Minimal residual disease assessment in B-cell precursor acute lymphoblastic leukemia by semi-automated identification of normal hematopoietic cells:A EuroFlow study

Presence of minimal residual disease (MRD), detected by flow cytometry, is an important prognostic biomarker in the management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, data-analysis remains mainly expert-dependent. In this study, we designed and validated an Automated Gating &amp; Identification (AGI) tool for MRD analysis in BCP-ALL patients using the two tubes of the EuroFlow 8-color MRD panel. The accuracy, repeatability, and reproducibility of the AGI tool was validated in a multicenter study using bone marrow follow-up samples from 174 BCP-ALL patients, stained with the EuroFlow BCP-ALL MRD panel. In these patients, MRD was assessed both by manual analysis and by AGI tool supported analysis. Comparison of MRD levels obtained between both approaches showed a concordance rate of 83%, with comparable concordances between MRD tubes (tube 1, 2 or both), treatment received (chemotherapy versus targeted therapy) and flow cytometers (FACSCanto versus FACSLyric). After review of discordant cases by additional experts, the concordance increased to 97%. Furthermore, the AGI tool showed excellent intra-expert concordance (100%) and good inter-expert concordance (90%). In addition to MRD levels, also percentages of normal cell populations showed excellent concordance between manual and AGI tool analysis. We conclude that the AGI tool may facilitate MRD analysis using the EuroFlow BCP-ALL MRD protocol and will contribute to a more standardized and objective MRD assessment. However, appropriate training is required for the correct analysis of MRD data.</p

Flow cytometric minimal residual disease assessment in B-cell precursor acute lymphoblastic leukaemia patients treated with CD19-targeted therapies — a EuroFlow study

The standardized EuroFlow protocol, including CD19 as primary B-cell marker, enables highly sensitive and reliable minimal residual disease (MRD) assessment in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) patients treated with chemotherapy. We developed and validated an alternative gating strategy allowing reliable MRD analysis in BCP-ALL patients treated with CD19-targeting therapies. Concordant data were obtained in 92% of targeted therapy patients who remained CD19-positive, whereas this was 81% in patients that became (partially) CD19-negative. Nevertheless, in both groups median MRD values showed excellent correlation with the original MRD data, indicating that, despite higher interlaboratory variation, the overall MRD analysis was correct.The EuroFlow Consortium received support from the FP6-2004-LIFESCIHEALTH-5 programme of the European Commission (grant LSHB-CT-2006-018708) as Specific Targeted Research Project (STREP). The EuroFlow Consortium is part of the European Scientific Foundation for Hemato-Oncology (ESLHO), a Scientific Working Group (SWG) of the European Hematology Association (EHA). TS and LS were supported by a Scientific Grant from the Medical University of Silesia Nr. PCN-1-050/K/0/K

An Extensive Quality Control and Quality Assurance (QC/QA) Program Significantly Improves Inter-Laboratory Concordance Rates of Flow-Cytometric Minimal Residual Disease Assessment in Acute Lymphoblastic Leukemia: An I-BFM-FLOW-Network Report

Monitoring of minimal residual disease (MRD) by flow cytometry (FCM) is a powerful prognostic tool for predicting outcomes in acute lymphoblastic leukemia (ALL). To apply FCM-MRD in large, collaborative trials, dedicated laboratory staff must be educated to concordantly high levels of expertise and their performance quality should be continuously monitored. We sought to install a unique and comprehensive training and quality control (QC) program involving a large number of reference laboratories within the international Berlin-Frankfurt-Münster (I-BFM) consortium, in order to complement the standardization of the methodology with an educational component and persistent quality control measures. Our QC and quality assurance (QA) program is based on four major cornerstones: (i) a twinning maturation program, (ii) obligatory participation in external QA programs (spiked sample send around, United Kingdom National External Quality Assessment Service (UK NEQAS)), (iii) regular participation in list-mode-data (LMD) file ring trials (FCM data file send arounds), and (iv) surveys of independent data derived from trial results. We demonstrate that the training of laboratories using experienced twinning partners, along with continuous educational feedback significantly improves the performance of laboratories in detecting and quantifying MRD in pediatric ALL patients. Overall, our extensive education and quality control program improved inter-laboratory concordance rates of FCM-MRD assessments and ultimately led to a very high conformity of risk estimates in independent patient cohorts

