Urinary Micrornas MiR-377, MiR-423, MiR-93 and MiR-199 As Biomarkers for Graft Versus Host Disease

Introduction: Graft-versus-host disease (GvHD) is a major complication of allogeneic hematopoietic stem cell transplantation (HSCT). Studies have investigated the potential of biological biomarkers to predict GvHD, however, to date no markers are clinically used that aid in early detection or severity monitoring.

MicroRNAs are small, single-stranded RNAs that regulate ~50% of all genes by binding to the mRNA 3’UTR and repressing translation. They have been demonstrated as informative biomarkers and can also be found in bodily fluids, where they are protected from RNases by encapsulation into exosomes. MicroRNAs present in the peripheral circulation demonstrate the capacity to regulate immune function.

Materials and Methods: The expression of a microRNA panel (miR-146a, miR-155, miR-423, miR-199a-3p, miR-93* and miR-377) was assessed in a series (n=30) of urine samples taken from HSCT patients every 7 days from pre to post-HSCT (day -7 (D-7) to D28) as well as healthy controls. Expression was assessed in whole urine and exosomal preparations by TaqMan® qRT-CPR. The expression of hemoxygenase-1 (HO-1) and TGF-β, putative targets or regulators of some of the microRNA panel, was also assessed by TaqMan® qRT-PCR.

Results: The 30 patient cohort (19 (63%) male, 11 (37%) female) comprised of 9 (30%) sibling (SIB) and 21 (70%) matched unrelated donor (MUD) allogeneic peripheral blood stem cell (PBSC) transplants. 6/30 (20%) were deceased at last follow up. Twenty (67%) patients developed acute GvHD (aGvHD) (9 grade I, 7 grade II, 4 grade III) and 12 (48%) developed chronic GvHD (cGvHD) (9 limited, 3 extensive, 5 cGvHD status unknown).

Significantly higher expression of miR-377 was observed in aGvHD vs. no GvHD at Day 0 (D0) (p=0.02) and D14 (p=0.04) and this was also demonstrated by ROC analysis (D0 p=0.03, AUC 0.78; D14 p=0.05, AUC 0.74). High expression of miR-377 was also observed in classic aGvHD (aGvHD <100 days post-HSCT) vs. no GvHD (p=0.04; ROC p=0.03, AUC=0.71).

Assessing aGvHD severity, miR-423 expression was significantly lower in severe (III-IV) vs. mild (I-II) aGvHD at D0 (p=0.004; ROC p=0.01, AUC=1.0), D7 (p<0.001; ROC p=0.005, AUC=0.98) and D28 (p=0.047; ROC p=0.05, AUC=0.95) and similarly, in severe vs. no aGvHD at the same time points (D0 p=0.014, D7 p<0.001 and D28 p=0.05). Expression of miR-93 was significantly lower in severe vs. mild aGvHD at D0 (p=0.047; ROC p=0.03, AUC=0.93).

No microRNA demonstrated differential expression between pre-HSCT and day of HSCT (D0) (p>0.05). Pre-HSCT expression of miR-146a, miR-199 and miR-377 was significantly higher in aGvHD vs. healthy controls (p<0.001, p=0.010, p<0.001, respectively) and HSCT patients vs. controls (p<0.001, p=0.007 and p<0.001, respectively).

With regard to cGvHD, miR-199 was expressed at a lower level in cGvHD compared to no cGvHD and this was significant at D-7 (p=0.01), D14 (p=0.04) and D28 (p=0.04).

Expression of the microRNAs in urinary exosomes differed significantly from that of whole urine, except for miR-199 and miR-155 which demonstrated positive correlation (p=0.002 and p=0.04, respectively). No microRNA was predictive for aGvHD or cGvHD in urinary exosomes (p>0.05).

The mRNA expression of HO-1 and TGF-β was assessed in urinary total RNA at D0 and D14. TGF-β was not significantly associated with aGvHD or cGvHD (p>0.05). Expression of HO-1 at D0 was significantly lower in cGvHD vs. no GvHD (p<0.01). With regard to microRNAs, there was a significant inverse correlation between miR-146a vs. TGF-β at D14 (p<0.001) and between miR-155 vs. TGF-β at D0 (p<0.001).

Conclusions: This investigation demonstrates the potential of urinary miR-377, miR-423, miR-199 and miR-93 as biomarkers for GvHD, as assessed using robust and non-invasive methodology. Results are being validated in an expanded independent cohort.Further studies into the function and targets of these microRNAs may advance understanding of GvHD pathobiology as well as aid in early detection and prophylactic treatment.