367Vol. 9 | No. 1 | Winter 2012 |U R O LO G Y J O U R N A L Detection of Recurrent Bladder Cancer NMP22 Test or Urine Cytology? Jalil Hosseini,1 Ali Reza Golshan,2 Mohammad Mohsen Mazloomfard,1 Abdolrasoul Mehrsai,3 Mohammad Ali Zargar,4 Mohsen Ayati,5 Saeed Shakeri,6 Majid Jasemi,7 Mahmoud Kabiri8 Purpose: To assess the accuracy of voided urine cytology versus urinary nu- clear matrix protein 22 (NMP22) qualitative assay in the diagnosis of various grades and stages of recurrent bladder transitional cell carcinoma (TCC). Materials and Methods: From July 2007 to February 2009, all patients with history of superficial bladder TCC were included in this multi-center study. Each patient provided three serial voided urine samples for cytologic examina- tion and one sample for the NMP22 qualitative assay prior to urethrocystos- copy. The sensitivity and specificity of urine cytology and the NMP22 test were determined. Results: The sensitivities of the NMP22 test and cytology for detection of re- currence were 78.8% and 44.2%, respectively (P = .001), while the specificities were 69.6% and 83.7%, respectively (P = .019). The NMP22 test showed sig- nificantly higher sensitivity than cytology in detecting recurrences in low-risk and intermediate-risk groups. Conclusion: The NMP22 assay could be used for detection of superficial blad- der cancer, especially in low- and intermediate-risk groups; however, the value of the test is limited by its low specificity. Keywords: transitional cell carcinoma, urinary bladder neoplasms, diagnosis, nuclear matrix proteins, tumor markers Corresponding Author: Jalil Hosseini, MD Infertility and Reproductive Health Research Center, Recon- structive Urology, Shohada-e- Tajrish Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran Tel: +98 21 2243 2558 Fax: +98 21 2203 8462 E-mail: jhosseinee@gmail.com Received March 2010 Accepted September 2010 1 Infertility and Reproductive Health Research Center, Recon- structive Urology, Shohada-e- Tajrish Hospital, Shahid Beheshti University of Medical Sciences, Tehran, Iran 2 North Khorasan University of Medical Sciences, Bojnurd, Iran 3 Sina Hospital, Tehran Univer- sity of Medical Sciences, Tehran, Iran 4 Hasheminejad Kidney Center, Tehran University of Medical Sciences, Tehran, Iran 5 Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran 6 Namazi Hospital, Shiraz University of Medical Sciences, Shiraz, Iran 7Golestan Hospital, Ahvaz University of Medical Sciences, Ahvaz, Iran 8Alzahra Hospital, Isfahan University of Medical Sciences, Isfahan, Iran Urological Oncology 368 | Urological Oncology INTRODUCTION The most common genitourinary cancer among our population is the bladder carci-noma with prevalence of 48.3%.(1) Super- ficial type accounts for 70% of the urothelial cell carcinoma of the bladder that has a high probabil- ity of recurrence (60% to 85%).(2,3) Long-term follow-up is recommended to detect any cancer recurrence or progression. Therefore, cystoscopy, the gold standard test, and urine cytol- ogy every 3 to 4 months for the first two years and at a longer interval in subsequent years are recom- mended as the current standard of care for detec- tion of tumor recurrence.(4,5) Due to invasiveness and high cost of this approach, new techniques and markers, including flow cytometry, quantitative fluorescence image analysis, and nuclear matrix protein 22 (NMP22), etc, have been introduced and studied for their accuracy in detection of re- current bladder cancers.(6-10) The NMP22 test detects the nuclear matrix protein qualitatively, which is part of the nuclear mitotic apparatus released from urothelial nuclei upon cel- lular apoptosis.(11-13) Nuclear matrix protein 22 is shed into the urine and has a 20 to 80-times higher concentration in the urine of the patients with blad- der cancer compared to noncancerous controls.(12) This test has been approved by the Food and Drug Administration (FDA) for patient surveillance.(14) This test has sensitivity and specificity as high as urine cytology. We compared the sensitivity and specificity of voided urine cytology in the diagnosis of various grades and stages of recurrent bladder transitional cell carcinoma (TCC) with the urinary NMP22 qualitative test. MATERIALS AND METHODS From July 2007 to February 2009, all patients with history of superficial bladder TCC from sev- en academic centers were enrolled in this study. Patients with urinary tract infections, concurrent urolithiasis, a history of bladder substitution, and other malignancies were excluded from the study. Written informed consent was obtained from each participant and the study was approved by the Eth- ics Committee of the Infertility and Reproductive