DNA methylation analysis by bisulfite next-generation sequencing for early detection of oral squamous cell carcinoma and high-grade squamous intraepithelial lesion from oral brushing

J Craniomaxillofac Surg. 2015 Oct;43(8):1494-500. doi: 10.1016/j.jcms.2015.07.028. Epub 2015 Aug 4.


Purpose: Oral squamous cell carcinoma (OSCC) is commonly preceded by oral potentially malignant lesions (OPML). The aim of the present study was to assess, by bisulfite next-generation sequencing (NGS), the methylation status of a list of candidate genes obtained from oral brushings to early detect OPML and OSCC.

Material and methods: Oral brushing specimens from 11 OSCC, 11 high-grade squamous intraepithelial lesions (HG-SIL), 9 low-grade SIL (LG-SIL), 9 oral lichen planus (OLP), and 8 healthy donors were included in this study. We investigated, by means of bisulfite NGS, the promoter of GP1BB, ZAP70, KIF1A, p16[CDKN2A], CDH1, miR137, and miR375. Statistical significance between lesions and a pool of healthy donors were evaluated with the Mann-Whitney U test.

Results: ZAP70 was found to be hypermethylated in 100% of OSCC and HG-SIL and in 28.6% of LG-SIL. GP1BB hypomethylation was detected in 90.9% OSCC and HG-SIL and in 37.5% of LG-SIL. MiR137 was hypermethylated in 100% of OLP, 44.4% of OSCC, 40% HG-SIL, and 25% LG-SIL. KIF1A hypermethylation was found to be associated with TP53 mutations (p < 0.0001).

Conclusion: In the present preliminary cohort of patients, DNA methylation analysis of GP1BB and ZAP70 seems to be a promising noninvasive tool for early detection of OSCC and HG SIL from oral brushing specimens.

Keywords: DNA methylation; Next generation sequencing; Oral scraping; Oral squamous cell carcinoma.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aged
  • Aged, 80 and over
  • Antigens, CD
  • Cadherins / genetics
  • Carcinoma in Situ / diagnosis*
  • Carcinoma in Situ / genetics
  • Carcinoma, Squamous Cell / diagnosis*
  • Carcinoma, Squamous Cell / genetics
  • Cohort Studies
  • CpG Islands / genetics
  • Cyclin-Dependent Kinase Inhibitor p16 / genetics
  • Cytodiagnosis / methods*
  • DNA Methylation* / genetics
  • Early Detection of Cancer / methods*
  • Exons / genetics
  • Female
  • Humans
  • Kinesins / genetics
  • Lichen Planus, Oral / genetics
  • Male
  • MicroRNAs / genetics
  • Middle Aged
  • Mouth Neoplasms / diagnosis*
  • Mouth Neoplasms / genetics
  • Mutation / genetics
  • Platelet Glycoprotein GPIb-IX Complex / genetics
  • Promoter Regions, Genetic / genetics
  • Sequence Analysis, DNA / methods*
  • Sulfites
  • Tumor Suppressor Protein p53 / genetics
  • ZAP-70 Protein-Tyrosine Kinase / genetics


  • Antigens, CD
  • CDH1 protein, human
  • Cadherins
  • Cyclin-Dependent Kinase Inhibitor p16
  • MIRN137 microRNA, human
  • MIRN375 microRNA, human
  • MicroRNAs
  • Platelet Glycoprotein GPIb-IX Complex
  • Sulfites
  • TP53 protein, human
  • Tumor Suppressor Protein p53
  • adhesion receptor
  • ZAP-70 Protein-Tyrosine Kinase
  • ZAP70 protein, human
  • Kinesins
  • hydrogen sulfite