Plasma DNA-based molecular diagnosis, prognostication, and monitoring of patients with <i>EWSR1</i> fusion-positive sarcomas.

Abstract

Ewing Sarcoma (ES) and Desmoplastic Small Round Cell Tumors (DSRCT) are aggressive sarcomas molecularly characterized by <i>EWSR1</i> gene fusions. As pathognomonic genomic events in these respective tumor types, <i>EWSR1</i> fusions represent robust potential biomarkers for disease monitoring. To investigate the feasibility of identifying <i>EWSR1</i> fusions in plasma derived cell-free DNA (cfDNA) from ES and DSRCT patients, we evaluated two complementary approaches in samples from 17 patients with radiographic evidence of disease. The first approach involved identification of patient-specific genomic <i>EWSR1</i> fusion breakpoints in formalin-fixed, paraffin-embedded tumor DNA using a broad, hybridization capture-based next generation sequencing (NGS) panel, followed by design of patient-specific droplet digital PCR (ddPCR) assays for plasma cfDNA interrogation . The second approach employed a disease-tailored targeted hybridization capture-based NGS panel applied directly to cfDNA which included <i>EWSR1</i> as well as several other genes with potential prognostic utility. <i>EWSR1</i> fusions were identified in 11/11 (100%) ES and 5/6 (83%) DSRCT samples by ddPCR, while 10/11 (91%) and 4/6 (67%) were identified by NGS. The ddPCR approach had higher sensitivity, ranging between 0.009-0.018% sensitivity. However, the hybrid capture-based NGS assay identified the precise fusion breakpoints in the majority of cfDNA samples, as well as mutations in <i>TP53</i> and STAG2<i>,</i> two other recurrent, clinically significant alterations in ES, all without prior knowledge of the tumor sequencing results. These results provide a compelling rationale for an integrated approach utilizing both NGS and ddPCR for plasma cfDNA-based biomarker evaluations in prospective cooperative group studies.