Urine Detection of Survivin and Diagnosis of Bladder Cancer PRELIMINARY COMMUNICATION
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Urine Detection of Survivin and Diagnosis of Bladder Cancer PRELIMINARY COMMUNICATION
PRELIMINARY COMMUNICATION Urine Detection of Survivin and Diagnosis of Bladder Cancer Shannon D. Smith, MD Marcia A. Wheeler, MS Janet Plescia, BS John W. Colberg, MD Robert M. Weiss, MD Dario C. Altieri, MD D EREGULATED EXPRESSION OF inhibitors of apoptosis is thought to contribute to cancer by abnormally extending cell viability, favoring the accumulation of mutations, and promoting resistance to therapy.1 A novel modulator of the cell death/viability balance in cancer was recently identified as survivin,2 a member of the inhibitor of apoptosis gene family.3 Undetectable in most normal adult tissues, survivin becomes the top fourth transcript expressed in common human cancers,2,4 in which it correlates with unfavorable disease and abbreviated overall survival.5-9 Urothelial (transitional cell) carcinoma of the bladder is the fourth most common cancer in men and the eighth most common cancer in women in the United States, accounting for more than 54000 new cases and 11200 deaths every year. Recurrences of bladder cancer occur in up to 80% of patients and constitute a formidable obstacle to longlasting remissions, frequently anticipating muscle invasion, and disseminated disease.10 Despite considerable efforts to develop safe, reliable, noninvasive screening strategies for bladder cancer, the identification of a single predictive/ prognostic marker of the disease has remained elusive.11 Consistent with a proposed role of deregulated apoptosis in urothelial cancer,12,13 survivin was found in 78% of bladder cancers, but not in nor324 Context Dysregulation of apoptosis may favor onset and progression of cancer and influence response to therapy. Survivin is an inhibitor of apoptosis that is selectively overexpressed in common human cancers, but not in normal tissues, and that correlates with aggressive disease and unfavorable outcomes. Objective To investigate the potential suitability of survivin detection in urine as a novel predictive/prognostic molecular marker of bladder cancer. Design, Setting, and Patients Survey of urine specimens from 5 groups: healthy volunteers (n = 17) and patients with nonneoplastic urinary tract disease (n = 30), genitourinary cancer (n = 30), new-onset or recurrent bladder cancer (n = 46), or treated bladder cancer (n = 35), recruited from 2 New England urology clinics. Main Outcome Measures Detectable survivin levels, analyzed by a novel detection system and confirmed by Western blot and reverse transcriptase polymerase chain reaction (RT-PCR), in urine samples of the 5 participant groups. Results Survivin was detected in the urine samples of all 46 patients with new or recurrent bladder cancer using a novel detection system (31 of 31) and RT-PCR (15 of 15) methods. Survivin was not detected in the urine samples of 32 of 35 patients treated for bladder cancer and having negative cystoscopy results. None of the healthy volunteers or patients with prostate, kidney, vaginal, or cervical cancer had detectable survivin in urine samples. Of the 30 patients with nonneoplastic urinary tract disease, survivin was detected in 3 patients who had bladder abnormalities noted using cystoscopy and in 1 patient with an increased prostate-specific antigen level. Patients with low-grade bladder cancer had significantly lower urine survivin levels than patients with carcinoma in situ (P = .002). Conclusions Highly sensitive and specific determination of urine survivin appears to provide a simple, noninvasive diagnostic test to identify patients with new or recurrent bladder cancer. mal urothelium, and its expression correlated with accelerated recurrences.14 Because of its expression in cancer but not in normal tissues, we investigated the