title: A 115-bp MethyLight assay for detection of p16 (CDKN2A) methylation as a diagnostic biomarker in human tissues.
abstract: BACKGROUND
  p16 Methylation is a potential biomarker for prediction of malignant transformation of epithelial dysplasia. A probe-based, quantitative, methylation-specific PCR (MSP) called MethyLight may become an eligible method for detecting this marker clinically. We studied oral mucosa biopsies with epithelial dysplasia from 78 patients enrolled in a published 4-years' followup cohort, in which cancer risk for patients with p16 methylation-positive dysplasia was significantly higher than those without p16 methylation (by 150-bp MSP and bisulfite sequencing; +133 ~ +283, transcription starting site, +1). The p16 methylation status in samples (N = 102) containing sufficient DNA was analyzed by the 70-bp classic (+238 ~ +307) and 115-bp novel (+157 ~ +272) MethyLight assays, respectively.
  
  
  RESULTS
  p16 Methylation was detectable in 75 samples using the classic MethyLight assay. The methylated-p16 positive rate and proportion of methylated-p16 by the MethyLight in MSP-positive samples were higher than those in MSP-negative samples (positive rate: 37/44 vs. 38/58, P=0.035, two-sided; proportion [median]: 0.78 vs. 0.02, P <0.007). Using the published results of MSP as a golden standard, we found sensitivity, specificity, and accuracy for this MethyLight assay to be 70.5%, 84.5%, and 55.0%, respectively. Because amplicon of the classic MethyLight procedure only partially overlapped with the MSP amplicon, we further designed a 115-bp novel MethyLight assay in which the amplicon on the sense-strand fully overlapped with the MSP amplicon on the antisense-strand. Using the 115-bp MethyLight assay, we observed methylated-p16 in 26 of 44 MSP-positive samples and 2 of 58 MSP-negative ones (P = 0.000). These results were confirmed with clone sequencing. Sensitivity, specificity, and accuracy using the 115-bp MethyLight assay were 59.1%, 98.3%, and 57.4%, respectively. Significant differences in the oral cancer rate were observed during the followup between patients (≥60 years) with and without methylated-p16 as detected by the 115-bp MethyLight assay (6/8 vs. 6/22, P = 0.034, two-sided).
  
  
  CONCLUSIONS
  The 115-bp MethyLight assay is a useful and practical assay with very high specificity for the detection of p16 methylation clinically.
authors.affiliation: Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, Department of Aetiology, Peking University Cancer Hospital & Institute, Beijing, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhou
authors.forename: Jie
authors.initials: J
authors.lastname: Cao
authors.forename: Zheming
authors.initials: Z
authors.lastname: Lu
authors.forename: Hongwei
authors.initials: H
authors.lastname: Liu
authors.forename: Dajun
authors.initials: D
authors.lastname: Deng
mesh_terms: D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D015331:Cohort Studies; D018899:CpG Islands / Q000235:genetics*; D019175:DNA Methylation / Q000235:genetics*; D004847:Epithelial Cells / Q000473:pathology; D019942:Genes, p16; D005819:Genetic Markers / Q000235:genetics*; D006801:Humans; D009061:Mouth Mucosa / Q000166:cytology; D009369:Neoplasms / Q000175:diagnosis* / Q000235:genetics / Q000473:pathology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011230:Precancerous Conditions / Q000235:genetics* / Q000473:pathology; D012680:Sensitivity and Specificity
chemical_list: D005819:Genetic Markers