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Distinct molecular subtypes of muscle-invasive bladder cancer (MIBC) may show different platinum sensitivities. Currently available data were mostly generated at transcriptome level and have limited comparability to each other. We aimed to determine the platinum sensitivity of molecular subtypes by using the protein expression-based Lund Taxonomy. In addition, we assessed the tumor heterogeneity within the primary tumor and between the primary and lymph node (LN) metastatic sites. Thirteen immunohistochemical markers were stained in a tissue microarray with an overall number of 1,508 cores. Statistical evaluation was performed in 199 patients divided into three chemo-naïve MIBC cohorts: (1) pT3/4 and/or LN+ patients who received radical cystectomy without platinum treatment, (2) patients who received adjuvant chemotherapy (AC), and (3) patients who underwent palliative platinum treatment for metastatic disease or postoperative progression. Overall survival (OS) was used as the primary endpoint. Patients with the genomically unstable (GU) subtype had significantly better OS in the AC group compared to the radical cystectomy group (HR: 0.395, 95% CI: 0.205-0.795, p = 0.005). In contrast, no such association was observed for the basal/squamous (Ba/Sq) subtype. Intratumor heterogeneity was present in 19% of cases, with the lowest level in the Ba/Sq and GU tumors (14% each) and the highest level of 43% in small-cell/neuroendocrine-like tumors. There was greater subtype heterogeneity between primary tumors and LN metastases. In conclusion, immunohistochemistry-based Lund Taxonomy subtypes remain stable within the same primary tumor, with the GU subtype deriving the greatest OS benefit from AC. However, high tumor heterogeneity between the primary tumor and metastatic sites can impact the effectiveness of therapies., (© 2025 The Author(s). The Journal of Pathology: Clinical Research published by The Pathological Society of Great Britain and Ireland and John Wiley & Sons Ltd.)

Aims: Adjuvant chemotherapy after radical cystectomy can reduce the risk of recurrence and death in advanced muscle-invasive urothelial bladder cancer (MIBC). Molecular subtypes have been shown to be associated with survival. However, their predictive value to guide treatment decisions is controversial and data to use subtypes as guidance for adjuvant chemotherapy is sparse. We aimed to assess survival rates based on MIBC consensus molecular subtypes with and without adjuvant chemotherapy., Methods: Gene expression profiles of 143 patients with MIBC undergoing radical cystectomy were determined from formalin-fixed, paraffin-embedded specimen to assign consensus molecular subtypes. Expression of programmed cell death ligand-1 (PD-L1) and immune cell infiltration were determined using multiplex immunofluorescence. Matched-pair analysis was performed to evaluate the effect of adjuvant chemotherapy on overall survival (OS) for molecular subtypes applying Kaplan-Meier and Cox regression survival analyses., Results: Samples were luminal papillary: 9.1% (n=13), luminal non-specified: 6.3% (n=9), luminal unstable: 4.9% (n=7), stroma-rich: 27.9% (n=40), basal/squamous (Ba/Sq): 48.9% (n=70) and neuroendocrine-like (NE-like): 2.8% (n=4). Ba/Sq tumours had the highest concentration of PD-L1+ tumour and immune cells. Patients with luminal subtypes had better OS than those with NE-like (HR 0.2, 95% CI 0.1 to 0.7, p<0.05) and Ba/Sq (HR 0.5, 95% CI 0.2 to 0.9, p<0.05). No survival benefit with adjuvant chemotherapy was observed for luminal tumours, whereas Ba/Sq had significantly improved survival rates with adjuvant chemotherapy. Retrospective design and sample size are the main limitations., Conclusion: Consensus molecular subtypes can be used to stratify patients with MIBC. Luminal tumours have the best prognosis and less benefit when receiving adjuvant chemotherapy compared with Ba/Sq tumours., Competing Interests: Competing interests: PW has received consulting fees and honoraria for lectures by Bayer, Janssen-Cilag, Novartis, Roche, MSD, Astellas Pharma, Bristol-Myers Squibb, Thermo Fisher Scientific, Molecular Health, Guardant Health, Sophia Genetics, Qiagen, Eli Lilly, Myriad, Hedera Dx and AstraZeneca. Research Support was provided by AstraZeneca and Roche. HTG Transcriptome Panel was funded by HTG Molecular Diagnostics. HR was on an advisory board of Bristol-Myers Squibb, received honoraria from Roche, Bristol-Myers Squibb, Janssen-Cilag, Novartis, AstraZeneca, MCI, CHOP GmbH and Diaceutics, received travel support from Philips, Roche, and Bristol-Myers Squibb, and received grants from Bristol-Myers Squibb., (© Author(s) (or their employer(s)) 2025. No commercial re-use. See rights and permissions. Published by BMJ Group.)

Lymphoepithelioma-like carcinoma of the bladder (LELC-B) is a rare histologic subtype characterized by strong immune cell (IC) infiltrates. A better prognosis and favorable response rates to immune checkpoint inhibitors have been described. We aimed to characterize the molecular profiles and IC infiltration of LELC-B for a better understanding of its therapeutic implications. We identified 11 muscle-invasive bladder cancer cases with pure and mixed LELC-B. Programmed cell death ligand-1 (PD-L1) expression and mismatch repair proteins were evaluated using immunohistochemistry. We calculated the tumor mutational burden and characterized mutational profiles using whole-exome DNA sequencing data. Transcriptomic signatures were detected using the NanoString nCounter PanCancer IO360 Panel. Multiplex immunofluorescence of tumor microenvironment (PD-L1, PanCK, α-SMA, vimentin, CD45, and Ki67) and T cells (CD4, CD3, PD-1, CD163, CD8, and FoxP3) was used to quantify cell populations. All LELC-B cases were highly positive for PD-L1 (median tumor proportion score/tumor cell, 70%; range, 20%-100%; median combined positive score, 100; range, 50-100) and mismatch repair proficient and negative for Epstein-Barr virus infection. IC infiltrates were characterized by a high CD8+ T-cell count and high PD-1/PD-L1 expression on immune and tumor cells. LELC-B showed upregulation of signaling pathways involved in IC response. Most common mutations were found in chromatin remodeling genes causing epigenetic dysregulation. All LELC-B cases showed high tumor mutational burden with a median of 39 mutations/Mb (IQR, 29-66 mutations/Mb). In conclusion, LELC-B is a highly immunogenic tumor, showing strong upregulation of PD-1/PD-L1 and making immune checkpoint inhibitors a promising treatment option., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The risk of recurrence in patients with muscle invasive bladder cancer (MIBC) after radical cystectomy depends on the pathological tumor stage. In particular, patients with lymph node metastasis (pN+), locally advanced (≥pT3), or residual muscle invasive tumor despite neoadjuvant chemotherapy are at high risk. Currently, the importance of adjuvant therapy with immune checkpoint inhibitors is increasing in the context of perioperative systemic therapeutic concepts. The indication for the PD‑1 inhibitor nivolumab currently approved in the European Union requires testing of PD-L1 (programmed cell death ligand 1) protein expression by immunochemistry in tumor tissue. Focusing on MIBC patients at high risk of recurrence, new questions arise regarding the implementation and interpretation of PD-L1 testing. An interdisciplinary group of experts from Germany has discussed relevant issues from a clinicopathological point of view and developed practical recommendations to facilitate the implementation of validated and quality-assured PD-L1 testing for the approved indications in daily clinical practice., (© 2024. The Author(s).)

The emergence of mutational signatures in cancer research represents a paradigm shift, offering profound insights into tumor biology, therapeutic strategies, and risk assessment. A recent article published in The Journal of Pathology delves into the significance of mutational signatures, assessing their role as molecular fingerprints that illuminate the etiology of individual cancer cases. By deciphering the diverse patterns of DNA alterations, in the context of urachal and nonurachal bladder adenocarcinomas, researchers can unravel intrinsic and environmental risk factors shaping cancer development. The study highlighted a predominance of environmental risk factors in Chinese populations affected by bladder adenocarcinomas. In this context, in this commentary, we highlight some of the potential applications of molecular signatures in differential diagnosis and therapy prediction. As the field continues to evolve, a deeper understanding of mutational signatures promises to revolutionize precision oncology by offering personalized approaches informed by comprehensive mutational patterns of tumors. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland., (© 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.)

