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Urothelial cancer (UC) care is moving toward precision oncology. For tumor biology-driven treatment of metastatic UC (mUC), molecular subtypes play a crucial role. However, it is not known whether subtypes change during metastatic evolution. To address this, we analyzed a UC progression cohort (N = 154 patients) with 138 matched primary tumors (PRIM) and synchronous or metachronous distant metastasis (MET) by immunohistochemistry, and mRNA sequencing in a subgroup of 20 matched pairs. Protein-based tumor cell subtypes and histomorphology remained stable during metastatic progression (concordance: 94%, 95% confidence interval [CI] 88-97%). In comparison, transcriptome-based molecular consensus subtypes exhibited higher heterogeneity between PRIM and MET (concordance: 45%, 95% CI 23-69%), with switches particularly occurring between luminal and stroma-rich tumors. Of note, all tumors classified as stroma rich showed luminal tumor cell differentiation. By an in-depth analysis, we found a negative correlation of luminal gene and protein expression with increasing desmoplastic stroma content, suggesting that luminal tumor cell differentiation of "stroma-rich tumors" is superimposed by gene expression signals stemming from the stromal compartment. Immunohistochemistry allows tumor cell subtyping into luminal, basal, or neuroendocrine classes that remain stable during metastatic progression. These findings expand our biological understanding of UC MET and have implications for future subtype-stratified clinical trials in patients with mUC. PATIENT SUMMARY: Urothelial carcinomas (UCs) occur in different appearances, the so-called molecular subtypes. These molecular subtypes will gain importance for the therapy of metastatic UCs in the future. We could demonstrate that the subtype remains stable during metastasis, which is highly relevant for future studies., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: The value of programmed cell death ligand-1 (PD-L1) to predict durable responses to immune checkpoint inhibitors (ICIs) in metastatic urothelial carcinoma (mUC) is inconsistent. We hypothesize that the use of archived primary tumor material (PRIM) for PD-L1 testing in clinical trials not properly reflecting the metastatic disease status (MET) contributes to this clinical issue., Objective: To analyze the predictive and prognostic value of PD-L1, spatial immunephenotypes, and major histocompatibility complex class I (MHC-I) determined in patient-matched PRIM/MET., Design, Setting, and Participants: PD-L1, spatial immunephenotypes, and MHC-I were examined in 154 mUC patients with at least one available pretreatment MET (138 patient-matched PRIM/MET pairs)., Outcome Measurements and Statistical Analysis: PD-L1, spatial immunephenotype, and MHC-I status of (patient-matched PRIM and) pretreatment MET were correlated with chemotherapy and ICI response and outcomes., Results and Limitations: Discordance rates in patient-matched PRIM/MET were 25/30%, 36%, and 49% for PD-L1 (CPS10/IC5%), immunephenotypes, and MHC-I (loss vs preserved), respectively. Correlations with chemotherapy and ICI responses were observed for immunephenotypes and MHC-I status determined in MET (not for PD-L1 alone), but not in PRIM. In case of ICIs, patients with cytotoxic tumor immune microenvironment (TIME) showed durable responses with disease control rates of 90% and a hazard ratio for disease progression/death of 0.05 (95% confidence interval: 0.01-0.65) versus patients with immunedepleted MET (disease control rate 29%). MET MHC-I status added an incremental value to predict durable ICI responses. Limitations include the partly retrospective design and the lack of MET multisampling on individual patient level., Conclusions: The TIME is subject to substantial dynamics during metastatic evolution. MET immunephenotypes and MHC-I statuses show promising potential to predict chemotherapy and durable ICI responses, while the PRIM TIME does not. Thus, future clinical trials should rather rely on pretreatment MET biopsies reflecting the current immunological disease state than on PRIM., Patient Summary: Prediction of chemotherapy and responses to immune checkpoint inhibitors might be possible using representative pretreatment metastatic biopsies., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Introduction: Urothelial bladder cancer (UBC) with clinical suspicion of locally advanced growth or pelvic lymphogenic spread has a high risk of progression and death., Patients and Methods: Bladder cancer patients with locally advanced (cT3/4) tumor growth or suspected pelvic lymphogenic spread (cN+) were treated with preoperative cisplatin-containing chemotherapy and consolidative cystectomy with pelvic lymphadenectomy. We aimed to identify prognostic factors and describe the patients' oncological outcome., Results: A complete dataset including follow-up data was available for 96 patients. In a univariate analysis, we identified cN stage (cN+ vs cN-, HR 2.7, 95% CI 1.3-6.0), response to chemotherapy (HR 0.2, 95% CI 0.1-0.5), ypT stage (ypT0/is/1 vs ypT2-4, HR 3.1, 95% CI 1.4-6.8), ypN stage (ypN + vs ypN-, HR 7.9, 95% CI 3.7-17.0), resection status (HR 4.4, 95% CI HR 1.5-13.0) as significantly associated with cancer-specific survival. In a multivariate regression analysis, both cN and ypN statuses were validated as independent prognostic factors for cancer-specific survival (cN: HR 2.6, 95% CI 1.1-6.1; ypN: HR 5.5, 95% CI 2.0-15.1)., Discussion: Lymph node status was identified as a prognostic marker in a high-risk cohort of UBC patients treated with inductive chemotherapy and cystectomy. Establishing cN status as a prognosticator underlines the necessity to aggressively treat these patients despite reported impreciseness of imaging procedures in UCB. Patients with histologically positive lymph nodes following preoperative chemotherapy have a very poor prognosis, and thus, the need for adjuvant systemic treatment is emphasized., Conclusion: Both clinically and pathologically affected lymph nodes convey a poor prognosis in bladder cancer and necessitate aggressive treatment., (© 2022. The Author(s).)

