Transcriptomic signatures related to the obesity paradox in patients with clear cell renal cell carcinoma: a cohort study.

BACKGROUND: Obesity is associated with an increased risk of developing clear cell renal cell carcinoma (RCC) but, paradoxically, obesity is also associated with improved oncological outcomes in this cancer. Because the biological mechanisms underlying this paradoxical association are poorly understood, we aimed to identify transcriptomic differences in primary tumour and peritumoral adipose tissue between obese patients and those at a normal weight. METHODS: In this cohort study, we assessed data from five independent clinical cohorts of patients with clear cell RCC aged 18 years and older. Overweight patients were excluded from each cohort for our analysis. We assessed patients from the COMPARZ phase 3 clinical trial, a cohort from the Cancer Genome Atlas (TCGA), and a Memorial Sloan Kettering (MSK) observational immunotherapy cohort for their inclusion into our study. We assessed overall survival in obese patients (those with a body-mass index [BMI] ≥30 kg/m2) and in patients with a normal weight (BMI 18·5-24·9 kg/m2, as per WHO's BMI categories), defined as the time from treatment initiation (in the COMPARZ and MSK immunotherapy cohorts) or surgery (in the TCGA cohort) to the date of any-cause death or of censoring on the day of the last follow-up. We also evaluated and validated transcriptomic differences in the primary tumours of obese patients compared with those of a normal weight. We compared gene-expression differences in peritumoral adipose tissue and tumour tissue in an additional, prospectively collected cohort of patients with non-metastatic clear cell RCC (the MSK peritumoral adipose tissue cohort). We analysed differences in gene expression between obese patients and those at a normal weight in the COMPARZ, TCGA, and peritumoral adipose tissue cohorts. We also assessed the tumour immune microenvironment in a prospective cohort of patients who had nephrectomy for localised RCC at MSK. FINDINGS: Of the 453 patients in the COMPARZ trial, 375 (83%) patients had available microarray data, pretreatment BMI measurements, and overall survival data for analyses, and we excluded 119 (26%) overweight patients, leaving a final cohort of 256 (68%) patients from this study for our analyses. From 332 patients in the TCGA cohort, we evaluated clinical and demographic data from 152 (46%) patients with advanced (ie, stages III and IV) clear cell RCC treated by nephrectomy; after exclusion of 59 (39%) overweight patients, our final cohort consisted of 93 (61%) patients. After exclusion of 74 (36%) overweight patients from the initial MSK immunotherapy study population of 203 participants, our final cohort for overall survival analysis comprised 129 (64%) participants. We found that overall survival was longer in obese patients than in those with normal weight in the TCGA cohort, after adjustment for stage or grade (adjusted HR 0·41, 95% CI 0·22-0·75), and in the COMPARZ clinical trial after adjustment for International Metastatic RCC Database (IMDC) risk score (0·68, 0·48-0·96). In the MSK immunotherapy cohort, the inverse association of BMI with mortality (HR 0·54, 95% CI 0·31-0·95) was not significant after adjustment for IMDC risk score (adjusted HR 0·72, 95% CI 0·40-1·30). Tumours of obese patients showed higher angiogenic scores on gene-set enrichment analysis-derived hallmark gene set angiogenesis signatures than did those of patients at a normal weight, but the degree of immune cell infiltration did not differ by BMI. We found increased peritumoral adipose tissue inflammation in obese patients relative to those at a normal weight, especially in peritumoral fat near the tumour. INTERPRETATION: We found aspects of the tumour microenvironment that vary by BMI in the tumour and peritumoral adipose tissue, which might contribute to the apparent survival advantage in obese patients with clear cell RCC compared with patients at a normal weight. The complex interplay between the clear cell RCC tumour and peritumoral adipose tissue microenvironment might have clinical relevance and warrants further investigation. FUNDING: Ruth L Kirschstein Research Service Award, American Society of Clinical Oncology Young Investigator Award, MSK's Ludwig Center, Weiss Family Kidney Research Fund, Novartis, The Sidney Kimmel Center for Prostate and Urologic Cancers, and the National Institutes of Health (National Cancer Institute) Cancer Center Support Grant.

