Heterogeneity Of Bladder Cancer Research Articles

Recent contributions of single-cell and spatial profiling to the understanding of bladder cancer.

Current risk stratification and treatment decision-making for bladder cancer informed by histopathology as well as molecular diagnostics face limitations. This review summarizes recent advancements in single-cell and spatial omics methodologies for understanding bladder cancer biology and their potential impact on development of novel therapeutic strategies. Single-cell RNA sequencing and spatial omics techniques offer unprecedented insights into various aspects of tumor microenvironment (TME), bladder cancer heterogeneity, cancer stemness, and cellular plasticity. Studies have identified multiple malignant cell subpopulations within tumors, revealing diverse transcriptional states and clonal evolution. Additionally, intratumor heterogeneity has been linked to tumor progression and therapeutic response. Immune cell composition analysis has revealed immunosuppressive features in the TME, impacting treatment response. Furthermore, studies have elucidated the role of cancer-associated fibroblasts and endothelial cells in shaping the tumor immune landscape and response to therapy. Single-cell and spatial omics technologies have revolutionized our understanding of bladder cancer biology, uncovering previously unseen complexities. These methodologies provide valuable insights into tumor heterogeneity and microenvironmental interactions, with implications for therapeutic development. However, challenges remain in translating research findings into clinical practice and implementing personalized treatment strategies. Continued interdisciplinary collaboration and innovation are essential for overcoming these challenges and leveraging the full potential of single-cell and spatial omics in improving bladder cancer diagnosis and treatment.

Advances in preclinical assessment of therapeutic targets for bladder cancer precision medicine.

Bladder cancer incidence is on the rise, and until recently, there has been little to no change in treatment regimens over the last 40 years. Hence, it is imperative to work on strategies and approaches to untangle the complexity of intra and inter-tumour heterogeneity of bladder cancer with the aim of improving patient-specific care and treatment outcomes. The focus of this review is therefore to highlight novel targets, advances, and therapy approaches for bladder cancer patients. The success of combining an antibody-drug conjugate (ADC) with immunotherapy has been recently hailed as a game changer in treating bladder cancer patients. Hence, interest in other ADCs as a treatment option is also rife. Furthermore, strategies to overcome chemoresistance to standard therapy have been described recently. In addition, other studies showed that targeting genomic alterations (e.g. mutations in FGFR3, DNA damage repair genes and loss of the Y chromosome) could also be helpful as prognostic and treatment stratification biomarkers. The use of single-cell RNA sequencing approaches has allowed better characterisation of the tumour microenvironment and subsequent identification of novel targets. Personalized functional precision medicine could be another avenue to improve and guide personalized treatment options. Several novel preclinical targets and treatment options have been described recently. The validation of these advances will lead to the development and implementation of robust personalized treatment regimens for bladder cancer patients.

Integrative Single Cell Atlas Revealed Intratumoral Heterogeneity Generation from an Adaptive Epigenetic Cell State in Human Bladder Urothelial Carcinoma.

Intratumor heterogeneity (ITH) of bladder cancer (BLCA) contributes to therapy resistance and immune evasion affecting clinical prognosis. The molecular and cellular mechanisms contributing to BLCA ITH generation remain elusive. It is found that a TM4SF1-positive cancer subpopulation (TPCS) can generate ITH in BLCA, evidenced by integrative single cell atlas analysis. Extensive profiling of the epigenome and transcriptome of all stages of BLCA revealed their evolutionary trajectories. Distinct ancestor cells gave rise to low-grade noninvasive and high-grade invasive BLCA. Epigenome reprograming led to transcriptional heterogeneity in BLCA. During early oncogenesis, epithelial-to-mesenchymal transition generated TPCS. TPCS has stem-cell-like properties and exhibited transcriptional plasticity, priming the development of transcriptionally heterogeneous descendent cell lineages. Moreover, TPCS prevalence in tumor is associated with advanced stage cancer and poor prognosis. The results of this study suggested that bladder cancer interacts with its environment by acquiring a stem cell-like epigenomic landscape, which might generate ITH without additional genetic diversification.

