Tumor Markers in the Detection of Recurrent Transitional Cell Carcinoma of the Bladder DO NOT DUPLICATE

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1 Tumor Markers in the Detection of Recurrent Transitional Cell Carcinoma of the Bladder A Brief Review Aiman Zaher, M.D., and Todd Sheridan, B.S. It is unlikely that any of these tests, despite the cost benefits and ease of performance, will replace cytology or cystoscopy soon. OBJECTIVE: To determine the status of the most promising tumor markers of bladder cancer, including comparison with cytology, technical complexity and utility in patient management. STUDY DESIGN: An extensive literature search was performed, and multiple markers were evaluated. The markers with the greatest potential for use as an adjunct to cytology were reviewed to determine the value of clinical implementation. Markers with a paucity of clinical research and poor results in clinical trials were omitted from review, as were genetic and cytologic prognostic determinants. RESULTS: NMP22, bladder tumor antigen, fibrin/ fibrinogen degradation products, telomerase and QUANTICYT image analysis cytometry produced the most favorable and reproducible results. Each test obtained favorable sensitivities in comparison with cytology, especially in the detection of low grade lesions. Many also retrospectively placed patients in high- and low-risk groups based on the test results, allowing increased follow-up time between cystoscopies. However, inability to detect some high grade lesions reduces their utility. CONCLUSION: Continued clinical trials using these and other predictors of bladder cancer will eventually find a test that is suitable, in sensitivity and specificity, for use in urology clinics. Until that time, these tests may be useful in conjunction with cytology to prolong the interval between cystoscopies. (Acta Cytol 2001;45: ) Keywords: tumor markers, biochemical; carcinoma, transitional cell; bladder neoplasms. Bladder cancer is the fourth most common cancer in the United States, with an estimated 53,200 new cases in It affects men three times more than women 1 and is most common in the Caucasian population. Most patients evaluated for transitional cell carcinoma (TCC) of the bladder present with gross From the Department of Pathology, Medical College of Ohio, Toledo, Ohio, U.S.A. Dr. Zaher is Associate Professor and Director of Cytopathology. Mr. Sheridan is Medical Student and Student Fellow in Pathology. The abstract was presented at the 14th International Congress of Cytology, Amsterdam, the Netherlands, May 27 31, Address reprint requests to: Aiman Zaher, M.D., Department of Pathology, Medical College of Ohio, 3000 Arlington Avenue, Toledo, Ohio Financial Disclosure: The authors have no connection to any companies or products mentioned in this article. Received for publication June 13, Accepted for publication October 19, /01/ /$19.00/0 The International Academy of Cytology 575

2 576 Zaher and Sheridan hematuria, though some present with dysuria, nocturia, or increased urinary frequency or urgency. 2 In others, microhematuria is detected during a routine physical examination. Subsequent evaluation of these patients involves cystoscopy, regarded as the gold standard, with or without urinary cytology, obtained via voided urine or bladder wash Implementation of one of these simple assays might prolong the interval of cystoscopy in patients monitored for bladder TCC recurrence. specimens. Cystoscopy is invasive, causing significant discomfort and cost to the patient, and the specificity in patients with microhematuria is < 10%. 3 Flexible cystoscopes are less uncomfortable but remain invasive. Cytology, while quite sensitive in detecting high grade lesions and carcinoma in situ (CIS), varies in the detection of low grade lesions. The effectiveness of cytology depends greatly on the training of the cytologist, and the technique suffers from low interobserver and intraobserver reproducibility. 4 Moreover, its dependence on proper sampling and interpretation in the presence of degenerative changes and benign processes (stones, infection, radiation, chemotherapy, catheterization effects), which can mimic TCC, 5 reduces the utility of cytology. At the time of diagnosis, 80% of patients present with superficial tumors, grade 1 or 2, while 20% present with grade 3 tumors. 6 However, 50 70% of tumors recur within five years of diagnosis, 90% recur within 15 years, and 10 20% of patients eventually develop invasive disease. 