Urinary biomarkers of non-muscle-invasive bladder cancer: Current status and future potential

Expert Review of Anticancer Therapy

Volumn 12, Issue 6, 2012, Pages 743-752

  Calistri, Daniele ^a   Casadio, Valentina ^a   Bravaccini, Sara ^a   Zoli, Wainer ^a   Amadori, Dino ^a  

^a DEPARTMENT OF MEDICAL ONCOLOGY   (Italy)

Author keywords

early diagnosis; epigenetic; FDA marker; FISH; gene mutation; immunoassay; molecular marker; noninvasive bladder tumor; surveillance; urine

Indexed keywords

AURORA A KINASE; BIOLOGICAL MARKER; CARCINOEMBRYONIC ANTIGEN RELATED CELL ADHESION MOLECULE 1; CELL DNA; CYTOKERATIN; CYTOKERATIN 20; FAS LIGAND; FIBROBLAST GROWTH FACTOR RECEPTOR 3; GELATINASE A; GELATINASE B; HYALURONIC ACID; HYALURONIDASE; MESSENGER RNA; MICRORNA; MICRORNA 126; MICRORNA 182; MICRORNA 183; MICRORNA 96; NUCLEAR MATRIX PROTEIN 22; PROTEIN BLCA 1; PROTEIN BLCA 4; SIALYL LEWIS X ANTIGEN; SURVIVIN; TELOMERASE; TELOMERASE REVERSE TRANSCRIPTASE; TISSUE INHIBITOR OF METALLOPROTEINASE 2; TUMOR ANTIGEN; TUMOR MARKER; UNCLASSIFIED DRUG;

CANCER CYTODIAGNOSIS; CANCER GRADING; CANCER INVASION; CANCER STAGING; CELL FREE SYSTEM; CYSTOSCOPY; CYTOKERATIN 20 GENE; DIAGNOSTIC ACCURACY; EARLY DIAGNOSIS; EPIGENETICS; EVIDENCE BASED MEDICINE; FIBROBLAST GROWTH FACTOR RECEPTOR 3 GENE; FLUORESCENCE IN SITU HYBRIDIZATION; GENE EXPRESSION REGULATION; GENE MUTATION; HUMAN; HYALURONIC ACID GENE; IMMUNOASSAY; MARKER GENE; MICROSATELLITE MARKER; NON INVASIVE MEASUREMENT; NON MUSCLE INVASIVE BLADDER CANCER; PATIENT MONITORING; PREDICTION; RELIABILITY; REVIEW; SENSITIVITY AND SPECIFICITY; TELOMERASE REVERSE TRANSCRIPTASE GENE; TUMOR VASCULARIZATION; URINALYSIS;

HUMANS; NEOPLASM INVASIVENESS; TUMOR MARKERS, BIOLOGICAL; URINARY BLADDER NEOPLASMS;

EID: 84862862840     PISSN: 14737140     EISSN: 17448328     Source Type: Journal    
DOI: 10.1586/era.12.50     Document Type: Review

References (98)

