Comprehensive Molecular Characterization of Muscle-Invasive Bladder Cancer.

We report a comprehensive analysis of 412 muscle-invasive bladder cancers characterized by multiple TCGA analytical platforms. Fifty-eight genes were significantly mutated, and the overall mutational load was associated with APOBEC-signature mutagenesis. Clustering by mutation signature identified a high-mutation subset with 75% 5-year survival. mRNA expression clustering refined prior clustering analyses and identified a poor-survival "neuronal" subtype in which the majority of tumors lacked small cell or neuroendocrine histology. Clustering by mRNA, long non-coding RNA (lncRNA), and miRNA expression converged to identify subsets with differential epithelial-mesenchymal transition status, carcinoma in situ scores, histologic features, and survival. Our analyses identified 5 expression subtypes that may stratify response to different treatments.

Comparison of clinicopathological characteristics, gene expression profiles, mutational analysis, and clinical outcomes of pure and mixed small-cell carcinoma of the bladder.

AIMS: Small cell bladder carcinoma (SCBC) is a rare, divergent form of urothelial carcinoma (UC). We aimed to determine whether pure (n = 16) and mixed (SCBC and UC; n = 30) tumours differed in pathology, gene expression characteristics, genetic alterations, and clinical outcomes. METHODS AND RESULTS: Forty (87%) patients received first-line chemotherapy. Twenty-nine patients had no metastatic disease at diagnosis and underwent radical cystectomy. There were no differences in age, sex, race distribution, tumour size, stage at presentation, therapy response with pathological downstaging to ≤ypT1N0, or overall or progression-free survival (PFS) between pure and mixed tumours. There was a longer PFS among downstaged chemotherapy-responding tumours ≤ypT2N0M0 than among unresponsive tumours ≥ypT2 ≥ yN1M1 (P = 0.001). Patients who achieved pathological downstaging with neoadjuvant chemotherapy (n = 10) were stage cT2N0M0 at the time of diagnosis and were alive at the last follow-up (median 37 months), while 46% of patients who failed to achieve pathological downstaging were alive at the last follow-up (median 38 months; P = 0.008). RNA sequencing showed that the UC of mixed SCBC had similar neural expression signatures to pure SCBC. DNA sequencing revealed alterations in TERT (83%), P53 (56%), ARID1A (28%), RB1 (22%), and BRCA2 (11%). Immunohistochemistry for RB1 showed loss of expression in 18/19 (95%) patients, suggesting frequent pathway downregulation despite a low prevalence of RB1 mutation. CONCLUSION: Patients with pure and mixed SCBC have similar outcomes and these outcomes are determined by the pathological stage at RC and are best among patients who have pathological downstaging after NAC.

BCG invokes superior STING-mediated innate immune response over radiotherapy in a carcinogen murine model of urothelial cancer.

Radiation and bacillus Calmette-Guérin (BCG) instillations are used clinically for treatment of urothelial carcinoma, but the precise mechanisms by which they activate an immune response remain elusive. The role of the cGAS-STING pathway has been implicated in both BCG and radiation-induced immune response; however, comparison of STING pathway molecules and the immune landscape following treatment in urothelial carcinoma has not been performed. We therefore comprehensively analyzed the local immune response in the bladder tumor microenvironment following radiotherapy and BCG instillations in a well-established spontaneous murine model of urothelial carcinoma to provide insight into activation of STING-mediated immune response. Mice were exposed to the oral carcinogen, BBN, for 12 weeks prior to treatment with a single 15 Gy dose of radiation or three intravesical instillations of BCG (1 × 108 CFU). At sacrifice, tumors were staged by a urologic pathologist and effects of therapy on the immune microenvironment were measured using the NanoString Myeloid Innate Immunity Panel and immunohistochemistry. Clinical relevance was established by measuring immune biomarker expression of cGAS and STING on a human tissue microarray consisting of BCG-treated non-muscle-invasive urothelial carcinomas. BCG instillations in the murine model elevated STING and downstream STING-induced interferon and pro-inflammatory molecules, intratumoral M1 macrophage and T-cell accumulation, and complete tumor eradication. In contrast, radiotherapy caused no changes in STING pathway or innate immune gene expression; rather, it induced M2 macrophage accumulation and elevated FoxP3 expression characteristic of immunosuppression. In human non-muscle-invasive bladder cancer, STING protein expression was elevated at baseline in patients who responded to BCG therapy and increased further after BCG therapy. Overall, these results show that STING pathway activation plays a key role in effective BCG-induced immune response and strongly indicate that the effects of BCG on the bladder cancer immune microenvironment are more beneficial than those induced by radiation. © 2021 The Pathological Society of Great Britain and Ireland.

