Urothelial Bladder Cancer Research Articles

IDMIR: identification of dysregulated miRNAs associated with disease based on a miRNA-miRNA interaction network constructed through gene expression data.

Micro ribonucleic acids (miRNAs) play a pivotal role in governing the human transcriptome in various biological phenomena. Hence, the accumulation of miRNA expression dysregulation frequently assumes a noteworthy role in the initiation and progression of complex diseases. However, accurate identification of dysregulated miRNAs still faces challenges at the current stage. Several bioinformatics tools have recently emerged for forecasting the associations between miRNAs and diseases. Nonetheless, the existing reference tools mainly identify the miRNA-disease associations in a general state and fall short of pinpointing dysregulated miRNAs within a specific disease state. Additionally, no studies adequately consider miRNA-miRNA interactions (MMIs) when analyzing the miRNA-disease associations. Here, we introduced a systematic approach, called IDMIR, which enabled the identification of expression dysregulated miRNAs through an MMI network under the gene expression context, where the network's architecture was designed to implicitly connect miRNAs based on their shared biological functions within a particular disease context. The advantage of IDMIR is that it uses gene expression data for the identification of dysregulated miRNAs by analyzing variations in MMIs. We illustrated the excellent predictive power for dysregulated miRNAs of the IDMIR approach through data analysis on breast cancer and bladder urothelial cancer. IDMIR could surpass several existing miRNA-disease association prediction approaches through comparison. We believe the approach complements the deficiencies in predicting miRNA-disease association and may provide new insights and possibilities for diagnosing and treating diseases. The IDMIR approach is now available as a free R package on CRAN (https://CRAN.R-project.org/package=IDMIR).

The Association of Death Receptors and TGF-β1 Expression in Urothelial Bladder Cancer and Their Prognostic Significance.

Death receptor signalization that triggers the extrinsic apoptotic pathway and TGF-β1 have important roles in urothelial carcinogenesis, with a complex interplay between them. The aim of this research was to assess the association of death receptors DR4, DR5, and FAS as well as TGF-β1 immunohistochemical expression with the clinicopathological characteristics of urothelial bladder cancer (UBC) and to evaluate their prognostic significance. The decrease or loss of death receptors' expression was significantly associated with muscle-invasive tumors, while non-invasive UBC often retains the expression of death receptors, which are mutually strongly linked. High DR4 expression is a marker of low-grade tumors and UBC associated with exposition to known carcinogens. Conversely, TGF-β1 was significantly associated with high tumor grade and advanced stage. High expression of DR4 and FAS indicates longer overall survival. High TGF-β1 signifies an inferior outcome and is an independent predictor of adverse prognosis in UBC patients. This study reveals the expression profile of death receptors in UBC and their possible interconnection with TGF-β1 and indicates independent prognostic significance of high FAS and TGF-β1 expression in UBC, which may contribute to deciphering the enigma of UBC heterogeneity in light of the rapid development of novel and effective therapeutic approaches, including targeting of the TRAIL-induced apoptotic pathway.

Comparative Analysis of Clinicopathologic Characteristics and Outcomes of Urothelial Bladder Cancer Between Young and Older Adults-Experience from a Tertiary Care Center.

The evidence on bladder cancer in the young population remains fragmented due to lack of literature and conflicting results from the existing studies. We aim to elucidate such conflicting data and define the clinicopathologic characteristics, management trends, and outcomes of urothelial bladder carcinoma in young adults as compared to their older counterparts. This was a retrospective, single-center study involving patients with primary urothelial bladder cancer who underwent treatment at our center from March 2017 to March 2022. For analysis, patients were stratified into three subgroups based on age: group A, 18-40years; group B, > 40years; and group C, > 60years. Group A with younger patients was compared with groups B and C. A total of 471 eligible patients (422 males and 49 females) were included in the study with a median age of 44years. Group A had significantly lower recurrence and progression rates as compared to group B (31% vs 57.1%, p = 0.002 and 9.5% vs 19.2%, p = 0.04, respectively). Group A had significantly more recurrence-free survival (RFS) than group B (5-year-RFS = 68.03% vs 32.58%, p = 0.01). Similarly, group A also had lower recurrence (31% vs 62.6%, p < 0.001) and progression (9.5% vs 28.6%, p = 0.015) rates as compared to group C as well as better RFS (5-year-RFS = 68.03% vs 19.00%, p = 0.04) and progression-free survival (5-year-PFS = 83.1% vs 62.8%, p = 0.03) in comparison to group C. Age and tumor grade were found to be independent predictors of recurrence-free and progression-free survival. We concluded that high-grade disease is more common than low-grade disease both in younger and older patients. Younger patients fare better in terms of recurrence and progression when compared to their older counterparts.

