Bladder cancer: allelic deletions at and around the retinoblastoma tumor suppressor gene in relation to stage and grade.

Inhibition of the retinoblastoma tumor suppressor gene (RB) is probably important in the pathogenesis of urinary bladder cancer. Little information is available concerning allelic loss on 13q11 to 13q32 and its relation to grade and stage. In a population-based study, freshly frozen tissue was collected from all new cases of urinary bladder cancer in the Stockholm region during 1995-1996. Here we report the occurrence of loss of heterozygosity (LOH) at seven sites in 13q11 to 13q32 as analyzed in 236 cases by a fluorescent multiplex PCR-based on tumor DNA and peripheral blood. For each site, about 30% of the cases were not informative because of homozygosity. Replication errors were detected in 4% (17 cases). LOH was found in 21 (at 13q11-12.1) to 32% (at 13q14.3 in RB) of the informative cases. A correlation was found between the prevalence of LOH at all observed loci and stage and grade, respectively, and it was statistically significant for 13q14.3. LOH at RB was found in Ta as well as grade 1 tumors. Also, a statistically significant correlation was found between the number of loci with LOH at 13q and tumor stage and grade, respectively. Typically an altered RB function is related to the expected clinical course of urinary bladder cancer, but allelic loss including the gene also occurs in low grade and low stage tumors. An altered RB function probably is not necessary for a malignant transformation of urothelial cells. The causal direction of the relation between the quantity of the deleted DNA and tumor aggressiveness is not clear.

Initiation-development modelling of allelic losses on chromosome 9 in multifocal bladder cancer.

Multiple low-grade, low-stage superficial tumours were analysed for loss of heterozygosity (LOH) on chromosome 9 with 29 markers. Three consensus regions were found, one at 9p (9p21-22) and two at 9q (9q21-31 and 9q32-34). Phylogenetic trees were calculated for each patient using both designated chromosome 9 regions and, separately, using individual microsatellite data. Regional analysis suggested that multiple, equally important regions for bladder tumour initiation exist on chromosome 9. During the development of tumours all regions were eventually affected. The phylogenetic analyses with individual markers were used as molecular clocks to trace the ordering of tumours. The results were compared with the physical locations of the tumours and a hypothetical development model was built. These are novel approaches which, to our knowledge, have not been used before.

Loss of heterozygosity in tumour-adjacent normal tissue of breast and bladder cancer.

Normal tumour-adjacent breast tissue samples from 12 breast cancer patients forming six monozygotic twin pairs were analysed for loss of heterozygosity (LOH) on chromosomes 1, 13 and 17. 7 patients showed LOH at one or more markers. Each of them had a different LOH pattern. Only one twin pair showed LOH at the same locus, but the twins had lost a different allele. Multiple (n=1-13), histologically normal samples were collected from 6 bladder cancer patients and analysed for LOH on chromosomes 3 and 9. On chromosome 9, all 6 patients analysed showed LOH in at least one sample and one marker. Four of them also showed LOH on chromosome 3. Samples surrounding different tumours of a given patient resembled each other. More heterogeneity was seen between the patients, even though they shared some similarities in LOH clustering. The results demonstrate that tumour-adjacent normal tissues already harbour genetic changes typical for tumours. These alterations can reveal the earliest changes leading to tumorigenesis.

Multiple regions with allelic loss at chromosome 3 in superficial multifocal bladder tumors.

We have examined six patients with multiple low grade, low stage superficial multifocal bladder tumors with surrounding tissues for loss of heterozygosity (LOH) and microsatellite instability at chromosome 3, totaling 76 samples. The majority (4/5) of the patients had LOH at or close to the fragile histidine triad (FHIT) gene (3p14.2; D3S1300), which is a candidate tumor suppressor gene for many cancer types. One patient showed a consistent LOH with four adjacent markers around FHIT region in all the tumors whereas in the corresponding surrounding tissues the heterozygosity was retained. In addition to the region near FHIT, two other regions had frequent allelic losses - one near the p telomere (3p25-26; D3S3050) and another near the q telomere (3q27; D3S2418). The largest numbers of LOH in the surrounding tissues were found at these regions (3/5 at D3S3050 and 2/5 at D3S2418). The D3S3050 marker is located at 3p26-3p25, near the Von Hippel-Lindau (VHL) tumor suppressor gene locus. LOH that were more random were found at 3q21.3-25.2 (D3S1744) and at 3p12-3p11 (D3S2465). Taken together, at least three regions at chromosome 3p25-26, 3p14.2 and 3q27 seem to have frequent loss of heterozygosity in multifocal superficial bladder tumors. We also performed a phylogenetic-type analysis to find out common changes and the degree of heterogeneity. The overall heterogeneity was low within a given patient: in all cases the majority of the tumor samples arranged in a single branch with a common origin. This point of origin varied from patient to patient, which is compatible with the earlier studies demonstrating the heterogeneity of the single primary bladder tumors. However, the phylogenetic-type analysis suggests that the FHIT region contains often the very first alterations at chromosome 3.

