Papillary Bladder Cancer Research Articles

SUPERFICIAL BLADDER TUMOR;

Introduction: Bladder cancer is the second most common urologic cancer. Approximately 90% are transitional cellcarcinoma among which superficial bladder cancer constitutes about 50-70%. It is usually treated by transurethral resection withadjuvant intravesical instillations of chemotherapy or immunotherapy. Primary problems in superficial bladder cancers are its tendency torecur, about 50-80%, following surgical ablation alone, with progression to muscle invasive disease in 20-25% cases. Intravesicalchemotherapy appears to have major impact on decreasing chances of recurrence of superficial bladder cancer. Objective: To determinethe efficacy of single dose perioperative intravesical mitomycin C in reducing recurrence of superficial bladder tumor. Study Design:Comparative study. Settings: Department of Urology Shaikh Zayed Hospital Lahore. Duration of Study: One year.13-04-2009 to 13-04-2010. Methodology: Patients were divided into two groups randomly by using random numbers i.e. 40 patients in group A and 40 patientsin group B. Group A (40 patients) of bladder tumor received post TURBT single dose Mitomycin-C 40 mg/40ml N/Saline intravesically andGroup B (40 patients) was control group i.e. TURBT alone without Mitomycin-C. Results: The recurrence at first year follow up in bothgroups were showed a significant difference (p<0.05) as shown in Table No V: There were only 10% recurrence in Group A i.e. patientswho had intravesical Mitomycin –C as compared to 55% recurrence in Group B, who did not received postoperative intravesicalMitomycin–C. Conclusions: It is concluded that one perioperative (within 6 hours of TURBT) intravesical instillation of chemotherapysignificantly decreases the risk of recurrence after TURBT in patients with stage Ta T1, single and multiple papillary bladder cancer in lowrisk as well as high risk tumors. One immediate instillation after TUR reduces the recurrence.

1141 EXPRESSION MICROARRAY META-ANALYSIS IDENTIFIES GENES ASSOCIATED WITH RAS/MAPK AND RELATED PATHWAYS IN PROGRESSION OF MUSCLE-INVASIVE BLADDER TRANSITIONAL CELL CARCINOMA

You have accessJournal of UrologyBladder Cancer: Basic Research (III)1 Apr 20131141 EXPRESSION MICROARRAY META-ANALYSIS IDENTIFIES GENES ASSOCIATED WITH RAS/MAPK AND RELATED PATHWAYS IN PROGRESSION OF MUSCLE-INVASIVE BLADDER TRANSITIONAL CELL CARCINOMA Jonathan Ewald, Tracy M. Downs, Jeremy Cetnar, and William A. Ricke Jonathan EwaldJonathan Ewald Madison, WI More articles by this author , Tracy M. DownsTracy M. Downs Madison, WI More articles by this author , Jeremy CetnarJeremy Cetnar Madison, WI More articles by this author , and William A. RickeWilliam A. Ricke Madison, WI More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2013.02.756AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The effective detection and management of muscle-invasive bladder Transitional Cell Carcinoma (TCC) continues to be an urgent clinical challenge. While some differences of gene expression and function in papillary (Ta), superficial (T1) and muscle-invasive (≥T2) bladder cancers have been investigated, the understanding of mechanisms involved in the progression of bladder tumors remains incomplete. METHODS Statistical methods of pathway-enrichment, cluster analysis and text-mining can extract and help interpret functional information about gene expression patterns in large sets of genomic data. The public availability of patient-derived expression microarray data allows open access and analysis of large amounts of clinical data. Using these resources, we investigated gene expression differences associated with tumor progression and muscle-invasive TCC. RESULTS Gene expression was calculated relative to Ta tumors to assess progression-associated differences, revealing a network of genes related to Ras/MAPK and PI3K signaling pathways with increased expression. Further, we identified genes within this network that are similarly expressed in superficial Ta and T1 stages but altered in muscle-invasive T2 tumors, finding 7 genes (COL3A1, COL5A1, COL11A1, FN1, ErbB3, MAPK10 and CDC25C) whose expression patterns in muscle-invasive tumors are consistent in 5 to 7 independent outside microarray studies. CONCLUSIONS Our results suggest that increased expression of genes involved in mitogenic signaling supports the progression of muscle-invasive bladder tumors, while expression changes of fibrillar collagens, fibronectin and specific signaling proteins are associated with muscle-invasive disease. These results identify potential biomarkers and targets for TCC treatments, and provide an integrated systems-level perspective of TCC pathobiology to inform future studies. © 2013 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 189Issue 4SApril 2013Page: e466 Advertisement Copyright & Permissions© 2013 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jonathan Ewald Madison, WI More articles by this author Tracy M. Downs Madison, WI More articles by this author Jeremy Cetnar Madison, WI More articles by this author William A. Ricke Madison, WI More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Expression Microarray Meta-Analysis Identifies Genes Associated with Ras/MAPK and Related Pathways in Progression of Muscle-Invasive Bladder Transition Cell Carcinoma

