ABSTRACT
Background Exosomes are nano-sized extracellular vesicles released by many cells that contain molecules characteristic of their cell-of-origin, including microRNA. Exosomes released by glioblastoma cross the blood-brain-barrier into the peripheral circulation, and carry molecular cargo distinct to that of ‘free-circulating’ miRNA.
Methods Serum exosomal-microRNAs were isolated from glioblastoma (n=12) patients and analyzed using unbiased deep sequencing. Results were compared to sera from age- and gender-matched healthy controls, and to grades II-III (n=10) glioma patients. Significant differentially expressed microRNAs were identified, and the predictive power of individual and subsets of microRNAs were tested using univariate and multivariate analyses. Additional sera from glioblastoma patients (n=4) and independent sets of healthy (n=9) and non-glioma (n=10) controls were used to further test the specificity and predictive power of this unique exosomal-microRNA signature.
Results Twenty-six microRNAs were differentially expressed in serum exosomes from glioblastoma patients’ relative to healthy controls. Random forest modeling and data partitioning selected seven miRNAs (miR-182-5p, miR-328-3p, miR-339-5p, miR-340-5p, miR-485-3p, miR-486-5p and miR-543) as the most stable for classifying glioblastoma. Strikingly, within this model, two iterations of these miRNA classifiers could distinguish glioblastoma patients from controls with perfect accuracy. The seven-miRNA panel was able to correctly classify all specimens in validation cohorts (n=23). Also identified were 23 dysregulated miRNAs in IDHMUT gliomas; a partially overlapping yet distinct signature of lower grade glioma.
Conclusions Serum exosomal-miRNA signatures can accurately diagnose glioblastoma preoperatively. miRNA signatures identified are distinct from previously reported ‘free-circulating’ miRNA studies in GBM patients, and appear to be superior.
IMPORTANCE OF STUDY There is a real need for accurate biomarkers that can measure glioblastoma disease activity and treatment response in a safe, sensitive, affordable and timely manner. This study demonstrates that exosome-associated microRNAs isolated from patient sera have exceptional utility as blood-based biomarkers in glioma patients. These tests have the potential to provide objectively measured surrogate endpoints to allow clinical trial protocols to be more dynamic and adaptive.
Footnotes
Funding: This work was supported by grants provided by Brainstorm, Brain Foundation and Pratten Foundation as well as career support from Australian Postgraduate Awards (S.E. and S. H.), Australian Rotary Health Postgraduate Award (S. H.), and Cancer Institute New South Wales (K.L.K.). Many thanks to the wonderful and dedicated staff at RPAH, with special thanks to Mary Lordan, Jane Raftesath, Audrey Caudon, Shu Wang and Ladan Noroozi.
Conflict of Interest: The authors declare that they have no competing interests.
Authorship: All authors contributed to manuscript preparation and approve the submission of the work presented here. Specific contributions are as follows: S.E. performed technical work including serum processing, exosome purification, electron microscopy, small RNA sequencing, data analysis, manuscript preparation. F.V. and P.Y. developed and performed the bioinformatics analytical pipeline. S.H. performed technical work including serum processing, nanosight particle tracking, and mass spectrometry. H.W characterized clinical cases, including molecular characterizations of tumour tissue. L.S and B.S assisted with clinical sample procurement and case characterization. C.M.S. assisted with small RNA sequencing protocols and data interpretation. M.E.B. provided experimental design and data interpretation. K.L.K provided experimental design, cohort characterisation, proteomics methods, bioinformatics, data interpretation and presentation.