Cytogenetic analysis of 62 transitional cell bladder carcinomas.

Chromosome analysis of biopsy material obtained after vinblastine pretreatment was carried out in 108 specimens from 89 patients with transitional cell carcinoma of the urinary bladder. Analyzable metaphases were obtained in 62 tumors, but only in nine tumors could karyotypes be analyzed by banding; a conventional technique was used in all others. Ploidy and occurrence of markers corresponded with tumor morphology and invasion and sometimes aided in the clinical evaluation; chromosome anomalies specific for bladder cancer were not revealed. In noninvasive tumors of WHO grade 1 and 2, near-diploid karyotypes with occasional marker chromosomes dominated. Grade 3 tumors showed a variety of grossly aneuploid karyotypes, with an almost constant occurrence of different markers. Superficially invasive G2 tumors had moderately pronounced aberrations with more deviations than non-invasive tumors but without the great variety of G3 tumors.

Chromosome and DNA cytometric study of a papillary carcinoma of the bladder with a high stemline and numerous double minutes.

A papillary cancer of the bladder [WHO grade 3] was studied with chromosome and DNA analysis. A high frequency of mitoses was noted, and analysis of 151 metaphases revealed a dominating stemline in the pentaploid region. More than 95% of the cells contained double minutes (dm). Flow cytometry showed the dominant stemline to be in the hexaploid region, that is, about 20% above the numerical chromosome value. This difference is generally obtained when these two methods are compared and is not influenced by the presence of dm. Both methods have been complementary in the evaluation of this tumor.

Prevention and treatment of urothelial premalignant and malignant lesions.

Bladder cancer is believed to develop through reversible premalignant stages followed by irreversible steps, and ending in invasive cancer giving rise to distant metastases. Because of the variation in the clinical course it has also been suggested that different forms of cancer develop along different molecular pathways leading to tumor presentations of various malignant potential. Today we treat and prognosticate bladder cancer on the basis of clinical and histologic findings that are insufficient to assess all the biologic potential of these tumors. Understanding the pathogenesis of bladder cancer might lead to a more precise identification of particular tumors with regard to clinical aggressiveness, resulting in individualized strategies for treatment and prophylaxis. Bladder cancer is seldom diagnosed in its preclinical stage, it is instead detected at cystoscopy and virtually never recognized as an incidental finding on autopsy. Therefore its "natural history" largely reflects that of "treated" disease. The true incidence of premalignant and malignant epithelial changes is not known. Incidences of hyperplasia and dysplasia of approximately 10% and approximately 5%, respectively and only occasional findings of cancer itself were reported in two autopsy series. Urothelial dysplasia is generally believed to be premalignant and a putative precursor of invasive cancer but unfortunately there has been a lack of standardization in terms of terminology and diagnosis. There is also a need for an agreed definition of the boundary between premalignancy, i.e. urothelial changes that have some but not all the features of carcinoma in situ, and malignancy, especially when considering potentially harmful treatments to prevent this transition. Most new diagnostic tools available and being tested today compare new detection techniques with traditional methods such as cytology or conventional histology of malignant rather than premalignant changes. There is probably also a short preclinical latency, as implied by the incidental findings of bladder cancer at autopsy, which makes it necessary to define how and when to promote early detection and treatment. Future studies therefore have to concentrate on methods for early detection of disease as well as characterization of host susceptibility, evaluation of exposure to carcinogens and potential effects of preventive measures. It is also likely that the improved tools of molecular prognostication will allow us to design trials more precisely in order to tailor therapeutic strategies.

Prophylactic instillation therapy of superficial bladder cancer. A randomized study comparing mitomycin C and adriamycin with special reference to DNA ploidy.

Sixty patients with Ta and T1 bladder cancer were randomized between treatment with resection only and resection and instillations with either Adriamycin or Mitomycin C. Treatment lasted for one year and patients were evaluated after a mean follow-up of 35 to 47 months if progression had not occurred. Mitomycin C was superior in reducing the recurrence rate. Progressive disease was observed in 17 patients regardless of therapy but in all patients DNA aneuploidy could be identified at a risk factor.

