Abstract Annually 650,000 new patients are diagnosed with Head and Neck cancer, making it the sixth most common cancer worldwide. The 5-year survival is only 60% due to inaccurate detection of nodal metastases in the cervical or submandibular region (N-status). Our goal is to find new DNA methylation markers in the primary tumor that predict nodal metastases to improve diagnostics and treatment. For biomarker discovery we used a new approach combining global methylation assessment, next generation sequencing, quantitative methylation and microarray expression profiling. Here we report the success of this new approach and the further validation of novel biomarkers. We performed MethylCap-Seq on DNA from 6 oral squamous cell carcinomas (OSCC) with nodal metastases (N+) and 6 OSCC without nodal metastases (N0) to measure global methylation levels. After comparison of the sequenced reads a list was composed of the 5000 most differentially methylated genes between the two groups. The most significantly differentially methylated genes were validated using Quantitative Methylation Specific PCR (QMSP) and Pyrosequencing on a independent series of 20 N+ and 20 N0 OSCC. In addition, the MethylCap-Seq methylation data was combined with microarray expression data of 696 genes for a N-status validation cohort containing 222 OSCC (Hoof et al.; J Clin Oncol. 2012 Oct 8). Based upon the assumption that hypermethylation is associated with downregulation of gene expression, we selected hypermethylated and downregulated genes distinguishing between the two groups. The effect of DNA methylation on gene expression will be measured by (Q)RT-PCR and immunohistochemistry on a N-status validation panel. The most promising methylation markers will be further validated by Pyrosequencing and QMSP on DNA of a N-status validation cohort containing 463 cases for which complete clinicopathological and follow-up data are available. These markers might contribute to better diagnosis, improved quality of life and may be associated with increased survival. This research was supported by the Center for Translational Molecular Medicine (project AIRFORCE). Citation Format: Martijn Clausen, Lieuwe J. Melchers, Leonie Bruine de Bruin, Mirjam F. Mastik, Lorian Slagter-Menkema, Harry J. Groen, Bert van der Vegt, Bernard F. Van der Laan, Tim De Meyer, Wim Van Criekinge, G Bea Wisman, Jan L. Roodenburg, Ed Schuuring. DNA methylation marker discovery for the prediction of nodal metastases in head and neck cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 645. doi:10.1158/1538-7445.AM2013-645