To extend our previous findings, we used the HM450 array to study genome-wide DNA methylation in a discovery cohort of 21 FP tumors (12 PAX3-FOXO1 and 9 PAX7-FOXO1) and 17 FN tumors. Unsupervised hierarchical analysis confirmed our previous finding of a close association of methylation pattern and fusion status. To gain further insight into the DNA methylation patterns in RMS, we noted that our DNA methylation analysis identified two major subsets (FP-1 and FP-2) within the FP cluster, and two major subsets (FN-1 and FN-2) within the FN cluster. Our evaluation of the association between DNA methylation-defined subsets FP-1 and FP-2 and fusion subtype showed that the PAX3-FOXO1 and PAX7-FOXO1 fusions were significantly enriched in the FP-2 and FP-1 subsets, respectively. In addition, evaluation of RAS mutation status in methylation-defined subsets FN-1 and FN-2 revealed that RAS gene mutations were differentially distributed between these subsets such that cases with RAS gene mutations were preferentially found in the FN-2 subset and cases with wild-type RAS status wre preferentially found in the FN-1 subset. To validate these findings, we analyzed DNA methylation in an additional cohort of 21 FP tumors (14 PAX3-FOXO1, 5 PAX7-FOXO1, and 2 alternative fusions) and 27 FN tumors. Our comparison of the PAX3-FOXO1 and PAX7-FOXO1 gene fusions in the two FP subsets confirms the significant associations between these two gene fusions and DNA methylation pattern in this validation cohort. Furthermore, analysis of the two methylation-defined FN subsets in the validation cohort also confirms the statistically significant association between RAS mutation status and DNA methylation pattern. We further explored DNA methylation differences between fusion subtypes in FP RMS tumors by combining cases from the discovery and validation cohorts. A supervised analysis of the FP cases indicated that 1688 probes that were significantly hypermethylated (corresponding to 184 promoter-hypermethylated genes) and only 97 probes that were significantly hypomethylated (corresponding to 6 promoter-hypomethylated genes) in PAX3-FOXO1- compared with PAX7-FOXO1-positive tumors. Examination of the differentially methylated genes between the two FP subtypes demonstrated that CDKN1C, a gene with potential importance in RMS tumor biology, exhibited promoter hypermethylation in PAX3-FOXO1- compared to PAX7-FOXO1-positive tumors. RNA sequencing studies revealed that CDKN1C was significantly under-expressed in PAX3-FOXO1- compared to PAX7-FOXO1-positive tumors. Finally, immunohistochemistry studies on TMAs containing FP RMS tumors confirmed that there was a significant difference in CDKN1C expression between PAX3-FOXO1- and PAX7-FOXO1-positive tumors at the protein level. To further investigate DNA methylation differences between RAS mutant and wild-type FN RMS tumors, we combined FN RMS tumors from the discovery and validation cohorts. A supervised analysis of DNA methylation differences identified 117 hypermethylated CpG sites (corresponding to 28 promoter-hypermethylated genes) and 77 hypomethylated CpG sites (corresponding to 7 promoter-hypomethylated genes) in RAS mutant compared to RAS wild-type FN RMS tumors. We used these CpG sites to compare DNA methylation in FN RMS tumors and normal skeletal muscle to further examine the relationship between FN tumors and this normal tissue. Clustering analysis of FN tumors and muscle defined two main subgroups, which closely correspond to the RAS wild-type-enriched FN-1 and RAS mutant-enriched FN-2 subsets. The normal muscle samples clustered with the FN-1 subset, thus indicating that normal developing muscle has a DNA methylation pattern more similar to the RAS wild-type-enriched subset than the RAS mutant-enriched subset. Further examination of the DNA methylation pattern within the RAS mutant-enriched subset revealed two smaller subgroups, one subgroup (FN-2L) consisting of most RAS mutant tumors and no RAS wild-type tumors, and a second subgroup (FN-2R) consisting of a mixture of RAS mutant and RAS wild-type tumors. In support of the hypothesis that additional RAS pathway alterations occur in RAS wild-type tumors in the FN-2R subgroup, we found that mutations of two additional RAS pathway genes (NF1 and SOS1) were present at a high frequency in these RAS wild-type tumors. We next evaluated whether differential methylation in FN tumors with mutant RAS pathway genes versus wild-type RAS pathway genes corresponds to gene expression differences. Differential methylation analysis between the two FN subsets identified 48 hypomethylated CpG sites (corresponding to 5 promoter-hypomethylated genes) and only 3 hypermethylated CpG sites in mutant RAS pathway compared to wild-type RAS pathway tumors. Examination of these differentially methylated genes demonstrated that ALDH1A3, a gene previously reported as a potential marker for cancer stem cells in embryonal RMS, was promoter-hypomethylated in RAS pathway mutant versus wild-type FN tumors. Furthermore, ALDH1A3 was overexpressed in RAS pathway mutant versus wild-type tumors. To examine whether any RMS model systems faithfully recapitulate DNA methylation patterns found in RMS primary tumors, we compared genome-wide methylation patterns in 11 RMS cell lines, 12 cell line-derived xenografts (CDXs) and 14 patient-derived xenografts (PDXs) to patterns in primary tumors. Hierarchical clustering analysis of these samples identified a FP cluster and a FN cluster. Further examination of the FP and FN clusters showed that nearly all cell lines and CDXs form one subset in each cluster whereas nearly all PDXs and primary tumors form a second subset. Most pairs of cell line and corresponding CDX (or pairs of subcutaneous and intramuscular CDXs) cluster together closely. The finding of two PDXs clustering with cell lines and CDXs suggests that PDXs may lose the native methylation pattern under some unknown circumstances. An examination of the heat maps in our clustering analysis reveals that the vast majority of analyzed CpG sites are hypermethylated in cell lines and CDXs in contrast to the wider distribution of hypo- and hypermethylation found in primary tumors and PDXs. Finally, overall methylation levels in FP cell lines and CDXs are significantly higher than methylation levels in primary tumors whereas there is no statistical differences in overall methylation levels between PDXs and primary tumors. A similar pattern is found in the FN samples.
