Koos Boeve

146 Chapter 8 markers might also be useful for the early detection of tumor cells or be detectable in shed DNA fragments in liquid biopsies such as plasma and sputum [10] and has been reported in lung [15], breast [16], colorectal [16] and hepatocellular cancer [17]. The detection of tumor cells in saliva of patients with head and neck SCC has been reported as well [18-20] and requires markers with high sensitivity and high specificity. In patients with OSCC, only few markers that are methylated in tumor tissue but not in normal epithelium, have been reported [10]. To identify newmethylationmarkers in patients with OSCC that are associated with lymph node status, we recently used a genome-wide methylation screening method based on MethylCap-Seq analysis [21] and reported a methylome of several OSCC cases and numerous new differentially methylated tumor markers [22,23]. In the current study, to identify new biomarkers associated with OSCC and not with other tissue, we assessed the available methylome of a series of OSCC cases generated by MethylCap-Seq analysis [23] with the methylome of 80 control tissues. We describe the identification of several new markers which are significantly hypermethylated in OSCC and not in non-cancer control samples. We validated the performance of these OSCC specific DNA hypermethylation markers using quantitative methylation specific PCR (QMSP) in a proof of principle pilot study with saliva of OSCC patients and non-cancer controls. In addition, we included five DNA methylation markers previously reported to be associated with OSCC[19,20,24]. The aim of this study was to identify methylation markers with a high sensitivity and a high negative predictive value (NPV) for the detection of tumor cells in saliva from patients with OSCC. MATERIALS AND METHODS Identification of novel methylation markers using MethylCap-seq analysis The strategy of methylation marker selection is summarized in Figure 1. To identify genomic loci hypermethylated in OSCC and not in normal tissue, in silico analysis was performed of MethylCap-Seq data [25] as reported previously [21,26]. In summary, 12 OSCC samples and two pools of leukocytes of 500 ng DNA each were fragmented using Covaris S2 (Covaris, Woburn, MA, USA). Subsequently, methylated DNA fragments were separated from unmethylated fragments by enrichment with the MethylCap kit (Diagenode, Belgium), paired-end sequenced using the Illumina Genome Analyzer II and mapped to the human reference genome (NCBI build 37.3). For further analysis, only pair-end sequenced fragments (reads) were included that could be mapped to unique specific loci, and summarized using an in house generated . “Map of the Human Methylome” for MethylCap-seq data [27].