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.

A TRP Channel Senses Lysosome Neutralization by Pathogens to Trigger Their Expulsion.

Vertebrate cells have evolved elaborate cell-autonomous defense programs to monitor subcellular compartments for infection and to evoke counter-responses. These programs are activated by pathogen-associated pattern molecules and by various strategies intracellular pathogens employ to alter cellular microenvironments. Here, we show that, when uropathogenic E. coli (UPEC) infect bladder epithelial cells (BECs), they are targeted by autophagy but avoid degradation because of their capacity to neutralize lysosomal pH. This change is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized to lysosomes. TRPML3 activation then spontaneously initiates lysosome exocytosis, resulting in expulsion of exosome-encased bacteria. These studies reveal a cellular default system for lysosome homeostasis that has been co-opted by the autonomous defense program to clear recalcitrant pathogens.

Creation of bladder assembloids mimicking tissue regeneration and cancer.

Current organoid models are limited by their inability to mimic mature organ architecture and associated tissue microenvironments1,2. Here we create multilayer bladder 'assembloids' by reconstituting tissue stem cells with stromal components to represent an organized architecture with an epithelium surrounding stroma and an outer muscle layer. These assembloids exhibit characteristics of mature adult bladders in cell composition and gene expression at the single-cell transcriptome level, and recapitulate in vivo tissue dynamics of regenerative responses to injury. We also develop malignant counterpart tumour assembloids to recapitulate the in vivo pathophysiological features of urothelial carcinoma. Using the genetically manipulated tumour-assembloid platform, we identify tumoural FOXA1, induced by stromal bone morphogenetic protein (BMP), as a master pioneer factor that drives enhancer reprogramming for the determination of tumour phenotype, suggesting the importance of the FOXA1-BMP-hedgehog signalling feedback axis between tumour and stroma in the control of tumour plasticity.

Ubiquitination of Innate Immune Regulator TRAF3 Orchestrates Expulsion of Intracellular Bacteria by Exocyst Complex.

Although the intracellular trafficking system is integral to most physiologic activities, its role in mediating immune responses to infection has remained elusive. Here, we report that infected bladder epithelial cells (BECs) mobilized the exocyst complex, a powerful exporter of subcellular vesicles, to rapidly expel intracellular bacteria back for clearance. Toll-like receptor (TLR) 4 signals emanating from bacteria-containing vesicles (BCVs) were found to trigger K33-linked polyubiquitination of TRAF3 at Lys168, which was then detected by RalGDS, a guanine nucleotide exchange factor (GEF) that precipitated the assembly of the exocyst complex. Although this distinct modification of TRAF3 served to connect innate immune signaling to the cellular trafficking apparatus, it crucially ensured temporal and spatial accuracy in determining which among the many subcellular vesicles was recognized and selected for expulsion in response to innate immune signaling.

Mutational signature and prognosis in adenocarcinoma of the bladder.

Adenocarcinoma of the bladder is a rare urinary bladder carcinoma with limited therapy options due to lack of molecular characterization. Here, we aimed to reveal the mutational and transcriptomic landscapes of adenocarcinoma of the bladder and assess any relationship with prognosis. Between February 2015 and June 2021, a total of 23 patients with adenocarcinoma of the bladder were enrolled. These included 16 patients with primary bladder adenocarcinomas and seven patients with urachal adenocarcinoma. Whole exome sequencing (16 patients), whole genome sequencing (16 patients), bulk RNA sequencing (RNA-seq) (19 patients), and single-cell RNA-seq (5 patients) were conducted for the specimens. Correlation analysis, survival analysis, and t-tests were also performed. Prevalent T>A substitutions were observed among somatic mutations, and major trinucleotide contexts included 5'-CTC-3' and 5'-CTG-3'. This pattern was mainly contributed by COSMIC signature 22 related to chemical carcinogen exposure (probably aristolochic acid), which has not been reported in bladder adenocarcinoma. Moreover, genes with copy number changes were also enriched in the KEGG term 'chemical carcinogenesis'. Transcriptomic analysis suggested high immune cell infiltration and luminal-like features in the majority of samples. Interestingly, a small fraction of samples with an APOBEC-derived mutational signature exhibited a higher risk of disease progression compared with samples with only a chemical carcinogen-related signature, confirming the molecular and prognostic heterogeneity of bladder adenocarcinoma. This study presents mutational and transcriptomic landscapes of bladder adenocarcinoma, and indicates that a chemical carcinogen-related mutational signature may be related to a better prognosis compared with an APOBEC signature in adenocarcinoma of the bladder. © 2024 The Pathological Society of Great Britain and Ireland.