The effective detection and management of muscle-invasive bladder Transition Cell Carcinoma (TCC) continues to be an urgent clinical challenge. While some differences of gene expression and function in papillary (Ta), superficial (T1) and muscle-invasive (≥T2) bladder cancers have been investigated, the understanding of mechanisms involved in the progression of bladder tumors remains incomplete. Statistical methods of pathway-enrichment, cluster analysis and text-mining can extract and help interpret functional information about gene expression patterns in large sets of genomic data. The public availability of patient-derived expression microarray data allows open access and analysis of large amounts of clinical data. Using these resources, we investigated gene expression differences associated with tumor progression and muscle-invasive TCC. Gene expression was calculated relative to Ta tumors to assess progression-associated differences, revealing a network of genes related to Ras/MAPK and PI3K signaling pathways with increased expression. Further, we identified genes within this network that are similarly expressed in superficial Ta and T1 stages but altered in muscle-invasive T2 tumors, finding 7 genes (COL3A1, COL5A1, COL11A1, FN1, ErbB3, MAPK10 and CDC25C) whose expression patterns in muscle-invasive tumors are consistent in 5 to 7 independent outside microarray studies. Further, we found increased expression of the fibrillar collagen proteins COL3A1 and COL5A1 in muscle-invasive tumor samples and metastatic T24 cells. Our results suggest that increased expression of genes involved in mitogenic signaling may support the progression of muscle-invasive bladder tumors that generally lack activating mutations in these pathways, while expression changes of fibrillar collagens, fibronectin and specific signaling proteins are associated with muscle-invasive disease. These results identify potential biomarkers and targets for TCC treatments, and provide an integrated systems-level perspective of TCC pathobiology to inform future studies.

The association between the recurrence of solitary non-muscle invasive bladder cancer and tumor infiltrating lymphocytes

AimTo evaluate whether tumor infiltrating lymphocytes (TIL) in biopsy specimens are associated with the clinical outcome of non-muscle invasive bladder cancer.MethodsWe retrieved tumor specimens from 115 patients with solitary papillary non-muscle invasive bladder cancer treated between 1996 and 2006 and constructed tissue microarrays. Patients were divided in two groups: those with recurrent disease (N = 69) and those without recurrent disease (N = 46) during the follow up of minimum 5 years. All patients were treated with initial transurethral resection and none received adjuvant therapy. Immunhistochemical staining was performed with anti-CD3, CD4, CD8, and Granzyme B (GrB). The CD4+:CD8+ and GrB+:CD8 ratios were determined.ResultsTumor infiltrating lymphocytes were predominantly observed within cancer stroma, and only rare individual cells were observed intraepithelially. The group without recurrent disease had lower levels of CD3+ and CD8+ lymphocytes than the group with recurrent disease (P = 0.0001, P = 0.0002, respectively). The CD4+:GrB+ and GrB+:CD8+ ratios were significantly higher in patients without recurrent disease (P = 0.0002, P = 0.039, respectively).ConclusionThis study revealed a possible connection between TIL number and bladder cancer recurrence. TIL subset ratio showed different patterns in recurrent and non-recurrent tumors, which is why it could become a useful a prognostic clinical index if our findings are confirmed in randomized trials.

Lower cyclooxygenase-2 expression is associated with recurrence of solitary non-muscle invasive bladder carcinoma.

