Ore we decided to explore if DNA methylation, a well known epigenetic marker, may play a role in chordoma development and if hypermethylation of specific CpG islands may serve as potential biomarkers correlated with single nucleotide polymorphisms (SNP) analyses in chordoma.Materials and Methods Patient samplesThe Caucasian study-group included ten chordoma specimens obtained from four male and six female patients. The age of patients at time of diagnosis was between 25 to 75 years (average age 59.7). Tumors were located in the skull, the sacrum/coccyx and the mobile spine. Tumor-volume ranged from 1.5 toDNA Methylation and SNP Analyses in Chordoma668.2 cm3 (average 146). All ten chordomas were morphological and histological classified as classic chordomas. The follow-up period ranged from 1 to 113 months (average 41.9). All patients included in the present study were treated by surgery. Seven patients had an intralesional resection, two patients a wide, and one patient a marginal resection. Three out of ten patients received an irradiation-therapy. During the follow-up half of the patients developed a chordoma recurrence. Two patients showed lung metastases. At the end of the follow-up period four patients were DOD (death of disease), one patient suffered a DOC (death of other cause), three patients were AWD (alive with disease), and two patients had NED (no evidence of disease). The research is an original one, presently not under consideration for publication elsewhere, free of conflict of interest and conducted by the 
highest principles of human subjects. The study protocol and the consent of the informed patients were approved by the ethics committee of the Medical University Graz (vote #18-192ex06/07; valid until 17.04.2013). No research outside Austria was conducted. All patients were informed in detail and have given their written approval.normalized using the Genotyping Console 4.0 program default settings. All samples passing QC criteria were subsequently genotyped using the Birdseed (v2) algorithm. We used 60 raw HapMap data generated with the Epigenetics Affymetrix Genome-Wide Human SNP Array 6.0 as reference. Data were obtained from Affymetrix web site and used for normalization. For visualization of Copy Number state and LOH Chromosome Epigenetics Analysis Suite 1.1 software was used.DNA methylation analysesThe digestion of 600 ng genomic DNA with methylationsensitive restriction enzymes (MSRE) was performed overnight at 37uC by employing a mixture of 6 units of each AciI (New England Biolabs, Frankfurt, Germany), Hin6I (Fermentas, St. Leon-Rot, Germany) and HpaII (Fermentas). Completion of digestion was confirmed by using a control PCR covering known differentially methylated and cancer gene regions (DMRs; H19, IGF2, ABL1, PITX2, XIST and FMR1) as published [8]. Then restriction enzymes were heat inactivated at 65uC for 20 min and digested DNA was amplified in 16 multiplex reactions covering a total of 360 59UTR targets using biotinylated reverse primers. Amplicons of the 16 multiplex PCRs were pooled and upon agarose-gel-control mixed with 16574785 hybridization buffer and hybridized onto the AIT-CpG360 microarray, presenting triplicate spots of amplicon-specific DNA probes. Upon hybridization and stringency washings, the hybridized amplicons were detected via streptavidin-Cy3 fluorescence. Microarrays were scanned and intensity data extracted from images using Genepix6.0 softwareAffymetrix SNP 6.0 array processing and analysisGenomic DNA was isolated f.Ore we decided to explore if DNA methylation, a well known epigenetic marker, may play a role in chordoma development and if hypermethylation of specific CpG islands may serve as potential biomarkers correlated with single nucleotide polymorphisms (SNP) analyses in chordoma.Materials and Methods Patient samplesThe Caucasian study-group included ten chordoma specimens obtained from four male and six female patients. The age of patients at time of diagnosis was between 25 to 75 years (average age 59.7). Tumors were located in the skull, the sacrum/coccyx and the mobile spine. Tumor-volume ranged from 1.5 toDNA Methylation and SNP Analyses in Chordoma668.2 cm3 (average 146). All ten chordomas were morphological and histological classified as classic chordomas. The follow-up period ranged from 1 to 113 months (average 41.9). All patients included in the present study were treated by surgery. Seven patients had an intralesional resection, two patients a wide, and one patient a marginal resection. Three out of ten patients received an irradiation-therapy. During the follow-up half of the patients developed a chordoma recurrence. Two patients showed lung metastases. At the end of the follow-up period four patients were DOD (death of disease), one patient suffered a DOC (death of other cause), three patients were AWD (alive with disease), and two patients had NED (no evidence of disease). The research is an original one, presently not under consideration for publication elsewhere, free of conflict of interest 
and conducted by the highest principles of human subjects. The study protocol and the consent of the informed patients were approved by the ethics committee of the Medical University Graz (vote #18-192ex06/07; valid until 17.04.2013). No research outside Austria was conducted. All patients were informed in detail and have given their written approval.normalized using the Genotyping Console 4.0 program default settings. All samples passing QC criteria were subsequently genotyped using the Birdseed (v2) algorithm. We used 60 raw HapMap data generated with the Affymetrix Genome-Wide Human SNP Array 6.0 as reference. Data were obtained from Affymetrix web site and used for normalization. For visualization of Copy Number state and LOH Chromosome Analysis Suite 1.1 software was used.DNA methylation analysesThe digestion of 600 ng genomic DNA with methylationsensitive restriction enzymes (MSRE) was performed overnight at 37uC by employing a mixture of 6 units of each AciI (New England Biolabs, Frankfurt, Germany), Hin6I (Fermentas, St. Leon-Rot, Germany) and HpaII (Fermentas). Completion of digestion was confirmed by using a control PCR covering known differentially methylated and cancer gene regions (DMRs; H19, IGF2, ABL1, PITX2, XIST and FMR1) as published [8]. Then restriction enzymes were heat inactivated at 65uC for 20 min and digested DNA was amplified in 16 multiplex reactions covering a total of 360 59UTR targets using biotinylated reverse primers. Amplicons of the 16 multiplex PCRs were pooled and upon agarose-gel-control mixed with 16574785 hybridization buffer and hybridized onto the AIT-CpG360 microarray, presenting triplicate spots of amplicon-specific DNA probes. Upon hybridization and stringency washings, the hybridized amplicons were detected via streptavidin-Cy3 fluorescence. Microarrays were scanned and intensity data extracted from images using Genepix6.0 softwareAffymetrix SNP 6.0 array processing and analysisGenomic DNA was isolated f.