UPK3A+ umbrella cell damage mediated by TLR3-NR2F6 triggers programmed destruction of urothelium in Hunner-type interstitial cystitis/painful bladder syndrome.

Urothelial damage and barrier dysfunction emerge as the foremost mechanisms in Hunner-type interstitial cystitis/bladder pain syndrome (HIC). Although treatments aimed at urothelial regeneration and repair have been employed, their therapeutic effectiveness remains limited due to the inadequate understanding of specific cell types involved in damage and the lack of specific molecular targets within these mechanisms. Therefore, we harnessed single-cell RNA sequencing to elucidate the heterogeneity and developmental trajectory of urothelial cells within HIC bladders. Through reclustering, we identified eight distinct clusters of urothelial cells. There was a significant reduction in UPK3A+ umbrella cells and a simultaneous increase in progenitor-like pluripotent cells (PPCs) within the HIC bladder. Pseudotime analysis of the urothelial cells in the HIC bladder revealed that cells faced challenges in differentiating into UPK3A+ umbrella cells, while PPCs exhibited substantial proliferation to compensate for the loss of UPK3A+ umbrella cells. The urothelium in HIC remains unrepaired, despite the substantial proliferation of PPCs. Thus, we propose that inhibiting the pivotal signaling pathways responsible for the injury to UPK3A+ umbrella cells is paramount for restoring the urothelial barrier and alleviating lower urinary tract symptoms in HIC patients. Subsequently, we identified key molecular pathways (TLR3 and NR2F6) associated with the injury of UPK3A+ umbrella cells in HIC urothelium. Finally, we conducted in vitro and in vivo experiments to confirm the potential of the TLR3-NR2F6 axis as a promising therapeutic target for HIC. These findings hold the potential to inhibit urothelial injury, providing promising clues for early diagnosis and functional bladder self-repair strategies for HIC patients. © 2024 The Pathological Society of Great Britain and Ireland.

Mechanosensitive Piezo1 channels promote neurogenic bladder fibrosis via regulating TGF-ß1/smad and Hippo/YAP1 pathways.

Bladder fibrosis is the final common pathway of neurogenic bladder (NB), and its underlying mechanisms are not fully understood. The current study aims to evaluate the involvement of Piezo1, a mechanosensitive channel, in bladder fibrosis. A full-thickness bladder specimen was taken during ileocystoplasty or ureteral reimplantation from the surgical cut's edge. By chopping off the bilateral lumbar 6 (L6) and sacral 1 (S1) spinal nerves, NB rat models were produced. Utilizing both pharmacological inhibition and Piezo1 deletion, the function of Piezo1 in the TGF-ß1-induced fibrosis model of SV-HUC-1 cells was delineated. RNA-seq, immunofluorescence, immunohistochemistry (IHC), and Western blotting were used to evaluate the degrees of fibrosis and biochemical signaling pathways. Piezo1 protein expression was noticeably elevated in the human NB bladder. The abundance of Piezo1 protein in bladder of NB rats was significantly increased. RNA-seq analysis revealed that the ECM-receptor interaction signaling pathway and collagen-containing ECM were increased in spinal cord injury (SCI)-induced bladder fibrosis. Moreover, the bladder of the NB rat model showed activation of YAP1 and TGF-ß1/Smad. In SV-HUC-1 cells, siRNA suppression of Piezo1 led to profibrotic responses and activation of the TGF-ß1/Smad pathway. However, Yoda1, a Piezo1-specific agonist, significantly reduced these effects. TGF-ß1 increased Piezo1 activation and profibrotic responses in SV-HUC-1 cells. In the TGF-ß1-induced fibrosis model of SV-HUC-1 cells, the TGF-ß1/Smad pathway was activated, whereas the Hippo/YAP1 signal pathway was blocked. Inhibition of Piezo1 further prevented this process. Piezo1 is involved in the progression of NB bladder fibrosis and profibrotic alterations in SV-HUC-1 cells, likely through regulating the TGF-ß1/Smad and Hippo/YAP1 pathways.

