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Urinary liquid biopsy (LB) tests have contributed to reducing overtreatment and improving the detection of bladder and prostate cancer. Circulating tumor cells, DNA, and RNA are the focus of next-generation blood-based LB tests. These tests aim to stratify risk according to the detection of resistance and recurrence markers in patients with metastatic disease. The costs, lack of standardization, reimbursement challenges, and specialized workflows associated with characterization of blood-derived biomarkers are the primary barriers to their clinical implementation. This review provides an overview of the successes and pitfalls of LB-based tests for bladder and prostate cancer. PATIENT SUMMARY: Urinary and blood-based liquid biopsy tests are minimally invasive and highly efficacious in detecting clinically significant cancer., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Purpose: The risk of muscle invasive disease in a high risk patient with superficial bladder cancer is up to 50%. Identifying patients at risk for progression remains an unsolved problem. A suggested prognosticator is mutations in the p53 tumor suppressor gene. We determined the value of p53 mutation, as demonstrated by mutation analysis, in a clinically selected group of high risk patients with superficial bladder cancer., Materials and Methods: p53 Mutation analysis was performed by automated sequencing of bladder wash samples of 105 patients with high risk superficial bladder cancer. The mutation and WT groups were subsequently compared with regard to mortality, progression, disease worsening and the recurrence-free period., Results: A total of 29 patients had a mutation and 76 had WT. Median followup was 58.3 months (range 3 to 161). A total of 13 patients died of bladder cancer, including 6 of 29 with a mutation and 7 of 76 patients in the WT group. p53 Mutation had no significant prognostic value for decreased survival, progression or disease worsening. Recurrence-free survival was significantly lower in the WT group., Conclusions: We observed a trend toward a worse clinical outcome in high risk patients with a p53 mutation in the bladder wash. However, no significant differences were seen in clinical outcome parameters. Based on these data we conclude that the prognostic value of a p53 mutation is insufficient for individual policy making.

Introduction: Superficial bladder cancer can be treated by transurethral resection and additional intravesical therapy. Although agents like Mitomycin C, Epirubicin and BCG are routinely used, there is a need for more potent and/or less toxic agents. Gemcitabine is a deoxycytidine analogue, used systemically for several tumours, such as non-localised bladder cancer, where it is effective and well tolerated. We investigated the use of three dose levels of gemcitabine when given intravesically in humans for safety and pharmacokinetic research., Material and Methods: Patients with superficial bladder cancer, except pT1G3 or CIS were included. Six weekly instillations of 1000, 1500 or 2000 mg gemcitabine were given in 50 ml saline for one hour. Dose modifications were defined in case of dose limiting toxicities. Blood samples were taken before, and 5, 30, 60 (= evacuation) and 120 minutes after instillations 1, 3 and 6. Samples were used for blood counts and pharmacokinetics. Side effects were noted., Results: 3, 4 and 3 patients were treated with 1000, 1500, and 2000 mg gemcitabine respectively, of which 2, 3 and 1 patients had highly recurrent tumours before treatment. Seven patients experienced side effects: 2 with dysuria after the first instillation, 2 after instillations 3-6 and 4-6 and in 3 patients headache, fatigue and heavy legs were experienced once. All side effects were reversible, non-limiting and WHO 1. No macroscopic hematuria was seen. Haematology showed only one case of drop in white blood cell count (lowest dose level, after the first instillation). Gemcitabine plasma levels were immeasurable or low, with peak levels between 30 and 60 minutes, decreasing after more instillations. The metabolite difluorodeoxyuridine reached levels of at most 5 microM, indicating a very low passage of the drug to the systemic circulation., Conclusion: Intravesical gemcitabine in the dose used has minimal and reversible side effects. Plasma evaluation indicates that its intravesical use is safe.

Background: Expression of the inhibitor of apoptosis protein survivin is up-regulated in many tumors of epithelial origin and frequently shows a relationship with disease prognosis., Materials and Methods: We investigated survivin mRNA expression in 32 urothelial cell carcinomas by use of real-time quantitative PCR. Expression values were normalized to transcript levels of the housekeeping gene cyclophilin., Results: All bladder tumor tissues expressed survivin mRNA. The median normalized survivin mRNA expression values were 0.26 for superficial tumors (n = 17) and 0.78 for invasive tumors (n = 15). A significant relationship with increasing pathological stage (p < 0.001) and grade (p < 0.001) was observed. Although survivin mRNA expression did not relate to disease progression or the patient survival period, patients with superficial bladder tumors and normalized survivin values over 0.26 had an increased risk of recurrence (log-rank test: p = 0.018)., Conclusion: Our results suggest that quantitative measurement of survivin mRNA 1) can identify invasive and high-grade urothelial cell carcinomas and 2) may be used as an indicator for early recurrence of superficial tumors.

Objectives: The polymorphic enzyme alcohol dehydrogenase (ADH) catalyses the conversion of ethanol into the carcinogenic metabolite acetaldehyde which is partly excreted into the urine. Objectives of this pilot study are to determine whether this polymorphism may be related to bladder cancer and whether it modifies the relation between alcohol intake and bladder cancer., Methods: 120 bladder cancer patients and 133 convenience controls were recruited at the University Medical Centre Nijmegen. A self-administered questionnaire was used to assess alcohol intake and potential confounders. DNA was isolated from peripheral blood, and ADH3 genotype was determined using PCR/RFLP. People with the ADH3 genotype gamma1gamma1 were compared to people with other ADH3 genotypes. Above moderate drinkers (>14 glasses of alcohol per week) were compared to moderate drinkers. Odds ratios (ORs) and 95% confidence intervals were computed using logistic regression analyses., Results: After correction for sex, age and smoking, ORs for ADH3 genotype and alcohol intake were 2.10 (1.05-4.22) and 1.21 (0.60-2.44), respectively. Moderate drinkers with the 'high-risk' (gamma1gamma1) ADH3 genotype appeared to have a threefold higher risk of bladder cancer compared to moderate drinkers with a 'low-risk' (gamma1gamma2 or gamma2gamma2) genotype. Surprisingly, the ORs for above moderate drinkers with the low-risk genotype (1.95; 95% CI: 0.85-4.48) and with the high-risk genotype (2.18; 95% CI: 0.82-5.77) were almost similar, suggesting no interaction., Conclusions: ADH3 genotype is a possible risk factor for bladder cancer. Although moderate drinkers with the gamma1gamma1 genotype seem to have the highest risk, we did not get a clear indication that ADH3 genotype modifies the relationship between alcohol intake and bladder cancer.

