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Alexandra Böhm, Karoline Sonneck,
Karoline V Gleixner,
Karina Schuch,
Winfried F Pickl,
Katharina Blatt,
Barbara Peter,
Harald Herrmann,
Gerit-Holger Schernthaner,
Hubert Pehamberger,
Werner Rabitsch,
Wolfgang R Sperr,
Peter Valent
[show abstract]
[hide abstract]
ABSTRACT: In most patients with systemic mastocytosis (SM), including aggressive SM (ASM) and mast cell (MC) leukemia (MCL), neoplastic cells express the oncogenic KIT mutation D816V, which confers resistance to imatinib. Cladribine (2CdA) is a nucleoside analog that has been introduced as a promising agent for treatment of advanced SM.
We examined the in vitro effects of 2CdA on growth of neoplastic MC, and the in vivo effects of 2CdA (0.13 mg/kg/day intravenously, days 1-5; three to eight cycles) in seven patients with advanced SM.
Cladribine was found to inhibit growth of primary MC and the MC line HMC-1 in a dose-dependent manner, with lower IC(50) values recorded in HMC-1.2 cells harboring KIT D816V (IC(50): 10 ng/mL) compared to HMC-1.1 cells lacking KIT D816V (IC(50): 300 ng/mL). In two patients with progressive smoldering SM, 2CdA produced a long-lasting response with a sustained decrease in serum tryptase levels, whereas in patients with progressive ASM or MCL, 2CdA showed little if any effects. The drug was well-tolerated in most cases. However, one patient developed a massive generalized purulent long-lasting skin rash. The antiproliferative effects of 2CdA on MC were found to be associated with morphologic signs of apoptosis and caspase cleavage. Cladribine did not counteract the kinase activity of KIT D816V or KIT-downstream signaling molecules.
Cladribine may be a promising agent for treatment of progressive smoldering KIT D816V(+) SM. In rapidly progressing ASM or MCL, additional or alternative drugs are required to induce long-lasting antineoplastic effects.
Experimental hematology 09/2010; 38(9):744-55. · 3.11 Impact Factor
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Christian Baumgartner,
Sabine Cerny-Reiterer, Karoline Sonneck,
Matthias Mayerhofer,
Karoline V Gleixner,
Richard Fritz,
Marc Kerenyi,
Cedric Boudot,
Fabrice Gouilleux,
Jan-Wilhelm Kornfeld,
Christian Sillaber,
Richard Moriggl,
Peter Valent
[show abstract]
[hide abstract]
ABSTRACT: Recent data suggest that the signal transducer and activator of transcription (STAT)5 contributes to differentiation and growth of mast cells. It has also been described that constitutively phosphorylated STAT5 (pSTAT5) plays a pro-oncogenic role in various myeloid neoplasms. We examined the expression of pSTAT5 in neoplastic mast cells in systemic mastocytosis and asked whether the disease-related oncoprotein KIT D816V is involved in STAT5 activation. As assessed by immunohistochemistry using the anti-pSTAT5 antibody AX1, neoplastic mast cells were found to display pSTAT5 in all SM patients examined (n = 40). Expression of pSTAT5 was also demonstrable in the KIT D816V-positive mast cell leukemia cell line HMC-1. Using various staining-protocols, pSTAT5 was found to be located in both the cytoplasmic and nuclear compartment of mast cells. To define the functional role of KIT D816V in STAT5-activation, Ba/F3 cells with doxycycline-inducible expression of KIT D816V were used. In these cells, induction of KIT D816V resulted in an increased expression of pSTAT5 without substantial increase in total STAT5. Moreover, the KIT D816V-targeting kinase-inhibitor PKC412 was found to counteract expression of pSTAT5 in HMC-1 cells as well as doxycycline-induced expression of pSTAT5 in Ba/F3 cells. Finally, a dominant negative STAT5-construct was found to inhibit growth of HMC-1 cells. Together, our data show that neoplastic mast cells express cytoplasmic and nuclear pSTAT5, that KIT D816V promotes STAT5-activation, and that STAT5-activation contributes to growth of neoplastic mast cells.
