The study of p16 and p15 gene methylation in head and neck squamous cell carcinoma and their quantitative evaluation in plasma by real-time PCR.

Epigenetic silencing of the p16 and p15 genes by promoter methylation are commonly observed in human epithelial malignancies, including head and neck squamous cell carcinomas (HNSCC). In this study, a methylation-specific polymerase chain reaction (MSP) was used to evaluate the methylation status of the p16 and p15 genes in 73 HNSCC surgical specimens. p16 and p15 gene methylation was also examined in 29 paired metastatic lymph nodes and 29 paired histologically, normal resection margin mucosae. The quantity of cell-free methylated p16 and p15 DNA in the plasma samples of 20 HNSCC patients and 24 healthy controls was also examined using a fluorescence-based real-time PCR assay. The frequencies of p16 and p15 methylation in the primary tumour were 49% and 60%, respectively. Concordant methylation of p16 and p15 in tumour samples and metastatic lymph nodes was found in 59 and 38% of cases, respectively. A significantly higher prevalence of p15 methylation was found in histologically-normal surgical margin epithelia of HNSCC patients with chronic smoking and drinking habits compared with non-smokers and non-drinkers. In addition, methylated p16 and p15 DNA levels were significantly higher in the plasma of HNSCC patients (mean 56 copies/ml plasma and 65 copies/ml plasma, respectively) compared with normal controls (mean 6 copies/ml plasma and 16 copies/ml plasma, respectively). In conclusion, promoter methylation of the p16 and p15 genes is involved in the pathogenesis of HNSCC and may be related to chronic smoking and drinking. The differential levels of methylated p16 and p15 DNA in plasma might be potential useful markers in screening high-risk populations for early HNSCC and monitoring their treatment response.