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Purpose:A minority of patients currently respond to single-agent immune-checkpoint blockade (ICB), and strategies to increase response rates are urgently needed. AXL is a receptor tyrosine kinase commonly associated with drug resistance and poor prognosis in many cancer types, including in clear-cell renal cell carcinoma (ccRCC). Recent experimental cues in breast, pancreatic, and lung cancer models have linked AXL with immune suppression and resistance to antitumor immunity. However, its role in intrinsic and acquired resistance to ICB remains largely unexplored.Experimental Design:In this study, tumoral expression of AXL was examined in ccRCC specimens from 316 patients who were metastatic receiving the PD-1 inhibitor nivolumab in the GETUG AFU 26 NIVOREN trial after failure of antiangiogenic therapy. We assessed associations between AXL and patient outcomes following PD-1 blockade, as well as the relationship with various markers, including PD-L1; VEGFA; the immune markers CD3, CD8, CD163, and CD20; and the mutational status of the tumor-suppressor gene von Hippel-Lindau (VHL).Results:Our results show that high AXL-expression level in tumor cells is associated with lower response rates and a trend to shorter progression-free survival following anti–PD-1 treatment. AXL expression was strongly associated with tumor–PD-L1 expression, especially in tumors with VHL inactivation. Moreover, patients with tumors displaying concomitant PD-L1 expression and high AXL expression had the worst overall survival.Conclusions:Our findings propose AXL as candidate factor of resistance to PD-1 blockade, and provide compelling support for screening both AXL and PD-L1 expression in the management of advanced ccRCC.See related commentary by Hahn et al., p. 6619

This file contains 6 supplementary figures and 5 supplementary Tables. Supplementary Figure S1 - Protein expression pattern of AXL in ccRCC specimens. Supplementary Figure S2 - Response rates, Survival and Biomarker analyses according to AXLneg, AXLlow and AXLhigh expression Supplementary Figure S3 - ORR and Progression-Free Survival according to PD-L1 Status alone or by grouping AXL expression plus PD-L1 status Supplementary Figure S4 - High AXL expression plus PD-L1 TC positivity is associated with worse OS in Nivolumab-treated patients in IMDC intermediate-risk/poor-risk group Supplementary Figure S5 - VHL status does not interfere with outcomes or AXL expression upon treatment with Nivolumab Supplementary Figure S6 - Biomarker distribution across AXL groups stratified based on VHL status

Purpose: A minority of patients currently respond to single-agent immune-checkpoint blockade (ICB), and strategies to increase response rates are urgently needed. AXL is a receptor tyrosine kinase commonly associated with drug resistance and poor prognosis in many cancer types, including in clear-cell renal cell carcinoma (ccRCC). Recent experimental cues in breast, pancreatic, and lung cancer models have linked AXL with immune suppression and resistance to antitumor immunity. However, its role in intrinsic and acquired resistance to ICB remains largely unexplored. Experimental Design: In this study, tumoral expression of AXL was examined in ccRCC specimens from 316 patients who were metastatic receiving the PD-1 inhibitor nivolumab in the GETUG AFU 26 NIVOREN trial after failure of antiangiogenic therapy. We assessed associations between AXL and patient outcomes following PD-1 blockade, as well as the relationship with various markers, including PD-L1; VEGFA; the immune markers CD3, CD8, CD163, and CD20; and the mutational status of the tumor-suppressor gene von Hippel-Lindau (VHL). Results: Our results show that high AXL-expression level in tumor cells is associated with lower response rates and a trend to shorter progression-free survival following anti–PD-1 treatment. AXL expression was strongly associated with tumor–PD-L1 expression, especially in tumors with VHL inactivation. Moreover, patients with tumors displaying concomitant PD-L1 expression and high AXL expression had the worst overall survival. Conclusions: Our findings propose AXL as candidate factor of resistance to PD-1 blockade, and provide compelling support for screening both AXL and PD-L1 expression in the management of advanced ccRCC. See related commentary by Hahn et al., p. 6619

