Alexandra Böhm

Immunosuppression and atypical infections in CML patients treated with dasatinib at 140 mg daily

Background  The multikinase inhibitor dasatinib exerts growth‐inhibitory effects in patients with imatinib‐resistant chronic myeloid leukaemia (CML). In first clinical trials, side effects of dasatinib, 140 mg daily, were reported to be mild and tolerable.

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.

Hematopoietic Stem-Cell Transplantation for Advanced Systemic Mastocytosis

PURPOSE Advanced systemic mastocytosis (SM), a fatal hematopoietic malignancy characterized by drug resistance, has no standard therapy. The effectiveness of allogeneic hematopoietic stem-cell transplantation (alloHCT) in SM remains unknown. PATIENTS AND METHODS In a global effort to define the value of HCT in SM, 57 patients with the following subtypes of SM were evaluated: SM associated with clonal hematologic non-mast cell disorders (SM-AHNMD; n = 38), mast cell leukemia (MCL; n = 12), and aggressive SM (ASM; n = 7). Median age of patients was 46 years (range, 11 to 67 years). Donors were HLA-identical (n = 34), unrelated (n = 17), umbilical cord blood (n = 2), HLA-haploidentical (n = 1), or unknown (n = 3). Thirty-six patients received myeloablative conditioning (MAC), and 21 patients received reduced-intensity conditioning (RIC). RESULTS Responses in SM were observed in 40 patients (70%), with complete remission in 16 patients (28%). Twelve patients (21%) had stable disease, and five patients (9%) had primary refractory disease. Overall survival (OS) at 3 years was 57% for all patients, 74% for patients with SM-AHNMD, 43% for those with ASM, and 17% for those with MCL. The strongest risk factor for poor OS was MCL. Survival was also lower in patients receiving RIC compared with MAC and in patients having progression compared with patients having stable disease or response. CONCLUSION AlloHCT was associated with long-term survival in patients with advanced SM. Although alloHCT may be considered as a viable and potentially curative therapeutic option for advanced SM in the meantime, given that this is a retrospective analysis with no control group, the definitive role of alloHCT will need to be determined by a prospective trial.

Evaluation of antileukaemic effects of rapamycin in patients with imatinib-resistant chronic myeloid leukaemia.

Background  Recent data suggest that the mammalian target of rapamycin (mTOR) is involved in the regulation of growth of neoplastic cells in chronic myeloid leukaemia (CML).

Identification of basogranulin (BB1) as a novel immunohistochemical marker of basophils in normal bone marrow and patients with myeloproliferative disorders.

In myeloproliferative disorders (MPDs), basophils typically increase in number in the bone marrow (BM) and blood. In chronic myeloid leukemia (CML), basophilia is a diagnostic and prognostic variable. However, no reliable approach for routine detection and enumeration of basophils in BM sections is available. We applied the antibasogranulin antibody BB1 on paraffin-embedded BM sections in 21 control samples (normal BM), 45 patients with CML, 9 with chronic idiopathic myelofibrosis, 11 with polycythemia vera, 19 with essential thrombocythemia, and 7 with indolent systemic mastocytosis. As assessed by immunostaining of serial BM sections, BB1+ cells coexpressed myeloperoxidase, histidine decarboxylase, and leukosialin but did not express B- or T-cell-restricted antigens. BB1+ BM cells were found to be highly elevated in patients with CML compared with normal BM or other MPDs, with maximum counts found in accelerated phase CML (median, 160 cells/mm(2)). In summary, BB1 (basogranulin) is a new immunohistochemical basophil marker that should allow quantification of basophils in CML at diagnosis and during therapy.

Immunohistochemical detection of vascular endothelial growth factor (VEGF) in the bone marrow in patients with myelodysplastic syndromes: correlation between VEGF expression and the FAB category.

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.

In vitro and in vivo growth-inhibitory effects of cladribine on neoplastic mast cells exhibiting the imatinib-resistant KIT mutation D816V

OBJECTIVE 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. MATERIALS AND METHODS 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. RESULTS 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. CONCLUSIONS 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.