Race reporting and disparities regarding clinical trials in bladder cancer: a systematic review.

PURPOSE: To systematically review the literature to investigate racial disparities among bladder cancer clinical trial enrollees. METHODS: A systematic review was conducted using Ovid, MEDLINE® to identify clinical trials between 1970 and 2020. Articles were reviewed and were included if they assessed race in their outcomes reporting among bladder cancer patients enrolled in clinical trials. The review was conducted in accordance with the PRISMA statement. RESULTS: We identified 544 clinical trials meeting our initial search criteria, with only 24 (4.4%) studies reporting racial demographic data. Enrollees were largely Caucasian (81-98%), with a strikingly small proportion of enrolled patients consisting of African-Americans (2-8%) and Hispanics (2-5%). Only one of the studies reported results on the efficacy and safety/tolerability of the tested treatment separately for racial groups and performed analyses stratified by race. CONCLUSION: Race is poorly studied in bladder cancer clinical trials. Trial cohorts may not reflect multicultural populations. The potential association between race and efficacy, safety or tolerability of the tested interventions is unknown. Given the up to twofold increase in bladder cancer-specific death among African-Americans, further research is needed to address the impact of race in clinical trials, while encompassing socioeconomic factors and disease risk factor exposures.

A miRNA signature predicts benefit from addition of hypoxia-modifying therapy to radiation treatment in invasive bladder cancer.

BACKGROUND: miRNAs are promising biomarkers in oncology as their small size makes them less susceptible to degradation than mRNA in FFPE tissue. We aimed to derive a hypoxia-associated miRNA signature for bladder cancer. METHODS: Taqman miRNA array cards identified miRNA seed genes induced under hypoxia in bladder cancer cell lines. A signature was derived using feature selection methods in a TCGA BLCA training data set. miRNA expression data were generated for 190 tumours from the BCON Phase 3 trial and used for independent validation. RESULTS: A 14-miRNA hypoxia signature was derived, which was prognostic for poorer overall survival in the TCGA BLCA cohort (n = 403, p = 0.001). Univariable analysis showed that the miRNA signature predicted an overall survival benefit from having carbogen-nicotinamide with radiotherapy (HR = 0.30, 95% CI 0.094-0.95, p = 0.030) and performed similarly to a 24-gene mRNA signature (HR = 0.47, 95% CI 0.24-0.92, p = 0.025). Combining the signatures improved performance (HR = 0.26, 95% CI 0.08-0.82, p = 0.014) with borderline significance for an interaction test (p = 0.065). The interaction test was significant for local relapse-free survival LRFS (p = 0.033). CONCLUSION: A 14-miRNA hypoxia signature can be used with an mRNA hypoxia signature to identify bladder cancer patients benefitting most from having carbogen and nicotinamide with radiotherapy.

Expression Analysis of Same-Patient Metachronous and Synchronous Upper Tract and Bladder Urothelial Carcinoma.

