Methylation of p16^INK4a is a non-rare event in cervical intraepithelial neoplasia

The cell cycle inhibitor, p16^INK4a may be a useful surrogate biomarker of cervical intraepithelial neoplasia (CIN); however, there is currently no consensus of p16^INK4a genetic alterations throughout the multiple step process of CIN. Our goal was to identify the methylation frequency of p16^INK4a in each step of CIN that is associated with human papillomavirus (HPV) infection, using several different detection methods of p16^INK4a methylation to correlate the data. The present study included a total of 43 patients, including 38 with CIN, and 5 normal patients. Three different methods were used to detect hypermethylation of CpG islands, methylation-specific PCR (MSP) amplification of different primer sets of M1, M2, and M3, pyrosequencing of each forward primer region, and immunohistochemistry of p16^INK4a. Analysis of MSP showed that 20 of the 38 CIN patients (52.6%) revealed hypermethylation in at least 1 primer set of the p16 ^INK4a promoter. A complete loss of p16^INK4a protein expression was observed in 11 cases (28.9%). There was no observed association of methylation of the p16^INK4a gene with either CIN grading (P=0.0698) or HPV status (P=0.2811): specifically 42.9% (3/7) was found in CIN 1, 57.1% (8/14) in CIN 2, and 52.9% (9/17) in CIN 3. In concordance with immunohistochemistry results, hypermethylation of the p16^INK4a promoter was significantly correlated with a lack of p16 protein expression (P=0.0145). All positive peaks from pyrosequencing matched the MSP results, which ranged from 6.3% to 24.5%. In conclusion, p16^INK4a gene silencing during CIN was not determined to be a particularly rare event; however, it does not correlate with either HPV status or CIN grading.