Literature on the relationship between recurrent urinary tract infections and urinary bladder carcinoma risk has been inconsistent. Therefore, we carried out this systematic review of observational studies to ascertain if there is any association between chronic urinary tract infection and urinary bladder carcinoma. A total of 10 databases were searched using Boolean: CINAHL, PUBMED, Google Scholar, Medline, Science Direct, SCIRUS, Cochrane, UK PubMed central, NHS evidence and WHO-website. The search yielded an initial hit of 3,518 articles and after screening and critical appraisal, seven studies were included for this review. Four articles reported an association between chronic urinary tract infections and bladder cancer while three concluded a weak or no association at least in one gender. Main findings in this review were that most of the studies reported an association between chronic urinary tract infections and bladder cancer risk. However, inferences about the causal association between chronic urinary tract infections and bladder cancer risk should be drawn cautiously considering the methodological limitations of case-control studies included in this review. Therefore, more empirical evidence is needed to determine the causal nature of relationships between chronic urinary tract infections and bladder cancer risk.

PURPOSE: Image guided adaptive radiation therapy offers individualized solutions to improve target coverage and reduce normal tissue irradiation, allowing the opportunity to increase the radiation tumor dose and spare normal bladder tissue.
METHODS AND MATERIALS: A library of 3 intensity modulated radiation therapy plans were created (small, medium, and large) from planning computed tomography (CT) scans performed at 30 and 60 minutes; treating the whole bladder to 52 Gy and the tumor to 70 Gy in 32 fractions. A "plan of the day" approach was used for treatment delivery. A post-treatment cone beam CT (CBCT) scan was acquired weekly to assess intrafraction filling and coverage.
RESULTS: A total of 18 patients completed treatment to 70 Gy. The plan and treatment for 1 patient was to 68 Gy. Also, 1 patient's plan was to 70 Gy but the patient was treated to a total dose of 65.6 Gy because dose-limiting toxicity occurred before dose escalation. A total of 734 CBCT scans were evaluated. Small, medium, and large plans were used in 36%, 48%, and 16% of cases, respectively. The mean ± standard deviation rate of intrafraction filling at the start of treatment (ie, week 1) was 4.0 ± 4.8 mL/min (range 0.1-19.4) and at end of radiation therapy (ie, week 5 or 6) was 1.1 ± 1.6 mL/min (range 0.01-7.5; P=.002). The mean D98 (dose received by 98% volume) of the tumor boost and bladder as assessed on the post-treatment CBCT scan was 97.07% ± 2.10% (range 89.0%-104%) and 99.97% ± 2.62% (range 96.4%-112.0%). At a median follow-up period of 19 months (range 4-33), no muscle-invasive recurrences had developed. Two patients experienced late toxicity (both grade 3 cystitis) at 5.3 months (now resolved) and 18 months after radiation therapy.
CONCLUSIONS: Image guided adaptive radiation therapy using intensity modulated radiation therapy to deliver a simultaneous integrated tumor boost to 70 Gy is feasible, with acceptable toxicity, and will be evaluated in a randomized trial.

BACKGROUND: Highly sensitive and specific urine-based tests to detect either primary or recurrent bladder cancer have proved elusive to date. Our ever increasing knowledge of the genomic aberrations in bladder cancer should enable the development of such tests based on urinary DNA.
METHODS: DNA was extracted from urine cell pellets and PCR used to amplify the regions of the TERT promoter and coding regions of FGFR3, PIK3CA, TP53, HRAS, KDM6A and RXRA which are frequently mutated in bladder cancer. The PCR products were barcoded, pooled and paired-end 2 x 250 bp sequencing performed on an Illumina MiSeq. Urinary DNA was analysed from 20 non-cancer controls, 120 primary bladder cancer patients (41 pTa, 40 pT1, 39 pT2+) and 91 bladder cancer patients post-TURBT (89 cancer-free).
RESULTS: Despite the small quantities of DNA extracted from some urine cell pellets, 96% of the samples yielded mean read depths >500. Analysing only previously reported point mutations, TERT mutations were found in 55% of patients with bladder cancer (independent of stage), FGFR3 mutations in 30% of patients with bladder cancer, PIK3CA in 14% and TP53 mutations in 12% of patients with bladder cancer. Overall, these previously reported bladder cancer mutations were detected in 86 out of 122 bladder cancer patients (70% sensitivity) and in only 3 out of 109 patients with no detectable bladder cancer (97% specificity).
CONCLUSION: This simple, cost-effective approach could be used for the non-invasive surveillance of patients with non-muscle-invasive bladder cancers harbouring these mutations. The method has a low DNA input requirement and can detect low levels of mutant DNA in a large excess of normal DNA. These genes represent a minimal biomarker panel to which extra markers could be added to develop a highly sensitive diagnostic test for bladder cancer.

BACKGROUND AND PURPOSE: Adaptive radiotherapy (ART) using plan selection is being introduced clinically for bladder cancer, but the challenge of how to compensate for intra-fractional motion remains. The purpose of this study was to assess target coverage with respect to intra-fractional motion and the potential for normal tissue sparing in MRI-guided ART (MRIGART) using isotropic (MRIGARTiso), an-isotropic (MRIGARTanIso) and population-based margins (MRIGARTpop).
MATERIALS AND METHODS: Nine bladder cancer patients treated in a phase II trial of plan selection underwent 6-7 weekly repeat MRI series, each with volumetric scans acquired over a 10 min period. Adaptive re-planning on the 0 min MRI scans was performed using density override, simulating a hypo-fractionated schedule. Target coverage was evaluated on the 10 min scan to quantify the impact of intra-fractional motion.
RESULTS: MRIGARTanIso reduced the course-averaged PTV by median 304 cc compared to plan selection. Bladder shifts affected target coverage in individual fractions for all strategies. Two patients had a v95% of the bladder below 98% for MRIGARTiso. MRIGARTiso decreased the bowel V25 with 15-46 cc compared to MRIGARTpop.
CONCLUSION: Online re-optimised ART has a considerable normal tissue sparing potential. MRIGART with online corrections for target shift during a treatment fraction should be considered in ART for bladder cancer.

