Non Invasive Bladder Cancer Research Articles

FGFR3 Mutation Status and FGFR3 Expression in a Large Bladder Cancer Cohort Treated by Radical Cystectomy: Implications for Anti-FGFR3 Treatment?†

Fibroblast growth factor receptor 3 (FGFR3) is an actionable target in bladder cancer (BC). FGFR3 mutations are common in noninvasive BC and associated with favorable BC prognosis. Overexpression was reported in up to 40% of FGFR3 wild-type muscle-invasive BC. We analyzed FGFR3 mutations, FGFR3, and p53 protein expression and assessed their prognostic value in a cohort of 1000 chemotherapy-naive radical cystectomy specimens. FGFR3 mutations were found in 11%, FGFR3 overexpression was found in 28%, and p53 overexpression was found in 69% of tumors. Among FGFR3 mutant tumors, 73% had FGFR3 overexpression versus 22% among FGFR3 wild-type tumors. FGFR3 mutations were significantly associated with lower pT stage, tumor grade, absence of carcinoma in situ, pN0, low-level p53, and longer disease-specific survival (DSS). FGFR3 overexpression was associated only with lower pT stage and tumor grade. Moreover, FGFR3 overexpression was not associated with DSS in patients with FGFR3 wild-type tumors. In conclusion, FGFR3 mutations identified patients with favorable BC at cystectomy. Our results suggest that FGFR3 mutations have a driver role and are functionally distinct from FGFR3 overexpression. Hence, patients with FGFR3 mutations would be more likely to benefit from anti-FGFR3 therapy. Ideally, further research is needed to test this hypothesis. Patient summaryOncogenic fibroblast growth factor receptor 3 (FGFR3) mutations are very common in bladder cancer. In this report, we found that these FGFR3 mutations were associated with favorable features and prognosis of bladder cancer compared with patients with FGFR3 overexpressed tumors only. As a consequence, patients with FGFR3 mutant tumors would be more likely to benefit from anti-FGFR3 therapy than patients with FGFR3 protein overexpression only.

Comprehensive assessment of oncological outcomes in 186 patients with high‑risk non‑muscle‑invasive bladder cancer: A single institution retrospective study

The aims of the current study were to analyze the oncological outcomes of patients with high-risk non-invasive bladder cancer (NMIBC) and to identify prognostic factors in these patients. The present study included 186 consecutive patients who underwent transurethral resection of the bladder tumor (TURBT) between January 2007 and June 2017 at Hamamatsu University School of Medicine and were subsequently diagnosed with high-risk NMIBC according to the classification of the European Urological Association guidelines. The oncological outcomes, including recurrence-free survival (RFS), progression-free survival (PFS) and overall survival (OS) in the 186 patients were evaluated. Additionally, the effects of several clinicopathologiocal parameters on these outcomes were investigated. After the initial TURBT, the second transurethral resection and intravesical bacillus of Calmette-Guerin (BCG) therapy were performed for 47 (25.3%) and 108 (58.1%) patients, respectively. During the observation period of the current study, disease recurrence, disease progression and overall deaths occurred in 54 (29.0%), 14 (7.5%) and 19 (10.2%) patients, respectively. The 5-year RFS, PFS and OS rates in the 186 patients were 66.6, 90.2 and 87.2%, respectively. Multivariate analyses using the Cox proportional hazards regression model identified the following independent factors for the oncological outcomes: Tumor multiplicity and introduction of BCG therapy for RFS (P=0.018 and P<0.008, respectively), tumor multiplicity and recurrence status for PFS (P=0.043 and P=0.029, respectively), and age and tumor multiplicity for OS (P<0.008 and P=0.041, respectively). Although management following initial TURBT was insufficient, the oncological outcomes in the present series were comparable to those in previous studies targeting high-risk patients with NMIBC. However, attention should be paid to patients with factors independently associated with poor prognostic outcomes, particularly those with multiple tumors.

Mycobacteria-Based Vaccines as Immunotherapy for Non-urological Cancers.

