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


CONCLUSIONS
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.
Guo
B
B
Key Laboratory of Clinical Laboratory Diagnostics, Ministry of Education, Chongqing Medical University, Chongqing 400016, China. hexiujian123@163.com
Luo
C
C
Xun
C
C
Xie
J
J
Wu
X
X
Pu
J
J
D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D014408:Biomarkers, Tumor / Q000235:genetics* / Q000652:urine*; D002295:Carcinoma, Transitional Cell / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D005260:Female; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D053554:Keratin-20 / Q000235:genetics* / Q000652:urine; D008297:Male; D008875:Middle Aged; D009367:Neoplasm Staging; D012333:RNA, Messenger / Q000235:genetics / Q000652:urine*; D012372:ROC Curve; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D001749:Urinary Bladder Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D014556:Urine / Q000737:chemistry / Q000166:cytology
D014408:Biomarkers, Tumor; D053554:Keratin-20; D012333:RNA, Messenger