Genomically annotated risk model for advanced renal-cell carcinoma: a retrospective cohort study.

BACKGROUND: The Memorial Sloan Kettering Cancer Center (MSKCC) risk model is an established prognostic tool for metastatic renal-cell carcinoma that integrates clinical and laboratory data, but is agnostic to tumour genomics. Several mutations, including BAP1 and PBRM1, have prognostic value in renal-cell carcinoma. Using two independent clinical trial datasets of patients with metastatic renal-cell carcinoma, we aimed to study whether the addition of the mutation status for several candidate prognostic genes to the MSKCC model could improve the model's prognostic performance. METHODS: In this retrospective cohort study, we used available formalin-fixed paraffin-embedded tumour tissue and clinical outcome data from patients with metastatic renal-cell carcinoma assigned to treatment with tyrosine kinase inhibitors in the COMPARZ trial (training cohort; n=357) and RECORD-3 trial (validation cohort; n=258). Eligible patients in both trials were treatment-naive; had histologically confirmed, advanced, or metastatic renal-cell carcinoma; and a Karnofsky performance status score of at least 70. For each cohort, data from patients in all treatment groups (sunitinib and pazopanib in the training cohort, and everolimus and sunitinib in the validation cohort) were pooled for this analysis. In the training cohort, tumour tissue was used to evaluate somatic mutations by next-generation sequencing, and the association between cancer-specific outcomes (overall survival, progression-free survival, and overall response) and the mutation status of six genes of interest (BAP1, PBRM1, TP53, TERT, KDM5C, and SETD2) was tested. Only those genes with prognostic value in this setting were added to the MSKCC risk model to create a genomically annotated version. The validation cohort was used to independently test the prognostic value of the annotated model compared with the original MSKCC risk model. FINDINGS: 357 (32%) of 1110 patients assigned to protocol treatment in the COMPARZ study between August, 2008, and September, 2011, were evaluable for mutation status and clinical outcomes in the training cohort. The independent validation cohort included 258 (55%) of 471 evaluable patients, enrolled between October, 2009, and June, 2011, on the RECORD-3 study. In the training cohort, the presence of any mutation in BAP1 or TP53, or both, and absence of any mutation in PBRM1 were prognostic in terms of overall survival (TP53wt/BAP1mut, TP53mut/BAP1wt o TP53mut/BAP1mut vs TP53wt/BAP1wt hazard ratio [HR] 1·57, 95% CI 1·21-2·04; p=0·0008; PBRM1wt vs PBRMmut, HR 1·58, 1·16-2·14; p=0·0035). The mutation status for these three prognostic genes were added to the original MSKCC risk model to create a genomically annotated version. Distribution of participants in the training cohort into the three risk groups of the original MSKCC model changed from 87 (24%) of 357 patients deemed at favourable risk, 217 (61%) at intermediate risk, and 53 (15%) at poor risk, to distribution across four risk groups in the genomically annotated risk model, with 36 (10%) of 357 deemed at favourable risk, 77 (22%) at good risk, 108 (30%) at intermediate risk, and 136 (38%) at poor risk. Addition of genomic information improved model performance for predicting overall survival (C-index: original model, 0·595 [95% CI 0·557-0·634] vs new model, 0·637 [0·595-0·679]) and progression-free survival (0·567 [95% CI 0·529-0·604] vs 0·602 [0·560-0·643]) with adequate discrimination of the proportion of patients who achieved an objective response (Cochran-Armitage one-sided p=0·0014). Analyses in the validation cohort confirmed the superiority of the genomically annotated risk model over the original version. INTERPRETATION: The mutation status of BAP1, PBRM1, and TP53 has independent prognostic value in patients with advanced or metastatic renal-cell carcinoma treated with first-line tyrosine kinase inhibitors. Improved stratification of patients across risk groups by use of a genomically annotated model including the mutational status of these three genes warrants further investigation in prospective trials and could be of use as a model to stratify patients with metastatic renal-cell carcinoma in clinical trials. FUNDING: Novartis Pharmaceuticals Corporation, MSKCC Support Grant/Core Grant, and the J Randall & Kathleen L MacDonald Research Fund.

A Targetable Myeloid Inflammatory State Governs Disease Recurrence in Clear-Cell Renal Cell Carcinoma.

