High-grade Muscle-invasive Bladder Cancer Research Articles

Transcriptome Analysis Reveals Anti-Cancer Effects of Isorhapontigenin (ISO) on Highly Invasive Human T24 Bladder Cancer Cells.

Bladder cancer, the most common malignancy of the urinary tract, has a poor overall survival rate when the tumor becomes muscle invasive. The discovery and evaluation of new alternative medications targeting high-grade muscle invasive bladder cancer (MIBC) are of tremendous importance in reducing bladder cancer mortality. Isorhapontigenin (ISO), a stilbene derivative from the Chinese herb Gnetum cleistostachyum, exhibits a strong anti-cancer effect on MIBCs. Here, we report the whole transcriptome profiling of ISO-treated human bladder cancer T24 cells. A total of 1047 differentially expressed genes (DEGs) were identified, including 596 downregulated and 451 upregulated genes. Functional annotation and pathway analysis revealed that ISO treatment induced massive changes in gene expression associated with cell movement, migration, invasion, metabolism, proliferation, and angiogenesis. Additionally, ISO treatment-activated genes involved in the inflammatory response but repressed genes involved in hypoxia signaling, glycolysis, the actin cytoskeleton, and the tumor microenvironment. In summary, our whole transcriptome analysis demonstrated a shift in metabolism and altered actin cytoskeleton in ISO-treated T24 cells, which subsequently contribute to tumor microenvironment remodeling that suppresses tumor growth and progression.

Artificial Intelligence Reveals Distinct Prognostic Subgroups of Muscle-Invasive Bladder Cancer on Histology Images.

Muscle-invasive bladder cancer (MIBC) is a highly heterogeneous and costly disease with significant morbidity and mortality. Understanding tumor histopathology leads to tailored therapies and improved outcomes. In this study, we employed a weakly supervised learning and neural architecture search to develop a data-driven scoring system. This system aimed to capture prognostic histopathological patterns observed in H&E-stained whole-slide images. We constructed and externally validated our scoring system using multi-institutional datasets with 653 whole-slide images. Additionally, we explored the association between our scoring system, seven histopathological features, and 126 molecular signatures. Through our analysis, we identified two distinct risk groups with varying prognoses, reflecting inherent differences in histopathological and molecular subtypes. The adjusted hazard ratio for overall mortality was 1.46 (95% CI 1.05-2.02; z: 2.23; p = 0.03), thus identifying two prognostic subgroups in high-grade MIBC. Furthermore, we observed an association between our novel digital biomarker and the squamous phenotype, subtypes of miRNA, mRNA, long non-coding RNA, DNA hypomethylation, and several gene mutations, including FGFR3 in MIBC. Our findings underscore the risk of confounding bias when reducing the complex biological and clinical behavior of tumors to a single mutation. Histopathological changes can only be fully captured through comprehensive multi-omics profiles. The introduction of our scoring system has the potential to enhance daily clinical decision making for MIBC. It facilitates shared decision making by offering comprehensive and precise risk stratification, treatment planning, and cost-effective preselection for expensive molecular characterization.

BTA stat®, NMP22® BladderChek®, UBC® Rapid Test, and CancerCheck® UBC® rapid VISUAL as urinary marker for bladder cancer: Final results of a German multicenter study

Introduction and objectiveBTA stat®, NMP22® BladderChek®, UBC® Rapid Test, and CancerCheck® UBC® rapid VISUAL are urinary-based rapid tests. This multicenter study is the first study comparing all available rapid tests on a large cohort of bladder cancer patients and healthy controls in one setting. MethodsIn total 732 urine samples (second morning urine) in a real-world assessment have been analyzed. We evaluated clinical samples from 464 patients with histologically confirmed urothelial tumors of the urinary bladder (17 solitary CIS, 189 low-grade, 187 high-grade nonmuscle invasive, 71 high-grade muscle invasive), 77 patients with No Evidence of Disease (NED), and from 191 healthy controls. Urine samples were analyzed by the BTA stat®, NMP22® BladderChek®, UBC® Rapid Test point-of-care (POC) system using the concile Omega 100 POC reader, and CancerCheck® UBC® rapid VISUAL. Sensitivities and specificities were calculated by contingency analyses. ResultsAll investigated urinary markers detected more pathological concentrations in urine of bladder cancer patients compared to tumor-free patients. The calculated diagnostic sensitivities for BTA stat®, NMP22® BladderChek®, UBC® Rapid Test, CancerCheck® UBC® rapid VISUAL, and cytology were 62.4%, 13.4%, 58.2%, 28.6%, 36.2% for low-grade, 83.4%, 49.5%, 84.5%, 63.1%, 71.2% for high-grade nonmuscle invasive, and 95.8%, 35.2%, 76.1%, 50.7%, 67.7% for high-grade muscle-invasive bladder cancer. The specificity was 67.9%, 95.5%, 79.4%, 94.4%, and 83.7%, respectively. The area under the curve (AUC) after receiver operating characteristics (ROC) analysis for high-grade non–muscle-invasive tumors was 0.757, 0.725, 0.819, 0.787, and 0.774, respectively. ConclusionsThe analysis of more than 700 urine samples offers an objective view on urine-based rapid diagnostics. Elevated pathological concentrations of markers in urine of bladder cancer patients were detected in all investigated tests. The highest sensitivities for high-grade non–muscle-invasive tumors were calculated for BTA stat® and UBC® Rapid Test, whereas NMP22® BladderChek®, and cytology showed the highest specificities. BTA stat® and UBC® Rapid Test have the potential to be used as a clinical valuable urinary protein biomarker for the detection of high-grade non–muscle-invasive bladder cancer patients and could be included in the management of these tumors.

