Integrated Molecular Profiling of Rhabdomyosarcoma Subtypes by Targeted RNAseq

All subtypes of rhabdomyosarcoma (RMS) show skeletal muscle differentiation but each has different oncogenic mechanisms including recurrent PAX3 and PAX7 gene fusions (alveolar), a hotspot mutation in MYOD1 (spindle/sclerosing) and mutations in other oncogenes and tumor suppressors (embryonal, pleomorphic). This range of genomic findings typically requires several different DNA and RNA-based diagnostic assays. Here, we utilize a clinically validated, targeted RNA next-generation sequencing (RNAseq) panel to simultaneously detect MYOD1 transcript levels for lineage assignment, PAX3 and PAX7 translocations and mutations in MYOD1 and other RMS-related genes for definitive subtyping in a single assay. RNA-based detection of MYOD1 p.L122R and other mutations were orthogonally validated with digital droplet (dd)PCR or DNA-based NGS. Expanding the utility of clinical-grade RNAseq data beyond the detection of gene fusions is a cost-effective and time-efficient approach for more comprehensive screening of RMS. Impact statement Comprehensive and rapid molecular diagnostics is a key driver in the selection and initiation of targeted therapies.