Tracking Cancer Evolution Reveals Constrained Routes to Metastases: TRACERx Renal.

Clear-cell renal cell carcinoma (ccRCC) exhibits a broad range of metastatic phenotypes that have not been systematically studied to date. Here, we analyzed 575 primary and 335 metastatic biopsies across 100 patients with metastatic ccRCC, including two cases sampledat post-mortem. Metastatic competence was afforded by chromosome complexity, and we identify 9p loss as a highly selected event driving metastasis and ccRCC-related mortality (p = 0.0014). Distinct patterns of metastatic dissemination were observed, including rapid progression to multiple tissue sites seeded by primary tumors of monoclonal structure. By contrast, we observed attenuated progression in cases characterized by high primary tumor heterogeneity, with metastatic competence acquired gradually and initial progression to solitary metastasis. Finally, we observed early divergence of primitive ancestral clones and protracted latency of up to two decades as a feature of pancreatic metastases.

Tumor-associated macrophages expressing the transcription factor IRF8 promote T cell exhaustion in cancer.

Tumors are populated by antigen-presenting cells (APCs) including macrophage subsets with distinct origins and functions. Here, we examined how cancer impacts mononuclear phagocytic APCs in a murine model of breast cancer. Tumors induced the expansion of monocyte-derived tumor-associated macrophages (TAMs) and the activation of type 1 dendritic cells (DC1s), both of which expressed and required the transcription factor interferon regulatory factor-8 (IRF8). Although DC1s mediated cytotoxic T lymphocyte (CTL) priming in tumor-draining lymph nodes, TAMs promoted CTL exhaustion in the tumor, and IRF8 was required for TAMs' ability to present cancer cell antigens. TAM-specific IRF8 deletion prevented exhaustion of cancer-cell-reactive CTLs and suppressed tumor growth. Tumors from patients with immune-infiltrated renal cell carcinoma had abundant TAMs that expressed IRF8 and were enriched for an IRF8 gene expression signature. Furthermore, the TAM-IRF8 signature co-segregated with CTL exhaustion signatures across multiple cancer types. Thus, CTL exhaustion is promoted by TAMs via IRF8.

TGF-ß suppresses type 2 immunity to cancer.

The immune system uses two distinct defence strategies against infections: microbe-directed pathogen destruction characterized by type 1 immunity1, and host-directed pathogen containment exemplified by type 2 immunity in induction of tissue repair2. Similar to infectious diseases, cancer progresses with self-propagating cancer cells inflicting host-tissue damage. The immunological mechanisms of cancer cell destruction are well defined3-5, but whether immune-mediated cancer cell containment can be induced remains poorly understood. Here we show that depletion of transforming growth factor-ß receptor 2 (TGFBR2) in CD4+ T cells, but not CD8+ T cells, halts cancer progression as a result of tissue healing and remodelling of the blood vasculature, causing cancer cell hypoxia and death in distant avascular regions. Notably, the host-directed protective response is dependent on the T helper 2 cytokine interleukin-4 (IL-4), but not the T helper 1 cytokine interferon-γ (IFN-γ). Thus, type 2 immunity can be mobilized as an effective tissue-level defence mechanism against cancer.

Nivolumab plus Cabozantinib versus Sunitinib for Advanced Renal-Cell Carcinoma.