1. Williams SG, Stein JP. Molecular pathways in bladder cancer. Urol. Res. 32(6), 373-385 (2004).
2. Sciarra A, De Matteis A, Mariotti G, Voria G, Lucera R, Di Silverio F. Histopathological aspects of transitional cell carcinoma of the bladder: analysis of 20 years experience. Int. J. Urol. 11(7), 467-475 (2004).
3. Denzinger S, Burger M, Walter B et al. Clinically relevant reduction in risk of recurrence of superficial bladder cancer using 5-aminolevulinic acid-induced fluorescence diagnosis: 8-year results of prospective randomized study. Urology 69(4), 675-679 (2007).
4. Zaak D, Kriegmair M, Stepp H et al. Endoscopic detection of transitional cell carcinoma with 5-aminolevulinic acid: results of 1012 fluorescence endoscopies. Urology 57(4), 690-694 (2001).
5. Adachi J, Kumar C, Zhang Y, Olsen JV, Mann M. The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins. Genome Biol. 7(9), R80 (2006).
6. Coon JJ, Zürbig P, Dakna M et al. CE-MS analysis of the human urinary proteome for biomarker discovery and disease diagnostics. Proteomics Clin. Appl. 2(7-8), 964 (2008).
7. Bastacky S, Ibrahim S, Wilczynski SP, Murphy WM. The accuracy of urinary cytology in daily practice. Cancer 87(3), 118-128 (1999).
8. van Rhijn BW, van der Poel HG, van der Kwast TH. Urine markers for bladder cancer surveillance: a systematic review. Eur. Urol. 47(6), 736-748 (2005).
9. Lotan Y, Shariat SF, Schmitz-Dräger BJ et al. Considerations on implementing diagnostic markers into clinical decision making in bladder cancer. Urol. Oncol. 28(4), 441-448 (2010).
10. Tilki D, Burger M, Dalbagni G et al. Urine markers for detection and surveillance of non-muscle-invasive bladder cancer. Eur. Urol. 60(3), 484-492 (2011).
11. Babjuk M, Oosterlinck W, Sylvester R et al.; European Association of Urology (EAU). EAU guidelines on non-muscle-invasive urothelial carcinoma of the bladder, the 2011 update. Eur. Urol. 59(6), 997-1008 (2011).
12. Lotan Y, Svatek RS, Malats N. Screening for bladder cancer: a perspective. World J. Urol. 26(1), 13-18 (2008).
13. Messing EM, Young TB, Hunt VB et al. Comparison of bladder cancer outcome in men undergoing hematuria home screening vs. those with standard clinical presentations. Urology 45(3), 387-396 (1995).
14. Madeb R, Messing EM. Long-term outcome of home dipstick testing for hematuria. World J. Urol. 26(1), 19-24 (2008).
15. Abogunrin F, O'Kane HF, Ruddock MW et al. The impact of biomarkers in multivariate algorithms for bladder cancer diagnosis in patients with hematuria. Cancer 118(10), 2641-2650 (2012).
16. Roobol MJ, Bangma CH, el Bouazzaoui S, Franken-Raab CG, Zwarthoff EC. Feasibility study of screening for bladder cancer with urinary molecular markers (the BLU-P project). Urol. Oncol. 28(6), 686-690 (2010).
17. Raitanen MP; FinnBladder Group. The role of BTA stat test in follow-up of patients with bladder cancer: results from FinnBladder studies. World J. Urol. 26(1), 45-50 (2008).
18. Sarosdy MF, Hudson MA, Ellis WJ et al. Improved detection of recurrent bladder cancer using the Bard BTA stat test. Urology 50(3), 349-353 (1997).
19. Saad A, Hanbury DC, McNicholas TA, Boustead GB, Morgan S, Woodman AC. A study comparing various noninvasive methods of detecting bladder cancer in urine. BJU Int. 89(4), 369-373 (2002).
20. Grossman HB, Messing E, Soloway M et al. Detection of bladder cancer using a point-of-care proteomic assay. JAMA 293(7), 810-816 (2005).
21. Grossman HB, Soloway M, Messing E et al. Surveillance for recurrent bladder cancer using a point-of-care proteomic assay. JAMA 295(3), 299-305 (2006).
22. Atsü N, Ekici S, Oge OO, Ergen A, Hasçelik G, Ozen H. False-positive results of the NMP22 test due to hematuria. J. Urol. 167(2 Pt 1), 555-558 (2002).