American Journal Of Pathology 11/2009; 175(6):2416-29. · 4.89 Impact Factor
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Noria Harir,
Cédric Boudot,
Katrin Friedbichler, Karoline Sonneck,
Rudin Kondo,
Séverine Martin-Lannerée,
Lukas Kenner,
Marc Kerenyi,
Saliha Yahiaoui,
Valérie Gouilleux-Gruart,
Jean Gondry,
Laurence Bénit,
Isabelle Dusanter-Fourt,
Kaïss Lassoued,
Peter Valent,
Richard Moriggl,
Fabrice Gouilleux
[show abstract]
[hide abstract]
ABSTRACT: The D816V-mutated variant of Kit triggers multiple signaling pathways and is considered essential for malignant transformation in mast cell (MC) neoplasms. We here describe that constitutive activation of the Stat5-PI3K-Akt-cascade controls neoplastic MC development. Retrovirally transduced active Stat5 (cS5(F)) was found to trigger PI3K and Akt activation, and to transform murine bone marrow progenitors into tissue-infiltrating MCs. Primary neoplastic Kit D816V(+) MCs in patients with mastocytosis also displayed activated Stat5, which was found to localize to the cytoplasm and to form a signaling complex with PI3K, with consecutive Akt activation. Finally, the knock-down of either Stat5 or Akt activity resulted in growth inhibition of neoplastic Kit D816V(+) MCs. These data suggest that a downstream Stat5-PI3K-Akt signaling cascade is essential for Kit D816V-mediated growth and survival of neoplastic MCs.
Blood 07/2008; 112(6):2463-73. · 9.90 Impact Factor
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Kuan-Wei Chen,
Gudrun Fuchs, Karoline Sonneck,
Anna Gieras,
Ines Swoboda,
Nikolas Douladiris,
Birgit Linhart,
Marija Jankovic,
Tea Pavkov,
Walter Keller,
Nikolaos G Papadopoulos,
Peter Valent,
Rudolf Valenta,
Susanne Vrtala
[show abstract]
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ABSTRACT: The major allergen of the house-dust mite Dermatophagoides pteronyssinus, Der p 2, is recognized by approximately 90% of mite-allergic patients. We have produced two recombinant fragments of Der p 2 comprising aa 1-53 and aa 54-129 and a hybrid molecule (aa 54-129+1-53), combining the two fragments in inverse order, by genetic engineering. The recombinant Der p 2 derivatives were expressed in E. coli and purified to homogeneity. rDer p 2 derivatives (fragments and hybrid) showed a considerably reduced beta sheet structure and IgE reactivity compared to the Der p 2 wild-type allergen. The allergenic activity of the Der p 2 derivatives was reduced more than tenfold as evaluated in vitro in basophil activation assays and in vivo by skin prick testing of mite-allergic patients. Immunization of mice and rabbits with rDer p 2 derivatives induced Der p 2-specific IgG antibodies, which inhibited the binding of allergic patients' IgE to Der p 2. Immunization of mice with rDer p 2 derivatives induced less allergenic IgE responses than immunization with rDer p 2. Thus the rDer p 2 derivatives exhibited less in vivo allergenic activity and allergenicity than the Der p 2 allergen but preserved immunogenicity and may hence represent candidates for specific immunotherapy of house-dust mite allergy.
Molecular Immunology 06/2008; 45(9):2486-98. · 2.90 Impact Factor
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Karoline V Gleixner,
Matthias Mayerhofer, Karoline Sonneck,
Alexander Gruze,
Puchit Samorapoompichit,
Christian Baumgartner,
Francis Y Lee,
Karl J Aichberger,
Paul W Manley,
Doriano Fabbro,
Winfried F Pickl,
Christian Sillaber,
Peter Valent
[show abstract]
[hide abstract]
ABSTRACT: In a majority of all patients with systemic mastocytosis (SM) including those with mast cell leukemia (MCL), neoplastic mast cells (MC) display the D816V-mutated variant of KIT. The respective oncoprotein, KIT D816V, exhibits constitutive tyrosine kinase (TK) activity and has been implicated in malignant cell growth. Therefore, several attempts have been made to identify KIT D816V-targeting drugs.
We examined the effects of the novel TK-inhibitor dasatinib alone and in combination with other targeted drugs on growth of neoplastic MC.
Confirming previous studies, dasatinib was found to inhibit the TK activity of wild type (wt) KIT and KIT-D816V as well as growth and survival of neoplastic MC and of the MCL cell line, HMC-1. The growth-inhibitory effects of dasatinib in HMC-1 cells were found to be associated with a decrease in expression of CD2 and CD63. In addition, we found that dasatinib blocks KIT D816V-induced cluster-formation and viability in Ba/F3 cells. In drug combination experiments, dasatinib was found to co-operate with PKC412, AMN107, imatinib, and 2CdA in producing growth-inhibition and apoptosis in neoplastic MC. In HMC-1.1 cells lacking KIT D816V, all drug interactions were found to be synergistic in nature. By contrast, in HMC-1.2 cells exhibiting KIT D816V, only the combinations dasatinib+PKC412 and dasatinib+2CdA were found to produce synergistic effects.
