Differential gene expression in leukemic blasts and leukemic stem cells (LSC) sorted from pediatric AML patients compared to control myeloblasts and hematopoietic stem cells (HSC) sorted from cord blood. Differential gene expression in leukemic blasts and leukemic stem cells (LSC) sorted from pediatric AML patients compared to control myeloblasts and hematopoietic stem cells (HSC) sorted from cord blood

Purpose: Currently, only few reports address the molecular abnormalities of LSC compared to HSC in pedAML. Identifying LSC aberrations is crucial to tackle the high relapse rate and to develop therapeutic targeting strategies for LSC elimination, while ensuring salvage of normal HSCs. Methods: Blasts and stem cells were separated based on CD38 expression (positive and negative, respectively). Leukemic blasts (n=4), LSCs (n=3) and lymphocytes (n=4) were sorted from 4 de novo pedAML patients (two FLT3-ITD, two FLT3 WT). As control, CD34+CD38+ (n=3) and CD34+CD38- (n=2) cells were sorted from cord blood (CB). RNA was extracted using the miRNeasy Mini Kit (Qiagen) in combination with on-column DNase I digestion (RNase-Free DNase set, Qiagen). Mean RIN of all sorted fractions was 9.3 (95% CI 9.1-9.5). All sorted cell fractions (n=15) were profiled on a custom 8x60K human Gene expression micro-array, containing probes for all human protein-coding genes with lncRNA content based on LNCipedia 2.13 (Biogazelle), as follows: 20 ng RNA was pre-amplified using the Complete Whole Transcriptome Amplification Kit (Sigma-Aldrich). Amplified RNA was subsequently labelled using the Genomic DNA ULS Labeling Kit (Agilent) and hybridized to the array in combination with CGH blocking to reduce background signaling. Micro-arrays were analyzed using an Agilent micro-array scanner and Feature Extraction software (v12.0). Probe intensities were background subtracted, quantile-normalised and log2-based probe intensities were calculated. Overall design: To examine differential gene expression between LSC-HSC and leukemic blasts-control myeloblasts within pediatric AML.