Molecular classification of nonurothelial histologic subtypes of bladder cancer.

INTRODUCTION AND OBJECTIVE: Molecular subtypes have been suggested to predict outcome in patients with muscle invasive bladder cancer (MIBC). The major subtypes reported have been “luminal” and “basal”; however, there is little consensus regarding subtype-signatures and the clinical parameters used to evaluate outcome. The objective of this study was to examine using multiple datasets, if molecular subtypes are distinct entities within MIBC and whether they predict clinical outcome. METHODS: We analyzed transcriptome data from all 402 MIBC samples in The Cancer Genome Atlas (TCGA) and 151 high-grade MIBC samples from three datasets in Oncomine™. Transcript levels were also measured in 52 bladder tumor specimens with clinical follow-up (cohort-1). Samples were subtype-scored using gene panels (GP-11, GP-30, BASQ) consisting only of those genes common among published studies. Correlation of subtypes to recurrence/progression-free survival (R/PFS), metastasis, cancer-specific survival (CSS) and overall-survival (OS) was examined by logistic regression, Cox proportional Hazards model and Kaplan-Meier analyses. RESULTS: Based on GP-11 subtype-scoring, >75% of the MIBC TCGA-dataset samples were mixed, i.e. neither pure-luminal nor pure-basal. When tumors were categorized as pure-luminal, pure-basal, or mixed, the subtypes could not predict OS or R/PFS. Consistent with published studies, when tumors were categorized as luminal or basal the subtypes predicted OS (P=0.051); sensitivity: 53.9%; specificity: 62%. Subtypes also correlated with tumor-grade (P=0.0005); most low-grade MIBC cases (16/21) in the TCGA-dataset were luminal. However, MIBC is rarely low-grade and subtypes could not predict OS (P=0.131) when only high-grade cases were included. Subtypes were not significant prognosticators in multivariate analyses. GP-30 and BASQ panels validated these results. In the Oncomine-dataset and cohort-1, subtypes could not predict metastasis, CSS, or OS. CONCULUSIONS: Our study of multiple datasets reveals that molecular subtypes may reflect tumor-heterogeneity but are likely not distinct entities within MIBC. Furthermore, molecular subtypes have limited prognostic capability for MIBC patients. Although there is a need to individualize patient care, further examination into the molecular subtypes of MIBC is needed before their incorporation into clinical practice. Citation Format: Sarrah S. Lahorewala, Daley S. Morera, Jiaojiao Wang, Vinata B. Lokeshwar. Results from multiple datasets including the TCGA reveal limited clinical significance of molecular subtypes in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 462.

Introduction: Using RNA-seq (HiSeq) transcriptome data in The Cancer Genome Atlas (TCGA) bladder cancer (BCa) dataset two molecular subtypes, basal and luminal, were identified in muscle invasive BCa (MIBCa). Recurring reported markers of the basal subtype are KRT5, KRT6A, KRT14 high and KRT20, GATA3, UPK3A, FOXA1 low. The luminal subtype is the opposite pattern of expression. Double-Negative (DN) subtype was defined as no expression of the 7 markers. Luminal, basal and DN subtypes were reported as predictors of better, poor and worst prognosis, respectively, based on overall survival (OS). EGFR is defined as a squamous differentiation marker associated with poor prognosis. The objective was to assess the clinical significance of these subtypes in TCGA data with validation in datasets from Oncomine. Methods: TCGA dataset containing 407 MIBCa patients was accessed through Xena Browser. The dataset included patient demographics, clinical parameters, OS, recurrence-free survival (RFS) and the transcript levels (log2(nor._count+1) of basal and luminal markers. All available follow-up data was included (27.9±28.49; max: 166 months). 25 BCa datasets (n=360 MIBCa patients) containing these same variables were accessed through Oncomine. High and low levels were stratified by median. Association of individual basal and luminal markers and the subtypes with clinical and outcome variables was analyzed by univariate and multivariate analyses. Kaplan-Meier analysis was performed to stratify patients into risk groups for OS and RFS. Results: In TCGA data, neither basal nor luminal markers levels significantly correlated with metastasis or lymphovascular invasion (LVI); P = 0.2 to 0.9. Only KRT5 significantly but inversely correlated with lymph node (LN) positivity; P=0.011. EGFR levels did not correlate with metastasis, LVI, or LN positivity; P > 0.05. In TCGA dataset 77 (18.9%), 50 (12.3%) and 14 (3.4%) expressed basal, luminal and DN subtypes, respectively; 266 (66.4%) patients did not conform to any group. In univariate or multivariate analyses, the subtypes also did not correlate with metastasis, LVI or LN status. Only luminal subtype associated with better OS; P=0.003. However no subtype significantly correlated with RFS. In KM analysis no subtype stratified patients regarding RFS (P>0.2). Oncomine BCa datasets validated these results. Conclusion: TCGA and Oncomine datasets show that the majority of MIBCa tissues express a mixed pattern of basal and luminal markers. Furthermore, basal, luminal or DN subtypes do not associate with clinical parameters or prognosis of MIBCa patients. Citation Format: Daley S. Morera, Daniel Belew, Andre R. Jordan, Vinata B. Lokeshwar. Molecular subtypes in muscle invasive bladder cancer: Evaluation of clinical significance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4614.

