Molecular characterisation of longitudinally collected circulating cell-free DNA in HPV+ve and HPV-ve oropharyngeal cancer

Oropharyngeal squamous cell carcinoma (OPSCC) is an increasing global health problem and is divided into two types dependent on association with human papillomavirus (HPV), with a more favourable prognosis in virus-associated tumours. Current methods of establishing viral aetiology, assessing response to therapy and clinical monitoring rest on tissue biopsy, clinical examination and post-treatment imaging. However, tissue biopsy is invasive and carries significant risk of morbidity, and post-treatment scans are frequently indeterminate. Analysis of cell-free DNA (cfDNA) from the circulation provides a minimally invasive method for detecting and monitoring cancer-derived DNA fragments, with the potential for enhancing clinical care. Through the longitudinal collection of 166 blood samples in 67 OPSCC patients we evaluate the utility of three cfDNA analysis methods: droplet digital PCR (ddPCR) and fragment size analysis in both HPV+ve and HPV-ve disease, and ultra-deep sequencing in patients with HPV-ve disease. We show that ddPCR analysis of cfDNA for five HPV types (16, 18, 31, 33 & 35) is strongly concordant with existing clinical assays (p16 immunohistochemistry (IHC) and quantitative PCR analysis of solid tumour tissue) and that cfDNA fragment size was reduced in OPSCC patients compared to healthy controls. Sequential ddPCR measurements of cfDNA HPV copy number showed a decrease to undetectable levels in all 30 HPV+ve patients in at least one of their post-treatment samples and a corresponding increase in cfDNA fragment size in patients who had a complete response to chemoradiotherapy. In two HPV+ve patients, clinical decision-making based on HPV ddPCR of cfDNA may have led to earlier detection of relapse in one patient or avoided surgical exploration in a second patient, which led to resection of tissue that did not harbour malignancy. In HPV-ve disease, ultra-deep sequencing identified tumour-derived somatic mutations of circulating cfDNA in genes such as TP53 and members of the ERBB family that are potential markers of therapeutic responsiveness and patient prognosis. Together our data suggest that analysis of circulating cfDNA can enhance current clinical strategies for assessing therapeutic response and disease monitoring in both HPV+ve and HPV-ve OPSCC.