Classification Of Muscle-invasive Bladder Cancer Research Articles

Molecular classification of muscle-invasive bladder cancer based on a simplified immunohistochemical panel using GATA3, CK5/6 and p16.

The choice of therapy for muscle-invasive bladder cancer (MIBC) could be influenced by the tumor's molecular subtype. Currently, well-defined consensus subtypes are based on tumor microarray mRNA data. Clearly defined and easy-to-use surrogate molecular subtypes, based on immunohistochemistry (IHC) performed on whole slides, are needed to make subtyping cost-effective and useful in routine work and future research. To aid in the development of a simple immunohistochemical classifier, a retrospective single-center series of 92 cases of localized bladder cancer was identified. Routine IHC for GATA3, cytokeratins 5 and 6 (CK5/6), and p16 was performed on whole tissue blocks containing muscle-invasive disease. Electronic medical records were retrieved and searched for clinical variables, treatment, and survival data. The mean age was 69.6 years, and 73% were males. Conservative treatment was used in 55% of cases, while cystectomy with chemotherapy was used in 45%. GATA3 and CK5/6 expression divided cases into broad luminal and basal subtypes, respectively, while p16 expression was used to subclassify luminal cases into luminal papillary and luminal unstable types according to the consensus molecular classification. When subtyped in this way, GATA3 and CK5/6 negative cases showed worse overall survival. Molecular subtyping of MIBC on whole slides containing muscle-invasive tumor using only three commonly used, consensus-based antibodies, is a feasible and cost-effective method for detecting subtypes of invasive bladder cancer. Future work combining morphological analysis and IHC is needed to fully translate the consensus molecular classification into a comprehensive, cost-effective subtyping strategy.

Multilayer spectrum of intratumoral heterogeneity in basal bladder cancer.

Basal/squamous (Ba/Sq) subtype represents an intrinsic and robust group in the consensus molecular classification of muscle-invasive bladder cancer (MIBC), with poor outcome and controversial chemosensitivity. We aimed to investigate the spectrum of intratumor heterogeneity (ITH) in the Ba/Sq subtype. First, we validated a 29-gene NanoString CodeSet to predict the Ba/Sq subtype for FFPE samples. We identified heterogeneous Ba/Sq tumors in a series of 331 MIBC FFPE samples using dual GATA3/KRT5/6 immunohistochemistry (IHC). Heterogeneous regions with distinct immunostaining patterns were studied separately for gene expression using the 29-gene CodeSet, for mutations by targeted next-generation sequencing, and for copy number alteration (CNA) by microarray hybridization. Among 83 Ba/Sq tumors identified by GATA3/KRT5/6 dual staining, 19 tumors showed heterogeneity at the IHC level. In one third of the 19 cases, regions from the same tumor were classified in different distinct molecular subtypes. The mutational and CNA profiles confirmed the same clonal origin for IHC heterogeneous regions with possible subclonal evolution. Overall, two patterns of intratumoral heterogeneity (ITH) were observed in Ba/Sq tumors: low ITH (regions with distinct immunostaining, but common molecular subtype and shared CNA) or high ITH (regions with distinct immunostaining, molecular subtype, and CNA). These results showed multilayer heterogeneity in Ba/Sq MIBC. In view of personalized medicine, this heterogeneity adds complexity and should be taken into account for sampling procedures used for diagnosis and treatment choice. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Abstract 2476: The oncogenic role of p63 in altered-FGFR3 bladder tumors

