Oral squamous cell carcinoma (OSCC) is usually diagnosed at an advanced stage and is commonly preceded by oral premalignant lesions. The mortality rates have remained unchanged (50% within 5 years after diagnosis), and it is related to tobacco smoking and alcohol intake. Novel molecular markers for early diagnosis are urgently needed. The purpose of this study was to evaluate the diagnostic value of methylation level in a set of 18 genes by bisulfite next-generation sequencing. With minimally invasive oral brushing, 28 consecutive OSCC, one squamous cell carcinoma with sarcomatoid features, six high-grade squamous intraepithelial lesions (HGSIL), 30 normal contralateral mucosa from the same patients, and 65 healthy donors were evaluated for DNA methylation analyzing 18 target genes by quantitative bisulfite next-generation sequencing. We further evaluated an independent cohort (validation dataset) made of 20 normal donors, one oral fibroma, 14 oral lichen planus (OLP), three proliferative verrucous leukoplakia (PVL), and two OSCC. Comparing OSCC with normal healthy donors and contralateral mucosa in 355 CpGs, we identified the following epigenetically altered genes: ZAP70, ITGA4, KIF1A, PARP15, EPHX3, NTM, LRRTM1, FLI1, MIR193, LINC00599, PAX1, and MIR137HG showing hypermethylation and MIR296, TERT, and GP1BB showing hypomethylation. The behavior of ZAP70, GP1BB, H19, EPHX3, and MIR193 fluctuated among different interrogated CpGs. The gap between normal and OSCC samples remained mostly the same (Kruskal-Wallis P values < 0.05), but the absolute values changed conspicuously. ROC curve analysis identified the most informative CpGs, and we correctly stratified OSCC and HGSIL from normal donors using a multiclass linear discriminant analysis in a 13-gene panel (AUC 0.981). Only the OSCC with sarcomatoid features was negative. Three contralateral mucosa were positive, a sign of a possible field cancerization. Among imprinted genes, only MIR296 showed loss of imprinting. DNMT1, TERC, and H19 together with the global methylation of long interspersed element 1 were unchanged. In the validation dataset, values over the threshold were detected in 2/2 OSCC, in 3/3 PVL, and in 2/14 OLP. Our data highlight the importance of CpG location and correct estimation of DNA methylation level for highly accurate early diagnosis of OSCC.

MicroRNAs have recently been proposed as non-invasive biomarkers in Oral Squamous Cell Carcinoma (OSCC). The aim of this study was to analyze the expression of a panel of miRNAs in epithelial cells collected by oral brushing from OSCCs from regenerative areas after OSCC surgical resection and from their respective normal distant mucosa. Oral brushing specimens were collected from 24 healthy donors, 14 OSCC patients with specimens from tumour and normal distant mucosa, and from 13 patients who had OSCC resection, with samples from regenerative areas after OSCC resection and normal distant mucosa. Expression levels of eight targets (miR-21, miR-375, miR-345, miR-181b, miR-146a, miR-649, miR-518b, and miR-191) were evaluated by real-time Polymerase Chain Reaction (PCR). A highly significant between-group difference was found for miR-21 (F = 6.58, p < 0.001), miR-146a (F = 6.974, p < 0.001), and miR-191 (F = 17.07, p < 0.001). The major difference was observed between samples from healthy donors and from OSCC brushing, whereas no significant differences were observed between areas infiltrated by OSCC and their respective normal distant mucosa. Furthermore, altered expression of miR-146a and miR-191 was also observed in regenerative areas after OSCC resection. Conclusions: Oral brushing could be proposed as a noninvasive method to study microRNA expression in oral mucosa in OSCC patients.

Background: Prognosis of oral squamous cell carcinoma (OSCC) is difficult to exactly assess on pre-operative biopsies. Since OSCC DNA methylation profile has proved to be a useful pre-operative diagnostic tool, the aim of the present study was to evaluate the prognostic impact of DNA methylation profile to discriminate OSCC with high and low aggressive potential. Methods: 36 OSCC cases underwent neoplastic cells collection by gentle brushing of the lesion, before performing a pre-operative biopsy. The CpG islands methylation status of 13 gene (ZAP70, ITGA4, KIF1A, PARP15, EPHX3, NTM, LRRTM1, FLI1, MiR193, LINC00599, MiR296, TERT, GP1BB) was studied by bisulfite Next Generation Sequencing (NGS). A Cox proportional hazards model via likelihood-based component-wise boosting was used to evaluate the prognostic power of the CpG sites. Results: The boosting estimation identified five CpGs with prognostic significance: EPHX3-24, EPHX3-26, ITGA4-3, ITGA4-4, and MiR193-3. The combination of significant CpGs provided promising results for adverse events prediction (Brier score = 0.080, C-index = 0.802 and AUC = 0.850). ITGA4 had a strong prognostic power in patients with early OSCC. Conclusions: These data confirm that the study of methylation profile provides new insights into the molecular mechanisms of OSCC and can allow a better OSCC prognostic stratification even before surgery.