e14563 Background: Bladder cancer is a molecularly diverse disease with heterogeneous clinical outcomes. Transcriptome-based molecular subtypes of muscle invasive bladder cancer (MIBC) and non-muscle invasive bladder cancer (NMIBC) have been shown to be both prognostic and predictive. However, its predictive role in Chinese bladder cancer remains unclear. The aim of this study was to identify the predictive role of molecular subtypes in China bladder cancer with neoadjuvant therapy. Methods: The study was conducted from May 2020 to August 2021. Patients who were age 18 years or older, were diagnosed with NMIBC or MIBC, and neoadjuvant tislelizumab combining nab-paclitaxel followed by surgery were included. Multigenomic sequencing was performed. Molecular subtypes were identified by published consensus. Results: We prospectively recruited 30 patients for neoadjuvant tislelizumab combining nab-paclitaxel, including 14 patients with NMIBC and 16 patients with MIBC. For bladder cancer, 73.3% (22/33) patients responded to neoadjuvant tislelizumab combining nab-paclitaxel. In NMIBC groups, 64.3% (9/14) patients were pCR. The objective remission rate ( ORR) was 71.4%. 4 molecular classes were identified in Chinese NMIBC, including class 1 (7.1%), class 2a (35.7%), class 2b (50%), class 3 (7.1%). Interesting, ORR was significantly higher for patients with class 2b (5/7, 71.4%) as compared to class 2a (3/5,60%) before neoadjuvant. Molecular classes changed in 35.7% (5/14) of patients before and after neoadjuvant. After neoadjuvant, 80% of class 2b patients responded to neoadjuvant, and all class 2a patients did not respond to neoadjuvant. In MIBC groups, a pPR was achieved by 37.5% patients (6/16). The ORR was 75%. We found 4 molecular classes in Chinese MIBC: basal/squamous (43.8%), luminal unstable (25%), luminal papillary (25%), and stroma-rich (6.2%). Patients with basal/squamous (85.7%) and luminal unstable (75%) had significantly higher ORR than luminal papillary (50%) before neoadjuvant. Molecular classes changed in 44.4% (4/9) of patients before and after neoadjuvant. After neoadjuvant, all patients with stroma-rich responded to neoadjuvant. Conclusions: Molecular classification could predict the efficacy of neoadjuvant tislelizumab combining nab-paclitaxel in Chinese bladder cancer, which may be preferable when studying biomarkers of bladder cancer in the future.

Feasibility and Impact of Immunohistochemistry-based Molecular Subtyping for Muscle-invasive Bladder Cancer in Patients Treated with Radiation-based Therapy

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.

Bladder cancers have high total mutation burdens resulting in genomic diversity and intra- and inter-tumor heterogeneity that may impact the diversity of gene expression, biologic aggressiveness, and potentially response to therapy. To compare bladder cancers among patients, an organizational structure is necessary that describes the tumor at the histologic and molecular level. These "molecular subtypes", or "expression subtypes" of bladder cancer were originally described in 2010 and continue to evolve secondary to next generation sequencing (NGS) and an increasing public repository of well-annotated cohorts. To review the history and methodology of expression-based subtyping of non-muscle invasive (NMIBC) and muscle invasive bladder cancer (MIBC). A literature review was performed of primary papers from PubMed that described subtyping methods and their descriptive feature including search terms of "subtype", and "bladder cancer". 21 papers were identified for review. Tumor subtyping developed from N = 2 to N = 6 subtyping schemes with most subtypes comprised of at least luminal and basal tumors. Most NMIBCs are luminal cancers and luminal MIBCs may be associated with less aggressive features, while one study of basal tumors identified a better clinical outcome with systemic chemotherapy. Tumors with a P53-like may have intrinsic resistance to chemotherapy. The heterogeneity of tumors, which is likely derived from stromal components and immune cell infiltration, affect subtype calls. Subtyping, while still evolving, is ready for testing in clinical trials. Improved patient selection with tumor subtyping may help with tumor classification and potentially match patient or tumor to therapy.

