Bladder Urothelial Cell Research Articles

Low Frequency of Epigenetic Events in Urothelial Tumors in Young Patients

Bladder urothelial cell carcinoma is uncommon in young patients. We recently reported a series of tumors in patients younger than 20 years at diagnosis and performed exhaustive genetic screening for molecular alterations. Few events typical of bladder urothelial cell carcinoma were detected. Since many carcinogenic events occur at the epigenetic rather than the genetic level, we analyzed the same tumors for alterations in DNA hypermethylation. We compared our findings with those in tumors in older patients with similar pathological profiles. We analyzed 76 bladder urothelial cell carcinomas from 3 groups stratified by age at diagnosis, including less than 19, 20 to 45 and greater than 46 years (median 78), and matched for low grade and nonmuscle invasive stage. We used quantified methyl specific polymerase chain reaction to investigate promoter methylation for 8 tumor suppressor genes implicated in urothelial carcinogenesis. Tumors in the youngest age group had the lowest incidence of global hypermethylation compared to the other tumors with a methyl index of 37.5% vs 62.5% and 50%, respectively (ANOVA p = 0.009). When individual loci were analyzed, younger patients had a significantly lower rate and concentration of methylation at APC, Bcl2, MGMT and E-cadherin promoters than in the older groups (p <0.05). Few differences were present between the 2 older cohorts but the APC and MGMT methylation concentration increased with age. Urothelial tumors in patients younger than 19 years have a low rate of epigenetic alteration. Tumors in patients older than 20 years have epigenetic profiles similar to those of tumors in patients within the typical bladder urothelial cell carcinoma age range.

Narrow-band imaging flexible cystoscopy in the detection of bladder urothelial cell carcionma

Objective To study the sensitivity and specifity for detection of bladder tumor by Narrow-band imaging flexible cystoscopy compared with WLI flexible systoscopy. Methods Between February 2009 and July 2009, NBI flexible cystoscopy and conventional WLI flexible cystoscopy with the same instrument (Olympus Exera Ⅱ endoscopy system) were both performed on 31 patients highly suspect of bladder neoplasm with same observed time and in a randomized sequenced paradigm. Every suspect mucosa lesion was biopsied in both NBI and WLI image to compare the diagnostic accuracy between them. Results Twenty-eight patients(90%) were pathologically bladder urothelial cell carcinoma (UCC). Of 28 patients 3 were Tis, 15 were Ta, 7 were T1, and 3 were T2. Twenty were low grade carcinom, 8 were high grade carcinoma and 16 had multiple tumors, 12 had a single tumor.Of 73 biopsied lesions, 61 were diagnosed UCC under WLI image with 84% sensitivity, while 80 of 91 diagnosed under NBI image with 88% sensitivity. WLI detected 23 patients with bladder UCC while NBI detected all 28 patients. NBI detected 19 additional UCC lesions in 15 of 28 patients, as compared with WLI(P＜0. 05). Conclusion NBI flexible cystoscopy can detect more bladder urothelial cell carcinoma than WLI flexible cystoscopy. Key words: Bladder neoplasms; Narrow-band image; Fexible cystoscopy

Gene Expression Signature in Urine for Diagnosing and Assessing Aggressiveness of Bladder Urothelial Carcinoma

To develop an accurate and noninvasive method for bladder cancer diagnosis and prediction of disease aggressiveness based on the gene expression patterns of urine samples. Gene expression patterns of 341 urine samples from bladder urothelial cell carcinoma (UCC) patients and 235 controls were analyzed via TaqMan Arrays. In a first phase of the study, three consecutive gene selection steps were done to identify a gene set expression signature to detect and stratify UCC in urine. Subsequently, those genes more informative for UCC diagnosis and prediction of tumor aggressiveness were combined to obtain a classification system of bladder cancer samples. In a second phase, the obtained gene set signature was evaluated in a routine clinical scenario analyzing only voided urine samples. We have identified a 12+2 gene expression signature for UCC diagnosis and prediction of tumor aggressiveness on urine samples. Overall, this gene set panel had 98% sensitivity (SN) and 99% specificity (SP) in discriminating between UCC and control samples and 79% SN and 92% SP in predicting tumor aggressiveness. The translation of the model to the clinically applicable format corroborates that the 12+2 gene set panel described maintains a high accuracy for UCC diagnosis (SN = 89% and SP = 95%) and tumor aggressiveness prediction (SN = 79% and SP = 91%) in voided urine samples. The 12+2 gene expression signature described in urine is able to identify patients suffering from UCC and predict tumor aggressiveness. We show that a panel of molecular markers may improve the schedule for diagnosis and follow-up in UCC patients.