PURPOSE: We compared upper tract urothelial carcinoma (UTUC) and bladder urothelial carcinoma (BUC) in same-patient metachronous UTUC and synchronous UTUC and BUC using next-generation sequencing. MATERIALS AND METHODS: Consecutive untreated same-patient samples of UTUC and BUC were macrodissected from unstained formalin-fixed, paraffin-embedded slides after quality control. Samples were divided into 4 groups: 1) UTUC-metachronous BUC, 2) BUC-metachronous UTUC, 3) synchronous UTUC-BUC, 4) UTUC without BUC. Exclusions were inadequate clinical data or histological tumor purity <30%. Whole transcriptome RNA sequencing was performed. After quality assessment, gene expression clusters using unsupervised hierarchical consensus clustering and correlation with pertinent clinicopathologic variables, a prior RNASeq data set and other published data were performed. RESULTS: RNAseq was performed on 95 samples (UTUC=61, BUC=34) from 40 untreated patients. Unsupervised consensus clustering segregated the tumors into 2 clusters that were enriched with BASE47 basal-like or luminal-like gene expression. Almost two-thirds (61.9%) of Group 2 tumors were basal-like, while the majority of Groups 1, 3, 4 (80.6%, 70.0% and 69.6%, respectively) were luminal-like (p=0.017). Further analyses revealed that the differences in basal-like and luminal-like gene expression were associated with differential fibroblast and immune cell gene expression signatures. In all, 87.5% of metachronous tumors maintained subtype membership. CONCLUSIONS: Gene expression analysis of same-patient metachronous UTUC-BUC suggests that the majority of mUTUC developing after BUC appear more basal-like, while synchronous and initial UTUC tumors appear luminal-like. Metachronous tumors largely maintain molecular subtype membership of the initial tumor regardless of chronologic development or anatomical origin.

Mutational Landscape and Environmental Effects in Bladder Cancer.

Bladder cancer is the most common cancer of the urinary tract. Although nonmuscle-invasive bladder cancers have a good prognosis, muscle-invasive bladder cancers promote metastases and have a poor prognosis. Comprehensive analyses using RNA sequence of clinical tumor samples in bladder cancer have been reported. These reports implicated the candidate genes and pathways that play important roles in carcinogenesis and/or progression of bladder cancer. Further investigations for the function of each mutation are warranted. There is suggestive evidence for several environmental factors as risk factors of bladder cancer. Environmental factors such as cigarette smoking, exposure to chemicals and gases, bladder inflammation due to microbial and parasitic infections, diet, and nutrition could induce several genetic mutations and alter the tumor microenvironment, such as immune cells and fibroblasts. The detailed mechanism of how these environmental factors induce carcinogenesis and/or progression of bladder cancer remains unclear. To identify the relationship between the mutations and the lifestyle could be useful for prevention and treatment of bladder cancer.

SIU-ICUD consultation on bladder cancer: basic science.

PURPOSE: To provide a condensed summary of the Basic Science chapter that was included in the Third International Consultation on Bladder Cancer. METHODS: World bladder cancer basic science experts used the published literature to create summaries of recent progress in their areas of expertise. RESULTS: The completion of several large-scale genomics projects coupled with a strong collaborative culture within the research community and the exciting clinical activity of immune checkpoint blockade have combined to transform the bladder cancer research landscape. Bladder cancer molecular subtypes and the presence of specific DNA alterations provide important information about disease heterogeneity that has direct implications for clinical management, and some can be targeted by compounds that are already clinically available. Tests are being developed that can measure many of these alterations non-invasively in peripheral blood or urine, raising confidence that they could be used as biomarkers for surveillance and monitoring the effects of local and systemic therapies. CONCLUSIONS: Although the bulk of the mechanistic work lies ahead, the genomics results have created a hypothesis-generating description of bladder cancer heterogeneity that has set the stage for deeper mechanistic studies, and they have already provided us with extremely attractive candidate biomarkers to guide clinical practice. Here, we will summarize the recent progress in basic bladder cancer research and highlight near-term opportunities for the future.

The development of interferon-based gene therapy for BCG unresponsive bladder cancer: from bench to bedside.

BACKGROUND AND PURPOSE: BCG unresponsive bladder cancer is an inherently resistant disease state for which the preferred treatment is radical cystectomy. To date, no effective intravesical therapies exist for patients who possess these resistant tumors. For this reason, many research groups are actively investigating/testing novel therapeutic agents to aid in bladder preservation for this patient population. This review article describes our 15-year experience developing and testing IFN-based gene therapy. METHODS: A comprehensive review was performed of all studies pertaining to IFN-based gene therapy for non-muscle invasive bladder cancer from 2003 to 2018. RESULTS AND CONCLUSIONS: Over the past two decades, gene therapy has evolved into a powerful tool in our fight against cancer. After overcoming the initial barriers associated with gene delivery to the bladder, we have made significant strides forward in developing this novel therapeutic strategy for the treatment of this inherently resistant disease state. Our results to date are very encouraging; however, much work lies ahead to better understand and optimize this novel approach for treating non-muscle invasive bladder.

Clinical implications of molecular subtyping in bladder cancer.

