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.