The arsenal against different types of cancers has increased impressively in the last decade. The detailed knowledge of the tumor microenvironment enables it to be manipulated in order to help the immune system fight against tumor cells by using specific checkpoint inhibitors, cell-based treatments, targeted antibodies, and immune stimulants. In fact, it is widely known that the first immunotherapeutic tools as immune stimulants for cancer treatment were bacteria and still are; specifically, the use of Mycobacterium bovis bacillus Calmette-Guérin (BCG) continues to be the treatment of choice for preventing cancer recurrence and progression in non-invasive bladder cancer. BCG and also other mycobacteria or their components are currently under study for the immunotherapeutic treatment of different malignancies. This review focuses on the preclinical and clinical assays using mycobacteria to treat non-urological cancers, providing a wide knowledge of the beneficial applications of these microorganisms to manipulate the tumor microenvironment aiming at tumor clearance.

Do repeated transurethral procedures under general anesthesia influence mortality in patients with non-invasive urothelial bladder cancer? A Danish national cohort study

Purpose: To investigate the effect of repeated transurethral procedures under general anesthesia on overall mortality in patients with non-invasive bladder cancer.Materials and methods: All Danish residents with non-invasive papillary urothelial carcinoma or primary urothelial carcinoma in situ diagnosed between 1 January 2000 and 1 January 2011 were included and followed until death or 31 March 2017. All transurethral procedures under general anesthesia, intravesical instillation therapy, recurrences and progression to invasive disease or cystectomy were recorded during follow-up. Associations between treatments and overall mortality were evaluated using multivariable regression analysis adjusted for age, gender, comorbidities and socioeconomic status. The effect of disease progression on mortality was removed by censoring patients at the time of progression or cystectomy.Results: Risk of death increased with the number of transurethral procedures under general anesthesia for Ta low- and high-grade tumors compared to patients who had only one procedure; after eight or more procedures the risk of death increased by 28% and 83%, respectively. There was no similar relationship for carcinomas in situ. In total, 36–52% of procedures under general anesthesia did not identify urothelial neoplasia.Conclusions: Repeated transurethral procedures under general anesthesia appear to be associated with increased risk of death in patients with primary non-invasive papillary urothelial carcinoma. Furthermore, a substantial number of procedures were without findings of neoplasia, indicating that too many patients are admitted for transurethral procedures under GA. Attempts should be taken to reduce unnecessary transurethral procedures under GA, e.g. by improved outpatient diagnosis of urothelial neoplasia.

Abstract B35: Isolation and characterization of circulating tumor cells (CTCs) from bladder cancer patients using a highly sensitive graphene oxide-based microfluidic device (GO chip)

Abstract Introduction: A critical research topic in bladder cancer is determining the key regulators in the progression from noninvasive tumor to invasive and ultimately metastatic disease. Although patients with noninvasive bladder cancer can be treated through transurethral resection of bladder tumor and have a high survival rate (~95%), noninvasive bladder tumors recur in 50%-70% of cases. Of these, 10-15% of cases progress to invasive disease. Circulating tumor cells (CTCs) represent the transitional phase of progression between lymphovascular intravasation and distant metastases. Molecular profiling of these CTCs, beyond enumeration, can inform the molecules/pathways important in the invasion process, and potentially help unravel the mechanism of metastasis and provide new targets for therapy. Here we report the application of a highly sensitive graphene oxide-based microfluidic device to isolate and characterize CTCs from bladder cancer patients for the expression of markers implicated in an invasive phenotype. Methods: An anti-EpCAM and anti-EGFR biotinylated antibody cocktail was immobilized onto the graphene oxide chip (GO chip) to capture CTCs from whole blood. In patient samples, immunofluorescence staining for DAPI (nuclei), cytokeratin (cancer cell marker), EGFR and HER2 (markers of invasive phenotype), and CD45 (white blood cell marker) was used to enumerate CTCs. Cells with a DAPI+CK+CD45- phenotype were counted as CTCs. Blood samples from ten metastatic bladder cancer patients and two healthy controls were processed, stained, and analyzed. Results: CTCs were found in 10/10 (100%) patients, with an average of 17.70 cells/mL (s = 10.69 cells/mL). 9/10 patients exhibited CTCs that express EGFR, but only one patient had one CTC express HER2. Conclusion: The antibody-functionalized GO chips were able to successfully capture and enable characterization of CTCs from metastatic bladder cancer patients. EGFR potentially plays an important role in the vascular invasion process. Citation Format: Zeqi Niu, Molly A. Kozminsky, Kathleen C. Day, Phillip L. Palmbos, Mark L. Day, Sunitha Nagrath. Isolation and characterization of circulating tumor cells (CTCs) from bladder cancer patients using a highly sensitive graphene oxide-based microfluidic device (GO chip) [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr B35.

