Journal articles on the topic 'Granulocyte-macrophage colony-stimulating factor; imatinib; chronic myeloid leukemia gene therapy'
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1
Maifrede, Silvia, Dan Liebermann, and Barbara Hoffman. "Stress Response Transcription Factor Egr-1 As Tumor Suppressor Of CML." Blood 122, no. 21 (November 15, 2013): 4905. http://dx.doi.org/10.1182/blood.v122.21.4905.4905.
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The transcription factor early growth response 1 (Egr-1) gene was identified as a macrophage differentiation primary response gene, shown to be essential for and to restrict differentiation along the macrophage lineage. There’s evidence consistent with Egr-1 behaving as a tumor suppressor of leukemia, both in vivo and in vitro, including (1) loss of Egr-1 associated with treatment derived AMLs; (2) deregulated Egr-1 overriding blocks in myeloid differentiation, and (3) haplo-insufficiency of Egr-1 in mice leading to increased development of myeloid disorders following treatment with the potent DNA alkylating agent, N- ethyl-nitrosourea (ENU). BCR-ABL driven leukemia (Chronic Myelogenous Leukemia [CML]) was chosen as a model system to investigate the role of Egr-1 as a tumor suppressor for different leukemias. CML is a disease resulting from the neoplastic transformation of hematopoietic stem cells (HSC) with the BCR-ABL oncogene. The BCR-ABL protein is a constitutively active tyrosine kinase, which promotes cell survival and proliferation by means of diverse intracellular signaling pathways, thereby being the culprit for malignant transformation. Although the Tyrosine Kinase Inhibitor (TKI) imatinib mesylate (Gleevec, Novartis) is effectively used on CML patients, resistance to imatinib has been described. Thus there is a high priority to enhance our understanding of how BCR/ABL subverts normal hematopoiesis and to identify novel targets for therapy. It was observed that Egr-1 expression is reduced in bone marrow (BM) of CML patients, and its expression is further reduced in more advanced stages of CML. Consistent with this data, Egr-1 expression is reduced in BCR-ABL-expressing murine BM. The tumor suppressor role of Egr-1 in CML was validated using mouse models. Lethally irradiated syngeneic wild type mice were reconstituted with bone marrow (BM) from either wild type or Egr-1 null mice transduced with a 210-kD BCR-ABL-expressing MSCV-retrovirus (bone marrow transplantation {BMT}). Loss of Egr-1 accelerated the development of BCR-ABL driven leukemia in recipient mice. Furthermore, no statistically significant difference in the percentage of stem cells (Lin-Sca+c-Kit+, LSK) was observed between Egr-1 WT and Egr-1-/- BM. Thus, the BM stem cell compartment of the Egr-1-/- mice does not offer a quantitative advantage to justify the faster development of leukemia compared to Egr-1 WT mice. An increased population of lineage negative BM cells was observed in Egr-1-/- BCR-ABL recipients when compared to animals transplanted with WT BCR-ABL BM, consistent with more rapid development of disease. Preliminary results from serial BMT has shown that Egr-1-/- BCR-ABL BM has an increased leukemic burden when compared to the WT counterpart. Data from serial colony transfer and studies using spleens from diseased mice as well as BCR-ABL-expressing BM will be presented. These data could result in novel targets for diagnosis, prognosis, and targeted therapeutics, including strategies for activating Egr-1 expression, that can be used to treat CML, as well as other leukemic diseases. Disclosures: No relevant conflicts of interest to declare.
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Kim, Theo D., Michaela Schwarz, Karl-Anton Kreuzer, Jaspal Kaeda, Kamran Movassaghi, Peggy Grille, Peter Daniel, Bernd Dörken, and Philipp le Coutre. "Long-Term Follow-up of Philadelphia Chromosome-Positive Chronic Myeloid Leukemia Patients After Stem Cell Mobilisation Under Imatinib." Blood 116, no. 21 (November 19, 2010): 3418. http://dx.doi.org/10.1182/blood.v116.21.3418.3418.
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Abstract Abstract 3418 Introduction: Mobilisation and collection of in vivo-purged CD34+ stem cells for chronic myeloid leukemia (CML) patients while in complete cytogenetic remission (CCyR) may offer a therapeutic option for those who develop resistance to the tyrosine kinase inhibitor imatinib mesylate (IM), the recommended first line therapy. Indeed, several groups have reported on retrospective studies showing autologous stem cell mobilisation to be safe and efficacious during IM therapy. However, assessing the clinical utility of managing patients using this approach is difficult as long-term follow-up is lacking. Here, we report the clinical outcome for 22 CML patients with a median of 99.5 months follow-up post G-CSF induced stem cell mobilisation. We assessed the efficacy and the the impact on BCR-ABL1 expression levels in these individuals. Material and Methods: Stem cell mobilisation was achieved by administering 10 mg/kg body weight (bw)/day filgrastim (granulocyte-colony stimulating factor, G-CSF) subcutaneously. Stem cell apheresis was performed if the absolute number of circulating CD34+ cells exceeded 5/ml. Target stem cell yield was 32×106 CD34+ cells/kg body weight. BCR-ABL1 transcripts were quantified by real time PCR (Q-PCR) of reverse transcribed total RNA using LightCycler® with β-actin as endogenous control gene. Results: For the twenty-two patients included in the present study, median age was 47 years (range, 24 – 71) and median disease duration was 20 months (range, 1 – 77). At the start of IM, 19 patients were in chronic phase (CP), and 3 in accelerated phase (AP). With a median duration on IM of 16 months (range, 2 – 29), time to first CCyR was 6 months (range, 0 – 25) and time from CCyR to apheresis was 8.5 months (range, 1 – 20). Mobilisation was performed once in 16 patients and twice in 6 patients, of whom eighteen patients (82 %) proceeded to apheresis. Overall, mobilisation was successful in 17 of 22 patients (78 %) with a median CD34+ cell number of 3.1×106/kg body weight (range, 0,7 - 6). BCR-ABL1 transcripts were undetectable in 12 of 22 peripheral blood samples (55 %) at the time of apheresis and 7 of 14 stem cell harvests (50 %) without discernible correlation with matched peripheral blood samples. Progression-free survival at 5 and 8 years was determined to be 63 %, and overall survival at 5 years and 8 years was 95 % and 90 %, respectively. Sixteen of 22 patients (73 %) continue to be treated with IM and twelve of 22 patients (55 %) are without detectable BCR-ABL1 transcripts, i.e. in complete molecular remission (CMR). Of the six patients that stopped IM (3 resistant and 3 intolerant), all received nilotinib as second-line TKI. Five of these six patients are still alive (1 in CHR; 1 in MCyR; 1 in MMR; and 2 in CMR), and one patient who subsequently received dasatinib died after progressing to blast crisis. Conclusion: These observations show G-CSF induced stem cell mobilisation and collection during IM therapy for autologous transplantation to be safe and efficient with a favourable long-term outcome. Disclosures: No relevant conflicts of interest to declare.
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Kassem, Neemat M., Alya M. Ayad, Noha M. El Husseiny, Doaa M. El-Demerdash, Hebatallah A. Kassem, and Mervat M. Mattar. "Role of Granulocyte-Macrophage Colony-Stimulating Factor in Acute Myeloid Leukemia/Myelodysplastic Syndromes." Journal of Global Oncology, no. 4 (December 2018): 1–6. http://dx.doi.org/10.1200/jgo.2017.009332.
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Purpose Granulocyte-macrophage colony-stimulating factor (GM-CSF) cytokine stimulates growth, differentiation, and function of myeloid progenitors. We aimed to study the role of GM-CSF gene expression, its protein, and antibodies in patients with acute myeloid leukemia/myelodysplastic syndromes (AML/MDS) and their correlation to disease behavior and treatment outcome. The study included 50 Egyptian patients with AML/MDS in addition to 20 healthy volunteers as control subjects. Patients and Methods Assessment of GM-CSF gene expression was performed by quantitative real-time polymerase chain reaction. GM-CSF proteins and antibodies were assessed by enzyme-linked immunosorbent assay. Results There was significant decrease in GM-CSF gene expression ( P = .008), increase in serum level of GM-CSF protein ( P = .0001), and increase in anti–GM-CSF antibodies ( P = .001) in patients with AML/MDS compared with healthy control subjects. In addition, there was a significant negative correlation between serum levels of GM-CSF protein and initial peripheral blood blasts, percentage as well as response to therapy. Conclusion Any alteration in GM-CSF gene expression could have implications in leukemogenesis. In addition, GM-CSF protein serum levels could be used to predict outcome of therapy. GM-CSF antibodies may also play a role in the pathogenesis of AML/MDS. The use of these GM-CSF parameters for disease monitoring and as markers of disease activity needs further research.
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Dewar, Andrea L., Antony C. Cambareri, Andrew C. W. Zannettino, Bernadette L. Miller, Kathleen V. Doherty, Timothy P. Hughes, and A. Bruce Lyons. "Macrophage colony-stimulating factor receptor c-fms is a novel target of imatinib." Blood 105, no. 8 (April 15, 2005): 3127–32. http://dx.doi.org/10.1182/blood-2004-10-3967.
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AbstractImatinib is a tyrosine kinase inhibitor that suppresses the growth of bcr-abl–expressing chronic myeloid leukemia (CML) progenitor cells by blockade of the adenosine triphosphate (ATP)–binding site of the kinase domain of bcr-abl. Imatinib also inhibits the c-abl, platelet-derived growth factor (PDGF) receptor, abl-related gene (ARG) and stem-cell factor (SCF) receptor tyrosine kinases, and has been used clinically to inhibit the growth of malignant cells in patients with CML and gastrointestinal stromal tumors (GISTs). Although initially considered to have minimal effects of normal hematopoiesis, recent studies show that imatinib also inhibits the growth of some nonmalignant hematopoietic cells, including monocyte/macrophages. This inhibition could not be attributed to the known activity profile of imatinib. Here, we demonstrate for the first time that imatinib targets the macrophage colony-stimulating factor (M-CSF) receptor c-fms. Phosphorylation of c-fms was inhibited by therapeutic concentrations of imatinib, and this was not due to down-regulation in c-fms expression. Imatinib was also found to inhibit M-CSF–induced proliferation of a cytokine–dependent cell line, further supporting the hypothesis that imatinib affects the growth and development of monocyte and/or macrophages through inhibition of c-fms signaling. Importantly, these results identify an additional biologic target to those already defined for imatinib. Imatinib should now be assessed for activity in diseases where c-fms activation is implicated, including breast and ovarian cancer and inflammatory conditions.
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Borrello, Ivan M., Hyam I. Levitsky, Wendy Stock, Dorie Sher, Lu Qin, Daniel J. DeAngelo, Edwin P. Alyea, et al. "Granulocyte-macrophage colony-stimulating factor (GM-CSF)–secreting cellular immunotherapy in combination with autologous stem cell transplantation (ASCT) as postremission therapy for acute myeloid leukemia (AML)." Blood 114, no. 9 (August 27, 2009): 1736–45. http://dx.doi.org/10.1182/blood-2009-02-205278.
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AbstractPreclinical models have demonstrated the efficacy of granulocyte-macrophage colony-stimulating factor-secreting cancer immunotherapies (GVAX platform) accompanied by immunotherapy-primed lymphocytes after autologous stem cell transplantation in hematologic malignancies. We conducted a phase 2 study of this combination in adult patients with acute myeloid leukemia. Immunotherapy consisted of autologous leukemia cells admixed with granulocyte-macrophage colony-stimulating factor-secreting K562 cells. “Primed” lymphocytes were collected after a single pretransplantation dose of immunotherapy and reinfused with the stem cell graft. Fifty-four subjects were enrolled; 46 (85%) achieved a complete remission, and 28 (52%) received the pretransplantation immunotherapy. For all patients who achieved complete remission, the 3-year relapse-free survival (RFS) rate was 47.4% and overall survival was 57.4%. For the 28 immunotherapy-treated patients, the RFS and overall survival rates were 61.8% and 73.4%, respectively. Posttreatment induction of delayed-type hypersensitivity reactions to autologous leukemia cells was associated with longer 3-year RFS rate (100% vs 48%). Minimal residual disease was monitored by quantitative analysis of Wilms tumor-1 (WT1), a leukemia-associated gene. A decrease in WT1 transcripts in blood was noted in 69% of patients after the first immunotherapy dose and was also associated with longer 3-year RFS (61% vs 0%). In conclusion, immunotherapy in combination with primed lymphocytes and autologous stem cell transplantation shows encouraging signals of potential activity in acute myeloid leukemia (ClinicalTrials.gov: NCT00116467).
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Schuster, Christine, Karin Forster, Henning Dierks, Annika Elsässer, Gerhard Behre, Nicola Simon, Susanne Danhauser-Riedl, Michael Hallek, and Markus Warmuth. "The effects of Bcr-Abl on C/EBP transcription-factor regulation and neutrophilic differentiation are reversed by the Abl kinase inhibitor imatinib mesylate." Blood 101, no. 2 (January 15, 2003): 655–63. http://dx.doi.org/10.1182/blood-2002-01-0043.
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The clinical progression of chronic myeloid leukemia (CML) from chronic phase to blast crisis is characterized by the increasing failure of myeloid precursors to differentiate into mature granulocytes. This study was undertaken to investigate the influence of Bcr-Abl and of the small molecule Abl tyrosine–kinase inhibitor imatinib mesylate on granulocyte colony-stimulating factor (G-CSF)–induced neutrophilic differentiation. We show that differentiation of 32Dcl3 cells into mature granulocytes is accompanied by the increased expression of the antigens macrophage adhesion molecule–1 (Mac-1) and Gr-1, of the G-CSF receptor (G-CSFR), of myeloid transcription factors (CCAAT/enhancer-binding protein–α [C/EBPα], C/EBPε, and PU.1), and of the cyclin-dependent kinase inhibitor p27Kip1. In 32Dcl3 cells transfected with thebcr-abl gene (32DBcr-Abl), G-CSF did not trigger either granulocytic differentiation or the up-regulation of C/EBPα, C/EBPε, and the G-CSFR. This could be correlated to a defect in c-Myc down-regulation. In contrast, the up-regulation of PU.1 and p27Kip1 by G-CSF was not affected by Bcr-Abl. Importantly, incubation of 32DBcr-Ablwtcells with the kinase inhibitor imatinib mesylate prior to G-CSF stimulation completely neutralized the effects of Bcr-Abl on granulocytic differentiation and on C/EBPα and C/EBPε expression. Taken together, the results suggest that the Bcr-Abl kinase induces a reversible block of the granulocytic differentiation program in myeloid cells by disturbing regulation of hematopoietic transcription factors such as C/EBPα and C/EBPε.
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Lewis, Ian D., Louise A. McDiarmid, Leanne M. Samels, L. Bik To, and Timothy P. Hughes. "Establishment of a Reproducible Model of Chronic-Phase Chronic Myeloid Leukemia in NOD/SCID Mice Using Blood-Derived Mononuclear or CD34+ Cells." Blood 91, no. 2 (January 15, 1998): 630–40. http://dx.doi.org/10.1182/blood.v91.2.630.
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Abstract An animal model of chronic myeloid leukemia (CML) will help characterize leukemic and normal stem cells and also help evaluate experimental therapies in this disease. We have established a model of CML in the NOD/SCID mouse. Infusion of ≥4 × 107chronic-phase CML peripheral blood cells results in engraftment levels of ≥1% in the bone marrow (BM) of 84% of mice. Engraftment of the spleen was seen in 60% of mice with BM engraftment. Intraperitoneal injection of recombinant stem cell factor produced a higher level of leukemic engraftment without increasing Philadelphia-negative engraftment. Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor did not increase the level of leukemic or residual normal engraftment. Assessment of differential engraftment of normal and leukemic cells by fluorescence in situ hybridization analysis with bcr and abl probes showed that a median of 35% (range, 5% to 91%) of engrafted cells present in the murine BM were leukemic. BM engraftment was multilineage with myeloid, B-cell, and T-cell engraftment, whereas T cells were the predominant cell type in the spleen. BM morphology showed evidence of eosinophilia and increased megakaryocytes. We also assessed the ability of selected CD34+ CML blood cells to engraft NOD/SCID mice and showed engraftment with cell doses of 7 to 10 × 106 cells. CD34− cells failed to engraft at cell doses of 1.2 to 5 × 107. CD34+ cells produced myeloid and B-cell engraftment with high levels of CD34+ cells detected. Thus, normal and leukemic stem cells are present in CD34+ blood cells from CML patients at diagnosis and lead to development of the typical features of CML in murine BM. This model is suitable to evaluate therapy in CML.
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Lewis, Ian D., Louise A. McDiarmid, Leanne M. Samels, L. Bik To, and Timothy P. Hughes. "Establishment of a Reproducible Model of Chronic-Phase Chronic Myeloid Leukemia in NOD/SCID Mice Using Blood-Derived Mononuclear or CD34+ Cells." Blood 91, no. 2 (January 15, 1998): 630–40. http://dx.doi.org/10.1182/blood.v91.2.630.630_630_640.
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An animal model of chronic myeloid leukemia (CML) will help characterize leukemic and normal stem cells and also help evaluate experimental therapies in this disease. We have established a model of CML in the NOD/SCID mouse. Infusion of ≥4 × 107chronic-phase CML peripheral blood cells results in engraftment levels of ≥1% in the bone marrow (BM) of 84% of mice. Engraftment of the spleen was seen in 60% of mice with BM engraftment. Intraperitoneal injection of recombinant stem cell factor produced a higher level of leukemic engraftment without increasing Philadelphia-negative engraftment. Granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor did not increase the level of leukemic or residual normal engraftment. Assessment of differential engraftment of normal and leukemic cells by fluorescence in situ hybridization analysis with bcr and abl probes showed that a median of 35% (range, 5% to 91%) of engrafted cells present in the murine BM were leukemic. BM engraftment was multilineage with myeloid, B-cell, and T-cell engraftment, whereas T cells were the predominant cell type in the spleen. BM morphology showed evidence of eosinophilia and increased megakaryocytes. We also assessed the ability of selected CD34+ CML blood cells to engraft NOD/SCID mice and showed engraftment with cell doses of 7 to 10 × 106 cells. CD34− cells failed to engraft at cell doses of 1.2 to 5 × 107. CD34+ cells produced myeloid and B-cell engraftment with high levels of CD34+ cells detected. Thus, normal and leukemic stem cells are present in CD34+ blood cells from CML patients at diagnosis and lead to development of the typical features of CML in murine BM. This model is suitable to evaluate therapy in CML.