23. Lotan Y, Elias K, Svatek RS et al. Bladder cancer screening in a high risk asymptomatic population using a point of care urine based protein tumor marker. J. Urol. 182(1), 52-57 (2009).
24. Barbieri CE, Cha EK, Chromecki TF et al. Decision curve analysis assessing the clinical benefit of NMP22 in the detection of bladder cancer: secondary analysis of a prospective trial. BJU Int. 109(5), 685-690 (2012).
25. Shariat SF, Savage C, Chromecki TF et al. Assessing the clinical benefit of nuclear matrix protein 22 in the surveillance of patients with nonmuscle-invasive bladder cancer and negative cytology: a decision-curve analysis. Cancer 117(13), 2892-2897 (2011).
26. Mian C, Pycha A, Wiener H, Haitel A, Lodde M, Marberger M. Immunocyt: a new tool for detecting transitional cell cancer of the urinary tract. J. Urol. 161(5), 1486-1489 (1999).
27. Vriesema JL, Atsma F, Kiemeney LA, Peelen WP, Witjes JA, Schalken JA. Diagnostic efficacy of the ImmunoCyt test to detect superficial bladder cancer recurrence. Urology 58(3), 367-371 (2001).
28. Olsson H, Zackrisson B. ImmunoCyt a useful method in the follow-up protocol for patients with urinary bladder carcinoma. Scand. J. Urol. Nephrol. 35(4), 280-282 (2001).
29. Lodde M, Mian C, Negri G et al. Role of uCyt+ in the detection and surveillance of urothelial carcinoma. Urology 61(1), 243-247 (2003).
30. Halling KC. Vysis UroVysion for the detection of urothelial carcinoma. Expert Rev. Mol. Diagn. 3(4), 507-519 (2003).
31. Skacel M, Fahmy M, Brainard JA et al. Multitarget fluorescence in situ hybridization assay detects transitional cell carcinoma in the majority of patients with bladder cancer and atypical or negative urine cytology. J. Urol. 169(6), 2101-2105 (2003).
32. Gofrit ON, Zorn KC, Silvestre J et al. The predictive value of multi-targeted fluorescent in-situ hybridization in patients with history of bladder cancer. Urol. Oncol. 26(3), 246-249 (2008).
33. Sarosdy MF, Schellhammer P, Bokinsky G et al. Clinical evaluation of a multi-target fluorescent in situ hybridization assay for detection of bladder cancer. J. Urol. 168(5), 1950-1954 (2002).
34. Bravaccini S, Casadio V, Gunelli R et al. Combining cytology, TRAP assay, and FISH analysis for the detection of bladder cancer in symptomatic patients. Ann. Oncol. 22(10), 2294-2298 (2011).
35. Konety BR, Nguyen TS, Dhir R et al. Detection of bladder cancer using a novel nuclear matrix protein, BLCA-4. Clin. Cancer Res. 6(7), 2618-2625 (2000).
36. Van Le TS, Myers J, Konety BR, Barder T, Getzenberg RH. Functional characterization of the bladder cancer marker, BLCA-4. Clin. Cancer Res. 10(4), 1384-1391 (2004).
37. Van Le TS, Miller R, Barder T, Babjuk M, Potter DM, Getzenberg RH. Highly specific urine-based marker of bladder cancer. Urology 66(6), 1256-1260 (2005).
38. Zuiverloon TC, van der Aa MN, van der Kwast TH et al. Fibroblast growth factor receptor 3 mutation analysis on voided urine for surveillance of patients with low-grade non-muscle-invasive bladder cancer. Clin. Cancer Res. 16(11), 3011-3018 (2010).
39. van Oers JM, Lurkin I, van Exsel AJ et al. A simple and fast method for the simultaneous detection of nine fibroblast growth factor receptor 3 mutations in bladder cancer and voided urine. Clin. Cancer Res. 11(21), 7743-7748 (2005).
40. Zuiverloon TC, Tjin SS, Busstra M, Bangma CH, Boevé ER, Zwarthoff EC Optimization of nonmuscle invasive bladder cancer recurrence detection using a urine based FGFR3 mutation assay. J. Urol. 186(2), 707-712 (2011).
41. Miyake M, Sugano K, Sugino H et al. Fibroblast growth factor receptor 3 mutation in voided urine is a useful diagnostic marker and significant indicator of tumor recurrence in non-muscle invasive bladder cancer. Cancer Sci. 101(1), 250-258 (2010).
