Chromosomal alterations in exfoliated urothelial cells from bladder cancer cases and healthy men: a prospective screening study.

BACKGROUND: Chromosomal instability in exfoliated urothelial cells has been associated with the development of bladder cancer. Here, we analyzed the accumulation of copy number variations (CNVs) using fluorescence in situ hybridization in cancer cases and explored factors associated with the detection of CNVs in tumor-free men. METHODS: The prospective UroScreen study was designed to investigate the performance of UroVysion™ and other tumor tests for the early detection of bladder cancer in chemical workers from 2003-2010. We analyzed a database compiling CNVs of chromosomes 3, 7, and 17 and at 9p21 that were detected in 191,434 exfoliated urothelial cells from 1,595 men. We assessed the accumulation of CNVs in 1,400 cells isolated from serial samples that were collected from 18 cancer cases up to the time of diagnosis. A generalized estimating equation model was applied to evaluate the influence of age, smoking, and urine status on CNVs in cells from tumor-free men. RESULTS: Tetrasomy of chromosomes 3, 7 and 17, and DNA loss at 9p21 were the most frequently observed forms of CNV. In bladder cancer cases, we observed an accumulation of CNVs that started approximately three years before diagnosis. During the year prior to diagnosis, cells from men with high-grade bladder cancer accumulated more CNVs than those obtained from cases with low-grade cancer (CNV < 2: 7.5% vs. 1.1%, CNV > 2: 16-17% vs. 9-11%). About 1% of cells from tumor-free men showed polysomy of chromosomes 3, 7, or 17 or DNA loss at 9p21. Men aged ≥50 years had 1.3-fold more cells with CNVs than younger men; however, we observed no further age-related accumulation of CNVs in tumor-free men. Significantly more cells with CNVs were detected in samples with low creatinine concentrations. CONCLUSIONS: We found an accumulation of CNVs during the development of bladder cancer starting three years before diagnosis, with more altered cells identified in high-grade tumors. Also, a small fraction of cells with CNVs were exfoliated into urine of tumor-free men, mainly exhibiting tetraploidy or DNA loss at 9p21. Whether these cells are preferentially cleared from the urothelium or are artifacts needs further exploration.

Screening for bladder cancer with urinary tumor markers in chemical workers with exposure to aromatic amines.

PURPOSE: To validate urinary markers for the early detection of bladder cancer (BC) in chemical workers. METHODS: UroScreen was conducted as a validation study for tumor markers within the frame of a health surveillance program of the German Social Accident Insurance for active or retired workers with former exposure to aromatic amines. From 2003 to 2010, 1,609 men took part in voluntary annual screens. Cytology, the quantitative NMP22(®) assay, and UroVysion™ were applied to 7,091 urine samples. RESULTS: Fifteen out of 21 tumors were detected following test positivity. The UroVysion/NMP22 panel detected 14 out of 21 tumors versus 8 tumors with cytology alone (sensitivity 66.7 vs. 44.4 %, specificity 94.5 vs. 98.5 %). The sensitivity of the panel increased to 85.7 % in samples collected ≤12 months before diagnosis and when papillomas were excluded, compared to 58.3 % with cytology. About 3 % of NMP22 tests were false-positive. UroVysion results overlapped with cytology due to the preselection of atypical cells. NMP22 was less and UroVysion more frequently positive in diluted urine samples. Leukocytes confounded NMP22 but not UroVysion. The low incidence of BC in this study population yielded low positive predictive values of the markers and high costs per tumor detected with screening. CONCLUSIONS: UroVysion in combination with NMP22 detected more cases than cytology alone, at the expense of a lower specificity. High costs per detected case resulted from a lower BC incidence than in the past when levels of occupational exposure to aromatic amines were higher. Currently, it cannot be recommended to apply these markers for screening in asymptomatic workers. The increase in sensitivity is not balanced by the high costs of UroVysion and the false-positive tests of NMP22.

Chromosomal instability and bladder cancer: the UroVysion(TM) test in the UroScreen study.

