Development Of Bladder Tumors Research Articles

Bladder tumour development after urothelial carcinoma of the upper urinary tract is related to primary tumour location

To better define the predictors of bladder tumour development in patients operated for upper urinary tract urothelial cancer (UT-UC). Surgical specimens from 191 consecutive patients with no history of bladder cancer and operated for UT-UC were chosen for analysis. Bladder tumour development was assessed in relation to UT-UC location, tumour multiplicity, stage and grade, margin status, mode of operation, age and gender. Overall, 51 of 191 (27%) patients developed subsequent bladder tumours, including 25 of 123 (20%) with pelvic, 19 of 47 (40%) with ureteric and seven of 21 (33%) with multifocal tumours (P = 0.04 for all subgroups; P = 0.01 for pelvic vs ureteric). There was no influence of the other variables. The median (mean, range) time to recurrence was 12 (18, 3-64) months. In a multivariate analysis, ureteric tumour location was an independent predictor (P = 0.02; risk ratio, RR, 2.0, 95% confidence interval, CI, 1.1-3.7). After excluding 68 patients with systemic disease progression, bladder tumour development was noted in 39 of 123 (32%), including 18 of 76 (24%) with pelvic, 16 of 34 (47%) with ureteric and five of 13 with multifocal tumours (P = 0.06 for all subgroups; P = 0.02 for pelvic vs ureteric). In a multivariate analysis, ureteric location (P = 0.03; RR 2.1, 95% CI 1.1-4.2) and high tumour grade (P = 0.04; RR 2.2, 95% CI 1.03-4.7) were independent predictors of subsequent bladder tumour development. The risk of developing a bladder tumour after surgery for UT-UC is significantly related to ureteric tumour location and high tumour grade. Clinical trials to evaluate a possible reduction of bladder cancer risk by intraoperative ureteric ligation and/or peri-operative topical intravesical chemotherapy instillation are justified.

UP-03.99: Bladder tumor development after transitional cell carcinoma of the upper urinary tract is related to tumor location

UP-03.99: Bladder tumor development after transitional cell carcinoma of the upper urinary tract is related to tumor location

Effects of ginger (Zingiber officinale Roscoe) on DNA damage and development of urothelial tumors in a mouse bladder carcinogenesis model.

Extracts of the spice ginger (Zingiber officinale Roscoe) are rich in gingerols and shogaols, which exhibit antioxidant, anti-inflammatory, antifungal, antimycobacterial, and anticarcinogenic proprieties. The present study evaluated the chemoprotective effects of a ginger extract on the DNA damage and the development of bladder cancer induced by N-butyl-N-(4-hydroxibutyl) nitrosamine (BBN)/N-methyl-N-nitrosourea (MNU) in male Swiss mice. Groups G1-G3 were given 0.05% BBN in drinking water for 18 weeks and four i.p. injections of 30 mg/kg body weight MNU at 1, 3, 10, and 18 weeks. Group G4 and G5 received only the BBN or MNU treatments, respectively, and groups G6 and G7 were not treated with BBN or MNU. Additionally, Groups G2, G3, and G6 were fed diets containing 1, 2, and 2% ginger extract, respectively, while Groups G1, G4, G5, and G7 were fed basal diet. Samples of peripheral blood were collected during the experiment for genotoxicity analysis; blood collected 4 hr after each MNU dose was used for the analysis of DNA damage with the Comet assay (assay performed on leukocytes from all groups), while reticulocytes collected 24 hr after the last MNU treatment of Groups G5-G7 were used for the micronucleus assay. At the end of the experiment, the urinary bladder was removed, fixed, and prepared for histopathological, cell proliferation, and apoptosis evaluations. Ginger by itself was not genotoxic, and it did not alter the DNA damage levels induced by the BBN/MNU treatment during the course of the exposure. The incidence and multiplicity of simple and nodular hyperplasia and transitional cell carcinoma (TCC) were increased by the BBN/MNU treatment, but dietary ginger had no significant effect on these responses. However, in Group G2 (BBN/MNU/2% ginger-treated group), there was an increased incidence of Grade 2 TCC. The results suggest that ginger extract does not inhibit the development of BBN-induced mouse bladder tumors.

