Plasma extracellular vesicle sampling from high grade gliomas demonstrates a small RNA signature indicative of disease and identifies lncRNA RPPH1 as a high grade glioma biomarker.

Background: High grade gliomas (HGGs) and cells of the tumour microenvironment (TME) secrete extracellular vesicles (EVs) into the plasma that contain genetic and protein cargo which function in paracrine signaling. Isolation of these EVs and their cargo from plasma could lead to a simplistic tool that can inform on diagnosis and disease course of HGG.Methods: In the present study, plasma EVs were captured utilizing a peptide affinity method (Vn96 peptide) from HGG patients and normal controls followed by next generation sequencing (NovaSeq6000) to define a small RNA (sRNA) signature unique to HGG.Results: Over 750 differentially expressed sRNA (miRNA, snoRNA, lncRNA, tRNA, mRNA fragments and non-annotated regions) were identified between HGG and controls. MiEAA 2.0 pathway analysis of the miRNA in the sRNA signature revealed miRNA highly enriched in both EV and HGG pathways demonstrating the validity of results in capturing a signal from the TME. Also revealed were several novel HGG plasma EV sRNA biomarkers including lncRNA RPPH1 (Ribonuclease P Component H1), RNY4 (Ro60-Associated Y4) and RNY5 (Ro60-Associated Y5). Furthermore, in paired longitudinal patient plasma sampling, RPPH1 informed on surgical resection (decreased on resection) and importantly RPPH1 increased again on clinically defined progression.Conclusions: The present study supports the role of plasma EV sRNA sampling (and particularly RPPH1) as part of a multi-pronged approach to HGG diagnosis and disease course surveillance. Overall design: Extracellular vesicles were captured by peptide affinity captured methods (Vn96 peptide), extracted with miRVana Total RNA kit (Thermo Fisher) and small RNA sequencing was performed using NEXTFLEX small RNA sequencing (v3; Perkin Elmer).