Urothelial Bladder Research Articles

Urothelium removal does not impact mucosal activity in response to muscarinic or adrenergic receptor stimulation

ABSTRACT The inner lining of the urinary bladder (urothelium and lamina propria, or bladder mucosa) has an important role as a tissue barrier between stored urine and the underlying smooth muscle, as well as in the modulation and regulation of bladder contractility. However, the individual influence of the apical urothelial layer on the contractile activity of this tissue is uncertain. The aim of this experiment was to identify the contractile activity of the lamina propria after removal of the urothelium. Several methods were used to mechanically disrupt the urothelium, including dabbing the tissue with a paper towel, longitudinal swipes with a cotton bud, or a longitudinal scrape with the edge of a scalpel. Hematoxylin-eosin staining was utilized to determine the level of removal of the apical urothelial cells. Spontaneous contractile activity was measured in organ baths, and responses to the agonists carbachol and isoprenaline were obtained. Three longitudinal swipes with a cotton bud was found to be the optimal method to remove the majority of the urothelium without damaging the lamina propria. Upon removal of the urothelium, the spontaneous activity of the tissue was unaltered. Similarly, responses to carbachol (1 µM) and isoprenaline (1 µM) were not affected after removal of the urothelium. The urothelium can be effectively removed without damaging the lamina propria. This apical tissue layer is not responsible for mediating the increases to spontaneous phasic activity or tonic contractions of the bladder mucosa (urothelium with lamina propria) when muscarinic or adrenergic receptors are stimulated. This research presents the lamina propria as the important cell layer mediating the overall contractile activity of the bladder wall.

Mannose-targeting Concanavalin A-Epirubicin Conjugate for Targeted Intravesical Chemotherapy of Bladder Cancer.

Intravesical instillation of chemotherapeutic drugs such as epirubicin (EPI) is routinely used to prevent tumor recurrence and progression after transurethral resection of bladder tumor. However, the lack of tumor selectivity often causes severe damage to normal bladder urothelium leading to intolerable side effects. Here, we analyzed abnormal changes in glycosylation in bladder cancer and identified mannose as the most aberrantly expressed glycan on the surface of bladder cancer cell lines and human bladder tumor tissues. We then constructed a lectin-drug conjugate by linking concanavalin A (ConA) - a lectin that specifically binds to mannose, with EPI through a pH-sensitive linker. This ConA-EPI conjugate conferred EPI with mannose-targeting ability and selectively internalized cancer cells in vitro. This conjugate showed selective cytotoxicity to cancer cells in vitro and better antitumor activity in an orthotopic mouse model of bladder cancer. Our lectin-drug conjugation strategy makes targeted intravesical chemotherapy of bladder cancer possible.

Predicting Prognosis and Distinguishing Cold and Hot Tumors in Bladder Urothelial Carcinoma Based on Necroptosis-Associated lncRNAs.

BackgroundIn reference to previous studies, necroptosis played an important role in cancer development. Our team decided to explore the potential prognostic values of long non-coding RNAs (lncRNAs) associated with necroptosis in bladder urothelial carcinoma (BLCA) and their relationship with the tumor microenvironment (TME) and the immunotherapeutic response for accurate dose.MethodsTo obtain the required data, bladder urothelial carcinoma transcriptome data were searched from Cancer Genome Atlas (TCGA) (https://portal.gdc.cancer.gov/). We used co-expression analysis, differential expression analysis, and univariate Cox regression to screen out prognostic lncRNAs associated with necroptosis in BLCA. Then the least absolute shrinkage and selection operator (LASSO) was conducted to construct the necroptosis-associated lncRNAs model. Based on this model, we also performed the Kaplan–Meier analysis and time-dependent receiver operating characteristics (ROC) to estimate the prognostic power of risk score. Multivariate and univariate Cox regression analysis were performed to build up a nomogram. Calibration curves, and time-dependent ROC were also conducted to evaluate nomogram. Principal component analysis (PCA) revealed a difference between high- and low-risk groups. In addition, we explored immune analysis, gene set enrichment analyses (GSEA), and evaluation of the half-maximal inhibitory concentration (IC50) in constructed model. Finally, the entire samples were divided into three clusters based on model of necroptosis-associated lncRNAs to further compare immunotherapy in cold and hot tumors.ResultsA model was built up based on necroptosis-associated lncRNAs. The model revealed good consistence between calibration plots and prognostic prediction. The area of 1-, 3-, and 5-year OS under the ROC curve (AUC) were 0.707, 0.679, and 0.675. Risk groups could be helpful for systemic therapy due to the markedly diverse IC50 between risk groups. To our delight, clusters could effectively identify cold and hot tumors, which would be beneficial to accurate mediation. Clusters 2 and 3 were considered the hot tumor, which was more sensitive to immunotherapeutic drugs.ConclusionsThe outcomes of our study suggested that necroptosis-associated lncRNAs could effectively predict patients with BLCA prognosis, which may be helpful for distinguishing the cold and hot tumors and improving individual treatment of BLCA.

