A Consensus Molecular Classification of Muscle-invasive Bladder Cancer

Background: Muscle-invasive bladder cancer (MIBC) is a molecularly diverse disease with heterogeneous clinical outcomes. Several molecular classifications have been proposed, but the diversity of their subtype sets impedes their clinical application. Objective: To achieve an international consensus on MIBC molecular subtypes that reconciles the published classification schemes. Design, setting, and participants: We used 1750 MIBC transcriptomic profiles from 16 published datasets and two additional cohorts. Outcome measurements and statistical analysis: We performed a network-based analysis of six independent MIBC classification systems to identify a consensus set of molecular classes. Association with survival was assessed using multivariable Cox models. Results and limitations: We report the results of an international effort to reach a consensus on MIBC molecular subtypes. We identified a consensus set of six molecular classes: luminal papillary (24%), luminal nonspecified (8%), luminal unstable (15%), stroma-rich (15%), basal/squamous (35%), and neuroendocrine-like (3%). These consensus classes differ regarding underlying oncogenic mechanisms, infiltration by immune and stromal cells, and histological and clinical characteristics, including outcomes. We provide a single-sample classifier that assigns a consensus class label to a tumor sample's transcriptome. Limitations of the work are retrospective clinical data collection and a lack of complete information regarding patient treatment. Conclusions: This consensus system offers a robust framework that will enable testing and validation of predictive biomarkers in future prospective clinical trials. Patient summary: Bladder cancers are heterogeneous at the molecular level, and scientists have proposed several classifications into sets of molecular classes. While these classifications may be useful to stratify patients for prognosis or response to treatment, a consensus classification would facilitate the clinical use of molecular classes. Conducted by multidisciplinary expert teams in the field, this study proposes such a consensus and provides a tool for applying the consensus classification in the clinical setting. An international consortium of bladder cancer expert teams establishes a consensus reconciling the diverse molecular classifications of muscle-invasive bladder cancer. This work offers a robust framework that will enable testing and validating predictive biomarkers in future prospective clinical trials

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Perfis de atividade de regulon : inferência e aplicações

Orientador: Prof. Dr. Mauro Antônio Alves Castro.Dissertação (mestrado) - Universidade Federal do Paraná, Setor de Educação Profissional e Tecnológica, Programa de Pós-Graduação em Bioinformática. Defesa : Curitiba, 26/03/2019.Inclui referências: p. 63-67.Resumo: O entendimento do câncer é um dos desafios cruciais da ciência atual. Em busca desta compreensão, governos e institutos em todo o mundo estão gerando uma abundância de dados biológicos. Dentre os tipos de dados, transcriptomas se provaram úteis, mas de interpretação complicada. Redes transcricionais provêm uma estrutura para organizar os dados transcricionais ao redor de elementos regulatórios. Neste trabalho, descrevo um método para inferir estados regulatórios de uma coorte a partir de dados de transcriptoma. Proponho perfis de atividade de regulon (RAPs), implementados no pacote de linguagem R RTNsurvival. O RTNsurvival já foi utilizado para criar modelos de desfecho de paciente em câncer de mama e fígado, avaliar subtipos de câncer de bexiga, e entender efeitos de interações entre co-reguladores de alvos em variáveis como sobrevida. Finalmente, eu demonstro como atividade de regulon reflete acessibilidade de cromatina em pontos distais, potencialmente enhancer, de alvos positivos e negativos de regulons em câncer de mama. Perfis de atividade de regulon estão se provando uma importante ferramenta estatística para comparação de estados regulatórios de amostras em uma coorte.Abstract: Understading cancer is one of the crucial challenges of contemporary science. In pursuit of this understanding, governments and institutions around the world are generating a wealth of biological data. Among the datatypes, transcriptomes have proven themselves useful, but are many times unwieldy to interpret. Transcriptional networks provide a framework to organize the transcriptional data around regulatory elements. In this work, I describe a method to infer regulatory state of a cohort from transcriptome data. I propose regulon activity profiles (RAPs), implemented in the R language package RTNsurvival. RTNsurvival has been used to make models of patient outcome in breast and liver cancer, evaluate bladder cancer subtyping results, and understand interactions effects between co-regulators of targets on a variable like survival. I also demonstrate how regulon activity reflects chromatin accessibility at distal, enhancer points of regulon positive and negative targets in breast cancer. Regulon activity profiles are proving to be an important statistical tool in comparing regulatory states of samples within a cohort