potential suitability of urine survivin as a new molecular marker for detection of bladder cancer. METHODS Urine Specimens One hundred fifty-eight urine specimens were collected at the urology clinics at Yale-New Haven Hospital and at the Veterans Affairs, New England Health Care Systems, West Haven, Connecticut, Division. Random clean-catch or JAMA, January 17, 2001—Vol 285, No. 3 (Reprinted) Downloaded From: http://www.jamafacial.com/ on 06/15/2014 www.jama.com JAMA. 2001;285:324-328 straight catheter urine samples were obtained from individuals who were categorized into 5 different groups: group 1 included healthy volunteers with a mean (SD) age of 47.6 (20.8) years who were not taking any medication (n=17); group 2 patients had a mean (SD) age of 60.0 (18.1) years with diagnosis of nonAuthor Affiliations: Departments of Surgery (Drs Smith, Colberg, and Weiss, and Ms Wheeler) and Pathology (Ms Plescia and Dr Altieri), Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Conn. Corresponding Author and Reprints: Dario C. Altieri, MD, Yale University School of Medicine, BCMM436B, 295 Congress Ave, New Haven, CT 06536 (e-mail: [email protected]). ©2001 American Medical Association. All rights reserved. SURVIVIN AND BLADDER CANCER neoplastic urinary tract disease or hematuria (n = 30); group 3 patients had a mean (SD) age of 71.5 (9.9) years with diagnosis of genitourinary cancer, excluding bladder cancer (n=30); group 4 patients had a mean (SD) age of 69.7 (8.7) years with diagnosis of new-onset or recurrent bladder cancer (n=46); and group 5 patients had a mean (SD) age of 76.1 (8.9) years and were undergoing treatment or had already received treatment for bladder cancer and had negative cytoscopic findings on the day of urine collection (n=35). Treatment measures in group 5 included intravesical bacillus Calmette-Guerin, thiotepa, transurethral resection, partial cystectomy, and radiation. Group 4 included patients who, after urine collection, underwent similar treatment measures and/or salvage cystectomy or radical cystectomy. Urine Detection of Survivin Urine specimens were filtered onto a nitrocellulose membrane using a microfiltration apparatus in a module providing a 48-wells-lot format. The blot was analyzed for the presence of survivin using a polyclonal antibody. The protocol is as follows: urine was collected and stored at −80°C until analysis. On the day of analysis, urine samples were centrifuged at 20000g for 20 minutes. Meanwhile, the Bio-Dot microfiltration apparatus was assembled with a 0.2-µm nitrocellulose membrane (Bio-Rad Laboratories, Hercules, Calif) and moistened in 20-mmol Tris-buffered saline (pH, 7.5). Then, the urine supernatant (300 µL), along with increasing concentrations of Escherichia coli–expressed recombinant survivin15 as a standard (0.001-1.0 µg/mL) in 300 µL of Trisbuffered saline, were filtered onto the membrane. After filtration, the membrane was dried then blocked in 5% dried milk plus 0.01% sodium azide in phosphate-buffered saline (PBS) (pH, 7.4) for 12 hours at 4°C. After washing in PBSTween 20 (0.25%), the membrane was incubated with 2 µg/mL of a rabbit antibody to survivin16 for 3 hours at 22°C, washed in PBS-Tween, and incubated with a 1:1000 dilution of horseradish peroxidase-conjugated donkey antirabbit IgG (Amersham Biotech, Piscataway, NJ) for 1 hour at 22°C. After washes in PBS twice for 10 minutes, PBS-Tween twice for 5 minutes, and PBS twice for 5 minutes, binding of the primary antibody was detected by enhanced chemiluminescence (Amersham Biotech) and autoradiography. Bands were quantitated by densitometry and a weighted survivin score was calculated on the basis of the antibody reactivity with increasing concentrations of recombinant survivin as follows: 0 for not detectable; 1 for 0.0010.25 µg/mL; 2 for 0.25 to 1 µg/mL; and 3 for more than 1 µg/mL. Each urine specimen was analyzed at least twice on 2 different occasions