Role of ERK signaling in bladder urothelium in response to cyclophosphamide injury.

Mice with inducible urothelial deletion of fibroblast growth factor receptor 2 (ShhCreERT2;Fgfr2 Fl/Fl ) injured with cyclophosphamide had aberrant basal cell endoreplication and poor regeneration. The endoreplication correlated with an absence of phosphorylated (activated) ERK expression in urothelium. We assessed whether inhibiting ERK activity phenocopied the urothelial defects in injured Fgfr2 mutant mice. We co‐administered cyclophosphamide and an ERK inhibitor (ERKi) systemically in mice and assessed general histology and immunofluorescence for various markers post injury. Since AKT also signals downstream of FGFR2, we assessed effects of an AKT inhibitor (AKTi) on cyclophosphamide injury. ERK knockdown did not affect urothelial injury or proliferation 24 h after cyclophosphamide. Conversely, ERK inhibition led to larger basal cell nuclei, more submucosal hemorrhage and attenuated uroplakin staining 3 days after injury versus vehicle‐treated mice. Compared to vehicle‐treated mice, ERKi‐treated mice had a trend for more Ki67+ urothelial cells and had statistically fewer phospho‐Histone H3+ cells normalized to Ki67 and higher basal cell DNA content, consistent with endoreplication 3 days after injury. Ten days after injury, ERKi‐treated mice still had signs of poor urothelial regeneration with absent or aberrant expression of differentiation markers and ectopic lumenal expression of keratin 14 (basal progenitor marker). Co‐administration of the AKTi led to no apparent urothelial defects 3 days after cyclophosphamide. Thus, ERK knockdown (but not AKT knockdown) leads to urothelial regenerative responses after cyclophosphamide reminiscent of Fgfr2 mutant mice. Together, it appears that FGFR2 acts through ERK to prevent aberrant urothelial basal cell endoreplication and ensure normal regeneration after cyclophosphamide.

Persistent γ-H2AX Formation and Expression of Stem Cell Markers in N-Butyl-N-(4-Hydroxybutyl)Nitrosamine-Induced Bladder Carcinogenesis in Rats.

We investigated γ-H2AX formation, a biomarker of DNA damage, and expression of stem cell markers (SCMs), including cytokeratin 14, aldehyde dehydrogenase 1A1 (ALDH1A1), and CD44, in the development of rat bladder tumors induced by short-term administration of N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Histopathological examination showed that diffuse simple hyperplasia of the bladder urothelium induced by BBN recovered to the normal-appearing urothelium after withdrawal, whereas focal proliferative lesions were newly developed and subsequently progressed to benign papilloma and carcinoma. Immunohistochemical analysis revealed that BBN-induced γ-H2AX formation and ALDH1A1 and CD44 expression persisted at higher levels in the normal-appearing urothelium than those in the control group for long periods after withdrawal. Since persistent chronic inflammation was observed even after withdrawal, targeted gene expression analysis of inflammation-related factors revealed 101 genes, including Stat3 and Myc, that showed persistent high expression. Pathway analysis suggested that Stat3 and/or Myc activation may be associated with SCM expression. We focused on hepatocyte growth factor (Hgf), one of the genes predicted in relation to Stat3/Myc, and confirmed that HGF-positive cells increased by BBN persisted in the normal-appearing urothelium after withdrawal and colocalized with γ-H2AX and SCMs. These results suggested that the long-term persistence of γ-H2AX formation and SCM expression, which occurred during the early stages of bladder tumorigenesis, is not a transient response to exposure and might contribute to bladder tumorigenesis. Although further studies are needed, BBN-induced rat bladder tumors may originate from focal hyperplasia arising from SCM-positive cells via activation of the STAT3/MYC pathway after DNA damage involving γ-H2AX formation.

Aging-Associated Changes in Oxidative Stress Negatively Impacts the Urinary Bladder Urothelium.