Paysages moléculaires du cancer de la vessie : études multiomiques de l'hétérogénéité des tumeurs de la vessie en tant que biomarqueurs de réponse au traitement

Le cancer de la vessie est le 10ème cancer le plus diagnostiqué dans le monde, avec ~75 % des cas se présentant comme une tumeur de vessie n’infiltrant pas le muscle (TVNIM) et environ 25 % comme infiltrant le muscle (TVIM). Maladie agressive, le TVIM a un taux de survie à 5 ans de 60%. Bien que les tumeurs de la vessie présentent une hétérogénéité génétique et phénotypique significative, les options thérapeutiques actuelles ne sont pas suffisamment adaptées à leurs vulnérabilités spécifiques. Afin d’atteindre une médecine stratifiée, je présente ici trois études établissant un lien entre les données moléculaires et l’action thérapeutique par la poursuite et la validation des biomarqueurs:- Les options thérapeutiques évoluent rapidement avec l'apparition de thérapies ciblées et d'inhibiteurs de points de contrôle immunitaire (immunothérapies). L’hypothèse émise est que la présence de structures lymphoïdes tertiaires (SLT) pourrait être un biomarqueur spécifique de la réponse à l'immunothérapie dans le cancer de la vessie, tels d'autres cancers, et que cela pourrait être étudié à l'aide d'un proxy moléculaire. Nous avons découvert que l'expression du gène CXCL13 est un biomarqueur fort des TLS et prédit la réponse à l'immunothérapie dans le cadre adjuvant dans le TVIM à un stade avancé. Combinés à d'autres biomarqueurs proposés, TLS/CXCL13 sont des prédicteurs utiles dans ce contexte.- Le FGFR3, un des gènes mutés les plus courants dans les TVNIM, est également présent dans environ 10 à 15 % des TVIM. Le ciblage avec l'inhibiteur du FGFR erdafitinib a un taux de réponse d'environ 40 %, mais les tumeurs deviennent souvent résistantes. Ayant intégré des données provenant d'altérations du nombre de copies, transcriptomique et protéomique des TVNIM et TVIM, nous avons découvert que les patients présentant des altérations du FGFR3 avaient une expression plus élevée de protéines clés de l'apoptose et que les modèles précliniques ayant des altérations du FGFR3 étaient très sensibles à l'apoptos, qui pouvait être inversée par la régulation négative du FGFR3 et potentialisée par birinapant. Cette analyse, débutée par l'exploration de données multimodales, a abouti à une étude préclinique d'une nouvelle option thérapeutique pour les tumeurs FGFR3 mutées- La chimiothérapie néoadjuvante (NAC) est le traitement de référence pour le TVIM mais ne profite qu'à 1/3 des patients qui la reçoivent. Les classes moléculaires basées sur la transcriptomique ont été associées précédemment à la réponse à la NAC, mais les études ont des conclusions contradictoires : des études antérieures ont affirmé que les tumeurs de type basal sont plus sensibles à la NAC, tandis que certaines études récentes soutiennent que les tumeurs de type basal ont de pire résultats après la NAC. Nous avons exploité des échantillons de 293 patients de la cohorte VESPER, qui ont été colorés avec H&E et des marqueurs immunohistochimiques pour détecter l'hétérogénéité spatiale intra-tumorale. Une fois trouvée, plusieurs échantillons ont été prélevés. Après l'extraction d'ARN et le séquençage des résections (TURBT), nous avons classé les patients en une classe moléculaire de TVIM. Ceux atteints de tumeurs de tumeurs hétérogènes peuvent soit avoir une molécule spatialement conservée, soit être de type « Mixte » (45,8% dans les tumeurs hétérogènes). Nous avons constaté que les patients de type basal et mixte ont une réponse pathologique et des résultats plus mauvais. Nous confirmons les résultats d'études récentes et identifions l'hétérogénéité intra-tumorale comme une caractéristique importante à considérer dans ce contexte.Grâce à des collaborations étroites avec des cliniciens, des statisticiens et des biologistes moléculaires, et à l'intégration de données omiques