Signoretti, Sabina

SNF'ing out antitumor immunity Immune checkpoint inhibitors induce durable tumor regressions in some, but not all, cancer patients. Understanding the mechanisms that determine tumor sensitivity to these drugs could potentially expand the number of patients who benefit (see the Perspective by Ghorani and Quezada). Pan et al. discovered that tumor cells in which a specific SWI/SNF chromatin remodeling complex had been experimentally inactivated were more sensitive to T cell–mediated killing. The cells were more responsive to interferon-γ, leading to increased secretion of cytokines that promote antitumor immunity. Miao et al. examined the genomic features of tumors from patients with metastatic renal cell carcinoma who had been treated with immune checkpoint inhibitors. Tumors harboring inactivating mutations in PBRM1 , which encodes a subunit of the same SWI/SNF complex, were more likely to respond to the drugs. Science , this issue p. 770 , p. 801 ; see also p. 745

424 Background: Programmed death-1 (PD-1) receptor negatively regulates T cell-mediated responses.PD-1 ligand (PD-L1) is aberrantly expressed in clear cell renal cell carcinoma (ccRCC) and is associated with worse prognosis. Levels of PD-L1 expressions in non-ccRCC and its association with clinicopathological features and survival are unknown. Methods: Formalin-fixed paraffin-embedded (FFPE) specimens were obtained from 97 patients with chromophobe (CHR), papillary (PAP), translocation Xp11.2 (TrL) RCC and oncoytoma (ONC) and were included in the analysis. PD-L1 expression was evaluated by immunohistochemistry using a mouse monoclonal anti-PD-L1 antibody (405.9A11). The assay was validated using FFPE cell line controls known to be positive or negative for PD-L1 expression by flow cytometry. PD-L1 tumor positivity (PD-L1+) was defined as ≥5% tumor cell membrane staining. For PD-L1 expression in immune cells, a combined score based on the intensity of infiltrate and percentage of positive cells was used. Baseline characteristics including stage/grade, and survival data were collected. Comparisons between PD-L1 expression and clinicopathological features were evaluated using chisq or wilcoxon rank sum tests. Cox model tests for association of PD-L1 expression with OS in univariate and multivariable analysis. Results: Among 97 patients, 12 (12.4%) were considered PD-L1+ in tumor cells: 2/36 (5%) of CHR RCC, 5/50 (10%) of PAP RCC, 3/7 (43%) of TrL RCC, and 2/4 (50%) of ONC. PD-L1 positivity in tumor cells was significantly associated with higher stage (p=0.026) and grade (p=0.046), as well as lower OS on univariate (p=0.02) but not multivariate analysis (p=0.29). On the other hand, PD-L1 positivity by immune cells was observed in 50 (51.5%) patients: 13/36 (36%) of CHR RCC, 30/50 (60%) of PAP RCC, 6/7 (86%) of TrL RCC, and 1/4 (25%) of ONC. PD-L1 positivity in immune cells did not correlate with stage (p=0.7), grade (p=0.1) or OS (p=0.8). Conclusions: PD-L1 expression is variable in non-ccRCC and depends on histology and tumor vs. immune cells scoring. Only PD-L1 positivity in tumors cells was associated with aggressive features. Patients with non-ccRCC should not be automatically excluded from trials of agents that target the PD-1/PD-L1 pathway.

422 Background: There is significant variability in the response to VEGF-TT in mRCC with no validated predictive biomarkers. We hypothesized that whole exome analysis might identify markers of response and resistance to VEGF-TT in mRCC. Methods: mRCC patients who received first-line sunitinib or pazopanib and were in two extreme phenotypes of response were identified. Extreme responders (ER) were defined as PR or CR for ≥3 years (n=10) and primary refractory patients (PRP) were defined as PD within the first 3 months of therapy (n=10). WES was performed in pre-treatment specimens and established Broad Institute analytical pipelines were utilized to identify point mutations and copy number alterations across the exome. ER (n=4) and PRP (n=4) who were part of TCGA project for clear-cell RCC were included in this analysis. Nonsense, missense and indel mutations in established or novel RCC genes were investigated. Results: IMDC prognostic scores were not significantly different between the two groups. VHL mutations were observed at similar frequency in ER and PRP, overall 57%. Mutations in PBRM1 were identified in 7 ER (50%) vs. 1 PRP (7%) (p=0.03). In addition, mutations in TP53 were only found in PRP (p=0.09). No other gene had mutations that were associated with either response or primary refractory disease (Table). Conclusions: In this pilot study, PBRM1 mutations were associated with extreme response to VEGF-TT. However, this was exploratory, and multivariable analysis was not performed. Analysis of additional patient samples is ongoing to confirm or refute this association. If true it suggests that epigenetic effects of PBRM1 mutation may contribute to response to VEGF-TT in mRCC. [Table: see text]