Background: Urachal cancer (UrC) is a rare disease with limited availability of representative incidence and clinical data. Although, the prevalence is accounting for less than 1% of bladder tumors, the 5-year survival rate is around only 50% for patients with resectable tumors, and even worse for patients with metastatic disease. Due to the lack of comprehensive prospective studies, our current knowledge of UrC is still limited., Objective: The present study aimed to summarize the available registry-based studies with unselected UrC patients to evaluate its incidence and clinicopathological characteristics., Material and Methods: We conducted a systematic literature search of registry-based UrC publications on the 15th of May 2023 in 5 databases, which identified 4,748 publications. After duplicate removal and selection by 2 independent investigators, 6 publications proved to be appropriate for the final meta-analysis. Estimated incidence and clinicopathological parameters were extracted., Results: Estimated incidence ranged between 0.022 and 0.060/ 100.000 person-years, with the highest occurrence in Japan and the lowest in Canada, while the random effect model calculated an overall incidence rate of 0.04 (95%CI: 0.03-0.05) 100.000 person-years. The median age at first diagnosis was 60 years (range: 58-64). The female to male ratio was 2:3. Lymph node or distant metastases were present in 9% and 14% of patients. The predominant tumour type was adenocarcinoma (86%) followed by urothelial carcinoma (12%) and squamous cell carcinoma (2%). The 5-year survival rate was 51.0% with 95%CI: 45.2-57.4., Conclusions: Our study provides an up-to-date comparison of estimated incidence rates between 6 countries of 3 continents based on rigorously selected registry-based studies. The results suggest low incidence rates for UrC with considerable geographic differences. The present meta-analysis provides unbiased registry-based data on the incidence, clinicopathological parameters and survival of UrC., Competing Interests: Declaration of competing interest The authors declare no potential conflicts of interest., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: The extent of pelvic lymphadenectomy (PLND) as part of radical cystectomy (RC) for bladder cancer (BC) remains unclear. Sentinel-based and lymphangiographic approaches could lead to reduced morbidity without sacrificing oncologic safety., Objective: To evaluate the feasibility and diagnostic value of fluorescence-guided template sentinel region dissection (FTD) using a handheld near-infrared fluorescence (NIRF) camera in open radical cystectomy., Design, Setting, and Participants: After peritumoral cystoscopic injection of indocyanine green (ICG) 21 patients underwent open RC with FTD due to BC between June 2019 and June 2021. Intraoperatively, the FIS-00 Hamamatsu Photonics® NIRF camera was used to identify and resect fluorescent template sentinel regions (FTRs) followed by extended pelvic lymphadenectomy (ePLND) as oncological back-up., Outcome Measurement and Statistical Analysis: Descriptive analysis of positive and negative results per template region., Results and Limitations: FTRs were identified in all 21 cases. Median time (range) from ICG injection to fluorescence detection was 75 (55-125) minutes. On average (SD), 33.4 (9.6) lymph nodes were dissected per patient. Considering template regions as the basis of analysis, 67 (38.3%) of 175 resected regions were NIRF-positive, with 13 (7.4%) regions harboring lymph node metastases. We found no metastatic lymph nodes in NIRF-negative template regions. Outside the standard template, two NIRF-positive benign nodes were identified., Conclusion: The concept of NIRF-guided FTD proved for this group all lymph node metastases to be found in NIRF-positive template regions. Pending validation in a larger collective, resection of approximately 40% of standard regions may be sufficient and may result in less morbidity., (© 2024 The Authors. Journal of Surgical Oncology published by Wiley Periodicals LLC.)

Background: Of all urothelial carcinomas (UCs), 25% are muscle invasive and associated with a 5-year overall survival rate of 50%. Findings regarding the molecular classification of muscle-invasive urothelial carcinomas (MIUCs) have not yet found their way into clinical practice., Objectives: Prediction of molecular consensus subtypes in MIUCs with artificial intelligence (AI) based on histologic hematoxylin-eosin (HE) sections., Methods: Pathologic review and annotation of The Cancer Genome Atlas (TCGA) Bladder Cancer (BLCA) Cohort (N = 412) and the Dr. Senckenberg Institute of Pathology (SIP) BLCA Cohort (N = 181). An AI model for the prediction of molecular subtypes based on annotated histomorphology was trained., Results: For a five-fold cross-validation with TCGA cases (N = 274), an internal TCGA test set (N = 18) and an external SIP test set (N = 27), we reached mean area under the receiver operating characteristic curve (AUROC) scores of 0.73, 0.8 and 0.75 for the classification of the used molecular subtypes "luminal", "basal/squamous" and "stroma-rich". By training on correlations to individual molecular subtypes, rather than training on one subtype assignment per case, the AI prediction of subtypes could be significantly improved., Discussion: Follow-up studies with RNA extraction from various areas of AI-predicted molecular heterogeneity may improve molecular classifications and thereby AI algorithms trained on these classifications., (© 2024. The Author(s).)

Glandular lesions in the urinary tract or their associated pathologies can pose a diagnostic challenge. There is a variety of benign alterations and tumor types that need to be taken into account in differential diagnostic considerations. In recent times, efforts for better defining these alterations or lesions both on the histopathological and molecular levels have been undertaken. This article will provide an update on current diagnostic and molecular considerations of these lesions., Competing Interests: The authors have no funding or conflicts of interest to disclose., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Molecular subtypes predict prognosis in muscle-invasive bladder cancer (MIBC) and are explored as predictive markers. To provide a common base for molecular subtyping and facilitate clinical applications, a consensus classification has been developed. However, methods to determine consensus molecular subtypes require validation, particularly when FFPE specimens are used. Here, we aimed to evaluate two gene expression analysis methods on FFPE samples and to compare reduced gene sets to classify tumors into molecular subtypes., Methods: RNA was isolated from FFPE blocks of 15 MIBC patients. Massive analysis of 3' cDNA ends (MACE) and the HTG transcriptome panel (HTP) were used to retrieve gene expression. We used normalized, log2-transformed data to call consensus and TCGA subtypes with the consensusMIBC package for R using all available genes, a 68-gene panel (ESSEN1), and a 48-gene panel (ESSEN2)., Results: Fifteen MACE-samples and 14 HTP-samples were available for molecular subtyping. The 14 samples were classified as Ba/Sq in 7 (50%), LumP in 2 (14.3%), LumU in 1 (7.1%), LumNS in 1 (7.1%), stroma-rich in 2 (14.3%) and NE-like in 1 (7.1%) case based on MACE- or HTP-derived transcriptome data. Consensus subtypes were concordant in 71% (10/14) of cases when comparing MACE with HTP data. Four cases with aberrant subtypes had a stroma-rich molecular subtype with either method. The overlap of the molecular consensus subtypes with the reduced ESSEN1 and ESSEN2 panels were 86% and 100%, respectively, with HTP data and 86% with MACE data., Conclusion: Determination of consensus molecular subtypes of MIBC from FFPE samples is feasible using various RNA sequencing methods. Inconsistent classification mainly involves the stroma-rich molecular subtype, which may be the consequence of sample heterogeneity with (stroma)-cell sampling bias and highlights the limitations of bulk RNA-based subclassification. Classification is still reliable when analysis is reduced to selected genes., (© 2023. The Author(s).)

Urachal carcinoma is a rare malignancy that is uniquely associated with the field of urology. Urachal carcinoma is mostly diagnosed in urological care centers due to its most frequently presenting symptom that is hematuria. Currently available diagnostic and therapeutic knowledge is solely based on case reports and single center series, while no prospective clinical studies are carried out due to the modest number of patients. These circumstances have made creating professional guidelines challenging, hence the treatment of urachal carcinoma is commonly based on individual clinical decisions. In this review, we summarize the epidemiology, diagnostic modalities, prognosis as well as local and systemic therapeutic approaches of urachal carcinoma. Furthermore, we aim to draw conclusions from this knowledge base that may guide clinical decision-making. Finally, we highlight some of the novel therapeutic strategies that hold the potential to improve urachal carcinoma patients' prognosis and quality of life. Orv Hetil. 2023; 164(16): 602-609.