7 Cytology is the gold standard for noninvasive detection of bladder cancer. The sensitivity of cytology, highly dependent on the study and institution, approaches merely 50% and falls to 30% for tumors of low grade and stage. 3 High grade lesions differ from low grade tumors in biologic behavior, and the aggressive nature of high grade tumors requires prompt diagnosis. Low grade, papillary tumors are usually superficial or minimally invasive at the time of discovery, with a favorable prognosis. 8 Flat lesions, more difficult to detect by cystoscopy, more frequently represent high grade lesions or CIS, both of which carry a greater chance of invasiveness and a poorer prognosis. 8 Superficial transitional cell neoplasms, despite minimal initial risk, require early detection and frequent follow-up testing. The recurrence rate of these lesions is > 60%, and progression to a higher grade or stage is found in 42% of cases within 10 years. 9 In general, the higher the grade of the initial lesion, the greater the chance of recurrence and progression. 9 The standard of care for follow-up of patients with a history of TCC is cytology and cystoscopy at three-month intervals for two years and six-month intervals up to five years, and yearly cystoscopy until 10 years after resection. A noninvasive, sensitive and specific means of detecting de novo and recurrent TCC is therefore necessary to reduce the number of cystoscopies and supplement the limited utility of cytology. Recently a number of tests became available for use in the detection of TCC. Most exploit the differences in tumor cells in expression of surface antigens, ploidy and enzymes. Most tests are not yet commercially available, and many antibodies have not been fully antigenically characterized, but a significant amount of literature exists regarding the potential use of these markers in distinguishing bladder cancer from coexistent neoplastic disease, inflammatory disorders and normal bladder urothelium. A multitude of monoclonal antibodies have been developed, including 486p 3/12, T138, T43, 9A7, AN43, 14III, AUA1, HMFG1, BL2-10D1, Due ABC3, OM5, GF26.7.3, BO 3.2, LBS8, LBS17, LBS19, LBS2, M344, LDQ10, 19A211 and P12. Most of these antibodies detect surface glycoproteins, mucins, complement factors, growth factors, tumor-related proteins and other differentially expressed antigens as well as products of enzymatic digestion, such as cytokeratin fragments. Some of these antibodies are named for the antigen they bind with, while with other antibodies the antigen has not been determined. Notable antigens proposed as tumor markers, for which the reverse transcriptase/polymerase chain reaction or enzymelinked immunosorbent assay is the usual method of detection, include CD44, cytokeratin 20, TATI, TPA, CYFRA21-1, E cadherin, hyaluronic acid, MAUB, CEA, AFP, β-hcg, PSA, SCC and CA19.9. The vast majority of the antibodies are not available commercially, and virtually all of them and antigens lack sufficient evidence for utility. Therefore, this review focuses on five methods: telomerase, NMP22 (nuclear membrane protein), fibrinogen/fibrin degradation products, bladder

3 Volume 45, Number 4/July August Tumor Markers in TCC tumor antigen (BTA) and image analysis cytometry. These tests were selected for their relative abundance in the literature, their availability, their potential for office use and their contrasting features involving expense, labor and turnaround time. However, a number of other antibodies and antigens may become equally or more useful in the detection of bladder cancer after further characterization. In fact, many of these markers have already produced very promising results, though more extensive studies must be performed. Characteristics of an ideal tumor marker for bladder cancer (or any cancer) include a rapid, officebased, inexpensive test that is noninvasive and possesses high sensitivity and specificity. The test should add to or, in certain cases, replace cystoscopy, cytology and urinalysis. It should be useful to screen at-risk populations, test symptomatic patients and monitor for recurrence in those with prior cancer. To date, these goals have not been achieved for bladder tumors. The particular studies selected for this review were chosen for their large patient population, potential insights into the use of the tests for detection of recurrence or for screening symptomatic patients, method of sampling and comparison of multiple