The preliminary efficacy of "quadri-combination" therapy on the treatment of bladder cancer

To investigate the safety and efficacy of "quadri-combination" therapy including maximal transurethral resection of bladder tumor (mTURBT), combined with systemic chemotherapy and immunotherapy, concurrent radiotherapy, and immune maintenance therapy. The clinical data of 8 patients with bladder cancer who could not tolerate or refused radical cystectomy at the Department of Urology, Peking University Cancer Hospital from November 2019 to October 2021 were retrospectively analyzed. There were 5 males and 3 females with a mean age of 69 years. The Eastern Cooperative Oncology Group(ECOG) score was 0 in 6 cases and 1 in 2 cases. There were 5 cases of high-grade urothelial carcinoma (1 case of T3b; 2 cases were T2; 2 cases of T1 stage, with multiple tumors and repeated recurrence), 1 case of high-grade urothelial carcinoma with carcinoma in situ (T1/Tis stage), 1 case of high-grade urothelial carcinoma with squamous differentiation (T3b stage), and 1 case of high-grade urothelial carcinoma with glandular differentiation (T2). All patients underwent "quadri-combination" therapy.The patient's tolerance, success rate of bladder preservation and prognosis were evaluated.The median follow-up time was 22.5 (12-35) months. One patient with high-grade muscle-invasive bladder cancer (T2) received mTURBT, albumin-bound paclitaxel and durvalumab combined therapy for 3 cycles, concurrent radiotherapy, and immune maintenance therapy for 18 months, and the tumor recurrence was found. The pathology was high-grade urothelial carcinoma. Salvage radical cystectomy combined with pelvic lymph node dissection is recommended. The remaining 7 patients were regularly reexamined, and no recurrence or metastasis was found.The 2-year progression-free survival rate was 80%, and the success rate of bladder preservation was 87.5%(7/8). Treatment-related adverse reactions were resolved by symptomatic treatment, and patients' compliance and tolerance were acceptable.The "quadri-combination" bladder-preserving therapy is feasible and well tolerated, but further studies are needed.

Abstract 6032: Establishment of a bladder cancer model using organoids derived from bladder epithelium in genetically engineered mice

Abstract Despite recent advances in molecular profiling of bladder cancer, molecular mechanisms for urothelial carcinogenesis have not fully understood. It is partly because the lack of faithful disease models that accurately recapitulate human bladder cancer. In our previous study, we have analyzed mutation landscape of chemical-induced mouse bladder cancer and human bladder cancer by exome-sequence and found that some components of histone methyltransferase complex like Kmt2c, Kmt2d, Kdm6a were highly prevalent as common mutations and associated with Trp53. Additionally, the downregulation of tumor suppressor gene PTEN is known in human muscle invasive bladder cancer (MIBC). We also investigated cell of origin of chemical-induced mouse bladder cancer using lineage tracing method and found that Krt5 expressing cells with Trp53 alteration efficiently give a rise of high-grade MIBC with squamous differentiation. Based on these results, we have tried to make MIBC mouse model using genetically engineered mouse in which Cas9 and target gene mutation express specifically in Krt5 expressing cells by Cre-LoxP system, combined with CRISPR/Cas9 gene editing technique and 3D organoid culture system. Eight-week-old mice harboring Krt5CreERT2/+; LSL-Cas9; LSL-Trp53+/R172H were used in this study. After introducing gene recombination by administration of tamoxifen, we generated organoids from bladder epithelial cells. For further gene editing, we infected adeno-associated virus with sgPten and sgMll3 to the organoids, which successfully edited the target genes. There seemed to be no significant differences in growth of the organoids in culture between the wild-type and gene-edited organoids. However, gene-edited organoids showed tumorigenesis in vivo when inoculated in subcutaneous space, renal subcapsular space or bladder submucosa of athymic mice, whereas wild-type organoids did not show tumorigenesis in vivo. Histologically, the organoid-derived tumors showed morphological similarity to human urothelial carcinoma with squamous differentiation and intra-tumoral heterogeneity. Furthermore, tumor derived organoid has a potential of tumor development in immune-competent mice. These tumors also maintain squamous phenotype and tumor heterogeneity. These findings suggest that the technique is a promising approach toward the establishment of a useful model of human bladder cancer for the elucidation of bladder carcinogenesis including driver genes. In addition, we think our syngeneic mouse model may be useful tool for further research of tumor immunity and immune checkpoint inhibitor drugs. Citation Format: Akihiro Hamada, Yuki Kita, Hideaki Takada, Kenji Nakamura, Toru Sakatani, Takeshi Sano, Takayuki Goto, Atsuro Sawada, Shusuke Akamatsu, Takashi Kobayashi. Establishment of a bladder cancer model using organoids derived from bladder epithelium in genetically engineered mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6032.

