High-grade Bladder Cancer Research Articles

Abstract 5274: Multi-omics integration analysis robustly predicts high grade patient survival and identified altered fatty acid metabolism in high grade bladder cancer

Abstract Cancer metabolism varies depending on tumor grade. Currently, there is an absence of integration of multi-omics data to predict Bladder cancer (BLCA) survival. We aimed to identify a metabolic signature in high-grade BLCA by integration of unbiased metabolomics, lipidomics and transcriptomics to predict patient survival and to discover novel therapeutic targets. In the current study, using a robust mass spectrometry platform, we conducted global unbiased metabolic and lipid profiling analysis. We identified 518 differential metabolites and 19 lipids of various classes using the NIST MS metabolomics compendium and lipidblast MS/MS libraries, respectively, between low-grade and high-grade BLCA then mapped them to associated genes. Pathway analysis revealed a unique set of biochemical pathways highly deregulated in high-grade BLCA. Integromics analysis identified a molecular gene signature associated with patient poor survival. Importantly, low expression of fatty acid associated enzymes in high-grade tumors was associated with low fatty acid β- oxidation and acyl carnitines in high-grade BLCA and confirmed using tissue microarray. Our key finding is that impaired fatty acid β-oxidation (FAO) by downregulation of fatty acid associate enzymes suggests a crucial role in the progression of low grade to high grade BLCA. Using a metabolic-centered multi-omics based integrative analysis provided a system-level perspective of BLCA that would facilitate the development of novel therapeutics for high-grade BLCA. Note: This abstract was not presented at the meeting. Citation Format: Venkata Rao Vantaku, Jianrong Dong, Chandrashekar R Ambati, Dimuthu Perera, Sri Ramya Donepudi, Chandra Sekhar Amara, Vasanta Putluri, Ravi Shiva Shankar, Matthew J Robertson, Danthasinghe Waduge Badrajee Piyarathna, Mariana Villanueva, Friedrich-Carl von Rundstedt, Balasubramanyam Karanam, Leomar Y Ballester, Martha K. Terris, Seth P. Lerner, Apolo B Andrea, Hugo Villanueva, Andrew Sikora, Yair Lotan, Arun Sreekumar, Cristian Coarfa, Nagireddy Putluri. Multi-omics integration analysis robustly predicts high grade patient survival and identified altered fatty acid metabolism in high grade 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 5274.

Abstract CT047: Phase I trial of intravesical Bacillus Calmette-Guérin combined with intravenous Pembrolizumab in high grade nonmuscle invasive bladder cancer

Abstract PURPOSE: A phase I trial of intravesical bacillus Calmette-Guérin (BCG) in combination with systemic pembrolizumab was conducted in patients with high grade non-muscle invasive bladder cancer who had persistent or recurrent disease after failing treatment with at least 2 courses of intravesical therapy (one of which had to contain BCG) or BCG followed by maintenance BCG. The primary objective was to determine the safety of this combination. Secondary end points included response to treatment at 19 weeks (end of treatment) and 3 months post treatment. MATERIALS AND METHODS: A total of 9 patients with recurrent/persistent high-grade non-muscle invasive bladder cancer after at least two courses of intravesical therapy or one course of BCG treatment followed by one course of maintenance BCG were enrolled in the study. Six doses of pembrolizumab were given every 3 weeks over 16 weeks given concurrently with 6 weekly doses of BCG beginning at week 7. Patient safety was evaluated during and for 30 days following pembrolizumab treatment. Preliminary combination efficacy was determined at 19 weeks using cystoscopy. Bladder biopsy was performed in patients with suspicious lesions. RESULTS: The combination of BCG and pembrolizumab was well tolerated at both 100mg and 200mg fixed doses. Fatigue and cystitis-type symptoms (burning, spasm, urgency, sensitivity, and frequency) were the most common adverse events and all cases were either grade 1 or 2. Two patients died during the trial period. One patient died due to progression of low grade upper urinary tract transitional cell carcinoma. The other patient died after cystectomy (for progressive disease) due to causes unrelated to the trial treatment. Of the 9 patients treated, 6 (78%) had no evidence of disease in the bladder and 1 had a mixed response (progression in the upper urinary tract only) at 19 weeks (end of treatment) while 5 (56%) remained disease-free at the 3 month follow up. CONCLUSIONS: The combination of BCG and pembrolizumab was well tolerated with subjects exhibiting the expected adverse effects associated with the use of both medications. Intravesical BCG and pembrolizumab may have clinical activity in non-muscle invasive bladder cancer recurring after repeated intravesical therapy. .A phase III trial is now open and enrolling in the United States and other countries (NCT03711032). Citation Format: Shaheen Alanee, Ahmed El Zawahry, Sherjeel Sana, Kevin McVary, Kathy Robinson, Krishna A. Rao. Phase I trial of intravesical Bacillus Calmette-Guérin combined with intravenous Pembrolizumab in high grade nonmuscle invasive 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 CT047.