Bilateral Metachronous Ureteral and Renal Pelvic Carcinomas: Incidence, Clinical Presentation, Histopathology, Treatment and Outcome

Metachronous bilateral UUTTs are rare. The authors reported baseline and long-term followup data for all patients diagnosed in Western Sweden during a 28-year period. We performed a clinical and histopathological analysis of all patients in Western Sweden surgically treated for ureteral and renal pelvic tumors from 1971 to 1998. Of 768 patients a contralateral UUTT developed in 24 (3.1%) after a median of 46 months (range 2 to 232). The projected incidence after initial UUTT diagnosis was 2.7%, 5.8% and 6.5% at 5, 10 and 15 years, respectively. Median age of the 24 patients at initial UUTT diagnosis was 67 years and the median age at death was 77 years. Bladder cancer was significantly more common among patients with bilateral UUTT compared to those with unilateral UUTT (83% vs 31%, p <0.0001). Routine followup urography was normal in 9 patients 2 to 11 months before the diagnosis of the contralateral UUTT. Nine patients died of UUTT and 5 patients died of bladder cancer. Bilateral subsequent upper tract tumors are rare, in general diagnosed at an older age and associated with short survival. Many patients die of bladder cancer and it may be possible to improve survival if the bladder cancer is treated early and aggressively. Routine followup urography is not indicated among patients with a tumor-free bladder and a history of UUTT.

Role of Activating Fibroblast Growth Factor Receptor 3 Mutations in the Development of Bladder Tumors

Bladder tumors develop through different molecular pathways. Recent reports suggest activating mutations of the fibroblast growth factor receptor 3 (FGFR3) gene as marker for the "papillary" pathway with good prognosis, in contrast to the more malignant "carcinoma in situ" (CIS) pathway. The aim of this clinical follow-up study was to investigate the role of FGFR3 mutations in bladder cancer development in a longitudinal study. We selected 85 patients with superficial bladder tumors, stratified into early (stage T(a)/grade 1-2, n = 35) and more advanced (either stage T(1) or grade 3, n = 50) developmental stages. The patients were followed prospectively, and metachronous tumors were included. We did screening for FGFR3 and TP53 mutations by direct bidirectional sequencing and for genome-wide molecular changes with microarray technology. A total of 43 of 85 cases (51%) showed activating mutations of FGFR3. The mutations were associated with papillary tumors of early developmental stage. However, after stratifying for developmental stage, FGFR3-mutated tumors showed the same malignant potential as wild-type tumors. Tumors with concomitant CIS were generally FGFR3 wild type. They were characterized by different patterns of chromosomal changes and gene expression signatures compared with FGFR3-mutated tumors, indicating different molecular pathways. FGFR3 mutations seem to have a central role in the early development of papillary bladder tumors. These tumors follow a common molecular pathway, which is different from tumors with concomitant CIS. FGFR3 mutations do not seem to play a role in bladder cancer progression.

RUNX3 Inactivation by Point Mutations and Aberrant DNA Methylation in Bladder Tumors

RUNX3 is inactivated at high frequency in many tumors. However, in most cases, inactivation is caused by silencing of the gene due to promoter hypermethylation. Because epigenetic silencing is known to affect many major tumor suppressor genes in cancer cells, it is not clear whether RUNX3 is primarily responsible for the induction of carcinogenesis in these cases, except for the gastric cancer cases that we reported previously. We investigated genetic and epigenetic alterations of RUNX3 in 124 bladder tumor cases and seven bladder tumor-derived cell lines. Here we show that RUNX3 is inactivated by aberrant DNA methylation in 73% (90 of 124) of primary bladder tumor specimens and 86% (six of seven) of bladder tumor cell lines. In contrast, the promoter regions of 20 normal bladder mucosae were unmethylated. Importantly, one patient bore missense mutations, each of which resulted in amino acid substitutions in the highly conserved Runt domain. The mutations abolished the DNA-binding ability of RUNX3. A second patient had a single nucleotide deletion within the Runt domain coding region that resulted in truncation of the protein. RUNX3 methylation was a significant risk factor for bladder tumor development, superficial bladder tumor recurrence, and subsequent tumor progression. These results strongly suggest that inactivation of RUNX3 may contribute to bladder tumor development and that promoter methylation and silencing of RUNX3 could be useful prognostic markers for both bladder tumor recurrence and progression.