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Li, Shaoguang, Silke Gillessen, Michael H. Tomasson, Glenn Dranoff, D. Gary Gilliland, and Richard A. Van Etten. "Interleukin 3 and granulocyte-macrophage colony-stimulating factor are not required for induction of chronic myeloid leukemia-like myeloproliferative disease in mice by BCR/ABL." Blood 97, no. 5 (March 1, 2001): 1442–50. http://dx.doi.org/10.1182/blood.v97.5.1442.
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Primitive hematopoietic progenitors from some patients with Philadelphia chromosome (Ph)–positive chronic myeloid leukemia (CML) express aberrant transcripts for interleukin 3 (IL-3) and granulocyte colony-stimulating factor (G-CSF), and exhibit autonomous proliferation in serum-free cultures that is inhibited by anti–IL-3 and anti–IL-3 receptor antibodies. Expression of the product of the Ph chromosome, the BCR/ABL oncogene, in mice by retroviral bone marrow transduction and transplantation induces CML-like leukemia, and some leukemic mice have increased circulating IL-3, and perhaps granulocyte-macrophage colony-stimulating factor (GM-CSF). These observations raise the possibility of autocrine or paracrine cytokine production in the pathogenesis of human CML. Mice with homozygous inactivation of the Il-3 gene, the Gm-csf gene, or both, were used to test the requirement for these cytokines for induction of CML-like disease by BCR/ABL. Neither IL-3 nor GM-CSF was required in donor, recipient, or both for induction of CML-like leukemia by p210 BCR/ABL. Use of novel mice deficient in both IL-3 and GM-CSF demonstrated that the lack of effect on leukemogenesis was not due to redundancy between these hematopoietic growth factors. Analysis of cytokine levels in leukemic mice where either donor or recipient was Il-3−/−indicated that the increased IL-3 originated from the recipient, suggestive of a host reaction to the disease. These results demonstrate that IL-3 and GM-CSF are not required for BCR/ABL-induced CML-like leukemia in mice and suggest that autocrine production of IL-3 does not play a role in established chronic phase CML in humans.
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Shaknovich, Rita, Patricia L. Yeyati, Sarah Ivins, Ari Melnick, Cheryl Lempert, Samuel Waxman, Arthur Zelent, and Jonathan D. Licht. "The Promyelocytic Leukemia Zinc Finger Protein Affects Myeloid Cell Growth, Differentiation, and Apoptosis." Molecular and Cellular Biology 18, no. 9 (September 1, 1998): 5533–45. http://dx.doi.org/10.1128/mcb.18.9.5533.
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ABSTRACT The promyelocytic leukemia zinc finger (PLZF) gene, which is disrupted in therapy-resistant, t(11;17)(q23;q21)-associated acute promyelocytic leukemia (APL), is expressed in immature hematopoietic cells and is down-regulated during differentiation. To determine the role of PLZF in myeloid development, we engineered expression of PLZF in murine 32Dcl3 cells. Expression of PLZF had a dramatic growth-suppressive effect accompanied by accumulation of cells in the G0/G1 compartment of the cell cycle and an increased incidence of apoptosis. PLZF-expressing pools also secreted a growth-inhibitory factor, which could explain the severe growth suppression of PLZF-expressing pools that occurred despite the fact that only half of the cells expressed high levels of PLZF. PLZF overexpression inhibited myeloid differentiation of 32Dcl3 cells in response to granulocyte and granulocyte-macrophage colony-stimulating factors. Furthermore, cells that expressed PLZF appeared immature as demonstrated by morphology, increased expression of Sca-1, and decreased expression of Gr-1. These findings suggest that PLZF is an important regulator of cell growth, death, and differentiation. Disruption of PLZF function associated with t(11;17) may be a critical event leading to APL.
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Zhang, You-Yan, Terry A. Vik, John W. Ryder, Edward F. Srour, Tyler Jacks, Kevin Shannon, and D. Wade Clapp. "Nf1 Regulates Hematopoietic Progenitor Cell Growth and Ras Signaling in Response to Multiple Cytokines." Journal of Experimental Medicine 187, no. 11 (June 1, 1998): 1893–902. http://dx.doi.org/10.1084/jem.187.11.1893.
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Neurofibromin, the protein encoded by the NF1 tumor-suppressor gene, negatively regulates the output of p21ras (Ras) proteins by accelerating the hydrolysis of active Ras-guanosine triphosphate to inactive Ras-guanosine diphosphate. Children with neurofibromatosis type 1 (NF1) are predisposed to juvenile chronic myelogenous leukemia (JCML) and other malignant myeloid disorders, and heterozygous Nf1 knockout mice spontaneously develop a myeloid disorder that resembles JCML. Both human and murine leukemias show loss of the normal allele. JCML cells and Nf1−/− hematopoietic cells isolated from fetal livers selectively form abnormally high numbers of colonies derived from granulocyte-macrophage progenitors in cultures supplemented with low concentrations of granulocyte-macrophage colony stimulating factor (GM-CSF). Taken together, these data suggest that neurofibromin is required to downregulate Ras activation in myeloid cells exposed to GM-CSF. We have investigated the growth and proliferation of purified populations of hematopoietic progenitor cells isolated from Nf1 knockout mice in response to the cytokines interleukin (IL)-3 and stem cell factor (SCF), as well as to GM-CSF. We found abnormal proliferation of both immature and lineage-restricted progenitor populations, and we observed increased synergy between SCF and either IL-3 or GM-CSF in Nf1−/− progenitors. Nf1−/− fetal livers also showed an absolute increase in the numbers of immature progenitors. We further demonstrate constitutive activation of the Ras-Raf-MAP (mitogen-activated protein) kinase signaling pathway in primary c-kit+ Nf1−/− progenitors and hyperactivation of MAP kinase after growth factor stimulation. The results of these experiments in primary hematopoietic cells implicate Nf1 as playing a central role in regulating the proliferation and survival of primitive and lineage-restricted myeloid progenitors in response to multiple cytokines by modulating Ras output.
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Tamura, Tomohiko, Hee Jeong Kong, Chainarong Tunyaplin, Hideki Tsujimura, Kathryn Calame, and Keiko Ozato. "ICSBP/IRF-8 inhibits mitogenic activity of p210 Bcr/Abl in differentiating myeloid progenitor cells." Blood 102, no. 13 (December 15, 2003): 4547–54. http://dx.doi.org/10.1182/blood-2003-01-0291.
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AbstractInterferon consensus sequence binding protein/interferon regulatory factor 8 (ICSBP/IRF-8) is a transcription factor that controls myeloid cell development. ICSBP-/- mice develop a chronic myelogenous leukemia (CML)-like syndrome. Several observations on patients and mouse models have implicated ICSBP in the pathogenesis of CML. In this paper, we investigated whether ICSBP modulates the growth-promoting activity of Bcr/Abl, the causal oncoprotein for CML. When transformed with p210 Bcr/Abl, ICSBP-/- myeloid progenitor cells lost growth factor dependence and grew in the absence of granulocyte-macrophage colony-stimulating factor. When ICSBP was ectopically expressed, Bcr/Abl-transformed cells underwent complete growth arrest and differentiated into mature, functional macrophages without inhibiting the kinase activity of Bcr/Abl. Providing a mechanistic basis for the growth arrest, ICSBP markedly repressed c-Myc messenger RNA (mRNA)-expression, a downstream target of Bcr/Abl. A further analysis with the ICSBP/estrogen receptor chimera showed that ICSBP repression of c-Myc is indirect and is mediated by another gene(s). We identified Blimp-1 and METS/PE1, potent c-Myc repressors, as direct targets of ICSBP activated in these cells. Consistent with this, ectopic Blimp-1 repressed c-Myc expression and inhibited cell growth. These results indicate that ICSBP inhibits growth of Bcr/Abl-transformed myeloid progenitor cells by activating several genes that interfere with the c-Myc pathway. (Blood. 2003;102:4547-4554)
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Stripecke, Renata, Angelo A. Cardoso, Karen A. Pepper, Dianne C. Skelton, Xiao-Jin Yu, Leo Mascarenhas, Kenneth I. Weinberg, Lee M. Nadler, and Donald B. Kohn. "Lentiviral vectors for efficient delivery of CD80 and granulocyte-macrophage– colony-stimulating factor in human acute lymphoblastic leukemia and acute myeloid leukemia cells to induce antileukemic immune responses." Blood 96, no. 4 (August 15, 2000): 1317–26. http://dx.doi.org/10.1182/blood.v96.4.1317.
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Abstract Cell vaccines engineered to express immunomodulators have shown feasibility in eliminating leukemia in murine models. Vectors for efficient gene delivery to primary human leukemia cells are required to translate this approach to clinical trials. In this study, second-generation lentiviral vectors derived from human immunodeficiency virus 1 were evaluated, with the cytomegalovirus (CMV) promoter driving expression of granulocyte-macrophage–colony-stimulating factor (GM-CSF) and CD80 in separate vectors or in a bicistronic vector. The vectors were pseudotyped with vesicular stomatitis virus G glycoprotein and concentrated to high titers (108-109 infective particles/mL). Human acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic myeloid leukemia cell lines transduced with the monocistronic pHR-CD80 vector or the bicistronic pHR-GM/CD vector became 75% to 95% CD80 positive (CD80+). More important, transduction of primary human ALL and AML blasts with high-titer lentiviral vectors was consistently successful (40%-95% CD80+). The average amount of GM-CSF secretion by the leukemia cell lines transduced with the pHR-GM-CSF monocistronic vector was 2182.9 pg/106 cells per 24 hours. Secretion was markedly lower with the bicistronic pHR-GM/CD vector (average, 225.7 pg/106 cells per 24 hours). Lower amounts of CMV-driven messenger RNA were detected with the bicistronic vector, which may account for its poor expression of GM-CSF. Primary ALL cells transduced to express CD80 stimulated T-cell proliferation in an autologous mixed lymphocyte reaction. This stimulation was specifically blocked with monoclonal antibodies reactive against CD80 or by recombinant cytotoxic T-lymphocyte antigen 4–immunoglobulin fusion protein. These results show the feasibility of efficiently transducing primary leukemia cells with lentiviral vectors to express immunomodulators to elicit antileukemic immune responses.
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Stripecke, Renata, Angelo A. Cardoso, Karen A. Pepper, Dianne C. Skelton, Xiao-Jin Yu, Leo Mascarenhas, Kenneth I. Weinberg, Lee M. Nadler, and Donald B. Kohn. "Lentiviral vectors for efficient delivery of CD80 and granulocyte-macrophage– colony-stimulating factor in human acute lymphoblastic leukemia and acute myeloid leukemia cells to induce antileukemic immune responses." Blood 96, no. 4 (August 15, 2000): 1317–26. http://dx.doi.org/10.1182/blood.v96.4.1317.h8001317_1317_1326.
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Cell vaccines engineered to express immunomodulators have shown feasibility in eliminating leukemia in murine models. Vectors for efficient gene delivery to primary human leukemia cells are required to translate this approach to clinical trials. In this study, second-generation lentiviral vectors derived from human immunodeficiency virus 1 were evaluated, with the cytomegalovirus (CMV) promoter driving expression of granulocyte-macrophage–colony-stimulating factor (GM-CSF) and CD80 in separate vectors or in a bicistronic vector. The vectors were pseudotyped with vesicular stomatitis virus G glycoprotein and concentrated to high titers (108-109 infective particles/mL). Human acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and chronic myeloid leukemia cell lines transduced with the monocistronic pHR-CD80 vector or the bicistronic pHR-GM/CD vector became 75% to 95% CD80 positive (CD80+). More important, transduction of primary human ALL and AML blasts with high-titer lentiviral vectors was consistently successful (40%-95% CD80+). The average amount of GM-CSF secretion by the leukemia cell lines transduced with the pHR-GM-CSF monocistronic vector was 2182.9 pg/106 cells per 24 hours. Secretion was markedly lower with the bicistronic pHR-GM/CD vector (average, 225.7 pg/106 cells per 24 hours). Lower amounts of CMV-driven messenger RNA were detected with the bicistronic vector, which may account for its poor expression of GM-CSF. Primary ALL cells transduced to express CD80 stimulated T-cell proliferation in an autologous mixed lymphocyte reaction. This stimulation was specifically blocked with monoclonal antibodies reactive against CD80 or by recombinant cytotoxic T-lymphocyte antigen 4–immunoglobulin fusion protein. These results show the feasibility of efficiently transducing primary leukemia cells with lentiviral vectors to express immunomodulators to elicit antileukemic immune responses.
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Pietsch, T., U. Kyas, U. Steffens, E. Yakisan, MR Hadam, WD Ludwig, K. Zsebo, and K. Welte. "Effects of human stem cell factor (c-kit ligand) on proliferation of myeloid leukemia cells: heterogeneity in response and synergy with other hematopoietic growth factors." Blood 80, no. 5 (September 1, 1992): 1199–206. http://dx.doi.org/10.1182/blood.v80.5.1199.1199.
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Abstract A novel hematopoietic growth factor, the stem cell factor (SCF), for primitive hematopoietic progenitor cells has recently been purified and its gene has been cloned. In this study we tested the mitogenic activity of recombinant human SCF on myeloid leukemia cells as well as the expression of its receptor. We have investigated the proliferation of 31 myeloid leukemia cell lines as well as fresh myeloid leukemic blasts from 17 patients in a 72-hour 3H-thymidine uptake assay in the presence of various concentrations of recombinant human (rh) SCF alone or in combination with saturating concentrations of granulocyte- macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, interleukin-3 (IL-3), or erythropoietin (EPO). Only five of 31 lines, but fresh leukemic blasts from 12 of 17 patients with acute myeloid leukemia (AML), significantly responded to SCF. The responding cell lines were of the acute promyelocytic, chronic myeloid, megakaryoblastic, and erythroleukemia origin, the responding blast preparations of all French-American-British subtypes. Synergistic activities of SCF were found with G-CSF, GM-CSF, EPO, and IL-3. To determine the SCF binding sites on leukemic cells, we used 125I- radiolabeled SCF in Scatchard analysis and cross-linking studies. The leukemic cell lines responding to SCF expressed from 2,300 up to 29,000 binding sites per cell. The SCF receptor expression was downregulated in vitro by the presence of its ligand. Cross-linking studies demonstrated a 150-Kd SCF receptor on the surface of all responding myeloid leukemias. This study suggests that SCF may be an important factor for the growth of myeloid leukemia cells, either as a direct stimulus or as a synergistic factor for other cytokines. Furthermore, using polymerase chain reaction analysis of total RNA from the myeloid leukemia lines, we found expression of SCF-mRNA in 17 of 30 lines, suggesting autocrine mechanisms in the growth of a subgroup of leukemic cells by coexpression of SCF and its receptor.
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Pietsch, T., U. Kyas, U. Steffens, E. Yakisan, MR Hadam, WD Ludwig, K. Zsebo, and K. Welte. "Effects of human stem cell factor (c-kit ligand) on proliferation of myeloid leukemia cells: heterogeneity in response and synergy with other hematopoietic growth factors." Blood 80, no. 5 (September 1, 1992): 1199–206. http://dx.doi.org/10.1182/blood.v80.5.1199.bloodjournal8051199.
Full textAbstract:
A novel hematopoietic growth factor, the stem cell factor (SCF), for primitive hematopoietic progenitor cells has recently been purified and its gene has been cloned. In this study we tested the mitogenic activity of recombinant human SCF on myeloid leukemia cells as well as the expression of its receptor. We have investigated the proliferation of 31 myeloid leukemia cell lines as well as fresh myeloid leukemic blasts from 17 patients in a 72-hour 3H-thymidine uptake assay in the presence of various concentrations of recombinant human (rh) SCF alone or in combination with saturating concentrations of granulocyte- macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, interleukin-3 (IL-3), or erythropoietin (EPO). Only five of 31 lines, but fresh leukemic blasts from 12 of 17 patients with acute myeloid leukemia (AML), significantly responded to SCF. The responding cell lines were of the acute promyelocytic, chronic myeloid, megakaryoblastic, and erythroleukemia origin, the responding blast preparations of all French-American-British subtypes. Synergistic activities of SCF were found with G-CSF, GM-CSF, EPO, and IL-3. To determine the SCF binding sites on leukemic cells, we used 125I- radiolabeled SCF in Scatchard analysis and cross-linking studies. The leukemic cell lines responding to SCF expressed from 2,300 up to 29,000 binding sites per cell. The SCF receptor expression was downregulated in vitro by the presence of its ligand. Cross-linking studies demonstrated a 150-Kd SCF receptor on the surface of all responding myeloid leukemias. This study suggests that SCF may be an important factor for the growth of myeloid leukemia cells, either as a direct stimulus or as a synergistic factor for other cytokines. Furthermore, using polymerase chain reaction analysis of total RNA from the myeloid leukemia lines, we found expression of SCF-mRNA in 17 of 30 lines, suggesting autocrine mechanisms in the growth of a subgroup of leukemic cells by coexpression of SCF and its receptor.
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Wang, Ying, Dali Cai, Cornelia Brendel, Christine Barett, Philipp Erben, Paul W. Manley, Andreas Hochhaus, Andreas Neubauer, and Andreas Burchert. "Adaptive secretion of granulocyte-macrophage colony-stimulating factor (GM-CSF) mediates imatinib and nilotinib resistance in BCR/ABL+ progenitors via JAK-2/STAT-5 pathway activation." Blood 109, no. 5 (November 7, 2006): 2147–55. http://dx.doi.org/10.1182/blood-2006-08-040022.