tests using the same population. A large literature search revealed striking differences in study methodology, sensitivities and specificities, and perspective on the results. These variations suggest that great caution should be used in evaluating these markers for clinical use. Table I Table I Comparison of Reported Sensitivity and Specificity of Telomerase, NMP22, Fibrinogen/FDP, BTA, IAC and Cytology Fibrinogen/ Telomerase NMP22 FDP BTA IAC Cytology Authors Sens Spec Sens Spec Sens Spec Sens Spec Sens Spec Sens Spec Stampfer et al 10 NS NS NS NS NS NS NS NS Zippe et al 12 NS NS NS NS NS NS NS NS Schmetter et al 13 NS NS NS NS NS NS NS NS Nasuti et al 15 NS NS NS NS NS NS NS NS van der Poel et al 4 NS NS NS NS NS NS NS NS Kavaler et al NS NS NS NS NS NS NS NS 51 NR Pode et al 17 NS NS NS NS NS NS NS NS van der Poel et al 19 NS NS NS NS NS NS Wiener et al 20 NS NS NS NS Sharma et al 3 NS NS NS NS NS NS Ramakumar et al NS NS Johnston et al 22 NS NS NS NS NS NS Sensitivity (Sens) and specificity (Spec) in percentages. NMP = nuclear matrix protein, IAC = image analysis cytometry, NS = not studied, NR = not reported. summarizes the sensitivities and specificities of these tests in comparison with those of cytology. The NMP22 test kit, an enzyme immunoassay for the detection of nuclear matrix protein/nuclear mitotic apparatus protein in voided urine, has recently been approved by the United States Food and Drug Administration (FDA) for use in the detection of recurrent disease after resection of bladder tumors. 10 It detects fragmented and complexed forms of this protein in urine samples. Nuclear matrix proteins are an integral part of the structural scaffold of the nucleus, and they are involved in replication and transcription of DNA as well as gene regulation and RNA processing. 11 As the name suggests, the nuclear mitotic apparatus protein (NMAP) associates with the mitotic spindle apparatus during mitosis, allowing normal distribution of chromatids to daughter cells. 11 Studies show the NMAP concentration is 25-fold greater in bladder cancer lines than in normal urothelial cells and that a 10-fold difference exists between cancer-derived and normal cells from other tissues. 11 Stampfer and associates 10 measured the NMP22 level in urine samples from patients with a history of TCC, with favorable results over cytology, but the lower specificity and positive predictive values detract from the results. Due to the cost and high sensitivity, however, the authors suggested the use of these tests to extend the interval between cystoscopies, which would greatly reduce patient cost. Lesions of higher grade and stage, including CIS, were detected more often than were lesions of lower grade and

4 578 Zaher and Sheridan stage. The authors also noted slightly elevated values in inflammatory conditions. A study by Zippe et al 12 chose a population of symptomatic patients (predominantly hematuria) to screen urine samples for NMP. Their results were quite favorable, and they achieved a negative predictive value of 100%, which is equal to or greater than that of cytology. Furthermore, they showed that 267 unnecessary cystoscopies could have been avoided by using this test, resulting in a savings of $28, ,072. Both studies agreed that a value of 6.4 U/mL should be the cutoff value. Fibrin/fibrinogen degradation products (FDP) are protein fragments formed by action of the fibrinolytic system on fibrin and fibrinogen. The increased vascularity of tumors, induced by tumorproduced angiogenic factors, creates neovessels and increases the permeability of microvasculature. 13 Plasma proteins, including clotting factors, leak out and form a fibrin clot. This is subsequently broken down by the fibrinolytic system, causing the release of FDP, detectable in the urine in increased quantities. AuraTek FDP (Intracel Corporation, Rockville, Maryland, U.S.A.) is an immunoassay for these products in the urine, designed for office use and approved by the FDA. Schmetter et al 13 compared AuraTek FDP results with hemoglobin dipstick and urinary cytology in patients undergoing cystoscopic examination for bladder cancer as well as patients with a wide spectrum of other urologic diseases and healthy individuals. The sensitivity of FDP was significantly better than the that of cytology and hemoglobin dipstick. Furthermore, this study looked at the sensitivity of the test in detecting both grade and stage independently. All T2, T3 and T4 lesions were detected, but