BackgroundThe phase II NIVOREN GETUG-AFU 26 study reported safety and efficacy of nivolumab in patients with metastatic clear cell renal cell carcinoma (m-ccRCC) in a ‘real-world setting’. We conducted a translational-research program to determine whether specific circulating immune-cell populations and/or soluble factors at baseline were predictive of clinical outcomes in patients with m-ccRCC treated with nivolumab within the NIVOREN study.MethodsAbsolute numbers of 106 circulating immune-cell populations were prospectively analyzed in patients treated at a single institution within the NIVOREN trial with available fresh-whole-blood, using dry formulation panels for multicolor flow cytometry. In addition, a panel of 14 predefined soluble factors was quantified for each baseline plasma sample using the Meso-Scale-Discovery immunoassay. The remaining patients with available plasma sample were used as a validation cohort for the soluble factor quantification analysis. Tumor immune microenvironment characterization of all patients included in the translational program of the study was available. The association of blood and tissue-based biomarkers, with overall survival (OS), progression-free survival (PFS) and response was analyzed.ResultsAmong the 44 patients, baseline unswitched memory B cells (NSwM B cells) were enriched in responders (p=0.006) and associated with improved OS (HR=0.08, p=0.002) and PFS (HR=0.54, p=0.048). Responders were enriched in circulating T follicular helper (Tfh) (p=0.027) and tertiary lymphoid structures (TLS) (p=0.043). Circulating NSwM B cells positively correlated with Tfh (r=0.70, pConclusionWe report the first fresh blood immune-monitoring of patients with m-ccRCC treated with nivolumab. Baseline blood concentration of NSwM B cells was associated to response, PFS and OS in patients with m-ccRCC treated with nivolumab. BCA-1/CXCL13 and BAFF, inversely correlated to NSwM B cells, were both associated with worse OS in discovery and validation cohorts. Our data confirms a role for B cell subsets in the response to immune checkpoint blockade therapy in patients with m-ccRCC. Further studies are needed to confirm these findings.

618 Background: The NIVOREN GETUG-AFU 26 study reported safety and efficacy of N in mccRCC pts in a “real world setting”. A translationnal research program was launched to characterize immune cell populations in the tumor by immunohistochemistry (IHC) and correlate them with outcome on N. Methods: All pts treated with N in the GETUG AFU 26 NIVOREN trial who consented for translational program and with available archived paraffin- embedded (FFPE) tumor tissue samples were eligible. Tumor were centrally reviewed. Using IHC we quantified main immune populations (B-cells, CD8 T-cells, macrophages), and immune checkpoints receptors (TIM-3, LAG-3, PD-1) at the invasive margin (IM) and at the core of the tumor (CT). We also identified AXL and PBRM1/BAP1 expression. Results: Overall 324 pts were included. Pts had similar baseline characteristics (IMDC Good, Intermediate, Poor in 18%, 60% and 22%, respectively) and comparable outcomes than overall trial population (PFS/OS = 4.5 / 25.4 months). PD-1 (IM) expression was associated with better PFS whereas AXL expression by tumor cells (TC) was associated with worse PFS (table). LAG-3 expression tend to be associated with worse OS. PBRM-1 loss (15%) was associated with better OS and PFS and with a higher density of CD8 T-cells (p = 0.001) and CD163-macrophages (p = 0.01) (CT) and a higher expression of LAG-3 (CT) (p = 0.01) and PD-1 (CT) (p = 0.02). BAP-1 loss was not associated with PFS (p = 0.6) nor OS (p = 0.9) in this cohort. Conclusions: We report the largest translational analysis supporting that PD-1 and AXL expression are associated with PFS in pts with mccRCC receiving N. We further confirm that PBRM-1 loss is a strong prognostic factor in this setting.[Table: see text]