Objective: Administration of targeted therapies provides a promising treatment strategy for urachal adenocarcinoma (UrC) or primary bladder adenocarcinoma (PBAC); however, the selection of appropriate drugs remains difficult. Here, we aimed to establish a routine compatible methodological pipeline for the identification of the most important therapeutic targets and potentially effective drugs for UrC and PBAC., Methods: Next-generation sequencing, using a 161 cancer driver gene panel, was performed on 41 UrC and 13 PBAC samples. Clinically relevant alterations were filtered, and therapeutic interpretation was performed by in silico evaluation of drug-gene interactions., Results: After data processing, 45/54 samples passed the quality control. Sequencing analysis revealed 191 pathogenic mutations in 68 genes. The most frequent gain-of-function mutations in UrC were found in KRAS (33%), and MYC (15%), while in PBAC KRAS (25%), MYC (25%), FLT3 (17%) and TERT (17%) were recurrently affected. The most frequently affected pathways were the cell cycle regulation, and the DNA damage control pathway. Actionable mutations with at least one available approved drug were identified in 31/33 (94%) UrC and 8/12 (67%) PBAC patients., Conclusions: In this study, we developed a data-processing pipeline for the detection and therapeutic interpretation of genetic alterations in two rare cancers. Our analyses revealed actionable mutations in a high rate of cases, suggesting that this approach is a potentially feasible strategy for both UrC and PBAC treatments., (© 2023 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Objective: Although targeted approaches have become available in second- and third-line settings, platinum-based chemotherapy remains the standard first-line treatment for advanced muscle-invasive bladder cancer (MIBC). Therefore, the prediction of platinum resistance is of utmost clinical importance., Methods: In this study, we established a routine compatible method for the molecular classification of MIBC samples according to various classification systems and applied this method to evaluate the impact of subtypes on survival after adjuvant chemotherapy. This retrospective study included 191 patients with advanced MIBC (pT≥3 or pN+) who underwent radical cystectomy, with or without adjuvant chemotherapy. A 48-gene panel and classifier rule set were established to determine molecular subtypes according to TCGA, MDA, LundTax, and Consensus classifications. Additionally, 12 single platinum-predictive candidate genes were assessed. The results were correlated with patients' clinicopathological and follow-up data and were validated using independent data sets., Results: Our final evaluation of 159 patients demonstrated better survival in the luminal groups for those who received chemotherapy compared with those who did not. In contrast, no such differences were observed in basal subtypes. The use of chemotherapy was associated with better survival in patients with high APOBEC3G expression (p < 0.002). This association was confirmed using an independent data set of patients who received neoadjuvant platinum therapy., Conclusions: The proposed method robustly replicates the most commonly used transcriptome-based subtype classifications from paraffin-embedded tissue samples. The luminal, but not basal, molecular subtypes had the greatest benefit from adjuvant platinum therapy. We identified and validated APOBEC3G as a novel predictive marker for platinum-treated patients., (© 2022 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Introduction and Objective: Muscle-invasive urothelial bladder cancer (MIBC) is associated with limited response rates to systemic therapy, risk of recurrence and death. Tumor infiltrating immune cells have been associated with outcome and response to chemo-and immunotherapy in MIBC. We aimed to profile the immune cells in the tumor microenvironment (TME) to predict prognosis in MIBC and responses to adjuvant chemotherapy., Methods: We performed multiplex immunohistochemistry (IHC) profiling and quantification of immune and stromal cells (CD3, CD4, CD8, CD163, FoxP3, PD-1, and CD45, Vimentin, αSMA, PD-L1, Pan-Cytokeratin, Ki67) in 101 patients with MIBC receiving radical cystectomy. We used uni- and multivariate survival analyses to identify cell types predicting prognosis. Samples were subdivided using K-means clustering for Treg and macrophage infiltration resulting in 3 clusters, Cluster 1: Treg high, cluster 2: macrophage high, cluster 3: Treg and macrophage low. Routine CD68 and CD163 IHC were analyzed with QuPath in an extended cohort of 141 MIBC., Results: High concentrations of macrophages were associated with increased risk of death (HR 10.9, 95% CI 2.8-40.5; p < 0.001) and high concentrations of Tregs were associated with decreased risk of death (HR 0.1, 95% CI 0.01-0.7; p = 0.03) in the multivariate Cox-regression model adjusting for adjuvant chemotherapy, tumor and lymph node stage. Patients in the macrophage rich cluster (2) showed the worst OS with and without adjuvant chemotherapy. The Treg rich cluster (1) showed high levels of effector and proliferating immune cells and had the best survival. Cluster 1 and 2 both were rich in PD-1 and PD-L1 expression on tumor and immune cells., Conclusion: Treg and macrophage concentrations in MIBC are independent predictors of prognosis and are important players in the TME. Standard IHC with CD163 for macrophages is feasible to predict prognosis but validation to use immune-cell infiltration, especially to predict response to systemic therapies, is required., (© 2023. The Author(s).)

Muscle-invasive bladder cancer (MIBC) is associated with limited response rates to systemic therapy leading to a significant risk of recurrence and death. A recently discovered histone methyltransferase KMT9, acts as an epigenetic regulator of carcinogenesis in different tumor entities. In this study, we investigated the presence and association of histological and molecular subtypes and their impact on the survival of KMT9α in MIBC. We performed an immunohistochemical (IHC) analysis of KMT9α in 135 MIBC patients undergoing radical cystectomy. KMT9α was significantly overexpressed in the nucleus in MIBC compared to normal urothelium and low-grade urothelial cancer. Using the HTG transcriptome panel, we assessed mRNA expression profiles to determine molecular subtypes and identify differentially expressed genes. Patients with higher nuclear and nucleolar KMT9α expression showed basal/squamous urothelial cancer characteristics confirmed by IHC and differentially upregulated KRT14 expression. We identified a subset of patients with nucleolar expression of KMT9α, which was associated with an increased risk of death in uni- and multivariate analyses (HR 2.28, 95%CI 1.28-4.03, p = 0.005). In conclusion, basal-like MIBC and the squamous histological subtype are associated with high nuclear KMT9α expression. The association with poor survival makes it a potential target for the treatment of bladder cancer.

Cell death and injury at the site of tumor ablation attracts macrophages. We sought to understand the status and activity of these cells while focusing on transforming growth factor-β1 (TGF-β1), a potent immunosuppressive and tumorigenic cytokine. Patients with urothelial cancer who underwent ablation using electrocautery or laser demonstrated increased infiltration and numbers of CD8+ T cells, along with FoxP3+ regulatory T cells, CD68+ macrophages and elevated levels of TGF-β1 in recurrent tumors. Similar findings were reproduced in a mouse model of urothelial cancer (MB49) by partial tumor ablation with irreversible electroporation (IRE). Stimulation of bone marrow derived macrophages with MB49 cell debris produced using IRE elicited strong M2 polarization, with exuberant secretion of TGF-β1. The motility, phenotypic markers and cytokine secretion by macrophages could be muted by treatment with Pirfenidone (PFD), a clinically approved drug targeting TGF-β1 signaling. MB49 cancer cells exposed to TGF-β1 exhibited increased migration, invasiveness and upregulation of epithelial-mesenchymal transition markers α-Smooth Muscle Actin and Vimentin. Such changes in MB49 cells were reduced by treatment with PFD even during stimulation with TGF-β1. IRE alone yielded better local tumor control when compared with control or PFD alone, while also reducing the overall number of lung metastases. Adjuvant PFD treatment did not provide additional benefit under in vivo conditions., Competing Interests: GS has stock options in Aperture medical and is a board member of the Society for Interventional Radiology Foundation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could construed as a potential conflict of interest., (Copyright © 2023 Kimura, Fujimori, Rajagopalan, Poudel, Kim, Nagar, Vroomen, Reis, Al-Ahmadie, Coleman and Srimathveeravalli.)

Introduction: Guideline recommendations are meant to help minimize morbidity and to improve the care of nonmuscle invasive bladder cancer (NMIBC) patients but studies have suggested an underuse of guideline-recommended care. The aim of this study was to evaluate the level of adherence of German and Austrian urologists to German guideline recommendations., Methods: A survey of 27 items evaluating diagnostic and therapeutic recommendations (15 cases of strong consensus and 6 cases of consensus) for NMIBC was administered among 14 urologic training courses. Survey construction and realization followed the checklist for reporting results of internet e-surveys and was approved by an internal review board., Results: Between January 2018 and June 2019, a total of 307 urologists responded to the questionnaire, with a mean response rate of 71%. The data showed a weak role of urine cytology (54%) for initial diagnostics although it is strongly recommended by the guideline. The most frequently used supporting diagnostic tool during transurethral resection of the bladder was hexaminolevulinate (95%). Contrary to the guideline recommendation, 38% of the participants performed a second resection in the case of pTa low-grade NMIBC. Correct monitoring of Bacille Calmette-Guérin (BCG) response with cystoscopy and cytology was performed by only 34% of the urologists., Conclusions: We found a discrepancy between certain guideline recommendations and daily routine practice concerning the use of urine cytology for initial diagnostics, instillation therapy with a low monitoring rate of BCG response, and follow-up care with unnecessary second resection after pTa low-grade NMIBC in particular. Our survey showed a moderate overall adherence rate of 73%. These results demonstrate the need for sharpening awareness of German guideline recommendations by promoting more intense education of urologists to optimize NMIBC care thus decreasing morbidity and mortality rates., (© 2021 S. Karger AG, Basel.)

Transcriptome-based molecular subtypes of muscle-invasive bladder cancer (MIBC) have been shown to be both prognostic and predictive, but are not used in routine clinical practice. We aimed to develop a feasible, reverse transcription quantitative polymerase chain reaction (RT-qPCR)-based method for molecular subtyping. First, we defined a 68-gene set covering tumor intrinsic (luminal, basal, squamous, neuronal, epithelial-to-mesenchymal, in situ carcinoma) and stromal (immune, extracellular matrix, p53-like) signatures. Then, classifier methods with this 68-gene panel were developed in silico and validated on public data sets with available subtype class information (MD Anderson [MDA], The Cancer Genome Atlas [TCGA], Lund, Consensus). Finally, expression of the selected 68 genes was determined in 104 frozen tissue samples of our MIBC cohort by RT-qPCR using the TaqMan Array Card platform and samples were classified by our newly developed classifiers. The prognostic value of each subtype classification system and molecular signature scores were assessed. We found that the reduced marker set combined with the developed classifiers were able to reproduce the TCGA II, MDA, Lund and Consensus subtype classification systems with an overlap of 79%, 76%, 69% and 64%, respectively. Importantly, we could successfully classify 96% (100/104) of our MIBC samples by using RT-qPCR. Neuronal and luminal subtypes and low stromal gene expressions were associated with poor survival. In conclusion, we developed a robust and feasible method for the molecular subtyping according to the TCGA II, MDA, Lund and Consensus classifications. Our results suggest that stromal signatures have a superior prognostic value compared to tumor intrinsic signatures and therefore underline the importance of tumor-stroma interaction during the progression of MIBC., (© 2021 The Authors. International Journal of Cancer published by John Wiley & Sons Ltd on behalf of UICC.)

