Aberrant p16INK4a methylation is a frequent event in colorectal cancers: prognostic value and relation to mRNA expression and immunoreactivity

Aberrant p16(INK4a) promoter methylation is common in colorectal cancer (CRC), but its clinicopathological significance remains controversial. The present study was therefore conducted to analyze p16(INK4a) methylation and its relationship to clinicopathological features, mRNA levels and immunoreactivity in a series of lesions. p16(INK4a) methylation was assessed for normal mucosa (n = 30) and CRC samples (n = 212) by methylation-specific real-time quantitative PCR, and p16(INK4a) expression by immunostaining in formalin-fixed paraffin-embedded specimens. In addition, fresh DNA (n = 61) was analyzed for relationships to p16(INK4a) mRNA by reverse-transcription PCR. The p16(INK4a) methylation index of normal mucosa samples ranged from 0 to 2% (mean, 0.23%; median, 0.02%), while the values for tumor samples varied widely from 0 to 100% (mean, 25.7%; median, 7.1%), the difference being statistically significant (P < 0.001). Of 151 paraffin-embedded CRC tissue samples, 51 (34%), 54 (36%), and 46 (30%) were classified as low, intermediate, and high for aberrant methylation of p16(INK4a). High p16(INK4a) methylation was significantly associated with large tumor size (P = 0.025). Patients with higher methylation further showed more frequent recurrence as compared with the low-methylation group, and shortened cancer-related survival (Hazard ratio [HR], 3.379; P < 0.001) and recurrence-free survival (HR, 3.962; P < 0.001 on multivariate analysis). A significant inverse relationship was apparent between the p16(INK4a) methylation and immunoreactivity (P = 0.017). A similar tendency was also observed for the methylation status and the mRNA level (P = 0.195). We conclude that p16(INK4a) methylation results in transcriptional silencing and defines a group of CRCs with a poor prognosis.