Combinations of targeted drugs may represent an interesting pharmacologic approach for the treatment of aggressive SM or MCL.
Haematologica 12/2007; 92(11):1451-9. · 6.42 Impact Factor
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Karoline V Gleixner,
Laura Rebuzzi,
Matthias Mayerhofer,
Alexander Gruze,
Emir Hadzijusufovic, Karoline Sonneck,
Anja Vales,
Michael Kneidinger,
Puchit Samorapoompichit,
Tuddow Thaiwong,
Winfried F Pickl,
Vilma Yuzbasiyan-Gurkan,
Christian Sillaber,
Michael Willmann,
Peter Valent
[show abstract]
[hide abstract]
ABSTRACT: Aggressive mast cell (MC) tumors are hematopoietic neoplasms characterized by uncontrolled growth of MC and resistance to conventional drugs. In most cases, the tyrosine kinase (TK) receptor KIT is involved in malignant cell growth. Therefore, several KIT TK-targeting drugs are currently being tested for their ability to block growth of neoplastic MC. We examined the effects of four TK inhibitors (imatinib, midostaurin, nilotinib, and dasatinib) on C2 canine mastocytoma cells, as well as primary neoplastic canine MC. As assessed by (3)H-thymidine incorporation experiments, all TK inhibitors produced dose-dependent inhibition of proliferation in C2 cells with the following IC(50) values: imatinib: 269 +/- 180 nM, midostaurin: 157 +/- 35 nM, nilotinib: 55 +/- 24 nM, dasatinib: 12 +/- 3 nM. Growth-inhibitory effects of TK inhibitors were also observed in primary neoplastic mast cells, although IC(50) values for each drug varied from patient to patient, with midostaurin being the most potent agent in all samples tested. In consecutive experiments, we were able to show that TK inhibitors cooperate with each other in producing growth inhibition in C2 cells with synergistic effects observed with most drug combinations. In flow cytometry and TUNEL assay experiments, growth-inhibitory effects of TK inhibitors were found to be associated with cell-cycle arrest and apoptosis. Together, these data show that several TK-targeting drugs induce apoptosis and inhibit proliferation in canine mastocytoma cells in vitro, and that synergistic drug interactions can be obtained. Clinical trials are now warranted to explore whether these TK inhibitors also counteract growth of neoplastic cells in vivo in patients with aggressive MC tumors.
Experimental Hematology 11/2007; 35(10):1510-21. · 2.90 Impact Factor
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Laura Rebuzzi,
Michael Willmann, Karoline Sonneck,
Karoline V Gleixner,
Stefan Florian,
Rudin Kondo,
Matthias Mayerhofer,
Anja Vales,
Alexander Gruze,
Winfried F Pickl,
Johann G Thalhammer,
Peter Valent
[show abstract]
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ABSTRACT: Vascular endothelial growth factor (VEGF) is a major regulator of angiogenesis and a potential autocrine growth factor for neoplastic cells in various malignancies. In the present study, we have investigated expression of VEGF and VEGF receptors in canine mastocytomas and the canine mastocytoma cell line C2. As assessed by immunostaining of tissue sections and cytospin slides, primary neoplastic mast cells (MC) and C2 cells were found to express the VEGF protein. In Northern blot and RT-PCR experiments, C2 cells expressed VEGF mRNA in a constitutive manner. VEGF mRNA expression in C2 cells was counteracted by LY294002 and rapamycin, suggesting involvement of the PI3-kinase/mTOR pathway. Moreover, C2 cells were found to express VEGF receptor-1 (Flt-1) and VEGF receptor-2 (KDR). However, recombinant VEGF failed to promote (3)H-thymidine uptake in C2 cells, and a neutralizing anti-VEGF antibody (bevacizumab) failed to downregulate spontaneous proliferation in these cells. In addition, rapamycin decreased the expression of VEGF in C2 cells at the mRNA and protein level without suppressing their proliferation. Together, canine mastocytoma cells express VEGF as well as VEGF receptors. However, despite co-expression of VEGF and its receptors, VEGF is not utilized as an autocrine growth regulator by canine mastocytoma cells.