BackgroundA new modality is necessary to prevent recurrence of superficial bladder cancer after complete transurethral resection because of the high recurrence rate even with current prophylaxis protocols.MethodsIn order to analyze the predictive value of cyclooxygenase-2 (COX-2) expression and tumor infiltrating lymphocytes (TILs) in recurrence of this disease tumor specimens from 127 patients with solitary papillary non-muscle invasive bladder cancer (NMIBC), 78 with recurrent disease and 49 without recurrence during follow up of minimum 5 years, were retrieved for tissue microarrays construction and immunohistochemical analysis. COX-2 expression was scored according to Allred’s scoring protocol, while presence of TILs was categorized as absent (no) or present (yes) on whole tissue sections.ResultsCOX-2 immunoreactivity was presented in 70 (71%), weak in 16% and strong in 55% of cases, while 29 (29%) tumors were negative. TILs were present in 64 (58%) NMIBC, while 44 cases (41%) did not reveal mononuclear infiltration in tumoral stroma. Statistical analysis demonstrated a higher proportion of patients with recurrence in the group with the COX-2 score 0, and lower in the group with score 2 (p=0.0001, p=0.0101, respectively). In addition, a higher proportion of recurrent patients in the group with no TILs, and lower proportion in the group with TILs were found (p=0.009, p=0.009, respectively). Univariate and multivariate analysis revealed overexpression of COX-2 and presence of TILs as negative predictors.ConclusionPatients with lower COX-2 expression and absence of TILs in NMIBC need to be followed up more vigorously and probably selected for adjuvant therapy.Virtual slideThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1411318819790406

Proteomic tissue profiling for the improvement of grading of noninvasive papillary urothelial neoplasia

ObjectivesIn 2004, a novel grading system for papillary non-invasive bladder cancer was introduced; low grade (LG) and high grade (HG) in lieu of the former G1, G2, G3. This change allowed for increased reproducibility as well as diminished interobserver variability in histopathological grading among individual pathologists. Matrix Assisted Laser Desorption/Ionization Time of Flight Imaging Mass Spectrometry (MALDI TOF IMS) was evaluated as an automatic and objective tool to assist grading of urothelial neoplasms and to facilitate accuracy. Design and methodsTo separate G1 (LG, n=27) and G3 (HG, n=21) papillary tumors MALDI TOF IMS was performed using an appropriate algorithm. Thereafter, the automatic assignment of a separate G2 (n=31) group was completed. ResultsG1 (LG) and G3 (HG) tumors were separated with an overall cross validation of 97.18%. G2 tumors indicated a true positive rate of 78.3% for LG and 87.5% for HG, respectively. ConclusionsMALDI TOF IMS is a powerful support tool to ascertain pathological diagnosis/grading.

Abstract 3862: Functional characterization of sFRP1 expression loss in human papillary bladder cancer

Abstract Introduction: Secreted frizzled-related protein 1 (sFRP1) is an inhibitor of the Wnt-signaling pathway and expression loss is associated with e.g. colon and breast cancer. Our previous studies revealed an involvement of sFRP1 loss in progression of papillary bladder cancer. We therefore performed functional studies to characterise the role of sFRP1 in papillary bladder cancer cell lines. Material and methods: Human papillary bladder cancer cell lines RT112 and BFTC 905 were used for the analyses. mRNA-expression status of sfrp1 and Wnt-target genes c-myc and cyclin D1 was determined using qRT-PCR. Methylation status of sfrp1 promoter was analysed with MS-PCR. sFRP1 knockdown was performed using sFRP1-specific siRNAs. 5’ Aza-2’deoxycytidine (5’Aza) treatment was used for promoter demethylation and restoration of sFRP1 expression. To determine Wnt pathway activity a luminescence-based TOP flash/FOP flash assay was used. Proliferation level was investigated with ELISA-based BrdU Cell Proliferation assay. Results: RT112 is strongly expressing sfrp1 on mRNA level, whereas BFTC905 showed no sfrp1 expression. MS-PCR revealed sfrp1-promoter methylation in BFTC905. This methylation could be reversed after 5’-Aza-treatment and led to re-expression of sfrp1 on mRNA level. sfrp1 siRNA transfection in RT112 led to ∼80% gene silencing. sfrp1 knockdown resulted in an upregulation of c-myc (515%) and cyclin D1 (51,5%) on mRNA level, however TCF/LEF-presence could not be detected, neither in untreated nor in siRNA transfected RT112 cells. Untreated BFTC905 cells showed a very weak Wnt signalling activitiy, compared to positive control cell line SW480, but 7-fold higher than in untreated and siRNA treated RT112. sfrp1 knowdown in RT112 had no influence on cell proliferation. Conclusion: sfrp1 knockdown in RT112 cells is associated with an up-regulation of proliferation-associated genes c-myc and cyclin D1, whereas this increased expression showed no effects on proliferation behaviour of the cells. Knockdown of sFRP1 did not result in significant Wnt-signaling activity in RT112 cells. Reduced sFRP1 expression might increase the proliferative potential of the cells; however additional genetic events might have to occur to induce increased proliferation of the cells. Ongoing analyses of BFTC905 with transient restoration of sFRP1 expression will give further insights into the role of sFRP1 in papillary bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3862. doi:10.1158/1538-7445.AM2011-3862