Hsa-miR-30a-3p overcomes the acquired protective autophagy of bladder cancer in chemotherapy and suppresses tumor growth and muscle invasion

Bladder cancer (BC) is the second most common urologic cancer in western countries. New strategies for managing high-grade muscle-invasive bladder cancer (MIBC) are urgently required because MIBC has a high risk of recurrence and poor survival. A growing body of evidence indicates that microRNA has potent antitumorigenic properties in various cancers, and thus, therapeutic strategies based on microRNA may show promising results in cancer therapy. Analysis of The Cancer Genome Atlas (TCGA) database indicated that hsa-miR-30a-3p is downregulated in human BC. Our in vitro investigation demonstrated that hsa-miR-30a-3p suppresses the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 and reduces the cell invasive potential of BC cells. Furthermore, hsa-miR-30a-3p directly targets ATG5, ATG12, and Beclin 1; this in turn improves the chemosensitivity of BC cells to cisplatin through the repression of protective autophagy. In a tumor-xenograft mice model, hsa-miR-30a-3p suppressed muscle invasion. Cotreatment with hsa-miR-30a-3p enhanced the antitumor effect of cisplatin in reducing tumor growth in BC. The current study provides a novel strategy of using hsa-miR-30a-3p as an adjuvant or replacement therapy in future BC treatment.

Fibroblast growth factor receptor 3 gene (FGFR3) mutations in high-grade muscle-invasive urothelial bladder cancer in a Brazilian population: evaluation and prevalence.

ABSTRACTObjective To understand the feasibility of FGFR3 tests in the Brazilian public health context, and to sample the mutational burden of this receptor in high-grade muscle invasive bladder cancer.Methods A total of 31 patients with high-grade muscle-invasive bladder cancer were included in the present study. Either transurethral resection of bladder tumor or radical cystectomy specimens were analyzed. Formalin-fixed paraffin-embedded tissue blocks were sectioned, hematoxylin and eosin stained, and histologic sections were reviewed. Total RNA was extracted using the RNeasy DSP formalin-fixed paraffin-embedded kit. Qualitative results were displayed in Rotor-Gene AssayManager software.Results Six patients were excluded. From the samples analyzed, four (16.7%) were considered inadequate and could not have their RNA extracted. Two patients presented FGFR3 mutations, accounting for 9.5% of material available for adequate analysis. The two mutations detected included a Y373C mutation in a male patient and a S249C mutation in a female patient.Conclusion FGFR3 mutations could be analyzed in 84% of our cohort and occurred in 9.5% of patients with high-grade muscle invasive bladder cancer in this Brazilian population. FGFR3 gene mutations are targets for therapeutic drugs in muscle-invasive bladder cancer. For this reason, know the frequency of these mutations can have a significant impact on public health policies and costs provisioning.

Upregulation of IFN\u0263-mediated chemokines dominate the immune transcriptome of muscle-invasive urothelial carcinoma

Tumor inflammation is prognostically significant in high-grade muscle-invasive bladder cancer (MIBC). However, the underlying mechanisms remain elusive. To identify inflammation-associated immune gene expression patterns, we performed transcriptomic profiling of 40 MIBC archival tumors using the NanoString nCounter Human v.1.1 PanCancer Panel. Findings were validated using the TCGA MIBC dataset. Unsupervised and supervised clustering identified a distinctive immune-related gene expression profile for inflammation, characterized by significant upregulation of 149 genes, particularly chemokines, a subset of which also had potential prognostic utility. Some of the most enriched biological processes were lymphocyte activation and proliferation, leukocyte adhesion and migration, antigen processing and presentation and cellular response to IFN-γ. Upregulation of numerous IFN-γ-inducible chemokines, class II MHC molecules and immune checkpoint genes was detected as part of the complex immune response to MIBC. Further, B-cell markers linked to tertiary lymphoid structures were upregulated, which in turn is predictive of tumor response to immunotherapy and favorable outcome. Our findings of both an overall activated immune profıle and immunosuppressive microenvironment provide novel insights into the complex immune milieu of MIBC with inflammation and supports its clinical significance for predicting prognosis and immunotherapeutic responsiveness, which warrants further investigation. This may open novel opportunities to identify mechanisms for developing new immunotherapeutic strategies.