42. Serizawa RR, Ralfkiaer U, Steven K et al. Integrated genetic and epigenetic analysis of bladder cancer reveals an additive diagnostic value of FGFR3 mutations and hypermethylation events. Int. J. Cancer 129(1), 78-87 (2011).
43. Knudson W. Tumor-associated hyaluronan. Providing an extracellular matrix that facilitates invasion. Am. J. Pathol. 148(6), 1721-1726 (1996).
44. Lokeshwar VB, Obek C, Soloway MS, Block NL. Tumor-associated hyaluronic acid: a new sensitive and specific urine marker for bladder cancer. Cancer Res. 57(4), 773-777 (1997).
45. Pham HT, Block NL, Lokeshwar VB. Tumor-derived hyaluronidase: a diagnostic urine marker for high-grade bladder cancer. Cancer Res. 57(4), 778-783 (1997).
46. Hautmann SH, Lokeshwar VB, Schroeder GL et al. Elevated tissue expression of hyaluronic acid and hyaluronidase validates the HA-HAase urine test for bladder cancer. J. Urol. 165(6 Pt 1), 2068-2074 (2001).
47. Hautmann S, Toma M, Lorenzo Gomez MF et al. Immunocyt and the HA-HAase urine tests for the detection of bladder cancer: a side-by-side comparison. Eur. Urol. 46(4), 466-471 (2004).
48. Eissa S, Kassim SK, Labib RA et al. Detection of bladder carcinoma by combined testing of urine for hyaluronidase and cytokeratin 20 RNAs. Cancer 103(7), 1356-1362 (2005).
49. Saad A, Hanbury DC, McNicholas TA, Boustead GB, Woodman AC. The early detection and diagnosis of bladder cancer: a critical review of the options. Eur. Urol. 39(6), 619-633 (2001).
50. Golijanin D, Sherman Y, Shapiro A, Pode D. Detection of bladder tumors by immunostaining of the Lewis X antigen in cells from voided urine. Urology 46(2), 173-177 (1995).
51. Pode D, Golijanin D, Sherman Y, Lebensart P, Shapiro A. Immunostaining of Lewis X in cells from voided urine, cytopathology and ultrasound for noninvasive detection of bladder tumors. J. Urol. 159(2), 389-392 (1998).
52. von Knobloch R, Hegele A, Brandt H, Olbert P, Heidenreich A, Hofmann R. Serum DNA and urine DNA alterations of urinary transitional cell bladder carcinoma detected by fluorescent microsatellite analysis. Int. J. Cancer 94(1), 67-72 (2001).
53. van Rhijn BW, Lurkin I, Kirkels WJ, van der Kwast TH, Zwarthoff EC. Microsatellite analysis - DNA test in urine competes with cystoscopy in follow-up of superficial bladder carcinoma: a Phase II trial. Cancer 92(4), 768-775 (2001).
54. Bartoletti R, Cai T, Dal Canto M, Boddi V, Nesi G, Piazzini M. Multiplex polymerase chain reaction for microsatellite analysis of urine sediment cells: a rapid and inexpensive method for diagnosing and monitoring superficial transitional bladder cell carcinoma. J. Urol. 175(6), 2032-2037 (2006).
55. van der Aa MN, Zwarthoff EC, Steyerberg EW et al. Microsatellite analysis of voided-urine samples for surveillance of low-grade non-muscle-invasive urothelial carcinoma: feasibility and clinical utility in a prospective multicenter study (Cost- Effectiveness of Follow-Up of Urinary Bladder Cancer Trial [CEFUB]). Eur. Urol. 55(3), 659-668 (2009).
56. de Bekker-Grob EW, van der Aa MN, Zwarthoff EC et al. Non-muscle-invasive bladder cancer surveillance for which cystoscopy is partly replaced by microsatellite analysis of urine: a costeffective alternative? BJU Int. 104(1), 41-47 (2009).
57. Grégoire M, Fradet Y, Meyer F et al. Diagnostic accuracy of urinary cytology, and deoxyribonucleic acid flow cytometry and cytology on bladder washings during follow-up for bladder tumors. J. Urol. 157(5), 1660-1664 (1997).
58. Moonen PM, Merkx GF, Peelen P, Karthaus HF, Smeets DF, Witjes JA. UroVysion compared with cytology and quantitative cytology in the surveillance of non-muscle-invasive bladder cancer. Eur. Urol. 51(5), 1275-1280 (2007).
59. Shariat SF, Casella R, Khoddami SM et al. Urine detection of survivin is a sensitive marker for the noninvasive diagnosis of bladder cancer. J. Urol. 171(2 Pt 1), 626-630 (2004).