Abstract 234: Fibroblast growth factor receptor (FGFR)-3 as a suitable target in bladder cancer

Abstract Introduction and Objective: Mutations in the FGFR3 gene, which can confer constitutive activity, are found in up to 80% of low grade papillary bladder cancers (BC). Overexpression of FGFR3 has been detected in approximately half of high grade and invasive tumors. In this study we evaluate FGFR3 in a cohort of American patients and investigate the effect of targeting FGFR3 in pre-clinical models of bladder cancer. Materials and Methods: DNA was extracted from 170 fresh frozen BC specimens using a commercial kit and PCR of exons 7 and 10 was performed. Mutation analysis was performed by ABI Big Dye Terminator Cycle Sequencing. FGFR3 expression was analyzed by immunohistochemistry in an overlapping cohort of 143 patients with BC. FGFR3 expression, phosphorylation and downstream signaling were measured in a panel of BC cell lines, and the efficacy of two small molecule inhibitors (TKI258 and BGJ398) was tested on signaling and 3H-thymidine incorporation. A specific inhibitory monoclonal antibody targeting FGFR3 was evaluated in an orthotopic xenograft model of BC. Tumor burden was measured with bioluminescent imaging. Results: FGFR3 mutations were identified in 44 (26%) BC samples, including 28/50 (56%) of low grade papillary lesions. Overexpression of FGFR3 was noted in 53% of all BC specimens regardless of stage and grade, but not in normal urothelium. FGFR3 inhibition effectively abrogated constitutive phosphorylation of FGFR3 and p42/44MAPK in a subset of bladder cancer cell lines. A growth inhibitory effect was observed in the same cell lines. The growth of UC14 xenografts in the bladder of nude mice was inhibited by 40%. Conclusion: These studies underline the potential clinical relevance of FGFR3 mutation and expression in bladder cancer. Furthermore, the preclinical investigations offer proof of principle for moving forward with clinical trials studying the administration of specific FGFR3 inhibitors in patients with bladder cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 234. doi:10.1158/1538-7445.AM2011-234

966 SH3GL2 POTENTIALLY SUPPRESSES BLADDER CANCER THROUGH INHIBITION OF EGFR INTERNALIZATION

You have accessJournal of UrologyBladder Cancer: Basic Research II1 Apr 2010966 SH3GL2 POTENTIALLY SUPPRESSES BLADDER CANCER THROUGH INHIBITION OF EGFR INTERNALIZATION Edward M. Gong, Dolores Di Vizio, and Rosalyn M. Adam Edward M. GongEdward M. Gong More articles by this author , Dolores Di VizioDolores Di Vizio More articles by this author , and Rosalyn M. AdamRosalyn M. Adam More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.1913AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Bladder cancer is the fourth most common cancer in men, causing over 14,000 deaths per year in both men and women. However, the molecular mechanisms underlying urothelial carcinogenesis are still largely unknown. Recent informatics analysis in our group identified Sh3gl2 as a bladder urothelium-enriched transcript and protein. Sh3gl2, a mediator of endocytosis of cell surface receptors, including the epidermal growth factor receptor (EGFR), has been implicated as a possible tumor suppressor gene in breast and laryngeal cancers. However its role in bladder cancer pathogenesis is unknown. The objective of the study was to determine the role of Sh3gl2 in urothelial carcinoma and explore its association with the EGFR, a protein over-expressed in bladder cancer and associated with increased progression and mortality. METHODS Sh3gl2 levels in normal bladder tissues and bladder cancer specimens were determined by immunohistochemistry (IHC) of human bladder cancer tissue microarrays (TMA). Sh3gl2 expression and EGFR activation in bladder cancer cell lines were determined by immunoblotting. Interaction between Sh3gl2 and EGFR was assessed by reciprocal co-immunoprecipitation (IP). Sh3gl2 was knocked down using RNA interference and the impact on the EGFR determined by indirect immunofluorescence (IF) and immunoblotting. RESULTS TMA IHC demonstrated statistically significant loss of Sh3gl2 expression in bladder cancer compared to matched normal controls, as well as loss of expression with increased tumor grade. Sh3gl2 expression was also lost in aggressive human bladder cancer lines, but preserved in RT4, a grade 1 papillary bladder cancer cell line. In RT4, Sh3gl2 and EGFR formed a protein complex, with increased binding evident after EGF stimulation, as determined by co-IP. SiRNA-mediated Sh3gl2 knockdown inhibited ligand-induced EGFR internalization in RT4 cells, consistent with sustained EGFR-dependent signaling. Immunoblot data demonstrated increased phosphorylation of EGFR Y1045, EGFR 1086, and ERK with Sh3gl2 knockdown. CONCLUSIONS Sh3gl2 is a potential tumor suppressor gene in bladder cancer. Sh3gl2 forms a protein complex with EGFR, and knockdown of Sh3gl2 prevents EGF-induced receptor downregulation. Thus, loss of Sh3gl2 with tumor progression may lead to persistent EGFR-dependent signaling. This may result in increased downstream activation of the Erk-MAPK pathway and subsequent cell cycle alterations. Boston, MA© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e375 Peer Review Report Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Edward M. Gong More articles by this author Dolores Di Vizio More articles by this author Rosalyn M. Adam More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