Objectives: To study the diagnostic performance of the ImmunoCyt test in patients in follow-up for superficial urothelial cell carcinoma (UCC) of the bladder., Methods: Voided urine samples were collected from all included patients. Samples were processed with the ImmunoCyt test. The ImmunoCyt slides were scored under a fluorescence microscope by 3 observers. The ImmunoCyt test was considered positive if one or more observers scored the test positive. Urethrocystoscopy (and additional histologic examination in the case of suspicious cystoscopic findings) was used as the reference standard. To investigate the validity of ImmunoCyt, sensitivity, specificity, positive predictive value, negative predictive value, area under the receiver operating characteristic curve, and diagnostic odds ratios were determined. To investigate the reproducibility of ImmunoCyt, kappa values (measure of agreement) were computed. The observers' findings were analyzed in pairs., Results: One hundred four patients in follow-up after primary superficial UCC of the bladder were included. Samples of 18 patients had to be excluded because of low cellularity (ie, insufficient assessable urothelial cells). Tumor recurrence was found in 22 of the remaining 86 patients (17 pTa, 3 pT1, 1 carcinoma in situ, 1 pT2 or higher). The test had a sensitivity of 50%, specificity of 73%, positive predictive value of 39%, and negative predictive value of 81%. The diagnostic odds ratio was 2.8 (95% confidence interval 1.0 to 7.5). The area under the curve for the different observers varied between 0.54 and 0.60. The kappa values were low (0.05 to 0.45), representing high interobserver variability., Conclusions: The promising results from other studies could not be confirmed in this specific group of patients in follow-up for superficial UCC of the bladder. The validity of ImmunoCyt was insufficient to justify the omission of cystoscopy in patients in follow-up for superficial UCC.

Synchronous and metachronous tumors are frequently observed in the urinary tract and may be explained by the concept of 'field cancerization,' i.e., exposure to carcinogens leading to the independent transformation of many urothelial cells resulting in oncogenetically unrelated tumors. Increasing evidence, however, supports the concept of clonality, i.e., the progeny of a single transformed cell spreads through the urinary system resulting in genetically related tumors. The aim of our study was to investigate the putative clonal origin of invasive urothelial cell carcinomas (UCCs) of the bladder from a prior superficial tumor. We selected 6 patients (5 males and 1 female) with superficial and subsequent invasive UCC tumors. All patients were previously diagnosed with a p53 mutation in their invasive tumor. At least 1 superficial and 1 invasive tumor of the same patient were analyzed for mutations in the p53 tumor suppressor gene by PCR-SSCP and, in case of a band shift, followed by direct sequencing. In all patients the same p53 mutation was found in the superficial and subsequent invasive tumor(s). All tumors arose from the same progenitor cell. These results support the concept of a clonal origin of superficial and metachronous invasive bladder UCCs., (Copyright 2001 Wiley-Liss, Inc.)

Synchronous and metachronous tumours are frequently observed in the urinary tract and may be explained by the concept of field cancerisation, i.e. exposure to carcinogens leads to independent transformation of many urothelial cells resulting in genetically unrelated tumours. However, increasing evidence supports the concept of clonality, i.e. the progeny of a single transformed cell spreads through the urinary system resulting in genetically related tumours. The aim of this study was to review the molecular biological evidence for both concepts and to assess the consequences of a clonality assumption on the incidence of urothelial cell carcinoma (UCC). In total 1198 non-invasive and 1113 invasive (> or = T1) UCCs of the bladder were registered as incident tumours in 1996-1997 by three Dutch cancer registries following the current registration rules of the International Association of Cancer Registries (IACR). Assuming clonality, the number of non-invasive and invasive bladder UCCs decreased by 10.9% and 11.5% respectively. A decline of 8.5% and 9.5% was found for UCCs of the ureter and renal pelvis, respectively. Current registration rules have substantial impact on the incidence estimates of UCC. New insights into the molecular biology of UCC should be translated into registration rules.

Objectives: Quantitative cytology (Quanticyt) provides an objective reproducible alternative for routine cytology. To increase the sensitivity of cytology, we studied the application of consecutive cytology in patients in follow-up for superficial bladder cancer., Methods: Between 1991 and 1998, a set of five or more bladder wash samples was obtained from 614 patients. These patients were retrospectively studied for follow-up data. Each sample was scored according to the Quanticyt risk score., Results: In 614 patients (508 men, 106 women), 5832 bladder wash samples were taken. The mean interval between the first and fifth sample per patient was 21.5 months (SD 13.8 months). The mean number of tumor recurrences per patient was 2.17 (SD 1.82). The risk score of the first sample was not predictive of recurrence. Invasive disease was found in 0%, 0%, and 0.8% of patients with one low, intermediate, and high-risk sample, respectively. After five samples, the corresponding rate was 0%, 0%, and 10%. A comparison of visual cytology and quantitative cytology revealed that the false-negative rate was significantly different (17% versus 3.8%). The positive predictive value was highest for visual cytology (17% versus 9.2%)., Conclusions: Adding consecutive quantitative cytology to urine cytopathologic evaluation improves the detection rate of high-grade lesions. Combining quantitative cytology and visual cytology provided a more accurate prediction of tumor stage.