PurposeLower urinary tract symptoms are known to significantly increase with age, negatively impacting quality of life and self-reliance. The urothelium fulfills crucial tasks, serving as a barrier protecting the underlying bladder tissue from the harsh chemical composition of urine, and exhibits signaling properties via the release of mediators within the bladder wall that affect bladder functioning. Aging is associated with detrimental changes in cellular health, in part by increasing oxidative stress in the bladder mucosa, and more specifically the urothelium. This, in turn, may impact urothelial mitochondrial health and bioenergetics.MethodsWe collected mucosal tissue samples from both young (3–4 months old) and aged (25–30 months old) rats. Tissue was evaluated for p21-Arc, nitrotyrosine, and cytochrome C expression by western immunoblotting. Urothelial cells were cultured for single-cell imaging to analyze basal levels of reactive oxygen species and the mitochondrial membrane potential. Mitochondrial bioenergetics and cellular respiration were investigated by the Seahorse assay, and measurements of adenosine triphosphate release were made using the luciferin-luciferase assay.ResultsAging was associated with a significant increase in biomarkers of cellular senescence, oxidative stress, and basal levels of reactive oxygen species. The mitochondrial membrane potential was significantly lower in urothelial cell cultures from aged animals, and cultures from aged animals showed a significant decrease in mitochondrial bioenergetics.ConclusionsAging-related increases in oxidative stress and excessive reactive oxygen species may be contributing factors underlying lower urinary tract symptoms in older adults. The mechanisms outlined in this study could be utilized to identify novel pharmaceutical targets to improve aging-associated bladder dysfunction.

Downregulation of PTCD1 in Bladder Urothelial Carcinoma Predicts Poor Prognosis and Levels of Immune Infiltration.

Pentatricopeptide repeat domain 1 (PTCD1) was reported to regulate mitochondrial metabolism and oxidative phosphorylation. However, the effect and mechanism of PTCD1 in the development of bladder urothelial carcinoma (BLCA) remain unclear. The databases from The Cancer Genome Atlas (TCGA) and Human Protein Atlas (HPA) were used to analyze the expression changes, clinical features, and prognostic values of PTCD1. A nomogram was built to predict the prognostic outcomes of BLCA cases. The potential genes interacting with PTCD1 were explored by Weighted Gene Coexpression Network Analysis (WGCNA). The estimation of associations between PTCD1 and tumor mutations, tumor immunities, and m6A methylations was performed. The study found that the gradual decrease of PTCD1 expression was observed with the increase of stage and grade. Low PTCD1 expression was greatly correlated with higher pathological stage, N stage, and poor prognosis in TCGA cohorts; interestingly, low-grade BLCA cases all exhibited high expression of PTCD1. HPA database analysis implied that the expression of PTCD1 protein in BLCA was lower than that in normal bladder tissue, and the protein expression of PTCD1 in high-grade BLCA was lower than that in low-grade BLCA. Multivariate Cox regression analysis indicated that PTCD1 may serve as an independent factor influencing prognosis of BLCA. Mechanistically, PTCD1 played a regulatory role in BLCA progression through multiple tumor-related pathways containing PI3K-Akt signaling, ECM-receptor interaction, oxidative phosphorylation, and extracellular matrix organization. WGCNA reported that PTCD1 had a strong positive correlation with POLR2J, ZNHT1, ATP5MF, PDAP1, BUD31, and COPS6. Besides, the mRNA expression of PTCD1 was negatively associated with immune cells' infiltrations, immune functions, and checkpoints, especially with some m6A methylation regulators in BLCA. In sum, downregulation of PTCD1 expression may be involved in the development of BLCA and remarkably correlated with poor prognosis. Meantime, it showed an influence in immune cell infiltration and may serve as an agreeable prognostic indicator in BLCA.

A Necroptosis-Related lncRNA to Develop a Signature to Predict the Outcome, Immune Landscape, and Chemotherapeutic Responses in Bladder Urothelial Carcinoma.

