The immediate goal of this project is to investigate differences in DNA methylation between FP and FN RMS tumors. In previous studies, we used Illumina methylation arrays to examine DNA methylation in 20 FP and 17 FN RMS samples and demonstrated that methylation pattern permits these cases to be clustered into two distinct groups. To investigate whether any methylation difference represents an aberrant event in FP or FN RMS, we compared methylation in RMS with two normal tissues, skeletal muscle and bone marrow. Using the CpG sites that are differentially methylated between the two RMS subtypes, hierarchical clustering revealed that the normal tissue samples were tightly clustered, and were grouped together on one main branch with the FN RMS samples while all FP samples were on the other main branch. Though the methylation status of numerous differentially methylated sites is similar in FN tumors and the normal tissues, there are also differentially methylated sites for which the methylation status in FP tumors closely resembles either or both normal tissues. These methylation studies show similarities of both fusion subtypes to normal tissues for different sets of genes, and we propose that there are aberrant hypo- and hypermethylation events occurring in FP tumors, and other aberrant hyper- and hypomethylation events occurring in FN tumors. In more recent studies, DNA methylation was examined in a new cohort of 48 RMS tumors (21 FP and 27 FN) as well as 10 RMS cell lines (5 FP and 5 FN) on the Illumina HumanMethylation450 BeadChip platform. Unsupervised clustering analysis using the most variable probes (top 1%) in the 48 RMS tumors revealed that patterns of DNA methylation segregated these tumors into two distinct subgroups; one subgroup contains all 21 FP cases along with 2 FN cases and a second subgroup contains 25 of the 27 FN cases. A principal component analysis confirmed this close association of methylation pattern and fusion status, and showed that the two discordant FN cases map in a region between the FP and FN clusters. The FP tumors showed substantially lower overall levels of methylation compared to FN tumors. A supervised analysis identified probes that were significantly hypermethylated and other probes that were significantly hypomethylated in FP compared to FN RMS tumors. Application of an 11-gene methylation signature developed in our earlier study classified these 48 cases into FP and FN categories with 95% accuracy. In contrast to our previous findings, there was a significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation. Though unsupervised clustering analysis indicated that FP tumors and cell lines cluster as do the FN tumors and cell lines, a principal component analysis clarified these relationships by showing that the two groups of cell lines are located at a considerable distance from the two tumor subtypes. Analysis of the most varied probes in the tumors indicated that the vast majority of these probes are hypermethylated in all RMS cell lines. In a complementary analysis with the most variable probes (top 1%) in the cell lines, most of these probes are hypomethylated in all RMS tumors, though two smaller groups of probes show differential methylation between both groups of FP and FN samples. This study provides an independent validation that FP and FN RMS tumors possess distinct and characteristic methylation profiles. The enrichment of PAX3-FOXO1 binding sites in genes that are differentially methylated between these FP and FN tumors suggests that the PAX3-FOXO1 fusion protein may contribute to this methylation pattern. These analyses also indicate that RMS cell lines do not faithfully recapitulate the DNA methylation patterns that characterize primary tumors, and indicate that cell line models may have limited utility in the study of epigenetic alterations present in the RMS tumor samples.
