Diagnostic accuracy of urine cytokeratin 20 for bladder cancer: A meta-analysis.

AIM: Urine cytokeratin 20 (CK20) has been reported as a novel diagnostic biomarker for bladder cancer (BC). This meta-analysis aims to evaluate the diagnostic value of urine CK20 for BC. METHODS: A systematic search for literatures was performed till March 31, 2018. Sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR), diagnostic odds ratio (DOR) and the area under the summary receiver operator characteristic curve (AUC) were analyzed. RESULTS: Twenty-seven studies from 22 articles met the eligible criteria. The pooled estimates were as follows: sensitivity, 0.79; specificity, 0.90; PLR, 8.17; NLR, 0.23; DOR, 35.29 and; AUC, 0.90. CK20 was more sensitive for the diagnosis of bladder urothelial carcinoma (BUC) than all types of BC (0.83 vs 0.75). For Tis/Ta tumors, the values were inferior to T1 and ≥T2 stage tumors, that is, the sensitivity (0.74 vs 0.81 vs 0.86), PLR (7.95 vs 9.97 vs 10.75) and DOR (27.81 vs 47.09 vs 69.63). For grade 1 tumors, the values were inferior to grade 2 and ≥grade 3 tumors, that is, the sensitivity (0.70 vs 0.82 vs 0.87), PLR (11.86 vs 13.63 vs 14.26) and DOR (36.63 vs 72.00 vs 101.71). RT-PCR or PCR showed superior sensitivity and specificity (0.80 vs 0.76, 0.91 vs 0.89) than immunostaining in detection of urine CK20 for BC. CONCLUSION: Urine CK20 might be a potential noninvasive biomarker for BC, especially for BUC. The diagnostic accuracy of urine CK20 is improved with the progression of tumor stage and grade. In addition, RT-PCR or PCR shows better diagnostic performance in detecting urine CK20 than immunostaining.

Cytokeratin 20 immunocytochemistry on urine sediments: A potential low-cost adjunct to cytology in the diagnosis of low-grade urothelial carcinoma.

BACKGROUND: Urine cytology is the corner-stone for the diagnosis of urothelial neoplasia; however, a substantial proportion of low-grade carcinomas are reported as inconclusive owing to scant cellularity and subtle cytological features. Biomarkers applied on urine sediment smears of such patients are likely to be clinically relevant. Access to Food and Drug Administration approved urinary biomarkers in resource limited setting is poor. Detection of cytokeratin 20 (CK20) in urine sediments, although still a research tool, is a promising marker as immunocytochemistry is performed regularly in several Indian laboratories. OBJECTIVE: We tested the clinical utility of CK20 immunocytochemistry as a potential low-cost adjunct to urine cytology in diagnosis of low-grade urothelial carcinoma. One hundred and fifty fresh, voided urine specimens from 42 cases of biopsy proven urothelial neoplasia (14 high grade, 28 combined low-grade [n=26]) and low malignant potential [n=2]), and 20 non-neoplastic lesions were included in the study sample. RESULTS: Confident diagnosis of malignancy was possible in five (17.8%) low-grade malignancies. Thirteen of 16 (81.3%) low-grade malignancies with inconclusive cytology showed positive CK20 expression. This reduced the proportion of low-grade cases with inconclusive cytology from 57.1% to 10.7% (P=.021). In addition, we could correctly classify one case of bladder lithiasis with false positive urine cytology. Discrepant CK20 staining (positive) was seen in one patient with acute cystitis. CONCLUSIONS: CK20 expression in non-umbrella cells is a robust marker of urinary bladder carcinoma. It has potential clinical utility for identification of low-grade urothelial malignancy with inconclusive cytological diagnosis.

Urinary transcript quantitation of CK20 and IGF2 for the non-invasive bladder cancer detection.

