Computational protocol: Selective inhibition of BCL-2 is a promising target in patients with high-risk myelodysplastic syndromes and adverse mutational profile

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Protocol publication

[…] BMMNC were stained with AnnexinV-FITC in AnnexinV staining solution (0.1 M HEPES/NaOH, pH 7.4, 1.4 M NaCl 0.9%, 25 mM CaCl2), followed by staining with fluorescently labelled antibodies against CD34 (clone 4H11). For intracellular staining, cells were stained against CD34, followed by fixation in 2% paraformaldehyde, permeabilization using perm/wash buffer (BD Bioscience, Franklin Lakes, NJ, USA) and subsequent staining with fluorescently labeled antibodies against BCL-2 (clone Bcl-2/100, BD Bioscience) or respective isotype controls (Cat.: 556357, BD Bioscience; clone DA1E). Dead cells were excluded by Fixable Viability Dye staining. If not otherwise stated, reagents and antibodies were purchased from eBioscience. Flowanalysis was performed on a BD FACS Canto II (BD Bioscience) and data were analyzed using FlowJo software (TreeStar Inc., Ashland, OR, USA). [...] Gene expression analysis was performed using the Human Genome U133 Plus 2.0 Array from Affymetrix (Santa Clara, CA, USA). The Affymetrix normalization method was used. All expression measurements of each array are divided by the median (calculated across all calls (present, mixed and absent)) and plotted on a logarithmic scale to normalize the data and show a log2 median-centered intensity blot. BMMNCs of 90 MDS patients (MDS with del (5q) n = 23; MDS-SLD n = 4, MDS-RS-SLD n = 6, MDS-MLD n = 6, MDS-RS-MLD n = 6, MDS-EB-1 (refractory anemia with blast excess 5–9%) n = 23, MDS-EB-2 (refractory anemia with blast excess ≥10%) n = 22) and peripheral blood mononuclear cells from 110 healthy controls were analyzed. To test for any significant differences of gene expression between the MDS groups, ‘early’, ‘intermediate’ and ‘late’ and in between the groups “proven mutation” vs “no mutation detectable” Student’s T Test was used. All reported P-values are two-sided, with a significance level of 0.05 and have not been adjusted for multiple testing (unpaired Student’s t-test). Pairwise differences are presented with 95% confidence intervals (CIs). Statistical analyses were performed using GraphPadPrism version 5.01 (Graphpad Software, Inc., San Diego, CA, USA). […]

Pipeline specifications

Software tools FlowJo, GraphPad Prism
Applications Miscellaneous, Flow cytometry
Organisms Homo sapiens