Veterinary Immunology and Immunopathology 03/2007; 115(3-4):320-33. · 2.08 Impact Factor
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Noria Harir,
Christian Pecquet,
Marc Kerenyi, Karoline Sonneck,
Boris Kovacic,
Remy Nyga,
Marie Brevet,
Isabelle Dhennin,
Valerie Gouilleux-Gruart,
Hartmut Beug,
Peter Valent,
Kaiss Lassoued,
Richard Moriggl,
Fabrice Gouilleux
[show abstract]
[hide abstract]
ABSTRACT: Persistent activation of Stat5 is frequently found in hematologic neoplasms. Studies conducted with constitutively active Stat5 mutants (Stat51*6 and cS5F) have shown that deregulated Stat5 activity promotes leukemogenesis. To investigate the oncogenic properties of these mutants, we used cS5F-expressing bone marrow cells which induce a multilineage leukemia when transplanted into recipient mice. Here, we show by immunocytochemistry that cS5F is localized mainly in the cytoplasmic compartment of leukemic cells, suggesting that the transforming nature of cS5F may be associated with a cytoplasmic function. In support of this hypothesis, we found that cS5F forms a complex with the p85 subunit of the phosphatidylinositol 3-kinase (PI3-K) and the scaffolding adapter Gab2 in leukemic bone marrow cells, resulting in the activation of Akt/PKB, a crucial downstream target of PI3-K. By using transducible TAT-Gab2 or TAT-Akt recombinant proteins, we were able to demonstrate that activation of the PI3-kinase/Akt pathway by cS5F molecules through Gab2 is essential for induction of cell growth. We also found that persistently phosphorylated Stat5 in primary cells from patients with myeloid leukemias has a cytoplasmic localization. These data suggest that oncogenic Stat5 proteins exert dual transforming capabilities not only as transcriptional activators but also as cytoplasmic signaling effectors.
Blood 03/2007; 109(4):1678-86. · 9.90 Impact Factor
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[show abstract]
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ABSTRACT: Mylotarg (gemtuzumab ozogamicin [GO]) has recently been introduced as a novel CD33-targeting drug in clinical hematology. However, despite efficacy, GO produces significant side effects including an infusion syndrome. We have recently shown that mast cells (MCs) and basophils (BAs) express CD33. In the present study, we investigated the effects of GO on growth and mediator secretion in MCs and BAs.
Growth-inhibitory effects of GO on neoplastic MCs (HMC-1) and BAs (KU812) as well as cord blood-derived MC and BA progenitor cells were determined by counting cell numbers and the numbers of apoptotic cells. The amount of histamine secreted from primary MCs and BAs was measured by radioimmunoassay after incubation of cells with GO alone or GO together with an anti-immunoglobulin E (IgE) antibody.
MCs and BAs as well as HMC-1 cells and KU812 cells were found to express CD33 mRNA and the CD33 protein. GO was found to inhibit the growth of HMC-1 cells and KU812 cells as well as stem cell factor-dependent differentiation of MCs and interleukin-3-induced growth of BAs from their cord blood-derived progenitors. The GO-induced inhibition of growth of neoplastic cells was found to be associated with induction of apoptosis. GO neither induced secretion of histamine from MCs or BAs nor upregulated the anti-IgE-induced release of histamine in these cells.
GO counteracts growth of normal and neoplastic MCs and BAs without inducing rapid release of histamine. The exact value of GO as a targeted drug for the treatment of high-grade MC or BA neoplasms remains to be determined.
Experimental Hematology 02/2007; 35(1):108-16. · 2.90 Impact Factor
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[show abstract]
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ABSTRACT: Patients with mastocytosis may suffer from severe hypotension after wasp or bee stings. In these patients, no specific IgE is detectable, but they usually have skin lesions and an elevated serum tryptase level.
We report on 6 patients who were referred to our department because of severe hypotension following bee or wasp stings without cutaneous lesions.
In 3 patients, the baseline serum tryptase level was elevated (26, 36, and 67 ng/ml, respectively), and investigation of their bone marrow revealed systemic mastocytosis (SM). In the remaining 3 patients, serum tryptase levels were <20 ng/ml, and bone marrow histology and tryptase immunohistochemistry did not reveal diagnostic mast cell infiltrates. However, in 1 patient, three minor SM criteria were demonstrable leading to the diagnosis SM, and in the 2nd patient, two minor SM criteria, including an aberrant mast cell phenotype, were found. In the 3rd patient, no minor SM criteria were detected.
