Clinical validation of a tissue-agnostic genome-wide methylome enrichment molecular residual disease assay for head and neck malignancies

Background: Outcomes for patients with locally advanced head and neck cancer (HNC) treated with curative intent remain disappointing, with 5-year survival rates at 50%. Most recurrences occur within the first 2 years after treatment, providing a window of opportunity to identify patients with molecular residual disease (MRD). A tissue-agnostic test for MRD detection in patients with human papillomavirus (HPV) positive and negative HNC, where tissue is often scarce, is needed.

Patients and methods: Patients with stage I-IVB HNC, including patients positive and negative for HPV, were enrolled and peripheral blood plasma was collected longitudinally at diagnosis and ∼3, 12, and 24 months after curative intent treatment. The full cohort includes 325 patients with 1155 samples. Samples were split into distinct sets to train and validate a classifier capable of identifying MRD using a tissue-agnostic genome-wide methylome enrichment platform. The primary endpoint was recurrence-free survival (RFS).

Results: With a median follow-up of 60 months, patients in the blinded validation set with MRD positivity experienced significantly worse RFS with a hazard ratio (HR) of 35.7 [95% confidence interval (CI) 10.8-117.8; P < 0.0001]. For patients with HPV negativity, HR was 42.3 (95% CI 9.8-182.3; P < 0.0001); for patients with HPV-positive oropharyngeal cancer, HR was 24.1 (95% CI 3.0-196.8; P < 0.0001). Moreover, the lead time between MRD positivity and clinical recurrence was up to 14.9 months, with a mean lead time of 4.1 months. Surveillance sensitivity was 91% (95% CI 77% to 97%) and specificity was 88% (95% CI 80% to 93%).

Conclusions: Here we validate the clinical performance characteristics of a tissue-agnostic genome-wide methylome enrichment assay for MRD detection in patients with HNC. The MRD detection test showed high sensitivity for identifying recurrence at high specificity across different anatomical sites, HPV status, and treatment regimens, highlighting the broad applicability for MRD detection in patients with HNC.