It is poorly understood how the tumor immune microenvironment influences disease recurrence in localized clear-cell renal cell carcinoma (ccRCC). Here we performed whole-transcriptomic profiling of 236 tumors from patients assigned to the placebo-only arm of a randomized, adjuvant clinical trial for high-risk localized ccRCC. Unbiased pathway analysis identified myeloid-derived IL6 as a key mediator. Furthermore, a novel myeloid gene signature strongly correlated with disease recurrence and overall survival on uni- and multivariate analyses and is linked to TP53 inactivation across multiple data sets. Strikingly, effector T-cell gene signatures, infiltration patterns, and exhaustion markers were not associated with disease recurrence. Targeting immunosuppressive myeloid inflammation with an adenosine A2A receptor antagonist in a novel, immunocompetent, Tp53-inactivated mouse model significantly reduced metastatic development. Our findings suggest that myeloid inflammation promotes disease recurrence in ccRCC and is targetable as well as provide a potential biomarker-based framework for the design of future immuno-oncology trials in ccRCC. SIGNIFICANCE: Improved understanding of factors that influence metastatic development in localized ccRCC is greatly needed to aid accurate prediction of disease recurrence, clinical decision-making, and future adjuvant clinical trial design. Our analysis implicates intratumoral myeloid inflammation as a key driver of metastasis in patients and a novel immunocompetent mouse model. This article is highlighted in the In This Issue feature, p. 2221.

A Phase I/II Study to Assess the Safety and Efficacy of Pazopanib and Pembrolizumab Combination Therapy in Patients with Advanced Renal Cell Carcinoma.

BACKGROUND: This study assessed whether antiangiogenic treatment may potentiate immune checkpoint blockade in patients with advanced renal cell carcinoma. PATIENTS AND METHODS: This was an open-label, two-part, multicenter study involving treatment-naïve patients with advanced renal cell carcinoma. Part 1 consisted of a phase I dose escalation and expansion of pazopanib plus pembrolizumab (combination therapy). Cohorts A and B received pazopanib in combination with pembrolizumab, whereas Cohort C received pazopanib monotherapy for 9 weeks before receiving the combination therapy. Part 2 was planned as a randomized three-arm study but was not conducted. RESULTS: Overall, 42 patients were enrolled (10 each in Cohorts A and B, 22 in Cohort C). The maximum tolerated dose was not reached and the recommended phase II dose was not declared, as Cohort C was closed early because of safety concerns. The overall response rates were 60% and 20% in Cohorts A and B, respectively. In Cohort C, the overall response rates were 33%, 25%, and 0% in the combination therapy, pembrolizumab monotherapy, and pazopanib monotherapy groups, respectively. The median progression-free survival rates were 21.95 months and 41.40 months in Cohorts A and B, respectively. Grade 3 or 4 adverse events (AEs) were observed in 90% of patients in Cohorts A and B. In Cohort C, the frequencies of grade 3 or 4 AEs, serious adverse events, and AEs leading to dose reduction were typically high in the combination therapy group. CONCLUSIONS: Despite preliminary signs of efficacy, significant hepatotoxicity was observed in Cohorts A and B. The sequential schedule of pazopanib followed by pazopanib plus pembrolizumab showed reduced hepatotoxicity; however, other safety issues emerged with this approach.

Spartalizumab in metastatic, well/poorly-differentiated neuroendocrine neoplasms.

