Non-muscle-invasive bladder cancer (NMIBC) is a heterogeneous disease with widely different outcomes. We performed a comprehensive transcriptional analysis of 460 early-stage urothelial carcinomas and showed that NMIBC can be subgrouped into three major classes with basal- and luminal-like characteristics and different clinical outcomes. Large differences in biological processes such as the cell cycle, epithelial-mesenchymal transition, and differentiation were observed. Analysis of transcript variants revealed frequent mutations in genes encoding proteins involved in chromatin organization and cytoskeletal functions. Furthermore, mutations in well-known cancer driver genes (e.g., TP53 and ERBB2) were primarily found in high-risk tumors, together with APOBEC-related mutational signatures. The identification of subclasses in NMIBC may offer better prognostication and treatment selection based on subclass assignment.

PURPOSE Novel sensitive methods for early detection of relapse and for monitoring therapeutic efficacy may have a huge impact on risk stratification, treatment, and ultimately outcome for patients with bladder cancer. We addressed the prognostic and predictive impact of ultra-deep sequencing of cell-free DNA in patients before and after cystectomy and during chemotherapy. PATIENTS AND METHODS We included 68 patients with localized advanced bladder cancer. Patient-specific somatic mutations, identified by whole-exome sequencing, were used to assess circulating tumor DNA (ctDNA) by ultra-deep sequencing (median, 105,000×) of plasma DNA. Plasma samples (n = 656) were procured at diagnosis, during chemotherapy, before cystectomy, and during surveillance. Expression profiling was performed for tumor subtype and immune signature analyses. RESULTS Presence of ctDNA was highly prognostic at diagnosis before chemotherapy (hazard ratio, 29.1; P = .001). After cystectomy, ctDNA analysis correctly identified all patients with metastatic relapse during disease monitoring (100% sensitivity, 98% specificity). A median lead time over radiographic imaging of 96 days was observed. In addition, for high-risk patients (ctDNA positive before or during treatment), the dynamics of ctDNA during chemotherapy was associated with disease recurrence ( P = .023), whereas pathologic downstaging was not. Analysis of tumor-centric biomarkers showed that mutational processes (signature 5) were associated with pathologic downstaging ( P = .024); however, no significant correlation for tumor subtypes, DNA damage response mutations, and other biomarkers was observed. Our results suggest that ctDNA analysis is better associated with treatment efficacy compared with other available methods. CONCLUSION ctDNA assessment for early risk stratification, therapy monitoring, and early relapse detection in bladder cancer is feasible and provides a basis for clinical studies that evaluate early therapeutic interventions.

Exosomes are small secreted vesicles that can transfer their content to recipient cells. In cancer, exosome secretion has been implicated in tumor growth and metastatic spread. In this study, we explored the possibility that exosomal pathways might discard tumor-suppressor miRNA that restricts metastatic progression. Secreted miRNA characterized from isogenic bladder carcinoma cell lines with differing metastatic potential were uncoupled from binding to target transcripts or the AGO2-miRISC complex. In metastatic cells, we observed a relative increase in secretion of miRNA with tumor-suppressor functions, including miR23b, miR224, and miR921. Ectopic expression of miR23b inhibited invasion, anoikis, angiogenesis, and pulmonary metastasis. Silencing of the exocytotic RAB family members RAB27A or RAB27B halted miR23b and miR921 secretion and reduced cellular invasion. Clinically, elevated levels of RAB27B expression were linked to poor prognosis in two independent cohorts of patients with bladder cancer. Moreover, highly exocytosed miRNA from metastatic cells, such as miR23b, were reduced in lymph node metastases compared with patient-matched primary tumors and were correlated with increments in miRNA-targeted RNA. Taken together, our results suggested that exosomemediated secretion of tumor-suppressor miRNA is selected during tumor progression as a mechanism to coordinate activation of a metastatic cascade. Cancer Res; 74(20); 5758-71. Ó2014 AACR.

The molecular landscape in non-muscle-invasive bladder cancer (NMIBC) is characterized by large biological heterogeneity with variable clinical outcomes. Here, we perform an integrative multi-omics analysis of patients diagnosed with NMIBC (n = 834). Transcriptomic analysis identifies four classes (1, 2a, 2b and 3) reflecting tumor biology and disease aggressiveness. Both transcriptome-based subtyping and the level of chromosomal instability provide independent prognostic value beyond established prognostic clinicopathological parameters. High chromosomal instability, p53-pathway disruption and APOBEC-related mutations are significantly associated with transcriptomic class 2a and poor outcome. RNA-derived immune cell infiltration is associated with chromosomally unstable tumors and enriched in class 2b. Spatial proteomics analysis confirms the higher infiltration of class 2b tumors and demonstrates an association between higher immune cell infiltration and lower recurrence rates. Finally, the independent prognostic value of the transcriptomic classes is documented in 1228 validation samples using a single sample classification tool. The classifier provides a framework for biomarker discovery and for optimizing treatment and surveillance in next-generation clinical trials.

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Overtreatment with cisplatin-based chemotherapy is a major issue in the management of muscle-invasive bladder cancer (MIBC), and currently none of the reported biomarkers for predicting response have been implemented in the clinic. Here we perform a comprehensive multi-omics analysis (genomics, transcriptomics, epigenomics and proteomics) of 300 MIBC patients treated with chemotherapy (neoadjuvant or first-line) to identify molecular changes associated with treatment response. DNA-based associations with response converge on genomic instability driven by a high number of chromosomal alterations, indels, signature 5 mutations and/or BRCA2 mutations. Expression data identifies the basal/squamous gene expression subtype to be associated with poor response. Immune cell infiltration and high PD-1 protein expression are associated with treatment response. Through integration of genomic and transcriptomic data, we demonstrate patient stratification to groups of low and high likelihood of cisplatin-based response. This could pave the way for future patient selection following validation in prospective clinical trials.