4CD163+ Tumor-Associated Macrophages and Clinical Outcomes to First-Line Nivolumab Therapy in Patients with Metastatic Clear Cel Renal Cell Carcinoma: Insights from the HCRN GU16-260 Trial

Tumor-associated macrophage (TAM) infiltration has been shown to modulate response to immune checkpoint inhibitors in various cancers, but its role in metastatic clear cell renal cell carcinoma (mccRCC) remains unclear. Here, we investigated the role of CD163+ TAMs as a potential determinant of clinical outcomes to first-line anti-PD-1 therapy (nivolumab) in patients with mccRCC enrolled in the HCRN GU16-260 trial. Moreover, as recent data suggest that the interaction between TAMs and tumor infiltrating lymphocytes (TILs) promotes T cell exhaustion, we explored the spatial relationship between CD163+ TAMs and CD8+ TILs in different states of exhaustion (ie, terminally exhausted (TE) and non-terminally exhausted (NTE) CD8+ TILs). Pre-treatment tumor samples from 67 patients were analyzed by multiplex immunofluorescence to identify CD163+ TAMs, CD8+PD-1+TIM-3+ and/or LAG-3 + (TE CD8+), and CD8+PD-1+TIM-3−LAG-3− (NTE CD8+) TILs. Associations between the natural log of density of CD163+ TAMs with progression-free survival (PFS) and objective response rate (ORR) were assessed using univariable Cox and logistic regression models, respectively. An optimized cutoff was determined using minimum p value for ORR. For each tumor, the density of TE CD8+ TILs and the density of NTE CD8+ TILs were calculated within a 30 µm radius area centered on CD163+ TAMs (proximal area) and outside of this area (non-proximal area), using the ‘sf’ package within R software. The densities of TE and NTE CD8+ TILs were compared in proximal versus non-proximal areas across all tumor samples using the Wilcoxon signed-rank test. For each CD8+ TIL population (TE and NTE), the enrichment in proximity of CD163+ TAMs was assessed by calculating the difference in densities in proximal and non-proximal areas normalized by the density in the overall tumor area. The level of enrichment in TE CD8+ TILs versus NTE CD8+ TILs in proximity of CD163+ TAMs was compared using the Wilcoxon signed-rank test. The density of CD163+ TAMs, analyzed as a continuous variable was positively associated with ORR (OR: 2.21, 95% CI: 1.33 to 3.69, P = .002) and PFS (HR: 0.77, 95% CI: 0.61 to 0.97, P = .028). At an optimized cutoff, patients with high density of CD163+ TAMs (n = 34, 50.7%) had higher ORR (65% vs. 15%, P < .001) and longer median PFS (16.6 months, 95% CI: 5.5-32.9 vs. 5.5 months, 95% CI: 4.1-10.6, P = .009) compared to patients with low density of CD163+ TAMs (n = 33, 49.3%). The density of CD163+ TAMs was moderately correlated with the density of TE CD8+ TILs (Spearman correlation, r = 0.55) and weakly correlated with the density of NTE CD8+ TILs (r = 0.32). Proximity analysis showed that the density of TE CD8+ TILs was significantly higher in the area proximal to the CD163+ TAMs compared to the non-proximal area (median density: 123.3/mm2 vs. 37.2/mm2; P < .001). Similarly, the density of NTE CD8+ TILs was significantly higher in the area proximal to the CD163+ TAMs compared to the non-proximal area (median density: 127.2/mm2 vs. 66.8/mm2; P < .001). The level of enrichment in proximity of CD163+ TAMs was higher for TE CD8+ TILs compared to NTE CD8+ TILs (0.77 vs. 0.58; p = 0.0011). High levels of CD163+ TAMs are associated with improved outcomes to anti-PD-1 therapy in mccRCC. In addition, exhausted CD8+ TILs preferentially localize in proximity of CD163+ TAMs in ccRCC tissues, supporting that TAM-T cell interactions are critical for driving T cell dysfunction. Taken together, our data are consistent with the hypothesis that the efficacy of PD-1 blockade may be in part mediated by reprogramming TAMs from a pro-tumorigenic to an anti-tumorigenic state.