616 Background: Polybromo-1 (PBRM1) and BRCA1 associated protein-1 (BAP1) are genes commonly mutated in clear cell RCC (ccRCC) and have been associated with clinical outcome. This work aims to evaluate the impact of PBRM1 and BAP1 expression by IHC in mRCC patients treated with VEGF-TT. Methods: PBRM1 and BAP1 expression was evaluated by IHC in a TMA including 146 mRCC patients. PBRM1 and BAP1 IHC scores were dichotomized as a binary variable: negative (-) or positive (+) (weak positivity was excluded). The associations of PBRM1 or BAP1 expression with baseline clinico-pathological characteristics were evaluated, as well as with overall survival (OS) and time to treatment failure (TTF) usingCox proportional hazards models. Results: Out of 146 patients, 116 and 109 samples had available results for PBRM1 and BAP1 staining, respectively. Overall, 90% (n = 131) patients had ccRCC. 70/116 samples (60%) were PBRM1- and 26/109 patients (24%) were BAP1-. Only 12% (n = 13) of patients had simultaneous negative PBRM1 and BAP1. While there was no association between PBRM1 expression status and clinical factors, BAP1- samples were associated with poor IMDC prognostic risk score (p = 0.004) and higher Fuhrman grade (p = 0.012). PBRM1+ patients showed a trend towards an increased risk of death and a shorter TTF compared to PBRM1- patients (OS: HR = 1.38, 95%CI: 0.92-2.07, p = 0.1; TTF: HR = 1.39, 95%CI: 0.94-2.06, p = 0.1). BAP1 expression was not independently associated with OS or TTF. Conclusions: Loss of PBRM1, but not BAP1 expression showed a trend towards longer TTF and OS in mRCC patients treated with VEGF-TT.

501 Background: Reports have demonstrated an inverse relationship between suppression of immune surveillance mechanisms and activation of the vascular endothelial growth factor receptor (VEGFR) pathway suggesting that T cell repertoires may impact response to VEGFR blockade. We evaluated the association between clinical outcomes and T cell repertoire in metastatic RCC patients receiving a front-line anti-VEGFR, pazopanib. Methods: Pre-treatment RCC tumors were analyzed from VEG105192, a phase III study of mRCC patients randomized (2:1) to pazopanib (paz) 800 mg daily vs placebo (pbo) for TCR gamma (TCRG) and TCR beta (TCRB) CDR3 regions. Using the Adaptive Biotechnologies immunoSEQ Assay, we assessed TCR clonality, a measure of total repertoire represented by expanded clones, and entropy, a measure of evenness and diversity. PD-L1 was evaluated by immunohistochemistry (IHC) H-Scores. The goal of the study was to determine if the repertoire of T cell clones was associated with progression-free survival as a clinical endpoint. Results: In the cohort with available tissue, the median PFS was 10.8 and 5.5 months (mos) for paz and pbo, respectively. TCRB (n = 114) and TCRG (n = 43 pbo, 109 paz) clonality ranged from 0-0.31 and 0-0.98, and entropy from 1-12.1 and 0-10.37, respectively. TCRB and TCRG entropy were highly correlated (Spearman’s R = 0.92, n = 114). Samples from the pbo-treated group with higher TCRG entropy, defined as the top 25th percentile, were associated with an improved median PFS (12.8 months) when compared to the lower 75th percentile (3.1 months, P = 0.023); similar trends were seen for TCRB entropy. Neither entropy nor clonality was associated with maximal reduction in tumor volume in the paz-treated group. PD-L1 H-scores were not associated with entropy or clonality (P > 0.05). Conclusions: Our data suggests that RCC samples with higher entropy are associated with a favorable prognosis. Identification of tumors with restricted TCRB/G chain usage and less diverse repertoire, as represented by lower entropy and higher clonality, may impact responses to VEGFR blockade and requires further study. Clinical trial information: NCT00334282.