Effect of epigenetic imprinting biomarkers in urine exfoliated cells (UEC) on the diagnostic accuracy of low-grade bladder cancer.

e17027 Background: Urine Exfoliated Cells (UEC) is the most widely used sample for noninvasive bladder cancer detection. However, current UEC-based methods including urinary cytology and other molecular detections showed low sensitivities especially for low-grade cancer (&lt; 50% sensitivity). Epigenetic alterations such as elevated allelic expression of imprinted genes usually occur at early stages of malignancy, and can be considered as potential bladder cancer biomarkers. In this study, Quantitative Chromogenic Imprinting Gene In-Situ Hybridization (QCIGISH) was conducted to analyze the biallelic expression (BAE) and multiallelic expression (MAE) in UEC, and develop a more accurate method for early stage bladder cancer. Methods: 53 patients with urinary system diseases including 13 low-grade bladder cancer, 24 high-grade bladder cancer and 16 benign lesions, as well as 28 healthy volunteers were recruited in clinical trial NCT03563443 for validation. The total expression (TE), BAE and MAE of imprinted genes GNAS, PEG10, GRB10, SNRPN and HM13 were blindly detected by QCIGISH. The severity of malignancy was predicted by a grading model previously established on 229 tissue FFPE and cystoscopic biopsy samples, and subsequently validated with the pathology and clinical diagnosis results. Results: The QCIGISH method achieved 84.6% and 95.8% sensitivities for low- and high-grade bladder cancers, respectively, with an equally high specificity of 97.7%. The sensitivity of QCIGISH for low-grade bladder cancer is much higher than standard urinary cytology (16%), and FDA-approved molecular tests such as Hemoglobin Dipstick (38%), BTA Stat (36%), NMP22 BladderChek (25%), and ImmunoCyt (47%). Conclusions: The high accuracy of QCIGISH especially for low-grade bladder cancer demonstrated its great potential in becoming an important and powerful clinical tool for noninvasive diagnosis and large-scale screening for early bladder cancer.

