Fusion-Negative Rhabdomyosarcoma: Clinical Application of Targeted RNA Sequencing

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. For stratification purposes, rhabdomyosarcoma is classified into fusion-positive RMS (alveolar rhabdomyosarcoma) and fusion-negative RMS (embryonal or spindle cell/sclerosing, FN-RMS) subtypes according to its PAX::FOXO1 fusion status. This study aims to highlight the pathologic and molecular characteristics of a cohort of FN-RMS using a targeted NGS RNA-Seq assay.

Twelve tumors were analyzed through targeted RNA-Seq using the Trusight Pancancer panel from Illumina. Molecular alterations were then correlated with the clinicopathological features.

Of the 12 tumors analyzed, we identified 6 embryonal rhabdomyosarcomas (ERMSs) harboring mutations in key signaling molecules (KRAS, HRAS, NRAS, and FGFR4), oncogenic DICER1 mutations in 2 ERMS, pathogenic TP53 and NF1 mutations in an ERMS with features of anaplasia, a TEAD1::NCOA2 gene fusion in a congenital spindle cell and sclerosing rhabdomyosarcoma (SSRMS), and a FUS::TFCP2 gene fusion in a skull base SSRMS. Only 1 ERMS in the bladder showed no reportable molecular alterations.

We illustrate case examples demonstrating how a combined morphological and molecular approach with targeted RNA-Seq can aid in diagnosis and identify clinically actionable alterations in pediatric FN-RMS.