Prognostic impact of DNA methylation analysis in adjacent area of surgically resected oral squamous cell carcinoma during follow up

Background & Objectives: Oral Squamous Cell Carcinoma (OSCC) showed a significant risk to develop local recurrences or second primary tumours during follow-up. Recently, we developed a non-invasive assay based on oral brushing and DNA methylation analysis to early detect OSCC. In the present study using this assay, we investigated the adjacent area of surgical resection in a series of OSCC during follow-up for prog- nostic purposes. Methods: 42 consecutive OSCC patients were sampled during routine follow-up after 6 months from surgical treatment, brushing the regenera- tive mucosa covering the region that underwent the surgical OSCC exci- sion. DNA methylation level of ZAP70, GP1BB, KIF1A, ITGA4, LINC00599, MIR193, MIR296, TERT, LRRTM1, NTM, EPHX3, FLI1 and PARP15 was evaluated by quantitative Bisulfite-NGS. After calcu- lating a score by Linear-Discriminant-Analysis, the samples were dichot- omized using a predefined threshold previously developed for early di- agnosis. One-Way-ANOVA and Kaplan-Meier curves served to evaluate any significant difference between patients who experienced a second neoplastic manifestation and the group who did not. Results: 6/42 (14,3%) patients developed a second neoplastic manifesta- tion during follow-up period (mean follow-up: 14.3 months), of which 5 showed a positive methylation score. Additional 11 patients exceeded the threshold but up to date they have not experienced any second manifes- tation. Among the remaining 26 negatives, only one developed a recur- rence. A positive score correlated with a worse locoregional control of disease (p<0.05). Conclusion: The DNA methylation analysis of 13 genes can be a useful non-invasive method to identify surgically treated OSCC patients at risk of developing a second neoplasia.