Bladder cancer after managing upper urinary tract transitional cell carcinoma: predictive factors and pathology

To evaluate patients with a history of transitional cell carcinoma (TCC) of the upper urinary tract (UUT) to determine the incidence, pathological distribution, and risk factors for developing subsequent bladder tumours. Between 1993 and 2003, 103 patients were treated at our institution for UUT-TCC. We reviewed demographic, clinical, surgical, and pathological data from these patients at a median follow-up of 38.7 months, and used univariate and multivariate analyses with logistic regression modelling to determine prognostic variables for bladder recurrences. In all, 51 (49.5%) patients developed bladder tumours after treatment for UUT-TCC, at a mean interval of 13.2 months. Patient age (P = 0.01), UUT tumour size (P = 0.03), UUT tumour multifocality (P = 0.05), a history of bladder tumours (P = 0.03), and the number of previous bladder tumours (P = 0.05) predicted the development of bladder recurrences on univariate analysis. On multivariate analysis, only a previous history of bladder tumours (odds ratio 2.6, P = 0.05) remained significant. Over 90% of the recurrent bladder tumours were superficial, with two-thirds of these being low to moderate grade. Six patients had muscle-invasive disease, and five had a cystectomy. Bladder tumours occurred in half the patients after treatment for UUT-TCC; > 60% of these subsequent bladder tumours were superficial, low- to moderate-grade lesions. Neither the pathology of the UUT tumours nor the method of treatment for the UUT disease was associated with recurrent bladder tumours. Only a history of bladder cancer predicted the development of subsequent bladder tumours.

MULTIVARIATE ANALYSIS OF CLINICAL PARAMETERS OF SYNCHRONOUS PRIMARY SUPERFICIAL BLADDER CANCER AND UPPER URINARY TRACT TUMOR

We determined the incidence and characteristic of synchronous upper urinary tract tumors (UUTTs) in patients with primary superficial bladder carcinoma and evaluated the characteristics of bladder tumors related to UUTTs. We performed a retrospective study of 1,529 patients with primary superficial bladder carcinoma who underwent initial examination of the upper urinary tract with excretory urography. Data were analyzed by multivariate analysis using logistic regression. Variables evaluated and related to the incidence of UUTT were multiplicity, carcinoma in situ, bladder tumor size, localization of tumor in the bladder, and tumor grade and stage. A total of 28 patients (1.8%) had simultaneous bladder tumor and UUTT. UUTTs showed no preferred location and 17.9% were multiple. Of UUTTs 46% were invasive and almost 87% were grade 2 or 3. The only significant variable related to UUTT was bladder tumor in the trigone (RR 5.8, 95% IC 2.18 to 15.9, p <0.0005). Of 147 tumors located in the trigone 11 (7.5%) were associated with UUTT, corresponding to 41% of the UUTTs first diagnosed. If multiplicity and tumors in the trigone (551 cases) had been considered, 66.7% of tumors would have been diagnosed. Synchronous UUTT and superficial bladder tumor are uncommon but 46% are invasive. Considering the possible examination of the upper urinary tract only in patients with tumor in the trigone or with multiple bladder tumors 41.4% or 69% of UUTTs, respectively, would have been diagnosed. Patients with tumor in the trigone are at almost 6-fold higher risk for a synchronous tumor in the upper urinary tract.

133Risk factors for bladder tumour development after transitional cell carcinoma of the upper urinary tract

133Risk factors for bladder tumour development after transitional cell carcinoma of the upper urinary tract

Association of bladder tumors and GA genotype of −308 nucleotide in tumor necrosis factor-alpha promoter with greater tumor necrosis factor-alpha expression

