A plasma microRNA signature as a biomarker for acquired aplastic anemia

Abstract

Aplastic anemia is an acquired bone marrow failure characterized by marrow hypoplasia, a paucity of hematopoietic stem and progenitor cells, and pancytopenia of the peripheral blood, due to immune attack on the bone marrow. In aplastic anemia, a major challenge is to develop immune biomarkers to monitor the disease. We measured circulating microRNAs in plasma samples of aplastic anemia patients in order to identify disease-specific microRNAs. A total of 179 microRNAs were analyzed in 35 plasma samples from 13 aplastic anemia patients, 11 myelodysplastic syndrome patients, and 11 healthy controls using the Serum/Plasma Focus microRNA Polymerase Chain Reaction Panel. Subsequently, 19 microRNAs from the discovery set were investigated in the 108 plasma samples from 41 aplastic anemia patients, 24 myelodysplastic syndrome patients, and 43 healthy controls for validation, confirming that 3 microRNAs could be validated as dysregulated (>1.5-fold change) in aplastic anemia, compared to healthy controls. MiR-150-5p (induction of T-cell differentiation) and miR-146b-5p (involvement in the feedback regulation of innate immune response) were elevated in aplastic anemia plasma, whereas miR-1 was decreased in aplastic anemia. By receiver operating characteristic curve analysis, we developed a logistic model with these 3 microRNAs that enabled us to predict the probability of a diagnosis of aplastic anemia with an area under the curve of 0.86. Dysregulated expression levels of the microRNAs became normal after immunosuppressive therapy at 6 months. Specifically, miR-150-5p expression was significantly reduced after successful immunosuppressive therapy, but did not change in non-responders. We propose 3 novel plasma biomarkers in aplastic anemia, in which miR-150-5p, miR-146b-5p, and miR-1 can serve for diagnosis and miR-150-5p for disease monitoring. Clinicaltrials.gov identifiers:00260689, 00217594, 00961064.

Trial registration: ClinicalTrials.gov NCT00260689 NCT00217594 NCT00961064.

Copyright© Ferrata Storti Foundation.

Figures

Figure 1.

Distinct circulating microRNA (miRNA) expression profiles of AA patients compared to MDS and HC in the discovery set. (A) A volcano plot of 178 miRNA expression levels in the plasma of AA patients (n=13) and HC (n=11) in the discovery set. The x-axis displays the estimated expression difference measured in log2. Vertical lines refer to a 1.5-fold expression difference between two groups, showing that miRNAs highly expressed in AA or HC are on the right or the left, respectively, in which 6 miRNAs with higher fold changes are depicted. The y-axis shows the significance of the expression difference measured in −log10 of the P-value. The horizontal red line represents our cut-off for the significance at P<0.05. (B) A heatmap analysis visualizes hierarchical clustering of 14 miRNAs in the plasma from 13 AA patients and 11 HC. A red-blue color scale indicates normalized miRNA expression levels (red: high, blue: low). (C) Principal component analysis (PCA) plots of significantly (P<0.05) and differentially expressed miRNAs from the discovery set. Blue circles = AA, red circles = MDS, and green circles = HC. AA: aplastic anemia; HC: healthy control; MDS: myelodysplastic syndrome.

Figure 2.

Validation of the circulating microRNA (miRNA) expression profiles in the validation set. (A-B) Volcano plots of 19 miRNA expression levels in the plasma of AA (n=41), MDS (n=24), and HC (n=43) in the validation set. The x-axis is the estimated difference in expression measured in log2; vertical lines refer to a 1.5-fold difference in expression between the two groups. MiRNAs highly expressed in AA (MDS) or HC are on the right or the left, respectively. The y-axis is the significance of the difference measured in −log10 of the P-value; the horizontal red line represents our cut-off for significance at P<0.05. (C) Volcano plots of 19 miRNA expression levels in the plasma of AA (n=41) and MDS patients (n=24) in the validation set. MiRNAs highly expressed in AA or MDS are on the right or the left, respectively. (D) miR-150-5p, miR-146b-5p, miR-1, miR-22-3p, and miR-424-5p expression in the plasma of AA (n=41), MDS (n=24), and HC (n=43). *P<0.05 (one-way ANOVA). AA: aplastic anemia; HC: healthy control; MDS: myelodysplastic syndrome.

Figure 3.

Receiver operating characteristic (ROC) curves of dysregulated miRNAs in the validation set. ROC curves for individual miRNAs in AA vs. HC (A), MDS vs. HC (B), and AA vs. MDS (C). Logistic regression demonstrated a linear combination of values of miRNAs for the compared groups: miR-150-5p, miR-146b-5p, and miR-1 produced the model for AA diagnosis (the equation of the Combined miRNA panel = 4.728 + 0.446 × miR-150-5p + 1.725 × miR-146b-5p − 1.022 × miR-1); miR-146b-5p and miR-22-3p produced the model for MDS diagnosis (the equation of Combined miRNA panel = 9.547 + 1.416 × miR-146b-5p + 1.089 × miR-22-3p), and miR-22-3p, miR-424-5p, and miR-1 produced the model for AA diagnosis compared to MDS (the equation of Combined miRNA panel = −13.117 − 2.046 × miR-22-3p − 1.384 × miR-424-5p − 1.221 × miR-1). The ROC curve of the miRNA panel was generated based on the predicted probability for each patient. Predicted probability = Exponential function (Exp) (Combined miRNA panel) / [1+ Exp (Combined miRNA panel)]. AA: aplastic anemia; HC: healthy control; MDS: myelodysplastic syndrome; AUC: area under the curve; miRNA: microRNA.

Figure 4.

MicroRNA (miRNA) expression changes after IST. (A) MiR-150-5p, miR-146b-5p, and miR-1 expression in the plasma of AA patients at onset (n=40) and after IST at 6 months (n=40). (B) MiR-150-5p, miR-146-5p, and miR-1 expression in the plasma of AA patients (responders) at onset (n=23) and after IST at 6 months (n=23). (C) MiR-150-5p, miR-146-5p, and miR-1 expression in the plasma of AA patients (non-responders) at onset (n=17) and after IST at 6 months (n=17). *P<0.05 (paired two-tailed t-test). AA: aplastic anemia; IST: immunosuppressive therapy; N.S: not significant.

Figure 5.

Ingenuity Pathway Analysis (IPA) to identify immune targets of the selected three microRNAs (miRNAs). Shown are network genes of the dysregulated 3 miRNAs (miR-1, miR-146b-5p, and miR-150-5p) in aplastic anemia (AA) plasma, compared to healthy control (HC). Color intensity indicates upregulation (red) and downregulation (green). Solid and dotted lines represent direct and indirect relationships between genes.