Enfortumab vedotin (EV) is an antibody-drug conjugate (ADC) targeting NECTIN4 (encoded by the PVRL4/NECTIN4 gene) approved for treatment-refractory metastatic urothelial cancer. Factors that mediate sensitivity or resistance to EV are unknown. In this study, we sought to (i) examine heterogeneity of NECTIN4 gene expression across molecular subtypes of bladder cancer and (ii) determine whether NECTIN4 expression mediates EV sensitivity or resistance. Molecular subtyping and NECTIN4 expression data from seven muscle-invasive bladder cancer clinical cohorts (n = 1,915 total specimens) were used to assess NECTIN4 expression across molecular subtypes. The outcome of the transcriptomic analysis was relative NECTIN4 expression in the consensus molecular subtypes of bladder cancer. Expression of NECTIN4 was validated in bladder cancer cell lines. NECTIN4 was stably overexpressed or knocked down in basal and luminal bladder cancer cell lines and EV drug sensitivity assays were performed, as measured by cell proliferation and clonogenic assays. NECTIN4 expression is heterogenous across molecular subtypes of bladder cancer and significantly enriched in luminal subtypes. NECTIN4 expression is positively correlated with luminal markers GATA3, FOXA1, and PPARG across all cohorts. NECTIN4 expression is both necessary and sufficient for EV sensitivity in luminal and basal subtypes of urothelial bladder cancer cells. Downregulation of NECTIN4 leads to EV resistance. Sensitivity to EV is mediated by expression of NECTIN4, which is enriched in luminal subtypes of bladder cancer. These findings may have implications for biomarker development, patient selection, and the inclusion of molecular subtyping in ongoing and future EV clinical trials.See related commentary by Teo and Rosenberg, p. 4950.

<div>AbstractPurpose:<p>Enfortumab vedotin (EV) is an antibody–drug conjugate (ADC) targeting NECTIN4 (encoded by the <i>PVRL4/NECTIN4</i> gene) approved for treatment-refractory metastatic urothelial cancer. Factors that mediate sensitivity or resistance to EV are unknown. In this study, we sought to (i) examine heterogeneity of <i>NECTIN4</i> gene expression across molecular subtypes of bladder cancer and (ii) determine whether NECTIN4 expression mediates EV sensitivity or resistance.</p>Experimental Design:<p>Molecular subtyping and <i>NECTIN4</i> expression data from seven muscle-invasive bladder cancer clinical cohorts (<i>n</i> = 1,915 total specimens) were used to assess <i>NECTIN4</i> expression across molecular subtypes. The outcome of the transcriptomic analysis was relative <i>NECTIN4</i> expression in the consensus molecular subtypes of bladder cancer. Expression of <i>NECTIN4</i> was validated in bladder cancer cell lines. NECTIN4 was stably overexpressed or knocked down in basal and luminal bladder cancer cell lines and EV drug sensitivity assays were performed, as measured by cell proliferation and clonogenic assays.</p>Results:<p><i>NECTIN4</i> expression is heterogenous across molecular subtypes of bladder cancer and significantly enriched in luminal subtypes. <i>NECTIN4</i> expression is positively correlated with luminal markers <i>GATA3, FOXA1</i>, and <i>PPARG</i> across all cohorts. <i>NECTIN4</i> expression is both necessary and sufficient for EV sensitivity in luminal and basal subtypes of urothelial bladder cancer cells. Downregulation of <i>NECTIN4</i> leads to EV resistance.</p>Conclusions:<p>Sensitivity to EV is mediated by expression of <i>NECTIN4</i>, which is enriched in luminal subtypes of bladder cancer. These findings may have implications for biomarker development, patient selection, and the inclusion of molecular subtyping in ongoing and future EV clinical trials.</p><p><i>See related commentary by Teo and Rosenberg, p. 4950</i></p></div>

