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 prognosti-cator 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 hypoth-esized that the tumor DNA methylation landscape might indicate epigenetic reprogramming induced by chronic intratumoral hypoxia. Experimental Design: A DNA-methylome-based tumor hyp-oxia 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 con-sisting 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 1/4 0.001) and overall survival (HR, 2.34; P 1/4 0.03) but not distant metastasis after RCHT in both cohorts. Hyp-oxia-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 1/4 0.04), 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 hyp-oxia for identifying high-risk features in patients with HNSCC tumors. See related commentary by Heft Neal and Brenner, p. 2954