DNA-Methylome–Based Tumor Hypoxia Classifier Identifies HPV-Negative Head and Neck Cancer Patients at Risk for Locoregional Recurrence after Primary Radiochemotherapy

Purpose: Tumor hypoxia is a paradigmatic negative prognosticator of treatment resistance in head and neck squamous cell carcinoma (HNSCC). The lack of robust and reliable hypoxia classifiers limits the adaptation of stratified therapies. We hypothesized that the tumor DNA methylation landscape might indicate epigenetic reprogramming induced by chronic intratumoral hypoxia. Experimental Design: A DNA-methylome–based tumor hypoxia classifier (Hypoxia-M) was trained in the TCGA (The Cancer Genome Atlas)-HNSCC cohort based on matched assignments using gene expression–based signatures of hypoxia (Hypoxia-GES). Hypoxia-M was validated in a multicenter DKTK-ROG trial consisting of human papillomavirus (HPV)–negative patients with HNSCC treated with primary radiochemotherapy (RCHT). Results: Although hypoxia-GES failed to stratify patients in the DKTK-ROG, Hypoxia-M was independently prognostic for local recurrence (HR, 4.3; P ¼0.001) and overall survival (HR, 2.34; P ¼ 0.03) but not distant metastasis after RCHT in both cohorts. Hypoxia-M status was inversely associated with CD8 T-cell infiltration in both cohorts. Hypoxia-M was further prognostic in the TCGA-PanCancer cohort (HR, 1.83; P ¼0.04), underscoring the breadth of this classifier for predicting tumor hypoxia status. Conclusions: Our findings highlight an unexplored avenue for DNA methylation–based classifiers as biomarkers of tumoral hypoxia for identifying high-risk features in patients with HNSCC tumors.