Importance:
Next generation sequencing (NGS) allows the detection of low abundance tumor specific genetic alterations in circulating cell-free DNA. Detectable circulating tumor DNA (ctDNA) in patients with head and neck squamous cell carcinoma (HNSCC), at the time of diagnosis, may indicate high-risk disease and prompt treatment escalation. 

Objective:
 ctDNA, containing tumor-specific mutational signature, has been suggested as a surrogate biomarker for early detection of cancer recurrence and for monitoring treatment response. We aimed to explore whether detecting ctDNA in newly diagnosed head and neck squamous cell carcinoma (HNSCC) patients can provide molecular risk stratification. 

Study design: A cohort of newly diagnosed HNSCC patients, treated with upfront surgical resection at a referal tertiary medical center between 2014 and 2021. Utilizing ultrasensitive NGS, TP53 coding regions were sequenced in HNSCC specimens. The utility of targeted sequencing of TP53 gene for the detection of tumor specific somatic alterations in circulating cell free DNA was assessed. Adverse pathological features and clinical outcomes were analyzed in relation to ctDNA detection.

Results: Overall, TP53 was sequenced in 45 tumor specimens. Somatic mutations in TP53 were identified in 82% of patients. Following TP53 profiling, circulating DNA were scrutinized for the presence of tumor specific mutations: cell free DNA was extracted from plasma samples (N=20), subjected to polymerase chain reaction (PCR) amplification of the amplicon containing the tumor-specific alternation and deeply sequenced. ctDNA, with the mutated TP53, was detected in 14 patients (70%), with allele frequency of 0.2%-2%. Advanced stage disease, perineural invasion and deep invasion were more frequently found in patients whose ctDNA was detected. Detectable ctDNA corresponded with regional spread (N stage ≥ 1, P = 0.04) and a tendency to worse disease-free survival (P = 0.066).

Conclusions and relevance: Targeted sequencing of TP53 gene in HNSCC using NGS technology can be utilized for the detection of low abundance tumor specific genetic alteration in circulating DNA. ctDNA detection correlated with pathological adverse features, indicating the potential implementation of ctDNA-based molecular profiling for identifying patients who may benefit from intensified treatment regimens.