title: Hypermethylation of CDKN2A exon 2 in tumor, tumor-adjacent and tumor-distant tissues from breast cancer patients.
abstract: BACKGROUND
  Breast carcinogenesis is a multistep process involving genetic and epigenetic changes. Tumor tissues are frequently characterized by gene-specific hypermethylation and global DNA hypomethylation. Aberrant DNA methylation levels have, however, not only been found in tumors, but also in tumor-surrounding tissue appearing histologically normal. This phenomenon is called field cancerization. Knowledge of the existence of a cancer field and its spread are of clinical relevance. If the tissue showing pre-neoplastic lesions is not removed by surgery, it may develop into invasive carcinoma.
  
  
  METHODS
  We investigated the prevalence of gene-specific and global DNA methylation changes in tumor-adjacent and tumor-distant tissues in comparison to tumor tissues from the same breast cancer patients (n = 18) and normal breast tissues from healthy women (n = 4). Methylation-sensitive high resolution melting (MS-HRM) analysis was applied to determine methylation levels in the promoters of APC, BRCA1, CDKN2A (p16), ESR1, HER2/neu and PTEN, in CDKN2A exon 2 and in LINE-1, as indicator for the global DNA methylation extent. The methylation status of the ESR2 promoter was determined by pyrosequencing.
  
  
  RESULTS
  Tumor-adjacent and tumor-distant tissues frequently showed pre-neoplastic gene-specific and global DNA methylation changes. The APC promoter (p = 0.003) and exon 2 of CDKN2A (p < 0.001) were significantly higher methylated in tumors than in normal breast tissues from healthy women. For both regions, significant differences were also found between tumor and tumor-adjacent tissues (p = 0.001 and p < 0.001, respectively) and tumor and tumor-distant tissues (p = 0.001 and p < 0.001, respectively) from breast cancer patients. In addition, tumor-adjacent (p = 0.002) and tumor-distant tissues (p = 0.005) showed significantly higher methylation levels of CDKN2A exon 2 than normal breast tissues serving as control. Significant correlations were found between the proliferative activity and the methylation status of CDKN2A exon 2 in tumor (r = -0.485, p = 0.041) and tumor-distant tissues (r = -0.498, p = 0.036).
  
  
  CONCLUSIONS
  From our results we can conclude that methylation changes in CDKN2A exon 2 are associated with breast carcinogenesis. Further investigations are, however, necessary to confirm that hypermethylation of CDKN2A exon 2 is associated with tumor proliferative activity.
authors.affiliation: Department of Analytical Chemistry, University of Vienna, Währinger Str. 38, 1090, Vienna, Austria.
authors.forename: Melanie
authors.initials: M
authors.lastname: Spitzwieser
authors.affiliation: Department of Analytical Chemistry, University of Vienna, Währinger Str. 38, 1090, Vienna, Austria.
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Entfellner
authors.affiliation: Department of Analytical Chemistry, University of Vienna, Währinger Str. 38, 1090, Vienna, Austria.
authors.forename: Bettina
authors.initials: B
authors.lastname: Werner
authors.affiliation: Molecular Diagnostics, Austrian Institute of Technology, Muthgasse 11, 1190, Vienna, Austria.
authors.forename: Walter
authors.initials: W
authors.lastname: Pulverer
authors.affiliation: Department of Obstetrics and Gynecology, Division of Gynecology and Gynecological Oncology, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria.
authors.forename: Georg
authors.initials: G
authors.lastname: Pfeiler
authors.affiliation: Department of Plastic and Reconstructive Surgery, Medical University of Vienna, Währinger Gürtel 18-20, 1090, Vienna, Austria.
authors.forename: Stefan
authors.initials: S
authors.lastname: Hacker
authors.affiliation: Department of Analytical Chemistry, University of Vienna, Währinger Str. 38, 1090, Vienna, Austria. margit.cichna@univie.ac.at.
authors.forename: Margit
authors.initials: M
authors.lastname: Cichna-Markl
mesh_terms: D000328:Adult; D000368:Aged; D001943:Breast Neoplasms / Q000235:genetics*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050763:Cyclin-Dependent Kinase Inhibitor p18 / Q000235:genetics*; D019175:DNA Methylation; D044127:Epigenesis, Genetic; D047629:Estrogen Receptor beta / Q000235:genetics*; D005091:Exons; D005260:Female; D006801:Humans; D061986:MCF-7 Cells; D008875:Middle Aged; D011401:Promoter Regions, Genetic; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: C000614131:CDKN2A protein, human; D019941:Cyclin-Dependent Kinase Inhibitor p16; D050763:Cyclin-Dependent Kinase Inhibitor p18; C000600034:ESR2 protein, human; D047629:Estrogen Receptor beta
keywords: Breast cancer; CDKN2A; DNA methylation; Exon 2; Field cancerization; Methylation-sensitive high resolution melting; Pyrosequencing; Tumor suppressor genes; Tumor-adjacent tissue; Tumor-distant tissue