Circulating tumour DNA in patients with advanced melanoma treated with dabrafenib or dabrafenib plus trametinib: a clinical validation study.

BACKGROUND: Melanoma lacks validated blood-based biomarkers for monitoring and predicting treatment efficacy. Cell-free circulating tumour DNA (ctDNA) is a promising biomarker; however, various detection methods have been used, and, to date, no large studies have examined the association between serial changes in ctDNA and survival after BRAF, MEK, or BRAF plus MEK inhibitor therapy. We aimed to evaluate whether baseline ctDNA concentrations and kinetics could predict survival outcomes. METHODS: In this clinical validation study, we used analytically validated droplet digital PCR assays to measure BRAFV600-mutant ctDNA in pretreatment and on-treatment plasma samples from patients aged 18 years or older enrolled in two clinical trials. COMBI-d (NCT01584648) was a double-blind, randomised phase 3 study of dabrafenib plus trametinib versus dabrafenib plus placebo in previously untreated patients with BRAFV600 mutation-positive unresectable or metastatic melanoma. Patients had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. COMBI-MB (NCT02039947) was an open-label, non-randomised, phase 2 study evaluating dabrafenib plus trametinib in patients with BRAFV600 mutation-positive metastatic melanoma and brain metastases. Patients in cohort A of COMBI-MB had asymptomatic brain metastases, no previous local brain-directed therapy, and an ECOG performance status of 0 or 1. Biomarker analysis was a prespecified exploratory endpoint in both trials and performed in the intention-to-treat populations in COMBI-d and COMBI-MB. We investigated the association between mutant copy number (baseline or week 4 or zero conversion status) and efficacy endpoints (progression-free survival, overall survival, and best overall response). We used Cox models, Kaplan-Meier plots, and log-rank tests to explore the association of pretreatment ctDNA concentrations with progression-free survival and overall survival. The effect of additional prognostic variables such as lactate dehydrogenase was also investigated in addition to the mutant copy number. FINDINGS: In COMBI-d, pretreatment plasma samples were available from 345 (82%) of 423 patients and on-treatment (week 4) plasma samples were available from 224 (53%) of 423 patients. In cohort A of COMBI-MB, pretreatment and on-treatment samples were available from 38 (50%) of 76 patients with intracranial and extracranial metastatic melanoma. ctDNA was detected in pretreatment samples from 320 (93%) of 345 patients (COMBI-d) and 34 (89%) of 38 patients (COMBI-MB). When assessed as a continuous variable, elevated baseline BRAFV600 mutation-positive ctDNA concentration was associated with worse overall survival outcome (hazard ratio [HR] 1·13 [95% CI 1·09-1·18], p<0·0001 by univariate analysis), independent of treatment group and baseline lactate dehydrogenase concentrations (1·08 [1·03-1·13], p=0·0020), in COMBI-d. A ctDNA cutoff point of 64 copies per mL of plasma stratified patients enrolled in COMBI-d as high risk or low risk with respect to survival outcomes (HR 1·74 [95% CI 1·37-2·21], p<0·0001 for progression-free survival; 2·23 [1·73-2·87], p<0·0001 for overall survival) and was validated in the COMBI-MB cohort (3·20 [1·39-7·34], p=0·0047 for progression-free survival; 2·94 [1·18-7·32], p=0·016 for overall survival). In COMBI-d, undetectable ctDNA at week 4 was significantly associated with extended progression-free and overall survival, particularly in patients with elevated lactate dehydrogenase concentrations (HR 1·99 [95% CI 1·08-3·64], p=0·027 for progression-free survival; 2·38 [1·24-4·54], p=0·0089 for overall survival). INTERPRETATION: Pretreatment and on-treatment BRAFV600-mutant ctDNA measurements could serve as independent, predictive biomarkers of clinical outcome with targeted therapy. FUNDING: Novartis.

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.

Ruxolitinib reduces JAK2 p.V617F allele burden in patients with polycythemia vera enrolled in the RESPONSE study.

