Many patients with locoregionally advanced human papillomavirus–negative head and neck squamous cell carcinoma (HNSCC) relapse. ctDNA has the potential to identify minimal residual disease, but its clinical utility for virus-negative HNSCC is not well understood.

Experimental Design:

We retrospectively evaluated a personalized, commercial ctDNA assay (Signatera, Natera) during clinical care of patients treated for predominantly newly diagnosed human papillomavirus–negative HNSCC. Signatera utilizes 16-plex PCR from matched tumor and blood. Objectives were to understand ctDNA detectability and correlate changes posttreatment with disease outcomes.


Testing was successful in 100/116 (86%) patients (median age: 65 years, 68% male, 65% smokers); testing failed in 16 (14%) because of insufficient tissue. Oral cavity (55, 47%) tumors were most common; most had stage III to IV disease (82, 71%), whereas 17 (15%) had distant metastases. Pretreatment, 75/100 patients with successful testing (75%) had detectable ctDNA (range: 0.03–4049.69 mean tumor molecules/mL). No clinical features predicted ctDNA detectability or levels (multivariate analysis). At a median follow-up of 5.1 months (range: 0.2–15.1), 55 (55%) had >1 test result (range: 1–7; 194 samples). Of 55 patients, 17 (31%) remained ctDNA positive after starting treatment. Progression-free survival was significantly worse for patients who were ctDNA positive versus ctDNA negative posttreatment (HR, 7.33; 95% confidence interval, 3.12–17.2; P < 0.001); 1-year overall survival was 89.1% versus 100%, respectively (HR, 7.46; 95% confidence interval, 0.46–119.5; P = 0.155).


Tumor-informed ctDNA testing is feasible in nonviral HNSCC. ctDNA positivity is an indicator of disease progression and associated with inferior survival. Further research is warranted to understand whether ctDNA may be leveraged to guide therapy in HNSCC.

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