Abstract 2388: In vitro assessment of tumor associated I.O. markers in cancer cell lines following Vicinium treatment

Abstract Vicinium is a fusion protein that comprises a scFv fragment specific for the Epithelial Cell Adhesion Molecule (EpCAM) genetically fused to a truncated form of Pseudomonas exotoxin A, ETA, via a flexible linker. Vicinium is used for the treatment of loco-regionally accessible tumors and is currently in a phase III clinical trial (VISTA) for the treatment of high-grade non-muscle invasive bladder cancer and a phase I study in combination with durvalumab for the same indication. In a different phase I study in late stage squamous cell carcinoma of the head and neck, direct injection of Vicinium led to shrinkage of the principal injected tumor as well as non-targeted tumors in some patients suggesting the activation of a T cell-mediated anti-tumor response through cross-priming. Supporting this hypothesis, in vitro studies have demonstrated that Vicinium mediated tumor cell killing elicits biological features of an immunogenic cell death (ICD). Tumor cells’ ability to evade the innate and adaptive immune response plays a major role in cancer development and progression. Altered expression of immune modulators is a key mechanism responsible for tumor escape. Recent studies have shown that exposure to chemotherapeutic drugs results in the selection of tumor cells expressing immunosuppressive markers such as PD-L1, CD47 and CD39. Following in vitro treatment with Vicinium, the enrichment of tumor cell population expressing I.O. markers was examined and compared to chemotherapeutic agents. Unlike small molecule chemotherapies, Vicinium treatment did not lead to an enrichment of CD47 or CD39 positive tumor cell populations. Taken together, the data suggests that Vicinium does not induce an immunosuppressive environment. Citation Format: Shilpa Chooniedass, Rachelle L. Dillon, Arjune Premsukh, Glen C. MacDonald, Jeannick Cizeau, Gregory P. Adams. In vitro assessment of tumor associated I.O. markers in cancer cell lines following Vicinium treatment [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 2388.

Abstract 785: The inhibitory effect of compound ChlA-F onhuman bladder cancer cell invasion can be attributed to its blockage of SOX2 protein

Abstract Sex-determining region Y-box 2 (SOX2), a well-known stemness biomarker, is highly expressed in a variety of cancers, including human highly invasive bladder cancer (BC). However, the role of SOX2 may vary in different kinds of malignancy. In the present study, we discovered that ChlA-F, a novel conformation derivative of isolate Cheliensisin A (Chel A), remarkably inhibits the invasive ability of human invasive BC cells through downregulation of SOX2 protein expression. We found that ChlA-F treatment dramatically decreases SOX2 protein expression in human high-grade invasive BC cells. Ectopic expression of SOX2 reversed ChlA-F inhibition of cell invasion ability in human bladder cancer cells, suggesting that SOX2 is a major target of ChlA-F during its inhibition of human BC invasion. Mechanistic studies revealed that SOX2 is downregulated by ChlA-F at both the protein degradation and protein translation levels. Further studies revealed that ChlA-F treatment induces HuR protein expression and that the increased HuR interacts with USP8 mRNA, resulting in elevation of USP8 mRNA stability and protein expression. Elevated USP8 subsequently acts as an E3 ligase to promote SOX2 ubiquitination and protein degradation. We also found that ChlA-F treatment substantially increases c-Jun phosphorylation at Ser63 and Ser73, initiating miR-200c transcription. The increased miR-200c directly binds to the 3’-UTR of SOX2 mRNA to suppress SOX2 protein translation. These results present novel mechanistic insight into understanding SOX2 inhibition upon ChlA-F treatment and provide important information for further exploration of ChlA-F as a new therapeutic compound for the treatment of highly invasive/metastatic human BC patients. Citation Format: Xiaohui Hua, Jiheng Xu, Chuanshu Huang, Jingxia Li, Zhongxian Tian, Haishan Huang. The inhibitory effect of compound ChlA-F onhuman bladder cancer cell invasion can be attributed to its blockage of SOX2 protein [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 785.