PURPOSE: Cytokeratin 20 (CK20) and insulin-like growth factor 2 (IGF2) were previously proposed to be elevated in clinical samples from patients with bladder cancer (BCa). A two cohort design validation study was used to assess the relevance for BCa detection by transcript quantitation of both markers in urine samples. Their diagnostic value was assessed in comparison with voided urine cytology (VUC). METHODS: RNA isolation was carried out using cellular sediments of urine samples from 196/103 histologically positive BCa patients, as well as 97/50 control subjects for the test (TC) and validation cohort (VC), respectively. Urinary transcript levels of CK20 and IGF2 were determined by qPCR. RESULTS: Relative transcript levels were significantly elevated 3.4/11-fold for CK20 and 188/64-fold for IGF2 (p < 0.001) in urine sediments of BCa patients compared to controls in the TC and VC, respectively. In a combined analysis, the resulting sensitivity (SN) (SNTC: 77.9; SNVC: 90.3%) and specificity (SP) (SPTC: 88.0; SPVC: 84.0%) were similar to that of VUC. The sensitivity of VUC in combination with CK20 and IGF2 was considerably increased (SNTC: 94.6; SNVC: 93.2%) while specificity was reduced (SPTC: 72.0; SPVC: 82.0%) compared to VUC alone in the test and validation cohort. CONCLUSIONS: Transcript levels of IGF2 and CK20 enabled the detection of BCa with a diagnostic performance similar to VUC. Combined analysis of voided urine cytology together with altered transcript levels of CK20 and IGF2 enhanced sensitivity, but did not improve overall test performance.

The diagnostic value of cytohistological urine analysis and cytokeratin 20 in malignant and atypical urothelial cells.

INTRODUCTION: To determine the ability of cytohistology and cytokeratin 20 (CK 20) expression in malignant and atypical cells (AUC) from urine to serve as a diagnostic tool for assessing urothelial carcinoma (UC). METHODS: Diagnoses from 55 urine cytological samples from 55 patients were analyzed and correlated with subsequent biopsy findings. A total of 50 archived urine slides from patients that received a cytological diagnosis and histological follow-up were selected for immunostaining with monoclonal CK 20 antibodies and elaborated by Z-test for proportions. RESULTS: The majority of all positive or atypical smears (24; 89%) were confirmed through histological analysis. The majority of urinary cytological diagnoses reported as negative (15; 54%) were also confirmed through biopsies. The overall sensitivity, specificity, PPV, and NPV were 65%, 83%, 89%, and 54%, respectively. All 13 smears cytologically determined to contain malignant cells, with subsequent biopsies confirming UC, exhibited strong positive staining with the CK 20 antibody. All cases evaluated as benign both cytologically and histologically had negative CK 20 staining. Of the 15 AUC cases with lesions confirmed through biopsies, 11 (73%) had atypical cells that stained positive for CK 20. DISCUSSION: Our results demonstrate the diagnostic value of urinary cytology and confirm CK 20 as an adjunct marker for the diagnosis of UC and for the triage of AUC.

The Effects of Instrumentation on Urine Cytology and CK-20 Analysis for the Detection of Bladder Cancer.

OBJECTIVE: To evaluate the effects of cystoscopy on urine cytology and additional cytokeratin-20 (CK-20) staining in patients presenting with gross hematuria. PATIENTS AND METHODS: For 83 patients presenting with gross hematuria, spontaneous and instrumented paired urine samples were analyzed. Three patients were excluded. Spontaneous samples were collected within 1 hour before cystoscopy, and the instrumented samples were tapped through the cystoscope. Subsequently, patients underwent cystoscopic evaluation and imaging of the urinary tract. If tumor suspicious lesions were found on cystoscopy or imaging, subjects underwent transurethral resection or ureterorenoscopy. Two blinded uropathological reviewers (DB, KK) evaluated 160 urine samples. Reference standards were results of cystoscopy, imaging, or histopathology. RESULTS: Thirty-seven patients (46.3%) underwent transurethral resection or ureterorenoscopy procedures. In 30 patients (37.5%) tumor presence was confirmed by histopathology. The specificity of urine analysis was significantly higher for spontaneous samples than instrumented samples for both cytology alone (94% vs 72%, P = .01) and for cytology combined with CK-20 analysis (98% vs 84%, P = .02). The difference in sensitivity between spontaneous and instrumented samples was not significant for both cytology alone (40% vs 53%) and combined with CK-20 analysis (67% vs 67%). The addition of CK-20 analysis to cytology significantly increases test sensitivity in spontaneous urine cytology (67% vs 40%, P = .03). CONCLUSION: Instrumentation significantly decreases specificity of urine cytology. This may lead to unnecessary diagnostic procedures. Additional CK-20 staining in spontaneous urine cytology significantly increases sensitivity but did not improve the already high specificity. We suggest performing urine cytology and CK-20 analysis on spontaneously voided urine.

Detection of circulating tumor cells in bladder cancer using multiplex PCR assays.

