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Bladder cancer (BC) is an epidemiological urologic malignancy that continues to increase each year. Early diagnosis and prognosis monitoring is always significant in clinical practice, especially in distinguishing non-muscle-invasive bladder cancer (NMIBC) from muscle-invasive bladder cancer (MIBC), due to the various depths of tumor invasion related to different therapeutic schedules and recurrence rates. Common diagnostic approaches are too invasive or generally inefficient in accuracy and specificity. In this work, a totally non-invasive and cost-effective method is established by investigating urine samples using surface-enhanced Raman spectroscopy (SERS) and multivariate statistical analysis. The comparison of urine SERS spectra shows the intensities of characteristic peaks for DNA/RNA, hypoxanthine, albumin, D-( +)-galactosamine, fatty acids, and some amino acids are distinguishable in BC occurrence and invasion progression. A PLS-LDA-based two-step binary classification scheme is performed on urine SERS spectra and the diagnostic accuracies were 97.7% and 96.3% for healthy individuals versus BC patients and NMIBC versus MIBC patients, respectively. Moreover, the impact of urine SERS spectral lengths in reaching high-precision recognition of BC is investigated. The results show that the Raman peaks at 803, 893, 1139, 1375, and 1466 cm -1 play an essential role in correctly categorizing healthy control, NMIBC, and MIBC patients, and SERS spectra ranges from 400 to 1600 cm -1 are enough for this identification task. These findings provide a sensitive, label-free, rapid, and totally non-invasive way for assessment of invasion depth of BC to its early diagnosis and prognosis monitoring, as well as valuable insights for selecting reasonable spectral range to enhance the measurement efficiency especially in large-scale sample datasets., Competing Interests: Declarations. Ethical approval: The urine samples were provided by the First Affiliated Hospital of Ningbo University. Ethical approvals have been approved by the medical ethics committee of the First Affiliated Hospital of Ningbo University, and the relevant informed consent form was signed. Conflict of interest: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Introduction: Urothelial tract cancer (UTC) ranks as the tenth most prevalent cancer and holds the seventh position in terms of mortality worldwide. Despite its prevalence and mortality ranking, there are still gaps in the knowledge of the mutational landscape in patients with advanced disease who have limited therapeutic options after multiple lines of prior treatment. This study compares the genomic and transcriptomic landscape, and targeted treatment options between metastatic UTC (mUTC) patients treated with multiple lines of therapy compared to newly diagnosed, untreated Muscle Invasive Bladder Cancer (MIBC)., Methods: We compared genomic and clinical data from two cohorts: mUTC patients who received multiple lines of therapy and were referred to the Copenhagen Prospective Personalized Oncology (CoPPO) project at Rigshospitalet, University of Copenhagen. Data for MIBC UTC patients were acquired from the Cancer Genome Atlas Bladder Cancer (TCGA BLCA) cohort. Biopsies in CoPPO were performed at the time of enrollment. 523 highly important cancer-related genes (TrueSight Oncology-500 targeted sequencing panel) were used from both cohorts for comparative analysis. Analyses included RNA count data to compare predicted molecular subtypes in each cohort separately., Results: Patients from the CoPPO cohort had a lower median age at first-line treatment than the TCGA BLCA cohort, with no significant gender disparity. The predominant histology was urothelial cell carcinoma in both cohorts. Genomic analysis revealed no significant difference between the top mutated genes in the two cohorts, specifically looking into DNA damage repair genes. Molecular subtyping indicated a higher frequency of neuroendocrine differentiation in the CoPPO cohort. 13% of patients in the CoPPO cohort received targeted therapy based on genomic findings, and 16% received non-targeted treatment, totaling 29% receiving CoPPO treatment (9 patients). The remaining 71% received best supportive care. Kaplan-Meier analysis showed a non-significant survival benefit for the intervention group in the CoPPO cohort., Conclusion: When focusing on 523 highly relevant cancer genes, the mutational profile of mUTC patients who have undergone numerous treatment lines resembles that of newly diagnosed MIBC. These alterations can be targeted, indicating the potential advantage of early genomic testing for personalized treatment within clinical trials., (© 2024. The Author(s).)

Background: Bladder cancer (BCa) is among the most prevalent malignant tumors affecting the urinary system. Due to its highly recurrent nature, standard treatments such as surgery often fail to significantly improve patient prognosis. Our research aims to predict prognosis and identify precise therapeutic targets for novel treatment interventions., Methods: We collected and screened genes related to the TGF-β signaling pathway and performed unsupervised clustering analysis on TCGA-BLCA samples based on these genes. Our analysis revealed two novel subtypes of bladder cancer with completely different biological characteristics, including immune microenvironment, drug sensitivity, and more. Using machine learning classifiers, we identified SMAD6 as a hub gene contributing to these differences and further investigated the role of SMAD6 in bladder cancer in the single-cell transcriptome data. Additionally, we analyzed the relationship between SMAD6 and immune checkpoint genes. Finally, we performed a series of in vitro assays to verify the function of SMAD6 in bladder cancer cell lines., Results: We have revealed two novel subtypes of bladder cancer, among which C1 exhibits a worse prognosis, lower drug sensitivity, a more complex tumor microenvironment, and a 'colder' immune microenvironment compared to C2. We identified SMAD6 as a key gene responsible for the differences and further explored its impact on the molecular characteristics of bladder cancer. Through in vitro experiments, we found that SMAD6 promoted the prognosis of BCa patients by inhibiting the proliferation and migration of BCa cells., Conclusion: Our study reveals two novel subtypes of BCa and identifies SMAD6 as a highly promising therapeutic target., (© 2024. The Author(s).)

Background: Based on liquid-based cytology, we performed an enzyme histochemical staining using acid phosphatase as a marker and termed it ELLBC. The aim of this study was to investigate the value of ELLBC in the diagnosis of bladder cancer., Methods: Fifty patients who were initially diagnosed with suspected bladder cancers (hematuria or bladder irritation symptoms, urinary ultrasound suggestive of bladder mass) at the Second Affiliated Hospital of Anhui Medical University (Anhui, China) from January 2022 to December 2022 were selected as the study subjects, all of whom underwent ELLBC, CC, and histopathology Histopathology was used as the gold standard to calculate the diagnostic efficacy of ELLBC, CC and ELLBC combined with CC in bladder cancer., Results: Histopathological examination revealed 35 positive cases in 50 patients, including 15 cases of high-grade uroepithelial carcinoma (HGUC) and 20 cases of low-grade uroepithelial carcinoma (LGUC.) The sensitivity of ELLBC was 82.86%, the specificity was 93.33%, the positive predictive value (PPV) was 96.67%, the negative predictive value (NPV) was 70.00%, and the accuracy was 86.00%; CC had a sensitivity of 37.14%, specificity of 80.00%, PPV of 81.25%, NPV of 35.29%, and accuracy of 50%; ELLBC combined with CC had a sensitivity of 88.57%, specificity of 73.33%, PPV of 88.57%, NPV of 73.33%, and accuracy of 84.00%. The sensitivity and specificity of ELLBC were higher than that of CC, and the difference was statistically significant (p < 0.05), ELLBC combined with CC achieved higher sensitivity, but the diagnostic accuracy decreased. For clinical staging, the diagnostic accuracy was 86.36% for ELLBC and 40.91% for CC in patients in Stage I, and 90.91% for ELLBC and 36.36% for CC in patients in Stage II., Conclusion: ELLBC has high clinical application value for the diagnosis of bladder cancer and can provide new options and methods for the early screening of bladder cancer., (© 2024. The Author(s).)