60. Wang H, Xi X, Kong X, Huang G, Ge G. The expression and significance of survivin mRNA in urinary bladder carcinomas. J. Cancer Res. Clin. Oncol. 130(8), 487-490 (2004).
61. Weikert S, Christoph F, Schrader M, Krause H, Miller K, Müller M. Quantitative analysis of survivin mRNA expression in urine and tumor tissue of bladder cancer patients and its potential relevance for disease detection and prognosis. Int. J. Cancer 116(1), 100-104 (2005).
62. Kenney DM, Geschwindt RD, Kary MR, Linic JM, Sardesai NY, Li ZQ. Detection of newly diagnosed bladder cancer, bladder cancer recurrence and bladder cancer in patients with hematuria using quantitative rt-PCR of urinary survivin. Tumour Biol. 28(2), 57-62 (2007).
63. Horstmann M, Bontrup H, Hennenlotter J et al. Clinical experience with survivin as a biomarker for urothelial bladder cancer. World J. Urol. 28(3), 399-404 (2010).
64. Eissa S, Swellam M, Shehata H, El-Khouly IM, El-Zayat T, El-Ahmady O. Expression of HYAL1 and survivin RNA as diagnostic molecular markers for bladder cancer. J. Urol. 183(2), 493-498 (2010).
65. Kim NW, Piatyszek MA, Prowse KR et al. Specific association of human telomerase activity with immortal cells and cancer. Science 266(5193), 2011-2015 (1994).
66. Bravaccini S, Casadio V, Amadori D, Calistri D, Silvestrini R. The current role of telomerase in the diagnosis of bladder cancer. Indian J. Urol. 25(1), 40-46 (2009).
67. Sanchini MA, Bravaccini S, Medri L et al. Urine telomerase: an important marker in the diagnosis of bladder cancer. Neoplasia 6(3), 234-239 (2004).
68. Sanchini MA, Gunelli R, Nanni O et al. Relevance of urine telomerase in the diagnosis of bladder cancer. JAMA 294(16), 2052-2056 (2005).
69. Casadio V, Bravaccini S, Gunelli R et al. Accuracy of urine telomerase activity to detect bladder cancer in symptomatic patients. Int. J. Biol. Markers 24(4), 253-257 (2009).
70. Bravaccini S, Sanchini MA, Granato AM et al. Urine telomerase activity for the detection of bladder cancer in females. J. Urol. 178(1), 57-61 (2007).
71. Eissa S, Swellam M, Ali-Labib R, Mansour A, El-Malt O, Tash FM. Detection of telomerase in urine by 3 methods: evaluation of diagnostic accuracy for bladder cancer. J. Urol. 178(3 Pt 1), 1068-1072 (2007).
72. 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. 167(5), 2001-2006 (2002).
73. Wu XX, Kakehi Y, Nishiyama H, Habuchi T, Ogawa O. Telomerase activity in urine after transurethral resection is not a predictive marker for recurrence of superficial bladder cancer. Int. J Urol. 10(2), 117-118 (2003).
74. de Kok JB, van Balken MR, Ruers TJ, Swinkels DW, Klein Gunnewiek JM. Detection of telomerase activity in urine as a tool for noninvasive detection of recurrent bladder tumors is poor and cannot be improved by timing of sampling. Clin. Chem. 46(12), 2014-2015 (2000).
75. Volpi A, Bravaccini S, Medri L, Cerasoli S, Gaudio M, Amadori D. Usefulness of immunological detection of the human telomerase reverse transcriptase. Cell. Oncol. 27(5-6), 347-353 (2005).
76. Khalbuss W, Goodison S. Immunohistochemical detection of hTERT in urothelial lesions: a potential adjunct to urine cytology. Cytojournal 3, 18 (2006).
77. Kameshima H, Yagihashi A, Yajima T, Kobayashi D, Hirata K, Watanabe N. Expression of telomerase-associated genes: reflection of telomerase activity in gastric cancer? World J. Surg. 25(3), 285-289 (2001).
78. Heicappell R, Schostak M, Müller M, Miller K. Evaluation of urinary bladder cancer antigen as a marker for diagnosis of transitional cell carcinoma of the urinary bladder. Scand. J. Clin. Lab. Invest. 60(4), 275-282 (2000).
79. Mian C, Lodde M, Haitel A, Egarter Vigl E, Marberger M, Pycha A. Comparison of two qualitative assays, the UBC rapid test and the BTA stat test, in the diagnosis of urothelial cell carcinoma of the bladder. Urology 56(2), 228-231 (2000).