BACKGROUND: The efficacy and safety of nivolumab plus cabozantinib as compared with those of sunitinib in the treatment of previously untreated advanced renal-cell carcinoma are not known. METHODS: In this phase 3, randomized, open-label trial, we randomly assigned adults with previously untreated clear-cell, advanced renal-cell carcinoma to receive either nivolumab (240 mg every 2 weeks) plus cabozantinib (40 mg once daily) or sunitinib (50 mg once daily for 4 weeks of each 6-week cycle). The primary end point was progression-free survival, as determined by blinded independent central review. Secondary end points included overall survival, objective response as determined by independent review, and safety. Health-related quality of life was an exploratory end point. RESULTS: Overall, 651 patients were assigned to receive nivolumab plus cabozantinib (323 patients) or sunitinib (328 patients). At a median follow-up of 18.1 months for overall survival, the median progression-free survival was 16.6 months (95% confidence interval [CI], 12.5 to 24.9) with nivolumab plus cabozantinib and 8.3 months (95% CI, 7.0 to 9.7) with sunitinib (hazard ratio for disease progression or death, 0.51; 95% CI, 0.41 to 0.64; P<0.001). The probability of overall survival at 12 months was 85.7% (95% CI, 81.3 to 89.1) with nivolumab plus cabozantinib and 75.6% (95% CI, 70.5 to 80.0) with sunitinib (hazard ratio for death, 0.60; 98.89% CI, 0.40 to 0.89; P = 0.001). An objective response occurred in 55.7% of the patients receiving nivolumab plus cabozantinib and in 27.1% of those receiving sunitinib (P<0.001). Efficacy benefits with nivolumab plus cabozantinib were consistent across subgroups. Adverse events of any cause of grade 3 or higher occurred in 75.3% of the 320 patients receiving nivolumab plus cabozantinib and in 70.6% of the 320 patients receiving sunitinib. Overall, 19.7% of the patients in the combination group discontinued at least one of the trial drugs owing to adverse events, and 5.6% discontinued both. Patients reported better health-related quality of life with nivolumab plus cabozantinib than with sunitinib. CONCLUSIONS: Nivolumab plus cabozantinib had significant benefits over sunitinib with respect to progression-free survival, overall survival, and likelihood of response in patients with previously untreated advanced renal-cell carcinoma. (Funded by Bristol Myers Squibb and others; CheckMate 9ER ClinicalTrials.gov number, NCT03141177.).

Lenvatinib plus pembrolizumab in patients with either treatment-naive or previously treated metastatic renal cell carcinoma (Study 111/KEYNOTE-146): a phase 1b/2 study.

BACKGROUND: Despite advances in the first-line treatment of metastatic renal cell carcinoma (RCC), there is an unmet need for options to address disease progression during or after treatment with immune checkpoint inhibitors (ICIs). Pembrolizumab and lenvatinib are active as monotherapies in RCC; thus, we aimed to evaluate the combination of lenvatinib plus pembrolizumab in these patients. METHODS: We report results of the metastatic RCC cohort from an open-label phase 1b/2 study of lenvatinib plus pembrolizumab in patients aged at least 18 years with selected solid tumours and an Eastern Cooperative Oncology Group performance status of 0-1. Oral lenvatinib at 20 mg was given once daily along with intravenous pembrolizumab at 200 mg once every 3 weeks. Patients remained on study drug treatment until disease progression, development of unacceptable toxicity, or withdrawal of consent. Efficacy was analysed in patients with clear cell metastatic RCC receiving study drug by previous therapy grouping: treatment naive, previously treated ICI naive (previously treated with at least one line of therapy but not with an anti-PD-1 or anti-PD-L1 ICI), and ICI pretreated (ie, anti-PD-1 or anti-PD-L1) patients. Safety was analysed in all enrolled and treated patients. The primary endpoint was the objective response rate at week 24 per immune-related Response Evaluation Criteria In Solid Tumors (irRECIST) by investigator assessment. This trial is registered with ClinicalTrials.gov (NCT02501096) and with the EU Clinical Trials Register (EudraCT2017-000300-26), and is closed to new participants. FINDINGS: Between July 21, 2015, and Oct 16, 2019, 145 patients were enrolled in the study. Two patients had non-clear cell RCC and were excluded from the efficacy analysis (one in the treatment-naive group and one in the ICI-pretreated group); thus, the population evaluated for efficacy comprised 143 patients (n=22 in the treatment-naive group, n=17 in the previously treated ICI-naive group, and n=104 in the ICI-pretreated group). All 145 enrolled patients were included in the safety analysis. The median follow-up was 19·8 months (IQR 14·3-28·4). The number of patients with an objective response at week 24 by irRECIST was 16 (72·7%, 95% CI 49·8-89·3) of 22 treatment-naive patients, seven (41·2%, 18·4-67·1) of 17 previously treated ICI-naive patients, and 58 (55·8%, 45·7-65·5) of 104 ICI-pretreated patients. Of 145 patients, 82 (57%) had grade 3 treatment-related adverse events and ten (7%) had grade 4 treatment-related adverse events. The most common grade 3 treatment-related adverse event was hypertension (30 [21%] of 145 patients). Treatment-related serious adverse events occurred in 36 (25%) patients, and there were three treatment-related deaths (upper gastrointestinal haemorrhage, sudden death, and pneumonia). INTERPRETATION: Lenvatinib plus pembrolizumab showed encouraging antitumour activity and a manageable safety profile and might be an option for post-ICI treatment of metastatic RCC. FUNDING: Eisai and Merck Sharp & Dohme.