Racial differences in aneuploidy in high-grade muscle-invasive bladder cancer.

400 Background: Bladder cancer is marked by racial disparities in stage at presentation, treatment, and survival. It is unknown how somatic tumor genomes differ by race. Tumor aneuploidy, defined as aberrant counts of chromosome arms, is pervasive in tumors including bladder cancer. Its associations with race are incompletely understood. We sought here to characterize the relation of race with aneuploidy in high-grade muscle-invasive bladder cancer. Methods: To quantify how aneuploidy differs by race, we leveraged the cohort of patients with high-grade, muscle-invasive bladder cancer from The Cancer Genome Atlas. Chromosome arm gains and losses were identified based on Affymetrix SNP 6.0 arrays (Taylor 2018). We focused on aneuploidy burden defined as the count of chromosome arms altered by gains and losses per tumor, which was based on both p and q chromosome arms of all autosomes except for acrocentric autosomes (q arms only) and ranged from 0 to 34 altered chromosome arms. We used multivariable linear regression to obtain mean differences and 95% confidence intervals (CIs) in the number of altered chromosome arms between self-reported racial groups, adjusting for demographics (sex, age at diagnosis, smoking status) and tumor characteristics (grade, histology). We also evaluated associations between race and number of altered chromosome arms stratified by papillary/non-papillary histology and sex. Results: Of 362 participants, 315 self-identified as White (87%), 25 as Asian (7%), and 22 as Black (6%). 73% were men, 21% were current smokers, and 52% former smokers. Median age at diagnosis was 69 years (interquartile range [IQR]: 61 to 76). Asians and Blacks tended to be younger at diagnosis than Whites, with more never-smokers among Asians. More profiled tumors were of papillary histology among Asians (44%), and fewer among Blacks (18%), than among Whites (30%). Aneuploidy burden was high overall (median, 14 altered chromosome arms; IQR: 8 to 19); only 17% of tumors had five or fewer altered chromosome arms. Compared to Whites, aneuploidy burden overall was similar in tumors from Asians (adjusted mean difference, –2.3 fewer altered chromosome arms; 95% CI: –5.4 to 0.9) and Blacks (–1.7; 95% CI: –5.1 to 1.7). Asian race appeared more strongly associated with lower aneuploidy burden in non-papillary tumors (–3.9 altered chromosome arms; 95% CI: –7.7 to –0.1) than in papillary tumors (–0.3; 95% CI: –5.6 to 5.0), and in men (–3.5; 95% CI: –7.0 to 0.1) than in women (–0.8; 95% CI: –6.8 to 5.2). Conclusions: These findings suggest potential differences in aneuploidy burden between Asians and Whites among high-grade muscle-invasive bladder cancers. They also highlight a need for validation in cohorts that are more racially diverse and have a well-defined source population (study base) with detailed data on cancer risk factors and histopathology.

Abstract A13: Entinostat enhances antitumor immune responses and cooperates with PD-1 blockade in bladder cancer mouse models

Abstract Bladder cancer harbors a high frequency of somatic mutations that occur randomly across the genome. A fraction of these mutations can be expressed and processed into peptides called neoantigens. When presented on the surface of tumor cells by the major histocompatibility complexes (MHC), these neoantigens can invoke an adaptive immune response. In general, a high neoantigen burden is associated with a significant increase in overall survival. However, tumor cells can avoid the detection of immune cells by silencing the expression of the neoantigens. The post-translational acetylation of histone tails is catalyzed by histone acetyltransferases (HATs) and is counterbalanced by histone deacetylases (HDACs), which mediate their removal. Histone acetylation is associated with open chromatin and active transcription. HDAC inhibition would, therefore, promote histone acetylation and restore the expression of suppressed genes. Many of the frequently mutated genes in bladder cancer are involved in chromatin remodeling, such as KMT2D, KDM6A, and KMT2C, suggesting the contribution of epigenetic dysregulation to disease progression. Therefore, therapy that targets epigenetic processes such as HDAC inhibition is a rational treatment approach for bladder cancer. Here, we evaluated the antitumor efficacy and mechanisms of entinostat, a selective HDAC1 and HDAC3 inhibitor, in a preclinical murine model of high-grade muscle-invasive bladder cancer, BBN963. In this study, BBN963 cells were implanted subcutaneously into immunocompetent C57BL/6 (B6) mice or immunodeficient NOD/scid/gamma (NSG) mice. Entinostat exhibited robust in vivo activity in B6 mice implanted with BBN963 cells with a number of complete responses. However, the effect was much weaker in BBN963 cells grown in NSG mice. RNAseq analysis demonstrated increased immune gene signature expression in entinostat treated tumors from B6 (but not NSG) mice. Flow cytometry verified the increased numbers of CD8+ effector memory T cells. Most interestingly, the expressions of predicted neoantigens were almost completely lost in the entinostat-treated tumors in B6 mice but were unchanged in tumors from NSG mice. Additionally, the in vivo antitumor effect of entinostat in the BBN963 cells lacking MHC class 1 was significantly reduced. Finally, the combination of anti-PD1 therapy and entinostat robustly out-performed each agent alone in inhibiting BBN963 tumor growth with 67% incident of complete response. These data support the hypothesis that entinostat mediates antitumor effect through enhanced neoantigen expression and antigen-driven immune response. Citation Format: Andrew S. Truong, Mi Zhou, Takanobu Utsumi, Bhavani Krishnan, Kyle G. Stewart, Ryoichi Saito, Ujjawal Manocha, Sean T. Bailey, William Y. Kim. Entinostat enhances antitumor immune responses and cooperates with PD-1 blockade in bladder cancer mouse models [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2019 May 18-21; Denver, CO. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(15_Suppl):Abstract nr A13.

