Alexander W. Hauswirth

Standards and standardization in mastocytosis: Consensus Statements on Diagnostics, Treatment Recommendations and Response Criteria

Although a classification for mastocytosis and diagnostic criteria are available, there remains a need to define standards for the application of diagnostic tests, clinical evaluations, and treatment responses. To address these demands, leading experts discussed current issues and standards in mastocytosis in a Working Conference. The present article provides the resulting outcome with consensus statements, which focus on the appropriate application of clinical and laboratory tests, patient selection for interventional therapy, and the selection of appropriate drugs. In addition, treatment response criteria for the various clinical conditions, disease‐specific symptoms, and specific pathologies are provided. Resulting recommendations and algorithms should greatly facilitate the management of patients with mastocytosis in clinical practice, selection of patients for therapies, and the conduct of clinical trials.

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.

Expression of the target receptor CD33 in CD34+/CD38-/CD123+ AML stem cells.

Background  CD33 (Siglec‐3) is becoming increasingly important as a target of antibody‐mediated therapy in acute myeloid leukaemia (AML). In normal myelopoiesis, expression of CD33 is restricted to advanced stages of differentiation, whereas primitive stem cells do not express CD33. In the present study, we asked whether leukaemic stem cells in patients with AML express CD33.

Formation of Disulfide Bonds and Homodimers of the Major Cat Allergen Fel d 1 Equivalent to the Natural Allergen by Expression in Escherichia coli

Dander from the domestic cat (Felis domesticus) is one of the most common causes of IgE-mediated allergy. Attempts to produce tetrameric folded major allergen Fel d 1 by recombinant methods with structural features similar to the natural allergen have been only partially successful. In this study, a recombinant folded Fel d 1 with molecular and biological properties similar to the natural counterpart was produced. A synthetic gene coding for direct fusion of the Fel d 1 chain 2 N-terminally to chain 1 was constructed by overlapping oligonucleotides in PCR. Escherichia coli expression resulted in a non-covalently associated homodimer with an apparent molecular mass of 30 kDa defined by size exclusion chromatography. Furthermore, each 19,177-Da subunit displayed a disulfide pattern identical to that found in the natural Fel d 1, i.e. Cys3(1) Cys73(2), Cys44(1)-Cys48(2), Cys70(1)-Cys7(2), as determined by electrospray mass spectrometry after tryptic digestion. Circular dichroism analysis showed identical folds of natural and recombinant Fel d 1. Furthermore, recombinant Fel d l reacted specifically with serum IgE, inducing expression of CD203c on basophils and lymphoproliferative responses in cat-allergic patients. The results show that the overall fold and immunological properties of the recombinant Fel d 1 are very similar to those of natural Fel d 1. Moreover, the recombinant Fel d 1 construct provides a tool for defining the three-dimensional structure of Fel d 1 and represents a reagent for diagnosis and allergen-specific immunotherapy of cat allergy.

Detection of novel CD antigens on the surface of human mast cells and basophils.

Background: Mast cells (MC) and basophils are effector cells of allergic reactions. Growth and function of these cells are regulated by a network of cytokines, other ligands, and respective cell surface membrane receptors. Methods: In the present study, we examined the expression of novel CD antigens on human lung MC, skin MC, blood basophils, the MC line HMC-1, and the basophil cell line KU-812. Expression of surface antigens was analyzed by monoclonal antibodies (mAbs) and indirect immunofluorescence staining techniques. Results: Primary MC were found to react with mAbs against KIT (CD117), the signal regulatory protein SIRP-α (CD172a), and the ectoenzyme E-NPP3 (CD203c). Human basophils were found to express large amounts of E-NPP3 and lower levels of Siglec-5 (CD170), CXCR4 (CD184) and SIRP-α. The HMC-1 cell line was recognized by mAbs against SIRP-α, CXCR4, endothelial protein C receptor (CD201) and E-NPP3. KU-812 cells were found to react with mAbs against E-NPP3, CXCR4 and glycophorin C (CD236R), but did not react with mAb against endothelial protein C receptor. Most of the other CD antigens tested disclosed negative results. Conclusions: In summary, our data provide further evidence that MC and basophils express a unique composition of surface antigens. The use of novel CD antibodies may help to isolate MC and basophils and to study their functional properties.

