Methylation and immunoexpression of p16^ink4a tumor suppressor gene in primary breast cancer tissue and their quantitative p16^ink4a hypermethylation in plasma by real-time PCR

Background: The p16^INK4a gene methylation has been reported to be a major tumorigenic mechanism. Methods: We evaluated the methylation status of the p16^INK4a genes in 231 invasive breast cancer and 90 intraductal carcinoma specimens using a methylation-specific polymerase chain reaction and p16 protein expression using immunohistochemistry. The quantity of cell-free methylated p16^INK4a DNA in the plasma samples of 200 patients with invasive breast cancer was also examined using a fluorescence-based real-time polymerase chain reaction assay. Results: The frequencies of p16^INK4a methylation in invasive and intraductal tumors were 52.8% (122/231) and 57.8% (52/90), respectively. The p16 protein was overexpressed in 145 of the 231 invasive carcinomas (62.8%) and 63 of the 90 intraductal carcinomas (70%). High p16 expression in invasive carcinomas correlated significantly with a high histologic grade, a negative estrogen receptor and progesterone receptor status, p53 immunoreactivity and high Ki-67 expression with immunohistochemistry. In addition, the methylation index of p16^INK4a was significantly higher in the cancer patients than the normal controls (p<0.001). Conclusions: High p16 immunoreactivity correlated with a loss of differentiation in breast carcinomas and high frequency of p16^INK4a promoter methylation in both invasive and intraductal carcinomas, suggesting it may be involved in the pathogenesis of breast cancer.