Overcome tumor heterogeneity-imposed therapeutic barriers through convergent genomic biomarker discovery: A braided cancer river model of kidney cancer.

Tumor heterogeneity, encompassing genetic, epigenetic, and microenvironmental variables, is extremely complex and presents challenges to cancer diagnosis and therapy. Genomic efforts on genetic intratumor heterogeneity (G-ITH) confirm branched evolution, support the trunk-branch cancer model, and present a seemingly insurmountable obstacle to conquering cancers. G-ITH is conspicuous in clear cell renal cell carcinoma (ccRCC), where its presence complicates identification and validation of biomarkers and thwarts efforts in advancing precision cancer therapeutics. However, long-term clinical benefits on targeted therapy are not uncommon in metastatic ccRCC patients, implicating that there are underlying constraints during ccRCC evolution, which in turn force a nonrandom sequence of parallel gene/pathway/function/phenotype convergence within individual tumors. Accordingly, we proposed a "braided cancer river model" depicting ccRCC evolution, which deduces cancer development based on multiregion tumor genomics of exceptional mTOR inhibitor (mTORi) responders. Furthermore, we employ an outlier case to explore the river model and highlight the importance of "Five NGS Matters: Number, Frequency, Position, Site and Time" in assessing cancer genomics for precision medicine. This mutable cancer river model may capture clinically significant phenotype-convergent events, predict vulnerability/resistance mechanisms, and guide effective therapeutic strategies. Our model originates from studying exceptional responders in ccRCC, which warrants further refinement and future validation concerning its applicability to other cancer types. The goal of this review is employing kidney cancer as an example to illustrate critical issues concerning tumor heterogeneity.

Comprehensive Molecular Characterization of Papillary Renal-Cell Carcinoma.

BACKGROUND: Papillary renal-cell carcinoma, which accounts for 15 to 20% of renal-cell carcinomas, is a heterogeneous disease that consists of various types of renal cancer, including tumors with indolent, multifocal presentation and solitary tumors with an aggressive, highly lethal phenotype. Little is known about the genetic basis of sporadic papillary renal-cell carcinoma, and no effective forms of therapy for advanced disease exist. METHODS: We performed comprehensive molecular characterization of 161 primary papillary renal-cell carcinomas, using whole-exome sequencing, copy-number analysis, messenger RNA and microRNA sequencing, DNA-methylation analysis, and proteomic analysis. RESULTS: Type 1 and type 2 papillary renal-cell carcinomas were shown to be different types of renal cancer characterized by specific genetic alterations, with type 2 further classified into three individual subgroups on the basis of molecular differences associated with patient survival. Type 1 tumors were associated with MET alterations, whereas type 2 tumors were characterized by CDKN2A silencing, SETD2 mutations, TFE3 fusions, and increased expression of the NRF2-antioxidant response element (ARE) pathway. A CpG island methylator phenotype (CIMP) was observed in a distinct subgroup of type 2 papillary renal-cell carcinomas that was characterized by poor survival and mutation of the gene encoding fumarate hydratase (FH). CONCLUSIONS: Type 1 and type 2 papillary renal-cell carcinomas were shown to be clinically and biologically distinct. Alterations in the MET pathway were associated with type 1, and activation of the NRF2-ARE pathway was associated with type 2; CDKN2A loss and CIMP in type 2 conveyed a poor prognosis. Furthermore, type 2 papillary renal-cell carcinoma consisted of at least three subtypes based on molecular and phenotypic features. (Funded by the National Institutes of Health.).