Signaling properties of murine MPL and MPL mutants after stimulation with thrombopoietin and romiplostim

Thrombopoietin (THPO) and its receptor myeloproliferative leukemia virus oncogene (MPL) regulate hematopoietic stem cell (HSC) quiescence and maintenance, but also megakaryopoiesis. Thrombocytopenias or aplastic anemias can be treated today with THPO peptide mimetics (romiplostim) or small-molecule THPO receptor agonists (e.g., eltrombopag). These THPO mimetics were designed for human application; however, many preclinical studies are performed in murine models. We investigated the activation of wild-type murine MPL (mMPL) by romiplostim. Romiplostim stimulated AKT, ERK1/2, and STAT5 phosphorylation without preference for one of these pathways, however, with a four- to fivefold lower phosphorylation intensity at high concentration. Faster internalization of mMPL after romiplostim binding could be one explanation of reduced signaling. In vitro megakaryocyte differentiation, proliferation, and maturation by romiplostim was less efficient compared with stimulation with mTHPO. We further dissected mMPL signaling by lentiviral overexpression of mMPL mutants with tyrosine (Y)-to-phenylalanine (F) substitutions in the distal cytoplasmic tyrosines 582 (Y582F), 616 (Y616F), and 621 (Y621F) individually and in combination (Y616F_Y621F) and in truncated receptors lacking 53 (Δ53) or 69 (Δ69) C-terminal amino acids. Mutation at tyrosine residue Y582F caused a gain-of-function with baseline activation and increased ERK1/2 phosphorylation upon stimulation. In agreement with this, proliferation in Y582F-32D cells was increased, yet did not rescue in vitro megakaryopoiesis from Mpl-deficient cells. Y616F and Y621F mutated receptors exhibited strongly impaired ERK1/2 and decreased AKT signaling and conferred reduced proliferation to 32D cells upon mTHPO stimulation but a partial correction of immature megakaryopoiesis in vitro

Inhibition of Thrombopoietin/Mpl Signaling in Adult Hematopoiesis Identifies New Candidates for Hematopoietic Stem Cell Maintenance

<div><p>Thrombopoietin (Thpo) signals via its receptor Mpl and regulates megakaryopoiesis, hematopoietic stem cell (HSC) maintenance and post-transplant expansion. Mpl expression is tightly controlled and deregulation of Thpo/Mpl-signaling is linked to hematological disorders. Here, we constructed an intracellular-truncated, signaling-deficient Mpl protein which is presented on the cell surface (dnMpl). The transplantation of bone marrow cells retrovirally transduced to express dnMpl into wildtype mice induced thrombocytopenia, and a progressive loss of HSC. The aplastic BM allowed the engraftment of a second BM transplant without further conditioning. Functional analysis of the truncated Mpl <i>in vitro</i> and <i>in vivo</i> demonstrated no internalization after Thpo binding and the inhibition of Thpo/Mpl-signaling in wildtype cells due to dominant-negative (dn) effects by receptor competition with wildtype Mpl for Thpo binding. Intracellular inhibition of Mpl could be excluded as the major mechanism by the use of a constitutive-dimerized dnMpl. To further elucidate the molecular changes induced by Thpo/Mpl-inhibition on the HSC-enriched cell population in the BM, we performed gene expression analysis of Lin-Sca1+cKit+ (LSK) cells isolated from mice transplanted with dnMpl transduced BM cells. The gene expression profile supported the exhaustion of HSC due to increased cell cycle progression and identified new and known downstream effectors of Thpo/Mpl-signaling in HSC (namely TIE2, ESAM1 and EPCR detected on the HSC-enriched LSK cell population). We further compared gene expression profiles in LSK cells of dnMpl mice with human CD34+ cells of aplastic anemia patients and identified similar deregulations of important stemness genes in both cell populations. In summary, we established a novel way of Thpo/Mpl inhibition in the adult mouse and performed in depth analysis of the phenotype including gene expression profiling.</p></div

Gene expression analysis of LSK cells from dnMpl mice.

