title: Promoter Methylation of CDKN2A, RARβ, and RASSF1A in Non-Small Cell Lung Carcinoma: Quantitative Evaluation Using Pyrosequencing.
abstract: BACKGROUND
  While qualitative analysis of methylation has been reviewed, the quantitative analysis of methylation has rarely been studied. We evaluated the methylation status of CDKN2A, RARβ, and RASSF1A promoter regions in non-small cell lung carcinomas (NSCLCs) by using pyrosequencing. Then, we evaluated the association between methylation at the promoter regions of these tumor suppressor genes and the clinicopathological parameters of the NSCLCs.
  
  
  METHODS
  We collected tumor tissues from a total of 53 patients with NSCLCs and analyzed the methylation level of the CDKN2A, RARβ, and RASSF1A promoter regions by using pyrosequencing. In addition, we investigated the correlation between the hypermethylation of CDKN2A and the loss of p16(INK4A) immunoexpression.
  
  
  RESULTS
  Hypermethylation of CDKN2A, RARβ, and RASSF1A promoter regions were 16 (30.2%), 22 (41.5%), and 21 tumors (39.6%), respectively. The incidence of hypermethylation at the CDKN2A promoter in the tumors was higher in undifferentiated large cell carcinomas than in other subtypes (p=0.002). Hyperrmethylation of CDKN2A was significantly associated with p16(INK4A) immunoexpression loss (p=0.045). With regard to the clinicopathological characteristics of NSCLC, certain histopathological subtypes were found to be strongly associated with the loss of p16(INK4A) immunoexpression (p=0.016). Squamous cell carcinoma and undifferentiated large cell carcinoma showed p16(INK4A) immunoexpression loss more frequently. The Kaplan-Meier survival curves analysis showed that methylation level and patient survival were barely related to one another.
  
  
  CONCLUSIONS
  We quantitatively analyzed the promoter methylation status by using pyrosequencing. We showed a significant correlation between CDKN2A hypermethylation and p16(INK4A) immunoexpression loss.
authors.affiliation: Department of Pathology, St. Mary's Hospital, The Catholic University of Korea School of Medicine, Daejeon, Korea.
authors.forename: Jung Uee
authors.initials: JU
authors.lastname: Lee
authors.forename: Hae Joung
authors.initials: HJ
authors.lastname: Sul
authors.forename: Ji Woong
authors.initials: JW
authors.lastname: Son
keywords: Carcinoma, Non-Small Cell Lung; DNA Methylation; Genes, p16; RASSF1 Protein, Human; Receptors, Retinoic Acid; Sequence Analysis, DNA