MP17-04 NOVEL HYALURONIDASE TMEM2 EXPRESSION OF BLADDER TUMOR IS ALTERED BY INDUCING EMT-MET PROCESS

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II (MP17)1 Apr 2020MP17-04 NOVEL HYALURONIDASE TMEM2 EXPRESSION OF BLADDER TUMOR IS ALTERED BY INDUCING EMT-MET PROCESS Mihoko Yoneyama*, Tohru Yoneyama, Hayato Yamamoto, Yuki Tobisawa, Shingo Hatakeyama, Senji Hoshi, Shigeru Tsuboi, Tadashi Suzuki, and Chikara Ohyama Mihoko Yoneyama*Mihoko Yoneyama* More articles by this author , Tohru YoneyamaTohru Yoneyama More articles by this author , Hayato YamamotoHayato Yamamoto More articles by this author , Yuki TobisawaYuki Tobisawa More articles by this author , Shingo HatakeyamaShingo Hatakeyama More articles by this author , Senji HoshiSenji Hoshi More articles by this author , Shigeru TsuboiShigeru Tsuboi More articles by this author , Tadashi SuzukiTadashi Suzuki More articles by this author , and Chikara OhyamaChikara Ohyama More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000000842.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: As a major component of ECM, Hyaluronic acid (HA) fragments different molecular sizes exhibit different biological actions and biological responses. While high molecular weight (HMW) HA inhibits the cell motility and tumor proliferation, it has been reported that low MW HA has enhanced the cell migration/invasion and increased apoptosis. Recently, we identified transmembrane protein 2 (TMEM2) as a novel hyaluronidase that degraded HMW-HA into ∼5-kDa HA fragments. There are few data on TMEM2 involvement in human cancer. In this study, we investigated that association between TMEM2 expression and bladder cancer (BCa) progression. METHODS: TMEM2 expression of BCacells were examined by western blotting and droplet digital PCR. To examine the association of TMEM2 expression during EMT-MET process,we performed spheroid formation assay by using RT4-EMT and, 5637-EMT cell lines which isolated from the noninvasive RT4 and 5637 BCa cell line. To assess hyaluronidase activity of TMEM2, we examined that BCa cells put on FITC-HMW-HA1500. To examine TMEM2 expression in radical cystectomy tissues, we performed immunohistochemical staining by using anti-TMEM2 polyclonal antibody and evaluated association between TMEM2 expression and pathological features. RESULTS: We showed TMEM2 expression were significantly lower invasive BCa cells than non-invasive BCa cells. BCa cells expressing TMEM2 exhibit hyaluronidase activity depending on its expression level. Although TMEM2 expression of EMT induced BCa cells (RT4-EMT and 5637-EMT) were significantly decreased and little hyaluronidase activity was observed, the TMEM2 expression of EMT induced BCa cells increased during spheroid formation and its expression level was positively correlated with spheroid volume. In addition, immunohistochemical analyze revealed that TMEM2 expression were significantly higher in expansive growth lesion (eg, papillary or local invasive lesion, metastatic colonization lesion) and significantly lower in diffuse infiltrative lesion of muscle or stroma of bladder. CONCLUSIONS: Our results revealed TMEM2 expression of bladder tumor is changed by inducing EMT-MET process. Source of Funding: none © 2020 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 203Issue Supplement 4April 2020Page: e226-e227 Advertisement Copyright & Permissions© 2020 by American Urological Association Education and Research, Inc.MetricsAuthor Information Mihoko Yoneyama* More articles by this author Tohru Yoneyama More articles by this author Hayato Yamamoto More articles by this author Yuki Tobisawa More articles by this author Shingo Hatakeyama More articles by this author Senji Hoshi More articles by this author Shigeru Tsuboi More articles by this author Tadashi Suzuki More articles by this author Chikara Ohyama More articles by this author Expand All Advertisement PDF downloadLoading ...

Synthesis and biological evaluation of new antioxidant and antiproliferative chalcogenobiotin derivatives for bladder carcinoma treatment

Approximately 90% of bladder carcinomas are of the urothelial carcinoma type, which are characterized by high rates of recurrence and predisposition to progress to invasive tumors, representing one of the most costly neoplasms for health systems. Intravesical chemotherapy is a standard for the treatment of non-invasive bladder cancer. However, chemotherapy is usually aggressive and cytotoxic, which increases the death rates caused by cancer. Heterocyclic compounds which exhibit favorable pharmacokinetic and pharmacodynamic properties may enhance drug affinity for a target protein by targeting the treatment. Thus, this work presents the synthesis, characterization, and in vitro biological evaluation of new antioxidant (inhibition of lipid peroxidation, scavenging of free radical DPPH, and thiol peroxidase-like activity) and antiproliferative chalcogenobiotin derivatives and tests them against bladder carcinoma 5637 cells. A prominent response was obtained for the selected compounds, with tellurium biotin derivatives displaying effective antioxidant and antiproliferative activity. The effective compounds also demonstrated no toxicity in in vitro or in vivo studies.

Impact of bladder cancer stage on work impairment and financial toxicity.

