for TAMs] and longer PFS [hazard ratio: 0.79 (0.67-0.95) for TILs; 0.92 (0.85-0.99) for total TLSs; 0.94 (0.90-0.98) for peritumoral TLSs; and 0.77 (0.61-0.97) for TAMs]. Although the percentage of PD-1+ Tregs as a continuous variable was not associated with outcomes, at an optimal cutoff, a high percentage of PD-1+ Tregs tended to be associated with lower ORR (12.5% vs. 43.6%, P = 0.093) and was associated with shorter PFS (3.4 vs. 10.9 months, P < 0.001). The biomarkers were not strongly correlated with each other, and their integration in multibiomarker models further stratified outcomes. CONCLUSIONS: Individual immune cell populations within the ccRCC microenvironment are associated with response/resistance to first-line anti-PD-1 therapy. Our findings support the development of combined immune marker models to identify patients with divergent outcomes.