ObjectiveMany studies have drawn their attention to the immunotherapy of bladder urothelial carcinoma in terms of immunologic mechanisms of human body. These include immunogenicity of the tumor cells and involvement of long non-coding RNA (lncRNA). We constructed a necroptosis-related long noncoding RNA (nrlncRNA) risk factor model to predict BLCA outcomes and calculate correlations with chemosensitivity and immune infiltration.MethodsTranscriptomic data from BLCA specimens were accessed from The Cancer Genome Atlas, and nrlncRNAs were identified by performing co-expression analysis. Univariate analysis was performed to identify differentially expressed nrlncRNA pairs. We constructed least absolute contraction and selector operation regression models and drew receiver operating characteristic curves for 1-, 3-, and 5-year survival rates. Akaike information criterion (AIC) values for survival over 1 year were determined as cutoff values in high- and low-risk subgroups. We reassessed the differences between subgroups in terms of survival, clinicopathological characteristics, chemotherapy efficacy, tumor-infiltrating immune cells, and markers of immunosuppression.ResultsWe identified a total of 260 necroptosis-related lncRNA pairs, of which we incorporated 13 into the prognostic model. Areas under the curve of 1-, 3-, and 5- year survival time were 0.763, 0.836, and 0.842, respectively. We confirmed the excellent predictive performance of the risk model. Based on AIC values, we confirmed that the high-risk group was susceptible to unfavorable outcomes. The risk scores correlated with survival were age, clinical stage, grade, and tumor node metastases. The risk model was an independent predictor and demonstrated higher predictive power. The risk model can also be utilized to determine immune cell infiltration status, expression levels of immune checkpoint genes, and the sensitivity to cisplatin, doxorubicin, and methotrexate.ConclusionWe constructed a novel necroptosis-related signature that predicts BLCA outcomes and performs satisfactorily in the immune landscape and chemotherapeutic responses.

Abstract 942: Development of customizable bladder cancer mouse model using CRISPR

Abstract Introduction: In forward genetics, a phenotype (sensitivity to therapy) is attributed to a genotype (mutation in relevant gene) based on findings in anecdotal cases (e.g., extreme responders) with little systematic/experimental study. Although powerful, this approach is not widely applicable due to the limited availability of clinical materials and throughput thus limiting the number and type of hypotheses that can be rigorously tested. However, a genotype-therapy sensitivity relationship could be measured in isogenic systems using reverse genetics, whereby mutations in relevant genes are introduced followed by evaluation of a phenotype in wild-type and mutant contexts. Since no such pre-clinical system exists for bladder cancer, we developed an immunocompetent mouse model to induce customizable isogenic tumor formation in bladder urothelium using CRISPR. Methods: Bladder urothelium was harvested from transgenic mice harboring LSL-Cas9/GFP in Rosa26 locus (as described by Platt et al) and grown as organoids. These organoids had a basal phenotype. AAV harboring Cre and sgRNA(s) were used to induce Cas9 and engineer mutations. Based on their frequent somatic alteration pathway in TCGA-BLCA, Tp53 and Rb1 were chosen for knockout first to establish ‘base organoids’. Additional genes related to PI3K/AKT/mTOR pathway were edited in base organoids to develop a series of organoids with different genotypes predicted to activate this signaling pathway. Sanger sequencing and western blot were used to confirm knockout. Kinase inhibitors targeting the mTOR pathway at different levels were assessed in organoids with different genotypes. The edited urothelial cells were grafted in mouse bladder to develop isogenic tumor to assess in vivo therapeutic vulnerability of in-vitro-prioritized kinase inhibitors matching the genotype of the allografted tumor. Results: Rb1, Tp53, and Pten were successfully knocked out in urothelial organoids. When treated with Ipataseritib, an AKT inhibitor, the triple knockout organoid was found to be 9X more sensitive than Trp53/Rb1 knockout organoids. In contrast, triple knockout organoids were 15X less sensitive to Gefitinib, an EGFR inhibitor, than Trp53/Rb1 knockout organoids, in line with published data. Edited organoids generate a neo-urothelium in murine bladder in short term assays. Conclusions: We developed a flexible bladder cancer mouse model to study mutation-therapy relationship using reverse genetics. This system is a plausible way to uncover unappreciated therapeutic vulnerabilities that probably exist in bladder cancer. Citation Format: Henkel L. Valentine, Uttam Satyal, Philip H. Abbosh. Development of customizable bladder cancer mouse model using CRISPR [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 942.

Abstract 4058: Second generation imidazolium compounds as tumor-specific intravesical therapeutic agents for non-muscle invasive bladder cancer

