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Purpose: PD-L1 expression on tumor cells (TC) is associated with response to anti-PD-1-based therapies in some tumor types, but its significance in clear cell renal cell carcinoma (ccRCC) is uncertain. We leveraged tumor heterogeneity to identify molecular correlates of TC PD-L1 expression in ccRCC and assessed their role in predicting response to anti-PD-1 monotherapy., Experimental Design: RNA sequencing was performed on paired TC PD-L1 positive and negative areas isolated from eight ccRCC tumors and transcriptomic features associated with PD-L1 status were identified. A cohort of 232 patients with metastatic ccRCC from the randomized CheckMate-025 (CM-025) trial was used to confirm the findings and correlate transcriptomic profiles with clinical outcomes., Results: In both the paired samples and the CM-025 cohort, TC PD-L1 expression was associated with combined overexpression of immune- and cell proliferation-related pathways, upregulation of T-cell activation signatures, and increased tumor-infiltrating immune cells. In the CM-025 cohort, TC PD-L1 expression was not associated with clinical outcomes. A molecular RCC subtype characterized by combined overexpression of immune- and cell proliferation-related pathways (previously defined by unsupervised clustering of transcriptomic data) was enriched in TC PD-L1 positive tumors and displayed longer progression-free survival (HR, 0.32; 95% confidence interval, 0.13-0.83) and higher objective response rate (30% vs. 0%, P = 0.04) on nivolumab compared with everolimus., Conclusions: Both TC-extrinsic (immune-related) and TC-intrinsic (cell proliferation-related) mechanisms are likely intertwined in the regulation of TC PD-L1 expression in ccRCC. The quantitation of these transcriptional programs may better predict benefit from anti-PD-1-based therapy compared with TC PD-L1 expression alone in ccRCC., (©2022 American Association for Cancer Research.)