Background: Radical cystectomy (Cx) is the standard of care for muscle-invasive bladder cancer (BCa). In locally advanced pT4 BCa the oncologic outcome is inexplicit but Cx may be necessary for palliation., Objectives: The aim of this retrospective study was to evaluate the outcomes of Cx performed in patients with locally advanced pT4 BCa and to identify patient subgroups with improved outcome., Methods: Between 2008 and 2017, we identified 76 of 905 patients who underwent Cx for pT4 BCa at a single tertiary referral center. The physical patients' status was estimated according to the American Society of Anesthesiologists (ASA) classification. For the classification of postoperative complication rates, the Clavien-Dindo grading was used. Time-to-event variables with log-rank statistics were calculated with the use of the Kaplan-Meier method., Results: Median age was 74 years (range 42-90). Preoperatively, the physical status was estimated poor in 40 (52%) patients (ASA-score of ≥3). Overall, 19 (25%) patients had pT4b BCa, 41 (54%) patients were lymph node positive (c/pN+) and 14 (18%) patients had distant metastases (c/pM+). Within 30 and 90 days after surgery, 21% and 30% of the patients, respectively, developed severe complications (Clavien-Dindo grade ≥3). Overall, 30- and 90-day mortality rates were 9% and 11%, respectively. Moreover, 86% and 75% of patients who died within 30 and 90 days after surgery, respectively, had an ASA-score ≥3. At a median postoperative follow-up of 8 months (range 0-85), 53 (70%) patients have died. During the follow-up period, 46% of the patients died due to progressive disease, 16% died of a noncancer-specific cause, and for 8% of the patients, the reason remains unknown. Median overall survival (OS) and cancer-specific survival were 13.0 and 16.0 months, respectively. In subgroup analyses ASA-score ≥3 and hemoglobin <11.7 g/dl was significantly associated with poor OS. No statistically significant differences were detected between subgroups., Conclusion: Cx performed in patients with locally advanced pT4 BCa is associated with an increased mortality rate within 90 days postoperatively. Our study revealed that the ASA-score is a relevant and easily available tool to rate the patient´s condition and estimate postoperative outcome., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The JAK-STAT signalling pathway regulates cellular processes like cell division, cell death and immune regulation. Dysregulation has been identified in solid tumours and STAT3 activation is a marker for poor outcome. The aim of this study was to explore potential therapeutic strategies by targeting this pathway in bladder cancer (BC). High STAT3 expression was detected in 51.3% from 149 patient specimens with invasive bladder cancer by immunohistochemistry. Protein expression of JAK, STAT and downstream targets were confirmed in 10 cell lines. Effects of the JAK inhibitors Ruxolitinib and BSK-805, and STAT3/5 inhibitors Stattic, Nifuroxazide and SH-4-54 were analysed by cell viability assays, immunoblotting, apoptosis and cell cycle progression. Treatment with STAT3/5 but not JAK1/2 inhibitors reduced survival, levels of phosphorylated STAT3 and Cyclin-D1 and increased apoptosis. Tumour xenografts, using the chicken chorioallantoic membrane (CAM) model responded to Stattic monotherapy. Combination of Stattic with Cisplatin, Docetaxel, Gemcitabine, Paclitaxel and CDK4/6 inhibitors showed additive effects. The combination of Stattic with the oncolytic adenovirus XVir-N-31 increased viral replication and cell lysis. Our results provide evidence that inhibitors against STAT3/5 are promising as novel mono- and combination therapy in bladder cancer.

