A 115-bp MethyLight assay for detection of p16 (CDKN2A) methylation as a diagnostic biomarker in human tissues
1 Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Department of Aetiology, Peking University Cancer Hospital & Institute, Beijing, 100142, China
2 Department of Oral Medicine, Peking University School of Stomatology, Beijing 100081, China
BMC Medical Genetics 2011, 12:67 doi:10.1186/1471-2350-12-67Published: 13 May 2011
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.
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).
The 115-bp MethyLight assay is a useful and practical assay with very high specificity for the detection of p16 methylation clinically.