Automatic detection method of bladder tumor cells based on color and shape features

Bladder urothelial carcinoma is the most common malignant tumor disease in urinary system, and its incidence rate ranks ninth in the world. In recent years, the continuous development of hyperspectral imaging technology has provided a new tool for the auxiliary diagnosis of bladder cancer. In this study, based on microscopic hyperspectral data, an automatic detection algorithm of bladder tumor cells combining color features and shape features is proposed. Support vector machine (SVM) is used to build classification models and compare the classification performance of spectral feature, spectral and shape fusion feature, and the fusion feature proposed in this paper on the same classifier. The results show that the sensitivity, specificity, and accuracy of our classification algorithm based on shape and color fusion features are 0.952, 0.897, and 0.920, respectively, which are better than the classification algorithm only using spectral features. Therefore, this study can effectively extract the cell features of bladder urothelial carcinoma smear, thus achieving automatic, real-time, and noninvasive detection of bladder tumor cells, and then helping doctors improve the efficiency of pathological diagnosis of bladder urothelial cancer, and providing a reliable basis for doctors to choose treatment plans and judge the prognosis of the disease.

Adenoid cystic/basal-cell carcinoma of the prostate following high-grade urothelial bladder cancer: a case report

Adenoid cystic/basal-cell carcinoma of the prostate following high-grade urothelial bladder cancer: a case report

Molecular profiling of a bladder cancer with very high tumour mutational burden

The increasing incidence of urothelial bladder cancer is a notable global concern, as evidenced by the epidemiological data in terms of frequency, distribution, as well as mortality rates. Although numerous molecular alterations have been linked to the occurrence and progression of bladder cancer, currently there is a limited knowledge on the molecular signature able of accurately predicting clinical outcomes. In this report, we present a case of a pT3b high-grade infiltrating urothelial carcinoma with areas of squamous differentiation characterized by very high tumor mutational burden (TMB), with up-regulations of immune checkpoints. The high TMB, along with elevated expressions of PD-L1, PD-L2, and PD1, underscores the rationale for developing a personalized immunotherapy focused on the use of immune-checkpoint inhibitors. Additionally, molecular analysis revealed somatic mutations in several other cancer-related genes, including TP53, TP63 and NOTCH3. Mutations of TP53 and TP63 genes provide mechanistic insights on the molecular mechanisms underlying disease development and progression. Notably, the above-mentioned mutations and the elevated hypoxia score make the targeting of p53 and/or hypoxia related pathways a plausible personalized medicine option for this bladder cancer, particularly in combination with immunotherapy. Our data suggest a requirement for molecular profiling in bladder cancer to possibly select appropriate immune-checkpoint therapy.

Targeted and immunotherapy for the management of advanced urothelial carcinoma of the bladder.

In an effort to expedite the publication of articles, AJHP is posting manuscripts online as soon as possible after acceptance. Accepted manuscripts have been peer-reviewed and copyedited, but are posted online before technical formatting and author proofing. These manuscripts are not the final version of record and will be replaced with the final article (formatted per AJHP style and proofed by the authors) at a later time. The activity of targeted and immunotherapy for the management of advanced bladder cancer is reviewed. Platinum-based chemotherapy is standard first-line treatment for advanced bladder cancer. Pembrolizumab is approved alone as first-line therapy for patients who are ineligible for any platinum-based chemotherapy and with enfortumab for patients ineligible for cisplatin-based chemotherapy. Avelumab is approved for maintenance therapy in patients who have not progressed with first-line platinum-containing therapy. Pembrolizumab, avelumab, and nivolumab are approved second-line therapy in patients who experience progression during or after platinum-containing chemotherapy. Erdafitinib is indicated for advanced disease that has susceptible FGFR2 or FGFR3 genetic alterations and has progressed during or after treatment with at least one line of platinum-containing chemotherapy. Enfortumab vedotin and sacituzumab govitecan are antibody-drug conjugates. They are both approved for patients who have received anti-PD-L1 or anti-PD-1 therapy and treatment with platinum-containing chemotherapy. Enfortumab is also indicated for patients who are ineligible to receive cisplatin-based therapy and have received one or more prior lines of therapy. Six targeted and immunotherapeutic agents have been approved for patients with advanced urothelial bladder cancer. They all have demonstrated activity in patients for whom disease has progressed during or after platinum-based therapy. Pembrolizumab, with and without enfortumab, has demonstrated first-line activity, and avelumab is a key maintenance therapy after first-line treatment. The results of additional clinical trials should provide evidence to establish the exact role in therapy of each agent in patients with advanced disease.

Treatment of Non-Metastatic Muscle-Invasive Bladder Cancer: AUA/ASCO/SUO Guideline (2017; Amended 2020, 2024).