Although aromatic amines are widely used as raw materials for dyes, some, such as o-toluidine and o-anisidine, have shown concerning results regarding carcinogenicity in the urinary bladder. We have recently developed a short-term detection method for bladder carcinogens using immunohistochemistry for γ-H2AX, a DNA damage marker. Here, using this method, we evaluated aromatic amines with structures similar to o-toluidine and o-anisidine for bladder mucosal damage and potential carcinogenicity. In total, 17 aromatic amines were orally administered to male F344 rats for 28 days, and histopathological examination and γ-H2AX immunostaining of the urinary bladder were performed. Histopathological analysis revealed that seven aromatic amines, including 4-chloro-o-toluidine (4-CT), o-aminoazotoluene, 2-aminobenzyl alcohol (ABA), o-acetotoluidine (o-AT), 3,3'-dimethoxybenzidine, 4-aminoazobenzene (AAB), and 4,4'-methylenedianiline (MDA), induced various bladder lesions, such as hemorrhage, necrosis, and urothelial hyperplasia. The morphological characteristics of mucosal damage induced by these substances were divided into two major types: those resembling o-toluidine and those resembling o-anisidine. Six of these aromatic amines, excluding MDA, also caused significant increases in γ-H2AX formation in the bladder urothelium. Interestingly, 4-CT did not cause mucosal damage or γ-H2AX formation at the lower dose applied in previous carcinogenicity studies. These results showed for the first time that o-AT and ABA, metabolites of o-toluidine, as well as AAB caused damage to the bladder mucosa and suggested that they may be bladder carcinogens. In addition, 4-CT, which was thought to be a noncarcinogen, was found to exhibit bladder toxicity upon exposure to high doses, indicating that this compound may contribute to bladder carcinogenesis., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The study was designed to detect the expression and clinical significance of the HEATR3 gene in bladder cancer (BCa) and to preliminarily explore whether this gene can affect the occurrence and development of BCa through the AKT/ERK signaling pathway. The expression and prognostic value of HEATR3 were explored based on The Cancer Genome Atlas (TCGA) and Genotypic Tissue Expression (GTEx) databases. Microarray immunohistochemical analysis was performed in 30 BCa cases to investigate the level of HEATR3 protein and to explore the relationship between HEATR3 and the clinicopathological features of BCa. Western Blot and qRT-PCR were used to detect HEATR3 protein and mRNA in BCa cell lines (5637, TCCSUP, SW780) and fallopian tube epithelial cell (SV-HUC-1). CCK8 method was employed to study the proliferation of BCa cells after heat treatment. Transwell assay was conducted to analyze the effect of HEATR3 on cell migration and invasion. And cell cycle and apoptosis were detected by flow cytometry. Furthermore, Western Blot assay was used to probe the effects of down-regulation of HEATR3 expression on the expression and phosphorylation levels of AKT and ERK proteins in BCa cells. Bioinformatics analysis showed that HEATR3 was significantly up-regulated in BCa, and high HEATR3 expression was associated with poor prognosis of BCa patients. In vitro experiments demonstrated that HEATR3 expression was up-regulated in BCa tissues compared with that in adjacent tissues. HEATR3 protein was also up-regulated in malignant cell lines. HEATR3 knockdown in BCa cells could inhibit cell proliferation, invasion and migration, block cell cycle and promote cell apoptosis. At the same time, HEATR3 knockdowns reduced the expression levels of p-AKT and p-ERK proteins. HEATR3 knockdown inhibits the development of BCa cells through the AKT/ERK signaling pathway. and it may become one of the most promising molecular targets for BCa treatment., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Introduction: The molecular mapping of cancers by the Cancer Genome Atlas Project has accelerated the quest for new therapeutic targets for urinary bladder cancer, including sex steroid receptors. Previous studies have demonstrated conflicting results on their relationship with bladder cancer, and there is sparse data on their expression in the Indian population. The aim of our study is to examine the expression of androgen receptors (AR) and estrogen receptors (ERα and ERβ) in patients with bladder cancer and their correlation with clinicopathologic features., Materials and Methods: In this retrospective cohort study, a total of 132 patients, who were surgically managed for urinary bladder mass by transurethral resection or radical cystectomy in our institute, with transitional cell carcinoma on histopathology and with at least two years of follow-up were included. Their demographic and treatment details were obtained, histopathology blocks were retrieved and immunohistochemical staining for androgen and estrogen receptors was performed. Then, the relationship between their expression and clinicopathologic features was studied., Results: A total of 3.79% of patients showed estrogen receptor alpha positivity, 51.52% estrogen receptor beta positivity and 63.64% androgen receptor positivity. No statistically significant correlation was found between age of patients (p = 0.75/0.52/0.87), tumour stage and grade (0.71/0.3/0.21), pathological variant (p = 1/0.58/0.38) and overall survival (p = 0.70/0.052/0.45 for NMIBC and p = 0.82/0.36/0.22) and estrogen receptor alpha, estrogen receptor beta and androgen receptor-positive status, respectively. Estrogen receptor beta positivity was significantly higher in patients with unifocal (p = 0.015) and small tumours (< 5 cm) (p = 0.03), and its expression was associated with better disease-free survival (DFS) (p = 0.046) in patients of non-muscle invasive bladder cancer (NMIBC)., Conclusion: Our study has the largest sample size conducted on Indian population with results differing from previous studies conducted on western population. Estrogen receptor beta expression was significantly associated with small unifocal tumours and better DFS. Estrogen receptor alpha and androgen receptor expression were not found to be associated with the clinicopathologic features of the study population., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: At present, the prediction of bladder tumor nature during cystoscopy is partially dependent on the clinician's own experience. Subjective factors may lead to excessive biopsy or delayed treatment. The purpose of our study is to establish a reliable model for predicting the nature of bladder tumors using narrow band imaging., Methods: From November 2021 to November 2022, the clinical data of 231 patients who required a cystoscopy were prospectively collected at our center. Cystoscopy was performed in 219 eligible patients, in which both tumor and vascular morphology characteristics were recorded. Pathological results were used as the diagnostic standard. A logistic regression analysis was used to screen out factors related to tumor pathology. Bootstrap resampling was used for internal validation. A total of 71 patients from four other centers served as an external validation cohort., Results: The following diagnostic factors were identified: tumor morphology (cauliflower-like or algae-like lesions), vascular morphology (dotted or circumferential vessels), tumor boundary (clear or unclear), and patients' symptoms (gross hematuria) and were included in the prediction model. The internal validation results showed that the area under the curve was 0.94 (95% CI 0.92-0.97), and the P value from the goodness-of-fit test was 0.97. After external validation, the results showed the area under the curve was 0.89 (95% CI 0.82-0.97) and the P value of the goodness-of-fit test was 0.24., Conclusion: A diagnostic prediction nomogram was established for bladder cancer. The verification results showed that the prediction model has good prediction performance., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: This study aimed to establish and validate nomograms to predict the probability of intravesical recurrence (IVR) after radical nephroureterectomy (RNU) for upper urinary tract epithelial carcinoma (UTUC)., Methods: Clinical data of 528 patients with UTUC after RNU were collected from two medical centers between 2009 and 2020. We used the least absolute shrinkage and selection operator (LASSO) regression to select variables for multivariable Cox regression analysis in the training cohort and included independent risk factors into nomogram models predicting IVR-free survival (IVRFS). Another center was applied as the external cohort to validate the predictive accuracy and discriminative ability of the nomogram by performing area under the receiver operating curve (AUC), consistency index (C-index), and calibration curve., Results: History of bladder cancer, tumor size, preoperative urine cytology, postoperative instillation, Ki-67, and platelet-to-lymphocyte ratio (PLR) were identified as independent risk factors for IVR. The prognosis model including these predictors demonstrated excellent discriminatory performance in both the training cohort (C-index, 0.814) and external validation cohort (C-index, 0.748). The calibration plots of the nomogram revealed good consistency in both cohorts. Finally, patients could be classified into two risk groups based on scores obtained from the nomogram, with significant differences in IVRFS., Conclusion: Our study provided a reliable nomogram for predicting the probability of IVR in patients with UTUC after RNU. Risk stratification based on this model may assist urologists make optimal clinical decisions on the management of UTUC., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objective: To investigate the value of gemcitabine and pirarubicin in patients with non-muscle-invasive bladder cancer (NMIBC)., Methods: 405 patients with non-muscle invasive bladder cancer admitted to our hospital from January 2012 to December 2020 who underwent transurethral bladder tumor electronic resection were studied. 177 patients were treated with gemcitabine (Gemcitabine group) and 228 patients were treated with pirarubicin (Pirarubicin group) after surgery. The efficacy and adverse effects of the two groups were observed and the patients were followed up., Results: No differences were found when comparing age, gender, smoking, bladder mass, number of masses, hypertension, diabetes, coronary artery disease, hematuria and tumor diameter between the 2 groups (P > 0.05). In the Gemcitabine group, bladder irritation signs, meatus hematuria, fever, nausea and vomiting were lower than those in the Pirarubicin group (P < 0.05). The recurrence rates were 6.21% and 12.28% at 1 year, 11.86% and 23.68% at 2 years, 15.82% and 25.88% at 3 years in the Gemcitabine and Pirarubicin groups respectively, with the Gemcitabine group having a significantly lower recurrence rate than the Pirarubicin group (P < 0.05). The tumor recurrence-free survival rate for 5 years of gemcitabine was significantly higher than that of the Pirarubicin group (P < 0.05)., Conclusion: Gemcitabine and pirarubicin are both effective in treating patients with non-muscle invasive bladder cancer, with gemcitabine having a lower incidence of adverse reactions, a higher safety rating, a lower recurrence rate and an improved survival outcome., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: Urothelial carcinoma (UC) of the bladder (BUC) and the upper urinary tract (UTUC) are the two most common UCs. The incidence of UTUC in Taiwan is the highest worldwide. Aristolochic acid (AA) was identified as the main cause of UTUC in Taiwan. To explore trends in the incidence of UC in Taiwan after the ban on Chinese herbal preparations containing AA in 2003., Methods: We used data from the Taiwanese National Health Insurance Research Database-linked Taiwanese National Cancer Registry for 2001-2018. UC was defined in accordance with the International Classification of Disease for Oncology. The age-standardized incidence was calculated on the basis of the World Health Organization standard population. Trends in the incidence were calculated as the annual percent change (APC) by using the Joinpoint regression program., Results: Over the investigated period, the incidence of UC decreased at an average annual percent change (AAPC) of - 1.19% (95% CI - 1.47 ~ - 0.91, P < 0.001). However, the incidence in UTUC significantly increased, with the AAPC being 1.47% (95% CI 1.03 ~ 1.90, P < 0.001). In contrast, the incidence of BUC significantly decreased, with the overall AAPC being - 1.92% (95% CI - 2.3 ~ - 1.54, P < 0. 001). From 2001 to 2018, the overall incidence of UCs and BUC decreased in Taiwan, but the incidence of UTUC significantly increased., Conclusion: We suggest to apply the same review standards of new drug development process to herbal preparations and incorporate them into the adverse drug reaction or poison surveillance system. Most importantly, raise public awareness of the potential toxicity of phytotherapy., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: Predicting lymph node metastasis (LNM) in patients with bladder urothelial carcinoma (BUC) before radical cystectomy aids clinical decision making. Here, we aimed to develop and validate a nomogram to preoperatively predict LNM in BUC patients., Methods: Patients with histologically confirmed BUC, who underwent radical cystectomy and bilateral lymphadenectomy, were retrospectively recruited from two institutions. Patients from one institution were enrolled in the primary cohort, while those from the other were enrolled in the external validation cohort. Patient demographic, pathological (using transurethral resection of the bladder tumor specimens), imaging, and laboratory data were recorded. Univariate and multivariate logistic regression analyses were performed to explore the independent preoperative risk factors and develop the nomogram. Internal and external validation was conducted to assess nomogram performance., Results: 522 and 215 BUC patients were enrolled in the primary and external validation cohorts, respectively. We identified tumor grade, infiltration, extravesical invasion, LNM on imaging, tumor size, and serum creatinine levels as independent preoperative risk factors, which were subsequently used to develop the nomogram. The nomogram showed a good predictive accuracy, with area under the receiver operator characteristic curve values of 0.817 and 0.825 for the primary and external validation cohorts, respectively. The corrected C-indexes, calibration curves (after 1000 bootstrap resampling), decision curve analysis results, and clinical impact curves demonstrated that the nomogram performed well in both cohorts and was highly clinically applicable., Conclusion: We developed a nomogram to preoperatively predict LNM in BUC, which was highly accurate, reliable, and clinically applicable., (© 2023. The Author(s).)

