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

Abstract

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 Validation Cohort. In the Discovery Cohort, associations between genome-wide methylation change (posttreatment minus pretreatment) 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 regulatory 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 negative 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 significantly 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 replicated in the Validation Cohort, for which high MRS also independently predicted worse RFS (HR, 10.2; 95%, CI 2.4-43.4; P = .002) and OS (HR, 3.7; 95% CI, 1.3-10.4; P = .015).

Conclusions: We successfully trained and validated a signature of DNA methylation in peripheral blood before and after radiation therapy that stratified outcomes among patients with HNSCC, implicating the potential for genomics-tailored surveillance and consolidation treatment.

Fig. 1.

Study schema.

Fig. 2.

Kaplan-Meier plots of recurrence-free survival and overall survival for patients in the (A) Discovery Cohort and (B) Validation Cohort.

Fig. 3.

Forest plot of (A) recurrence-free survival and (B) overall survival log hazard ratios with 95% confidence intervals and cumulative event rates at 5 years for high versus low methylation risk score. In comparisons for which at least one group has zero events, hazard ratios could not be confidently estimated using Cox proportional hazards regression.

Fig. 3.

Forest plot of (A) recurrence-free survival and (B) overall survival log hazard ratios with 95% confidence intervals and cumulative event rates at 5 years for high versus low methylation risk score. In comparisons for which at least one group has zero events, hazard ratios could not be confidently estimated using Cox proportional hazards regression.