Event Abstract Back to Event Analysis of factors that may influence the methylation pattern of oral mucosa Sara Grillini1*, Andrea Gabusi1, Roberto Rossi1, Linda Sozzi1, Luca Morandi1 and Davide Bartolomeo Gissi1 1 University of Bologna, Department of Biomedical and Neuromotor Sciences, Section of Oral Sciences, Italy Aim. Different studies have recently proposed an attractive strategy to reduce the burden of OSCC by analyzing the methylation status of a panel of genes starting from saliva and/or brushing specimens. In a recent paper, using the oral brushing as a non-invasive sampling method, we analyzed the mehtylation level of a pre-selected panel of 13 genes and we correctly stratified OSCC from a selected group of healthy donors (sensitivity 97.1%, specificity 100%, AUC 0.981). However, the oral cavity is characterized by the presence of different types of conditions: benign inflammatory conditions, infective or reactive lesions that may potentially influence the methylation level of one or more genes. In the present pilot study we investigated the variables that may influence our non invasive procedure based on quantitative DNA methylation analysis. For this reason we analyzed three different groups of patients: a group of 10 patients with periodontitis, a group of 10 patients with benign reactive lesions and a group of 18 smokers. Materials and Methods. Epithelial cells from oral brushing were collected in three different groups of patients: 10 patients with diagnosis of periodontitis, 10 patients with presence of benign reactive lesions (6 fibroma, 1 papilloma, 3 aspecific ulcerated lesions) and 18 heavy smokers without presence of lesions in oral mucosa. Epithelial cells were collected in patients with periodontisis only in gingival area before any surgical and/or non surgical treatment. In the patients with benign reactive lesions brushing cell collection was always performed before incisional and/or excisional biopsy without any local anesthetic and lesions were included in the study group only after histological analysis. Study population was also composed by 29 patients with a histological diagnosis of OSCC, considered as positive control and 65 healthy donors, considered as negative control. In all brushing specimens the DNA methylation level of 13 genes (ZAP70, GP1BB, KIF1A, ITGA4, LINC00599, MIR193, MIR296, TERT, LRRTM1, NTM, EPHX3, FLI1 and PARP15) was evaluated by quantitative Bisulfite-Next Generation Sequencing (NGS). Each sample was considered either as a numeric and a dichotomic variable (pos/neg) in relationship to a pre-definite cut off value based on our developed algorithm. Statistical analysis: Chi Square analysis and Kruskall Wallis test with multiple range test were used to evaluate the presence of any between-group significant difference. Kruskall Wallis test with multiple range test was also used to evaluate the presence of any between group difference in the methylation level of each gene. The outcome of interest of this second analysis was the identification of one or more genes with a similar methylation pattern between studies groups (periodontitis, smokers and reactive lesions) and OSCC group (positive control). P values <0.05 were considered statistically significant in all analyses. Results. Chi Square analysis and Kruskall Wallis test taking into account the panel of 13 genes: 0/65 healthy donors, 0/18 smoking patients, 0/10 patients with benign reactive lesions, 2/10 patients with periodontitis, 28/29 OSCCs and 0/65 healthy donors were detected as positive. Kruskall Wallis test identified a significant between group difference (T 67,3341; p<.05). OSCC group showed significant higher values with respect to other groups; but a significant difference was also found between group of patients with periodontitis and group of healthy donors, smokers and benign reactive lesions. Kruskall Wallis analysis and (MRT) applied to each single gene: The analysis identified the presence of three genes (GP1BB, miR193 and miR296) without any significant difference in methylation level between OSCC group and the group of patients with periodontitis. Chi Square analysis and Kruskall Wallis test taking into account a panel of 10 genes (without GP1BB, miR193, miR296): In order to increase the specificity, a new algorithm and a new cut off level based on methylation level of 10 remaining genes without GP1BB, miR193 and miR296 was performed. This new analysis identified 26/29 (89,6%) OSCC, 0/65 healthy donors, 0/18 smokers, 0/10 benign reactive lesions and 0/10 periodontitis as positive. Kruskall Wallis analysis showed a significant between group difference(T 63,3275; p<.05) and MRT identified a significant difference between OSCC group and all other groups. No significant differences were found between healthy donors, smokers and reactive lesions. Discussion. These data confirmed the value of our novel assay in the analysis of a suspected lesion. Indeed, none of benign reactive lesions showed a positive value, in contrast with 28/29 malignant lesions detected as positive. At the same time, these preliminary data suggested the possibility that not only OSCC patients but also a part of patients with chronic periodontitis may be characterized by an altered methylation pattern. (2) In particular in our study population three genes GP1BB, miR193 and miR296 showed a similar methylation pattern between chronic periodontitis and OSCC. The development of a new algorithm and a new cut off based on a panel of 10 genes without GP1BB, miR193 and miR296 allowed us to identify all patients with chronic periodontitis as negative, even if the sensitivity of the procedure became lower (26/29 OSCC detected as positive with the new algorithm, sensitivity 89.7%). Large multicenter studies are necessary to evaluate the meaning of these data and the meaning of an altered methylation pattern in the pathogenesis of periodontitis.