more superficial lesions, including CIS, showed lower sensitivities (62 67%). Lesions of higher grade were not consistently detected, most probably because of the presence or absence of invasion. The authors noted lower specificities, however, and the positive and negative predictive values were not significantly greater. Given these results, the authors suggested the use of AuraTek FDP as a diagnostic adjunct, particularly regarding more careful cystoscopic examination in patients with a positive FDP. BTA has been characterized as complement factor H, a negative regulator of the alternative complement pathway, 14 or a related protein (human complement factor H related protein). 15,16 It binds C3b and also serves as a cofactor for complement factor I, allowing degradation of C3b. Antibodies to factor H were used initially to create the Bard BTA test (Bion Diagnostic Sciences Inc., Redmond, Washington, U.S.A.), involving latex agglutination. 17 This test has since been replaced by the BTA stat test (Bion), an enzyme immunoassay using lateral flow chromatography, and the BTA TRAK test (Bion), a quantitative enzyme immunoassay. 14 Both detect the antigen in voided urine and have been approved by the FDA for detection of recurrence of bladder cancer. Multiple studies on each test are available. However, Pode and associates 17 studied the use of the test in detecting recurrent bladder cancer and with de novo symptomatic patients. They achieved favorable results in both populations, especially in regard to high grade lesions. Progressively higher sensitivities were noted with increasing size, grade and stage of the lesion, including all lesions of high grade and stage, CIS, and invasive disease. They suggested use of the test in detecting primary bladder cancer in symptomatic patients as well as in patients with previous bladder cancer and no history of intravesical bacillus Calmette- Guerin treatment due to false positive results created by inflammation. A study by Nasuti et al compared the results of voided urine cytology to the BTA stat test for the detection of primary TCC of the bladder. 15 Using samples from 100 patients with no history of urinary tract malignancies or symptoms, both methods obtained 100% sensitivity. However, the BTA stat test produced 16 false positive results, with a specificity of 84% and a positive predictive value of only 16%. All false positive cases involved gross hematuria. Despite the small patient population, the study elegantly demonstrated a major drawback of the BTA stat test. The authors provided supplementary information in the form of a side experiment, showing that the confounding factor is the positive BTA reaction when exposed to sanguineous fluid regardless of the presence of malignant cells. The study also showed the value of a skilled cytologist, as the values were markedly greater than in some reports. The authors concluded that the BTA stat test, despite the low cost, could be used only as an adjunct to cytology. In terms of cost reduction, the study results support initial screening with the BTA stat test, sending positive results to cytology for a more thorough evaluation. Raitanen and Tammela also looked at the utility of the BTA stat test in the healthy population, aiming to determine the actual test-dependent false positive rate using additional studies. 16 Of 100 sub-

5 Volume 45, Number 4/July August Tumor Markers in TCC jects with no history of urinary tract cancer or current urinary tract disease, only two cases had false positive BTA stat test results, confirmed by an extensive workup. The same two cases tested with the BTA stat test three months later had negative results. The authors concluded that the BTA stat test should be further evaluated as a screening method, possibly proving to be an effective test to reduce the number of cystoscopies. Telomerase is an enzyme that maintains telomeres after replication. The enzyme has been proposed to allow cells to escape senescence, especially in the case of germ cells, which maintain the integrity of telomeres throughout multiple rounds of replication. 18 Furthermore, the enzyme is hypothesized to be essential for cell immortality, as has been borne out in studies of multiple cancer cell lines. 