AIM: The aim of this study was to develop multiplex-PCR assays for the detection of circulating tumor cells in peripheral blood and urine samples of patients with bladder cancer. MATERIALS AND METHODS: Peripheral blood and urine samples were collected from 208 patients (169 patients and 39 healthy volunteers). After RNA extraction and cDNA synthesis, the samples were analyzed for the expression of cytokeratin 19 (CK19), CK20 and epidermal growth factor receptor (EGFR) mRNA in blood and for SURVIVIN, human telomerase reverse transcriptase (hTERT), cytokeratin 20 (CK20) mRNA in urine, using multiplex-PCR assays. RESULTS: EGFR and CK20 alone or in combination as well as all urine markers correlated well with histological grade. hTERT correlated well with primary tumor size T≥3. Patients with positive urine markers had significantly worse progression-free survival. CONCLUSION: Multiplex-PCR assays can be a useful tool for staging and monitoring purposes in patients with bladder cancer.

Cytokeratin-20 immunocytochemistry in voided urine cytology and its comparison with nuclear matrix protein-22 and urine cytology in the detection of urothelial carcinoma.

This study was done on 59 subjects (42 urinary bladder carcinoma patients and 17 non-neoplastic controls). Urine cytology and bladder chek NMP22 test was done on all cases. CK20 immunostaining was performed on archived papanicolaou stained urine cytology smears in 34 cases (27 bladder carcinoma and 7 negative controls). Results of all three tests (cytology, NMP22, and CK20 immunostaining) were compared with histopathology to evaluate the accuracy of individual test. The combination of cytology and NMP22 was compared with combination of cytology and CK20 immunostaining for detection of bladder carcinoma. NMP22 had sensitivity of 92.9% and specificity of 70.6%, as compared with voided urine cytology (sensitivity of 76.2% and specificity of 76.5%) and CK20 immunostaining (sensitivity of 70.4% and specificity of 71.4%). Combination of cytology and NMP22 gave better results (sensitivity of 88.1% and specificity of 88.2%) than combination of cytology and CK20 immunostaining or any other test in isolation.

The clinical relevance of urine-based markers for diagnosis of bladder cancer.

The aim of the present study was to evaluate the diagnostic relevance of urinary fibronectin (FN), telomerase (RTA), and cytokeratin 20 (CK20) mRNA in comparison with voided urine cytology (VUC). The study included 132 patients with bladder cancer, 60 patients with benign bladder lesions, and 48 healthy individuals. All were subjected to urine cytology, estimation of fibronectin by ELISA, RTA by TRAP, and CK20 mRNA by conventional RT-PCR in urothelial cells from voided urine. The best cutoff point for FN was determined by receiver operating characteristic curve (41.7 ng/mg protein) revealed the highest sensitivity for malignant (80%) followed by the benign (70%) than the healthy individuals (4.1%) at P < 0.001. Also, RTA and VUC showed significant difference among the three investigated groups (P < 0.001). The overall sensitivity (89.3%) and specificity (98.4%) were the highest for CK20 mRNA. Combined sensitivity of VUC with FN, RTA, and CK20 mRNA together (98.4%) was higher than either the combined sensitivity of VUC with any of them or than that of the biomarker alone. Accordingly, when the diagnostic efficacy was considered, CK20 mRNA had the highest sensitivity and specificity compared to all investigated markers.

Quantitative detection of cytokeratin 20 mRNA in urine samples as diagnostic tools for bladder cancer by real-time PCR.

AIM: To determine and compare cytokeratin 20 (CK20) mRNA expression in urine of patients with transitional cell carcinomas of bladder (TCCB), urological benign diseases, and healthy volunteers. METHODS: Taqman probe was designed according to the sequence of CK20 cloned gene. The quantitative PCR reaction system was optimized and evaluated. The CK20 mRNA level was screened by real-time polymerase chain reaction (RT-PCR) in 95 urine samples and analyzed according to the following parameters: urinary cytology, nuclear matrix protein 22 (NMP22) expression, tumor stage and grade. RESULTS: For 60 TCCB patients urinary cytology was positive in 28 (46.7%), control group had no false-positive results (specificity 100%). CK20 expression was positive in RT-PCR of 51 cases (85%) of TCCB, but control group was positive in 2 cases (specificity 94.3%) with a cutoff value of crossover point (CT) = 30. Two methods have significant variation in sensitivity (p < 0.001), NMP22 expression was positive in 47 cases (78.3%), but control group was positive in 9 cases (specificity 85%). In the simultaneous evaluation of CK20 and NMP22 mRNA expression, there were 54 positive cases (90%). CK20 mRNA values in TCCB group (mean 27712.57 copies/microl) were significantly higher than in non-cancer disease urological group (mean 74.45 copies/microl) and control group (mean 8.47 copies/microl) (p < 0.001, p < 0.001, respectively). CK20 mRNA values increased gradually with higher tumor grade and stage: G1 differs significantly from G2 (p = 0.016); T(is) /T(a) differs significantly from T(1-2) (p = 0.022). CONCLUSION: Our results indicate that CK20 mRNA expression could be regarded as a potential marker for TCCB. We demonstrated correlation between CK20 expression and the clinicopathologic features of TCCB (tumor stage and grade); simultaneous use of CK20 and NMP22 markers will elevate the sensitivity of the method. CK20 RT-PCR is a sensitive, quantitative, rapid and specific method to detect free cancer cells in the urine, and could be recommended for be wide application in the diagnostics of TCCB and evaluation of therapeutic effect.

