Low IL-1alpha expression in bladder cancer tissue and survival.

OBJECTIVE: To investigate whether the previously reported association between IL-1alpha mRNA levels and survival in urinary bladder cancer remains in an extended patient material and to search a mechanism behind a possible antitumoral activity of IL-1alpha. PATIENTS AND METHODS: IL-1alpha mRNA levels were determined in 164 tumors with quantitative TaqMan PCR. RESULTS: A large variation was found in mRNA levels of IL-1alpha. We found, by immunohistochemistry, that IL-1alpha is expressed by tumor rather than stromal cells. In a univariate Cox proportional hazards model, low levels (median split) of IL-1alpha mRNA were associated with a relative hazard ratio (RHR) of 1.7 (95% CI: 1.0-2.9) for cancer-specific death (n=157); a restriction to muscle invasive tumors (n=63) resulted in an RHR of 1.8 (0.9-3.3). In bivariate analyses, adjustment for age, stage and grade respectively, decreased the RHR and the association between IL-1alpha expression and cancer-specific survival was not statistically significant. Which factors to regard as confounders remains unclear. CONCLUSIONS: Low levels of IL-1alpha mRNA expression are associated with an increased risk for cancer-specific death in the investigated material. However, confounding is an issue and to determine whether or not the observed association is causal, we need a defined mechanism and data from other studies.

Time after surgery, symptoms and well-being in survivors of urinary bladder cancer.

OBJECTIVE: To evaluate how an increasing burden of symptoms influences well-being, anxiety and depression at different intervals after a radical cystectomy with urostomy for bladder cancer, as this therapy can induce long-term distressful symptoms. PATIENTS AND METHODS: Patients with bladder cancer undergoing radical cystectomy in Stockholm between 1969 and 1995 were matched with 434 controls from the normal population; all 404 patients operated on between 1985 and 1995 at three other hospitals in Sweden were invited to enter the study. The final analysis included 306 patients and 310 controls, all assessed for symptoms and well-being. RESULTS: A low or moderate level of well-being was reported by 35% of the patients having none or one of the symptoms studied, by 39% with two symptoms, by 45% with three symptoms and by 66% of those with four or more symptoms. The values, irrespective of symptom burden, were 45% after 2-5 years of follow-up, 58% after 6-10 years and 38% at>10 years after surgery. The total symptom burden also influenced the risk of anxiety and depression. Symptom prevalence remained largely unaffected by the duration of follow-up, except for defecation urgency. CONCLUSIONS: The number of long-term symptoms after radical surgery with a urostomy for urinary bladder cancer affects the risk of anxiety, depression and low or moderate well-being.

Distressful symptoms after radical cystectomy with urinary diversion for urinary bladder cancer: a Swedish population-based study.

OBJECTIVE: To study the excess prevalence of distressful symptoms after radical surgery for urinary bladder cancer. METHODS: We included all patients who underwent cystectomy due to bladder cancer before 1996 in Stockholm County. A control group was randomly selected from the general population. Information was collected by means of an anonymous postal questionnaire. RESULTS: Completed questionnaires were returned by 310 (71%) controls and 251 (85%) cystectomized individuals. A 5-fold (reservoir) and 9-fold (conduit) increase in defecation urgency and a 4-fold (reservoir) and 6-fold (conduit) increase in faecal leakage were reported in individuals operated on. Urinary tract infection was increased 3-fold in cystectomized individuals compared with controls, during the previous year 26% of the patients reported a symptomatic infection. The perception of a reduced physical attractiveness due to disease was more than 5-fold increased in the men operated on compared to the controls. The majority, 135 out of 201 (67%), reported that they would have refused alternative bladder-sparing procedures if they decreased the prospects of survival by even as little as 1%. CONCLUSIONS: The patient's situation after cystectomy is considerably impaired due to changed bowel and sexual function, urinary tract infections and a sense of decreased attractiveness. However, most patients are in spite of this unwilling to compromise survival.

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.

p53 mutations in urinary bladder cancer.