UNLABELLED: What's known on the subject? and what does the study add?: UroVysion™ is a multicolour fluorescence in situ hybridisation assay that detects DNA gain at chromosomes 3, 7 and 17 and loss at the 9p21 locus in exfoliated urothelial cells. This cell-based test is time-consuming and costly compared with voided urine cytology or other molecular markers for the early detection of bladder cancer. We determined copy number changes at chromosomes 3, 7 and 17 and at the 9p21 locus with UroVysion in a prospective screening study among chemical workers. Strong correlations between DNA gains yield a similar performance in detecting bladder cancer with just one of the probes for chromosomes 3, 7 or 17 instead of all, supporting the development of a simpler and cheaper assay. OBJECTIVE: To explore changes at chromosomes 3, 7, 17 and 9p21 in order to assess associations with bladder cancer for possible improvements of the UroVysion™ assay regarding screening. SUBJECTS AND METHODS: In all, 1609 men took part in the prospective study UroScreen. Annual screening for bladder cancer was offered to male chemical workers with former exposure to aromatic amines as a voluntary surveillance programme between 2003 and 2010. In all, 191 434 cells in 6517 UroVysion tests were analysed for copy number variations (CNV) at chromosome 3, 7, 17 (gains) and 9p21 (deletions) in 1595 men. We assessed CNVs at single or multiple loci using polysomy indices (PIs, called multiple PI and PI 3, PI 7 and PI 17). We calculated Spearman's rank correlation coefficients (rs ) between these PIs and receiver operating characteristic (ROC) curves with areas under the curves (AUCs). We applied Cox regression to estimate hazard ratios (HRs) to assess the risk of developing bladder cancer. RESULTS: Nine out of 21 bladder tumours detected in 20 participants ('cases') had a positive UroVysion test, including seven high-grade carcinomas and seven overlapping results with a positive cytology. Four cases with negative test results did not attend screening annually. No case was found because of a complete loss of 9p21 in at least 12 cells. There were strong correlations between pairwise combinations of gains at chromosome 3, 7 or 17, ranging between rs = 0.98 and rs = 0.99 in cases and between rs = 0.84 and rs = 0.88 in non-cases (P < 0.001). Associations were less pronounced with CNVs at 9p21 among cases and were lacking in non-cases. Estimates of the relative risk of DNA gain for developing a bladder tumour assessed with PIs (threshold 10% of cells) were 47.7 (95% confidence interval [CI] 18.3-124.1) for the multiple PI, 44.5 (95%CI 16.5-119.9) for PI 3, 34.7 (95%CI 13.1-92.1) for PI 7 and 52.4 (95%CI 20.7-132.6) for PI 17, as well as 7.9 (95%CI 3.0-20.6) for a complete loss of 9p21 (threshold 2.5% of cells), respectively. ROC analyses showed similar AUCs for multiple PI compared with PIs of single chromosomes 3, 7 and 17 (all AUCs between 0.79 and 0.80) and a lower AUC for a homozygous loss of 9p21 (AUC 0.72). CONCLUSIONS: The UroVysion assay showed a reasonable performance in detecting bladder cancer in the present study population and shared positive test results with cytology, which is much cheaper. A simpler, faster and cheaper version of the UroVysion assay might rely on the very strong correlations between gains at chromosomes 3, 7 and 17, resulting in a similar performance in detecting bladder cancer with single-probe PIs compared with the full set of these probes. Loss of 9p21 was less predictive for developing bladder cancer in UroScreen.

Performance of survivin mRNA as a biomarker for bladder cancer in the prospective study UroScreen.

BACKGROUND: Urinary biomarkers have the potential to improve the early detection of bladder cancer. Most of the various known markers, however, have only been evaluated in studies with cross-sectional design. For proper validation a longitudinal design would be preferable. We used the prospective study UroScreen to evaluate survivin, a potential biomarker that has multiple functions in carcinogenesis. METHODS/RESULTS: Survivin was analyzed in 5,716 urine samples from 1,540 chemical workers previously exposed to aromatic amines. The workers participated in a surveillance program with yearly examinations between 2003 and 2010. RNA was extracted from urinary cells and survivin was determined by Real-Time PCR. During the study, 19 bladder tumors were detected. Multivariate generalized estimation equation (GEE) models showed that ß-actin, representing RNA yield and quality, had the strongest influence on survivin positivity. Inflammation, hematuria and smoking did not confound the results. Survivin had a sensitivity of 21.1% for all and 36.4% for high-grade tumors. Specificity was 97.5%, the positive predictive value (PPV) 9.5%, and the negative predictive value (NPV) 99.0%. CONCLUSIONS: In this prospective and so far largest study on survivin, the marker showed a good NPV and specificity but a low PPV and sensitivity. This was partly due to the low number of cases, which limits the validity of the results. Compliance, urine quality, problems with the assay, and mRNA stability influenced the performance of survivin. However, most issues could be addressed with a more reliable assay in the future. One important finding is that survivin was not influenced by confounders like inflammation and exhibited a relatively low number of false-positives. Therefore, despite the low sensitivity, survivin may still be considered as a component of a multimarker panel.