Abstract Introduction: Intravesical therapy is critical in management of high-risk non-muscle invasive bladder cancer. The most effective intravesical therapy, BCG, has an ongoing shortage, while other second line therapies are not very effective, leaving radical cystectomy (RC) as the most effective option for cancer control. However, RC is a morbid, life-altering, and expensive procedure. Unfortunately, high-grade superficial disease is associated with cancer-specific mortality in 1/3 of the patients. Improved second line therapies or development of a BCG alternative would increase the rate of bladder preservation and survival, especially in light of the recurrent BCG shortages which have plagued bladder cancer patients. We earlier demonstrated that our first-generation imidazolium compounds are effective exfoliants that induce apoptosis via mitochondrial pathway and had potential to be used as intravesical therapy for bladder cancer. In this study, we developed and investigated second generation imidazolium compounds that have selective toxicity towards tumor tissues. We also investigated the mitochondrial target(s) of these imidazolium compounds. Methods: Second generation imidazolium compounds were screened for their toxicity against various bladder cancer cell lines using CellTiter-Glo and colony forming assay. Apoptosis induction was confirmed using western blot and propidium-iodide incorporation analysis. In vitro toxicity comparison between BBN-tumor organoids and normal organoids was performed using confocal microscopy. In vivo toxicity of these compounds was studied in normal mice. For candidate target identification, mitochondria from cells grown in SILAC heavy/light media were treated with biotinylated imidazolium compound or a mixture of biotinylated compound plus TPP1 (a first-generation imidazolium compound). Lysates were subjected to streptavidin pull down followed by LC-MS/MS proteomics analysis. Shortlisted target candidates were knocked down using siRNA followed by imidazolium treatment to assess their candidacy as the imidazolium target. Results: All second-generation imidazolium compounds had significantly higher toxicity to bladder cancer cell lines compared to first generation compounds. Compounds IS154 and IS155 were shown to be cancer selective. These two compounds showed no change in histopathology of murine bladder urothelium 24 h after intravesical instillation. Proteomics data from SILAC experiments prioritized three possible targets of the imidazolium compounds which are currently being explored. Conclusions: Second generation imidazolium compounds are tumor-specific and highly potent compounds that are worthy of further study as an intravesical agent for the treatment of high grade superficial bladder cancer. Citation Format: Uttam Satyal, David Weader, Henkel Valentine, Michael Stromyer, Wiley J. Youngs, Philip H. Abbosh. Second generation imidazolium compounds as tumor-specific intravesical therapeutic agents for non-muscle invasive bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4058.

Lentiviral interferon: A novel method for gene therapy in bladder cancer

Interferon alpha (IFNα) gene therapy is emerging as a new treatment option for patients with non-muscle invasive bladder cancer (NMIBC). Adenoviral vectors expressing IFNα have shown clinical efficacy treating bacillus Calmette-Guerin (BCG)-unresponsive bladder cancer (BLCA). However, transient transgene expression and adenoviral immunogenicity may limit therapeutic activity. Lentiviral vectors can achieve stable transgene expression and are less immunogenic. In this study, we evaluated lentiviral vectors expressing murine IFNα (LV-IFNα) and demonstrate IFNα expression by transduced murine BLCA cell lines, bladder urothelium, and within the urine following intravesical instillation. Murine BLCA cell lines (MB49 and UPPL1541) were sensitive to IFN-mediated cell death after LV-IFNα, whereas BBN975 was inherently resistant. Upregulation of interleukin-6 (IL-6) predicted sensitivity to IFN-mediated cell death mediated by caspase signaling, which when inhibited abrogated IFN-mediated cell killing. Intravesical therapy with LV-IFNα/Syn3 in a syngeneic BLCA model significantly improved survival, and molecular analysis of treated tumors revealed upregulation of apoptotic and immune-cell-mediated death pathways. In particular, biomarker discovery analysis identified three clinically actionable targets, PD-L1, epidermal growth factor receptor (EGFR), and ALDHA1A, in murine tumors treated with LV-IFNα/Syn3. Our findings warrant the comparison of adenoviral and LV-IFNα and the study of novel combination strategies with IFNα gene therapy for the BLCA treatment.

89Zirconium-labelled girentuximab (89Zr-TLX250) PET in patients with urothelial cancer (ZiPUP): A phase I trial of a novel staging modality for urothelial carcinoma.