We have screened for mutations in exons 5-8 of the p53 gene in a series consisting of 189 patients with urinary bladder neoplasms. 82 (44%) neoplasms were lowly malignant (Ta, G1-G2a) and 106 (56%) were highly malignant (G2b-G4 or > or = T1). Only one mutation was in a lowly malignant urinary bladder neoplasm, in total we found p53 mutations in 26 (14%) of the 189 patients. 30% of the samples had loss of heterozygosity (LOH) for one or both of the p53 exogenic (CA)n repeat and the p53 intragenic (AAAAT)n repeat markers. 31 samples (21%) showed LOH but were not mutated, suggesting other mechanisms inactivating p53 than mutations. 4 mutations were found at codon 280 and 2 mutations were found at codon 285, 2 previously reported hot spots for urinary bladder cancer. The study indicate a boundary between G2a and G2b tumours concerning the occurrence of genetic events affecting p53 function; moderately differentiated (G2) urinary bladder neoplasms probably are genetically heterogeneous which supports the suggestion that they should not be grouped together but instead, for example, be categorized as either lowly or highly malignant.

The prevalence of loss of heterozygosity in chromosome 3, including FHIT, in bladder cancer, using the fluorescent multiplex polymerase chain reaction.

OBJECTIVE: To determine some of the genetic alterations involved in the pathogenesis and progression of transitional cell carcinoma of the bladder. Materials and methods In a population-based study, freshly frozen tissue was collected from all patients newly diagnosed with urinary bladder cancer in the Stockholm region during 1995-1996. The prevalence of loss of heterozygosity (LOH) was assessed at seven sites on chromosome 3, analysed in 151 patients, using a fluorescent multiplex polymerase chain reaction based on DNA from the tumour and peripheral blood. RESULTS: LOH was detected in 12.1% (at 3q25-26.2) to 22.1% (at 3p11-12) of the informative cases. Relatively frequent LOH was detected at 3p22-24.2 (21.6%), at 3p14.2 within FHIT (21.5%), and at 3p11-12 (22.1%). Of 151 tumours, 72 (47.7%) showed LOH at one or more loci on chromosome 3. LOH on chromosome 3 was weakly associated with tumour grade (P = 0.095), but not with tumour stage (P = 0.701). However, when the frequency of LOH was analysed individually at each site, the prevalence of LOH at 3p11-12 was closely correlated with higher tumour stage (P = 0.011). Replication errors were detected in only four of 151 (2.6%) tumours. Conclusion These findings suggest that the 3p11-12 locus may involve a putative candidate tumour-suppressor gene which might be associated with bladder tumour invasiveness. The FHIT gene locus showed a relatively high frequency of LOH even in Ta tumours.

Low interleukin-1alpha messenger RNA levels predict decreased overall survival time of patients with urinary bladder carcinoma.

Due to our inability to exactly characterize tumours, many patients with urinary bladder cancer undergo unnecessary surgery or cytostatic therapy. We have here studied the expression of the cytokine interleukin-1alpha (IL-1alpha ) in 73 human bladder carcinomas in relation to patient survival, and examined possible relationships between IL-1alpha and urokinase plasminogen activator (uPA) expression. Expression levels of IL-1alpha and uPA mRNA were determined by RT-PCR using the quantitative TaqMan technique. The levels of IL-1alpha mRNA expression did not differ significantly between tumours of different grade or stage. Calculation of the overall survival rates showed a decreased overall survival time for patients with low levels of IL-1alpha mRNA in their tumours (log rank; P = 0.0002, median follow up: 37 months). Low tumoral IL-1alpha expression predicted decreased survival of patients with poorly differentiated tumours (P< 0.005) and of patients with invasive tumours (P = 0.02). uPA expression was about 4-fold increased in poorly differentiated tumours. High levels of uPA mRNA were associated with decreased overall survival times (log rank; P = 0.032, n = 60). We conclude that IL-1alpha is important for bladder cancer biology, and that measurements of this cytokine may be useful in pre-treatment characterization of urinary bladder cancer.

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.

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.

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.

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.

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.

5-year followup of a randomized prospective study comparing mitomycin C and bacillus Calmette-Guerin in patients with superficial bladder carcinoma. Swedish-Norwegian Bladder Cancer Study Group.