Abstract 2558: CEBPD induces SOD1 and cisplatin resistance in urothelial carcinoma

Abstract Bladder cancer is the fourth most common type of cancer in men (ninth in women) in the United States. Cisplatin is an effective agent against the most common subtype, urothelial carcinoma. However, the development of chemotherapy resistance is a severe clinical problem for the successful treatment of cancers. A better understanding of the cellular and molecular events in response to cisplatin treatment and the development of resistance are critical to improve the therapeutic options for patients. Here, we report that expression of the CCAAT/enhancer binding protein delta (CEBPD, C/EBPδ, NFIL-6β) is induced by cisplatin in the human bladder urothelial carcinoma NTUB1 cell line and is specifically elevated in a cisplatin resistant subline. Expression of CEBPD reduces cisplatin-induced reactive oxygen species (ROS) and apoptosis in NTUB1 cells by inducing the expression of Cu/Zn-superoxide dismutase (SOD1) via direct promoter trans-activation. Several reports have implicated CEBPD as a tumor suppressor gene. This study reveals a novel role for CEBPD in conferring drug resistance, suggesting that it can also be pro-oncogenic. Furthermore, our data suggest that SOD inhibitors, which are already used as antiangiogenic agents, may be suitable for combinatorial chemotherapy to prevent or treat cisplatin resistance in bladder and possibly other cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2558.

Transcriptional up-regulation of SOD1 by CEBPD: A potential target for cisplatin resistant human urothelial carcinoma cells

Bladder cancer is the fourth most common type of cancer in men (ninth in women) in the United States. Cisplatin is an effective agent against the most common subtype, urothelial carcinoma. However, the development of chemotherapy resistance is a severe clinical problem for the successful treatment of this and other cancers. A better understanding of the cellular and molecular events in response to cisplatin treatment and the development of resistance are critical to improve the therapeutic options for patients. Here, we report that expression of the CCAAT/enhancer binding protein delta (CEBPD, C/EBPδ, NF-IL6β) is induced by cisplatin in the human bladder urothelial carcinoma NTUB1 cell line and is specifically elevated in a cisplatin resistant subline. Expression of CEBPD reduced cisplatin-induced reactive oxygen species (ROS) and apoptosis in NTUB1 cells by inducing the expression of Cu/Zn-superoxide dismutase (SOD1) via direct promoter transactivation. Several reports have implicated CEBPD as a tumor suppressor gene. This study reveals a novel role for CEBPD in conferring drug resistance, suggesting that it can also be pro-oncogenic. Furthermore, our data suggest that SOD inhibitors, which are already used as anti-angiogenic agents, may be suitable for combinatorial chemotherapy to prevent or treat cisplatin resistance in bladder and possibly other cancers.