Plant phenolics inhibit focal adhesion kinase and suppress host cell invasion by uropathogenic Escherichia coli.

Traditional folk treatments for the prevention and management of urinary tract infections (UTIs) and other infectious diseases often include plants and plant extracts that are rich in phenolic compounds. These have been ascribed a variety of activities, including inhibition of bacterial interactions with host cells. Here, we tested a panel of four well-studied phenolic compounds-caffeic acid phenethyl ester (CAPE), resveratrol, catechin, and epigallocatechin gallate-for the effects on host cell adherence and invasion by uropathogenic Escherichia coli (UPEC). These bacteria, which are the leading cause of UTIs, can bind and subsequently invade bladder epithelial cells via an actin-dependent process. Intracellular UPEC reservoirs within the bladder are often protected from antibiotics and host defenses and likely contribute to the development of chronic and recurrent infections. In cell culture-based assays, only resveratrol had a notable negative effect on UPEC adherence to bladder cells. However, both CAPE and resveratrol significantly inhibited UPEC entry into the host cells, coordinate with attenuated phosphorylation of the host actin regulator Focal Adhesion Kinase (FAK or PTK2) and marked increases in the numbers of focal adhesion structures. We further show that the intravesical delivery of resveratrol inhibits UPEC infiltration of the bladder mucosa in a murine UTI model and that resveratrol and CAPE can disrupt the ability of other invasive pathogens to enter host cells. Together, these results highlight the therapeutic potential of molecules like CAPE and resveratrol, which could be used to augment antibiotic treatments by restricting pathogen access to protective intracellular niches.IMPORTANCEUrinary tract infections (UTIs) are exceptionally common and increasingly difficult to treat due to the ongoing rise and spread of antibiotic-resistant pathogens. Furthermore, the primary cause of UTIs, uropathogenic Escherichia coli (UPEC), can avoid antibiotic exposure and many host defenses by invading the epithelial cells that line the bladder surface. Here, we identified two plant-derived phenolic compounds that disrupt activation of the host machinery needed for UPEC entry into bladder cells. One of these compounds, resveratrol, effectively inhibited UPEC invasion of the bladder mucosa in a mouse UTI model, and both phenolic compounds significantly reduced host cell entry by other invasive pathogens. These findings suggest that select phenolic compounds could be used to supplement existing antibacterial therapeutics by denying uropathogens shelter within host cells and tissues and help explain some of the benefits attributed to traditional plant-based medicines.

Long-term follow-up of TREK-1 KO mice reveals the development of bladder hypertrophy and impaired bladder smooth muscle contractility with age.