Health Research Center (IRHRC). Each patient provided three serial voided urine samples within 3 days for cytologic examination and one sample for the NMP22 qualitative assay. In each center, one cytopathologist performed cy- tologic examination, who was unaware of the cys- toscopy and NMP22 results. Malignant and sus- picious results for malignancy were classified as positive. The NMP22 assay was performed according to the instructions provided in the NMP22 point-of-care device (Matritech Inc, 330 Nevada St, Newton, MA). The quality of NMP22 in patients’ urine was assessed using a lateral flow immunochromato- graphic strip. Four drops of urine at room tempera- ture were added to the point-of-care device and re- sults were interpreted within 30 minutes. Positive result yielded a colored band in the test position. All the NMP22 test results were interpreted by a single observer at each center, who was blind to the cystoscopy and cytology results. Urethrocystoscopy, using a rigid cystoscope and video camera, was performed for all the patients. Any visible tumor or suspicious lesion was biop- sied for histopathologic examination, using the TNM staging system(15) and World Health Organi- zation grading.(16) Findings from histopathologic evaluation of biopsies were considered as a gold base for comparing the results of other two tests. Either normal appearance in endoscopy or histo- pathologically nonmalignant tissue in biopsy was defined as negative for TCC. If no tumor was ob- served endoscopically, patients with positive iso- lated cytology or NMP22 test were further evalu- ated by random biopsies from the bladder and urethra and by imaging, like intravenous urogra- phy or contrast-enhanced computed tomography, 369Vol. 9 | No. 1 | Winter 2012 |U R O LO G Y J O U R N A L NMP22 and Bladder Cancer | Hosseini et al in order to rule out any missed lesion in the geni- tourinary system. Patients with a higher index of suspicion were re-evaluated every 4 weeks until 6 months. Patients who were found to have an upper tract lesion or a bladder lesion in the next cystos- copy were considered true positives for the test. Data analysis was performed by using SPSS soft- ware (the Statistical Package for the Social Scienc- es, Version 15.0, SPSS Inc, Chicago, Illinois, USA) using Chi-Square, Fisher’s Exact, and Mann-Whit- ney U tests and by calculating the 95% confidence interval (CI) to determine the sensitivity and speci- ficity of urine cytology and the NMP22 assay. RESULTS Of 320 recruited participants, 144 patients, 125 men and 19 women, met the inclusion criteria, di- agnostic tests, and follow-up period. The mean age of the entire group was 61.8 years (range, 26 to 86 years). Of 144 patients, 52 (36.11%) were diagnosed with recurrent bladder TCC; 48 patients were detected with cystoscopy and 4 after the first follow-up cys- toscopy. One of these 4 patients had carcinoma in situ (CIS) in his pathology and was positive for cy- tology; but other 3 subjects were NMP22-positive with small tumor size, 2 of them had T1G2 and the other one had T1G3. Of 52 patients with tumor recurrence, 16 had grade I, 20 had grade II, and 16 had grade III disease, while 18 had stage Ta, 22 had stage T1, 3 had CIS, and 9 had stage T2 or more. On histopathology, 41 were positive for the NMP22 test and 23 were positive for cytology. The sensitivity, specificity, positive predictive val- ue, and negative predictive value of the NMP22 test and voided urine cytology for detection of re- currence are presented in Table 1. The sensitivities of urine cytology and urinary NMP22 regarding the stage, grade, and risk stratification are shown in Table 2. Pathological data were grouped according to risk for recurrence, progression, and invasion into a low-risk group (Single Ta, G1 < 3 cm), a high-risk group (Multiple T1G2, Tis, T1G3, and TaG3), and an intermediate-risk group (rather than two other groups). The NMP22 test showed significantly higher sensitivity than cytology in detecting low- risk and intermediate-risk groups recurrences. However, this test detects recurrence as same as cytology in the high-risk group (Table 2). After combining the results of the NMP22 test and cytology, NMP22 test and cystoscopy, and cytology and cystoscopy, the overall sensitivity increased to 88.5% (46/52), 98.1% (51/52), and 94.2% (49/52), respectively, rather than cytology or NMP22 test alone. DISCUSSION Cystoscopy is the gold standard modality for the diagnosis of bladder carcinoma; however, it is in- vasive and relatively expensive.