This review summarises the treatment strategies of the last five decades for metastatic urothelial cancer. The introduction of combination chemotherapy in the mid-1980s led to clinically significant response rates and prolonged survival. Two years ago, the results of a phase-3 clinical trial with the PD1 inhibitor pembrolizumab for second-line treatment of metastatic urothelial carcinoma were published. These data were the first to show an overall survival benefit in comparison with a conventional chemotherapy with vinflunine, docetaxel or paclitaxel. Currently, PD1/PD-L1 inhibitors are also tested within randomized phase-3-trials for first-line treatment using different approaches either as a monotherapy or a combination with conventional chemotherapy or CTLA-4 inhibitors. Whereas data from single-arm phase-2 clinical trials have already been published, first phase-3 data are expected in 2019., Competing Interests: Die Autoren geben an, dass kein Interessenkonflikt besteht., (© Georg Thieme Verlag KG Stuttgart · New York.)

Positron-emission tomography/computed tomography combining both functional and morphological information has emerged as a powerful tool in oncological imaging within the past decades. The most commonly used radiotracer in oncology visualizing metabolic information is 2-[18F]fluoro-2-deoxy-d-glucose. However, the use of 2-[18F]fluoro-2-deoxy-d-glucose in urological oncology is challenging, as it is limited by physiological excretion through the urinary system. Therefore, it is only useful when applied to specific indications in selected patients with urological malignancy; for example, for detection of residual disease in the post-chemotherapy management of patients with seminoma. Despite initial promising results in bladder cancer, no relevant additional diagnostic value with positron-emission tomography using 2-[18F]fluoro-2-deoxy-d-glucose or choline-based tracers could be obtained, and should therefore be used with caution or only within clinical trials. In prostate cancer, however, a paradigm shift in imaging can be observed after development of new tracers that target the prostate-specific membrane antigen. Biochemical recurrent prostate cancer has become a clinically widely accepted indication for prostate-specific membrane antigen ligand positron-emission tomography/computed tomography, with several studies showing superior detection efficacy compared with conventional imaging. For primary high-risk prostate cancer, growing evidence suggests well-improved staging. The present review aimed to provide an overview of the current status of positron-emission tomography imaging in cancer of the urogenital system including the latest advances in 68 Ga-labeled and 18 F-labeled positron-emission tomography agents targeting the prostate-specific membrane antigen for positron-emission tomography imaging of prostate cancer., (© 2018 The Japanese Urological Association.)

Purpose: Patients with carcinoma in situ (CIS) of the bladder refractory to bacillus Calmette-Guérin (BCG) treatment are usually treated with cystectomy. Therefore, new treatment options with preservation of the urinary bladder are needed. The objective of the study was to investigate the feasibility, safety and efficacy of a novel targeted alpha-emitter immunotherapy for CIS after BCG treatment failure., Methods: A pilot study was conducted in 12 patients (age range 64-86 years, ten men, two women) with biopsy-proven CIS of the bladder refractory to BCG treatment. The patients were treated intravesically with a single instillation (one patient was treated twice) of the alpha-emitter 213 Bi coupled to an anti-EGFR antibody (366-821 MBq). The primary aims of the study were to determine the feasibility of treatment with the 213 Bi-immunoconjugate and evaluation of adverse effects. Therapeutic efficacy was monitored by histological mapping of the urinary bladder 8 weeks after treatment and at different time points thereafter., Results: The study proved that intravesical instillation of the 213 Bi-immunoconjugate targeting EGFR is feasible. No adverse effects were observed and all blood and urine parameters determined remained in their normal ranges. Therapeutic efficacy was considered satisfactory, in that three of the 12 patients showed no signs of CIS 44, 30 and 3 months after treatment., Conclusion: Intravesical instillation of 213 Bi-anti-EGFR monoclonal antibody was well tolerated and showed therapeutic efficacy. Repeated instillation and/or instillation of higher activities of the 213 Bi-immunoconjugate might lead to better therapeutic outcomes. A phase I clinical trial is planned.