The inhibitory effect of compound ChlA-F on human bladder cancer cell invasion can be attributed to its blockage of SOX2 protein.

Sex-determining region Y-box 2 (SOX2), a well-known stemness biomarker, is highly expressed in a variety of cancers, including human highly invasive bladder cancer (BC). However, the role of SOX2 may vary in different kinds of malignancy. In the present study, we discovered that ChlA-F, a novel conformation derivative of isolate Cheliensisin A (Chel A), remarkably inhibits the invasive ability of human invasive BC cells through downregulation of SOX2 protein expression. We found that ChlA-F treatment dramatically decreases SOX2 protein expression in human high-grade invasive BC cells. Ectopic expression of SOX2 reversed ChlA-F inhibition of cell invasion ability in human bladder cancer cells, suggesting that SOX2 is a major target of ChlA-F during its inhibition of human BC invasion. Mechanistic studies revealed that ChlA-F downregulates SOX2 at both the protein degradation and protein translation levels. Further studies revealed that ChlA-F treatment induces HuR protein expression and that the increased HuR interacts with USP8 mRNA, resulting in elevation of USP8 mRNA stability and protein expression. Elevated USP8 subsequently acts as an E3 ligase to promote SOX2 ubiquitination and protein degradation. We also found that ChlA-F treatment substantially increases c-Jun phosphorylation at Ser63 and Ser73, initiating miR-200c transcription. The increased miR-200c directly binds to the 3'-UTR of SOX2 mRNA to suppress SOX2 protein translation. These results present novel mechanistic insight into understanding SOX2 inhibition upon ChlA-F treatment and provide important information for further exploration of ChlA-F as a new therapeutic compound for the treatment of highly invasive/metastatic human BC patients.

Multi-omics Integration Analysis Robustly Predicts High-Grade Patient Survival and Identifies CPT1B Effect on Fatty Acid Metabolism in Bladder Cancer.

The perturbation of metabolic pathways in high-grade bladder cancer has not been investigated. We aimed to identify a metabolic signature in high-grade bladder cancer by integrating unbiased metabolomics, lipidomics, and transcriptomics to predict patient survival and to discover novel therapeutic targets. We performed high-resolution liquid chromatography mass spectrometry (LC-MS) and bioinformatic analysis to determine the global metabolome and lipidome in high-grade bladder cancer. We further investigated the effects of impaired metabolic pathways using in vitro and in vivo models. We identified 519 differential metabolites and 19 lipids that were differentially expressed between low-grade and high-grade bladder cancer using the NIST MS metabolomics compendium and lipidblast MS/MS libraries, respectively. Pathway analysis revealed a unique set of biochemical pathways that are highly deregulated in high-grade bladder cancer. Integromics analysis identified a molecular gene signature associated with poor patient survival in bladder cancer. Low expression of CPT1B in high-grade tumors was associated with low FAO and low acyl carnitine levels in high-grade bladder cancer, which were confirmed using tissue microarrays. Ectopic expression of the CPT1B in high-grade bladder cancer cells led to reduced EMT in in vitro, and reduced cell proliferation, EMT, and metastasis in vivo. Our study demonstrates a novel approach for the integration of metabolomics, lipidomics, and transcriptomics data, and identifies a common gene signature associated with poor survival in patients with bladder cancer. Our data also suggest that impairment of FAO due to downregulation of CPT1B plays an important role in the progression toward high-grade bladder cancer and provide potential targets for therapeutic intervention.

Is Repeat Transurethral Resection Always Needed in High-Grade T1 Bladder Cancer?