(17) Voided urine cytology is based on morphologic assessment in intact cells shed in urine; hence, small tumors Table 1. Sensitivity, specificity, positive predictive value, and negative predictive value of the NMP22 test and voided urine cytology Urine Cytology NMP22 Test P Sensitivity (%) 44.2 (23/52) 78.8 (41/52) .001 Specificity (%) 83.7 (77/92) 69.6 (64/92) .019 Negative Predictive Value (%) 72.6 (77/106) 85.3 (64/75) NS* Positive Predictive Value (%) 60.5 (23/38) 59.4 (41/69) NS *NS indicates non-significant 370 | or well-differentiated ones are difficult to recog- nize because cells less likely exfoliate spontane- ously. This fact explains its low sensitivity (15% to 30%) in low-stage cancers.(13) Therefore, many new urine-based tests for substitution of urine cy- tology have been developed. Of which, BTA stat, BTA trak, NMP22, FDP, ImmunoCyt, and FISH (UroVysion) have been approved by the FDA.(18) Nuclear matrix, first described in 1974, is a non- chromatin structure that supports nuclear shape, organizes DNA, and takes part in DNA replication and transcription, and in RNA processing. Nuclear matrix protein 22 is a nuclear protein which plays a role in control of the chromatid regulation and cell separation during replication. The NMP22 test is an office-based procedure that can be interpreted by a urologist within 30 minutes; therefore, it can be used as an alternative to urine cytology.(19,20) Various published studies have reported NMP22 (sensitivity: 70% to 80%) to be at least twice more sensitive than urine cytology (sensitivity: 10% to 40%) in detecting bladder cancer.(8,21-23) In our study, the sensitivity of the NMP22 test was significantly higher, but the specificity was lower than that for cytology (77.8% versus 44.2% and 69.6% versus 83.7%, respectively). These find- ings are compatible with the quantitative analyses of NMP22 performed by other investigators.(8,22,23) It was demonstrated that the quantitative NMP22 test had an overall sensitivity of 70% to 80% for the detection of recurrent superficial bladder TCC in approximately 400 patients. In comparison, cy- tology showed sensitivity of 10% to 40%.(20,24,25) A higher sensitivity for urine cytology rather than other published articles was detected in this study, may be due to examining of three urine samples instead of one. Few drawbacks have been mentioned for NMP22, including lack of exact cutoff point for quantita- tive assessment.(26) Jamshidian and colleagues suggested a cutoff point of 10.1 U/mL for Iranian patients for detection of the bladder cancer.(19) Us- ing NMP22 point-of-care device (Matritech Blad- derChek Test), bladder cancer can be evaluated qualitatively by cutoff point of 10 U/mL, as a posi- tive result for test. Our study showed that the NMP22 test had consist- ently higher sensitivity than cytology in detecting Urological Oncology Table 2. Diagnostic value of urine cytology and NMP22 test in each tumor grade and stage, and risk of recurrence and progression Tumor Class (n = 52) Sensitivity of Urine Cytology (%) Sensitivity of NMP22 Test (%) P Stage CIS (n = 3) Ta (n = 18) T1 (n = 22) ≥ T2 (n = 9) 100 (3/3) 11.1 (2/18) 45.5 (10/22) 88.9 (8/9) 66.7 (2/3) 61.1 (11/18) 95.5 (21/22) 77.8 (7/9) .00 .002 .021 NS Grade G I (n = 16) G II (n = 20) G III (n = 16) 0.0 (0/16) 40.0 (8/20) 93.8 (15/16) 68.8 (11/16) 85.0 (17/20) 81.3 (13/16) .00 .022 NS Risk stratification Low (n = 15) Intermediate (n = 20) High (n = 8) 0.0 (0/15) 40.0 (8/20) 87.5 (7/8) 66.7 (10/15) 85.0 (17/20) 87.5 (7/8) .00 .022 NS * CIS indicates carcinoma in situ; and NS, non-significant. 371Vol. 9 | No. 1 | Winter 2012 |U R O LO G Y J O U R N A L different stages and grades of recurrence in patients with a history of superficial bladder cancer. But in detection of high-grade or advanced tumor, there was no significant difference between two tests. It was already demonstrated that there is no signifi- cant difference in risk of progression in the low- and intermediate-risk groups. Kumar and associ- ates showed that the NMP22 test was eight times more sensitive than cytology to detect the low-risk group, but not intermediate- and high-risk groups. (20) Tendency of the NMP22 assay to detect recur- rent bladder tumor in patients with intermediate- risk in our study is in contrast to Kumar’s study. Traditionally, diagnosis of these groups has been the greatest challenge for non-invasive assays. By use of NMP22 test as a surveillance marker, repeti- tion of cystoscopy or transurethral resection of the bladder tumors may be avoided or can be delayed in such patients. CONCLUSION The NMP22 test is a non-invasive and rapid test for the diagnosis of low-stage bladder cancer. ACKNOWLEDGEMENTS This study was approved in Infertility and Repro- ductive Health research Center (IRHRC, SBMU) as a research project. 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