The tumor immune microenvironment plays a critical role in cancer progression and response to immunotherapy in clear cell renal cell carcinoma (ccRCC), yet the composition and phenotypic states of immune cells in this tumor are incompletely characterized. We performed single-cell RNA and T cell receptor sequencing on 164,722 individual cells from tumor and adjacent non-tumor tissue in patients with ccRCC across disease stages: early, locally advanced, and advanced/metastatic. Terminally exhausted CD8 + T cells were enriched in metastatic disease and were restricted in T cell receptor diversity. Within the myeloid compartment, pro-inflammatory macrophages were decreased, and suppressive M2-like macrophages were increased in advanced disease. Terminally exhausted CD8 + T cells and M2-like macrophages co-occurred in advanced disease and expressed ligands and receptors that support T cell dysfunction and M2-like polarization. This immune dysfunction circuit is associated with a worse prognosis in external cohorts and identifies potentially targetable immune inhibitory pathways in ccRCC., Competing Interests: Declaration of interests D.A.B. reports nonfinancial support from Bristol Myers Squibb, honoraria from LM Education/Exchange Services, and personal fees from Octane Global, Defined Health, Dedham Group, Adept Field Solutions, Slingshot Insights, Blueprint Partnerships, Charles River Associates, Trinity Group, and Insight Strategy, outside of the submitted work. S.H.G. has patents licensed to Novalgen Inc, outside of the submitted work. Z.B. reports nonfinancial support from Bristol Myers Squibb and Genentech/imCore. D.B.K. has acted as an advisor to and has received consulting fees from Neon Therapeutics and owns equity in Agenus, Armata pharmaceuticals, Breakbio, Biomarin Pharmaceutical, Bristol Myers Squibb, Celldex Therapeutics, Chinook Therapeutics, Editas Medicine, Exelixis, Gilead Sciences, IMV, Lexicon Pharmaceuticals, Moderna, and Regeneron Pharmaceuticals. K.M.M. reports research support from Bristol Myers Squibb. B.A.M. reports consulting fees from Bayer, Astellas, Astra Zeneca, Seattle Genetics, Dendreon, Calithera, Exelixis, Nektar, Pfizer, Janssen, Genentech, Eisai, and EMD Serono, and research support to DFCI from Bristol Myers Squibb, Calithera, Exelixis, Seattle Genetics. D.F.M. has received consulting fees from Bristol Myers Squibb, Pfizer, Merck, Alkermes, EMD Serono, Eli Lilly, Iovance, and Eisai, and research support from Bristol Myers Squibb, Merck, Genentech, Pfizer, Exelixis, X4 Pharma, and Alkermes. S.A.S. reports nonfinancial support from Bristol Myers Squibb outside the submitted work. S.A.S. previously advised and has received consulting fees from Neon Therapeutics. S.A.S. reports nonfinancial support from Bristol Myers Squibb, and equity in Agenus Inc., Agios Pharmaceuticals, Breakbio Corp., Bristol Myers Squibb, and Lumos Pharma, outside the submitted work. S.S. reported personal fees from Merck, AstraZeneca, Bristol Myers Squibb, CRISPR Therapeutics AG, AACR, and NCI, grants from Bristol Myers Squibb, AstraZeneca, Novartis, and Exelixis, and royalties from Biogenex. A.H.S. has patents/pending royalties on the PD-1 pathway from Roche and Novartis. A.H.S. is on advisory boards for Bicara, Janssen Immunology, Surface Oncology, Elstar, SQZ Biotechnologies, Elpiscience, Selecta, and Monopteros, and consults for Novartis. A.H.S. has received research funding from AbbVie, Novartis, Roche, UCB, Ipsen, Quark, and Merck. D.F.M. reports personal fees from Bristol Myers Squibb, Pfizer, Merck, Novartis, Exelixis, Array BioPharm, Genentech, Alkermes, Jounce Therapeutics, X4 Pharma, Peloton, EMD Serono, and Eli Lilly, and research support from Bristol Myers Squibb, Prometheus Laboratories, Merck, Genentech, Pfizer, Exelixis, Novartis, X4 Pharma, Alkermes, and Peloton. T.K.C. reports grants and personal fees from Astra Zeneca; personal fees from Bayer; grants and personal fees from Bristol Myers Squibb; personal fees from Cerulean; grants and personal fees from Eisai; personal fees from Foundation Medicine Inc.; grants and personal fees from Exelixis; grants and personal fees from Genentech; personal fees from Roche; grants and personal fees from GlaxoSmithKline; grants and personal fees from Merck, from Novartis, Peloton, and Pfizer; personal fees from Prometheus Labs; grants and personal fees from Corvus; personal fees from Ipsen; grants from Tracon; and grants from Astellas outside the submitted work. The other authors declare no potential conflicts of interest. C.J.W. is an equity holder of BioNTech., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Purpose: We sought to validate levels of CD8 + tumor-infiltrating cells (TIC) expressing PD-1 but not TIM-3 and LAG-3 (IF biomarker; Pignon and colleagues, 2019) and to investigate human endogenous retroviruses (hERV) as predictors of response to anti-PD-1 in a randomized trial of nivolumab (nivo) versus everolimus (evero) in patients with metastatic clear cell renal cell carcinoma (mccRCC; CheckMate-025)., Experimental Design: Tumor tissues (nivo: n = 116, evero: n = 107) were analyzed by multiparametric immunofluorescence (IF) and qRT-PCR. Genomic/transcriptomic analyses were performed in a subset of samples. Clinical endpoints included objective response rate (ORR), progression-free survival (PFS), overall survival (OS), and durable response rate (DRR, defined as complete response or partial response with a PFS ≥ 12 months)., Results: In the nivo (but not evero) arm, patients with high-IF biomarker density (24/116, 20.7%) had higher ORR (45.8% vs. 19.6%, P = 0.01) and DRR (33.3% vs. 14.1%, P = 0.03) and longer median PFS (9.6 vs. 3.7 months, P = 0.03) than patients with low-IF biomarker. By RNA sequencing, several inflammatory pathways ( q < 0.1) and immune-related gene signature scores ( q < 0.05) were enriched in the high-IF biomarker group. When combined with the IF biomarker, tumor cell (TC) PD-L1 expression (≥1%) further separated clinical outcomes in the nivo arm. ERVE-4 expression was associated with increased DRR and longer PFS in nivo-treated patients., Conclusions: High levels of CD8 + TIC expressing PD-1 but not TIM-3 and LAG-3 and ERVE-4 expression predicted response to nivo (but not to evero) in patients with mccRCC. Combination of the IF biomarker with TC PD-L1 improved its predictive value, confirming our previous findings., (©2020 American Association for Cancer Research.)