In patients with polycythemia vera (PV), an elevated JAK2 p.V617F allele burden is associated with indicators of more severe disease (e.g., leukocytosis, splenomegaly, and increased thrombosis risk); however, correlations between allele burden reductions and clinical benefit in patients with PV have not been extensively evaluated in a randomized trial. This exploratory analysis from the multicenter, open-label, phase 3 Randomized Study of Efficacy and Safety in Polycythemia Vera With JAK Inhibitor INCB018424 Versus Best Supportive Care trial evaluated the long-term effect of ruxolitinib treatment on JAK2 p.V617F allele burden in patients with PV. Evaluable JAK2 p.V617F-positive patients randomized to ruxolitinib (n = 107) or best available therapy (BAT) who crossed over to ruxolitinib at week 32 (n = 97) had consistent JAK2 p.V617F allele burden reductions throughout the study. At all time points measured (up to weeks 208 [ruxolitinib-randomized] and 176 [ruxolitinib crossover]), mean changes from baseline over time in JAK2 p.V617F allele burden ranged from -12.2 to -40.0% (ruxolitinib-randomized) and -6.3 to -17.8% (ruxolitinib crossover). Complete or partial molecular response was observed in 3 patients (ruxolitinib-randomized, n = 2; ruxolitinib crossover, n = 1) and 54 patients (ruxolitinib-randomized, n = 33; ruxolitinib crossover, n = 20; BAT, n = 1), respectively. Among patients treated with interferon as BAT (n = 13), the mean maximal reduction in allele burden from baseline was 25.6% after crossover to ruxolitinib versus 6.6% before crossover. Collectively, the data from this exploratory analysis suggest that ruxolitinib treatment for up to 4 years provides progressive reductions in JAK2 p.V617F allele burden in patients with PV who are resistant to or intolerant of hydroxyurea. The relationship between allele burden changes and clinical outcomes in patients with PV remains unclear.

Circulating biomarkers and outcome from a randomised phase II trial of sunitinib vs everolimus for patients with metastatic renal cell carcinoma.

BACKGROUND: RECORD-3 assessed non-inferiority of progression-free survival (PFS) with everolimus vs sunitinib in previously untreated patients with metastatic renal cell carcinoma. Baseline plasma sample collection and randomised design enabled correlation of circulating biomarkers with efficacy. METHODS: Samples were analysed for 121 cancer-related biomarkers. Analyses of biomarkers categorised patients as high or low (vs median) to assess association with first-line PFS (PFS1L) for each treatment arm. A composite biomarker score (CBS) incorporated biomarkers potentially predictive of PFS1L with everolimus. RESULTS: Plasma samples from 442 of the 471 randomised patients were analysed. Biomarkers were associated with PFS1L for everolimus alone (29), sunitinib alone (9) or both (12). Everolimus-specific biomarkers (CSF1, ICAM1, IL-18BP, KIM1, TNFRII) with hazard ratio ⩾ 1.8 were integrated into a CBS (range 0-5). For CBS low (0-3, n = 291) vs high (4-5, n = 151), PFS1L differed significantly for everolimus but not for sunitinib. There was no significant difference in PFS1L between everolimus and sunitinib in the high CBS patient cohort. CONCLUSIONS: Baseline levels of multiple soluble biomarkers correlated with benefit from everolimus and/or sunitinib, independent of clinical risk factors. A similar PFS1L was observed for both treatments among patients with high CBS score.

Dovitinib versus sorafenib for third-line targeted treatment of patients with metastatic renal cell carcinoma: an open-label, randomised phase 3 trial.

