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Introduction MicroRNAs are expressed in body fluids and have recently been associated with the etiology of acute graft versus host disease (GvHD), but global expression profiling in a haematopoietic stem cell transplant (HSCT) setting is lacking. An improved understanding of the molecular pathogenesis of aGvHD may allow for improved therapeutic options, or guide personalised prophylactic protocols. Methods Mature microRNA serum expression (n=799) was assessed using nCounter technology (NanoString) in a training cohort of diagnostic aGvHD samples (n=12). Analysis assessed for microRNAs that were dysregulated in patients who developed aGvHD compared to no aGvHD. Signature microRNAs were validated in an independent cohort of serum samples taken at aGvHD diagnosis (n=42) and prior to disease onset (day 14 (D14) post-HSCT, n=47). Results NanoString profiling identified 61 microRNAs that were differentially expressed at aGvHD onset, of which n=27 were downregulated (fold change (FC) -6.94 - -1.75; p MicroRNA expression was also assessed at D14 post-HSCT in an independent cohort (n=47) to investigate their prognostic marker potential. MiR-146a (p=0.01), miR-20a (p=0.03), miR-18a (p=0.03), miR-19a (p=0.03), miR-19b (p=0.02) and miR-451 (p=0.01) were expressed at significantly higher levels in patients who developed aGvHD vs. no aGvHD. In ROC analysis, miR-146a (A=0.68, p=0.03), miR-19b (A=0.70, p=0.02) and miR-451 (A=0.69, p=0.03) had diagnostic ability with regards to aGvHD incidence, whilst miR-18a (A=0.65, p=0.09), miR-19a (A=0.65, p=0.08) and miR-20a (A=0.67, p=0.06) were approaching significance. Conclusions Circulating microRNAs have the capacity to act as prognostic and diagnostic biomarkers for aGvHD and might assist in developing personalised therapeutic approaches. Their dysregulated expression suggests a role in aGvHD pathology, which warrants further investigation. Disclosures No relevant conflicts of interest to declare.

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 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 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.