PURPOSE OF REVIEW: The purpose of this review is to examine and evaluate similarities and differences in bladder cancer expression subtypes and to understand the clinical implications of the molecular subtyping. RECENT FINDINGS: Four independent classification systems have been described, and there are broad similarities among the subtyping callers. Two major subtypes have been identified, that is, luminal and basal, with underlying subcategories based on various distinct characteristics. Luminal tumors generally bear a better prognosis and increased survival than basal tumors, although there is subtle variation in prognosis among the different subtypes within the luminal and basal classifications. Clinical subtyping is now commercially available, although there are limitations to its generalizability and application. SUMMARY: Expression subtyping is a new method to personalize bladder cancer management. However, there is probably not sufficient evidence to incorporate use into current standards-of-care. Validation cohorts with clinically meaningful outcomes may further establish the clinical relevance of molecular subtyping of bladder cancer. Additionally, genetic alterations in bladder cancer may 'color' the interpretation of individual tumors beyond the expression subtype to truly personalize care for bladder cancer.

Predictive biomarkers for drug response in bladder cancer.

Bladder cancer is a heterogeneous disease. Interpatient heterogeneity in response to a drug limits treatment options and impairs improvement of patient survival. For example, approximately half of patients do not respond to cisplatin-based combination chemotherapy, although it is the standard of care for muscle-invasive and metastatic bladder cancer. The development of robust predictive biomarkers is expected to improve outcomes by enabling clinicians to use chemotherapy only in the patients who will benefit from it. Recent advances in the molecular characterization of bladder cancer showed that the basal subtype of bladder cancer and tumors with inactivating mutations in DNA damage repair genes were associated with greater benefit from cisplatin-based chemotherapy. The present review summarizes current efforts to develop predictive biomarkers for drug response in bladder cancer, focusing on those that predict the response to cisplatin-based chemotherapy for advanced bladder cancer. We also review the current situation with regard to the identification of predictive biomarkers for response to intravesical therapy, immune checkpoint inhibitors and molecularly-targeted drugs. We also discuss the future applications of new technologies, including liquid biopsies and patient-derived organoids that will also serve as resources for the identification of biomarkers in bladder cancer.

CD24 regulates cancer stem cell (CSC)-like traits and a panel of CSC-related molecules serves as a non-invasive urinary biomarker for the detection of bladder cancer.

BACKGROUND: CD24 is a cornerstone of tumour progression in urothelial carcinoma of the bladder (UCB). However, its contribution to cancer stem cell (CSC)-like traits and the clinical utility of CD24 as a urinary biomarker for cancer detection have not been determined. METHODS: The functional relevance of CD24 was evaluated using in vitro and in vivo approaches. The clinical utility of CSC-related molecules was assessed in urine samples by quantitative RT-PCR. RESULTS: The knockdown of CD24 attenuated cancer stemness properties. The high-CD24-expressing cells, isolated from patient-derived UCB xenograft tumours, exhibited their enhanced stemness properties. CD24 was overexpressed not only in primary tumours but also in urine from UCB subjects. By assessment of 15 candidate CSC-related molecules in urine samples of a training cohort, a panel of three molecules (CD24, CD49f, and NANOG) was selected. The combination of these three molecules yielded a sensitivity and specificity of 81.7% and 74.3%, respectively, in an independent cohort. A combined set of 84 cases and 207 controls provided a sensitivity and specificity of 82% and 76%, respectively. CONCLUSION: CD24 has a crucial role in maintaining the urothelial cancer stem-like traits and a panel of CSC-related molecules has potential as a urinary biomarker for non-invasive UCB detection.

Comparison of Pathological Stage in Patients Treated with and without Neoadjuvant Chemotherapy for High Risk Upper Tract Urothelial Carcinoma.