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Abstract Overcoming imatinib mesylate (IM) resistance and disease persistence in patients with chronic myeloid leukemia (CML) is of considerable importance to the issue of potential cure. Here we asked whether autocrine signaling contributes to survival of BCR/ABL+ cells in the presence of IM and nilotinib (NI; AMN107), a novel, more selective Abl inhibitor. Conditioned media (CM) of IM-resistant LAMA84 cell clones (R-CM) was found to substantially protect IM-naive LAMA cells and primary CML progenitors from IM- or NI-induced cell death. This was due to an increased secretion of the granulocyte-macrophage colony-stimulating factor (GM-CSF), which was identified as the causative factor mediating IM resistance in R-CM. GM-CSF elicited IM and NI drug resistance via a BCR/ABL-independent activation of the janus kinases 2 (JAK-2)/signal transducer and activator of transcription 5 (STAT-5) signaling pathway in GM-CSF receptor α receptor (CD116)–expressing cells, including primary CD34+/CD116+ GM progenitors (GMPs). Elevated mRNA and protein levels of GM-CSF were detected in IM-resistant patient samples, suggesting a contribution of GM-CSF secretion for IM and NI resistance in vivo. Importantly, inhibition of JAK-2 with AG490 abrogated GM-CSF–mediated STAT-5 phosphorylation and NI resistance in vitro. Together, adaptive autocrine secretion of GM-CSF mediates BCR/ABL-independent IM and NI resistance via activation of the antiapoptotic JAK-2/STAT-5 pathway. Inhibition of JAK-2 overcomes GM-CSF–induced IM and NI progenitor cell resistance, providing a rationale for the application of JAK-2 inhibitors to eradicate residual disease in CML.
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Newburger, Peter E. "Disorders of Neutrophil Number and Function." Hematology 2006, no. 1 (January 1, 2006): 104–10. http://dx.doi.org/10.1182/asheducation-2006.1.104.
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Abstract This review of disorders of neutrophil number and function will discuss important research advances in the field and then provide a clinical diagnostic approach. The focus will be on two recent clinical developments in the field of phagocyte disorders. First, an important natural history study from the Severe Chronic Neutropenia International Registry has recently quantitated the incidence and risk factors for death from sepsis and for progression to myelodysplastic syndrome and acute myeloid leukemia in a large cohort of severe chronic neutropenia patients, many of whom were followed 10 or more years on treatment with granulocyte colony-stimulating factor. Second, in the past year, a multinational group has announced successful gene therapy of two adults with chronic granulomatous disease, the most common disorder of neutrophil function. However, monitoring of retroviral insertion sites revealed expansion of the multiclonal population of gene-modified cells, raising concerns about eventual leukemogenesis. The review also provides a pragmatic approach to the evaluation of a patient with a suspected disorder of neutrophil number or function.
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Fang, Baijun, Ning Li, Yongping Song, Qin Han, and Robert Chunhua Zhao. "Standard-dose imatinib plus low-dose homoharringtonine and granulocyte colony-stimulating factor is an effective induction therapy for patients with chronic myeloid leukemia in myeloid blast crisis who have failed prior single-agent therapy with imatinib." Annals of Hematology 89, no. 11 (May 25, 2010): 1099–105. http://dx.doi.org/10.1007/s00277-010-0991-4.
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Cho, Eunpi, Hyam I. Levitsky, Jeanne Kowalski, Hua-Ling Tsai, Lu Qin, Christopher Gocke, and B. Douglas Smith. "K562/GM-CSF Immunotherapy as a “Booster” Vaccine In Chronic Myeloid Leukemia." Blood 116, no. 21 (November 19, 2010): 4347. http://dx.doi.org/10.1182/blood.v116.21.4347.4347.
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Abstract Abstract 4347 Background: Chronic myeloid leukemia (CML) is a disease that is responsive to T-cell-mediated immunity. K562/granulocyte macrophage-colony stimulating factor (GM-CSF) is a vaccine derived from a CML cell line that produces GM-CSF and expresses several CML-associated antigens. An initial pilot study was performed in 19 patients in chronic phase CML who had achieved at least a major cytogenetic response on imatinib mesylate (IM) but had measurable molecular disease. The results suggested that immunotherapy resulted in a lowering tumor burden in the majority of patients (n = 13) with a total of 7 patients achieving undetectable bcr-abl levels by QT-PCR. (Smith, Clinical Cancer Research, 2010). This extension study evaluated the biologic impact of K562/GM-CSF given as a “booster” in subjects who completed the pilot study's scheduled 4 vaccinations and continued to have have persistent measurable disease or who lost their best response to the original therapy. Methods: This is a single-institution pilot study using open-label K562/GM-CSF vaccination as a “booster” in chronic phase CML patients who were previously treated with a full 4-vaccination course of therapy and continued to have measurable CML disease. Patients were stratified as having either responded to the initial set of vaccines and had subsequent loss of response or as having had no measurable response to therapy. None experienced significant adverse events in the first set of vaccinations. “Booster” treatment consisted of 4 doses of 1 × 108 irradiated K562/GM-CSF given every 3 weeks. Imatinib was continued through the study at the patients’ starting doses. Disease burden was measured using peripheral blood RT-PCR and FISH was every 3 months throughout the course of planned booster vaccinations for up to a year following the vaccination boost. Patients with evidence of advancing disease requiring adjustments to their IM dosing or those progressing to accelerated or blast phase were taken off study. Results: 11 patients met the eligibility to receive the booster vaccinations. Mean age was 54 (range 38–78) years. Median dose of IM therapy was 600 mg daily (range 400–800 mg). The median time from final vaccination in the pilot study to first booster vaccination was 24 (range 18–24) months. Duration of follow up was a median of 36 months (range 6–42 months). In general, the trend for lower disease burden following booster vaccines was significant by Generalize Estimating Equation using all study measures (p<0.009). Seven patients (63%) achieved their lowest levels of disease burden to date at a median of 5 (range 3–39) months following the initiation of their booster vaccinations with only 1 patient losing their “best” response as defined by a 10-fold increase in PCR value. One of the responding patients who had lost her complete molecular remission and became positive for 5 consecutive PCR readings prior to starting the booster immunizations was found to be undetectable after starting the booster treatments and has maintained negative PCR after the completion of the booster series (now at 1 year post booster). Three patients, all of whom qualified for the booster vaccines as “non-responders” to the initial immunizations, went off study secondary to progression of disease. Using sera from patients on the initial pilot study, antibodies against a total of 24 newly identified CML associated antigens were detected in post-vaccination > pre-vaccination samples. Of these, 15 antigens were recognized in 2 or more subjects. The influence of the vaccine boost series on antibody titer and on the induction of antibodies to antigens not previously recognized is under study. K562/GM-CSF immunotherapy given as a “booster” vaccination was associated with the lowering tumor burdens in 7 of 11 treated patients. Ongoing efforts to identify tumor-associated antigens that may be serving as immunologic targets is ongoing. Disclosures: Levitsky: HL vaccine: Patents & Royalties.
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Cignetti, A., E. Bryant, B. Allione, A. Vitale, R. Foa, and M. A. Cheever. "CD34+ Acute Myeloid and Lymphoid Leukemic Blasts Can Be Induced to Differentiate Into Dendritic Cells." Blood 94, no. 6 (September 15, 1999): 2048–55. http://dx.doi.org/10.1182/blood.v94.6.2048.
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Abstract CD34+ hematopoietic stem cells from normal individuals and from patients with chronic myelogenous leukemia can be induced to differentiate into dendritic cells (DC). The aim of the current study was to determine whether acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cells could be induced to differentiate into DC. CD34+ AML-M2 cells with chromosome 7 monosomy were cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), and interleukin-4 (IL-4). After 3 weeks of culture, 35% of the AML-M2 cells showed DC morphology and phenotype. The DC phenotype was defined as upmodulation of the costimulatory molecules CD80 and CD86 and the expression of CD1a or CD83. The leukemic nature of the DC was validated by detection of chromosome 7 monosomy in sorted DC populations by fluorescence in situ hybridization (FISH). CD34+ leukemic cells from 2 B-ALL patients with the Philadelphia chromosome were similarly cultured, but in the presence of CD40-ligand and IL-4. After 4 days of culture, more than 58% of the ALL cells showed DC morphology and phenotype. The leukemic nature of the DC was validated by detection of the bcr-abl fusion gene in sorted DC populations by FISH. In functional studies, the leukemic DC were highly superior to the parental leukemic blasts for inducing allogeneic T-cell responses. Thus, CD34+ AML and ALL cells can be induced to differentiate into leukemic DC with morphologic, phenotypic, and functional similarities to normal DC.
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Cignetti, A., E. Bryant, B. Allione, A. Vitale, R. Foa, and M. A. Cheever. "CD34+ Acute Myeloid and Lymphoid Leukemic Blasts Can Be Induced to Differentiate Into Dendritic Cells." Blood 94, no. 6 (September 15, 1999): 2048–55. http://dx.doi.org/10.1182/blood.v94.6.2048.418k21_2048_2055.
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CD34+ hematopoietic stem cells from normal individuals and from patients with chronic myelogenous leukemia can be induced to differentiate into dendritic cells (DC). The aim of the current study was to determine whether acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) cells could be induced to differentiate into DC. CD34+ AML-M2 cells with chromosome 7 monosomy were cultured in the presence of granulocyte-macrophage colony-stimulating factor (GM-CSF), tumor necrosis factor (TNF), and interleukin-4 (IL-4). After 3 weeks of culture, 35% of the AML-M2 cells showed DC morphology and phenotype. The DC phenotype was defined as upmodulation of the costimulatory molecules CD80 and CD86 and the expression of CD1a or CD83. The leukemic nature of the DC was validated by detection of chromosome 7 monosomy in sorted DC populations by fluorescence in situ hybridization (FISH). CD34+ leukemic cells from 2 B-ALL patients with the Philadelphia chromosome were similarly cultured, but in the presence of CD40-ligand and IL-4. After 4 days of culture, more than 58% of the ALL cells showed DC morphology and phenotype. The leukemic nature of the DC was validated by detection of the bcr-abl fusion gene in sorted DC populations by FISH. In functional studies, the leukemic DC were highly superior to the parental leukemic blasts for inducing allogeneic T-cell responses. Thus, CD34+ AML and ALL cells can be induced to differentiate into leukemic DC with morphologic, phenotypic, and functional similarities to normal DC.
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Okabe, Seiichi, Tetsuzo Tauchi, Seiichiro Katagiri, Yuko Tanaka, and Kazuma Ohyashiki. "Combining ABL1 Kinase Inhibitor, Imatinib and the Jak Kinase Inhibitor TG101348: A Potential Treatment for Residual BCR-ABL Positive Leukemia Cells." Blood 120, no. 21 (November 16, 2012): 3737. http://dx.doi.org/10.1182/blood.v120.21.3737.3737.
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Abstract Abstract 3737 ABL kinase inhibitor, imatinib is highly effective therapy against chronic myeloid leukemia (CML) patients and eliminates disease progression and transformation. However, imatinib is not curative for most CML patients. Residual CML cells are present in bone marrow microenvironment. Bone marrow microenvironment is a source of soluble factors and regulates the proliferation of leukemia cells. These leukemia cells are contained within a niche in the bone marrow and are often impervious to current treatments, thus maintaining their proliferative activity when the treatment is ceased, suggests that the new therapeutic strategies designed to override stroma-associated drug resistance are required to treat against Philadelphia (Ph)-positive leukemia patients. The hematopoietic cytokine receptor signaling is mediated by tyrosine kinases termed Janus kinases (Jaks) and downstream transcription factors, signal transducers and activators of transcription (STATs). Jak-STAT signaling is also activated in CML cells. One of the Jak kinase inhibitor, TG101348 (SAR302503) is an orally available inhibitor of Jak2 and developed for the treatment of patients with myeloproliferative diseases. Therefore, combination therapy using a BCR-ABL tyrosine kinase inhibitors and a Jak inhibitor, TG101348 may help prevent stroma-associated drug resistance and these approaches may be expected to improve the outcomes of CML patients. In this study, we investigated the ABL tyrosine kinase inhibitor, imatinib and TG101348 efficacy by using the BCR-ABL positive cell lines, K562 and primary CML samples when leukemic cells were protected by the feeder cell lines (HS-5 and S9). 72 hours treatment of imatinib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. However, the treatment of imatinib exhibits cell growth inhibition partially against K562 cells in the presence of HS-5 conditioned media. We found that the treatment of TG101348 did not exhibit cell growth inhibition against K562 cells directly, but the combination treatment with imatinib and TG101348 abrogated the protective effects of HS-5 conditioned media in K562 cells. We next investigated the intracellular signaling of imatinib and TG101348. Phosphorylation of BCR-ABL, Crk-L was not reduced after TG101348 treatment. However, phosphorylation of BCR-ABL, Crk-L was significantly reduced and increased apoptosis after combination treatment with imatinib and TG101348. We next investigated the efficacy between imatinib and TG101348 by using CD34 positive primary CML samples. The treatment of imatinib exhibits cell growth inhibition partially against CD34 positive CML samples in the presence of feeder cells. Combined treatment of CD34 positive primary samples with imatinib and TG101348 caused significantly more cytotoxicity and induced apoptosis. We also found that mitogen-activated protein kinase (MAPK) was also inhibited by imatinib and TG101348 treatment. We next investigated the intracellular signaling of imatinib and TG101348 by using the CD34 positive primary samples. Phosphorylation of BCR-ABL, Crk-L was significantly reduced and increased apoptosis after treatment with imatinib and TG101348. Moreover, combination of imatinib and TG101348 inhibited the colony growth of Ph-positive primary samples. We also investigated the TG101348 activity against feeder cell. Phosphorylation of STAT5 was reduced by TG101348 in a dose dependent manner. The cytokine production was analyzed by using cytokine array systems. The cytokine production such as granulocyte macrophage colony-stimulating factor (GM-CSF) from HS-5 was also reduced by TG101348 treatment. Data from this study suggested that administration of the imatinib and Jak inhibitor, TG101348 may be a powerful strategy against stroma-associated drug resistance of Ph-positive cells and enhance cytotoxic effects of imatinib in those residual CML cells. Disclosures: No relevant conflicts of interest to declare.
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Dale, David C., Audrey Anna Bolyard, Merideth L. Kelley, Vahagn Makaryan, Mary Ann Bonilla, Laurence A. Boxer, Sabine Mellor-Heineke, Karl H. Welte, Peter E. Newburger, and Cornelia Zeidler. "Long Term Outcomes for Patients with Cyclic Neutropenia Treated with Granulocyte Colony-Stimulating Factor (G-CSF)." Blood 126, no. 23 (December 3, 2015): 996. http://dx.doi.org/10.1182/blood.v126.23.996.996.
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Cyclic neutropenia (CyN) is a rare hematological condition with neutrophil counts decreasing to <0.2 x 109/L, usually at 21 day intervals, accompanied by fever, mouth ulcers and the risk of severe sepsis during the neutropenic periods. Twenty-six years ago we reported results of treating six patients with CyN on G-CSF (N Engl J Med. 1989;320: 1306-1311). We have now followed these patients and many other CyN on G-CSF for up to 28 years through the Severe Chronic Neutropenia International Registry (SCNIR). The original six patients (2 male, 4 female, ages 8.8 to 65) are all living, now ages 36 to 92 years. All of the original six had documented fever and recurring infections prior to treatment (i.e. mouth ulcers, gingivitis, lymphadenopathy, cellulitis, abscesses, pharyngitis, otitis, and pneumonia). Five were found to have mutations in ELANE after the discovery of mutations in this gene as the cause for CyN. The sixth patient, in retrospect, probably more appropriately should have be given the diagnosis of severe idiopathic neutropenia, is now age 92, off G-CSF and residing at home with skilled nursing care. The other five have maintained good health on G-CSF treatment and are now employed as teachers, working in a business, or retired. None have developed notable infections, hematological or other sequelae except for one patient with decreased bone density and one patient with idiopathic thrombocytopenia purpura (ITP) responding to splenectomy. In aggregate these six patients have received approximately 6.6 grams of G-CSF over 143 patient-years (median dose of G-CSF per patient per year is 0.035 gm, range 0.018 to 0.075 gm, treatment period: 7-28 years). Our broader experience with 308 patients with the diagnosis of CyN based on serial blood counts and/or genotyping and treated with G-CSF is similar to these original six patients. However, we are aware of at least 40 cases of severe sepsis and 17 deaths in patients with CyN not on G-CSF. We are not aware of any such severe infections in CyN patients consistently receiving G-CSF (i.e. at least two or three times per week). We have recorded one case on chronic myeloid leukemia in a CyN patient never on G-CSF. For patients receiving G-CSF we have recorded one case of non-Hodgkin's lymphoma, one case of AML in patient with probable CyN having 2 ELANE mutations and a secondary CSF3R mutation, and one case of AML after chronic immunosuppressive therapy with cyclophosphamide. Conclusion: Observations in 308 patients for 2993 years demonstrate that G-CSF is a very beneficial treatment for patients with CyN. There is good evidence that this treatment prevents severe infections and death from infections and there is a very low risk myeloid malignancies, and specifically conversion to MDS or AML. Disclosures Dale: Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau. Boxer:Amgen: Equity Ownership.
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Qian, SiXuan, Peng Liu, and Jianyong Li. "Effect of Low-Dose Cytarabine and Aclarubicin in Combination with Decitabine and Granulocyte Colony-Stimulating Factor Priming on the Outcome of Patients with Myeloid neoplasms." Blood 118, no. 21 (November 18, 2011): 5047. http://dx.doi.org/10.1182/blood.v118.21.5047.5047.
