Background: Despite improvements in multimodal treatment options for patients with head and neck squamous cell carcinoma (HNSCC), survival has only improved modestly over the past decades as patients frequently develop recurrences. Detection of cell-free circulating tumor DNA (ctDNA) post-operatively and during clinical follow-up has the potential to identify patients with molecular residual disease (MRD) or who are at an increased risk of relapse, and who may therefore benefit from personalized treatment strategies. Methods: We conducted LIONESS, a single-center prospective cohort study to investigate ctDNA in patients with HNSCC who received primary surgical treatment with curative intent. 58 patients have been recruited as of January 2022, with disease stage I (15.5%), II (13.8%), III (36.2%) and IV (34.5%). Whole exome sequencing was performed on FFPE tissue. RaDaR, a highly sensitive personalized assay using deep sequencing of tumor-specific variants, was used to analyze serial pre- and post-operative plasma samples for evidence of molecular residual disease and recurrence. In addition, pre-operative saliva samples were collected for detection of tumor DNA in saliva. Results: 236 longitudinal plasma samples from 35 patients have been analyzed so far, using personalized panels designed targeting a median 48 (20-60) somatic variants. Preliminary data shows 94.28% ctDNA detection in baseline samples taken prior to surgery. In post-surgery samples, ctDNA could be detected at levels as low as 0.0005% variant allele fraction (eVAF). Survival analysis showed a significant difference (p-value = 7e-7) in recurrence rates between patients who tested ctDNA+ during follow-up (8/11, 72.7%) and those who were negative for ctDNA (0/24, 0%). In all eight cases with clinical recurrence, ctDNA was detected prior to progression, with lead times ranging from 56 to 265 days. Patients were followed for clinical recurrence with a median follow-up of 9.5 months to date, but longer follow-up is necessary for patients who may be at increased risk of recurrence. Work is ongoing for analysis of the full cohort of plasma and saliva samples, which will be presented at the congress. Conclusions: In this prospective observational study, detection of residual disease using ctDNA was associated with poorer progression-free survival and much earlier detection of disease prior to clinical relapse. The implementation of a highly sensitive ctDNA assay such as RaDaR into clinical practice has the potential to tailor personalized treatment strategies for molecular residual disease and recurrence in patients with HNSCC. In future, ctDNA analysis with subsequent ctDNA-guided treatment may reduce morbidity for HNSCC patients.