Distinct Methylation Profiles Characterize Fusion-Positive and Fusion-Negative Rhabdomyosarcoma

Wenyue Sun; Bishwanath Chatterjee; Yonghong Wang; Holly S Stevenson; Daniel C Edelman; Paul S Meltzer; Frederic G Barr

Disclosures

Mod Pathol. 2015;28(9):1214-1224. 

In This Article

Abstract and Introduction

Abstract

Rhabdomyosarcoma comprises two major subtypes, fusion positive (PAX3–FOXO1 or PAX7–FOXO1) and fusion negative. To investigate the significance of DNA methylation in these subtypes, we analyzed methylation profiles of 37 rhabdomyosarcoma tumors and 10 rhabdomyosarcoma cell lines, as well as 8 normal tissues. Unsupervised clustering of DNA methylation clearly distinguished the fusion-positive and fusion-negative subsets. The fusion-positive tumors showed substantially lower overall levels of methylation compared with fusion-negative tumors. Comparison with the methylation pattern of normal skeletal muscle and bone marrow indicates that fusion-negative rhabdomyosarcoma is more similar to these normal tissues compared with fusion-positive rhabdomyosarcoma, and suggests that many of the methylation differences between these subtypes arise from 'aberrant' hyper- and hypomethylation events in fusion-positive rhabdomyosarcoma. Integrative methylation and gene expression analysis revealed that methylation differences between fusion-positive and fusion-negative tumors could either be positively or negatively associated with mRNA expression. There was no significant difference in the distribution of PAX3–FOXO1-binding sites between genes with and without differential methylation. However, the finding that PAX3–FOXO1-binding sites were enriched among genes that were both differentially methylated and differentially expressed suggests that the fusion protein interacts with DNA methylation to regulate target gene expression. An 11-gene DNA methylation signature, classifying the rhabdomyosarcoma tumors into fusion-positive and fusion-negative subsets, was established and validated by pyrosequencing assays. Notably, EMILIN1 (part of the 11-gene signature) showed higher methylation and lower mRNA expression in fusion-positive compared with fusion-negative tumors, and demonstrated demethylation and re-expression in multiple fusion-positive cell lines after treatment with 5-aza-2′-deoxycytidine. In conclusion, our study demonstrates that fusion-positive and fusion-negative rhabdomyosarcoma tumors possess characteristic methylation profiles that contribute to the expression differences between these fusion subtypes. These findings indicate an important relationship between fusion status and epigenetic changes in rhabdomyosarcoma, present a novel approach for ascertaining fusion status, and may identify new therapeutic targets in rhabdomyosarcoma.

Introduction

The pediatric soft tissue cancer rhabdomyosarcoma has been traditionally classified by histology into two major subtypes, alveolar (~30%) and embryonal (~70%). Most alveolar rhabdomyosarcoma cases (~80%) have chromosomal translocations that join the DNA-binding domain of PAX3 or PAX7 to the transactivation domain of FOXO1. The PAX3–FOXO1 and PAX7–FOXO1 fusions (henceforth referred to as PAX3/7–FOXO1) encode potent transcription factors that contribute to tumorigenesis by altering growth and apoptotic pathways, modulating myogenic differentiation, and stimulating metastatic pathways.[1,2] Several studies have shown that the PAX3/7–FOXO1 fusion is a predictor of unfavorable outcome in children with rhabdomyosarcoma, and that fusion status is superior to the histologic subtype in predicting rhabdomyosarcoma outcome.[3–5] In addition, subcategorization by fusion status more accurately captures the genomic landscape, expression pattern and biology of rhabdomyosarcoma.[4,6,7]

Epigenetic alterations such as DNA methylation can also influence tumor development by altering the expression of tumor suppressor genes and oncogenes. DNA methylation occurs in CpG islands, CpG shores and distal regulatory regions. Previous methylation studies identified genes epigenetically silenced in rhabdomyosarcoma tumors, such as RASSF1 (ref[8]) and HIC1.[9] In addition, other studies suggested that there may be distinct DNA methylation patterns in alveolar and embryonal rhabdomyosarcoma.[10,11]

In this study, we undertook the first systematic, genome-wide comparison of DNA methylation profiles between fusion-positive and fusion-negative rhabdomyosarcoma. We delineated the association between PAX3/7–FOXO1 fusion status and DNA methylation through a combination of DNA methylation and gene expression assays, including array-based profiling. In addition, we report an 11-probe DNA methylation signature that is sufficient to classify fusion-positive and fusion-negative rhabdomyosarcoma tumors. These methylation findings were validated by pyrosequencing and extended by 5-aza-2′-deoxycytidine inhibition studies.

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