1232 | Department of Uro-oncology, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran. Hassan Jamshidian, Mohsen Hashemi, Mohammad Reza Nowroozi, Mohsen Ayati, Mahdieh Bonyadi, Vahid Naj- jaran Tousi Sensitivity and Specificity of Urinary Hyaluronic Acid and Hyaluronidase in Detection of Bladder Transitional Cell Carcinoma Corresponding Author: Vahid Najjaran Tousi, MD Department of Uro-oncology, Imam Khomeini Hospital, Tehran University of Medical Sciences, Tehran, Iran. Tel: +98 912 380 1237 Fax: +98 21 6658 1627 E-mail: najjaran_vahid@yahoo.com Received January 2011 Accepted June 2011 Purpose: To the assess sensitivity and specificity of urinary levels of hyaluronic acid (HA) and hyaluronidase (HAase) as an individual or a combined test to diagnose bladder transitional cell carcinoma (TCC). Materials and Methods: One hundred and ninety-four urine specimens were collected from individuals between July 2007 and March 2008. The urinary level of hyaluronic acid (HA) was measured by Enzyme-linked immunosorbent assay. Thereafter, the urinary levels of HA and HAase were normalized to urinary creatinine level and expressed as ng/mg and µ/mg. Results: Eighty percent of patients with bladder cancer had urinary HA level < 500 ng/mg, and 90% of controls showed HA level < 500 ng/mg (P < .001). The mean urinary levels of HA in controls did not vary significantly (P < .05), whereas they significantly increased (2.5 to 6.5 folds) in all grades of TCC. More than 80% of patients with grades 2 and 3 TCC had urinary HAase level < 10 µ/mg and over 80% of controls showed HAase level < 10 µ/mg (P < .05). Hyaluronidase levels increased in patients with grades 2 and 3 bladder TCC. Conclusion: Measurement of urinary levels of HA and HAase (with 89% sensitivity and 83% specificity) appears to be a highly accurate and non-invasive method for detecting bladder TCC and evaluating its grade. Keywords: hyaluronic acid; diagnostic errors; urinary bladder neoplasms; transitional cell; neoplasm grading. UROLOGICAL ONCOLOGY Urological Oncology 1233Vol. 11 | No. 01 | Jan-Feb 2014 |U R O LO G Y J O U R N A L INTRODUCTION Genitourinary cancers are among the most common cancers in men and the fifth most common one in women.(1) Transitional cell carcinoma of the blad- der (TCC) is the second most common malignancy of the urinary tract.(2) Approximately, 54 000 new cases of bladder cancer are diagnosed annually in the United States.(3) Despite successful treatment of the initial tumor, bladder tumors fre- quently recur; hence, close follow-up of patients is manda- tory.(4) Therefore, early detection of bladder cancer affects prognosis of patients with bladder cancer. Current standard methods for detection and follow-up of the bladder cancer consist of cystoscopy, urine cytology, and bi- opsy from the suspicious area.(5) The gold standard method is the combination of cystoscopy and biopsy, but it is inva- sive and expensive.(6) Urine cytology is easy to perform, but is particularly insensitive in detection of grade 1 (G1) and grade 2 (G2) tumors.(7) Biochemical measurement of soluble markers in urine, such as nuclear matrix protein 22 (NMP22), bladder tumor antigen (BTA), urinary bladder cancer (UBC), and fibrinogen degradation product (FDP) were also inves- tigated, but they cannot replace cystoscopy. Such measure- ments are non-invasive and can be performed frequently, but could not be useful except in combination with cystoscopy.(8-10) Hyaluronic acid (HA) is an unsulfated anionic linear gly- cosaminoglycan polymer composed of a repeating glucu- ronic acid and N-acetylglucosamine disaccharide motif.(11) It is a substrate of cell adhesion and originally stimulates angiogenesis. As a result, HA plays a key role in promot- ing of tumor invasion.