Abstract 4632: Lineage plasticity as a determinant of antibody-drug conjugate target expression in urothelial bladder cancer

Abstract Bladder cancers display a wide spectrum of morphologies that frequently co-exist within individual tumors. Several histologic variants, including plasmacytoid, neuroendocrine, and micropapillary subtypes, are associated with an increased bladder cancer recurrence risk and cancer-specific mortality. The molecular basis and therapeutic implications of this phenotypical plasticity remain poorly understood. Enfortumab vedotin (EV), an antibody-drug conjugate targeting Nectin-4, has also emerged as a new standard-of-care for metastatic bladder cancer patients, but the association between lineage plasticity as manifested by morphologic heterogeneity in bladder cancer, Nectin-4 expression and ADC response remains poorly defined. Previous DNA-based molecular profiling studies of histologic variants revealed that CDH1 mutations were the pathognomonic molecular alteration in plasmacytoid urothelial carcinoma, but no recurrent driver mutations have been found to be unique to the aberrant histology regions of other mixed histology subtypes. Thus, to explore the molecular basis for the lineage plasticity of bladder cancers with aberrant histology and to define the association between lineage plasticity manifested as morphologic heterogeneity and the expression of Nectin-4, HER2 and other potential ADC targets, we performed RNA-seq of pure urothelial carcinomas (n=118, not otherwise specified, UC-NOS) and bladder tumors with divergent differentiation or histologic subtypes (n=199). Our cohort included a subset of tumors in which RNA-seq was performed on microdissected UC-NOS and variant histology regions of the same tumor. Tumors with neuroendocrine, sarcomatoid and squamous histology had the lowest Nectin-4 expression, whereas histologic subtypes that retained a luminal transcriptional profile such as micropapillary, plasmacytoid and nested histology had higher levels of Nectin-4 expression. In mixed histology tumors, Nectin-4 and/or HER2 expression was often restricted to only one of the two histologic components. Gene Set Enrichment Analysis noted enrichment of distinct oncogenic pathway gene sets in different histologic variants. Preliminary investigation into the mechanism(s) of Nectin-4 regulation using bladder cancer cell lines and patient-derived organoids (PDOs) has identified targetable pathways which when inhibited revert lineage plasticity and induce Nectin-4 expression. In sum, lineage plasticity manifested as heterogenous histomorphologies was associated with heterogeneous Nectin-4 expression in some but not all histologic variants of bladder cancer. Ongoing studies using PDOs from bladder cancer patients with mixed histology will seek to identify molecular pathways whose inhibition leads to increased expression of Nectin-4 or other ADC targets such as HER2 and thus increased sensitivity of ADCs targeting these cell surface proteins. Citation Format: Jiaqian Luo, Sizhi P. Gao, Fengshen Kuo, Gamze Gokturk Ozcan, Merve Basar, Florestan Koll, Jacob E. Tallman, Syed M. Alam, Carissa E. Chu, Ziyu Chen, Eugene J. Pietzak, Gopakumar Iyer, Jonathan E. Rosenberg, David B. Solit, Hikmat Al-Ahmadie. Lineage plasticity as a determinant of antibody-drug conjugate target expression in urothelial bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4632.

Assessment of aggressive bladder cancer mutations in plasma cell-free DNA

Background and aimsThe spatial and temporal genetic heterogeneity of bladder cancer (BC) makes challenging to find specific drivers of metastatic disease, thus preventing to determine those BC patients at high risk of tumor progression. Our aim was to identify DNA mutations providing aggressive behavior to bladder tumors and analyze them in patients’ cell-free DNA (cfDNA) during their follow-up after radical cystectomy (RC) in order to monitor tumor evolution.MethodsSix BC patients who underwent RC and presented disease progression during their follow-up were included. Next-generation sequencing was used to determine somatic mutations in several primary tumor and metastatic specimens from each patient. Shared DNA mutations between primary bladder tumor and metastatic sites were identified in cfDNA samples through droplet digital PCR.ResultsBesides BC genetic heterogeneity, specific mutations in at least one of these genes —TERT, ATM, RB1, and FGFR3— were found in primary tumors and their metastases in all patients. These mutations were also identified in the patients’ cfDNA at different follow-up time points. Additionally, the dynamic changes of these mutations in cfDNA allowed us to determine tumor evolution in response to treatment.ConclusionThe analysis of BC mutations associated with poor prognosis in plasma cfDNA could be a valuable tool to monitor tumor evolution, thus improving the clinical management of BC patients.