UP-3.006: Association of Low Expression of Notch-1 and Jagged-1 in Human Papillary Bladder Cancer and Shorter Survival

UP-3.006: Association of Low Expression of Notch-1 and Jagged-1 in Human Papillary Bladder Cancer and Shorter Survival

Predicting Recurrence and Progression of Noninvasive Papillary Bladder Cancer at Initial Presentation Based on Quantitative Gene Expression Profiles

BackgroundCurrently, tumor grade is the best predictor of outcome at first presentation of noninvasive papillary (Ta) bladder cancer. However, reliable predictors of Ta tumor recurrence and progression for individual patients, which could optimize treatment and follow-up schedules based on specific tumor biology, are yet to be identified. ObjectiveTo identify genes predictive for recurrence and progression in Ta bladder cancer at first presentation using a quantitative, pathway-specific approach. Design, setting, and participantsRetrospective study of patients with Ta G2/3 bladder tumors at initial presentation with three distinct clinical outcomes: absence of recurrence (n=16), recurrence without progression (n=16), and progression to carcinoma in situ or invasive disease (n=16). MeasurementsExpressions of 24 genes that feature in relevant pathways that are deregulated in bladder cancer were quantified by real-time polymerase chain reaction on tumor biopsies from the patients at initial presentation. Results and limitationsCCND3 (p=0.003) and HRAS (p=0.01) were predictive for recurrence by univariate analysis. In a multivariable model based on CCND3 expression, sensitivity and specificity for recurrence were 97% and 63%, respectively. HRAS (p<0.001), E2F1 (p=0.017), BIRC5/Survivin (p=0.038), and VEGFR2 (p=0.047) were predictive for progression by univariate analysis. Multivariable analysis based on HRAS, VEGFR2, and VEGF identified progression with 81% sensitivity and 94% specificity. Since this is a small retrospective study using medium-throughput profiling, larger confirmatory studies are needed. ConclusionsGene expression profiling across relevant cancer pathways appears to be a promising approach for Ta bladder tumor outcome prediction at initial diagnosis. These results could help differentiate between patients who need aggressive versus expectant management.

A phase I study of intravesical Ad-IFN in superficial bladder cancer patients

e16048 Background: Intravesical BCG (Bacillus Calmette-Guérin) is the gold standard for treatment of superficial bladder cancer. Recurrence is a significant problem. Intravesical interferon α (IFN) demonstrated complete response (CR) in ∼40% patients with most relapsing &lt;1 year. Intravesical Ad-IFN takes advantage of the bladder to allow prolonged exposure to high concentrations of IFN; this is expected to potentiate durable therapeutic responses. Syn3 is an excipient used to increase adenoviral vector (Ad) transfection. Methods: The phase I study is a nonrandomized, open-label, rising-dose, multi-center study of Ad-IFN in patients with papillary bladder cancer, or carcinoma in situ refractory to BCG. Patients with T1 were not enrolled unless they refused cystectomy. A minimum of 3 subjects were enrolled at each dose level. Adverse events and lab abnormalities were graded according to CTC version 3 criteria. The occurrence of treatment-related Grade 3 or Grade 4 toxicity is considered dose-limiting toxicity (DLT). The maximum tolerated dose is defined as the dose which results in DLT in ≤1/6 subjects. Urine from pre-treatment, posttreatment days 0–7, 10, 14, and 28 were tested for IFN, Syn3, Ad-IFN DNA, and research biomarkers. Blood was assayed for IFN, Syn3, Ad-IFN DNA, anti- adenovirus and anti-IFN antibodies. Results: Intravesical administration of 3×109, 1×1010, 3x1010, 1×1011, or 3×1011 particles/ml of Ad-IFN (75ml) in 1mg/ml Syn3 is safe with only minor initial urgency controlled with anticholinergic medication. Of the 13 evaluable patients, 6 are CR, defined as a negative cytology and biopsy at 3 months. CR patients were offered a second dose. Response duration is variable with longest &gt;1 year. Urinary IFN was seen for all patients in a dose-dependent fashion. TRAIL, IP-10, M65, and M30 were also seen. Conclusions: Initial safety and response activity justify further clinical development. [Table: see text]