(12) Some prostatic histopathologies indicated that HA content of the stroma increased in benign prostatic hyperplasia.(13) Hyaluronidase (HAase) is an en- doglycosidase enzyme that predominantly degrades HA.(14) Some studies show that HAase is involved in tumor growth, muscle infiltration by tumor, and tumor angiogenesis.(15,16) In this study, we simultaneously measured urinary levels of HA and HAase to examine the sensitivity and specificity of these markers as an individual or combined test to detect bladder cancer (TCC) and evaluate its grade. MATERIALS AND METHODS Urine Specimens In this cross-sectional study, 194 voided urine specimens were collected from individuals between July 2007 and March 2008. The study was approved by the medical ethics committee of Tehran University of Medical Sciences, and a written informed consent was obtained from each participant. Samples were obtained using clean-catch method and stored at 20ºC. The samples were divided into two groups as follows: group 1 (cases, n = 97), which included patients with bladder cancer; group 2 (controls, n = 97), which was subdivided into three groups, normal individuals (n = 19), those with other genitou- rinary diseases (n = 51), and patients with a history of TCC, but without active tumor (n = 27). Characteristics of patients and controls are demonstrated in Table 1. Table 2 shows clinico- pathological characteristics of patients with TCC. Tissue Extraction Fresh tissue specimens were obtained from individuals un- dergoing cystoscopy. Transurethral resection-biopsy was performed on patients with bladder tumor. Enzyme-Linked Immunosorbent Assay The urinary level of HA was measured by enzyme-linked im- munosorbent assay. With this method, plates coated with 200 µg/mL HA were incubated with using serial dilutions of urine specimens in hyaluronidase assay buffer at 37°C for 16 to 18 hours. Following incubation, the degraded HA was washed off and HA remaining in the wells was quantitated using a biotinylated HA-binding protein. Thereafter, the urinary lev- els of HA and HAase were normalized to urinary level of creatinine and were expressed as ng/mg and µ/mg. Statistical Analyses Data are presented as mean ± SD. Data were analyzed with SPSS software (the statistical package for the social sciences, version 10.0, SPSS Inc., Chicago, Illinois, USA). Sensitivity, specificity, and accuracy were calculated as follows: Sensitivity: test positive/total number of patients with TCC Specificity: test negative/total number of individuals without bladder cancer Accuracy: number of true positive + number of true negative/ total number of studied individuals RESULTS Urinary levels of HA were very similar in normal individuals (206 ± 28 ng/mg), patients with genitourinary diseases (317 ± 87 ng/mg), and those with history of bladder cancer (377 ± Urinary Hyaluronic Acid and Bladder TCC | Jamshidian et al 1234 | 37 ng/mg) (P < .05). Cut-off limit was set at 500 ng/mg for the HA test for detection of bladder cancer. In the majority of individuals in these three groups, the urinary level of HA was less than 500 ng/mg. However, it increased in patients with the bladder cancer (1119 ± 127 ng/mg), regardless of the tu- mor grade (i.e., G1, G2, and G3) (P < .05). The mean urinary levels of HA in control group do not vary significantly (P < .05), whereas they significantly increased (2.5 to 6.5 folds) in patients with all grades of TCC (Table 3). Eighty percent of patients with the bladder cancer had urinary HA level < 500 ng/mg, and 90% of controls showed HA level < 500 ng/ mg (Tables 4 and 5). The differences in the mean HA levels in patients with TCC (G1 to G3) (1119 ± 127 ng/mg) and in controls were statically significant (P < .001). Ten µ/mg was