TPS4609 Background: Bladder cancer is a lethal disease with a rising incidence. The current standard imaging modalities for staging are either CT of the chest, abdomen and pelvis or FDG PET/CT. However, there are issues with using these modalities for staging. CT is known to have relatively low sensitivity for detecting low volume metastatic disease, while FDG is predominantly renally excreted and therefore has intense activity in the urinary tract, which limits its utility to detect bladder or upper tract lesions, or nodal metastases in close proximity to the urinary tract. Zirconium-89-Girentuximab (89Zr-TLX250) may have utility in the accurate staging of bladder and urothelial cancer, with less renal excretion as compared to FDG, however this has not previously been investigated. ZipUp is an investigator initiated trial. South Metropolitan Health Service WA is the trial sponsor. The study received funding and study drug support from Telix Pharmaceuticals Methods: ZiPUP is a single-arm, phase I trial examining the feasibility, safety, and utility of 89Zr-TLX250 PET/CT in patients either undergoing pre-operative staging of urothelial carcinoma or bladder cancer for curative intent, or with known metastatic urothelial carcinoma or bladder cancer. All participants will undergo 89Zr-TLX250 PET/CT and will need to have undergone recent FDG PET/CT for means of comparison. This trial aims to recruit 10 participants undergoing staging and 10 participants with known metastatic disease. The primary endpoint is effectiveness by assessing sensitivity and specificity in detecting lymph node metastases compared to FDG PET/CT; secondary endpoints are safety, tolerability, and feasibility. The first patient was enrolled in this single site study and the study is expected to be completed by December 2022. If 89Zr-TLX250 PET/CT is proven to be feasible, safe, and effective in staging urothelial cancer, it could improve the appropriate selection of treatment for patients with metastatic or primary urothelial carcinoma or bladder cancer. Clinical trial information: NCT05046665.

S100A1 expression characterizes terminally differentiated superficial cells in the urothelium of the murine bladder and ureter.

The urothelium is a stratified epithelium that lines the inner surface of the components of the urinary drainage system. It is composed of a layer of basal cells, one or several layers of intermediate cells, and a layer of large luminal superficial or umbrella cells. In the mouse, only a small set of markers is available that allows easy molecular distinction of these urothelial cell types. Here, we analyzed expression of S100A1, a member of the S100 family of calcium-binding proteins, in the urothelium of the two major organs of the murine urinary tract, the ureter and the bladder. Using RNA in situ hybridization analysis, we found exclusive expression of S100a1 mRNA in luminal cells of the ureter from embryonic day (E)17.5 onwards and of the bladder from E15.5 to adulthood. Immunofluorescence analysis showed that expression of S100A1 protein is confined to terminally differentiated superficial cells of both the ureter and bladder where it localized to the nucleus and cytoplasm. We conclude that S100A1 is a suitable marker for mature superficial cells in the urothelial lining of the drainage system of the developing and mature mouse.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00418-022-02120-1.

A phase 3 study of the subcutaneous programmed cell death protein 1 inhibitor sasanlimab as single agent for patients with bacillus Calmette-Guérin, unresponsiv,e high-risk, non-muscle invasive bladder cancer: CREST Study Cohort B.

TPS4614 Background: Bacillus Calmette-Guérin (BCG) therapy is the standard of care for high-risk non-muscle invasive bladder cancer (NMIBC) after transurethral resection of bladder tumor. However, disease recurrence or progression is common and patients with BCG-unresponsive disease are unlikely to respond to further BCG therapy. In these patients, the current standard of care is radical cystectomy and bladder-preserving treatment options, limited to intravesical chemotherapy or intravenous pembrolizumab. In a phase 1 study, sasanlimab (PF-06801591), a monoclonal antibody to programmed cell death protein 1 (PD-1), was administered subcutaneously at 300 mg every 4 weeks. Sasanlimab had an acceptable safety profile and showed clinical activity aligned to other anti-PD-1/PD-ligand 1 (PD-L1) agents in patients with advanced urothelial carcinoma and non-small cell lung cancer, while offering the convenience of subcutaneous administration. Therefore, CREST Study Cohort B aims to evaluate sasanlimab administered subcutaneously in patients with BCG-unresponsive NMIBC. Methods: CREST Study Cohort B is a non-randomized, multicenter, multinational, open-label, phase 3 study and will enroll ̃160 patients with histologically confirmed BCG-unresponsive, high-risk, non-muscle invasive transitional cell carcinoma of the bladder urothelium (high-grade Ta or T1 tumor, or carcinoma in situ [CIS]) in 2 separate Cohorts, B1 and B2 (̃110 and ̃50 patients, respectively). Cohort B1 will enroll patients with persistent or recurrent CIS with or without concomitant recurrent high-grade Ta/T1 disease, within 12 months of completing adequate BCG therapy. Cohort B2 will enroll patients with recurrent high-grade Ta/T1 disease within 6 months of completing adequate BCG therapy. All patients will receive subcutaneous sasanlimab as a single agent. Efficacy will be assessed at regular intervals by cystoscopy, urine cytology, biopsy, and imaging. The primary endpoint is complete response (CR) and event-free survival (EFS) for Cohort B1 and B2, respectively. Secondary endpoints include duration of CR (Cohort B1 only), EFS (Cohort B1 only), overall survival, time to cystectomy, safety, health-related quality of life, pharmacokinetic parameters, PD-L1 expression, and incidence of anti-drug antibodies. Recruitment of patients in CREST Study Cohort B will be opened in Canada and the United States of America, with other sites in Asia, Australia, and Europe. Clinical trial information: NCT04165317.