<div>AbstractPurpose:<p>Enfortumab vedotin (EV) is an antibody–drug conjugate (ADC) targeting NECTIN4 (encoded by the <i>PVRL4/NECTIN4</i> gene) approved for treatment-refractory metastatic urothelial cancer. Factors that mediate sensitivity or resistance to EV are unknown. In this study, we sought to (i) examine heterogeneity of <i>NECTIN4</i> gene expression across molecular subtypes of bladder cancer and (ii) determine whether NECTIN4 expression mediates EV sensitivity or resistance.</p>Experimental Design:<p>Molecular subtyping and <i>NECTIN4</i> expression data from seven muscle-invasive bladder cancer clinical cohorts (<i>n</i> = 1,915 total specimens) were used to assess <i>NECTIN4</i> expression across molecular subtypes. The outcome of the transcriptomic analysis was relative <i>NECTIN4</i> expression in the consensus molecular subtypes of bladder cancer. Expression of <i>NECTIN4</i> was validated in bladder cancer cell lines. NECTIN4 was stably overexpressed or knocked down in basal and luminal bladder cancer cell lines and EV drug sensitivity assays were performed, as measured by cell proliferation and clonogenic assays.</p>Results:<p><i>NECTIN4</i> expression is heterogenous across molecular subtypes of bladder cancer and significantly enriched in luminal subtypes. <i>NECTIN4</i> expression is positively correlated with luminal markers <i>GATA3, FOXA1</i>, and <i>PPARG</i> across all cohorts. <i>NECTIN4</i> expression is both necessary and sufficient for EV sensitivity in luminal and basal subtypes of urothelial bladder cancer cells. Downregulation of <i>NECTIN4</i> leads to EV resistance.</p>Conclusions:<p>Sensitivity to EV is mediated by expression of <i>NECTIN4</i>, which is enriched in luminal subtypes of bladder cancer. These findings may have implications for biomarker development, patient selection, and the inclusion of molecular subtyping in ongoing and future EV clinical trials.</p><p><i>See related commentary by Teo and Rosenberg, p. 4950</i></p></div>

Molecular classifications for urothelial bladder cancer appear to be promising in disease prognostication and prediction. This study investigated the novel molecular subtypes of muscle invasive bladder cancer (MIBC). Tumor samples and normal tissues of MIBC patients were submitted for transcriptome sequencing. Expression profiles were clustered using K-means clustering and principal component analysis. The molecular subtypes were also applied to The Cancer Genome Atlas (TCGA) dataset and analyzed for clinical outcome correlation. Three molecular subtypes of MIBC were discovered, clusters A, B, and C. The most differentially upregulated genes in cluster A were BDKRB1, EDNRA, AVPR1A, PDGFRB, and TNC, while the most upregulated genes in cluster C were collagen-related genes, PDGFRB, and PRKG1. For cluster B, COL6A3, COL1A2, COL6A2, tenascin C, and fibroblast growth factor 2 were statistically suppressed. When the centroids of clustering on PCA were applied to TCGA data, the clustering significantly predicted survival outcomes. Cluster B had the best overall survival (OS), and cluster C was associated with poor OS but exhibited the best response to perioperative chemotherapy. Among all groups, cluster B had a better pathologic response to neoadjuvant chemotherapy (40%). Based on the results of the present study, the novel clusters of subtype MIBC appear potentially suitable for integration into clinical practice.

Transcriptome-based molecular subtypes of muscle-invasive bladder cancer (MIBC) have been shown to be both prognostic and predictive, but are not used in routine clinical practice. We aimed to develop a feasible, reverse transcription quantitative polymerase chain reaction (RT-qPCR)-based method for molecular subtyping. First, we defined a 68-gene set covering tumor intrinsic (luminal, basal, squamous, neuronal, epithelial-to-mesenchymal, in situ carcinoma) and stromal (immune, extracellular matrix, p53-like) signatures. Then, classifier methods with this 68-gene panel were developed in silico and validated on public data sets with available subtype class information (MD Anderson [MDA], The Cancer Genome Atlas [TCGA], Lund, Consensus). Finally, expression of the selected 68 genes was determined in 104 frozen tissue samples of our MIBC cohort by RT-qPCR using the TaqMan Array Card platform and samples were classified by our newly developed classifiers. The prognostic value of each subtype classification system and molecular signature scores were assessed. We found that the reduced marker set combined with the developed classifiers were able to reproduce the TCGA II, MDA, Lund and Consensus subtype classification systems with an overlap of 79%, 76%, 69% and 64%, respectively. Importantly, we could successfully classify 96% (100/104) of our MIBC samples by using RT-qPCR. Neuronal and luminal subtypes and low stromal gene expressions were associated with poor survival. In conclusion, we developed a robust and feasible method for the molecular subtyping according to the TCGA II, MDA, Lund and Consensus classifications. Our results suggest that stromal signatures have a superior prognostic value compared to tumor intrinsic signatures and therefore underline the importance of tumor-stroma interaction during the progression of MIBC.