PURPOSE: Although cigarette smoking is a well established risk factor for urothelial cancer, its role in urothelial cancer prognosis is still undetermined. In this meta-analysis we quantify the role of lifetime smoking history in bladder cancer recurrence, progression and survival by pooling available data on nonmuscle invasive bladder cancer, muscle invasive bladder cancer and upper tract urothelial carcinoma.
MATERIALS AND METHODS: A total of 24 studies, comprising data from 13,114 patients with bladder cancer and 2,259 patients with upper tract urothelial carcinoma, were included in this meta-analysis. Publication bias was addressed through Egger's test, and the heterogeneity among studies was assessed by the I(2) test statistic and subgroup analyses.
RESULTS: Current smokers at diagnosis are at increased risk for local recurrence in nonmuscle invasive bladder cancer (HR 1.27, 95% CI 1.09-1.46) and smokers with muscle invasive bladder cancer have an increased risk of dying of bladder cancer (HR 1.23, 95% CI 1.02-1.44). In the upper tract urothelial carcinoma population smokers have an increased risk of recurrence in the operative bed (HR 1.57, 95% CI 1.19-1.95) and of death from upper tract urothelial carcinoma (HR 1.53, 95% CI 1.13-1.92). We did not identify significant heterogeneity among included studies.
CONCLUSIONS: The body of evidence is limited due to the absence of prospective studies. However, the results from this meta-analysis unambiguously support the hypothesis that lifetime cigarette smokers are at increased risk for a more malignant type of urothelial carcinoma associated with a worse prognosis.

INTRODUCTION: Inappropriate DNA methylation is frequently associated with human tumour development, and in specific cases, is associated with clinical outcomes. Previous reports of DNA methylation in low/intermediate grade non-muscle invasive bladder cancer (NMIBC) have suggested that specific patterns of DNA methylation may have a role as diagnostic or prognostic biomarkers. In view of the aggressive and clinically unpredictable nature of high-grade (HG) NMIBC, and the current shortage of the preferred treatment option (Bacillus:Calmette-Guerin), novel methylation analyses may similarly reveal biomarkers of disease outcome that could risk-stratify patients and guide clinical management at initial diagnosis.
METHODS: Promoter-associated CpG island methylation was determined in primary tumour tissue of 36 initial presentation high-grade NMIBCs, 12 low/intermediate-grade NMIBCs and 3 normal bladder controls. The genes HOXA9, ISL1, NKX6-2, SPAG6, ZIC1 and ZNF154 were selected for investigation on the basis of previous reports and/or prognostic utility in low/intermediate-grade NMIBC. Methylation was determined by Pyrosequencing of sodium-bisulphite converted DNA, and then correlated with gene expression using RT-qPCR. Methylation was additionally correlated with tumour behaviour, including tumour recurrence and progression to muscle invasive bladder cancer or metastases.
RESULTS: The ISL1 genes' promoter-associated island was more frequently methylated in recurrent and progressive high-grade tumours than their non-recurrent counterparts (60.0% vs. 18.2%, p = 0.008). ISL1 and HOXA9 showed significantly higher mean methylation in recurrent and progressive tumours compared to non-recurrent tumours (43.3% vs. 20.9%, p = 0.016 and 34.5% vs 17.6%, p = 0.017, respectively). Concurrent ISL1/HOXA9 methylation in HG-NMIBC reliably predicted tumour recurrence and progression within one year (Positive Predictive Value 91.7%), and was associated with disease-specific mortality (DSM).
CONCLUSIONS: In this study we report methylation differences and similarities between clinical sub-types of high-grade NMIBC. We report the potential ability of methylation biomarkers, at initial diagnosis, to predict tumour recurrence and progression within one year of diagnosis. We found that specific biomarkers reliably predict disease outcome and therefore may help guide patient treatment despite the unpredictable clinical course and heterogeneity of high-grade NMIBC. Further investigation is required, including validation in a larger patient cohort, to confirm the clinical utility of methylation biomarkers in high-grade NMIBC.

OBJECTIVE: To compare early recurrence between good-quality white-light transurethral resection of bladder tumor (GQ-WLTURBT) and photodynamic diagnosis-assisted (PDD) transurethral resection of bladder tumor (TURBT) in a real-life controlled setting.
METHODS: A prospective controlled study was conducted commencing with a planned prospective cohort of patients with new tumors undergoing white-light TURBT in 2007-2008. Previously defined principles of GQ-WLTURBT for standardization and comparison of TURBT techniques, which are (1) cystoscopic mapping using a bladder diagram, (2) documented complete resection of the tumor, (3) resection performed or supervised by an experienced surgeon, (4) presence of detrusor muscle in the specimen, and (5) patient receiving mitomycin C within 24 hours of the resection, were applied. This was followed by a prospective cohort of new patients undergoing PDD-TURBT in 2009-2011. Only patients with new non-muscle-invasive bladder cancer (NMIBC) deemed to have had complete first TURBT were included for analysis. Tumor features and findings at first check cystoscopy and early re-TURBT (in high-risk NMIBC) were evaluated. Early recurrence (for calculating recurrence rate at first follow-up cystoscopy) was defined as pathologically confirmed tumor on early re-TURBT or recurrence at the first check cystoscopy. Comparison was analyzed between GQ-WLTURBT and good-quality PDD-TURBT (GQ-PDDTURBT).
RESULTS: A total of 808 patients were evaluated. The overall RRFFCs for GQ-WLTURBT and GQ-PDDTURBT were 30.9% and 13.6%, respectively (odds ratio = 2.9; 95% CI = 1.6-5.0; P <.001), with statistically significant lower recurrence rates in low- and intermediate-risk NMIBC after GQ-PDDTURBT.
CONCLUSION: Hexvix PDD-assisted TURBT is associated with a significantly lower risk of early recurrence compared with GQ-WLTURBT in a real-life clinical setting.