Focus on urinary bladder cancer markers: a review.

Finding and development of new bladder cancer markers is still a very dynamic field. Because of the mass of all these markers it is impossible to report all of them. This paper reviews the role of bladder cancer markers in diagnosis and highlights the most important biomarkers studied and reported recently. A medline based literature search was performed to examine the field of bladder cancer markers. Major topics focus on selected bladder cancer markers from nearly all categories of the wide field of bladder cancer markers: Hematuria, FISH, FGFR3, SURVIVIN, u-PAR, TP53 mutation, HER-2/neu, TPA, NMP22, CK-19, CK-20, CYFRA 21-1. The use and clinical importance as diagnostic help are discussed. In this review a highlight to some of the most important markers was made. Further determination of recurrence and progression marker will contribute to establish better treatments for the individual patient. Molecular staging of urological tumors will allow selecting cases that will require systemic treatment. It is necessary and important to integrate under the same objectives basic and clinical research.

Comparison of CD44 and cytokeratin 20 mRNA in voided urine samples as diagnostic tools for bladder cancer.

OBJECTIVES: We evaluated the diagnostic efficacy of urinary CD44 and cytokeratin 20 (CK20) mRNA in comparison with voided urine cytology (VUC) for the detection of bladder cancer. DESIGN AND METHODS: A total of 136 Egyptian patients provided a single voided urine sample for CD44, CK20 mRNA and VUC before cystoscopy. Of the 136 cases, 111 were histologically diagnosed as bladder cancer whereas the remaining 25 had benign urological disorders. A group of 20 healthy volunteers was also included in this study. Voided urine was centrifuged and the urine sediment was used for cytology, estimation of CD44 by ELISA and RNA extraction. CK20 mRNA was detected by conventional RT-PCR and quantitative real-time RT-PCR. RESULTS: The best cutoff values for CD44 and relative CK20 mRNA detected by real-time RT-PCR were calculated by receiver operating characteristic curve. The positivity rates and the mean ranks for CD44 and CK20 mRNA showed significant difference among the three investigated groups (p=0.001). Quantitative real-time RT-PCR results were comparable to conventional RT-PCR for the detection of CK20 mRNA. The positivity rate of CD44 was significantly associated with schistosomiasis and urine cytology. The overall sensitivity and specificity were 52.3% and 88.9% for VUC, 63.1% and 88.9% for CD44, and 82.0% and 97.8% for CK20 mRNA. Combined sensitivity of VUC with CD44 and CK20 mRNA together (95.5%) was higher than either the combined sensitivity of VUC with CD44 (78.4%) or with CK20 mRNA (91.0%) or than that of the biomarker alone. CONCLUSION: Urinary CD44 and CK20 mRNA had higher sensitivities compared to VUC. However, when the diagnostic efficacy was considered, CK20 mRNA by either conventional RT-PCR or real-time RT-PCR had the highest sensitivity and specificity compared to CD44 and VUC.

[Clinical value of combined detection with urinary bladder cancer antigen, hyaluronic acid and cytokeratin 20 in diagnosis of bladder cancer].