All patients with unexplained hypotension after hymenoptera stings should undergo a thorough investigation for major and minor SM criteria regardless of the tryptase level or presence of skin lesions, in order to diagnose or exclude SM or a related subdiagnostic condition (1 or 2 minor SM criteria) tentatively termed monoclonal mast cell activation syndrome.
International Archives of Allergy and Immunology 01/2007; 142(2):158-64. · 2.40 Impact Factor
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Karl J Aichberger,
Matthias Mayerhofer,
Anja Vales,
Maria-Theresa Krauth,
Karoline V Gleixner,
Martin Bilban,
Harald Esterbauer, Karoline Sonneck,
Stefan Florian,
Sophia Derdak,
Winfried F Pickl,
Hermine Agis,
Andras Falus,
Christian Sillaber,
Peter Valent
[show abstract]
[hide abstract]
ABSTRACT: Basophil numbers are typically elevated in chronic myeloid leukemia (CML) and increase during disease progression. Histamine is an essential mediator and marker of basophils and is highly up-regulated in CML. We examined the biochemical basis of histamine synthesis in CML cells. The CML-specific oncoprotein BCR/ABL was found to promote expression of histidine decarboxylase (HDC) and synthesis of histamine in Ba/F3 cells. Moreover, the BCR/ABL tyrosine kinase inhibitors imatinib (STI571) and nilotinib (AMN107) decreased histamine levels and HDC mRNA expression in BCR/ABL-transformed Ba/F3 cells, in the CML-derived basophil cell line KU812, and in primary CML cells. Synthesis of histamine was found to be restricted to the basophil compartment of the CML clone and to depend on signaling through the PI3-kinase pathway. CML cells also expressed histamine receptors (HRs), including HR-1, HR-2, HR-4, and histamine-binding CYP450 isoenzymes which also serve as targets of HR antagonists. The HR-1 antagonists loratadine and terfenadine, which bind to CYP450, were found to counteract proliferation of CML cells, whereas no growth inhibition was observed with the HR-1 antagonist fexofenadine which is not targeted or metabolized by CYP450. Moreover, DPPE, an inhibitor of histamine-binding CYP450 isoenzymes, produced growth inhibition in CML cells. Together, these data show that BCR/ABL promotes histamine production in CML cells and that certain HR-targeting drugs exert antileukemic effects on CML cells.
Blood 12/2006; 108(10):3538-47. · 9.90 Impact Factor
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Karoline Sonneck,
Stefan Florian,
Alexandra Böhm,
Maria-Theresa Krauth,
Rudin Kondo,
Alexander W Hauswirth,
Karoline V Gleixner,
Karl J Aichberger,
Sophia Derdak,
Winfried F Pickl,
Wolfgang R Sperr,
Lawrence B Schwartz,
Peter Valent
[show abstract]
[hide abstract]
ABSTRACT: A number of autocrine and paracrine growth regulators are considered to be involved in the survival and proliferation of blast cells in acute myeloid leukemia (AML). We have recently shown that blast cells in a group of patients with AML produce and secrete the mitogenic enzyme tryptase. In the present study, we examined functional effects of tryptase in the context of AML. As assessed by 3H-thymidine uptake experiments, tryptase-containing serum from patients with AML as well as heparin-complexed recombinant tryptase were found to promote the proliferation of cultured bone marrow- and lung fibroblasts in a dose-dependent manner. A neutralizing antibody against human beta-tryptase was found to diminish these growth-stimulatory effects of serum-tryptase in all patients examined. Tryptase also induced the expression of mRNA for GM-CSF and SCF, two cytokines known to promote growth of AML cells, in cultured bone marrow fibroblasts. Neither recombinant tryptase nor tryptase-rich serum of AML patients, showed an effect on the growth of leukemic blast cells irrespective of the FAB category or expression of protease-activated receptor (PAR)-2, a putative molecular target of tryptase. Together, tryptase is secreted from AML blasts as a biologically active molecule that may exhibit paracrine rather than autocrine effects in AML.
