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 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.

Introduction

Advances in treatment and personalized tumor characterization during the past few decades have offered tremendous improvement in outcomes for patients with head and neck squamous cell carcinoma (HNSCC). However, more than half of all patients continue to experience locoregional and metastatic recurrence, which is associated with significant morbidity and mortality.¹ Salvage treatment is often complex or infeasible. This challenge has motivated a wide breadth of innovation in imaging, biomarkers, and prognostic risk scores to facilitate earlier detection of recurrence with greater salvage amenability.²

Physical examination and ¹⁸F-fluorodeoxyglucose positron emission tomography (PET)/computed tomography (CT) scans remain the standard of care for surveillance of HNSCC recurrence. Among HNSCC biomarkers directly gleaned from tumor, the most effective so far has been human papilloma virus (HPV) status in patients with oropharyngeal SCC.¹ Higher density of tumor-infiltrating lymphocytes has also been shown to portend superior overall survival among patients with HNSCC.^3,4 Tumor hypoxia has long been recognized as a hallmark of radiation therapy resistance and an overall negative prognostic factor,⁵ although its direct detection by electrodes is invasive and without spatial resolution.⁶ On the other hand, indirect detection of hypoxia by molecular and polygenic expression signatures have led to conflicting results.⁷ Nevertheless, when patients with highly hypoxic HNSCCs are successfully identified, the addition of radiosensitizers improves locoregional control.^8,9 The importance of circulating biomarkers such as Epstein-Barr virus (EBV) ctDNA¹⁰ and HPV ctDNA¹¹ is currently an active topic of investigation.

In this study, we sought to overcome the limitations of prognostic biomarkers for HNSCC that apply to only a select subset of patients (eg, oropharyngeal SCCs, highly hypoxic tumors) or that are measurable from one specimen at one time point (eg, immunologic markers, genomic classifiers). Through its appeal as a reflection of longitudinal environmental effects, DNA methylation in peripheral blood has emerged as a potentially valuable prognostic biomarker in several cancers, most notably colorectal cancer.¹² In head and neck cancer, studies have focused on methylation involving only small panels of genes or cell-free DNA so far.¹³ We report the first use of epigenome-wide data from peripheral blood cells collected before and after radiation treatment for HNSCC to construct a prognostic signature that discerns poor recurrence-free survival and overall survival, and may help guide closer posttreatment follow-up.