Tumor microenvironment heterogeneity in bladder cancer identifies biologically distinct subtypes predicting prognosis and anti-PD-L1 responses

Bladder cancer (BCa) is heterogeneous in the tumour microenvironment (TME). However, the role of the TME in BCa in modulating the response to immunotherapy has not been fully explored. We therefore analysed fractions of immune cells using CIBERSORTx and clustered BCa into subtypes. We also analyzed weighted correlation networks to generate immunotherapy-related hub genes that we used to construct a prediction model using multivariate Cox and LASSO regression analyses. We found that BCa comprised three subtypes (C1‒C3). The prognosis of the patients was the most favourable and the response rate to anti-programmed death ligand 1 (PD-L1) was the highest in C1 among the three subtypes. Immune cells, including CD8+, CD4+ memory activated, and follicular helper T cells, activated NK cells, and M1 macrophages infiltrated the C1 subtype. The C2 subtype was enriched in M0 macrophages and activated mast cells, and the C3 subtype was enriched in B and resting immune cells. Mechanistically, the enhanced immunogenicity of subtypes C1 and C2 correlated positively with a higher response rate, whereas the dysregulated ECM-related pathways in the C2 subtype and glycolytic and fatty acid metabolic pathways in the C3 subtype impaired the responses of patients to anti-PD-L1 therapy. We also constructed a TME-related signature based on 18 genes that performed well in terms of overall survival. In conclusion, we determined prognoses and anti-PD-L1 responses by analysing TME heterogeneity in BCa.

Longitudinal Analyses of Mutational Subclonal Architecture and Tumor Subtypes in Recurrent Bladder Cancer.

Longitudinal tumor sequencing of recurrent bladder cancer (BC) can facilitate the investigation of BC progression-associated genomic and transcriptomic alterations. In this study, we analyzed 18 tumor specimens including distant and locoregional metastases obtained during tumor progression for five BC patients using whole-exome and transcriptome sequencing. Along with the substantial level of intratumoral mutational heterogeneity across the cases, we observed that clonal mutations were enriched with known BC driver genes and apolipoprotein B mRNA editing enzyme, catalytic polypeptide (APOBEC)-associated mutation signatures compared with subclonal mutations, suggesting the genetic makeup for BC tumorigenesis associated with APOBEC deaminase activity was accomplished early in the cancer evolution. Mutation-based phylogenetic analyses also revealed temporal dynamics of mutational clonal architectures in which the number of mutational clones varied along the BC progression and notably was often punctuated by clonal sweeps associated with chemotherapy. The bulk-level transcriptome sequencing revealed frequent subtype switching in which transcriptionally defined BC subtypes may vary during tumor progression. Longitudinal whole-exome and transcriptome sequencing of recurrent BC may advance our understanding into the BC heterogeneity in terms of somatic mutations, cell clones and transcriptome-based tumor subtypes during disease progression.

Single-nucleus and Spatially Resolved Intratumor Subtype Heterogeneity in Bladder Cancer

BackgroundCurrent bulk transcriptomic classification systems for bladder cancer do not consider the level of intratumor subtype heterogeneity. ObjectiveTo investigate the extent and possible clinical impact of intratumor subtype heterogeneity across early and more advanced stages of bladder cancer. Design, setting, and participantsWe performed single-nucleus RNA sequencing (RNA-seq) of 48 bladder tumors and additional spatial transcriptomics for four of these tumors. Total bulk RNA-seq and spatial proteomics data were available from the same tumors for comparison, along with detailed clinical follow-up of the patients. Outcome measurements and statistical analysisThe primary outcome was progression-free survival for non–muscle-invasive bladder cancer. Cox regression analysis, log-rank tests, Wilcoxon rank-sum tests, Spearman correlation, and Pearson correlation were used for statistical analysis. Results and limitationsWe found that the tumors exhibited varying levels of intratumor subtype heterogeneity and that the level of subtype heterogeneity can be estimated from both single-nucleus and bulk RNA-seq data, with high concordance between the two. We found that a higher class 2a weight estimated from bulk RNA-seq data is associated with worse outcome for patients with molecular high-risk class 2a tumors. The sparsity of the data generated using the DroNc-seq sequencing protocol is a limitation. ConclusionsOur results indicate that discrete subtype assignments from bulk RNA-seq data may lack biological granularity and that continuous class scores may improve clinical risk stratification of patients with bladder cancer. Patient summaryWe found that several molecular subtypes can exist within a single bladder tumor and that continuous subtype scores can be used to identify a subgroup of patients with poor outcomes. Use of these subtype scores may improve risk stratification for patients with bladder cancer, which can help in making decisions on treatment.