Paragangliomas of the urinary bladder are very rare. They are extra-adrenal autonomic nervous system tumors. This report presents a case of a 13-year-old boy with urinary bladder paraganglioma and the role of somatostatin receptor imaging with simultaneous positron emission tomography magnetic resonance (PET/MR). We have also described appearances of iodine-123 metaiodobenzylguanidine ((123)I-MIBG) single photon emission computed tomography (SPECT/CT) in comparison to somatostatin receptor imaging.

BACKGROUND: Gemcitabine plus cisplatin (GC) has been adopted as a neoadjuvant regimen for muscle-invasive bladder cancer despite the lack of Level I evidence in this setting.
METHODS: Data were collected using an electronic data-capture platform from 28 international centers. Eligible patients had clinical T-classification 2 (cT2) through cT4aN0M0 urothelial cancer of the bladder and received neoadjuvant GC or methotrexate, vinblastine, doxorubicin, plus cisplatin (MVAC) before undergoing cystectomy. Logistic regression was used to compute propensity scores as the predicted probabilities of patients being assigned to MVAC versus GC given their baseline characteristics. These propensity scores were then included in a new logistic regression model to estimate an adjusted odds ratio comparing the odds of attaining a pathologic complete response (pCR) between patients who received MVAC and those who received GC.
RESULTS: In total, 212 patients (146 patients in the GC cohort and 66 patients in the MVAC cohort) met criteria for inclusion in the analysis. The majority of patients in the MVAC cohort (77%) received dose-dense MVAC. The median age of patients was 63 years, they were predominantly men (74%), and they received a median of 3 cycles of neoadjuvant chemotherapy. The pCR rate was 29% in the MVAC cohort and 31% in the GC cohort. There was no significant difference in the pCR rate when adjusted for propensity scores between the 2 regimens (odds ratio, 0.91; 95% confidence interval, 0.48-1.72; P = .77). In an exploratory analysis evaluating survival, the hazard ratio comparing hazard rates for MVAC versus GC adjusted for propensity scores was not statistically significant (hazard ratio, 0.78; 95% confidence interval, 0.40-1.54; P = .48).
CONCLUSIONS: Patients who received neoadjuvant GC and MVAC achieved comparable pCR rates in the current analysis, providing evidence to support what has become routine practice.

UNLABELLED: Targeted cancer therapies offer great clinical promise, but treatment resistance is common, and basic research aimed at overcoming this challenge is limited by reduced genomic and biologic complexity in artificially induced rodent tumors compared with their human counterparts. Animal models that more faithfully recapitulate genotype-specific human pathology could improve the predictive value of these investigations. Here, a newly identified animal model for oncogenic BRAF-driven cancers is described. With 20,000 new cases in the United States each year, canine invasive transitional cell carcinoma of the bladder (InvTCC) is a common, naturally occurring malignancy that shares significant histologic, biologic, and clinical phenotypes with human muscle invasive bladder cancer. In order to identify somatic drivers of canine InvTCC, the complete transcriptome for multiple tumors was determined by RNAseq. All tumors harbored a somatic mutation that is homologous to the human BRAF(V600E) mutation, and an identical mutation was present in 87% of 62 additional canine InvTCC tumors. The mutation was also detectable in the urine sediments of all dogs tested with mutation-positive tumors. Functional experiments suggest that, like human tumors, canine activating BRAF mutations potently stimulate the MAPK pathway. Cell lines with the mutation have elevated levels of phosphorylated MEK, compared with a line with wild-type BRAF. This effect can be diminished through application of the BRAF(V600E) inhibitor vemurafenib. These findings set the stage for canine InvTCC as a powerful system to evaluate BRAF-targeted therapies, as well as therapies designed to overcome resistance, which could enhance treatment of both human and canine cancers
IMPLICATIONS: This study demonstrates the activating BRAF mutation (V600E), which is found in multiple human cancers, is a driver of canine InvTCC, and highlights a urine-based test for quick diagnosis.

Muscle invasive bladder carcinoma is a complex, multifactorial disease caused by disruptions and alterations of several molecular pathways that result in heterogeneous phenotypes and variable disease outcome. Combining this disparate knowledge may offer insights for deciphering relevant molecular processes regarding targeted therapeutic approaches guided by molecular signatures allowing improved phenotype profiling. The aim of the study is to characterize muscle invasive bladder carcinoma on a molecular level by incorporating scientific literature screening and signatures from omics profiling. Public domain omics signatures together with molecular features associated with muscle invasive bladder cancer were derived from literature mining to provide 286 unique protein-coding genes. These were integrated in a protein-interaction network to obtain a molecular functional map of the phenotype. This feature map educated on three novel disease-associated pathways with plausible involvement in bladder cancer, namely Regulation of actin cytoskeleton, Neurotrophin signalling pathway and Endocytosis. Systematic integration approaches allow to study the molecular context of individual features reported as associated with a clinical phenotype and could potentially help to improve the molecular mechanistic description of the disorder.

INTRODUCTION: Because of the lack of published evidence, this study was done to explore the decisions and rationale of uro-oncology consultants regarding the treatment of patients with muscle-invasive bladder cancer who have positive lymph nodes after radical cystectomy (RC) and neoadjuvant chemotherapy (NAC).
MATERIALS AND METHODS: An electronic survey was sent to UK pelvic cancer centers regarding: (1) choice of NAC regimen; (2) indications for reimaging; (3) choice and indication of adjuvant chemotherapy (AC) for patients with nodal disease after NAC and RC; (4) choice and indication of chemotherapy regimen if disease continues to progress in patients with advanced bladder cancer; and (5) guidelines used by those surveyed.
RESULTS: Consultant uro-oncologists from 77% of UK pelvic cancer centers responded, who treated a median of 13 patients per year with NAC before RC. Three cycles of gemcitabine and cisplatin was the most common NAC regimen, with 93% and 67% respondents giving it for downstaging of cN1- and cN2- and 3-positive patients, respectively. Forty-five percent would not give AC after NAC and RC in patients with positive lymph nodes. The patient's performance status, followed by response to NAC were key factors in dictating the use of AC. In the presence of disease progression, 46% of participants would use a taxane. Fifty-two percent of responders do not follow any guidelines.
CONCLUSION: In the United Kingdom, the treatment of patients with nodal disease after NAC and RC is variable. There is little evidence on which to base the management of such patients. The creation of national and international guidelines might help clinicians to optimize care for these patients.

