Analysis of genetic variants of frequently mutated genes in human papillomavirus-negative primary head and neck squamous cell carcinoma, resection margins, local recurrences and corresponding circulating cell-free DNA.

Head and neck squamous cell carcinoma remains a substantial burden to global health. Despite evolving therapies, 5-year survival is <50% and unlike in other cancers, reliable molecular biomarkers to guide treatment do not exist. We performed targeted panel next-generation sequencing to analyse somatic variants from primary and recurrent tumour tissue, corresponding resection margins and cell-free DNA from intra-operatively collected plasma samples from eight patients with human papillomavirus-negative head and neck squamous cell carcinoma. Patients were primarily treated with curative-intent surgery and received subsequent adjuvant treatment. The most frequently mutated gene was TP53. Other mutated genes included NOTCH1, NF1 and CDKN2A among others. A total of 20.8% of variants were shared between primary tumour and resection margin. Out of all the variants detected, 37.5% were shared between cell-free DNA and primary tumour, whereas 12.5% were commonly found in cell-free DNA, primary tumour and resection margin. Mutational profiling was able to distinguish between a locoregional recurrence and a second primary tumour by identifying a different TP53 mutation in the primary tumour compared to the recurrent tumour in addition to private FBXW7 and CTNNB1 mutations. We also identified identical TP53 and PIK3CA mutations in another primary tumour and corresponding recurrence. Molecular profiling of cell-free DNA and resection margins has potential applications in clinical practice to guide future treatment decisions.