Abstract Recently, a new classification of muscle-invasive bladder cancer (MIBCs) has been proposed (Kamoun, 2020). There are 6 classes of MIBC: the over-expression or activating mutations in Fibroblast Growth Factor Receptor 3 (FGFR3) are associated with luminal subtypes. As typically expected, basal tumors exhibit increased TP63 activity, but interestingly, this is also observed in luminal-papillary (LumP) tumors. LumP are enriched in both, FGFR3 mutations and p63 activation. The regulatory gene pathways and p63 functional role in these tumors remains poorly characterized. Therefore, this is our main objective, together with the study of the link with FGFR3 mutations. We hypothesize that p63 drives a protumoral program in bladder cancer (BC) commanded by the regulation of altered FGFR3. We used human BC cell lines bearing FGFR3 mutations with inducible silencing for p63 in order to identify the functional role of p63 in this context. Loss of p63 led to a decreased 2D and 3D growth (p<0.001). Gelatin degradation and migration ability were significantly lower in p63 depleted cells compared to CRL (p<0,01). In vivo assays showed that tumor volume was significantly lower in p63 depleted tumors compared to CRL (p<0.01), accompanied by a reduction of proliferating cell nuclear antigen (PCNA) expression, indicating that p63 is an essential protein through which the cell mediates its proliferative and migratory mechanism. In order to study the link between both, FGFR3 alterations and p63 activity, we used FGFR3 Knocked down cells and pharmacological inhibitors. FGFR3 inhibition decreased p63 expression associated with a reduction in TP63 activity. It reduced proliferation and cell migration in similar level to those observed by directly silencing p63 in FGFR3 altered cells. Conversely, FGFR3 wt cells were not affected by the inhibitor. Overall, these results show that FGFR3 inhibition blunted the carcinogenic program carried out by p63, suggesting there is a regulation of FGFR3 over p63. Supporting the regulation of p63 by FGFR3, analysis of mRNA levels in human bladder tumors showed that p63 expression was significantly higher in FGFR3 mutated tumors in two different sets of samples. In summary, we experimentally demonstrated the regulation of FGFR3 over the protumoral transcription factor p63 that induces proliferation, migration and tumor growth in bladder cancer. Citation Format: Macarena Zambrano, Aura I. Moreno-Vega, Florent Dufour, Marianela Sciacca, Yanina V. Langle, François Radvanyi, Ana M. Eiján, Isabelle Bernard-Pierrot, Catalina Lodillinsky. The oncogenic role of p63 in altered-FGFR3 bladder tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2476.

Vesical Imaging-Reporting and Data System (VI-RADS®): Initial experience in the classification of muscle-invasive bladder cancer

ObjectiveTo analyze the diagnostic accuracy of the VI-RADS® system in the differentiation of non-muscle-invasive bladder tumors (NMIBT) from muscle-invasive bladder tumors (MIBT) in suspicious cystoscopic findings without prior transurethral resection (TUR) evaluated by radiologists with no prior experience in its use. Material and methodsRetrospective study carried out with 18 patients with suspicious lesions in cystoscopy. All of them underwent MRI of the bladder. Two radiologists with no prior experience in the use of the VI-RADS® system evaluated the results. All patients underwent TUR of the suspicious lesions after MRI. The sensitivity and specificity of the system were analyzed for VI-RADS® values ​​≥3 or VI-RADS® ≥4, as well as the Cohen’s kappa coefficient between both radiologists. ResultsThe mean values ​​of sensitivity and specificity of both radiologists considering both the VI-RADS® ≥3 or VI-RADS® ≥4 values were 91.7% and 87.5%, respectively. The kappa coefficient considering the VI-RADS® ≥3 as positive, was 0.551 (P < .05), while considering the VI-RADS® ≥4 as positive, it was 0.571 (P < .05). ConclusionThe VI-RADS® system presents excellent sensitivity ​​ (91.7%) and specificity (87.5%) values in the classification of MIBT performed by radiologists with no prior experience in its use, with a moderate interobserver agreement.

Integrative multi-omics analysis of muscle-invasive bladder cancer identifies prognostic biomarkers for frontline chemotherapy and immunotherapy

Only a subgroup of patients with muscle-invasive bladder cancer (MIBC) are responders toward cisplatin-based chemotherapy and PD-L1 blockade immunotherapy. There is a clinical need to identify MIBC molecular subtypes and biomarkers for patient stratification toward the therapies. Here, we performed an integrative clustering analysis of 388 MIBC samples with multi-omics data and identified basal and luminal/differentiated integrative subtypes and derived a 42 gene panel for classification of MIBC. Using nine additional gene expression data (n = 844), we demonstrated the prognostic value of the 42 basal-luminal genes. The basal subtype was associated with worse overall survival in patients receiving no neoadjuvant chemotherapy (NAC), but better overall survival in patients receiving NAC in two clinical trials. Each of the subtypes could be further divided into chr9 p21.3 normal or loss subgroup. The patients with low expression of MTAP/CDKN2A/2B (indicative of chr9 p21.3 loss) had a significantly lower response rate to anti-PD-L1 immunotherapy and worse survival than the patients with high expression of MTAP/CDKN2A/2B. This integrative analysis reveals intrinsic MIBC subtypes and biomarkers with prognostic value for the frontline therapies.

Classification of Muscle-Invasive Bladder Cancer Based on Immunogenomic Profiling.