PURPOSE: High risk upper tract urothelial carcinoma has been associated with poor survival outcomes. Limited retrospective data support neoadjuvant chemotherapy prior to radical nephroureterectomy. To validate prior findings we evaluated differences in the pathological stage distribution in patients with high risk upper tract urothelial carcinoma based on the administration of neoadjuvant chemotherapy before radical nephroureterectomy. MATERIALS AND METHODS: We retrospectively analyzed the records of 240 patients with upper tract urothelial carcinoma at The Johns Hopkins Hospital from 2003 to 2017. Patients with biopsy proven high grade disease and a visible lesion on cross-sectional imaging were offered neoadjuvant chemotherapy prior to radical nephroureterectomy. A control group of a time matched cohort of patients with biopsy proven high grade disease underwent extirpative surgery alone. The chi-square and Fisher exact tests were used to evaluate clinical and pathological variables between the cohorts. RESULTS: There were 32 patients in the study group and 208 in the control group. Significantly lower pathological stage was noted in the study group than in the control group (p <0.001). Significantly fewer patients with pT2 disease or higher were treated with neoadjuvant chemotherapy (37.5% vs 59.6%, p = 0.02). There was a 46.5% reduction in the prevalence of pT3 disease or higher in study group patients without clinically node positive or low volume metastatic disease (25.9% vs 48.4%, p = 0.04). A 9.4% complete remission rate was observed in patients who underwent neoadjuvant chemotherapy. CONCLUSIONS: Patients with high risk upper tract urothelial carcinoma treated with neoadjuvant chemotherapy were noted to have a lower pathological stage distribution than patients treated with radical nephroureterectomy alone.

Effects of thiazolidinedione in patients with active bladder cancer.

OBJECTIVE: To examine the influence of perioperative thiazolidinedione (TZD) on cancer-specific outcomes in patients with diabetes mellitus (DM) undergoing radical cystectomy (RC) for urothelial carcinoma (UC). PATIENTS AND METHODS: A retrospective cohort of 173 patients with DM undergoing RC from 2005 to 2010 was identified. Of those, 53 were on TZD treatment at the time of RC, with 33 patients taking pioglitazone. Baseline clinicopathological characteristics, as well as cancer-specific survival (CSS), recurrence-free survival (RFS), and overall survival (OS) were compared between the patients on and off TZD therapy at the time of RC. In subgroup analysis, outcomes in patients specifically taking pioglitazone at the time of RC were compared to those not on a TZD. RESULTS: Baseline clinicopathological characteristics were similar between patients on and off TZD therapy at the time of RC. Overall, the median CSS rate was not reached in either group (P = 0.7). The estimated 5-year CSS was 67.8% in the non-TZD group and 66.3% in the TZD group. On multivariate analysis incorporating patient age, pathological T-staging, and adjuvant chemotherapy, TZD use was found not to be a significant predictor for CSS (hazard ratio 1.20, 95% confidence interval 0.66-2.17; P = 0.5). Additionally, RFS (P= 0.3) and OS (P = 0.2) were also similar between the two groups without adjusting for other variables. Comparison between patients taking pioglitazone vs patients not taking TZD yielded similar CSS (P = 0.2), RFS (P = 0.5), and OS (P= 0.2). CONCLUSIONS: CSS, as well as RFS and OS after RC were not compromised in patients on TZD therapy at the time of RC. Additional investigation is warranted in patients with non-muscle-invasive bladder cancer and muscle-invasive bladder cancer undergoing bladder-sparing procedures to assess the safety of using TZD in the setting of active UC.

Molecular Subtypes of Bladder Cancer.

PURPOSE OF REVIEW: Recent whole genome characterizations of primary human bladder cancers revealed that they can be grouped into "intrinsic" basal and luminal molecular subtypes. Here, we provide an overview of the subtypes and discuss their biological and clinical properties. RECENT FINDINGS: Basal cancers are characterized by advanced stage and metastatic disease at presentation. They tend to be enriched with squamous and small cell/neuroendocrine features and inactivating mutations and deletions of TP53 and RB1. Basal cancers can be divided into "epithelial" and "mesenchymal" (also known as "claudin low") subsets, and a portion of the latter form a "neuroendocrine/neuronal" subset that is associated with particularly poor survival. Luminal cancers are often enriched with papillary histopathological features and activating mutations in FGFR3, and they can also be divided into additional subsets based on differential stromal cell infiltration, relative genomic instability, and high- versus low-level expression of carcinoma in situ (CIS) gene expression signatures. Importantly, the bladder cancer molecular subtypes display differential sensitivities to neoadjuvant chemotherapy and immune checkpoint blockade, and preliminary data also suggest that they respond differently to radiation with or without hypoxia modulation. Ongoing studies are investigating the relevance of the molecular subtypes to the bladder cancer histopathological variants and to upper tract urothelial cancer. The bladder cancer molecular subtypes were associated with different prognoses and responses to conventional and targeted therapies in retrospective studies. If validated in prospective studies, molecular subtyping will be integrated into bladder cancer clinical management.