Identification of mTOR as a novel bifunctional target in chronic myeloid leukemia: dissection of growth-inhibitory and VEGF-suppressive effects of rapamycin in leukemic cells

The mammalian target of rapamycin (mTOR) has recently been described to be constitutively activated in Bcr‐Abl‐transformed cells and to mediate rapamycin‐induced inhibition of growth in respective cell lines. We have recently shown that rapamycin down‐regulates expression of vascular endothelial growth factor (VEGF), a mediator of leukemia‐associated angiogenesis, in primary CML cells. In the present study, we analyzed growth‐inhibitory in vitro and in vivo effects of rapamycin on primary CML cells and asked whether rapamycin‐induced suppression of VEGF in leukemic cells is related to growth inhibition. Rapamycin dose dependently inhibited growth of primary CML cells obtained from patients with imatinib‐responsive or imatinib‐resistant disease as well as growth of Bcr‐Abl‐transformed imatinib‐resistant cell lines. Moreover, we observed potent cytoreductive effects of rapamycin in a patient with imatinib‐resistant Bcr‐Abl+ leukemia. The growth‐inhibitory effects of rapamycin on CML cells were found to be associated with G1 cell cycle arrest and with induction of apoptosis. In all cell types tested, rapamycin was found to down‐regulate expression of VEGF. However, exogenously added VEGF did not counteract the rapamycin‐induced decrease in proliferation. In conclusion, rapamycin inhibits growth of CML cells in vitro and in vivo and, in addition, down‐regulates expression of VEGF. Both effects may contribute to the antileukemic activity of the drug in CML.

Autoimmune thrombocytopenia in non-Hodgkin’s lymphomas

Autoimmune thrombocytopenia is a potentially life-threatening complication in non-Hodgkin’s lymphomas. This study focuses on the temporal relationship between autoimmune thrombocytopenia and lymphoma, the frequency in lymphoma subtypes, laboratory data and treatment outcome. Autoimmune thrombocytopenia is a common immunehematologic complication in non-Hodgkin’s lymphomas and may complicate the treatment. We analyzed an original series from our institute as well as published cases of non-Hodgkin’s lymphomas (excluding chronic lymphocytic leukemia) associated with autoimmune thrombocytopenia with regard to demographic factors, prevalence in non-Hodgkin’s lymphoma subtypes and treatment outcome. The male/female ratio is 1.75. Half of the cases occurred prior to diagnosis of lymphoma. Chemotherapy is the best treatment in many non-Hodgkin’s lymphomas patients with autoimmune thrombocytopenia compared with standard treatment of autoimmune thrombocytopenia. Splenectomy is effective in splenic marginal zone lymphoma. Autoimmune thrombocytopenia in patients with non-Hodgkin’s lymphomas is potentially life-threatening and difficult to treat.

Myelomastocytic Leukemia: Evidence for the Origin of Mast Cells from the Leukemic Clone and Eradication by Allogeneic Stem Cell Transplantation

Purpose: Myelomastocytic leukemia is a term used for patients with advanced myeloid neoplasms, in whom elevated numbers of immature atypical mast cells are found, but criteria for a primary mast cell disease are not met. The origin of mast cells in these patients is presently unknown. Patient and Methods: We have analyzed clonality of mast cells in an 18-year-old patient suffering from acute myeloid leukemia with a complex karyotype including a t(8;21) and mastocytic transformation with a huge increase in immature mast cells and elevated serum tryptase level, but no evidence for a primary mast cell disease/mastocytosis. Results: As assessed by in situ fluorescence hybridization combined with tryptase staining, both the tryptase-negative blast cells and the tryptase-positive mast cells were found to contain the t(8;21)-specific AML1/ETO fusion gene. Myeloablative stem cell transplantation resulted in complete remission with consecutive disappearance of AML1/ETO transcripts, decrease of serum tryptase to normal range, and disappearance of neoplastic mast cells. Conclusion: These data suggest that mast cells directly derive from the leukemic clone in patients with myelomastocytic leukemia.

Detection of differentiation- and activation-linked cell surface antigens on cultured mast cell progenitors.

Background:  Mast cells (MC) are multifunctional effector cells of the immune system. They derive from uncommitted CD34+ hemopoietic progenitor cells (HPC). Depending on the stage of maturation and the environment, MC variably express differentiation‐ and activation‐linked antigens. Little is known, however, about the regulation of expression of such antigens in immature human MC.

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.