Stretch-activated two-pore domain K+ (K2P) channels play important roles in many visceral organs, including the urinary bladder. The TWIK-related K+ channel TREK-1 is the predominantly expressed K2P channel in the urinary bladder of humans and rodents. Downregulation of TREK-1 channels was observed in the urinary bladder of patients with detrusor overactivity, suggesting their involvement in the pathogenesis of voiding dysfunction. This study aimed to characterize the long-term effects of TREK-1 on bladder function with global and smooth muscle-specific TREK-1 knockout (KO) mice. Bladder morphology, bladder smooth muscle (BSM) contractility, and voiding patterns were evaluated up to 12 mo of age. Both sexes were included in this study to probe the potential sex differences. Smooth muscle-specific TREK-1 KO mice were used to distinguish the effects of TREK-1 downregulation in BSM from the neural pathways involved in the control of bladder contraction and relaxation. TREK-1 KO mice developed enlarged urinary bladders (by 60.0% for males and by 45.1% for females at 6 mo; P < 0.001 compared with the age-matched control group) and had a significantly increased bladder capacity (by 137.7% at 12 mo; P < 0.0001) and compliance (by 73.4% at 12 mo; P < 0.0001). Bladder strips isolated from TREK-1 KO mice exhibited decreased contractility (peak force after KCl at 6 mo was 1.6 ± 0.7 N/g compared with 3.4 ± 2.0 N/g in the control group; P = 0.0005). The lack of TREK-1 channels exclusively in BSM did not replicate the bladder phenotype observed in TREK-1 KO mice, suggesting a strong neurogenic origin of TREK-1-related bladder dysfunction.NEW & NOTEWORTHY This study compared voiding function and bladder phenotypes in global and smooth muscle-specific TREK-1 KO mice. We found significant age-related changes in bladder contractility, suggesting that the lack of TREK-1 channel activity might contribute to age-related changes in bladder smooth muscle physiology.

Effects of oral administration of nonselective Trk inhibitor on bladder overactivity in rodent models of prostatic inflammation.

BACKGROUND: Our research focused on the assessment of the impact of systemic inhibition of Trk receptors, which bind to nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), on bladder hypersensitivity in two distinct rodent models of prostatic inflammation (PI). METHODS: Male Sprague-Dawley rats were divided into three groups (n = 6 each): the control group (no PI, vehicle administration), the untreated group (PI, vehicle administration), and the treated group (PI, nonselective Trk inhibitor, GNF 5837, administration). PI in rats was induced by a intraprostatic injection of 5% formalin. Posttreatment, we carried out conscious cystometry and a range of histological and molecular analyses. Moreover, the study additionally evaluated the effects of a nonselective Trk inhibitor on bladder overactivity in a mouse model of PI, which was induced by prostate epithelium-specific conditional deletion of E-cadherin. RESULTS: The rat model of PI showed upregulations of NGF and BDNF in both bladder and prostate tissues in association with bladder overactivity and inflammation in the ventral lobes of the prostate. GNF 5837 treatment effectively mitigated these PI-induced changes, along with reductions in TrkA, TrkB, TrkC, and TRPV1 mRNA expressions in L6-S1 dorsal root ganglia. Also, in the mouse PI model, GNF 5837 treatment similarly improved bladder overactivity. CONCLUSIONS: The findings of our study suggest that Trk receptor inhibition, which reduced bladder hypersensitivity and inflammatory responses in the prostate, along with a decrease in overexpression of Trk and TRPV1 receptors in sensory pathways, could be an effective treatment strategy for male lower urinary tract symptoms associated with PI and bladder overactivity.

Urine-derived bladder cancer organoids (urinoids) as a tool for cancer longitudinal response monitoring and therapy adaptation.

BACKGROUND: Bladder cancer is one of the most common cancer types worldwide. Generally, research relies on invasive sampling strategies. METHODS: Here, we generate bladder cancer organoids directly from urine (urinoids). In this project, we establish 12 urinoid lines from 22 patients with non-muscle and muscle-invasive bladder tumours, with an efficiency of 55%. RESULTS: The histopathological features of the urinoids accurately resemble those of the original bladder tumours. Genetically, there is a high concordance of single nucleotide polymorphisms (92.56%) and insertions & deletions (91.54%) between urinoids and original tumours from patient 4. Furthermore, these urinoids show sensitivity to bladder cancer drugs, similar to their tissue-derived organoid counterparts. Genetic analysis of longitudinally generated tumoroids and urinoids from one patient receiving systemic immunotherapy, identify alterations that may guide the choice for second-line therapy. Successful treatment adaptation was subsequently demonstrated in the urinoid setting. CONCLUSION: Therefore, urinoids can advance precision medicine in bladder cancer as a non-invasive platform for tumour pathogenesis, longitudinal drug-response monitoring, and therapy adaptation.