Although representing approximately 25% of patients diagnosed with bladder cancer, muscle-invasive bladder cancer (MIBC) carries a significant risk of death that has not significantly changed in decades. Increasingly, clinicians and patients recognize the importance of multidisciplinary collaborative efforts that take into account survival and quality of life concerns. This guideline provides a risk-stratified, clinical framework for the management of muscle-invasive urothelial bladder cancer. In 2024, the MIBC guideline was updated through the AUA amendment process in which newly published literature is reviewed and integrated into previously published guidelines in an effort to maintain currency. The amendment allowed for the incorporation of additional literature released since the previous 2020 amendment. The updated search gathered literature from May 2020 to November 2023. This review identified 3739 abstracts, of which 46 met inclusion criteria.When sufficient evidence existed, the body of evidence was assigned a strength rating of A (high), B (moderate), or C (low) for support of Strong, Moderate, or Conditional Recommendations. In the absence of sufficient evidence, additional information is provided as Clinical Principles and Expert Opinions. Updates were made regarding neoadjuvant/adjuvant chemotherapy, radical cystectomy, pelvic lymphadenectomy, multi-modal bladder preserving therapy, and future directions. Further revisions were made to the methodology and reference sections as appropriate. This guideline seeks to improve clinicians' ability to evaluate and treat patients with MIBC based on currently available evidence. Future studies will be essential to further support or refine these statements to improve patient care.

Microbiome Sex-Related Diversity in Non-Muscle-Invasive Urothelial Bladder Cancer.

Sex-specific discrepancies in bladder cancer (BCa) are reported, and new studies imply that microbiome may partially explain the diversity. We aim to provide characterization of the bladder microbiome in both sexes diagnosed with non-muscle-invasive BCa with specific insight into cancer grade. In our study, 16S rRNA next-generation sequencing was performed on midstream urine, bladder tumor sample, and healthy-appearing bladder mucosa. Bacterial DNA was isolated using QIAamp Viral RNA Mini Kit. Metagenomic analysis was performed using hypervariable fragments of the 16S rRNA gene on Ion Torrent Personal Genome Machine platform. Of 41 sample triplets, 2153 taxa were discovered: 1739 in tumor samples, 1801 in healthy-appearing bladder mucosa and 1370 in midstream urine. Women were found to have smaller taxa richness in Chao1 index than men (p = 0.03). In comparison to low-grade tumors, patients with high-grade lesions had lower bacterial diversity and richness in urine. Significant differences between sexes in relative abundance of communities at family level were only observed in high-grade tumors.

A Novel Decision Tree Model for Predicting the Cancer-Specific Survival of Patients with Bladder Cancer Treated with Radical Cystectomy.

Background/Objectives: The aim was to develop a decision tree and a new prognostic tool to predict cancer-specific survival in patients with urothelial bladder cancer treated with radical cystectomy. Methods: A total of 11,834 patients with bladder cancer treated with radical cystectomy between 2004 and 2019 from the SEER database were randomly split into the derivation (n = 7889) and validation cohorts (n = 3945). Survival curves were estimated using conditional decision tree analysis. We used Multiple Imputation by Chained Equations for the treatment of missing values and the pec package to compare the predictive performance. We extracted data from our model following CHARMS and assessed the risk of bias and applicability with PROBAST. Results: A total of 4824 (41%) patients died during the follow-up period due to bladder cancer. A decision tree was made and 12 groups were obtained. Patients with a higher AJCC stage and older age have a worse prognosis. The risk groups were summarized into high, intermediate and low risk. The integrated Brier scores between 0 and 191 months for the bootstrap estimates of the prediction error are the lowest for our conditional survival tree (0.189). The model showed a low risk of bias and low concern about applicability. The results must be externally validated. Conclusions: Decision tree analysis is a useful tool with significant discrimination. With this tool, we were able to stratify patients into 12 subgroups and 3 risk groups with a low risk of bias and low concern about applicability.

Cisplatin-Resistant Urothelial Bladder Cancer Cells Undergo Metabolic Reprogramming beyond the Warburg Effect.

Advanced urothelial bladder cancer (UBC) patients are tagged by a dismal prognosis and high mortality rates, mostly due to their poor response to standard-of-care platinum-based therapy. Mediators of chemoresistance are not fully elucidated. This work aimed to study the metabolic profile of advanced UBC, in the context of cisplatin resistance. Three isogenic pairs of parental cell lines (T24, HT1376 and KU1919) and the matching cisplatin-resistant (R) sublines were used. A set of functional assays was used to perform a metabolic screening on the cells. In comparison to the parental sublines, a tendency was observed towards an exacerbated glycolytic metabolism in the cisplatin-resistant T24 and HT1376 cells; this glycolytic phenotype was particularly evident for the HT1376/HT1376R pair, for which the cisplatin resistance ratio was higher. HT1376R cells showed decreased basal respiration and oxygen consumption associated with ATP production; in accordance, the extracellular acidification rate was also higher in the resistant subline. Glycolytic rate assay confirmed that these cells presented higher basal glycolysis, with an increase in proton efflux. While the results of real-time metabolomics seem to substantiate the manifestation of the Warburg phenotype in HT1376R cells, a shift towards distinct metabolic pathways involving lactate uptake, lipid biosynthesis and glutamate metabolism occurred with time. On the other hand, KU1919R cells seem to engage in a metabolic rewiring, recovering their preference for oxidative phosphorylation. In conclusion, cisplatin-resistant UBC cells seem to display deep metabolic alterations surpassing the Warburg effect, which likely depend on the molecular signature of each cell line.