and comparable results were obtained. Western Blot Urine specimens (100 mL) were centrifuged at 1200g for 10 minutes at 22°C, and the cell pellet was washed twice in Tris-buffered saline and made soluble in 0.5% Triton X-100 (Sigma, St Louis, Mo) in the presence of protease inhibitors for 30 minutes at 4°C. Samples were separated by SDS gel (BioRad Laboratories) electrophoresis, transferred to nylon membranes (Millipore Corp, Bedford, Mass), and further incubated with 1 µg/mL of an antibody to survivin 1 6 followed by horseradish peroxidase–conjugated goat antirabbit IgG and chemiluminescence. Reverse Transcriptase Polymerase Chain Reaction Fifty milliliters of clean-catch urine was obtained from 15 patients with new or recurrent urothelial cancer, 2 patients with treated bladder cancer, 1 patient with prostate cancer, 1 patient with nonneoplastic urinary tract disease, and 1 healthy volunteer. Total RNA was isolated from urine pellets using the Trizol reagent (Life Technologies Inc, Gaithersburg, Md). Single-strand complementary DNA (cDNA) was synthesized by random priming of 1-5 µg total RNA using 1 µL of RT (Gibco BRL, Life Technologies Inc) for 1 hour at 43°C. After heating at 70°C for 15 minutes, a first amplification reaction was ©2001 American Medical Association. All rights reserved. Downloaded From: http://www.jamafacial.com/ on 06/15/2014 carried out with survivin primers 59CTGCCTGGCAGCCCTTTCTCAA-39 (forward) and 59AATAAACCCTGGAAGTGGTGCA-39 (reverse) with denaturation at 94°C for 15 seconds, annealing at 53°C for 15 seconds, and extension at 72°C for 1 minute for 20 cycles, followed by incubation at 72°C for 5 minutes. A 463-base pair fragment of the survivin cDNA was subjected to a second round of amplification with nested survivin primers 59CCGCATCTCTACATTCAAGAAC-39 (forward) and 59-CTTGGCTCTTTCTCTGTCC-39 (reverse), with denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, and extension at 72°C for 45 seconds for 30 cycles, followed by incubation at 72°C for 5 minutes. The amplified survivin cDNA of 279 base pair was separated on a 2.0% solution of agarose gel and visualized by ethidium bromide staining. Control reactions were amplified using b-actin–specific primers 59-AGCGGGAAATCGTGCGTG-39 (forward) and 59-CAGGGTACATGGTGGTGCC-39 (reverse) with generation of a 309– base pair fragment. Statistical Analysis The relationship between urine survivin and patients’ diagnosis was analyzed by a x2 test. Nonparametric statistical analysis was used to compare the weighted urine survivin score with the grading classification system performed at the Yale-New Haven Hospital. The calculation of predictive accuracy is not appropriate for this study since the diagnosis was known at the time of urine collection. RESULTS A representative experiment of detection of urine survivin using the BioDot test is shown in FIGURE 1. Determination of urine survivin with the BioDot method was carried out in 138 of the 158 specimens collected for this study (TABLE 1). Twenty additional urine samples were analyzed for survivin expression by reverse transcriptase polymerase chain reaction (RTPCR) to independently evaluate the (Reprinted) JAMA, January 17, 2001—Vol 285, No. 3 325 SURVIVIN AND BLADDER CANCER specificity of the new method. Survivin was not detected in urine of the 16 volunteers, 6 patients with benign pros- tatic hyperplasia, 2 with interstitial cystitis, 3 with renal calculi, 6 with urinary tract infection, or 6 with other nonneoplastic urinary tract disease (Table 1). Urine survivin was detected in 3 of 5 patients with cryptogenic hematuria (weighted survivin score, 2), who presented with a history of retention and dysuria post-transurethral prostate resection, and revealed a trabeculated, irregularly thickened bladder, by cystoscopy (see “Comment” section). One patient with increased prostatespecific antigen levels but without diagnosis of prostate cancer was positive for