Leukemia and Lymphoma 06/2006; 47(5):897-906. · 2.58 Impact Factor
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Friedrich Wimazal,
Maria-Theresa Krauth,
Anja Vales,
Alexandra Böhm,
Hermine Agis, Karoline Sonneck,
Karl J Aichberger,
Matthias Mayerhofer,
Ingrid Simonitsch-Klupp,
Leonhard Müllauer,
Wolfgang R Sperr,
Peter Valent
[show abstract]
[hide abstract]
ABSTRACT: Recent data suggest that vascular endothelial growth factor (VEGF) is produced in neoplastic cells in various myeloid neoplasms and plays a key role as an autocrine regulator and mediator of angiogenesis. We examined the expression of VEGF in paraffin-embedded bone marrow sections obtained from normal donors (n = 5) and 46 patients with myelodysplastic syndromes [MDS, French-American-British (FAB)-type refractory anemia (RA), n = 10; refractory anemia with ringed sideroblasts (RARS), n = 10; refractory anemia with excess blasts (RAEB), n = 10; RAEB in transformation (RAEB-T), n = 8; chronic myelomonocytic leukemia (CMML), n = 8] by immunohistochemistry using an anti-VEGF antibody. In normal bone marrow, the anti-VEGF antibody was found to react with myeloid progenitor cells, immature monocytic cells, plasma cells and megakaryocytes, but not with erythroid cells or mature granulocytic cells. Higher levels of VEGF were found in patients with MDS, subtypes RAEB, RAEB-T and CMML, compared to patients with RA or RARS, or the normal bone marrow. These differences were found to result from expression of VEGF in immature myeloid cells in RAEB, RAEB-T and CMML. The microvessel density was also higher in patients with RAEB-T and CMML compared to RA and RARS or the normal bone marrow. Expression of VEGF mRNA was demonstrable in isolated neoplastic cells by reverse transcriptase-polymerase chain reaction in all patients examined. In aggregate, these data show that VEGF is expressed in bone marrow cells in patients with MDS. The amount of expressed VEGF is related to the percentage of immature myeloid cells (blasts and monocytic progenitors) and correlates with the FAB category.
Leukemia and Lymphoma 04/2006; 47(3):451-60. · 2.58 Impact Factor
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[show abstract]
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ABSTRACT: Recent data suggest that myeloid neoplasms are organized hierarchically in terms of self-renewal and maturation of early progenitor cells, similar to normal myelopoiesis. In acute myeloid leukemia (AML), the NOD/SCID mouse-repopulating leukemic stem cells usually co-express CD123 with CD34, but lack CD38. So far, however, little is known about expression of other markers and targets on these progenitors. In the present study, expression of target antigens on CD34+/CD38- cells was analysed by multi-color flow cytometry in patients with AML (n = 18), myelodysplastic syndromes (MDS, n = 6), chronic myeloid leukemia (CML, n = 8) and systemic mastocytosis (SM, n = 9). The IL-3Ralpha chain (CD123) was found to be expressed on CD34+/CD38- cells in a majority of the patients in all disease categories. Independent of the type of disease, the vast majority of these stem cells co-expressed aminopeptidase-N (CD13) and CD44 in all patients. By contrast, the CD34+/CD38- progenitor cells expressed variable amounts of the target receptor CD33, c-kit (CD117) and AC133 (CD133). In conclusion, neoplastic stem cells in various myeloid neoplasms appear to express a similar phenotype including target antigens such as CD13, CD33 and CD44. Since many of these targets are not expressed on all stem cells in all patients, the elimination of the entire clone may require combinations of targeted antibodies or use of additional drugs.
Leukemia and Lymphoma 03/2006; 47(2):207-22. · 2.58 Impact Factor
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ABSTRACT: High-dose cytosine arabinoside (HiDAC) and intermediate-dose cytosine arabinoside (IDAC) have been introduced as effective and safe consolidation chemotherapy in acute myeloid leukemia, with relatively low rates of life-threatening infections despite the high total dose of the cytostatic drug. To explore the biological background of low toxicity, we examined the numbers, immunophenotype, and functional properties of granulocytes in patients with acute myeloid leukemia receiving HiDAC or IDAC. Interestingly, the absolute numbers of neutrophils remained >500/microl until day 10 in 92 of 125 (74%) HiDAC cycles and in 106 of 113 (94%) IDAC cycles. As assessed by electron microscopy, these day-10 granulocytes surviving chemotherapy were found to be mature cells containing secondary granules and phagolysosomes. They also expressed opsonization- and phagocytosis-linked surface Ags (C3biR, CR1, C1qR, C5aR, FcgammaRI, FcgammaRII, FcgammaRIII, and G-CSF and GM-CSF receptors) like neutrophils in healthy controls. Moreover, these day-10 neutrophils exhibited oxidative burst activity and took up and digested bacteria in the same way as neutrophils in healthy controls. There was a negative correlation between absolute neutrophil counts and severe infections in HiDAC- and IDAC-treated patients with a later onset of infections in IDAC patients (median: IDAC, day 18; HiDAC, day 16). Together, functionally mature neutrophils are detectable at least until day 10 in patients treated with HiDAC or IDAC, and may explain the relatively low hematologic toxicity of these consolidation protocols. IDAC is a superior protocol in this regard and may therefore be most suitable for elderly patients and those at high risk for severe infections.
