The simultaneous use of telomerase, cytokeratin 20 and CD4 for bladder cancer detection in urine.

OBJECTIVE: Because of the low sensitivity of urinary cytological diagnosis of urinary bladder carcinoma, new molecular diagnostic methods have been proposed. We decided to verify the expression of telomerase mRNA coding for the catalytic component (hTRT), cytokeratin 20 (CK20) and CD4 antigen mRNAs in urine as possible diagnostic tool. METHODS: Evaluation of hTRT, CK20, CD4 mRNAs was performed in 50 ml of naturally voided urine of 205 patients of which 153 with bladder cancer (Tis, n = 11; TaGx, n = 4; TaG1, n = 25; TaG2, n = 26; TaG3, n = 8; T1G1, n = 16; T1G2, n = 17; T1G3, n = 20; T2G2, n = 6; T2G3, n = 13; T3G3, n = 7) and 52 controls. A quantitative expression of hTRT at mRNA level versus TRAP (telomeric repeat amplification protocol) assay was performed in 20 patients and 14 controls. The expression of RT-PCR for hTRT, CK20, CD4 versus urinary cytology was analysed in 44 patients with bladder cancer. Evaluating the three molecular markers together, the result was considered correct when at least two of the markers were positive, suspected when only one marker was positive and negative for diagnosis of tumour when all markers were negative. The performance of the diagnostic model resulted from the logistic analysis evaluated with receiver operating characteristics (ROC) curve analysis. RESULTS: The sensitivity detected for each tumour marker was as follows: for hTRT 90.8%, for CK20 84.3% and for CD4 was 64.7%, while the specificity was 94.2% for CD4 and 78.8% for both hTRT and CK20. When a simultaneous evaluation of the three tumour markers was considered, 88.2% of the diagnoses were correct, 11.8% were suspected for tumour and none were mistaken. When compared with cytology, the simultaneous use of the three markers allowed reaching a correct diagnosis in 88% of the cases in comparison to 25% by urinary cytology. The sensitivity in the detection of bladder cancer was higher for hTRT at mRNA level in comparison with the enzymatic activity detection with TRAP (90% vs. 35%) while the specificity for both markers resulted very high (100%). CONCLUSIONS: These data show that in the future the diagnostic improvement of urine based molecular markers for the detection of bladder cancer in the urine could improve the sensitivity of urinary cytology reducing the need of a cystoscopy.

Detection of bladder carcinoma by combined testing of urine for hyaluronidase and cytokeratin 20 RNAs.

BACKGROUND: A new, sensitive, noninvasive method for the detection of urothelial carcinomas of the urinary bladder would open new possibilities in both the diagnosis and followup of patients. METHODS: This study included 228 patients diagnosed with bladder carcinoma, 68 patients with benign bladder lesions, and 44 healthy persons served as the control group. All were subjected to: serologic schistosomiasis antibody assay in serum, urine cytology, estimation of urine hyaluronic acid (HA) by enzyme-linked immunosorbent assay, and detection of CK-20 and hyaluronidase (HAase) by reverse transcription polymerase chain reaction (RT-PCR) in urothelial cells from voided urine. RESULTS: HA mean rank was higher in benign and malignant groups than in the healthy group (P < 0.0001) and was significantly related to tumor grade (P = 0.021). HA best-cutoff, determined using receiver operating characteristic curve to discriminate between malignant and nonmalignant groups, was 58.5 units/mg protein at 85.8% sensitivity and 60.7% specificity. HAase RNA showed superior sensitivity (90.8%) over cytology (68.9%) and CK-20 (78.1%) with specificity of 93.4%, 98.1% and 80.2%, respectively. The sensitivity reached 94.7% at a specificity of 91.5% when combined with CK-20. All 4 of the investigated markers were related to grade at P <0.05. Whereas only HAase and CK-20 were significantly related to stage (P < 0.05). As to schistosomiasis, only HAase RNA positivity was significantly associated (P = 0.038). CONCLUSIONS: HAase RNA is a promising noninvasive test with high sensitivity and specificity in bladder carcinoma detection.

Comparison of cytokeratin 20 RNA and angiogenin in voided urine samples as diagnostic tools for bladder carcinoma.