In recent years, the number and type of treatment options in advanced bladder cancer (BC) have been rapidly evolving. To select an effective therapy and spare unnecessary side effects, predictive biomarkers are urgently needed. As the host's anti-cancer immune response is by far the most effective system to impede malignant tumor growth, immune system-based biomarkers are promising. We have recently described altered proteasomal epitope processing as an effective immune escape mechanism to impair cytotoxic T-cell activity. By altering the neoantigens' characteristics through different proteasomal peptide cleavage induced by non-synonymous somatic mutations, the ability for T-cell activation was decreased ("processing escapes"). In the present study, we analyzed primary chemo-naïve tissue samples of 26 adjuvant platinum-treated urothelial BC patients using a targeted next-generation sequencing panel followed by the epitope determination of affected genes, a machine-learning based prediction of epitope processing and proteasomal cleavage and of HLA-affinity as well as immune activation. Immune infiltration (immunohistochemistries for CD8, granzyme B, CD45/LCA) was digitally quantified by a pathologist and clinico-pathological and survival data were collected. We detected 145 epitopes with characteristics of a processing escape associated with a higher number of CD8-positive but lower number of granzyme B-positive cells and no association with PD-L1-expression. In addition, a high prevalence of processing escapes was associated with unfavorable overall survival. Our data indicate the presence of processing escapes in advanced BC, potentially creating a tumor-promoting pro-inflammatory environment with lowered anti-cancerous activity and independence from PD-L1-expression. The data also need to be prospectively validated in BC treated with immune therapy.

Purpose: To compare clinical outcome and quality of life (QoL) in octogenarian patients with muscle-invasive urothelial carcinoma (MIBC) either treated by radical cystectomy (RC) or transurethral resection of the tumor (TURBT)., Methods: We identified octogenarian patients with MIBC in our institutions since 2005. Clinical treatment outcomes and QoL were analyzed. Uni- and multivariable Cox regression analyses, two-tailed Wilcoxon test, Mann-Whitney test and Fisher's exact test were assessed as appropriate. QoL was evaluated using FACT-G (Functional Assessment of Cancer Therapy-General) questionnaire., Results: 143 patients were identified (RC: 51 cases, TURBT: 92 cases). Mean follow-up was 14 months (0-100 months). Median overall survival (OS) was 12 months in the RC group and 7 months in the TURBT group. TURBT and low preoperative hemoglobin were independent risk factors for reduced cancer-specific survival (CSS) (TURBT: p = 0.019, Hb: p = 0.008) and OS (TURBT: p = 0.026, Hb: p = 0.013) in multivariable analyses. Baseline QoL was low throughout the whole cohort. There was no difference in baseline FACT-G scoring comparing RC and TURBT (FACT-G total score (median): RC 43.7/108 vs. TURBT 44.0/108, p = 0.7144). Increased FACT-G questionnaire scoring was assessed for RC patients (median percentage score change RC 22.9%, TURBT 2.3%, p < 0.0001)., Conclusion: RC and TURBT are feasible treatment options for MIBC in octogenarian patients. In our cohort, RC was associated with increased CSS, OS and QoL. QoL in general was low throughout the whole cohort. Interdisciplinary decision-making has to be improved for these critically ill patients., (© 2021. The Author(s).)

Objective: Patients with bladder cancer (BC) are at risk of developing upper tract urothelial carcinoma (UTUC). Therefore, CT urography is recommended for follow-up. To avoid intravenous contrast agents, retrograde pyelography (RPG) is an alternative. However, it is still unclear whether RPG increases the incidence of UTUC. The aim of this study was to investigate the impact of RPG in the presence of BC on the risk of developing UTUC., Patients and Methods: Retrospectively analysing a total of 3,680 RPGs between 2009 and 2016, all patients with simultaneous BC (group 1) and those without synchronous BC (group 2) during RPG were compared. All patients were risk stratified according to the EORTC bladder calculator. In patients without BC during RPG, risk stratification was based on the worst prior tumour characteristics., Results: A total of 145 patients with a history of BC were analysed. Of these, 112 patients underwent RPG with simultaneous BC. UTUC developed in 6 of 112 patients (5.4%) and 58.9% (66/112) had high-risk BC according to the EORTC bladder calculator. In the control group, one out of 33 (3%) patients with metachronous high-risk BC developed UTUC., Conclusions: Using RPG in the presence of BC did not increase the risk of UTUC. Due to the predominant number of high-risk/high-grade tumours, individual tumour biology appears to be the primary driver for the development of UTUC., (© 2021 S. Karger AG, Basel.)

Background: Expression levels of collagens have been implicated in the progression of various cancers and interaction with cytokeratins but are not well studied in bladder cancer (BC). Therefore, we analyzed the gene and protein expression levels of collagen 1A1 (Col1a1/COL1A1), collagen 3A1 (col3a1/COL3A1), collagen 5A2 (col5a2/COL5A2), cytokeratin 14 (krt14/CK14), and cytokeratin 17 (krt17/CK17) in urothelial BC samples of different stages., Methods: In total, 102 fresh frozen and 190 formalin-fixed and paraffin-embedded (FFPE) samples were tested using immunohistochemistry and RT-qPCR. Expression levels were correlated with clinicopathological and follow-up data., Results: Col1a1, col3a1, col5a2 and krt14 mRNA levels were significantly overexpressed in high-grade and muscle-invasive BC (MIBC) compared to low-grade and non-muscle invasive BC (NMIBC) cases. Disease-specific survival (DSS) was shorter in patients with high expression levels of col1a1 (p = 0.004), col3a1 (p = 0.004), and col5a2 (p = 0.028). CK14 (p = 0.020), COL3A1 (p = 0.006), and Col5A2 (p = 0.006) protein expression levels were significantly higher and protein expression levels of CK17 (p = 0.05) were significantly lower in MIBC compared to NMIBC. Furthermore, CK14 (p = 0.002) and COL5A2 (p = 0.006) protein expression level were significantly higher in high-grade compared to low-grade BC. DSS was shorter in patients with high expression levels of COL5A2 (p = 0.033) and CK14 (p = 0.042)., Conclusion: Expression changes of collagens and cytokeratins are univariable prognostic markers in BC., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Urachal adenocarcinomas (UrC) are rare but aggressive. Despite being of profound therapeutic relevance, UrC cannot be differentiated by histomorphology alone from other adenocarcinomas of differential diagnostic importance. As no reliable tissue-based diagnostic biomarkers are available, we aimed to detect such by integrating mass-spectrometry imaging-based metabolomics and digital pathology, thus allowing for a multimodal approach on the basis of spatial information. To achieve this, a cohort of UrC (n = 19) and colorectal adenocarcinomas (CRC, n = 27) as the differential diagnosis of highest therapeutic relevance was created, tissue micro-arrays (TMAs) were constructed, and pathological data was recorded. Hematoxylin and eosin (H&E) stained tissue sections were scanned and annotated, enabling an automized discrimination of tumor and non-tumor areas after training of an adequate algorithm. Spectral information within tumor regions, obtained via matrix-assisted laser desorption/ionization (MALDI)-Orbitrap-mass spectrometry imaging (MSI), were subsequently extracted in an automated workflow. On this basis, metabolic differences between UrC and CRC were revealed using machine learning algorithms. As a result, the study demonstrated the feasibility of MALDI-MSI for the evaluation of FFPE tissue in UrC and CRC with the potential to combine spatial metabolomics data with annotated histopathological data from digitalized H&E slides. The detected Area under the curve (AUC) of 0.94 in general and 0.77 for the analyte taurine alone (diagnostic accuracy for taurine: 74%) makes the technology a promising tool in this differential diagnostic dilemma situation. Although the data has to be considered as a proof-of-concept study, it presents a new adoption of this technology that has not been used in this scenario in which reliable diagnostic biomarkers (such as immunohistochemical markers) are currently not available., (© 2021. The Author(s).)