1158 IDENTIFICATION OF A GENE SET PANEL ON URINE SAMPLES FOR THE NONINVASIVE DIAGNOSIS AND AGGRESSIVENESS ASSESSMENT OF BLADDER UROTHELIAL CARCINOMA

You have accessJournal of UrologyBladder Cancer: Detection and Screening1 Apr 20101158 IDENTIFICATION OF A GENE SET PANEL ON URINE SAMPLES FOR THE NONINVASIVE DIAGNOSIS AND AGGRESSIVENESS ASSESSMENT OF BLADDER UROTHELIAL CARCINOMA Lourdes Mengual, Moises Burset, Maria J. Ribal, Elisabet Ars, Mercedes Marín-Aguilera, Manuel Fernández, Mercedes Ingelmo-Torres, Humberto Villavicencio, and Antonio Alcaraz Lourdes MengualLourdes Mengual More articles by this author , Moises BursetMoises Burset More articles by this author , Maria J. RibalMaria J. Ribal More articles by this author , Elisabet ArsElisabet Ars More articles by this author , Mercedes Marín-AguileraMercedes Marín-Aguilera More articles by this author , Manuel FernándezManuel Fernández More articles by this author , Mercedes Ingelmo-TorresMercedes Ingelmo-Torres More articles by this author , Humberto VillavicencioHumberto Villavicencio More articles by this author , and Antonio AlcarazAntonio Alcaraz More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.658AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES To develop an accurate and noninvasive method for bladder cancer diagnosis and prediction of disease aggressiveness based on the gene expression patterns of urine samples. METHODS Gene expression patterns of 341 urine samples from bladder urothelial cell carcinoma (UCC) patients and 235 controls were analyzed by means TaqMan Arrays. In a first phase of the study, 3 consecutively gene selection steps were performed to identify a gene set expression signature to detect and stratify UCC in urine. Subsequently, those genes more informative for UCC diagnosis and prediction of tumor aggressiveness were combined to obtain a classification system of bladder cancer samples. In a second phase, the obtained gene set signature was evaluated in a clinical routine scenario analyzing only voided urine samples. RESULTS We have identified a 12+2 gene expression signature for UCC diagnosis and prediction of tumor aggressiveness on urine samples. Overall, this gene set panel had 98% sensitivity (SN) and 99% specificity (SP) in discriminating between UCC and control samples and 79% SN and 92% SP in predicting tumor aggressiveness. The translation of the model to the clinically applicable format corroborate that the 12+2 gene set panel described maintains a high accuracy for UCC diagnosis (SN=89% and SP=95%) and tumor aggressiveness prediction (SN=79% and a SP=91%) in voided urine samples. CONCLUSIONS The 12+2 gene expression signature described in urine is able to identify patients suffering from UCC and predict tumor aggressiveness. We show that a panel of molecular markers can improve the schedule for diagnosis and follow-up in UUC patients. Barcelona, Spain© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e448 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Lourdes Mengual More articles by this author Moises Burset More articles by this author Maria J. Ribal More articles by this author Elisabet Ars More articles by this author Mercedes Marín-Aguilera More articles by this author Manuel Fernández More articles by this author Mercedes Ingelmo-Torres More articles by this author Humberto Villavicencio More articles by this author Antonio Alcaraz More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Aldehyde Dehydrogenase 1 A1–Positive Cell Population Is Enriched in Tumor-Initiating Cells and Associated with Progression of Bladder Cancer

Aldehyde dehydrogenase 1 A1 (ALDH1A1) has recently been suggested as a marker for cancer stem or stem-like cancer cells of some human malignancies. The purpose of this study was to investigate the stem cell-related function and clinical significance of the ALDH1A1 in bladder urothelial cell carcinoma. Aldefluor assay was used to isolate ALDH1A1+ cells from bladder cancer cells. Stem cell characteristics of the ALDH1A1+ cells were then investigated by in vitro and in vivo approaches. Immunohistochemistry was done for evaluating ALDH1A1 expression on 22 normal bladder tissues and 216 bladder tumor specimens of different stage and grade. The ALDH1A1+ cancer cells displayed higher in vitro tumorigenicity compared with isogenic ALDH1A1- cells. The ALDH1A1+ cancer cells could generate xenograft tumors that resembled the histopathologic characteristics and heterogeneity of the parental cells. High ALDH1A1 expression was found in 26% (56 of 216) of human bladder tumor specimens and significantly related to advanced pathologic stage, high histologic grade, recurrence and progression, and metastasis of bladder urothelial cell carcinomas (all P < 0.05). Furthermore, ALDH1A1 expression was inversely associated with cancer-specific and overall survivals of the patients (P = 0.027 and 0.030, respectively). Therefore, ALDH1A1+ cell population could be enriched in tumor-initiating cells. ALDH1A1 may serve as a useful marker for monitoring the progression of bladder tumor and identifying bladder cancer patients with poor prognosis who might benefit from adjuvant and effective treatments.

