ALBERT

Description: Hematopoietic stem cell (HSC) function is critical in maintaining hematopoiesis continuously throughout the lifespan of an organism and any change in their ability to self-renew and/or to differentiate into blood cell lineages induces severe diseases. Postnatally, HSC are mainly located in bone marrow where their stem cell fate is regulated through a complex network of local influences, thought to be concentrated in the bone marrow (BM) niche. Despite more than 30 years of research, the precise location of the HSC niche in human BM remains unclear because most observations were obtained from mice models. BM harvesting collects macroscopic coherent tissue aggregates in a cell suspension variably diluted with blood. The qualitative interest of these tissue aggregates, termed hematons, was already reported (first by I. Blaszek's group (Blaszek et al., 1988, 1990) and by our group (Boiret et al., 2003)) yet they remain largely unknown. Should hematons really be seen as elementary BM units, they must accommodate hematopoietic niches and must be a complete ex vivo surrogate of BM tissue. In this study, we analyzed hematons as single tissue structures. Biological samples were collected from i) healthy donor bone marrow (n= 8); ii) either biological samples collected for routine analysis by selecting bone marrow with normal analysis results (n=5); or iii) from spongy bone collected from the femoral head during hip arthroplasty (n=4). After isolation of hematons, we worked at single level, we used immunohistochemistry techniques, scanning electronic microscopy, confocal microscopy, flow cytometry and cell culture. Each hematon constitutes a miniature BM structure organized in lobular form around the vascular tree. Hematons are organized structures, supported by a network of cells with numerous cytoplasmic expansions associated with an amorphous structure corresponding to the extracellular matrix. Most of the adipocytes are located on the periphery, and hematopoietic cells can be observed as retained within the mesenchymal network. Although there is a degree of inter-donor variability in the cellular contents of hematons (on average 73 +/- 10 x103 cells per hematon), we observed precursors of all cell lines in each structure. We detected a higher frequency of CD34+ cells than in filtered bone marrow, representing on average 3% and 1% respectively (p

Description: GD is a genetic lysosomal disease characterized by deficiency in glucocerebrosidase (Glc) particularly affecting macrophage-like cells where glucocerebroside accumulates in the lysosomes. Alglucerase then imiglucerase (IMI, Genzyme-Sanofi Corporation), a recombinant human glucocerebrosidase, improved or reversed many type 1 GD symptoms; Two new biosimilar agents are now available: velaglucerase-alfa (Shire Human Genetic Therapies) and taliglucerase-alfa (Biotherapeutics, Pfizer). All three recombinant enzymes are administered with identical therapeutic regimens, i.e. one infusion (30-60 IU/kg) every two weeks. However, disease responses remain variable and there are no pharmacokinetic explanations justifying this dosage regimen since plasma half-life is estimated to be less than 10 min. in murine models and in humans We compared endogenous glucocerebrosidase (Glc) activity evaluated by flow cytometry in the blood monocyte (Mo) compartment of 20 patients with 71 healthy donors, and we then followed residual intra-Mo Glc activity in patients starting imiglucerase (n=8). By using the only technique able to identify blood leukocyte subsets, we confirmed the marked higher value in Mo (×43) as compared to lymphocytes and polymorphonuclear (PMN) cells. In the 20 type 1 GD patients, we confirmed that enzyme deficiency was clear in Mo (p = 10-11 compared to HD), residual Glc activity in Mo from type 1 GD representing only 10% of normal Glc activity. We then followed trough Glc activity in 8 patients starting imiglucerase treatment de novo (n=1) or restarting imiglucerase after shortage (n=7), at 40 (n=1) or 60 U (n=7) /Kg/2 weeks. We observed progressive uptake over 6 months and a significant cumulative persistence of enzyme activity in monocyte compartment even 14 days after infusion. The estimated increase of residual intra-monocyte enzyme activity at M6 was 1.4 of endogenous level (p=.04) leading to a steady state value. The estimated rate constant of increase corresponded to an initial half-life of 2.8 days. These results explain for the first time