BACKGROUND: An unmet medical need exists for patients with metastatic renal cell carcinoma who have progressed on VEGF-targeted and mTOR-inhibitor therapies. Fibroblast growth factor (FGF) pathway activation has been proposed as a mechanism of escape from VEGF-targeted therapies. Dovitinib is an oral tyrosine-kinase inhibitor that inhibits VEGF and FGF receptors. We therefore compared dovitinib with sorafenib as third-line targeted therapies in patients with metastatic renal cell carcinoma. METHODS: In this multicentre phase 3 study, patients with clear cell metastatic renal cell carcinoma who received one previous VEGF-targeted therapy and one previous mTOR inhibitor were randomly assigned through an interactive voice and web response system to receive open-label dovitinib (500 mg orally according to a 5-days-on and 2-days-off schedule) or sorafenib (400 mg orally twice daily) in a 1:1 ratio. Randomisation was stratified by risk group and region. The primary endpoint was progression-free survival (PFS) assessed by masked central review. Efficacy was assessed in all patients who were randomly assigned and safety was assessed in patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT01223027. FINDINGS: 284 patients were randomly assigned to the dovitinib group and 286 to the sorafenib group. Median follow-up was 11·3 months (IQR 7·9-14·6). Median PFS was 3·7 months (95% CI 3·5-3·9) in the dovitinib group and 3·6 months (3·5-3·7) in the sorafenib group (hazard ratio 0·86, 95% CI 0·72-1·04; one-sided p=0·063). 280 patients in the dovitinib group and 284 in the sorafenib group received at least one dose of study drug. Common grade 3 or 4 adverse events included hypertriglyceridaemia (38 [14%]), fatigue (28 [10%]), hypertension (22 [8%]), and diarrhoea (20 [7%]) in the dovitinib group, and hypertension (47 [17%]), fatigue (24 [8%]), dyspnoea (21 [7%]), and palmar-plantar erythrodysaesthesia (18 [6%]) in the sorafenib group. The most common serious adverse event was dyspnoea (16 [6%] and 15 [5%] in the dovitinib and sorafenib groups, respectively). INTERPRETATION: Dovitinib showed activity, but this was no better than that of sorafenib in patients with renal cell carcinoma who had progressed on previous VEGF-targeted therapies and mTOR inhibitors. This trial provides reference outcome data for future studies of targeted inhibitors in the third-line setting. FUNDING: Novartis Pharmaceuticals Corporation.

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.

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.

Genomic Biomarkers of a Randomized Trial Comparing First-line Everolimus and Sunitinib in Patients with Metastatic Renal Cell Carcinoma.

BACKGROUND: Metastatic renal cell carcinoma (RCC) patients are commonly treated with vascular endothelial growth factor (VEGF) inhibitors or mammalian target of rapamycin inhibitors. Correlations between somatic mutations and first-line targeted therapy outcomes have not been reported on a randomized trial. OBJECTIVE: To evaluate the relationship between tumor mutations and treatment outcomes in RECORD-3, a randomized trial comparing first-line everolimus (mTOR inhibitor) followed by sunitinib (VEGF inhibitor) at progression with the opposite sequence in 471 metastatic RCC patients. DESIGN, SETTING, AND PARTICIPANTS: Targeted sequencing of 341 cancer genes at ∼540× coverage was performed on available tumor samples from 258 patients; 220 with clear cell histology (ccRCC). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Associations between somatic mutations and median first-line progression free survival (PFS1L) and overall survival were determined in metastatic ccRCC using Cox proportional hazards models and log-rank tests. RESULTS AND LIMITATIONS: Prevalent mutations (≥ 10%) were VHL (75%), PBRM1 (46%), SETD2 (30%), BAP1 (19%), KDM5C (15%), and PTEN (12%). With first-line everolimus, PBRM1 and BAP1 mutations were associated with longer (median [95% confidence interval {CI}] 12.8 [8.1, 18.4] vs 5.5 [3.1, 8.4] mo) and shorter (median [95% CI] 4.9 [2.9, 8.1] vs 10.5 [7.3, 12.9] mo) PFS1L, respectively. With first-line sunitinib, KDM5C mutations were associated with longer PFS1L (median [95% CI] of 20.6 [12.4, 27.3] vs 8.3 [7.8, 11.0] mo). Molecular subgroups of metastatic ccRCC based on PBRM1, BAP1, and KDM5C mutations could have predictive values for patients treated with VEGF or mTOR inhibitors. Most tumor DNA was obtained from primary nephrectomy samples (94%), which could impact correlation statistics. CONCLUSIONS: PBRM1, BAP1, and KDM5C mutations impact outcomes of targeted therapies in metastatic ccRCC patients. PATIENT SUMMARY: Large-scale genomic kidney cancer studies reported novel mutations and heterogeneous features among individual tumors, which could contribute to varied clinical outcomes. We demonstrated correlations between somatic mutations and treatment outcomes in clear cell renal cell carcinoma, supporting the value of genomic classification in prospective studies.