Interview: Timothy Clinton

Interview: Timothy Clinton

A prognostic model based on necroptosis-related genes for prognosis and therapy in bladder cancer

Bladder cancer, one of the most prevalent malignant cancers, has high rate of recurrence and metastasis. Owing to genomic instability and high-level heterogeneity of bladder cancer, chemotherapy and immunotherapy drugs sensitivity and lack of prognostic markers, the prognosis of bladder cancer is unclear. Necroptosis is a programmed modality of necrotic cell death in a caspase-independent form. Despite the fact that necroptosis plays a critical role in tumor growth, cancer metastasis, and cancer patient prognosis, necroptosis-related gene sets have rarely been studied in bladder cancer. As a result, the development of new necroptosis-related prognostic indicators for bladder cancer patients is critical. Herein, we assessed the necroptosis landscape of bladder cancer patients from The Cancer Genome Atlas database and classified them into two unique necroptosis-related patterns, using the consensus clustering. Then, using five prognosis-related genes, we constructed a prognostic model (risk score), which contained 5 genes (ANXA1, DOK7, FKBP10, MAP1B and SPOCD1). And a nomogram model was also developed to offer the clinic with a more useful prognostic indicator. We found that risk score was significantly associated with clinicopathological characteristics, TIME, and tumor mutation burden in patients with bladder cancer. Moreover, risk score was a valid guide for immunotherapy, chemotherapy, and targeted drugs. In our study, DOK7 was chosen to further verify our prognosis model, and functional assays indicated that knockdown the expression of DOK7 could prompt bladder cancer proliferation and migration. Our work demonstrated the potential role of prognostic model based on necroptosis genes in the prognosis, immune landscape and response efficacy of immunotherapy of bladder cancer.

Characterization of Epithelial-Mesenchymal Transition Identifies a Gene Signature for Predicting Clinical Outcomes and Therapeutic Responses in Bladder Cancer.

The complex etiological variables and high heterogeneity of bladder cancer (BC) make prognostic prediction challenging. We aimed to develop a robust and promising gene signature using advanced machine learning methods for predicting the prognosis and therapy responses of BC patients. The single-sample gene set enrichment analysis (ssGSEA) algorithm and univariable Cox regression were used to identify the primary risk hallmark among the various cancer hallmarks. Machine learning methods were then combined with survival and differential gene expression analyses to construct a novel prognostic signature, which would be validated in two additional independent cohorts. Moreover, relationships between this signature and therapy responses were also identified. Functional enrichment analysis and immune cell estimation were also conducted to provide insights into the potential mechanisms of BC. Epithelial-mesenchymal transition (EMT) was identified as the primary risk factor for the survival of BC patients (HR=1.43, 95% CI: 1.26-1.63). A novel EMT-related gene signature was constructed and validated in three independent cohorts, showing stable and accurate performance in predicting clinical outcomes. Furthermore, high-risk patients had poor prognoses and multivariable Cox regression analysis revealed this to be an independent risk factor for patient survival. CD8+ T cells, Tregs, and M2 macrophages were found abundantly in the tumor microenvironment of high-risk patients. Moreover, it was anticipated that high-risk patients would be more sensitive to chemotherapeutic drugs, while low-risk patients would benefit more from immunotherapy. We successfully identified and validated a novel EMT-related gene signature for predicting clinical outcomes and therapy responses in BC patients, which may be useful in clinical practice for risk stratification and individualized treatment.

CK5/6 and GATA3 Defined Phenotypes of Muscle-Invasive Bladder Cancer: Impact in Adjuvant Chemotherapy and Molecular Subtyping of Negative Cases.