Natural History and Genomic Landscape of Chemotherapy-Resistant Muscle-Invasive Bladder Cancer.

Patients with residual invasive bladder cancer after neoadjuvant chemotherapy (NAC) and radical cystectomy have a poor prognosis. Data on adjuvant therapy for these patients are conflicting. We sought to evaluate the natural history and genomic landscape of chemotherapy-resistant bladder cancer to inform patient management and clinical trials. Data were collected on patients with clinically localized muscle-invasive urothelial bladder cancer treated with NAC and cystectomy at our institution between May 15, 2001, and August 15, 2019, and completed four cycles of gemcitabine and cisplatin NAC, excluding those treated with adjuvant therapies. Survival was estimated using the Kaplan-Meier method, and multivariable Cox proportional hazards models were used to identify predictors of recurrence-free survival (RFS). Genomic alterations were identified in targeted exome sequencing (Memorial Sloan Kettering Integrated Mutation Profiling of Actionable Cancer Targets) data from post-NAC specimens from a subset of patients. Lymphovascular invasion (LVI) was the strongest predictor of RFS (hazard ratio, 2.15 [95% CI, 1.37 to 3.39]) on multivariable analysis. Patients with ypT2N0 disease without LVI had a significantly prolonged RFS compared with those with LVI (70% RFS at 5 years). Lymph node yield did not affect RFS. Among patients with sequencing data (n = 101), chemotherapy-resistant tumors had fewer alterations in DNA damage response genes compared with tumors from a publicly available chemotherapy-naïve cohort (15% v 29%; P = .021). Alterations in CDKN2A/B were associated with shorter RFS. PIK3CA alterations were associated with LVI. Potentially actionable alterations were identified in more than 75% of tumors. Although chemotherapy-resistant bladder cancer generally portends a poor prognosis, patients with organ-confined disease without LVI may be candidates for close observation without adjuvant therapy. The genomic landscape of chemotherapy-resistant tumors is similar to chemotherapy-naïve tumors. Therapeutic opportunities exist for targeted therapies as adjuvant treatment in chemotherapy-resistant disease.

The Usefulness of Positron Emission Tomography/Computed Tomography in the Diagnosis of Metastasis in Patients with Urothelial Carcinoma — A Secondary Publication

Purpose: This study examined the usefulness of positron emission tomography (PET)/computed tomography (CT) in the diagnosis of metastasis in patients with urothelial carcinoma. Materials and methods: The subjects were patients who were newly diagnosed with urothelial carcinoma in our department on whom we performed CT and PET/CT to search for metastasis. Results: The median age of the 92 subjects was 71 years, and bladder and upper tract urothelial cancer were underlying diseases in 41 (46%) and 51 (54%) patients, respectively. In 66 (72%) of the 92 cases, no metastasis was observed by CT, while PET/CT revealed metastasis in 9 (14%). The 57 (86%) patients in whom both CT and PET/CT showed no metastasis underwent radical surgery, while 2 patients (4%) exhibited pathological lymph node metastasis. Of the 26 patients in whom CT revealed metastasis, PET/CT showed no metastasis in 3 (12%), and the absence of pathological metastasis was confirmed in all patients. Of the 23 patients found to have metastasis in both CT and PET/CT, metastasis that could not be identified by CT was discovered by performing PET/CT in 10 (43%) patients. PET/CT showed significantly higher diagnostic accuracy than CT alone (P &lt; 0.01), with sensitivities of 94.1% and 67.6%, specificities of 100% and 94.8%, and accuracy rates of 97.8% and 84.7%, respectively. Conclusions: PET/CT of patients with urothelial cancer revealed that metastases that cannot be diagnosed by CT alone are found at a significant frequency. Since these metastases can affect treatment choices in patients with urothelial cancer, PET/CT is considered to be useful in diagnosing metastases in patients with urothelial cancer.

Trichostatin C Synergistically Interacts with DNMT Inhibitor to Induce Antineoplastic Effect via Inhibition of Axl in Bladder and Lung Cancer Cells.

Aberrant epigenetic modifications are fundamental contributors to the pathogenesis of various cancers. Consequently, targeting these aberrations with small molecules, such as histone deacetylase (HDAC) inhibitors and DNA methyltransferase (DNMT) inhibitors, presents a viable strategy for cancer therapy. The objective of this study is to assess the anti-cancer efficacy of trichostatin C (TSC), an analogue of trichostatin A sourced from the fermentation of Streptomyces sp. CPCC 203909. Our investigations reveal that TSC demonstrates potent activity against both human lung cancer and urothelial bladder cancer cell lines, with IC50 values in the low micromolar range. Moreover, TSC induces apoptosis mediated by caspase 3/7 and arrests the cell cycle at the G2/M phase. When combined with the DNMT inhibitor decitabine, TSC exhibits a synergistic anti-cancer effect. Additionally, protein analysis elucidates a significant reduction in the expression of the tyrosine kinase receptor Axl. Notably, elevated concentrations of TSC correlate with the up-regulation of the transcription factor forkhead box class O1 (FoxO1) and increased levels of the proapoptotic proteins Bim and p21. In conclusion, our findings suggest TSC as a promising anti-cancer agent with HDAC inhibitory activity. Furthermore, our results highlight the potential utility of TSC in combination with DNMT inhibitors for cancer treatment.