Urachal carcinoma (UrC) is a rare tumor with remarkable histological and molecular similarities to colorectal cancer (CRC). Adenomatous polyposis coli (APC) is the most frequently affected gene in CRC, but the prevalence and significance of its alterations in UrC is poorly understood. In addition, loss of phosphatase and tensin homologue (PTEN) was shown to be associated with therapy resistance in CRC. Our primary aim was to assess specific genetic alterations including APC and PTEN in a large series of UrC samples in order to identify clinically significant genomic alterations. We analyzed a total of 40 UrC cases. Targeted 5-gene (APC, PTEN, DICER1, PRKAR1A, TSHR, WRN) panel sequencing was performed on the Illumina MiSeq platform (n = 34). In addition, ß-catenin (n = 38) and PTEN (n = 30) expressions were assessed by immunohistochemistry. APC and PTEN genes were affected in 15% (5/34) and 6% (2/34) of cases. Two of five APC alterations (p.Y1075*, p.K1199*) were truncating pathogenic mutations. One of the two PTEN variants was a pathogenic frameshift insertion (p.C211fs). In 29% (11/38) of samples, at least some weak nuclear ß-catenin immunostaining was detected and PTEN loss was observed in 20% (6/30) of samples. The low prevalence of APC mutations in UrC represents a characteristic difference to CRC. Based on APC and ß-catenin results, the Wnt pathway seems to be rarely affected in UrC. Considering the formerly described involvement of PTEN protein loss in anti-EGFR therapy-resistance its immunohistochemical testing may have therapeutic relevance.

Primary glandular bladder tumours (bladder adenocarcinoma [BAC], urachal adenocarcinoma [UAC], urothelial carcinoma with glandular differentiation [UCg]) are rare malignancies with histological resemblance to colorectal adenocarcinoma (CORAD) in the majority of this subgroup. Definite case numbers are very low, molecular data are limited and the pathogenesis remains poorly understood. Therefore, this study was designed to complement current knowledge by in depth analysis of BAC (n = 12), UAC (n = 13), UCg (n = 11) and non-invasive glandular lesions (n = 19). In BAC, in addition to known alterations in TP53, Wnt, MAP kinase and MTOR pathway, mutations in SMAD4, ARID1A and BRAF were identified. Compared to published data on muscle invasive bladder cancer (BLCA) and CORAD, UCg exhibited frequent "urothelial" like alterations while BAC and UAC were characterised by a more "colorectal" like mutational pattern. Immunohistochemically, there was no evidence of DNA mismatch repair deficiency or PD-L1 tumour cell positivity in any sample. Depending on the used antibody 0-45% of BAC, 0-30% of UCg and 0% UAC cases exhibited PD-L1 expressing tumour associated immune cells. A single BAC (9%, 1/11) showed evidence of ARID1A protein loss, and two cases of UCg (20%, 2/10) showed loss of SMARCA1 and PBRM1, respectively. Taken together, our data suggest at least in part involvement of similar pathways driving tumourigenesis of adenocarcinomas like BAC, UAC and CORAD independent of their tissue origin. Alterations of TERT and FBXW7 in single cases of intestinal metaplasia further point towards a possible precancerous character in line with previous reports.

To optimize treatment decisions in advanced bladder cancer (BC), we aimed to assess the therapy predictive value of STIP1 with regard to cisplatin therapy. Cisplatin-based chemotherapy represents the standard first-line systemic treatment of advanced bladder cancer. Since novel immunooncologic agents are already available for cisplatin-resistant or ineligible patients, biological markers are needed for the prediction of cisplatin resistance. STIP1 expression was analyzed in paraffin-embedded bladder cancer tissue samples of 98 patients who underwent adjuvant or salvage cisplatin-based chemotherapy by using immunohistochemistry. Furthermore, pre-chemotherapy serum STIP1 concentrations were determined in 48 BC patients by ELISA. Results were correlated with the clinicopathological and follow-up data. Stronger STIP1 nuclear staining was associated with worse OS in both the whole patient group (p = 0.034) and the subgroup of patients who received at least 2 cycles of chemotherapy (p = 0.043). These correlations remained significant also in the multivariable analyses (p = 0.035 and p = 0.040). Stronger STIP1 cytoplasmatic immunostaining correlated with shorter PFS both in the whole cohort (p = 0.045) and in the subgroup of patients who received at least 2 cycles of chemotherapy (p = 0.026). Elevated STIP1 serum levels were associated with older patient's age, but we found no correlation between STIP1 serum levels and patients' outcome. Our results suggest that tissue STIP1 analysis might be used for the prediction of cisplatin-resistance in BC. In contrast, pretreatment STIP1 serum levels showed no predictive value for chemotherapy response and survival.

The Slit-Robo pathway has shown to be altered in several malignant diseases. However, its role in bladder cancer is poorly understood. Therefore, we aimed to assess the tissue expression of Robo1 and Robo4 as well as their ligand Slit2 in different stages of bladder cancer to explore possible changes of Slit-Robo signalling during the progression of bladder cancer. Robo1, Robo4 and Slit2 gene expressions were analyzed in 92 frozen bladder cancer tissue samples by using reverse transcription quantitative real-time PCR. Immunohistochemical analyses were performed on 149 formalin-fixed and paraffin-embedded bladder cancer tissue samples. Results were correlated with the clinical and follow-up data by performing both univariable and multivariable analyses. Robo1 and Robo4 nuclear staining intensitiy was significantly higher in low stage and low grade bladder cancer. Elevated Robo1 nuclear staining was associated with better disease-specific survival (DSS) (p = 0.045). Similarly, stronger Robo4 nuclear staining tended to be associated with longer DSS (p = 0.061). We found higher Robo1 and Slit2 gene expression levels in advanced stages of bladder cancer (p = 0.007 and p < 0.001). High Slit2 gene expression was correlated with significantly shorter DSS (p < 0.005), while Robo1 and Robo4 gene expressions were not associated with patients' prognosis. Our results demonstrate that the nuclear expression of Robo1 and Robo4 is associated with a favourable prognosis suggesting that its translocation into the nucleus represent a posttranslational regulation process which may exhibit an antitumor effect in bladder cancer.

Introduction: Urachal cancer (UrC) is a rare but aggressive cancer. Due to its low incidence, structured epidemiological data have only rarely been reported. To date, no valid data on UrC are available for the German population., Methods: Data on incidence and relative 5-year survival of urachal lesions (ICD-10: C67.7) were collected from all population-based cancer registries in Germany, provided by the Robert Koch-Institut (RKI). Data were anonymized and included age, sex, and general histology (ICD-O-3). For comparison, a similar inquiry of the "Surveillance, Epidemiology, and End Results program" (SEER-18) database for the USA was conducted., Results: From 2011 to 2015, a total of 154 and 152 cases of UrC were reported for Germany (RKI) and the USA (SEER-18 area), respectively. Age-standardized incidence was 0.32/1,000,000 age-standardized cases/year in both cohorts, and elderly persons were more often affected. The major histological type was adenocarcinoma (64.9 and 81.6%). Relative 5-year survival was 54.8% (CI: 45.0-64.6) in Germany (RKI) and 64.4% (54.1-72.1) in the USA (SEER-18 cohort). Discusssion/Conclusion: The collected data demonstrate low incidence rates and similar epidemiological and clinicopathological characteristics of UrC for both registries. This is the first report of structured epidemiological data for UrC for the German population., (© 2020 S. Karger AG, Basel.)

Background: Urachal cancer is a rare type of cancer, often following a clinically aggressive course. Due to its rarity, knowledge about its molecular background is still limited. In addition, no sufficiently reliable diagnostic markers are available., Objectives: The aim of the present study is to give an overview of our recent molecular projects on urachal cancer and to connect it with current literature in the field., Materials and Methods: Three projects are introduced. The first project identified and validated diagnostic biomarkers in urachal adenocarcinomas compared to colorectal adenocarcinomas and primary adenocarcinomas of the bladder using various proteomic methods. In the second project, the most relevant differential diagnostic markers between urachal adenocarcinomas and colorectal adenocarcinomas compared to normal tissue (urachal remnants) were determined by analyzing a miRNA panel. Sequence analyses were performed in the third project. The focus was on molecular differences to colorectal adenocarcinomas and urothelial carcinomas., Results and Conclusions: We detected potential biomarker candidates for the immunohistochemical differential-diagnosis and generated a miRNA-based diagnostic scoring system with a potentially high differential-diagnostic significance. The sequence analyses data confirm the molecular autonomy of the urachal adenocarcinomas compared to other entities.

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.

