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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.

PDF file - 82K, Response kinetics in treated patients. Baseline tumor measurements are standardized to zero. A. Entire treatment population (cohorts 1-4). B. Cohort 2 patients (MTD). Horizontal line PD = progressive disease representing 20% increase. Horizontal line PR represents 30% decrease from baseline. Filled in circle depicts radiographic development of new sites of disease.

Supplementary Figure S1. Ipi-Bev treatment altered circulating cytokine/chemokine expression. Supplementary Figure S2. Pre-treatment and post-treatment serum levels of IP-10, TNFa, IL-1a, INFa2, GRO, and IL-8 in Ipi-Bev-treated patients.

PDF file - 68K, A. Axial CT section of the chest at baseline demonstrates two nodules in the right lung, the dominant one in the lower lobe (white arrows). B. Follow-up CT 3 months after start of treatment demonstrates resolution of one of the nodules and no change in the other nodule. C. Axial CT section 8 months after start of treatment demonstrates complete resolution of the dominant lung nodule.

PDF file - 74K, High-power H&E and CD31 stained post treatment biopsy of intratumoral vessels. The endothelial cells appear morphologically columnar. CD31 staining is concentrated at the interendothelial junctions (arrows).

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Supplementary Figure S1. Gal-1 antibodies were identified in the posttreatment plasma samples from melanoma patients on Ipi-Bev and pembrolizumab treatment. Supplementary Figure S2. Gal-1 antibody titers in pretreatment and posttreatment plasma samples from melanoma patients receiving ipilimumab and PD-1 blockade. Supplementary Figure S3. Longitudinal analyses of Gal-1 antibody responses to Ipi-Bev in metastatic melanoma patients. Supplementary Figure S4. Purification of recombinant human Gal-1 with His, biotinylated Avi, and SUMO tags at the N terminus (HAS-Gal-1). Supplementary Figure S5. Enrichment of Gal-1 antibodies from patient plasma. Supplementary Figure S6. Binding of Gal-1 to CD45. Supplementary Figure S7. Immunohistochemistry of Gal-1 expression in pretreatment and posttreatment tumor biopsies of patients on Ipi-Bev therapy. Supplementary Figure S8. Kaplan-Meier survival curves of patients based on pretreatment circulating Gal-1 levels. Supplementary Figure S9. Kaplan-Meier survival curves of patients based on circulating Gal-1 fold changes as a result of ipilimumab treatment.

PDF file - 64K, Illustrations of the variation in pathologic responses to treatment. Examples of intermediate and no response.

PDF file - 76K, CONSORT Diagram.

PDF file - 125K, Individual response of CCR7+CD45RO+ and CCD7-CD45RO+ of CD4+ and CD8+ T cells in melanoma patients treated with ipilimumab (3mg/kg), ipilimumab (3mg/kg) plus bevacizumab, or ipilimumab (10mg/kg) plus bevacizumab.

Immune checkpoint therapies targeting CTLA-4 and PD-1 have proven effective in cancer treatment. However, the identification of biomarkers for predicting clinical outcomes and mechanisms to overcome resistance remain as critical needs. Angiogenesis is increasingly appreciated as an immune modulator with potential for combinatorial use with checkpoint blockade. Angiopoietin-2 (ANGPT2) is an immune target in patients and is involved in resistance to anti-VEGF treatment with the monoclonal antibody bevacizumab. We investigated the predictive and prognostic value of circulating ANGPT2 in metastatic melanoma patients receiving immune checkpoint therapy. High pretreatment serum ANGPT2 was associated with reduced overall survival in CTLA-4 and PD-1 blockade–treated patients. These treatments also increased serum ANGPT2 in many patients early after treatment initiation, whereas ipilimumab plus bevacizumab treatment decreased serum concentrations. ANGPT2 increases were associated with reduced response and/or overall survival. Ipilimumab increased, and ipilimumab plus bevacizumab decreased, tumor vascular ANGPT2 expression in a subset of patients, which was associated with increased and decreased tumor infiltration by CD68+ and CD163+ macrophages, respectively. In vitro, bevacizumab blocked VEGF-induced ANGPT2 expression in tumor-associated endothelial cells, whereas ANGPT2 increased PD-L1 expression on M2-polarized macrophages. Treatments elicited long-lasting and functional antibody responses to ANGPT2 in a subset of patients receiving clinical benefit. Our findings suggest that serum ANGPT2 may be considered as a predictive and prognostic biomarker for immune checkpoint therapy and may contribute to treatment resistance via increasing proangiogenic and immunosuppressive activities in the tumor microenvironment. Targeting ANGPT2 provides a rational combinatorial approach to improve the efficacy of immune therapy. Cancer Immunol Res; 5(1); 17–28. ©2016 AACR.

Supplementary Figure S4. IL-1a, TNFa and IL-1b induce adhesion molecule expression and promote T cell adhesion onto human dermal microvascular endothelial cells (HDMEC). Supplementary Figure S5. VEGF inhibits TNFa-mediated ICAM-1expression in TEC and HUVEC. Supplementary Figure S6. IP-10 induces migration of activated T cells across HDMEC. Supplementary Figure S7. Kaplan-Meier survival curves based on fold-changes of circulating IL-1a, TNFa, IFNa2, IL-8, IP-10, and GRO in response to Ipi-Bev treatment. Supplementary Figure S8. Cytokines/chemokines and clinical outcomes in Ipi-Bev treated patients. Supplementary Figure S9. Ipi-Bev therapy influenced circulating endothelial cells (CEC) in melanoma patients.

Culture and treatment of endothelial cells and melanoma cells. Generation and polarization of monocyte derived macrophages (MDM). Purification of ANGPT2 antibodies from plasma. Immunoblot analyses.

The combination of anti-VEGF blockade (bevacizumab) with immune checkpoint anti–CTLA-4 blockade (ipilimumab) in a phase I study showed tumor endothelial activation and immune cell infiltration that were associated with favorable clinical outcomes in patients with metastatic melanoma. To identify potential immune targets responsible for these observations, posttreatment plasma from long-term responding patients were used to screen human protein arrays. We reported that ipilimumab plus bevacizumab therapy elicited humoral immune responses to galectin-1 (Gal-1), which exhibits protumor, proangiogenesis, and immunosuppressive activities in 37.2% of treated patients. Gal-1 antibodies purified from posttreatment plasma suppressed the binding of Gal-1 to CD45, a T-cell surface receptor that transduces apoptotic signals upon binding to extracellular Gal-1. Antibody responses to Gal-1 were found more frequently in the group of patients with therapeutic responses and correlated with improved overall survival. In contrast, another subgroup of treated patients had increased circulating Gal-1 protein instead, and they had reduced overall survival. Our findings suggest that humoral immunity to Gal-1 may contribute to the efficacy of anti-VEGF and anti–CTLA-4 combination therapy. Gal-1 may offer an additional therapeutic target linking anti-angiogenesis and immune checkpoint blockade. Cancer Immunol Res; 5(6); 446–54. ©2017 AACR.

PDF file - 39K, Summary of grade 3 and grade 4 events incidence at each dose level for all events and those events possibly, probably, or definitively related.