Abstract 462: Results from multiple datasets including the TCGA reveal limited clinical significance of molecular subtypes in bladder cancer

Abstract INTRODUCTION AND OBJECTIVE: Molecular subtypes have been suggested to predict outcome in patients with muscle invasive bladder cancer (MIBC). The major subtypes reported have been “luminal” and “basal”; however, there is little consensus regarding subtype-signatures and the clinical parameters used to evaluate outcome. The objective of this study was to examine using multiple datasets, if molecular subtypes are distinct entities within MIBC and whether they predict clinical outcome. METHODS: We analyzed transcriptome data from all 402 MIBC samples in The Cancer Genome Atlas (TCGA) and 151 high-grade MIBC samples from three datasets in Oncomine™. Transcript levels were also measured in 52 bladder tumor specimens with clinical follow-up (cohort-1). Samples were subtype-scored using gene panels (GP-11, GP-30, BASQ) consisting only of those genes common among published studies. Correlation of subtypes to recurrence/progression-free survival (R/PFS), metastasis, cancer-specific survival (CSS) and overall-survival (OS) was examined by logistic regression, Cox proportional Hazards model and Kaplan-Meier analyses. RESULTS: Based on GP-11 subtype-scoring, >75% of the MIBC TCGA-dataset samples were mixed, i.e. neither pure-luminal nor pure-basal. When tumors were categorized as pure-luminal, pure-basal, or mixed, the subtypes could not predict OS or R/PFS. Consistent with published studies, when tumors were categorized as luminal or basal the subtypes predicted OS (P=0.051); sensitivity: 53.9%; specificity: 62%. Subtypes also correlated with tumor-grade (P=0.0005); most low-grade MIBC cases (16/21) in the TCGA-dataset were luminal. However, MIBC is rarely low-grade and subtypes could not predict OS (P=0.131) when only high-grade cases were included. Subtypes were not significant prognosticators in multivariate analyses. GP-30 and BASQ panels validated these results. In the Oncomine-dataset and cohort-1, subtypes could not predict metastasis, CSS, or OS. CONCULUSIONS: Our study of multiple datasets reveals that molecular subtypes may reflect tumor-heterogeneity but are likely not distinct entities within MIBC. Furthermore, molecular subtypes have limited prognostic capability for MIBC patients. Although there is a need to individualize patient care, further examination into the molecular subtypes of MIBC is needed before their incorporation into clinical practice. Citation Format: Sarrah S. Lahorewala, Daley S. Morera, Jiaojiao Wang, Vinata B. Lokeshwar. Results from multiple datasets including the TCGA reveal limited clinical significance of molecular subtypes in bladder cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 462.

Stem-Like Signature Predicting Disease Progression in Early Stage Bladder Cancer. The Role of E2F3 and SOX4.