The orthotopic Fischer/AY-27 rat bladder urothelial cell carcinoma model to test the efficacy of different apaziquone formulations

Objectives Apaziquone used intravesically showed significant activity in phase I and II marker lesion studies in non-muscle-invasive bladder cancer. The objective of this study was to assess antitumor activity and safety of 3 different formulations of intravesical apaziquone in an orthotopic rat bladder cancer model. Materials and methods Female Fischer F344 rats were instilled with 1.5 × 10 6 AY-27 urothelial cell carcinoma cells and divided in 3 treatment groups ( n = 10) and 1 placebo group ( n = 6). Intravesical treatment was administered for 1 hour on days 2 and 5. Rats were treated with apaziquone in the formulation used in phase I/II clinical trials (group 1); apaziquone with an altered buffering capacity being used in phase III clinical trials (group 2), and apaziquone as in group 2, but without propylene glycol in the diluent (group 3). On days 5 and 14, the bladder wall was inspected by cystoscopy and evaluated for macroscopic tumor growth. After sacrificing the rats (day 14), cystectomy was performed and the bladders were investigated. Results There were no signs of any toxicity due to the study drug. On histopathologic examination of the bladders 0, 1, and 2 tumors per group were found in group 1, 2, and 3, respectively. In the placebo-treated group, 60% of animals developed tumor, which is comparable to untreated animals. Conclusions Apaziquone showed an excellent antitumor activity. The effectiveness of apaziquone in this orthotopic rat bladder tumor model corroborates the clinical observations and implies the validity of this model.

Taurine modulates neutrophil function but potentiates uropathogenic E. coli infection in the murine bladder

Eradication of a urinary tract infection (UTI) appears to be related to a number of innate host defence mechanisms and their interactions with invading bacteria. Recurrent UTIs (rUTIs) pose a difficult problem in that these bacteria use both host and bacterial factors to evade elimination. Neutrophil bactericidal function is depressed, both systemically and in urine, in patients with a history of recurrent UTI. Taurine is a semi-essential amino acid and is successful in preserving neutrophil bactericidal function in urine. Taurine may preserve neutrophil function at the urothelium and thus aid UTI resolution. Adult female (6 weeks old) C57Bl/6 mice were randomised into three groups: a saline gavage only control group, a saline gavage + E. coli group, and a taurine gavage + E. coli group [21 g/70 kg taurine in 0.9% normal saline (N/S) for 5 days]. Whilst taurine gavage pre-treatment resulted in increased serum neutrophils respiratory burst activity, at the urothelial-endothelial interface it caused higher colony forming units in the urine and a higher incidence of E. coli invasion in the bladder wall with no evidence of increased bladder wall neutrophils infiltration on MPO assay of histological assessment. Histologically there was also evidence of reduced bladder inflammation and urothelial cell apoptosis. In conclusion, taurine effectively increases neutrophils activity but given its anti-inflammatory properties, at the expense of decreased urothelial-endothelial activation thus preventing clearance of active E. coli infection in the bladder. Despite the negative results, this study demonstrates the importance of modulating interactions at the urothelial interface.

Experimental rat bladder urothelial cell carcinoma models

Bladder cancer is a major public health problem. Currently available therapeutic options seem to be unable to prevent bladder cancer recurrence and progression. To enable preclinical testing of new intravesical therapeutic agents, a suitable bladder tumor model that resembles human disease is highly desirable. The aim of this topic paper was to discuss the problems associated with current in vivo animal bladder tumor models, focusing on the orthotopic syngeneic rat bladder tumor model. In the second part of the paper the development of a potential new orthotopic rat bladder tumor model is described.

