Koos Boeve

147 Methylation biomarkers for the detection of tumor DNA in saliva For further analyses only the MCs that are located either in a promoter region, between 2000 bp upstream to 500 bp downstream of the Transcription Start Site (TSS) or in the first exon of an Ensemble (v65), gene were selected and statistically compared using R with R-package Bayseq [28]. The most equally methylated MCs amongst all 12 OSCC were ranked according the likelihood of equal methylation. Additionally, an approximate false discovery rate (FDR) was calculated. The 5000 most equally methylated MCs with the lowest FDR were used for further analysis. These highest ranked 5000 MCs in OSCC were compared to the 2276 MCs available in the MethylCap-Seq data of the two leukocyte pools, by the Mann- Whitney U test (wilcox.test function in R). All MCs with a p-value <0.05 were selected for further analyses (n = 335, Supplementary data 1). In the next step, all MCs were selected with a 100% positive and negative predictive value defined by ≤2 reads in both leukocytes pools as well as ≥3 reads in all 12 OSCC (Supplementary data 1). Finally, the MCs were compared to the semi-quantitative methylation data of the“Map of the Human Methylome” (non-OSCC primary tumor samples (n = 32), non-OSCC cancer stem cells (n = 11), normal tissue (n = 22), stem cells (n = 6) and normal cell lines (n = 9)) [27] to select MCs without methylation detected in the average methylome. Selected Genes : C11orf85, KCNA5, SIPA1 MethylCap-Seq analysis 12 OSCC and 2 leukocyte pools compared to 80 samples of tumors and healthy tissue, cell lines and stem cells Literature Genes hypermethylated in saliva of HNSCC patients versus healthy controls Selected Genes: EDNRB, HOXA9, NID2, TIMP3 Technical validation pilot using QMSP Comparison of saliva from 10 OSCC patients versus 10 healthy controls Figure 1. Study design. Methylation markers were selected using a MethylCap-Seq protocol. Selected genes were technically validated in a pilot study with saliva from 10 OSCC patients and 10 healthy controls (five younger and age-matched controls) and compared to methylation markers associated with OSCC and selected from literature. Abbreviations: OSCC, oral squamous cell carcinoma; HNSCC, head and neck squamous cell carcinoma; QMSP, quantitative methylation-specific PCR.