Serum and Extracellular Vesicle Micrornas MiR-423, MiR-199 and MiR-93* As Biomarkers for Acute Graft Versus Host Disease
Authors: Crossland Rachel E , Norden Jean , Pearce Kim F , Kralj Juric Mateja , Lendrem Clare , Bibby Louis A , Collin Matthew , Greinix Hildegard T. , Dickinson Anne M 
Published: October 2019

Abstract
Introduction
MicroRNAs are small, non-coding single-stranded RNAs and regulate approximately 50% of all genes by repressing translation. They are present in bodily fluids, where they are protected from RNase-mediated degradation by encapsulation into extracellular vesicles (EVs) and demonstrate a novel capacity to regulate the cellular differentiation of blood cells and immune function. Candidate microRNAs miR-377, miR-199, miR-93* and miR-423 have previously been associated with acute graft versus host disease (aGvHD) in post-hematopoietic stem cell transplant (HSCT) patient plasma. However, validation in independent cohorts is necessary, as well as further exploration to assess expression in the EV fraction of the blood.
Methods
MicroRNA expression was evaluated in early HSCT time point exploratory (n=34), validation (n=47) and diagnostic (n=65) serum cohorts by TaqMan qRT-PCR. Expression was also assessed in serum EVs (exploratory n=16 and validation n=47 cohorts) by EV isolation, RNA extraction and TaqMan qRT-PCR analysis.
Results
In sequential pre- and post-HSCT serum samples (n=34; pre-HSCT, Day0 (D0), D7, D14 & D28), miR-423 (p=0.03), miR-199 (p=0.06), miR-93* (p=0.04) and miR-377 (p=0.03) were upregulated at D14 in patients who developed aGvHD vs. no aGvHD. MiR-423 was also significantly upregulated at D0 (p=0.04), D7 (p=0.03) and D28 (p=0.03) in aGvHD patients. In relation to aGvHD severity, miR-423 (p=0.05), miR-199 (p=0.007) and miR-93* (p=0.09) were differentially expressed at D14 according to aGvHD grade. MiR-423 (p=0.02), miR-199 (p=0.07) and miR-93* (p=0.01) expression was validated at D14 in an independent cohort (n=47).
When the exploratory and validation D14 samples were combined (n=81), miR-423 (p<0.001), miR-199 (p=0.04) and miR-93* (p<0.001) expression was upregulated in patients that developed aGvHD vs. no GvHD and ROC analysis identified miR-423 (p<0.001, AUC=0.75), miR-199 (p=0.09, AUC=0.62) and miR-93* (p<0.001, AUC=0.74) to have diagnostic ability. MiR-423 (p=0.001), miR-199 (p=0.01) and miR-93* (p<0.001) expression was higher in severe (III-IV) vs. no aGvHD, miR-423 (p=0.006) and miR-93* (p=0.01) were higher in mild (I-II) vs. no aGvHD and miR-199 was higher in severe (III-IV) vs. mild (I-II) aGvHD (p=0.002). All microRNAs demonstrated significant positive correlation (p<0.001), thus, principle component analysis (PCA) was performed. The PC1 composite score was used for ROC analysis and showed diagnostic ability for aGvHD incidence (p<0.001, AUC=0.73).
In an independent diagnostic cohort (n=65), from a separate Institution, miR-423 (p=0.02), miR-199 (p=0.007) and miR-93* (p=0.004) expression was higher at aGvHD onset and showed diagnostic ability by ROC analysis (miR-423 p=0.03 AUC=0.66; miR-199 p=0.04 AUC=0.65; and miR-93* p=0.01 AUC=0.68). The three microRNAs had diagnostic ability with respect to aGvHD incidence based on composite ROC analysis of PC1 (p=0.019, AUC=0.68).
MicroRNAs were also investigated within serum EVs (n=15). MiR-199 (p=0.008), miR-93* (p=0.001) and miR-423 (p=0.09) expression was lower at D14 in patients who developed aGvHD. Results were confirmed in a D14 validation cohort (n=47), with lower EV miR-423 (p=0.02) and miR-199 (p=0.04), but not miR-93* (p=0.15) expression in patients who developed aGvHD. MiR-423 and miR-199 had diagnostic ability based on composite PC1 ROC analysis (p=0.06, AUC=0.69). By D14, patients remaining aGvHD free had higher expression of miR-423 (p=0.03), miR-199 (p=0.05) and miR-93* (p<0.001) in the EV fraction compared to whole serum.
Conclusions
Results validate the capacity for circulating serum miR-423, miR-199 and miR-93* to act as diagnostic and prognostic biomarkers for aGvHD. Novel findings of differential expression between whole serum and the EV compartment prior to disease onset suggest a role for EV microRNAs in the biology of aGvHD, which warrants further investigation.

Disclosures
No relevant conflicts of interest to declare.