urine survivin (Table 1). This patient also had a trabeculated, thickened bladder, by cystoscopy. Survivin was not detected in urine specimens of 19 patients with prostate, 8 with renal, 1 with vaginal, or 1 with cervical cancer (Table 1). In contrast, urine survivin was detected in all 31 patients with new-onset or recurrent bladder cancer (Table 1). Histopathologic grading (grades I through IV) of the 31 patients in group 4 analyzed for urine survivin by the novel method included 13 patients with grade II, 7 patients with grade III, and Figure 1. Urine Detection of Survivin Patient Group Survivin, µg/mL 0 RCC TCC TCC/R TCC/R Control 0.001 PC TCC TCC/R TCC/R Control 0.005 PC TCC TCC/R TCC/R Control 0.01 PC TCC TCC/R TCC/R Control 0.05 PC TCC TCC/R TCC/R Control 0.25 PSA TCC/R TCC/T TCC/R Control 1 BPH TCC TCC/R TCC/R Control Control BPH RCC PC TCC TCC/R TCC/T Healthy Volunteers (Group 1) Benign Prostatic Hyperplasia (Group 2) Renal Cell Carcinoma (Group 3) Prostate Cancer (Group 3) Bladder Cancer (Group 4) Bladder Cancer In Remission (Group 5) Bladder Cancer Receiving Treatment (Group 5) Increasing concentrations of recombinant survivin or urine specimens from the indicated patient groups were applied to a slot-blot apparatus. The membrane was incubated with an antibody to survivin followed by horseradish peroxidase–conjugated goat antirabbit IgG. Bands were visualized by chemiluminescence and quantitated by densitometry. Table 1. Survivin Detection in 138 Urine Specimens Using a Novel Detection Method Urine Specimens Group 1 (control healthy volunteers) Group 2 (nonneoplastic urinary tract diseases) Total No. of Patients 16 No. of Patients Survivin-Negative 16 No. of Patients Survivin-Positive 0 29 Hematuria 5 2 3 Urinary tract infection 6 6 0 Benign prostatic hyperplasia 6 6 0 Increased prostate specific antigen 1 0 1 Interstitial cystitis 2 2 0 Renal calculi 3 3 0 6 29 6 0 19 19 0 Renal 8 8 0 Vaginal 1 1 0 1 31† 33 1 0 30 0 31 3§ Other* Group 3 (genitourinary cancers except bladder) Prostate Cervical Group 4 (new or recurrent bladder cancer) Group 5 (treated bladder cancer)‡ *Includes 1 patient with papillary necrosis, 2 with prostatitis, 1 with vesicoureteral reflux, and 2 with renal transplant with elevated creatinine. †Includes 1 patient with urothelial cancer of the ureter. ‡Patients had normal cystoscopy results. §Two of these patients were treated with transurethral resection of the bladder tumor, and 1 with fulguration. One of these patients had urine cytology positive for bladder cancer. 326 JAMA, January 17, 2001—Vol 285, No. 3 (Reprinted) Downloaded From: http://www.jamafacial.com/ on 06/15/2014 5 patients with grade IV tumors. Carcinoma in situ was found in association with the papillary and invasive carcinomas of 5 patients and in association with high-grade urothelial cancer of the ureter in 1 patient. Thirty of 33 patients in group 5 analyzed by the novel system had no detectable urine survivin (Table 1). Five of these 30 patients were receiving bacillus Calmette-Guerin and had completed 3 to 5 treatments, the other 25 were status posttreatment with negative cystoscopy findings. Three patients in group 5 with initial diagnosis of grade II noninvasive bladder cancer had positive test results for urine survivin after undergoing negative cystoscopic examination. One of the 3 patients had urine cytology positive for bladder cancer. Two of the 3 patients were treated with transurethral resection of the bladder tumor and 1 was treated with fulguration. When normalized for a weighted mean (SD) survivin score, patients with carcinoma in situ had considerably higher survivin score (2.5 [0.5]; n=6) than patients with grade II bladder cancer (1.3 [0.6]; n=13). The correlation between weighted survivin score and histopathology or grading of the various bladder cancer cases is shown in TABLE 2 and TABLE 3, respectively. By Western blot, a single survivin band of 16.5 