Evaluation of DNA methylation levels of SEPT9 and SHOX2 in plasma of patients with head and neck squamous cell carcinoma using droplet digital PCR

Oncol Rep. 2024 Mar;51(3):52. doi: 10.3892/or.2024.8711. Epub 2024 Feb 1.

Abstract

Head and neck squamous cell carcinoma (HNSCC) is the seventh most commonly diagnosed cancer globally. HNSCC develops from the mucosa of the oral cavity, pharynx and larynx. Methylation levels of septin 9 (SEPT9) and short stature homeobox 2 (SHOX2) genes in circulating cell‑free DNA (ccfDNA) are considered epigenetic biomarkers and have shown predictive value in preliminary reports in HNSCC. Liquid biopsy is a non‑invasive procedure that collects tumor‑derived molecules, including ccfDNA. In the present study, a droplet digital PCR (ddPCR)‑based assay was developed to detect DNA methylation levels of circulating SEPT9 and SHOX2 in the plasma of patients with HNSCC. The assay was first set up using commercial methylated and unmethylated DNA. The dynamic changes in the methylation levels of SEPT9 and SHOX2 were then quantified in 20 patients with HNSCC during follow‑up. The results highlighted: i) The ability of the ddPCR‑based assay to detect very low copies of methylated molecules; ii) the significant decrease in SEPT9 and SHOX2 methylation levels in the plasma of patients with HNSCC at the first time points of follow‑up with respect to T0; iii) a different trend of longitudinally DNA methylation variations in small groups of stratified patients. The absolute and precise quantification of SEPT9 and SHOX2 methylation levels in HNSCC may be useful for studies with translational potential.

Keywords: DNA methylation; cell free DNA; droplet digital PCR; head and neck squamous cell carcinoma; liquid biopsy.

MeSH terms

  • Biomarkers, Tumor / metabolism
  • Carcinoma, Squamous Cell* / pathology
  • Cell-Free Nucleic Acids* / genetics
  • Cytoskeletal Proteins / genetics
  • DNA Methylation
  • Genes, Homeobox
  • Head and Neck Neoplasms* / genetics
  • Homeodomain Proteins / genetics
  • Homeodomain Proteins / metabolism
  • Humans
  • Polymerase Chain Reaction
  • Squamous Cell Carcinoma of Head and Neck / genetics

Substances

  • Homeodomain Proteins
  • Cytoskeletal Proteins
  • Cell-Free Nucleic Acids
  • Biomarkers, Tumor
  • SHOX2 protein, human

Grants and funding

The present study was supported by Fondazione Spedali Civili. The research was also funded by CIB (Biotechnology Interuniversity Consortium, Italy) grant no. 12/10/2020, and by the University of Brescia (local grants; nos. 60/2021 and 60/2022). The funding bodies played no role in the design of the study and in the collection, analysis, and interpretation of data, or in the writing of the manuscript.