Abstract 5131: TYMS overexpression - but not copy number alteration - is associated with cancer aggressiveness in urothelial bladder cancer

Abstract Background: Thymidylate Synthetase (TYMS) is an import enzyme in the early stage of DNA biosynthesis. It is coded by the TYMS gene at 18p11.32, a region which has been found amplified in urothelial carcinoma. TYMS is a target enzyme for 5-fluorouracil (5-FU) and methotrexate. Elevated TYMS expression has been associated with poor prognosis and unfavorable therapy response in different cancer types. The aim of this study was to clarify the clinical relevance of TMYS expression in urothelial carcinoma and to determine the relationship between TYMS expression and gene amplification. Design: TYMS expression was analyzed by immunohistochemistry and TYMS copy number status was determined by fluorescence in-situ hybridization on a tissue microarray (TMA) containing more than 2,700 urothelial carcinomas of the urinary bladder including &amp;gt;600 patients who had undergone cystectomy for muscle-invasive disease and for which follow-up data were available. Results: TMYS immunostaining was detectable in 83.1% of 1,799 analyzable bladder cancers, including 56.4% with weak, 14.4% with moderate, and 12.3% with strong staining. The rate of strongly TMYS positive cancers increased markedly from pTaG2 low (0.3%) to pTaG2 high (4.1%), and pTaG3 cancers (11.8%; p&amp;lt;0.0001) and was even somewhat higher in pT2-4 cancers (16.7%; p=0.6401; pTaG3 vs pT2-4). In muscle-invasive cancers, the frequency of strong TMYS immunostaining increased with tumor stage (p=0.0011) and grade (p=0.0007) but was lower in lymph node positive cancers (p=0.0018). No association was found between TYMS expression and patient prognosis. TYMS amplification was found in 3.1% and TMYS deletion in 1.2% of 1,775 analyzable bladder cancers. The rate of TYMS amplification increased from pTa (0.6%) to pT2-4 (3.7%; p&amp;lt;0.0001) cancers whereas TYMS deletion was only found in pT2-4 (1.4%) cancers. In muscle invasive-cancers, TYMS copy number alterations were unrelated to tumor phenotype and patient prognosis. Strong TYMS positivity was significantly associated with TYMS amplification (p=0.0096) but only 28.6% of 42 amplified cancers showed a strong TMYS staining and only 7.2% of strongly TYMS positive cancers had a TYMS amplification. Conclusion: The significant association between TYMS overexpression and parameters of aggressive phenotype are consistent with a role of TYMS in bladder cancer progression. The incomplete association between TYMS amplification and overexpression demonstrates that TYMS is not the critical target of 18p11 amplification in bladder cancer and that TYMS upregulation is in most cases due to other mechanisms than amplification. Citation Format: Henning Plage, Sebastian Hofbauer, Kira Furlano, Sarah Weinberger, Antonia Franz, Annika Fendler, Florian Roßner, Simon Schallenberg, Sefer Elezkurtaj, Adrian Frericks, Maximilian Lennartz, Andreas H. Marx, Henrik Samtleben, Margit Fisch, Michael Rink, Marcin Slojewski, Krystian Kaczmarek, Thorsten Ecke, Stefan Koch, Nico Adamini, Ronald Simon, Guido Sauter, Henrik Zecha, Joachim Weischenfeldt, Tobias Klatte, Sarah Minner, David Horst, Thorsten Schlomm, Martina Kluth. TYMS overexpression - but not copy number alteration - is associated with cancer aggressiveness in urothelial bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5131.

Abstract 1048: Loss of MTAP expression is strongly linked to homozygous 9p21 deletion, unfavorable tumor phenotype and non-inflamed microenvironment in urothelial bladder cancer