SETD2 alterations impair DNA damage recognition and lead to resistance to chemotherapy in leukemia.

Mutations in SETD2, encoding the histone 3 lysine 36 trimethyltransferase, are enriched in relapsed acute lymphoblastic leukemia and MLL-rearranged acute leukemia. We investigated the impact of SETD2 mutations on chemotherapy sensitivity in isogenic leukemia cell lines and in murine leukemia generated from a conditional knockout of Setd2. SETD2 mutations led to resistance to DNA-damaging agents, cytarabine, 6-thioguanine, doxorubicin, and etoposide, but not to a non-DNA damaging agent, l-asparaginase. H3K36me3 localizes components of the DNA damage response (DDR) pathway and SETD2 mutation impaired DDR, blunting apoptosis induced by cytotoxic chemotherapy. Consistent with local recruitment of DDR, genomic regions with higher H3K36me3 had a lower mutation rate, which was increased with SETD2 mutation. Heterozygous conditional inactivation of Setd2 in a murine model decreased the latency of MLL-AF9-induced leukemia and caused resistance to cytarabine treatment in vivo, whereas homozygous loss delayed leukemia formation. Treatment with JIB-04, an inhibitor of the H3K9/36me3 demethylase KDM4A, restored H3K36me3 levels and sensitivity to cytarabine. These findings establish SETD2 alteration as a mechanism of resistance to DNA-damaging chemotherapy, consistent with a local loss of DDR, and identify a potential therapeutic strategy to target SETD2-mutant leukemias.

Genomic classifications of renal cell carcinoma: a critical step towards the future application of personalized kidney cancer care with pan-omics precision.

Over the past 20 years, classifications of kidney cancer have undergone major revisions based on morphological refinements and molecular characterizations. The 2016 WHO classification of renal tumors recognizes more than ten different renal cell carcinoma (RCC) subtypes. Furthermore, the marked inter- and intra-tumor heterogeneity of RCC is now well appreciated. Nevertheless, contemporary multi-omics studies of RCC, encompassing genomics, transcriptomics, proteomics, and metabolomics, not only highlight apparent diversity but also showcase and underline commonality. Here, we wish to provide an integrated perspective concerning the future 'functional' classification of renal cancer by bridging gaps among morphology, biology, multi-omics, and therapeutics. This review focuses on recent progress and elaborates the potential value of contemporary pan-omics approaches with a special emphasis on cancer genomics unveiled through next-generation sequencing technology, and how an integrated multi-omics approach might impact precision-based personalized kidney cancer care in the near future. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

In Vivo PET Assay of Tumor Glutamine Flux and Metabolism: In-Human Trial of 18F-(2S,4R)-4-Fluoroglutamine.