Sarcomatoid and rhabdoid (S/R) renal cell carcinoma (RCC) are highly aggressive tumors with limited molecular and clinical characterization. Emerging evidence suggests immune checkpoint inhibitors (ICI) are particularly effective for these tumors, although the biological basis for this property is largely unknown. Here, we evaluate multiple clinical trial and real-world cohorts of S/R RCC to characterize their molecular features, clinical outcomes, and immunologic characteristics. We find that S/R RCC tumors harbor distinctive molecular features that may account for their aggressive behavior, including BAP1 mutations, CDKN2A deletions, and increased expression of MYC transcriptional programs. We show that these tumors are highly responsive to ICI and that they exhibit an immune-inflamed phenotype characterized by immune activation, increased cytotoxic immune infiltration, upregulation of antigen presentation machinery genes, and PD-L1 expression. Our findings build on prior work and shed light on the molecular drivers of aggressivity and responsiveness to ICI of S/R RCC.

PD-1 blockade has transformed the management of advanced clear cell renal cell carcinoma (ccRCC), but the drivers and resistors of the PD-1 response remain incompletely elucidated. Here, we analyzed 592 tumors from patients with advanced ccRCC enrolled in prospective clinical trials of treatment with PD-1 blockade by whole-exome and RNA sequencing, integrated with immunofluorescence analysis, to uncover the immunogenomic determinants of the therapeutic response. Although conventional genomic markers (such as tumor mutation burden and neoantigen load) and the degree of CD8 + T cell infiltration were not associated with clinical response, we discovered numerous chromosomal alterations associated with response or resistance to PD-1 blockade. These advanced ccRCC tumors were highly CD8 + T cell infiltrated, with only 27% having a non-infiltrated phenotype. Our analysis revealed that infiltrated tumors are depleted of favorable PBRM1 mutations and enriched for unfavorable chromosomal losses of 9p21.3, as compared with non-infiltrated tumors, demonstrating how the potential interplay of immunophenotypes with somatic alterations impacts therapeutic efficacy.

Despite remarkable success of immune checkpoint inhibitors, the majority of cancer patients have yet to receive durable benefits. Here, in order to investigate the metabolic alterations in response to immune checkpoint blockade, we comprehensively profile serum metabolites in advanced melanoma and renal cell carcinoma patients treated with nivolumab, an antibody against programmed cell death protein 1 (PD1). We identify serum kynurenine/tryptophan ratio increases as an adaptive resistance mechanism associated with worse overall survival. This advocates for patient stratification and metabolic monitoring in immunotherapy clinical trials including those combining PD1 blockade with indoleamine 2,3-dioxygenase/tryptophan 2,3-dioxygenase   (IDO/TDO) inhibitors.

Purpose: Immune-related RECIST (irRECIST) were designed to capture atypical responses seen with immunotherapy. We hypothesized that, in patients with metastatic clear cell renal cell carcinoma (mccRCC), candidate biomarkers for nivolumab response would show improved association with clinical endpoints capturing atypical responders (irRECIST) compared with standard clinical endpoints (RECISTv1.1)., Experimental Design: Endpoints based on RECISTv1.1 [objective response rate (ORR)/progression-free survival (PFS)] or irRECIST [immune-related ORR (irORR)/immune-related PFS (irPFS)] were compared in patients enrolled in the CheckMate-010 trial. Pretreatment tumors were analyzed by PD-L1 and PD-L2 IHC, and by multiplex immunofluorescence for CD8, PD-1, TIM-3, and LAG-3. T-cell activation signatures were assessed by RNA sequencing., Results: Median irPFS was significantly longer than median PFS. irORR was not significantly different from ORR, but immune-related progressive disease (irPD) rate was significantly lower than progressive disease (PD) rate. Tumor cell (TC) PD-L1 expression was not associated with PFS or ORR, but patients with TC PD-L1 ≥1% had longer median irPFS and higher irORR. High percentage of CD8 + tumor-infiltrating cells (TIC) that are PD-1 + TIM-3 - LAG-3 - (% CD8 + PD-1 + TIM-3 - LAG-3 - TIC) correlated with high levels of T-cell activation and was associated with longer median irPFS and higher irORR. Notably, combination of TC PD-L1 expression with % CD8 + PD-1 + TIM-3 - LAG-3 - TIC identified three groups of patients for which irPFS and irORR were significantly different., Conclusions: Atypical responders to nivolumab were identified in the CheckMate-010 trial. We observed improved association of candidate biomarkers for nivolumab response with endpoints defined by irRECIST compared with RECISTv1.1. TC PD-L1 expression in combination with PD-1 expression on CD8 + TIC may predict outcome on nivolumab in mccRCC., (©2019 American Association for Cancer Research.)