Objective: To investigate the impact of histological variants (HV) in patients with upper tract urothelial carcinoma (UTUC) and analyze the potential association between HV and postoperative bladder recurrence., Materials and Methods: The medical records of UTUC patients treated with RNU at our center from January 2012 to December 2019 were retrospectively analyzed. Patients were grouped according to the types of HV. Clinicopathological features and prognostic factors were compared among groups., Results: A total of 629 patients were included in the study: 458 (73%) patients had pure urothelial carcinoma (PUC) and 171 (27%) patients had UTUC with HV. Squamous differentiation was the most common type (124 cases, 19%), followed by glandular differentiation (29 cases, 5.0%). Patients with HV had a higher proportion of T3 and T4 pathologic stages (P < 0.001) as well as high-grade disease (P = 0.002). In the univariate analysis, squamous differentiation and glandular differentiation were significantly associated with worse cancer-specific survival (CSS) (HR 2.22, 95% CI 1.62-3.04, P < 0.001; HR 1.90, 95% CI 1.13-3.20, P = 0.016). However, the multivariate analysis showed that this association became non-significant. We found that HV were associated with recurrent muscle-invasive bladder cancer (MIBC) after RNU and all patients had T2 and T3 initial tumor stages (P = 0.008, P < 0.001)., Conclusion: We found that UTUC patients with HV were associated with biologically aggressive disease and recurrent MIBC after RNU. The detection of bladder recurrence following surgery needs to be given more attention in advanced UTUC patients with HV., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: To evaluate the efficacy of repeat transurethral resection on restaging, preventing tumor recurrence and progression in high-risk non-muscle invasive bladder cancer patients who received initial en bloc resection., Methods and Patients: We reviewed retrospectively the clinical records of 330 consecutive patients who received en bloc resection for non-muscle invasive bladder cancer. Eligible patients with and without repeat transurethral resection were matched 1:1 by propensity score. Important covariates were balanced between the two groups. We compared the recurrence-free survival, progression-free survival, recurrence rate, and progression rate between groups. And the perioperative results regarding residual tumors and the safety of the repeat resection were also evaluated., Results: Finally, there are 245 patients included in our analysis with a median follow-up duration of 19 months (range 3-50). Detrusor muscle presented in 244 (99.6%) specimens at initial en bloc resection. And among them, 30 (12.2%) patients had undergone a repeat resection and 215 (87.8%) did not. After 1:1 propensity score matching, 30 pairs were further analyzed.No case of upstaging was identified in repeat resection. During the follow-up, recurrence was observed in 5 (16.7%) and 7 (23.3%) patients in reresection group and non-reresection group, respectively. And progression was found only in 1 (3.3%) patient in each group. The 1-year recurrence-free survival estimates were comparable (86.7% vs 83.3%, p = 0.86) between groups., Conclusion: Our study demonstrates that repeat resection after initial transurethral en bloc resection for bladder tumor appears not to improve staging accuracy, recurrence, and progression., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: There is gender-specific disparity in bladder cancer (BlCa) prognosis. Female BlCa patients present with more advanced tumor and have higher risks of disease recurrence, progression, and mortality than males. Since gonadotropin-releasing hormone (GNRH) family genes were critical genes in gender-related biological activity and could be detected in BlCa specimens, this study aimed to explore potential roles of GNRH1 and GNRHR in gender disparity of BlCa., Methods: RNA-sequencing data from The Cancer Genome Atlas Bladder Urothelial Carcinoma dataset, IMvigor210 immunotherapy cohort and Cancer Cell Line Encyclopedia database were used to compare potential roles of GNRH1 and GNRHR in males and females, respectively. Gene set enrichment analysis was used to analyze the biological functions., Results: Males with higher GNRH1 and GNRHR have better overall survival (P < 0.05, HR < 1), while females with higher expression have a trend toward worse overall survival (P < 0.05, HR > 1). Gene set enrichment analysis identified GNRH1 and GNRHR exert opposite regulatory roles in myogenesis (M5909), interferon-α response (M5911), interferon-γ response (M5913), inflammatory response (M5932) and TNF-α signaling via NF-κβ (M5890) between males and females. The five functions are up-regulated in females (NES > 0), while down-regulated in males (NES < 0). GNRH1 in females was positively correlated with CD3D (R-value > 0 and P < 0.05), while GNRHR in males was negatively correlated with CD247, CD3D and CD3E (R-value < 0 and P < 0.05)., Conclusion: GNRH1 and GNRHR have opposite effects on overall survival in different genders, and exert opposite roles in immune-related functions between different genders, which could emerge as a contributor to gender disparity of BlCa prognosis., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: In this study, we aimed to identify prognostic factors of cancer mortality in patients who received radical cystectomy and to identify genomic alterations in a sub-cohort of patients with locally advanced (pT3-4) and/or positive lymph nodes bladder cancer (BC)., Methods: We collected 101 BC samples from 2010 to 2018 who previously received radical cystectomy. Immunohistochemical slides were evaluated for PPAR, cAMP, IMP3, Ki67, CDK4, POU5F1, Cyclin E and MDM2, p65, CD3, CD4, CD8, CD20, CD68, CD163, FOXP3, PD-1 and PD-L1 expression. We calculated a prognostic score (PS) based on the positivity to PD-1, PD-L1 and of cAMP (final score ranging from 0 to 3). DNA of each sample have been used for sequencing by NGS in a sub-cohort of 6 patients with locally advanced (pT3-4) and/or positive lymph nodes BC., Results: PD-1 + (HR [hazard ratio] 2.59; p = 0.04), PD-L1 + (HR = 6.46; p < 0.01) and cAMP + (HR 3.04; p = 0.02) were independent predictors of cancer-specific mortality (CSM). Increase of PS (score = 0 as reference) was associated with CSM, 0.81 (p = 0.80), 4.72 (p = 0.01) and 10.51 (p < 0.0) for PS 1, 2 and 3, respectively. ERBB2 was the gene most frequently mutated., Conclusion: BC exhibited heterogenous protein expression and variable genomic features. Identification of expression of PD-1, PD-L1 and cAMP could help in predicting oncological outcomes., (© 2022. The Author(s).)