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Abstract Abstract 5047 Purpose Patients older than 60 years with acute myeloid leukemia (AML) or with chronic myeloid leukemia in blast phase (CML-BC) or myelodysplastic syndrome (MDS) have non-intensive treatment options because of the lack of effectiveness and the toxicity of available therapies. We investigated the efficacy and toxicity of the low-dose cytarabine and aclarubicin in combination with hypomethylating agent decitabine and granulocyte colony-stimulating factor priming as induction therapy in MDS, CML-BC and older patients with AML. Patients and Methods We described 28 consecutive patients (median age 65.5 years, range 49–78 years with MDS (n= 7) or AML (n= 17), or CML-BC (n=4). Among them, Seventeen were cases of AML (10 previously untreated AML, 3 bone marrow relapse, 2 extramedullary relapse and 1 MDS-AML); 4 were cases of CML-BC who were resistant to imatinib or nilotinib therapy and 7 were cases of MDS. Cytogenetic analysis was performed in all the patients, showing 7 patients had cytogenetic aberrations. All patients were treated with decitabine 15 mg/m2 intravenously for 5 consecutive days (d1-5) in combination with cytarabine 10 mg/m2 q12h for 7 days (d3-9) and aclarubicin 10mg every day for 4 days (d3-6) and granulocyte colony-stimulating factor (G-CSF) 300μg every day (d 0–9) priming (D-CAG). G-CSF was withdrawn when the white blood cell count (WBC) >20×109/L. Hydroxyurea or in combination with homoharringtonine 1mg/d intravenously was treated until WBC was <10×109/L before decitabine was given. CML-BC patients continued to be treated with imatinib or nitotinib during the D-CAG. Response was assessed by weekly complete blood count and bone marrow biopsy was taken at the time of recovery of peripheral hemogram or 3–4 weeks later after each course. Patients with MDS and AML continued to receive the second cycle. All of patients underwent baseline and efficacy evaluations. Results Fifteen older patients with AML (median 68 years, range 60–78 years) were enrolled. Among those patients, 10 were in previously untreated AML, 3 bone marrow relapse, 1 extramedullary relapse and 1 secondary AML. The overall response rate (ORR) was 86.7% (13/15) in all older patients, 80.0% (8/10) in patients with previously untreated AML, and the complete rate (CR) was 73.3% after one cycle. 3 older patients with bone marrow relapse achieved. Two patients with extramedullary relapse achieved partial remission (PR) after one cycle. Two fourth of patients with high WBC more than 100×109/L at the time of diagnosis achieved CR. 7 patients with MDS (6 high-risk MDS, 1 RCMD) achieved the therapy. 3/5 patients achieved the CR or 2/5 PR. Other 2 high-risk MDS patients with refractory to convenient regimens such as CAG and HA had non-response to D-CAG regimen. All of the CML-BC patients received the one cycle of D-CAG regimen. Three fourth CML-BC patients achieved the CR and one stopped disease progression. All patients completed the treatment and there was no induction mortality. Grade 4 neutropenia and thrombocytopenia was universal. Median duration of granulocyte recovery (0.5×109/L) was 13 days(4–20), and that of platelet recovery (×109/L) was 10 days(2–16).The platelets rose above 600×109/L in 5 patients and all counts returned to the normal range with a week and there were no thrombotic complications including one patient with atrial fibrillation. The most common toxicities were myelosuppression, febrile neutropenia. Conclusion D-CAG regimen as the induction therapy seemed to be a safe and effective regimen for patients with untreated/relapsed AML, MDS and CML-BC, including older patients, and it has well tolerance. Disclosures: No relevant conflicts of interest to declare.
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Gabriele, Lucia, Paola Borghi, Carmela Rozera, Paola Sestili, Mauro Andreotti, Anna Guarini, Enrico Montefusco, Robert Foà, and Filippo Belardelli. "IFN-α promotes the rapid differentiation of monocytes from patients with chronic myeloid leukemia into activated dendritic cells tuned to undergo full maturation after LPS treatment." Blood 103, no. 3 (February 1, 2004): 980–87. http://dx.doi.org/10.1182/blood-2003-03-0981.
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AbstractChronic myelogenous leukemia (CML) is a malignant myeloproliferative disease arising from the clonal expansion of a stem cell expressing the bcr/abl oncogene. CML patients frequently respond to treatment with interferon-α (IFN-α), even though the mechanisms of the response remain unclear. In the present study, we evaluated the role of IFN-α in differentiation and activity of monocyte-derived dendritic cells (DCs) from CML patients as well as in modulation of the cell response to lipopolysaccharide (LPS). Treatment of CML monocytes with IFN-α and granulocyte-macrophage colony-stimulating factor (GM-CSF) resulted in the rapid generation of activated DCs (CML-IFN-DCs) expressing interleukin-15 (IL-15) and the antiapoptotic bcl-2 gene. These cells were fully competent to induce IFN-γ production by cocultured autologous T lymphocytes and expansion of CD8+ T cells. LPS treatment of CML-IFN-DCs, but not of immature DCs generated in the presence of IL-4/GM-CSF, induced the generation of CD8+ T cells reactive against autologous leukemic CD34+ cells. Altogether, these results suggest that (1) the generation of highly active monocyte-derived DCs could be important for the induction of an antitumor response in IFN-treated CML patients and (2) IFN-α can represent a valuable cytokine for the rapid generation of active monocyte-derived DCs to be utilized for vaccination strategies of CML patients. (Blood. 2004;103:980-987)
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Burchert, Andreas, Ying Wang, Cornelia Brendel, Philipp Erben, Paul W. Manley, Andreas Hochhaus, Dali Cai, and Andreas Neubauer. "Adaptive Autocrine Secretion of the Granulocyte Macrophage Colony Stimulating Factor (GM-CSF) Mediates Imatinib- and Nilotinib-Resistance in BCR/ABL-Positive Progenitors Via JAK-2/STAT-5 Pathway Activation." Blood 108, no. 11 (November 1, 2006): 2187. http://dx.doi.org/10.1182/blood.v108.11.2187.2187.
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Abstract Overcoming disease persistence in chronic myeloid leukemia (CML) patients is of considerable importance to the issue of potential cure. Here we asked whether autocrine signaling contributes to survival of BCR/ABL positive cells in presence of imatinib mesylate (IM, Gleevec™) and nilotinib (AMN107, NI), a novel more selective Abl inhibitor. Conditioned media (CM) of IM-resistant LAMA84-cell clones (R-CM) was found to substantially protect IM-naïve LAMA cells and primary CML progenitors from IM- or NI-induced cell death. This was due to an increased secretion of the granulocyte-macrophage colony stimulating factor (GM-CSF) which was identified as the causative factor mediating IM resistance in R-CM. GM-CSF elicited IM and NI drug resistance via a BCR/ABL-independent activation of the JAK-2/STAT-5 signaling pathway in GM-CSF-receptor alpha receptor (CD116) expressing cells, including primary CD34+/CD116+ GM-progenitors (GMP). In a cell based resistance induction assay, GM-CSF enabled the evolution of IM- and NI-resistant colonies. When analysing GM-CSF expression in over 120 patient samples of first diagnosis CML patients and resistant patient, elevated mRNA and protein levels of GM-CSF were detected exclusively in IM-resistant patient samples, suggesting a contribution of GM-CSF secretion for IM and NI resistance in vivo. Importantly, inhibition of JAK-2 with AG490 abrogated GM-CSF-mediated STAT-5 phosphorylation and NI resistance in vitro. Together, this suggests that adaptive autocrine secretion of GM-CSF and cytokines in general may be a novel IM and NI resistance mechanism, which may also contribute to CML-persistence. GM-CSF protects CML-progenitors via a BCR/ABL-independent activation of the anti-apoptotic JAK-2/STAT-5 pathway. Inhibition of JAK-2 overcomes GM-CSF-induced IM and NI progenitor cell resistance providing a rationale for the application of JAK-2 inhibitors to eradicate residual disease in CML.
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Hawley, R. G., A. Z. Fong, B. Y. Ngan, V. M. de Lanux, S. C. Clark, and T. S. Hawley. "Progenitor cell hyperplasia with rare development of myeloid leukemia in interleukin 11 bone marrow chimeras." Journal of Experimental Medicine 178, no. 4 (October 1, 1993): 1175–88. http://dx.doi.org/10.1084/jem.178.4.1175.
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Post 5-fluorouracil-treated murine bone marrow cells infected with a recombinant retrovirus (murine stem cell virus-interleukin 11 [MSCV-IL-11]) bearing a human IL-11 gene were transplanted into lethally irradiated syngeneic mice. Analysis of proviral integration sites in DNA prepared from hematopoietic tissues and purified cell populations of long-term reconstituted primary and secondary recipients demonstrated polyclonal engraftment by multipotential stem cells. High levels (100-1,500 U/ml) of IL-11 were detected in the plasma of the MSCV-IL-11 mice. Systemic effects of chronic IL-11 exposure included loss of body fat, thymus atrophy, some alterations in plasma protein levels, frequent inflammation of the eyelids, and often a hyperactive state. A sustained rise in peripheral platelet levels (approximately 1.5-fold) was seen throughout the observation period (4-17 wk). No changes were observed in the total number of circulating leukocytes in the majority of the transplanted animals (including 10 primary and 18 secondary recipients) despite a > 20-fold elevation in myeloid progenitor cell content in the spleen. The exceptions were members of one transplant pedigree which presented with myeloid leukemia during the secondary transplant phase. A clonal origin of the disease was determined, with significant expansion of the MSCV-IL-11-marked clone having occurred in the spleen of the primary host. Culturing of leukemic spleen cells from a quaternary recipient led to the establishment of a permanent cell line (denoted PGMD1). IL-11-producing PGMD1 myeloid leukemic cells are dependent on IL-3 for continuous growth in vitro and they differentiate into granulocytes and macrophages in response to granulocyte/macrophage colony-stimulating factor. The inability of autogenously produced IL-11 to support autonomous growth of PGMD1 cells argues against a mechanism of transformation involving a classical autocrine loop.
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Touw, Ivo P. "Game of clones: the genomic evolution of severe congenital neutropenia." Hematology 2015, no. 1 (December 5, 2015): 1–7. http://dx.doi.org/10.1182/asheducation-2015.1.1.
Full textAbstract:
Abstract Severe congenital neutropenia (SCN) is a genetically heterogeneous condition of bone marrow failure usually diagnosed in early childhood and characterized by a chronic and severe shortage of neutrophils. It is now well-established that mutations in HAX1 and ELANE (and more rarely in other genes) are the genetic cause of SCN. In contrast, it has remained unclear how these mutations affect neutrophil development. Innovative models based on induced pluripotent stem cell technology are being explored to address this issue. These days, most SCN patients receive life-long treatment with granulocyte colony-stimulating factor (G-CSF, CSF3). CSF3 therapy has greatly improved the life expectancy of SCN patients, but also unveiled a high frequency of progression toward myelodysplastic syndrome (MDS) and therapy refractory acute myeloid leukemia (AML). Expansion of hematopoietic clones with acquired mutations in the gene encoding the G-CSF receptor (CSF3R) is regularly seen in SCN patients and AML usually descends from one of these CSF3R mutant clones. These findings raised the questions how CSF3R mutations affect CSF3 responses of myeloid progenitors, how they contribute to the pre-leukemic state of SCN, and which additional events are responsible for progression to leukemia. The vast (sub)clonal heterogeneity of AML and the presence of AML-associated mutations in normally aged hematopoietic clones make it often difficult to determine which mutations are responsible for the leukemic process. Leukemia predisposition syndromes such as SCN are unique disease models to identify the sequential acquisition of these mutations and to interrogate how they contribute to clonal selection and leukemic evolution.
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Dale, David C., James A. Shannon, Audrey Anna Bolyard, Jim Connelly, Daniel C. Link, Mary Ann Bonilla, and Peter E. Newburger. "Severe Chronic Neutropenia in the Large Granular Lymphocyte Syndrome: Outcomes in Response to Granulocyte Colony Stimulating Factor (G-CSF) and Immunosuppressive Therapies." Blood 134, Supplement_1 (November 13, 2019): 3589. http://dx.doi.org/10.1182/blood-2019-126181.
Full textAbstract:
The large granular lymphocyte (LGL) syndrome and large granular lymphocytic leukemia (LGLL) are rare hematological disorders. Through the Severe Chronic Neutropenia International Registry (SCNIR) we have followed the clinical course and treatment responses of 27 LGL patients presenting with severe chronic neutropenia without evidence of leukemia, rheumatoid arthritis or autoimmune diseases. This report focuses on their treatment responses to G-CSF and outcomes with and without other immunosuppressive therapies. Methods: Patients with severe chronic neutropenia and a diagnosis of LGL syndrome (at least 3 ANC values < 0.5 x 109/L over a 3 month period) were enrollment in the SCNIR if they do not have concomitant systemic autoimmune disease, myeloid or lymphoid malignancy or drug-induced neutropenia. Findings: The 27 LGL patients (8 males, median age 53; 19 females, median age 57) had chronic neutropenia for approximately 4 years (range 0.4 - 25.6 years) before enrollment. Diagnoses were primarily based on FACS analysis (12 bone marrow [50%]; 11 blood and bone marrow [46%]; 1 blood only [4%]). None of these patients presented with lymphocytosis, i.e., ALC > 5.0 x 109/L. Before enrollment, 7 patients had anti-neutrophil antibody tests (3 positive, 4 negative), 4 patients had antinuclear antibody tests (2 positive, 2 negative), and ANCA was positive for the one patient tested. After enrollment, additional results were: 1 patient had a negative anti-neutrophil antibody, 8 patients had antinuclear antibody tests (2 positive, 6 negative), and ANCA was positive in two additional patients. The diagnosis of LGL was based on increased LGL cells in the blood or bone marrow, most frequently increased CD 3+, CD 8+, CD 57+ cells. Increases in lymphocytes in the marrow, often in clusters were a common finding; lymphocytes in the marrow ranged from 6-79%. The range of CD 57+ in blood 11-19%, bone marrow 19-61%; CD 56+/NK cells in blood 0.06-14%, bone marrow <1-76.8%; increased CD 8+/CD 3+ in blood 1.5-90%, bone marrow <1-91%. Ten patients have gene rearrangement studies, 7 prior to enrollment (3 negative, 4 positive) and 3 patients had these studies after enrollment, all positive. Because most patients had neutropenia, fevers and infections we evaluated the effectiveness and safety of G-CSF therapy. Treatments were G-CSF alone (13), G-CSF with prednisone (5), G-CSF with methotrexate and prednisone (4), G-CSF with methotrexate alone (2), G-CSF with cyclosporine (1), G-CSF with cyclosporine and prednisone (1), and G-CSF with prednisone, methotrexate and cyclosporine (1). Prior to G-CSF treatment the median ANC was 0.54 x 109/L, mean 1.48 +/- 0.35 SEM. The ALC median was 1.67 x 109/L, mean 2.74 +/- 0.74. The G-CSF dosing median was 1.44 mcg/kg/day, mean 2.27 +/- 0.31. All responded to G-CSF alone or in combination therapy to achieve a mean ANC greater than 1.0 x 109/L. After G-CSF treatment the median ANC was 2.23 x 109/L, mean 3.78 +/- 0.40. The ALC median was 2.24 x 109/L, mean 3.17 +/- 0.48. Time period for SCNIR observation median years is 9.6 years; mean 10.0 +/- 1.2, range 0.4 to 23.1 years. There was a total 270 years of observation in the SCNIR and 307 years of G-CSF administration in this group of 27 patients. The increase in neutrophils was consistently associated with fewer episodes of fever, infections and antibiotic use. One of 27 patients had episodic thrombocytopenia (<50 x 109/L) before G-CSF and continued while on G-CSF. Seven of 26 patients reported episodic thrombocytopenia after starting G-CSF. There were no report episodes of significant bleeding. Fourteen of 27 are now living, ages 17 to 72, and all have continued on treatment. Over the 24 year observation period, 5 patients developed Hodgkin disease (1) or non-Hodgkin lymphoma (5); and 5/6 died. Five other LGL patients died of sepsis or pneumonia. Other deaths were attributed to a T-cell lymphoproliferative disorder (1), chronic pancreatitis (1) and lung cancer (1). There were no myeloid malignancies. Conclusions: Our long term observations suggests that the combination of low dose G-CSF plus methotrexate or cyclosporine appears to be more effective than either drug alone, titrated each to minimal effective doses, and continued indefinitely. There was no increased frequency of opportunistic infections. The expected course is a long period of normal ANCs without other autoimmune diseases. Evolution to lymphoid, not myeloid, malignancies is the greatest concern. Disclosures Dale: Coherus: Consultancy; x4pharma: Consultancy, Honoraria, Research Funding; Athelas: Equity Ownership; Prolong: Consultancy; Cellerant: Other: Scientific Advisory Board; Hospira: Consultancy; Amgen: Consultancy, Research Funding; Sanofi Aventis: Consultancy, Honoraria; Beheringer/Ingelheim: Consultancy. Newburger:TransCytos LLC: Consultancy; X4 Pharmaceuticals: Consultancy, Honoraria.
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Wisniewski, David, Annabel Strife, Steve Swendeman, Hediye Erdjument-Bromage, Scott Geromanos, W. Michael Kavanaugh, Paul Tempst, and Bayard Clarkson. "A Novel SH2-Containing Phosphatidylinositol 3,4,5-Trisphosphate 5-Phosphatase (SHIP2) Is Constitutively Tyrosine Phosphorylated and Associated With src Homologous and Collagen Gene (SHC) in Chronic Myelogenous Leukemia Progenitor Cells." Blood 93, no. 8 (April 15, 1999): 2707–20. http://dx.doi.org/10.1182/blood.v93.8.2707.408k17_2707_2720.
