Abstract
Purpose: Ewing Sarcoma Family of Tumors (ESFT), the second most common pediatric osseous malignancy, are characterized by the pathognomonic chromosomal EWS-ETS translocation. Outside of tumor biopsy, no clinically relevant ESFT biomarkers exist. Additionally, tumor burden assessment at diagnosis, monitoring of disease responsiveness to therapy, and detection of disease recurrence are limited to radiographic imaging. To identify new, clinically relevant biomarkers we evaluated the proteome of a subset of ESFT-derived small extracellular vesicles (sEVs).Materials and Methods: We performed the first high quality proteomic study of ESFT-derived sEVs from 5 ESFT cell lines representing the most common EWS-ETS fusion types and identified 619 proteins composing the core ESFT sEV proteome. We compared these core proteins to databases of common plasma-based proteins and sEV-associated proteins found within healthy plasma to identify proteins unique or enriched within ESFT.Results: From these analyses, two membrane bound proteins with biomarker potential were selected, CD99/MIC2 and NGFR, to develop a liquid-based assay enriching of ESFT-associated sEVs and detection of sEV mRNA cargo (i.e., EWS-ETS transcripts). We employed this immuno-enrichment approach to diagnosis of ESFT utilizing plasma (250 μl) from both localized and metastatic ESFT pediatric patients and cancer-free controls, and showed significant diagnostic power [AUC = 0.92, p = 0.001 for sEV numeration, with a PPV = 1.00, 95% CI = (0.63, 1.00) and a NPV = 0.67, 95% CI = (0.30, 0.93)].Conclusions: In this study, we demonstrate utilization of circulating ESFT-associated sEVs in pediatric patients as a source of minimally invasive diagnostic and potentially prognostic biomarkers.
Highlights
Ewing Sarcoma Family of Tumors (ESFT) encompass a group of highly aggressive pediatric osseous and soft tissue malignancies thought to originate from primordial bone marrow-derived mesenchymal stem cells and consists of small round blue cells with minimal stroma and differentiation [1]
In this study we report for the first time the proteome of ESFT-derived small extracellular vesicles (sEVs)/exosomes and develop a clinically useful test based on immuno-enrichment of ESFT-sEVs and detection of the EWS-ETS fusion transcripts
T, a benign osteoid osteoma cell line known to lack EWS-ETS fusions was included as a negative control (Supplementary Table 1) [37]. sEVs from each of the ESFT and control cell lines were isolated using ultracentrifugation [38] and the contents of the resulting 120,000 × g pellet were characterized for markers and size distribution (Figure 1 and Supplementary Figure 1)
Summary
Ewing Sarcoma Family of Tumors (ESFT) encompass a group of highly aggressive pediatric osseous and soft tissue malignancies thought to originate from primordial bone marrow-derived mesenchymal stem cells and consists of small round blue cells with minimal stroma and differentiation [1]. Ewing sarcoma of the bone, extraosseous Ewing sarcoma, and peripheral primitive neuroectodermal tumors (pPNET) are all considered manifestations of a single neoplastic entity. With a peak incidence within the second decade of life, slight male preponderance, high incidence in those of European ancestry and approximately 3 cases/million/year [2], this malignancy continues to remain the second most prevalent pediatric bone tumor after osteosarcoma. Overt metastatic disease is prognostic, with approximately 25% of newly diagnosed ESFT being affected [4]. Of the ESFT metastatic patients, 37% (or 9% of all ESFT patients) have metastases confined to the lung or pleura [5]
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