18 - Ex vivo analysis of bladder urothelium and bladder cancer tissues by means of fiber based attenuated total reflection infrared spectroscopy

18 - Ex vivo analysis of bladder urothelium and bladder cancer tissues by means of fiber based attenuated total reflection infrared spectroscopy

Identification of molecular feature in tumor immune microenvironment of bladder cancer patients.

e16500 Background: Bladder cancer (BC) is the most common malignant tumor of the urinary system, with various heterogeneity. Immunotherapy has become one of the hot areas of cancer research. Tumor immune microenvironment is closely related to prognosis and immunotherapy effect. Therefore, Immune cell infiltration analysis is necessary for the prognosis and treatment of BC. Methods: In this study, we analyzed IMvigor210 dataset, which included complete expression data and detailed clinical information from a cohort associated with Bladder Urothelial Carcinoma (BLCA) immunotherapy. The dataset was divided into two groups based on tumor remission after immunotherapy, including 68 remission samples and 230 stable samples. The CIBERSORT program package was used for immune invasion analysis to compute the relative proportion of immune cells in the tumor tissue from tissue gene expression profiles. Results: Significant results of differentially immune cells proportion were obtained in 126 BC patients by CIBERSORT analysis ( p< 0.05). Compared with the stable group, the number of resting memory CD4+ T cells decreased and the number of CD8+T cells increased in the remission group. The relative proportion of immune cells in tumor microenvironment has certain influence on the occurrence and development of tumor. Conclusions: Memory CD4+ T cells in the resting state did not promote the activation and proliferation of CD8+ T cells, whereas resting memory CD4+ T cells were associated with poor outcome. The increased level of CD8+ T cells in tumor microenvironment was associated with the anti-tumor effect and improved prognosis of various cancers. The results indicate that resting memory CD4+ T cells and CD8+ T cells may be the target of immunotherapy.

Digital score of lymphocytic infiltration in tumor-associated stroma in relation to overall survival in bladder cancer.

4576 Background: Bladder Urothelial Carcinoma (BLCA) is the most common type of bladder cancer and the sixth most frequent cancer in the US. High numbers of lymphocytes colocated with tumour-associated stromal (TAS) tissue has demonstrated prognostic significance for overall survival in multiple cancers. Our goal in this study was to explore the prognostic significance of an automated score to quantify the colocalisation of lymphocytes and TAS for overall survival (OS) in BLCA patients from Haematoxylin & Eosin (H&E) stained Whole Slide Images (WSIs). Methods: Two cohorts of BLCA patients were included in this study. From the UK, a cohort of 67 BLCA patients constituted cohort A. We also evaluated our method on The Cancer Genome Atlas (TCGA) bladder cancer cohort of 453 cases, which we refer to as cohort B. We developed a two-stage method for digital quantification of lymphocytic infiltrates in TAS. First, we employed an AI algorithm to recognise and classify different regions as areas of high concentration of tumour, lymphocytes and stroma for each WSI creating a segmentation map of the different tissue types. For patients with multiple WSIs, we used the slide with the highest percentage of tumour to predict survival. This algorithm had been pre-trained on a cohort of Oral cancer and was fine-tuned using annotations from 4 BLCA WSIs, 3 from cohort A and 1 from cohort B. These WSIs were excluded from the final survival analysis. Using the segmentation maps, a statistical measure for the colocalisation of TAS and lymphocytes termed the tumour-associated stroma infiltrating lymphocytes or (TASIL) score was computed. Finally, for each cohort, data was right-censored at 10 years and the digital BLCA-TASIL (BT) score’s prognostic significance for OS was investigated by fitting a Kaplan-Meier estimator and Cox proportional hazard (PH) analysis, stratifying patients into two groups based on the BT score. In each cohort, two thirds of the data was used as the discovery set to determine the best cut-off for the TASIL Score and the remaining third was used as the validation set. Results: Our classification algorithm achieved high average F1-score of 0.88 on a held-out set of unseen data for the classification of tumour, lymphocytic and stromal regions. In cohort A, higher BT score was strongly associated with better OS ( P= 0.00906) on the unseen validation data. This significant association was also found in the validation data of cohort B ( P< 0.001). Using the BT score as the only covariate, a Cox PH model for the validation data resulted in a C-index of 0.73 for cohort A and 0.57 for cohort B, respectively. Conclusions: The digital BT score showed significant prognostic value for overall survival in both BLCA cohorts, reinforcing the findings of prior work. We intend to further validate these findings on another cohort. To the best of our knowledge, this is the first attempt to predict survival solely from H&E slides in BLCA.