18 This idea was tested on voided urine by Kavaler et al 18 using the telomeric repeat amplification protocol assay. They were able to detect telomerase activity in 79% of patients with grade 1 disease, including 15 samples that were negative using cytology. Sensitivities of 84% and 87.5% were obtained for grade 2 and 3 lesions, respectively, and all patients with CIS were detected. Sixty-six percent of patients with inflammatory conditions were negative by telomerase assay, with only severe inflammatory conditions causing false positive results, and all healthy volunteers were negative for telomerase activity. Though objective, the test suffers from technician requirements and slow turnaround time. The QUANTICYT system (H. G. van der Poel, Department of Urology, University Hospital, Nijmegen, the Netherlands) is a recently described light microscopic image analysis method of interpreting features of cellular atypia while allowing ploidy analysis of specified cells. 4 Bladder wash material is centrifuged and placed on a slide, and 50 randomly selected images with nuclei are analyzed and recorded. For each nucleus, the DNA content and nuclear shape features are analyzed and compared to reference lymphocyte nuclei, and a deviation index is determined. van der Poel and others 4 evaluated 4,137 bladder wash samples from 1,412 patients for the diagnostic and prognostic value of the QUANTICYT test. They classified lesions into low, intermediate and high risk based on data from previous studies correlating nuclear deviation index with histology, assuming the presence of tumor for intermediate- and high-risk specimens. In 200 samples, the specimen could not be evaluated due to the paucity of urothelial cells or abundance of neutrophils. The authors obtained comparably better results than cytology, and they were able to correlate their results with the risk of tumor recurrence within one year. Overall, the technical complexity, combined with specimen requirements and probable high cost (no specific information given), substantially reduces the utility of this method. Without procedural standardization, additional testing and low implementation costs, the QUANTICYT system probably offers a glimpse of futuristic, mathematical medicine. Further testing focuses on comparison of these tests on the same patient population. In 1998, van der Poel et al 19 compared the ability of the Bard BTA test, cytology and the QUANTICYT system to detect bladder cancer in voided urine and bladder wash specimens. They analyzed specimens from patients with previous bladder TCC and from symptomatic patients with no history of TCC, with the results listed above. They were unable to detect the presence of CIS in all three cases using the BTA test, while the QUANTICYT system detected all cases of CIS. Furthermore, image analysis cytometry detected more lesions of high grade and stage, similar to cytology. The BTA test showed no improvement in detecting higher grade lesions over those of low grade, but progression to a higher stage resulted in higher sensitivities. Despite the relatively lower specificity of image analysis cytometry, however, the authors found better prediction of tumor recurrence after normal cystoscopy. Wiener and associates 20 looked at the use of NMP22, BTA stat, QUANTICYT and cytology in the detection of TCC in 291 patients, 101 of whom were being followed for recurrence. BTA suffered from low sensitivity, and false positive results were commonly achieved in patients with inflammatory conditions. The NMP22 test achieved similar results, but the authors noted the potential value of both tests to detect a risk of tumor recurrence. The QUANTICYT method fared better than the other tests but was limited by its time-consuming nature and the requirement for adequate numbers of cells in the specimen, as 30% of the specimens were insufficient for evaluation. Cytology and QUANTI- CYT were able to detect high grade and stage lesions, while BTA stat and NMP22 results showed little correlation with grade or stage. The researchers concluded that the tests, using immunologic markers, were useful in the detection of low grade tumors but that the low sensitivity in grade 3