PURPOSE: We report the 5-year followup of a randomized comparison of mitomycin C and bacillus Calmette-Guerin (BCG) in patients with superficial bladder carcinoma. Recurrence, progression and survival rates, crossover results, prognostic factors and long-term side effects were analyzed. MATERIALS AND METHODS: A total of 261 patients were enrolled in the study, and the inclusion criteria were primary Tis, dysplasia G2, T1 G3 and multiple recurrent Ta/T1 G1-2 disease. Intravesical instillations of 40 mg. mitomycin C and 120 mg. Pasteur BCG, Danish strain 1331, were given for 2 years. RESULTS: After a median followup of 64 months 101 of the 250 evaluable patients (42%) were disease-free. A significant difference was noted in disease-free survival with BCG (p = 0.04), which was most pronounced for stage Tis disease. No difference in tumor progression, or crude or corrected survival was found between the 2 arms. Crossover treatment was successful in 39% of patients with second line BCG and 19% with second line mitomycin C. Independent risk factors for progression were initial p53 status and stage. Only the completion of treatment was predictive of outcome for patients treated with BCG. Bladder shrinkage occurred in 2.4% of patients. CONCLUSIONS: Therapy with BCG was superior to mitomycin C for recurrence prophylaxis but no difference was found for progression and survival.

Evaluation of clinical staging before cystectomy in transitional cell bladder carcinoma: a long-term follow-up of 276 consecutive patients.

OBJECTIVE: To evaluate retrospectively the clinical staging in a consecutive series of patients selected for cystectomy and to define its limitations with a view to possible improvements. PATIENTS AND METHODS: From 1979 to 1988, 276 patients with newly detected or recurring transitional cell carcinoma (TCC) of the bladder, were offered pre-operative irradiation (20 Gy) and cystectomy. The patients were assessed during 1995 and the outcome related to both clinical and surgical data. Survival was analysed on the basis of 'intention to treat'. Estimates of survival probabilities were calculated by the method of Kaplan and Meier. Differences in survival among subgroups were assessed using the log rank test and Cox stepwise regression analysis. RESULTS: Cancer-specific actuarial survival for the whole series was 68% at 5 years and 63% at 10 years. Survival was closely related to the depth of invasion found at surgery, clearly discriminating those with tumours confined to the bladder wall (< or = P3A) from those with extravesical extension (> or = P3B). The cancer-specific survival at 5 years for patients with < or = P3A tumours was 85% and for those with > or = P3B tumours was 50%. This important distinction was anticipated accurately using bimanual palpation before surgery, those patients with no palpable mass after transurethral resection of bladder tumour (TURBT) having an actuarial survival of 83%, and those with a residual mass a survival of 50% at 5 years. In the multivariate analysis, increasing clinical stage was the only pretreatment variable with significant prognostic value for survival. However, this variable was highly dependent on the palpatory findings after TURBT, the presence of a residual mass being a prerequisite for the clinical stage T3 in case of muscle-invasive tumour. CONCLUSION: Bimanual palpation remains crucially important in clinical staging, and there is a need for further standardization and refinement of this procedure.

A randomized prospective study comparing long-term intravesical instillations of mitomycin C and bacillus Calmette-Guerin in patients with superficial bladder carcinoma.

PURPOSE: We compared the efficacy and toxicity of long-term mitomycin C versus bacillus Calmette-Guerin (BCG) instillation in patients at high risk for recurrence and progression of superficial bladder carcinoma. MATERIALS AND METHODS: Our randomized comparison study included 261 patients with primary dysplasia, or stage Tis, stage T1, grade 3 and multiple recurrent stage Ta/T1, grade 1 or 2 disease. Mitomycin C (40 mg.) or Pasteur strain BCG (120 mg.) was instilled weekly for 6 weeks, then monthly for up to 1 year and every 3 months during year 2. RESULTS: After a median followup of 39 months 49% of the patients given BCG and 34% given mitomycin C were disease-free (p < 0.03), compared to 48 and 35%, respectively, of those with stage Ta or T1 disease, and 54 and 33%, respectively, of those with dysplasia or stage Tis tumor. Tumor progressed in 13% of patients, with no statistically significant difference observed regarding progression between the mitomycin C and BCG groups. Side effects were more common after BCG instillation, with 5 cases of severe side effects compared to 1 in the mitomycin C group. Treatment was stopped due to toxicity in 10% of the patients. CONCLUSIONS: The majority of patients tolerated long-term intravesical therapy well. BCG instillation was hampered by more frequent side effects. BCG was superior regarding recurrence prophylaxis, since patients given BCG had fewer recurrences and a significantly longer time to treatment failure compared to those treated with mitomycin C. No statistically significant difference was observed regarding progression.