BackgroundRecently, erdafitinib (Balversa), the first targeted therapy drug for genetic alteration, was approved to metastatic urothelial carcinoma. Cancer genomics research has been greatly encouraged. Currently, a large number of gene regulatory networks between different states have been constructed, which can reveal the difference states of genes. However, they have not been applied to the subtypes of Muscle-invasive bladder cancer (MIBC).ResultsIn this paper, we propose a method that construct gene regulatory networks under different molecular subtypes of MIBC, and analyse the regulatory differences between different molecular subtypes. Through differential expression analysis and the differential network analysis of the top 100 differential genes in the network, we find that SERPINI1, NOTUM, FGFR1 and other genes have significant differences in expression and regulatory relationship between MIBC subtypes.ConclusionsFurthermore, pathway enrichment analysis and differential network analysis demonstrate that Neuroactive ligand-receptor interaction and Cytokine-cytokine receptor interaction are significantly enriched pathways, and the genes contained in them are significant diversity in the subtypes of bladder cancer.

BackgroundMuscle-invasive bladder carcinomas (MIBCs) exhibit significant heterogeneity, with diverse histopathological features associated with varied prognosis and therapeutic response. Although genomic profiling studies have identified several molecular subtypes of MIBC, two basic molecular subtypes are identified - luminal and basal, differing in biological behaviour and response to treatment. As molecular subtyping is complex, surrogate immunohistochemical (IHC) markers have been used to determine the molecular subtypes with good correlation to genomic profiling.MethodsWe analysed the clinicopathological features of 66 cases of MIBCs received over a 5-year study period. IHC expression was determined using GATA3 and CK5/6 to classify MIBC into luminal, basal and double-negative subtypes. The association between clinicopathologic variables and molecular subtypes were analysed using Chi-square test.ResultsThe mean age at diagnosis of MIBC was 65.91 years with a male predominance. Based on IHC expression of GATA3 and CK5/6, MIBCs were classified into luminal, basal and double negative subtypes in 62.1%, 30.3% and 7.6% respectively. The luminal subtype occurred at an older age and showed predominantly conventional urothelial carcinoma with papillary morphology. Basal subtype occurred at earlier age, showed greater association with smoking and was more commonly associated with urothelial carcinoma with non -papillary morphology and exhibiting divergent differentiation as well as pure squamous cell carcinoma on histopathological examination. The double-negative subtype was found exclusively in males and exhibited a non-papillary morphology. Notably, all diagnosed neuroendocrine carcinomas were classified as double-negative type. While there was no statistically significant difference in tumour stage in cystectomy specimens between the molecular subtypes, lympho-vascular invasion and lymph node metastasis was more commonly associated with the basal type (p < 0.05) There was no significant difference in recurrence rates, metastasis and death between luminal and basal subtypes.ConclusionA simple two-antibody panel using GATA3 and CK5/6 could help in classifying MIBC into basic molecular subtypes of MIBC with distinctive histopathological features that can provide insights into the corresponding molecular subtype. Greater association of lymphovascular invasion and lymph nodal involvement in cystectomy specimens in basal type and distant metastasis in the double-negative subtype suggests a more aggressive clinical behaviour of these, necessitating more intensive treatment.

Notch Pathway: Bioinformatic Analysis of Related Transcription Factors within Bladder Cancer Types and Subtypes

Introduction. Bladder cancer (BCa) is one of the most common oncological diseases with many relapses and progressions, which requires more and more new ways to solve this problem.Objective. To analyse of existing data on the molecular and genetic features of BCa classification based on them, assessment of the possibility of its introduction into clinical practice as a criterion for recurrence and progression.Materials &amp; methods. We analysed the data of studies on molecular subtypes of BCa published in PubMed, Scientific Electronic Library of Russia (eLibrary), Scopus, Medline, EMBASE, websites of professional associations. We searched the following keywords: "molecular subtypes of bladder cancer", "non-muscle invasive bladder cancer", "molecular subtypes of bladder cancer" "non-muscle invasive bladder cancer". The paper focuses on the current classifications and the April 2020 consensus, which can be universal and applied in everyday practice.Results. During the analysis of the obtained data, it was revealed that the molecular and genetic heterogeneity of BCa is reflected not only in the proteins expressed on tumor cells, but also in the response to treatment and prognosis of the disease.Conclusion. Molecular and genetic markers seem promising for introduction into wide clinical practice to adjust treatment and assess the risks of recurrence and progression.