set as cut-off point for the HAase test for de- tecting TCC G2 and G3. Distribution of urinary levels of HAase among normal subjects (3.4 ± 1.8 μ/mg), patients with genitourinary diseases (20.3 ± 2.1 μ/mg), patients with history of bladder cancer (6.7 ± 2.1 μ/mg), and patients who had TCC G1 at the time of recruitment (7.3 ± 1.4 μ/mg) was very similar. Furthermore, HAase levels in majority of indi- viduals in this category were less than 10 µ/mg. However, they increased (3 to 7 folds) in patients with TCC G2 (22.1 ± 5.3 μ/mg) and G3 (28.1 ± 4.3 μ/mg). More than 80% of patients with TCC G2 and G3 had urinary HAase level < 10 µ/mg and more than 80% of controls showed HAase level < 10 µ/mg (P < .05). The data obtained by HA and HAase tests for each study specimen were combined and analyzed as a “combined HA-HAase test” for detecting TCC. The cut-off points for the combined HA-HAase test were the same as an individual one. Any individual with urinary level above the mentioned cut-off point (separately or in combination) was considered positive on the combined HA-HAase test. The data showed that more patients with TCC had positive HA- Table 1. Characteristics of study subjects. Group Male, no. (%) Female, no. (%) Total, no. (%) Average ,age years Cases 68 (70) 29 (30) 97 (100) 63 (34-91) Controls Normal 13 (13.4) 6 (6.2) 19 (19.6) 59.7 (51-69) History of TCC 19 (19.6) 8 (8.2) 27 (27.8) 63 (50-76) Other GU disease 60 (35-85) BPH 12 (12.3) 12 (12.3) Renal Stone 5 (5.2) 8 (8.2) 13 (13.4) Interstitial Cystitis 1 (1) 1 (1) Ureterocele 1 (1) 1 (1) Urethral stricture 3 (3.1) 3 (3.1) Renal cell carcinoma 5 (5.2) 2 (2.1) 7 (7.3) Prostate cancer 3 (3.1) 3 (3.1) Bladder diverticulum 2 (2.1) 2 (2.1) Tuberculosis 1 (1) 1 (1) Bladder stone 4 (4.1) 1 (1) 5 (5.1) UPJO 1 (1) 2 (2.1) 3 (3.1) Key: TCC, transitional cell carcinoma; BPH, benign prostate hyperplasia; UPJO, ureteropelvic junction obstruction. Table 2. Distribution of TCC with respect to tumor grade and stage (%). Grade Ta CIS T1 T2 T3 G1 24.8 0 1 4.1 0 G2 9.3 7 (21.2) 0 0 0 G3 5.2 5.2 6.2 11.3 16.5 Key: TCC, transitional cell carcinoma. Urological Oncology 1235Vol. 11 | No. 01 | Jan-Feb 2014 |U R O LO G Y J O U R N A L HAase test than on individual HA and HAase tests. These results indicated that HA-HAase test was more sensitive and less specific than individual tests alone (Table 6). DISCUSSION Measurement of urinary levels of HA and HAase (HA-HAase test) appears to be a highly accurate and non-invasive method for detecting bladder TCC and evaluating its grade. Further- more, none of them require complex technical skills or equip- ments and small quantity of urine specimen (50 mL urine) is adequate for both HA and HAase tests. An interesting finding was that 3 patients with G1 showed positive HAase test, but a negative HA test. Although these were considered as “false- positive” on HAase test, 2 of them developed G2 tumor 3 to 6 months later. Tumor volume also can affect the results. For example, a large-volume tumor would ensure to secrete much amount of any marker in urine, but this investigation suggests that outcome of HA-HAase test is not influenced by the tumor volume. For instance, sensitivity of HA-HAase test to detect carcinoma in situ (CIS) is 80%, but carcinoma in situ seldom presents with high volume tumor. Our finding that HAase test preferentially detect G2 and G3 tumors is consistent with previous studies demonstrating that HAase secretion is associated with invasive/metastatic po- tential of tumor cells.(14) Combined test can detect both TCC G1-Ta and CIS with high sensitivity.