Re-staging transurethral resection, the so-called repeat TUR (Re-TUR), is mandatory in case of incomplete first transurethral resection of bladder tumor (TURBT). In completely resected high grade T1 tumors, Re-TUR is recommended but question remains whether it provides advantages in terms of recurrence-free survival (RFS), progression-free survival (PFS), and cancer specific survival (CSS). The present study aimed to determine whether Re-TUR improves such outcomes in patients with completely resected high-grade T1 bladder cancer (BC). We queried our prospectively maintained database to identify patients with completely resected high-grade T1 BC who underwent (Group A) or not (Group B) Re-TUR before starting intravesical instillations of Bacillus Calmette-Guerin (BCG). The impact of Re-TUR as well as of other tested variables on RFS, PFS, and CSS was tested by Kaplan-Meier method and Log-rank testing. A total of 118 patients underwent Re-TUR, which pointed out no BC in 61 (51.7%), NMIBC in 54 (45.8%) and pT2 disease in 3 (2.5%). The 3 patients with pT2 disease underwent cystectomy, whereas all others were offered BCG treatment. Forty-two patients refused BCG treatment while 2 did not complete it; therefore, Group A (Re-TUR before BCG treatment) consisted of 71 patients whereas Group B consisted of 40 patients who refused Re-TUR but completed BCG treatment. Mean follow-up was 60 months (range 12-142). Kaplan-Meier curves and Log-rank testing showed no difference in RFS, PFS and CSS between patients who had (Group A) or had not (Group B) Re-TUR before starting BCG treatment. Our findings suggest that a Re-TUR in patients with a completely resected high-grade T1 BC does not translate into a better oncological outcome. Given its impact on both patients and healthcare system, the need for Re-TUR in completely resected high grade T1 BC should be further investigated into the framework of a randomized study.

Percutaneous BCG enhances innate effector antitumor cytotoxicity during treatment of bladder cancer: a translational clinical trial

ABSTRACT Background: Intravesical bacillus Calmette-Guérin (BCG) is the gold standard immunologic agent for treating patients with high-grade non-muscle invasive bladder cancer (NMIBC). Nevertheless, relapse rates remain high and BCG unresponsive NMIBC often requires bladder removal. Preclinical data suggest that priming with percutaneous BCG vaccine could improve response to intravesical BCG. Methods: A single-arm trial (NCT02326168) was performed to study the safety, immunogenicity, and preliminary efficacy of priming. Percutaneous BCG was given 21 days prior to intravesical BCG instillation in patients (n = 13) with high-risk NMIBC. Immune responses were monitored and compared to a sequentially enrolled cohort of nine control patients receiving only intravesical BCG. The effect of BCG on natural killer (NK) and γδ T cell in vitro cytotoxicity was tested. γδ T cell subsets were determined by T cell receptor gene expression with NanoString. Results: Priming was well tolerated and caused no grade ≥3 adverse events. The 3-month disease-free rate for prime patients was 85% (target goal ≥ 75%). Priming boosted BCG-specific immunity at 3 months and increased the activation status of in vitro expanded circulating NK and γδ T cells and their cytotoxicity against bladder cancer cells through receptor NKG2D. BCG enhanced the cytotoxicity of NK and γδ T cells against K562, T24, and UM-UC6 but not against UM-UC-3 or UM-UC-14 cells. Infiltrating γδ T cell subsets identified in the bladder includes γ9δ2 and γ8δ2. Conclusions: BCG priming is safe and tolerable. Poor sensitivity to NK and γδ T cell cytotoxicity by some bladder tumors represents a potential BCG-resistance mechanism.

Second Transurethral Resection of Bladder Tumor: Is it Necessary in All T1 and/or High-Grade Tumors?

To evaluate the role of second transurethral resection of bladder tumor (TURBT) in patients with T1 and/or high-grade bladder tumor regarding tumor size, multiplicity, and presence or absence of muscle in specimens of initial resection. A total of 107 patients with either primary T1 or high-grade urothelial bladder cancer underwent second TURBT within 6 weeks after initial surgery and prior to starting intravesical immunotherapy. We assessed the incidence of residual disease and upstaging in second TURBT. Upstaging was noted in 11 (10.3%) patients and residual tumor was evident in 29 (27%) patients. Disease upstaging had a statistically significant association with tumor size, multifocality, and absence of muscle at initial resection in univariate analysis. Presence of residual tumor in second resection also showed significant association with tumor size and absence of muscle at initial resection but not multifocality. Multivariate logistic regression analysis revealed that absence of muscle at initial resection independently predicts disease upstaging during second TURBT (OR = 8.123, 95% CI: 1.478-44.632). Furthermore, both tumor size (OR = 13.573, 95% CI: 3.104-59.359) and absence of muscle (OR = 21.214, 95% CI: 6.062-74.244) were independent predictors of residual disease in second TURBT. We showed that second TURBT in a subset of patients with single, small T1 and/or high-grade tumor who underwent complete initial resection might be of limited value.