ObjectivesTo investigate whether the genetic polymorphism of the −308 nucleotide in the tumor necrosis factor-alpha (TNF-α) promoter is associated with bladder cancer. MethodsDNA samples from blood and tumor were analyzed by polymerase chain reaction-based restriction fragment length polymorphism to characterize the genetic polymorphism of the −308 nucleotide in the TNF-α promoter. TNF-α mRNA expression levels were assessed by quantitative competitive polymerase chain reaction, and the serum concentrations of TNF-α were measured using enzyme-linked immunosorbent assay. ResultsPatients with bladder tumor and control subjects did not differ in their genetic polymorphism of the −308 nucleotide (P = 0.259). However, the relation of the tumor grade with the GA phenotype was statistically significant (P = 0.04). TNF-α mRNA concentrations were also significantly greater in the GA genotype than in the GG genotype (P = 0.022). The TNF-α serum levels of the GA genotype were significantly greater than those of the GG genotype for both patients and controls. However, patients with bladder tumor had significantly greater TNF-α serum levels than did the controls for both the GG and the GA genotypes (GG type, P = 0.001; GA type, P = 0.009). ConclusionsThe genotype of the −308 nucleotide in the TNF-α promoter had a statistically significant effect on TNF-α production and was related to the bladder tumor grade. The GA polymorphism might be associated with a statistically significant increase in gene transcription. That the TNF-α serum levels were greater in the patients with bladder tumor compared with controls suggests that high TNF-α production is associated with bladder tumor development and that bladder tumors may secrete TNF-α.

The development of bladder tumors and contralateral upper urinary tract tumors after primary transitional cell carcinoma of the upper urinary tract

Contralateral, metachronous upper urinary tract (UUT) tumors after primary transitional cell carcinoma (TCC) of the UUT are reported rarely, and to the authors' knowledge the risk factors have not been determined to date. In addition, few reports have described the characteristics of recurrent bladder tumors and contralateral UUT tumors and any relation between theses tumor types. Statistical analysis of data from 223 patients with documented primary UUT-TCC was undertaken. After excluding bilateral involvement and distant metastases, 12 variables were analyzed by multivariate analysis in 189 patients to determine the risk factors for recurrent urothelial tumors. The incidence rates of recurrent bladder tumors and contralateral UUT tumors were 31.2% and 5.8%, respectively. Multiplicity was determined as a risk factor for recurrent bladder tumors. Renal insufficiency, uremia, and concurrent bladder tumors significantly predisposed patients to develop contralateral UUT tumors after primary UUT-TCC. The time intervals and stage distributions differed significantly between recurrent bladder tumors and contralateral UTT tumors. Patients who had recurrent bladder tumors had earlier stage tumors and had a shorter time to recur compared with patients who had contralateral, metachronous UUT tumors. For patients with primary UUT-TCC, regular follow-up by cystoscopy is necessary to detect recurrent bladder tumors. Intravenous urography or retrograde pyelography should be performed for patients who have a high risk of developing contralateral UUT tumors.

Alteration of the PATCHED locus in superficial bladder cancer.

Chromosome 9 alterations are the most frequently encountered cytologic anomalies in urothelial carcinoma (UC). We previously screened 139 low-stage UCs for loss of heterozygosity on chromosome 9, and identified five distinct regions likely to harbour tumour-suppressor genes. The present study focused on deletion mapping in the 9q22 region with 11 additional microsatellite markers. New deletions in the 9q22 region were found in five tumours. Deletion mapping allowed us to identify a 0.5 CM common minimal region of deletion between markers D9S280 and D9S1809, encompassing PATCHED (PTC), a gene identified as a tumour suppressor in basal cell carcinoma and in medulloblastoma. A marker located in the first intron of this gene showed the highest percentage of deletion (45%). cDNA sequencing in 15 tumours with deletion of PTC showed no mutation in the remaining allele. However, average expression of PTC mRNA measured by semiquantitative RT-PCR was significantly decreased in tumours with LOH in the 9q22 region, compared to normal urothelium (P=0.04), while it showed marked fluctuations in tumours without deletion. Our results suggest that the PTC gene is a putative suppressor at the 9q22 locus and that haploinsufficiency of this gene may be an early event in the development of papillary bladder tumours.