Objectives: Cisplatin-based chemotherapy represents the gold standard in the treatment of advanced bladder cancer (BC) both in the neoadjuvant and adjuvant setting. Since novel immunooncologic agents are available for cisplatin-resistant or ineligible patients, biological markers for the prediction of cisplatin resistance become more important in treatment decisions. Therefore, we aimed to assess the therapy predictive value of 8 promising tissue biomarkers with regard to cisplatin therapy., Methods: Emmprin, survivin, HMGA2, MTA1, RhoGDI, PEG10, TGM2, and TLN1 expressions were analyzed in paraffin-embedded bladder cancer tissue samples of 106 patients who underwent adjuvant or salvage cisplatin-based chemotherapy by using immunohistochemistry. Results were correlated with the clinicopathological and follow-up data by performing both univariable and multivariable survival analyses., Results: Higher HMGA2 nuclear staining intensity and positive survivin nuclear staining were associated with worse overall survival (OS) (P = 0.045 and P = 0.002, respectively). In accordance, survivin nuclear staining also significantly correlated with shorter progression free survival (PFS, P = 0.024), while HMGA2 nuclear positivity tended to correlate with shorter PFS (P = 0.069) after at least 2 cycles of chemotherapy. In the multivariable analyses only survivin remained as an independent predictor of both OS and PFS (P = 0.008 and P = 0.025). None of the other markers proved to be significant predictors of adjuvant or salvage cisplatin-based chemotherapy., Conclusions: Our results demonstrate that survivin represents a promising marker for the prediction of cisplatin resistance in BC. In addition the therapy predictive role of HMGA2 should be further investigated. Immunohistochemical analysis of BC samples provides a feasible way for the prediction of cisplatin-resistance and may therefore provide a valuable tool for optimizing treatment decisions in advanced BC., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Urachal cancer is a rare but aggressive disease. In addition to the non-glandular tumors, non-cystic urachal adenocarcinomas are nowadays distinguished from the primary cystic variant. (Immunohistochemical) markers are only of minor differential diagnostic value and, therefore, the diagnosis is primarily established in a multidisciplinary approach. The non-cystic variant accounts for the majority of cases (83%), is more common in men (63%), shows a median age at diagnosis of 51 years and has a 5-year survival rate of about 50%. In organ-confined disease, usually a partial cystectomy of the tumor in the bladder dome, including the median umbilical ligament and umbilicus, is performed. In advanced stages, systemic therapy is needed while 5‑fuorouracil (5-FU) containing regimes have been shown to be more effective. Due to the rarity of the tumor, targeted therapy approaches based on a biological rationale are becoming increasingly relevant. As molecular data are still sparse, we compiled and analyzed the largest urachal cancer cohort to date. In 31% of the cases, MAPK-/PI3K signaling pathway alterations were detected (especially in K-/NRAS) with implications for anti-EGFR therapy approaches. Further potentially therapeutic alterations were detected in FGFR1, MET, PDGFRA, and erbB2/HER2. Additionally, PD-L1 tumor cell expression (clone: 22C3) was demonstrated in 16% of cases, therefore making anti-PD-1/PD-L1 immuno-oncological approaches worth considering despite the absence of mismatch repair deficiency (MMR-d) and/or high microsatellite instability (MSI-h). Finally, urachal adenocarcinomas seem to be a distinct entity on the molecular level with closer resemblance to colorectal adenocarcinomas than to urothelial carcinomas.

The bladder exstrophy-epispadias complex (BEEC) represents the severe end of uro-rectal malformation spectrum involving aberrant embryonic morphogenesis of the cloacal membrane and the urorectal septum. The most common form of BEEC is isolated classic bladder exstrophy (CBE). Long-term complications in CBE are malignancies of the bladder with 95% of them being adenocarcinomas. Since CBE and adenocarcinoma of the bladder are rare entities, their frequent co-occurrence suggests a common etiology. Recent studies suggest that promoter methylation of various genes play a crucial role during the phenotypical morphogenesis of adenocarcinomas of urinary bladder. To examine, whether epigenetic processes such as DNA methylation patterns are potentially associated with CBE, we performed Illumina 450 K methylation arrays in blood (n = 10) and tissue samples (n = 2) of CBE patients and healthy matched controls (n = 12). In our analysis, we found total lack of methylation in the blood and methylation differences were restricted to 10 CpG sites in the tissue samples. In comparison to other bladder anomalies, CBE tissue methylation profiles differ from those of adenocarcinoma, adenocarcinoma with CBE, urothelial carcinoma and urachal carcinoma. In this preliminary study, we did not provide any strong evidence of major DNA methylation alterations which would be suggestive for strong underlying epigenetic mechanism. However, larger studies are required to provide more robust statistical evidence to exclude smaller effects in the tissues., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Tumors of the genitourinary system are common. In recent years, our understanding of their molecular background and therefore the number of potential predictive biomarkers has massively increased., Objectives: The aim of the current work is to give an overview of recent (molecular) developments and predictive biomarkers in urologic oncology and to give a perspective of what might become relevant in the future of the field., Material and Methods: We considered the recent literature and study data and combined it with our own expertise in tumors of the urinary system, kidneys, and prostate., Results and Conclusions: The molecular subtypes of muscle-invasive urothelial bladder cancer (MIBC) hold a predictive and prognostic significance and correlate with clinicopathological features. Immune therapy with checkpoint inhibitors (CPI) has a major role in urothelial carcinoma (UC), but also in renal cell carcinoma and a subgroup of prostate cancers. The first-line use in UC is restricted to PD-L1-"positive" cases (≥IC2/3, CPS ≥ 10). Further predictive markers are currently under evaluation, while the predictive significance of tumor mutational burden (TMB) is under debate. In addition to a subgroup of renal cell carcinomas, a subgroup of prostate carcinomas with alterations in the DNA repair system might benefit from a customized therapy approach (PARP inhibitors, platin-containing chemotherapy). The multitude of potentially therapy-relevant molecular alterations and related predictive biomarkers calls for the implementation of sophisticated molecular analyses in daily routine. This will lead to an even more rapid dynamic in the field of genitourinary pathology.

Urachal cancer is a rare but aggressive disease. In addition to the non-glandular tumors, non-cystic urachal adenocarcinomas are nowadays distinguished from the primary cystic variant. (Immunohistochemical) markers are only of minor differential diagnostic value and, therefore, the diagnosis is primarily established in a multidisciplinary approach. The non-cystic variant accounts for the majority of cases (83%), is more common in men (63%), shows a median age at diagnosis of 51 years and has a 5-year survival rate of about 50%. In organ-confined disease, usually a partial cystectomy of the tumor in the bladder dome, including the median umbilical ligament and umbilicus, is performed. In advanced stages, systemic therapy is needed while 5‑fuorouracil (5-FU) containing regimes have been shown to be more effective. Due to the rarity of the tumor, targeted therapy approaches based on a biological rationale are becoming increasingly relevant. As molecular data are still sparse, we compiled and analyzed the largest urachal cancer cohort to date. In 31% of the cases, MAPK-/PI3K signaling pathway alterations were detected (especially in K-/NRAS) with implications for anti-EGFR therapy approaches. Further potentially therapeutic alterations were detected in FGFR1, MET, PDGFRA, and erbB2/HER2. Additionally, PD-L1 tumor cell expression (clone: 22C3) was demonstrated in 16% of cases, therefore making anti-PD-1/PD-L1 immuno-oncological approaches worth considering despite the absence of mismatch repair deficiency (MMR-d) and/or high microsatellite instability (MSI-h). Finally, urachal adenocarcinomas seem to be a distinct entity on the molecular level with closer resemblance to colorectal adenocarcinomas than to urothelial carcinomas.

Urachal cancer (UrC) is a rare but aggressive malignancy often diagnosed in advanced stages requiring systemic treatment. Although cytotoxic chemotherapy is of limited effectiveness, prospective clinical studies can hardly be conducted. Targeted therapeutic treatment approaches and potentially immunotherapy based on a biological rationale may provide an alternative strategy. We therefore subjected 70 urachal adenocarcinomas to targeted next-generation sequencing, conducted in situ and immunohistochemical analyses (including PD-L1 and DNA mismatch repair proteins [MMR]) and evaluated the microsatellite instability (MSI) status. The analytical findings were correlated with clinicopathological and outcome data and Kaplan-Meier and univariable/multivariable Cox regression analyses were performed. The patients had a mean age of 50 years, 66% were male and a 5-year overall survival (OS) of 58% and recurrence-free survival (RFS) of 45% was detected. Sequence variations were observed in TP53 (66%), KRAS (21%), BRAF (4%), PIK3CA (4%), FGFR1 (1%), MET (1%), NRAS (1%), and PDGFRA (1%). Gene amplifications were found in EGFR (5%), ERBB2 (2%), and MET (2%). We detected no evidence of MMR-deficiency (MMR-d)/MSI-high (MSI-h), whereas 10 of 63 cases (16%) expressed PD-L1. Therefore, anti-PD-1/PD-L1 immunotherapy approaches might be tested in UrC. Importantly, we found aberrations in intracellular signal transduction pathways (RAS/RAF/PI3K) in 31% of UrCs with potential implications for anti-EGFR therapy. Less frequent potentially actionable genetic alterations were additionally detected in ERBB2 (HER2), MET, FGFR1, and PDGFRA. The molecular profile strengthens the notion that UrC is a distinct entity on the genomic level with closer resemblance to colorectal than to bladder cancer., (© 2018 UICC.)