privées et publiques, ma thèse a contribué à la découverte de biomarqueurs des thérapies existantes et a introduit une nouvelle option de traitement pour un sous-groupe de patients atteints de cancer de la vessie.Bladder cancer is the 10th most commonly diagnosed cancer in the world, with ~75% of cases presenting as non-muscle invasive (NMIBC) and ~25% as muscle-invasive bladder cancer (MIBC). MIBC is an aggressive disease currently with 60% survival rates at 5 years. Although bladder tumors exhibit significant genetic and phenotypic heterogeneity, current therapeutic options are not sufficiently tailored for their distinctive vulnerabilities. Further investigation and validation of biomarkers are necessary to achieve the goal of stratified medicine. Here, I present three studies linking molecular data to treatment action.• The treatment landscape for MIBCs particularly is rapidly evolving with emerging targeted therapies and immune checkpoint inhibitors (immunotherapies). We hypothesized that the presence of tertiary lymphoid structures (TLS) could be a specific biomarker of immunotherapy response in bladder cancer like found in other cancer types, and that this could be investigated using a proxy molecular marker. After testing TLS markers, we found that CXCL13 gene expression is a strong biomarker of TLS and predicts response to immunotherapy in the adjuvant setting in advanced-stage MIBC. Combined with other biomarkers proposed, TLS/CXCL13 are useful predictors in this setting.• FGFR3 is one of the most common mutated genes in NMIBC but also present in ~10-15% of MIBC. Targeting with FGFR inhibitor erdafitinib has a response rate of ~40% with tumors often becoming resistant. We integrated data from copy number alterations, transcriptomics and proteomics data from a set of bladder tumors and found that patients harboring FGFR3 alterations had higher expression of key proteins of apoptosis, something not detected using transcriptomics alone, and that pre-clinical with FGFR3 alterations models were highly sensitive to TRAIL-induced apoptosis, which could be reversed by FGFR3 downregulation and potentialized by XIAP inhibitor birinapant. This analysis started from data mining of multi-modal data and culminated in a pre-clinical study of a new treatment option for FGFR3 mutated tumors.• Neo-adjuvant chemotherapy (NAC) is the gold standard treatment for MIBC, but it only benefits 1/3 of patients who receive it. Transcriptomic-based molecular classes were previously associated with NAC response, but studies report conflicting conclusions: earlier studies claimed basal-like tumors are more sensitive to NAC while some recent studies assert basal-like have the worse outcomes after NAC. Though these conclusions are not mutually exclusive, they suggest distinct clinical paths. In my thesis, we leveraged samples from 293 patients enrolled in the VESPER prospective cohort, which were stained with H&E and immunohistochemical markers to detect spatial intra-tumor heterogeneity. When heterogeneity was found, multiple samples were taken. After RNA extraction and sequencing of the resections (TURBTs), we classified the patients into a consensus molecular class of MIBC. Patients with heterogeneous tumors could either have a spatially conserved molecular or be of “Mixed” subtype (45.8% within heterogeneous). We found that basal-like and mixed subtype patients have worse pathological response and outcomes, particularly for those mixed where one area is basal-like. We confirm the findings of recent studies and identify intra-tumor heterogeneity as an important feature to consider in this context.Through close collaborations with clinicians, statisticians, and molecular biologists, and the integration of private and public omics data, my thesis contributes to the discovery of biomarkers of existing therapies and introduces a novel treatment option for a subgroup of bladder cancer patients