Intoduction: Bladder cancer is the second most common urinary tract cancer. Above 70% of the occurrence of bladder cancer is superficial (pTis, pTa, and pT1), non-muscle invasive tumor (NMIBC), and the incidence of invasive disease is occasional. Treatments for NMIBC consist of transurethral resection (TUR) and subsequently intravesical immunotherapy with Bacillus Calmette-Guérin (BCG), intending to prevent tumor progression and decrease recurrence. However, 20-30% of these tumors have progression, and 70% have a recurrence after exclusive TUR treatment. The immunomodulator of biological response, OncoTherad ® , is an attractive potential to revolutionize cancer therapy. In our previous studies with mice, the results showed that treatment with OncoTherad ® reduced 100% of tumor progression in NMIBC through the activation of Toll-Like Receptors' non-canonical pathway., Materials and Methods:  In the present study, 36 female C57Bl/6J mice were divided into 6 groups (n = 6/group): Control, Cancer, Cancer + BCG, Cancer + OncoTherad ® (MRB-CFI-1), Cancer + P14-16 and Cancer + CFI-1. NMIBC was chemically induced and the treatments were followed for 6 weeks. A week after the last dose of treatment, animals were euthanized, the bladder was collected and routinely processed for immunohistochemical analyses of RANK, RANKL, FOXP3, and PD-1/PD-L1, such as PD-1/PD-L1 western blotting., Conclusion: The immunohistochemical results showed that OncoTherad ® reduced RANK and RANKL immunoreactivities compared to the cancer group, which indicates a good prognosis. Immunohistochemical and western blotting analyses confirmed that OncoTherad ® modulated PD-1/PD-L1 immune checkpoint., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: To identify pre-surgical imaging predictive value and factors associated with the clinicopathologic discrepancy for implication of definitive pelvic radiotherapy in clinically node-negative bladder cancer., Method: The documented data of bladder cancer patients who underwent radical cystectomy plus pelvic lymphadenectomy were collected retrospectively. Patients' characteristics, last imaging, pathology reports, disease-specific survival and overall survival were retrieved., Results: From 142 patients, pre-surgical imaging had a sensitivity of 76.4%, specificity of 73.7%, positive predictive value (PPV) of 94.9%, and negative predictive value (NPV) of 32.6% (p value < 0.0001) for detection of muscle invasion. Also, for detection of positive lymph nodes, imaging had a sensitivity of 31.8%, specificity of 85.7%, PPV of 50%, and NPV of 73.7% (p value: 0.022). 44.4% of study population were upstaged after surgery (24.6% associated with N-upstaging) and 18.3% were downstaged (12% associated with N-downstaging). Receipt of neoadjuvant chemotherapy and T-stage were not correlated with N-upstaging. On multivariate analysis, lymphovascular invasion (LVI) maintained its significance for independent prediction of upstaging (OR 3.3, 95% CI 1.5-7.5, p value: 0.004) and inversely with downstaging (OR 0.34, 95% CI 0.12-0.96, p value: 0.04). Older age (OR 1.03, 95% CI 1.0-1.05, p value 0.047), positive margins (OR 2.1, 95% CI 1.2-3.8, p value 0.011), presence of LVI (OR 2.5, 95% CI 1.4-4.7, p value 0.003), perineural invasion (OR 2.0, 95% CI 1.2-3.4, p value 0.013), and lymph node ratio (OR 1.011, 95% CI 1.001-1.021, p value 0.03) were associated with worse survival. Also, N-upstaging independently predicted a worse survival after controlling for surgical pathology stage (OR 2.3, 95% CI 1.2-4.5, p value 0.011)., Conclusions: The optimal target volume in definitive chemoradiotherapy of node-negative bladder cancer patients remains to be established. Since then, customizing the treatment is considered especially for positive LVI in TURBT specimen., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: The HGF/MET pathway is involved in cell motility, angiogenesis, proliferation, and cancer invasion. We assessed the clinical utility of plasma HGF level as a prognostic biomarker in patients with MIBC., Methods: We retrospectively analyzed 565 patients with MIBC who underwent radical cystectomy. Logistic regression and Cox regression models were used, and predictive accuracies were estimated using the area under the curve and concordance index. To estimate the clinical utility of HGF, DCA and MCID were applied., Results: Plasma HGF level was significantly higher in patients with advanced pathologic stage and LN metastasis (p = 0.01 and p < 0.001, respectively). Higher HGF levels were associated with an increased risk of harboring LN metastasis and non-organ-confined disease (OR1.21, 95%CI 1.12-1.32, p < 0.001, and OR1.35, 95%CI 1.23-1.48, p < 0.001, respectively) on multivariable analyses; the addition of HGF improved the predictive accuracies of a standard preoperative model (+ 7%, p < 0.001 and + 8%, p < 0.001, respectively). According to the DCA and MCID, half of the patients had a net benefit by including HGF, but the absolute magnitude remained limited. In pre- and postoperative predictive models, a higher HGF level was significant prognosticator of worse RFS, OS, and CSS; in the preoperative model, the addition of HGF improved accuracies by 6% and 5% for RFS and CSS, respectively., Conclusion: Preoperative HGF identified MIBC patients who harbored features of clinically and biologically aggressive disease. Plasma HGF could serve, as part of a panel, as a biomarker to aid in preoperative treatment planning regarding intensity of treatment in patients with clinical MIBC., (© 2022. The Author(s).)

We sought to examine epigenetic inactivation of DNA damage repair (DDR) genes as prognostic and predictive biomarkers for urothelial bladder cancer (UBC) as there are currently no reliable prognostic biomarkers that identify UBC patients who would benefit from chemotherapy. Genome-wide DNA methylome using the cancer genome atlas-bladder cancer (TCGA-BLCA) datasets (primary tumors = 374 and normal tissues = 37) was performed for 154 DDR genes. The most two significant differentially methylated genes, Retinoblastoma binding protein 8 (RBBP8) and MutS homologue 4 (MSH4), between primary tumors and normal tissues of TCGA-BLCA were validated by methylation-specific PCR (MSP) in UBC (n = 70) compared to normal tissues (n = 30). RBBP8 and MSH4 expression was measured using qRT-PCR. We developed a predictive model for therapeutic response based on the RBBP8- and MSH4-methylation along with patients' clinical features. Then, we assessed the prognostic significance of RBBP8 and MSH4. RBBP8- and MSH4 methylation and corresponding gene downregulation significantly associated with muscle-invasive phenotype, prolonged progression-free survival (PFS) and increased susceptibility to cisplatin chemotherapy in UBC. Promoter methylation of RBBP8 and MSH4 was positively correlated with each other and with their corresponding gene repression. The best machine-learning classification model predicted UBC patients' response to cisplatin-based chemotherapy with an accuracy of 90.05 ± 4.5%. Epigenetic inactivation of RBBP8 and MSH4 in UBC could sensitize patients to DNA-damaging agents. A predictive machine-learning modeling approach based on the clinical features along with RBBP8- and MSH4-methylation might be a promising tool for stratification of UBC responders from nonresponders to chemotherapy., (© 2022. The Author(s).)

Choline metabolism alteration is considered as a metabolic hallmark in cancer, reflecting the complex interactions between carcinogenic signaling pathways and cancer metabolism, but little is known about whether genetic variants in the metabolism pathway contribute to the susceptibility of bladder cancer. Herein, a case-control study comprising 580 patients and 1,101 controls was carried out to analyze the association of bladder cancer with genetic variants on candidate genes involved in the choline metabolism pathway using unconditional logistic regression. Gene expression data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) database were applied for differential gene expression analysis. Cox regression was also applied to estimate the role of candidate genes on bladder cancer prognosis. Our results demonstrated that C allele of rs6810830 in ENPP6 was a significant protective allele of bladder cancer, compared to the T allele [Odds ratio (OR) = 0.74, 95% confidence interval (CI) = 0.64-0.86, P = 7.14 × 10 -5 in additive model]. Besides, we also found that the expression of ENPP6 remarkably decreased in bladder tumors compared with normal tissues. Moreover, high expression of ENPP6 was associated with worse overall survival (OS) in bladder cancer patients [hazard ratio (HR) with their 95% CI 1.39 (1.02-1.90), P = 0.039]. In conclusion, our results suggested that SNP rs6810830 (T > C) in ENPP6 might be a potential susceptibility loci for bladder cancer, and these findings provided novel insights into the underlying mechanism of choline metabolism in cancers., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Concerning follow-up in bladder cancer, it must be distinguished between superficial, muscle-invasive, and metastatic tumors. In superficial bladder cancer, urethrocystoscopy is still standard for follow-up. Frequency depends on the risk classification. Even muscle-invasive carcinomas, which underwent a R0 resection, will metastasize in about 30% of cases. These tumors as well as primarily metastasized cancer cannot be cured. Therefore, in these cases, one should not speak about follow-up but therapeutic control. Nonetheless, even in these cases the S3 guideline recommends regular follow-up examinations because new therapeutic options can clearly improve patient survival. Possible complications of urinary diversions need consideration during follow-up., (© 2022. The Author(s), under exclusive licence to Springer Medizin Verlag GmbH, ein Teil von Springer Nature.)