The role of polymer nanosurface roughness and submicron pores in improving bladderurothelial cell density and inhibiting calcium oxalate stone formation

Synthetic polymers have been proposed for replacing resected cancerous bladder tissue.However, conventional (or nanosmooth) polymers used in such applications (such aspoly(ether) urethane (PU) and poly-lactic-co-glycolic acid (PLGA)) often fail clinically dueto poor bladder tissue regeneration, low cytocompatibility properties, and excessive calciumstone formation. For the successful reconstruction of bladder tissue, polymer surfacesshould be modified to combat these common problems. Along these lines, implementingnanoscale surface features that mimic the natural roughness of bladder tissue on polymersurfaces can promote appropriate cell growth, accelerate bladder tissue regeneration andinhibit bladder calcium stone formation. To test this hypothesis, in this study,the cytocompatibility properties of both a non-biodegradable polymer (PU) anda biodegradable polymer (PLGA) were investigated after etching in chemicals(HNO3 and NaOH, respectively) to create nanoscale surface features. After chemical etching, PUpossessed submicron sized pores and numerous nanometer surface features while PLGApossessed few pores and large amounts of nanometer surface roughness. Results from thisstudy strongly supported the assertion that nanometer scale surface roughness produced onPU and PLGA promoted the density of urothelial cells (cells that line the interior of thebladder), with the greatest urothelial cell densities observed on nanorough PLGA. Inaddition, compared to respective conventional polymers, the results providedevidence that nanorough PU and PLGA inhibited calcium oxalate stone formation;submicron pored nanorough PU inhibited calcium oxalate stone formation themost. Thus, results from the present study suggest the importance of nanometertopographical cues for designing better materials for bladder tissue engineeringapplications.

Cell Discohesion and Multifocality of Carcinoma In situ of the Bladder: New Insight From the Adhesion Molecule Profile (e-Cadherin, Ep-CAM, and MUC1)

Urothelial cell carcinoma in situ (CIS) of the bladder is a superficially diffusive and highly discohesive disease. The authors analyzed the expression of some adhesion molecules (e-cadherin and Ep-CAM) and MUC1 in 32 unifocal and multifocal bladder urothelial cell CIS in an attempt to clarify this discohesion. E-cadherin was strongly expressed, in more than 75% of the cases. The presence of methylation of the CDH1 e-cadherin promoter gene was also investigated, but methylation was found in only one case. Ep-CAM was present in all the cases with a heterogeneous staining pattern. Similarly, MUC1/episialin was variously present in 94% of the cases without a polarized staining pattern and was expressed more strongly in cases with multifocal disease. Because loss of MUC1 polarization leads to interference with cell-cell adhesion mechanisms mediated by cadherins, these findings help explain why bladder urothelial cell CIS often shows a discohesive morphology and multifocality despite a strongly expressed adhesion molecule profile. Finally, Ep-CAM expression might provide some support for future target therapy trials.

Structure−Activity Relationship Studies for the Peptide Portion of the Bladder Epithelial Cell Antiproliferative Factor from Interstitial Cystitis Patients

We performed comprehensive structure-activity relationship (SAR) studies on the peptide portion of antiproliferative factor (APF), a sialylated frizzled-8 related glycopeptide that inhibits normal bladder epithelial and urothelial carcinoma cell proliferation. Glycopeptide derivatives were synthesized by solid-phase methods using standard Fmoc chemistry and purified by RP-HPLC; all intermediate and final products were verified by HPLC-MS and NMR analyses. Antiproliferative activity of each derivative was determined by inhibition of (3)H-thymidine incorporation in primary normal human bladder epithelial cells. Structural components of the peptide segment of APF that proved to be important for biological activity included the presence of at least eight of the nine N-terminal amino acids, a negative charge in the C-terminal amino acid, a free amino group at the N-terminus, maintenance of a specific amino acid sequence in the C-terminal tail, and trans conformation for the peptide bonds. These data provide critical guidelines for optimization of structure in design of APF analogues as potential therapeutic agents.

