1. 1. UROTHELIAL CARCINOMA: URINARY BIO-MARKERS Gaurav Nahar DNB Urology (Std.) MMHRC, Madurai
2. 2. INTRODUCTION Urothelial carcinoma: MC malignancy of urinary tract and 2nd MC cause of death among genitourinary tumors. Gross, painless hematuria- primary symptom in 85% of patients with a newly diagnosed bladder tumor, and microscopic hematuria occurs in virtually all patients.
3. 3. DIAGNOSIS Full hematuria evaluation for bladder cancer includes cystoscopy, urine cytology, upper-tract imaging (CT scan of abdomen & pelvis), and a prostate-specific antigen (PSA) blood test. A PSA blood test is recommended, because 10% of patients with recurrent gross hematuria will have prostate cancer. AUA guidelines for microscopic hematuria evaluation include a cystoscopy, upper tract imaging, and urine cytology.
4. 4. DIAGNOSIS Microscopic hematuria requires a full evaluation, but low-risk patients do not require repeat evaluations. High-risk individuals primarily are those with a smoking history and should be evaluated every 6 months.
5. 5. DIAGNOSIS Main diagnostic test for bladder cancer is cystoscopy and biopsy. White Light Cystoscopy(rigid/flexible)- Gold standard. Blue light Cystoscopy- using porphyrin(5-hexa ALA) dye. Narrow Band Imaging(NBI). Random bladder biopsies.
6. 6. Urine cytology. Urine markers.
7. 7. URINE CYTOLOGY Voided urine cytology is the standard noninvasive method for diagnosis in the detection of bladder carcinoma. Microscopic examination of voided urine or bladder- washing specimens(barbotage) for exfoliated cancer cells. Cytology is used to assess morphologic changes in intact cells- cellular clumping, a high nuclear-to- cytoplasmic ratio, nucleoli, and atypia.
8. 8. URINE CYTOLOGY At least 100 mL of a freshly voided specimen required. First morning sample should not be used because of frequent presence of cytolysis. Bladder wash cytology yields more tumor cells in the sample and is more sensitive in identifying cancer, especially for high-grade tumors, but it also yields a higher false-positive rate than voided urine cytology.
9. 9. URINE CYTOLOGY High sensitivity in high-grade tumours but low sensitivity in low-grade tumours. Useful for detection of CIS, where its sensitivity is 28- 100%. Useful as an adjunct to cystoscopy, when a high-grade malignancy or CIS is present. Positive voided urinary cytology can indicate an urothelial tumour anywhere in the urinary tract, from the calyx to the ureters, bladder, and proximal urethra. Negative cytology does not exclude the presence of a tumour in the urinary tract.
10. 10. URINE CYTOLOGY Evaluation can be hampered by low cellular yield, UTIs, stones, or intravesical instillations, but in experienced hands, specificity exceeds 90%. Positive cytology + negative cystoscopy, it is necessary to exclude a tumour in the upper tract (CT-urography), CIS in the bladder (random biopsies or photodynamic diagnosis (PDD) targeted biopsies) and tumour in prostatic urethra (prostatic urethral biopsy).
11. 11. URINARY MOLECULAR MARKERS Characteristics of a good urinary bladder cancer marker: Technically simple(a point-of-care test, with readily available results, easy to perform); Low cost; Good reliability and reproducibility; high specificity( to avoid unnecessary work-up because of false-positive results) & high sensitivity (to avoid the risk of missing a tumour); Ability to detect high-risk urothelial cancer before it escapes curative treatment.
12. 12. POTENTIAL APPLICATIONS 1. Screening of high-risk population for bladder cancer; not recommended routinely. 2. Exploration of patients after haematuria or other symptoms suggestive of BC (primary detection). 3. Surveillance of NMIBC(non muscle-invasive bladder cancer).