Full textAbstract:
Because of the probable causal relationship between constitutive p210bcr/abl protein tyrosine kinase activity and manifestations of chronic-phase chronic myelogenous leukemia (CML; myeloid expansion), a key goal is to identify relevant p210 substrates in primary chronic-phase CML hematopoietic progenitor cells. We describe here the purification and mass spectrometric identification of a 155-kD tyrosine phosphorylated protein associated with src homologous and collagen gene (SHC) from p210bcr/abl-expressing hematopoietic cells as SHIP2, a recently reported, unique SH2-domain–containing protein closely related to phosphatidylinositol polyphosphate 5-phosphatase SHIP. In addition to an N-terminal SH2 domain and a central catalytic region, SHIP2 (like SHIP1) possesses both potential PTB(NPXY) and SH3 domain (PXXP) binding motifs. Thus, two unique 5-ptases with striking structural homology are coexpressed in hematopoietic progenitor cells. Stimulation of human hematopoietic growth factor responsive cell lines with stem cell factor (SCF), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) demonstrate the rapid tyrosine phosphorylation of SHIP2 and its resulting association with SHC. This finding suggests that SHIP2, like that reported for SHIP1 previously, is linked to downstream signaling events after activation of hematopoietic growth factor receptors. However, using antibodies specific to these two proteins, we demonstrate that, whereas SHIP1 and SHIP2 selectively hydrolyze PtdIns(3,4,5)P3 in vitro, only SHIP1 hydrolyzes soluble Ins(1,3,4,5)P4. Such an enzymatic difference raises the possibility that SHIP1 and SHIP2 may serve different functions. Preliminary binding studies using lysates from p210bcr/abl-expressing cells indicate that both Ptyr SHIP2 and Ptyr SHIP1 bind to the PTB domain of SHC but not to its SH2 domain. Interestingly, SHIP2 was found to selectively bind to the SH3 domain of ABL, whereas SHIP1 selectively binds to the SH3 domain of Src. Furthermore, in contrast to SHIP1, SHIP2 did not bind to either the N-terminal or C-terminal SH3 domains of GRB2. These observations suggest (1) that SHIP1 and SHIP2 may have a different hierarchy of binding SH3 containing proteins and therefore may modulate different signaling pathways and/or localize to different cellular compartments and (2) that they may be substrates for tyrosine phosphorylation by different tyrosine kinases. Because recent evidence has clearly implicated both PI(3,4,5)P3 and PI(3,4)P2 in growth factor-mediated signaling, our finding that both SHIP1 and SHIP2 are constitutively tyrosine phosphorylated in CML primary hematopoietic progenitor cells may thus have important implications in p210bcr/abl-mediated myeloid expansion.
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Jenal, Mathias, Venkateshwar A. Reddy, Judith Laedrach, Deborah Shan, Andreas Tobler, Bruce E. Torbett, Martin F. Fey, and Mario P. Tschan. "The Anti-Apoptotic Gene BCL2A1 Is Transcriptionally Regulated by PU.1." Blood 112, no. 11 (November 16, 2008): 3579. http://dx.doi.org/10.1182/blood.v112.11.3579.3579.
Full textAbstract:
Abstract PU.1 is a hematopoietic transcriptional regulator that is necessary for the development of both myeloid and B cells. To identify new PU.1 target genes in neutrophil development PU.1 was introduced into mouse 503 PU.1-null cells using lentiviral gene transfer and microarray analyses of two independent 503 PU.1-rescued and parental 503 cells were compared. The BCL2A1 gene was found to be more than 50-fold induced in 503 PU.1- restored as compared to the parental 503-null cells. BCL2A1 (also known as BFL-1/A1) is an anti-apoptotic member of the BCL2 family. BCL2A1 was initially identified as a tissue-specific BCL2-related factor that is induced by different reagents such as granulocyte macrophage colony-stimulating factor (GM-CSF) or all-trans retinoic acid (ATRA) during myeloid differentiation. Upregulation of BCL2A1 in granulocytes may promote a time-dependent survival. To follow up on our microarray findings we evaluated loss of PU.1 function in human NB4 acute promyelocytic leukemia (APL) cells using lentivector delivered, short hairpin (sh) RNAs targeting PU.1. Knockdown efficacy upon ATRA-treatment in the two shPU.1 expressing NB4 cell lines was 67 and 30%, respectively. Silencing of PU.1 markedly reduced BCL2A1 mRNA induction upon ATRA-treatment from 167-fold in control cells to 47- and 112-fold in the two PU.1 knockdown NB4 cell lines, respectively (Figure A). Co-transfection of PU.1 with a human BCL2A1 promoter reporter resulted in a 7-fold activation, suggesting PU.1 can directly regulate BCL2A1. Co-transfection with NF-kappaB, used as positive control, induced the BCL2A1 promoter 14.5-fold. Moreover, in vivo binding of the transcription factor PU.1 to 2/8 putative PU.1 binding sites in the BCL2A1 promoter was shown by chromatin immunoprecipitation in HL60 promyelocytic cells further supporting a role for PU.1 regulation of BCL2A1. Evaluation of BCL2A1 and PU.1 mRNA expression in CD34+ hematopoietic progenitors, granulocytes, and primary acute myeloid leukemia (AML) cells was assessed using real-time quantitative RT-PCR. BCL2A1 and PU.1 mRNA levels were significantly lower in primary AML patient samples (n=80; p<0.0001) and in CD34+ progenitor cells (n=4; p=0.0095) than in granulocytes (n=6; Figure B). In line with this observation, we found that upon ATRA therapy BCL2A1 levels were increased in 5/5 APL patients and PU.1 mRNA levels in 4/5 APL cases, respectively. Altogether, these results clearly indicate that PU.1 and BCL2A1 are co-regulated during granulocyte differentiation. Lastly, we confirmed earlier data showing that ATRA-pretreatment of NB4 cells and thus induction of PU.1 and BCL2A1, rendered these cells less sensitive to arsenic trioxide (As2O3)- induced cell death. Conversely, NB4 PU.1 knockdown cells were markedly more sensitive to As2O3 -induced cell death upon ATRA-pretreatment than the parental NB4 control cells. The increase in sensitivity to As2O3 correlated with the lower BCL2A1 levels found in the PU.1 knockdown cells. In summary, we identified the anti-apoptotic BCL2A1 gene as direct, transcriptional target of PU.1 in myeloid leukemic cells. We hypothesize that PU.1-dependent induction of BCL2A1 is necessary for the survival of normal, terminally differentiated myeloid cells. Furthermore, aberrant expression of PU.1 in erythroleukemia may result in elevated BCL2A1 levels that support increased survival of erythroblasts in this particular type of leukemia. Figure Figure
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Wisniewski, David, Annabel Strife, Steve Swendeman, Hediye Erdjument-Bromage, Scott Geromanos, W. Michael Kavanaugh, Paul Tempst, and Bayard Clarkson. "A Novel SH2-Containing Phosphatidylinositol 3,4,5-Trisphosphate 5-Phosphatase (SHIP2) Is Constitutively Tyrosine Phosphorylated and Associated With src Homologous and Collagen Gene (SHC) in Chronic Myelogenous Leukemia Progenitor Cells." Blood 93, no. 8 (April 15, 1999): 2707–20. http://dx.doi.org/10.1182/blood.v93.8.2707.
Full textAbstract:
Abstract Because of the probable causal relationship between constitutive p210bcr/abl protein tyrosine kinase activity and manifestations of chronic-phase chronic myelogenous leukemia (CML; myeloid expansion), a key goal is to identify relevant p210 substrates in primary chronic-phase CML hematopoietic progenitor cells. We describe here the purification and mass spectrometric identification of a 155-kD tyrosine phosphorylated protein associated with src homologous and collagen gene (SHC) from p210bcr/abl-expressing hematopoietic cells as SHIP2, a recently reported, unique SH2-domain–containing protein closely related to phosphatidylinositol polyphosphate 5-phosphatase SHIP. In addition to an N-terminal SH2 domain and a central catalytic region, SHIP2 (like SHIP1) possesses both potential PTB(NPXY) and SH3 domain (PXXP) binding motifs. Thus, two unique 5-ptases with striking structural homology are coexpressed in hematopoietic progenitor cells. Stimulation of human hematopoietic growth factor responsive cell lines with stem cell factor (SCF), interleukin-3 (IL-3), and granulocyte-macrophage colony-stimulating factor (GM-CSF) demonstrate the rapid tyrosine phosphorylation of SHIP2 and its resulting association with SHC. This finding suggests that SHIP2, like that reported for SHIP1 previously, is linked to downstream signaling events after activation of hematopoietic growth factor receptors. However, using antibodies specific to these two proteins, we demonstrate that, whereas SHIP1 and SHIP2 selectively hydrolyze PtdIns(3,4,5)P3 in vitro, only SHIP1 hydrolyzes soluble Ins(1,3,4,5)P4. Such an enzymatic difference raises the possibility that SHIP1 and SHIP2 may serve different functions. Preliminary binding studies using lysates from p210bcr/abl-expressing cells indicate that both Ptyr SHIP2 and Ptyr SHIP1 bind to the PTB domain of SHC but not to its SH2 domain. Interestingly, SHIP2 was found to selectively bind to the SH3 domain of ABL, whereas SHIP1 selectively binds to the SH3 domain of Src. Furthermore, in contrast to SHIP1, SHIP2 did not bind to either the N-terminal or C-terminal SH3 domains of GRB2. These observations suggest (1) that SHIP1 and SHIP2 may have a different hierarchy of binding SH3 containing proteins and therefore may modulate different signaling pathways and/or localize to different cellular compartments and (2) that they may be substrates for tyrosine phosphorylation by different tyrosine kinases. Because recent evidence has clearly implicated both PI(3,4,5)P3 and PI(3,4)P2 in growth factor-mediated signaling, our finding that both SHIP1 and SHIP2 are constitutively tyrosine phosphorylated in CML primary hematopoietic progenitor cells may thus have important implications in p210bcr/abl-mediated myeloid expansion.
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Deliliers, Giorgio Lambertenghi, Nicola S. Fracchiolla, Federica Servida, Silvano Bosari, and Davide Soligo. "Effect of Dioxin on Normal Human Hematopoietic and Leukemic Cells." Blood 104, no. 11 (November 16, 2004): 4354. http://dx.doi.org/10.1182/blood.v104.11.4354.4354.
Full textAbstract:
Abstract 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is the prototypical agonist of the aryl hydrocarbon receptor (AhR), a member of the erb-A family that also includes receptors for steroids, thyroid hormones, peroxisome proliferators and retinoids. When bound to dioxin, the AhR can bind to DNA and alter the expression of various genes, including cytokines and growth factors. TCDD has a large number of biological effects, such as long-lasting skin disease, cardiovascular disease, diabete and cancer. An increase risk of Hodgkin’s disease, non-Hodgkin’s lymphoma and myeloid leukemia was observed after 15 years in the population exposed to dioxin following the 1976 accident in Seveso, Italy. In this study, we analysed the effect of escalating doses of TCDD on the human myeloid leukemic cell lines HL60 (promyelocytic leukemia) and K562 (chronic myelogenous leukemia), as well as on human CD34+ progenitor cells from the leukapheresis of normal donors stimulated with G-CSF. The possible specific modulation of gene expression induced by TCDD was then tested by means of macroarray analyses (Clontech), the results of which were successfully validated by real-time quantitative PCR on five candidate genes. The dioxin dose capable of inhibiting the growth of 50% of the colonies in semisolid medium (IC50 calculated using the CalcuSyn computer program) was 33 nM for HL60, 28 nM for K562, and 20 nM for the CD34+ cells. The HL60, K562 and CD34+ cells tested before and after exposure to 20 nM of TCDD allowed us to identify a series of modulated genes. The up-regulated genes included LUCA15 putative tumour suppressor, cyclin-D binding Myb-like protein (hDMP1), MAPK/ERK kinase 6, ras-related protein RAB2, RalB GTP-binding protein, transcription factor ZFM1, and BRCA1-associated ring domain protein (BARD1). The down-regulated genes included found c-myc, TNF alpha precursor, ICAM1, MIP1-beta, interferon gamma-induced protein precursor (gamma-IP10), granulocyte-macrophage colony stimulating factor (GM-CSF), monocyte chemotactic protein 1 precursor (MCP1). All of these genes are variously involved in the processes of proliferation, differentiation and transformation in hematological and tumoral cell models. On the basis of these data, we can hypothesise that the inhibition of clonogenic potential and the gene expression induced by TCDD exposure on normal CD34+ progenitor cells plays a role in the neoplastic transformation of hemopoietic stem cells. This supports the epidemiological data indicating an increased hematological cancer risk in the population accidentally exposed to TCDD.
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Pang, Qishen, June Li, Xiaoling Zhang, Daniel P. Sejas, and Grover C. Bagby. "Nucleophosmin Protects Hematopoietic Cells from Stress-Induced Apoptosis through Inhibition of P53." Blood 104, no. 11 (November 16, 2004): 2558. http://dx.doi.org/10.1182/blood.v104.11.2558.2558.
Full textAbstract:
Abstract Nucleophosmin (NPM) is a multifunctional protein frequently overexpressed in actively proliferating cells including tumor and hematopoietic stem cells. Here we report that NPM protects hematopoietic cells from stress-induced apoptosis through inhibition of the tumor suppressor p53. Specifically, we forced expression of wild-type (WT) NPM or a mutant variant with a deletion of the C-terminal 120 aa of NPM (NPMΔC) by retroviral gene transfer in the granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent myeloid cell line MO7e (expresses low level of NPM) and the lymphoblast HSC536 cells derived from a Fanconi anemia (FA) patient in the C complementation group (expresses essentially undetectable NPM). Using a flow cytometric method for caspase 3 activation (early apoptosis), we demonstrated that overexpression of NPM but not the mutant NPMΔC confers MO7e and HSC536 cells resistance to apoptosis induced by growth factor deprivation and treatment with the chemotherapeutic drug etoposide. In addition, suppression of NPM expression by small interfering RNA targeting NPM in chronic myelogenous leukemia line K562 and FA-associated acute myelogenous leukemia cell line UoC-M1 increases etoposide-induced apoptosis, thus providing proof of concept evidence that the pathological elevations of NPM found in cancers and leukemias are important for maintaining cell survival and resistance to apoptosis. Because overexpression of the mutant NPMΔC, which lacks the p53-interacting domain, fails to confer cellular resistance to stress-induced apoptosis, we determined whether NPM protects cells from apoptotic cell death through a mechanism involving p53. We used the genetically matched p53 WT and null mouse bone marrow (BM) cells to show that overexpression of WT NPM protects against ionizing irradiation (IR)-induced apoptosis of wild-type but not p53-null BM cells. Moreover, NPM inhibits IR-induced p53 phosphorylation at Ser18 and transactivation, and interacts with p53 in bone marrow hematopoietic cells. Thus, this study not only demonstrates anti-apoptotic function of a proliferation-promoting protein but also suggests that cancer progression may require increased expression of NPM to suppress p53 activation and maintain cell survival.
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Bund, Dagmar, Raymund Buhmann, and Hans Jochem Kolb. "IFN-Alpha and gm-CSF Can Reverse Immunomodulatory Effects of Imatinib." Blood 112, no. 11 (November 16, 2008): 2903. http://dx.doi.org/10.1182/blood.v112.11.2903.2903.
Full textAbstract:
Abstract Presently, Imatinib (IM) is the first line therapy for chronic myeloid leukaemia (CML), but in most patients CML recurs after discontinuation of IM. In contrast, allogeneic stem cell transplantation (ASCT) is considered to be the only curative treatment for this malignancy curing by the immune effects of donor T-cells against CML progenitor cells. Recent reports have described that IM has inhibitory effects on both the function of T-cells and antigen presenting cells (APCs) like dendritic cells. In the present study we further characterized the immunomodulatory effects of IM on T-cells and APCs and analysed whether IFN-alpha (interferon alpha, IFN-a) alone or in combination with GM-CSF (granulocyte-macrophage colony-stimulating factor) can influence the negative IM effects in vitro. Patient derived CML cells were examined to stimulate allogeneic HLA-mismatched T-cells without and with Imatinib (1, 2 or 5 micro M). The activation profile (CD25) and the expression of the TCR-alpha (T-cell receptor) on the T-cells was determined by FACS after 5 days in presence of IM as well as the proliferative T-cell response which was evaluated using CFSE (Carboxy Fluoroscein Succinimidyl Ester). In [51Cr]-release assays the cytotoxicity of CD8+ T-cells was assessed. The cytokine profile of the culture supernatants was detected by CBA (Cytokine bead array, Beckton Dickinson, USA). We also characterized the antigen profile (HLA-DR, CD40, CD54, CD58, CD80 and CD86) of the CML cells as APCs over a 5 day culture with and without IM. The rescue of the CML cells was initiated by IFN-a or IFN-a/GM-CSF after 3 days incubation with IM. On day 6 the APC profile of the CML cells was obtained and cells were used as stimulators in terms of T-cell proliferation which was determined by CFSE. We could show that the proliferation of allogeneic T-cell was inhibited in the presence of IM in a dose-dependent manner. In addition, the T-cell activation marker CD25 and the TCR-alpha expression were significantly downregulated by different concentrations of IM. Moreover, IM impaired the cytotoxic function of allogeneic T-cells in a dose-dependent manner. Interestingly, after stimulation with CML-cells cytotoxic T-cell activity could only be induced in those cases that did not secrete IL-6. Down-regulation of the APC profile on CML-cells (adhesion/costimulatory molecules) by increasing concentrations of IM could be in part reversed by the addition of IFN-a and completely restored by the combination of IFN-a and GM-CSF. Although, the proportion of proliferating T-cells could not be increased further by the combination of IFN-a ± GM-CSF, the number of T-cells was increased in the presence of IFN-a + GM-CSF. In conclusion, the down-modulating effects of IM could be almost completely reversed by the addition of IFN-a and GM-CSF. It remains to be seen whether these findings can be successfully applied to the treatment of CML-patients.
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Stieglitz, Elliot, Y. Lucy Liu, Peter D. Emanuel, Robert P. Castleberry, Todd Michael Cooper, Kevin Shannon, and Mignon L. Loh. "Mutations In GATA2 Are Rare In Juvenile Myelomonocytic Leukemia." Blood 122, no. 21 (November 15, 2013): 1526. http://dx.doi.org/10.1182/blood.v122.21.1526.1526.