460 Background: Bladder cancer has been linked to financial toxicity with conflicting results regarding the impact of stage. Our objective was to evaluate financial toxicity and work impairment among bladder cancer patients in a large cross-sectional bladder cancer cohort. Methods: We surveyed bladder cancer patients in the Bladder Cancer Advocacy Network Patient Survey Network and Inspire platforms to determine financial toxicity and work impairment. We measured financial toxicity with the COST measure and Work Productivity and Activity Impairment General Health questionnaires. Patients were also queried regarding demographic, socioeconomic and clinical characteristics with specific attention to insurance status, income, and education. Results: 972 respondents self-identified as patients with bladder cancer. Among respondents, 41% were female and 97% were white. The mean age was 67.6 years, ranging from 29 to 93 years. Respondents were highly educated (67% completed college). Most patients identified as having non-invasive bladder cancer (NMIBC, n=578 [63%]); 30% (n=270) had MIBC, 7% (n=63) had metastatic cancer. Although patients with metastatic cancer had higher average COST scores (worse financial toxicity, Table below), stage was not significantly associated with COST on bivariable (p=0.07) or multivariable analysis (p=0.14). Patients with metastatic disease were more likely to report more work time missed and activity impairment due to health on bivariable analysis; only activity impairment was statistically associated with stage on multivariable analysis (p&lt;0.001) when controlling for age, gender, race, comorbidity, insurance, and education. Conclusions: Bladder cancer stage significantly impacts activity impairment but not percentage work time missed due to health nor financial toxicity. [Table: see text]

Comprehensive targeted gene profiling to determine the genomic signature likely to drive progression of high-grade nonmuscle invasive bladder cancer to muscle invasive bladder cancer.

568 Background: Bladder cancer is the fifth most common cancer and eighth leading cause of cancer related-death in North America. It can present as non-muscle invasive bladder cancer (NMIBC) and/or muscle invasive bladder (MIBC). Although genomic profiling studies have established that low-grade NMIBC and MIBC are genetically distinct, high-grade NMIBC can recur and progress to MIBC [ Knowles, M.A. and C.D. Hurst, 2015]. Low grade, non-invasive bladder cancers are characterized by activating mutations in fibroblast growth factor receptor 3 (FGFR3), HRAS or other pathways of receptor kinase activation. High-grade disease, which is often becomes invasive, is characterized by inactivation of TP53 and Rb pathways [Kim, J., et al.]. Finding a subtype of invasive carcinoma with FGFR3 mutation may suggest an alternate pathway by which low grade, non-invasive pathology could transform into invasive disease [Knowles, M.A. and C.D. Hurst, 2015]. Methods: In this study, using a total of 30 bladder cancer (NMIBC and MIBC) patient samples from Windsor Regional Hospital Cancer Program, we performed comprehensive targeted gene sequencing to identify single nucleotide variants, small insertions / deletions, copy number variants and splice variants in over 500 common tumor genes panel. Results: Preliminary data from our study correlates with previously published mutation landscape for NMIBC and MIBC, and includes mutations in EGFR, FGFR3, FGFR4, PIK3CA, CDK6, ALK, JAK, as well as RET. While mutations in AKT1, BRCA1, CCND1, ERBB2, FGFR1, FGFR2, HRAS, and MET appear to be prevalent in NMIBC, mutations in IDH1 and MAP2K2 appear to be more common in MIBC. Three of the samples used in the study are from patients who progressed from high-grade NMIBC to MIBC. Conclusions: Therefore, have the genomic profiling performed at these two stages, which provides a unique ability to identify the potential “genomic triggers” for the transition.

Discoidin Domain Receptor 1 functionally interacts with the IGF-I system in bladder cancer

Bladder cancer is one of the most common and aggressive cancers and, regardless of the treatment, often recurs and metastasizes. Thus, a better understanding of the mechanisms regulating urothelial tumorigenesis is critical for the design and implementation of rational therapeutic strategies. We previously discovered that the IGF-IR axis is critical for bladder cancer cell motility and invasion, suggesting a possible role in bladder cancer progression. However, IGF-IR depletion in metastatic bladder cancer cells only partially inhibited anchorage-independent growth. Significantly, metastatic bladder cancer cells have decreased IGF-IR levels but overexpressed the insulin receptor isoform A (IR-A), suggesting that the latter may play a more prevalent role than the IGF-IR in bladder tumor progression. The collagen receptor DDR1 cross-talks with both the IGF-IR and IR in breast cancer, and previous data suggest a role of DDR1 in bladder cancer. Here, we show that DDR1 is expressed in invasive and metastatic, but not in papillary, non-invasive bladder cancer cells. DDR1 is phosphorylated upon stimulation with IGF-I, IGF-II, and insulin, co-precipitates with the IGF-IR, and the IR-A and transient DDR1 depletion severely inhibits IGF-I-induced motility. We further demonstrate that DDR1 interacts with Pyk2 and non-muscle myosin IIA in ligands-dependent fashion, suggesting that it may link the IGF-IR and IR-A to the regulation of F-actin cytoskeleton dynamics. Similarly to the IGF-IR, DDR1 is upregulated in bladder cancer tissues compared to healthy tissue controls. Thus, our findings provide the first characterization of the molecular cross-talk between DDR1 and the IGF-I system and could lead to the identification of novel targets for therapeutic intervention in bladder cancer. Moreover, the expression profiles of IGF-IR, IR-A, DDR1, and downstream effectors could serve as a novel biomarker signature with diagnostic and prognostic significance.

