Genome Wide Assessment Of Epigenetic Alterations In Ewing Sarcoma

Background: Epigenetic alterations are common in many cancer types and play an important role in their pathogenesis. However, the role of aberrant DNA methylation in Ewing Sarcoma (ES), a malignant neoplasm of bone and soft tissues in adolescents and young adults, is currently poorly understood. Aim: To determine the role of aberrant DNA methylation in the molecular pathology of ES Methods: We used a genome wide approach to identify genes hypermethylated in these neoplasms. Genomic DNA was isolated from formalin fixed, paraffin embedded primary ES tumors and cell lines. We compared the methylation signatures of 13 tumors and 3 ES cell lines with human mesenchymal stem cell primary cultures (hMSCs). Illumina Goldengate methylation analysis was used to determine the methylation status of 800 genes known to be altered in human cancer. Validation of the array results was performed with methylation specific PCR and sodium bisulfite sequencing. Results: The Illumina Goldengate arrays were found to be both sensitive and reproducible. After filtering out genes methylated in hMSCs, we determined that primary ES tumors and cell lines could be stratified into low and high methylator groups. In addition, we identified a group of genes that were methylated in >30% of ES primary tumors (range 30-100%) but not in hMSCs. Known tumor suppressor genes and genes methylated in other cancers were included in this group, including RASSF1A, a known tumor suppressor gene methylated in both adult and pediatric cancers. Methylation specific PCR and bisulfite sequencing analysis of RASSF1A confirmed the aberrant methylation detected by the methylation arrays in the ES cell lines. Furthermore, treatment of the cell lines with 5-aza-2\#8217;deoxycytidine induced RASSF1A expression in the cell lines with methylated RASSF1A demonstrating that RASSF1A is epigenetically silenced in ES cell lines due to DNA hypermethylation. Conclusions: Aberrant DNA methylation occurs in ES, demonstrating that epigenetic alterations play a role in pediatric tumors of mesenchymal origin. Determination of the genes affected by this process will provide further insights into the molecular pathology of ES. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 5182.