There is a significant heterogeneity in the immunotherapeutic responsiveness of each muscle-invasive bladder cancer (MIBC) patient. In our research, we aimed to identify a novel classification of MIBC based on immunogenomic profiling that may facilitate the reasonable stratification of prognosis and response to immunotherapy. The single-sample gene-set enrichment analysis (ssGSEA) was used to analyze the RNA-seq data of 29 important immune signatures from TCGA. Unsupervised hierarchical clustering was performed to identify an immunogenomic classification of MIBC. Then, we assessed the features of the classification in prognosis, immune infiltration, tumor-infiltration lymphocytes, HLA genes, and PD-L1 expression level. A total of 399 MIBC samples were included and three subtypes named Immunity_High, Immunity_Medium, and Immunity_Low were identified. The Immunity_High had a significant advantage in overall survival over the Immunity_Medium and Immunity_Low (p = 0.046 and p = 0.024). From Immunity_Low to Immunity_High, immune cell infiltration and stromal content showed an upward trend (p < 0.001). Meanwhile, Immunity_High was associated with a significantly higher proportion of TILs including dendritic cells resting, macrophages M1, mast cells resting, T cells CD4 memory activated, and T cells CD8+. And the expression levels of all HLA genes and PD-L1 of Immunity_High were the highest, consistent (p < 0.001). Two hundred ninety eight MIBC patients treated with immunotherapy from the IMvigor210 were included to form an independent validation cohort to verify the robustness of immunogenomic classification and the ability to predict the response to immunotherapy. This classification had potential clinical implications for predicting prognosis and immunotherapeutic responsiveness of MIBC patients.

A Consensus Molecular Classification of Muscle-invasive Bladder Cancer

A Consensus Molecular Classification of Muscle-invasive Bladder Cancer

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.

Biological molecular layer classification of muscle-invasive bladder cancer opens new treatment opportunities

BackgroundMuscle-invasive bladder tumors are associated with a high risk of relapse and metastasis even after neoadjuvant chemotherapy and radical cystectomy. Therefore, further therapeutic options are needed and molecular characterization of the disease may help to identify new targets. The aim of this study was to characterize muscle-invasive bladder tumors at the molecular level using computational analyses.MethodsThe TCGA cohort of muscle-invasive bladder cancer patients was used to describe these tumors. Probabilistic graphical models, layer analyses based on sparse k-means coupled with Consensus Cluster, and Flux Balance Analysis were applied to characterize muscle-invasive bladder tumors at a functional level.ResultsLuminal and Basal groups were identified, and an immune molecular layer with independent value was also described. Luminal tumors showed decreased activity in the nodes of epidermis development and extracellular matrix, and increased activity in the node of steroid metabolism leading to a higher expression of the androgen receptor. This fact points to the androgen receptor as a therapeutic target in this group. Basal tumors were highly proliferative according to Flux Balance Analysis, which makes these tumors good candidates for neoadjuvant chemotherapy. The Immune-high group showed a higher degree of expression of immune biomarkers, suggesting that this group may benefit from immune therapy.ConclusionsOur approach, based on layer analyses, established a Luminal group candidate for therapy with androgen receptor inhibitors, a proliferative Basal group which seems to be a good candidate for chemotherapy, and an immune-high group candidate for immunotherapy.

A Consensus Molecular Classification of Muscle-Invasive Bladder Cancer

Muscle-Invasive Bladder Cancer (MIBC) is a molecularly diverse disease with heterogeneous clinical outcomes. Several molecular classifications have been proposed, yielding diverse sets of subtypes. This diversity hampers the clinical application of such knowledge. Here, we report the results of a large international effort to reach a consensus on MIBC molecular subtypes. Using 1750 MIBC transcriptomes and a network-based analysis of six independent MIBC classification systems, we identified a consensus set of six molecular classes: Luminal Papillary (24%), Luminal Non-Specified (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. This consensus system offers a robust framework that will enable testing and validating predictive biomarkers in future clinical trials.

Molecular tumor heterogeneity in muscle invasive bladder cancer: Biomarkers, subtypes, and implications for therapy.

Despite years of slow progress, muscle invasive bladder cancer (MIBC) is finally entering the era of molecularly guided targeted therapy. However, tumor heterogeneity is high in MIBC and may impact treatment response and resistance. The objective of this review is to dissect recent insights into inter- and intratumor heterogeneity (ITH) in MIBC, with emphasis on the clinical implications of this heterogeneity for biomarker-driven strategies and the development of new therapies. A nonsystematic review was performed in PubMed and EMBASE using the terms "tumor heterogeneity" and "bladder cancer." Intertumor heterogeneity, as reflected by different clinical phenotypes in different patients, has been partially explained with next generation sequencing and other molecular profiling technologies. RNA-based molecular subtyping, for example, provides a classification of MIBC into distinct categories that can be used for further molecular analysis, biomarker discovery, risk stratification, and treatment selection. Molecular subtyping and specific genomic alterations, especially in DNA damage repair genes, may help explain why some patients respond better to systemic chemotherapy and immunotherapy. Conversely, spatial and temporal ITH threaten to confound attempts to target specific molecular lesions since not all tumor cells within a patient may carry the relevant lesion. Improved understanding and management of ITH is required for the most effective use of biomarker-driven targeted therapies. Strategies to assess and overcome intertumor and ITH in MIBC will be critical steps toward realizing the objectives of precision oncology. Novel techniques such as analysis of circulating tumor DNA and single cell sequencing are likely to revolutionize our understanding of tumor heterogeneity.