The integrated stress response and proteotoxicity in cancer therapy.

A variety of different forms of cellular stress can cause protein misfolding and aggregation and proteotoxicity. The cytoprotective response to proteotoxicity is termed the integrated stress response and involves 4 distinct serine/threonine protein kinases that converge on the translation initiation factor eIF2α, resulting in phosphorylation at S51, cell cycle arrest, and a general inhibition of global protein synthesis. Phosphorylation of eIF2α also promotes translation of ATF4 and the expression of ATF4 target genes that ameliorate proteotoxic stress but can also promote apoptosis. This mini review provides a general overview of these mechanisms and discusses how the inter-tumor heterogeneity that involves them affects sensitivity and resistance to proteasome inhibitors, a new class of cancer therapeutics that promotes tumor cell killing via proteotoxic stress.

Clinical risk stratification in patients with surgically resectable micropapillary bladder cancer.

OBJECTIVE: To analyse survival in patients with clinically localised, surgically resectable micropapillary bladder cancer (MPBC) undergoing radical cystectomy (RC) with and without neoadjuvant chemotherapy (NAC) and develop risk strata based on outcome data. PATIENTS AND METHODS: A review of our database identified 103 patients with surgically resectable (≤cT4acN0 cM0) MPBC who underwent RC. Survival estimates were calculated using Kaplan-Meier method and compared using log-rank tests. Classification and regression tree (CART) analysis was performed to identify risk groups for survival. RESULTS: For the entire cohort, estimated 5-year overall survival and disease-specific survival (DSS) rates were 52% and 58%, respectively. CART analysis identified three risk subgroups: low-risk: cT1, no hydronephrosis; high-risk: ≥cT2, no hydronephrosis; and highest-risk: cTany with tumour-associated hydronephrosis. The 5-year DSS for the low-, high-, and highest-risk groups were 92%, 51%, and 17%, respectively (P < 0.001). Patients down-staged at RC

Genetic subtypes of invasive bladder cancer.

PURPOSE OF REVIEW: Recently completed cancer genomics projects identified intrinsic subtypes in muscle-invasive bladder cancers. Here we will describe the studies that led to their discovery and review their biological and clinical properties. RECENT FINDINGS: Whole genome mRNA expression profiling and unsupervised hierarchical cluster analyses identified intrinsic basal and luminal subtypes in muscle-invasive bladder cancers that are similar to the ones found in breast cancer. Tumors within each subtype have distinct responses to conventional cisplatin-based combination chemotherapy, and they contain gene expression signatures and DNA alterations that may render them vulnerable to clinically available targeted therapies. SUMMARY: Like their breast cancer counterparts, basal bladder cancers are characterized by poor clinical outcomes in the absence of effective systemic therapy, but a large fraction of them do respond to neoadjuvant chemotherapy, suggesting that the tumors should be managed aggressively. On the contrary, tumors that belong to the 'p53-like' subtype tend to be chemoresistant, so patients with these tumors should probably be managed differently. It seems likely that prospective identification of tumor intrinsic subtype membership could complement the use of DNA-based biomarkers to identify the groups of patients who will benefit the most from chemotherapy and targeted agents.

Targeting Signaling Transduction Pathways in Bladder Cancer.

Systemic therapy for urothelial carcinoma (UC) of the bladder has largely revolved around cytotoxic chemotherapy regimens. However, several recent clinical trials have explored the roles of targeted therapies which specifically inhibit signal transduction pathways. Simultaneously, a rationale for such therapies has come to the forefront of management of this disease because an overabundance of signaling pathways are genetically deranged as a result of point mutation or copy number alteration (CNA) as identified by several recent next generation sequencing (NGS) studies. Importantly, these derangements are found in all stages of disease, and therefore targeted therapies hold promise as a next step in the evolution of the medical management of both localized and metastatic UCC. We review the rationale for and progress in studying inhibition of signal transduction as a means of treatment of UCC.