Deoxyribonucleic acid flow cytometry in predicting response to radical radiotherapy of bladder cancer.

Using flow cytometry tumors can be classified according to their deoxyribonucleic acid content as diploid or aneuploid with 1 or several cell lines and to the ploidy level of the aneuploid cell lines, as well as to the proportion of proliferating cells. We used this technique in 73 patients with invasive transitional cell carcinoma of the bladder who received radical radiation therapy with followup of surviving patients for at least 5 years. The effects of therapy were related to the deoxyribonucleic acid patterns before irradiation. All diploid tumors disappeared, while aneuploid tumors with 1 cell line disappeared in 55% and those with several cell lines in 30% of the patients. Aneuploid tumors with cell lines exceeding 4c appeared to be more radiosensitive than those in the triploid region. Thus, tumors unresponsive to therapy as well as those that recurred after irradiation were mostly triploid. Since most of the tumors had high proliferation rates the proportion of S-phase cells was of only limited value for further classification of the various tumors. In the choice between radical radiotherapy, in which bladder function is maintained, and cystectomy an analysis of tumor ploidy provides at least some basis on which radiation response can be predicted. In the choice of therapy we must also consider the fact that almost 20% of the patients who failed to respond to therapy revealed aneuploidy only in bladder washings after irradiation, indicating existence of concomitant carcinoma in situ.

Allelic losses demonstrate monoclonality of multifocal bladder tumors.

The clonality of multifocal bladder tumors has been studied over the years with some controversial results. We have examined 5 patients with 2-11 low-grade superficial multifocal bladder tumors for loss of heterozygosity (LOH) at 87 loci on 9 chromosomes. When LOH was detected at a given marker, the tumors consistently showed deletion of a specific allele, suggesting the monoclonality of the patients' tumors. No allelic imbalancies were detected between heterozygote alleles, and the allelic losses were only slightly biased toward the loss of the shorter alleles as the overall ratio was 0.48 +/- 0.10 (0.50 for nonbiased). We calculated the probabilities for monoclonality using binomial distribution. The use of multiple tumors with multiple microsatellite markers gives high statistical power for the calculation. The combined probabilities for monoclonality varied from 0.984 to (1-4 x 10(-28)). Thus, in most (4/5) cases, the probability for polyclonality was <2 x 10(-16). These results demonstrate that superficial multifocal bladder tumors are most likely of monoclonal origin.

Treatment of invasive grade 3 transitional cell bladder cancer. A retrospective study of 10 years' experience with external irradiation, and preoperative irradiation and cystectomy.

Three hundred and seventy-four patients with invasive grade 3 bladder cancer treated with either external irradiation or preoperative irradiation and cystectomy were re-evaluated. Cancer-free survival was markedly better for patients who underwent preoperative irradiation and operation than irradiation only, even if a complete clinical response had occurred initially.

Urine or bladder washings in the cytologic evaluation of transitional cell carcinoma of the urinary tract. A comparison made under routine conditions supplemented by flow cytometric DNA analysis.

The usefulness of urine and bladder washings for cytological purposes and routine conditions was compared in a reevaluation of 192 specimens from urothelial tumors. To supplement the cytological interpretation by more objective means urine and bladder washings from the same tumours were analyzed by flow cytometry. The cytological reevaluation demonstrated the necessity for extensive experience in this kind of material. No real difference was noted between urine and bladder washings in the cytological interpretation but bladder washings were superior to urine in displaying aneuploidy. The results illustrate the need for a precise sampling technique and for the supportive role of flow cytometry in the management of urothelial tumors.

DNA analysis in predicting survival of irradiated patients with transitional cell carcinoma of bladder.

A total of 115 patients with invasive transitional cell carcinoma of the bladder underwent radical radiotherapy between 1975 and 1986 and were followed up until the end of 1990. Apart from routine clinical observations, flow cytometric DNA measurements made on fresh tumour material were available for analysis. Actuarial cancer-free survival controlling for response to treatment was analysed with the log-rank test, bivariate and multivariate analyses using Cox's stepwise regression model on probable prognostic factors. The overall actuarial 5-year cancer-free survival rate was 30%. Survival was significantly correlated with response to treatment: 59% for patients with complete regression and 5% for those with residual tumour. Prognostic factors that significantly correlated with death from cancer were advanced stage, large size, incomplete resection, ureteric obstruction, anaemia, carcinoma in situ grade 3 and occurrence of more than one aneuploid cell population. However, only 3 of these factors were of independent power in the multivariate analysis: stage, size and carcinoma in situ. Of 21 patients with a history of primary or secondary carcinoma in situ, 19 died from cancer during follow-up: 18 of the 21 patients had tumours that were aneuploid with more than one aneuploid cell population. It is concluded that curative radiotherapy can be successful only in patients with less advanced tumours assessed according to clinical stage and size, aneuploid tumours with not more than one aneuploid cell line, no carcinoma in situ, no ureteric obstruction, and in whom a complete transurethral resection of the exophytic tumour is possible.