PO-509 Novel molecular classification of muscle-invasive bladder cancer opens new treatment opportunities

IntroductionMuscle-invasive bladder cancer (MIBC) is associated with a high risk of relapse and metastasis. The standard treatment consists of radical cystectomy followed by cisplatin-based chemotherapy. Therefore, the molecular characterisation of MIBC in order to find new therapeutic targets is still necessary.Material and methodsIn this study, using The Cancer Genome Atlas (TCGA) cohort, functional structure and layer analyses were done to characterise MIBC tumours at functional level. Functional structure was developed using probabilistic graphical models and gene ontology analyses. Layer analyses were based on sparse k-means and Consensus Cluster Algorithm (CCA). Sparse k-means assigns a weight to each variable, based on its relevance in the sample classification. Then, a CCA using variables that were selected by the sparse K-means method was applied to define the optimum number of groups for each case. This sparse k-means-CCA workflow was performed successive times to explore the existence of independent informative molecular layers. Once relevant genes for one molecular layer are identified, these genes were removed from the dataset and the sparse k-means-CCA workflow was performed again, allowing the identification of different layers of molecular information and establishing different classifications based on various molecular features. Gene ontology analyses were done for each layer.Results and discussionsA probabilistic graphical model with a functional structure was obtained. Sixteen layers were functionally characterised and the first three layers were analysed in depth. The first layer defined luminal and basal groups. Luminal tumours had a better prognosis than basal ones. Strikingly, luminal tumours showed higher expression of the androgen receptor, suggesting that this group is a good candidate for receiving treatment with androgen receptor inhibitors. The second layer established two groups based on genes related to extracellular matrix function. Lastly, the third layer defined two groups on the basis of immune information. The immune high group had a better prognosis and was characterised by higher expression of CTLA4 and PDL1, so this group may be a candidate for receiving immunotherapy.ConclusionUsing probabilistic graphical models and layer analyses it is possible to establish independently a molecular and an immune classification. Using these approaches, we also proposed a specific treatment for luminal tumours (androgen receptor inhibitors) and for immune high tumours (immunotherapy).

Prognostic classification of muscle-invasive bladder cancer (MIBC) following radical cystectomy (RC) alone or with perioperative chemotherapy: Analysis of the U.S. National Cancer Database (NCDB).

372 Background: While the role of adjuvant therapy for MIBC is being evaluated in randomized trials of patients (pts) following RC with or without neoadjuvant chemotherapy (NC), pts receiving adjuvant chemotherapy (AC) have been excluded. Understanding the prognosis of a broad spectrum of pts undergoing RC with or without perioperative chemotherapy may aid patient selection, stratification and interpretation of nonrandomized data. We conducted a retrospective analysis of the NCDB to construct a comprehensive prognostic nomogram in this broad population. Methods: Data from NCDB was obtained for all patients diagnosed with clinical (c)-T2-T4aN0M0 urothelial carcinoma of bladder who underwent RC in the US from 2004-2013. Those who underwent RC &gt; 6 months (mo) after initiating NC, received AC &gt; 4 mo after RC were excluded to allow only those receiving optimal therapy. Pts with unknown race and number of lymph nodes (LNs) examined were also excluded. Multivariate analyses were conducted to determine the impact on survival of: clinical stage, pathologic (p)-stage, treatment group (RC alone, NC, AC), age, year of diagnosis, Charlson Comorbidity Index (CCI), race, number of examined LNs examined and sex. Results: A total of 10,256 pts were evaluable for analysis: 6864 in the RC group, 1380 in the AC group and 2012 in the NC group. On multivariate analysis, the following variables were significantly (p &lt; 0.001) associated with survival: treatment group, age ( &lt; 65 vs. ≥ 65 years), CCI, p-stage and number of examined LNs. Calibration was performed and the c-index was 0.79 (95% CI: 0.76-0.81). Limitations of a retrospective analysis apply. Conclusions: A comprehensive prognostic nomogram to predict the survival of pts with MIBC undergoing RC with or without NC or AC was proposed. The estimation of residual risk of mortality after RC alone or with AC or NC aids optimal stratification of pts in more inclusive randomized trials of subsequent adjuvant therapy. The nomogram also highlights the impact of comorbidities and age, and helps interpret non-randomized data evaluating perioperative regimens.