Histopathological changes in androgen-deprived localized prostatic cancer. A study in total prostatectomy specimens.

Androgen deprivation with triptorelin treatment prior to total prostatectomy gave the opportunity of studying consequent morphological changes in the surgical specimens from 38 men with localized prostatic cancer. Multiple core-needle biopsies were taken prior to treatment and compared to the step-sectioned surgical specimens. The histological changes in the prostate following androgen deprivation include glandular atrophy, nuclear pyknosis, cytoplasmic vacuolation, squamous metaplasia and an increase in the relative amount of stroma. None of these changes could be correlated to pretreatment tumour grade, nor to the volume of the residual tumour. A positive correlation was found between tumour grade prior to therapy and volume of residual tumour after treatment. The treatment was not associated with downgrading of the malignancy. On the contrary, in 16% of the cases, foci of a higher grade were found in the surgical specimens compared to the pretreatment core-needle biopsies.

Neoadjuvant GnRH-agonist treatment (triptorelin and cyproterone acetate for flare protection) and total prostatectomy.

The effects of 3 months treatment with the GnRH agonist triptorelin as a neoadjuvant to total prostatectomy in 40 men with localized prostatic cancer have been evaluated. The study included 1 patient with a stage T1b tumour, 25 patients with stage T2 tumours and 14 with stage T3 tumours. The patients were examined by digital rectal examination, transrectal ultrasound before and after treatment. Serum testosterone and prostate-specific antigen (PSA) levels were followed. The totally removed prostate gland was step-sectioned at 5-mm intervals and the whole-mount sections were assessed for tumour pathology stage (pT stage). Triptorelin treatment resulted in a significant decrease in total gland and tumour volume and in a reduction in the serum levels of PSA and testosterone. In comparison with the findings from a previous study, in which neoadjuvant treatment was not used, it appears that the proportion of tumours invading the margins of the surgical specimen decreased.

Deoxyribonucleic acid profile and tumor progression in primary carcinoma in situ of the bladder: a study of 63 patients with grade 3 lesions.

In 63 patients with primary grade 3 carcinoma in situ of the bladder flow cytometric deoxyribonucleic acid (DNA) analysis was performed at diagnosis and during an average followup of 63 months. The results of DNA measurements were related to disease progression, that is invasive tumor and/or metastatic disease. The DNA histograms were classified as diploid (2 patients) or aneuploid (61). A total of 3 categories of aneuploid tumors with different prognostic significance could be defined: 1) carcinoma in situ with 1 aneuploid cell population at diagnosis and with no change to multiple aneuploid cell populations throughout observation, 2) carcinoma in situ with 1 aneuploid cell population at diagnosis but with a later change to multiple aneuploid cell populations and 3) carcinoma in situ with multiple aneuploid cell populations already at diagnosis. At 5 years the progression-free survival for the 3 categories was 94%, 43% and 20%, respectively. Over-all, of the patients with multiple aneuploid cell populations (categories 2 and 3) 76% had progression, in contrast to 19% of those in category 1 (p less than 0.0005). In category 2 development of multiple aneuploid cell populations preceded progression in 8 of 11 progressive cases by an average of 20 months. Therefore, the occurrence of multiple aneuploid cell populations must be considered as a sign of high aggressiveness. We conclude that flow cytometric DNA analysis is a potent predictor of prognosis in cases of primary carcinoma in situ of the bladder.