Spartalizumab, a humanized anti-programmed death protein 1 (PD-1) monoclonal antibody, was evaluated in patients with well-differentiated metastatic grade 1/2 neuroendocrine tumors (NET) and poorly-differentiated gastroenteropancreatic neuroendocrine carcinomas (GEP-NEC). In this phase II, multicenter, single-arm study, patients received spartalizumab 400 mg every 4 weeks until confirmed disease progression or unacceptable toxicity. The primary endpoint was confirmed overall response rate (ORR) according to blinded independent review committee using response evaluation criteria in solid tumors 1.1. The study enrolled 95 patients in the NET group (30, 32 and 33 in the thoracic, gastrointestinal, and pancreatic cohorts, respectively), and 21 patients in the GEP-NEC group. The ORR was 7.4% (95% confidence interval [CI]: 3.0, 14.6) in the NET group (thoracic, 16.7%; gastrointestinal, 3.1%; pancreatic, 3.0%), which was below the predefined success criterion of ≥10%, and 4.8% (95% CI: 0.1, 23.8) in the GEP-NEC group. In the NET and GEP-NEC groups, the 12-month progression-free survival was 19.5% and 0%, respectively, and the 12-month overall survival was 73.5% and 19.1%, respectively. The ORR was higher in patients with ≥1% PD-L1 expression in immune/tumor cells or ≥1% CD8+ cells at baseline. The most common adverse events considered as spartalizumab-related included fatigue (29.5%) and nausea (10.5%) in the NET group, and increased aspartate and alanine aminotransferases (each 14.3%) in the GEP-NEC group. The efficacy of spartalizumab was limited in this heterogeneous and heavily pre-treated population; however, the results in the thoracic cohort is encouraging and warrants further investigation. Adverse events were manageable and consistent with previous experience.

PTEN Expression, Not Mutation Status in TSC1, TSC2, or mTOR, Correlates with the Outcome on Everolimus in Patients with Renal Cell Carcinoma Treated on the Randomized RECORD-3 Trial.

PURPOSE: Genomic alterations in key components of PI3K/mTOR pathway have been proposed as candidate predictive markers for rapalog therapy in renal cell carcinoma (RCC). We tested this hypothesis in patients from a randomized phase II trial of everolimus versus sunitinib. PATIENTS AND METHODS: Archival specimens collected at baseline were analyzed with targeted next-generation sequencing (NGS). Focus of interest were alterations in key PI3K pathway components. PTEN expression was assessed by IHC. Association between molecular findings and treatment outcomes was investigated; same associations were tested for 2 everolimus-treated trial cohorts in gastric and hepatocellular carcinoma (HCC). RESULTS: Among 184 everolimus-treated patients with RCC with NGS data, mutation rates in genes of interest were 6% (TSC1), 4.4% (TSC2), and 8.2% (mTOR); 44% harbored alterations in ≥1 PI3K pathway component. For subjects with presence versus absence of mutations in TSC1, TSC2, or mTOR progression-free survival (PFS) neither differed on univariate analysis (HR, 1.0; P = 0.895) nor on multivariate testing stratified by MSKCC risk group and other established prognostic factors (HR, 1.1; P = 0.806). Everolimus-treated patients with retained (n = 50) versus lost (n = 50) PTEN IHC expression had median PFS of 5.3 months versus 10.5 months (HR, 2.5; P < 0.001). Such differences were not seen with sunitinib (10.9 months vs. 10.3 months; HR, 0.8; P = 0.475). Molecular findings did not correlate with outcomes in gastric and HCC cohorts. CONCLUSIONS: Association between mutation status for TSC1/TSC2/mTOR and therapeutic outcome on everolimus was not confirmed. Clinically meaningful differences in PFS were seen based on PTEN expression by IHC, lost in >50% of patients.

Transcriptomic Profiling of the Tumor Microenvironment Reveals Distinct Subgroups of Clear Cell Renal Cell Cancer: Data from a Randomized Phase III Trial.

Metastasis remains the main reason for renal cell carcinoma (RCC)-associated mortality. Tyrosine kinase inhibitors (TKI) impart clinical benefit for most patients with RCC, but the determinants of response are poorly understood. We report an integrated genomic and transcriptomic analysis of patients with metastatic clear cell RCC (ccRCC) treated with TKI therapy and identify predictors of response. Patients in the COMPARZ phase III trial received first-line sunitinib or pazopanib with comparable efficacy. RNA-based analyses revealed four distinct molecular subgroups associated with response and survival. Characterization of these subgroups identified mutation profiles, angiogenesis, and macrophage infiltration programs to be powerful predictors of outcome with TKI therapy. Notably, predictors differed by the type of TKI received. Our study emphasizes the clinical significance of angiogenesis and immune tumor microenvironment and suggests that the critical effects its various aspects have on TKI efficacy vary by agent. This has broad implications for optimizing precision treatment of RCC. SIGNIFICANCE: The determinants of response to TKI therapy in metastatic ccRCC remain unknown. Our study demonstrates that key angiogenic and immune profiles of the tumor microenvironment may affect TKI response. These findings have the potential to inform treatment personalization in patients with RCC.This article is highlighted in the In This Issue feature, p. 453.