Data published in November 2016 showed a significant survival benefit for the PD-1 antibody Pembrolizumab in the second-line treatment of metastatic urothelial cancer in comparison to standard chemotherapy. Other PD-1/PD-L1 antibodies are being tested in advanced clinical trials. This class of substances will become standard of care from the time of their approval which is expected for 2017 and will replace vinflunine, which is currently recommended in the German guideline. PD-1/PD-L1 directed substances are also being tested as a first line treatment of metastatic urothelial carcinoma. Monotherapy is evaluated as well as combination treatments with CTLA-4 inhibitors of conventional chemotherapy. First data are expected at the end of 2017.A new strategy is the application of PD-1/PD-L1 directed substances in the perioperative treatment of patients with a muscle-invasive urothelial carcinoma after complete surgical resection (cystectomy or nephroureterectomy). Two international phase III trials evaluate an adjuvant immunotherapy with nivolumab and atezolizumab. Recruitment will be completed in 2018., Competing Interests: Interessenkonflikt: Thomas Horn: MSD Mitarbeit, Berater- und Vortragstätigkeit Jansson, Astellas, Medac, BMSMargitta Retz: Astellas, AstraZeneca, Bayer, BMS, Janssen, MSD, Novartis, Pfizer, Roche, Sanofi-AventisRobert Tauber: Berater- und Vortragstätigkeit Astellas, Bayer, BMS, Ipsen, Janssenn, Novartis, Pierre-Fabre, Pfizer, Roche, Sanofi-Aventis, (© Georg Thieme Verlag KG Stuttgart · New York.)

Purpose: Computed tomography (CT) is current standard-of-care for preoperative staging in patients with invasive bladder cancer before radical cystectomy (RC). There are only sparse data on the association between preoperative CT findings and postoperative survival of patients., Methods: We retrospectively evaluated preoperative CTs of 206 patients with invasive bladder cancer undergoing RC in an academic tertiary referral center. CTs were analyzed retrospectively for relative bladder wall thickness (BWT) and size of lymph nodes (LN). Associations between CT findings and risk of death from any cause (AC) as well as risk of death from bladder cancer (BC) were assessed by Kaplan-Meier estimates, cumulative incidence curves and multivariable Cox regression analysis., Results: The median follow-up was 40 months. Increased BWT was significantly correlated with higher risk of death (AC: HR 1.68; p = 0.043; BC: HR 2.00; p = 0.027), as well as LN with a size of 6-10 mm (AC: HR 2.13; p = 0.002; BC: HR 2.77; p = 0.002) and >10 mm (AC: HR 2.47; p = 0.018; BC: HR 3.66; p = 0.007) when compared to LN ≤ 5 mm., Conclusion: Our data showed a significant correlation of bladder wall thickness and LN size with the risk of death. Also lymph nodes >5 mm but ≤ 10 mm (resp. ≤ 8 mm)-usually considered non-pathologic-were associated with a significantly worse prognosis. This information can be used to counsel patients preoperatively. It might also be useful for a risk-adapted approach in regard to neoadjuvant chemotherapy.

Objectives: To retrospectively evaluate the value of CT for lymph node (LN) staging in bladder cancer., Methods: Two uroradiologists reviewed CT scans of 231 patients who underwent radical cystectomy and pelvic lymphadenectomy according to a predefined 12-field template. A 5-step model was used to grade the radiological likelihood of a LN to represent malignant spread based on size, configuration and structure as well as regional clustering. Statistical analyses were performed both on patient- and field-based levels., Results: LN metastases were found in 59 of 231 patients (25.5%). On a patient-based level, sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy were 52.6, 93.6, 73.2, 85.6 and 83.4%, respectively. Using the field-based approach, a total of 1,649 anatomical fields were evaluable, of which 114 fields showed malignancy (6.9%). On a field basis, sensitivity, specificity, PPV, NPV and accuracy were 30.2, 98, 51.5, 94.5 and 93.3%, respectively. Concerning local staging (pT category), the overall accuracy was 78%; overstaging occurred in 6% and understaging in 16%., Conclusions: In line with prior studies, the sensitivity of CT imaging for the detection of LN metastases was low, while high values for specificity were achieved. This was further underlined by analyzing standardized anatomical fields. Concerning local staging, postoperative changes after TURB-T rarely led to overstaging., (© 2015 S. Karger AG, Basel.)