The rapid development of the cancer stem cells (CSC) field, together with powerful genome-wide screening techniques, have provided the basis for the development of future alternative and reliable therapies aimed at targeting tumor-initiating cell populations. Urothelial bladder cancer stem cells (BCSCs) that were identified for the first time in 2009 are heterogenous and originate from multiple cell types; including urothelial stem cells and differentiated cell types—basal, intermediate stratum and umbrella cells Some studies hypothesize that BCSCs do not necessarily arise from normal stem cells but might derive from differentiated progenies following mutational insults and acquisition of tumorigenic properties. Conversely, there is data that normal bladder tissues can generate CSCs through mutations. Prognostic risk stratification by identification of predictive markers is of major importance in the management of urothelial cell carcinoma (UCC) patients. Several stem cell markers have been linked to recurrence or progression. The CD44v8-10 to standard CD44-ratio (total ratio of all CD44 alternative splicing isoforms) in urothelial cancer has been shown to be closely associated with tumor progression and aggressiveness. ALDH1, has also been reported to be associated with BCSCs and a worse prognosis in a large number of studies. UCC include low-grade and high-grade non-muscle invasive bladder cancer (NMIBC) and high-grade muscle invasive bladder cancer (MIBC). Important genetic defects characterize the distinct pathways in each one of the stages and probably grades. As an example, amplification of chromosome 6p22 is one of the most frequent changes seen in MIBC and might act as an early event in tumor progression. Interestingly, among NMIBC there is a much higher rate of amplification in high-grade NMIBC compared to low grade NMIBC. CDKAL1, E2F3 and SOX4 are highly expressed in patients with the chromosomal 6p22 amplification aside from other six well known genes (ID4, MBOAT1, LINC00340, PRL, and HDGFL1). Based on that, SOX4, E2F3 or 6q22.3 amplifications might represent potential targets in this tumor type. Focusing more in SOX4, it seems to exert its critical regulatory functions upstream of the Snail, Zeb, and Twist family of transcriptional inducers of EMT (epithelial–mesenchymal transition), but without directly affecting their expression as seen in several cell lines of the Cancer Cell Line Encyclopedia (CCLE) project. SOX4 gene expression correlates with advanced cancer stages and poor survival rate in bladder cancer, supporting a potential role as a regulator of the bladder CSC properties. SOX4 might serve as a biomarker of the aggressive phenotype, also underlying progression from NMIBC to MIBC. The amplicon in chromosome 6 contains SOX4 and E2F3 and is frequently found amplified in bladder cancer. These genes/amplicons might be a potential target for therapy. As an existing hypothesis is that chromatin deregulation through enhancers or super-enhancers might be the underlying mechanism responsible of this deregulation, a potential way to target these transcription factors could be through epigenetic modifiers.

Abstract 2748: Antitumor activity of HDAC inhibition in bladder cancer mouse models correlates with enhanced immune response

Abstract Aberrant chromatin remodeling by epigenetic modifier proteins such as histone deacetylases (HDACs) is common within many types of cancer, including muscle-invasive bladder cancer (MIBC). The removal of acetyl groups from the lysine residues of histones leads to transcriptional silencing that promotes tumor growth and potentially enhances cancer cells' ability to evade the host immune response. Therefore, HDACs present themselves as attractive targets for cancer therapy. Histone deacetylase inhibitors (HDACi) demonstrate broad anti-cancer activity. Many proposed mechanisms exist to explain their effects, including those that involve the immune system. Here, we evaluated the efficacy and transcriptional effects of entinostat, a selective class 1 HDAC inhibitor (HDAC1 and HDAC3), in a novel preclinical murine model of high-grade MIBC. BBN963 cells were derived from a primary bladder cancer GEM, cultured, and implanted subcutaneously into immunocompetent C57BL/6 mice and immunodeficient NOD scid gamma (NSG) mice. Animals were randomized to control or entinostat treatment. Tumor volume was recorded weekly and fresh tissue collected for RNAseq and Ingenuity Pathway Analysis (IPA). Entinostat exhibited in vivo activity in the BBN963 model in both C57BL/6 and NSG mice, however the anti-tumor response in B6 mice was significantly greater than the observed response in NSG mice. RNAseq analysis on tumor tissue collected from NSG mice indicated that entinostat treated tumors had distinct gene expression changes. Comparison of control to entinostat treated tumors revealed 4987 genes were significantly upregulated and 4112 genes were significantly downregulated (p-value < 0.05). IPA contextualized these changes as an enhanced inflammatory response, as indicated by increased expression of upstream regulators TGFβ, LPS, and IFNγ. Additionally, hierarchical clustering using established immune gene signatures stratified entinostat treated and untreated tumors into two distinct groups. Our results suggest that the anti-cancer properties of entinostat are in part immunologically mediated. BBN963 tumors in B6 mice displayed a more robust therapeutic response compared to their NSG counterparts. Transcriptional analysis of the tumor tissue indicated significant differences in immune gene signature levels as well as inflammatory pathway activation. Therefore, an intact immune system appears critical to achieve a maximal therapeutic response observed with HDAC inhibition. Together these results lay the foundation for further elucidating entinostat's mechanism of action in the context of high-grade MIBC. Citation Format: Kyle G. Stewart, Andrew S. Truong, Bhavani Krishnan, Mi Zhou, Ryoichi Saito, Jordan Kardos, Ujjawal Manocha, Sean T. Bailey, William Y. Kim. Antitumor activity of HDAC inhibition in bladder cancer mouse models correlates with enhanced immune response [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2748.

Curative radiation therapy for very elderly bladder cancer patients with localized disease.