Abstract 9p21 deletions are common in urothelial carcinoma and mostly include S-methyl-5′-thioadenosine phosphorylase (MTAP) a critical enzyme for DNA and RNA synthesis. Homozygous MTAP deletions result in a complete loss of expression which can be visualized by immunohistochemistry (IHC). Evidence is emerging that MTAP deficiency results in a critical vulnerability of cancer cells towards drugs targeting multiple different pathways that depend on a functional MTAP gene and causes resistance to immune checkpoint inhibitors. To determine the prevalence and clinical significance of MTAP deficiency and its relationship with the tumor microenvironment in urothelial bladder carcinomas, more than 2,500 tumors were analyzed by fluorescence in-situ hybridization (FISH) and IHC in a tissue microarray format, and the data were compared with existing data on intratumoral lymphocyte subsets and PD-L1 expression. The cohort included 1,826 patients that underwent radical cystectomy for muscle-invasive disease (pT2-4). 9p21 deletion was homozygous in 364 (21.3%) and heterozygous in 334 (19.5%) of 1,711 analyzable tumors. The rate of 9p21 deleted tumors increased from pTa G2 low (9.2% homozygous, 25.8% heterozygous) to pTa G2 high (32.6%, 20.9%; p&amp;lt;0.0001) but was slightly lower in pTa G3 (16.7% each). In pT2-pT4 carcinomas, 9p21 deletions were homozygous in 23.3% and heterozygous in 17.9%. Within muscle-invasive cancers, 9p21 deletions were tied to high pT (p=0.0014) and poor overall survival (p=0.0461). MTAP expression loss was strongly linked to homozygous 9p21 deletions. Complete absence of MTAP staining was seen in 96.1% of homozygous deleted cancers while only 4 of 249 MTAP negative cancers had retained at least one 9p21 copy (p&amp;lt;0.0001). Accordingly, absence of MTAP staining was also linked to advanced pT status and poor overall survival (p&amp;lt;0.05 each). Tumors with heterozygous 9p21 deletion did often show preserved but low-level expression of MTAP. 9p21 deletions were associated with low numbers of PD-L1 positive tumor cells (p&amp;lt;0.0001), CD8 (p=0.01) and CD4 positive lymphocytes (p=0.009), M2-macrophages (p&amp;lt;0.0001), and dendritic cells (p=0.0007). A similar tendency was observed for MTAP expression loss, but the level of significance was only reached for PD-L1 positive tumor cells (p&amp;lt;0.0001), M2 macrophages (p=0.001) and dendritic cells (p=0.012). Complete MTAP expression loss is common in urinary bladder cancer and strongly tied to homozygous 9p21 loss, aggressive disease, and non-inflamed microenvironment. Drugs targeting MTAP-deficiency may be highly useful in bladder cancer. MTAP IHC is an excellent surrogate for the detection of MTAP deficiency in this tumor entity. Citation Format: Natalia Gorbokon, Niklas Wößner, Viktoria Ahlburg, Simon Schallenberg, Henning Plage, Sebastian Hofbauer, Kira Furlano, Sarah Weinberger, Paul G. Bruch, Florian Roßner, Sefer Elezkurtaj, Maximilian Lennartz, Niclas C. Blessin, Andreas H. Marx, Henrik Samtleben, Margit Fisch, Michael Rink, Marcin Slojewski, Krystian Kaczmarek, Thorsten Ecke, Steffen Hallmann, Stefan Koch, Nico Adamini, Sarah Minner, Ronald Simon, Guido Sauter, Henrik Zecha, David Horst, Tobias Klatte, Thorsten Schlomm, Martina Kluth. Loss of MTAP expression is strongly linked to homozygous 9p21 deletion, unfavorable tumor phenotype and non-inflamed microenvironment in urothelial bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1048.

Abstract 883: Genetic characteristics between males and females with urothelial bladder cancer

Abstract Urothelial bladder cancer (UBC) affects the epithelial cells of the urinary tract and is the most common type of bladder cancer worldwide. Statistically, males are four times more likely to develop UBC than females. It is known that decreased expression of the KDM6A gene, coded on the X chromosome, corresponds with a higher incidence of UBC. KDM6A mutations are known to be more prevalent in females with non-muscle-invasive UBC. These mutations are targetable by inhibiting EZH2, an enzyme coded by its namesake gene. On the environmental side, males are at a higher risk of developing carcinogenic complications due to smoking and environmental toxins. However, this discrepancy fails to exist when comparing males and females who smoke similar levels. Though environmental factors may play a role in influencing behavior and altering levels of risk to develop UBC, research suggests that they do not single-handedly explain sex-based disparities in UBC. In this study, we characterize the molecular mechanisms associated with altered overall survival by uncovering previously unreported data on the expression of KDM6A and FGFR3, a commonly altered oncogene, in UBC patients. Our in silico approach consisted of broad survival data analysis through cBioPortal (n=1486), sequencing-based gene expression data through ICGC Portal (n=295), and single nucleotide polymorphism identification with the PLCO Atlas (n=69,429). The 5-year overall survival (OS) curves of 13 therapeutic and/or significantly altered genes between males and females were analyzed. BRCA2 was the only gene associated with worse survival for females with alterations. Interestingly, males with alterations to the following genes had significantly better OS outcomes: ERCC2, KDM6A, BRCA2, FGFR3. However, there was no gene whose alterations were associated with significantly better OS outcomes for females. KDM6A and FGFR3, two genes whose alterations are typically associated with worse OS outcomes in males, exhibit the opposite effect. Additionally, males exhibited significantly worse OS outcomes when presenting with CDKN2A and CDKN2B alterations, while females exhibited significantly worse OS outcomes when presenting with CDKN2A and NTRK1. A majority of European males had SNPs on the following genes: CDKN2B, ERBB2, CREBBP, while a majority of European females had SNPs on BRCA2. Differential expression also revealed 714 genes that were significantly expressed in one sex when compared to the other. Therefore, we suggest that there are unique molecular differences between sex that conflict with previous analyses; further research should be conducted to explore the effect of sex via the tumor-suppressive characteristics of KDM6A as well as oncogenic properties of FGFR3. Citation Format: Rishi Nair, Roy Khalife, Anthony Magliocco. Genetic characteristics between males and females with urothelial bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 883.

