Nested quantitative PCR approach for urinary cell-free EZH2 mRNA and its potential clinical application in bladder...

EZH2 is overexpressed in bladder cancer (BC) and plays important roles in tumor development and progression. Recent studies show cell free (cf) RNAs released from cancer cells can reflect tissues changes and are stable and detectable in urine. Although conventional quantitative real-time PCR (qPCR) is highly sensitive, low abundances of urinary cf-RNAs usually result in false-negatives. Thus, this study develops a nested qPCR (nqPCR) approach to quantify cf-EZH2 mRNA in urine and further assess its clinical significance for BC. Forty urine samples were first selected to evaluate feasibility of nqPCR. Then, levels of urinary cf-EZH2 mRNA were detected using developed method in an independent cohort of subjects with 91 healthy, 81 cystitis, 169 nonmuscle invasive BC (NMIBC) and 103 muscle-invasive BC (MIBC). In cf-EZH2 mRNA detection, nqPCR method was significantly associated with qPCR, but it could detect more urine samples and increase detection limit three orders of magnitude. Based on nqPCR method, cf-EZH2 mRNA levels have been found to be increased in urine of NMIBC and MIBC patients (p < 0.001). Compared with cytology, cf-EZH2 mRNA showed higher diagnostic ability for MIBC (p < 0.001) while not for NMIBC (p > 0.05). Moreover, it also could distinguish MIBC from NMIBC, with AUC of 0.787. For MIBC patients, high expression of cf-EZH2 mRNA associated with advanced stage and was an independent predictor of reduced disease free survival or overall survival. In conclusion, detection of cf-EZH2 mRNA in urine by nqPCR is a sensitive and noninvasive approach and may be used for diagnosis and prognosis prediction of MIBC.