In this pilot study, we tested whether it is possible to apply neural network-based diagnostics on bladder washings to detect urothelial cell carcinoma of the bladder. Eighty-five bladder wash (BW) samples were chosen at random from our own database. Cystoscopy, histology, and follow-up data concerning tumor recurrence were available. Each slide was scanned by the neural network-based digitized cell image system. The neural network-based diagnosis (NNBD) was based on 128 digitized cell images provided by the system. The light microscopic diagnosis (LMD) was rendered by an experienced cytopathologist using the same terminology, i.e., negative, low-grade tumor, and high-grade tumor. Finally, an automatic QUANTICYT analysis was performed on the same material, with as classification low, intermediate, and high risk. The sensitivity for diagnosing a histologically confirmed tumor was for NNBD 92%, for LMD 50%, and for QUANTICYT 69%. For the three methods, receiver operating characteristic (ROC) curves were made for the thresholds low grade/intermediate risk and high grade/high risk. For the prediction of a positive cystoscopy, the highest area under the curve (AUC) was found for NNBD, being 0.71. The AUC for LMD was 0.58. QUANTICYT analysis had the highest AUC value (0.62) for predicting tumor recurrence after a negative cystoscopy, with a lower value for NNBD (0.50). These findings indicate that neural network-based diagnosis of bladder washing samples is highly promising., (Copyright 2000 Wiley-Liss, Inc.)

Telomerase reverse transcriptase (hTERT) messenger RNA has been detected in 95% of bladder tumors using RT-PCR. In this study, we quantified the expression of hTERT in 35 bladder urothelial cell carcinomas and in 6 normal bladder epithelia using a real-time quantitative PCR assay. hTERT expression was detected in all 35 urothelial cell carcinomas of varying grade and stage, but not in normal tissue samples. An increase in both pathological grade and clinical stage as prognostic parameters correlated with increased hTERT expression. Using different cutoff values for grades and stages, normalized hTERT expression values could discriminate among low, medium, and high grade tumors and between superficial and muscle-invasive tumors. We conclude that standardized real-time measurement of hTERT expression can be used for early tumor detection and may be used for determination of prognosis in urothelial cell carcinomas of the bladder., (Copyright 2000 Wiley-Liss, Inc.)

Objectives: Karyometric analysis (Quanticyt) has proved of value as a cytologic marker for bladder cancer. This study was conducted to identify diagnostic and prognostic factors in a high-risk Quanticyt population to predict the prognosis of transitional cell carcinoma (TCC) of the bladder., Methods: Quanticyt is a karyometric system for quantitative bladder wash cytologic findings based on two nuclear features: the 2c-deviation index (2cDI) and the mean of nuclear shape. Samples are scored as low, intermediate, or high risk. Before 1995, 109 patients with high-risk quantitative bladder wash cytologic findings were identified at our clinic. Four patients with previous invasive tumors were excluded., Results: Histologically proven malignancy was found in 54 of 105 patients at first high-risk quantitative bladder wash cytologic findings. Invasive TCC was found in 16 patients, and another 10 patients had progression during a median follow-up of 3.7 years. In univariate analysis, the presence of carcinoma in situ (CIS), highest tumor grade, 2cDI, and highest tumor stage were significant predictors of progression. The presence of CIS proved to be the only predictor of progression in the multivariate analysis. A 2cDI of 2.00 c(2) or higher was a significant predictor of CIS, invasive TCC, and progression. At follow-up analysis after negative cystoscopy, 2cDI showed a tendency toward predicting progression., Conclusions: These data confirm earlier findings that CIS is an important marker of progression. 2cDI as assessed by quantitative cytology is a practical tool to predict CIS, invasive TCC, and subsequent progression. A 2cDI of 2. 00 c(2) can be used to further stratify high-risk quantitative bladder wash cytologic findings.

The involvement of E-cadherin in the progression of carcinoma is supported by a large number of studies showing an inverse relationship between E-cadherin immunoreactivity and tumor aggressiveness. However, the mechanisms leading to decreased E-cadherin immunoreactivity are still unclear. Comparison of Northern blotting and immunohistochemistry in a series of 49 frozen bladder tumors revealed that, in 16 of 23 tumors with abnormal staining, clear mRNA down-regulation occurred. In the 7 cases without mRNA down-regulation, no structural anomalies of E-cadherin could be detected by Southern blotting, Western blotting or PCR-SSCP. Western blotting confirmed that, in 6 of these tumors, E-cadherin was down-regulated at the protein level. This down-regulation was accompanied by down-regulation of alpha-catenin and, to a lesser extent, of beta- or gamma-catenin. However, Northern-blot analysis indicated that expression of the 3 catenins is maintained at the mRNA level. Thus our data show that, in bladder tumors, mRNA down-regulation accounts for about two thirds (16/23) of tumors with abnormal staining and that post-transcriptional down-regulation of E-cadherin occurs in 6/23 of these tumors., (Copyright 1999 Wiley-Liss, Inc.)

We are investigating the hypothesis that cancer progression involves the formation of abnormal cadherin-catenin complexes. The detailed analysis of cadherins and catenins expressed in a panel of 17 human bladder-cancer cell lines revealed that E-cadherin was down-regulated at the mRNA level in 5 cell lines. Interestingly, plakoglobin was also down-regulated at the mRNA level in these 5 cell lines only. Furthermore, a slower migrating form of pp120 was detected in these cell lines and in 2 cell lines with heterogeneous E-cadherin expression. Cloning of the cadherins expressed in the bladder lines revealed that P-cadherin is expressed in the lines expressing E-cadherin and down-regulated at the mRNA level in lines devoid of E-cadherin. N-cadherin was expressed in the 5 lines with reduced E-cadherin expression, in the 2 lines with heterogeneous E-cadherin expression and in 2 other cell lines. Thus, we showed that catenin changes occur in correlation with lack of E-cadherin expression and that N-cadherin becomes predominantly expressed in cells that have lost E-cadherin expression. Our data suggest that co-regulation of the expression of genes encoding different members of the classical cadherins occurs during tumor progression and that expression of some catenins is also coordinated with cadherin expression.