6 580 Zaher and Sheridan TCC and low specificity limit their usefulness. BTA was the fastest and cheapest test to use, and all compared favorably with cytology, but no test was able to replace cystoscopy. Sharma et al 3 evaluated the use of NMP22, BTA stat and cytology on voided urine specimens from 278 symptomatic patients. Their study differed from others in using easily assessed exclusion criteria to enhance the specificity of the tests. This resulted in improvement in the specificity to 95% with NMP22 and 92% with BTA. Moreover, the positive and negative predictive values improved significantly for both. The inexpensive nature and relative lack of need for technical expertise make these tests potentially useful for screening at-risk populations. Ramakumar and others 21 evaluated the use of cytology, BTA stat, NMP22, fibrin/fdp, telomerase, chemiluminescent hemoglobin and hemoglobin dipstick on voided urine in the detection of bladder cancer. One hundred ninety-six patients, 57 with bladder cancer and 139 without, were studied, and the researchers achieved the best results with the telomerase assay. These results were further improved when the specimen was obtained using a bladder wash specimen. The authors achieved marginal results with both BTA and NMP22 and noted that NMP22 alone was not a statistically significant predictor of bladder TCC. The BTA test in this series showed improved detection of TCC with increasing grade and stage, detecting all grade 3 lesions, all stage pt1-pt3b lesions and 82% of CIS. The authors obtained high specificity using the FDP test, but the sensitivity was lower than in other studies. As expected, the chemiluminescent hemoglobin assay and hemoglobin dipstick were quite nonspecific, but the dipstick, when added to the telomerase assay, produced statistically significant results. Overall, the authors found the telomerase assay to be the most cost-effective addition to monitoring for bladder cancer. Johnston et al 22 studied the use of point of care tests in 130 patients (60 with bladder cancer). They determined that FDP was a more valuable test than Bard BTA, cytology or hemoglobin dipstick. The results of the BTA test in that study were disappointing, but all tests obtained false positives in patients with inflammatory conditions. Conclusion The heterogeneity of the results in this small comparison of studies suggests that much more evidence is needed before any of the above tests will become practical. The striking differences in statistical values throughout the large body of literature require a proper metaanalysis of each method to sort out the discrepancies. Such an analysis could only hope to dissect out and weigh the potential, hidden benefits of these methods, which differ between individual studies and methods of tumor detection. Perhaps the issue of greatest concern is the confounding factor of inflammatory conditions, as it highlights the drawbacks of these methods. The contribution of Nasuti and others suggest that the BTA stat test may do nothing more than detect blood in the urine. While this statement may be an oversimplification, it certainly does not allow classification of the BTA test as a specific test for bladder tumors, regardless of the stage or grade. QUANTICYT, though combining the proven strengths of cytology with mathematics and statistics to average out human error, does not provide overwhelming results that outweigh the costs of implementation, quality control and operating costs. Further research using other markers may eventually find a more characteristic marker of bladder cancer. It is unlikely that any of these tests, despite the cost benefits and ease of performance, will replace cytology or cystoscopy soon. However, implementation of one of these simple assays might prolong the interval of cystoscopy in patients monitored for bladder TCC recurrence. This would significantly reduce the cost to patients, who, by current standards, must undergo cystoscopy every three months for the first two years following resection of the tumor. Provided that these tests absolutely detect all potentially invasive, high grade lesions, this protocol would be acceptable. Low grade lesions are difficult to detect, even on cystoscopy, and the risk of early invasion is low. The search for a simple and cost-effective marker of bladder TCC is ongoing. References 1. Greenlee RT, Murray T, Bolden S, Wingo P: Cancer statistics, CA Cancer J Clin 2000;49: Thomas L, Leyh H, Marberger M, Bombardieri E, Bassi P, Pagano F, Pansadoro V, Sternberg CN, Boccon-Gibod L, Ravery V, Le Guludec D, Meulemans A, Conort P, Ishak L: Multicenter trial of the quantitative BTA TRAK assay in the detection of bladder cancer. Clin Chem 1999;45: Sharma S, Zippe CD, Pandrangi L, Nelson D, Agarwal A: Exclusion criteria enhance the specificity and positive predictive value of NMP22 and BTA stat. J Urol 1999;162: van der Poel HG, Witjes JA, van Stratum P, Boon ME, De-

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