Figure 1.
BCL6 expression in AML subclasses is heterogeneous. (A) BCL6 expression was compared between normal human hematopoietic cells (GSE42519 and human primary cell atlas) and human AML cells using 2 different AML cohorts. Using 2 independent cohorts of AML from Oregon Health & Science University (OHSU; 531 cases) and Erasmus (520 cases), BCL6 expression level normalized by housekeeping genes (RRN18S, ACTB, GAPDH, PGK1, PPIA, RPL13A, RPLP0, ARBP, B2M, YWHAZ, SDHA, TFRC, GUSB, HMBS, HPRT1, and TBP) was compared between molecular markers. Trends for biomarkers and BCL6 expression level were compared between 2 cohorts, each of which was compared with normal hematopoietic cells. Red and blue dotted lines are placed as a reference for the levels of expression for HSC and GMP, respectively. Statistical differences for BCL6 expression were calculated between each of the AML categories vs median values of either normal HSCs or GMPs. Asterisks represent the significance in either red or blue in comparisons vs HSCs or GMPs, respectively. (B) Expression level of BCL6 was compared in the same AML cohorts between different FAB subclasses. (C) Primary AML cells were subjected to quantitative reverse transcription polymerase chain reaction to compare expression levels of BCL6 either among primary AML cells or to reference normal donor blood cells. For primary AML specimens, bulk AML cells were subjected to blast purification (MACS sorting to exclude CD3 positive and CD19 positive cells), and BM cells were sorted for CD34 positive cells (using MACS). Relative expression of BCL6 was calculated by normalization to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Histograms represent averages of duplicates, and error bars are standard deviations. (D) Expression levels of BCL6 were analyzed by intracellular staining and flow cytometry. Expression levels of AML cells were compared vs those of normal BM cells. Mean fluorescence intensity (MFI) values were normalized to isotype control. Experiments were done in triplicates, and the mean from 3 independent experiments was plotted. Error bars indicate standard deviations. BC, band cell; CMP, common myeloid progenitor cell; MEP, megakaryocyte-erythroid progenitor cell; MM, metamyelocytes; MPP, multipotential progenitors; MY, myelocyte; nBMC (CD34+), CD34 positive normal bone marrow cells; PM, promyelocyte; PMN, polymorphonuclear cells.

BCL6 expression in AML subclasses is heterogeneous. (A) BCL6 expression was compared between normal human hematopoietic cells (GSE42519 and human primary cell atlas) and human AML cells using 2 different AML cohorts. Using 2 independent cohorts of AML from Oregon Health & Science University (OHSU; 531 cases) and Erasmus (520 cases), BCL6 expression level normalized by housekeeping genes (RRN18S, ACTB, GAPDH, PGK1, PPIA, RPL13A, RPLP0, ARBP, B2M, YWHAZ, SDHA, TFRC, GUSB, HMBS, HPRT1, and TBP) was compared between molecular markers. Trends for biomarkers and BCL6 expression level were compared between 2 cohorts, each of which was compared with normal hematopoietic cells. Red and blue dotted lines are placed as a reference for the levels of expression for HSC and GMP, respectively. Statistical differences for BCL6 expression were calculated between each of the AML categories vs median values of either normal HSCs or GMPs. Asterisks represent the significance in either red or blue in comparisons vs HSCs or GMPs, respectively. (B) Expression level of BCL6 was compared in the same AML cohorts between different FAB subclasses. (C) Primary AML cells were subjected to quantitative reverse transcription polymerase chain reaction to compare expression levels of BCL6 either among primary AML cells or to reference normal donor blood cells. For primary AML specimens, bulk AML cells were subjected to blast purification (MACS sorting to exclude CD3 positive and CD19 positive cells), and BM cells were sorted for CD34 positive cells (using MACS). Relative expression of BCL6 was calculated by normalization to GAPDH (glyceraldehyde-3-phosphate dehydrogenase). Histograms represent averages of duplicates, and error bars are standard deviations. (D) Expression levels of BCL6 were analyzed by intracellular staining and flow cytometry. Expression levels of AML cells were compared vs those of normal BM cells. Mean fluorescence intensity (MFI) values were normalized to isotype control. Experiments were done in triplicates, and the mean from 3 independent experiments was plotted. Error bars indicate standard deviations. BC, band cell; CMP, common myeloid progenitor cell; MEP, megakaryocyte-erythroid progenitor cell; MM, metamyelocytes; MPP, multipotential progenitors; MY, myelocyte; nBMC (CD34+), CD34 positive normal bone marrow cells; PM, promyelocyte; PMN, polymorphonuclear cells.

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