Will bacille Calmette-Guerin immunization arrest the COVID-19 pandemic?

Will bacille Calmette-Guerin immunization arrest the COVID-19 pandemic?

Clinical value of microRNA-19a-3p and microRNA-99a-5p in bladder cancer.

MicroRNAs (miRNA) are small (approximately 17 to 25 nucleotides in length), single stranded, non-coding RNAs that play an important role in the control of gene expression at the post-transcriptional stage, by inhibiting protein translation or promoting mRNA degradation. The main aim of the study was to evaluate the clinical utility of the tested markers (miRNAs 19a-3p and 99a-5p), which might be important in the diagnostics of non-invasive bladder cancer (BC). The study involved a group of 60 patients suffering from BC (histopathologically confirmed), in which 20 patients were diagnosed with muscle invasive BC (INBC) and 40 patients with non-muscle invasive BC (NINBC). The control group consisted of 20 samples of normal urothelium, which did not show any cancerous changes during histopathological examination. We assessed the expression of microRNA, using real-time PCR and the miRCURY LNA Universal RT microRNA PCR Kit by Exiqon, Denmark. Reduced expression of both analyzed markers was observed in most cases: miR-19a-3p in 51.8% and miR-99a-5p in 65.5% (as follows Mann-Whitney U test p < 0.000001 and Student's t test p = 0.034262). Moreover, miR-19a-3p in our tested group was useful to differentiate between low and high grade disease in non-invasive stages (t test p = 0.0315435). Furthermore, miR-19a-3p and miR-99a-5p were able to discriminate patients in low grade for groups with or without recurrence. Our data indicated that miR-19a-3p and miR-99a-5p were significantly altered in bladder cancer samples and useful as diagnostic markers.

P54 - MicroRNA for differentiation low and high grade of noninvasive bladder cancer

P54 - MicroRNA for differentiation low and high grade of noninvasive bladder cancer

Molecular identification of telomerase reverse transcriptase (TERT) promotor mutations in primary and recurrent tumors of invasive and noninvasive urothelial bladder cancer

ObjectiveTelomerase reverse transcriptase (TERT) promotor mutations occur in majority of cancers and are of notably meaning for tumor research. In bladder cancer the TERT promotor mutations exist in all stages and grades and are more frequent than other earlier reported genetic alterations. Here we report TERT promotor mutation status in primary bladder cancer samples related to survival and the risk of recurrence. Additionally we want to compare the TERT promotor mutation status from primary tumor and their recurrent tumor. Material and methodsFor this purpose, we detect TERT promotor mutation status in tumor tissues from 25 invasive and 50 noninvasive urothelial bladder cancer (UBC) patients using Sanger sequencing. Moreover we detect TERT promotor mutation status in the recurrent tumors (n = 21) of these UBC patients. ResultsIn 84% (63/75) of our UBC patients we found TERT promotor mutations: 80% (20/25) of invasive and 86% (43/50) of noninvasive tumors. In the recurrent tumors, 2 TERT promotor mutations differ from the primary tumor. Our study reveal, that TERT promotor mutations not significant correlate but show tendency with reduced overall survival and disease specific survival. ConclusionsOur findings suggest TERT promotor mutations were frequent in invasive and noninvasive UBC, making them potential therapeutic targets and useful as a biomarker in UBC. Detection of tumor cells with TERT promotor mutation in blood or urine could be efficient for the purpose of early detection of UBC and to predict survival of UBC patients.