Radiomics analysis of multiparametric MRI for the preoperative evaluation of pathological grade in bladder cancer tumors.

To develop and validate an MRI-based radiomics strategy for the preoperative estimation of pathological grade in bladder cancer (BCa) tumors. A primary cohort of 70 patients (31 high-grade BCa and 39 low-grade BCa) with BCa were retrospectively enrolled. Three sets of radiomics features were separately extracted from tumor volumes on T2-weighted imaging (T2WI), diffusion-weighted imaging (DWI), and apparent diffusion coefficient (ADC) maps. Two sets of multimodal features were separately generated by the maxout and concatenation of the abovementioned single-modality features. Each feature set was subjected to a two-sample t test and the least absolute shrinkage and selection operator (LASSO) algorithm for feature selection. Multivariable logistic regression (LR) analysis was used to obtain five corresponding radiomics models. The diagnostic abilities of the radiomics models were evaluated using receiver operating characteristic (ROC) curve analysis and compared using the DeLong test. Validation was performed on a time-independent cohort containing 30 consecutive patients. The areas under the ROC curves (AUCs) of single-modality T2WI, DWI, and ADC models in the training cohort were 0.7933 (95% confidence interval [CI] 0.7471-0.8396), 0.8083 (95% CI 0.7565-0.8601), and 0.8350 (95% CI 0.7924-0.8776), respectively. Both multimodality models achieved higher AUCs (maxout 0.9233, 95% CI 0.9001-0.9466; concatenation 0.9233, 95% CI 0.9001-0.9466) than single-modality models. The AUCs of the maxout and concatenation models in the validation cohort were 0.9186 and 0.9276, respectively. The MRI-based multiparametric radiomics approach has the potential to be used as a noninvasive imaging tool for preoperative grading of BCa tumors. Multicenter validation is needed to acquire high-level evidence for its clinical application. • Multiparametric MRI may help in the preoperative grading of BCa tumors. • The Joint_Model established from T2WI, DWI, and ADC feature subsets demonstrated a high diagnostic accuracy for preoperative prediction of pathological grade in BCa tumors. • The radiomics approach has the potential to preoperatively assess tumor grades in BCa and avoid subjectivity.

Absolute basophil count is associated with time to recurrence in patients with high-grade T1 bladder cancer receiving bacillus Calmette-Guérin after transurethral resection of the bladder tumor.

Basophils, eosinophils and monocytes may be involved in BCG-induced immune responses and be associated with outcomes of bladder cancer patients receiving intravesical BCG. Our objective was to explore the association of baseline counts of basophils, eosinophils and monocytes with outcomes of patients with high-grade T1 bladder cancer receiving a standard course of intravesical BCG. We retrospectively reviewed medical records of patients with primary T1 HG/G3 bladder cancer. After re-TURBT, patients were treated with a 6-week course of intravesical BCG induction followed by intravesical BCG every week for 3weeks given at 3, 6, 12, 18, 24, 30 and 36months from initiation of therapy The analysis of potential risk factors for recurrence, muscle invasion and cancer-specific and overall survival was performed using univariable Cox regression models. Those factors that presented, at univariate analysis, an association with the event at a liberal p < 0.1, have been selected for the development of a multivariable model. A total of 1045 patients with primary T1 HG/G3 were included. A total of 678 (64.9%) recurrences, 303 (29.0%) progressions and 150 (14.3%) deaths were observed during follow-up. Multivariate analysis showed that logarithmic transformation of basophils count was associated with a 30% increment in the hazard of recurrence per unit increase of logarithmic basophils count (HR 1.30; 95% confidence interval 1.09-1.54; p = 0.0026). Basophil count modeled by quartiles was also significantly associated with time to recurrence [second vs. lower quartile HR 1.42 (1.12-1.79); p = 0.003, third vs. lower quartile HR 1.26 (1.01-1.57); p = 0.041; upper vs. lower quartile HR 1.36 (1.1-1.68); p = 0.005]. The limitations of a retrospective study are applicable. Baseline basophil count may predict recurrence in BCG-treated HG/G3 T1 bladder cancer patients. External validation is warranted.