Reduced bladder cancer recurrence rate with cardioprotective aspirin after intravesical bacille Calmette‐Guérin

To evaluate the recurrence-free survival (RFS) rate of patients taking cardioprotective aspirin after intravesical bacille Calmette-Guérin (BCG) for high-grade noninvasive urothelial carcinoma of the bladder, as preventing the recurrence of superficial bladder cancer might decrease patient morbidity and mortality from this disease, and nonsteroidal anti-inflammatory agents (NSAIDs) have shown promise in preclinical prevention through inhibition of the prostaglandin pathway and other mechanisms. In all, 43 patients with carcinoma in situ (CIS) and/or high-grade papillary bladder cancer were treated with intravesical BCG. Patients were stratified according to whether they took cardioprotective aspirin after treatment, and Kaplan-Meier curves of RFS were compared by log-rank analysis. Multivariable analysis was used for potentially confounding factors, including maintenance BCG, the presence of CIS, and smoking status. Of patients taking cardioprotective aspirin, the 5-year RFS rate was 64.3%, compared with 26.9% for patients not taking aspirin, with a significantly higher RFS by univariable log rank analysis (P = 0.03). Even after adjusting for the other factors by multivariable analysis, aspirin seems to affect recurrence (hazard ratio 0.179, P = 0.001). Maintenance BCG (hazard ratio 0.233, P = 0.02) and smoking history (hazard ratio 3.199, P = 0.05) also significantly affected recurrence. There was a significantly higher RFS rate in patients taking cardioprotective aspirin after intravesical BCG therapy for bladder cancer. The results of this study support the further investigation of aspirin and other NSAIDs as preventive agents in patients being treated for superficial bladder cancer.

Carcinoma in situ of the Urinary Bladder: Review of Clinicopathologic Characteristics with an Emphasis on Aspects Related to Molecular Diagnostic Techniques and Prognosis

Carcinoma in situ (CIS) of the urinary bladder is defined as a flat lesion comprising of cytologically malignant cells which may involve either full or partial thickness of the urothelium. De novo CIS constitutes less than 3% of all urothelial neoplasms; however, CIS detected concurrently or secondarily during follow-up of urothelial carcinoma constitutes 45% and 90%, respectively, of bladder cancer. CIS is noted predominantly in male smokers in the sixth or seventh decade. Patients may present with dysuria, nocturia, and urinary frequency and urgency with microscopic hematuria. Cystoscopic findings may range from unremarkable to erythema or edema. Urine cytology is an important diagnostic tool. Cellular anaplasia, loss of polarity, discohesion, nuclear enlargement, hyperchromasia, pleomorphism, and atypical mitoses are the histopathologic hallmarks of CIS. Extensive denudation of the urothelium, monomorphic appearance of the neoplastic cells, inflammatory atypia, radiation induced nuclear smudging, multinucleation, and pagetoid spread of CIS may cause diagnostic difficulties. Together with clinical and morphologic correlation, immunostaining with CK 20, p53 (full thickness), and CD44 (absence of staining) may help accurately diagnose CIS. Fluorescent in situ hybridization analysis of voided urine for amplification of chromosomes 3, 7, and 17 and deletion of 9p has high sensitivity and specificity for diagnosing CIS in surveillance cases. Several other molecular markers, such as NMP 22 and BTA, are under evaluation or used variably in clinical pathology. Intravesical bacillus Calmette-Guerin (BCG) instillation is considered the preferred treatment, with radical cystectomy being offered to refractory cases. Chemotherapy, alpha-interferon, and photodynamic therapy are other modalities that can be considered in BCG-refractory cases. Multifocality, involvement of prostatic urethra, and response to BCG remain the most important prognostic factors, although newer molecular markers are being evaluated for this entity. Patient outcome varies based on whether it is de novo development or diagnosed secondary to prior or concomitant papillary bladder cancer. From a clinical perspective, the principal determinants of outcome are extent of disease, involvement of prostatic urethra, response to therapy, and time to recurrence.

