Online Resource Table 1 Description of the 24 Tumor Suppressor Genes Included in the ME001-C2

Online Resource table 1Description of the 24 tumor suppressor genes included in the ME001-C2 kit (MRC-Holland). Information about chromosome location, CpG site location and relative location near important gene regions is supplied in Online Resource table 9.

Online Resource table 2Description of the 18 tumor suppressor genes included in the ME003-A1 kit (MRC-Holland). Information about chromosome location, CpG site location and relative location near important gene regions is supplied in Online Resource table 9.

Online Resource table 3 Significant differences seen in methylation status of normal tissue between various locations. Breast n=25, brain n=5, liver n=5, lung n=5. Lung, liver and brain tissue seems often more methylated than breast tissue.

Online Resource table 4Overview of molecular subtype and location of dissemination of the 53 selected patients. Luminal A tumors preferentially appear to disseminate to liver over lung. luminal B tumors to skin over liver and triple negative tumors to lung over skin. The group of HER2 enriched tumors is too small to perform statistics on.

Online Resource table 5Association between methylation status of 53 patients for 40 tumor suppressor genes (53 loci) and classical clinicopathological characteristics. Only significant correlations (p<0.05) were shown.

Online Resource table 6 mRNA expression data of 1038 breast cancer patients from TCGA (RNA Seq V2RSEM). Z-values of mRNA sequencing data for 40 tumor suppressor genes were compared to percentages of DNA methylation (beta values by the IlluminaInfiniumHumanMethylation27 platform). TCGACpG-sites that matched closest to our MS-MLPACpG-sites (criteria for matching: <1000 bp between CpG-sites, significant inverse correlation, Pearson’s r>-20), were shown when possible. Significant inverse correlations with Pearson’s r>-0.20 are depicted in bold.

Online resource table 7 Raw methylation percentages of 53 patients for 40 tumor suppressor genes (53 loci) by MS-MLPA. P: primary breast tumors. M: metastases. N: normal tissues.

Online Resource table 8Cut-offs for dichotomization of methylation values for the 40 tested tumor suppressor genes (53 loci). to distinguish between hypermethylated and unmethylated. ROC curves were used to find the most appropriate cut-off between normal tissue (n=25 normal breast) and tumor tissue (n=53 primary breast tumors). The appropriate cut-offs varied between 0.5 and 22.75% for the 40 genes.

Online Resource table9List of 53 loci of 40 tumor suppressor genes tested in this study and their most important characteristics. includingCpG site of MS-MLPA probe, chromosome location, transcription and translation start sites and #bp between MS-MLPA probe and TSS/ATG. Also, information used to correlate MS-MLPA data to TCGA data were depicted, including CpG site of IlluminaTCGA methylation probe, #bp between TCGA methylation probe and TSS/ATG, #bp between TCGA methylation probe and MS-MLPA probe and correlation between TCGAmRNA expression z-values and TCGA methylation bèta values.