Purpose To assess the clinical safety, pharmacokinetics, and tumor imaging characteristics of fluorine 18-(2S,4R)-4-fluoroglutamine (FGln), a glutamine analog radiologic imaging agent. Materials and Methods This study was approved by the institutional review board and conducted under a U.S. Food and Drug Administration-approved Investigational New Drug application in accordance with the Helsinki Declaration and the Health Insurance Portability and Accountability Act. All patients provided written informed consent. Between January 2013 and October 2016, 25 adult patients with cancer received an intravenous bolus of FGln tracer (mean, 244 MBq ± 118, <100 µg) followed by positron emission tomography (PET) and blood radioassays. Patient data were summarized with descriptive statistics. FGln biodistribution and plasma amino acid levels in nonfasting patients (n = 13) were compared with those from patients who fasted at least 8 hours before injection (n = 12) by using nonparametric one-way analysis of variance with Bonferroni correction. Tumor FGln avidity versus fluorodeoxyglucose (FDG) avidity in patients with paired PET scans (n = 15) was evaluated with the Fisher exact test. P < .05 was considered indicative of a statistically significant difference. Results FGln PET depicted tumors of different cancer types (breast, pancreas, renal, neuroendocrine, lung, colon, lymphoma, bile duct, or glioma) in 17 of the 25 patients, predominantly clinically aggressive tumors with genetic mutations implicated in abnormal glutamine metabolism. Acute fasting had no significant effect on FGln biodistribution and plasma amino acid levels. FGln-avid tumors were uniformly FDG-avid but not vice versa (P = .07). Patients experienced no adverse effects. Conclusion Preliminary human FGln PET trial results provide clinical validation of abnormal glutamine metabolism as a potential tumor biomarker for targeted radiotracer imaging in several different cancer types. © RSNA, 2018 Online supplemental material is available for this article. Clinical trial registration no. NCT01697930.

Molecular Subtypes Improve Prognostic Value of International Metastatic Renal Cell Carcinoma Database Consortium Prognostic Model.

INTRODUCTION: Gene-expression signatures for prognosis have been reported in localized renal cell carcinoma (RCC). The aim of this study was to test the predictive power of two different signatures, ClearCode34, a 34-gene signature model [Eur Urol 2014;66:77-84], and an 8-gene signature model [Eur Urol 2015;67:17-20], in the setting of systemic therapy for metastatic disease. MATERIALS AND METHODS: Metastatic RCC (mRCC) patients from five institutions who were part of TCGA were identified and clinical data were retrieved. We trained and implemented each gene model as described by the original study. The latter was demonstrated by faithful regeneration of a figure and results from the original study. mRCC patients were dichotomized to good or poor prognostic risk groups using each gene model. Cox proportional hazard regression and concordance index (C-Index) analysis were used to investigate an association between each prognostic risk model and overall survival (OS) from first-line therapy. RESULTS: Overall, 54 patients were included in the final analysis. The primary endpoint was OS. Applying the ClearCode34 model, median survival for the low-risk-ccA (n = 17)-and the high-risk-ccB (n = 37)-subtypes were 27.6 and 22.3 months (hazard ratio (HR): 2.33; p = .039), respectively. ClearCode34 ccA/ccB and International Metastatic Renal Cell Carcinoma Database Consortium (IMDC) classifications appear to represent distinct risk criteria in mRCC, and we observed no significant overlap in classification (p > .05, chi-square test). On multivariable analyses and adjusting for IMDC groups, ccB remained independently associated with a worse OS (p = .044); the joint model of ccA/ccB and IMDC was significantly more accurate in predicting OS than a model with IMDC alone (p = .045, F-test). This was also observed in C-Index analysis; a model with both ccA and ccB subtypes had higher accuracy (C-Index 0.63, 95% confidence interval [CI] = 0.51-0.75) and 95% CIs of the C-Index that did not include the null value of 0.5 in contrast to a model with IMDC alone (0.60, CI = 0.47-0.72). The 8-gene signature molecular subtype model was a weak but insignificant predictor of survival in this cohort (p = .13). A model that included both the 8-gene signature and IMDC (C-Index 0.62, CI = 0.49-0.76) was more prognostic than IMDC alone but did not reach significance, as the 95% CI included the null value of 0.5. These two genomic signatures share no genes in common and are enriched in different biological pathways. The ClearCode34 included genes ARNT and EPAS1 (also known as HIF2a), which are involved in regulation of gene expression by hypoxia-inducible factor. CONCLUSION: The ClearCode34 but not the 8-gene molecular model improved the prognostic predictive power of the IMDC model in this cohort of 54 patients with metastatic clear cell RCC. The Oncologist 2017;22:286-292 IMPLICATIONS FOR PRACTICE: The clinical and laboratory factors included in the International Metastatic Renal Cell Carcinoma Database Consortium model provide prognostic information in metastatic renal cell carcinoma (mRCC). The present study shows that genomic signatures, originally validated in localized RCC, may add further complementary prognostic information in the metastatic setting. This study may provide new insights into the molecular basis of certain mRCC subgroups. The integration of clinical and molecular data has the potential to redefine mRCC classification, enhance the understanding of mRCC biology, and potentially predict response to treatment in the future.