<p>(A) Schematic picture of the performed experiment. Wildtype Lin- BM cells were transduced with either GFP or dnMpl.IRES.GFP expressing vectors and transplanted into lethally irradiated wt recipients. Eight weeks after transplantation LSK cells were sorted using flow cytometry. (B) Representative FACS blots of the BM cells of GFP control and dnMpl.IRES.GFP transplanted mice. In control mice GFP positive and negative LSK cells were pooled, whereas for dnMpl.IRES.GFP mice GFP positive (47%) and negative (53%) LSK cells were separately subjected to transcriptome analysis. (C) Principal component analysis (PCA) on genes differentially expressed between dnMpl-GFP+ and control LSK cells. (D) Heatmap of selected genes found to be deregulated in the gene expression analysis. dnMpl positive and negative LSK cells from the same mice (dnMpl pos (4–6) and neg (7–9)) were compared to the expression in LSK of GFP control transplanted mice (control 1–3). (blue: donwregulated genes, red: upregulated genes). (E) Gene set enrichment analysis (GSEA) of dnMpl versus GFP control transplanted mice. The expression matrix of dnMpl positive and dnMpl negative cells from the same mice were either used in combination or separately for the GSEA. The normalized enrichment scores (NES) of gene sets are displayed that are significantly enriched in the dnMpl phenotype. Most of the gene sets were part of the gene set collection of the GSEA tool and depicted based on the following publications [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131866#pone.0131866.ref013" target="_blank">13</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131866#pone.0131866.ref015" target="_blank">15</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131866#pone.0131866.ref034" target="_blank">34</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131866#pone.0131866.ref035" target="_blank">35</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131866#pone.0131866.ref046" target="_blank">46</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131866#pone.0131866.ref047" target="_blank">47</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0131866#pone.0131866.ref054" target="_blank">54</a>] or based on the KEGG database.</p

Impact of dnMpl expression on the HSC compartment.

<p>(A) Survival curve of primary transplanted mice of the dnMpl or the control groups (experiment 1–3, dnMpl n = 25, control n = 20). Of experiment three, the BM was transplanted into two secondary recipients each (3 dnMpl donors, 2 cd-dnMpl donors, 3 GFP control donors). There was no difference in survival in primary recipients but a significantly reduced survival of secondary recipients (10 dnMpl recipient mice and 6 control recipient mice, p<0.05). (B) Percentage of LSK cells in primary recipients (dnMpl and trCD34 control black filled circles, cd-dnMpl and GFP control in grey filled circles) or in steady state hematopoiesis (wt, <i>Mpl-/-</i>, <i>Thpo-/-</i>). (*p<0.05, Students t-test). (C) Percentage of LSK-CD34 negative cells in primary recipients of GFP control (n = 3) or dnMpl mice (n = 5) (***p<0.005, Students t-test). (D) Histological analysis of the BM of dnMpl and control transplanted mice. Primary (1°) dnMpl chimeric mice had reduced numbers and smaller megakaryocytes similar to <i>Mpl-/-</i> mice. Secondary dnMpl recipient mice (2°) had a hypocellular BM in agreement with the symptoms of BM failure. BM sections were Hematoxylin/Eosin stained and microscopic images were taken at 200x magnification. (E) CD45.2 wildtype C57Bl/6 mice were transplanted with dnMpl, cd-dnMpl or GFP control transduced CD45.2 wildtype lin- BM cells (experiment 3). 19 weeks after the first transplantation, two dnMpl, two cd-dnMpl and two GFP mice were infused with a second graft of 2x10⁷ CD45.1 whole BM cells without further conditioning. The chimerism of CD45.1 in the blood leukocytes was analyzed over a period of 16 weeks. dnMpl conditioned mice allowed the engraftment of a second wt graft without further conditioning. (F) In experiment 5, CD45.2 wildtype C57Bl/6 mice were transplanted with dnMpl or GFP control transduced CD45.2 wildtype lin- BM cells, however, with intended lower chimerism. 19 weeks after the first transplantation mice were given a second graft of 2 x10⁷ CD45.1 whole BM cells without further conditioning. The chimerism of CD45.1 in the blood leukocytes was analyzed over a period of 20 weeks. While the two mice exhibiting the highest percentage of dnMpl expression (4 and 14%) allowed stable engraftment of the second graft, the mice with < 1% dnMpl expression in the periphery failed to do so and presented with the low engraftment levels similar to the four GFP control mice.</p

Comparison of dnMpl LSK cells and of CD34+ cells of aplastic anemia patient.

