DNA methylation heterogeneity in Ewing sarcoma defines an epigenetic disease spectrum underlying a genetically homogeneous developmental cancer.

We performed reduced representation bisulfite seqeuncing (RRBS) and ChIP-seq of histone modification marks on three Ewing sarcoma tumor samples, and we quantified epigenetic heterogeneity on three levels. First, we identified a Ewing sarcoma specific hypomethylation signature at EWS-FLI1 regulated enhancers, showing that epigenetic enhancer reprogramming is a defining feature of Ewing sarcoma. Second, inter-individual DNA methylation differences in Ewing sarcoma samples identified a continuous disease spectrum with two dimensions: the strength of the EWS-FLI1 regulatory signature and the balance of mesenchymal versus stem cell regulatory signatures. Third, we observed substantial epigenetic heterogeneity within individual tumors. In summary, our study provides a comprehensive assessment of epigenetic heterogeneity in Ewing sarcoma, highlighting its importance as a source of variability for genetically homogeneous tumors.