BACKGROUND: We evaluated the diagnostic efficacy of urinary angiogenin (ANG) and cytokeratin 20 (CK-20) mRNA in comparison with voided urine cytology in the detection of bladder cancer patients. OBJECTIVES AND METHODS: A total of 97 Egyptian patients provided a single voided urine sample for ANG, CK-20 and cytology before cystoscopy. Of the 97 cases, 63 were histologically diagnosed as bladder cancer; 33 with transitional cell carcinoma (TCC) and 30 with squamous cell carcinoma (SCC), whereas the remaining 34 had benign urological disorders. A group of 46 healthy volunteers were also included in this study. Voided urine was centrifuged and the supernatant was used for estimation of ANG by EIA and confirmed by Western blotting (WB). The urine sediment was used for cytology and RNA extraction. CK-20 RNA was detected by RT-PCR. RESULTS: The best cutoff value for ANG was calculated by a ROC curve as 322.7 ng/mg protein. The median urinary ANG level in bladder carcinoma, benign urological disorders and healthy volunteer groups was: 802.7, 425 and 33 pg/mg protein, respectively. The positivity rate for urinary CK-20 mRNA of the control, benign and malignant groups was 0%, 2.9% and 82.3%, respectively (P = 0.000); while the rates for ANG were 11.6%, 54.8% and 75.4%, respectively (P = 0.000). There was no significant difference in positivity rates of CK-20 and ANG with respect to sex, smoking, schistosomiasis, urine cytology, tumor grade, tumor stage, hematuria or pus cells. The overall sensitivity and specificity were 71.4% and 90% for voided urine cytology, 75.4% and 70.3% for ANG, and 82.3% and 98.8% for CK-20. Combined sensitivity of voided urine cytology with ANG and CK-20 together (98.2%) was higher than either the combined sensitivity of voided urine cytology with ANG (96.5%) or with CK-20 (91.6%) or than that of the biomarker alone. We demonstrated significant positive correlation between CK-20 positivity with age (P = 0.043) and nodal involvement (P = 0.037); however, there was no significant correlation between CK-20 and ANG with the other clinicopathological parameters. CONCLUSIONS: Our data indicate that CK-20 and ANG in voided urine had higher sensitivities compared to voided urine cytology. However, when specificity was considered, CK-20 alone had superior sensitivity and specificity compared to ANG and voided urine cytology.

Mucin 7 and cytokeratin 20 as new diagnostic urinary markers for bladder tumor.

PURPOSE: We determine the sensitivity and specificity of cytokeratin 20 (CK-20) and mucin 7 (MUC7) gene expression in voided urine samples taken from patients with bladder tumor and from control groups to investigate putative, noninvasive urinary markers for bladder tumor detection and monitoring. MATERIALS AND METHODS: Voided urine samples were collected from 50 patients with histologically proven bladder neoplasms (pTaN0M0G1-3 in 19 and pTisN0M0G3-pT4pN1M1G3 in 31), 20 patients with urolithiasis, 20 patients with urinary tract infection, 20 patients with other urological neoplasms and 20 healthy volunteers. Total RNA was extracted from exfoliated cells collected from 200 ml. voided urine. All RNA samples were investigated by a specific CK-20 and MUC7 nested reverse transcriptase polymerase chain reaction. RESULTS: The overall sensitivity of CK-20 gene expression in voided urine samples for the detection of bladder neoplasms was 78%. In contrast, voided urine samples from control patients and healthy volunteers showed a high rate of false-positive CK-20 detection resulting in a low specificity of 36%. The overall sensitivity of the MUC7 test for all bladder tumor cases was 66%. The sensitivity for papillary urothelial neoplasms (pTaN0M0G1-3) was 42% whereas analysis of the carcinoma in situ and invasive bladder cancer group (pTisN0M0G3-pT4pN1M1G3) yielded a sensitivity of 81%. The overall specificity of the MUC7 nested reverse transcriptase polymerase chain reaction method in the control groups was 80%. CONCLUSIONS: A high positive CK-20 detection rate was found not only in voided urine samples from patients with bladder tumor, but also in urine specimens from control groups. Therefore, CK-20 is not a reliable urinary tumor marker for bladder neoplasms. In contrast to CK-20, analysis of MUC7 demonstrated a high sensitivity and high specificity for carcinoma in situ and invasive bladder cancer, thus fulfilling the criteria of a urinary tumor marker.

Urine: beyond cytology for detection of malignancy.

In the present review we discuss various ancillary modalities for detection of malignancies in urine samples, with an emphasis on urothelial carcinomas. Flow cytometry, bladder tumor antigen (BTA), nuclear matrix protein (NMP), matrix metalloproteinase (MMP), human chorionic gonadotrophic (HCG), telomerase, and other techniques are discussed. DNA FCM is a relatively costly and sophisticated technique. It has a practical application in the diagnosis of bladder cancer among subjects at high risk and is of value in monitoring the course of the disease and anticipating recurrence following conservative treatment. The BTA test is a simple, rapid, and inexpensive adjunct to cystoscopy and the results of the test are equivalent or superior to those of voided urinary cytology. NMP-22 immunoassay is a useful diagnostic test for predicting recurrence of urothelial malignancy. It is also a cost-effective and sensitive screening test for detecting tumor in patients with urothelial carcinoma. Beta-HCG estimation in urine samples appears to be an efficient diagnostic marker for the assessment of distant metastasis in bladder carcinoma rather than a screening test. Other ancillary techniques such as detection of expression of cytokeratin 20 by RT-PCR, MMP-9 estimation, and fluorescent in situ hybridization and telomerase activity are rarely applied clinically in routine urinary samples and are not cost-effective.