Mutations in the tumor suppressor gene p53, analyzed in bladder washings, have positive predictive value for the progression of superficial bladder cancer to invasive disease. Bladder washings reflect the general status of the urothelium, and because sampling of bladder washings can be performed as an outpatient procedure, patients can be monitored more carefully. To determine the actual value of bladder washing specimens in assessing the p53 status of histologic specimens, we used the technique of polymerase chain reaction-single-strand conformation polymorphism to analyze bladder washings and the synchronous tumors of 15 patients for the presence of p53 mutations. A significant correlation (2-tailed Fisher's exact test) between the p53 status of bladder washings and histologic specimens was observed if the 2 were compared among the specimens of a single patient. Overall, in 2 patients the mutation present in the tumors was not detected in the bladder washings, and in 1 patient the mutation in the bladder washing was not detected in the histologic specimens. These conflicting results obtained with bladder washings and histologic specimens could be explained mainly by the architecture of the tumors. The observed specificity of 86% and sensitivity of 75% emphasizes that although the correlation between the 2 methods is good, in a number of cases they are complementary to each other. The analysis of p53 mutations in at least 2 bladder washings gives insight into the p53 status of the synchronous tumors.

Objectives: Application of immunocytology directed against antigens of urothelial tumor cells or tumor-associated breakdown products intends to improve the sensitivity of the diagnosis of superficial transitional cell carcinoma (TCC) of the urinary bladder. In this study, the mode of action, sensitivity and specificity of the Bard(R) BTA test, detecting a tumor-associated release of a basement membrane complex, was addressed and compared with voided urine cytology (VUC)., Results: Contrary to grade, a significant (p = 0. 003) relationship between tumor stage and BTA test sensitivity was observed, being 23.8, 33.3 to 100% for Ta (n = 42), T1 (n = 6) to >/=T2 (n = 5), respectively. These data suggest an association between an increase of the BTA test sensitivity with an increase of basement membrane degradation or interruption. With regard to this mechanism, the BTA test may be of special importance for monitoring tumor progression or increase in tumor invasiveness. Detection of low-stage, low-grade tumors by noninvasive techniques remains a challenge. The sensitivity of the BTA test for the presence of TCC was 32.3%, while that of VUC was 17.7%, but in this study the difference was not statistically significant. Furthermore, the BTA test was not more effective in identifying the various tumor grades, stages or stage/grade groupings. However, dividing the patients in two groups of low risk (TaG1/TaG2) and high risk (TaG3 to >/=T2) leads to a significant (p = 0.008) increased sensitivity of the BTA test (27.3%) in detecting patients with low-risk tumors compared to VUC (3.0%). The specificity of the BTA test in patients with a history of TCC was 81.6%, while that of VUC was 98.9%., Conclusion: The sensitivity of the BTA test is at least equivalent to VUC and may be suited to monitor increase in stage in patients suffering from bladder carcinoma, but cannot replace cystoscopy in patients suspected for a bladder tumor.

Objective: To study the value of two outpatient urine tests with regard to the diagnosis and recurrence of bladder tumors., Methods: Fifty patients with a history of superficial bladder cancer were evaluated with urinary NMP22 levels (cutoff level 10 U/ml), bladder wash karyometry (low versus intermediate and high risk) and cystoscopy. All patients were followed for 1 year., Results: Diagnostic negative and positive predictive values (NPV and PPV) of the tests were, respectively: NMP22 91.2 and 56.3%, and karyometry 80 and 33.3%. Prognostic NPV and PPV with regard to a subsequent recurrence were, respectively: NMP22 77.8 and 27.3%, and karyometry 82.6 and 50%., Conclusion: The diagnostic value of NMP22 is good. Since the 3 false-negative results were in low-stage and low-grade lesions, this test could be used as a prescreening for cystoscopy. The NPV of these tests with regard to tumor recurrence is around 80%, but only karyometry has a significant PPV. Change in the follow-up policy on the basis of these tests remains difficult. In patients with neobladders NMP22 appears to be of little use, because of the high urinary NMP22 levels in the absence of malignancy.

Objectives: Two new methods for the detection of transitional tumor cells in bladder wash (karyometry: QUANTICYT) and voided urine material (BARD BTA test) were compared with bladder wash cytology for the prediction of histology and tumor recurrence., Methods: Bladder wash material and voided urine were sampled from 138 patients. Bladder wash karyometric (BWK) image analysis and the BTA test were applied. A subsequent urethrocystoscopy was performed and a bladder tumor, when present, was resected. Moreover, each patient was followed for tumor recurrence and progression., Results: Sensitivities for the detection of tumors were 34.4%, 44.8%, and 69.0% for the BTA test, bladder wash cytology (BWC), and BWK, respectively (BTA versus BWC, P = 0.64; BTA versus BWK, P = 0.0002; BWC versus BWK, P = 0.0001, using the McNemar test). Specificities for the different tests were 81.3%, 92.5%, and 72.5%, respectively (BTA versus BWC, P = 0.096; BTA versus BWK, P = 0.031; BWC versus BWK, P = 0.001, using the McNemar test). Combinations of tests did not result in better prediction of the presence of tumor. Sensitivity of carcinoma in situ for the three tests was 0 of 3, 3 of 3, and 3 of 3, respectively. Follow-up analysis after a negative cystoscopy revealed comparable predictive values for BWC and BWK., Conclusions: The BTA test may be useful for patients with recurrent, low-grade papillary lesions. However, sensitivity for detection of these lesions, although higher than that for BWC, was only 42.9%. The highest specificity was found for BWC; however, this was accompanied by the lowest sensitivity of all three tests. The lower specificity of BWK was accompanied by a better prediction of tumor recurrence after a normal urethrocystoscopy. BWK is particularly sensitive for the recurrence of high-grade bladder lesions.

Bladder wash cytology provides superior results for the detection of bladder malignancies than does voided urine analysis. Image analysis systems have been developed for quantification of cytologic features. In this study, routine bladder wash cytology is compared with an automated image analysis system (QUANTICYT). We studied a random set of 100 bladder wash samples from a population of 1614 patients in follow-up after bladder cancer. Four experienced pathologists interpreted the same 100 Papanicolaou-stained slides. Cytologic and image analysis results were compared for prediction of a cystoscopic lesion, histologic abnormalities, and tumor recurrence. After application of receiver operating characteristic curves, prediction of a cystoscopic lesion by cytology and image analysis was comparable. Both the image analysis system and the cytologic examination detected all of the high-grade lesions. Image analysis was superior to cytologic analysis for the prediction of tumor recurrence after normal findings at cystoscopic examination.

