MicroRNA signatures associated with cytogenetics and prognosis in acute myeloid leukemia

Abstract

MicroRNAs (miRNAs) are small RNAs of 19 to 25 nucleotides that are negative regulators of gene expression. To determine whether miRNAs are associated with cytogenetic abnormalities and clinical features in acute myeloid leukemia (AML), we evaluated the miRNA expression of CD34(+) cells and 122 untreated adult AML cases using a microarray platform. After background subtraction and normalization using a set of housekeeping genes, data were analyzed using Significance Analysis of Microarrays. An independent set of 60 untreated AML patients was used to validate the outcome signatures using real-time polymerase chain reaction. We identified several miRNAs differentially expressed between CD34(+) normal cells and the AML samples. miRNA expression was also closely associated with selected cytogenetic and molecular abnormalities, such as t(11q23), isolated trisomy 8, and FLT3-ITD mutations. Furthermore, patients with high expression of miR-191 and miR-199a had significantly worse overall and event-free survival than AML patients with low expression (overall survival: miR-191, P = .03; and miR-199a, P = .001, Cox regression). In conclusion, miRNA expression in AML is closely associated with cytogenetics and FLT3-ITD mutations. A small subset of miRNAs is correlated with survival.

Figure 1

miRNAs down-regulated in AML samples with respect to CD34⁺cells and mature and hematopoietic precursors. (A,B) We selected the most differentiated miRNAs according to SAM score and fold change and measured them in a random group of 6 AML patients and 4 CD34 samples obtained from healthy donors by quantitative RT-PCR. Results are presented as fold change of the miRNA expression in AML samples with respect to the CD34⁺ expression from one healthy donor after normalization with let-7i and 2^Δ Ct conversion (thin bars represent standard deviations). The difference in miRNA expression between the 4 CD34s and all the 6 AML patients was statistically significant by the t test: miR-106a (P = .001), miR125a (P = .001), miR-126 (P = .001), miR-93(P = .001), miR-130a (P = .006), miR-146 (P = .001), except for miR-135 (P = .38). (C) Average miRNA expression (from 4 different healthy donors) of peripheral blood mature granulocytes and monocytes and bone marrow committed (erythrocytic and megakaryocytic) precursors and 6 AML patients compared with that of CD34⁺ cells after normalization and 2^Δ Ct conversion. The results are presented as fold change, with respect to the CD34⁺ cells, average miRNA expression. The down-regulation of miRNA expression in mature peripheral blood cells and committed precursors with respect to CD34 cells was statistically significant by t test (P < .05).

Figure 2

MiR-155 expression in AML with FLT3-ITD mutations. Average miR-155 expression in AML patients with FLT3-WT (n = 12) and FLT3-ITD positive mutations (n = 4) measured by quantitative RT-PCR. The miRNA expression between the different groups was compared using t test (SPSS).

Figure 3

miRNAs associated with overall survival in newly diagnosed patients with AML. Kaplan-Meier estimates of overall survival for 60 AML patients with high or low expression of miR-191(A) and miR-199a (B) detected by quantitative RT-PCR. The log-rank test was used to compare differences between survival curves.