Moreau Strain Bacillus Calmette-Guérin Low Versus Standard Dose in the Treatment of High-Grade T1 Bladder Cancer: A Retrospective Observational Cohort Study

To evaluate recurrence, progression, and cancer-specific mortality of high-grade T1 non-muscle-invasive bladder cancer by assessing receipt of a low dose of the underexplored bacillus Calmette-Guérin (BCG) Moreau strain in a retrospective observational cohort study. From January 2006 to December 2015, a total of 219 consecutive patients with high-grade T1 non-muscle-invasive bladder cancer received half-dose (40 mg; n=109) or standard-dose (80 mg; n= 110) BCG Moreau strain after transurethral resection of the bladder. BCG therapy was initiated 2 or 3 weeks after transurethral resection of the bladder using the following protocol: 6 weekly doses, 12 monthly, 4 once every 3 months, and 2 once every 6 months, with a total of 24 doses. Comparing the half-dose and standard-dose treatment groups, in a median follow-up of 74.6 months, recurrence (n= 51, 46.8% vs. n= 60, 54.5%, P= .28), progression (n= 18, 16.5% vs. n= 16, 14.5%, P= .69), and disease-specific mortality (n= 9, 8.3% vs. n= 5, 4.5%, P= .26) were not significantly different on Kaplan-Meier curves and log-rank test, respectively. Charlson comorbidity index was an independent predictor of death from disease (hazard ratio= 1.341; 95% confidence interval, 1.033-1.740; P= .0274); no predictor of recurrence or progression was identified. Treatment intolerance occurred in 1 (0.9%) versus 6 (5.4%) patients (P= .12), respectively. No hospital admission or systemic BCG toxicity was reported. To our knowledge, this is the largest low-dose Moreau BCG strain study in high-grade T1 scenario. A half dose of BCG Moreau strain might be safe and effective in terms of tumor control, progression, or cancer-specific mortality with a low complication rate, which is central to the worldwide scenario of BCG shortage, and can help regulatory agencies approve efficient daughter BCG strains.

Association between AXIN1 Gene Polymorphisms and Bladder Cancer in Chinese Han Population.

Background Previous evidence has indicated that the reduction of axis inhibition protein 1 (AXIN1) expression is related with the poor differentiation of non-small-cell lung cancer (NSCLC). However, the potential association between AXIN1 and bladder cancer (BC) is unknown. We aimed to initially explore the relevance of AXIN1 gene polymorphisms (rs12921862 C/A, rs1805105 T/C, and rs370681 C/T) and BC. Methods Three hundred and sixteen BC patients and 419 healthy controls had been enrolled. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used for genotyping three tag single-nucleotide polymorphisms (SNPs) of AXIN1. The SNPstats online analysis software and SPSS software were used for statistical analysis. Results Our data revealed that three tag SNPs were associated with an increased risk of BC (rs12921862: P < 0.001, OR (95%CI) = 4.61 (3.13-6.81); rs1805105: P = 0.046, OR (95%CI) = 1.35 (1.00-1.82); and rs370681: P = 0.004, OR (95%CI) = 1.56 (1.15-2.10)). For rs12921862, A allele was an independently protective factor which correlated with a better prognosis in non-muscle-invasive bladder cancer (NMIBC) patients (P = 0.03, OR (95%CI) = 0.10 (0.01-0.84)). Stratification analysis demonstrated that rs370681 polymorphism was related with high-grade bladder cancer (P = 0.04, OR (95%CI) = 1.85 (1.04-3.23)). Conclusion The AXIN1 gene polymorphisms might implicate in BC risk, and rs12921862 could be a potential forecasting factor for prognosis of BC patients.

Investigation of Clinical Outcome and Prognosis of 2nd TUR-Bt for T1 High Grade Bladder Cancer

Second transurethral resection of bladder tumor (TUR-Bt) is a standard treatment for high grade T1 initial, non-muscle invasive bladder cancer. In our hospital from October 2008 to April 2017, 2nd TUR-Bt was performed in 51 cases of initial T1 bladder cancer. The risk factors of residual tumors on 2nd TUR-Bt and the clinical outcome were examined retrospectively. Twenty two of the 51 cases (43.1%) had residual tumors on 2nd TUR-Bt, and upstaging was not admitted. To determine the risk factor for residual tumors in 2nd TUR-Bt, we examined gender, tumor morphology (papillary/non-papillary, pedunculated/ nonpedunculated), number of tumors (single/frequent), and tumor size (≧20 mm/＜20 mm), but none of these were significant risk factors for tumor residue. The recurrence free survival (RFS) of the 51 cases was 86.0% after 1 year, and 77.0% after 3 years. There tended to be a higher RFS in the pedunculated tumor group, pT0 group on 2nd TUR-Bt, intravesical BCG therapy group, but no statistically significant difference was observed. The progression free survival (PFS) was 90.6% after 3 years, and 87.3% after 5 years. These values were similar to those reported previously.