Introduction and ObjectiveIdentifying patients that benefit from cisplatin-based adjuvant chemotherapy is a major issue in the management of muscle-invasive bladder cancer (MIBC). The purpose of this study is to correlate “luminal” and “basal” type protein expression with histological subtypes, to investigate the prognostic impact on survival after adjuvant chemotherapy and to define molecular consensus subtypes of “double negative” patients (i.e., without expression of CK5/6 or GATA3).Materials and MethodsWe performed immunohistochemical (IHC) analysis of CK5/6 and GATA3 for surrogate molecular subtyping in 181 MIBC samples. The mRNA expression profiles for molecular consensus classification were determined in CK5/6 and GATA3 (double) negative cases using a transcriptome panel with 19.398 mRNA targets (HTG Molecular Diagnostics). Data of 110 patients undergoing radical cystectomy were available for survival analysis.ResultsThe expression of CK5/6 correlated with squamous histological subtype (96%) and expression of GATA3 was associated with micropapillary histology (100%). In the multivariate Cox-regression model, patients receiving adjuvant chemotherapy had a significant survival benefit (hazard ratio [HR]: 0.19 95% confidence interval [CI]: 0.1–0.4, p < 0.001) and double-negative cases had decreased OS (HR: 4.07; 95% CI: 1.5–10.9, p = 0.005). Double negative cases were classified as NE-like (30%), stroma-rich (30%), and Ba/Sq (40%) consensus molecular subtypes and displaying different histological subtypes.ConclusionImmunohistochemical-based classification was associated with histological subtypes of urothelial MIBC. IHC markers like CK5/6 and GATA3 that are used in pathological routine could help to identify patients with basal and luminal tumor characteristics. However, a two-sided classification system might not sufficiently reflect the heterogeneity of bladder cancer to make treatment decisions. Especially the group of IHC-double negative cases, as further analyzed by mRNA expression profiling, are a heterogeneous group with different implications for therapy.

Identification of glycolysis-associated long non-coding RNA regulatory subtypes and construction of prognostic signatures by transcriptomics for bladder cancer.

Glycolysis-targeted cancer therapy based on long non-coding RNAs (lncRNAs), owing to its high specificity and less toxicity, is at the preclinical stages. Our study aimed to examine the roles of the core glycolysis-associated lncRNAs in bladder cancer (BC). Glycolysis scores of BC were computed by single-sample gene set enrichment analysis (ssGSEA). Glycolysis-associated lncRNAs were screened by Pearson's correlation analysis. Unsupervised consensus clustering using ConsensusClusterPlus assessed the glycolysis-associated lncRNAs for the identification of molecular subtypes of BC. The Kaplan-Meier survival analysis, genomic mutations, and tumor microenvironment (TME) analysis were used to compare the characteristics of different subtypes. Key glycolysis-associated lncRNAs were screened by first-order partial correlation and univariate Cox proportional-hazards model analyses; finally, the lncRNA signature was constructed. Four glycolysis-associated lncRNA-regulated subtypes having differential overall survival (OS), clinical features, genomic mutation profiles, and TME profiles along with nuclear immunotherapeutic responses were identified. Nine lncRNAs localized in the nucleus were identified and transcription factors (TFs) significantly negatively associated with these were found to be enriched in multiple oncogenic signaling pathways. Among them, three lncRNAs (AC093673.5, AC034220.3, and RP11-250B2.3) exerted the most profound effects on glycolysis and constituted the lncRNA signature, which could substantially distinguish the risk levels among different BC patients. Four glycolysis-associated lncRNA-regulated subtypes were identified in this study, reflective of the biological characteristics and heterogeneity of BC. Three key glycolysis-associated lncRNA constituting a signature could predict the risk levels in BC, provide a reference for stratification, and be used as prognostic markers for BC diagnosis and treatment.

Multi-Omics Characterization of Tumor Microenvironment Heterogeneity and Immunotherapy Resistance Through Cell States-Based Subtyping in Bladder Cancer.