Phase II trial of afatinib in patients with advanced urothelial carcinoma with genetic alterations in ERBB1-3 (LUX-Bladder 1).

BACKGROUND: Preclinical and early clinical data suggest that the irreversible ErbB family blocker afatinib may be effective in urothelial cancers harbouring ERBB mutations. METHODS: This open-label, phase II, single-arm trial (LUX-Bladder 1, NCT02780687) assessed the efficacy and safety of second-line afatinib 40 mg/d in patients with metastatic urothelial carcinoma with ERBB1-3 alterations. The primary endpoint was 6-month progression-free survival rate (PFS6) (cohort A); other endpoints included ORR, PFS, OS, DCR and safety (cohorts A and B). Cohort A was planned to have two stages: stage 2 enrolment was based on observed antitumour activity. RESULTS: Thirty-four patients were enroled into cohort A and eight into cohort B. In cohorts A/B, PFS6 was 11.8%/12.5%, ORR was 5.9%/12.5%, DCR was 50.0%/25.0%, median PFS was 9.8/7.8 weeks and median OS was 30.1/29.6 weeks. Three patients (two ERBB2-amplified [cohort A]; one EGFR-amplified [cohort B]) achieved partial responses. Stage 2 for cohort A did not proceed. All patients experienced adverse events (AEs), most commonly (any/grade 3) diarrhoea (76.2%/9.5%). Two patients (4.8%) discontinued due to AEs and one fatal AE was observed (acute coronary syndrome; not considered treatment-related). CONCLUSIONS: An exploratory biomarker analysis suggested that basal-squamous tumours and ERBB2 amplification were associated with superior response to afatinib. CLINICAL TRIAL REGISTRATION: NCT02780687.

Tumor-associated neutrophils suppress CD8+ T cell immunity in urothelial bladder carcinoma through the COX-2/PGE2/IDO1 Axis.

BACKGROUND: Many urothelial bladder carcinoma (UBC) patients don't respond to immune checkpoint blockade (ICB) therapy, possibly due to tumor-associated neutrophils (TANs) suppressing lymphocyte immune response. METHODS: We conducted a meta-analysis on the predictive value of neutrophil-lymphocyte ratio (NLR) in ICB response and investigated TANs' role in UBC. We used RNA-sequencing, HALO spatial analysis, single-cell RNA-sequencing, and flow cytometry to study the impacts of TANs and prostaglandin E2 (PGE2) on IDO1 expression. Animal experiments evaluated celecoxib's efficacy in targeting PGE2 synthesis. RESULTS: Our analysis showed that higher TAN infiltration predicted worse outcomes in UBC patients receiving ICB therapy. Our research revealed that TANs promote IDO1 expression in cancer cells, resulting in immunosuppression. We also found that PGE2 synthesized by COX-2 in neutrophils played a key role in upregulating IDO1 in cancer cells. Animal experiments showed that targeting PGE2 synthesis in neutrophils with celecoxib enhanced the efficacy of ICB treatment. CONCLUSIONS: TAN-secreted PGE2 upregulates IDO1, dampening T cell function in UBC. Celecoxib targeting of PGE2 synthesis represents a promising approach to enhance ICB efficacy in UBC.

Presence, Subtypes, and Prognostic Significance of Tertiary Lymphoid Structures in Urothelial Carcinoma of the Bladder.