To identify molecular markers with predictive value for the progression of superficial bladder cancer we used the differential hybridization analysis approach. Since primary tumor material is heterogeneously composed of subpopulations that are poorly characterized, we used in this study a rat progression model system that phenotypically and cytogenetically resembles human superficial bladder cancer. In the differential hybridization analysis we compared the mRNA populations of low and high metastatic tumor lines. We observed an overexpression of ferritin Heavy chain (ferritin H) in the tumor line with the lower metastatic capacity and better differentiated phenotype. The exact clinical relevance for the differential expression of ferritin H in human bladder cancer remains to be determined.

We have studied p16(INK4) mutation (by PCR-SSCP) and deletion (by Southern blotting and/or multiplex PCR) in a series of 47 bilharziasis-associated tumors from Egypt and compared the results with those obtained on a series of 17 established bladder cell lines and non-bilharziasis-associated bladder cancers from the Netherlands. In the cell lines we found 9 homozygous deletions and 1 mutation (59% of p16(INK4) alterations in cell lines), whereas in cases from the Netherlands deletions were found in 4 of 22 samples. No mutations were detected in the 46 samples screened. Interestingly, in bilharziasis-associated bladder cancer, deletions were present in 23 samples and mutations in a further 2 cases (53% of p16(INK4) alteration in bilharziasis-associated bladder cancer). No correlation was found between p16(INK4) alteration and histopathological data. Likewise, the same frequency of alteration was found in tumors with different differentiation patterns (squamous, transitional or adenocarcinoma). Three conclusions can be drawn from our findings: (i) p16(INK4) alterations are more frequent in cell lines than in primary tumors; (ii) in primary bladder tumors (bilharziasis-associated or not), p16(INK4) deletions are much more frequent than p16(INK4) mutations; (iii) p16(INK4) alterations are more frequent in bilharziasis-associated bladder tumors than in other bladder tumors. This high frequency of deletion is not related to a specific histological type but to the specific etiology of these tumors.

Loss of E-cadherin-mediated adhesion is an important step in the progression of many carcinomas. In model systems, it has been shown that cadherin function requires not only proper E-cadherin expression but also its linkage to the cytoskeleton through catenins. Hence, defects in catenins may cause defective E-cadherin function, and catenins as well as E-cadherin might constitute prognostic indicators. Here, we extend our previous study on E-cadherin in bladder cancer (Cancer Res., 53: 3241-3245, 1993). We have evaluated the expression of E-cadherin-associated cytoplasmic molecules (alpha-, beta-, and gamma-catenins and p120cas) to clarify whether or not the pattern of their expression could provide additional prognostic information beyond that from E-cadherin alone. Forty-eight frozen bladder tumor specimens and 9 samples of normal urothelium were studied by immunohistochemistry. A discrepancy between the E-cadherin and catenin expression pattern was seen in 20.8% of cases. Abnormal expression of each molecule is significantly correlated with tumor grade (P < 0.01) and stage (P < 0.01). Reduced expression of all of the molecules correlates with poor survival (P < 0.01 for each variable). Proportional hazard regression analysis showed that beta-catenin, E-cadherin, and alpha-catenin have strong predictive value, whereas plakoglobin and p120cas have a somewhat lower predictive value. Within patients with invasive tumors, those with a normal staining for either E-cadherin, alpha-catenin, or beta-catenin show a trend toward better survival. However, the difference in survival is significant only for E-cadherin (P < 0.05). Thus, beta-catenin, E-cadherin, and alpha-catenin have similar prognostic values. Therefore, from a practical point of view, the expression of any of these proteins can be of prognostic value for patients with bladder cancer.

Objectives: Quantitative cytology by image-analysis techniques enables objective interpretation of nuclear features in light microscopic images. QUANTICYT, a quantitative karyometric cytology system, was used in the follow-up of patients with superficial bladder cancer., Methods: From 1992 to 1995, 4137 samples from 1412 patients were obtained. At 1-year follow-up after the initial bladder washing, a tumor recurrence rate of 21% was found. In this period, tumor progression to invasive disease occurred in 1.6% of patients. Scoring of tumor by the QUANTICYT system was based on two nuclear features: the 2c deviation index and the mean of a nuclear shape feature: MPASS., Results: The method was found to be reproducible and superior to visual cytologic interpretation. QUANTICYT analysis of the bladder washings resulted in a score of low, intermediate, and high risk. In a multivariate analysis, highest grade of earlier tumor and QUANTICYT risk score were the best predictors of tumor recurrence and progression. For the easy application of QUANTICYT analysis in daily routine, a report form that included patient history and DNA histogram was developed., Conclusions: QUANTICYT karyometric analysis of bladder-wash material proved a useful, clinically applicable grading tool in the follow-up of patients with superficial bladder cancer, with sufficient power to be used in decision-making in the individual patient.

To assess the value of p53 mutations in predicting the progression of superficial bladder cancer [transitional cell carcinoma (TCC)] and to define exactly when p53 mutations occur in the process of tumor progression, 80 consecutive bladder washings from 26 high-risk (indicated by quantitative karyometric analysis) superficial TCC patients were examined by single-strand conformation polymorphism. Six of 13 patients who experienced clinical progression (progression to T2 or higher) were found to have a p53 mutation in one or more of their bladder washings. In the control group (no progression to invasive disease), only 1 of 13 patients had a p53 mutation. For these high-risk superficial TCC patients, the occurrence of a p53 mutation has a positive predictive value of 86% for the progression of disease. A negative predictive value of 63% was observed. Moreover, because p53 mutations were found in samples prior to progression (mean, 8 months), they could identify patients who need changes in their treatment strategies to prevent progression to invasive disease. Despite these promising results, it is obvious that to increase not only the positive predictive value but especially the negative predictive value of this procedure to predict progression, additional prognostic markers are still needed.