Abstract 4632: Lineage plasticity as a determinant of antibody-drug conjugate target expression in urothelial bladder cancer

Abstract Bladder cancers display a wide spectrum of morphologies that frequently co-exist within individual tumors. Several histologic variants, including plasmacytoid, neuroendocrine, and micropapillary subtypes, are associated with an increased bladder cancer recurrence risk and cancer-specific mortality. The molecular basis and therapeutic implications of this phenotypical plasticity remain poorly understood. Enfortumab vedotin (EV), an antibody-drug conjugate targeting Nectin-4, has also emerged as a new standard-of-care for metastatic bladder cancer patients, but the association between lineage plasticity as manifested by morphologic heterogeneity in bladder cancer, Nectin-4 expression and ADC response remains poorly defined. Previous DNA-based molecular profiling studies of histologic variants revealed that CDH1 mutations were the pathognomonic molecular alteration in plasmacytoid urothelial carcinoma, but no recurrent driver mutations have been found to be unique to the aberrant histology regions of other mixed histology subtypes. Thus, to explore the molecular basis for the lineage plasticity of bladder cancers with aberrant histology and to define the association between lineage plasticity manifested as morphologic heterogeneity and the expression of Nectin-4, HER2 and other potential ADC targets, we performed RNA-seq of pure urothelial carcinomas (n=118, not otherwise specified, UC-NOS) and bladder tumors with divergent differentiation or histologic subtypes (n=199). Our cohort included a subset of tumors in which RNA-seq was performed on microdissected UC-NOS and variant histology regions of the same tumor. Tumors with neuroendocrine, sarcomatoid and squamous histology had the lowest Nectin-4 expression, whereas histologic subtypes that retained a luminal transcriptional profile such as micropapillary, plasmacytoid and nested histology had higher levels of Nectin-4 expression. In mixed histology tumors, Nectin-4 and/or HER2 expression was often restricted to only one of the two histologic components. Gene Set Enrichment Analysis noted enrichment of distinct oncogenic pathway gene sets in different histologic variants. Preliminary investigation into the mechanism(s) of Nectin-4 regulation using bladder cancer cell lines and patient-derived organoids (PDOs) has identified targetable pathways which when inhibited revert lineage plasticity and induce Nectin-4 expression. In sum, lineage plasticity manifested as heterogenous histomorphologies was associated with heterogeneous Nectin-4 expression in some but not all histologic variants of bladder cancer. Ongoing studies using PDOs from bladder cancer patients with mixed histology will seek to identify molecular pathways whose inhibition leads to increased expression of Nectin-4 or other ADC targets such as HER2 and thus increased sensitivity of ADCs targeting these cell surface proteins. Citation Format: Jiaqian Luo, Sizhi P. Gao, Fengshen Kuo, Gamze Gokturk Ozcan, Merve Basar, Florestan Koll, Jacob E. Tallman, Syed M. Alam, Carissa E. Chu, Ziyu Chen, Eugene J. Pietzak, Gopakumar Iyer, Jonathan E. Rosenberg, David B. Solit, Hikmat Al-Ahmadie. Lineage plasticity as a determinant of antibody-drug conjugate target expression in urothelial bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4632.

Abstract 5219: Loss of chromosome Y is unrelated to the composition of the tumor microenvironment and patient prognosis in muscle-invasive urothelial bladder cancers