Immunotherapy has revolutionized the systemic treatment of solid tumors, including advanced urothelial carcinoma (aUC), providing durable responses with a favorable safety profile. Multiple immune checkpoint inhibitor agents have been approved in monotherapy in second-line setting, and for a selected group of chemo-naïve cisplatin-ineligible patients with high PD-L1 expression. Despite the incorporation of immunotherapy to the systemic treatment landscape of aUC, platinum-based chemotherapy remains the standard of care in frontline setting for vast majority of patients. Urothelial carcinoma is a chemosensitive disease with response rates of up to 50% to frontline chemotherapy. However, the response to chemotherapy is short lasting with vast majority of patients experiencing disease progression and death within months. In this context, maintenance therapy constitutes an attractive therapeutic strategy to maximize the time to treatment failure. Different cytotoxic and targeted agents have been investigated as maintenance therapy for aUC but have not shown an impact on survival. Avelumab has become the first and only drug to improve overall survival as maintenance therapy after frontline platinum-based therapy in aUC patients and the first drug to be approved in this setting. This article will review the rational for maintenance therapy, the different drugs investigated as maintenance therapy for aUC, and the impact of avelumab maintenance therapy as a new standard of care in the management of aUC., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Autophagy plays a critical role in cancer, since it can either suppress tumorigenesis by inhibiting cancer cell survival, or facilitate tumorigenesis by promoting cancer cell proliferation and tumor growth. However, the role of genetic variants of autophagy-regulated key genes for bladder cancer risk remained unclear. Here, we aimed to explore the association of bladder cancer with genetic variants on genes involved in autophagy pathway. Gene-based analysis was performed with multi-marker analysis of genomic annotation (MAGMA) in 580 bladder cancer cases and 1101 controls. The logistic regression model was used to calculate the SNP effects on bladder cancer susceptibility. Expression quantitative trait loci (eQTL) analysis was conducted by the genotype-tissue expression (GTEx) project. Gene expression was evaluated based on the Cancer Genome Atlas (TCGA) database. Three potentially functional SNPs RPS6KB1 rs1292038, PIK3R1 rs34303, and rs56352616 were demonstrated to be associated with risk of bladder cancer (OR = 0.71, 95% CI = 0.61-0.82, P = 7.88 × 10 -6 for rs1292038; OR = 1.25, 95% CI = 1.09-1.45, P = 2.11 × 10 -3 for rs34303; OR = 0.74, 95% CI = 0.62-0.90, P = 2.47 × 10 -3 for rs56352616). An increasing number of risk genotypes of these three SNPs were associated with a higher risk of developing bladder cancer. Besides, rs1292038 exhibited an eQTL effect for RPS6KB1 in whole blood (P = 3.90 × 10 -7 ). Furthermore, the higher expression of RPS6KB1 and lower expression of PIK3R1 were both significantly associated with bladder cancer risk. Our findings indicated that genetic variants in autophagy pathway genes RPS6KB1 and PIK3R1 confer bladder cancer susceptibility., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Rho-associated protein kinase 1 (ROCK1) has been reported to be overexpressed in many types of tumors, but its role in urothelial bladder cancer is poorly understood. The study aims to investigate the role of ROCK1 in urothelial bladder cancer and explored the underlying mechanism. Protein and mRNA levels of ROCK1 were detected in 64 urothelial bladder cancer patients using western blot, immunohistochemistry and qRT-PCR. Relationships between ROCK1 expression and clinicopathological factors and survival rate were analyzed. ROCK1 was silenced by shRNA in multiple urothelial bladder cancer cells to explore its function and underlying mechanism. ROCK1 expression was significantly increased in tumor tissues compared with the paired adjacent healthy tissues of patients. Higher ROCK1 expression of tumor tissues positively correlated with poor prognosis of patients (p = 0.0435). ROCK1 silence significantly inhibited cell proliferation and colony formation, and enhanced activation of apoptotic pathway in urothelial bladder cancer cells. High ROCK1 expression predicts poor prognosis of urothelial bladder cancer. ROCK1 silence inhibit cell proliferation and promote apoptosis, which may be of value as a therapeutic target for bladder cancer treatment., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Purpose: To compare the efficacy and safety of high dose-intensity combination of methotrexate, vinblastine, adriamycin and cisplatin (HD MVAC) with gemcitabine plus cisplatin (GC) as a neoadjuvant chemotherapy (NAC) in muscle-invasive bladder cancer (MIBC) or locally advanced upper tract urothelial cancer (UTUC)., Patients and Methods: A retrospective analysis was conducted for patients with UC (cT2-4aN0-1M0) who received NAC from January 2011 and December 2017 at Asan Medical Center. Pathologic complete response (pCR), down-staging (< ypT2 and no N upstaging), disease-free survival (DFS), OS and safety were compared for each regimen., Results: Out of a total of 277 patients, 176 patients received GC and 41 patients received HD MVAC. With the exception of age (patients receiving HD MVAC were younger; p = 0.002), other baseline characteristics were well balanced between groups. pCR rates were 27.0% for GC and 22.6% for HD MVAC (p = 0.62), and down-staging rate was 50.8% for GC and 58.1% for HD MVAC (p = 0.47). There were no differences in OS (72.1% vs 73.1% for GC vs HD MVAC; p = 0.58) and DFS (54.9% vs 63.3% for GC vs HD MVAC; p = 0.21) at 3 years. HD MVAC with prophylactic G-CSF was associated with a higher incidence of febrile neutropenia (p < 0.001) than GC. The NAC regimen was not an independent prognostic factor for OS., Conclusion: Oncologic outcomes were not significantly different between the GC and HD MVAC when used as NAC in MIBC/UTUC., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: (I) To evaluate the clinical efficacy of three different BCG strains in patients with intermediate-/high-risk non-muscle-invasive bladder cancer (NMIBC). (II) To determine the importance of performing routine secondary resection (re-TUR) in the setting of BCG maintenance protocol for the three strains., Methods: NMIBCs who received an adjuvant induction followed by a maintenance schedule of intravesical immunotherapy with BCG Connaught, TICE and RIVM. Only BCG-naïve and those treated with the same strain over the course of follow-up were included. Cox proportional hazards model was developed according to prognostic factors by the Spanish Urological Oncology Group (CUETO) as well as by adjusting for the implementation of re-TUR., Results: n = 422 Ta-T1 patients (Connaught, n = 146; TICE, n = 112 and RIVM, n = 164) with a median (IQR) follow-up of 72 (60-85) were reviewed. Re-TUR was associated with improved recurrence and progression outcomes (HR RFS : 0.63; 95% CI 0.46-0.86; HR PFS : 0.55; 95% CI 0.31-0.86). Adjusting for CUETO risk factors and re-TUR, BGC TICE and RIVM provided longer RFS compared to Connaught (HR TICE : 0.58, 95% CI 0.39-0.86; HR RIVM : 0.61, 95% CI 0.42-0.87) while no differences were identified between strains for PFS and CSS. Sub-analysis of only re-TUR cases (n = 190, 45%) showed TICE the sole to achieve longer RFS compared to both Connaught and RIVM., Conclusion: Re-TUR was confirmed to ensure longer RFS and PFS in intermediate-/high-risk NMIBCs but did not influence the relative single BCG strain efficacy. When routinely performing re-TUR followed by a maintenance BCG schedule, TICE was superior to the other strains for RFS outcomes., (© 2021. The Author(s).)

Purpose: Our study was aimed to understand the importance of LIMD1-VHL-HIF1α pathway in development of bladder carcinoma (BlCa) in association with arsenic prevalence., Methods: At first, the mRNA expression pattern of the genes of this pathway (LIMD1, VHL and HIF1α) was checked in GEO datasets and in our samples. Next, genetic and epigenetic profiling of LIMD1 and VHL was done in our sample pool, validated in T24 BlCa cell line. The results were next correlated with various clinico-pathological parameters., Results: Differential under-expression of LIMD1 and VHL genes was found in muscle-invasive BlCa (MIBC) in comparison to non-muscle-invasive BlCa (NMIBC). However, HIF1α protein, but mRNA, was found to be overexpressed among the MIBC samples; depicting the probability of HIF1α protein stabilization. Analysis of genetic and epigenetic profiles of LIMD1 and VHL exposed a frequent promoter methylation of LIMD1 gene in MIBC samples. Further, in-depth look into the results unveiled that the high nuclear expression of HIF1α was significantly correlated with genetic alterations of LIMD1, alone or in combination with VHL. Moreover, treating the T24 cells with a de-methylating agent (5-aza-2'-deoxycytidine) re-expressed the methylated LIMD1 and VHL genes, which in turn, reduced the HIF1α protein level significantly. Additionally, patients with high arsenic content (> 112 ng/g, AsH) seemed to have recurrent promoter methylation in LIMD1, as well as co-methylation/alteration of LIMD1 and VHL gene. Lastly, high nuclear expression of HIF1α in association with co-alteration of VHL and LIMD1 showed the worst overall survival (OS) among the patients., Conclusion: To conclude, MIBC samples portrayed higher alterations in VHL and LIMD1, thereby, stabilizing HIF1α protein and lowering the OS of patients.

