Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood and comprises two major subtypes: fusion-positive (FP, most commonly PAX3-FOXO1 [P3F] or PAX7-FOXO1 [P7F] resulting from 2;13 and 1;13 chromosomal translocations) and fusion-negative (FN). Our previous study demonstrated that FP and FN RMS tumors exhibit distinct DNA methylation profiles. To further examine the significance of DNA methylation, we generated genome-wide DNA methylation profiles for a new cohort of 48 RMS tumors for which we previously assessed mutation, copy number and expression status. Investigation of the RMS subsets defined by methylation clustering revealed a significant association of methylation with P3F versus P7F fusion status in the FP subset, and an association of methylation with RAS mutation status in the FN subset. Localization studies of differentially methylated probes showed these probes were not evenly distributed with respect to annotated genomic features. In particular, hypomethylated probes were enriched in FP tumors in the promoter region and in the intergenic region, whereas hypermethylated probes were enriched in these regions in FN tumors. In contrast, hypermethylated probes were enriched in FP tumors in the 3' UTR region whereas hypomethylated probes were enriched in FN tumors in these regions. In our new larger cohort of cases, there was a significant difference in the distribution of P3F binding sites between genes with and without differential methylation. Integrative analysis of P3F binding sites, promoter methylation and gene expression demonstrated that genes with P3F binding sites tended to be more highly expressed in FP tumors (compared to FN tumors) than genes without P3F binding sites regardless of promoter methylation status. Though promoter hypomethylation is most highly associated with enhanced expression among genes with P3F binding sites, the group of genes with P3F binding sites and promoter hypomethylation is small in number compared to the much larger group of genes with P3F binding sites but without promoter hypomethylation. In conclusion, these results demonstrate the interaction of these epigenetic changes with mutational alterations and transcriptional organization in RMS tumors and provide a direction for future studies of these epigenetic events.

Citation Format: Wenyue Sun, Bishwanath Chatterjee, Jack F. Shern, Sivasish Sindiri, Yonghong Wang, Holly S. Stevenson, Daniel C. Edelman, Paul S. Meltzer, Javed Khan, Frederic G. Barr. Relationship of DNA methylation to mutational changes and transcriptional organization in fusion-positive and fusion-negative rhabdomyosarcoma tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3877. doi:10.1158/1538-7445.AM2017-3877