p16INK4a and p14ARF methylation as a potential biomarker for human bladder cancer

Promoter hypermethylation is one of the putative mechanisms underlying the inactivation of negative cell-cycle regulators. We examined whether the methylation status of p16INK4a and p14ARF, genes located upstream of the RB and p53 pathway, is a useful biomarker for the staging, clinical outcome, and prognosis of human bladder cancer. Using methylation-specific PCR (MSP), we examined the methylation status of p16INK4a and p14ARF in 64 samples from 45 bladder cancer patients (34 males, 11 females). In 19 patients with recurrent bladder cancer, we examined paired tissue samples from their primary and recurrent tumors. The methylation status of representative samples was confirmed by bisulfite DNA sequencing analysis. The median follow-up duration was 34.3 months (range 27.0–100.1 months). The methylation rate for p16INK4a and p14ARF was 17.8% and 31.1%, respectively, in the 45 patients. The incidence of p16INKa and p14ARF methylation was significantly higher in patients with invasive (⩾pT2) than superficial bladder cancer (⩽pT1) (p = 0.006 and p = 0.001, respectively). No MSP bands for p16INK4a and p14ARF were detected in the 8 patients with superficial, non-recurrent tumors. In 19 patients with tumor recurrence, the p16INK4a and p14ARF methylation status of the primary and recurrent tumors was similar. Of the 22 patients who had undergone cystectomy, 8 (36.4%) manifested p16INKa methylation; p16INK4a was not methylated in 23 patients without cystectomy (p = 0.002). Kaplan–Meier analysis revealed that patients with p14ARF methylation had a significantly poorer prognosis than those without (p = 0.029). This is the first study indicating that MSP analysis of p16INK4a and p14ARF genes is a useful biomarker for the pathological stage, clinical outcome, and prognosis of patients with bladder cancer.