Introduction: Patients with lymph node-positive urothelial carcinoma of the bladder generally have a poor prognosis. Nevertheless, long-term survival in up to 30% of patients is reported. In the absence of established prognostic molecular markers, an assessment of the prognosis with clinical parameters is mandatory., Patients and Methods: All patients from one high-volume center with a curatively intended cystectomy for lymph node-positive urothelial carcinoma were evaluated. Patients' overall and cancer-specific survival were correlated with clinicopathological parameters. Pathological lymph node staging was performed with both the 2002 and 2010 TNM classification of the AJCC., Results: Lack of a perioperative chemotherapy (p < 0.001), higher numbers of positive nodes (p = 0.002), a higher lymph node density (p = 0.003), a higher pathological T stage (p = 0.006) and urinary diversion with an ileal conduit compared to an ileal neobladder (p = 0.023) were prognostic of a shorter overall survival while the number of removed lymph nodes showed no significant association with survival. Both with the 2002 and 2010 TNM classifications patients staged pN1 had a longer overall survival and time to cancer-specific death in comparison to patients with more extensive lymph node disease. According to the 2002 classification, there was a significant survival difference between patients with lymph node metastases in regional and distant lymph nodes., Discussion: Patients with a low lymph node density and an early pT stage present with the best prognosis among LN positive patients. The value of perioperative chemotherapy is emphasized. Which lymph node metastases are to be considered regional or distant remains a matter of debate.

Purpose: To determine the optimum dosage and instillation time for water-soluble polyvinylpyrrolidone (PVP)-hypericin for photodynamic diagnosis of bladder cancer and to monitor its use in regard to patient safety., Patients and Methods: Forty patients with a cystoscopically suspected bladder neoplasm were enrolled in this prospective phase IIA study. Different combinations of PVP-hypericin dosage (225 μg and 75 μg and instillation time (120, 60, 30, 15 min) were used to evaluate the optimal conditions. After a run-in cohort of five patients to validate the test method, each group comprised seven patients. All intravesical lesions were documented, and their fluorescence characteristics were recorded. Dose finding was the primary, safety the secondary end point., Results: Fluorescence intensities for the first two groups (225 μg PVP-hypericin for 120 and 60 min, respectively) were not different. For group three (225 μg for 30 min), both specific fluorescence and background noise were reduced. A shorter instillation time (225 μg for 15 min) or lower dose (75 μg for 30 min) was considered insufficient for lesion identification. A dose of 225 μg PVP-hypericin instilled for 30 minutes was determined as appropriate for the detection of lesions. Of the total 93 identified lesions, 62 were detected with both white light and fluorescence, 25 were seen with blue light only, and six with white light only. It was possible to identify additionally two carcinoma in situ, eight pTa, and one pT1 lesions with PVP-hypericin and blue light. PVP-hypericin was safe and well tolerated., Conclusion: The optimum combination of dosage of PVP-hypericin and its instillation time was established and will be used to determine sensitivity and specificity of PVP-hypericin cystoscopy in a larger multicenter phase IIB study. The preliminary data of this study hint to a higher sensitivity of hypericin-assisted fluorescence cystoscopy.

