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Updated: Jun 2, 2026
Genome-Wide Analysis of DNA Methylation in Gastrointestinal Cancer
Published on: September 18, 2020
A 115-bp MethyLight assay for detection of p16 (CDKN2A) methylation as a diagnostic biomarker in human tissues
Jing Zhou1, Jie Cao, Zheming Lu
1Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, Department of Aetiology, Peking University Cancer Hospital & Institute, Beijing, China.
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.
Insights
A novel 115-bp MethyLight assay offers high specificity for detecting p16 methylation in oral dysplasia, aiding in early cancer risk prediction. This method proves practical for clinical use in identifying patients at higher risk of malignant transformation.
Area of Science:
- Oncology
- Molecular Diagnostics
- Biomarker Discovery
Background:
- p16 methylation is a potential biomarker for predicting malignant transformation of epithelial dysplasia.
- Quantitative methylation-specific PCR (MSP) methods like MethyLight show promise for clinical detection of p16 methylation.
- Previous studies linked p16 methylation to significantly higher oral cancer risk in epithelial dysplasia patients.
Purpose of the Study:
- To evaluate the clinical utility of a novel 115-bp MethyLight assay for detecting p16 methylation in oral dysplasia.
- To compare the performance of the 115-bp MethyLight assay against a classic 70-bp MethyLight assay and established MSP methods.
- To assess the correlation between p16 methylation detected by the novel assay and oral cancer development in a patient cohort.
Main Methods:
- Analysis of 102 oral mucosa biopsy samples from 78 patients with epithelial dysplasia using 70-bp and 115-bp MethyLight assays.
- Comparison of MethyLight assay results with a 150-bp MSP and bisulfite sequencing as the golden standard.
- Evaluation of assay sensitivity, specificity, and accuracy, and correlation with oral cancer development over a 4-year follow-up period.
Main Results:
- The 115-bp MethyLight assay detected p16 methylation in 26 of 44 MSP-positive and 2 of 58 MSP-negative samples (P = 0.000).
- The 115-bp MethyLight assay demonstrated high specificity (98.3%) and accuracy (57.4%) compared to MSP.
- A significant association was found between p16 methylation detected by the 115-bp MethyLight assay and oral cancer development in older patients (≥60 years) (P = 0.034).
Conclusions:
- The 115-bp MethyLight assay is a highly specific and practical method for clinical detection of p16 methylation.
- This assay can aid in identifying patients with oral epithelial dysplasia at higher risk for malignant transformation.
- The findings support the use of the 115-bp MethyLight assay for improved clinical management and early cancer prediction.