Full textAbstract:
Abstract Germline mutations in GATA2, a gene that encodes for transcription factors involved in hematopoiesis and vascular development, have recently been described in MonoMAC syndrome, Emberger syndrome and in select cases of mild chronic neutropenia. These disorders are unified by their predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Patients with MonoMAC syndrome have also been noted to display monosomy 7 in their bone marrows in up to 50% of cases. Overexpression of GATA2 due to somatic mutations in cases of de novo pediatric AML, has also been shown to be a negative predictor of outcome. Juvenile myelomonocytic leukemia is a rare childhood malignancy with overlapping features of MDS and myeloproliferative neoplasm (MPN) that can transform to AML and is characterized by hyperactive RAS signaling. Mutations in NF1, NRAS, KRAS, PTPN11, and CBL are found in 85-90% of newly diagnosed patients, and monosomy 7 is the most common recurrent karyotypic abnormality seen in JMML. We therefore hypothesized that mutations in GATA2 may play a role in the development of JMML. Samples from 57 patients with JMML were screened for GATA2 mutations. Patient samples and clinical data were collected from the Children's Oncology Group (COG) trial AAML0122. DNA was extracted as per previous protocols from peripheral blood or bone marrow and whole genome amplified using Qiagen REPLI-g kit according to manufacturer specifications. We performed bidirectional Sanger sequencing (Beckman Coulter Genomics) of the entire coding region of GATA2 (NM_001145661.1) and aligned the sequences using CLC Workbench software (CLC Bio, Aarhus, Denmark). Only missense, splice site or nonsense mutations were evaluated using SIFT (Sorting Tolerant From Intolerant) to predict the impact on the structure and function of identified mutations on the protein. Patient J384 was found to have a nonsense point mutation at c.988C>T (R330X) in the N-terminal region of the zinc finger portion of the protein (Figure 1a). This hotspot mutation has been reported in several patients with mild chronic neutropenia who displayed a predisposition to developing MDS and AML. The patient was also found to have a missense point mutation at c.962T>G (L321R) predicted to be damaging by SIFT. Subcloning of the gene using a TA cloning kit with pCR 2.1 vector (Invitrogen), followed by direct sequencing of individual colony picks, revealed that the two sequence variants only occurred in a trans configuration. Out of 40 amplicons sequenced, 20 were found to have the c.988C>T transition, 16 were found to be have the c.962T>G variant, and four were found to be wild type. We therefore hypothesize that the c.988C>T was inherited as a germline event and that c.962T>G was somatically acquired in the majority of the remaining wild type alleles. No other point mutations or insertions/deletions were discovered in this cohort.Figure 1Identification of 2 distinct GATA2 mutations in patient J384.Figure 1. Identification of 2 distinct GATA2 mutations in patient J384. This patient was previously identified to have a KRAS G12D mutation (c.35G>A) as well as monosomy 7. This patient died prior to undergoing transplant within months of diagnosis. While the patient technically met criteria for the diagnosis of JMML, it should be noted there were several atypical features, including older age at diagnosis (4 years and 10 months), and absence of hypersensitivity in myeloid progenitor cells to the cytokine granulocyte–macrophage colony stimulating factor (GM-CSF) in colony assay. This raises the possibility that patient J384 actually had MonoMAC syndrome with MDS and not JMML. This represents the first description of a GATA2 mutation in a patient suspected of having JMML. To our knowledge, this is the first report of a biallelic mutation in GATA2, combining a germline mutation with somatic acquisition. In addition, MonoMAC syndrome has not been reported to be associated with KRAS mutations to date. GATA2 mutations should therefore be considered in patients with atypical features of MDS or JMML. Panel (a) Bidirectional sequencing of patient sample J384 revealed two distinct sequence variants in both the forward (shown here) and reverse strands. Panel (b) Sequencing of 40 individual colony picks revealed that each sequence variant occurred in a trans configuration (CP 9 and CP13 are shown here as examples). In addition, 10% of colony picks (i.e. CP 32) revealed a wild type sequence, indicating that at least one of the two variants was a somatic event. Disclosures: No relevant conflicts of interest to declare.
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Zeidler, Cornelia, Peter Lohse, Beate Schwinzer, Gusal Pracht, Karl-Walter Sykora, and Karl H. Welte. "Shwachman-Diamond-Bodian Syndrome (SBDS): Long-Term Follow-Up of 31 Patients." Blood 108, no. 11 (November 16, 2006): 1282. http://dx.doi.org/10.1182/blood.v108.11.1282.1282.
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Abstract Shwachman-Bodian-Diamond syndrome (SBDS) is an autosomal recessive multisystem disorder primarily affecting the bone marrow, exocrine pancreas, and skeleton. As with other constitutional bone marrow failure syndromes, there is a predisposition to malignant myeloid transformation. The risk can only be estimated and varies considerably in the literature. Significant hematologic abnormalities can be present from early childhood with requirement of growth factor treatment (granulocyte-colony stimulating factor, erythropoietin). Since the first description of mutations in the SBDS gene, most clinically diagnosed patients have been genetically analyzed meanwhile, which now allows for genotype-phenotype correlations. Here, we report long-term clinical data of 31 SBDS patients (10 female, 21 male; 27 patients alive, 3 expired, 1 lost to follow up) with regard to different genotypes collected by the European Branch of the Severe Chronic Neutropenia International Registry since 1994: Severe Neutropenia (ANC <500/μl) is present in 11 patients, of whom 9 receive G-CSF treatment. In 16 of the remaining 22 untreated patients, the median ANC is below 1500/μl. G-CSF doses range from 0.4 to 7.7 μg/kg/day (mean G-CSF dose: 3.15 μg/kg/d). One patient is treated with granulocyte-macrophage colony stimulating factor (GM-CSF). Two of the G-CSF-treated patients have received a combination of G-CSF plus erythropoietin prior to stem cell transplantation. In 4 patients, G-CSF therapy was started within the first year of life, in 1 patient at the age of 20 months, and in 2 patients at the age of 8 and 13 years, respectively. MDS-like leukemic transformation occurred in 2 (1 male/1 female) out of 31 patients (6.5 %) at an age of 5.3 and 7.0 years. Both patients had additional cytogenetic abnormalities (monosomy 7 and complex aberrant karyotype). One patient had received G-CSF for 22 months at a dose of 3.8 μg/kg/d prior to SCT. In the other patient, pancytopenia including severe neutropenia started at the time cytogenetic aberrations were detectable. Both patients received stem cell transplantation (SCT) from unrelated donors (1 MUD, 1 haploident.). 14 months after MUD-SCT the girl is in a good clinical condition with full hematologic reconstitution, whereas the boy died within the first month after haploident. SCT from septicemia. SCT was also performed in 4 non-leukemic patients due to pancytopenia and prior to lung transplantation (2 HLA ident SCT: alive, 2 MUD SCT: 1 alive, 1 expired). SBDS gene analysis has been carried out in 14 of 19 German patients. Ten of 14 patients (71%) were compound heterozygous for 183–184TA>CT/258+2T>C. The rare genotypes 199A>G/258+2T>C, 297–300delAAGA/258+2T>C, and TGC>TGG/258+2T>C were observed in single patients. In another individual, a complex aberration with 7 mutations (Rosendahl et al, 2006) was found. Interestingly, two of the latter four patients presented with severe hematological anomalies early in life. One of them died from SCT after leukemic transformation. The third patient has developed severe osteoporosis and diabetes mellitus type 1. The fourth is on continuous G-CSF treatment. Our data demonstrate the importance of hematologic follow-up and regular cytogenetic evaluation of the bone marrow in SBDS patients. Patients with rare SBDS gene mutations seem to develop a more severe phenotype, but it requires a larger patient cohort for statistical proof.
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Alves, Marcia Dias, Marta M. Lemos, and Carla Boquimpani. "Resource Use and Costs Associated With The Treatment Of Hematologic and NON-Hematologic Adverse Events In Chronic Myeloid Leukemia (CML) Brazilian Patients." Blood 122, no. 21 (November 15, 2013): 5620. http://dx.doi.org/10.1182/blood.v122.21.5620.5620.
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Abstract Introduction Chronic myeloid leukemia (CML) is a clonal malignant disease characterized by excessive proliferation of myeloid lineage, followed by a progressive loss of cell differentiation, with evolution including progression from chronic phase to accelerated or blastic phase. The disease is associated with a specific cytogenetic abnormality, the Philadelphia chromosome, which results from a reciprocal translocation between the long arms of chromosomes 9 and 22 [t(9;22)(q34;q11)], leading to the generation of the new leukemia-specific gene BCR-ABL, resulting in the production of an oncoprotein with tyrosine kinase activity, responsible for the pathophysiology of disease. Effective treatments with tyrosine kinase inhibitors (TKI) are available for CML patients and differences in adverse events (AE) and incidences occurring during therapy are reported in the literature depending on the type of TKI. Objective Improve the standard of resources used and reduce later treatment costs of treatment-related haematological and non-hematological adverse events of CML patients, from the perspective of the Brazilian National Health System. Cost of treatment per event will be presented as a result. Method A Delphi methodology was applied in Brazil to study the management of adverse events related to CML treatment by collecting data on resource used., considering the resource use and its cost associated. Based on the literature, a questionnaire was developed on the available resources for the treatment of select adverse events. Ten experts completed the questionnaires and the results were compiled and validated. The data collected were related to percentage of patients affected by each AE and percentage of each resource used to manage the AE. A consensus meeting was held after questionnaire completion. The unit cost of resources was based on the Bank Price and Health Management System Procedures Table of SUS (SIGTAP). Cost of treatment of the AEs consisted of the cost of the treatment options and monitoring costs (e.g., outpatient visits, laboratory tests and procedures, hospitalizations) in the Brazilian Public Health System. Results Among non-hematologic adverse events grade 3-4, gastrointestinal bleeding and central nervous system (CNS) hemorrhage presented the highest costs of treatment per event (R$ 1.022,90 and R$ 1.173,34, respectively). Hospitalization is the resource with more impact; mainly in CNS hemorrhage, with an average length of hospitalization reported by the experts of 1 week and also being demanded by 100% of patients affected by this AE. Management of cardiac events is R$973,35 and Pericardial effusion is R$3.896,55. Cytopenias are the most commonly occurring events in patients with CML receiving BCR–ABL inhibitors. Experts suggest the use of growth factors, such as granulocyte colony-stimulating factor, interleukin-11 and erythropoietin in patients with CML receiving TKI therapy. The Panel experts agreed the cost associated with the management the hematologic adverse events are: Anemia: R$14,93, Thrombocytopenia: R$14,47 and neutropenia: R$211,54. Conclusion All adverse events related to treatment of CML, with differences in the occurrence and intensity should be valued and recorded as contributing to the estimated total cost of treating the disease. Adverse event management in CML patients has important clinical and economic impact. I Banco de Preço em Saúde. Disponível em: http://bps.saude.gov.br/visao/consultapublica/publico_interno_item.cfm - Accessed in 08/04/2013 II Sistema de Gerenciamento da Tabela de Procedimentos, Medicamentos e OPM do SUS. DATASUS – SIGTAP. Available in http://sigtap.datasus.gov.br/tabela-unificada/app/sec/inicio.jsp - Accessed in: 08/04/2013 Disclosures: Alves: Novartis: Employment. Lemos: Novartis: Employment. Boquimpani: Novartis: Consultancy.
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Jahns-Streubel, Gerlinde, Christoph Reuter, Ulrike Auf der Landwehr, Michael Unterhalt, Eberhard Schleyer, Bernhard Wörmann, Thomas Büchner, and Wolfgang Hiddemann. "Activity of Thymidine Kinase and of Polymerase α as Well as Activity and Gene Expression of Deoxycytidine Deaminase in Leukemic Blasts Are Correlated With Clinical Response in the Setting of Granulocyte-Macrophage Colony-Stimulating Factor–Based Priming Before and During TAD-9 Induction Therapy in Acute Myeloid Leukemia." Blood 90, no. 5 (September 1, 1997): 1968–76. http://dx.doi.org/10.1182/blood.v90.5.1968.
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Abstract The present study was undertaken to assess the predictive value of pretherapeutic determinants of ara-C metabolism and proliferative activity of leukemic blasts for early response to antileukemic therapy in the setting of granulocyte-macrophage colony-stimulating factor (GM-CSF )–based priming before and during TAD-9 induction in 36 consecutive patients with de novo acute myeloid leukemia (AML). Ara-C metabolism was assessed by the activities of deoxycytidine kinase (DCK), deoxycytidine deaminase (DCD), DNA polymerase α (Poly α), and overall polymerase (overall Poly). The fraction of cells in S phase (%S phase) and thymidine kinase (TK) activity were determined as a measure of proliferative activity. Early response to therapy was defined by the percentage of leukemic blasts in the bone marrow 5 to 7 days after completion of TAD-9 with less than 5% signaling an adequate response and greater than 5% indicating an inadequate early reduction, respectively. While neither %S phase, DCK, nor overall Poly activity were predictive for early response, TK and Poly α activities were significantly higher for cases with adequate blast cell clearance. The respective median values were for TK 3.8 versus 1.85 pmol/min/mg protein (P = .012), and for Poly α 1.9 versus 0.69 pmol/min/mg protein (P = .014). An inverse relation was detected for DCD activity which was significantly lower in responding patients with a median of 0.33 nmol/min/mg protein (range, 0.0 to 29.5) as compared to a median of 5.1 nmol/min/mg protein (range, 0.11 to 8.45) in early nonresponders, (P = .009). Taking the respective median values as arbitrary cut-points for high or low enzyme activities, responders and nonresponders could be discriminated prospectively. Hence, 14 of 16 cases (88%) with DCD activities below the median of 1.56 nmol/min/mg protein responded as compared to only 3 of 14 (22%) patients with higher DCD activities (P = .0004). From the 15 patients with TK activity above the overall median of 3.2 pmol/min/mg protein, 11 cases (73%) achieved an adequate blast cell clearance while only 6 of 17 cases (35%) with lower values responded (P = .035). Similarly, 12 of 15 patients (80%) with high Poly α levels (>1.22 pmol/min/mg protein) responded to induction therapy as compared to only 5 of 14 patients (36%) with lower enzyme activities (P = .02). By logistic regression analysis of enzyme activities, DCD activity was found to be the most sensitive parameter to predict an adequate blast cell clearance (P = .032). Activities of DCD and TK were not only associated with initial response but were also found predictive for remission duration. Hence, from 11 patients with low TK levels 8 (73%) relapsed within 1 year, whereas only 2 of 11 (18%) patients with high TK activity experienced a recurrence of their disease (P = .015). Six of 9 (66%) patients with higher than median DCD levels relapsed within 1 year, whereas 10 of 14 patients (71%) with lower DCD levels had a longer remission duration (P = .085). Analysis of DCD gene expression at the mRNA level by a semi-quantitative reverse transcriptase-polymerase chain reaction method showed that a high transcription rate of the DCD gene was associated with high enzyme activities and vice versa. Hence, the observed intraindividual differences in DCD activity are a reflection of differences in gene activity and transcription rate rather than of variants in translation. Although further analyses are needed to elucidate the molecular mechanisms that determine the variation of enzyme activities in individual patients, the present study strongly suggests that pretherapeutic determination of TK and Poly α as well as of DCD allows to predict response to TAD-9 + GM-CSF induction therapy and may provide the means for the development of a risk adapted treatment strategy.
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Asari, Kartini, Susan L. Heatley, Teresa Sadras, Tamara M. Leclercq, Stephen Fitter, Chung Hoow Kok, Andrew C. W. Zannettino, Timothy P. Hughes, and Deborah L. White. "In Vitro Modeling of Ph-like ALL Fusions Identifies Novel Kinase-Domain Mutations As Mode of TKI-Resistance - Implications for Targeted Therapy." Blood 128, no. 22 (December 2, 2016): 3957. http://dx.doi.org/10.1182/blood.v128.22.3957.3957.