Deadly splicing: Bax becomes Almighty.

Activation of Bax and Bak by BH3-only molecules triggers mitochondrial apoptosis. In a recent issue of Molecular Cell, Fu et al. (2009) identify a constitutively active isoform of Bax, Baxbeta, whose activity is tightly controlled by the ubiquitin-proteasome system.

Stepwise activation of BAX and BAK by tBID, BIM, and PUMA initiates mitochondrial apoptosis.

While activation of BAX/BAK by BH3-only molecules (BH3s) is essential for mitochondrial apoptosis, the underlying mechanisms remain unsettled. Here we demonstrate that BAX undergoes stepwise structural reorganization leading to mitochondrial targeting and homo-oligomerization. The alpha1 helix of BAX keeps the alpha9 helix engaged in the dimerization pocket, rendering BAX as a monomer in cytosol. The activator BH3s, tBID/BIM/PUMA, attack and expose the alpha1 helix of BAX, resulting in secondary disengagement of the alpha9 helix and thereby mitochondrial insertion. Activator BH3s remain associated with the N-terminally exposed BAX through the BH1 domain to drive homo-oligomerization. BAK, an integral mitochondrial membrane protein, has bypassed the first activation step, explaining why its killing kinetics are faster than those of BAX. Furthermore, death signals initiated at ER induce BIM and PUMA to activate mitochondrial apoptosis. Accordingly, deficiency of Bim/Puma impedes ER stress-induced BAX/BAK activation and apoptosis. Our study provides mechanistic insights regarding the spatiotemporal execution of BAX/BAK-governed cell death.

Validation and genomic interrogation of the MET variant rs11762213 as a predictor of adverse outcomes in clear cell renal cell carcinoma.

BACKGROUND: The exonic single-nucleotide variant rs11762213 located in the MET oncogene has recently been identified as a prognostic marker in clear cell renal cell carcinoma (ccRCC). This finding was validated with The Cancer Genome Atlas (TCGA) cohort, and the biologic implications were explored. METHODS: The genotype status for rs11762213 was available for 272 patients. Paired tumor-normal data, genomic data, and clinical information were acquired from ccRCC TCGA data sets. Cancer-specific survival (CSS) was analyzed with the competing risk method, and Cox proportional hazards regression was used for the analysis of the time to recurrence (TTR). Multivariate competing risk models were fitted to adjust for the validated Mayo Clinic Stage, Size, Grade, and Necrosis (SSIGN) score. RESULTS: The variant allele of rs11762213 was detected in 10.3% of the cohort. After adjustments for the SSIGN score, the risk allele remained a significant predictor for adverse CSS (hazard ratio [HR], 3.88; 95% confidence interval [CI], 1.99-7.56; P < .0001) and for TTR (OR, 2.97; 95% CI, 1.43-6.2; P = .003). The mapping of rs11762213 to regulatory regions within the genome suggested that it might affect a DNA enhancer region. RNA and protein sequencing data for MET did not reveal differences in steady-state expression with stratification by risk allele. CONCLUSIONS: The exonic MET variant rs11762213 is an independent predictor of adverse CSS and TTR in ccRCC and should be integrated into clinical practice for prognostic stratification. Genomic analysis suggests that the single-nucleotide polymorphism may affect an enhancer region located in the coding region of MET. Further biological mechanistic interrogation is currently underway.

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.

A Phase Ib Study of BEZ235, a Dual Inhibitor of Phosphatidylinositol 3-Kinase (PI3K) and Mammalian Target of Rapamycin (mTOR), in Patients With Advanced Renal Cell Carcinoma.

