Despite high remission rates after therapy, 60% to 70% of patients with acute myeloid leukemia (AML) do not survive 5 years after their initial diagnosis. The main cause of treatment failures may be insufficient eradication of a subpopulation of leukemic stem-like cells (LSC), which are thought to be responsible for relapse by giving rise to more differentiated leukemic progenitors (LP). To address the need for therapeutic targets in LSCs, we compared microRNA (miRNA) expression patterns in highly enriched healthy CD34

<p>XLSX file - 11KB, MicroRNA-126 is highly expressed in core binding factor AML: qRT-PCR results of miR-126 in CD34+CD38-ALDHlow/dim LSC and CD34+CD38+ALDHlow/dim leukemic progenitors in 18 AML patients. Cytogenetics indicates the presence of a CBF karyotype. Values are calculated using the dCT method with the mean of the LSC ct-values set to 1. Average and ratio, respectively indicates the averages and ratios of miR-126 expression in the LSC and progenitor fractions. CBF leukemias show higher expression of miR-126 compared to non-CBF leukemias. However the ratio of miR-126 expression between LSC and leukemic progenitors is not different in CBF and non-CBF leukemias.</p>

<p>XLSX file - 15KB, MicroRNAs differentially expressed between LSC and HSC (at least 2 out of 3 patients): Upper: MicroRNA expression ratios between CD34+CD38-ALDHlow/dim(LSC) and CD34+CD38-ALDHhigh (HSC) populations obtained from microarray analysis. The average ratio is calculated over all 3 analyzable patients. Number of patients indicate the number of patients that show significant differential expression for that microRNA. Lower: Average and standard deviations of all ratios between LSC and HSC per patient. 'Average + Stdev' and 'Average - Stdev', average plus or minus one time the standard deviation for each individual patient indicate the values used as cutoff for significant microRNAs per patients. Italic printed ratios represent non-significant microRNAs for that individual patient.</p>

<p>PDF file - 93KB, Validation of microarray by qRT-PCR: Surplus RNA from LSC and progenitor populations that remained after array hybridization was used for the validation of the microarray results. MicroRNA expression analysis was performed by qRT-PCR using microRNA specific primers and RNU48 as internal control. Fold change was calculated using ddCT-method. qRT-PCR validated the results obtained from microarray analysis. CD34+CD38- LSC show higher expression of miR-22, miR-126, miR-150, miR-335 and lower expression of miR-886-3p compared to CD34+CD38+ leukemic progenitors. Blue bars, LSC; Red bars, leukemic progenitors.</p>

<p>XLSX file - 9KB, Patient characteristics and stem cell frequencies: Patient characteristics and stem cell frequencies of samples used for miRNA expression profiling using microarray. Molecular aberrancy indicates the aberrancy as detected by routine molecular analysis of the patient at diagnosis. '-' indicates no abnormalities found in all molecular analysis tested (e.g. t(8;21), t(15;17), inv(16), NPM1, EVI1, CEBPA, and MLL). The fraction HSC within the CD34+ population indicates the CD34+CD38-ALDHhighLAPneg cells within the CD45+CD34+ cell compartment. For LSC the fraction of CD34+CD38-ALDHlow/dim cells were used. F, female; M, male; WBC, white blood cell count.</p>

<p>XLSX file - 14KB, MicroRNAs differentially expressed between LSC and progenitors (at least 4 out of 6 patients): Upper: MicroRNA expression ratios between CD34+CD38-ALDHlow/dim (LSC) and CD34+CD38+ALDHlow/dim (malignant progenitor) populations obtained from microarray analysis. The average ratio is calculated over all 6 patients. Number of patients indicate the number of patients that show significant differential expression for that microRNA. Lower: Average and standard deviations of all ratios between LSC and malignant progenitors per patient. 'Average + Stdev' and 'Average - Stdev', average plus or minus one time the standard deviation for each individual patient indicate the values used as cutoff for significant microRNAs per patients. Italic printed ratios represent non-significant microRNAs for that individual patient.</p>

<p>XLSX file - 11KB, MicroRNA-126 is highly expressed in core binding factor AML: qRT-PCR results of miR-126 in CD34+CD38-ALDHlow/dim LSC and CD34+CD38+ALDHlow/dim leukemic progenitors in 18 AML patients. Cytogenetics indicates the presence of a CBF karyotype. Values are calculated using the dCT method with the mean of the LSC ct-values set to 1. Average and ratio, respectively indicates the averages and ratios of miR-126 expression in the LSC and progenitor fractions. CBF leukemias show higher expression of miR-126 compared to non-CBF leukemias. However the ratio of miR-126 expression between LSC and leukemic progenitors is not different in CBF and non-CBF leukemias.</p>

<p>XLSX file - 54KB, Genes correlated with microRNA-126 expression in 163 AML patients show overlap with stem cell genes: Genes positively and negatively associated with miR-126 expression in 163 AML patients as published by (34). 854 genes were significantly correlated in a Spearman's rank correlation analysis with a significance threshold of univariate tests <0.001. Correlation coefficient, Parametric p-value and FDR are shown. Correlated genes were compared with the previously published HSC and LSC gene signatures of Eppert et al. (7). Genes that are associated with miR-126 and are also found in the LSC signature are shown in columns LSC-R FDR <0.05 and FDR <.0.01. Correlated genes that overlap with HSC signatures are shown under HSC-R <0.05. N/A, no overlap.</p>