Smoking is estimated to cause about half of all bladder cancer cases. Case-control studies have provided evidence of an inverse association between fruit and vegetable intake and bladder cancer risk. As part of the World Cancer Research/American Institute for Cancer Research Continuous Update Project, we conducted a systematic review and meta-analysis of prospective studies to assess the dose-response relationship between fruit and vegetables and incidence and mortality of bladder cancer. We searched PubMed up to December 2013 for relevant prospective studies. We conducted highest compared with lowest meta-analyses and dose-response meta-analyses using random effects models to estimate summary relative risks (RRs) and 95% confidence intervals (CIs), and used restricted cubic splines to examine possible nonlinear associations. Fifteen prospective studies were included in the review. The summary RR for an increase of 1 serving/day (80 g) were 0.97 (95% CI: 0.95-0.99) I(2)  = 0%, eight studies for fruits and vegetables, 0.97 (95% CI: 0.94-1.00, I(2)  = 10%, 10 studies) for vegetables and 0.98 (95% CI: 0.96-1.00, I(2)  = 0%, 12 studies) for fruits. Results were similar in men and women and in current, former and nonsmokers. Amongst fruits and vegetables subgroups, for citrus fruits the summary RR for the highest compared with the lowest intake was 0.87 (95% CI: 0.76-0.99, I(2)  = 0%, eight studies) and for cruciferous vegetables there was evidence of a nonlinear relationship (P = 0.001). The current evidence from cohort studies is not consistent with a role for fruits and vegetables in preventing bladder cancer.

BACKGROUND: Outcomes with cisplatin-based first-line therapy for advanced UC after previous perioperative cisplatin-based chemotherapy are unclear. In this study we evaluated outcomes with a focus on the effect of time from previous cisplatin-based perioperative chemotherapy.
PATIENTS AND METHODS: Data were collected for patients who received cisplatin-based first-line therapy for advanced UC after previous perioperative cisplatin-based therapy. Cox proportional hazards models were used to investigate the prognostic ability of visceral metastasis, ECOG PS, TFPC, anemia, leukocytosis, and albumin on overall survival (OS).
RESULTS: Data were available for 41 patients from 8 institutions including 31 men (75.6%). The median age was 61 (range, 41-77) years, most received gemcitabine plus cisplatin (n = 26; 63.4%), and the median number of cycles was 4 (range, 1-8). The median OS was 68 weeks (95% confidence interval [CI], 48.0-81.0). Multivariable Cox regression analysis results showed an independent prognostic effect on OS for PS > 0 versus 0 (hazard ratio [HR], 4.56 [95% CI, 1.66-12.52]; P = .003) and TFPC ≥ 78 weeks versus < 78 weeks (HR, 0.48 [95% CI, 0.21-1.07]; P = .072). The prognostic model for OS was internally validated with c-index = 0.68. Patients with TFPC < 52 weeks, 52 to 104 weeks, and ≥ 104 weeks had median survival of 42, 70, and 162 weeks, respectively.
CONCLUSION: Longer TFPC ≥ 78 weeks and ECOG PS = 0 were independently prognostic for better survival with cisplatin-based first-line chemotherapy for advanced UC after previous perioperative cisplatin-based chemotherapy. The data support using TFPC ≥ 52 weeks to rechallenge with cisplatin-based first-line chemotherapy for metastatic disease.

OBJECTIVE: To determine the rates of the available urinary diversion options for patients treated with radical cystectomy for bladder cancer in different settings (pioneering institutions, leading urologic oncology centers, and population based).
METHODS: Population-based data from the literature included all patients (n = 7608) treated in Sweden during the period 1964-2008, from Germany (n = 14,200) for the years 2008 and 2011, US patients (identified from National Inpatient Sample during 1998-2005, 35,370 patients and 2001-2008, 55,187 patients), and from Medicare (n = 22,600) for the years 1992, 1995, 1998, and 2001. After the International Consultation on Urologic Diseases-European Association of Urology International Consultation on Bladder Cancer 2012, the urinary diversion committee members disclosed data from their home institutions (n = 15,867), including the pioneering institutions and the leading urologic oncology centers. They are the coauthors of this report.
RESULTS: The receipt of continent urinary diversion in Sweden and the United States is <15%, whereas in the German high-volume setting, 30% of patients receive a neobladder. At leading urologic oncology centers, this rate is also 30%. At pioneering institutions up to 75% of patients receive an orthotopic reconstruction. Anal diversion is <1%. Continent cutaneous diversion is the second choice.
CONCLUSION: Enormous variations in urinary diversion exist for >2 decades. Increased attention in expanding the use of continent reconstruction may help to reduce these disparities for patients undergoing radical cystectomy for bladder cancer. Continent reconstruction should not be the exclusive domain of cystectomy centers. Efforts to increase rates of this complex reconstruction must concentrate on better definition of the quality-of-life impact, technique dissemination, and the centralization of radical cystectomy.