Background: In patients with bladder cancer (BCa) preoperative staging with (11)C-choline positron emission tomography-computed tomography (PET/CT) could be used to derive prognostic information and hence stratify patients preoperatively with respect to disease management., Methods: From June 2004 to May 2007, 44 patients with localized BCa were staged with (11)C-choline PET/CT before radical cystectomy. The results of imaging were correlated to overall survival (OS) and cumulative incidence of cancer-specific death (CSD)., Results: There was no statistically significant difference in OS and CSD between the patient groups when stratified for organ-confined versus non-organ-confined disease or lymph node involvement defined by either (11)C-choline PET/CT (OS: p = 0.262, hazard ratio [HR] = 1.60; p = 0.527, HR = 0.76; CSD: p = 0.144, HR = 2.25; p = 0.976, HR = 0.98) or CT (OS: p = 0.518, HR = 1.34; p = 0.228, HR = 1.67; CSD: p = 0.323, HR = 1.90; p = 0.136, HR = 2.38). The limitation of this study is the small number of included patients., Conclusion: In our prospective trial neither CT nor (11)C-choline PET/CT were able to sufficiently predict OS or CSD in BCa patients treated with radical cystectomy albeit trends and moderately increased HRs could be demonstrated without significant differences between CT or (11)C-choline PET/CT. However, these trends might prove statistically significant in bigger patient cohorts. Therefore initial transsectional imaging might be of clinical relevance in respect to prognosis and could play a role in the counseling of BCa patients., (2014 S. Karger AG, Basel.)

Background: Platinum-based chemotherapy is the treatment of choice for metastatic urothelial carcinoma, which is limited by primary and secondary resistance of the tumour. The Cu(2+) transporting beta polypeptide ATPase (ATP7B) is believed to play a role in this resistance. The aim of this study was to screen the ATP7B gene for mutations and loss of heterozygosity in bladder cancer and to evaluate their impact on chemotherapy resistance., Materials and Methods: DNA extracted from 17 patients with metastatic bladder cancer was analyzed by DNA sequencing, and microsatellite analysis., Results: We found 12 non-synonymous mutations and 20 synonymous mutations out of which 11 and 15, respectively, have not been previously described. Results were correlated with response to platinum-based chemotherapy: 65% of patients exhibited LOH of the ATP7B locus on chromosome 13q14.3, with a tendency to have a better response to chemotherapy., Conclusion: Although resistance is complex, LOH at the ATP7B locus might be useful in predicting chemotherapy response and needs further evaluation.

The bladder cancer genome harbors numerous oncogenic mutations and aberrantly methylated gene promoters. The aim of our study was to generate a profile of these alterations and investigate their use as biomarkers in urine sediments for noninvasive detection of bladder cancer. We systematically screened FGFR3, PIK3CA, TP53, HRAS, NRAS and KRAS for mutations and quantitatively assessed the methylation status of APC, ARF, DBC1, INK4A, RARB, RASSF1A, SFRP1, SFRP2, SFRP4, SFRP5 and WIF1 in a prospective series of tumor biopsies (N = 105) and urine samples (N = 113) from 118 bladder tumor patients. We also analyzed urine samples from 33 patients with noncancerous urinary lesions. A total of 95 oncogenic mutations and 189 hypermethylation events were detected in the 105 tumor biopsies. The total panel of markers provided a sensitivity of 93%, whereas mutation and methylation markers alone provided sensitivities of 72% and 70%, respectively. In urine samples, the sensitivity was 70% for all markers, 50% for mutation markers and 52% for methylation markers. FGFR3 mutations occurred more frequently in tumors with no methylation events than in tumors with one or more methylation events (78% vs. 33%; p < 0.0001). FGFR3 mutation in combination with three methylation markers (APC, RASSF1A and SFRP2) provided a sensitivity of 90% in tumors and 62% in urine with 100% specificity. These results suggest an inverse correlation between FGFR3 mutations and hypermethylation events, which may be used to improve noninvasive, DNA-based detection of bladder cancer., (Copyright © 2010 UICC.)

Ligation of two oligonucleotide probes hybridized adjacently to a DNA template has been widely used for detection of genome alterations. The multiplex ligation-dependent probe amplification (MLPA) technique allows simultaneous screening of multiple target sequences in a single reaction by using pairs of probes that carry tails for binding of common amplification primers. Resolution of the various targets is achieved by electrophoresis on the basis of predefined differences in amplicon length. In the conventional MLPA approach, one of the two target probes is generated by cloning in a single-stranded bacteriophage vector to introduce a sequence of defined length between the primer binding site and the specific target sequence. Here we demonstrate that differences in amplicon length can be achieved by using multiple short synthetic probes for each target sequence. When joined by a DNA ligase, these probes will form a single amplifiable template whose length is defined by the number and lengths of the individual probes. We have used this principle to establish a methylation-specific MLPA (MS-MLPA) assay that simultaneously determines the methylation status of five promoter CpG islands, and we have used this assay to analyze DNA from tumor tissue and corresponding urine samples from patients with bladder cancer. Our data show that the use of multiple short synthetic probes provides a simple means for custom-designed MS-MLPA analysis.