Aristolochic acid (AA-I) induces upper urothelial tract cancer (UUTC) and bladder cancer (BC) in humans. AA-I forms the 7-(2'-deoxyadenosin-N 6 -yl)aristolactam I (dA-AL-I) adduct, which induces multiple A:T-to-T:A transversion mutations in TP53 of AA-I exposed UTUC patients. This mutation is rarely reported in TP53 of other transitional cell carcinomas and thus recognized as an AA-I mutational signature. A:T-to-T:A transversion mutations were recently detected in bladder tumors of patients in Asia with known AA-I-exposure, implying that AA-I contributes to BC. Mechanistic studies on AA-I genotoxicity have not been reported in human bladder. In this study, we examined AA-I DNA adduct formation and mechanisms of toxicity in the human RT4 bladder cell line. The biological potencies of AA-I were compared to 4-aminobiphenyl, a recognized human bladder carcinogen, and several structurally related carcinogenic heterocyclic aromatic amines (HAA), which are present in urine of smokers and omnivores. AA-I (0.05-10 µM) induced a concentration- and time-dependent cytotoxicity. AA-I (100 nM) DNA adduct formation occurred at over a thousand higher levels than the principal DNA adducts formed with 4-ABP or HAAs (1 µM). dA-AL-I adduct formation was detected down to a 1 nM concentration. Studies with selective chemical inhibitors provided evidence that NQO1 is the major enzyme involved in AA-I bio-activation in RT4 cells, whereas CYP1A1, another enzyme implicated in AA-I toxicity, had a lesser role in bio-activation or detoxification of AA-I. AA-I DNA damage also induced genotoxic stress leading to p53-dependent apoptosis. These biochemical data support the human mutation data and a role for AA-I in BC.

Objective: To compare the efficacy and safety of intra-arterial chemotherapy (IAC) combined with intravesical chemotherapy (IVC) against intravesical BCG immunotherapy in high-risk non-muscle-invasive bladder cancer (NMIBC) after transurethral resection of the bladder tumor (TURBT)., Materials and Methods: 130 patients with high-risk NMIBC who had underwent TURBT were divided into two groups, of which IAC + IVC group received four courses of IAC (cisplatin and epirubicin) combined with IVC (epirubicin or pirarubicin) after surgery and BCG group received intravesical BCG immunotherapy. Recurrence rate and progression rate were assessed by Chi-square test, while recurrence-free survival and progression-free survival were calculated using the Kaplan-Meier method., Results: In this study, the recurrence rate was 27.9% (12/43) in IAC + IVC group and 26.4% (14/53) in BCG group, while progression rate was 9.3% (4/43) in IAC + IVC group and 9.4% (5/53) in BCG group. Both of the recurrence and progression rate did not show a significant difference. In the Kaplan-Meier plot, no difference was found with respect to recurrence-free survival and progression-free survival. Moreover, 46.5% (20/43) patients suffered from adverse events of IAC and 83.1% (49/59) patients suffered from adverse events associated with BCG, of which 6 patients discontinued treatment due to serious adverse events of BCG. Univariate analysis suggested that only recurrent tumor could be an independent risk factor related to recurrence., Conclusions: IAC combined with IVC used in high-risk NMIBC could reduce the recurrence and progression as effective as BCG instillation with lower adverse events.

Bladder cancer, which is a complex disease, can be treated with a variety of stage-oriented treatment options. In this article, the treatment recommendations of the German S3 guideline "Early detection, diagnosis, treatment and aftercare of bladder cancer" are applied in a fictitious case study. In a patient with invasive transitional cell carcinoma, the treatment options-ranging from bladder preservation to radical cystectomy with neoadjuvant chemotherapy-are discussed on the basis of the current literature.

Objective: The malignant transformation of normal bladder cells (SV-HUC-1) was induced by arsenite to explore the possible mechanism of circRNA-100284 influencing bladder cancer cell proliferation., Methods: Normal bladder SV-HUC-1 cells were cultured with 2 μM arsenite to induce malignant transformation. After 0, 3, 6, 12, and 24 h of culture, the expression level of circRNA-100284 in cells was detected by quantitative real-time PCR. Western blotting assays were used to detect the expression levels of EZH2 and cyclin-D1 proteins in cells treated with different media. Cell cycle was analyzed by flow cytometry. In addition, through cell transfection and CCK-8 experiments, the effect and mechanism of circRNA-100284 targeting microRNA-217 on proliferation was determined. The interaction between HSP70 methylation and Aurora-B was determined by Western blotting and immunoprecipitation experiments., Results: With prolonged contact time with arsenite, the expression level of circRNA-100284 in cells increased continuously (P < 0.05). Western blotting assays showed that the expression levels of EZH2 and cyclin-D1 proteins in arsenite-transformed cells increased. Flow cytometry and CCK-8 showed that circRNA-100284 accelerated cell cycle transition and cell proliferation through miR-217. Finally, after culturing human bladder cancer T24 cells, combined with immunoprecipitation and in vitro kinase experiments, it was found that K561- dimethyl HSP70 activated Aurora-B, thus promoting the proliferation of bladder cancer cells., Conclusion: CircRNA-100284 activates aurora kinase B by inducing methylation of HSP70 via microRNA-217 to promote the proliferation of bladder cancer cells.

We previously demonstrated that immunohistochemistry for γ-H2AX, a biomarker of DNA damage, is useful for early detection of urinary bladder carcinogens in rats. In a 28-day repeated-dose study, γ-H2AX was shown to have high sensitivity for detection of bladder carcinogens. However, no reports have evaluated whether a combination of multiple biomarkers may further improve sensitivity. Accordingly, in this study, we aimed to evaluate the applicability of bladder tissue and cancer stem cell markers, including cytokeratin 14 (KRT14), aldehyde dehydrogenase 1A1 (ALDH1A1), and cluster of differentiation 44 (CD44), as complementary markers for early detection of bladder carcinogens. Bladder samples obtained from male F344 rats orally treated with 14 bladder carcinogens and five nonbladder carcinogens for 28 days were used for immunohistochemical analysis of stem cell markers. In the bladder carcinogen-treated rats, increases in KRT14, ALDH1A1, and CD44 expression were observed in 9, 10, and 10 out of 14 groups, respectively, whereas the five nonbladder carcinogens did not cause upregulation of these markers. Although most epithelial cells with KRT14 or ALDH1A1 expression were also positive for CD44, KRT14 and ALDH1A1 expression were mutually exclusive. Twelve bladder carcinogens showed increases in at least one of the three markers, indicating that the combined evaluation showed higher sensitivity than the use of individual markers alone. Importantly, two of three bladder carcinogens that did not induce γ-H2AX immunostaining showed stem cell marker expression. Our results demonstrated that these stem cell markers may be useful as complementary markers for γ-H2AX in evaluation of bladder carcinogens.

Introduction: After transurethral resection of a bladder tumor, patients frequently have a recurrence of the disease, thereby requiring adjuvant therapy., Purpose: The study aimed to determine the prognostic value of expression levels of p53, Ki-67, and survivin, and to develop a new prognostic model for patients with non-muscle-invasive bladder cancer (NMIBC) after transurethral resection of a bladder tumor., Methods: The study group consisted of 101 patients with primary NMIBC. Univariate followed by multivariate Cox proportional hazard regression analysis was performed to obtain a model including the smallest possible number of descriptive variables with the highest statistical significance and impact on risk., Results: The RECINT model (RECurrence In Not Treated) including factors independently associated with cancer recurrence (tumor size [HR 1.148; p = 0.034], intensity of the color reaction for p53 [HR 1.716; p = 0.008], Ki-67 [HR 3.001; p = 0.022], and survivin [HR 1.461; p = 0.021]) adequately stratified recurrence free-survival (R 2  = 0.341, p < 0.001) in patients with primary NMIBC. Patients with the lowest RECINT score (0-6) had the lowest probability of cancer recurrence (1- and 5-year recurrence of 16%) in comparison with other groups (p < 0.001)., Conclusions: The RECINT model may be useful for stratifying the risk of recurrence in patients with non-muscle-invasive bladder cancer and may allow for identification of those who may benefit the most from adjuvant BCG immunotherapy.

Background: Great advances have been made for the treatment of urothelial carcinoma by the introduction of checkpoint inhibitors (CPI). Single-agent immunotherapy with CPIs has been approved for patients with metastatic or locally advanced inoperable urothelial carcinoma who have either progressed during or after platinum-based chemotherapy or who are cisplatin-ineligible. For cisplatin-ineligible patients, approval is restricted to patients with high programmed cell death ligand 1 (PD-L1) expression. For patients with nonmuscle invasive bladder cancer (NMIBC) or patients with muscle invasive bladder cancer (MIBC) who receive curative therapy, no CPIs have received approval in Germany., Objectives: To provide an overview of the current landscape of immunotherapy in patients with urothelial carcinoma., Methods: Summary of the therapeutic landscape and resulting challenges based on currently published data using a PubMed search., Results: In the treatment of metastatic or inoperable urothelial carcinoma, CPIs represent standard treatment. Depending on the results of currently performed trials, an extension of its use to the perioperative setting (neoadjuvant/adjuvant) and to patients with Bacillus Calmette Guérin (BCG) unresponsive NMIBC in the near future is currently being discussed., Conclusions: Immuno-oncologic treatment using CPIs has become an integral part of the management of patients with advanced bladder cancer. For biomarker-based patient selection and combination therapies, there is an urgent need for further investigations within clinical trial protocols.