13. 13. More than 30 urinary biomarkers reported for use in bladder cancer diagnosis. Only a few are commercially available; remainder are still being tested. Sensitivity higher and specificity lower compared to urine cytology. Benign conditions and BCG influence many urinary marker tests.
14. 14. URINE BIOMARKERS Bladder tumor antigen (BTA) assays- BTA stat & BTA TRAK Nuclear matrix protein-22 NMP 52 BLCA-4 and BLCA-1 UroVysion (FISH) Cytokeratins(CK 20 & CYFRA 21- 1) HA and HAase Telomerase Survivin Quanticyt nuclear karyometry Fibrin degradation products Microsatellite analysis Immunocytology (ImmunoCyt/uCyt+) CertNDx/FGFR3 CxBladder DD23 monoclonal antibody Lewis X antigen Automated image cytometry Aurora kinase A Carcinoembryonic antigen- related cell adhesion molecule (CEACAM1)
15. 15. NUCLEAR MATRIX PROTEIN-22 (NMP-22)
16. 16. NMP-22 a nonchromatin nuclear matrix protein used to form cell nuclei; supports nuclear shape and organizes DNA. also takes part in DNA replication, transcription, & RNA processing. released from the nuclei of tumor cells after they die and can be detected in the urine.
17. 17. NMP-22 NMP-22 is shed into urine and has a 20-times higher concentration in urine of bladder cancer patients than in noncancer controls. A cut-off of 10 units/mL is used to identify patients with or without cancer. A lower cutoff level of 5 units/mL improves sensitivity but significantly worsens specificity. Cutoff level not related to stage or grade of disease. False positives: active UTI or significant hematuria.
18. 18. Using a cutoff level of 10 units/mL, overall sensitivity and specificity- 49% and 87%, respectively. The sensitivity for Ta, T1, and T2 tumors was 36%, 65%, and 88%, respectively.
19. 19. NMP-22 BladderChek test (Alere; Waltham, Mass) is an in vitro immunoassay for qualitative detection of NMP-22 in urine. Does not depend on intact cells and does not require expert analysis or laboratory time. Provides an absolute positive or negative test result, similar to a pregnancy test. Painless and noninvasive assay. Provides results within 30 minutes (thus allowing performance during an office visit). Cost is less than half that of cytology.
20. 20. When combined with cystoscopy, NMP-22 test improves the detection of recurrence in patients with a history of bladder cancer. Combination of the NMP-22 test with cystoscopy increases overall sensitivity to 99%(from initial 91% with cystoscopy alone) Significantly more sensitive than urine cytology.
21. 21. BLADDER TUMOR ANTIGEN(BTA) -STAT & TRAK
22. 22. BLADDER TUMOR ANTIGEN(BTA)-STAT & TRAK Uses monoclonal antibodies to detect complement factor H-related protein and complement factor H in voided urine specimens. These factors are found in bladder cancer cell lines and inhibit the complement cascade to prevent cell lysis.
23. 23. BLADDER TUMOR ANTIGEN(BTA)
24. 24. BLADDER TUMOR ANTIGEN(BTA) BTA STAT is a point of care qualitative assay with an average sensitivity and specificity of 68.7% (53-89%) and 73.7% (54-93%), respectively. BTA TRAK is a quantitative enzyme-linked immunosorbent assay with similar sensitivity and specificity of 62% (17-78%) and 73.6% (51-95%), respectively.
25. 25. Specificity of both tests can be significantly decreased, as false positives have been noted to occur in the setting of hematuria, urolithiasis, inflammation, recent instrumentation, other genitourinary malignancies, and intravesical BCG therapy.
26. 26. IMMUNOCYTOLOGY (Immunocyt/uCyt+) An immunohistochemical test with 3 fluorescent monoclonal antibodies directed at mucin-based urothelial cell antigens found on exfoliated cells. Two antibodies, LDQ10 and M344, are directed against mucins, specifically glycoproteins found on epithelial cell surfaces in malignancy, and labeled with fluorescein. The other antibody is labeled Texas red and directed against a high molecular weight glycosylated form of carcinoembryonic antigen CEA 19A211. M344 and CEA 19A211 expressed in 71% and 90% of Ta-T1 tumors, respectively.