Interphase cytogenetics by in situ hybridization (ISH) using a panel of centromere-associated DNA probes for chromosomes 1, 7, 9, 10, 11, 16, 17, and 18 was performed on 5 microns thick frozen tissue sections of transitional cell carcinomas (TCCs) of the urinary bladder. By this approach, chromosome ploidy, numerical chromosome aberrations, imbalance between chromosomes, and heterogeneity of aberrations within individual tumours were determined. In 15 of 24 TCCs, loss or underrepresentation of chromosome 9, compared with the ISH copy numbers of at least five other chromosomes, was demonstrated. Independently, RFLP analysis were performed on the same cases to detect loss of heterozygosity (LOH) of chromosome loci 9q34, 11p15, 16q22-24, 17p13, and 18q21. LOH was found in 9 of 19 informative cases for chromosome locus 9q34. Comparison of the ISH and RFLP results showed no correlation between numerical aberration and LOH for the loci on chromosomes 11, 16, 17, and 18. However, numerical loss of chromosome 9 was found in 89 per cent (eight of nine cases) with LOH for 9q34. Conversely, LOH at 9q34 was observed in only 67 per cent (eight of 12 cases) with underrepresentation of chromosome 9. Moreover, in 60 per cent of the non-informative cases (three of five cases), underrepresentation for chromosome 9 was observed. These results indicate that the heterochromatin probe for chromosome 9 can be reliably used in TCC tissue sections for the detection of chromosomal loss. In aneuploid TCCs, this DNA probe can be used for the detection of chromosomal underrepresentation only in combination with other centromere-associated DNA probes.

11q13 amplifications have been found in several cancers, including bladder tumours. However, the biological significance of this genetic alteration is not yet fully understood. To get more insight into the role of 11q13 amplification in bladder tumour development, we have studied the level of amplification and expression of 4 (protoonco)genes lying within the amplicon; cyclin D1, FGF3, FGF4 and EMS1 DNA amplification was found in 5/46 tumours. There was no correlation between amplification and clinico-pathological data. No expression of FGF3 and FGF4 was detected whereas both cyclin D1 and EMS1 were expressed at higher level in tumours with amplifications. Thus cyclin D1 and EMS1, but not FGF3 and FGF4, are likely to play a pathogenic role in the 11q13 amplification in bladder cancer. However, amplification is not the unique way of activation of these genes. Indeed, in situ hybridisation and Northern blot analysis have shown that most bladder tumours have a fair to high expression of cyclin D1 and EMS1 in contrast to normal urothelium with a moderate expression. Interestingly, a trend towards higher expression occurs in superficial versus invasive tumours (8.8 +/- 2.0 versus 1.9 +/- 0.4; P approximately equal to 13% for cyclin D1 and 4.5 +/- 1.4 versus 2.0 +/- 0.4; P approximately equal to 8% for EMS1). Moreover, the 9 tumours with low expression are all highly malignant, leading to the hypothesis that the tumours developing through a cyclin D1/EMS1 independent pathway are more aggressive.

Background: Alterations of the tumor suppressor gene p53 are known to occur in bladder cancer. Although p53 overexpression is associated with mutation of the p53 gene, a substantial discrepancy between molecular genetic alteration in p53 and overexpression of the protein has been found., Experimental Design: Tumor specimens of 39 bladder cancer patients were immunohistochemically analyzed for p53 overexpression, and the results were compared with the presence of a mutation as assessed by single strand conformation polymorphism (SSCP) and direct sequencing. Both clinical and biologic aspects were studied., Results: A significant correlation between p53 overexpression and poor survival in the whole group studied was found (p < 0.01). No association between p53 overexpression and decreased survival was found for invasive tumors in contrast with other studies. Differences in treatment of the patients and different Ab and scoring systems used might explain these differences. In our study, the Kaplar-Meier curves showed the same result for p53 overexpression and p53 mutation when the whole group and the invasive tumors were studied. However, in the group of superficial tumors, which was unfortunately too small for statistical analysis, we found p53 overexpression in three tumors, and no p53 mutations were found. A good concordance between p53 mutation and p53 overexpression was found (p < 0.02). However, two out of eight tumors with an SSCP-proven p53 mutation showed no p53 immunoreactivity, probably as a result of loss of the nuclear localization signal. Twenty three percent (7/31) of the tumors showed p53 overexpression without any sign of a mutation., Conclusions: Our results indicate that, despite good concordance between p53 mutation and p53 overexpression, there is no direct casual relationship between mutation and protein accumulation. Apparently, other events than mutation can trigger p53 stability.

Purpose: Because we have found that random mucosal biopsies have no additional prognostic value to conventional histopathology, we studied biopsies of histologically normal bladder mucosa with several molecular markers believed to be associated with the development of transitional cell carcinoma., Materials and Methods: Six groups of patients with an increasing stage of bladder tumor were selected: (1) benign disease (for example, benign prostatic hyperplasia, n = 8); (2-4) low (n = 10), intermediate (n = 9) and high risk (n = 7) superficial tumors; (5) progressive superficial tumors resistant to optimal conservative therapy (n = 6); (6) invasive or disseminated tumors at presentation (n = 5). We studied the expression of cytokeratin (used as an epithelial marker), fibronectin, E-cadherin (HECD-1), I-CAM, human leukocyte antigen (HLA)-I, HLA-II and epidermal growth factor receptor (EGF-R) in cold-cup biopsies of normal mucosa., Results: Fibronectin, HECD-1, I-CAM and HLA-II expression showed no significant changes in the different groups. There was a significant increase in the expression of HLA-I (p = .003) and EGF-R (p = .0001) with a higher stage of the tumor., Conclusion: An increasing EGF-R expression in normal looking mucosa of patients with increasing stages of bladder tumors could be a prognostic factor or might indicate that this increase in expression is not tumor specific but is seen in the whole bladder.

We have induced tumors by feeding guinea pigs with a diet containing 25 or 30% dried bracken fern for 100 or 150 days. A high incidence of bladder tumors was obtained. All but one animal had preneoplastic or neoplastic lesions after 4 months; after one year, 24 or 25 exposed animals had carcinoma. Bladder tumors obtained were essentially pure transitional cell carcinomas, although 4 cases (7% of the exposed animals and 10% of the 39 transitional cell carcinoma observed) showed areas of focal squamous metaplasia. Immunohistological detection of cytokeratins 10, 13, and 18 confirmed the transitional nature of these tumors. Tumor development can be followed by ultrasonography and cytology. Bladder tumors arose through several steps. Dysplasia and preneoplastic hyperplasia were seen after 4 months and papillary carcinomas appeared after 6 months, whereas muscle-invasive carcinomas required 1 year. Thus this model reproduces the full spectrum of preneoplastic and neoplastic bladder lesions observed in humans. Interestingly, when tumors were induced in older guinea pigs, none of them progressed to a muscle-invasive stage. This phenomenon should provide the opportunity to study the molecular mechanisms associated with these two different growth patterns, a major issue in understanding human bladder tumor progression.