kd was detected in the urine cell pellet from a patient with bladder canTable 2. Correlation Between Weighted Urine Survivin Score and Bladder Cancer Histopathology* Histopathology Not determined Noninvasive papillary carcinoma No detrusor muscle invasion Muscle invasion Carcinoma in situ No. of Cases Tested 3 Mean (SD) Survivin Score 1.7 (1.2) 4 1 (0) 12 1.6 (0.8) 6 6 1.7 (0.8) 2.5 (0.5)† *The weighted survivin score was calculated using a standard curve with increasing concentrations of recombinant survivin as follows: 0, not detectable; 1, 0.001 to 0.25 µg/mL; 2, 0.25 to 1 µg/mL; and 3, more than 1 µg/mL. †Significantly greater than either grade II or noninvasive papillary carcinoma (P,.02). ©2001 American Medical Association. All rights reserved. SURVIVIN AND BLADDER CANCER COMMENT In this study, we describe a simple, antibody-based test to identify the ap- optosis inhibitor survivin2,4 in urine of patients with bladder cancer. Survivin was found in urine samples of all 46 patients with new or recurrent bladder cancer, but not in any of the 17 healthy volunteers, or in any of the 30 patients with other urologic cancers, and only in 4 of 30 patients with nonneoplastic genitourinary disorders. Importantly, of the 3 patients with hematuria who tested positive for urine survivin, 1 had a positive cytology result for bladder cancer and another was diagnosed with bladder cancer within 6 months of survivin detection. Moreover, 32 of 35 patients treated for bladder cancer and achieving cystoscopic remission had negative test results for urine survivin. There is a positive correlation between a weighted urine survivin score and more advanced histopathologic tumor grading. For its overexpression in cancer but not in normal tissues,2,4 and its unfavorable predictive and/or prognostic significance in various malignancies,5-9 survivin may constitute a useful molecular marker in cancer. This may be particularly relevant in bladder cancer,11,17 in which simple and noninvasive diagnostic means to monitor response to therapy and simplify follow-up protocols are urgently needed. Although regarded as the criterion standard,18 urine cytology has low sensi- tivity (30%-40%) in bladder cancer, and fails to detect superficial, low-grade lesions. In this context, several urine Table 3. Correlation Between Weighted Urine Survivin Score and Bladder Cancer Grading* Grade II III IV IV No. of Cases Tested Mean (SD) Survivin Score 13 7 5 1 1.3 (0.6) 1.5 (0.8) 2 (1) 3† *The weighted survivin score was calculated using a standard curve with increasing concentrations of recombinant survivin as follows: 0, not detectable; 1, 0.001 to 0.25 µg/mL; 2, 0.25 to 1 µg/mL; and 3, more than 1 µg/mL. Histopathological analysis was carried out using the Broader cytologic grading system for the classification of papillary transitional cell tumors, grades I through IV. †One of the 6 patients with associated carcinoma in situ had urothelial cancer of the ureter (grade IV; survivin score, 3). Figure 2. Western Blot of Urine Survivin TCC Relative Molecular Weight, ×10 –3 cer but not in that from a healthy volunteer (FIGURE 2). To independently evaluate the results obtained with the new method, 15 additional patients with new or recurrent bladder cancer were analyzed for urine survivin by RT-PCR. A 279-base pair survivin cDNA was amplified from urine cell pellets of all the 15 new patients with bladder cancer (FIGURE 3 and data not shown). In contrast, urine cell pellets from 5 additional individuals, 1 with urinary tract infection, 2 with treated bladder cancer and negative cystoscopy results, 1 with prostate cancer, and 1 from a volunteer, had no survivin cDNA (Figure 3). In control experiments, a 309–base pair b-actin– cDNA fragment was indistinguishably amplified from urine of controls and patients with bladder cancer (Figure 3). Histopathologic cases of bladder cancer analyzed by RT-PCR included 5 patients with grade