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Abstract Introduction Treatment-resistant acute lymphoblastic leukemia (ALL) remains a significant clinical issue. Recently, genomic profiling has identified a new subtype of high-risk ALL termed Philadelphia-chromosome-like (Ph-like) ALL, associated with a poor outcome1. Ph-like ALL has a gene expression profile similar to Ph+ (BCR-ABL1+) ALL, characterized by the presence of fusion genes converging on kinase and cytokine signaling pathways. These pathways have been shown to be targetable both in vitro and in case reports by tyrosine kinase inhibitors (TKIs). Despite well-documented efficacy profiles, it is known from TKI-use in chronic myeloid leukemia (CML) andPh+ ALL that resistance is likely, resulting in relapse. Our study aims to model and understand mechanisms of TKI-resistance inPh-like ALL, informing future therapeutic strategies that may avert or overcome resistance, potentially improving patient outcomes. Methods Three Ph-like ALL lines were generated via retroviral-transduction from plasmids of fusion genes identified in patient cohorts (RANBP2-ABL1, SSBP2-CSF1R and PAX5-JAK2, a kind gift from C. Mullighan)2 into Ba/F3 pro-B cells. Transformation was confirmed via growth of cells in the absence of IL-3. Cells were tested for sensitivity to a panel of TKIs (imatinib, dasatinib, ponatinib, ruxolitinib and BMS-911543) via Annexin-V/7-AAD flow-cytometry and western blotting of downstream effector proteins. Drug resistance was generated through exposure of cells to incrementally increasing concentrations of TKIs over a period of 3-6 months, and cell death LD50 determined byAnnexin-V/7-AAD. Sanger sequencing of the 3-prime partner gene of each fusion was performed to identify the emergence of any kinase-domain mutations. Results Ba/F3 Ph-like cells demonstrated sensitivity to TKIs at clinically relevant doses (RANBP2-ABL1: 1 μM imatinib, 5 nM dasatinib & 5 nM ponatinib; SSBP2-CSF1R: 1 μM imatinib, 6 nM dasatinib; PAX5-JAK2: 1 μM ruxolitinib & 2 μM BMS-911543). This correlated with decreased levels of relevant downstream signaling proteins including p-Stat5, p-Erk and p-CrkL. TKI-resistant Ph-like ALL lines were tolerant to a significantly higher concentration of TKIs compared to control (RANBP2-ABL1: 10 μM imatinib, 200 nM dasatinib & 200 nM ponatinib; SSBP2-CSF1R: 10 μM imatinib, 200 nM dasatinib; PAX5-JAK2: 10μMruxolitinib & 10μM BMS-911543; Table 1). Sequencing analysis revealed that Ba/F3 RANBP2-ABL1 imatinib and dasatinib resistant cells acquired the clinically significant ABL1 T315I (c.944C>T) kinase-domain mutation, which was ultimately targetable using the third-generation TKI ponatinib (LD50: 25 nM). An ABL1 E255K (c.763G>A) and c-terminus deletion was discovered in the ponatinib-resistant line. In Ba/F3 SSBP2-CSF1R cells, a novel CSF1R L785M (c.2566C>A) mutation was identified in imatinib-resistant cells whereas a deletion spanning the SSBP2-CSF1R breakpoint was acquired in the dasatinib-resistant line. A JAK2 Y931C (c.3286A>G) point mutation previously associated with resistance to ATP-competitive inhibitors was acquired in Ba/F3 PAX5-JAK2ruxolitinib and BMS-911543 resistant lines. Conclusion In vitro modeling of Ph-like ALL resistance has identified novel kinase domain mutations and deletions that may arise as a result of targeted TKI therapy. In addition, previously identified mutations (T315I and E255K) were also identified. Detection of these mutations is important because alterations in drug-binding regions are known to result in significantly reduced TKI sensitivity, leading clinically to relapse3. This study describes an in vitro platform that can be utilized to inform future clinical approachesincluding the development of rational therapeutic approaches (and/or combination therapies) to avert resistance inPh-like ALL cases treated with rationally targeted therapies. Abbreviations: ABL1 - Abelson tyrosine protein kinase 1 CSF1R - Colony stimulating factor 1 receptor JAK2 - Janus kinase 2 PAX5 - Paired box 5 RANBP2 - RAN-binding protein 2 SSBP2 - Single-stranded DNA binding protein 2 References: 1 Den Boer et al, Lancet Oncology 2009; 10(2):125-34 2 Roberts et al, Cancer Cell 2012; 22(2):153-66 3 Barouch-Bentov & Sauer, Expert Opinion on Investigational Drugs 2011; 20(2);153-208 Disclosures Hughes: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Australasian Leukaemia and Lymphoma Group (ALLG): Other: Chair of the CML/MPN Disease Group. White:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria, Research Funding.
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Collins, Shannon, Audrey Anna Bolyard, Tracy M. Marrero, Lan Phan, and David C. Dale. "Barth Syndrome and Severe Chronic Neutropenia." Blood 116, no. 21 (November 19, 2010): 3787. http://dx.doi.org/10.1182/blood.v116.21.3787.3787.
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Abstract Abstract 3787 Barth syndrome is an X-linked recessive genetic condition characterized by neutropenia, cardiomyopathy, growth delay, muscle weakness, and 3-methylglutaconic aciduria (an increase in organic acid caused by abnormal mitochondria). Its clinical manifestations are variable and can include fatigue, hypotonia, and dilated cardiomyopathy. Symptoms usually appear at birth or within the first few months of life. Mutations in the tafazzin (TAZ) gene cause Barth syndrome. The Severe Chronic Neutropenia International Registry (SCNIR) is building a base of information about the natural history and response to granulocyte colony stimulating factor (G-CSF) treatment for patients with Barth syndrome and other causes of chronic neutropenia. Through the SCNIR, we follow the clinical course and treatment of seven male subjects (median age 17 years, range: 6–28 years) with Barth syndrome observed for up to 11 years (median 9, range 3–11). Six of these seven subjects were neutropenic prior to G-CSF treatment, the median absolute neutrophil count (ANC) was 0.293 × 109/L (range 0 – 1.260). The seventh subject was not consistently neutropenic, median ANC 2.107 × 109/L (range 0.779 – 3.520). Sixteen bone marrow evaluations were performed on four of the seven subjects. Four of 16 bone marrows were prior to G-CSF exposure (3 subjects). Two of three subjects manifest eosinophilia in the marrow but not in the blood. Marrow exams for two of the three subjects' evaluations were read as normocellular marrow, and one of the three was read as hypocellular with a decrease in cells of the myeloid series. Twelve of the 16 bone marrow evaluations were performed in two subjects who were receiving G-CSF. One of the four subjects had bone marrow evaluations both before and after G-CSF exposure. His pre G-CSF evaluations displayed hypocellular bone marrow, and his post G-CSF evaluations showed normocellular bone marrow and eosinophilia. None of the marrow evaluations before or on G-CSF suggested myelodysplasia or showed evidence of acute myeloid leukemia. The six neutropenic subjects have all received G-CSF for a median of 96 months (range 28 – 137) at a median dose of 1.57 mcg/kg/day (range 0.43 to 2.18). The total G-CSF exposure for all six subjects is 507 months. The median ANC of the six subjects prior to G-CSF treatment was 0.293 × 109/L (range 0–1.260). The median ANC on G-CSF was 2.056 × 109/L (range 1.640–3.403). Prior to receiving G-CSF, three of the seven subjects reported mouth ulcers. Two of seven subjects reported skin infections, including one subject who reported infections around the G-tube used to maintain his nutritional status. One of seven subjects reported an episode of bacteremia. Of the six subjects who received G-CSF, three reported a reduced number of mouth ulcers and two of six reported reduced skin infections (G-tube, port-a-cath). None of the subjects experienced unusual side effects or clinically significant complications associated with G-CSF therapy. These data indicate that patients with Barth syndrome and neutropenia have ulcers and patterns of infections similar to other patients with chronic neutropenia. They are responsive to G-CSF treatment and it appears to be safe and effective to reduce their predisposition to bacterial infections. Disclosures: Dale: Amgen: Consultancy, Research Funding.
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Hayashi, Yoshihiro, Hideyo Hirai, Hisayuki Yao, Satoshi Yoshioka, Sakiko Satake, Naoka Kamio, Yasuo Miura, et al. "BCR/ABL-Mediated Myeloid Expansion Is Promoted by C/EBPβ, a Regulator of Emergency Granulopoiesis,." Blood 118, no. 21 (November 18, 2011): 3747. http://dx.doi.org/10.1182/blood.v118.21.3747.3747.
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Abstract Abstract 3747 Chronic phase chronic myeloid leukemia (CP-CML) is characterized by the increase of myeloid cells in the peripheral blood (PB) and bone marrow (BM). We have previously shown that the C/EBPβ transcription factor is required for emergency granulopoiesis, increased proliferation and differentiation of granulocytic precursors in emergency situations such as infection (Hirai H et al., Nature Immunol. 2006). Enhanced myelopoiesis is a common feature between emergency situations and CP-CML. However, little is known about the roles of C/EBPβ in the pathogenesis of CP-CML. The aim of this study is to elucidate the regulation and function of C/EBPβ in BCR/ABL-mediated myeloid expansion. We first assessed the expression level of C/EBPβ in hematopoietic stem cells and myeloid progenitors in BM obtained from healthy donors or CP-CML patients. The transcript of C/EBPβ is expressed at significantly higher level in common myeloid progenitors (CMPs) and granulocyte-macrophage progenitors (GMPs) in CP-CML BM than those in normal BM. When BCR/ABL was retrovirally transduced into a mouse hematopoietic stem cell line, EML, C/EBPβ expression was significantly upregulated. Treatment of the EML-BCR/ABL cells with imatinib mesylate normalized the expression level of C/EBPβ. These data suggested that C/EBPβ was upregulated in response to the downstream signaling of BCR/ABL. In order to investigate the function of C/EBPβ in BCR/ABL-mediated myeloid expansion, BCR/ABL was retrovirally introduced into BM cells obtained from 5-FU treated C/EBPβ knockout (KO) mice and their properties were compared with those of BCR/ABL-transduced BM cells from wild type (WT) mice. When the transduced cells were cultured in cytokine-free semisolid methylcellulose medium, the number and the size of the colonies of C/EBPβ KO cells were significantly smaller. Flow cytometric analysis of the colony-forming cells revealed that the BCR/ABL-transduced C/EBPβ KO BM cells gave rise to higher frequency of c-kit+ cells and lower CD11b+ cells than BCR/ABL-transduced WT BM cells (%c-kit+ cells=8.2±3.0% vs. 11.3±3.5%, p=0.002, %CD11b+ cells=75.1±2.1% vs. 90.0±4.2%, p=0.003). In addition, BCR/ABL-transduced C/EBPβ KO BM cells revealed higher replating efficiency than BCR/ABL-transduced WT BM cells. To investigate the role of C/EBPβ in leukemogenesis, BCR/ABL-transduced BM cells from C/EBPβ KO mice or WT mice were transplanted into lethally irradiated recipient mice. In mice transplanted with BCR/ABL-transduced C/EBPβ KO cells, the increase of white blood cell count was delayed (Figure) and higher frequency of c-kit+ cells were observed in the BM at day 19 post transplantation (16.0±2.6% vs. 5.5±4.6%, p=0.01). Spleen size of mice transplanted with BCR/ABL-transduced WT cells is much larger than that of BCR/ABL-transduced C/EBPβ KO cells (Figure). The median survival of mice transplanted with BCR/ABL-transduced WT cells was 19 days. In contrast, the median survival of mice transplanted with BCR/ABL-transduced C/EBPβ KO cells was 31 days (p=0.0005). In summary, C/EBPβ is upregulated by BCR/ABL and the absence of C/EBPβ resulted in delayed proliferation and differentiation of myeloid cells both in vitro and in vivo. Our results suggest that C/EBPβ is involved in the BCR/ABL-mediated myeloid expansion in CP-CML and that C/EBPβ can be the novel molecular target for the therapy of CML. We are currently investigating the molecular mechanisms which mediate the upregulation of C/EBPβ and the direct targets of C/EBPβ in CP-CML. Disclosures: No relevant conflicts of interest to declare.
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Saito, Shoji, Aiko Hasegawa, Mika Nagai, Yoichi Inada, Hirokazu Morokawa, Ikumi Nakashima, Daisuke Morita, et al. "Mutated GM-CSF-Based CAR T-Cells Targeting CD116/CD131 Complexes Exhibit Enhanced Anti-Tumor Effects Against Acute Myeloid Leukemia." Blood 136, Supplement 1 (November 5, 2020): 36–37. http://dx.doi.org/10.1182/blood-2020-134395.
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Background: The prognosis of relapsed/refractory (R/R) acute myeloid leukemia (AML) remains poor; therefore, novel treatment strategies are required urgently. Meanwhile, recent clinical trials have demonstrated that CAR-T cells for AML have been less successful than those targeting CD19 for B cell malignancies. Recently, we developed piggyBac-modified ligand-based CAR-T cells that target CD116, also called granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor (GMR) α chain, for treating juvenile myelomonocytic leukemia (Nakazawa, et al. J Hematol Oncol. 2016). Since CD116 is overexpressed in 60%-80% of AML cases, the present study aimed to develop a novel therapeutic method for R/R AML using GMR CAR-T cells. Methods: CD116 expression in AML cell lines or primary leukemia cells were examined using flow cytometry. The original piggyBac transposon plasmid for GMR CAR comprises GM-CSF as an antigen recognition site, IgG1 CH2CH3 hinge region, CD28 costimulatory domain, and CD3ζ chain. To improve the in vivo persistency and anti-tumor effects, two types of spacer (∆CH2H3 and G4S) that lack CH2CH3 lesion were newly constructed. In order to modulate the antigen recognition ability, mutated ligand-based GMR CAR vectors were constructed with a mutation at residue 21 of GM-CSF that is reported to play a critical role in its biological activity (Lopez, et al. Embo j. 1992). All the GMR CAR-T cells were generated with piggyBac gene modification. To investigate the in vitro anti-tumor activity, GMR CAR-T cells were co-cultured with AML cell lines. In order to evaluate the in vivo anti-tumor effects, NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice were intravenously injected with THP-1, THP1-ffLuc, or MV4-11 and then treated with GMR CAR-T cells. To characterize the safety profile of GMR CAR-T cells, peripheral blood mononuclear cells or polymorphonuclear cells were co-cultured with GMR CAR-T cells at an effector:target ratio of 1:1 for 3 days. Thereafter, B cells, NK cells, neutrophils, and monocytes were quantified using flow cytometry using counting beads. Results: Approximately 80% of the AML cells predominant in myelomonocytic leukemia expressed CD116. PiggyBac-modified GMR CAR-T cells displayed a favorable CD45RA+CCR7+-dominant phenotype, consistent with our previous findings. GMR CAR-T cells exhibited potent cytotoxic activities against CD116+ AML cells in vitro. GMR CAR-T cells incorporating a G4S spacer significantly improved the long-term in vitro and in vivo anti-tumor effects as compared to those incorporating a ∆CH2CH3 spacer. Furthermore, by employing a mutated GM-CSF at residue 21 (E21K and E21R) as an antigen recognition site, the in vivo anti-tumor effects were also substantially improved along with prolonged survival (Figure 1) over controls (PBS or CD19.CAR-T cells) (all, p < 0.01) as well as over GMR CAR-T cells with a wild-type GM-CSF ligand (E21R: p < 0.01; E21K: p = 0.02), with 4 out of 5 mice surviving for > 150 days. Safety tests revealed that the toxicity of GMR CAR-T cells was restricted to normal monocytes. It is noteworthy that the cytotoxic effects of GMR CAR-T cells on normal neutrophils, T cells, B cells, and NK cells were minimal. Conclusions: GMR CAR-T cell therapy appears to be a potentially useful strategy for CD116+ R/R AML. Based on the promising results, we plan to perform the first-in-human clinical trial of GMR CAR-T cells. Disclosures Saito: Toshiba Corporation: Research Funding. Hasegawa:Toshiba Corporation: Research Funding. Inada:Kissei Pharmaceuticals: Ended employment in the past 24 months. Nakashima:Toshiba Corporation: Research Funding. Yagyu:Toshiba Corporation: Research Funding. Nakazawa:Toshiba Corporation: Research Funding.
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Trottier, Amy M., Ira L. Kraft, Lawrence J. Druhan, Amanda Lance, Belinda R. Avalos, and Lucy A. Godley. "New Germline Syndrome Discovery: Heterozygous CSF3R Mutations May Predispose to Myeloid and Lymphoid Malignancies." Blood 134, Supplement_1 (November 13, 2019): 2543. http://dx.doi.org/10.1182/blood-2019-129492.
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Introduction Colony stimulating factor 3 receptor (CSF3R) plays an important role in granulocyte proliferation and differentiation. Acquired mutations in CSF3R are found in the majority of cases of chronic neutrophilic leukemia and in a minority of atypical chronic myeloid leukemia and acute myeloid leukemia patients, whereas inherited biallelic mutations have been identified as a cause of severe congenital neutropenia (SCN). CSF3R variants have also been detected in lymphoid cells in patients with SCN. However, whether germline monoallelic CSF3R variants predispose to myeloid and/or lymphoid malignancies is unknown. Methods We analyzed variants detected by a clinical next generation sequencing (NGS) panel of about 150 genes important in oncogenesis (OncoPlus) performed on peripheral blood and/or bone marrow of patients with hematopoietic malignancies at The University of Chicago Medical Center from June 2017 to February 2019. We prioritized genes for which variants were likely of germline origin based on variant allele fraction (VAF) > 0.4 and persistence over multiple tests. Variant germline status was determined by extracting DNA from cultured bone marrow-derived mesenchymal stromal cells and/or from cultured skin fibroblasts. Variants in CSF3R [transcript NM_000760.3] were ranked by predicted pathogenicity using standard American College of Medical Genetics criteria. Variants of uncertain significance (VUS) were evaluated using population frequency data, in silico functional prediction, and REVEL scores. Results A total of 620 OncoPlus tests were conducted on 496 patients (ages 1 month - 96 years; median 64 years), and 824 unique variants were identified within 33 genes. Among these patients, 89 (17.9%) had an invariant variant, one that was found at similar VAFs across multiple testing time points. These invariant variants involved 31 different genes with a total of 203 unique variants. Variants with a VAF less than 0.4 were excluded from further study, leaving 81 patients with 164 invariant variants among 30 genes (Figure 1). The genes with the most invariant variants included known germline predisposition genes, such as ATM, BRCA2, DDX41, ETV6, and TP53, as well as CSF3R, which was not previously recognized as an autosomal dominant germline cancer susceptibility gene. Among the 81 patients, 8 (9.9%) were found to have invariant variants in CSF3R, with 7 unique variants identified. Including patients for whom only a single OncoPlus test was conducted in addition to those with invariant variants, there were a total of 43 patients with 20 unique variants in CSF3R among the entire 496 patient cohort (Figure 2). Of these 20 CSF3R variants, 8 (40%) were found to be germline, and among those, one was classified as likely pathogenic, one was classified as a VUS, and six were classified as likely benign. We focused our attention on the likely pathogenic and VUS germline variants. These two variants were among those with the highest predicted pathogenicity scores. The likely pathogenic variant, W547*, is a nonsense mutation in the extracellular domain of CSF3R that was identified in a patient with a history of bladder cancer treated with surgery and chemotherapy who subsequently developed therapy-related myelodysplastic syndrome. W547* has previously been found to be pathogenic in a compound heterozygous state in an infant with SCN. The germline VUS, P784T, is a missense variant identified in a patient with multiple myeloma. This variant localizes to the cytoplasmic domain of CSF3R and is not listed in the COSMIC or gnomAD databases. Functional studies are underway to assess the oncogenic potential of the W547* and P784T variants. Conclusion Detection of invariant variants on clinical, tumor-based NGS panels can be used as a way to identify potential germline mutations to aid in new germline syndrome discovery. Using this approach, CSF3R was identified as a candidate gene for which monoallelic pathogenic variants may predispose to both myeloid and lymphoid malignancies. Future work is ongoing to assess the functional consequences of germline CSF3R variants and the frequency of such mutations. Disclosures Avalos: Best Practice-Br Med J: Patents & Royalties: receives royalties from a coauthored article on evaluation of neutropenia; Juno: Membership on an entity's Board of Directors or advisory committees. Godley:Invitae, Inc.: Membership on an entity's Board of Directors or advisory committees; UpToDate, Inc.: Patents & Royalties: receives royalties from a coauthored article on inherited hematopoietic malignancies .