MicroRNA expression profiling of human metastatic cancers

1059 Small non-coding microRNAs (miRNAs) contribute to cancer development and progression and are differentially expressed in normal tissues and cancers. To test the hypothesis that a specific miRNA expression signature characterizes the metastatic phenotype of solid tumors, we performed miRNA micro-array analysis on 43 paired primary tumors and lymphnode metastasis. Specimens were collected at the Department of Pathology, Thomas Jefferson University from 43 patients consisting in 13 breast carcinomas, 10 lung cancers, 10 bladder urothelial cell carcinomas and 10 colon cancers. 5 μg of total RNA from each sample were used. Hybridization of biotin-labeled complementary DNA was performed on a two new miRNA microarray chip (OSU_CCC version 3.0 and 4.0), which contains ∼1,100 miRNA probes (326 human and 249 mouse miRNA genes, spotted in duplicates). We identified a common metastatic cancer miRNA signature mainly composed by over-expressed miRNAs. Some of these miRNAs (miR-10b, miR-20a, miR-21) have a well characterized association with cancer progression. In particular, in metastatic breast cancers there was an up-regulation of miR-450a, miR-148a and a down-regulation of miR- 205, miR-130b. In metastatic lung cancer there was an up-regulation of miR-369–3p and a down-regulation of miR-138–1. In metastatic bladder cancer there was an up-regulation of miR-29a, miR-10b, miR-126 and a down-regulation of miR-145, miR-143, and in metastatic colon cancer there was an up-regulation of miR-138, miR-21 and a down-regulation of miR-20a and miR-204. Moreover, in the metastatic samples we found that miRNAs associated to inflammation and immune response were over-expressed due to the partial contamination of our samples by the lymphocytes. The over-expression of most miRNAs that we found over-expressed in the primary tumors had been previously reported in published signatures of the corresponding normal tissues, thus confirming that miRNA signatures are a potent tool in classifying tissues of different origin.Our results indicate that specific miRNAs may be directly involved in cancer metastasis and that they may represent a novel diagnostic tool in the characterization of metastatic cancer gene targets. No significant financial relationships to disclose.

EVALUATION OF AN ORTHOTOPIC RAT BLADDER UROTHELIAL CELL CARCINOMA MODEL BY CYSTOSCOPY

OBJECTIVES: To enable preclinical testing of intravesical therapies against non-muscle-invasive bladder cancer (NMIBC) in an orthotopic rat bladder tumour model, augmented by the use of serial cystoscopy for in vivo tumour assessment and follow-up. MATERIALS AND METHODS: Fischer F344 rats had a 16-G transurethral cannula placed. The bladder mucosa was conditioned with an acid rinse, followed by a 1-h instillation of 1.5 x 10(6) AY-27 rat bladder urothelial cell carcinoma (UCC) cells (day 0). Cystoscopy (1 mm) was done on day 0 (control) and at 3, 4, 5, 6, 7, 10, 13 and 17 days. At the scheduled times the rats were killed after cystectomy (four at each time) for histopathological examination of the bladder. RESULTS: Overall, tumour establishment was >80%, with predominantly carcinoma in situ preceding or concomitant with invasive tumour growth. All tumours were formed at 3-5 days, and remained non-muscle-invasive up to 5 days. From 6 days, tumours progressed to muscle-invasive disease in 40% of the rats. Visibility at cystoscopy was excellent and tumours were apparent in >90% of rats from 5 days on, with a specificity and sensitivity of >90%. Cystoscopy could not distinguish NMIBC from muscle-invasive disease. CONCLUSIONS: This is a reliable model of orthotopic rat bladder UCC, with early high-grade NMIBC growth, immediately followed by muscle-invasive growth, i.e. the recommended time to start intravesical therapy would be 5 days after tumour cell inoculation. Tumour growth can easily be monitored by cystoscopy, but cannot be used to distinguish NMIBC from muscle-invasive bladder cancer.

