Plasma DNA-based molecular diagnosis, prognostication, and monitoring of patients with fusion-positive sarcomas.

PURPOSE

Ewing Sarcoma (ES) and Desmoplastic Small Round Cell Tumors (DSRCT) are aggressive sarcomas molecularly characterized by gene fusions. As pathognomonic genomic events in these respective tumor types, fusions represent robust potential biomarkers for disease monitoring.

PATIENTS AND METHODS

To investigate the feasibility of identifying 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 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 as well as several other genes with potential prognostic utility.

RESULTS

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 and STAG2 two other recurrent, clinically significant alterations in ES, all without prior knowledge of the tumor sequencing results.

CONCLUSION

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.