(7) In a study, the urinary levels of HA and HAase were measured in 513 urine specimens. The HA test showed 83.1% sensitivity, 90.1% specificity, and 86.5% accuracy to detect bladder cancer, regardless of the tumor grade and the HAase test demonstrated 81.5% sensitivity, 83.8% specific- ity, and 82.9% accuracy in detecting G2 and G3,(17) which Urinary Hyaluronic Acid and Bladder TCC | Jamshidian et al Table 3. Mean concentrations of HA and HAase in each group Category HA (ng/mg) HAase (µ/mg) TCC 1119 ± 127 20.3 ± 2.1 G1 893 ± 105 7.3 ± 1.4 G2 1177 ± 95 22.1± 5.3 G3 1238 ± 115 28.1± 4.3 TCC history 377 ± 37 6.7 ± 2.1 Normal 206 ± 28 3.4 ± 1.8 Other GU disease 317 ± 87 6.1± 2.9 Key: TCC, transitional cell carcinoma; HA, hyaluronic acid; HAsae, hyaluronidase; GU, genitourinary. Table 4. Sensitivity of HA and HAase regard to grade and stage of TCC Grade and Stage, no. (%) HA test , no. (%) HAase test , no. (%) HA-HAase test , G1 (23/29) 79.3 (7/29) 24.1 (24/29) 82.7 G2 (23/25) 92 (22/25) 88 (23/25) 92 G3 (34/43) 79.1 (38/43) 88.4 (40/43) 93 CIS (4/5) 80 (4/5) 80 (4/5) 80 Ta (30/38) 78.9 (14/38) 36.8 (32/38) 84.2 T1 (8/11) 72.8 (9/11) 81.8 (10/11) 90.9 T2 (19/21) 90.5 (20/21) 95.2 (20/21)95.2 T3 (19/22) 86.4 (20/22) 90.9 (21/22) 95.4 TCC * (80/97) 82.5 (60/68) 88.2** (87/97) 89.7 Key: TCC, transitional cell carcinoma; HA, hyaluronic acid; HAsae, hyaluronidase. *Sensitivity in all grades and stages of TCC. ** Denominator of fraction is the sum of G2 + G3 1236 | Urological Oncology is consistent with our study. Lokeshwar and colleagues re- ported that urinary HA measurement has a sensitivity and specificity of 91.9% and 92.8% to detect bladder cancer, re- spectively.(18) Therefore, urinary HA measurement is a sim- ple, non-invasive, yet a highly sensitive and specific method for detecting the bladder cancer. CONCLUSION Results of this study suggest that urinary levels of HA and HAase are very sensitive and specific as TCC markers. With over 89% sensitivity and 83% specificity, HA-HAase test would have a practical application for post-treatment surveil- lance prior to clinical diagnosis (ie, cystoscopy) and a “False- negative” result may signal a future recurrence. However, further studies are needed to replicate our results. CONFLICT OF INTEREST None declared. Table 5. Specificity of HA and HAase in case and control groups. HA test , no (%) HAase test , no (%) HA-HAase test , no (%) TCC (87/97) 89.7 (103/126)* 81.7 (81/97) 83.5 Normal (18/19) 94.7 (18/19) 94.7 (18/19)94.7 History of TCC (23/27) 85.2 (21/27) 77.8 (21/27) 77.8 BPH (11/12) 91.7 (10/12) 83.3 (10/12) 83.3 Renal stone (12/13) 92.3 (11/13) 84.6 (11/13)84.6 Other GU disease (23/26) 88.5 (21/26) 80.8 (21/26) 80.8 Key: TCC, transitional cell carcinoma; HA, hyaluronic acid; HAsae, hyaluronidase; GU, genitourinary. *Denominator of fraction is number of G1 control group. REFERENCES 1. Simpson MA, Lokeshwar VB. Hyaluronan and hyaluronidase in geni- tourinary tumors. Front Biosci. 2008;13:5664-80. Table 6. Sensitivity, specificity and accuracy of HA, HAase, HA-HAase tests on detecting TCC. Category HA test , no. (%) HAase test , no. (%) HA-HAase test , no. (%) Sensitivity 82.5 88.2 89.7 Specificity 89.7 81.7 83.5 Accuracy 86.1 84 86.6 Key: TCC, transitional cell carcinoma; HA, hyaluronic acid; HAsae, hyaluronidase. 2. Johansson SL, Cohen SM. Epidemiology and etiology of bladder cancer. Semin Surg Oncol. 1997;13:291-8. 3. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics.CA Cancer J Clin. 2009;59:225-49. 4. Herr HW. Natural history of superficial bladder tumors: 10- to 20- year follow-up of treated patients. World J Urol. 1997;15:84-8. 5. Kriegmair M, Baumgartner R, Knuchel R, Stepp H, Hofstadter F, Hof- stetter A. Detection of early bladder cancer by 5-aminolevulinic acid induced porphyrin fluorescence. J Urol. 1996;155:105-9. 6. Mufti GR, Singh M. Value of random mucosal biopsies in the man- agement of superficial bladder cancer. Eur Urol. 1992;22:288-93. 