Abstract PR04: Progress towards development of an exosome-based biomarker assay in Ewing sarcoma
Authors: Samuel Glenson , Crow Jennifer , Atay Safinur , Godwin Andrew K. 
Published: August 2018

Abstract
Background: Ewing sarcoma (EWS) is the second most common primary pediatric bone malignancy in the United States. Despite significant advancements made in the management/treatment of localized EWS, the 5-year overall survival for patients with metastatic or recurrent EWS is less than 30%. Sadly, outside of scans, no clinically relevant predictive biomarkers currently exist; therefore, there is a glaring need for complementary biomolecular approaches to help predict drug resistance, metastasis, and recurrence, with the overall aim of enhancing the management of EWS patients. Recent advancements in the field of biomarker development have demonstrated that nano-sized vesicles, termed exosomes, are actively released by tumor cells and carry content (such as proteins and RNAs) which are representative of their cell of origin. Importantly, these vesicles can be isolated from various biologic fluids including plasma and have the potential to provide a minimally invasive, real-time “shapshot” of the genetic and proteomic content of these tumor cells, thereby providing a rich source of potential diagnostic and predictive biomarkers. We hypothesize that analysis of the proteomic content of EWS-derived exosomes may result in the ability to selectively enrich for EWS-specific exosomes from patient plasma and, upon analysis of the content, provide a novel and clinically relevant source of disease-specific biomarkers.
Methods: We isolated exosomes from five EWS cell line derived exosomes with varying EWS-Ets fusions (TC-71, CHLA-258, RD-ES, SK-ES-1, and COG-E-352). We characterized the proteomic and nucleic acid content of these extracellular vesicles using Western blot analysis, Nanoparticle Tracking Analysis, and real-time PCR in order to establish exosome purity and identified already established EWS markers such as the CD99/MIC2 protein and the EWS-Ets fusion transcripts. We next conducted mass spectrometry to obtain a global proteomic assessment of these EWS cell line derived exosomes. Longitudinal pediatric clinical samples collected through an established pediatric sarcoma biobank (16 Ewing sarcoma, 5 rhabdomyosarcoma and 7 osteosarcoma pediatric patients) were utilized for this study. Through the use of this proteomic data set and a portion of the pediatric sarcoma clinical sample set, we have developed a magnetic bead-based immunoprecipitation approach requiring the use of only 250 µL of patient plasma to isolate tumor-derived exosomes in pediatric EWS patients (with localized, metastatic or recurrence of disease). These isolated extracellular vesicles were then evaluated for the presence of EWS markers by Western blot and the EWS-Ets fusion transcripts by nested qPCR.
Results: Exosomes from each of the experimental cell lines contained typical exosomal markers (i.e., ALIX and CD81) and had a mean size of 170 nm. EWS cell line derived exosomes demonstrated the presence of EWS-Ets fusion transcripts at both the mRNA level and proteomic level. The analysis of the mass spectrometry data revealed 619 commonly shared proteins. Upon comparison with the content of plasma proteomic and exosomal specific databases, 3 unique proteins of interest were identified. Lastly, exosomes immunoprecipitated from both patient plasma and exosome-spiked plasma displayed EWS markers such as MIC2/CD99 and EWS-FLI1, and contained the EWS-Ets mRNA transcripts.
Conclusions: For the first time reported, tumor-derived exosomes from EWS pediatric patients are demonstrated to be isolated by immunoprecipitation techniques and express presence of EWS-specific markers. The future evaluation and analysis of these exosomal EWS markers should provide a simple and cost effective “liquid biopsy” that would allow clinicians to diagnose and monitor tumor burden, detect tumor responsiveness to therapy, monitor changes within the tumor including determining drug sensitivity alterations, and thus enable clinicians to better manage patients with EWS.
This abstract is also being presented as Poster A31.
Citation Format: Glenson Samuel, Jennifer Crow, Safinur Atay, Andrew K. Godwin. Progress towards development of an exosome-based biomarker assay in Ewing sarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr PR04.