Due to the strong heterogeneity of bladder cancer (BC), there is often substantial variation in the prognosis and efficiency of immunotherapy among BC patients. For the precision treatment and assessment of prognosis, the subtyping of BC plays a critical role. Despite various subtyping methods proposed previously, most of them are based on a limited number of molecules, and none of them is developed on the basis of cell states. In this study, we construct a single-cell atlas by integrating single cell RNA-seq, RNA microarray, and bulk RNA-seq data to identify the absolute proportion of 22 different cell states in BC, including immune and nonimmune cell states derived from tumor tissues. To explore the heterogeneity of BC, BC was identified into four different subtypes in multiple cohorts using an improved consensus clustering algorithm based on cell states. Among the four subtypes, C1 had median prognosis and best overall response rate (ORR), which characterized an immunosuppressive tumor microenvironment. C2 was enriched in epithelial-mesenchymal transition/invasion, angiogenesis, immunosuppression, and immune exhaustion. Surely, C2 performed the worst in prognosis and ORR. C3 with worse ORR than C2 was enriched in angiogenesis and almost nonimmune exhaustion. Displaying an immune effective environment, C4 performed the best in prognosis and ORR. We found that patients with just an immunosuppressive environment are suitable for immunotherapy, but patients with an immunosuppressive environment accompanied by immune exhaustion or angiogenesis may resist immunotherapy. Furthermore, we conducted exploration into the heterogeneity of the transcriptome, mutational profiles, and somatic copy-number alterations in four subtypes, which could explain the significant differences related to cell states in prognosis and ORR. We also found that PD-1 in immune and tumor cells could both influence ORR in BC. The level of TGFβ in a cell state can be opposite to the overall level in the tissues, and the level in a specific cell state could predict ORR more accurately. Thus, our work furthers the understanding of heterogeneity and immunotherapy resistance in BC, which is expected to assist clinical practice and serve as a supplement to the current subtyping method from a novel perspective of cell states.

Unique somatic variants in DNA from urine exosomes of individuals with bladder cancer

Bladder cancer (BC), a heterogeneous disease characterized by high recurrence rates, is diagnosed and monitored by cystoscopy. Accurate clinical staging based on biopsy remains a challenge, and additional, objective diagnostic tools are needed urgently. We used exosomal DNA (exoDNA) as an analyte to examine cancer-associated mutations and compared the diagnostic utility of exoDNA from urine and serum of individuals with BC. In contrast to urine exosomes from healthy individuals, urine exosomes from individuals with BC contained significant amounts of DNA. Whole-exome sequencing of DNA from matched urine and serum exosomes, bladder tumors, and normal tissue (peripheral blood mononuclear cells) identified exonic and 3′ UTR variants in frequently mutated genes in BC, detectable in urine exoDNA and matched tumor samples. Further analyses identified somatic variants in driver genes, unique to urine exoDNA, possibly because of the inherent intra-tumoral heterogeneity of BC, which is not fully represented in random small biopsies. Multiple variants were also found in untranslated portions of the genome, such as microRNA (miRNA)-binding regions of the KRAS gene. Gene network analyses revealed that exoDNA is associated with cancer, inflammation, and immunity in BC exosomes. Our findings show utility of exoDNA as an objective, non-invasive strategy to identify novel biomarkers and targets for BC.

Tumor heterogeneity and the potential role of liquid biopsy in bladder cancer.

Bladder cancer (BC) is a heterogeneous disease that characterized by genomic instability and a high mutation rate. Heterogeneity in tumor may partially explain the diversity of responses to targeted therapies and the various clinical outcomes. A combination of cytology and cystoscopy is the standard methodology for BC diagnosis, prognosis, and disease surveillance. However, genomics analyses of single tumor‐biopsy specimens may underestimate the mutational burden of heterogeneous tumors. Liquid biopsy, as a promising technology, enables analysis of tumor components in the bodily fluids, such as blood and urine, at multiple time points and provides a minimally invasive approach that can track the evolutionary dynamics and monitor tumor heterogeneity. In this review, we describe the multiple faces of BC heterogeneity at the genomic and transcriptional levels and how they affect clinical care and outcomes. We also summarize the outcomes of liquid biopsy in BC, which plays a potential role in revealing tumor heterogeneity. Finally, we discuss the challenges that must be addressed before liquid biopsy can be widely used in clinical treatment.

TRNA-Derived Fragments (tRFs) in Bladder Cancer: Increased 5'-tRF-LysCTT Results in Disease Early Progression and Patients' Poor Treatment Outcome.