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Fasan, Annette, Claudia Haferlach, Karolína Perglerová, Wolfgang Kern, and Torsten Haferlach. "CSF3R Mutations Are Predominantly Subclonal Events in Intermediate Risk Karyotype AML and Prevalently Occur with CEBPA Mutations." Blood 128, no. 22 (December 2, 2016): 1658. http://dx.doi.org/10.1182/blood.v128.22.1658.1658.
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Abstract Background: The colony stimulating factor 3 receptor(CSF3R) plays an important role in granulocyte differentiation and acquired mutations have been described in different myeloid malignancies. Besides chronic neutrophilic leukemia (CNL) and severe congenital neutropenia (SCN), mutations in CSF3R (CSF3Rmut) have also been reported in acute myeloid leukemia (AML) at low frequency and recently have been associated with CEBPA mutations (Lavallèe et al., Blood 2016; Maxson et al., Blood 2016) in adult and pediatric AML. First studies suggest sensitivity of CSF3Rmut CNL against JAK kinase inhibitors suggesting the possibility for targeted therapy for a subset of CSF3Rmut AML patients. Aims: The evaluation of CSF3Rmut in AML with intermediate-risk karyotype for frequency and association with other mutations with special focus on CEBPA mutation status. Methods: We analyzed 274 cases with intermediate risk de novo AML for CSF3Rmut by next generation sequencing (Illumina, San Diego, CA) of exon 14 and 17 (sensitivity: 1%), which contain the mutational hotspot regions of CSF3R. As CSF3R mutations were associated with CEBPA mutations in the above-mentioned studies, we analyzed a selected cohort of 179 CEBPA mutated cases in comparison to 95 cases with CEBPA wild type.202 cases (74%) had normal karyotype (CN-AML) and 72 (26%) had intermediate-risk aberrant cytogenetics according to MRC criteria. Female/male ratio was 127/147 and age ranged from 16- 88 y (median: 64 y). Mutation status of the following genes were available in all cases: ASXL1, CEBPA, FLT3-ITD, FLT3-TKD, GATA2, IDH1/2, KRAS, KMT2A-PTD, NPM1, NRAS, RUNX1 and WT1. All mutations with a variant allele frequency (VAF) < 10% were defined as subclonal. 95 cases had wild type CEBPA (CEBPAwt), 92 cases were CEBPA single mutated (CEBPAsm), 81 cases were CEBPA double mutated (CEBPAdm), 6 cases showed CEBPA mutations withloss of heterozygosity (CEBPA LOH). Results: Overall, in 7/274 patients (3%) CSF3Rmut were detected. This is slightly higher than expected in an unselected AML cohort (Sano et al., Br J Haematol 2015). Cytomorphology revealed AML M0 (n=1), AML M1 (n=2), AML M2 (n=3) and AML M4 (n=1). With regard to immunophenotype CSF3Rmut cases did not differ from CSF3Rwt cases which may be due to limited case numbers. All CSF3Rmut cases had CN-AML. The activating transmembrane mutation p.Thr618Ile, which has been identified as common mutation in CNL was the most frequent mutation (4/7). In addition two mutations leading to a truncated receptor (p.Ser715* and p.Gln749*) and a previously not described frame-shift mutation leading to a defecting splice variant (p.Ser783Glnfs*6) were identified. The median CSF3R VAF was 7% (range 2-45%). In 5/7 cases, CSF3Rmut was subclonal (VAF <10%, see figure). CSF3Rmut cases showed a median of 2 concomitant mutations (range 1-4). The most frequent concomitantly mutated gene was CEBPA (5/7 cases; 2 cases CEBPAsm, 1 case CEBPAdm and 2 cases with CEBPA LOH), followed by NRAS (3/7), ASXL1 (2/7) and RUNX1 (2/7). IDH1, TET2 and WT1 were mutated in one case each (see figure). Mutations in NPM1, FLT3, KMT2A or GATA2 were never detected. Interestingly, in CEBPAdm and CEBPA LOH cases, CSF3R was the only concomitantly mutated gene, whereas CEBPAsm cases harbored mutations in at least 1 gene in addition to CSF3R. In 2/7 CSF3Rmut cases we analyzed changes in the patterns of genetic lesions between diagnosis and relapse. Case 1 harbored CEBPAdm and a subclonal CSF3Rmut (VAF: 7%) at diagnosis. At relapse the CEBPAdm persisted, whereas CSF3Rmut was lost. Case 2 harbored two germline CEBPAmut and one somatic CEBPAmut at diagnosis. In addition, mutations in CSF3R and WT1 were identified. At relapse both the somatic CEBPAmut and WT1mut persisted, whereas CSF3Rmut was lost. These results indicate again that the CSF3Rmut was present only in a subclone of CEBPAmut AML in both cases. Due to the limited number of CSF3Rmut cases, we did not perform survival analysis in this study. Summary: 1) CSF3R mutations are rare and predominantly subclonal events in AML and therefore may not be a suitable target for directed therapy.2) CSF3Rmut prevalently occur with CEBPA mutations but are not limited to CEBPAdm. Figure Molecular abnormalities and cytogenetics in CSF3R mutated AML. Figure. Molecular abnormalities and cytogenetics in CSF3R mutated AML. Disclosures Fasan: MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Perglerová:MLL2 s.r.o: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Other: Part Owner MLL Munich Leukemia Laboratory.
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Matsumura, Risa, Shiho Nishimura, Yoko Mizoguchi, Mizuka Miki, Maki Taniguchi, Maiko Shimomura, Shuhei Karakawa, et al. "Successful Bone Marrow Transplantation Using an Immunomyelosuppressive Conditioning in Patients with Severe Congenital Neutropenia: The Results of a Single-Institute." Blood 134, Supplement_1 (November 13, 2019): 1035. http://dx.doi.org/10.1182/blood-2019-126653.
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Severe congenital neutropenia (SCN) is a rare heterogeneous genetic disorder characterized by recurrent bacterial infections from early infancy due to severe chronic neutropenia. Majority of SCN patients have benefitted by the treatment with granulocyte colony-stimulating factor (G-CSF). However, patients on long-term G-CSF therapy have a relative risk of developing myelodysplastic syndrome/acute myeloid leukemia. The only curable treatment for SCN patients is hematopoietic stem cell transplantation (HSCT). Recently, HSCTs with reduced intensity conditioning regimens have been applied to the treatment for SCN patients prior to malignant transformation. However, the optimal conditioning of HSCT for SCN patients has not been established. In this study, we conducted bone marrow cell transplantations (BMT) in 16 patients with SCN using an immunomyelosuppressive conditioning regimen to minimize early and late transplant-related morbidity in Hiroshima University Hospital. A total of 17 BMT procedures were performed in 16 patients with SCN from 2008 to 2019. Five of 16 patients had experienced the engraftment failure of initial HSCT and 4 of them were referred to our hospital for re-transplantation. Fifteen of 16 patients had a heterozygous mutation in the ELANE gene. Bone marrow cells (BM) were obtained from 6 HLA-matched related, 3 HLA-matched unrelated, and 8 HLA-mismatched unrelated (7/8 antigens) donors, respectively. Conditioning regimen consisted of fludarabine, cyclophosphamide, melphalan, total body irradiation (3.6 Gy) with or without antithymocyte globulin. Short-term methotrexate and tacrolimus were administered for the prophylaxis of graft-versus-host disease (GVHD). Engraftment of neutrophils was observed within post-transplant 24 days in all patients. Two patients developed graft failure on day 40 and day 90, respectively, after the temporal engraftment. However, both patients were rescued by second BMT from different HLA-matched unrelated donors receiving the same conditioning regimen. Four patients who received BMT from HLA-matched related donors developed stable mixed chimerism without neutropenia in peripheral blood for 3 to 10 years. Although one patient who received donor lymphocyte infusion due to mixed chimerism developed grade II acute GVHD and limited chronic GVHD, the others did not develop severe GVHD. All patients are alive for 6 months to 11 years after BMT with no signs of severe infections or transplantation-related morbidity. Similar conditioning regimen has been applied to BMT for 35 patients with chronic granulomatous disease (CGD) in our hospital. In that study 4 male adulthood patients with CGD already fathered each child by their wives through spontaneous pregnancy, implying the successful preservation of patients' fertility. Collectively, our results demonstrate that BMT with a sufficient immunosuppressive conditioning regimen may be a feasible and effective treatment for SCN patients, irrespective of initial engraftment failure. The excellent results in our cohort suggest that indications for proceeding to HSCT could be extended to patients without malignant transformation.The further analyses of accumulated cases are necessary to assess the efficacy, safety, and less late adverse effects related to HSCT including fertility. Disclosures No relevant conflicts of interest to declare.
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Jamieson, Catriona, Jorge E. Cortes, Vivian Oehler, Michele Baccarani, Hagop M. Kantarjian, Cristina Papayannidis, Kristen N. Rice, et al. "Phase 1 Dose-Escalation Study of PF-04449913, An Oral Hedgehog (Hh) Inhibitor, in Patients with Select Hematologic Malignancies." Blood 118, no. 21 (November 18, 2011): 424. http://dx.doi.org/10.1182/blood.v118.21.424.424.
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Abstract Abstract 424 Background: Hh signaling is activated in a variety of human cancers and is required for maintenance of leukemic stem cell (LSC) populations in several hematologic malignancies. PF-04449913 selectively inhibits Hh signaling by binding Smoothened, a membrane protein that regulates the Hh pathway. PF-04449913 significantly reduced LSC burden following xenotransplantation of patient-derived CD34+ imatinib-resistant chronic myeloid leukemia (CML) cells in preclinical models. Methods: This first-in-patient Phase 1a dose-escalation study is assessing first-cycle dose-limiting toxicities (DLTs) and the recommended Phase 2 dose (RP2D) of PF-04449913 in patients with select hematologic malignancies (primary endpoint). Secondary endpoints include safety, pharmacokinetics (PK), pharmacodynamics, and preliminary signs of efficacy as defined by disease-specific guidelines. Patients had refractory, resistant, or intolerant select hematologic malignancies and could be previously untreated but not candidates for standard therapies: CML including T315I mutations (any phase), acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), myelofibrosis (MF), or chronic myelomonocytic leukemia (CMML). Cohorts of ≥3 patients received PF-04449913 alone, administered continuously in 28-day cycles, starting at a dose of 5 mg orally once daily. Results: Thirty-two patients have been enrolled at doses up to 270 mg: 18 males/14 females; AML, 18; MF, 6; CML, 5; MDS, 3; with a median age of 68.5 (35–79) years. ECOG PS was 0/1/2: n=11/16/5. Treatment duration ranged from 1 to 387 days (AML: 1–266; CML: 1–281; MDS: 2–335; MF: 44–387 days). One patient discontinued the study due to a treatment-related adverse event (AE) after 137 days of therapy at the 10 mg dose level (grade [G] 3 hemorrhagic gastritis in the setting of chronic proton pump inhibitor administration prior to and during the study). One AML patient evolved from CMML on 80 mg had a DLT comprising G3 hypoxia and G3 pleural effusion. The majority of AEs were of G1/2 severity; the most frequent treatment-related AEs included dysguesia (16%), alopecia (6%), arthralgia (6%), decreased appetite (6%), nausea (6%), and vomiting (6%). Preliminary indications of efficacy were observed across all hematologic diseases studied. One patient with AML (evolved from CMML and with a concurrent diagnosis of systemic mastocytosis) achieved a complete remission with incomplete blood count recovery; bone marrow blast count decreased from 92% to 1%. Five AML patients had a ≥50% reduction in bone marrow blast counts (20% to 10%, 70% to 20%, 44% to 8%, 14% to 7%, 40% to 10%). One patient with low-risk MDS, currently remaining on study after 335 days, achieved significant reduction in spleen size and a hematologic improvement in platelets (from 98.5 to 369 ×109/L) and neutrophils (ANC from 410 to 5490), and is no longer granulocyte colony-stimulating factor (G-CSF) dependent. Five patients with MF attained stable disease; an additional MF patient, currently remaining on study after 385 days, achieved clinical improvement with a >50% reduction in extramedullary disease (spleen size decreased from 10 cm to 3.5 cm sustained over 8 weeks). One patient with T3151 lymphoid blast crisis CML on study for 115 days achieved a major cytogenetic response with loss of their T3151 mutation. PF-04449913 PK data (5–180 mg) indicated a dose-proportional profile, with a median time to maximum concentration (Tmax) of 1–2 hours, a mean half-life ranging from 17 to 35 hours, and a large volume of distribution (mean range 250–480 L). Following daily dosing, steady state was achieved by Day 8 and the median accumulation ratio ranged from 1.3 to 2.9. Conclusions: PF-04449913 was safe and well tolerated, with early signs of efficacy observed in all hematologic diseases studied. Several patients with aggressive malignancies remained on trial for prolonged durations with improved quality of life; some exhibited cellular differentiation as determined by flow cytometry. On-target AEs (e.g. dysgeusia and alopecia) were observed at multiple dose levels. Pharmacokinetics were linear, predictable, and compatible with once-daily dosing. On the basis of these encouraging results, a Phase 1b study of PF-04449913 in combination with bosutinib and dasatinib is planned in patients with CML. Disclosures: Jamieson: Pfizer Oncology: Research Funding; Celgene: Research Funding; Novartis: Honoraria; Wintherix: Equity Ownership. Cortes:Pfizer Oncology: Consultancy; Novartis: Consultancy; BMS: Consultancy; Pfizer Oncology: Research Funding; Novartis: Research Funding; BMS: Research Funding; Infinity: Research Funding. Oehler:Pfizer Oncology: Research Funding. Baccarani:Pfizer Oncology: Consultancy; Novartis: Consultancy; BMS: Consultancy; Ariad: Consultancy; Novartis: Research Funding; Pfizer Oncology: Honoraria; Novartis: Honoraria; BMS: Honoraria; Ariad: Honoraria; Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Ariad: Membership on an entity's Board of Directors or advisory committees. Zhang:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Shaik:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Courtney:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Levin:Pfizer Oncology: Employment; Pfizer Oncology: Equity Ownership. Martinelli:Novartis: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees.
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Mizoguchi, Yoko, Mizuka Miki, Aya Furue, Shiho Nishimura, Maiko Shimomura, Keita Tomioka, Sonoko Sakata, et al. "Successful Hematopoietic Stem Cell Transplantation Using an Immunosuppressive Conditioning Regimen in Ten Patients with Severe Congenital Neutropenia: A Single-Institute Experience." Blood 128, no. 22 (December 2, 2016): 3688. http://dx.doi.org/10.1182/blood.v128.22.3688.3688.
Full textAbstract:
Abstract Severe congenital neutropenia (SCN) is a rare heterogeneous genetic disorder characterized by severe chronic neutropenia, with absolute neutrophil counts below 0.5×109/L, and by recurrent bacterial infections from early infancy. Granulocyte colony-stimulating factor (G-CSF) is widely used for the treatment of neutropenia in patients with SCN. However, the long-term G-CSF therapy has a relative risk of developing myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). The only curative treatment available for SCN patients is hematopoietic stem cell transplantation (HSCT). Recently, HSCTs with reduced intensity conditioning (RIC) regimens have been applied to the treatment of SCN patients without malignant transformation who have become G-CSF refractory. However, the optimal conditions of HSCT for SCN patients have not been established. In this study, we conducted bone marrow cell transplantations (BMT) in ten patients with SCN using an immunosuppressive conditioning regimen to minimize early and late transplant-related morbidity in Hiroshima University Hospital. Ten patients with a total of 11 HSCT procedures in our institution (performed from 2007 to 2015) were enrolled in this study. Four of the ten patients had experienced engraftment failure of the initial HSCT and three of them were referred to our hospital for re-transplantation. Heterozygous mutation inthe ELANE gene was identified in nine of ten patients. These nine patients received BMT less than 10 years of age. All ten patients had recurrently experienced moderate to severe bacterial or fungal infection before HSCT and received temporal or regular administration of G-CSF. Bone marrow cells (BM) were obtained from five HLA-matched related (MRD), three HLA-matched unrelated (MUD), and three HLA-mismatched unrelated (7/8) donors (MMUD), respectively. The conditioning regimen basically consisted of fludarabine (100 to 125 mg/m2), cyclophosphamide (100 to 150 mg/kg), melphalan (70 to 90 mg/m2), total body irradiation (3 to 3.6 Gy), and/or anti-thymocyte globulin (10 to 12 mg/kg). Short-term methotrexate and tacrolimus were administered for the prophylaxis of graft-versus-host disease (GVHD). Engraftment of neutrophils was successfully observed within 24 days of post-transplantation in all patients. All patients achieved complete chimerism at the time of engraftment. Two patients who underwent BMT from MRD and one patient who underwent BMT from MUD showed the gradual decrease of donor-derived cells. Donor lymphocyte infusion treatment successfully achieved the complete chimerism or stable mixed chimerism in these 3 patients. Although 3 patients experienced the acute GVHD (Grade I-II), the addition of glucocorticoids to tacrolimus prevented the extension of acute GVHD. Only one patient developed mild chronic GVHD presenting limited type of skin involvement. All patients are alive for 9 months to 9 years after HSCT with no signs of severe infections or transplantation-related morbidity. Our results demonstrate that BMT together with a sufficient immunosuppressive conditioning regimen may be a feasible and effective treatment for SCN patients, irrespective of initial engraftment failure. Although our results through the small number of cohort is limited to conclude, the BMT with the optimal donors may lead to the increased opportunity for lower risk of SCN patients especially at younger age as a curative treatment. The further analyses of accumulated cases are necessary to assess the efficacy, safety, and less late adverse effects related to HSCT including fertility. Disclosures No relevant conflicts of interest to declare.