A genome-wide association study (GWAS) of bladder cancer identified a genetic marker rs8102137 within the 19q12 region as a novel susceptibility variant. This marker is located upstream of the CCNE1 gene, which encodes cyclin E, a cell-cycle protein. We performed genetic fine-mapping analysis of the CCNE1 region using data from two bladder cancer GWAS (5,942 cases and 10,857 controls). We found that the original GWAS marker rs8102137 represents a group of 47 linked SNPs (with r(2) ≥ 0.7) associated with increased bladder cancer risk. From this group, we selected a functional promoter variant rs7257330, which showed strong allele-specific binding of nuclear proteins in several cell lines. In both GWASs, rs7257330 was associated only with aggressive bladder cancer, with a combined per-allele OR = 1.18 [95% confidence interval (CI), 1.09-1.27, P = 4.67 × 10(-5)] versus OR = 1.01 (95% CI, 0.93-1.10, P = 0.79) for nonaggressive disease, with P = 0.0015 for case-only analysis. Cyclin E protein expression analyzed in 265 bladder tumors was increased in aggressive tumors (P = 0.013) and, independently, with each rs7257330-A risk allele (P(trend) = 0.024). Overexpression of recombinant cyclin E in cell lines caused significant acceleration of cell cycle. In conclusion, we defined the 19q12 signal as the first GWAS signal specific for aggressive bladder cancer. Molecular mechanisms of this genetic association may be related to cyclin E overexpression and alteration of cell cycle in carriers of CCNE1 risk variants. In combination with established bladder cancer risk factors and other somatic and germline genetic markers, the CCNE1 variants could be useful for inclusion into bladder cancer risk prediction models.

PURPOSE: In a recent phase II clinical trial, low-dose (100 mg/m(2)) gemcitabine showed promise as a radiosensitizer in bladder cancer, but underlying mechanisms lack elucidation. Here, we investigated the mechanism of radiosensitization by low-dose gemcitabine in bladder cancer cell lines.
EXPERIMENTAL DESIGN: Four bladder cancer cell lines were screened for radiosensitization by low-dose gemcitabine using clonogenic assay, and gemcitabine-resistant RT112gem and CALgem cells created by exposure to increasing gemcitabine doses. Four key gemcitabine-regulatory genes were knocked down by transient siRNA. Nude mice carrying CALgem subcutaneous xenografts were exposed to 100 mg/kg gemcitabine ± ionizing radiation (IR) and response assessed by tumor growth delay.
RESULTS: Gemcitabine was cytotoxic in the low nanomolar range (10-40 nmol/L) in four bladder cancer cell lines and radiosensitized all four lines. Sensitizer enhancement ratios at 10% survival were: RT112 1.42, CAL29 1.55, T24 1.63, and VMCUB1 1.47. Transient siRNA knockdown of deoxycytidine kinase (dCK) significantly reduced radiosensitization by gemcitabine (P = 0.02). RT112gem and CALgem cells displayed robust decreases of dCK mRNA and protein levels; reexpression of dCK restored gemcitabine sensitivity. However, CALgem xenografts responded better to combination gemcitabine/IR than either treatment alone (P < 0.001) with dCK strongly expressed in the tumor vasculature and stroma.
CONCLUSIONS: Gemcitabine resistance in bladder cancer cell lines was associated with decreased dCK expression, but gemcitabine-resistant xenografts were responsive to combination low-dose gemcitabine/IR. We propose that dCK activity in tumor vasculature renders it gemcitabine sensitive, which is sufficient to invoke a tumor response and permit tumor cell kill in gemcitabine-resistant tumors.

UNLABELLED: Activating mutations of FGFR3 are a common and early event in bladder cancer. Ectopic expression of mutant FGFR3 in normal urothelial cells has both pro-proliferative and antiapoptotic effects at confluence, suggesting that mutant cells are insensitive to cell-cell contact inhibition. Herein, detailed analysis revealed that these cells have reduced cell-cell adhesion, with large intercellular spaces observable at confluence, and diminished cell-substrate adhesion to collagen IV, collagen I, and fibronectin. These phenotypic alterations are accompanied by changes in the expression of genes involved in cell adhesion and extracellular matrix remodeling. Silencing of endogenous mutant FGFR3 in bladder cancer cells induced converse changes in transcript levels of CDH16, PLAU, MMP10, EPCAM, TNC, and HAS3, confirming them as downstream gene targets of mutant FGFR3. Overexpression of EPCAM, HAS3, and MMP10 transcripts was found in a large fraction of primary bladder tumors analyzed, supporting their key role in bladder tumorigenesis in vivo. However, no correlation was found between their protein and/or mRNA expression and FGFR3 mutation status in tumor specimens, indicating that these genes may be targeted by several converging oncogenic pathways. Overall, these results indicate that mutant FGFR3 favors the development and progression of premalignant bladder lesions by altering key genes regulating the cell-cell and cell-matrix adhesive properties of urothelial cells.
IMPLICATIONS: The ability of mutant FGFR3 to drive transcriptional expression profiles involved in tumor cell adhesion suggests a mechanism for expansion of premalignant urothelial lesions.

OBJECTIVES: To summarize the current status of clinicopathological and molecular markers for the prediction of recurrence or progression or both in non-muscle-invasive and survival in muscle-invasive urothelial bladder cancer, to address the reproducibility of pathology and molecular markers, and to provide directions toward implementation of molecular markers in future clinical decision making.
METHODS AND MATERIALS: Immunohistochemistry, gene signatures, and FGFR3-based molecular grading were used as molecular examples focussing on prognostics and issues related to robustness of pathological and molecular assays.
RESULTS: The role of molecular markers to predict recurrence is limited, as clinical variables are currently more important. The prediction of progression and survival using molecular markers holds considerable promise. Despite a plethora of prognostic (clinical and molecular) marker studies, reproducibility of pathology and molecular assays has been understudied, and lack of reproducibility is probably the main reason that individual prediction of disease outcome is currently not reliable.
CONCLUSIONS: Molecular markers are promising to predict progression and survival, but not recurrence. However, none of these are used in the daily clinical routine because of reproducibility issues. Future studies should focus on reproducibility of marker assessment and consistency of study results by incorporating scoring systems to reduce heterogeneity of reporting. This may ultimately lead to incorporation of molecular markers in clinical practice.

