DNA Methylation Analysis By Bisulfite Next Generation Sequencing To Early Detect Oral Squamous Cell Carcinoma From Oral Scrapings

Background: Oral squamous cell carcinoma (OSCC) is the most frequent neoplastic disease in head and neck region and it is commonly preceded by potentially malignant lesions. The discovery of highly sensitive and specific biomarkers to identify those lesions having a high risk to undergo malignant transformation is urgently required. Aim of the present study is to evaluate the methylation status of a list of candidate genes from oral scraping specimens to improve the current strategies for early cancer detection with non-invasive methods. Design: Oral scraping from 11 OSCC, 9 High Grade Squamous intraepithelial Lesion (HG-SIL), 9 Low Grade SIL( LG-SIL), 9 oral lichen planus (OLP) and 8 healthy donors were included in this study. PAP smear evaluation was done to confirm the presence of lesional cells within the brush. DNA was purified and bisulfite treated. A set of previously described differentially methylated genes in OSCC (GP1BB, ZAP70, KIF1A, p16[CDKN2A], CDH1, miR137, miR375) were investigated by bisulfite-Next Generation Sequencing (GSJunior, Roche, Branford, CT). ReadSeqs in Fasta format were analyzed by QuMA (http://quma.cdb.riken.jp/). The statistical significance between lesions and normal epithelia from the same patient and from a pool of healthy donors were evaluated with the Mann-Whitney U-test. Additionally TP53 mutation analysis for exon 4-9 were performed by the same NGS platform. Results: ZAP70 was found to be hypermethylated in 100% of OSCC and HG-SIL cases, in 28.5% of LG-SIL and in none of OLP. GP1BB hypomethylation was detected in 90.9% OSCC, in 88.8% of HG-SIL, 37.5% of LG-SIL and in none of OLP. MIR137 was hypermethylated in 100% of OLP, while only in 44.4% of OSCC, 50% in HG-SIL, 25% in LG-SIL. Hypermethylation in proximal promoter of KIF1A was detected in 54.5% OSCC, 33.3% HG-SIL, 50% LG-SIL and 0% in OLP. No epigenetic aberrations were detected in normal healthy donors. p16, CDH1 and miR375 did not revealed variations in the methylation pattern for all the classes. Conclusions: In our preliminary results, Bisulfite-NGS analysis of GP1BB, ZAP70 and miR137 promoters from oral scrapings allows to discriminate OSCC and HG-SIL from LG-SIL, OLP and normal oral mucosa. HG-SILs share the same epigenetic modifications of OSCC. These data confirm that CpG methylation changes may play a role in oral cancer progression and that DNA methylation analysis may have significant utility in early detection of OSCC. Furthermore the method here proposed is non invasive and can be applied to screen patients.