To report the outcomes of a cohort of very elderly muscle-invasive bladder cancer (MIBC) patients treated with contemporary 3D-conformal radiation therapy (3D-CRT) with or without concurrent chemotherapy, after transurethral resection of bladder tumor (TURBT). From February 2010 to January 2014, a total of 41 patients older than 75years, with T2-3 N0-1 high-grade MIBC, a Karnofsky index (KI) of at least 90% and/or a Barthel scale score of at least 95, were treated with TURBT followed by radiotherapy (RT) with or without chemotherapy, and were prospectively followed-up. The mean age of patients was 82years (range 76-88). Median follow-up was 47months for surviving patients. Mean Charlson Comorbidity Index (CCI) score was 5 points. 28 patients (68.29%) were T2N0. All received 3D-CRT to a mean dose of 60Gy (range 48.6-66Gy), and chemotherapy was delivered to 34 patients (83%). Cause-specific survival (CSS) was 86 and 78.8% at 1 and 5years, respectively. Patients achieving a complete response lived longer (48 vs 14m, p=0.036) than those with a progressive disease, who were more likely to die from cancer than from other causes (HR 3.865, IC95% 1.562-9.562). Dead patients had a longest treatment time (mean 56.78 vs 48.91days, p=0.019) than survivors. RT with contemporary 3D-CRT techniques after TURBT for MIBC in elderly patients is feasible and well-tolerated. Achieving a maximal response and shortening the total radiation treatment time may improve outcomes and quality of life.

Prognostic and Predictive Value of O6-methylguanine Methyltransferase for Chemotherapy in Patients with Muscle-Invasive Bladder Cancer.

DNA repair genes are potential biomarkers for chemotherapy in muscle-invasive bladder cancer (MIBC). O6-methylguanine methyltransferase (MGMT) is involved in DNA repair and is found to affect the efficacy of platinum-based chemotherapy. However, the prognostic or predictive value of MGMT expression in chemotherapy for MIBC is unknown. Immunohistochemical staining for MGMT was performed in paraffin-embedded tumor tissue of high-grade MIBC patients who underwent cystectomy in two independent cohorts [n=74 for Fudan University Shanghai Cancer Center (FUSCC) cohort and n=115 for Zhongshan Hospital (ZS) cohort]. MGMT messenger RNA (mRNA) analysis was conducted using patients' clinical and fragments per kilobase of exon model per million mapped fragments mRNA data from The Cancer Genome Atlas (TCGA) database (n=245). In our cohorts, high MGMT expression was significantly correlated with shorter overall survival (OS) in patients with platinum-based adjuvant chemotherapy [hazard ratio (HR) 2.386, p=0.048; HR 2.920, p=0.007; HR 2.324, p=0.004, respectively, in FUSCC, ZS, and combination sets], but not in patients without chemotherapy. These findings were corroborated by the TCGA set (HR 1.952 and 0.697 for patients with and without chemotherapy, respectively). The chemotherapy-MGMT interaction for OS was significant in both the surgery set (p=0.045) and TCGA set (p=0.034). We demonstrated that high MGMT expression is an independent poor prognostic factor in MIBC patients with platinum-based adjuvant chemotherapy, but not in patients without chemotherapy. MGMT expression may be a potential predictor for administration of adjuvant chemotherapy.

Robotic Radical Cystectomy and Extended Pelvic Lymph Node Dissection in a Female Patient

Abstract Introduction: Open radical cystectomy remains the standard of care in muscle invasive bladder cancer (MIBC). However, in recent years, robotic radical cystectomy has gained in popularity and shown acceptable long-term survival outcomes in a multi-institutional series.1 Female robotic radical cystectomy presents unique challenges to the urologist, particularly in those unfamiliar with female pelvic anatomy. Reproductive organ preservation may improve sexual function and quality-of-life outcomes postoperatively.2 Herein, we will highlight our procedure for robotic radical cystectomy and extended pelvic lymph node dissection in a female patient, with emphasis on the extirpative component. Materials and Methods: A 52-year-old, otherwise healthy, female with high-grade MIBC, status post-neoadjuvant chemotherapy, underwent robotic radical cystectomy with intracorporeal neobladder urinary diversion and extended pelvic lymphadenectomy. Standard six trocar configuration is utilized, with an additional 15 ...

Overexpression of long non-coding RNA TUG1 predicts poor prognosis and promotes cancer cell proliferation and migration in high-grade muscle-invasive bladder cancer.

Long non-coding RNA TUG1 is involved in the development and progression of a variety of tumors. Little is known about TUG1 function in high-grade muscle-invasive bladder cancer (MIBC). The aims of our study were to determine expression levels of long non-coding RNA TUG1 in tumor tissue, to evaluate its relationship with clinico-pathological features of high-grade MIBC, and to describe its function in MIBC cells in vitro. TUG1 expression levels were determined in paired tumor and adjacent non-tumor bladder tissues of 47 patients with high-grade MIBC using real-time PCR. Cell line T-24 and siRNA silencing were used to study the TUG1 function in vitro. We observed significantly increased levels of TUG1 in tumor tissue in comparison to adjacent non-tumor bladder tissue (P<0.0001). TUG1 levels were significantly increased in metastatic tumors (P=0.0147) and were associated with shorter overall survival of MIBC patients (P=0.0241). TUG1 silencing in vitro led to 34% decrease in cancer cell proliferation (P=0.0004) and 23% reduction in migration capacity of cancer cells (P<0.0001). We did not observe any significant effects of TUG1 silencing on cell cycle distribution and number of apoptotic cells. Our study confirmed overexpression of TUG1 in MIBC tumor tissue and described its association with worse overall survival in high-grade MIBC patients. Together with in vitro observations, these data suggest an oncogenic role of TUG1 and its potential usage as biomarker or therapeutic target in MIBC.

