Abstract C018: DNA methylation profiling reveals novel rhabdomyosarcoma subsets with distinct histologic, genetic and clinical properties

Abstract

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma and can be divided into two major subtypes: fusion-positive (FP) RMS, which most commonly involves the PAX3::FOXO1 or PAX7::FOXO1 fusion gene, and fusion-negative (FN) RMS, which lacks these gene fusions. Previous DNA methylation studies have shown different patterns in the FP and FN subtypes. We hypothesized that there are subsets within the FP and FN subtypes that are associated with distinct clinical and biological characteristics and can be identified by DNA methylation profiling. To test this, we determined genome-wide DNA methylation profiles for two large independent cohorts of RMS: a Children’s Oncology Group cohort of n=223 patients and a Molecular Characterization Initiative cohort of n=172 patients. DNA methylation in the tumors was analyzed using the Illumina Infinium HumanMethylation450K or EPIC BeadChips. Unsupervised hierarchical clustering and t-distributed stochastic neighbor embedding (t-SNE) analyses were conducted to segregate patients into DNA methylation-defined subsets, in which we assessed histology, fusion status, mutations, copy number and clinical parameters. The DKFZ sarcoma DNA methylation classifier (PMID: 33479225) was also used to explore the significance of these DNA methylation-defined subsets. Our study confirms the presence of two major subsets (FN1 and FN2) in FN RMS as well as PAX3::FOXO1- and PAX7::FOXO1-enriched subsets in FP RMS. The larger FN2 subset shows typical embryonal histology, frequent mutations in the RAS signaling pathway and chromosome 8 gain. In contrast, the FN1 subset is heterogeneous and consists of multiple distinct groups, including the previously recognized MYOD1-mutant group as well as several novel groups. The AFN group consists of FN tumors with alveolar histology and frequent MYCL or MYCN amplification. The FN1/E group consists of FN tumors with embryonal histology and a higher frequency of botryoid histology and MDM2 amplification and a lower frequency of RAS pathway mutations, BCOR mutations, and chromosome 8 gain than the FN2 subset. The FN1/O group is a heterogeneous group of tumors and can be further divided into groups with high-level normal cell contamination, rare RMS tumors and non-RMS tumors. In summary, DNA methylation profiling provides a robust approach for subcategorizing the RMS family of soft tissue tumors into more homogeneous subsets. This framework will enable progress in understanding the distinct subsets of RMS, thereby guiding future advances in clinical and biological studies of RMS.