27. 27. Immunocyt/uCyt+ Use of Immunocyt/uCyt+ improves sensitivity at a minimum of 15% over cytology alone. Improved sensitivity in low-grade tumors; improves further with combination of cytology and Immunocyt/uCyt+; Specificity is slightly lower than that of cytology.
28. 28. Positive
29. 29. Immunocyt/uCyt+ LIMITATIONS: Requires processing in laboratories with properly trained personnel. Requires a minimum of 500 negative cells on the slide in order for the sample to be deemed negative. As is common with other protein-based assays, false positives are common in UTIs, urolithiasis & BPH.
30. 30. FGFR3/CertNDx
31. 31. FGFR3/CertNDx Fibroblast growth factor receptor 3 (FGFR3) belongs to a family of tyrosine kinase receptors; encoded by the FGFR3 gene. Specific point mutations in various domains result in constitutive activation of the receptor; found in approximately 50% of urothelial carcinomas. Frequency of these mutations is high in low-grade pTa tumors and low in pT1-4 tumors. Presence of the FGFR3 mutation is a selective marker for favorable disease, with a low recurrence rate and improvement in disease-specific survival.
32. 32. FGFR3/CertNDx CertNDx- a multianalyte diagnostic assay used for the evaluation of hematuria and monitoring bladder cancer recurrence. Analyze urine for presence of mutant FGFR3, quantified matrix metalloproteinase 2 (MMP-2), and hypermethylation of TWIST1 and NID2. This allows for the presence of 2 biomarker cutoff values. Unlike other assays, this is not affected by the degree of hematuria or presence of other urinary tract diseases.
33. 33. CxBladder
34. 34. CxBladder CxBladder (PacificEdge, Dunedin, New Zealand) is a recently released urine-based assay consisting of 5 mRNA markers, CDC2, HOXA13, MDK, IGFBP5, and CXCR5. sensitivity superior to that of NMP-22 and cytology, at 83%, with a specificity of 85%. Interestingly, specificity for high-grade tumors was 97% while the specificity for low-grade tumors was 69%. This offers a potential adjunct to cystoscopy for the diagnosis of urothelial carcinoma.
35. 35. LEWIS BLOOD GROUP X ANTIGEN Lewis blood group antigen X is usually absent from urothelial cells in adults except for occasional umbrella cells. There is increased Lewis X expression in bladder cancers, and it is independent of secretor status, grade, and stage. The sensitivity and specificity for the detection of bladder cancer is 75% and 85%, respectively. There is no commercially available test to date.
36. 36. CYTOKERATINS (CK 20 & CYFRA 21-1) CK 20 and CYFRA 21.1 are fragments of cytoskeletal proteins; can be detected in urine of bladder cancer patients by either protein or mRNA detection. CK 20 has a sensitivity and specificity of 85% and 76%, respectively.
37. 37. CYFRA 21.1, with a cutoff value of 4 ng/mL, found a sensitivity and specificity of 43% and 68%, respectively. Unfortunately, none of the Ta tumors were identified at the 4 ng/mL cutoff. Decreasing the CYFRA 21.1 cutoff to 1.5 ng/mL increased Ta detection to 33%, but the specificity dropped to an unacceptable 43%. Therefore it is not a useful marker in the current form, or at least for low-grade disease.
38. 38. FISH (UroVysion)
39. 39. FISH (UroVysion) Fluorescence in-situ hybridization (FISH) identifies fluorescently labeled DNA probes (for the centromeres of chromosomes 3, 7, and 17 and a locus-specific probe for 9p21) that bind to intranuclear chromosomes. The current commercially available probes evaluate aneuploidy for chromosomes 3, 7, and 17 and homozygous loss of 9p21. The median sensitivity and specificity of FISH analysis is 79% and 70%, respectively.
