De r 11

Archival and consultation material from adult and pediatric patients with a diagnosis of ERMS where additional testing identified RAS / RAF mutations was retrieved from the pathology files at Memorial Sloan Kettering Cancer Center. Overall, 93 cases of ERMS were identified in which next-generation targeted DNA sequencing (MSK-IMPACT) was performed. In order to increase the study group, archival material from an additional cohort of 5 ERMS, which preceded the clinical MSK-IMPACT testing (2010-2014), was studied by targeted DNA PCR and Sanger sequencing for hot spot mutations in the 3 RAS genes. Overall, 26 ERMS were identified harboring RAS mutations, with 22 cases being detected by MSK-IMPACT and 4 by Sanger sequencing. The pathologic features of these cases were re-reviewed and analyzed for cell morphology, presence of rhabdomyoblasts, pleomorphism or anaplasia, mitotic activity, and necrosis. The diagnosis of RMS was confirmed by positivity for desmin (Ventana, DE-R-11, RTU) and myogenin (Ventana, F5D, RTU) in all cases. Clinical Risk Group was assessed based on current guidelines^{5 (link), 6 (link)} and follow-up data was collected by review of clinical records. The study was approved by the Institutional Review Board at MSKCC (IRB# 02-060).

The relevant antibodies and the dilutions used in this study are as follows: ALK1 (Ventana #ATK01, undiluted), ALK-D5F3 (Cell Signaling #DF53, 1:400), pan-cytokeratin (AE1/AE3) (Dako #M3515, 1:1600), CD31 (Ventana #JC70, undiluted), CD34 (Ventana #QBEnd10, undiluted), ERG (Ventana #EPR3864, undiluted), S100 (Cell Marque #4C4.9, 1:600), SOX10 (Biocare #BC34, 1:50), SMA (Cell Marque #1A4, undiluted), desmin (Ventana #DE-R-11, undiluted).