High susceptibility of p53 knockout mice to esophageal and urinary bladder carcinogenesis induced by N, N-dibutylnitrosamine

In human cancer, alterations in the p53 tumor suppressor gene are the most common genetic alterations. The aim of the present study was to detect sensitivity of the p53 (+/−) mice and their littermates p53 (+/+) mice to N, N-dibutylnitrosamine (DBN) carcinogenicity. In experiment 1, 6–7-week-old p53 (+/−) and p53 (+/+) mice were treated with 0, 0.025 and 0.05% DBN, respectively, in drinking water for 20 weeks. Esophageal squamous cell and urinary bladder transitional cell carcinomas (TCCs) and fibrosarcomas were found to be significantly increased in p53 (+/−) mice treated with doses of DBN compared to p53 (+/+) mice administered similar doses. In experiment 2, 6–7-week-old p53 (+/−) and p53 (+/+) mice were administered 0 or 0.05 % DBN in drinking water for 8 weeks. Immunohistochemical staining revealed a significant increase in numbers of p53 and bromodeoxyuridine (BrdU) positive cells in the esophageal and urinary bladder epithelia of DBN-treated p53 (+/−) mice compared to p53 (+/+) mice administered DBN. Molecular analysis revealed point mutations in the residual p53 allele in four of eight (50%) esophageal mucosa of DBN-treated p53 (+/−) mice, and in three of eight (38%) of treated p53 (+/+) mice. The results show that p53 (+/−) mice were sensitive to DBN treatment with respect to esophageal and bladder tumor development, with a mechanism that could be confined to early mutations of the residual p53 allele and increased cellular proliferation in the target organs.

Promoting Effect of Sodium L-Ascorbate on N-Butyl-N-(4-hydroxybutyl)nitrosamine-induced Renal Pelvic Carcinogenesis in SD/cShi Rats of Both Sexes

Susceptibility to the promoting effects of sodium L-ascorbate (Na-AsA) on the development of pelvis and urinary bladder tumors in male and female SD/cShi rats, featuring spontaneous hydronephrosis, was investigated. Rats received 0.05% N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN) in their drinking water for 4 weeks and subsequently given basal diet with or without a 5% Na-AsA supplement for 32 weeks. Histopathological examination revealed the promoting effect of Na-AsA on not only the development of urinary bladder tumors but also renal pelvic tumors in the animals of both sexes in this two-stage carcinogenesis experiment, the effect being more prominent in males. Administration of either BBN or Na-AsA alone also induced papillomas and papillary or nodular hyperplasia of renal pelvis or urinary bladder, respectively, in male but not female rats. However, the 5-bromo-2'-deoxyuridine-labeling index of urothelium in the pelvis and bladder increased slightly in male rats and significantly in female rats given Na-AsA alone for 8 weeks. N-butyl-N-(3-carboxypropyl)nitrosamine, which is a metabolite of BBN and proximate carcinogen, was found more in the urine of urinary bladder than that of renal pelvis. These results indicate that the urothelium of the renal pelvis and urinary bladder in SD/cShi rats is susceptible to promoting effects of Na-AsA in the present two stage model urinary tract carcinogenesis, with the urinary bladder of male rats as the most sensitive organ.

Effects of Isoliquirithigenin on the Development of Preneoplastic Liver Lesions Caused by a Choline-Deficient, L-Amino Acid-Defined Diet and on the Urinary Bladder Carcinogenesis by N-Butyl-N-(4-hydroxybutyl)nitrosamine in Rats

Cancer chemopreventive efficacy of a chalcone, isoliquiritigenin (ISO), was assessed on the rat hepatocarcinogenesis associated with fibrosis caused by a choline-deficient, L-amino acid-defined (CDAA) diet, using glutathione S-transferase placental form (GST-P)-positive preneoplastic foci as the end point lesion, and on the rat superficial bladder carcinogenesis initiated by N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). ISO, when given to F344 male rats at the doses of 12.5, 50, 100, and 200 ppm in the CDAA diet for 15 weeks, did not significantly affect the number and size of GST-P-positive foci and the % liver area occupied by the foci. However, it tended to decrease the grade of liver fibrosis. ISO, which was given in a basal diet at doses of 12.5, 25, and 100 ppm for 12 weeks to F344 male rats initiated by 0.05% BBN in drinking water for 12 weeks, also exhibited no clear chemopreventive effects on the development of urinary bladder tumors. Nevertheless, it tended to decrease the multiplicity of nodulo-papillary hyperplasia but not of transitional cell carcinoma (TCC), and differentiation grade of TCCs. The results indicate that ISO at least at the doses used in the present study, possesses no clear cancer chemopreventive efficacy on rat hepatocarcinogenesis caused by a CDAA diet and on rat superficial urinary bladder carcinogenesis by BBN.

Novel fibroblast growth factor receptor 3 (FGFR3) mutations in bladder cancer previously identified in non-lethal skeletal disorders.

Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) gene are responsible for several autosomal dominant craniosynostosis syndromes and chondrodysplasias i.e. hypochondroplasia, achondroplasia, SADDAN and thanatophoric dysplasia--a neonatal lethal dwarfism syndrome. Recently, activating FGFR3 mutations have also been found to be present in cancer, i.e. at high frequency in carcinoma of the bladder and rarely in multiple myeloma and carcinoma of the cervix. Almost all reported mutations in carcinomas corresponded to the mutations identified in thanatophoric dysplasia. We here screened a series of 297 bladder tumours and found three FGFR3 somatic mutations (G380/382R; K650/652M and K650/652T) that were not previously identified in carcinomas or thanatophoric dysplasia. Another novel finding was the occurrence of two simultaneous FGFR3 mutations in four tumours. Two of the three new mutations in bladder cancer, the G380/382R and the K650/652M mutations, were previously reported in achondroplasia and SADDAN, respectively. These syndromes entail a longer life span than thanatophoric dysplasia. The K650/652T mutation has not previously been detected in patients with skeletal disorders, but affects a codon that has been shown to be affected in some cases of thanatophoric dysplasia, SADDAN and hypochondroplasia. From a clinical perspective, the patients with FGFR3-related, non-lethal skeletal disorders might be at a higher risk for development of bladder tumours than the general population.

Inhibitory effects of 2-mercaptoethane sulfonate and 6-phenylhexyl isothiocyanate on urinary bladder tumorigenesis in rats induced by N-butyl- N-(4-hydroxybutyl)nitrosamine

The effects of 2-mercaptoethane sulfonate (MESNA) and phenethyl and 6-phenylhexyl isothiocyanates (PEITC and PHITC) on urinary bladder tumorigenesis were investigated in Wistar rats treated with N-butyl- N-(4-hydroxybutyl)nitrosamine (BHBN). A total of 190 male rats were divided into eight groups. Animals in groups 1, 3 and 4 were administered BHBN in drinking water (0.025%) and group 2 rats were given 0.025% BHBN plus 0.05% MESNA solution for 15 weeks. Rats in groups 3 and 4 were fed a diet supplemented with PEITC or PHITC at a dose level of 0.5 μmol/g for 16 weeks starting 1 week prior to the BHBN treatment. Rats in groups 5–7 served as MESNA, PEITC or PHITC alone controls. Group 8 served as a non-treatment control. The chromatographic profile of drinking water for group 2 animals indicated no chemical interaction between BHBN and MESNA. The incidences of papillomas in the bladder and total bladder tumors including transitional cell carcinomas were significantly ( P<0.01) reduced in group 2 as compared to that in group 1. The multiplicities of papillomas or total bladder tumors were significantly ( P<0.01) lower in group 2 and 4 rats treated with MESNA and PHITC, respectively, than in group 1 rats. The PEITC treatment, however, did not affect the BHBN-induced bladder tumorigenesis. No bladder tumors were found in animals of groups 5–8. The results in the present study clearly indicate that both MESNA and PHITC are potent chemopreventive agents against bladder tumor development in rats induced by BHBN.

Transgenic rats carrying copies of the human c-Ha- ras proto-oncogene exhibit enhanced susceptibility to N -butyl- N -(4-hydroxybutyl)nitrosamine bladder carcinogenesis

We have established a transgenic rat line carrying three copies of the human c-Ha-ras proto-oncogene with its own original promoter region, Jcl/SD-TgN(HrasGen)128Ncc (Hras128) rat. c-Ha-ras protein from expression of transduced and endogenous c-Ha-ras genes could be detected in the bladder epithelium of untreated transgenic rats. To examine their susceptibility to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced urinary bladder carcinogenesis, male transgenic and wild-type littermates were treated with 0.05% BBN in their drinking water for 10 weeks and then killed at week 20. The numbers and volumes of total macroscopic bladder tumors including both transitional cell papillomas and carcinomas (TCC) per rat were much greater in Hras128 rats than in their wild-type counterparts. The numbers of carcinomas per rat were also significantly greater in Hras128 rats. Two cases of TCC exhibiting invasion of the bladder muscle layer, which is extremely rare in the wild-type animals under the experimental conditions used, were also observed in Hras128 rats. The GGC-->GAC mutations at codon 12 of the transgene were observed in only two TCC out of 21 bladder tumors (9.5%), assessed by RFLP analysis and direct sequencing. SSCP analysis did not show any endogenous c-Ha-ras gene mutations. One of 25 tumors (4.0%) in wild-type rats had an endogenous c-Ha-ras gene mutation at codon 12 that was detected (GGA-->GAA) by single-strand conformation polymorphism and direct sequencing. These results indicate that the Hras128 rat is highly susceptible to BBN carcinogenesis and may be utilized as a rat model for analysis of bladder tumor development. The mutation findings indicate that the enhanced tumor development is not primarily due to mutations occurring in the transgene.