80. May M, Hakenberg OW, Gunia S et al. Comparative diagnostic value of urine cytology, UBC-ELISA, and fluorescence in situ hybridization for detection of transitional cell carcinoma of urinary bladder in routine clinical practice. Urology 70(3), 449-453 (2007).
81. Babjuk M, Soukup V, Pesl M et al. Urinary cytology and quantitative BTA and UBC tests in surveillance of patients with pTapT1 bladder urothelial carcinoma. Urology 71(4), 718-722 (2008).
82. Eissa S, Swellam M, Amin A, Balbaa ME, Yacout GA, El-Zayat TM. The clinical relevance of urine-based markers for diagnosis of bladder cancer. Med. Oncol. 28(2), 513-518 (2011).
83. Eissa S, Ali-Labib R, Swellam M, Bassiony M, Tash F, El-Zayat TM. Noninvasive diagnosis of bladder cancer by detection of matrix metalloproteinases (MMP-2 and MMP-9) and their inhibitor (TIMP-2) in urine. Eur. Urol. 52(5), 1388-1397 (2007).
84. Eissa S, Swellam M, Ali Labib R, El-Zayat TM, El Ahmady O. A panel of angiogenic factors for early bladder cancer detection: enzyme immunoassay and western blot. J. Urol. 181(3), 1353-1360 (2009).
85. Park HS, Park WS, Bondaruk J et al. Quantitation of Aurora kinase A gene copy number in urine sediments and bladder cancer detection. J. Natl. Cancer Inst. 100(19), 1401-1411 (2008).
86. Tilki D, Singer BB, Shariat SF et al. CEACAM1: a novel urinary marker for bladder cancer detection. Eur. Urol. 57(4), 648-654 (2010).
87. Szarvas T, Kovalszky I, Bedi K et al. Deletion analysis of tumor and urinary DNA to detect bladder cancer: urine supernatant versus urine sediment. Oncol. Rep. 18(2), 405-409 (2007).
88. Chang HW, Tsui KH, Shen LC, Huang HW, Wang SN, Chang PL. Urinary cell-free DNA as a potential tumor marker for bladder cancer. Int. J. Biol. Markers 22(4), 287-294 (2007).
89. Zancan M, Galdi F, Di Tonno F et al. Evaluation of cell-free DNA in urine as a marker for bladder cancer diagnosis. Int. J. Biol. Markers 24(3), 147-155 (2009).
90. Chung W, Bondaruk J, Jelinek J et al. Detection of bladder cancer using novel DNA methylation biomarkers in urine sediments. Cancer Epidemiol. Biomarkers Prev. 20(7), 1483-1491 (2011).
91. Reinert T, Modin C, Castano FM et al. Comprehensive genome methylation analysis in bladder cancer: identification and validation of novel methylated genes and application of these as urinary tumor markers. Clin. Cancer Res. 17(17), 5582-5592 (2011).
92. Eissa S, Swellam M, El-Khouly IM et al. Aberrant methylation of RARbeta2 and APC genes in voided urine as molecular markers for early detection of bilharzial and nonbilharzial bladder cancer. Cancer Epidemiol. Biomarkers Prev. 20(8), 1657-1664 (2011).
93. Weber JA, Baxter DH, Zhang S et al. The microRNA spectrum in 12 body fluids. Clin. Chem. 56(11), 1733-1741 (2010).
94. Hanke M, Hoefig K, Merz H et al. A robust methodology to study urine microRNA as tumor marker: microRNA-126 and microRNA-182 are related to urinary bladder cancer. Urol. Oncol. 28(6), 655-661 (2010).
95. Yamada Y, Enokida H, Kojima S et al. MiR-96 and miR-183 detection in urine serve as potential tumor markers of urothelial carcinoma: correlation with stage and grade, and comparison with urinary cytology. Cancer Science 102(3), 522-529 (2010).
96. Svatek RS, Herman MP, Lotan Y et al. Soluble Fas - a promising novel urinary marker for the detection of recurrent superficial bladder cancer. Cancer 106(8), 1701-1707 (2006).
97. Shariat SF, Karam JA, Lerner SP. Molecular markers in bladder cancer. Curr. Opin. Urol. 18(1), 1-8 (2008).
98. Schrag D, Hsieh LJ, Rabbani F, Bach PB, Herr H, Begg CB. Adherence to surveillance among patients with superficial bladder cancer. J. Natl Cancer Inst. 95(8), 588-597 (2003).