Modulation of marrow proliferation and chemosensitivity by tumor-produced cytokines from syngeneic pancreatic tumor lines.

PURPOSE: A dynamic process exists in which hematopoietic progenitor andstromal cells interact to maintain normal hematopoiesis or to adjust to hematopoietic needs under "stress" situations. The effect that tumor-produced growth factors have on hematopoiesis has not been addressed. We postulate that an excess of tumor-produced stimulatory or inhibitory cytokines could impact marrow proliferation and sensitivity to cytotoxic agents. METHODS: We used two tumor lines (TGP47 and TGP51) taken from a panel of syngeneic murine pancreatic carcinomas, in which each produces a unique array of cytokines, and evaluated their effect in vitro on marrow proliferation and chemosensitivity. RESULTS: TGP51- and TGP47-conditioned medium increased [3H]thymidine incorporation into cultured marrow cells by approximately 12-fold and 4.8-fold, respectively. The percent of cells in the S + G2-M phases of the cell cycle increased by 110% (TGP51) and 44% (TGP47), and the MCF for proliferating cell nuclear antigen expression increased by 104% (TGP51) and 45% (TGP47). Marrow proliferation of untreated cells could be reduced by interleukin 6 but not by granulocyte macrophage colony-stimulating factor neutralization. Conditioned medium-induced stimulation was unchanged by either interleukin 6alpha or granulocyte macrophage colony-stimulating factor alpha. FLT3-Lalpha reduced marrow proliferation induced by TGP51 medium. Addition of FLT3-L to TGP47 medium additionally enhanced the marrow proliferation. Antitumor necrosis factor alpha additionally increased marrow proliferation induced by TGP47 and TGP51 conditioned medium, whereas addition of tumor necrosis factor alpha reduced marrow proliferation associated with TGP51 medium. The TGP51-induced increase in marrow proliferation resulted in increased marrow chemosensitivity to three myelosuppressive drugs: doxorubicin, cyclophosphamide, and CPT-11, decreasing the IC50 by 46%, 38%, and 95%, respectively. CONCLUSION: Tumor-produced cytokines can affect marrow proliferative activity and, thus, chemosensitivity to three distinct classes of chemotherapeutics.

The use of rat lens explant cultures to study the mechanism of drug-induced cataractogenesis.

Lens explant cultures were used to assess the mechanism of drug-induced cataractogenic potential of NVS001, a peroxisome proliferator-activated receptor delta (PPARδ) agonist, which resulted in cataract in all treated animals during a 13-week rat study. Ciglitazone, a PPARγ agonist and cataractogenic compound, was used as a positive control to validate this model. Rat lenses were extracted and cultured in medium supplemented with antibiotics for 24-h preincubation pretreatment. Lenses showing no signs of damage at the end of the preincubation pretreatment period were randomized into five experimental groups, (1) untreated control, (2) 0.1% dimethyl sulphoxide control, (3) 10µM NVS001, (4) 10µM ciglitazone, and (5) 10µM acetaminophen (negative control). Lenses were treated every 24 h after preincubation pretreatment for up to 48 h. Samples for viability, histology, and gene expression profiling were collected at 4, 24, and 48 h. There was a time-dependent increase in opacity, which correlated to a decrease in viability measured by adenosine triphosphate levels in NVS001 and ciglitazone-treated lenses compared with controls. NVS001 and ciglitazone had comparable cataractogenic effects after 48 h with histology showing rupture of the lens capsule, lens fiber degeneration, cortical lens vacuolation, and lens epithelial degeneration. Furthermore, no changes were seen when lenses were treated with acetaminophen. Gene expression analysis supported oxidative and osmotic stress, along with decreases in membrane and epithelial cell integrity as key factors in NVS001-induced cataracts. This study suggests that in vitro lens cultures can be used to assess cataractogenic potential of PPAR agonists and to study/understand the underlying molecular mechanism of cataractogenesis in rat.