Supersensitive and robust disease monitoring in oropharyngeal cancer patients by circulating tumor HPV-DNA sequencing (ctHPV-DNAseq)

• Detection of ctHPV-DNA by target-enrichment DNA sequencing including selected human cancer genes (ctHPV-DNAseq) allows determination of robust statistical cut-offs and provides a variety of quality controls that will facilitate clinical implementation of liquid biopsy disease monitoring. • Detection of ctHPV-DNA by target-enrichment DNA sequencing in plasma of HPV-positive oropharyngeal cancer patients and non-cancer controls is 100% accurate at baseline. • Detection of ctHPV-DNA by target-enrichment DNA sequencing demonstrates a 10-100x higher analytical sensitivity than digital PCR. • Plasma ctHPV-DNA detection by target-enrichment DNA sequencing provides an extremely valuable tool for the detection of recurrent and residual oropharyngeal cancer, outperforming standard clinical practice. Post-treatment disease monitoring of HPV-positive oropharyngeal squamous cell carcinoma (OPSCC) is challenging. Liquid biopsies could improve disease monitoring, but the variety in methods hampers clinical implementation. In this study, target-enrichment sequencing to detect circulating tumor HPV DNA (ctHPV-DNA) was applied in liquid biopsies of HPV-positive OPSCC patients, and robust statistical readouts determined. Next, it was investigated whether longitudinal plasma monitoring could accurately diagnose residual and recurrent disease. The target-enrichment panel included 29 cancer genes and high-risk HPV genomes. The assay was tested on plasma from 30 non-cancer controls and 33 patients with HPV-positive tumors, 15 of whom had residual or recurrent disease, and 18 who remained disease-free. Samples were analyzed from baseline to 24 months after treatment. By determining and applying robust statistical cut-off values, ctHPV-DNA could be detected in plasma of all patients with HPV-positive OPSCC at baseline, and was absent in plasma of all non-cancer controls. In OPSCC patients who remained disease-free, post-treatment plasma samples were negative for ctHPV-DNA. In contrast, ctHPV-DNA was detected in plasma of all OPSCC patients with recurrent disease to a year before clinical diagnosis. Cases suspect for residual disease in the neck, but with a necrotic metastasis without vital tumor after resection, tested correctly negative for ctHPV-DNA in plasma. Target-enrichment sequencing of plasma shows 100% accurate detection of ctHPV-DNA at baseline. Longitudinal monitoring enables early recurrence detection and correct diagnosis of non-vital residual disease. The data indicate that liquid biopsy could improve post-treatment follow-up in HPV-positive OPSCC patients.