40. 40. FISH (UroVysion) FISH analysis is moderately useful for high-grade disease and may be anticipatory of new tumor formation. Approved by the US Food and Drug Administration (FDA) in 2005 as an aid for initial diagnosis of bladder cancer in patients with hematuria and subsequent monitoring for tumor recurrence in patients previously diagnosed with bladder cancer.
41. 41. FISH (UroVysion) FISH is considerably more sensitive and only slightly less specific than cytology in diagnosing urothelial carcinoma. A useful initial diagnostic tool in patients suspected of both new and recurrent bladder cancer.
42. 42. MICROSATELLITE ANALYSIS
43. 43. MICROSATELLITE ANALYSIS short DNA repeats present throughout the chromosomes; lost in some tumor cells- multiple markers available to identify these. Microsatellite analysis amplifies these repeats in the genome that are highly polymorphic, and PCR amplification can detect tumor-associated loss of heterozygosity by comparing peak ratio of two alleles in tumor DNA in urine sample with the presence of alleles in a blood sample from the same individual. The sensitivity and specificity range from 72% to 97% and 80% to 100%, respectively. Microsatellite analysis is the most promising marker of all.
44. 44. if the microsatellite analysis is persistently positive, there was an 83% 2-year recurrence rate, but if the analysis was persistently negative, only 22% of patients had recurrent tumors.
45. 45. DNA METHYLATION ANALYSIS CpG dinucleotide islands cluster around promoters in an unmethylated state to allow gene expression . Methylation of CpG islands shuts down the promoter, and if the promoter in question is part of a tumor suppressor gene then cancer can form. Examples of promoter methylation of CpG islands causing epigenetic changes in urothelial cancer include the P16/CDKN2A gene. Sensitivity of gene methylation - 75%. Methylated CpG islands can be found in normal urothelial cells of older patients.
46. 46. SURVIVIN Survivin is an antiapoptotic protein that has a high expression in urothelial cancer. Survivin is found in 10% to 30% of bladder cancers and is readily shed into urine. Sensitivity and specificity in the detection of urothelial tumors is 64% to 100% and 87% to 93%, respectively. May be useful in predicting which patients will respond to intravesical therapy. Survivin was relatively poor at detecting advanced-stage or high-grade tumors, with a sensitivity of 71% for stage T2 tumors and 80% for high-grade cancers.
47. 47. HYLAURONIC ACID Hylauronic acid controls intercellular communications and cell replication. Urothelial cancer induces hylauronic acid production from fibroblasts, and the amount correlates with the stage of the disease. Sensitivity and specificity is 91% to 100% and 84% to 90%, respectively. Sensitivity and specificity for discriminating between low-grade and high-grade lesions is unclear.
48. 48. TELOMERASE Telomerase resides at the terminal ends of chromosomes and duplicates random DNA repeats to prevent cell death. Telomerase activity is measured in telomeric repeat application protocol (TRAP) and is detected in 80% of urine from patients with bladder cancer with no grade differential. The sensitivity and specificity is 90% and 88%, respectively.
49. 49. BCLA-4 BCLA-4 is a nuclear transcription factor present in bladder tumors and adjacent benign areas of the bladder, but not in benign urothelium. sensitivity of 89%-96% with a specificity of 100% for bladder cancer. BLCA-4 has a potential role in tumor development or progression. high expression level of BLCA-4 identifies patients at high risk.
50. 50. ROLE OF URINARY MARKERS No urinary biomarker is sensitive and specific enough to replace cystoscopy in the primary detection or follow-up of bladder cancer. Adjunctive role of a non-invasive urine test performed before follow-up cystoscopy. Combination of cystoscopy with urine markers, in select situations, is appropriate for surveillance of patients with nonmuscle-invasive bladder cancer.
51. 51. Thank you