the clinical efficacy of the routine infusion regimen, since intra-cellular pharmacokinetics are longer than plasma pharmacokinetics. Furthermore, we can hypothesize that intra-cellular pharmacokinetics after infusion may vary between patients, as suggested by the different peaks of activity and variable residual levels of enzyme activity observed in this study. In this limited series, we observed several profiles: i) Four patients ( #1,2,3, 8) of whom one received 40 U/kg/2 weeks, showed progressive Glc uptake correlated with improvement of clinical and biological parameters; this corresponds to patients with hematological phenotype only (Figure S1 A). ii) Other patients showed variability of Glc uptake (Figure S1 B); these patients were more heterogenous, with bone disease (n=2), splenectomy (n=2), or BM disorders (myelodysplasia, n=1). Consequently intra-Mo imiglucerase uptake appears to be variable, suggesting for the first time a possible relation between Glc activity in cell compartments, phenotype and clinical efficacy. In summary, the persistence of imiglucerase in monocyte compartments explains the efficacy of 2-week infusion rates and variability may be a factor of ERT efficiency Disclosures Stirnemann: Genzyme/Sanofi: Consultancy. Belmatoug:Genzyme/Sanofi and Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees. Rose:Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees. Berger:Genzyme/Sanofi and Shire: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Description: Introduction Chronic Phase - Chronic Myeloid Leukemia (CP-CML) is a myeloproliferative disorder characterized by malignant proliferation of the granulocytic lineage without the arrest of cell differentiation. Tyrosine Kinase Inhibitors (TKI) have revolutionized CML treatment but several studies showed that a combination of TKI and Interferon alpha (IFNα) provides better clinical response. Myeloid Nuclear Differentiation Antigen (MNDA), which belongs to the hematopoietic interferon-inducible nuclear proteins with the 200-amino-acid repeat (HIN200) gene family, encodes a protein expressed in myeloid cells but whose function remains poorly understood. Because of its high expression in polymorphonuclear cells, its involvement in cell differentiation and apoptosis, and its induction by IFNα, we evaluated MNDA expression in CML cells and its modulation after incubation with IFNα. Material and methods We tested MNDA expression in several cell lines (K562, KCL22, LAMA84, TF1 and U937 (positive control)), in polymorphonuclear cells from healthy donors (HD-PMN, n=13) and in primary cells from patients with CP-CML at diagnosis (CP-CML; n=17). The relative expression of the MNDA transcript was analyzed using the 2-ΔΔCt method and was normalized to the endogenous reference gene GAPDH. HD-PMN were used as calibrator. We developed a multiparametric flow cytometry assay (CD45-V500/CD14-APC-H7/CD15-PerCpCy5.5/CD34-PC7/CD38-V450/MNDA-FITC) to detect MNDA protein in the different cell subsets, particularly in CD34+cells. Results As previously described, MNDA was poorly expressed in the K562 cell line. Similarly, mRNA was detected at low levels in two other CML cell lines (KCL22, LAMA84) and in TF1 cells, but at a high level in the U937 cell line, used as a positive control. In each cell line, the transcript expression was correlated to the protein level, as evaluated by flow cytometry (MFI ratio: 2.04±0.21, 2.36±0.24, 1.59±0.14, 1.88±0.11 and 8.77±0.54 for K562, KCL22, LAMA84, TF1 and U937, respectively (n=3)). In CP-CML primary cells, MNDA expression was greatly diminished as compared with HD-PMN in both mRNA (0.20±0.08 (n=17) vs. 1.32±0.21 (n=10); p=1.52x10-6) and protein (MFI ratio: 6.9±0.98 vs. 16.31±1.25, p=0.001). After having verified that IFNα (2000 U/ml, 16 hours) induced MNDA expression in HD mononuclear cells but not in PMN, we observed that induction of MNDA was moderate in CML cell lines K562 and LAMA84 (2-fold increase, n=3) whereas the level of MNDA mRNA was significantly increased in TF1 cells (28-fold increase, n=4). Induction in primary CML cells was variable (3/5 patients). Aiming to evaluate the expression of MNDA in leukemic stem cells (LSC), we first analyzed MNDA expression in CD34+ and CD34+/CD38- cells from HD. We observed that MNDA is down-regulated in healthy CD34+ and CD34+/CD38- cells compared to mature cells (mRNA: about 4 logs, protein: 8-10 fold lower, n=4), but we always detected a significant signal in CD34+cells (MFI ratio: 2.76±0.46, n=3). However, MNDA was not expressed by CML cells from the LSC compartment (n=4). This inhibition does not seem to be antagonized by nilotinib or IFNα (n=2). Discussion/Conclusion MNDA expression appears to be clearly down-regulated in CP-CML cells and dramatically so in the LSC compartment. In some patients, we observed sustained sensitivity to IFNα, but only in the compartment of more mature cells. This suggests early deregulation of MNDA expression which seems to be only partially dependant on differentiation. The mechanisms involved in this down-regulation remain to be elucidated but could be independent to TK activity of BCR-ABL protein and resistant to IFNα in the LSC compartment. This marked deregulation of MNDA in the LSC compartment is an additional argument in favor of intrinsic changes specific to primitive cells. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4425 Introduction: Despite the major benefit of TKI in the treatment of Chronic Myeloid Leukemia (CML) chronic phase, some of patients are resistant or progress to blast phase (BP) becoming not very accessible to therapy. We have been interested in the Syk molecule as a potential marker for CML progression for several reasons: i) its potential interaction with Src kinases, activated by BCR-ABL, and tyrosine kinase receptors, ii) its involvement in the molecular complexes activating actin and the cytoskeleton and integrin signalling pathways, regulating cell adhesion, a property that is impaired in CML, iii) its interaction with the PI3K/Akt pathway, activated by BCR-ABL. Furthermore, resistance to nilotinib was recently showed dependent on Syk expression. Method: The amount of Syk transcript was analyzed in primary cells using the 2-ΔΔ Ct method and was normalized to the endogenous reference gene (β2-microglobuline) and K562 cells as the calibrator. Using flow cytometry, we evaluated the expression of Syk and pSyk348 in K562 cells and in polymorphonuclear cells from 3 healthy donors (HD-PMN), primitive CML cells from 15 patients in chronic phase (CP) (patients #1 to #15) at diagnosis, in the blast cells from 4 patients in accelerated phase (AP) (patient#16, #17, #18 and #19) and from 2 patients in blast crisis (BC) (patient#20, #21). The level of intracellular dasatinib (DAS) was evaluated by an original flow cytometry method (Bourgne et al. Cytometry Part A, in press). Results: We observed a significant over expression of Syk mRNA in BP-CML cells, whereas there is no difference between HD-PMN and CP-CML cells. At the protein level we detected a decrease (2 times; p

Description: Abstract 2747 Introduction Despite the major benefit of TKI in the treatment of Chronic Myeloid Leukemia (CML), patient response is heterogeneous and it is generally accepted that residual disease and relapse are due to persistent CML cells, considered as leukemic stem cells. Their resistance has been related to lower TKI uptake. The amount of drug penetrating the targeted cells is most likely a major parameter of targeted therapy efficacy since it is essential that the therapeutic molecule be as close as possible to the target molecule. We developed a flow cytometry technique to analyze primary cells. Method To evaluate intracellular imatinib (ICIM) uptake, we developed a patented method based on natural UV fluorescence related to chemical structure. Consequently, since the difference in UV fluorescence units between treated and control cells is proportional to the amount of intra-cellular drugs, we validated this method after incubating K562 and KCL22 cell lines with TKI. The flow cytometry technique was standardized by using Flow-Check Fluorosphere calibrated beads immediately before, and at the end of, each series of analyses with a Coulter Epics Elite™ flow cytometer (Beckman Coulter) equipped with an Innova I90C-4 UV laser (Coherent). Then we analyzed primary blood cells from CML patients in chronic phase before any treatment. After lysis of erythrocytes, nucleated cells were incubated at 1.106 cells/ml with different doses of imatinib (IMA) (n=22), Nilotinib (NIL) (n=20) and Dasatinib (DAS) (n=20) at different times. Whenever possible, CML stem cells were analyzed using CD34-FITC staining. Results In preliminary assays, we checked that there was a significant correlation between additional fluorescence measured by flow cytometry and the amount quantified by physico-chemical analysis after lysing a known number of cells (n=57, r2=0.73, p