Perioperative chemotherapy has become a standard treatment for muscle invasive bladder cancer and is recommended by national and international guidelines. The treatment of metastatic urothelial cancer evolved by the use of immune-modulating therapies like checkpoint inhibitors. Many clinical trials have been initiated which try to evaluate the role of immune checkpoint inhibition in the neoadjuvant and adjuvant setting. These trials focus not only on monotherapy, but also on the combination of checkpoint inhibitors with classical chemotherapy or with local radiation therapy (radioimmunotherapy). In neoadjuvant radioimmunotherapy, the radiation is supposed to act as a sensitizer for the systemic effects of checkpoint inhibition, in addition to the local effects. This review presents and discusses current trials and published results for perioperative immunomodulating treatment-alone or in combination-in muscle invasive bladder cancer.

The first-line therapy of metastatic bladder cancer (urothelial carcinoma, UC) depends on whether a patient is cisplatin-fit or not. Cisplatin-fit patients should be treated with the standard chemotherapy protocol GC (gemcitabine/cisplatin) or alternatively MVAC (methotrexate/vinblastine/doxorubicin/cisplatin). The optimal first-line therapy for cisplatin-unfit patients remains unclear due to the lack of high level of evidence. One criterion for selecting therapy can be the PD-L1 (programmed cell death ligand 1) status of the tumor. The PD-L1-negative patients (PD-L1 <5% for atezolizumab and combined positivity score [CPS] <10 for pembrolizumab) seem to have a greater benefit from the combination chemotherapy GCa (carboplatin/gemcitabine). The PD-L1-positive patients (PD-L1 ≥5% or CPS ≥10) on the other hand may have a greater benefit from and a longer response to the two immune checkpoint inhibitors that are currently approved for this indication, namely atezolizumab and pembrolizumab. Two phase 3 trials that compare head-to-head immunotherapy alone or in combination with chemotherapy vs. chemotherapy alone may help to define the optimal first-line therapy for metastatic UC. Preliminary data from one of these studies indicate an advantage for the combination of immunotherapy with chemotherapy in all subgroups.

The approval of the PD‑1 and PD-L1 (programmed cell death [ligand] 1) antibodies pembrolizumab, nivolumab, and atezolizumab has fundamentally changed the therapeutic landscape of locally advanced or metastatic urothelial carcinoma. Checkpoint inhibitors (CPI) are the standard of care in second-line treatment if not already used in first line. They replace conventional chemotherapeutics such as vinflunine, paclitaxel, or docetaxel and offer a superior toxicity profile. This article provides an overview of current second-line treatment strategies for locally advanced or metastatic urothelial carcinoma.

Background: There is to date no convincing literature that has assessed the association between traumatic spinal cord injury (SCI) and the later development of urinary bladder cancer. The aim of this work is to present medical experts as well as the national accident insurance and the social courts decision-making aids based on the latest medical scientific knowledge, for assessment of this causal association., Materials and Methods: A study conducted between April 1998 and March 2017 in the BG Trauma Hospital Hamburg forms the basis for the decision-making aids. Urinary bladder cancer was diagnosed in 32 out of 6432 treated outpatient and inpatient SCI patients. Furthermore, relevant published literature was taken into consideration for the decision-making aids., Results: It was found that urinary bladder cancer in SCI patients occurs at a considerably younger age as compared to the general population, more frequently shows muscle invasive carcinoma with a higher grade at first diagnosis and a higher proportion of the more aggressive squamous cell carcinoma than that of the general population. Correspondingly, the survival time is extremely unfavorable. For medical experts a matrix was compiled where the various influencing factors, either for or against the recognition of an association between SCI and urinary bladder cancer, were weighted according to their relevance., Conclusion: The results showed that urinary bladder cancer in SCI patients differs considerably from that of able-bodied patients. These differences drastically shorten the survival time. A study on patients with spina bifida, i.e., a congenital spinal cord disorder, corroborates these observations. They indicate histopathological differences that have so far been intangible.

Background: Technical advancement and availability of high-throughput analysis has advanced molecular subtyping of most cancers. Thus, new possibilities for precision oncology have emerged., Aim: Therefore, we aimed to collect data regarding availability and use of next generation sequencing (NGS) for urothelial cancer within the uropathology working group of the German Society of Pathology., Methods: We collected data by questionnaires and additionally asked for sequencing results of bladder cancers in the participating institutions., Results: A total of 13 university-affiliated institutes of pathology took part in the survey. All university institutes offer NGS-based molecular panel diagnostics and provide panels covering between 15 and 170 genes. Altogether, only 20 bladder cancers were sequenced in routine diagnostics and for 10 cancers potential targeted treatment options were available., Discussion: So far, despite availability of NGS diagnostics at university institutes of pathology, only few bladder cancer samples have been sequenced. Based on current data from the molecular subtyping of bladder cancers, we recommend a step-by-step protocol with basic immunohistochemistry analysis and subsequent subtype-dependent analyses, e.g., alterations of the fibroblast growth factor receptors (FGFR) or comprehensive gene panel analyses.

Introduction: Prostate specific membrane antigen (PSMA) has become a target for radionuclide imaging and therapy. Previous studies have shown that the expression of PSMA is not specific to prostate tissue. In this study we examine the expression of PSMA in urothelial cell carcinoma (UCC)., Methods: Immunhistochemical PSMA-staining was performed in 89 UCC samples. PSMA expression in tumor tissue, adjacent healthy tissue and blood vessels was examined. We furthermore analyzed PSMA-mRNA expression in nine human UCC cell lines. We correlated our findings with clinical data regarding recurrence and progression of UCC., Results: UCC tissue showed a significantly higher PSMA expression compared to healthy urothelial tissue (p < 0.001). Non muscle invasive bladder cancer revealed significantly higher PSMA expression compared to muscle invasive bladder cancer (p < 0.05). PSMA expression significantly differed between various T-stages (p < 0.05) and tumor differentiation (p < 0.001). In four human UCC cell lines PSMA-mRNA was detectable. Those patients who suffered recurrence showed a higher rate of PSMA expression but no correlation to recurrence-free survival was evident. Progression of disease correlated significantly with a higher PSMA expression (p = 0.036)., Conclusions: Both UCC tissue and healthy urothelial tissue express PSMA, with significantly higher levels in UCC. We confirmed these findings in human UCC cell lines. In this small first cohort expression of PSMA correlates significant with progression of disease but not with recurrence and recurrence-free survival. These first results make PSMA a promising target for future diagnosis and therapy of UCC.

Purpose: Bladder cancer represents a major cause of malignancy-related morbidity and the most expensive per-patient-to-treat cancer, due to the lifelong surveillance of the patients. Accurate disease prognosis is essential in establishing personalized treatment decisions; yet optimum tools for precise risk stratification remain a competing task. In the present study, we have performed the complete evaluation of TP63 clinical significance in improving disease prognosis., Methods: The levels of ΔNp63 and TAp63 transcripts of TP63 were quantified in 342 bladder tissue specimens of our screening cohort (n = 182). Hedegaard et al. (Cancer Cell 30:27-42. doi:10.1016/j.ccell.2016.05.004, 2016) (n = 476) and TCGA provisional (n = 413) were used as validation cohorts for NMIBC and MIBC, respectively. Survival analysis was performed using recurrence and progression for NMIBC or mortality for MIBC as endpoint events. Bootstrap analysis was performed for internal validation, while decision curve analysis was used for the evaluation of the clinical net benefit on disease prognosis., Results: ΔNp63 was significantly expressed in bladder tissues, and was found to be over-expressed in bladder tumors. Interestingly, reduced ΔNp63 levels were correlated with muscle-invasive disease, high-grade tumors and high-EORTC-risk NMIBC patients. Moreover, ΔNp63 loss was independently associated with higher risk for NMIBC relapse (HR = 2.730; p = 0.007) and progression (HR = 7.757; p = 0.016). Hedegaard et al. and TCGA validation cohorts confirmed our findings. Finally, multivariate models combining ΔΝp63 loss with established prognostic markers led to a superior clinical benefit for NMIBC prognosis and risk stratification., Conclusions: ΔΝp63 loss is associated with adverse outcome of NMIBC resulting in superior prediction of NMIBC early relapse and progression.

