Functional epigenetic approach identifies frequently methylated genes in Ewing sarcoma

Using a candidate gene approach we recently identified frequent methylation of the RASSF2 gene associated with poor overall survival in Ewing sarcoma (ES). To identify effective biomarkers in ES on a genome-wide scale, we used a functionally proven epigenetic approach, in which gene expression was induced in ES cell lines by treatment with a demethylating agent followed by hybridization onto high density gene expression microarrays. After following a strict selection criterion, 34 genes were selected for expression and methylation analysis in ES cell lines and primary ES. Eight genes (CTHRC1, DNAJA4, ECHDC2, NEFH, NPTX2, PHF11, RARRES2, TSGA14) showed methylation frequencies of>20% in ES tumors (range 24-71%), these genes were expressed in human bone marrow derived mesenchymal stem cells (hBMSC) and hypermethylation was associated with transcriptional silencing. Methylation of NPTX2 or PHF11 was associated with poorer prognosis in ES. In addition, six of the above genes also showed methylation frequency of>20% (range 36-50%) in osteosarcomas. Identification of these genes may provide insights into bone cancer tumorigenesis and development of epigenetic biomarkers for prognosis and detection of these rare tumor types

Genetic and epigenetic alterations of sarcoma

Primary malignant bone tumours are rare cancers that are characterised by different genetic and epigenetic alterations. A functional epigenomic approach was combined with the Illumina HumanHT-12.v4-BeadChip expression microarray in three Ewing Sarcoma (ES) cell lines to identify genome-wide functional methylation changes in these cells and ES primary samples. This study revealed eight frequently methylated genes in ES patients’ samples, where NPTX2 and PHF11 promoter methylation was associated with poor patient prognosis. The second methylation study involved genome-wide DNA methylation profiling of chordoma samples using the Infinium-HumanMethylation450-BeadChip microarray. This study identified a list of 8,819 loci which were differentially methylated between chordomas and controls and eight genes which were differentially methylated between recurrent and non-recurrent chordoma samples. RNA sequencing (RNA-seq) analysis of primitive small blue round cell tumour (SBRCT) samples was also carried out in order to identify gene fusions in this type of cancer. Three different somatic gene fusions in SBRCT samples were identified using RNA-Seq (CRTC1-SS18;BCR-UPB1 and KHDRBS2-CIC). Moreover, two other gene fusions were identified in unpaired SBCRT samples. Overall, this study used high-throughput technologies to identify novel genetically and epigenetically altered genes in different types of bone sarcoma which may, therefore, provide unique insight into bone sarcoma tumorigenesis

Functional epigenetic approach identifies frequently methylated genes in Ewing sarcoma

Using a candidate gene approach we recently identified frequent methylation of the RASSF2 gene associated with poor overall survival in Ewing sarcoma (ES). To identify effective biomarkers in ES on a genome-wide scale, we used a functionally proven epigenetic approach, in which gene expression was induced in ES cell lines by treatment with a demethylating agent followed by hybridization onto high density gene expression microarrays. After following a strict selection criterion, 34 genes were selected for expression and methylation analysis in ES cell lines and primary ES. Eight genes (CTHRC1, DNAJA4, ECHDC2, NEFH, NPTX2, PHF11, RARRES2, TSGA14) showed methylation frequencies of>20% in ES tumors (range 24-71%), these genes were expressed in human bone marrow derived mesenchymal stem cells (hBMSC) and hypermethylation was associated with transcriptional silencing. Methylation of NPTX2 or PHF11 was associated with poorer prognosis in ES. In addition, six of the above genes also showed methylation frequency of>20% (range 36-50%) in osteosarcomas. Identification of these genes may provide insights into bone cancer tumorigenesis and development of epigenetic biomarkers for prognosis and detection of these rare tumor types