Karoline Sonneck

Detection of molecular targets on the surface of CD34+/CD38− stem cells in various myeloid malignancies

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-3Rα 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.

Identification of CD13, CD107a, and CD164 as novel basophil-activation markers and dissection of two response patterns in time kinetics of IgE-dependent upregulation.

ABSTRACTUsing 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 (FcεRI). 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 FcεRI-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.

Diagnostic and Subdiagnostic Accumulation of Mast Cells in the Bone Marrow of Patients with Anaphylaxis: Monoclonal Mast Cell Activation Syndrome

Background: 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. Methods: We report on 6 patients who were referred to our department because of severe hypotension following bee or wasp stings without cutaneous lesions. Results: 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. Conclusions: 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.

Oncogenic Kit controls neoplastic mast cell growth through a Stat5/PI3-kinase signaling cascade

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.

Synergistic growth-inhibitory effects of two tyrosine kinase inhibitors, dasatinib and PKC412, on neoplastic mast cells expressing the D816V-mutated oncogenic variant of KIT

Background and Objectives 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. Design and Methods We examined the effects of the novel TK-inhibitor dasatinib alone and in combination with other targeted drugs on growth of neoplastic MC. Results 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. Interpretation and Conclusions Combinations of targeted drugs may represent an interesting pharmacologic approach for the treatment of aggressive SM or MCL.

Indolent Systemic Mastocytosis with Elevated Serum Tryptase, Absence of Skin Lesions, and Recurrent Severe Anaphylactoid Episodes

Background: In contrast to aggressive mastocytosis, patients with indolent systemic mastocytosis (ISM) usually present with urticaria pigmentosa-like skin lesions. In those who lack skin lesions, mastocytosis is often overlooked or confused with endocrinologic, allergic, or other internal disorders. Case Report and Results: We report on a 33-year-old male patient in whom severe hypotensive episodes occurred after contact with ants or yellow jackets. Since no specific IgE was detected, the serum tryptase concentration was measured and found to be clearly elevated (70 ng/ml). Consecutive staging and examination of the bone marrow revealed ISM. The patient was advised to circumvent insect contact, to take antihistamines on demand, and to carry an epinephrine self-injector for emergency events. In a retrospective analysis of 40 patients seen between 1988 and 2003, only 2 had a life-threatening mediator-related episode before ISM was diagnosed. Conclusions: Our report confirms the diagnostic value of tryptase in patients with suspected mastocytosis. In addition, the report suggests that the lack of typical skin lesions does not exclude an indolent form of mastocytosis even if the serum tryptase is clearly elevated. Finally, our case further shows that mastocytosis can be an important differential diagnosis to be considered in patients with unexplained anaphylactoid or other mediator-related symptoms.

Expression of activated STAT5 in neoplastic mast cells in systemic mastocytosis: subcellular distribution and role of the transforming oncoprotein KIT D816V.

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.

Interleukin-3 promotes the expression of E-NPP3/CD203C on human blood basophils in healthy subjects and in patients with birch pollen allergy.

We recently identified the ectoenzyme CD203c as a novel basophil activation antigen that is up-regulated in response to FceRI cross-linkage. We investigated the effects of various interleukins (ILs) on expression of CD203c on blood basophils using an antibody against CD203c and flow cytometry. Of all cytokines tested, only IL-3 was found to upregulate expression of CD203c on basophils above baseline levels. The effects of IL-3 were dose- and time-dependent (EC50: 0.1–1 ng/ml) without differences observed between healthy and allergic donors. Whereas anti-IgE induced maximum upregulation of CD203c within 15 minutes, the IL-3-induced upregulation showed a maximum after 180 minutes. IgE-receptor cross-linking resulted in enhanced expression of both CD63 and CD203c, whereas IL-3 enhanced the levels of CD203c without promoting expression of CD63. The IL-3-induced upregulation of CD203c was also observed in highly enriched basophils and was counteracted by a blocking antibody against the alpha chain of the IL-3 receptor (CD123). The IL-3-induced upregulation of CD203c was also found to depend on the presence of calcium. To analyze signaling pathways involved in IL-3-induced upregulation of CD203c, pharmacologic inhibitors were applied. The PI3-kinase inhibitors, wortmannin and LY294002 counteracted the IL-3-induced expression of CD203c, whereas MEK- and PKC inhibitors showed no effects. In conclusion, IL-3 upregulates expression of CD203c on basophils through a specific receptor and via a PI3-kinase-dependent signaling-pathway. Compared to FceRI-mediated cell activation, IL-3-induced upregulation of CD203c is a late(r) event and is not accompanied by upregulation of CD63.

Detection of novel leukocyte differentiation antigens on basophils and mast cells by HLDA8 antibodies

Background:  Basophils (BA) and mast cells (MC) are important effector cells in allergic reactions. Development, growth and effector cell functions are regulated by a network of cytokines, other ligands, and respective cell surface antigens.

Reducing allergenicity by altering allergen fold: a mosaic protein of Phl p 1 for allergy vaccination.

Background:  The major timothy grass pollen allergen, Phl p 1, resembles the allergenic epitopes of natural group I grass pollen allergens and is recognized by more than 95% of grass‐pollen‐allergic patients. Our objective was the construction, purification and immunologic characterization of a genetically modified derivative of the major timothy grass pollen allergen, Phl p 1 for immunotherapy of grass pollen allergy.