Effects of the Kava Chalcone Flavokawain A Differ in Bladder Cancer Cells with Wild-type versus Mutant p53

Flavokawain A is the predominant chalcone from kava extract. We have assessed the mechanisms of flavokawain A's action on cell cycle regulation. In a p53 wild-type, low-grade, and papillary bladder cancer cell line (RT4), flavokawain A increased p21/WAF1 and p27/KIP1, which resulted in a decrease in cyclin-dependent kinase-2 (CDK2) kinase activity and subsequent G(1) arrest. The increase of p21/WAF1 protein corresponded to an increased mRNA level, whereas p27/KIP1 accumulation was associated with the down-regulation of SKP2, which then increased the stability of the p27/KIP1 protein. The accumulation of p21/WAF1 and p27/KIP1 was independent of cell cycle position and thus not a result of the cell cycle arrest. In contrast, flavokawain A induced a G(2)-M arrest in six p53 mutant-type, high-grade bladder cancer cell lines (T24, UMUC3, TCCSUP, 5637, HT1376, and HT1197). Flavokawain A significantly reduced the expression of CDK1-inhibitory kinases, Myt1 and Wee1, and caused cyclin B1 protein accumulation leading to CDK1 activation in T24 cells. Suppression of p53 expression by small interfering RNA in RT4 cells restored Cdc25C expression and down-regulated p21/WAF1 expression, which allowed Cdc25C and CDK1 activation, which then led to a G(2)-M arrest and an enhanced growth-inhibitory effect by flavokawain A. Consistently, flavokawain A also caused a pronounced CDK1 activation and G(2)-M arrest in p53 knockout but not in p53 wild-type HCT116 cells. This selectivity of flavokawain A for inducing a G(2)-M arrest in p53-defective cells deserves further investigation as a new mechanism for the prevention and treatment of bladder cancer.

Association of Low Expression of Notch-1 and Jagged-1 in Human Papillary Bladder Cancer and Shorter Survival

We investigated the expression of Notch receptors and ligands in normal bladder transitional epithelium and transitional cell carcinoma of the bladder. We also explored its clinical and pathological implications. The expression of Notch-1 to 3, Jagged-1 and Delta-like-1 was detected respectively in 70 cases of bladder carcinoma, 10 of normal urothelium and the 2 cell lines T24 and BIU-87 using immunohistochemistry. Reverse transcriptase-polymerase chain reaction and Western blot were used to assay the expression level of Notch-1 and Jagged-1. The predictive value of this expression for prognosis was investigated by Kaplan-Meier curves and Cox proportional hazards analysis in a multivariate model. All 5 kinds of Notch factors were intensively stained in normal bladder transitional epithelium immunohistochemically but expression was significantly decreased in tumor tissues. Moreover, expression of the 5 genes in papillary tumors was lower than in invasive tumors but only Notch-1 and Jagged-1 showed a statistically significant difference. Postoperative disease-free survival time in patients with low Notch-1 plus Jagged-1 expression was significantly shorter than that in patients with other expression patterns in papillary tumors (p = 0.014). Multivariate Cox proportional hazards model analysis identified Jagged-1 expression as an independent prognostic factor for disease-free survival (RR 3.09, p = 0.011). The Notch family expression pattern in papillary bladder transitional cell carcinoma is different from that in invasive bladder transitional cell carcinoma. Low expression of Notch-1 as well as Jagged-1 is potentially a useful marker for survival in patients with papillary bladder transitional cell carcinoma.

Radical Cystectomy for Carcinoma of the Bladder: 2,720 Consecutive Cases 5 Years Later

We performed a critical analysis of the results of radical cystectomy for invasive bladder carcinoma treated at 1 center. Between 1970 and 2000, 2,090 men and 630 women with invasive bladder cancer were treated with 1-stage radical cystectomy and urinary diversion. Followup ranged from 0 to 34.2 years with a mean of 5.5 +/- 5.7. Survival data were correlated to patient and tumor characteristics using univariate and multivariate analysis. Postoperative mortality was 2.6%. Squamous tumors accounted for 49.4% of cases, transitional cell carcinoma for 36.4% and adenocarcinoma for 9.6%. Regional lymph nodes were involved in 20.4% of cases. The 5 and 10-year disease-free survival rates were 55.5% and 50.03%, respectively. Evidence was provided that tumor stage, histological grade and lymph node status are the only independent variables which affect survival probability. Contemporary cystectomy can be performed with minimal mortality. Radical cystectomy for organ confined disease is followed by good therapeutic results and enhances the possibilities for functional restoration. With stage progression there is a stepwise reduction in survival probability. The radical operation can provide disease-free survival for an important subgroup of node positive cases (27.3%). Additional therapy is needed to improve the oncological outcome for advanced locoregional disease.