OBJECTIVE: To evaluate the presence and subtypes of tertiary lymphatic structures (TLSs) in urothelial carcinoma of the bladder (UCB) and to analyze their associated clinicopathological characteristics and prognostic significance. METHODS: The study enrolled 580 patients with surgically treated UCB, including 313 non-muscle invasive bladder cancer (NMIBC) and 267 muscle-invasive bladder cancer (MIBC). The presence and subtypes of TLSs were identified by immunohistochemistry (CD20, CD3, Bcl-6, and CD21). TLSs were classified into non-GC (nGC) TLS and GC TLS subtypes based on germinal center (GC) formation. Disease-free survival (DFS) was used as an endpoint outcome to evaluate the prognostic significance of TLS and its subtypes in UCB. RESULTS: TLSs were more common in MIBC than in NMIBC (67.8% vs 48.2%, P < .001), and the tumor-infiltrating lymphocyte (TIL) mean density was significantly higher in MIBC than in NMIBC (24.0% vs 17.5%, P < .001). Moreover, a positive correlation was found between TLS presence and GC structure formation and TIL infiltration in UCB. Endpoint events occurred in 191 patients. Compared to patients with endpoint events, patients without disease progression exhibited higher TIL density and more TLSs (P < .05). Kaplan-Meier curves showed that TLS was associated with better DFS in NMIBC (P = .041) and MIBC (P = .049). However, the Cox multivariate analysis did not demonstrate the prognostic significance of TLS. CONCLUSIONS: TLS is heterogeneous in UCB, and that TLS and GC structures are related to TIL density and prognostic events. However, TLS as a prognostic indicator remains unclear, warranting further investigation.

Development and Validation of a Contrast-Enhanced US VI-RADS for Evaluating Muscle Invasion in Bladder Cancer.

Background Contrast-enhanced US (CEUS) can be used preoperatively for evaluating muscle invasion in bladder cancer, which is important for determining appropriate treatment. However, diagnostic criteria for assessing this at CEUS have not been standardized. Purpose To develop and validate a CEUS Vesical Imaging Reporting and Data System (VI-RADS) for evaluating muscle invasion in bladder cancer. Materials and Methods This single-center prospective study consecutively enrolled patients with suspected bladder cancer. Participants underwent transabdominal or intracavity CEUS between July 2021 and May 2023. Participants were divided into a training set and a validation set at a 2:1 ratio based on the chronologic order of enrollment. The training set was used to identify major imaging features to include in CEUS VI-RADS, and the likelihood of muscle invasion per category was determined using a pathologic reference standard. The optimal VI-RADS category cutoff for muscle invasion was determined with use of the maximum Youden index. The validation set was assessed by novice and expert readers and used to validate the diagnostic performance and interreader agreement of the developed system. Results Overall, 126 participants (median age, 64 years [IQR, 57-71 years]; 107 male) and 67 participants (median age, 64 years [IQR, 56-69 years]; 49 male) were included in the training and validation set, respectively. In the training set, the optimal CEUS VI-RADS category cutoff for muscle invasion was VI-RADS 4 or higher (Youden index, 0.77). In the validation set, CEUS VI-RADS achieved good performance for both novice and expert readers (area under the receiver operating characteristic curve, 0.80 [95% CI: 0.70, 0.90] vs 0.88 [95% CI: 0.80, 0.97]; P = .09). The interreader agreement regarding the evaluation of CEUS VI-RADS category was 0.77 (95% CI: 0.65, 0.85) for novice readers, 0.87 (95% CI: 0.79, 0.92) for expert readers, and 0.78 (95% CI: 0.70, 0.84) for all readers. Conclusion The developed CEUS VI-RADS showed good performance and interreader agreement for the assessment of muscle invasion in bladder cancer. Chinese Clinical Trial Registry no. ChiCTR2100049435 © RSNA, 2024 Supplemental material is available for this article. See also the editorial by Morrell in this issue.

KLHDC7B as a novel diagnostic biomarker in urine exosomal mRNA promotes bladder urothelial carcinoma cell proliferation and migration, inhibits apoptosis.