PURPOSE: Loss of epigenetic gene regulation through altered long noncoding RNA (lncRNA) expression seems important in human cancer. LncRNAs have diagnostic and therapeutic potential, and offer insights into the biology disease, but little is known of their expression in urothelial cancer. Here, we identify differentially expressed lncRNAs with potential regulatory functions in urothelial cancer.
EXPERIMENTAL DESIGN: The expression of 17,112 lncRNAs and 22,074 mRNAs was determined using microarrays in 83 normal and malignant urothelial (discovery) samples and selected RNAs with qPCR in 138 samples for validation. Significantly differentially expressed RNAs were identified and stratified according to tumor phenotype. siRNA knockdown, functional assays, and whole-genome transcriptomic profiling were used to identify potential roles of selected lncRNAs.
RESULTS: We observed upregulation of many lncRNAs in urothelial cancer that was distinct to corresponding, more balanced changes for mRNAs. In general, lncRNA expression reflected disease phenotype. We identified 32 lncRNAs with potential roles in disease progression. Focusing upon a promising candidate, we implicate upregulation of AB074278 in apoptosis avoidance and the maintenance of a proproliferative state in cancer through a potential interaction with EMP1, a tumor suppressor and a negative regulator of cell proliferation.
CONCLUSIONS: We report differential expression profiles for numerous lncRNA in urothelial cancer. We identify phenotype-specific expression and a potential mechanistic target to explain this observation. Further studies are required to validate lncRNAs as prognostic biomarkers in this disease.

UNLABELLED: Robotic radical cystectomy (RRC) has been growing in popularity across the world as a treatment option for bladder cancer.
OBJECTIVES: To compare early surgical outcomes for RRC and open radical cystectomy (ORC) with an emphasis on complications and postoperative mortality rates.

PURPOSE: FGFR3 is considered a good therapeutic target for bladder cancer. However, to our knowledge it is unknown whether the FGFR3 status of primary tumors is a surrogate for related metastases, which must be targeted by FGFR targeted systemic therapies. We assessed FGFR3 protein expression in primary bladder tumors and matched nodal metastases.
MATERIALS AND METHODS: We examined matched primary tumor and nodal metastases from 150 patients with bladder cancer clinically staged as N0M0. Four samples per patient were incorporated into a tissue microarray and FGFR3 expression was assessed by immunohistochemistry. FGFR3 expression was tested for an association with categorical clinical data using the Fisher exact test, and with overall and recurrence-free survival by Kaplan-Meier analysis.
RESULTS: Duplicate spots from primary tumors and lymph node metastases were highly concordant (OR 8.6 and 16.7, respectively, each p <0.001). Overall FGFR protein expression levels did not differ between primary and metastatic lesions (p = 0.78). Up-regulated expression was recorded in 53 of 106 evaluable primary tumor spots and 56 matched metastases. Concordance of FGFR3 expression levels in 79 matched primary tumor and metastasis specimens was high (OR 8.45, p <0.001). In 15 and 12 patients expression was up-regulated in only metastasis and in only the primary tumor, respectively. Overall and recurrence-free survival was not related to FGFR3 expression.
CONCLUSIONS: FGFR3 expression in matched primary and metastasized bladder cancer specimens showed good but not absolute concordance. Thus, in most patients primary tumor FGFR3 status can guide the selection of FGFR targeted therapy.

The aim of this study was to test the utility of AIMP3, an upstream regulator of DNA damage response following genotoxic stress, as a clinical biomarker in muscle-invasive bladder cancer (MIBC). AIMP3 was identified from a meta-analysis of a global gene-expression dataset. AIMP3 protein expression was determined by immunohistochemistry on a customised bladder cancer tissue-microarray (TMA). The mechanism of gene silencing was probed using methylation-specific PCR. The association between AIMP3 expression, Tp53 transactivity and genomic stability was analysed. In vitro AIMP3 translocation to the nucleus in response to ionising radiation was demonstrated using immunofluorescence. Radiosensitisation effects of siRNA-mediated AIMP3-knockdown were measured using colony forming assays. TMAs derived from patients enrolled in BCON, a Phase III multicentre radiotherapy trial in bladder cancer (ISRCTN45938399) were used to evaluate the association between AIMP3 expression and survival. The prognostic value of AIMP3 expression was determined in a TMA derived from patients treated by radical cystectomy. Loss of AIMP3 expression was frequent in MIBC and associated with impaired Tp53 transactivity and genomic instability. AIMP3-knockdown was associated with an increase in radioresistance. Loss of AIMP3 expression was associated with survival in MIBC patients following radiotherapy (HR = 0.53; 95% CI: 0.36 to 0.78, p = 0.002) but was not prognostic in the cystectomy set. In conclusion, AIMP3 expression is lost in a subset of bladder cancers and is significantly predictive of survival following radiotherapy in MIBC patients.