Simple SummaryBladder cancer (BlCa) management relies on lifelong surveillance strategies with invasive interventions that adversely affect patients’ quality-of-life and lead to a high economic burden for healthcare systems. Exploitation of bladder tumors’ molecular background could lead to modern precision medicine. tRNA-derived fragments (tRFs), rather than degradation debris, are novel functional small ncRNAs that have emerged as key regulators of cellular homeostasis. This is the first study of the clinical utility of tRFs in BlCa. Using in silico analysis of the TCGA-BLCA project, we identified 5′-tRF-LysCTT (5′-tRF of tRNALysCTT) to be significantly deregulated in BlCa, and we have studied its clinical value in our cohort of 230 BlCa patients. Elevated 5′-tRF-LysCTT levels were significantly associated with aggressive tumor phenotype as well as early disease progression and poor treatment outcome. Integration of 5′-tRF-LysCTT with established disease markers resulted in superior prediction of patients’ prognosis, supporting personalized treatment and monitoring decisions.The heterogeneity of bladder cancer (BlCa) prognosis and treatment outcome requires the elucidation of tumors’ molecular background towards personalized patients’ management. tRNA-derived fragments (tRFs), although originally considered as degradation debris, represent a novel class of powerful regulatory non-coding RNAs. In silico analysis of the TCGA-BLCA project highlighted 5′-tRF-LysCTT to be significantly deregulated in bladder tumors, and 5′-tRF-LysCTT levels were further quantified in our screening cohort of 230 BlCa patients. Recurrence and progression for non-muscle invasive (NMIBC) patients, as well as progression and patient’s death for muscle-invasive (MIBC) patients, were used as clinical endpoint events. TCGA-BLCA were used as validation cohort. Bootstrap analysis was performed for internal validation and the clinical net benefit of 5′-tRF-LysCTT on disease prognosis was assessed by decision curve analysis. Elevated 5′-tRF-LysCTT was associated with unfavorable disease features, and significant higher risk for early progression (multivariate Cox: HR = 2.368; p = 0.033) and poor survival (multivariate Cox: HR = 2.151; p = 0.032) of NMIBC and MIBC patients, respectively. Multivariate models integrating 5′-tRF-LysCTT with disease established markers resulted in superior risk-stratification specificity and positive prediction of patients’ progression. In conclusion, increased 5′-tRF-LysCTT levels were strongly associated with adverse disease outcome and improved BlCa patients’ prognostication.

Abstract A29: Multiplex functional genomics assay to deconvolute compound genetic drivers of bladder cancer

Abstract Next-generation sequencing has led to substantial insights into the genetic and molecular heterogeneity of bladder cancer. However, the functional consequences of many of these genetic abnormalities have not been explored. Current genetically defined models of tumorigenesis are poorly suited for systematic functional interrogation of the diverse genetic abnormalities in bladder cancer. To address this issue, we are developing an approach that is readily scalable and leverages an established organoid transformation assay, multiplex transduction of organoids by a lentiviral pool, and digital profiling by single-cell sequencing. We found that C57BL/6J mouse basal urothelial cells (Lin−EpCAM+CD49fhigh) efficiently initiated organoids in 3D culture in defined media conditions. Urothelial organoids were readily transduced with lentivirus to achieve viral copy numbers (VCNs) of up to 20 with a linear relationship between VCN and lentiviral dose. As lentiviral transduction follows a Poisson distribution, we reasoned that a lentiviral pool could be used to introduce random combinations of genetic alterations into urothelial organoids. We selected 33 recurrently altered genes in bladder cancer and categorized them as gain of function (genomic amplification and activating mutations) and loss of function (genomic deletion and inactivating mutations). We cloned lentiviral constructs that express open reading frames to recapitulate gain-of-function events and short hairpin RNAs to model loss-of-function events. Each lentiviral clone has also been barcoded with matching 10-nucleotide sequences at both the 5' and 3' ends to facilitate barcode enumeration by DNA amplicon sequencing and RNA sequencing. In preliminary experiments, we have transduced mouse urothelial organoids with the barcoded lentiviral pool to target a mean viral copy number of 4. After a brief period of culture, organoids were grafted subcutaneously into immune-deficient mice to biologically select for tumorigenic clones. Resultant tumors will be isolated for single-cell DNA amplicon sequencing to enable the enumeration of combinations of oncogenic events within each tumor clone that induce bladder cancer tumorigenesis. Optimization of single-cell RNA sequencing is ongoing to allow direct, massively parallel association of genotype to bladder cancer phenotype within this assay. Overall, we are establishing a functional cancer genomics platform to provide rapid functional annotation of genetic alterations and their roles in bladder cancer initiation. Another goal of these studies is the generation of multiple genetically defined models of bladder cancer that recapitulate the spectrum of human disease. Citation Format: Alicia Wong, Huiyun Sun, Sujata Jana, Andrew C. Hsieh, John K. Lee. Multiplex functional genomics assay to deconvolute compound genetic drivers of bladder cancer [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A29.