LESSONS LEARNED: Our results highlight additional toxicities of dual PI3K/mTOR inhibition in the clinical setting that were unforeseen from preclinical models.Because of toxicity and lack of efficacy, BEZ235 should not be further developed in the current formulation for patients with renal cell carcinoma. BACKGROUND: Allosteric inhibitors of the mammalian target of rapamycin complex 1 (mTORC1) are approved for advanced renal cell carcinoma (RCC). Preclinical models have suggested that dual inhibition of phosphatidylinositol 3-kinase (PI3K) and mTOR kinase may establish superior anticancer effect. We aimed to establish safety for BEZ235, a potent inhibitor of both PI3K and mTOR, in advanced RCC. METHODS: Patients with advanced RCC who had previously failed standard therapy received escalating doses of BEZ235 in sachet formulation twice daily until progression or unacceptable toxicity. Primary endpoints were to identify the maximally tolerated dose (MTD) and to determine the recommended dose for the phase II study. RESULTS: The study was terminated early because of high incidence of dose-limiting toxicities (DLTs) across all dose levels tested. Ten patients were treated with BEZ235-six with clear cell and four with non-clear cell subtypes. Five of these patients suffered DLTs: 2 of 2 patients in the original 400 mg b.i.d. cohort, 1 of 6 in the 200 mg b.i.d. cohort, and 2 of 2 in the 300 mg b.i.d. COHORT: DLTs included fatigue, rash, nausea and vomiting, diarrhea, mucositis, anorexia, and dysgeusia. Five patients were evaluable for response: Two had stable disease as best response, and three had progressive disease. CONCLUSION: BEZ235 twice daily resulted in significant toxicity without objective responses; further development of this compound will not be pursued in this disease.

Phosphorylation of MLL by ATR is required for execution of mammalian S-phase checkpoint.

Cell cycle checkpoints are implemented to safeguard the genome, avoiding the accumulation of genetic errors. Checkpoint loss results in genomic instability and contributes to the evolution of cancer. Among G1-, S-, G2- and M-phase checkpoints, genetic studies indicate the role of an intact S-phase checkpoint in maintaining genome integrity. Although the basic framework of the S-phase checkpoint in multicellular organisms has been outlined, the mechanistic details remain to be elucidated. Human chromosome-11 band-q23 translocations disrupting the MLL gene lead to poor prognostic leukaemias. Here we assign MLL as a novel effector in the mammalian S-phase checkpoint network and identify checkpoint dysfunction as an underlying mechanism of MLL leukaemias. MLL is phosphorylated at serine 516 by ATR in response to genotoxic stress in the S phase, which disrupts its interaction with, and hence its degradation by, the SCF(Skp2) E3 ligase, leading to its accumulation. Stabilized MLL protein accumulates on chromatin, methylates histone H3 lysine 4 at late replication origins and inhibits the loading of CDC45 to delay DNA replication. Cells deficient in MLL showed radioresistant DNA synthesis and chromatid-type genomic abnormalities, indicative of S-phase checkpoint dysfunction. Reconstitution of Mll(-/-) (Mll also known as Mll1) mouse embryonic fibroblasts with wild-type but not S516A or ΔSET mutant MLL rescues the S-phase checkpoint defects. Moreover, murine myeloid progenitor cells carrying an Mll-CBP knock-in allele that mimics human t(11;16) leukaemia show a severe radioresistant DNA synthesis phenotype. MLL fusions function as dominant negative mutants that abrogate the ATR-mediated phosphorylation/stabilization of wild-type MLL on damage to DNA, and thus compromise the S-phase checkpoint. Together, our results identify MLL as a key constituent of the mammalian DNA damage response pathway and show that deregulation of the S-phase checkpoint incurred by MLL translocations probably contributes to the pathogenesis of human MLL leukaemias.

TCEB1-mutated renal cell carcinoma: a distinct genomic and morphological subtype.