Real time reverse transcriptase polymerase chain reaction of urinary cytokeratin 20 detects transitional cell carcinoma cells.

PURPOSE: We evaluate the diagnostic use of cytokeratin 20 messenger (m) RNA quantitation in urine as a marker of urothelial transitional cell carcinoma using the real time reverse transcriptase polymerase chain reaction (RT-PCR). MATERIALS AND METHODS: Spontaneously voided urine was obtained from 47 patients with urothelial transitional cell carcinoma (carcinoma group), 19 other urological diseases (noncarcinoma group) and 27 healthy volunteers (control group). Quantification of cytokeratin 20 was performed with mRNA extracted from urine samples with primers and hybridization probes specific for cytokeratin 20 on a LightCycler instrument (Roche Diagnostics Corp., Indianapolis, Indiana). RESULTS: This method allowed reproducible quantitation of 10 to 106 cytokeratin 20 expressing colon carcinoma cells per 107 peripheral blood leukocytes, comparable to the sensitivity of conventional RT-PCR with a wide linear measuring range. Cytokeratin 20 mRNA values in the carcinoma group (mean 35,850) were significantly higher than noncarcinoma (171) and control groups (4.55, p <0.0001 and <0.0001, respectively). Urinary cytokeratin 20 mRNA values significantly correlated with tumor grade, urinary cytological class, immunostaining pattern and depth of tumor invasion. Sensitivity and specificity of real time RT-PCR with a cutoff value of 15 were 81% and 83%, whereas those of conventional cytology were 28% and 100%, respectively. CONCLUSIONS: These results indicate that real time cytokeratin 20 RT-PCR is a sensitive, quantitative, rapid and specific method to detect free cancer cells in the urine, with good potential for monitoring recurrence of urothelial transitional cell carcinoma.

Cytokeratin 20 as an immunocytochemical marker for detection of urothelial carcinoma in atypical cytology: preliminary retrospective study on archived urine slides.

Previous studies have shown that expression of cytokeratin 20 (CK20), a constituent of intermediate filaments, is increased in malignant versus benign urine samples. To evaluate whether immunocytochemical staining of CK20 on archived urine slides could be used as a potential adjunct marker for triage of atypical urine cytology, we analyzed a total of 77 archived urine slides obtained from a spectrum of patients with various risks of developing urothelial carcinoma. These patients were divided into four groups on the basis of initial urine cytologic results and subsequent follow-up biopsy findings; group 1 had negative results in both evaluations, whereas the results in group 4 were positive for both cytology and biopsy. Groups 2 and 3 had a diagnosis of atypical urine cytology; however, patients in group 3 had a positive follow-up biopsy, and patients in group 2 did not. The Papanicolaou-stained archived urine slides were destained and then restained immunocytochemically with monoclonal antibody against CK20. With 5% positively stained nonumbrella cells as a threshold, CK20 was positive in 94.4% of group 3 or 4 patients. In contrast, CK20 was positive in 27.3% of group 2 patients and in 10.5% of group 1 patients. The overall sensitivity and specificity for CK20 for the detection of urothelial carcinoma in this population of patients were 94.4% and 80.5%, respectively. This study demonstrated that immunocytochemical analysis of CK20 on archived urine slides could be used to triage atypical urine cytology into low- and high-risk categories and that CK20 might be a simple and useful early detection marker for urothelial carcinoma.

Urinary cytokeratin 20 as a marker for transitional cell carcinoma.

OBJECTIVES: To determine if detection of cytokeratin 20 (CK20) gene expression, by reverse transcriptase-polymerase chain reaction (RT-PCR) in urine from transitional cell carcinoma (TCC) patients, can provide a new noninvasive tool for the follow-up of patients with urothelial carcinoma of the bladder. METHODS: Urine was collected from 95 patients previously diagnosed as TCC during their follow-up, and from 27 healthy volunteers. All patients had a transurethal resection of tumor or biopsies obtained from 'suspicious' areas in the bladder. RNA was extracted from cells collected from the urine and RT-PCR was performed with specific primers for the amplification of cytokeratin 8, a general marker for epithelial cells, and of CK 20, a marker for TCC urothelium. RESULTS: CK20 expression was detected in 86.7% of TCC patients, and only in 3.3% of healthy volunteers (specificity 96.7%). Strong correlation was found between tumor grade and expression of CK20 in urine. All grade III and IV tumors demonstrated positive CK20 expression (100% sensitivity), whereas the sensitivity for lower grades was between 71 and 80%. Among 11 patients with a previous biopsy-proven diagnosis of TCC and a current negative biopsy, in 9 patients CK20 expression was detected. Further follow-up of these patients for a period of 6 months revealed recurrence of TCC in 4 patients. CONCLUSION: CK20 detection in urine cells is a simple, noninvasive method with a high potential to become the marker of choice for monitoring and follow-up of TCC patients. More information is needed regarding CK20 expression in nonmalignant urological disease, to evaluate its use for routine screening purposes.