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Acute graft-versus-host disease (aGvHD) is a major cause of adverse outcome in hematopoietic stem cell transplantation (HSCT), with a high incidence (20–50%). A novel, non-invasive diagnostic test to predict for prevalence and severity would enable improved prophylaxis and reduce morbidity. Circulatory microRNAs (miRNAs) miR-423, miR-199, miR-93*, and miR-377 have previously been associated with aGvHD in post-HSCT patient plasma, but validation is lacking and their expression within extracellular vesicles (EVs) has not been explored. This study replicated elevated serum expression of miR-423 ( p  < 0.001), miR-199 ( p  = 0.04), miR-93* ( p  < 0.001), and miR-377 ( p  = 0.03) in aGvHD, using a prognostic cohort of day 14 (D14) post-HSCT patient samples ( n  = 81). Expression also associated with disease severity. Further analysis at aGvHD diagnosis in an independent cohort ( n  = 65) confirmed high miR-423 ( p  = 0.02), miR-199 ( p  = 0.007), and miR-93* ( p  = 0.004) expression at disease onset. Investigation of expression patterns during early HSCT sequential timepoints (pre-HSCT to D28) identified elevated miRNAs at D7 post-HSCT in all transplant patients. In a novel investigation of miRNA expression in serum EVs ( n  = 15), miR-423 ( p  = 0.09), miR-199 ( p  = 0.008), and miR-93* ( p  = 0.001) levels were lower at D14 in patients who later developed aGvHD, and this was replicated for miR-423 ( p  = 0.02) and miR-199 ( p  = 0.04) ( n  = 47). Comparing serum to circulating EVs, at D14 patients remaining aGvHD-free had higher expression of miR-423 ( p  = 0.03), miR-199 ( p  = 0.009), and miR-93* ( p  = 0.002) in the EV fraction. Results verify the capacity for circulating miR-423, miR-199, and miR-93* as diagnostic and prognostic aGvHD biomarkers. The novel finding of their differential expression in EVs suggests a potential role in aGvHD etiology.

Serum and Extracellular Vesicle MicroRNAs miR-423, miR-199, and miR-93* As Biomarkers for Acute Graft-versus-Host Disease