CD163+ macrophages predict a poor prognosis in patients with primary T1 high-grade urothelial carcinoma of the bladder.

Macrophages are a major cell type that can infiltrate solid tumors and exhibit distinct phenotypes in different tumor microenvironments. This study investigates the prognostic value of tumor-infiltrated CD163+ macrophages in patients with T1 high-grade (T1HG) bladder cancer. CD163+ macrophages were assessed by immunohistochemistry in 94 T1HG bladder cancer samples. Kaplan-Meier analyses and Cox proportional hazards' regression models were applied to evaluate recurrence-free survival, progression-free survival and disease-specific survival. With a median follow-up of 60months, 37 (39.4%) patients experienced disease recurrence, 14 (14.9%) progression, 11 (11.7%) disease-specific mortality. High CD163+ macrophages were associated with higher risk of disease recurrence and progression (P < 0.05, for both). In multivariate Cox proportional hazards regression analysis, high CD163+ macrophages were a significant negative predictor of recurrence-free survival, progression-free survival and disease-specific survival (P < 0.05 for all). CD163+ macrophages are a poor prognostic factor in T1HG bladder cancer. This finding provide the ground for further testing it in predicting the outcome of this challenging disease.

The rational and benefits of the second look transurethral resection of the bladder for T1 high grade bladder cancer.

The second look transurethral resection of the bladder (TURB) represents a fundamental step in the treatment of papillary non-muscle invasive bladder cancer (NMIBC); it is therefore recommended by all guidelines. However, not all the literature agrees on its staging value and its ability to improve oncological outcomes of patients. Therefore, the purpose of this review is to evaluate the strengths and weaknesses of second look TURB, trying to depict its evolving role in the management of high grade NMIBC. Using Medline, a non-systematic review was performed including articles between January 2000 and June 2018. English language original articles, reviews and editorials were selected based on their clinical relevance. To date, TURB seems to be largely inadequate in retrieving a correct diagnosis and in removing all tumor tissue. Second look TURB maximizes staging accuracy, allows to clear residual cancer and yields prognostic advantages allowing key information to identify possible candidates for immediate radical cystectomy for very high risk T1HG tumors. Moreover, it seems to have a therapeutic benefit by improving recurrence- and progression-free survivals. However, few recent large studies showed that these advantages seem to be limited to patients without detrusor muscle present at first resection. Similarly, the presence of residual disease and the risk of upstaging are related to the presence of detrusor muscle in specimen. It could well be that in the future the presence of detrusor muscle would be a quality criteria to avoid an unnecessary second look TURB as shown by recent studies using the en-bloc resection technique. Finally, it has to be underlined that this is a surgical procedure not free of risks and complications and with a non-negligible impact on patients’ quality of life, waiting lists and healthcare costs. Therefore, future studies trying to identify the criteria that may better allow which patients to select for a second look TURB while avoiding an unnecessary intervention with possible risks and associated cost are needed to allow a personalized approach to even this one size fits all strategy.

PT212 - Narrow band imaging reduces persistence of cancer in patients with pT1 high grade bladder cancer

PT212 - Narrow band imaging reduces persistence of cancer in patients with pT1 high grade bladder cancer

Micropipette Aspiration of Single Cells for Both Mechanical and Electrical Characterization.

Cellular physical properties have been identified to reflect cell states. Existing techniques are able to characterize either mechanical or electrical properties of a cell. This paper presents a micropipette aspiration technique that enables the characterization of both mechanical (instantaneous elastic modulus, equilibrium elastic modulus, and viscosity), and electrical (specific membrane capacitance) properties of the same single cell. Two bladder cancer cell lines (RT4 and T24) with different metastatic potential were used to evaluate the technique. The results showed that high-grade bladder cancer cells (T24, grade III) possess lower viscosity, lower elastic modulus, and larger SMC than the low-grade cancer cells (RT4, grade I). The Naive Bayes classifier was utilized to assess the classification accuracy using single-physical and multi-physical parameters. The classification results confirmed that the use of multi-biophysical parameters resulted in higher accuracy (97.5%), sensitivity (100%), and specificity (95.2%) than the use of a single-physical parameter for distinguishing T24 and RT4 cells.