BACKGROUND & OBJECTIVE: Bladder cancer is the most common malignancy of all genitourinary tumors. Urine cytology is the "gold standard" for non-invasive diagnosis of bladder cancer, but its sensitivity is low. This study was to explore the clinical value of combined detection with urinary bladder cancer antigen (UBC), hyaluronic acid (HA) and cytokeratin 20 (CK20) in the diagnosis of bladder cancer. METHODS: Urine samples were obtained from 64 patients with bladder cancer and 20 patients with benign urological disease. Urine UBC, HA and CD20 were measured using enzyme-linked immunosorbent assay (ELISA), radioimmunology assay, and reverse transcription polymerase chain reaction (RT-PCR) respectively, before cystoscopy. RESULTS: UBC, HA and CK20 yielded significantly higher sensitivity in detecting bladder cancer compared to urinary cytology (85.9%, 89.1% and 78.1% vs. 40.6%, P<0.01). The specificity of UBC, HA, CK20 and urinary cytology for the detection of bladder cancer were 85.0%, 80.0%, 80.0% and 95.0%, respectively. The sensitivity of UBC, HA and CK20 were all significantly higher than that of urinary cytology in detecting different histological stages and grades of bladder cancer (P<0.01). The value of UBC had no significant difference in different histological stages and grades of bladder cancer (P>0.05). The sensitivity of HA was significantly higher in G2 and G3 grades than in G1 grade (P<0.01), but not different between G2 and G3 grades(P>0.05). No difference in HA values was observed between different histological types (P>0.05) regarding to HA. The sensitivity of CK20 was significantly elevated with the increase of histological grades and stages. Combined use of UBC, HA and CK20 improved the sensitivity and specificity of detecting bladder cancer to 96.9% (62/64) and 100.0%, respectively. CONCLUSIONS: Combined use of UBC, HA and CK20 can improve the sensitivity and specificity for the detection of bladder cancer in urine, thus it may replace conventional cystoscopy in the primary diagnosis.

Expression of cytokeratin 20 in urine cytology smears: a potential marker for the detection of urothelial carcinoma.

BACKGROUND: Urine cytomorphology is one of the oldest methods for screening and monitoring patients with transitional cell carcinoma (TCC). Sensitivity of urine cytology is relatively low. Ancillary techniques on urine sample may increase the sensitivity. AIM: To explore the utility of cytokeratin 20 (CK20) immunostaining in identifying malignant cells in urine cytology smears. MATERIALS AND METHODS: Fourteen cases each of confirmed TCC and benign urinary cytology along with five cases of atypical cells in urine were immunostained with a monoclonal CK20 antibody. Of 14 cases of TCC, 12 showed strong positive staining with the antibody. All benign cases were negative except for a few cases in which the umbrella cells were weakly to moderately positive. In all five cases of atypical urine cytology the atypical cells stained positive with the antibody. These cases were later confirmed as TCC on histopathology of bladder wall biopsy. CONCLUSION: CK20 is an important biomarker that can be used to identify TCC in urine cytology smears. It is particularly useful in those cases where malignancy cannot be confirmed by morphology alone.

Telomerase activity, cytokeratin 20 and cytokeratin 19 in urine cells of bladder cancer patients.

AIM OF THE STUDY: This work aims to search for markers suitable for the screening of bladder cancer, which should be specific, sensitive, reproducible, non-invasive and at acceptable cost. PATIENTS AND METHODS: The study included 50 patients diagnosed as bladder cancer (35 TCC, 15 SCC) of different stages and grades, 30 patients with various urothelial diseases, besides 20 apparently healthy subjects of matched age and sex to the malignant group. A random midstream urine sample was collected in a sterile container for the determination of telomerase by RT-PCR, keratin 19 by ELSA CYFRA 21-1 IRMA kit, keratin 20 by RT-PCR and immunohistochemical staining, and urine cytology. RESULTS: For all parameters (telomerase, K19, K20 and cytology) the malignant group was significantly different from both the benign and the control groups. None of the four studied parameters was correlated to the stage of the disease, and when it comes to grade, only K19 showed a significant positive correlation with grade both in TCC and SCC. When ROC curves for all parameters were compared, K19 had the largest area under the curve, and then comes K20. CONCLUSION: K 19 may be used as a biological marker for the diagnosis of bladder cancer. K19 could not be used for differential diagnosis of different types of bladder cancer, meanwhile it could be a marker for differentiation that decreases in less differentiated tumors. As a tumor marker, K20 reflects inability to differentiate tumor type or grade in TCC, while in SCC of the bladder it is correlated with the grade. As a method, RT-PCR is superior to immunostaining for the detection of bladder cancer, meanwhile K20 immunohistochemistry (IHC) results were much better than urine cytology as a bladder cancer screening test. Haematuria and inflammation reduced the specificity of telomerase assay, which reduced its validity as a tumor marker of bladder cancer. K19 and K20 are the best candidates as screening tests for the diagnosis of bladder cancer, representing the highest sensitivity and specificity, beside the radiological and histopathology. Meanwhile, telomerase, although it was a sensitive enough marker, it reflected a high false positive rate.