<p>(A) Expression scores of each gene in murine dnMpl mice and human RC and SAA patients CD34+ cells were compared. Negative scores in both cases (lower left quadrant) reflect the downregulation of genes typically associated with a healthy phenotype. Differentially expressed genes (lower right and upper left quadrant) may reflect species/disease differences. Score: (expression sample–expression control)/(SD sample + SD control). Gene lists referring to each quadrant in the supplements.</p

dnMpl does not transmit Thpo induced signals.

<p>(A) The gammaretroviral LTR vector encoded the full length or the intracellular truncated, dominant-negative (dn)Mpl cDNA. For detection of the Mpl proteins a hemagglutinin (HA)Tag was added at the N-terminus between the signal peptide and the ECD. The vector also co-expressed GFP using an internal ribosomal entry site (IRES). As control the retroviral vector only containing IRES.GFP or a truncated form of human CD34 was used. (LTR: long terminal repeat, ψ: packaging signal, SD: splice donor, SA: splice acceptor, wPRE: Woodchuck hepatitis virus posttranscriptional regulatory element, SP: signal peptide, ECD: extracellular domain, TMD: transmembrane domain, ICD: intracellular domain). (B) Western blot analysis of Mpl downstream signaling proteins in 32D cells that were transduced with wtMpl, dnMpl or GFP as a control. Transduced cells were stimulated with mThpo (20ng/mL), IL-3 (5ng/ml) or fixed without stimulation. Activation of STAT3 and STAT5 was analyzed by EMSA. No phosphorylation of ERK1/2, AKT and STATS was detected in dnMpl expressing 32D cells after Thpo stimulation similar to the GFP control transduced cells.</p

Systemic and cell intrinsic effects of dnMpl expression.

<p>(A) wtMpl expressing 32D cells (aHA-PE positive) were transduced with dnMpl.IRES.GFP, constitutive dimerized (cd)-dnMpl.IRES.GFP or GFP encoding vectors to establish cultures with single and double positive cells. Cells were starved for any cytokine stimuli for 16 hrs and stimulated with 2.5 or 5 ng/mL mThpo for 15 minutes the next day. Unstimulated (negative control) and stimulated cells were fixed and permeabelized to allow intracellular staining of phosphorylated signaling molecules. Anti-phosphoERK1/2 or phosphoSTAT5 antibodies conjugated to Alexa Fluor 647 (BD Biosciences) were used. Shown are histogram overlays of pERK1/2 and pSTAT5 activation from wtMpl/GFP negative cells and wtMpl/GFP, wtMpl/dnMpl, wtMpl/cd-dnMpl double positive cells. (Dashed line—activation border; solid line—mean fluorescence intensity of wtMpl/GFP double positive cells). (B) Human erythroid leukemia (HEL) cells were transduced with retroviral vectors encoding dnMpl, cd-dnMpl or GFP as control. wtMpl expressing 32D cells (100% positive) were mixed with untransduced, GFP control, dnMpl or cd-dnMpl expressing HEL cells in different ratios (1:1, 1:2, 2:1). Cells were co-cultured in murine Thpo supplemented medium (6ng/mL). The percentage of 32D cells three and six days after co-culture was measured. In the absence of dnMpl or cd-dnMpl wtMpl/32D cells grew faster than HEL cells. But in the presence of dnMpl (1:1, 1:2 and 2:1) or cd-dnMpl (1:2) wtMpl/32D cells stopped proliferating and were diminished over time in the culture. (C) Blood counts of mice transplanted with dnMpl, cd-dnMpl or GFP control transduced wildtype Lin- BM cells six and twelve weeks after transplantation (*p<0.05, ***p<0.005 Student’s t-test, n = 4–5). One of the cd-dnMpl mice had to be killed at 12 weeks due to the severe anemia.</p