Three Prognostic Biomarkers Correlate with Immune Checkpoint Blockade Response in Bladder Urothelial Carcinoma.

Aim We aim to develop a signature that could accurately predict prognosis and evaluate the response to immune checkpoint blockade (ICB) in bladder urothelial carcinoma (BLCA). Methods Based on comprehensive analysis of public database, we identified prognosis-related hub genes and investigated their predictive values for the ICB response in BLCA. Results Among 69 common DEGs, three genes (AURKA, BIRC5, and CKS1B) were associated with poor prognosis, and which were related to histological subtypes, TP53 mutation status, and the C2 (IFN-gamma dominant) subtype. Three genes and their related risk model can effectively predict the response of immunotherapy. Their related drugs were identified through analysis of drug bank database. Conclusions Three genes could predict prognosis and evaluate the response to ICB in BLCA.

Changes in the Ultrastructure of the Bladder Urothelium in Patients with Interstitial Cystitis after Intravesical Injections of Platelet-Rich Plasma.

Urothelial dysfunction is considered a key pathological mechanism of interstitial cystitis/bladder pain syndrome (IC/BPS). Intravesical platelet-rich plasma (PRP) injections might be effective for treating IC/BPS. This prospective study investigated the changes in electron microscopic findings among IC/BPS patients after intravesical PRP injections. Twenty-six patients with refractory non-ulcer IC/BPS underwent monthly intravesical PRP injections for 4 months. Changes in clinical symptom scores and video urodynamic study parameters were assessed from baseline to after the PRP injections. A post-treatment Global Response Assessment (GRA) score ≥ 2 was considered a successful outcome. The mean GRA score was significantly higher after 4 PRP injections than at baseline. Approximately 42% of patients experienced successful outcomes after PRP treatment. Urothelial ultrastructural defects showed no significant differences between baseline and after the PRP injections. However, patients showed variable improvements in different urothelial defects (grade improvements: urothelium cell layers, 31%; umbrella cell integrity, 42%; umbrella cell surface uroplakin plaque, 54%; tight junctions between adjacent umbrella cells, 46%; lysed organelles, 58%; inflammatory cell infiltration, 31%). Patients with successful treatment outcomes showed significant improvements in urothelial tight junction defects. Repeated intravesical PRP injections are effective for improving IC/BPS symptoms as they promote urothelial ultrastructural defect recovery.

Activation of the chemokine receptor 3 pathway leads to a better response to immune checkpoint inhibitors in patients with metastatic urothelial carcinoma

Immune checkpoint inhibitors (ICIs) have made important breakthrough in anti-tumor therapy, however, no single biomarker can accurately predict their efficacy. Studies have found that tumor microenvironment is a key factor for determining the response to ICI therapy. Cytokine receptor 3 (C-X-C Motif Chemokine Receptor 3, CXCR3) pathway has been reported to play an important role in the migration, activation, and response of immune cells. We analyzed survival data, genomics, and clinical data from patients with metastatic urothelial carcinoma (mUC) who received ICI treatment to explore the relationship between CXCR3 pathway activation and the effectiveness of ICIs. The Cancer Genome Atlas Bladder Urothelial Carcinoma cohort and six other cohorts receiving ICI treatment were used for mechanism exploration and validation. In the ICI cohort, we performed univariate and multivariate COX analyses and discovered that patients in the CXCR3-high group were more sensitive to ICI treatment. A Kaplan–Meier analysis demonstrated that patients in the high CXCR3-high group had a better prognosis than those in the CXCR3-low group (P = 0.0001, Hazard Ratio = 0.56; 95% CI 0.42−0.75). CIBERSORT analysis found that mUC patients in the CXCR3-high group had higher levels of activated CD8+ T cells, M1 macrophages, and activated NK cells and less regulatory T cell (Treg) infiltration. Immunogenicity analysis showed the CXCR3-high group had higher tumor neoantigen burden (TNB). Our study suggests that CXCR3 pathway activation may be a novel predictive biomarker for the effectiveness of immunotherapy in mUC patients.