Optical sensory arrays for the detection of urinary bladder cancer-related volatile organic compounds.

Non‐invasive detection of urinary bladder cancer remains a significant challenge. Urinary volatile organic compounds (VOCs) are a promising alternative to cell‐based biomarkers. Herein, we demonstrate a novel diagnosis system based on an optic fluorescence sensor array for detecting urinary bladder cancer VOCs biomarkers. This study describes a fluorescence‐based VOCs sensor array detecting system in detail. The choice of VOCs for the initial part was based on an extensive systematic search of the literature and then followed up using urinary samples from patients with urinary bladder transitional cell carcinoma. Canonical discriminant analysis and partial least squares discriminant analysis (PLS‐DA) were employed and correctly detected 31/48 urinary bladder cancer VOC biomarkers and achieved an overall 77.75% sensitivity and 93.25% specificity by PLS‐DA modelling. All five urine samples from bladder cancer patients, and five healthy controls were successfully identified with the same sensor arrays. Overall, the experiments in this study describe a real‐time platform for non‐invasive bladder cancer diagnosis using fluorescence‐based gas‐sensor arrays. Pure VOCs and urine samples from the patients proved such a system to be promising; however, further research is required using a larger population sample.

Rewiring E2F1 with classical NHEJ via APLF suppression promotes bladder cancer invasiveness

BackgroundBladder cancer progression has been associated with dysfunctional repair of double-strand breaks (DSB), a deleterious type of DNA lesions that fuel genomic instability. Accurate DSB repair relies on two distinct pathways, homologous recombination (HR) and classical non-homologous end-joining (c-NHEJ). The transcription factor E2F1 supports HR-mediated DSB repair and protects genomic stability. However, invasive bladder cancers (BC) display, in contrast to non-invasive stages, genomic instability despite their high E2F1 levels. Hence, E2F1 is either inefficient in controlling DSB repair in this setting, or rewires the repair apparatus towards alternative, error-prone DSB processing pathways.MethodsRT-PCR and immunoblotting, in combination with bioinformatics tools were applied to monitor c-NHEJ factors status in high-E2F1-expressing, invasive BC versus low-E2F1-expressing, non-invasive BC. In vivo binding of E2F1 on target gene promoters was demonstrated by ChIP assays and E2F1 CRISPR-Cas9 knockdown. MIR888-dependent inhibition of APLF by E2F1 was demonstrated using overexpression and knockdown experiments, in combination with luciferase assays. Methylation status of MIR888 promoter was monitored by methylation-specific PCR. The changes in invasion potential and the DSB repair efficiency were estimated by Boyden chamber assays and pulse field electrophoresis, correspondingly.ResultsHerein, we show that E2F1 directly transactivates the c-NHEJ core factors Artemis, DNA-PKcs, ligase IV, NHEJ1, Ku70/Ku80 and XRCC4, but indirectly inhibits APLF, a chromatin modifier regulating c-NHEJ. Inhibition is achieved by miR-888-5p, a testis-specific, X-linked miRNA which, in normal tissues, is often silenced via promoter methylation. Upon hypomethylation in invasive BC cells, MIR888 is transactivated by E2F1 and represses APLF. Consequently, E2F1/miR-888/APLF rewiring is established, generating conditions of APLF scarcity that compromise proper c-NHEJ function. Perturbation of the E2F1/miR-888/APLF axis restores c-NHEJ and ameliorates cell invasiveness. Depletion of miR-888 can establish a ‘high E2F1/APLF/DCLRE1C’ signature, which was found to be particularly favorable for BC patient survival.ConclusionSuppression of the ‘out-of-context’ activity of miR-888 improves DSB repair and impedes invasiveness by restoring APLF.

Inhibition of GLI2 with antisense-oligonucleotides: A potential therapy for the treatment of bladder cancer.