FGFR3 mutations and a normal CK20 staining pattern define low-grade noninvasive urothelial bladder tumors: van Oers JM, Wild PJ, Burger M, Denzinger S, Stoehr R, Rosskopf E, Hofstaedter F, Steyerberg EW, Klinkhammer-Schalke M, Zwarthoff EC, van der Kwast TH, Hartmann A, Department of Pathology, Erasmus MC, Rotterdam, The Netherlands.

Objectives Molecular markers superior to conventional clinicopathologic parameters are needed to predict disease courses in bladder cancer patients. In this study, we investigated four markers (Ki-67, TP53, CK20, FGFR3) in primary urothelial bladder tumors and compared them with traditional pathologic features. Methods Tissue microarrays were used to analyze CK20, TP53, and Ki-67 expression immunohistochemically in 255 unselected patients. FGFR3 mutations were detected by SNaPshot analysis. Results Abnormal CK20 expression was strongly associated with higher tumor grades and stages (P Conclusions Dysregulation of CK20 expression is an early event in the carcinogenesis of papillary noninvasive bladder cancer, but occurs later than FGFR3 mutations. The group of low-grade noninvasive papillary tumors is defined by the presence of an FGFR3 mutation and a normal CK20 expression pattern.

Feasibility of early intravesical instillation chemotherapy after transurethral resection of the bladder: A prospective evaluation in a consecutive series of 210 cases

Early instillation chemotherapy (less than 6 h after tumour resection) is an accepted adjuvant treatment after transurethral resection of non-muscle-invasive papillary bladder tumours. Because most studies have reported on selected patients fulfilling specific eligibility criteria, this study investigated the feasibility of this therapy in a non-selected, consecutive series of patients who had undergone transurethral surgery to the bladder at a single institution. All transurethral resections of the bladder were prospectively evaluated. In patients with assumed non-muscle-invasive papillary bladder cancer, resection was followed by early instillation of 50mg epirubicin. Practical problems, staging and cystoscopic follow-up were systematically registered and evaluated. From October 2002 to February 2005, 210 transurethral resections (including 31 diagnostic biopsies) were performed in 163 patients (median age at resection 73.8 years). The following pathological T-stages were found: pT0 27.6%, pTa 39.0%, pT1 8.6%, > or =pT2 19.0% and pTis 5.7%. Patients received early instillation chemotherapy in 110 cases, which was generally well tolerated, but was prevented in four patients by intense bleeding or perforation. The treatment decision was correct in 82.8% and positively correlated with the experience of the treating urologist. Cumulative incidence rates of first postoperative tumour recurrence in the pTa group at 6, 12 and 24 months were 6.7, 24.5 and 52.0%, respectively. Early instillation chemotherapy with epirubicin after transurethral resection of bladder tumours is generally feasible and usually has no major side-effects, but evaluation of intraoperative T-stage can be a problem and depends on experience.

Methylation of the TPEF‐ and PAX6‐promoters is increased in early bladder cancer and in normal mucosa adjacent to pTa tumours

To evaluate CpG island methylation patterns of cancer-associated genes for their applicability as molecular biomarkers for the detection of superficial bladder cancer and for the discrimination of invasive from noninvasive tumours. We analysed the methylation status of CpG islands in the promoter region of the cancer-associated genes GSTP1, DAPK, MDR1, TPEF, PAX6, and TSLC1 in primary papillary bladder cancer specimens from 39 patients (pT1 10, pTis one, pTa 20, pT2 five). Tumour-adjacent normal mucosa served as the control. The DNAs were bisulphite-treated and submitted to methylation-specific real-time polymerase chain reactions. Only TPEF and PAX6 had substantial CpG island methylation percentages. The TPEF- and PAX6-promoters also had significantly higher methylation rates in tumour tissue compared with the normal tumour-adjacent tissue. Interestingly, the methylation rates of the TPEF- and the PAX6-promoter were higher in adjacent normal tissues from bladders with pTa then in those with pT1 tumours. Our results shed a critical light on the hypothesis that CpG island hypermethylation of the GSTP1-, DAPK-, MDR1- and TSLC1-promoter could represent molecular biomarkers for bladder cancer diagnosis and detection. However, methylated PAX6- or TPEF-promoters could represent biomarkers for this disease. Additional studies are needed to evaluate whether methylation rates of these genes in normal bladder tissues are applicable as accessory markers for the tumour state or its invasive behaviour.