RTNduals an R/Bioconductor package for analysis of co-regulation and inference of dual regulons

MOTIVATION: Transcription factors (TFs) are key regulators of gene expression, and can activate or repress multiple target genes, forming regulatory units, or regulons. Understanding downstream effects of these regulators includes evaluating how TFs cooperate or compete within regulatory networks. Here we present RTNduals, an R/Bioconductor package that implements a general method for analyzing pairs of regulons. RESULTS: RTNduals identifies a dual regulon when the number of targets shared between a pair of regulators is statistically significant. The package extends the RTN (Reconstruction of Transcriptional Networks) package, and uses RTN transcriptional networks to identify significant co-regulatory associations between regulons. The Supplementary Information reports two case studies for TFs using the METABRIC and TCGA breast cancer cohorts. AVAILABILITY AND IMPLEMENTATION: RTNduals is written in the R language, and is available from the Bioconductor project at http://bioconductor.org/packages/RTNduals/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online

Novel lncRNAs Co-Expression Networks Identifies LINC00504 with Oncogenic Role in Luminal A Breast Cancer Cells

Long non-coding RNAs (lncRNAs) are functional transcripts with more than 200 nucleotides. These molecules exhibit great regulatory capacity and may act at different levels of gene expression regulation. Despite this regulatory versatility, the biology of these molecules is still poorly understood. Computational approaches are being increasingly used to elucidate biological mechanisms in which these lncRNAs may be involved. Co-expression networks can serve as great allies in elucidating the possible regulatory contexts in which these molecules are involved. Herein, we propose the use of the pipeline deposited in the RTN package to build lncRNAs co-expression networks using TCGA breast cancer (BC) cohort data. Worldwide, BC is the most common cancer in women and has great molecular heterogeneity. We identified an enriched co-expression network for the validation of relevant cell processes in the context of BC, including LINC00504. This lncRNA has increased expression in luminal subtype A samples, and is associated with prognosis in basal-like subtype. Silencing this lncRNA in luminal A cell lines resulted in decreased cell viability and colony formation. These results highlight the relevance of the proposed method for the identification of lncRNAs in specific biological contexts

Transcriptomic Profiling of Upper Tract Urothelial Carcinoma: Bladder Cancer Consensus Classification Relevance, Molecular Heterogeneity, and Differential Immune Signatures

International audienceAnalyses of large transcriptomics data sets of muscle-invasive bladder cancer (MIBC) have led to a consensus classification. Molecular subtypes of upper tract urothelial carcinomas (UTUCs) are less known. Our objective was to determine the relevance of the consensus classification in UTUCs by characterizing a novel cohort of surgically treated ≥pT1 tumors. Using immunohistochemistry (IHC), subtype markers GATA3-CK5/6-TUBB2B in multiplex, CK20, p16, Ki67, mismatch repair system proteins, and PD-L1 were evaluated. Heterogeneity was assessed morphologically and/or with subtype IHC. FGFR3 mutations were identified by pyrosequencing. We performed 3'RNA sequencing of each tumor, with multisampling in heterogeneous cases. Consensus classes, unsupervised groups, and microenvironment cell abundance were determined using gene expression. Most of the 66 patients were men (77.3%), with pT1 (n = 23, 34.8%) or pT2-4 stage UTUC (n = 43, 65.2%). FGFR3 mutations and mismatch repair-deficient status were identified in 40% and 4.7% of cases, respectively. Consensus subtypes robustly classified UTUCs and reflected intrinsic subgroups. All pT1 tumors were classified as luminal papillary (LumP). Combining our consensus classification results with those of previously published UTUC cohorts, LumP tumors represented 57.2% of ≥pT2 UTUCs, which was significantly higher than MIBCs. Ten patients (15.2%) harbored areas of distinct subtypes. Consensus classes were associated with FGFR3 mutations, stage, morphology, and IHC. The majority of LumP tumors were characterized by low immune infiltration and PD-L1 expression, in particular, if FGFR3 mutated. Our study shows that MIBC consensus classification robustly classified UTUCs and highlighted intratumoral molecular heterogeneity. The proportion of LumP was significantly higher in UTUCs than in MIBCs. Most LumP tumors showed low immune infiltration and PD-L1 expression and high proportion of FGFR3 mutations. These findings suggest differential response to novel therapies between patients with UTUC and those with MIBC. Copyrigh

PPARγ is a tumor suppressor in basal bladder tumors offering new potential therapeutic opportunities

PPARactivation is a critical event in luminal muscle-invasive bladder cancer (MIBC) tumorigenesis, favoring both tumor cell growth and microenvironment modulation toward tumor immune escape. Conversely, the down-regulation of PPARactivity in basal MIBC suggests tumor suppressive effects in this subgroup. Here, we report genetic, epigenetic and functional evidence to support the tumor suppressor role for PPAR in basal bladder tumors. We identified hemizygous deletions, DNA hyper-methylation and loss-of-function mutations of PPARin basal MIBC, associated with PPAR under-expression and its decreased activity. Re-expression of PPARin basal tumor cells resulted in the activation of PPAR-dependent transcription program that modulated fatty acid metabolism and cell differentiation and decreased cell growth, which could partly rely on EGFR down-regulation. Structure-function studies of two PPAR mutant revealed a destabilization of a region important for coactivator recruitment and should help develop potent molecules to activate PPAR as a therapeutic strategy for basal MIBC. The identification of this subtype-dependent dual role of PPAR in MIBC strengthens the critical role of PPAR in bladder tumorigenesis and reinforces the interest in stratified medicine based on tumor molecular subtyping