The Journal of Immunology 03/2006; 176(3):1759-68. · 5.79 Impact Factor
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Karoline V Gleixner,
Matthias Mayerhofer,
Karl J Aichberger,
Sophia Derdak, Karoline Sonneck,
Alexandra Böhm,
Alexander Gruze,
Puchit Samorapoompichit,
Paul W Manley,
Doriano Fabbro,
Winfried F Pickl,
Christian Sillaber,
Peter Valent
[show abstract]
[hide abstract]
ABSTRACT: In most patients with systemic mastocytosis (SM), including aggressive SM and mast cell leukemia (MCL), neoplastic cells express the oncogenic KIT mutation D816V. KIT D816V is associated with constitutive tyrosine kinase (TK) activity and thus represents an attractive drug target. However, imatinib and most other TK inhibitors fail to block the TK activity of KIT D816V. We show that the novel TK-targeting drugs PKC412 and AMN107 counteract TK activity of D816V KIT and inhibit the growth of Ba/F3 cells with doxycycline-inducible expression of KIT D816V as well as the growth of primary neoplastic mast cells and HMC-1 cells harboring this KIT mutation. PKC412 was a superior agent with median inhibitory concentration (IC(50)) values of 50 to 250 nM without differences seen between HMC-1 cells exhibiting or lacking KIT D816V. By contrast, AMN107 exhibited more potent effects in KIT D816V(-) HMC-1 cells. Corresponding results were obtained with Ba/F3 cells exhibiting wild-type or D816V-mutated KIT. The growth-inhibitory effects of PKC412 and AMN107 on HMC-1 cells were associated with induction of apoptosis and down-regulation of CD2 and CD63. PKC412 was found to cooperate with AMN107, imatinib, and cladribine (2CdA) in producing growth inhibition in HMC-1, but synergistic drug interactions were observed only in cells lacking KIT D816V. Together, PKC412 and AMN107 represent promising novel agents for targeted therapy of SM.
Blood 02/2006; 107(2):752-9. · 9.90 Impact Factor
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ABSTRACT: It is generally appreciated that bone marrow function and growth of myelopoietic cells depends on an intact microvasculature. A pivotal regulator of angiogenesis is vascular endothelial growth factor (VEGF). Here, we describe analysis of VEGF expression and microvessel density in the bone marrow of patients with aplastic anemia by immunohistochemistry. Bone marrow was examined at diagnosis and at the time of hematological remission after immunosuppressive therapy using anti-thymocyte globulin, cyclosporin A, and glucocorticoids or allogeneic stem cell transplantation. At diagnosis, both VEGF expression and microvessel density were found to be significantly lower in aplastic anemia compared to normal bone marrow (aplastic anemia, 1.1 +/- 0.7 events per field, versus controls, 5.9 +/- 3.0 events per field; P < 0.05). In response to successful therapy, VEGF and microvessel density in the bone marrow increased substantially. Serum VEGF levels were also found to be significantly lower at diagnosis in aplastic anemia compared to healthy controls (aplastic anemia, 51 +/- 35 pg/ml versus controls, 444 +/- 220 pg/ml; P < 0.05). VEGF in the serum increased substantially after successful immunosuppressive therapy or stem cell transplantation (P < 0.05). Taken together, these data show that aplastic anemia is associated with reduced angiogenesis and reduced VEGF expression.
American Journal Of Pathology 01/2006; 168(1):123-30. · 4.89 Impact Factor
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ABSTRACT: An unusual case of secondary acute myeloid leukemia (AML) with indolent clinical course is described. The patient, a 67-yr-old female, had first been diagnosed to suffer from low-risk myelodysplastic syndrome, subtype refractory anemia with ringed sideroblasts, in 1992. In 2001, transformation to secondary AML with an increase in bone marrow blasts (>20%) and thrombocytopenia, was found. The patient did not require cytoreductive drugs. Rather, during the following months, spontaneous improvement of peripheral blood cells with normalization of platelets and decrease in the red cell transfusion frequency, were noted. In October 2002, she even became transfusion independent. However, the bone marrow still showed AML with >20% blasts. These blast cells exhibited a monoclonal pattern in the human androgen receptor (HUMARA) assay. However, no chromosomal defects occurred during a total observation period of 14 yr. We hypothesize that clonal stability may have contributed to the indolent course of the disease in this patient. The exact mechanisms underlying clinical and genetic stability remain unknown, however.
