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.

What ' s known on the subject? and What does the study add?The prognosis of bladder cancer signifi cantly depends on tumour stage and time of diagnosis so early diagnosis is desirable to decrease mortality and treatment costs. The NMP22 test is approved for clinical application by the Food and Drug Administration (FDA) of the US. Previous studies have reported values of 47 -100% for sensitivity and 58 -91% for specifi city with this test, but there is no new data on the predictive value of NMP22 for screening bladder cancer (BC). The most important risk factor for BC is the tobacco consumption but occupational exposure to carcinogenic substances, especially aromatic amines, is regarded as another risk factor.The UroScreen study is a prospective longitudinal study for the early detection of BC. To our knowledge, it is the largest prospective validation study conducted over the longest period of time. The study results led us to conclude that, based on the currently available data, NMP22 should not be regarded as an alternative to endoscopy, and we could not make a general recommendation for screening or follow-up. The UroScreen results indicate that urine-based molecular markers could be a suitable addition to urine cytology and the detection of microhaematuria. OBJECTIVE• To evaluate the value of nuclear matrix protein-22 (NMP22) in bladder cancer (BC) screening, and its effect on variables in a prospective study in a high-risk population. PATIENTS AND METHODS• A total of 1772 chemical workers (mean age 62 years) exposed to carcinogenic aromatic amines were enrolled in the study.• In all, 7091 screening check-ups in 1609 subjects were performed.• Urine samples were collected for a quantitative NMP22 immunoassay, urine analysis and creatinine concentration assessment.• Cystoscopy and subsequent transurethral resection were performed where there were suspicious fi ndings. RESULTS• Histopathological analysis found three papillary urothelial neoplasms of low malignant potential, fi ve recurrent BCs and 13 primary BCs. Three tumours were at a muscle-invasive stage (pT2, pT3a or pT3b).• We found higher NMP22 concentrations ( > 10 U/mL) in 224 patients, which correctly predicted BC in six cases (sensitivity 97.29%, specifi city 28.57%; negative predictive value 99.04%, positive predictive value 12.24%).• Gross haematuria affected NMP22 results (odd ratio [ OR ] 3.49, 95% confi dence interval [ CI ] 1.81 -6.73). Infection also affected NMP22 results (OR 4.13,).• NMP22 was more frequently positive in urine with creatinine concentration > 2.5 g/L (OR 1.61, 95% CI 0.91 -2.86). CONCLUSIONS• NMP22 outcomes are affected by haematuria, infection and concentrated urine.• NMP22 alone cannot be recommended for primary screening in a high-risk population nor as an alternative to cystoscopy during follow-up.• A NMP22 test might be a useful adjunct to urine cytology. KEYWORDSbladder cancer , early detection , renal function , NMP22 , haematuria Study Type -Diagnostic (non-consecutive cohort without consistently applied reference st...

RESULTS• As of July 2010, 15 bladder tumours were detected in 14 participants.• GH was found in four out of nine highgrade tumours and associated with a rate ratio of 3.82, 95% confidence interval (CI) 0.50-29.15 for the development of bladder lesions.• The PPV of GH was 11.4%, but only 1.2% for μ H. μ H occurred in 18.8% of urine samples and was not associated with bladder cancer [rate ratio (RR) 0.72, 95% CI 0.11-4.78].• Abundant urinary leukocytes were associated with μ H [odds ratio (OR) CONCLUSION• While the PPV of μ H for bladder cancer was low, there was a strong influence of haematuria and leukocytes on the proteinbased tumour test NMP22®.• Erythrocytes and leukocytes should be determined at least semi-quantitatively for the interpretation of positive NMP22 test results.• In addition, a panel of tumour tests that includes methods not affected by the presence of erythrocytes or leukocytes such as cytology and UroVysion TM would improve bladder cancer screening.

BackgroundUrinary 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/ResultsSurvivin 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 β-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%.ConclusionsIn 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.

• 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. Results • 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.

BackgroundChromosomal 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.MethodsThe 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.ResultsTetrasomy 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.ConclusionsWe 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.

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