Basic research is currently investigating the molecular cascade associated with bladder cancer development. Many new findings are potential leads towards the improvement of the diagnosis and prognosis of this disease. Special care, however, should be taken in the design of protocols for clinical evaluation of the value of these markers. Some initial guidelines have been put forward in this report.

Comparative genomic hybridization (CGH) was applied for a comprehensive screening of chromosomal aberrations in 14 transitional cell carcinomas of the bladder of different grade and stage. The results were compared in a number of selected cases with those obtained by restriction fragment length polymorphism analyses and targeted fluorescence in situ hybridization. Distinct amplifications, found with CGH, were located on 3p22-24, 10p13-14, 12q13-15, 17q22-23, 18p11, and 22q11-13. These high copy number amplifications and the frequency of imbalances involving chromosome 5, occurring in 4 of 14 cases, have not yet been identified in transitional cell carcinomas. Apart from these new aberrations, imbalances were detected in 3 or more cases for chromosomes 9 and 11, as already described previously in the literature. In four tumors, the copy number of specific chromosomal regions was also analyzed by interphase cytogenetics. Although in most instances the CGH data were confirmed, in one tumor, distinct differences were observed, possibly a result of heterogeneity of the tumor cell population. Furthermore, the CGH data were compared with loss of heterozygosity as revealed by restriction fragment length polymorphism analysis in the same tumors. In 80% of informative cases, no loss was detected by restriction fragment length polymorphism or by CGH. Of the 15 cases of loss of heterozygosity, 7 showed a loss also with CGH, whereas in 8 cases no loss was observed. In summary, CGH is a fast method to obtain a comprehensive picture of chromosomal imbalances in transitional cell carcinomas, including a number of previously unknown genomic alterations such as high level amplifications.

Evidence is accumulating that the tumour-suppressor gene p53 is involved in the development of bladder cancer. Therefore we studied p53 mutations in 47 bladder cancers obtained from 45 patients using polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analysis. Eight out of 24 invasive tumours appeared to have a p53 mutation, while no p53 mutations were found in the superficial tumours. All the p53 mutations were found in grade 3 tumours. The tumours with altered p53 showed a higher frequency of allelic loss (FAL) than the tumours without a mutation (55.8% vs 21.1%, P < 0.05, chi 2 test). This increase in FAL suggests a correlation between p53 mutations and genetic instability. A significant correlation between mutated p53 and poor survival in the whole group studied was found (P < 0.001, log-rank test). However, within the group of muscle-invasive tumours the occurrence of p53 mutations had no additional prognostic value. Therefore, even though p53 mutations were found in aggressive tumours, the clinical usefulness of its detection seems limited. Nevertheless, these results imply that p53 is involved in the clinical behaviour of bladder cancer; its role in the progression of superficial cancer to invasive disease merits further attention.

In this paper the predictive value of molecular prognostic parameters for bladder cancer is discussed. DNA ploidy has additional prognostic value for grade 2 tumors, irrespective of stage, with aneuploid tumors having a poor prognosis. Overexpression of the epidermal growth factor receptor (EGFR) can be used as a prognostic factor for the group of superficial tumors. Both abnormal E-cadherin and retinoblastoma (RB) expression have additional prognostic value for invasive tumors. The exact predictive value for the superficial tumors needs further study. The results with respect to p53 are conflicting and its exact role especially in the progression of pT1g3 tumors has to be clarified. In view of the discordance concerning its prognostic value, c-erbB-2 overexpression also needs further study. It appears that at this moment only a few molecular markers seem to have potential prognostic value, but their precise clinical relevance has to be studied more extensively. In particular the value of progression markers in the superficial TCC needs more attention.

Decreased E-cadherin expression assessed by immunohistochemistry correlates with poor survival of bladder and prostate cancer patients. The clinical usefulness of this parameter should therefore be evaluated in a large-scale prospective study. E-cadherin is an epithelial cell-cell adhesion molecule and impaired function presumably leads to increased invasive capacity of the cells. It has been shown that defective function can result from several mechanisms: mutation of the gene, alteration of transcription, posttranslational modification or changes in the interaction of E-cadherin with cytoskeleton anchoring proteins--the catenins. A major mechanism leading to decreased E-cadherin expression in tumors lies in decreased transcription of the gene. Hence, a better understanding of the regulation of E-cadherin transcription might open avenues for therapy by restoring normal expression.

E-cadherin, an intercellular adhesion molecule, has been shown to behave like an invasion suppressor gene in vitro. This may explain the inverse relation between expression of E-cadherin and tumor grade that was found in certain cancers. We therefore examined E-cadherin expression in bladder cancer samples from patients with known clinical follow-up. Forty-nine snap-frozen specimens (24 superficial and 25 invasive tumors) and 4 samples of normal urothelium were retrospectively analyzed with anti-E-cadherin monoclonal antibodies. In normal urothelium E-cadherin is expressed homogeneously with a typical membranous staining at cell-cell borders. Decreased expression is found in 5 of 24 superficial tumors and in 19 of 25 invasive cancers. Completely negative tumors are infrequent (4 cases). Most of the time a heterogeneous staining, which may correspond to an unstable E-cadherin expression during tumor development, is seen. Decreased E-cadherin expression correlates with both increased grade and stage (chi 2 = 9.5, P < 0.01, and chi 2 = 14.9, P < 0.005, respectively). More importantly, abnormal E-cadherin expression correlates with shorter survival (log rank test: chi 2 = 16.5, P < 0.001). In keeping with its in vitro invasion suppressor function, decreased E-cadherin expression correlates with the clinical aggressiveness of bladder tumors. This is the first report of E-cadherin as a marker with prognostic value. This parameter must now be tested in a large prospective study to assess its precise clinical relevance.