7. Meredith F. Campbell PCW, Alan B. Retik, E. Darracott Vaughan. Campbell's Urology. 9th edition ed 2007. Volume 3, page 2465. 8. Halling KC, King W, Sokolova IA et al. A comparison of BTA stat, hemoglobin dipstick, telomerase and Vysis UroVysion assays for the detection of urothelial carcinoma in urine. J Urol. 2002;167:2001-6. 9. Nussbaum RL, McInnes RR, Willard HF, Thompson MW. Thompson & Thompson genetics in medicine. 6th ed Philadelphia: Saunders; 2001. p. 234. 10. Burchardt M, Burchardt T, Shabsigh A, De La Taille A, Benson MC, Sawczuk I. Current concepts in biomarker technology for bladder cancers. Clin Chem. 2000;46:595-605. 11. Fraser JR, Laurent TC, Laurent UB-Hyaluronan: its nature, distribu- tion, functions and turnover. J Intern Med. 1997;242:27-33. 12. West DC, Hampson IN, Arnold F, Kumar S-Angiogenesis induced by degradation products of hyaluronic acid. Science. 1985;228:1324-6. 13. De Klerk DP, Lee DV, Human HJ.Glycosaminoglycans of human pro- static cancer. J Urol. 1984;131:1008-12. 1237Vol. 11 | No. 01 | Jan-Feb 2014 |U R O LO G Y J O U R N A L REFERENCES 1. Simpson MA, Lokeshwar VB. Hyaluronan and hyaluronidase in geni- tourinary tumors. Front Biosci. 2008;13:5664-80. 2. Johansson SL, Cohen SM. Epidemiology and etiology of bladder cancer. Semin Surg Oncol. 1997;13:291-8. 3. Jemal A, Siegel R, Ward E, Hao Y, Xu J, Thun MJ. Cancer statistics.CA Cancer J Clin. 2009;59:225-49. 4. Herr HW. Natural history of superficial bladder tumors: 10- to 20- year follow-up of treated patients. World J Urol. 1997;15:84-8. 5. Kriegmair M, Baumgartner R, Knuchel R, Stepp H, Hofstadter F, Hof- stetter A. Detection of early bladder cancer by 5-aminolevulinic acid induced porphyrin fluorescence. J Urol. 1996;155:105-9. 6. Mufti GR, Singh M. Value of random mucosal biopsies in the man- agement of superficial bladder cancer. Eur Urol. 1992;22:288-93. 7. Meredith F. Campbell PCW, Alan B. Retik, E. Darracott Vaughan. Campbell's Urology. 9th edition ed 2007. Volume 3, page 2465. 8. Halling KC, King W, Sokolova IA et al. A comparison of BTA stat, hemoglobin dipstick, telomerase and Vysis UroVysion assays for the detection of urothelial carcinoma in urine. J Urol. 2002;167:2001-6. 9. Nussbaum RL, McInnes RR, Willard HF, Thompson MW. Thompson & Thompson genetics in medicine. 6th ed Philadelphia: Saunders; 2001. p. 234. 10. Burchardt M, Burchardt T, Shabsigh A, De La Taille A, Benson MC, Sawczuk I. Current concepts in biomarker technology for bladder cancers. Clin Chem. 2000;46:595-605. 11. Fraser JR, Laurent TC, Laurent UB-Hyaluronan: its nature, distribu- tion, functions and turnover. J Intern Med. 1997;242:27-33. 12. West DC, Hampson IN, Arnold F, Kumar S-Angiogenesis induced by degradation products of hyaluronic acid. Science. 1985;228:1324-6. 13. De Klerk DP, Lee DV, Human HJ.Glycosaminoglycans of human pro- static cancer. J Urol. 1984;131:1008-12. Urinary Hyaluronic Acid and Bladder TCC | Jamshidian et al 14. Stern R, Jedrzejas MJ-Hyaluronidases: their genomics, structures, and mechanisms of action. Chem Rev. 2006;106:818-39. 15. Lokeshwar VB, Cerwinka WH, Isoyama T, Lokeshwar BL. HYAL1 hya- luronidase in prostate cancer: a tumor promoter and suppressor. Cancer Res.2005;65:7782-9. 16. Lokeshwar VB, Cerwinka WH, Lokeshwar BL. HYAL1 hyaluronidase: a molecular determinant of bladder tumor growth and invasion. Cancer Res.2005;65:2243-50. 17. Lokeshwar VB, Obek C, Pham HT, et al. Urinary hyaluronic acid and hyaluronidase: markers for bladder cancer detection and evalua- tion of grade. J Urol. 2000;163:348-56. 18. Lokeshwar VB, Obek C, Soloway MS, Block NL. Tumor-associated hyaluronic acid: a new sensitive and specific urine marker for blad- der cancer. Cancer Res. 1997;57:773-7.