Urachal cancer (UrC) is a rare but aggressive cancer. Due to overlapping histomorphology, discrimination of urachal from primary bladder adenocarcinomas (PBAC) and adenocarcinomas secondarily involving the bladder (particularly colorectal adenocarcinomas, CRC) can be challenging. Therefore, we aimed to give an overview of helpful (immunohistochemical) biomarkers and clinicopathological factors in addition to survival analyses and included institutional data from 12 urachal adenocarcinomas. A PubMed search yielded 319 suitable studies since 1930 in the English literature with 1984 cases of UrC including 1834 adenocarcinomas (92%) and 150 nonadenocarcinomas (8%). UrC was more common in men (63%), showed a median age at diagnosis of 50.8 years and a median tumor size of 6.0 cm. No associations were noted for overall survival and progression-free survival (PFS) and clinicopathological factors beside a favorable PFS in male patients ( p = 0.047). The immunohistochemical markers found to be potentially helpful in the differential diagnostic situation are AMACR and CK34 β E12 (UrC versus CRC and PBAC), CK7, β -Catenin and CD15 (UrC and PBAC versus CRC), and CEA and GATA3 (UrC and CRC versus PBAC). Serum markers like CEA, CA19-9 and CA125 might additionally be useful in the follow-up and monitoring of UrC.

High rates of telomerase reverse transcriptase (TERT) promoter mutations have recently been described in urothelial carcinoma (UC). Unlike UC in the bladder, adenocarcinomas account for the majority of urachal cancer (UrC) cases. As data in UrC is unclear, we analyzed TERT promoter mutations in a large cohort of UrC for its differential diagnostic, clinicopathological and prognostic significance. UrC cases from six academic centers were analyzed for c.-146C>T (C250T) and c.-124C>T (C228T) TERT promoter mutations by PCR and Sanger sequencing. Clinicopathological and survival data were collected. The cohort consisted of 15 men (56%) and 12 women (44%) with a median age of 50 years including 23 adenocarcinomas, two squamous cell carcinomas (SCC), one UC and one undifferentiated carcinoma. In one case of (mucinous) urachal adenocarcinoma a C228T mutation was detected (1/23; 4%), like in a case of SCC in addition to one C250T mutation in the UC case. TERT promoter mutations are very rare in urachal adenocarcinomas (unlike in UC) with differential diagnostic implications. Additionally, the low TERT promoter mutation rate in urachal adenocarcinomas is more comparable to colorectal adenocarcinomas than to UC, giving further support to recent genetic findings and therapeutic considerations., (© 2017 Japanese Society of Pathology and John Wiley & Sons Australia, Ltd.)

Purpose: To evaluate the impact of three BCL2 single-nucleotide polymorphisms, i.e., c.-938C>A (rs2279115), c.21G>A (rs1801018), and c.*2203A>G (rs4987853) on survival in patients with transitional cell carcinoma of the bladder., Methods: We analyzed 179 patients who underwent surgical treatment for bladder cancer at the Clinic of Urology, University Hospital Essen, Germany. Genomic DNA was extracted and genotyped for the polymorphisms. For all polymorphisms, linkage analysis was performed. Kaplan-Meier and Cox regression analyses were used to determine the putative impact of the three polymorphisms on outcome., Results: c.-938C>A and c.21G>A, but not c.*2203A>G, are in strong linkage disequilibrium (D' 0.96). We found a significant association between c.-938C>A and relapse-free survival (p = 0.024) with an allele dose effect. In the same way, c.21G>A had a significant impact on both relapse-free survival (p = 0.009) and progression-free survival (p = 0.012), as well as a pronounced allele dose effect. Regression analysis proved c.21G>A and c.-938C>A, to be an independent risk factor in univariate and multivariable analyses., Conclusions: In our cohort, both c.-938C>A and c.21G>A showed a significant impact on outcome with TCC of the bladder. Due to the linkage disequilibrium of both SNPs, maybe, only one of them could mediate this effect. In multivariable analysis, however, both proved to be independently associated with overall survival. Contrary to other findings which found the c.-938C>A mainly influencing outcome, our data may suggest that the main effect on TCC could be due to the c.21G>A polymorphism.

Background and Objectives: Urachal carcinoma (UrC) is a rare and poorly investigated disease. Our current knowledge is mainly based on single-institutional studies. Despite growing interest in UrC, the included case numbers in recently published studies are still low. Therefore, we aimed to provide a comprehensive meta-analysis on the clinical, prognostic, and therapeutic aspects of UrC., Methods: A systematic Medline/PubMed search was performed on UrC using the terms "urachal carcinoma," "urachal cancer," and "urachus." Original articles and reviews in English language with case numbers>10 were selected., Results: The vast majority (91%, 489/532) of UrCs are diagnosed at later stages (Sheldon≥III) when the tumor invades the urinary bladder. About 21% (136/646) of UrC patients have distant metastasis at first presentation. Although for patients with non-metastatic UrC surgical treatment provides an acceptable disease control, the systemic treatment of patients with progressed/metastatic UrC-in lack of prospective clinical trials-are less well established. Comparing cisplatin-based and 5-FU-based therapies in 74 published UrC cases, we found the latter to be superior in terms of radiographic response rates (9% vs. 44%, P = 0.043), but the combination of these 2 therapies provided the lowest progression rate (14%) with a similarly high response rate (43%)., Conclusions: Owing to the lack of evidence-based guidelines, the therapy of UrC remains challenging. Given the infrequency of UrC, large prospective studies comparing different systemic therapies can hardly be conducted. Our metadata indicates that 5-FU-containing chemotherapy regimens are more effective than cisplatin-based treatment modalities, whereas their combination seems to provide the strongest antitumor effect. Nevertheless, in the lack of evidences from prospective clinical trials, therapeutic decision-making necessarily remains on an individual basis. In this situation, targeted therapies may provide a reasonable alternative. Therefore, better understanding of the molecular background of UrC is needed to rationalize treatment decisions in UrC., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Purpose: Targeted therapy represents an attractive alternative for rare tumors such as urachal carcinoma (UrC). The aim of this study was to assess the mutations of the most commonly affected 5 genes in the targetable EGFR-pathway in UrC and comapre their frequencies to those of found in urothelial and colorectal cancer., Materials and Methods: Mutational hot-spots of selected genes were tested in 22 UrC samples by pyrosequencing. Mutational patterns were compared to those published for colorectal and urothelial cancers. Furthermore, we sought correlations between mutations and clinicopathological and follow-up data., Results: We found 11 mutations in 10 of 22 (45%) patients. The most frequently mutated gene was KRAS (27%) followed by BRAF (18%) and NRAS (5%), while no mutations were detected in the EGFR and PIK3CA genes. No correlation was found between the mutation status and clinicopathological parameters (Sheldon/Mayo stage, tumor grade, metastases). Furthermore, none of the mutations correlated with progression-free or overall survival., Conclusions: The mutation pattern of UrC is more similar to colorectal than to urothelial cancer. However, the mutation characteristics of UrC seems to be unique suggesting that clinical decision-making for UrC cannot be simply adopted from urothelial or colorectal carcinoma. The high occurence of EGFR-pathway mutations warrants the testing for KRAS and BRAF mutations when considering anti-EGFR therapy in UrC., Competing Interests: CONFLICTS OF INTERESTS None of the authors reported any financial interests or potential conflicts of interest related to this study.

Purpose: Urachal carcinoma of the bladder is a rare malignancy. Its histological phenotype is similar to that of primary bladder and colorectal adenocarcinoma. The aim of this study was to explore the expression and prognostic relevance of 6 select protein markers of urachal carcinoma of the bladder, including p53, Ki67, RHAMM, BGN, IMP3 and MMP-7, which were formerly shown to be prognostic in urothelial carcinoma and colorectal adenocarcinoma., Materials and Methods: Clinical and followup data were obtained on a total of 26 patients with urachal carcinoma of the bladder treated at 2 university hospitals. Immunohistochemical analysis of p53, Ki67, RHAMM, BGN, IMP3 and MMP-7 expression was performed in samples from 15 patients. Clinicopathological parameters and immunohistochemical results were tested for prognostic value on univariable and multivariable analyses., Results: Followup was 50 months. Five-year overall and progression-free survival was 46% and 32%, respectively. On multivariable analysis a positive resection margin was an independent predictor of poor overall survival (p = 0.025). RHAMM (p = 0.0431), IMP3 (p = 0.0052), Ki67 (p = 0.0006) and p53 (p = 0.0024) expression rates were significantly increased in urachal carcinoma of the bladder cells compared to normal urothelium. IMP3 was elevated in Sheldon tumor stage IIIA compared to IIIB or greater (p = 0.0048). None of the analyzed protein markers was associated with survival., Conclusions: The independent prognostic value of a positive resection margin underlines the importance of complete surgical removal of urachal carcinoma of the bladder combined with en bloc resection of the median umbilical ligament and umbilicus. Our results in a limited number of samples show that Ki67, p53, RHAMM and IMP3 expression is enhanced but has no prognostic significance in urachal carcinoma of the bladder., (Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.)