A Hypoallergenic Vaccine Obtained by Tail-to-Head Restructuring of Timothy Grass Pollen Profilin, Phl p 12, for the Treatment of Cross-Sensitization to Profilin

Profilins are highly cross-reactive allergens in pollens and plant food. In a paradigmatic approach, the cDNA coding for timothy grass pollen profilin, Phl p 12, was used as a template to develop a new strategy for engineering an allergy vaccine with low IgE reactivity. Non-IgE-reactive fragments of Phl p 12 were identified by synthetic peptide chemistry and restructured (rs) as a new molecule, Phl p 12-rs. It comprised the C terminus of Phl p 12 at its N terminus and the Phl p 12 N terminus at its C terminus. Phl p 12-rs was expressed in Escherichia coli and purified to homogeneity. Determination of secondary structure by circular dichroism indicated that the restructuring process had reduced the IgE-reactive α-helical contents of the protein but retained its β-sheet conformation. Phl p 12-rs exhibited reduced IgE binding capacity and allergenic activity but preserved T cell reactivity in allergic patients. IgG Abs induced by immunization of mice and rabbits with Phl p 12-rs cross-reacted with pollen and food-derived profilins. Recombinant Phl p 12-rs, rPhl p 12-rs, induced less reaginic IgE to the wild-type allergen than rPhl p 12. However, the rPhl p 12-rs-induced IgGs inhibited allergic patients’ IgE Ab binding to profilins to a similar degree as those induced by immunization with the wild type. Phl p 12-rs specific IgG inhibited profilin-induced basophil degranulation. In conclusion, a restructured recombinant vaccine was developed for the treatment of profilin-allergic patients. The strategy of tail-to-head reassembly of hypoallergenic allergen fragments within one molecule represents a generally applicable strategy for the generation of allergy vaccines.

Targeting of mTOR is associated with decreased growth and decreased VEGF expression in acute myeloid leukaemia cells.

Background  The mammalian target of rapamycin (mTOR) has recently been implicated in leukaemic cell growth, tumour‐associated angiogenesis and expression of vascular endothelial growth factor (VEGF). We examined whether mTOR plays a role as regulator of growth and VEGF‐expression in acute myeloid leukaemia (AML). Three mTOR‐targeting drugs, rapamycin, everolimus (RAD001) and CCI‐779, were applied. The effects of these drugs on growth, survival, apoptosis and VEGF expression in primary AML cells and various AML cell lines were examined.

Enumeration and immunohistochemical characterisation of bone marrow basophils in myeloproliferative disorders using the basophil specific monoclonal antibody 2D7

Background: Basophils are highly specialised granulocytes that express a unique profile of antigens and increase in myeloproliferative disorders (MPD). In chronic myeloid leukaemia (CML), basophilia is a diagnostic and prognostic determinant. So far, however, no reliable approach for routine detection and enumeration of bone marrow basophils has become available. Objective: To detect and enumerate basophils in bone marrow sections in patients with CML and other MPD Methods: The anti-basophil antibody 2D7 was applied to paraffin embedded bone marrow sections from normal/reactive subjects (n = 31), patients with CML (chronic phase, n = 37; accelerated phase, n = 9), and other MPD (chronic idiopathic myelofibrosis (CIMF), n = 20; polycythaemia vera (PV), n = 20; essential thrombocythaemia (ET), n = 20; indolent systemic mastocytosis (ISM), n = 7). Results: As assessed by serial section staining, 2D7+ cells were found to co-express myeloperoxidase, histidine decarboxylase, CD9, and CD43, but did not express B cell or T cell restricted antigens. 2D7+ bone marrow cells were found to increase in CML compared with normal/reactive bone marrow and other MPD (median numbers of 2D7+ cells/mm2: CML, 33; normal/reactive bone marrow, 6; CIMF, 10; PV, 6; ET, 5; ISM, 3; p<0.05). The highest basophil counts were recorded in accelerated phase CML (115/mm2). Conclusions: A novel immunohistochemical procedure has been established for basophil detection in normal bone marrow and MPD. This approach should help in the quantification of bone marrow basophils at diagnosis and during anti-leukaemic treatment.