Outcomes Stratification of Head and Neck Cancer Using Pre- and Post-treatment DNA Methylation From Peripheral Blood

Purpose: Established prognostic factors for head and neck squamous cell carcinoma (HNSCC) mostly consist of clinical and tumor features assessed before treatment. We report a novel application of DNA methylation in peripheral blood before and after radiation therapy to further improve outcomes stratification.Methods and Materials: Peripheral blood samples from patients with nonmetastatic HNSCC were obtained for methylation analysis 1 week before and 1 month after radiation therapy. Patients were randomized 1:1 to a Discovery Cohort or a Valida-tion Cohort. In the Discovery Cohort, associations between genome-wide methylation change (posttreatment minus pretreat-ment) and recurrence-free survival (RFS) as well as overall survival (OS) were evaluated using Cox regression. A methylation risk score (MRS) was then constructed from methylation levels at the top associated sites, filtered for residing within the regula-tory regions of genes expressed in cells of hematopoietic lineage. The prognostic value of MRS was separately assessed in the Discovery and Validation Cohorts.Results: Between December 2013 and September 2018, 115 patients participated in this study. Human papilloma virus nega-tive status, oral cavity cancer, gastrostomy tube insertion, and higher neutrophil count before radiation therapy were associated with shorter RFS and OS (P < .05). Genes downstream of the methylation sites comprising MRS are HIF1A, SF1, LGALS9, and FUT5, involved in hypoxia response, blood cell maturation, and immune modulation. High MRS (in the top third) was signifi- cantly associated with worse RFS (hazard ratio [HR], 7.1; 95% confidence interval [CI], 1.4-35.5; P = .016) and OS (HR, 15.9; 95% CI, 1.6-153.6; P = .017) in the Discovery Cohort, independent of the aforementioned risk factors. These findings were