MicroRNA Content of Ewing Sarcoma Derived Extracellular Vesicles Leads to Biomarker Potential and Identification of a Previously Undocumented EWS-FLI1 Translocation

Jennifer Crow; Glenson Samuel; Emily Farrow; Margaret Gibson; Jefferey Johnston; Erin Guest; Neil Miller; Dong Pei; Devin Koestler; Harsh Pathak; Xiaobo Liang; Cooper Mangels; Andrew K Godwin

doi:10.1177/11772719221132693

MicroRNA Content of Ewing Sarcoma Derived Extracellular Vesicles Leads to Biomarker Potential and Identification of a Previously Undocumented EWS-FLI1 Translocation

Biomark Insights. 2022 Oct 31:17:11772719221132693. doi: 10.1177/11772719221132693. eCollection 2022.

Authors

Jennifer Crow¹, Glenson Samuel^{2

3}, Emily Farrow⁴, Margaret Gibson⁴, Jefferey Johnston⁴, Erin Guest^{2

4}, Neil Miller⁴, Dong Pei^{5

1}, Devin Koestler^{3

5

1

6}, Harsh Pathak^{1

6}, Xiaobo Liang¹, Cooper Mangels¹, Andrew K Godwin^{1

3

6}

Affiliations

¹ Department of Pathology and Laboratory Medicine, University of Kansas Medical Center, Kansas City, KS, USA.
² Children's Mercy Kansas City, Kansas City, MO, USA.
³ The University of Kansas Cancer Center, University of Kansas Medical Center, Kansas City, KS, USA.
⁴ The Center for Pediatric Genomic Medicine at Children's Mercy, Kansas City, MO, USA.
⁵ The Department of Biostatistics, University of Kansas Medical Center, Kansas City, KS, USA.
⁶ Kansas Institute for Precision Medicine, University of Kansas Medical Center, Kansas City, KS, USA.

Abstract

Objective: Ewing Sarcoma Family of Tumors (ESFT) are a highly aggressive pediatric bone and soft tissue malignancy with poor outcomes in the refractory and recurrent setting. Over 90% of Ewing Sarcoma (ES) tumors are driven by the pathognomonic EWS-ETS chimeric transcripts and their corresponding oncoproteins. It has been suggested that the EWS-ETS oncogenic action can mediate microRNA (miRNA) processing. Importantly, small extracellular vesicles (sEVs), including those frequently referred to as exosomes have been shown to be highly enriched with tumor-derived small RNAs such as miRNAs. We hypothesized that ESFT-specific sEVs are enriched with certain miRNAs which could be utilized toward an exo-miRNA biomarker signature specific to this disease. Methods: We performed miRNAseq to compare both the exo-derived and cell-derived miRNA content from 8 ESFT, 2 osteosarcoma, 2 non-cancerous cell lines, and pediatric plasma samples. Results: We found that sEVs derived from ESFT cells contained nearly 2-fold more number of unique individual miRNAs as compared to non-ESFT samples. Quantitative analysis of the differential enrichment of sEV miRNAs resulted in the identification of 62 sEV-miRNAs (exo-miRNAs) with significant (P < .05) enrichment variation between ESFT and non-ESFT sEV samples. To determine if we could utilize this miRNA signature to diagnose ESFT patients via a liquid biopsy, we analyzed the RNA content of total circulating sEVs isolated from 500 µL plasma from 5 pediatric ESFT patients, 2 pediatric osteosarcoma patients, 2 pediatric rhabdomyosarcoma patients, and 4 non-cancer pediatric controls. Pearson's clustering of 60 of the 62 candidate exo-miRNAs correctly identified 80% (4 of 5) of pathology confirmed ESFT patients. Importantly, RNAseq analysis of tumor tissue from the 1 outlier, revealed a previously uncharacterized EWS-FLI1 translocation.Conclusions: Taken together, these findings support the development and validation of an exo-miRNA-based liquid biopsy to aid in the diagnosis and monitoring of ESFT.

Keywords: EWS-FLI1 fusions; Ewing Sarcoma; Ewing Sarcoma Family of Tumors; Small extracellular vesicles; biomarkers; exo-miRNAs; exosomes; liquid biopsy; microRNA.

Abstract

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