Evaluation of an orthotopic rat bladder urothelial cell carcinoma model by cystoscopy

To enable preclinical testing of intravesical therapies against non-muscle-invasive bladder cancer (NMIBC) in an orthotopic rat bladder tumour model, augmented by the use of serial cystoscopy for in vivo tumour assessment and follow-up. Fischer F344 rats had a 16-G transurethral cannula placed. The bladder mucosa was conditioned with an acid rinse, followed by a 1-h instillation of 1.5 x 10(6) AY-27 rat bladder urothelial cell carcinoma (UCC) cells (day 0). Cystoscopy (1 mm) was done on day 0 (control) and at 3, 4, 5, 6, 7, 10, 13 and 17 days. At the scheduled times the rats were killed after cystectomy (four at each time) for histopathological examination of the bladder. Overall, tumour establishment was >80%, with predominantly carcinoma in situ preceding or concomitant with invasive tumour growth. All tumours were formed at 3-5 days, and remained non-muscle-invasive up to 5 days. From 6 days, tumours progressed to muscle-invasive disease in 40% of the rats. Visibility at cystoscopy was excellent and tumours were apparent in >90% of rats from 5 days on, with a specificity and sensitivity of >90%. Cystoscopy could not distinguish NMIBC from muscle-invasive disease. This is a reliable model of orthotopic rat bladder UCC, with early high-grade NMIBC growth, immediately followed by muscle-invasive growth, i.e. the recommended time to start intravesical therapy would be 5 days after tumour cell inoculation. Tumour growth can easily be monitored by cystoscopy, but cannot be used to distinguish NMIBC from muscle-invasive bladder cancer.

Role of Urinary Cathepsin B and L in the Detection of Bladder Urothelial Cell Carcinoma

We tested the hypothesis that urinary cathepsin B and L are associated with bladder cancer recurrence and invasiveness in patients with a history of nonmuscle invasive urothelial carcinoma of the bladder. Cathepsin B and L, and NMP22 were determined in the urine specimens of 188 consecutive subjects with a history of treated urothelial carcinoma of the bladder, 31 with noncancerous urological conditions and 10 healthy subjects. Cathepsin B and L were analyzed as continuous and categorical variables based on their quartile distribution. Urinary cathepsin L was higher in the 122 patients with cystoscopic evidence of bladder tumor compared with levels in 107 with normal cystoscopy (median 5.9, IQR 4.4 vs 3.0, IQR 3.2, p <0.001). Higher levels of cathepsin L were associated with positive cytology assay results, higher NMP22 and T1 or greater pathological stage (each p <0.001). Area under the ROC curves of NMP22 and cathepsin L for bladder cancer detection were 0.704 (95% CI 0.637-0.772) and 0.793 (95% CI 0.736-0.850), respectively. On multivariate analysis cathepsin L, NMP22 and cytology were associated with invasive pathological stage (OR 1.29, 2.42 and 2.76, respectively, p </=0.002). Urinary cathepsin B was not associated with any outcome variables. Urinary cathepsin L is an independent predictor of bladder cancer presence and invasiveness in patients with a history of urothelial carcinoma of the bladder. Further evaluation of this marker is necessary before its use as an adjunct to cystoscopy for urothelial carcinoma of the bladder.