Abstract PR02: Profiling intratumoral heterogeneity of bladder cancer subtypes at the single-cell level using machine-learning assisted histopathology

Abstract Purpose: Machine-learning assisted histopathology using markers of basal and luminal differentiation was employed to profile the intratumoral heterogeneity of bladder cancer from cystectomy patients and predict disease-free survival in this high-risk patient population. Methods: Urothelial carcinomas are biologically heterogeneous and vary greatly in clinical progression as well as treatment response. Delineation of molecular subtypes by gene expression analysis of luminal and basal markers has indicated differential outcomes associated with basal and luminal subtypes. However, histologic validation of this classification using protein markers (basal = KRT5/6, P63; luminal = KRT20/GATA3) has been challenging. While using multiplex-immunofluorescence to subtype a retrospective cystectomy cohort (a TMA of 380 patients), we determined that nearly 50% of tumors did not exhibit cytokeratin markers. Subtyping was further confounded by frequent loss of basal-to-luminal stratification and the emergence of intratumoral spatial heterogeneity with the basal and luminal subtypes being completely intermixed throughout the tumor. These observations caused us to hypothesize that previously undefined but clinically relevant subtypes might exist. To address this challenge we developed a single-cell image analysis pipeline that leveraged machine learning to classify molecular subtype and spatial heterogeneity within each tumor. Using the informatics software KNIME we achieved single-cell segmentation and extracted 285 features for 5 protein markers (P63, GATA3, collagen, nuclear stain, and pan-cytokeratin) from each ~20,000 cells contained in 2 cores of tumor and adjacent benign for each patient. Under guidance from a pathologist, definitive urothelial cells (luminal, intermediate, and basal cells) as well as stromal cells were selected from 25 cores normal urothelium to form the ground truth for XGboost-based machine-learning. Summary Findings: Single-cell profiling with machine learning on transcription factors could classify basal and luminal subtypes with greater than 97% accuracy according to validation in normal urothelium using keratin markers. While we were able to recapitulate differential survival associated with a pure basal subtype, it was the intratumoral heterogeneity of basal and luminal cells that was the predominant driver of disease-free survival. Conclusion: A newly identified bladder cancer subtype defined by intratumoral heterogeneity is a clinically relevant driver of disease-free survival. This abstract is also being presented as Poster B22. Citation Format: Adel Eskaros, Tatiana Novitskaya, Andries Zijlstra. Profiling intratumoral heterogeneity of bladder cancer subtypes at the single-cell level using machine-learning assisted histopathology [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr PR02.

Three-Dimensional Microtumors for Probing Heterogeneity of Invasive Bladder Cancer.

Bladder cancer is an increasingly common malignancy, and muscle invasive bladder cancer is associated with particularly high rates of morbidity and mortality. The morphologic and molecular diversity of bladder cancer poses significant challenges in elucidating the invasion mechanisms responsible for disease progression. Furthermore, conventional invasion assays do not provide a physiological context for studying bladder cancer invasion within 3D microenvironments and have limited ability to capture the contribution of cellular phenotypic heterogeneity to disease progression. Here, we describe the development of a 3D microtumor invasion model suitable for the analysis of cellular phenotypic heterogeneity in cell lines and primary tumor cells from bladder cancer patients. This model incorporates a self-assembly approach for recapitulating features of bladder cancer invasion in 3D microenvironments and probing the invasive cell subpopulations. The gene expression profiles of invading microtumors were analyzed by incorporating a gold nanorod-locked nucleic acid biosensor. The incorporation of the single cell biosensor and transient gene knockdown into the system revealed the formation of invasive leader cells with upregulated Delta-like ligand 4 (DLL4) expression as well as the role of NOTCH1-DLL4 signaling in collective bladder cancer invasion. The involvement of DLL4 expressing cells in bladder cancer invasion was also observed in patient samples obtained from transurethral resection. Collectively, our study demonstrates a 3D microtumor invasion model for investigating intracellular heterogeneity of bladder cancer invasion and analyzing patient derived samples toward personalized medicine applications.