Transgenic rats carrying copies of the human c-Ha-ras proto-oncogene exhibit enhanced susceptibility to N-butyl-N-(4-hydroxybutyl)nitrosamine bladder carcinogenesis

We have established a transgenic rat line carrying three copies of the human c-Ha-ras proto-oncogene with its own original promoter region, Jcl/SD-TgN(HrasGen)128Ncc (Hras128) rat. c-Ha-ras protein from expression of transduced and endogenous c-Ha-ras genes could be detected in the bladder epithelium of untreated transgenic rats. To examine their susceptibility to N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN)-induced urinary bladder carcinogenesis, male transgenic and wild-type littermates were treated with 0.05% BBN in their drinking water for 10 weeks and then killed at week 20. The numbers and volumes of total macroscopic bladder tumors including both transitional cell papillomas and carcinomas (TCC) per rat were much greater in Hras128 rats than in their wild-type counterparts. The numbers of carcinomas per rat were also significantly greater in Hras128 rats. Two cases of TCC exhibiting invasion of the bladder muscle layer, which is extremely rare in the wild-type animals under the experimental conditions used, were also observed in Hras128 rats. The GGC→GAC mutations at codon 12 of the transgene were observed in only two TCC out of 21 bladder tumors (9.5%), assessed by RFLP analysis and direct sequencing. SSCP analysis did not show any endogenous c-Ha-ras gene mutations. One of 25 tumors (4.0%) in wild-type rats had an endogenous c-Ha-ras gene mutation at codon 12 that was detected (GGA→GAA) by single-strand conformation polymorphism and direct sequencing. These results indicate that the Hras128 rat is highly susceptible to BBN carcinogenesis and may be utilized as a rat model for analysis of bladder tumor development. The mutation findings indicate that the enhanced tumor development is not primarily due to mutations occurring in the transgene.

DNA Copy Number Changes in Schistosoma-Associated and Non- Schistosoma-Associated Bladder Cancer

DNA copy number changes were investigated in 69 samples of schistosoma-associated (SA) and non-schistosoma-associated (NSA) squamous cell carcinoma (SCC) and transitional cell carcinoma (TCC) of the bladder by comparative genomic hybridization (CGH). DNA copy number changes were detected in 47 tumors. SA tumors had more changes than NSA tumors (mean, 7 vs. 4), whereas the number of changes in SCC and TCC tumors was similar. SA tumors displayed more gains than losses (1.7:1), whereas NSA tumors showed an equal number of gains and losses. Changes that were observed at similar frequencies in SCC and TCC, irrespective of the schistosomal status, included gains and high-level amplifications at 1q, 8q, and 20q and losses in 9p and 13q. These changes may be involved in a common pathway for bladder tumor development and progression independent of schistosomal status or histological subtype. Losses in 3p and gains at 5p were seen only in SCC (P < 0.01) and losses in 5q were more frequent in SA-SCC than in other tumors (P < 0.05). However, changes that were more frequent in TCC than those in SCC included gains at 17q (P < 0.01) and losses in 4q (P < 0.05) and 6q (P < 0.01). Gains and high-level amplifications at 5p were seen only in SA-SCC (P < 0. 01), whereas gains and high-level amplifications with minimal common overlapping regions at 11q13 were more frequently seen both in SA-SCC and SA-TCC tumors (P < 0.01). In addition to the above mentioned alterations, several other changes were also seen at lower frequencies. The variations in the DNA copy number changes observed in TCC, SCC, SA, and NSA bladder carcinomas suggest that these tumors have different genetic pathways.