Transitional cell bladder tumors (TCT) is prone to recurrence (60-80%) after tumor resection. Up to 25% of these patients will progress, so it is important to find reliable predictive markers. We analyzed for loss of heterozygosity (LOH) with respect to 13 microsatellites located on 10 different chromosomal arms. This analysis was performed on the urine sediment and tumor tissue from 59 patients with bladder TCT and on the urine and normal-looking mucosa from 25 patients with a history of bladder TCT but no evidence of disease at the time of the study inclusion. The median follow-up period was 23.1 months (range, 2-48 months) for the 59 patients with bladder TCT and 25 months (range, 4-57 months) for the 25 patients without evidence of ongoing active disease. Correlation between LOH and eventual recurrence, progression, and mortality was investigated. In patients with noninvasive TCT, correlation between 11p tumor tissue LOH and recurrence was found. Similarly, 8p LOH in both urine sediment and tumor tissue correlated with progression. Finally, in the group of patients with a history of bladder TCT, normal tissue 8p and/or 11p LOH correlated with recurrence.

Background: To investigate whether the recently reported evidence of differences in the overall loss of heterozygosity (LOH) frequency between urine and tumour tissue in patients with transitional cell tumours (TCT) of the urinary bladder involved specific chromosomal sites, and their impact in diagnosis., Materials and Methods: Blood, tissue and urine specimens were obtained from 55 patients and 25 controls. Sixteen microsatellites were PCR-amplified and blindly analyzed for LOH through a laser-based capillary electrophoresis system., Results: Significant frequence differences between tumour tissue and urine sediment LOH were found in 9q and 11p in non-invasive disease and 14q in invasive disease. There was no significant difference for all the other chromosomal arms analyzed., Conclusion: The contribution in the urine sediment of cells belonging to tumours of the same histological classification differs according to the specific genetic alterations these cells carry. Furthermore, the location regarding these differences could indicate regions involved in tumour exfoliation or apoptosis.

Background: To determine the diagnostic value of plasma DNA microsatellite analysis in patients with transitional cell carcinoma (TCC) of the urinary bladder, by redefining plasma LOH from the equivalent analysis in controls. The method was further tested for MSI (microsatellite instability) and compared with tissue DNA analysis., Materials and Methods: Sixteen microsatellites were amplified in leukocyte, plasma and tissue DNA from 40 patients and 28 controls, and analysed in a laser-based, capillary electrophoresis system. Plasma LOH was determined from the controls' cut-off values., Results: The difference between plasma LOH frequency in patients (25% (10/40)) and controls (14% (4/28)) was not significant. Nevertheless, it occurred significantly more often in low rather than high-grade tumors (p=0.03) and controls (p=0.04). Plasma MSI was dependent upon the number of PCR cycles. Tissue LOH was present in 78% (31/40) of the patients and in none of the controls. Tissue MSI was uncommon., Conclusion: The results of plasma DNA microsatellite analysis in TCC need cautious interpretation.

The aim was to evaluate microsatellite analysis of urine sediment (MAUS) as an alternative method to urine cytology for routine diagnosis of patients with transitional cell tumors (TCT) of the urinary bladder. Urine cytology has the advantage of being non-invasive, fast and cheap, but is of limited value because of its low sensitivity. MAUS has previously been found to be a successful alternative method. However, the experimental set-up of such investigations implied exclusion of samples with unfavorable characteristics and use of a large number of markers. In the present study, MAUS was tested on all samples routinely available and a small panel of markers was selected. The urine sediments of 66 TCT patients and 24 controls were analyzed by MAUS with 16 fluorescent markers and by urine cytology. All samples were analyzed, including the ones of later micturition, with gross hematuria, leukocyturia or absence of visible sediment. In patients with tumors of low grade (grades I-II), MAUS was significantly more sensitive than urine cytology. The two methods were of equivalent diagnostic power in high-grade (grades III-IV), high-stage (pT1-pT4) tumors. A panel of the six most informative markers for MAUS was selected. Although MAUS has an advantage over routine cytology in low-grade, low-stage tumors, an overall sensitivity of 45% is not sufficient for routine clinical use.