Bladder cancer is a common kind of urinary system cancer, in which bladder urothelial carcinoma (BLCA) comprises approximately 90% of all bladder cancer types. In our previous study, we discovered KLHDC7B in urine exosomal messenger RNA (mRNA) as a prospective molecular marker for bladder cancer detection. To systematically study the role and mechanism of KLHDC7B in BLCA, we focused on the most common type of BLCA in this study. First, we used RNA sequencing to discover that KLHDC7B was considerably increased in BLCA patients' urine exosomes compared to healthy controls. Then, we validated this result in an independent cohort and identified it as an effective tool for diagnosing and distinguishing high-grade and low-grade BLCA. Finally, we studied the role and mechanism of KLHDC7B in BLCA at the cellular level, providing a functional basis for its expression as a novel laboratory diagnostic biomarker for BLCA exosomal mRNA, which has important theoretical and clinical significance.

Characterizing the Genomic Landscape of the Micropapillary Subtype of Urothelial Carcinoma of the Bladder Harboring Activating Extracellular Mutations of ERBB2.

The micropapillary subtype of urothelial carcinoma (MPUC) of the bladder is a very aggressive histological variant of urothelial bladder cancer (UBC). A high frequency of MPUC contains activating mutations in the extracellular domain (ECD) of ERBB2. We sought to further characterize ERBB2 ECD-mutated MPUC to identify additional genomic alterations that have been associated with tumor progression and therapeutic response. In total, 5,485 cases of archived formalin-fixed, paraffin-embedded UBC underwent comprehensive genomic profiling to identify ERBB2 ECD-mutated MPUC and evaluate the frequencies of genomic co-alterations. We identified 219 cases of UBC with ERBB2 ECD mutations (74% S310F and 26% S310Y), of which 63 (28.8%) were MPUC. Genomic analysis revealed that TERT, TP53, and ARID1A were the most common co-altered genes in ERBB2-mutant MPUC (82.5%, 58.7%, and 39.7%, respectively) and did not differ from ERBB2-mutant non-MPUC (86.5%, 51.9%, and 35.3%). The main differences between ERBB2 ECD-mutated MPUC compared with non-MPUC were KMT2D, RB1, and MTAP alterations. KMT2D and RB1 are tumor-suppressor genes. KMT2D frequency was significantly decreased in ERBB2 ECD-mutated MPUC (6.3%) in contrast to non-MPUC (27.6%; P < .001). RB1 mutations were more frequent in ERBB2 ECD-mutated MPUC (33.3%) than in non-MPUC (17.3%; P = .012). Finally, MTAP loss, an emerging biomarker for new synthetic lethality-based anticancer drugs, was less frequent in ERBB2 ECD-mutated MPUC (11.1%) than in non-MPUC (26.9%; P = .018). Characterizing the genomic landscape of MPUC may not only improve our fundamental knowledge about this aggressive morphological variant of UBC but also has the potential to identify possible prognostic and predictive biomarkers that may drive tumor progression and dictate treatment response to therapeutic approaches.

Diabetes compromises tight junction protein claudin 14 in the urinary bladder.

Infections are common in patients with diabetes. Moreover, increasing incidence of antibiotic resistance impedes the complete bacterial clearance and calls for alternative treatment strategies. Along with antibacterial resistance, compromised host conditions create a favorable condition for the disease progression. In particular, cell junction proteins are of major importance as they contribute to a tight cell barrier, protecting against invading pathogens. However, the impact of high glucose on cell junction proteins has received little attention in the urinary bladder but merits closer investigation. Here, we report that during diabetes the expression of cell junction protein, claudin 14 is compromised in the human urine exfoliated cells and in the urinary bladder of type 2 diabetic mouse. Further in vitro analysis confirmed a direct correlation of lower intracellular calcium levels with claudin 14 expression in high glucose-treated human uroepithelial cells. Moreover, external calcium supplementation in high glucose-treated cells significantly affected the cell migration and restored the claudin 14 expression through focal adhesion and ß-1 integrins. Strengthening the epithelial barrier is essential, especially in individuals with diabetes where basal calcium levels could contribute.