Abstract Purpose: We examined the hypothesis that mutations in mTOR pathway genes are associated with response to rapalogs in metastatic renal cell carcinoma (mRCC). Experimental Design: We studied a cohort of mRCC patients who were treated with mTOR inhibitors with distinct clinical outcomes. Tumor DNA from 79 subjects was successfully analyzed for mutations using targeted next-generation sequencing of 560 cancer genes. Responders were defined as those with partial response (PR) by RECIST v1.0 or stable disease with any tumor shrinkage for 6 months or longer. Nonresponders were defined as those with disease progression during the first 3 months of therapy. Fisher exact test assessed the association between mutation status in mTOR pathway genes and treatment response. Results: Mutations in MTOR, TSC1, or TSC2 were more common in responders, 12 (28%) of 43, than nonresponders, 4 (11%) of 36 (P = 0.06). Mutations in TSC1 or TSC2 alone were also more common in responders, 9 (21%), than nonresponders, 2(6%), (P = 0.05). Furthermore, 5 (42%) of 12 subjects with PR had mutations in MTOR, TSC1, or TSC2 compared with 4 (11%) of 36 nonresponders (P = 0.03). Eight additional non-mTOR pathway genes were found to be mutated in at least 4 of 79 tumors (5%); none were associated positively with response. Conclusions: In this cohort of mRCC patients, mutations in MTOR, TSC1, or TSC2 were more common in patients who experienced clinical benefit from rapalogs than in those who progressed. However, a substantial fraction of responders (24 of 43, 56%) had no mTOR pathway mutation identified. Clin Cancer Res; 22(10); 2445–52. ©2016 AACR. See related commentary by Voss and Hsieh, p. 2320

Abstract Purpose: The interaction of programmed death-1 ligand (PD-L1) with its receptor (PD-1) on T cells inactivates antitumor immune responses. PD-L1 expression has been associated with poor outcomes in renal cell carcinoma (RCC) but has not been investigated in advanced RCC patients receiving VEGF-targeted therapy. Experimental Design: Formalin-fixed paraffin-embedded specimens were collected at baseline from patients in the COMPARZ trial. Tumor cell PD-L1 expression by IHC was evaluated using H-score (HS). Dual PD-L1/CD68 staining was used to differentiate PD-L1 tumor expression from tumor-associated macrophages. Intratumor CD8-positive T cells were quantified morphometrically. Associations between biomarkers and survival were investigated using the log-rank test. Results: HS data were available from 453 of 1,110 patients. Sixty-four percent of patients had negative PD-L1 expression (HS = 0). Patients with HS > 55 (n = 59, 13%) had significantly shorter overall survival (OS) than those with HS ≤ 55 in both pazopanib and sunitinib arms (median 15.1 vs. 35.6 and 15.3 vs. 27.8 months, respectively, P = 0.03). In both arms, median OS was shortest in patients with HS > 55 and intratumor CD8-positive T-cell counts > 300 (9.6 and 11.9 months with pazopanib and sunitinib, respectively). Median OS in patients with HS ≤ 55 and CD8-positive T-cell counts ≤ 300 was 36.8 and 28.0 months with pazopanib and sunitinib, respectively. Progression-free survival results were similar to OS results. Conclusions: Increased tumor cell PD-L1, or PD-L1 plus tumor CD8-positive T-cell counts, were associated with shorter survival in patients with metastatic RCC receiving VEGF-targeted agents. These findings may have implications for future design of randomized clinical trials in advanced RCC. Clin Cancer Res; 21(5); 1071–7. ©2014 AACR.

Abstract Purpose: High-dose aldesleukin (HD IL2) received FDA approval for the treatment of metastatic renal cell carcinoma (MRCC) in 1992, producing a 14% objective response rate (ORR) and durable remissions. Retrospective studies suggested that clinical and pathologic features could predict for benefit. The Cytokine Working Group conducted this prospective trial to validate proposed predictive markers of response to HD IL2. Experimental Design: Standard HD IL2 was administered to prospectively evaluate whether the ORR of patients with mRCC with “good” predictive pathologic features based on an “integrated selection” model [ISM (e.g., clear-cell histology subclassification and carbonic anhydrase-9 (CA-9) IHC staining] was significantly higher than the ORR of a historical, unselected population. Archived tumor was collected for pathologic analysis including tumor programmed death-ligand 1 (PD-L1) expression. Results: One hundred and twenty eligible patients were enrolled between June 11 and September 7; 70% were Memorial Sloan Kettering Cancer Center (New York, NY) intermediate risk, 96% had clear cell RCC, and 99% had prior nephrectomy. The independently assessed ORR was 25% (30/120, 95% CI, 17.5%–33.7%, P = 0.0014; 3 complete responses, 27 partial responses) and was higher than a historical ORR. Thirteen patients (11%) remained progression free at 3 years and the median overall survival was 42.8 months. ORR was not statistically different by ISM classification (“good-risk” 23% vs. “poor-risk” 30%; P = 0.39). ORR was positively associated with tumor PD-L1 expression (P = 0.01) by IHC. Conclusions: In this prospective, biomarker validation study, HD IL2 produced durable remissions and prolonged survival in both “good” and “poor-risk” patients. The proposed ISM was unable to improve the selection criteria. Novel markers (e.g., tumor PD L1 expression) appeared useful, but require independent validation. Clin Cancer Res; 21(3); 561–8. ©2014 AACR.