The sonic hedgehog (SHH) signaling pathway plays an integral role in the maintenance and progression of bladder cancer (BCa) and SHH inhibition may be an efficacious strategy for BCa treatment. We assessed an in-house human BCa tissue microarray and found that the SHH transcription factors, GLI1 and GLI2, were increased in disease progression. A panel of BCa cell lines show that two invasive lines, UM-UC-3 and 253J-BV, both express these transcription factors but UM-UC-3 produces more SHH ligand and is less responsive in viability to pathway stimulation by recombinant human SHH or smoothened agonist, and less responsive to inhibitors including the smoothened inhibitors cyclopamine and SANT-1. In contrast, 253J-BV was highly responsive to these manipulations. We utilized a GLI1 and GLI2 antisense oligonucleotide (ASO) to bypass pathway mechanics and target the transcription factors directly. UM-UC-3 decreased in viability due to both ASOs but 253J-BV was only affected by GLI2 ASO. We utilized the murine intravesical orthotopic human BCa (mio-hBC) model for the establishment of noninvasive BCa and treated tumors with GLI2 ASO. Tumor size, growth rate, and GLI2 messenger RNA and protein expression were decreased. These results suggest that GLI2 ASO may be a promising new targeted therapy for BCa.

MP63-14 EIGHT GENE EXPRESSION CLASSIFIER FOR NON INVASIVE BLADDER CANCER SURVEILLANCE

INTRODUCTION AND OBJECTIVES:To develop a high accuracy non-invasive bladder cancer (BC) surveillance test focusing on the detection of non high-risk non-muscle invasive BC (NMIBC) tumours.METHODS:A...

Understandings of Immunomodulatory Pathways of Indoleamine 2,3‐dioxygenase (IDO) in Bladder Cancer Cells

Bladder cancer (BC) is the fourth most common cancer between men and the ninth in women, with a high recurrence rate and progression after conventional treatments. A growing body of evidence suggests that immunomodulatory molecules protect bladder tumors against immune surveillance by triggering signaling pathways that modulate locally the immune system. One important molecule is indoleamine 2,3‐dioxygenase (IDO), which promotes tryptophan breakdown, activating sensitive pathways such as GCN2 in immune cells. In addition, tryptophan breakdown leads to the production of kynurenine compounds, which activate aryl hydrocarbon receptor (AHR), promoting anergy and apoptosis on T cells and Natural killer cells. Although these IDO‐mediated pathways are well recognized in immune cells, they were not yet analyzed in bladder cancer cells. The use of IDO inhibitors arises as a promise anti‐tumor strategy, however, they have divergent effects depending on the activated pathway. Here, we analyzed the expression of IDO and the activation of its pathways in non‐invasive muscle and in invasive muscle bladder cancer cells, and the effect of two IDO inhibitors on these pathways.RT4 and T24 cells were maintained in RPMI medium and incubated for 48h with the following stimuli: IFN‐γ (1000U/mL), Methyl‐D‐tryptophan (MT) (500μM), Incb024360 analog (INCB) (1μM). Control cells were maintained in RPMI medium. After the stimuli, RNA was extracted and qRT‐PCR for IDO, CHOP (a marker of GCN2 activation), CYP1A1 (an AHR activation marker), and AHR was performed.T24 cells constitutively express higher levels of IDO when compared to RT4 cells (relative expression (RE) of 1.0±0.1 in RT4 vs. 73.8±17.0 in T24), accompanied by high expression of CHOP (RE of 1.0±1.2 in RT4 vs. 10.5±4.5 in T24) and CYP1A1 (RE 1.0±0.0 in RT4 vs. 28.2±1.5 in T24), with no differences in AHr expression. When stimulated with IFN‐γ, both RT4 and T24 cells showed increased IDO expression, CHOP and CYP1A1 (p&lt;0.001). While INCB significantly reduced the IDO‐mediated pathways (CHOP and CYP1A1 expression), MT treatment increased CYP1A1 expression (p&lt;0.05).IDO‐regulated pathways are differentiated in invasive and noninvasive bladder cancer cells. As such, adjuvant treatments to modulate IDO are indeed promising. The use of certain inhibitors, however, such as MT, while may inhibit tryptophan depletion, also lead to activation of AHR, promoting kynurenine‐like effects. The use of non AHR binding compounds, such as Incb024360 analogs are promising then proposed as an alternative and safer approach for regulating IDO‐mediated events without nonspecific bindings.Support or Funding InformationFundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.