OP003: Discovery of DNA methylation markers that predict nodal metastases in oral squamous cell carcinoma.

About halve of all oral squamous cell carcinoma (OSCC) patients have occult nodal metastases (N-status) due to the inaccuracy of current detection methods. The aim of our study is to find DNA methylation markers in the DNA of the primary tumor that predict N-status. For biomarker discovery we used a new approach combining a global DNA methylation screen, next generation sequencing, quantitative methylation detection and microarray expression profiling. Here we report the success of this new approach and the further validation of novel methylation markers. We assessed global methylation levels on DNA extracted from 6 oral squamous cell carcinomas (OSCC) with nodal metastases (N+) and 6 OSCC without nodal metastases (N0) by MethylCap-Seq. Isolated methylated DNA fragments were sequenced by Illumina GA II and computationally mapped back to the genome. A list was composed of the 5000 most differentially methylated genes between the N+and the N0 group. The most significantly differentially methylated genes were selected for further validation by Quantitative Methylation Specific PCR and Pyrosequencing on an independent series of 20N+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., 2012, J. Clin. Oncol.). The effect of DNA methylation on gene expression will be measured by (Q)RT-PCR and immunohistochemistry on genes differently hypermethylated and downregulated. The most promising methylation markers will be further validated on 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. [Copyright &y& Elsevier]