Quantitative detection of cytokeratin 20 mRNA expression in bladder carcinoma by real-time reverse transcriptase-polymerase chain reaction

Objectives. To determine cytokeratin 20 (CK 20) expression in tumor tissue, normal urothelial tissue, bladder washings, and urine from patients with urothelial carcinoma by real-time reverse transcriptase-polymerase chain reaction using the LightCycler Instrument. Methods. Urine, bladder washings, tumor, and normal urothelial tissue were obtained from 33 patients with bladder carcinoma. RNA was subsequently extracted. Twenty tissue samples from healthy volunteers were used as controls. Real-time reverse transcriptase-polymerase chain reaction with the LightCycler was performed using fluorescence-labeled CK 20 primers as the target gene and the porphobilinogen deaminase housekeeping gene as the internal standard. Results. CK 20 mRNA expression was detected in all samples investigated. CK 20 expression in the tissue and urine samples from patients with tumor was significantly increased compared with that in normal urothelial tissue (P = 0.028) or urine from the negative control group (P = 0.012). The average CK 20 expression was 70,140 arbitrary units (AU) in tumor tissue (30,533 AU in urine from patients with tumor) and 8460 AU in normal urothelial tissue (2234 AU in normal urine). A stage-related difference in CK 20 mRNA expression was observed in tumor tissue (P = 0.027) and for tumor grade G1-G2 versus G3 [P = 0.03). Conclusions. CK 20 mRNA expression levels in tissue and urine were elevated in patients with urothelial carcinoma compared with the levels in healthy individuals. CK 20 expression was also detectable in normal urine or normal urothelial tissue, but at significantly lower levels than in tumor samples. Fluorescence-labeled quantitative polymerase chain reaction using the LightCycler helped to differentiate between low-level CK 20 expression in normal urothelial tissue and the levels in tumor tissue samples. CK 20 might be an excellent and sensitive marker for tumor monitoring and routine follow-up in patients with transitional cell carcinoma. (C) 2004 Elsevier Inc.