Microrna Expression In Patients With Myelodysplastic Syndromes Treated With Demethylating Agents

Myelodysplastic syndromes (MDS) comprise a heterogeneous group of hematopoietic stem cell disorders characterized by bone marrow failure and dysplasia of one or more blood cell lineages resulting in peripheral blood cytopenias with frequent progression to AML. Treatment with azanucleosides improves survival and quality of life without the toxicities associated with intensive chemotherapy programs. The azanucleosides are DNA demethylating agents that may improve hematopoiesis in MDS by releasing tumor suppressor genes from silencing due to promoter hypermethylation. MicroRNAs (miRNAs) play key roles in cell growth and differentiation and oncogenic transformation. Dysregulation of miRNA expression may contribute to the development of MDS, as expression of several miRNAs has been reported to be downregulated in bone marrow cells from MDS patients. We performed the current study to test the hypothesis that treatment with demethylating agents modulates miRNA expression in hematopoietic tissue from MDS patients and to characterize the specific changes in miRNA expression.

This was a two-center, prospective cohort study to evaluate miRNA expression signatures in hematopoietic progenitor cells isolated from the bone marrow of subjects undergoing evaluation for peripheral blood cytopenias. Subjects were included for analysis if RNA of sufficient quality was available and the final pathologic diagnosis was one of the following: 1) normal (served as controls), 2) dyspoiesis without meeting criteria for MDS and without cytogenetic abnormalities, 3) MDS and 4) acute myeloid leukemia (AML) with myelodysplasia-related changes. Subjects who underwent treatment with a demethylating agent had bone marrow collected after treatment for before and after comparisons of miRNA signatures. Mononuclear cells were isolated from bone marrow aspirate specimens by density gradient centrifugation using a Ficoll-Paque technique. MicroRNA was isolated using the miRNeasy kit (Qiagen) and was analyzed using miScript miRNA PCR Array Human Cell Development & Differentiation (MIHS-103Z, Qiagen). Isolated microRNA was checked for quality control using miScript miRNA QC PCR array. Data were analyzed using Qiagen analysis software for microRNA, and miRNA levels are expressed as 2^-ΔΔC_T. P-values<0.05 were considered statistically significant.

Forty subjects were enrolled in the study. Eight subjects were excluded after the initial bone marrow biopsy for poor quality RNA (n = 3) or inappropriate diagnoses (lymphoid (n = 3) or myeloproliferative (n = 2) neoplasms). The remaining 32 subjects comprised 5 groups: 1) normal (n = 6), 2) dyspoiesis but not meeting criteria for MDS and normal cytogenetics (n = 5), 3) low-intermediate risk MDS (n = 8), 4) high-risk MDS (n = 6) and 5) AML with myelodysplasia-related changes (n = 7). Bone marrow samples were obtained from 5 patients after treatment with azanucleosides. The miRNA expression pattern in subjects with normal bone marrow and in those with no clear morphological evidence of MDS (groups 1 & 2) was similar. Conversely, subjects with low-intermediate risk MDS showed decreased expression of multiple miRNAs compared to controls (> 2 fold decrease in 22/84 miRNAs), most notably miR-126-3p (p = 0.02) and miR-20a-5p (p = 0.04). In contrast, high-risk MDS patients demonstrated a significant overall increase in microRNA expression (>2-fold increase in 33/84 miRNAs), especially miR-20b-5p (p = 0.04) and miR-125b-5p (p = 0.01). Treatment with demethylating agents led to decreased expression of most miRNAs (>2-fold decrease in 14/84 miRNAs), including miR-181a and let-7a, but an increase in others, such as miR-22 (2.3-fold increase). We obtained similar results in experiments conducted with primary bone marrow samples that were treated with demethylating agents ex vivo.

Our study is the first to characterize the changes in miRNA expression patterns in patients with MDS treated with demethylating agents. We also identify novel miRNA candidates to be further evaluated as biomarkers for assessing response to treatment with demethylating agents (e.g., miR-22, which has been described as possibly having a role in regulating TET2). We also demonstrate the striking difference in the pattern of miRNA expression between low-risk and high-risk MDS patients, a feature that may one day be exploited to assist with making treatment decisions.

No relevant conflicts of interest to declare.