II tumors, 1 patient with grade III, 6 patients with grade IV, and 3 patients with carcinoma in situ. These experiments suggest that exfoliated cancer cells may passively release survivin in the extracellular milieu (ie, urine) during tumor progression. Control 30 21 14.5 Urine cell pellets from a healthy volunteer and a group 4 patient with bladder cancer (TCC) were electrophoresed, transferred to nylon membranes, and immunoblotted with an antibody to survivin followed by chemiluminescence. Figure 3. Reverse Transcriptase Polymerase Chain Reaction Amplification of Survivin Messenger RNA in Urine Specimens TCC Control Molecular Weight, Base Pair M TCC M 400 300 200 M Survivin Control M 400 300 β-Actin 200 Total RNA was extracted from urine cell pellets and reverse-transcribed by random priming. Amplification reactions were carried out with survivin-specific nested primers (279 bp) or b-actin–specific primers (309 bp). M indicates molecular weight markers in base pair; TCC, analysis of 5 representative patients with new or recurrent bladder cancer (group 4). ©2001 American Medical Association. All rights reserved. Downloaded From: http://www.jamafacial.com/ on 06/15/2014 (Reprinted) JAMA, January 17, 2001—Vol 285, No. 3 327 SURVIVIN AND BLADDER CANCER markers including bladder tumor antigen, nuclear matrix protein, telomerase activity, hyaluronic acid/hyaluronidase, and fibrin degradation products have been characterized for their potential diagnostic/predictive value in bladder cancer.19,20 In this patient series, the sensitivity of the urine survivin test for new or recurrent bladder cancer was 100%, and its specificity for other neoplastic and nonneoplastic genitourinary tract diseases was 95% (P,.02). However, the overall specificity of the test is likely to vary depending on which patient population is the focus of clinical interest. A screening test for group 1 individuals will have a false-positive rate of essentially zero, whereas patients with clinical symptoms in groups 2 and 3 will likely have a combined false-positive rate of 5% to 10%. However, similarly to the 2 patients with hematuria described above, these individuals should Author Contributions: Dr Smith participated in study concept and design, acquisition of data, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript for important intellectual content, provided statistical expertise, obtained funding, and provided administrative, technical, or material support. Ms Wheeler participated in study concept and design, acquisition of data, analysis and interpretation of data, drafting of the manuscript, critical revision of the manuscript for important intellectual content, and provided administrative, technical, or material support. Ms Plescia participated in study concept and design, acquisition of data, analysis and interpretation of data, critical revision of the manuscript for important intellectual content, and provided administrative, technical, or material support. Dr Colberg participated in study concept and design and acquisition of data. Dr Weiss participated in study concept and design, acquisition of data, analysis and interpretation of data, critical revision of the manuscript for important intellectual content, obtained funding, provided administrative, technical, or material support, and supervised conduct of the study. Dr Altieri participated in study concept and design, analysis and interpretation of data, drafting of the manuscript, and obtained funding. Funding/Support: Our work was supported by National Institutes of Health grants DK02499 (Dr Smith); DK47548 and DK38311 (Dr Weiss); and CA78810 and CA82130 (Dr Altieri). Acknowledgment: We thank all the patients for their participation in the study. We also thank Bernard Lytton, MD, Harris Foster, Jr, MD, Hubert Swana, MD, Greg Barme, MD, Ithaar Derweesh, MD, and Luke Cho, MD, and Sharyn Jones, PCA, Kathy Olsen, RN, and Claire Puklin, RN, for devoting their time for sample collection and their practical perspective of patient care. 8. Monzo M, Rosell R, Felip E, et al. A novel antiapoptosis gene: re-expression of survivin messenger RNA as a prognosis marker in non-small-cell lung cancers. J Clin Oncol. 