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BACKGROUND AND PURPOSE: Next-generation sequencing (NGS) assays are being evaluated to measure circulating tumor DNA (ctDNA) in human papilloma virus-driven oropharynx squamous cell carcinoma (HPV-OPSCC). This technology has not yet been explored in patients receiving definitive therapy. Here, we employ HPV-DeepSeek, a whole-genome NGS assay, to interrogate ctHPVDNA levels in patients receiving chemoradiation for HPV-OPSCC.MATERIALS AND METHODS: This was a secondary analysis of plasma samples from a single-institution prospective biomarker study. Plasma samples from adults treated with definitive chemoradiation for HPV-OPSCC were analyzed using HPV-DeepSeek. Clinical and patient factors were abstracted from the electronic medical record. Spearman correlation, Kruskal-Wallis/Mann-Whitney U testing and univariable linear regression were used to test the association between baseline ctHPVDNA reads and clearance rate by patient and disease features.RESULTS: Twenty-three patients with AJCC 8th edition clinical stage I-II HPV-OPSCC were included. HPV-DeepSeek detected ctHPVDNA in 100% of samples collected in week 1. Twenty-six percent of patients cleared ctHPVDNA by week 4, 52% cleared by week 6, and 17% cleared by first follow-up. One patient did not clear ctHPVDNA and was lost to follow-up. On univariable regression, ctHPVDNA level was associated with AJCC 8th edition nodal stage (coeff = 44831, p = 0.022), overall stage (coeff = 43907, p = 0.05), and viral integration (coeff = 61813, p = 0.008). Median follow-up was 7.3 years. There were no recurrences.CONCLUSIONS AND RELEVANCE: HPV-DeepSeek, an ultra-sensitive NGS assay, can detect ctHPVDNA clearance in patients receiving definitive chemoradiation for HPV-OPSCC.