title: Prognostic significance of p16(INK4a) hypermethylation in non-small cell lung cancer is evident by quantitative DNA methylation analysis.
abstract: BACKGROUND
  p16(INK4a) is a tumor suppressor gene frequently inactivated by aberrant promoter hypermethylation. In the present study, p16(INK4a) methylation was evaluated in non-small cell lung cancer (NSCLC) using a quantitative assay and the clinical significance of the methylation was explored.
  
  
  METHODS
  A total of 244 tumor samples from formalin-fixed paraffin-embedded archives were examined in this study. p16(INK4a) methylation was analyzed by the fluorescence-based, real-time methylation-specific PCR assay, MethyLight. The quantitative methylation value was expressed as the percentage of methylated reference (PMR).
  
  
  RESULTS
  The median level of p16(INK4) methylation was 0.55 PMR (range 0.00-503.4). The p16(INK4) methylation value was significantly higher in males (p = 0.005) and in squamous cell carcinoma (p = 0.018). Prognostic analysis using the Cox proportional hazard model showed that the p16(INK4a) methylation value was a significant prognostic factor (odds ratio, 1.005; 95% CI, 1.003 to 1.008; p < 0.0001). The p16(INK4a) methylation value remained a significant prognostic factor (p = 0.0004) in multivariate analysis including age, gender, histological type and clinical stage. Specimens were then classified into hypermethylated or non-hypermethylated groups based on the p16(INK4a) methylation value using various cut-offs from 1 to 100 PMR. There was no significant difference in prognosis between the two groups using a cut-off value of 1 PMR. On the other hand, there was a significant difference using 6 PMR or more as the cut-off value (p < 0.01).
  
  
  CONCLUSIONS
  These results provide clear evidence for the prognostic significance of p16(INK4a) methylation in NSCLC using quantitative DNA methylation analysis. Careful assessment of DNA methylation is needed because qualitative methylation analysis may overestimate low levels of methylation, which have less clinical significance.
authors.affiliation: Department of Surgery, Kanazawa University School of Medicine, Ishikawa 920-8641, Japan.
authors.forename: Naohiro
authors.initials: N
authors.lastname: Ota
authors.forename: Kazuyuki
authors.initials: K
authors.lastname: Kawakami
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Okuda
authors.forename: Akira
authors.initials: A
authors.lastname: Takehara
authors.forename: Chikashi
authors.initials: C
authors.lastname: Hiranuma
authors.forename: Kaeko
authors.initials: K
authors.lastname: Oyama
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Ota
authors.forename: Makoto
authors.initials: M
authors.lastname: Oda
authors.forename: Go
authors.initials: G
authors.lastname: Watanabe
mesh_terms: D000368:Aged; D002289:Carcinoma, Non-Small-Cell Lung / Q000235:genetics* / Q000473:pathology; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D019175:DNA Methylation; D005260:Female; D006801:Humans; D008175:Lung Neoplasms / Q000235:genetics* / Q000473:pathology; D008297:Male; D008875:Middle Aged; D011379:Prognosis; D011401:Promoter Regions, Genetic; D016019:Survival Analysis
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16