Identification of a risk dependent microRNA expression signature in myelodysplastic syndromes

Abstract

The myelodysplastic syndromes (MDS) display both haematological and biological heterogeneity with variable leukaemia potential. MicroRNAs play an important role in tumour suppression and the regulation of self-renewal and differentiation of haematopoietic progenitors. Using a microarray platform, we evaluated microRNA expression from 44 patients with MDS and 17 normal controls. We identified a thirteen microRNA signature with statistically significant differential expression between normal and MDS specimens (P < 0·01), including down-regulation of members of the leukaemia-associated MIRLET7 family. A unique signature consisting of 10 microRNAs was closely associated with International Prognostic Scoring System (IPSS) risk category permitting discrimination between lower (Low/Intermediate-1) and higher risk (Intermediate-2/High) disease (P < 0·01). Selective overexpression of MIR181 family members was detected in higher risk MDS, indicating pathogenetic overlap with acute myeloid leukaemia. Survival analysis of an independent cohort of 22 IPSS lower risk MDS patients revealed a median survival of 3·5 years in patients with high expression of MIR181 family compared to 9·3 years in patients with low MIR181 expression (P = 0·002). Our pilot study suggested that analysis of microRNA expression profile offers diagnostic utility, and provide pathogenetic and prognostic discrimination in MDS.

Fig 1

MicroRNAs can differentiate MDS and normal controls. Two-way hierarchical clustering of 44 MDS and 17 normal controls (NC) was performed after quality filtering. Hierarchical clustering showed clustering of the samples according to disease status. A heat map was generated using the expression ratios of 15 microRNAs selected out of total 73 that differed significantly (P < 0·001). Red: up-regulated microRNAs, green: downregulated microRNAs. Each column represents a MDS or NC sample, and each row represents a single microRNA. Patient samples are grouped by IPSS. The heatmap colour key gradient ranging from global minimum (green) −3 to global maximum (red) +3 is on the right side.

Fig 2

MicroRNAs can differentiate MDS according to prognosis. Hierarchical clustering of 10 high risk and 10 low risk MDS samples revealed a clustering according to prognostic group. A heat map was generated using the expression ratios of 30 microRNAs selected out of total 68 that differed significantly (P < 0·05). Red: up-regulated microRNAs, green: down-regulated microRNAs. Each column represents a patient sample, and each row represents a single microRNA. Patient samples are grouped by IPSS. The heatmap colour key gradient ranging from global minimum (green) −3 to global maximum (red) +3 is on the right side.

Fig 3

Validation of microRNA expression using independent sample set by Real -Time RT-PCR. Samples from 18 independent MDS patients (HR = 6, LR = 12) were analysed. Seven differentially expressed microRNAs were randomly selected for Real-Time RT-PCR. Significant correlation was demonstrated for all tested microRNA between microRNA microchip platform and qRT-PCR.

Fig 4

MicroRNA181B locked nucleic acid in situ hybridization (LNA-ISH) of formalin-fixed, paraffin-embedded bone marrow biopsy sections. We examined six patients with MDS and two controls. MIR181B is present in the cytoplasm of both (A) HR (+++) and (B) LR MDS (++) cells arranged in clusters on the background of normal (MIR181B negative) haematopoietic cells. (C) Negative control – scrambled microRNA probe. (D) Positive control –MIR328 (A 200×, B 400×, C 400×, D 200×).

Fig 5

Kaplan–Meier estimate of overall survival in 22 IPSS lower-risk MDS patients by MIR181 family expression. Four microRNAs (MIR181A, MIR181B, MIR181C, MIR181D) from the 10 microRNA prognostic signature were selected due to the wide variance in gene expression (>10-fold). Since family member expression levels were highly correlated (r > 0·8), a composite score was generated by averaging four microRNAs separate standardized scores. Anticipating that about one-third of the patients should be considered as having ‘high risk’ due to high microRNA expression, we divided the patients into two groups, comparing the 14 patients with lower scores, to the eight patients with higher scores. Results showed a significant difference in survival from date of diagnosis to date of death or last visit.