OBJECTIVES: Cisplatin is the key systemic chemotherapeutic agent used for bladder cancer, but chemoresistance is a major clinical problem. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) regulates various critical cellular processes, including cellular antioxidant response, cellular detoxification, and drug uptake/efflux. These processes, and the expression of multiple Nrf2 target genes, have been found to be associated with bladder cancer prognosis and chemotherapy resistance. We, therefore, investigated whether Nrf2 might regulate cisplatin resistance in bladder cancer.
MATERIALS AND METHODS: We first used bladder cancer cell lines, including a cisplatin-resistant RT112 subline (RT112-CP), to investigate Nrf2 expression and activation and its association with cisplatin response. We then undertook immunohistochemical analysis of a tissue microarray of archival bladder cancer radical cystectomy specimens to test the relevance of clinical Nrf2 expression to outcomes following either neoadjuvant chemotherapy and cystectomy or cystectomy alone.
RESULTS: Bladder cancer cell lines showed variable Nrf2 expression. Nrf2 expression was greater in RT112-CP cisplatin-resistant cells compared with that in parental RT112 cells. Nrf2 overexpression was functional in this model as it was associated with increased antioxidant response element reporter construct activity, Nrf2 target gene expression (metallothionein and glutathione reductase), and basal glutathione levels. Cisplatin resistance was associated with Nrf2 expression, and in RT112-CP cells, its depletion partially restored cisplatin sensitivity. We demonstrated increased cytoplasmic or nuclear Nrf2 expression or both in 32% of clinical bladder cancer samples compared with that in normal tissue samples. Expression of Nrf2 in bladder cancer following radical cystectomy was associated with unfavorable overall (median = 0.65 vs. 2.11 y, P = 0.045), bladder cancer-specific, and recurrence-free survival in those patients who also received neoadjuvant cisplatin-based chemotherapy but not in those treated with cystectomy alone.
CONCLUSIONS: Nrf2 overexpression in bladder cancer is associated with clinically relevant cisplatin resistance that is reversible in experimental models and should now be tested in prospective studies.

UNLABELLED: Activating mutations and/or overexpression of FGFR3 are common in bladder cancer, making FGFR3 an attractive therapeutic target in this disease. In addition, FGFR3 gene rearrangements have recently been described that define a unique subset of bladder tumors. Here, a selective HSP90 inhibitor, ganetespib, induced loss of FGFR3-TACC3 fusion protein expression and depletion of multiple oncogenic signaling proteins in RT112 bladder cells, resulting in potent cytotoxicity comparable with the pan-FGFR tyrosine kinase inhibitor BGJ398. However, in contrast to BGJ398, ganetespib exerted pleiotropic effects on additional mitogenic and survival pathways and could overcome the FGFR inhibitor-resistant phenotype of FGFR3 mutant-expressing 97-7 and MHG-U3 cells. Combinatorial benefit was observed when ganetespib was used with BGJ398 both in vitro and in vivo. Interestingly, two additional FGFR3 fusion-positive lines (RT4 and SW480) retained sensitivity to HSP90 inhibitor treatment by the ansamycins 17-AAG and 17-DMAG yet displayed intrinsic resistance to ganetespib or AUY922, both second-generation resorcinol-based compounds. Both cell lines, compared with RT112, expressed considerably higher levels of endogenous UGT1A enzyme; this phenotype resulted in a rapid glucuronidation-dependent metabolism and subsequent efflux of ganetespib from SW780 cells, thus providing a mechanism to account for the lack of bioactivity.
IMPLICATIONS: Pharmacologic blockade of the molecular chaperone HSP90 represents a promising approach for treating bladder tumors driven by oncogenic gene rearrangements of FGFR3. Furthermore, UDP-glucuronosyltransferase enzyme expression may serve as a predictive factor for clinical response to resorcinol-based HSP90 inhibitors.

PURPOSE: Low, intermediate and high risk categories have been defined to help guide the treatment of patients with nonmuscle invasive bladder cancer (Ta, T1, CIS). However, while low and high risk disease has been well classified, the intermediate risk category has traditionally comprised a heterogeneous group that does not fit into either of these categories. As a result, many urologists remain uncertain about the categorization of patients as intermediate risk as well as the selection of the most appropriate therapeutic option for this patient population. We review the current literature and clinical practice guidelines on intermediate risk nonmuscle invasive bladder cancer and, based on our findings, provide urologists with a better understanding of this heterogeneous risk group as well as practical recommendations for the treatment of intermediate risk patients.
MATERIALS AND METHODS: The IBCG analyzed published clinical trials, meta-analyses and current clinical practice guidelines on intermediate risk nonmuscle invasive bladder cancer available as of September 2013. The definitions of intermediate risk, patient outcomes and guideline recommendations were considered, as were the limitations of the available literature and additional parameters that may be useful in guiding treatment decisions in intermediate risk patients.
RESULTS: Current definitions and management recommendations for intermediate risk nonmuscle invasive bladder cancer vary. The most simple and practical definition is that proposed by the IBCG and the AUA of multiple and/or recurrent low grade Ta tumors. The IBCG suggests that several factors should be considered in clinical decisions in intermediate risk disease, including number (greater than 1) and size (greater than 3 cm) of tumors, timing (recurrence within 1 year) and frequency (more than 1 per year) of recurrence, and previous treatment. In patients without these risk factors a single, immediate instillation of chemotherapy is advised. In those with 1 to 2 risk factors adjuvant intravesical therapy (intravesical chemotherapy or maintenance bacillus Calmette-Guérin) is recommended, and previous intravesical therapy should be considered when choosing between these adjuvant therapies. For those patients with 3 to 4 risk factors, maintenance bacillus Calmette-Guérin is recommended. It is also important that all intermediate risk patients are accurately risk stratified at initial diagnosis and during subsequent followup. This requires appropriate transurethral resection of the bladder tumor, vigilance to rule out carcinoma in situ or other potential high risk tumors, and review of histological material directly with the pathologist.
CONCLUSIONS: Intermediate risk disease is a heterogeneous category, and there is a paucity of independent studies comparing therapies and outcomes in subgroups of intermediate risk patients. The IBCG has proposed a management algorithm that considers tumor characteristics, timing and frequency of recurrence, and previous treatment. Subgroup analyses of intermediate risk subjects in pivotal EORTC trials and meta-analyses will be important to validate the proposed algorithm and support clear evidence-based recommendations for subgroups of intermediate risk patients.