Elucidation of the pathophysiology of interstitial cystitis/bladder pain syndrome via experimental autoimmune cystitis rat model.

Although the cause of interstitial cystitis/painful bladder syndrome (IC/PBS) remains unknown, autoimmune involvement has been strongly suggested to be a contributing factor. To elucidate the pathophysiology of IC/PBS, we characterized the experimental autoimmune cystitis (EAC) in rats. Adult female Sprague-Dawley rats were divided into the EAC and control groups. The EAC rats were generated by administrating a homogenate of donor rat bladder tissue as a bladder antigen. The characteristics of the two groups were determined by evaluating pain behavior and conducting cystometry, histopathology, and molecular analyses. The EAC rats showed: 1) a decreased paw withdrawal threshold, 2) a reduced intercontraction interval on cystometry, 3) the irregular surfaces of the umbrella cells of epithelium throughout the bladder wall, 4) accumulation of stress granules in the bladder and vascular endothelium, 5)the increased expression of genes related to inflammation and ischemia at the mRNA and protein levels, 6) a significantly increased paw withdrawal threshold with pain treatment, and 7) the induction of glomerulation of the bladder wall, epithelium denudation, and lymphocyte infiltration in the interstitium by bladder distension. These results suggest that the EAC rats showed pain and frequent urination with the overexpression of inflammatory chemokines, reflecting clinical IC/BPS, and the bladder epithelium and vascular endothelium may be the primary sites of IC/BPS, and bladder injury, such as bladder distension, can cause progression from BPS to IC with Hunner lesions.NEW & NOTEWORTHY The experimental autoimmune cystitis model rats showed pain and frequent urination with the overexpression of inflammatory chemokines, reflecting clinical interstitial cystitis/painful bladder syndrome (IC/PBS), and the bladder epithelium and vascular endothelium may be the primary sites of IC/BPS, and bladder injury, such as bladder distension, can cause progression from BPS to IC with Hunner lesions.

Pattern of Expression of MicroRNA in Patients with Radiation-Induced Bladder Injury.

INTRODUCTION: Bladder cancer (BC) is sensitive to radiation treatment and a subset of patients experience radiation-induced injuries including shrinkage of bladder due to bladder fibrosis. METHODS: This study is a retrospective cohort study. Three Japanese BC patients were randomly selected. Using a microRNA (miRNA) array, comparing their samples with or without radiation-induced injuries, we have checked the clustering of miRNA expression. RESULTS: Hsa-miR-130a, hsa-miR-200c, hsa-miR-141, and hsa-miR-96 were found to be highly expressed (>50 times) in patients with fibrotic bladder shrinkage (FBS) compared to those with intact bladder (IB) function. In patients with FBS, hsa-miR-6835, hsa-miR-4675, hsa-miR-371a, and hsa-miR-6885 were detected to have lesser than half expression to IB patients. We have analyzed the significance of these genes in relation to overall survival of 409 BC patients retrieved from the Cancer Genome Atlas data set. All available cutoff values between the lower and upper quartiles of expression are used for the selected genes, and false discovery rate using the Benjamini-Hochberg method is computed to correct for multiple hypothesis testing. We have run combined survival analysis of the mean expression of these four miRNAs highly expressed in FBS patients. 175 patients with high expression had a longer median survival of 98.47 months than 23.73 months in 233 patients with low expression (hazard ratio [HR]: 0.53; 0.39-0.72, log-rank p value: 7.3e-0.5). Combination analysis of all 8 genes including hsa-miR-6835, hsa-miR-4675, hsa-miR-371a, and hsa-miR-6885 showed the same HR for OS. Target scanning for these miRNAs matched specific cytokines known as an early biomarker to develop radiation-induced fibrosis. CONCLUSIONS: BC patients with fibrotic radiation injury have specific miRNA expression profile targeting profibrotic cytokines and these miRNAs possibly render to favorable survival.