The value of preoperative classification according to the TNM system. A critical examination of Category T2 in 96 patients with grade 3 transitional cell bladder carcinoma subjected to preparative irradiation and cystectomy.

A critical reevaluation of the preoperative classification in 96 patients with transitional cell bladder carcinomas of grade 3 (WHO) and clinical category T2 has been performed. As a result of incomplete biopsies and clinical data, it was possible to reclassify only 49 tumors (51%). 43 of these seemed to be of a lower stage than originally proposed. In addition downstaging after preoperative irradiation occurred in 77 patients (80%). In view of the uncertainties in the present series, a return to a simplified preoperative classification for clinical purposes is proposed--e.g. tumors with mucosal growth, tumors with muscle infiltration, tumors extending outside the bladder.

Deoxyribonucleic acid analysis in the evaluation of transitional cell carcinoma before cystectomy.

Analysis of cellular deoxyribonucleic acid content via flow cytometry was done on tumor biopsies and bladder washings from 61 patients with transitional cell carcinoma who underwent preoperative irradiation (36 or 20 Gy) and cystectomy. Classification only of tumors as diploid or various forms of aneuploid did not facilitate the separation of these advanced tumors into different subgroups. In addition, the proliferation rates, which also were high, did not facilitate further subclassification. On the other hand, more than 1 aneuploid cell line in the preoperative biopsies was linked more closely to pathological than to clinical stage and was found particularly if the stage was unchanged or increased after irradiation. The deoxyribonucleic acid pattern generally was the same after irradiation in patients with residual tumor and persistent aneuploidy. However, a diploid deoxyribonucleic acid value was noted in a number of patients with residual tumors, which indicated the existence of diploid and aneuploid tumor cells, and a preferential irradiation effect on the aneuploid portion of the tumor cell population.

Flow cytometric DNA and cytogenetic studies in human tumors: a comparison and discussion of the differences in modal values obtained by the two methods.

The numerical chromosome values in 53 human tumors were determined and compared with the modal DNA values as measured by flow cytometry. In tumors with chromosome counts in the diploid and tetraploid range, the modal DNA values were found to correspond to the modal values based on the chromosome counts. In tumors with chromosome counts in the triploid range, however, the modal DNA values were about 15% higher than expected. In order to explain this difference, the ratio between large and small chromosomes in the karyotyped metaphases was assessed. In addition, the DNA content of individual chromosomes, including markers and minutes, was calculated as a reflection of the DNA content of the whole cell. The ratio of large to small chromosomes did not deviate from the normal ratio found in cells with diploid, triploid, and tetraploid chromosome counts. Neither difficulties in karyotyping nor short-comings in the flow cytometric methodology could be used to explain the discrepancy between the expected and empirical modal DNA values. Some of the chromosomes in triploid tumors may, therefore, contain an increased amount of DNA.

Chromosomal and DNA patterns in transitional cell bladder carcinoma. A comparative cytogenetic and flow-cytofluorometric DNA study.

Biopsy tissue from 71 bladder tumors in 57 patients was studied by chromosome analysis and flow cytofluorometry. Chromosome analysis was hampered by preparation difficulties and was successful in only 53% of the analyzed tumors. DNA analysis failed to reveal near-diploid deviations, while the grossly aneuploid tumors generally had DNA values 10% to 15% above the numerical chromosome counts. Noninvasive tumors were diploid-near-diploid with occasional markers. Superficially invasive tumors were both diploid-near-diploid and near-tetraploid, but with a chromosome count of only 80 in the latter cases. Deeply invasive tumors tended to be triploid on DNA analysis, and had chromosome counts in the vicinity of 60. In addition to single chromosome abnormalities, increased malignancy apparently is also associated with an increased total number of chromosomes. A tetraploid DNA level seems to represent a more stable genome, although chromosomes show gross abnormalities including markers.

Computed tomography in staging of bladder carcinoma.

The value of computed tomography in staging of bladder carcinoma was evaluated by comparison with clinical and histopathologic stage according to the TNM system. The CT results seem to indicate a possibility to distinguish between superficially and deeply invasive tumours.