PURPOSE: Resistance to cisplatin-based chemotherapy is a major obstacle to bladder cancer treatment. We aimed to identify microRNAs (miRNA) that are dysregulated in cisplatin-resistant disease, ascertain how these contribute to a drug-resistant phenotype, and how this resistance might be overcome.
EXPERIMENTAL DESIGN: miRNA expression in paired cisplatin-resistant and -sensitive cell lines was measured. Dysregulated miRNAs were further studied for their ability to mediate resistance. The nature of the cisplatin-resistant phenotype was established by measurement of cisplatin/DNA adducts and intracellular glutathione (GSH). Candidate miRNAs were examined for their ability to (i) mediate resistance and (ii) alter the expression of a candidate target protein (SLC7A11); direct regulation of SLC7A11 was confirmed using a luciferase assay. SLC7A11 protein and mRNA, and miRNA-27a were quantified in patient tumor material.
RESULTS: A panel of miRNAs were found to be dysregulated in cisplatin-resistant cells. miRNA-27a was found to target the cystine/glutamate exchanger SLC7A11 and to contribute to cisplatin resistance through modulation of GSH biosynthesis. In patients, SLC7A11 expression was inversely related to miRNA-27a expression, and those tumors with high mRNA expression or high membrane staining for SLC7A11 experienced poorer clinical outcomes. Resistant cell lines were resensitized by restoring miRNA-27a expression or reducing SLC7A11 activity with siRNA or with sulfasalazine.
CONCLUSION: Our findings indicate that miRNA-27a negatively regulates SLC7A11 in cisplatin-resistant bladder cancer, and shows promise as a marker for patients likely to benefit from cisplatin-based chemotherapy. SLC7A11 inhibition with sulfasalazine may be a promising therapeutic approach to the treatment of cisplatin-resistant disease.

PURPOSE: To estimate the radiation equivalent of the chemotherapy contribution to observed complete response rates in published results of 1-phase radio-chemotherapy of muscle-invasive bladder cancer.
METHODS AND MATERIALS: A standard logistic dose-response curve was fitted to data from radiation therapy-alone trials and then used as the platform from which to quantify the chemotherapy contribution in 1-phase radio-chemotherapy trials. Two possible mechanisms of chemotherapy effect were assumed (1) a fixed radiation-independent contribution to local control; or (2) a fixed degree of chemotherapy-induced radiosensitization. A combination of both mechanisms was also considered.
RESULTS: The respective best-fit values of the independent chemotherapy-induced complete response (CCR) and radiosensitization (s) coefficients were 0.40 (95% confidence interval -0.07 to 0.87) and 1.30 (95% confidence interval 0.86-1.70). Independent chemotherapy effect was slightly favored by the analysis, and the derived CCR value was consistent with reports of pathologic complete response rates seen in neoadjuvant chemotherapy-alone treatments of muscle-invasive bladder cancer. The radiation equivalent of the CCR was 36.3 Gy.
CONCLUSION: Although the data points in the analyzed radio-chemotherapy studies are widely dispersed (largely on account of the diverse range of chemotherapy schedules used), it is nonetheless possible to fit plausible-looking response curves. The methodology used here is based on a standard technique for analyzing dose-response in radiation therapy-alone studies and is capable of application to other mixed-modality treatment combinations involving radiation therapy.

Bladder cancers commonly show genetic aberrations in the phosphatidylinositol 3-kinase signaling pathway. Here we have screened for mutations in PIK3R1, which encodes p85α, one of the regulatory subunits of PI3K. Two hundred and sixty-four bladder tumours and 41 bladder tumour cell lines were screened and 18 mutations were detected. Thirteen mutations were in C-terminal domains and are predicted to interfere with the interaction between p85α and p110α. Five mutations were in the BH domain of PIK3R1. This region has been implicated in p110α-independent roles of p85α, such as binding to and altering the activities of PTEN, Rab4 and Rab5. Expression of these mutant BH-p85α forms in mouse embryonic fibroblasts with p85α knockout indicated that all forms, except the truncation mutants, could bind and stabilize p110α but did not increase AKT phosphorylation, suggesting that BH mutations function independently of p110α. In a panel of 44 bladder tumour cell lines, 80% had reduced PIK3R1 mRNA expression relative to normal urothelial cells. This, along with mutation of PIK3R1, may alter BH domain functioning. Our findings suggest that mutant forms of p85α may play an oncogenic role in bladder cancer, not only via loss of ability to regulate p110α but also via altered function of the BH domain.

Targeted therapies have yet to have significant impact on the survival of patients with bladder cancer. In this study, we focused on the urea cycle enzyme argininosuccinate synthetase 1 (ASS1) as a therapeutic target in bladder cancer, based on our discovery of the prognostic and functional import of ASS1 in this setting. ASS1 expression status in bladder tumors from 183 Caucasian and 295 Asian patients was analyzed, along with its hypothesized prognostic impact and association with clinicopathologic features, including tumor size and invasion. Furthermore, the genetics, biology, and therapeutic implications of ASS1 loss were investigated in urothelial cancer cells. We detected ASS1 negativity in 40% of bladder cancers, in which multivariate analysis indicated worse disease-specific and metastasis-free survival. ASS1 loss secondary to epigenetic silencing was accompanied by increased tumor cell proliferation and invasion, consistent with a tumor-suppressor role for ASS1. In developing a treatment approach, we identified a novel targeted antimetabolite strategy to exploit arginine deprivation with pegylated arginine deiminase (ADI-PEG20) as a therapeutic. ADI-PEG20 was synthetically lethal in ASS1-methylated bladder cells and its exposure was associated with a marked reduction in intracellular levels of thymidine, due to suppression of both uptake and de novo synthesis. We found that thymidine uptake correlated with thymidine kinase-1 protein levels and that thymidine levels were imageable with [(18)F]-fluoro-L-thymidine (FLT)-positron emission tomography (PET). In contrast, inhibition of de novo synthesis was linked to decreased expression of thymidylate synthase and dihydrofolate reductase. Notably, inhibition of de novo synthesis was associated with potentiation of ADI-PEG20 activity by the antifolate drug pemetrexed. Taken together, our findings argue that arginine deprivation combined with antifolates warrants clinical investigation in ASS1-negative urothelial and related cancers, using FLT-PET as an early surrogate marker of response.