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

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.

Circulating and extracellular vesicle-derived microRNAs as biomarkers in bone-related diseases

“…Using miRNA sequencing, Crow et al. ( 158 ) identified a disease-specific signature set of 62 exosomal miRNAs able to differentiate between ESFT and non-ESFT samples. Similarly, Kosela-Patercyzk et al.…”

Exosome-Based Liquid Biopsy Approaches in Bone and Soft Tissue Sarcomas: Review of the Literature, Prospectives, and Hopes for Clinical Application

“…Additionally, the quantitative measurement of EWS-FLI1 mRNA copy numbers in pPNET-derived exosomes represents an effective biomarker signature with respect to total cell RNA content, increasing the sensitivity for MRD identification during therapy and post-therapy [ 86 ]. Analogously, ESFT patients have been proven to present a higher content of exosomal miRNAs, with an average of 275 exo-miRNAs identified in ESFT pediatric patients and <100 exo-miRNAs identified in pediatric non-cancer, rhabdomyosarcoma, and OS samples [ 87 ]. Of note, Pearson’s clustering of 46 exo-miRNAs correctly identified 80% (4 of 5) of pathology-confirmed ESFT patients, with respect to healthy controls and 75% (3/4) of the non-ESFT sarcoma samples [ 87 ].…”

“…Analogously, ESFT patients have been proven to present a higher content of exosomal miRNAs, with an average of 275 exo-miRNAs identified in ESFT pediatric patients and <100 exo-miRNAs identified in pediatric non-cancer, rhabdomyosarcoma, and OS samples [ 87 ]. Of note, Pearson’s clustering of 46 exo-miRNAs correctly identified 80% (4 of 5) of pathology-confirmed ESFT patients, with respect to healthy controls and 75% (3/4) of the non-ESFT sarcoma samples [ 87 ]. Importantly, RNAseq analysis of tumor tissue from the one outlier revealed a previously uncharacterized EWS-FLI1 translocation [ 87 ].…”

“…Of note, Pearson’s clustering of 46 exo-miRNAs correctly identified 80% (4 of 5) of pathology-confirmed ESFT patients, with respect to healthy controls and 75% (3/4) of the non-ESFT sarcoma samples [ 87 ]. Importantly, RNAseq analysis of tumor tissue from the one outlier revealed a previously uncharacterized EWS-FLI1 translocation [ 87 ].…”

Identification of small extracellular vesicle protein biomarkers for pediatric Ewing Sarcoma

“…Specifically, EWS derived sEVs have been shown to mediate cross-talk with the surrounding tumor microenvironment and promote immunosuppressive phenotypes (Gassmann et al, 2021;Pachva et al, 2021). We have recently reported that circulating sEVs can be used to accurately diagnose EWS patients using a panel of exo-miRNAs (Crow et al, 2022). sEVs have great potential to be explored as biomarkers to detect early-stage cancers and also monitor tumor progression and predict outcomes (Xu et al, 2018;Möller and Lobb, 2020).…”