A cohort of 300 ACI rats was kept under standard laboratory conditions. After 30 months or upon natural death, complete autopsy was performed. In the genitourinary tract four kidney and five bladder tumors were found. Two of these bladder tumors, RBT323 and RBT157, are serially transplantable. In the fifth transplant generation the RBT323 tumor becomes metastatic to the lungs in more than 90% of animals. The metastatic ability of the RBT157 tumor changes from low to intermediate (50% of the rats have lung metastases) in the fourth passage. Histologically, the initial passages of the RBT323 and 157 tumors are grade II transitional cell carcinoma (TCC). The histological pattern of the RBT157 tumor remains essentially unchanged, whereas the RBT323 tumor progresses to a grade III tumor in the third passage. Electron microscopical studies reveal oblong elliptical and round vesicles lined by an asymmetrical unit membrane in the tumor cells, which stresses the urothelial origin of the tumors. Immunohistochemically both tumors show expression of cytokeratin 5, 7, 8 and 18. The progression of the tumors to a metastatic phenotype, however, is not associated with a specific change in the morphological characteristics. Cytogenetic analysis shows that both tumors are peridiploid with few marker chromosomes. Interestingly, both of these independently arising tumors exhibit a loss of chromosome 5. Rat chromosome 5 is syntenic to the major portion of human chromosome 9 (p23-qter). Loss of chromosome 9 is a cytogenetic trait of human superficial TCC, hence the RBT model is also in cytogenetic respect similar to human TCC.(ABSTRACT TRUNCATED AT 250 WORDS)

We have studied the antitumor effects of Bacillus Calmette-Guerin (RIVM strain) in the syngeneic rat bladder tumor model RBT323. In an immunohistochemical infiltrate study we compared the antitumor effects of BCG with the immunopathological findings in order to get more insight into the possible effector mechanisms of BCG. The antitumor effects of BCG appeared not to be dose-dependent, in the dose range tested. In rechallenge experiments no difference in growth of control tumors was seen between rats pretreated- or not pretreated with BCG. There was, however, a significant increase in antitumor effect of BCG after pretreatment with BCG. Immunohistological examination of BCG treated tumors revealed infiltrates consisting of macrophages, B-cells and T-cells. These results imply that whereas no specific response against the RBT323 cells is generated by BCG treatment, possibly BCG induced antigens do serve as immunogens in this nonimmunogenic syngeneic rat bladder tumor model system.

For transitional cell carcinoma of the bladder, clinical data indicate that invasive, metastatic tumors can arise through at least two different progression pathways. The majority of invasive, metastatic bladder neoplasms clinically present de novo, i.e., the patients have no history of malignant bladder disease. This implies that the highly malignant tumor cells either arise de novo or have undergone a rapid progression. Alternatively, a considerable fraction of patients with superficial bladder cancer process to invasive disease after a history of relatively benign superficial TCC. The molecular and cell biological basis of tumor progression is only poorly understood. Clearly, a better understanding of this progress could have profound clinical implications, since patients with superficial TCC with a high risk for progression would have to be treated more aggressively. We discuss the problems that are associated with tumor biological studies on early steps in the progression of TCC, especially from a "model system point of view."

The antiproliferative activities of recombinant rat gamma-interferon and recombinant human tumor necrosis factor alpha were evaluated in a new rat bladder tumor model, RBT323. The RBT323 is a spontaneously arisen, serially transplantable, bladder tumor in ACI rats. Histologically and ultrastructurally this tumor is a grade 2-3 transitional tumor without squamous cells. The RBT323 appears to be non-immunogenic in tumor excision and rechallenge experiments. The tumor was transplanted subcutaneously and the drugs were administered peritumorally. Gamma-interferon and tumor necrosis factor monotherapy starting two days after tumor implantation showed dose-dependent growth inhibiting effects in the dose range tested. Combination treatment with gamma-interferon and the highest dose of tumor necrosis factor resulted in synergistic antiproliferative effects. After cessation of the combination treatment all tumors resumed growth, initially with a low doubling time, but finally with their original growth rate. Gamma-interferon treatment initiated at a tumor volume of 0.2 to 0.5 cm.3 had no antitumor effects, whereas treatment with 100 micrograms. tumor necrosis factor per rat inhibited tumor growth significantly. Different combinations of the cytokines had synergistic effects against established tumors. These studies demonstrate antitumor effects of gamma-interferon and tumor necrosis factor in a rat bladder tumor model system. Combination therapy has synergistic effects and prolongs the time to tumor recurrence.

Transitional cell carcinoma of the bladder has a high recurrence rate after local treatment. Progression to a higher stage occurs in 10-30% of the recurrent tumors, and early detection of potentially progressive tumors is important. In the current study morphometric, densitometric, and chromatin textural features of nuclei of superficial bladder tumors (pTa-T1) were studied to determine the value of karyometric features in the prediction of tumor progression. Seventy-two histological samples from 36 patients, consisting of both the primary and the first recurrent superficial tumor, were analyzed. Patients were divided into two groups: those with tumor progression, defined as an increase in tumor stage or occurrence of metastatic disease, and those without. Discriminant analysis on four karyometric features resulted in correct prediction of prognosis of 78% and 97% in the primary and recurrent tumors, respectively (P < 0.001). Tumor grade and stage did not offer additional information concerning prognosis. Karyometric analysis of recurrent superficial transitional cell tumors can be useful in selecting patients who need a more aggressive therapy. However, tumor characteristics of recurrent tumors varied and continuous evaluation of the karyometric features is necessary for early detection of an increase in the malignant potential of the tumor.

The mechanisms that control the biological behaviour of urothelial cancer are complex, and many regulative interactions are involved. So far, few aspects of these control mechanisms have been recognized and characterized. A precondition for better understanding is knowledge the interaction of this factors. Some markers (e.g., chromosomal aberrations, EGFR expression) are correlated with progression of tumour. Whether they are the cause or the result of the aggressive behaviour of growth remains unknown. Only a few markers, especially in flow cytometry, will have any benefit in clinical routine. Whether it is possible to find a marker with prognostic value remains uncertain.