European Journal Of Haematology 08/2005; 75(1):73-7. · 2.61 Impact Factor
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Stefan Florian,
Minoo Ghannadan,
Matthias Mayerhofer,
Karl J Aichberger,
Alexander W Hauswirth,
Gerit-Holger Schernthaner,
Dieter Printz,
Gerhard Fritsch,
Alexandra Böhm, Karoline Sonneck,
Maria-Theresa Krauth,
Michael R Müller,
Christian Sillaber,
Wolfgang R Sperr,
Hans-Jörg Bühring,
Peter Valent
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ABSTRACT: Signal regulatory proteins (SIRPs) and tyrosine phosphatases have recently been implicated in the control of receptor tyrosine kinase (RTK)-dependent cell growth. In systemic mastocytosis (SM), neoplastic cells are driven by the RTK KIT, which is mutated at codon 816 in most patients. We examined expression of SIRPalpha, SIRPalpha ligand CD47, and Src homology 2 domain-containing protein tyrosine phosphatase-1 (SHP-1), a tyrosine phosphatase-type, negative regulator of KIT-dependent signaling, in normal human lung mast cells (HLMC) and neoplastic MC obtained from nine patients with SM. As assessed by multicolor flow cytometry, normal LMC expressed SIRPalpha, CD47, and SHP-1. In patients with SM, MC also reacted with antibodies against SIRPalpha and CD47. By contrast, the levels of SHP-1 were low or undetectable in MC in most cases. Corresponding data were obtained from mRNA analysis. In fact, whereas SIRPalpha mRNA and CD47 mRNA were detected in all samples, the levels of SHP-1 mRNA varied among donors. To demonstrate adhesive functions for SIRPalpha and CD47 on neoplastic MC, an adhesion assay was applied using the MC leukemia cell line HMC-1, which was found to bind to immobilized extracellular domains of SIRPalpha1 (SIRPalpha1ex) and CD47 (CD47ex), and binding of these cells to CD47ex was inhibited by the CD172 antibody SE5A5. In summary, our data show that MC express functional SIRPalpha and CD47 in SM, whereas expression of SHP-1 varies among donors and is low compared with LMC. It is hypothesized that CD172 and CD47 contribute to MC clustering and that the "lack" of SHP-1 in MC may facilitate KIT-dependent signaling in a subgroup of patients.
Journal of Leukocyte Biology 07/2005; 77(6):984-92. · 4.99 Impact Factor
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ABSTRACT: Using two-colour flow cytometry >200 antibodies submitted to the 8th International Workshop of Human Leukocyte Differentiation Antigens (HLDA8) have been analyzed for their reactivity with resting and activated CD203c+ basophils. Four antibodies either non-reactive or weakly reactive with resting basophils exhibited an increased reactivity with basophils activated by anti-IgE-mediated cross-linking of the high affinity IgE receptor (FceRI). These include antibodies against CD164 (WS-80160, clone N6B6 and WS-80162, clone 67D2), as well as two reagents with previously unknown specificities that were identified as CD13 (WS-80274, clone A8) and CD107a (WS-80280, clone E63-880). The activation patterns followed either the "CD203c-like" or "CD63-like" activation profile. The CD203c profile is characterized by a rapid and significant upregulation (of CD13, CD164, and CD203c), reaching maximum levels after 5-15 min of stimulation. The Phosphoinositide-3-kinase (PI3K)-specific inhibitor Wortmannin inhibited the upregulation of these markers whereas 12-O-tetradecanoyl-phorbol-13-acetate (TPA) induced a rapid and FceRI-independent upregulation within 1-2 min. In the CD63 profile, maximum upregulation (of CD63 and CD107a) was detected only after 20-40 min, and upregulation by TPA reached maximum levels after 60 min. In summary, our data identify CD13, CD107a, and CD164 as novel basophil-activation antigens. Based on time kinetics of upregulation, we hypothesize that molecules of the "CD203c group" and the "CD63 group" are linked to two different mechanisms of basophil activation.
Cell Research 06/2005; 15(5):325-35. · 8.19 Impact Factor
