Plasma DNA-Based Molecular Diagnosis, Prognostication, and Monitoring of Patients With EWSR1 Fusion-Positive Sarcomas

Abstract

Purpose Ewing Sarcoma (ES) and Desmoplastic Small Round Cell Tumors (DSRCT) are aggressive sarcomas molecularly characterized by EWSR1 gene fusions. As pathognomonic genomic events in these respective tumor types, EWSR1 fusions represent robust potential biomarkers for disease monitoring. Patients and Methods To investigate the feasibility of identifying EWSR1 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 EWSR1 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 EWSR1 as well as several other genes with potential prognostic utility. Results EWSR1 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 TP53 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.

Detection of Somatic Structural Variants Enables Quantification and Characterization of Circulating Tumor DNA in Children With Solid Tumors

“…36–38 To compare TranSS-Seq with this previously validated approach, patient-specific polymerase chain reaction primers were developed for a subset of Ewing sarcoma and alveolar rhabdomyosarcoma samples (Data Supplement). The percentage of ctDNA levels identified by TranSS-Seq correlated with the percentage of ctDNA detected by ddPCR (Fig 3E).…”

“…36–38 Although this approach has proven to be highly sensitive and quantitative, the development of each assay requires genomic profiling of large amounts of tumor biopsy material to establish the patient-specific oncogenic translocation. One recent study used a combination of ddPCR and hybrid capture sequencing to detect EWSR1 translocations in Ewing sarcoma and desmoplastic small round-cell tumors.…”

“…One recent study used a combination of ddPCR and hybrid capture sequencing to detect EWSR1 translocations in Ewing sarcoma and desmoplastic small round-cell tumors. 38 Results showed that it is feasible to detect EWSR1 translocations directly from the plasma of these patients without first sequencing the tumor sample. In our study, we developed a unique hybrid capture sequencing assay that targets a wider range of oncogenic translocations observed in pediatric sarcomas.…”

Genotyping of circulating cell‐free DNA enables noninvasive tumor detection in myxoid liposarcomas

“…As amplification of the target region is performed with primers binding to separate chromosomal regions and detection of the target amplicon occurs by a probe that is placed on the breakpoint region, these assays offer unprecedented specificity. The feasibility of this approach has also recently been confirmed in Ewing sarcomas . The assays to detect synovial sarcoma t(X;18) ctDNA from cell‐lines showed similar sensitivity and specificity than with myxoid liposarcomas (Fig.…”

“…Targeted massive parallel sequencing of the breakpoint regions might overcome these limitations and allow breakpoint sequencing from FFPE tumor samples or directly from plasma samples. Maybe a combinatorial approach of massive parallel sequencing to detect multiple targetable mutations in conjunction with individual, highly sensitive assays to quantify ctDNA might provide the best performance …”