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 (in) RN A quantitation in urine as a marker of urothelial transitional cell carcino ma using the real time reverse transcriptase polymerase chain reaction (RT- PCR). Materials and Methods: Spontaneously voided urine was obtained from 47 pati ents with urothelial transitional cell carcinoma (carcinoma group), 19 othe r urological diseases (noncarcinoma group) and 27 healthy volunteers (contr ol group). Quantification of cytokeratin 20 was performed with mRNA extract ed from urine samples with primers and hybridization probes specific for cy tokeratin 20 on a LightCycler instrument (Roche Diagnostics Corp., Indianap olis, Indiana). Results: This method allowed reproducible quantitation of 10 to 10(6) cytok eratin 20 expressing colon carcinoma cells per 10(7) peripheral blood leuko cytes, comparable to the sensitivity of conventional RT-PCR with a wide lin ear measuring range. Cytokeratin 20 mRNA values in the carcinoma group (mea n 35,850) were significantly higher than noncarcinoma (171) and control gro ups (4.55, p <0.0001 and <0.0001, respectively). Urinary cytokeratin 20 mRN A values significantly correlated with tumor grade, urinary cytological cla ss, immunostaining pattern and depth of tumor invasion. Sensitivity and spe cificity of real time RT-PCR with a cutoff value of 15 were 81% and 83%, wh ereas 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 uroth elial transitional cell carcinoma.