1999;17:2100-2104. 9. Kawasaki H, Altieri DC, Lu CD, Toyoda M, Tenjo T, Tanigawa N. Inhibition of apoptosis by survivin predicts shorter survival rates in colorectal cancer. Cancer Res. 1998;58:5071-5074. 10. Dawson C, Whitfield H. ABC of urology: urological malignancies, II: urothelial tumors. BMJ. 1996; 312:1090-1094. 11. Stein JP, Grossfeld GD, Ginsberg DA, et al. Prognostic markers in bladder cancer: a contemporary review of the literature. J Urol. 1998;160:645659. 12. Gazzaniga P, Gradilone A, Vercillo R, et al. Bcl2/bax mRNA expression ratio as prognostic factor in low-grade urinary bladder cancer. Int J Cancer. 1996; 69:100-104. 13. Lara PC, Perez S, Rey A, Santana C. Apoptosis in carcinoma of the bladder: relation with radiation treatment results. Int J Radiat Oncol Biol Phys. 1999;43: 1015-1019. 14. Swana HS, Grossman D, Anthony JN, Weiss RM, Altieri DC. Tumor content of the antiapoptosis molecule survivin and recurrence of bladder cancer. N Engl J Med. 1999;341:452-453. 15. Li F, Ambrosini G, Chu EY, et al. Control of apoptosis and mitotic spindle checkpoint by survivin. Nature. 1998;396:580-584. 16. Grossman D, McNiff JM, Li F, Altieri DC. Expression and targeting of the apoptosis inhibitor, survivin, in human melanoma. J Invest Dermatol. 1999; 113:1076-1081. 17. Ozen H. Bladder cancer. Curr Opin Oncol. 1998; 10:273-278. 18. Brown FM. Urine cytology: it is still the gold standard for screening? Urol Clin North Am. 2000;27:2537. 19. Ramakumar S, Bhuiyan J, Besse JA, et al. Comparison of screening methods in the detection of bladder cancer. J Urol. 1999;161:388-394. 20. Lokeshwar VB, Obek C, Pham HT, et al. Urinary hyaluronic acid and hyaluronidase: markers for bladder cancer detection and evaluation of grade. J Urol. 2000;163:348-356. be closely followed up because they may subsequently develop bladder cancer. Because of its high specificity, the urine survivin test may be useful to complement cytology and/or other diagnostics markers19,20 to better monitor bladder cancer patients and identify early recurrences or de novo neoplasms. Other potential advantages of the urine survivin test include its simplicity, suitability as a point-of-service procedure, and its cost-effectiveness, using 1-step detection with a single antibody to survivin that has now become commercially available. Analysis of a larger patient series may establish the general suitability of urine survivin detection for monitoring response to therapy and follow-up protocols in bladder cancer. REFERENCES 1. Reed JC. Dysregulation of apoptosis in cancer. J Clin Oncol. 1999;17:2941-2953. 2. Ambrosini G, Adida C, Altieri DC. A novel antiapoptosis gene, survivin, expressed in cancer and lymphoma. Nat Med. 1997;3:917-921. 3. Deveraux QL, Reed JC. IAP family proteins— suppressors of apoptosis. Genes Dev. 1999;13:239252. 4. Velculescu VE, Madden SL, Zhang L, et al. Analysis of human transcriptomes. Nat Genet. 1999;23: 387-388. 5. Adida C, Berrebi D, Peuchmaur M, Reyes-Mugica M, Altieri DC. Anti-apoptosis gene, survivin, and prognosis of neuroblastoma. Lancet. 1998;351:882-883. 6. Islam A, Kageyama H, Takada N, et al. High expression of survivin, mapped to 17q25, is significantly associated with poor prognostic factors and promotes cell survival in human neuroblastoma. Oncogene. 2000;19:617-623. 7. Tanaka K, Iwamoto S, Gon G, Nohara T, Iwamoto M, Tanigawa N. Expression of survivin and its relationship to loss of apoptosis in breast carcinomas. Clin Cancer Res. 2000;6:127-134. 328 JAMA, January 17, 2001—Vol 285, No. 3 (Reprinted) Downloaded From: http://www.jamafacial.com/ on 06/15/2014 ©2001 American Medical Association. All rights reserved.