Experience with Sequential Intravesical Gemcitabine and Docetaxel as Salvage Therapy for Non-Muscle Invasive Bladder Cancer.

Patients with high-grade muscle invasive bladder cancer (NMIBC) receive intravesical therapy with bacillus Calmette-Guérin (BCG) as the well-established standard-of-care. However, even with prompt induction of intravesical therapy, approximately 40 % of patients will recur within 2 years. For patients who fail BCG, options include radical cystectomy, repeat BCG therapy, or alternative intravesical salvage therapy. In this review, we will discuss the most recent published evidence on salvage intravesical therapy with an emphasis on a more in-depth report of our therapeutic strategy with sequential gemcitabine and docetaxel intravesical therapy for this treatment-refractory population. In addition, we will provide practical advice on our approach to this challenging patient population including the use of operative staging to aid early identification of treatment failures.

Open radical cystectomy: still the gold standard for muscle invasive bladder cancer.

Muscle invasive bladder cancer is an unforgiving disease, and if untreated, it leads to death within 2years of the diagnosis in >85% of the patients. Long-term oncologic efficacy remains the ultimate standard that all procedures have to be measured by. In the past decades, open radical cystectomy (RC), extended pelvic lymph node dissection (PLND), and urinary diversion have been established as the gold standard. In the last few years, however, growing attention has been set on robotic-assisted radical cystectomy (RARC). Even in the very long term, open RC has good oncological results and if an ileal neobladder is performed excellent functional results. Follow-up of patients after open RC exceeds more than a decade which is unsurpassed by any other technique. Its outcomes have been proven to be durable and cost-effective. Least perioperative complications as well as best oncological and functional results can be achieved if open RC and urinary diversion were performed in a high-volume hospital by high-volume surgeons and an experienced team. Despite upcoming new technologies such as RARC, open RC following extended (PLND) remains the gold standard treatment for high-grade muscle invasive bladder cancer.

MP46-01 AKR1C3 ACTIVATION AND INTRACRINE ANDROGENS CONFER RESISTANCE TO ENZALUTAMIDE

INTRODUCTION AND OBJECTIVES: The metabolism of cancer cells is fundamentally different from that of their normal counterparts. The M2 isoenzyme of pyruvate kinase (PKM2), expressed primarily during embryogenesis, is switched back on and exerts two major effects during oncogenesis: (i) slowing down glycolysis thus shunting glycolytic intermediates for anabolic process necessary for cell proliferation; and (ii) interacting with transcriptional factors thus upregulating growth-promoting genes. The role of PKM2 in bladder cancer formation and progression is presently unknown. METHODS: Human specimens and cultured cells from normal urothelia, low-gradenon-invasive and high-grademuscle-invasive bladder cancerwereassessed forPKM2expressionby immunohistochemistryand immunoblotting. Selected cancer cell lines over-expressing PKM2 were treated with shikonin, a naphthoquinone inhibitor of PKM2 found in medicinal plants, alone or in combination with rapamycin, an mTOR inhibitor, and their effects on cell proliferation, metabolism, apoptotic, autophagic and unfolded protein responses were determined using biochemical and molecular approaches. Target-specific effects of shikonin were verified by transfection with PKM2-specifc small interfering RNA (siRNA). RESULTS: Compared to normal urothelia, both low-grade noninvasive and high-grade muscle-invasive bladder cancer markedly over-express PKM2. Accompanying this is the hyper-phosphorylation of tyrosine residue at codon Y105 and nuclear translocation of PKM2. Cultured bladder cancer cell lines including UroTsa, RT4, RT112, J82, T24 and UMUC3 also express significantly more PKM2 than primarycultured urothelial cells. Exposure of RT112 and T24 to shikonin results in a dose-dependent inhibition of proliferation, reduced oxygen consumption and marked upregulation of signals indicative of apoptosis, autophage and unfolded protein response. A greater inhibition of cell proliferation is observed when shikonin is combined with rapamycin, suggesting a synergism between PKM2 and mTOR inhibition. Enforced expression of siRNA of PKM2 produces a similar inhibitory effect on cell line proliferation, establishing target-specific effects of shikonin. CONCLUSIONS: These data demonstrate for the first time an important role of PKM2 overexpression in bladder cancer formation and progression, and suggest a potentially unconventional way to therapeutically inhibit bladder cancer via specific alteration of tumor-promoting metabolism.