Abstract 3278: Distinct methylation profiles characterize fusion-positive and fusion-negative rhabdomyosarcoma

Abstract

Abstract BACKGROUND: Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma of childhood. Based on the presence or absence of a characteristic fusion gene (either PAX3-FOXO1 or PAX7-FOXO1), RMS comprises two major subtypes: fusion-positive (FP) and fusion-negative (FN). The goal of this study is to analyze DNA methylation differences between FP and FN tumors and to determine how these methylation differences contribute to biological differences between FP and FN RMS. METHODS: DNA methylation profiles of 37 RMS tumors (20 FP and 17 FN) and 10 RMS cell lines (5 FP and 5 FN) as well as 8 normal tissues (4 muscles and 4 bone marrows) were assessed using DNA methylation arrays. Expression array analysis was performed on 58 RMS tumors (33 FP and 25 FN, including 16 with matched methylation data). Bioinformatic methods including classification, differential methylation, differential expression, methylation-expression correlation and gene set analysis were carried out. RESULTS: Unsupervised clustering clearly distinguished the FP and FN subsets in 37 RMS primary tumors and in 10 RMS cell lines. The FP tumors showed substantially lower overall levels of methylation compared to FN tumors. Integrative methylation and gene expression analysis revealed that methylation differences between FP and FN tumors could either be positively or negatively associated with mRNA expression. Though there was no significant difference in the distribution of PAX3-FOXO1 binding sites between genes with and without differential methylation, PAX3-FOXO1 binding sites were enriched among genes that were both differentially methylated and differentially expressed. An 11-gene DNA methylation signature, classifying the RMS tumors into FP and FN subsets, was established in 37 RMS primary tumors and validated in 10 RMS cell lines. Pyrosequencing studies of 6 of the methylation signature genes independently confirmed differential methylation between FP and FN cases for the CpG site queried on the methylation array as well as other nearby CpG sites. To further investigate the relationship between DNA methylation and expression, we focused on the EMILIN1 gene, which shows higher methylation and lower mRNA expression in FP tumors compared to FN tumors. After treating RMS cell lines with 5-Aza-2′-deoxycytidine, the EMILIN1 gene demonstrated decreased DNA methylation and increased mRNA expression in multiple FP cell lines. CONCLUSION: FP and FN RMS tumors possess characteristic methylation profiles that contribute to the expression differences between these fusion subtypes. These findings indicate an important role of epigenetics in RMS tumorigenesis, suggest novel approaches for identifying fusion status, and may find new therapeutic targets in RMS. Citation Format: Wenyue Sun, Bishwanath Chatterjee, Yonghong Wang, Holly S. Stevenson, Daniel C. Edelman, Paul S. Meltzer, Frederic G. Barr. Distinct methylation profiles characterize fusion-positive and fusion-negative rhabdomyosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3278. doi:10.1158/1538-7445.AM2015-3278