Abstract Background. Aplastic anemia (AA) is an acquired bone marrow (BM) disease characterized by marrow hypoplasia, a paucity of hematopoietic stem and progenitor cells, and pancytopenia of the peripheral blood, due to immune attack on the BM. Although the immune pathophysiology of AA is well characterized, there are no biomarkers that would allow a better understanding of the immunological status of an individual AA patient. Extracellular miRNAs are cell-free circulating molecules residing in microvesicles, exosomes, and microparticles. Recent studies have reported circulating miRNAs as biomarkers to monitor T cell mediated autoimmune diseases. Our work aimed to analyze the circulating miRNA profile in the plasma of AA patients and to assess whether specific miRNAs could serve as new biomarkers for AA. Method. Human blood samples were obtained from patients (n = 129) and healthy controls (HC) (n = 54) who were enrolled on clinical protocols between 2006 and 2015 at NIH. All AA patients were diagnosed as severe AA and received immunosuppressive therapy (IST) on a clinical research protocol (#NCT00260689). Plasma samples from myelodysplastic syndrome (MDS) patients and HC were used for comparison (NCT00217594 and NCT00961064). RNA was isolated from EDTA plasma using the miRCURY RNA Isolation Kit - Biofluids (Exiqon). A total of 179 miRNAs were analyzed in 35 plasma samples from 13 AA, 11 MDS, and 11 HC (a discovery set) using the Serum/Plasma Focus microRNA PCR Panel (Exiqon). Subsequently, 19 miRNAs from the discovery set were investigated in the 108 plasma samples from 41 AA, 24 MDS, and 43 HC (a validation set) for validation using a custom PCR panel. To assess the effect of IST, 40 out of 41 AA patients with plasma samples available both before and after 6 months of IST were further analyzed. Results and Discussion . We first analyzed 35 discovery-set plasma samples. Between AA and HC, 14 miRNAs displayed more than 1.5 fold change (FC) (P < 0.05): seven miRNAs were upregulated (miR-501-3p, miR-146b-5p, miR-150-5p, let-7b-5p, miR-200a-3p, miR-1260a, and miR-424-5p) and seven miRNAs were downregulated (miR-1, miR-29b-3p, miR-30e-5p, miR-143-3p, let-7e-5p, let-7c-5p, and let-7f-5p) in AA, compared to HC. In the validation set, group comparison revealed distinct expression profiles (> 1.5 FC, P < 0.05): upregulated miR-150-5p and miR-146b-5p and downregulated miR-1 in AA, compared to HC; upregulated miR-146b-5p and miR-22-3p in MDS, compared to HC; and downregulated miR-1, miR-22-3p, and miR-424-5p in AA, compared to MDS. By multivariate analysis, miR-1 [odds ratio (OR) = 0.34, adjusted P = 0.005], miR-146b-5p (OR = 3.82, adjusted P = 0.03), and miR-150-5p (OR = 2.19, adjusted P = 0.04), independently associated with the AA diagnosis. A receiver operating characteristic curve analysis indicated strong association of miR-146b-5p [95% confidence interval (CI), 0.65-0.86, P = 0.0001], miR-150-5p (95% CI, 0.63-0.85, P = 0.0002), and miR-1 (95% CI, 0.62-0.85, P = 0.0004) with AA [area under the curve (AUC) of 0.76, 0.74, and 0.73, respectively). Further, logistic regression demonstrated that a linear combination of expression levels of miR-150-5p, miR-146b-5p, and miR-1 produced the best model. A miRNA biomarker panel composed of miR-150-5p, miR-146b-5p, and miR-1 provided significantly increased AUC of 0.86 (95% CI, 0.78-0.94, P < 0.00001), compared with use of each miRNAs alone. At onset of AA, miR-150-5p and miR-146b-5p showed modest but significant negative correlations with platelet counts and absolute reticulocyte counts, respectively. Significant changes after IST were detected in all three miRNA (miR-150-5p, miR-146b-5p, and miR-1) levels, suggesting restoration of dysregurated miRNA expressions after therapy (P = 0.0001 for miR-150-5p, P = 0.0263 for miR-146b-5p, and P= 0.0003 for miR-1). Specifically, miR-150-5p expression was significantly reduced after successful IST but did not change in non-responders. Conclusion. We demonstrate that expression levels of three dysregulated miRNAs in AA plasma associated with clinical parameters and normalized after IST, suggesting them as potential biomarkers in AA. We developed a diagnostic logistic model using combined miRNA panels. Additional studies in larger patient cohorts are required to validate miRNAs as disease biomarkers for diagnostic and therapeutic purposes in BM failure. Disclosures Hosokawa: Aplastic Anemia and MDS International Foundation: Research Funding. Desierto:GSK/Novartis: Research Funding. Rios:GSK/Novartis: Research Funding. Weinstein:GSK/Novartis: Research Funding. Townsley:GSK/Novartis: Research Funding.