Tissue Inhibitor of Metalloproteinases-3 Promoter Methylation is an Independent Prognostic Factor for Bladder Cancer

TIMP-3 (tissue inhibitor of metalloproteinases-3) is 1 of 4 members of a family of proteins that were originally classified according to their ability to inhibit matrix metalloproteinases. We analyzed TIMP-3 methylation in 175 urine sediment DNA samples from patients with bladder cancer with well characterized clinicopathological parameters, including patient outcome. We examined urine sediment DNA for aberrant methylation of 9 genes, including TIMP-3, by quantitative fluorogenic real-time polymerase chain reaction. Using an optimal cutoff value by TaqMan(R) quantitation we found that the risk of death was statistically significantly higher in patients with higher TIMP-3 and ARF methylation (HR 1.99, 95% CI 1.12 to 3.27, p = 0.01 and HR 1.66, 95% CI 1.00 to 2.76, p = 0.05, respectively) than in patients without/lower TIMP3 and ARF methylation in urine. A significant correlation was also seen between the risk of death and stage 3 tumor (HR 2.73, 95% CI 1.58 to 4.72, p = 0.003) and metastasis (HR 3.32, 95% CI 1.98 to 5.57, p = 0.0001). Multivariate analysis subsequently revealed that TIMP-3 methylation was an independent prognostic factor for bladder cancer survival with stage and metastasis (p = 0.001 and 0.02, respectively). These results suggest that TIMP-3 promoter methylation could be a clinically applicable marker for bladder cancer progression.

Overexpression of CDC91L1 (PIG‐U) in bladder urothelial cell carcinoma: correlation with clinical variables and prognostic significance

To investigate cell division cycle 91-like 1 (CDC91L1; also called phosphatidylinositol glycan class U, PIG-U) expression in bladder cancer at both the mRNA and protein levels, and to study its clinical and prognostic significance, as CDC91L1 was recently identified as a new oncogene in human bladder cancer and its role in the biological behaviour of bladder cancer is largely unknown. In all, 73 bladder tumours and 14 samples of normal bladder urothelium were studied by reverse-transcription polymerase chain reaction (PCR), real-time quantitative PCR and immunohistochemistry. The normalized CDC91L1 mRNA copy number in tumours was significantly greater than in normal controls (P < 0.05). There was overexpression of CDC91L1 mRNA in 30.1% (22/73) of the bladder tumours compared with the normal urothelium. At the protein level, 75.3% (55/73) of the bladder tumours and two of 14 of the normal urothelium had high expression of CDC91L1 protein, which is statistically significant (P < 0.001). The correlation between CDC91L1 protein and tumour grade, and muscle invasion of tumour was significant (both P < 0.05). In addition to tumour extent and tumour grade, CDC91L1 protein was an independent predictor of recurrence for superficial bladder cancer and had a trend to predict tumour progression. CDC91L1 (PIG-U) plays a role in the development of bladder urothelial cell carcinoma. CDC91L1 protein might be a potential biomarker for prediction of recurrence and a therapeutic target in bladder cancer.

Artificial intelligence for predicting recurrence-free probability of non-invasive high-grade urothelial bladder cell carcinoma

The objective of our study was to define a neural network for predicting recurrence and progression-free probability in patients affected by recurrent pTaG3 urothelial bladder cancer to use in everyday clinical practice. Among all patients who had undergone transurethral resection for bladder tumors, 143 were finally selected and enrolled. Four follow-ups for recurrence, progression or survival were performed at 6, 9, 12 and 108 months. The data were analyzed by using the commercially available software program NeuralWorks Predict. These data were compared with univariate and multivariate analysis results. The use of Artificial Neural Networks (ANN) in recurrent pTaG3 patients showed a sensitivity of 81.67% and specificity of 95.87% in predicting recurrence-free status after transurethral resection of bladder tumor at 12 months follow-up. Statistical and ANN analyses allowed selection of the number of lesions (multiple, HR=3.31, p=0.008) and the previous recurrence rate (>or=2/year, HR=3.14, p=0.003) as the most influential variables affecting the output decision in predicting the natural history of recurrent pTaG3 urothelial bladder cancer. ANN applications also included selection of the previous adjuvant therapy. We demonstrated the feasibility and reliability of ANN applications in everyday clinical practice, reporting a good recurrence predicting performance. The study identified a single subgroup of pTaG3 patients with multiple lesions, >or=2/year recurrence rate and without any response to previous Bacille Calmette-Guérin adjuvant therapy, that seem to be at high risk of recurrence.