Prognostic significance of tumor-related genes hypermethylation detected in cancer-free surgical margins of oral squamous cell carcinomas

Oral squamous cell carcinoma (OSCC) is characterized by high mortality rates. High incidence of local recurrences in the normal-appearing surgical margins of OSCC patients indicates the existence molecular alterations, including DNA methylation, which could not be detectable by histopathologic analysis. The objective of our study was to determine correlation of tumor-related genes hypermethylation detected in histopathologically negative surgical margins with clinical and prognostic parameters. The genes selected for methylation analysis covered a wide range cellular processes including cell cycle control (p16), apoptosis (DAPK and RASSF1A), Wnt signaling (APC, WIF1 and RUNX3), cell-cell adhesion (E-cad), and DNA repair (MGMT and hMLH1). All of 47 patients had histologically confirmed negative surgical margins. For each of patient, samples from primary malignant tissue and the two consecutive surgical margins were taken at the time of surgery. DNA methylation was determined by multiplex nested methylation-specific PCR. Twenty-seven patients were margin-positive for promoter hypermethylation of at least one gene under study. The presence of DAPK promoter hypermethylation detected in surgical margins was associated with the decreased overall survival (p = 0.004, log rank test). Multivariate analysis revealed that DAPK promoter hypermethylation in surgical margins is an independent prognostic factor for overall survival, HR = 4.105 (1.458-11.555, 95% CI, p = 0.007). Hypermethylation of other tumor-related genes under study did not have prognostic significance. These results demonstrate that DNA hypermethylation in histologically negative surgical margins is a frequent event. Promoter hypermethylation of DAPK gene detected in surgical margins may be a useful molecular marker for the poor survival in OSCC patients. Further investigation into the therapeutic potential of these findings in OSCC is warranted.