Background: In the last few years, significant progress has been achieved in the therapeutic options for advanced urothelial bladder cancer., Objectives: The aim of this work was to give an overview of the status and future perspective of the therapeutic options in this setting. Its focus is on the discussion of tissue-based therapy-predictive markers, which are evaluated through (molecular) pathology and thereby strengthening the role of pathology itself., Materials and Methods: Current (clinical study) data, the literature, and our own expertise were considered and summarized in the areas of therapy prediction of platinum-based chemotherapy, immunotherapy, and other therapeutic approaches., Results and Conclusions: Molecular subtypes exhibit a predictive value both in platinum-based chemotherapy as well as in immunotherapy. However, further work is required to elucidate the predictive role of molecular subtypes in both settings. Changes in the DNA damage repair enzyme (DDR) genes, ERCC2, and ERBB2 as well as differences in the expression of EMMPRIN, survivin, and HMGA2 show promising results as further markers of chemotherapy efficacy. In the prediction of immunotherapy success, this mainly relates to the evaluation of the tumor mutation burden (TMB), tumor neoantigen burden (TNB), APOBEC signatures (MSig1; 3A/3B), and CD8-positive T‑effector cell signature. When using the fibroblast growth factor receptor (FGFR) inhibitor erdafitinib, which has not yet been approved in Germany, the evaluation of specific FGFR mutations and/or gene fusions by a companion diagnostic test is mandatory in the USA.

Recent epidemiological studies have indicated that occupational exposure to the aromatic amine acetoaceto-o-toluidide (AAOT) was associated with a marked increase in urinary bladder cancers in Japan. However, little is known about the carcinogenicity of AAOT. To evaluate the urinary bladder carcinogenicity of AAOT, male and female F344 rats were treated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) for 4 weeks followed by dietary administration of 0, 0.167, 0.5, or 1.5% AAOT for 31 weeks. The incidences and multiplicities of bladder tumors were significantly increased in the 0.5 and 1.5% groups of male and female rats in a dose-response manner. AAOT and seven downstream metabolites were detected in the urine of the male and female rats administered AAOT with levels increasing in a dose-dependent manner. The most abundant urinary metabolite of AAOT was the human bladder carcinogen o-toluidine (OTD), which was at least one order of magnitude higher than AAOT and the other AAOT metabolites. In a second experiment, male F344 rats were administered 0, 0.167, or 1.5% AAOT for 4 weeks. Gene expression analyses revealed that the expression of JUN and its downstream target genes was increased in the urothelium of male rats treated with 1.5% AAOT. These results demonstrate that AAOT promotes BBN-induced urinary bladder carcinogenesis in rats and suggest that overexpressed of JUN and its downstream target genes may be involved the bladder carcinogenicity of AAOT. In conclusion, AAOT, like other carcinogenic aromatic amines, is likely to be a carcinogen to the urinary bladder, and OTD metabolized from AAOT is the ultimate carcinogen.

Purpose: The incidence of Urothelial carcinoma of bladder (UBC) is gradually increasing by changing lifestyle and environment. The development of a tumor has been noted to be accompanied by modifications in the extracellular matrix (ECM) consisting of CD44, hyaluronic acid (HA) and its family members. The importance of CD44 splice variants and HA family members has been studied in UBC., Methods: The cohort of study included 50 UBC patients undergoing radical cystectomy and 50 healthy subjects. The molecular expression of CD44 and HA family members was determined. Effect of CD44 variant-specific silencing on downstream signaling in HT1376 cells was investigated. Combinatorial treatment of 4-MU (4-methylumbelliferone) with cisplatin or doxorubicin on chemosensitivity was also explored., Results: Higher expression of HA, HAS2, and CD44 was observed in Indian UBC patients which also showed the trend with severity of disease. Splice variant assessment of CD44 demonstrated the distinct role of CD44v3 and CD44v6 in bladder cancer progression. shRNA-mediated downregulation of CD44v3 showed an increase effect on cell cycle, apoptosis and multiple downstream signaling cascade including pAkt, pERK and pSTAT3. Furthermore, 4-MU, an HA synthesis inhibitor, observed to complement the effect of Cisplatin or Doxorubicin by enhancing the chemosensitivity of bladder cancer cells., Conclusions: Our findings exhibit involvement of CD44 splice variants and HA family members in UBC and significance of 4-MU in enhancing chemosensitivity suggesting their novel therapeutic importance in disease therapeutics.

Follow-up care of patients with muscle-invasive bladder cancer is subdivided into oncological and functional surveillance. More than 80% of local relapses and distant metastases occur within the first 2 years. Recurrences in the remnant urothelium also occur several years after radical cystectomy. Urinary cytology and a computed tomography (CT) scan of the abdomen and thorax including a urography phase are the standard diagnostics for tumor follow-up. There is no clear evidence for a survival benefit for the detection of asymptomatic vs. symptomatic recurrences. After partial cystectomy or trimodal treatment, there is no established follow-up schedule; however, the relatively high incidence of intravesical recurrences should be considered as there are curative treatment approaches including salvage cystectomy. Functional surveillance, which should be carried out lifelong, encompasses prevention and diagnostics of metabolic complications, urethral/ureteral strictures, problems with the urinary stoma, urinary incontinence, sexual dysfunction and urinary tract infections.

Purpose: To investigate the role of sonic hedgehog (Shh) signaling and epithelial-mesenchymal transition (EMT) in bladder cancer progression and invasion., Methods: We cultured three bladder cancer cell lines, muscle-invasive T24 and 5637, and non-muscle-invasive KK47, in the presence of a recombinant-Shh (r-Shh) protein or cyclopamine, a Shh signaling inhibitor, to investigate proliferation and expression of EMT markers. Wound-healing assays and transwell assay were performed to evaluate cell invasion and migration. Mice were then inoculated with bladder cancer cells and treated with cyclopamine. Mouse tumor samples were stained for Shh signaling and EMT markers., Results: R-Shh protein enhanced cell proliferation, whereas cyclopamine significantly suppressed cell proliferation, especially in invasive cancer (5637 and T24) (p < 0.05). R-Shh protein promoted EMT, suppressed E-cadherin and enhanced N-cadherin and vimentin and Gli1, an Shh downstream molecule, while cyclopamine blocked EMT, especially in 5637 and T24. Cyclopamine also inhibited cell invasion and migration in vitro. In the animal study, intraperitoneal injection of cyclopamine significantly suppressed tumor growth in 5637 and T24 in mice (p = 0.01 and p = 0.004, respectively) and slightly suppressing KK47 tumor growth (p = 0.298). Significant cyclopamine-induced suppression of Gli1 in 5637 and T24 mouse tumors (both p = 0.03) was seen, suggesting that muscle-invasive bladder cancer may be more dependent on Shh signaling than non-muscle-invasive bladder cancer., Conclusions: Shh signaling and EMT were especially enhanced in muscle-invasive bladder cancer progression and invasion, and suppressed by the inhibition of Shh signaling.

Purpose: This study aimed at determining the relationship between classification according to the papillary tumor pattern of carcinoma in situ (CIS) of the bladder and prognosis, as this has not yet been well established., Methods: This study comprised a consecutive cohort of 254 patients (primary CIS: 66 patients, stage Ta-CIS: 52 patients, and stage T1-CIS: 136 patients) with CIS-associated bladder cancer. We classified CIS according to the pathological pattern of papillary tumors and analyzed prognostic factors, including CIS classification, for progression. We evaluated progression using two endpoints: infiltrative tumors detected at stage T1 or higher or at stage T2 or higher. Bacillus Calmette-Guerin (BCG) immunotherapy response was defined as no recurrence within 6 months., Results: Both the BCG immunotherapy response and CIS classification were significant prognostic factors for both the endpoints. Patients with CIS-associated stage Ta urinary bladder cancer had better prognosis for both the endpoints than those with stage T1 cancer or those with primary CIS. BCG immunotherapy response (p < 0.001) and age (p = 0.007) were also significant prognostic factors for the progression of stage T2 or higher infiltrative tumors. The prognosis of patients with recurrent primary CIS (12/26, 46.2%) and T1-CIS (25/45, 55.6%) was poor for progression; distant metastasis occurred in approximately 40% of these patients., Conclusions: Clinicians should consider radical surgery for poor prognosis in patients with recurrent CIS-associated T1 cancer or primary CIS. The CIS classification according to the tumor pattern reflects the prognosis.

Background: Molecular tumor boards (MTB) are becoming more common. There are several molecular alterations in urothelial cancer a molecular tumor board can potentially rely on., Objectives: The aim is to specify molecular alterations and their correlations with different clinical endpoints and to highlight potential questions addressed to a MTB for urothelial cancer., Materials and Methods: Descriptive review of the literature based on PubMed., Results: The landscape of molecular alterations in urothelial cancer is heterogeneous. Thus, recent biomarker research has been focusing on biomarker panels and classifiers instead of single biomarkers. Recently, molecular subtypes of urothelial cancer have been identified and correlated with different clinical endpoints. Furthermore, circulating tumor cells and tumor DNA are under investigation as potential biomarkers. In addition to treatment response and prognosis, molecular markers are also needed to improve clinical staging prior to radical cystectomy or for proper patient selection for neoadjuvant chemotherapy. Erdafitinib is the first targeted therapy (fibroblast growth factor receptor [FGFR] alteration) in urothelial cancer that was recently approved (in the USA)., Conclusions: Due to the lack of external validation, none of the identified biomarkers is currently established in clinical routine. In addition, there is no single driver mutation in urothelial cancer that facilitates the development of biomarkers and targeted therapies.