Abstract Background: Loss of chromosome Y (LOY) occurs commonly in cancers of male patients. It was recently proposed to be associated with cancer aggressiveness and altered T-cell function in bladder cancer and it was found to be linked to poor prognosis in a TCGA bladder cancer patient cohort of 300 patients based on a combined analysis of RNAs from Y-chromosome genes. In this study, we aimed to clarify the clinical relevance of LOY in a large cohort of urothelial bladder cancer patients by using fluorescence in-situ hybridization (FISH). Design: X and Y chromosomes were analyzed by FISH on a tissue microarray (TMA) containing 1,819 urothelial carcinomas of the urinary bladder from male patients including 487 patients who had undergone cystectomy for muscle-invasive disease and for which follow-up data were available. Data of tumor microenvironment parameters were obtained in a previous study. Results: LOY was found in 26.0% of 1,479 analyzable cancers. In the subset of non-invasive cancers, the frequency of LOY increased from pTaG2 (14.5%) to pTG3 (28.6%) but these differences were not significant (p=0.1015). In muscle-invasive cancers, the frequency of LOY increased slightly from pT2 (25.4%) to pT4 cancers (32.8%) but this association was again not significant (p=0.1868). Within pT2-4 cancers, LOY was associated with venous invasion (p=0.0010) but unrelated to pT, pN, and L-status, as well as to overall, recurrence-free, and cancer-specific survival. Muscle-invasive urothelial carcinomas with and without LOY did not show significant differences in the number of CD8 positive lymphocytes, the fraction of CD8 positive intraepithelial lymphocytes, the number of macrophages and dendritic cells, as well as the fraction of t helper and t regulatory cells. Conclusion: FISH is the gold standard for determining LOY. That LOY was neither associated with histopathological parameters of cancer aggressiveness and patient prognosis nor with any parameters describing the tumor microenvironment strongly argues against a driving role of LOY for bladder cancer progression and cancer associated immune reaction. The slight increase of LOY from pTaG2 to pT4 cancers is consistent with a continuous (but clinically irrelevant) loss of the dispensable Y chromosomes during tumor evolution over time. Citation Format: Martina Kluth, Henning Plage, Sebastian Hofbauer, Kira Furlano, Sarah Weinberg, Antonia Franz, Annika Fendler, Florian Roßner, Simon Schallenberg, Sefer Elezkurtaj, Niclas C. Blessin, Maximilian Lennartz, Andreas H. Marx, Henrik Samtleben, Margit Fisch, Michael Rink, Marcin Slojewski, Krystian Kaczmarek, Thorsten Ecke, Stefan Koch, Nico Adamini, Ronald Simon, Guido Sauter, Joachim Weischenfeldt, Tobias Klatte, Sarah Minner, David Horst, Thorsten Schlomm, Dr. med Henrik Zecha Zecha. Loss of chromosome Y is unrelated to the composition of the tumor microenvironment and patient prognosis in muscle-invasive urothelial bladder cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5219.

Abstract 5220: Reduced expression of Glutathione peroxidase 2 (GPX2) is an independent predictor of poor prognosis in muscle-invasive urothelial carcinoma of the bladder

Abstract Background: The Glutathione peroxidase 2 (GPX2) is a selenium-dependent glutathione peroxidase with a pivotal role in removing potentially harming reactive oxygen species (ROS) from cells by catalyzing the reduction process of hydrogen peroxide to water. GPX2 is strongly expressed in normal urothelium but can be reduced in urothelial cancer. The aim of the study was to evaluate the potential clinical significance of GPX2 expression in urothelial bladder cancer. Design: GPX2 was analyzed by immunohistochemistry (IHC) in more than 2,500 urothelial bladder carcinomas in a tissue microarray format. Follow-up data were available for more than 600 patients with muscle-invasive disease. Results: GPX2 staining occurred in 67.5% of urothelial carcinomas including 11.4% with weak, 11.9% with moderate, and 44.2% with strong staining. While GPX2 staining was always strong in normal urothelium, it decreased markedly during grade and stage progression. Within pTa tumors, GPX2 positivity continuously decreased from pTaG2 low grade (99.4% positive) to pTa G2 high grade (97.2% positive) and to pTa G3 (90.5%; p=0.0013) while there was a marked further drop of GPX2 positivity from pTaG3 to muscle-invasive cancers (56.6%; p&amp;lt;0.0001 for pT2-4 vs. pTa G3). Within pT2-4 carcinomas, low or absent GPX2 staining was significantly linked to poor overall survival (p=0.0140), V1 (p&amp;lt;0.0001), L1 (p=0.0014), and high grade (p=0.0091), but statistically unrelated to pT and pN. In a multivariate analysis, the prognostic impact of reduced GPX2 expression (p=0.0081) was independent of pT (p&amp;lt;0.0001) and pN (p&amp;lt;0.0001) in pT2-4 tumors. GPX2 expression loss was inversely related to the expression of the luminal subtype markers GATA3 (p&amp;lt;0.0001), uroplakin 1b (p&amp;lt;0.0001) and cytokeratin 20 (p&amp;lt;0.0001) in pT2-4 carcinomas. Conclusion: GPX2 expression loss is a common event during bladder cancer progression which is strongly associated with poor prognosis in patients with pT2-4 urothelial carcinomas. GPX2 expression measurement - optimally together with other markers - could be useful for clinical risk assessment of bladder cancer patients. Citation Format: Henning Plage, Sebastian Hofbauer, Kira Furlano, Sarah Weinberger, Antonia Franz, Annika Fendler, Joachim Weischenfeldt, Florian Roßner, Simon Schallenberg, Sefer Elezkurtaj, Martina Kluth, Andreas H. Marx, Henrik Samtleben, Margit Fisch, Marcin Slojewski, Krystian Kaczmarek, Thorsten Ecke, Stefan Koch, Nico Adamini, Ronald Simon, Guido Sauter, Henrik Zecha, Tobias Klatte, Sarah Minner, David Horst, Thorsten Schlomm, Michael Rink, Maximilian Lennartz. Reduced expression of Glutathione peroxidase 2 (GPX2) is an independent predictor of poor prognosis in muscle-invasive urothelial carcinoma of the bladder [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5220.