p16 hypermethylation is observed as an early event in mammary carcinogenesis.

B127

Background: p16(INK4a) is a key regulator of cell-cycle progression and p16(INK4a) loss promotes carcinogenesis. Models of early mammary carcinogenesis predict that histologically normal-appearing human mammary epithelial cells with silenced p16(INK4a) represent early precursors of breast cancer [Reynolds, et al., JBC, 2006]. Methods: The frequency of p16(INK4a) promoter hypermethylation was tested in the initial Random Periareolar Fine Needle Aspiration (RPFNA) samples obtained from 107 asymptomatic women at high risk for development of breast cancer. RPFNA specimens were stratified using the Masood Cytology Index. Results: p16(INK4a) promoter hypermethylation was observed throughout all early stages of intraepithelial neoplasia, and importantly, in morphologically normal-appearing mammary epithelial cells from high-risk women: 1) 24% (4/17) non-proliferative (normal) cytology (Masood ≤10), 2) 26% (16/62) hyperplastic cytology (Masood 11-13), and 3) 39% (16/41) atypical cytology (Masood 14-17). Loss of heterozygosity at the INK4a/ARF locus was an infrequent event (1/23 atypical specimens). The frequency of p16(INK4a) promoter hypermethylation was not associated with age, atypia, family history of breast/ovarian cancer, or Gail risk score; however, there was an association with decreasing BRCAPRO risk scores (p=0.027) and the combined frequency of promoter hypermethylation of retinoic acid receptor-beta2, estrogen receptor-alpha, and breast cancer associated-1 genes (p=0.001). Significance: Since p16(INK4a) promoter hypermethylation does not increase with age but does increase with the frequency of other methylation events, we predict that p16(INK4a) promoter hypermethylation may serve as a marker of global methylation dysregulation and early predictor of short-term progression.