Integrated sequencing analysis identified a group of tumors among clear cell renal cell carcinomas characterized by hotspot mutations in TCEB1 (a gene that contributes to the VHL complex to ubiquitinate hypoxia-inducible factor). We analyzed 11 tumors from two distinct cohorts with TCEB1 mutations along with an expanded cohort to assess whether these should be considered an entity distinct from clear cell renal cell carcinoma and clear cell papillary renal cell carcinoma. All tumors were characterized by hotspot mutations in TCEB1 Y79C/S/F/N or A100P. Morphological and immunohistochemical characteristics of the tumors were assessed by two experienced genitourinary pathologists. Clinical and pathological variables, copy number alterations, mutations, and expression signatures were compared with a cohort of TCEB1 wild-type tumors. All TCEB1-mutated tumors were VHL and PBRM1 wild type and contained distinct copy number profiles including loss of heterozygosity of chromosome 8, the location of TCEB1 (8q21.11). All tumors lacked the clear cell renal cell carcinoma signature 3p loss and contained distinct gene expression signatures. None of the clear cell papillary tumors harbored TCEB1 mutations. Pathologically, all TCEB1-mutated tumors shared characteristic features including thick fibromuscular bands transecting the tumor, pure clear cell cytology frequently with cells showing voluminous cytoplasm, and clear cell renal cell carcinoma-like acinar areas associated with infolding tubular and focally papillary architecture. The presence of voluminous cytoplasm, absence of luminal polarization of tumor nuclei, and lack of extensive cup-like distribution of carbonic anhydrase-IX expression distinguish it from clear cell papillary carcinoma. None of the patients developed metastases at last follow-up (median 48 months). In sum, TCEB1-mutated renal cell carcinoma is a distinct entity with recurrent hotspot mutations, specific copy number alterations, pathway activation, and characteristic morphological features. Further clinical follow-up is needed to determine whether these tumors are more indolent compared with the conventional clear cell renal cell carcinoma.

Radiogenomics of clear cell renal cell carcinoma: associations between CT imaging features and mutations.

PURPOSE: To investigate associations between computed tomographic (CT) features of clear cell renal cell carcinoma (RCC) and mutations in VHL, PBRM1, SETD2, KDM5C, or BAP1 genes. MATERIALS AND METHODS: The institutional review board approved this retrospective, hypothesis-generating study of 233 patients with clear cell RCC and waived the informed consent requirement. The study was HIPAA compliant. Three radiologists independently reviewed pretreatment CT images of all clear cell RCCs without knowledge of their genomic profile. One radiologist measured largest diameter and enhancement parameters of each clear cell RCC. Associations between CT features and mutations in VHL, PBRM1, SETD2, KDM5C, and BAP1 genes were tested by using the Fisher exact test. Associations between mutations and size and enhancement were assessed by using the independent t test. Interreader agreement was calculated by using the Fleiss κ. RESULTS: Mutation frequencies among clear cell RCCs were as follows: VHL, 53.2% (124 of 233); PBRM1, 28.8% (67 of 233); SETD2, 7.3% (17 of 233); KDM5C, 6.9% (16 of 233); and BAP1, 6.0% (14 of 233). Mutations of VHL were significantly associated with well-defined tumor margins (P = .013), nodular tumor enhancement (P = .021), and gross appearance of intratumoral vascularity (P = .018). Mutations of KDM5C and BAP1 were significantly associated with evidence of renal vein invasion (P = .022 and .046, respectively). The genotype of solid clear cell RCC differed significantly from the genotype of multicystic clear cell RCC. While mutations of SETD2, KDM5C, and BAP1 were absent in multicystic clear cell RCC, mutations of VHL (P = .016) and PBRM1 (P = .017) were significantly more common among solid clear cell RCC. Interreader agreement for CT feature assessments ranged from substantial to excellent (κ = 0.791-0.912). CONCLUSION: This preliminary radiogenomics analysis of clear cell RCC revealed associations between CT features and underlying mutations that warrant further investigation and validation.