CCDC6 and USP7 expression levels suggest novel treatment options in high-grade urothelial bladder cancer

BackgroundThe muscle invasive form of urothelial bladder cancer (UBC) is a deadly disease. Currently, the therapeutic approach of UBC is mostly based on surgery and standard chemotherapy.Biomarkers to establish appropriate drugs usage are missing. Deficiency of the tumor suppressor CCDC6 determines PARP-inhibitor sensitivity. The CCDC6 levels are modulated by the deubiquitinase USP7. In this work we scored CCDC6 and USP7 expression levels in primary UBC and we evaluated the expression levels of CCDC6 in correlation with the effects of the PARP-inhibitors combined with the USP7 inhibitor, P5091, in vitro. Since PARP-inhibitors could be enhanced by conventional chemotherapy or DNA damage inducers, we tested the new agent RRx-001, able to induce DNA damage, to prove the benefit of combined treatments in bladder cancer cells.MethodsThe J82, T24, 5637 and KU-19-19 bladder cancer cells were exposed to USP7 inhibitor P5091 in presence of cycloheximide to analyse the CCDC6 stability. Upon the CCDC6 degradation induced by P5091, the cells sensitivity to PARP-inhibitor was evaluated by cell viability assays. The ability of the DNA damage inducer RRx-001 to modulate CCDC6 protein levels and H2AX phosphorylation was detected at immunoblot. The combination of USP7 inhibitor plus RRx-001 enhanced the PARP-inhibitor sensitivity, as evaluated by cell viability assays. The results of the scores and correlation of CCDC6 and USP7 expression levels obtained by UBC primary biopsies staining were used to cluster patients by a K-mean cluster analysis.ResultsP5091 determining CCDC6 degradation promoted bladder cancer cells sensitivity to PARP-inhibitor drugs. RRx-001, by inducing DNA damage, enhanced the effects of the combined treatment. The immunohistochemical staining of both CCDC6 and USP7 proteins allowed to cluster the high grade (G3) UBC patients, on the basis of CCDC6 expression levels.ConclusionsIn high grade UBC the identification of two clusters of patients based on CCDC6 and USP7 expession can possibly indicate the use of PARP-inhibitor drugs, in combination with USP7 inhibitor in addition to the DNA damage inducer RRx-001, that also acts as an immunomodulatory agent, offering novel therapeutic strategy for personalized medicine in bladder cancer patients.

Probe-Based Confocal Laser Endomicroscopy During Transurethral Resection of Bladder Tumors Improves the Diagnostic Accuracy and Therapeutic Efficacy

PurposeThis study was designed to assess the diagnostic accuracy and therapeutic efficacy of probe-based confocal laser endomicroscopy (pCLE), which provides real-time, in vivo histological information during transurethral resection of bladder tumors.MethodsWe performed a prospective study between August 2013 and August 2014. pCLE was performed on a total of 119 lesions in 75 patients. We analyzed the diagnostic accuracy of pCLE by comparing the confocal image reports with the pathology reports of surgical specimen. Confocal images were interpreted by a single urologist blinded to the pathology reports. The therapeutic efficacy was analyzed by comparing the outcomes in pCLE and non-pCLE groups.ResultsIn a total of 119 lesions, 23 were benign and 96 were malignant. The detection accuracy for malignant lesions with pCLE was determined with a sensitivity and a positive predictive value (PPV) of 91.7% and 93.6%, respectively. For high-grade versus low-grade bladder cancer, sensitivity and PPV of pCLE were 94.5% and 89.7%, respectively. Distinguishing carcinoma in situ from inflammatory lesions also was accurate with sensitivity, specificity, and PPV of 71.4%, 81.3%, and 83.3%, respectively. The Kaplan–Meier curves revealed that the recurrence-free survival rate was significantly higher in the pCLE group than in the non-pCLE group (p = 0.031).ConclusionsProbe-based confocal laser endomicroscopy is a promising method to provide the surgeon during the transurethral resection of a bladder tumor with real-time tumor histology, regardless of the tumor’s gross appearance. Furthermore, it also may improve the therapeutic efficacy with longer recurrence-free periods.