Objectives: YKL-40 is a novel inflammatory serum protein shown to be associated with the presence and prognosis of several malignancies. However, its prognostic relevance has not yet been analyzed in bladder cancer (BC). Therefore, the aim of this study was to assess the tissue, serum, and urinary levels of YKL-40 and their prognostic value in BC., Methods and Materials: YKL-40 gene expression levels were analyzed in frozen tissue samples of 91 patients with BC; YKL-40 concentrations were measured in 120 serum (101 patients with BC and 19 controls) and 154 urine samples (125 patients with BC and 29 controls). In 16 cases, corresponding serum samples collected before and after radical cystectomy were analyzed for YKL-40. Results were correlated with clinicopathological parameters and follow-up data., Results: YKL-40 gene expressions and serum concentrations were higher in patients with BC compared with controls; however, urinary YKL-40 levels remained under the detection limit in both patients and controls. Higher tissue gene expressions and serum concentrations were associated with poor patients' survival in the univariable analysis (P = 0.037 and 0.022, respectively), but only high YKL-40 serum levels proved to be independent prognostic factors in BC (hazard ratio = 1.755, 95% CI: 1.014-3.039, P = 0.045). We found no significant difference between preoperative and postoperative serum concentrations of YKL-40., Conclusions: YKL-40 serum levels are associated with the presence of BC and poor patients' survival. The independent prognostic relevance of YKL-40 is of particular interest in patients with muscle-invasive BC treated with radical surgery. Our data suggest that BC tissue is not the main source of serum YKL-40 levels., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In this study, we assessed the changes and prognostic relevance of syndecan-1 (SDC1) tissue and serum levels in bladder cancer (BC). SDC1 levels were analyzed in 213 samples (119 paraffin-embedded and 79 serum samples of BC patients and 15 controls) using immunohistochemistry and enzyme-linked immunosorbent assay. Results were correlated with clinicopathological characteristics and follow-up data, as well as previously determined serum levels of angiogenic factors (basic fibroblast growth factor, endostatin, angiostatin, angiopoietin, vascular endothelial growth factor, Tie2 and MMP-7). SDC1 staining was present in the cell membrane of normal bladder epithelium and non-muscle-invasive BC cells but was absent in a significant proportion of muscle-invasive carcinomas (P < .001). In contrast, stromal SDC1 expression was enhanced in muscle-invasive compared to non-muscle-invasive BCs (P = .001). Serum concentrations of the SDC1 ectodomain were higher in muscle-invasive BCs compared to controls or non-muscle-invasive carcinomas (P < .001 each). Lymph node-positive cases had the highest SDC1 serum concentrations (P < .001). SDC1 expression in stromal cells was independently associated with survival (hazard ratio = 2.034, 95% confidence interval 1.176-3.519, P = .011). SDC1 serum concentrations correlated with those of endostatin and matrix metalloproteinase 7. Loss of SDC1 in tumor cells and the parallel increase of serum SDC1 ectodomain concentration in high-stage, high-grade BCs suggest the involvement of SDC1 shedding in BC progression. In addition, high preoperative SDC1 serum levels may help to identify patients with lymph node metastases, supporting therapeutic decision-making. Presence of SDC1 in tumor stroma is an independent risk factor for patient survival and may therefore be used to select patients for more aggressive therapy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background: Urothelial bladder cancer is the ninth most common cancer. Despite surgical and chemotherapeutic treatment the prognosis is still poor once bladder cancer progresses to a muscle-invasive state. Discovery of new diagnostic markers and pathophysiologic effectors might help to contribute to novel diagnostic and therapeutic options. The extracellular matrix microenvironment shaped by the extracellular matrix critically affects tumor cell and stroma cell functions. Therefore, aim of the present study was to assess the possible implication of the small leucine-rich proteoglycan biglycan in progression of human urothelial bladder cancer., Methods and Results: For this purpose tumor biopsies of 76 bladder cancer patients with different tumor stages (pTa, pT1-T4) were investigated with respect to biglycan expression and correlated with a long-term (10 years) clinical follow-up. Interestingly, higher biglycan mRNA expression was associated with higher tumor stages and muscle invasiveness. In vitro knock-down of endogenous biglycan in human urothelial carcinoma cells (J82 cells) increased proliferation, whereas addition of recombinant biglycan and overexpression of biglycan inhibited tumor cell proliferation. In line with this growth-inhibitory effect of biglycan, transplantation of J82 cells after knock-down of biglycan resulted in significantly increased growth of subcutaneous xenograft tumors in nude mice in vivo. Furthermore, treatment with two anti-proliferative, multi-receptor tyrosine kinase inhibitors-sunitinib and sorafenib-strongly upregulated biglycan expression. Collectively, the experimental data suggest that high biglycan expression is associated with reduced tumor cell proliferation. In accordance, Kaplan-Meier analysis revealed higher 10-year survival in patients with high biglycan mRNA expression in tumor biopsies., Conclusion: In conclusion, the present data suggest that biglycan is an endogenous inhibitor of bladder cancer cell proliferation that is upregulated in response to anti-proliferative tyrosine kinase inhibitors. In addition, high biglycan expression is associated with favorable prognosis.

Unlabelled: What's known on the subject? and What does the study add? Insulin-like growth factor mRNA-binding protein 3 (IMP3) is an oncofetal protein found to be re-expressed in a series of human cancers including bladder cancer. In vitro analyses showed an invasion and proliferation promoting effect for IMP3. Further in vitro studies suggested that IMP3 is able to bind to the mRNAs of CD44 and insulin-like growth factor 2 (IGF2), enhancing their stability and expression. However, this molecular interaction has not yet been analysed in tumour samples. In the present study, we identified for the first time high IMP3 tissue protein expression as an independent predictor of poor patients' survival in muscle-invasive bladder cancer. Furthermore, there was no correlation between IMP3 and its molecular targets in bladder carcinoma specimens and concluded that the tumour-promoting effect of IMP3 is not related to its regulatory action on IGF2 and CD44., Objective: To assess the prognostic value and molecular actions of the oncofetal protein insulin-like growth factor mRNA-binding protein 3 (IMP3) in muscle-invasive bladder cancer (BC)., Patients and Methods: IMP3 expression was analysed by immunohistochemistry, real-time polymerase chain reaction and Western blot analysis in 224 patients with BC. The molecular targets of IMP3; CD44, insulin-like growth factor 2 (IGF2) and its receptor the IGF1 receptor (IGF1-R) were also investigated. Expression levels were correlated with clinical follow-up data by using both univariate and multivariate Cox regression analyses., Results: IMP3 mRNA and protein levels were significantly elevated in high-stage and high-grade muscle-invasive BC. In muscle-invasive BC IMP3 protein but not gene expression proved to be an independent predictor of disease-specific (hazard ratio [HR] 2.58, 95% confidence interval [CI] 1.28-4.56, P = 0.004) and overall survival (HR 2.07, 95% CI 1.12-3.82, P = 0.020). The expression levels of IGF2 and CD44 showed no correlation with that of IMP3., Conclusions: High IMP3 protein levels may identify patients with BC at high risk of disease progression and may therefore select patients for a more intensive therapy or for a strict follow-up. Its high expression in high-grade bladder carcinoma cells makes IMP3 for an attractive target for therapy. The tumour promoting effect of IMP3 is independent from its regulatory action on IGF2 and CD44 expression., (© 2012 THE AUTHORS. BJU INTERNATIONAL © 2012 BJU INTERNATIONAL.)

Endostatin, the proteolytic fragment of collagen XVIII, is an inhibitor of angiogenesis and tumor growth. Interestingly, elevated circulating endostatin levels have been found to correlate with poor patients' prognosis in several cancers. The aim of this study was to assess the prognostic value of endostatin in bladder cancer (BC) and to gain insight into the mechanisms involved in its production. This retrospective study included a total of 337 patients with BC and 103 controls. Collagen XVIII gene expression was analyzed using real-time PCR (n = 82). Endostatin tissue localization was assessed by immunohistochemistry (n = 27). Endostatin serum (n = 87) and urine (n = 153) levels were determined by ELISA. In 12 cases, both serum and paraffinized tissue samples from the same patients were available. We found decreased collagen XVIII tissue expression and increased endostatin urine and serum concentration in samples of patients with BC compared to controls. High serum endostatin levels correlated with the presence of lymph node metastases and MMP-7 concentrations and were independently associated with poor metastasis-free and disease-specific survival. Immunohistochemical analysis revealed a strong endostatin staining in the wall of tumor associated blood vessels in superficial but not in muscle-invasive BCs. Based on these, we concluded that elevated endostatin levels in patients with BC are the consequence of enhanced extracellular matrix degradation and are independent from collagen XVIII expression. Furthermore, serum endostatin levels may provide prognostic information independent from histopathological parameters and may therefore help to optimize therapy decisions. Loss of endostatin expression in tumor associated blood vessels might represent an important step supporting tumor-induced angiogenesis., (Copyright © 2011 UICC.)